Identification and Characterisation of Potential Sources of Resistance to Ascochyta Blight Within the Exotic Germplasm of Lentil

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IDENTIFICATION AND CHARACTERISATION OF POTENTIAL SOURCES OF RESISTANCE TO ASCOCHYTA BLIGHT WITHIN THE EXOTIC GERMPLASM OF LENTIL

Presented by

Rama Harinath Reddy Dadu

Submitted in total fulfilment of the requirements of the degree of

Doctor of Philosophy

Faculty of Veterinary and Agricultural Sciences

The University of Melbourne

December 2018 Declaration

I declare that this thesis comprises only my original work towards the degree of Doctor of Philosophy. Due acknowledgement has been made in the text to all other material used. This thesis does not exceed 100,000 words and complies with the stipulations set out for the degree of Doctor of Philosophy by The University of Melbourne.

Rama Harinath Reddy Dadu

December 2018

Acknowledgements

I feel immense pleasure to express my heart-felt gratitude and deep sense of indebtedness and hearty thanks to my supervisor Dr Dorin Gupta for her immense knowledge, patience, untiring help and constant encouragement throughout the period of study. I appreciate the effort and the time spared reading and editing my manuscripts. I have learned so much from you regarding lentil, research and life in the process since I arrived in Melbourne. It would not be possible to finish my PhD without your support.

I am indebted to my co-supervisor Professor Rebecca Ford. She has mentored, supported and inspired me throughout my PhD candidature. Her invaluable support and suggestions have shaped my project and allowed me to be a better researcher. Additionally, I would like to appreciate her help and guidance towards publishing scientific papers.

Thanks to my other co-supervisor Dr Prabhakaran Sambasivam for his help, support and positive impact throughout my PhD more specifically with disease bioassays and histopathology.

I would like to thank my Chairman of the advisory committee Dr Graham Brodie for his insightful comments and encouragement during my PhD.

Warmest regards to Dr Jenny Davidson and Sara Blake from South Australian Research and Development Institute, Adelaide, SA; Dr Janine Croser and Dr Federico Ribalta from University of Western Australia, Crawley, WA; Dr Kenneth Street from International Center for Agricultural Research in the Dry Areas, Morocco and Dr. Sukhjiwan Kaur from AgriBio, Bundoora, VIC for their invaluable contribution to my research.

I would like to specially thank Dr Ido Bar, Griffith University, Brisbane, for helping me with the bioinformatic analysis towards the end of my PhD candidature.

I would like to appreciate my lab manager Dr Ravneet Kaur Jhajj for her continuous support and assistance throughout my PhD. A massive thanks to my fellow lab mates Muhammad Jamal Khan, Humayun Kabir, Sajitha Biju, Sally Foletta, Charlotte Aves and Waseem Ashfaq

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for their support, help and friendship, and for all the hard work and fun we had for the last three and half years.

I gratefully thank the Victorian Government for awarding me the prestigious Victoria-India Doctoral Scholarship (VIDS) and The University of Melbourne for Melbourne International Fee Remission Scholarship (MIFRS), without this support I would not have been able to achieve my dream. I would also like to thank Faculty of Veterinary and Agricultural Sciences, The University of Melbourne for Graduate Research Scholarship-2018. Partial funding support for this project was provided by The University of Melbourne through Richard W S Nicholas Trust fund-2015, Research Initiative Fund (RIF)-2018 and travel funding-Faculty of Veterinary and Agricultural Sciences-2016 and Elizabeth Ann Crespin Scholarship Award-2018 are gratefully acknowledged.

I am forever indebted to my parents for their understanding, endless patience and encouragement when it was most required. I also thank them for allowing me what I am interested at. I would like to thank my brother Sekhar, sister-in-law Anusha, parent-in-laws, brother-in-law’s Nani and Rukku for their love and support.

Finally, I owe my loving thanks to my wife Anitha for her endless understanding, love and support. I wouldn’t want to miss a second of the good times we had together and the memories we made.

Abstract

Lentil (Lens culinaris Medikus. ssp. culinaris) is high value cool season legume staple food and cash crop, cultivated globally including in Australia. However, the crop is often challenged by biotic and abiotic stresses that reduce the full yield potential. Among these, Ascochyta blight (AB) caused by Ascochyta lentis affects gross profits and yield stability in Australia. Resistant cultivars are the most viable, long-term sustainable option among the recommended disease management strategies. However, the susceptibility of previously released resistant cultivars (Northfield and Nipper) and a future uncertainty over the resistance status of the few remaining available resistance sources has necessitated an immediate influx of novel and diverse resistance sources into the Australian lentil breeding program. To aid in this, the potential of exotic germplasm of lentil including landraces and wild species to provide such new resistance to AB has been evaluated.

To the time, space and financial commitment, a focused identification of germplasm strategy (FIGS) was applied to aid in the identification of novel AB resistance sources from the lentil landrace collection of the International Center for Agricultural Research in Dry Areas (ICARDA), Morocco. This entailed a systemic filtering of germplasm collection sites against environmental variables favouring AB progression to predict regions harbouring AB resistant genotypes. Accordingly, a subset (87 accessions) with the highest probabilities for harbouring AB resistance was selected from a collection of 4576 accessions. The subset was tested against a highly aggressive Australian isolate of A. lentis (FT13037) in a completely randomised and replicated bioassay, which resulted in identification of a highly resistant accession (IG 207) and twelve (IG 96, IG 712, IG 914, IG 1687, IG 1735, IG 5911, IG 7104, IG 7593, IG 7731, IG 8218, IG 8360 and IG 8550) moderately resistant accessions. Additionally, screening of 30 accessions from five-wild species against aggressive isolates (FT13037 and FT13038) revealed two highly resistant Lens orientalis accessions (ILWL 180 and ILWL 7) and a ten (L. nigricans (6), L. odomensis (1), L. ervoides (1), L. lamottei (1), and L. orientalis (1)) moderately resistant accessions. Subsequent screening of the resistant accessions IG 207 (L. culinaris) and ILWL 180 (L. orientalis) against a group of geographically diverse isolates with variable level of aggressiveness revealed superior resistance of both accessions over current resistance sources such as ILL 7537 and Indianhead.

Replicated histopathological studies of the lentil/A. lentis pathosystem validated the results of the bioassays and the AB resistance of IG 207 and ILWL 180. These are proposed to be, more effective than existing sources through strong and early activation of defence mechanisms. Further evidence of higher levels of reactive oxygen species (ROS; hydrogen

peroxide and superoxide) and phenolic compounds from ILWL 180 in response to AB infection added to this conclusion.

Introgression of novel and diverse resistance genes into the cultivated genetic background will likely improve lentil resistance to AB and minimise yield losses. Therefore, an interspecific cross between ILWL 180 and ILL 6002 was attempted. Despite, being a wild

relative, ILWL 180 was crossed with the cultivar ILL 6002 and produced healthy F1 seeds. The segregation of the subsequent F2 population indicated two recessive genes confer resistance to

AB. Additionally, the genetic analysis of other desirable traits within the F2 population revealed beneficial variants for traits such as days to first flower, plant height at flowering, number of nodes below first flower node, seed diameter, 100-seed weight and seed yield that may be helpful in widening the genetic base of cultivated lentil.

To select the physical location of the gene effects and possible candidate genes on the genome, a linkage map was constructed using 815 high quality single nucleotide polymorphism

(SNP) markers generated from a transcriptome sequencing of the F5 recombinant inbred line (RIL) population (N = 140). The map stretched 488.02 centiMorgan (cM) along eight linkage groups (LGs) with an 0.66 cM average marker-marker distance. Genetic dissection of the RIL population detected a quantitative trait loci (QTL) on LG5 which harbored nine putative candidate genes linked to AB resistance. Of these, five candidate genes were directly related to plant defence responses. Moreover, two of the three nonsynonymous mutations within the coding sequences of the putative candidate genes (Uroporphyrinogen decarboxylase (UROD) and Glutathione - S – transferase, DHAR3, chloroplastic (GST-DHAR3)) were predicted to modify the 3D structure of the corresponding proteins and ultimately the phenotype of the cultivar ILL 6002. Overall, the disease symptomatology, physiological and biochemical responses, and genetic evidence of resistance against AB infection support the conclusion that a stable and novel AB resistance was identified and characterised from ILWL 180 that offers significant potential to improve AB resistance of the existing cultivars within the Australian lentil breeding program.

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Table of contents

Declaration ...... ii Acknowledgements ...... iii Abstract ...... vi Table of contents...... viii List of figures ...... xiii List of tables ...... xviii List of appendices ...... xx List of abbreviations ...... xxv Preface and dissemination of research findings ...... xxix Chapter 1 - Introduction ...... 1 Chapter 2 - Literature review ...... 4 2.1. Lentil ...... 4 2.1.1. Origin and domestication history ...... 4 2.1.2. Classification of genus Lens ...... 4 2.1.3. Botany and agronomy ...... 5 2.1.4. Lentil industry and economic importance ...... 6 2.1.5. Constraints to lentil production ...... 6 2.2. Ascochyta blight ...... 7 2.2.1. Disease cycle and infection process ...... 7 2.2.2. Disease management ...... 9 2.3. Resistance in L. culinaris to A. lentis ...... 10 2.3.1. Genetics of disease resistance ...... 13 2.3.2. Evidence of resistance breakdown ...... 15 2.4. Utilisation of exotic germplasm in widening genetic diversity ...... 16 2.4.1. Value of wild relatives and landraces of crops ...... 16 2.4.2. Evaluation and selection of vast germplasm collections for beneficial traits ...... 18 2.5. Increasing the reliability of resistance through understanding the mechanisms of plant pathogen defence ...... 19 2.5.1. Plant-pathogen interactions ...... 20 2.5.2. Key defence related responses involved in the lentil – A. lentis pathosystem .... 21

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2.5.2.1. Recognition and structural defence responses ...... 21 2.5.2.2. Biochemical defence responses and oxidative burst ...... 23 2.5.2.3. Effector triggered immunity and programmed cell death (PCD) ...... 24 2.5.2.4. Phytohormone based signaling and SAR ...... 25 2.6. Introgression of beneficial traits from exotic germplasm and broadening the genetic base of cultivated lentil ...... 26 2.7. Marker-assisted selection (MAS) ...... 28 2.7.1. Status of MAS in lentil breeding for AB resistance ...... 28 2.7.1.1. Construction of genetic linkage maps in lentil ...... 28 2.7.1.2. Mapping QTLs conferring resistance to AB ...... 31 2.8. Transcriptome sequencing or genotyping-by-sequencing through transcriptomics (GBS-t) ...... 32 2.9. Conclusion and aims of the research ...... 34 Chapter 3 - Identification of novel Ascochyta lentis resistance in a global lentil collection using a focused identification of germplasm strategy (FIGS) ...... 35 3.1. Abstract ...... 35 3.2. Introduction ...... 36 3.3. Materials & methods ...... 38 3.3.1. FIGS and plant materials ...... 38 3.3.2. Fungal materials ...... 40 3.3.3. Preparation of inoculum ...... 41 3.3.4. Inoculation process ...... 41 3.3.5. Evaluation of FIGS set for AB resistance ...... 42 3.3.6. Assessment of the stability of resistant accessions ...... 42 3.3.7. Disease assessment and data analysis...... 42 3.3.8. Evaluation of physiological defence responses by detached leaf assay ...... 43 3.4. Results ...... 44 3.4.1. Assessment of FIGS set for resistance to A. lentis isolate FT13037 ...... 44 3.4.2. IG 207 as a potential novel resistance source to A. lentis ...... 48 3.4.3. Histopathological assessment of early defence responses to A. lentis within IG 207 ...... 51 3.5. Discussion ...... 54 3.5.1. Application of FIGS for the identification of best-bet AB resistance sources in the global lentil collection ...... 54

3.5.2. Evidence of better resistance to A. lentis in IG 207 than ILL 7537 and Indianhead ...... 56 3.5.3. Evidence of better defence through early physical containment A. lentis in IG 207 ...... 57 Chapter 4 - A novel Lens orientalis resistance source to the recently evolved highly aggressive Australian Ascochyta lentis isolates ...... 59 4.1. Abstract ...... 59 4.2. Introduction ...... 60 4.3. Materials and methods ...... 62 4.3.1. Plant and fungal materials ...... 62 4.3.2. Experimental design ...... 62 4.3.3. Preparation of inoculum and bioassay ...... 63 4.3.4. Disease assessment ...... 64 4.3.5. Statistical analysis ...... 64 4.4. Results ...... 65 4.4.1. Phenotyping of wild genotype resistance to two most aggressive isolates of A. lentis ...... 65 4.4.2. L. orientalis ILWL 180 as a potential novel resistance source ...... 66 4.5. Discussion ...... 69 Chapter 5 - Evidence of early defence to Ascochyta lentis within the recently identified Lens orientalis resistance source ILWL 180 ...... 72 5.1. Abstract ...... 72 5.2. Introduction ...... 73 5.3. Materials and Methods ...... 75 5.3.1. Plant materials ...... 75 5.3.2. Fungal materials ...... 76 5.3.3. Experimental design for evaluating fungal structures ...... 76 5.3.4. Evaluation of infection process by detached leaf assay ...... 76 5.3.5. Evaluation of infection process by attached/intact leaf assay ...... 77 5.3.6. Experimental design for biochemical analysis of ROS and phenolic compounds ...... 77

5.3.7. Histochemical localisation of hydrogen peroxide (H2O2) ...... 77

5.3.8. Quantification of H2O2 ...... 78 - 5.3.9. Histochemical localisation of superoxide anion (O2 ) ...... 78

5.3.10. Histochemical localisation of phenolic compounds ...... 79 5.3.11. Statistical analysis ...... 79 5.4. Results ...... 79 5.4.1. Infection process/physiological differences ...... 79 5.4.1.1. Spore germination percentage ...... 79 5.4.1.2. Germ tube length ...... 80 5.4.2. Appressorium percentage ...... 81 5.4.2.1. Timing and percent of appressorium formation on detached leaflets ...... 81 5.4.2.2. Timing and percent of appressorium formation on intact leaflets ...... 82 5.4.3. Biochemical analysis of ROS ...... 83

5.4.3.1. Localisation of H2O2 ...... 83

5.4.3.2. Quantification of H2O2 ...... 83 - 5.4.3.3. Localisation of O2 ...... 84 5.4.3.4. Phenolic compounds localisation ...... 84 5.5. Discussion ...... 88 Chapter 6 - Genetics of resistance to ascochyta blight and an assessment of agro- morphological traits in an interspecific hybrid population of lentil ...... 93 6.1. Abstract ...... 93 6.2. Introduction ...... 94 6.3. Materials and Methods ...... 96 6.3.1. Plant materials and interspecific crossing ...... 96 6.3.2. Plant propagation and experimental design ...... 96 6.3.3. Fungal material, preparation of inoculum and bioassay ...... 97 6.3.4. Disease assessment ...... 98 6.3.5. Resistance as a Mendelian trait ...... 98 6.3.6. Evaluation of genetic variation within the interspecific hybrid population for agronomical traits ...... 98 6.4. Results ...... 99 6.4.1. Interspecific crossing ...... 99 6.4.2. Genetic basis of resistance to A. lentis in L. orientalis accession ILWL 180 .... 100 6.4.3. Evaluation of interspecific progeny for agronomical traits ...... 103 6.5. Discussion ...... 107 Chapter 7 - Identification of quantitative trait loci and candidate genes associated with ascochyta blight resistance in the interspecific RIL population ...... 111

7.1. Abstract ...... 111 7.2. Introduction ...... 112 7.3. Materials and Methods ...... 114 7.3.1. Development of bi-parental mapping populations ...... 114 7.3.2. Phenotypic assessment of AB resistance under controlled growth conditions .. 114 7.3.3. RNA extraction, cDNA library construction and Illumina sequencing ...... 115 7.3.4. Variant calling and filtering of single nucleotide polymorphisms (SNP) ...... 115 7.3.5. Genetic linkage map construction ...... 116 7.3.6. QTL analysis and identification of candidate genes ...... 117 7.3.7. Prediction of candidate gene associated mutations and consequences ...... 117 7.4. Results ...... 118 7.4.1. Phenotypic assessment of LA-2 RIL population derived from cross between ILL 6002 × ILWL 180 for AB resistance ...... 118 7.4.2. Transcriptome sequencing and SNP discovery ...... 119 7.4.3. Construction of a genetic linkage map ...... 120 7.4.4. QTL detection...... 122 7.4.5. Identification of candidate genes and associated mutations ...... 125 7.5. Discussion ...... 129 Chapter 8 - General discussion and future directions ...... 134 8.1. Identification of new resistance sources to ascochyta blight (AB) in a global collection of lentil ...... 134 8.2. Physiological and biochemical evidence of better resistance within the genus Lens ...... 135 8.3. Generation of lentil interspecific population, and its assessment for agro- morphological traits and genetics of AB resistance ...... 137 8.4. Identification of QTLs and putative candidate genes controlling AB resistance ...... 139 8.5. Suggestions for future research ...... 140 References ...... 143 Appendices ...... 176

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List of figures

Figure 2.1: Disease cycle of Ascochyta lentis as illustrated by Kylie Fowler (adapted from (Kaiser, 1997)) ...... 8

Figure 2.2: A hypothetical model of the molecular lentil defence response system to A. lentis. Adapted from Khorramdelazad et al. (2018) ...... 22

Figure 3.1: Geographical distribution of lentil FIGS set for ascochyta blight resistance. FIGS set comprised of 87 landraces with highest likelihood for ascochyta blight resistance and were detected from ICARDA germplasm collection of 4576 accessions. The collection sites are indicated by red triangles ...... 47

Figure 3.2: Geographical distribution of moderately resistant to highly resistant lentil accessions detected within the FIGS set of 81 accessions against aggressive Ascochyta lentis isolate FT13037. Disease severity was assessed as percent APD. The collection sites of moderately resistant accessions and highly resistant, IG 207 are indicated by orange and green triangles, respectively ...... 48

Figure 3.3: Response of accession IG 207 to five Ascochyta lentis isolates ...... 50

Figure 3.4: Spore germination percentage of isolate FT13038 on the leaflets of accessions IG 207 and Nipper at 6, 12, 24 and 36 hours post inoculation (hpi). Bar at 36 hpi on the leaflets of Nipper represent an estimated value of spore germination percent. Estimates of means are derived from three replicates at each time point using a repeated measure analysis. For each replicate 100 conidia were assessed. Error bars show standard error of the means ...... 52

Figure 3.5: Germ tube length of isolate FT13038 on the leaflets of accessions IG 207 and Nipper at 6, 12, 24 and 36 hours post inoculation (hpi). Bar at 36 hpi on the leaflets of Nipper represent an estimated value of germ tube length. Estimates of means are derived from three replicates at each time point using a repeated measure analysis. For each replicate 100 germinated conidia were assessed. Error bars show standard error of the means ...... 52

Figure 3.6: Appressoria formation percentage of isolate FT13038 on the leaflets of accessions IG 207 and Nipper at 6, 12, 24 and 36 hours post inoculation (hpi). Bar at 36 hpi on the

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leaflets of Nipper represent an estimated value of percent of appressoria formed. Estimates of means are derived from three replicates at each time point using a repeated measure analysis. For each replicate 100 germinated conidia were assessed. Error bars show standard error of the means ...... 53

Figure 3.7: Histological analysis of pre-penetration behaviour of Ascochyta lentis isolate FT13038 on accessions IG 207 and Nipper at 6, 12, 24 and 36 hours post inoculation (hpi). Arrowheads indicate appressorium...... 53

Figure 4.1: Response of wild Lens ILWL 180 to five isolates ...... 68

Figure 5.1: Spore germination percent (%) of isolates FT13037 and F13082 at 6, 12, 20 and 30 hours post inoculation (hpi) on three lentil genotypes ILWL 180, ILL 6002 and ILL 7537 in a) detached assay and b) intact assay. Estimates of means are derived from four biological replicates at each time point using a repeated measure analysis. For each replicate 100 conidia were assessed. Error bars show standard error of the means ...... 80

Figure 5.2: Germ tube length of isolates FT13037 and F13082 at 6, 12, 20 and 30 hours post inoculation (hpi) on three lentil genotypes ILWL 180, ILL 6002 and ILL 7537 in a) detached assay and b) intact assay. Estimates of means are derived from four biological replicates at each time point using a repeated measure analysis. For each replicate 100 germinated conidia were assessed. Error bars show standard error of the means ...... 81

Figure 5.3: Appressorium percent (%) of isolates FT13037 and F13082 at 6, 12, 20 and 30 hours post inoculation (hpi) on three lentil genotypes ILWL 180, ILL 6002 and ILL 7537 in a) detached assay and b) intact assay. Estimates of means are derived from four biological replicates at each time point using a repeated measure analysis. For each replicate 100 germinated conidia were assessed. Error bars show standard error of the means ...... 82

Figure 5.4: Histochemical localisation of biochemical defence responses elicited within the leaflets of lentil genotypes ILWL 180, ILL 7537 and ILL 6002 in response to A. lentis

isolate FT13037. (a–d) Detection of hydrogen peroxide (H2O2) production by DAB- uptake method. Arrowheads indicate accumulation of reddish-brown deposits beneath - the appresoria and surrounding cells. (e-h) Accumulation of superoxide (O2 ) analysed - by NBT method. Dark blue deposits beneath the appresoria represent O2 production

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(arrowheads). (i-l) Bright-light micrographs of phenolic compounds detection by staining with toluidine blue. Arrowheads indicate greenish-blue deposits due to accumulation of phenolic compounds within the infected and surrounding cells...... 85

Figure 5.5: Histochemical localisation of biochemical defence responses elicited within the leaflets of lentil genotypes ILWL 180, ILL 7537 and ILL 6002 in response to A. lentis

isolate F13082. (a–d) Detection of hydrogen peroxide (H2O2) production by DAB-uptake method. Arrowheads indicate accumulation of reddish-brown deposits beneath the - appresoria and surrounding cells. (e-h) Accumulation of superoxide (O2 ) analysed by - NBT method. Dark blue deposits beneath the appresoria represent O2 production (arrowheads). (i-l) Bright-light micrographs of phenolic compounds detection by staining with toluidine blue. Arrowheads indicate greenish-blue deposits due to accumulation of phenolic compounds within the infected and surrounding cells...... 86

Figure 5.6: Standard calibration curve of absorbance vs concentration of hydrogen peroxide

(H2O2; µM) ...... 87

Figure 5.7: Production of hydrogen peroxide (H2O2) in response to the aggressive isolate FT13037 at 12, 24 and 48 hours post inoculation (hpi). Estimates of means are generated from three biological replicates using repeated measure analysis. Error bars indicate standard error of the means ...... 87

Figure 5.8: Production of hydrogen peroxide (H2O2) in response to the non aggressive isolate F13082 at 12, 24 and 48 hours post inoculation (hpi). Estimates of means are generated from three biological replicates using repeated measure analysis. Error bars indicate the standard error of the means ...... 88

Figure 6.1: Interspecific hybridisation between a) L. orientalis accession ILWL 180 (male parent) and L. culinaris cultivar ILL 6002 (female parent); b) Variation in seed cotyledon

colour of parents and F1 seed...... 100

Figure 6.2: Segregation of ascochyta blight resistance at 21 dpi in F2 (a) and F5 (b) generations of LA-1 and LA-2 populations, respectively, derived from an interspecific cross between L. culinaris ILL 6002 and L. orientalis ILWL 180. ILWL 180 (RP) and ILL 6002 (SP) were included as resistant and susceptible checks, respectively. Red arrows indicate the

position of the parents in the distribution. Disease severity was assessed as percent APD...... 102

Figure 6.3: Frequency distribution of agronomical traits in the F2 generation of LA-2 population derived from interspecific cross between L. culinaris ILL 6002 and L. orientalis ILWL 180. a) days to first flower b) plant height at flowering c) plant height below first flowering node d) number of nodes below first flowering node e) peduncle length f) seed diameter g) 100-seed weight and h) seed yield ...... 106

Figure 7.1: Frequency distribution of LA-2 RIL population derived from the cross between accessions ILL 6002 and ILWL 180 for a) leaf lesion score and b) stem lesion score; as demonstrated by violin plots. ILWL 180 and ILL 6002 were included as resistant and susceptible checks, respectively ...... 119

Figure 7.2: Heatmap demonstrating the estimated recombination fractions and corresponding LOD scores. SNP markers are lined up against each other. A red diagonal block indicate the tightly linked SNP markers with low recombinations ...... 121

Figure 7.3: Illustration of density of the markers within each linkage group of the map derived from a cross between ILL 6002 and ILWL 180 ...... 122

Figure 7.4: QTL peak for leaf lesion score at 21 days post inoculation (dpi) on linkage group 5 (LOD > 3) as demonstrated by R/qtl2 package ...... 123

Figure 7.5: QTL detection for stem lesion score at 14, 21, 28 days post inoculation (dpi) as demonstrated by R/qtl2 package on the linkage map derived from cross between ILL 6002 and ILWL 180 ...... 123

Figure 7.6: Linkage map of the interspecific LA-2 recombinant inbred line (RIL) population derived from a cross between ILL 6002 and ILWL 180, and localisation of the QTL controlling the resistance to A. lentis. The bars within each linkage group (LG) depict the position of the SNP markers. The loci within the QTL region are named to the left of the linkage group and the positions of the corresponding loci on the right. The QTL controlling A. lentis resistance is shown with a red bar...... 124

Figure 7.7: Predicted 3D model structures of proteins a) UROD (PDB ID: 5ECS) and b) GST- DHAR3 (PDB-1J93) from Arabidiopsis thaliana and Nicotinia tabacum, respectively.

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The green annotations indicate the positions of the mutations within the 3D structures of the proteins...... 128

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List of tables

Table 2.1: Some sources of resistance within the cultivated gene pool to Ascochyta lentis described in different lentil growing countries ...... 11

Table 2.2: Lentil cultivar collection of Australia and disease ratings at the time of release ... 12

Table 2.3: QTL's/genes conferring resistance to ascochyta blight (AB) resistance and corresponding markers ...... 14

Table 3.1: Details of Ascochyta lentis isolates (including the lentil cultivar from which they were collected, location and year of collection) used in the study ...... 41

Table 3.2: Ascochyta lentis isolate/lentil FIGS set interactions at 7, 14 and 21 days post inoculation (dpi) ...... 45

Table 3.3: Percent area of plant diseased (% APD) scores for lentil accessions at 21 days post inoculation (dpi) with four Ascochyta lentis isolates. Estimates of means are derived from three biological replicates using linear mixed model analysis...... 49

Table 4.1: Details of A. lentis isolates (including the lentil cultivar from which they were collected, location and year of collection) used in the study ...... 63

Table 4.2: Modal disease score of three host differentials at 14 and 21 days post inoculation (dpi) for A. lentis isolates FT13038 and FT13037 ...... 65

Table 4.3: Details of the Lens spp. genotypes used in the study along with corresponding modal disease scores at 14 and 21 days post inoculation (dpi) and area under disease progress curve (AUDPC) ...... 67

Table 4.4: Modal disease scores of ILWL 180 and controls at 14 and 21 days post inoculation (dpi) against five A. lentis isolates ...... 69

Table 6.2: Segregation pattern for ascochyta blight seedling, foliar, stem and broken stem

resistance in the F2 and F5 generation of LA-1 and LA-2 populations, respectively,

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derived from the interspecific cross (L. culinaris ILL 6002 × L. orientalis ILWL 180) ...... 102

Table 6.3: Ascochyta blight resistance status and agronomical trait description of L. orientalis

ILWL 180, L. culinaris ILL 6002 and F1 derived from the cross ...... 103

Table 6.4: Segregation ratios for various morphological traits in the F2 generation of LA-2 population derived from the interspecific cross (L. culinaris ILL 6002 × L. orientalis ILWL 180) ...... 104

Table 6.5: Descriptive statistics of morphological traits of F2 generation of LA-2 population derived from the interspecific cross (L. culinaris ILL 6002 × L. orientalis ILWL 180) ...... 105

Table 7.1: Marker distribution over the linkage groups of the linkage map derived from a cross between ILL 6002 and ILWL 180 ...... 121

Table 7.2: Details of markers and corresponding loci identified within the QTL region on linkage group 5 ...... 126

Table 7.3: Details of putative candidate genes and corresponding SNP effects ...... 127

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List of appendices

Appendix 3.1: Results of analysis of variance obtained from linear mixed model procedure of GenStat for the reaction of FIGS lentil subset to aggressive Ascochyta lentis isolate FT13037 ...... 176

Appendix 3.2: Comparison of means (percent area of plant diseased) of FIGS lentil subset for the reaction to aggressive Ascochyta lentis isolate FT13037 at 7, 14 and 21 days post inoculation (dpi) based on least significant differences with the Tukey’s adjustment (α = 0.05) ...... 177

Appendix 3.3: Results of analysis of variance obtained from linear mixed model procedure of GenStat for the reaction of FIGS and host differential lentil accessions to Ascochyta lentis isolates FT13038, FT15160, FT16112 and FT16299-2 at 7, 14 and 21 days post inoculation (dpi) ...... 179

Appendix 3.4: Comparison of means (percent area of plant diseased) of FIGS and host differential lentil accessions to Ascochyta lentis isolates FT13038, FT15160, FT16112 and FT16299-2 at 21 days post inoculation (dpi) based on least significant differences with the Tukey’s adjustment (α = 0.05) ...... 180

Appendix 3.5: Results of analysis of variance obtained from repeated measure analysis of GenStat for comparison of spore germination percentage, percentage of appressoria formation and germ tube length by Ascochyta lentis isolate FT13038 at 6, 12 and 24 hours post inoculation (hpi) on lentil genotypes IG 207 and Nipper ...... 181

Appendix 3.6: Comparison of means of spore germination percentage, percentage of appressoria formation and germ tube length by Ascochyta lentis isolate FT13038 at 6, 12 and 24 hours post inoculation (hpi) on lentil genotypes IG 207 and Nipper based on least significant differences with the Tukey’s adjustment (α = 0.05) ...... 181

Appendix 4.1: Results of analysis of variance obtained from Friedman’s non-parametric test of IBM SPSS Statistic software for the reaction of Lens spp. genotypes to Ascochyta lentis ...... 182

Appendix 4.2: Results of pairwise comparison of mode disease scores of lentil genotypes from preliminary screening using Wilcoxon signed ranks test of IBM SPSS Statistic software ...... 182

Appendix 4.3: Results of pairwise comparison of mode disease scores of wild lentil genotypes using Wilcoxon signed ranks test of IBM SPSS Statistic software...... 183

Appendix 4.4: Results of pairwise comparison of mode disease scores of lentil genotypes from stability analysis using Wilcoxon signed ranks test of IBM SPSS Statistic software .. 184

Appendix 5.1: Results of analysis of variance obtained from repeated measure analysis of GenStat for comparison of spore germination percentage by Ascochyta lentis isolates FT13037 and F13082 at 6, 12, 20 and 30 hours post inoculation (hpi) on lentil genotypes ILL 6002, ILWL 180 and ILL 7537 ...... 185

Appendix 5.2: Comparison of means of spore germination percentage by Ascochyta lentis isolates FT13037 and F13082 at 6, 12, 20 and 30 hours post inoculation (hpi) on detached leaflets of lentil genotypes ILL 6002, ILWL 180 and ILL 7537 based on least significant differences with the Tukey’s adjustment (α = 0.05) ...... 185

Appendix 5.3: Comparison of means of spore germination percentage by Ascochyta lentis isolates FT13037 and F13082 at 6, 12, 20 and 30 hours post inoculation (hpi) on attached leaflets of lentil genotypes ILL 6002, ILWL 180 and ILL 7537 based on least significant differences with the Tukey’s adjustment (α = 0.05) ...... 186

Appendix 5.4: Results of analysis of variance obtained from repeated measure analysis of GenStat for comparison of germ tube length by Ascochyta lentis isolate FT13037 and F13082 at 6, 12, 20 and 30 hours post inoculation (hpi) on lentil genotypes ILL 6002, ILWL 180 and ILL 7537 ...... 186

Appendix 5.5: Comparison of means of germ tube length by Ascochyta lentis isolates FT13037 and F13082 at 6, 12, 20 and 30 hours post inoculation (hpi) on detached leaflets of lentil genotypes ILL 6002, ILWL 180 and ILL 7537 based on least significant differences with the Tukey’s adjustment (α = 0.05)...... 187

Appendix 5.6: Comparison of means of germ tube length by Ascochyta lentis isolates FT13037 and F13082 at 6, 12, 20 and 30 hours post inoculation (hpi) on attached leaflets of lentil

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genotypes ILL 6002, ILWL 180 and ILL 7537 based on least significant differences with the Tukey’s adjustment (α = 0.05)...... 187

Appendix 5.7: Results of analysis of variance obtained from repeated measure analysis of GenStat for comparison of percent appressorium formed by Ascochyta lentis isolate FT13037 and F13082 at 6, 12, 20 and 30 hours post inoculation (hpi) on lentil genotypes ILL 6002, ILWL 180 and ILL 7537 ...... 188

Appendix 5.8: Comparison of means of percent appressorium formed by Ascochyta lentis isolates FT13037 and F13082 at 6, 12, 20 and 30 hours post inoculation (hpi) on detached leaflets of lentil genotypes ILL 6002, ILWL 180 and ILL 7537 based on least significant differences with the Tukey’s adjustment (α = 0.05) ...... 188

Appendix 5.9: Comparison of means of percent of appressorium formed by Ascochyta lentis isolates FT13037 and F13082 at 6, 12, 20 and 30 hours post inoculation (hpi) on attached leaflets of lentil genotypes ILL 6002, ILWL 180 and ILL 7537 based on least significant differences with the Tukey’s adjustment (α = 0.05) ...... 189

Appendix 5.10: Results of analysis of variance obtained from repeated measure analysis of

GenStat for comparison of hydrogen peroxide (H2O2) concentration accumulated by lentil genotypes ILL 6002, ILWL 180 and ILL 7537 in response to Ascochyta lentis isolates (FT13037 and F13082) infection at 12, 24 and 48 hours post inoculation (hpi) ...... 189

Appendix 5.11: Comparison of means of hydrogen peroxide (H2O2) concentration accumulated by lentil genotypes ILL 6002, ILWL 180 and ILL 7537 in response to Ascochyta lentis isolates (FT13037 and F13082) infection at 12, 24 and 48 hours post inoculation (hpi) based on least significant differences with the Tukey’s adjustment (α = 0.05) ...... 190

Appendix 5.12: Histochemical localisation of hydrogen peroxide (H2O2) accumulation in the leaflets of lentil genotypes ILWL180, ILL7537 and ILL6002 in response to aggressive isolate A. lentis isolate FT13037 using DAB-uptake method. a, d and g: accumulation of

H2O2 on detached leaflets of ILWL180 beneath appressoria (arrow) at 12, 24 and 48 hpi.

b, e and h: accumulation of H2O2 on detached leaflets of ILL7537 beneath appressoria

(arrow) at 12, 24 and 48 hpi. c, f and i: accumulation of H2O2 on detached leaflets of

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ILL6002 beneath appressoria (arrow) at 12, 24 and 48 hpi. Scale bars represent 50 µm (a-h plates) and 100 µm (plate i)...... 191

Appendix 5.13: Histochemical localisation of hydrogen peroxide (H2O2) accumulation in the leaflets of lentil genotypes ILWL180, ILL7537 and ILL6002 in response to non- aggressive isolate A. lentis isolate FT3082 using DAB-uptake method. a, d and g:

accumulation of H2O2 on detached leaflets of ILWL180 beneath appressoria (arrow) at

12, 24 and 48 hpi. b, e and h: accumulation of H2O2 on detached leaflets of ILL7537

beneath appressoria (arrow) at 12, 24 and 48 hpi. c, f and i: accumulation of H2O2 on detached leaflets of ILL6002 beneath appressoria (arrow) at 12, 24 and 48 hpi. Scale bars represent 50 µm (a-h plates) and 100 µm (plate i)...... 192

- Appendix 5.14: Histochemical localisation of superoxide (O2 ) accumulation in the leaflets of lentil genotypes ILWL180, ILL7537 and ILL6002 in response to aggressive isolate A. - lentis isolate FT13037 using NBT method. a, d and g: accumulation of O2 on detached leaflets of ILWL180 beneath appressoria (arrow) at 12, 24 and 48 hpi. b, e and h: - accumulation of O2 on detached leaflets of ILL7537 beneath appressoria (arrow) at 12, - 24 and 48 hpi. c, f and i: accumulation of O2 on detached leaflets of ILL6002 beneath appressoria (arrow) at 12, 24 and 48 hpi. Scale bars represent 50 µm (a-h plates) and 100 µm (plate i)...... 193

- Appendix 5.15: Histochemical localisation of superoxide (O2 ) accumulation in the leaflets of lentil genotypes ILWL180, ILL7537 and ILL6002 in response to aggressive isolate A. - lentis isolate F13082 using NBT method. a, d and g: accumulation of O2 on detached leaflets of ILWL180 beneath appressoria (arrow) at 12, 24 and 48 hpi. b, e and h: - accumulation of O2 on detached leaflets of ILL7537 beneath appressoria (arrow) at 12, - 24 and 48 hpi. c, f and i: accumulation of O2 on detached leaflets of ILL6002 beneath appressoria (arrow) at 12, 24 and 48 hpi. Scale bars represent 50 µm (b-e and g plates) and 100 µm (a, f, h and i plates)...... 194

phenolic compounds on detached leaflets of ILWL180 beneath appressoria (arrow) at 12, 24 and 48 hpi. b, e and h: accumulation of phenolic compounds on detached leaflets of

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ILL7537 beneath appressoria (arrow) at 12, 24 and 48 hpi. c, f and i: accumulation of phenolic compounds on detached leaflets of ILL6002 beneath appressoria (arrow) at 12, 24 and 48 hpi. Scale bars represent 50 µm (a-c, e, f and i plates) and 100 µm (d, g and h plates)...... 195

Appendix 5.17: Histochemical localisation of phenolic compounds deposition in the leaflets of lentil genotypes ILWL180, ILL7537 and ILL6002 in response to aggressive isolate A. lentis isolate F13082 using toluidine blue staining method. a, d and g: accumulation of phenolic compounds on detached leaflets of ILWL180 beneath appressoria (arrow) at 12, 24 and 48 hpi. b, e and h: accumulation of phenolic compounds on detached leaflets of ILL7537 beneath appressoria (arrow) at 12, 24 and 48 hpi. c, f and i: accumulation of phenolic compounds on detached leaflets of ILL6002 beneath appressoria (arrow) at 12, 24 and 48 hpi. Scale bars represent 50 µm (a-d, f, h and i plates) and 100 µm (e and g plates)...... 196

Appendix 6.1: Results of analysis of variance obtained from ANOVA tests of GenStat for the

reaction of lentil genotypes ILWL 180, ILL 6002 and interspecific F1 of the cross ILL 6002 × ILWL 180 to A. lentis isolate FT13038 and various agro-morphological traits ...... 197

Appendix 6.2: Correlation coefficients (R) among various agro-morphological traits in the

interspecific segregating F2 population (N = 199) derived from the cross ILWL 180 × ILL 6002 ...... 198

Appendix 7.1: Details of SNPs used in the linkage map and their corresponding position on different linkage groups ...... 199

Appendix 7.2: BLASTx-based sequence analysis of contigs/scaffold sequences underlying SNP markers against protein sequences of Fabaceae family ...... 221

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List of abbreviations

% APD area of a plant that showed disease symptoms 6PGDH 6 - phosphogluconate dehydrogenase A Alanine AB Ascochyta blight ABA Abscisic acid AFLP Amplified fragment length polymorphism AGGB Australian Grain Gene bank ANOVA Analysis of variance APRT Anthranilate phosphoribosyltransferase, chloroplastic ARP Auxin – repressed protein ATG Autophagy-related AUD Australian Dollar AUDPC Area under disease progress curve Avr Avirulence B Boron BAK1 BR11-associated receptor kinase BAM Binary format BLASTN Basic local alignment-nucleotide BLASTx Basic local alignment search tool BWA Burrows wheeler aligner CALS Callose synthase CDD Conserved domain database CDK Cyclin-dependent kinase CDPK Calmodulin domain protein kinase CDS Coding sequences CESA Cellulase synthase CHR Chalcone reductase CHS Chalcone synthase CIPAL Coordinated improvement program for Australian lentil cM centiMorgan CRISPR Clustered regularly interspaced palindromic repeats CSB3 Constitutive subtilisin 3 CSLC6 Xyloglucan glycotransferase CuAO Copper containing amine oxidase South Asia and China, North America, Central Asia CWANA and West Asia and North Africa

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CWDE Cell wall degrading enzymes DAB 3, 3-diaminobenzidine DArT Diversity arrays technology DAS days after sowing DE Differential expression DEDJTR Department of economic development, jobs, transport and resources Dpi days post inoculation E3 UPL-RHC2A E3 ubiquitin-protein ligase RHC2A EFR3 EFR3 receptor kinase ERF Ethylene responsive transcription factor EST Expressed sequence tag EST-SSR Expressed sequence tag-simple sequence repeats ET Ethylene ETI Effector triggered immunity Exo70A1 Exocyst subunit 70A1 family protein F Phenylalanine FDSS Fungicide decision support system FIGS Focused identification of germplasm strategy FOX Ferrous xylenol orange FVAS Faculty of veterinary and agricultural sciences

GA3 Gibberellic acid GAGA-TF ERF1B and gaga transcription factor GBS Genotyping-by-sequencing GBS-RAD Genotyping-by-sequencing - restriction site associated DNA GBS-t Genotyping-by-sequencing through transcriptomics GIS Geographic information system GPS Global positioning system GST Glutathione s transferase GST-DHAR3 Glutathione - S – transferase, DHAR3, chloroplastic

H2O2 Hydrogen peroxide Ha hectare Hpi hours post inoculation HR Hypersensitive reaction I Isoleucine ICARDA International center for agricultural research in dry areas ImageJ Image processing and analysis in java ISR Induced systemic resistance ISSR Inter simple sequence repeats

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ITAP Intron targeted amplified polymorphism JA Jasmonic acid L Leucine LECRK Lectin s-receptor-like serine threonine protein kinase LGs Linkage Groups LOX Lipoxygenase LRR-RK Leucine – rich repeat receptor kinase LSD Least significant difference M ha million hectares MAB Marker assisted breeding MAS Marker assissted selection MBC Methyl benzimidazole carbamate Mlo Mutation induced recessive alleles MR Moderately resistant MS Moderately susceptible Mt million tonnes NB-ARC NB - ARC domain disease resistance protein NBT Nitro-blue tetrazolium NCBI National center for biotechnology information NEK6 NIMA – related kinase 6 NGS Next generation sequencing Nr Non redudant - O2 Superoxide anion ORF Open reading frame PAMP Pathogen associated molecular patterns PARP Poly polymerase like PBA Pulse Breeding Australia PCD Programmed cell death PCR Polymerase chain reaction PDA Potato dextrose agar PDB Protein data bank P-EXE2 Protein EXECUTER 2, chloroplastic isoform X1 PGIP Polygalacturonase inhibitor PMEI Plant invertase pectin methylesterase inhibitor PPOI Laccase diphenol oxidase PR proteins Pathogenesis related proteins PRRs Pattern recognition receptors PTI PAMP triggered immunity

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QTL Quantitative trait loci R Resistant R gene Resistance gene RAPD Random amplified polymorphic DNA RFLP Restriction fragment length polymorphism RGAs Resistance gene analogues RH Relative humidity RIL Recombinant inbred line RING/U - box RING/U – box protein RLK Receptor like kinase ROS Reactive oxygen species S Susceptible SA Salicylic acid SA South Australia SAG Senescence associated gene SAM Sequence alignment and mapping SAR Systemic acquired resistance SARDI South Australian research and development institute SNP Single nucleotide polymorphism SOD Superoxide dismutase SSR Simple sequence repeats STPK Serine threonine protein kinase T Threonine TILLING Targeting induced local lesions in genomes UROD Uroporphyrinogen decarboxylase UTR Untranslated region UV Ultra violet V Valine WAK1 Wall associated protein kinase 1 XTH Xyloglucan endotransglucosylase/hydrolase

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Preface and dissemination of research findings

Scientific papers published or submitted by the author in collaboration with supervisors and other scientists are listed in the following:

Peer-reviewed papers published in international scientific journals:

Dadu RHR, Ford R, Sambasivam P, Gupta D, 2017. A novel Lens orientalis resistance source to the recently evolved highly aggressive Australian Ascochyta lentis isolates. Frontiers in Plant Science 8, 1038.

