A Dissertation

Entitled

Stress Induced Silicon Accumulation in the Inducible Accumulator Nicotiana tabacum by

Wendy L. Zellner

Submitted to the Graduate Faculty as partial fulfillment of the requirements for Doctor of Philosophy degree in Biology

______

Dr. Scott M. Leisner Committee Chair

______

Dr. Patricia R. Komuniecki, Dean College of Graduate Studies

The University of Toledo August 2012

Copyright © 2012, Wendy Louise Zellner This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author.

An Abstract of

Stress Induced Silicon Accumulation in the Inducible Accumulator Nicotiana tabacum by

Wendy L. Zellner

Submitted to the Graduate Faculty as partial fulfillment of the requirements for

Doctor of Philosophy degree in Biology

The University of Toledo August 2012

While Silicon (Si) is not a panacea for stress resistance in plants, the element has a number of beneficial effects against both abiotic and biotic stress.

Most research to date has been inundated with salt, cold, and fungal resistance induced by Si in high accumulators, with little work performed on viral infections and low accumulators. The low accumulator, N. tabacum infected with Tobacco ringspot virus and supplemented with Si showed a reduction in viral systemic symptoms and a increase in foliar Si levels compared to controls. Si supplementation did not influence the systemic symptoms induced by the unrelated pathogen, Tobacco mosaic virus, nor did infections influence foliar Si levels. Si accumulation in the TRSV-N. tabacum pathosystem was quite variable, so to study stress induced Si accumulation (SISA), a more consistent system was developed. Copper (Cu) toxicity increases foliar Si accumulation in a low accumulator and in N. tabacum. The consistency of this response indicates that it is a good model to study SISA. In addition to Cu, hormones involved in defense iii

and environmental perception were tested to determine if they too influenced Si accumulation in leaves. N. tabacum were exposed to exogenous abscisic acid, methyl jasmonate, or salicylic acid and foliar Si content was determined. Plants treated with ABA showed a significant reduction in foliar Si levels, while the other two hormones had no effect. This suggests the ABA has an antagonistic effect on SISA. Along with physiological work, we examined factors involved in the transport of Si into the plant. Aquaporins, belonging to the major intrinsic protein (MIP) family, are involved in water and/or solute transport into and throughout the plant. We identified putative MIPs in N. tabacum, including a novel ntNIP3;1. This protein is likely a Si transporter based on sequence homology to documented transporters. In addition, Si caused a reduction in expression of ntNIP3;1as well as a root-specific tonoplast intrinsic protein (TIP), ntRT-TIP1, which correlates with the expression of other Si transporters. This leads us to believe that in addition to the classical NIP family members involved in Si transport, TIPs may also play a role. Taken together, these data suggest that characterizing plants as low-, intermediate- or high-accumulators can be somewhat misleading, since the ability to accumulate the element is dependent on not just the tissue assayed, but can also be influenced by the environmental conditions in which the plants are grown. In summary, Cu toxicity is a reliable system to begin studying the molecular aspects of SISA in the inducible- accumulator N. tabacum. This process may be under hormonal regulation, since

ABA reduces Si levels. With the heavy use of hormone regulators (such as

ABA), especially in the floriculture industry, could lead to lowered resistance

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against a number of abiotic and biotic stressors, because of reduced foliar Si levels.

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For my husband who encouraged me to pursue my degree, my children who remind me to question the world around me and enjoy all the little discoveries, my family and friends who have supported me throughout my entire schooling and to my teachers and professors who have added to my excitement and love for plant biology.

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Acknowledgments

This work could not have been accomplished without the encouragement and guidance from my advisor, Dr. Scott M. Leisner, who gave me the freedom to pursue my interests, while working toward my degree. I also would like to thank the members of my graduate committee Dr. Jonathan Frantz, Dr. John Gray, Dr.

James Locke, and Dr. Lirim Shemshedini, who have helped answer questions and shared with me a wide spectrum of skills and knowledge throughout the years.

Not a committee member, but equally helpful in my success here at UT is Dr.

Charles Krause. I also would like to thank many of my lab members, past and present who have reminded me that there is no better job than a scientist. I especially would like to thank Genevieve Okenka, Dr. Gaurav Raikhy, Dr.

Sushant Khandekar, Dr. Jie Li, and Lindy Lutz who have provided great debates and troubleshooting ideas for all the mishaps along the way. In addition, I would like to thank the USDA-ARS technicians Douglas Sturtz, Russell Friedrich, and

Alycia Pittenger who were responsible for compiling the ICP-OES data for these studies. Lastly, but not anywhere near the least, I would like to thank Lynn Biltz for everything she has taught me in regards to horticulture and identification of all my unknown species in addition to all that she did keeping plants pest-free.

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Table of Contents

Abstract………………………………………………………...……….……...... iii

Acknowledgments…………………………………………...…………..……....vii

Table of Contents…………………………………………...……………..….....viii

List of Tables………………………………………….…...……………….….....xi

List of Figures ………………………………………….…………...……..…....xiii

List of Abbreviations………………………………………………………...... xvii

1 Introduction………………………………………………………….…...1

2 Silicon Delays Tobacco Ringspot Virus Systemic Symptoms In Nicotiana tabacum ………………………..…..…………….....7 2.1 List of Abbreviations……………………………..…………...……....8 2.2 Summary…………………………………………..…..…………..…10 2.3 Materials and Methods………………….………...…………………11

2.4 Results and Discussion………………………………………………14

2.5 Acknowledgments………………………………...…………………19

2.6 References ………………………………………...…………………20

3 Induced Silicon Accumulation in N. tabacum…………...………………24

3.1 Abstract………………………………………………………24

3.2 Introduction……………………………………….…………25

3.3 Materials and Methods………………………………………28

3.4 Results……………………………………………………..…30

3.5 Discussion ………………………………………….…..……43 viii

4 Silicon Regulation of Aquaporin Gene Expression in N. tabacum……...47

4.1 Abstract………………………………………………………47

4.2 Introduction………………………………………….….....…48

4.3 Materials and Methods……………………………….………55

4.4 Results…………………………………………...... ……59

4.5 Discussion …………………………………...... ……………72

5 Additional Si Experiments....…………....………….....…………………75

5.1 Abstract……………………....……………...... …………75

5.2 Introduction………………....……………………...... ………77

5.3 Materials and Methods……………………………….....……80

5.4 Results………………………………………………...... 84

5.5 Discussion ……………………………………………...... …93

6 Discussion/Future Work……………………………………………....…96

References ………………………………………………...... 106

A Bioinformatic Codes………………………………………………...... 126

A.1. Introduction...... 126

A.2 AUDPC………………………………………………...... 126

A.3 Statistical analysis……………………………………….…128

B Identification and analysis of viruses infecting Pelargonium …………..129

B.1 Abstract………………………………………………...... 129

B.2 Introduction………………………………………………...130

B.3Materials and Methods……………………………………...132

B.4 Results………………………………………………...... 136

B.5 Discussion……………………………………………….....146

C Contig Sequences………………………………………………...... 150

ix

C.1 Contig Accessions…………………………………….……150

C.2 Contig FASTA Sequences…………………...…….………158

x

List of Tables

3.S1: Statistical Parameters of TRSV Symptomatic Area...... 21

3.S2: Statistical Parameters of TRSV AUDPC...... 21

3.S3: Statistical Parameters of foliar Si in TRSV infected N. tabacum...... 22

3.S4: Statistical Parameters of foliar Si in TMV infected N. tabacum...... 22

3.S5: Statistical Parameters of root Si in TRSV infected N. tabacum...... 23

3.1: Total elemental analysis of N. tabacum treated with 75 μM Cu……………37

3.2: Total elemental analysis of ABA and SA treated N. tabacum…………..….42

3.3: Total elemental analysis of JA treated N. tabacum…………………..……..43

4.1: MIP ar/R selectivity filter amino acid residues……………………..………52

4.2: Websites used for Computational Analysis……………………………..…..55

4.3: Primers used for Real-Time PCR……………………………..…………….58

4.4: Accession number of Si transporters…………………………..……………60

4.5: Selectivity filter and NPA residues for putative

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N. tabacum MIPs………………………………………………...64

4.6: Accession numbers of A. thaliana and N. tabacum TIPs………..………….67

4.7: Accession number of putative Si transporters……………………..………..70

5.1: Local and systemic TRSV symptoms in A. thaliana ecotypes………..…….84

B.1: Primer sequences for virus detection in OPGC Pelargoniums……...... 134

B.2 Viruses detected in OPGC Pelargoniums in 2006……………………...….139

B.2: PFBV ecotype study in A. thaliana……………………………..…………146

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List of Figures

3-1: TRSV systemic symptom spread and detection in N. tabacum……………..16

3-2: Si concentration (mg/kg) within virus-infected plants

determined by ICP-OES…………………………………………18

3-1: Symptoms on N. tabacum treated with 50 μM Cu………………………….31

3-2: Total Si in N. tabacum treated with Si+Cu+, Cu+, Si+

and control……………………………………………………….33

3-3: Symptoms on N. tabacum treated with 75 μM Cu……………...…………..34

3-4: N. tabacum roots treated with 75 μM Cu…………………...………………35

3-5: Total foliar Si in N. tabacum treated with 75 μM Cu……………………….36

3-6: Fresh weight of N. tabacum treated with hormones………………………...38

3-7: Si accumulation in MeJA treated N. tabacum………………………………39

3-8: N. tabacum foliar Si following ABA and SA treatment………………….…40

3-9: Foliar Si in N. tabacum sprayed with dH2O…………………...……………40

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4-1: MIP schematic for tomato NIP2;1…………………………………………..49

4-2: Subunit of MIP relative to cellular membrane………………...……………54

4-3: MSA of Si transporters in plants……………………….……..…………….61

4-4: Phylogenetic tree of Si transporters in plants…………….…..……………..61

4-5: Phylogenetic tree of putative N. tabacum MIPs……………..…..………….63

4-6: Relative expression of N. tabacum aquaporins…………………...………...66

4-7: TIP phylogenetic tree……………………………………………...………..68

4-8: MSA of putative Si transporters from various plant

EST sequences…………………………………………………...71

4-7: Phylogenetic tree of putative Si transporters………………………………..70

5-1: Systemic, local or no TRSV symptoms in A. thaliana ecotypes……………85

5-2: Local and systemic AUDPC for TRSV infected Sf-1………...…………….86

5-3: Sf-1 TRSV symptoms with varying B and Si treatments...... 87

5-4: PCR using PS60 designed primers...... 88

5-5: Average slop of POD activity in turnip extract………………...…………...90

5-6: Si accumulation in yeast expressing ntRT-TIP1 or empty vector...... 91

5-7: Si concentration comparison between ICP-OES and the

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Molybdenum Blue Colorimetric Assay………………………….92

5-8: The Molybdenum Blue Method detection of Si in

different phosphate buffers………………………………………93

6-1: Model for SISA...... 98

6-2: Phylogenetic tree of identified and putative N. tabacum Lsi2...... 102

6-3: Cladogram of Angiosperm orders containing ornamental plants

identified as low-, intermediate- or high-accumulators………...104

A-1: Excel spreadsheet of CSV file…………………………...………………..128

B-1: PFBV genome organization……………………………………………….131

B-2: Symptoms on OPGC Pelargonium cuttings rooted plants………...……...138

B-3: Symptoms on OPGC 230 infected with PLPV

and later PFBV/PLPV …………………………………………..139

B-4: PFBV symptoms on OPGC Pelargoniums………………………………..140

B-5: PFBV symptoms on OPGC 1084 treated with varying levels of B...... 142

B-6: Bleaching symptoms on OPGC 1084 treated with boron…………………143

B-7: MSA of PFBV p12 MP from various OPGC clones……………...………144

B-9: Amino acid differences in CP regions of PFBV from

OPGC isolated populations……………………………………..145 xv

B-10: PFBV symptoms on A. thaliana……………………………...………….146

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List of Abbreviations

ABA…………………Abscisic acid AILV………………..Artichoke Italian latent virus ANOVA…………….Analysis of variance APS….………………American Phytopathological Society AQP…………………Aquaporin Ar/R………....………Aromatic/arginine AsA………….………Ascorbic Acid ATCC ………………American Type Culture Collection AUDPC……………..Area Under the Disease Progression Curve

β-ME…………………2-Mercaptoethanol B……………………..Boron BLAST………………Basic local alignment search tool BoMV………………..Belladonna mottle virus

CHI………………….Chitinase CMV……….………..Cucumber mosaic virus CP……………………Coat protein CSV………………….Comma delimited file Cu……………………Copper

DPI………………….Days post-inoculation

ELV…………………Elderberry latent virus EST………………….Expressed sequence tags Et……………………Ethylene

GA…………………..Gibberellic acid GAST1………………Gibberellic acid stimulated transcript 1 GFP………………….Green fluorescent protein GIP…………………..GlpF-like intrinsic protein

H2…………………...Helix 2 H5…………………...Helix 5 HIP………………….Hybrid intrinsic protein

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ICP-OES…………….Inductively coupled plasma-optical emission spectroscopy

JA……………………Jasmonic acid

LE1………………….Loop E residue 1 LE2………………….Loop E residue 2 Lsi……………………Low silicon in rice

MeJA………………...Methyl jasmonate MIP………………….Major intrinsic protein MMLV………………Moloney murine leukemia virus MP…………………...Movement protein MPV…………………Moroccan pepper virus MSA…………………Multiple sequence alignment

NCBI………………..National Center for Biotechnology Information NIP………………….Nodulin-26 like protein NPA…………………Asparagine-proline-alanine

OPGC……………….Ornamental Plant Germplasm Center OTU…………………Operational Taxonomical Unit

P-domain……………Protruding domain PAL…………………Phenylalanine ammonia lyase PCR…………………Polymerase chain reaction PFBV………………..Pelargonium flowerbreak virus PIP…………………..Plasma membrane intrinsic protein PLCV………………..Pelargonium leaf curl virus PLPV………………..Pelargonium line pattern virus PMT…………………Putrescine N-methyltransferase POD…………………Peroxidase PPM…………………Parts per million PPO………………….Polyphenol oxidase PR1a…………………Pathogenesis related protein PS60…………………60 kDa Pollen-specific ascorbic oxidase protein PZSV………………...Pelargonium zonate spot virus

R-domain……………RNA-binding domain ROS…………………Reactive oxygen species RT…………………...Reverse transcriptase RT-PCR……………..Reverse transcriptase-polymerase chain reaction

S-domain……………Central shell domain SA…………………...Salicylic acid Si…………………….Silicon SIP…………………..Small basic intrinsic proteins

xviii

SISA………………...Stress-induced silicon accumulation SKS7………………...Skewed root 5 similar 7 protein

TIP…………………..Tonoplast intrinsic protein TMV………………....Tobacco mosaic virus ToRSV……………....Tomato ringspot virus TRSV………………..Tobacco ringspot virus TSWV……………….Tomato spotted wilt virus Tukey’s HSD………..Tukey’s honestly significantly different test

XIP………………….Uncharacterized X intrinsic protein

xix

Chapter 1

Introduction

Si is the second most abundant element in the lithosphere, being only surpassed by oxygen (1). However, the amount of Si available in a soil solution may range from 0.1-0.6 mM (2). For example, the soil-less media, Sunshine Mix, provides less than 0.11 mM Si in the extracted aqueous solution (3). Si is absorbed by plant roots as silicic acid (Si(OH)4 and deposited in cell walls as amorphous silica, also known as opal or phytoliths (1, 4). Phytoliths add support to stalks and increase leaf erectness, permitting better exposure of photosynthetic cells to light (1, 5).

While many plants have the ability to uptake Si, accumulation in higher plants varies greatly (6). Thus, plants have been characterized based on foliar Si content. The three categories are plants that acquire greater than 10%, 1-3% and less than 1% that are classified as high-, intermediates and low-accumulators, respectively (7, 8). The uptake of Si by accumulator plants (Oryza sativa and

Zea mays) and intermediates (Benincase hispida and Helianthus annuus) is both passive and active. Passive Si transport allows the plants to acquire the element, while transport into the vascular system involves active transport (9).

1

The use of the term “non-accumulators” is somewhat misleading. While plants under this category contain lower concentrations of Si in their xylem sap compared to the available nutrient solution. These plants still have the ability to accumulate Si in quantities greater than any micronutrient. Non-accumulators just do not accumulate foliar Si to the same degree as accumulators, which can have as much as 10% Si per kg dry weight (10).

Si is unevenly distributed throughout plants (11). In the low-accumulator, tomato, Si concentrations are similar between the roots and shoots (12, 13), while in accumulators, shoots tend to have a higher concentration than roots (14). In leaf tissue, Si is stored in the vacuole as well as being deposited in the cell wall

(15). Leaf age also influences the elemental concentration, with older leaves containing higher Si levels compared to younger (16, 17). It is interesting to note that in cucumber and rice xylem sap, concentrations of 6 mM and 18 mM silicic acid were reported, respectively (18, 19), even though silicic acid polymerizes around 3 mM at neutral pH (20). This suggests that plant factors likely maintain the element in the soluble state while it is mobile and during storage in plant tissue, prior to deposition.

Si deposition has been suggested to confer a number of beneficial effects on plants (21). For example, increased Si accumulation can lead to resistance.

Recently, Belenger et. al reported a difference in Si accumulation between soybean cultivars (22). A cultivar that had the ability to absorb four times the amount of Si than a lower accumulating counterpart showed a significantly higher resistance to rust. 2

Originally, increased pathogen resistance in plants treated with Si was believed to only occur due to deposition of amorphous silica in the cell wall, inhibiting fungal penetration (23). Erysiphe cichoracearum, a fungus causing powdery mildew in Arabidopsis, penetrates leaves, but when the plants were supplied with Si, collapsed haustoria (invasion structures) were detected surrounded by dark toluidine blue O-staining, suggesting the presence of phenolic compounds (24). Haustoria in treatments lacking Si were significantly larger and not collapsed. A study done by Fawe et. al (25) showed that in cucumbers inoculated with the powdery mildew fungus, S. fuliginea, a similar incidence, where one of the stained compounds was the phytoalexin, rhamnetin. This suggests that Si can create a physical barrier not merely by deposition of amorphous silica, but also by influencing the deposition of phenolic compounds.

Not only does Si protect against fungal infections, but also plays a protective role against bacterial disease in plants. Ralstonia solanacearum infection of susceptible tomato genotypes showed a delay in tomato wilt death with Si treatment (26). Thus Si can protect plants against more than one type of pathogen, indicating that it has multiple modes of action.

In addition to use as a physical barrier, Si plays a more dynamic role in the activation of various defense pathways. Cucumis sativus infected with

Podosphaera xanthii showed a significant increase in resistance as measured by peroxidase (POD), polyphenol oxidase (PPO), and chitinase (CHI) activity

(defense related enzymes) when treated with Si (27). PPO, POD and CHI activity did not change in non-inoculated plants when treated with Si, suggesting 3

that the effect of this element on enzymatic products was only activated when plants were stressed. Similarly, Si supplementation lead to altered expression of nearly 4000 genes when A. thaliana were challenged with Erysiphe cichoracearum DC, compared to only two genes in non-inoculated plants. This further supports the hypothesis that Si can influence events at the biochemical and molecular level in addition to acting as a physical barrier, and that Si effects are most pronounced when plants are stressed (28).

However, Si has not always been shown to confer a beneficial effect on plants with respect to some pathogens. Tobacco inoculated with Belladonna mottle virus (BoMV) and treated with Si showed a four-fold increase in leaf systemic coverage (29). It is also interesting to note that the Si treatment showed a more drastic effect in experiments conducted during the winter then those executed during the spring and summer months, suggesting that the effect is influenced by light levels and/or temperature.

In addition to cell wall reinforcement and activation of defense pathways, another mechanism by which Si may have its effect is by the induction of plant hormones or secondary metabolites, specifically phenolics (23). Phenolics play at least two roles in plant defense (30). First, some phenolics act as signaling molecules (31). Second, certain phenolics act directly as antipathogen molecules

(23).

Three hormones implicated in defense pathways are abscisic acid (ABA), jasmonic acid (JA) and salicylic acid (SA) (32). ABA is responsible for

4

environmental perception and in some cases, enhances pathogens ability to invade plants. JA is induced through wounding, necotrophic fungi and herbivore feeding and also works with ethylene to mediate some of the responses. SA is involved in systemic acquired resistance (SAR) where leaves distal from the initial infection are more resistant to the pathogen. However, at this time it is unclear whether

ABA, JA or SA are influenced by Si treatment.

In addition to its effects on biotic challenge, Si also protects plants from a number of abiotic stresses including salt, drought and cold. It is likely that some of these beneficial effects against abiotic stress are due to Si induced changes in hormone levels. In wheat plants sprayed with Si under varying salt concentrations, gibberellic acid (GA) levels increased while abscisic acid (ABA) levels decreased compared to control treated plants (33). Si treatment, via

Diatomites de Mozambique diatomite amended garden soil, enhanced the growth of water stressed Lupinus albus L. in addition to increasing endogenous levels of phenolics (PPO, POD, SOD and catalase) as well as hormones (auxin, GA, and cytokinin) (34). Control and one day water-stressed plants showed an increase in

ABA, while at four days of water starvation, there was no change.

As indicated above, Si provides a number of beneficial effects to plants, but most of these studies have been done with high-accumulators. However, evidence suggests that low-accumulators can benefit from Si supplementation.

To perform more mechanistic studies, it is important to use a versatile, model system.

5

One such low accumulator is the cigarette tobacco, Nicotiana tabacum

(35). N. tabacum is a tetraploid derived from a cross between N. sylvestris and N. tomentosiformis (36). The transcriptome has been sequenced, and is now accessible through the Michigan State University website

(http://solanaceae.plantbiology.msu.edu/species/overview/4097), in addition to the National Center for Biotechnology Information (NCBI)

(http://www.ncbi.nlm.nih.gov/ ).

A series of experiments was employed to study the effects of Si on stress in N. tabacum. While Si enhanced infection by BoMV in N. tabacum, whether this was a general principle or specific for a virus-host system was unclear.

Therefore, we examined the effects of Si on TRSV infection in N. tabacum. If Si does influence TRSV infection, it is critical to determine if Si levels correlate with virus infection within these plants. Tobaccos have the ability to acquire Si from growth media. However, how foliar levels of Si respond under virus infection is unclear. Analysis of regulation of Si accumulation was tested by both metal toxicity and hormone treatment. Si is acquired by N. tabacum in response to stress. Therefore, we sought putative transporters within the expressed sequence tag database. Further, we examined the regulation of some of these putative transporters by Si. Lastly, we examined a potential role for ROS in Si-mediated responses.

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Chapter 2

Silicon Delays Tobacco Ringspot Virus Systemic Symptoms In

Nicotiana tabacum

1Wendy Zellner, 2Jonathan Frantz, and 1Scott Leisner*

1Department of Biological Sciences, The University Of Toledo, 2801 West

Bancroft Street, Toledo, OH 43606, Phone: (419) 530-1550 Fax: (419)

530-7737.

2USDA-ARS, New England Plant, Soil, and Water Laboratory, Orono, ME 04469

*Corresponding author email: [email protected]

Phone: (419) 530-1550

Fax: (419) 530-7737

Zellner, W., J. Frantz and S. Leisner. 2011. Silicon delays Tobacco ringspot virus systemic symptoms in Nicotiana tabacum. Journal of Plant Physiology. 168: 1866-1869. 7

LIST OF ABBREVIATIONS

AUDPC Area under the disease progress curve

C Control (0.1 mM soluble silicon, K2SiO4)

DPI Days post-inoculation

ICP-OES Inductively coupled plasma-optical emission spectroscopy

PCR Polymerase chain reaction

RT Reverse transcriptase

RT-PCR Reverse transcriptase-polymerase chain reaction

SEM Standard error of the mean

Si Silicon

Si+ 1.0 mM soluble silicon (K2SiO4)

TMV Tobacco mosaic virus

TRSV Tobacco ringspot virus

8

SUMMARY:

Soluble silicon (Si) provides protection to plants against a variety of abiotic and biotic stress. However, the effects of Si on viral infections are largely unknown. To investigate the role of Si in viral infections, hydroponic studies were conducted in Nicotiana tabacum with two pathogens: Tobacco ringspot virus (TRSV) and Tobacco mosaic virus (TMV). Plants grown in elevated Si showed a delay in TRSV systemic symptom formation and a reduction in symptomatic leaf area, compared to the non-supplemented controls. TRSV- infected plants showed significantly higher levels of foliar Si compared to mock- inoculated plants. However, the Si effect appeared to be virus-specific, since the element did not alter TMV symptoms nor did infection by this virus alter foliar Si levels. Hence, increased foliar Si levels appear to correlate with Si-modulated protection against viral infection. This is all the more intriguing since N. tabacum is classified as a low Si accumulator.

9

Key words: Nepovirus, plant nutrition, Si tobamovirus INTRODUCTION

Silicon (Si) is the second most abundant element in the lithosphere and is absorbed by plants to varying degrees (Frantz et al., 2010; Marschner, 2005). The range of Si uptake in dicot plants varies substantially from more than 12,000 mg

Si per kg of dry tissue for plants such as Zinnia elegans (a high Si accumulator), to <300 mg Si per kg of dry tissue in plants such as Nicotiana tabacum (a low Si accumulator). Si provides many benefits to stressed plants including protection against attack from certain fungal and bacterial pathogens. For example, treatment of tomato varieties and Cucumis sativus with Si reduced infection by

Ralstonia solanacearum and Podosphaera xanthii, respectively (Dannon and

Wydra, 2004; Datnoff et al., 2007; Liang et al., 2005). Interestingly, defense enzyme activity did not change in plants in the absence of the pathogen, whether or not Si was applied. This suggests that the effect of Si on host gene expression only occurs when plants are stressed (Fauteaux et al., 2006; Liang et al., 2005).

While Si protects plants against fungal and bacterial pathogens, the effects of this element on viral infections are unclear. Si was shown to increase viral incidence in tobacco infected with Belladonna mottle virus (BMoV) (Bengsch et al., 1989). However, N. tabacum are susceptible to a wide variety of viruses

(Brunt et al., 1997) and it is unclear if Si would have the same effect on all of these pathogens. Tobacco ringspot virus (TRSV) is a single-stranded, positive- sense, RNA nepovirus that causes systemic chlorotic and necrotic ringspots in N.

10

tabacum (Rezaian and Franki, 1973). Tobacco mosaic virus (TMV) is another single-stranded, positive-sense RNA virus that infects N. tabacum (Brunt et al.,

1997; Chapman, 1998). However, TMV belongs to the tobamovirus family and is only distantly related to TRSV. In this study, the ability of Si to influence TRSV and TMV infection of N. tabacum, was investigated. Our hypothesis was that like

BMoV, Si treatment of N. tabacum would render the plants more susceptible to

TRSV and TMV infection. In addition, we expected Si concentrations in leaves to remain unchanged in response to either virus since N. tabacum is a low Si accumulator (Frantz et al., 2010).

MATERIALS AND METHODS:

Propagation of viruses and N. tabacum plants.

All experiments were performed using Nicotiana tabacum L. cv.

Wisconsin 38 as the viral host. TRSV Rubus strain (American Type Culture

Collection PV-172) was propagated in N. tabacum by serial passage and virus was purified as described by Rezaian and Franki (1973). TRSV particles were mixed with washed celite and used for inoculation. TMV was propagated in that same N. tabacum cultivar by serial passage and virus was isolated as described by

Chapman (1998). TMV was resuspended in sodium phosphate buffer (pH 7.0) mixed with washed celite and used for inoculum.

N. tabacum seeds were germinated hydroponically in nutrient solution containing 0.1 mM soluble K2SiO4 as described by Li et al. (2008). Plants were hydroponically propagated in a growth chamber at 20ºC under a 16 h light:8 h dark photoperiod at a light intensity of 70 mol m-2 s-1 of photosynthetically

11

active radiation. After reaching the four-leaf stage, seedlings were transferred to

4 L buckets containing nutrient solution. Immediately prior to virus inoculation,

K2SiO4 concentrations were changed to 1.0 mM (Si+), or maintained at 0.1 mM

(C) for elevated Si and control hydroponic conditions, respectively. Three leaves per plant (at the five to six leaf stage) were rub-inoculated with purified TRSV or

TMV. Plants were propagated in a growth chamber under the conditions described above. Nutrient solution was changed every 5-7 days. The solution pH was monitored weekly and did not change during the course of the experiments.

Plants were examined daily for the formation of symptoms up to 15 days post- inoculation (DPI). Seven-to-nine plants were examined per treatment.

Analysis of viral infection and Si content.

Digital images were taken of the plants (Nikon D40; resolution of 3008 X

2000 pixels), once systemic viral symptoms appeared (about 9 days post- inoculation; DPI). Percent symptomatic leaf area was calculated using the Assess

Program (American Phytopathological Society Press). The pixel value for the sum of local lesions present was divided by the pixel value of the entire leaf area and converted to percentage. Digital image analysis was repeated three times for each leaf image. The total symptom coverage of each plant was determined by dividing the sum of the percent symptom area of each leaf by the total number of leaves present on each plant. Each individual plant was treated as a sample. The average and standard error of the mean (SEM) values for each set of treatments as well as ANOVA were calculated (R program; R Development Core Team, 2005).

Tukey’s HSD test was then used to indicate significant differences among

12

treatment means. The F, Df and P values were given in the Fig.1 legend and provided in Supplementary Table 1. Area under the disease progress curve

(AUDPC) for the time points indicated in Fig.1 was calculated by the method described by Sparks et al. (2008).

To detect TRSV, total RNA was isolated from systemic leaves of N. tabacum plants at 15 DPI using the RNeasy Kit and reverse transcriptase (RT) reactions were performed with the Moloney murine leukemia virus enzyme according to the manufacturer’s specifications (Promega, Madison, WI). Duplex polymerase chain reactions (PCR) were then performed using the Go Taq Master

Mix (Promega) according to the manufacturer’s specifications. In the same PCR reaction mixtures the TRSV-RNA1-nsp1F (5’

CCGCGAGGAGGGTCTTTCTTTTAG 3’), TRSV-RNA1-nsp1R (5’

CGGGGTGGCAGCGGTCTTC 3’), TRSV-RNA2-nsp1F (5’

AAGGCGCTCCGGGCTGCTCT 3’), and TRSV-RNA2-nsp1R (5’

CATGAAGGCGGGCTGCTGAA 3’) (synthesized by Integrated DNA

Techologies Inc., Coralville, IA) were added. The TRSV-RNA1-nsp1F and

TRSV-RNA1-nsp1R primers were designed based on the TRSV RNA 1 sequence

(GenBank accession number: NC_005097) to amplify a 471 base pair (bp) fragment (from nucleotide positions 166-637) in PCR reactions. The TRSV-

RNA2-nsp1F and TRSV-RNA2-nsp1R primers were designed based on the

TRSV RNA 2 sequence (GenBank accession number: NC_005096) to amplify a

383 bp fragment (from nucleotide positions 414-797). PCR reactions were performed in a BioRad (BioRad Laboratories Inc, Hercules, CA) iCycler Personal

13

using an initial thermal denaturation step at 92ºC for 1 min, followed by 25 cycles each of: a 30 s denaturation step at 92ºC, a 30 s annealing step at 50ºC, and an elongation step of 72ºC for 30 s. PCR products were separated by electrophoresis through a two percent agarose gel, stained with ethidium bromide and photographed under ultraviolet light.

The influence of Si on the stability/infectability of purified TRSV preparations was tested by incubating the inoculum overnight at 4°C with 0.1 mM or 1.0 mM K2SiO4. The pH did not change with the addition of the higher level of K2SiO4. The next day, washed celite was added to the inoculum and three N. tabacum plants (three leaves per plant) were rub-inoculated as before. Plants were then observed for symptoms daily.

For Si measurements, plants were harvested at the end of each experiment

(18 DPI) and separated into leaf and root tissue, dried in an oven at 60°C for 7 days, and ground. Si concentrations in tissue samples were determined as described in Frantz et al. (2008). ANOVA was performed on the averages for each of treatments, plant organs were analyzed separately and statistical analyses were performed based on Tukey’s HSD test as above. Statistical parameters are included in the Fig. 2 legend and provided in Supplementary Tables 2A-C.

RESULTS AND DISCUSSION

The effects of Si on TRSV systemic symptom spread in N. tabacum

At nine DPI, viral systemic symptoms just started to appear on most of the control (C, 0.1 mM K2SiO4) plants and some of the tobaccos treated with elevated

Si (Si+, 1.0 mM K2SiO4) (Fig. 3.1A). However, by 11 DPI, an obvious difference

14

was observed between the C and Si+ plants. Systemic TRSV symptoms covered about 30% average leaf area on C plants compared to approximately 2% leaf coverage in Si+ plants. By 13 DPI, C plants averaged about 37% systemic leaf coverage, while that of the Si+ tobaccos averaged about 10%. By 15 DPI, C plants showed approximately 34% leaf coverage, while the Si+ plants showed on average approximately 19% leaf symptoms. Overall, the majority of the Si+ plants never exhibited levels of symptomatic leaf coverage to the same extent as the controls. To examine the effectiveness of Si on the control of TRSV systemic infection, the area under the disease progress curve (AUDPC) was calculated for the four time points (Fig 3.1B). The AUDPC values show that Si confers a beneficial effect on tobacco by reducing the systemic TRSV symptomatic leaf area. Against our hypothesis, treatment of tobacco with increased Si lead to a reduction in TRSV systemic symptom distribution in infected plants. Systemic viral symptoms eventually appeared on the Si treated plants. Therefore, Si does not eradicate TRSV, but likely helps tobaccos mount a more effective defense delaying both the onset and appearance of systemic symptoms. To be certain that symptomatic plants were infected with TRSV, total RNA was isolated from all of the N. tabacum and analyzed by RT-PCR. All samples from infected plants were positive for TRSV (Fig. 3.1C).

15

C.

Fig. 3-1: TRSV systemic symptom spread and detection in N. tabacum. (A) Average percent TRSV symptomatic leaf area (Y-axis) on N. tabacum treated with 0.1 mM (C, white) or 1.0 mM K2SiO4 (Si+, black) at 9, 11, 13, and 15 DPI; the time points are indicated in the figure (X-axis). Average values and SEM are given, asterisks indicate significant difference between the C and Si+ plants at a particular time point (P<0.05). Df =1; F values are 0.663 for 9 DPI, 34.85 for 11, 6.555 for 13 and 1.4 for 15. Other statistical parameters are given in Supplementary Table 3.S1. (B) AUDPC average and SEM is given for C (white) and Si+ plants (black); asterisk indicates a significant difference between the values (P<0.05) with Df=1 and an F value of 12.127, as given in Supplementary Table 3.S2. (C) Detection of TRSV by RT-PCR in infected N. tabacum plants grown under C (lanes 1 and 3) or Si+ (lanes 2 and 4) conditions. Note, lanes 1 and 2 were from plants infected with TRSV, while 3 and 4 were from mock-inoculated controls. A marker (exACTGene 100 bp DNA ladder; Fisher Scientific, Pittsburgh, PA; lane M) and no cDNA PCR control (lane 5). The solid and dashed arrows indicate TRSV RNA 1 and RNA 2 PCR products, respectively.

The mechanism(s) by which Si delays viral systemic symptom formation is/are unclear. One possibility was that the element destabilized TRSV particles.

To test this, isolated TRSV was co-incubated with 0.1 or 1.0 mM K2SiO4 and inoculated to N. tabacum. The onset and spread of TRSV symptoms in tobaccos was unchanged by the co-incubation.

16

Since Si does not directly influence the infectivity of TRSV particles, it seemed more likely that the element modulates host defenses. Arabidopsis plants provided with Si induce a variety of genes in response to mildew that are not up- regulated in the absence of the element (Fauteaux et al., 2006). Perhaps Si enhanced tobacco defenses to provide protection against TRSV. Differences in defenses modulated by Si could lead to variations in plant physiological responses. One possibility is that Si may facilitate its own uptake into infected leaves. Therefore, at 18 DPI, leaves and roots of mock-inoculated and TRSV infected plants were harvested, dried, and analyzed for Si content.

Leaf Si levels were influenced by TRSV infection. Plants inoculated with

TRSV and supplemented with Si contained four-fold higher Si concentrations

(approximately 440 mg Si per kg leaf dry weight) compared to Si-supplemented, mock-inoculated tobaccos (approximately 110 mg per kg) (Fig. 3.2A). Since leaf

Si levels in Si+ TRSV-infected tobaccos were significantly higher than mock- inoculated plants, this suggests that foliar accumulation of Si is regulated in tobacco and may be part of a defense response in tobacco to TRSV. This is intriguing since tobacco is a low Si accumulator (Frantz et al., 2010). Leaves of

TRSV-infected plants provided with control levels of Si (0.1 mM) contained an average of approximately 280 mg Si per kg dry weight compared to 80 mg per kg in mock-inoculated controls.

17

Fig. 3-2: Si concentration (mg/kg) within virus-infected plants determined by ICP-OES. Plants infected with TRSV (A, C) or TMV (B). Si levels in leaves (A, C) and roots (B) of N. tabacum; white bars, control plants (0.1 mM K2SiO4); black bars, treated with elevated Si (1.0 mM K2SiO4). Bars indicate average, error bars, SEM; letters indicate statistically significant differences as determined by Tukey’s HSD test (P<0.05). Other statistical parameters are given in Supplementary Tables 3.S3-5.

In contrast, N. tabacum root Si levels were dependent upon the supply of

Si but independent of TRSV-infection (Fig. 3.2B). TRSV and mock-inoculated plants supplemented with Si contained about 990 and 1100 mg Si per kg root dry weight, respectively, which were not significantly different. However, root Si levels in plants not supplemented with Si were approximately 120 and 230 mg per kg in TRSV and mock-inoculated control plants, respectively. One might speculate that virus-infected leaves produce a signal that is sent to the roots, either

18

releasing Si from internal stores or inducing an increase in root uptake for transport to the leaves.

To determine if the beneficial effects of Si on TRSV were virus-specific, the effects of this element were tested on TMV infection. Interestingly, no obvious differences were observed in TMV systemic spread or symptom distribution between the control plants and those provided with additional Si (data not shown). Si accumulation in TMV-infected leaves at 18 DPI was entirely dependent upon Si application but independent of viral infection (Fig. 3.2C). The fact that Si did not protect plants against TMV infection suggests that the role of

Si in virus protection is specific to TRSV. Such pathogen selectivity has also been shown for certain fungal infections (Rogers-Gray and Shaw, 2000, 2004).

Our data suggest that leaf Si uptake is part of the responses that tobaccos use as a specific defense mechanism against TRSV but these mechanisms are different from those to defend the plant against TMV. In summary, Si specifically delays

TRSV systemic symptom formation and this response correlates with higher

Si levels in virus-infected leaves.

ACKNOWLEDGMENTS

The authors thank Dr. Gaurav Raikhy (Department of Biological Sciences at the University of Toledo), the University of Toledo Plant Science Research

Center, as well as USDA researchers Douglas Sturtz, Russell Friedrich, and

Alycia Pittenger for their assistance. This work was supported by USDA-ARS

Specific Cooperative Agreement 58-3607-1-193.

19

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Bengsch E, Korte F, Polster J, Schwenk M, Zinkernagel V. Zeit für Natur 1989;

44c:777-80.

Brunt AA, Crabtree K, Dallwitz MJ, Gibbs AJ, Watson L, Zurcher EJ. 1997;

http://biology.anu.edu.au/Groups/MES/vide/.

Chapman SN. In: Foster GD, Taylor SC, editors. Plant Virology Protocols: from

Virus Isolation to Transgenic Resistance. Totowa: Humana Press; 1998, p

123-9.

Dannon EA, Wydra K. Physiol Mol Plant Pathol 2004; 64:233-43.

Datnoff L, Rodrigues FA, Seebold KW. In: Datnoff L, Elmer WH, Huber DM,

editors. Mineral Nutrition and Plant Disease. St. Paul: APS Press; 2007, p

233-46.

Fauteux F, Chain F, Belzile F, Menzies JG, Belanger RR. Proc Natl Acad Sci

USA 2006; 103:17554-9.

Frantz JM, Locke JC Datnoff L, Omer M, Widrig, A, Sturtz, D, Horst L, Krause

CR. Comm Soil Sci and Pl Anal 2008; 39:2734-51.

Frantz JM, Locke JC, Sturtz D, Leisner S. In: Rodriguez F, editor. Silicio na

Agricultura: Anais do V Simposio Brasileiro Sobre Silicio Agricultura.

Minas Gerais: Universidade Federal de Viçosa; 2010, p 111-34.

Li J, Frantz JM, Leisner SM. J Amer Soc Hort Sci, 2008; 133: 670-7.

Liang Y, Si J, Römheld V. New Phytol 2005; 167:797-804.

Marschner H. Mineral Nutrition of Higher Plants. San Diego: Academic Press;

2002.

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R Development Core Team. R: A language and environment for statistical

computing, reference index version 2.2.1. R Foundation for Statistical

Computing, 2005;Vienna, Austria. ISBN 3-900051-07-0, URL

http://www.R-project.org.

Rezaian MA, Francki RIB. Virology 1973; 56: 238-46.

Rodgers-Gray BS, Shaw MS. Plant Pathol 2000; 49:590-9.

Rodgers-Gray BS, Shaw MS. Plant Pathol 2004; 53:733-40.

Sparks AH, Esker PD, Bates M, Dall Acqua W, Guo Z, Segovia V, Silwal SD,

Tolos S, Garrett KA. Ecology and Epidemiology in R: disease progress

over time. The Plant Health Ins 2008; DOI:10.1094/PHI-A-2008-0129-02.

Table 3.S1: Statistical Parameters of TRSV Symptomatic Area. Values were from ANOVA performed using the R program. Statistical Parameters of Symptomatic Area dpi Df Fvalue Pvalue 9 Treatment 1 0.663 0.446 Residuals 6 11 Treatment 1 34.85 0.00105 Residuals 6 13 Treatment 1 6.555 0.04289 Residuals 6 15 Treatment 1 1.4714 0.2707 Residuals 6

Table 3.S2: Statistical Parameters of TRSV AUDPC. Values were from ANOVA performed using the R program. Statistical Parameters of AUDPC Df Fvalue Pvalue AUDPC Treatment 1 12.127 0.013104 Residuals 6

21

Table 3.S3: Statistical Parameters of foliar Si in TRSV infected N. tabacum. Values were from ANOVA performed using the R program. Individual p- values were calculated using Tukey’s HSD post hoc test. Statistical Parameters of Foliar Si in TRSV infected N. tabacum Df Fvalue Pr(>F) Treatment 3 7.2247 0.0164 Residuals 11 Individual P values Mock Si+ 0.7326333 Mock C TRSV C 0.1003034 Mock C TRSV Si+ 0.0019755* Mock C TRSV C 0.5106569 Mock Si+ TRSV Si+ 0.0156328* Mock Si+ TRSV Si+ 0.1580609 TRSV C

Table 3.S4: Statistical Parameters of foliar Si in TMV infected N. tabacum. Values were from ANOVA performed using the R program. Individual p- values were calculated using Tukey’s HSD post hoc test. Statistical Parameters of Foliar Si in TMV infected N. tabacum Df Fvalue Pr(>F) Treatment 3 30.82 3.122e-08 Residuals 23 Individual P values Mock Si+ 0.0000081* Mock C TRSV C 0.5567984 Mock C TRSV Si+ 0.000003* Mock C TRSV C 0.0000970* Mock Si+ TRSV Si+ 0.9681127 Mock Si+ TRSV Si+ 0.0000037* TRSV C

22

Table 3.S5: Statistical Parameters of root Si in TRSV infected N. tabacum. Values were from ANOVA performed using the R program. Individual p- values were calculated using Tukey’s HSD post hoc test. Statistical Parameters of Root Si in TRSV infected N. tabacum Df Fvalue Pr(>F) Treatment 3 60.973 1.554e-07 Residuals 12 Individual P values Mock Si+ 0.0000949* Mock C TRSV C 0.7348685 Mock C TRSV Si+ 0.0000401* Mock C TRSV C 0.000047* Mock Si+ TRSV Si+ 0.0.7207956 Mock Si+ TRSV Si+ 0.0000003* TRSV C

23

Chapter 3

Induced Silicon Accumulation in N. tabacum

3.1 Abstract

Low accumulators have an ability to accumulate higher levels of silicon

(Si) when challenged with abiotic and biotic stress. In addition to showing

Tobacco ringspot virus infection induced Si accumulation in N. tabacum leaves, other studies have shown copper (Cu) induced Si accumulation in snapdragon

(37). Here we studied the effect of Cu and exogenous hormone application on Si accumulation in leaves of N. tabacum. Two Cu levels were independently tested.

