Autophagy-Related (Atg) Genes in Rainbow Trout (Oncorhynchus Mykiss) and Their Role in Nutrient Restriction in Vivo and in Vitro

Autophagy-related (Atg) genes in rainbow trout (Oncorhynchus mykiss) and their role in nutrient restriction in vivo and in vitro

by Jaramar Balmori-Cedeño

A Thesis Presented to The University of Guelph

In partial fulfillment of requirements for the degree of Master of Science in Pathobiology

Guelph, Ontario, Canada © Jaramar Balmori-Cedeño, September 2017 ABSTRACT Autophagy-related (Atg) genes in rainbow trout (Oncorhynchus mykiss) and their role in

nutrient restriction in vivo and in vitro

Jaramar Balmori-Cedeño Advisor: University of Guelph, 2017 Professor John S. Lumsden

The expression of ten autophagy-related genes (ATG4, ATG5, ATG7, ATG9, ATG12, ATG13,

ATG16, BECN-1, GABARAP and LC3) was examined in RTgill-W1 (gill epithelial) cells and in muscle and liver from rainbow trout (Oncorhynchus mykiss) undergoing serum nutrient restriction and compared to control groups. Six of these sequences, ATG5, ATG7, ATG9, ATG13,

ATG16 and BECN-1, were previously unknown in rainbow trout. The rainbow trout sequences had highest nucleotide sequence similarity with Atlantic salmon and other fish species used for comparison in this study. In serum-restricted RTgill-W1 cells, most autophagy-related genes were significantly expressed at Day 3. In liver from feed-restricted fish, most Atg were significantly upregulated within the first 4 days. Autophagy-related gene expression in muscle was significantly upregulated between Day 8 and 15. Both tissues had a second peak of Atg upregulation at Day 21 suggesting that autophagy is maintained for longer periods of time in fish than previously understood.

ACKNOWLEDGEMENTS

I would like to thank my advisor Dr. John Lumsden for his professional academic support throughout my MSc. program and for shaping my work to its best form. I would also like to express my gratitude to my committee Dr. Brandon Lillie for his guidance and contribution through the last two years. I would also like to thank Mr. R. Frank and M. Cornish from the

Hagen Aqualab. None if this would be possible without your dedication.

A very special thank you to Dr. Ehab Misk, Paul Huber, Leah Read and Pat Bell Rogers for teaching me all the techniques I needed, and for having the patience to answer all my doubts.

Your support made this work possible. To my graduate student colleagues, Ehab Misk, Ryan

Horricks, Doran Kirkbright, Juan-Ting Liu, Maureen Jarau and Paige Vroom, thank you for our talks and support when things didn’t go as planned. To Elein Hernandéz and Wendy Xiao, thank you for your friendship. A special thank you to Donna Kangas, Karla de Uslar, Marny Struyk and Cathy Bernardi, who helped me through all the administrative process and always had a smile when I needed it.

I would like to thank my funding sources NSERC, the Ontario Veterinary College (OVC) and

Mitacs Inc.

Finally, I’d like to thank my parents and sister, who have always pushed me forward and have never let me fall, Amanda Macfarlane, for making me part of her friend-family group, and to my boyfriend, Wes Darrah, for his kind love and infinite patience. You have all helped me get to where I am.

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DECLARATION OF WORK PERFORMED

All worked reported in this thesis was performed by Jaramar Balmori-Cedeño under the supervision of Dr. John Lumsden and my advisory committee Dr. Brandon Lillie, with the following exceptions:

The fish trial from which the muscle and liver samples were obtained for Chapter 3 was performed by Ian Ryerse and Juan-Ting Liu. The RT-qPCR for autophagy-related genes protocols were developed by Dr. Ehab Misk and adapted and optimized by myself. The Western blot used for Chapter 2 was developed and performed by Juan-Ting Liu.

The statistical analysis was done with the help of Dr. William Sears, from the Department of

Population Medicine, University of Guelph.

Table of Contents

CHAPTER 1: LITERATURE REVIEW ...... 1 1.1 AUTOPHAGY ...... 1 1.1.1 Macroautophagy...... 3 1.1.2 Microautophagy ...... 3 1.1.3 Chaperone Mediated Autophagy...... 4 1.1.4 Autophagosome ...... 4 1.1.5 Molecular Machinery of Autophagy ...... 6 1.1.6 Core Components of Autophagy Machinery ...... 6 1.2 THE ROLE OF AUTOPHAGY IN NUTRIENT RESTRICTION ...... 11 1.3 THE EFFECT OF AUTOPHAGY IN FISH DURING FEED RESTRICTION ...... 11 1.4 AUTOPHAGY IN FISH DURING INFECTIOUS DISEASE ...... 13 1.5 TECHNIQUES TO STUDY AUTOPHAGY ...... 15 1.6 TRANSCRIPTIONAL STUDIES OF AUTOPHAGY-RELATED GENES ...... 16 RATIONALE ...... 19 HYPOTHESES AND OBJECTIVES ...... 21 CHAPTER 2: Autophagy-related (Atg) genes in rainbow trout (Oncorhynchus mykiss) and their role in nutrient restriction in RTgill-W1...... 23 2.1 ABSTRACT ...... 24 2.2 INTRODUCTION ...... 25 2.2 MATERIAL AND METHODS ...... 30 2.2.1 Autophagy-Related Genes Sequences in Rainbow Trout ...... 30 2.2.2. Primer Design ...... 30 2.2.3. PCR Validation and Optimization ...... 35 2.2.4. Cell Culture ...... 35 2.2.5. Cell Experimental Conditions ...... 36 2.2.6. RNA Extraction...... 37 2.2.7 RNA Column Purification ...... 37 2.2.8. Reverse Transcription Polymerase Chain Reaction (RT-PCR) ...... 38 2.2.9. qPCR Primer Standardization ...... 38 2.2.10. Reference Gene Validation ...... 39 2.2.11 Western Blot Assay ...... 44 2.2.12 Statistical Analysis ...... 45

2.3 RESULTS ...... 46 2.3.1 Autophagy-Related Gene Selection ...... 46 2.3.2. PCR Validation and qPCR Primer Assessment ...... 57 2.3.3. Selection of Reference Gene(s) ...... 71 2.3.4. Quantitative Real-Time PCR Assay...... 73 Figure 2.8. a) Western blot representing the effect of serum restriction (1%, 2%, and 10% serum percentages) on RTgill-W1 cells after incubation for 48 hours. Percentage for stacking gel was 4% and for resolving gel 15%. b) After densitometry the ratio of LC3 to β-tubulin was used to measure relative changes of LC3 expression and of expressed as means +- SD (n=2) and were analyzed with a one-way ANOVA Different letters represent significantly different values (p < 0.05). The graphs represent LC3/β-tubulin ratio.2.4 DISCUSSION ...... 81 Chapter 3. Autophagy-related (Atg) gene expression in the liver and muscle of rainbow trout (Oncorhynchus mykiss) undergoing feed restriction ...... 87 3.1 ABSTRACT ...... 88 3.2 INTRODUCTION ...... 89 3.3 MATERIAL AND METHODS ...... 94 3.2.1 Fish Trials ...... 94 3.2.2 RNA Extractions ...... 95 3.2.3 RNA Column Purification ...... 95 3.2.4 Reverse Transcription Polymerase Chain Reaction (RT-PCR) ...... 97 3.2.5 Reference Gene Validation ...... 98 3.2.6 Statistical Analysis ...... 98 3.4 RESULTS ...... 100 3.3.1 Reference Gene Selection ...... 100 3.3.2 Semi-quantitative Reverse Transcription- PCR Assay in Liver...... 102 3.3.3 Quantitative Real-Time PCR Assay in Muscle ...... 107 3.5 DISCUSSION ...... 112 GENERAL DISCUSSION AND CONCLUSIONS ...... 117 REFERENCES ...... 123 APPENDIX ...... 134 Appendix 2.1: Rainbow trout Atg gene sequences of Atg4, Atg12, LC3 and GABARAO from NCBI (Madden, 2002)...... 134 Appendix 2.2a-f) Clustal alignments between rainbow trout Atg genes and sequences from 14 other species...... 136 Appendix 2.2b) Clustal alignments between rainbow trout Atg genes and sequences from 14 other species...... 150

Appendix 2.3a-b) BLASTN results of forward a) and reverse b) sequences from PCR product of Atg gene primers. Table A. Forward sequence ...... 249 Appendix 2.4. Relative expression of Atg genes under different degrees of serum restrictions251 Appendix 2.5a-c) Statistical analysis of Atg gene expression in RTgill-W1 cells. Three-factorial- factor (gene, time, treatment) analysis was performed using SAS ...... 252 Appendix 3.1a-c: Statistical analysis of Atg gene expression in liver and muscle. Three-factorial- factor (gene, time, treatment) analysis was performed using SAS ...... 261

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List of Tables

Table 2.1a-b Characteristics of rainbow trout (Oncorhynchus mykiss) autophagy-related gene primers used in the present study………………………………………………………….……..32

Table 2.2 Autophagy-related gene primer sequences and NCBI gene sequence accession number…………………………………………………………………………………………...34

Table 2.3a-b Characteristics of rainbow trout (Oncorhynchus mykiss) reference gene primers used in the present study…………………………………………………………………………41

Table 2.4 Reference gene primer sequences and rainbow trout gene sequence accession number from NCBI web site…………………………………………………………...…………………43

Table 2.5 Autophagy-related genes elected as possible molecular biomarkers………………….47

Table 2.6 Percent identity matrix of autophagy-related genes between rainbow trout

(Oncorhynchus mykiss) and selected species………………………………………………….....48

Table 2.7 Organisms included in the phylogenetic trees and autophagy-related gene information……………………………………………………………………………………….49

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List of Figures

Figure 1.1 Types of autophagy in mammalian cells…………………………...…………….……2

Figure 1.2 Overview of the general autophagy pathway……………………………………….…5

Figure 1.3. Autophagy molecular machinery……………………………………………………10

Figure 2.1 Phylogeny trees of ATG5, ATG7, ATG9, ATG13, ATG16 and BECN1 a-e….……....51

Figure 2.2 Agarose gel for reference genes 18S, EF1, rs11, β-actin and α-tubulin………….….58

Figure 2.3 Agarose gel for reference genes ATG4, ATG5, ATG9, ATG13, LC3 and BECN1…...59

Figure 2.4 Atg gene (ATG5, ATG7, ATG9, ATG13, ATG16 and BECN1) sequences aligned to primers………………………………………………………………………...…………………60

Figure 2.5 Ct of reference genes…………………….………………………..………………….72

Figure 2.6 Atg expression during feed restriction in RTgill-W1 cells…………….……….……75

Figure 2.7 a-d Ratio of gene expression (1.5/ 10% FBS) in RTgill-W1…………...……………77

Figure 2.8 Western blot representing effect of serum restriction………………………….…….80

Figure 3.1 Ct values of reference genes………………………………………..……………….101

Figure 3.2 a-d Atg gene expression of liver from rainbow trout under feed restriction………..103

Figure 3.3 a –d Ratio of Atg gene expression (feed restricted/control) in liver...... …….....105

Figure 3.4 a-d Atg gene expression of muscle from rainbow trout under feed restriction……..108

Figure 3.5 a –d Ratio of gene expression (feed restricted/control) in muscle ………………....110

List of Abbreviations

Atg autophagy-related

BLAST basic local alignment tool

BLASTn basic local alignment search tool - nucleotide

Bp base pair cDNA complementary deoxyribonucleic acid

Ct crossing point value

DNA deoxyribonucleic acid

FBS fetal bovine serum

LL low level

PBS phosphate-buffered saline

PCR polymerase chain reaction

Prob probability

QC query coverage

RNA ribonucleic acid

RoR report of odds ratio

RT-qPCR quantitative reverse transcription polymerase chain reaction

RTgill-W1 rainbow trout gill epithelial cell line

Tm melting temperature

TBE tris-borate-EDTA buffer

UL upper level

CHAPTER 1: LITERATURE REVIEW

Salmonids, including rainbow trout, are known to go through periods of feed restriction in natural and farmed conditions, managing to survive for long periods of time (Seiliez et al., 2010).

Proteins are an important source of energy during this period (Seiliez et al., 2012).

One of the pathways feed restriction activates is the autophagy/lysosomal pathway, which has been shown to partly control protein catabolism (Seiliez et al., 2012). Additionally, feed restriction triggers a defense response against certain pathogens, which can result in a reduction in mortalities (Mohapatra et al., 2016). Aquaculture operations have been known to restrict feed intake in order to manage diseases. This strategy has been gaining importance as an alternative to drug use in disease management (Vojo, 2011). The following literature review examines some of these aspects of food restriction, autophagy and its importance in fish.

1.1 AUTOPHAGY

Autophagy is a highly conserved pathway that exists in organisms as diverse as yeast and mammals. The purpose of autophagy is to clear cells of long-lived proteins, damaged organelles, invading microorganisms and apoptotic bodies. It also has a role in development, cell survival, nutrient deprivation, immunology and aging. Alterations in autophagy are important in numerous pathological situations such as neoplasia, neurodegenerative diseases and muscle diseases. There are three major types of autophagy: macroautophagy, chaperone mediated autophagy (CMA) and microautophagy (Cuervo and Macian, 2012) (Figure 1.1).

Fig. 1.1 Redrawn from: Types of autophagy in mammalian cells (Singh & Cuervo, 2011)

1.1.1 Macroautophagy

Macroautophagy, referred as autophagy for the remainder of this thesis, is highly upregulated during stress conditions and nutrient limitations (Bernard et al., 2015). Autophagy degrades dysfunctional organelles, portions of the cytoplasm and pathogens through double membrane structures called autophagosomes. The phagophore sequestrates the cargo and elongates. Once it fuses at both ends and completely engulfs the cargo, it is called an autophagosome. The autophagosome fuses with the vacuole (yeast) or lysosome (mammals), forming the single membrane autophagolysosome. The cytoplasmic cargo is then degraded by hydrolases, forming molecules that are recycled to the cytosol (Ferraro & Cecconi, 2007). These molecules are reused by the cell, maintaining cellular homeostasis (Burman and Ktistakis, 2010).

1.1.2 Microautophagy

Microautophagy involves the direct engulfment of cytoplasmic cargo at the surface of vacuoles in yeast and lysosomes in mammals. Microautophagy machinery uses both macroautophagy and microautophagy specific genes. Its primary function is membrane homeostasis, cell survival under nitrogen restriction and maintenance of organelle size (Li et al., 2012). It can be non- selective or selective. Selective microautophagy sequesters specific organelles for their degradation. (Cuervo & Macian, 2012).

1.1.3 Chaperone Mediated Autophagy

Chaperone mediated autophagy (CMA) is a selective type of autophagy that degrades soluble proteins. Cytosolic proteins with an aminoacid sequence for a pentapeptide motif biochemically related to KFERQ (peptide sequences that target cytosolic proteins for lysosomal proteolysis) are recognized by heat shock protein hsc70. They are delivered to a lysosome-associated protein

(LAMP-2A). The substrate is then translocated and degraded in the lysosomal lumen

(Bandyopadhyay et al., 2008). Stressors like starvation and oxidative stress can upregulate CMA.

The regulation pathway is directly linked to LAMP 2A and degradation depends on accessibility to hsc70 (Cuervo & Macian, 2012).

1.1.4 Autophagosome

Autophagosomes are double-membrane vesicles that sequester cytoplasmic cargo and deliver it to the lysosome for degradation. This results in macromolecules which are released for use by the cell through the action of permeases (Rubinsztein et al., 2012).

The major steps in the autophagy cycle are cargo recognition, cytoplasmic cargo sequestration, elongation and expansion of the phagophore, lysosomal delivery, degradation and recycling of macromolecules (Yang & Daniel, 2010) (Figure 1.2). Formation of a phagophore in mammals requires a set of molecular machinery, which include autophagy-related (Atg) proteins and genes

(Galluzzi et al., 2012; Rubinsztein, Shpilka & Elazar, 2012).

Fig. 1.2 Redrawn from: Overview of the general autophagy pathway (Green & Levine 2014).

1.1.5 Molecular Machinery of Autophagy

Autophagy requires a core molecular machinery for autophagosome formation. There are 33 identified Atg proteins, 18 of which are involved in starvation-induced autophagy in yeast

(Klionskky et al., 2016). The proteins that regulate the autophagosome formation are divided into the following major groups (Figure 1.3) (Daniel and Zhifen, 2010):

 Atg1 kinase complex

 Phosphatidylinositol 3-kinase (PI3K) complex

 Atg9

 Atg2-Atg18 complex

 Two ubiquitin-like conjugation systems (Atg12-Atg5 and Atg8/LC3)

1.1.6 Core Components of Autophagy Machinery

Atg1 complex

Atg13, Atg17, Atg11, Atg29 and Atg31 proteins compose the core components of the autophagy machinery in yeast. In mammals, it is composed of serine/threonine protein kinase ULK1 (the mammalian orthologue of Atg1), Atg13, FIP200 and Atg101. The Atg1 complex is present in the prephagosomal structures (PAS) in yeast and would be the one of the first proteins to activate, with Atg1 and Atg13 mediating PAS organization. Its functions include a kinase-dependent role in autophagosome formation and a kinase-independent recruitment of downstream Atg factors

(Cheong et al., 2008). Atg13 is needed for autophagy induction, Atg1 activity, and cytoplasm to vacuole targeting. During starvation, the hypo-phosphorylated Atg13 induces localization of

Atg1 and Atg17 (Jung et al., 2010). In mammals ULK1 is present in the cytosol and translocates

6 to the ER when autophagy is activated, probably forming PAS-like structures (Itakura and

Mizushima, 2010b). In mammals, ULK1, Atg13, FIP200 and Atg101 form a complex under normal conditions, and under autophagy induction, translocate to the autophagosome formation site (Egan et al., 2011).

PI3K complex

The PI3K complex is composed of proteins Vps34, Vps15, Atg6 (Beclin-1 is the mammalian orthologue) and Atg14; in mammals, it also includes AMBRA1. The PI3K complex is needed for autophagy induction, as it is responsible for phosphatidylinositol 3-phosphate (PI3P) formation.

PI3P is contained in autophagosome membranes and is essential for autophagosome formation.

Atg6/Beclin 1 functions in the initial phases of autophagosome formation. It promotes nucleation of autophagic vesicles and increases the size of pre-autophagosomal membranes by recruiting proteins from the cytosol (Ferraro et al., 2007).

Atg2 and Atg18 complex

The Atg2-Atg18 complex acts downstream of the Atg1 and the PI3K complexes. Atg2-Atg18 binds with PI3P through the Arg-Arg-Gly motif. Atg2 is needed for PAS localization of Atg18, leading to Atg18 binding with PI3P and regulating the autophagy pathway. It has recently been suggested that the Atg2-Atg18 complex also functions at the final steps of autophagosome formation, inhibiting Atg8 deconjugation by Atg4 and facilitating Atg8- phosphatidylethanolamine recruitment to the PAS (Nair et al., 2010).

Mammalians have 2 orthologues of Atg18:WIPI1 and WIPI2. They possibly have an important role in maturation of the phagosome into an autophagosome (Itakura and Mizushima, 2010a).

Atg9

In PAS organization, hierarchical studies have shown that Atg9 is downstream of the Atg1 complex. In yeast, Atg9 containing structures localize to PAS structures in an Atg17-dependant manner under nutrient restriction conditions (Sekito et al., 2009). It is hypothesized that Atg9 structures are delivered to the PAS, supplying critical factors and lipids to the autophagosome

(Ferraro et al., 2007). Atg9 later localizes to the outer membrane of the autophagosome and is absent in mature autophagosomes. In mammals, Atg9 localizes to autophagy related structures, but how it interacts with other Atg proteins is not well known (Takahashi et al., 2011).

Ubiquitin-like conjugation systems

The two ubiquitin-like conjugation systems work in the expansion and closure of the membrane, during the late steps of autophagosome formation (Suzuki, Kirisako, Kamada, Mizushima, Noda

& Ohsumi, 2007). Atg12-Atg5 localizes to the autophagosome membrane and Atg8 can be used to trace the formation of the autophagosome as well as of the autophagolysosome (Kabeya et al.,

2000).

Atg8/LC3

Atg8 has several mammalian orthologues, which form a multigene family and include LC3 and

Gabarap. Atg4 functions as a deconjugation enzyme, producing the active form of LC3-I. It is then transferred to Atg10 (E2 enzyme), forming LC3-II, which can localize to the autophagosome membrane (Itakura and Mizushima, 2010a). Atg4 cleaves off LC3-PE from the outer surface of the autophagosome after it fuses with the lysosome, and LC3-PE is subsequently degraded by hydrolases in the inner membrane. LC3-II reflects autophagosome formation

(Ferraro et al., 2007).

Atg12-Atg5 Conjugation System

Atg7 activates Atg12 and is then transferred to Atg10 to later conjugates with Atg5 (Mizushima et al., 1998). This complex is formed regardless of nutrient conditions. The pre-autophagosome membrane needs to elongate to form the autophagosomal membrane. This happens when Atg16 binds to Atg12-Atg5. This complex is found in the outer membrane of the pre-autophagosome and disassociates near the end of the maturation of the autophagosome (Suzuki, Kubota, Sekito

& Ohsumi, 2007).

Figure 1.3 Redrawn from: Autophagy machinery (Ding, Manley & Ni, 2011)

1.2 THE ROLE OF AUTOPHAGY IN NUTRIENT RESTRICTION

Autophagy provides energy and nutrients as an adaptive response to stress and starvation.

Various experiments indicate that nutrient status and autophagy are regulated by intracellular nutrient sensors with low nutrient levels activating the autophagy complex and autophagosome formation (Singh et al., 2011). Studies have also shown that glucagon stimulates autophagy and insulin inhibits it in livers of rats (Mortimore &Mondon, 1907). The end product of autophagy are amino acids, which can be used as an energy source (Zhifen & Klionsky 2010).

Amino acids can be used in the Krebs cycle to generate ATP during times of increased stress, like exercise, starvation or disease. Lipids are a better source of energy under nutritional deprivation. Studies in wild-type and ATG5 gene knock-out mouse embryonic fibroblasts and hepatocyte cell line RALA255-10G found that autophagy also regulates lipid breakdown in the liver, although it is not the main pathway. Lipid breakdown results in free fatty acids that can generate energy through mitochondrial β-oxidation. Lipid degradation by autophagy also occurs when intracellular lipids increase (Singh et al., 2009).

1.3 THE EFFECT OF AUTOPHAGY IN FISH DURING FEED RESTRICTION

During starvation, mammalians use glucose and fatty acids and ketone bodies as energy sources, while proteins are considered a last resource. Fish tend to use proteins and lipids earlier in starvation than mammals, and as the starvation period increases, protein becomes an important source of energy (Seiliez et al., 2008). A study in Pacific salmon showed that during spawning

11 migration, fats are used during the first stages of feed-restriction, followed by protein catabolism, with proteins coming from the gut and white muscle tissue first (Mommsen et al., 1980).

Muscle protein degradation in fish is dependent on proteolytic enzyme activity, which is highly regulated and controlled. In the skeletal muscle of vertebrates, protein degradation is thought to be regulated by two conserved pathways (Mohamed et al., 2007):

1) The autophagic/lysosomal pathway

2) The ubiquitin-proteasome pathway

The autophagic/lysosomal pathway is induced during stress situations, like nutrient, nitrogen and/or oxygen restriction. Amino acid sensing and other signals like growth factors, regulate protein kinase TOR, an inhibitor of autophagy. Once TOR is inhibited by nutrient restriction, the autophagic/lysosomal pathway is induced and controls autophagosome formation through autophagy-related proteins and genes that make the molecular machinery required for autophagosome formation. While autophagy-related proteins and their functions have been highly studied in yeast and mammals, autophagy-related genes in fish have just started to be studied in the last years. In fish, both autophagy-related proteins and genes have not been as well studied as in other species and the autophagy/lysosomal pathway is not completely understood in fish (Mohamed et al., 2007).

1.4 AUTOPHAGY IN FISH DURING INFECTIOUS DISEASE

Besides examining the role of autophagy during feed restriction, studies in fish have also focused on autophagy during disease, in particular during virus and bacterial infections. A zebrafish model was used to determine if autophagy inhibited rhabdovirus replication to determine if it is possible to prevent or treat rhabdoviral infections by modulating autophagy (García-Valtanen et al., 2014). Zebrafish embryonic fibroblasts (ZF4) were infected with vesicular stomatitis virus

(VSV) and spring viremia of carp virus (SVCV). To determine if autophagy inhibited replication of either of the viruses, cells were treated with 3-methyladenine (3MA), an autophagy inhibitor, and the amount of viral particles released to the culture media after 24 h was titrated. Cells treated with 3MA had reduced virus compared to cells that were not been treated with 3MA, supporting the idea that autophagy increases replication of these two rhaboviruses (García-

Valtanen et al., 2014).

Studies were also conducted to determine if rhabdoviral G-protein induces autophagy in vivo.

Zebrafish were immunized with the G-protein-encoding plasmids and 3 days after immunization gene expression using microarray was examined. Zebrafish immunized with G-protein-encoding plasmids showed significant upregulation of LC3 in the transcriptomic analysis. At least in this zebrafish model autophagy induction appears to be a response to rhabdovirus infection (García-

Valtanen et al., 2014).

In a different study, salmon cells were infected with infectious salmon anemia virus (ISAV) and cells were observed after 3 d and the number of puncta (autophagosomes) counted; ISAV-

13 infected cells had increased puncta compared to controls, indicating that autophagosomes play a role in ISAV replication. (Schiotz et al., 2010).

The lysosomal degradation pathway of autophagy could prove to play a role in defense against viruses. Studies in the plant Arabidopsis thaliana infected with tobacco mosaic virus (TMV) found that genes ATG7, ATG3 and BECN1 function in limiting TMV replication (Liu et al.,

2005). Autophagy studies in Drosophila compared replication of vesicular stomatitis virus (VSV) between Drosophila cells where autophagy had been inhibited and control cells, suggesting that autophagy activation supressed VSV replication (Shelly et al., 2009). In contrast, studies in rat primary neurons found that upon coxsackievirus infection, autophagy was activated and supported the replication of the virus (Seung et al., 2008). This study is similar to other research in human mammary gland epithelial cells infected with foot-and-mouth disease virus (FMDV) that found that autophagy induction enhanced replication (O´Donnell et al., 2010). Further studies are required to better understand the role of autophagy during viral infections, since the impact may depend on the virus and cellular systems used.

The relationship between bacterial infection and autophagy has received only limited study in fish. Zebrafish have been used as a model to study bacteria-host interaction and the autophagy response. A study with Salmonella typhimurium investigated BECN1 expression and LC3 protein turnover to find a potential relationship between the gene associated with Salmonella proliferation in macrophages (SPVB) and autophagy. The SPVB gene was amplified and introduced to the Salmonella typhimurium strain STM-WT, constructing the SPVB-mutant strain

(STM-SPVB). Zebrafish larvae were immersed in water containing S. typhimurium strain (STM-

WT) or STM-SPVB. At 72 hpi (hours post infection), expression of LC3-II and BECN1 in zebrafish infected with STM-WT were lower than in zebrafish infected with STM-SPVB.

Transmission electron microscopy was used to observe autophagosome membranes in the intestinal epithelial cells of zebrafish, finding them increased upon infection. The results of this study indicated that SPVB can inhibit autophagosome formation in zebrafish (Li et al., 2015).

Studies in autophagy induction in rainbow trout are important to understand its role in host immune response against certain pathogens and how autophagy can work as a defense mechanism against some diseases (Li et al., 2015).

1.5 TECHNIQUES TO STUDY AUTOPHAGY

The main techniques to study autophagy are Western blot, immunofluorescence and electron microscopy. Western blotting and immunofluorescence are used to detect LC3-II, which is currently the only reliable marker of autophagy, while electron microscopy is used to observe autophagosomes in cells. Western blotting has been used in salmonids like Atlantic salmon

(Salmo salar) (Schiotz et al., 2010) and rainbow trout (Onchorhynchus mykiss) (Seiliez et al.,

2010), and cyprinids like zebrafish (Danio rerio) (Yabu et al., 2012) to detect protein LC3-II.

During autophagy, there is change in the LC3-I/LC3-II ratio or an increase in LC3-II is determined relative to a housekeeping gene like β-tubulin. Subcellular localization of LC3 in cells using immunofluorescence has also been used to determine autophagy induction and

15 subcellular localization of LC3 protein in cells of zebrafish embryos and myoblasts of rainbow trout under serum-restricted condition. (Congcong et al., 2010; Seiliez et al., 2012).

Microarray experiments have been used in salmonids, including rainbow trout, to identify the pathways and genes involved during starvation. Studies have focused on evaluating changes in response to starvation in hepatic gene expression in rainbow trout. Seiliez et al. (2008) demonstrated that during amino-acid withdrawal, autophagosome formation increased, inducing expression of GABARAP, LC3 and ATG4 in the white muscle of trout. Critically, neither insulin or IGF1 had an effect on autophagy-related genes suggesting that, at least in rainbow trout white muscle, the pathway differs from that commonly referenced in mammals (Seiliez et al., 2008;

Zhifeng, et al., 2010). The microarray results were confirmed with quantitative real time PCR analysis (Seiliez et al., 2012). Amino acids seem to have a stronger impact in long-term autophagy genes in fish than in mammals (McDue, 2010) and similar effect could be induced by diet (Seiliez et al., 2010; Seiliez et al., 2012). These genes have high potential to serve as molecular biomarkers to study autophagy and protein turnover in starvation (Mohamed et al.,

2007).

1.6 TRANSCRIPTIONAL STUDIES OF AUTOPHAGY-RELATED GENES

Autophagy research has mainly focused on the induction, restriction, and effects of autophagy- related proteins (Klionsky et al., 2007). Researchers have questioned mRNA levels of Atg genes as good markers to study autophagy as they are a second signal for autophagy induction

(Mizushima et al., 2010). However, to better interpret autophagy in nutrient restriction and

16 pathological circumstances, mRNA levels should also be studied (Karim et al., 2014; Bernard et al., 2015).

Most recent studies have focussed on studying transcriptional regulation of Atg genes in yeast and humans. Transcriptional regulation of Atg genes is highly important for autophagy. A study in wild-type cells and a collection of DNA-binding mutants analyzed the expression of ATG1,

ATG7, ATG8, ATG9, ATG14, ATG29 and ATG32. They found that ATG7, ATG8 and ATG9 are upregulated during autophagic activity. They also found that ATG7, ATG8 and ATG9 modulate the autophagosome in different parts of its formation cycle; ATG9 is needed to supply lipids to the expanding phagophore while ATG7 is needed for vesicle expansion and ATG8 for vesicle completion. (Bernard et al., 2014).

A study in rat hepatoma H4-II-E cells studied LC3 protein and LC3 gene levels. They observed enhanced levels of LC3 after 15 min of amino acid restriction and found Atg gene levels increased at 3 h after aminacid restriction, after protein levels were depleted. Their results suggested that the first signal during starvation-mediated activation of autophagy goes to the cytoplasmic protein levels. After protein levels decrease, the second signal activates transcription of Atg genes, maintaining autophagy (Karim et al., 2014).

A study done in human athletes focussed on Atg gene expression changes after periods of intense exercise. Humans ran a marathon of 28 h average, with samples taken 3 h after the marathon

17 finished. They found the expression of ATG4, ATG12, BECN-1, LC3 and ULK2 were upregulated after ultra-endurance in muscle (Jamart et al., 2012).

Studies in fish have focussed on the transcriptional levels of a few Atg genes in myoblasts in vivo and in vitro during serum and nutrient withdrawal in rainbow trout (Yabu et al., 2012;

Seiliez et al., 2012). They discovered significant changes in expression levels of ATG4, LC3 and

GABARAP after 24 h of 0.5% FBS serum restriction, in the case of in vitro experiments, and after 14 d of nutrient restriction in the case of in vivo experiment. They also found LC3 to the most upregulated gene (3.25-fold). They corroborated autophagy induction by the appearance of punctate LC3-containing structures using immunofluorescence and LC3-II induction by Western blot after 4 h of nutrient restriction.

More studies that focus on transcriptional changes in autophagy are needed to understand the role Atg genes play in autophagy maintenance, protein turn-over and pathological events.

RATIONALE

Autophagy likely plays an important role in starvation-associated defense against diseases and muscle mass regulation in rainbow trout (Yabu et al., 2012). Although it has been highly studied in mammals and yeast, it is not until recently that autophagy has been studied in other species, like teleosts and invertebrates (Moreau et al., 2015).

Salmonids experience long periods of feed-restriction that represent a model to study and monitor autophagy induction over a longer period of time. In mammals autophagy had been studied in a period of days (1 week max), but in salmonids, it had been studied for a period of 2 weeks. Rainbow trout, a member of the salmonid group of teleosts, have a higher dietary protein requirement than mammals (Salem et al., 2007) using amino acids as an important energy source. Because of this, fish could be a valuable model to study and understand nutritional regulation of the autophagy pathway. The most common techniques to study autophagy in fish have been Western blots for LC3 and immunofluorescence, although new methods, as mentioned above, are starting to be used. Genetic advances have led to identifying some of the key Atg genes in rainbow trout. ATG4, ATG12, LC3 and GABARAP were upregulated during nutrient and serum restriction in rainbow trout (Seiliez et al., 2012). Further studies should focus on studying gene expression under different conditions to understand how they are regulated.

Atg gene regulation studies may allow us to understand autophagy in this species better, as well as to develop techniques that detect autophagy in a faster, more efficient way, and to understand the role of autophagy during diseases.

I propose that understanding expression of Atg genes, which are crucial for autophagosome formation will allow their use as molecular biomarkers to study and detect autophagy in teleosts, providing better tools to expand our knowledge on how autophagy works in them.

HYPOTHESES AND OBJECTIVES

Hypothesis 1. Since autophagy is so highly conserved, Atg genes of rainbow trout can be identified by aligning their genome to other species and used to study autophagy through long periods of feed-restriction

Objectives

a) From the literature, select essential Atg genes for autophagosome formation that can be

used as molecular biomarkers in rainbow trout.

b) Align chosen Atg gene sequences from Atlantic salmon to the rainbow trout reference

genome and other species to look for regions of high sequence similarity using

phylogenetic analysis.

c) Develop and validate RT-qPCR for 10 selected Atg genes.

Hypothesis 2. Autophagy genes selected at key stages in autophagosome development can be used as molecular markers in rainbow trout under conditions of nutrient restriction in vitro and in vivo.

Objectives

a) Establish a model for nutrient restriction in RTgill-W1 to evaluate selected Atg gene

expression and compare to Western blot for LC3-II.

b) Examine the effect of feed restriction in rainbow trout on Atg gene expression in muscle

and liver.

21 c) Study the autophagosome steps through the expression of Atg genes and their role in the

autophagy machinery.

CHAPTER 2: Autophagy-related (Atg) genes in rainbow trout (Oncorhynchus mykiss) and their role in nutrient restriction in RTgill-W1.

This chapter is formatted according to guidelines of the Journal of Fish Diseases

2.1 ABSTRACT

Autophagy is primarily an adaptive response to provide nutrients and energy following exposure to stress and starvation but can also regulate muscle mass. This project focuses on detecting gene expression of a number of autophagy-related genes in rainbow trout (Oncorhynchus mykiss) throughout the autophagosome formation cycle. Autophagy-related genes (ATG5, ATG7, ATG9,

ATG13, ATG16 and BECN1) from Atlantic salmon (Salmo salar) were aligned to the reference genome (NCBI) of rainbow trout. The sequences obtained were compared to other species to identify highly conserved regions. Phylogeny trees were created with rainbow trout sequences and other 14 species (mammalians, avian, reptile, fungus, fish and plant), finding autophagy- related genes of rainbow trout highly conserved, with greatest similarities to Atlantic salmon and other fish species. RTgill-W1 cells were subjected to nutrient restriction (1.5% FBS) and compared to cells under normal nutrient conditions (10% FBS). Quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) was used to detect autophagy-related gene expression. Nutrient restriction has a direct impact on autophagy related gene expression, with genes ATG4, ATG9, ATG12, LC3, GABARAP and BECN1 undergoing the greatest statistically significant differential expression and most dramatically on Day 3. Autophagy was corroborated by Western blot, which also showed a peak of autophagy activity at Day 3 post-nutrient restriction. This study showed qPCR can be used to study autophagy flux and give important information on how autophagy-related genes behave over time as well as identify which genes are significantly expressed.

2.2 INTRODUCTION

Autophagy is a highly conserved pathway that plays an important role in cellular physiology, adaptive responses to stress and the immune response (Singh & Cuervo, 2011). Many fish, like rainbow trout, go through periods of feed restriction during their life, which is one source of biological stress (Behnke & Coad, 2002). Fish, unlike mammals, preferentially use protein over carbohydrates as a dietary energy source as they are able to derive more metabolizable energy from the catabolism of proteins than from carbohydrates (Cowey, Adron & Knox, 1975). This is especially important during periods of reduced nutritional intake, as proteins are considered the primary gluconeogenic precursors and amino acids become the main source of energy, with carbohydrates been used intermittently (Seiliez et al., 2011). Feed restriction in fish has also been associated with increased resistance to certain infectious pathogens. Rainbow trout

(Oncorhynchus mykiss Walbaum) experimentally infected with Flavobacterium psychrophilum after a period of 4 weeks of pre-infection nutrient restriction that continued for 3 weeks post- infection had a significantly higher survival rate than fully fed controls (Ryerse, Hooft, Bureau,

Hayes and Lumsden, 2104, 2015). Feed restriction has also been demonstrated to reduce the number of mortalities in rainbow trout infected with Flavobacterium branchiophilum. The fed groups showed high mortality 24 h post-infection while the unfed groups only showed mild clinical signs of the disease (MacPhee, Ostland, Lumsden, Derksen and Ferguson, 1995).

Similarly, catfish infected with Edwardsiella ictaluri (Wise, Greenway, Li, Camus and

Robinson, 2007) and Atlantic salmon (Salmo salar) infected with Vibrio salmonicida

(Damsgård, Sorum, Ugelstad and Eliassen, 2004) were more resistant than fully-fed fish.

Macroautophagy, hereafter referred to as autophagy, is highly important in starvation-related protein turnover. Autophagy uses the autophagosome, a double-membrane vesicle, to deliver cytoplasmic cargo to the lysosome for degradation (Burman and Ktistakis, 2010). The major steps in the autophagy cycle are cargo recognition, cytoplasmic cargo sequestration, lysosomal delivery, degradation and recycling of macromolecules, which require a core molecular machinery of autophagy-related (Atg) proteins and genes (Yang and Klionsky, 2010) The main techniques used to study autophagy are detection of LC3-II protein by Western blot and immunofluorescence or observation of autophagosome formation by electron microscopy

(Itakura & Mizushima, 2010). These methods monitor static levels of autophagy-related proteins and are highly labour-intensive, providing semi-quantitative data.

Messenger RNA (mRNA) levels of Atg genes have not been considered good markers to study autophagy as they are a secondary signal for autophagy induction (Mizushima et al., 2010).

However, to better interpret autophagy in nutrient restriction and pathological circumstances gene expression via mRNA levels should also be studied, to provide an insight in autophagy flux and maintenance of autophagy over time. Monitoring temporal Atg gene expression can provide insight into the time point at which autophagy is activated, when peak expression occurs and if more than one round of gene expression occurs (Karim, Kawanago, & Kadowkai, 2014).

Transcriptional regulation of Atg genes in yeast and humans under conditions of nitrogen restriction found that ATG7, ATG8 and ATG9 were upregulated, and that ATG8 expression was correlated with autophagosome size (Jin et al., 2014). To identify the earliest time points during

26 the course of starvation that changes in LC3-mRNA as well as post-translational modification of

LC3 protein occur, complete medium was replaced by Hank's balanced salt solution (HBSS) for

3 h in a rat hepatoma cell line (Karim, Kawanago & Kadowaki, 2014). After 15 min of feed- restriction, they observed an increase in LC3 protein, followed by LC3 gene expression at 3 h post feed restriction. Their results suggested that the first signal, at 15 min, during starvation- mediated activation of autophagy is directed to the cytoplasmic protein levels to initiate the autophagosome formation. This is followed by transcriptional regulation of Atg genes, which would maintain autophagy activity (Karim et al., 2014). This study sets a possible time frame for studying Atg gene expression in vitro. A large collection of yeast DNA-binding mutants were used to study transcriptional control of ATG1, ATG7, ATG8, ATG9, ATG14, ATG29 and ATG32 under conditions of amino acid and glucose restriction, finding upregulation of ATG7, ATG8 and

ATG9 after 2 h of amino acid and glucose restriction (Bernard & Klionsky, 2015). They also studied proteins in the Gata1 family (gcn4, gln3 and gat1), which are known activators of autophagy in yeast and found that their deletion reduced expression of all Atg genes, compared to the controls, confirming the need for Atg proteins for Atg gene expression, and Atg genes as a second signal in autophagy. Vigorous exercise and muscle catabolism can mimic nutrient restriction and Atg gene expression in human athletes was examined after the subjects ran a 200- km running race (Jamart et al., 2012). Periods of high-energy demand stimulate protein catabolism, as proteins become an alternate energy substrate. Skeletal muscle cells degrade proteins and release amino acids through the autophagy-lysosomal pathway (Jamart et al., 2012).

Muscle samples were taken from the subjects 3 h after the marathon was over and gene upregulation of ATG4, ATG12, BECN1 and LC3 occurred, strongly suggesting that these genes

27 are activated in response to muscle catabolism caused by ultra-endurance exercise (Jamart et al.,

2012).

Transcription of Atg genes have not been studied often in fish. Most Atg genes have been characterized in zebrafish and Atlantic salmon, with a limited number in trout. The Atg gene sequences available in GenBank for rainbow trout are ATG4, ATG12, LC3 and GABARAP.

Studies in trout to date have focussed on the transcriptional levels of the above-mentioned Atg in myoblasts during serum withdrawal. They starved the cells using 0.5% FBS for 0.5, 4 and 24 h and compared them to control cells provided 10% FBS (Seiliez et al., 2012). Slight upregulation of these 4 ATG was seen at 4 h with a peak of expression at 24 h and LC3 had the highest level of upregulation (3.25-fold). They corroborated these results by demonstrating the appearance of punctate LC3-containing structures using immunofluorescence and LC3-II induction with

Western blot by 4 h after nutrient restriction (Seiliez et al., 2012). This experiment demonstrated that autophagy can be induced rapidly in trout, like mammals, after nutrient restriction. The increase at 4 h and then at 24 h demonstrates Atg gene expression has an important role in autophagy maintenance, as LC3 and Gabarap proteins need to be replenished to form more autophagosomes (Seiliez et al., 2012. Further studies of the effect of nutrient restriction at short and long term time points in Atg gene expression are needed to fully understand autophagy maintenance and activation.

The purpose of this study was therefore to expand the repertoire of rainbow trout autophagy- associated genes to study their role in nutrient restriction in vitro. Atg genes known to be

28 involved with different stages of autophagosome formation were selected as potential molecular biomarkers in rainbow trout. These included ATG9, BECN1 and ATG13 for autophagy induction,

ATG5, ATG12 and ATG16 were selected for their role in autophagosome formation, ATG7 and

ATG4 for elongation of the autophagosome and GABARAP and LC3 for their role in vesicle completion. RT-qPCR was used to study autophagy flux over longer periods of time (6 d), as well as the impact nutrient restriction has over Atg gene expression in rainbow trout gill cells

(RTgill-W1).

2.2 MATERIAL AND METHODS

2.2.1 Autophagy-Related Genes Sequences in Rainbow Trout

The Atg genes in the present study were chosen based on genes available from other studies and species and their role in the autophagosome cycle. Ten Atg genes were selected based on their demonstrated association within the cycle of autophagosome formation. The Atg gene sequences of Atlantic salmon were blasted against rainbow trout genome (Oncorhynchus mykiss Omyk_1.0,

USDA/ARS) using the software tool-nucleotide BLAST (Madden, 2009) to obtain the most similar Atg gene sequences in rainbow trout. The analysis tool web services from the EMBL-

EBI (McWilliam et al., 2013) was used to align Atg gene sequences of rainbow trout, Atlantic salmon, mice, yeast and human and the determine percent identity matrix.

Phylogenetic trees were created on the six Atg gene sequences from rainbow trout and for the selected sequences from 14 organisms, including six types of fish, four mammalian, one reptile, one avian, one fungal, one fly and one plant. Sequences from start to stop codon were used. The web service Phylogeny.fr was used to create phylogenetic trees. A bootstrapping procedure with

100 bootstraps was used as a statistical test for branch support.

2.2.2. Primer Design

Primers were designed using the tool Primer-BLAST (Ye et al., 2012) with the following target parameters; melting temperatures between 55.0-60.0 °C, a GC content of 50-60% and length of

18-22 bp. Amplicon sizes ranged between 100-150 bp. Primers were analyzed for non-target annealing, secondary structures, self-dimers and hetero-dimers using the software analyzers 30

Oligoanalyzer (Integrated DNA Technologies (IDT); Coralville, USA) and UNAfold (IDT).

Parameters used for each primer can be found in Table 2.1a-b. Primer sequences can be found in

Table 2.2. The specificity of the primers was theoretically assessed by inputting reverse, forward and primer total product sequences in tool-nucleotide BLAST (Madden, 2009) and searching for specific sequences housed at the National Center for Biotechnology Information (NCBI).

Table 2.1a: Characteristics of rainbow trout (Oncorhynchus mykiss) autophagy-related gene primers used in the present study.

Reverse Forward Forward Reverse PCR primer primer primer primer product (R) Tm (F) Tm GC% GC% (bp) (°C) (°C)

ATG4 124 60.81 59.79 52.38 52.38

ATG5 117 60.04 60.04 55.00 55.00

ATG7 111 59.97 59.67 55.00 55.00

ATG9 119 59.96 60.11 55.00 50.00

ATG12 132 60.20 60.34 52.38 57.14

ATG13 126 58.19 58.27 50.00 55.00

ATG16 139 60.25 59.40 60.00 50.00

LC3 135 59.11 58.62 55.00 50.00

GABARAP 108 59.74 60.18 55.00 55.00

BECN1 109 58.63 58.44 52.63 58.44

Table 2.1b: Characteristics of rainbow trout (Oncorhynchus mykiss) autophagy-related gene primers used in the present study (continued).

Self-dimer Hetero-dimer Hairpin score (Max. score (Max. Tm (°C) delta G delta G [kcal/mole]) [kcal/mole])

ATG4 37.5 -3.61 -3.61

ATG5 24.4 -3.61 -6.62

ATG7 30.3 -3.61 -6.14

ATG9 33 -3.14 -5.09

ATG12 43.6 -3.14 -1.94

ATG13 4.6 -4.85 -6..46

ATG16 10.5 -3.65 -4.64

LC3 25.5 -3.14 -3.9

GABARAP 29.5 -3.14 -5.02

BECN1 2.6 -6.76 -6.61

Table 2.2: Autophagy-related gene primer sequences for rainbow trout Atg genes

Autophagy related gene primers for rainbow trout

Gene Forward primer Reverse primer sequence 5ʹ – 3ʹ sequence 5ʹ – 3ʹ

ATG4 GCTGCGATGTGGACAGATGAT AAGGAAGGCGTTGAGGATACC

ATG5 ATTTCCCAGAGCGTGACCTG TGTCGTTGATGACCTGGCTC

ATG7 TTCTGTTCCCTCAGCGTGTC GCCAGTCTCTTTGGGTCCAT

ATG9 TGGGCAATCTGGTGTTCCTC TGCACGTTTGTACTTGGGGT

ATG12 CTTCAACCCCACAACAGCCTA TCCTCCCTTTCTCTACCGACC

ATG13 GGACTTTAAGCCTGCGTTCT GCCATGGACTGAGAACTGAC

ATG16 GAAGGATGAGTACGACGCCC TTCCGCATTGAGCTTGTTGG

LC3 CAGCACCCCAACAAGATTCC GCGCCTCCTGATGATTTTGA

GABARAP AGCCCCCAAAGCAAGGATAG CGCAGGTGGATTCGTTTTCG

BECN1 TGGATGTCGAGAAGGGCAA ATGAACTTGAGCGCCTTGG

2.2.3. PCR Validation and Optimization

DNA from the untreated rainbow trout gill cell line (RTgill-W1) (Bols et al., 1994) was used to test all primers. 20 µl of template were added to 25 µl of TopTaq Master Mix

(QIAGEN), 2.5 µl of primer mix and 2.5 µl of water for a total reaction volume of 50 µl.

The PCR protocol was optimized for initialization at 94.0 °C for 30 s, denaturation at 94.0

°C for 30 s, annealing at 59 °C for 30 s, extension at 72 °C for 1 min for 34 cycles, a final elongation at 72.0 °C for 10 min, and a hold at 4.0 °C. The PCR products were run on a 1% w/v tris-borate (TBE) agarose gel with 10 µl of SYBR Safe gel stain (Invitrogen;

Burlington, ON). The first lane of the agarose gels was loaded with a Quick-Load Purple

Low Molecular Weight Ladder [New England Biolab Inc., Ipswich MA]. Subsequent lanes were loaded with 10 µl of PCR sample with 2 µl of Promega Blue/Orange Loading Dye

[Madison, WI] and negative controls were loaded on another lane. Electrophoresis was performed at 120 V for 45 min, and results visualized by UV transillumination. If there was a single band observed, the PCR products were submitted to the University of Guelph’s

Laboratory Services, Agriculture and Food Laboratory [Guelph, ON] for direct sequencing on the 2720 Thermal Cycler with the ABI Prism BigDye® Terminator Cycle Sequencing

Ready Reaction kit v3.1 [Applied Biosystems, Foster City, CA].

2.2.4. Cell Culture

RTgill-W1 cells were maintained and grown using Leibovitz´s L15 media at a pH of 7.6 after filtration at 0.22 µm. The media was supplemented with 1% antibiotic-antimycotic

(10,000 IU penicillin, 10,000 µg streptomycin, and 24 µg amphotericin B) (Gibco, ON,

Canada) and 10% fetal bovine serum (FBS, certified, heat inactivated, Gibco, ON) was

35 used for cell propagation and 5% FBS for maintenance. Cells were kept and grown in 175 cm polystyrene tissue culture flasks at 15.0 °C (Corning®, Sigma-Aldrich).

2.2.5. Cell Experimental Conditions

The RTgill-W1 cell were cultured in 175 cm tissue culture flasks (Corning®, Sigma-

Aldrich). Cells were kept in Leibovitz’s 15 Medium (L15) (Gibco Life technologies

Corporation, Grand Island, NY). RTgill-W1 cells were washed with versene solution (0.2 g EDTA (Na4) per liter of phosphate buffered saline (PBS) for 4-6 min before been subjected to nutrient restriction to activate autophagy. For a preliminary trial, RTgill-W1 cells were subjected to different serum percentages (0.5, 1.5 and 2%) to identify which one affected Atg gene expression the most. Three biological replicates were used for each serum percentage.

For the main trial, 5 biological replicates were used for control (10% FBS) and 5 biological replicates were used for nutrient restriction (1.5% FBS) at each time point (12 h, 1 d, 3 d, 6 d). TrypLe Reagent (TrypLETM Express Enzyme (1X), Gibco, ON) was used for 3-4 min to split the cells and used once 90% of the flask surface was covered (5 days). Cells were washed with Versene solution for 4-6 min to ensure a reduction in FBS before subjecting them to nutrient restriction or control conditions. Cells were also washed with Versene for

4 min before harvesting using TrypLe Reagent (TrypLETM Express Enzyme (1X), Gibco,

ON) for 3-4 min at each time point.

2.2.6. RNA Extraction

Total RNA was extracted from RTgill-W1 cell samples using Trizol reagent (Invitrogen,

ON, Canada) according to the manufacture’s protocol. Cell samples were homogenized with 1 ml of Trizol reagent in 1.5 ml centrifuge tubes (InvitrogenTM AmbionTM RNase-free

Microfuge Tubes, 1.5 mL). Chloroform (200 µl) was added to the mix which was vortexed, incubated at 5 °C for 5 min and then centrifuged (9600 g, 4 °C, 15 min). Then, the aqueous phase (550 µl) was obtained and deposited into new tubes and isopropanol (550 µl) was added. The mix was gently homogenized (inverted 2-3 times), incubated at 5 °C for 5 min then centrifuged (9400 g, 4.0 °C, 30 min). The pellet was washed and centrifuged again

(3200 g, 4.0 °C, 5 min) with 75% ethanol and re-suspended with PCR grade nuclease-free water (32 µl at 54.0 °C). The extracted RNA concentrations were measured using the

Nanodrop ND-1000 (Nanodrop Tachnologies, DE, USA) and stored at- 80.0 °C until use.

2.2.7 RNA Column Purification

RNA samples were treated with RNase-Free DNase Set (QIAGEN) following the manufacturer’s protocol. Samples were then purified using the RNeasy Midi Kit

(QIAGEN) using a slightly modified protocol. Briefly, a mix of 350 µl of lysis buffer RLT

Buffer and 250 µl of 100% ethanol were added to samples and mixed (up and down). The total volume was then transferred to the columns and centrifuged at 9600 g for 1 min.

Columns were washed twice with RPE solution (500 µl) and centrifuged at 9600 g for 1 min for the first wash and 10,000 g for 2 min for the second wash. The column was transferred to a new collection tube and 40 µl of RNase free water at 54.0 °C was added to the column and centrifuged at 10,000 g for 2 min. The purified RNA concentrations were 37 measured using the Nanodrop ND-1000 (Nanodrop Technologies, DE, USA) and stored at -

80.0 °C.

2.2.8. Reverse Transcription Polymerase Chain Reaction (RT-PCR)

RT-PCR reaction was performed using the High-Capacity cDNA Reverse Transcription Kit

(Applied Biosystems) and RNase OUT (5000 U) (ThermoFisher Scientific). The master mix contained 2 µl of 10x RT Buffer, 0.8 µl of 25x dNTP mix, 2 µl of 10x RT Primers, 1.0

µl of reverse transcriptase, 0.4 µl of RNase inhibitor and 0.6 µl of RNase free water per sample. Samples were normalized to 250 ng/µl of RNA and 6.8 µl of Master Mix was added to each sample to obtain a total volume of 20 µl. Thermal cycling conditions were programmed for a first step of 25 °C for 10 min, a second step of 37 °C for 120 min, a third step of 85 °C for 4 min. and a pause step at 4 °C. cDNA samples were then diluted with

180 µl of RNase free water to obtain a final volume of 200 µl. Samples were stored at -20

°C until use.

2.2.9. qPCR Primer Standardization

Five-fold dilutions of cDNA from untreated cultured cells (RTgill-W1 with 10% FBS) were used as template to assess the efficiency of each primer set. Gene-specific amplification was confirmed by obtaining a single peak in the melting curve. The reaction was prepared using LightCycler 480 SYBR Green, qPCR Master Mix (Roche, Applied

Sciences) containing 5 µl of Master mix SYBR Green, 2.5 µl of primer mix (forward and reverse) and 2.5 µl of sampled cDNA, for a total volume of 10 µl. The cycling conditions

38 were adjusted in the Roche LightCycler® 489 II to a pre-incubation at 95.0 °C for 5 min, an amplification at 95 °C for 10 s, 59 °C for 20 s and 72.0 °C for 15 s for 45 cycles, melting at 95 °C for 5 s, 65 °C for 1 min, with a final cooling step of 40 °C for 10 s. RNA extracted from cultured cells (RTgill-W1 10% FBS) was used as a calibrator (positive control) and water was used as a negative control. Each sample had three technical replicates and qPCR was performed in 384 well plates (Roche, Applied Sciences). Five reference genes were tested in order to select the genes not affected by nutrient restriction. Quantification of gene expression was obtained using standard curve efficiencies (Eq. 1) with the instrument software (the Roche LightCycler® instrument II). The same formula was used to calculate the ratio of 1.5 over 10% serum using the relative calculation procedure REST program

(http://REST.gene-quantification.info/). Samples were normalized using reference genes and calibrator values.

∆퐶푝푡푎푟푔푒푡(푐표푛푡푟표푙−푠푎푚푝푙푒) (퐸푡푎푟푔푒푡) 푟푎푡푖표 = ∆퐶푃푟푒푓(푐표푛푡푟표푙−푠푎푚푝푙푒) (퐸푟푒푓) Eq. (1)

2.2.10. Reference Gene Validation

Five reference genes were selected and designed to follow the same parameters as autophagy-related genes using Primer-BLAST, except for α-tubulin (Misk et al., 2016).

Primers were analyzed for non-target annealing, secondary structures, self-dimers and hetero-dimers using the software analyzers Oligoanalyzer (Integrated DNA Technologies

(IDT); Coralville, USA) and UNAfold (IDT). Parameters for each primer can be found in

Table 2.3a-b and primer sequences can be found in Table 2.4.

The stability of the 5 reference genes was assessed using the web-based tool RefFinder

(http://www.leonxie.com/referencegene.php), which integrates the algorithims for

Normfinder, BestKeeper, geNorm and the comparative ∆Ct method to rank reference genes from most stable to least stable.

Table 2.3a: Characteristics of rainbow trout (Onchorhynchus mykiss) reference gene primers used in the present study.

Forward Forward Reverse PCR Reverse primer primer primer product primer (R) (F) GC% GC% (bp) Tm (°C) Tm (°C)

18S 111 60.25 59.96 55 55

EF1 116 59.96 60.04 55 55

β-actin 100 58.92 58.99 52.63 57.89

rs11 102 59.67 59.82 55 55

α-tubulin 88 59.01 58.97 57.89 57.89

Table 2.3b: Characteristics of rainbow trout (Onchorhynchus mykiss) reference gene primers used in the present study (continued).

Self-dimer Hairpin Hetero-dimer score score (Max. Tm (Max. delta G delta G (°C) [kcal/mole]) [kcal/mole])

18S 29.5 -5.19 -9.31

EF1 27.9 -3.9 -3.9

β-actin 31 -3.14 -8.16

rs11 25.1 -4.16 -5.02

α-tubulin 2.0 -4.41 -9.31

Table 2.4: Reference gene primer sequences and rainbow trout gene sequence accession number from NCBI web site.

Accession Forward primer Reverse primer Gene number sequence 5ʹ – 3ʹ sequence 5ʹ – 3ʹ

18S TCGGGGAGGTAGTGACGAAA ACCAGACTTGCCCTCCAAT AF308735.1 G

β-actin TTTGAGCAGGAGATGGGCA CTCGTTGCCGATGGTGATG AF157514.1

EF-1 TTGAGGATGCCCCCAAGTTC AGCGAAACGACCAAGAGG AF498320.1 AG

rs11 CAAGAGTGTTGGGCTAGGCT CGACCACGGATGGAGACAT NM_001246324. T 1

α-tubulin GGTTCACCTCCCTGCTGAT ACCTGGGGAGCTGGATAGA AY150303.1

2.2.11 Western Blot Assay

To verify the induction of autophagy; conversion of LC3-I to LC3-II protein was detected using Western Blot using LC3B and β-tubulin antibodies (New England Biolabs). Proteins

(20-30 µg/well) were treated with DNase 1 and incubated in a 95 °C water bath for 5 min.

Samples were loaded to SDS-PAGE gel along with a molecular weight marker

(PageRulerTM PlusPrestained Protein Ladder, 10 to 250 kDa, ThermoFisher). The gel for electrophoresis was stacked at 4% SDS-gel electrophoresis at 64.67 V, 0.01-0.03 A for 12 min and resolved using 15% SDS-gel at 150 V, 0.02-0.05 A for 80 min. The gel was then washed with Milli-Q (MQ) water for 1 min and put in cathode buffer and ice bath (0-3° C) for 15 min. Immunodetection included transfer (PierceTM Power Blotter ThermoFisher) at 4

V, 0.07 A for 70 min to PVDF, 0.45 µm transfer membrane (Thermo Scientific Pierce semi-dry blotter). The membrane was then blocked with 5% skim milk in 0.05% phosphate buffer saline with Tween 20 (PBST) on a shaker for 10 min. Membranes were then washed in MQ water and air-dried. The blots were then incubated with the primary antibody (LC3B antibody, New England Biolabs) (1:10,000) diluted in 0.05% PBST) on shaker at 4 °C overnight. The blots were washed with 0.05% PBST 3 times, 5 min/time and the second antibody (1:10,000) diluted in 0.05% PBST) was incubated with agitation at room temperature for 1 h. The membrane was then washed with 0.05% PBST 5 times (5 min/wash) and enhanced chemiluminescence (ECL) prime detection kit (RPN2232,

SIGMA-ALDRICH) was used for detection. Image lab ™ software was used to measure photodensity and a ratio of LC3 to β-tubulin was calculated to measure relative changes of

LC3 expression. The average was taken for 2, 1 and 10% serum respectively. A one-way

ANOVA was performed to evaluate significant data. P values greater than 0.05 were not considered significant.

2.2.12 Statistical Analysis

Stability of the five candidate reference genes was evaluated using the software tool

RefFinder, which compares geNorm, NormFinder5, BestKeeper, and the ΔCt method and gives a ranking of the best genes to use.

For RT-qPCR data analysis, CT values were normalized to the selected reference gene

(EF1) and calibrator values. Quantification of gene expression was obtained using standard curve efficiencies with the instrument software (Roche LightCycler® instrument II) and the relative calculation procedure REST program (http://REST.gene-quantification.info/).

Effect of time and treatment on Atg genes were compared using a three factorial-factor

(gene, time, treatment) ANOVA (SAS). Comparisons between treatment and time with values less than p<0.05 were considered statistically significant and unless stated otherwise significance reported in the present study is with this value. Post-doc analysis was performed using a t-test (least significant difference test). P values greater than 0.05 were not considered significant. The RTqPCR results were normally and symmetrical distributed. For the gene GABARAP, sample 2E1 was eliminated from the analysis as it was

13 standard deviations from its group and was considered to be an error.

2.3 RESULTS

2.3.1 Autophagy-Related Gene Selection

Ten potential Atg genes were chosen as biomarkers at different stages of the autophagy cycle based on previous studies in rainbow trout (Seiliez et al., 2012) and other species

(Bernard et al., 2015; Jamart et al., 2012; Li et al., 2011). The Atg genes used to date in other studies in rainbow trout are ATG4, ATG12, LC3 and GABARAP. The selected genes are indicated in Table 2.5.

A percent identity matrix (Table 2.6) was created between trout sequences (Appendix 2.1).

Alignments showed Atlantic salmon had the closest identity match (78.72 – 98.55%). Most genes were above 84% similarity to Atlantic salmon except ATG4, which ranked the lowest similarity percentage across all species. Zebrafish was the second closest (65.85-84.01%), followed by human (61.05- 81.07%) and finally yeast (49.17-55.93%). Phylogenetic trees were also created for ATG5, ATG7, ATG9, ATG13, ATG16 and BECN1 from sequences from a diverse group of organisms from within and outside the Kingdom Animalia

(mammals, reptiles, avian, fish, plant, insect and fungi) (Table 2.7) (Figures 2.1a-f).

Rainbow trout and Atlantic salmon were within the same clade and close to other fish species. Mammalians clustered together, and fruit fly, sorghum, yeast, red junglefowl and clawed frog diverged in their location depending on the Atg gene. Clustal alignments for the sequences can be found in Appendix 2.2 a-e.

Table 2.5. Autophagy-related genes selected as possible molecular biomarkers.

Autophagy-related genes selected as possible molecular biomarkers

Autophagy-related gene Position in autophagy cycle

ATG9, ATG13, BECN1 Autophagy induction

ATG5, ATG12, ATG16 Autophagosome formation

ATG7, ATG4 Elongation of autophagosome

GABARAP, LC3 Vesicle completion

Table 2.6. Percent identity matrix between rainbow trout (Onchorhynchus mykiss) sequences for autophagy-related and selected species.

Yeast Atlantic Human Black Rainbow trout (Saccharomyces Salmon (Homo Rat Zebrafish autophagy related cerevisiae) (Salmo sapiens) (Rattus (Danio gene sequences* salar) rattus) rerio)

ATG4 49.17 78.72 68.36 64.73 68.93 ATG5 49.85 97.89 70.71 72.95 78.86 ATG7 50.69 84.80 68.28 69.97 75.52 ATG9 44.99 93.01 66.98 66.09 65.85 ATG12 47.01 91.85 61.05 62.55 72.22 ATG13 49.76 98.24 72.17 73.39 76.16 ATG16 50.60 95.12 66.42 65.79 75.56 LC3 49.85 98.41 75.96 74.59 82.66 GABARAP 55.93 97.83 81.07 79.66 84.01 BECN1 50.64 98.44 75.37 74.96 76.64 *Rainbow trout sequences are available in Appendix 2.1

Table 2.7. Organisms included in the phylogenetic trees and autophagy-related gene information

Genus/ Organism Accession Number used for phylogenetic trees and sequence length (bp) Species ATG5 ATG7 ATG9 ATG13 ATG16 BECN1 gi|620601 gi|620635850:<27 gi|620599678:c1086 gi|620599740:6892 gi|620599759:3374 gi|185133437:38- Rainbow Oncorhynchus 708:59912 21-2833, 2931- 695-1086580 97-689365 11-337516 1381 trout mykiss -60019 3117 (2343 bp) (1647 bp) (1197 bp) (1344 bp) (828 bp) (300 bp) gi|291190 gi|929240355:342- gi|929306140:253- gi|929277354:317- gi|929274495:43- Atlantic 515:111- gi|259155099:46- Salmo salar 2786 1902) 2137) 1398 Salmon 938 2196 (230 bp) (2445 bp) (1650 bp) (1821 bp) (1356 bp) (828 bp) XM_004084929.3: NM_0011 XM_004068648.3: AM085511.1) XM_004066617.3: NM_001104778.1: Japanese 200-2011 Oryzias latipes 04780.1 533-2683 (2571 bp) 174-1679 41-1381 rice fish (1812 bp) (828 bp) (2151 bp) (1506 bp) (1341 bp)

XM_0174 XM_017450797.1: (XM_017458742.1: XM_017483306.1: Channel cat Ictalurus 95706.1:4 DQ381245.1 XR_001815151.1 917-3055 184-1722 141-1538 fish punctatus 08-1181 (344 bp) (2487 bp) (2139 bp) (1539 bp) (1398 bp) (774 bp) XM_0116 XM_003966383.2: XM_003973434.2: AM085515.1 XM_011617923.1: Japanese Takifugu 05230.1:1 162-1682 NM_001037874.1 656-2818 (2565 bp) 282-2111) pufferfish rubripe 70-997 (1521 bp) (1344 bp) (2163 bp) (1830 bp) (828 bp) BC077086 NM_200433.2:202- XM_017358254.1: NM_001083031.1:2 NM_001017854.1: NM_200872.1:44- .1:190- 1713 Zebrafish Danio rerio 47-2161 15-2755 177-1169 1387 1011 (1512 bp) (2115 bp), (2418 bp) (993 bp) (1344 bp) (822 bp) NM_0010 NM_001076812.1: NM_001142967.1: AM085499.1:141- NM_001191389.1: AM051355.1:92- Domestic 34579.2:1 266-1708 Bos Taurus 60-2171 2660 149-1972 1438 cattle 04-931 (1443 bp), (2112 bp) (2520 bp) (1824 bp) (1347 bp) (828 bp) gi|620792 gb|BC074011.1|:9 NM_001012097.1: gi|62079162:530- NM_001271212.1: gi|215272400:2- Rattus 30:228- 0-1436 Brown rat 16-2112 3049) 305-1744 1825 norvegicus 881 (1347 bp) (2097bp) (2520 bp) (1440 bp) (1824 bp) (645 bp)

Table 2.7. Organisms included in the phylogenetic trees and autophagy-related gene information (continued)

Organis Genus/ Accession Number used for phylogenetic trees and sequence length (bp) m Species ATG5 ATG7 ATG9 ATG13 ATG16 BECN1 NM_001142673.2: gi|442092991|gb|JQ NM_001144912.1: gi|544583527:236- gi|298229004:508- gb|AF139131.1| 685-2238 Human Homo sapiens 924061.1 118-1989 2755 2079 :121-1473 (1554 bp) (828 bp) (1872 bp) (2520 bp) (1572 bp) (1353 bp)

NM_001257895.1: XM_015137018.1:3 NM_001265972.1: XM_015434821.1: NM_001261111.1: NM_00126668 Rhesus Macaca 30-2549 49-990 89-2200 674-2116 160-1926 5.1:148-1500 monkey mulatta (2520 bp) (642 bp) (2112 bp) (1443 bp) (1767 bp) (1353 bp)

AM085507.1:55- Red XM_015284523.1:1 NM_001030592.1: XM_015287234.1: XM_015277206.1: NM_00100633 2577 junglefo Gallus gallus 63-861 175-2304 347-1687 180-1877 2.1:67-1410 (2253bp), wl (699 bp) (275bp) (1341 bp) (1698 bp) (1344 bp)

NM_001016445.2:5 NM_001129922.1: XM_012970949.2: NM_001102950.1: NM_001102950.1: NM_00103394 Clawed Xenopus 4-755 62-2179 621-3101 292-1824 292-1824 0.2:129-1466 frog tropicalis (702 bp) (230 bp) (2481 bp) (1533 bp) (1869 bp) (1338 bp) NM_141539.4:320 Drosophila XM_001355109.2 NM_137506.4 EU313735.1 NM_001144652.2 AM051363.1 Fruit fly -1891 pseudoobscura (812 bp) (230 bp) (648 bp) (2374 bp) (1257 bp) (1572 bp) XM_002453141.2:1 AM085512.1:181- XM_002448197.2: AM075825.1:1 Sorghu Sorghum XM_021456620.1: KXG37566 12-1236 2847 325-1851) 72-1674 m bicolor 111-3182 (266 bp) (1728 bp) (454 bp) (2667 bp) (1527 bp) (1503 bp) ref|NM_00118266 ref|NM_001183 Saccharomyces NM_0011883963.1 (NM_001179302.) NM_001180209.1 NM_001184282.1 Yeast 3.1 934.11) cerevisiae 847 bp (234 bp) (2994 bp) (2217 bp) (453 bp) (1674 bp)

Figure 2.1a) Phylogeny tree of ATG5 from rainbow trout (Oncorhynchus mykiss) and 14 species: yeast, sorghum, fruit fly, clawed frog, red junglefowl, Atlantic salmon, rainbow trout,, Japanese rice fish, Japanese pufferfish, cat fish, zebrafish, domestic cattle, brown rat, human and rhesus monkey. Sequences were used from start to stop codon. A procedure with 100 bootstraps was used as value support for the branches.

Figure 2.1b) Phylogeny tree of ATG7 from rainbow trout (Oncorhynchus mykiss) and 14 species: yeast, sorghum, fruit fly, clawed frog, red junglefowl, Atlantic salmon, rainbow trout,, Japanese rice fish, Japanese pufferfish, cat fish, zebrafish, domestic cattle, brown rat, human and rhesus monkey. Sequences were used from start to stop codon. A procedure with 100 bootstraps was used as value support for the branches.

Figure 2.1c) Phylogeny tree of ATG9 from rainbow trout (Oncorhynchus mykiss) and 14 species: yeast, sorghum, fruit fly, clawed frog, red junglefowl, Atlantic salmon, rainbow trout,, Japanese rice fish, Japanese pufferfish, cat fish, zebrafish, domestic cattle, brown rat, human and rhesus monkey. Sequences were used from start to stop codon. A procedure with 100 bootstraps was used as value support for the branches.

Figure 2.1d Phylogeny tree of ATG13 from rainbow trout (Oncorhynchus mykiss) and 14 species: yeast, sorghum, fruit fly, clawed frog, red junglefowl, Atlantic salmon, rainbow trout,, Japanese rice fish, Japanese pufferfish, cat fish, zebrafish, domestic cattle, brown rat, human and rhesus monkey. Sequences were used from start to stop codon. A procedure with 100 bootstraps was used as value support for the branches.

Figure 2.1e) Phylogeny tree of ATG16 from rainbow trout (Oncorhynchus mykiss) and 14 species: yeast, sorghum, fruit fly, clawed frog, red junglefowl, Atlantic salmon, rainbow trout,, Japanese rice fish, Japanese pufferfish, cat fish, zebrafish, domestic cattle, brown rat, human and rhesus monkey. Sequences were used from start to stop codon. A procedure with 100 bootstraps was used as value support for the branches.

Figure 2.1f) Phylogeny tree of BECN1 from rainbow trout (Oncorhynchus mykiss) and 14 species: yeast, sorghum, fruit fly, clawed frog, red junglefowl, Atlantic salmon, rainbow trout,, Japanese rice fish, Japanese pufferfish, cat fish, zebrafish, domestic cattle, brown rat, human and rhesus monkey. Sequences were used from start to stop codon. A procedure with 100 bootstraps was used as value support for the branches.

2.3.2. PCR Validation and qPCR Primer Assessment

Conventional PCR’s were run for all primer sets to test their specificity using cDNA from

RTgill-W1 cells under 10% FBS (maintenance percentage). All PCR products amplified a single band (Figure 2.2 and 2.3) at the correct molecular weight. The PCR products were sent for sequencing and the results obtained were then verified by using BLAST-n online

(Appendix 2.3) and found to have a nucleotide similarity of 89-100% percentage to the expected Atg gene, with ATG13 having the lowest identity percentage of 85%. The results showed primers were target-specific for the desired Atg genes. The Atg gene sequences were aligned to their primers and the PCR products and can be found in Figures 2.4a-f.

Figure 2.2. Agarose gel (1%) of 100-150bp products generated from RTgill-W1 cells (10% FBS) with primer sets for reference genes. a) 18S, EF1, rs11, β-actin, b) α-tubulin. PCR were run at 3 different annealing temperatures, 58.0, 59.0 and 60.0 °C.

Figure 2.3. Agarose gel (1%) of 100-150bp products generated from RTgill-W1 cells (10% FBS). a) ATG7, ATG12, ATG16 and GABARAP, b) ATG4, ATG13, LC3 and BECN1, c) ATG5 and ATG9. PCR were run at 3 separate annealing temperatures, 58, 59 and 60 °C.

Figure 2.4a) Rainbow trout (Oncorhynchus mykiss) ATG5 (gi|620601708:59912-60019. The < and > signs represent places where primers aligned to the sequence. The – sign represent places where the PCR product aligned to the sequence. The N represents missing nucleotides on the sequence.

ATGGCAGATGACAAGGACGTGCTGCGGGATGTTTGGTTTGGTCGGATTCCGACCTGCTTC 60 1 M A D D K D V L R D V W F G R I P T C F

ACACTTTACCAGGATGAGATCACAGAGAGAGAGGCCGAACCCTTCTATCTCCTCCTGCCA 120 21 T L Y Q D E I T E R E A E P F Y L L L P

AGGGTGAGCTACCTGACTCTGGTCACAGACAAGGTGAAAAAGCACTTCCTGAAGGTCATG 180 41 R V S Y L T L V T D K V K K H F L K V M

AAGGCCGAAGACGTAGAGGAGATGTGGTTTGATTTCGAAGGAACGCCACTCAAATGGCAC 240 61 K A E D V E E M W F D F E G T P L K W H

TATCCAATCGGATTGCTGTTCGACCTACATGCCTCCAACACTGCCCTGCCATGGAGTATC 300 81 Y P I G L L F D L H A S N T A L P W S I

>>>>>>>>>>>>>>>>>>>> ACTGTGCACTTTAAGAATTTCCCAGAGCGTGACCTGCTCCACTGCCCCTCTAACTCTGTG 360 101 T V H F K N F P E R D L L H C P S N S V ------

<<<<<<< ATCGAGGCCCACTTCATGTCCAGCATCAAGGAGGCGGATGCCCTCAAACACAAGAGCCAG 420 121 I E A H F M S S I K E A D A L K H K S Q ------

<<<<<<<<<<<<< GTCATCAACGACATGCAGAAGAAAGACCACAAGCAGCTGTGGATGGGTCTGCAGAACGAT 480 141 V I N D M Q K K D H K Q L W M G L Q N D ------

AAGTTTGACCAGTTCTGGGCCATGAACCGTAAGCTGATGGAATACCCCACAGAGGAGGGA 540 161 K F D Q F W A M N R K L M E Y P T E E G

GGCTTTCGCTACATCCCCTTCAGGATATACCTGACGATGAGTGACAGGCCATTCATCCAG 600 181 G F R Y I P F R I Y L T M S D R P F I Q

AAGTTGTTTCGCCCCATCTCGTCCGACGGCTACACACACACGCTGGGGGATCTGCTGAAG 660 201 G F R Y I P F R I Y L T M S D R P F I Q

GAGGTGTACCCCATGGCCATATCCAGCGACGACGAGTCCAAGCGCTACCAGGTGGTGATC 720 221 K L F R P I S S D G Y T H T L G D L L K

CATGGGATTGAGCCCCTGCTGGAGACCCCTTTGCAGTGGCTGAGCGAGCACCTCAGTCAC 780

CCTGATAACTTCCTCCACATCAGCGTCATACCCGCGCCTAGCGACTGA 828

Figure 2.4b) Rainbow trout (Oncorhynchus mykiss) ATG7 (gi|620635850:<2721-2833, 2931-3117). The < and > signs represent places where primers aligned to the sequence. The – sign represent places where the PCR product aligned to the sequence. The N represents missing nucleotides on the sequenced.

>>>>>>>>>>>>>>>>>>>> TCTTTGCGTTCTGTTCCCTCAGCGTGTCCAAAGAATGTAGGATGGGAGAAGAACCCTAAA 60 1 M G P R M V N L S E C M D P K R L A E S ------

<<<<<<<<<<<<<<<<<<<< GGAGCCATGGGACCCCGGATGGTCAACCTCAGTGAATGTATGGACCCAAAGAGACTGGCC 120 21 S V D L N L K L M R W R L V P S L N L D ------

GAGTCCTCGGTGGACCTCAACCTGAAGTTGATG-CGCTGGAGGCTGGTACCTTCTCTGAA 180 41 K V T S T K C L L L G A G T L G C N V A

TCTGGACAAAGTGACTTCAACTAAGTGTCTGCTGCTGGGAGCTGGTACACTGGGCTGCAA 240 61 R T L M V S A C D V D M I Q L D F

CGTAGCCAGGACTCTAATGGTGAGTGCCTGCGATGTTGACATGATTCAATTAGATTTCTAG 300

Figure 2.4c) Rainbow trout (Oncorhynchus mykiss) ATG9 (gi|620599678:c1086695- 1086580. The < and > signs represent places where primers aligned to the sequence. The – sign represent places where the PCR product aligned to the sequence. The N represents missing nucleotides on the sequenced.

ATGAGATGGAATTTCCCTTCAATTCTTGTTTTTCTTTACTTGAATTTCCACATCTATCAG 60 1 M R W N F P S I L V F L Y L N F H I Y Q

TTCCATCAGAAGAATGGCTTTGCCTGTATGATGTTATCAGAGTTCTTTGAACTTTTTCAG 120 21 F H Q K N G F A C M M L S E F F E L F Q

TTCCTGTTTGTGGTCACATTCACAACATTCCTGTTCAACTGTGTGGAGTATGATATCCTG 180 41 F L F V V T F T T F L F N C V E Y D I L

TTTGCCAATCGGGCAGTGAACCATACAGGACCTGGCCAGAACCCCCTGGACAGAAACAAG 240 61 F A N R A V N H T G P G Q N P L D R N K

GTTACTCTGCCTGATGCCATCCTACCAAGCCAGCAGTGCACTGAGAGGATCCAGGAGAAC 300 81 V T L P D A I L P S Q Q C T E R I Q E N

AGTTGGATCATCTTCCTGCTCATCATGGCAGCCATCTTCTGGGTCTACCGGATGATCAAG 360 101 S W I I F L L I M A A I F W V Y R M I K

GTCTTCTGCAACGTCCTCCACTACTGGGAGATCCGACAGTTCTACATCAAAGCACTGAAG 420 121 V F C N V L H Y W E I R Q F Y I K A L K

ATCAGAATGGGCGAGCTGTGTAACTTCACGTGGCAGGAGGTGCAGGACCGTCTGATCAGC 480 141 I R M G E L C N F T W Q E V Q D R L I S

CTGCAGCGGGAGCAGCAGATGTGCATCCACAAGAAGGAGCTAACTGAGCTGGACATCTAC 540 161 L Q R E Q Q M C I H K K E L T E L D I Y

CACCGCATCCTGCGCTTCAAGAACTACATGGTAGCCATGGTCAACAAGTCCCTGTTACCT 600 181 H R I L R F K N Y M V A M V N K S L L P

>>>>>>>>>>>>>>>>>>>> GTACAGTTACAGCTCCCCCTGTTGGGCAATCTGGTGTTCCTCACCCAGGGCCTCAAGTAC 660 201 V Q L Q L P L L G N L V F L T Q G L K Y ------

AACTTTGAGCTGATTCTCTTCTGGGGTCCGGGGTCGCTTTTCCAAAACAAATGGAACCTG 720 221 N F E L I L F W G P G S L F Q N K W N L ------

<<<<<<<<<<<<<<<<<<<< CACCCCAAGTACAAACGTGCAGGCAACCGTCTGGAGCTGGCCCAGCAGCTGAGCCGGGTC 780 241 H P K Y K R A G N R L E L A Q Q L S R V ------

ATTCTTCTGATGGGCGTGGCCAACCTGCTCCTCTGCCCCTTCATCCTGGTGTGGCAGGTG 840 62

261 I L L M G V A N L L L C P F I L V W Q V

CTCTACGCCTTCTTCAGCTACACCGAGGTCATCAGGAGGGAGCCGGGGAGTCTGGGAGCA 900 281 L Y A F F S Y T E V I R R E P G S L G A

CGCCGCTGGTCCCTGTACGGCCGCCTCTACCTGCGCCACTTCAACGAGCTGGACCACGAG 960 301 R R W S L Y G R L Y L R H F N E L D H E

CTGCACGGCAGACTGGGTCGCGGCTACAAGCCCACCTCCAAGTACATGAACTCGTTCACC 1020 321 L H G R L G R G Y K P T S K Y M N S F T

TCACCGCTGCTGACCGTGCTGGCCAAGAACGTGGCGTTTTTCTCTGGCTCGGTGCTGGCC 1080 341 S P L L T V L A K N V A F F S G S V L A

GTGCTCATCGCGCTGACCGTGTACGACGAGGATGTTTTGACCGTGCAGCATATCCTGACC 1140 361 V L I A L T V Y D E D V L T V Q H I L T

GCCATCACCGTGCTGGGAGTGGTCATCACCATCACCAGGTCGTTCATCCCAGACGAACAC 1200 381 A I T V L G V V I T I T R S F I P D E H

ATGGTGTGGTGCCCGGAGCAGCTGCTGCAGTGTGTGCTGGCTCACATCCACTACATGCCA 1260 401 M V W C P E Q L L Q C V L A H I H Y M P

GACCACTGGAGGGATAACGCCAACAAGAGTGAGACCCGGGACGAGGTGGCACAGCTGTTT 1320 421 D H W R D N A N K S E T R D E V A Q L F

CAGTACAAAGCGGTGTTCATCCTGGAGGAGCTGCTGAGCCCCATCGTCACGCCCTTCATC 1380 441 Q Y K A V F I L E E L L S P I V T P F I

CTCATCTTCCTCCTGAGGAACAAGTCCCTGGAGATCATCGACTTCTTCAGGAACTTCACC 1440 461 L I F L L R N K S L E I I D F F R N F T

GTGGAGGTGGTCGGAGTTGGCGACATCTGCTCCTTCGCACAGATGGACATCCGTCGCCAC 1500 481 V E V V G V G D I C S F A Q M D I R R H

GGAAACCCACTGTGGATGTCAGAGGGCCAGACAGAGGCATCTGTGTACCAGCAGGCTGAG 1560 501 G N P L W M S E G Q T E A S V Y Q Q A E

AACGGCAAGACTGAGCTGTCCCTGATGCACTTCACCATAAAGAACCCCCACTGGCAACCC 1620 521 N G K T E L S L M H F T I K N P H W Q P

CCACAGGAGAGCTCAGTGTTCATCAGCCATCTAAAGGAGAAGGTGCACCAGGACGCTCAG 1680 541 P Q E S S V F I S H L K E K V H Q D A Q

GGAGGACCCTCCACCCAGCTGCTGCTCTCTGAGGCCCCGCTCTGCTCCTCACTGCTCTCC 1740 561 G G P S T Q L L L S E A P L C S S L L S

AACGAGTCCGGCAACGCACCTGACAACCTATTGGCCAGTGTCCTGGCCCACCCTATTCTG 1800 581 N E S G N A P D N L L A S V L A H P I L

ACCGCGTCAGGCCTACCCGGCCGAGACCGCCGCTTCGTCCCTCCCAGCACGGCTGCCTCA 1860 601 T A S G L P G R D R R F V P P S T A A S

GCCGCCGCCAGCGTCCTGGCCTCGCTGTCCATCTCCCACTCCCAGCTCCCCCACGCCAGC 1920 621 A A A S V L A S L S I S H S Q L P H A S

CGATCCCACTCGCATGTTCTCCTGCCCTCCACCCACCCCGACAGCACCATGTACTGCAGT 1980 641 R S H S H V L L P S T H P D S T M Y C S

GACCGCACTGTCATTGACAGCATGTCAGCCAGTGACTCTCGGATCAGGAGCACGACAATG 2040 661 D R T V I D S M S A S D S R I R S T T M

CTTTCAGAGTTTGCCTTGGCCGAGATGAGTCTCCATGCTATTTACATGCATGAGGTCCAC 2100 681 L S E F A L A E M S L H A I Y M H E V H

CAACAGAACTCGCTCCCCCACCAGAGGACATCAGGCCAGTGGCAGAACCCAATGCCAATG 2160 701 Q Q N S L P H Q R T S G Q W Q N P M P M

AGAGATCTAATCAGCAACACGGGTTCGCAGACGCACAGCGGCCACACAGCAGTCCACCTG 2220 721 R D L I S N T G S Q T H S G H T A V H L

TCTAGCATGCCCACTCCGCTGCGTCTGGGAGGCTGGACGGAGGAAGAGGAGGAGGATGCA 2280 741 S S M P T P L R L G G W T E E E E E D A

GAGGAAGAGGAAGTCCTCAACAGGGGATCGACTCCTAAACAGGACTCCAGGAGTAGTTGT 2340 761 E E E E V L N R G S T P K Q D S R S S C

TAA 2343

Figure 2.4d) Rainbow trout (Oncorhynchus mykiss) ATG13 (gi|620599740:689297- 689365). The < and > signs represent places where primers aligned to the sequence. The – sign represent places where the PCR product aligned to the sequence. The N represents missing nucleotides on the sequence.