Dadu RHR, Ford R, Sambasivam P, Gupta D, 2018. Evidence of early defence to Ascochyta lentis in a novel primary Lens genepool accession. Plant Pathology 67, 1492-1501.

Dadu RHR, Ford R, Sambasivam P, Street K, Gupta D, 2018, ‘Identification of novel Asochyta lentis resistance in the global lentil collection using a focused identification of germplasm strategy (FIGS)’, Australasian Plant Pathology. doi: 10.1007/s13313-018-0603-7.

Conference proceedings

Dadu RHR, Ford R, Sambasivam P, Gupta D, 2015, ‘Unlocking lentil genetic resources for the identification of resistant sources to ascochyta blight and molecular mapping of QTL for ascochyta blight resistance’, FVAS Post Graduate Research symposium, The University of Melbourne, Parkville, Australia, 3-4th December 2015.

Dadu RHR, Ford R, Sambasivam P, Gupta D, 2016, ‘Screening of wild lentil germplasm to identify novel Ascochyta lentis resistance sources’, IV International Ascochyta Workshop, Lisbon, Portugal 10-11th October 2016.

Dadu RHR, Ford R, Sambasivam P, Gupta D, 2016, ‘Screening of wild lentil germplasm and confirmation of novel wild resistance source to Ascochyta lentis’, FVAS Post Graduate Research Symposium, The University of Melbourne, Parkville, Australia 30th November-2nd December 2016.

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Dadu RHR, Ford R, Sambasivam P, Gupta D, 2017, ‘Evidence of early defence to Ascochyta lentis in a novel primary Lens genepool accession’, Science Protecting Plant Health 2017, Brisbane, Australia, 26-28th September 2017.

Dadu RHR, Ford R, Sambasivam P, Gupta D, 2017, ‘Evidence of early defence to Ascochyta lentis in a novel primary Lens genepool accession’, FVAS Post Graduate Research Symposium, The University of Melbourne, Werribee, Australia, 24-25th October 2017.

Dadu RHR, Ford R, Sambasivam P, Gupta D, 2018, ‘Identification of novel Asochyta lentis resistance in the global lentil collection using a focused identification of germplasm strategy (FIGS)’, 7th International Food Legume Research Conference, Marrakech, Morocco 6-8th May 2018.

Dadu RHR, Ford R, Sambasivam P, Gupta D, 2018, ‘Identification and characterisation of novel ascochyta blight resistance resources in lentil’, Australian Pulse Pathology Workshop, Clare, Australia, 18-20th September 2018.

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Chapter 1 - Introduction

Lentil is a major export crop for Australia but is greatly constrained by disease Ascochyta blight (AB) caused by a necrotrophic fungus Ascochyta lentis (Murray and Brennan, 2012; Rodda et al., 2017). The disease may be effectively controlled by chemical methods such as seed treatment and foliar sprays (Tripathi, 2015). However, an indiscriminate use of fungicides contaminates the crop products and results in a negative impact on the environment (Komárek et al., 2010). Additionally, the efficacy of fungicides has been affected in some situations due to the development of resistant strains within the target pathogen populations (Owati et al., 2017) as evident a Carbendazim-resistant strain within the Australian A. lentis population (Lopez and Kay, 2017). Therefore, instead of a complete reliance on chemical control, resistant cultivars integrated with other management practices such as crop rotation, burial of infested residue, use of disease-free seed, and weather based sowing dates are more environmentally friendly and sustainable to combat AB (Davidson and Kimber, 2007; Hawthorne et al., 2015b). This thesis is focused on identifying long-term sustainable answers to manage the disease AB through introduction of novel and diverse resistance genes to the genome of Lens culinaris Medikus. ssp. culinaris.

In light of the above, the origin, taxonomy, biology and production constraints of lentil with a detailed description of AB, its lifecycle and disease management strategies are presented in the Chapter 2. The importance of host plant resistance with emphasis on exotic germplasm and plant defence mechanisms, particularly in lentil-A. lentis interaction are discussed. Additionally, the status of marker-assisted breeding (MAB) and next generation (NGS) application in lentil is updated.

Success in breeding improved crop cultivars is determined by the amount of genetic gain achieved through production of superior genotypes which possess agronomically stable traits and increased yield potential (Barker et al., 2005; Koester et al., 2014; Bankole et al., 2017). However, the amount of genetic gain achieved within a progeny is always dependent on the amount of variation existing between the parents that are selected for hybridisation (Roy et al., 2013). Incidentally, phenotyping the current genetic diversity of the Australian lentil breeding program revealed an increased genetic similarity among the elite cultivars for

resistance to AB (Ford et al., 1997; Lombardi et al., 2014). This is suspected to pose a significant risk to sustainable resistance highly aggressive isolates of A. lentis (Davidson et al., 2016). One strategy to improve genetic gain towards AB resistance is through the introduction of germplasm from a wider genetic background into the cultivated background (Tullu et al., 2010a). In this context, Chapters 3 and 4 examine the variation within the wide germplasm of lentil including landraces and wild species for resistance to AB. Identification of resistance sources within wild accessions collected from the Australian Gene Bank, Horsham, was performed through conventional screening techniques. However, a larger collection of landraces collected from the International Center for Agricultural Research in Dry Areas (ICARDA) was narrowed down using a focused identification of germplasm strategy (FIGS) approach to screen a small subset of 87 landraces with highest likelihood for AB resistance.

An understanding of the defence mechanisms within the new resistance sources is necessary prior to establishing them as reliable sources to improve the resistance breeding strategies (Armstrong-Cho et al., 2015; Sari et al., 2017). Histopathology and transcriptome profiling of lentil-A. lentis interactions revealed that the resistance within lentil cultivars against A. lentis infection is determined through a strong and early activation of defence mechanisms (Mustafa et al., 2009; Sambasivam et al., 2016; Sari et al., 2017; Khorramdelazad et al., 2018; Sari et al., 2018). Thus, Chapter 5 highlights the use of microscopy techniques to dissect the physiological and biochemical defence mechanisms within the resistant L. orientalis accession ILWL 180. This provides evidence of a superior and useful resistance through an early and fast recognition of A. lentis.

Chapter 6 demonstrates the use of traditional techniques of crossing supplemented with

gibberellic acid (GA3) application after fertilization to eliminate any post-fertilization barriers for normal growth of embryo and successful introgression of AB resistance from L. orientalis accession ILWL 180 into L. culinaris cultivar ILL 6002. This was achieved using an F1 selfing

produced segregating F2 population, to determine the genetic mechanism of AB resistance within accession ILWL 180. Additionally, the F2 population was used to evaluate the frequency of 17 agronomically desirable traits that either individually or collectively are expected to improve the recovery of elite phenotypes through selection. Useful variation within the interspecific crosses increase the frequency of desirable genes and broadens the genetic base of the cultivated lentil (Tullu et al., 2013; Singh et al., 2018).

Tightly linked disease resistance markers would greatly facilitate the development of durable resistance within the breeding program through selection of the resistant germplasm in the absence of the pathogen (Collard and Mackill, 2008; Nadeem et al., 2018). Efforts to identify quantitative trait loci (QTLs) controlling resistance to AB provided successful and effective marker-trait associations (Ford et al., 1999; Chowdhury et al., 2001; Rubeena et al., 2006; Gupta et al., 2012a; Sudheesh et al., 2016a). With advances in NGS technologies, the resolution of QTL mapping was greatly improved and thus the availability of several potentially reliable markers within or nearby the genic regions of the desirable traits (Kaur et al., 2011; Sharpe et al., 2013; Kaur et al., 2014; Temel et al., 2015). Chapter 7 discusses the feasibility of transcriptome sequencing in identifying gene based single nucleotide polymorphism (SNP) markers to develop a high-density linkage map, and QTLs controlling AB resistance within the RIL population derived from an interspecific cross between accessions ILWL 180 and ILL 6002.

In summary, this thesis identified new sources of resistance to A. lentis within a wide lentil germplasm collection. Physiological, biochemical and genetic evidence indicated that the resistance within the wild lentil genotype ILWL 180 is useful and superior compared to that of the currently used resistance source ILL 7537. The markers identified as closely linked to a QTL and the identified putative candidate genes underpinning the resistance to AB infection may assist in future selective breeding with accession ILWL 180 as a source of resistance. Additionally, evidence of useful variation within the wild accession is likely to aid efforts to broaden the genetic base of Australian lentil cultivars.

Chapter 2 - Literature review

2.1. Lentil

2.1.1. Origin and domestication history

Lentil (Lens culinaris Medikus. ssp. culinaris) is one of the oldest domesticated crops and “the history of lentil dates back as far as the history of agriculture itself” (Helbaek, 1963; Ladizinsky, 1979). Based on the archaeological remains, lentil is widely accepted to have originated and to have been domesticated in the Fertile Crescent in the Near East region (Zohary, 1972). The oldest remains date back to 11000 BC and 8500-7500 BC in Greece and Syria, respectively (Erskine, 1997). Pod dehiscence and seed shattering (up to 2 meters radius) are considered important lentil plant traits for their spread to the Mediterranean, Europe and Asia (Erskine, 1997; Ljuština and Mikić, 2010). Furthermore, an excellent ability for adaptation helped lentil move to new climatic zones and it is now cultivated in all continents (Erskine, 2009).

2.1.2. Classification of genus Lens

The genus Lens along with genera Pisum, Vicia and Lathyrus is phylogenetically nested within the tribe Vicieae, sub-family Papilionoideae of the family Fabaceae (Ladizinsky and Muehlbauer, 1993). Lens was initially proposed to have seven taxa, L. culinaris Medik. subsp. culinaris (cultivated species), progenitor L. culinaris subsp. orientalis (Boiss.) Ponert, L. culinaris subsp. tomentosus (Ladiz.), L. culinaris subsp. odemensis (Ladiz.), L. lamottei (Czefr.), L. ervoides (Brign.) Grande and L. nigricans (M.Bieb.) Godr. (Ladizinsky et al., 1984; Van Oss et al., 1997). Subsequently, Ferguson et al. (2000) reported the first comprehensive classification of Lens and divided the genus into seven taxa with four species, namely, L. culinaris (including subsp. culinaris, orientalis, tomentosus and odemensis), L. lamottei, L. ervoides and L. nigricans based on morphological, cytological and molecular marker studies. However, the genetic relatedness among taxa assessed through either morphological, isozyme or molecular markers have been frequently disagreed (Havey and Muehlbauer, 1989a; Abo- Elwafa et al., 1995; Ahmad and McNeil, 1996; Sonnante et al., 2003). More recently, a re-

organized classification of genus Lens was proposed based on phylogenetic and structural analysis using genotyping-by-sequencing (GBS) enriched with single nucleotide polymorphism (SNP) markers and alignment with the CDC Redberry draft genome-derived exome array (Wong et al., 2015; Ogutcen et al., 2018). The new classification included four gene pools, namely primary (L. culinaris/L. orientalis/L. tomentosus), secondary (L. lamottei/L. odomensis), tertiary (L. ervoides) and quaternary gene (L. nigricans). These studies also re-confirmed L. orientalis as the immediate progenitor of the cultivated lentil (Ladizinsky et al., 1984; Ferguson et al., 2000)

2.1.3. Botany and agronomy

Lentil is a self-pollinated, diploid (2n=14), herbaceous annual plant with slender, erect to semi-erect stem, erect compact to prostrate or spreading branches and compound leaves (Muelhbauer and Cubero, 1985). The plant has a slender, shallow to deep tap root system with fibrous lateral roots carrying numerous nodules when grown with appropriate Rhizobium strains (Saxena and Hawtin, 1981). Although the plant structure varies with genotype, environmental conditions at the cultivation site also influence the physiology (Saxena and Hawtin, 1981). Lentil have an indeterminate growth habit with an acropetal flowering pattern and hence plants close to maturity may bear nodes with pods and flowers. Papilionaceous flowers range from small to large in size (4-9 mm long) with varied colors such as white, pink, purple, blue or pale blue to pale purple (Muelhbauer and Cubero, 1985). Pods are oblong and laterally compressed with one or two lens shaped seeds (Saxena and Hawtin, 1981).

Lentil requires a cold climate and is grown in areas with temperatures ranging from 18◦C to 30◦C and an annual rainfall as little as 250 mm to a maximum of >1000 mm (Ali et al., 2009; Materne and Siddique, 2009; Choudhury et al., 2012). Lentil is cultivated on a wide range of soil types; however, higher yields are realized when grown on sandy loam to clay loam soils that are fertile, have good drainage, water holding capacity and neutral to alkaline pH (6-7) (Ali et al., 2009). Lentil do not grow well in soils with high boron (B) content, sodicity or salinity, which cause plant death and substantially limits yields (Pulse Australia, 2016).

2.1.4. Lentil industry and economic importance

Lentil is cultivated in ~52 countries worldwide and ranks fifth among all the legumes grown globally with 6.31 million tonnes (Mt) production (FAOSTAT, 2016). Major lentil growing areas include South Asia and China, North America, Central Asia and West Asia and North Africa (CWANA), Sub-Saharan Africa and Australia. Canada is the largest producer and exporter of lentil in the world. World lentil production has nearly six-fold from 0.95 Mt in 1961-63 to 6.31 Mt in 2016 (FAOSTAT, 2016).

Lentil is an important pulse crop of Australia and has risen into prominence since the late 1990s, cultivated in an area of about 0.22 million hectares (M ha) with an annual production of 0.181 Mt, thus making Australia the sixth largest producer of lentil in the world (FAOSTAT, 2016). Lentil is mainly grown in semi-arid regions of Victoria and South Australia and most is exported to the Mediterranean (16%) and the Indian subcontinent (78%), valued at $1.2 billion AUD in 2015 (Pulse Australia, 2016).

Lentil is considered as an alternative to expensive non-vegetarian food especially in the Mediterranean and on the Indian subcontinent due to its high dietary protein (28%) containing all of the essential amino acids except the sulfur-containing amino acids, methionine and cysteine (Grusak, 2009). Lentil also possesses high levels of lysine and thus compliments a cereal-(rice and wheat) based diet (Shewry and Halford, 2002). Lentil seed is used in and consumed as snacks, soups, stews, purees or eaten as sprouts (Raghuvanshi and Singh, 2009). Among other crop benefits, lentil straw is used as animal feed (Erskine et al., 1990b), nodulated lentil fixes atmospheric nitrogen (Taha et al., 2018) and lentil provides a disease break in cereal cropping systems (Stagnari et al., 2017).

2.1.5. Constraints to lentil production

Lentil yields have been considerably low for the past two decades for various reasons including cultivation in marginal lands especially in developing countries (Yadav et al., 2009), narrow genetic diversity of the cultivar base (Alo et al., 2011) and various abiotic and biotic stresses (Kumar et al., 2013). Losses due to abiotic and biotic stresses are as high as of 28% and 52%, respectively, in lentil growing areas leading to huge economic losses (Kumar et al.,

2013). Often, lentil is exposed to abiotic (terminal drought, heat stress, cold stress, frost injuries, salinity, nutrient deficiency and nutrient toxicity) and biotic (fungal, bacterial, viral diseases, weeds and insect pests) stresses at various stages of crop growth. Among the biotic stresses, fungal diseases such as AB (caused by Ascochyta lentis), Botrytis grey mold (Botrytis cinereal, B. fabae), Anthracnose, (Colletotrichum lentis), Stemphylium blight (Stemphylium botryosum), Fusarium wilt (Fusarium oxysporum) and Rust (Uromyces viciae fabae) cause substantial yield losses (Chen et al., 2009).

2.2. Ascochyta blight

AB, a fungal disease caused by the necrotrophic fungus A. lentis Vassilievsky (teleomorph: Didymella lentis), is a serious constraint in lentil production ever since it was detected (Muehlbauer and Chen, 2007). AB has been detected in all major lentil producing countries around the world (Morrall and Sheppard, 1981; Kaiser and Hannan, 1986; Erskine et al., 1994; Nasir and Bretag, 1997a). In Australia, an estimated 49% of cultivation is affected by A. lentis infection in years with favorable conditions (Murray and Brennan, 2012). The seed harvested from AB-infected plants are poor in quality and appear small, discolored and shriveled, which makes them unmarketable (Gossen and Morrall, 1983; Sahi et al., 2018). Additionally, the subsequent use of infected seed serves as primary inoculum for the spread of the disease (Kaiser, 1997).

2.2.1. Disease cycle and infection process

The fungus may complete both sexual (teleomorph) and asexual (anamorph) stages in its life cycle (Kaiser, 1997) (Figure 2.1). Upon favorable climatic conditions, either of the stage can infect lentil and produce symptoms. The asexual stage produces a fruiting body known as a pycnidium within the lesions on the lentil plant, crop debris or seeds and releases pycnidiospores upon rain splash/heavy winds (Kaiser, 1997). The dispersed spores settle and germinate on healthy lentil plants under favorable conditions (temperatures within a range 20◦C-24◦C and availability of moisture) and spread the disease (Pedersen and Morrall, 1994).

Figure 2.1: Disease cycle of Ascochyta lentis as illustrated by Kylie Fowler (adapted from (Kaiser, 1997))

A. lentis is a heterothallic fungus with two different mating types (MAT 1-1 and MAT 1-2) (Kaiser, 1997). The presence of two mating types help to reproduce sexually and produce a teleomorph stage (Didymella lentis) (Galloway et al., 2004). The teleomorph produces ascospores within a psuedothecium which are carried by rain/wind onto new lentil plants for a fresh infection (Skiba and Pang, 2003). Ascospores survive on crop debris and stubble until next season (Kaiser and Hannan, 1986).

Roundhill et al. (1995) described the process of A. lentis infection and first appearance of lesions on plant surface in lentil. Aided by favourable conditions, conidia germinate within 6 hours post inoculation (hpi) on the leaf surface and produces germ tubes of varying lengths before appresorium formation within 10 hpi. Later, a penetration peg pierces the cuticle and pass through or between the epidermal cells and reach the mesophyll cells. Further, hyphae spread into and colonize the cells, which leads to necrosis of host tissue and subsequent production of pycnidia within 10-14 days post inoculation (dpi). Eventually, necrotic lesions speckled with black pycnidia spread across stems, pods and seeds causing the plant to collapse (Morrall and Sheppard, 1981).

2.2.2. Disease management

In severe conditions, AB infected crops may lose up to 70% grain yield (Gossen and Morrall, 1983), leading to estimated potential losses of up to $16.2 million AUD annually (Murray and Brennan, 2012). Although the losses cannot be completely eliminated, preventive and control measures can minimize losses significantly (Murray and Brennan, 2012). The best possible management strategy to suppress disease infection and increase production efficiency includes a combination of cultural and chemical methods (Davidson and Kimber, 2007). Among cultural practices, removal of infested residue and stubble, paddock selection, use of disease-free seed and crop rotations with non-host crops are suggested to reduce the likelihood of primary infection. The use of resistant cultivars, seed treatment and application of foliar fungicides may reduce the disease establishment and spread.

Under Australian conditions, a three-year rotation between two lentil crops, with 250 metre separation from the adjacent past lentil paddocks, seed testing, weather forecast-based manipulation of sowing dates and fungicidal seed treatment and foliar sprays are recommended (Lindbeck et al., 2002; Materne, 2003; Hawthorne et al., 2015a). Current chemical control includes seed dressing with thiram® and foliar spraying (chlorothalonil® or mancozeb ®) at critical stages of crop development (seedling, canopy closure, flowering and podding) and especially before a potential rain event (Hawthorne et al., 2015a).

Although the use of chemicals is an immediate and effective measure, it is costly and not environmentally sustainable. Moreover, the ability of A. lentis to evolve and develop resistance to the application of fungicides such as Carbendazim makes control management even more complex (Lopez and Kay, 2017). In the near future, the events of fungicidal resistance are expected to be more frequent due to an increased reliance on fungicides to control AB. A possible solution to this situation could be strategising the foliar sprays in line with the epidemiological knowledge of the pathogen, otherwise known as creating a fungicide decision support system (FDSS) (Davidson and Kimber, 2007; Buchwaldt et al., 2018). This would help to direct the management efforts towards the vulnerable stages of the pathogen and thus control the disease more efficiently. In Canada, FDSS was reported to control ascochyta and anthracnose diseases in lentil, and 85% of the assessments demonstrated significant yield increase following FDSS (Buchwaldt et al., 2018). However, among all the control measures

available, host plant resistance has been a strong choice over the years among breeders and growers for its economical and effective management of AB (Davidson and Kimber, 2007; Tullu et al., 2010a; Rubiales and Fondevilla, 2012).

2.3. Resistance in L. culinaris to A. lentis

To increase and stabilise yields using host plant resistance, a well-planned plant breeding program is necessary that includes germplasm evaluation and introgression of resistance into adapted material (Muehlbauer and Kaiser, 1993; Mundt, 2014). Accordingly, several sources of resistance to AB have been reported within the cultivated germplasm (Table 2.1) and later deployed in the breeding programs globally along with other desirable traits. (Erskine et al., 1996; Vandenberg et al., 2002; Vandenberg et al., 2006; Pulse Australia, 2016; Rodda et al., 2017; Kumar et al., 2018a).

In Australia, the cultivation of lentil started in 1995 in an area of about 6000 ha in Yorke Peninsula, South Australia. Lentil breeding investment was subsequently conducted through the “Coordinated Improvement Program for Australian Lentil” (CIPAL) until 2006 when “Pulse Breeding Australia” (PBA) introduced a revised breeding program (GRDC, 2013). Utilising germplasm mainly sourced from the International Center for Agricultural Research in Dry Areas (ICARDA) and Canada, the first Australian-bred lentil cultivars were released in 2005 for commercial production (Table 2.2). The lentil winter cropping season in Australia generally has wet and cold temperatures, which are conducive to AB epidemics. Thus, breeding for AB resistance, along with other desirable traits, has been a major priority within the Australian breeding program (Rodda et al., 2017). Lentil cultivars Northfield (a pure line selection of ILL 5588), CDC Matador (a Canadian cultivar) and the Canadian landrace Indianhead are the major sources of AB resistance used in Australian cultivars. Cultivar PBA Jumbo2, which is resistant to highly aggressive isolates of A. lentis both in field and controlled conditions, has most likely obtained resistance from CIPAL205, one of the parent (Rodda et al., 2017)

Table 2.1: Some sources of resistance within the cultivated gene pool to Ascochyta lentis described in different lentil growing countries

S. No Resistant cultivar Country Reference 1 169, 170, 172, 173, 174, 186, 191, 192, 204, 209 and 210 India Singh et al. (1982) 2 Primera New Zealand Cromey et al. (1987) 3 Mansehra – 89 Pakistan Iqbal et al. (1990) 4 Talya – 2 Lebanon Abi-Antoun et al. (1990) 5 SS-159 and L-3328 India Sugha et al. (1991) 6 ILL 5588 Syria Erskine et al. (1996) 7 Precoz Canada Ahmed and Morrall (1996) 8 Indianhead, PI 339283, PR86-360 and PI 374118 Canada Andrahennadi et al. (1996) 9 ILL 7537, ILL 358 Australia Nasir and Bretag (1997b) 10 26 accessions (Pakistan (23), Greece (1) and Turkey (2) Australia Nasir and Bretag (1998) 11 35 accessions Ethiopia Ahmed and Beniwal (1991) 12 5473, 5490, 5499, 5569, 5545, 5547, 5548, and 5570 Pakistan Iqbal et al. (2010)

Table 2.2: Lentil cultivar collection of Australia and disease ratings at the time of release

Ascochyta blight S. No Cultivar Year released Pedigree Seedling Pod 1 Nugget 2006 Northfield/ILL5714 MR-MS MR-MS 2 Nipper 2008 Indianhead & Northfield MR-MS MR 3 Boomer 2008 Digger and Palouse MR MR-MS 4 PBA Flash 2009 ILL7685/Nugget MS MS 5 PBA Blitz 2010 Cumra/Indianhead/Cassab MR MR-MS 6 PBA Bounty 2010 ILL6788/Nugget MR-MS MS 7 PBA Jumbo 2010 Aldinga/CDC Matador MR-MS S 8 PBA Herald XT 2011 96-047L*99R060-EMSO2 R R 9 PBA Ace 2012 CIPAL501/96-047L*99R099 R R 10 PBA Bolt 2012 ILL7685/96-047L*99R060 MR R 11 PBA Hurricane XT 2013 PBA Flash/96-047L*99R060 MR R 12 PBA Jumbo2 2014 Boomer/CIPAL205 & CIPAL401 R R 13 PBA Greenfield 2014 PBA Flash/Boomer & CIPAL205 MR-MS MR-MS 14 PBA Giant 2014 Boomer and PBA Flash MR MS Note: cultivars in pedigree with bold font are the sources of AB resistance to the respective cultivar

R: Resistant; MR: Moderately Resistant; MS: Moderately Susceptible; S: Susceptible

2.3.1. Genetics of disease resistance

Mendelian studies on the inheritance of specific disease resistances were initially done via recombinant segregation analysis. Accordingly, the resistance to AB was proposed to be governed by either a single major dominant gene to underpin the seedling (AbR1) and pod (Ral2) resistance to AB in cultivar ILL 5588 (Andrahennadi, 1994; Ford et al., 1999); or by a single recessive gene conditioning pod resistance in cultivars Laird (ral1) (Tay, 1989; Ye et al., 2003) and Indianhead (ral2) (Andrahennadi, 1994; Chowdhury et al., 2001), respectively. In addition, two dominant and two recessive genes were proposed to confer seedling resistance in cultivars ILL 7537 and Indianhead, respectively (Nguyen et al., 2001; Ye et al., 2001). Likewise, in wild species, two complementary gene pairs in L. ervoides and L. odemensis controlled seedling resistance to AB (Ahmad et al., 1997). Seedling resistance to AB in wild progenitor L. orientalis was controlled by a single dominant gene (Ahmad et al., 1997) or two major genes (Ye et al., 2000).

With the advent of marker technology, many independent studies have demonstrated the quantitative nature of AB resistance both at seedling and podding stage (Table 2.3). Also, the position of the quantitative trait loci (QTL) (a section of genomic region which correlates with the phenotypic variation of a quantitative trait in a population) conferring resistance to AB at different host growth stages and in different genetic backgrounds were mapped on different chromosomal/linkage groups (LGs) (Rodda et al., 2017). Whilst the resistance may indeed be conferred by different genes, the uncertainty of determining this was compounded by a lack of common markers (Sudheesh et al., 2016a) and the use of different nomenclatures to label LGs (Sharpe et al., 2013). Nevertheless, a common SSR locus was reported to harbor three QTLs identified in three independent studies (QTL5 on LG1, (Rubeena et al., 2006), QTL1 on LG1, (Gupta et al., 2012a), AB_NF1 on LG6, (Sudheesh et al., 2016a)).

Table 2.3: QTL's/genes conferring resistance to ascochyta blight (AB) resistance and corresponding markers

Phenotypic Mapping population Number of QTLs Marker types Reference variation %

Single dominant gene ILL 5588 × ILL 6002 RAPD 89 Ford et al. (1999) (AbR1) ILL 5588 × L692-16-1 2 RAPD, ISSR, RFLP and AFLP 36 Tar’an et al. (2002) ILL 5588 × ILL 7537 5 RAPD, AFLP and ISSR Up to 69 Rubeena et al. (2006) ILL 7537 ×ILL 6002 9 RAPD, AFLP and ISSR Up to 34 Rubeena et al. (2006) Eston × PI320937 1 AFLP, RAPD and SSR 41 Tullu et al. (2006b) Northfield × Digger (ILL 5722) 6 EST-SSR/SSR, ITAP, RAPD and ISSR Up to 61 Gupta et al. (2012a) Indianhead × Northfield 3 EST-SSR, SSR and SNP Up to 47 Sudheesh et al. (2016a) Indianhead × Digger (ILL 5722) 2 EST-SSR, SSR and SNP Up to 30 Sudheesh et al. (2016a) RAPD – Random amplification of polymorphic DNA; AFLP – Amplified fragment of length polymorphism; ISSR – Inter – simple sequence repeats; ITAP – Intron targeted amplified polymorphism; EST-SSR – Expressed sequence tags – simple sequence repeats; SSR – Simple sequence repeats; SNP – Single nucleotide polymorphism

2.3.2. Evidence of resistance breakdown

Host plant resistance to a particular pathogen enhances the agronomic performance of the plant and maintains consistency in yields during disease epidemics (Tullu et al., 2010a; Rubiales and Fondevilla, 2012). As a result, resistant cultivars are routinely preferred by farmers and are cultivated in larger areas compared to the cultivars that are susceptible. Nonetheless, this intensity may threaten the durability of the resistance through adaptive selection of more aggressive isolates of the pathogen (Pariaud et al., 2009). Incidentally, popular cultivars such as Northfield and Nipper released as resistant to AB in Australia have been revised as susceptible within 4 years after their commercial release and changes in aggressiveness of isolates within the A. lentis population was determined to have caused their demise (Davidson et al., 2016). A similar incidence of change in resistance response was reported in a Canadian cultivar Laird, which resulted in 50% yield loss (Morrall, 1997; Banniza and Vandenberg, 2006). Analysis of a historic collection of A. lentis recovered from Nipper since commercialization and until the first evidence of a susceptible reaction revealed that the isolates that were aggressive, particularly on Nipper, were detected more frequently after its cultivation (Davidson et al., 2016).

Although resistance status of cultivar Northfield (one of the parents of the cultivar Nipper) has been questioned previously by few isolates (Nasir and Bretag, 1997b), significant number of isolates were able to cause a disease in cultivar Northfield at around the time when Nipper was found susceptible (Davidson et al., 2016). This potentially confirms that the selective adaptation of the pathogen may extend to the cultivars that share common genetic background. Unlike, Northfield, cultivar Indianhead (the other parent of cultivar Nipper) showed a resistant reaction to the Nipper-virulent isolates (Davidson et al., 2016; Sudheesh et al., 2016a). However, some isolates collected recently showed a susceptible reaction in cultivars (PBA Jumbo and PBA Blitz) with Indianhead pedigree (Davidson et al., 2016). In addition, some of these isolates were also able to overcome the resistance of cultivar Indianhead too (Dadu et al., 2018b). Therefore, widespread planting of cultivars with Indianhead heritage are likely also under selection pressure. Thus, suggesting a diversification of the Australian lentil industry particularly with narrow genetic base for resistance to AB.

2.4. Utilisation of exotic germplasm in widening genetic diversity

With the increase in the frequency of unpredictable climatic events, climate resilient crops are sought to sustain agricultural production (McCouch et al., 2013). Breeding programs have been focused on high yielding cultivars for the past years and decades, which has increased productivity. However, a consequence has been a dramatic decrease of diversity among the elite cultivars of many crops and thus a narrow genetic base for breeding, which is vulnerable to various abiotic and biotic stresses (Palmgren et al., 2015). Unsurprisingly, an estimated ~75% of the genetic diversity within agricultural crops has already been lost since the 1990’s, and an estimated 60000 plant species will disappear by 2025 (Moreta et al., 2013). Meanwhile, continuous development of new high yielding climate resilient lentil cultivars is required to meet the food demand of the growing human population. This objective could be achieved by engaging the largely untapped germplasm (wild relatives and diverse landrace population) in the breeding programs to potentially regain the lost variation and increase genetic gain (Wong et al., 2015; Caldu-Primo et al., 2017; Thomas et al., 2017).

2.4.1. Value of wild relatives and landraces of crops

Wild relatives of many crops have been studied for the presence of genes that improve quality, yield, and resistance to various abiotic and biotic stresses (Brummer et al., 2011; Dempewolf et al., 2014). Sources of resistance to Phytophthora infestans (the cause of the famous potato Irish famine) and Puccinia graminis ssp. graminis (the cause of wheat stem rust) were discovered in wild relatives of potato (Solanum demissum) and wheat (Aegilops tauschii), respectively (Prescott-Allen and Prescott-Allen, 1986; Kilian et al., 2010). A recent survey conducted by Dempewolf et al. (2017) reported a total of 4157 confirmed or potential uses of wild relatives (spread across 127 different crops) in plant breeding. As observed with wild relatives of various crops, several useful traits were identified also from landraces, more closely related to the cultigen, and these have been deployed in breeding programs globally. For example: Sources of resistance to Blumeria graminis f.sp. tritici and Puccinia graminis Pers. f. sp. tritici (causal agents of powdery mildew and stem rust, respectively) were identified within the wheat landrace germplasm (Bhullar et al., 2009; Endresen et al., 2012).

Nearly 40% of the available genetic diversity was lost during lentil domestication (Alo et al., 2011) and a narrow genetic base of cultivars resulted within breeding programs due to the reliance on a small number of initial genotypes (Ford et al., 1997; Lombardi et al., 2014; Khazaei et al., 2016). Therefore, a broader genetic diversity is required for improvement of several adaptive traits (Singh et al., 2018). Incidentally, the potential of Lens germplasm (including wild species and landraces) for various morphological and adaptive traits has been reported (Tullu et al., 2010b; Singh et al., 2018). For example: exotic germplasm of lentil exhibited a greater variation for morphological, yield and yield contributing traits (Erskine and Choudhary, 1986; Tullu et al., 2001; Zaccardelli et al., 2012; Singh et al., 2014; Gaad et al., 2018). Likewise, a number of sources of tolerance to abiotic stresses such as drought (Gupta and Sharma, 2006; Gorim and Vandenberg, 2017), cold (Hamdi et al., 1996), heat (Choudhury et al., 2012; Delahunty et al., 2015; Bhandari et al., 2016) and salinity (Kumawat et al., 2017) have been identified through phenotyping of Lens germplasm. Similarly, sources of resistance to various diseases including fusarium wilt (Bayaa et al., 1995; Gupta and Sharma, 2006), anthracnose (Buchwaldt et al., 2004; Tullu et al., 2006a; Vail et al., 2012), stemphylium blight (Podder et al., 2013), rust (Gupta and Sharma, 2006; Singh et al., 2014), and the parasitic weed Orobanche (Fernández‐Aparicio et al., 2008; Fernández‐Aparicio et al., 2009) were reported within the Lens germplasm. Meanwhile, efforts to enhance the biofortification potential of cultivated lentil has been expedited through the identification of mineral rich germplasm in wild lentil (Kumar et al., 2018c).

Bayaa et al. (1994) was first to evaluate ICARDA’s wild germplasm of lentil for resistance to AB. Although the majority of the genotypes were found to be highly susceptible, considerable variation was revealed for AB resistance in the wild population. Genotypes belonging to L. orientalis, L. nigricans, L. ervoides and L. odemensis had resistance to AB and one genotype of L. ervoides (ILWL 138) had combined resistance to fusarium wilt and AB. Phenotypic variability for AB in wild lentil population was further assessed by Tullu et al. (2010a). Meanwhile, in 1998, 488 L. culinaris accessions collected from 25 countries and screened with three Australian isolates, revealed 26 resistant accessions with a potential to be deployed into the breeding program Nasir and Bretag (1998). More recently, twelve genotypes resistant to Nipper-virulent isolates of A. lentis were reported within the wild germplasm (Dadu et al., 2017).

2.4.2. Evaluation and selection of vast germplasm collections for beneficial traits

Although there is much evidence for high genetic diversity and potential uses of exotic germplasm, wild species and landraces are still underutilised in breeding programs (Prohens et al., 2017) due to lack of efficient strategies to select and introgress the trait of interest into the elite genepool (Wang et al., 2017). At the same time, the germplasm collections of various crops continue to grow in systematic germplasm collection programs globally, with approximately 7.4 million accessions being conserved at various genebanks worldwide (Wang et al., 2017). Lentil germplasm alone comprises approximately 43,200 accessions (Global Crop Diversity Trust, 2008) and mining for the adaptive traits/rare alleles from such a large collection is resource intensive. Therefore, considering economic feasibilities, evaluating a sub set representing maximum genetic diversity of the total collection would be beneficial (Glaszmann et al., 2010).

Core collection (Brown, 1989) and mini-core collections (Upadhyaya and Ortiz, 2001) that comprise 10% and 1% of the total collections, respectively, are constructed based on the origin, geographical distribution and genetic characterisation that is valuable to consumers. The core collection approach has shown great promise in major crops including rice (Kim et al., 2016), wheat (Sehgal et al., 2015), barley (Muñoz-Amatriaín et al., 2014), soybean (Qiu et al., 2013) and chickpea (Upadhyaya and Ortiz, 2001). In lentil, a core set with 96 accessions was developed based on the quantitative and qualitative variables and is suggested as a starting material for the targeted broadening of the genetic base of cultivated lentil (Singh et al., 2014).

However, as Bari et al. (2012) suggested, rare traits such as disease resistance may not be captured in fixed representative subsets (core collections) since these are built without bias towards a specific allele/s. Focused identification of germplasm strategy (FIGS) was proposed by Mackay (1990) and developed by ICARDA as a response to the conundrum. FIGS approach is based on the premise that the adaptive traits displayed by the germplasm are resultant of the selection pressures of the environment in which the germplasm was developed (Street et al., 2016). The FIGS approach is rapid and productive and is preferred to maximise the likelihood of the specific traits of interest in the targeted subset. As FIGS explores the trait-collection site- environmental relationship to capture the material with desirable traits, only geo-referenced germplasm is suitable for such studies, wherein a long history of environmental profile is

available. With the advances in global positioning system (GPS) and geographic information system (GIS) technology, coordinates and environmental profiles of collection sites were determined with high accuracy (Street et al., 2016).

In practice, the FIGS approach follows two pathways to identify desirable traits. One way to detect the germplasm with specific traits of interest is by simply filtering the collection sites using environmental variables such as climatic data that mimic the selection pressures for a given trait. For example: Using climatic and topographical filters, a population of 16000 accessions was reduced to a workable set of 534 accessions within which nine Sunn pest resistant accessions were detected in wheat (El Bouhssini et al., 2009). Based on historic trait - evaluation data, the second method uses statistical models to identify a putative ‘trait – environment’ relationship and predict the collection sites with the highest likelihood for the best-bet accessions to contain that trait. Using the collection site information of 400 bread wheat landraces with known resistance to powdery mildew, a total of 212 resistant landraces were identified from a population of 16000 unevaluated accessions (Bhullar et al., 2009). Thus far, FIGS has been deployed successfully to identify several rare traits in wheat such as resistances to powdery mildew (Bhullar et al., 2009; Bhullar et al., 2010), sunn pest (El Bouhssini et al., 2009; El Bouhssini et al., 2013), stem rust (Bari et al., 2012), Russian wheat aphid (El Bouhssini et al., 2011; El Bouhssini et al., 2013), Hessian fly (El Bouhssini et al., 2013) and stripe rust (Bari et al., 2014). More recently, FIGS has been applied to select the accessions with drought tolerance in faba bean (Khazaei et al., 2013).