At 50 μM Cu, a slight but statistically marginal increase (p-value = 0.055) in Si was observed, while at 75 μM Cu a significant increase of Si in Cu stressed plants was recorded. We also tested the ability of a number of hormones involved in defense and environmental perception to induce foliar Si accumulation. Abscisic acid (ABA), methyl jasmonate and salicylic acid in addition to water were sprayed onto plants. Only ABA treatment significantly decreased leaf Si concentration compared to controls, while the other hormones had no influence on

Si. These data suggest that inducible Si accumulation in N. tabacum leaves is

24

caused by a specific stress defense and ABA may play an antagonistic role in the pathway.

3.2 Introduction

Silicon (Si) has the ability to protect plants against both abiotic and biotic stress, which likely uses multiple pathways, including those involved in both environmental perception and pathogen resistance (38). As shown previously

(39), Tobacco ringspot virus (TRSV) infection increased foliar Si concentrations in tobacco. However, employing a pathosystem to study Stress Induced Si

Accumulation (SISA) resulted in large variability both within and between studies, as seen with N. tabacum-TRSV. Finding a less variable system would allow for an easier approach to determining pathway(s) involved in SISA.

Copper (Cu), an essential micronutrient in plants, causes a number of physiological and chemical changes within a plant in addition to altering the levels of other nutrients. Recent studies with snapdragon hydroponically grown with excessive copper (Cu) have shown a significant increase in foliar Si content compared to controls, suggesting that the plants have SISA in response to Cu stress (37). Interestingly, snapdragon, like tobacco, is considered a low- accumulator. In the same study, the high-accumulator, Zinnia elegans, showed no change in foliar Si content, suggesting SISA is plant specific. SISA was not observed in A. thaliana treated with 30 μM Cu (40).

Toxic levels of Cu also have adverse effects on a number of metabolic processes. Cu has the ability to induce reactive oxygen species in plants (41). At

25

high concentrations, Cu displaces key enzymatic elements rendering enzymes inactive. Cu-induced oxidative damage can activate a number of defense pathways including the conjoined jasmonic acid (JA)/ethylene (Et) pathway (41).

Jasmonic acid (JA), typically in the form of methyl jasmonate (MeJA) is a mobile plant hormone that has been implemented in plant defense (42). While JA plays a role in wound-induced responses and fungal protection, little data is available for its role in virus infection. TMV was proposed to inhibit JA signaling and nicotine production in N. tabacum (43). Upon fungal infection in

Arabidopsis, the JA/Et pathway induced the activation of the defensin, PDF1.2, independent of SA (44, 45). Thus, PDF1.2 has been used as a marker for activation of the JA/Et pathway (46). Recently, in tomato, a significant increase in JA/Et marker genes was observed following infection with Ralstonia solanacearum in Si treated plants, suggesting that a JA-dependent pathway is involved in the resistance (47).

The phytohormone salicylic acid (SA), similar to JA, plays a vital role in plant defense (48-50). SA is produced via the phenylpropanoid pathway and is responsible for the induction of systemic acquired resistance, where leaves distal from the site of inoculation are primed to fight off infection. SA production increases during pathogen infection and travels throughout the plant (51). Not only is the hormone involved in systemic signaling, but there is evidence of its suppression of viral infection. In transgenic tobacco constitutively expressing SA,

Tobacco mosaic virus (TMV) infection was inhibited by both repression of local

26

movement between epidermal cells and suppression of replication in mesophyll cells (52).

A dynamic and intertwined web of signaling between SA, jasmonic acid

JA, and ethylene (Et) takes place to elicit specific defense responses in plants

(53). SA and JA/Et can be both antagonistic and synergistic in amplification of defense responses. In particular, SA production is induced by biotrophic fungi, while JA/Et synthesis are induced by necotrophic fungi, herbivore feeding, and wounding (54).

Abscisic acid (ABA) is a plant hormone involved in developmental, environmental perception and stress pathways (55). ABA suppresses phenylalanine ammonia lyase (PAL) activity in soybeans thereby increasing their susceptibility to the fungal pathogen, Phytophthora (56). This is not too surprising, since some pathogenic fungi have the ability to produce ABA (57).

Nicotiana infected with TMV showed increased levels of ABA (58). ABA does not act alone but also is intertwined with other hormone pathways. In A. thaliana, a MYB-related protein gene, containing ABA responsive elements, was induced through the JA pathway when plants were infected with Botrytis, suggesting that there is both antagonistic and mutual signaling between the hormones (59). The tomato mRNA, Gibberellic acid stimulated transcript 1 (GAST1) was shown to be reduced by ABA, while induced with gibberellic acid (GA) treatment (60), and so serves as a good marker for these hormones.

27

Induced Si accumulation in low accumulators has now been documented with an abiotic stress, Cu, and a biotic stress, TRSV. Since working with a pathosystem has its limitations, one of our goals was to develop a model system to study SISA. Cu treatment of plants takes less time and effort compared to propagation and maintenance of a virus. In these studies, we wanted to determine if Cu and hormone treatment of N. tabacum would result in SISA.

3.3 Materials and Methods

3.3.1 Nutrient Solution

The hydroponic solution was designed by Futong Yu as described in Li et. al (40). Nutrient solution stock solution was made 1000 times the concentration and diluted to the final concentrations indicated below. K2SiO3·4H2O (0.1 mM

Si, 0.2 mM KOH), KNO3 (1.25mM), KH2PO4 (0.5 mM), Ca(NO3)2·4H2O (0.5 mM), MgSO4·7H2O (0.5 mM), NH4NO3 (0.1 mM), ZnSO4·7H2O (0.5μM),

CuSO4·5H2O (0.12 μM), MnSO4·H2O (5 μM), KCl (4 μM), (NH4)6Mo7O24·4H2O

(0.08 μM), Co(NO3)2 (0.01 μM), NiSO4 (0.1 μM), Fe-EDTA (50 μM), H3BO3 (30

μM), MES (0.1 mM).

3.3.2 Hydroponic growth of plants

Eppendorf tubes (1.5 ml) with the bottoms removed were filled with black packaging foam and soaked in nutrient solution. Tubes were then floated in nutrient solution. One seed was placed onto the foam in each tube and they were placed directly into the growth chamber. Once roots were present, typically 2-3 weeks, tubes were transferred to 4 L buckets connected to a pump, using tubing containing a porous rod at the end submerged in the solution allowing for 28

aeration. The nutrient solution was maintained at pH 6.3 and changed every seven days.

3.3.3 Cu-Induced Si Accumulation

In the initial study, N. tabacum were sown hydroponically with 0 mM Si and 0.1 μM Cu (Control). Once plants reached the 4-6 true leaf stage, they were transplanted to 4 L buckets containing nutrient solution with three plants per bucket. After one week, nutrient solution were changed to 0 mM Si/ 0.1 μM Cu

(Control), 2 mM Si/ 0.1 μM Cu (Si+), 0 mM Si/ 50 μM Cu (Cu+) or 2 mM Si/ 50

μM Cu (Si+Cu+). Nutrient solution was changed every seven to eight days for three weeks. Leaves were harvested on the 21st day, dried, and then ground for

ICP-OES. In a second set of experiments, the Cu concentration was increased to

75 μM Cu, while all other procedures remained constant.

3.3.4 Hormone Study

N. tabacum were treated with 50 μM MeJA, 25 μM ABA, 50 μM SA, H2O or EtOH. Since MeJA and EtOH are volatile, 500 μl of the appropriate solution were placed in the top of a 15 mL culture tube lid and the buckets were encased in plastic wrap to concentrate the vapors around the plants. Buckets for different hormone treatment were kept in separate growth chambers with all ventilation accesses closed to ensure no cross-contamination of the treatments (61). Growth conditions remained constant at 16:8 light:dark 65 μmol·m-2s-1 at 25°C. ABA, SA and H2O were sprayed onto plants until runoff occurred (typically 10 sprays for the first two weeks and 15 sprays the last week due to the increase in plant size).

29

Plants were sprayed every two days for three weeks. Following treatment, leaves were collected, weighed, dried, then ground for ICP-OES. A small amount of tissue from a mature, expanded green leaf was collected from each plant, flash frozen, and stored at -80 °C until RNA isolation was performed.

3.4 Results

3.4.1 Cu-Induced Si Accumulation

N. tabacum were grown hydroponically with either 0 mM Si/ 0.1 μM Cu

(control), 0 mM Si/ 50 μM Cu (Cu+), 2 mM Si/ 0.1μM Cu (Si+) or 2 mM Si/ 50

μM Cu (Si+Cu+) for three weeks. Plants grown under Cu+ and Si+Cu+ conditions showed slight chlorosis and stunting with little difference in symptoms

(Fig. 3-1). Si+ and control treated plants exhibited no chlorosis and no significant difference in development.

30

Figure 3-1: Symptoms on N. tabacum treated with 50 μM Cu. Plants were treated for three weeks with 2 mM Si/ 50 μM Cu (Si+Cu+), 0 mM Si/ 50 μM Cu (Cu+), 2 mM Si/ 0.1 μM Cu (Si+) or 0 mM Si/ 0.1 μM Cu (Control) prior to ICP-OES analysis. The arrows represent stunted tobacco plants that were pooled with other tissue that resulted in higher Si values determine.

31

Si accumulation in N. tabacum leaves was determined by ICP-OES performed by the USDA-ARS at the University of Toledo. Total foliar Si in N. tabacum was significantly higher in two of the 12 Si+Cu+ plants compared to Si+

(Fig. 3-2). The higher values were from tissue samples that had been pooled together, which included stunted plants. However, when taken together, the difference between Si+Cu+ and Si+ had a p-value equal to 0.055, based on

Tukey’s HSD, slightly above the 0.05 cut-off value. Cu+ and control plants both had similar Si concentrations that were significantly lower compared to Si+ and

Si+Cu+ plants, with a number of plants showing undetectable Si concentrations.

32

A. 2500.00

2000.00

1500.00

1000.00 mg kg Si/ leafmg tissue

500.00

0.00 Si+Cu+ Cu+ Si+ Control

B. 1400 a

1200

1000

800 a* 600

400 mg kg Si/ drymg tissue leaf

200 b b 0 Si+Cu+ Cu+ Si+ Control

Figure 3-2: Total Si in N. tabacum treated with (A) 50 μM Cu/ 2 mM Si (Si+Cu+, white), 50 μM Cu/ 0 mM Si (Cu+, black), 0.1 μM Cu/ 2 mM Si (Si+, light gray) or 0.1 μM Cu/ 0 mM Si (control, dark gray). All individual plants and (B) average Si content ± SEM. Different letters represent a p-value 0.001. The * represents a difference between Si+Cu+ and Si+ that was just beyond a significant difference (p- value= 0.055).

33

Since only a few plants exhibited Cu toxicity symptoms at 50 μM concentration, and those that did had higher Si concentrations, Cu levels were increased to 75 μM. In addition to increasing Cu levels, 0 mM Si treatments were not conducted, since many of the plants from the previous study at 0 mM Si showed non-detectable levels of Si regardless of Cu treatment. Higher Cu levels resulted in more plants developing chlorosis (Fig. 3-3). Interestingly, none of the plants exhibited the extreme stunting observed during the 50 μM trial. As observed in other Cu toxicity studies, roots of Cu+ treated plants were compacted and brown compared to controls (Fig. 3-4).

Figure 3-3: N. tabacum treated with 2 mM Si and either 0.1 μM Cu (Si+) or 75 μM Cu (Si+Cu+) for three weeks.

34

Figure 3-4: N. tabacum roots treated with 0.1 μM Cu (Si+) or 75 μM Cu (Si+Cu+)

Leaves again were collected and dried for total Si analysis. The Si levels were both consistently and significantly higher in Si+Cu+ plants compared to Si+ with a p-value of 4.9X10-12 (Fig. 3-5). Interestingly, the variability of foliar Si between plants was much smaller compared to TRSV infected tobacco, suggesting this system is better suited to begin looking at molecular aspects of

SISA.

35

A. 1400.00

1200.00

1000.00

800.00

600.00

mg kg Si/ leafmg tissue 400.00

200.00

0.00 Si+Cu+ Si+

B. 1200 *

1000

800

600

400 mg kg Si/ drymg tissue leaf 200

0 Si+Cu+ Si+

Figure 3-5: Total foliar Si in N. tabacum treated with 2 mM Si and either 75 μM Cu (Si+Cu+, gray) or 0.1 μM Cu (Si+, black) for three weeks of (A) individual plants or (B) average ± SEM. The * represents a significant difference with pV-value = 4.852X10-12. To ensure the increase in Si concentration among Cu treatments was not due to an increase in membrane permeability due to the toxic effects of heavy metal, total elemental analysis from collected tissue was conducted. Ca and K were significantly lower in Si+Cu+ treatment compared to Cu+ (Table 3.1). B

36

and Cu were the only elements that were significantly higher in Si+Cu+ treated tobacco, while Mg, Mn and Mo were not significantly different between the two treatments. In fact, a number of the elements were significantly higher in the

Si+Cu+ plants, compared to Si+, including P, S, Fe and Zn. Most interestingly, Fe was almost eight fold higher in the Cu+ compared to Si+Cu+ treated plants.

Table 3.1: Total elemental analysis of N. tabacum plants treated with 2 mM Si and 75 μM Cu (Cu+) or 0.1 μM Cu (C) with SEM in parentheses. Different letters represent a significant difference for each individual element based on Tukey’s HSD analysis with a p-value 0.05.

P K Ca Mg S % dry weight 0.59 5.91 1.72 1.09 0.36 Cu+ (0.02) (0.26) (0.10) (0.06) (0.02) b b b a b 1.26 9.41 2.89 1.01 0.55 C (0.09) (0.38) (0.10) (0.04) (0.01) a a a a a B Cu Fe Mn Mo Zn mg/kg 56.70 49.37 96.32 226.68 11.45 45.89 Cu+ (2.88) (2.53) (8.13) (15.60) (0.62) (6.90) a a b a a a 46.12 15.63 765.61 261.93 17.33 52.26 C (1.38) (0.50) (87.78) (19.05) (0.52) (4.68) b b a a a a

3.4.2 Hormone Study

It is apparent, based on our TRSV data, that a mobile signal is involved in

SISA. To test the effect of hormones on Si accumulation in tobacco, ABA, MeJA and SA were exogenously applied to tobacco grown in separate growth chambers.

Since MeJA is a volatile hormone, in addition to keeping treatments in separate growth chambers, buckets were surrounded with plastic wrap to ensure the 37

hormone did not dissipate before it could be taken up by the plant. As a control,

EtOH was used and buckets were similarly wrapped with the plastic. Plants treated with MeJA and EtOH appeared shorter compared to controls, however, there was no significant difference in the weights compared to control or SA treated plants (Fig. 3-6). ABA treated N. tabacum were significantly larger than the other four treatments. MeJA treated plants were extremely chlorotic compared to EtOH treated N. tabacum. ABA and SA plants appeared similar to

H2O sprayed plants with the exception of the larger size of ABA treated plants.

Interestingly, when collecting ABA and MeJA treated plants tissue, the leaves had a brittle feel compared to SA, EtOH and H2O.

16 b 14

12

10 a 8 a 6 fresh weightfresh (g) a a 4

2

0 Control SA ABA EtOH MeJA

Figure 3-6: Fresh weight (g) ±SEM of N. tabacum sprayed with H2O (Control), SA, ABA or exposed to EtOH or MeJA for three weeks. Different letters represent a significant difference based on Tukey’s HSD analysis with a p-value 0.05.

38

No difference in foliar Si accumulation was observed between MeJA and

EtOH (Fig. 3-7). ABA treated plants showed significantly lower Si concentrations in their leaves compared to SA and H2O. The level of Si in H2O sprayed leaves was higher than expected for non-challenged N. tabacum.

Previous studies with SA resulted in inconsistent Si accumulation with SA treatment, compared to controls. Interestingly, in one study, using both dH2O- sprayed and non-sprayed N. tabacum, dH2O-sprayed plants showed significantly higher concentrations of Si in leaves compared to non-sprayed plants (Fig. 3-8).

This suggests that in N. tabacum, H2O application to leaves may significantly increase foliar Si, independent of any other treatment.

A. 700 a a 600

500

400

300 mg kg Si/ tissue mg 200

100

0 EtOH MeJA

Figure 3-7: Si accumulation in EtOH and MeJA sprayed N. tabacum. Same letters represent no statistical difference based on ANOVA analysis.

39

B. 800 a a 700

600

b 500

400

300 mg kg Si/ tissue mg

200

100

0 Control SA ABA

Figure 3-8: N. tabacum foliar Si following hormone application. Total Si ±SEM for N. tabacum exposed to EtOH or MeJA (A) or sprayed with H2O, SA or ABA (B) for three weeks. Different letters represent a significant difference with p-value < 0.05 based on Tukey’s HSD test. Hydroponic solution contained 2 mM Si.

250

a

200

b

150

100 mg kg Si/ foliartissue mg 50

0 Foliar H2O Control

Figure 3-9: Foliar Si in N. tabacum sprayed with dH2O. Total Si in N. tabacum sprayed with dH2O (Foliar H2O) or no treatment (Control) ±SEM for three weeks. Different letter represent a significant difference with a p-value < 0.05 based on Tukey’s HSD test. Hydroponic solution contained 1 mM Si.

40

To ensure that the decrease in Si for ABA treatment was not due to a decrease in membrane permeability, a total elemental analysis was also conducted on foliar tobacco tissue. K and Ca levels for ABA were significantly higher compared to control treated plants (Table 3.2). A significant increase in Mg, S

Cu, Mo and Zn concentrations were also seen for ABA compared to control treated plants. Interestingly, exogenous SA treatment resulted in tobacco plants having significantly higher levels of Fe, compared to control and ABA and control treatments. An increase was also seen in K, Ca, Cu, and Mn, while Mg, S,

Mo and Zn concentrations were not significantly altered with SA treatment compared to control. P concentrations did not change with ABA or SA treatment compared to controls.

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Table 3.2: Total elemental analysis of ABA and SA treated N. tabacum. Plants were sprayed with water (Control), 50 μM SA, or 10μM ABA and total elemental analysis was performed by ICP-OES. Element concentrations as % dry weight for the macronutrients and mg of the element/kg of dry tissue for micronutrients are reported with SEM in parentheses. Different letters represent statistically significant differences of p-value 0.05 based on Tukey’s HSD analysis.

P K Ca Mg S % dry weight 0.87 7.24 1.64 0.85 0.53 Control (0.36) (1.87) (0.36) (0.18) (0.11) a a a a a 0.81 10.14 2.41 1.06 0.49 SA (0.05) (0.32) (0.14) (0.05) (0.03) a b b a a 0.97 9.25 2.71 1.12 0.63 ABA (0.04) (0.44) (0.14) (0.06) (0.03) a b c b b Cu Fe Mn Mo Zn mg/kg 6.61 243.59 113.56 15.47 16.89 Control (0.87) (130.64) (24.10) (3.36) (6.60) a a a a a 14.37 876.94 220.46 16.44 20.94 SA (0.41) (192.2) (37.17) (0.86) (3.03) b b b a a,b 20.94 144.55 112.30 20.31 22.18 ABA (0.56) (20.08) (21.29) (1.04) (3.21) b a a b b

Total elemental analysis was also done with EtOH and MeJA treated tobacco. MeJA treated plants had significantly higher concentrations of S and Mo and significantly lower levels of P, K and Fe compared to EtOH treatment (Table

3.3). Ca, Mg, Cu, Mn and Zn were not significantly different between the two treatments.

42

Table 3.3: Total elemental analysis of MeJA treated N. tabacum. Plants were treated with EtOH or 50 μM MeJA and total elemental analysis was performed using ICP-OES. Element concentrations as % dry weight for macronutrients and mg of the element/kg of dry tissue for micronutrients are reported with SEM in parentheses. ANOVA analysis was performed and p- value 0.05; 0.01; 0.005; 0.001; 0.00004 represents *, *2, *3, *4, and *5, respectively.

P*5 K*5 Ca Mg S*3 % dry weight 0.89 10.21 2.66 1.05 0.59 EtOH (0.13) (0.57) (0.44) (0.06) (0.07) 0.55 7.66 2.65 0.99 0.74 MeJA (0.3) (0.69) (0.15) (0.07) (0.07) Cu Fe*4 Mn Mo*2 Zn mg of the element/ kg dry tissue 11.64 535.52 208.40 19.22 21.66 EtOH (4.11) (182.27) (54.79) (1.31) (5.44) 15.49 219.26 183.57 23.20 26.63 MeJA (3.24) (65.83) (26.36) (2.70) (3.11)

3.5 Discussion

Si enhances resistance to a number of stressors (38). However, in order for a plant to reap the benefits of this element, they need to first have the ability to accumulate Si to some degree in the stressed tissue. In the previous chapter, we concluded that TRSV has the ability to induce Si accumulation in the inducible accumulator, N. tabacum (39). In combination of having to deal with problems of a variable virus population and distribution, propagation of a virus, TMV contamination, and the overall variability in Si accumulation of individual plants infected with TRSV, this system is less than perfect to begin to understand the underlying mechanisms of SISA as a defense mechanism in plants.

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Work on snapdragon that also resulted in an inducible Si accumulation in the classically defined low-accumulator prompted the hypothesis that N. tabacum may also possess this response. In contrast to dealing with the highly variable

TRSV system, metal toxicity is perceived more uniformly by the plants. At a lower Cu concentration, a marginal increase (P=0.055) in Si accumulation occurred, similar to previous work with A. thaliana (40). When Cu was increased to 75 μM, a consistent and significant increase in foliar Si concentration between

Cu+ and control plants was observed. With numerous studies done to date examining Si accumulation, the Cu data has one of the least variable Si levels between treatments, making this an ideal system to study the mechanisms behind

SISA. Since Ca or K levels did not increase, but significantly decreased, in contrast to Si levels, our data suggest that Si accumulation was not due to an overall increase in membrane permeability, which supports the idea that Cu- induced Si accumulation in N. tabacum is a regulated process.

The significant decrease in Fe for Cu+ treated plants could be a result from the formation of Fe plaques on the root surface. In some rice genotypes, Cu increased the Fe content of the plaques (62). This would also explain the brown roots present for Cu+ treated plants compared to controls. Along with Fe, the fact that other elements are changing with hormone treatment compared to controls suggests the hormones are having an effect on the plants and thus are being perceived.

In addition to creating a model system to study SISA, we also found that hormones may play a role in the event as well. JA and SA are two important 44

hormones implicated in a number of defense mechanisms (cite). However, no difference in foliar Si levels was observed in MeJA- and SA-treated N. tabacum compared to water sprayed plants. Consistent with our results, salt stress in soybeans supplemented with Si showed reduced stress damage with no significant difference in endogenous JA or SA concentrations (63). Interestingly, ABA significantly reduced Si concentration in N. tabacum. Some fungal pathogens have the ability to produce ABA, which could help them survive in a low- accumulator (57). Since TMV also has the ability to increase endogenous ABA, this could help explain why a significant increase in Si was observed in TRSV- infected and not in TMV-infect N. tabacum (39).

How might ABA reduce foliar Si concentratoin? Si transport in plants falls into two categories; active and passive transport (64). Passive transport of Si in plants is regulated by a number of aquaporins belonging to the Nodulin-26 like major intrinsic protein family (65). In experiments testing aquaporin gating through mechanical stimulation, Wan et. al speculates that extracellular ABA has the ability to bind to the extracellular face of aquaporins, blocking the pore while binding of the hormone to the cytosolic face would maintain the pore in an open position (66). If true, this could account for the reduction in foliar Si. Since ABA has the ability to influence stomatal closure (67), one might argue that Si diffusion through the transpiration stream is inhibited leading to lower foliar Si; however, since other elemental levels do not significantly decrease in ABA treatment, we conclude that disruption of passive diffusion is not occurring in the plants.

45

The data suggesting application of water alone to the leaf surface could mean defense mechanisms involved in Si accumulation are similar to those involved in perception of leaf wetness (68). In the study, they found that the five calmodulin-like genes induced after GA application were similarly induced after spraying plants with water, suggesting that the perception of water possibly through touch leads to induction of pathway(s) that parallels GA-induced pathways. The ability to send Si into leaves in the presence of moisture, such as rain, could be a defense mechanism to protect against a number of pathogens present in rain droplets, or those that thrive on damp vegetation.

Most molecular aspects of the Si research have been done in high accumulators, leaving a sizable gap in available data for low accumulators. SISA in N. tabacum induced by Cu toxicity shows potential for a powerful model system to further study how Si leads to stress resistance in low accumulators. In addition, exogenous application of ABA, a hormone involved in environmental perception, significantly reduces foliar Si levels. Since pathogens have the ability to produce or induce ABA as is the case for pathogenic fungi (67) and TMV (39), respectively, this could explain why Si accumulation is not seen in all pathosystems, resulting in no protection from elevated Si application.

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Chapter 4

Silicon Regulation of Aquaporin Gene Expression in N. tabacum

4.1 Abstract

Movement of Si from the environment into roots then subsequently into shoots presumably is regulated by a number of factors, including Si transporters.

Si transporters are typically divided into two major classes; active and passive.

All the characterized passive Si transporters to date have been found in high accumulators and are Nodulin-26 like intrinsic protein (NIP) family members.

Through computational analysis we have identified a putative NIP that contains the classical Si transporter residues, along with identifying over 60 other undefined aquaporin sequences. Expression of ntNIP3;1 and a root-specific tonoplast intrinsic protein (ntRT-TIP1) was decreased in plants treated with Si for seven days, in a manner similar to the rice Lsi1 Si transporter. This is the first indication that a major intrinsic protein outside of the NIP subfamily is Si- regulated. In addition to the identification of a putative Si transporter in N. tabacum, a number of other sequences were identified in a number of

47

dicotyledonous plants. This suggests that even low accumulators have the ability to selectively accumulate Si from its environment.

4.2 Introduction

Si is beneficial against a number of plant stresses (38). For the element to alleviate stress, it first needs to be absorbed by the plant from its environment. Si transporters were initially identified and characterized in rice that had low silicon content and were thus named Lsi for “low silicon” (69). There are two identified types of Si transporter proteins, an active efflux transporter and a passive aquaporin that is a member of a larger family of Nodulin-26 like intrinsic proteins

(NIPs). Here we focused our efforts on understanding the use of aquaporins as Si transporters.

NIP family members contain six-transmembrane helices, two conserved

NPA (asparagine-proline-alanine) motifs and an aromatic/arginine (ar/R) selectivity filter (Fig. 4-1) (70). These polypeptides form a homo- or heterotetramer with other aquaporin subunits to create a functional protein. The ar/R amino acid residues function to provide specificity to the solutes transported through the channel. Hence, the motifs can be used to distinguish between members of NIP subgroup 1 (W-V(I)-A-R) and subgroup 2 (A-I(V)-A-R).

Subgroup 1 NIPs are considered to be general aquaporins, while subgroup 2 members are more selective to their particular substrate. Lsi1 and Lsi6 are classified as NIP group 2 proteins. However, Mitani et. al (71) argued that Lsi1 should be placed in a third subgroup since its ar/R selectivity filter residues, G-S-

G-R, are unique. Xenopus oocytes microinjected with the rice Lsi1 RNA 48

acquired higher amounts of Si compared to water injected controls. Interestingly,

Lsi1 mRNA is Si regulated (69). Lsi1 expression decreases over time in rice supplemented with Si, while control plants sustained consistent expression levels.

Expression of Lsi1GFP-fusion proteins was observed in main and lateral roots but not root hairs (69). Homologs in barley, corn, and, most recently, pumpkin, have also been identified (72-74). Lsi1, 2 and 6 are the three major transporters in rice involved in Si transport into the roots, xylem, and leaves, respectively (14, 75,

76).

Figure 4-1: Schematic for secondary structure of the monomeric subunit of tomato NIP2;1. The protein contains the six transmembrane helices. A. Illustrates how loop B and E fold inward where the NPA motif helps restrict pore size. B. Shows the secondary structure of the pore having cytosolic residues in both the loops and the N- and C- terminal in red, while the extracellular loops are in gray. Loop B and E contain the conserved NPA motif (yellow), while the selectivity filter residues (green) are contained in helix 2 (H2), helix 5 (H5) and loop E (LE1 and LE2).

49

A. thaliana do not have any identified NIPs containing the G-S-G-R selectivity filter indicative of Si transporters in other plant species (77).

Transporter studies were performed to using the rice osLsi1 Si transporter and the

A. thaliana atNIP5;1 boric acid transporter to determine if mutating the ar/R residues would change Si transport capabilities (65). When the H5 residue in osLsi1 was altered from S to I, a significant reduction in Si transport was observed. However, in atNIP5;1 when the H5 I residue was mutated to S, no Si transport was observed, indicating that other residues in addition to the ar/R selectivity filter play a role in excluding Si transport in A. thaliana. Over expression of the wheat Si transporter under the regulation of a constitutive promoter in A. thaliana plants not only significantly increased Si content, but also negatively impacted growth of the plants resulting in necrotic lesions on leaves

(78). When the transporter expression was under the control of a root-specific promoter, a significant increase in shoot Si content was still observed with a reduction in necrotic lesions.

Tonoplast intrinsic proteins (TIP) could be important for Si transport since

Si is stored in vacuoles. TIPs have structures similar to NIPs and are involved in the transport of materials into the vacuole (79). TIPs have an ar/R selectivity filter amino acid sequence H(T/Q)-I (M/T/V/S)-A (G)-V(R) (80). Two seperate

N. tabacum root specific TIP gene products (ntRT-TIP 1&2) are nearly identical, differing at only two amino acid positions (residues 3 and 140) (81). The ntRT-

TIP1 is expressed in the meristematic and immature central cylinder regions of 50

the root (81). Expression of TobRB7, here referred to as ntRT-TIP, was induced upon root-knot nematode feeding in tobacco roots (82). Upon infection, expression patterns of the protein are also altered, in that β-galactosidase activity was observed in mature portions of the roots containing the nematode.

Interestingly, expression is not induced by the Tobacco cyst nematode, NaCl, cytokines, auxin or wounding. It is important to keep in mind that even though the proteins are referred to as TIPs, no studies on their intracellular localization have been reported. The N. tabacum TIPa was identified from tonoplast enriched

N. tabacum suspension culture fractions (83).

NIPs and TIPs are not the only major intrinsic proteins (MIPs) found in higher plants (84). There are five other distinct MIP families, including GlpF-like intrinsic proteins (GIPs), plasma membrane intrinsic proteins (PIPs), small basic intrinsic proteins (SIPs) and the more recently identified hybrid intrinsic proteins

(HIPs) and uncategorized X intrinsic proteins (XIPs) (85). Mosses contain all seven types of MIPs, while GIPs and HIPs were apparently lost during the evolution of higher plants, and XIPs were lost during the evolution between monocots and dicots. The A. thaliana genome encodes 35 aquaporin genes (9

NIPs, 10 TIPs, 13 PIPs, and 3 SIPs), but no predicted XIP proteins have been detected (86). More recently through computational analysis, 37 aquaporins have been identified in S. lycopersicum (6 NIPs, 9 TIPs, 18 PIPs, 3 SIPs and 1 XIP)

(87).

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Table 4.1: MIP ar/R selectivity filter amino acid residues. H2 (helical 2), H5 (helical 5), LE1 (loop E residue 1) and LE2(loop E residue 2) are the four amino acid residues that make up MIP selectivity filters. The characterized Si transporters belong to the NIP III subgroup (red).

Aquaporin H2 H5 LE1 LE2 PIP1 F H T R PIP2 NIP I W I/V A R NIP II A I/V A/G R NIP III G S G R TIP I H I A V TIP IIA H I G R TIP IIB H I A R TIP III N V G C XIP A T A R SIP I T F/V/I P I SIP II S H G A

XIPs are a unique group of aquaporins found in dicots and lower plants that contain four subgroups dependent on the ar/R selectivity filter (86). XIP genes in Solanaceae, including N. tabacum and S. lycopersicum, have been identified through expressed sequence tags (EST) searches (88). A subgroup I gene was identified producing two splice variants (XIP1:1α, XIP1:1β) in both tobacco and tomato. The N. tabacum XIP1:1 protein was localized to the plasma membrane in the roots, shoots, leaves, and inflorescence. Beta–galactosidase

(GUS) driven by the expression of NtXIP1:1 promoter was absent in all vascular tissue of the plants. In the roots, GUS expression was detected in the mature

(upper) sections along with primary and lateral roots but absent from the elongation zone. In cross sections of the roots, expression was detected in the cortex. Expression was observed in the epidermal and sub-epidermal cells in both 52

stems and leaves in addition to leaf guard cells. Flowers expressed the protein in the upper portions of the petals and the surface of the peduncle. When expressed in xenopus and yeast, NtXIP1:1 was shown to transport glycerol and urea, as well as increasing yeast sensitivity to boric acid and H2O2, respectively. Most interestingly, NtXIP1:1 was not shown to transport water, which is chemically similar to H2O2. H2O2 is an important signaling molecule in ROS mediated defense responses (89).

Gating of aquaporins involves a number of processes. The NPA motif contained in loops B and E fold toward the interior of the pore restricting the solute movement based on size (Fig. 4-2). In addition to the NPA motif, key amino acid residues within the ar/R filter and possibly others, contribute to the solute specificity of the pore. A third tier to aquaporin gating is conformational changes effected through phosphorylation of conserved serine residues located on the N- and C- terminal.

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Figure 4-2: Subunit of MIP relative to the cellular membrane. The figure was adapted from Forrest and Bhave (80). The red loops B and E contain the NPA motifs that fold into the pore and are involved in exclusion of larger residues.

Hormones play a role in expression and aquaporin activity. Abscisic acid

(ABA) is an important plant hormone involved in environmental perception (90).

Application of exogenous ABA to maize roots resulted in significant reduction of

PIPs (91). ABA has also been shown to alter phosphorylation of certain proteins, including decreasing phosphorylation of a subset of A. thaliana PIP2s (92).

Phosphorylation of aquaporins leads to an open configuration allowing for movement of solutes, if ABA reduces phosphorylation of MIPs, this could lead to a reduction in solute transport. In A. thaliana, ABA application only effects certain PIPs with some specificity between roots and leaves, suggesting that these

MIPs are regulated by both ABA-dependent and –independent pathways (93).

Si transport is currently being studied in high accumulators, which only accounts for a limited number of plants. The majority of dicots are intermediate

54

to low Si accumulators that may have different mechanisms to uptake and accumulate or exclude Si, so the need to study movement of the element in low accumulators is crucial. N. tabacum is an excellent model plant for the study due to the recent release of expressed sequence tag (EST) data in combination with the already available mutants and experimental analysis in a number of defense and stress pathways.

4.3 Materials and Methods

4.3.1 Websites used for Bioinformatic Analyses

Table 4.2: Databases used for Computational Analyses. Tool web address Angiosperm http://www.mobot.org/mobot/research/apweb/ Phylogeny BLAST search http://blast.ncbi.nlm.nih.gov/Blast.cgi ClustalW http://www.genome.jp/tools/clustalw/ EXPASY translate http://web.expasy.org/translate/ Primer3 http://biotools.umassmed.edu/bioapps/primer3_www.cgi Solanaceae genomics resource (Michigan http://solanaceae.plantbiology.msu.edu/species State University)

4.3.2 MSA and construction of phylogenetic trees

Some analyses were performed using free bioinformatic programs available from Kyoto University Bioinformatics Center (Table 4.2). FASTA sequences were saved in a notepad program. Using the ClustalW program (Table

4.2), the sequences loaded and a multiple sequence alignment (MSA) was performed and a phylogenetic tree constructed, using the default settings.

55

For DNA star, sequences were first saved as single files using EditSeq

(Lasergene 9). The individual sequences were then added to the MegAlign program and an MSA using CLUSTALW was performed. The order of the sequences was then changed to reflect the order in the phylogenetic tree and the alignment was repeated. The phylogenetic tree was constructed by the MegAlign program using default settings.

4.3.3 Real Time PCR

RNA was isolated using the RNeasy Kit (Qiagen, Madison WI). Briefly, roots were ground in liquid nitrogen, then transferred to a 1.5 ml Eppendorf tube.

Buffer RLT with β-ME (450 μl) was then added and the tube was vortexed and incubated at room temperature while the remaining tissue was homogenized.

Eight reactions were done at a time to ensure that the RNA extraction procedure would take less than an hour. All following steps were carried out at room temperature. Once all samples were ground, the homogenate was transferred to the supplied tubes, centrifuged for 2 min at 13,000 rpm and the supernatant was transferred to a new Eppendorf tube. Room-temperature ethanol (250 μl) was added to each tube, and the contents were pippetted up and down three times to mix and transferred to the second tube provided by the kit. Extracts were allowed to incubate at room temperature for 2 min, then centrifuged for 15 sec at 13,000 rpm. The flow through was discarded and 700 μl RW1 was added to each tube, and centrifuged for another 15 sec. The flow through was discarded and 500 μl

RPE with EtOH was added to each tube and centrifuged for 15 sec. The flow through was discarded and another 500 μl RPE with EtOH was added to each tube

56

and centrifuged at 13,000 rpm for 2 min. A clean collection tube was then placed under the column and they were centrifuged for an additional two min to ensure the column was dry. The columns were then placed in clean Eppendorf tubes and

40 μl of nuclease free water was added. The columns incubated at room temperature for 5 min and were centrifuged for one min. An additional 30 μl of nuclease free water was added to each column and centrifuged for one min. The

RNA was then either placed in the -80°C freezer prior to use for subsequent analyses. Prior to Real-Time PCR, RNA concentrations were determined using the nanodrop spectrophotometer.

Real-Time PCR was performed following the protocol from iSCRIPT one- step (Promega). Briefly, 25μl reactions were set up for each individual target gene. A master mix was made containing of 10 μl H2O, 12.5 μl 2X SYBR green,

1 μl 2X10-5M forward primer, 1 μl 2X10-5M reverse primer and 1 μl reverse transcriptase per reaction. To the appropriate well, 22.9 μl of the mix and 2.1 μl of RNA were added. The wells were then covered and briefly centrifuged to remove any bubbles. The PCR reaction was done with a Bio-Rad CFX96 thermocycler using the suggested protocol. Incubation at 50°C for 10 min was followed by a denaturation step at 95C for 5 min. Then, 39 cycles followed with a 10 sec 95°C and 30 sec 60°C, followed by a plate read. At the end of the 39th cycle, a melt curve was performed by increasing the temperature from 55°C to

95°C at 0.5°C increments with a plate read every 5 sec. Data analysis was performed by the Bio-Rad Manager Program.

57

Table 4.3: Primers used for Real-Time PCR. Primer names followed by * were taken from Mahdieh et. al. (94). The primer positions refer to the nucleotide position indicates the base pair location of the primer sequence from the respective NCBI number.

primer Primer Name Gene NCBI number position 5’-3’ Sequence s (bp) CAA CCG GTA Act-3F Actin NM_112764.3 699-723 TTG TGC TGG ATT CTG G GCA AGG TCA Act-3R Actin NM_112764.3 778-798 AGA CGG AGG ATG TTT CCT CAA ntAQP1RT-F* Aquaporin AJ0014016.1 938-961 GAA GCC TTA

ATC TTT TTG AAC ACA 1031- ntAQP1RT-R* Aquaporin AJ0014016.1 AGA AAA TCC 1053 ACA TT ntNIP3;1RT- putative CGG GGG AGC DV160518.1 5-23 1F NIP3;1 TAA AAG TTC A TCT CCA TTG ntNIP3;1RT- putative DV160518.1 202-176 ATG AAC TAG 1R NIP3;1 ATA GAC AAA CTG GGT TGG ntPIP1;1RT-F* PIP1;1 AF440271.1 849-867 ACC ATT CAT T TTG ACA GAC ntPIP1;1RT- PIP1;1 AF440271.1 977-998 ACC AGC AAG R* AGA 1017- TTG CAA CCA ntPIP2;1RT-F* PIP2;1 AF440272.1 1035 CTA CCA GAA A AAT TTG GAC ntPIP2;1RT- 1071- PIP2;1 AF440272.1 AGA TGA ATG R* 1085 CAA TTG TTT GTT ntRT-TIP1-6F RT-TIP1 X54855.1 146-165 GGG GTT TCC AT AAC TCC ATG ntRT-TIP1-6R RT-TIP1 X54855.1 446-465 GGT TGG AAC AG TACTCCCTCCGG ntTIP1;1-2F TIP1;1 AB371711.1 236-255 CCTAATCT GGAAGCAAGCA ntTIP1;1-2R TIP1;1 AB371711.1 435-414 ACAGTGGAT

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Table 4.3: continued CCA TGA AAA ntXIP1-2F XIP1 HM475295.1 743-762 AGG GCT ACG CC CTT GTA TCC ntXIP1-2R XIP1 HM475295.1 913-930 ATC TGC ATG

4.4 Results

4.4.1 Identification of putative N. tabacum MIPs

While a number of studies have been carried out on the ability of Si transporters to transport the element and other solutes, such as arsenic (95), no reports have examined whether other aquaporins are regulated by Si treatment.

Most recently, 37 tomato aquaporins were identified through bioinformatic approaches (87). One identified aquaporin sequence, slNIP2:1 (AK321561.1), contains the selectivity filter residues identical to known Si transporters (65).

Using the tomato sequence along with other known Si transporters in rice, wheat, barley and pumpkin, an MSA was performed. From that, a conserved region containing eight identical nucleotides was identified and subsequently used to search the Ntaseq data file from the sequenced tobacco database (Table 4.2).

This search identified Nta#S33531779 (DV160518.1). A discontiguous megaBLAST search of this sequence resulted with 71-62% identity to known Si transporters in Cucumis maschata, Zea mays and Oryza sativa. Unfortunately, the

EST was lacking the 3’ end of the mRNA sequence.

An MSA including the characterized and putative Si transporters (castor oil, cumber, field pumpkin, tomato, and the recently identified tobacco) was

59

performed using MegAlign (Fig. 4-3). Construction of a phylogenetic tree showed two clades that grouped the mono- and dicot putative NIP proteins.

While the 3’ end of the predicted tobacco NIP sequence was missing, the remaining residues did, however, contain the conserved amino acid sequences in the ar/R selectivity filter indicative of Si transporters, and thus we refer to the sequence as ntNIP3;1, since it is the first identified subgroup three member of the family in N. tabacum.

Table 4.4: Accession number of Si transporters used for alignments. Putative Si transporters are in grey.