ATGGACAGTGATCTAAGTCCTCAGGATAAAAAAGACTTGGACAAGTTCATAAAGTTTTTC 60 1 M D S D L S P Q D K K D L D K F I K F F

GCTTTGAAGACAGTGCAGGTGATAGTCCAAGCGCGACTTGGGGAAAAAGTATGCACACGC 120 21 A L K T V Q V I V Q A R L G E K V C T R

TCCTTGTCTTCACCTACTGGCTCTGATTGGTTCAATCTGGCAATCAAAGATATCCCAGAA 180 41 S L S S P T G S D W F N L A I K D I P E

GTTACACACGAGGCAAAGAAAGCTCTGTCCGGGCAGCTTCCTGGCATCGGACGATCCATG 240 61 V T H E A K K A L S G Q L P G I G R S M

TGTGTTGAGATCTCCCTCAAAACCTCAGAGGGAGACTCAATGGAATTGGAGACATGGTGT 300 81 C V E I S L K T S E G D S M E L E T W C

CTGTCATTACTGCTCAAGTCACTGCTGGCCATTACAAGGGTAACCCCAGCCTACAAACTC 360 101 L E M N E K C D K D I K V S Y T V Y N R

TCACGAAAGCAAGGCCATGACTATGTCATACTGTACAGGATATATTTTGGAGAGGTTCAA 420 121 L S L L L K S L L A I T R V T P A Y K L

CTCACAGGATTGGGAGAAGGGTTCCAGACAGTGCGTGTAGGAGTTGTGGGAACTCCCATT 480 141 S R K Q G H D Y V I L Y R I Y F G E V Q

GGAACAATCACCTTGTCTTGTGCTTACAGGACAAACCTGGCCTTCATGTCCACCAGGCAG 540 161 L T G L G E G F Q T V R V G V V G T P I

TTTGAGAGGACGGCTCCGATTATGGGCATAATTATTGACCACTTCGTTGAGCGCCCCTTT 600 181 G T I T L S C A Y R T N L A F M S T R Q

GGCAACATGGGACACATGCGTCCATGTAACTACAGAGCACCCGGGGAAGATGAAGGAGCA 660 201 F E R T A P I M G I I I D H F V E R P F

TACAATGGGGTGGAAGATTCTCAGGAAGTTGCACCACGTCCTTCTCCACTTCTCCACCC 720 221 G N M G H M R P C N Y R A P G E D E G A

TCACAATGTGTGTTCACTGTAAGTAAGGCACATTTAAAGACCCCTAAACCTGCAGTGATG 780 241 Y N G V E D S Q E V C T T S F S T S P P

GACACTCTGAAAGTCCCCATGATGGGCCTGGCCTTCTCACACCAACTCTACTGCTCTCGT 849 261 S Q C V F T V S K A H L K T P K P A V M

CTTTCTTATCAGCCTCCTGTTCTTGTCGGAGCTGCTGACTTCTGCCACCCTTCCACCTTA 900 281 D T L K V P M M G L A F S H Q L Y C S R

AACGCTGCCAACCCACACCAGGTAACCACCCAGATGGGAATCCCAGGGAAGGAGGGGGGC 960 301 L S Y Q P P V L V G A A D F C H P S T L

GTACCCCAGCAGGTGCCTGTGCAGCCTTGTCATGGTGCCCAGGCAGAGCATGGACGAGTG 1020 321 N A A N P H Q V T T Q M G I P G K E G G

TCCTCATGCCCCCCCGCTGATCATGTTCCCGCCACTCCCTCCAGCAGTGGCGAGGGTGCA 1080 341 V P Q Q V P V Q P C H G A Q A E H G R V

GAGACCTTGTCGAGGAGCATTGAGGTGAAGGGTGTCTCTCCCTGTGATGTACTGGAGACC 1140 361 S S C P P A D H V P A T P S S S G E G A

ACCTTCACCAGGAAAGTGGGTGCCTTTGTCAACAAGCCAGGCACACAGGTAACCACAGCA 1200 381 E T L S R S I E V K G V S P C D V L E T

AGCCTGGACTTACCATTTGCAGCGTTTGCTCCCAGAGCCTATGACCTCGAGGAAAATGAC 1260 401 T F T R K V G A F V N K P G T Q V T T A

CCTATGGTTCAGCCGCCGGAGTCCCCAGCCACGTCCTCTCCCCTGCAGGGCAGTTTACAC 1320 421 S L D L P F A A F A P R A Y D L E E N D

>>>>>>> TCCCAGGGCTCTGGTGAGAGTGGTGGCCCGGCACAGGACGACTTTGTTATGGTGGACTTT 1380 441 P M V Q P P E S P A T S S P L Q G S L H ------

>>>>>>>>>>>>> AAGCCTGCGTTCTCTAAGGATGACCTGCTTCCCATGGACCTTGGCACATTCTACAGAGAG 1440 461 S Q G S G E S G G P A Q D D F V M V D F ------N------

<<<<<<<<<<<<<<<<<<<< TTCCAGAACCCCCCACAGCTCGCCAGCCTCACAATTGATGTCAGTTCTCAGTCCATGGCA 1500 481 K P A F S K D D L L P M D L G T F Y R E ------NN-NNN-N------

GAAGACCTGGACTCTCTTCCCGAAAAACTTGCAGTATACGAGAAGAACATTGATGAATTT 1560 501 F Q N P P Q L A S L T I D V S S Q S M A

GATGCATTTGTGGACACATTGCAGTAG 1587 521 E D L D S L P E K

Figure 2.4e) Rainbow trout (Oncorhynchus mykiss) ATG16 (gi|620599759:337411- 337516). The < and > signs represent places where primers aligned to the sequence. The – sign represent places where the PCR product aligned to the sequence. The N represents missing nucleotides on the sequence.

ATGGCAGAACGTCGTGTGGAGTGTTCATGGAAGAGGCACGTCGGAGAGCAGCTGAAGCAG 60 1 M A E R R V E C S W K R H V G E Q L K Q

AGGGACCGTGTGCAGAGGCAGGCCTTCGAGGAGATCATCCACCAATATAACCGTCTCTTG 120 21 R D R V Q R Q A F E E I I H Q Y N R L L

GAGAAGTCCGATCTACAGGCTGTTCTTTCAGAGAGATACCAGTCGGACAAATATGACTTT 180 41 E K S D L Q A V L S E R Y Q S D K Y D F

CAGAACAGACATGACGGCAGCCCGGGGGCAGACTCAAGTCGCAGCGACAACCTACAGCAG 240 61 Q N R H D G S P G A D S S R S D N L Q Q GAGATGGCCCAGATGCGAATCAGGCACCAGGAGGAGCTGACAGAGCTTCATAAGAAAAGG 300 81 E M A Q M R I R H Q E E L T E L H K K R

GGCGAGCTGGCCCAGAGTGTGATTGAGCTGAACCACCAGATTCAGCAGAAGGACAAGGAG 360 101 G E L A Q S V I E L N H Q I Q Q K D K E

ATTCAGCACAATGAGGCCAAGATGCAGGAGTACCAGCGGCAGATTGCCGAGCTGGAGGGA 420 121 I Q H N E A K M Q E Y Q R Q I A E L E G

>>>>> GAGTGCCGGGAGCTGCGTGGACAGCTGCAGGACCTGGAGCAGGCTAACCAGACGCTGAAG 480 141 E C R E L R G Q L Q D L E Q A N Q T L K ----- >>>>>>>>>>>>>>>> GATGAGTACGACGCCCTGCAGATCACCTTCGGTGCCCTGGAGGAGAAGCTGCGGCGCACC 540 161 D E Y D A L Q I T F G A L E E K L R R T ------<<<<< ACCGATGACAACCAGGAGTTGGTGTCGCGTTGGATGGCTGAGAAATCCCAAGAGGCCAAC 600 181 T D D N Q E L V S R W M A E K S Q E A N ------N------

<<<<<<<<<<<<<<< AAGCTCAATGCGGAAAATGAGAAAGACTGCAAGCGCAGACAAGCCAAGCTTCAGAAGGAG 660 201 K L N A E N E K D C K R R Q A K L Q K E ------N-

CTTGCAGACGCTGCAAAGGAGCCCCTGCCCTTGGACCCGGACGACGACATCGAGGTTCTA 720 221 L A D A A K E P L P L D P D D D I E V L

GCTGAGGATTCTGGGAAAGGCACTAGAGAGGCCTCTCCCAACCGGCCGCTCAGCCGCACC 780 241 A E D S G K G T R E A S P N R P L S R T CCCAGTAAACGTATTTCCGGACAGCCTCCTCAAAGCGGCCTGTTGGACTCAATCTCCAAC 840 261 P S K R I S G Q P P Q S G L L D S I S N

ATATTTGGGCGTCGTCCTGGGAACTCGTACGGAACATCACCTGAAAACACGGAGACGACC 900 67

281 I F G R R P G N S Y G T S P E N T E T T TCCGGAGTGTGTGCCGATGTCCGCGTTCCCTCCACTGCCCTCCATGTGTTTGAAGCCCAT 960 301 S G V C A D V R V P S T A L H V F E A H

GATGGGGAAGTGAATGCGGTGCGGTTCAGCCCAGGCTCCCGACTCCTGGCCACCGGAGGG 1020 321 D G E V N A V R F S P G S R L L A T G G

ATGGACCGCAGGGTCAAACTGTGGGAAGTGGTCTCAGGTCGCTGTGAGCCTAAGGGTGCT 1080 341 M D R R V K L W H V V S G R C E P K G A

CTGACTGGCAGCAACGCCGGGATCACCAGTATAGAGTTTGACAGTGCTGTGAGTGTCATC 1140 361 L T G S N A G I T S I E F D S A V S V I

CTGATTAATCTCCTAGCCTGTCTAAAATGGCACCCTATATCATGGATCATCAACTAG 1197 381 L I N L L A C L K W H P I S W I I N

Figure 2.4f) Rainbow trout (Oncorhynchus mykiss) BECN1 (gi|185133437:38-1381). The < and > signs represent places where primers aligned to the sequence. The – sign represent places where the PCR product aligned to the sequence. The N represents missing nucleotides on the sequence.

ATGGAGGGCTCCAAGTCCTCTAGTACCACCATGCAGGTCAGCTTTGTGTGTCAGCGCTGC 60 1 M E G S K S S S T T M Q V S F V C Q R C AGTCAGCCTCTAAAGCTGGATACATCCTTCAATGTGCTCGACCGTGTCACCATCCAGGAA 120 21 S Q P L K L D T S F N V L D R V T I Q E CTTATTGCTCCTCTGGTCACAGTGACACCAAGCAAGCAGACAGAAAGCAATGAGGCGGAA 180 41 L I A P L V T V T P S K Q T E S N E A E

AGTGCTCCAGAGGAAACCTTTGTGGAAACAAAGCAAGATGGAGTCGCAAGAAAATACATC 240 61 S A P E E T F V E T K Q D G V A R K Y I

CCTCCTGCAAGGATGATGTCTACAGAGAGCGCCAACAGCTTCACTCTGATTGGAGAAGCG 300 81 P P A R M M S T E S A N S F T L I G E A

TCGGACGGAGGCACCATGGAGAACCTCAGTCGGAGGCTGAAGGTGACCAGCGATCTGTTT 360 101 S D G G T M E N L S R R L K V T S D L F

GACATCATGTCGGGCCAGACCGACGTAGACCACCCGCTGTGCGAGGAGTGTACTGACACC 420 121 D I M S G Q T D V D H P L C E E C T D T

CTGCTAGACCACCTGGACACGCAGCTCAACATCACAGAGAACGAGTGCCAGAACTACAAG 480 141 L L D H L D T Q L N I T E N E C Q N Y K

AACTGCCTGGAGCTGCTGTCCCAGCTGAAGGAGGAGGAGGAGGACAGCCTGTTGCTGGAG 540 161 N C L E L L S Q L K E E E E D S L L L E

CTCCAGAAACTGGGCGAGGAGGAGTCGTCACTGGTGGGGGAGCTGGAGGCGGTGGAAGAG 600 181 L Q K L G E E E S S L V G E L E A V E E CAGAGGGCTGCCGTGGCCGAGGAACTGGTCCAGGGACGCAGCCACTCCCAGCAGCTAGAT 660 201 Q R A A V A E E L V Q G R S H S Q Q L D ACTGAGGAACTGCAGTACCAGAAGGAGTACAGTGAGTTCCAGCGGCAGCAGCTGGAGCTG 720 221 T E E L Q Y Q K E Y S E F Q R Q Q L E L

GATGACGAGCTGAAGAGTGTGGACAACCAGATGCGATACTGCCAGATTCAACTGGACCGC 780 241 D D E L K S V D N Q M R Y C Q I Q L D R CTGAAGAAGACCAACGTCTTCAACGCCACCTTCCACATCTGGCACAGTGGCCAGTTTGGC 840 261 L K K T N V F N A T F H I W H S G Q F G

ACCATCAATAACTTCCGTCTGGGCCGACTACCCAGTGTCCCCGTCGAGTGGAACGAGATC 900 281 T I N N F R L G R L P S V P V E W N E I

AACGCCGCCTGGGGTCAGACCGTGCTACTGCTCCACGCCCTCGCCAACAAGATGGGGCTA 960 301 N A A W G Q T V L L L H A L A N K M G L CGCTTCCAGAGATACCGCCTGGTCCCATATGGAAACCACTCTTACCTGGAGTCACTTACA 1020 321 R F Q R Y R L V P Y G N H S Y L E S L T

GACAAGTCCAAGGAGCTACCTCTGTACTGTTCAGGGGGCCTGCGCTTCTTCTGGGACAAC 1080 341 D K S K E L P L Y C S G G L R F F W D N

AAGTTTGACCATGCCATGGTGGCCTTCCTGGACTGTGTGCAGCAGTTCAAGGAGGAGGTG 1140 361 K F D H A M V A F L D C V Q Q F K E E V

>>>>>>>>>>>>>>>>>>> GAGAAGGGAGACACGGGATTCTGCCTGCCTTACAGGATGGATGTCGAGAAGGGCAAGATT 1200 381 E K G D T G F C L P Y R M D V E K G K I ------

GAGGACACGGGAGGCAGCGGCGGCTCCTACTCCATCAAGACCCAGTTCAACTCGGAGGAG 1260 401 E D T G G S G G S Y S I K T Q F N S E E ------N-N------

<<<<<<<<<<<<<<<<<<< CAGTGGACCAAGGCGCTCAAGTTCATGTTGACCAACCTCAAGTGGGGTCTGGCGTGGGTG 1320 421 Q W T K A L K F M L T N L K W G L A W V ------

TCGTCTCAGTTTTACAACCGATAG 1344 441 S S Q F Y N R

2.3.3. Selection of Reference Gene(s)

RefFinder, an integrative software tool, was used to select the best candidate reference genes based on their stability for the experimental conditions used. The genes were ranked in declining order of preference: 18S, EF1, rs11, β-actin and α-tubulin.

Reference gene 18S, however, had early cycles in the qPCR machine and inaccurate Ct values and therefore was not used. α-tubulin was also discarded as it was the least stable under the experimental conditions used. EF1 was therefore used as the reference gene for

RTgill-W1 cell line. All Ct values are shown in Figure 2.5.

30 Reference Gene – Rtgill-W1

15 CP Value CP Ct Value 10

1 day 1

1 day 1 day 1 day 1 day 1

3 days 3 days 3 3 days 3 days 6 days 3 days 6 days 3 days 6 days 6 days 6

12 hours 12 hours 12 hours 12 hours 12 hours 12 EF1 sp11rs11 β-actin α-tubulin 18S

10% 1.5%

Figure 2.5. Ct of reference genes (EF1, 18S, rs11, β-actin and α-tubulin) are shown above. RTgill-W1 cells under the experimental conditions (10% FBS and 1.5% FBS) at different time points (12 h, 1, 3, and 6 days) were used as template.

2.3.4. Quantitative Real-Time PCR Assay

The preliminary trial to screen different serum percentages (2%, 1.5% and 0.5% of FBS) to achieve nutrient restriction demonstrated that all percentages of serum used affected Atg gene upregulation to some extent, but 1.5% FBS resulted in a slightly higher increase in

Atg expression and was therefore selected for this study. Results of this preliminary trial can be seen in Appendix 2.4.

RTgill-W1 cells were then subjected to nutrient restriction using 1.5% FBS to induce autophagy with regular nutrient levels (10% FBS) as a control. The common pattern for the majority of Atg genes examined was of continuous upregulation of gene expression of cells exposed to 1.5% FBS to a significant time at Day 3 followed by a significant down regulation in expression by Day 6. The exception to this pattern was ATG4, which had a significant peak at 12 h and was then downregulated by Day 1. Atg gene expression curves are shown in Figure 2.6a-d.

Relative fold changes (Figure 2.7a-d) were calculated as a ratio between serum-restricted and control samples. The pattern of significant upregulation to a peak at Day 3 followed by a decrease in expression or significant downregulation compared with controls at Day 6 was demonstrated for most genes. GABARAP had a higher relative expression at Day 3 than all other genes. ATG12 and ATG7 were the exceptions, for which there were no significant changes in relative gene expression except at Day 6 for ATG12 and 12 h for ATG7 at which time points expression was significantly less than controls. ATG5 was significantly

73 downregulated except at Day 3 when there was no difference from controls. ATG13 did not show significant change at 12 hours and 1 day, but did at day 3 and 6. Results looking at the effect time had over Atg genes expression in feed restricted (1.5% FBS) cells were significant in most cases (p < 0.05) except for ATG12 time point 12h to 6 d, ATG16, ATG9 and ATG5 at time point 1 d to 6 d, and LC3 time point 12 h to 1 d. The results from the statistical analysis and the effect of time and treatments on Atg gene regulation can be found in Appendix 2.7a-c.

Autophagy induction *

* * * *

mRNA/EF1 gene Atg

ATG13 BECN1 ATG9

Autophagosome formation *

* * * * *

Atg gene mRNA/EF1 gene Atg

ATG5 ATG12 ATG16

Figure 2.6a, b. Atg gene expression during serum-restriction in RTgill-W1 cells over the course of 6 days. Atg genes have been separated depending on the autophagy phase they interact at, with a) showing autophagy induction and b) autophagosome formation. Expression values were normalized with EF1 expressed values. Results are expressed as means +- SD (n=5). The * represents statistical (p < 0.05) significance between serum- restricted and control groups at each time point.

Elongation

* * *

Atg gene mRNA/EF1 gene Atg

ATG7 ATG4

Vesicle completion

* *

* * * *

Atg gene mRNA/EF1 gene Atg

LC3ATG7 GABARAP

Figure 2.6c, d. Atg gene expression during serum-restriction in RTgill-W1 cells over the course of 6 days. Atg genes have been separated depending on the autophagy phase they interact at, with c) showing autophagy elongation and d) vesicle completion. Expression values were normalized with EF1 expressed values. Results are expressed as means +- SD (n=5). The * represents statistical significance (p < 0.05) between serum-restricted and control groups at each time point.

Autophagy induction

Ratio of gene expresión (1.5/ 10% FBS) 10% (1.5/ expresión ofRatiogene ATG13 BECN1 ATG9

Autophagosome formation

FBS) 10% (1.5/ expresión ofRatiogene ATG5 ATG12 ATG16

Figure 2.7a, b. Effect of serum-restriction over the course of 6 days in RTgill-W1 cells. Data was normalized with EF1 gene expression. Each bar represents the ratio of the mean expression of 1.5 over 10% FBS. The * represents significant change between controls and feed restricted groups (p < 0.05, three factorial factor ANOVA). Data over 1 signifies upregulation of 1.5% Atg genes over 10% while data under 1 represents downregulation of Atg genes. Figure a shows the autophagy induction step and Figure b shows results for the autophagosome formation step.

Elongation of autophagosome

Ratio of gene expresión (1.5/ 10% FBS) 10% (1.5/ expresión gene of Ratio

ATG7 ATG4

Vesicle completion

FBS) 10% (1.5/ expresión ofgene Ratio LC3 GABARAP

Figure 2.7c, d. Effect of feed restriction over the course of 6 days in RTgill-W1 cells. Data was normalized with EF1 gene expression. Each bar represents the ratio of the mean expression of 1.5 over 10% FBS. The * represents significant change between controls and feed restricted groups (p < 0.05, Factorial ANOVA). Data over 1 signifies upregulation of 1.5% Atg genes over 10% while data under 1 represents downregulation of Atg genes. Figure c shows the elongation of autophagosome step and Figure d shows results for the vesicle completion step.

2.3.5. Western-Blot Assay

Cell lysates (20-30 µg/well) were analyzed by Western blot with an anti-LC3B antibody and anti-β-actin as internal loading controls. The Western blot bands were more intense for

RTgill-W1 cells under 2% and 1% serum restriction than in cells under 10% serum restriction. A representative blot (Figure 2.8) is presented.

b) 48 h nutrient restriction LC3/β-Tubulin change 3.50000 a a b

3.00000 2.50000 2.00000 1.50000

1.00000

Tubulin change Tubulin -

β 0.50000 0.00000

LC3/ 1% 2% 10% Serum percentage %

Figure 2.8. a) Western blot representing the effect of serum restriction (1%, 2%, and

10% serum percentages) on RTgill-W1 cells after incubation for 48 hours.

Percentage for stacking gel was 4% and for resolving gel 15%. b) After

densitometry the ratio of LC3 to β-tubulin was used to measure relative changes of

LC3 expression and of expressed as means +- SD (n=2) and were analyzed with a

one-way ANOVA Different letters represent significantly different values (p < 0.05).

The graphs represent LC3/β-tubulin ratio.2.4 DISCUSSION

Autophagy research has recently increased significantly, especially with regards to its role in growth development and disease in mammals. In teleosts, like the rainbow trout, manipulation of autophagy has potential as a tool to influence protein turnover and defense against infectious agents, but there are very few studies to date (Zhang et al., 2015).

The first objective of this study was therefore to identify potential molecular biomarkers to study autophagy gene expression in rainbow trout. In addition to four autophagy-related genes previously investigated in rainbow trout, six genes not yet studied were investigated here, including: ATG5 and ATG16, as Atg5 and Atg16 proteins are involved in autophagy induction and forms part of the Atg12-Atg5-Atg16 complex; BECN-1, as beclin-1 protein is involved in autophagy induction and opposes the cellular apoptosis pathway (Cao and DJ,

2007); ATG13, which is involved in the early phagosome; and finally ATG7 and ATG9, as

Atg7 and Atg9 proteins are necessary for autophagosome membrane elongation (Suzuki and Ohsumi, 2007). In total, 10 genes were selected, 6 for the first two phases of autophagy and 4 for the last two phases, providing a range of genes to span the autophagy cycle and to investigate gene expression during nutrient restriction through time. 81

Atg gene sequences from other species (mice, yeast, human, zebrafish and Atlantic salmon) were aligned to the rainbow trout genome to obtain sequences of Atg genes in rainbow trout. The percent identity rates for the 10 Atg genes of rainbow trout ranged from 78-98% with Atlantic salmon, 65-84% with zebrafish, 68-81% with humans, 62-79% with rat and

44-55% with yeast, depending on the gene. The degree of similarity obtained are in accordance with homology-based searches in other trout studies for ATG4, ATG12, LC3 and GABARAP (Seiliez et al., 2012) and with phylogenetic trees created for yellowtail

(Seriola quinqueradiata) heat shock gene HSC70/HSP70 involved in chaperone-mediated autophagy (Yabu et al., 2011). For these genes, highest homology was found between fish species but with significant homology to mammalian Atg gene sequences. Phylogeny studies of autophagy genes GABARAP and LC3 in zebrafish also found high homology to mammalian proteins (He et al., 2009). Since the Atg gene sequences in rainbow trout were highly similar to those of the other fish species examined this suggests that trout may be a useful model to study autophagy function and stimulation in teleosts. Further genomic studies, including complete sequences and promotor regions will be essential to better understand autophagy gene expression and maintenance.

The second purpose of this study was to assess RT-qPCR as a reliable technique to study autophagy under nutrient restriction. Depending on the type of cell, tissue and conditions, reference genes are not always stable, and so must be validated for each experimental system. The change in gene expression observed in some of our reference genes could be due to cell lysis, catabolic effects or the involvement of the genes in other cellular processes 82 apart from basic function of the cell (Rebrikov et al., 2006; Andersen et al., 2004; Pfaffl et al., 2004).

The percentage of fetal bovine serum used as nutrient source for the cells was based on the literature and on a preliminary trial, where different serum percentages (2, 1.5 and 0.5%

FBS) were used to identify which concentration affected Atg gene expression the most.

Unlike a previous study with a rainbow trout cell line that used 0.5% FBS (Seiliez et al.,

2010), our cells had higher Atg gene upregulation at 1.5%. This could be due to differences in the cell line, as studies of nutrient restriction vary from 0.5% to 2% (Seiliez et al., 2011;

Bernard et al., 2015; He et al., 2009) while demonstrating diverse Atg gene upregulation.

An additional preliminary trial also was conducted with a similar design as the trial presented here in detail but using three biological replicates instead of five. Atg gene expression trends were similar to those obtained in the final trial presented in this chapter and can be seen in Appendix 2.6, however few statistically significant differences were found. Therefore, five biological replicates for each treatment were used for the final trial presented in detail in this chapter. Most importantly, the trends in Atg gene expression were similar across all trials performed.

After serum-restriction, expression of most Atg genes examined peaked at Day 3 with downregulation by Day 6. An outlier from this pattern was ATG4, which was upregulated as early as 12 hours, peaked by Day 3 and was still upregulated, although to a lesser degree, by Day 6. Genes ATG5 and ATG12 stayed within basal levels of 1 – 1-5 fold change, and

83 can be upregulated even if there is no nutrient restriction (Salem et al., 2007, Jamart et al.,

2012). As such, nutrient expression should be less likely to consistently affect them, which is consistent with the present study. Gene ATG16, which is part of the complex, is affected by nutrient restriction, in other models, and was upregulated. The time points selected for the present study were based on analysis of LC3II protein in RTgill-W1 after various treatments from other studies in our laboratory and the Western blot presented in Figure.

2.8 is representative. LC3II protein as detected by Western blot was consistently highest at

Day 2 or 3 in most experiments involving nutrient restriction in RTgill-W1.

Previous experiments in yeast and in other cell lines showed that some Atg genes could be significantly upregulated within one h after nutrient or nitrogen restriction (Bernard et al.,

2015). Studies on rat hepatocyte cell lines however, showed prominent macroautophagy vacuoles using electron microscopy after 24 h of starvation (de Waal et al., 1986), an increase in LC3 protein within 15 min using Western blot and of LC3 gene expression within 3 h post-starvation (Razaul et al., 2014). Other studies using epithelial cell lines from mice under amino acid starvation did not detect an increase of protein GFP-LC3 levels until 48 hours (Mizushima et al., 2004). The time to detect autophagy induction therefore varies with the technique and the biological system used for study. Studies in rainbow trout myocytes with ATG12, GABARAP, LC3 and ATG4 showed basal levels of expression for the first four h after nutrient restriction and an increase at 24 hours (Seiliez et al., 2010) which is similar to the results observed in the present study. In rainbow trout myocytes, LC3 had the highest upregulation (3.25 fold change) at 24 h (Iban et al., 2010), which is also similar to the results we obtained at 24 h in LC3 and GABARAP with 3-fold

84 levels of expression. That study did not go beyond the 24 h time point, however (Iban et al.,

2010). The highest upregulation we obtained was GABARAP, with an 8-fold change at Day

3, corresponding with the peak in LC3II protein detected by Western blot. The lowest degree of upregulation obtained in trout myocytes was for ATG4 (2 fold-change) and

ATG12 (1 fold-change) at 24 h (Iban et al., 2010). This is also similar to our results in

RTgill-W1 (1.7 fold for ATG4 and 1.5 fold for ATG12) at 24 h. However, the Atg genes that were upregulated the least in our trial were ATG5 and ATG7 with a 1 and 1.2 fold change, respectively, at Day 3. Studies in zebrafish embryos also found roughly similar results to the present study with increased LC3-II by 48 h post-fecundation, accompanied by ATG9 and LC3 transcriptional upregulation, by the same time point (He et al., 2009).

The current study also let us determine which genes had the greatest level of upregulation as compared to non-nutrient restricted controls: BECN1 and LC3 were upregulated 2-fold;

ATG4 and ATG9 were upregulated 2.5-fold; and GABARAP had the highest relative expression with an 8-fold increase. This degree of gene expression roughly corresponded to those obtained in other experiments (Karim et al., 2014; Li et al., 2011; Seiliez et al., 2012).

This present study has therefore corroborated that ATG4, ATG12, LC3 and GABARAP are genes expressed during autophagy following nutrient restriction. In addition, BECN1 and

ATG9 also showed significant upregulation and deserve further investigation.

One of the issues of our trial was the upregulation at 12 h of all control Atg genes except

ATG4, and of ATG13 control at Day 6. As the cells were under normal nutrient conditions,

85 a possibility for this phenomena was that the flasks endured additional stress while been handled, although this seems an unlikely explanation, since all flask were treated the same.

Further studies would be needed to fully understand why control cells could experience greater upregulation than nutrient-restricted cells at this time point. The use of time points before and after 12 h might also be illustrative.

In fish species, there seems to be an increase in Atg gene expression between 24 h to 48 h post-nutrient-restriction. Our findings indicate that nutrient restriction has a direct impact on Atg gene expression, showing upregulation from 24 h, with a peak at 3 d post nutrient restriction, and this is corroborated by the Western blot results. Our data also establishes a connection between LC3 protein and LC3 gene, which is needed in order to maintain the autophagy signal. GABARAP, which belongs to the ATG8 family, had the same pattern of expression as LC3, but with a higher degree of expression, suggesting that GABARAP expression is more affected than LC3 under in vitro nutrient restriction conditions in

RTgill-W1. Further studies are needed to fully understand the connection between autophagy-related proteins and autophagy related genes.

Chapter 3. Autophagy-related (Atg) gene expression in the liver and muscle of rainbow trout (Oncorhynchus mykiss) undergoing feed restriction

This chapter is formatted according to guidelines of the Journal of Aquaculture

3.1 ABSTRACT

Salmonids, which include rainbow trout (Oncorhynchus mykiss), undergo long periods of nutrient restriction, in which lipids and white muscle are degraded. Protein becomes an important source of energy, and the autophagic pathway is critical in protein degradation.

We studied 10 autophagy-related genes throughout the autophagosome cycle. To induce autophagy, rainbow trout were subjected to feed restriction for a period of 21 days and compared to controls. Samples of muscle and liver were taken at day 1, 2, 7, 15 and 21 days and reverse transcription quantitative polymerase chain reaction was performed. Several autophagy-related genes were significantly affected by nutrient restriction. In liver, autophagy-related genes were significantly upregulated (p<0.05) within the first 4 days, with ATG5, ATG7 and ATG12 having the greatest differential expression. The autophagy- related genes in muscle were significantly upregulated later than in liver; between Day 8 and 15, with ATG4, ATG12, GABARAP and LC3 having the greatest differential expression. There was a second peak of upregulation at Day 15 and/or 21 in both tissues, which could mean autophagy is maintained for at least 21 days in rainbow trout if the stressor is maintained.

3.2 INTRODUCTION

Autophagy is a mechanism that plays a critical role in protein degradation, muscle mass regulation, and the immune response through formation of an intracellular vesicle called the autophagosome (Jamart et al., 2012). All organisms maintain basal levels of autophagy, which supports proper cell function and degrades damaged organelles and proteins (Nixon,

2013; Bernard, Jin & Klionsky, 2015). During stress, such as nutrient and nitrogen restrictions or pathological events, autophagy can be highly upregulated (Mohapatra et al.,

2016a), playing a key role in protein turn-over and in resistance against certain pathogens

(Mizushima et al., 1998).

Salmonids, which include rainbow trout (Oncorhynchus mykiss), undergo long periods of nutrient restriction caused by environmental, physiological or anthropogenic causes (Yabu et al., 2012). Salmonids use proteins as their main source of energy, and as starvation periods increase in length, amino acids become an even more important source of energy, as they are gluconeogenic precursors (Mohamed, Silverstain, Rexroad & Yao, 2007;

Seiliez, Dias & Cleveland, 2014). The autophagic pathway is essential for protein and muscle maintenance. As such, it is important to understand this pathway at a molecular level to improve muscle growth and reduce muscle wasting under stress conditions. In previous studies, ATG7 knockout mice and wild-type controls were subjected to fasting for

24 h to induce autophagy, and muscle samples were taken. Morphological analysis found that the lack of ATG7 led to atypical giant mitochondria accumulation and a decrease in myofiber size. Furthermore, the degradation of muscle in ATG7 knockout mice was significantly higher compared to wild type mice (Masiero et al., 2009). Rats subjected to 89 endurance exercise (1 h treadmill, 6 d/week, for 8 weeks) saw an increase in expression of

ATG7, as well as BECN1 and LC3 in rat muscles (Feng et al., 2011. This experiment suggests that ATG7 and BECN1 are highly important in muscle maintenance during exercise and could potentially be affected by nutrient restriction. A study in humans observed changes in ATG4, ATG12, GABARAP, LC3 and BECN1 after ultra-endurance, where subjects ran a marathon with an average time of 28 h. Samples were taken a week before the marathon and 3 hours after completion. Subjects had an increase in mRNA expression in ATG4 (1.5 fold change), ATG12 (1.5 fold change), GABARAP (3.7 fold change) and LC3 (2.0 fold change) compared to control samples, with GABARAP having the highest fold change. This suggests that these genes are highly important for autophagy during exercise and might also be upregulated during nutrient restriction (Jamart et al.,

2011). Finally, the muscle of rainbow trout that were starved for 14 days showed increased expression of ATG4 (19 fold change), ATG12 (2 fold change), GABARAP (1.75 fold change) and LC3 (2.7 fold change) (Seiliez et al., 2010). These were the same genes that were upregulated in the humans after ultra-endurance, but to a different degree. Some variation is not surprising given the difference in organism, time-frame and the stressor used to induce autophagy. Nevertheless, further studies are necessary to understand the effects nutrient restriction have in these genes.

Autophagy also influences innate and adaptive immunity, modulating the presentation of antigens from intracellular pathogens and enhancing priming of CD4+ T cell responses against intracellular pathogens (Miller and Krijnse-Locker, 2008). Phagocytic cells can also use macroautophagy to target intracellular pathogens and degrade them, including some

90 bacteria, parasites and virus (Nakagawa et al., 2004). Nutritional factors also influence the innate and adaptive responses, with nutrient restriction, obesity and other nutrition-related circumstances having a direct impact. Feed restriction can decrease mortality rates and increase resistance, in some cases, while in other situations, starvation can be damaging.

Studies in mice infected with Listeria monocytogenes found that parenteral supplementation of glucose twice daily 8 h post-infection killed infected mice, while the mice that were fasted had higher rates of survival. However, the opposite was true in mice infected with influenza and mice supplemented with glucose had higher survival rates than fasting mice (Wang et al., 2016). Feed restriction seems to have a different effect depending on the pathogen and the level of infection. As autophagy is highly induced by the levels of by nutrient factors, it is important to understand the relation between nutritional stress, autophagy and the immune response (Wen et al., 2011). Studies in different aquatic species suggest that feed restriction can result in a reduction in mortality and increase resistance to infectious disease (Mohapatra et al., 2016b; Vojo, 2011).

Feed restriction has been found to reduce mortality rates of channel catfish infected with

Edwarsiella ictaluri. After infection was confirmed, fish were separated into different groups: medicated and non-medicated groups fed every day, every other day and every third day, as well as a completely feed-restricted group. Survival was found to be greatest in the completely feed-restricted group, followed by the medicated groups every other day and every third day. This experiment indicated that feeding frequency is an important factor in the defense against E. ictaluri and feed restriction may be a potential tool against this pathogen (Wise et al., 1998).

A study in Atlantic salmon infected with Vibrio salmonicida demonstrated that feed restriction can greatly affect survival rates. Fish were separated in optimal, feed restricted and starved groups after infection was confirmed. Starved fish had a 98.8% survival rate compared to 64.5% in the optimal group and 42.5% in the feed-restricted group (Damsgard et al., 2004). The study demonstrated increased resistance to V. salmonicida following feed deprivation, but showed higher mortalities in feed-restricted groups. Levels of feed restriction seem to influence the susceptibility of fish to diseases, but further studies are needed to explain this phenomena (Damsgard et al., 2004).

Red sea bream were infected with Edwarsiella tarda and separated into two groups; starved-infected and fed-infected. Fish tissues were sampled at 0 h, 5 d and 10 d. Fish that underwent short term-starvation had lower E. tarda loads in spleen and muscle that the fed fish and had enhanced survival (Mohapatra et al., 2008). Rainbow trout were treated with

DON, a mycotoxin that reduces feed intake, and compared to fed and feed-restricted groups and then all groups were infected with Flavobacterium psychrophilum. There was a significant reduction in mortalities in the DON and feed-restricted groups compared to the fed group after 21 days (Ryerse et al., 2014, 2015).

Due to the increased importance attached to autophagy in development, growth and protein maintenance under feed restriction, and as a mechanism to combat infections in fish, additional methods to study autophagy are needed (Schiotz et al., 2010). The following trial aimed to study the pattern of expression of autophagy-related (Atg) genes in rainbow trout

92 under feed-restriction conditions that have been previously demonstrated to provide resistance from experimental infection (Ryerse et al., 2014). Gene expression of Atg genes representing all stages of the autophagy cycle were examined in muscle and liver tissue from rainbow trout provide normal and restricted feed at intervals up to 21 days.

3.3 MATERIAL AND METHODS

3.2.1 Fish Trials

All studies involving live fish were performed following the University of Guelph’s animal care guidelines. Rainbow trout fingerlings from Lyndon Fish Hatcheries (New Dundee,

ON, Canada) were housed at the University of Guelph Hagen Aqualab. Fish had an initial average weight of 7.5 g and were treated with 1:10,000 v/v buffered formaldehyde for 1 h upon arrival. Forty fish were randomly assigned to one of the six 125 L tanks. Trout were fed with commercial trout feed (Profishent, Martin Mills Inc, Elmira, Ontario, Canada) at

2% of biomass once per day for a one-week period of acclimation. Once fish were screened

(bacteriological and histological studies) and deemed healthy, tanks were randomly assigned to control and feed-restricted diets, with three tank replicates for each. Control groups of fish were fed an average of 6.4 g/fish/feeding while feed-restricted groups were fed an average of 3.3 g/fish/feeding. Both groups were fed twice daily at 09:00 and 17:00 hours during weekdays and once on weekends for 21 days.

Fish were fasted for a period of 24 h prior to sampling and euthanized with an overdose of benzocaine (Sigma-Aldrich Inc, St. Louis, MO, USA), and muscle and liver samples were collected and stored in RNAlater (40 mL 0.5M EDTA, 25 mL 1 M sodium citrate, 700 g ammonium sulfate and 935 mL ultrapure water at 5.2 pH) at -80° C.

3.2.2 RNA Extractions

Total RNA was extracted from muscle and liver samples using Trizol reagent (Invitrogen,

ON, Canada) according to the manufacture’s protocol. Tissue samples were homogenized with 1 ml of Trizol reagent in 1.5 ml centrifuged tubes. Chloroform (200 µl) was added to the mix which was then vortexed, incubated at 5 °C for 5 min and finally centrifuged (9600 g, 4°C, 15 min). The aqueous phase was then obtained (550 µl) and deposited into new tubes and isopropanol (550-600 µl) was added. The mix was gently homogenized (inverted

2-3 times) and incubated at 5 °C for 5 min. Samples were then centrifuged (9600 g, 4 °C,

30 min). The pellet was washed and centrifuged (3200 g, 4 °C, 5 min) with 75% ethanol and re-suspended with PCR grade nuclease-free water (32 µl at 54 °C). The extracted RNA concentrations were measured using the Nanodrop ND-1000 (Nanodrop Tachnologies, DE,

USA) and stored at -80 °C.

3.2.3 RNA Column Purification

RNA samples were treated with RNase-Free DNase Set (QIAGEN) and purified using the

RNeasy Midi Kit (QIAGEN) following the manufacturer´s protocol. A mix of 350 µl of

RLT Buffer provided in the RNeasy Midi Kit (QIAGEN) and 250 µl of ethanol 100% were added to samples and mixed (up and down). The total volume was then transferred to the columns and centrifuged (9600 g, 1 min, 4 °C). The supernatant was then discarded and columns were washed twice with RPE solution (500 µl) and centrifuged twice (9600 g, for

1, 4 °C and 10,000 g, 2 min, 4 °C, respectively). The column was transferred to a new collection tube and 40 µl of RNase free water at 54.0 °C was added to the column and

95 centrifuged at 10,300 g, 2 min, 4 °C. The purified RNA concentrations were measured using the Nanodrop ND-1000 (Nanodrop Technologies, DE, USA) and stored at -80 °C.

3.2.4 Reverse Transcription Polymerase Chain Reaction (RT-PCR)

RT-PCR reaction was performed using the High-Capacity cDNA Reverse Transcription

(RT) Kit (Applied Biosystems) and RNase OUT (5000 U) (ThermoFisher Scientific). The master mix contained 2 µl of 10x RT Buffer, 0.8 µl of 25x dNTP mix, 2 µl of 10x RT primers, 1.0 µl of RT, 0.4 µl of RNase inhibitor and 0.6 µl of RNase free water per sample.

Samples were normalized to 250 ng/µl of RNA and 6.8 µl of master mix was added to each sample to obtain a total volume of 20 µl. Thermal cycling conditions were programmed to a first step of 25 °C for 10 min., second step of 37 °C for 120 min, third step of 85 °C for 4 min and a pause step at 4 °C. cDNA samples were then diluted with 180 µl of RNase-free water to obtain a final volume of 200 µl. Samples were stored at -20 °C.

Primers designed previously (Chapter 2) were used to test RT-qPCR in trout tissues. The reaction for RT-qPCR was prepared using LightCycler 480 SYBR Green, qPCR Master

Mix (Roche, Applied Sciences) containing 5 µl of master mix SYBR Green, 2.5 µl of primer mix (forward and reverse) and 2.5 µl of sampled cDNA, for a total reaction volume of 10 µl. PCR was performed in a Roche LightCycler® 489 II with the following cycling conditions: pre-incubation at 95 °C for 5 min, an amplification at 95 °C for 10 s, 60 °C for

20 s and 72 °C for 15 s for 45 cycles, melting at 95 °C for 5 s, 65 °C for 1 min, with a final cooling step of 40 °C for 10 s. RNA extracted from liver and muscle tissues pooled together were used as a calibrator (positive control) and water was used as a negative control.

Samples were run in 384 well plates (Roche, Applied Sciences) with each sample run in triplicate. As was done previously with RTgill-W1 cells, quantification of gene expression was obtained using standard curve efficiencies and normalized to the two reference genes selected (Eq. 1) with the instrument software (the Roche LightCycler® instrument II). The

97 same formula was used to calculate the ratio of 1.5 over 10% serum using the relative calculation procedure REST program (http://REST.gene-quantification.info/). Samples were normalized using reference genes and calibrator values.

∆퐶푝푡푎푟푔푒푡(푐표푛푡푟표푙−푠푎푚푝푙푒) (퐸푡푎푟푔푒푡) 푟푎푡푖표 = ∆퐶푃푟푒푓(푐표푛푡푟표푙−푠푎푚푝푙푒) (퐸푟푒푓)

(1) 3.2.5 Reference Gene Validation

Five reference genes, designed in the previous chapter, were tested for their stability in conditions of feed restriction for muscle and liver tissues of rainbow trout. The stability of the five reference genes was assessed using the web-based tool RefFinder

(http://www.leonxie.com/referencegene.php), which integrates the algorithms for

Normfinder, BestKeeper, geNorm and the comparative ∆Ct method to rank reference genes from most to least stable.

3.2.6 Statistical Analysis

For qPCR data analysis, CT values were normalized to selected reference gene (EF1 and rs11) and calibrator values. Quantification of gene expression was obtained using standard curve efficiencies with the instrument software (the Roche LightCycler® instrument II) and the relative calculation procedure REST program (http://REST.gene-quantification.info/).

Effect of time and treatment were compared using a three factorial-factor ANOVA (SAS) in a mixed model analysis of variance (MMV). The post-hoc test was a simple t-test (least significant difference test). Data was transformed to log2 for the statistical analysis and outliers (standard deviation of more than 12 degrees) were taken out of the analysis. All

98 samples in the study were given a unique sample ID compromised of a number

(representing a time point), a letter (representing the biological replicate, and a number, representing nutrient restriction conditions). The data points removed were 2D1 and 21A2 for liver and 2B2, 2D2, 2C2 for muscle. Comparisons between treatment and time p < 0.05 were considered statistically significant.

3.4 RESULTS

3.3.1 Reference Gene Selection

The genes EF1 (elongation factor 1) and rs11 (ribosomal protein s11) were chosen as reference genes for liver and muscle as they proved to be the most stable under conditions of feed restriction. Gene 18S had inaccurate Ct values and was left out of the study. Genes

α-tubulin and β-actin were both unstable in trout undergoing feed restriction. Ct values are shown in Figure 3.1.

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Reference Genes - Tissues

Ct Value Ct 15

1 day 1 day 1 day 1 day 1 day 1

8 days 8 2 days 2 days 4 days 8 days 2 days 4 days 8 days 2 days 4 days 8 days 2 days 4 days 2 days 4 days 8 rs11 EF1 β-actin α-tubulin 18S Control Restricted

Figure 3.1. Ct values of genes rs11, EF1, α-tubulin and β-actin are shown above. Tissues were pooled together (liver and muscle) from control rainbow trout fed normally (6.4 g/fish) and under feed-restricted conditions (3.3 g/fish) at different time points (Day 1, 2, 4 and 8).

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3.3.2 Semi-quantitative Reverse Transcription- PCR Assay in Liver

The results obtained in the present study provide insight into autophagy flux in liver of fish undergoing feed restriction. Of the genes in the induction phase of autophagy, BECN1 was highly but variably expressed and on Day 1, 4 and 15 was significantly upregulated compared to controls but was significantly downregulated at Day 8. Both ATG13 and ATG9 were minimally expressed until Day 15 and 21, however there were rarely significant differences between feed-restricted and control fish at these time points.

Of the genes involved in the autophagosome formation step, ATG5 was highly expressed at all-time points, while ATG12 and ATG16 were minimally expressed. ATG5 had significantly increased expression in feed-restricted fish compared with controls at all days except Day 1 and 8. ATG12 was significantly expressed in feed-restricted fish only at Day 1, as was

ATG16, however increased expression of this gene was also noted at Day 2, 15 and 21.

The elongation gene ATG7 was significantly upregulated compared to controls at all-time points, while ATG4 was only significantly increased at Day 4. LC3 was significantly upregulated on Day 1, 4, 15 and 21, while GABARAP was on Day 2, 4, and 15 but was significantly downregulated on Day 8 (Figure 3.2 a-d and 3.3 a-d).

Fixed effects of gene, treatment and time were found to be highly significant (all p<0.001).

Tables with the complete statistical data can be found in Appendix 3.1 a-c.

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Liver:Autophagy Autophagy induction induction 1.8

1.6 * 1.4 1.2 * 1 * * * 0.8

Expression 0.6 * Gene Expression Gene *

0.4 * * * 0.2 * 0

1 day 1 day 1 day 1

4 days 4 days 8 days 4 days 8 days 4 days 8

2 days 2 days 2 days 2

21 days 21 days 15 days 21 days 15 days 21 15 days 15 ATG13ATG13 BECNBECN-11 ATG9ATG9

Control Feed-Restricted

b) Liver: AutophagosomeAutophagosome formation formation

7 *

6 5 * 4

3 * * Expression * *

2 * Gene Expression Gene

* 1 * * * 0

1 day 1 day 1 day 1

4 days 4 4 days 4 days 8 days 4 days 8 days 8

2 days 2 days 2 days 2

21 days 21 15 days 15 days 21 days 15 days 21 days 15

ATG5 ATG12 ATG16ATG16 ATG5 ATG12 Control Feed-Restricted

Figure 3.2a, b. Atg gene expression of liver from rainbow trout under feed restriction and controls). Atg genes are shown in their respective step in the autophagosome cycle. Expression values were normalized with EF1 and rs11 expressed values. Results are expressed as means +- SD (n=5). The * represents statistical significance between restricted and control groups.

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c) Liver: Elongation Elongation

7 * 6 5

4 3 * * * 2 * * Gene Expression Gene * * 1

1 day 1 day 1

4 days 4 days 8 days 4 days 8

2 days 2 days 2

21 days 21 days 15 days 21 days 15 ATG7ATG7 ATG4ATG4 Control Feed-Restricted

d) Liver:Vesicle Vesicle completion Completition

7 * 6 5 4 3 Expression 2 * * Gene Expression Gene 1 * * * * 0

1 day 1 day 1

4 days 4 4 days 4 days 8 days 8

2 days 2 days 2

15 days 15 days 21 days 15 days 21 LC3LC3 GABARAPGABARAP

Control Feed-Restricted

Figure 3.2c, d. Atg gene expression of liver from rainbow trout under feed restriction and controls. Atg genes are shown in their respective step in the autophagosome cycle. Expression values were normalized with EF1 and rs11 expressed values. Results are expressed as means + SD (n=5). The * represents statistical significance between restricted and control groups. 104

AutophagyAutophagy induction induction

feed restricted/control) feed ( expressio ofRatiogene

ATG13 BECN-1 ATG9

b) Autophagosome formation

n n

restricted/control)

feed feed ( expressio ofRatiogene

ATG5 ATG12 ATG16

Figure 3.3.a, b. Atg gene expression of liver from rainbow trout under feed restriction and controls. Data was normalized with EF1 and rs11 gene expression. Each bar represents the ratio of the mean expression of feed-restricted over control groups. The * represents significant change between normal and feed restricted fish (p<0.05). Data over 1 signifies upregulation of feed restricted Atg genes over control while data under 1 represents downregulation of Atg genes at each time point.

105

Autophagosome elongation

n n

feed restricted/control) feed ( expressio ofRatiogene

ATG7 ATG4 Vesicle completion d)

feed restricted/control) feed

Ratio of gene expressio ofRatiogene (

LC3 GABARAP

Figure 3.3c, b. Atg gene expression of liver from rainbow trout under feed restriction and controls. Data was normalized with EF1 and rs11 gene expression. Each bar represents the ratio of the mean expression of feed-restricted over control groups. The * represents significant change between normal and feed restricted fish (p<0.05). Data over 1 signifies upregulation of feed restricted Atg genes over control while data under 1 represents downregulation of Atg genes at each time point.

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3.3.3 Quantitative Real-Time PCR Assay in Muscle

The gene expression study performed provided insight into autophagy flux and possible key Atg genes for muscle under feed-restriction. ATG13 and BECN-1 were downregulated compared to controls at Day 1, 2 and 4 and were significantly upregulated at Day 8. ATG13 was also upregulated significantly at Day 21. ATG9 was variably expressed, with significant upregulation at Day 1, 8 and 15 but significant downregulation at Day 2 and 4 compared to controls. Of the genes involved in autophagosome formation, ATG5 was significantly upregulated at Day 4 and 8 compared to controls, while ATG12 and ATG16 were significantly downregulated at Day 1, 2 and 4 compared to controls and significantly upregulated at Day 8. All of the genes in autophagosome elongation were downregulated compared to controls at Day 1, 2 and 4, but were significantly upregulated at Day 8 and

ATG7 was significantly upregulated at Day 21. In the vesicle completion phase, LC3 had a variable expression pattern. It was downregulated, sometimes significantly, at Day 1, 4, 15 and 21 and significantly upregulated at Day 2 and 8. GABARAP, on the contrary, was significantly downregulated compared to controls until Day 8 and after was not different from controls (Figure 3.4 a-d and Figure 3.5 a-d).

While most genes were significantly upregulated compared to controls at Day 8, the magnitude of upregulation varied widely, with ATG13, ATG16, ATG7 and LC3 showing the highest levels of upregulation. A three-factorial factor (genes, time and treatment) analysis was performed, finding most data significant (p<0.05). Complete statistical data can be found in Appendix 3.2 a-c.

107

Autophagy induction

* * * * * * * * * * *

Atg gene mRNA/EF1 & & rs11 mRNA/EF1 gene Atg

ATG13 BECN-1 ATG9

b) Autophagosome formation

* * * * * * *

* * mRNA/EF1 & & rs11 mRNA/EF1

Atg gene gene Atg

ATG5 ATG12 ATG16

Figure 3.4a, b. Atg gene expression of muscle from rainbow trout under feed restriction and controls. Atg genes are shown in their respective step in the autophagosome cycle a) showing autophagy induction and b) autophagosome formation. Expression values were normalized with EF1 and rs11 expressed values. Results are expressed as means + SD (n=5). The * represents statistical significance between restricted and control groups.

108

c) Elongation

* * * * * *

Atg gene mRNA/EF1 & rs11 & rs11 mRNA/EF1 gene Atg ATG5 ATG4

d) Vesicle complition

* * * * * *

Atg gene mRNA/EF1 & rs11 & rs11 mRNA/EF1 gene Atg LC3 GABARAP

Figure 3.4c, d. Atg gene expression of muscle from rainbow trout under feed restriction (pair-fed) and controls. Atg genes are shown in their respective step in the autophagosome cycle c) showing elongation and d) vesicle completion. Expression values were normalized with EF1 and rs11 expressed values. Results are expressed as means +- SD (n=5). The * represents statistical significance between restricted and control groups at each time point.

109

Autophagy induction

Autophagosome formation

Figure 3.5 a b. Atg gene expression of muscle from rainbow trout under feed restriction and controls. Data was normalized with EF1 and rs11 gene expression. Each bar represents the ratio of the mean expression of feed-restricted over control groups. The * represents significant change between normal and feed restricted fish (p < 0.05). Data over 1 signifies upregulation of feed restricted Atg genes over control while data under 1 represents downregulation of Atg genes at each time point.

110

Elongation of autophagosome

restricted/control)

Ratio of gene expresión (feed (feed expresión ofRatiogene

Vesicle completion

restricted/control)

Ratio of gene expresión (feed (feed expresión ofRatiogene

Figure 3.5 c, d. Atg gene expression of muscle from rainbow trout under feed restriction and controls. Data was normalized with EF1 and rs11 gene expression. Each bar represents the ratio of the mean expression of feed-restricted over control groups. The * represents significant change between normal and feed restricted fish (p < 0.05). Data over 1 signifies upregulation of feed restricted Atg genes over control while data under 1 represents downregulation of Atg genes.

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3.5 DISCUSSION

The purpose of the present study was to examine the expression of ten Atg genes in rainbow trout liver and muscle over a period of 21 days under conditions of feed restriction.

Feed restriction was performed in trout over a longer time period than reported previously and it affected the expression of Atg genes in a tissue-dependent manner. During starvation, glycogen and protein are mobilized and lipids are reduced in the liver (Guderley et al.,

2003). As the period of starvation increases, proteolysis of muscle fibres become essential for protein turnover. White muscle is catabolized and muscle glycogen and protein are used as energy sources (Guderley et al., 2003). As such, one could predict that autophagy would be induced strongly in liver first and then in muscle.

Experiments in mice and rats have shown up to 25-40% of liver protein can be lost within the first 48 h after nutrient restriction (Mortimore, 1987) with autophagy undergoing rapid upregulation in the liver and with autophagosome structures appearing as early as 12 h post feed-restriction in mouse hepatocytes (Yin et al., 2008). This suggests that in mammals, autophagy induction in liver is highly active during the first 48 h and autophagy gene expression would most likely be upregulated during the first 48 h (Äijälä et al., 2013).

Results in vitro in trout myoblasts under nutrient restriction, however, showed only moderately increased upregulation at 24 h (the last experimental time point) with very slight levels of upregulation at 4 h (Iban et al., 2010). This makes some sense since the metabolic activity of rainbow trout at 12 °C is much less rapid than in mammals (Clarke,

2003; Johnston, 1987) and peak expression of all genes in fish would take longer to occur.

Peak Atg gene expression in liver occurred at 2 to 4 d, similar to the results obtained in 112

RTgill-W1 cells (Chapter 2), in which peak expression occurred at Day 3; and this latter result was corroborated by Western blot at 48 h. In muscle tissue, on the other hand, Atg gene expression was occasionally upregulated by Day 4 but most commonly at Day 8; later than in liver. The only other study performed to date in trout muscle, studied the effect of nutrient restriction only at Day 14 and found that expression of ATG4, ATG12, LC3 and

GABARAP was upregulated. In our experiment, ATG4, ATG12 and GABARAP were upregulated at Day 8, while LC3 was upregulated at Day 2 and 8. Overall, the results from the present study suggest that autophagy gene expression occurs in the liver before the muscle. Further assays on autophagy flux, perhaps over even longer time periods are needed in order to understand autophagy flux in liver and muscle and in other tissues as well. Parallel studies are also required to confirm induction of autophagy related proteins in these tissues.

To our knowledge, the present study ran for a longer period of time than any other trials, and with more time points assayed between 24 h and 21 d. For numerous Atg genes the present study demonstrated a phenomena of an initial peak of expression followed by a subsequent peak many days later. This occurred for Atg gene expression at Day 15 for

BECN1 and ATG16, and at Day 21 for ATG5, ATG13 and ATG16 in liver after initial peaks between Day 2 and 4. This phenomenon also occurred in muscle, which also showed a second peak of expression for ATG5, ATG13, ATG16 (similar to liver) but also ATG7 at

Day 21 after initial peaks between Day 4 and 8. This could suggest that if the stressor is maintained, autophagy can be continually or cyclically induced in both liver and muscle,

113 but further studies are needed to confirm this and to determine if the autophagy signal is exhausted at some point.

Although levels of Atg gene expression and the Atg genes that are upregulated seem to vary between experimental models, there are a certain Atg genes that seem to be upregulated consistently during feed restriction in the liver: ATG4, ATG7, ATG9, ATG16 and LC3 have been found to be consistently upregulated in several different trials. Liver from mice fasted for 24 h saw an increase In ATG4, ATG7, ATG16, LC3 and ATG9, with an average of 1 to 2 relative fold change (Lee et al., 2014), while livers from male rats given a fructose diet had increased in ATG7 (1.25 fold change) (Äijälä, et al. 2013), and livers from mice treated with lipopolysaccharides showed high levels of LC3, BECN1 and ATG7 expression (4.5 and 1.5 fold) (Di Ma et al., 2013). These results are similar to some of ours, which also showed increased expression of ATG4, ATG16 and LC3, plus ATG5 and

ATG13, with the highest fold change at Day 21 in Atg5 (7 fold change), which suggests that these genes are highly regulated by nutrient restriction and could be the focus of future studies of autophagy under different stress conditions.

In the muscle of rainbow trout, ATG4, ATG12, LC3 and GABARAP were upregulated under conditions of feed restriction after 14 d (Seiliez et al., 2012). These same genes plus

BECN1 were also found to be upregulated under conditions of ultra-endurance in humans

28 h after a marathon (Jamart et al., 2012). Studies in the muscle of rats subjected to endurance exercise for an hour also saw an increase of LC3 and BECN1, as well as ATG7

114

(Feng et al., 2011). The results of the present study, however, are not completely in agreement. While ATG7, BECN1 and LC3 were significantly upregulated at some time points as in the previous studies, ATG4 and ATG12 were only slightly differentially expressed (1.5 – 2), while GABARAP was no different or was downregulated in contrast to

Seiliez et al. (2012). Additionally ATG5, ATG9, ATG13 and ATG16, which were not included in the previous studies, were upregulated significantly. There does however, appear to be a core group of Atg genes affected under stress conditions in muscle that are important for autophagy regulation, however confirmation of the consistent involvement of the additional genes is needed.

Of the genes that were identified to be significantly expressed in the present study, ATG13 was upregulated only in the liver. Atg13 protein plays an important role in the pre-initiation of autophagy, activating during low energy situations and located at the upstream in the autophagy pathway (Egan et al., 2011). Pre-initiation is followed by the initiation complex, in which beclin-1 and Atg9 proteins work downstream of Atg13, promoting nucleation of autophagic vesicles and increase the size of pre-autophagosomal membranes (Ferraro,

2007). Both BECN1 and ATG9 were upregulated in muscle but not liver. We predicted that

BECN1, given the position of beclin-1 protein in the autophagy cycle, would be significantly differently expressed by nutrient restriction. This was true for the muscle of rainbow trout and in RTgill-W1 cells in our previous experiment, but not for liver. After this, the Atg12-Atg5-Atg16 protein complex drives formation and expansion of the autophagosome (Itakura & Mizushina, 2010). However, the only genes that showed upregulation in both tissues were ATG5 and ATG16. In the case of RTgill-W1 cells, ATG12

115 and ATG16 were the genes that were upregulated. Atg4 protein is essential for the elongation phase and deconjugates LC3, producing its active form (Itakura & Mizushima,

2010). ATG4, in our trial, was upregulated in liver and RTgill-W1 cells, but not in muscle.

ATG7, which in contrast was upregulated in muscle, is also essential for elongation, as

Atg7 protein activates LC3 and interacts with the Atg5-Atg12-Atg16 protein complex for further autophagosome elongation (Suzuki et al., 2007). Finally, LC3 was upregulated in both tissues (muscle at earlier time points and liver) as well as the previously investigated

RTgill-W1 cells. LC3 protein is involved in cargo recruitment and vesicle completion, as well as serving as the main molecular marker for autophagy induction (Ferraro, 2007).

There were some marked differences in gene expression between the liver and muscle, suggesting that transcriptional factors are essential to regulate autophagy and this may help to explain the differences seen. Prolonged autophagic induction requires gene expression in order to renew LC3 and Gabarap, which are proteins that are destroyed during the autophagolysosome formation. This could suggest that short-term and long-term autophagy have different signalling pathways. Further studies are definitely needed to better understand this phenomenon. It will be critical to expand the range of tissues examined under nutrient restriction and to perform Western blot at various time points to confirm autophagy induction.

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GENERAL DISCUSSION AND CONCLUSIONS

This thesis has increased the number of known Atg genes in rainbow trout to 10 and provided valuable insight into autophagy flux and gene expression during nutrient restriction conditions in vivo and in vitro. Atg gene activation and regulation occur under a variety of stresses and in infectious disease. As such, having a wider range of Atg genes available to study autophagy will be useful in future investigations. This thesis also provided insight into the use of RT-qPCR as a technique to study autophagy flux over time.

Salmonids can go for long periods without eating yet there is only one other study that has examined autophagy expression over a period of weeks (Seiliez et al., 2008). Mammalian studies typically spam a few days at most (Masiero et al., 2009; Mizushima et al., 2004;

Äijälä et al., 2013). There is significant knowledge to be gained regarding the impact of feed restriction on fish physiology and resistance to disease.

This thesis provides a direction for future research of autophagy studies in rainbow trout.

The second chapter of this thesis determined 3 d as the peak for autophagy in the RTgill-

W1 cell line. The genes with the greatest upregulation under conditions of serum restriction were ATG4, ATG9, BECN1 and LC3. This thesis also provides insight into optimal serum restriction conditions and time for autophagy induction that can be used in future research.

Studies should focus on the effect that nutrient restriction has on RTliver cells (rainbow trout hepatocyte cell line) and on other tissues, such as the gill and intestine. Studies should also focus on the effect other stress conditions have on Atg genes and how they differ from our results, such as following experimental infection. Investigating autophagy gene expression proved challenging since the number of known Atg gene sequences in rainbow 117 trout were limited. The rainbow trout genome is available (NCBI), however is not completely annotated. There have been a few studies of Atg genes in fish species, with

ATG4, ATG12, LC3 and GABARAP identified previous to this study (Seiliez et al., 2012).

Atg gene sequences for ATG5, ATG7, ATG9, ATG13, ATG16 and BECN1 were obtained through alignment of rainbow trout genome to Atlantic salmon, and phylogeny analysis and percentage matrix confirmed these sequences to be highly homologous to those in other group of species.

The third chapter of this research provided insight into autophagy gene induction in rainbow trout tissues under nutrient restriction. Liver had a significant upregulation in many Atg genes within the first four days. The genes that had the most significant upregulation were ATG7, ATG5 and ATG12, suggesting that these genes are important in liver under conditions of nutrient restriction. The Atg7 protein activates Atg12 for its conjugation with Atg5, as well as for elongation and closure of the autophagosome. Atg5 and Atg12 form a complex important for vesicle formation. The Atg12-Atg5 proteins are also important for Atg8 conjugation to the lipid phosphatidylethanolamine (PE) and for autophagosome maturation. For muscle, significant upregulation was found at d 8, with

ATG7, ATG16, and LC3 being the most upregulated. The Atg16 protein interacts with

Atg5-Atg12 complex to mediate the conjugation of LC3 to PE and helps control vesicle formation and elongation of the autophagosome. Gabarap and LC3 proteins, are orthologues of Atg8 and are necessary for autophagosome closure. How LC3 and

GABARAP are upregulated and why they are differentially regulated in muscle, at least in the present study, requires further study.

118

Another phenomenon that occurred in both tissues and requires further investigation was the decrease of Atg gene expression at d 14, followed by an increase at d 21. Gene expression is a second signal for autophagy induction and maintenance (Mizushima et al.,

2014). The second peak of Atg gene expression that was observed in our trial would support the maintenance of autophagy for at least 21 days in the face of continued nutrient stress. This raises questions as to how autophagy gene expression is maintained and for how long in can be maintained after the stressor is introduced. Future studies are needed to understand the significance of this secondary peak in Atg gene expression, and how it affects muscle maintenance or immunity on long term starvation. Examination of the expression of apoptosis genes in parallel to Atg genes is required since prolonged autophagy is understood to induce apoptosis (Klionsky et al., 2012).

There were some technical challenges experienced during this research however, resulting in a reduction in sample numbers in some instances. One tank from the feed-restricted groups had to be taken out of the analysis, since the results of this group were extremely different from all other groups, and one tank from the control groups was also taken out of the analysis, as the RNA extractions were not done properly. Additionally, for Day 21 there were only 3 replicates available (not 5 as for other samples). Although we still obtained highly statistically significant results for Day 21, further analysis should be performed to corroborate the results obtained. My results do still give valuable insight into Atg gene pattern behaviour throughout time, as well as which Atg genes could be used in future nutrient restriction trials.

119

Although gene expression is a secondary signal to protein expression in autophagy, it is needed to maintain autophagy flux. Future studies should focus on the effect that different stressful conditions have in Atg gene expression, as well as the relation between protein and gene expression. It will be necessary to observe autophagy protein and gene expression in parallel throughout time under different stress conditions and analyse how protein levels affect gene expression. This could give further insight into how the autophagy pathway is regulated and how muscle is maintained in rainbow trout muscle and other salmonids.

Measurement of muscle protein and tissue loss should also be examined in parallel to gene expression, perhaps over an even longer period than used in the present study. As salmonids depend significantly on protein as an energy source, it is important to understand how nutrient restriction affects the autophagy pathway. Past studies (Ryerse et al., 2014,

2015) have also concluded that feed restriction could help improve resistance against some infectious agents, and Atg gene expression should be examined before and after infection following feed restriction.

My first hypothesis was that Atg genes of rainbow trout could be identified by aligning their genome to other species allowing construction of primers to examine gene expression in feed restriction. In Chapter 2, we managed to align six Atg gene sequences of Atlantic salmon to rainbow trout and design primers based on them. The Atg genes in rainbow trout were highly conserved compared to other species, as verified by percent identity matrix and phylogenetic trees. Atlantic salmon and rainbow trout shared the most similarity between each other, followed by other species like catfish, Japanese rice fish and Japanese

120 pufferfish. The sequences obtained in rainbow trout proved to be specific to the desired Atg genes, and primers were successfully designed.

The second hypothesis was that autophagy genes selected at key stages in autophagosome development could be used as molecular markers in rainbow trout under conditions of nutrient restriction in vitro and in vivo. Chapter 2 demonstrated that most Atg genes were affected by serum restriction and a clear pattern was observed, with a significant upregulation after three d. We were also able to identify the Atg genes that were most affected by serum restriction. The in vivo trials in Chapter 3 demonstrated visible Atg gene patterns throughout time, identified the Atg genes most affected by nutrient restriction in liver and muscle and that there was distinct tissue-specific expression in some cases. A curious phenomenon, which is difficult to explain and was observed in in vivo trials in muscle, were the high levels of Atg gene expression in control groups relative to feed restricted groups in the first 4 days.

This thesis has provided important information regarding Atg gene expression under conditions of nutrient restriction in vitro and in vivo. Further studies are obviously still needed to understand the autophagy pathway and how it affects fish under different stressors.

121

The following conclusions can be drawn:

Chapter 2:

1) Autophagy related genes can be used as molecular biomarkers to study autophagy

flux following nutrient restriction.

2) In RTgill-W1 cells, expression of the majority of Atg genes peaked at Day 3,

roughly in agreement with LC3II detected using western blot.

3) ATG4, ATG9, BECN1 and LC3 underwent highly significant expression during

conditions of serum restriction.

4) Atg genes cannot be used to sequentially study individual stages of the autophagy

cycle as, at least in RTgill-W1, as Atg gene expression occurs at similar time

regardless of the gene position in the cycle.

Chapter 3:

1) The expression of most Atg in liver were significantly increased within the first 4 d,

with ATG5, ATG7 and ATG12 the most dramatically upregulated. The Atg genes

that did not show major changes were ATG4 and ATG16.

2) The expression of most Atg genes in muscle were significantly upregulated at Day

8, with ATG7, ATG16 and LC3 the most dramatically affected. The Atg genes that

did not show major changes were ATG4, ATG12 and GABARAP.

3) Liver and muscle samples both had a second significant peak of upregulation of Atg

genes at Day 21, suggesting can autophagy might still be activated after a longer

period of time than previously understood in rainbow trout.

122

REFERENCES

Äijälä, M., Malo, E., Ukkola, O., Bloigu, R., Lehenkari, P., Autio-Harmainen, H.,

Santaniemi, M., Kesäniemi, Y. A. (2013). Long-term fructose feeding changes the expression of leptin receptors and autophagy genes in the adipose tissue and liver of male rats: a possible link to elevated triglycerids. Genes & Nutrition, 8 (6), 623-635.

Andersen, C.L., Jensen, J.L., Orntoft, T.F. (2004). Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets.

Cancer Research Journal, 64 (15), 5245-5250.

Bandyopadhyay, U., Kaushik, S., Varticovski, L., Cuervo, M. A. (2008). The

Chaperone-mediated autophagy receptor organizes in dynamic protein complexes at the lysosomal membrane. Molecular and Cellular Biology, 28, 5747-5763.

Behnke, J., Coad, B.W. (2002). Trout and salmon of North America. The Canadian

Field-Naturalist, 117 (2), 218.

Bernard, A., Jin, M., Xu, Z., Klionsky, D.J. (2015). A large-scale analysis of autophagy-related gene expression indentifies new regulators of autophagy. Autophagy, 11,

2114-2122.

Bols, N.C., Barlian, A., Chirino-Trejo, M., Caldwell, S.J., Goegan, P., Lee, L.E.J.

(1994) Development of a cell line from primary cultures of rainbow trout, Oncorhynchus mykiss (Walbaum), gills. Journal of Fish Diseases, 17, 601–611.

Burman, C. & Ktistakis, N.T. (2010). Autophagosome formation in mammalian cells. Seminars in Immunopathology, 32, 397-413.

123

Cao, Y. & Klionsky, D.J. (2007). Physiological functions of Atg6/Beclin 1: a unique autophagy-related protein. Cell Research, 10, 839-49.

Cheong, H., Nair, U., Geng, J., Klionsky, D.J. (2008). The Atg1 kinase complex is involved in the regulation of protein recruitment to initiate sequestering vesicle formation for nonspecific autophagy in Saccharomyces cerevisiae. Molecular Biololy of the Cell, 19,

668-681.

Clarke, A. (2003). Costs and consequences of evolutionary temperature adaptation.

Trends in Ecology & Evolution, 18, 573-581.

Cowey, C. B., Adron, J. W., Knox, D. (1975). Studies on the nutrition of marine flatfish. The thiamin requirement of turbot (Scophthalmus maximus). British Journal of

Nutrition, 34 (03), 383-390.

Cuervo, A.M., & Macian, F. (2012). Autophagy, nutrition and immunology.

Molecular Aspects of Medicine, 33, 2-13.

Damsgard, S., Sorum, U., Ugelstad, I., Eliassen, R.A., Mortensem, A. (2004). Effects of feeding regime on susceptibility of Atlantic salmon (Salmo salar) to cold water water vibriosis. Aquaculture, 239, 37-46.

Dereeper, A., Guignon, V., Blanc G., Audic S., Buffet S., Chevenet F., Dufayard

J.F., Guindon S., Lefort V., Lescot M., Claverie J.M., Gascuel O. (2008). Phylogeny.fr: robust phylogenetic analysis for the non-specialist. Nucleic Acids Research, 36, 465-469.

Deretic, V. (2011). Autophagy in immunity and cell-autonomous defense against intracellular microbes. Immunological Reviews, 240, 92-104.

de Waal, E.J., Vreeling-Sindelárová, H., Schellens, J.P., Houtkooper, J.M., James, J.

(1986). Quantitative changes in the lysosomal vacuolar system of rat hepatocytes during short-term starvation. Cell Tissue Research, 243, 641-648. 124

Dice, J.F., Walker, C.D., Byrne, B., Cardiel, A. (1978). General characteristics of protein degradation in diabetes and starvation. Proceedings of the National Academy of

Sciences, 75 (5), 2093-2097.

Ding, W., Manley S., Ni, H. (2011). The emerging role of autophagy in alcoholic liver disease. National Institute of Health, 236 (5), 546-556.

Green, R.D., Levine, B. (2014). To be or not to me? How selective autophagy and cell death govern cell fate. Cell, 157, 65-75.

Guderley, H., Lapointe, D., Bédard, M., Dutil J.D. (2003). Metabolic priorities during starvation: enzyme sparing in liver and white muscle of Atlantic cod, Gadus morhua

L. Comparative Biochemistry and Physiology Part A, 135, 347-356.

Egan, D.F., Shackelfold, D.B., Mihaylova, M.M., Gelino, S., Kohnz, R.A., Mair,

W., Vasquez D.S., Joshi, A., Gwinn, D.M., Taylor, R., Asara, J.M., Fitzpatrick, J., Dillin,

A., Viollet, B., Kundu, M., Hansen, M., Shaw, R. J. (2011). Phosphorylation of ULK1

(hATG1) by AMP-activated protein kinase connects energy sensing to mitophagy. Science,

331, 456-461.

Feng Z, Bai L, Yan J, Li Y, Shen W, Wang Y. (2011). Mitochondrial dynamic remodeling in strenuous exercise induced muscle and mitochondrial dysfunction: regulatory effects of hydroxytyrosol. Free Radical Biology & Medicine, 50(10), 1437–46.

Ferraro, E. & Cecconi F. (2007). Autophagic and apoptotic response to stress signals in mammalian cells. Archives of Biochemistry and Biophysics, 462, 210-219.

Galluzzi, L., Vitale, I., Abrams, J.M., Alnemri, E. H., Baehrecke, E.H.,

Blagosklonny, M.V., Dawson, T.M., Dawson, V.L., El-Deiry, W.S., Fulda, S., Gottlieb, E.,

Green, D.R., Hengartner, M.O., Kepp, O., Knight, R.A., Kumar, S., Lipton, S.A., Lu, X.,

Madeo, F., Malorni, W., Mehlen, P., Nuñez, G., Peter, M.E., Piacentin, M., Rubinsztein, 125

D.C., Shi, Y., Simon, H.U., Vandenabeele, P., White, E., Yuan, J., Zhivotovsky, B.,

Melino, G., Kroemer, G. (2012). Molecular definitions of cell death subroutines: recommendations of the nomenclature committee on cell death. Cell Death and

Differentiation, 19, 107-120.

García-Valtanen, P., Ortega-Villaizan, M.M., Martinez-Lopez, A., Medina-Gali, R.,

Perez, L., Mackenzie, S., Fiqueras, A., Coll, J. M., Estepa, A. (2014). Autophagy-inducing peptides from mammalian VSV and fish VHSV rhabdoviral G glycoproteins (G) as models for the development of new therapeutic molecules. Autophagy, 10, 1666-1680.

Guderley, H., Lapointe, D., Bédard, M., Dutil, J.D. (2003). Metabolic priorities during starvation: enzyme sparing in liver and white muscle of Atlantic cod, Gadus morhua

L. Comparative Biochemistry and Physiology (Part A), 135, 347-356.

Gutierrez, M.G., Saka, H.A., Chinen, I., Zoppino, F.C. M., Yoshimori, T., Bocco,

J.L., Colombo, M.I., (2007). Protective role of autophagy against Vibrio cholerae cytolysin, a pore-forming toxin from V. cholerae. Proceedings of the National Academy of Sciences,

104 (6), 1829-1834.

He, C., Bartholomew, C.R., Zhou, W., Klionsky, D.J. (2009). Assaying autophagic activity in transgenic GFP-Lc3 and GFP-Gabarap zebrafish embryos. Autophagy, 5, 520-

526.

Itakura, E. & Mizushima, N. (2010). Characterization of autophagosome formation by a hierarchical analysis of mammalian Atg proteins. Autophagy, 6, 764-767.

Jamart, C., Benoit, N., Raymackers, J.M., Kim, H.J., Kim, C.K., Francaux, M.

(2012). Autophagy-related and autophagy-regulatory genes are induced in human muscle after ultra-endurance exercise. European Journal of Applied Physiology, 112, 3173-3177.

126

Jin, M., He, D., Backues, S.K., Freeberg, M.A., Liu X., Kim, J. K., Klionsky, D.J.

(2014). Transcriptional regulation by pho23 modulates the frequency of autophagosome formation. Current Biology, 24, 1314-1322.

Johnston, I.A., Dunn, J. (1987). Temperature acclimation and metabolism in ectotherms with particular reference to teleost fish. Symposia of the Society for

Experimental Biology, 41, 67-93.

Jung, C. H., Ro, S.H., Cao, J., Otto, N. M., Kim, D.H., (2010). mTOR regulation of autophagy. Federation of European Biochemical Societies Letters, 584, 1287-1295.

Kabeya, Y., Mizushima, N., Ueno, T., Yamamoto, A., Kirisako, T., Noda, T.,

Kominami, E., Ohsumi, Y., Yoshimori, T. (2000). LC3, a mammalian homologue of yeast

Apg8p, is localized in autophagosome membrane after processing. The European

Molecular Biology Organization Journal, 19, 5720-5728.

Karim, R. M., Kawanago, H., Kadowaki, M. (2014). A quick signal of starvation induced autophagy: transcription versus post-translational modification of LC3. Analytical

Byochemistry, 465, 28-34.

Klionsky, D.J., Cregg, J.M., Dunn, W.A. Jr., Emr, S.D., Sakai, Y., Sandoval, I.V.,

Sibirny, A., Subramani, S., Thumm, M., Veenhuis, M., Ohsomi, Y. (2003). A unified nomenclature for yeast autophagy-related genes. Development Cell, 5 (4), 539-545.

Klionsky, D.J. & Yang, Z. (2010). Eaten alive: a history of macroautophy. Nature

Cell Biology, 12, 814-822.

Kundu, M. & Thompson, C.B. (2008). Autophagy: Basic principles and relevance to disease. Annual Review of Pathology, 3, 427-455.

127

Lee, J. M., Wagner, M., Xiao, R., Kim, K.H., Feng, D., Lazar, M.A., Moore, D.D.

(2014). Nutrient-sensing nuclear receptors coordinate autophagy. Nature, 516 (7529), 112-

115.

Li, Q., Deng, X., Huang, Z., Zheng, S., Tettamanti, G., Cao, Y., Ferg, Q. (2011).

Expression of autophagy-related genes in the anterior silk gland of the silkworm (Bombyx mori) during metamorphosis. Canadian Journal of Zoology, 89, 1019-1026.

Li, W.W., Li J., Bao, J.K. (2012) Microautophagy: lesser-known self-eating.

Cellular and Molecular Life Sciences, 69, 1125-1136.

Li, Y.Y., Wang, T., Gao, S., Xu, G. M., Niu, H., Huang, R., Wu, S. Y.

(2015). Salmonella plasmid virulence gene spvB enhances virulence by inhibiting autophagy in zebrafish model. Fish & Shellfish Immunology, 49, 252-259.

Ma, D., Molusky, M.M., Song, J., Hu, C.R., Fang, F., Rui, C., Mathew, A.V.,

Pennathur, S., Liu, F., Cheng, J.X., Guan, J. L., Lin, J.D. (2013). Autophagy deficiency by hepatic FIP200 deletion uncouples steatosis from liver injury in NAFLD. Molecular

Endocrinology, 27, 1643-1654.

Madden T. (2002). The BLAST Sequence Analysis Tool. Retrieved from: http://www.ncbi.nlm.nih.gov/books/NBK21097/

MacPhee DD, Ostland VE, Lumsden JS, Derksen J, Ferguson HW. (1995).

Influence of feeding on the development of bacterial gill disease in rainbow trout

Oncorhynchus mykiss. Diseases of Aquatic Organisms, 21, 163-170.

McCue, M.D. (2010). Starvation physiology: reviewing the different strategies animals use to survive a common challenge. Comparative Biochemistry and Physiology

Part A, 156, 1-18.

128

McWilliam H., Li, W., Uludag, M., Squizzato, S., Park, Y.M., Buso, N., Cowley,

A.P., Lopez, R (2013). Nucleic acids research. Retrieved from: https://www.ncbi.nlm.nih.gov/pubmed/23671338.

Mizushima, N., Noda, T., Yoshimori, T., Tanaka, Y., Ishii, T., George, M.D.,

Klionsky J.D., Ohsumi, M., Ohsumi, Y. (1998). A protein conjugation system essential for autophagy. Nature, 395, 395-398.

Mizushima, N., Yoshimori, T., Levine, B. (2010). Methods in mammalian autophagy research, Cell, 140, 313-326.

Mohapatra, S., Chakraborty, T., Shimizu, S., Matsubara, T., Ohta, K., Kacem, R. B.

H. (2016). Starvation: An alternate measure to improve immunity and physiology of red sea bream during Edwardsiella tarda infection. Journal of Aquaculture Research &

Development, 2, 2-7.

Moreau, P., Moreau, K., Segarra, A., Tourbiez, D., Travers, M.A., Rubinsztein, D.

C., Renault, T. (2015). Autophagy plays an important role in protecting Pacific oysters from OsHV-1 and Vibrio aestuarianus infections. Autophagy, 11 (3), 516-526.

Mortimore, G.E., Mondon, C.E. (1970). Inhibition by insulin of valine turnover in liver. Evidence for a general control of proteolysis. Journal of Biological Chemistry, 245

(9), 2375-2383.

Nair, U., Cao, Y., Xie, Z., Klionsky, D.J. (2010). Roles of the lipid-binding motifs of Atg18 and Atg21 in the cytoplasm to vacuole targeting pathway and autophagy. Journal of Biological Chemistry, 285, 11476-11488.

Nixon, R. (2013). The role of autophagy in neurodegenerative disease. Nature

Medicine, 19, 983-997.

129

O'Donnell, V., Pacheco, J. M., LaRocco, M., Burrage, T., Jackson, W., Rodriquez,

L.L., Borca, M.V., Baxt, B., (2010). Foot-and-mouth disease virus utilizes an autophagic pathway during viral replication. Virology, 410, 142-150.

Pfaffl, M. W., Tichopad, A., Prgomet, C., Neuvians, T.P. (2004) Determination of stable housekeeping genes, differentially regulated target genes and sample integrity:

BestKeeper-Excel-based tool using pair-wise correlations. Biotechnology, 26 (6), 509-515.

Rebrikov, D.V. & Trofimov, D. (2206) Real-time PCR: approaches to data analysis

(a review). Prikladnaia Biokhimiia i Mikrobiolgiia, 42 (5), 520-528.

Ryerse, I.a., Hooft, J.M., Bureau, D.P., Hayes, M.A., Lumsden, J.S. (2014). Diets containing corn naturally contaminated with deoxynivalenol reduces the susceptibility of rainbow trout (Oncorhynchus mykiss) to experimental Flavobacterium psychrophilum infection. Aquaculture Research, 47 (3), 787-796.