2.5. Increasing the reliability of resistance through understanding the mechanisms of plant pathogen defence

Resistance breeding is often challenging due to the dynamic and complex nature of the plant pathogen interactions. Breeding of disease-resistant cultivars is either based on the presence of highly specific, complete resistance (qualitative) or non-specific, incomplete resistance (quantitative). Qualitative resistance controlled by a single dominant gene is often short-lived, while the resistance regulated by the cumulative effect of multiple genes is relatively active for a longer period of time (Ayliffe et al., 2008). Nevertheless, the effectiveness of both types of resistance relies on the underpinning defence mechanisms.

Knowledge of such mechanisms may help to increase the efficiency of selective breeding programs through targeted selection of novel resistance alleles/genes.

2.5.1. Plant-pathogen interactions

Several studies have detailed the mechanisms underpinning disease resistance in plants as reviewed by (Jones and Dangl, 2006). Pathogen infection on hosts provoke activation of an inter-connected network of constitutive/preformed and inducible responses to help plants defend themselves against pathogens as briefly described below:

Prior to the pathogen’s entry into the host tissue, the pre-formed defence barriers of the host plant including both physiological/structural (vertical leaf attitude, leaf pubescence, stomatal openings, waxing on the leaf surface, cuticle, and cell wall) and biochemical features (secondary metabolites/anti-microbial compounds such as constitutive phytoanticipins and inducible phytoalexins) determine the pathogen‘s establishment on the host surface (Dixon, 2001; Garcion et al., 2007). Either instantaneously or simultaneously, inducible defence responses within the host are activated upon the recognition of elicitors released by the pathogen, often referred to as pathogen associated molecular patterns (PAMP) (Nürnberger et al., 2004). The recognition of PAMPs by membrane-associated conserved pattern recognition receptors (PRRs) on the host triggers the first line of defence responses in the host known as PAMP triggered immunity (PTI). To overcome PTI, pathogens release effector proteins/avirulence proteins (encoded by Avr gene) into plant cells. Recognition of these effectors by a corresponding resistance (R) protein (encoded by a R gene) triggers a second line of defence system in plants termed as effector triggered immunity (ETI), which determines further resistance to the pathogen (Wu et al., 2014).

The major defence responses include elevation of cytoplasmic Ca2+ (Nicaise et al., 2009), accumulation of reactive oxygen species (ROS) such as superoxide (O-2) and hydrogen

peroxide (H2O2) (Torres et al., 2006), callose deposition (Luna et al., 2011), phenolic based lignification (Huckelhoven, 2007), cell wall strengthening (Huckelhoven, 2007), synthesis of pathogenesis related (PR) proteins and transcription of defence-related genes (Bindschedler et al., 2006; Hancock et al., 2006). Both, PTI and ETI may use common signaling networks to transfer the signals to the nucleus and thereby activate these defence mechanisms (Tsuda et al.,

2008). The hormonal signals activated after pathogen infection include mainly salicylic acid (SA), jasmonic acid (JA), abscisic acid (ABA) and ethylene (ET) (Pieterse et al., 2012). They activate both local defence responses and a broad-spectrum defence responses such as systemic acquired resistance (SAR) and induced systemic resistance (ISR) away from the site of infection (Durrant and Dong, 2004; Conrath et al., 2006).

2.5.2. Key defence related responses involved in the lentil – A. lentis pathosystem

Until recently, lentil-A. lentis interactions were poorly understood. However, recent advancements in histopathology and gene expression studies have revealed the complex defence mechanisms underlying lentil-A. lentis interactions to some extent. Defence mechanisms in lentil are featured by the presence of preformed barriers and simultaneous activation of physiological, biochemical and molecular responses. Integration of the results gathered from different studies was used to construct a hypothetical model representing the host defence response system within the lentil-A. lentis pathosystem as described below (Figure 2.2).

2.5.2.1. Recognition and structural defence responses

In lentil, defence mechanisms were activated as soon as 2 hpi following the recognition of A. lentis through the PRRs. The cluster of PRRs included leucine – rich repeat receptor kinase (LRR-RK), calmodulin domain protein kinase (CDPK), serine threonine protein kinase (STPK), NIMA – related kinase 6 (NEK6), receptor like kinase (RLK), lectin s-receptor-like serine threonine protein kinase (LECRK), EFR3 receptor kinase (EFR3), BR11-associated receptor kinase (BAK1) and wall associated protein kinase 1 (WAK1) (Mustafa et al., 2009; Sari, 2014; Khorramdelazad et al., 2018). The presence of A. lentis was communicated through MAPK signal transducers such as MEK2, MAPKL, MAPKK and MAPK-ntf6 (Sari, 2014). Alerted by the signalling, lentil strengthened structural barriers to cease fungal development through the activation of xyloglucan endotransglucosylase / hydrolase (XTH), laccase diphenol oxidase (PPOI), Exocyst subunit 70A1 family protein (Exo70A1) Delta (12)-FAD, cellulase synthase (CESA), xyloglucan glycotransferase (CSLC6) and callose synthase (CALS) genes involved in cell wall restructuring and papillae formation (Sari, 2014; Khorramdelazad et al., 2018).

Figure 2.2: A hypothetical model of the molecular lentil defence response system to A. lentis. Adapted from Khorramdelazad et al. (2018)

These results further validated the microscopic evidence of cytoplasmic aggregation and papillae formation (Roundhill et al., 1995; Sambasivam et al., 2016; Sari et al., 2017). However, as observed in the secretome of Ascochyta rabiei (the causal agent of AB of chickpea), A. lentis is assumed to release cell wall degrading enzymes (CWDE) to break- through the physical barriers (Fondevilla et al., 2015). Lentil counters the action of pathogen induced CWDE through the release of enzymes such as subtilisin inhibitor, plant invertase pectin methylesterase inhibitor (PMEI), polygalacturonase inhibitor (PGIP) and auxin – repressed protein (ARP) that limit the potential of fungal CWDE (Sambasivam et al., 2016; Khorramdelazad et al., 2018).

2.5.2.2. Biochemical defence responses and oxidative burst

A. lentis also generates phytotoxins such as lentisone, tyrosol and/or pseurotin A to penetrate the host cells (Andolfi et al., 2013). Consequently, this may prompt the lentil immunity system through phytohormone signalling to synthesise PR proteins (involved in permeabilization of fungal membranes and reduction in hyphal growth) to restrict the fungal growth within the tissue. Elevated transcript levels of PR proteins such as PR-1, 1a, PR-2, PR- 3, PR-4, 4a, PR-5, PR-9, PR-10, 10a, thaumatin-like protein, hevein-like protein and defensin- like protein were found within the ascochyta infected lentil plants (Mustafa et al., 2009; Vaghefi et al., 2013; Sari, 2014; Ford et al., 2017; Sari et al., 2017; Khorramdelazad et al., 2018). Additionally, lentil was shown to synthesise anti-microbial compounds such as phytoalexins, to counter the fungal penetration. Enzymes, Chalcone reductase (CHR) and chalcone synthase (CHS) involved in the production of phytoalexin precursors were upregulated in the lentil cultivar ILL 7537 in response to A. lentis infection (Sambasivam, 2011). As a consequence of the faster kinetics and spatial-temporal expression of these defence responses, lentil may effectively restrict the establishment and development of A. lentis through reduced spore germination percentage, shortened germ tubes and delayed appresorium formation (Sambasivam et al., 2016; Sari et al., 2017; Dadu et al., 2018a).

Simultaneously, A. lentis infection also induces lentil to produce ROS such as H2O2 and - O2 within the apoplast (Sambasivam et al., 2016; Dadu et al., 2018a). Histopathological observations were further supported by the elevated levels of genes such as superoxide

dismutase (SOD), lipoxygenase (LOX), glutathione s transferase (GST), 6 - phosphogluconate dehydrogenase (6PGDH), NADH dehydrogenase and copper containing amine oxidase (CuAO). These were associated with pathogen-induced oxidative burst in previous studies (Mustafa et al., 2009; Sambasivam, 2011). However, like A. rabiei, the A. lentis secretome is expected to release proteins with oxidoreductase activity that are used to detoxify the host- generated oxidative burst (Fondevilla et al., 2015; Verma et al., 2016).

2.5.2.3. Effector triggered immunity and programmed cell death (PCD)

To enforce additional pressure on the lentil defence system, A. lentis may secrete large numbers of host-specific effector proteins, which are proposed to induce cell death and thereby promote fungal growth (Sari et al., 2017; Sari et al., 2018). A number of necrotrophic effectors (orthologs to host-specific toxins within Stagonospora nodorum – wheat pathosystem) were identified within A. lentis secretome that are specific to lentil (Lichtenzveig et al., 2012). These effectors are then perceived by host-generated cognate R proteins and successful recognition triggers a second layer of defence response i.e., ETI. Several novel R proteins, mainly encoded with NLR genes along with several resistance gene analogues (RGAs) and a Cf-9 protein gene (member of LRR (R) gene family), were identified within the lentil – A. lentis interaction (Yaish et al., 2004; Sambasivam, 2011; Sari, 2014). Further, ubiquitination of the pathogen- induced effector proteins may inhibit the A. lentis spread within lentil (Mustafa et al., 2009).

The alterations caused due to A. lentis at the site of infection on the lentil leaflet results in an hypersensitive reaction (HR) or PCD, which was demonstrated through the expression of the genes related to cell death (Khorramdelazad et al., 2018). Cell death-associated genes that were reportedly up-regulated upon A. lentis infection included senescence associated gene (SAG), RING/U – box protein (RING/U - box) and a NB - ARC domain disease resistance protein (NB-ARC) (Khorramdelazad et al., 2018). An HR response is argued to contain the spread of a biotrophic pathogen by limiting nutrient access to the pathogen from the healthy cells. However, necrotrophic pathogens are suggested to utilise the cell death mechanism to proliferate and further colonize the plant tissues (Hammond-Kosack and Rudd, 2008; Grant and Jones, 2009). It is most likely that A. lentis hijacks the host generated cell death mechanism to promote its growth within the host tissue (Andolfi et al., 2013). Nonetheless, it has also been revealed that lentil resists the pathogen’s advance through the expression of genes that function

to inhibit programmed cell death. Sari (2014) reported genes such as mutation induced recessive alleles (mlo), poly polymerase like (PARP), autophagy-related (ATG)18g, ATG8d, cyclin-dependent kinase (CDK), PCD and constitutive subtilisin 3 (CSB3) playing a key role in cell death inhibition mechanism in A. lentis infected lentil cultivars.

2.5.2.4. Phytohormone based signaling and SAR

The efficiency of pathogen-induced defence responses depends on phytohormone signaling. Convincing evidence for the involvement of phytohormones such as SA, JA, ET and ABA are reported in lentil – A. lentis interactions (Vaghefi et al., 2013; Sari, 2014). Likewise, the induction of PR proteins including β-1,3-glucanse (Mustafa et al., 2009), PR-1 and PR-5 (Sari et al., 2017) suggested involvement of SA based signalling, the expression of PR-4, PR- 10 (Mustafa et al., 2009; Sambasivam, 2011) and allene oxidase cyclase (Sari et al., 2017) demonstrated the active participation of JA signalling in lentil in response to A. lentis infection. Furthermore, the involvement of ET in lentil – A. lentis interactions was indicated through the expression of genes associated with ET such as ethylene responsive transcription factor (ERF), ERF1B and gaga transcription factor (GAGA-TF) (Sari, 2014). ABA-mediated regulation of defence responses was shown through the expression of genes such as ABIlb and ABI5 (Sari, 2014). Then again, the expression of PR-4 and PR-10 was also shown to be dependent on an ABA pathway as demonstrated through over-expression in plants treated with exogenous application of ABA (Ford et al., 2017).

Crosstalk between the signaling pathways has also been reported to tailor the defence responses in lentil – A. lentis interactions. Antagonistic effects of JA on the SA pathway might have reduced levels of SA dependent PR proteins including PR-1 and PR-5, with a simultaneous rise in the JA dependent PR-4 protein in cultivar 964a-46 (Sari et al., 2017) whilst an antagonistic effect of ABA on JA/ET signaling pathways was suggested through the suppression of JA-associated PR-4a gene and ET related ERF and ERF1b genes (Sari, 2014). In contrast, the uniform expression of ET/JA-related genes, including defensin-like protein, indicated a synergistic relationship between JA and ET signaling pathways (Sari, 2014). In addition, the up-regulation of three genes including gibberellin signalling DELLA protein, gibberellin receptor and an E3 ubiquitin ligase indicated the active role of SAR signalling

within lentil-A. lentis interactions. SAR may provide a broad spectrum and long-lasting resistance against the subsequent invasion by the pathogen (Khorramdelazad et al., 2018).

2.6. Introgression of beneficial traits from exotic germplasm and broadening the genetic base of cultivated lentil

Controlled pollination is often chosen to introgress beneficial alleles identified within exotic germplasm into the cultivated gene pool. All of the wild species of lentil are considered cross-compatible with the cultivated species L. culinaris but with variable hybrid fertility due to pre- and post-fertilization barriers (Ladizinsky et al., 1988; Fratini et al., 2004; Gupta and Sharma, 2007). Fertile hybrids have been produced between genotypes within the primary gene pool using conventional crossing techniques (L. culinaris × L. orientalis) (Muehlbauer et al., 1989; Vandenberg and Slinkard, 1989; Vaillancourt and Slinkard, 1992; Fratini et al., 2004; Gupta and Sharma, 2007). Crosses between genotypes of L. culinaris and the secondary gene pool (L. odemensis) formed partial fertile hybrids due to reduced pollen fertility and chromosomal aberrations (Ahmad et al., 1997; Fratini et al., 2004; Gupta and Sharma, 2007). As expected with distant crosses, aborted pods were produced as a result of embryo abortion in the interspecific hybrids developed between the crosses involving L. culinaris × L. ervoides or L. nigricans belonging to the most distant tertiary and quaternary gene pool species (Abbo and Ladizinsky, 1991; 1994; Gupta and Sharma, 2007).

Nonetheless, an array of methods has been developed to overcome pre and post fertilization barriers and produce successful hybrids (Cohen et al., 1984; Liu et al., 2005; Van de Wiel et al., 2010). In lentil, viable hybrids were produced between L. culinaris x L. ervoides or L. nigricans) using embryo rescue protocols (Cohen et al., 1984; Fratini and Ruiz, 2006; Fiala et al., 2009; Yuan et al., 2011; Saha et al., 2014). An ovule rescue protocol was applied to overcome crossing incompatibilities between L. culinaris and L. tomentosus (Suvorova, 2014). Furthermore, fertile hybrids were also produced between L. culinaris and L. odemensis, L. ervoides or L. nigricans after the interspecific crosses were aided by the application of gibberellic acid (GA3) post pollination (Ahmad et al., 1995; Gupta and Sharma, 2007). Meanwhile, Gulati et al. (2001) used a micro-grafting approach to produce hybrids between different genotypes of L. culinaris. Later, Yuan et al. (2011) proposed a fusion of embryo

rescue method and micrografting to improve the rooting of the rescued embryo hybrids. They used regenerated shoots of all six Lens species as scions and grafted these scions onto the rootstocks of faba bean to produce efficient interspecific hybrids. A similar approach was used to produce successful interspecific hybrids between L. culinaris and L. tomentosus, L. odemensis or L. lamottei (Saha et al., 2014).

Advanced interspecific hybrid material derived from crosses between L. culinaris and L. orientalis, L. odemensis, L. ervoides and L. lamottei revealed useful variations for agronomical traits (plant height, days to flowering, maturity), yield and yield related traits (number of branches / plant, number of pods / plant, seed yield / plant, biological yield / plant and harvest index) (Gupta and Sharma, 2007; Singh et al., 2013). Tullu et al. (2013) evaluated a recombinant inbred line (RIL) population obtained from a cross between L. culinaris and L. ervoides and substantial variation was revealed within the RIL population for almost 18 agronomically important traits, including yield related traits and resistance to aggressive race 0 of C. lentis. More recently, evaluation of interspecific advanced lines derived from a cross between L. culinaris and L. orientalis revealed higher variability for various agro- morphological traits and resistance to rust (Uromyces viciae-fabae) (Kumari et al., 2018). In a separate study, Fiala et al. (2009) achieved successful transfer of anthracnose resistance genes from the anthracnose resistant L. ervoides accession L-01-827A to Eston, a cultivar of L. culinaris. RILs derived from this cross exhibited consistently higher resistance levels to both races of Colletotrichum lentis in field conditions and thus were backcrossed to the recessive parent to produce valuable breeding material (Vail et al., 2012). Similarly, resistance to AB has been introgressed from wild species including L. orientalis, L. odemensis and L. ervoides into the cultivated genepool (Ahmad et al., 1997; Ye et al., 2000).

Although useful variations have been reported through wide crosses, they are generally avoided by breeders due to linkage drag. Nevertheless, an assessment for linkage drag effect on the yield in the crosses developed between L. culinaris and L. ervoides showed that approximately 13% of backcrossed L. culinaris lines performed better than the cultivars for yield (Tullu et al., 2011). This study demonstrated that repeated backcrossing could produce a reduced impact from linkage drag within the interspecific bred cultivars. Alternatively, molecular markers may be used to select and deploy the trait of interest within the backcrosses

and thus reduce the impact of linkage drag and increase the efficiency of the introgression process (Wang et al., 2017).

2.7. Marker-assisted selection (MAS)

MAS employs DNA markers associated with traits of interest to select a plant for inclusion in a breeding program early in their development. By doing so, this approach greatly reduces the time required to identify cultivars which express desirable trait in a breeding program (Collard and Mackill, 2008). However, availability of appropriate markers linked as closely as possible to the desired gene(s), ideally situated on the gene sequence (perfect marker), is a critical point for successful MAS. Quantitative resistance is inherited by many genes with each contributing a minor effect, molecular markers should be a great benefit for selection (Miedaner and Korzun, 2012). Since the development of MAS, several successful selections based on marker information have been made in different crops for different traits that include morphologically important characters, biotic stress resistance, abiotic stress resistance, quality traits and yield attributes (Ragot et al., 2000; Baliyan et al., 2018; Hossain et al., 2018; Sundaram et al., 2018).

2.7.1. Status of MAS in lentil breeding for AB resistance

2.7.1.1. Construction of genetic linkage maps in lentil

Genetic linkage maps are valuable resources in genomic studies, which assist in the identification of the physical location of the gene effects or traits of interest and permit identification of markers tightly linked to the desirable traits for use as diagnostic tools in breeding programs. Both intra and interspecific populations were used in lentil to construct linkage maps, however, to achieve maximum polymorphism, linkage maps in lentil were initially constructed using interspecific mapping populations. The reported interspecific linkage maps are mostly derived from crosses between L. orientalis and L. culinaris. The first genetic linkage map was constructed using allozyme and morphological markers within the mapping populations derived from crosses between L. culinaris and L. orientalis (Zamir and Ladizinsky, 1984). The allozyme markers segregated in co-dominant fashion and along with morphological marker (epicotyl color) reported two LGs with 5 loci. In subsequent studies, the

linkage map was further strengthened by using additional number of allozyme and morphological markers (Tadmor et al., 1987; Muehlbauer et al., 1989). However, because of their limited availability and dependency on the environment, allozyme and morphological markers produced low-density linkage maps.

The discovery of DNA based markers led to an increase in the number of available markers in lentil, which accelerated the development of genetic linkage maps. The DNA markers were more polymorphic compared to morphological and allozyme markers (Paterson et al., 1991). Subsequently, the first DNA marker based genetic linkage map was developed using RFLP markers (Havey and Muehlbauer, 1989b). This map was further expanded by using an additional number of restriction fragment length polymorphism (RFLP) markers, which discerned ten LGs and stretched 560 centiMorgan (cM) in length (Weeden et al., 1992). Tahir et al. (1993) then reviewed all the earlier maps and constructed a preliminary linkage map of Lens with ten LGs. However, the first comprehensive linkage map including polymerase chain reaction (PCR)-based markers (random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP)) along with RFLP and morphological markers was developed using a RIL population derived from a cross between L. culinaris and L. orientalis. Eujayl et al. (1998) reported a dense linkage map with seven LGs covering 1073 cM of the lentil genome.

With the increase in the availability of PCR-based markers that could detect higher amounts of polymorphism between parental and recombinant genomes, the first intraspecific linkage map was developed using a RIL population derived from cultivars ILL 5588 and ILL 7537 (Rubeena et al., 2003). Duran et al. (2004) employed highly polymorphic and abundant SSR markers for the first time in lentil along with RAPD, AFLP, inter simple sequence repeats (ISSR) and morphological loci to produce a highly dense interspecific linkage map, which spanned a distance of 2172.4 cM. Hamwieh et al. (2005) developed a set of new simple sequence repeats (SSR) markers and improved the map of Eujayl et al. (1998) by adding 41 new SSR and 45 AFLP markers, that mapped on to 14 LGs. This was followed by the development of more linkage maps in lentil (Tullu et al., 2008; Kahraman and Muehlbauer, 2010; Saha et al., 2010; De la Puente et al., 2012; Gupta et al., 2012b). Efforts were also made to develop linkage maps using the synteny between lentil and the model species Medicago truncatula to produce gene-based markers. Accordingly, 79 cross genera intron targeted

amplified polymorphism (ITAP) gene based markers along with SSR markers were used to develop the first synteny-based intraspecific linkage map in lentil (Phan et al., 2007). The map was further extended by employing 15 new expressed sequence tag - simple sequence repeats (EST-SSR/SSR) markers generated from M. truncatula EST database and with a total of 196 markers, and spread across 11 LGs and 1156.4 cM (Gupta et al., 2012a).

The introduction of next generation sequencing technologies (NGS) permitted the sequencing of the lentil transcriptome and discovery of functional markers such as SSRs in large scale. Kaur et al. (2011) generated a large collection of EST-SSR markers from the sequence analysis of lentil genotypes (Northfield, ILL2024, ILL 7537, ILL6788, Digger, Indianhead) using Roche 454 GS-FLX Titanium technology. Likewise, Verma et al. (2013) assembled a de novo transcriptome assembly of lentil using short reads generated by Illumina GA II and detected large scale functional SSR markers. Subsequently dense linkage maps were developed by deploying these informative SSR markers (Verma et al., 2015; Sudheesh et al., 2016a).

More recently, PCR-based markers were quite rapidly replaced by DNA chip-based markers such as single nucleotide polymorphisms (SNPs), which represent common sequence differences between allelic forms of a gene. SNPs are abundant and ubiquitous in nature, thus enabling the development of dense linkage maps, high resolution QTL analysis and fine- mapping (Rafalski, 2002). A large number of high quality and informative SNP markers were generated in lentil using NGS and high throughput genotyping technologies (Fedoruk et al., 2013; Sharpe et al., 2013; Kaur et al., 2014; Temel et al., 2015; Ates et al., 2016; Aldemir et al., 2017; Ates et al., 2018a). These markers were then compiled along with other informative SSR markers to develop high density linkage maps in lentil (Fedoruk et al., 2013; Sharpe et al., 2013; Kaur et al., 2014; Temel et al., 2015; Ates et al., 2016; Sudheesh et al., 2016a; Aldemir et al., 2017; Ates et al., 2018a; Subedi et al., 2018). Using the advantages offered by NGS, a high density genetic map with 543 SNP markers was produced in a RIL population developed from a cross between two L. ervoides accessions (Bhadauria et al., 2017). The maps reported by Ates et al. (2016) and Aldemir et al. (2017) included the highest number of markers (1,784 and 4,177 diversity arrays technology (DArT) markers, respectively) within a lentil linkage map to date. Furthermore, the development of SNP markers led to the identification of common markers across the RIL populations derived from different crosses, and enabled the

construction of consensus linkage maps in lentil (Sudheesh et al., 2016a; Ates et al., 2018a). The most recent consensus map included a total of 9,793 DArT markers with seven LGs and 977.42 cM (Ates et al., 2018a). Consensus maps provide greater resolution in terms of genome coverage and enable detection of potential chromosomal rearrangements aiding in the draft lentil genome assembly (Sudheesh et al., 2016a).

2.7.1.2. Mapping QTLs conferring resistance to AB

QTL mapping provides a means to dissect genomic regions that control the phenotypic variation of a quantitative trait and thereby allow identification of markers tightly linked to the trait (Collard and Mackill, 2008). Using the above-mentioned maps as a reference, several closely linked markers to the genes of interest including AB resistance were identified with greater potential for MAS in lentil (Kumar et al., 2015; Rodda et al., 2017).

Prior to the introduction of molecular markers, an AB resistance locus was tagged using less effective morphological and isozyme markers. Resistance locus (Ral1) within cultivar ILL 5588 was found to be linked to morphological markers such as cotyledon colour (Yc, 10.5 cM) and seed coat spotting (Sakr, 1994) whereas a distant linkage was observed to an isozyme marker Aat-p (29 cM) (Andrahennadi, 1994). Meanwhile, another resistance locus (ral1) within cultivar Indianhead was flanked by an isozyme marker at 28 cM (Andrahennadi, 1994). However, the advance of DNA-based marker technology offered great potential for identifying markers closely linked to desirable traits in a more rapid and efficient way. RAPD markers were associated with another AB resistance locus (ral2) in cultivar Indianhead with the closest

marker (UBC2271290) at 14 cM (Andrahennadi, 1997). Ford et al. (1999) using bulk segregant analysis, identified RAPD markers, RV01 and RB18 flanking a major dominant AB resistance locus (AbR1) at a distance of 6 and 14 cM, respectively, on a chromosome of ILL 5588.

Likewise, Chowdhury et al. (2001) reported two RAPD markers, UBC2271290 and OPD-10870 flanking the resistance locus (ral2) of cultivar Indianhead at 12 and 16 cM, respectively. Later,

QTL analysis of an F2 mapping population (ILL 7537 × ILL 6002) revealed several markers flanking the QTLs conferring resistance to AB; however, an AFLP marker among them, C-

TTA/M-AC285 was found just 3.4 cM away from the resistant QTL6 (Rubeena et al., 2006).

The introduction of SSRs and SNPs enabled high resolution genetic mapping of AB resistance loci (Tullu et al., 2006b; Gupta et al., 2012b; Sudheesh et al., 2016a). Gupta et al. (2012a) reported eight markers (EST-SSR/SSR, ISSR, RAPD and ITAP) flanking QTLs conferring seedling and pod/maturity resistance. More recently, high resolution QTL analysis led to identification of AB resistance linked markers (SSR and SNP) within the cultivars Indianhead and Northfield (Sudheesh et al., 2016a). Among the markers identified, marker (SNP20005010) flanking QTL (AB_IH1) accurately predicted field resistance of 68 of the 79 genotypes and clearly differentiated resistance from susceptible genotypes.

Closely linked markers may be employed in a MAS program and further pyramiding of potentially different disease resistance genes into elite backgrounds will help new cultivars to sustain the multiple disease pressures and produce higher yields. For example: Tar'an et al. (2003) used a QTL pyramiding approach to combine resistance to AB and anthracnose in a CDC Robin × 964a-46 RIL population in a bid to develop cultivars with resistance to the two most devastating diseases of lentil. It was revealed that 82% of the lines that showed positive

for either or both markers (UBC2271290 and RB18680) were resistant to A. lentis, while susceptibility was observed in the 80% of the lines that lacked both the markers.

2.8. Transcriptome sequencing or genotyping-by-sequencing through transcriptomics (GBS-t)

The advances in NGS and genotyping technologies in the past two decades have increased our understanding of several traits in crops (Varshney et al., 2015). Genome sequences of various food legumes are currently available. For example, soybean (Schmutz et al., 2010), chickpea (Varshney et al., 2013), pigeon pea (Varshney et al., 2012), common bean (Schmutz et al., 2014) and adzuki bean (Yang et al., 2015) have been released for use, while work is in progress to determine the genetic code of the other remaining legumes by respective international genome sequence consortia (Varshney et al., 2018). Resequencing of several germplasm lines in food legumes led to the identification of large number of genome wide variants (Kumar et al., 2016; Thudi et al., 2016). Discovery of these high-density markers assisted in the construction of high-density linkage maps, high-resolution QTL analysis, fine mapping and candidate gene identification. For example, the large-scale identification of SNPs

has resulted in mapping of several QTLs and trait linked markers in food legumes (Jaganathan et al., 2015; Kale et al., 2015; Valdisser et al., 2017).

Nevertheless, in crops with no genome sequence available and potentially large genome size, genome complexity reduction approaches such as genotyping-by-sequencing - restriction site associated DNA (GBS-RAD) (Poland et al., 2012) and transcriptome sequencing or genotyping-by-sequencing through transcriptomics (Wang et al., 2009; Sharpe et al., 2013; Kaur et al., 2014) were used to enable the reduction of the genome size and assist the high throughput sequencing. Consequently, large sequence data sets and molecular markers including SSRs and SNPs have been made available in crop species by aligning the sequence data to the de novo reference assemblies. However, among the two, transcriptome sequencing has been widely used recently because of the following reasons: 1) ability to access the information of an annotation of the genomic region 2) less laborious 3) cost effective and 4) less missing data compared to GBS – RAD (Malmberg et al., 2018). GBS-t has been applied for genotyping in crops with large genomes such as wheat (Miller et al., 2016), maize (Hansey et al., 2012), rapeseed (Trick et al., 2009), field pea (Duarte et al., 2014) and faba bean (Kaur et al., 2012).

Similarly, lentil possess a large genome of approximately 4 Gbp because of which, it has long been considered an ‘orphan’ crop in terms of available genomic resources (Arumuganathan and Earle, 1991). Nevertheless, both GBS-RAD (Ates et al., 2016; Aldemir et al., 2017; Bhadauria et al., 2017; Ates et al., 2018a) and GBS-t (Sharpe et al., 2013; Kaur et al., 2014) have been implemented in lentil to produce large sets of genomic resources including markers (SSRs and SNPs) and genetic linkage maps to assist in trait dissection. Using GBS-t, QTLs responsible for resistance to A. lentis (Sudheesh et al., 2016a), tolerance to B toxicity (Kaur et al., 2014), flowering time (Fedoruk et al., 2013) and seed quality related traits (Fedoruk et al., 2013) have been tagged with closely linked markers in various lentil cross combinations. Moreover, comprehensive transcriptome reference assemblies for lentil have enabled the identification of genes of key metabolic pathways (Sharpe et al., 2013; Kaur et al., 2014; Sudheesh et al., 2016a). Candidate genes for B toxicity tolerance and flowering time were identified by comparing the sequences underlying the flanking markers of both traits with known genes from model species M. truncatula through basic local alignment-nucleotide (BLASTN) similarity search (Sudheesh et al., 2016b).

2.9. Conclusion and aims of the research

Resistance to A. lentis in cultivated lentil is eroding due to increased aggressiveness in the pathogen population. New resistance sources are required, and these would require characterisation for future potential use in selective breeding. Therefore, the aims of this thesis are

1. To evaluate landraces and wild relatives of the cultivated lentil species against recently evolved highly aggressive Australian A. lentis isolates for novel resistance sources; 2. To observe the microscopic and biochemical histopathological evidence, and characterise the functional defence responses underpinning the novel resistant sources against A. lentis; 3. To understand the genetics of AB resistance within the novel resistance source ILWL 180 by developing an interspecific hybrid population, to further investigate the variation for agro-morphological traits; 4. To generate a genetic linkage map using SNP markers and identify the physical location of the QTL(s) regulating the genomic regions controlling AB resistance.

Chapter 3 - Identification of novel Ascochyta lentis resistance in a global lentil collection using a focused identification of germplasm strategy (FIGS)

Manuscript published in Australasian Plant Pathology

https://link.springer.com/article/10.1007%2Fs13313-018-0603-7

3.1. Abstract

The Australian lentil breeding program is historically genetically narrow and recent reports suggests a loss of resistance to Ascochyta lentis within resistant cultivars such as Northfield and Nipper. There is evidence that the pathogen population is becoming more virulent on other widely adopted cultivars, thus there is an urgent need to identify novel resistance sources that may be transferred into the cultigen. To reduce the substantial economic and time commitment in this search, a focused identification of germplasm strategy (FIGS) was applied. This entailed exploring potential association between collection site environment and climatic conditions favouring A. lentis development, to predict regions imposing favourable selection towards A. lentis resistance. Accordingly, a subset of 87 landraces (originating from 16 countries) with highest likelihood for A. lentis resistance was selected from 4576 accessions held by the International Center for Agricultural Research in the Dry Areas (ICARDA), Morrocco collection. Significant variation for resistance was detected within the subset using completely randomised and replicated controlled climate bioassays with a highly virulent Australian A. lentis isolate, FT13037. Genotype IG 207 expressed the lowest percent area of symptomatic tissue and a further 12 genotypes demonstrated moderate resistance. Furthermore, IG 207 recorded lowest mean disease score against four other highly aggressive isolates and performed better than the currently used best resistance sources, ILL 7537 and Indianhead. In addition, delayed pre-penetration behaviour of isolate FT13038 on IG 207 leaflets indicated discovery through the FIGS technique of a novel and highly effective A. lentis resistance source.

Keywords: FIGS, Lentil, Landraces, Ascochyta lentis, Resistance, Histopathology

3.2. Introduction

Driven by dietary benefits and high export market returns, lentil cultivation has extended to non-native regions including Canada, Australia and Europe, growing six-fold in production from 0.85 Mt to 6.31 Mt within five decades (FAOSTAT, 2016). However, a significant reduction in lentil productivity (21%) was reported during the 2015-2016 season in Australia (FAOSTAT, 2016). One of the diseases responsible for the drop-in productivity was the fungal disease AB, caused by Ascochyta lentis. This disease is of a global concern (Muehlbauer and Chen, 2007), as it reduces yields and causes poor seed quality (Morrall and Sheppard, 1981; Gossen and Morrall, 1983). In Australia alone, approximately $15.3 million AUD is spent annually to control AB disease (Murray and Brennan, 2012).

In an effort to minimize the risk of AB infection, an integrated disease management approach is in place at the farm level that involves a combination of best cultural practices, cultivation of resistant cultivars and chemical control (Tivoli et al., 2006). However, the use of fungicides is reported to drive selection and emergence of pathogen resistance, and resistance to the fungicide Carbendazim (methyl benzimidazole carbamate (MBC) Fungicide Resistance Action Committee group 1) was recently confirmed in the Australian A. lentis population (Lopez and Kay, 2017). Simultaneously, the mono-cropping of cultivars with poor rotation and/or cultural disease management strategies was proposed to have caused the selection of highly aggressive isolates within local populations (Davidson et al., 2016). Therefore, breeding for high yielding cultivars with novel resistance genes or gene combinations remains a priority for the sustainability of the lentil industry.

In this context, several sources of AB resistance were identified and deployed in breeding programs globally along with other desirable traits (Nasir and Bretag, 1998; Hussain et al., 2000; Iqbal et al., 2010; Dadu et al., 2017). Resistance to AB in Australia has been introgressed into elite backgrounds from the cultivars Northfield, a selection from International Center for Agricultural Research in the Dry Areas (ICARDA) breeding line ILL 5588 (Ali, 1995) and Indianhead (Ye et al., 2001). However, the resistance conditioned by the single dominant gene within Northfield, (Ford et al., 1999), has been compromised and this cultivar is now vulnerable to the majority of isolates within the 2013-14 population it was assessed

against (Davidson et al., 2016). Although the resistance in Indianhead is still intact (Davidson et al., 2016), some isolates collected within Australia caused a susceptible disease reaction in previous controlled bioassays (Nguyen et al., 2001). Consequently, resistance of several Australian cultivars, such as Nipper, which is a descendant of Northfield and Indianhead, has also been compromised (Davidson et al., 2016). Therefore, the Australian lentil industry requires the urgent identification of alternative sources of AB resistance to diversify the genetic base of cultivars deployed by the industry.

The currently used AB resistance sources were discovered through screening of random selections from genetic resource collections (Iqbal et al., 2010; Dadu et al., 2017) or by screening a set of accessions collected from specific locations (Bayaa et al., 1994; Tullu et al., 2010a). Meanwhile, a large proportion of the global lentil collections, with potential useful variations, remain unexplored for potential A. lentis resistance. This presents an opportunity and a challenge since large genetic resource collections such as that housed at ICARDA comprises nearly 11000 lentil accessions. Mining these for novel and rare alleles, such as those contributing to AB resistance is resource intensive. As an interim measure, many studies have made use of core-collections, supposedly representative of the entire genome but these are without bias towards the alleles specifically sought (Tullu et al., 2001; Singh et al., 2014).

To tailor the search towards potentially appropriate genes, alleles and combination, a method was proposed by Mackay (1990) and developed by ICARDA termed focused identification of germplasm strategy (FIGS) (Mackay, 1990; Street et al., 2008). FIGS is based on the premise that the emergence of specific adaptive traits within in situ populations is determined by the selection pressures present in the environment in which the population is grown, given that these factors influence gene flow, and thus spatial/geographic differentiation (Vavilov, 1957; Lin et al., 1975). Thus, if a relationship exists between environmental factors and adaptive traits, it can be used to predict geographic locations where specific traits are likely to emerge. In practice, the FIGS approach uses environmental variables, such as climatic data, to filter germplasm collections sites for probable locations where a selection pressure is likely to have occurred for a given trait (e.g. El Bouhssini et al. (2009)) or uses historic evaluation and site environmental data to develop statistical models that assign collection sites with probabilities for the presence of a desired trait (e.g. Bari et al. (2014)).

FIGS has been used to identify novel sources of resistance in wheat to biotic stresses such as powdery mildew (Bhullar et al., 2009), sunn pest (El Bouhssini et al., 2009; El Bouhssini et al., 2013), stem rust (Bari et al., 2012), Russian wheat aphid (El Bouhssini et al., 2011; El Bouhssini et al., 2013), hessian fly (El Bouhssini et al., 2013) and stripe rust (Bari et al., 2014). FIGS was also used to identify drought adaptation traits for faba bean (Khazaei et al., 2013). These studies demonstrate that FIGS can be used to identify subsets of germplasm that contain novel trait variation for biotic and abiotic stresses from relatively large genetic bases. Thus, given that there are approximately 43,200 accessions in the global lentil collection (Global Crop Diversity Trust, 2008), FIGS may be an effective means to mine novel AB resistance sources from this collection.

Therefore, the objectives of the study were to 1) Create a subset of ICARDA lentil collection using FIGS approach to capture potentially novel resistance sources to an Australian- derived aggressive AB isolate (FT13037); 2) Determine the stability of the resistance(s) to a group of A. lentis isolates; and 3) Assess the physiological defence responses deployed by the most resistant identified source against A. lentis.

3.3. Materials and methods

3.3.1. FIGS and plant materials

The FIGS approach used to identify a best-bet subset of lentil landraces with potential resistance to AB involved filtering out landrace collection sites that are most likely to have, over the long-term, favoured development of the disease during the growing season. Environmental factors, defined as critical to AB infection, were temperature ranges from 15 to 25°C together with 24 h to 48 h of leaf wetness and 98% relative humidity (RH) (Kaiser et al., 1994; Pedersen and Morrall, 1994). The value of these parameters informed the construction of the site filter.

Since the approach focuses on identifying conditions favorable to disease development within the growing season of each collection site being considered, it necessitates long-term average estimates for 1) data of onset of the growing period, and 2) for the dates of the beginning of each crop development stage. An initial estimate for the onset of the growing

period was derived using a temperature and moisture limiting model described by De Pauw et al. (1996). Where possible, the estimates were checked against published data for long-term average planting dates and if necessary were adjusted. Crop development phases were estimated using a growing degree day model such as that described by Lancashire et al. (1991). The estimation of the long-term average crop development phase dates, as well as the filtering process required estimations for long-term average daily climatic data for each collection site. This data was generated by the ICARDA geo informatic systems (GIS) unit as described by (Biradar, 2016a; b).

The following step-wise FIGS methodology was developed and applied by Dr Kenneth Street, ICARDA, Morocco:

1. For each collection site, and for the period between 10% into the vegetative phase and to the end of pod filling, the number of days where the average daily temperatures fell between 15 and 20°C was counted. 2. Sites were discarded where the day count was zero. 3. For the remaining sites and for the same period in the growing season, the number of rainy days was counted. 4. Sites were discarded if there were no rainy days. 5. For each remaining site, the total precipitation that fell during the same period in the growing season was summed. 6. Sites were ranked from highest to lowest based on the precipitation data and the bottom 30% sites were discarded. 7. A Z-score was calculated for each of the elements determined in steps 1, 3 and 5 such that Z = (X - μ) / σ, where Z is the Z-score, X is the value of the element, μ is the population mean, and σ is the standard deviation. 8. Z-scores, calculated for each element of respective collection site were summed. 9. An index was developed using the combined Z-score and then the sites were ranked from highest to lowest based on the index. 10. Top 200 sites in the ranking were retained and we randomly chose one accession per site. 11. From the selected 200 accessions, we chose 100 accessions based on geolocation and elevation of site such that the geographic diversity is maximized.