Plant Short Name NCBI number Rice (Oryza sativa) osLsi1 BAE92561.1 Corn (Zea mays) zmLsi1 NP_001105637.1 Barley (Hordeum vulgare) hvLsi1 BAH24163.1 Wheat (Triticum aestivum) taLsi1 ADM47602.1 Crookneck Squash cmLsi1 BAK09175.1 (Cucurbita maschata) Field Pumpkin cpLsi1 CAD67694.1 (Cucurbita pepo) Cucumber csLsi1 ACU29604.1 (Cucumis sativus) Castor Oil rcLsi1 XP002534417.1 (Ricinus communis) Tomato slNIP2;1 AK321561.1 (Solanaceae lycopersicum) Tobacco ntNIP3;1 X54855.1 (Nicotiana tabacum)

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Figure 4-3: MSA of Si transporters in plants. Characterized (black) and putative (purple) Si transporters were aligned using the MegAlign program (Lasergene 9) (Table 4.2). The ar/R selectivity filter residues (red) and NPA motifs (blue) are highlighted.

Figure 4-4: Phylogenetic Tree of Si transporters. Construction of the tree was performed by the MegAlign program (Lasergene 9) of the putative N. tabacum Si transporter, ntNIP3;1, and other monocot (red) and dicot (green) identified and putative Si transporters (Table 4.2).

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With MIPs being a diverse family, we wanted to expand our study to include other proteins that could transport Si, since they have not been previously tested. In addition to ntNIP3;1, the available EST data set for N. tabacum was searched for additional previously unpublished aquaporins. Using the 36 identified S. lycopersicum aquaporins, a BLAST search of the N. tabacum EST database was performed. FASTA sequences of the top 100 hits were complied into a notepad file. Duplicate sequences were removed, resulting in 750 individual sequences. The sequences were entered into EditSeq (Lasergene 9) and then loaded into SeqMan where 67 individual contigs were identified

(Appendix C). The consensus sequence for each contig was then translated using

EXPASY translate tool (Table 4.2) and an MSA was performed using MegAlign

(Lasergene 9) with an operational taxonomical unit (OTU) including the residues from the first methionine to the last amino acid residue before the stop codon.

Two of the contigs were not aquaporins and a BLASTp search resulted in 98 and

88 % sequence identity to a predicted uridine diphosphate galactose transporter in

Vitis vinifera and Glycine max for contig two and 67, respectively. These sequences were obtained because they shared some amino acid similarities to a number of various MIPs. The remaining contigs were true aquaporins. The constructed phylogenetic tree shows diverse putative MIPs that group into PIPs,

SIPs, XIPs, TIPs and NIPs (Fig. 4-5). The ar/R selectivity filter residues and

NPA motifs correspond to the phylogenetic groupings as well (Table 4.5).

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Figure 4-5: Phylogenetic tree of putative N. tabacum MIPs identified through computational analysis. PIPs (blue), SIPs (pink), XIP (gray), TIPs (green) and NIPs (orange) including the putative NIP3;1 (yellow star) were identified using MegAlign package from DNA star Lasergene. Contigs 2 and 67 were used as outliers since they were identified in the BLAST search but do not have the conserved residues found in MIPs.

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Table 4.5: Selectivity filter and NPA motif residues for putative N. tabacum MIPs.

Contig MIP H2 H5 LE1 LE2 NPA motif number 3, 4, 8, 11- 16, 18-20, PIPs 25-30, 32, F H L/T R NPA/NPA 47-50, 57, 62, 66 SIPs 35 V V P N NPT/NPA 24, 59 L V P N NPA/NPA 45, 52, 64 V H G S NPL/NPA XIP 44 I I A Y NPV/NPA 10, 23, 31, TIP I 39, 40, 60, H I A V NPA/NPA 61 17, 38, 42, TIP IIA 43, 46, 53, H I A R NPA/NPA 65 TIP IIB 5, 6 H I G R NPA/NPA 7, 56, 58, NIP 1 W I A R NPA/NPA 63 NIP 2 1, 51, 55 S I A R NPS/NPV 36 R F A R NPS/NPA 34 I I A R NPA/NPV 41 T I A R NPA/NPV NIP 3 9 G S G R NPA/NPA NIP ? 21 A V R R NPA/VPA

If MIPs transport Si, we would expect their expression to be regulated similarly to the characterized Lsi1s (69). In addition to ntNIP3;1, four other known N. tabacum transporters (ntXIP1, ntAQP1, ntPIP1;1, ntPIP2;1, ntRT-TIP1 and ntTIP1;1) were examined for Si regulation (88, 94, 96, 97). Real-Time primers were designed using the 3’UTR sequences with the exception of ntNIP3;1 and ntXIP1. Since the 3’ UTR was not available primers from other portions of the genes had to be developed for ntNIP3;1 and ntXIP1. The 5’ UTR for ntNIP3;1 was used for primer design, whereas for ntXIP1, a variable region 64

identified from an MSA was used. All PCR products for selected primers were sequenced to confirm the correct aquaporin was being specifically amplified.

4.4.2 N. tabacum transporter study with Si treatment

For the transporter study, N. tabacum were sown in a flat containing hydroponic solution with 0 mM Si. Once roots were protruding from the foam, the solution was either maintained at 0 mM Si (Si-) or increased to 2 mM Si (Si+).

Roots were collected at seven days post treatment and placed in liquid nitrogen then stored at -80°C. RNA was extracted from roots using the RNeasy kit

(Qiagen, Madison, WI) and one-step Real Time PCR was performed. NIP3;1 and

RT-TIP1 normalized expression (to total RNA) was lower in Si+ treated plants compared to Si-. AQP, PIP1;1, PIP2;1, TIP1;1 and XIP1 did not have any difference in normalized expression between treatments (Fig. 4-6).

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2 * ** 1.8

1.6

1.4

1.2

1 Si+ Si- 0.8

0.6 Normalized Expression Normalized

0.4

0.2

0 ntAQP ntNIP3;1 ntPIP1;1 ntPIP2;1 ntRT-TIP1 ntTIP1;1 ntXIP1

Figure 4-6: Relative expression of N. tabacum aquaporins. Plants were treated with 2 mM (Si+, gray) or 0 mM (Si-, light gray) Si for 7 days (±SEM). The average of four plants in triplicate for each reaction is shown. P-value =0.037 (*), =0.0056 (**) based on Tukey’s HSD test.

With the identification of an aquaporin other then NIP changing in response to Si, we investigated the relationship of ntRT-TIP1 to TIPs in other plant species, including A. thaliana, since there are no identified NIP III proteins

(77). An MSA in MegAlign was performed using TIP sequences from A. thaliana, S. lycopersicum (87) and the recently identified in N. tabacum along with the ntRT-TIP1 (Table 4.6). The OTU included residues from the first methionine to the end of the protein, when available. The phylogenetic tree constructed by the MegAlign program resulted in clades based on the TIP subgroups (Fig. 4-7). The ntRT-TIP1 grouped with TIP II proteins and was more closely related to A. thaliana TIP 2;2 and TIP 2;3.

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Table 4.6: Accession numbers of A. thaliana and N. tabacum TIPs.

Organism Name Accession Number atTIP1;1 NP_181221.1 atTIP1;2 NP_189283.1 atTIP2;1 NP_188245.1 atTIP2;2 NP_193465.1 A. thaliana atTIP2;3 NP_199556.1 atTIP3;1 NP_177462.1 atTIP3;2 NP_173223.1

atTIP4;1 NP_18052.1 atTIP5;1 NP_190328.1 N. tabacum ntRT-TIP1 X54855.1*

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Figure 4-7: TIP Phylogenetic tree. Tomato (sl), A. thaliana (at), and published (nt) and putative (contig #) N. tabacum TIPs (Table 4.5) sequences were aligned using Megalign (DNAstar). The arrow indicates the N. tabacum TIP whose expression decreased with Si treatment after 7 d. TIP I (blue), TIP II (red), TIP III (orange) and TIP 4 (purple) subgroups grouped together while atTIP5;1 was an out group.

4.4.3 Identification of putative NIP-type Si transporters in other plants

After identifying putative Si transporters in tomato and tobacco, we examined the presence and conservation of the genes in other plant species.

Therefore, a BLAST search of EST sequences using the slNIP2;1 sequence was performed. The tomato sequence was used in place of the tobacco since the 3’ portion of the latter sequence was unknown. From this search, a number of putative Si transporters containing the conserved G-S-G-R filter residues were

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identified in plants belonging to various dicotyledonous families, including

Solanales, Asterales, Lamales, Fabales, Magaphylaes and Mesophyls. An MSA using ClustalW (Table 4.2) was performed using an OTU that included residues spanning the ar/R selectivity filter (Fig. 4-8).

A similar EST search was done using the rice Lsi1 sequence; the top 100 hits were sequences exclusively from grasses belonging to the order Poaceae (data not shown). However, sequences outside of the order Poaceae for monocots were not identified in this search; therefore, we limited our analysis to the previous search data.

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Table 4.7: Accession number of putative Si transporters.

Organism Name Accession Number Solanum tuberosum Potato DN849108.1 Solanum lycopersicum Tomato AK321561.1 Manihot esculenta Cassava DV452155.1 Triphysaria vesicolor T. vesicolor EX987787.1 Triphysaria pusilla T. pusilla EY143324.1 Siraitia grosvenorii Siriatia HS402303.1 Cucumis melo Muskmelon AM727967.1 Mimulus guttatus Monkeyflower GR132013.1 Mimulus guttatus altMonkeyflower GR119051.1 Antirrhinum majus Snapdragon AJ796939.1 Citrullus lanatus Watermelon GD179982.1 Nicotiana tabacum Tobacco DV160518.1 Gossypium hirsutum Cotton DW484352.1 Citrus sinensis Sweet Orange DC900105.1 Citrus reticulate Mandarin EY769777.1 Arachis hypogaea Peanut GO265110.1 Helianthus petiolaris H. petiolaris DY978984.1 Helianthus annuus H. annus DY924133.1 Guizotia abyssinica Niger GE554188.1 Artemisia annua Artemisia EL110061.1 Zinnia violacea Zinnia FM881058.1 Helianthus paradoxus H. paradoxus EL477091.1 Helianthus cilaris H. cilaris EL421136.1 Helianthus tuberosus H. tuberosus EL457080.1 Cichorium endivia Cichorium EL355421.1 Centaurea solstitialis Centarurea EH766476.1 Ipomea nil MorningGlory CJ755915.1 Citrus japonica Kumquat GW689843.1

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Potato ------HVVHQLLVGTDGHKVSKLGASVAGGLIVTVMIYAVGHISGAHMNPAVTFAFAAV Tomato YLLVFVTCGAASLSWSDEHKVSKLGASVAGGLIVTVMIYAVGHISGAHMNPAVTFAFAAV Cassava ------ISASDEQRISKLGASIAGGLIVTVMIYAVGHVSGAHMNPAVTAAFAAV T.vesicolor -LLVFVTCGSAALSASDEHKVARLGASIAGGLIVTVMIYAVGHISGAHMNPAVTLAFAAV T.pusilla YLLVFVTCGSAALSASDEHKVARLGASIAGGLIVTVMIYAVGHISGAHMNPAVTLAFAAV Siraitia YLLVFVTCGAAALSARDERQVSKLGASIAGGLIVTVMIYAVGHISGAHMNPAVTFAFAAV Muskmelon ------SKLGASITCGLIVTVMIYAVGHISGAHMNPAVTIAFAAV Monkeyflower FLLVFVTCGTATISGSDEHKVSKLGASVAGGLIVTVMIYAVGHISGAHMNPAVTLAFAAV altMonkeyflower FLLVFVTCGTATISGSDEHKVSKLGASVAGGLIVTVMIYAVGHISGAHMNPAVTLAFAAV Snapdragon YLLVFVTCGAAAISAVDEHKVSRLGASVAGGLIVTAMIYAVGHISGAHMNPAVTLAFAAV Watermelon YLLVFVTCGAGALSASDERRVSQLGASVAGGLIVTVMIYAVGHISGAHMNPAVTMAFAAT Tobacco YLLVFVTCGAAAISASDEHKVSRLGASVAGGLIVTVMIYAVGHISGAHMNPAVTFAFAAV Cotton YLLVFVTCGSAAISSVDEHKISRLGASVAGGLIVTVMIYAVGHVSGAHMNPAVTLAFAAV Sweet Orange YLLVFVTCGSAALSAYDEHRVSKLGASVAGGLIVTVMIYAVGHISGAHMNPAVTLAFAAV Mandarin YLLVFVTCGSAALSAYDEHRVSKLGASVAGGLIVTVMIYAVGHISGAHMNPAVTLAFAAV Peanut YLLVFVGSGSAGLSAIDENKVSKLGASLAGGFIVTVMIYSIGHISGAHMNPAVSLAFASI H.petiolaris FLLVFVTCGAASLTTSDDRKVSQLGASVAGGLIVTVMIYAVGHISGAHMNPAVTLAFATV H.annus FLLVFMTCGAASLTTSDNRKVSQLGASIVGGLIVTVMIYSVGHISGAHMNPAVTFAFATG Niger FLMVFVTCGSAALTTSDERKVSQLGASVAGGLIVTVMIYAVGHISGAHMNPAVTLAFAAV Artemisia FVLVFVSCGCAALGASDEPKVSQLGISVASGLIVTVMIYAVGHISGAHMNPAVTIAFATV Zinnia ------H.paradoxus FMLVFVTCGSAALTKSDEHKVSQLGASVAGGLIVTVMIYAVGHISGAHMNPAVTLAFASI H.cilaris FMLVFVTCGSAALTKSDEHKVSQLGASVAGGLIVTVMIYAVGHISGAHMNPAVTLAFASI H.tuberosus FMLVFVTCGSAALTKSDEHKVSQLGASVAGGLIVTVMIYAVGHISGAHMNPAVTLAFASI Cichorium FLLVFVTCGSAALATSNEHRVSQLGASLAGGLIVTVMIYAVGHISGAHVNPAVTLAFAVI Centarurea FLLVFVACGSAALGASNEHKVSQLGASVAGGLVVTVMIYAVGHISGAHMNPAVTIAFATV MorningGlory ------AHMNPAVTFXFAAF Kumquat ------LLLLLDIFPGNRSQ------FMHAAS

Potato MMFITSAVATDTKAIGELAGIAVGSAVCITSILAGPVSGGSMNPARTIGPAMASNDYRAI Tomato MMFITSAVATDTKAIGELAGIAVGSAVCITSILAGPVSGGSMNPARSIGPAMASNDYRAI Cassava MMFVTSAVATDTKAIGELAGIAVGSAVCITSILAGPVSGGSMNPARTLGPAIASAHYKGI T.vesicolor MMFITSAVATDTKAIGELAGIAVGSAVCITSILAGPVSXGSMNPARTIGPAIASNYYNGI T.pusilla MMFITSAVATDTKAIGELAGIAVGSAVCITSILAGPVSGGSMNPARTIGPAIASNYYNGI Siraitia MMFVTSAVASDTKAVGELAGIAVGSAVCITSIFAGPISGGSMNPARSIGPAIASSHYEGI Muskmelon MMFVTSAVATDTKAIGELGGIAVGSAVCISSIFAGPISGGSMNPARSIGPAIASSRYEGI Monkeyflower MMFITSAVATDTKAIGELAGIAVGSAVCITSILAGPISRGSMNPARTIGPAIASNYYKVF altMonkeyflower MMFITSAVATDTKAIGELAGIAVGSAVCITSILAGPISGGQ------Snapdragon MMFITSAVATDTKAIGELAGIAVGSAVCISSVLGGPVSGGSMNPARTIGPAIASNYYKGV Watermelon MMFVTAAVATDTKA------Tobacco MMFITSAVATDTKAIGELAGMAVGSAVCITSILAGPVSGGSMNPARTIGPAMASNDYRG- Cotton MMFITSAVATDTKAIGELAGIAVGSAVCITSILAGVGIQTNIRWINEPSKEHRASNS--- Sweet Orange MMFVTSAVATDTKAIGELAGIAVGSAVCITSVLAGPVSGGSMNPARTVGPAIASSFYKGI Mandarin Orange MMFVTSAVATDTKAIGELAGMAVGSAVCITSVFAG------Peanut RVFISAAVATDPKAIGELSGVAVGSSVSIASIVAGPISGGSMNPARSLGPAIATASYKGI H.petolaris MMFVTSAVATDSKAVGELAGIAVGSAVCITSILAGPVSGGSMNPARTIGPALASNNYXAI H.annus MMFVTSAVATDAKAVGELAGIAVGSAVCITSIFAGPVSGGSMNPARTIGPAIASNNYKGI Niger MMVVTSAVAADSKAVGDLAGMAVGSAVCITSILAGPVSGGSMNPARTIGPALASNNYKGI Artemisia MMFVTSAVATDCKAVGELAGIAVGSAVCINSILAGPISGGSMNPARTIGPALASNTYKGI Zinnia MMFVTSAVATDSKAVGELAGMAVGSAVCITSILAGPVSGGSMNPARTIGPALASNNYKGI H.paradoxus MMFVTSAVATDSKAVGELAGIAVGSAVCITSILAXPVSGGSMNPARTIGPALASNNYKGL H.cilaris MMFVTSAVATDSKAVGELAGIAVGSAVCITSILAGPVSGGSMNPARTIGPAIASNNYXGL H.tuberosus MMFVASAVATDSKAVGELAGIAVGSAVCITSILAGPVSGGSMNPARTIGPALASNNYKGL Cichorium MMFVTSAVATDSKAIGELAGIAVGSAVCITSILAGPVSGGSMNPARTIGPALASNNYKGI Centarurea MMFVTSAVATDSKAVGELAGIAVGSAVCISSILAGPVSGGSMNPARTIGPALASNTYKGI MorningGlory MMFVTSAVATDTKAIGELAGIAVGSAVCITSIFAGPISGGSMNPARTIGPAIASNDYKGI Kumquat MMFVTSAVATDTKAIGQLPGIAVGSAVGLSSAVAGPVSGGSMNPARAVRAASASSIYSGI

Figure 4-8: MSA of putative Si transporter proteins from various plants EST sequences. Conserved NPA motifs are highlighted in yellow, while the selectivity filter residues are highlighted in red. Sequence conserved with the N. tabacum NIP3;1 are highlighted in gray.

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4.5 Discussion

Silicon transporters have been identified and characterized in a number of high accumulators including rice, corn, barley, wheat, cucumber and pumpkin

(69, 73, 74). While aquaporins have been identified in a number of intermediate and low accumulators, including moss, A. thaliana, and tomato, no one has examined their potential role as Si-regulated genes. After examining the 36 tomato aquaporins identified by Sade et. al , a putative Si transporter, termed

NIP2;1 was identified (87). This lead to the identification of a putative tobacco Si transporter, ntNIP3;1. Both have the conserved aromatic/arginine selectivity filter residues in addition to NPA motifs. Unfortunately, the data for the complete sequence of ntNIP3;1 lacked the 3’ end and our efforts to amplify this end using

3’ RACE were unsuccessful. While examining other putative Si transporters, a number of the available EST sequences were also lacking the last transmembrane domain suggesting there might be elements that inhibit amplification of that region.

In addition to the putative ntNIP3;1, more than 60 separate contigs from

750 sequences were identified as putative aquaporins in N. tabacum. Tomato and

A. thaliana contain 37 and 36 aquaporins, respectively (77, 87). Since tobacco is an allotetraploid (98), it is likely that they would contain a larger number of aquaporins.

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Real-Time PCR analysis of ntNIP3;1 expression with Si treatment for seven days was statistically significantly lower compared to controls. This mirrors the Si transporter expression in other high accumulators, further suggesting ntNIP3;1 is a putative Si transporter. The sequence also contains the conserved selectivity filter residues characteristic of Si transporters.

More importantly, ntRT-TIP1 expression was statistically significantly decreased with Si treatment. To date, only NIPs have been investigated as putative Si transporters. The fact that a TIP is behaving in a similar manner to known Si transporters could suggest that there are regulated Si transporters that have been overlooked. With Si being stored in the vacuole it makes sense that transporters localized to the tonoplast would have the ability to transport the element. Hence, the ntRT-TIP1 may be involved in release of Si from internal stores, like the vacuole, in roots. This could also explain why A. thaliana, an intermediate accumulator, does not contain a predicted NIP protein with the identified ar/R selectivity filter present in all classified Si transporters, but still has the ability to accumulate the element.

A. thaliana TIP2 family members were closely related to N. tabacum ntRT-TIP1 sequences (Fig. 4-6). The A. thaliana TIP2 proteins have been implicated in a number of processes in addition to solute transport. A portion of

Cucumber mosaic virus replicase 1a protein has been shown to interact with atTIP2 proteins, which suggests its involvement in either CMV replication or movement of viral RNAs (99). A. thaliana TIP2;1 and TIP2;2 have been shown

+ to transfer NH4 in xenopus oocytes and are localized mainly in root tonoplasts 73

and are diurnally regulated with expression rising at the onset of light and lower as the light diminishes (100). This could indicate that an A. thaliana TIP2 protein is also involved in Si accumulation since the subgroup may be involved in pathogen and nutritional interactions.

The identification and classification of other plant putative Si transporters mirrors the angiosperm phylogenetic relationship

(http://www.mobot.org/mobot/research/apweb/). These putative NIP III proteins are commonly found amongst various angiosperm taxa, including those which accumulate low levels of Si. Hence, the ability of plants to acquire Si is likely an ancient process, since the proteins involved appear to be present in a wide variety of angiosperms.

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Chapter 5

Additional Si Experiments

5.1 Abstract

Previously, we showed that treatment of Nicotiana tabacum with Si reduced Tobacco ringspot virus (TRSV) systemic infection. Because of its utility, we desired to perform similar experiments in Arabidopsis thaliana. It was first necessary to identify A. thaliana ecotypes that were susceptible to TRSV. In a large ecotype screen, we identified the ecotype Sf-1 as susceptible to TRSV. In contrast to N. tabacum, Sf-1 plants supplemented with Si showed an increase in incidence in systemic symptoms. Hence, host and possibly environmental conditions influence the ability of Si to influence TRSV infection. The effects of the environment on plant growth and development are often influenced by reactive oxygen species (ROS). Indeed the Si literature suggests that the beneficial effects of this element may be through signaling by ROS. Therefore, we examined this in two ways: by determining the effects of an ROS pathway on a Si-mediated response and by studying the direct effects of Si on an enzyme regulating ROS detoxification. Ascorbic acid (AsA) is an important molecule in

ROS detoxification. Intriguingly in A. thaliana, only two genes were found to be

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regulated by elevated Si alone, one of which was a putative ascorbate oxidase,

SKS7. To study this gene in N. tabacum, we performed a BLAST search to identify potential homologs and identified a gene named PS60. Unfortunately,

PS60 levels did not change in response to Si in N. tabacum as they did in A. thaliana, providing further evidence that although Si can have a beneficial effect in both plants, they respond to the element in different ways. Further analyses indicated that AsA did not influence TRSV infection in N. tabacum, suggesting that Si-mediated resistance does not operate through this pathway. While these data appear to exclude the AsA pathway, they do not rule out a potential role of

ROS in Si-mediated processes. Our preliminary data suggest that Si can directly inhibit the major peroxidase activity in turnip extracts. Thus, the presence of Si in

Brassicas may cause an increase in ROS directly. However, it is unclear if Si needs to be taken up by cells to influence peroxidase activity or if it is the apoplastic peroxidase that is involved. Our prior work indicated that there are putative Si transporters in the characterized low-accumulator species N. tabacum.

Therefore, we chose to determine if one of the putative N. tabacum aquaporins was capable of transporting Si. Yeast expressing ntRT-TIP1 did not aquire additional Si compared to control, suggesting that either ntRT-TIP1 is not a Si transporter, or it is incorrectly processed in yeast. The use of the Molybdenum

Blue Colorimetric Method in place of ICP-OES was used to determine Si concentrations since a smaller concentration of yeast are needed. This is a common method for determining the amount of Si in samples and can use smaller quantities of sample to detect the element. However, we discovered that Si

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concentrations determine by this method were grossly overestimated, as we detected phosphorous instead of Si. Our findings suggest that the method needs to be improved to be more specific for Si.

5.2 Introduction

Plants, unlike animals, are unable to move from their location and have thus adapted complex mechanisms that allow them to perceive and alter the environment that they inhabit. Environmental factors play an important role in plant growth and overall health (48). This can be particularly problematic for disease detection, since plants, such as tolerant hosts, can contain large amounts of pathogen and served as a reservoir for infection of other hosts.

Tobacco ringspot virus (TRSV) has a wide host range and can be seed transmitted (101). However, not all plants infected with TRSV are symptomatic.

In a published study of 97 A. thaliana ecotypes infected with TRSV, all plants tested were susceptible to the virus; however, only 12 exhibited symptoms (102).

A single gene (TTR1) is responsible for the tolerance effect. Plant nutrition may also play a role in virus infection in plants. Boron treatment reduced necrosis in

N. tabacum infected with Belladonna mottle virus (29).

Antioxidants are involved in tolerance to disease and environmental stress in plants (103). In tomato challenged with root-knot nematode (Meloidogyne incognita), treatment with 45 mM ascorbic acid reduced the number of nematodes by 96.5 % compared to controls (104). In the same study, addition of lycorine to inhibit AsA synthesis increased susceptibility by 31-116% depending on the

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tomato cultivar. In SA-deficient and wild-type A. thaliana, the application of

AsA reduced Cucumber mosaic virus and Turnip crinkle virus symptoms (105).

The reduction correlated to a decrease in the viral coat protein (CP). ROS scavengers were also applied to the plants but did not alleviate symptoms or reduce CP levels, suggesting that AsA involvement in reduction of viral symptoms is more complex than its role as an antioxidant. AsA can also permit plants to better respond to abiotic stress. Application of AsA increased tolerance of tomato to salt stress without significantly altering the accumulation of sodium or membrane permeability (106).

Peroxidase (POD) is a central enzyme, regulating ROS levels in plants

(103). Si has been shown to induce the expression of PODs in response to both biotic and abiotic stress (107). These enzymes also play a role in plant responses to pathogens (108). In a study examining Mn toxicity in cucumber, endogenous

POD levels in leaf apoplastic fluid significantly decreased with Si treatment

(109). In vitro analysis of Si on a commercial POD also showed a significant reduction in peroxidase activity, suggesting the element directly interacts with the enzyme.

Multicopper oxidases are thought to regulate ROS levels present within plants. Skewed 5 (SKU 5), a multicopper oxidase gene, was first discovered in an

Arabidopsis thaliana mutant that had slanted root growth (1). The A. thaliana genome encodes eighteen SKU 5 similar (SKS) proteins. The only protein that has been studied other than SKU5 is SKS6, which is involved in vascular patterning in cotyledons (2). SKS7 expression increases in A. thaliana plants 78

amended with increased silicon (28), but the function of the protein is not known.

An increase in an SKS-like protein in rice has also been observed (110).

For Si to have effects on plants, it needs to be acquired from its environment (38). Si transporters, that are members of the aquaporin family, are important players in this process. To examine the function of these proteins, assay systems are used to measure either uptake or export of the element from cells expressing a particular transporter. The use of a yeast-based system has been previously employed to determine solute transport by various aquaporins.

Studies done on the transport of boron, urea, arsenic and a number of other solutes has been carried out with a combination of xenopus expression and yeast transporter assays (95, 111, 112). However, sometimes yeast systems have limitations and under these circumstances, studies done in Xenopus oocytes are more appropriate.

To determine if a protein is an Si transporter, it is important to have a method for quantifying Si. Several ways to quantify Si from plants have been developed (35). One of the earliest and cheapest methods was the Molybdenum

Blue Colorimetric Method (113). Si interacts with the molybdenum complex in this assay, forming a heterpoly complex, which can be reduced, resulting in the formation of a blue color that is proportional to the amount of Si present (113).

However, this method can also detect phosphorus (P), germanium, and arsenic.

The use of HCl and tartaric acid have been employed to reduce P contamination

(114). A more specific test to quantify Si is inductively coupled plasma-optical emission spectroscopy (ICP-OES), which determines Si levels above 3 mg Si/kg 79

tissue at a wavelength of 212.412 nm (158) (35). P is not detectable at this wavelength (115).

5.3 Materials and Methods

5.3.1 Plant material

Zinnia elegans and N. tabacum were grown hydroponically from seed in large walk-in growth chambers 16:8 light:dark photoperiod with 50 μmol·m-2s-1 at

19°C. Nutrient solution was changed every 7-8 days. Higher light growth chamber conditions increased light levels to 75 μmol·m-2s-1.

A. thaliana were grown from seed both in soil-less media and hydroponically. Briefly, plants were sown onto presoaked media and placed at

4°C for two weeks, then placed in a growth chamber with 16:8 light:dark photoperiod with 60 μM·m-2s-1 at 20°C. Continual light growth chamber had 24 hr light at 35 μmol·m-2s-1 with the temperature held at 19°C. For hydroponics, seeds were incubated in an Eppendorf tube with nutrient solution at 4°C for two days. The seeds were then planted on presoaked foam and floated in nutrient solution until roots were present, after which they were transferred to 4 L buckets containing nutrient solution with 0.1 mM Si.

5.3.2 Ascorbic Acid Effects on Plants

N. tabacum were grown hydroponically from seed and after they reached the 6-8 leaf stage were transferred to 4 L buckets with 1.0 mM Si. AsA was added to hydroponic solutions to a final concentration of 1.5 mM.

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5.3.3 Semiquantitative PCR

Primers (PS60RT-1F; 5’-GCGTTCCTTGGAATTGGATA-3’, nt 1383-

1402 and PS60RT-1R; 5’- GCGTTCCTTGGAATTGGATA-3’, nt1558-1577) were designed or amplify a conserved region between tobacco, tomato and A. thaliana, RNA was isolated using the RNeasy kit (Qiagen, Madison, WI). A reverse-transcriptase reaction was then performed and the subsequent cDNA was used for PCR. For semiquantitative analysis, samples were run for 15, 20, 25 and

30 cycles and the PCR products were separated on an agar gel stained with ethidium bromide.

5.3.5 Modified Yeast Transformation

Primers (ntRT-TIP1-4F; 5’-AAGCTTATGGTGAGGATTGCCTTTGGT-3’, nt

1954-1974 ntRT-TIP1-4R; 5’-GACAAGCTTGTCTTCTTTTAAG-3’, nt 3290-

3311) were designed to amplify the entire ntRT-TIP1 protein coding sequence from mRNA with Hin dIII sites at each end. Phusion PCR was used to amplify the cDNA, followed by blunt end ligation into the pCR Blunt vector (Invitrogen).

The construct was transformed into DH5α E. coli, and the presence of the insert was confirmed by sequencing. The ntRT-TIP1 protein coding sequence was then inserted into the Hin dIII sites of the pGAD424 yeast expression vector.

YPH499 yeast were spread on YPD plates and incubated at 30°C for 2-3 days. Once colonies were an appropriate size, 50 ml YPD media was inoculated with 3-5 EGYH8 colonies and incubated at 30°C shaking overnight. The solution was then transferred to 50 ml falcon tubes and centrifuged at 740·g for 5 min.

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The supernatant was discarded and the pellet resuspended in 30 ml sterile water.

The tubes were then centrifuged at 700·g for 5 min and the pellet was resuspended in 1.5 ml 1.1X TE/LiAc. The solution was transferred to 2 ml

Eppendorf tubes and centrifuged at 14,000 rpm for 15 sec. The solution was discarded. The pellet was resuspended in 600 μl 1.1 xTE/LiAc (competent cells).

Carrier DNA (5 μl/ reaction) was heated at 95-100°C for 5-10 min then quick chilled on ice for 5-10 min. Next, 5 μl carrier DNA and 5 μl plasmid DNA were mixed and 50 μl of the competent cell solution was added to the DNA and gently mixed. Then 500 μl PEG/LiAc was added and the solution was incubated at 30°C for 30 min, mixing every 10 min. DMSO (25 μl) was added, tubes were vortexed for about two sec and then incubated at 42°C for 15 min, mixing every 5 min.

Tubes were then centrifuged at 14,000 rpm for 15 sec. The supernatant was discarded and the pellet resuspended in 1 ml –L media. The solution was incubated in a 30°C shaker for 1 hr. The solution (100 μl) was spread onto –L plates and incubated at 30°C for 3-5 days.

5.3.6 Silicon Accumulation in transgenic yeast

Transgenic yeast colonies were inoculated into 3 ml –L yeast media and incubated shaking at 30°C for 7 hrs. Then 10-50 μl yeast/-L media was transferred to 50 ml –L media and incubated shaking at 30°C for 16-20 hrs until absorbance at 595 nm reached 0.5. The solution was then centrifuged at 740·g for

5 min and the solution was discarded. Transgenic yeast were incubated in solution containing 2 mM silicic acid or KOH for 30 min then centrifuged for 10

min to collect the yeast. The cells were rinsed twice with dH2O. The solution was

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then centrifuged at 740·g for 5 min and resuspended in 50 ml dH2O, and repeated three times. Cells were then homogenized in 5 ml dH2O. Cells were sonicated for 90 sec in 15 sec pulses on ice. Tubes were then centrifuged at 740·g for 5 min. A small amount of solution was set aside for a Bradford Assay and the remaining was used for the Molybdenum Colorimetric Assay.

5.3.7 Molybdenum Blue Method

To 15 ml culture tubes, 0.5 ml yeast solution was added to 0.4 ml H2O.

The addition of 0.1 μl 1:1 HCl and 0.2 μl 10% (NH4)6Mo7O2 (pH 7.0) followed, the tubes were vortexed, and incubated at room temperature for 10 min. Then 200

μl of 20% tartaric acid was added, tubes were vortexed, and incubated for 3 min at room temperature. The addition of 282.3 μl 0.5% ascorbic acid followed and tubes were vortexed. After 30 min of incubation at room temperature, absorbance readings at 650 nm were recorded.

5.3.9 Peroxidase Assay

Fresh turnips purchased from a local grocery store were ground in phosphate buffer, pH 5.0, with sand. The extract was kept on ice. In a test tube,

2.4 ml phosphate buffer, pH 5.0, 1 ml 1% H2O2, 0.5 ml 0.25% guaiacol, and 0.1 ml of the turnip extract were added to the tube, quickly mixed by inversion and the absorbance was measured at 500 nm. Si was added to the buffer to a final volume of 2 μM (Si1) or 20 μM (Si2). KOH was also added to buffer for a final concentration of 20 μM to match the KOH present in the Si2 solution.

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5.4 Results

5.4.1 A. thaliana ecotype study on TRSV susceptibility

The effects of Si on TRSV were tested using the model plant, A. thaliana.

An ecotype study was initially performed to find a susceptible variety. Ten ecotypes were inoculated with semi purified TRSV. Col-0, Estland, Lc-0, Np-0,

No-0, Ita-0, Mh-0, and Wil-2 did not develop systemic symptoms. Sf-1 had a higher TRSV infectivity rate with identifiable symptoms, compared to Rsch4, and unlike Est-0, was able to survive the infection. Typically symptoms of TRSV infection were partial to total leaf chlorosis, stunting of the plants and death in some cases. TRSV infection in Sf-1 plants was confirmed by RT-PCR (data not shown).

Table 5.1: Local and systemic TRSV symptoms in A. thaliana ecotypes with (+) or without (-) chlorosis.

Col- Lc- Np- No- Ita- Mh- Wil- Estland Rsch4 Sf-1 0 0 0 0 0 0 2 Local - + - - - + - + + + Systemic - + - - - - - + + -

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Figure 5-1: Systemic (Sf-1, RSCH4, Est-0), local (Wil-2 & Ita-0) or no (Col-0) TRSV symptoms in A. thaliana ecotypes.

5.4.2 Si Increases Susceptibility of Sf-1 to TRSV

Sf-1 was grown hydroponically in nutrient solution containing 0.1 mM Si in a continual light growth chamber. Solution was changed every seven days.

Once plants reached the 6-8 leaf stage, 12 plants were transferred to 4 L buckets containing nutrient solution with 0.1 mM Si. Just prior to inoculation with semi purified TRSV, nutrient solution was maintained at 0.1M Si (C) or changed to 1.0 mM Si (Si+). Symptoms were tracked over time. The infection rate was low, infecting only one plant per bucket in control and two plants for Si+. Using the data, however, we did observe a delay in symptom formation in C plants relative to Si treated plants. When these studies were repeated in a new growth chamber,

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no differences in the onset or the number of symptomatic plants were observed, suggesting that the effect was environmentally dependent.

45

40 * 35

30

25 Control 20 AUDPC Si+ 15

10

5

0 Local Systemic

Figure 5-2: Local and systemic AUDPC for TRSV infected Sf-1. Plants were treated with 0.1 mM (control, light gray) or 1.0 mM (Si+, dark gray) Si. The asterisk indicates a significant difference based on ANOVA analysis with a p-value < 0.004.

We also tested the effect of both B and Si treatment on TRSV infection in

A. thaliana Sf-1. Plants were grown in nutrient solution containing 1 μM B and

0.1 mM Si. Just prior to TRSV inoculation, nutrient solution was changed to 0

μM B/ 0.1 mM Si (B-), 30 μM B/ 0.1 mM Si (B+), 1 μM B/1.0 mM Si (Si+), or remained constant at 1 μM B/0.1 mM Si (C). Twenty-one percent of soil-less media (S) grown Sf-1 showed TRSV symptoms, which was significantly less than

C, B-, B+ and Si+ having 61, 84, 81 and 91 percent symptomatic plants, respectively (Fig. 5-3). The difference between S and C had a p-value of 0.084, making it not a statistically significant difference.

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120

b 100 b b a,b 80

60

40 a

percent symptomatic plants symptomatic percent 20

0 S C B- B+ Si+

Figure 5-3: Sf-1 TRSV symptoms with varying B and Si treatment. The average number of symptomatic plants from three buckets ±SEM of soil-less media (S), 1 μM B/ 0.1 mM Si (C), 0 μM B/ 0.1 mM Si (B-), 30 μM B/ 0.1 mM Si (B+) or 1 μM B/ 1.0 mM Si (Si+) treated Sf-1. Different letters represent a statistically significant difference with a p-value < 0.0084 based on Tukey’s HSD test.

5.4.4 Treatment of N. tabacum with Ascorbic Acid did not alter TRSV infection

Since Si has been shown to influence antioxidant capabilities of plants

(116), a study was performed to test whether the addition of ascorbic acid (AsA) would enhance Si-mediated TRSV protection. N. tabacum plants were grown hydroponically under control conditions. Prior to TRSV inoculation, the Si content of the nutrient solution was elevated to 1.0 mM and AsA was added at 0 mM (control) and 1.5 mM (AsA+). The onset and spread of symptoms were followed. No differences were observed in the onset or severity of plants treated with or without AsA. However, the plants present in one of the AsA+ buckets

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developed darker green leaves compared to the controls. This suggests that the

Si-mediated delay in TRSV infection likely does not utilize this pathway.

5.4.5 No changes in transcript expression of putative Si-induced genes

From the microarray study done by Fauteux et al., the A. thaliana SKS7 gene was described as changing between Si-treated and non-treated plants (28).

Using the SKS7 sequence (NM_102034), a BLAST search was performed to identify N. tabacum homologues. PS60 (X969332.1) was the only hit (70%, 75% nucleotide and protein sequence identity, respectively). The search also identified the tomato LEPE transcript (CAB08077) with 90 % identity to PS60 and 73% identity to SKS7.

Primers were designed to amplify a portion of the transcript conserved between PS60, LEPE, SKS5, SKS7 for the amplification of the transcript in tobacco, tomato and A. thaliana with a single primer pair. PCR using these primers resulted in a triplet for A. thaliana and N. tabacum and a doublet for S. lycopersicum (Fig. 5-4).

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Figure 5-4: PCR using PS60 designed primers. Lanes 1-4 are A. thaliana mock inoculated (1) or TRSV inoculated under control (2-3) or increased silicon (4). Lanes 5-7 are N. tabacum infected with TRSV (5 and 6) or TRSV and TMV(7) . Lane 8 is a negative control. Lane 9 is cDNA from a tomato from my home garden.

Semi-quantitative PCR was performed on RNA isolated from Si+ and C treated N. tabacum 25 dpi with TRSV. No significant changes in PS60 expression were observed between the Si treatments (data not shown). Since ROS production is an early response to infection (117), changes in PS60 expression influenced by Si may be an earlier event. In addition, at that time, the N. tabacum transcriptome had not been released, so it is likely that PS60 is not the only ascorbic peroxidase with sequence similarities to A. thaliana SKS7.

5.4.6 Peroxidase Study

Si has been implicated in pathways associated with ROS. Crude turnip extract was used to examine the effect of Si on POD activity. Enzymatic activity 89

was determined using the crude extract with the addition of Si at 2 μM (Si1) or 20 mM (Si2). An extract incubated with KOH equivalent to the concentration used in Si2 was used as a control in addition to the use of water (C), since the reduction in pH could have affected enzymatic activity independent of Si treatment. In Si2 treated extract, a significant reduction in the slope of POD activity was recorded compared to Si1, KOH and C (Fig. 5-5).

0.045 a a

0.04

0.035 a 0.03

0.025 b 0.02

0.015

0.01

Average Slope Average of Enzymatic Activity 0.005

0 C K Si1 Si2

Figure 5-5: Average slope of peroxidase activity in turnip extract. KOH (K), 2 μM Si (Si1), 20 μM Si (Si2) or nothing (C) was added to the extract. Different letters represent a statistically significant difference with a Tukey’s HSD p-value < 0.0034. 5.4.7 Yeast Expression of RT-TIP1

A transporter assay using transgenic yeast expressing the ntRT-TIP1 transcript was employed to test for Si transport. The yeast were grown in media containing 2 mM Si. After one hour, cells were sonicated. A portion was used to measure protein content, using the BioRad protein assay, while the rest was used to measure Si content. The sample was digested following the standard

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autoclave-digest protocol (35). The values were reported as a ratio of Si/protein

(Fig. 5-6). While the first set of experiments suggested that yeast expressing ntRT-TIP1 had a higher Si/protein ratio, there was no consistent difference in Si content in the extract of ntRT-TIP1 yeast compared to yeast containing an empty vector across experiments. Furthermore, in later experiments, the Si content detected by the Colorimetric Assay was returning values significantly higher than those expected, which raised the question of how accurate this method was.

40

35

30 g/ml) g/ml) μ 25

20

15

Si (mM)/ (mM)/ Protein Si ( 10

5

0 RT-TIP1 RT-TIP1 RT-TIP1 Empty Empty Empty Empty

Figure 5-6: Si accumulation in Yeast expressing ntRT-TIP1 or an empty vector. Yeast were treated with 2 mM Si in –L yeast media for one hour prior to the assay. The calculated Si content was divided by the protein concentration determined by the Bradford Assay.

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An experiment was conducted to test the accuracy of the Colorimetric

Assay. Si content of digest N. tabacum leaf tissue was determined using ICP-

OES and the Colorimetric Assay. When the Si concentrations were converted to mM units from ICP-OES data, we found that levels reported by the colorimetric assay were significantly higher (above 2500 mM) compared to IPC-OES, which showed levels similar to previously reported Si concentrations in tobacco (Fig. 5-

7).

3500.00

3000.00

2500.00

2000.00 ICP 1500.00 Colorimetric

1000.00 "Si" Concentration (mM) Concentration "Si" 500.00

0.00 P1 P2 P3 P4 Figure 5-7: Si concentration comparison between ICP-OES and the Molybdenum Blue Colorimetric Assay. ICP-OES (ICP, black) or the Molybdenum Blue Colorimetric Assay (Colorimetric, gray) was performed on the same sample for four different N. tabacum plants (P1-P4).

At first, it seemed that there was an error in the conversion from ppm to mM. However, after showing the data to Dr. Jonathan Frantz, the suggestion was made that the levels appeared similar to those present for phosphorus (P). Since the colorimetric method has been used for P detection, an experiment was 92

developed to determine if the element was reacting with the reagents. Common phosphate buffers were used in place of digested tissue. When the colorimetric assay was performed, in the absence of Si, high “Si” concentrations were observed, suggesting that P was reacting with the reagents (Fig. 5-8).