Ryerse, I., Hooft, J.M., Bureau, D.P., Hayes, M.A., Lumsden, J.S. (2015). Purified deoxynivalenol or feed restriction reduces mortality in rainbow trout, Oncorhynchus mykiss

(Walbaum), with experimental bacterial coldwater disease but biologically relevant concentrations of deoxynivalenol do not impair the growth of Flavobacterium psychrophilum. Journal of Fish Diseases, 38 (9), 809-819.

Rubinsztein, D.C., Shpilka, T., Elazar, Z. (2012). Mechanisms of autophagosome biogenesis. Current Biology, 22, 29-34.

Salem, M. Silverstein, J., Rexroad, J., Yao, J. (2007). Effect of starvation on global gene expression and proteolysis in rainbow trout (Oncorhynchus mykiss). Biomedical

Central Genomics, 8, 328.

Seiliez, I., Gutierrez, J., Salmeron, C., Skiba-Cassy, S., Chauvin, C., Dias, K.,

Kaushik, S., Tesseraud, S., Panserat, S. (2010). An in vivo and in vitro assessment of 130 autophagy-related gene expression in muscle of rainbow trout (Oncorhynchus mykiss).

Comparative Biochemistry and Physiology Part B, 157, 258-266.

Seiliez, I., Gabillard, J.C., Riflade, M., Sadoul, B., Dias, K., Avérous, J., Tesseraud,

S., Skiba, S., Panserat, S. (2012). Amino acids downregulate the expression of several autophagy-related genes in rainbow trout myoblasts. Autophagy, 8, 364-375.

Seiliez. I, Panserat, S., Lansard, M., Polakof, S., Plagnes J.,E., Surget, A., Dias, K.,

Larquier, M., Kaushik, S., Skiba-Cassy, S. (2011). Dietary carbohydrate-to-protein ratio affects TOR signaling and metabolism-related gene expression in the liver and muscle of rainbow trout after a single meal. The American Journal of Physiology, 300 (3), 733-743.

Sekito, T., Kawamata, T., Ichikawa, R., Suzuki, K., Ohsumi, Y. (2009). Atg17 recruits Atg9 to organize the pre-autophagosomal structure. Genes to Cells, 14, 525-538.

Settembre, C., Di Malta, C., Polito, V.A., Arencibia, M. G., Vetrini, F., Erdin, S.,

Erdin, S.U., Huynh, T., Medina, D., Colella, P., Sardiello, M., Rubinsztein, D.C., Ballabio,

A., (2011). TFEB links autophagy to lysosomal biogenesis. Science, 332 (6036), 1429-

1433.

Schiotz, B.L., Roos, N., Rishovd, A., Gjoen, T. (2010). Formation of autophagosome and redistribution of LC3 upon in vitro infection with infectious salmon anemia. Virus Research, 151, 104-107.

Shin, H, R., Kim, K. L., Baek, S. H. (2016) Epigenetic and transcriptional regulation of autophagy. Autophagy, 12, 2248-2249.

Singh, R. & Cuervo, A. M., (2011). Autophagy in the cellular energetic balance.

Cell Metabolism, 13, 495-504.

131

Singh, R., Kaushik, S., Wang, Y., Xiang, Y., Novak, I., Komatsu, M., Tanaka, K.,

Cuervo, M.A., Czaja, M. J. (2009). Autophagy regulates lipid metabolism. Nature, 458,

1131-1135.

Suzuki, K., Kirisako, T., Kamada, Y., Mizushima, N., Noda, T., Ohsumi, Y. (2001).

The pre-autophagosomal structure organized by concerted functions of APG genes is essential for autophagosome formation. European Molecular Biology Organization

Journal, 20, 5971-5981.

Suzuki, K., Kubota, Y., Sekito, T., Ohsumi, Y. (2007). Hierarchy of Atg proteins in pre-autophagosomal structure organization. Genes to Cells, 12, 209-218.

Takahashi, Y., Meyerkord, C.L., Hori, T., Runkle, K., Fox, T.E., Kester, M.,

Loughran, T.P., Wang, H. G. (2011). Bif-1 regulates Atg9 trafficking by mediating the fission of golgi membranes during autophagy. Autophagy, 7, 61-73.

Wang, A., Huen, S.C., Laun, H.H., Yu, H., Zhang, C., Gallezot, J.D., Booth, C.J.,

Medzhitov, R. (2016). Opposing effects of fasting metabolism on tissue tolerance in bacterial and viral inflammation. Cell, 166 (6), 1512-1525.

Wise, D.J., Greenway, T., Li, M. H., Camus, A., Robinson, C. (2007). Effects of variable periods of food deprivation on the development of enteric septicemia in channel catfish. Journal of Aquatic Animal Health, 20, 39-44.

Yabu, T., Imamura, S., Mohammed, M., S., Touhata, K., Minami, T., Terayama,

M., Yamashita, M. (2011) Differential gene expression of HSC70/HSP70 in yellowtail cells in response to chaperone-mediated autophagy. Federation of European Biochemical

Societies Journal, 278, 673-685.

132

Yabu, T., Imamura, S., Mizusawa, N., Touhata, K., Yamashita, M. (2012).

Induction of autophagy by amino acid starvation in fish cells. Marine Biotechnology

Journal, 14, 491-501.

Yang, Z. and K. J. Daniel. (2010). Mammalian autophagy: core molecular machinery and signaling regulation. Cellular and Developmental Biology, University of

Michigan, 124-131.

Ye, J., Coulouris, G., Zaretskaya, I., Cutcutache, I., Rozen, S., Madden, T. L.

(2012). Primer-BLAST: A tool to design target-specific primers for polymerase chain reaction. Biomed Central Bioinformatics, 13, 134.

Xie, Z., Nair, U., Klionsky, J.D. (2008). Atg8 controls phagophore expansion during autophagosome formation. Molecular and Cellular Biology, 19 (8), 3290-3298.

Zhang, Z., Guo, M., Zhao, S., Xu, W., Shao, J., Zhang, F., Wu, L., Zheng, S.

(2015). The update on transcriptional regulation of autophagy in normal and pathological cells: A novel therapeutic target. Biomediine & Pharmacotherapy Journal, 74, 17-29.

Zhifen, Y., Klionsky, D.J. (2010). Mammalian autophagy: core molecular machinery and signaling regulation. Current Opinion in Cell Biology, 22 (2), 124-131.

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APPENDIX

Appendix 2.1: Rainbow trout Atg gene sequences of Atg4, Atg12, LC3 and GABARAO from NCBI (Madden, 2002).

Rainbow trout ATG4 gene sequence >(gi|620602803:c59722-59690) ATGTTGTTGTTGTTGTTGTTTACACCTGCAGAAATGCACTTTTATATTTCAGCTA CTCTCACATACGACACCCTTGGCTTTGGAGAGTTTGACGATTTTCCAGAGACCT CAGAGCCAGTGTGGATCTTAGGGAAAGAATTCAATGCACTCACAGAAAAGGAA GACATTCTTTCACATGTCACTTCACGACTATGGTTCACCTACAGAAAAAACTTC CCGCCCATTGGAGGGACAGGGCCTACGTCCGACACAGGCTGGGGCTGCATGCT GCGATGTGGACAGATGATCCTAGGCGAGGCCCTGGTCCGCAGGCATCTAGGCC GAGACTGGAGATGGGTGAGGAGCCAGTCTCAGAGAGAAGACTACATCGGTATC CTCAACGCCTTCCTTGACAAGAAAGACGGCTACTATTCTTTACATCAAATCGGT CAAATTAAGATCCTACATCTGTACTTGTGTGATTGCGTTTAA

Rainbow trout ATG12 gene sequence >(gi|620628870:2737-2839) ATGTCTGCCAACGCAGAGTCTCCTACAGAAACGCAAAAAGATGAGCCTTCAAC CCCACAACAGCCTACGGAAGACTCGGGAACGGCAGACGAGAAGAAAAAAATT GATGTGTTGTTGAAGGCAGTAGGAGACACCCCCATCATGAAGACAAAGAAATG GTCGGTAGAGAAAGGGAGGACAGTGCAATCACTCTCTCAATTCATCTCTCGATT CCTCAAGATGGAGGCCAATGAACAGCTGGTGAGTTATTGTGTGTGTGCTTCTCA ATAG

Rainbow trout LC3 gene sequence >(gi|620599730:c670512-670473) ATGCCTTCAGAGAAGACATTCAAACAAAGGAGGACATTTGAGCAGAGAGTAG ATGATGTACGACTGATCCGAGAGCAGCACCCCAACAAGATTCCGGTCATCATT GAGAGATACAAGGGAGAGAAGCAGCTGCCCATCCTGGATAAAACCAAGTTCCT GGTTCCTGACCATGTCAACATGAGTGAACTGATCAAAATCATCAGGAGGCGCC TCCAGCTGAACTCCAACCAGGCCTTTTTCCTGCTGGTGAACGGCCACAGCATGG TGTCCGTGTCGGCAGCCATCGCTGAGGTATACGAGCGCGAGAAGGATGACGAT GGCTTCCTCTACATGGTGTACGCCTCGCAGGAGACCTTCGGAACCATGGCCCCC CAGTAA

134

Appendix 2.1: Rainbow trout Atg gene fragment sequences (continue) Rainbow trout GABARAP gene sequence >gb|BT073647.1|:65-433 ATGAAGTTTCAATACAAAGAAGAGCACCCATTTGAGAAGAGACGGTCTGAGGG CGAGAAAATAAGAAAGAAGTATCCGGACAGGGTACCCGTAATTGTGGAAAAA GCCCCCAAAGCAAGGATAGGAGATCTGGACAAGAAGAAATACCTTGTGCCCTC TGACCTCACAGTGGGCCAGTTCTACTTCCTCATCCGAAAACGAATCCACCTGCG GGCCGAGGACGCCCTGTTCTTCTTTGTAAACAACGTCATTCCTCCCACCTCAGC CACTATGGGCCTTCTATACCAGGAGCACCATGAAGAGGACTTTTTCCTCTACAT TGCCTACAGTGATGAGAGTGTCTATGGAGACGGCCAAACGGAAGTCTAA

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Appendix 2.2a-f) Clustal alignments between rainbow trout Atg genes and sequences from 14 other species. CLUSTAL O(1.2.4) multiple sequence alignment for ATG5. Yeast ------ATGA------60 Sorghum ------ATGG-----CTG Fruit fly ATAAATCCGCGGTCATACTGGTTCGAGGCCAAAAACAAAAAAATTATAATAATTAAAAGT Clawed Frog ------Red junglefowl ------Brown rat ------Cattle ------Human ------Rhesus macaque ------Cat ------Zebrafish ------Atlantic salmon------Rainbow trout ------Rice fish ------Pufferfish ------

Yeast ------ATGACATTAAACAATTACTTTGGAATGGTGAGCTTAATGTGC-----TG 120 Sorghum CCCCGCACGACGAGGCCGCGGCGTGGTCCGAGGAGGCGGCGCGCCGGGTCTGGGCCGGCG Fruit fly TTAAGCAAAAGATAGCCATGGCCCACGACCGCGAGGTGTTGCGCATGATATGGGAGGGTC Clawed frog ------ATGACAGATGACAAAGATGTTCTTCGTGATGTCTGGTTTGGCC Red junglefowl ------ATGACAGATGACAAAGATGTGCTTCGAGATGTGTGGTTTGGAC Brown rat ------ATGACAGATGACAAAGATGTGCTTCGAGACGTGTGGTTTGGAC Cattle ------ATGACAGATGACAAAGATGTGCTTCGAGATGTGTGGTTTGGAC Human ------ATGACAGATGACAAAGATGTGCTTCGAGATGTGTGGTTTGGAC Rhesus macaque ------ATGACAGATGACAAAGATGTGCTTCGAGATGTGTGGTTTGGAC Cat ------ATGGCAGATGACAAAGATGTACTTCGAGACGTGTGGTTCGGCC Zebrafish ------ATGGCAGATGACAAGGATGTGCTTCGAGATGTTTGGTTTGGAA Atlantic salmon------ATGGCAGATGACAAGGACGTGCTGCGAGATGTTTGGTTTGGTC Rainbow trout ------ATGGCAGATGACAAGGACGTGCTGCGGGATGTTTGGTTTGGTC Rice fish ------ATGGCAGATGACAAAGACGTGCTGAGGGATGTCTGGTTCGGCC Pufferfish ------ATGGCAGATGACAAGGATGTGTTGAGAGATGTTTGGTTCGGCC * * * * **

136

Yeast GTATCGATCGATCCTTCATTTTTGATGAAA-GGAAGTCCAAGAGAAATTGCGGTGCTACG 180 Sorghum CCGTGCCGCTCCAGGTCCACCTCCACGACGCCGACGTCACCGCACTGCCCC-CGCCGCC- Fruit fly AGATTGGCATCTGCTTCCAGGCGGACCGCGACGAGATAGTGGGCATCAAGC-CAGAGCC- Clawed frog GAATACCAACCTGCTTCACTTTGTACCAAGATGAATTAACAGAAAGGGAAG-CTGAACC- Red junglefowl GCATACCAACCTGCTTCACTCTGTATCAGGATGAGATAACTGAGAGGGAAG-CTGAACC- Brown cat GGATTCCAACGTGCTTTACTCTCTATCAGGATGAGATAACTGAACGAGAAG-CAGAGCC- Cattle GAATTCCAACTTGCTTCACCCTGTATCAGGATGAGATAACTGAACGCGAGG-CAGAGCC- Human GAATTCCAACTTGTTTCACGCTATATCAGGATGAGATAACTGAAAGGGAAG-CAGAACC- Rhesus macaque GTATTCCAACTTGCTTCACGCTATATCAGGATGAGATAACTGAAAGGGAAG-CAGAACC- Cat GGATACCGGCGTGCTTCATGCTTTATCCAGATGAAGTCACGGAGAGAGAAG-CAGAACC- Zebrafish GGATACCCGCCTGTTTCACACTGTCTCCAGACGAAACCACAGAGAGAGAGG-CAGAACC- Atlantic salmonGGATTCCGACCTGCTTCACACTTTACCAGGATGAGATCACCGAGAGAGAGG-CCGAACC- Rainbow trout GGATTCCGACCTGCTTCACACTTTACCAGGATGAGATCACAGAGAGAGAGG-CCGAACC- Rice fish GTATACCCACCTGCTTCACCCTTAACCCGGATGAAGTCACTGAGAGAGAGG-CAGAGCC- Pufferfish GGATCCCCACCTGTTTCACCCTCAACCAGGACGAGGTCACCGAGAGAGAGG-CCGAGCC- * * ** * *

Yeast AATAAGGGTACCA-AGGGAAACATATTTAGTCAATTATATGCCCCTCATTTGGAACAAGA 240 Sorghum ----GGCCTTCCTGACTTTGGGGCCAAGAATTGGGTACTTGCCCCTCTTGATACCTGTTA Fruit fly ------CTTCTACCTGATGGTCTCCCGCCTCAGCTACTTGCCGCTGGTCACCGATAAGG Clawed frog ------CTATTATTTGCTTCTACCTCGCATCAGCTATTTGACACTAGTGACTGACAAAG Red junglefowl ------TTATTATTTGCTTTTGCCAAGAATAAGTTACTTGACACTGGTAACAGACAAAG Brown rat ------ATACTATTTGCTTTTGCCAAGAGTCAGCTATTTGACGCTGGTAACTGACAAAG Cattle ------ATACTATTTGCTTTTGCCAAGAGTAAGTTATTTGACGTTGGTAACTGACAAAG Human ------ATACTATTTGCTTTTGCCAAGAGTAAGTTATTTGACGTTGGTAACTGACAAAG Rhesus macaque ------ATACTATTTGCTTTTGCCAAGAGTAAGTTATTTGACGTTGGTAACTGACAAAG Cat ------GTTTTATCTCCTCCTTCCGAGGATCAGTTATCTGACGCTAGTGACCGACAAGG Zebrafish ------CTACTATCTGCTCCTCCCACGGGTCAGTTACCTGACACTAGTCACTGATAAAG Atlantic salmon------CTTCTATCTCCTCCTGCCAAGGGTGAGCTACCTGACTCTGGTCACAGACAAGG Rainbow trout ------CTTCTATCTCCTCCTGCCAAGGGTGAGCTACCTGACTCTGGTCACAGACAAGG Rice fish ------ATACTATCTGCTGTTGCCCAGGGTGAGTTACCTGCCTCTGGTCACAGACAAGG Pufferfish ------TTACTATCTGTTGCTCCCGAGGGTGAGCTACCTGACTCTGGTTACAGACAAGG * * ** ** * * *

Yeast TTAAAAGCTT---TCTTTCTTTTGACCCATTGACCGACAGTGAGAAGTATTTCTGGTTTG 300 Sorghum TAAAGGCTCATTTCAGCAAT------GCGCTCCCACCTGGTGTTGACACTGTTTGGTTTG Fruit fly TGCGAAAGTACTTCACCCGCTACATTGCCGCCGAGCATCAGGATGGTGCTGTCTGGTTCG Clawed frog TGAAAAAACATTTTCAGAAAGTCATGAGGACAGA---AGATATAAGTGAAATTTGGTTTG Red Junglefowl TGAAGAAACACTTCCAGAAAGTTATGAGACAAGA---GGAAGTTAATGAGATATGGTTTG

137

Brown rat TGAAAAAGCACTTTCAGAAGGTTATGAGACAAGA---AGATGTTAGTGAGATTTGGTTTG Cattle TGAAAAAGCACTTTCAGAAGGTTATGAGACAAGA---GGACATTAGTGAGATATGGTTTG Human TGAAAAAGCACTTTCAGAAGGTTATGAGACAAGA---AGACATTAGTGAGATATGGTTTG Rhesus macaque TGAAAAAGCACTTTCAGAAGGTTATGAGACAAGA---AGACATTAGTGAGATATGGTTTG Cat TCAAGAAGCACTTTCTGAAAGTCCTAAAAGCCGA---AGACGTAGAGGAAATGTGGTTTG Zebrafish TCAAGAAACATTTTCTCAAAGTCATGAAGGCAGA---GGATGTGGAGGAAATGTGGTTTG Atlantic salmonTGAAAAAGCACTTCCTGAAGGTCATGAAGGCCGA---AGACGTAGAGGAGATATGGTTTG Rainbow trout TGAAAAAGCACTTCCTGAAGGTCATGAAGGCCGA---AGACGTAGAGGAGATGTGGTTTG Rice fish TGAAAAAACACTTCCTTAAAGTGATGAGGACAGA---AGATGTGGAGGAGATGTGGTTTG Pufferfish TGAAGAAACACTTCCTCAAAGTGATGAAGGCGGA---CGATGTGGAGGAGATGTGGTTTG * * ***** *

Yeast AGCATAATAAGACGCCTATTCCGTGGAATTACCCAGTAGGTGTTTTGTTTGACTGCCTAG 360 Sorghum AATATAAAGGGCTGCCTTTAAAATGGTATGTGCCAATTGGTGCTCTCTTTGACCTTCTAT Fruit fly ACTACAATGGCATACCGCTGCGACTGCACTATCCAACTGGTGTGCTCTACGATCTGCTGC Clawed frog AATTTGAAGGGATTCCTCTAAAATGGCATTACCCAATTGGTTTGCTGTTTGATTTACATG Red junglefowl AATATGAAGGTACGCCACTGAAATGGCATTACCCAATTGGTTTGCTGTTTGATTTGCATG Brown rat AATATGAAGGCACACCCCTGAAATGGCATTATCCAATTGGCCTACTGTTCGATCTTCTTG Cattle AATATGAAGGCACACCACTGAAATGGCATTATCCAATTGGTTTGCTATTTGATCTTCTTG Human AATATGAAGGCACACCACTGAAATGGCATTATCCAATTGGTTTGCTATTTGATCTTCTTG Rhesus macaque AATATGAAGGCACACCACTGAAATGGCATTATCCAATTGGTTTACTATTTGATCTTCTTG Cat AATACGAAGGCACACCTCTGAAATGGCACTATCCAATCGGAGTATTGTTCGATCTCCATG Zebrafish AACACGAGGGAACGCCTCTTAAATGGCACTATCCCATTGGTGTGTTGTTCGACCTCCATG Atlantic salmonATTTCGAAGGAACGCCACTCAAATGGCACTATCCAATCGGATTGCTGTTTGACCTGCATG Rainbow trout ATTTCGAAGGAACGCCACTCAAATGGCACTATCCAATCGGATTGCTGTTCGACCTACATG Rice fish AGCATGAAGGAACACCTCTCAAATGGCACTACCCGATTGGAGTTCTGTTCGACTTCCATG Pufferfish AGTATGAAGGGACGCCGCTGAAATGGCACTATCCAATTGGAGTCCTGTTTGACTTTCATG * * ** * * * ** ** * * ** *

Yeast CCGGAAAAAGCGCTACTTTTACC------420 Sorghum GCGCA---GATCCAGAAAGACCATGGAATCTAATAGTCCATTTTAGGGGATATCCCTCAG Fruit fly ATCCAGAGGAGGACTGCACGCCCTGGGGTCTGACAATACACTTTTCCAAATTCCCGGAGG Clawed frog CATCA---AATACATCTCTGCCTTGGAGCATCACTGTCCATTTTAAGAACTTTCCAGCTA Red junglefowl CATCA---AATACAGCCCTTCCTTGGAGCATCACAGTGCATTTCAAGAATTTTCCGGAAA Brown rat CATCA---AGCTCAGCTCTGCCTTGGAACATCACAGTACATTTCAAGAGTTTTCCAGAAA Cattle CATCA---AGTTCAGCTCTTCCTTGGAACATCACAGTACATTTTAAGAGTTTTCCAGAAA Human CATCA---AGTTCAGCTCTTCCTTGGAACATCACAGTACATTTTAAGAGTTTTCCAGAAA Rhesus macaque CATCA---AGTTCAGCTCTTCCTTGGAACATCACAGTACATTTTAAGAGTTTTCCAGAAA Cat CTTCA---AATACTGCTCTCCCTTGGAGTATCACTGTACACTTTAAGCATTTTCCTGATC

138

Zebrafish CTTCA---AACTCCGCTCTGCCCTGGAATGTCACAGTGCACTTCAAGAACTTCCCAGAAC Atlantic salmonCCTCC---AACACTGCCCTGCCGTGGAGCATCACTGTGCACTTTAAGAATTTCCCAGAGC Rainbow trout CCTCC---AACACTGCCCTGCCATGGAGTATCACTGTGCACTTTAAGAATTTCCCAGAGC Rice fish CCTCA---AACACGGTCTTACCCTGGAGCATCACGGTGCACTTTAAGAATTTTCCAGATG Pufferfish CGTCC---AACACTGTATTACCATGGAGCATCACAGTGCACTTTAAGAATTTTCCAGATC *

Yeast ------480 Sorghum AAATCTTGTCACCATGCGAGGGTGAAGATAGCGTGAAGTGGAGCTACATGAATTCCCTGA Fruit fly AGACGCTGGTCAAAATGAATTCAAAGGAGCTGTTGGAATCGCACTACATGTCCTGCTTGA Clawed frog AAGACCTTCTCCATTGCCAGTCTAAAGATGTTATAGAAGCTCATTTCATGTCATCTGTTA Red junglefowl AGGATCTTCTACACTGCCATTCTAAGGATGTGATTGAAGCTCATTTTATGGCTTGCATAA Brown rat AGGACCTTCTGCACTGTCCGTGCAAGGATGCAGTTGAGGCTCACTTTATGTCATGTGTGA Cattle AGGACCTTCTACACTGTCCATCTAAGGATGTAATTGAAGCTCATTTTATGTCTTGTGTGA Human AAGACCTTCTGCACTGTCCATCTAAGGATGCAATTGAAGCTCATTTTATGTCATGTATGA Rhesus macaque AAGACCTTCTGCACTGTCCATCTAAGGATGCAATTGAAGCTCATTTTATGTCATGTATGA Cat GAGACTTGCTCCGCTGCTCCTCGAGCGTCGTGATAGAAGCCCATTTTATGTCCACCGTTA Zebrafish AAGACTTGCTTCACTGCTCAACAAACTCTGTGATAGAAGCACATTTCATGTCTTGCATTA Atlantic salmonGTGACTTGCTCCACTGCCCCTCTAACTCTGTGATCGAGGCCCACTTCATGTCCAGCATCA Rainbow trout GTGACCTGCTCCACTGCCCCTCTAACTCTGTGATCGAGGCCCACTTCATGTCCAGCATCA Rice fish TAGACCTCCTTCACTGCCCCACCAACTCCATGGTAGAGGCCCACTTCATGTCCAGCATCA Pufferfish GTGACCTGCTCCACTGTCCGTCCAGCTCTGTGGTGGAGGCTCACTTCATGTCCTGCATCA

Yeast ------540 Sorghum AAGAGGCC------Fruit fly AGGAGGCCGATGTGCTGAAACATCGCGGTCTGGTCATCTCGGCCATGCAAAAGAAAGACC Clawed frog AGGAAGCCGATGCTTTGAAGCATAAAAGTCAAGTTATTAATGAAATGCAGAAGAAGGACC Red junglefowl AAGAAGCAGATGCTTTAAAGCACAAAAGTCAAGTCATCAATGAGATGCAAAAAAAGGATC Brown rat AGGAAGCTGACGCTTTAAAGCACAAAAGTCAGGTGATCAACGAAATGCAGAGAAAAGACC Cattle AAGAAGCTGATGCTTTAAAGCACAAAAGTCAAGTAATCAATGAAATGCAGAAAAAAGATC Human AAGAAGCTGATGCTTTAAAACATAAAAGTCAAGTAATCAATGAAATGCAGAAAAAAGATC Rhesus macaque AAGAAGCTGATGCTTTAAAACATAAAAGTCAAGTAATCAATGAAATGCAGAAAAAAGATC Cat AAGAAGCAGACGCATTCAAGCACAAGAGCCAGGTCATCAACGACATGCAGAAGAAAGATC Zebrafish AAGAGGCCGATGCACTCAAACATAAAGGCCAGGTCATCAACGATATGCAGAAGAAAGACC Atlantic salmonAGGAGGCGGATGCCCTCAAACACAAGAGCCAGGTCATCAACGACATGCAGAAGAAAGACC Rainbow trout AGGAGGCGGATGCCCTCAAACACAAGAGCCAGGTCATCAACGACATGCAGAAGAAAGACC Rice fish AGGAGGCCGATGCCCTGAAGCACAAGAGTCAAGTGGTTAACGACATGCAGAAGAAAGACC Pufferfish AGGAGGCAGATGCCCTAAAGCACAAGGGCCAAGTCATCAATGACATGCAGAAGAAAGACC

139

Yeast ------600 Sorghum ------Fruit fly ACAATCAGCTTTGGCTGGGCCTTATCAATGATAAGTTCGATCAGTTTTGGGCCGTCAACA Clawed frog ATAAACAGTTGTGGATGGGTTTACAAAATGATAAGTTTGAACAGTTTTGGGCCATAAACC Red junglefowl ATAAGCAACTGTGGATGGGGTTACAGAATGATAAATTTGAGCAATTTTGGGCTATAAATC Brown rat ACAAGCAGCTCTGGATGGGACTGCAG----AATGATTTGACCAGTTTTGGACCATCAACC Cattle ACAAGCAACTCTGGATGGGTTTACAAAATGACAGATTTGACCAGTTTTGGGCCATCAATC Human ACAAGCAACTCTGGATGGGATTGCAAAATGACAGATTTGACCAGTTTTGGGCCATCAATC Rhesus Macaque ACAAGCAACTCTGGATGGGATTACAAAA----TGATTTGACCAGTTTTGGGCCATCAATC Cat ACAAGCAGCTGTGGATGGGCCTGCAGAACGATAAATTTGACCAGTTCTGGGCCATAAATC Zebrafish ATAAACAGCTGTGGATGGGTCTGCAGAATGATAAATTTGACCAGTTCTGGGCCATGAATC Atlantic salmonACAAGCAGCTGTGGATGGGTCTGCAGAACGATAAGTTTGACCAGTTCTGGGCCATGAACC Rainbow trout ACAAGCAGCTGTGGATGGGTCTGCAGAACGATAAGTTTGACCAGTTCTGGGCCATGAACC Rice fish ACAAACAGCTGTGGATGGGCCTGCAGAACGATAAGTTTGACCAGTTCTGGGCCATGAACA Pufferfish ACAAACAGCTGTGGATGGGCCTGCAGAACGATAAGTTTGACCAGTTCTGGGCCATGAACA

Yeast ------660 Sorghum ------Fruit fly GACGACTCATGGAGCCGTACGGCGACCAGGAGTCCTTCAAAAACATTCCCGTTCGCCTCT Clawed frog GAAAACTTATGGAAGTCCCCCCAGAAGATGGTGGATTTCGATATATACCATTTAGAATAT Red junglefowl GAAAACTCATGGAGTATCCTCCAGAAGATAGTGGATTTCGGTACATCCCATTTAGAATTT Brown rat GGAAACTCATGGAATACCCTCCAGAAGAAAATGGATTTCGTTATATCCCTTTTAGAATAT Cattle GGAAACTCATGGAATATCCTGCAGAAGAAAATGGATTTCGTTATATCCCTTTTAGAATAT Human GGAAACTCATGGAATATCCTGCAGAAGAAAATGGATTTCGTTATATCCCCTTTAGAATAT Rhesus macaque GGAAACTCATGGAATATCCTGCAGAAGAAAATGGATTTCGTTATATCCCCTTTAGAATAT Cat GCAAACTGATGGAATATCCAACTGAGGAGGGCGGCTTTCGATATATTCCTTTCAGGATAT Zebrafish GCAAACTCATGGAGTATCCCACCGAAGAGGGAGGCTTTCGGTATATCCCCTTTAGAATAT Atlantic salmonGTAAGCTGATGGAATACCCCACAGAGGAGGGAGGCTTCCGCTACATCCCCTTCAGGATAT Rainbow trout GTAAGCTGATGGAATACCCCACAGAGGAGGGAGGCTTTCGCTACATCCCCTTCAGGATAT Rice fish GGAAGCTGATGGAGTATCCCACAGAGGAAGGCGGCTTCAGATACATTCCCTTCAGGATAT Pufferfish GGAAGCTAATGGAATGTTCCACAGAGGAGGGAGGCTTTAGATACATCCCCTTCAGAATAT

Yeast ------720 Sorghum ------Fruit fly ACAACGATGATGATTTCA---CGTACACGCAAAAACTGATCTCGCCCATAAGCGAAAGTG

140

Clawed frog ATCAGGCAATCAATGAACGACCCTTTATTCAGAAGTTATTTCGACCAGTGGCCAATGATG Red junglefowl ATCAGGCAACAACAGAGAGACCTTTTATACAAAAGCTGTTTCGACCAATTGCTTCTGGAG Brown rat ATCAGACCACAACTGAACGGCCTTTCATTCAGAAGCTGTTCCGTCCTGTGGCCGCAGATG Cattle ACCAGACAACAACTGAACGGCCTTTCATTCAGAAGCTTTTTCGACCTGTGTCTACAGATG Human ATCAGACAACGACTGAAAGACCTTTCATTCAGAAGCTGTTTCGTCCTGTGGCTGCAGATG Rhesus macaque ATCAGACAACGACTGAAAGACCTTTCATTCAGAAGCTCTTTCGTCCTGTGGCTGCAGATG Cat ACCAGACGCTGAGCGACAGGCCGTTCATTCAGAAGCCGTTCCGGCCCGTTTCTTCAGAAG Zebrafish ATCAGACTATGAGTGACAGACCATTCATCCAGACGCTCTTCCGACCAGTGTCTTCTGAAG Atlantic salmonACCTGACGATGAGTGACAGGCCGTTCATCCAGAAGTTGTTTCGCCCCATCTCGCCCGACG Rainbow trout ACCTGACGATGAGTGACAGGCCATTCATCCAGAAGTTGTTTCGCCCCATCTCGTCCGACG Rice fish ACCAGACAACAAACGACAGACCGTTCATCCAAAGACTGTTTCGGCCCGTTTCGACCGAAG Pufferfish ACCAGACAATGATTGAGAGGCCGTTCATTCAAAAACTGTTTCGACCTGTCTCACCTGAAG

Yeast ------780 Sorghum ------Fruit fly GACAAAAGAAGAGCCTCGCCGACCTGATGGCCGAATTATCAACACCTGAACGC------Clawed frog GAAGGCCTTATACACTTGGTGATCTTATTAGAGAAGTGTGTCCTGCAGCAGTTCCAGCAG Red junglefowl GACAATTGCACACTTTGGGAGATCTTCTAAAAGATGTATGTCCTAGTGCTATCACCCCTG Brown rat GACAGCTGCATACCCTTGGAGATCTCCTAA------Cattle GACAATTGCATACACTAGGAGATCTCCTCAAAGAAGTCTGTCCTTCTGCAGTTGCTCCTG Human GACAGTTGCACACACTAGGAGATCTCCTCAAAGAAGTTTGTCCTTCTGCTATTGATCCTG Rhesus macaque GACAGTTGCACACACTAG------Cat GCCACGCCCACACCTTGGGAGATCTGCTCAAAGAGATGTGCCCTGCTGCCGTATGCCAGG Zebrafish GCCAAGCGCTTACACTGGGAGACCTGCTGAAAGAGCTGTTTCCTGCTGCCA------TTG Atlantic salmonGCCACACACACACGCTGGGGGATCTGCTGAAGGAGGTGTACCCCGTGGCCATATCCAATG Rainbow trout GCTACACACACACGCTGGGGGATCTGCTGAAGGAGGTGTACCCCATGGCCATATCCAGCG Rice fish GCAATCCGCACACGCTGTTTGACCTGCTGAAGGAGATGTGCCCAGATGCTCTGACAAAAG Pufferfish GCACCGTGCACAGTCTGGGTGACCTGCTGAAGGAAGTGTACCCGGCTGCTTTGCCCAGCG

Yeast ------840 Sorghum ------Fruit fly ------AAAGCGGTTGGATTTCGCACCCATGGAATCGATCTGCACGAGGAGACAC Clawed frog AAGGACATGTTTGA------Red junglefowl AAGATGCCTAA------Brown rat ------Cattle AAGATGGGGAAAAAAAGAACCAAGTAATGATTCATGGAATTGAACCAATGTTGGAAACAC Human AAGATGGGGAAAAAAAGAATCAAGTGATGATTCATGGAATTGAGCCAATGTTGGAAACAC

141

Rhesus macaque ------Cat ATGATGGCGTATACGGTACTACGCATGAATCTGACACCACAAAC-CCAAACGACATGCTG Zebrafish AGGATGAGCCAAAGAAATTCCAGGTTATGATTCACGGCATTGAACCCCTGCTAGAGACGC Atlantic salmonACGACGAGTCCAAGCGCTACCAGGTGGTGATCCATGGGATTGAGCCCCTGCTGGAGACCC Rainbow trout ACGACGAGTCCAAGCGCTACCAGGTGGTGATCCATGGGATTGAGCCCCTGCTGGAGACCC Rice fish ATGGCGAGGAAAAGCGTTTCCAGGTCGTGATCCACGGAATCGAACCTCTGCTGGAGACTC Pufferfish ATGGTAAGCAGAAACACTTCCAGGTGGTGATCCACGGCATCGAGCCGCTGCTGGAGACGC

Yeast ------ACCTCTTTT--- 900 Sorghum ------ACCTTCATCATTACTGG----- Fruit fly AGGTGCAATGGATGTCCGAGCACCTGAGCTATCCAGACAATTTTCTGCATTTATCTGTGG Clawed frog ------Red junglefowl ------Brown rat ------Cattle CTTTGCAGTGGCTGAGTGAACATCTGAGCTACCCGGATAATTTTCTTCATATTAGCATCA Human CTCTGCAGTGGCTGAGTGAACATCTGAGCTACCCGGATAATTTTCTTCATATTAGTATCA Rhesus macaque ------Cat C-AGGCAATGGGTT------CTTCAGTCTGCTAG------Zebrafish CCATCCAGTGGCTAAGTGAACACTTGAGCCACCCCGACAACTTTCTCCACATCAGCATCA Atlantic salmonCGTTGCAGTGGCTGAGCGAGCACCTCAGTCACCCTGATAACTTCCTCCACATCAGCATCA Rainbow trout CTTTGCAGTGGCTGAGCGAGCACCTCAGTCACCCTGATAACTTCCTCCACATCAGCGTCA Rice fish CTTTGCAGTGGCTCAGCGAACACCTCAGCCACCCGGACAACTTCCTCCACATTTGCATCA Pufferfish CCCTGCAGTGGCTGAGTGAACACCTGAGTCACCCCGACAACTTCCTGCACATCTGTATCA

Yeast ---GAGAACCAGGTAAAGGATG---TTCTCACTTTTTT------960 Sorghum ---GAACAGTAAGAGTGTGATGAATATGTCACATGCTGATCAAGTCGCTCTGTGGGAATC Fruit fly ACTACAAGGATGTCTAGCCATAACTTTTCCTCTTTGTGTGTG--TGTGTGTGTGTGTATA Clawed frog ------Red junglefowl ------Brown rat ------Cattle TCCCGCAACCAACAGATTGA------Human TCCCACAGCCAACAGATTGA------Rhesus macaque ------Cat ------Zebrafish TCCCTGCACCGAGTGACTGA------Atlantic salmonTACCCGCGCCTAGCGACTGA------Rainbow trout TACCCGCGCCTAGCGACTGA------

142

Rice fish TCCCCGCTCCCACTGACTGA------Pufferfish CCCCGGTCCCCTCTGACTGA------

Yeast ------GAGAATTCACTTGGTTATGGGCGATTCGTTG 1020 Sorghum TGTAATGAAAGGTAACTTAGATGGGT--ATAAA----AGTATCTCCACCAGGCTTAAGAT Fruit fly TATACTCGAACTCGTATTGTCCCAGTCCATATATTTATGTATATTTATGGTGTTTTTTTT Clawed frog ------Red junglefowl ------Brown rat ------Cattle ------Human ------Rhesus Macaque ------Cat ------Zebrafish ------Atlantic salmon------Rainbow trout ------Rice Fish ------Pufferfish ------

Yeast CCACCAACTATCATTCCCATCGCATCTAGCAAAACGCAAG------CG------GAA 1080 Sorghum TGGACCATTCGAAGATGATGGGTTAGTACGAACAGCCTCCGCAGAGCGGAAACGTCAACA Fruit fly TCACCCATATTCGATTCCAGCGTTCCCAGGGCGATCCAAT------GCGATCGATTTATA Clawed frog ------Red junglefowl ------Brown rat ------Cattle ------Human ------Rhesus macaque ------Cat ------Zebrafish ------Atlantic salmon------Rainbow trout ------Rice fish ------Pufferfish ------

Yeast AAGTTTTGGTTTCACCAATGGAAACAAGTTTGCTTCATACTGA------1140

143

Sorghum AAATTCTGATGAACCTGAATCACCTGGATCTAGCAAACCATGCAGGGTCCCTGTTCGA-- Fruit fly TAGTAATTTGTACACATATACACATATGTATACACCTCCATGTATGTATAGTTTTCTAAG Clawed frog ------Red junglefowl ------Brown rat ------Cattle ------Human ------Rhesus macaque ------Cat ------Zebrafish ------Atlantic salmon------Rainbow trout ------Rice fish ------Pufferfish ------

Yeast ------1200 Sorghum CTATATGTACGCAATGTTCAAGAAGACCTTGAGTATATAGAAGATGCAGTACCTGTTAGT Fruit fly CAAAAAGTGTGTAATATTTATGA---CCCTAGCATTTAATGAAAGCCACAAACAATAAGC Clawed frog ------Red junglefowl ------Brown rat ------Cattle ------Human ------Rhesus macaque ------Cat ------Zebrafish ------Atlantic salmon------Rainbow trout ------Rice fish ------Pufferfish ------

Yeast ------ATGGTTCCTCAAAAGCTA------TTATGTCTTTATCGG 1260 Sorghum GACT------Fruit fly AACCGAAAACCAAAACGATTACTGCAACTACATATAGTCACGGATGGATGGATGGATGGA Clawed frog ------Red Junglefowl ------Brown rat ------

144

Cattle ------Human ------Rhesus macaque ------Cat ------Zebrafish ------Atlantic salmon------Rainbow trout ------Rice fish ------Pufferfish ------

Yeast TCAACGAAGCTCGAAAATTTTGGGGCAGTGTTATTACGAGAAATTTCCAAGATTTCATTG 1320 Sorghum ------GGGAAGGTGTG---TCCTACATAAATCGGCCATTTGAGATT Fruit fly TGGATGGATCGGGCCACTGGAGAAGATCTAGCATTCATCGTTCATCGATCATTTTCTATT Clawed frog ------Red junglefowl ------Brown rat ------Cattle ------Human ------Rhesus macaque ------Cat ------Zebrafish ------Atlantic salmon------Rainbow trout ------Rice fish ------Pufferfish ------

Yeast AAATATCTAACAAGATAAGTTCATCAAGACCGCGACATATACCGCTCATCATTCAAACCT 1380 Sorghum CGCAAGGTTGAAGGT-A-----GAAGCTACATCACCCTGGAACACGCATTG------Fruit fly AGATATCTA------TAAGCCGCCGGAAACTGGTCAAGCCCTTGCACTAACAA Clawed frog ------Red junglefowl ------Brown rat ------Cattle ------Human ------Rhesus macaque ------Cat ------Zebrafish ------

145

Atlantic salmon------Rainbow trout ------Rice fish ------Pufferfish ------

Yeast CAAGGACATCAGGAACTTTTCGAATATCACAAC------1440 Sorghum ------CAA------ACATTGCTTCCAGAGTTCTTCAGCTCAA--- Fruit fly GCGAACAAACAGAAAGTTTTCAGCTATCAATTTCGATTTATCCGTTTGTCAAGATAGTGA Clawed frog ------Red junglefowl ------Brown rat ------Cattle ------Human ------Rhesus macaque ------Cat ------Zebrafish ------Atlantic salmon------Rainbow trout ------Rice fish ------Pufferfish ------

Yeast ------CAACTATCAGTATGACTGGAGTCAATCCTACGTTGAAGGATATT------1500 Sorghum ------AGGCCGCAAGTAGAGC--CGACGGCTCCCACCCTGCAGGGGCACTGGACT Fruit fly CTGACGGAACGGGACAGAATTGGGTCTGGAATGGCTTCTACCCGG-TCTCGGTCTGGGCT Clawed frog ------Red junglefowl ------Brown rat ------Cattle ------Human ------Rhesus macaque ------Cat ------Zebrafish ------Atlantic salmon------Rainbow trout ------Rice fish ------Pufferfish ------

146

Yeast --GAAGGCGATATTCTG------GACG-TAAAGGAAGGAATCAATGGTAATG 1560 Sorghum CAGCTGCAGACAGTTCAGACGCCACCAACTCTTCAAGAAGTTCTAAGGAGGCAGAACAAG Fruit fly TTTGGGCGGACCTTGCA------CAATATTTTAGCCAAGGCCCAAAATCGATGATCTTG Clawed frog ------Red junglefowl ------Brown rat ------Cattle ------Human ------Rhesus macaque ------Cat ------Zebrafish ------Atlantic salmon------Rainbow trout ------Rice fish ------Pufferfish ------

Yeast ATGTCATGGTTATTTGTCAAGGAATTGAAATTCCTTGGCATAT------GCTCCTGT 1620 Sorghum CC---C---TGGCAAGTCCACGAGAGGTGGGTGCTGCCAAGAAAACGAAAGTGAAGCTGG Fruit fly TT---TT-GTTGCCACTCCACGAACTT------ATTTGAAGAGCATCGATTTGACACTGT Clawed frog ------Red junglefowl ------Brown rat ------Cattle ------Human ------Rhesus macaque ------Cat ------Zebrafish ------Atlantic salmon------Rainbow trout ------Rice fish ------Pufferfish ------

Yeast ATGA-TTTGTATTCTAAATTGCGAAGTTTTGATG------GCTTTTTGTACATAACTCTT 1680 Sorghum TAAGGGTGCAAGGCATCGAGCTGGACATGGACATCCCGTTCCTTTGGGTCGCCAA----- Fruit fly CA---GTAGAGTGGAGGAATCGTACCATGGGCTGCCTGATC-TATAATTAGTTAACGCTT Clawed frog ------

147

Red junglefowl ------Brown rat ------Cattle ------Human ------Rhesus macaque ------Cat ------Zebrafish ------Atlantic salmon------Rainbow trout ------Rice fish ------Pufferfish ------

Yeast GTTCCTATAAAAGGCGGCGATAA------1740 Sorghum -----TAACCTGAAGAACCCTGAATACTACCTCCACATCTGTGTA-TACGTCGGCACTAG Fruit fly TTGCCCGCACTCGAGAATGATTTATTCGATTGTTATTTGTATCGTTTGTCTGGGCCCAAA Clawed frog ------Red junglefowl ------Brown rat ------Cattle ------Human ------Rhesus macaque ------Cat ------Zebrafish ------Atlantic salmon------Rainbow trout ------Rice fish ------Pufferfish ------

Yeast ----AGCTTCCTCTGAGCTCTAA------1769 Sorghum AAAACAATAG------Fruit fly AATAAACTGCAAATCTGATAAAAAGCAAA Clawed frog ------Red junglefowl ------Brown rat ------Cattle ------Human ------Rhesus macaque ------

148

Cat ------Zebrafish ------Atlantic salmon------Rainbow trout ------Rice Fish ------Pufferfish ------

149

Appendix 2.2b) Clustal alignments between rainbow trout Atg genes and sequences from 14 other species.

CLUSTAL O(1.2.4) multiple sequence alignment ATG7

Sorghum ------ATGG------CGGG------60 Yeast ------ATGTCG Fruit fly CTCATTGGTCTATCGCCATCGAGAGCTTTGTTTGTTTATAATTTGCAGCCAAACGTTTGA Red junglefowl ------ATGGCA Brown rat ------Cattle ------Human ------Rhesus macaque ------Clawed frog ------ATGGCG Pufferfish ------Atlantic salmon ------Rainbow trout ------Zebrafish ------Pufferfish ------

Sorghum --T------GCCGGTGCGGTGGGCATCCCAAGGGAGCTGATGGTTCA 120 Yeast TCAGA------AAGGGTCTTAAGTTATGCAC------C Fruit fly ACGTAAACAACGCCCTAAGCAAAACATGAGCACGGAAAAAGAGATAATTTTGCAATTTGC Red junglefowl GCAGTCAGTA-ATGAGTCCCAGAACCCTGTGGATCCTGGAAGTTCAAAGCTGCAGTTTGC Brown rat ------ATGGGGGACCCTGGACTGTCCAAGCTGCAGTTTGC Cattle ------ATGGCAGCAGCTATGGGGGACCTCGGCCTCTCCAGATTGCAGTTCGC Human ------ATGGCGGCAGCTACGGGGGATCCTGGACTCTCTAAACTGCAGTTTGC Rhesus macaque ------ATGGCAGCAGCTACGGGGGATCCTGGACTCTCTAAACTGCAGTTTGC Clawed frog TCTGGGACCG-ACCCCCCCAGTGATCTCCTCGCTGCCGACGATGCCAAGTTACAGTTTGT Pufferfish ATGGCATCTG-ACACCAATGAGGCATCGTCCTCCGCTGCAGACCTGAAGCTGCAGTTTGC Atlanitc ------ATGG-CTTCTGTGCCCGTAGACAAAGACAGCGCAGATCAAAAGCTGCAGTTTGC Rainbow Trout ------Zebrafish ------ATGGCGGAATCCAGTCTGAAGCTTCAGTTCGC Fish ------ATGG-CAGCTGAATCTGTGGAAAAGGGCGCAGCTCTTCCCAAGTTGCAGTTTGC

Sorghum GCGCATCCAGAGCTTAGTGGAGGAGGGGTTCTGGGACGCGCTCCGCCGCCTCAAGCTCGA 180

150

Yeast AGCTTTTAAATCATTTCTGGATACATCTTTTTTCCAAGAATTATCGAGATTGAAACTCGA Fruit fly TCCGTGGGAATCTTTTGTGTCGCCCACTTTCTGGCACAAGCTAGCGGAGCTTAAGCTCGA Red junglefowl TCCCTTCAGTAGTGCCTTGAACGTAGGATTCTGGCATGAACTCACCCAGAAGAAGCTCAA Brown rat CCCCTTTAATAGTGCCCTGGACGTTGGCTTCTGGCACGAACTGACCCAGAAGAAGTTGAA Cattle CCCCTTCAGCAGCGCCTTGGATGTTGGCTTCTGGCATGAGTTGACCCAGAAGAAGCTGAA Human CCCTTTTAGTAGTGCCTTGGATGTTGGGTTTTGGCATGAGTTGACCCAGAAGAAGCTGAA Rhesus macaque CCCCTTTAGCAGTGCCTTGGATGTTGGGTTCTGGCATGAGTTGACCCAGAAGAAGCTGAA Clawed frog GCCGTTCACCAGCGCCCTGGATGCCGGCTTCTGGCACCAACTGACTCAGAAGAAACTCAA Pufferfish ACCCTTCAGCAGTGCTCTGGAGGCAGGATTCTGGCATCAGCTTACGCAGAAGAAACTTAA Atlantic salmon TCCTTTCTCCAGTGCCTTAGAGGCAGGCTTCTGGCACCAGCTCACTCAGAAGAAACTCAA Rainbow trout ------Zebrafish CCCGTTCTGCAGTGCTCTGGAGGCCGGATTCTGGCATCAGCTCACACAGAAGAAACTCAA Rice fish CCCTTTCTCTAGTGCCCTTGAGGCAGGATTCTGGCATCAGCTCACCCAGAAGAAACTCAA

Sorghum CGTCCTCGGCACCGACGACTCGCCCATCCCAATCACCG------GTTATTATACTCCCCG 240 Yeast TGTTCTGAAATTAGATTCAACATGCCAACCACTTACTGTGAATCTAGATTTACACAACAT Fruit fly CCACGATCGCCTGTCCGACTCGAAACGCTCCATTACTG------GACACTACACAAATCG Red junglefowl TGAGTACCGATTAGATGAGACTCCAAAAGTTATCAAAG------GATACTACTACAATGG Brown rat CGAGTATCGCCTGGACGAGGCACCCAAAGACATCAAGG------GCTATTACTACAATGG Cattle TGAGTACCGGCTAGACGAAGCTCCCAAGGACATTAAGG------GTTATTACTACAACGG Human CGAGTATCGGCTGGATGAAGCTCCCAAGGACATTAAGG------GTTATTACTACAATGG Rhesus macaque TGAGTATCGGTTGGATGAAGCTCCCAAGGACATTAAGG------GTTATTACTACAATGG Clawed frog CGAGTACAGACTGGACGAGACCCCCAAGGAAATAAAGG------GACATTATTATAATGG Pufferfish TGACTACAAATTGGATGAAAGCCCTAAATGCATCAAAG------GATATTACTATAACGG Atlantic salmon CGACTACCGACTGGATGAAAGCCCCAAGAATATCAAAG------GCTACTACTACAATGG Rainbow trout ------Zebrafish CGAGTACCGCTTAGACGAAAGTCCAAAAAACATCAAGG------GATATTACTATAACGG Fish TGACTACCGGCTGGATGAAAGTCCAAGAAGCATCAAGG------GTTATTATTATAACGG

Sorghum CCAGCGTCGCCCGATGGCAAGCTTCTTCAACCTACGGTCAGGATCATTGGTGCCGCCATC 300 Yeast TCCTAAATCTGCTGATCAAGTTCCATTATTCTTAACAAATAGAAGCTTTGAA------Fruit fly TAATGCAAGTGGA------TGCCTTTTGGAAGTAGACTACACGGCCTACAAC------Red junglefowl CGATCCATCAGGTTTCCCAGCTCGTTTGACGTTGGAATATAGTGCCTTTGAT------Brown rat TGACTCTGCCGGCCTCCCCACCCGCTTGACATTGGAGTTCAGTGCTTTTGAC------Cattle TGACTCTGTGGGGCTGCCAGCTCGCTTAACGTTGGAGTTCAGTGCTTTCGAC------Human TGACTCTGCTGGGCTGCCAGCTCGCTTAACATTGGAGTTCAGTGCTTTTGAC------Rhesus macaque TGACTCTGCTGGGCTGCCAGCTCGCTTAACATTGGAGTTCAGTGCTTTTGAC------

151

Clawed frog GGATCCAGCCGGACTCCCAACCCGTCTCACTCTGGAATTCAGCGCCTTCGAT------Pufferfish TGATCCACTTGGTTTGCCCACTCGCCTGACATTAGAGTTTAGTGCTTTTGAA------Atlanitc CGATCCGGTCGGCCTCCCCACTCGCCTGACTTTGGAGTTTAGTGCATTTGAG------Rainbow trout ------Zebrafish TGATGCTGTTGGTCTGCCGGCGCGCTTGACACTGGAGTTCAGCGCATTTGAT------Fish TGACCCATCTGGCCTGCCGACTCGTTTGACCCTGGAGTTCAGTGCATTTGAG------

Sorghum TCTCAATTCTGTTGGCGACAGAAATAATTGTCCAGTCCCAGGGACCCTCATAAACACTAA 360 Yeast -AAACACAATAATAAGCGTACTAATGAAGTGCCTTTACAGGGCAGTATTTTCAATTTTAA Fruit fly -AGAATGGCAAAGCCTCCAAAATTCAGCCATTCCGCTATAGGCACCATCTACAATAAGAA Red junglefowl -ATCAATGCTTCAATACCAGCACGTTGCTGTCCTGCTTTTGGAACATTGTATAATACCAA Brown rat -ATGAGCGCCCCCACGCCTGCCCGCTGCTGCCCGGCCATGGGAACCCTGCACAACACCAA Cattle -ATGAGTGCACCCACCCCAGCCCGCTGCTGCCCGGCGGTCGGCATACTGTATAATACCAA Human -ATGAGTGCTCCCACCCCAGCCCGTTGCTGCCCAGCTATTGGAACACTGTATAACACCAA Rhesus macaque -ATGAGTGCTCCCACCCCAGCCCGTTGCTGCCCAGCTGTTGGAACACTGTATAACACCAA Clawed frog -GTTAATACCCCCACCCCTGCCCGCTGCTGCCCGGCGCTTGGCTCGCTGTACAACACCAA Pufferfish -GTGGACGGTCCGACTCCAGCTCACTGCTGTCCAGTTACTGGAACACTTTTCAACACCAA Atlantic salmon -GCGGATGGCCCCAGCCCAGCCCGCTGCTGTCCCGTCACAGGGACCCTGTACAACACCAA Rainbow trout ------Zebrafish -GCGGATGGACCCACGCCGGCGCGCTGCTGTCCCGCATCAGGAACTCTGTACAACACAAA Fish -GTTGATGGACCCACTCCAGCTTACTGCTGTGCTGCTAAGGGAACTCTGTACAACACCAA

Sorghum TAACATGAGGGGATTCCAAAACCTAGATATAGAACAGCTTCTGAAAGCAGAAGCAAAGAA 420 Yeast TGTATTAGACGAGTTTAAGAATCTTGATAAGCAACTATTTTTACATCAAAGAGCATTGGA Fruit fly CACAATCGAAGAGTTCAAGGCCCTGGACAAATTACAACTGCTGGCCGATGAGGGCAAGGA Red junglefowl CACTTTTGAGACTTTCAAGTCCTGTGATAAAAAATCACTTTTGGAAAAAGAAGCAAATGA Brown rat CACACTTGAGGCTTTTAAGACTGCAGACAAGAAGCTCCTTCTGGAGCAGTCAGCCAATGA Cattle CACACTCGAGGCTTTCAAGGCTGCAGATAAGAAGCTACTTCTGGAAGAAGCGGCAAACGA Human CACACTCGAGTCTTTCAAGACTGCAGATAAGAAGCTCCTTTTGGAACAAGCAGCAAATGA Rhesus macaque CACACTCGAGTCTTTCAAGACTGCAGATAAGAAGCTCCTTTTGGAACAAGCAGCAAATGA Clawed frog CACTCTGGAGTCATTCAAATCGTGCGATAAGAAGGCGCTTTTGGATCGAGCAGCCAATGA Pufferfish TACACTCGATGCCTTTAAGACTACCGATAAGAAGGCTCTGCTGGAGAAAGAAGCCAAAGA Atlantic salmon CACGCTAGAGGCCTTCAAGACCTCAGACAAAAAAGCACTGCTGGAACAACAGGCCACGGA Rainbow trout ------Zebrafish CACACTCGAAGCCTTCAAATCCACCGACAAAAAAGCCCTGCTGGACAAAGCAGCCAATGA Fish CACGCTGGAAGCCTTTAAATCCACCGACAAGAAAGCACTTCTAGAAAAAGCAGCTAATGA

152

Sorghum GATCTTGGATGACATTGTGTCTGGTAAAGTAGAAGAGGATCCTTCTGTTCTACTTAGGTT 480 Yeast ATGCTGGG------AAGATGGAATAAAGGATATCAATAAGTGTGTTTCTTT Fruit fly ACTGCTGGCTGATATGTGCAGTGGTGGCGCCTTGAGAGATCCCAGTCTATTGACCCGGTT Red junglefowl GATATGGGAATCAATAAAATCTGGAGCTGCACTTGAAAACCCTATGCTCTTGAACAGGTT Brown rat GATCTGGGAAGCCATAAAGTCAGGTGCTGCTCTCGAAAACCCCATGCTCCTCAACAAGTT Cattle GATCTGGGAGTCCATCAAATCGGGGGCCGCCCTGGACAACCCCGTGCTCCTCAACAAGTT Human GATATGGGAATCCATAAAATCAGGCACTGCTCTTGAAAACCCTGTACTCCTCAACAAGTT Rhesus macaque GATATGGGAATCCATAAAATCAGGCACTGCTCTTGAAAATCCTGTACTCCTCAACAAGTT Clawed frog GATTTGGGAGGCGATTCAGTCGGGAGCTGCCATTGAGGATCCGACGCATCTGACTAAGTT Pufferfish GATATGGGATGCCATACAGTCTGGGAGTGCAATTAACGACCCCTCCATCCTCTGCAGATT Atlantic salmon GATTTGGGATTCCATACAGTCCGGTGATGCTCTCAAGGACCCATCCCTCCTCTGCAAATT Rainbow trout ------Zebrafish GATCTGGAGCGCTATTCAGTCCGGCGCTGCTCTCGAAGACTCTTCAATCCTCAACAAGTT Fish GATTTGGACAGCCATACAGTCAGGTGCTGCAGTGGAAGACCCTTCAGTCCTGAGCAAATT

Sorghum CCTTGTGACATCATTTGCCGATTTAAAGAATTGGAAGGTCTATTACAATGTTGCATTCCC 540 Yeast TGTGATTATTAGTTTTGCTGACCTAAAAAAGTACAGGTTTTATTACTGGTTAGGTGTTCC Fruit fly CTTTGTTCTCTCCTTTGCCGATTTAAAGTGTCACAGCTACTACTATTGGTTCGCCTTTCC Red junglefowl CCTGCTGTTGACATTTGCAGATTTGAAAAAGTATCATTTTTATTACTGGTTTTGCTACCC Brown rat TCTGCTCTTGACCTTCGCGGACCTAAAGAAGTACCACTTCTACTACTGGTTTTGCTGCCC Cattle CCTCCTCTTGACATTTGCGGATCTAAAGAAGTACCATTTCTACTACTGGTTCTGCTCCCC Human CCTCCTCTTGACATTTGCAA------Rhesus macaque CCTCCTCTTGACATTTGCAGATCTAAAGAAGTACCACTTCTACTATTGGTTTTGCTATCC Clawed frog CCACCTCCTCACATTCTCAGATTTAAAGAAATACCGCTTCTACTACTGGTTTTGTTTCCC Pufferfish TATCCTACTGACCTTTGCTGATTTGAAAAAATACCATTTCTACTACTGGTTCTGCTTCCC Atlantic salmon CCTTTTGCTGACTTATGCAGACCTGAAGAAGTACCACTTCTACTACTGGTTCTGTTTCCC Rainbow trout ------Zebrafish CATCCTCTTGACATTTGCGGATCTGAAGAAATATCACTTCTATTATTGGTTCTGCTTTCC Fish CATCATTTTGACATTTGCGGATCTGAAGAAATACCAGTTTTATTACTGGTTCTGCTTCCC

Sorghum CTCATTGGTTTTCAACTCAAGAATGACCC---TGCTCAACCTGCAACC------TGCC 600 Yeast CTGTTTTCAAAGACCCTCTTCAACAGTTTTACATGTGCGGCCAGAGCC---GAGTTTAAA Fruit fly GTGCCCGCTGACGCCCACCTTAAAGCTT-CAGGGAGCCGTCCAAAAACTGCGGGACTTGC Red junglefowl TGCTCTCTGCTTCCCTGATGGAATAC------ATGTAATTCAGAAACCGGTGTGTCTTGG Brown rat TGCCCTCTGTCTTCCTGAGAGCATCC------CTCTAATCCGGGGACCTGTGGGCTTGGA Cattle AGCTCTCTGCCTTCCAGAGAGCATAC------CCCTCATTCAGGGGCCAGTGGCCTTGGA

153

Human ------Rhesus macaque TGCCCTCTGCCTTCCAGAGAGTTTAC------CTCTCATTCAGGGGCCAGTGGGTTTGGA Clawed frog TGCCCTGTGCCTTCAGGAGGGGGTCC------GTCTCCTACAGCCCCCGGCTTCACTTGC Pufferfish TGCTCTTTGTTTCCCCGAGGGAATAA------AGATAATCCAGCAACCTTCCCTCCTGGA Atlantic salmon AGCTCTCTGTTTCTCTGAGGGAATTA------AGATCCTCAAAGAGCCTGCTACGTTAGA Rainbow Trout ------Zebrafish TGCGCTCTGCTTTGTGGAGGGCATCC------AGCTGCTGCGCGCGCCGCTGTCATTAGA Fish TGCTCTCTGCTTCTTGGAAGGAATTC------AGCTTGTGACGGATCCGGTGTTGTTAGA

Sorghum TCAAAAGTGCTCACTAAAGAAGAGGCAGCATCCATATATACATCATTGCAAAAGTGGCGC 660 Yeast AGGGCTGTTCTCAAAG------TGTCAAAAATGGTTTGA------Fruit fly CCAATAGTAGTAG------CTATATAATGGCTCTAA------AGGC Red junglefowl TGACAGGTTCTCTTTA--AATCAGATTCAAGCACTTCAGAAAGCATATGATGAACTTTGC Brown rat TCAAAGGCTTTCACCA--AAACAGATCCAGGCCCTGGAGCATGCCTACGATGACCTGTGT Cattle TCAATGGTTTTTGCCA--AAACAGATTCAAGCCCTCGAGCACGCGTATGATGCCCTTTGT Human ------TTGAAGCACTAGAGTGTGCATATGATAATCTTTGT Rhesus macaque TCAAAGGTTTTCACTA--AAACAGATTGAAGCATTAGAGTGTGCATATGATAATCTTTGT Clawed frog CCAACGTTTCTCTGAG--CTTCAGGTGGGGGCGCTCCAGAGATCTTATGATGAGTTATGG Pufferfish GGCCGTGTTCTCACCA—AAACAGGTTATAGCACTGCAGGAGGCCTATGATGACCTGTGGC Atlantic salmon ACAAGTCTTCTCTTCT--AAACAGACCTCAGCCTTACAGGCAGCCTATGACAGTCTGTGC Rainbow Trout ------Zebrafish GCAGCATTTCTCTGAC--AAACAGATATCGAGTTTGCAATCGGCGTATGATAATCTCTGT Fish GCGGAAATTCTCCCCT--CGGCAGGTGTCTGCTTTTCAAGCAGCCTACGATGAGTTATGT

Sorghum GCTTCTAGTGAAACAACAGTTGTTCCATTCTTTCTGGTTAGTATATCCTCAGATTCCTCT 720 Yeast ------TGTCAATTACTCAA-AATGGGTGTGTATACTGG Fruit fly CTTACCCACTGAGTCACAGAACTTCTTCATTCTGTATGCTA-ATGTGGAGAAGAACATTT Red junglefowl CAGACAGAAGGGGTTACAGCCTTTCCTTATTTTTTAATCAA-GTATCATGACAACTCTGT Brown rat CGAACTGAAGGCGTCACAGCCCTGCCATACTTCTTATTCAA-GTACGATGACGACACTGT Cattle CAGACAGAAGGAGTCCCAGCACTGCCTTACTTCCTAATCAA-GTATGACGAGACCACGGT Human CAAACAGAAGGAGTCACAGCTCTTCCTTACTTCTTAATCAA-GTATGATGAGAACATGGT Rhesus macaque CAAACAGAAGGAGTCACAGCTCTTCCTTACTTCTTAATCAA-GTATGATGAGAACACGGT Clawed frog CAGAAGG---AAGGGACCCCCCCACCTTATTTCCTGGTGAA-ATACACTGAGACTTCCTG Pufferfish GTCACTAAAGGCAATACTGCGGTTCCATATTTCCTGATAAA-GTACACTGATCACACTGT Atlantic salmon AGTGAAACAGGGACCTCAGCTGTTCCTTACTTCCTGATCAA-GTACACAGAGGATGCTGT Rainbow Trout ------Zebrafish GCCTCCAGCGGAACGACGGCAGTCCCGCATTTTCTGCTGAA-ATACTCTGAGGAAAGTGT

154

Fish TCAGCGAGTGGAGCCACAGCTGTGCCGTATTTCCTTGTGAA-GTACTCTGAGGAGAACGT

Sorghum GCCTCTATAAGGCAACTTAAGGACTGGAAAGCTTG---CCAGGGGAACTATAAAAAGCTA 780 Yeast ATG-CAGACGATGAAATAGTAAATTATGACAAGTGT------ATTATTCGAAAAACTAA Fruit fly TC--GAAGCC------CGTAGTTTAAGTTCCCTT-GACGATAAAAATGTTGAGTTCTGT Red junglefowl GG--TTGTATCTCCTCTCAAAAAGTGGGATGGTTTCTTCCAAGATCAAGGGGGAAAGGTG Brown rat GC--TGGTCTCCTTGCTCAAACACTACAGTGATTTCTTCCAAGGTCAAAGGACAAAGTTA Cattle GC--TGGTTTCCTCGCTCAAACACTACAGCGATTTCTTCCAGGGTCAAAGGACAAAGATA Human GC--TGGTTTCCTTGCTTAAACACTACAGTGATTTCTTCCAAGGTCAAAGGACGAAGATA Rhesus macaque GC--TGGTTTCCTTGCTTAAACACTACAGTGATTTCTTCCAAGGTCAAAGGATGAAGATA Clawed frog CG--CTGTGGCCCCGCTCCGTGCGTTCCCCGACTTCTATA---AGGATGGAGACAAAGTG Pufferfish TC--AGATGGCTCCACTTGAAGACTGGGAGAGCTTTTTCA---CTATGACGAAGAAGGTT Atlantic salmon CC--AGGTGGCCCAGCTACGAGACTGGGACAGCTTCAACA---CTGATCTCAGCAAGGTA Rainbow trout ------Zebrafish TG--AAGTGGCTCCTCTGAAAGAGCTGAACTCCTTCTTCC---CGGACCTAAAGAGGGTG Fish TG--AAGTGGCTCTGCTGAAAGACTGGGGTTCATTCTTCA---CAGATCTAAAGAAGGTA

Sorghum CTATTTGGATTTTATGATCATGGATGCCGCTCAGATTGCCCTGGCTGGGTAATTAGAAAC 840 Yeast AGTTTTGGCGATAAGGGACACTAGTACGATGGAAAACGTCCCTTCTGCTCTAACCAAAAA Fruit fly TATTTTGGTTTTGCTGATCC---CAGCGAGTATGAGCATCCAGCTTGGATAATGCGAAAC Red junglefowl ACAGTTGGAGTTTATGATCCGTGTAATTTATCTCACTATCCAGGATGGCCACTGCGGAAC Brown rat ACAGTCGGTGTGTACGATCCCTGTAACCTAACCCAGCACCCTGGATGGCCTTTGAGGAAT Cattle ACTATTGGCGTGTACGATCCCTGTAACTTAGCACAGTACCCCGGATGGCCTTTGAGGAAC Human ACAATTGGTGTATATGATCCCTGTAACTTAGCCCAGTACCCTGGATGGCCTTTGAGGAAT Rhesus macaque ACAATTGGTGTATATGATCCCTGTAACTTAGCCCAGTATCCTGGATGGCCTTTGAGGAAT Clawed frog ACATTGGGGTTCTGTGACCCCTGCACTTTACCCCAGTACCCGGGGTGGCCCCTGCGCAAT Pufferfish ACCATAGGAGTCTATGATCCGTGTACTTTATCTCAACACCCAGGCTGGCCCCTGAGAAAT Atlantic salmon ACCGTGGGTGTGTATGACCCTTGCACTCTGCCCCAACATCCAGGATGGCCTCTGAGGAAC Rainbow trout ------Zebrafish GCTGTGGGTGTGTACGACCCATGCACGCTACCTCAGCATCCCGGATGGCCTCTCAGAAAC Fish ACCGTGGGTGTCTATGACCCATGCACGCTGCCTCAACATCCTGGCTGGCCTCTTCGTAAT

Sorghum TATGTTACCTTTCTGAGCATCCGATGGAAGATTGAGA------AAGTCCAGT 900 Yeast TTTTCTCAGCGTGTTGCAATACGATGTTCCTGACTTGA---TAG--ATTTCAAA-CTATT Fruit fly TATGCTGCCTTTCTGCTTCAGCAATGTCCCTCTTTTGTTGGAAAACCATTGAAG------Red junglefowl TTCCTGATCTTGGCATCGCATAAATGGGGCAATATTCT--CCAGTCAATTGAAGTGCTCT

155

Brown rat TTTTTGGTCCTGGCAGCCCACAGATGGAGCGGCAGTTT--CCAGTCTGTTGAAGTTCTCT Cattle CTTTTGGTCCTAGCAGCCCACAGATGGAGTGGCAGTTT--CCAGTCCGTGGAAGTGGTCT Human TTTTTGGTCCTAGCAGCCCACAGATGGAGTAGCAGTTT--CCAGTCTGTTGAAGTTGTTT Rhesus macaque TTTTTGGTCCTAGCAGCCCACAGATGGAGTAGCAGTTT--CCAGTCTGTTGAAGTTCTTT Clawed frog CTCCTGGTCCTGGCGGCGTATCATTGGGGAAGCCGCGT--GCGGGAAGCTGAGGTTCTGT Pufferfish CTGTTGGCTCTCTTGGCTAACCAATGGGGTTCAAAGCT--GGACGTGGTAGAGGTGCTGT Pufferfish CTCCTGGTCCTCTTAGCAAACCGATGGGGTTCAAAGTT--AGACATAGTGGAGGTGCTAT Atlantic salmon CTGCTGTTTCTCCTAGCCAGCAAATGGGGCTCCCAAAT--AGATGTGATGGAGGTGATGT Rainbow Trout ------Zebrafish CTCCTCGTCCTCCTCGCTAAAAAGTGGGCTTCACAGCT--GGATGTTGTGGAGGTTCTGT Fish TTTCTGATCCTCCTCGCCAGTAAATGGAGTTCACAGCT--GGACGTGCTTGAAATTGTGT

Sorghum TTTTCTGCTACCGTGAATATAAAGGGAACCCTGATCTAGAACAGTCCCTCATTGGTGAAG 960 Yeast AATTATTAGACAGAACGAAGGTAGCTTTGCATTAAATGCTACATTTGCTTCTATTGACCC Fruit fly --TTTCTTGGCCTGCGACACAATCAACAAATGAACATCGATGACAGCCTTGTATGGAAGG Red junglefowl GCTTCAGAGACAGGACCATGCAAGGGGTGAGAGACATAACACACAGCATTATCTTTGAAA Brown rat GCTTTCGGGACCGCACCATGCAGGGAGCAAGAGATGTGACACACAGCATCATCTTTGAAG Cattle GCTTCCGGGACCGCACCCTGCAGGGGGTGAGAGACGTCACCCACAGCATCGTCTTCGAAG Human GCTTCCGTGACCGTACCATGCAGGGGGCGAGAGACGTTGCCCACAGCATCATCTTCGAAG Rhesus macaque GCTTCCGTGACCGTACCATGCAGGGGGCGCGAGATGTTGCCCACAGCATCATCTTCGAAG Clawed frog GCTTTCGGGACCGGACCCTCCAGGGGGAACGAGATGTCACCCACAGCCTCATCTTCCACA Pufferfish GTTTCCGAGACAGAACCCTGCAGGGAAGCCGCTCCATTCAGCACAGCGTCGTGTTCCAGA Pufferfish GTTTCAGGGACCGAACTCTGCAGGGGAACCGCTCCATTCAGCACAGTCTGATCTTTCACA Atlantic salmon GTTTCAGAGACAGGACTCTTCAGGGTAGTAGGAACATTCAGCACAGCATCGTGTTCCAGG Rainbow Trout ------Zebrafish GCTTCAGAGACCGGACTCTCCAAGGCGTTCGCTCCGTCCAGCACAGCATCATCTTTCAGC Fish GCTTTAGAGACAGGACACTCCAGGGATGTCGCTCAGTGCAGCACAGCCTTGTTTTTGAGG

Sorghum CATCATTTCCATCACCTTGTGGTGTGGATGACCCTGATTTTCTACCTGATGCTATTGGAT 1020 Yeast AC------AATCATCCTCAAGTAACCCAGA---TATGAAAGTTTCTGGAT Fruit fly TGATTCAGACTGAAGCTTGCGATCTTAGCCAAAGCGAAAA---TATAAAGTTCGTGGGCT Red junglefowl TAAAACTTCCACA------AGGAGCATTTGGCCCAGATTG---TCCAAAAGCTGTTGGAT Brown rat TGAAACTTCCAGA------AATGGCGTTTAGCCCAGATTG---TCCTAAGGCAGTTGGCT Cattle TGAAGCTTCCAGA------GATGGCGTTCAGCCCAGATTG---TCCCAAGGCGGTCGGAT Human TGAAGCTTCCAGA------AATGGCATTTAGCCCAGATTG---TCCTAAAGCAGTTGGAT Rhesus macaque TGAAGCTTCCAGA------AATGGCATTTAGCCCAGATTG---TCCTAAAGCAGTTGGAT Clawed frog TTCAACTCCCAGA------GATGCCAGCCAATCAGGAGCT---CCCAAAAGCAGTTGGAT

156

Pufferfish TCAAGTTACCTGG------ACTCTCCAGCAATTCCGGTTG---TCCTAAAAGTGTTGGTT Pufferfish TCAAACTTCCAGA------ACTTCCACTCAACTCTGCATG---TCCTAAAAGTGTTGGCT Atlantic salmon TCAAACTGCCGGTCCCTGAGCAGTCTGTCAATGCAGTGTG---TCCAAAGAATGTAGGAT Rainbow trout ------Zebrafish TCAGACTCTCAGA------CCCGGCGCCTTCTGCAGTGTG---TCCGAAGAGTGTGGGCT Fish TCAAGCTTCCCAG------TCTGTCCGTGTCTGCAGCTTG---TCCAAAGAGTGTGGGCT

Sorghum GGGAAGGAATAAACCCAAGGAAGGGAACAAAAGAAATGAAACCAAAAGAGATAGA---TC 1080 Yeast GGGAAAGGAA-----TGTGCAA------GGTAAGCTAGCACCACGTGTTGTTGATTTAA Fruit fly GGGAGCTAAA-----TAAAAAT------GGCAAAATGGGGCCAAGAATGGTGTGCATGA Red junglefowl GGGAGAAAAA-----CCAAAAG------GGAGGCATGGGTCCAAGGGTGGTCAATCTCA Brown rat GGGAGAAGAA-----CCAGAAA------GGAGGCATGGGTCCGAGGATGGTGAACCTCA Cattle GGGAAAAGAA-----CCAGAAG------GGAGGCATGGGACCGAGGATGGTGAACCTCA Human GGGAAAAGAA-----CCAGAAA------GGAGGCATGGGACCAAGGATGGTGAACCTCA Rhesus macaque GGGAAAAGAA-----CCAGAAA------GGAGGCATGGGACCAAGGATGGTGAACCTCA Clawed frog GGGAGAAGAA-----CCAGAAA------GGGCAAATGGGCCCCAGGATGGTGAATCTCA Pufferfish GGGAAAAAAA-----TGCAAAG------GGAGCCATGGGACCCAGGATGGTCAACCTGA Pufferfish GGGAGAAGAA-----CTCTAAG------GGGGCCATGGGACCCAGGATGGTCAACCTGA Atlantic salmon GGGAGAAGAA-----CCCTAAG------GGAGCAATGGGACCCCGGATGGTCAACCTCA Rainbow trout ------GACCCCGGATGGTCAACCTCA Zebrafish GGGAGAAAAA-----CACTAAA------GGAGCGATGGGACCCAGAAGTGTTAATCTGA Fish GGGAGAAGAA-----CCCCAAA------GGAGCAATGGGACCTAGAAGTGTCAATCTCA ** *

Sorghum TTCAATCAATGAACCCGGCAAGTCAGGATGAAGAAAAACAATTGATGCACTTAAAGCTTA 1140 Yeast GTTCATTGTTAGATCCTTTAAAAATTGCTGACCAATCCGTGGATTTAAATTTGAAGCTAA Fruit fly GGGACAGCATGGATCCAGCCAAACTCGCTGAGAACTCTGTAAACCTGAACCTGAAACTAA Red junglefowl GTGAGTGTATGGATCCAAAAAGGTTAGCAGAATCATCAGTGGATCTTAATTTGAAATTGA Brown rat GCGGATGTATGGACCCCAAAAGGCTGGCTGAGTCATCCGTGGATCTGAATCTCAAGCTGA Cattle GCGAGTGTATGGACCCTAAAAGGTTAGCTGAGTCATCGGTTGATCTCAACCTCAAGCTGA Human GTGAATGTATGGACCCTAAAAGGTTAGCTGAGTCATCAGTGGATCTAAATCTCAAACTGA Rhesus macaque GTGAATGTATGGACCCTAAAAGGTTAGCTGAGTCATCAGTGGATCTAAATCTCAAACTGA Clawed frog GTGAATGTATGGATCCAAAAAGGTTGGCCGAATCCTCCGTTGACCTGAACCTGAAGCTGA Pufferfish GCGAGTGCATGGACCCCAAAAGGCTTGCAGAGTCATCAGTGGACCTGAACCTGAAGCTGA Pufferfish GTGAATGCATGGATCCGTGCAGGCTTGCAGAGTCATCTGTGGACCTGAATCTGAAGTTAA Atlantic salmon GTGAATGTATGGACCCAAAGAGACTGGCGGAGTCATCAGTGGATCTCAACCTGAGGTTGA Rainbow trout GTGAATGTATGGACCCAAAGAGACTGGCCGAGTCCTCGGTGGACCTCAACCTGAAGTTGA Zebrafish GCGAATGCATGGACCCCAAGAGACTGGCAGAATCGTCTGTGGACCTGAACCTGAAGCTGA

157

Fish GTGAATGCATGGACCCAAAAAGACTTGCAGAATCATCAGTCGATCTTAATCTGAAGTTGA * * ** * * ** * * * * * *

Sorghum TGGGGTGGCG---CCACTTTCCTGTGAATATAGACAAGTTATCTCATGTTCGAGTTCTTC 1200 Yeast TGAAATGGAGAATTCTTCCTGACTTAAATCTAGATATCATCAAAAACACAAAAGTACTAC Fruit fly TGAAGTGGCGCCTTGTTCCCGATCTCAATCTCGAAATCATCTCCCAAACAAAATGCCTGC Red junglefowl TGTGCTGGCGGTTGGTCCCTACTCTTGATTTGGAAAAAATTGTGTCTGCCAAGTGTCTGT Brown rat TGTGCTGGCGGTTGGTCCCCACCTTGGACTTGGACAAGGTCGTGTCTGTCAAGTGCCTGC Cattle TGTGTTGGAGATTGGTCCCTACTCTGGACTTAGACAAGGTTGTGTCTGCCAAGTGTCTGC Human TGTGTTGGAGATTGGTTCCTACTTTAGACTTGGACAAGGTTGTGTCTGTCAAATGTCTGC Rhesus macaque TGTGTTGGAGATTGGTTCCTACTTTAAACTTGGACAAGGTTGTGTCTGTCAAATGTCTGC Clawed frog TGCGCTGGAGACTCGTCCCAACTCTGGACTTGGATAAAGTGATTCACACCAAGTGTTTGC Pufferfish TGAGGTGGAGGCTGGTTCCTTCCCTGGACCTGGACAAAGTGGTCAGCACCAAGTGTCTCC Atlantic salmon TGCGTTGGAGGCTGGTGCCTTCTCTGGATCTGGACAAAGTGACTTCTACTAAATGTCTGC Rainbow trout TGCGCTGGAGGCTGGTACCTTCTCTGAATCTGGACAAAGTGACTTCAACTAAGTGTCTGC Zebrafish TGCGCTGGAGGCTGGTGCCGGCTCTGGATCTGGAGAAAGTGGTGTCTACGAGGTGTTTAC Fish TGCGCTGGCGGCTTGTCCCCTCTCTGGATTTGGATAAAGTTGTGTCCACGAAGTGTCTCT ** *** * * * * ** * * *

Sorghum TCTTGGGTGCTGGAACTCTTGGATGTGAAGTTGCTCGTCTTCTTACGACCTGGGGTGTAC 1260 Yeast TACTAGGTGCTGGTACACTAGGTTGTTATGTTTCACGCGCATTGATAGCATGGGGGGTTA Fruit fly TCTTTGGAGCTGGTACTTTAGGATGCGCTGTAGCTAGAAATCTGCTGTCCTGGGGATTTA Red junglefowl TGCTAGGAGCTGGTACTCTGGGTTGTAGTGTGGCAAGGACTCTGATGGGTTGGGGTGTCA Brown rat TGCTGGGAGCTGGTACCTTGGGGTGTAATGTGGCTAGAACACTGATGGGCTGGGGCGTCA Cattle TGCTTGGAGCCGGCACCTTGGGTTGTAACGTAGCCCGGACGTTGATGGGTTGGGGTGTCA Human TGCTTGGAGCCGGCACCTTGGGTTGCAATGTAGCTAGGACGTTGATGGGTTGGGGCGTGA Rhesus macaque TGCTTGGAGCCGGCACCTTGGGTTGCAATGTAGCTAGGACGTTGATGGGTTGGGGCGTGA Clawed frog TGCTGGGGGCTGGCACGCTGGGCTGCAATGTGGCGCGGGCACTAATGGGCTGGGGAGTCA Pufferfish TTCTAGGTGCTGGGACATTGGGCTGCAATGTAGCCCGCACTCTGATGGGATGGGGCGTGA Atlantic salmon TGTTGGGGGCTGGTACACTGGGTTGCAACGTAGCCAGGACTCTAATGGGCTGGGGAGTGA Rainbow trout TGCTGGGAGCTGGTACACTGGGCTGCAACGTAGCCAGGACTCTAATGGTGAGTGCCTGCG Zebrafish TGCTGGGAGCCGGAACACTGGGCTGCAATGTGGCCAGAACACTGATGGGCTGGGGTGTGC Fish TGCTTGGAGCTGGTACACTGGGCTGCAATGTAGCAAGGACACTCATGGGTTGGGGAGTGA * * ** ** ** ** * ** ** ** * * * * *

Sorghum GGAAACTAACGGTTGTTGATAGTAATTGTGTTGCAACGTCTGACCTTGTCAAGCAATCAC 1320 Yeast GAAAAATAACATTTGTGGATAACGGTACAGTTTCATATTCAAATCCAGTGAGGCAAGCGT Fruit fly AGCACATAACCCTTTTGGACAGCGGCAAGGTTGGGTTCTCTAATCCAGTGCGTCAGAATC Red junglefowl GGAAGATAACATTTGTTGACAATGCAAGGATTTCATACTCCAACCCAGTACGACAGCCAC

158

Brown rat GACACGTCACGTTTGTGGACAACGCCAAGATCTCCTACTCCAATCCCGTGAGGCAGCCTC Cattle GACACATCACGTTCGTGGACAATGCCAAGATCTCCTACTCTAACCCTGTGAGGCAGCCTC Human GACACATCACATTTGTGGACAATGCCAAGATCTCCTACTCCAATCCTGTGAGGCAGCCTC Rhesus macaque GACACATCACATTTGTGGACAATGCCAAGATCTCCTACTCCAATCCTGTGAGGCAGCCTC Clawed frog GACACATAACCTTCGTGGACAACGCCAAGATCTCCTACTCTAACCCTGTGCGGCAGCCCC Pufferfish GACACATCACATTTGTGGACAACGCAAAGATCTCCTACTCCAATCCTGTCCGCCAGCCGC Pufferfish GACACATCACGTTCGTAGACAATGCAAAGATTTCATATTCCAACCCAGTTCGCCAGCCGC Atlantic salmon GACACATCACGTTTGTGGACAACGCTAAGATCTCTTACTCCAACCCAGTGAGACAGCCAC Rainbow trout ATGTTGACATGATTCAATTAGATTTCTAG------Zebrafish GGCACATCACGTTTGTGGACAACGCAAAGATCTCGTACTCAAACCCGGTGCGGCAGCCGC Fish GACACATCACATTTGTGGACAATGCAAAGGTCTCCTACTCGAACCCAGTGCGTCAGGCTC * *