12. Finally, 87 accessions were resourced from the ICARDA genebank in Rabat, Morocco.

Of the 87 accessions received from the ICARDA collection, 81 were available for screening after passing through quarantine at the Department of Economic Development, Jobs, Transport and Resources, (DEDJTR), Horsham, VIC, Australia. AB bioassays were conducted at Dookie Campus, University of Melbourne, VIC, Australia as follows: For each accession, six 10 cm pots (three inoculated and three non-inoculated), filled with pine bark potting mix (Australian Growing Solutions, Tyabb, VIC, Australia, 3913) were sown with three seeds and seedlings were maintained at 20 ± 0.5°C under 12 h/12 h day/night photoperiod and 60% RH in a Conviron growth cabinet. Pots were watered on every alternative day and fertilised weekly with Nitrosol, Amsgrow ® (4.5 mL/L) to induce germination and aid in plant development. Two cultivars, ILL 6002 (susceptible) and ILL 7537 (resistant) routinely used in such bioassays were included as experimental controls (Davidson et al., 2016; Dadu et al., 2017). Cultivars with known reaction (ILL 7537, Indianhead, ILL 6002 and Nipper) were included in differential host set (Sudheesh et al., 2016a; Dadu et al., 2017).

3.3.2. Fungal materials

Single-spored cultures of five isolates (FT13037, FT13038, FT15160, FT16112 and FT16299-2) of A. lentis with variable aggressiveness (Table 3.1) (Dadu et al., 2017) were supplied by the South Australian Research and Development Institute (SARDI). Aggressiveness of isolates FT13037 and FT13038 was confirmed earlier by Dadu et al. (2017) on a differential host set. Both isolates were aggressive and induced a highly susceptible reaction on genotypes ILL 6002 and Nipper.

The most aggressive isolate FT13037 was used to evaluate the FIGS set initially before assessing the stability of the identified resistant and moderately resistant accessions using isolates FT13038, FT15160, FT16112 and FT16299-2. Aggressive isolate FT13038 was chosen to evaluate the physiological defence responses because of its ability to discriminate resistance (ILL 7537) from susceptibility (ILL 6002 and Nipper). These isolates were sub- cultured on potato dextrose agar media (PDA) plates and were incubated for 14 days at 22°C, 12 h/12 h dark/light cycle under near Ultra Violet (UV) light (PHILIPS BLB/18W).

Table 3.1: Details of Ascochyta lentis isolates (including the lentil cultivar from which they were collected, location and year of collection) used in the study

Lentil - Location in Year Isolate Nature of isolate Comments cultivar Australia collected Urania, South FT13037 Aggressive Flash 2013 (Dadu et al., 2017) Australia (SA)

FT13038 Aggressive Cumra Urania, SA 2013 (Dadu et al., 2017)

Aggressive on FT15160 Aggressive Nipper Laura, SA 2015 Nipper and Cumra Aggressive only on FT16112 Non-aggressive Nugget Kadina, SA 2015 Cumra Aggressive on PBA PBA- FT16299-2 Aggressive Mallala, SA 2016 HurricaneXT and HurricaneXT Indianhead

3.3.3. Preparation of inoculum

Spore suspensions were prepared by harvesting pycnidiospores from fourteen-day-old fungal cultures by flooding with sterile water and gently scrapping the surface with a sterile glass rod (Ford et al., 1999; Davidson et al., 2016). Spores were separated from mycelium using a 250-mm pore sized sieve and concentrations were adjusted to 1×106 spores/mL using a haemocytometer. Prior to inoculation, two to three drops of Tween 20 (0.02% v/v) per 100 mL was added to the spore suspension as a surfactant.

3.3.4. Inoculation process

Fourteen-day-old seedlings were uniformly inoculated with a fine mist of inoculum using an air pressurized hand sprayer until run off occurred. The non-inoculated control pots were sprayed with water mixed with two to three drops of Tween 20 (0.02% v/v). To induce germination of the spores, soon after inoculation, pots were covered with an inverted paper cup coated with wax (In Hospitality, Shepparton, Australia) and placed randomly in plastic crates covered with wet hessian bags to maintain 100% RH (Chen and Muehlbauer, 2003; Dadu et al., 2017). After 48 h of inoculation, the paper cups were removed. To maintain adequate RH, the plants were misted with water three times a day until the disease assessment at 21 dpi.

3.3.5. Evaluation of FIGS set for AB resistance

Experiments were conducted in a completely randomised design with three replications and two treatments (inoculated and non-inoculated). Initially, 81 accessions were evaluated for AB resistance against the most aggressive isolate FT13037 (Dadu et al., 2017). Three pots per accession were inoculated with the isolate while the remaining three pots were sprayed with water.

3.3.6. Assessment of the stability of resistant accessions

The stability of resistance in the five resistant accessions (IG 207, IG 96, IG 1687, IG 7104 and IG 8550) to FT13037 was assessed against a group of A. lentis isolates (FT13038, FT15160, FT16112 and FT16299-2) with different levels of aggressiveness. At the same time, a host differential set (ILL 7537, Indianhead, Nipper and ILL 6002) was assessed for disease reaction against the same set of isolates. Each treatment combination (genotype × isolate) was replicated three times and was compared to an uninoculated control.

3.3.7. Disease assessment and data analysis

At 7, 14 and 21 days post inoculation (dpi), plants were assessed for A. lentis infection (Ford et al., 1999; Sambasivam et al., 2016) as the percent area of a plant that showed disease symptoms (% APD) (Davidson et al., 2016). Percent APD was calculated as the mean of percent leaf and stem area with AB lesions for the four nodes and internodes that were inoculated (Davidson et al., 2016). Later, percent APD was square root transformed to normalize the data. The data was analyzed using the GenStat software package (Version 16.1.0.10916 (64-bit edition), VSN International Limited, United Kingdom). Homogeneity of variances was tested by plotting a graph between residuals and fitted values. Data analysis was performed using a linear mixed model in GenStat’s Mixed Model (Residual maximum likelihood (REML)) procedure. Genotypes and dpi were considered as fixed factors and replicates as random factors. Likewise, stability experiment data was subjected to mixed model (REML) analysis with genotypes, isolates and dpi assigned as fixed and replicates as random factors. Differences among genotypes at each time point (7, 14 and 21 dpi) and for each isolate (FT13037, FT13038, FT15160, FT16112 and FT16299-2) were assessed based on Tukey’s

least significant difference at 95% confidence interval. Least significant difference (LSD) between the mean disease scores was used (Davidson et al., 2016; Sudheesh et al., 2016a) to place the genotype × isolate reaction into the following categories: Resistant (0 - 2.58% APD), Moderately Resistant (2.59 - 5.16% APD), Moderately Susceptible (5.17 - 7.74% APD) or Susceptible (>7.75% APD).

3.3.8. Evaluation of physiological defence responses by detached leaf assay

Seed of the most resistant FIGS landrace IG 207 and popular Australian cultivar Nipper were grown in a controlled growth chamber (20 ± 0.5°C, 60% RH and 12 h/12 h day/night photoperiod) to assess the physiological defence mechanism employed using the method described by Sambasivam et al. (2016). Fourteen days after emergence, 12 fully expanded leaflets from each accession were detached and sterilized in 70% ethanol for 1 min. One droplet (10 µl, 1 × 106 spores/mL) of aggressive isolate FT13038 (Dadu et al., 2017) was applied on the abaxial side of each leaflet. The leaflets were then placed in separate Petri dishes lined with moist filter paper. The sealed Petri dishes, were then incubated at 20◦C in the dark for 6, 12, 24 and 36 hours post inoculation (hpi).

After each incubation treatment, three leaflets per accession were collected and immersed in a leaf clearing solution of ethanol and glacial acetic acid (1:2 v/v) for a minimum of 36 h with one change in solution after 24 h to remove the chlorophyll. The cleared leaflets were stained with lacto-phenol cotton blue (Sigma Aldrich) and examined for spore germination, germ tube length and the percent of germinated spores that produced appressoria. In total, 100 spores (25 spores per corner of each leaflet) were assessed (Dadu et al., 2018a) using an Olympus BHC light microscope fitted with a Nikon digital slight DS-Fi2 camera to capture images.

Spores with germ tubes were considered as germinated. The length of germ tube was measured using Image Processing and Analysis in Java (ImageJ) software (version 1. 50i, Rueden et al. (2017)). Globular structures larger than the diameter of the germ tube and found at the tip of the germ tube were considered as appressoria (Dita et al., 2007). Data analysis was conducted in GenStat software package, (Version 16.1.0.10916, 64-bit edition, VSN International Limited, United Kingdom). Prior to the analysis, percent spore germination and

percent appressoria formation data were square root transformed to normalize the date. Homogeneity of variances was tested by plotting a graph between residuals and fitted values. The data were then subjected to a repeated measure analysis conducted with the Mixed Model (REML) procedure. Genotypes were considered as fixed, replicates as subjects and hpi as time points. Differences among the two genotypes (IG 207 and Nipper) at each time point (6, 12 and 24 hpi) for percent spore germination, germ tube length and percent appressorium formation were compared based on Tukey’s least significant difference at p = 0.05.

3.4. Results

3.4.1. Assessment of FIGS set for resistance to A. lentis isolate FT13037

The origin of the best-bet set of 87 accessions chosen for evaluation using the FIGS filtering method had a broad geographic distribution that included Ethiopia, countries of north Africa, west and central Asia, north and south Europe and India (Figure 3.1). Genotypes and dpi had significant effect on percent APD (p<0.001) (Appendix 3.1). Eighty-one accessions were tested for reaction to isolate FT13037 and the mean disease scores ranged from 1.39 to 9.51% APD 21 dpi with an LSD (genotype × dpi) of 2.58 (Appendix 3.2). Several genotypes showed either resistant or moderately resistant reactions at 7 dpi, as the incubation period progressed, these became moderately susceptible or susceptible at 21 dpi. Accession IG 207 had the lowest disease severity with a mean disease score of 1.39% APD at 21 dpi, while accession ILL 7537 was moderately resistant with 4.81% APD at 21 dpi. The susceptible control accession ILL 6002 had a score of 8.02% APD at 21 dpi (Table 3.2). A further, twelve accessions (IG 96, IG 712, IG 914, IG 1687, IG 1735, IG 5911, IG 7104, IG 7593, IG 7731, IG 8218, IG 8360 and IG 8550) were identified as moderately resistant, with nine of these exhibiting a lower mean disease score than the resistant control ILL 7537 (Table 3.2). The geographical distribution of these twelve accessions along with most resistant accession IG 207 is shown in Figure 3.2.

Table 3.2: Ascochyta lentis isolate/lentil FIGS set interactions at 7, 14 and 21 days post inoculation (dpi)

S. Square root transformed percent area of plant diseased (% APD) Accession ID Country No 7 dpi Category 14 dpi Category 21 dpi Category 1 IG 7883 Nepal 3.47 MR 8.71 S 9.51 S 2 IG 2305 Iran 2.27 R 8.66 S 9.15 S 3 IG 82 Ukraine 1.36 R 8 S 9.08 S 4 IG 2228 Russia 0 R 8.37 S 8.97 S 5 IG 7592 Morocco 3.73 MR 9.15 S 8.94 S 6 IG 341 Italy 2.85 MR 7.26 MS 8.85 S 7 IG 8243 Russia 0.9 R 8.46 S 8.83 S 8 IG 8248 Russia 2.68 MR 8.77 S 8.8 S 9 IG 642 Turkey 2.55 R 8.21 S 8.78 S 10 IG 8355 Russia 1.53 R 7.95 S 8.69 S 11 IG 600 Russia 2.77 MR 7.98 S 8.68 S 12 IG 6538 Egypt 4.06 MR 8.32 S 8.68 S 13 IG 8234 Russia 0.45 R 6.41 MS 8.65 S 14 IG 8547 Azerbaijan 1.36 R 7.79 S 8.61 S 15 IG 171 Turkey 4.28 MR 8.08 S 8.6 S 16 IG 7851 Nepal 3.52 MR 8.17 S 8.4 S 17 IG 615 Russia 0.78 R 7.69 MS 8.38 S 18 IG 7591 Morocco 1.07 R 7.75 S 8.32 S 19 IG 1699 Ethiopia 1.3 R 7.15 MS 8.27 S 20 IG 507 Tunisia 4.09 MR 8.05 S 8.25 S 21 IG 8254 Russia 2.61 MR 7.64 MS 8.24 S 22 IG 8315 Russia 1.81 R 7.29 MS 8.24 S 23 IG 641 Turkey 2.96 MR 6.99 MS 8.14 S 24 IG 6486 Morocco 4.52 MR 7.91 S 8.08 S 25 IG 298 Greece 1.77 R 7.67 MS 8.08 S 26 ILL 6002a Argentina 3.54 MR 6.15 MS 8.02 S 27 IG 911 Spain 1.49 R 7.79 S 8.01 S 28 IG 8401 Russia 0.68 R 6.34 MS 7.94 S 29 IG 504 Russia 0.39 R 5.82 MS 7.9 S 30 IG 132877 Georgia 2.49 R 6.82 MS 7.88 S 31 IG 94 Russia 0.85 R 8.14 S 7.85 S 32 IG 301 Greece 3.49 MR 6.13 MS 7.83 S 33 IG 920 Tunisia 0.78 R 7.16 MS 7.77 S 34 IG 7228 Nepal 2.49 R 6.97 MS 7.59 MS 35 IG 292 Algeria 3.2 MR 7.39 MS 7.53 MS 36 IG 7253 Nepal 3.84 MR 7.46 MS 7.47 MS 37 IG 8246 Russia 0.45 R 6.69 MS 7.42 MS 38 IG 1720 Ethiopia 2.36 R 6.71 MS 7.4 MS 39 IG 5928 Ethiopia 3.15 MR 6.58 MS 7.13 MS 40 IG 342 Italy 3.39 MR 7.19 MS 7.05 MS 41 IG 603 Russia 2.49 R 6.72 MS 7.05 MS 42 IG 4369 Tunisia 2.83 MR 6.69 MS 7.01 MS 43 IG 6487 Morocco 2.1 R 6.27 MS 6.73 MS 44 IG 8314 Russia 0 R 5.59 MS 6.72 MS 45 IG 1719 Ethiopia 2.91 MR 6.06 MS 6.66 MS 46 IG 8158 Ethiopia 2.17 R 5.14 MR 6.65 MS 47 IG 7852 Nepal 1.13 R 6.49 MS 6.56 MS 48 IG 4849 Greece 2.07 R 6.29 MS 6.5 MS 49 IG 921 Tunisia 2.72 MR 6.28 MS 6.49 MS 50 IG 4851 Greece 2.6 MR 5.92 MS 6.49 MS 51 IG 6491 Morocco 1.29 R 6.31 MS 6.31 MS 52 IG 506 Ukraine 3.1 MR 5.8 MS 6.28 MS Continued over the page …

Table 3.2 continued … 53 IG 597 Russia 3.52 MR 6.28 MS 6.28 MS 54 IG 6485 Morocco 3.82 MR 6.24 MS 6.24 MS 55 IG 606 Ukraine 3.89 MR 5.75 MS 6.17 MS 56 IG 5418 Italy 0.79 R 6.07 MS 6.07 MS 57 IG 611 Russia 0.45 R 4.64 MR 6.03 MS 58 IG 915 Spain 3.28 MR 6.32 MS 6 MS 59 IG 8232 Russia 2.12 R 5.62 MS 5.85 MS 60 IG 859 Algeria 1.14 R 5.69 MS 5.83 MS 61 IG 640 Turkey 1.66 R 4.95 MR 5.79 MS 62 IG 7623 Morocco 2.08 R 5.73 MS 5.73 MS 63 IG 7622 Morocco 1.96 R 5.84 MS 5.7 MS 64 IG 7599 Tunisia 1.37 R 5.17 MS 5.56 MS 65 IG 8363 Russia 1.57 R 4.77 MR 5.37 MS 66 IG 7422 Nepal 0.91 R 4.73 MR 5.34 MS 67 IG 8282 Russia 0.9 R 5.38 MS 5.31 MS 68 IG 4853 Greece 2.18 R 5.07 MR 5.3 MS 69 IG 5233 Jordan 1.96 R 4.96 MR 5.21 MS 70 IG 7731 Morocco 0.95 R 4.7 MR 5.04 MR 71 IG 8218 Russia 2.75 MR 3.68 MR 5.03 MR 72 IG 5911 Ethiopia 1.57 R 4.84 MR 4.82 MR 73 ILL 7537b Jordan 3.14 MR 3.81 MR 4.81 MR 74 IG 712 Morocco 0.59 R 2.84 MR 4.54 MR 75 IG 914 Spain 2.71 MR 4.76 MR 4.5 MR 76 IG 7593 Morocco 1.36 R 2.94 MR 3.72 MR 77 IG 8360 Russia 0.59 R 2.87 MR 3.71 MR 78 IG 1687 Ethiopia 1.57 R 2.58 R 3.39 MR 79 IG 1735 Ethiopia 1.66 R 2.93 MR 3.3 MR 80 IG 7104 Russia 0.9 R 3.17 MR 3.17 MR 81 IG 8550 Russia 0 R 2.15 R 2.81 MR 82 IG 96 Morocco 0.45 R 3.13 MR 2.61 MR 83 IG 207 Ethiopia 0.39 R 1.24 R 1.39 R Accessions are arranged in descending order of overall percent APD scores at 21 dpi. Disease severity in the FIGS set was assessed based on percent APD and to find significant differences among the genotypes, the data was square root transformed. Estimates of means are derived from three biological replicates using linear mixed model analysis. The analysis revealed that genotypes and days post inoculation (dpi) had significant effect on percent APD. asusceptible control; bresistant control; R: Resistant; MR: Moderately resistant; MS: Moderately susceptible; S: Susceptible

3.4.2. IG 207 as a potential novel resistance source to A. lentis

The resistant (IG 207) and moderately resistant accessions (IG 96, IG 1687, IG 7104 and IG 8550) were tested for resistance stability against a group of A. lentis isolates (FT13038, FT15160, FT16299-2 and FT16112). All factors and interactions had significant effect on percent APD (Appendix 3.3) Accession IG 207 remained highly resistant and did not show any significant difference for percent APD to all four isolates at 21 dpi (Figure 3.3; Appendix 3.4). By contrast, accessions IG 96, IG 8550 and IG 1687 were moderately resistant to isolates FT16299-2 and FT16112 and moderately susceptible to susceptible when challenged with isolates FT13038 and FT15160. On the other hand, IG 7104 was moderately susceptible to susceptible to all four isolates assessed (Table 3.3). The resistance status of cultivars Indianhead and Nipper as moderately susceptible and susceptible to the isolates FT16299-2

and FT15160, respectively, reflected the aggressive nature of these isolates on both the genotypes as previously determined by J. Davidson (SARDI, Adelaide, personal communication) (Table 3.1). Meanwhile, expected reactions by the cultivars ILL 7537 and ILL 6002 to all the four isolates demonstrated the repeatability of the bioassay (Table 3.3).

Based on the collective mean disease scores for the isolates tested, the accessions could be grouped into four different resistance classes such as resistant, moderately resistant, moderately susceptible and susceptible (Table 3.3). Among the accessions evaluated, IG 207 was the only accession to be placed in the group resistant with a mean disease score of 1.21% APD. The two currently employed resistant cultivars ILL 7537 and Indianhead were grouped moderately resistant with mean disease scores 4.07 and 4.92% APD, respectively. FIGS accessions IG 96, IG 8550 and IG 1687 along with cultivars Nipper and ILL 6002 are placed into group moderately susceptible, while susceptible group included accession IG 7104.

FT13038 FT15160 FT16112 FT16299-2 S. No Accession % % % % APD Category APD Category APD Category APD Category 1 IG 207 1.47 R 1.28 R 1.28 R 0.84 R

2 IG 96 5.45 MS 7.19 MS 4.28 MR 4.48 MR

3 IG 8550 6.21 MS 8.14 S 4.57 MR 4.97 MR

4 IG 1687 5.41 MS 8.90 S 3.77 MR 3.26 MR

5 IG 7104 9.04 S 9.33 S 6.34 MS 7.71 MS

6 Nipper 7.88 S 8.62 S 6.60 MS 4.60 MR

7 Indianhead 4.60 MR 5.60 MS 3.84 MR 5.65 MS

8 ILL 7537 5.35 MS 4.80 MR 1.78 R 4.36 MR

9 ILL 6002 8.09 S 8.33 S 6.38 MS 5.97 MS

R: Resistant; MR: Moderately resistant; MS: Moderately susceptible; S: Susceptible

Figure 3.3: Response of accession IG 207 to five Ascochyta lentis isolates (a) control, (b-f) response of accession IG 207 to isolates FT13037, FT13038, FT15160, FT16112 and FT16299-2 at 21 days post inoculation (dpi)

3.4.3. Histopathological assessment of early defence responses to A. lentis within IG 207

Leaf tissues of resistant accessions IG 207 and Nipper were assessed histopathologically to determine and compare their physiological defence responses to A. lentis infection. Genotypes had significant effect on percent spore germination, germ tube length and percent appressoria formed (p<0.001) (Appendix 3.5). Spore germination, germ tube length, timing and percent of appressoria formation at each time point differed between IG 207 and Nipper, however, both the genotypes did not show significant difference for germ tube length at 6 hpi (Figures 3.4, 3.5 and 3.6; Appendix 3.6). Only 22.7% of spores of isolate FT13038 successfully germinated on the leaflets of accession IG 207 by 36 hpi, whereas they quickly germinated on the leaflets of cultivar Nipper and gradually reached 76.59% within 24 hpi (Figures 3.4 and 3.7).

Likewise, germ tubes were significantly shorter at 12, 24 and 36 hpi on accession IG 207 leaflets compared to on Nipper leaflets (Figures 3.5 and 3.7; Appendix 3.6). The maximum mean germ tube length of isolate FT13038 incubated on the leaflets of accession IG 207 was 8.88 µm at 36 hpi. This was almost equivalent to the minimum mean germ tube length (8.83 µm) produced on the Nipper leaflets at only 6 hpi.

The timing and percent of appressorium formed was also significantly different between the genotypes. Spores of isolate FT13038 produced the first appressorium at 6 hpi on Nipper compared to 12 hpi on IG 207 leaflets (Figure 3.7). Likewise, the percent of appressoria was higher on Nipper leaflets at all time points (Figure 3.6; Appendix 3.6).

Figure 3.6: Appressoria formation percentage of isolate FT13038 on the leaflets of accessions IG 207 and Nipper at 6, 12, 24 and 36 hours post inoculation (hpi). Bar at 36 hpi on the leaflets of Nipper represent an estimated value of percent of appressoria formed. Estimates of means are derived from three replicates at each time point using a repeated measure analysis. For each replicate 100 germinated conidia were assessed. Error bars show standard error of the means

3.5. Discussion

3.5.1. Application of FIGS for the identification of best-bet AB resistance sources in the global lentil collection

The predictions of finding useful resistance to AB through a FIGS approach revealed 17.2% accessions within the subset of lentil as resistant or moderately resistant at 21 dpi following expected responses of control cultivars ILL 7537 and ILL 6002 to inoculations with isolate FT13037 as previously established (Dadu et al., 2017). Further, evaluation of physiological defence responses of the highly resistant accession IG 207 indicated that the approach captured novel, highly effective, and potentially useful A. lentis resistance mechanisms. Thus, a possible conclusion would be that the FIGS approach for mining gene banks is an effective alternative compared to random selection or arduous and costly large collection screening. Certainly, this has been the conclusion of other authors who found multiple sources for rare resistance to Russian wheat aphid (Diuraphis noxia) and Sunn pest (Eurygaster integriceps) in relatively small FIGS subsets after unsuccessful screening of thousands of wheat landraces conserved in the ICARDA gene bank (El Bouhssini et al., 2009; El Bouhssini et al., 2011; El Bouhssini et al., 2013).

However, a cautionary note must be added here. While this study, like the others cited above, does show that FIGS was successful at identifying useful trait variation, it did not directly compare the FIGS set to a subset constructed using different methods, such as random selection or a core collection. In fact, it could be the case that the frequency of resistant accessions in the FIGS set used is merely a reflection of the native frequency of resistance present in the genepool at large. For example, 17% of total wild lentil accessions screened have shown resistant reaction against the virulent A. lentis isolate FT13038 (Dadu et al., 2017). Likewise, Bayaa et al. (1994) reported that 27% of L. orientalis genotypes were resistant to the tested A. lentis isolates. By contrast an evaluation of 188 Indian lentil genotypes revealed only 5% of the accessions were resistant to the tested isolates (Singh et al., 1982). Meanwhile in Australia, of 488 lentil accessions only 5% were resistant to the three tested isolates, while 29% showed variable reactions (Nasir and Bretag, 1998). Although it is difficult to draw firm conclusions by comparing such studies due to different lentil genotypes and A. lentis isolates

used, the cited studies do demonstrate considerable variation in resistance reactions to A. lentis. Thus, to confirm the utility of the FIGS approach for gene bank mining would require a comparative study where the approach is compared to at least multiple random selections.

This study, however, did demonstrate that the incidence of resistance to A. lentis does not seem to be randomly distributed geographically. Rather, the bulk of the resistant and moderately resistant accessions came from relatively small pockets that included northern Morocco and southern Spain, Ethiopia and south west Russia (trans-Caucuses) (Figure 2). This link to geographic locations has been noted in previous studies. Bayaa et al. (1994) observed that the majority of resistant wild lentil genotypes came from Syria and Turkey as did Tullu et al. (2010a) and Dadu et al. (2017), who studied wild lentil resistance to Canadian and Australian A. lentis isolates. This is not surprising given that wild lentils are native to west Asia. However, geographic specificity has also been observed in cultivated lentils. Nasir and Bretag (1998), who evaluated 488 lentil accessions from 25 different countries for resistance to three Australian isolates of A. lentis, found that almost all the resistant genotypes came from Pakistan. Further, Iqbal et al. (2010), who studied resistance of Pakistani lentils to A. lentis, found that most of the resistant genotypes were from the Punjab province. The prevalence of resistance found in Punjab material was proposed to be due to a strong positive selection pressure imposed by the wide distribution and occurrence of A. lentis in the province (Singh et al., 1982).

In the above context, the reader is reminded that the FIGS selection strategy used for this study was designed to capture collection sites that have climatic profiles most likely to favour A. lentis development and thus impose a selection pressure for resistance. However, because there are multiple factors that influence seasonal population dynamics of the pathogen, it is likely that the selection process could be improved. For example, it is suggested that the selection algorithm could include measures of seasonal variation for each variable. The rationale is that, environments that have a high degree of inter-seasonal stability and popular cultivars with widespread planting, populations of the pathogen are likely to evolve and produce aggressive isolates, that can infect even the best of the resistant cultivars. Thus, the selection process would be designed to favour environments with higher inter-seasonal stability for climatic variables critical to disease development. Further improvements could be made by

combining the results of this study with previous studies to refine the environmental profiles (including geographic locations) most likely to predict resistant genotypes.

3.5.2. Evidence of better resistance to A. lentis in IG 207 than ILL 7537 and Indianhead

The evolution of A. lentis pathogenicity is important to consider when developing breeding strategies. In Australia, the presence of both mating types (MAT1-1 and MAT1-2) of A. lentis (Skiba and Pang, 2003) leads to sexual ascospores, which is assumed to quickly increase the genetic diversity of the fungal populations (Martin et al., 2013). Indeed, an assessment of genetic diversity within the Australian A. lentis populations showed that there were large variations among isolates collected from different parts of Australia (Ford et al., 2000). Likely, this variation in aggressiveness within the pathogen genepool has helped it to rapidly overcome simply inherited resistances. Therefore, it is important that new resistances be stable and quantitative to withstand the pressure created by the aggressive and evolving pathogen population.

The consistent resistance reaction demonstrated by IG 207 when challenged with four aggressive isolates (FT13038, FT15160, FT16299-2 and FT16112) indicates that its resistance to A. lentis is likely to be stable. These findings are in broad agreement with the hypothesis that resistance to A. lentis is probably far more polygenic than originally proposed (Gupta et al., 2012a), although further research is required to understand the genetics of resistance displayed by IG 207.

The literature suggests that most resistance sources to AB used in advanced lentil cultivars are derivatives of a single, or at most a few major resistance loci, and thus are likely to be vulnerable to the pathogen population at large (Ford et al., 1999; Nguyen et al., 2001; Sudheesh et al., 2016a). To sustain the lentil industry, it is suggested that multiple resistance genes need to be pyramided into elite cultivars (Sari, 2014). It is worth noting that the resistance genes derived from sources including CDC-Robin, ILL 7537, 964a-46 and ILL 1704 are proposed as non-allelic and therefore provide a unique opportunity to stack the different genes shared among them (Sari, 2014). In this study, clear discrimination of disease reactions to A. lentis (isolates FT13038, FT15160 and FT16299-2) between highly resistant IG 207 and currently employed resistance sources (Indianhead, ILL 7537 and Nipper) provided an initial

indication that IG 207 carries resistance that is putatively different. The number of resistance genes and the nature of the resistance within the accession IG 207 and current resistance sources (Indianhead and ILL 7537) could be revealed through genetic analysis and allelism tests (Ye et al., 2001; Sari, 2014). Following confirmation of the allelic nature of these resistances and mapping of the genes, it would be desirable to pyramid these genes together in an effort to breed for durable resistance and benefit Australian lentil industry.

3.5.3. Evidence of better defence through early physical containment A. lentis in IG 207

The efficacy of a resistance source is dependent on the spatial and temporal distribution of defence responses to the invading pathogen, which are largely genotype-dependent (Khorramdelazad et al., 2018; Sari et al., 2018). Characterising such defence responses could help understand the resistance mechanism adopted by the newly identified resistance sources as well as indicate its novelty. This can be achieved by undertaking time-course histopathological assays and gene-expression studies. Such a study was undertaken by Sambasivam et al. (2016) and Dadu et al. (2018a) who histopathologically quantified and characterised A. lentis resistance of genotypes IG 7537 and ILWL 180. A more recent RNA- sequencing study revealed differential expression (DE) of defence related genes within lentil cultivars ILL 7537 and ILL 6002 and concluded that faster and stronger amplitudes in expression of these genes were critical for resistance to A. lentis (Khorramdelazad et al., 2018).

In this study, pre-penetration behavior including spore germination percentage, germ tube length, timing and formation percentage of appressoria of aggressive isolate FT13038 revealed significant differences between the resistance mechanisms deployed by accession IG 207 and Nipper. This again is in acceptance with the results of disease symptomology at all time intervals as mentioned above. Upon inoculation, spores of isolate FT13038 produced maximum germination within 24 hpi as previously observed on the leaflets of accession ILL 6002 (Sambasivam et al., 2016; Dadu et al., 2018a). It is worth noting that the spore germination percentage recorded for accession IG 207 was lower than well-known resistant accession ILL 7537 (Sambasivam et al., 2016; Sari et al., 2017) and though different isolates were tried, they were known to have similar aggressiveness (Dadu et al., 2017). The lower spore germination percentage for accession IG 207 might be due to early recognition of the pathogen associated molecular patterns (PAMPs) released by A. lentis assisted by pattern

recognition receptors (PRRs) including receptor-like kinase proteins and receptor-like proteins (Dangl et al., 2013). By contrast, the delayed recognition by PRRs in cultivar Nipper might be responsible for the increased germination percentage.

Germinated isolate FT13038 produced significantly shorter germ tubes on IG 207 leaflets than those of cultivar Nipper at each time point except at 6 hpi. This hindrance of germ tube growth has also been observed on the leaflets of other resistant accessions including ILL 7537 (Sambasivam et al., 2016), CDC Robin, L-01-827A (Sari et al., 2017) and ILWL 180 (Dadu et al., 2018a). By contrast, the germ tubes grew extensively on the leaflets of cultivar Nipper, which indicates a delayed activation of defence mechanisms. On the other hand, it is proposed that the ability of IG 207 to recognize the presence of the fungus earlier than Nipper may result in the activation of the downstream defence responses in the host and provide an extended resistance against the pathogen infection.

Reduced germination and restricted growth of isolate FT13038 on the leaflets of accession IG 207 failed to produce adequate appressoria for tissue penetration. Conversely, spores of isolate FT13038 produced appressoria within 6 hpi on the leaflets of cultivar Nipper, which led to active penetration of tissue and colonization within 36 hpi. These findings are in agreement with previous studies Sambasivam et al. (2016) and Dadu et al. (2018), who reported similar differences in the timing and percent of appressoria produced between resistant and susceptible accessions (ILL 7537 and ILL 6002).

In conclusion, this study indicates that FIGS is an efficient sampling strategy to mine large germplasm collections for A. lentis resistance. However, studies that compare FIGS to alternate sampling strategies are required to support this assertion with more authority. Accession IG 207 was identified with higher and stable resistance to AB infection than resistant controls. A histopathological study suggested that relatively faster recognition of A. lentis presence on the leaflets is likely to contribute to the resistance displayed by IG 207. However, differential molecular studies would provide further evidence and a more complete understanding of the defence mechanisms within accession IG 207 to A. lentis infection.

Chapter 4 - A novel Lens orientalis resistance source to the recently evolved highly aggressive Australian Ascochyta lentis isolates

Manuscript published in Frontiers of Plant Science

https://doi.org/10.3389/fpls.2017.01038

4.1. Abstract

Substantial yield losses and poor seed quality are frequently associated with AB infection of lentil caused by Ascochyta lentis. Recently reported changes in aggressiveness of A. lentis have led to decreased resistance within cultivars such as Northfield and Nipper in Australia. Furthermore, the narrow genetic base of the current breeding program remains a risk for further selective pathogen evolution to overcome other currently used resistances. Therefore, incorporation of potentially novel and diverse resistance genes into the advanced lines will aid to improve cultivar stability. To identify these, 30 genotypes sourced from five wild species (Lens orientalis, L. odomensis, L. ervoides, L. nigricans and L. lamottei), including eight previously reported resistance sources, were screened for disease reaction to two recently isolated and highly aggressive isolates. Subsequently, two L. orientalis accessions were found highly resistant and a further six L. nigricans, one L. odemensis, one L. ervoides, one L. lamottei and one L. orientalis accessions were moderately resistant. Several of these were more resistant than the currently deployed resistance source, ILL 7537. Furthermore, L. orientalis accession ILWL 180 was consistently resistant against other highly aggressive isolates recovered from diverse geographical lentil growing regions and host genotypes, suggesting stability and potential for future use of this accession in the Australian lentil breeding program.

Keywords: Ascochyta blight, Ascochyta lentis, lentil, wild lentils, screening

4.2. Introduction

Lentil (Lens culinaris Medikus ssp. culinaris) (2n =14), a cool season high protein (28%) food legume cultivated around the world, is ranked fifth in size of global production among legumes at 6.31 million tonnes (Mt) (FAOSTAT, 2016). However, a significant reduction in lentil productivity (21%) was reported during 2015-2016 in Australia (FAOSTAT, 2016), largely due to the disease AB, caused by necrotrophic fungus Ascochyta lentis. This disease is of global concern (Kaiser and Hannan, 1986; Erskine et al., 1993; Nasir and Bretag, 1997a; Muehlbauer and Chen, 2007), reducing yields and seed quality (Morrall and Sheppard, 1981; Gossen and Morrall, 1983). It causes an estimated $16.2 million AUD in losses to the Australian lentil industry alone due to reduced production and disease management costs (Murray and Brennan, 2012).

To date, integrated disease management approaches combining best cultivation practices, application of fungicides and cultivars with moderately resistant or resistant ratings have sustained the industry in the presence of A. lentis (Hawthorne et al., 2012). However, continuous cultivation of relatively few resistant cultivars with a narrow genetic base has likely led to episodes of resistance breakdown through selection of adapted and aggressive isolates (Nasir and Bretag, 1997b; Davidson et al., 2016; Sambasivam et al., 2016). This has also occurred for several Canadian cultivars including Laird (Ahmed and Morrall, 1996) and breeding line ILL 5588 (Tullu et al., 2010a). ILL 5588 was also introduced into Australia after its success in Canada and Northfield, a selection from ILL 5588 (Ali, 1995) along with Indianhead were employed either individually or in combination to breed resistant cultivars. However, an increased susceptibility of Northfield to the Australian A. lentis population was detected within six seasons after its commercialization (Nasir and Bretag, 1997b). Consequently, this most likely led to the demise of the new Australian cultivar Nipper with Northfield × Indianhead pedigree after just four seasons (Davidson et al., 2016). Meanwhile, other Australian cultivars such as PBA Ace, PBA Blitz, PBA Bolt, PBA Jumbo, PBA Jumbo2, PBA Herald XT and PBA Hurricane XT, were developed containing a CDC Matador pedigree with A. lentis resistance from Indianhead (Pulse Australia, 2016). Several of these were found susceptible or moderately susceptible to recently detected highly aggressive Australian isolates, with predicted increasing industry reliance on those that remained somewhat resistant,

such as PBA Jumbo2 and PBA Hurricane XT (Davidson et al., 2016). This will again likely lead to increased selection pressure on the highly variable pathogen population (Nasir and Bretag, 1997b; 1998; Davidson et al., 2016; Sambasivam et al., 2016), to evolve and overcome the relatively few resistance sources upon which the industry is currently reliant.

Therefore, a major goal for the Australian lentil breeding program remains to introgress novel resistance genes/alleles or combinations thereof to improve the stability and further enhance durability of resistance to A. lentis within elite cultivated backgrounds. Several previous investigations have uncovered sources for novel A. lentis resistance in all five wild relative Lens taxa (L. orientalis, L. odomensis, L. ervoides, L. nigricans and L. lamottei) (Bayaa et al., 1994; Ahmad et al., 1997; Tullu et al., 2010a). Although crossing incompatibility exists among such broad germplasm (Ladizinsky, 1979; Ladizinsky et al., 1984), inter-specific fertile hybrids were produced through conventional techniques between accessions of L. culinaris and L. orientalis within the primary gene pool (Ladizinsky, 1999; Fratini et al., 2004; Gupta and

Sharma, 2007). Success was achieved with the aid of gibberellic acid (GA3) application and embryo rescue techniques for the more incompatible crosses (Cohen et al., 1984; Ahmad et al., 1995; Tullu et al., 2013). Subsequently, segreggating populations for ascochyta resistance were successfully produced from L. culinaris x L. orientalis and L. culinaris x L. ervoides crosses, within which resistance was simply inherited (Ahmad et al., 1997). More recently, Fiala et al. (2009) successfully transferred anthracnose resistance from L. ervoides to L. culinaris and developed a recombinant inbred line (RIL) population which was later evaluated by Vail (2010). This cross was also used to generate backcrosses which were reported to be stable and without any phenotypic linkage drag with yield. Selected breeding lines evaluated under field conditions were reported to be highly resistant to anthracnose under high disease pressure.

The hypothesis is that the wild species of lentil possess novel and diverse resistance alleles/genes to AB and the resistance conferred is potentially durable. Therefore, the aim of the current study was to 1) uncover potentially novel wide germplasm sources of resistance to the most aggressive isolates of A. lentis recently detected in Australia and 2) determine potential stability of the resistance(s) through screening against a diverse collection of isolates from the current population.

4.3. Materials and methods

4.3.1. Plant and fungal materials

Thirty wild lentil accessions were provided by the Australian Grain Gene bank (AGGB), Horsham, Victoria (Table 4.3). Two cultivars routinely used to discriminate the reaction of A. lentis (Davidson et al., 2016; Sambasivam et al., 2016), ILL 6002 (susceptible) and ILL 7537 (resistant) were included as controls. Three seeds per genotype were sown in 10 cm pots filled with pine bark potting mix, fertilised with Nitrosol, Amsgrow ® (4.5 mL/L) on a weekly basis and watered on every alternate day. Three replications (three inoculated and three non-inoculated/control pots) were included per treatment combination (genotype x isolate). After sowing, pots were placed in a glass house at Dookie Campus, University of Melbourne, Victoria maintained at 20 ± 5°C with a 16 h/8 h day/night photoperiod until inoculation. Considering germination inhibition in wild species, 21-day-old seedlings were used for bioassay, such that the leaf number and number of nodes were a minimum of 8-10 and a minimum of 4, respectively, in both wild species and 14-day old cultivars. Post-inoculation, pots were moved into a Conviron growth cabinet replicating glass house conditions.

Single spore cultures of four highly aggressive isolates (FT13037, FT13038, FT13050 and FT13027) and one low aggressive isolate (F13082) of A. lentis were obtained from the South Australian Research and Development Institute (SARDI) (Table 4.1). These were sub- cultured onto potato dextrose agar media (PDA) plates 8 days after seeding of the wild lentil genotypes and incubated for 14 days at 22°C, 12 h/12 h dark/light cycle under florescent (OSRAM TLD/18W) and near Ultra Violet (UV) lights (PHILIPS BLB/18W).

4.3.2. Experimental design

Preliminary bioassays were conducted to reconfirm the aggressiveness of the two isolates (FT13037 and FT13038) by screening them against three host differentials with known resistance levels comprising ILL 7537 (resistant), Nipper (moderately resistant-moderately susceptible) and ILL 6002 (susceptible) (Davidson et al., 2016; Sambasivam et al., 2016).

Table 4.1: Details of A. lentis isolates (including the lentil cultivar from which they were collected, location and year of collection) used in the study

Nature of Location in Isolate Lentil - cultivar Date collected isolate Australia Maitland, South FT13027 Aggressive Blitz 02/08/2013 Australia (SA) FT13037 Aggressive Flash Urania, SA 29/07/2013 FT13038 Aggressive Cumra Urania, SA 29/07/2013 FT13050 Aggressive Nipper Mallala, SA 30/08/2013 F13082 Non-aggressive Nipper Pinery, SA 24/09/2013

Later, experiments were carried out in two stages. Initially, all 30 genotypes were screened against isolates FT13037 and FT13038 to determine disease responses and identify those with lowest disease severity. Subsequently, the highly resistant genotype (ILWL 180) identified was assessed for its reaction to all five isolates. All experiments were set out in a completely randomised design under controlled conditions with 3 replications. Initial screening included 120 treatment combinations (30 genotypes x 2 isolates x 2 inoculation treatments (inoculated or non-inoculated)), whereas stability experiments included 10 treatment combinations (1 genotype x 5 isolates x 2 inoculation treatments (inoculated or non- inoculated)).