8000

7000

6000 5000 4000 3000

2000 "Si" concentration (mM) concentration "Si" 1000 0 Sodium Phosphate 0.1 M phosphate 0.05 M phosphate 10X PBS Dibasic buffer, pH 7.0 buffer, pH 7.0 heptahydrate

Figure 5-8: The Molybdenum Blue Method detection of Si in different phosphate buffers. Phosphate buffers at indicated pH were used in place of tissue solution.

5.5 Discussion

Preliminary data suggest that Si affects plants differently both dependent on the species and environmental conditions. Light levels influence a number of pathways within a plant, such as the induction of ABA in tobacco three hours after dark (118), which may influence Si movement into and within a plant.

In addition, A. thaliana appeared to become more susceptible to TRSV in the presence of Si, which is similar to N. tabacum infected with Belladonna

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mottle virus (29). Since Si did not lead to resistance in A. thaliana, this may suggest that the pathways triggered by the element are different between the two hosts. We already know that A. thaliana acquire the element in an unknown manner, since no identified NIP III proteins have been found, so the plants may utilize Si in a different way. Taken together, this suggests that the use of Si for a beneficial effect needs to be carefully evaluated in the plant one is interested in protecting.

Si appears to operate through alternative mechanisms, since various hosts respond to the same pathogen differently (39). ROS have been proposed to play a role in Si-mediated responses (38). An important regulator of ROS levels is the

AsA pathways (103). AsA treatment may have not worked since the compound oxidizes quickly and was only replenished every seven days, resulting in little to no changes in plants treated with the vitamin. Another possibility is that the form of AsA was not acquired by the plant and therefore was incapable of inducing a response. Therefore, the ROS pathway in Si-mediated responses to virus infection is still an open question.

In order for Si to have an effect, it needs to be acquired from plants. Our preliminary data (Ch. 4) suggested that ntRT-TIP1 expression decreases after Si treatment, while its ability to function as a Si transporter was tested. No consistent changes in Si concentration between the yeast lines expressing ntRT-

TIP1 versus empty vector were observed. ICP-OES was performed on a KOH digested experiment and Si levels were barely detectable, again with no differences between ntRT-TIP1 or empty vector. While Ma et al. used a yeast 94

expression system to identify arsenic and other nutrient transporters from rice, a yeast system was not employed to identify the Si transporter (95). This may suggest that the Si transporter is not expressed or does not function properly in yeast.

Preliminary data suggests that POD activity is inhibited by the presence of

Si. This supports data from Maksimovic et. al that shows POD activity is reduced both in vitro and in vivo with Si treatment (109). The inhibition of an antioxidant, may help stabilize ROS, allowing for a more robust signal, with H2O2, but may also lead to irreversible oxidative damage within plants, depending on the degree of inhibition.

Yeast-expressing ntRT-TIP1 did not show reproducibly higher Si levels then control yeast transformants harboring an empty vector, using the colorimetric assay. Yeast contain a transporter to pump B out of the cell (111). It is possible that while the Si was being acquired through the ntRT-TIP1 transporter, the efflux

B transporter was moving the element back into the media. Also, the fact that we were getting P contamination while using the colorimetric assay could suggest that our reagents may have been faulty. It is possible that the tartaric acid used in these assays had degraded during the course of our work. Or, the reducing agent may have been oxidized during storage and was not functioning appropriately.

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Chapter 6

Discussion / Future Work

Classification of higher plants based on Si accumulation has divided these organisms into three groups based on their foliar Si content (7, 21) . High-, intermediate- and low-accumulators were characterized as containing greater than

10 %, 1-3 % , or less than 0.5 %, respectively. In 1977, Takahshi et. al introduced the terms as simply accumulators or non-accumulators based on whether the Si content exceeded or was less than/equal to water uptake (21). To date, the use of foliar Si levels to classify plants has been the standard protocol. However, this classification can be misleading. The studies presented above provide evidence that Si accumulation can vary greatly between roots and shoots in N. tabacum.

Not only is Si content dependent on tissue, but is also influenced by the environmental conditions in which the plants are grown. In our studies, both an abiotic (Cu) and biotic (TRSV) stress significantly increased foliar Si concentrations in the same plant species, suggesting the need to re-evaluate how we classify plants based on their ability to accumulate the element. Here we suggest the use of inducible- and constitutive-accumulators to describe plants that regulate the movement of Si from roots to shoots (N. tabacum) in contrast to those 96

that allow for the continual movement of Si into the shoots (Z. elegans), respectively.

Our data suggests that stress-induced Si-accumulation (SISA) for N. tabacum, has at least four steps (Fig. 6-1). First, a specific stress is recognized by the plant. This then leads to the activation of pathways, possibly through a signaling mechanism, resulting in the release of Si from the roots either from internal stores or new uptake from the media. This release then allows for the movement of Si into the leaves, presumably aiding the plant in resistance.

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Figure 6-1: Model For SISA. In N. tabacum, a specific stress is first perceived by the plant (1), which allows for a signal to move to the roots (2). The roots then allow for the release of Si from internal stores or new uptake from the growth media (3), leading to Si accumulation in leaves of the plant (4).

We have data which suggest that a specific stress is an important step in

SISA in N. tabacum. TMV infection did not alter Si concentrations in leaves, nor did Si treatment reduce viral symptoms in tobacco. This suggests not just any

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stress induces SISA. It is still unclear what role the increase in foliar Si plays in disease resistance for N. tabacum. It is well documented that pathogens induce specific pathways in plants (119). TMV may not trigger a portion of the pathway that TRSV induces, resulting in lack of Si accumulation and symptom tolerance.

Alternatively, TMV may have a mechanism for shutting down foliar Si uptake in response to stress. If our data on ABA are correct, since TMV induces ABA production (58), this could inhibit foliar Si uptake in response to viral infection.

To test if a higher foliar Si concentration has a similar effect on viral symptom spread, plants grown with high Si could be treated with higher Cu levels to allow for the release of the element into the leaves. Following treatment, Cu levels would be reduced and plants would be infected with TMV. A change in symptom formation between treatments would suggest that foliar Si concentration is involved in the reduction of symptoms. If leaf Si levels can be elevated by Cu treatment but TMV infection is not affected, then merely increasing foliar Si levels is not sufficient to provide resistance to this virus.

The use of Cu for SISA could also be used to determine if the release of Si from roots to shoots is from internal stores or the result of new uptake from the media. Plants again would be treated with high Si concentrations in the nutrient solution allowing for Si to accumulate in the roots. Following this treatment, Si would be removed from the media, plants would be treated with high or low Cu, and foliar Si levels would be measured. An increase in foliar Si would suggest that Si is being released from stores. It would be important to determine Si content in both roots and shoots to ensure that root Si content in control is higher

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than Cu-treated plants. Analysis of Si-regulated aquaporins may also give insight as to whether the plant is trying to take up additional Si. An increase in transcript levels of Si-transporters would suggest a mechanism for uptake from the media, while no change would suggest the release from stores. However, this experiment would only provide preliminary data for answering the question of internal release or new uptake. It is likely that Si uptake could be very complex and may involve both mechanisms. It is clear that mechanisms responding to Cu toxicity and virus infection are extremely complex. Therefore, identifying signaling components common to these two major defense pathways is unclear. However, ROS signaling has been implicated in both responses to Cu toxicity and to virus infection.

In addition to the idea that Si accumulation is inducible, another significant discovery made in this work is that a TIP is Si-regulated. All the published research to date on Si transport in plants has identified a small NIP family subgroup as Si transporters (69, 72, 74). These are the only MIPs shown to be Si-regulated. The identification of a tonoplast, root-specific aquaporin that is Si-regulated may not be too surprising, since Si is stored in the vacuole (120).

Perhaps this MIP helps release Si from the vacuole to allow the plant to deal with stress. This does, however, open the question of what other MIPs may be regulated by Si. In our study, we only tested seven of the possible 65 putative transporters. The classical NIP III transporters are not present in the intermediate- accumulator, A. thaliana aquaporins, but other MIPs such as TIPs may play a role in their ability to accumulate Si. However, to determine if the Si-regulated

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aquaporins actually transport the element, Xenopus expression assays would be done for both ntRT-TIP1 and ntNIP3;1. This will allow us to further study the role of these aquaporins in SISA.

Since active Si-transporters have been identified in a number of high- accumulators (121), there is possibly a similar protein present in N. tabacum.

Supporting this hypothesis is the observation that the root Si concentration of tobacco plants suggests that Si can accumulate to higher levels than previously documented. This suggests Si must move against a concentration gradient and thus, an active transporter is present.

The Lsi2 anion transporter is important for the movement of Si across casparian strips and into the xylem cells, against the concentration gradient (75,

121). The Solanaceae have been proposed to only accumulate Si through passive diffusion and thus, may not contain an active transporter. However, bioinformatic approaches suggest otherwise. Through searching the available tobacco EST data sets against a conserved region of the known Lsi2-like proteins, a putative efflux

Si transporter in N. tabacum was identified (Fig. 6-2). Further characterization of this transporter would include cloning the gene for the putative transporter and testing the protein using a Xenopus oocyte expression study. This is examined by injecting Si into oocytes expressing the transporter to determine the ability to move the element out of the cell. To confirm that in vitro transport studies correspond to endogenous function of the transporter, transgenic N. tabacum will be made reducing ntLsi2 expression through an shRNA mediated process. Plants

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will then be grown in the presence of high copper to analyze whether foliar Si levels are altered by the absence of a protein.

Figure 6-2: Phylogenetic tree of identified (wheat/hv, rice/os, corn/zm and pumpkin/cm) and the putative N. tabacum Lsi2.

In addition to Si transporter regulation and SISA, there is growing evidence that ROS may play a vital role in Si-mediated defenses in plants (109).

Preliminary data suggest that Si inhibited POD activity from a crude turnip extract while having no effect on H2O2 stability. This is further supported by a recent study performed by Maksimovic et. al who showed that both in vivo with a commercial peroxidase and in vitro in cucumber, Si inhibits peroxidase activity leading to an increase in ROS (109). If Si has the ability to prolong the existence of ROS, by reducing their degradation, this could enhance oxidation events that have the potential to further inhibit bacterial, fungal and viral propagation either directly by inhibition of propagation or indirectly by activating defenses. It would be interesting to determine if the application of chemicals inducing ROS in plants, such as certain algaecides, can enhance the ability of Si to protect plants against stress.

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The data presented above along with previously published research suggest inducible Si accumulation in at least two different plant species spanning divergent phylogenetic taxa. Work done by Frantz et. al has determined Si content in a number of important floriculture crops (35). Examining phylogenetic relationship with respect to Si accumulation has identified angiosperm orders containing the three categories of accumulators (Fig. 6-3). It would be interesting to study the ability of Cu to induce Si accumulation in a number of more closely related plants, such as those belonging to the order, Solanaceae, that have been previously classified as “low accumulators” or even “excluders”(21). To determine how broadly SISA is distributed amongst these species. In addition, the study could be expanded to include a number of other plants from various taxa to determine if the phenomenon is a general mechanism or has evolved with certain plant species. The order Asterales would be interesting to examine, because a number of the plants contained within this group are the classical, high- intermediate- and low- accumulators.

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Figure 6-3: Cladogram of Angiosperm orders containing ornamental plants identified as “low”, “intermediate” (single underlined) or “high” accumulators (double underlined). Bold orders contain “high” accumulators such as Rice, corn and barley in Poales and cucumber and pumpkin in Cucurbitales.

Taken together, these data support the growing body of knowledge that Si can benefit plants regardless of whether they are high- or low-accumulators. It also supports the notion that Si may not be beneficial for all plants under all conditions, including A. thaliana. With circumstantial data suggesting environmental conditions in addition to hormones effect Si regulation within a plant, one must take into consideration a number of factors when determining if, when, and how Si fertilization should be used. Still, the element if beneficial in

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most cases and it will be critical to understand why the exceptions exist and what they mean.

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125

Appendix A

Bioinformatics

A.1 Introduction

The following appendix contains the R codes used for various analyses from the above (and below) studies. The R program is a free bioinformatics tool that can be downloaded from http://www.r-project.org/. The program allows for a number of bioinformatic analyses including searching a given gene for repetitive sequence all the way to complex analysis of microarray data. One can choose to use packages available for certain analyses or write individual codes. In the above studies, R was used to determine the Area Under the Disease Progression

Curve (AUDPC) in addition to conducting statistical analysis of data using

ANOVA along with Tukey’s Honestly Significantly Different (HSD) post hoc test.

A.2 AUDPC

The American Phytopathological Society website has a number of useful bioinformatic information, one of which involves using the R program to determine the AUDPC for a given study (122). Calculating the AUDPC employs 126

the tetrazoidal method to determine the area between adjacent time points in a given study; http://www.apsnet.org/EDCENTER/ADVANCED/TOPICS/ECOLOGYANDEPI

DEMIOLOGYINR/DISEASEPROGRESS/Pages/AUDPC.aspx. The following code was taken from the above website and adjusted for the specific parameters of the study. The disease severity represented the ASSESS data for percent symptomatic symptoms and the time represented dpi. The last step, rm(ds1,ds2,ds3,t0,t1,t2,t3), removed the previous code, allowing for multiple analysis of subsequent treatments in a single R window.

>ds0<-0 >ds1<-22.25 >ds2<-33.48 >ds3<-34.85 >disease.severity<-c(ds0,ds1,ds2,ds3) >t0<-9 >t1<-11 >t2<-13 >t3<-15 >time.period<-c(t0,t1,t2,t3) >audpc <- function(disease.severity,time.period){ n <- length(time.period) meanvec <- matrix(-1,(n-1)) intvec <- matrix(-1,(n-1)) for(i in 1:(n-1)){ meanvec[i] <- mean(c(disease.severity[i],disease.severity[i+1])) intvec[i] <- time.period[i+1] - time.period[i]} >infprod <- meanvec * intvec >sum(infprod)} >audpc(disease.severity,time.period)->AUDPCc1 >print(AUDPCc1) >rm(ds1,ds2,ds3,t0,t1,t2,t3)

127

A.3 Statistical Analysis

To perform statistical analysis, the data was placed into an excel file with the first row containing the term “trt”, short for treatment, in column A and a short description of the data (i.e. Si for silicon) in column B. The descending rows contained the corresponding treatment and data for each individual point.

The data file was saved as a comma delimited (CSV) file (Fig. A-1).

Figure A-1: Excel spreadsheet of CSV file

The next step is to open the R program. And use the following code:

>si.data=read.csv(file.choose()) >attach(si.data) >si.aov=aov(Si~trt) >summary(si.aov) >TukeyHSD(si.aov)

The portion in red can be changed as long as it is kept consistent throughout the code. The highlighted code must represent the title of the columns exactly, including the case. Tukey’s HSD was only performed on data that contained more than two comparisons that had an ANOVA Pr(>F) value less than 0.05.

128

Appendix B

Identification and analysis of viruses infecting Pelargonium spp.

B.1 Abstract

Pelargoniums are a staple for the Ohio floriculture industry. A number of pathogens are able to infect these plants and cause foliar damage. Here we sought out to determine what viruses were present within Pelargonium species housed at the Ornamental Plant Germplasm Center (OPGC) in Columbus, Ohio. We found that the majority of infected plants had the Carmovirus, Pelargonium flowerbreak virus (PFBV). Pelargonium line pattern virus was also found in a number of plants, while Tomato ringspot virus was found in only one. Different

Pelargonium cultivars exhibited variable PFBV symptoms that were either year round or seasonal. To further study PFBV, we cloned and sequenced a portion of the two movement proteins (MP) and the entire coat protein (CP) from a number of infected varieties and sent a subset of the clones for sequencing. While the

MPs were highly conserved, the CPs exhibited more differences compared to other varieties, but were more conserved when compared to the same variety.

Since Pelargoniums are a challenging organism to work with at the molecular 129

level, we wanted to adapt a model system to study PFBV infection. An A. thaliana study was performed with 22 ecotypes to find susceptible species. In this study, all ecotypes were susceptible, 13 were tolerant while nine were locally and systemically symptomatic. This suggests that A. thaliana could be a useful system to further study molecular aspects of PFBV-host interactions.

B.2 Introduction

Pelargoniums are an important local ornamental that are plagued by many viral diseases (123). Carmoviruses, including Pelargonium flower break virus

(PFBV) and Pelargonium line pattern virus (PLPV) are prevalent in asymptomatic Pelargonium species in Spain and other countries (123, 124).

PFBV is mainly spread through vegetative cuttings and plant contact, but has also been shown to be transmitted through irrigation systems and possibly via pollen aided by thrip feeding (125). Symptoms were also reported to vary seasonally or were not present. PFBV is a positive-sense, single-stranded, unipartite RNA virus with a genome of five open reading frames encoding a 27/86 kDa protein

(p27/86), RNA-dependent RNA polymerase, two movement proteins (MP) p7 and p12, and a capsid protein (CP) (Fig. B-1) (126). The CP can be further subdivided into a putative N- terminal RNA-binding domain (R), a central shell domain (S), and a C-terminal protruding domain (P) (127). The CP, along with the p7 and p12 MPs of PFBV are under high selection pressure containing 24; 3; and 3 negatively selected amino acid substitution sites, respectively (128).

Recently, the PFBV CP has been shown to be a silencing suppressor (129). The

CP constructs from an infectious clone, along with a five amino acid covariant

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sequence from C. quinoa were both able to suppress N. benthamiana suppression of GFP. Interestingly, an isolate containing a single amino acid mutation (P323L) resulted in an inhibition of the silencing suppressor.

Figure B-1: PFBV genome organization. The genome contains an RNA-dependent RNA polymerase (p86, white box), two putative movement proteins (p7 and p12, green boxes) and a coat protein (CP, yellow box)

Of the known viruses to infect Pelargoniums, the majority are single-stranded

RNA viruses belonging to the carmovirus, nepovirus, and tombusvaridae families.

Virus infection in Pelargoniums are problematic for two reasons: 1) many infections are symptomless and those that do cause visible disease can take years to develop and 2) Pelargoniums are propagated through vegetative cuttings, which allows for further spread of the pathogen. Pelargoniums infected with

PLPV can be asymptomatic for over three years (124).

Auxin is an important plant hormone involved in advantageous root formation, which is important for vegetative propagation of a number of plants

(130). Repression of auxin by an miRNA resulted in resistance of A. thaliana to

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the bacterium Pseudomonas syringae (131). In both tomato and A. thaliana, the replicase protein of TMV has been shown to interact with auxin/IAA proteins, specifically ARF8, presumably inhibiting interaction with endogenous proteins and leading to an enhancement of viral disease (132). More recently, misregulation of ARF8 in A. thaliana was shown to be involved in plant developmental abnormalities during viral infection (133).

B.3 Materials and Methods

B.3.1 ConcertTM Plant RNA Reagent small scale RNA Isolation

Less than 0.1 g of plant tissue was homogenized in liquid nitrogen and added to 1.5 ml Eppendorf tubes containing 500 µl cold Plant RNA reagent.

Tubes containing the reagent were vortexed and incubated flat for 5 min at room temperature then centrifuged for 2 min at top speed at room temperature. The supernatant was then transferred to RNase-free tubes. NaCl (100 µL of 5M) was added to each tube and mixed. Next, 300 µL of chloroform was added, the tubes were mixed by inversion, and centrifuge for 10 min at 4º C. The aqueous solution was transferred to new tubes and an equal volume of isopropyl alcohol was added, mixed and incubated for 10 min at room temp. The tubes then were centrifuged for 10 min at 4º C and the supernatant was decanted. One ml of 75% EtOH was added to the pellet and the tubes were centrifuged at room temp for 1 min. The liquid was decanted and the tubes were briefly centrifuge to collect the residual liquid, which was removed with a pipette. The pellet was resuspended with 30

µL RNase-free water, which was pippetted up and down over the pellet to dissolve the RNA. 132

PCR

PCR was performed using a similar protocol to the above procedure.

Primers were designed using the DNAstar PrimerSelect program where viral sequences were saved in EditSeq and then loaded into the program. The top primer pair was chosen (Table B.1) and a BLAST search was ran to ensure the oligos would not amplify any subset of the plants genome. The PCR reaction

-5 consisted of 12.5 μl GoTaq, 9.5 μl dH2O, 1 μl 2X10 M forward primer, 1 μl

2X10-5 M reverse primer and 1 μl cDNA. The PCR program had an initial denaturation at 94 °C for 2 min, followed by 28 cycles including 30 sec each denaturation, annealing and elongation at 94; 55; and 72 °C, respectively. The reactions were then ran on a two percent agarose gel containing 1.5μl ethidium bromide/ 50μl gel and observed under UV light for the presence of bands.

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Table B.1: Primer sequences for virus detection in OPGC Pelargoniums

NCBI primer Primer Name Virus Name 5’-3’ Sequence number positions Artichoke GCA CCC CAA AILV-2F Italian latent X87254.1 566-585 CAA CTT GCT AT virus Artichoke GGC TGT GTT AILV-2R Italian latent X87254.1 766-785 CCC AAA GAA AA virus Elderberry TTA CAC TGT ELV-1F AY038066 534-553 latent virus CCC GGT TGA CA Elderberry CTG ATG TAT ELV-1R AY038066 709-728 latent virus TGC GTG GAT CG Moroccan CCC GAG TTA MPV-1F AF540886 876-895 Pepper virus CTT GAC GGT GT Moroccan GGC TAA ACC MPV-1R AF540886 1028-1047 Pepper virus AAC CGT ATC CA Pelargonium TGG ATC GCA PFBV-1F flowerbreak NC_005286 1871-1890 ATA CGT CAT GT virus Pelargonium TGG GCT AAC PFBV-1R flowerbreak NC_005286 2100-2119 CTG TAC GTT CC virus Pelargonium ATC GTT CCA PFBV-3F flowerbreak NC_005286 2303-2322 GAT CTG GGT AG virus Pelargonium ACT ACT CCT PFBV-5R flowerbreak NC_005286 3714-3733 CGA GGA ACC TC virus Pelargonium CCT CAA TTC PLPV-1F line pattern NC_007017 558-577 AAC CGA GGT GT virus Pelargonium GAG GCA TCC PLPV-1R line pattern NC_007017 719-738 CTA TTT GAC GA virus Pelargonium TTG GAG AGG PLCV-1F AF290026 500-519 leaf curl virus AGG GTG TTG AC Pelargonium CAG CTA CCG PLCV-1R AF290026 653-672 leaf curl virus CAG TAC CAA CA Pelargonium ACC GTC AGA PZSV-1F zonate spot NC_003649 1102-1121 GAA GTC GAG GA virus Pelargonium TTT CTC CGG PZSV-1R zonate spot NC_003649 1236-1255 CCA TCA TAG TC virus

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Table B.1 (cont.)

Tomato GCA GCG ATT ToRSV-1F NC_003840 4909-4928 ringspot virus TTG GTT TCA AT Tomato AAC TTC AGT ToRSV-1R NC_003840 5107-5126 ringspot virus ACG CGC CTG TT Tobacco CCG CGA GGA TRSVnspRNA1- ringspot virus NC_005097 166-189 GGG TCT TTC TTT 1F RNA1 TAG Tobacco TRSVnspRNA1- CGG GGT GGC ringspot virus NC_005097 619-637 1R AGC GGT CTT C RNA1 Tobacco TRSVnspRNA2- AAG GCG CTC ringspot virus NC_005096 414-433 1F CGG GCT GCT CT RNA2 Tobacco TRSVnspRNA2- CAT GAA GGC ringspot virus NC_005096 778-797 1R GGG CTG CTG AA RNA2 TTA GCA TTC Tomato spotted TSWVL-1F NC_002052 2329-2348 AAA GGG GAT wilt virus GG Tomato spotted TTA GTC TCT TSWVL-1R NC_002052 2484-2503 wilt virus GCG GCC TCA TT

B.3.4 Cloning of PFBV MP/CP into pCR2.1

RNA was isolated from OPGC 382; 453; 604; 1321; and 1325. Using

PFBV-3F,5R primer pairs, a 1.5 kb fragment including a portion of the MP, p7 and p12, and the entire CP , was amplified and gel purified. The cDNA was phenol extracted and ethanol precipitated. The cDNA was then pelleted by

® centrifugation, dried and resuspended in sterile H2O. A TOPO TA Cloning

(Invitrogen) reaction was then performed following the recommended protocol.

Briefly, 4 μl of cDNA, 1 μl salt solution and 1 μl pCR2.1 vector were placed into an Eppendorf tube and incubated at room temperature for five min. Top 10 competent cells were thawed on ice and 2 μl of the ligation reaction was added.

The solution incubated on ice for 30 min. While waiting for the reaction to finish,

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40 μl x-gal was added to L-carb plates and placed at 37 °C. After incubation, 250

μl S.O.C. media was added to the tubes containing the competent cells/ligation reaction and the tube was placed flat on a shaker at 37 °C for 1 hr. After which

200 μl of the transformation reaction was spread onto the L-carb/x-gal plates and placed in a 37 °C incubator overnight. White colonies were streaked onto new L- carb plates and colony PCR was performed to check for colonies containing inserts. The PCR products were then sent for sequencing.

B.3.2 PFBV Inoculum

Infected tissue was ground in 0.03 M phosphate buffer, pH 7.6, with 2.5%

PEG (1:3 tissue: buffer). Washed celite was added to the homogenate and used to inoculate 2-3 leaves per plant. The leaves were then rinsed with dH2O.

B.3.3 Phosphate Buffer

To make a pH 7.6 phosphate buffer, 0.42588g Na2HPO4·7H2O was added to 100 ml H2O in one flask and 0.41397g NaH2PO4·H2O was added to 100 ml

H2O in another. The stock solutions were autoclaved. In a clean 100 ml bottle,

13 ml of stock NaH2PO4·H20 to 87 ml of stock Na2HPO4·7H2O was added and mixed.

B.4 Results

B.4.1 PFBV was the most prevalent virus in OPGC Pelargonium Accessions

While studies using TRSV were being performed, we were interested in identifying problematic viruses from ornamentals, mainly Pelargoniums. Plants were obtained from the Ornamental Plant Germplasm Center (OPGC, Columbus, 136

OH) that exhibited possible viral symptoms including chlorosis, necrosis and flower break (Fig. B-2). A few asymptomatic plants were also examined. The

OPGC propagated their Pelargonium stock plants via vegetative cuttings. At the time, the facility maintained hundreds of individual varieties that were kept in close contact to one-another. It was observed that chlorotic plants would appear grouped together on benches. Cuttings were collected and taken back to UT where they were rooted hydroponically in nutrient solution. Cuttings of 23 accessions (OPGC 223; 230; 239; 264; 337; 344; 364; 366; 382; 453; 479; 485;

569; 604; 799; 1063; 1084; 1107; 1281; 1321; 1325; 1482; 1724 and 1728), including P. x hortorum, P. zonale and P. spp. were aquired and rooted in nutrient solution.. Interestingly, symptoms dissipated on cuttings, only to reappear after roots were present for 5-10 days. Once robust roots were present, plants were transplanted to soil-less media.

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Figure B-2: Symptoms on OPGC Pelargoniums on cuttings and rooted plants. Symptoms faded on OPGC cuttings after cuttings were rooted in nutrient solution. The pictures are representative of all symptomatic cuttings.

RNA was isolated from each accession and reverse transcriptase reactions were performed to obtain cDNA. A series of PCR reactions were performed to test for viral transcripts of AILV, CMV, ELV, MPV, PFBV, PLPV, PLCV,

PZSV, ToRSV, TRSV, and TSWV. Of the 11 viruses tested, only PFBV, PLPV and ToRSV were detected in 19 of the 23 varieties, as confirmed through PCR 138

(Table B.2). PFBV by far was the most prevalent virus, being detected in 17 varieties either alone or in combination with PLPV and ToRSV. Interestingly,

PFBV was not detected in OPGC 230 until a year later, when it was retested due to the appearance of green islands in contrast to the previous large white lesions symptoms (Fig. B-3).

Table B.2: Viruses detected in OPGC Pelargoniums in 2006. Using RT-PCR with virus-specific primers, Pelargonium flower break virus (PFBV), Pelargonium line pattern virus (PLPV), Tomato ringspot virus (ToRSV) were either detected (+) or no band was amplified (-).

OPGC Number 223 230 239 344 366 382 453 479 485 PFBV + - + + - + + + + PLPV - + + - + - - - - ToRSV ------OPGC Number 569 604 1084 1107 1281 1321 1325 1724 1728 PFBV + + + + + + + + - PLPV - + ------+ ToRSV - - - - - + - - -

Figure B-3: Symptoms on OPGC 230 infected with PLPV (A) and both PLPV and PFBV (B).

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B.4.2 Seasonal Symptoms and RNA isolation interference

Symptom expression of infected Pelargoniums varied between plants and was seasonal. Different varieties infected with PFBV contained foliar necrotic and or chlorotic lesions (Fig. B-4). Floral symptoms varied among varieties as well with deformed flower stalks and petals to the classical flower break symptom, where the petal color is interrupted by a white segment as seen in the

“Peppermint Stick” OPGC 1281. Some plants developed abnormal leaves, while others showed no symptoms. During late fall through early spring, symptom formation on OPGC 453; 1107 and 1325 were the most prevalent and faded throughout late spring into summer, only to reappear the following fall. Other plants, such as OPGC 1084, maintained chlorotic lesions throughout the year.

Figure B-4: PFBV symptoms on OPGC Pelargoniums.

Since most of the viruses infecting Pelargoniums were RNA viruses, RNA extraction was necessary to isolate viral transcripts. The use of the RNeasy kit

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(Qiagen) was employed when we first began our studies. The kit soon was unable to isolate RNA from Pelargoniums during the months of September to March.

Once April arrived, RNA again was able to be isolated from the plants. The following year, similar observations with isolation of RNA from Pelargonium using the RNeasy kit was observed. This led us to abandon the kit method and use a Trizol based RNA purification.

B.4.3 Boron toxicity changes PFBV symptoms in OPGC 1084

Recent studies on boron (B) deficiency in Pelargoniums were being conducted by Deng et. al (134). Since B and Si have similar characteristics, we wanted to see what effect B treatments might have on symptom formation in Pelargoniums and A. thaliana.

OPGC 1084 harbored a natural PFBV infection. Cuttings of soil-less media grown plants

(S) were taken and rooted in hydroponic solution. Once roots were present in all the plants, B concentrations were changed to 0 μM (B-), 1 μM (control), and 30 μM (B+).

Symptoms were followed for 36 days. B- treated plants exhibited small chlorotic lesions about 1mm in diameter, similar to C and S plants. B+ treated plants, however, developed vein clearing in addition to larger circular chlorotic lesions compared to the B-, C and S

(Fig. B-5). B+ plants showed bleaching of the upper leaves that began in the center of the leaf, spreading to the edges (Fig. B-6). Soil-less media and C treated plants had some bleaching of older leaves, but it occurred at the edges and never in the center or encompassing the entire leaf area. None of the B- plants developed leaves with bleaching at the center or leaf edges.

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Figure B-5: PFBV symptoms on OPGC treated with varying levels of boron (B). Cuttings of the parent plant (S) were rooted in control nutrient solution, than B concentrations were changed to 0 μM (B-), 1 μM (C) or 30 μM (B+).

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Figure B-6: Bleaching symptoms on OPGC 1084 treated with varying levels of boron (B). Cuttings from plants in soil-less media (S) were rooted in hydroponic solution, then B concentrations were changed to 0 μM (B-), 1 μM (C), or 30 μM (B+). Bleaching in S and C plants began at the leaf edges, while B+ bleaching began in the center of the leaf and spread outwards to the edges. None of the B- plants developed leaves with bleaching.

B.4.4 Sequencing of PFBV movement and CP from different Pelargoniums A portion of the PFBV genome including the movement proteins p7 and p12, and the CP was amplified and cloned from five OPGC varieties (OPGC 382,

453, 604, 1321 and 1325). Sequences were obtained and a MSA was performed using the published PFBV sequence (AJ514833) as a reference.

The p7 MP was essentially identical between all varieties, excluding

OPGC 1325 that differed at two amino acid positions. The sequence alignment revealed that all but OPGC 1325 were 100% identical at the amino acid level to the p7 published sequence.

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The p12 MP was also highly conserved with clones from OPGC 382 containing a serine to leucine change at amino acid position 48 (Fig. B-7). Again,

OPGC 1325 had differences at three amino acid positions, compared to the other sequences. Sequence identities for p12 were between 98.1 and 96.3% identical to the published sequence with OPGC 1325 having the lowest identity.

M R L T C Q W E D V G I G E N R R V R Y P S Q R T L S P N G H L M V V M G V L G L L W L R P F R S I Y T S T F S M P P L Majority 10 20 30 40 50 60 1 M R L T C Q W E D V G T G V N R R V R Y P S Q R T L S P N G H L M V V M G V L G L L W L R P F R S I Y T S T F S M P P L p12PublishedSequence 1 M K L T C Q W E D V G I G E N R R V R Y P S Q R T L S P N G H L M V V M G V L G L L W L R P F R L I Y T S T F S M P P L 382-1.p12 1 M R L T C Q W E D V G I G E N R R V R Y P S Q R T L S P N G H L M V V M G V L G L L W L R P F R L I Y T S T F S M P P L 382-2.p12 1 M R L T C Q W E D V G I G E N R R V R Y P S Q R T L S P N G H L M V V M G V L G L L W L R P F R L I Y T S T F S M P P L 382-3.p12 1 M R L T C Q W E D V G I G E N R R V R Y P S Q R T L S P N G H L M V V M G V L G L L W L R P F R S I Y T S T F S M P P L 453-1.p12 1 M R L T C Q W E D V G I G E N R R V R Y P S Q R T L S P N G H L M V V M G V L G L L W L R P F R S I Y T S T F S M P P L 453-2.p12 1 M R L T C Q W E D V G I G E N R R V R Y P S Q R T L S P N G H L M V V M G V L G L L W L R P F R S I Y T S T F S M P P L 453-3.p12 1 M R L T C Q W E D V G I G E N R R V R Y P S Q R T L S P N G H L M V V M G V L G L L W L R P F R S I Y T S T F S M P P L 1321-1.p12 1 M R L T C Q W E D V G I G E N R R V R Y P S Q R T L S P N G H L M V V M G V L G L L W L R P F R S I Y T S T F S M P P L 1321-3.p12 1 M R L T R Q W E D V G I G E N R R V R Y P S Q R T L S P N G R L M V V M G V L G L L W L K P F R S I Y T S T F S M P P L 1325-1.p12

I N L Q H I L N L T L L S L I L S S F I L A E R V T H N H Y S N D N S K A Q Y I R I S T G Q - Majority 70 80 90 100 181 I N L Q H I L N L T L L S L I L S S F I L A E R V T H N H Y S N D N S K A Q Y I R I S T G Q . p12PublishedSequence 181 I N L Q H I L N L T L L S L I L S S F I L A E R V T H N H Y S N D N S K A Q Y I R I S T G Q . 382-1.p12 181 I N L Q H I L N L T L L S L I L S S F I L A E R V T H N H Y S N D N S K A Q Y I R I S T G Q . 382-2.p12 181 I N L Q H I L N L T L L S L I L S S F I L A E R V T H N H Y S N D N S K A Q Y I R I S T G Q . 382-3.p12 181 I N L Q H I L N L T L L S L I L S S F I L A E R V T H N H Y S N D N S K A Q Y I R I S T G Q . 453-1.p12 181 I N L Q H I L N L T L L S L I L S S F I L A E R V T H N H Y S N D N S K A Q Y I R I S T G Q . 453-2.p12 181 I N L Q H I L N L T L L S L I L S S F I L A E R V T H N H Y S N D N S K A Q Y I R I S T G Q . 453-3.p12 181 I N L Q H I L N L T L L S L I L S S F I L A E R V T H N H Y S N D N S K A Q Y I R I S T G Q . 1321-1.p12 181 I N L Q H I L N L T L L S L I L S S F I L A E R V T H N H Y S N D N S K A Q Y I R I S T G Q . 1321-3.p12 181 I N L Q H I L N L T L L S L I L S S F I L A E R V T H N H Y S N D N S K A Q Y I R I S T G Q . 1325-1.p12 Figure B-7: MSA of PFBV p12 MP from various OPGC clones. Residues matching the consensus sequence are in black.

The CP, however, exhibited the lowest percent identity compared to the published sequence, ranging from 98.0- 93.2% identity at the amino acid level.

Multiple amino acid covariant differences among OPGC varieties occurred in the

R and P domain, while single amino acid variations from different varieties occurred in the S domain (Fig. B-8). This supports the idea that the P and R domains are more variable compared to the S domain (135). It is also interesting to note that OPGC 604, which is infected with both PFBV and PLPV, shows its highest level of conserved amino acid sequences in the R domain, compared to the published sequence. Preliminary data suggests this domain is involved in cross association with other Carmovirus CPs (unpublished data, G. Raikhy and S.

M. Leisner). OPGC 1325 was the only isolate found to contain an amino acid

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difference at position 323, which in N. benthamiana resulted in suppression of the

PFBV CP silencing suppressor (129).

Figure B-8: Amino acid differences in CP regions of PFBV from OPGC isolated populations. P323L mutation position resulting in suppression of CP-mediated silencing suppression in N. benthamiana (*) (129).

B.4.5 PFBV ecotype study in A. thaliana

Since PFBV symptoms are problematic in Pelargoniums (123) and symptoms can take years to develop, a model system to study the virus needed to be designed. A. thaliana was chosen to investigate its susceptibility to the virus, and a study was performed with 22 ecotypes (Table B.2). Of these, nine were susceptible while the remainders were tolerant. TRSV symptoms caused chlorotic vein netting with purple development between (Fig. B-9).

Unfortunately, infectivity in A. thaliana was extremely low in the symptomatic ecotypes.

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Table B.2: A. thaliana PFBV ecotype Study. PFBV produced chlorotic symptoms (YES) in A. thaliana plants or no symptoms were observed (-) on inoculated (Local, blue) and upper, non-inoculated (Systemic, red) leaves. Asymptomatic plants (white).

A. thaliana ecotype Aa- Ag- Be- Bla-4 Col- Di-0 Dy-0 En- Est- Ita-0 Kas- 0 0 0 0 2 0 1 Local + + + + + + - - + + + Systemic - - + - + + - - + - + A. thaliana ecotype La- Lc- Lou- Mh- No- Np- RSCH4 Sf-1 Tsu- Wil- Zu-0 er 0 2 0 0 0 0 2 Local + + + - + + + + + + + Systemic + - - - + - + - - + +

Figure B-9: PFBV symptoms on A. thaliana.

B.5 Discussion

Pelargoniums are an important floriculture crop for both the United States and Ohio (136). In 2011, they accounted for a wholesale value of $7.3 and $110 million locally and nationally, with Pelargoniums from vegetative cuttings being

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responsible for more than half. It is imperative that the strictest sterile techniques be used when propagating plants through cuttings to decrease the spread of pathogens. In Pelargoniums, the incidence of disease is so common that even artificial plants have chlorotic rings painted onto their leaves (personal observation).

Our goal for these studies was to determine what, if any viral infections were present and common in Ohio Pelargoniums. From the symptomatic plants obtained from a single facility, PFBV and PLPV were the most prevalent viruses found. This makes sense since PFBV is easily spread through plant contact and plants were housed in close proximity at the OPGC.

Pelargoniums are not the best plant to use when trying to understand the molecular characteristics of a viral infection for multiple reasons. There are limited molecular tools, such as sequence data and available mutants, in addition to the fact that symptoms may take years to develop (124). The use of a model system would allow for an easier analysis of various virus-host interactions. A. thaliana is a promising system to study various aspects of PFBV infection, since ecotypes tested were tolerant or susceptible to the virus. This system could be used to study symptom formation, among other things.

While performing these studies, we ran across an interesting physiological observation where symptoms would dissipate as Pelargonium cuttings were rooting, but would reappear following transplanting to either soil-less media or hydroponics. A reduction in foliar Auxin/IAA levels during development of

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advantageous roots may lead to the suppression of PFBV symptoms. It would be interesting to test viral levels in the upper portions of the shoot to see if they decrease during the development of the roots.

In addition to decreasing symptoms following rooting of Pelargoniums, this might also be an addition tool to clear PFBV infection from Pelargoniums. A method used to remove viral infection from plants is a combination of thermotherapy and meristem-tip culture (137). If virus titer decreases during root formation, taking cuttings prior to excising the meristem may decrease the viral titer enough to allow from the production of virus-free stock without the use of heat.

In addition to studying PFBV from the plant perspective, experiments were also carried out examining the population of the virus. Sequencing of MP and CP from different Pelargonium varieties showed more variability in CP sequences compared to MP. OPGC 604, which was infected with both PFBV and

PLPV, had the least variable CP sequence compared to the published sequence.

This could suggest that in a co-infection, there is less variability in CP sequences within the virus population allowing for more stable association between PFBV and PLPV CPs.

In addition to Si research being conducted, our lab was also investigation boron and its involvement in plant disease. Boron treatment exhibited varying effects in Pelargonium and A. thaliana. B toxicity changed PFBV symptoms in

OPGC 1084 while B deficiency showed no change between control and soil-less

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media grown plants. This was opposite then expected since data at the time for

Nittney Lion Red suggests that Pelargoniums are sensitive to B deficiency (134).

In A. thaliana, B treatment in addition to Si did not decrease TRSV symptoms compared to control and significantly increased susceptibility of the plants compared to soil-less media. This was opposite then expected since Si has a number of beneficial effects in plants (38).

These studies were performed to develop a system to examine virus infections present in Pelargoniums in Ohio. While we were expecting to find more types of viral infections in the plants, we were pleased to be able to set up a model system to begin studying PFBV from both the pathogen and plant angles.

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Appendix C

Contig Sequences

The following are the accession numbers along with consensus nucleotide sequences from NCBI in FASTA format used from the identification of N. tabacum MIPs in chapter 5.