Sorghum TCTACATAGATAAGGACTGTGGAGTTCCAAGAGT------AACTGCAATAGTTACAC 1380 Yeast TATATAATTTTGAGGACTGTGGAAAGCCAAAGGCAGAA------CTTGCGGCTGCGT Fruit fly TCTACACGCATGCAGATGCTGTGGCAGGCAATCGGATGAAGGCGACTACAGCTGCTCAGA Red junglefowl TGTATGAATTTGAAGACTGTCTCAGTGGTGGGAAGCCAAAGGCACTTGCAGCAGCAGAGA Brown rat TGTATGAATTTGAAGATTGTCTAGGGGGTGGCAAGCCCAAGGCCCTGGCTGCAGCAGAGC Cattle TGTATGAATTTGAAGATTGCCTGGCGGGTGGGAAGCCCAAGGCCCTGGCAGCCGCAGACC Human TCTATGAGTTTGAAGATTGCCTAGGGGGTGGTAAGCCCAAGGCTCTGGCAGCAGCGGACC Rhesus macaque TCTATGAGTTTGAAGATTGCCTAGCGGGTGGTAAGCCCAAGGCTCTGGCTGCAGCAGACC Clawed frog TCTATGACTTCTCCGACTGCCTGGGCGGGGGCAACCCCAAAGCTGAGGTGGCAGCGGCAA Pufferfish TGTACGAGTTTGAGGATTGTCTTGGAGGAGGAAAGTCCAAAGCCACCGCAGCAGCTGAGC Pufferfish TCTACGAGTTTGAGGACTGTCTCGGAGGAGGGAAATCCAAAGCAATGGCAGCTGTTGAAC Atlantic salmon TGTATGAGTTTGAAGACTGTCTGGGAGGAGGCAAGTCCAAGGCCCTCGCTGCCGTTGACA Rainbow trout ------Zebrafish TCTACGAGTTTGAAGACTGTCTGAGTNGAGGAAAGTCTAAAGCGCTCGCCGCCGTGGACA Fish TCTATGAGTTTGAGGACTGCCTGAGTGGAGGGAAAACTAAAGCCTTGGCTGCAGTGGATA

Sorghum ATCTAAAAGAAAGATGTCCTGCAGTGGAGGTTGAAGGCATCCAAATGGAAATACCGGTGC 1440 Yeast CTTTGAAAAGAATATTTCCTTTAATGGACGCTACTGGTGTGAAATTAAGTATTCCTATGA Fruit fly GATTGAAAGAGATCAATCCGTCTGCGGAAACGGCGGGTTATGTGCTGGAGATTCCCATGC Red junglefowl GACTTCAGAAAATCTTCCCAGGAGTGAATTCAGAAGGCTATAACATGAGCATCCCTATGC Brown rat GGCTCCAGAAAATATTTCCCGGAGTGAATGCCAGCGGGTTCAACATGAGCATCCCCATGC Cattle GGCTGCAGAAGATATTCCCCGGAGTGAACGCCAGAGGGTTCAACATGAGCATCCCCATGC Human GGCTCCAGAAAATATTCCCCGGTGTGAATGCCAGAGGATTCAACATGAGCATACCTATGC Rhesus macaque GGCTCCAGAAAATATTCCCCGGTGTGAATGCCAGAGGATTCAACATGAGCATACCCATGC Clawed frog AGCTGGAGAAGATATTCCCTGGCGTGAATGCCAGAGGATTTAACCTGAGCATCCCCATGC

159

Pufferfish GTCTTTCCAAAATCTTTCCCGGTGTTAATGCAAAGGGTTACAATATGAGCATCCCGATGC Pufferfish GACTTAGCAAAATCTTTCCTGGCGTGAATGCTGTAGGACACAACATGAGCATCCCCATGC Atlantic salmon GACTGGGCAAGATCTTCCCTGGAGTGAATTCAGAGGGCCACGTGATGAGCATCCCTATGC Rainbow trout ------Zebrafish GACTCAAGAAGATCTTTCCTGGAGTGAATGCAGAAGGCTTCAACATGAGCATCCCGATGC Fish GATTAAAGAAGATCTTCCCCGGTGTGAATACTGAAGGATTCAACATGAGCATCCCCATGC

Sorghum CTGGGCATCCTGTTTCTTCC------AGCAAAATGGCAAGTGTGCTTGATGACTTCAAGC 1500 Yeast TCGGTCATAAATTAGTAAAC------GAGGAGGCTCAGCATAAGGATTTTGATA Fruit fly CTGGGCATACTATTGGAGAA------TCGCTTCTAGCCCAGACGAAGGAGCATTTGAAGG Red junglefowl CAGGCCATCCAGTGAATTTTTCAGAAGTGACAATGGCGCAGGCTCGGAAGGATGTGGCTA Brown rat CCGGACACCCTGTGAACTTCTCTGACGTCACGATGGAGCAGGCCCGCAGAGATGTGGAGC Cattle CGGGGCACCCCGTGAACTTCTCCAGCGTCACCCTGGAGCAGGCCCGCAGGGACGTGGAAC Human CTGGGCATCCAGTGAACTTCTCCAGTGTCACTCTGGAGCAAGCCCGCAGAGATGTGGAGC Rhesus macaque CTGGGCATCCAGTGAACTTCTCCAGTGTCACTCTGGAGCAAGCCCGCAGAGATGTGGAGC Clawed frog CGGGGCATCCGGTCCATTTCTCCCAGGAGACCGTG---CAGGCCCAGAGGGACGTAGAGA Pufferfish CCGGCCACCCAGTGAATTTCTCACAGGCCACTCTTTCTCTGGCCCAAATGGATGTGGAGC Pufferfish CTGGTCACCCGGTCAGTTTCTCTCAGGCTACGCTGTCGCAGGCACAGGAGGACGTAGAGC Atlantic salmon CAGGACACCCGGTCAACTTCTCTGAGGCGACCATGTCCCAGGCCAGGCAGGACGTGGAAC Rainbow trout ------Zebrafish CCGGACACCCTGTCAACTTCTCAGATCTGACCGTAGCTCAAGCGCAGCAGGACGTGGAGC Fish CTGGCCACCCTGTGAGTTTTTCTGAAGCGATGATTGCTCAGACGCAGAAAGACGTGGAAC

Sorghum ATCTGCAAACATTAGTGGCTGCCAATGATGCAGTCTTCTTGTTAACTGACACATGGGAGA 1560 Yeast GATTAAGAGCATTAATAAAAGAGCATGATATCATTTTTTTATTGGTGGATTCTCGAGAAA Fruit fly TTATCGAAAAGCTTGTGCAGGACCATGATGTCATCTTTCTGCTCACAGATTCGCGCGAAA Red junglefowl CACTTGAAGAGCTTATTGATGCTCATGATGTTGTTTTCCTACTAATGGACACTAGAGAGA Brown rat AGCTGGAGGAACTCATCGATAGCCACGATGTCATCTTCCTGCTAATGGACACCAGGGAGA Cattle AGCTGGAGCAGCTCATTGACAGCCACGACGTCGTCTTCCTGTTGATGGACACCCGGGAGA Human AACTGGAGCAGCTCATCGAAAGCCATGATGTCGTCTTCCTATTGATGGACACCAGGGAGA Rhesus macaque AACTGGAGCAGCTCATCGAAAGCCATGATGTCATTTTCCTGTTGATGGACACCAGGGAGA Clawed frog AGCTGGAAGCTCTGATTGGCCAACACGATGTTGTGTTTCTGCTGATGGACACGCGGGAGA Pufferfish AGCTGGAGAAGCTCATCTCAGAGCACGATGTCATCTTCTTACTGATGGACACGCGAGAGA Atlantic salmon ACCTGGAGAAACTGATCTCGGAGCACGACGTCGTCTTCCTGTTGATGGACACCAGGGAGA Rainbow trout ------Zebrafish AGCTGAAAAAACTCATTTCTGAACACGATGTGGTTTTTCTGCTGATGGACACCAGAGAGA Fish AGTTGGAGTCACTAATCTCAGCACATGACGTTATCTTCCTGCTTATGGACACACGGGAGA

160

Sorghum GCAGGTGGCTTCCAACTCTTCTCTGTGCAAGTGAAAACAAGATTGCTATTAGTGCAGTAT 1620 Yeast GCAGATGGCTTCCGTCGTTATTGAGCAACATCGAAAACAAAACCGTTATTAATGCTGCTC Fruit fly GCCGCTGGCTGCCCACCCTGCTTGGAGCGGCAAAGGAAAAAATTGTCATCAACGCAGCTT Red junglefowl GTCGATGGCTTCCTGCTGTTATTGCAGCCAGCAAGAGGAAGCTGGTCATCAATGCTGCCC Brown rat GCCGATGGCTTCCTACTGTTATTGCAGCCAGCAAGCGAAAGCTGGTCATCAATGCTGCCT Cattle GCCGGTGGCTGCCTGCCGTCATCGCCGCGAGCAAGAGAAAGCTGGTCATCAATGCTGCCT Human GCCGGTGGCTTCCTGCCGTCATTGCTGCAAGCAAGAGAAAGCTGGTCATCAATGCTGCTT Rhesus macaque GCAGGTGGCTTCCTGCTGTCATTGCTGCAAGCAAGAGAAAGCTGGTCATCAATGCTGCTT Clawed frog GCCGATGGCTGCCTACAGTGATCGCCGCCAGCAAGAGGAAGCTGGTGGTGAACGCTGCTC Pufferfish GCCGCTGGCTGCCCACAGTAATTGCTGCCAGCAAGAGGAAGCTTGTGGTAAATGCAGCTC Atlantic salmon GTCGATGGCTGCCGACTGTTATAGCTGCCAGCAACAGGAAGTTGGTAGTGAATTCTGCCC Rainbow trout ------Zebrafish GCCGCTGGCTGCCCACCGTCATCGCTGCCAGCCAGAGAAAGTTAATAGTAAACGCTGCTC Fish GTCGATGGCTCCCCACAGTCATCGCAGCTAGCCAGAGAAAGCTTGTAGTTAATGCAGCTC

Sorghum TAGGATGTGACAGTTACCTTGTCATGCGACATGGTGCTGGACCAGGAACAAGTGGGGGTA 1680 Yeast TGGGGTTTGATAGCTACTTAGTTATGAGGCATGGTAATAGAGATGAACAGTCTTCAAA-- Fruit fly TAGGCTTTGACAGTTATCTGGTAATGCGACATGGCACTACGCGTAAGGAAGCAGGCGACG Red junglefowl TGGGGTTTGACACGTTTGTTGTTATGAGACACGGACTAAAGAAACCCAAACAGCAAGAAA Brown rat TGGGGTTTGACACCTTTGTTGTCATGAGACATGGCCTGAAGAAACCCAAGCAGCAGGGAG Cattle TGGGATTTGACACCTTCGTTGTCATGAGACACGGCCTGAAGAAGCCCAGGCATCAGGGAG Human TGGGATTTGACACATTTGTTGTCATGAGACATGGTCTGAAGAAACCAAAGCAGCAAGGAG Rhesus macaque TGGGATTTGACACATTTGTTGTCATGAGACATGGCCTGAAGAAACCAAAGCAGCAGGGAG Clawed frog TGGGCTTTGACACGTTCGTAGTGATGAGGCACGGCCTGAAGATGCCAAGGAAGGCGGGCG Pufferfish TTGGCTTTGACACGTTTGTTGTGATGCGTCACGGCCTGAAGAAGCCTCCCGCGACTCTGA Atlantic salmon TGGGCTTTGACACCTTCGTGGTGATGAGACACGGTCTGAAGAAACCTAAACCCTGTGGAC Rainbow trout ------Zebrafish TGGGATTCGACACATTTGTGGTAATGAGACACGGCCTGAAGAAACCCAGAGAGTCTGAAG Fish TGGGGTTTGACACCTTTGTTGTAATGAGGCATGGCCTTAAGAAACCCAAAGACTCCGACG

Sorghum CAGATGAAGTGATTACTCAGATAGAAAATTTGTCCACAGA----AGATGCTCCTGGTCAT 1740 Yeast ------Fruit fly ATGGCCAAGAAATCG------Red junglefowl CTGGCAATGCGTGTTT------CA Brown rat CCGGGGACCTGTGCCC------AA

161

Cattle CTGGGGACTTGTGTCC------CA Human CTGGGGACTTGTGTCC------AA Rhesus macaque CTGGGGACTTGTGTCC------AA Clawed frog CGGCTGAGTCGTGGG------Pufferfish GTGTC------GACTCCTCCCCTTCCTGCTCGTCTGCTTCTTCGTCACCGTCCA Pufferfish GCGTTTCTGCAGGCCCGGACTCATCTCCTTCCTGTTCTTCCGCTTCGTCTTCATCGTCCA Atlantic salmon CAGGAGACTCCTCG------GACCCCTCTTCCTCCTCCGCCTCATCCTCTTCCT Rainbow trout ------Zebrafish AGTCCAGTCCGATG------TCTGCATCTTCCAGCTC---CTC------GTCTT Fish CTGAAGACTCCAGT------GCATCATCATCCTTGTGTGCATC------AGACT

Sorghum CAAAGATTGGGCTGTTGTTTCTGCAATGATGCCTCTTCCCTTATTAAGCCAATTCCTTAT 1800 Yeast ------Fruit fly ------AAGGACTTAAGTGCATTAATGGC Red junglefowl GCACTGCCCCTGGTCCT-TCTGACCTTTTGGGTTCATCGCTCTTTTCAAATATCCCTGGC Brown rat GCCATCTTGTAGCACCT-GCT---GACCTGGGCTCCTCACTTTTTGCCAACATCCCTGGA Cattle GCTACCCCATGGCGTCT-GCCGACCTCCTGAGCTCATCGCTGTTTGCCAACATCCCTGGC Human ACCACCCTGTGGCATCT-GCTGACCTCCTGGGCTCATCGCTTTTTGCCAACATCCCTGGT Rhesus macaque ACCACCCTGTGGCATCT-GCTGACCTCCTGGGCTCATCACTTTTTGCCAACATCCCTGGT Clawed frog CAGACGGTGCCAACTCC-TCCGATCTTCTGGGCTCCTCCCTTTTCTCCAACATCCCTGGG Pufferfish CGCCAGCTGGTTCAGCT-CCCAGCGTCCCAGGCTCCTCCCTCTTCTCCAACATCCCAGGA Pufferfish CGCCAGCTGGGGGGGCA-GCTTCTTTTTCAGGATCGTCTCTCTTCTCCAACATTCCCGGA Atlantic salmon CCACGCCTGCCGAGCCC-GCCCAGCTCCCAGCAGCCTCTCTGTTCTCCAACATCCCCGGA Rainbow trout ------Zebrafish CCAACACACCGGCCGCC-ACTGTTACTGCAGGATCCTCTCTGTTCTCCAACATCCCCGGT Fish CCTCTTCAGCCTCGACT-CCAGCGACACCAGGAATGTCTCTGTTCTCCAACATACCTGGC

Sorghum GAAACACTACCTGGACTTACCTCAGTTGC---ATCTGGCAAAGCAGTGGAGCTCTTTGCC 1860 Yeast --ACAACTGGGCTGTTATTTTTGCCATGATGTGGTAGCACCAACTGACAGTTTAACTGAC Fruit fly GATCAATTAGGTTGCTACTTTTGTAACGATGTTACGGCCCCTGGAAATTCGCTCAAAGAC Red junglefowl TACAAACTGGGCTGCTATTTCTGCAATGATGTCGTGGCACCAGGGGATTCTACCAGGGAT Brown rat TACAAGCTTGGCTGCTACTTCTGCAATGATGTGGTGGCTCCAGGAGATTCCACCAGAGAC Cattle TATAAACTCGGCTGCTACTTCTGCAACGATGTGGTGGCCCCAGGGGATTCAACCAGAGAC Human TACAAGCTTGGCTGCTACTTCTGCAATGATGTGGTGGCCCCAGGAGATTCAACCAGAGAC Rhesus macaque TACAAGCTTGGCTGCTATTTCTGCAATGATGTGGTGGCCCCAGGAGATTCAACCAGAGAC Clawed frog CACCGGCTTGGCTGCTATTTCTGTAACGACGTGGTGGCACCAGGAGATTCCACTAGGGAC Pufferfish CACAAGCTGGGATGTTACTTCTGTAATGATGTTGTGGCCCCAGGAGATTCGACCAGGGAT

162

Atlantic salmon CACCGCCTCGGATGCTACTTCTGTAACGACGTTGTCGCCCCCGGAGACTCCACCAGAGAC Rainbow trout ------Zebrafish CACCGTCTCGGCTGTTACTTCTGCAATGATGTGGTGGCTCCTGGAGACTCCACCAGAGAC Fish TATCGACTGGGCTGTTACTTCTGCAATGATGTTGTTGCCCCTGGAGATTCGACCAGGGAC

Sorghum AGGATGTTACATCACCCTGATGAGATACATGCCCCAGGC------GATACTGCT---GGT 1920 Yeast AGGACTTTGGATCAAATGTGCACAGTAACTAGACCCGGCGTTGCTATGATGGCCTCTTCT Fruit fly CGAACTTTGGATCAACAGTGCACAGTGACACGACCCGGAGTATCCAACATTGCGGCCAGC Red junglefowl CGGACATTGGATCAGCAGTGTACAGTCAGTCGACCTGGATTAGCCATGATAGCTGGAGCT Brown rat CGGACTCTGGACCAGCAGTGCACAGTGAGCCGCCCAGGGCTGGCCGTGATTGCAGGAGCC Cattle CGGACCTTGGACCAGCAGTGCACTGTGAGCCGTCCAGGACTAGCCATGATCGCAGGAGCC Human CGGACCTTGGACCAGCAGTGCACTGTGAGTCGTCCAGGACTGGCCGTGATTGCAGGAGCC Rhesus macaque CGGACCTTGGACCAGCAGTGCACTGTGAGTCGTCCAGGATTGGCCATGATTGCAGGAGCC Clawed frog CGCACTCTGGACCAGCAGTGCACGGTGAGCCGACCGGGACTGGCTATGATCGCCGGAGCC Pufferfish CGGACGCTAGATCAGCAGTGCACGGTCAGCAGACCAGGTCTGGCCATGATAGCGGGAGCT Atlantic salmon CGGACCCTGGACCAGCAGTGTACAGTGAGTCGTCCAGGCCTGGCCATGATAGCAGGAGCC Rainbow trout ------Zebrafish CGGACTCTGGACCAGCAGTGTACAGTGAGCCGCCCGGGTCTGGCCATGATTGCTGGAGCT Fish AGGACTCTGGATCAGCAGTGTACGGTGAGCCGGCCTGGTTTGGCCATGATTGCCGGCGCG

Sorghum ATGGAAACAGAACATCAGCTTGGTCTATTGCCGCATCAGCTACGAGGGTCACTCCCAAAG 1980 Yeast TTAGCAGTTGAATTGATGACTTCCTTACTACAGACCAAATACTCTGGTTCAGAAA----- Fruit fly TATGCGGTGGAATTGTTGGTGGCTTTGCTGCAGCATCCTCGTAAGGAGCTGGCAC----- Red junglefowl CTTGCGGTGGAATTAATGGTTTCTGTTTTACAGCATCCAGAAGGTGGTTATGCTG----- Brown rat CTGGCTGTGGAGCTGATGGTTTCTGTCTTGCAGCATCCTGAGGGGGGCTACGCCA----- Cattle CTGGCGGTGGAGTTGATGGTTTCCGTTTTGCAGCATCCAGAAGGGGGCTATGCCA----- Human CTGGCCGTGGAATTGATGGTATCTGTTTTGCAGCATCCAGAAGGGGGCTATGCCA----- Rhesus macaque CTGGCCGTGGAATTGATGGTATCTGTTTTGCAGCATCCAGAAGGGGGCTATGCCA----- Clawed frog CTCGCCGTGGAGCTCATGGTGTCCGTTATCCAGCACCCCGAAGGAGGTTACGCAG----- Pufferfish TTGGCTGTGGAGCTCATGGTGTCCATGTTACAGCACAGTGAAGGGGGATATGCAG----- Pufferfish CTGGCTGTGGAGATGATGGTGTCCATCCTGCAGCATAGCGAAGGAGGCTATGCCG----- Atlantic salmon CTGGCTGTAGAACTCATGGTGTCCATACTCCAGCATACTGAAGGAGGTTATGCGG----- Rainbow trout ------Zebrafish CTGGCTGTGGAGCTGATGGTGTCTGTACTGCAGCATCCAGAAGGGGGTTATGCAG----- Fish CTGGCTGTTGAGCTGATGGTCTCCATCCTGCAGCACCCAGAAGGAGGCTACGCGG-----

163

Sorghum TGTGGTTTATCTATGGAACTGAGCAATTCCTCAGTCAATTGTATGGCATGTTCTATTGCT 2040 Yeast ------CAACAGTATTA------G------Fruit fly ------CCGCCTACTAT------GCACA-----GAGTGGGCGTGGCAGGTCGG Red junglefowl ------TGGCCAGCAGT------A------Brown rat ------TCGCCAGCAGC------A------Cattle ------TTGCCAGCAGC------A------Human ------TTGCCAGCAGC------A------Rhesus macaque ------TTGCCAGCAGC------A------Clawed frog ------TCGCTAGCAGC------A------Pufferfish ------TGGCCAGCAGC------A------Pufferfish ------TGGCCAGCAGC------A------Atlantic salmon ------TGGCCAGCAGT------A------Rainbow trout ------Zebrafish ------TGGCCAGCAGC------A------Fish ------TGGCGAGTAGC------A------

Sorghum GTATTGTCAGAGTATAGAAGAAGAGGATTGGATTTTGTCATGCAGGCCATCAACTATCCA 2100 Yeast ------GGGATATACCTCATCAAATACGTG Fruit fly AGGAGACGGAAGAAAAGGTGCCGGAGGGTCTGCTAGGAATTTTGCCTCACTCCATCCGTG Red junglefowl GTGATGATAGAATGAATGAGCCACCTACTTCTCTTGGACTTGTTCCTCATCAGATCCGAG Brown rat GTGATGACCGCATGAATGAGCCTCCCACCTCGCTGGGGCTTGTACCTCACCAGATCCGGG Cattle GTGACGACCGCATGAATGAGCCGCCAACGTCCCTCGGCCTCGTGCCTCACCAGATACGGG Human GTGACGATCGGATGAATGAGCCTCCAACCTCTCTTGGGCTTGTGCCTCACCAGATCCGGG Rhesus macaque GTGACGATCGGATGAATGAGCCTCCAACCTCTCTTGGGCTCGTGCCTCACCAGATCCGGG Clawed frog GTGACGACCGTATGAATGAGCCCCCAACTTCCCTTGGGCTTGTGCCCCATCAGATCCGAG Pufferfish GTGATGACCGAATGAATGAACCTCCCACATCGCTGGGTCTGGTGCCACATCAGATTCGAG Pufferfish GTGATGACAGAATGAACGAACCACCCACCTCTCTCGGTCTGGTGCCGCATCAGATTCGAG Atlantic salmon GTGATGACAGGATGAATGAACCTCCAACCTCCCTGGGTCTCGTCCCTCATCAGATCAGAG Rainbow trout ------Zebrafish GTGACGATCGCATGAATGAACCTCCGACCTCTCTGGGTCTCGTCCCTCATCAGATTCGAG Fish GCGATGATCGCATGAACGAACCGCCGACCTCACTTGGCCTCGTCCCACATCAGATTCGAG

Sorghum ACATATCTGAAAGATTTTACAGG------CG-TTTCTGATTTAAAAATGCCAGAT 2160 Yeast GGTTTTTGCATAATTTTTCCATCTTAAAATTAGAAACTCCAGCCTATGAGCATTGTCCAG Fruit fly GAATGCTGTGCAACTACGAGAATATTCTGCCCGCCACTCAAAAGTTTGCTCAGTGCATAG Red junglefowl GCTTTTTATCAAGATTTGATAATGTTCTTCCAGTCAGTTTGGCATTTGATAAATGCACAG Brown rat GCTTTCTGTCACGGTTCGATAATGTCCTTCCTGTCAGCCTGGCATTTGATAAATGTACAG

164

Cattle GATTTCTGTCACGGTTTGATAACGTCCTTCCTGTCAGCCTGGCCTTTGACAAATGCACGG Human GATTTCTTTCACGGTTTGATAATGTCCTTCCCGTCAGCCTGGCATTTGACAAATGTACAG Rhesus macaque GATTTCTGTCACGGTTTGATAATGTCCTTCCCGTCAGCCTGGCATTTGACAAATGTACAG Clawed frog GCTTTTTGTCGAGGTTTGATAATGTGCTGCCAGTGAGCCTGGCGTTCGACAAGTGCACTG Pufferfish GCTTTCTTTCAAGATTTGACAATGTCCTGCCTGCCAGCCTGGCCTTTGACAAATGCACCG Pufferfish GCTTTCTTTCAAGGTTTGACAACGTCCTGCCAGCCAGCCTGGCCTTTGACAAATGCACCG Atlantic salmon GGTTTCTGTCCAGATTTGACAACGTTCTACCGGCCAGTCTGGCGTTCGACAAATGCACCG Rainbow trout ------Zebrafish GATTCCTGTCCAGATTTGACAACGTTCTCCCTGCCAGTTTGGCCTTCGACAAATGCACCG Fish GCTTCCTCTCCAGATTTGACAATGTTCTTCCTGCCAGCTTGGCCTTTGATAAATGCACTG

Sorghum AC------TTGTCCTAAAATACCAGCTAGCATCTCTGTAAATTCAGACAAGGT 2220 Yeast CATGTAGCCCTAAAGTTATCGAAGCATTTACAGACTTAGGTTGGGAATTTGTTAAAAAGG Fruit fly CCTGTTCAGCGGCCGTCCTAAATGAATATAAAAAAGAGGGTCATGCTTTCCTGTTTAAAA Red junglefowl CCTGTTCCCCCAAAGTCCTTGACCAATATGAACGTGAAGGATTTAATTTCCTAGCTAAAG Brown rat CCTGTTCATCCAAAGTTCTTGATCAATATGAGCAAGAAGGATTCACCTTCCTGGCCAAGG Cattle CTTGTTCTTCCAAAGTTCTCGATCAGTATGAACGAGAAGGATTTAATTTCCTGGCGAAGG Human CTTGTTCTTCCAAAATCTGGGACATGAGCGATGATGAGACCATCTGA------Rhesus macaque CTTGTTCTTCCAAAGTTCTTGATCAATATGAACGAGAAGGATTTAACTTCCTAGCCAAGG Clawed frog CCTGTTCTCCCAAGGTTCTTGATCAGTACGAGAGGGACGGATTCCAGTTCTTGGCCAAAG Pufferfish CCTGCTCACCCATCGTCTTGGACAACTATGAGAAGGAAGGTTTCGACTTTCTGTCCAAGG Pufferfish CCTGCTCAGCCACTGTTTTGGACCACTATGAGAGAGAAGGCTTCAGCTTCCTGTCCAAGG Atlantic salmon CCTGCTCACAAATCGTCCTGGACAGCTATGAGAAGGATGGTTTCAACTTCCTGTCCAAGG Rainbow trout ------Zebrafish CATGTTCACCCATAGTTCTGGAGAACTACGAGCGAGAAGGTTTCCAATTTCTAGCGAAGG Fish CCTGCTCACAAGTGGTCCTGGACCATTACGAGCGTGAAGGATTCCAGTTTTTGTCCAAGG

Sorghum TTCTGATGTCCGATGTCTACTATTGGGTGCTGGAACTCTTGGATGTGATGTTGCACGCAT 2280 Yeast CCTTGGAGC------ATCCACTTTAC---CTTGAAGAAATTAGCGGTTT------GTCAG Fruit fly CTTTTGAAA------CAGCCAAGTTT---CTGGAGGATTTGACGGGAAT------CTCCG Red junglefowl TTTTTAATT------CCTCGCATTCCTTCTTAGAAGACCTGACAGGTCT------AACTT Brown rat TGTTTAACT------CTTCACATTCCTTCTTAGAAGATTTGACTGGTCT------TACAC Cattle TGTTTAATT------CTTCGCACTCCTTCTTAGAGGACTTGACCGGTCT------TACAT Human ------Rhesus macaque TGTTTAATT------CTTCACATTCCTTCTTGGAAGACTTGACTGGTCT------TACAT Clawed frog CTTTCAACT------CCTCTCATTCATTCCTGGAGGATCTGACCGGCCT------GACGC Pufferfish TTTTTAATT------CATCCCATTCGTTTCTAGAAGACTTGACAGGCCT------TACTC

165

Atlantic salmon TGTTTAACT------CCTCCCACTCCTTCCTGGAGGACCTGACTGGTTT------GACTC Rainbow trout ------Zebrafish TCTTCAACT------CGTCCCATTCCTTCCTGGAGGATCTGACCGGCTT------AACCT Fish TGTTCAACT------CGTCTCACTCCTTCCTGGAGGATTTGACGGGTCT------GACCC

Sorghum TCTTATGGACTGTGGTGTACGGAAGCTAACAATAGTTGATAGTGGTTGTGTAGTTGTG-- 2340 Yeast TCATAAA--GCAGGAGGTCGAACGACTAGGCAACGATGTTTTTGAATGGGAAGATGATGA Fruit fly AATTCAA--GCGGTTAAACAG------CGAGATTATCGATTTTGATGATGAAGA Red junglefowl TATTACA--TCAAGAGACACA------GGCTGC-TGAGATCTG------Brown rat TGCTCCA--CCAAGAGACCCA------AGCTGC-TGAGATCTG------Cattle TGCTGCA--TCAAGAGACCCA------GGCTGC-TGAGATCTG------Human ------Rhesus macaque TGCTGCA--TCAAGAAACCCA------AGCTGC-CGAGATCTG------Clawed frog TGCTGCA--CCAGGAGACCCA------GGCTGC-GGAGATCTG------Pufferfish TGTTGCA--TCAAGAGACACA------AGCTGC-AGAGATTTG------Atlantic salmon AGCTACA--CCAGGAGACACA------GAACGC-TGAGATTTG------Rainbow trout ------Zebrafish TACTCCA--TCAGGAAACACA------GGCCGC-TGAGATCTG------Fish TGCTGCA--TCAGGAGACACA------GGCTGC-TGAGATTTG------

Sorghum --TCAAATTTAGCGAGACAGTCACTCTATAC------TTCTGATGACCGTGGTGCTC 2400 Yeast ATCTGATGAGATTGCTTAA------Fruit fly ATTTGACATGTCCGATAGCGACGAGGATTAGGTTCCACAAAATTGGTCAAGTTTTATCTT Red junglefowl ---GGACATGAGTGATGATGAAACAGTCTGA------Brown rat ---GGACATGAGTGACGAGGAGACCGTCTGA------Cattle ---GGACATGAGTGACGACGAGACCATCTGA------Human ------Rhesus macaque ---GGACATGAGCGACGATGAGACCATCTGA------Clawed frog ---GGACATGAGCGACGATGAGATTGTGTGA------Pufferfish ---GGATATGAGCGACGATGAGAGCATCTGA------Pufferfish ---GGACATGAGCGACGACGAAAGCATCTGA------Atlantic salmon ---GGACATGAGTGATGATGAGAGCATCTGA------Rainbow trout ------Zebrafish ---GGACATGAGTGACGACGAGAGCATCTGA------Fish ---GGACATGAGTGATGATGAAAGTATCTGA------

166

Sorghum CAAAAGCAACTGCAATACTTAGGCATCTAGTGGAGAGATGTCCTTCAGTGGATGCACAAG 2460 Yeast ------Fruit fly CAAAGCAAAATATCTTAAATATGAAATTCAGCTTATTGTGGCTTTAATTTTCTGCA---- Red junglefowl ------Brown rat ------Cattle ------Human ------Rhesus macaque ------Clawed frog ------Pufferfish ------Pufferfish ------Atlantic salmon ------Rainbow trout ------Zebrafish ------Fish ------

Sorghum GCATTAGAATGGAAATACCTATGCCTGGGCATCCTGTGTCTCCTGGTGAAGCAGCTGGTG 2520 Yeast ------Fruit fly GCATTTATAGTTCAATGTCTATACCAAAA----ATGTGTGTAC------TTCAA Red junglefowl ------Brown rat ------Cattle ------Human ------Rhesus macaque ------Clawed frog ------Pufferfish ------Pufferfish ------Atlantic salmon ------Rainbow trout ------Zebrafish ------Fish ------

Sorghum TGCTCCAAGATTGTGAGCGCCTTAAGGAATTAGTGGCTTCCCATGATGCTGTCTTCTTGT 2580 Yeast ------Fruit fly TGCTTTTATTCTATCTAGTCGTTAAGCAAATTGTGAATCATTCAAATGAAATTGATGTGA Red junglefowl ------Brown rat ------

167

Cattle ------Human ------Rhesus macaque ------Clawed frog ------Pufferfish ------Pufferfish ------Atlantic salmon ------Rainbow trout ------Zebrafish ------Fish ------

Sorghum TGACTGACACAAGAGAAAGTAGGTGGCTTCCAACTCTCCTCTGCACTAATGAAAACAAGA 2640 Yeast ------Fruit fly -AAATTAGGCATAAGAAAAATGTGAATTTACTATTGAAAATTGTAATAATAAAAGTGT-- Red junglefowl ------Brown rat ------Cattle ------Human ------Rhesus macaque ------Clawed frog ------Pufferfish ------Pufferfish ------Atlantic salmon ------Rainbow trout ------Zebrafish ------Fish ------

Sorghum TTGCTATTACTGCAGCATTAGGGTATGATAGTTACCTTGTCATGCGACATGGGGCTGGTC 2700 Yeast ------Fruit fly --GAATTTACT------Red junglefowl ------Brown rat ------Cattle ------Human ------Rhesus macaque ------Clawed frog ------Pufferfish ------

168

Pufferfish ------Atlantic salmon ------Rainbow trout ------Zebrafish ------Fish ------

Sorghum CTGGCATAAGCTGTGATGCTTCCAGTGTGGCCACTGCCACAGATAAGCTATCCACAGAGG 2760 Yeast ------Fruit fly ------Red junglefowl ------Brown rat ------Cattle ------Human ------Rhesus macaque ------Clawed frog ------Pufferfish ------Pufferfish ------Atlantic salmon ------Rainbow trout ------Zebrafish ------Fish ------

Sorghum ATGCCCTTGGGCGTCAAAGGCTGGGGTGTTATTTCTGCAATGACGTCATTGCTCCTGTTG 2820 Yeast ------Fruit fly ------Red junglefowl ------Brown rat ------Cattle ------Human ------Rhesus macaque ------Clawed frog ------Pufferfish ------Pufferfish ------Atlantic salmon ------Rainbow trout ------Zebrafish ------Fish ------

169

Sorghum ATTCAGTTTCCAATCGGACACTGGATCAACAATGTACAGTGACACGACCTGGGTTGGCCT 2880 Yeast ------Fruit fly ------Red junglefowl ------Brown rat ------Cattle ------Human ------Rhesus macaque ------Clawed frog ------Pufferfish ------Pufferfish ------Atlantic salmon ------Rainbow trout ------Zebrafish ------Fish ------

Sorghum CTATTGCATCTGGACGTGCCGCAGACCTTTTCACAAGAATGTTACATCATCCAGATGGCA 2940 Yeast ------Fruit fly ------Red junglefowl ------Brown rat ------Cattle ------Human ------Rhesus macaque ------Clawed frog ------Pufferfish ------Atlantic salmon ------Rainbow trout ------Zebrafish ------Fish ------

Sorghum TACATGCTCCAGGAGAGATTGCTGGTACAAGCAGTGGTCATCAGCATGGTATATTACCTC 3000 Yeast ------Fruit fly ------Red junglefowl ------

170

Brown rat ------Cattle ------Human ------Rhesus macaque ------Clawed frog ------Pufferfish ------Pufferfish ------Atlantic salmon ------Rainbow trout ------Zebrafish ------Fish ------

Sorghum ACCAGATGCGTGGATCACTATCGCAGTACAACTTACTAACCCTGTTGGGCTATTCCTCGA 3060 Yeast ------Fruit fly ------Red junglefowl ------Brown rat ------Cattle ------Human ------Rhesus macaque ------Clawed frog ------Pufferfish ------Pufferfish ------Atlantic salmon ------Rainbow trout ------Zebrafish ------Fish ------

Sorghum GTAATTGCACGGCATGTTCCAATGCGGTATTGTCTGAATATCGGAGGAGAGGAATGGACT 3120 Yeast ------Fruit fly ------Red junglefowl ------Brown rat ------Cattle ------Human ------Rhesus macaque ------Clawed frog ------

171

Pufferfish ------Atlantic salmon ------Rainbow trout ------Zebrafish ------Fish ------

Sorghum TTGTGATGCAAGTGATCAATGAACCAACTTATTTGGAAGACCTAACCGGCCTCACGGACT 3180 Yeast ------Fruit fly ------Red junglefowl ------Brown rat ------Cattle ------Human ------Rhesus macaque ------Clawed frog ------Pufferfish ------Pufferfish ------Atlantic salmon ------Rainbow trout ------Zebrafish ------Fish ------

Sorghum TGATGAAATCCACATCTTACTCGCAGGTCGAGTGGGTAGATGAAACTGATGAAGACGAAT 3240 Yeast ------Fruit fly ------Red junglefowl ------Brown rat ------Cattle ------Human ------Rhesus macaque ------Clawed frog ------Pufferfish ------Atlantic salmon ------Rainbow trout ------Zebrafish ------Fish ------

172

Sorghum TTGTCGAGGTCTGA 3254 Yeast ------Fruit fly ------Red junglefowl ------Brown rat ------Cattle ------Human ------Rhesus macaque ------Clawed frog ------Pufferfish ------Pufferfish ------Atlantic salmon ------Rainbow trout ------Zebrafish ------Fish ------

173

Appendix 2.2c) Clustal alignments between rainbow trout Atg genes and sequences from 14 other species.

CLUSTAL O(1.2.4) multiple sequence alignment ATG13

Yeast TACTAATATGCTCCACCGAATCAAGTCGATACCAGTCTTCTACAGAAAATATATTCCTAT 60 Sorghum ------Fruit fly ------Rice Fish ------Pufferfish ------Atlantic Salmon ------Rainbow trout ------Zebrafish ------Cat ------Clawed frog ------Red junglefowl ------Brown rat ------Cattle ------Human ------Rhesus macaque ------

Yeast TTGACGACACATGGTTTGAAGATCACTCAGAATTAGTGAGTGAGCTACCCGAGATAATAT 120 Sorghum ------Fruit fly ------ATGTCGGCGCAGCGTCTAAAT------Rice Fish ------ATGGACAGTGACCTGAGTCCA------Pufferfish ------ATGGAGAGTGGCTTGAGTCCT------Atlantic Salmon ------ATGATGGACAGTGATCTAAGTCCT------Rainbow trout ------ATGGACAGTGATCTAAGTCCT------Zebrafish ------ATGGATAGTGATCTGAGCCCA------Cat ------ATGATGGACAGCGATCTGAGCCCA------Clawed frog ------ATGGATACCGACCTCAGCCCC------Red junglefowl ------ATGGACACTGATCTCAGTTCG------Brown rat ------ATGGAAACTGATCTCAGTTCC------Cattle ------ATGGAAACTGATCTTAATTCC------Human ------Rhesus macaque ------

174

Yeast CAAAATGGTCTCACTACGATGGTCGAAAAGAGTTGCCACCCTTAGTGGTAGAGACATATT 180 Sorghum ------Fruit fly ------GCAGCGGAACGGGATTTGGAGAAGTTC-----ATCAAGTTCCT Rice Fish ------CAGGATAAAAAAGACCTGGACAAGTTC-----ATTAAATTCTT Pufferfish ------CAAGATAAAAAAGATTTGGACAAGTTC-----ATCAAGTTTTT Atlantic Salmon ------CAGGATAAAAAAGACTTGGACAAGTTC-----ATCAAGTTTTT Rainbow trout ------CAGGATAAAAAAGACTTGGACAAGTTC-----ATAAAGTTTTT Zebrafish ------CAGGACAAGAAAGACTTAGATAAATTC-----ATCAAATTCTT Cat ------CAGGACAGGAAAGACTTGGACAAATTC-----ATCAAATTTTT Clawed frog ------CAGGACCGGAAGGACTTGGACAAGTTT-----ATCAAATTCTT Red junglefowl ------CAGGACAGGAAGGACCTGGACAAGTTC-----ATCAAATTTTT Brown rat ------CAGGACAGAAAGGACCTGGACAAGTTT-----ATTAAGTTTTT Cattle ------CAGGACAGAAAGGACCTGGACAAGTTC-----ATTAAGTTTTT Human ------Rhesus macaque ------

Yeast TGGATTTAAGACAGTTAAACTCGTCTCATTTAGTTAGATTAAAGGACCACGAAGGCCATT 240 Sorghum -----TCAAGGTGCTCCACGCCGT------CCTCGCCGTGCGCACGC Fruit fly GGTCTTGAAGTCCACCCAGGTGGT------GGTGCAGTCCCGACTGG Rice Fish TGCTTTGAAGACTGTCCAGGTGAT------AGTCCAAGCCCGCCTAG Pufferfish CGCCTTGAAGACCTTCCAGGTGAT------TGTACAAGCTCGTCTTG Atlantic Salmon TGCTTTGAAGACAGTGCAGGTGAT------AGTCCAAGCACGACTTG Rainbow trout CGCTTTGAAGACAGTGCAGGTGAT------AGTCCAAGCGCGACTTG Zebrafish TGCTTTGAAGACAGTGCAGGTTAT------AGTGCAAGCCCGGCTTG Cat CGTCTTGAAGACAGTGCAGGTGAT------AGTGCAGGCTCGTCTGG Clawed frog TGCGCTAAAGAGCGTTCAGGTGAT------TGTACAAGCTCGACTCG Red junglefowl TGCTCTAAAGACGGTACAAGTAAT------TGTCCAGGCCCGACTTG Brown rat TGCCCTCAAGACTGTCCAAGTGAT------TGTCCAGGCTCGACTTG Cattle TGCCCTCAAGACTGTCCAAGTGAT------TGTCCAGGCTCGACTGG Human ------CTGTCCAAGTGAT------TGTCCAGGCTCGGCTTG Rhesus macaque ------CTGTCCAAGTGAT------TGTCCAGGCTCGTCTTG * * *

Yeast TGTGGAACGTTTGCAAAGGAACTAAGAAG-CAGGAAATCGTGATGGAACGTTGGCTTATC 300 Sorghum CGCGCCCGCTCGCCGCCGCCGCGGCGTCGTTCAGGCGGAGGGACAGGTGGTTCCAT-CTC Fruit fly GCGAGAAGATGCAGACACAGTGCAATCC---ACTGGCGGGCAGTGATTGGTTTAAC-ATA Rice Fish GAGAGAAGATTTGCACTCGGTCCTCTTCATCACCCACTGGTTCTGATTGGTTTAAT-CTG Pufferfish GAGAGAAGATCTGCACATGCTCTTCCTCGTCACCCACTGGCTCTGACTGGTTTAAC-CTG

175

Atlantic Salmon GGGAAAAAGTATGCACACGCTCCTTGTCTTCACCTACTGGCTCTGATTGGTTCAAT-CTG Rainbow trout GGGAAAAAGTATGCACACGCTCCTTGTCTTCACCTACTGGCTCTGATTGGTTCAAT-CTG Zebrafish GAGAGAAGATCAGCACTTGCTCGTCTTCATCACCGACAGGTTCGGACTGGTTTAAT-TTG Cat GCGAGAAGATCTGCACTCGCTCCTCCTCTTCCCCCACAGGCTCAGACTGGTTCACC-CTG Clawed frog GAGATAAGATCTGCACTCGCTCCTCCTCATCTCCTACAGGCTCGGACTGGTTCAAC-TTG Red junglefowl GAGAGAAAATCTGTACCCGATCGTCGTCCTCCCCAACAGGCTCCGACTGGTTCAAT-TTG Brown rat GAGAAAAGATTTGCACCCGTTCATCATCTTCCCCGACGGGTTCAGATTGGTTCAAT-TTA Cattle GAGAGAAGATTTGTACTCGTTCGTCTTCTTCCCCAACGGGTTCAGACTGGTTCAAT-TTA Human GTGAAAAGATTTGCACTCGTTCATCATCTTCTCCAACGGGTTCAGATTGGTTCAAC-TTA Rhesus macaque GTGAAAAGATTTGCACTCGTTCATCATCATCTCCAACGGGTTCAGATTGGTTCAAC-TTA * * *** *

Yeast GAATTAGATAATTCATCCCCAACTTTCAAATCATACAGTGAAGATGAGACTGATGTTAAT 360 Sorghum CCG----CTCCATGACCCGCCGCCGCCGCCCGAGGCGGCCGACCGCCTGGACG-----AG Fruit fly GCC----GTGCAGGATCATCCGGAGGTCCTGGACGAGACCAAGCGGGCTCTGA-----AC Rice Fish GCC----ATCAAGGACATCCCAGAGGTGAGTCATGAAGCGAAGAAGGCTCTGG------Pufferfish GCC----ATTAAAGACATTCCAGAGGTCACTCATGAAGCTAAAAAGGCACTAG------Atlantic Salmon GCA----ATCAAAGATATCCCAGAAGTTACACATGAGGCAAAGAAAGCTCTGT------Rainbow trout GCA----ATCAAAGATATCCCAGAAGTTACACACGAGGCAAAGAAAGCTCTGT------Zebrafish GCA----ATAAAGGACATCCCCGAGGTCACTCATGAAGCAAAAAAAGCTCTTG------Cat GCA----ATTAAAGACATTCCAGAGGTGACCCATGAAGCTAAGAAAGCTCTGG------Clawed frog GCC----ATCAAGGATATCCCAGAGGTGACCCATGAAGCAAAGAAAGCTCTAT------Red junglefowl GCC----ATCAAAGATATACCAGAGGTTACTCATGAAGCAAAGAAAGCCTTGG------Brown rat GCA----ATCAAAGATATCCCAGAAGTCACCCATGAAGCAAAGAAGGCCCTGT------Cattle GCA----ATCAAAGACATCCCAGAGGTTACACATGAAGCAAAGAAGGCCCTGG------Human GCA----ATCAAAGACATCCCAGAGGTTACACATGAAGCAAAGAAGGCACTGG------Rhesus macaque GCA----ATCAAAGACATCCCAGAGGTTACACATGAAGCAAAGAAGGCACTGG------* * * * *

Yeast GAACTTTCTAAACAGCTAGTCCTTCTCTTCCGTTATTTGTTGACTTTAATACAGTTACTA 420 Sorghum CTGGCCCCCGGGGAGCCGCTGGTGGTGGAC------GTCCTCCTGTGCCCC Fruit fly CTAAAGACAGGAGAGAGCATTCTGCAGCGCCTGC---CGTTGTGCGTGGAAATATCCCTG Rice Fish ------CCGGCCAACTCCCGGGCATCGGGCGCT---CCATGTGCGTGGAGATCTCCCTG Pufferfish ------CTGGGCAACTTCCAGGCATAGGTCGCT---CCATGTGTGTGGAGATCTCCTTG Atlantic Salmon ------CCGGGCAGCTTCCTGGCATCGGACGAT---CCATGTGTGTTGAGATCTCCCTC Rainbow trout ------CCGGGCAGCTTCCTGGCATCGGACGAT---CCATGTGTGTTGAGATCTCCCTC Zebrafish ------CTGGACAACTGCCTGGCATCGGGCGAT---CTATGTGTGTGGAGATCTCCCTC Cat ------CTGGGCAGCTGCCCAGTGTCGGGCGCT---CCATGTGTGTGGAGATTTCCCTG Clawed frog ------CAGGGCAGCTGCCTGCAGTGGGACGTT---CCATGTGTGTGGAGATTTCTCTG

176

Red junglefowl ------CAGGACAGCTACCCGCTGTTGGACGGT---CTATGTGTGTGGAGATTTCTCTT Brown rat ------CTGGGCAGCTGCCTGCTGTGGGGCGAT---CTATGTGTGTGGAGATCTCACTC Cattle ------CAGGGCAGCTGCCTGCTGTTGGGAGGT---CTATGTGTGTGGAGATCTCACTC Human ------CAGGACAGCTGCCTGCAGTCGGGAGGT---CCATGTGTGTGGAGATTTCACTT Rhesus macaque ------CAGGGCAGCTGCCTGCGGTCGGGAGGT---CTATGTGTGTGGAGATTTCACTT * * * *

Yeast CCCACAACAGAATTATACCAATTATTAATAAAGTCTTATAACGGCCCGCAAAATGAAGGA 480 Sorghum GCCGGTGGTGTGGGAGCCCGAGGGGAGGTGGTGGAGAGGTGGACTGTGGCGTGCGAGCCC Fruit fly AAAACGACTGAGGGTGACCAGATGGTGTTGGAGGTGTGGTCCCTAGACCTGCTCCAGCCC Rice Fish AAGACGTCGGAGGGCGACTCAATGGAATTAGAGACTTGGTGCCTGGAAATGAATGAAAAG Pufferfish AAAACATCAGAGGGAGACTCCATGGAGTTGGAGACGTGGTGCCTGGAAATGAATGAAAAG Atlantic Salmon AAAACCTCAGAGGGAGACTCAATGGAATTGGAGACATGGTGTCTCGAAATGAACGAGAAA Rainbow trout AAAACCTCAGAGGGAGACTCAATGGAATTGGAGACATGGTGTCTTGAAATGAACGAGAAG Zebrafish AAAACCTCAGAGGGAGACTCAATGGAGCTGGAAACATGGTGTTTGGAAATGAATGAGAAG Cat AAAACATCAGAGGGGGATTCAATGGAGCTGGAAACATGGTGCTTGGAGATGAACGAGAAG Clawed frog AAGACATCAGAGGGAGATTCCATGGAGCTTGAAGTCTGGTGTCTGGAAATGAATGAAAAG Red junglefowl AAAACCTCAGAGGGGGATTCCATGGAGCTAGAAATCTGGTGTCTAGAAATGAATGAGAAG Brown rat AAGACTTCTGAGGGAGATTCCATGGAGTTGGAAATTTGGTGTCTTGAAATGAATGAAAAG Cattle AAGACTTCTGAGGGAGATTCCATGGAGCTAGAAATCTGGTGTCTGGAAATGAATGAAAAG Human AAGACTTCTGAGGGAGATTCCATGGAGCTGGAAATATGGTGTCTTGAAATGAATGAAAAG Rhesus macaque AAGACTTCTGAGGGAGATTCCATGGAGCTGGAAATATGGTGTCTTGAAATGAACGAAAAG * * *

Yeast AGTTCCAATCCAAT------AACTTCCACGGGCCCACTAGTAAGTATCCGGACGTGT 540 Sorghum TGGCCGGACGCCGC------GGGCGGCG-AGGA------GGTTGC Fruit fly CAGAATGGTGCCTCCCCGGCCACCAATGACCTGAACCCCG-AAGGCCAGACCCTAAAGGC Rice Fish TGTGATAAAGACAT------AAAGGT Pufferfish TGTGATAAAGACAT------TAAAGT Atlantic Salmon TGTGACAAAGATAT------TAAGGT Rainbow trout TGTGACAAAGATAT------TAAGGT Zebrafish TGTGATAAGGACAT------TAAAGT Cat TGTGAAAAGGACAT------TAAAGT Clawed frog TGTGACCGAGACAT------TAAGGT Red junglefowl TGTGACAAAGAAAT------CAAAGT Brown rat TGTGACAAAGAAAT------CAAAGT Cattle TGTGATAAAGAAAT------CAAAGT Human TGTGATAAAGAAAT------CAAAGT Rhesus macaque TGTGATAAAGAAAT------CAAAGT

177

Yeast GTCCTTGACGGATCTAAACCAAT------TTTATCGAAGGGGAGAATAG 600 Sorghum CGTGAACCGGGCGTACAAGCACTGCTTCACGCTGCTCAGGTCCGTCTACGCCGCCCTCCG Fruit fly AGCCCACGCCATCTACAATCGCATGGGCATCATGCTCAAGTCCCTCATATCGCTGACCCG Rice Fish GTCGTACACGGTCTACAACCGTCTGTCTGTCCTTCTGAAGTCGCTCATGGCCATAACCAG Pufferfish GTCGTATACAGTCTACAATCGTCTGTCTGTCCTTCTGAAATCTCTGCTGGCTATCACAAG Atlantic Salmon GTCCTACACCGTCTATAACCGCCTGTCGTTACTGCTCAAGTCACTGCTGGCCATTACAAG Rainbow trout GTCCTACACTGTCTACAACCGCCTGTCATTACTGCTCAAGTCACTGCTGGCCATTACAAG Zebrafish GTCCTATACAGTTTATAATCGACTGTCTCTGCTGCTCAAGTCTTTACTGGCCATCACAAG Cat CTCCTACACGGTTTATACGCGTCTGTCCCTGCTTCTCAAATCTCTCCTGGCCATTACGAG Clawed frog CTCCTATACTGTATATAACAGATTGTCATTGCTGCTAAAGTCATTACTTGCTGTCACCAG Red junglefowl CTCATACACAGTATACAACAGACTGTCTCTACTACTGAAGTCTTTGCTTGCTATAACCAG Brown rat TTCCTACACTGTATACAACCGATTGTCACTGCTGCTGAAGTCTCTCCTTGCTATTACGAG Cattle TTCCTATGCTGTGTACAACAGATTGTCGTTGCTGCTGAAGTCTCTGCTTGCTATAACTAG Human TTCCTACACGGTGTACAACAGACTGTCATTGCTGCTGAAGTCCCTTCTTGCTATAACTAG Rhesus macaque TTCCTACACGGTGTACAACAGACTGTCATTGCTGCTGAAGTCTCTTCTTGCTATAACTAG * * * *

Yeast GGTTGAGCAAACCGATTATTAATACATATTCCAATGCGCTTAACGAATCAAACCTGCCAG 660 Sorghum CGTCCTCCCGGCGTACCGCATCTTCCGCCTCCTCTGCGCCAACC-----CCTCCTACAAC Fruit fly CACCACGCCCGCCTACAAATTATCCCGGCGCCAGTGCCCCGACT------CG Rice Fish GGTGACGCCTGCCTACAAACTGTCCAGGAAGCAGGGACACG------AC Pufferfish AGTAACACCTGCGTACAAACTGTCCAGAAAGCAGGGACATG------AC Atlantic Salmon GGTAACCCCAGCCTACAAACTCTCACGAAAGCAAGGCCATG------AC Rainbow trout GGTAACCCCAGCCTACAAACTCTCACGAAAGCAAGGCCATG------AC Zebrafish GGTGACTCCTGCTTACAAGCTTTCACGTCAACAAGGCCATG------AT Cat AGTTACTCCTGCCTACAAACTGTCACGCAAACAAGGCCACG------AT Clawed frog AGTGACCCCAGCTTACAGACTCTCCAGGAAGCAAGGACATG------AA Red junglefowl GGTAACTCCAGCCTACAGACTCTCAAGAAAGCAAGGCCATG------AA Brown rat GGTGACACCAGCCTATAGACTCTCCAGGAAGCAAGGGCATG------AA Cattle GGTGACACCAGCTTACAGACTCTCCAGAAAGCAGGGGCATG------AA Human GGTGACACCAGCCTATAGGCTCTCCAGGAAACAAGGGCATG------AA Rhesus macaque GGTGACACCAGCCTATAGGCTCTCCAGAAAACAAGGGCATG------AA * * * * * *

Yeast CCCATTTAGATCAAAAGAAGATCACACCTGTATGGACAAAGTTTGGACTCTTAAGAGTCT 720 Sorghum TACGAGATGGGCCATCGCGTTGACACCTTCGCTGAGCCCTTCTCGCGCCCCCAGGAGGCC Fruit fly TATGGCATCTTCTACAGGATCTACG--TGGACAGGCCGCAGGTCCACACCCTG-GGCGAG

178

Rice Fish TACGTCATATTATACAGGATCTACT--TTGGGGAGGTCCAGCTGAGTGGATTA-GGAGAA Pufferfish TATGTCATACTCTATAGGATTTATT--TTGGGGAAGTGCAGCTCGGTGGATTA-GGTGAA Atlantic Salmon TATGTCATCCTGTACAGGATATATT--TTGGAGAGGTTCAACTCACAGGATTG-GGAGAA Rainbow trout TATGTCATACTGTACAGGATATATT--TTGGAGAGGTTCAACTCACAGGATTG-GGAGAA Zebrafish TATGTAATACTCTACAGGATCTATT--TTGGAGATGTGCAACTGACAGGTCTT-GGGGAA Cat TATGTCATCCTGTATAGGATATATT--TTGGTGATGTACAGTTAACAGGACTT-GGTGAA Clawed frog TATGTCATTCTATACAGGATTTACT--TTGGAGACGTTCAGCTCCTCGGACTT-AAGGAA Red junglefowl TACGTGATATTGTACAGGATATATT--TTGGTGAAGTACAACTGAGCGGCTTG-GGAGAA Brown rat TATGTAATTTTGTACAGGATTTATT--TTGGAGAAGTTCAACTGAATGGCTTA-GGAGAA Cattle TATGTCATCTTATACAGAATATATT--TTGGGGACGTTCAGCTGAATGGCTTA-GGGGAA Human TATGTCATATTATACAGGATATATT--TTGGAGAAGTTCAGCTGAGTGGCTTA-GGAGAA Rhesus macaque TATGTCATATTATACAGGATATATT--TTGGGGAAGTTCAGCTGAATGGCTTA-GGAGAA * * *

Yeast CGGTATCATACAGACGTGATTGGAAGTTTGAAATTAACAATACAAACG-ACGAATTATTT 780 Sorghum GCCATGCGTTCCCAACGCTTTGTTCCCGTCGAAACCCAGCTCGGTCGCCTCGTCGTTTCC Fruit fly GGTCACAAGCACGTAAAGATCGGACAACTCAGCACGATTGTTGGCTCGCTGGTCATGTCC Rice Fish GGTTTCCAGACGGTGCGCGTCGGGGTGGTGGGCACCCCCGTGGGGACGGTCACCCTCTCC Pufferfish GGTTTCCAGACAGTGCGTGTTGGTGTTGTTGGTACCCCAGTTGGCACGGTCACGCTGTCA Atlantic Salmon GGGTTCCAGACAGTGCGTGTAGGAGTTGTGGGAACTCCCATTGGAACAATCACCTTGTCT Rainbow trout GGGTTCCAGACAGTGCGTGTAGGAGTTGTGGGAACTCCCATTGGAACAATCACCTTGTCT Zebrafish GGCTTTCAGACAGTGCGAGTGGGTATTGTTGGCACTCCCATTGGCACCATCACTTTGTCT Cat GGTTTCCAGACAGTACGTGTAGGAGTTGTAGGTACTCCCATAGGAACCCTCACACTCTCC Clawed frog GGTTTCCAGGCTGTACGGGTTGGAACTGTTGGAACACCAGTCGGAACACTCACACTTACA Red junglefowl GGTTTTCAAACAGTTCGTGTTGGGACAGTGGGTACCCCAGTGGGCACCATCACTTTGTCT Brown rat GGCTTCCAGACAGTTCGTGTTGGAACAGTGGGCACCCCCGTGGGTACCCTCACTCTTTCC Cattle GGTTTCCAGACAGTTCGTGTTGGGACAGTGGGTACCCCTGTGGGCACCATCACTCTTTCC Human GGCTTCCAGACAGTTCGTGTTGGGACAGTGGGCACCCCTGTGGGCACCATCACTCTTTCT Rhesus macaque GGCTTCCAGACAGTTCGTGTTGGGACAGTGGGCACCCCTGTGGGCACCATCACTCTTTCT * * * * *

Yeast T--CAGCTCGACATGCATCTGTCTCACATA------ACTCACAAGGACCCCAG-- 840 Sorghum GTCCAGTACCTCCCCAGCCTTGCCGCCTT-----CAA----CCTCGAGATCACCTCCCTT Fruit fly GTGGCCTACCGCACAAAGCTGACCATTTCGCCCACGGCTGCCCAGTCAGAGAGCAA---- Rice Fish TGCGCTTACCGCACCAACCTGGCCTTCATGTCCAGCAGGCAGTTTGACCGGTCGGCCCC- Pufferfish TGTGCTTACCGCACCAACCTTGCATTCATGTCCAACAGGCAGTTTGAGCGTTTAGCCCC- Atlantic Salmon TGTGCTTACAGGACAAACCTGGCCTTCATGTCCACCAGGCAGTTTGAGAGGACGGCTCC- Rainbow trout TGTGCTTACAGGACAAACCTGGCCTTCATGTCCACCAGGCAGTTTGAGAGGACGGCTCC- Zebrafish TGTGCATATCGCACTAATTTGGCTCTTGTGTCTTCAAGACAATTTGAGAGAACAGGCCC-

179

Cat TGTGCATACCGCACCAATCTGGCTCTCATGTCCTCCAGGCAGTTTGAGAGGACGGGCCC- Clawed frog TGTGCCTACAGAACCAACCTGGCTTTTATGTCAACCAGGCAGTATGAGTGGACCCAGCC- Red junglefowl TGTGCCTACAGAATCAACCTTGCTTTCATGTCAACCAGAGCTGGTGAAGACAA------Brown rat TGTGCTTATAGAATTAACTTGGCGTTCATGTCCACCAGGCAATTTGAGAGGACCCCACC- Cattle TGTGCTTACAGGATTAACTTGGCATTCATGTCCACCAGGCACTTTGAGAGGACCCCACC- Human TGTGCTTACAGAATTAACTTGGCATTCATGTCTACCAGGCAATTTGAGAGGACCCCACC- Rhesus macaque TGTGCTTACAGAATTAACTTGGCATTCATGTCTACCAGGCAATTTGAGAGGACCCCACC- * *

Yeast --AATCAGCCAGAACAAGAAGGACAAAGT---GATCAAGACATAGGGAAACGCCAACCAC 900 Sorghum TCGCCCTCCGTGATAATCCCAGACTAC------Fruit fly --CACCATAATGCTGAAGTCGGATCACTTTAGACCCGCCACGGATGC------Rice Fish --CATCCTGGGGATCATCGTGGACCACTTTGTGGACCCCCCCTGCAGCAGCCAGCGCCCC Pufferfish --CATCATGGGAATCATTGTAGATCACTTTGTGGACCCCCCCTGTAGCAACCAGCGTCCA Atlantic Salmon --GATTATGGGCATAATTATTGACCACTTCGTTGAGCGCCCCTTTGGCAACATAGGACAC Rainbow trout --GATTATGGGCATAATTATTGACCACTTCGTTGAGCGCCCCTTTGGCAACATGGGACAC Zebrafish --TATCATGGGTATTATCATTGATCATTTTGTGGAGCGTCCCTTCACTAATATGGCACAC Cat --AATCATGGGGATCATCATAGACCACTTCGTGGAGCGTCCACTTGGGAACACGGCCCTC Clawed frog --CATCATGGGGATTATAATTGATCACTTTGTTGACCGACCCTTCCCCAGCACCTCACAC Red junglefowl ------Brown rat --TATCATGGGGATTATCATTGATCACTTTGTTGACCGTCCCTATCCCAGCTCCTCTCCT Cattle --TATCATGGGCATTATTATTGATCACTTTGTGGACCGTCCCTATCCCAGCTCCTCGCCC Human --TATCATGGGGATTATTATTGATCACTTTGTGGACCGTCCCTATCCCAGCTCCTCTCCC Rhesus macaque --TATCATGGGGATTATTATTGATCACTTTGTGGACCGTCCCTATCCCAGCTCCTCTCCC

Yeast AATTTCAACAGCAGCAGCAGCCCCAACAGCAGCAGCAGCAGCAGCAACAGCAACAGAGAC 960 Sorghum ------ATCGGCAGCC-CAGCCGCTGAGCCCATGCGAGC Fruit fly ------CAATACACCTGGCAAC-CAGCAGCAGA------CTCAAA Rice Fish GCAGTC------CTGGGACAGCCCTGCAACTACAGAG-CCCCCGACGAGGATGACGGGGG Pufferfish GTAAAC------TTTGGACAGCCCTGCAACTACAAAG-CTCCAGAGGA---GGACAGAGG Atlantic Salmon ATG------CGTCCATGTAACTACAGAG-CACCCGGGGAAGATGAAGGA-- Rainbow trout ATG------CGTCCATGTAACTACAGAG-CACCCGGGGAAGATGAAGGA-- Zebrafish ACA------CATCCCTGCAGCTACAGAG-CACCTGGGGAAGATGATGGAGG Cat ATG------CACCCCTGCAACTACAGAG-CTCCAGGTGAGGAAGAGGGAGG Clawed frog ATG------CATCCCTGCAACTACAGGG-GCGCTGAAGAGCA---AGTGCT Red junglefowl ------TGGTGC Brown rat ATG------CACCCTTGCAATTACAGAA-CTGCTGAGGA---TGCTGGAGT Cattle ATG------CACCCCTGCAATTACAGAA-CCGCCGGCGAGGACACTGGAGT

180

Human ATG------CACCCCTGCAATTACAGAA-CTGCTGGTGAGGACACTGGAGT Rhesus macaque ATG------CACCCCTGCAATTACAGAA-CTGCTGGTGAGGACACTGGAGT

Yeast AACACCAGGTCCAGACACAACAACAAAGACAGATACCTGATAGGAGATCTCTTTCACTTT 1020 Sorghum CTTCCCGGCCTCCCTTACA-GAAGCCACGGGCTCCGGCTTCCCGCCGTC-CTATCAGCAG Fruit fly ATGGCACTGTCGTGGC----CAAGAAACTGGGCTTGGGTGCCCTAAATCCCGCCCAGGGT Rice Fish TGCCTTCGCAGGAGTC----CAGGACTCCCAGGAGGTCTGCACCACCTCCTTCTCCACCT Pufferfish AGCATTTGCAGGGGTT----CAGGATTCACAGGAGGTCTGCACCACCTCCTTCTCAACCT Atlantic Salmon -GCATACAATGGGGTG----GAGGATTCTCAGGAAGTGTGCACCACGTCCTTCTCCACTT Rainbow trout -GCATACAATGGGGTG----GAAGATTCTCAGGAAGTGTGCACCACGTCCTTCTCCACTT Zebrafish GACTTACGCAGGGATA----GAAGACTCTCAGGAGGTCTGCACCACATCCTTTTCCACCT Cat GGTTTACGCTGGAGTG----GAGGATTCACAGGAAGTGTGCACCACCTCCTTTTCCACTT Clawed frog TACCTACCCTGCAGTG----GAGGAGTCTCAAGAAGTCTGCACAACATCGTTTTCCACTT Red junglefowl AGTATACCCCTCAGTA----GAAGATTCCCAAGAAGTGTGTACTACATCATTCTCCACCT Brown rat CGCATACCCATCAGTG----GAAGACTCTCAGGAAGTGTGTACCACATCTTTTTCCACAT Cattle AGCGTGTCCTTCTGTG----GAAGACTCTCAGGAAGTGTGCGCCACCTCCTTTTCCACCT Human AATATACCCGTCTGTA----GAAGACTCTCAAGAAGTGTGTACCACCTCTTTTTCCACCT Rhesus macaque AATATACCCATCTGTG----GAAGACTCTCAAGAAGTGTGTACCACCTCTTTTTCCACCT * ** *

Yeast CTCCTTGTACAAGAGCC-AATTCTTTTG------AACCAC------AATC 1080 Sorghum CAGCGCCCACACAGCTGGGCTTCGCCTGCTTTCTGGCCGCACACGC------CGGC Fruit fly ACAGCCGATCGGCGGTTTATCGATATAGAGAAGCCGTTGCGACCGGGAGCCTTCACAGAT Rice Fish CCCCGCCCTCCCAGATGTGCTCCTCCAGGTTATCGTAC-CAGCCTGGAGCTCTGCCCGGC Pufferfish CTCCTCCATCTCAGCTGTACTCATCCAGATTATTATAC-CAGCCTTCTGCCGTGGCAGGA Atlantic Salmon CTCCACCCTCACAATGTGTGTTCACTGTAAGTAAGGCA-CATTTAAAGACCCCTAAACCT Rainbow trout CTCCACCCTCACAATGTGTGTTCACTGTAAGTAAGGCA-CATTTAAAGACCCCTAAACCT Zebrafish CCCCACCATCACAGCTGTACAGCTCGCGTCTG------Cat CTCCACCTTCTCAGCTTTGCACTTCTCGTCTG------Clawed frog CCCCTCCTTCACAGCTCTGCAGCTCTCGCCTCTCCTAT-CAACCAGCTGTGCCAGGAGCA Red junglefowl CTCCTCCATCTCAGTTGATCGGTCCA------Brown rat CCCCTCCC------Cattle CGCCTCCCT------Human CCCCACCATCCCAGCTCTCAAGCTCTCGCCTTTCCTAT-CAGCCTGCTGCCCTGGGCGTT Rhesus macaque CCCCACCATCCC------

Yeast TTGGCAGAAGAAAGTCTATCC------AAT------ATCGAGAC 1140

181

Sorghum ACACCAGGCCAGGTTCTCGCCGCCTCC----AGTGTTTTACGCG-TCACCG-ACGCCGTC Fruit fly ATGGGC----AAACTAAAGCAGTACACTGAAGATGACTTT-GTGTTGCCGGAAACCCCGC Rice Fish GCCGCCGAGGTCCATCCCACC------Pufferfish ACAGCAGACCTTTGTCAGCCT------Atlantic Salmon GCAGTGATGGACACTCTGAAAGTCCCCATGATGGGCCTGGCCTTCTCACAGCAACTCTAC Rainbow trout GCAGTGATGGACACTCTGAAAGTCCCCATGATGGGCCTGGCCTTCTCACACCAACTCTAC Zebrafish ------Cat ------Clawed frog GGACCC------TCCGAAC------TGGGTT------Red junglefowl ------Brown rat ------Cattle ------Human GGATCA------GCTGACC------TGGCTT------Rhesus macaque ------

Yeast CTGTTCAACCATTTAAAGTTGGTTCAATTGGAAGT------CAAAGTGCGAGCAGAAAT 1200 Sorghum GCCTCCCCACTTTCCACCCCGTCTCATGAGGTGGG----AGTCTGCAC------CAAT Fruit fly CT------TTCGAGTGGCTTTTGCGAGGACGCGGCTCTGTGGAGTCCCTCAATCGA Rice Fish ------Pufferfish ------Atlantic Salmon TGCTCTCGTCTTTCTTATCAGCCTCCTGTTCTTGTTGGAGCTGCTGACTTCTGCCACCCC Rainbow trout TGCTCTCGTCTTTCTTATCAGCCTCCTGTTCTTGTCGGAGCTGCTGACTTCTGCCACCCT Zebrafish ------TCATATCAGACTCCTCCTCTGGG---AACTGTGGATTTGTGCCATCCT Cat ------TCATATCAGACTCCTCTCCTCGG---GCCAGTAGATTTGTGCCATCCT Clawed frog ------ACCCAGTGGCCTTGGCTACTGGA------GTGAGC------Red junglefowl ------Brown rat ------Cattle ------Human ------ATCCAGTAGTGTTTGCTGCTGGC------TTAAAT------Rhesus macaque ------

Yeast CCCTCTAATTCATCGTTTTTCAACCAACCACCTGTTCATAGGCCAAGTATGAGCTCCAAC 1260 Sorghum GCC------CATACCACAGGTGAGCG Fruit fly CTGGATAACAATTCGGTCGCCAG-TGTAAAC------ATCAGTAATAACAACAATAG Rice Fish ------GCGGCCAA-CGCCGCC------CAGGTGCTGCATGCTGG Pufferfish ------GCTGCCAA-TCCAAAC------CCGATGTTGCATGCTGG Atlantic Salmon TCCACCTTAAATG---CTGCCAA-CCCACACCAGGTAACCACCCAGATGGGAATTCCAGG

182

Rainbow trout TCCACCTTAAACG---CTGCCAA-CCCACACCAGGTAACCACCCAGATGGGAATCCCAGG Zebrafish ACAGCTTGTACTGGTGCTGCACA-TCCTCAC------CAGATGGTTGTTCCGGG Cat ACTAACTGCACTGCTGCTGCCCA-CCCACAC------CAGATTGCTGTTCCGGG Clawed frog ------GCAGGCCA-TCCGCAC------CAGATGATGGCACCAGG Red junglefowl ------GG Brown rat ------TCC------CAGCTGATGGTTCCTGG Cattle ------CC------CAGCTAATGGTCCCTGG Human ------GCTACACA-CCCTCAC------CAGCTGATGGTTCCTGG Rhesus macaque ------AGCTGATGGTTCCTGG

Yeast TACGGGCCACAAATGAATATTGAAGGTACCAGTGTTGGAAGCACCTCAAAGTATTCCTCC 1320 Sorghum AGAGGAGGAGCCCCGCACACCGCCAGAATAT--GCTGCCACCTC------CTTCGCCGA Fruit fly TACGCAGGATAGTAAATTCA------ACCAGATCAGCAATCTC------AACAACA Rice Fish GAAAGA---AGGCGGAGTCGGAGTGGTTCCCTGTCAGCCTCACGGGGCGGAGACCCACCT Pufferfish GAAGGA---GGGTGGAGTGGTGCTGGTTCCGGCCCAGCCTTCTCATGGAGCCGATGCGCA Atlantic Salmon GAAGGAGGGGGGCGTACCCCAGCAGGTGCCTGTGCAGCCTTGTCATGGTGCCCAGGCAGA Rainbow trout GAAGGAGGGGGGCGTACCCCAGCAGGTGCCTGTGCAGCCTTGTCATGGTGCCCAGGCAGA Zebrafish TAAAGA---TGGAGGAATCCCACAGGTGCCAGCTCAGCCTAATCACGGCACTGGCGCTGA Cat GAAAGA---AGGTGGAGTCCCGCAGGTGCTGGTTCAGCCCAGTCATGGCACTCAAGCAGA Clawed frog AAAAGA---AAGCGGTTTGCCTGCTGTTAGTGTTCAGCCTGCCCACGGAAACCAGGCAGA Red junglefowl CAAAGA---GGGGGGAGTTCCCCCAGTTCCTAGCCAGCCAGCACATGGCACTCAAGCTGA Brown rat GAAGGA---AGGTGGGGTGCCTCTTGCTCCCAACCACCCTGCTCATGGTGCCCAGGCTGA Cattle GAAGGA---AGGTGGGGTACCCCTTGGTCCCAGCCAGCCTGCCCATGCTGCCCAGGCTGA Human GAAGGA---AGGTGGGGTACCCCTTGCTCCCAACCAGCCTGTCCATGGTACCCAGGCTGA Rhesus macaque GAAGGA---AGGTGGGGTACCCCTTGCTTCCAACCAGCCTGTCCATGGTGCCCAGGCTGA

Yeast T------CCTTTGGGAA---CATTCGTCGTCACTCAAGTGTAAA 1380 Sorghum GAAGGGGAGACATGGGGGCAGCAGGTGCTCTAGAGTC--TCCATCTGAGAGTGGCCGGTT Fruit fly AC------TCGGC------AGGCTTCAAGAGTTTC Rice Fish GACTGGGGAGCCCCCCCTCAACACCCCCGGCAG---CAGTGGTGATGATGAGGGTCTGTC Pufferfish TT---TTGAGAATGTAACAAACACACCTGGAAG---CAGTGGTGAGGATGACGGCCTCTT Atlantic Salmon GCATGGACGAGTGTCCTCATGCCCCCCTGCTGA------TCATGTTCTCGCCACGCCCTC Rainbow trout GCATGGACGAGTGTCCTCATGCCCCCCCGCTGA------TCATGTTCCCGCCACTCCCTC Zebrafish GCAAGGTCGTATCCCATCGTGCCCCACTGGACA------GCCCCCTCAGTTGCCTC---- Cat CCATGGGCGTGTCCCGTCCTGCCCCTCAGCAGA------CACACCCCATCCTCCGCCCTC Clawed frog GCATGACCGGACCCTGTCTGTGTCTCC------CGCCAATGTAGCAGGGACAACATC Red junglefowl CCAAGAGAGGATGTGCACCC---CACTAGATGGAGTCCACTACTCAGCAGCTACTCCTTC

183

Brown rat TCAGGAGAGACTGGTGGTGCACATGCCTTCTGATGGAACCCACTGTGCAGCCACACCCTC Cattle CCAGGAGAGACTGGCAACCTACACCCCTTCTGATGGGGCCCACTGCGCCGCCACGCCGTC Human CCAGGAGAGACTGGCAACCTGCACCCCTTCTGACAGAACCCACTGTGCTGCCACACCCTC Rhesus macaque CCAGGAGAGACTGGCAACCTGCACCCCTTCTGACGGAACCCACTGTGCTGCCACACCCTC

Yeast GACGACAGAGAATGCTGAAAAAGTATCAA----AAGCTGTAAAGAGCCCACTA---CAAC 1440 Sorghum GATTGG----GAGGATGGAGGAACTTCGGATAGCTGATCCA--TATGCAACCTCGTCACC Fruit fly GAGAAGAAC-TCGGAAAACTCAGTATCGC------CAATA-AAGAGCCTGCTGATCCCC Rice Fish GCGGAGCGCCG------AAGAGCGAAGCGTCTCCCCGTCTGA Pufferfish GCAAAGTGGGGAAGGCAAGAGGGAAGAAG---GGAGGTGCCGGAGTTCCCCCACCTCTGA Atlantic Salmon CAGCAGTGGTGAGGATGCAGAGACCTCGTCGAGGAGCATCGAGGTGAAGAGTGTCTCTCC Rainbow trout CAGCAGTGGCGAGGGTGCAGAGACCTTGTCGAGGAGCATTGAGGTGAAGGGTGTCTCTCC Zebrafish ------CACCCACATCTTGTAGCAGTGAGGTGAAGACTGTGTCCCC Cat CAACTGCAGCAGTGGTGATACAGATGTGTTGTTGAGAAGTGAAGGGAAGAGTGTGTCCCC Clawed frog CAGCAGCGAGGAAGCAGACTCCCCCTCAGGTAGCAGCACCACTGGGGCAAAAAGCTCGCC Red junglefowl CAGTAGCGAGGACACGGAAACAGTATCAA---ACAGCAGTGAAGGGAAATGTGGCTCCCC Brown rat CAGCAGTGAGGACACTGAGACTGTATCTA---ACAGCAGTG---AAGGACGGGCCTCACC Cattle CAGCAGTGAGGATGCTGAAACTGTATCAA---ACAGCAGCG---AGGGACGGGCCTCCCC Human CAGTAGTGAGGATACTGAAACCGTATCAA---ACAGCAGTG---AGGGACGGGCCTCCCC Rhesus macaque CAGTAGTGAGGATACTGAAACTGTATCAA---ATAGCAGCG---AGGGACGGGCCTCCCC *

Yeast C—TCAAGAATCACAAGAAGATTTAATGGATTTTGTTAAATTACTCGAAGAAAAACCCGA 1500 Sorghum C--AGACATAAGGGCAAGGACAATAAAGATGAATCTGGCAGATTCTCTGCACTCTCCTCA Fruit fly GCCAGCGCCA-----CAGCCACATATCGTCACCATTCAGAGCCTTCGCTACAACCACC-- Rice Fish CCCGGTGGAGTACCTCAACGCTTTCACCCGGAAAGTCGGCGCCTTCGTGAACAAACCCA- Pufferfish CCCTGTCCAGGCAATAAACGCCTTCACGAGAAAGACGGGGGCCTTTGTAAATAAACCCA- Atlantic Salmon CTGTGATGTACTGGAGACCACCTTCACCAGGAAAGTGGGTGCCTTTGTCAACAAGCCAG- Rainbow trout CTGTGATGTACTGGAGACCACCTTCACCAGGAAAGTGGGTGCCTTTGTCAACAAGCCAG- Zebrafish CTCTGATGTTCTAGAGACAACCTTCACCAGGAAAGTTGGAGCGTTTGTCAACAAAGCCA- Cat TTCAGATGCCCCCGAGACAACCTTCACCAGGAAAGTGGGAGCCTTCGTCAACAAACCCA- Clawed frog CAACAAGAACACCCATGTACTATTTACAAGGAAAGTGGGAGCTTTTGTTGATAAACCTT- Red junglefowl GCATGACCTTTTGGAGACTATCTTTATCCGGAAGGTGGGAGCTTTTGTCAACAAACCTA- Brown rat CCATGACATCCTGGAAACTATCTTTGTCCGAAAAGTGGGAGCATTTGTCAACAAACCCA- Cattle CCACGATGTCTTGGAGACCATCTTTGTCCGGAAAGTGGGGGCTTTTGTCAACAAGCCCA- Human TCACGATGTCTTGGAGACCATCTTTGTCCGAAAAGTGGGGGCTTTTGTCAACAAACCCA- Rhesus macaque CCATGATGTCTTGGAGACCATCTTTGTCCGAAAAGTGGGGGCTTTTGTCAACAAACCCA- * *

184

Yeast TCTAACTATAAAGAAGACAAGTGGAAATAATCCACCCAATATCAATATTTCTGATTCTCT 1560 Sorghum TGTGATTCACCACG------GCAAGATGATCTGGATGATGTGGATTACCATTTTGACGT Fruit fly -----GCCTGATGA------TGATAATCTGCTAAAAGAGCTCCACTTCCCTTTCGCCTC Rice Fish -----GCACGCAGA------TAACGG-----CCGCCAACCTGGACCTGCCGTTCGCCGC Pufferfish -----GCACACAAA------CACTGA-----CAGCCAATCTGGACCTGCCCTTCTCTGA Atlantic Salmon -----GCACACAGG------TAACCA-----CAGCAAGCCTGGACTTACCATTTGCAGC Rainbow trout -----GCACACAGG------TAACCA-----CAGCAAGCCTGGACTTACCATTTGCAGC Zebrafish -----CCACTCAGG------TAACAA-----CAACAGGCCTGGATCTTCCTTTTGCTGC Cat -----CCACACAGG------TGACCA-----CTGCCAGTCTGGATCTGCCGTTTGCTGC Clawed frog -----CCCCCCAGG------TCACCT-----ATGCCAGTACTGATATACCGTTTGCCGT Red junglefowl -----TTAACCAGG------TGACCA-----TGGCCAACTTAGACATTCCTTTTGCTAT Brown rat -----TCAATCAGG------TGACAC-----TGACGAGTTTGGACATACCCTTTGCCAT Cattle -----TCAACCAGG------TGACCC-----TGACCAGTTTGGACATACCCTTTGCCAT Human -----TTAACCAGG------TGACCC-----TGACGAGTTTGGATATACCCTTTGCCAT Rhesus macaque -----TTAATCAGG------TGACCC-----TGACGAGTTTGGATATACCCTTTGCCAT *

Yeast AATCAGATATCAGAATTTGAAGCCAAGTAATGACTTATTAAGTGAAGATTTATCCGTAAG 1620 Sorghum T------GATGATGTTGACACTCCA------GTCTCCCAGC--- Fruit fly G------C-CCACGTCGCACGTAAA------CGATCTGGCCAA- Rice Fish T------T------TCGCCGCTCGG------GGCCTGGACT--- Pufferfish G------T------TTGCTCCTCGA------GGCCTGGACT--- Atlantic Salmon G------T------TTGCTCCCAGA------GCCTATGACC--- Rainbow trout G------T------TTGCTCCCAGA------GCCTATGACC--- Zebrafish A------T------TTGCTCCAAGA------TCTTACGACA--- Cat A------T------TTGCTCCCAGA------GGTTATGACC--- Clawed frog G------T------TTGTGCCAAAG------CTTGCAGACA--- Red junglefowl G------T------TTGCTCCCAAG------AATGTTGAGC--- Brown rat G------T------TTGCTCCCAAG------AATTTGGAGC--- Cattle G------T------TTGCTCCCAAG------AACCTGGAGC--- Human G------T------TTGCTCCCAAG------AATTTGGAGC--- Rhesus macaque G------T------TTGCTCCCAAG------AATTTGGAGC--- * *

Yeast TTTATCCATGGATCCAAATCATACATATCACAGAGGCAGATCAGATTC----CCACTCAC 1680 Sorghum ------CTCGGAGCGCTGATG---GGAA-GGAAACAGGTGA------TCAGGTTG Fruit fly GTTCTACAGGGAATGCTATCATGCGCCACC-GCTGAAGGGACTTAATGAACTGCAGGCAG Rice Fish ------CGGA-GGGGAGCGATCCCATGGTTCAGCCCCCGG