4.3.3. Preparation of inoculum and bioassay

Preparation of spore suspension and subsequent inoculation of pots was conducted as described in Chapter 3. Three-week-old seedlings of each wild lens genotype and 2-week-old seedlings of both controls were uniformly inoculated using an air pressurized hand sprayer until run off. Control/non-inoculated pots were sprayed with water mixed with Tween 20 (0.02% v/v). Post inoculation, all pots were covered with long inverted solid paper cups, randomly placed in plastic crates and moved to a Conviron growth chamber, Dookie College, The University of Melbourne, and maintained as described in Chapter 3, to stimulate the development of blight symptoms on plants.

4.3.4. Disease assessment

Each of the three seedlings per pot was scored for symptoms of A. lentis infection at 14 and 21 days-post inoculation (dpi) (Ford et al., 1999; Sambasivam et al., 2016) using a non- destructive 1-9 scoring scale specifying a size limit on leaf and stem lesions and percentage leaf drop (Ford et al., 1999; Davidson et al., 2016; Sambasivam et al., 2016). The scores were 1 = no disease symptoms; 3 = leaf lesions only, chlorosis of affected leaves, < 10% leaf drop; 5 = leaf lesions, up to 25% leaf drop, stem flecks or lesions < 2 mm; 7 = leaf lesions, up to 50% leaf drop, stem flecks or lesions > 2 mm; 9 = leaf lesions, potential defoliation, stem girdling and potential plant death (adapted from Davidson et al. (2016)).

4.3.5. Statistical analysis

Statistical analysis was performed using IBM SPSS statistics software. Data from all controls (non-inoculated) were excluded from analysis since plants were symptom free with a consistent score of 1. Modes of disease scores of each pot were calculated to study each genotype x isolate interaction and Friedman’s non-parametric analysis of variance (ANOVA) was used to assess the modal variances among them. Most frequently observed scores pooled from three inoculated replicates were used to calculate modal disease score at 14 and 21 dpi. Post-hoc analysis with Wilcoxon signed-rank tests was conducted with a Bonferroni correction applied to evaluate significant differences among the genotypes for AB resistance. Modal disease scores were used to categorise the genotypes into Resistant (1-3), Moderately Resistant (5) and Susceptible (7-9) (Ford et al., 1999; Nguyen et al., 2001; Rubeena et al., 2006). Area under disease progress curve (AUDPC) was used to summarise the disease intensity over time and was estimated as described by Campbell and Madden (1990).

+ AUDPC = 𝑛𝑛 ( ) 2 𝑦𝑦𝑖𝑖 𝑦𝑦𝑖𝑖+1 � � � 𝑡𝑡𝑖𝑖+1 − 𝑡𝑡𝑖𝑖 𝑖𝑖=1 th Where; n = total number of observations, yi = modal disease score at the i observation and t = time at the ith observation.

4.4. Results

4.4.1. Phenotyping of wild genotype resistance to two most aggressive isolates of A. lentis

From preliminary screening on ILL 7537, Nipper and ILL 6002, both isolates FT13038 and FT13037 were deemed aggressive, producing a susceptible reaction on ILL 6002 and Nipper with extensive leaf lesions, stem girdling and subsequent plant death at 21 dpi (Appendix 4.1 and 4.2). Further, both isolates produced leaf lesions on ILL 7537, with isolate FT13037 (Modal disease score of 7) more aggressive than isolate FT13038 (Modal disease score of 3) (p ≤ 0.001) (Table 4.2).

Table 4.2: Modal disease score of three host differentials at 14 and 21 days post inoculation (dpi) for A. lentis isolates FT13038 and FT13037

Isolate / FT13037 FT13038 S. No Genotype 14 dpi 21 dpi 14 dpi 21 dpi 1 ILL 6002 7 9 7 9 2 ILL 7537 5 7 3 3 3 Nipper 7 9 7 9 Disease severity was assessed using 1-9 scale, where 1 represents no disease symptoms and 9 denotes leaf lesions, potential defoliation, stem girdling and potential plant death. Estimates of modes are derived from three biological replicates. Friedman’s non-parametric analysis of variance (ANOVA) revealed a significant effect of genotypes on fungal disease score (p ≤ 0.001).

Following inoculation of the 30 wild Lens genotypes with these isolates, first visual symptoms (leaf lesions) occurred from 7 dpi and stem lesions coalesced leading to stem girdling and plant death by 21 dpi on the most susceptible genotypes. Disease symptoms did not appear until 11 dpi on other genotypes and several were observed to overcome the infection. This demonstrated a range of disease reactions, from susceptible to resistant based on Friedman’s test (p = 0.002) (Appendix 4.1).Two genotypes of L. orientalis, ILWL 180 and ILWL 7, were resistant to both isolates at 21 dpi with modal disease scores of 1 and 3, respectively, whereas five genotypes of L. nigricans, ILWL 37, PI 572348, PI 572351, PI 572359 and PI 615677, were resistant to just isolate FT13038 (Table 4.3).

Unsurprisingly, disease severity increased significantly between 14 and 21 dpi for most of the genotypes assessed and when inoculated with either isolate. However, disease severity on ILWL 221, ILWL 235, ILWL 261, ILWL 325, PI 572334, PI 572345, PI 572347, PI 572348 and PI 572360, to isolate FT13038 did not progress after 14 dpi, potentially indicating stability of the resistance response(s) to this isolate. These accessions did however become susceptible at 21 dpi following inoculation with isolate FT13037. Similarly, ILL 7537 was resistant to isolate FT13038 but susceptible to isolate FT13037 at 21 dpi (Table 4.3). Isolate FT13037 was able to cause significantly more disease on 25 of the genotypes, and on the two controls, compared to isolate FT13038. The remaining five genotypes, ILWL 70, ILWL 160, PI 572347, ILWL 7 and ILWL 180, had equal or significantly higher disease over time when inoculated with isolate FT13038 compared to isolate FT13037 (Appendix 4.3). The highest and lowest disease severity and AUDPC was observed on genotypes ILWL 206 (90.41) and ILWL 180 (19.46), respectively when inoculated with isolate FT13038. Meanwhile, the highest and lowest disease severity and AUDPC was observed on genotypes PI 572362 (123.66) and ILWL 180 (18.69), respectively when inoculated with isolate FT13037.

Five genotypes, PI 572348, PI 572359, PI 615677, PI 572351 and ILWL 180, were more resistant than ILL 7537 to isolate FT13038, and 11 genotypes, ILWLW 146, ILWL 160, PI 572333, PI 572348, PI 572347, PI 572359, ILWL 37, PI 615677, PI 572351, ILWL 7 and ILWL 180, were more resistant than ILL 7537 to isolate FT13037 (Appendix 4.3). Likewise, two genotypes, ILWL 206 and PI 572342, were more susceptible than ILL 6002 to isolate FT13038, and six genotypes, PI 572362, ILWL 172, PI 572330, PI 572317, ILWL 116 and ILWL 206, were more susceptible than ILL 6002 to isolate FT13037 (Table 4.3; Appendix 4.3).

4.4.2. L. orientalis ILWL 180 as a potential novel resistance source

The genotype ILWL 180 remained resistant at 21 dpi following repeated screening with the initial two isolates as well as three further isolates FT13027, FT13050 and FT13082 (p = 0.534). This was the case even when challenged with the most aggressive isolate FT13037, which was able to overcome resistance in ILL 7537 (p ≤ 0.001) (Figure 4.1; Table 4.4; Appendix 4.1 and 4.4).

Table 4.3: Details of the Lens spp. genotypes used in the study along with corresponding modal disease scores at 14 and 21 days post inoculation (dpi) and area under disease progress curve (AUDPC)

FT13038 FT13037 S. No Isolate/Genotype Species Country 14 dpi 21 dpi AUDPC Category 14 dpi 21 dpi AUDPC Category 1 ILL 6002a culinaris a pure line selection from Precoz 5 9 79.94 S 7 9 100.38 S 2 ILL 7537b culinaris Jordan 3 3 46.66 R 5 7 71.75 S 3 PI 572330 ervoides Israel 5 7 74.69 S 7 9 107.1 S 4 PI 572317 ervoides Italy 5 7 73.89 S 7 9 106.54 S 5 PI 572362 odemensis Unknown 5 5 76.23 MR 9 9 123.66 S 6 PI 572336 ervoides Turkey 5 7 79.35 S 5 9 84 S 7 ILWL 172 odemensis Syria 3 7 57.54 S 9 9 120.54 S 8 ILWL 206 ervoides Bosnia and Herzegovina 7 7 90.41 S 7 9 105.14 S 9 ILWL 116 odemensis Syria 5 5 72.35 MR 7 9 105.77 S 10 PI 572342 nigricans France 5 9 82.81 S 7 9 100.28 S 11 ILWL 221 odemensis Turkey 5 5 75.46 MR 5 9 77.77 S 12 ILWL 235 odemensis Syria 5 5 73.89 MR 5 7 80.12 S 13 PI 572345 nigricans Italy 5 5 72.35 MR 5 7 77.77 S 14 PI 572334 ervoides Turkey 5 5 68.46 MR 7 7 87.89 S 15 ILWL 261 ervoides Turkey 5 5 70.81 MR 5 7 75.43 S 16 ILWL 325 orientalis Jordan 5 5 70 MR 5 7 75.81 S 17 ILWL 69 orientalis Former Soviet Union 3 5 52.12 MR 5 7 75.08 S 18 ILWL 70 orientalis Iran 5 7 77 S 5 7 77 S 19 ILWL 146 orientalis Syria 3 5 49 MR 5 5 70 MR 20 PI 572360 odemensis Israel 5 5 66.12 MR 5 5 74.66 MR 21 ILWL 437 lamottei Turkey 5 5 71.96 MR 5 5 74.66 MR 22 PI 572399 orientalis Bosnia and Herzegovina 3 7 59.12 S 5 5 73.89 MR 23 ILWL 160 odemensis Syria 5 7 76.97 S 5 5 69.97 MR 24 PI 572348 nigricans Yugoslavia 3 3 45.12 R 3 5 53.69 MR 25 PI 572347 nigricans Italy 5 5 66.89 MR 3 5 52.92 MR 26 PI 572359 nigricans Turkey 3 3 44.31 R 3 5 52.12 MR 27 PI 572333 ervoides Turkey 3 5 49.39 MR 5 5 66.12 MR 28 PI 615677 nigricans Yugoslavia 3 3 45.12 R 3 5 49.77 MR 29 ILWL 37 nigricans Turkey 3 3 48.23 R 3 5 50.58 MR 30 PI 572351 nigricans Bosnia and Herzegovina 3 3 41.23 R 3 5 48.62 MR 31 ILWL 7 orientalis Turkey 3 3 50.58 R 3 3 44.31 R 32 ILWL 180 orientalis Syria 1 1 19.46 R 1 1 18.69 R Genotypes are arranged in descending order of overall resistance for FT13037 isolate. Scores 0 = no disease to 9 = plant death; a ILL 6002 - susceptible control; b ILL 7537 – resistant control. Estimates of modes are derived from three biological replicates. Friedman’s non-parametric analysis of variance (ANOVA) revealed a significant effect of genotypes on fungal disease score (p = 0.002).

Figure 4.1: Response of wild Lens ILWL 180 to five isolates a: control pot of wild Lens ILWL 180 b, c, d, e, f: Response of wild Lens ILWL 180 to isolates FT13050, FT13027, F13082, FT13037 and FT13038 21 days post inoculation (dpi)

Table 4.4: Modal disease scores of ILWL 180 and controls at 14 and 21 days post inoculation (dpi) against five A. lentis isolates

ILL 6002 ILWL 180 ILL 7537 S. No Isolates 14 dpi 21 dpi 14 dpi 21DP1 14 dpi 21 dpi 1 FT13037a 7 7 3 3 5 7 2 FT13038a 5 7 1 3 3 3 3 FT13050a 7 7 1 3 3 3 4 FT13027a 7 7 3 3 3 3 5 FT13082b 5 5 1 1 1 3 aaggressive; bnon-aggressive

Disease severity was assessed using 1-9 scale, where 1 represents no disease symptoms and 9 denotes leaf lesions, potential defoliation, stem girdling and potential plant death. Estimates of modes are derived from three biological replicates. Friedman’s non-parametric analysis of variance (ANOVA) revealed a significant effect of genotypes on fungal disease score (p ≤ 0.001), whereas isolates did not show a significant effect on the modal disease score of ILWL180 (p = 0.534).

4.5. Discussion

Evolution of the pathogen population towards more highly aggressive isolates has likely contributed to failure and reclassification of the resistance status of widely grown cultivars such as Laird and breeding line ILL 5588 in Canada (Morrall, 1997; Morrall et al., 2004), and Northfield and Nipper in Australia (Nasir and Bretag, 1997b; Davidson et al., 2016), although this requires further spatial and temporal population assessment for validation. Furthermore, the broad diversity within the A. lentis population will likely maintain pressure on the few remaining resistance sources within the Australian cultivars (Davidson et al., 2016). Hence, introduction of potentially novel resistance sources from diverse germplasm such as wild relatives is pivotal for maintaining production stability within the lentil industry.

The recent inclusion of ILL 7537 as a resistance source in the Australian breeding program was largely consistent with the findings of this study, whereby this accession was resistant against the majority of isolates assessed. Although none of the existing cultivars have ILL 7537 as one of the parent in their pedigree, isolate FT13037, which was isolated in 2013 from Urania, the Yorke Peninsula of South Australia, was able to cause severe disease on ILL 7537 under the bioassay conditions. Therefore, caution should be taken when relying upon this source of resistance for future resistance breeding strategies. The resistance status of this source

and Indianhead was previously questioned following controlled bioassays (Nguyen et al., 2001; Davidson et al., 2016).

The quantitative summary of disease severity and progression in this study identified resistant genotypes from L. orientalis (2) and L. nigricans (5) but not from L. odemensis, L. ervoides or L. lamottei. This agreed with the findings of Tullu et al. (2010a), who reported ILWL 206 (L. ervoides) as susceptible and ILWL 146 (L. orientalis) as moderately resistant against Canadian isolates. However, this was in contrast to the previous findings of Bayaa et al. (1994), who reported that ILWL 69, ILWL 116, ILWL 172, ILWL 206 and ILWL 261 were resistant to Syrian isolates, potentially indicating a higher aggressiveness of isolates within the current Australian population. The two L. orientalis genotypes identified in this study as resistant (ILWL 180 and ILWL 7) and moderately resistant (ILWL 146) were previously also reported to be resistant to Syrian isolates (Bayaa et al., 1994), potentially highlighting the stability of these resistance sources. Similarly, Tullu et al. (2010a) identified L. ervoides, L. nigricans and L. orientalis genotypes resistant to both Canadian and Syrian isolates, also highlighting that the wild species may possess broad resistances.

Interestingly, the resistant genotypes ILWL 180 and ILWL 146 originated from a common geographical region of Syria and other moderately resistant genotypes originated from Turkey (Bayaa et al., 1994). Associations between geographical origin and the A. lentis resistance trait have previously been reported in larger germplasm collections representative of different geographical regions (Bayaa et al., 1994), indicating potential co-evolution of resistance mechanisms with selection from regional populations. Given that these accessions are also resistant to the most aggressive Australian isolates, shared environmental-trait (resistance) based relationships would be useful to consider when seeking further resistance sources within germplasm collections. For this, researchers at the International Center for Agricultural Research in Dry Areas (ICARDA) have developed a focused identification of germplasm strategy (FIGS) (Mackay, 1990; 1995; Street et al., 2008; Mackay, 2011).

After identification of resistance in a wild relative species (subspecies), the next hurdle is to bring the desirable genes/alleles across to an elite cultivated background. For Lens, interspecies crossing has been encumbered with pre - and post-fertilization barriers such as reduced pollen fertility, chromosomal aberrations and embryo abortion (Abbo and Ladizinsky, 1991;

1994; Gupta and Sharma, 2007). To date, no successful deployment of wild relative-derived resistance for improved A. lentis resistance has been reported. Nevertheless, fertile and phenotypically normal hybrids have been created between primary gene pool species such as L. culinaris and L. orientalis through conventional techniques (Wong et al., 2015). Thus, exploiting the resistance detected in L. orientalis would be a practical choice rather than pursuing that detected in secondary, tertiary or quaternary gene pools, which would be time consuming and laborious.

In conclusion, substantial variation for resistance to A. lentis is present in wild relative genepools and the L. orientalis accession ILWL 180 was most resistant to the most highly aggressive isolates detected in the recent Australian population. Further investigation into this resistance source is required to validate its stability against the breadth of the pathogen population and to identify resistance loci for selective breeding purposes.

Chapter 5 - Evidence of early defence to Ascochyta lentis within the recently identified Lens orientalis resistance source ILWL 180

Manuscript published in journal Plant pathology

https://onlinelibrary.wiley.com/doi/abs/10.1111/ppa.12851

5.1. Abstract

In plant-pathogen interactions, strong structural and biochemical barriers may induce a cascade of reactions in planta, leading to host resistance. The kinetic speed and amplitudes of these defence mechanisms may discriminate resistance from susceptibility to necrotrophic fungi. The infection processes of two Ascochyta lentis isolates (FT13037 and F13082) on the recently identified AB resistant Lens orientalis genotype ILWL 180 and two cultivated genotypes, ILL 7537 (resistant) and ILL 6002 (susceptible), was assessed. Using histopathological methods, significant differences in early behaviour of the isolates and the subsequent differential defence responses of the hosts were revealed. Irrespective of virulence, both isolates had significantly lower germination, shorter germ tubes and delayed appressorium formation on the resistant genotypes (ILWL 180 and ILL 7537) compared to the susceptible genotype (ILL 6002). Further, these were more pronounced on genotype ILWL 180 than on genotype ILL 7537. Subsequently, host perception of pathogen entry led to a faster accumulation and notably higher amounts of reactive oxygen species (ROS) and phenolic compounds at the penetration sites of the resistant genotypes ILWL 180 and ILL 7537. In contrast, genotype ILL 6002 responded slowly to the A. lentis infection and reaffirmed previously reported symptoms of the disease as highly susceptible. Interestingly, quantification

of H2O2 was markedly higher in the L. orientalis ILWL 180 particularly at 12 hpi compared to landrace ILL 7537, potentially indicative of its superior resistance capability. In conclusion, faster recognition of A. lentis is likely to be a major contribution to the superior resistance observed in genotype ILWL 180 to the highly aggressive isolates of A. lentis assessed.

Keywords: Lentil, Ascochyta lentis, Lens orientalis, histopathology, reactive oxygen species, phenolic compounds

5.2. Introduction

AB of lentil, caused by Ascochyta lentis, is the most widespread fungal disease across lentil cultivating regions globally (Ye et al., 2002). Seedling infection can destroy an entire crop whilst infection at podding/maturity results in poor seed quality (Gossen and Morrall, 1983) and substantial yield losses (Brouwer et al., 1995). In Australia alone, the epidemics of AB itself has caused an estimated $16.2 million AUD losses to the lentil industry (Murray and Brennan, 2012). Although fungicides are mostly effective, improving lentil resistance is vital to manage the disease more sustainably.

Significant numbers of resistance sources have been identified and deployed in resistance breeding programs around the world (Ali, 1995; Erskine et al., 1996; Nasir and Bretag, 1998; Vandenberg et al., 2001). However, continuous reliance on relatively few resistance sources in Australia and Canada, that also contain desirable agronomic and yield characteristics, has likely led to erosion of resistance genes in elite cultivars (Laird, Northfield and Nipper) through selection of more aggressive isolates (Ahmed and Morrall, 1996; Nasir and Bretag, 1997b; Davidson et al., 2016). Therefore, there is an urgent need to identify and introgress novel AB resistance genes/alleles to enhance the longevity of defence mechanisms within elite Australian cultivars. For this, wild species of lentil have been identified as potential reservoirs of useful resistance genes/alleles (Tullu et al., 2010a; Dadu et al., 2017) and inter - specific fertile hybrids were successfully produced using conventional techniques (Gupta and Sharma, 2007; Tullu et al., 2013). To aid in this, an understanding of the key defence responses within target resistant wild accessions is necessary.

A. lentis is a necrotrophic fungus and uses an appressorium based penetration peg to penetrate the external cuticular layers before colonising the host tissue. Successful invasion and colonisation ultimately leads to disease symptoms of necrotic lesions speckled with pycnidia containing pycnidiospores (Roundhill et al., 1995). During the process of infection, conidia of A. lentis germinate within 2 hours post inoculation (hpi) and develop a bulb like appressorium at the tip of germ tube in the following 6- 8 hours for further penetration into the host cell (Roundhill et al., 1995). This then induces a series of defence mechanisms in plants that include physiological, biochemical and molecular responses.

Roundhill et al. (1995) suggested a mere contact of the pathogen on the leaf surface is sufficient to regulate the first line of defences in lentil. The initial response of the infected cell following tissue penetration is an aggregation of cytoplasm and development of papillae, which interrupts further spread of the pathogenic hyphae (Roundhill et al., 1995). Microarray profiling of resistant and susceptible lentil genotypes ILL 7537 and ILL 6002, respectively, revealed the first transcriptional responses of lentil to A. lentis (Mustafa et al., 2009). The results explained most of the defence responses in resistant ILL 7537 were expressed within 6 – 48 hpi whereas in susceptible ILL 6002, they extended up to 96 hpi. The short period of transcriptional regulation in ILL 7537 compared to ILL 6002 may indicate rapid recognition of A. lentis infection. The DE profile of both genotypes (ILL 7537 and ILL 6002) included genes related to biochemical and structural defences such as reactive oxygen species (ROS) expression, synthesis of pathogenesis related (PR) proteins and lignification. However, the delayed activation of defence responses, particularly after 72 and 96 hpi in genotype ILL 6002, may have led to susceptibility (Mustafa et al., 2009).

The upregulation of these genes within the genotype ILL 7537 may have caused reduction in conidial germination percentage, germ tube length and appressoria formation percentage, thereby restricting disease development (Sambasivam et al., 2016). In addition to the physical reasons for delayed fungal establishment in genotype ILL 7537, these histopathological studies also reported the accumulation of ROS, cell wall thickening and cytoplasmic aggregation as part of biochemical and structural defence responses in genotype ILL 7537 (Sambasivam et al., 2016). Conversely, the absence of these events in genotype ILL 6002 may have been the reasons for the observed susceptibility. Also, ROS are well known to trigger a cascade of defence responses such as cell wall strengthening, transcription of defence related genes and hypersensitive cell death which subsequently restrict pathogen growth (Hückelhoven and Kogel, 2003; Lin et al., 2005).

The most recent study on the lentil-A. lentis interaction reported defence-related differential gene expression using RNA-Sequencing within 2 hpi with a single aggressive isolate (AL4) from Australia (Khorramdelazad et al., 2018). Several genes that are representative of key defence functions such as fungal elicitors recognition and early signalling

(2 hpi), structural response (6 hpi), biochemical response (6 hpi), hypersensitive reaction (HR) and cell death (24 hpi), and systemic acquired resistance (24 hpi) were uncovered. Several of these responses validated the previous findings of Mustafa et al. (2009) and provided the genetic components potentially underpinning the defence-related histopathological observations by Sambasivam et al. (2016). In general, the resistant ILL 7537 was able to initiate key defence-related genetic responses faster and at higher amplitudes than the susceptible ILL 6002. However, efficacy of this timing may be largely dependent on the host genotype involved. Therefore, there is a need to fully understand the diversity in timing of previously well characterised defence responses prior to establishing it as a reliable resistance source.

Thus, the aims of the study were to assess the physiological and biochemical defence responses employed by ILWL 180 with reportedly higher resistance than ILL 7537 in response to A. lentis infection. Also, to characterise the timing of these responses in comparison to the resistant ILL 7537 and determine if L. orientalis ILWL 180 resistance is discriminant.

5.3. Materials and Methods

5.3.1. Plant materials

L. orientalis accession ILWL 180 was procured from the Australian Grains Gene bank (AGGB), Horsham, Victoria. Two L. culinaris genotypes ILL 7537 (resistant control) and ILL 6002 (susceptible control) were supplied by the Faculty of Veterinary and Agriculture Sciences (FVAS), Dookie Campus, The University of Melbourne. Three seed of each genotype replicated four times (four pots) were sown in 10 cm diameter pots filled with pine bark soil, fertilised with Nitrosol, Amsgrow ® (4.5 mL/L) weekly and watered on alternate days. Prior to sowing, seeds of ILWL 180 were soaked in water overnight to enhance germination and were also sown 7 days earlier than the control genotypes to ensure all seedlings were at the same physiological stage at inoculation on 14 and 21 days after sowing (DAS) for controls and wild genotype, respectively. Pots were placed in a Conviron growth cabinet at Dookie campus, The University of Melbourne and maintained at 18 ± 1°C, 12 h/12 h day/night photoperiod with 300 μE m-2 s-1 of light.

5.3.2. Fungal materials

Single spore cultures of one aggressive isolate (FT13037) and one non-aggressive isolate (F13082) were supplied by the South Australian Research and Development Institute (SARDI), South Australia. Aggressiveness of isolate FT13037 was previously identified by Dadu et al. (2017) and isolate F13082 was identified as non-aggressive on a set of host differentials in a controlled bioassay (Davidson, SARDI, Adelaide, personal communication). Spore suspensions were prepared from 14-day-old fungal cultures as described previously by Dadu et al. (2017).

5.3.3. Experimental design for evaluating fungal structures

Spore germination, germ tube length and appressorium formation of isolates FT13037 and F13082 were assessed to evaluate the infection process at 6, 12, 20 and 30 hpi in all three genotypes using detached and attached leaf assays. Experiments were conducted in a completely randomised design with four replicates sown at each time point/isolate and one leaflet from each replicate was used to assess the development of the infection structures. A total of 100 spores (25 spores per corner) per replication were examined to calculate spore germination and percent appressorium. To measure germ tube length, 100 randomly germinated spores from each replicate were observed. Image processing and analysis was carried out in Java (ImageJ) software version 1. 50i to measure germ tube length. Criteria for determining spore germination, germ tube length and appressoria formation were followed as described in Chapter 3.

5.3.4. Evaluation of infection process by detached leaf assay

Fully expanded 21-day-old leaflets from ILWL 180, and 14-day-old leaflets from ILL 7537 and ILL 6002 were detached separately for each isolate and time point. Immediately after excision, one leaflet from each of four replicates of each host/isolate interaction collected at specific time points was sterilized, inoculated and incubated as described in Chapter 3. Leaflets were then fixed and cleared to remove chlorophyll by immersing them in ethanol: glacial acetic acid (1:2 v/v) for 36 h with at least one change in clearing solution at 24 h (Sambasivam et

al., 2016). Cleared leaflets were stained with lacto-phenol cotton blue (Sigma Aldrich) for 5 min and visualised for fungal structures using an Olympus BHC light microscope and images were captured using a Nikon digital slight DS-Fi2 camera.

5.3.5. Evaluation of infection process by attached/intact leaf assay

In attached assay, pots of all genotypes were inoculated using an air pressurized hand- held sprayer at a concentration of 2 x 106 spores/mL. To enable uniform spread of inoculum droplets on the abaxial side of leaflets, pots were tilted slightly at a 45° angle and then sprayed with a fine mist of inoculum until run-off. Soon after inoculation, plants were covered with paper cups coated with wax (In Hospitality, Shepparton, Australia) and arranged randomly before carefully placing them in the growth cabinet under dark conditions at 16-18°C and 98% relative humidity (RH). One leaflet from each of four replicates of each host/isolate interaction was selected and detached carefully at each time point. Subsequently, the detached leaflets were fixed, cleared and stained as described above to visualise fungal structures.

5.3.6. Experimental design for biochemical analysis of ROS and phenolic compounds

- Histochemical localisation of ROS species (O2 and H2O2) and phenolic compounds were assessed at 12, 24 and 48 hpi in the detached leaflets of all three genotypes. Experiments - were conducted in a completely randomised design. One leaflet each for O2 , H2O2 and phenolic compounds and two leaflets each for quantifying H2O2 were assessed from each of the four replications at each time point to detect the responses of lentil genotypes to isolates FT13037 and F13082.

5.3.7. Histochemical localisation of hydrogen peroxide (H2O2)

H2O2 release in detached leaflets of lentil was examined using a modified 3, 3- diaminobenzidine (DAB) staining method as described by Thordal‐Christensen et al. (1997). Detached leaflets of all three genotypes were inoculated as described in section 5.3.4 and incubated for 12, 24 and 48 h. Inoculated leaflets were immediately immersed in 1 mg mL-1

HCl acidified DAB (pH 3.8) solution and incubated in the dark at RT for 8h. As described above in section 5.3.4, leaflets were fixed, cleared and stained before detecting accumulation

of H2O2 as reddish-brown colouration at the sites of hyphal penetration and neighbouring cells using Olympus BHC light microscope. The images were captured using a Nikon digital slight DS-Fi2 camera.

5.3.8. Quantification of H2O2

H2O2 produced in response to A. lentis infection was quantified using a modified ferrous xylenol orange (FOX) assay (Bellincampi et al., 2000). Briefly, point inoculated detached leaflets, measuring a minimum of 50 mg, each of three genotypes were homogenized in 10 mL 10 mM phosphate buffer (pH 7.0) using a mortar and pestle. Later, the homogenate was filtered through a 250 mm sieve and centrifuged at 7000 rpm for 13 min. The resultant supernatant (1.5 mL) was then added to an equal volume of assay reagent (500 µM ammonium ferrous sulphate,

50 mM H2SO4, 200 µM xylenol orange and 200 mM sorbitol). Subsequently, the assay mixture was incubated for 45 min and absorbance of the FOX complex was measured at 560 nm using

Novaspec® II spectrophotometer. To determine the amount of H2O2 in the leaflets, standard

solutions of different concentrations of H2O2 ranging from 5 µM to 80 µM were prepared in distilled water. An aliquot of 1.5 mL of each concentration was added to an equal volume of assay reagent and incubated for 45 min. The absorbance of the FOX complex was measured at

560 nm. A standard calibration curve was plotted for concentrations of H2O2 and absorbance

A560 to estimate the amount of H2O2 from the resultant regression equation (Figure 5.6). The

concentration of H2O2 was expressed as µM/g FW.

- 5.3.9. Histochemical localisation of superoxide anion (O2 )

- O2 production by lentil in response to A. lentis infection was determined using a nitro- blue tetrazolium (NBT) staining method as described by Ge et al. (2013). Leaflets from all three genotypes were detached, inoculated and incubated as described above. Post incubation at 12, 24 and 48 hpi, leaflets were vacuum infiltrated in 50 mM sodium phosphate buffer (pH 7.5) containing 0.2% NBT for 1 h at room temperature. The leaflets were then fixed, cleared and stained as described above in section 5.3.4. Subsequently, the leaflets were examined for

superoxide release using an Olympus BHC light microscope fitted with a Nikon digital slight DS-Fi2 camera. Dark blue deposits at the hyphal penetration sites and close by cells indicated - O2 release.

5.3.10. Histochemical localisation of phenolic compounds

To detect phenolic compound deposition, inoculated leaflets at 12, 24 and 48 hpi were cleared using ethanol: glacial acetic acid (1:2 v/v) as described in section 5.3.4. Cleared leaflets were stained using 0.05% toluidine blue in 0.1 M phosphate buffer (pH 5.5) for 1 h and examined under Olympus BHC light microscope for greenish–blue deposits corresponding to phenolic compound accumulation (Ge et al., 2013). The images were captured using a Nikon digital slight DS-Fi2 camera.

5.3.11. Statistical analysis

Spore germination percent and percent appressorium formation data were square root transformed prior to statistical analysis and graphs were prepared with original back transformed percent data. Data analysis was conducted in GenStat software package, (Version 16.1.0.10916, 64-bit edition, VSN International Limited, United Kingdom). Homogeneity of variances was tested by plotting a graph between residuals and fitted values. The data were then subjected to a repeated measure analysis conducted with the Mixed Model (REML) procedure. Genotypes, isolates and assay were considered as fixed, replicates as subjects and hpi as time points. Differences among the three genotypes (ILL 6002, ILWL 180 and ILL 7537) at each time point (6, 12 and 24 hpi) for percent spore germination, germ tube length, percent appressorium formation and H2O2 quantification (12, 24 and 48 hpi) were compared based on Tukey’s least significant difference at p = 0.05. Localisation of ROS and phenolic compounds was recorded as either present or absent.

5.4. Results

5.4.1. Infection process/physiological differences

5.4.1.1. Spore germination percentage

In both assays and at all time points, genotypes had a significant effect on the spore germination percent of isolates FT13037 and F13082 (p < 0.001) (Appendix 5.1). Independent of whether the leaf was detached or not, both isolates germinated within 6 hpi on all three genotypes. However, the rate of germination on all three genotypes differed for both isolates and gradually increased between 6 hpi and 30 hpi (p = 0.035) (Appendix 5.1). At 30 hpi, a mean of 75.86% and 69.37% spores of isolates FT13037 and F13082, respectively, had germinated on the detached leaflets of the three genotypes (Figure 5.1). In contrast, only 25.77% of FT13037 and 19.69% of F13082 spores had germinated when the leaflets were attached to the plants when inoculated. Further, among the three genotypes, the lowest spore germination percent for the aggressive isolate FT13037 at 30 hpi with a mean of 70.77% and 15.49% on detached and attached leaflets, respectively, was observed for ILWL 180 (Appendix 5.2 and 5.3).

5.4.1.2. Germ tube length

Genotypes, assays, and isolates had a significant effect on germ tube length of isolates FT13037 and F13082 (p < 0.001) (Appendix 5.4). This suggested that the length of the germ tube is a genotype-dependent trait. For all genotypes, the length of germ tube increased over

time and both isolates produced shorter germ tubes when inoculated on leaflets attached to plants compared to detached leaflets. However, slightly longer germ tubes were observed on intact leaflets of genotype ILWL 180 for isolate FT13037 at 20 and 30 hpi as compared to detached leaflets. In both assays, significant differences in the length of germ tubes produced by aggressive isolate FT13037 were detected between the resistant genotypes (ILWL 180 and ILL 7537) and the susceptible genotype (ILL 6002) at 20 hpi and 30 hpi (Appendix 5.5 and 5.6); however, the shortest germ tubes for both isolates were produced on genotype ILWL 180 (Figure 5.2).

5.4.2. Appressorium percentage

5.4.2.1. Timing and percent of appressorium formation on detached leaflets

Like germ tube length, all factors including genotypes, assays and isolates had significant effect on appressorium formation (p < 0.001) and indicated that appressorium formation is a genotype-dependent trait (Appendix5.7). By 6 hpi, spores of the aggressive isolate FT13037 produced appressoria on genotypes ILL 6002 and ILL 7537 and this increased gradually up to 27.13% and 9.8%, respectively, of germinating spores that had produced appressoria by 30 hpi. In contrast, no appressoria were produced until 12 hpi on genotype ILWL 180, but, a maximum mean of 8.79% had formed by 30 hpi. Meanwhile, spores of the

non-aggressive isolate F13082 did not produce appressoria until 12 hpi on genotypes ILWL 180 and ILL 7537, and just 2.69% of them produced appressoria on ILL 6002 at 6 hpi. Further, a significant difference in this trait was observed for isolate FT13037 among resistant genotypes (ILL 7537 and ILWL 180) and susceptible genotype (ILL 6002) at each time point (Appendix 5.8). The most appressoria were observed on ILL 6002 (11.68%) and the least on ILWL 180 (7.57%), at 30 hpi (Figure 5.3a).

Unlike on detached leaflets, spores of the aggressive isolate FT13037 did not produce appressoria until 20 hpi on genotypes ILWL 180 and ILL 7537 and until 12 hpi on genotype ILL 6002 (Figure 5.3b). Interestingly, spores of the non-aggressive isolate F13082 did not produce appressoria until 30 hpi on genotypes ILWL 180 and ILL 7537 and on genotype ILL 6002 until 12 hpi. However, mean comparisons showed no significant difference in the formation of appressoria for both isolates among the three genotypes (Appendix 5.9). The highest and lowest percent of appressoria formation by isolate FT13037 was observed on genotypes ILL 6002 (10.46%) and ILWL 180 (1.47%), respectively, at 30 hpi. Similarly, leaflets of genotypes ILL 6002 and ILWL 180 were identified with highest (5.11%) and lowest (1.47%) percent of appressoria formation by isolate F13082 at 30 hpi.

5.4.3. Biochemical analysis of ROS

5.4.3.1. Localisation of H2O2

The release of H2O2 in ascochyta infected lentil leaflets of all three genotypes coincided with the formation of appressoria and was detected underneath the appressoria (Figures 5.4a-

d and 5.5a-d; Appendix 5.12 and 5.13). However, visible differences in the intensity of H2O2 production were detected between the three genotypes (Figures 5.4a-d and 5.5a-d).

Accumulation of H2O2 gradually increased in the cells surrounding the infected region over time in the two resistant genotypes, ILWL 180 and ILWL 7537 (Figures 5.4a-c and 5.5a-c). However, this response was, weaker and restricted directly to the infected cells in the susceptible genotype ILL 6002 (Figures 5.4d and 5.5d).

5.4.3.2. Quantification of H2O2

The production of H2O2 in all three lentil genotypes was detected as early as 12 hpi (Figures 5.7 and 5.8) in response to A. lentis infection. Genotypes had a significant effect on the accumulation of H2O2 (p < 0.001) whereas isolates (p = 0.715) and interaction (p = 0.768) showed no significant effect (Appendix 5.10). In all the genotypes, the accumulation of H2O2 increased over time, but at different rates, hence the different concentrations at each time point (Figures 5.7 and 5.8). Interestingly, the wild genotype ILWL 180 responded quickly to the infection and subsequently produced significantly higher concentrations of H2O2 ranging from 43.92 to 73.12 µM/g FW within 24 hpi compared to the resistant control ILL 7537, which had lower concentrations ranging from 5.85 to 65.82 µM/g FW by 24 hpi (Appendix 5.11). Meanwhile leaflets of the susceptible genotype ILL 6002 contained negligible concentrations of H2O2 at 12 hpi with a maximum of only 57.42 µM/g FW H2O2 at 24 hpi.

The concentration of H2O2 in the leaflets of all three genotypes was determined using a 2 standard curve produced from known concentrations of H2O2 at A560 (R = 0.9857; Figure 5.6). The trends of production over the time were then determined in all three genotypes in response to the aggressive and non-aggressive isolate (Figures 5.7 and 5.8).

- 5.4.3.3. Localisation of O2

- Similar to the release of H2O2, O2 production also coincided with the formation of appressoria for both isolates on three genotypes (Appendix 5.14 and 5.15) and was detected underneath the appressoria as early as 12 hpi (Figure 5.4e). However, the response was delayed on ILWL 180 until 24 hpi for isolate F13082 (Figure 5.5e), likely related to the delayed appressorium formation as reported above. Further, a gradual and sustained increase in the - accumulation of O2 at the infection sites was observed in the resistant genotypes ILWL 180 and ILL 7537 (Figures 5.4e-g and 5.5e-g), unlike in the susceptible genotype ILL 6002 (Figures 5.4h and 5.5h).

5.4.3.4. Phenolic compounds localisation

Visible differences were observed among the three genotypes for production of phenolic compounds and their localisation (Figures 5.4i-l and 5.5i-l; Appendix 5.16 and - 5.17). Much like H2O2 and O2 production, accumulation of phenolic compounds was also detected beneath the appressoria and lining the cell walls of infected cells. Visible differences were apparent for the intensity and timing of accumulation. In the resistant genotypes, ILWL 180 and ILL 7537, accumulation of phenolic compounds was detected as early as 12 hpi and by 24 hpi in response to isolate FT13037 and isolate F13082, respectively, before gradually increasing at 48 hpi (Figures 5.4i-k and 5.5i-k). In contrast, phenolic compounds were not detected on the susceptible genotype ILL 6002 until 24 hpi for both isolates. Following inoculation with isolate FT13037 the amount of phenolic compounds surrounding the infected region increased at 48 hpi (Figure 5.4l). This was not observed for isolate F13082 (Figure 5.5l).

Figure 5.4: Histochemical localisation of biochemical defence responses elicited within the leaflets of lentil genotypes ILWL 180, ILL 7537 and ILL 6002 in response to A. lentis isolate FT13037. (a–d) Detection of hydrogen peroxide (H2O2) production by DAB-uptake method. Arrowheads indicate accumulation of reddish-brown - deposits beneath the appresoria and surrounding cells. (e-h) Accumulation of superoxide (O2 ) analysed by NBT method. Dark blue deposits beneath the appresoria - represent O2 production (arrowheads). (i-l) Bright-light micrographs of phenolic compounds detection by staining with toluidine blue. Arrowheads indicate greenish-blue deposits due to accumulation of phenolic compounds within the infected and surrounding cells.

Figure 5.5: Histochemical localisation of biochemical defence responses elicited within the leaflets of lentil genotypes ILWL 180, ILL 7537 and ILL 6002 in response to A. lentis isolate F13082. (a–d) Detection of hydrogen peroxide (H2O2) production by DAB-uptake method. Arrowheads indicate accumulation of reddish-brown - deposits beneath the appresoria and surrounding cells. (e-h) Accumulation of superoxide (O2 ) analysed by NBT method. Dark blue deposits beneath the appresoria - represent O2 production (arrowheads). (i-l) Bright-light micrographs of phenolic compounds detection by staining with toluidine blue. Arrowheads indicate greenish-blue deposits due to accumulation of phenolic compounds within the infected and surrounding cells.