C.1 Accession numbers used to construct contigs

Contig 1

AM806600.1, BP533341.1, DW002048.1, DW004211.1, DW004490.1, EB426773.1, EB426872.1, EB428694.1, EB440301.1, EB445576.1, EB446011.1, FG140623.1, FG141795.1, FG143579.1, FG145547.1, FG150838.1, FG151394.1, FG155266.1, FG157236.1, FG160069.1, FG168262.1, FG174086.1, FG175179.1, FG175885.1, FG185837.1, FG186947.1, FG187035.1, FG187794.1, FG190967.1, FG193439.1, FG195279.1, FG198270.1,

Contig 2

DW004892.1, EB440432.1, EB450197.1, FG637387.1, FS415532.1, FS427703.1,

Contig 3

CK720596.1, CK720598.1, CV016693.1, DV158887.1, EB427807.1, EB427808.1, EB428025.1, EB430265.1, EB438535.1, EB439056.1, EB439236.1, EB439527.1, EB439882.1, EB440282.1, EB441806.1, EB447509.1, EB447643.1, FG146811.1, FG191503.1, FG194945.1, FG622661.1, FG627103.1, FG627964.1, FG632449.1, FS395150.1, FS397162.1, FS430977.1, FS431153.1, FS436186.1, 150

Contig 4

FG176532.1, FG186262.1, FG195809.1, FG199168.1, FG199254.1,

Contig 5

DW003590.1, EB425560.1, EB425695.1, EB425776.1, EB426165.1, EB439507.1, EB448144.1, EB449066.1, EB449305.1, EB451071.1, EB451097.1, EB451157.1, EB451784.1, FG628390.1, FG636139.1, FG640702.1, FS429513.1,

Contig 6

BP131058.1, DW000053.1, EB425012.1, EB426217.1, EB434571.1, FG628111.1, FS413503.1,

Contig 7

FG154063.1, FG185269.1, FG185360.1, FG630405.1, FS397674.1, FS398876.1, FS402870.1,

Contig 8

DV999429.1, DV999619.1, DV999839.1, EB429973.1, EB439016.1, EB441109.1, EB449156.1, EB449298.1, EB449868.1, EB449931.1, EB451430.1, EB680957.1, EB682943.1, EH619664.1, FG159063.1, FG161154.1, FG165931.1, FG190086.1, FG629297.1, FS419611.1,

Contig 9

AM811259.1, DV160518.1, FS408078.1,

Contig 10

DV162255.1, DW001631.1, EB424726.1, EB424782.1, EB424869.1, EB425051.1, EB425060.1, EB425280.1, EB425386.1, EB425610.1, EB425653.1, EB425954.1, EB426176.1, EB426223.1, EB426271.1, EB426521.1, EB426594.1, EB426690.1, EB426708.1, EB427852.1, EB428155.1, EB428311.1, EB428322.1, EB428758.1, EB429000.1, EB429074.1, EB429443.1, EB430126.1, EB433601.1, EB434287.1, EB437964.1, EB438125.1, EB438695.1, EB438741.1, EB439459.1, EB439583.1, EB441429.1, EB447744.1, EB447771.1, EB447846.1, EB448069.1, EB449769.1, EB450906.1, EB451199.1, EB451419.1, EB451756.1, EH614241.1, EH614397.1, EH615394.1, EH615425.1, FG135442.1, FG150214.1,FG150806.1, FG154254.1, FG159571.1, FG164794.1, FG168061.1, FG168129.1, FG172136.1, FG172218.1, FG176969.1, FG186188.1, FG186277.1, FG191302.1, FG194838.1, 151

FG196637.1, FG196985.1, FG197003.1, FG197076.1, FG200414.1, FG200423.1, FG200510.1, FG621591.1, FG622001.1, FG623565.1, FG623846.1, FG624887.1, FG625224.1, FG625396.1, FG626635.1, FG628950.1, FG630216.1, FG631652.1, FG631789.1, FG633438.1, FG635706.1, FG637269.1, FG637701.1, FS401522.1, FS410529.1, FS415520.1, FS418864.1, FS430679.1, FS434054.1,

Contig 11 DV159703.1, DW003292.1, DW004999.1, EB426354.1, EB439015.1, EB445876.1, EB449389.1, EB450978.1, EH616437.1, EH616439.1, EH616444.1, EH616453.1, EH616460.1, EH616475.1, FG151539.1, FG173003.1, FG173068.1, FG176460.1, FG186171.1, FG195718.1, FG621330.1, FG628191.1, FG628840.1, FG644747.1, FS374805.1, FS380089.1, FS381422.1, FS381982.1, FS384080.1, FS384421.1, FS387685.1, FS389493.1, FS406492.1, FS407906.1, FS415842.1, FS418486.1, FS424287.1, FS428483.1, FS429202.1, FS431032.1, FS431568.1, FS434300.1, FS437828.1,

Contig 12

BP529677.1, CK720585.1, CK720587.1, CK720594.1, CV016972.1, CV018730.1, CV018915.1, DV160101.1, DV160566.1, DV160774.1, DV160834.1, DV160980.1, DV161833.1, DV162466.1, DV999131.1, DV999317.1, DV999572.1, DV999587.1,DV999675.1, DV999905.1, DW003118.1, DW003537.1, EB427803.1, EB427918.1, EB438483.1, EB438734.1, EB439825.1, EB440399.1, EB447519.1, EB447900.1, EB449217.1, EB449319.1, EB449371.1, EB449429.1, EB449776.1, EB679082.1, EB679529.1, EB680258.1, EB680869.1, EB681430.1, EB683063.1, EB683244.1, EH615222.1, EH615277.1, EH615340.1, FG155278.1, FG186960.1, FG190295.1, FG192180.1, FG195466.1, FG197097.1, FG197186.1, FG198583.1, FG198672.1, FG623221.1, FG623756.1, FG627347.1, FG629600.1, FG630601.1, FG636535.1, FS377879.1, FS378427.1, FS380343.1, FS392728.1, FS402895.1, FS406797.1, FS407146.1, FS423139.1, FS423886.1, FS425473.1, HO845403.1

Contig 13

EB445130.1, EB445635.1, EB447939.1,

Contig 14

FG168969.1, FG189742.1, FG625154.1,

Contig 15 DV999499.1, EB443112.1, EB449153.1, FS376756.1, FS391834.1, FS407384.1, FS410665.1, FS416829.1,

152

Contig 16

AM785237.1, AM826126.1, AM845529.1, CK720591.1, CV016493.1, CV016796.1,CV018000.1, DV158831.1, DV162521.1, DW001459.1, DW001703.1, EB427871.1, EB427904.1, EB428034.1, EB429149.1, EB429155.1, EB429619.1, EB429980.1, EB430125.1, EB430660.1, EB430841.1, EB433052.1, EB435219.1, EB435598.1, EB438093.1, EB438842.1, EB438906.1, EB438915.1, EB439431.1, EB439688.1, EB440021.1, EB440039.1, EB440051.1, EB440120.1, EB440263.1, EB440268.1, EB442803.1, EB443489.1, EB447430.1, EB447512.1, EB447624.1, EB448934.1, EB681672.1, FG155152.1, FG159906.1, FG179837.1, FG179930.1, FG186546.1, FG186639.1, FG195480.1, FG196095.1, FG196179.1, FG198407.1, FG198497.1, FG202330.1, FG202418.1, FG588722.1, FG621637.1, FG621743.1, FS379934.1, FS388748.1, FS398478.1, FS402892.1, FS411325.1, FS416231.1, FS430911.1, FS435005.1,

Contig 17

DW004942.1, EB445103.1, EB445529.1, EB447272.1, FG642817.1, FS391131.1, FS406932.1, FS412195.1, FS437702.1,

Contig 18 DW003416.1, EB426225.1, EB447860.1, EB447944.1, EB451420.1, FG621528.1, FS378333.1,

Contig 19

AM789783.1, CK720593.1, DV160095.1, DV160719.1, DV161034.1, DV999302.1,DV999402.1, DV999661.1, DV999922.1, DV999959.1, DW000449.1, DW004732.1, EB427872.1, EB432792.1, EB432794.1, EB439354.1, EB443390.1, EB449938.1, EB683426.1, EH615062.1, EH617377.1, FG137715.1, FG169647.1, FG184684.1, FG184776.1, FG199326.1, FG199415.1, FG621714.1, FG626082.1, FS394114.1,

Contig 20

FG630771.1, FS407225.1,

Contig 21

CN949759.1, GR977060.1,

Contig 22

FG191215.1, FG631276.1,

153

Contig 23

AM841196.1, BJ999181.1, BP530787.1, EB434264.1, EB437489.1, EB441720.1, FG150891.1, FG153095.1, FG154328.1, FG162180.1, FG177028.1, FG191390.1, FG196724.1, FG200502.1,

Contig 24

AM816965.1, BP535473.1, EB428280.1, EB451173.1, FG623994.1, FG642620.1, FS420626.1,

Contig 25 CK720589.1, DV160688.1, DV160839.1, EB438200.1, EB447935.1, EB449986.1, EB451666.1, EB677385.1, EB677394.1, EB683471.1, EH615844.1, FG623130.1, FG625619.1, FG628534.1, FG633267.1, FG640053.1, FS409849.1, FS427841.1, FS432991.1,

Contig 26 DW000629.1, EB680590.1,

Contig 27

EB683097.1, FG182869.1, FG182957.1, FS433855.1, FG191594.1

Contig 28 FG141184.1, FG631972.1,

Contig 29

DW002052.1, EB447547.1, EB683013.1, FS378994.1, FS379842.1, FS381884.1, FS382683.1, FS391368.1, FS402820.1, FS403155.1,

Contig 30 CK720597.1, DV160563.1

Contig 31 BP529542.1, EB443618.1, EB448262.1, EB448825.1, EB449597.1, EB451871.1, FG162699.1, FG167236.1, FG639955.1, FG640504.1, FS393913.1, FS408241.1,

Contig 32

EB449867.1, FG161084.1, FG165862.1,

Contig 33

CV019344.1, FG146251.1,

154

Contig 34 EB426739.1, EB439651.1, EB439907.1, FG144599.1, FG165069.1, FG165137.1, FG176605.1, FG639226.1, FS375485.1, FS404271.1, FS407972.1, FS409617.1, FS409788.1,

Contig 35

FG644061.1, FS377707.1, FS394631.1, FS403706.1, FS428295.1, FS436307.1,

Contig 36

FG141061.1, FG143916.1, FG146711.1, FG146929.1, FG147206.1, FG151362.1, FG152830.1, FG153672.1, FG160928.1, FS379534.1,

Contig 37 FG192275.1, FG195559.1

Contig 38

AM848901.1, CV019641.1, DV160340.1, DW004730.1, EB450656.1,EB451121.1,FG152816.1, FG627552.1, FG640875.1, FG640889.1, FS377053.1, FS378602.1, FS378950.1, FS395238.1, FS399023.1, FS401801.1, FS403433.1, FS406484.1, FS409394.1, FS417181.1, FS426896.1, FS431398.1,

Contig 39 DV159730.1, DW004478.1, EB424652.1, EB444684.1, EB449182.1, EB682870.1, EB682872.1, EG650383.1,

Contig 40

EB425270.1, EB682906.1, FG640378.1, FG641591.1, FG641594.1,

Contig 41

EB447741.1, FG176668.1, FG643862.1,

Contig 42

DW001956.1, DW002490.1, DW004193.1, EB445911.1, EB679110.1, FS380146.1, FS411708.1,

Contig 43

AM791979.1, EB683176.1,

Contig 44 FG137598.1, FG185847.1, FG185939.1, FG635594.1, FS381059.1, 155

Contig 45

EB437260.1, EB445778.1, FG159950.1, FG160007.1,

Contig 46

AJ937856.1, CV016921.1, DV159837.1, DV160183.1, DV160579.1, DV160835.1, DV161030.1, DV161361.1, DV999167.1, DW000225.1, DW004798.1, EB425000.1, EB426621.1, EB428297.1, EB439088.1, EB439239.1, EB439772.1, EB441580.1, EB447793.1,EB447836.1, EB447913.1, EB448438.1, EB448858.1, EB448938.1, EB449072.1,EB449340.1, EB449402.1, EB449472.1, EB449483.1, EB449513.1, EB449693.1, EB451069.1, EB451137.1, EB451207.1, EB451214.1, EB451520.1, EB451549.1, EB451638.1, EB451922.1, EB452194.1, EB680822.1, EB681808.1, EB682098.1, FG161500.1, FG161575.1, FG629010.1, FG632157.1, FG636895.1, FG640779.1, FS373753.1, FS375919.1, FS379113.1, FS383150.1, FS391895.1, FS394102.1, FS401600.1, FS404455.1, FS405692.1, FS410603.1, FS412088.1, FS416218.1, FS418483.1, FS433817.1, FS436081.1,

Contig 47

DV999473.1, DW000295.1, DW003899.1, DW004201.1, EB438296.1, EB439142.1, EB439218.1, EB451577.1, EB683536.1, FG183990.1, FG184076.1, FG624381.1, FG630559.1, FS385599.1, FS392100.1, FS437147.1,

Contig 48 CK720599.1, DW002645.1, EB425288.1, EB449084.1, EB450245.1, EB450406.1, EH665974.1,

Contig 49 DV999814.1, DW001985.1, EB445427.1, EB448868.1, EB450913.1, EB451104.1, EB451289.1, EB451793.1, FG640536.1

Contig 50

AM805610.1, FG629967.1

Contig 51

AM827884.1, AM836835.1, EB426672.1, FG628552.1

Contig 52

FG162438.1, FS390494.1, FS434377.1

156

Contig 53

FG165891.1, FG165957.1, FG635838.1, FS379984.1

Contig 54

EB440523.1, FS423070.1, FS429823.1

Contig 55

FG160052.1, FG160999.1

Contig 56

FG192012.1, FG192104.1

Contig 57

DV159920.1, EB446671.1

Contig 58

FG196196.1, FG196290.1

Contig 59 BP533505.1, DV160046.1, DV160304.1, EB425876.1, EB435691.1, EH618318.1, FG639664.1, FS375803.1, FS381235.1, FS384920.1, FS406187.1, FS417850.1, FS435565.1, FS437882.1, HO841531.1

Contig 60

AM833294.1, FS391736.1

Contig 61

AM827162.1, DW002328.1, DW003232.1, EB443312.1, EB446121.1, EB682985.1, EB683703.1, EB684075.1, FG148015.1, FG162767.1, FG167305.1, FS403583.1, FS408458.1, FS427071.1

Contig 62

CK720590.1, DV159802.1, EB438787.1, FG150435.1, FG191121.1, FS435212.1

Contig 63

EH620132.1, FG154120.1, FS385424.1

157

Contig 64 BP133418.1, FG162513.1

Contig 65

DV159665.1, EB429136.1, EB449687.1, EB681717.1, EB683092.1, EB683313.1, FS424715.1

Contig 66

FS387924.1, FS391474.1, FS397556.1, FS431252.1, FS437878.1

Contig 67

EB438829.1, FS375899.1, FS378504.1, FS400287.1, FS403546.1, FS422883.1, FS426864.1, FS435207.1

C.2 Contig consensus sequences

>ntMIPcontig1.seq ACAAGTCGGTCCCCAAWMCATRTAACCCACTGCCTGTCCTTGCCTAAA ATCATTTCTGGTTCTTGTTTCCACTGTTTCG TTAATCTATTTCTTCTTCTTCTTTATCTTTTTTGTCTGTTACTATCAAAA GTTGGATCCTCTTCAATTTATTCATTAAG CAGTCTGAGTTAGTCATCGTACTGGTTTCTTGTTTTGTACTGTAATTCA GATCCTATATATGCTCAAGGTTTCGCCATC ATCTTAATTAGTGCATATATAGTTAAGCTGGATAGCTTCAGTTCTTGTA TTCCTTAGTGGCACAATTATAAAAACCTAT AAAGCATGTAACTTTTTTCTCCTAGCTTCTTGTTCCCACCACAAAAAAG AAAATAAAAAAAAAACTTTTTTTTCCATTT GTTATGTTTGCTGCCGGATTCTGATTATTCTGAGGATCAAACGATCTGG CTGTTAAACTGAATATCGTGTAGCTAAACA TTTAAAAAAGCCAAAAAATCATGTAATACTTCTCCATTGAAAGAAACC TAGCTTATCTCTCTTTCCAAAAAACCAAAAA AAAAAAAAAAAAAAAAAAACCAAAAAAAAAAAAGAAAGGAAAAGG ATCGGAGATGCCGGAATTTGAATCACCAGTATCG GCGCCGGCAACGCCGGGGACACCAACGCCGCTATTCTCGTCGATTCGA GTGGACTCAATGGGGTCTAATTATGATCGAA AGTCAATGCCCCGATGCAAGTGCTTGCCTTTGGATGCTCCAACATGGG GCACTCCTCACACGTGTCTTTcCGACTTcCC CGCACcAGATGTCTCCCTCACTCGCaAGTTGGGAGCAGAGTTCGTGGGA ACATTTATcCTTATATTTGCTGCAACAGCC GGGCCAATTGTGAACCAAAAGTACAACGGAGCCGAATCTCTAATCGG AAATGCAGCTTGCTCTGGGCTGGCCGTTATGA 158

TCGTGATTCTGTCGACGGGCCATATTTCTGGAGCACATCTTAATCCGTC GCTCACCATTGCATTTGCAGCACTTCGTCA TTTTCCGTGGGTTCAAGTGCCGGCCTATGTTGCAGCGCAGGTTTCAGC ATCAGTTTGTGCTTCTTTTGCTCTCAAGGGT GTTTTTCATCCTTTCATGTCTGGTGGCGTTACTGTTCCTTCTGTAAACAC TGGCCAGGCTTTTGCTCTCGAATTCCTCA TCACATTCAATCTCCTTTTTGTTGTCACTGCTGTTGCTACCGACACCCG CGCGGTGGGAGAGTTGGCGGGCATTGCAGT TGGAGCTACAGTCATGCTCAATATTCTAGTGGCTGGGCCATCAAGTGG TGCTTCCATGAATCCAGTAAGAACTTTGGGG CCAGCCGTTGCAGCAGGAAATTACAAGTCATTGTGGATATACATAGTG GCTCCAACTCTGGGGGCTCTTGCAGGGGCAG CTGTTTATACGCTCGTCAAACTTCGAGGAGATGAYAGTWCTGAGACAC CACGCCAGGTTAGGAGCTTCCGCCGCTAGCC TGCTGAAGGAAGGRTGTTGYTCCAACTTCTAATAGCTTATCTTATTRTA CTATTGCGTGCTTGAAAATAAAGGTGGAGA CTCGGAAAATGTGACTTCCAGTCTCACTACCAAAAACAATTTACAGTT GCATTTGGYACTCTTTCTCCTCATWCGCTRT GTGTGGATGRCTTGAGGAAGGGGCTGATATTCGCTTGTGGTCTGCGAS CYTTTTTCCTTTTYTTATGGTTTGCTGTGTG WGAAACTTTGCTACGTTATAATGTGTGATGGTAATAAGAAGCTTTCTG AGCTGCTACTTGTTTATGAKCKGCCGCATGA

>ntMIPcontig2.seq GATGAAGTAAAAACGCAATCTTTCCTTAGTCCACGTCTTACTTAATGG TGCAAAAGATCCAAACTTTTTCCATTCTTCA CCAATTTTTTCTCTGTATTTATTCAACACATTCAATTCACATATAATTA AACCCACTTTCCATTTTCTCCAAAGCTTCT TGATTTTTTCTSAGYGGAGTAGCAGCTGCTCTTGTTACTATTTTGGCGC TTTTTTTTGGATTCATTTGAAAATTTTTGG TGATCATTCTGAAGGGCAAACGTTAAAGAATTTTCTATGCTTTAGATG CGGTCAAAGAAAATGTCCACGACTGTCAAGG ATGAGAAGAAAATGTCTCTTGATGTGGCAGCATGGGCATTCAATATTG TCACTTCAGTTGGAATTATTATTGTTAATAA AGCCTTAATGGCTACATATGGTTTCAGTTTCGCGACAACCTTAACTGGT CTACATTTTGCCACGACGACATTGATGACC TTTTTCCTTAAATGGCTTGGGCATATCCAGAATTCCCAACTTCCTTGGT CTGAACGACTGAAATTTGTATTGTTTGCAA ACTTTTCTATTGTCGGAATGAATGTGAGTTTAATGTGGAACTCTGTCGG ATTCTATCAGATYGCAAAGCTAAGTATGAT ACCAGTGTCGTGCTTTYTGGAAATTGTGCTGGACAATGTGCGATACTC AAGGGACACCAAATTAAGCATTTTGTTGGTC CTACTAGGTGTTGCAATCTGTACTGTTACTGATGTWAGTGTAAATGCA AAGGGTTTTATTGCTGCCTTCATTGCSGTCT GGAGCACTGCCCTRCAGCAATATTATGTACATTTTCTTCAGCGTAAAT ATTCMYTGGGATCATTCAACCTGTTGGGGCA

159

TACYGCACCAATACAGGCKACATCMCTGCTGTTAACGGGACCCCTTGT AGACTACTGGTTGACTGAGAAGAGGGTCGAT GCCTATAACTATACCTCAATATCACTATTTTTCATCATCCTATCATGTA CAATAGCGATAGGGACRAACCTCAGCCAAT TCATCTGCATTGGTAGATTTACAGCAGTGACATTTCAAGTGCTTGGTCA TATGAAGACAATTCTTGTCCTGATTTTGGG TTTCCTCTTCTTCGGGAAAGAGGGACTCAATCTACAYGTTGTCTTCGGA ATGTCTATWGCAATCGTTGGCATGATATGG TATGGTAATGCTTCCTCACAACCCGGTGGAAAAGAGCGGYTACCACCT CCWTCTACCATCAAACCTGAAAAACAAAATC GCTTACTAGCAACTGAGCTCGACGAGAAAGTATAGTGAGTCATTTYCA AGAAATCAAGCAGAGTAATTGACAATTTCTT ATTTCTTTTCTGCCATATAGTCACAAAGTGTTTAATTCAATCATATGAT TTGATCTATCACCATCTAAwTTTTTTTTTC TTCTTTTTGACAGTGAGCTTTTCATTTCATTTTCCCTTTTCCCCCTTCTA ATTTATGTTGAAATTCACATGTTTAAACT ACAA

>ntMIPcontig3.seq GATKGACCCAACTTCCTTTTTTGGATTCCATTTACAGAGAGAaGAAAAC TCAGTTGAGTGACTGAAGAAAAAAAAATGG CAGAGAACAAGGAAGAGGATGTTAAGCTAGGAGCAAACAAGTACAG AGAAACACARCCTTTRGGTACAGCAGCTCAAAC AGACAAGGATTATAAGGAGCCACCACCAGCTCCTTTGTTTGARCCAGG RGAGTTGTCGTCATGGTCTTTTTACAGAGCT GGAATTGCAGAATTCATGGCCACTTTCTTGTTCTTGTACATCACTATCT TGACTGTTATGGGTCTTAAAAGATCTGATA GTTTGTGTTCTTCTGTTGGTATTCAAGGAGTTGCTTGGGCTTTTGGTGG TATGATCTTTGCCCTTGTCTACTGCACTGC TGGTATCTCAGGAGGACACATTAACCCAGCAGTGACATTTGGTCTGTT CTTGGCAAGAAAGTTGTCYTTAACAAGGGCT CTGTTCTACATGGTGATGCAGTGCCTWGGTGCAATCTGTGGTGCTGGT GTTGTTAAAGGTTTYATGGTGGGTCCATACC AGAGACTTGGTGGTGGGGCCAACGTGGTTCAACCTGGCTACACTAAAG GTGATGGACTTGGTGCTGAGATTGTTGGCAC CTTTGTCCTTGTTTACACTGTTTTCTCTGCCACTGATGCCAAGAGAAAT GCTAGAGATTCACATGTTCCTATTTTGGCA CCTCTTCCTATTGGATTCGCGGTGTTCTTGGTTCATTTGGCCACCATCC CAATCACCGGAACCGGTATCAACCCCGCCC GGAGCCTTGGAGCTGCTATCATCTTCAACCAAGACCGGGCATGGGATG ATCACTGGATCTTCTGGGTTGGACCATTCAT TGGAGCTGCACTTGCTGCAGTTTACCACCAGATAATCATCAGAGCCAT TCCATTCAAGAGCAAATCTTAATTGTCTTTG TCTTATGTCTCTTCCCCTGTTATATTTCAAGACTCCTCCACCCTTTTCCC CCTTTTCTGATGCTGTTGTYTCATGTAAT

160

TTTCTTYTCTTTTGATGTCAATTATTTGTGTAAATTATCAGGTTAATGTT CTATcCTAAAGCTTTCTTGCTCCAWAWMA ARWRAAARAWWWCTCGTGCC

>ntMIPcontig4.seq TTTTTTTTTTTTTTKKYTKKKYTTTKTKYWTKWTTTYWTYTKTTACTGC ACCGCCGGTATCTCCGGAGGACATATAAAC CCAGCAGTGACATTTGGTCTGTTTTTGGCAAGGAAAGTATCACTAATC AGAGCAGTTTTGTACATGGTCGCACAGTGTT TGGGTGCAATCTGTGGTGTGGGTTTGGTGAAGGGATTCCAGAGTGCTT ATTATGTTAGGTATGGTGGAGGTGCTAATGT CATGGCTCCTGGCCATACCAAAGGTGTTGGATTAGCTGCTGAGATTAT TGGTACTTTTGTTTTGGTTTATACTGTCTTC TCTGCCACTGATCCAAAGAGAAATGCTAGAGACTCCCATGTCCCTGTG TTGGCACCACTTCCAATTGGATTTGCTGTGT TCATGGTTCACTTAGCTACCATACCAATTACTGGRACTGGCATCAATCC TGCTAGGAGTTTTGGTGCAGCAGTTATTTA CAATCAGGACAAAGCTTGGGATGAACACTGGATTTTCTGGGTGGGTCC CTTCATCGGAGCCTTCGCCGCCGCCGTCTAC CACCAGTTCATCCTCCGTGCCGGTGCCATCAAAGCTCTTGGTTCCTTCA GGAGCAATGCCTAAAATTTTCCAATCCAAT TGAATAAAACCAGCCATGAAAATTTGTAGATATTTCTTTCCCAATGGG AAAAGTTTTTTAAAAATGTACTGTTTGTTTT TTCTATGAAWTTCCCTCTTTTGTGTATTTTTCTACTTCTTTTCCCTTTTTT ATTTTTAATTCCTTTCTCCTTTTTTGCT TTTTTCAATCTAGTCTcTTGTATCTTTGAATTATT

>ntMIPcontig5.seq AAAGCCAAAATAAACCACAGCCATTCTTCATTTCTTTATAGTTATTAAC CTTTCATTTGCCAGTGTAATTTGATCAGAG AAGGCCAATGGCGAAGATTGCTGTTGGAAGTAGCCGTGAGGCTATTCA GCCTGACTGCATCCAAGCACTCATTGTTGAG TTTATTTGCACTTTTCTCTTTGTTTTTGCTGGTGTTGGATCTGCCATGGC TGCCAACAAGCTAAATGGAGATCCACTGG TGAGTTTATTTTTTGTGGCAATGGCACATGCATTGGTGGTGGCAGTGA CCATCTCTGCTGGTTTCAGAATTTCTGGTGG ACACCTCAACCCTGCTGTCACACTTGGCCTTTGTATGGGTGGTCATATC ACTGTCTTCAGATCAATCCTTTACTGGATT GACCAATTATTGGCTTCTGTTGCTGCTTGTGCTTTGCTCAATTACCTCA CTGCTGGATTGGAAACACCAGTTCACACAC TAGCAAATGGAGTGAGCTATGGCCAAGGGATAATTATGGAGGTGATTT TGACCTTTTCTTTGTTGTTCACTGTCTATAC TACCATTGTGGACCCCAAGAAGGGAATTCTTGAAGGGATGGGCCCACT TCTAACTGGGCTTGTTGTTGGGGCCAACATC ATGGCTGGAGGGCCTTTTTCAGGAGCTTCAATGAACCCAGCAAGATCA TTTGGGCCAGCTTTTGTTAGTGGAATCTGGA

161

CTGACCATTGGGTTTACTGGGTTGGGCCTTTGATTGGTGGTGGGCTTGC TGGCTTTATCTGTGAAAACTTTTTCATTGT TAGAACTCATGTTCCTCTTCCTAGTGATGAATCTTTCTAGGTTTTGTGT CAAAGGGTCCCTGGCTAGTTTGCAGTCTGC CTTGCTATGCATGTATTTTCTATGTTTCAGTTTTGTAAGTTTCATATATA ATAACACTTTGTATRAAATTATG

>ntMIPcontig6.seq GGGAACCAAAGCAAACCATAGCCATTCCTCATTTCTTTTCTTCTTAGTT TTTAATTAGCCTTTCATTTGTCTGCAAATT TATCAGTGTTTGATCAGAAAAGGGCCAATGGCGAAGATTGCTGTTGGA AATAGCCGTGAGGCTATTCAGCCAGACTGCA TCCAAGCACTTATTGTTGAGTTTATTGTCACTTTTCTCTTTGTTTTTGCT GGTGTTGGATCTGCCATGGCTGCCAATAA GCTAAATGGAGATCCACTAGTGAGCTTATTTTTTGTGGCAATGGCACA TGCATTGGTGGTGGCTGTGACAATCTCTGCT GGCTTCCGAATCTCTGGTGGGCACCTCAACCCCGCTGTCACGCTTGGC CTTTGTATGGGTGGTCATATCACTGTCTTCA GATCAATCCTTTACTGGATTGACCAATTATTGGCTTCTGTTGCTGCTTG TGCTTTGCTCAATTACGTCACTGCTGGGTT GGAAACACCAGTTCACACACTAGCAAATGGAGTGAGCTATAGTCAAG GGATAATTATGGAGGTGATTTTGACATTTTCT CTATTGTTCACTGTCTATACAACCATTGTGGACCCCAAGAAGGGAGTT CTTGAAGGGATGGGCCCACTTCTAACTGGGC TTGTGGTTGGGGCCAACATCATGGCTGGAGGGCCTTTTTCAGGAGCTT CAATGAACCCAGCAAGATCATTTGGGCCGGC TTTTGTGAGTGGAATCTGGACTGACCACTGGGTTTACTGGGTTGGGCC TTTGATTGGTGGTGGGCTTGCTGGCTTTATC TGTGAAAACTTTTTCATTGTTAGAACTCATGTTCCTCTTCCTAGTGATG AATCTTTCTAGGTTTTGTATCAAATGCCCT CTTCCTATTGTGCAGTATTGATGTGGTTTTGCTAGTATTTCTTGCTATGC ATGTACTATTTTCTATGTTTCCTTTTTGT AAGTTTATATGTAATAACACTATCTATGCAATTGTGcTATCTAANAAAA AAAAAA

>ntMIPcontig7.seq GATCAGCTAGAAGATCTCAGAGTGAAAAGCATKGATcTCYCCGAAAA GAAAGTACAAACAAAATTAAAAAAGTCCATCT CTCTCTCTCTCCCCGGCCCCCTTCACTTTTATCTTTCTGGATTTGTATAT ATAAATTGGCGAAGCTGTAATGTTTGTTT GCTCGCTTATTCTTCCTATGCAAACAATAAGCTGATCATCTTGCACAAC AACCCCCCAAATCTACACATCTTAATTAGT AGGAGTTAAAAGTAATTAAGGATATACAATTATGGGTGATCTGCAGAC AGCAGAGGCTAATGGAAACCATGCATCAGTG AGTTTGAATATTAGAGATAATGATATGAACAACAACAAGACTTCTGCA CATGAAGATTCTTCATCAAGCTGTTGCTTTG

162

TCACTGTTCCCTTCATCCAAAAGATAATAGCGGAGACATTAGGGACGT ACTTCTTGATATTTGCGGGGTGTGGTTCAGT GGCGGTGAATGCAGACRAAGGAATGGTTACTTTCCCTGGTATCTCCAT TGTCTGGGGGCTGGTGGTTATGGTCATGGTT TACTCTGTTGGSCACATTTCTGGTGCTCATTTTAACCCTGCTGTTACCAT TTCCTTTGCCACTTGCAAAAGGTTCCCAT GGAAACAGGTACCAGCTTACGTGGCAGCTCAAGTGATTGGATCAACCC TAGCAAGCGGAACCCTACGACTAATATTCAA TGGCAAACATGATCATTTTCTTGGAACTTCACCCTCTGGATCAGATATC CAATCTCTTGTTCTGGaATTTAtTATCACA TTTTATCTTATGtTTGTCGtTTCTGGTGTCGCAACTGATAATCGAGCKAT MGGAGAACTTGCTGGTCTTGCTGTGGGGG CAACCGTGTTGCTTAATGTGATGTTTGCCGGRCCGATATCAGGAGCGT CGATGAACCCAGCAAGGAGTTTGGGCCCAGC AATAGTRTGGAGCCATTACAGARGTATATGGGTTTAYATGTTAGGCCC AACAGCTGGGGCCATATCAGGTGCTTGGGTC TATAACATCATCAGATTCACWGACAAGCCTTTACGTGAGATTACCAAA AGTGGATCTTTCCTCAAGTCCATCAGATCAA GCAAGTCCCTCAGATCTAGCACGTAACAAACAGCTCCAACTGAAAAG GGGAGTAATTGTTTTTTTTCTTCTTCTTCTGA AATATATATAGACAAAAGAAAGAAAAATAGGAATGAGAAAAGGGGG AAAAGCATGTGTTCCTAGCTATTAGTTTCCTTA ACTTTTGTTACAAAAGAAACGACTTCTCTC

>ntMIPcontig8.seq CGGGGATCCTCACTATTTCCTCTGTTTCTTTTCTACAGTTTTTTAAATTT TTCTTAGTAATGTCTAAAGAAGTAGAGGC AGTGTCTGAGCAGCCGGCGGAGTATTCTGCTAAGGATTACACTGATCC ACCACCAACTCCGCTTATTGATTTTGAGGAG TTAACTAAATGGTCACTTTACAGAGCTTGTATTGCTGAGTTTATTGCTA CTTTGTTGTTTCTTTATGTAACTGTTTTGA CTGTGATTGGGTATAAGCATCAGTCGGATACTAAAGATGGCGGCGATA TCTGTGGCGGCGTTGGTATTCTTGGTATTGC TTGGGCTTTTGGTGGTATGATCTTTGTTCTTGTTTACTGCACTGCTGGT ATTTCTGGTGGACACATCAACCCTGCTGTG ACATTTGGGCTATTTTTGGCAAGGAAAGTATCATTGATCAGGGCAGTA TTATACATGGTATCACAGTGCTTAGGTGCAA TATGTGGTGTGGGTTTGGTAAAGGCTTTCCAAAAGGCATATTTCAATA GATATGGTGGTGGTGTTAATGTTATGGCAGG TGGACACAACAAAGGTGTTGGTTTGGGTGCTGAGATTATTGGTACCTT TGTTTTGGTCTACACTGTCTTCTCTGCTACT GACCCTAAAAGGAGTGCCAGAGACTCCCATGTCCCTGTATTGGCACCA CTTCCAATCGGATTCGCTGTATTCATGGTCC ACCTTGCCACCATTCCGATCACCGGAACCGGAATCAACCCGGCAAGA AGTTTTGGAGCAGCCGTGATTTACAACCAGGA

163

TAAGGCTTGGGATGAGCACTGGATCTTCTGGGTCGGACCATTCGTCGG AGCTTTCGCCGCCGCCGTCTACCATCAGTAC ATCCTCCGAGCTGGCGCCCTTAAAGCTCTTGGTTCCTTCAGGAGCAAT GCTTAGAGATTTTCATGCATGAGAAAATTTG TAAATAAAATGTTTGGATGGGAAAAGGGTGTATTTTCTGTTCTTTTGA AATTTTTCTATCTTTTCTTTTTTGGATATTG TACTTAAATTAAATCTGTTTGTATGTTTGCTTCCTTCTATTTTAATTTCC TTATCCTTTGtTTTTTTTAATCTTGTTGG CCTTTTGTAtTTTTTGTTTAtTTAATTACTGGTTcCTCGTACATTATTATGT aAAAWRYWRWRWMCMTMWMWWGTTTTT TTTTGGTTTTTTTGCAAAGTACGCTTTATTCTC

>ntMIPcontig9.seq CGGCCGGGGGAGCTAAAAGTTCAAAGCACAAGTTTATTCCGCCTCCTT AATTAAAACCTCTTCAACTTCACATATTCAC GSCTCCTTTCCCAACTACAAACATTTTAAAAGGAGGAAGAAGAGATTC AATTTTCTGTTTTATTCTTCTATAGCACTAT TTGTTAACCAGTTTCTATTTGTCTATCTAGTTCATCAATGGAGAGTGAA CGAGGAAACTCTACAGAAAACAAAAAACCA AATGAATTGGTGTCCGTCGAAAATCCAAAGTCCAACTTGTCCTTCCGT TACATATTGATATTTTTTCAAGAACATTACC CACCTGGGTTTCTCAAGAAGGTGATAGCGGAGGTAATCGCGACGTATC TAtTGGTGtTTGTGACATGTGGTGCAGCTGC AATTAGTGCGAGCGACGAACACAAAGTGTCGAGACTTGGAGCCTCCG TTGCGGGTGGACTCATTGTGACAGTCATGATA TATGCTGTTGGACATATCTCTGGTGCGCATATGAATCCTGCTGTTACCT TTGCTTTTGCCGCCGTTAGACATTTCCCAT GGAGACAGGTACCACCGTATGCAGCAGCACAACTTACAGGGGCGACC TCAGCTGCATTCACACTACGGGTATTGCTTCA CCCAATAAAACATGTGGGAACTACAACTCCTTCAGGCTCAGACATTCA AGCTTTAATCATGGAAATTGTGGTCACCTTC TCTATGATGTTCATCACCTCTGCAGTCGCCACTGATACCaAAGCTATCG GCGAGCTGGCAGGCATGGCAGTTGGTTCGG CTGTATGTATTACTTCTATCTTGGCGGGGCCGGTGTCAGGAGGATCAA TGAACCCAGCCAGAACCATTGGACCTGCAAT GGCTAGCAACGACTACAGAGGA

>ntMIPcontig10.seq GTCGGTCCGGtSACTCCCYTTKCCTWTAGTGACTYTGTAACACTAGAAA ATTTTCATTTTCTCTATYTYCAGATCCTGT TTTTCCGRCCATGCCGATCCACCAAATTGCTGTTGGAAGCCATGAGGA ACTCCGCCAAYCAGGGACGCTCAAGGCGGCC TTRGCGGAGTTCATCTGTACCCTGATCTTCGTKTTCGCAGGTCAGGGTT CTGGCATGGCTTTCAACAAGCTATCAGYTG ACGGTACCGCTACTCCCKCCGGCCTMATCTCTGCCTCTATAGCGCATG CCTTCGGGCTKTTYGTGGCYGTCTCCGTCGG

164

TGCTAACATCTCCGGCGGCCAYGTTAATCCCGCCGTTACCTTCGGTGC TTTCGTTGGTGGAAACATCACTTTGTTYCGT GGSATTCTCTACATTRTYGCACAGTTGCTTGGATCCACTGTTGCTTGCT TCCTCCTTGAATTTGCCACTGGTGGCATGA GCACAGGAGCATTTGCWTTGTCAGCTGGTGTATCAGTATGGAATGCW TTTGTCTTTGAAATAGTGATGACTTTTGGACT TGTTTACACTGTGTATGCCACTGCTRTTGACCCAAAGAAGGGAGACTT GGGWGTAATTGCACCAATTGCCATTGGTTTT ATTGTTGGTGCCAACATTTTAGCTGGTGGGGCCTTTACTGGAGCTTCAA TGAACCCTGCTGTCTCATTTGGKCCWGCTT TGGTTAGCTGGACCTGGACCCACCAATGGGTCTACTGGGCTGGRCCCC TTGTTGGTGGTGGGATTGCTGGTGTTGTCTA TGAACTCATCTTCATCAACCACTCCCATGAGCCACTCCCCAGTGGAGA TTTTTAAGATATTCAtgAaATTTCAACCTCT atCTGTTGCTTTCATTTTCTCTTTGGAGTTTTCCGTGTTTTGTCTATGTTT ATTGGACGCTGTTGAATTGTACTGTGTA TTCAGACGGCTTTTATGTTTCCTATATTAGCCTTCTTTAATTTCCTGGTG GTAAATAAGGTATTTTAGATTTTCAAAAA TATTGCAWWMAAAAcAAAAAAAAAAggAAAAAAAG

>ntMIPcontig11.seq CGGGGACTCTAAAATAAATCATTAAAAGTCCTCTCTTTTCTCCCATAA ATACCTATCCATTTTACACTCACWTCWCGAT CATCCATATCCCTTGTTTCAAATTTTCTTTCTTTTTCGTYAGCCATGACT AAAGAAGTWGAGGTAGCAASAGAGCAASC ARCAGAGTTTTCAGCAAAAGACTATACTGACCCTCCACCAGCTCCTTT AGTAGACTTTGAGGAGCTGACACAATGGTCA CTTTACAGRGCTGTTATTGCTGAGTTCATTGCCACTTTGCTTTTCCTTTA TGTTACTGTTTTGACTGTGATTGGRTAYA AGGTCCAGTCAGATGTCAAAGCCGACGGTGATATCTGTGGCGGCGTTG GTATTCTTGGTATTGCTTGGGCTTTTGGTGG CATGATTTTCATTCTTGTTTACTGCACCGCCGGTATCTCCGGAGGACAC ATAAACCCAGCAGTGACATTTGGKCTGTTT TTGGCAAGGAAAGTATCAYTRATCAGAGCAGTWTTGTACATGGTGGC ACAGTGTTTGGGTGCAATCTGTGGTGTGGGTT TKGTGAAGGSATTCCAGAGTGCTTATTATGTTAGGTATGGTGGAGGTG CTAATGTCATGGCTCCTGGCCATACCAAAGG TGTTGGATTAGCTGCTGARATTATTGGTACTTTTGTTTTGGTTTACACT GTCTTCTCTGCCACTGACCCWAAGAGAAAT GCTAGAGACTCCCATGTCCCTGTGTTGGCACCACTTCCAATTGGATTTG CTGTGTTCATGGTTCACTTAGCYACCATAC CAATTACTGGAACTGGCATCAATCCTGCTAGRAGTTTTGGTGCAGCAG TTATTTACAATCAKGACAAAGCTTGGGATGA ACACTGGATTTTCTGGGTGGGTCCCTTCATCGGAGCCTTCGCCGCCGC CGTCTACCACCAGTTCATCCTCCGTGCTGGT

165

GCCATCAAAGCTCTTGGTTCCTTCAGGAGCAATGCCTAAAAAATTTCA ATCCAATTGAATAAAACCAGCCATGAAAAAT TGTAGATATTTCCCAATGGGAAAAGAAAAAAAAATGTACTGTTTGTTT TTCCTATGAATTTCCCTCTTTTGTGTATTTT TCTACTTGTAATCTTCATTTCCCTTTTTTATTTTTACTTCCTTGCTCTCTT TTTTCAATCTTGTCTCTTGTATCTTTGA AT

>ntMIPcontig12.seq GACCAGAACCCATCCTCTTCTCTCATACRcgGGAACAAATCACTTTTAA CTCWTCTTCTTCAGAAAATCAGAAGAAAGA AAMAATGTCAAAGGACGTGATWGAAGAAGGACAAGTTCATCAACAR CAYGGGAARGATTACGTGGACCCACCACCAGCW CCTTTGCTTGATTTTGCAGAACTCAAGCTCTGGTCTTTTYACAGAGCTC TTATTGCTGAGTTCATTGCTACTCTTCTTT TCCTTTACGTYACTGTWGCAACTGTAATTGGTCACAAGAAGTTGAATG GTGCTGATAAATGTGATGGGGTTGGKATTCT TGGYATTTCTTGGGCTTTTGGTGGCATGATTTTTGTTCTTGTTTACTGCA CTGCYGGTATCTCTGGWGGACAYATTAAC CCAGCAGTGACATTTGGGTTRTTCTTAGCAAGAAAAGTGTCATTRTTA AGRGCAGTGGGATATATTATTGCMCARTCWT TAGGTGCAATTTGTGGTGTTGGTTTAGTGAAAGGTTTCATGAAACATT ACTACAACACATTAGGTGGTGGTGCTAATTT TGTGCAACCTGGTTATAACAAGGGCACWGCTTTGGGTGCTGAGATTAT TGGAACTTTTGTTCTTGTTTACACTGTTTTC TCTGCTACTGACCCTAAAAGAAGTGCCCGTGACTCMCATGTCCCTGTK TTGGCCCCWCTGCCAATTGGWTTTGCTGTKT TCATGGTTCATTTGGCTACTATTCCTATTACTGGAACTGGTATTAACCC TGCTAGGAGCTTTGGAGCTGCTGTYATTTA CAACACTGAAAAARTCTGGGATGAYCAATGGATTTTCTGGGTTGGACC ATTTGTGGGAGCATTGGTAGCAGCAGTATAT CATCAGTATATCTTGAGAGGTTCAGCAATTAAGGCATTGGGTTCTTTCC GCAGTAACCCAACCAACTAAAACAACTTTT GCAACCACTACCAGAAAAAAGAAAAGAAGTGGAAAAAGAATTGCATT CATCTGTCCAAATTATCTTTGTTTATTTATTT GATTCTATGTGTGTAAGAGGAACCTATGGAACTCTCCCTTTTACTTTTT ATGTATTGCTTATAATAGTTTTAATCCTTA ATGTTAGTGTAAGAGGAGCCTTGGATGTGTATTTATTTGTATGAGTAG TGTTGGTGATGTTTTTCTTTTTAATT