185

Pufferfish ------CAGA-GGAGAATGATCCTATGGTGCAGCCGCCAG Atlantic Salmon ------TCGA-GGAAAATGACCCTATGGTTCAGCCGCCGG Rainbow trout ------TCGA-GGAAAATGACCCTATGGTTCAGCCGCCGG Zebrafish ------TGGA-GGAAAATGACCCAATGGTGCATCCTCCTC Cat ------TGGA-GGAGAACGACCCGATGGTACAGCCGCCAG Clawed frog ------ATGA-GGACACTGACCCTATGGTTGTCTACCCTG Red junglefowl ------TGGA-AGATAACGACCCCATGGTCAATCCTCCTG Brown rat ------TGGA-GGATGCTGATCCTATGGTGAACCCTCCAG Cattle ------TGGA-GGATGCAGATCCCATGGTGAATCCCCCAG Human ------TGGA-GGATACCGATCCAATGGTGAATCCTCCAG Rhesus macaque ------TGGA-GGATACCGATCCGATGGTGAATCCTCCAG *

Yeast CATTGCCTTCAATATCCCCTTCG-ATGCATTATGGATCGTTGAACTCGAGAATGTCTCAA 1740 Sorghum GTTCATCATCCCATAAATCTCAAGACGCACAAGTCGGTTCTCTAGTCAACCTGCTGA--- Fruit fly AGATCTCATCGATTTCCTCGACC------CCGCCAGCATCCAGTG-----GTTCCGGC Rice Fish AGTCCCCCCCCAGCCAGTCCCCC----CTGCAGGCCAGCCTTCACTCTCAGGGCTCA--- Pufferfish ACTCTCCACTTTGCACATCCCCC----CTCCAGCGCAGCCTTCATTCTCAGGGCTCA--- Atlantic Salmon AGTCCCCAGCCACGTCCTCTCCC----CTGCAGGGCAGTTTACACTCCCAGGGCTCTGGT Rainbow trout AGTCCCCAGCCACGTCCTCTCCC----CTGCAGGGCAGTTTACACTCCCAGGGCTCTGGT Zebrafish CATCTCCAATTCCCTCTTCTCCA----CTACAAGGGAGCCTTCACTCAAACAACTCAAGC Cat CATCACCCACCCCCTCCTCTCCA----TTACAAGACAGTCTTCACTCACAGAGCTCCAGC Clawed frog AGACCCCCGAAGAGGAGTCGCCT----GAAGCCAGGAGCCTGCACAGTT------CC Red junglefowl ATTCCCCAGAAACTGAATCTCCT----TTACAAGGCAGCTTACACTCAGAGGGCTCCAGT Brown rat ACTCCCCAGAGACCACGTCTCCC----CTCCATGGCAGTCTGCACTCAGAGGGCTCCAGC Cattle ATTCCCCAGAGACTACATCTCCT----CTCCAGGGCAGCCTGCACTCTGATGGCTCCAGC Human ATTCCCCAGAGACTGAATCTCCT----CTCCAGGGCAGCCTGCACTCAGATGGCTCCAGC Rhesus macaque ATTCCCCACAGACTGAATCTCCT----CTCCAGGGCAGCCTGCACTCAGATGGCTCCAGC * * * *

Yeast GGCGCCAATGCAAGCCATT------TGATTGC-AAGAGGCGGTGGGAATTCATCT---- 1800 Sorghum ------GAAA---CGCCTGTCCGCTGCGAAATCCTAGCAATTCATCA---- Fruit fly GGTGTAGCAGCATGCGGTCCGACGGCGGCGGCCACAGCGATCGCAACCAGTTCCGCTGAT Rice Fish ------GAGGGATCCGGGC---CGCAGGACGACTTCGTCATGGTGGACTTTCGTCCGGCC Pufferfish ------GAGGGCTCTGGAC---CACACGATGACTTTGTCATGGTGGACTTTCGACCAGCT Atlantic Salmon ---GAGAGTGGTGGCCCGG---CACAGGACGACTTTGTTATGGTGGACTTTAAGCCTGCG Rainbow trout ---GAGAGTGGTGGCCCGG---CACAGGACGACTTTGTTATGGTGGACTTTAAGCCTGCG Zebrafish ---CACAGTGGGGGGCAGC---CAAATGATGACTTTGTCATGGTTGACTTTAAACCAGCA Cat ---CAGAGTGGAGGCCAAG---CACAAGACGATTTTGTCATGGTTGATTTTAAACCAGCG

186

Clawed frog TGTTCCAGCAGTGGGACAC---CCCAAGATGATTTTGTGATGGTCGACTTTAAACCTGCC Red junglefowl GGCAGCAGCGCAGGGAACA---CCCACGATGACTTTGTTATGATTGATTTTAAACCAGCA Brown rat GGGGGCAGCAGTGGCAATG---CACATGATGACTTTGTCATGATTGACTTTAAACCAGCT Cattle GGGGGCAGCAGCGGCCATA---CCCAGGACGACTTCGTCATGATCGACTTTAAACCAGCT Human GGGGGCAGCAGTGGCAATA---CCCATGATGACTTTGTTATGATAGACTTTAAACCAGCT Rhesus macaque GGGGGCAGCAGTGGCAATA---CCCATGATGACTTTGTTATGATAGACTTTAAACCAGCT * * *

Yeast ------ACT----AGTGCCTTGAATAGTAGAAGGAATTCTTTAGATAAGAGCTCAAACAA 1860 Sorghum -----CAAACATCAAGGGCTGAGTCTAGCGAAGTAGCCTCAGCAAGCTCAGTTACGTCCC Fruit fly GCCAGCGCCATGGACGATCTGTCCCGGCAACTGGAGCAGTTCGAGACCTCGCTGGAGGAC Rice Fish TTCTCCAAGGACGACCTCCTGCCGATGGACCTGGGCACGTTCTACAGAGAGTTCCAGAAC Pufferfish TTTTCAAAAGATGATCTGTTGCCAATGGATCTCGGTACTTTCTACAGAGAGTTTCAGAAT Atlantic Salmon TTCTCCAAGGATGACCTCCTTCCCATGGACCTTGGCACATTCTACAGAGAGTTCCAGAAC Rainbow trout TTCTCTAAGGATGACCTGCTTCCCATGGACCTTGGCACATTCTACAGAGAGTTCCAGAAC Zebrafish TTCTCTAAGGATGACCTTTTGCCAATGGATCTGGGCACATTCTACAGAGAGTTTCAAAAC Cat TTCTCTAAGGACGACCTCCTCCCGATGGACCTGGGCACTTTTTACCGAGAGTTTCAGAAT Clawed frog TTCTCCAAAG---ACGACCTTCCCATGGATGTGGGGACCTTCTACCGGGAGTTCCAGAAC Red junglefowl TTTTCAAAAGATGACATTCTTCCAATGGACCTGGGGACATTTTACCGGGAGTTTCAGAAC Brown rat TTTTCTAAAGACGACATTCTTCCAATGGACTTGGGGACCTTTTACCGTGAATTTCAGAAT Cattle TTTTCTAAAGACGACATCCTTCCGATGGACTTGGGGACCTTCTATCGTGAATTTCAGAAC Human TTTTCTAAAGATGACATTCTTCCGATGGACCTGGGGACCTTCTATCGGGAGTTTCAGAAC Rhesus macaque TTTTCTAAAGATGACATTCTTCCGATGGACCTGGGGACCTTCTATCGGGAGTTTCAGAAC * * *

Yeast GCAGGGTAT—GTCAGGCTTACCTCCTATTTTTGGTGGAGAGAGTACTTCATATCACCACC 1920 Sorghum GCAGGACATCTGATGCGCTTGAGGAGCTC------CAGTCCTTCAAAGAGATAAGGGAGC Fruit fly TACGACAAGCTGGTCTC------GCAGTTCGGGCTAACGGGCTCCTCATC Rice Fish CCCCCCCAGCTGACCAGCCTGTCGCTCCACATCAGCTCCCAGTCCATGGCCGACGACCTG Pufferfish CCCCCGCAACTAGCTAGTCTCACGCTGGACATTAGTTCCCAATCAATGGCTGATGACCTG Atlantic Salmon CCCCCACAGCTCGCCAGCCTCACTATTGATGTCAGTTCTCAGTCCATGGCAGAAGACCTG Rainbow trout CCCCCACAGCTCGCCAGCCTCACAATTGATGTCAGTTCTCAGTCCATGGCAGAAGACCTG Zebrafish CCTCCTCAACTTGCGAGCTTGTCCATTGATGTCAGCGCTCAGTCTATGGCTGAAGACTTG Cat CCTCCCCAACTCACAAGCCTTTCCATCGATGTCAGTGCGCAGTCTATGGCTGAGGACTTG Clawed frog CCCCCACAGCTAAGCAGCTTATCCATAGATATCGCTGTGCAGTCGATGGCAGAGGACTTG Red junglefowl CCCCCTCAACTCAGCAGCCTCTCCATTGACATAGGAGCTCAGTCCATGGCAGAGGATTTG Brown rat CCCCCTCAGCTGAGCAGCCTCTCCATAGATTTTGGAGCTCAGTCTATGGCAGAAGACTTG Cattle CCGCCTCAGCTGAGCAGCCTCTCCCTCGACATCGGGGCCCAGTCCATGGCCGAGGACTTG Human CCACCTCAGCTGAGCAGCCTCTCCATAGATATTGGAGCACAGTCCATGGCTGAAGACTTG

187

Rhesus macaque CCCCCTCAGCTGAGCAGTCTCTCCATAGATATTGGAGCACAGTCCATGGCTGAAGACTTG * *

Yeast GACAACAAAATACAAAAGTACAACCAATTAGGAGTAGAAGAAGATGATGATGACGAGAAT 1980 Sorghum AACTGCTG--GCC------CGGAGCAGCTCAAA------ACATCA Fruit fly GACGGGT---AGC------CGCAGCA------GTGGTGG Rice Fish GACTCGC---TCC------CGGAGAAGCTGCGCGTCTACGAGAAGAACATCGACGAGTTC Pufferfish GACTCGC---TTC------CGGAAAAACTGGCGGTGTATGAGAAAAACATCGATGAGTTT Atlantic Salmon GACTCTC---TTC------CCGAAAAACTGGCAGTATACGAGAAGAACATTGATGAATTT Rainbow trout GACTCTC---TTC------CCGAAAAACTTGCAGTATACGAGAAGAACATTGATGAATTT Zebrafish GACTCTC---TTC------CAGAGAAACTGGCTGTTTATGAGAAGAACATTGATGAGTTT Cat GACTCTC---TGC------CAGAGAAGCTGGCCATCTACGAGAAGAACATTGATGAGTTC Clawed frog GACTCTC---TGC------CGGAGAAACTGGCCATGCACGAAAAGAACATGAAAGAGTTT Red junglefowl GACTCGT---TAC------CAGAGAAGCTGGCAGTCCATGAGAAAAATGTCAAAGAGTTT Brown rat GACTCCT---TAC------CAGAGAAGCTGGCTGTGCATGAGAAGAATGTCCGAGAATTT Cattle GACTCAC---TCC------CAGAGAAGCTGGCTGCGCACGAGAAGAATGTCCGAGAATTT Human GACTCAT---TAC------CAGAGAAGCTGGCTGTGCATGAGAAGAATGTCCGCGAGTTT Rhesus macaque GACTCAT---TAC------CAGAGAAGCTGGCTGTGCATGAGAAGAATGTCCGCGAGTTT ** * * *

Yeast GACCGTTTGCTCAACCAAATGGGAAACAGTGCTACAAAATTCAAAAGTTCAATATCTCCA 2040 Sorghum AGAGCCACCGGG------AAAGCCATAG------Fruit fly GCTCCAAATGAG------CAACT---GA------Rice Fish GACGCGTTTGTG------GACATGCTCCAGTAG------Pufferfish GATGCTTTTGTG------GATACACTCCAGTAG------Atlantic Salmon GATGCATTTGTG------GACACATTGCAGTAG------Rainbow trout GATGCATTTGTG------GACACATTGCAGTAG------Zebrafish GATGCATTTGTG------GACACTCTGCAATAG------Cat GATGCATTTGTG------GACACACTACAGTAG------Clawed frog GATGCATTTGTT------GATTCTCTGTAG------Red junglefowl GATGCCTTTGTA------GAAACCTTGCAGTGA------Brown rat GATGCCTTCGTC------GAAACCCTGCAGTAA------Cattle GATGCCTTTGTA------GAAACCCTGCAGTAA------Human GATGCCTTTGTG------GAAACCCTGCAGTAA------Rhesus macaque GATGCCTTTGTG------GAAACCCTGCAGTAA------*

Yeast AGATCAATTGATAGCATTTCAAGTTCTTTCATAAAAAGTAGGATACCTATCAGACAACCA 2100

188

Sorghum ------Fruit fly ------Rice Fish ------Pufferfish ------Atlantic Salmon ------Rainbow trout ------Zebrafish ------Cat ------Clawed frog ------Red junglefowl ------Brown rat ------Cattle ------Human ------Rhesus macaque ------

Yeast TACCATTACTCTCAACCAACTACTGCGCCCTTTCAAGCTCAGGCGAAATTTCATAAACCT 2160 Sorghum ------Fruit fly ------Rice Fish ------Pufferfish ------Atlantic Salmon ------Rainbow trout ------Zebrafish ------Cat ------Clawed frog ------Red junglefowl ------Brown rat ------Cattle ------Human ------Rhesus macaque ------

Yeast GCAAATAAGTTAATCGATAATGGTAATAGGAGTAATAGTAACAATAACAATCATAATGGG 2220 Sorghum ------Fruit fly ------Rice Fish ------Pufferfish ------Atlantic Salmon ------

189

Rainbow trout ------Zebrafish ------Cat ------Clawed frog ------Red junglefowl ------Brown rat ------Cattle ------Human ------Rhesus macaque ------

Yeast AATGATGCAGTTGGTGTGATGCATAATGACGAGGATGATCAAGATGATGATCTAGTATTT 2280 Sorghum ------Fruit fly ------Rice Fish ------Pufferfish ------Atlantic Salmon ------Rainbow trout ------Zebrafish ------Cat ------Clawed frog ------Red junglefowl ------Brown rat ------Cattle ------Human ------Rhesus macaque ------

Yeast TTCATGAGTGATATGAACCTTTCTAAAGAAGGTTAA 2316 Sorghum ------Fruit fly ------Rice Fish ------Pufferfish ------Atlantic Salmon ------Rainbow trout ------Zebrafish ------Cat ------Clawed frog ------Red junglefowl ------

190

Brown rat ------Cattle ------Human ------Rhesus macaque ------

191

Appendix 2.2d) Clustal alignments between rainbow trout Atg genes and sequences from 14 other species.

CLUSTAL O(1.2.4) multiple sequence alignment ATG9

Fruit fly ------60 Yeast ATGGAGAGAGATGAATACCAGTTACCCAACTCTCATGGGAAGAATACTTTCTTATCGCGA Sorghum ------Zebrafish ------Atlantic Salmon ------Rainbow trout ------Pufferfish ------Cat ------Rice Fish ------Clawed frog ------Red junglefowl ------Brown rat ------Cattle ------Humaun ------Rhesus macaque ------

Fruit fly ------120 Yeast ATATTTGGTTTACAATCGGATGAAGTTAATCCTTCTCTTAATAGCCAGGAGATGAGCAAC Sorghum ------ATGATGTCC Zebrafish ------Atlantic Salmon ------Rainbow trout ------Pufferfish ------Cat ------Rice Fish ------Clawed frog ------Red junglefowl ------Brown rat ------Cattle ------Humaun ------Rhesus macaque ------

192

Fruit fly ------180 Yeast TTCCCTTTACCAGACATAGAGAGAGGCTCATCTTTATTGCATTCTACTAACGACAGCCGC Sorghum TTCCTTTCGAAGGA------CACACATGCACAAA Zebrafish ------Atlantic Salmon ------Rainbow trout ------Pufferfish ------Cat ------Rice Fish ------Clawed frog ------Red junglefowl ------Brown rat ------Cattle ------Humaun ------Rhesus macaque ------

Fruit fly ------240 Yeast GAAGATGTAGATGAGAATGACTTACGTGTTCCTGAGTCTGACC-AAGGCACTAGTACAGA Sorghum CAAGACTTAACTGGCCATGGAGAAGCCAATCGCCGTTATCAGCGCAGCTGCTTGTTGATA Zebrafish ------ATGGCTGGTTTTGAGAC------A--TA Atlantic Salmon ------ATGGCGAACTTTGAAGC------T--TA Rainbow trout ------Pufferfish ------ATGGCGCACAT------T--GA Cat ------Rice Fish ------ATGGCACACTT------T--GA Clawed frog ------ATGGCTATGTA------C--GA Red junglefowl ------ATGGCCCACCT------G--GA Brown rat ------ATGGCACAGTT------T--GA Cattle ------ATGGCGCAGTT------T--GA Humaun ------ATGGCGCAGTT------T--GA Rhesus macaque ------ATGGCGCAGTT------T--GA

Fruit fly ------300 Yeast AGAAGAGGATGAAGTAGATGAAGAGCAAGTCCAGGCGTATGCTCCACAGATTAGTGATGG Sorghum TCCCACCTGAAATAGAGTT------ATCTGATTACCGGAGATTGCCAAGTTCTG- Zebrafish CCAGGAGTATCAGCG-GAT------TCACGATTATGATGAAGACTCGCCTCCAG- Atlantic Salmon CCAGGAGTACCAGCG-GAT------TGATGACTTTGAGGAGGACTCACCTCCCG- Rainbow trout ------

193

Pufferfish CACAGAGTATCAGCG-CCT------GGAGGCATCCTACAGTGACTCTCCCCCTG- Cat ------Rice Fish CACGGAGTACCAGCG-TCT------GGAAGCGTCCTACAGCGACTCTCCTCCTG- Clawed frog CACCCCATACCAGAG-GCT------GGAGGCTTCCTACACTGACTCGCCGCTGG- Red junglefowl GACGCAGTACCAGCG-GCT------GGAGAGCTCCTCCACCGAGTCCCCGCCCG- Brown rat CACTGAATACCAGCG-CCT------AGAGGCCTCTTATAGCGATTCACCCCCAG- Cattle CACTGAATACCAGCG-CCT------AGAGGCCTCCTACAGCGATTCACCCCCTG- Humaun CACTGAATACCAGCG-CCT------AGAGGCCTCCTATAGTGATTCACCCCCAG- Rhesus macaque CACTGAATACCAGCG-CCT------AGAGGCCTCCTACAGTGATTCACCCCCAG-

Fruit fly ------360 Yeast ATTGGATGGAGACCACCAGCTAAATTCTGTAAC-GAGCAAAGAAAATGTACTTGAAACGG Sorghum -----GAAGTGAGAGCCCATCTGGACTTCTC---CATGGCGAAGGCATCAAGGAAGAACA Zebrafish -----GAGAGGAAGATTTACTCATCCACGTGCCAGAGGGAAGAGGAGACCCATGGCACCA Atlantic Salmon -----GGGAGGAGGACCTATTAGTGCATGTGCCAGAGGGCCTGAGTGACTCATGGCACCA Rainbow trout ------ATGAGATGGAATTTCCCTTC Pufferfish -----GAGAGGAGAACCTGTTGATGCATGTGCCAGAAGGAGCCAAATCCCAGTGGCACCA Cat ------Rice Fish -----GAGAGGAGAACCTGCTAATGCATGTCGCAGAAGGAGCCAAATCCCACTGGCACCA Clawed frog -----GGGAAGATGATCTGCTGGTGCACGTGCCAGAGGGCAGCAAATCTCCCTGGCATCA Red junglefowl -----GCGGCGGCGATCTCCTCGTGCACGTCCCGGAGGGCGCTAAGTCTCCCTGGCACCA Brown rat -----GGGAAGAGGACCTGTTGGTGCATGTGGCTGAGGGGAGCAAATCACCTTGGCACCA Cattle -----GAGAGGAGGACCTGTTGGTGCATGTTCCTGAGGGGAGCAAGTCACCTTGGCACCA Humaun -----GGGAGGAGGACCTGTTGGTGCACGTCGCCGAGGGGAGCAAGTCACCTTGGCACCA Rhesus macaque -----GGGAGGAGGACCTGTTGGTGCACGTCGCTGAGGGGAGCAAGTCACCGTGGCACCA

Fruit fly ------420 Yeast AAAAAAGCAATTTAGAAAGACTGGTT-GAAGGCTCTACCGATGATTCTGTGCCCAAAGTC Sorghum TATCC-CTGATTTGGACATTTTCTTTGAAAGGCTTTACGAATATTTCTGTGCAAAAGGGC Zebrafish TATCA-AGAACCTCGACAATTTCTTCACAAGAATCTATCACTTCCATCAGAAGAACGGAT Atlantic Salmon TATCA-AGAATCTGGACAACTTCTTTACAAGAATCTATCAGTTTCATCAGAAAAATGGCT Rainbow trout AATTC-TTGTTTTTCTTTACTTGAATTTCCACATCTATCAGTTCCATCAGAAGAATGGCT Pufferfish CATAG-AAAATCTGGACCTGTTCTTTCAGATCTATAAT---CTGCACCAGAAGAATGGCT Cat ------Rice Fish CATAG-AGAACCTGGATCTGTTTTTCCAAAGAATATCCTTATACTTAATTAAAAATGGAT Clawed frog CATAG-AAAACCTCGACCTCTTCTTCTCTCGTGTGTACAATCTGCACCAAAAGAACGGCT Red junglefowl CATAG-AGAACCTAGACCTCTTCTTCTCTCGGGTCTATAATTTGCATCAGAAGAATGGCT

194

Brown rat CATTG-AAAACCTTGACCTCTTCTTCTCTCGAGTTTATAATCTACATCAGAAGAATGGGT Cattle CATCG-AAAATCTTGACCTCTTCTTCTCTCGAGTTTATAATCTACACCAGAAGAATGGTT Humaun TATTG-AAAACCTTGACCTCTTCTTCTCTCGAGTTTATAATCTGCACCAGAAGAATGGCT Rhesus macaque TATTG-AAAACCTTGACCTCTTCTTCTCTCGAGTTTATAATCTGCACCAGAAGAATGGCT

Fruit fly ------480 Yeast GGACAGCTTTCGTCAGAAGAAGAAGAGGATAATGAGTTCATAAATAATGATGGATTTGAT Sorghum TCAGGTGTATCATTACCA----AATGGATAATAGAGGTCCTTAATG-TACTTTTTATGGT Zebrafish TCGCCTGCATGGTGTTAT----CAGAGTTTTTTGAACTTGTGCAAT-TCCTGTTTGTGGT Atlantic Salmon TTGCTTGTATGATGTTAT----CAGAGTTCTTTGAACTTTTTCAGT-TCCTGTTTGTGGT Rainbow trout TTGCCTGTATGATGTTAT----CAGAGTTCTTTGAACTTTTTCAGT-TCCTGTTTGTGGT Pufferfish TCACCTGCATGCTGCTGG----GAGAGATCTTTGAACTGGTGTATT-TGCTCTTTGTGGT Cat ------Rice Fish TCACCTGCATGCTGTTGG----GAGAGCTCTTTGAGTTGGTGTACT-TGCTGTTTGTGGT Clawed frog TTTCCTGTATGCTGATTG----GAGAAATCTTCGAGTTGCTGCAGT-TCATCTTTATTGT Red junglefowl TCACCTGCATGCTCATCG----GGGAGATCTTCGAGCTCATGCAGT-TCATCTTTGTGGT Brown rat TCACATGTATGCTCATTG----GAGAGATTTTTGAACTCATGCAGT-TCCTCTTTGTGGT Cattle TCACTTGTATGCTCATCG----GGGAGATCTTTGAGCTCATGCAGT-TCCTCTTTGTGGT Humaun TCACATGTATGCTCATCG----GGGAGATCTTTGAGCTCATGCAGT-TCCTCTTTGTGGT Rhesus macaque TCACATGTATGCTCATCG----GGGAGATCTTTGAGCTCATGCAGT-TCCTCTTTGTGGT

Fruit fly ------540 Yeast GACGATA-CGCCCCTTTTTCAAAAAAGCAAGATTCATGAATTTAGCTCTAAGAAAAGCAA Sorghum ATGCTGTATCGGGTTCTTTTTCTTATTTGTTGATTGGGATACTCTTATTCATTTGAAATG Zebrafish CACGTTTACAACTTTTCTCTTCAACTGTGTGGAATATGATGTTCTCTTTGCCAATCGAGC Atlantic Salmon CACTTTCACAACATTCCTCTTCAACTGTGTGGAGTATGATATACTGTTTGCCAACCGGGC Rainbow trout CACATTCACAACATTCCTGTTCAACTGTGTGGAGTATGATATCCTGTTTGCCAATCGGGC Pufferfish TGGCTTCACAGTTTTCCTCGCTAACTGTGTGGATTATGACATCCTTTTTGCCAACAAGTT Cat ------TGTGTGGACTACGACATCCTGTTTGCCAACAAGTT Rice Fish TTGCTTCACTGTGTTCCTTGCCAACTGTGTGGATTATGACGTCCTCTTTGCCAACAAGTT Clawed frog CAGTTTTACCACCTTACTGGTGAGCTGTGTGGATTATGATATTCTCTTTGCCAACAAGAT Red junglefowl GGCGTTTACCACCTTTCTTATTAGCTGCGTTGATTATGATATTCTTTTTGCCAACAAAGC Brown rat TGCCTTCACCACCTTCCTGGTTAGCTGTGTGGACTATGACATCCTATTTGCCAACAAGAT Cattle TGCCTTTACCACCTTCCTGGTCAGCTGTGTGGACTACGACATCCTATTCGCCAACAAGAT Humaun TGCCTTCACTACCTTCCTGGTCAGCTGCGTGGACTATGACATCCTATTTGCCAACAAGAT Rhesus macaque TGCCTTCACTACCTTCCTGGTCAGCTGCGTGGACTATGACATCCTATTTGCCAACAAGAT

195

Fruit fly ------600 Yeast TACTATAGAGGACGGTAAACGACCTTTGTTTTTCAGGCATATCTTACAGAATAACCGCCC Sorghum CGGTGTGGAGGCACT------AGAATCTGGGGAGAA Zebrafish CGTCA---ATCACAC------GGGTCAGAGCCTCGG Atlantic Salmon AGTGA---ACCACAC------AGGACCTAGCCAGAA Rainbow trout AGTGA---ACCATAC------AGGACCTGGCCAGAA Pufferfish TGTAA---ATCATGT------T------Cat CGTCA---ATCATAC------C------Rice Fish TGTAA---ACCACAC------A------Clawed frog GGTGA---ACCACAG------TCAGAG------Red junglefowl GGTAA---ATCACAG------CCAGCAT------Brown rat GGTGA---ACCACAG------TCTGCAT------Cattle GGTGA---ACCACAG------TCTTCAC------Humaun GGTGA---ACCACAG------TCTTCAC------Rhesus macaque GGTGA---ACCACAG------TCTTCAC------

Fruit fly ------660 Yeast TCAACGTGATACCCAAAAGCTATTTACTTCGTCGAACGCCATTCACCATGATAAGGACAA Sorghum ACCATGTGATCTGATGAAGG---TCATTAAGCATGACCCATTAGTCCCCTTCACACTGCC Zebrafish GCCTCTGGACAGGAGCAAAG---TCACCCTTCCTGATGCTATTTTACCTAGTGAGCAATG Atlantic Salmon CCCCCTGGACAGAAACAAGG---TCTCTCTGCCTGATGCCATCCTACCAAGCCAGCAGTG Rainbow trout CCCCCTGGACAGAAACAAGG---TTACTCTGCCTGATGCCATCCTACCAAGCCAGCAGTG Pufferfish ------GACTCATCTAAAG---TCACGTTGCCTGATGCCTTTTTGCCTATGGATGTCTG Cat ------GACTCATCCAAGG---TGACTCTTCCTGATGCGTTTCTTCCCGTGGATGTGTG Rice Fish ------GATTCTTCTAAAG---TCACTTTGCCAGATGCGTTGATTCCTATGGATGTTTG Clawed frog ------TGACCACGTCAAGG---TGACACTTCCTGATGCCTTCCTGCCCCCTGGGGTCTG Red junglefowl -CCCAGTGAGCCCATTAAGG---TGACTCTACCAGATGCTTTCCTGCCTCCAAATGTCTG Brown rat -CCTACCGAGCCTGTCAAGG---TCACTCTGCCAGATGCCTTTTTGCCTGCCCAAGTTTG Cattle -CCGACCGAGCCCGTCAAGG---TCACTCTGCCAGATGCCTTTCTGCCTGCCCAAGTCTG Humaun -CCTACTGAACCCGTCAAGG---TCACTCTGCCAGACGCCTTTTTGCCTGCTCAAGTCTG Rhesus macaque -CCTACTGAACCTGTCAAGG---TCACTCTGCCAGACGCCTTTTTGCCTGCTCAGGTCTG

Fruit fly ------720 Yeast GAGTGCAAATAATGGTCCCCGTAACATTAATGGTAATCAAAAGCATGGCACTAAATACTT Sorghum AAAAA------TGATAACTGTTG------GATCAATGGTCATAATGACAG Zebrafish CACTGAGAGGAT----TCAAGGAAACAGCTGGATCATCTTTCTCTTGATAATGGCAGCCA

196

Atlantic Salmon CACTGAGAGGAT----CCAGGAGAACAGTTGGATCATCTTCCTGCTCATCATGGCGACCA Rainbow trout CACTGAGAGGAT----CCAGGAGAACAGTTGGATCATCTTCCTGCTCATCATGGCAGCCA Pufferfish CAGTGCTCGGAT----TCGAGGTAATGCATTTGTGATCTTCGTCCTGATTATCTCTGGAG Cat TCATGCTCGGAT----TCGTGATAACGTCTTTGTTATTTTCATTCTGGTGATATCTGGGG Rice Fish CAGTGCTCGTAT----TCGAGATAATGGATTTGTGATCTTTGTCCTGATCATCTCCGGGG Clawed frog CAGAGACAGAAT----ACAAGAAAATGGCTTCCTCGTCTGTTTACTGGCCATTGCTGGAG Red junglefowl CAGTGCAAGAAT----CCAGGCAAACAGCTTCCTCATCTGCATCCTGGTGATAGCTGGGG Brown rat CAGTGCCAGGAT----TCAGGAAAACGGCTCCCTCATCACCATCCTGGTCATCGCTGGTG Cattle TAGTGCCAGGAT----TCAGGAAAATGGCTCCCTTATCACCATCCTGGTCATCGCTGGTG Humaun TAGTGCCAGGAT----TCAGGAAAATGGCTCCCTTATCACCATCCTGGTCATTGCTGGTG Rhesus macaque TAGTGCCAGGAT----TCAGGAAAATGGCTCCCTTATCACCATCCTGTTCATTGCTGGTG

Fruit fly ------780 Yeast TGGTAGTGCTACACAACCTCGG------TTTACCGGTTCGCCCTTAAATAATACAAAC Sorghum CTTATGGGCTTACCAACTTCCTCAAGTTCTTTGTACAACTCAGAAGTACATTGAATATTC Zebrafish TTTTCTGGGTGTACCGGCTGGTGAAAGTGATCTGTAACGTCCTCAGCTACTGGGAGATCC Atlantic Salmon TCTTCTGGGTCTACCGGATGATCAAGGTCTTCTGCAACATCCTCAGCTACTGGGAGATAC Rainbow trout TCTTCTGGGTCTACCGGATGATCAAGGTCTTCTGCAACGTCCTCCACTACTGGGAGATCC Pufferfish TGTTTTGGCTGCACCGGCTCGTCAAATTCCTCTACAATGTTTGTTGTTACTGGGAGATCC Cat TGTTCTGGCTCCATCGTTTGGTCAAATTCATCTACAACGTCTGCTGCTACTGGGAGATCC Rice Fish TATTTTGGCTCCATCGCCTGGTCAAATTCTTCTACAACGTCTGCTGTTACTGGGAGATCC Clawed frog TCTTCTGGATTCACCGACTCATAAAATTTATATATAATATCTGCTGCTACTGGGAGATCC Red junglefowl TTTTCTGGATCCACAGACTTGTCAAATTTATCTACAACATTTGCTGCTACTGGGAGATTC Brown rat TCTTCTGGATCCACCGGCTTATCAAGTTTATCTATAACATTTGCTGCTATTGGGAGATCC Cattle TCTTCTGGGTCCACCGGCTCATCAAGTTTATCTATAACATTTGCTGCTACTGGGAGATCC Humaun TCTTCTGGATCCACCGGCTTATCAAGTTCATCTATAACATTTGCTGCTACTGGGAGATCC Rhesus macaque TCTTCTGGATCCACCGGCTTATCAAGTTCATCTATAACATTTGCTGCTACTGGGAGATCC

Fruit fly ------840 Yeast AGGTTCACTAAATTGTTTCCATTAAGAAAGCCAAATTTACTGAGCAATATATCCGTTTTA Sorghum GTGACTTCTACTGTAACAGTCTTAAGGTCACAGATCTGGAG------ATTCAGACTA Zebrafish GGCAGTTTTATATCAAAGCACTGAAAATACAGATGGATGAG------TTGTGCAACT Atlantic Salmon GCCAGTTCTACATCAAAGCACTGAAGATCAGCATGGACGAG------CTGTGTAACT Rainbow trout GACAGTTCTACATCAAAGCACTGAAGATCAGAATGGGCGAG------CTGTGTAACT Pufferfish GGTCATTCTACAGCCATGCACTTAAAATGACCATGGTGAGG------CAGAAATATG Cat GCTCTTTCTACATCAACGCTCTGAAAATGTCAATGGCAGAT------CTTCCTTATT Rice Fish GATCCTTCTACAAAAACGCCCTGAAGATGTCTATGGTGAGACGGACAAATCTTCCCTACG

197

Clawed frog ACAACTTCTATATACACGCTCTGCGCATCCCTATGTCAGAC------CTTCCGTATT Red junglefowl ACTCTTTCTACATCAATGCCCTCAGAATCCCCATGTCCAAC------CTGCCCTACT Brown rat ACTCCTTCTACCTACATGCTCTGCGGATCCCAATGTCTGCC------CTTCCGTATT Cattle ACTCCTTCTACCTGCATGCTCTGCGCATCCCCATGTCGGCA------CTTCCATACT Humaun ACTCCTTCTACCTGCACGCTCTGCGCATCCCTATGTCTGCC------CTTCCGTATT Rhesus macaque ACTCCTTCTACCTGCATGCTCTGCGCATCCCTATGTCTGCC------CTTCCGTATT

Fruit fly ------900 Yeast AACAATACCCCGGAAGATAGAATCAATACGTT--GAGCGTGAAAGAGCGGGCTCTGTGGA Sorghum TATCATGGCCCAAAGTAGTTGAGAAGGTTGTCCTCCTCCAGAAGTCACAACAACTTTGCG Zebrafish TCACATGGCAGGAAGTCCAGAACCGTCTCATCCTCCTCCAGCGTGAGCATCCCATGTGTG Atlantic Salmon TCACATGGCAGGAGGTCCAGGACCGTCTGATCAGCCTGCAGCGGGAGCAGCAGATGTGTA Rainbow trout TCACGTGGCAGGAGGTGCAGGACCGTCTGATCAGCCTGCAGCGGGAGCAGCAGATGTGCA Pufferfish CCACATGGCAAGAGGTTCAAGCAAGGATCGTGGAGATCCAGAAAGAGCATCAGATCTGTA Cat TCACCTGGCAAGAGGTGCAGGCACGGATCATCGAGATCCAAAAAGAGCATCAGATTTGCA Rice Fish CAACATGGCAGGAAGTTCAGGCCAGGATCATAGAGATTCAGAAGGAGCACCAGATCTGCA Clawed frog ACACCTGGGAGGAGGTTCAGAGCCGCATTGTGCAGATCCAGAAAGAGCACCAAATCTGCA Red junglefowl ACACTTGGCAGGAGGTACAGGCCCGCATCGTGCAGATCCAGAAAGAGCACCAGATCTGCA Brown rat GCACGTGGCAGGAAGTGCAGGCCCGGATTGTGCAGACCCAGAAAGAGCATCAGATCTGCA Cattle GCACATGGCAAGAAGTGCAGGCCCGGATCGTGCAGACCCAGAAGGAGCACCAGATATGCA Humaun GCACGTGGCAAGAAGTGCAGGCCCGGATCGTGCAGACGCAGAAGGAGCACCAGATCTGCA Rhesus macaque GCACGTGGCAGGAAGTGCAGGCCCGGATCGTGCAGACTCAGAAGGAGCACCAGATCTGCA

Fruit fly ------960 Yeast AATGGGCAAACGTAGAAAACCTCGATATATTTCTTCAAGATGTTTACAATTATTATCTGG Sorghum T------TGTT---AAGGACCTTTCAGAGCATGATATTATCATGAGAT Zebrafish T------CCAGAAGCGTGAACTCTCTGAACTGGACATTTACCACCGCA Atlantic Salmon T------CCACAAGAAGGAGCTAACTGAGCTGGACATCTACCACCGCA Rainbow trout T------CCACAAGAAGGAGCTAACTGAGCTGGACATCTACCACCGCA Pufferfish T------CCACAAAAGAGAACTGACTGAGCTTGATATTTACCACCGCA Cat T------CCACAAAAAGGAACTCAC------Rice Fish T------TCACAAGAAGGAACTGACAGAGCTCGACATTTATCATCGCA Clawed frog T------TCACAAAAAGGAGCTCTCGGAACTTGATGTGTCTCACAGAA Red junglefowl T------CCACAAGAAGGAACTGACAGAGCTGGACATCTACCACCGCA Brown rat T------CCACAAGCGTGAGCTGACAGAGTTGGACATCTACCATCGCA Cattle T------CCACAAGCGTGAGTTGACAGAGCTGGACATCTACCACCGAA Humaun T------CCACAAACGTGAGCTGACAGAACTGGACATCTACCACCGCA

198

Rhesus macaque T------CCACAAACGTGAGCTGACAGAACTGGACATCTACCACCGCA

Fruit fly ------1020 Yeast GAAATGGGTTCTATTGCATCATACTGGAAAAAATATTGAATATATGCACTTTGCTTTTTG Sorghum TAATGCGGAAACAGAATTACTTAATT---GGGATGGTTAATAAAGGCATTATTGCATTTC Zebrafish TCCTGCGCTTCAAGAACTACACAGTG---GCCATGATCAACAAATCCCTGCTG-CCTGTC Atlantic Salmon TCCTGCGATTCAAGAACTACATGGTG---GCCATGGTCAATAAGTCCCTGTTA-CCTGTA Rainbow trout TCCTGCGCTTCAAGAACTACATGGTA---GCCATGGTCAACAAGTCCCTGTTA-CCTGTA Pufferfish TCCTGCGCTTTAAAAACTACATGGTAGTTGCCATGGTGAATAAATCACTCCTG-CCTGTT Cat ------Rice Fish TCCTCCGCTTTAAAAACTACATGGTGGTTGCCATGGTGAATAAATCTCTTCTG-CCTGTG Clawed frog TCCTGCGCTTCAAGAACTATATGGTA---GCCATGGTAAATAAGAACCTGCTG-CCCCTT Red junglefowl TCCTCCGCTTCAAGAACTACATGGTG---GCCATGGTGAACAAGTCACTGCTG-CCAATC Brown rat TCCTCCGTTTCCAGAACTACATGGTG---GCACTGGTGAACAAATCCCTCTTG-CCTCTG Cattle TCCTCCGGTTCCAGAACTACATGGTG---GCCCTGGTGAACAAGTCCCTCCTG-CCCCTA Humaun TCCTCCGTTTCCAGAACTACATGGTG---GCACTGGTTAACAAATCCCTCCTG-CCTCTG Rhesus macaque TCCTCCGTTTCCAGAACTACATGGTG---GCACTGGTTAACAAATCCCTCCTG-CCTCTG

Fruit fly ------1080 Yeast TTGTTTTTGTTT-----CTACATATATGGGTCACTGTGTTGACTACTCCAA------Sorghum CGGTTGCCTGTTGGCTGCCTGGAGTAGGACCAACTGTCAGTTCGCGTTTCCATGGAAGGA Zebrafish AGGCTCCGAGTG-----CCCTTCATAGGTGACGTCATCTTCCTCACGCAGGGCCTCAAAT Atlantic Salmon CAGCTTCAGCTC-----CCCCTTCTGGGAAACCTGGTGTTCCTCACCCAGGGCCTCAAGT Rainbow trout CAGTTACAGCTC-----CCCCTGTTGGGCAATCTGGTGTTCCTCACCCAGGGCCTCAAGT Pufferfish CGATTTGGACTT-----CCTGTAATTGGAGAATATGTTTTCTACACCAGGGGTCTCAAAT Cat ------Rice Fish CGATTTCGACTT-----CCTGTTCTCGGAGAAGTTGTGTTTTTCACTCGGGGTCTCAAAT Clawed frog CAGCACAGGCTC-----CCCCTGCTGGGCAACACTGTCTTCTACACCCGAGGCCTCAAGT Red junglefowl CGCTTCCGCCTG-----CCCCTGCTGGGAGACACCGTATTCTACACGCGTGGGCTCAAGT Brown rat CGCTTCCGTCTG-----CCCGGCCTCGGAGAGGTTGTCTTCTTCACCCGTGGCCTCAAGT Cattle CGCTTCCGCCTG-----CCCGGTCTCGGGGAGGTTGTCTTCTTCACCCGGGGCCTCAAGT Humaun CGCTTCCGCCTG-----CCTGGCCTCGGGGAAGCTGTCTTCTTCACCCGTGGTCTCAAGT Rhesus macaque CGCTTCCGCCTG-----CCTGGCCTCGGGGAGGCCGTCTTCTTCACCCGTGGCCTCAAGT

Fruit fly ------1140 Yeast ------ATTACCGACTAGTC--ACCGGGTTTCGGATATTATTAT-----CGA

199

Sorghum AAAGCTATCTGATGCTTCCAAAGACGCTAGAGTGGGCATTAAATTGGTGCATATTTCATA Zebrafish ACAACTTCGAGCTCATCCTCTTCTGGGG--CCCGTTGTCTCTCTTCCAGAAC---A-AAT Atlantic Salmon ACAACTTTGAGCTGATTCTCTTCTGGGG--TCCGGGGTCGCTGTTCCAGAAC---A-AAT Rainbow trout ACAACTTTGAGCTGATTCTCTTCTGGGG--TCCGGGGTCGCTTTTCCAAAAC---A-AAT Pufferfish ACAACTTTGAGCTCATCTTCTTCTGGGG--CCCAGGGTCTCTGTTTGAGAAT---G-AGT Cat ------Rice Fish ACAACTTTGAGCTCATCTTCTTTTGGGG--CCCAGGCTCTCTTTTTGAGAAT---G-AGT Clawed frog ACAACTTCCAGCTGATTTTTTTCTGGGG--CCCGGGCTGCCTCTTCCAAAAT---G-AAT Red junglefowl ATAACTTTGAGCTCATCTTCTTCTGGGG--GCCCGGCTCACTCTTTGAGAAT---G-AGT Brown rat ACAACTTTGAGCTCATCCTCTTCTGGGG--ACCCGGCTCTCTGTTTCTCAAT---G-AAT Cattle ACAACTTCGAGCTGATCCTCTTCTGGGG--ACCTGGCTCTCTCTTTCTCAAT---G-AAT Humaun ACAACTTTGAGCTGATCCTCTTCTGGGG--ACCTGGCTCTCTGTTTCTCAAT---G-AAT Rhesus macaque ACAATTTTGAGCTGATCCTCTTCTGGGG--ACCTGGCTCTCTGTTTCTCAAT---G-AAT

Fruit fly ------1200 Yeast CAAGTGTTATTCTAATAGTATAACGGGGTTCACAAAG------TTTTTCC Sorghum GCATGTTTGATAGTAAATTTTGTGTCAGGAAAGACTTTCTAACAGGCCCATCTGTTTTGA Zebrafish GGAGCCTGCATCCCAAATACAAGCGGGCAGCAAACCGCCACGATCTGGCCAAGCAGCTCA Atlantic Salmon GGAACCTGCACCCTAAGTACAAGCGAGCAGGGAACCGTCTGGAGCTGGCTCAGCAGCTGA Rainbow trout GGAACCTGCACCCCAAGTACAAACGTGCAGGCAACCGTCTGGAGCTGGCCCAGCAGCTGA Pufferfish GGAGTCTGAAGCCAGAATACAAGCGAGGAAGCAACAGACTGGAGCTGGCAGACAGACTGG Cat ------Rice Fish GGAGTTTAAAGTCAGAGTATAAGAGAGGAGGAAACAGGCTTGAGCTTGCAGACAGGCTGG Clawed frog GGAGCCTTAAGCCTGAGTATAAGCGCGCAGGAGCCCGCCTGGAGCTGTCTGAAAAGCTGG Red junglefowl GGAGCTTGAAGGCTGAATACAAGCGGGCTGGAAACCGCCTGGAGCTGGCTGAGAAGATCA Brown rat GGAGCCTCAAGGCCGAGTACAAACGTGGAGGGCAACGGCTAGAGCTGGCCCAGCGTCTCA Cattle GGAGCCTCAAGGCTGAGTACAAACGTGGGGGGCAACGGCTGGAGCTGGCCCAGCGCCTCA Humaun GGAGCCTCAAGGCCGAGTACAAACGTGGGGGGCAACGGCTAGAGCTGGCCCAGCGCCTCA Rhesus macaque GGAGCCTCAAGGCCGAGTACAAACGTGGGGGGCAACGGCTAGAGCTGGCCCAGCGCCTCA

Fruit fly ------1260 Yeast TTTGGATGTTTTATTTCTTCGTGATTCTCAAAATTGTTCAACTCTACTTTGATGTTCAAA Sorghum CAAAGCGACTTGTATTTATGGGCAT-TGCAATGCTTTTGCTGTCGCCCTGCCTTGTC--- Zebrafish GTCGGGTTATTCTGCTGACTGGACT-GGTCAATCTGCTGCTGTGCCCATTTGTGTTA--- Atlantic Salmon GCCGGGTCATTCTTCTACTAGGCGT-GGCCAACCTGCTCCTCTGCCCCTTCATCCTG--- Rainbow trout GCCGGGTCATTCTTCTGATGGGCGT-GGCCAACCTGCTCCTCTGCCCCTTCATCCTG--- Pufferfish CATCCCGAATTCTCTGGATTGGGAT-TGCTAATCTGTTGCTGTGTCCAGTCATCCTT---

200

Cat ------Rice Fish CATCCCGGATTCTGTGGATTGGAAT-TGCCAATCTGCTTCTGTGTCCGGTCATTTTG--- Clawed frog CTCAGCGCATCCTGTGGATCGGGCT-TGCCAATCTGGTATTGTGCCCACTTGTAATA--- Red junglefowl GCACTCGCATCCTTTGGATTGGCAT-CGCCAACTTCCTCCTCTGCCCCCTCATCCTC--- Brown rat GCAACCGCATCCTGTGGATTGGCAT-CGCCAACTTCCTGCTCTGTCCCCTCATCCTC--- Cattle GCAACCGCATCCTGTGGATCGGCAT-TGCCAACTTCCTGCTGTGCCCCCTCATCCTC--- Humaun GCAACCGCATCCTGTGGATTGGCAT-CGCTAACTTCCTGCTGTGCCCCCTCATCCTC--- Rhesus macaque GCAACCGCATCCTGTGGATTGGCAT-CGCTAACTTCCTGCTGTGCCCCCTCATCCTC---

Fruit fly ------1320 Yeast AATTATCAGAATTACAAAACTTTTACAAGTATCTTTTAAATATATCGGATGACGAACTTC Sorghum ------ATCTTCCCATTGGTATACATGTTTCTGAGACATGCT----GAAGAAT-TC Zebrafish ------GTCTGGCAGGTGTTATACGCGTTTTTTAGCTACGCA----GAAGTGA-TC Atlantic Salmon ------GTGTGGCAGGTGCTCTACGCCTTCTTCAGCTACACC----GAGGTCA-TC Rainbow trout ------GTGTGGCAGGTGCTCTACGCCTTCTTCAGCTACACC----GAGGTCA-TC Pufferfish ------GTCTGGCAGATCCTTTATGCCTTCTTCAGTTATACG----GAAGTGA-TC Cat ------Rice Fish ------GTTTGGCAAATTCTTTATGCTTTCTTCAGCTATACT----GAGGTGA-TC Clawed frog ------GTCTGGCAGATTCTTTATGCCTTCTTCAGCTACACT----GAGGTGC-TT Red junglefowl ------ATCTGGCAGATCCTCTACGCCTTCTTCAGCTACACA----GAGATCC-TG Brown rat ------ATCTGGCAGATCCTCTATGCCTTCTTCAGCTATGCC----GAGGTGC-TG Cattle ------ATCTGGCAGATCCTCTACGCCTTCTTCAGTTATGCC----GAGGTGC-TC Humaun ------ATATGGCAAATCCTCTATGCCTTCTTCAGCTATGCT----GAGGTGC-TG Rhesus macaque ------ATATGGCAAATCCTCTATGCCTTCTTCAGCTATGCT----GAGGTGC-TG

Fruit fly ------1380 Yeast AAACTTTGCCCTGGCAAAATGTGATACAGCAGCTAATGTACTTGAA------Sorghum TACAATAATCCCAGTACAGCATCATCTCGAAGATGGTCAAATTTGTCAAGGTGGATTTTG Zebrafish AAGCGCGAGCCGGGGAGTTTGGGGGCTCATCGATGGTCCCTGTTTGGACGTCTTTATCTG Atlantic Salmon AGGAGGGAGCCAGGGAGTCTGGGAGCACGCCGCTGGTCTATGTTCGGCCGCCTCTACCTG Rainbow trout AGGAGGGAGCCGGGGAGTCTGGGAGCACGCCGCTGGTCCCTGTACGGCCGCCTCTACCTG Pufferfish AAGCGAGAGCCTGGCTCTCTCGGAGCGAGATGCTGGTCTCTTTATGGTCGATGTTACCTT Cat ------Rice Fish AAGCGAGAGCCCGGTTCTCTTGGGGCAAGATGTTGGTCTCTCTACGGTCGGTGTTACTTG Clawed frog CGGAGGGAGCCTGGCAGTCTAGGAGCACGCTGCTGGTCCCTCTATGGTCGCTGCTACCTT Red junglefowl AAGCGGGAACCGGGCAGCCTGGGTGCCCGCTGCTGGTCTCTGTACGGCCGCTGTTACCTC Brown rat AAGAGAGAGCCGGGAGCCCTGGGAGCGCGTTGCTGGTCACTCTATGGCCGTTGCTACCTC

201

Cattle AAGCGGGAGCCGGGTGCCCTGGGTGCGCGCTGCTGGTCGCTCTACGGCCGCTGCTACCTC Humaun AAGCGGGAGCCGGGGGCCCTGGGAGCACGCTGCTGGTCACTCTATGGCCGCTGCTACCTC Rhesus macaque AAGCGGGAGCCGGGGGCCCTGGGAGCACGCTGCTGGTCACTCTATGGCCGCTGCTACCTC

Fruit fly ------1440 Yeast ----GGATCAAAACGCTATGACGGCAAATGTTGTGGAAGTCAAAGCCAAGAATAGAAT-- Sorghum CGGGAGTACAACGAGGTAGATCAGTTCTTCAGACAT-AGGATGAACAACAGCGCTGTGCA Zebrafish CGGCATTTCAATGAGCTGGACCACGAGCTGCAGGGC-AGGCTGGGTCGTGGGTATAAACC Atlantic Salmon CGCCACTTCAATGAGCTGGACCACGAGCTGCATGGC-AGGCTAGGCCGCGGCTACAAGCC Rainbow trout CGCCACTTCAACGAGCTGGACCACGAGCTGCACGGC-AGACTGGGTCGCGGCTACAAGCC Pufferfish CGTCATTTTAATGAGTTGGACCATGAACTCATGTCA-CGCCTCAGCAAAGGCTACAAGGC Cat ------Rice Fish CGTCATTTTAACGAGCTGGACCATGAGCTCATGTCA-CGCCTCAGTAAAGGCTACAAGGC Clawed frog CGCCATTTTAATGAACTGGATCATCAGCTGCAAGCC-AGGCTAAGCCGAGCATATAAGCC Red junglefowl CGTCACTTCAATGAGCTAGACCATGAACTGCAGTCA-CGTCTCAGCAAAGGTTACAAGCC Brown rat CGCCACTTCAATGAGCTAGAACACGAGCTGCAGTCC-CGCCTCAACCGAGGATACAAGCC Cattle CGCCACTTCAACGAGCTGGAGCACGAGCTGCAGTCC-CGCCTCAACCGAGGCTACAAGCC Humaun CGCCACTTCAACGAGCTGGAGCACGAGCTGCAGTCC-CGCCTCAACCGTGGCTACAAGCC Rhesus macaque CGCCACTTCAACGAGCTGGAGCACGAGCTGCAGTCC-CGCCTCAACCGTGGCTACAAGCC

Fruit fly ------1500 Yeast ------CGACGCGCATGACGTTGCCAATAGAATCATGAGAAGAGAAAACTATCTCAT Sorghum CTCTTTAAATTACCTGAAGCAATTTCCAACTCCACTGATTTCTATAATGGCAAAATTCAT Zebrafish CGCTGCTAAGTACATGAACGCTTTCGTGTCTCCTCTGTTAGCGGTGCTGGCCAAAAATGT Atlantic Salmon CATCTCCAAGTACATGAACTCGTTCACCTCGCCGCTCCTCACCGTGCTGGCCAAGAACGT Rainbow trout CACCTCCAAGTACATGAACTCGTTCACCTCACCGCTGCTGACCGTGCTGGCCAAGAACGT Pufferfish TGCGTCTAAGTATATGAATTGTTTCCTCTCACCATTGTTGACTGTGGTTGCAAAAAATCT Cat ------Rice Fish TTCCTCTAAGTACATGAACTGTTTTCTTTCCCCGCTGCTGACAGTTGTTGCCAAAAATAT Clawed frog AGCTAGCAAGTATATGAACTGCTTCCTCTCCCCGCTGCTGGCTGTAGTTGCCAAACACTT Red junglefowl AGCTTCCAAGTATATGAACTGCTTCATCTCCCCGCTCCTCACTATTGTGGCCAAGAACGT Brown rat CGCCTCCAAGTACATGAATTGCTTCTTGTCACCGCTACTGACTCTGCTGGCCAAGAATGG Cattle TGCGTCCAAGTACATGAATTGCTTCTTGTCACCGCTGCTGACACTGCTGGCCAAGAACTG Humaun CGCCTCCAAGTACATGAATTGCTTCTTGTCACCTCTTTTGACACTGCTGGCCAAGAATGG Rhesus macaque CGCCTCCAAGTATATGAATTGCTTCTTGTCACCTCTTTTGACACTGCTGGCCAAGAATGG

202

Fruit fly ------1560 Yeast AGCACTTTACAATAGCGACATCCTGAATTTATCCTTGCCTATTCCATTATTTAGGACCAA Sorghum ATCTTTTGTTTCTGGTGGTTTGGCTGGTGCTCTTATAATCATTGGATTTCTGGGTGAATC Zebrafish GGCTTTCTTTTCTGGCTCAGTGCTGGCGGTGCTCATCGCTCTGACGGTGTACGATGAAGA Atlantic Salmon GGCGTTCTTCTCGGGCTCGGTGCTGGCCGTGCTCATCGCACTGACCGTATATGATGAGGA Rainbow trout GGCGTTTTTCTCTGGCTCGGTGCTGGCCGTGCTCATCGCGCTGACCGTGTACGACGAGGA Pufferfish TGCATTTTTTGCCGGATCACTGCTGGCTGTCCTCATTGCCCTGACTATCTACGACGAGGA Cat ------Rice Fish TGCGTTTTTTGCTGGATCTCTTCTGGCCGTTCTCATTGCTCTGACCATCTACGATGAGGA Clawed frog GGGCTTTCTCGCAGGCTCTATACTGGCAGTGCTGATATCACTTACTGTATATGATGAGGA Red junglefowl GGCCTTCTTTGCTGGCTCCATCCTGGCAGTGCTCATCGCTCTCACCATCTACGATGAGGA Brown rat TGCCTTCTTCGCGGGCTCTATCCTGGCCGTGCTTATTGCTCTCACGATCTACGATGAAGA Cattle CGCCTTCTTCGCTGGCTCCATCCTGGCCGTGCTTATTGCCCTCACCATCTATGATGAAGA Humaun AGCCTTCTTCGCTGGCTCCATCCTGGCTGTGCTTATTGCCCTCACCATTTATGACGAAGA Rhesus macaque AGCCTTCTTCGCTGGCTCCATCCTGGCTGTGCTTATTGCCCTCACCATTTATGACGAAGA

Fruit fly ------1620 Yeast CGTTCTGACAAAAACACTAGAATGGAATATTAATTTATGCGTCATGGGCTTTGTGTTCAA Sorghum TGTTCTTGAGG------Zebrafish TGTGCTCACCG------Atlantic Salmon TGTCTTGACCG------Rainbow trout TGTTTTGACCG------Pufferfish CGTTCTTGCGG------Cat ------Rice Fish TGTTCTTGCAG------Clawed frog TGTGCTGGCAG------Red junglefowl CGTGCTGGCGG------Brown rat TGTGTTGGCTG------Cattle CGTGCTGGCTG------Humaun TGTGTTGGCTG------Rhesus macaque TGTGTTGGCTG------

Fruit fly ------1680 Yeast CGAGTCCGGATTTATTAAACAAAGTATTTTAAAACCTTCTCAAAGAGAATTCACGAGAGA Sorghum ----GCCATATCTTTGG------AAAGAATCTTCTCTGGTACACTATTGTTTT Zebrafish ----TCCAA------CACATCCTGACGGCCATCACTGTGCT Atlantic Salmon ----TGCAG------CACATCCTGACCGCCATCACCGTGCT

203

Rainbow trout ----TGCAG------CATATCCTGACCGCCATCACCGTGCT Pufferfish ----TGGAA------CACGTTCTCACATCCATCACACTGCT Cat ------Rice Fish ----TAGAG------CATGTGCTGTCATCCATTACTCTGCT Clawed frog ----TGGAA------CATGTGTTGAGTGCTGTCACCCTGCT Red junglefowl ----TTGAG------CACGTCCTGACCACGGTCACCCTTCT Brown rat ----TGGAG------CATGTCCTCACCACTGTCACCCTACT Cattle ----TGGAA------CACGTCCTCACTACCGTCACACTCCT Humaun ----TGGAA------CATGTGCTGACCACCGTCACACTCCT Rhesus macaque ----TGGAA------CACGTCCTGACCACCGTCACACTCCT

Fruit fly ------1740 Yeast AGAATTACAAAAAAGATTCATGCTGGCAGGATTT-CTTAATATTATATTGGCACCATTTC Sorghum TGGAACAATAGCAGCTATAAGTCGGAAAGTAGTGGCTGATGAGCTTCAAGTT-----ATT Zebrafish GGGGGTCGTCATCACCGTGACCAGGTCTTTTATTCCTGATGAGCATATGGTG-----TGG Atlantic Salmon GGGAGTGGTTATCACCATCACCAGGTCCTTCATCCCAGACGAACACATGGTG-----TGG Rainbow trout GGGAGTGGTCATCACCATCACCAGGTCGTTCATCCCAGACGAACACATGGTG-----TGG Pufferfish CGGAGTCTGCATCACAGTCTGCAGATCATTTATTCCTGATAAGAACATGGTG-----TTT Cat ------Rice Fish TGGAGTGTGTATCACCATTTGTCGATCTTTTATCCCGGATAAGCACATGGTG-----TTT Clawed frog GGGAGTTGTAGTGACAGTCTGTAGGTCGTTCATCCCTGACCAGCACTTGGTG-----TAC Red junglefowl CGGGGTGGGCATCACAGTATGCAGGTCTTTCATCCCCGATCAGCACCTGGTG-----TTT Brown rat GGGAGTCACGGTGACTGTGTGCAGGTCTTTCATCCCAGACCAGCACATGGTG-----TTC Cattle GGGGGTCACCGTGACCGTGTGCAGGTCCTTTATCCCAGACCAGCACATGGTG-----TTC Humaun GGGGGTCACCGTGACCGTGTGCAGGTCCTTTATCCCGGACCAGCACATGGTG-----TTC Rhesus macaque GGGGGTCACCGTGACCGTGTGCAGGTCCTTTATCCCGGACCAGCACATGGTG-----TTC

Fruit fly ------1800 Yeast TGGTCACATACTTTGTTTTGCTTTATTTTTTCAGATATTTTAATGAGTATAAAACTTCTC Sorghum GACCCAGAAGGGGCCATGTGCCTCGCTGTTCACCAAACAC-ATTACATGCCAAAGCGGTG Zebrafish TGTCCTGAACAGCTGCTTCAGTGTGTCCTGGCACACATTC-ACTACATGCCCGACCACTG Atlantic Salmon TGCCCAGAGCAGCTGCTGCAGTGTGTGCTGGCTCACATCC-ACTACATGCCAGACCACTG Rainbow trout TGCCCGGAGCAGCTGCTGCAGTGTGTGCTGGCTCACATCC-ACTACATGCCAGACCACTG Pufferfish TGTCCAGAGCAGCTTCTGCGGGTTATCCTGGCTCATATCC-ACTACATGCCTGACCACTG Cat ------Rice Fish TGTCCTGAGCAGCTGCTGCGGGTCATCCTGGCTCACATTC-ACTACATGCCGGACCACTG Clawed frog TGCCCTGAGCAGCTTCTCCGTGTTATTCTGGCCCATATCC-ACTATATGCCAGACCACTG

204

Red junglefowl TGCCCTGAGCAGCTGCTGCGAGTCATCCTGGCACACATCC-ACTACATGCCTGACCACTG Brown rat TGCCCTGAGCAGCTGCTCCGAGTGATCCTTGCCCACATCC-ACTACATGCCTGACCACTG Cattle TGCCCTGAGCAGCTGCTCCGCGTGATCCTCGCTCACATCC-ACTACATGCCTGACCACTG Humaun TGCCCTGAGCAGCTGCTCCGCGTGATCCTCGCTCACATCC-ACTACATGCCTGACCACTG Rhesus macaque TGCCCTGAGCAGCTGCTCCGCGTGATCCTCGCTCACATCC-ACTACATGCCTGACCACTG

Fruit fly ------1860 Yeast CTGGATCTATTGGTGCTCGTCAATACACACCAATTGCTGAATGGAAATTCCGTGAGTACA Sorghum GCGTGGTAAA-GAGAACAGCGAACTTGTCCGAAGAGAATTTGAGACATTATTTCAGTACA Zebrafish GCAGGGCAGC-GCTAACAAGAGCGAGACCCGAGACGAGATGGCACAGCTTTTCCAGTATA Atlantic Salmon GAGGGGCAAC-GCCAACAAGAGCGAGACCCGGGACGAGGTGGCACAGCTGTTTCAGTACA Rainbow trout GAGGGATAAC-GCCAACAAGAGTGAGACCCGGGACGAGGTGGCACAGCTGTTTCAGTACA Pufferfish GCAAGGAAAT-GCACACAGATATGAGACCCGTGACCAGTTCTCCCAGCTCTTTCAGTACA Cat ------Rice Fish GCAGGGCAAT-GCGCACAGATATGAGACGCGAGACCAGTTCTCACAGCTCTTCCAGTACA Clawed frog GCAGCGCAGC-GCTCACCGTGCAGAGACACGCGGAGAGTTTTCACAACTCTTCCAATACA Red junglefowl GCAGGGCAAT-GCCCACCGCTATGAGACAAGGGACGAGTTTGCCCAGCTCTTCCAGTACA Brown rat GCAGGGTAAT-GCCCACCGCTCGCAGACCCGGGACGAGTTTGCCCAGCTCTTCCAGTACA Cattle GCAGGGTAAT-GCCCACCGCTCGCAGACCCGGGACGAGTTTGCCCAGCTCTTCCAGTACA Humaun GCAGGGTAAT-GCCCACCGCTCGCAGACCCGGGACGAGTTTGCCCAGCTCTTCCAGTACA Rhesus macaque GCAGGGTAAT-GCCCACCGCTCACAGACCCGGGACGAGTTTGCCCAGCTCTTCCAGTACA

Fruit fly ------1920 Yeast ATGAACTTTACCATATTTTTAAGAAAAGAATAAGTTTGAGCACAACCTTAGCTAACAAGT Sorghum CTATAATT------ATGCTACTTGAAGAGATGG--CCTCCATTTTCATCAC-CCCTTAC Zebrafish AAGCGGTC------TTCATCTTGGAGGAGCTGC--TCAGTCCCATCATTAC-CCCCTTT Atlantic Salmon AAGCGCTG------TTTATCCTGGAGGAGCTGC--TTAGCCCCATCGTCAC-GCCCTTC Rainbow trout AAGCGGTG------TTCATCCTGGAGGAGCTGC--TGAGCCCCATCGTCAC-GCCCTTC Pufferfish AAGCGGTAAGTGTT-TTTATTCTAGAGGAGCTGC--TCAGCCCAGTGGTGAC-ACCGATC Cat ------Rice Fish AGGCAGTGAGTGTG-TTTATTTTAGAAGAGCTGC--TAAGTCCTGTGGTGAC-TCCCATC Clawed frog AGGCAGTA------TTTATCCTAGAAGAACTTC--TGAGTCCGCTGTTAAC-ACCCCTT Red junglefowl AAGCGGTC------TTCATCCTGGAGGAGCTTC--TGAGTCCCATCATTAC-CCCCCTG Brown rat AGGCAGTG------TTCATCTTGGAGGAGTTGC--TGAGTCCCATTGTTAC-ACCCCTC Cattle AGGCAGTG------TTCATCCTGGAGGAGTTAC--TGAGCCCCATTGTCAC-ACCTCTC Humaun AGGCAGTG------TTCATTTTGGAAGAGTTGC--TGAGCCCCATTGTCAC-ACCCCTC Rhesus macaque AGGCAGTG------TTCATTTTGGAAGAGTTGC--TGAGCCCCATTGTCAC-ACCCCTC

205

Fruit fly ------1980 Yeast ATGTTGATCAGTTCCCGAAGGAAAAGACCAATTTG------TTTCTGAAATTTGTTT Sorghum TTGCTTATTTTTGAGGTACCAAAGCGTGTTGATGACATTCTGCGCTTCATCTCAGACTTC Zebrafish ATCCTTATCTTCCCCCTGAGAAGCAAATCTCTGGAGATCATCGACTTCTTCCGCAACTTC Atlantic Salmon ATTCTCATCTTCCTCCTGAGGAACAAGTCCCTGGAGATCATTGACTTCTTCAGGAACTTC Rainbow trout ATCCTCATCTTCCTCCTGAGGAACAAGTCCCTGGAGATCATCGACTTCTTCAGGAACTTC Pufferfish ATCCTGATCTTCTGTCTGAGGAGGAAGTCTCTGGAGATTATTGACTTCTTTAGGAACTTC Cat ------Rice Fish ATTCTCATCTTCTGTCTGAGGAGGAAATCTCTGGAGATCATCGATTTCTTCAGAAACTTT Clawed frog GTCCTGATTTTCTGCCTCCGCCCCAAATCGCTGGAGATTATTGACTTTTTCCGAAACTTC Red junglefowl ATCCTCATCATCTGCCTGCGGCCCAAATCTTTGGACATCGTTGACTTTTTCCGTAACTTC Brown rat ATTCTCATCTTCTGCCTACGCCCACGGGCCCTGGAGATCATAGACTTCTTCCGAAACTTT Cattle ATCCTCATCTTCTGTCTGCGCCCACGGGCCCTGGAGATTATAGACTTCTTCCGCAATTTC Humaun ATCCTCATCTTCTGCCTGCGCCCACGGGCCCTGGAGATTATAGACTTCTTCCGAAACTTC Rhesus macaque ATCCTCATCTTCTGCCTGCGCCCACGGGCCCTGGAGATTATAGACTTCTTCCGAAACTTC

Fruit fly ------2040 Yeast CCTTTATTTGTGGATCATTTGTGGCCATTTTAGCTTTTCTCACTGTATTCGATCCAGAAA Sorghum ACAATTTATGTAGACGGAGTGGGAGATGTATGCAGTCTAAGCATGTTTGACTTCAAAAGA Zebrafish ACTGTGGATGTGGCAGGAGTCGGGGACATTTGCTCATTCGCACAGATGGACATCAGGAGG Atlantic Salmon ACTGTGGAGGTAGTTGGGGTCGGAGACATCTGCTCCTTCGCACAGATGGACATCCGTCGT Rainbow trout ACCGTGGAGGTGGTCGGAGTTGGCGACATCTGCTCCTTCGCACAGATGGACATCCGTCGC Pufferfish ACTGTGGAGGTGGTTGGGGTCGGAGACACGTGTTCCTTTGCCCAGATGGACATTCGGCAG Cat ------Rice Fish ACTGTAGAAGTTATTGGAGTCGGAGACACTTGCTCTTTTGCTCAGATGGACATTAGGCAG Clawed frog ACAGTGGAGGTGATAGGAGTGGGAGATACCTGCTCATTTGCACAAATGGATGTCCGTCAG Red junglefowl ACTGTGGAGGTGGTGGGGGTGGGTGACACCTGCTCCTTTGCCCAGATGGATGTGCGCCAG Brown rat ACTGTGGAGGTCGTCGGTGTGGGGGACACCTGCTCCTTTGCGCAGATGGACGTTCGCCAG Cattle ACCGTGGAAGTTGTCGGTGTTGGAGACACCTGCTCCTTCGCTCAGATGGATGTTCGCCAG Humaun ACCGTGGAGGTCGTTGGTGTGGGAGATACCTGCTCCTTTGCTCAGATGGATGTTCGCCAG Rhesus macaque ACCGTGGAGGTCGTTGGTGTGGGAGATACCTGCTCCTTTGCTCAGATGGATGTTCGCCAG

Fruit fly ------2100 Yeast ACTT---TTTAAACTTTGAAATCACCTCGG-----ACAGATCTGTCA--TTTTCTACATT Sorghum CATGGAAATAGAAACTATGGGTCGCCGTTCAATGGTCTTAAAGGTCTCAGGA------

206

Zebrafish CACGGAAACCCACAGTGGATGTCTGAAGGGCAG--ACGGAGGCCTCGGTGTACCAGCAGG Atlantic Salmon CATGGAAACCCTCTGTGGATGTCTGAGGGCCAG--ACGGAGGCGTCTGTGTACCAGCAGG Rainbow trout CACGGAAACCCACTGTGGATGTCAGAGGGCCAG--ACAGAGGCATCTGTGTACCAGCAGG Pufferfish CACGGACACCCTGCGTGGATGTCAGAGGGGAAG--ACAGAGGCTTCTATTTACCAACAAG Cat ------Rice Fish CATGGACATCCTGCGTGGATGTCGGAGGGAAAG--ACGGAGGCCTCTATCTACCAACAAG Clawed frog CATGGAAATCCCGCGTGGATGTCTGCAGGCAAG--ACTGAAGCGTCCGTGTACCAACAGG Red junglefowl CATGGCCACCCAGCGTGGATGTCAGCAGGGAAG--ACGGAGGCCTCCATTTACCAGCAGG Brown rat CACGGTCATCCTCAGTGGCTGTCTGGAGGGCAG--ACTGAGGCCTCAGTGTACCAGCAAG Cattle CATGGGCATCCCCAGTGGCTGTCCGGTGGGCAG--ACGGAGGCCTCAGTGTACCAGCAGG Humaun CATGGTCATCCCCAGTGGCTATCTGCTGGGCAG--ACAGAGGCCTCAGTGTACCAGCAAG Rhesus macaque CATGGTCATCCCCAGTGGCTGTCTGCTGGGCAG--ACAGAGGCCTCAGTGTACCAGCAAG

Fruit fly ------GCCAAGCA------GTTC 2160 Yeast ACTATTCTGGGTGCTATATGGTCTGTAAGTAGAAATACAATAACCCAGGAGTACCATGTT Sorghum GCTCCCAAGGGAAGATGGAGAAATCATTCTT-----AAGTTTTCA------GAGT Zebrafish CGGAGAACGGGAAGACGGAGCTCTCTCTCAT-----GCACTTCAC------CATC Atlantic Salmon CTGAGAACGGCAAGACTGAGCTGTCCCTGAT-----GCACTTCAC------CATC Rainbow trout CTGAGAACGGCAAGACTGAGCTGTCCCTGAT-----GCACTTCAC------CATA Pufferfish CAGAGGACGGCAAGACAGAGTTATCTCTGAT-----GCATTTTGC------AATC Cat ------Rice Fish CAGAGGATGGGAAAACAGAGTTATCGCTGAT-----GCACTTTGC------CATC Clawed frog CAGAGGATGGGAAGACGGAGCTCTCGCTCAT-----GCACTTTGC------TATT Red junglefowl CTGAGGATGGCAAGACGGAGCTGTCCCTCAT-----GCACTTTGC------CATC Brown rat CCGAGGATGGGAAGACCGAGTTGTCACTCAT-----GCACTTTGC------CATC Cattle CCGAGGATGGGAAGACAGAGTTGTCACTCAT-----GCACTTTGC------CATC Humaun CTGAGGATGGAAAGACAGAGTTGTCACTCAT-----GCACTTTGC------CATC Rhesus macaque CCGAGGATGGAAAGACAGAGTTGTCACTCAT-----GCACTTTGC------CATC

Fruit fly CTGCGCGGCGTCAGGGAG-CAC------GCCGTGGGTGAACTAGTCCAGGCCAAGAC 2220 Yeast TTCGACCCCGAAGAGA---CGCTCAAGGAGTTGTATGAATATACACACTATCTACCAAAG Sorghum GTCTATCCATCATGGGAGCCAAATGCTGATGGCAAACAGTTCTTATCTAACCTCCAAAAG Zebrafish AAGAACCCGCAGTGGCAGCCGCCGCAGGACAGCTCCATCTTCATCAGTCACCTTAAAGAG Atlantic Salmon AAGAACCCCCGTTGGCAGCCCCCCCAGGAGAGCTCAGTGTTCATCAGCCATCTGAAGGAG Rainbow trout AAGAACCCCCACTGGCAACCCCCACAGGAGAGCTCAGTGTTCATCAGCCATCTAAAGGAG Pufferfish ACCAATCCCCAATGGCAGCCTCCTCGTGAGACGACACACTTTATCAGCCAGCTGAAAGAA Cat ------

207

Rice Fish ACCAACCCACACTGGCAGCCTCCTCAGGAGACCACACACTTCATTAGCCAGCTGAAAGAG Clawed frog ACCAACCCTCGCTGGCAGCCTCCTAGGGAATGCACAGCCTTTCTGAGCCAGCTAAGGGAG Red junglefowl ACCAACCCCAAGTGGCAGCCACCCCGCGAGAGCACGGCCTTCATCGGCTTCCTGAAGGAA Brown rat ACCAATCCTGGCTGGCAGCCCCCTCGTGAGAGCACAGCCTTCCTGGGCTTCCTCAAGGAG Cattle ACCAACCCTGGCTGGCAGCCACCACGTGAGAGCACGGCCTTCCTAGGTTTCCTGAAGGAG Humaun ACCAACCCTGGCTGGCAGCCACCACGTGAGAGCACAGCCTTCCTAGGCTTCCTCAAGGAG Rhesus macaque ACCAACCCTGGCTGGCAGCCACCGCGTGAGAGCACAGCCTTCCTAGGCTTCCTCAAGGAG

Fruit fly GTCGATGGTGCAGGAGAACCCGCTGA—CCAACAGCCTCATCTCATTTGGTACCATGGGA 2280 Yeast GAATGGGAAGGAAGATATCACAAAGAGGAAATCAAACTA----GAGTTC-TGTAAATTGT Sorghum -TTCAAAGAAAAGCAAATACGTCAACAAGCGCTTGCACAATACCAAGCAATGGAAGCTTC Zebrafish -AAGGTGCATCAGGATGCCCAGACCGGCCCATCAACACAACTGCTGCTG-TCTG---AGG Atlantic Salmon -AAGGTGCACCAGGACGCCCAGGGAGGACCCCCTACCCAGCTGCTGCTC-TCTG---AGG Rainbow trout -AAGGTGCACCAGGACGCTCAGGGAGGACCCTCCACCCAGCTGCTGCTC-TCTG---AGG Pufferfish -AGAGTCCACAGAGAGGCTACAGGGACTTCTACAGAAACACACCGTCTC-TCAATATCTG Cat ------Rice Fish -CGAGTTCACAGAGAGGCTGCAGGGGCCCCTTCTGAAACACATCCGCTC-TCTCTGTCAG Clawed frog -AGAGTTCAGCGAGACAGCACTGTCAGCAACCAGAAACACCCCGCTGGC-CTAA---TGC Red junglefowl -CGGGTGCACCGGGACAGCAGCGTG---GCATTGGCCCAGCAGGCTGTG-CTCCCCGAAA Brown rat -CAGGTGCAGCGAGATGGAGCGGCTGCTGGCCTTGCCCAGGGTGGTCTG-CTCCCAGAGA Cattle -CAAGTTCAGCGGGATGGAGCAGCTGCTGGCCTTGCCCAAGGGGGTCTG-CTCCCGGAAA Humaun -CAGGTTCAGCGGGATGGAGCAGCTGCTAGCCTCGCCCAAGGGGGTCTG-CTCCCTGAAA Rhesus macaque -CAGGTTCAGCGGGATGGAGCAGCTGCTAGCCTCGCCCAAGGGGGTCTG-CTCCCTGAAA

Fruit fly GCGGACTATTGTTCCATTGCGAACTCTGTGCTGACCGCTCAGGTGA------CGCCC 2340 Yeast ACAACTTGAGAATAGTAAT--ACTTTTGAGGGAACTCACTAGCCTGATGATAACACCGTT Sorghum AGGCTTTGTGGCAGGCACTAGAGGTCAAAGGGATGATAC------C----TTCCAACAAC Zebrafish CTCCGCTCTGCTCCTCGCT-GCTGTCCAATGAGTCAGGCACCGGGCCT--GACAACCTGT Atlantic Salmon CCCCGCTCTGCTCCTCACT-GCTCTCCAACGGGTCCGGCAATGAACCT--GACAACCTAT Rainbow trout CCCCGCTCTGCTCCTCACT-GCTCTCCAACGAGTCCGGCAACGCACCT--GACAACCTAT Pufferfish AGTCT-----GAGCCACAA-AGTCTCATTGCAAACCTTCTTGGTGGCCCCTCGACGCTGG Cat ------Rice Fish AGTCT-----GAACCAAGG-AGCCTTGTAGCAAACCTCTTAGCAGGACCCTCCAGTCTGG Clawed frog ATCCTCTGCAGTGCTCAGG-CCTCTCTATTCAGTCCGACCCTGAGCCG--CACAGCCTGA Red junglefowl ATGCCCTCTTCAGCTCCAT-CCAGTCCCTGCAGTCAGAGTCAGAGCCT--CACAGCCTGA Brown rat ATGCCCTTTTCACATCCAT-CCAGTCCTTACAGTCTGAGTCTGAGCCA--CTGAGCCTCA Cattle ATGCCCTCTTTACGTCCAT-CCAGTCCTTACAATCCGAGTCTGAGCCA--CTGAGCCTTA

208

Humaun ATGCCCTCTTTACGTCTAT-CCAGTCCTTACAATCTGAGTCTGAGCCC--CTGAGCCTTA Rhesus macaque ATGCCCTCTTTACGTCTAT-CCAGTCCTTACAATCTGAGTCTGAGCCC--CTGAGCCTTA

Fruit fly CAGCAACTGGAGATCTCACAGTCGCTGCGTCCGGGTCTTGG------ACCGGT—ATC 2400 Yeast TGTACTTTGGTTTTCGTTGCCCTCGTCAGCCGGCAGGATTG------TAGATTTCTTCA Sorghum TCAATAGTGATATTCATG---GCCATGCTGAGACCACTTTGCCTCCTGTTTATAACTTGA Zebrafish TAGCCAGTG-TTTTGGCTCACCCTGTCCTGACGGCCTCGGGTCTGCCTGGAAGAA--ACC Atlantic Salmon TGGCAAGTG-TCCTGGCCCACCCTGTGCTGACCGCGTCAGGCCTACCAGGCCGAG--ACC Rainbow trout TGGCCAGTG-TCCTGGCCCACCCTATTCTGACCGCGTCAGGCCTACCCGGCCGAG--ACC Pufferfish AATCAATTCATTTCTGCAGGGACGGCTCTCTGGCCGGTCAC------ACAGCA--GCT Cat ------Rice Fish CCTCAGTTCATTTTGGTTGTGACAACTCTTTGATCAACCAT------GCAGTG--GCT Clawed frog TGGCGAATA-TGTTCTCTGGAGTGTCCCCTGCAGGGTTCCC---TGCTCACTCTG--AGA Red junglefowl TTGCCAATG-TGATAGCGGGCTCCTCGGTGCTGGGGTTCCA---CATGGGCCGTG--ATG Brown rat TTGCAAATG-TGGTAGCAGGCTCGTCCTGCCGAGGACCCCC---ACTGTCCAGAG--ACC Cattle TTGCAAATG-TGGTAGCTGGCTCATCCTGCCGGGGACCCCC---ACTGCCTAGAG--ACC Humaun TCGCAAATG-TGGTAGCTGGCTCATCCTGCCGGGGCCCTCC---ACTGCCCAGAG--ACC Rhesus macaque TTGCAAATG-TAGTAGCTGGCTCATCCTGCCGGGGCCCTCC---ACTGCCCAGAG--ACC

Fruit fly TGGAGGATTCCCCGTCGCAGCC------AGCGACTTCCG------CCAGATGTTGCAGC 2460 Yeast GAGAGAATTCTGAATATGTGGACGGATTAGGTTACGTTTGCAAGTATGCTATGTTTAACA Sorghum GCCCCTTGGGA-TTGCTCGACACGGACCAAAGAACTCATCCATATATCCTGGACTGGTAT Zebrafish GGCGCTTCATT-TCTCCCAGCAGCGCGGCGTCAGCGGCTGCTAGCGTCCTAGCGTCTCTC Atlantic Salmon GCCGCTTCGTT-CCTCCCAGCACGGCTGCCTCTGCCGCCGCCAGCGTCCTGGCCTCGCTA Rainbow trout GCCGCTTCGTC-CCTCCCAGCACGGCTGCCTCAGCCGCCGCCAGCGTCCTGGCCTCGCTG Pufferfish GGCATAATAGA-TGGGGCGTCCGCGCTACGCTCTCTATCCCCACTTACCAACAGTGTCCA Cat ------Rice Fish GGCAG---TGA-TGGAGCATCTGCCTTGCGTTCCCTTTCTCCTGTCAGCAGCAGTCTTCA Clawed frog TTCTGGCCTCT-CCTCATGTATCTGCTGCATCAGAGGTGGCTTCTGCTTTACGTTCTTTA Red junglefowl GACAGGCCTCT-CGTCATCTCTCT------GAAGTCGCTTCAGCCCTGCGTTCCTTC Brown rat TGCAGGGTTCC-AGGCACAGGGCT------GATGTTGCTTCTGCCCTTAGATCCTTC Cattle TGCAGGGCTCC-AGGCACAGGGCT------GAAGTTGCCTCTGCTCTGCGCTCCTTC Humaun TGCAGGGCTCC-AGGCACAGGGCT------GAAGTCGCCTCTGCCCTGCGCTCCTTC Rhesus macaque TGCAGGGCTCC-AGGCACAGGGCT------GAAGTGGCCTCTGCCCTGCGCTCCTTC

Fruit fly AGAATCTGTCCGCTAGCGTCGGCCCGC--TGGACAGTATGCGCCGCTTGCGCCT------2520

209

Yeast TGAAAAACATAGACG------GTGAAGATACACACAGCATGGATGAAGACAG Sorghum TACGTGTGTCATCCA-CCACACCAAGATAGAGCTGAGGCCCCCCAATTTG------Zebrafish TCATCTTCCCAGCAGCCTCA------CGCCAGCCGGTCGCGCTCACACACACTCCTGCCG Atlantic Salmon TCCAGCTCCCACTCTCAGCTCCCCCACGCCAGCCGCTCCCGCTCGCATGTCCATCTGCCT Rainbow trout TCCATCTCCCACTCCCAGCTCCCCCACGCCAGCCGATCCCACTCGCATGTTCTCCTGCCC Pufferfish GCTAAGGGGGAGTCTGAGTGTTGCTCACAGGCCTGCTGGTCACAACTTAGCCAT------Cat ------Rice Fish TCTGAGAGGCAGTCTGAGTATGGCTCAGAGAGCTAGTGGCT------Clawed frog TCCCCTCAGCAG----TCTTGTCACCC------Red junglefowl TCCCCACTCCAG----TCTGCCCAGCAGCCCTCTGCTGGCTTTCAGACAGGTGG------Brown rat TCCCCTCTACAG----CCTGGACAG------GCCCCTCAAGGCCGTGT------Cattle TCGCCTCTGCAG----CCTGGTCAG------GCTCCCACGGGCCGGGC------Humaun TCCCCGCTGCAA----CCCGGGCAG------GCGCCCACAGGCCGGGC------Rhesus macaque TCCCCGCTGCAA----CCCGTGCAG------GCGCCCACAGGCCGGGC------