Figure 5.6: Standard calibration curve of absorbance vs concentration of hydrogen peroxide (H2O2; µM)

5.5. Discussion

Upon necrotrophic fungal pathogen recognition, plants display strong and complex defence responses which deter pathogen entry. In lentil, several structural; biochemical and molecular responses were previously reported to underpin defence to A. lentis (Roundhill et al., 1995; Mustafa et al., 2009; Sambasivam et al., 2016). In this study, physiological and biochemical defence responses to A. lentis were evaluated to determine the early defence mechanisms within the L. orientalis resistance source ILWL 180.

Previous histopathology assays employed detached leaf tissues for evaluating plant- pathogen interactions (Armstrong-Cho et al., 2012; Sambasivam et al., 2016) and the conclusions were subsequently applied to whole plants. In contrast, attached leaflets were assessed along with detached leaflets in the present work and the comparison of results showed significant differences between the two assays. For all three host genotypes when challenged with each of the two fungal isolates, detached leaflets displayed higher conidial germination, longer germ tubes and higher appressorial percentages than that of attached leaflets. Similarly, differences were detected between detached and attached leaf assays for length of primary

hyphae produced by Colletotrichum lentis on lentil (Armstrong-Cho et al., 2012). The detachment process may affect systemic hormone defence-related signalling and likely initiates senescence in the leaflets, contributing to the faster invasion of the pathogen (Liu et al., 2007). Gaining from the compromised defence responses, A. lentis infected detached leaflets much earlier than intact leaflets regardless of resistance status of the genotypes. These observed differences in resistance between detached and intact leaflets to AB could be further validated either through the use of defence-impaired mutants or by gene expression studies. By doing so, it would be possible to detect the defence responses with uneven contribution or less responsiveness between detached and intact leaflets. Previously, detachment was reported to compromise the salicylic acid and jasmonate/ethylene dependent signalling pathways of Arabidopsis thaliana leading to easier penetration and aggressive colonization by Colletotrichum linicola A1 and Colletotrichum higginsianum (Liu et al., 2007). Furthermore, these compromised defence responses may potentially bridge the knowledge gap regarding the genes that are involved in the early defence responses and thereby may assist the efforts to identify and develop AB resistant cultivars through marker-assisted selection (MAS).

As with previous reports, pre-penetration events were genotype-specific (Sambasivam et al., 2016; Sari et al., 2017) with both isolates exhibiting differential germination percentages, germ tube lengths, as well as timing and percentage of appressoria formation on both the attached and detached leaflets of the three genotypes assessed. These results provide further evidence of the presence of different defence mechanisms among lentil genotypes. In the present work, and as previously reported (Sambasivam et al., 2016; Sari et al., 2017), conidial germination of both isolates was significantly higher on the attached leaflets of the susceptible genotype ILL 6002 than on the resistant genotype ILL 7537. However, lowest conidial germination percentages were observed on the leaflets of the genotype ILWL 180 for both isolates. This may be due to the early recognition of pathogen through pathogen activated molecular patterns (PAMPs) and pattern recognition receptors (PRRs) including receptor-like kinase proteins and receptor-like proteins (Dangl et al., 2013). In the ILL 7537-A. lentis interaction, protein kinases such as leucine-rich repeat receptor kinase (LRR-RK) and calmodulin domain protein kinase (CDPK) were previously reported to play key roles in early signalling of downstream defence responses (Khorramdelazad et al., 2018). In addition, the dense hairy nature of genotype ILWL 180 leaflets might also have aided in reducing

germination percentages by obstructing the deposition of spores on the leaf surface, as similarly observed in the Masoor-93, a resistant cultivar of Pakistan (Sahi et al., 2000).

Successfully germinated conidia develop germ tubes which help the penetration into host tissues by producing an appressorium at their tip (Roundhill et al., 1995). In previous reports, longer germ tubes and higher appressoria frequencies were determined as indicators of host susceptibility to A. lentis (Sambasivam et al., 2016; Sari et al., 2017). Similarly, conidia of both isolates produced longer germ tubes and higher appressorial percentages on the detached and intact leaflets of genotype ILL 6002 compared to that of genotypes ILL 7537 and ILWL 180. Further, shorter germ tubes and lower appressoria frequencies were detected on genotype ILWL 180 compared to genotype ILL 7537, as similarly reported on Lens ervoides accession L-01-827A (Sari et al., 2017). This is likely due to the timing of activation of structural and biochemical defence responses. Structural defence responses include papillae formation at the point of penetration (Roundhill et al., 1995) to entrap the penetration peg. Sambasivam et al. (2016) demonstrated the earlier formation of papillae in resistant genotype ILL 7537 compared to genotype ILL 6002. Accordingly, differentially elevated transcript levels of laccase diphenol oxidase (PPOl) and exocyst subunit 70A1 family protein (Exo70A1), linked to papillae formation, were detected as early as 2 hpi in genotype ILL 7537, much earlier than in genotype ILL 6002 (Khorramdelazad et al., 2018).

Host biochemical defences include production of anti-fungal compounds such as proteinaceous inhibitors and pathogenesis related proteins (PR), to counteract the tissue penetration by the pathogen. Proteinaceous inhibitors such as pectin methylesterase inhibitor (PMEI), auxin-repressed protein (ARP) and polygalacturonase inhibitor (PGIP), involved in countering cell wall degrading enzymes (CWDE) released by fungi, were found significantly over-expressed in genotype ILL 7537 compared to genotype ILL 6002 (Khorramdelazad et al., 2018). Similarly, elevated levels of PR proteins PR2 and PR4, known for cell lysis and limiting the growth of fungi, respectively, were more highly expressed in genotypes ILL 7537 and 964A-46 than genotype ILL 6002 (Mustafa et al., 2009; Vaghefi et al., 2013). Based on the different defence mechanisms of different genotypes shown above, ILWL 180 may have different genes regulating more efficient molecular defence mechanism to counter A. lentis infection compared to that of ILL 7537.

Together with the differences observed in percentage of appressoria formed among the three genotypes, significant differences were also evident in time taken for the conidia of both isolates to develop first appressorium, which was 6 hpi and 12 hpi on the detached and attached leaflets, respectively, on genotype ILL 6002. However, this was delayed until 12 hpi and 20 hpi on the detached and attached leaflets, respectively on genotype ILWL 180. Similarly, Sambasivam et al. (2016) found differences in timing of appressorium formation on the detached leaflets of three genotypes with known resistances.

Following early recognition of the pathogen triggering signal transduction, plants activate a battery of defence mechanisms. Among them, the oxidative burst with a rapid - generation of ROS such as O2 and H2O2 is considered as one of the earliest defence response - (Doke, 1983). In the current study, all three genotypes produced H2O2 and O2 in response to appressorium-assisted penetration by both isolates at 12, 24 and 48 hpi. However, significant - differences in the magnitude of H2O2 and O2 production occurred between genotypes ILWL - 180 and ILL 7537. H2O2 and O2 production was lower in genotype ILL 6002. Quantification

of H2O2 showed a rise in concentrations at 12 and 24 hpi and a sudden decrease at 48 hpi in all three genotypes in response to the infection by both isolates. Overall, H2O2 production in genotype ILWL 180 was higher at all time points than in genotype ILL 7537 (particularly at 12 hpi). This provides further evidence that early release and accumulation of ROS is a major mechanism employed by resistant lentil accessions to limit the growth of A. lentis in planta (Sambasivam et al., 2016).

- This is the first histological report on O2 involvement in the defence response of lentil - to A. lentis. However, this is an intermediate response, since O2 is rapidly catalysed to H2O2 by superoxide dismutase (SOD), which was previously shown to be elevated during the

interaction (Mustafa et al., 2009). This would help to explain why the elevated levels of H2O2 - were detected more in the infected and neighboring cells compared to O2 , which was confined to the penetration sites on all genotypes. Apart from direct toxicity to the invading pathogen,

the diffusible nature of ROS species, particularly H2O2 across cell membranes, leads to signalling of downstream defence mechanisms including cell wall strengthening (Lin et al., 2005), synthesis of pathogenesis related (PR) proteins (Hancock et al., 2006), which together lead to the expression of the hypersensitive reaction (HR) (Lam, 2004). On the other hand,

elicitation of ROS species also induces the activation of phenylpropanoid metabolism in plants (Jabs et al., 1997) to synthesize phenolic compounds (Dalkin et al., 1990). Accordingly, comparatively higher ROS production in resistant genotypes such as ILWL 180 and ILL 7537 likely increases accumulation of phenolic compounds in the infected and surrounding cells leading to deposition of lignin for physical strengthening and synthesis of anti-microbial compounds such as phytoalexins at the infection site.

In conclusion, based on the physical and biochemical evidence presented, we propose L. orientalis accession ILWL 180 to be a superior and potentially useful source of resistance to A. lentis. This is achieved through delayed pre-penetration events and relatively faster and stronger accumulation of ROS and phenolic compounds to limit the growth and spread of A. lentis post penetration. Future investigations involving differential molecular studies of the specific biological steps within the defence mechanism will be helpful to elucidate the potential novelty of genotype ILWL 180. Additionally, analysis of segregating populations developed among the known resistant genotypes including ILWL 180 may decipher the allelic relationships among them and if the resistant genes are novel.

Chapter 6 - Genetics of resistance to ascochyta blight and an assessment of agro-morphological traits in an interspecific hybrid population of lentil

6.1. Abstract

Wild species and landraces of the cultigen are considered as potential genetic resources for the improvement of crops, particularly with limited genetic base. Introgression of novel genes from the distantly related accessions into the cultigen is desirable to increase the genetic gains in lentil. Incidentally, the limited genetic diversity within the Australian lentil breeding program could have led to the loss of AB resistance of the premium cultivars. This necessitated the urge to incorporate a broader spectrum of durable resistance genes within the cultivated genome. Accordingly, an interspecific hybrid population was developed from a cross between a recently identified ascochyta blight resistant Lens orientalis accession ILWL 180 (wild) and Lens culinaris accession ILL 6002 (cultivated) using conventional hybridisation supplemented

with gibberellic acid (GA3) application after pollination. Subsequently, the population was assessed for the genetic variations for AB seedling resistance and agronomically important

traits. Segregation patterns within the F2 and F5 populations suggested two recessive genes control AB resistance. Additionally, the interspecific hybrid population exhibited transgressive segregants for desirable agronomic traits. Beneficial variants for traits such as days to first flower, plant height at flowering (cm), number of nodes below first flower node, seed diameter (mm), 100-seed weight (g) and seed yield (g) were collected and may be utilised for broadening the genetic base of the Australian lentil breeding program.

Keywords: Lentil, Wild relatives, Interspecific cross, Ascochyta blight, Agro-morphological traits, Genetic diversity

6.2. Introduction

Lentil (Lens culinaris Medikus. ssp. culinaris), is a cool season legume acknowledged as an affordable source of protein (28%) (Grusak, 2009) and an important export cash crop of Australia since its introduction in the 1990’s. Although Australian lentil production reached a maximum of 0.38 million tonnes (Mt) in 2011 (FAOSTAT, 2016), a significant reduction has occurred in subsequent years that was, largely credited to various abiotic and biotic stresses. Among biotic stresses that are of particular significance, AB caused by the necrotrophic fungus Ascochyta lentis is a major disease (Erskine et al., 1994) resulting in poor yields and reduced seed quality (Morrall and Sheppard, 1981; Gossen and Morrall, 1983). Although fungicides along with best cultural practices and high yielding resistant cultivars form an effective strategy to control the disease, cultivation of resistant cultivars remains preferable for reducing impact on the environment and economic viability.

Breeding for AB resistance in modern cultivars has been one of the priorities in the Australian breeding program considering the prevalence of conducive environments for AB development during the lentil cropping season (Rodda et al., 2017). Therefore, several cultivars with resistance to AB have been released for commercial production over the years such as PBA Hurricane XT (2013) and PBA Jumbo2 2013 and 2014, respectively (Pulse Australia, 2016). However, the resistance within popular cultivars such as Northfield and Nipper has become compromised due to a selective adaptation and evolution of highly aggressive A. lentis isolates (Davidson et al., 2016). Furthermore, a larger threat looms over the Australian lentil industry considering the widespread presence of lentil cultivars sharing cultivars Northfield, CDC Matador and Indianhead in their pedigree and whose resistance has been questioned in recent times (Davidson et al., 2016). To avoid widespread resistance-breakdown, selection and deployment of novel resistance sources in the Australian lentil breeding program is necessary.

However, cultivated lentil germplasm over the years has been selected for higher yield and related traits, which has led to a loss of variability for other important traits including agronomical, resistance to various biotic or tolerance to abiotic stresses (Ford et al., 1997; Alo et al., 2011; Lombardi et al., 2014). Conversely, distant species of lentil possess useful variations for agronomical traits, disease resistance and abiotic stress tolerance (Gupta and

Sharma, 2005; Tullu et al., 2006a; Gupta and Sharma, 2007; Podder et al., 2013; Singh et al., 2013). Indeed, the wild genetic base of lentil was previously explored for sources of AB resistance and considerable variation was revealed (Bayaa et al., 1994; Tullu et al., 2010a; Dadu et al., 2017). Several accessions were consistently resistant to isolates of different geographical origin such as Syria, Canada and Australia, and their resistance may be considered more durable than that in existing cultivated lentil (Bayaa et al., 1994; Tullu et al., 2010a; Dadu et al., 2017). Therefore, novel resistance sources such as that detected in ILWL 180 should be further explored for incorporation into elite cultivated backgrounds (Dadu et al., 2017; Dadu et al., 2018a).

The usefulness of wild resource depends upon the crossability with the cultivated lentil and this varies considerably due to pre - and post - fertilization barriers (reduced pollen fertility, chromosomal aberrations and embryo abortion) (Abbo and Ladizinsky, 1991; 1994; Gupta and Sharma, 2007). Successful interspecific hybrids have been produced between extremely

diverse species through various interventions such as, post pollination GA3 application, ovule and embryo rescue techniques (Cohen et al., 1984; Ahmad et al., 1995; Fratini and Ruiz, 2006; Gupta and Sharma, 2007; Fiala et al., 2009; Tullu et al., 2013; Saha et al., 2014). Also, valuable breeding material have been generated with variation for agronomical and yield related traits through conventional interspecific hybridisation involving primary gene pool species (L. orientalis and L. odemensis) and cultivated lentil (Gupta and Sharma, 2007; Singh et al., 2013). So far, a small number of disease resistance genes have been introgressed from wild species into cultivated lentil including anthracnose resistance from L. ervoides using embryo rescue techniques (Fiala et al., 2009; Vail et al., 2012) and AB resistance genes from L. orientalis, L. odemensis and L. ervoides (Ahmad et al., 1997; Ye et al., 2000).

Therefore, wild lentil species and accessions offer great potential for widening the genetic base of the cultivated lentil. The aim of the present study was 1) to transfer AB resistance to cultivar ILL 6002 from resistant L. orientalis accession ILWL 180 using conventional methods and 2) to determine the genetics of inheritance of AB resistance in ILWL

180 using F2 and F5 populations and other agronomic traits using F2 population.

6.3. Materials and Methods

6.3.1. Plant materials and interspecific crossing

L. orientalis accession ILWL 180 (AB resistant) and L. culinaris subsp. culinaris accession ILL 6002 (AB susceptible) used in this study were kindly provided by the Australian Grain Gene Bank (AGGB), Horsham, Victoria. In general, ILWL 180 is a moderate to late flowering dwarf and profusely branched plant with prostate growth habit, dehiscent pods and small lens shaped tan coloured seeds. ILL 6002 is a tall, erect and early flowering plant, and produces indehiscent pods with large sized green coloured seeds. All crosses were done in the glass house, Dookie campus, The University of Melbourne, Australia in the month of September 2016. ILWL 180 was used as the male and cultivar ILL 6002 as the female parent. The crosses were attempted by manually emasculating selected ILL 6002 flower buds whose petals were as long as or at least 75% of the length of the sepals (Tullu et al., 2013). Care was taken not to damage the stigma while removing the anthers from the flower using a sharp needle with a tiny bend at the tip. Emasculations were performed in the evenings and the flowers to be pollinated were tagged for pollination the following mornings. Manual pollination was done by rubbing pollen collected from the male parent on the stigmas of female parent carefully with a needle. Pollinated flowers were tagged with a record of cross combination, date and number of crosses performed (Figure 6.1a). Immediately after pollination and until the day pod formation was initiated, a drop of GA3 was carefully placed on the pedicel of pollinated flowers to aid in successful embryo and pod development. Orange seed cotyledon colour was used as a morphological marker to acertain the true hybrids developed through successful crossing (Gupta et al., 2007; Singh et al., 2013) (Figure 6.1b).

6.3.2. Plant propagation and experimental design

Both parents and F1 seeds were raised in 25 cm diameter pots, filled with pine bark potting mix (Australian Growing Solutions, Tyabb, VIC, Australia, 3913), fertilised by Nitrosol, Amsgrow ® (4.5 mL/L) once in two weeks and watered as required. Pots were maintained in a Conviron growth cabinet, Dookie campus, The University of Melbourne at 18 -2 -1 ± 1°C, 12 h/12 h day/night photoperiod and 300 μE m s light intensity. F2 population was

produced from selfed F1 plants. A total of 282 F1:2 seeds (LA-1) were harvested and evaluated

for AB resistance. In addition, 199 (LA-2) and 442 (LA-3) F1:2 seeds derived from two other independent crosses were used for genetic analysis of various agronomic traits. The seeds

harvested from each F2 plant of LA-2 population was raised, selfed and advanced to 124

recombinant inbred lines (RIL; F5 generation) at the Centre for Plant Genetics and Breeding, The University of Western Australia, Crawley utilising an accelerated single seed descent

method as described by Croser et al. (2016).

To evaluate the mode of genetic inheritance for AB resistance, seeds of parents (ILWL

180 and ILL 6002), the F1, F2 (four seeds per pot) and F5 generation (four seeds per each RIL)

were sown in 10 cm diameter pots. Both the parents and F1 seeds were sown in triplicate pots (three seeds each) for inoculated and control (water) treatments and were arranged in a completely randomised design in a controlled Conviron growth cabinet at Dookie campus, The University of Melbourne, under conditions as described above. In order to assess the genetic

nature of different agronomic traits, F2 seeds (one seed/pot) of LA-2 were sown in 199 pots (15 cm diameter) and maintained in a glass house at 20 ± 5°C temperature and 16 h/8 h day/night photoperiod. Along with F2 of LA-2 population, both parents and F1 were sown in 15 cm diameter pots and in three replications.

6.3.3. Fungal material, preparation of inoculum and bioassay

A single spore culture of A. lentis isolate FT13038 was supplied by the South Australian Research and Development Institute (SARDI), South Australia. Isolate FT13038 was earlier shown to be highly aggressive and allowed to effectively distinguish between the AB resistance in ILWL 180 (resistant) and ILL 6002 (susceptible) (Dadu et al., 2017). A spore suspension was prepared and adjusted to 1x106 spores/mL as described by Davidson et al. (2016) and Sambasivam et al. (2016).

Three-week-old seedlings of wild parent ILWL 180, two-week-old F1, F2, F5 seedlings and cultivar ILL 6002 were uniformly inoculated using an air pressurized hand sprayer until run off and maintained as described in Chapter 3, to promote development of disease symptoms on inoculated plants.

6.3.4. Disease assessment

Each seedling of parents ILWL 180 and ILL 6002, F1, F2 and F5 generations of LA-1 was scored for AB at 7, 14 and 21 days post-inoculation (dpi) as % area of plant diseased (% APD), incorporating leaf and stem lesions of four nodes that were spray inoculated (Davidson et al., 2016). Square root transformed data were analysed using analysis of variance (ANOVA) in GenStat® 16 version.

6.3.5. Resistance as a Mendelian trait

Individuals whose resistance status was statistically non-significant from resistant parent ILWL 180 were rated as resistant. Plants with significantly different scores from resistant parent IL1L 180 were classified as susceptible (Fiala et al., 2009; Tullu et al., 2013).

Ratios from the observed segregations within the F2 and F5 populations were compared to the expected Mendelian ratios to establish the appropriate model for genetic control using Pearson’s chi-square test (Pearson, 1900).

6.3.6. Evaluation of genetic variation within the interspecific hybrid population for agronomical traits

Each pot with a single F1:2 seed of LA-2 population was considered as an individual experimental unit to assess the leaf, flowering and seed related traits. Data were recorded at four different growth stages: 1) appearance of first flower, 2) during flowering, 3) pod filling and 4) maturity. As soon as the first flower appeared, traits such as, plant height below first flowering node (cm), node number below first flowering node and days to first flower were recorded. Flower petal colour (violet, light violet or white with blue lines), and plant height at flowering (cm) were recorded during the flowering stage. At the initiation of the pod filling stage, data were collected for leaf size (small, medium or large), tendril length (rudimentary or

prominent) and peduncle length (cm). The harvested seeds from each F2 plant of LA-2 were collected, threshed, cleaned and traits such as 100-seed weight (g) and seed yield (g) were determined. Inheritance of traits such as cotyledon colour (orange or yellow), seed coat pattern (present or absent), seed coat colour (tan, brown, grey, or green) and seed diameter (mm) were

determined using F1:2 seeds of LA-3 population. Seed diameter was determined by measuring

the diameter of each individual F1:2 seed using a pair of Vernier callipers.

Chi-square tests were performed to acertain Mendelian segregation ratios for various morpho-agronomical traits. Significant effects of parents on each agro-morphological trait was analysed using ANOVA tests within the GenStat (16th edition software). Individuals within the population that showed statistically significant differences to the mean of the best performing parent for any trait measured based on Tukey’s least significant difference were determined as transgressive segregants as described by (Al-Bakry and Al-Naggar, 2011).

6.4. Results

6.4.1. Interspecific crossing

In total, 430 conventional crosses supplemented with GA3 were attempted between ILWL 180 and ILL 6002 during the month of September 2016. The month had a mean temperature of 16.4◦C, 72.8% mean RH and the day length increased from 12/12 h to 13/11 h light/dark as the month progressed. Within the crossing period, no clear relationship was observed between the environmental factors and the success of crossing (Table 6.1). Of the

430 crosses, 46.74% were successful and resulted in the production of 260 healthy F1 seeds, while the remaining attempts (53.26%) failed to produce hybrid seeds. Among these, 38.41% pollinations of the did not result in the development of pods and 44.10% had pods but with an aborted embryo. A minor fraction (17.46%) of the total crosses produced selfed seeds.

Figure 6.1: Interspecific hybridisation between a) L. orientalis accession ILWL 180 (male parent) and L.

culinaris cultivar ILL 6002 (female parent); b) Variation in seed cotyledon colour of parents and F1 seed.

6.4.2. Genetic basis of resistance to A. lentis in L. orientalis accession ILWL 180

Seedlings of both parents, the F1, F2 and F5 population were scored for AB symptoms on the 7th, 14th and 21st dpi with isolate FT13038. On the 7th dpi, symptoms were superficial on seedlings and were not considered for segregation analysis. On the remaining two assessment dates, mean disease scores did not differ significantly and hence disease scores recorded 21 dpi were used to calculate the segregation ratio to determine the inheritance model. The mean disease scores of parents ILWL 180 and ILL 6002 were 1.2% APD and 7.43% APD,

respectively, at 21 dpi and F1 showed a moderately susceptible reaction (6.12% APD). When

tested for AB resistance, the 282 F1:2, population segregated (Figure 6.2a) and was heavily skewed towards the mean of the susceptible parent (271S:11R) and significantly deviated from the expected Mendelian single gene model (3:1) (Figure 6.2b). However, chi-square analysis of the disease scores showed a segregation pattern not significantly from a 15S:1R which suggested that resistance in genotype ILWL 180 was controlled by two recessive genes (χ2 =

2.65, P > 0.05). Furthermore, a segregation pattern of 3S:1R fitted the ratio of AB of F5 seedlings resistance (111S:13R) validated the hypothetical two gene mechanism observed in 2 the F2 generation (χ = 13.93, P> 0.0001).

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Table 6.1: Details of attempted cross-pollinations, successful and unsuccessful pollinations between L. orientalis accession ILWL 180 (male parent) and L. culinaris cultivar ILL 6002 (female parent) Temperature (°C) Relative Humidity (%) Failed pollinations Number of Successful Number of Number of S. No Date pollinated crossed flowers pod set Number of Max Min Mean Max Min Mean pollinations flowers failed to develop without a selfed seeds into pods seed 1 30/08/2016 18.2 13.7 15.2 86.4 68.9 76.8 7 3 2 1 1 2 1/09/2016 24.8 14 17.4 84.9 56.5 73.6 8 0 5 0 3 3 2/09/2016 22.4 13.8 16.4 83.5 60.7 73.5 6 3 1 1 1 4 3/09/2016 25.1 13.1 16.4 81 45.8 71.3 15 4 5 4 2 5 5/09/2016 21.5 14.2 16.6 81.2 60 72.9 15 6 1 3 5 6 6/09/2016 21.8 13.9 16.2 79.4 65.6 75.5 23 2 10 6 5 7 7/09/2016 25.8 13.9 17.5 80.8 62.5 74.8 15 8 2 3 2 8 8/09/2016 24.3 14.4 18 86.1 66.3 78.7 23 10 5 6 2 9 10/09/2016 26.3 14.2 17.5 89.3 47.2 70.9 28 6 15 7 0 10 11/09/2016 22.1 13.8 16.4 71 58.2 66.6 18 11 1 3 3 11 12/09/2016 23 13.9 16.1 79.8 61 71.5 26 11 4 6 5 12 13/09/2016 24.6 14 17.1 83.2 61.1 77 21 18 0 1 2 13 14/09/2016 20.9 14.6 16.2 90.6 66 76 22 12 2 6 2 14 15/09/2016 21.4 14.3 15.9 80.7 62.2 73 15 15 0 0 0 15 18/09/2016 18 14.5 15.2 85.8 70.1 79.5 13 9 0 4 0 16 19/09/2016 21 14.1 16 71.5 63.1 67.3 20 13 0 5 2 17 20/09/2016 22.4 13.8 16.5 77.5 60.7 71.1 24 10 4 6 4 18 21/09/2016 15.7 13.9 14.7 86 77.8 81.8 20 13 0 7 0 19 22/09/2016 21 14.3 16.5 77.6 66 70.9 16 6 3 7 0 20 23/09/2016 23 13.7 17 80.4 61.1 70.9 19 8 3 7 1 21 26/09/2016 23.2 14.2 17.4 72.7 55.3 63.9 21 6 12 2 1 22 27/09/2016 20.8 14.4 16.4 74.6 59 67.9 23 11 5 4 3 23 28/09/2016 22.4 14 17 75.6 61.8 69.2 11 4 5 2 0 24 29/09/2016 21 15.1 16 81.4 61.5 73.3 21 12 2 7 0 Total 430 201 (46.74) a 87 (20.23) b 98 (22.79) c 44 (53.31) d a Percentage of successful pollination in parenthesis; b Percentage of crossed flowers failed to develop into seeds; c Percentage of pod set without a seed; d Percentage of selfed seeds

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Additionally, the major contributing factors of seedling resistance were evaluated for segregation in the F2 and the F5 generations (foliar resistance and stem resistance). A similar segregation pattern of seedling resistance was observed in both generations (Table 6.2).

Table 6.2: Segregation pattern for ascochyta blight seedling, foliar, stem and broken stem resistance in

the F2 and F5 generation of LA-1 and LA-2 populations, respectively, derived from the interspecific cross (L. culinaris ILL 6002 × L. orientalis ILWL 180)

Observed segregation Expected Trait χ2 value p> Resistance Susceptible ratio

F2 generation Seedling 11 271 1:15 2.65 0.05 resistance Foliar 20 262 1:15 0.3414 0.5 resistance Stem 12 270 1:15 1.914 0.1 resistance

F5 generation Seedling 13 111 1:3 13.93 0.0001 resistance Foliar 28 96 1:3 0.387 0.5 resistance Stem 26 98 1:3 1.0752 0.25 resistance

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6.4.3. Evaluation of interspecific progeny for agronomical traits

Apart from AB resistance, the parents also varied in various agronomically important traits. Fourteen traits were evaluated to understand respective segregation patterns and reveal

variability within the F2 populations. The analysis of F1 plants revealed the dominant nature of the respective traits (Table 6.3). Chi-square analysis of the phenotypic ratios for tendril length and cotyledon color indicated single gene control, while the phenotypic ratios for traits such as leaf size, flower petal color and seed coat pattern best fitted a two-gene model. However, the mode of the gene interaction varied between each trait (Table 6.4) and phenotypic ratios for the trait seed coat color did not fit the expected two gene model of inheritance (12:3:1).

Table 6.3: Ascochyta blight resistance status and agronomical trait description of L. orientalis ILWL

180, L. culinaris ILL 6002 and F1 derived from the cross

S. No Trait ILWL 180 ILL 6002 F1 1 AB seedling resistance (% APD) 1.2 7.43 6.22 2 Leaf resistance (% APD) 0 8 6.32 3 Stem resistance (% APD) 2.46 6.81 6.12 4 Cotyledon colour Orange Yellow Orange 5 Days to first flower 111 73 70 Plant height below first 6 15 30 25 flowering node (cm) Node number below first 7 4 6 7 flowering node 8 Flower petal colour Violet White with blue lines Violet 9 Plant height at flowering (cm) 30 56 60 10 Leaflet size Small Large Large 11 Tendril length Rudimentary Prominent Prominent 12 Peduncle length (cm) 1 3 3.2 13 Seed coat pattern Present Absent Present 14 Seed coat colour Tan Green Tan 15 Seed diameter (mm) 3.42 4.92 4.76 16 100-seed weight (g) 1.32 4.25 3.2 17 Seed yield (g) 2.67 5.21 5.76

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Table 6.4: Segregation ratios for various morphological traits in the F2 generation of LA-2 population derived from the interspecific cross (L. culinaris ILL 6002 × L. orientalis ILWL 180)

Trait Observed segregation Expected ratio Gene control χ2 value P>

Small Medium Large Leaf let size 9:3:4 Recessive epistasis 5.2661 0.05 63 106 30

Prominent Rudimentary Tendril length 3:1 Mendelian single gene 11.4501 0.0001 123 68

Flower petal Violet White Dominant and recessive 13:3 2.769 0.2 colour 165 27 epistasis

Cotyledon Orange Yellow 3:1 Mendelian single gene 0.8663 0.25 colour 340 102

Present Absent 13:3 Dominant and recessive Seed Pattern 11.538 0.0001 387 55 epistasis

Tan Brown Green Seed coat colour 12:3:1 Did not fit the expected ratio 315 118 9

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The phenotypic data for the traits of plant height below first flowering node, node number below first flowering node, plant height at flowering, seed diameter and 100-seed weight did not produce discrete classes but were instead distributed across the range of parental phenotypes and may be quantitatively inherited (Figure 6.3 and Table 6.5). Although traits such as days to first flower, peduncle length and seed yield revealed considerable variability in the F2 population beyond the range of the parental phenotypes, means were slightly skewed towards the phenotypic means of the parent ILWL 180 (Figure 6.3). Incidentally, the review

of the performance of F1 plants revealed that the phenotypic means of the F1’s were comparable with the phenotypic means of the female parent ILL 6002 for the traits of days to first flower, plant height below first flowering node, node number below first flowering node, plant height at flowering, peduncle length, seed diameter, 100-seed weight and seed yield (Table 6.3).

In the F2 population, phenotypes similar to the parental phenotypes were observed for all of the traits except for days to first flower for which phenotypes flowering at 73 days after planting as the female parent ILL 6002 were absent (Figure 6.3a). However, transgressive segregants that were significantly better than the best-performing parent were evident for traits

such as days to first flower (3 F2 plants), plant height below first flowering node (12), node number below first flowering node (55), peduncle length (4), seed diameter (53) and seed yield (3) (Figure 6.3; Appendix 6.1).

Table 6.5: Descriptive statistics of morphological traits of F2 generation of LA-2 population derived from the interspecific cross (L. culinaris ILL 6002 × L. orientalis ILWL 180)

Std. Trait Mean Median Range Variance Skewness Kurtosis Dev. Days to first flower 121.02 129.00 92.00 405.78 20.14 -1.08 0.31 Plant height at flowering (cm) 39.85 39.50 47.00 79.06 8.89 0.13 -0.04 Plant height below first flowering node (cm) 21.21 21.00 39.00 60.66 7.79 0.55 -0.24 Number of nodes below first flower node 6.94 6.00 15.00 10.33 3.21 0.54 -0.35 Peduncle length (cm) 1.85 1.50 4.70 0.84 0.91 0.67 -0.01 Seed diameter (mm) 4.73 4.74 2.98 0.20 0.48 0.13 0.86 100-seed weight (g) 2.39 2.35 4.39 0.43 0.65 0.29 1.07 Seed yield (g) 2.11 1.81 6.61 2.40 1.55 0.79 -0.06

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6.5. Discussion

The major objective of this study was to introgress and understand the inheritance of AB resistance from the recently identified highly resistant L. orientalis accession ILWL 180 into the cultivated species of lentil. Being an accession from the primary gene pool, ILWL 180 was readily crossable with the cultivar ILL 6002 with a success rate of 46.74%. Crossing was

aided by the exogenous application of GA3 soon after pollination and until the pod formation.

GA3 is required for healthy embryo development (Plackett and Wilson, 2018) and previous studies in lentil reported successful hybridisations supplemented with GA3 (Ahmad et al., 1995; Gupta and Sharma, 2007). Additionally, crossing during optimal conditions may have contributed to the hybridisation success (Singh et al., 2013).

Analysis of segregation ratios in the F2 population revealed that the resistance to AB may be conferred by two recessive genes since the segregation pattern (15:1) was found to best

fit. Assuming duplicate dominant epistasis, it was hypothesized that the susceptibility in the F2 population was determined when both alleles were dominant at both or at either of the loci. The condition in this study can be explained by representing two genes with letters A and L and alleles with Aa and Ll. Putatively the parental genotypes of ILWL 180 (aall) and ILL 6002 (AALL) were considered homozygous for resistance and susceptibility. Upon interspecific crossing, the possible genotypes for the susceptible phenotype in the segregating population

(F2) could be AALL / AAll / AALl / AaLL / Aall / AaLl /aaLL / aaLl, while recessive alleles at both loci (aall) may determine a resistant phenotype. As expected with the two-gene model,

the distribution pattern of AB resistance within the F5 families fitted a 3:1 segregation ratio and

thus validated the proposed two gene model. The difference in the number of F2 plants (282) /

F5 families (124) produced in this study was significant and could be a problem of self-fertility

as expected in wild crosses. This could be due to chromosomal rearrangements beyond the F4 generation (Fiala et al., 2009).

Ahmad et al. (1997) proposed a single dominant gene for resistance to AB in L.

orientalis, however this report was inconclusive because of the small number of F2 (48) plants tested (Ye et al., 2000). In a separate study, the same cross was assessed at F3 and reported to contain a two gene (dominant) inheritance model for the AB resistance (Ye et al., 2000).

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However, the findings of the current study are contradictory to the two dominant gene model by Ye et al. (2000). As different accessions were evaluated in these two studies, this could point to potentially different genetic mechanisms to resist AB infection. Nevertheless, this is the first report of a two recessive gene model for the inheritance of AB resistance in L. orientalis. A similar two recessive gene model was proposed to be conditioning the resistance to Collectotrichum lentis in L. ervoides (Fiala et al., 2009; Tullu et al., 2013).

The agronomic performance of the interspecific F2 population was evaluated under controlled conditions to reveal whether variations could offer an opportunity to widen the genetic base of the cultigens. As wider variation was observed among parents, higher levels of variations were evident in the F2 population for various traits. In addition, transgressive segregants were detected for traits such as days to first flower, plant height below first flowering node, node number below first flowering node, peduncle length, seed diameter and seed yield. This presented greater genetic variation outside the range of parental phenotypes that can be employed in future breeding programs to recover better phenotypes. The appearance of transgressive segregants were expected when more similar parental phenotypes were used for crossing (Rieseberg et al., 1999; Tullu et al., 2013). Accordingly, a higher number of transgressive segregants (55) were evident for the trait node number below first flowering node because of the narrow variation among the parents as similarly observed by Tullu et al. (2013). Conversely, the frequency of transgressive segregants was comparatively lower for the other traits tested because of the wider variation between parents. However, there were higher numbers of transgressive segregants for the trait days to first flower though parents exhibited wider variation. Complementary gene action, overdominance and/or epistasis between the genes among the parent species are shown as the reasons for the emergence of transgressive segregants (Rieseberg et al., 1999; Guindon et al., 2018). Similarly, transgressive segregants were detected for various traits in previous studies in interspecific hybrid populations derived from crosses involving L. culinaris and wild species of lentil (Singh et al., 2013; Tullu et al., 2013).

Flowering time in lentil is dependent on photoperiod and temperature and flowering is understood to accelerate with the increase in day length and temperature (Erskine et al., 1990a). With the long winter season allowing for a long vegetative period in Australia, cultivars with

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early to moderate flowering time are preferred for cultivation (Pulse Australia, 2016). The

flowering time in the F2 population ranged from 56-148 days and 23% of the population flowered in less than 100 days. Plant height was reported to be strongly associated with yield (Tullu et al., 2013; Kumar et al., 2018b). However, in this study, the correlation between traits plant height at flowering and seed yield showed a weaker association (r = 0.38; Appendix 6.2).

Consistent with the suggestion, seven lines of the F2 population performed better than the cultivar ILL 6002 for trait plant height at flowering and produced lower yields compared to

either the F1 or to cultivar ILL 6002. Another, four lines performed better than both the F1 and cultivar ILL 6002 but were shorter plants. The reason for the higher seed yield despite the shorter phenotypes might be because of the higher number of flowering nodes combined with the shorter internode length that are known to influence the seed yield (Kumar et al., 2018b). Alternatively, transfer of more plant assimilates to seed rather than the biomass may have contributed to the higher seed yield (Zamski, 2017). Although the trait number of flowering nodes were not investigated in this study, greater variability was recorded for number of nodes below first flower node which is highly associated with the total number of flowering nodes (Tullu et al., 2013). Interestingly, two lines that showed higher seed yield had higher numbers of nodes below the first flower node in the F2 population. Furthermore, 39% of the population performed better than the best parent and F1 for number of nodes below first flowering node. This variation could be the result of combination of positive alleles from both parents and the selection for such traits could benefit the breeding programs.

Seed quality characters determine the marketable value of lentil seeds (Khazaei et al., 2017). For example: small size, brown or tan colored testa, patternless seed coat and seeds with red cotyledon color are largely preferred by the Indian subcontinent (Muehlbauer et al., 2009).

An assessment of seed quality characters in the F2 population revealed ample variation for traits including seed diameter and 100-seed weight beyond and within the range of both the parents, respectively. Additionally, for seed diameter, a higher number of transgressive segregants (53)

were detected within the F2 population that performed better than the best parent, ILL 6002. Both traits are highly correlated with a correlation coefficient (R) of (r = 0.74), and individually were found to be positively correlated with total seed weight (Appendix 6.2). This indicated that seed weight and seed size may share the same genomic region and collectively influence the total seed weight in lentil. Accordingly in L. culinaris, two major QTLs responsible for

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seed weight (qSW) and seed size (qSS), were found co-localized on LG4 (Verma et al., 2015). Moreover, two QTLs controlling seed weight and seed size were found co-segregating on LG3 in the linkage map developed from an interspecific cross between L. orientalis and L. culinaris (Fratini et al., 2007).

Additionally, genetic control of qualitative traits of the seed such as cotyledon color and seed coat pattern revealed that they are controlled by a single and two genes, respectively. Orange cotyledon color was found dominant in this study as it was observed in previous inheritance studies involving L. orientalis and L. culinaris crosses (Gupta et al., 2007). The locus (Yc) responsible for cotyledon color was mapped in the interspecific populations derived from a cross between L. orientalis and L. culinaris (Fratini et al., 2007). The seed coat pattern locus (Scp) was mapped to a different LG than that of cotyledon color in the same interspecific populations (Fratini et al., 2007). Seed coat color was previously reported to be controlled by two dominant genes at two independent loci with a phenotypic ratio of 9 brown: 3 grey: 3 tan: 1 green (Vandenberg and Slinkard, 1990). In contrast, the segregation ratio for seed coat color in this study did not fit into the expected two gene model. This could be a possible case of segregation distortion, which is expected in interspecific crosses as a consequence of chromosomal rearrangements, abortion of male or female gametes and/or preferential fertilization of either male or female gametophyte (Reflinur et al., 2014).

In conclusion, it is evident from this study that L. orientalis accession ILWL 180 possesses favorable agronomic trait combinations along with AB resistance. However, further research using RILs will be needed to precisely estimate these genetic effects. Although it is suggestive to include accession ILWL 180 in the breeding programs to bring in new genes to the breeding pool, the potential of linkage drag needs to be estimated. The challenges of linkage drag, and selection of desirable traits may be overcome by employing molecular markers. With the availability of next generation sequencing (NGS) technology and large-scale SNP discovery, identification of markers closely linked to desirable trait has become achievable. The analysis of QTLs to AB resistance is discussed in the Chapter 7, phenotyping and genotyping of other useful traits will be followed to isolate beneficial alleles.