>ntMIPcontig13.seq CCGGGGGACCACAATCAATTCTACATATTTCTATCTAAAAGCTTTAAA AACAACATTATTCAAGATTAGCATAATCTTC TTTTGGTGAGCCTTAATTTAGAGAACAGAGTGCAAAAATGGCCAAAGA CATTGAGTATGGCACTGATCAATATGCCCCT

166

AATAAGGACTACCAAGATCCACCTCCAGCACCACTAATTGATGCAGA GGAACTTGGAAAATGGTCATTTTATAGAGCCA TTGTTGCTGAATTTATTGCAACTCTGTTATTTCTCTATATCACTGTCCTC ACTGTGATTGGCTACAAGAGCCAAAGTGA TACTAAACATAATGGTGATGAATGTGGTGGTGTTGGCATTCTTGGCAT TGCTTGGGCTTTTGGTGGCATGATTTTCGTT CTTGTTTACTGCACTGCTGGTATTTCTGGAGGACATATTAACCCAGCAG TGACATTTGGGCTCTTCTTGGCTAGAAAAA TCTCATTGGCCAGAGCCGTTATGTATATGATAGCACAGTGTTTAGGAG CCATTTGTGGTTGTGGTTTGGTGAAGGCATT TCAGAAGTCTTACTATGTTAATTATGGTGGTGGTGCCAATGAGCTTGC AACAGGCTACAGCACTGGCACTGGATTAGCT GCTGAAATCATTGGAACTTTTGTCCTTGTTTATACTGTCTTTTCGGCCA CTGACCCCAAGAGAAATGCCAGAGACTCTC ATGTTCCTGTATTGGCACCACTTCCAATTGGATTTGCTGTATTCATGGT TCACTTGGCTACAATCCCAATCACTGGAAC TGGTATTAATCCAGCTAGAAGTTTTGGAGCTGCAGTCATATATGGCAA

>ntMIPcontig14.seq GTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTA GTCATCGCTATTACCATGGTGATGCGGTTT TGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTT CCAAGTCTCCACCCCATTGACGTCAATGGGA GTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACA ACTCCGCCCCATTGACGCAAATGGGCGGTAG GCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCG TCAGATCGCCTGGAGACGCCATCCACGCTGT TTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGGACTCTA GCCTAGGCCGCGGGACGGATAACAATTTCAC ACAGGAAACAGCTATGACCATTAGGCCTATTTAGGTGACACTATAGAA CAASTYGKTYCRRAtWTTCYYKSKWTTTCGT YAGCCATGACTAAAGAAGTWGAGGTAGCAASAGAGCAASCARCAGAG TTTTCAGCAAAAGACTATACTGAYCCTCCACC AGCTCCTTTAGTAGACTTTGAGGAGCTGACACAATGGTCACTTTACAG RGCTGTTATTGCTGAGTTCATTGCCACTTTG CTTTTCCTTTATGTTACTGTTTTGACTGTGATTGGRTAYAAGGTCCAGT CAGATGTCAAAGCCGACGGTGATATCTGTG GCGGCGTTGGTATTCTTGGTATTGCTTGGGCTTTTGGTGGCATGATTTT CATTCTTGTTTACTGCACCGCCGGTATCTC CGGAGGACACATAAACCCAGCAGTGACATTTGGGCTGTTTTTGGCAAG GAAAGTATCATTGATCAGAGCAGTATTGTAC ATGGTGGCACAGTGTTTGGGTGCAATCTGTGGTGTGGGTTTTGTGAAG GCATTCCAGAGTGCTTATTATGTTAGGTATG GTGKMGGTSCKRATgTCMYGGSWYCTTGTTTCaAATTTTCTTTCTTTTT CGTCAGCCATGACTAAAGAAGTAGAGGTAG

167

CAACAGAGCAACCAACAGAGTTTTCAGCAAAAGACTATACTGACCCT CCACCAGCTCCTTTAGTAGACTTTGAGGAGCT GACACAATGGTCACTTTACAGAGCTGTTATTGCTGAGTTCATTGCCACT TTGCTTTTCCTTTATGTTACTGTTTTGACT GTGATTGGGTATAAGGTCCAGTCAGATGTCAAAGCCGACGGTGATATC TGTGGCGGCGTTGGTATTCTTGGTATTGCTT GGGCTTTTGGTGGCATGATTTTCATTCTTGTTTACTGCACCGCCGGTAT CTCCGGAGGACACATAAACCCAGCAGTGAC ATTTGGGCTGTTTTTGGCAAGGAAAGTATCATTGATCAGAGCAGTATT GTACATGGTGGCACAGTGTTTGGGTGCAATC TGTGGTGTGGGTTTTGTGAAGGCATTCCAGAGTGCTTATTATGTTAGGT ATGGTGGAGGTGCTAATGTCATGGCTCCTG GCCATACCAAAGGTGTTGGATTAGCTGCTGAAATTATTGGTACTTTTGT TTTGGTTTACACTGTCTTCTCTGCCACTGA CCCTAAGAGAAATGCTAGAGACTCCCATGTCCCTGTAAGTATTCTATT CTCTTCATATTTTTTAAGGCAAACCAATATT TGTAAATAGTGAAATTTTGCCCCGGTGCACTAAGCTCCCGCTCTTATCC TGCATTTTGCAGAGATCACGGCTCGAACCC ATGACTCTGATACATGACAATAACTCTCGTTCAAATTTTGTATTTACAA TACAAAAATATTATTTATTGTCAAAAGCCC AGTCTTATCTATCTCATTATATCAGTTCACCCATCT

>ntMIPcontig15.seq GGCCATTACGGCCGGGGACGRCAATCAATTTTACATATTTCTATCTAA ATAAAAGCTCTCAAAACCAACATTATTCAAG ATTAGCATAATCTTCTTTTGGTGAGCCTAATACAGAGACAGAGTGCAG GATTAATGGCCAAGGATATTGAATATGGCAC TGATCAGTACGCCCCTAATAAGGACTACCAAGATCCACCTCCAGCACC ACTAATTGATGCAGAGGAACTTGGAAAATGG TCATTTTATAGAGCCATTATTGCTGAATTTATTGCCACTCTCTTATTTCT CTACATCACTGTCCTCACTGTGATTGGCT ACAAGAGCCAAAGTGACACTAAACATAATGGTGATGAATGTGGTGGT GTTGGCATTCTTGGCATTGCTTGGGCCTTTGG TGGCATGATTTTCGTTCTTGTTTACTGCACTGCTGGTATTTCTGGAGGA CATATTAACCCAGCTGTGACATTTGGGCTG TTCTTGGCTAGAAAAATCTCATTGGCCAGAGCAGTAATGTATATGATA GCACAGTGCTTAGGAGCCATTTGTGGTTGTG GTTTGGTGAAGGCATTTCAGAAGTCTTaTTATGTTAGATATGGTGGTGG TGCCAATGAGCTTGCAACAGGCTACAGCAC TGGCACTGGATTAGCTGCTGAAATCATTGGCACTTTTGTCCTTGTTTAT ACTGTCTTCTCTGCCACTGACCCCAAGAGA AATGCCAGAGACTCCCATGTTCCTGTATTGGCACCACTTCCAATTGGA TTTGCTGTATTCATGGTTCACTTGGCCACAA TCCCWATCACTGGAACTGGTATTAATCCAGCTAGAAGTTTTGGAGCTG CAGTCATATATGGCAAAGAMAAAGCCTGGGA

168

TGACCAATGGATTTTCTGGGTTGGACCTTTTATTGGTGCTGCAATTGCT GCATTGTATCACCAATACATTCTGAGAGCT GGAGCAGTCAAAGCACTTGGCTCATTCAGGAGCAATGCCTAAATGATA AGAATTTAGAGCTATTTAAGGAGATGGAATG AATATTAAATGGTCAAAATTGTATAGTGGTATTGGTGGAGATGGCCTC TTTTAATTGTGCTTTTGTTTGCTTTGTGGGT TGGCACTTAATTTAAATGTGCCAAACATTGTCATTTTGAAGCTTTTCTT CTTGTTTGGTTGCATTTCCTACTTTTGTTT AATTCCTGTCCCCTGTAAATTTCCTTATTCTGTATTCAATAATAATACT TGATTGACCAGGGCTCTTTATATTC

>ntMIPcontig16.seq TCGGTCCGGATTCCCGGGATAATTTGTCTTCATAAATGAGTTCTCCACT ATAAAACAACCACAACTCTRKCmATTCGGC gGGGACCAAACTTGGTTTTTGWACTRTCCACTTAGCACAAWAAAGAG AGAAAAASAAGSKAAGTTTAGTGAGTGTTCAA ATGGCAGAAAACAAAGAAGAAGATGTTAAGCTTGGAGCTAACAAATT CAGAGAAACACAGCCATTAGGAACAGCTGCTC AAACAGACAAAGATTACAAAGARCCACCACCAGCTCCTTTGTTTGAAC CAGGGGARTTRTCATCWTGGTCWTTTTACAG AGCYGGAATTGCAGAATTTATGGCYACTTTCTTGTTYTTGTACATCACT ATCTTGACTGTKATGGGTCTTAAGAGATCT GATAGTYTRTGTWSTTCAGTTGGTATTCAAGGTGTTGCTTGGGCTTTTG GTGGTATGATCTTTGCTTTGGTTTACTGYA CTGCTGGTATCTCAGGAGGACACATCAACCCAGCYGTGACCTTTGGAT TGTTCTTGGCAAGGAAACTGTCCTTAACCAG GGCTRTTTTCTACATAGTGATGCAATGCCTTGGTGCAATYTGTGGTGCT GGTGTTGTGAAGGGATTCATGGTWGGTCCA TACCAGAGACWTGGTGGKGGTGCTAATGTTGTTAACYMTGGTTACAC CAAAGGTGATGGCCTTGGTGCTGAAATTATTG GCACTTTTGTCCTTGTTTACACTGTTTTCTCWGCTACTGATGCYAAGAG AAATGCCAGAGACTCACATGTTCCTATTTT GGCACCACTTCCCATYGGATTCGCGGTKTTCTTGGTTCATTTGGCCACC ATTCCCATCACCGGAACTGGCATCAACCCC GCTAGGAGTCTTGGAGCTGCGATCATCTACAACACAGAYCAGGCATG GGACGACCACTGGATCTTTTGGGTTGGACCAT TCATTGGAGCTGCACTTGCTGCAGTTTACCAYCAAATAATYATCAGAG CCATTCCATTCCACAAGTCKTCTTAAGTTYC CTCAAGAAGCCTTAATCTTTCTGAAKTTTCAAGACWCWSTACCTTTTT TTKGGGGGTTAAATTTTAATCCYMTCCTTCT GRTGTATTTGTAATGTGGATTTTCTTGTGTTCARATGTCTATTATGTGT GTAAATTATCAGGTTWTGTTGTACTGTACC CTRTYAATGAAGGTTCTTTATTTTATCCYTCAAA

>ntMIPcontig17.seq

169

GGGATCAMRTTTTAAGCAAAACCCTYTTAAGAACTTAAATTGAGCTTC TTTAGGGGCAtTTTatTttTCTAGTGAGAAC TAAAAATGGTGAAGATTGCCTTTGGTAGCATTGGTGACTCTTTTAGTGT TGGGTCATTGAAGGCCTATGTAGCTGAGTT TATTGCTACTCTTCTCTTTGTATTTGCTGGTGTTGGATCTGCTATAGCTT ACAATAAATTGACAGCAGATGCAGCTCTA GACCCAGCTGGTCTAGTAGCAGTAGCTGTGGCTCATGCATTTGCATTG TTTGTTGGGGTTTCCATAGCAGCCAATATTT CAGGTGGYCATTTGAATCCAGCTGTMACTTTGGGATTGGCTGTTGGTG GAAACATCACCATCTTGACTGGMTTCTTCTA CTGGATTGCCCAATTRCTTGGCTCYACAGTTGCTTGCCTCCTCCTCAAA TACGTTACTAATGGATTGGCTGTTCCAACC CATGGAGTTGCTGCTGGGCTCAATGGWTTMCAAGGAGTGGTGATGGA GATAATCATAACCTTTGCACTGGTSTACACTG TTTATGCAACAGCAGCAGACCCYAAAAAGGGYTCMCTTGGAACCATT GCMCCCATTGCAATTGGGTTCATTGTYGGGGC CAACATTTTGGCAGCTGGTCCATTCAGTGGTGGGTCAATGAACCCAGC TCGATCATTTGGGCCAGCTGTGGTTGCAGGA GACTTTTCYCARAACTGGATCTATTGGGCCGGCCCACTCATTGGTGGA GGATTAGCTGGGTTTATTTATGGAGATGTCT TTATTGGATGCCACACCCCACTTCCAACCTCAGAAGACTATGCTTAAA ACTTAAAAGAAGACAAGTCTGTCTTCAATGT TTCTTTGTGTGTTTTCAAATGCAATGTTGATTTTTAATTTAAGCTTTGTA TATTATGCTATGCAACAAGTTTGTTTCCA ATGAAATATCATGTTTTGGTTTCTTTTG

>ntMIPcontig18.seq RRTCAAATCACAAAGAAAAAGCAAAAGCTGCATTCTGTTGCATTCTGT TGAAGAAATCTAGAAGCTAAGAATGGAGGGG AAAGAAGAGGATGTGAAGGTAGGAGCAAACAAGTATTCAGAAAGGC AGCCATTAGGGACCTCAGCACAGAGCAAAGACT ATAAGGAACCACCACCAGCACCATTGTTTGAGCCTGGTGAGCTCCATT CTTGGTCCTTTTGGAGAGCTGGGATTGCTGA ATTTATGGCTACTTTCTTGTTCCTTTACATCACTGTCTTGACTGTCATGG GTTACTCTAGGGCTAACAGCAAATGTAGT ACTGTTGGTGTTCAAGGTATTGCTTGGGCTTTTGGTGGTATGATTTTTG CCCTTGTGTACTGTACTGCTGGTATCTCAG GTGGACACATTAACCCTGCTGTGACATTTGGTTTGTTTCTGGCAAGAA AATTGTCCCTAACAAGGGCAGTGTTCTACAT TGTGATGCAATGCCTTGGTGCAATCTGTGGTGCTGGTGTTGTCAAAGG GTTCCAGCCATCTTTGTATCAGGTTAAGGGT GGAGGTGCCAATGTTGTGAATCATGGTTATACCAAAGGAGATGGCCTT GGTGCTGAGATTGTTGGCACTTTTGTTCTTG TCTATACAGTCTTCTCTGCTACTGATGCCAAGAGAAATGCTAGAGACT CCCATGTCCCTATTTTGGCACCTCTCCCAAT

170

TGGATTTGCAGTGTTCTTAGTTCACTTAGCCACTATCCCAATCACAGGC ACAGGCATTAATCCTGCTAGAAGCCTAGGT GCTGCCATTATCTACAACAGAGATCAAGCATGGGATGACCATTGGATT TTCTGGGTGGGACCATTCGTTGGAGCTGCAC TTGCTGCCTTGTACCACCAGGTTATCATAAGAGCCATTCCATTCAAGA GTGGAAACTTGGCTTAATTATGAGCTATGAT GGCTATGCCTTGGTTTCTGTAATTGGTTAGTTTGCTATGATTGGCAATG ATGTTCAATGTTGATTATGTACAAAAGCTG TTGTATTGCTTTATTTATCGTATTTGGTTTTGAATTGATCTTATTTATTA CAAAAAAAAAAAAAAAAG

>ntMIPcontig19.seq ACAGTCGGTCCGGATCAACGGGtaAACATWAATYCTTYCTSAAGTYGA GTAAWTTTTaSCTGTGAATTGAGAAAAAARM ATGGAGAACAAAGAAGAAGATGTAAGATTAGGAGCAAACAAATATTC AGAGAGGCARGCRATTGGGACGGCMGCGCAGA GTGACAAGGATTATAMGGAGCCACCACCAGCGCCACTGTTTGAGGCA GGAGAATTGACGTCTTGGTCATTCTATCGTGC TGGAATTGCTGAGTTYATGGCCACTTTCCTCTTCCTTTACATCACTATC TTAACAGTGATGGGTGTTTCAAAGTCTGAR TCCAAATGCTCCACTGTTGGTATTCAAGGCATTGCTTGGGCTTTTGGGG GCATGATYTTTGCCCTTGTYTACTGTACTG CTGGCATTTCTGGAGGACACATAAATCCAGCAGTGACYTTTGGGCTGT TCTTGGCAAGGAAAYTGTCGTTGACAAGGGC AGTGTTCTACATGGTGATGCAGTGTCTTGGWGCTATCTGTGGTGCTGG TGTGGTCAAAGGGTTYGGCAAAACTCTTTAT CAAACAAAGGGTGGTGGTGCCAATGTTGTAAATCTTGGTTACACAAAG GGATCAGGTCTTGGTGCTGAGATCGTTGGYA CTTTCGTYCTTGTTTACACTGTTTTCTCTGCYACTGATGCTAAGCGTAG TGCTAGGGATTCACATGTCCCTMTTTTGGC ACCATTGCCTATTGGATTTGCTGTGTTCYTGGTACACTTGGCCACAATT CCAATCACTGGAACTGGTATYAACCCAGCT AGGAGTCTTGGCGCAGCCATCATCTACAACCAAGACCACGCATGGGA TGACCACTGGATATTCTGGGTTGGACCATTTA TTGGAGCAGCACTAGCAGCTCTATACCACCAGGTGGTGATCCGGGCAA TCCCATTCAAGTCTAAGTGAAGTCTCTTTTA ACCACAGATTTCTCAAGCTKAATCRTCATSTGGCMAAAGATCAAAAGC CTCATTTCTGYCCATTTCCCCTTTGCATTAG ATCCTCTTTTCTTTTTGTTTTGTTTTTTGTATCTTGTATTTGTAAGTTACA TAAGTGGGAGAATGTATCTGATCGGGGC ATATTTTCTATAGAAGGACCTTCGCTTTTCATTYCATTTGTCCAGAGAA AATGTGCAAATGGGCCAAAGAGAAACGTTG GCGGGCTCGTGCATCATCATTCTCC

>ntMIPcontig20.seq

171

GATCAATTCTTACTTAGGAATCAACACTTATTGTTAGAGAAATGGAGC ACAGAGAAGAGGATGTTAGAGTGGGAGCTAA TAAATATTCAGAGAGGCAAGCGATTGGCACGGCGGCACATAGTCAAG ACAAGGATTACAAGGAGCCACCACCTGCGCCC TTGTTTGAGCCAGGGGAGTTGATGTCCTGGTCCTTCTATCGTGCTGGAA TTGCAGAGTTCATGGCCACTTTTCTCTTTC TTTATATCACTGTTTTAACTGTCATGGGAGTGTCCAAATCTGAATCTAA ATGTCCAACTGTTGGAATTCAAGGCATTGC TTGGGCTTTTGGCGGCATGATTTTCGCTCTAGTTTACTGCACTGCAGGA ATTTCAGGGGGACACATAAACCCGGCAGTG ACATTTGGGTTGTTCTTAGCAAGGAAATTGTCGGTGACAAGGGCATTG TTCTACATGGTAATGCAGTGCCTGGGAGCCA TATGTGGTGCTGGTGTTGTCMAAGGGTTCGGGAAGACCCTTTACCAAA CTAAAGGTGGTGGTGCTAATGTTGTAAA

>ntMIPcontig21.seq CGACTCACTATAGGGNTCATTGGGCCNAACGTCGCATGCTCCCGGTCC CCATGGCGGCCNCGGGAATTCGATTAGCGTG GTCGCGGCCGAGGTACGCGGGGGTTTNGGCAGAAGCATTGGGGACTT TTATGCTGATGTTTTGTATATGTGGGATAATG GCAAGCATGGAAATAATGGGAGTGAGGGTGGGGCTTATGGAATATGC AGCCACAGCAGCACTAACAGTAGTAGTTGTAG TCTTCTCAATAGGCCCCATTTCTGGGGCTCACATTAATCCTGCTGTCAC ATTAGCATTTGCAGCTGTTGGTCATTTCCC ATGGTCCAAGGTTCCATTTTATGTAGTGGCACAAGTGGGGGGTTCTAT ATTGGCTACATATACAGGGAAATTGGTGTAT GGATTAAAAGCAGAATTTGTGATAACAAGACCTCTCCATGGTAGCACT TCAGCATTTTTTGTGGAGCTTTTGGCTACCT TCATTGTGCTCTTCCTAACTGCATCATTGACAAATGATCCTCAATCAAC AGGGCCATTGTCCGGATTTGTTGTTGGAGT GGCCATTGGACTGGCTGTACCTGCCCGGGCCGGCCGCTCGAATCACTA GT

>ntMIPcontig22.seq AAGGCtWWKWWWWTWWWWWTACAGGGGAAAACAGCCGTCACAAT ACATTAAAAAAGTATCCAATCAGAAACATAGACAC AACAAAGTTTTAAAATAAAAAATAAAAACTCATAGGAAGTAAATACA AAAGCAAGAAATTTAAATTTTCATTTCCTTAA TACTCAGCAGTAGGCACTTGCTCATGAGAGTGATTGATGAAGAAAAGT TCATAGACAACACCAGCAAGCCCAGCACCAA TTATGGGCCCGGCCCAGTAAACCCACTGATGAGTCCAAGTCCAGCTAA CAAAAGCAGGCCCAAAAGAAACAGCAGGGTT CATTGATGCTCCATCAAATGGCCCACCAGCAAGAATGTTGGCCCCAAC AATGAAACCAATAGAAATTGGAGCAATCACA CCTAATTCTCCTTTTTTTGGGTCAATAGCAGTGGCATAAACAGTGTAAA CAAGCCCAAAAGTCATCACAATCTCAAATA

172

CTAAGGCATTCCATACTGATAATCCAGAACACAATGCAAATGCCCCTG TGCTCAATCCACCAGTGGCGAACTTAAGGAG CAAGCRAGCAACAGTGGATCCAAGTAACTGTGCAATAATATAGAGAA TACCACGGAAGAAAGTGATGTTTCCACCAATA AATGCACCGAAAGTAACGGCGGGGGTAACGTGGCCGCCGGAGATGTT AAAGC

>ntMIPcontig23.seq CAGATCTGTTTTCGCATGCGATcCAcCAAAtTGCTGTGAGcCATGAGaAC TcCGCMACCMRGGACGcTCAAGGCGCTaG CGAGTCATCTGTACCcTGATCTCGTGtTCGCAgGTCAGGGTCTGgCATgG CTTTCAACAAGCTATCAGTTGACGGTACC GCTACTCCCTCCGGCCTAATCTCTGCCTCTATAGCGCATGcCTTCGGGC TTTTCGTGGCTGTCTCCGTCGGTGCTAACA TCTCCGGCGGCCACGTTAATCCCGCCGTTACCTTCGGTGCTTTCGTTGG TGGAAACATCACTTTGTTTCGTGGGATTCT CTACATTATTGCACAGTTGCTTGGATCCACTGTTGCTTGCTTCCTCCTT GAATTTGCCACTGGTGGCATGAGCACAGGA GCATTTGCATTGTCAGCTGGTGTATCAGTATGGAATGCTTTTGTCTTTG AAATAGTGATGACTTTTGGACTTGTTTACA CTGTGTATGCCACTGCTATTGACCCAAAGAAGGGAGACTTGGGAGTAA TTGCACCAATTGCCATTGGTTTTATTGTTGG TGCCAACATTTTAGCTGGTGGGGCCTTTACTGGAGCTTCAATGAACCC TGCTGTCTCATTTGGTCCTGCTTTGGTTAGC TGGACCTGGACCCACCAATGGGTCTACTGGGCTGGGCCCCTTGTTGGT GGTGGGATTGCTGGTGTTGTCTATGAACTCA TCTTCATCAACCACTCCCATGAGCCACTCCCAAGTGGAGATTTTTAAG ATATTCAAATTTCAACCTCTGTTGCTTTGAT TTTCTCTTTGGAGTTTTCCTTGTTGTGTCTATGTTTATTGGACGCCGTTG AATTGTACTGTGTATTCAGACGGCTTTTA TTATTTACTATATTAGCCTTCTTTAATTTCtCTGGTGGTAAATAAGGTAT TTTAGATTTTCAAAAAAAAAAAAAAAAAA TCCCGGGAATCCGGACCGA

>ntMIPcontig24.seq ACCAAATATACTTCTAATGAATCCCTCAATTTATATTTATATACGTCCT TATCCCCTATTACTATTAGGTTGATCATCA TCTTAGAATTCAGTAGCATCGACAAATTTCTACAGTCGATTGATTGATT GAAAATCAAAATGGGTGCTGTAAAAGCAGC AGTGGCTGATTTTGTGTTGACATCGATGTGGGTATTTTGTTCATCAACA CTTGGGGTTTTCACTTACCTAATTGCTTCT GCTTTTGGAATTGCTCAAGGAATCACTACTCTGTTTATTACTACTGTTC TTCTCTTTGTTCTGTTTTTTGTGTTTGGGA TTATTGGTGACGCTTTGGGTGGTGCCGCTTTTAATCCTGCTGGTACGGC CGCCTTTTATGCTGCCGGTGTTGGAACCGA

173

CTCTCTCTTCTCTGTTGCCGCTCGATTTCCTGCTCAGGCAGCTGGCGCA GTTGCTGGTGCATTGGCGATATTGGAGGTT ATTCCTACGCAGTACAAGCACATGCTAGGTGGACCTTCATTGAAAGTT GACTTGCATAACGGAGCCATTGCTGAGGGGA TCTTGACTTTCATAATGACCTTTTTGGTCTTTCTTATTGTACTGAGGGGT CCTAGAAATGCACTTCTAAAGAATTGGTT ACTTGCAATGTCAACTGTTACCATGGTAGTTACAGGTTCAAAATACAC CGGACCGTCTATGAATCCCGCCAATGCATTT GGTTGGGCGTACATAAACAATTGGCACAACACATGGGAGCAATTTTAC GTCTACTGGATCTGTCCCTTCGTAGGAGCAA TAATGGCTGCATGGACTTTTCGTGCTCTGTTTCCGCCTCCAGTAAAGCa gaAGAAGCCTCAGAAGGCAAAGAAAAATTG AGAAGAACCTTCTGCTTCAATTACTTCATTAAACTAGGTTGCAATGTG GTTGAAGTATGCCCTTAAAGCTGAAAGCCAT TTTTARCTTCTAATGTGGAATCATAATGGATTCCTTTTGGATTTGTTGG AATTGTTAATAAACCCCCTTTTTTCACATT GTCCAAATTCAAGCAACTATTATTGCAAAAACTGAGTTGTCCAAAGTT GGATTCCCCTGATTATGTTCTTGTGCTTCTT TAGATATTTCCTTCGTGCTGTTTGAATGGTATGCTTGTGAGTTAAATTG GATAATTCTTTCAATAGTACTTTGCATATT TTATCAAGTCTAACAAGGATCATCTCTGGACTTTGCCCTTGGACCAAG TTCAATCAGGTGAAAGTTGGAACAAGTATTT CTACTCAGCAAGGTGGCATACTTTTGGATCAATGATATTAGCACTAGT AGTTTCCGCTTTTATTAATAAAAAAATTTGA ATCACTTTCCTGGTCCAGTTGTGTATTTTCCTGGGTTTTGTTTAAATACT GAGCGGCCGCATG

>ntMIPcontig25.seq TTTTTTTTTTTTTTTTTTAAACTAAAATTATTATCTTTATCGAGGAAATG AAAAGAGGAAAACACCACCAACACTAGTC ATACCAAATAAATACACATCCAATGCTAACATTAAAGATTAAACTGTA CCAAACGAACAAAAAGAGAATGGTTCGTACA TTCCTCATACACAGAATCAAACAATTAGACAAGAAAAATTTTGAACTT GTGAAGGCAATTTTCTTCTTCTCTGGTTGTG GGTAACTTTGCTCAATTGGTTGGGTTGCTGCGGAAAGATCCCAAAGCT TTAACAGCTCCTGCTCTCAATACAAACTGAT AGTATATTGCTGCTATCAAAGCTCCCACAAATGGTCCAACCCAGAAAA TCCATTGGTCATCCCAKATTTTYTCYTTGTT GTAAATGACAGCAGCTCCAAAGCTCCTAGCAGGGTTRATACCAGTTCC AGTAATTGGGATAGTTGCCAAATGAACCATG AATACAGCAAATCCAATTGGCAGAGGAGCCAAAACAGGGACGTGAGA GTCACGTGCRCTTCTCTTRGGGTCAGTAGCAG AGAAAACAGTGTAAACAAGAACAAAAGTTCCAATAATCTCAGCACCY AAAGCTGTACCCTTGTTGTAACCAGGTTGYAC AAAGTTAGCACCTCCACCTTCTAAGTTRTAGTAATGYTTCATAAAAGC CTTRACGAAACCAACACCACAAATAGCACCA

174

AGTGAYTGTGCWATTATGTATGCAACAGCTCTTATYARTGACACTTTC CTTGCCAASAACAAMCCWAATGTCACCGCTG GRTTAATATGACCACCAGAAATACCAGCAGTGCAGTARACAAGAACG AAAATCATGCCACCAAAAGCCCATGCRATACC AAGAATRCCWACACCATCACAATGGTCWGCRGCGTTCARYTTCTTGT GGCCAATGACAGTGGCRACRGTGACGTAAAGG AAKAGAAGAGTRGCAATRAACTCAGCAATAACAGCTCTRTAAAARGA CCATTTGGTAAGTTCAGCCATGTCAAGAAGAG GAGCTGGTGGAGGGTCAACATAGTCTTTTCCATGTTGATGAGTTTGTC CTTCYTCAATTACGTCCTTTGACATGTTTTC TTTGATGAAAGTAAARGTACTaTCGYTTAGAAGTTTGCAGTGAAAGAG AGMAGAGTTTTKAGTTTTTGGTTTKCTGCTT TTKTYTGWGCYYYKGYCTcAATGGCC

>ntMIPcontig26.seq CAGAGACCCCTTTAAAAGGTTTACATGAAATAAATGGTCGGTGATGTA CTATTATATGTTAAATTACGCTGATGGTATA AAAATCTCATAAGTTAAATACTTTCTAAAATAATAAATACACTTACTT GGTCATCCCATATTTTCTCTTTGTTGTAAAT GACAGCAGCTCCAAAGCTCCTAGCAGGGTTGATACCAGTTCCAGTAAT TGGGATAGTTGCCAAATGAACCATGAATACA GCAAATCCAATTGGCAGAGGAGCCAAAACAGGGACGTGAGAGTCACG TGCACTTCTCTTGGGGTCAGTAGCAGAGAAAA CAGTGTAAACAAGAACAAAAGTTCCAATAATCTCAGCACCCAAAGCT GTACCCTTGTTGTAACCAGGTTGCACAAAGTT AGCACCTCCACCTTCTAAGTTATAGTAATGTTTCATAAAAGCCTTAAC GAAACCAACACCACAAATAGCACCAAGTGAC TGTGCTATTATGTATGCAACAGCTCTTATCAATGACACTTTCCTTGCCA ACAACAAACCAAATGTCACCGCTGGATTAA TATGACCACCAGAAATACCAGCAGTGCAGTAGACAAGAACGAAAATC ATGCCACCAAAAGCCCATGCGATACCAAGAAT GCCAACACCATCACAATGGTCAGCGGCGTTCAGTTTCTTGTGGCTAAT GACAGTGGCGACGGTGACGTAAAGGAATAGA AGAGTGGCAATGAACTCAGCAATAACAGCTCTGTAAAAAGACCATTT GGTAAGTTCAGCCATGTCAAGAAGAGGAGCTG GTGGAGGGTCAACATAGTCTTTTCCATGTTGATGAGTTTGTCCTTCCTC AATTACGTCCTTTGACATGTTTTCTTTGAT GAAAGTAAAGGTACTTCTGCTTAGAAGTTTGCAGTGAAAGAGAGCAG AGTTTTTAAGTTTTTGGTTTTCTGCTTTTGTC TCCCCGGCCGTAATGGCC

>ntMIPcontig27.seq GATCTAACTTGCTTTTCTTGGCAACCCTCAAAGAGAGAAAAAAGAGAA AAGAACCCTTAGTGKGYSATTacggccgGRG TGATTAAAAATGGCAGAAAACAAAGAAGAAGATGTGAATCTTGGAGC AAACAAATACAGAGAAACACAACCCTTAGGAA

175

CAGCAGCACAAACAGAAAATAAGGATTATATTGAACCACCACCAGCA CCATTATTTGAACCTGGTGAAYTATCATCTTG GTCATTTTACAGAGCTGGGATTGCAGAATTTATGGCCACTTTCTTGTTC TTGTaCATTACAATCTTGACTGTAATGGGA CTTAAAAGGTCAGATAGTTTGTGTTCTTCTGTTGGTATTCAAGGWGTT GCTTGGGCTTTTGGTGGTATGATCTTTGCYC TTGTYTACTGCACTGCTGGTATCTCAGGAGGMCACATTAAYCCAGCW GTGACWTTTGGTCTGTTCTTRGCAAGAAASTT gTCTTTAACAAGGGCWSTRTTCTACATGGTRATGCARTGCCTWGGTGC WATYTGTGGTGCTGGTGTTGTTAAAGGTTTT ATGRWRGGWCCATACCARAGACTWGGTGGTGGKGCCAAYGTGGTTM AMCCTGGCTAYACTAAAGGTGATGGACTTGGTG CTGARATTRTTGGYACCTTTGTCCTTGTTTACACTGTTTTCTCTGCCACT GATGCCAAGAGAAATGCTAGAGATTCACA TGTTCCTATTTTGGCACCTCTTCCTATTGGATTCGCGGTGTTCTTGGTTC ATTTGGCCACCATCCCAATCACCGGAACC GGTATCAACCCCGCCCGGAGCCTTGGAGCTGCTATCATCTTCAACCAA GACCGGGCATGGGATGATCACTGGATCTTCT GGGTTGGACCATTCATTGGAGCTGCACTTGCTGCAGTTTACCACCAGA TAATCATCAGAGCCATTCCATTCAAGAGCAA ATCTTAATTATCTTGAAGAGTCTCATGTCCTATGTCTCTTCCCCTGTTAT ATTTCAAGACTCCTGTACCCCTTTTTCCC CCGTTTCTAATGCTGTTGTCTCATGTAATTTTCTTCTCTTTTGATGTTAA TTATGTGTGTAAATTATCAGGTTAATGAT GTATCTAAG

>ntMIPcontig28.seq TCGGTTCCGGAATCCCGGGATTCCATTTACAGAGAGAAGAAAACTCAG TTGAGTGACTGAAGAAAAAAAAATGGCAGAG AACAWKGAAGAGGATGTTAAGCTAGGAGCAAACAAGTACAGAGAAA CACAACCTTTGGGTACAGCAGCTCAAACAGACA AGGATTATAAGGAGCCACCACCAGCTCCTTTGTTTGAGCCAGGAGAGT TGTCGTCATGGTCTTTTTACAGAGCTGGAAT TGCAGAATTCATGGCCACTTTCTTGTTCTTGTACATCACTATCTTGACT GTTATGGGTCTTAAAAGATCTGATAGTTTG TGTTCTTCTGTTGGTATTCAAGGAGTTGCTTGGGCTTTTGGTGGTATGA TCTTTGCCCTTGTCTACTGCACTGCTGGTA TCTCAGGAGGACACATTAACCCAGCAGTGACATTTGGTCTGTTCTTGG CAAGAAAGTTGTCTTTAACAAGGGCTCTGTT CTACATGGTGATGCAGTGCCTAGGTGCAATCTGTGGTGCTGGTGTTGT TAAAGGTTTTATGGTGGGTCCATACCAGAGA CTTGGTGGTGGGGCCAACGTGGTTCAACCTGGCTACACTAAAGGTGAT GGACTTGGTGCTGAGATTGTTGGCACCTTTG TCCTTGTTTACACTGTTTTCTCTGCCACTGATGCCAAGAGAAATGCTAG AGATTCACATGTTCCTGTAAGTTCCGTCTT

176

GTCCTACCAATAACTTTAGTTTATAGTACTATCTTTTTGGCTCAATTTTT TATTTTTAGTGATGGTAACTGCTTATTGG ATGTAATATCTTATTCTGATTACTTTTGGTCTGAAAAGTAAGTGATCAT GCTAGTGTGTGGTGTTTTGTT

>ntMIPcontig29.seq CATTACGGCCGRGKACCTAACTTAGAAACAGAAAAAAAGAAAGAAGA AAAAACCTTCTAGTGTGTGTCTGTGTGTGAAA AAAATGGCAGAAAACAAAGAAGAAGATGTGAATCTTGGAGCAAACAA ATACAGAGAAACACAACCATTAGGAACAGCAG CACAAACAGAAAATAAAGATTATGTTGAACCACCACCAGCACCATTAT TTGAACCTGGTGAATTATCATCTTGGTCATT TTACAGAGCTGGAATTGCAGAATTTATGGCCACTTTCTTGTTTTTGTAT ATTACAATCTTGACTGTAATGGGACTTAAA AGATCAGATAGTTTGTGTTCTTCTGTTGGTATTCAAGGTGTTGCTTGGG CTTTTGGTGGTATGATCTTTGCTCTTGTTT ACTGTACTGCTGGTATCTCAGGAGGCCACATTAATCCAGCTGTGACCT TTGGTCTATTCTTAGCAAGGAAACTTTCCTT AACCAGGGCAGTATTCTACATGGTAATGCAATGCCTTGGTGCTATTTG TGGTGCTGGTGTTGTTAAAGGTTTCATGAAA GGTCCATACCAAAGACTTGGTGGTGGTGCCAATGTGGTTAACCCTGGC TATACTAAAGGTGATGGACTTGGTGCTGAAA TTATTGGTACTTTTGTTCTTGTTTACACTGTTTTCTCTGCTACTGATGCC AAGAGAAATGCCAGAGATTCACATGTTCC TATTTTGGCACCTCTTCCTATTGGATTTGCTGTGTTCTTGGTTCATTTGG CCACCATCCCAATTACTGGAACTGGCATC AACCCTGCCAGGAGTCTTGGAGCTGCTATTATCTTCAACAAAAAACAG GCATGGGATGACCATTGGATCTTCTGGGTTG GACCATTCATTGGAGCTGCTCTTGCTGCAGTTTATCACCAAATAATTAT CAGAGCCATTCCATTCAAGAGCAAGGCTTA AGATTCTCCTGCCATTTGCCtTTTTCAAGAYTCTGTATYCCATTTTCTTM TCTTKCTGGTGTCTGtCWAATGTGTAWTT TCTTCTATTMAGATGTCAAtTTATGTGAATAAATTATCAAGTTGTATCT

>ntMIPcontig30.seq GGCACGAGAAGGATGTTAAGCTAGGAGCAAACAAGTACAGAGAAACA CRRCCaTTaYGGSYRSRGCAGCTCAAACAGAC AAGGATTATAAGGAGCCACCACCAGCTCCTTTGTTTGAGCCAGGAGAG TTGTCGTCATGGTCTTTTTACAGAGCTGGAA TTGCAGAATTCATGGCCACTTTCTTGTTCTTGTACATCACTATCTTGAC TGTTATGGGTCTTAAAAGATCTGATAGTTT GTGTTCTTCTGTTGGTATTCAAGGAGTTGCTTGGGCTTTTGGTGGTATG ATCTTTGCCCTTGTCTACTGCACTGCTGGT ATCTCAGGAGGACACATTAACCCAGCAGTGACATTTGGTCTGTTCTTG RCAAGAAAGTTGTCTTTAACAAGGGCTCTGT

177

TCTACATGGTGATGCAGTGCCTAGGTGCAATCTGTGGTGCTGGTGTTG TTAAAGGTTTTATGGTGGGTCCATACCAGAG ACTTGGTGGTGGGGCCAACGTGGTTCAACCTGGCTACACTAAAGGTGA TGGACTTGGTGCTGAGATTGTTGGCACCTTT GTCCTTGTTTACACTGTTTTCTCTGCCACTGATGCCAAGAGAAATGCTA GAGATTCACATGTTCCTATTTTGGCACCTC TTCCTATTGGATTCGCGGTGTTCTTGGTTCATTTGGCCACCATCCCAAT CACCGGAACCGGTATCAACCCCGCCCGGAG CCTTGGAGCTGCTATCATCTTCAACCAAGACCGGGCATGGGATGATCA CTGGATCTTCTGGGTTGGACCATTCATTGGA GCTGCACTTGCTGCAGTTTACCACCAGATAATCATCAGAGCCATTCCA TTCAAGAGCAAATCTTAATTATCTTGAAGAG TCTCATGTCCTATGTCTCTTCCCCTGTTATATTTCAAGACTCCTGTACCC CTTTTTCCCCCGTTTCTAATGCTGTTGTC TCATGTAATTTTCTTTTCTTTTGATGTTAATTATGTGTGTAAATTATCAG GTTAATGATGTATCTAAGTCTTAAAAATT TTTAATATTAAATAATTACAATAAAAAAAAAAAAAAAAAAAAAAA

>ntMIPcontig31.seq GGCCATTACGGCCSGGGATAAACAGTCTCACAAATATAGAGCTTTTAA TTTCTTCTTCCTCAAAGAAAACATACAATTT TAAGAAGAAAAGATAATGCCGATTTCAAAAATTTCCCTCGGAAATTTA GCAGAGGCTAGTCAGCCTGATGCTCTCAAGG CTGCACTAGCTGAGTTCATTTCAATGCTCATTTTTGTTTTTGCCGGTGA AGGCTCTGGCATGGCCTTCGGTAAACTAAC AAATGGCGGAGCAGCCACGCCTGCTGGATTGATTTCGGCGGCTATAGC CCATGCCTTTGCACTTTTTGTGGCAGTTTCA GTAGGAGCAAATATTTCTGGAGGTCACGTAAATCCTGCGGTTACATTC GGTGCTTTCGTGGGAGGTCACATTACCCTTT TCAGAAGTGTTTTGTATTGGATTGCCCAATTGCTTGGATCTGTCGTCGC TTGCGTGCTCCTCAAGTTTGCTACTGGTGG ATTGGAAACATCAGCATTCGCACTCTCGACAGGAGTTACCCCATGGAA CGCAGTTGTTTTTGAGATAGTGATGACCTTC GGGCTTGTTTACACCGTTTACGCAACTGCAATTGATCCTAAGAGGGGC AATTTGGGAATTATTGCCCCAATTGCAATTG GTTTCATTGTAGGTGCGAACATTTTGGCTGGTGGAGCCTTTGATGGTGC ATCAATGAATCCTGCTGTGTCATTTGGTCC AGCAGTGGTTAGCTGGACATGGAACAGCCACTGGGTCTACTGGCTCGG ACCATTTGTTGGTGCTGCCATTGCTGCTTTG GTTTATGAAATTATTTTCATTgGTGACAaCACTCACGAGCAGCTCCCCA CCGCTGATTACTArGACATTTCTCtTTCTT CTCTAAAACAAATGTGGATAATATTAGCTATGTGCAGTGTGGTGTTGC TGT