Fruit fly ---CA--GC------CGGGCTGAAGGCCGCTTGGAAGGTCCGACGG------AT 2580 Yeast TTTAACGAAAAAGATTGCCGTGAACGGAAGTCATACACTCAACAGTAAACGAAGGAG--T Sorghum ---AA--CA------AGCATTCCCTGAGACTGGTGTGATCACAAGTCCACCAGCAAGGGA Zebrafish TCCCGACAG------CAACACGACGGACACATGTACAGCAGCGAGCACACAGTGGCGGAC Atlantic Salmon TCCA---CC------CACCCCGACAGCACCATGTACTGCAGTGACCGCACCATCATCGAC Rainbow trout TCCA---CC------CACCCCGACAGCACCATGTACTGCAGTGACCGCACTGTCATTGAC Pufferfish ---GAGCAA------AACACTGACCGGCTCTGGAACGGATGCACG------A Cat ------Rice Fish ------T------CACTGGCAAAGCAATGAGAACTGATGCTCA------C Clawed frog ---AGTGCA------GAACCAGAGTGGAGTCGGCACTGACTTCAG------A Red junglefowl ---TTCCAG------TGCCATGACAGCCTCTGGTGCTGATGCGAG------G Brown rat ---TCCCAG------TACCATGACAGGCTCTGGAGTGGACGCCAG------G Cattle ---TCCCAG------CACCATGACAGGCTCTGGGGTGGATGCCAG------G Humaun ---TCACAG------CACCATGACAGGCTCTGGGGTGGATGCCAG------G Rhesus macaque ---GCACAG------CACCATGACAGGCTCTGGGGTGGATGCCAG------G

Fruit fly ACACTGCTCTACGGACTCTGTGGAGTGGACCCGC--GCGTGGGCAGCACTCCCTTGAAC- 2640 Yeast AAATTTACCGCCGAAGACCATAGCGATAAAG-----ATTTGGCAAA------TAATA- Sorghum AACGTCTGAAATTGAAGAAGTTGGGGACTGG------GACTACGAG------TTGTACG Zebrafish AGCATGTCCGCTAGTGACCCCAGGATGCTCAGTCAGTCTCGTTCAGCGCTGGCGTCAGAG Atlantic Salmon AGCATGTCAGCCAGTGATTCTCGAATGA------AGAGCACTACATTGCTTTCAGAG Rainbow trout AGCATGTCAGCCAGTGACTCTCGGATCA------GGAGCACGACAATGCTTTCAGAG

210

Pufferfish ACTGTAAGCTCGGGCAGCAGTGCATGGGAGGGCCAGCTCACCAGTTTGGTACTTTCAGAG Cat ------Rice Fish ACTGTAAGCTCAGGCAGCAGTGCATGGGAGGGCCAGCTTACTAGCATGGTTTTATCAGAG Clawed frog AATGGCAGTTTTGGCAGCAATGTGTGGGAGGGCCCCATGAACAGCGGCACCCTGTCTGAA Red junglefowl ACCATGAGCTCAGGGAGCAGCGCCTGGGAGGGTCAGCTGCAGAGCATGATCCTGTCAGAG Brown rat ACAGCCAGCTCCGGGAGCAGCGTGTGGGAAGGACAGCTGCAAAGCCTGGTGCTGTCTGAA Cattle ACAGCCAGCTCCGGGAGCAGCGTGTGGGAAGGCCAACTGCAGAGCCTGGTGCTGTCAGAA Humaun ACAGCCAGCTCCGGGAGCAGCGTGTGGGAAGGACAGCTGCAGAGCCTGGTGCTGTCAGAA Rhesus macaque ACAGCCAGCTCCGGGAGCAGCGTGTGGGAAGGACAGCTGCAGAGCCTGATGCTGTCGGAA

Fruit fly GTGGGCGTGGCCGACATGTGCCTGAG-TGCCCTCTATTTGCACGAGCTGAACCAGCAGAA 2700 Yeast AAATGTTACAATCATATGTATATTTTATGGATGATTATTCCAACAG-TGAAAACTTAACA Sorghum AAAGGGTTCAAAGTCATCTGGGGGCA-TCCACACCAAGTGCTTTGT-----TCCGGAACA Zebrafish TTTGCGTCTGCAGAGATGAGTCTGCA-CGCAATATACATGCATGAGGTGCATCAGCAGAA Atlantic Salmon TTTGCCTCGGCTGAGATGAGTCTCCA-TGCTATTTACATGCATGAGGTCCACCAACAGAA Rainbow trout TTTGCCTTGGCCGAGATGAGTCTCCA-TGCTATTTACATGCATGAGGTCCACCAACAGAA Pufferfish TATGCATCCACTGAGATGAGCATTCA-TGCACTTTACATACATGAGCTACACAAGCAGCA Cat ------Rice Fish TACGCCTCTACTGAGATGAGCATTCA-TGCACTTTACATACATGAGTC------Clawed frog TACGCATCCACTGAAATGAGCCTGCA-CGCCCTGTACATGCACGAGTTGCACCAGCAGCA Red junglefowl TATGCCTCCACTGAGATGAGTCTCCA-CGCACTCTACATGCATGAGTTGCACAAGCAGCA Brown rat TATGCGTCCACTGAGATGAGCCTTCA-TGCCCTCTATATGCACCAGCTCCACAAGCAGCA Cattle TATGCATCCACTGAGATGAGCCTACA-TGCCCTCTACATGCACCAGCTCCACAAGCAGCA Humaun TATGCATCCACAGAGATGAGCCTGCA-TGCCCTCTATATGCACCAGCTCCACAAGCAGCA Rhesus macaque TACGCGTCCACAGAGATGAGCCTGCA-TGCCCTCTATATGCACCAGCTCCACAAGCAGCA

Fruit fly GCGCCAAGCG--CGACAGTCTCGCATCGACGAGGCCGAGGACGAGCGTC--CGGGTCCGA 2760 Yeast GGGAAGTACCAATTGCCTGCAAAAAAAGGCTATCCGAATAATGAAGGCGATTCATTTCTC Sorghum CTCCAGTGAA--GCACCATGGTACAGAAGATAAGACAAA------CA-GCAATTGGT Zebrafish GACCCATCAT--GC------ATCTGGACAGTTTCAG---GCCCCTGTGCCAATGAGG Atlantic Salmon CTCGCTCCCC--CACCAGAGGACATTCGGCCAGTGGCAG---AACCCAGTGCCAATGAGA Rainbow trout CTCGCTCCCC--CACCAGAGGACATCAGGCCAGTGGCAG---AACCCAATGCCAATGAGA Pufferfish GTCCCGTGGT--GAGCTATCCCGACACACATGGCACAGACAGGAGAGTGATGAAAGCAGT Cat ------Rice Fish ---TCGGGGT--GAGCTGTCCCGCCACACGTGGCACAGACGAGAGAGCGACGAAAGCAGC Clawed frog CACGCGCCTG--GCACCGGGGCGGCACATCTGGCACCGGCAGGAAAGTGATGAAAGCGGA Red junglefowl TGCCCAGCTG--GAGCCCGAGCGGCACACCTGGCACCGGCGAGAGAGTGACGAGAGCGGT

211

Brown rat GACCCAGGCT--GAGCCCGAGCGGCATGTGTGGCACCGCCGGGAGAGTGATGAGAGTGGA Cattle GGCCCAGGCT--GAACCTGAGCGGCATGTGTGGCACCGCCGGGAGAGTGATGAGAGTGGG Humaun GGCCCAGGCT--GAACCTGAGCGGCATGTATGGCACCGCCGGGAGAGTGATGAGAGTGGA Rhesus macaque GGCCCAGGCC--GAACCTGAGCGGCATGTATGGCACCGCCGGGAGAGTGATGAGAGTGGA

Fruit fly G----CCAC------TGGCCACCGCGACCACCGG------2820 Yeast AACAATAAGTATTCTTGGAGAAAGCAATTTC-AGCCAGGTCAAAAGCCGGAGCTATTT-- Sorghum G----GGCACATGC------CCCTGCATATCCTTCTGGTCCCCAGGGCAGCTTCAT Zebrafish G----ACATGAG------CGCCAGTGCTCCTCAGACGGTCACTCTGGTG-C Atlantic Salmon G----AGCTAGG------CAGCAACGCTGGTTCTCAGATGCACAGCGTCCA Rainbow trout G----ATCTAAT------CAGCAACACGGGTTCGCAGACGCACAGCGGCCA Pufferfish G----ACAGTGTCCCTGATGAAGTGAGGAGTGAACCCACTCCTCATTACAGGAATTTTCC Cat ------Rice Fish G----ACAGCATCCCAGACGAAGTAAGAAGCGACCACAATCCTCAGTCGAAAAGTTTCCC Clawed frog G----AAAGCTCACAGGGCGATGGCCTTATAAACACTACGTCTAACGTGCCA------Red junglefowl G----AGAGCACCCATGAGGAGCTGGACGCTCAGCGAGGTGCCCCCGTCCCCC---TCCC Brown rat G----AGAGTGCCCCTGAAGAGGGGGGAGAGGGTGCCCGGGCCCCCCAACCCA---TCCC Cattle G----AGAGTGCCCCTGAAGAGGGGGGAGAGGGGGCCCGGGCCACCCAGCCTA---TCCC Humaun G----AAAGCGCCCCTGATGAAGGGGGAGAGGGCGCCCGGGCCCCCCAGTCTA---TCCC Rhesus macaque G----AAAGTGCCCCTGATGAAGGGGGCGAGGGCGCCCGGGCCCCCCAGTCTA---TCCC

Fruit fly --CTGCTCCGTCGGCTGCCGCTGGCTCCGGCTCCC-GCCAT--AC------CGTGA 2880 Yeast --AGAATAGGTAAGCATGCTCTCGGCCCTGGCCAT-AATATTTCCCCTG------CTAT Sorghum CGAGCCTCCGGAATTTGGAAATCGATACATGACAGATCCTCATTCCAGTAATCACAGTGG Zebrafish ------CGCACACTTCAGGCCGTCTG------GGTGG Atlantic Salmon --CACAGCAGTCAGCC----TGTCTAATATGCCCACCCCGCTGTGTCTG------GGAGG Rainbow trout --CACAGCAGTCCACC----TGTCTAGCATGCCCACTCCGCTGCGTCTG------GGAGG Pufferfish --CCGTTCCTATACTTTCCCCGTGGCAGTTCCAAGCCCTAGTGCCATTCCAACGTCAGCC Cat ------Rice Fish --CCGCTCGCACACTTTTCCCACCGTGGTGTCCAGCCCAAGTGCCACTTCAACTTCAGCT Clawed frog ---TCCTCACTCAGCTGCCCCTCCCCTGCTGCCCCTGTTTGGGAAG------AGAGG Red junglefowl --TCGTTCTGCCAGCTACCCCTTCTCGTCGCGGCA---GCCTGCCGAGGAGACAGCCACG Brown rat --ACGCTCTGCCAGCTATCCCTGTGCTACTCCCCGGCCTGGAGCACCTGAGACCACCGCC Cattle --CCGCTCTGCCAGCTATCCCTGTGCTGCACCCCGGCCTGGAGCTCCTGAGACCACTGCC Humaun --TCGCTCTGCTAGCTATCCCTGTGCAGCACCCCGGCCTGGAGCTCCTGAGACCACTGCC Rhesus macaque --TCGCTCTGCCAGCTATCCCTGTGCAGCACCCCGGCCTGGAGCTCCCGAGACCACCGCC

212

Fruit fly TTACC--TCAAAGGCTGCCGAGAGCACGCCGCTTTTGGGT-AGCATCCGCTCA------2940 Yeast ATATTCTACAAGGAATCCCGGCAAGAACTGGGATAACAATAATAATGGTGATGACATTAA Sorghum CGATACATCAGAGGGAAGCATGGAGGATCTAGAGCAGAGCAATCGCAGAAGCAGCAGCGT Zebrafish ATGGG------CAGAGGAAGAGGAGGAGGAGCAGGGGGATGAAGAGGAGATCAACAGCAA Atlantic Salmon CTGGA------CGGAGGAAGAGGAGGGGGATGCAGAGGAAGAGGAAATCCTCAACAGCGG Rainbow trout CTGGA------CGGAGGAAGAGGAGGAGGATGCAGAGGAAGAGGAAGTCCTCAACAGGGG Pufferfish TCAGCCACCAGCAGTACCAGATGTGGCCAGGAGGTGGCCT---CATCACATAACTCTGGT Cat ------Rice Fish AACAG------CAGTAAGCCCAAAGGCCAGGAGGAAGAAAAATCACAGAGTCAGCGGCGC Clawed frog CTACC--TCCACTCAGCACACAC------AGATTTAGTGCCAGCACAG----ATCTTGGG Red junglefowl CTGCA--GACGGGCTTCCAGCGA------CGGTATGGCGGCATCACAG----ACCCAGGC Brown rat CTGCA--TGGGGGCTTCCAGAGG------CGCTACGGGGGCATCACAG----ATCCTGGC Cattle CTGCA--GGGGGGCTTCCAGAGG------CGTTATGGAGGCATCACAG----ATCCTGGC Humaun CTGCA--TGGGGGCTTCCAGAGG------CGCTACGGTGGCATCACAG----ATCCTGGC Rhesus macaque CTGCA--CGGGGGCTTCCAGAGG------CGCTACGGTGGCATCACAG----ATCCTGGC

Fruit fly ------3000 Yeast G------AACGGGACC----AATAATGCTACAGCTAAAAA--TGATGACAATAATGGCAA Sorghum GTGGAGGAGCCCGC------AAGCTTTGTCCAAGACAAGATATATGGACGACTCTGACAT Zebrafish C------CCTGAGCTC------CAGCACACCAGCAGAGGGAGC-AGCTGA--- Atlantic Salmon G------TCGACTCCT------AAACAGGACTCTAGGAGTAGC-TATTGA--- Rainbow trout A------TCGACTCCT------AAACAGGACTCCAGGAGTAGT-TGTTAA--- Pufferfish GCGTCTGGGAGCAAAGTAGTGAGGGTGGCACGTTCACCGATGGGTGGATG-GGCAGAGGA Cat ------Rice Fish CACAGTGGACCCGCCACAGTAAGGGTGGCTCGTGTACCTATGGGTGGATG-GGCAGTAGA Clawed frog TCTGTGCAAAGAACCT------CATTGGCTCGACAGCCAATGGGAGGGTG-GAGCGAGGA Red junglefowl ACAGTGCACAGAGCCCCATCACACTTCTCCCGCCTCCCACTGGGAGGCTG-GGCTGAGGA Brown rat ACAGTGCCCCGTGCTCCCTCTCACTTCTCCAGGCTGCCCTTGGGAGGATG-GGCAGAAGA Cattle ACAGTGCCCCGGGCTCCCTCTCACTTCTCTCGGCTGCCCCTGGGAGGATG-GGCTGAAGA Humaun ACAGTGCCCAGGGTTCCCTCTCATTTCTCTCGGCTGCCTCTTGGAGGGTG-GGCAGAAGA Rhesus macaque ACAGTGCCCCGGGCTCCCTCTCATTTCTCTCGGTTGCCTCTTGGAGGGTG-GGCGGAAGA

Fruit fly ------3060 Yeast TAATGATCATGAATACGTACTAACAGAGTCTTTCCTTGATTCGGGCGCATTCCCTAATCA Sorghum TGAGGAAGGGCTAAGCCTGCATTTTGCTGATGTCCATCAGAAGGATGAAGATGATAGACA Zebrafish ------

213

Atlantic Salmon ------Rainbow trout ------Pufferfish CAGCCAAGTGGCACCACGTCACCATGACCCTGTACCAGAGGGAAGCTCAGAGGACGAAAT Cat ------Rice Fish TGGTCAAGCAGCACCAAGACACCACGAGGCTGTGCCTGAAGAGAGTTCTGAGGATGAAAT Clawed frog TGTACA---GCCCAGCAGACACCCGGAAACTGTGCCAGAGGAAGGGTCTGAAGATGAGCT Red junglefowl CGGGCAGT---CAGCAAGACACCCAGAGCCTGTGCCAGAGGAGAGCTCAGAGGATGAGCT Brown rat TGGGCAGCCAGCATCAAGACACCCAGAGCCGGTGCCAGAGGAGGGCTCAGAGGATGAACT Cattle TGGGCAGTCAGCATCAAGGCACCCGGAGCCTGTGCCTGAAGAGGGCTCAGAGGATGAGCT Humaun TGGGCAGTCGGCATCAAGGCACCCTGAGCCCGTGCCCGAAGAGGGCTCGGAGGATGAGCT Rhesus macaque TGGGCAGTCGGCATCAAGGCACCCTGAGCCCGTGCCCGAAGAGGGCTCGGAGGATGAGCT

Fruit fly ------3120 Yeast TGATGTAATAGACCATAATAAAATGCTAAACTCAAATTACAATGGTAATGGCATACTCAA Sorghum CCTGGTCATGGA---TCATCAAGATCCCGCGCC---AGCTGGTCTTCCAGTAAGAATAA- Zebrafish ------Atlantic Salmon ------Rainbow trout ------Pufferfish ------GCC---TCCCCATGTCCACAAGGT---AAATACGTTTCACTGCAGTTTGTA Cat ------Rice Fish ------GCC---TCCTCACATCCACAAGGT---TAGCACCCTTAATGCAATAGATAT Clawed frog ------CCC---TCCGCAGGTTCAGAAGGT---ATAG------Red junglefowl ------TCC---ACCGCAGATCCACAAGGT---ATAG------Brown rat ------GCC---CCCTCAAGTGCACAAGGT---ATAG------Cattle ------CCC---CCCTCAGGTGCACAAGGT---ATAG------Humaun ------ACC---CCCTCAGGTGCACAAGGT---ATAG------Rhesus macaque ------ACC---CCCTCAGGTGCACAAGGT---ATAG------

Fruit fly ------3180 Yeast TAAGGGTGGTGTCT-TAGGACTTGTTAAAGAGTATTACAAGAAGTCTGACGTCGGAAGAT Sorghum ------TACCTAGAAGCAGTGACCCTGTGTAA------Zebrafish ------Atlantic Salmon ------Rainbow trout ------Pufferfish ---A------Cat ------Rice Fish ---AACTGCTTTGCTTGCATGTGCTGGACCTCAATAA------

214

Clawed frog ------Red junglefowl ------Brown rat ------Cattle ------Humaun ------Rhesus macaque ------

Fruit fly -- 3182 Yeast AA Sorghum -- Zebrafish -- Atlantic Salmon -- Rainbow trout -- Pufferfish -- Cat -- Rice Fish -- Clawed frog -- Red junglefowl -- Brown rat -- Cattle -- Humaun -- Rhesus macaque --

215

Appendix 2.2e) Clustal alignments between rainbow trout Atg genes and sequences from 14 other species.

CLUSTAL O(1.2.4) BECN1 multiple sequence alignment

Yeast ATGAAGTGCCAAACATGTCACTTACCCCTGCAACTAGACCCATCTTTAG-AAGGTCTTAG 60 Sorghum ATGAAGCCCCCG------GCGGGCGGCAGCGGCAAGAGCGGCGGGGTGGACC Fruit fly ------Clawed frog ------Red junglefowl ------Brown rat ------Cattle ------Human ------Rhesus macaque ------Zebrafish ------Cat ------ATGGTTTTGC Atlantic Salmon ------Rainbow trout ------Rice Fish ------Pufferfish ------

Yeast TTTAACCCAAAGAAATTTGCTACTTTCAAATAATTCAATTATCACTGCAACGAATGAAAA 120 Sorghum CTTCCCTGCCGCGGTTCAGGTGCCAGGAGTGCCGCCGCGCCCTCGTCGTCGTCGGGGTCG Fruit fly ------Clawed frog ------A Red junglefowl ------ATGGAGG Brown rat ------Cattle ------ATGGAGGGGT Human ------ATGGAAGGGT Rhesus macaque ------ATGGAAGGGT Zebrafish ------ATGGAGA Cat ------ATTACTTTAGCTGTAAAGATATTACTGAGGTATTAGAGAGGACCATGGAGG Atlantic Salmon ------ATGGGTGTTGCTATGGAGG Rainbow trout ------ATGGAGG Rice Fish ------ATGGA-- Pufferfish ------ATGGAGG Yeast TGTCA-TCAGCA----ACAAGGGCATCGAAGCCGCAGATAATTGTGGGCCTCAAATACCG 180

216

Sorghum ATTCCTACGCCGACAGGCTGCCCGCACATACCGCGCCCGGTATGCATGCATCTGCTGCTC Fruit fly ------ATGAGC----GAGGCGGAGAAACAGGCCGTGTCCTTCGCCTGCCAGCGCTGTCT Clawed frog TGGAG-ACCTCT----AAATCCTCTACAATGCAGGTCAGCTTTGTGTGTCAGCGATGCAG Red junglefowl GCGGA-CGTGCG----CCTGCCTGCACTACGCAGGTCAGCTTTGTGTGCCAGCGCTGCAG Brown rat ------Cattle ---CT-AAGACA----TCCAGCAGCACCATGCAGGTGAGCTTCGTGTGTCAGCGCTGCAG Human CTAAG-ACGTCC----AACAACAGCACCATGCAGGTGAGCTTCGTGTGCCAGCGCTGCAG Rhesus macaque CTAAG-ACGTCC----AACAACAGCACCATGCAGGTGAGCTTCGTGTGCCAGCGCTGCAG Zebrafish CTTTG-AGGTTT----TCTAGTAACACCATGCAGGTAAGTTTTGTGTGCCAGCGCTGCAA Cat GCTCC-AAGTCG----TCCAGCACCACCATGCAGGTGAGCTTCGTGTGTCAGCGCTGCAA Atlantic Salmon GCTCC-AAGTCC----TCTAGTACCACCATGCAGGTCAGCTTCGTGTGTCAGCGCTGCAG Rainbow trout GCTCC-AAGTCC----TCTAGTACCACCATGCAGGTCAGCTTTGTGTGTCAGCGCTGCAG Rice Fish -GGGC-TCCAAG----TTATCCAGCACAATGCAGGTGAGCTTCGTGTGTCAACGATGCTG Pufferfish TGTCC-AAATCA----TCGAGCACAACCATGCAGGTCAGCTTCGTGTGTCAGCGATGCTG

Yeast AAGGAGAGGCTGAGGCGATTAGGGGAGATTCAGAACATAAAAGACTTGAATTTAAAAGAT 240 Sorghum AGGGCAGTAC------TA--TGGGCGCAGGCAGCATGGACAGTTCTTATGTAGTAT- Fruit fly ------CCAGCCCATAGTGC-TGGACGAGCAACTGGAGAAG-- Clawed frog ------CCAGCCTCTGAAGT-TGGACACATCCTTCAAGATCC- Red junglefowl ------CCAGCCGCTGAAGC-TCGATACCTCCTTCAAGATCC- Brown rat ------TGAAAC-TGGACACGAGCTTCAAGATCC- Cattle ------CCAGCCTCTGAAAC-TGGACACGAGCTTCAAGATTC- Human ------CCAGCCCCTGAAAC-TGGACACGAGTTTCAAGATCC- Rhesus macaque ------CCAGCCCCTGAAAC-TGGACACGAGTTTCAAGATCC- Zebrafish ------TCAGCCTCTGAAGC-TCGACACATCCTTCAACGTCT- Cat ------CCAGCCTCTCAAAC-TCGACACTAGCTTCAACGTCC- Atlantic Salmon ------TCAGCCTCTAAAGC-TGGATACATCCTTCAATGTGC- Rainbow trout ------TCAGCCTCTAAAGC-TGGATACATCCTTCAATGTGC- Rice Fish ------CCAGCCGCTCAAAC-TGGACACGTCCTTCAATGTTC- Pufferfish ------TCAGCCGCTCAAGC-TGGACACGTCCTTTAATGTCC- *

Yeast GATAAATTAATCACTGATTCGTTTGTTTTTCTTAATC---ACGATGATGATGATAATGCT 300 Sorghum TATC----AAAGAACAATAGATCTCAAGGTCCTGGAATCCCCCCACGTCCACGAAGTGCA Fruit fly ------ATTAGCGTCCATGCCAT------Clawed frog --TG----GAT--AAGGTCACCATGCAAGAGCTGACAGCCCCTCTGGTGACAACAGCAGC Red junglefowl --TC----GAC--CGGCTCACCATCCAGGAGCTCACCGCGCCGCCACTGACCGCTGCACC Brown rat --TG----GAC--CGAGTGACCATTCAGGAACTCACAGCTCCATTACTTACCACAGCCCA

217

Cattle --TG----GAC--CGTGTCACCATCCAGGAGCTCACAGCTCCATTACTTGCCACAGCCCA Human --TG----GAC--CGTGTCACCATCCAGGAACTCACAGCTCCATTACTTACCACAGCCCA Rhesus macaque --TG----GAC--CGTGTCACCATCCAGGAACTCACAGCTCCATTACTTACCACAGCCCA Zebrafish --TG----GAC--AGAATGACTATCCACGAGCTCACTGCTCCGTTAGTGATGGTAACAGC Cat --TG----GAC--CGCCTCACCATCCAGGAGCTCATCGCTCCACTGGTCACTGTGACACA Atlantic Salmon --TC----GAC--CGTGTCACCATCCAGGAACTTATTGCTCCTCTGGTCACAGTGACACC Rainbow trout --TC----GAC--CGTGTCACCATCCAGGAACTTATTGCTCCTCTGGTCACAGTGACACC Rice Fish --TC----GAC--CGAGTCACCATCCTGGAGCTCACAGCGCCGCTGGTCACAGTGACACC Pufferfish --TC----GAC--CGCGTCACAGTCCATGAACTTACAGCACCGTTAGTCACAGTGACACC * * *

Yeast AA-TATTACGAGTAATAGCAGAGAGGACCAGCGTT---ATGGAAATGCCAACGGAAATGA 360 Sorghum G--CAGCCCCACATATTGAACCTAACCAACCACCAA--GGCCAATAGAGGGTTCATATAT Fruit fly ---GGTGCCCATATACAGTGACA------ATGGCAACACGCT Clawed frog AGTAAAGCCAGGTGACATCCAAGAAGTGGATTCAA------ATATAGAGGAAACCTTTGC Red junglefowl TGCCCGGCCCGGGGATGCGCAGGAGGAGAGCGCCC---TGAGCG---AGGAGGCCTTCAC Brown rat GGCGAAACCAGGAGAGAGCCAGGAGGAAGAGGCTAACTCAGGAGAGGAGCCATTTATTGA Cattle GCTGAAACCAGGAGAGACCCAGGAGGAAGAGGCTAACTCAGGAGAGGAGCCATTTATTGA Human GGCGAAACCAGGAGAGACCCAGGAGGAAGAGACTAACTCAGGAGAGGAGCCATTTATTGA Rhesus macaque GGCGAAACCAGGAGAGACCCAGGAGGAAGAGACTAACTCAGGAGAGGAGCCATTTATCGA Zebrafish CAATAAACAACAAGACAGTGGTGAGAGCAGCAGC------TTCCCTGAGGAGACCTTTTT Cat GAGCAAGCAAACAGAAAGCGGAAATGCAGACGCT------CCGGCCGAGGAGACGTTTGT Atlantic Salmon AAGCAAGCAGACAGAAAGCAATGAGGCAGAAAGT------GCTCCAGAGGAAACCTTTGT Rainbow trout AAGCAAGCAGACAGAAAGCAATGAGGCGGAAAGT------GCTCCAGAGGAAACCTTTGT Rice Fish CAGCAAGCAGGTTGACAGTAGCGACGGGGAGCCA------GCACCAGAGGAAACCTTTGA Pufferfish GAGTAAACAGGCGGAGAGCTCCGAGGGGGAGACG------GCACCAGAGGAGGCATTTGT *

Yeast TAATAAAAAAGCTAATAGTGATACTTCCGATGGAACCAGTACCTTTCGAGATCATGACGA 420 Sorghum TATGCTTCCACCTCCTGCTGCTTCCATATACAAGA--CATCTTCCTCTGAAGGAGTTGGT Fruit fly GGACACACAGGATGCCAG--CAGCTTTGACCACTN--TGTGCCGCCGTACAGGCTGACAG Clawed frog AGAAAACCGAACAGATGG--TGTATCCCGAAGACT--AATCCCTCCAGCCAGAATGATGT Red junglefowl GGAGGGTCGGCAGGACGG--CGTCTCCAGGAGGTT--CATCCCACCAGCCAGAATGATGT Brown rat AACT---CGCCAGGATGG--TGTCTCTCGAAGATT--CATCCCCCCAGCCAGGATGATGT Cattle AACT---CGCCAGGATGG--TGTCTCTCGCAGATT--CATCCCCCCAGCCAGGATGATGT Human AACTCCTCGCCAGGATGG--TGTCTCTCGCAGATT--CATCCCCCCAGCCAGGATGATGT Rhesus macaque AACTCCTCGCCAGGATGG--TGTCTCTCGCAGATT--CATCCCCCCAGCCAGGATGATGT Zebrafish GGAGAACAAACAAGATGG--CGTGGCTCGAAAATT--TATACCACCTGCAAGGATGATGT Cat GGAGAGTAAGCAGGACGG--CGTCTCGCGCAAGTC--CATCCCTCCTGCCAGGATGATGT

218

Atlantic Salmon GGAAACAAAGCAAGATGG--CGTCTCAAGAAAATA--CATCCCTCCTGCAAGGATGATGT Rainbow trout GGAAACAAAGCAAGATGG--AGTCGCAAGAAAATA--CATCCCTCCTGCAAGGATGATGT Rice Fish AGAAAACAAGCAAGATGG--AGTGTCAAGAAAGTA--CATTCCTCCTGCGCGAATGATGT Pufferfish AGAAAACAAACAAGATGG--CGTGTCAAGAAAGTA--CATCCCACCAGCACGCATGATGT *

Yeast AG-----AGGAACAAGAAGCTACGGATGAGGACGAAAACCAACAAATACAATTGAATTCC 480 Sorghum AGACAGCTGCTGCCACCAAGTG----TTAACTCCAGTAGCTCTATG-C-CAGGAAATAAT Fruit fly ACTCCATCAACGGAACTGGCTTCATGCTCG------TTTCGGATG-GCCGTGATAAC Clawed frog CCACAGAAAGTGCAACTAGTTTCACACTCATAGGAGAAGCATCCGATG-GAGGCACCATG Red junglefowl CAACAGAAAGCGCCAACAGCTTCACGCTGATTGGAGAGGCATCAGATG-GAGGCACGATG Brown rat CTACAGAAAGTGCTAATAGCTTCACTCTGATCGGGGAGGCATCTGATG-GTGGCACCATG Cattle CCACAGAAAGTGCCAACAGCTTCACTCTGATTGGGGAGGCATCTGATG-GCGGCACCATG Human CCACAGAAAGTGCCAACAGCTTCACTCTGATTGGGGAGGTATCTGATG-GCGGCACCATG Rhesus macaque CCACAGAAAGTGCCAACAGCTTCACTCTGATTGGGGAGGCATCTGATG-GCGGCACCATG Zebrafish CAGCTGAAAGCACAAACAGTTTCACACTTATTGGAGAAGCTTCTGATG-GTGGAACTATG Cat CCACAGAAAGCGCTAACAGCTTCACGCTGATCGGCGAGGCCTCGGACG-GAGGAACCATG Atlantic Salmon CCACAGAGAGTGCCAACAGCTTCACTCTGATTGGAGAAGCGTCGGACG-GAGGCACCATG Rainbow trout CTACAGAGAGCGCCAACAGCTTCACTCTGATTGGAGAAGCGTCGGACG-GAGGCACCATG Rice Fish CAACAGAGAGCGCGAACAGCTTCACACTGATAGGAGATGCATCAGACG-GAGGCACCATG Pufferfish CAACGGAGAGCGCCAACAGCTTCACGCTGATTGGAGAAGCATCTGATT-GTGGCAACATG * *

Yeast AAGACTTTGTCCACACAAGTCAACGCAATGACTAATGTATTTAACATTCTATCTTCTCAA 540 Sorghum TCTGGATTTTTCTCTAGTGTTACTGTATTGAAAAGAGCATTTGAGATTGCTACCTCACAA Fruit fly ACGAAGATGAGTGCTGCCTTCAATCTGAAGGCAGAGCTGTTCGACTGCTTGTCCTCCAAC Clawed frog GAGAATCTGAGCAGAAGGCTAAAGGTGACGGGGGATTTGTTTGACATTATGTCAGGGCAG Red junglefowl GAAAACCTCAGCAGAAGACTGAAGGTTACTGGTGATCTCTTTGATATCATGTCTGGGCAG Brown rat GAGAACCTCAGCCGGAGACTCAAGGTCACTGGAGACCTTTTTGACATCATGTCTGGCCAG Cattle GAGAACCTCAGCCGAAGACTAAAGGTCACTGGGGACCTTTTTGACATCATGTCAGGCCAG Human GAGAACCTCAGCCGAAGACTGAAGGTCACTGGGGACCTTTTTGACATCATGTCGGGCCAG Rhesus macaque GAGAACCTCAGCCGAAGACTGAAGGTCACTGGAGACCTTTTTGACATCATGTCGGGCCAG Zebrafish GAGAACTTGAGTCGCAGACTGAAAGTGACAAGCAATCTGTTCGACATCATGTCTGGTCAG Cat GAGAACCTGAGCCGCAGGTTAAAGGTAACGAGCGATCTGTTCGACATCATGTCGGGTCAG Atlantic Salmon GAGAACCTCAGTCGGAGGCTGAAGGTGACCAGCGATCTGTTTGACATCATGTCAGGTCAG Rainbow trout GAGAACCTCAGTCGGAGGCTGAAGGTGACCAGCGATCTGTTTGACATCATGTCGGGCCAG Rice Fish GAAAATCTTAGCCGAAGACTTAAGGTTACCAGTGATTTGTTTGACATCATGTCTGGCCAG Pufferfish GAACATCTCAGTCGTAGGCTGAAGGTCACCAGTGACCTGTTTGACATCATGTCAGGCCAG * * * ** * * *

219

Yeast ACAAACATAGACTTTCCTATCTGTCAGGACTGCTGTAACATCTTGATAAACAGACTAAAA 600 Sorghum ACTCAGCTTGAACAGCCACTTTGTCTGGAATGTATGAGGGTTCTTTCTGATAAGATGGAT Fruit fly TCGGAGATAGACCATCCCCTGTGCGAGGAGTGTGCCGACTCCATGCTGGAGATCATGGAT Clawed frog ACAGATGTGGACCATCCACTTTGTGAGGAATGCACAGATACTCTTTTGGACCAGCTGGAT Red junglefowl ACAGATGTGGATCACCCCTTGTGTGAGGAGTGCACAGATACTCTGCTAGACCAGCTAGAC Brown rat ACAGATGTGGATCACCCACTGTGTGAGGAATGCACAGATACTCTTTTAGACCAGCTGGAC Cattle ACAGATGTGGATCACCCCCTGTGTGAGGAATGCACAGACACTCTCCTAGACCAGCTGGAC Human ACAGATGTGGATCACCCACTCTGTGAGGAATGCACAGATACTCTTTTAGACCAGCTGGAC Rhesus macaque ACAGATGTGGATCACCCACTCTGTGAGGAATGCACAGATACTCTTTTAGACCAGCTGGAC Zebrafish ACAGATATTGATCACCCACTTTGTGAGGAGTGCACTGACACATTATTGGACCACTTGGAC Cat AGCGACGTGGATCACCCGCTGTGTGAGGAGTGTACAGACACGCTGCTCGATCACCTGGAC Atlantic Salmon ACCGACGTAGACCACCCGCTGTGCGAGGAGTGTACAGACACCCTGCTAGACCACCTGGAC Rainbow trout ACCGACGTAGACCACCCGCTGTGCGAGGAGTGTACTGACACCCTGCTAGACCACCTGGAC Rice Fish ACTGATGTGGATCACCCTCTGTGTGAGGAATGCACCGACACGCTATTGGATCACCTGGAC Pufferfish ACAGACGTGGACCACCCGCTCTGTGAGGAGTGTACCGACACACTGCTGGACCACCTCGAC * * ** ** * ** *** ** * * * *

Yeast AGTGAGTATGATGATGCAATAAAAGAGAGAGATACTTACGCTCAATTTTTATCGAAGTTA 660 Sorghum AAGGAGATTGAAGATGTTAATGCTGATATTAAAGCTTATGAGGCTTGTCTTCAACGTTTG Fruit fly CGAGAGCTGCGCATAGCCGAAGACGAGTGGGATGTGTACAAAACATATCTGGATGAGCTA Clawed frog ACTCAGTTAAACATCACTGAGAATGAGTGTCAAAATTACAAACGGTGCCTGGAGATTTTG Red junglefowl ACACAGCTTAATATCACAGAGAATGAATGCCAGAACTACAAGAGATGTCTGGAGATACTG Brown rat ACTCAGCTCAATGTTACTGAAAACGAGTGTCAGAACTACAAACGCTGTTTGGAGATGTTG Cattle ACTCAGCTCAACGTCACTGAAAATGAATGTCAGAACTACAAACGCTGTTTGGAGATCTTA Human ACTCAGCTCAACGTCACTGAAAATGAGTGTCAGAACTACAAACGCTGTTTGGAGATCTTA Rhesus macaque ACTCAGCTCAACGTCACTGAAAATGAGTGTCAGAACTACAAACGCTGTTTGGAGATCTTA Zebrafish ACACAACTCAACATCACTGAGAACGAGTGCCAGAATTACAAGAGCTGTTTAGAGCTGCTG Cat ACACAGCTTAACATCACCGAGAACGAGTGTCAGAACTACAAGAAGTGTCTGGAGCTTCTA Atlantic Salmon ACGCAGCTCAACATCACAGAGAACGAGTGCCAGAACTACAAGAACTGCCTGGAACTGCTG Rainbow trout ACGCAGCTCAACATCACAGAGAACGAGTGCCAGAACTACAAGAACTGCCTGGAGCTGCTG Rice Fish ACGCAGCTCAACATTACAGAGAACGAGTGCCAGAATTACAAACAATGTCTGGAGCTGCTG Pufferfish ACACAGCTCAATATCACAGAGAACGAATGCCAGAATTACAAGCAATGTCTGGAGCTGCTG * ** * ** * * *

Yeast GAGAGTCAAAACAAAGAAATATCAGAAAGCAATAAAGAAAAGCAGTATTCCCATAATCTT 720 Sorghum GAGCAGGAACCTTACAACATCCTC------AGTGA------AACAGATTTTCAA Fruit fly GAGCAGCAGCGGCAGGCCCCCAAT------GT------GGATGCCCTGGAC Clawed frog GAAAGAATGAATGAAGATGACAAA------GAGAAACTGGAA

220

Red junglefowl GAAAAAATGAACGAGGATGATAAG------GAGAAACTGCAA Brown rat GAGCAAATGAATGAGGGCGACAGT------GAACAGCTACAG Cattle GAGCAAATGAATGAAGATGACAGT------GAACAGCTTGGG Human GAGCAAATGAATGAGGATGACAGT------GAACAGTTACAG Rhesus macaque GAGCAAATGAATGAGGATGACAGT------GAACAGCTACAG Zebrafish TCTCAGCTTCCAGAAGAAGAGGAG------GCCAGTCTACTG Cat TCGCAGCTGCAGGAGGAAGACGAG------GACACGCTGCTG Atlantic Salmon TCCCAGCTGAAGGAGGAGGAGGAG------GACAGCCTGTTG Rainbow trout TCCCAGCTGAAGGAGGAGGAGGAG------GACAGCCTGTTG Rice Fish TCGAACCTCCAGGCAGAAGACGAG------AAGACCCTCCTG Pufferfish TCAAATCTGCAGGTGGAGGACGAG------GAAACCCTGCTG

Yeast TCGGAAAAAGAAAATTTGAAAAAGGAAGAGGAAAGACTCTTGGATCAATTATTACGTTTG 780 Sorghum AAGGAGAAACAAAAGATTGAGGAAGAGGAAAAGAAACTTAAAGCTGCTATTGAAGAAGCT Fruit fly AAGGAGCTGGACGAACTGAAGAAGAGTGAACAGCAGCTGCTGTCGGAGCTGGGTAAGCTA Clawed frog GCCAAACTGAAGGAGCTAGCCGAGGATGAGGATAGACTTATTCAGGAGTTAGAGGAGGTG Red junglefowl ACAGAACTGAAAGAACTTGCACTGGAAGAAGAACAACTGATTCAGGAGCTAGAAGATGTT Brown rat AGGGAGCTGAAGGAGTTGGCCTTGGAGGAGGAGAGGCTGATCCAGGAGCTGGAAGATGTG Cattle TTAGAGCTAAAGGAGCTGGCATTAGAGGAGGAGAGGCTGATCCAAGAGCTGGAAGATGTG Human ATGGAGCTAAAGGAGCTGGCACTAGAGGAGGAGAGGCTGATCCAGGAGCTGGAAGACGTG Rhesus macaque ATGGAGCTAAAGGAGCTAGCACTAGAGGAGGAGAGGCTGATCCAGGAGCTGGAAGACGTG Zebrafish AATGCATTGCAGCAGTTGAAACAAGAAGAGGAATCTCTGATCCAGGAACTAGAGAGCATT Cat AAGGAGCTGCAGCAGCTCAGGCAGGAGGAGGAGACGCTCATCCAGGAGCTGGAGGCCATC Atlantic Salmon CTGGAGCTCCAGAAACTGGGCGAGGAGGAGTCGTCACTGGTGGGGGAGCTGGAGGCGGTG Rainbow trout CTGGAGCTCCAGAAACTGGGCGAGGAGGAGTCGTCACTGGTGGGGGAGCTGGAGGCGGTG Rice Fish GCAGAATTGCAAGAGTTAAAGGATGAGGAGGCCTCTCTGGTCCAGGAGCTTGAGGCTGTG Pufferfish GCCGAGCTGCAGCAGCTGAAGGAGGAGGAGGCAGCGCTGGTGGAGGAACTGGAGACGGTG * * * ** ** *

Yeast GAAATGACAGATGATGACTTAGATGGAGAACTAGTTCGTTTACAAGAAAAGAAAGTTCAA 840 Sorghum GAAAAACAATATTCAGAAGTTAGATCTGAGATGGAAAATCTTGAAATAAAGTCTAAACAA Fruit fly AAGGAGGATGAACAATCACTGAACGCTGCCATAGCGCAAGAGGAGTTGGAGAAGGAGAAG Clawed frog GAACGAAACCGCGAGTTAGTTGCTAAGGACATCGAGAAAGTACGAGAGGAAGCAGAGAGG Red junglefowl GAGAAGAACCGCAAGATTGTGGCTGAAGACTTTGAGAGAGTCAGGGCAGAGGCAGAGCGA Brown rat GAAAAAAACCGAAAGGTGGTGGCAGAAAACCTGGAGAAGGTCCAGGCTGAGGCGGAGAGA Cattle GAAAAGAACCGGAAGATAGTGGCGGAAAATCTCGAGAAGGTCCAGGCTGAGGCTGAGAGG Human GAAAAGAACCGCAAGATAGTGGCAGAAAATCTCGAGAAGGTCCAGGCTGAGGCTGAGAGA Rhesus macaque GAAAAGAACCGCAAGATAGTGGCAGAAAATCTCGAGAAGGTCCAGGCTGAGGCTGAGAGA

221

Zebrafish GAGACAAAGCGTGAAGCCGTGGCCAAAGAGCTGGATGAGGGACGCAACCACAGCCAGCTG Cat GAGCAGCAGAGGGAATCCGTCGCCATGGAGATCACAGAGGCACGACGCCACGCCCAGCAG Atlantic Salmon GAAGAGCAGAGGGCTGCCGTGGCCGAGGACCTGGTCCAGGGACGCAGCCACTCCCAGCAG Rainbow trout GAAGAGCAGAGGGCTGCCGTGGCCGAGGAACTGGTCCAGGGACGCAGCCACTCCCAGCAG Rice Fish GAAGAGCAGAGAGCTGCTGTTGCCCAGGACTTAGCCCAAAGCAGGATCCAGTCCCAGCAG Pufferfish GAGGAGCAGAGGGCTGCTGTGGCCGACGACCTGGCCCAGTGCAGGGTCCACTCCCAACAG * * * *

Yeast CTGGAAAATGAGAAACTCCAAAAACTGAGTGATCAGAATTTAATGGATTTAAATAACATT 900 Sorghum TTTGAGGAATTGGAAGAGCGGTATTGGCATGAATTCAACAGTTTTCAGTTTCAGTTGACA Fruit fly CTGCACGAGCAGGAGGAGAGCTACTGGCGCGAGTACACCAAACACCGGCGTGAGTTGATG Clawed frog CTGGAGCAGGAAGAGGCTCGCTATCAGAAGGAGTACAGTGAGTTCAAGCGCCAACAGCTG Red junglefowl CTGGAGCAGGAAGAAGCTCAGTATCAGAAAGAATACTGTGAATTTAAGAGACAACAACTG Brown rat CTGGACCAGGAGGAAGCTCAGTACCAGCGAGAATATAGTGAATTTAAAAGGCAGCAGCTG Cattle TTGGATCAGGAGGAAGCTCAGTATCAAAGGGAATACAGTGAATTTAAAAGACAACAGCTG Human CTGGATCAGGAGGAAGCTCAGTATCAGAGAGAATACAGTGAATTTAAACGACAGCAGCTG Rhesus macaque CTGGATCAGGAGGAAGCTCAGTATCAGAGAGAATACAGCGAATTTAAACGGCAACAGCTG Zebrafish ATGGACACTGAAGAACTTCGGTATCAGAAGGAGTACTGTGAGTTTAAGAGACAGCAGCTG Cat CTGGATACCGAGGAGCTGCAGTACCAGAAGGAGTACTGCGAGTTTAAACGGCAGCAGCTG Atlantic Salmon CTAGACACTGAGGAACTGCAGTACCAGAAGGAGTACAGTGAGTTCAAGCGGCAGCAGCTG Rainbow trout CTAGATACTGAGGAACTGCAGTACCAGAAGGAGTACAGTGAGTTCCAGCGGCAGCAGCTG Rice Fish CTGGATACAGAGGAGCTACAGTACCAGAAAGAATACAGCGAGTTTAAACGACAGCAGCTA Pufferfish CTCGACACAGAGGAGCTCCAGTATCAGAAAGAGTACAGCGAGTTCAAACGGCAGCAGCTG * * * * ** *

Yeast CAATTCAACAAAAATTTACAGTCATTAAAACTGCAATACGAATTATCCTTGAATCAATTA 960 Sorghum TCTCACCAGGAAGAAAGAGACTCAGTTTTTGCCAAGATATATGTTTCCCAGGTTCATCTG Fruit fly CTCACCGAGGACGACAAACGGAGCCTGGAGAGCCAGATCAGCTACTCGAAGCAGCAGCTG Clawed frog GAGTTAGATGATGATTTAAAGAGTGTGGAGAATCAGATGAGATATGCTCAGATCCAGTTG Red junglefowl GAGCTAGATGATGAACTGAAAAGTGTGGAGAACCAGATGCGTTATGCTCAGATGCAACTG Brown rat GAGCTGGATGATGAGCTCAAGAGTGTAGAGAACCAGATGCGCTATGCCCAGATGCAGCTG Cattle GAACTGGATGATGAGCTGAAGAGTGTAGAAAACCAGATGCGTTATGCCCAGATGCAGCTG Human GAGCTGGATGATGAGCTGAAGAGTGTTGAAAACCAGATGCGTTATGCCCAGACGCAGCTG Rhesus macaque GAGCTGGATGATGAGCTGAAGAGTGTGGAAAACCAGATGCGTTATGCACAGATTCAGCTG Zebrafish GAGTTGGATGATGATCTTAAAAGCGTGGACAATCAGATGCGTTACTGTCAGATTCAACTT Cat GAGCTGGACGATGAACTGAAGAGTGTGGACAATCAGATGCGCTACTGCCAGGCTCAACTG Atlantic Salmon GAGCTGGATGACGAGCTGAAGAGTGTGGACAACCAGATGCGATACTGCCAGATCCAACTG Rainbow trout GAGCTGGATGACGAGCTGAAGAGTGTGGACAACCAGATGCGATACTGCCAGATTCAACTG Rice Fish GAGTTGGACGACGAGCTGAAGAGCGTCGACAACCAGATGCGATACTGTCAGATCCAGCTG

222

Pufferfish GAGCTGGACGACGAGCTGAAGAGTGTCGACAACCAGATGCGCTACTGCCAGATTCAGCTG * * * * * * ** *

Yeast GATAAGTTGAGGAAAATCAATATTTTCAATGCTACATTCAAAATCTCACATAGCGGCCCC 1020 Sorghum GAACTGTTGAAGCGTACTAATGTTCTTAATGATGCATTCTATATTTCACATGATGGAGTA Fruit fly GACAAGCTGCGCGACACGAACATCTTCAACATCACCTTTCACATCTGGCATGCCGGGCAC Clawed frog GACAAGCTTAAAAAGACCAATGTCTTCAATGCCACTTTTCATATCTGGCATAGCGGGCAG Red junglefowl GATAAATTAAAGAAAACTAATGTGTTCAATGCAACTTTCCACATCTGGCACAGTGGTCAG Brown rat GACAAGCTCAAGAAAACCAATGTCTTCAATGCGACCTTCCATATCTGGCACAGCGGACAA Cattle GACAAGCTGAAGAAAACCAACGTCTTTAATGCAACCTTCCACATTTGGCATAGTGGACAG Human GATAAGCTGAAGAAAACCAACGTCTTTAATGCAACCTTCCACATCTGGCACAGTGGACAG Rhesus macaque GATAAGCTGAAGAAAACCAATGTCTTTAATGCAACCTTCCACATCTGGCACAGTGGACAG Zebrafish GACAAACTCAAGAAGACCAACGTTTTCAATGCGACCTTTCATATCTGGCATAGTGGGCAG Cat GACCGGCTGAAGAAAACCAACGTCTTTAACGCCACCTTTCACATCTGGCACAGCGGGCAG Atlantic Salmon GACCGCCTGAAGAAGACCAACGTCTTCAACGCCACCTTCCACATCTGGCACAGTGGCCAG Rainbow trout GACCGCCTGAAGAAGACCAACGTCTTCAACGCCACCTTCCACATCTGGCACAGTGGCCAG Rice Fish GACCGACTGAAGAAAACCAACGTTTTTAATGCAACATTTCACATTTGGCATAGCGGCCAG Pufferfish GATCGTTTAAAAAAGACCAATGTCTTTAATGCCACGTTTCACATCTGGCACAGTGGCCAG ** * * ** * * ** * ** * ** * ** **

Yeast TTTGCCACTATAAATGGATTGAGATTAGGCAGTATACCAGAATCAGTGGTACCTTGGAAG 1080 Sorghum ATTGGAACAATAAATAATTTCCGCCTCGGCCGCCTTTCTAATGTAGAGGTTGAGTGGGAT Fruit fly TTTGGCACCATCAACAACTTCCGCCTGGGCCGCCTGCCCTCCGTTTCCGTCGACTGGTCG Clawed frog TTTGGCACAATTAACAACTTTCGTCTCGGCCGTCTGCCTAGTGTGCCAGTGGAATGGAAT Red junglefowl TTTGGCACAATAAATAACTTCAGACTTGGCCGTCTCCCCAGTGTTCCTGTAGAATGGAAT Brown rat TTTGGCACGATCAATAATTTCAGACTGGGTCGCTTGCCCAGTGCTCCTGTGGAATGGAAT Cattle TTTGGTACAATCAATAACTTCAGACTGGGTCGGCTGCCCAGTGTTCCTGTGGAATGGAAT Human TTTGGCACAATCAATAACTTCAGGCTGGGTCGCCTGCCCAGTGTTCCCGTGGAATGGAAT Rhesus macaque TTTGGCACAATCAATAACTTCAGGCTGGGTCGCCTGCCCAGTGTTCCCGTAGAATGGAAT Zebrafish TTTGGAACAATAAATAACTTCCGCCTTGGCAGATTACCCAGCGTACCAGTGGAATGGAAT Cat TTTGGGACGATAAATAATTTCCGTCTGGGGCGATTACCCAGCGTTCCTGTGGAGTGGAAC Atlantic Salmon TTTGGCACCATCAATAACTTCCGTCTGGGCCGACTACCCAGTGTCCCCGTCGAGTGGAAC Rainbow trout TTTGGCACCATCAATAACTTCCGTCTGGGCCGACTACCCAGTGTCCCCGTCGAGTGGAAC Rice Fish TTTGGTACCATCAATAACTTCAGACTGGGTCGACTTCCCAGCGTCCCTGTGGAGTGGAAT Pufferfish TTTGGCACAATCAACAACTTCCGCCTGGGTCGACTCCCCAGCGTCCCCGTGGAGTGGAAC *** ** ** ** ** * * ** * * * ** ***

Yeast GAAATCAATGCAGCACTGGGCCAATTGATTCTTTTACTGGCTACCATAAACAAAAACCTG 1140 Sorghum GAGATAAATGCTGCTTGGGGTCAGGCTGCACTGCTGTTGCATACCATGGCTCAGTATTTC

223

Fruit fly GAGATAAATGCCGCCTGGGGGCAGACGGTGCTGCTCCTCTCCGCTCTGGCCCGCAAGATT Clawed frog GAGATTAATGCAGCCTGGGGCCAGACTGTTCTACTGCTTCATGCACTAGCCAATAAGATG Red junglefowl GAGATCAATGCAGCCTGGGGGCAGACTGTGCTGTTACTGCATGCCCTTGCTAACAAAATG Brown rat GAAATCAATGCTGCCTGGGGCCAGACAGTGTTGTTGCTCCATGCTTTGGCCAATAAGATG Cattle GAGATTAATGCTGCTTGGGGCCAGACAGTGTTGCTGCTCCACGCCCTGGCCAATAAGATG Human GAGATTAATGCTGCTTGGGGCCAGACTGTGTTGCTGCTCCATGCTCTGGCCAATAAGATG Rhesus macaque GAGATTAATGCTGCTTGGGGCCAGACTGTGTTGCTGCTCCATGCTCTGGCCAATAAGATG Zebrafish GAGATTAATGCAGCCTGGGGTCAAACTGTTCTTCTGTTGCATGCACTTGCCAGCAAGATG Cat GAGATCAACGCTGCCTGGGGTCAGACTGTCCTGCTGCTGCACGCTCTCGCGAACAAGATG Atlantic Salmon GAGATCAACGCCGCCTGGGGTCAGACCGTGCTACTGCTCCACGCCCTCGCCAACAAGATG Rainbow trout GAGATCAACGCCGCCTGGGGTCAGACCGTGCTACTGCTCCACGCCCTCGCCAACAAGATG Rice Fish GAGATCAATGCAGCTTGGGGGCAGACGGTTCTTCTGCTGCATGCTCTGGCCAACAAAATG Pufferfish GAGATCAATGCAGCCTGGGGCCAGACGGTGCTGCTACTGCACGCTCTGGCCAATAAGATG ** ** ** ** ** *** ** * * * * * * *

Yeast AAGATAAATTTAGTAGACTACGAATTACAAC—CGATGGGTTCATTTTCCAAAATCAAGA 1200 Sorghum ACCCCAAAATTCCAATACCGGATCAAGATTCACCCTATGGGAAGCTATCCAAGAGT---- Fruit fly GGCCTCACCTCTGAGCGGTATCGCGTCAAAG--CGTTCGGCAATCACTCGTATGTGGAGG Clawed frog GGGCTGCAGTTCCAGAGGTACCGGTTAATGC--CATTTGGAAACCATTCCTATTTAGAGT Red junglefowl GGCCTGAAGTTTCAAAGATACCGTCTTGTAC--CGTATGGCAACCACTCGTATTTAGAGT Brown rat GGTCTGAAATTTCAGAGGTACCGACTTGTTC--CCTATGGAAATCACTCGTATCTGGAGT Cattle GGTCTGAAATTTCAGAGGTATCGACTTGTTC--CCTATGGAAACCATTCATATCTGGAGT Human GGTCTGAAATTTCAGAGATACCGACTTGTTC--CTTACGGAAACCATTCATATCTGGAGT Rhesus macaque GGTCTGAAATTTCAGAGATATCGACTTGTTC--CTTACGGAAACCATTCATATCTGGAGT Zebrafish GGGCTATGCTTTCAGAGATACCAATTAGTTC--CGTATGGAAATCATTCCTACTTGGAGT Cat GGGCTGCGCTTCCAGAGATATCGGCTCGTTC--CTTACGGAAACCACTCGTACCTGGAGT Atlantic Salmon GGGCTACGCTTCCAGAGATACCGCCTGGTCC--CATATGGAAACCACTCTTACCTGGAGT Rainbow trout GGGCTACGCTTCCAGAGATACCGCCTGGTCC--CATATGGAAACCACTCTTACCTGGAGT Rice Fish GGACTGTGTTTTCAAAGATACCGGCTTGTAC--CATATGGAAATCACTCCTACTTAGAGT Pufferfish GGGCTGCGCTTCAAAAGATATCGTCTTGTTC--CATATGGAAACCACTCATACTTGGAGT * * ** ** *

Yeast AAAGAATGGTTAACAGTGTCG-AATATAATAATTCCACTACGAACGCTCCCGGTGATTGG 1260 Sorghum CACAGACA------TCCACAATAATACA-TATGAACTGTTTGGTCCCGTGAATTTGTTCT Fruit fly TGCTGGGCGTG---AACGGCGAACTGCCGCTCTACGGAAGCGGGGGATTCAAGTTCTTTT Clawed frog CTCTTACAGACAAGTCTAAGGAATTGCCCCTGTACTGTTCAGGTGGTTTGCGGTTCTTCT Red junglefowl CCCTCACGGACAAATCGAAGGAGCTACCCTTGTACTGTTCTGGAGGCCTGAGGTTCTTCT Brown rat CCCTGACAGACAAATCTAAGGAGTTGCCGTTGTACTGTTCTGGGGGTTTGCGGTTTTTCT Cattle CTCTGACAGACAAATCTAAGGAGCTGCCGTTGTACTGTTCAGGGGGGCTTCGGTTTTTCT

224

Human CTCTGACAGACAAATCTAAGGAGCTGCCGTTATACTGTTCTGGGGGGTTGCGGTTTTTCT Rhesus macaque CTCTGACAGACAAATCTAAGGAGCTGCCGTTATACTGTTCTGGGGGGTTGCGGTTTTTCT Zebrafish CTCTCAGTGATAAGTCCAAGGAATTGCCATTGTATTGTTCAGGTGGTCTGCGTTTTTTCT Cat CGCTCACAGACAAGTCCAAGGAGTTGCCGCTGTACTGCTCGGGAGGGCTGCGCTTCTTCT Atlantic Salmon CACTTACAGACAAGTCCAAGGAGCTACCACTGTATTGTTCAGGGGGCCTGCGCTTCTTCT Rainbow trout CACTTACAGACAAGTCCAAGGAGCTACCTCTGTACTGTTCAGGGGGCCTGCGCTTCTTCT Rice Fish CACTGACAGACAAATCAAAGGAACTTCCATTGTACTGCTCTGGAGGTCTGAGGTTCTTCT Pufferfish CACTGACAGATAAGTCAAAGGAACTGCCTCTGTACTGTTCAGGAGGCTTGAGGTTCTTCT * * * **

Yeast TTGATTCTGCC------TGTCTACTATGATGAGAATTTCAATTTGGGG 1320 Sorghum GGAGCACCCGATTTGACAAAGCCATGACATGGTTTCTGACTTGCCTGCAAGAGTTCGCTG Fruit fly GGGATACCAAATTCGATGCGGCCATGGTGGCCTTTCTAGACTGCCTTACCCAGTTCCAGA Clawed frog GGGACAACAAGTTTGACCATGCCATGGTAGCGTTCCTGGACTGTGTGCAGCAGTTTAAGG Red junglefowl GGGACAATAAATTTGATCATGCGATGGTGGCTTTCTTGGACTGTGTGCAGCAATTTAAAG Brown rat GGGACAACAAGTTTGACCATGCAATGGTAGCTTTTCTGGACTGTGTGCAGCAGTTCAAAG Cattle GGGACAACAAGTTTGACCATGCAATGGTGGCTTTCCTGGACTGTGTGCAGCAGTTCAAAG Human GGGACAACAAGTTTGACCATGCAATGGTGGCTTTCCTGGACTGTGTGCAGCAGTTCAAAG Rhesus macaque GGGACAACAAGTTTGACCATGCAATGGTGGCTTTCCTGGACTGTGTGCAGCAGTTCAAAG Zebrafish GGGATAATAAATTTGATCATGCAATGGTGGCTTTCCTTGACTGTGTCCAGCAATTTAAAG Cat GGGATAACAAGTTCGACCATGCCATGGTGGCGTTTTTAGACTGCGTGCAGCAGTTTAAAG Atlantic Salmon GGGACAACAAGTTTGACCATGCCATGGTGGCCTTCCTGGACTGTGTGCAGCAGTTCAAGG Rainbow trout GGGACAACAAGTTTGACCATGCCATGGTGGCCTTCCTGGACTGTGTGCAGCAGTTCAAGG Rice Fish GGGACAATAAATTTGATCACGCCATGGTGGCTTTCCTGGATTGTGTCCAGCAGTTCAAAG Pufferfish GGGACAATAAATTCGACCACGCCATGGTGGCTTTTCTGGACTGTGTCCAGCAGTTCAAAG * * ** * **

Yeast AGAATCTTCCGCAAGGAAACGAAGTTTGATAAATCATT---AGAAACAACATTAGAAATA 1380 Sorghum AGTTTGCCATAAGTTTGGATAAGGAGAACAATGTTCCACCTGAAAAATCACTGAAGCTTC Fruit fly AGGAGGTGGAGAAACGCGACACCGAATTCCTGCTGCCCT--ACAAAATGGAAAAGGAC-A Clawed frog AGGAGGTCGAGAAAGGGGACACTGGTTTCTGCTTACCTT--ACAGGATGGATGTAGAA-A Red junglefowl AGGAAGTGGAAAAAGGTGAAACTCGGTTTTGTTTGCCTT--ATAGGATGGACGTGGAG-A Brown rat AAGAGGTGGAAAAAGGAGAGACTCGATTTTGTCTTCCGT--ACAGGATGGACGTGGAG-A Cattle AAGAAGTTGAGAAAGGCGAGACACGTTTTTGCCTTCCTT--ACAGGATGGATGTGGAG-A Human AAGAGGTTGAGAAAGGCGAGACACGTTTTTGTCTTCCCT--ACAGGATGGATGTGGAG-A Rhesus macaque AAGAGGTTGAGAAAGGCGAGACACGTTTTTGTCTTCCCT--ACAGGATGGATGTGGAG-A Zebrafish AGGAGGTGGAGAAGGATGACACTGGCTTCTGTTTGCCAT--ACAGAATGGATGTGGAC-A Cat AGGAAGTGGAGAAAGGCGACACGGGGTTCTGCCTACCGT--ACAGAATGGACGTGGAC-A Atlantic Salmon AGGAGGTGGAGAAGGGAGACACGGGATTCTGCCTGCCAT--ACAGGATGGATGTCGAG-A

225

Rainbow trout AGGAGGTGGAGAAGGGAGACACGGGATTCTGCCTGCCTT--ACAGGATGGATGTCGAG-A Rice Fish AAGAGGTGGAAAAAGGCGACACTGGTTTCTGCCTCCCTT--ATAGGATGGATGTGGAG-A Pufferfish AGGAGGTGGAGAAAGGAGACACTGGATTCTGCCTCCCGT--ACAGGATGGATGTGGAG-A * * * *

Yeast ATAAGCGA------AATCACTCGGCAGCTTTCTACGATAGCGTCATCATATTCA 1440 Sorghum CCTACAAGAT------TGATGGTGACAAAGTAGGGAGCCACACGATCGTCCTAAGTTTCA Fruit fly AAATCATT------GACCCATCCACTGGGAATTCCTACTCAATAAAGATACAGTTCA Clawed frog AAGGAAAAATTGAAGACACGGGAGGAAGTGGAGGTTCCTACTCCATCAAGACACAGTTCA Red junglefowl AGGGAAAGATTGAAGATACAGGTGGCAGTGGTGGTTCTTACTCTATTAAAACACAATTTA Brown rat AAGGCAAGATTGAAGACACTGGAGGCAGTGGCGGCTCCTATTCCATCAAAACCCAGTTTA Cattle AAGGCAAAATTGAAGACACAGGAGGCAGTGGTGGTTCCTATTCCATCAAAACTCAGTTTA Human AAGGCAAGATTGAAGACACAGGAGGCAGTGGCGGCTCCTATTCCATCAAAACCCAGTTTA Rhesus macaque AAGGCAAGATTGAAGACACAGGAGGCAGCGGCGGCTCCTATTCCATCAAAACCCAGTTTA Zebrafish AAGGAAAGATTGAGGACACAGGAGGAAGTGGAGGGTCTTACTCTATTAAGACTCAGTTTA Cat AGGGGAAGATCGAGGACACGGGCGGAAGCGGAGGATCGTACTCCATTAAAACGCAGTTCA Atlantic Salmon AGGGCAAGATTGAGGACACAGGAGGCAGCGGCGGCTCCTACTCCATCAAGACCCAGTTCA Rainbow trout AGGGCAAGATTGAGGACACGGGAGGCAGCGGCGGCTCCTACTCCATCAAGACCCAGTTCA Rice Fish AGGGAAAGATTGAGGACACAGGAGGCAGCGGCGGCTCCTACTCCATCAAAACCCAGTTCA Pufferfish AAGGGAAGATCGAGGACACCGGCGGCAGCGGCGGCTCGTACTCCATCAAAACCCAGTTCA * * * ** *

Yeast TCTCAAACACTCACAACAAGCCAAGATGAATCTTCAATGAATAACGCAAATGACGTTGAA 1500 Sorghum ATAAGAATGA------AAACTGGACC------AAGGCGCTAAA Fruit fly ACTCGGAGGA------ACAGTGGACG------AAGGCCTTAAA Clawed frog ACTCTGAGGA------ACAGTGGACC------AAGGCCCTAAA Red junglefowl ACTCTGAGGA------GCAATGGACA------AAAGCACTAAA Brown rat ACTCTGAGGA------GCAGTGGACA------AAGGCGCTCAA Cattle ACTCGGAGGA------ACAGTGGACA------AAAGCTCTCAA Human ACTCTGAGGA------GCAGTGGACA------AAAGCTCTCAA Rhesus macaque ACTCTGAGGA------GCAGTGGACA------AAAGCTCTCAA Zebrafish ACTCCGAGGA------ACAGTGGACC------AAAGCTCTCAA Cat ACTCCGAGGA------GCAGTGGACC------AAGGCGCTCAA Atlantic Salmon ACTCAGAGGA------GCAGTGGACC------AAGGCGCTCAA Rainbow trout ACTCGGAGGA------GCAGTGGACC------AAGGCGCTCAA Rice Fish ACTCTGAGGA------GCAGTGGACC------AAAGCACTCAA Pufferfish ACTCGGAGGA------GCAGTGGACC------AAAGCGCTCAA * * ** * * * * **

226

Yeast AATAGTACTTCCATTCTCGAGCTACCCTATATCATGAATAAGGACAAGATTAATGGATTA 1560 Sorghum GTACATGTTGTGCAATCTGAAGTGGGTTCTCTACTGGTTTATTGGCAATACAAGT-TTTG Fruit fly GTTCATGCTTACGAACCTGAAATGGGGTCTCGCCTGGGTCTCCTCGCAATTTGTG-TCGC Clawed frog ATTTATGCTGACAAATCTTAAATGGGGACTGGCCTGGGTTTCCTCTCAGTTCTAC-AATA Red junglefowl ATTCATGTTAACTAACCTAAAGTGGGGTCTTGCCTGGGTCTCATCCCAATTCTAT-AACA Brown rat GTTCATGCTGACGAATCTCAAGTGGGGTCTTGCTTGGGTGTCCTCACAGTTCTAT-AACA Cattle GTTCATGCTGACGAATCTTAAGTGGGGTCTTGCTTGGGTATCCTCACAGTTTTAT-AACA Human GTTCATGCTGACGAATCTTAAGTGGGGTCTTGCTTGGGTGTCCTCACAATTTTAT-AACA Rhesus macaque GTTCATGCTGACAAATCTTAAGTGGGGTCTTGCGTGGGTATCCTCACAATTTTAT-AACA Zebrafish GTTCATGCTCACTAACCTGAAGTGGGGTTTAGCCTGGGTTTCATCTCAGTTTTAC-AACC Cat GTTTATGCTCACCAATCTGAAATGGGGCCTGGCGTGGGTCTCGTCCCAATTCTAC-AACC Atlantic Salmon ATTCATGTTGACCAACCTCAAGTGGGGCCTGGCGTGGGTGTCGTCTCAGTTTTAC-AACC Rainbow trout GTTCATGTTGACCAACCTCAAGTGGGGTCTGGCGTGGGTGTCGTCTCAGTTTTAC-AACC Rice Fish ATTTATGCTTACCAATCTGAAATGGGGCCTGGCCTGGGTCACCTCTCAATTTTAC-AACA Pufferfish GTTCATGCTCACTAACCTGAAGTGGGGACTGGCCTGGGTCACTTCGCAGTTCTAC-AACA * * * * * ** *

Yeast TCCGTCAAATTGCATGGTAGCAGTCCCAACCTTGAATGGACAACGGCTATGAAATTTTTA 1620 Sorghum CACCACACTCGGGATCGCTGCACACACAATCTCTGAAGAACAAGAGTTGA------Fruit fly AATGA------Clawed frog AATGA------Red junglefowl AGTAA------Brown rat AGTGA------Cattle AATGA------Human AATGA------Rhesus macaque AATGA------Zebrafish GATAG------Cat GTTAA------Atlantic Salmon GATAG------Rainbow trout GATAG------Rice Fish GATAA------Pufferfish GATAG------

Yeast TTAACCAATGTAAAGTGGTTGTTAGCGTTTTCTTCCAATTTACTATCCAAGTCAATTACC 1680 Sorghum ------Fruit fly ------Clawed frog ------Red junglefowl ------

227

Brown rat ------Cattle ------Human ------Rhesus macaque ------Zebrafish ------Cat ------Atlantic Salmon ------Rainbow trout ------Rice Fish ------Pufferfish ------

Yeast TTAAGCCCTACAGTGAATTACAATGATAAGACCATCAGCGGAAACTAA 1728 Sorghum ------Fruit fly ------Clawed frog ------Red junglefowl ------Brown rat ------Cattle ------Human ------Rhesus macaque ------Zebrafish ------Cat ------Atlantic Salmon ------Rainbow trout ------Rice Fish ------Pufferfish ------

228

Appendix 2.2f) Clustal alignments between rainbow trout Atg genes and sequences from 14 other species. CLUSTAL O(1.2.4) ATG16 multiple sequence alignment

Sorghum ------ATGTTCGGGCCCAC------GCTATCCAGCACGAGTCCGAGTCCACTTG Yeast ------Fruit fly GTTAAAAACAATTATGCGAATAAACGCAGCCGCCTTATAGCGCCCGTGCCACACCCCCTG Zebrafish ------Pufferfish ------ATG------Cat ------AGTGATTTTGTGCACGAGCCAAGATGGCGGTTC-CGAATG--TGTGTGGAGTTG Rice Fish ------Atlantic Salmon------Rainbow trout ------Clawed frog ------Red junglefowl ------Cattle ------Brown rat ------Rhesus macaque ------Human ------

Sorghum CGGGCCCGGGCGCCGCCTCCGCTTCTTCGTCTTCCTCTTTCTTCTGGTTTCGGCTAGTCT Yeast ------ATGGGCAAT-----TTCATTATAACAGAAAGGAAGAAAGCAA Fruit fly CGATGCGAACAGGCGACCACGCCAAGG-----T---GTCGCCACGAATCCTG-CAAGCAA Zebrafish ------Pufferfish --CTGATGTCGGTTAACGAGACAAAAT-----AT--TTATCCTCTAATACCGAGACAAAA Cat GCTAGCTATCAGGCGACTTCATGTGAT-----TTCTTTCTTCGTTTTATTTTGCACGTAT Rice Fish ------Atlantic Salmon------Rainbow trout ------Clawed frog ------Red junglefowl ------Cattle ------Brown rat ------Rhesus macaque ------Human ------

229

Sorghum -TCAACGAAGACCCAACCGAGGCGCCCTGGCCGTGGCTGAGAACTGAAGCCACCGGACGC Yeast AGGAGGAAAGAAGCAATCCACAAACAGATA------GCATGGATGA Fruit fly -----GGAGAACCCAAGCCAAGAGTCCGGA------GTCCCCAGTACGGAGCACCCAGAA Zebrafish ------Pufferfish --CCACAGTG--GTTAACAGCAAGTGGATA------CTGTGCACGAACTCGGCTACATGG Cat --CAACAAGGATTCTCTCAAGAAGTGTTTA------G-AAATACTGGAGGAAACCTTTAA Rice Fish ------Atlantic Salmon------Rainbow trout ------Clawed frog ------AT Red junglefowl ------Cattle ------AT Brown rat ------AT Rhesus macaque ------Human ------

Sorghum GCGGCCT------CCATCTCCCCTGCTGCTGCCGAAATCCACGGGAGAATGACGA Yeast ----TTT------GTTAATTCGTAGACTAACA Fruit fly ATTCCGGGCAGAAACCCAATAAAAACATGTCTACGGAGGAGCATGTGTGGCGAGCGCACG Zebrafish ------ATGGCGGGACGCAGAGTGGAGTGTTTGTGGAAGCGTCACG Pufferfish --TGTGG------ACATGGCAGAGCGCTGGCTGGACTGTACCTGGAAGAGACATG Cat --CTCAC------CGATGGCAGGTCGCAGGGTGGAGTGTGTGTGGAAGAGACACG Rice Fish ------ATGGCGGAGCGGCGGGCAGAGTGCACGTGGAAGAGGCATG Atlantic Salmon------Rainbow trout ------ATGGCAGAACGTCGTGTGGAGTGTTCATGGAAGAGGCACG Clawed frog --GGCTG------CAGGTTGCCGGGGGCCCGCCTGCCCTTCCTGGAGGCTTCACA Red junglefowl ------Cattle --GTCGT------CGGGCCTCCGCGCTGCCGTCTTCCCGCGTTGGAAGCGCCACA Brown rat --GTCGT------CGGGCCTCCGCGCCGCAGACTTTCCCCGCTGGAAGCGCCACA Rhesus macaque ------Human ------

Sorghum TG------GCCGAGGC------CGAGGCTGGTAGGGCGGCGATCC Yeast G------ATCGAAACGACAAGGAGGCC---CATTTAAATGAGTTGTTTC Fruit fly TCGTGCGGCGCTTACGCGAGCGAAATCGCAAGGAGTGCGACAACTTCAAGGAGATCATCG

230

Zebrafish TTGTGGAGCAGCTGAAGCAGAGAGACAGAGTACAGCGTCAGGCCTTTGAGGAGATCATAC Pufferfish TGTCGGAGCAGCTGAAGCTCAGAGATCGACTCCAGAGACAGGCCTTCGAGGAGCTCATCC Cat TGGCGCAGCAGCTGAAGCAGAGGGACCGTGTGCAGCGACAGGCCTTTGAGGAGATCATCC Rice Fish TATTGGATCAGCTTAAGCTCCGAGACCGGATCCAGCGACAGACTTTCGAGGAGATCATCC Atlantic Salmon------TGCAGAGGCAGGCCTTCGAGGAGATCATCC Rainbow trout TCGGAGAGCAGCTGAAGCAGAGGGACCGTGTGCAGAGGCAGGCCTTCGAGGAGATCATCC Clawed frog TTTCCAGAGAGCTGAGGCGGAGAGACCGGGAGCAGCGGCAGGTGTTTGAGGAGCTCATCT Red junglefowl ------GGGACCGGCTGCAGCGGCAGGCCTTTGAGGAGATCGTCG Cattle TCTCGGAGGAGCTGAGGCGCCGGGACCGGCTGCAGAGGCAGGCGTTCGAGGAGATCATCC Brown rat TCGCGGAGGAACTGAGGCGCCGGGACCGGCTGCAGAGGCAGGCGTTCGAGGAGATCATTC Rhesus macaque ------GGGACCGGCTGCAGAGACAGGCGTTCGAGGAGATCATCC Human ------

Sorghum GGCGCGCCCTGCGGTCGCTGAAACGG------AGGCACC Yeast AAGATAA------Fruit fly AGCAAAACAACCGACTGATTGACCATGTGGCGCAACTGAAGGCGGACAACCTGAAGATCT Zebrafish ACCAATATAACCGGCTGTTGGAGAAGTCTGATCTTCAGGTTGTTTTTTCTGAACGTCTCC Pufferfish AACAGTATAACCGCCTCCTGGAGAAGTCAGACCTCCAGGCTGTTCTGTCTGAGCGATACC Cat AGCAGTATAACAGGTTGTTGGAGAAGTCAGACCTGCAGGCTGTGCTCTCAGAGCGACTGC Rice Fish ACCAATACAATCATCTATTGGAGAAGTCGGACTTTCAGACCGTTCTCACAGAGAGGTACC Atlantic SalmonACCAATATAACCGTCTCTTGGAGAAGTCCGATCTACAGGCTGTTCTTTCAGAGAGATACC Rainbow trout ACCAATATAACCGTCTCTTGGAGAAGTCCGATCTACAGGCTGTTCTTTCAGAGAGATACC Clawed frog CACAGTATAAAAGGCTGCTGGAAAAGTCCGACCTTCAATCTGTTTTGGCTGACAAACTGC Red junglefowl CGCAGTATAACAAGCTGCTAGAGAAGTCGGACCTCCACGCAGTGCTGGCTGATAAGCTGC Cattle TGCAGTATAACAAGTTGCTGGAAAAGTCAGATCTTCATTCAGTGCTGGCCCATAAGCTAC Brown rat TGCAGTATACCAAGTTGCTGGAGAAGTCAGATCTTCATTCAGTATTGACCCAGAAACTAC Rhesus macaque TGCAGTATAACAAATTGCTGGAAAAGTCTGATCTTCATTCAGTGTTGGCCCAGAAACTAC Human ------

Sorghum TCGCCGAGGAAGGGGCGC------ACAGCCCGGCCATCGAGGCGCTAACCCGTCCCTT-- Yeast ------TAGTGGCGCCATTGGTGGCAACATTGTCAGCC Fruit fly CCGTCGAAAACGAGCAGCTCCG------CAACGCGGTGT------CAACAGGTGGCACCG Zebrafish AAACTGAGAAATATGAGCAACAGAACAGACACGACCTCAGTCCGTGTGTGGACGGCGGGC Pufferfish AGACAGACAAATATGAGCTCCAGAGGGGACACGAGTGCAGTTTGGGCGGAGACCCGAGTC Cat AAACAGAGAAATATGAACTACAGAGCAGACATGACCTCAGCCCGGGGGCCGAGTTGAGCC Rice Fish AGGCCGACAAGTACGATGTGCAGAGAGGCCACGAGGCAGGGC---CTGTGGAGGCGGGTC Atlantic SalmonAGTCGGACAAATATGACTTTCAGAACAGACATGATGGCAGCCCGGGGGCGGACTCGAGTC

231

Rainbow trout AGTCGGACAAATATGACTTTCAGAACAGACATGACGGCAGCCCGGGGGCAGACTCAAGTC Clawed frog AAGCAGAGAAATATGAGCAGCAGAGCCGCCATGACAGCAGCCCTGGCCCAGATGGGATGC Red junglefowl AGGCAGAGAAGTATGACATGCAGAGCAGACATGAGATCAGTCCAGGACATGATGGCACAT Cattle AAGCCGAAAAGCATGACGTACCAAACAGGCATGAGATAAGTCCCGGACATGATGGTTCAT Brown rat AGGCTGAAAAGCACGACGTACCAAATAGGCATGAAATAAGTCCTGGGCACGATGGTGCAT Rhesus macaque AGGCTGAAAAACATGACGTACCAAACAGGCATGAGATAAGTCCCGGACATGACGGCACAT Human ------

Sorghum ----CGCCGCTCACGCTTTGGAGTGGAAAGAGAAGGCTGAGAAGCATGAGGTGGAGCTGC Yeast ATGACGATGCTTTATTGAATACACTGGCAATTCT------ACAAAAAGAACTGAAAA Fruit fly GCTCCAACGTGGCTATCGCCACCCTCGAAAAGAAGCTGCTCAGCCAGCAGGAGGAGCTCA Zebrafish GCAGTGATTCTCTGCAGCAGGAAATGTCTCAGATGCGGATCAGACATCAGGAAGAGCTGA Pufferfish AGAGAGATGCGCTACAACAGGAAATGGCTCAGATGAGGATTAAACATCAAGAGGAGCTGA Cat ACTGTGACCTGCTGCAGCAGGAAATGTCTCGGATGCGCATCAAACACCAAGAGGAGCTCA Rice Fish GCAGCGACGCTCTGTTGCAGGAAATGGCTCAGATGAGGATTCGCCACCAGGAGGAGCTGA Atlantic SalmonGCAGCGACAACCTACAGCAGGAGATGGCCCAGATGCGAATCAGGCACCAGGAGGAGCTGA Rainbow trout GCAGCGACAACCTACAGCAGGAGATGGCCCAGATGCGAATCAGGCACCAGGAGGAGCTGA Clawed frog GTAATGATATGCTATTGCAGGACATGGCCCAGATGAGGATAAAACACCAGGAGGAGCTCA Red junglefowl GGAATGATGCTCAGTTGCAGGAACTGGCACAGCTGAAGATAAAACATCAAGAGGAGCTGA Cattle GGAATGATAGTCAGCTGCAAGAAATGGCCCAGCTGAGAATTAAACACCAAGAAGAGCTGA Brown rat GGAATGACAGTCAGCTACAAGAAATGGCCCAGTTGAAGATGAAACACCAGGAAGAGCTGA Rhesus macaque GGAATGACAGTCAGCTACAAGAAATGGCCCAGCTGAGGATCAAGCACCAAGAAGAACTGA Human ------

Sorghum AGCACTGCTACAAGGCACAATCCCGGCTCTCTGAGCAGCTTGTAACCG--AGATAGAAGA Yeast GTAAGGAGCAAGAAATACGAAGATTGA------AAGAAGTTATTGCGTTGAAAAATAAGA Fruit fly CAGAGCTACACAAGCGCAAGGGCGAGAACTCCCAGATGATCGTGGACCTCAACCAGAAGG Zebrafish CGGAGCTCCACAAGAAAAGAGGAGAGCTGGCCCAGAGTGTGATTGAATTAAACAACCAGA Pufferfish CGGAGCTGCACAAAAAAAGAGGAGAGCTGGCTCAGAGCGTCATCGAACTGAACAACCAGA Cat CCGAGCTGCACAAAAAACGCGGAGAGCTGGCCCAGAGTGTAATCGAGCTGAATAACCAGA Rice Fish CAGAGCTTCACAAGAAACGAGGAGAGCTGGCTCAGAGCGTCATCGAGCTGAACAACCAGA Atlantic SalmonCAGAGCTTCATAAGAAACGGGGCGAGCTGGCCCAGAGTGTGATTGAGCTGAACCACCAGA Rainbow trout CAGAGCTTCATAAGAAAAGGGGCGAGCTGGCCCAGAGTGTGATTGAGCTGAACCACCAGA Clawed frog CCGAACTTCATAAAAAGAGAGGGGAGCTTGCTCAGACAGTCATAGAGCTGAACAATCAGA Red junglefowl CTGAGCTACATAAGAAACGTGGAGAGTTGGCCCAGTCTGTAATTGATCTGAATAACCAAA Cattle CTGAACTGCACAAGAAACGTGGGGAGTTAGCCCAGTTGGTGATTGACCTGAATAACCAAA Brown rat CTGAACTGCACAAGAAGCGTGGGGAGTTAGCTCAGTTGGTGATTGACCTGAATAACCAGA

232

Rhesus macaque CTGAATTACACAAGAAACGTGGGGAGTTAGCTCAATTGGTGATTGACCTGAATAACCAAA Human ------AACTGGTGATTGACCTGAATAACCAAA * * * * * *

Sorghum AGGTAAAGCATCAAA------AGCACTGCTCAAAGAGAAAGAAGCGA Yeast ATACGGAAA------Fruit fly TCGAGCAGCAGAGGATCATAATTTCCGAGAAGGAGCACAGCCTCGTCGAACAGCAAACGA Zebrafish TCCAGCAGAAAGACAAAGAGATCCAGAGTAACGAAGTCAAGATGCAGGAGTATCTGCAGC Pufferfish TTCAGCAGAAGGATAAAGAGATCCAGAGCAACACGGCCAGAATGTTGGAGTACCAGCAGC Cat TTCAACAGAAAGACAAAGAGATACAGAACAACGAGGCCAAGATGTTGGAGTACCAGCAGC Rice Fish TCCAGCAGAAGGACAAGGAGATTCAGAGCAATGAAGCCAGAATGTCTGAGCTCCAGCAGC Atlantic SalmonTTCAGCAGAAGGACAAGGAGATTCGGCGCAATGAGGCCAAGATGGAGGAGTACCAGCGGC Rainbow trout TTCAGCAGAAGGACAAGGAGATTCAGCACAATGAGGCCAAGATGCAGGAGTACCAGCGGC Clawed frog TGCAACAGAAGGACAAGGAAATCCAAGCCAATGAAGAAAAGATAGCGAAGTACTTGCAGA Red junglefowl TGCAGCAGAAGGACAAAGAGATGCAGATGAATGAAGCAAAGATTGCAGAGTATTTGCAAA Cattle TGCAGCAGAAGGACAAGGAGATGCAGATGAACGAAGCCAAAATTGCAGAATACCTGCAGA Brown rat TGCAGCAGAAGGACAAGGAGATACAGACGAATGAAGCAAAGATTGCAGAGTGTTTACAGA Rhesus macaque TGCAGCAGAAGGACAGGGAGATGCAGATGAATGAAGCAAAAATCGCAGAATGTTTGCAGA Human TGCAGCGGAAGGACAGGGAGATGCAGATGAATGAAGCAAAAATTGCAGAATGTTTGCAGA