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Chapter 7 - Identification of quantitative trait loci and candidate genes associated with ascochyta blight resistance in the interspecific RIL population

7.1. Abstract

The Australian lentil industry is affected by various biotic stresses; AB caused by Ascochyta lentis is one of the devastating fungal diseases that result in substantial yield losses. Cultivation of AB resistant cultivars remains the preferable long-term environmentally and economically viable strategy. However, the breakdown of AB resistance of cultivars such as Northfield and Nipper suggests the need for introgression of new and diverse resistance genes to widen the genetic base of cultivars and to improve cultivar stability against the disease. Successful introgression entails an understanding of the genetic basis of resistance. In this context, using a biparental mapping population derived from a cross between a recently identified AB resistant accession ILWL 180 (Lens orientalis) and susceptible cultivar ILL 6002, a genetic linkage map has been constructed using single nucleotide polymorphism (SNP) markers generated from transcriptome sequencing of the parents and recombinant inbred line (RIL) population. Genetic dissection of the RIL population revealed a QTL associated with resistance to AB on linkage group 5. The identified QTL region stretched 4.93 cM and harbored nine putative candidate defence-related genes linked to AB resistance. Furthermore, three nonsynonymous mutations within coding sequences of three putative candidate genes (Uroporphyrinogen decarboxylase (UROD); Glutathione S transferase DHAR3, chloroplastic (GST-DHAR3; and Protein EXECUTER 2, chloroplastic isoform X1 (PEXE2)) related to defence mechanisms against A. lentis were predicted. The QTL analysis and the candidate gene information are expected to contribute to the development of diagnostic markers and enable marker-assisted selection (MAS) to improve AB resistant cultivars.

Keywords: Lentil, Lens orientalis, Ascochyta blight, Genetic linkage map, QTL, Candidate genes

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7.2. Introduction

Lentil (Lens culinaris Medikus. ssp. culinaris), a member of Fabaceae is cultivated across the world for its high dietary benefits. However, a decline of up to 21% in lentil production has been reported in the recent years in Australia (FAOSTAT, 2016). Among many fungal diseases, AB caused by Ascochyta lentis is one of the major constraints affecting gross profits and yield stability globally. The disease causes an estimated $ 16.2 million AUD losses to the lentil industry (Murray and Brennan, 2012). AB resistant cultivars have been released in Australia (Rodda et al., 2017), however, increases in the aggressiveness of the A. lentis population have led to gradual susceptibility of resistant cultivars Northfield and Nipper within a few seasons of their commercial release (Davidson et al., 2016). This urges the need for inclusion of novel and diverse alleles/genes into the resistance breeding program to enhance durability of resistance sources to AB. Previous studies have revealed several resistance sources from wild accessions of lentil from, L. orientalis, L. odomensis, L. ervoides, L. nigricans and L. lamottei (Bayaa et al., 1994; Tullu et al., 2010a). More recently, accession ILWL 180 (L. orientalis) with novel and high resistance to recently evolved and most aggressive Australian A. lentis isolates, was reported (Dadu et al., 2017).

Genetic tagging of AB resistance genes with closely linked molecular markers would accelerate development of lentil cultivars with AB resistance through marker-assisted selection (MAS). For this, a dense linkage map is a pre-requisite to identify the physical location of the gene effects and markers tightly linked or ideally situated on the genome sequence (Sindhu et al., 2014). Consistent with the suggestion, several linkage maps using morphological, allozyme and molecular (hybridisation and DNA based) markers have been constructed in lentil using both intra and interspecific mapping populations (as reviewed by Kumar et al., 2015; Ates et al., 2018b). The length of the maps ranged from 333 – 3,843.4 centiMorgan (cM) and the average distance between two markers varied from 2.6 – 19.3 cM. Despite a low marker density in linkage maps, quantitative trait loci (QTLs) associated with various desirable traits including plant morphology and disease resistance have been mapped on lentil genetic maps as reviewed by Kumar et al. (2015). QTLs conferring resistance to AB from popular resistant cultivars such as ILL 5588 (Andrahennadi, 1994; Sakr, 1994; Ford et al., 1999; Rubeena et al., 2006), Indianhead (Andrahennadi, 1994; Chowdhury et al., 2001) and ILL 7537 (Rubeena et al., 2006)

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have also been detected. These QTLs were tagged using morphological, allozyme or DNA based markers such as random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), or inter simple sequence repeats (ISSR). However, these markers were considered anonymous since the genomic location of the amplified fragments was generally unknown (van Treuren and van Hintum, 2009).

Nevertheless, the advancement of transcriptome and genome-based sequencing technologies identified large numbers of expressed sequence tag-simple sequence repeats (EST-SSR) and single nucleotide polymorphisms (SNP) markers that were associated with putative gene (Kaur et al., 2011; Sharpe et al., 2013; Kaur et al., 2014; Temel et al., 2015; Ates et al., 2016; Aldemir et al., 2017; Bhadauria et al., 2017). Consequently, high density linkage maps were developed in lentil and sequence-related markers facilitated the identification of bridging loci across different maps and hence, high dense consensus linkage maps were developed (Sudheesh et al., 2016a; Ates et al., 2018b). The most recent consensus map included 9,793 diversity arrays technology (DArT)-derived SNP markers, which covered a distance of 977.42 cM with only 0.1 cM between adjacent markers (Ates et al., 2018b). Greater resolution of these maps from different mapping populations led to identification of QTLs and flanking markers associated with AB resistance (Gupta et al., 2012a; Sudheesh et al., 2016a). Sudheesh et al. (2016a) reported two and three QTLs explaining 52 and 69% of the phenotypic variation for resistance to AB in Indianhead × Digger and Indianhead × Northfield populations, respectively. However, none of the attempts have involved a linkage map derived from an interspecific mapping population involving wild species and using next generation sequencing (NGS). Furthermore, mapping QTLs controlling AB resistance in wild species has been particularly challenging due to a segregation distortion phenomenon (Bhadauria et al., 2017).

Apart from constructing high density linkage maps and identification of genomic regions, transcriptome sequencing or genotyping-by-sequencing through transcriptomics (GBS-t) facilitates identification of candidate genes associated with the trait of interest (Malmberg et al., 2018). Candidate genes associated with boron (B) toxicity and flowering time have been identified from the genomic regions associated with these traits on the linkage map developed from Cassab ×ILL 2024 and CDC Robin × 964a-46 recombinant inbred line (RIL) populations, respectively (Kaur et al., 2014; Sudheesh et al., 2016b). In this context, the

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aim of the present investigation is 1) to construct high-density genetic linkage map through a transcriptome sequencing approach for an interspecific population derived from a cross between L. orientalis ILWL 180 and L. culinaris ILL 6002; 2) to locate and characterise the QTLs and candidate genes associated with resistance to AB.

7.3. Materials and Methods

7.3.1. Development of bi-parental mapping populations

A segregating mapping population was developed from an interspecific cross between L. orientalis accession ILWL 180 and L. culinaris accession ILL 6002. The resistance status of accessions ILWL 180 and ILL 6002 as resistant and susceptible, respectively, was confirmed in Chapter 4. In the process of developing the mapping population (F2:5), 198 F2 plants of LA- 2 population were grown in the glasshouse in Dookie campus, The University of Melbourne, in 15 cm diameter pots filled with pine bark potting mix (Australian Grow Solutions, Tyabb, VIC, Australia). Seedlings were watered on alternative days and fertilised weekly using

nitrogen enriched liquid fertiliser Nitrosol, Amsgrow® (4.5 mL/L). Seeds from individual F2

plants were collected and advanced until F5 generation using an accelerated single seed descent method at Centre for Plant Genetics and Breeding, The University of Western Australia,

Crawley as described by Croser et al. (2016). During the process, 58 RILs (F2:5) were lost either

due to sterility, poor seed germination or pest infestation and thus a total of 140 RILs (F4:5) were developed.

7.3.2. Phenotypic assessment of AB resistance under controlled growth conditions

Resistance to AB was assessed in the LA-2 RIL population (N = 140) using an aggressive A. lentis isolate FT13038, which effectively discriminated the resistance between the accessions ILWL 180 and ILL 6002 (Dadu et al., 2017). The experiment was set out in a completely randomised design with two replicates of each RIL and three replicates of both parents. Two to four seeds per RIL (8 seeds in total) and three seeds from each parent (9 seeds in total) were sown in 10 cm diameter pots filled with pine bark potting mix and maintained at 18 ± 1°C, 12 h/12 h day/night photoperiod, 60% relative humidity (RH) and 300 μE m-2 s-1 light intensity in a Conviron growth chamber at Dookie college, The University of Melbourne.

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Seedlings were watered and fertilised as mentioned as in section 7.3.1, to promote germination and plant development. At 14 days after sowing, seedlings were inoculated with the spore suspension of isolate FT13038 prepared as described in Chapter 3. The inoculated plants were maintained, scored and analysed for AB resistance as described previously in Chapter 3.

7.3.3. RNA extraction, cDNA library construction and Illumina sequencing

Total RNA was extracted from selected young leaves of the parents (ILWL 180 and ILL 6002) and 140 RILs using the RNeasy® 96 kit (Qiagen Inc., Hilden, Germany) following the manufacturer’s instructions. The integrity and purity of the total RNA was determined by TapeStation 2200 platform (Agilent Technologies, Santa Clara, CA, USA) following the manufacturer’s instructions. The concentration of total RNA was then confirmed using a Nanodrop (Thermo-Scientific, Wilmington, DE, USA) at two wavelength ratios of A260/230 and A260/280 nm. RNA-Seq cDNA libraries were prepared using a SureSelect Strand-Specific mRNA Library Preparation kit (Agilent Technologies, Santa Clara, CA, USA). This entailed isolation of poly(A) RNA from total RNA, fragmentation of poly(A) RNA, synthesis of double stranded cDNA, adapter-ligated and preparations of indexed cDNA libraries. The quality of the libraries was assessed using TapeStation 2200 platform with D1000 Screen Tape System and equal samples of each cDNA library with a unique barcode were then multiplexed to create a single pooled sample suitable for NGS. The pooled sample was quantified by KAPA library quantification kit (KAPA Biosystems, Boston, MA, USA) and sequenced using the HiSeq 3000 system (Illumina Inc., San Diego, CA, USA).

7.3.4. Variant calling and filtering of single nucleotide polymorphisms (SNP)

The sequence output was demultiplexed and parsed into individual libraries according to the barcodes using a custom Perl script. Following fastq data generation, the raw sequence reads were filtered using a custom perl script and Cutadapt v1.4.1 (Martin, 2011). Reads were filtered by removing adaptor sequences along with reads and bases of low quality (containing more than 10% bases with Q ≤ 20). Reads were trimmed which had three consecutive N's and >3 consecutive nucleotides with Phred score ≤ 20. Finally, any reads that were shorter than 50 bp in length were removed from the final set. The cleaned and trimmed reads were then mapped onto the cultivar Cassab reference transcriptome (Sudheesh et al., 2016b) using Burrows

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Wheeler Aligner (BWA) v0.7.17 with the Mem algorithm (Li, 2013). SNPs between the parents were recorded in the sequence alignment and mapping (SAM) format. The SAM files were converted into binary format (BAM) files, sorted and indexed using SAMtools (version 1.9) sort and view options. BAM files were processed for variant calling through SAMtools mpileup and bcftools (version 1.9), and filtered using vcftools (v0.1.16) (Danecek et al., 2011). Variant calling in RILs was then performed using a SNP-list (biallelic, homozygote in each parent and polymorphic between parents) generated from the parents. Variants with minimum allele depth of 10, average mapping quality score of not less than 30 and minor allele frequency larger than 0.05 were retained. Simultaneously, markers differing from the above criteria were converted into missing values and those with >10% missing data were subsequently removed.

7.3.5. Genetic linkage map construction

Prior to the construction of a high-quality linkage map, markers and genotypes were verified for excessive segregation distortion (5% significance level), genotypes with a high proportion of matching alleles (>95%) and high missing rates (>20%). The filtered, high- quality SNPs were then clustered to linkage groups (LG) to construct a high-density linkage map using the ‘mstmap’ function from ASMap package (v1.0-4) (Taylor and Butler, 2017) in the R statistical computing environment (R Development Core Team, 2015). The ‘mstmap’ function was invoked with the following parameters: ‘Kosambi’ distance calculating function (dist.fun), missing threshold (miss.thresh) of 10% and population type (pop.type) RIL5 and p- value (p.value) of 5.12e-12. Unlinked markers or minor LGs that comprised less than 10 markers were discarded. The generated map was further tested to identify and remove erroneous markers and genotypes showing double crossovers, highly distorted segregation patterns and missing data using the ‘profileMark’ and ‘profileGen’ functions, respectively, from the ASMap package (v1.0-4) (Taylor and Butler, 2017). The map was then reconstructed and the correctness and reliability of the constructed map were confirmed by visually assessing a heatmap, produced by ASMap, showing the estimates of pairwise recombination fraction and LOD scores between each pair of markers (Taylor and Butler, 2017). The tightly linked markers with low recombination were represented by the red blocks along the diagonal line. The linkage map explaining the density within each group was plotted using the LinkageMapView R package (Version 2.1.2) (Ouellette et al., 2018).

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7.3.6. QTL analysis and identification of candidate genes

The constructed linkage map, filtered SNP data and the square-root transformed phenotypic data mentioned previously were used as input for QTL analysis using the R/qtl2 v0.12 package (Broman et al., 2018), an improved modern implementation of the original R/qtl (Broman et al., 2003). A genome scan approach was chosen to identify significant QTL regions, using a linear mixed model accounting for relationships among individuals using a random polygenic effect, supported by a permutation test (n=1000) to determine the LOD threshold for significance for each trait at each time point. A LOD score 3 was used as threshold to detect the QTLs in the linkage map constructed. LOD-1 confidence interval (CI) was used to identify the higher and lower limit CI for the QTLs (LOD > 3), determine the width of the QTL and detect the loci underlying the QTL regions (Snoek et al., 2018). Loci and QTL positions across the linkage map were prepared in R and exported for plotting in MapChart v2.32 (Voorrips, 2002). The sequences of the loci underpinning the QTL regions were extracted from the cultivar Cassab reference transcriptome (Sudheesh et al., 2016b) and annotated using the translated Basic Local Alignment Search Tool (BLASTx) against the non-redundant protein database (nr) at the National Center for Biotechnology Information (NCBI), limiting the search results to species of the Fabaceae family to reveal putative candidate genes as described by Leonforte et al. (2013).

7.3.7. Prediction of candidate gene associated mutations and consequences

Coding sequences (CDS) of each transcript of the putative candidate genes was predicted based on the best matching BLAST results. An in-house R script was used to determine each SNP location relative to the CDS as the 5’-Untranslated region (UTR), 3’-UTR or CDS as well as the position of the SNP relative to the open reading frame (ORF) as 1, 2 or 3 for CDS. For SNPs located in the coding region, the CDS for the reference and alternative alleles were translated to their amino acid sequences and compared to determine the effect of the mutation as synonymous, non-synonymous or non-sense (introduction of a stop codon). In the case of a non-synonymous mutation, the exact position and substitution of the amino acid was recorded as AxxB, where A is the reference amino acid, xx is the amino acid position in the predicted peptide and B is the alternative amino acid, as described by (Ogino et al., 2007).

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Further investigation of the possible effect of the substitution on the protein structure and function was performed by searching for conserved domains and their 3D structure using the conserved domain database (CDD) at NCBI and the Protein data bank (PDB). Crystal structures for the identified putative proteins are yet to be designed and hence the best representative protein structures within the plant species were used to predict the effects of the mutations.

7.4. Results

7.4.1. Phenotypic assessment of LA-2 RIL population derived from cross between ILL 6002 × ILWL 180 for AB resistance

Significant differences for AB resistance were reported between the parents, ILWL 180 (Leaf lesion score = 0.43% APD; Stem lesion score = 1.84% APD; Overall disease score = 1.35% APD) and ILL 6002 (Leaf lesion score = 7.76% APD; Stem lesion score = 7.90% APD; Overall disease score = 7.85% APD) following inoculation with the highly aggressive isolate FT13038 at 28 dpi (p>0.001). Overall disease severity within the RIL population ranged from 0 to 10% APD at 28 dpi. Segregation for AB resistance in the RIL population showed a monomodal and normal distribution following square-root transformation for leaf score ratio at 21 (Shapiro-Wilk test, W = 0.99, p>0.05) and 28 dpi (W = 0.99, p>0.05) and stem score ratio at 28 dpi (W = 0.98, p>0.05) and suggested that the resistance to AB in the wild accession ILWL 180 is polygenic and quantitatively inherited (Figure 7.1a and 7.1b). A small proportion of both the positive (5) and negative transgressive segregants (11) that significantly exceeded the range of parental mean based on Tukey’s Least significant difference were evident in the population.

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7.4.2. Transcriptome sequencing and SNP discovery

A total of 694,694,624 (150-bp) paired end reads were generated by sequencing multiplexed cDNA libraries on the Illumina HiSeq 3000 platform, and an average of 4,997,803

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reads per RIL were obtained. The reads were filtered for low quality, adaptor-free reads and those smaller than 50 bp prior to alignment with the reference transcriptome assembly of cultivar Cassab. On average, 98.5% of reads per sample mapped to the reference transcriptome. A total of 364,804 SNPs were identified from parents upon alignment of uniquely mapped reads to the reference transcriptome. The sequential filtering of the identified SNPs for allele depth, minor allele frequency, quality, heterozygosity and missing markers led to retaining only 25,591 SNPs. Among these, 1,137 high-confidence SNPs that were polymorphic, biallelic, homozygous within and heterozygous between the parents were selected for further analysis.

7.4.3. Construction of a genetic linkage map

Post-filtering of markers and genotypes for segregation distortion, double crossovers, and missing data, the linkage map was constructed using 815 markers (Appendix 7.1) and 92 genotypes (Figure 7.2). The linkage map spread across eight LGs and spanned a total distance of 488.02 cM with a mean marker-marker distance of 0.66 cM. The number of markers within LGs ranged from 32 (LG7) to 217 (LG1), and the genetic distance varied from 21.78 cM in LG8 to 101.66 cM in LG1. Mean marker-marker distances of the longest LG1 and smallest LG7 was 0.47 cM and 0.87 cM, respectively, and the longest distance between adjacent markers was found in LG6 (1.06 cM) (Table 7.1). Among the eight LGs, LG8 had the highest marker density (2.71/cM) and expectedly, LG6 had lowest marker density (1.30/cM) (Figure 7.3). The map had gaps across LGs and in total five gaps greater than 6 cM were found, but not larger than 12.77 cM in LG2 between 0.000001 cM and 12.770642 cM.

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Table 7.1: Marker distribution over the linkage groups of the linkage map derived from a cross between ILL 6002 and ILWL 180

Number of Mean marker- Average Length of Linkage group marker marker distance marker density linkage group numbers LG1 0.47 2.47 101.66 217 LG2 0.56 2.40 66.82 120 LG3 0.51 1.77 48.13 94 LG4 0.64 1.94 87.18 136 LG5 0.71 2.02 93.33 131 LG6 1.06 1.30 41.18 39 LG7 0.87 1.68 27.95 32 LG8 0.47 2.71 21.78 46 Total/Average 0.66 2.04 488.02 815

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Figure 7.3: Illustration of density of the markers within each linkage group of the map derived from a cross between ILL 6002 and ILWL 180

7.4.4. QTL detection

Since no-significant differences were detected for AB resistance within 7 dpi among the RILs and parents, leaf lesion and stem lesion scores at 14, 21 and 28 dpi were considered for QTL analysis. The LOD threshold for significance for each trait at each time point and at 5% confidence level was determined by a 1000 permutations test and using the linkage map. The LOD thresholds obtained for each trait and at each time point averaged at 2.8, so a more stringent LOD threshold was set at 3.0. A QTL with a significant LOD score of 3.20 was subsequently detected on LG5 (Figure 7.4). The QTL identified was associated to the leaf lesion score at 21 dpi and positioned on LG5 from 31.51 cM low CI to 36.44 cM high CI with the peak at 35.2 cM (Figure 7.5). The width of the QTL region was 4.93 cM. Conversely, no QTL was detected above the LOD threshold for trait stem lesion score at any dpi (Figure 7.6).

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Figure 7.4: QTL peak for leaf lesion score at 21 days post inoculation (dpi) on linkage group 5 (LOD > 3) as demonstrated by R/qtl2 package

Figure 7.5: QTL detection for stem lesion score at 14, 21, 28 days post inoculation (dpi) as demonstrated by R/qtl2 package on the linkage map derived from cross between ILL 6002 and ILWL 180

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7.4.5. Identification of candidate genes and associated mutations

A survey for QTL-linked markers revealed ten SNPs from nine loci within the QTL region (Table 7.2). The contig and scaffold sequences underpinning the QTL region were annotated by BLASTx similarity search against protein sequences of species of the Fabaceae family, returning 659 matches with an E-value < 1e-20 and percent identities ranging between 45.42 and 99.10% (Appendix 7.2). Among them, the top five annotations for each of the loci were filtered based on the highest percent identity and highest bit score. One best matching and fully characterised putative candidate gene was selected from the top five annotations for each of the nine loci (Table 7.3).

Post-confirmation of the SNP location on the CDS of each of the nine candidate loci, five markers were found positioned on the CDS and four other markers on the UTR of the putative candidate genes (Table 7.3). A comparison of the five SNPs located on the CDS with the reference protein sequence of the candidate genes revealed three non-synonymous and two synonymous mutations. The three non-synonymous mutations belonged to the putative candidate genes that are associated with stress response such as uroporphyrinogen decarboxylase (UROD; XP_003601037.1), glutathione-S-transferase DHAR3, chloroplastic (GST-DHAR3; XP_013460594.1) and protein EXECUTER 2, chloroplastic isoform X1 (P- EXE2; XP_003601283.1). The effects of the three non-synonymous mutations revealed substitution of an amino acid with the other (Threonine (T) 35 Alanine (A); Phenylalanine (F) 13 Leucine (L); Valine (V) 387 Isoleucine (I)) at specific positions on the coding sequence of the putative candidate genes. An investigation for the possible effects of the mutations on the 3D structure of putative proteins revealed that mutations T35A and F13L may affect the domain and substrate binding sites of the proteins UROD and GST-DHAR3, respectively (Figure 7.7). However, no relative crystal structure was identified for the protein P-EXE2 within the CDD and PDB of NCBI.

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Table 7.2: Details of markers and corresponding loci identified within the QTL region on linkage group 5

Position SNP REF ALT ILL ILWL Marker Locus (cM) position allele allele 6002 180

Scaffold14200_Locus_14227_0_59.0_FORK_762 Scaffold14200_Locus_14227_0_59.0_FORK 31.50977 762 T C 0/0 1/1

C205172_60.0_311 C205172_60.0 34.67857 311 C T 0/0 1/1

C210460_55.0_1836 C210460_55.0 35.24678 1836 G A 0/0 1/1

C199840_59.0_1368 C199840_59.0 35.24678 1368 G T 0/0 1/1

C196012_59.0_549 C196012_59.0 35.24678 549 C T 0/0 1/1

C196012_59.0_1181 C196012_59.0 35.24678 1181 A G 0/0 1/1

C197180_56.0_678 C197180_56.0 35.84922 678 A G 0/0 1/1

C207980_54.0_1302 C207980_54.0 35.84922 1302 T G 0/0 1/1

Scaffold25376_Locus_46472_0_39.2_LINEAR_2694 Scaffold25376_Locus_46472_0_39.2_LINEAR 35.84922 2694 C G 0/0 1/1

Scaffold20706_Locus_28526_0_45.3_LINEAR_1458 Scaffold20706_Locus_28526_0_45.3_LINEAR 36.44449 1458 G A 0/0 1/1

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Table 7.3: Details of putative candidate genes and corresponding SNP effects

Locus SNP location SNP effect Candidate gene Accession number

uroporphyrinogen decarboxylase [Medicago Scaffold14200_Locus_14227_0_59.0_FORK cds_2 Non-Synonymous (T35A) XP_003601037.1 truncatula] sedoheptulose-1,7-bisphosphatase, C205172_60.0 5'UTR N/A XP_003600853.1 chloroplastic [Medicago truncatula] probable E3 ubiquitin-protein ligase RHC2A C210460_55.0 3'UTR N/A XP_013460694.1 [Medicago truncatula] cytochrome c-type biogenesis ccda-like C199840_59.0 3'UTR N/A XP_013445560.1 chloroplastic protein [Medicago truncatula] glutathione S-transferase DHAR3, C196012_59.0 cds_3 Synonymous XP_013460594.1 chloroplastic [Medicago truncatula] glutathione S-transferase DHAR3, C196012_59.0 cds_2 Non-Synonymous (F13L) XP_013460594.1 chloroplastic [Medicago truncatula] C197180_56.0 cds_2 Synonymous ribonucleoprotein [Pisum sativum] CAA74889.1 60S ribosomal export protein nmd3-like C207980_54.0 cds_3 Synonymous PNX76336.1 [Trifolium pratense] anthranilate phosphoribosyltransferase, Scaffold25376_Locus_46472_0_39.2_LINEAR 3'UTR N/A XP_003601245.1 chloroplastic [Medicago truncatula] protein EXECUTER 2, chloroplastic isoform Scaffold20706_Locus_28526_0_45.3_LINEAR cds_3 Non-Synonymous (V387I) XP_003601283.1 X1 [Medicago truncatula]

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Figure 7.7: Predicted 3D model structures of proteins a) UROD (PDB ID: 5ECS) and b) GST-DHAR3 (PDB-1J93) from Arabidiopsis thaliana and Nicotinia tabacum, respectively. The green annotations indicate the positions of the mutations within the 3D structures of the proteins.

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7.5. Discussion

In the process of domestication, lentil has been estimated to have lost approximately 40% of its genetic diversity (Alo et al., 2011). Selection and breeding for higher yield and related traits in the cultivated germplasm eventually led to increased genetic similarity (Kumar et al., 2018a).Not surprisingly, breeding programs from around the world have reported limited diversity in their respective cultivated lentil gene pool (Ferguson et al., 1998; Lombardi et al., 2014; Khazaei et al., 2016). Particularly, the diversity of South Asian, Canadian and Australian germplasm is estimated as low (Ford et al., 1997; Lombardi et al., 2014; Khazaei et al., 2016; Kumar et al., 2018a). Therefore, interspecific introgression of new genes of interest such as disease resistance, abiotic stress tolerance and nutrient toxicities such as B is advocated and practiced (Gupta and Sharma, 2007; Tullu et al., 2010a; Bhadauria et al., 2017; Dadu et al., 2017). Moreover, the process is suggested to be fast-tracked through identification of trait- linked markers and marker-assisted breeding (MAS) (Bhadauria et al., 2017).

Lentil has a relatively large genome of approximately 4 Gb (Arumuganathan and Earle, 1991) and hence, genome complexity reduction-based methods were adapted in genomic studies of lentil (Sharpe et al., 2013; Kaur et al., 2014; Bhadauria et al., 2017). Likewise, genotyping of the RIL population in the current study was accessed through transcriptome sequencing, otherwise known as the GBS-t method. Particular advantages of employing a GBS-t approach include cost effective, reduction of the genome complexity, maintaining sufficient resolution of sequence assembly, conservation of gene sequences and SNP identification (Malmberg et al., 2018). Interestingly, 98.5% of reads per sample of the 694 million reads generated through the GBS-t approach in this study were successfully mapped to the reference transcriptome of lentil cultivar Cassab. In addition, a total of 671 of the 815 SNP markers identified within this RIL population were found to be common to the markers generated through transcriptome sequencing of six lentil cultivars (Kaur et al., 2014). These results show the extensive conservation for genes between L. orientalis and L. culinaris as established in previous studies (Wong et al., 2015; Ogutcen et al., 2018).

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The linkage map constructed in this study included 815 markers across eight LGs. The length of the map was shorter (488.02 cM) than many previously published maps in lentil (Eujayl et al., 1998; Rubeena et al., 2003; Duran et al., 2004; Hamwieh et al., 2005; Kahraman and Muehlbauer, 2010; De la Puente et al., 2012; Gupta et al., 2012b; Ates et al., 2016; Bhadauria et al., 2017; Ates et al., 2018b) but was very similar to other recently published maps using GBS-t (432.8 cM) and DArT (497.1 cM) derived SNP markers (Temel et al., 2015; Aldemir et al., 2017). However, as observed in these maps, the mean marker-marker distance of the map constructed was only 0.66 cM and is much shorter compared to the marker intervals of previously published maps that varied from 1.11 cM to 19.3 cM (Eujayl et al., 1998; Rubeena et al., 2003; Duran et al., 2004; Hamwieh et al., 2005; Kahraman and Muehlbauer, 2010; De la Puente et al., 2012; Gupta et al., 2012b; Sharpe et al., 2013; Gujaria-Verma et al., 2014; Ates et al., 2016; Sudheesh et al., 2016a; Bhadauria et al., 2017; Ates et al., 2018b). Nevertheless, there were a few gaps within the linkage map, potentially due to insufficient markers, low polymorphism in the regions with gaps or possibly because of genic markers that were interspersed across intergenic gaps on the genome. Also, this may be because of the under- representation of low expressed genes within the leaf tissue that was used for sequencing (Serin et al., 2017). Similarly, gaps were also evident in the maps previously constructed using GBS- t derived SNP markers (Sharpe et al., 2013; Temel et al., 2015).

QTL analysis identified one QTL on LG5 conferring leaf resistance to AB and, as expected, the resistance allele is contributed by ILWL 180 confirming previous symptomology, physical and biochemical evidences of resistance in this accession (Chapter 4; Chapter 5). Single QTL identification suggests a lack of selection pressure on ILWL 180 (L. orientalis) by A. lentis and thus a limited chance of coevolution (Madrid et al., 2014). Alternatively, potential QTLs might have remained undetected either due to incomplete map coverage or by falling below the statistically significant LOD threshold of 3. For example, two QTLs featured at exactly the same position on LG5 for the leaf lesion score at 14 and 28 dpi, however, failed to score a significant LOD threshold (2.54 and 2.90, respectively). Similarly, a QTL with a LOD score of 2.56 was found on LG3 for stem lesion score at 14 dpi. These QTLs without significant LOD scores are likely contributing minor effects to the cumulative resistance in accession ILWL 180 (Bhadauria et al., 2017).

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As one of the advantages of transcriptome sequencing, identified SNPs are most likely to be associated with the expressed trait (Kaur et al., 2014; Malmberg et al., 2018). Accordingly, ten markers from nine loci underpinning the QTL region were annotated for nine putative candidate genes following a BLASTx search for matching protein sequences. Among the nine loci, seven found best matching sequences from Medicago truncatula, a model species to which a direct relationship has been established with lentil in previous comparative mapping analysis studies (Sharpe et al., 2013; Kaur et al., 2014; Temel et al., 2015; Ates et al., 2018b). Among the nine putative candidate genes that were chosen based on the best matching annotations with highest percent identities and bit scores, five gene families showed evidence of direct involvement in the defence response. These included proteins UROD (XP_003601037.1), E3 ubiquitin-protein ligase RHC2A (E3 UPL-RHC2A; XP_013460694.1), GST–DHAR3 (XP_013460594.1), P-EXE2 (XP_003601283.1) and anthranilate phosphoribosyltransferase, chloroplastic (APRT; XP_003601245.1). Although the rest of the genes are reported to be part of various metabolic pathways and subsequently plant development, these may contribute indirectly to the defences against the pathogen (Miyagawa et al., 2001; Simon and Hederstedt, 2011; Wu et al., 2016; Ostendorp et al., 2017).

Among the defence related proteins putatively involved, UROD is involved in the pathway of chlorophyll biosynthesis and has been characterised to induce biochemical defence responses upon pathogen infection. Reduced activity of UROD in the transgenic tobacco leaves has been reported to result in antioxidative stress responses and necrotic leaf lesions (Mock et al., 1998; Mock et al., 1999). Additionally, UROD was also identified within the QTL region associated with resistance to AB caused by Peyronellaea pinodes in M. truncatula (Madrid et al., 2014). Considering the close relationship between barrel medic and lentil, it may be speculated that the candidate gene regulating the function of UROD in lentil may be an orthologue to the UROD gene of barrel medic. Therefore, a comparative mapping analysis of the linkage map constructed in this study to the genomic sequence of barrel medic may be beneficial. E3 UPL-RHC2A belongs to the family of ubiquitin ligases and is a component of the ubiquitin proteasome pathway, known for its role in plant defence responses (Craig et al., 2009). The role of E3 UPLs from recognition to downstream defence responses and cell death mechanisms is well established in Arabidiopsis thaliana (Bao et al., 2013; Zhou et al., 2015). E3 UPLs were also detected in the transcriptome of A. lentis infected lentil cultivars ILL 7537

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(AB resistant) and ILL 6002 (AB susceptible), however, showed completely opposite patterns of expression (Mustafa et al., 2009; Khorramdelazad et al., 2018). Protein APRT is considered as a branchpoint enzyme in the synthesis of tryptophan, which plays a direct role in plant defence responses through production of secondary metabolites that are anti-fungal such as indole-3-methyl-glucosinolates and indole phytoalexins (Niyogi and Fink, 1992; Asai et al., 2017).

Another defence related gene associated with the QTL region is GST-DHAR3, which belongs to a diverse protein family, gluatathione-S-transferases (GST), and has a role in pathogen-related stress responses such as oxidative stress and detoxification of a variety of compounds including microbial toxins (Ball et al., 2004). GST was up-regulated in lentil-A. lentis interactions (Sambasivam, 2011) as observed in chickpea – Ascochyta rabiei interactions (Fondevilla et al., 2011; Garg et al., 2018). P-EXE2, a plastid protein, enables higher plants to perceive the production of singlet oxygen, a reactive oxygen species (ROS) as a stress signal to activate a genetically determined programmed cell death (PCD) in response to pathogen infection (Lee et al., 2007; Kim et al., 2012). In summary, most of the candidate genes identified underpinning the QTL region on LG5 play a role in antioxidative stress and hypersensitive reaction (HR) or PCD to contain pathogen infection. This confirmed ILWL 180 as a relatively more durable resistance source to A. lentis along with previous evidences of physical and biochemical defence responses expressed during an interaction with A. lentis (Dadu et al., 2018a).

A further analysis of potential effects of SNPs, revealed three non-synonymous mutations causing a single amino acid substitution in the predicted protein sequence of the putative candidate genes including UROD (T35A), GST-DHAR3 (F13L) and P-EXE2 (V387I). These non-synonymous mutations with an amino acid substitution could potentially affect the structure and function of the corresponding protein and thereby determine the resistance of lentil to A. lentis. More recently, a non-synonymous SNP causing an amino acid substitution from arginine to lysine in a leucine rich repeat (LRR) domain was predicted based on a combination of genetic analysis to provide resistance to anthracnose in watermelon (Jang et al., 2018). The mutation of a single glycine to an arginine residue resulted in the drastic modification of the 3D protein structure of an effector AvrLm4-7 secreted by Leptosphaeria

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maculans (causal agent of stem canker disease in Brassica napus), which resulted in the loss of recognition specificity by two resistance genes (Rlm4 and Rlm7) (Blondeau et al., 2015). Likewise, the effects of mutations T35A and F13L are predicted to affect the domain and substrate binding sites of the proteins UROD and GST-DHAR3, respectively, and in turn may affect the structure and functions of the respective proteins as evidenced in other instances (Pham et al., 2011). Although it can be speculated that the origins of mutations may be from either of the parents, it is possible that the mutations might have been induced in ILL 6002 throughout the domestication process of the cultivated species and thus may have weakened the defence response system against A. lentis. However, this hypothesis may need further validation.

In conclusion, the present study has added a large number of SNPs to the long list of existing marker data available to the lentil community. Like others, these SNPs can be potentially useful in various future analyses including genetic characterisation, genetic linkage map analysis and comparative genomics. SNPs generated from transcriptome sequencing were used to construct an interspecific genetic linkage map that contained a QTL conferring resistance to AB on LG5. The markers within the QTL region and the corresponding candidate genes with functional importance in defence responses may permit development of precise diagnostic markers, which may be employed in the marker-assisted breeding (MAB) programs concerning AB resistance. To our knowledge, this is the first report of non-synonymous mutations and corresponding amino acid substitutions identified in lentil in response to A. lentis infection. However, an in-depth study is required to reveal the structural and functional changes of the prospective proteins and subsequently proposing reasons for the phenotype of either of the accessions ILWL 180 or ILL 6002 to AB resistance.

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Chapter 8 - General discussion and future directions

8.1. Identification of new resistance sources to ascochyta blight (AB) in a global collection of lentil

AB in lentil is a major production constraint globally including in Australia and affects both quantity and quality of the seed. The introduction of host plant resistance is the most acceptable and sustainable strategy to control AB. However, limited genetic diversity within the Australian lentil cultivars (Ford et al., 1997; Lombardi et al., 2014) together with selective adaptation of the A. lentis population to overcome resistance alleles has likely led to susceptibility of the resistant cultivars Northfield and Nipper (Nasir and Bretag, 1997b; Davidson et al., 2016). Meanwhile, several other commercial cultivars with a shared pedigree are under considerable risk of resistance breakdown due to pathogen population adaption and selection towards more aggressive isolates. Introgression of novel resistance alleles/genes from exotic germplasm may diversify the narrow genetic base and improve the stability and longevity of the resistance reaction of cultivars to the disease. In this context, this thesis examines the potential of global landraces in a wild germplasm collection of lentil for resistance to AB.

Sufficient variation for AB resistance was detected in the exotic germplasm of lentil. Utilising the relationship between trait and environment, the focused identification of germplasm strategy (FIGS) approach predicted a subset of 87 landraces with highest likelihood for AB resistance from 4576 accessions within International Center for Agricultural Research in Dry Areas (ICARDA) lentil genepool. Bioassays with the highly aggressive Australian A. lentis isolate FT13037 revealed moderate to high resistance for 17.2% of lentil subset accessions. Of the 30 wild accessions from five different species of lentil collected from the AGGB, Horsham, Australia, fourteen accessions showed resistance to AB. Three accessions (ILWL 180, ILWL 7 and ILWL 146) that were resistant to the Australian isolate had been reported previously to be resistant to Syrian A. lentis isolates (Bayaa et al., 1994). Tullu et al. (2010a) also, identified accessions resistant to both Canadian and Syrian A. lentis isolates. The

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inference from both studies highlighted that the wild species may possess a broader spectrum of resistance to AB than may be present in the cultigen.

Overall, IG 207 and L. orientalis ILWL 180 exhibited higher A. lentis resistance in comparison to remaining 109 accessions (landraces and wild relatives). Both IG 207 and ILWL 180 performed better than existing resistant cultivars ILL 7537 and Indianhead in response to a group of geographically diverse isolates with varying levels of aggressiveness. Additionally, a total of 25 accessions (wild and landraces) have been reported moderately resistant to A. lentis infection from this study. The genotypes with complete and higher resistance may be included in the AB resistance breeding as parents as similarly demonstrated through transfer of anthracnose resistance from L. ervoides to the cultivated background (Fiala et al., 2009). Meanwhile, accessions with moderate resistance may still be part of the lentil resistance breeding strategy because of their potential to limit the severity of infection and epidemic potential of the pathogen (Taylor and Ford, 2007).

8.2. Physiological and biochemical evidence of better resistance within the genus Lens

Histopathological findings of previous studies on temperate food legumes species confirmed that different sources of resistance underpinned by different mechanisms of resistance against ascochyta pathogens (Maurin et al., 1993; Ilarslan and Dolar, 2002; Armstrong-Cho et al., 2015). Sari et al. (2017) reported that an early and faster activation of phytohormone signalling pathways of cultivars CDC Robin and 964a-46 compared to that of the much slower response of cultivar Eston led to resistance against A. lentis infection. An understanding of the diversity among resistance mechanisms could improve the selection process for AB resistance through targeting most the effective resistance mechanisms and potentially also maximise the durability of that resistance.

Accordingly, resistant accessions IG 207 and ILWL 180 were assessed for underlying functional defence mechanisms using histopathological studies. Microscopic evidence of the infection process of A. lentis showed a significant variation between the resistant and susceptible lentil genotypes as previously reported (Sambasivam et al., 2016; Sari et al., 2017).

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Although tested independently with different isolates of similar aggressiveness level (FT13037 and FT13038), significant differences were detected between the resistant and susceptible genotypes. Germination percentage of A. lentis decreased with the change of genotypes in the order of Nipper (susceptible cultivar), ILL 6002 (susceptible cultivar), ILL 7537 (resistant cultivar), ILWL 180 (newly identified wild accession) and IG 207 (newly identified landrace). The differences in germination among the genotypes may directly reflect the temporal differential potential of the genotypes to recognise the pathogen and induce downstream defence responses to contain the progression of the pathogen at the entry point into the host tissue. Interestingly, molecular evidence in the lentil-A. lentis pathosystem suggested that the temporal differences in the expression of pathogen recognition receptors (PRRs) was associated with the resistance in cultivars ILL 7537, ILL 6002, CDC Robin, 964a-46 and Eston (Khorramdelazad et al., 2018; Sari et al., 2018). Likewise, a similar or even a much more robust expression of PRRs in accessions ILWL 180 and IG 207 may have created a non-conducive environment to cease the germination of the pathogen spores.

A successfully germinated conidia develops a germ tube with an appressorium at the tip, which subsequently using a penetration peg enters into host plant epidermis for further establishment. However, the ability of the host plant to allow development of the germ tube and formation of appressorium determines the status of its resistance against the pathogen infection (Sambasivam et al., 2016; Sari et al., 2017). Accordingly, A. lentis produced longer germ tubes and a higher number of appressoria in the susceptible cultivars (Nipper and ILL 6002). Accessions ILWL 180 and IG 207 performed better than resistant cultivar ILL 7537 by restricting the pathogen’s germ tube length and delaying the appressoria formation. This may be due to the early activation of basal chemical defence responses which are known to interfere with the fungal growth on the host’s surface (Prats et al., 2007).