>ntMIPcontig32.seq

178

GGATCCTCACTCTACACACACACACACAAAACAGAGMATYMYCKGK WTcTTTCTACAGTTTTTTAAATTTTTCTTAGTA ATGTCTAAAGAAGTAGAGGCAGTGTCTGAGCAGCCGGCGGAGTATTCT GCTAAGGATTACACTGATCCACCACCAACTC CGCTTATTGATTTTGAGGAGTTAACTAAATGGTCACTTTACAGAGCTTG TATTGCTGAGTTTATTGCTACTTTGTTGTT TCTTTATGTAACTGTTTTGACTGTGATTGGGTATAAGCATCAGTCGGAT ACTAAAGATGGCGGCGATATCTGTGGCGGC GTTGGTATTCTTGGTATTGCTTGGGCTTTTGGTGGTATGATCTTTGTTCT TGTTTACTGCACTGCTGGTATTTCTGGTG GACACATCAACCCTGCTGTGACATTTGGGCTATTTTTGGCAAGGAAAG TATCATTGATCAGGGCAGTATTATACATGGT ATCACAGTGCTTAGGTGCAATATGTGGTGTGGGTTTGGTAAAGGCTTT CCAAAAGGCATATTTCAATAGATATGGTGGT GGTGTTAATGTTATGGCAGGTGGACACAACAAAGGTGTTGGTTTGGGT GCTGAGATTATTGGTACCTTTGTTTTGGTCT ACACTGTCTTCTCTGCTACTGACCCTAAAAGGAGTGCCAGAGACTCCC ATGTCCCTGTATTGGCACCACTTCCAATCGG ATTCGCTGTATTCATGGTCCACCTTGCCACCATTCCGATCACCGGAACC GGAATCAACCCGGCAAGAAGTTTTGGAGCA GCCGTGATTTACACCAGGATAgGCTTGGATGAGCACTGGATCTTCTgGG TCGGAcCATTCGTCgGAGCtTTCGcCGcCG CGTCTACATCAGTACATCTCGAGCTGCGCCTTaAAGCTCTGTCTCAgGA GCaATGCTAAGAtTTCATGCATGAAAATTG TATAA

>ntMIPcontig33.seq GGGGAACAACTGGTYYKGatTcMCggKATCCACTTAGCAcWATaAAGAG AGaAAAACAAGGTaASTTTAGTGAGTGtTC AAATGGCAGAAAACAAAGAAGAAGATGTTAAGCTTGGAGCTAACAAA TTCAGAGAAACACAGCCATTAGGAACAGCTGC TCAAACAGACAAAGATTACAAAGAACCACCACCAGCTCCTTTGTTTGA ACCAGGGGAATTATCATCATGGTCATTTTAC AGAGCTGGAATTGCAGAATTTATGGCTACTTTCTTGTTTTTGTACATCA CTATCTTGACTGTTATGGGTCTTAAGAGAT CTGATAGTCTGTGTAGTTCAGTTGGTATTCAAGGTGTTGCTTGGGCTTT TGGTGGTATGATCTTTGCTTWGGTTTACTG TACTGCTGGTATCTCAGGAgGGACACATCAAtCCMAGCTGTGaACCTTT GGATTGKTCTTGSCARGGAAcACTGTCCTT AAcCCAGGRCTATTTaYCTACAtTARTGATGcCAATGcCCTTGGTgGCAcA TTTGTGGTGCTGGTGTTGTGAAGGGATT CATGGTTGGTCCATACCAGAGACTTGGTGGTGGTGCTAATGTTGTTAA CCATGGTTACACCAAAGGTGATGGCCTTGGT GCTGAAATTATTGGCACTTTTGTCCTTGTTTACACTGTTTTCTCTGCTAC TGATGCTAAGAGAAATGCCAGAGACTCAC

179

ATGTTCCTATTTTGGCACCACTTCCCATCGGATTCGCGGTTTTCTTGGT TCATTTGGCCACCATTCCATCACCGGAACT GGCATCAACCCCGCTAGGAGTCTTGAGCTGCGATCATCTACAACACAG ACCAGCATGGGACGACCACTGATCTTTGGGT TGGACATCATTGAGCTGCACTTGCTGCAGTTACATCAATATCATCGAG CATCATCACAGTCGTCTTAGTTTCCTCAGAG CTTATCTT

>ntMIPcontig34.seq GATRARGRMcaMMRGRaRRKKRAMCTCAAGTTTATCTRGCTGTTCATT AATTCTTTTTCCTATAATAYTAATAGTTTTT ATCTTTGAGCTATAGGTATGGACCCTGAAGATGGAKYYTCAYCCCCTT CAACACCAGCAACTCCAGGAACTCCTGGTGC TCCTCTTTTTGGTGGTTTCAAACATGAAAGAAACAGCAATGGCAGAAA CTCCCTYCTCAAGAGCTTAAAATGCTTCAGT GTRGAAGCATGGGCTTCAGAAGAAGGAAGCTTGCCMCCTGTTTCATGC GCGTTACCTCCTCCTCCTGTCTCACTAGCCA GAAAGGTGGGAGCAGAGTTCATAGGTACTATGATAYTAATTTTTGCAG GGACAGCCACAGCRATTGTGAACCARAAGAC ACAAGGCTCTGAAACCTTAATTGRWTKGSMRSCYTCCAYTGGTCTAGC TKTAATGATTGTCATTCKGTCAACAGGCCAC ATCTCTGGAGCTCATCTCAACCCAGCTGTGACCATTGCTTTTGCTGCTC TCAAGCATTTCCCcTGGAAAAATGTTCCTG TGTACATTGGAGCACAAATTATAGCATCATTYTGTGCTGCATTTACACT CAAGGTAGTTTTGCACCCAATAATGGGTGG TGGAGTCACTGTTCCGTCTGGTAGTTACCTTCAAGCTTTTGCTTTGGAG TTCATCATCAGCTTTAACCTCATGTTTGTT GTCACTGCCGTGGCCACCGACACTAGAGCTGTGGGAGAGCTTGCAGgA aTAGCAGTAgGAGCCACCGTCATGCTCaACA TTCTaATAGCTGGGGAGACaACTGGGGCTTCaATGaATCCAGTGAGAaCG CTGGGACCAGCAGTAGCAGCAgGAAACTA TAAAGCCATTTGGATCTATCTGACTGCTCCGATTCTTGGCGCTCTCGCT gGGGCAGGTRTTTACTCTGCAGTCAAACTG CCAAATGAAGATGACAACAATCATGGGAAGCCTTCACTGGAACATAG TTTCAGAAGGTGATTCATCAGTAATACCATTA GATCATGATAGTCACATACTTTAGTAGACAGTGTGCAGTTTTATCAAT GTAGAAACTTCTTGTACCATTTTGCATCTGC AAGGCAAGGGTGGATCGAATAAAACAAATCTGTCCACTCTCTCTCCAT ATGTACAGATTTTAGCAAGATATAATTTATC AAAGAACAGATTTTTAGCAAGATACAATGACCGAGAGATGGAAGGAA GGAAACCATATTAGGAAACTTATGCAATGTGT ACTATTAGTGAGGTTTTTACTGGATTGTAGAAGAGACTTCTGTGTAAC ATGTGTTTCCATTCATAGAACTAGTGCC

>ntMIPcontig35.seq

180

GGCCGTCCTCTATCTATTTGTAATACCAATACCAATTCAGGAGGAGTA CTTGGAGTTGACTTTTTTCTCCAGAGCTATC TCCTTCCTCTCTTCTCTGAGATGGGTGTGATTAAAGCAGCGATTGGTGA TGGGGCGTTAACTTTTTTATGGGTTTTATG CTCCTCTTGTATTGGGGTTTCTACTTACTTGGTAGCTACTACTTTTGGTG TTGTCAATGAAATGGGCAGCCTCTTCATC ACTACCCTTGTTGTTTTTCTTATATTCTTAGTGTTTGGATTTCTGGGTGA TGCATTGGGTGGTGCTGCTTTCAATCCAA CTGCCAATGCCGCCTTTTATGCAGCTGGTCTTGGTCACGATTCTCTTGT CTCAGCCGCCCTTCGTTGTCCTGCTCAGGT AGCAGGTGCAGTTGCCGGTTCGCTTGCAATAATGGAGCTTATGCCTAA GCAGTATCACCACATGCTTGAGGGGCCTGCT TTGAAGGTGGATGTACAAGCTGGAGCTATTGCGGAGGGTGTCTTGACC TTCATAATCACATTCATGGTCTTTGTCATTK TCCTTAGGGGTCCAAAAAGTGCACTTCTSAAGAATTGGTTGCTCACKTT GGTAACTGTCCCTTTGGTGGTTGCAGGTTC AAACTACACTGGACCTTCTATGAATCCAGCCAATGCATTTGGCTGGGC GTACCTGAGCAATGCACACAACACATgGGGA GCACTTTTATGTTTATTNN

>ntMIPcontig36.seq TCGGTCCGGATKCCCGKGCCTAAAATCATTTCTGTTTCTTGTTTCCGCT GTTTCGTTAATCTGTTTCTTCTTCTTCTTC TTCTTCTTCTTCTTCTTCTTCTTCTTCTTTTTTGTCTGTTACTCTCTGAAG CTGGATCCTCTTCAATTCATTCATTAAG CAGTCTGAGTTGTTGTTCTGTACTGTAATTCAGATCCTATATATGCTCA AGGTTTCGCCATCATCTTAATTAGTGCATA TATAGTCAAGCTGGATAGCTTCAGTTCTTGTAATCCTTAGTCGCACAAT TATAAAAAACCTCTAAAGCATGTAACTTTA GTTCTCCTAGCTTCTCCTTTCCACCAAAAAAATTAAAAAAAAATCTTTT TTTTTCCATTTGTTATGTTTCCTGCCGGAT TCTGATTATTCTGAGGATCAAACGATCTGGCCGTTAAACTGAATATCG TGTAGCTAAACATTTAAAAAAGCCAAAAAAT CATGTAATTACTTCTCCTTTGAAAGAAACCTAGCTCATCTAATAAAAC AAAAAACAAAAAAAAAACAAAGGAAAACTAT CGGAGATGCCGGAATTTGAATCACCAGTATCGGCGCCGGCAACGCCG GGGACACCAACGCCGCTATTCTCGTCGATTCG AGTGGACTCAATGGAGTCTAATTATGATCGAAAGTCAATGCCCCGATG CAAGTGCTTGCCTTTGGATGCTCCAACATGG GGCACCCCTCACACGTGTCTTTCTGACTTCCCTGCACcAGACGTCTCCC TCACCCGCAAGTTGGGAGCAGAGTTCGTGG GGACATTTATCCTTATATTTGCTGCACAGCTGGGCCaATTGTGaACCAGa AGTACAGCGgCGTAGaATCTCTaATAGAA ATGCAGCTTGCGCTGGGCTgGcCGTTATGATCGTGATTCTGTCACAGGC CATATTTCTGGAGCACATCtTAATcCGTCG

181

CTCAcCATCGCATTTGCaGCACTTCGTCACtTTcCGTgGGTTCAAGTGCCG GCCTATGTTGCAGCGCAGGTTTCAGCAT CCGTTTGTGCTTCTTTTGCTCTCAAGGGTGTTTTTCATCCTTTCATGTCT GGTGGCGTTACTGTTCCTTCCGTAAACAC TGGCCAGGCTTTTGCTCTCGAATTCCTCATCACATTCAATCTCCTTTTT GtTGTCACTGCTGTTGCTACCGACACCCGC GCGGTGGGAGAGTTGGCGGGCATTGCAGTTGGAGCTACAGTCATGCTC ATAtTCTAGTgGCGGGGcCATCAGTgGTGCT TCATGATCAGTAGACTTTGGGACAGCGTGCAGCAGAATACAGTCAtTG TGaATTACTAGTGCTCACTCGGGGCTCTGCA GGCAGCTGTTATCGCTCGTCACT

>ntMIPcontig37.seq GGAGGACATATTAACCCAGCAGTGACATTTGGGTTATTCTTAGCAAGA AAAGTGTCATTATTAAGGGCAGTGGGATATA TTATTGCCCAATCTTTAGGTGCAATTTGTGGTGTTGGTTTAGTGAAAGG TTTCATGAAACATTACTACAACACATTAGG TGGTGGTGCTAATTTTGTGCAACCTGGTTATAACAAGGGCACTGCTTT GGGTGCTGAGATTATTGGAACTTTTGTTCTT GTTTACACTGTTTTCTCTGCTACTGACCCTAAAAGAAGTGCCCGTGACT CACATGTCCCTGTGTTGGCCCCACTGCCAA TTGGATTTGCTGTGTTCATGGTTCATTTGGCTACTATTCCTATTACTGG AACTGGTATTAACCCTGCTAGGAGCTTTGG AGCTGCTGTTATTTACAACACTGAAAAAGTCTGGGATGACCAATGGAT TTTCTGGGTTGGACCATTTGTGGGAGCATTG GTAGCAGCAGTATATCATCAGTATATCTTGAGAGGTTCAGCAATTAAG GCATTGGGTTCTTTCCGCAGCAACCCCACCA ATTAAAACAACATTTGCAATCACAACCAGAAAAAAAGAAAAGAAAAG AACAAGAAAAAGAATTGCATTCATCTGTCCAA ATTATCTTTGTTTATTTATTTGATTCTATGTGTGTAAGAGGAAACTATG GAACTCTCCCTTTTACTTTTTATGTATTGC TTTTAATAGTTTAATCCTTAATGTTAACCTTGGATGTATATTTATTTGTA TGAGTAGTGTTGGTAATGTTTT

>ntMIPcontig38.seq GGTCATCAGAAAACATTGTCTCTGTTTCCATTTTCTTCAATTCTATTTA GAATCAATTTAATTAGTTTTTTGAAAATGC CTGCCATAGCTTTTGGTCGTTTCGATGATTCATTTAGTTTGGGGTCTAT TAAGGCCTACATTGCTGAATTCATCTCTAC ATTGCTCTTTGTCTTTGCTGGAGTTGGTTCAGCCATTGCTTACAACAAG TTGACAGCAGATGCTGCTCTTGATCCCGCG GGGCTTGTAGCAGTTGCAGTTTGCCATGGGTTCGCTCTGTTCGTGGCG GTTTCCGTTGGGGCTAACATCTCCGGTGGTC ACGTTAACCCCGCCGTTACTTTGGGATTGGCTCTTGGTGGCCAAATTAC AGTTCTTACTGGCCTCTTCTACTGGATTGC

182

TCAACTTATGGGCGCCACTGCTGCCTCCTACCTCCTCAAAGTTGTCACC GGAGGATTGGCTGTTCCAATCCACAGTGTA GCAGCTGGAGTAGGAGCTGCTGAAGGAGTAGTGATGGAAATAATCAT CACATTTGCATTGGTGTACACAGTGTACGCCA CAGCAGCTGACCCCAAGAAGGGTTCATTGGGCACAATTGCACCCATTG CCATTGGTTTCATTGTTGGTGCCAACATCTT GGCTGCTGGCCCATTCTCTGGTGGTTCAATGAACCCAGCTCGCTCCTTT GGACCTGCAGTGGCTAGTGGTAACTTCGCT GGTCACTGGATTTACTGGATTGGACCCCTTGTTGGTGGTGGCTTGGCTG GTCTTATCTACAGTAATGTGTTCATGAACC ACGACCATGCCCCTCTATCCACCGATTTCTAAGTTAGAATTTGATCATT GTGTCCAAAATCCATTTGCCTTTTGTAATA AAGGAAGAAAAAGGTAATATTTTGCTTTTTCTTTCTTTTGTATTAGTAA CTTTTGTTTTTGATTTTTTGTTTCTTTCTC TAGCTTTTGGTTTGCAGCTGTACACATCCATCTTTTGGTGACATGTTGC TGTCTACTTGATTTAYGAATTTGGGGAGAT CGAAAGGCAACCAtKCGGCCGCATGAGGGGTACCTGTCCTCTCCAAAT GAA

>ntMIPcontig39.seq CATTACGGSSGRGKATACTAGCCTCTAATCAAGGATCACCATATACCC ACCAAAACTCCTTCTTATTAAACTCTCTTCC ATTTCTTTGATCAACATTCATTCTTGATTTTCCGACGATGCCGATCAGC CGGATAGCAATCGGAAGRCCGGAGGAAGCC ACTCACCCCGACGCCTTAAAGGCGGCGTTGGCYGAGTTTATYTCCACC TTAATTTTCGTATTTGCAGGTTCAGGTTCAG GTGTAGCATTTAGCAAGTTGACTGGWGGTGGTGCTAACACMCCTGCC GGTCTYATYGCYGCYGCWATTGCTCATGCTTT TGGGTTGTTYGTGGCGGTTTCCGTCGGYGCTAACATTTCCGGYGGACA TGTTAACCCTGCTGTCACCTTTGGTGCSTTT GTTGGTGGTAATATYACACTTTTACGTGGAATTYTKTACTGGATTGCTC AGTTGCTTGGCTCTGTTGTTGCTTGCTTSC TTCTCAAGTTCACCACTGGTGGCTTGGAGATWGGTRCATTCGGCTTGT CTGATGGAGTGGGTGTAGGAAACGCATTGGT ACTTGAAATAGTAATGACATTTGGCCTAGTCTACACSGTGTACGCTAC CGCAGTGGATCCAAAKAAGGGCAGTTTGGGA ACAATTGCACCAATTGCAATTGGTTTYATTGTTGGAGCCAATATTTTGG CTGGTGGGGCCTTTGATGGRGCGTCWATGA ACCCAGCAGTTTCTTTCGGSCCAGCAGTTGTGAGCTGGAGCTGGRMCA ACCACTGGGTTTACTGGGCTGGGCCTTTGAT TGGTGGTGGGCTTGCTGGGCTTRTTTATGAGTTCTTCTTCATTAACCAG ACCCATGAACCTTTGCCCCAATAAGAAAAA ATGTAAATGGTGAATTTTCTGTGTGATTTTGTTCCAAGGGGTTTTGTTA TTCTCTGTTTGATTTCACAATTGCCCTTGA TTTGGGATGAACTTGGTTTTGTATTGTGTGATGCTGTTGGGGTGCGTGG GTATGAAGGGGTATTTATGTAATTCGTGTG

183

TTGGTTCCGTGTTTCAATTGTTCAGCTCTTTTCATTCTATTGCATTTTCC TTTCG

>ntMIPcontig40.seq AAGTATACTAGCCTTTAATTAAGGATCACCATATACGTACCCAAYAAA ACTCCTAAACTCTCTTCCATTTCTTTGATCA ACATTCATTCTTGATTTTCCGACGATGCCGATCAGCCGGAYAGCAATC GGAAGGCCGGAGGAAGCCACTCACCCCGACG CCTTAAAGGCGGCGTTGGCTGAGTTTATCTCCACCTTAATTTTCGTATT TGCAGGTTCAGGTTCAGGTGTAGCATTTAG CAAGTTGACTGGTGGTGGTGCTAACACACCTGCTGGTCTTATTGCTGCT GCTATTGCTCATGCTTTTGGGTTGTTTGTG GCGGTTTCCGTCGGCGCTAACATTTCCGGTGGACATGTTAACCCTGCT GTCACCTTTGGTGCGTTTGTTGGTGGTAATA TTACACTTTTACGTGGAATTCTTTACTGGATTGCTCAGTTGCTTGGCTC TGTTGTTGCTTGCTTGCTTCTCAAGTTCAC CACTGGTGGCTTGGAGATAGGTGCATTCGGCTTGTCTGATGGAGTGGG TGTAGGAAACGCATTGGTACTTGAAATAGTA ATGACATTTGGCCTAGTCTACACGGTGTACGCTACCGCAGTGGATCCA AATAAGGGCAGTTTGGGAACAATTGCACCAA TTGCAATTGGTTTTATTGTTGGAGCCAATATTTTGGCTGGTGGGGCCTT TGATGGAGCGTCAATGAACCCAGCAGTTTC TTTCGGCCCAGCAGTTGTGAGCTGGAGCTGGGCCAACCACTGGGTTTA CTGGGCTGGGCCTTTGATTGGTGGTGGGCTT GCTGGGCTTGTTTATGAGTTCTTCTTCATTAACCAGACCCATGAACCTT TGCCCCAATAAGCAAAGATGAAAATTGCAG AATGTAAATGGTGAATTTTCTCTGTGTGATTTTGGTCTTCTCTGTTTGA TTTCACAATTGCCCTTGATTTGGGATGAAG TTGGTTGTATTGTGTGATGCTGTTGGGGTGGGTTTGAAGGGGTATTTAT GTAATTCATGTGT

>ntMIPcontig41.seq TTCACCTACCGTGATTCTTTCCCTCATTTTTATTTAATTCATTTGGGAAG CAAAGATGGTGGCATGGAAAACCAAAATA CCTGGTGCAAAATGGTCAGCTCCATGATTGTAATGGTGAAGGATCTTG TGTTAAATAACAAGATCTAGTGGACCGGGAC AAGAACCACATGACTGATGTCTAGAGTTCAATGCTTCATTTAAATTCC AAATGCAAAGGTATGGACCCTGAAGATGGAG TCTCATCCCCTTCAACACCAGCAACTCCAGGAACTCCTGGTGCTCCTCT TTTTGGTGGTTTCAAACATGAAAGAAACAG CAATGGCAGAAACTCCCTTCTCAAGAGCTTAAAATGCTTCAGTGTAGA AGCATGGGCTTCAGAAGAAGGAAGCTTGCCA CCTGTTTCATGCGCGTTACCTCCTCCTCCTGTCTCACTAGCCAGAAAGG TGGGAGCAGAGTTCATAGGTACTATGATAC TAATTTTTGCAgGGACAGCCACAGCGATTGTGaACCAGaAGACACaAGG CTCKGAAAcCTTAAtTGGATTGGCAGcCTC

184

CACTGGTCTAGCTGTAATGATTGTCATTCTGTCAACAGGCCACATCTCT GGAGCTCATCTCAACCCAGCTGTGACCATT GCTTTTGCTGCTCTCAAGCATTTCCCCTGGAAAAATGTTCCTGTGTACA TTGGAGCACAAATTATAGCATCATTCTGTG CTGCATTTACACTCAAGGTAGTTTTGCACCCAATAATGGGTGGTGGAG TCACTGTTCCGTCTGGTAGTTACCTTCAAGC TTTTGCTTTGGAGTTCATCATCAGCTTTAACCTCATGTTTGTTRTCACTG CCGTGGCCACCGACACTAGAGCTGTGGGA GAGCTTGCAGGAATAGCAGTAGGAGCCACCGTCATGCTCAACATTYTA ATAGCTGGGGAGACAACTGGGGCTTCAATGA ATCCAGTGAGAACGCTGGGACCAGCAGTAGCAGCAGGAAACTATAAA GCCATTTGGATCTATCTGACTGCTCCGATTCT TGGCGCTCTCGCTGGGGCAGGTGTTTACTCTGCAGTCAAACTGCCAAA TGAAGATGACAACAATCATGGGAAGCCTTCA CTGGAACATAGTTTCAGAAGGTGATTCATCAGTAATACCATTAGATCA TGATAGTCACATACTTTAGTAGACAGTGTGC AGTTTTATCAATGTAGAAACTTCTTGTACCATTTWGCATCtGMATCAGA TAATTCCATTAAAACCAAGTAACCGACTAA ACCATCTGGAGTGCCTTAGCTTGAACAGATTGATCAGGGCTGCGGTGA AAGAAGGATGATTTGTAGAGAGAAAGAGCGA GAGCAGCGGTAAGAGTACCAAAGACGTCGGCGACAGCGTCCTTGGTA GATACGCCTGCTGACCTGAAATAACCTAATTC GTCGGCCACCTCCTTAGCGGCACCGGCAGCAAGTGAGACAATGGATC CGACCCAGATGCTCCGGCGGCGGATGAAAGGG TAACGAGCTTTTTCCGCGAGAAAGGAGGTAATGATAGCAATGAAGAA GCAGAAAAGGACGTGGTAAAACTTATCACGAG CTATCCAATCATCTCCGTCCTCCATTCTTTAAAAAAAAATAATTTATAG AAAAGAAT

>ntMIPcontig42.seq CGATCAAKTTTTTARCAAATACCATAATTGRTTAATTAGCAARTTTTTA GTTGGGAGCAAAAATGGYGAAGATTGCCTT TGGTAAYTATAATGACTCTGTTAGTGCAGCCTCAATCAAGGCATACCT AGCTGARTTCATTGCTACTCTTCTCTTTGTK TTTGCTGGTGTTGGATCTGYAATWTCTTACAATAAGTTGACAACAGAT GCAGCTCTAGACCCAGCTGGTTTAGTAGCAG TKGCKGTGGCWCATGCATTTGCATTGTTTGTTGGAGTTTCAATGGCAG CCAATATCTCAGGTGGCCATTTGAATCCAGC TGTCACCTTTGGATTGGCTGTTGGTGGCAATATAACTATCTTGACTGGT TTCTTCTACTGGATTGCCCAATTGCTTGGC TCTACAGTTGCTTGCCTTCTCCTCAAATTTGTTACTGGCGGATTGGCTG TTCCTACCCATGGAGTTGCTGCTGGGCTCA CTGGATTTGAGGGAGTGGTAATGGAGATAGTCATTACCTTTGCACTTG TCTACACTGTTTATGCTACAGCAGCAGACCC CCAAAAGGGTTCCCTTGGAACAATTGCACCAATTGCAATTGGGTTCAT TGTTGGAGCTAATATCTTGGCTGCTGGCCCA

185

TTTAGTGGTGGGTCAATGAACCCGGCTCGATCTTTTGGGCCTGCTGTG GTTTCTGGAGATTTCTCTCAAAACTGGATCT ATTGGGTCGGCCCACTTATTGGTGGAGGATTAGCTGGGCTTATTTATG GTGATGTGTTTATTGGATCACATGATCCACT TCCAGTCTCTGAAGACTATGCTTAGAGATCGTTCTCGGGCTAACACAT TGACCGACCATTCAAAAATAATTACATTTTT TAGTCAAGTATACAAAAAATATGTATTAAAAATATATATTTTATCAGC TATTATTTTTGGAAGCGGCTATATTGTGTAG TTTTCCCTCTTGTCATTTGTGTTTTTCAAATGTAACATTTAATTTTTTGT GAGCTTTGTAATTTCTGCTCTGCAACAAC TTGTACTTCATTTTATTTAAGAAAATGTGGRTATTTCCTTTTGTWATAT TTTGCAATTATTATTTTTCCTT

>ntMIPcontig43.seq CTGGTCTAGTAGCAGTAGCTGTGGCTCATGCATTTGCATTGTTTGTTGG GGTTTCCATAGCAGCCAATATTTCAGGTGG TCATTTGAATCCAGCTGTCACTTTGGGATTGGCTGTTGGTGGAAACATC ACCATCTTGACTGGATTCTTCTACTGGATT GCCCAATTACTTGGCTCTACAGTTGCTTGCCTCCTCCTCAAATACGTTA CTAATGGATTGGCTGTTCCAACCCATGGAG TTGCTGCTGGGCTCAATGGTTTCCAAGGAGTGGTGATGGAGATAATCA TAACCTTTGCACTGGTGTACACTGTTTATGC AACAGCAGCAGACCCCAAAAAGGGTTCCCTTGGAACCATTGCCCCCAT TGCAATTGGGTTCATTGTCGGGGCCAACATT TTGGCAGCTGGTCCATTCAGTGSCGGGTCAATGAACCCAGCTCGATCA TTTGGGCCAGCTGTGGTTGCAGGAGACTTTT CCCAGAACTGGATCTATTGGGCCGGCCCACTCATTGGTGGAGGATTGR CTGGGTTTATTTATGGAGATGTCTTTATTGG ATGCCACACCCCACTTCCTACCTCAGAAGACTATGCTTAWAAGAAGA GAACAGTTCTTCAATGTTTCTTTGTGTGTTTT CAAATGCAATGTTGATTTTTAATTTAAGCTTTGTATATTATGCTATGCA ACAAGTTTGTTTCCAATGAAATAKCATGTT TTGGWKYGGCCGCATG

>ntMIPcontig44.seq GAGTATTTATAACCAATCCATGCACACAATTTTAGAGAAAAATCATTA AAAGCTAAAGCATAAGCAACACTTTTCTCTC CCAAATATGGCTTCCAATGCTAGTCATGTTTTAGGCGATGAAGAAAGC CAACTTTCTGGTGGAAGTAATAGAGTTCAAC CTTTCTCTTCTACACCAAAAAAGAATATTGATGATGAGGGAAAGAAGC ATACTTCTCTCACAGTGGCACAAAGGCTGGG CATTTCTGACTTCTTTTCTTTGGATGTATGGCGAGCGTCAGTGGGAGAG CTCCTAGGCTCGGCGGTTCTTGTTTTTATG TTGGACACCATAGTGATCTCCACCTTTGAAAGTGATGTGAAAATGCCA AATTTGATCATGTCAATTCTCATAGCAATTG

186

TGATCACAATTCTACTCCTCGCCGTTGTTCCGGTGTCCGGTGGCCACAT AAACCCCGTCATCTCCTTCTCCGCCGCGCT TGTCGGAATTATATCCATGTCAAGAGCCATTATTTACATGGTGGCACA ATGTGTTGGAGCAATTTTAGGTGCACTAGCT CTAAAAGCAGTAGTTAGCTCTACTATTGCACAAACTTTCTCACTTGGTG GTTGTACCATAACAGTAATTGCACCGGGCC CAAATGGGCCCATTACAGTGGGCCTAGAAATGGCCCAAGCTTTGTGGC TTGAGATCTTTTGTACATTTGTTTTTCTTTT TGCTTCAATTTGGATGGCTTATGATCATAGGCAAGCTAAGGCCCTTGG CCTTGTCACTGTCTTGTCCATTGTTGGTATA GTYTTGGGcCtTCtTGTGTCATCTCGACTACgGTCAcCATGaAAAAGGGCT ACGcCGGAgCgGGGATGAATCCGGCGAG GTGTTTCGGGGCTGCTGTTGTTAGAGGAGGTCATCTTTGGGATGGGCA TTGGATCTTTTGGGTTGGGCCTACTATTGCT TGTGTAGCATTTTATGTGTACACAAAGATAATTCCACCAAAGCATTTTC ATGCAGATGGATACAAGTATGATTTTATTG GAGTTGTTAAGGCTTCGTTTGGGTTGCATGAATGAAGATTTGGTCCAG AAAAAAGTCCAGATGTAATAATGGGCTTTTT AGGTGATCATTTGGGTTCGTCCAATCATGGGCCGTTAATAAACTAATG TGTTATTAGCCTAATAGGATTTAAGTACCAA GTCAAAATGTGAAATAGAATATCTTTGATGTAATTTTAACTTGTCAAT GTTCCTGTAAGATTTAAATGTTCAGTATAGT TTCCTATAATCAACTTGTAATAGTATATCTTCGATGTAATTTTAACTTG TCAATGTTCCTGTAAGATTTAAATGTTCAG TATAGTCTCCTATAAT

>ntMIPcontig45.seq AGGAAAAGTTCTCGAGATTCTCAATCGCTCTTGAGAGAGGTTACAACA GAGACAAAACGTACATCCTTAACTGAACATT ATACATGTTAAGTTAAAAACAGAAAATTAAGTTCATGCAATCACACTG GAGAAAGACATGATACAAAATTCAAACTGTA CGATCACCAATAATTTAGGAATCCCCTTCATTCTGACTTTTTCTCTTTC ATCTTCTCCTTTTCGTCCTTTGGTGGGGAA ACTAGCAAATTGAAAGTCCACACAGCCAGCAAAGTTGCCTGTATTGGA GCAAGCCAATAAACCTGTATGTGCTCCTTGG TTATATGATCCCCCCGAGCATAAGCCCACCCCATCACAGAGGCTGGAT TCATGCACCCCCCTGTTAGATCAGAACCAAG TATGTGAAGAGTCAGCTTGGACAGACTGGATATCCACGTCTTCATCAA GGTACTCGCACGACTTCTTCTGGAAAGTCCA AGTGAAATGGTAACAATCGCAAATGTCAAGCACCCTTCAATCAGTGCA CCTCgGTGGATGTCGATgGTCAAACGAGGTc CACGcCCTATGTTTGGAAATGACGCAATGATGAGCCTAACCCCAGTAA TTGAACCAAAGACCTGAGCAGGAATTCTGGC ACCGACGGCGaAGAGGAAATTGGAGAGATCCCCAGAGATGGCACCGG ACaAGATAGTGAGAGGGTTGTAgGCCCCACCC

187

TTGGTAGCTTTAGCCaAGaAgGCAAAGaAGaACATAtTGATGACaGATAT TGaATGtTTGAGGAtTTCAcCTTTGAGAT cATGAGCCCCATAACCCAACATTGTGTGAACAAACATCTTAATAAGCA CACTTGACCAGACCCACATGAAGGACATGAT GAAGTCTGAAACCAGCAGCCTCCTCCTGCTTACCCCCATTTCTTGCTTC TTTTAGTTGATCCTCTTAGGTGTCGTGCTT TCTTTGGGTAGGAGGTGCGGGTAGAGGAGGTAAGAGTGTGCGGATTG AGGGGGGGGGGGGGGGGTAGGTCTAGGAACTT CTTATTAGTTAACTCCTTCCATCATCTTAATCCCTTATTTAAAAACTTAT ATCTGTCTCCGTCTTTTAATCTTCCACTA CTTCGTCTAAACCGGAATCAATTTCACCAAGTATTGGTAAGTCTACTCC ATGAACCTGGAGAAAGATTCAACACAATTG TTGCAATTCCAGGCAAGATTTCCGTCTCTTCGACAAAAACTTGATGTAT CGCGCCTGATTTCCCCCTTAGATCTCCCCA ATTTGTATATTAGGGTTCTGAAAAATTGGGGGAAAAGAAGATCCCGGG AATCCGGACCGA

>ntMIPcontig46.seq CCGGGTCTSATCCAAAAGCAATTTTCTCTCTAGTCTATTCTTGTCGTCC CTTCTCCTTTAGCTTCTAATTACAAGTAAA TAAATTATTTTAATTGTGAAAATGCCTGGCATAGCTTTTGGACGTATAG ATGATTCATTTAGCGTTGGGTCTCTTAAGG CCTATCTTGCTGAATTTATCTCCACCTTGCTCTTTGTCTTCGCCGGAGTT GGTTCTGCCATTGCTTACAATAAGTTGAC AGCAGATGCAGCTCTAGATCCGGCTGGGCTTGTAGCTGTTGCAGTTTG CCATGGATTTGGTCTATTCGTAGCCGTTGCC GTTGGCGCTAACATTTCCGGTGGACATGTTAACCCTGCTGTTACCTTCG GATTGGCTCTCGGCGGTCAAATTACAATAC TTACTGGCCTCTTTTACATCATTGCTCAGCTTTTGGGCTCCATTGTAGC TTGCTTGCTCCTCAAAGTTGTTACCGGTGG ATTGGCTGTTCCAATCCATAACGTGGCAGCTGGAGTGGGAGCTCTAGA AGGAGTTGTTATGGAAATTATTATCACCTTT GCTTTGGTATACACAGTGTATGCAACAGCAGCAGACCCAAAGAAGGG TTCATTGGGTACCATTGCACCCATTGCCATTG GTTTCATTGTTGGTGCCAACATTTTAGCCGCCGGCCCATTCTCTGGTGG TTCAATGAACCCGGCCCGTTCATTTGGGCC TGCTGTGGCCAGTGGCGACTTCACTAACAACTGGATTTACTGGGCTGG GCCTCTCGTTGGTGGTGGATTGGCTGGTCTT ACTTACAGCAACGTTTTCATGCAAAACGAGCACGCCCCTATCTCCAGC GATTTCTAAGTAAAAAATTGTTTGAGTTTGA TTTTGTAAAATAAAAAGGAAGAAAAAAGCAACATTAATTTTGCTCTTT CTTTCTTTTTGTTTGTTTGTTTTCCACTTTA TCCTTTGTTGTTTTCTTTCCCTTTATAGCTTTTGGGTTTGCAGCTGTACA TTCATCTTTGGTCCAATGTTGTCTACGTG ATGATGCTCGAATTTGGYGAAATCGCAACCATTTATTGTCG

188

>ntMIPcontig47.seq GGTCCGGAATTCCCGGGATCSACGACATTCTCCTCTCTCAAAATTAGC AAAGCTCAAATTTAACATCTTATGAGTCTAT TTTTTGGTGAACAAATGGCTAAGGACACTGAAGTTGGCACAGAATACG CCCCAAAAGACTACCAAGACCCACCCCCTGC ACCCTTAATTGACCCTGAGGAGCTAGGAAAATGGTCATTTTACAGAGC CATAATAGCTGAATTCATAGCCACTTTATTA TTTCTCTACATTACTGTCCTCACTGTGATTGGATACAAGAGCCAAATTG ACCCTGACCATAACGGTGAACAATGTGGTG GTGTTGGAATTCTTGGAATTGCATGGGCATTTGGGGGCATGATTTTTGT CCTTGTTTATTGTACTGCTGGTATTTCTGG TGGACATATTAACCCTGCTGTTACATTTGGACTATTCTTGGCTAGGAAA GTGTCGTTGGTTCGCGCAATTATGTATATG TTGGCTCAGTGTTTAGGAGCCATCTGTGGTTGTGGTTTGGTGAAGGCA TTTCAGAAGGCGTACTATGTTAAGTATGGTG GTGGTGCTAATACGTTGAATGATGGTTATARTACAGGTACCGGTTTGG GTGCTGAAATTATTGGCACATTTGTTCTTGT TTACACTGTTTTTGCTGCTACTGATCCTAAGAGAAATGCYAGAGACTC TCATGTTCCTGTGTTGGCACCACTTCCAATT GGATTTGCTGTGTTTATGGTTCATTTGGCYACAATTCCTGTAACTGGAA CTGGTATTAACCCWGCTAGAAGTTTTGGAG CTGCTGTTATTTATGGTAAAGAMAAAGCATGGGATGACCAATGGATW TTYTGGGTTGGACCTTTYATTGGAGCTGCAAT TGCTGCATTTTACCATCAATTCATATTGAGGGCTGGAGCAGTCAAAGC WCTTGGTTCATTCAGGAGCAATGCCTAATTT ATCACGAGAATCTGCAATTGTATATTTGAGAAgAAGTCTCTCTAAATTA TGCTTTTCATTTAGTAGAGTGKTCTTGGYT GTTTGRCACTTKTTTTaAAATGTGCCAAATATTTGTCAGYGCTGCTTTTC TTYTTGTTTCCCTTGCTTGTTTTYTGTTT GTGttTATGTAATTTTYYTTATGCTTGTTTGAGATTCAGAATAATCGAAT GAGCAAGGCTGTAATTTTCCTTATGCTTG AAATTTGAAAGTTATAATTATTTTGG

>ntMIPcontig48.seq CATTACGGCCGGGGACACTCTCAAATTAAAATTTCCAACTCCATAAAT ACCCCATCACTCTTCTATCAMCgGGGACTTC AGCCAAATATCAAACaAtATATTTTCAAGATTAGTCTTYCTTSCTTAGCC GAATTAAACWMAgggcAAAtATGGGCAAG GACATTGAAGTTGGCACAGAATATGCCCCAAAAGACTACCAAGACCC ACCCCCTGCACCYYTAATTGACCCWGAGGAGC TAGGAAAATGGTCATTTTACAGAGCCATAATAGCTGAATTCATAGCCA CTTTATTATTTCTCTACATTACTGTCCTCAC TGTGATTGGATACAAGAGCCAAATTGACCCTGACCATAACGGCGAAC AATGTGGTGGTGTTGGAATTCTWGGAATTGCA TGGGCATTTGGGGGCATGATTTTTGTCCTTGTTTATTGTACTGCTGGTA TTTCTGGTGGACATATTAACCCTGCTGTTA

189

CATTTGGACTATTCTTGGCTAGGAAAGTGTCGTTGGTTCGCGCAATTAT GTATATGTTGGCTCAGTGTTTAGGAGCCAT CTGTGGTTGTGGTTTGGTGAAGGCATTTCAGAAGGCGTACTATGTTAA GTATGGTGGTGGTGCTAATACGTTGAATGAT GGTTATAGTACAGGTACCGGTTTGGGTGCTGAAATTATTGGCACATTT GTTCTTGTTTACACTGTTTTTGCTGCTACTG ATCCTAAGAGAAATGCTAGAGACTCTCATGTTCCTGTGTTGGCACCAC TTCCAATTGGATTTGCTGTGTTTATGGTTCA TTTGGCTACAATTCCTGTAACTGGAACTGGTATTAACCCAGCTAGAAG TTTTGGAGCTGCTGTTATTTATGGTAAAGAA AAAGCATGGGATGACCAATGGATWTTYTGGGTTGGACCTTTYATTGG AGCTGCAATTGCTGCATTTTACCAYCAATTYA TATTGAGRGCTGGAGCAGTCAAAGCACTTGGTTCATTCAGGAGCAATG CCTAATTAATCACGAGAATCTGCAATTGTAT ATTTGAGAAAAGTCTYTYTAAATTATGCTTTTCATTTAGTAGAGTGGTC TTGGTTGTTTGGCACTTTTTTTAAAATGTG CCAAATATTTGTCAGTGCTGCTTTTCTTCTTGTTTCCCTTGCTTGTTTTT TGTTTGTGTATGTAATTTTTCTTATGCTT GTTTAAGATGCAAAATAATGGAATGTGTAAGACTATTATATTTAACTT TTTTTTTTCACCAAATTAATATACAAAATAG CATTTTGTTCACTTGGAAAAGGAATATGACAAATTATTCTTACTGGAA AAAAAAAAAAAACAAAAAAAAAAAAAAAAAA AAAA

>ntMIPcontig49.seq GCCKSACACCTAAAAACAGAGTTTTCAGCAACAGAGTACTGTTGAATA GATAGTGATGGGAAAAGACGTAGAAGCAGCA ACGGAGTTTTCAGCAAAGGACTACACGGACCCACCGCCAGCACCCTTG ATTGATTTTGAGGAGCTGAAACAATGGTCTT TTTACAGAGCTGCTATTGCTGAGTTTATTGCCACTTTGTTATTTCTTTAC GTAACTGTACTCACTGTGATTGGGTACAA GCACCAGTCGGACGTTGACGCTAATGGCGACGTCTGTGGCGGCGTTGG CATCCTTGGTATTGCTTGGGCATTTGGTGGC ATGATCTTTGTTCTTGTTTACTGCACTGCTGGTATCTCAGGGGGACACA TTAACCCGGCAGTGACATTTGGGCTGTTCT TGGCGAGGAAAGTGTCACTGATCAGGGCCTTGGTGTATATGGTAGCAC AGTGCTTGGGTGCAATATGTGGTGTGGGTTT TGTGAAGGCTTTTCAGAGCGCTTACTACGACAGATACGGTGGAGGTGC TAATGTGATGGCTGCTGGCCATACAAAGGGT GTCGGTCTTGCTGCTGAGATCATTGGTACCTTTGTTTTGGTCTACACTG TTTTCTCTGCCACTGACCCCAAAAGAAGCG CCAGAGACTCCCATGTCCCCGTATTGGCTCCACTTCCCATTGGATTCGC AGTATTCATGGTCCACTTAGCCACCATTCC AATCACCGGAACCGGTATCAATCCGGCTAGGAGTTTCGGAGCGGCCGT GATTTACAACCAGGACAAGGCATGGGATGAG

190

CACTGGATTTTCTGGGTCGGTCCATTTATCGGAGCCTTCGCCGCCGCCG CCTACCACCAGTATATCCTCCGAGCTGGTG CCGTCAAAGCTCTCGGTTCCTTCAGGAGCAATGCCTAAGACTTCTCATT GCTCAATATGAT