Sorghum TGGTTACAAGTTTGCAGGCTGAACTTGAGAAGTCAAG------CGAAGAGAATGTAC Yeast ------Fruit fly ACAACAACCGATTGCGCGCGGAGGTGCAGCTACTTCACTCGAGTTTGGAGGAACTCAAGA Zebrafish AGATCTCGCAGCTGGAAGGAGAGTGTCGTGAGCTCAGGAACTGTCTGGCGGATTTGGAAC Pufferfish AGATCAGCAGCCTGGAGGGGGAGTGTCGGGACCTGAAGGCGTGTTTACAGGACCTGGAGA Cat AGATCTCCCAGCTCGAAGGCGATTGCCGTGAGCTGCGCAACTGCTTGTCGGACCTGGAGC Rice Fish AGATCTCCAGCCTGGAAGGTGACTGTCGGGAGCTGATCAGCAGCCTGCAGGACCTGGAAC Atlantic SalmonAGATTGCCGAGCTGGAGGGAGAGTGCCGGGAGTTGCGTGGTCAGCTGCAGGACCTGGAGC Rainbow trout AGATTGCCGAGCTGGAGGGAGAGTGCCGGGAGCTGCGTGGACAGCTGCAGGACCTGGAGC Clawed frog CAATCCAAGATCTGGAAACTGAATGCCAGGATTTAAGGAACAAGCTGCAAGAACTTGAAC Red junglefowl AAATCTCTGAACTGGAAACTGAGTGCCAGGAACTACGCAGCAAGCTACAAGATCTTGAGC Cattle CTATCTCTGACCTGGAGACCGAGTGCCAGGAGCTACGCACTAAGCTTCAGGACCTTGAAA Brown rat CCATCTCTGACCTGGAGGCAGACTGCCTGGACCTGCGCACCAAACTGCAGGACCTTGAGG Rhesus macaque CTATCTCTGACCTGGAGACGGAGTGCCTAGAACTGCGCACTAAGCTTTGTGACCTTGAAA Human CTATCTCTGACCTGGAGACGGAGTGCCTAGACCTGCGCACTAAGCTTTGTGACCTTGAAA

Sorghum AACTGAAGCAGTCACTTGACGAAAAAACAAAAGCCTTAGATCTTCTCATTCAAGAGCATC

233

Yeast ------GGTTGAATGACGAATTGATTAGTGGAAC-CATTGAAAATAATGTTTTA Fruit fly AGCTGAACAACACAATGCTGGACGAACATACGGCGCTTCA-ATTGGCATTTAGCTCCCTC Zebrafish GAGCCAATCAGACGCTGCGAGACGAGTACGACGCCCTGCA-GATCACGTTCAGCGCTCTG Pufferfish AGGCCAACCAGACCCTGAAGGACGAGTACGACGCCCTGCA-GATAACCTTCTCCGCCCTG Cat GAGCCAATCAGATGCTACGGGACGAGTACGACGCCTTACA-GATCACGTTCAGCGCTCTG Rice Fish GGGCCAATCAGACACTGAAGGATGAGTACGACGCCTTGCA-GATCACCTTCTCAGCCCTG Atlantic SalmonAGGCCAACCAGACGCTGAAGGATGAGTACGACGCCCTGCA-GATCACCTTCGGTGCCCTG Rainbow trout AGGCTAACCAGACGCTGAAGGATGAGTACGACGCCCTGCA-GATCACCTTCGGTGCCCTG Clawed frog GTGCCAATCAGACCCTTAAAGACGAGTACGACGCTCTGCA-GATTACTTTTACTGCTCTG Red junglefowl GAGCTAATCAGACACTGAAAGATGAATATGATGCTCTCCA-GATCACCTTCAATGCCTTG Cattle GAGCCAACCAGACCCTGAAGGATGAATATGACGCCCTGCA-GATCACTTTCACCGCCCTC Brown rat TAGCCAACCAGACCCTGAAGGATGAGTATGATGCCCTGCA-GATTACTTTTACTGCCCTA Rhesus macaque GAGCCAACCAGACCCTGAAGGATGAATATGATGCCCTGCA-GATCACTTTTACTGCCTTG Human GAGCCAACCAGACCCTGAAGGATGAATATGATGCCCTGCA-GATCACTTTTACTGCCTTG * ** * * *

Sorghum AGACAGTGAAAGCCGAACATGAACAAGCATTGACGAAACTAAAAGCTGCAGAGGATGAGA Yeast ------CAACAAAAACTCTCGGATCTGAAAAAAGAAC Fruit fly ------GAGGAGAAGTTAAGGGGAGTGCAAGATGAAA Zebrafish ------GAGGAGAAACTGCGCAAAACCACCGAGGACA Pufferfish ------GAGGAGAAGCTGAGGAAGACGACGGAGGACA Cat ------GAGGAGAAACTGCGCAAAACCACAGAGGACA Rice Fish ------GAGGAGAAGCTGAGGAAGACCACAGAGGACA Atlantic Salmon------GAGGAGAAGCTGCGGCGCACCACTGATGACA Rainbow trout ------GAGGAGAAGCTGCGGCGCACCACCGATGACA Clawed frog ------GAAGATAAATTACGGAAGACTACAGAGGACA Red junglefowl ------GAGGAGAAACTGAGGAAAACAACTGAGGATA Cattle ------GAGGAGAAGCTGCGGAAAACTTCCGAGGAGA Brown rat ------GAAGAGAAACTGAGGAAAACTACTGAGGAGA Rhesus macaque ------GAGGAAAAACTGAGGAAAACTACGGAAGAGA Human ------GAGGGAAAACTGAGGAAAACTACGGAAGAGA ** * * **

Sorghum ACCAAAGTCTGATCGATCGATGGATGCTAGAGAAGATGAAGGACGCAGAGAAGCTCAACG Yeast ATTCCCAGCTAGTTGCGAGATGGCTAAAAAAGACAGAGAAAGAGACAGAAGCCATGAACA Fruit fly ATCGGCGTCTACTGGAGCGACTTATGCAATACAAGTCAAAGGATGCCGACAAGTTGAACG Zebrafish ATCAGGAGCTGGTGACGCGCTGGATGGCGGAGAAAGCCCAGGAGGCCAACAGACTCAATG Pufferfish ACCAGGAACTGGTGTCGCGCTGGATGGCAGAGAAGGCTCAGGAGGCCAACAAGCTGAACG Cat ATCAGGAGCTCGTCACACGCTGGATGGCCGAGAAGGCACAGGAGGCGAACCGGCTCAACG

234

Rice Fish ATCAGGAGCTGGTGTCCCGCTGGATGGCCGAGAAAGCCCAGGAGGCCAATCGTTTAAACG Atlantic SalmonACCAGGAGTTGGTGTCGCGCTGGATGGCTGAGAAATCCCAAGAGGCCAACAAGCTCAATG Rainbow trout ACCAGGAGTTGGTGTCGCGTTGGATGGCTGAGAAATCCCAAGAGGCCAACAAGCTCAATG Clawed frog ACCAGGAACTGGTGTCTCGCTGGATGGCAGAGAAAGCTCAGGAAGCTAACCGGCTCAATG Red junglefowl ACCAGGAGCTGGTCTCACGTTGGATGGCAGAGAAAGCACAGGAAGCCAATCGCTTGAATG Cattle ACCAGGAGCTGGTCACCCGGTGGATGGCCGAGAAAGCCCAGGAGGCCAACCGCCTAAACG Brown rat ACCAGGAACTGGTCACCAGGTGGATGGCTGAGAAGGCCCAAGAAGCCAATCGCCTTAATG Rhesus macaque ACCAGGAGCTGGTCACCAGGTGGATGGCTGAGAAAGCCCAGGAGGCCAATCGGCTCAATG Human ACCAGGAGCTGGTCACCAGATGGATGGCTGAGAAAGCCCAGGAAGCCAATCGGCTTAATG * * * * * * * * ** * * * **

Sorghum AGGCCAATGCAATGTATGAGGAGATGGTTC------TAAAGCTAAAGAC------Yeast GCGAAATAGATGGAACGAAATGA------Fruit fly AGGAAAACGAGAGCATAATTAGAAAAAGACTGCCGTCAATTTTCAGGAAACGTTCGGCTA Zebrafish CTGAGAACGAGAAGGACTCCAGACGCAGGCAGGCAAAGC--TGCAGAAGGAACTGGCAGA Pufferfish CAGAGAACGAGAAGGACAGCAGACGCAGGCAGGCCAAGC--TGCAGAAGGAGCTCGCCGA Cat CGGAGAACGAGAAGGACTGCAGGCGCAGACAAGCGAAGC--TGCAGAAAGAGTTGGCTGA Rice Fish CCGAGAATGAGAAGGACACCAGGCGTCGGCAGGCGAGGC--TGCAAAAGGAACTGGCGGA Atlantic SalmonCGGAAAACGAGAAAGACTGCAAGCGCAGGCAAGCCAAGC--TTCAGAAGGAGCTTGCAGA Rainbow trout CGGAAAATGAGAAAGACTGCAAGCGCAGACAAGCCAAGC--TTCAGAAGGAGCTTGCAGA Clawed frog CAGAAAACGAGAAGGACTCAAAGAGGAGACAGGCCCGAC--TTCAGAAAGAGTTAGCTGA Red junglefowl CAGAAAATGAAAAGGATTCAAGGAGGCGACAAGCAAGGC--TGCAGAAGGAGCTAGCAGA Cattle CGGAGAATGAAAAGGACTCAAGGAGGCGGCAAGCCCGGC--TGCAGAAGGAGCTCGCAGA Brown rat CGGAGAATGAGAAGGACTCCAGGAGGCGACAAGCACGGT--TGCAGAAGGAGCTTGCAGA Rhesus macaque CAGAGAATGAAAAAGACTCCAGGAGGCGGCAAGCCCGGC--TACAGAAAGAGCTTGCAGA Human CAGAGAATGAAAAAGACTCCAGGAGGCGGCAAGCCCGGC--TGCAGAAAGAGCTTGCAGA * * *

Sorghum AGCTGGAGTTGGTGGTATACAACTTAACGCACAGCAAGA------Yeast ------Fruit fly AACTGAAACGTGACCTAGAAGA-----CGCGGTTCGTGAGCCCAGTTCGTCCAGCAACGC Zebrafish TGCAGCAAAGGAGCCGCTTCCTATCGACCCAGATGATGACATCGAGGTTCTCACAGAGGA Pufferfish CGCCGCCAAGGAGCCGCTGCCCCTCGACCCGGACGATGACATCGAGGTTCTGACCGAGGA Cat CGCCGCAAAAGAACCACTTCCTGTTGATATGGATGATGACATTGAGGTGCTTTCAGAGGA Rice Fish CGCCGCCAAAGAGCCTCTACCCATCGAGCCGGATGATGACATCGAGGTTCTGCCTGAGGA Atlantic SalmonCGCTGCAAAGGAGCCCCTGCCCATGGACCCGGATGACGACATCGAGGTTCTAGCTGAGGA Rainbow trout CGCTGCAAAGGAGCCCCTGCCCTTGGACCCGGACGACGACATCGAGGTTCTAGCTGAGGA Clawed frog AGCAGCCAAGGAACCTTTGTCCTTTGAACATGATGATGATATAGAGGTGCTGAATGATAA Red junglefowl AGCTGCCAAAGAACCCCTGCCTGTTGAACCCAGGGATGATGACATTGAAGTGCTTGCAGA

235

Cattle AGCAGCGAAGGAGCCTCTGCCAGTTGAGCA---GGATGATGATATTGAAGTCCTCGTGGA Brown rat AGCAGCAAAGGAACCTCTACCTGTCGAACA---GGATGATGACATTGAAGTCATTGTAGA Rhesus macaque AGCAGCAAAGGAACCTCTACCAGTCGAACA---GGATGATGACATTGAGGTCATTGTGGA Human AGCAGCAAAGGAACCTCTACCAGTCGAACA---GGATGATGACATTGAGGTCATTGTGGA

Sorghum ------AGC------TGATG------Yeast ------Fruit fly CGCCTCCTCGCCCGGCGCCGCATCCTTGCAGCGCAACTCCAGTCCCGCCCAGTTCGTTGG Zebrafish ---TGCTGGGAAGGCAAC------GGGTG-----AAACGTCACCGAGCAGACAGCTCAG Pufferfish ---TGGAGGCAAAGGCGC------TGGAG-----AAGGGTCACCGAACAAACCTCTGAC Cat ---GACGGGGAAAGGGAC------AGGAG-----AGACGTCTCCCAACCACCCAGTGCG Rice Fish ---CGGGTCGAAGGCGGA------AGGGG-----AGACGTCACCCAGTCGGGGGCTCAG Atlantic Salmon---TTCTGGGAAAGGCAC------TAGAG-----AGGCCTCTCCTAACCGGCCGCTCAG Rainbow trout ---TTCTGGGAAAGGCAC------TAGAG-----AGGCCTCTCCCAACCGGCCGCTCAG Clawed frog TGCAGACCCTGTGGGAG------ACAGTGCGGGGGCACAAGCCACAGG Red junglefowl TGAAACCTCTGATACAGC------TGAGG-----AGACATCTCCAGTGCGAGCTGTCAG Cattle TGAAACCTCTGAGCACCC------TGAGG-----AGACCTCTCCCGTGCGAGCCATCAG Brown rat TGAGACCTCAGATCACAC------AGAAG-----AGACCTCTCCTGTGCGAGCCATCAG Rhesus macaque TGAAACCTCTGATCACAC------AGAAG-----AGACCTCTCCTGTGCGAGCCATCAG Human TGAAACTTCTGATCACAC------AGAAG-----AGACCTCTCCTGTGCGAGCCATCAG

Sorghum ------GC------Yeast ------Fruit fly CGGCCTCATCGGCGACGAGGATTTCGACGAGGCCGCGATAAATGGCGCCATGGAGGCCAT Zebrafish TCGCACACCCAGTAAACGGTTGTCTCAGCCCCCTCCTCCCGCAGGACTGCTGGACTCCAT Pufferfish CAGAACCGCCAGCAGCAAAAAAATCTCCCAGCCGCCCCCGGGCAGCCTGCTGGACTCCGT Cat CGGCACACCCAGTAGACGAGCGTCTCAGCTTCCTCC---TGGTAACCTCCTAGATTCCAT Rice Fish CAGAATCTCCAGGAA------ATCCTCGCAGGCGCCCCCTGCTGGGCTGCTTGACTCCAT Atlantic SalmonCCGCACCCCCAGTAAACGTATCTCCGGACAGCCTCCTCAAAGCGGTCTGTTGGACTCAAT Rainbow trout CCGCACCCCCAGTAAACGTATTTCCGGACAGCCTCCTCAAAGCGGCCTGTTGGACTCAAT Clawed frog CCGGAACATCAGTAAGCGAAACTC---TCAGCCTGCCGTAGGAGGCCTCCTGGACTCTAT Red junglefowl CCGAACACCCAGTAAGCGACTCTC---CCAGCC---AGCTGGAGGCCTTCTGGACTCTAT Cattle CAGAGCGGCCACTAAGCGACTCTC---GCAGCC---TGCTGGAGGCCTTCTGGATTCTAT Brown rat CAGAGCAGCTACTAAGCGACTCTC---GCAGCC---TGCTGGAGGCCTTCTGGATTCTAT Rhesus macaque CAGAGCAGCCAC------Human CAGAGCAGCCACTAAGCGACTCTC---GCAGCC---TGCTGGAGGCCTTCTGGATTCTAT

236

Sorghum ------ATCATACGGCGTTCTGAA------Yeast ------Fruit fly ------AGGTCTCGATGATAATGAATATATTAGCGCTCGTTTT------ACAG Zebrafish CTCTAATATGTTTGGA------A------G Pufferfish CTCCAACATCTTTGG------CA------G Cat CACCAATATATTTGGTGTGGTCA------G Rice Fish CTCCAACATCT------TTG------G Atlantic SalmonCTCCAACATAT------TTG------G Rainbow trout CTCCAACATAT------TTG------G Clawed frog CACTAATATCTTCGGCTTGTCTGAGTCTCCCTCCTTGAGTCATCGTTCTGACAGTTCCAG Red junglefowl CACTAATATCTTTGGTCTGT------Cattle CACTAATATCTTTGGGAGGC------Brown rat CACTAATATCTTTGGGAGAC------Rhesus macaque ------GAGAC------Human CACTAATATCTTTGGGAGAC------

Sorghum ------GC Yeast ------Fruit fly CAGGCGAAATCGCCG------AGAACTCGCGGGCTTCCATCGACACCCTGAAGGCCAC Zebrafish ACGTCGCTCCGTGAATTCGTTCAGCTCGTCTCCTGAAAACGCAGAAGT---GCCG---TC Pufferfish GCGTCGTAGCACCAACTCCTTCAGCACGTCACCAGAGAACGCCGAGGGTCCTTCT---GG Cat GCGGCGGTCACTGAACTCGCACGGCTCACCCCCGGACATGGCGGAGACTCCCTCC---GG Rice Fish GCGGCGGAGCACCAACTCCTTCAGCTCCTCGCCCGAATCCACGGAGACGCAGTCA---GG Atlantic SalmonGCGTCGTCCTGGGAACTCGTACGGAACATCACTCGAAAACACGGAAACGACCTCC---GG Rainbow trout GCGTCGTCCTGGGAACTCGTACGGAACATCACCTGAAAACACGGAGACGACCTCC---GG Clawed frog GAGACGGTCTTTAAATTCTTTCCCTGCCTCACCGGATTGTGCAGATACCCCCGGA---GC Red junglefowl -----CTGAGTCTCCCCTTTTGGGACAT---CAGTCTTC------C---GA Cattle -----GCTCTGTCTCTTCCTTTCCAGTTCCCCAGGATAACGTGGATCCTCATCCC---GG Brown rat -----GCTCTGTCTCTTCCATCCCAGTCCCCCAAGATGTCGTGGACACTCATCCT---GC Rhesus macaque -----GCTCTGTCTCTTCCTTCCCAGTCCCCCAGGACAATGTGGATACTCATCCT---GG Human -----GCTCTGTCTCTTCCTTCCCAGTCCCCCAGGACAATGTGGATACTCATCCT---GG

Sorghum TGGTTACATGGAAACATCAATCCCATCAACATGCACAGTAACTATTCGTGCTCATGATGG Yeast ------Fruit fly GGGCTACCTGGGCCAGGCCAATCCCACCAAGATCCTCATGAAGTTCGAGGCCCACGAGAA Zebrafish GGCGTGTGCGGATGTGCGTGTTCCCTCCACTGCACTCCACATCTTTGACGCTCATGATGG

237

Pufferfish GCTCTGCACGGAGGTGCGGGTTCCCTCCACAGCTCTGCACGTCTTTGAGGCCCATGACGG Cat TGTGTGCGCAGACGTCCGCGTCCCCTCCACTGCACTTCACTTGTTCGATGCCCATGAAGG Rice Fish AATGTGTGCTGAGGTTCGAGTCCCGTCCACAGCGCTGCACGTCTTTGAAGCCCATGATGG Atlantic SalmonAGTGTGTGCCGATGTTCGCGTTCCCTCCACTGCCCTCCATGTATTTGAAGCCCATGATGG Rainbow trout AGTGTGTGCCGATGTCCGCGTTCCCTCCACTGCCCTCCATGTGTTTGAAGCCCATGATGG Clawed frog TGGAAACAGAGAGGTCAGGGTTCCTGCTACAGCGGTTTATTCCTTTGATGCCCATGATGG Red junglefowl TGCTGCC------AG------GAGGCGTTCTTTGTCCTC------Cattle TGCTGGTAAAGAAGTGCGAGTGCCGACGACTGCAATGTCTGTCTTTGACGCGCATGACGG Brown rat TTCTGGTAAAGATGTGAGAGTCCCAACTACTGCCTCGTATGTCTTTGATGCACATGACGG Rhesus macaque TTCTGGTAAAGAAGTGAGGGTACCAACTACGGCCTTGTGTGTCTTCGATGCACATGATGG Human TTCTGGTAAAGAAGTGAGGGTACCAGCTACTGCCTTGTGTGTCTTCGATGCACATGATGG

Sorghum TGGGTGTGGATCTTTGATGTTTGAGCATAACTCAGACAAGCTAATTAGTGGGGGCCAGGA Yeast ------Fruit fly CGAGTCGCACGCGGTGCGCTGGAGTCCCGTGGAGCGCATGGTGGCCACCGGTGGCGCTGA Zebrafish AGAAGTGAACGCTGTGATTTGCCGGTAG------Pufferfish GGAGGTCAACGCCGTCAAGTTCAGCCCCGGCTCTCGTCTCTTAGCAACAGGAGGGATGGA Cat CGAGGTGAATGCAGTCAGGTTCAGTCCTGGCTCTCGTCAGTTCGCTACAGGAGGAATGGA Rice Fish CGAGGTGAATGCAGTCAGGTTCAGCCCTGGCTCTCGTCTCCTAGCAACAGGAGGGATGGA Atlantic SalmonGGAAGTGAATGCGGTGCGGTTCAGCCCAGGCTCCCGACTCCTGGCCACCGGAGGGATGGA Rainbow trout GGAAGTGAATGCGGTGCGGTTCAGCCCAGGCTCCCGACTCCTGGCCACCGGAGGGATGGA Clawed frog AGAAGTTAATGCTGTGCGATTCAGCCCAGGCTCCCGTTTACTGGCAACAGGTGGAATGGA Red junglefowl ------GTTTCCTCCTCCCCAGGATAATGCA Cattle GGAGGTCAACGCTGTGCAGTTCAGCCCAGGTTCCCGGCTCCTGGCCACGGGAGGCATGGA Brown rat AGAGGTCAATGCAGTACAGTTCAGTCCAGGCTCCCGGTTACTGGCCACTGGAGGCATGGA Rhesus macaque GGAAGTCAACGCTGTGCAGTTCAGTCCAGGTTCCCGGTTACTGGCCACCGGAGGCATGGA Human GGAAGTCAACGCTGTGCAGTTCAGTCCAGGTTCCCGGTTACTGGCCACTGGAGGCATGGA

Sorghum TCAAACTCTTAAGATATGGAGTGCATCTACTGGTGCCTTG------ACCTCCACCCTACA Yeast ------Fruit fly TCGCAAAGTCAAACTTTGGGACATTGGCAAAAATTCCACCGAACCACGTGCCGTTCTAAG Zebrafish ------Pufferfish CCGCAGAGTGAAGCTGTGGGAAGTCCTTGCTGGTCACTGCGAGCCCAAAGGCGCCCTGAC Cat CCGACGTGTAAAACTTTGGGAGGTGATTGCAGGTCACTGTGAGTGTAAAGGCACTTTGAT Rice Fish TCGCAGGGTGAAACTGTGGGAGGTCATTGCTGGTCGCTGTGAGACCAAAGGATCTTTGAC Atlantic SalmonCCGCAGGGTCAAACTGTGGGAGGTGGTCTCAGGTCGCTGTGAGCCGAAGGGTGCTCTGAC Rainbow trout CCGCAGGGTCAAACTGTGGGAAGTGGTCTCAGGTCGCTGTGAGCCTAAGGGTGCTCTGAC

238

Clawed frog CCGGAGGGTGAAACTCTGGGATGTGATTGGAAATAAATGTGAAGCAAAAGGTTCTTTAAC Red junglefowl GAGCCACATCC------AGGTGC Cattle TCGGAGGGTGAAGCTCTGGGAAGTGTTTGGAGACAAATGTGAGTTCAAGGGCTCCCTGTC Brown rat CCGCAGGGTTAAACTTTGGGAAGCATTCGGAGATAAATGTGAATTCAAGGGCTCCCTGTC Rhesus macaque CCGCAGGGTTAAGCTTTGGGAAGTATTTGGAGAAAAATGTGAGTTCAAGGGTTCCCTATC Human CCGCAGGGTTAAGCTTTGGGAAGTATTTGGAGAAAAATGTGAGTTCAAGGGTTCCCTATC

Sorghum TGGTTGCTTGGGGTCTGTTAATGATCTTGCCGTGACCAATGATAACAAGTTTGTGGTCGC Yeast ------Fruit fly CGGGAGCAGTGCTGGGATTAACTCCGTAGATTTCGATTCGACAGGCGCCTACATCCTGGG Zebrafish ------Pufferfish GGGGAGTAACGCAGGAATCACCAGCATCGACTTTGACAGCGCGGTGAGTCT------Cat GGGCAGTAACGCTGGAATCACCAGCATCGAGTTCGACAGCACGGGCTCTTACCTGTTGGC Rice Fish GGGCAGCAACGCCGGAATCACCAGCATCGAGTTTGACAGCGCCGGTTCGTGCCTGCTGGC Atlantic SalmonTGGCAGCAACGCCGGGATCACCAGTATAGAGTTTGACAGTGCTGGCTCCTACTTGCTGGC Rainbow trout TGGCAGCAACGCCGGGATCACCAGTATAGAGTTTGACAGTGCTGTGAGTGTCATCCTGAT Clawed frog AGGCAGCAATGCAGGAATTACCAGCATCGAATTTGACAGTGCTGGTTCCTACCTTTTGGC Red junglefowl CAGTAAAGAAGTGAGAGTGCCCACTACTGCCATATGTGTCTTTGGTTCTTACCTCTTAGC Cattle TGGCAGTAATGCAGGAATTACAAGCATTGAATTTGATAGTGCTGGATCTTACCTCTTAGC Brown rat TGGCAGCAATGCGGGAATTACAAGCATTGAATTTGATAGTGCTGGAGCTTACCTATTAGC Rhesus macaque TGGCAGTAATGCAGGAATTACAAGCATTGAATTTGATAGTGCTGGTTCTTACCTCTTAGC Human TGGCAGTAATGCAGGAATTACAAGCATTGAATTTGATAGTGCTGGATCTTACCTCTTAGC

Sorghum TGCCTGTAGCTCCAATAAG--CTGTTTGTGTGGGAAGTTAATGGGGGACGTCCTCGTCAC Yeast ------Fruit fly CACCTCCAATGACTACGGAGCCAGAGTATGGAC--AGTGATGGACAATCGCTTAAGACAC Zebrafish ------Pufferfish ------Cat TGCGTCCAATGACTTTGCGAGCCGAATCTGGAC--AGTAGACGACTACAGACTGAGGCAC Rice Fish AGCCTCCAATGACTTTGCCAGTCGGATTTGGAC--GGTGGAGGACTTTAGGCTCCGGCAC Atlantic SalmonTGCATCCAATGACTTTGCCAGTAGGATCTGGAC--TGTGGATGACTACAGACTGAGGCAC Rainbow trout TAATCTCCTAGCCTGTCTAAAATGGCACCCTATATCATGGATCATCAACTAG------Clawed frog TGCTTCGAATGACTTTGCCAGCAGAATCTGGAC--AGTGGATGATTACAGGCTTCGGCAC Red junglefowl AGCCTCAAATGACTTTGCCAGCAGAATCTGGAC--GGTTGATGACAATCGATTACGGCAC Cattle AGCTTCAAATGATTTTGCAAGCCGCATCTGGAC--CGTGGATGACTACCGGTTACGGCAC Brown rat AGCTTCAAATGATTTTGCAAGCCGAATCTGGAC--TGTGGATGATTATCGATTACGGCAC Rhesus macaque AGCTTCAAATGATTTTGCAAGCCGAATCTGGAC--TGTGGATGATTATCGGTTACGGCAC

239

Human AGCTTCAAATGATTTTGCAAGCCGAATCTGGAC--TGTGGATGATTATCGATTACGGCAC

Sorghum ACTCTGACTGGTCACACAAAAAATGTTTCATCTGTTGATGCAAGCTGGGTGAAGAGCTTT Yeast ------Fruit fly ACGCTGACGGGTCACAGCGGCAAGGTAATGGCCGCCAAGTACGTCCAGGAGCCCAT---C Zebrafish ------Pufferfish ------GCTGTTCTGGAACAACAC---C Cat ACGTTAACAGGTCACAGCGGGAAGGTTCTGTCTGCTCGCTTCCTGCTGGATAACGC---T Rice Fish ACCTTGACGGGCCACAGCGGGAAGGTTCTGTCTGCCCGCTTCCTGTTGGACAACGC---C Atlantic SalmonACGCTGACAGGCCACAGCGGCAAGGTCCTGGCGGCTCGCTTTCTCCTGGACAACGC---T Rainbow trout ------Clawed frog ACGCTAACTGGACATAGTGGGAAAGTTCTATCTGCCAAATTCCTACTGGACAATGC---C Red junglefowl ACTTTGACAGGTCACAGTGGTAAGGTTCTGTCTGCCAAGTTCCTGCTGGACAACGC---G Cattle ACGCTCACGGGACACAGTGGCAAAGTGCTGTCTGCCAAGTTCCTGCTGGACAATGC---G Brown rat ACACTTACCGGCCACAGCGGGAAAGTTCTCTCTGCCAAGTTCCTGCTGGACAATGC---A Rhesus macaque ACACTCACGGGACATAGTGGGAAAGTGCTGTCTGCTAAGTTCCTGCTGGACAATGC---G Human ACACTCACGGGACACAGTGGGAAAGTGCTGTCTGCTAAGTTCCTGCTGGACAATGC---G

Sorghum GTTGTTGCTAGTTCATCCAATGATCGTACTATCAAGACTTGGGATCTACAGACAGGTTTC Yeast ------Fruit fly AAAGTGGTGACCGGTAGCCACGATCGCACACTGAAGATCTGGGATTTGCGCAGCATCGCC Zebrafish ------Pufferfish CGGATCGTCTCTGGGAGCTACGACCGCACGCTGAAGCTGTGGGACCTGCGCAGCAAAGTG Cat CGCGTTGTCTCGGGAAGCTACGACAGAACGCTCAAACTGTGGGATCTGCGAAGCAAAGTC Rice Fish CGCATCGTGTCGGGCAGCTACGACCGCACGCTCAAGCTGTGGGACCTGCGCAGCAAAGTC Atlantic SalmonCGTATCGTCTCCGGGAGCTACGACAGGACTCTGAAACTGTGGGACCTGCGCAGCAAAGTC Rainbow trout ------Clawed frog CGCATTGTGTCTGGAAGTCACGACAGGACATTAAAGCTGTGGGACCTGCGCAGCAAAGTT Red junglefowl CGCATCGTTTCGGGAAGCCATGACCGGACCCTCAAGCTCTGGGACCTCCGCAGCAAAGTT Cattle CGCATCGTTTCTGGAAGTCACGACCGGACCCTCAAACTCTGGGATCTACGCAGCAAAGTC Brown rat CGGATAGTCTCAGGAAGTCATGACCGGACCCTCAAACTCTGGGATCTCCGCAGCAAAGTC Rhesus macaque CGGATCGTCTCAGGAAGTCACGACCGGACTCTCAAACTCTGGGATCTACGCAGCAAAGTC Human CGGATTGTCTCAGGAAGTCACGACCGGACTCTCAAACTCTGGGATCTACGCAGCAAAGTC

Sorghum TGCAAAAGCACCATAATGTCTGCAAGCAACCCCAACTCACTAGCTTTCATTGATGGTGA- Yeast ------

240

Fruit fly TGCATAGAGACGAAGTTCGCCGGCTCCAGCTGCAATGATCTAGTCACCACCGACAGCCTC Zebrafish ------Pufferfish TGTATCAAGACCGTGTTCGCCGGGTCCAGTTGCAACGACATCGTGTGCACAGAGCAGTG- Cat TGTACGAAGACTGTGTTTGCGGGCTCCAGCTGTAACGATATAGTGTGCACGGAGCAGTG- Rice Fish TGTATGAAGACGGTGTTTGCTGGCTCAAGCTGCAATGACATCGTGTGCACGGAGCAGTG- Atlantic SalmonTGCATGAAGACTGTGTTTGCTGGGTCCAGCTGTAATGACATAGTCTGCACAGAGCAGTG- Rainbow trout ------Clawed frog TGCATTAAGACTGTCTTTGCTGGTTCTAGCTGTAATGACATAGTTTGCACGGAGCAGTG- Red junglefowl TGTATAAAAACAGTGTTTGCAGGATCTAGCTGCAATGACATCGTATGCACTGAGCAATG- Cattle TGCATAAAGACAGTATTTGCGGGATCCAGCTGCAATGACATTGTCTGCACAGAGCAGTG- Brown rat TGTATCAAAACAGTGTTCGCAGGGTCCAGCTGCAATGACATTGTTTGCACTGAGCAATG- Rhesus macaque TGCATAAAGACAGTGTTTGCAGGATCCAGTTGCAATGATATTGTCTGCACAGAGCAATG- Human TGCATAAAGACAGTGTTTGCAGGATCCAGTTGCAATGATATTGTCTGCACAGAGCAATG-

Sorghum -----CATCATTTGTTCGGGCCATAGGGATGGAAATCTTCGGCTATGGGATGTCCGTAGT Yeast ------Fruit fly GGATCAACGATCATCAGTGGGCACTACGATAAGAAGATCCGGTTCTGGGACATACGCACC Zebrafish ------Pufferfish ------CGTCATGAGCGGACACTTCGATAAGAAGGTTCGGTTCTGGGACATCAGAGCG Cat ------TGTGATGAGTGGCCATTTTGATAAGAAAGTACGCTTCTGGGACATTCGTGCT Rice Fish ------CGTCATGAGCGGCCACTTCGACAAAAAGGTCCGCTTCTGGGACATCAGGGCT Atlantic Salmon------TGTCATGAGCGGCCATTTTGATAAGAAGGTGCGGTTCTGGGACATCAGAGCG Rainbow trout ------Clawed frog ------TGTCATGAGTGGTCATTTTGACAAAAAGATTCGGTTCTGGGATATAAGAACA Red junglefowl ------TGTAATGAGTGGACATTTTGATAAGAAAATCCGTTTCTGGGATATCAGGACT Cattle ------TGTGATGAGCGGGCATTTTGACAAGAAAATTCGTTTCTGGGACATCCGATCA Brown rat ------TGTAATGAGTGGACATTTTGACAAGAAAATTCGTTTCTGGGATATCCGGTCA Rhesus macaque ------TGTAATGAGTGGACATTTTGACAAGAAAATTCGTTTCTGGGACATTCGATCA Human ------TGTAATGAGTGGACATTTTGACAAGAAAATTCGTTTCTGGGACATTCGATCA

Sorghum GGGAAATGCACTACACAGATAGCTGCTCATCTAGATGTCACCTCGGTCTGCGTGTCACGG Yeast ------Fruit fly GAGAAGCAGGCGGACGACGTCTTAATGCCGGCCAAGATCACCTCTTTGGATTTGTCAAAA Zebrafish ------Pufferfish GAGAGCATTGTGAAGGAGCTGGAGCTGCTGGGTCGGGTTACGTCTCTGGACCTGAACCAT Cat GAGAGTACAGTGTGCGAGTTGGAACTGTTGGGTCGGGTCACGTCGTTGGACCTGAATCAC Rice Fish GAGAGCATTGTGCGGGAGCTGGAGCTGCTCGGCAGAGTCACCTCGTTGGACCTGAACCAC

241

Atlantic SalmonGAGAGTATTGTCCGTGAGCTGGAGCTCCTGGGCCGAGTCACATCTCTTGACCTGAACCAC Rainbow trout ------Clawed frog GAATGTATTGTGCGGGAACTGGAGTTGCAGGGCAGAATCACGGCTCTAGATCTGAATCCT Red junglefowl GAAAGCATAGTAAAAGAACTGGAGCTGCTTGGAAGGATCACAGCTCTGGATTTGAACTCG Cattle GAGAGTATTGTCCGAGAGATGGAGCTCTTGGGAAAGATCACTGCCCTGGACTTAAATCCA Brown rat GAGAGTGTGGTCCGAGAGATGGAACTGTTAGGGAAGATCACTGCTCTGGACTTAAACCCC Rhesus macaque GAGAGTATAGTTCGAGAGATGGAGCTGTTGGGAAAGATTACTGCCCTGGACTTAAACCCA Human GAGAGCATAGTTCGAGAGATGGAGCTGTTGGGAAAGATTACTGCCCTGGACTTAAACCCA

Sorghum AGCAAAAATTTTATTCTTACAAGTGGGAGAGATAATGTGCACAACCTTTTTGATGTTAGA Yeast ------Fruit fly GATTGCAATTACCTCATATGCTCTGTAAGAGACGATACTATTAAACTGTTAGATTTACGC Zebrafish ------Pufferfish GACCGCACTGAGCTGCTCACGTGTTCCAGAGACGACCTGGTGAAGATCATCGACCTGCGC Cat GACCGCACAGAGCTGCTCACCTGTTCCCGTGATGACCTCCTCAAAATCATCGACCTGCGC Rice Fish GACCGCACCGAGCTGCTCACCTGCTCCAGAGACGACCTGCTGAAGATCATCGACCTGCGC Atlantic SalmonGACCGCACCGAGCTGCTCACCTGCTCCCGGGACGACCTGGTTAAGATCATCGACCTGCGC Rainbow trout ------Clawed frog GAAAGGACACAGCTACTTAGCTGCTCTCGGGATGACCTTATCAAAATCACTGACTTGCGT Red junglefowl GAGCGAACAGAGCTTTTGACCTGTTCCCGTGATGATCTACTGAAGATCATTGATCTGCGG Cattle GAAAGAACTGAACTCCTCAGCTGCTCTCGAGATGACCTGCTGAAAATTATAGACCTACGA Brown rat GAGAGGACTGAGCTCCTGAGCTGCTCCCGTGATGACCTGCTAAAAATCATTGACCTGCGG Rhesus macaque GAAAGGACTGAGCTCCTGAGCTGCTCCCGGGATGACTTGCTAAAAGTTATTGATCTCCGA Human GAAAGGACTGAGCTCCTGAGCTGCTCCCGTGATGACTTGCTAAAAGTTATTGATCTCCGA

Sorghum ACATTGGAGATTTGTGGAACATTCAGGGCCATGGGTAACAAAGTGGTGGGCAGCTGGAGC Yeast ------Fruit fly AAGAACCAAGTTATATCCACGTTTACAAACGAACACTTTAAGATCAGCTGTGATTTTGCT Zebrafish ------Pufferfish ACCGACGCCATCAGACAGACCTTCAGCGCTCAGGGTTTTAAATGTGGAGCGGACTGGACC Cat AACAACGCTGTGCGACAAACGTTCAGTGCAGACCACTTCAAGTGTGGGTCAGACTTCACC Rice Fish ACCAACGCCGTCAGGCAGACTCTCAGCGCTCAGGGCTTCAAGTGTGGAGCCGACTGGACC Atlantic SalmonAGTAACGCGATCCGCCAGACCTTCAGTGCTCAGGGCTTCAAGTGTGGCGCTGACTGGACC Rainbow trout ------Clawed frog GCCAATGCTGTGCAGCAAACCTTCAGTGCCCCGGGCTTTAAGTGTGGATCAGACTGGACT Red junglefowl GTTGGTGCTGTCAAGCAAACATTCAGTGCCCAGGGATTCAAATGTGGCTCTGACTGGACA Cattle ATAAATGCTGTCAGACAGACATTCAGCGCGCCAGGCTTCAAGTGTGGCTCCGACTGGACT

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Brown rat ACAAACGCCATCAAGCAGTCATTCAGTGCACCTGGATTCAAGTGTGGCTCTGACTGGACC Rhesus macaque ACAAATGCTGTCAAGCAGACATTCAGTGCACCTGGGTTCAAGTGCGGCTCTGACTGGACC Human ACAAATGCTATCAAGCAGACATTCAGTGCACCTGGGTTCAAGTGCGGCTCTGACTGGACC

Sorghum AAACCTTGTATTAGCCCTGATGAAAATTGCATCGCTGCTGGTTCTTCTGATGGATCTGTT Yeast ------Fruit fly CGAGCTTCTTTCAACTCGAGCGGCCTAAAGATCGCTTGTGGATCAGCGGATGGCGCTATA Zebrafish ------Pufferfish AGAGTGACGTTCAGTCCTGACGGCGTTTATGTGGCTGGAGGATCAGCTGATGGCGCCCTC Cat AGAGTCACCTTCAGTCCTGATGGCAGTTACGTTGCGGGGGGTTCAGCAGATGGTGTTCTG Rice Fish AGAGTCACCTTCAGTCCGGATGGCAGCTACGTGGCTGGAGGCTCGGCTGACGGAGCTCTG Atlantic SalmonCGAGTTACCTTCAGTCCCGATGGCAGTTACGTGGCGGGGGGCTCTGCAGACGGCGCCTTG Rainbow trout ------Clawed frog CGGGTTATATTTAGCCCTGATGGCAGTTATGTGTCTGCTGGTTCGGCAGAAGGAACACTT Red junglefowl AGAGTTGTGTTCAGTCCTGATGGTAACTATGTGGCTGCTGGTTCAGCTGATGGTGCCCTC Cattle AGAGCTGTGTTCAGCCCTGACGGTAGTTACGTAGCAGCGGGCGCGGCCGAGGGCTCGCTG Brown rat CGAGTTGTCTTCAGCCCTGATGGCAGTTACGTGGCAGCAGGCTCAGCTGACGGCTCTCTT Rhesus macaque AGAGTTGTCTTCAGTCCTGACGGCAGTTACGTGGCGGCAGGCTCTGCTGAGGGCTCTCTC Human AGAGTTGTCTTCAGCCCTGATGGCAGTTACGTGGCGGCAGGCTCTGCTGAGGGCTCTCTG

Sorghum TACATATGGTCGAGACTGAAGAATGGAACACCAACCATCCTAGA---GGGTCATTCTTCA Yeast ------Fruit fly TACATTTGGAATGTGAATGGGTTCCTGGAAGCCA------CACTCAAGGGACACAGCACC Zebrafish ------Pufferfish TATGTTTGGAACGTGCTGACGGGGAAGCTTGATCGCACTCTGGACCGAAACCACAACTCT Cat TATGTGTGGAATGTTCTGACAGGGAAAGTGGAGATAAAACTCGACAAAGGGCACAGCTCC Rice Fish TACATCTGGAACGTCCTGACGGGGAAAGTGGAGCGCATTCTGGACCGGAACCACAACTCT Atlantic SalmonTATGTGTGGAACGTCCTGACGGGCAAGTTGGAGCGCACCCTGGACAAGAACCACTGCTCG Rainbow trout ------Clawed frog TACTTCTGGAACGTCCTTACAGGAAAAGTGGAGAGGATGTTTAGCAAGCAACACAGTTCC Red junglefowl TATGTTTGGAATGTACTCACTGGGAAGTTGGAGAGAACTCTTGCCAAGCATCACAGTTCT Cattle TACATCTGGAACGTGCTGTCTGGGAAAGTGGAGAAGGTCCTCTCAAAGCATCACAGCTCG Brown rat TATGTATGGAGTGTGCTCACGGGGAAAGTAGAGAAGGTTCTCTCCAAGCAGCACAGCTCT Rhesus macaque TACATCTGGAGTGTGCTCACAGGGAAAGTGGAAAAGGTTCTCTCAAAGCAGCACAGCTCG Human TATATCTGGAGTGTGCTCACAGGGAAAGTGGAAAAGGTTCTTTCAAAGCAGCACAGCTCA

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Sorghum CCTGTTCTCGCAAGTGCATGGTGCGGAC------TAGG Yeast ------Fruit fly GCCGTAAACGCGGTCAGTTGGAGTCCCAACAACAACATGCTGGCCTCCGTGGGAAAGAAC Zebrafish ------Pufferfish GCCATCAACTCGGTGTCGTGGTCGCCTTCGGGAACGTACGTGGCGAGCGTGGAGAGGGGC Cat TCCATTAACGCAGTGTCCTGGTCTCCATCTGGTTCCTACGTGCTCAGTGTGGAGAAGAGC Rice Fish GCCATCAACGCGGTGTCCTGGTCTCCGTCGGGGACCTACGTGGTCAGCGTGGAGAAGGGC Atlantic SalmonGCCATCAACTCTGTGTCCTGGTCACCATCGGGAGCCTACGTGGTCAGTGTGGAGAAGGGC Rainbow trout ------Clawed frog TCTATCAATGCTGTGGCTTGGTCACCCTCTGGGACACACGTGGTCAGCGTTGACAAAGGA Red junglefowl CCCATCAATGCAGTCGCGTGGTCCCCAGCGGGTGCCCACGTGGTCAGTGTGGACAAAGGA Cattle TCCATCAACGCGGTGGCCTGGTCCCCCGCCGGCTCCCACGTGGTCACTGTGGACAAAGGA Brown rat TCTATCAATGCGGTGGCGTGGGCCCCCTCTGGCTTACATGTTGTCAGTGTGGACAAAGGA Rhesus macaque TCCATCAATGCAGTGGCGTGGTCGCCCTCTGGCTCGCACGTTGTCAGCGTGGACAAAGGA Human TCCATCAATGCGGTGGCGTGGTCGCCCTCTGGCTCGCACGTTGTCAGTGTGGACAAAGGA

Sorghum A------Yeast ------Fruit fly AAGCG------CTGCACCAT Zebrafish ------Pufferfish AGTAAGACCATCCTCTGGTCCGACATGTGA------Cat AGCAAAGCCGTGCTCTGGTCTGACATGTGATTGGTCCAGCTCTGCCATGCTCTGATTGGT Rice Fish AGCCGCGCCATCCTGTGGTCTGACGTGTGA------Atlantic SalmonAGCAAGGCCGTCCTCTGGTCTGATATGTGA------Rainbow trout ------Clawed frog AGCCGTGGAGTGTTGTGGTCTGACTTTTGA------Red junglefowl AACAAGGCTGTCCTGTGGTCTGAGTTCTGA------Cattle AGTAAGGCCGTGCTGTGGTCGGAGTATTGA------Brown rat AGCAGAGCTGTGCTATGGGCACAGCCTTGA------Rhesus macaque TGCAAAGCTGTGCTGTGGGCACAGTATTGA------Human TGCAAAGCTGTGCTGTGGGCACAGTACTGA------

Sorghum ------Yeast ------Fruit fly CTACT------CGGAGTCATAGCCCTAATG------TTGCCA---TACACGTCAC Zebrafish ------Pufferfish ------

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Cat TCACTCACAGCCTGATCTGATTGGTTCAGCTCTGCTTCACCTTGCTCTGTTCGGCTCAGC Rice Fish ------Atlantic Salmon------Rainbow trout ------Clawed frog ------Red junglefowl ------Cattle ------Brown rat ------Rhesus macaque ------Human ------

Sorghum ------Yeast ------Fruit fly TCGAGCCAGTCGGATGCAATCCAATTCCGAA------ACTGAAACGCTTAAGAA----- Zebrafish ------Pufferfish ------Cat TCCAGCCTACAGGAGGACCCCACTTTTTACCTAGTACTCGTACAACATTTCCCAGGACGC Rice Fish ------Atlantic Salmon------Rainbow trout ------Clawed frog ------Red junglefowl ------Cattle ------Brown rat ------Rhesus macaque ------Human ------

Sorghum ------Yeast ------Fruit fly -----GTCAGAGCAGTGCTGCAATCTGCTGTACATAGTAAATTCGGTCGAC-GTTTTAAC Zebrafish ------Pufferfish ------Cat AGTGTGTCCGCGAGGCGTGGCCATCT---GTACAGAAGGATCCAACTTGCCCAATAGAAT Rice Fish ------Atlantic Salmon------Rainbow trout ------Clawed frog ------

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Red junglefowl ------Cattle ------Brown rat ------Rhesus macaque ------Human ------

Sorghum ------Yeast ------Fruit fly AACTTTA----CTTGATTATTTTATTTATACGTGAATCGGATATAGTATGCAAATTGTAT Zebrafish ------Pufferfish ------Cat GATTTTATTTTCCTGTCTCTTCTCTCTCCCACTCATTCTCATGCACACAATTTCTGATGG Rice Fish ------Atlantic Salmon------Rainbow trout ------Clawed frog ------Red junglefowl ------Cattle ------Brown rat ------Rhesus macaque ------Human ------

Sorghum ------Yeast ------Fruit fly CAATTCATATAGTGTTAGTCTAA------GTACATGTTTTCCTATTGTAAAAAA Zebrafish ------Pufferfish ------Cat AAATGAGTAATGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTTAATCAA Rice Fish ------Atlantic Salmon------Rainbow trout ------Clawed frog ------Red junglefowl ------Cattle ------Brown rat ------Rhesus macaque ------Human ------

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Sorghum ------Yeast ------Fruit fly --AAATAAACACACATGCGCATATTGGGTTG------CATATACGAGCAA Zebrafish ------Pufferfish ------Cat GTGTCTCTTAACAGCAATTAATAATGTCATGCCTTTTTAAAAGGCTTCACTTACTGTCTC Rice Fish ------Atlantic Salmon------Rainbow trout ------Clawed frog ------Red junglefowl ------Cattle ------Brown rat ------Rhesus macaque ------Human ------

Sorghum ------CCTCTTGCTACCTCGGACAGGAATCATATTT Yeast ------Fruit fly TCTAAGC------Zebrafish ------Pufferfish ------Cat CCTGACCACTTCTGGTCTGTACTCACTCTACCCTTGGCAATGTGGGAAAGGATTGTGTGT Rice Fish ------Atlantic Salmon------Rainbow trout ------Clawed frog ------Red junglefowl ------Cattle ------Brown rat ------Rhesus macaque ------Human ------

Sorghum ATCTCTGGTCTTGA------Yeast ------Fruit fly ------

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Zebrafish ------Pufferfish ------Cat GTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTG Rice Fish ------Atlantic Salmon------Rainbow trout ------Clawed frog ------Red junglefowl ------Cattle ------Brown rat ------Rhesus macaque ------Human ------

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Appendix 2.3a-b) BLASTN results of forward a) and reverse b) sequences from PCR product of Atg gene primers. Table A. Forward sequence

Atg Description of BLAST Max Total QC E value Identity genes match score score % (%) (Genbank accession #) ATG4 Oncorhynchus mykiss 128 128 100 1e-26 99% cysteine protease ATG4 % (XM_021604831.1) ATG5 Oncorhynchus mykiss 113 113 98% 3e-22 100% autophagy protein 5 (XM_021593753.1)

ATG7 Oncorhynchus mykiss 56.5 56.5 33% 8e-05 100% autophagy related 7 (XM_021610791.1) ATG9 Oncorhynchus kisutch 124 124 98% 1e-25 100% autophagy-related protein 9 (XM_020462642.1) ATG12 Oncorhynchus mykiss 156 276 100 1e-34 98% autophagy related 12 % (XM_021623074.1) ATG13 Oncorhynchus mykiss 106 106 100 6e-20 89% autophagy related protin % 13-like (XM_021585873.1) ATG16 Oncorhynchus mykiss 161 161 100 2e-36 98% autophagy related 16 % like 1 (XM_021587228.1) BECLIN-1 Oncorhynchus kisutch 100 100 96% 2e-18 95% beclin 1 (XM_020469225.1) LC3 Oncorhynchus mykiss 145 145 95% 2e-31 96% microtubule-associated proteins 1A71B light chain 3B (XM_021592266.1) GABARAP Oncorhynchus nykiss 50.0 99 83% 8e-04 94% GABA Type A receptor- associated protein (KX845473.1) EF1 Oncorhynchus kisutch 110 110 92% 4e-21 97% elongation factor 1- alpha (XM_020500543.1) RS11 Oncorhynchus mykiss 84.2 84.2 100 1e-13 98% 40S ribosomal protein % S11 (rs11) (NM_001246324.2)

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Appendix 2.3b) BLASTN results of forward a) and reverse b) sequences from PCR product of Atg gene primers Table B. Reverse sequences

Atg Description of BLAST match Max Total QC E Identity genes (Genbank accession #) score score % value (%) ATG4 Oncorhynchus mykiss cysteine protease 115 115 88% 1e-22 96% ATG4B-like (XM_021604831.1) ATG5 Oncorhynchus mykiss autophagy 113 113 92% 3e-22 97% protein 5-like (XM_021558888.1) ATG7 Salmo salat ATG7 autophagy related 7 69.4 125 63% 1e-08 100% homolog (NM_001165320.1) ATG9 Oncorhynchus mykiss autophagy- 91.5 91.5 100% 1e-15 95% related protein 9A-like (XM_021589202.1) ATG12 Oncorhynchus mykiss autophagy- 127 127 98% 3e-26 97% related 12 (atg12 (XM_021623074.1) ATG13 Oncorhynchus mykiss autophagy- 128 128 100% 1e-26 96% related 13-like (XM_021585873.1) ATG16 Oncorhynchus mykiss autophagy- 159 159 100% 6e-36 100% related 16 like 1 (XM_021587228.1) BECLIN-1 Oncorhynchus kisutch beclin 1 (becn1) 111 111 81% 1e-21 98% (XM_020469225.1) LC3 Oncorhynchus mykiss microtubule- 134 134 98% 3e-28 99% associated proteins 1A/1B light chain 3B (XM_021592266.1) GABARAP Oncorhynchus kisutch gamma- 50.0 50.0 83% 8e-04 94% aminobutyric acid receptor-associated protein-like (XM_021615250.1) EF1 Oncorhynchus kisutch elongation factor 124 124 98% 2e-25 99% 1-alpha (XM_020500543.1) RS11 Oncorhynchus mykiss 40S ribosomal 100 100 98% 2e-18 98% protein S11 (rs11) (NM_001246324.2)

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Appendix 2.4. Relative expression of Atg genes under different degrees of serum restrictions

Relative expression of Atg genes under different serum restrictions

Relative expression of ATG4, ATG5, ATG7 and ATG9 at different serum percentages (0.5, 1.5 and 2% FBS) and time points (12 h, 1 d, 3 d and 6 d). Values are represented in a log2 scale. Values above zero are upregulated and below zero are downregulated. The higher expression values were found at 1.5% FBS, with a peak at 3 days

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Appendix 2.5a-c) Statistical analysis of Atg gene expression in RTgill-W1 cells. Three- factorial-factor (gene, time, treatment) analysis was performed using SAS

THREE FACTORIAL FACTOR ANOVA

The Mixed Procedure

Model Information

Dependent Variable ratio

Covariance Structure diagonal

Estimation Method REML

Residual Variance Method Profile

Model- Fixed Effects SE Method Based

Type 3 Tests of Fixed Effects

Num Den

Effect DF DF F Value Pr > F

Target 9 319 2571.59 <.0001

Trt 1 319 3.97 0.0472

Time 3 319 673.86 <.0001

Target*Trt 9 319 33.17 <.0001

Target*Time 27 319 119.2 <.0001

Trt*Time 3 319 168.37 <.0001

Target*Trt*Time 27 319 10.93 <.0001

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Test for Normality

Test Statistic p value

Shapiro-Wilk W 0.99 Pr

Kolmogorov-Smirnov D 0.04 Pr>D 0.1153

W- Pr>W- Cramer-von Mises Sq 0.16 Sq 0.0199

A- Anderson-Darling Sq 1 Pr>A-Sq 0.0131

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Appendix 2.5b: Post-hoc statistical analysis of Atg gene expression in RTgill-W1 cells: Least Significant Difference test was performed in SAS (significant data is in blue)

Group 1 Group 2 Statistics Effect Target Treatment Time Target Treatment Time Estimate Probt LL RoR UL Time ATG12 Feed-Restricted 1 ATG12 Feed-Restricted 2 0.4651 0.002 1.186 1.592 2.137 Time ATG12 Feed-Restricted 1 ATG12 Feed-Restricted 3 -1.1001 <.0001 0.248 0.333 0.447 Time ATG12 Feed-Restricted 1 ATG12 Feed-Restricted 4 -0.01031 0.9451 0.737 0.99 1.328 Time ATG12 Feed-Restricted 2 ATG12 Feed-Restricted 3 -1.5653 <.0001 0.156 0.209 0.281 Time ATG12 Feed-Restricted 2 ATG12 Feed-Restricted 4 -0.4755 0.0016 0.463 0.622 0.834 Time ATG12 Feed-Restricted 3 ATG12 Feed-Restricted 4 1.0898 <.0001 2.216 2.974 3.991 Time ATG12 Control 1 ATG12 Control 2 0.6234 <.0001 1.39 1.865 2.503 Time ATG12 Control 1 ATG12 Control 3 -0.6695 <.0001 0.382 0.512 0.687 Time ATG12 Control 1 ATG12 Control 4 -0.7668 <.0001 0.346 0.465 0.623 Time ATG12 Control 2 ATG12 Control 3 -1.2929 <.0001 0.205 0.274 0.368 Time ATG12 Control 2 ATG12 Control 4 -1.3902 <.0001 0.186 0.249 0.334 Time ATG12 Control 3 ATG12 Control 4 -0.09731 0.5156 0.676 0.907 1.218 Time ATG13 Feed-Restricted 1 ATG13 Feed-Restricted 2 0.3831 0.0109 1.093 1.467 1.968 Time ATG13 Feed-Restricted 1 ATG13 Feed-Restricted 3 -1.2728 <.0001 0.209 0.28 0.376 Time ATG13 Feed-Restricted 1 ATG13 Feed-Restricted 4 -0.4825 0.0014 0.46 0.617 0.828 Time ATG13 Feed-Restricted 2 ATG13 Feed-Restricted 3 -1.6559 <.0001 0.142 0.191 0.256 Time ATG13 Feed-Restricted 2 ATG13 Feed-Restricted 4 -0.8656 <.0001 0.314 0.421 0.565 Time ATG13 Feed-Restricted 3 ATG13 Feed-Restricted 4 0.7903 <.0001 1.642 2.204 2.958 Time ATG13 Control 1 ATG13 Control 2 0.2406 0.1086 0.948 1.272 1.707 Time ATG13 Control 1 ATG13 Control 3 -0.8145 <.0001 0.33 0.443 0.594 Time ATG13 Control 1 ATG13 Control 4 -1.0138 <.0001 0.27 0.363 0.487 Time ATG13 Control 2 ATG13 Control 3 -1.0551 <.0001 0.259 0.348 0.467

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Group 1 Group 2 Statistics Effect Target Treatment Time Target Treatment Time Estimate Probt LL RoR UL Time ATG13 Control 2 ATG13 Control 4 -1.2544 <.0001 0.213 0.285 0.383 Time ATG13 Control 3 ATG13 Control 4 -0.1993 0.1836 0.611 0.819 1.1 Time ATG16 Feed-Restricted 1 ATG16 Feed-Restricted 2 -0.6462 <.0001 0.39 0.524 0.703 Time ATG16 Feed-Restricted 1 ATG16 Feed-Restricted 3 -3.0542 <.0001 0.035 0.047 0.063 Time ATG16 Feed-Restricted 1 ATG16 Feed-Restricted 4 -0.4528 0.0027 0.474 0.636 0.853 Time ATG16 Feed-Restricted 2 ATG16 Feed-Restricted 3 -2.408 <.0001 0.067 0.09 0.121 Time ATG16 Feed-Restricted 2 ATG16 Feed-Restricted 4 0.1935 0.1967 0.904 1.213 1.628 Time ATG16 Feed-Restricted 3 ATG16 Feed-Restricted 4 2.6015 <.0001 10.047 13.483 18.095 Time ATG16 Control 1 ATG16 Control 2 0.877 <.0001 1.791 2.404 3.226 Time ATG16 Control 1 ATG16 Control 3 -1.3265 <.0001 0.198 0.265 0.356 Time ATG16 Control 1 ATG16 Control 4 0.2621 0.0805 0.968 1.3 1.744 Time ATG16 Control 2 ATG16 Control 3 -2.2034 <.0001 0.082 0.11 0.148 Time ATG16 Control 2 ATG16 Control 4 -0.6148 <.0001 0.403 0.541 0.726 Time ATG16 Control 3 ATG16 Control 4 1.5886 <.0001 3.649 4.897 6.572 Time ATG4 Feed-Restricted 1 ATG4 Feed-Restricted 2 2.5128 <.0001 9.194 12.339 16.559 Time ATG4 Feed-Restricted 1 ATG4 Feed-Restricted 3 2.8633 <.0001 13.054 17.519 23.512 Time ATG4 Feed-Restricted 1 ATG4 Feed-Restricted 4 4.8231 <.0001 92.66 124.352 166.883 Time ATG4 Feed-Restricted 2 ATG4 Feed-Restricted 3 0.3505 0.0197 1.058 1.42 1.905 Time ATG4 Feed-Restricted 2 ATG4 Feed-Restricted 4 2.3103 <.0001 7.51 10.078 13.525 Time ATG4 Feed-Restricted 3 ATG4 Feed-Restricted 4 1.9598 <.0001 5.289 7.098 9.526 Time ATG4 Control 1 ATG4 Control 2 2.7182 <.0001 11.291 15.153 20.336 Time ATG4 Control 1 ATG4 Control 3 3.2241 <.0001 18.727 25.132 33.728 Time ATG4 Control 1 ATG4 Control 4 4.9634 <.0001 106.61 143.074 192.009 Time ATG4 Control 2 ATG4 Control 3 0.5059 0.0008 1.236 1.658 2.226 Time ATG4 Control 2 ATG4 Control 4 2.2451 <.0001 7.035 9.442 12.671 Time ATG4 Control 3 ATG4 Control 4 1.7392 <.0001 4.242 5.693 7.64

255

Group 1 Group 2 Statistics Effect Target Treatment Time Target Treatment Time Estimate Probt LL RoR UL Time ATG5 Feed-Restricted 1 ATG5 Feed-Restricted 4 -0.303 0.0435 0.55 0.739 0.991 Time ATG5 Feed-Restricted 2 ATG5 Feed-Restricted 3 -1.7946 <.0001 0.12384 0.1662 0.223 Time ATG5 Feed-Restricted 2 ATG5 Feed-Restricted 4 0.2843 0.0582 0.99014 1.3288 1.7833 Time ATG5 Feed-Restricted 3 ATG5 Feed-Restricted 4 2.0789 <.0001 5.95786 7.9956 10.7303 Time ATG5 Control 1 ATG5 Control 2 0.109 0.4666 0.83095 1.1152 1.4966 Time ATG5 Control 1 ATG5 Control 3 -0.7873 <.0001 0.3391 0.4551 0.6107 Time ATG5 Control 1 ATG5 Control 4 -0.4978 0.001 0.45294 0.6079 0.8158 Time ATG5 Control 2 ATG5 Control 3 -0.8963 <.0001 0.30408 0.4081 0.5477 Time ATG5 Control 2 ATG5 Control 4 -0.6068 <.0001 0.40617 0.5451 0.7315 Time ATG5 Control 3 ATG5 Control 4 0.2895 0.0538 0.9953 1.3357 1.7926 Time ATG7 Feed-Restricted 1 ATG7 Feed-Restricted 2 -0.9175 <.0001 0.2977 0.3995 0.5362 Time ATG7 Feed-Restricted 1 ATG7 Feed-Restricted 3 -2.0681 <.0001 0.09421 0.1264 0.1697 Time ATG7 Feed-Restricted 1 ATG7 Feed-Restricted 4 -0.3039 0.043 0.54989 0.738 0.9904 Time ATG7 Feed-Restricted 2 ATG7 Feed-Restricted 3 -1.1506 <.0001 0.23579 0.3164 0.4247 Time ATG7 Feed-Restricted 2 ATG7 Feed-Restricted 4 0.6136 <.0001 1.37635 1.8471 2.4789 Time ATG7 Feed-Restricted 3 ATG7 Feed-Restricted 4 1.7642 <.0001 4.34948 5.8371 7.8336 Time ATG7 Control 1 ATG7 Control 2 0.4647 0.0021 1.18595 1.5916 2.1359 Time ATG7 Control 1 ATG7 Control 3 -0.6214 <.0001 0.40027 0.5372 0.7209 Time ATG7 Control 1 ATG7 Control 4 0.7999 <.0001 1.65813 2.2253 2.9863 Time ATG7 Control 2 ATG7 Control 3 -1.0862 <.0001 0.25149 0.3375 0.4529 Time ATG7 Control 2 ATG7 Control 4 0.3351 0.0257 1.04181 1.3981 1.8763 Time ATG7 Control 3 ATG7 Control 4 1.4213 <.0001 3.08676 4.1425 5.5594

256

Group 1 Group 2 Statistics Effect Target Treatment Time Target Treatment Time Estimate Probt LL RoR UL Time ATG9 Feed-Restricted 1 ATG9 Feed-Restricted 4 -0.6976 <.0001 0.37093 0.4978 0.6681 Time ATG9 Feed-Restricted 2 ATG9 Feed-Restricted 3 -2.2544 <.0001 0.07819 0.1049 0.1408 Time ATG9 Feed-Restricted 2 ATG9 Feed-Restricted 4 0.1239 0.4078 0.84345 1.1319 1.5191 Time ATG9 Feed-Restricted 3 ATG9 Feed-Restricted 4 2.3783 <.0001 8.03787 10.787 14.4765 Time ATG9 Control 1 ATG9 Control 2 1.4991 <.0001 3.33639 4.4775 6.0089 Time ATG9 Control 1 ATG9 Control 3 -0.5134 0.0007 0.44594 0.5985 0.8032 Time ATG9 Control 1 ATG9 Control 4 1.8865 <.0001 4.91529 6.5965 8.8526 Time ATG9 Control 2 ATG9 Control 3 -2.0125 <.0001 0.0996 0.1337 0.1794 Time ATG9 Control 2 ATG9 Control 4 0.3875 0.01 1.09777 1.4732 1.9771 Time ATG9 Control 3 ATG9 Control 4 2.3999 <.0001 8.21314 11.0222 14.7921 Time BEC1 Feed-Restricted 1 BEC1 Feed-Restricted 2 -0.9282 <.0001 0.29452 0.3953 0.5304 Time BEC1 Feed-Restricted 1 BEC1 Feed-Restricted 3 -2.6937 <.0001 0.05039 0.0676 0.0908 Time BEC1 Feed-Restricted 1 BEC1 Feed-Restricted 4 -1.2942 <.0001 0.20425 0.2741 0.3679 Time BEC1 Feed-Restricted 2 BEC1 Feed-Restricted 3 -1.7655 <.0001 0.12749 0.1711 0.2296 Time BEC1 Feed-Restricted 2 BEC1 Feed-Restricted 4 -0.366 0.0149 0.51676 0.6935 0.9307 Time BEC1 Feed-Restricted 3 BEC1 Feed-Restricted 4 1.3995 <.0001 3.02028 4.0533 5.4396 Time BEC1 Control 1 BEC1 Control 2 1.1094 <.0001 2.25956 3.0324 4.0695 Time BEC1 Control 1 BEC1 Control 3 -0.3333 0.0265 0.53393 0.7166 0.9616 Time BEC1 Control 1 BEC1 Control 4 0.06891 0.6452 0.7983 1.0713 1.4378 Time BEC1 Control 2 BEC1 Control 3 -1.4427 <.0001 0.17608 0.2363 0.3171 Time BEC1 Control 2 BEC1 Control 4 -1.0404 <.0001 0.26326 0.3533 0.4741 Time BEC1 Control 3 BEC1 Control 4 0.4022 0.0075 1.11408 1.4951 2.0065

257

Group 1 Group 2 Statistics Effect Target Treatment Time Target Treatment Time Estimate Probt LL RoR UL Time GABA Feed-Restricted 1 GABA Feed-Restricted 4 -0.642 <.0001 0.39211 0.5262 0.7062 Time GABA Feed-Restricted 2 GABA Feed-Restricted 3 -1.2113 <.0001 0.22192 0.2978 0.3997 Time GABA Feed-Restricted 2 GABA Feed-Restricted 4 -0.657 <.0001 0.38629 0.5184 0.6957 Time GABA Feed-Restricted 3 GABA Feed-Restricted 4 0.5543 0.0002 1.29705 1.7407 2.336 Time GABA Control 1 GABA Control 2 1.5091 <.0001 3.37004 4.52268 6.06955 Time GABA Control 1 GABA Control 3 0.07973 0.6155 0.79271 1.08299 1.47957 Time GABA Control 1 GABA Control 4 0.2901 0.0532 0.99597 1.33662 1.79378 Time GABA Control 2 GABA Control 3 -1.4294 <.0001 0.17527 0.23946 0.32714 Time GABA Control 2 GABA Control 4 -1.219 <.0001 0.22022 0.29554 0.39662 Time GABA Control 3 GABA Control 4 0.2104 0.1855 0.90338 1.23419 1.68614 Time LC3 Feed-Restricted 1 LC3 Feed-Restricted 2 -0.1096 0.4641 0.66778 0.89618 1.2027 Time LC3 Feed-Restricted 1 LC3 Feed-Restricted 3 -2.0779 <.0001 0.09329 0.12519 0.16801 Time LC3 Feed-Restricted 1 LC3 Feed-Restricted 4 -1.4306 <.0001 0.17821 0.23916 0.32096 Time LC3 Feed-Restricted 2 LC3 Feed-Restricted 3 -1.9683 <.0001 0.10409 0.1397 0.18747 Time LC3 Feed-Restricted 2 LC3 Feed-Restricted 4 -1.321 <.0001 0.19886 0.26687 0.35814 Time LC3 Feed-Restricted 3 LC3 Feed-Restricted 4 0.6473 <.0001 1.42349 1.91037 2.56376 Time LC3 Control 1 LC3 Control 2 1.6409 <.0001 3.84495 5.16002 6.92488 Time LC3 Control 1 LC3 Control 3 -0.2928 0.0511 0.556 0.74617 1.00138 - Time LC3 Control 1 LC3 Control 4 0.05302 0.7231 0.70666 0.94836 1.27272 Time LC3 Control 2 LC3 Control 3 -1.9337 <.0001 0.10775 0.14461 0.19406 Time LC3 Control 2 LC3 Control 4 -1.694 <.0001 0.13695 0.18379 0.24665 Time LC3 Control 3 LC3 Control 4 0.2398 0.1098 0.94706 1.27097 1.70568

258

Appendix 2.5c: Post-hoc statistical analysis of Atg gene expression in RTgill-W1 cells: Least Significant Difference test was performed in SAS (significant data is in blue)

Effect Group 1 Group 2 Statistics

Treatment Target Treatment Time Target Treatment Time Estimate Probt LL RoR UL

Treatment ATG12 Feed-restricted 1 ATG12 Control 1 -0.174 0.2454 0.62614 0.8403 1.12771 - Treatment ATG12 Feed-restricted 2 ATG12 Control 2 0.01572 0.9163 0.73352 0.9844 1.32109 Treatment ATG12 Feed-restricted 3 ATG12 Control 3 0.2567 0.087 0.9632 1.29264 1.73475 Treatment ATG12 Feed-restricted 4 ATG12 Control 4 -0.9304 <.0001 0.29387 0.39438 0.52926 Treatment ATG13 Feed-restricted 1 ATG13 Control 1 -0.1373 0.3593 0.64956 0.87172 1.16988

Treatment ATG13 Feed-restricted 2 ATG13 Control 2 -0.2798 0.0622 0.56329 0.75595 1.01451 Treatment ATG13 Feed-restricted 3 ATG13 Control 3 0.321 0.0325 1.02722 1.37855 1.85005 Treatment ATG13 Feed-restricted 4 ATG13 Control 4 -0.6685 <.0001 0.38185 0.51245 0.68772 Treatment ATG16 Feed-restricted 1 ATG16 Control 1 -1.0971 <.0001 0.24876 0.33385 0.44803

Treatment ATG16 Feed-restricted 2 ATG16 Control 2 0.4261 0.0047 1.14103 1.53129 2.05503

Treatment ATG16 Feed-restricted 3 ATG16 Control 3 0.6307 <.0001 1.40005 1.8789 2.52154 Treatment ATG16 Feed-restricted 4 ATG16 Control 4 -0.3822 0.0111 0.50846 0.68237 0.91576 Treatment ATG4 Feed-restricted 1 ATG4 Control 1 0.4786 0.0015 1.20256 1.61387 2.16586 Treatment ATG4 Feed-restricted 2 ATG4 Control 2 0.6841 <.0001 1.47686 1.98198 2.65987 Treatment ATG4 Feed-restricted 3 ATG4 Control 3 0.8395 <.0001 1.72509 2.31511 3.10694 Treatment ATG4 Feed-restricted 4 ATG4 Control 4 0.6189 <.0001 1.38362 1.85685 2.49195 Treatment ATG5 Feed-restricted 1 ATG5 Control 1 -1.4439 <.0001 0.17586 0.23601 0.31674 Treatment ATG5 Feed-restricted 2 ATG5 Control 2 -0.7476 <.0001 0.35283 0.47351 0.63546

Treatment ATG5 Feed-restricted 3 ATG5 Control 3 0.1508 0.3141 0.86639 1.16272 1.5604

Treatment ATG5 Feed-restricted 4 ATG5 Control 4 -1.6387 <.0001 0.14474 0.19424 0.26067

259

Effect Group 1 Group 2 Statistics Treatment Target Treatment Time Target Treatment Time Estimate Probt LL RoR UL Treatment ATG7 Feed-restricted 3 ATG7 Control 3 0.1411 0.3459 0.85809 1.15158 1.54545 Treatment ATG7 Feed-restricted 4 ATG7 Control 4 -0.2018 0.1781 0.60897 0.81726 1.09678 Treatment ATG9 Feed-restricted 3 ATG9 Control 3 0.8878 <.0001 1.81054 2.4298 3.26085 Treatment ATG9 Feed-restricted 4 ATG9 Control 4 0.9094 <.0001 1.85002 2.48278 3.33195 Treatment BEC1 Feed-restricted 1 BEC1 Control 1 -1.5771 <.0001 0.15393 0.20657 0.27723 Treatment BEC1 Feed-restricted 2 BEC1 Control 2 0.4605 0.0023 1.18091 1.58481 2.12686 Treatment BEC1 Feed-restricted 3 BEC1 Control 3 0.7833 <.0001 1.63093 2.18875 2.93737 Treatment BEC1 Feed-restricted 4 BEC1 Control 4 -0.214 0.1534 0.60159 0.80736 1.08349 Treatment GABA Feed-restricted 1 GABA Control 1 -0.6595 <.0001 0.3853 0.51709 0.69395 Treatment GABA Feed-restricted 2 GABA Control 2 0.8346 <.0001 1.71674 2.3039 3.0919 Treatment GABA Feed-restricted 3 GABA Control 3 0.6165 0.0001 1.35589 1.8524 2.5307 Treatment GABA Feed-restricted 4 GABA Control 4 0.2726 0.0692 0.97868 1.3134 1.7626 Treatment LC3 Feed-restricted 1 LC3 Control 1 -0.9218 <.0001 0.29641 0.3978 0.5338 Treatment LC3 Feed-restricted 2 LC3 Control 2 0.8287 <.0001 1.70667 2.2904 3.0738 Treatment LC3 Feed-restricted 3 LC3 Control 3 0.8633 <.0001 1.76666 2.3709 3.1818 Treatment LC3 Feed-restricted 4 LC3 Control 4 0.4558 0.0025 1.17537 1.5774 2.1169

260

Appendix 3.1a-c: Statistical analysis of Atg gene expression in liver and muscle. Three- factorial-factor (gene, time, treatment) analysis was performed using SAS Factorial Factor Anova

The Mixed Procedure

Model Inforation

Dependent Variable Rratio

Covariance Structure Variance Components

Estimation Method REML

Residual Variance Method None

Fixed Effects SE Method Model-Based

The Mixed Procedure

Type 3 Tests of Fixed Effects

Num Den

Effect DF DF F Value Pr > F

Target 9 829 1193.95 <.0001

Trt 1 46 93.25 <.0001

Time 5 46 257.07 <.0001

Target*Trt 9 829 37.58 <.0001

Target*Time 45 829 261.12 <.0001

Trt*Time 5 46 28.4 <.0001

Target*Trt*Time 45 829 40.4 <.0001

Sourc*Targe*Trt*Time 45 829 34.89 <.0001

261

Tests for Normality Test Statistic p Value Shapiro-Wilk W 0.964132 Pr < W <0.0001 Kolmogorov-Smirnov D 0.038151 Pr < D <0.0100 Cramer-von Mises W-Sq 0.583099 Pr < W-Sq <0.0050

Anderson-Darling A-Sq 4.225707 Pr < A-Sq <0.0050

Extreme Observations

Lowest Highest

Value Obs Value Obs

-0.758017 677 0.531725 725

-0.63856 955 0.555757 64

-0.500421 295 0.602947 291

-0.494575 509 0.65007 707

-0.477193 836 0.660763 412

262

Appendix 3.1b: Post-hoc statistical analysis of Atg gene expression in liver: Least Significant Difference test was performed in SAS (significant data is in blue)

Group 1 Group 2 Statistics Effect Target Treatment Time Target Treatment Time Estimate Probt LL RoR UL Time ATG12 Control 1 ATG12 Control 2 -0.3924 0.0016 0.52922 0.67542 0.862 Time ATG12 Control 1 ATG12 Control 3 -0.586 <.0001 0.44036 0.55655 0.7034 Time ATG12 Control 1 ATG12 Control 4 -0.5885 <.0001 0.43862 0.55517 0.7027 Time ATG12 Control 1 ATG12 Control 5 -0.9195 <.0001 0.31333 0.39873 0.5074 Time ATG12 Control 1 ATG12 Control 6 0.5831 0.0004 1.29876 1.79156 2.4714 Time ATG12 Control 2 ATG12 Control 3 -0.1936 0.0487 0.67972 0.824 0.9989 Time ATG12 Control 2 ATG12 Control 4 -0.1961 0.048 0.67682 0.82196 0.9982 Time ATG12 Control 2 ATG12 Control 5 -0.527 <.0001 0.48296 0.59035 0.7216 Time ATG12 Control 2 ATG12 Control 6 0.9755 <.0001 1.97936 2.65251 3.5546 Time ATG12 Control 3 ATG12 Control 4 -0.00248 0.9787 0.83165 0.99753 1.1965 Time ATG12 Control 3 ATG12 Control 5 -0.3335 0.0006 0.59316 0.71644 0.8653 Time ATG12 Control 3 ATG12 Control 6 1.1691 <.0001 2.42163 3.21906 4.2791 Time ATG12 Control 4 ATG12 Control 5 -0.331 0.0007 0.5935 0.71822 0.8691 Time ATG12 Control 4 ATG12 Control 6 1.1716 <.0001 2.42465 3.22704 4.295 Time ATG12 Control 5 ATG12 Control 6 1.5025 <.0001 3.36091 4.49313 6.0068 Time ATG12 Feed-restricted 1 ATG12 Feed-restricted 2 0.8635 <.0001 1.85815 2.37146 3.0266 Time ATG12 Feed-restricted 1 ATG12 Feed-restricted 3 0.6338 <.0001 1.49126 1.88473 2.382 Time ATG12 Feed-restricted 1 ATG12 Feed-restricted 4 0.9316 <.0001 2.00568 2.53865 3.2132 Time ATG12 Feed-restricted 1 ATG12 Feed-restricted 5 0.538 <.0001 1.34723 1.71252 2.1768 Time ATG12 Feed-restricted 1 ATG12 Feed-restricted 6 1.9295 <.0001 4.99198 6.88615 9.499 Time ATG12 Feed-restricted 2 ATG12 Feed-restricted 3 -0.2297 0.0194 0.6556 0.79476 0.9635 Time ATG12 Feed-restricted 2 ATG12 Feed-restricted 4 0.06813 0.4915 0.88148 1.0705 1.3001 Time ATG12 Feed-restricted 2 ATG12 Feed-restricted 5 -0.3255 0.0014 0.59158 0.72214 0.8815

263

Effect Target Treatment Time TARGET Treatment Time Estimate Probt LL RoR UL Time ATG12 Feed-restricted 2 ATG12 Feed-restricted 6 1.066 <.0001 2.16684 2.90376 3.8913 Time ATG12 Feed-restricted 3 ATG12 Feed-restricted 4 0.2978 0.0014 1.12298 1.34696 1.6156 Time ATG12 Feed-restricted 3 ATG12 Feed-restricted 5 -0.09582 0.3158 0.75337 0.90863 1.0959 Time ATG12 Feed-restricted 3 ATG12 Feed-restricted 6 1.2957 <.0001 2.74856 3.65364 4.8568 Time ATG12 Feed-restricted 4 ATG12 Feed-restricted 5 -0.3937 <.0001 0.55824 0.67458 0.8152 Time ATG12 Feed-restricted 4 ATG12 Feed-restricted 6 0.9979 <.0001 2.03806 2.71252 3.6102 Time ATG12 Feed-restricted 5 ATG12 Feed-restricted 6 1.3915 <.0001 3.01065 4.02107 5.3706 Time ATG13 Control 1 ATG13 Control 2 -0.1762 0.1566 0.65695 0.83844 1.0701 Time ATG13 Control 1 ATG13 Control 3 -0.3752 0.0017 0.5437 0.68712 0.8684 Time ATG13 Control 1 ATG13 Control 4 -0.7576 <.0001 0.37039 0.46881 0.5934 Time ATG13 Control 1 ATG13 Control 5 -2.6385 <.0001 0.05616 0.07147 0.0909 Time ATG13 Control 1 ATG13 Control 6 -0.4331 0.0084 0.47013 0.6485 0.8946 Time ATG13 Control 2 ATG13 Control 3 -0.199 0.0427 0.67607 0.81953 0.9934 Time ATG13 Control 2 ATG13 Control 4 -0.5813 <.0001 0.46042 0.55915 0.679 Time ATG13 Control 2 ATG13 Control 5 -2.4623 <.0001 0.06973 0.08524 0.1042 Time ATG13 Control 2 ATG13 Control 6 -0.2569 0.0854 0.57718 0.77346 1.0365 Time ATG13 Control 3 ATG13 Control 4 -0.3823 <.0001 0.56883 0.68228 0.8184 Time ATG13 Control 3 ATG13 Control 5 -2.2633 <.0001 0.08611 0.10401 0.1256 Time ATG13 Control 3 ATG13 Control 6 -0.05785 0.6901 0.71001 0.9438 1.2546 Time ATG13 Control 4 ATG13 Control 5 -1.881 <.0001 0.12598 0.15244 0.1845 Time ATG13 Control 4 ATG13 Control 6 0.3245 0.0262 1.03934 1.3833 1.8411 Time ATG13 Control 5 ATG13 Control 6 2.2054 <.0001 6.78756 9.07408 12.1308 Time ATG13 Feed-restricted 1 ATG13 Feed-restricted 2 0.04577 0.7128 0.82024 1.04683 1.336 Time ATG13 Feed-restricted 1 ATG13 Feed-restricted 3 0.1027 0.3892 0.87689 1.10821 1.4006 Time ATG13 Feed-restricted 1 ATG13 Feed-restricted 4 -0.09541 0.427 0.71817 0.909 1.1505 Time ATG13 Feed-restricted 1 ATG13 Feed-restricted 5 -1.891 <.0001 0.11874 0.15092 0.1918

264

Effect Target Treatment Time TARGET Treatment Time Estimate Probt LL RoR UL Time ATG13 Feed-restricted 1 ATG13 Feed-restricted 6 -1.1228 <.0001 0.23586 0.32535 0.4488 Time ATG13 Feed-restricted 2 ATG13 Feed-restricted 3 0.05698 0.5613 0.87332 1.05864 1.2833 Time ATG13 Feed-restricted 2 ATG13 Feed-restricted 4 -0.1412 0.1541 0.71502 0.86834 1.05453 Time ATG13 Feed-restricted 2 ATG13 Feed-restricted 5 -1.9367 <.0001 0.11811 0.14417 0.17599 Time ATG13 Feed-restricted 2 ATG13 Feed-restricted 6 -1.1686 <.0001 0.23193 0.3108 0.41649 Time ATG13 Feed-restricted 3 ATG13 Feed-restricted 4 -0.1982 0.0327 0.68385 0.82024 0.98382 Time ATG13 Feed-restricted 3 ATG13 Feed-restricted 5 -1.9937 <.0001 0.11292 0.13619 0.16425 Time ATG13 Feed-restricted 3 ATG13 Feed-restricted 6 -1.2256 <.0001 0.22086 0.29358 0.39025 Time ATG13 Feed-restricted 4 ATG13 Feed-restricted 5 -1.7956 <.0001 0.1374 0.16603 0.20063 Time ATG13 Feed-restricted 4 ATG13 Feed-restricted 6 -1.0274 <.0001 0.26893 0.35792 0.47637 Time ATG13 Feed-restricted 5 ATG13 Feed-restricted 6 0.7681 <.0001 1.61405 2.15573 2.87921 Time ATG16 Control 1 ATG16 Control 2 -0.1763 0.1563 0.6569 0.83836 1.06995 Time ATG16 Control 1 ATG16 Control 3 -0.591 <.0001 0.43817 0.55375 0.69982 Time ATG16 Control 1 ATG16 Control 4 -0.9159 <.0001 0.31614 0.40015 0.50649 Time ATG16 Control 1 ATG16 Control 5 0.3861 0.0017 1.15612 1.47126 1.87231 Time ATG16 Control 1 ATG16 Control 6 -0.6927 <.0001 0.36264 0.50024 0.69005 Time ATG16 Control 2 ATG16 Control 3 -0.4147 <.0001 0.54489 0.66052 0.80068 Time ATG16 Control 2 ATG16 Control 4 -0.7396 <.0001 0.393 0.4773 0.57968 Time ATG16 Control 2 ATG16 Control 5 0.5624 <.0001 1.43561 1.75493 2.14526 Time ATG16 Control 2 ATG16 Control 6 -0.5164 0.0006 0.44525 0.59669 0.79963 Time ATG16 Control 3 ATG16 Control 4 -0.3249 0.0005 0.60245 0.72262 0.86676 Time ATG16 Control 3 ATG16 Control 5 0.9772 <.0001 2.19972 2.65689 3.20908 Time ATG16 Control 3 ATG16 Control 6 -0.1016 0.4836 0.67958 0.90337 1.20084 Time ATG16 Control 4 ATG16 Control 5 1.302 <.0001 3.03831 3.67676 4.44938 Time ATG16 Control 4 ATG16 Control 6 0.2232 0.1257 0.93929 1.25013 1.66385 Time ATG16 Control 5 ATG16 Control 6 -1.0788 <.0001 0.25432 0.34001 0.45456

265

Effect Target Treatment Time Target Treatment Time Estimate Probt LL RoR UL Time ATG16 Feed-restricted 1 ATG16 Feed-restricted 2 -0.0758 0.542 0.72635 0.927 1.18307 Time ATG16 Feed-restricted 1 ATG16 Feed-restricted 3 -0.2354 0.0487 0.6253 0.79024 0.99868 Time ATG16 Feed-restricted 1 ATG16 Feed-restricted 4 -0.1123 0.35 0.70615 0.89381 1.13134 Time ATG16 Feed-restricted 1 ATG16 Feed-restricted 5 0.5462 <.0001 1.36095 1.7266 2.19048 Time ATG16 Feed-restricted 1 ATG16 Feed-restricted 6 -0.9649 <.0001 0.27622 0.38103 0.52561 Time ATG16 Feed-restricted 2 ATG16 Feed-restricted 3 -0.1596 0.1039 0.70324 0.85247 1.03336 Time ATG16 Feed-restricted 2 ATG16 Feed-restricted 4 -0.03646 0.7128 0.7939 0.9642 1.17102 Time ATG16 Feed-restricted 2 ATG16 Feed-restricted 5 0.622 <.0001 1.52934 1.86257 2.2684 Time ATG16 Feed-restricted 2 ATG16 Feed-restricted 6 -0.8891 <.0001 0.30672 0.41104 0.55084 Time ATG16 Feed-restricted 3 ATG16 Feed-restricted 4 0.1232 0.1842 0.94297 1.13106 1.35667 Time ATG16 Feed-restricted 3 ATG16 Feed-restricted 5 0.7816 <.0001 1.81612 2.18491 2.62859 Time ATG16 Feed-restricted 3 ATG16 Feed-restricted 6 -0.7294 <.0001 0.36273 0.48218 0.64095 Time ATG16 Feed-restricted 4 ATG16 Feed-restricted 5 0.6584 <.0001 1.60255 1.93173 2.32853 Time ATG16 Feed-restricted 4 ATG16 Feed-restricted 6 -0.8526 <.0001 0.3203 0.4263 0.56738 Time ATG16 Feed-restricted 5 ATG16 Feed-restricted 6 -1.511 <.0001 0.16549 0.22068 0.29428 Time ATG4 Control 1 ATG4 Control 2 0.1938 0.1192 0.95114 1.2139 1.54926 Time ATG4 Control 1 ATG4 Control 3 0.03741 0.7539 0.82143 1.03812 1.31196 Time ATG4 Control 1 ATG4 Control 4 -0.311 0.0098 0.57887 0.7327 0.92742 Time ATG4 Control 1 ATG4 Control 5 -0.06365 0.6044 0.73735 0.93833 1.19409 Time ATG4 Control 1 ATG4 Control 6 0.0446 0.7856 0.75799 1.04561 1.44235 Time ATG4 Control 2 ATG4 Control 3 -0.1564 0.111 0.70548 0.85519 1.03667 Time ATG4 Control 2 ATG4 Control 4 -0.5049 <.0001 0.49699 0.60359 0.73306 Time ATG4 Control 2 ATG4 Control 5 -0.2575 0.012 0.63237 0.77299 0.94487 Time ATG4 Control 2 ATG4 Control 6 -0.1492 0.3173 0.64276 0.86136 1.1543 Time ATG4 Control 3 ATG4 Control 4 -0.3484 0.0002 0.58843 0.7058 0.84658 Time ATG4 Control 3 ATG4 Control 5 -0.1011 0.2937 0.74836 0.90388 1.0917

266

Effect Target Treatment Time Target Treatment Time Estimate Probt LL RoR UL Time ATG4 Control 3 ATG4 Control 6 0.007186 0.9605 0.75771 1.00721 1.33887 Time ATG4 Control 4 ATG4 Control 5 0.2474 0.0111 1.05832 1.28064 1.54965 Time ATG4 Control 4 ATG4 Control 6 0.3556 0.0148 1.07224 1.42705 1.89927 Time ATG4 Control 5 ATG4 Control 6 0.1083 0.4645 0.83352 1.11433 1.48974 Time ATG4 Feed-restricted 1 ATG4 Feed-restricted 2 0.4948 <.0001 1.2851 1.64012 2.09322 Time ATG4 Feed-restricted 1 ATG4 Feed-restricted 3 -0.424 0.0004 0.51784 0.65445 0.82708 Time ATG4 Feed-restricted 1 ATG4 Feed-restricted 4 -0.445 0.0002 0.50628 0.64083 0.81113 Time ATG4 Feed-restricted 1 ATG4 Feed-restricted 5 0.2248 0.0663 0.98499 1.25205 1.59152 Time ATG4 Feed-restricted 1 ATG4 Feed-restricted 6 -0.0702 0.6685 0.67578 0.93221 1.28592 Time ATG4 Feed-restricted 2 ATG4 Feed-restricted 3 -0.9187 <.0001 0.32917 0.39902 0.4837 Time ATG4 Feed-restricted 2 ATG4 Feed-restricted 4 -0.9398 <.0001 0.32172 0.39072 0.47453 Time ATG4 Feed-restricted 2 ATG4 Feed-restricted 5 -0.27 0.008 0.62537 0.76339 0.93187 Time ATG4 Feed-restricted 2 ATG4 Feed-restricted 6 -0.565 0.0002 0.42413 0.56838 0.76168 Time ATG4 Feed-restricted 3 ATG4 Feed-restricted 4 -0.02103 0.8205 0.81636 0.97919 1.17451 Time ATG4 Feed-restricted 3 ATG4 Feed-restricted 5 0.6488 <.0001 1.5863 1.91315 2.30736 Time ATG4 Feed-restricted 3 ATG4 Feed-restricted 6 0.3538 0.0149 1.07157 1.42442 1.89345 Time ATG4 Feed-restricted 4 ATG4 Feed-restricted 5 0.6698 <.0001 1.61696 1.9538 2.36083 Time ATG4 Feed-restricted 4 ATG4 Feed-restricted 6 0.3748 0.0102 1.093 1.45469 1.93606 Time ATG4 Feed-restricted 5 ATG4 Feed-restricted 6 -0.295 0.0458 0.55744 0.74454 0.99443 Time ATG5 Control 1 ATG5 Control 2 0.9378 <.0001 2.00137 2.55432 3.26003 Time ATG5 Control 1 ATG5 Control 3 0.04283 0.7196 0.82587 1.04376 1.31915 Time ATG5 Control 1 ATG5 Control 4 -0.4118 0.0006 0.52336 0.66243 0.83847 Time ATG5 Control 1 ATG5 Control 5 -0.7963 <.0001 0.35441 0.45101 0.57394 Time ATG5 Control 1 ATG5 Control 6 0.9496 <.0001 1.87377 2.58475 3.56552 Time ATG5 Control 2 ATG5 Control 3 -0.895 <.0001 0.33708 0.40863 0.49536 Time ATG5 Control 2 ATG5 Control 4 -1.3496 <.0001 0.21354 0.25934 0.31496

267

Effect Target Treatment Time Target Treatment Time Estimate Probt LL RoR UL Time ATG5 Control 2 ATG5 Control 5 -1.7341 <.0001 0.14445 0.17657 0.21583 Time ATG5 Control 2 ATG5 Control 6 0.01185 0.9367 0.75511 1.01192 1.35606 Time ATG5 Control 3 ATG5 Control 4 -0.4547 <.0001 0.52913 0.63466 0.76123 Time ATG5 Control 3 ATG5 Control 5 -0.8391 <.0001 0.35776 0.4321 0.52188 Time ATG5 Control 3 ATG5 Control 6 0.9068 <.0001 1.86294 2.47638 3.29181 Time ATG5 Control 4 ATG5 Control 5 -0.3844 <.0001 0.56264 0.68083 0.82386 Time ATG5 Control 4 ATG5 Control 6 1.3615 <.0001 2.93172 3.9019 5.19316 Time ATG5 Control 5 ATG5 Control 6 1.7459 <.0001 4.28685 5.73106 7.66182 Time ATG5 Feed-restricted 1 ATG5 Feed-restricted 2 -0.06253 0.615 0.73603 0.93938 1.19892 Time ATG5 Feed-restricted 1 ATG5 Feed-restricted 3 0.05513 0.6441 0.83609 1.05668 1.33547 Time ATG5 Feed-restricted 1 ATG5 Feed-restricted 4 -0.2429 0.0434 0.61967 0.78434 0.99277 Time ATG5 Feed-restricted 1 ATG5 Feed-restricted 5 -0.7641 <.0001 0.36642 0.46576 0.59204 Time ATG5 Feed-restricted 1 ATG5 Feed-restricted 6 -1.0835 <.0001 0.24532 0.33841 0.46681 Time ATG5 Feed-restricted 2 ATG5 Feed-restricted 3 0.1177 0.2305 0.92792 1.12487 1.36361 Time ATG5 Feed-restricted 2 ATG5 Feed-restricted 5 -0.7015 <.0001 0.40617 0.49582 0.60525 Time ATG5 Feed-restricted 2 ATG5 Feed-restricted 6 -1.021 <.0001 0.26882 0.36025 0.48276 Time ATG5 Feed-restricted 3 ATG5 Feed-restricted 4 -0.298 0.0013 0.61885 0.74227 0.8903 Time ATG5 Feed-restricted 3 ATG5 Feed-restricted 5 -0.8192 <.0001 0.36548 0.44078 0.53159 Time ATG5 Feed-restricted 3 ATG5 Feed-restricted 6 -1.1386 <.0001 0.24092 0.32026 0.42571 Time ATG5 Feed-restricted 4 ATG5 Feed-restricted 5 -0.5212 <.0001 0.49145 0.59383 0.71754 Time ATG5 Feed-restricted 4 ATG5 Feed-restricted 6 -0.8406 <.0001 0.32418 0.43146 0.57424 Time ATG5 Feed-restricted 5 ATG5 Feed-restricted 6 -0.3194 0.0306 0.54399 0.72657 0.97043 Time ATG7 Control 1 ATG7 Control 2 2.0068 <.0001 5.82921 7.43971 9.49515 Time ATG7 Control 1 ATG7 Control 3 -0.09438 0.429 0.72 0.90994 1.14998 Time ATG7 Control 1 ATG7 Control 4 -0.1933 0.1077 0.65118 0.82422 1.04325 Time ATG7 Control 1 ATG7 Control 5 -0.3801 0.002 0.53737 0.68381 0.87016

268

Effect Target Treatment Time Target Treatment Time Estimate Probt LL RoR UL Time ATG7 Control 1 ATG7 Control 6 -0.5464 0.0009 0.41978 0.57905 0.79876 Time ATG7 Control 2 ATG7 Control 3 -2.1012 <.0001 0.10089 0.12231 0.14827 Time ATG7 Control 2 ATG7 Control 4 -2.2001 <.0001 0.09122 0.11079 0.13455 Time ATG7 Control 2 ATG7 Control 5 -2.3869 <.0001 0.0752 0.09191 0.11235 Time ATG7 Control 2 ATG7 Control 6 -2.5532 <.0001 0.05808 0.07783 0.1043 Time ATG7 Control 3 ATG7 Control 4 -0.09894 0.286 0.75517 0.9058 1.08648 Time ATG7 Control 3 ATG7 Control 5 -0.2857 0.0031 0.62223 0.75149 0.90761 Time ATG7 Control 3 ATG7 Control 6 -0.452 0.0019 0.47873 0.63636 0.84591 Time ATG7 Control 4 ATG7 Control 5 -0.1868 0.0548 0.68565 0.82965 1.00388 Time ATG7 Control 4 ATG7 Control 6 -0.353 0.0156 0.52786 0.70254 0.93504 Time ATG7 Control 5 ATG7 Control 6 -0.1663 0.2612 0.63342 0.8468 1.13206 Time ATG7 Feed-restricted 1 ATG7 Feed-restricted 2 0.4058 0.0011 1.17573 1.50055 1.91513 Time ATG7 Feed-restricted 1 ATG7 Feed-restricted 3 0.1226 0.3042 0.89449 1.13047 1.42869 Time ATG7 Feed-restricted 1 ATG7 Feed-restricted 4 0.04532 0.7059 0.82668 1.04636 1.32443 Time ATG7 Feed-restricted 1 ATG7 Feed-restricted 5 -0.3802 0.0019 0.53791 0.68371 0.86904 Time ATG7 Feed-restricted 1 ATG7 Feed-restricted 6 -1.1201 <.0001 0.23652 0.32626 0.45006 Time ATG7 Feed-restricted 2 ATG7 Feed-restricted 3 -0.2832 0.004 0.62147 0.75337 0.91326 Time ATG7 Feed-restricted 2 ATG7 Feed-restricted 4 -0.3605 0.0003 0.57416 0.69732 0.84689 Time ATG7 Feed-restricted 2 ATG7 Feed-restricted 5 -0.7861 <.0001 0.37327 0.45564 0.55619 Time ATG7 Feed-restricted 2 ATG7 Feed-restricted 6 -1.5259 <.0001 0.16225 0.21743 0.29138 Time ATG7 Feed-restricted 3 ATG7 Feed-restricted 4 -0.07731 0.4043 0.77168 0.9256 1.11023 Time ATG7 Feed-restricted 3 ATG7 Feed-restricted 5 -0.5028 <.0001 0.50151 0.60481 0.72939 Time ATG7 Feed-restricted 3 ATG7 Feed-restricted 6 -1.2427 <.0001 0.21712 0.28861 0.38364 Time ATG7 Feed-restricted 4 ATG7 Feed-restricted 5 -0.4255 <.0001 0.54079 0.65342 0.78951 Time ATG7 Feed-restricted 4 ATG7 Feed-restricted 6 -1.1654 <.0001 0.23428 0.31181 0.41499 Time ATG7 Feed-restricted 5 ATG7 Feed-restricted 6 -0.7398 <.0001 0.35728 0.47719 0.63734

269

Effect Target Treatment Time Target Treatment Time Estimate Probt LL RoR UL Time ATG9 Control 1 ATG9 Control 2 1.2525 <.0001 2.74169 3.49922 4.46605 Time ATG9 Control 1 ATG9 Control 3 -0.3848 0.0013 0.53852 0.6806 0.86015 Time ATG9 Control 1 ATG9 Control 4 -0.2548 0.0341 0.61235 0.77506 0.98102 Time ATG9 Control 1 ATG9 Control 5 -1.1191 <.0001 0.25664 0.32659 0.4156 Time ATG9 Control 1 ATG9 Control 6 -1.3873 <.0001 0.18106 0.24976 0.34452 Time ATG9 Control 2 ATG9 Control 3 -1.6373 <.0001 0.16044 0.1945 0.23579 Time ATG9 Control 2 ATG9 Control 4 -1.5073 <.0001 0.18238 0.2215 0.269 Time ATG9 Control 2 ATG9 Control 5 -2.3716 <.0001 0.07635 0.09333 0.11409 Time ATG9 Control 2 ATG9 Control 6 -2.6398 <.0001 0.05326 0.07138 0.09565 Time ATG9 Control 3 ATG9 Control 4 0.13 0.1611 0.94943 1.1388 1.36594 Time ATG9 Control 3 ATG9 Control 5 -0.7343 <.0001 0.39728 0.47985 0.57959 Time ATG9 Control 3 ATG9 Control 6 -1.0025 <.0001 0.27607 0.36697 0.4878 Time ATG9 Control 4 ATG9 Control 5 -0.8642 <.0001 0.34821 0.42137 0.50989 Time ATG9 Control 4 ATG9 Control 6 -1.1325 <.0001 0.24212 0.32224 0.42888 Time ATG9 Control 5 ATG9 Control 6 -0.2682 0.0702 0.57203 0.76475 1.02239 Time ATG9 Feed-restricted 1 ATG9 Feed-restricted 2 0.9891 <.0001 2.10681 2.68892 3.43187 Time ATG9 Feed-restricted 1 ATG9 Feed-restricted 3 0.3081 0.01 1.07672 1.36078 1.71978 Time ATG9 Feed-restricted 1 ATG9 Feed-restricted 4 0.9122 <.0001 1.96711 2.48982 3.15143 Time ATG9 Feed-restricted 1 ATG9 Feed-restricted 5 -0.5919 <.0001 0.43526 0.55327 0.70327 Time ATG9 Feed-restricted 1 ATG9 Feed-restricted 6 -1.0082 <.0001 0.26453 0.36489 0.50334 Time ATG9 Feed-restricted 2 ATG9 Feed-restricted 3 -0.6811 <.0001 0.41746 0.50607 0.61349 Time ATG9 Feed-restricted 2 ATG9 Feed-restricted 4 -0.07693 0.4373 0.76243 0.92596 1.12456 Time ATG9 Feed-restricted 2 ATG9 Feed-restricted 5 -1.581 <.0001 0.16855 0.20576 0.25119 Time ATG9 Feed-restricted 2 ATG9 Feed-restricted 6 -1.9973 <.0001 0.10126 0.1357 0.18185 Time ATG9 Feed-restricted 3 ATG9 Feed-restricted 4 0.6042 <.0001 1.52543 1.8297 2.19465 Time ATG9 Feed-restricted 3 ATG9 Feed-restricted 5 -0.9 <.0001 0.33711 0.40658 0.49038

270

Effect Target Treatment Time Target Treatment Time Estimate Probt LL RoR UL Time ATG9 Feed-restricted 3 ATG9 Feed-restricted 6 -1.3162 <.0001 0.20173 0.26815 0.35644 Time ATG9 Feed-restricted 4 ATG9 Feed-restricted 5 -1.5041 <.0001 0.1839 0.22221 0.26851 Time ATG9 Feed-restricted 4 ATG9 Feed-restricted 6 -1.9204 <.0001 0.11011 0.14655 0.19505 Time ATG9 Feed-restricted 5 ATG9 Feed-restricted 6 -0.4162 0.0049 0.49379 0.65952 0.88088 Time BEC-1 Control 1 BEC-1 Control 2 0.3596 0.0039 1.12263 1.43279 1.82864 Time BEC1 Control 1 BEC1 Control 3 -0.00035 0.9976 0.79099 0.99965 1.26335 Time BEC1 Control 1 BEC1 Control 4 -0.1279 0.2871 0.69519 0.87994 1.11379 Time BEC1 Control 1 BEC1 Control 5 0.08785 0.4746 0.85794 1.09182 1.38947 Time BEC1 Control 1 BEC1 Control 6 -0.601 0.0003 0.39747 0.54828 0.7563 Time BEC1 Control 2 BEC1 Control 3 -0.36 0.0003 0.57555 0.69769 0.84576 Time BEC1 Control 2 BEC1 Control 4 -0.4875 <.0001 0.50568 0.61415 0.74589 Time BEC1 Control 2 BEC1 Control 5 -0.2718 0.0081 0.62335 0.76203 0.93156 Time BEC1 Control 2 BEC1 Control 6 -0.9606 <.0001 0.28555 0.38266 0.5128 Time BEC1 Control 3 BEC1 Control 4 -0.1275 0.1691 0.73385 0.88025 1.05587 Time BEC1 Control 3 BEC1 Control 5 0.0882 0.3595 0.90425 1.09221 1.31923 Time BEC1 Control 3 BEC1 Control 6 -0.6006 <.0001 0.41261 0.54847 0.72907 Time BEC1 Control 4 BEC1 Control 5 0.2157 0.0266 1.02537 1.24079 1.50147 Time BEC1 Control 4 BEC1 Control 6 -0.4731 0.0012 0.46816 0.62308 0.82928 Time BEC1 Control 5 BEC1 Control 6 -0.6888 <.0001 0.37562 0.50217 0.67135 Time BEC1 Feed-restricted 1 BEC1 Feed-restricted 2 1.4228 <.0001 3.2506 4.14868 5.29487 Time BEC1 Feed-restricted 1 BEC1 Feed-restricted 3 0.3218 0.0071 1.09165 1.37962 1.74356 Time BEC1 Feed-restricted 1 BEC1 Feed-restricted 4 1.5105 <.0001 3.57799 4.52885 5.73242 Time BEC1 Feed-restricted 1 BEC1 Feed-restricted 6 0.2036 0.2143 0.88868 1.22586 1.69097 Time BEC1 Feed-restricted 2 BEC1 Feed-restricted 3 -1.101 <.0001 0.27433 0.33255 0.40312

271

Effect Target Treatment Time Target Treatment Time Estimate Probt LL RoR UL Time BEC1 Feed-restricted 2 BEC1 Feed-restricted 4 0.08768 0.3761 0.89883 1.09164 1.3258 Time BEC1 Feed-restricted 2 BEC1 Feed-restricted 5 -0.9487 <.0001 0.31721 0.38725 0.47276 Time BEC1 Feed-restricted 2 BEC1 Feed-restricted 6 -1.2191 <.0001 0.2205 0.29548 0.39597

Time BEC1 Feed-restricted 3 BEC1 Feed-restricted 4 1.1887 <.0001 2.7367 3.28268 3.93758

Time BEC1 Feed-restricted 3 BEC1 Feed-restricted 5 0.1523 0.111 0.96552 1.16452 1.40453 Time BEC1 Feed-restricted 3 BEC1 Feed-restricted 6 -0.1182 0.4154 0.66844 0.88855 1.18113 Time BEC1 Feed-restricted 4 BEC1 Feed-restricted 5 -1.0364 <.0001 0.29358 0.35475 0.42866 Time BEC1 Feed-restricted 4 BEC-1 Feed-restricted 6 -1.3068 <.0001 0.20338 0.27068 0.36025

Time BEC1 Feed-restricted 5 BEC1 Feed-restricted 6 -0.2705 0.067 0.57127 0.76302 1.01913

Time GABA Control 1 GABA Control 2 0.1851 0.1368 0.94284 1.20334 1.53583 Time GABA Control 1 GABA Control 3 0.143 0.2309 0.91292 1.15374 1.45808 Time GABA Control 1 GABA Control 4 -0.2821 0.019 0.59586 0.75422 0.95467 Time GABA Control 1 GABA Control 5 -1.1037 <.0001 0.2606 0.33163 0.42202

Time GABA Control 1 GABA Control 6 0.04408 0.788 0.75761 1.04507 1.4416 Time GABA Control 2 GABA Control 3 -0.0421 0.6678 0.79091 0.95878 1.16227 Time GABA Control 2 GABA Control 4 -0.4672 <.0001 0.51606 0.62677 0.76124 Time GABA Control 2 GABA Control 5 -1.2888 <.0001 0.22546 0.27559 0.33688

Time GABA Control 2 GABA Control 6 -0.141 0.3447 0.64805 0.86847 1.16385

Time GABA Control 3 GABA Control 4 -0.4251 <.0001 0.545 0.65372 0.78414 Time GABA Control 3 GABA Control 5 -1.2467 <.0001 0.23799 0.28744 0.34717 Time GABA Control 3 GABA Control 6 -0.09893 0.4953 0.68142 0.90581 1.20408 Time GABA Control 4 GABA Control 5 -0.8217 <.0001 0.36337 0.4397 0.53207

Time GABA Control 4 GABA Control 6 0.3261 0.0254 1.04109 1.38562 1.84416

Time GABA Control 5 GABA Control 6 1.1478 <.0001 2.35713 3.15128 4.213 Time GABA Feed-restricted 1 GABA Feed-restricted 2 -0.2028 0.1031 0.6397 0.8164 1.042 Time GABA Feed-restricted 1 GABA Feed-restricted 3 0.1685 0.1581 0.9365 1.1835 1.4957

272

Effect Target Treatment Time Target Treatment Time Estimate Probt LL RoR UL

Time GABA Feed-restricted 1 GABA Feed-restricted 4 0.1623 0.177 0.9292 1.1762 1.4887

Time GABA Feed-restricted 1 GABA Feed-restricted 5 -1.0833 <.0001 0.2663 0.3385 0.4302 Time GABA Feed-restricted 1 GABA Feed-restricted 6 0.2254 0.1694 0.9082 1.2528 1.7282 Time GABA Feed-restricted 2 GABA Feed-restricted 3 0.3713 0.0002 1.1959 1.4497 1.7574 Time GABA Feed-restricted 2 GABA Feed-restricted 4 0.3651 0.0002 1.1862 1.4406 1.7497 Time GABA Feed-restricted 2 GABA Feed-restricted 5 -0.8804 <.0001 0.3396 0.4146 0.5061 Time GABA Feed-restricted 2 GABA Feed-restricted 6 0.4282 0.0042 1.1451 1.5346 2.0565

Time GABA Feed-restricted 3 GABA Feed-restricted 4 -0.00625 0.9462 0.8285 0.9938 1.192

Time GABA Feed-restricted 3 GABA Feed-restricted 5 -1.2518 <.0001 0.2371 0.286 0.3449 Time GABA Feed-restricted 3 GABA Feed-restricted 6 0.05689 0.6949 0.7963 1.0585 1.4071 Time GABA Feed-restricted 4 GABA Feed-restricted 5 -1.2455 <.0001 0.2382 0.2878 0.3477 Time GABA Feed-restricted 4 GABA Feed-restricted 6 0.06315 0.6647 0.8003 1.0652 1.4177 Time GABA Feed-restricted 5 GABA Feed-restricted 6 1.3087 <.0001 2.7711 3.7013 4.9436

Time LC3 Control 1 LC3 Control 2 0.06266 0.6143 0.8342 1.0647 1.3588

Time LC3 Control 1 LC3 Control 3 0.6003 <.0001 1.4422 1.8226 2.3035 Time LC3 Control 1 LC3 Control 4 -0.375 0.0018 0.543 0.6873 0.8699 Time LC3 Control 1 LC3 Control 5 -1.0505 <.0001 0.2748 0.3497 0.4451 Time LC3 Control 1 LC3 Control 6 -0.2104 0.1996 0.5874 0.8103 1.1177 Time LC3 Control 2 LC3 Control 3 0.5376 <.0001 1.4122 1.7119 2.0753

Time LC3 Control 2 LC3 Control 4 -0.4377 <.0001 0.5315 0.6455 0.784

Time LC3 Control 2 LC3 Control 5 -1.1132 <.0001 0.2687 0.3285 0.4016 Time LC3 Control 2 LC3 Control 6 -0.2731 0.0675 0.5679 0.7611 1.0199 Time LC3 Control 3 LC3 Control 4 -0.9753 <.0001 0.3144 0.3771 0.4523 Time LC3 Control 3 LC3 Control 5 -1.6508 <.0001 0.1589 0.1919 0.2318 Time LC3 Control 3 LC3 Control 6 -0.8107 <.0001 0.3344 0.4446 0.5909 Time LC3 Control 4 LC3 Control 5 -0.6755 <.0001 0.4206 0.5089 0.6158

273

Effect Target Treatment Time Target Treatment Time Estimate Probt LL RoR UL

Time LC3 Control 4 LC3 Control 6 0.1646 0.2586 0.8858 1.179 1.5691 Time LC3 Control 5 LC3 Control 6 0.8401 <.0001 1.7329 2.3167 3.0972 Time LC3 Feed-restricted 1 LC3 Feed-restricted 2 0.1261 0.3105 0.8889 1.1344 1.4479 Time LC3 Feed-restricted 1 LC3 Feed-restricted 3 -0.1462 0.2205 0.6836 0.8639 1.0919 Time LC3 Feed-restricted 1 LC3 Feed-restricted 4 -0.05732 0.6332 0.746 0.9443 1.1952 Time LC3 Feed-restricted 1 LC3 Feed-restricted 5 -0.9269 <.0001 0.3114 0.3958 0.5031 Time LC3 Feed-restricted 1 LC3 Feed-restricted 6 -0.5577 0.0007 0.4151 0.5725 0.7898

Time LC3 Feed-restricted 2 LC3 Feed-restricted 3 -0.2724 0.0056 0.6282 0.7616 0.9232 Time LC3 Feed-restricted 2 LC3 Feed-restricted 4 -0.1835 0.0642 0.6854 0.8324 1.0109 Time LC3 Feed-restricted 2 LC3 Feed-restricted 5 -1.053 <.0001 0.2858 0.3489 0.4259 Time LC3 Feed-restricted 2 LC3 Feed-restricted 6 -0.6838 <.0001 0.3766 0.5047 0.6764 Time LC3 Pair-fed 3 LC3 Pair-fed 4 0.08893 0.3374 0.9113 1.093 1.311 Time LC3 Feed-restricted 3 LC3 Feed-restricted 5 -0.7806 <.0001 0.3798 0.4581 0.5525 Time LC3 Feed-restricted 3 LC3 Feed-restricted 6 -0.4114 0.0047 0.4986 0.6627 0.8809

Time LC3 Feed-restricted 4 LC3 Feed-restricted 5 -0.8696 <.0001 0.3469 0.4191 0.5064 Time LC3 Feed-restricted 4 LC3 Feed-restricted 6 -0.5003 0.0006 0.4556 0.6063 0.807 Time LC3 Feed-restricted 5 LC3 Feed-restricted 6 0.3692 0.0125 1.0831 1.4466 1.9322

274

Appendix 3.1c: Post-hoc statistical analysis of Atg gene expression liver: Least Significant Difference test was performed in SAS (significant data is in blue)

Effect Group1 Group 2 Statistics Effect Target Treatment Time Target Treatment Time Estimate Probt LL RoR UL Treatment ATG12 Control 1 ATG12 Feed-Restricted 1 1.2577 <.0001 0.22 0.28 0.38 Treatment ATG12 Control 2 ATG12 Feed-Restricted 2 -0.00178 0.9864 0.81 1 1.22 Treatment ATG12 Control 3 ATG12 Feed-Restricted 3 -0.03791 0.6792 0.8 0.96 1.15 Treatment ATG12 Control 4 ATG12 Feed-Restricted 4 0.2624 0.0052 1.08 1.3 1.56 Treatment ATG12 Control 5 ATG12 Feed-Restricted 5 0.1997 0.0457 1 1.22 1.49 Treatment ATG12 Control 6 ATG12 Feed-Restricted 6 0.08872 0.6288 0.76 1.09 1.57 Treatment ATG13 Control 1 ATG13 Feed-Restricted 1 -0.7467 <.0001 0.36 0.47 0.63 Treatment ATG13 Control 2 ATG13 Feed-Restricted 2 -0.5247 <.0001 0.48 0.59 0.73 Treatment ATG13 Control 3 ATG13 Feed-Restricted 3 -0.2687 0.0035 0.64 0.76 0.92 Treatment ATG13 Control 4 ATG13 Feed-Restricted 4 -0.08454 0.367 0.76 0.92 1.1 Treatment ATG13 Control 5 ATG13 Feed-Restricted 5 0.000848 0.9932 0.82 1 1.22 Treatment ATG13 Control 6 ATG13 Feed-Restricted 6 -1.4364 <.0001 0.17 0.24 0.34 Treatment ATG16 Control 1 ATG16 Feed-Restricted 1 -0.6051 <.0001 0.4135 0.54601 0.72099 Treatment ATG16 Control 2 ATG16 Feed-Restricted 2 -0.5046 <.0001 0.49222 0.60374 0.74053 Treatment ATG16 Control 3 ATG16 Feed-Restricted 3 -0.2495 0.0066 0.65089 0.77919 0.93277 Treatment ATG16 Control 4 ATG16 Feed-Restricted 4 0.1985 0.0343 1.01482 1.21961 1.46573 Treatment ATG16 Control 5 ATG16 Feed-Restricted 5 -0.4451 <.0001 0.52805 0.64077 0.77755 Treatment ATG16 Control 6 ATG16 Feed-Restricted 6 -0.8773 <.0001 0.29013 0.4159 0.59618 Treatment ATG4 Control 1 ATG4 Feed-Restricted 1 -0.1822 0.1986 0.63115 0.83342 1.1005 Treatment ATG4 Control 2 ATG4 Feed-Restricted 2 0.1187 0.2542 0.91805 1.12604 1.38116

275

Effect Target Treatment Time Target Treatment Time Estimate Probt LL RoR UL

Treatment ATG4 Control 3 ATG4 Feed-Restricted 3 -0.6436 <.0001 0.43889 0.5254 0.62896

Treatment ATG4 Control 4 ATG4 Feed-Restricted 4 -0.3162 0.0008 0.60652 0.72891 0.87601 Treatment ATG4 Control 5 ATG4 Feed-Restricted 5 0.1062 0.2874 0.91425 1.11206 1.35267 Treatment ATG4 Control 6 ATG4 Feed-Restricted 6 -0.297 0.1058 0.51834 0.74303 1.06511 Treatment ATG5 Control 1 ATG5 Feed-Restricted 1 -0.3724 0.0087 0.52182 0.68905 0.90987 Treatment ATG5 Control 2 ATG5 Feed-Restricted 2 -1.3728 <.0001 0.2066 0.25341 0.31082 Treatment ATG5 Control 3 ATG5 Feed-Restricted 3 -0.3601 <.0001 0.58272 0.69758 0.83507

Treatment ATG5 Control 4 ATG5 Feed-Restricted 4 -0.2035 0.0301 0.67886 0.81586 0.9805 Treatment ATG5 Control 5 ATG5 Feed-Restricted 5 -0.3402 0.0007 0.58502 0.71159 0.86555 Treatment ATG5 Control 6 ATG5 Feed-Restricted 6 -2.4056 <.0001 0.06293 0.09021 0.12932 Treatment ATG7 Control 1 ATG7 Feed-Restricted 1 -0.7635 <.0001 0.35292 0.46603 0.61537 Treatment ATG7 Control 2 ATG7 Feed-Restricted 2 -2.3645 <.0001 0.07663 0.094 0.11529 Treatment ATG7 Control 3 ATG7 Feed-Restricted 3 -0.5465 <.0001 0.48364 0.57897 0.69309

Treatment ATG7 Control 4 ATG7 Feed-Restricted 4 -0.5249 <.0001 0.49228 0.59163 0.71102

Treatment ATG7 Control 5 ATG7 Feed-Restricted 5 -0.7637 <.0001 0.38308 0.46596 0.56677 Treatment ATG7 Control 6 ATG7 Feed-Restricted 6 -1.3372 <.0001 0.18318 0.26258 0.3764 Treatment ATG9 Control 1 ATG9 Feed-Restricted 1 -0.8955 <.0001 0.30927 0.40838 0.53926 Treatment ATG9 Control 2 ATG9 Feed-Restricted 2 -1.1589 <.0001 0.25585 0.31382 0.38492 Treatment ATG9 Control 3 ATG9 Feed-Restricted 3 -0.2027 0.0273 0.68208 0.81652 0.97746

Treatment ATG9 Control 4 ATG9 Feed-Restricted 4 0.2715 0.0038 1.09161 1.31189 1.57664

Treatment ATG9 Control 5 ATG9 Feed-Restricted 5 -0.3684 0.0002 0.56878 0.69184 0.84153 Treatment ATG9 Control 6 ATG9 Feed-Restricted 6 -0.5164 0.005 0.41622 0.59664 0.85527 Treatment BEC1 Control 1 BEC1 Feed-Restricted 1 -1.0087 <.0001 0.27619 0.36471 0.48158 Treatment BEC1 Control 2 BEC1 Feed-Restricted 2 0.0545 0.6005 0.86095 1.05602 1.29527 Treatment BEC1 Control 3 BEC1 Feed-Restricted 3 -0.6865 <.0001 0.42046 0.50333 0.60254

Treatment BEC1 Control 4 BEC1 Feed-Restricted 4 0.6297 <.0001 1.56187 1.87705 2.25584

276

Effect Target Treatment Time Target Treatment Time Estimate Probt LL RoR UL Treatment BEC1 Control 5 BEC1 Feed-Restricted 5 -0.6224 <.0001 0.4412 0.53666 0.65277 Treatment BEC1 Control 6 BEC1 Feed-Restricted 6 -0.204 0.2664 0.56885 0.81543 1.16889

Treatment GABA Control 1 GABA Feed-Restricted 1 -0.2822 0.0466 0.57109 0.75411 0.99578

Treatment GABA Control 2 GABA Feed-Restricted 2 -0.6702 <.0001 0.41712 0.51163 0.62755 Treatment GABA Control 3 GABA Feed-Restricted 3 -0.2567 0.0052 0.64622 0.77359 0.92607 Treatment GABA Control 4 GABA Feed-Restricted 4 0.1621 0.0838 0.97852 1.17599 1.41331 Treatment GABA Control 5 GABA Feed-Restricted 5 -0.2618 0.0089 0.63278 0.76969 0.93622

Treatment GABA Control 6 GABA Feed-Restricted 6 -0.1009 0.5825 0.63066 0.90403 1.29591 Treatment LC3 Control 1 LC3 Feed-Restricted 1 -0.3593 0.0114 0.52875 0.6982 0.92195 Treatment LC3 Control 2 LC3 Feed-Restricted 2 -0.2958 0.0046 0.60653 0.74395 0.91251 Treatment LC3 Control 3 LC3 Feed-Restricted 3 -1.1058 <.0001 0.27646 0.33095 0.39618

Treatment LC3 Control 4 LC3 Feed-Restricted 4 -0.04153 0.6576 0.79824 0.95933 1.15292

Treatment LC3 Control 5 LC3 Feed-Restricted 5 -0.2356 0.0185 0.64956 0.7901 0.96104 Treatment LC3 Control 6 LC3 Feed-Restricted 6 -0.7065 0.0001 0.34417 0.49336 0.70722

277

Appendix 3.2a-b: Post-hoc statistical analysis of Atg gene expression in muscle: Least Significant Difference test was performed in SAS (significant data is in blue)

Group 1 Group 2 Statistics Treatment Target Treatment Time Target Treatment Time Estimate Probt LL RoR UL Time ATG12 Control 1 ATG12 Control 2 0.5994 <.0001 1.4161 1.8211 2.3419 Time ATG12 Control 1 ATG12 Control 3 0.5149 <.0001 1.3241 1.6734 2.115 Time ATG12 Control 1 ATG12 Control 4 0.8502 <.0001 1.8489 2.3402 2.962 Time ATG12 Control 1 ATG12 Control 5 2.208 <.0001 7.1487 9.0973 11.5771 Time ATG12 Control 1 ATG12 Control 6 2.6819 <.0001 10.5933 14.6128 20.1575 Time ATG12 Control 2 ATG12 Control 3 -0.08456 0.4116 0.7508 0.9189 1.1246 Time ATG12 Control 2 ATG12 Control 4 0.2508 0.0159 1.0482 1.2851 1.5754 Time ATG12 Control 2 ATG12 Control 5 1.6085 <.0001 4.0495 4.9955 6.1625 Time ATG12 Control 2 ATG12 Control 6 2.0825 <.0001 5.9498 8.0242 10.8217 Time ATG12 Control 3 ATG12 Control 4 0.3354 0.0003 1.1659 1.3985 1.6774 Time ATG12 Control 3 ATG12 Control 5 1.6931 <.0001 4.5011 5.4363 6.566 Time ATG12 Control 3 ATG12 Control 6 2.167 <.0001 6.569 8.7323 11.6078 Time ATG12 Control 4 ATG12 Control 5 1.3577 <.0001 3.2126 3.8874 4.7039 Time ATG12 Control 4 ATG12 Control 6 1.8317 <.0001 4.6916 6.2442 8.3106 Time ATG12 Control 5 ATG12 Control 6 0.4739 0.0014 1.2015 1.6063 2.1474 Time ATG12 Feed-Restricted 1 ATG12 Feed-Restricted 2 0.6357 <.0001 1.4796 1.8883 2.41 Time ATG12 Feed-Restricted 1 ATG12 Feed-Restricted 3 0.6511 <.0001 1.5173 1.9177 2.4236 Time ATG12 Feed-Restricted 1 ATG12 Feed-Restricted 4 0.07924 0.5094 0.8552 1.0825 1.3701 Time ATG12 Feed-Restricted 1 ATG12 Feed-Restricted 5 1.4735 <.0001 3.4335 4.3644 5.5478 Time ATG12 Feed-Restricted 1 ATG12 Feed-Restricted 6 2.2272 <.0001 6.7228 9.2737 12.7924 Time ATG12 Feed-Restricted 2 ATG12 Feed-Restricted 3 0.0154 0.8752 0.8377 1.0155 1.2311 Time ATG12 Feed-Restricted 2 ATG12 Feed-Restricted 4 -0.5565 <.0001 0.472 0.5732 0.6962 Time ATG12 Feed-Restricted 2 ATG12 Feed-Restricted 5 0.8378 <.0001 1.8934 2.3113 2.8213

278

279

Treatment Target Treatment Time Target Treatment Time Estimate Probt LL RoR UL Time ATG12 Feed-Restricted 2 ATG12 Feed-Restricted 6 1.5915 <.0001 3.6647 4.911 6.5812 Time ATG12 Feed-Restricted 3 ATG12 Feed-Restricted 4 -0.5719 <.0001 0.4706 0.5645 0.6771 Time ATG12 Feed-Restricted 3 ATG12 Feed-Restricted 5 0.8224 <.0001 1.8871 2.2759 2.7449 Time ATG12 Feed-Restricted 3 ATG12 Feed-Restricted 6 1.5761 <.0001 3.638 4.8359 6.4285 Time ATG12 Feed-Restricted 4 ATG12 Feed-Restricted 5 1.3943 <.0001 3.3369 4.032 4.8719 Time ATG12 Feed-Restricted 4 ATG12 Feed-Restricted 6 2.1479 <.0001 6.437 8.5672 11.4024 Time ATG12 Feed-restricted 5 ATG12 Feed-restricted 6 0.7537 <.0001 1.5909 2.1248 2.8379 Time ATG13 Control 1 ATG13 Control 2 1.3108 <.0001 2.9064 3.7093 4.734 Time ATG13 Control 1 ATG13 Control 3 0.6264 <.0001 1.4803 1.8709 2.3644 Time ATG13 Control 1 ATG13 Control 4 1.2098 <.0001 2.6489 3.3527 4.2435 Time ATG13 Control 1 ATG13 Control 5 2.8148 <.0001 13.1158 16.6906 21.2398 Time ATG13 Control 1 ATG13 Control 6 2.1284 <.0001 6.0903 8.4011 11.5888 Time ATG13 Control 2 ATG13 Control 3 -0.6844 <.0001 0.4161 0.5044 0.6114 Time ATG13 Control 2 ATG13 Control 4 -0.1011 0.3075 0.7443 0.9039 1.0977 Time ATG13 Control 2 ATG13 Control 5 1.504 <.0001 3.6811 4.4997 5.5004 Time ATG13 Control 2 ATG13 Control 6 0.8175 <.0001 1.6901 2.2649 3.0352 Time ATG13 Control 3 ATG13 Control 4 0.5834 <.0001 1.4941 1.7921 2.1495 Time ATG13 Control 3 ATG13 Control 5 2.1884 <.0001 7.3865 8.9214 10.7751 Time ATG13 Control 3 ATG13 Control 6 1.502 <.0001 3.3781 4.4905 5.9692 Time ATG13 Control 4 ATG13 Control 5 1.6051 <.0001 4.1141 4.9783 6.0239 Time ATG13 Control 4 ATG13 Control 6 0.9186 <.0001 1.8827 2.5058 3.335 Time ATG13 Control 5 ATG13 Control 6 -0.6865 <.0001 0.3765 0.5033 0.6729 Time ATG13 Feed-Restricted 1 ATG13 Feed-Restricted 2 0.8477 <.0001 1.8291 2.3344 2.9792 Time ATG13 Feed-Restricted 1 ATG13 Feed-Restricted 3 0.7003 <.0001 1.5939 2.0144 2.5458 Time ATG13 Feed-restricted 1 ATG13 Feed-restricted 4 0.04442 0.7114 0.826 1.0454 1.3232

280

Treatment Target Treatment Time Target Treatment Time Estimate Probt LL RoR UL Time ATG13 Feed-Restricted 1 ATG13 Feed-Restricted 5 1.3384 <.0001 2.9996 3.8128 4.8466 Time ATG13 Feed-Restricted 1 ATG13 Feed-Restricted 6 0.2944 0.0728 0.9731 1.3423 1.8516 Time ATG13 Feed-restricted 2 ATG13 Feed-restricted 3 -0.1474 0.1331 0.7118 0.8629 1.0461 Time ATG13 Feed-Restricted 2 ATG13 Feed-Restricted 4 -0.8033 <.0001 0.3688 0.4478 0.5439 Time ATG13 Feed-Restricted 2 ATG13 Feed-Restricted 5 0.4906 <.0001 1.338 1.6334 1.9939 Time ATG13 Feed-restricted 2 ATG13 Feed-restricted 6 -0.5534 0.0002 0.4291 0.575 0.7706 Time ATG13 Feed-Restricted 3 ATG13 Feed-Restricted 4 -0.6559 <.0001 0.4327 0.519 0.6225 Time ATG13 Feed-Restricted 3 ATG13 Feed-Restricted 5 0.6381 <.0001 1.5695 1.8928 2.2828 Time ATG13 Feed-restricted 3 ATG13 Feed-restricted 6 -0.4059 0.0052 0.5013 0.6663 0.8858 Time ATG13 Feed-Restricted 4 ATG13 Feed-Restricted 5 1.294 <.0001 3.018 3.647 4.407 Time ATG13 Feed-Restricted 4 ATG13 Feed-Restricted 6 0.2499 0.0865 0.965 1.284 1.709 Time ATG13 Feed-restricted 5 ATG13 Feed-restricted 6 -1.044 <.0001 0.264 0.352 0.47 Time ATG16 Control 1 ATG16 Control 2 0.3719 0.0028 1.136 1.45 1.851 Time ATG16 Control 1 ATG16 Control 3 0.672 <.0001 1.549 1.958 2.475 Time ATG16 Control 1 ATG16 Control 4 1.0927 <.0001 2.356 2.982 3.775 Time ATG16 Control 1 ATG16 Control 5 5.1436 <.0001 133.698 171.324 219.539 Time ATG16 Control 1 ATG16 Control 6 -0.4211 0.0104 0.476 0.656 0.905 Time ATG16 Control 2 ATG16 Control 3 0.3001 0.0023 1.114 1.35 1.637 Time ATG16 Control 2 ATG16 Control 4 0.7209 <.0001 1.693 2.056 2.497 Time ATG16 Control 2 ATG16 Control 5 4.7717 <.0001 95.835 118.12 145.587 Time ATG16 Control 2 ATG16 Control 6 -0.7929 <.0001 0.338 0.453 0.606 Time ATG16 Control 3 ATG16 Control 4 0.4208 <.0001 1.27 1.523 1.827 Time ATG16 Control 3 ATG16 Control 5 4.4716 <.0001 71.809 87.494 106.606 Time ATG16 Control 3 ATG16 Control 6 -1.0931 <.0001 0.252 0.335 0.446 Time ATG16 Control 4 ATG16 Control 5 4.0508 <.0001 47.062 57.445 70.118

281

Treatment Target Treatment Time Target Treatment Time Estimate Probt LL RoR UL label Time ATG16 Control 4 ATG16 Control 6 -1.5138 <.0001 0.165 0.22 0.293 Time ATG16 Control 5 ATG16 Control 6 -5.5646 <.0001 0.003 0.004 0.005 Time ATG16 Feed-Restricted 1 ATG16 Feed-Restricted 2 0.593 <.0001 1.418 1.809 2.309 Time ATG16 Feed-Restricted 1 ATG16 Feed-Restricted 3 1.4224 <.0001 3.281 4.147 5.241 Time ATG16 Feed-restricted 1 ATG16 Feed-restricted 4 0.006464 0.9571 0.795 1.006 1.274 Time ATG16 Feed-Restricted 1 ATG16 Feed-Restricted 5 3.1293 <.0001 17.857 22.858 29.261 Time ATG16 Feed-Restricted 1 ATG16 Feed-Restricted 6 -1.006 <.0001 0.265 0.366 0.504 Time ATG16 Feed-Restricted 2 ATG16 Feed-Restricted 3 0.8294 <.0001 1.891 2.292 2.778 Time ATG16 Feed-restricted 2 ATG16 Feed-restricted 4 -0.5865 <.0001 0.458 0.556 0.676 Time ATG16 Feed-Restricted 2 ATG16 Feed-Restricted 5 2.5363 <.0001 10.262 12.633 15.552 Time ATG16 Feed-Restricted 2 ATG16 Feed-Restricted 6 -1.599 <.0001 0.151 0.202 0.271 Time ATG16 Feed-Restricted 3 ATG16 Feed-Restricted 4 -1.4159 <.0001 0.202 0.243 0.291 Time ATG16 Feed-restricted 3 ATG16 Feed-restricted 5 1.7069 <.0001 4.53 5.512 6.707 Time ATG16 Feed-Restricted 3 ATG16 Feed-Restricted 6 -2.4283 <.0001 0.066 0.088 0.117 Time ATG16 Feed-Restricted 4 ATG16 Feed-Restricted 5 3.1228 <.0001 18.63 22.711 27.686 Time ATG16 Feed-Restricted 4 ATG16 Feed-Restricted 6 -1.0124 <.0001 0.273 0.363 0.484 Time ATG16 Feed-restricted 5 ATG16 Feed-restricted 6 -4.1353 <.0001 0.012 0.016 0.021 Time ATG4 Control 1 ATG4 Control 2 0.742 <.0001 1.646 2.1 2.68 Time ATG4 Control 1 ATG4 Control 3 1.1946 <.0001 2.613 3.302 4.173 Time ATG4 Control 1 ATG4 Control 4 0.9491 <.0001 2.041 2.584 3.27 Time ATG4 Control 1 ATG4 Control 5 3.8744 <.0001 37.84 48.154 61.279 Time ATG4 Control 1 ATG4 Control 6 0.813 <.0001 1.634 2.255 3.11 Time ATG4 Control 2 ATG4 Control 3 0.4526 <.0001 1.297 1.572 1.906 Time ATG4 Control 2 ATG4 Control 4 0.2071 0.0367 1.013 1.23 1.494 Time ATG4 Control 2 ATG4 Control 5 3.1324 <.0001 18.757 22.929 28.028

282

Treatment Target Treatment Time Target Treatment Time Estimate Probt LL RoR UL Time ATG4 Control 2 ATG4 Control 6 0.07095 0.6344 0.801 1.074 1.439 Time ATG4 Control 3 ATG4 Control 4 -0.2455 0.0082 0.652 0.782 0.938 Time ATG4 Control 3 ATG4 Control 5 2.6798 <.0001 12.073 14.582 17.611 Time ATG4 Control 3 ATG4 Control 6 -0.3817 0.0086 0.514 0.683 0.908 Time ATG4 Control 4 ATG4 Control 5 2.9253 <.0001 15.404 18.639 22.553 Time ATG4 Control 4 ATG4 Control 6 -0.1362 0.35 0.656 0.873 1.161 Time ATG4 Control 5 ATG4 Control 6 -3.0614 <.0001 0.035 0.047 0.063 Time ATG4 Feed-Restricted 1 ATG4 Feed-Restricted 2 0.7868 <.0001 1.721 2.196 2.803 Time ATG4 Feed-Restricted 1 ATG4 Feed-Restricted 3 1.0521 <.0001 2.266 2.864 3.619 Time ATG4 Feed-restricted 1 ATG4 Feed-restricted 4 0.1034 0.3892 0.876 1.109 1.404 Time ATG4 Feed-Restricted 1 ATG4 Feed-Restricted 5 1.6669 <.0001 4.1661 5.2956 6.7314 Time ATG4 Feed-Restricted 1 ATG4 Feed-Restricted 6 1.7889 <.0001 4.3374 5.9831 8.2533 Time ATG4 Feed-Restricted 2 ATG4 Feed-Restricted 3 0.2653 0.0069 1.0756 1.3039 1.5806 Time ATG4 Feed-restricted 2 ATG4 Feed-restricted 4 -0.6833 <.0001 0.4158 0.5049 0.6132 Time ATG4 Feed-Restricted 2 ATG4 Feed-Restricted 5 0.8801 <.0001 1.9752 2.4112 2.9434 Time ATG4 Feed-Restricted 2 ATG4 Feed-Restricted 6 1.0022 <.0001 2.0328 2.7242 3.6507 Time ATG4 Feed-restricted 3 ATG4 Feed-restricted 4 -0.9487 <.0001 0.3229 0.3873 0.4645 Time ATG4 Feed-Restricted 3 ATG4 Feed-Restricted 5 0.6148 <.0001 1.5333 1.8492 2.2302 Time ATG4 Feed-Restricted 3 ATG4 Feed-Restricted 6 0.7368 <.0001 1.5718 2.0893 2.7773 Time ATG4 Feed-Restricted 4 ATG4 Feed-Restricted 5 1.5635 <.0001 3.9522 4.7753 5.7698 Time ATG4 Feed-restricted 4 ATG4 Feed-restricted 6 1.6855 <.0001 4.0537 5.3952 7.1807 Time ATG4 Feed-restricted 5 ATG4 Feed-restricted 6 0.1221 0.408 0.8459 1.1298 1.509 Time ATG5 Control 1 ATG5 Control 2 0.006952 0.9554 0.789 1.007 1.2852 Time ATG5 Control 1 ATG5 Control 3 0.5144 <.0001 1.3234 1.6726 2.1138 Time ATG5 Control 1 ATG5 Control 4 -0.179 0.1362 0.6606 0.8361 1.0582 Time ATG5 Control 1 ATG5 Control 5 2.4794 <.0001 9.3781 11.934 15.1865 Time ATG5 Control 1 ATG5 Control 6 -0.07591 0.6433 0.672 0.9269 1.2786

283

Treatment Target Treatment Time Target Treatment Time Estimate Probt LL RoR UL Time ATG5 Control 2 ATG5 Control 3 0.5074 <.0001 1.3702 1.661 2.0135 Time ATG5 Control 2 ATG5 Control 4 -0.186 0.0606 0.6837 0.8303 1.0083 Time ATG5 Control 2 ATG5 Control 5 2.4724 <.0001 9.6955 11.8513 14.4866 Time ATG5 Control 2 ATG5 Control 6 -0.08286 0.5786 0.6869 0.9205 1.2335 Time ATG5 Control 3 ATG5 Control 4 -0.6934 <.0001 0.4168 0.4999 0.5996 Time ATG5 Control 3 ATG5 Control 6 -0.5903 <.0001 0.4169 0.5542 0.7367 Time ATG5 Control 4 ATG5 Control 5 2.6584 <.0001 11.7962 14.2737 17.2716 Time ATG5 Control 4 ATG5 Control 6 0.1031 0.4792 0.833 1.1086 1.4755 Time ATG5 Control 5 ATG5 Control 6 -2.5553 <.0001 0.0581 0.0777 0.1038 Time ATG5 Feed-Restricted 1 ATG5 Feed-Restricted 2 0.3525 0.0047 1.1147 1.4226 1.8156 Time ATG5 Feed-Restricted 1 ATG5 Feed-Restricted 3 0.1465 0.2197 0.9161 1.1578 1.4632 Time ATG5 Feed-restricted 1 ATG5 Feed-restricted 4 -0.2544 0.0344 0.6127 0.7754 0.9814 Time ATG5 Feed-Restricted 1 ATG5 Feed-Restricted 5 1.7602 <.0001 4.5736 5.8135 7.3895 Time ATG5 Feed-Restricted 1 ATG5 Feed-Restricted 6 -0.07139 0.6632 0.675 0.9311 1.2844 Time ATG5 Feed-Restricted 2 ATG5 Feed-Restricted 3 -0.206 0.0359 0.6713 0.8138 0.9865 Time ATG5 Feed-Restricted 2 ATG5 Feed-Restricted 4 -0.6069 <.0001 0.4488 0.5451 0.6619 Time ATG5 Feed-Restricted 2 ATG5 Feed-Restricted 5 1.4077 <.0001 3.3476 4.0864 4.9882 Time ATG5 Feed-restricted 2 ATG5 Feed-restricted 6 -0.4239 0.0046 0.4884 0.6545 0.8771 Time ATG5 Feed-Restricted 3 ATG5 Feed-Restricted 4 -0.4008 <.0001 0.5584 0.6698 0.8033 Time ATG5 Feed-Restricted 3 ATG5 Feed-Restricted 5 1.6137 <.0001 4.1635 5.0213 6.0558 Time ATG5 Feed-Restricted 3 ATG5 Feed-Restricted 6 -0.2179 0.1333 0.605 0.8042 1.069 Time ATG5 Feed-Restricted 4 ATG5 Feed-Restricted 5 2.0145 <.0001 6.2049 7.4973 9.0588 Time ATG5 Feed-Restricted 4 ATG5 Feed-Restricted 6 0.183 0.2094 0.9022 1.2008 1.5981 Time ATG5 Feed-Restricted 5 ATG5 Feed-Restricted 6 -1.8316 <.0001 0.1199 0.1602 0.2139 Time ATG7 Control 1 ATG7 Control 2 0.671 <.0001 1.5327 1.9562 2.4965 Time ATG7 Control 1 ATG7 Control 3 -0.5053 <.0001 0.4774 0.6033 0.7625 Time ATG7 Control 1 ATG7 Control 4 0.9942 <.0001 2.1352 2.7025 3.4206

284

Treatment Target Treatment Time Target Treatment Time Estimate Probt LL RoR UL Time ATG7 Control 1 ATG7 Control 5 2.4702 <.0001 9.2922 11.8248 15.0478 Time ATG7 Control 1 ATG7 Control 6 0.05183 0.7519 0.7635 1.0532 1.4528 Time ATG7 Control 2 ATG7 Control 3 -1.1763 <.0001 0.2544 0.3084 0.3739 Time ATG7 Control 2 ATG7 Control 4 0.3232 0.0011 1.1376 1.3816 1.6778 Time ATG7 Control 2 ATG7 Control 5 1.7992 <.0001 4.9453 6.0449 7.3891 Time ATG7 Control 2 ATG7 Control 6 -0.6192 <.0001 0.4018 0.5384 0.7215 Time ATG7 Control 3 ATG7 Control 4 1.4995 <.0001 3.7346 4.4794 5.373 Time ATG7 Control 3 ATG7 Control 5 2.9755 <.0001 16.2275 19.5994 23.672 Time ATG7 Control 3 ATG7 Control 6 0.5571 0.0001 1.3132 1.7456 2.32 Time ATG7 Control 4 ATG7 Control 5 1.476 <.0001 3.616 4.3754 5.294 Time ATG7 Control 4 ATG7 Control 6 -0.9424 <.0001 0.2928 0.3897 0.519 Time ATG7 Control 5 ATG7 Control 6 -2.4184 <.0001 0.0666 0.0891 0.119 Time ATG7 Feed-Restricted 1 ATG7 Feed-Restricted 2 0.9104 <.0001 1.9474 2.4854 3.172 Time ATG7 Feed-Restricted 1 ATG7 Feed-Restricted 3 2.8693 <.0001 13.9459 17.6244 22.273 Time ATG7 Feed-restricted 1 ATG7 Feed-restricted 4 0.2723 0.0235 1.0374 1.313 1.662 Time ATG7 Feed-Restricted 1 ATG7 Feed-Restricted 5 1.3649 <.0001 3.0802 3.9153 4.977 Time ATG7 Feed-Restricted 1 ATG7 Feed-Restricted 6 -1.1714 <.0001 0.2247 0.3099 0.428 Time ATG7 Feed-Restricted 2 ATG7 Feed-Restricted 3 1.9589 <.0001 5.8499 7.0913 8.596 Time ATG7 Feed-Restricted 2 ATG7 Feed-Restricted 4 -0.6381 <.0001 0.435 0.5283 0.642 Time ATG7 Feed-restricted 2 ATG7 Feed-restricted 5 0.4545 <.0001 1.2905 1.5753 1.923 Time ATG7 Feed-Restricted 2 ATG7 Feed-Restricted 6 -2.0818 <.0001 0.0931 0.1247 0.167 Time ATG7 Feed-Restricted 3 ATG7 Feed-Restricted 4 -2.597 <.0001 0.0621 0.0745 0.089 Time ATG7 Feed-Restricted 3 ATG7 Feed-Restricted 5 -1.5044 <.0001 0.1842 0.2221 0.268 Time ATG7 Feed-Restricted 3 ATG7 Feed-Restricted 6 -4.0407 <.0001 0.0132 0.0176 0.023 Time ATG7 Feed-restricted 4 ATG7 Feed-restricted 5 1.0925 <.0001 2.4679 2.9819 3.603 Time ATG7 Feed-Restricted 4 ATG7 Feed-Restricted 6 -1.4437 <.0001 0.1774 0.2361 0.314

285

Treatment Target Treatment Time Target Treatment Time Estimate Probt LL RoR UL label Time ATG9 Control 1 ATG9 Control 2 -0.2744 0.0275 0.5955 0.76 0.97 Time ATG9 Control 1 ATG9 Control 3 0.1818 0.1279 0.949 1.1993 1.516 Time ATG9 Control 1 ATG9 Control 4 0.447 0.0002 1.2354 1.5635 1.979 Time ATG9 Control 1 ATG9 Control 5 4.2272 <.0001 53.8492 68.5269 87.205 Time ATG9 Control 1 ATG9 Control 6 -2.0944 <.0001 0.0893 0.1231 0.17 Time ATG9 Control 2 ATG9 Control 3 0.4562 <.0001 1.3018 1.5781 1.913 Time ATG9 Control 2 ATG9 Control 4 0.7214 <.0001 1.694 2.0573 2.498 Time ATG9 Control 2 ATG9 Control 5 4.5017 <.0001 73.761 90.1663 110.22 Time ATG9 Control 2 ATG9 Control 6 -1.8199 <.0001 0.1209 0.162 0.217 Time ATG9 Control 3 ATG9 Control 4 0.2652 0.0043 1.0869 1.3037 1.564 Time ATG9 Control 3 ATG9 Control 5 4.0455 <.0001 47.3052 57.137 69.012 Time ATG9 Control 3 ATG9 Control 6 -2.2761 <.0001 0.0772 0.1027 0.136 Time ATG9 Control 4 ATG9 Control 5 3.7803 <.0001 36.2192 43.8279 53.035 Time ATG9 Control 4 ATG9 Control 6 -2.5413 <.0001 0.0592 0.0788 0.105 Time ATG9 Control 5 ATG9 Control 6 -6.3216 <.0001 0.0013 0.0018 0.002 Time ATG9 Feed-Restricted 1 ATG9 Feed-Restricted 2 0.6658 <.0001 1.5248 1.946 2.484 Time ATG9 Feed-restricted 1 ATG9 Feed-restricted 3 1.6251 <.0001 4.0188 5.0789 6.419 Time ATG9 Feed-Restricted 1 ATG9 Feed-Restricted 4 0.2143 0.0746 0.9789 1.239 1.568 Time ATG9 Feed-Restricted 1 ATG9 Feed-Restricted 5 3.2693 <.0001 20.6856 26.294 33.423 Time ATG9 Feed-Restricted 1 ATG9 Feed-Restricted 6 -1.4516 <.0001 0.1698 0.2342 0.323 Time ATG9 Feed-Restricted 2 ATG9 Feed-Restricted 3 0.9593 <.0001 2.1529 2.6099 3.164 Time ATG9 Feed-restricted 2 ATG9 Feed-restricted 4 -0.4515 <.0001 0.5243 0.6367 0.773 Time ATG9 Feed-Restricted 2 ATG9 Feed-Restricted 5 2.6036 <.0001 11.0685 13.5118 16.495 Time ATG9 Feed-Restricted 2 ATG9 Feed-Restricted 6 -2.1174 <.0001 0.0898 0.1203 0.161 Time ATG9 Feed-Restricted 3 ATG9 Feed-Restricted 4 -1.4108 <.0001 0.2034 0.244 0.293 Time ATG9 Feed-Restricted 3 ATG9 Feed-Restricted 5 1.6443 <.0001 4.2925 5.1771 6.244 Time ATG9 Feed-restricted 3 ATG9 Feed-restricted 6 -3.0767 <.0001 0.0347 0.0461 0.061

286

Treatment Target Treatment Time Target Treatment Time Estimate Probt LL RoR UL Time ATG9 Feed-Restricted 4 ATG9 Feed-Restricted 5 3.055 <.0001 17.563 21.222 25.643

Time ATG9 Feed-Restricted 4 ATG9 Feed-Restricted 6 -1.6659 <.0001 0.142 0.189 0.252 Time ATG9 Feed-restricted 5 ATG9 Feed-restricted 6 -4.7209 <.0001 0.0067 0.0089 0.012 Time BEC1 Control 1 BEC1 Control 2 0.8064 <.0001 1.755 2.24 2.859 Time BEC1 Control 1 BEC1 Control 3 0.4632 0.0001 1.257 1.589 2.008 Time BEC1 Control 1 BEC1 Control 4 1.2636 <.0001 2.796 3.538 4.478 Time BEC1 Control 1 BEC1 Control 5 4.8716 <.0001 102.577 130.531 166.105 Time BEC1 Control 1 BEC1 Control 6 1.1927 <.0001 2.389 3.296 4.546

Time BEC1 Control 2 BEC1 Control 3 -0.3432 0.0005 0.585 0.709 0.86 Time BEC1 Control 2 BEC1 Control 4 0.4572 <.0001 1.301 1.58 1.918 Time BEC1 Control 2 BEC1 Control 5 4.0652 <.0001 47.676 58.278 71.238

Time BEC1 Control 2 BEC1 Control 6 0.3863 0.0098 1.098 1.472 1.972

Time BEC1 Control 3 BEC1 Control 4 0.8004 <.0001 1.856 2.227 2.671 Time BEC1 Control 3 BEC1 Control 5 4.4084 <.0001 68.008 82.141 99.212 Time BEC1 Control 3 BEC1 Control 6 0.7295 <.0001 1.56 2.074 2.757 Time BEC1 Control 4 BEC1 Control 5 3.608 <.0001 30.488 36.892 44.641 Time BEC1 Control 4 BEC1 Control 6 -0.07093 0.6264 0.7 0.932 1.24

Time BEC1 Control 5 BEC1 Control 6 -3.6789 <.0001 0.019 0.025 0.034 Time BEC1 Pair-fed 1 BEC1 Pair-fed 2 0.4336 0.0005 1.209 1.543 1.969 Time BEC1 Pair-fed 1 BEC1 Pair-fed 3 0.4726 <.0001 1.269 1.604 2.027 Time BEC1 Pair-fed 1 BEC1 Pair-fed 4 -0.1156 0.336 0.704 0.891 1.128 Time BEC1 Feed-Restricted 1 BEC1 Feed-Restricted 5 3.7971 <.0001 35.067 44.574 56.657

Time BEC1 Feed-Restricted 1 BEC1 Feed-Restricted 6 0.744 <.0001 1.526 2.104 2.903

Time BEC1 Feed-restricted 2 BEC1 Feed-restricted 3 0.03901 0.6909 0.858 1.04 1.26 Time BEC1 Feed-Restricted 2 BEC1 Feed-Restricted 4 -0.5492 <.0001 0.475 0.577 0.701 Time BEC1 Feed-Restricted 2 BEC1 Feed-Restricted 5 3.3635 <.0001 23.667 28.891 35.268 Time BEC1 Feed-restricted 2 BEC1 Feed-restricted 6 0.3104 0.0377 1.018 1.364 1.828

287

Treatment Target Treatment Time Target Treatment Time Estimate Probt LL RoR UL

Time BEC1 Feed-Restricted 1 BEC1 Feed-Restricted 5 3.7971 <.0001 35.067 44.574 56.657 Time BEC1 Feed-Restricted 1 BEC1 Feed-Restricted 6 0.744 <.0001 1.526 2.104 2.903 Time BEC1 Feed-restricted 2 BEC1 Feed-restricted 3 0.03901 0.6909 0.858 1.04 1.26 Time BEC1 Feed-Restricted 2 BEC1 Feed-Restricted 4 -0.5492 <.0001 0.475 0.577 0.701 Time BEC1 Feed-Restricted 2 BEC1 Feed-Restricted 5 3.3635 <.0001 23.667 28.891 35.268

Time BEC1 Feed-restricted 2 BEC1 Feed-restricted 6 0.3104 0.0377 1.018 1.364 1.828 Time BEC1 Feed-Restricted 3 BEC1 Feed-Restricted 4 -0.5882 <.0001 0.463 0.555 0.666 Time BEC1 Feed-Restricted 3 BEC1 Feed-Restricted 5 3.3245 <.0001 23.038 27.786 33.512 Time BEC1 Feed-restricted 3 BEC1 Feed-restricted 6 0.2714 0.0616 0.987 1.312 1.744 Time BEC1 Feed-restricted 4 BEC1 Feed-restricted 5 3.9127 <.0001 41.408 50.034 60.456

Time BEC1 Feed-Restricted 4 BEC1 Feed-Restricted 6 0.8596 <.0001 1.775 2.362 3.144

Time BEC1 Feed-Restricted 5 BEC1 Feed-Restricted 6 -3.0531 <.0001 0.035 0.047 0.063 Time GABA Control 1 GABA Control 2 0.3444 0.0057 1.106 1.411 1.801 Time GABA Control 1 GABA Control 3 0.05942 0.6185 0.84 1.061 1.341 Time GABA Control 1 GABA Control 4 0.9469 <.0001 2.037 2.578 3.263 Time GABA Control 1 GABA Control 5 3.7568 <.0001 33.644 42.813 54.481

Time GABA Control 1 GABA Control 6 1.815 <.0001 4.452 6.141 8.471

Time GABA Control 2 GABA Control 3 -0.285 0.0038 0.62 0.752 0.912 Time GABA Control 2 GABA Control 4 0.6025 <.0001 1.504 1.827 2.218 Time GABA Control 2 GABA Control 5 3.4124 <.0001 24.821 30.339 37.084 Time GABA Control 2 GABA Control 6 1.4706 <.0001 3.247 4.352 5.832 Time GABA Control 3 GABA Control 4 0.8875 <.0001 2.025 2.429 2.913

Time GABA Control 3 GABA Control 5 3.6974 <.0001 33.403 40.343 48.725

Time GABA Control 3 GABA Control 6 1.7555 <.0001 4.353 5.787 7.692 Time GABA Control 4 GABA Control 5 2.8099 <.0001 13.727 16.609 20.097 Time GABA Control 4 GABA Control 6 0.8681 <.0001 1.79 2.382 3.171 Time GABA Control 5 GABA Control 6 -1.9419 <.0001 0.107 0.143 0.192

288

Treatment Target Treatment Time Target Treatment Time Estimate Probt LL RoR UL Time GABA Feed-Restricted 1 GABA Feed-Restricted 2 0.007037 0.9549 0.789 1.007 1.285 Time GABA Feed-Restricted 1 GABA Feed-Restricted 3 0.09154 0.443 0.867 1.096 1.385 Time GABA Feed-Restricted 1 GABA Feed-Restricted 4 -0.1428 0.2345 0.685 0.867 1.097 Time GABA Feed-Restricted 1 GABA Feed-Restricted 5 1.0663 <.0001 2.269 2.905 3.718 Time GABA Feed-restricted 1 GABA Feed-restricted 6 1.1979 <.0001 2.402 3.313 4.57 Time GABA Feed-Restricted 2 GABA Feed-Restricted 3 0.08451 0.3891 0.898 1.088 1.319 Time GABA Feed-Restricted 2 GABA Feed-Restricted 4 -0.1498 0.1305 0.71 0.86 1.05 Time GABA Feed-restricted 2 GABA Feed-restricted 5 1.0593 <.0001 2.34 2.88 3.55 Time GABA Feed-Restricted 2 GABA Feed-Restricted 6 1.1909 <.0001 2.46 3.29 4.41 Time GABA Feed-Restricted 3 GABA Feed-Restricted 4 -0.2343 0.0116 0.66 0.79 0.95 Time GABA Feed-Restricted 3 GABA Feed-Restricted 5 0.9748 <.0001 2.18 2.65 3.23 Time GABA Feed-restricted 3 GABA Feed-restricted 6 1.1064 <.0001 2.27 3.02 4.02 Time GABA Feed-Restricted 4 GABA Feed-Restricted 5 1.2091 <.0001 2.75 3.35 4.08 Time GABA Feed-Restricted 4 GABA Feed-Restricted 6 1.3407 <.0001 2.8 3.82 5.09 Time GABA Feed-restricted 5 GABA Feed-restricted 6 0.1316 0.3818 0.85 1.14 1.53 Time LC3 Control 1 LC3 Control 2 0.9597 <.0001 2.05 2.61 3.33 Time LC3 Control 1 LC3 Control 3 -0.07441 0.5328 0.73 0.93 1.17 Time LC3 Control 1 LC3 Control 4 1.7363 <.0001 4.49 5.68 7.18 Time LC3 Control 1 LC3 Control 5 8.7065 <.0001 4747.95 6041.8 7688.23 Time LC3 Control 1 LC3 Control 6 0.3984 0.0152 1.08 1.49 2.05 Time LC3 Control 2 LC3 Control 3 -1.0341 <.0001 0.29 0.36 0.43 Time LC3 Control 2 LC3 Control 4 0.7766 <.0001 1.79 2.17 2.64 Time LC3 Control 2 LC3 Control 5 7.7468 <.0001 1893.2 2314.04 2828.44 Time LC3 Control 2 LC3 Control 6 -0.5613 0.0002 0.43 0.57 0.76 Time LC3 Control 3 LC3 Control 4 1.8108 <.0001 5.1 6.12 7.33 Time LC3 Control 3 LC3 Control 5 8.7809 <.0001 5388.97 6508.55 7860.73 Time LC3 Control 3 LC3 Control 6 0.4728 0.0012 1.21 1.6 2.13

289

Treatment Target Treatment Time Target Treatment Time Estimate Probt LL RoR UL

Time LC3 Control 4 LC3 Control 5 6.9701 <.0001 879.64 1064.34 1287.82 Time LC3 Control 4 LC3 Control 6 -1.3379 <.0001 0.2 0.26 0.35 Time LC3 Control 5 LC3 Control 6 -8.308 <.0001 0 0 0 Time LC3 Feed-restricted 1 LC3 Feed-restricted 2 -0.8727 <.0001 0.33 0.42 0.53 Time LC3 Feed-Restricted 1 LC3 Feed-Restricted 4 -0.2752 0.0221 0.6 0.76 0.96

Time LC3 Feed-restricted 1 LC3 Feed-restricted 5 5.976 <.0001 307.69 393.85 504.15 Time LC3 Feed-Restricted 1 LC3 Feed-Restricted 6 -0.7103 <.0001 0.36 0.49 0.68 Time LC3 Feed-Restricted 2 LC3 Feed-Restricted 3 0.9524 <.0001 2.14 2.59 3.14 Time LC3 Feed-Restricted 2 LC3 Feed-Restricted 4 0.5975 <.0001 1.5 1.82 2.21 Time LC3 Feed-Restricted 2 LC3 Feed-Restricted 5 6.8487 <.0001 765.79 942.66 1160.37

Time LC3 Feed-restricted 2 LC3 Feed-restricted 6 0.1624 0.2764 0.88 1.18 1.58

Time LC3 Feed-Restricted 3 LC3 Feed-Restricted 4 -0.3548 0.0001 0.58 0.7 0.84 Time LC3 Feed-Restricted 3 LC3 Feed-Restricted 5 5.8964 <.0001 298.9 363.71 442.58 Time LC3 Feed-Restricted 3 LC3 Feed-Restricted 6 -0.7899 <.0001 0.34 0.45 0.6 Time LC3 Feed-restricted 4 LC3 Feed-restricted 5 6.2512 <.0001 425.46 518.63 632.21 Time LC3 Feed-Restricted 4 LC3 Feed-Restricted 6 -0.4351 0.0029 0.49 0.65 0.86

Time LC3 Feed-Restricted 5 LC3 Feed-Restricted 6 -6.6863 <.0001 0 0 0

290

APPENDIX 3.2b: Post-hoc statistical analysis of Atg gene expression in muscle: Least Significant Difference test was performed in SAS (significant data is in blue)

Effect Group 1 Group 2 Statistics Effect Target Treatment Time Target Treatment Time Estimate Probt LL RoR UL Treatment ATG12 Control 1 ATG12 Feed-Restricted 1 0.4206 0.0031 1.5328 1.52287 2.109 Treatment ATG12 Control 2 ATG12 Feed-Restricted 2 0.4569 <.0001 1.27588 1.5791 1.95439 Treatment ATG12 Control 3 ATG12 Feed-Restricted 3 0.5568 <.0001 1.45778 1.74511 2.08909 Treatment ATG12 Control 4 ATG12 Feed-Restricted 4 -0.3504 0.0002 0.5861 0.7044 0.8465 Treatment ATG12 Control 5 ATG12 Feed-Restricted 5 -0.3139 0.0017 0.6006 0.7306 0.8887 Treatment ATG12 Control 6 ATG12 Feed-Restricted 6 -0.03413 0.8525 0.6742 0.9665 1.3854 Treatment ATG13 Control 1 ATG13 Feed-Restricted 1 0.4717 0.0009 1.2137 1.6026 2.1162 Treatment ATG13 Control 2 ATG13 Feed-Restricted 2 0.008551 0.9345 0.8223 1.0086 1.2371 Treatment ATG13 Control 3 ATG13 Feed-Restricted 3 0.5456 <.0001 1.4415 1.7256 2.0657 Treatment ATG13 Control 4 ATG13 Feed-Restricted 4 -0.6937 <.0001 0.4158 0.4997 0.6006 Treatment ATG13 Control 5 ATG13 Feed-Restricted 5 -1.0048 <.0001 0.301 0.3661 0.4453 Treatment ATG13 Control 6 ATG13 Feed-Restricted 6 -1.3623 <.0001 0.1786 0.2561 0.3671 Treatment ATG16 Control 1 ATG16 Feed-Restricted 1 0.3896 0.0061 1.1181 1.4764 1.9495 Treatment ATG16 Control 2 ATG16 Feed-Restricted 2 0.6107 <.0001 1.5016 1.8418 2.259 Treatment ATG16 Control 3 ATG16 Feed-Restricted 3 1.14 <.0001 2.6119 3.1267 3.743 Treatment ATG16 Control 4 ATG16 Feed-Restricted 4 -0.6967 <.0001 0.4146 0.4982 0.5988 Treatment ATG16 Control 5 ATG16 Feed-Restricted 5 -1.6246 <.0001 0.1593 0.197 0.2436 Treatment ATG16 Control 6 ATG16 Feed-Restricted 6 -0.1953 0.2874 0.5738 0.8226 1.1792 Treatment ATG4 Control 1 ATG4 Feed-Restricted 1 0.398 0.0051 1.1275 1.4888 1.966 Treatment ATG4 Control 2 ATG4 Feed-Restricted 2 0.4428 <.0001 1.2694 1.557 1.9098 Treatment ATG4 Control 3 ATG4 Feed-Restricted 3 0.2555 0.0054 1.0785 1.2911 1.5455 Treatment ATG4 Control 4 ATG4 Feed-Restricted 4 -0.4477 <.0001 0.5318 0.6391 0.7681 Treatment ATG4 Control 5 ATG4 Feed-Restricted 5 -1.8095 <.0001 0.1346 0.1637 0.1992

291

Effect Target Treatment Time Target Treatment Time Estimate Probt LL RoR UL Treatment ATG4 Control 6 Atg4 Feed-Restricted 6 1.374 <.0001 2.7563 3.9511 5.6638 Treatment ATG5 Control 1 Atg5 Feed-Restricted 1 -0.2215 0.1182 0.6068 0.8013 1.0581 Treatment ATG5 Control 2 Atg5 Feed-Restricted 2 0.124 0.2335 0.923 1.1321 1.3886 Treatment ATG5 Control 3 Atg5 Feed-Restricted 3 -0.5894 <.0001 0.4633 0.5547 0.664 Treatment ATG5 Control 4 Atg5 Feed-Restricted 4 -0.2968 0.0016 0.6184 0.7432 0.8931 Treatment ATG5 Control 5 Atg5 Feed-Restricted 5 -0.9407 <.0001 0.3209 0.3903 0.4748 Treatment ATG5 Control 6 Atg5 Feed-Restricted 6 -0.217 0.2372 0.5615 0.8049 1.1539 Treatment ATG7 Control 1 Atg7 Feed-Restricted 1 0.1536 0.2784 0.883 1.166 1.5397 Treatment ATG7 Control 2 Atg7 Feed-Restricted 2 0.393 0.0002 1.2078 1.4815 1.8171 Treatment ATG7 Control 3 Atg7 Feed-Restricted 3 3.5282 <.0001 28.4536 34.062 40.7757 Treatment ATG7 Control 4 Atg7 Feed-Restricted 4 -0.5683 <.0001 0.4714 0.5665 0.6808 Treatment ATG7 Control 5 Atg7 Feed-Restricted 5 -0.9517 <.0001 0.3174 0.3861 0.4696 Treatment ATG7 Control 6 Atg7 Feed-Restricted 6 -1.0696 <.0001 0.2394 0.3431 0.4919 Treatment ATG9 Control 1 Atg9 Feed-Restricted 1 -0.4694 0.001 0.4736 0.6254 0.8258 Treatment ATG9 Control 2 Atg9 Feed-Restricted 2 0.4708 <.0001 1.3056 1.6013 1.9642 Treatment ATG9 Control 3 Atg9 Feed-Restricted 3 0.974 <.0001 2.2123 2.6484 3.1704 Treatment ATG9 Control 4 Atg9 Feed-Restricted 4 -0.702 <.0001 0.4124 0.4956 0.5956 Treatment ATG9 Control 5 Atg9 Feed-Restricted 5 -1.4272 <.0001 0.1973 0.24 0.2919 Treatment ATG9 Control 6 Atg9 Feed-Restricted 6 0.1734 0.3449 0.8297 1.1893 1.7049 Treatment BECN1 Control 1 BECN1 Feed-Restricted 1 0.8233 <.0001 1.7251 2.278 3.008 Treatment BECN1 Control 2 BECN1 Feed-Restricted 2 0.4505 <.0001 1.2793 1.5691 1.9246 Treatment BECN1 Control 3 BECN1 Feed-Restricted 3 0.8327 <.0001 1.921 2.2996 2.7528 Treatment BECN1 Control 4 BECN1 Feed-Restricted 4 -0.5559 <.0001 0.4773 0.5736 0.6893 Treatment BECN1 Control 5 BECN1 Feed-Restricted 5 -0.2512 0.012 0.6395 0.7779 0.9462 Treatment BECN1 Control 6 BECN1 Feed-Restricted 6 0.3746 0.0415 1.0146 1.4544 2.0849 Treatment GABA Control 1 GABA Feed-Restricted 1 0.5465 0.0001 1.308 1.7272 2.2807

292

Effect Target Treatment Time Target Treatment Time Estimate Probt LL RoR UL

Treatment GABA Control 2 GABA Feed-Restricted 2 0.2091 0.0448 1.0049 1.2326 1.5119 Treatment GABA Control 3 GABA Feed-Restricted 3 0.5786 <.0001 1.4899 1.7836 2.1351 Treatment GABA Control 4 GABA Feed-Restricted 4 -0.5432 <.0001 0.4833 0.5809 0.6981 Treatment GABA Control 5 GABA Feed-Restricted 5 -2.144 <.0001 0.0955 0.1172 0.1438 Treatment GABA Control 6 GABA Feed-Restricted 6 -0.07052 0.7008 0.6501 0.9319 1.3359 Treatment LC3 Control 1 LC3 Feed-Restricted 1 1.183 <.0001 2.472 3.2642 4.3103 Treatment LC3 Control 2 LC3 Feed-Restricted 2 -0.6494 <.0001 0.4259 0.5224 0.6407 Treatment LC3 Control 3 LC3 Feed-Restricted 3 1.3371 <.0001 3.1809 3.8079 4.5584 Treatment LC3 Control 4 LC3 Feed-Restricted 4 -0.8285 <.0001 0.3634 0.4367 0.5248 Treatment LC3 Control 5 LC3 Feed-Restricted 5 -1.5475 <.0001 0.1734 0.2128 0.2611 Treatment LC3 Control 6 LC3 Feed-Restricted 6 0.07432 0.6855 0.7514 1.0771 1.5441

293