>ntMIPcontig50.seq GGCCGCTCCAAATAAATACACATCCAATGCTAACATTAAAGATTTAAC TGTACCAAACGAACAAAAAGAGAATGGTTCA TACATTCCTCATACACAAAATCAAACAATTAGACTTGAAAAATTTGAA CTCGTGAACTCAATTTTCTTCTTCTCTGGTT TGTGAGGGTGGTGGAATAACTTTTGCTTAGTTGGTTGGGTTGCTGCGG AAAGATCCCAAAGCTTTAACAGCTCCAGCTC TCAATACAAACTGATGGTAAATTGCTGCTATCAAAGCTCCCACAAATG GTCCAACCCAGAAAATCCATTGGTCATCCCA GATTTTTTCCTTGTTGTAAATGACAGCAGCTCCAAAGCTCCTAGCAGG GtTAATACCAGTTCCAGTAATTGGAATAGTT GCCAAATGAACCATGAATACAGCAAATCCAATTGGCAGAGGAGCCAA AACAGGGACGTGAGAGTCACGTGCGCTTCTCT TAGGGTCAGTAGCAGAGAAAACAGTGTAAACAAGAACAAAAGTTCCA ATAATCTCAGCACCTAAAGCTGTACCCTTGTT GTAACCAGGTTGTACAAAGTTAGCACCTCCACCTTCTAAGTTGTAGTA ATGCTTCATAAAAGCCTTGACGAAACCAACA CCACAAATAGCACCAAGTGATTGTGCAATTATGTATGCAACAGCTCTT ATTAGTGACACTTTCCTTGCCAAGAACAACC CTAATGTCACCGCTGGGTTAATATGACCACCAGAAATACCAGCAGTGC AGTAAACAAGAACGAAAATCATGCCRCCAAA AGCCCATGCAATACCAAGAATACCTACACCATCACAATGGTCTGCAGC GTTCARMTTCTTGCGGACGCG

>ntMIPcontig51.seq GATAGAACAAGCCACGTACTCAGCTACATTTGATGTGGACTAAGGTTG ATTAAGGGTATGGATGCTGAAGATGGAACGT CAGCCCCTTCAACACCAGCAACTCCAGGAACTCCTGGTGCTCCTCTTTT TGGTGGTTTCAAACATGAAAGAAACAGCAA TGGCAGAAACTCACTCCTCAAGAGCTTAAAATGCTTCAGTGTGGAAGC ATGGGCTTCAGAAGAAGGAAGCTTGCCGCCT GTTTCATGCGCGTTACCTCCTCCTGTCTCACTAGCCAGAAAGGTGGGA GCAGAGTTCATAGGTACTATGATATTGATCT TTGCAGGGACAGCCACAGCAATTGTGAACCAAAAGACACAAGGCTCT GAAACCTTAATTGGATTGGCAGCCTCCAGTGG TCTAGCTGTAATGGTTGTCATTCTGTCAACTGGCCACATCTCTGGAGCT CATCTCAACCCAGCTGTGACCATTGCTTTT GCTGCTCTCAAGCATTTCCCATGGAAAAATGTTCCTGTGTACATTGGA GCACAAATTATAGCATCATTTTGTGCTGCAT TCACACTCAAGGTAGTTTTGCACCCAATAATGGGTGGTGGAGTCACTG TTCCGTCTGGTAGTTACCTTCAAGCTTTTGC

191

TTTGGAATTCATCATCAGCTTTAACCTCATGTTTGTTATCACTGCCGTG GCCACCGACACTAGAGCTGTGGGAGAGCTT GCAGGAATAGCAGTAGGAGCCACCGTCATGCTCAACATTCTAATAGCT GGDGAGACAACTGGGGCTTCAATGAATCCAG tGAGAACGCTGGGACCAGCAGTAGCAGCAGGAAACTATAAAGCCATTT GGATCTACCTGACTGCTCCGATTCTTgGcGC TCTTGCTGGGGCAGGTATTTACTCTGCAGTCAAACTGCCAAAYGAAGA TGACAACAATCACGGGAAGCCTTCAGTGGAA CATAGTTTCAGAAGGTGATTTCATCAGTAATACCATCAGACCATGATA GTCACATACTTTAGTAGAAAATGTGCAGTTT TATGAATGTAAAAACTTCTGTAYCATATTGCATCTGGAAGGCAAGGGT GAGCTGAATARAACAAACTCTTTCCACTCTC TCCAAATGTACAGATTTTAGCAAGATAGGAGTATAACTTATCAAGGAA CAGATTTTTAGCAAGATACAATAACGGAGAG AGGGAAGGAAGGAAATGATTAAAGCCCAAGTTTGAGAAGAGAACTAA TATTGGCAA

>ntMIPcontig52.seq AGTATGTGAAGTGTCAGCTTGACAGACTGATATCCACGTCTTCATTAA GTACTCGCACGACTCTCTGAAAGTCCAAGTG AAATGGTAACAATCGCAAATGTCAAGCACCTTCAATCAGTGCACCTCG GTGGATGTCAATGGTCAAACGAGGTCCTCGT CCTATGTTTGGAAATGCTGCAATGATGAGCCTAACCCCAGTAATTGAA CCAAAGACTTGAGCAGGGATTCTGGCACCGA CAGTGAAGAGGAAATTGGAGAGATCCCCGGAGATGGCACCGGACAAG ATAGTGAGAGGGTTGTAGGCCCCACCCTTGGT AGCTTTCGCCAAGAAGGCAAAGAAGAACATATTGATGACAGATATTG AATGTTTGAGGATTTCACCTTTGAGATCATGA GCCCCATAACCCAACATTGTGTGAACAAACATCTTGATAAGCACACTT GACCAGACCCACATGAAGGACATGATGAAAT CTGAAACCAGCAGCCTCCTCCTGCTTACCCCCATTTCTTGCTTCTTTTG ATTGATCCTCTTAGGTGTCCTGCTTTCTTT GGGTAGGAGGTGCGGGTAGAGGAGTTAAGAGTGTGCGTCTTATTAGTT ATAGTCCTTCCATCATCTTAATCCCTTATTT AAAAACTTATATCTGTCTCCGTCCTTTACTCTTCCACAACTTAGTCTAA ACCGGAATCAATTTCGCCCAGTATTGGTAA GTCTACTCCATGAACCTGGAGAAAGATTCCACACAATTGTTGTAACAA GATTTCGCTCTCTTCGACAAAAACTCTATGT TTCGCTTCCTATTTCCCCCTTAGATCTCCCCAATTTGTACGTTAGGGTTC TGAAAAATTGGGGGAAAAGAAGGGAACAA TGTCAATCTTTTTTTGTTCAAAGATgCagACAGGACTCCGGACCG

>ntMIPcontig53.seq GATCCAAAAGCTTTATTTGCTATCTGTCTATTAATTTGTTTCTTAGAAG AAATTATTTATTCTCTGAAAATGCCTTGCA

192

TAGCTTTGGGACGTTTTGACGATTCATTCAGCTCAGGGTCTATTAAGGC TTATATTGCTGAATTTATCTCTACGTTGCT TTTCGTCTTCGCCGGAGTTGGTTCCGCCATTGCCTACAACAAGTTGACA GCAGATGCTGCTCTTGATCCGGCGGGACTA GTAGCAGTTGCAGTTTGCCATGGATTGGCTCTGTTCGTGGCGGTTGCTA TTGCCGCTAACATCTCCGGTGGTCACGTTA ACCCTGCGGTTACTTTTGGTTTAGCTCTTGGCGGTCAAATCACCATTAT TACCGGCTTGTTCTACTGGATTGCTCAGGT TTTGGGCGCCATTGCAGCTTCCTACCTCCTCAAATTTGTCACTGGAGGA CTAGCTGTTCCAATTCACGGCGTGGCAGCA GGAGTGGGAGCAACTGAAGGAGTTGTAATGGAAATCATTATCACCTTT GCATTAGTGTACACAGTGTTCGCAACAGCAG TTGACCCAAAGAAGGGAACATTGGGCACCATTGCACCCATTGCCATTG GTTTCATTGTTGGTGCTAACATCTTGGCTGC CGGTCCATTCTCCGGTGGTTCAATGAACCCGGCCCGCTCTTTTGGTCCA GCTGTGGCCAGTGGTAACTTCGCTGGAAAC TGGATTTACTGGGTTGGAcCACTAGTTGGTGGTGGTTTGGCTGGTTTAA CTTACAGCAATGTGTTCATGAACTACGACC ACGCCCCTCTCGTTAGTGAATTCTaAAAAATTGTTTGAATTTGATTGKG TTGTGaACTCCGTTTGCCTTTTGTaATaAA AAgGaAGACaAGCaATACTAACATAtTGCTGAtTCtTTCTTTTTTCATATTT ACCTATTTGTATTGTTTTTCTTCTTCC TTTTAAGCCTTTGTAAAGATCCATCCAACTTTGATGAAATGTTCGAAAT GATGCATGAGATTTGATGAGATTGCTTTCA TTCATTGATATTGGTGTGCTTTATATTCTGAA

>ntMIPcontig54.seq GACTTGTATTTTTGCCGTCCTCTATCTATTTGTAATASSAATACCAATTC AGTAGGAGTTGGAGTTGACTCTTTTCTCT TCAGCTATCTCCTTCCTCTCTTCTCTCAGATGGGTGTGATTAAAGTAGC AATTGCTGATGGGGCGTTAACTTTTTTATG GGTTTTATGCTCTTCTTGTATTGGGGTTTCTACTTACTTTGTAGCTACTA CTTTTGGTGTTGTCAATGAAATGGGCAGC CTCTTCATCACTACCCTTGTTGTTTTTCTTATGTTCTTGGTTTTTGGATT TCTGGGTGATGCATTGGGTGGTGCTGSTT TCAATCCARCYGSCAATGCCGCCTTTTATGCAGCTGGTCTTGGTSACGA TTCTCTTGTCTCAGCCGCCGTTCGTTGTCC TGCTCAGGTTCTTCAATTTTTATTTTTTTTTTAATAGAGGAGGTGAATA GAATAAGCACAATCCGCTAGTCTGATAGTG TTTTGTCTTAAGTTGCTAGTAGCTCCTGTGGTGTCCATACTACTTCATT AGGGTTGTAGGTTAGGTTGTAGAAAGCTAG TTTGATAGTGTTGTCTTAGGATGCTAGTGGCTCTTGTTGTGTCCTTATT ACTTCCGTTGCGTCTGTAGTACTGTGCCAT TGTTACTGCTTATTGTTATACGTTGCTGGTGTTATCTTTGTGGCTTCCTT TGGTGTTACTGCTCCACTGTCTCTTTCCA

193

TTGTCGTTGTCGTGAACCGAGGGTCTATTGGAAACAGCTTCTCTGCTCC TCCGGAATAGGGATAAGGTCTGCGTAAACA CTACTCTCCCTAGACCCCATTTGTGTGATTCTACTGGGTTGTTGTTATT GTTGTTGTAAAAT

>ntMIPcontig55.seq GGATTCCCGGGATCTCCTCACACGTGTCTTTCCGACTTCCCCGCACCAG ATGTCTCCCTCACTCGCAAGAACTAATTGT CATACTTCCAACGTCCACTTCCAACCAAGAGGTTGTGAGTTCCAGTCT ACCCAAGAGCAAGGTGAGAAGTTCTTGGAGG GAAGGATGCCGAGGGTCTATTTGGAAACAGCCTCTCTACCCCAGAGTA GGGGTAAGTTGGGAGCAGAGTTCGTGGGAAC ATTTATCCTTATATTTGCTGCAACAGCCGGGCCAATTGTGAACCAAAA GTACAACGGAGSCGWAKMTCTWATcRGAAAT GCAGCtTGCKCTGGGCTgGcCGtTATGATCGTGAtTCTGTCRACRGGCCAT AtTTCTGGAGCACATCtTAATCCGTCGC TCACCATYGCAtTTGCAGCACTTCGTCAYTTTCCGTGGGTTCAAGTGCC GGCMTATGTTGCAGCGCAGGTTTCAGCATC MGTTTGTGCTTCTTTTGCTCTCAAGGGTGTTTTTCATCCTTTCATGTCTG GTGGCGTTACTGTTCCTTCYGTAAACACT GGCCAGGCTTTTGCTCTCGAATTCCTCATCACATTCAATCTCCTTTTTG TTGTCACTGCTGTTGCTACCGACACCCGCG CGGTGGGAGAGTTGGCGGGCATTGCAGTTGGAGCTACAGTCATGCTCA ATATTCTAGTGGCKGGGCCATCAAGTGGTGC TTCCATGAATCCAGTaAGAACTTTGGGRCCAGCCGTTGCAGCAgGAAAT TACaAGTCATTGTGGATATACWTAGTGGCT CCaACTYTGGGGGCTCTTGCAgGGGCAGCTGTTTATACGCTCGTCaAAC TTCGAGGRRATGAtMGTWCTGAGACaCCAC GCCAgGTTAgGAGCTTcCGcCGCTAGCCTGCTGAAGGAAGGGTGTTGTT CCAACTTCTAATAGCTTATCTTATTATACT ATTGCGTGCTTGAAAATAAAGGTGGAGGACTCGGAAAATGTGACTTCC AGTCTCACTACCAAAAACAATTTACAGTTGC ATTTGGCACTCTTTCTCCTCATTCGCTATGTGTGGATGGCTTGAGGAAG GGGCTGATATTCGCTTGTGGTCTGCGACCT TTTTTCCTTTTTTTATGGTAGCCACTGAAAACCATCACACAAAATTAAA CGAGC

>ntMIPcontig56.seq GTCGGTCCGGAATTCCCGGGATGGAGTTTTGTTCGAAAGTGCAGGATG GAGTTTCAATATTAATAAGCTGCAAACTCTA ATCCGTAATATCCCTTTGAATTTGCACTCTATATATATATATATATATA TATATATATATATATATATATATATATATA TCCGATATTTCAATTTAATAAGCTGCAAATTAACTCCAATCCGTAATAT CCCTTTGAATTTGCACTATTAACTAGATAA CTATCAAATACGGGATTAGTCAGCATTTTGTAAAACTCCACCCTCCAA AAAACTCTCCAACTATGAATCGTCCTATCCT

194

TATTTAGGTAATCTAAAGCAAAACCTATCATATTTAAATCTAAGTACT ATTATTATTATTATCAAATAAGCCTTTTGGC AGACATCCTGAAGAAAAGAAAAAAAAACCCTTATATCTTCCGAACTTA TCAACTCTTTCAATTAAGAACAATGGCAAAG AAGGATGGCAATCGAGAAGAAGAAATCTCCCAAATGGAAGAAGGCAA TATTCATTCAGCCTCCAAGTCCGATTCCAACG TCGGATTTTGTTCATCAGTTTCTGTCGTTGTCATCTTACAAAAGTTGAT AGCGGAGGCTATAGGAACGTACTTTGTAAT ATTTGCAGGGTGTGGATCAGTTGCTGTGAATAAGATCTATGGGAGCGT TACGTTTCCAGGGATATGTGTGACATGGGGA CTAATCGTGATGGTGATGGTTTACACCGTGRGACACATATCTGGAGCT CATTTTAATCCTGCTGTCACTATTACTTTCA GCATCCTCGGCCACTTCCCATGGAAACAAGTACCTTTGTACATAATAG CTCAACTGATGGGTTCGATTTTGGCCAGTGG AACTCTGGCTCTATTGTTTGACGTAACTCCTCAAGCTTATTTTGGTACT GTTCCAGTTGGATCTAATGGCCAATCACTT GCTATCGAAATCATCATTTCTTTCCTTCTCATGTTTGTCATTTCTGGCGT TGCTACAGATGATAGAGCAATTGGACAGG TTGCTGGAATAGCAGTGGGAATGACCATAACTTTGAATGTCTTTGTAG CAGGGCCAATTTCAGGAGCATCAATGAATCC AGCAAGAAGCATTGGTCCAGCAATAGTGAAGCATGTTTATACAGGTCT TTGGGTGTATATAGTTGGTCCAATTATCGGA ACACTAGCCGGAGCATTTGTATATAACTTGATTCGGTCCACAGACAAA CCACTTCGTGAGTTGGCCAAAAGTGCATCAT CTCTTCGAAGTTGAGAGCAAATTAAATTCAATATATTTAACTGAAATT TTAGGAAATTATCATCCCAGTTGTCCCTAAA TCGTACATTTTTAGGCATTTTGAGCCATTGGAGTATTTTGAGCCATGAA CATAAGTATTGGAGCATTTGAGG

>ntMIPcontig57.seq GGGCATTTGGGGGCATGATTTTTGTCCTTGTTTATTGTACTGCTGGTAT TTCTGGTGGACATATTAACCCTGCTGTTAC ATTTGGACTATTCTTGGCTAGGAAAGTGTCGTTGGTTCGYGCAATTAT GTATATGTTGGCTCAGTGTTTAGGAGCCATY YGTGGTTGYGGKTTGGTKAAGGCATTTCAGAAGGCGTACTATGTTAAG TATGGTGGTGGTGCTAATAYGTTGAATGATG GTTATAGTACAGGTACCGGTTTGGGTGCTGAAATTATTGGCACATTTG TTCTTGTTTACACTGTTTTTGCTGCTACTGA TCCTAAGAGAAATGCTAGAGACTCTCATRTTCCTGTGTTGGCACCACTT CCAATTGGATTTGCTGTGTTYATGGTTCAT TTGGCYACAATWCCTGTAACTGGAACTGGTATTAACCCWGCTAGAAG TTTTGGAGCTGCTGTTATTTATGGTAAAGAAA AAGCATGGGATGACCAATGGATWTTCTGGGTTGGACCTTTTATTGGAG CTGCAATTGCTGCATTTTACCAYCAATTTAT ATTGAGAGCTGGAGCAGTYAAAGCACTTGGTTCCTTYAGGAGCAATGC ttaaYTggaagAATYTGCAATTATGAAGTAC

195

TTAAGGAAATGGAGYCAATGAATATAAGGTCTTAAMTTCAGAGTTCA MRTTWTATAAATTGCTCCaaatAAMTGTAAAT AAGWATTCYAGAAGAAGAGGGTCYCTTCAARTTGTGTTTACTYSTAGC CTTTTGTCCTTTCTcCtaTTGTATTTTTTTT TTCTCCAGTTTTTCCAGTCGTTTTTCCTTGTAGTTTTCATTTTCTCCTCCT GTCACCATTATCCAAATAATATAATGAA ATGACT

>ntMIPcontig58.seq TATAGAACAAGTCGGTCCGGAATTCCCGGGATTTGCTCGATAGTGCAG GATGGAGTTTCAATATTAGTGATAAGCTGCA AACTCCAATCCGTAATATCCCTTTGAATTTGCACTATATCCGATATTTC AATATTAATAAGGTGAAAATTAACTCCAAT CCGTAATATCCCTTTGAATTTGCACCATTAACTAGATAACTATCAAATA CGAGATTAGTCAGCATTTTGTAAAACTCCA CCCTCCAAAAAACTCTCCAACTATGAATCTTATCCTTATTTAGGTAATC TAAAGCAAAACCTATCATATTTAAATCTAA GTACTATTATTATCATCAAATAAGCCTTTTGGCAGACATCCTGAAGAA AAAAACCCTTGTATCTTCCTAACTTTTCAAC TCTTTGAATTAAGAACAATGGCAAAGAAGGATGGTAATCGAGAAGAA GAAATCTCCCAGATGGAAGAAGGCAATATTAC CCATTCAGCCTCCCAGTCCGATTCCAATGTCGGATTTTGTTCATCACTT ACTGTCGTTGTCATCGCACAAAAGTTGATA GCGGAGGCTATAGGAACGTACTTTGTAATATTTGCAGGGTGTGGATCA GTTGCTGTGAATAAGATCTATGGGAGCGTTA CGTTTCCAGGGATATGTGTAACATGGGGTCTAATCGTGATGGTGATGG TTTACACCGTGGGACATATATCAGGAGCTCA TTTTAACCCTGCTGTCACTATTACTTTCACCATCTTCGGCCGCTTCCCAT GGAAACAAGTACCTTTGTACATAATAGCT CAACTAATGGGTTCGATTTTGGCCAGTGGAACTCTGGCTCTATTGTTTG ACGTAACTCCTCAAGCTTATTTTGGTACTG TTCCAGTTGAATCTAATGGCCAATCACTTGCTATCGAAATCATCATTTC TCTCCTTCTCATGTTTGTCATTTCTGGCGT TGCTACAGATGATAGAGCAATTGGACAGGTTGCTGGAATAGCTGTGGG AATGACCATAACTTTGAATGTCTTTGTAGCA GGGCCAATTTCAGGAGCATCAATGAATCCAGCAAGAAGCATTGGTCC AGCAATAGTGAAGCATGTTTATACAGGTCTTT GGGTGTATATAGTTGGTCCAATCATCGGAACACTAGCCGGAGCATTTG TATACAATTTGATTCGATCCACAGATAAACC ACTTCGGGAGTTGGCCAAAACTGCATCATCTCTTCGAAGTTGAGAGCA AATTCAATATATTTAACTGAAATTTTAGGCC ATTCACTTTTTATCATCCCAGTTCGGATGTCCCTAAATCGTACATTTTT AGGTATTTTGAGCCATGAACATAAGTATGG AGCA

>ntMIPcontig59.seq

196

CTTTTTAAAATTATGCGTCCTTATCCCCATTAGCTGGAATATTAGGTGG ATCATCATCTCAGTATTCAGTACCTTAGTA TAATTGACGAAATTCTACAGCCGATTGATTCATTAAAAATGGGTGCTG TAAAAGCAGCTGTGGCTGATTTTGTGTTAAC ATCGATGTGGGTATTTTGTTCATCAACACTTGGGGTTTTCACTTACCTA ATTGCTTCTGCTTTTGGAATTGCTCAAGGA ATTACTACTCTGTTTATTACTACTGTGCTTCTCTTTATTCTGTTTTTTGT GTTTGGGATTATTGGTGATGCTTTGGGTG GTGCCGCTTTTAATCCTGCTGGTACGGCCGCCTTTTATGCTGCCGGTGT TGGAACGGACTCTCTCTTCTCTGTTGCCGC TCGATTTCCTGCTCAGGCAGCTGGCGCAGTTGCTGGTGCATTGGCGAT ATTGGAGGTTATTCCTACGCAGTACAAGCAC ATGCTAGGTGGGCCTTCATTGAAAGTTGACTTGCATAACGGAGCCATT GCTGAGGGCATCTTGACTTTCACAATGACCT TTCTGGTCTTTCTTATTGTACTGAGGGGTCCTAGAAATGCACTTCTAAA GAATTGGTTACTTGCAATGTCAACTGTTAC CATGGTAGTTACAGGTTCAAAATACACYGGACCGTCTATGAATCCMGC CAATGCATTTGGTTGGGCGTACATAAACAAT TGGCACAACACATGGGAGCAATTTTACGTCTACTGGATCTGTCCCTTC GTAGGRGCAATAATGGCTGCATGGACTTTTC GTGCTCTGTTTCCRCCTCCAGTAAAGCAGAAGCCTCAGAAGGCAAAGA AGAATTGAGAAAGAACACCTTCGCGCTGCTT CAATTACTTCACTGAACTAGGTTGCAATGTGGTTGAAGTATGCCCTTA GAGCTGAAAGCCATTTTTAACTTCTAATGTG GAATCATAATCGATTCCTTTTGGATTTGWTGGAATTGTTAATAAATCC CCTTTTTCMCATTGATCCAAATTCAAGCAAC TATTAGTGCAAAAACTGAGTTGTCCAAAAGCTGGATGCCCCTGAATAT TTTCTTATGCTTTTTTAGATGCTTCCTTCGT TCTGTTTTAGTGGTGTTCTTGAGAATTAAATTGGATAATTCTTCC

>ntMIPcontig60.seq GGCTCTTCACTTGTATTTTTGCCGTCCTCTATCTATTTGTAATACCAATA CCAATTCAGTAGGAGTACTTGGAGTTGAC TTTTTTCTCCAGAGCTATCTCCTTCCTCTCTTCTCTGAGATGGGTGTGAT TAAAGCAGCGATTGGTGATGGGGCGTTAA CTTTTTTATGGGTTTTATGCTCCTCTTGTATTGGGGTTTCTACTTACTTG GTAGCTACTACTTTTGGTGTTGTCAATGA AATGGGCAGCCTCTTCATCACTACCCTTGTTGTTTTTCTTATATTCTTAG TGTTTGGATTTCTGGGTGATGCATTGGGT GGTGCTGCTTTCAATCCAACTGCCAATGCCGCCTTTTATGCAGCTGGTC TTGGTCACGATTCTCTTGTCTCAGCCGCCC TTCGTTGTCCTGCTCAGGTAGCAGGTGCAGTTGCCGGTTCGCTTGCAAT AATGGAGCTTATGCCTAAGCAGTATCACCA CATGCTTGAGGGGCCTGCKYTGAAGGTGGAYGTACAAGCTGGAGcCTA TTGCGGAGGGTGTCTTGACCTTCATAATCAC

197

ATTCATGGTCTTTGTCATTTTCCTTAGGGGTCCAAAAAGTGCACTTCTG AAGAATTGGTTGCTCACTATGGTAACTGTC CCTTTGGTGGTTGCTGGTTCAAAGTACACTGGACCTTCTATGAATCCAG CCAATGTAAGTCTTGCTTTTGCATCTTGGA ACATTAACATATTTTTTTGGTAGTCTCTTTCATGTATTTGGAGGACATG ACCCACGATAGGAAGGTGTGGAGGTTGAGG ATTAGGGTTGTAGGTTGACAAATAGTAGTGTATTCCTCCTAGGCCATT CAATAGTACTAGGACTATTTTTGTAATTTCC TATTTATGGTTCTTTATTATGCATGTTGCGGCATTCGCACCCGTTGCGA TGTTACATTGTTAGCGGCCGCATGAGGGGA TCCTCTAGAGTCGACGGCATGCCCTGCTTTAATGAGATATGCGAGACG CCTATGATCGCATGATATTTGCTTTCAATTC TGTTGTGCACGTTGTAAAAAAACTGAGCATGTG

>ntMIPcontig61.seq GGCCATTACGGCCgGGGGaaATAAACAGAGTCACAATCAAAGAAAACA TAAAATTTTAAGAAGAAAAGATAATGCCGAT TTCAAAAATTACCCTCGGAAATTTAGCAGAGGCTAGCCAGCCTGATGC TCTCAAGGCTGCACTAGCTGAGTTCATTTCA ATGCTCATCTTTGTTTTTGCCGGTGAAGGCTCTGGCATGGCCTTCGGTA AGCTAACAAATGGAGGAGCAGCCACGCCTG CTGGATTGATTTCGGCGGCTATAGCCCATGCCTTTGCCCTTTTTGTGGC AGTTTCAGTAGGAGCAAACATTTCTGGAGG TCATGTAAATCCTGCGGTCACATTTGGTGCTTTCGTGGGAGGTCACATT ACCCTTTTCAGGAGTGTTTTGTATTGGATT GCCCAATTGCTTGGATCTGTCGTCGCTTGCGTGCTCCTCAAGTTTGCYA CTGGTGGATTGGAAACATCRGCATTCGCAC TCTCRACAGGAGTTACCCCATGGAACGCAGTTGTTTTTGAGATAGTGA TGACCTTYGGSCTTGTTTACACCGTYTACGC AACTGCARTTGATCCTAAGAGGGGCAATTTGGGAATTATTGCCCCAAT TGCWATTGGTTTCATTGTRGGTGCSAAYATY TTGGCYGGTGGAGCCTTTGATGGTGCATCAATGAAYCCTGCTGTGTCW TTTGGTCCAGCAGTGGTTAGCTGGACATGGA ACAGCCACTGGGTCTACTGGCTCGGACCATTTGTTGGTGCTGCCAYTG CTGCTTTGGTTTATGAAATTATYTTCATTGG TGACAACACTCACGAGCAGCTCCCCACCGCTGATTACTAARGAACAAT TTCCTCCTTTCTTTCTCTAAAACCAAATGTT GGATATAATTAATTTAAGCTATGGTTTGCAAGGTTGTGGTTTGTTTTGC TGTTCGCTCTTTGTTTTTCGTTTCAGTACA TGGTAAAAGTTCTGGGTTTTCTTAATTGTAAAATCGTGAAAAATCAAG AGATGCAAGTGTCGTGTTTAATGTTCAGGAG TTTGACTTCTCTTTGATATGG

>ntMIPcontig62.seq GCCGDCACTCTTATCTGCCTTCTCTCTCATATTAATCAAGAAAGAAAAT AGTCCCAATTTAGCATAGTGAGAAATGGCA

198

AAGAACGTGGGTAGTGAAGGGTCGTTCACGACCAAAGACTACCAAGA TCCTCCGCCAGCACCACTGATTGACCCAGAGG AGCTAACCCAATGGTCTTTTTACAGAGCACTCATTGCTGAATTCATAG CCACTCTTCTCTTCCTTTACATAACGGTGCT GACTGTCATTGGTTACAAGAGCCAAAGTGCTACGGCAACTGATCCTTG TGCTGGTGTTGGGATCCTTGGTATTGCTTGG GCATTTGGTGGCATGATTTTCGTTCTTGTCTACTGCACCGCTGGTATTT CTGGTGGTCATATAAATCCAGCAGTCACAT TTGGACTATTCTTGGCGAGGAAAGTATCACTAATAAGGGCAGTTATGT ATATGGTGGCTCAGTGTTTGGGAGCCATTTG TGGCGTTGGATTAGTTAAGGCTTTCCAATCAGCTTACTACCACCGCTAT GACGGCGGAGCCAATATGCTCTCCGACGAT TACAGTCACGGCGTCGGGTTGTCGGCGGAGATCATCGGAACTTTTGTT CTTGTTTACACTGTTTTCTCTGCCACTGATC CCAAGAGGAATGCTAGAGATTCTCACGTCCCCGTGTTGGCACCACTCC CAATTGGATTTGCAGTGTTCATGGTTCACTT AGCCACCATTCCGATCACCGGTACCGGAATCaACCCCGCTAGGAGTCT TGGCGCTGcCGTTATCtTCAAcCAACAcAAA GcCTgGaAAGACCACTGGATMTTTTKGGTAGGACCTTTCATTGGAGCAG CCATTGCAGCATTCTACCATCAATTCATAT TGAGAGCAGGTGCAGCTAAAGCCCTTGGTTCATTCAGGAGCAGTTCTC AAGTTTGATGATTATGAATGATAAATTATTG TTGTACTCTATTTATGTCAATGTTTTGTTTTAGTGGTTTTAATTTCTAGC TCTCCTTGTCCCAAGTTATGATGGTTTAG AATATCATGCGCAATAGTGGTGTTGTACTTGAGAATTTGAGAGCCAAA GATGTGTAGATTACAAATGTMWWGTCggTYY GGAAttcMSGGRATTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTcAAYYY GAAccKAcATRRTKKTYYAAWRSKKW WAWTMAMTTAtWMTcSGTRRMMAAKTTAAggYWAgAAAAtAAAAAYM CAKKKKAAAcACgARYCGWSAgCMATacWKTA AAAWRGTRTcSRATCRSWARMATAgAYWCARCaAAGTTgTYAWWWTR WWWMRTRWMMRCTcWTWGGaWKTRMRWASWRR AGCRAGAAATWKAMMYKKTSATTggTgSSTTWRKAcKMAGCtWKTAGa GMAgSTTaGSTCaYGAgWGTGaTKSATGaaG WARRGKKSATRGAYAWYRSYWGcaAGcccAGcAYSRAWWATGGKYSYG GcSSAgTaAACCMASTaGATcGAGtcCRAGY YYAGCTAWYWWWMGcAgRCCCaaRAGaAAYAgcWGKGWTCgATKGRT KMTYYWTCaAAtgGCCMAtYYAGSARRAATGT TggccCSRASAATSARASCaWTWGARATTSGAgCAWYCRSACaSTARYKC TcMWWKTWYYGRKTgCMAKARCWGWGSCg ATWARYWGWGYWAWYAWGYMCAAARGTaCtaATYWCMATSKSAART gACgKAAGRYAKTSCAWASTGATWRTcMASARC ASRATKMAAATGaCCtCCTGaTRCTcWRTcCaCCARTGaGYRAACttMAKS ASCAtMGCAAgCTGCTCCCCATGTCATG CTTATCCCTGGAGATGTTAAGCTCTCATCATCGTATAGCTTATTCATGG CAACCGATCCACACCATGAAAATATAATGA

199

AGTACGCGCCAATGGCCTCTGCAATCAACTTTTGTGCGACTGCAACAA CCGAAGCTGATGAATACAGCCTTGTGTTAGT ATCAGAGTGAGACTTAAGGATATTGGCTGCAGGGGCACCTTCTTCCAT TGTATATGGTGATTTCTTCTTCTCAGTTACC ATCCATTTTCGCCCTCTTTGGCTCGTGCC

>ntMIPcontig63.seq GGGATTATTCTTCCTATGTAACAATAAGCTGATCATCTCTTATCTCCTT GCACAACACCCCCCCAACTCTACAAATCTT GTTTTCTTGAACTATTAATTAGTAGGAGTTAAAAGTAATTAAGGATAT ACAATTATGGGTGATCTGCAGATAGCAGAGG CTAATGGAAACCATGCATCAGTGAGTTTGAATATTAGAGATAATGATA TGAACAGCAACAACAAGACTTCTGCACATGA AGATTCTTCATCAAGCTGTTGCTTTGTCACTGTTCCCTTCATCCAAAAG ATAATAGCGGAGACATTAGGGACGTACTTC TTGATATTTGCGGGGTGTGGTTCAGTGGCGGTGAATGCAGACAAAGGA ATGGTTACTTTCCCTGGTATCTCCATTGTCT GGGGGCTgGTgGtTATgGTCATgGTTTACTCtGTTgGMCACAtTTCTgGTGC TCAtTTTaAcCCTGCTGTTACCATTTC CTTTGcCACtTGCaAAAKGtTCCCATGGAAACAGGTACCAGCTTACGTGG CAGCTCAAGTKATTGGATCAACCCTAGCA AGYGGAACCCTACGACTAATATTCAATGGCAAACATGATCAtTTTCTTG GAACYTCRCCCTCTGGATCAGATATCCAAT CTCTTGTTCTGGAATTTATTATCACATTTTATCTTATGTTTGTCGTTTCT GGTGTCGCAACTGATAATCGAGCKATMGG AGAACTTGCTGGTCTTGCTGTGGGGGCAACCGTGTTGCTTAATGTGAT GTTTGCCGGRCCGATATCAGGAGCGTCGATG AACCCAGCAAGGAGTTTGGGCCCAGCAATAGTATGGAGCCATTACAG AGGTATATGGGTTTACATGTTAGGCCCAACAG CTGGGGCCATATCAGGTGCTTGGGTCTATAACATCATCAGATTCACAG ACAAGCCTTTACGTGAGATTACCAAAAGTGG ATCTTTCCTCAAGTCCATCAGATCAAGCAAGTCCCTCAGATCTAGCAC GTAACAAACAGCTCCAACTGAAAAGGGGAGT AATTGTTTTTTTTCTTCTTCTTCTGAAATATATATAGACAAAAGAAAGA AAAATAGGAATGAGAAAAGGGGGAAAAGCA TGTGTTCCTAGCTATTAGTTTCCTTAACTTTTGTTTACAAAAGAAACGA GTTTTATTTTTTGTAATATAAACT

>ntMIPcontig64.seq ATTTTTTTAACGAAAAAAAAATTTATATTTCATCTCTATGTTTTTACAT AGCAATTTTGGGTGGGTCATCACTTGTAGG AATGATATAATGCTAATATCCATGTTTCACGCGGCCAAGGAAGCATTC GTTTTTAATCTCATTATGACTTGGTCGTTCA GAAGAGCAATTACTTCAATCAGAATAGTGGAACGAAAGGTAGTAAAA GAATTAAATACTCCTCTCTTTGCAAATCGCCC

200

CGTGAAGACAGGCATTTGGAAAGGTTCTCGAGATTCTCAATCGCTCTT GAGAGGCTACAACATTGAGACAAAACGTACA TCCCTTAACTGAACATTATACGTGTTGTAGTTAAAAACAGAAAATTAA GTTCATGCAATCATACTGGAGAAAGACATAA TACATAATCCAAACTGTACGATCGCCAATAATTTAGGAATCTCCTTCA TTCTGACTTTTTCTCTATCATCTTCTCCCTT TCGTCCTTTGGGGGGGAAACTAGCAAGTTGAAAGTCCACACAgGCCAG CAAAGTTGCCTGTATTGGAGCAAGCCAATAA ACATGTATGTGCTCCYTGGTTATATGATCCCCACGCGCATAAGCCCAT CCCATCACAGAGGCTGGATTCATGCAGCCCC CTGTTAGATCAGAACCAAGTATGTGAAGTGTCAGCTTGGACAGACTGG ATATCCACGTCTTCATTAAGGTACTCGCACG ACTTCTTCTGGAAAGTCCAAGTGAAATGGTAACAATCGCAAATGTCAA GCACCCTTCAATCAGTGCACCTCGGTGGATG TCAATGGTCAAACGAGGTCCTCGTCCTATGTTTGGAAATGCTGCAATG ATGAGCCTAACCCCAGTAATTGAACCAAAGA CTTGAGCAGGGATTCTGGCACCGACAGTGAAGAGGAAATTGGAGAGA TCCCCGGAGATGGCACCGGACAAGATAGTGAG AGGGTTGTAGGCCCCACCCTTGGTAGCTTTCGCCAAGAAGGCAAAGAA GAACATATTGATGACAGATATTGAATGTTTG AGGATTTCACCTTTGAGATCATGAGCCCCATAACCCAACATTGTGTGA ACAAACATCTTGATAAGCACACTTGACCAGA CCCACATGAAGACATGATGAAATCTGAAACCAGCAGCCTCTCCTGCTT ACCCCATTCTGCTCTTTGATGATCCTCTAGT GTCTGCTTCTTGGGTAGGGGTGCGGTAGAGGAGTAGAGTGTGCGTCTT TTAGTTAAGTCTC

>ntMIPcontig65.seq GGCCATTACGGCCGGGGGTCATCAGAAAACATTGTCTCTGTTTCTATTT TCCTCTAATTCTTAGGATCAATTTAATTAG TTTTCTGAAAATGCCTGCCATAGCTTTTGGTCGTTTCGATGATTCATTT AGCTTAGGGTCTATTAAGGCCTACATTGCT GAATTCATCTCTACATTGCTCTTTGTCTTCGCTGGAGTTGGTTCAGCCA TTGCTTACAACAAGTTGACAGCAGATGCTG CTCTTGATCCGTCGGGGCTTGTAGCGGTTGCAGTTTGCCATGGGTTCGC TCTGTTCGTGGCAGTTTCAGTCGGGGCTAA CATCTCCGGTGGTCACGTCAACCCTGCCGTTACTTTCGGATTAGCTCTT GGTGGCCAAATTACTCTTCTTACTGGCCTC TTCTACTGGATTGCTCAACTTTTGGGCGCCACTGTTGCCTCTTACCTCC TCAAAGTTGTCACCGGAGGATTGGCTGTTC CAATCCACAGTGTAGCAGCTGGAGTAGGAGCTGTTGAAGGAGTAGTG ATGGAAATTATCATCACATTTGCATTGGTTTA CACAGTGTACGCCACAGCAGCTGACCCAAAGAAGGGTTCATTGGGCA CCATTGCACCCATTGCCATTGGTTTCATTGTT GGTGCCAACATCTTGGCTGCTGGCCCGTTCTCTGGTGGTTCAATGAAC CCAGCTCGGTCCTTTGGACCTGCAGTGGCTA

201

GTGGTAACTTCGCCGGTCACTGGATTTACTGGGTTGGACCCCTTGTTGG TGGTGGTTTGGCTGGTCTTATTTACAGTAA TGTGTTCATGAACCATGACCATGCCCCTCTATCCACCGATTTTTAAGTT TTAATTAATTTCTATGTTGTTAGAATTTGA TTATTGTGTTTGAAAATGTATTTTGCATTATTTTCTTTGTAATAAAAGG AAGAAAAAAGCAATATTTTGCTCTTTCTTT CTTTTGTAGTAGTACCTTTTGTTTTTTGTTTTTTGTTTTTTGTCTCTTTGT AGCTTTTGGTTTGCAGCTGTACACATTT GGTGACATGTTGTTGTCGAATTGATTTTATGGATTTGATGAGATC

>ntMIPcontig66.seq GGCATGTGTTGTTGCCTTTTTGCTACATCAATCTATTCACACCTTTAAC TTGTAACCACACAGCGTAATCTCCCTTGTA GATATCGTTTTCCCTGCTAGGCTGCTATTTAATTATTAATACCCAGGCC ACCATCACTCTcAAAGCGGRdTCGGGATAA CTAGCTAGCTAGCAAGATATGGTGAAAGACTACGTAGATAAGCCAGC AGCACCGCTGTTTGATACGGTGGAGGTGAAGA AATGGTCTTTCTACAGAGCCCTCATTGCTGAATTTGTTGCCACACTTCT TTTTCTTTACGTTAGTGTTGCTACTGTTAT TGGCCACAAGAAGCAAGTTGGTCCCTGCGACGGCGTTGGACTTCTTGG TATTGCATGGGCTTTTGGCGGCATGATTTTC GTTCTTGTCTATTGCACTGCTGGGATTTCTGGTGGGCATATAAACCCAG CAGTAACATTTGGGCTATTACTGGCAAGGA AGGTTTCATTACTGAGAGCGGTGGGGTATATGGTGGCGCAGTGCTTAG GAGCCATTTGCGGCGTTGGCTTAGTGAAAGG GTTCATGAAGCATGACTATAACACGTATGGCGGCGGTGCTAATACCGT TGCAGTTGGCTACTCAACCGGCACAGCCTTG GGTGCTGAGATCATTGGCACTTTCGTCCTTGTCTATACCGTCTTCTCCG SCACTGACGCCRAAAGCAAAGCGCGTGATT CTCACGTCCCCGTACTAGCACCATTGCCAATTGGATTCTCGGGTGTTAT GGTTCATTTGGGCACTATCCCATTACTGGC ACTGGGATCAACCCT

>ntMIPcontig67.seq GACACTTAARKMGcTTAATACAGTTGtTGTTTCTTGAACTTGYACTTCTT ATTTGTCAATTGTTGCTGACAAAAACAAG AAMAAGGGCCAATAAAGCAGCATCTTCCCTCATACTCTGTGGGGAAG AGCAAATAGAAGATTTGGAAATTTTGATGTGG AAGTGTGAATGCTTCTTTTTGCTGCTTGGAGGGTAAAACTTGTTTCTCG CAGCAAAAGATAAAGGCAAATGGCTCCTGC AAGCAARGCTGATAAGAAGGCTGCAGGTGATGTGGCTGCATGGATGT TCAATGTGGTCACTTCAGTTGGAATYATTATT GTCAACAAGGCYTTAATGGCTAACTATGGCTTTTTCTTTGCAACAACA TTAACCGGGATGCATTTTGCCACAACMACCT TGATGACTATTGTTCTTAGGTGGCTCGGRTACATCCAAGCTTCTCATTT ACCCCTTCCAGATCTTCTAAAATTTGTACT

202

GTTTGCAAACTTCTCTATCGTTGGGATGAACATCAGTTTAATGTGGAA CTCGGTGGGATTCTACCAGATTGCAAAGTTG AGTATGATCCCTGTGTCCTGCTTACTAGAAGTTGCCTTTGACAAGATCC GTTATTCAAGGGACACAAAGCTGAGTATTG TTGTGGTACTCCTAGGTGTTGCTGTGTGCACAGTTACTGACGTGAGTGT TAATACCAAAGGCTTCATTGCTGCCTTTGT AGCTGTATGGAGCACTGCACTGCAACATATGTTCATTACCTTCAAAGG AAATACTCCCTTAGTTCTTTCAATCTGCTGG GACATACTGCACCAGTTCAGGCTGCA

203