At the biochemical level, the differential AB resistance reaction of the genotypes was supported by the accumulation of reactive oxygen species (ROS) species such as hydrogen ̶ peroxide (H2O2) and superoxide (O2 ). Following early recognition of the pathogen and signal transduction, resistant accessions ILWL 180 and ILL 7537 accumulated higher amounts of ROS species compared to that of the susceptible cultivar ILL 6002. Apart from the direct toxicity, the production of ROS species at the site of invasion is suggested to act as an

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independent signalling pathway to induce a further set of downstream defence responses against the pathogen growth and development. This may include reinforcement of cell walls (Lin et al., 2005), phenolic-based lignification (Huckelhoven, 2007), activation of the phenylpropanoid pathway (Jabs et al., 1997), synthesis of PR proteins (Hancock et al., 2006) and ultimately rapid cell death or otherwise hypersensitive reaction (HR) (Lam, 2004). This is evident through the accumulation of higher deposits of phenolic compounds in the leaflets of resistant accessions ILL 7537 and ILWL 180.

Evidence for the accumulation of ROS species in response to A. lentis infection has been reported previously at biochemical and molecular levels in cultivar ILL 7537 (Mustafa et al., 2009; Sambasivam et al., 2016; Khorramdelazad et al., 2018). At the molecular level, the upregulation of genes including superoxide dismutase (SOD), glutathione – s - transferase (GST), 6- phosphogluconate (6PGDH), NADH dehydrogenase and copper containing amine oxidase (CuAO) are associated with the expression of ROS species in the lentil-A. lentis interaction (Mustafa et al., 2009; Sambasivam et al., 2016; Khorramdelazad et al., 2018). Although several other novel defence mechanisms might be playing a role in the resistant accessions (IG 207 and ILWL 180), discovery and understanding of these potential defence mechanisms of the resistant genotypes necessitates exploration of their transcriptomic analysis (Sari, 2014).

8.3. Generation of lentil interspecific population, and its assessment for agro-morphological traits and genetics of AB resistance

AB symptomatology associated with seedling resistance, and physiological and biochemical defense responses against AB infection provide evidence of better/higher resistance from L. orientalis accession ILWL 180, that may be employed to improve the AB resistance of existing Australian lentil cultivars. Despite being an accession of a wild species,

ILWL 180 was readily crossable with the cultivar ILL 6002 and an application of GA3 soon

after pollination improved the success of crossing resulting in the production of healthy F1 seeds. However, successful introgression of the resistance into the cultivars will require information regarding number and mode of inheritance of the resistance genes. Segregation

analysis of the F2 and F5 generation revealed that the resistance of accession ILWL 180 against

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A. lentis may be conferred by two recessive genes. Furthermore, the two putative genes segregated in a duplicate dominant epistasis pattern (15S:1R) and suggested a two-gene model

(1S:3R) in F2 and F5 generations, respectively. Interestingly, the condition was equally supported by an independent assessment of leaf and stem lesion scores (major contributing

factors of seedling resistance), which segregated in a similar segregation pattern at the F2

(15S:1R) and F5 (1S:3R) generations. This may indicate that the genes controlling seedling, stem and leaf resistance share a similar genomic region and act cohesively to control the infection caused by AB. However, further investigations and validation will be required to prove this hypothesis.

Wild crosses are usually avoided by breeders because of the linkage drag of unwanted genes along with the desirable trait(s). Therefore, several agronomically important traits of this

population at F2 generation were assessed to ascertain the existence of useful variation. In future when ILWL 180 will be used in lentil breeding programs, this information will be useful to improve the agronomic performance of hybrids. Higher levels of variation were evident for all quantitative traits (days to first flower, plant height at flowering (cm), number of nodes below first flower node, seed diameter (mm), 100-seed weight (g) and seed yield (g)) and in addition, transgressive variants were found for traits (days to first flower (3 F2 plants), plant height below first flowering node (12), node number below first flowering node (55), peduncle length (4), seed diameter (53) and seed yield (3)) as previously reported from different interspecific crosses of lentil (Singh et al., 2013; Tullu et al., 2013). The variation outside the parental boundaries resulted from the combination of positive alleles from both parents offers an opportunity to recover better phenotypes that can be used in breeding programs (Guindon et al., 2018). Four lines of the segregating F2 population performed better than the best parent

and F1 for seed yield which suggested that the wild accession ILWL 180 might possess positive alleles for seed yield and selection for such alleles could benefit the breeding programs.

Overall, accession ILWL 180 possesses some useful variations for agronomical traits along with resistance to AB. The use of molecular markers associated with traits of interest might help to avoid non-desirable traits and thus reduce the linkage drag associated with interspecific hybridisation involving wild species of lentil. Although two major genes are proposed to have influenced the resistance to AB, many other minor genes with much lesser effect compared to major genes could be involved in the resistance against A. lentis as

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suggested by Ye et al. (2001). Therefore, more careful genetic dissection of the resistance to AB is required to unveil the complexity of its genetic mechanisms from accession ILWL 180. This was achieved through the identification of quantitative trait loci (QTL) that harbor genes controlling resistance against A. lentis.

8.4. Identification of QTLs and putative candidate genes controlling AB resistance

Next generation sequencing (NGS) technologies have opened avenues for genome- complexity reduction approaches for crops like lentil with a large genome size ( ̴ 4 Gb). (Sharpe et al., 2013; Kaur et al., 2014; Bhadauria et al., 2017). Among the most common genome- complexity reduction approaches, genotyping-by-sequencing – restriction site associated DNA (GBS-RAD) (Poland et al., 2012) and transcriptome sequencing or genotyping-by-sequencing through transcriptomics (GBS-t) (Malmberg et al., 2018) have generated high quality and large numbers of single nucleotide polymorphism (SNPs) for linkage and QTL mapping in lentil (Sudheesh et al., 2016a; Bhadauria et al., 2017; Ates et al., 2018b). Likewise, transcriptome sequencing of the F5 recombinant inbred line (RIL) population of interspecific cross (ILWL 180 and ILL 6002) led to the identification of 815 high quality SNP markers. Subsequently, these markers allowed construction of a genetic linkage map with a genetic distance of 488.02 centimorgan (cM) and eight linkage groups (LGs). The map was quite similar to the previously published intraspecific Precoz × WA8649041 RIL map (genetic distance = 432.8 cM; nine LGs) based on SNP markers derived from transcriptome sequencing (Temel et al., 2015). The mean marker-marker distance of the current map (0.66 cM) ranks second to the best map (0.12 cM) (Aldemir et al., 2017) constructed to date.

A single QTL conferring resistance to AB was identified on LG5. The QTL region stretched 4.93 cM and included ten SNP markers associated with the resistance to AB. The corresponding loci of the SNP markers identified nine putative candidate genes upon comparison for matching sequences through BLASTx similarity search. The putative protein products of these genes indicated that functionally, five, including uroporphyrinogen decarboxylase (UROD; XP_003601037.1); E3 ubiquitin-protein ligase RHC2A (E3 UPL- RHC2A; XP_013460694.1); glutathione - S – transferase, DHAR3, chloroplastic (GST– DHAR3; XP_013460594.1); protein EXECUTER 2, chlorplastic isoform X1 (P-EXE2;

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XP_003601283.1) and anthranilate phosphoribosyltransferase, chloroplastic (APRT; XP_003601245.1) are directly involved in plant defence responses to disease.

Further analysis of potential effects of SNPs from the identified QTL region revealed three non-synonymous mutations with substitutional effects within the amino acid sequence of the putative candidate proteins UROD (Threonine (T) 35 Alanine (A)), GST-DHAR3 (Phenylalanine (F) 13 Leucine (L)) and P-EXE2 (Valine (V) 387 Isoleucine (I)). These substitutions may potentially modify the 3D structure of the protein and thus affect its ability to interact with other proteins, denaturation or loss of function, and could be significantly influencing the host phenotype (Blondeau et al., 2015; Jang et al., 2018). It is speculated that these mutations have most likely originated during the domestication process of the cultivated species resulting in susceptibility of ILL 6002 to A. lentis. Alternatively, as demonstrated with histopathological studies, identification of candidate genes with a major role in antioxidant stress responses and HR indicated the durability of the AB resistance detected in the accession ILWL 180.

8.5. Suggestions for future research

In this study, FIGS was applied for the first time to lentil to identify AB resistance. Although few resistance sources were recovered, the predictions were expected to be more accurate. Precision of the selection process may be further improved by including other factors that influence the seasonal population dynamics of the pathogen in the selection algorithm such as an estimate of aggressiveness of the pathogen and inter-seasonal stability. Wild accessions have shown sufficient variation for resistance to AB and thus, further investigations can include more wild accessions in the screening process and may characterise the remaining resistant and moderately resistant accessions identified from this study for novel defense responses.

Although few defence responses were detected in the resistant accession ILWL 180 against A. lentis using histopathological studies, it is most likely that this accession harbours few more novel defence mechanisms involved in the higher level of resistance as evident in the bioassays. To obtain greater insights into currently unknown defence responses, future investigations such as transcriptome profiling of the resistant accession ILWL 180 in a time- course experiment capturing the different steps of A. lentis interaction. Also, an understanding

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of the pathogenesis of A. lentis on lentil is required through determining the key virulence genes (such as, effector proteins) and corresponding host receptors or resistance genes (R genes) that determine the resistance of the host. This information may consequently enable an effector- based screening process for A. lentis resistance in lentil and potentially isolate novel R genes that may be used in future selective breeding programs as practised in wheat against Pyrenophora tritici repentis (causal agent of wheat tan spot disease) (Vleeshouwers and Oliver, 2014).

Successful introgression of AB resistance from wild accession ILWL 180 to the cultivar ILL 6002 has been achieved. However, further consideration should be given to identify the allelic nature and copy number of the resistance genes to allow gene pyramiding including the already identified candidate resistance genes. This may be achieved by developing and analysing segregating populations among the known resistant accessions including accession

ILWL 180 as described by Sari (2014). Analysis of various agronomical traits of F2 generation of RIL population revealed favourable trait combinations from wild accession ILWL 180 and cultivated ILL 6002 parents. However, few undesirable traits such as short stature, delayed flowering, small seed size and seed shattering may delay the inclusion of accession ILWL 180 into the Australian lentil breeding program. Therefore, future research should aim at reducing the impact of linkage drag due to undesirable traits in the crosses involving wild relatives through marker-assisted backcrossing (MAB) once associated markers/sequences are determined (Hajjar and Hodgkin, 2007).

The interspecific linkage map developed from the F5 RIL population comprised some gaps despite a comprehensive markers density. These gaps on the map may affect future QTL analysis of other desirable traits. Hence, to increase the coverage of the map, inclusion of additional markers is suggested. Additionally, aligning QTL region/the markers on the lentil reference genome once it becomes available will resolve the limitation of an unsaturated map and the discrepancy for the number of LGs obtained. Nevertheless, QTL analysis in the current study led to the discovery of a single QTL on LG5, ten SNP markers and nine putative candidate genes associated with defence to AB. Future research should consider fine mapping the genomic region of identified QTLs at high resolution and validation across multiple genetic backgrounds of intra and interspecific crosses to determine their utility in marker- assisted

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selection (MAS). Additionally, the functional characterisation of each of the identified candidate genes can be targeted using reverse genetic approaches such as transposon insertional mutagenesis, gene silencing by RNA interference, targeting induced local lesions in genomes (TILLING) and clustered regularly interspaced palindromic repeats (CRISPR) (Raingam et al., 2018). These approaches knock out the putative candidate genes and may help to identify the impact of a specific candidate gene on the phenotype of the host organism. Upon validation of their involvement in defence mechanisms against A. lentis, these candidate genes may be included in selective breeding programs for differential resistance and development of durable resistance sources to A. lentis through hybridisation or gene pyramiding.

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Appendices

Chapter 3 - Appendices

Appendix 3.1: Results of analysis of variance obtained from linear mixed model procedure of GenStat for the reaction of FIGS lentil subset to aggressive Ascochyta lentis isolate FT13037

Effects DF F Statistic P value

Genotype 82 10.25 <0.001

Days post inoculation (dpi) 2 809.12 <0.001

Genotype.dpi 164 1.75 <0.001

176

S. No Accession ID 7 dpi 14 dpi 21 dpi 1 IG 7883 3.47ab 8.71o 9.51a 2 IG 2305 2.27ab 8.66no 9.15a 3 IG 82 1.36ab 8.00klmno 9.08a 4 IG 2228 0b 8.37mno 8.97a 5 IG 7592 3.73ab 9.15o 8.94a 6 IG 341 2.86ab 7.26efghijklmno 8.86a 7 IG 8243 0.91ab 8.46mno 8.84ab 8 IG 8248 2.68ab 8.77o 8.80ab 9 IG 642 2.56ab 8.21lmno 8.78ab 10 IG 8355 1.53ab 7.95jklmno 8.69abc 11 IG 6538 4.06ab 8.32mno 8.68abc 12 IG 600 2.77ab 7.98jklmno 8.68abc 13 IG 8234 0.45ab 6.41abcdefghijklmno 8.66abc 14 IG 8547 0.45ab 2.6abcde 8.61fghi 15 IG 171 4.29ab 8.09lmno 8.6abc 16 IG 7851 3.52ab 8.17lmno 8.4abcd 17 IG 615 0.79ab 7.69ghijklmno 8.39abcd 18 IG 7591 1.07ab 7.75hijklmno 8.32abcd 19 IG 1699 1.3ab 7.15efghijklmno 8.27abcde 20 IG 8315 1.81ab 7.29efghijklmno 8.25abcde 21 IG 507 4.09ab 8.05lmno 8.25abcde 22 IG 8254 2.62ab 7.65ghijklmno 8.24abcde 23 IG 641 2.96ab 6.99defghijklmno 8.14abcde 24 IG 6486 4.52a 7.92jklmno 8.08abcde 25 IG 298 0.79ab 7.67abcdefghijkl 8.08cdefghi 26 ILL 6002 3.54ab 6.16abcdefghijklmno 8.02abcde 27 IG 911 1.50ab 7.79ijklmno 8.01abcde 28 IG 8401 0.68ab 6.34abcdefghijklmno 7.94abcdef 29 IG 504 0.39ab 5.82abcdefghijklmno 7.9abcdef 30 IG 132877 2.49ab 6.82defghijklmno 7.87abcdef 31 IG 94 0.85ab 8.14lmno 7.85abcdef 32 IG 301 1.92ab 6.13abcdef 7.83defghi 33 IG 920 0.79ab 7.17efghijklmno 7.77abcdef 34 IG 7228 2.50ab 6.97defghijklmno 7.59abcdefg 35 IG 292 3.20ab 7.39efghijklmno 7.53abcdefg 36 IG 7253 3.84ab 7.47fghijklmno 7.47abcdefg 37 IG 8246 0.45ab 6.69cdefghijklmno 7.42abcdefg 38 IG 1720 2.36ab 6.71cdefghijklmno 7.40abcdefg 39 IG 5928 3.16ab 6.59bcdefghijklmno 7.13abcdefg 40 IG 342 3.40ab 7.19efghijklmno 7.05abcdefgh

177

41 IG 603 2.49ab 6.72cdefghijklmno 7.05abcdefgh 42 IG 4369 2.83ab 6.67cdefghijklmno 7.01abcdefgh 43 IG 6487 2.1ab 6.28abcdefghijklmno 6.73abcdefgh 44 IG 8314 0b 5.59abcdefghijklmno 6.72abcdefgh 45 IG 1719 2.91ab 6.06abcdefghijklmno 6.66abcdefgh 46 IG 8158 2.17ab 5.14abcdefghijklmno 6.65abcdefgh 47 IG 7852 1.13ab 6.49abcdefghijklmno 6.56abcdefgh 48 IG 4849 2.07ab 6.29abcdefghijklmno 6.49abcdefghi 49 IG 921 2.72ab 6.28abcdefghijklmno 6.49abcdefghi 50 IG 4851 2.60ab 5.92abcdefghijklmno 6.49abcdefghi 51 IG 6491 1.29ab 6.31abcdefghijklmno 6.31abcdefghi 52 IG 506 3.1ab 5.81abcdefghijklmno 6.28abcdefghi 53 IG 597 3.52ab 6.28abcdefghijklmno 6.28abcdefghi 54 IG 6485 3.82ab 6.25abcdefghijklmno 6.25abcdefghi 55 IG 606 3.89ab 5.75abcdefghijklmno 6.17abcdefghi 56 IG 5418 0.79ab 6.07abcdefghijklmno 6.07abcdefghi 57 IG 611 0.45ab 4.64abcdefghijklmno 6.03abcdefghi 58 IG 915 3.29ab 6.32abcdefghijklmno 6abcdefghi 59 IG 8232 2.13ab 5.62abcdefghijklmno 5.85abcdefghi 60 IG 859 1.15ab 5.70abcdefghijklmno 5.84abcdefghi 61 IG 640 0.56ab 4.95abcdefghijklmno 5.79abcdefghi 62 IG 7623 2.09ab 5.73abcdefghijklmno 5.73abcdefghi 63 IG 7622 1.96ab 5.84abcdefghijklmno 5.70abcdefghi 64 IG 7599 1.37ab 5.17abcdefghijklmno 5.56abcdefghi 65 IG 8363 1.57ab 4.77abcdefghijklmno 5.38abcdefghi 66 IG 7422 0.91ab 4.73abcdefghijklmno 5.34abcdefghi 67 IG 8282 0.91ab 5.39abcdefghijklmno 5.31abcdefghi 68 IG 4853 2.18ab 5.07abcdefghijklmno 5.3abcdefghi 69 IG 5233 1.96ab 4.96abcdefghijklmno 5.21abcdefghi 70 IG 7731 0.95ab 4.70abcdefghijklmno 5.04abcdefghi 71 IG 8218 2.75ab 3.68abcdefghijklm 5.03abcdefghi 72 IG 5911 1.57ab 4.84abcdefghijklmno 4.82abcdefghi 73 ILL 7537 3.15ab 3.82abcdefghijklmn 4.82abcdefghi 74 IG 712 0.59ab 2.84abcdefg 4.54abcdefghi 75 IG 914 2.72ab 4.76abcdefghijklmno 4.5abcdefghi 76 IG 7593 1.35ab 2.94abcdefghi 3.72bcdefghi 77 IG 8360 0.39ab 1.92abc 3.71ghi 78 IG 1687 1.57ab 2.58abcde 3.39defghi 79 IG 1735 1.66ab 2.93abcdefgh 3.30defghi 80 IG 7104 0.91ab 3.17abcdefghijk 3.17efghi 81 IG 8550 0b 2.15abcd 2.82fghi 82 IG 96 0.45ab 3.13abcdefghij 2.61ghi 83 IG 207 0.39ab 1.79ab 1.39i Means with one letter in common are not significantly different.

178

Effects DF F statistic P value Genotype 8 131.61 <0.001 dpi 2 587.47 <0.001 Isolate 3 58.21 <0.001 Genotype.dpi 16 9.91 <0.001 Genotype.Isolate 24 5.32 <0.001 dpi.Isolate 6 19.25 <0.001 Genotype.dpi.Isolate 48 1.64 0.01

179

Genotype/isolate FT13038 FT15160 FT16112 FT16299-2

IG 207 1.47abc 1.28ab 1.28ab 0.83a

IG 96 5.45efghijk 7.19hijklmnop 4.28cdefg 4.48defgh

IG 8550 6.21fghijklmn 8.14klmnop 4.57efgh 4.97efghi

IG 1687 5.41efghijk 8.9nop 3.77bcdef 3.26abcde

IG 7104 9.04op 9.33p 6.34fghijklmno 7.71ijklmnop

Nipper 7.88jklmnop 8.62mnop 6.6ghijklmnop 4.6efgh

Indianhead 4.60efgh 5.6efghijkl 3.84bcdefg 5.65efghijkl

ILL 7537 5.35efghij 4.8efgh 1.78abcd 4.36cdefg

ILL 6002 8.09jklmnop 8.33lmnop 6.38fghijklmno 5.97efghijklm

Means with one letter in common are not significantly different.

180

Effects Germination % Appressoria % Germ tube length

F P F P F P DF DF DF statistic value statistic value statistic value

Genotype 1 39.43 <0.001 1 79.49 <0.001 1 16.28 0.001

Germination % Appressoria % Germ tube length Genotype 6 hpi 12 hpi 24 hpi 6 hpi 12 hpi 24 hpi 6 hpi 12 hpi 24 hpi

IG 207 2.343d 2.984d 3.43cd 0d 1.454cd 1.839bcd 5.89c 6.63c 7.34c

Nipper 5.295bc 6.731b 8.73a 2.785bc 3.776ab 5.773a 8.84c 17.95b 44.77a Means with one letter in common are not significantly different.

181

Chapter 4 - Appendices

Appendix 4.1: Results of analysis of variance obtained from Friedman’s non-parametric test of IBM SPSS Statistic software for the reaction of Lens spp. genotypes to Ascochyta lentis Preliminary bioassay Phenotyping of wilds Stability analysis

Chi-Square 14.815 50.45 47.211 df 2 31 2

Significance level .001 .002 .001

Appendix 4.2: Results of pairwise comparison of mode disease scores of lentil genotypes from preliminary screening using Wilcoxon signed ranks test of IBM SPSS Statistic software

Pairwise combinations ILL7537 - ILL6002 Nipper - ILL7537 Nipper - ILL6002

Z statistic -2.147 -3.275 -2.309

Significance level .032 .001 .021

182

Appendix 4.3: Results of pairwise comparison of mode disease scores of wild lentil genotypes using Wilcoxon signed ranks test of IBM SPSS Statistic software

Pairwise combination Z statistic Significance level PI572330 - ILL6002 .000 1.000 PI572330 - ILL7537 -3.051 .002 PI572317 - ILL6002 -.333 .739 PI572317 - ILL7537 -2.887 .004 PI572362 - ILL6002 -.144 .885 PI572362 - ILL7537 -2.762 .006 PI572336 - ILL6002 -1.265 .206 PI572336 - ILL7537 -2.333 .020 ILWL172 - ILL6002 -.378 .705 ILWL172 - ILL7537 -2.640 .008 ILWL206 - ILL6002 -.302 .763 ILWL206 - ILL7537 -2.972 .003 ILWL116 - ILL6002 -.866 .386 ILWL116 - ILL7537 -2.530 .011 PI572342 - ILL6002 .000 1.000 PI572342 - ILL7537 -2.299 .022 ILWL221 - ILL6002 -1.725 .084 ILWL221 - ILL7537 -1.000 .317 ILWL235 - ILL6002 -1.867 .062 ILWL235 - ILL7537 -1.000 .317 PI572345 - ILL6002 -2.332 .020 PI572345 - ILL7537 -.378 .705 PI572334 - ILL6002 -2.420 .016 PI572334 - ILL7537 -.333 .739 ILWL261 - ILL6002 -2.008 .045 ILWL261 - ILL7537 -1.000 .317 ILWL325 - ILL6002 -2.309 .021 ILWL325 - ILL7537 .000 1.000 ILWL69 - ILL6002 -2.495 .013 ILWL69 - ILL7537 .000 1.000 ILWL70 - ILL6002 -2.739 .006 ILWL70 - ILL7537 -1.414 .157 ILWL146 - ILL6002 -2.111 .035 ILWL146 - ILL7537 -1.414 .157 PI572360 - ILL6002 -2.456 .014 PI572360 - ILL7537 -1.000 .317 ILWL437 - ILL6002 -2.521 .012 ILWL437 - ILL7537 -.333 .739 PI572399 - ILL6002 -2.495 .013 PI572399 - ILL7537 .000 1.000

183

ILWL160 - ILL6002 -2.310 .021 ILWL160 - ILL7537 -.368 .713 PI572348 - ILL6002 -2.836 .005 PI572348 - ILL7537 -2.887 .004 PI572347 - ILL6002 -2.642 .008 PI572347 - ILL7537 -1.732 .083 PI572359 - ILL6002 -2.836 .005 PI572359 - ILL7537 -2.887 .004 PI572333 - ILL6002 -2.850 .004 PI572333 - ILL7537 -2.126 .033 PI615677 - ILL6002 -2.969 .003 PI615677 - ILL7537 -2.810 .005 ILWL37 - ILL6002 -2.873 .004 ILWL37 - ILL7537 -2.165 .030 PI572351 - ILL6002 -2.969 .003 PI572351 - ILL7537 -2.810 .005 ILWL7 - ILL6002 -2.994 .003 ILWL7 - ILL7537 -2.724 .006 ILWL180 - ILL6002 -3.097 .002 ILWL180 - ILL7537 -2.965 .003

Appendix 4.4: Results of pairwise comparison of mode disease scores of lentil genotypes from stability analysis using Wilcoxon signed ranks test of IBM SPSS Statistic software

Pairwise combinations ILL7537 - ILL6002 ILL6002 - ILWL180 ILWL180 - ILL7537

Z statistic -4.174b -4.396c -2.968b

Significance level .000 .000 .003

184

Chapter 5 - Appendices

Effects DF F statistic P value Genotype 2 112.3 <0.001 Assay 1 1288.44 <0.001 Isolate 1 4.56 0.035 Genotype.Assay 2 24.79 <0.001 Genotype.Isolate 2 2.91 0.037 Assay.Isolate 1 17.11 <0.001 Genotype.Assay.Isolate 2 2.09 0.105

FT13037 F13082

Genotype/Isolate/hpi 6 hpi 12 hpi 20 hpi 30 hpi 6 hpi 12 hpi 20 hpi 30 hpi

ILWL 180 4.52a 6.01cde 7.41fgh 8.41ijk 6.32cde 7.27fg 8.29ijk 8.54ijk

ILL 7537 5.12ab 7.79ghi 8.16hijk 8.76jk 5.62bc 6.64def 7.41fgh 8.06ghij

ILL 6002 5.84bcd 6.74ef 8.07ghij 8.94k 6.06cde 6.87ef 8.31ijk 8.37ijk Means with one letter in common are not significantly different.

185

FT13037 F13082 Genotype/Isolate/hpi 30 6 hpi 12 hpi 20 hpi 6 hpi 12 hpi 20 hpi 30 hpi hpi

ILWL 180 3.47ab 3.65abc 3.54ab 3.93bc 2.68a 3.55ab 3.60abc 3.7abc

ILL 7537 3.99bcd 4.62cdefgh 5.06efghi 5.30ghi 3.79bc 3.87bc 4.32bcdefg 4.45bcdefgh

ILL 6002 4.21bcdef 5.16fghi 5.47hi 5.77i 3.74bc 4.05bcde 5.14fghi 5.05defghi Means with one letter in common are not significantly different.

Effects DF F statistic P value Genotype 2 67.99 <0.001 Isolate 1 49.46 <0.001 Assay 1 59.4 <0.001 Genotype.Isolate 2 10.11 <0.001 Genotype.Assay 2 11.58 <0.001 Isolate.Assay 1 1.96 0.164 Genotype.Isolate.Assay 2 2.82 0.063

186

FT13037 F13082 Genotype/Isolate/hpi 6 hpi 12 hpi 20 hpi 30 hpi 6 hpi 12 hpi 20 hpi 30 hpi

ILWL 180 8.66ab 9.82abcd 12.72abcde 21.09fgh 7.03a 10.04abcd 12.84abcde 15.64cdefg

ILL 7537 9.12abc 12.32abcde 16.37defgh 21.9fgh 6.49a 11.41abcde 13.29abcde 17.68efgh

ILL 6002 12.36abcde 23.28h 32.08i 47.02j 12.17abcde 15.24bcdef 22.48gh 21.79fgh Means with one letter in common are not significantly different.

Appendix 5.6: Comparison of means of germ tube length by Ascochyta lentis isolates FT13037 and F13082 at 6, 12, 20 and 30 hours post inoculation (hpi) on attached leaflets of lentil genotypes ILL 6002, ILWL 180 and ILL 7537 based on least significant differences with the Tukey’s adjustment (α = 0.05)

FT13037 F13082 Genotype/Isolate/hpi 6 hpi 12 hpi 20 hpi 30 hpi 6 hpi 12 hpi 20 hpi 30 hpi

ILWL 180 5.77ab 10.6abcde 12.29cdef 17.63fg 4.77a 8.1abcd 9.46abcde 11.73bcdef

ILL 7537 7.21abcd 11.76bcdef 11.76bcdef 15.14ef 6.47abc 8.98abcde 10.87abcde 12.41cdef

ILL 6002 6.89abc 13.25def 21.65gh 27.33h 5.42a 10.34abcde 14.78ef 17.46fg

187

Effects DF F statistic P value Genotype 2 53.29 <0.001 Isolate 1 27.95 <0.001 Assay 1 138.55 <0.001 Genotype.Isolate 2 12.19 <0.001 Genotype.Assay 2 12.67 <0.001 Isolate.Assay 1 2.28 0.133 Genotype.Isolate.Assay 2 8.57 <0.001

FT13037 F13082 Genotype/Isolate/hpi 6 hpi 12 hpi 20 hpi 30 hpi 6 hpi 12 hpi 20 hpi 30 hpi

ILWL 180 0a 1.44bc 2.74ef 2.96fg 0a 1.47bcd 2.43cdef 2.73def

ILL 7537 0.66ab 2.74ef 2.82ef 3.11fg 0a 3.08fg 2.99fg 3.07fg

ILL 6002 2.23cdef 4.21gh 4.69h 5.20h 1.64bcde 3.06fg 3.13fg 3.38fg

Means with one letter in common are not significantly different.

188

FT13037 F13082 Genotype/Isolate/hpi 6 hpi 12 hpi 20 hpi 30 hpi 6 hpi 12 hpi 20 hpi 30 hpi

ILWL 180 0a 0a 2.28bcd 1.95abcd 0a 0a 0a 0.7ab

ILL 7537 0a 0a 1.29abcd 2.19bcd 0a 0a 0a 1.27abcd

ILL 6002 0a 1.52abcd 2.78cd 3.22d 0a 0.75abc 2.02abcd 2.20bcd

Means with one letter in common are not significantly different.

Appendix 5.10: Results of analysis of variance obtained from repeated measure analysis of GenStat for comparison of hydrogen peroxide (H2O2) concentration accumulated by lentil genotypes ILL 6002, ILWL 180 and ILL 7537 in response to Ascochyta lentis isolates (FT13037 and F13082) infection at 12, 24 and 48 hours post inoculation (hpi)

Effects DF F statistic P value

Genotype 2 26.11 <0.001

Isolate 1 0.14 0.715

Genotype.Isolate 2 0.27 0.768

189

FT13037 F13082 Genotype/Isolate/hpi 12 hpi 24 hpi 48 hpi 12 hpi 24 hpi 48 hpi

ILWL 180 43.92cde 73.12a 50.75bcd 45.45cd 68.72a 47.45cd

ILL 7537 5.85gh 65.82ab 37.95de 20.15fg 57.35abc 37.59de

ILL 6002 -5.78h 51.82bcd 50.75bcd 1.55h 57.42abc 29.22ef

Means with one letter in common are not significantly different.

190

DAB-uptake method. a, d and g: accumulation of H2O2 on detached leaflets of ILWL180 beneath appressoria (arrow) at 12, 24 and 48 hpi. b, e and h: accumulation of H2O2 on detached leaflets of ILL7537

191

using DAB-uptake method. a, d and g: accumulation of H2O2 on detached leaflets of ILWL180 beneath appressoria (arrow) at 12, 24 and 48 hpi. b, e and h: accumulation of H2O2 on detached leaflets of ILL7537 beneath appressoria (arrow) at 12, 24 and 48 hpi. c, f and i: accumulation of H2O2 on detached leaflets of ILL6002 beneath appressoria (arrow) at 12, 24 and 48 hpi. Scale bars represent 50 µm (a-h plates) and 100 µm (plate i).

192

- Appendix 5.14: Histochemical localisation of superoxide (O2 ) accumulation in the leaflets of lentil genotypes ILWL180, ILL7537 and ILL6002 in response to aggressive isolate A. lentis isolate FT13037 using - NBT method. a, d and g: accumulation of O2 on detached leaflets of ILWL180 beneath appressoria (arrow) - at 12, 24 and 48 hpi. b, e and h: accumulation of O2 on detached leaflets of ILL7537 beneath appressoria - (arrow) at 12, 24 and 48 hpi. c, f and i: accumulation of O2 on detached leaflets of ILL6002 beneath appressoria (arrow) at 12, 24 and 48 hpi. Scale bars represent 50 µm (a-h plates) and 100 µm (plate i).

193

- Appendix 5.15: Histochemical localisation of superoxide (O2 ) accumulation in the leaflets of lentil genotypes ILWL180, ILL7537 and ILL6002 in response to aggressive isolate A. lentis isolate F13082 using - NBT method. a, d and g: accumulation of O2 on detached leaflets of ILWL180 beneath appressoria (arrow) - at 12, 24 and 48 hpi. b, e and h: accumulation of O2 on detached leaflets of ILL7537 beneath appressoria - (arrow) at 12, 24 and 48 hpi. c, f and i: accumulation of O2 on detached leaflets of ILL6002 beneath appressoria (arrow) at 12, 24 and 48 hpi. Scale bars represent 50 µm (b-e and g plates) and 100 µm (a, f, h and i plates).

194

Appendix 5.16: Histochemical localisation of phenolic compounds deposition in the leaflets of lentil genotypes ILWL180, ILL7537 and ILL6002 in response to aggressive isolate A. lentis isolate FT13037 using toludine blue staining method. a, d and g: accumulation of phenolic compounds on detached leaflets of ILWL180 beneath appressoria (arrow) at 12, 24 and 48 hpi. b, e and h: accumulation of phenolic compounds on detached leaflets of ILL7537 beneath appressoria (arrow) at 12, 24 and 48 hpi. c, f and i: accumulation of phenolic compounds on detached leaflets of ILL6002 beneath appressoria (arrow) at 12, 24 and 48 hpi. Scale bars represent 50 µm (a-c, e, f and i plates) and 100 µm (d, g and h plates).

195

196

Chapter 6 - Appendices

Appendix 6.1: Results of analysis of variance obtained from ANOVA tests of GenStat for the reaction of lentil genotypes ILWL 180, ILL 6002 and interspecific F1 of the cross ILL 6002 × ILWL 180 to A. lentis isolate FT13038 and various agro-morphological traits

S. No Trait ILWL 180 ILL 6002 F1 MSS P value LSD Value

1 AB seedling resistance 1.2 7.43 6.22 59.69 0.001 1.689 2 Leaf resistance 0 8 6.32 245.91 <0.001 1.037 3 Stem resistance 2.46 6.81 6.12 46.34 0.002 1.358 4 Cotyledon colour Orange Yellow Orange na na na 5 Days to first flower 111 73 70 109.33 <.001 8.58 Plant height below first flowering node 6 (cm) 15 30 25 40.38 0.002 4.719 7 Node number below first flowering node 4 6 7 2.25 0.221 2.618 White with blue 8 Flower petal colour Violet lines Violet na na na 9 Plant height at flowering (cm) 30 56 60 614.25 <0.001 2.618 10 Leaflet size Small Large Large na na na 11 Tendril length Rudimentary Prominent Prominent na na na 12 Peduncle length (cm) 1 3 3.2 27.18 0.005 0.916 13 Seed coat pattern Present Absent Present na na na 14 Seed coat colour Tan Green Tan na na na 15 Seed size (mm) 3.42 4.92 4.76 99.02 <0.001 0.3259 16 100-seed weight (g) 1.32 4.25 3.2 254 <0.001 0.3631 17 Seed yield (g) 2.67 5.21 5.76 52.21 0.001 0.897

197

Appendix 6.2: Correlation coefficients (R) among various agro-morphological traits in the interspecific segregating F2 population (N = 199) derived from the cross ILWL 180 × ILL 6002

Plant No. of Plant Height nodes Peduncle 100 seed Seed size Total seed Days to Days to 50 % Height at Traits below 1st below 1st length weight (g) (mm) weight (g) flowering flowering flowering flowering flower (cm) (cm) node (cm) node 100 seed weight (g) 1 Seed size (mm) 0.74** 1.00 Total seed weight (g) 0.31** 0.04 1.00 Days to flowering -0.20 -0.01 -0.16 1.00 Days to 50 % flowering -0.20 -0.01 -0.14 0.98** 1.00 Plant Height at flowering (cm) 0.31** 0.14 0.38** -0.49 -0.48 1.00 Plant Height below 1st flowering node 0.12 0.08 0.23** 0.11 0.13 0.26** 1.00 (cm) No. of nodes below 1st flower node 0.09 0.03 0.20** 0.20** 0.21** 0.08 0.63** 1.00 Peduncle length (cm) 0.05 -0.01 0.12 -0.51 -0.52 0.42** -0.11 -0.27 1

** Significance at 0.01 level of probability

198

Chapter 7 - Appendices

Appendix 7.1: Details of SNPs used in the linkage map and their corresponding position on different linkage groups

S. No Locus SNP Linkage Marker position position group 1 C199356_58.0_826 0.00 826 LG1 2 C199356_58.0_1000 0.00 1000 LG1 3 scaffold22710_Locus_33736_1_49.0_BUBBLE_2086 4.01 2086 LG1 4 Locus_2441_Contig1_1229 14.30 1229 LG1 5 Locus_2441_Contig1_1009 14.30 1009 LG1 6 Locus_2441_Contig1_1724 14.30 1724 LG1 7 Locus_2441_Contig1_2546 14.30 2546 LG1 8 Locus_2441_Contig1_2819 14.30 2819 LG1 9 Locus_2441_Contig1_2950 14.30 2950 LG1 10 Locus_2441_Contig1_3500 14.30 3500 LG1 11 Locus_2441_Contig1_4565 14.30 4565 LG1 12 Locus_2441_Contig1_4652 14.30 4652 LG1 13 Locus_2441_Contig1_4804 14.30 4804 LG1 14 Locus_2441_Contig1_5084 14.30 5084 LG1 15 scaffold21725_Locus_31098_0_17.9_LINEAR_1624 14.30 1624 LG1 16 C144708_55.0_342 15.47 342 LG1 17 C210784_62.0_767 16.07 767 LG1 18 C210784_62.0_174 16.07 174 LG1 19 C210784_62.0_492 16.07 492 LG1 20 C210784_62.0_813 16.07 813 LG1 21 C210784_62.0_1865 16.07 1865 LG1 22 C210784_62.0_1898 16.07 1898 LG1 23 scaffold15430_Locus_16627_1_54.3_FORK_198 16.65 198 LG1 24 C206640_56.0_697 16.65 697 LG1 25 C206640_56.0_853 16.65 853 LG1 26 C215184_59.0_1452 17.25 1452 LG1 27 C215184_59.0_1557 17.25 1557 LG1 28 C215184_59.0_1890 17.25 1890 LG1 29 C215184_59.0_1974 17.25 1974 LG1 30 scaffold15256_Locus_16262_1_54.1_FORK_220 17.25 220 LG1 31 scaffold15256_Locus_16262_1_54.1_FORK_470 17.25 470 LG1 32 C214468_61.0_2904 17.85 2904 LG1 33 C197842_60.0_1067 20.29 1067 LG1

199

34 C197842_60.0_719 20.29 719 LG1 35 C197842_60.0_746 20.29 746 LG1 36 C197842_60.0_926 20.29 926 LG1 37 C197842_60.0_932 20.29 932 LG1 38 C197842_60.0_1016 20.29 1016 LG1 39 C197842_60.0_1097 20.29 1097 LG1 40 C197842_60.0_1107 20.29 1107 LG1 41 C197842_60.0_1118 20.29 1118 LG1 42 C197842_60.0_1120 20.29 1120 LG1 43 scaffold24847_Locus_41925_0_58.6_LINEAR_3686 20.29 3686 LG1 44 C194526_56.0_698 21.51 698 LG1 45 C194526_56.0_707 21.51 707 LG1 46 Locus_2398_Contig1_3020 30.64 3020 LG1 47 Locus_2398_Contig1_3050 30.64 3050 LG1 48 Locus_2398_Contig1_3132 30.64 3132 LG1 49 Locus_2398_Contig1_3206 30.64 3206 LG1 50 C171726_50.0_89 31.83 89 LG1 51 scaffold4380_Locus_1553_1_56.4_COMPLEX_1223 32.43 1223 LG1 52 C201416_47.0_738 32.43 738 LG1 53 Locus_20288_Contig1_298 34.26 298 LG1 54 Locus_20288_Contig1_866 34.26 866 LG1 55 Locus_20288_Contig1_977 34.26 977 LG1 56 Locus_20288_Contig1_1043 34.26 1043 LG1 57 C185874_55.0_124 35.45 124 LG1 58 C190724_54.0_762 35.45 762 LG1 59 C215884_60.0_896 36.67 896 LG1 60 C197654_62.0_558 36.67 558 LG1 61 Locus_1486_Contig1_2914 38.57 2914 LG1 62 scaffold20759_Locus_28652_1_44.5_COMPLEX_1589 41.04 1589 LG1 63 C175144_61.0_134 42.92 134 LG1 64 C175144_61.0_288 42.92 288 LG1 65 scaffold5459_Locus_2008_1_56.8_BUBBLE_1472 43.53 1472 LG1 66 scaffold5459_Locus_2008_1_56.8_BUBBLE_1532 43.53 1532 LG1 67 scaffold21828_Locus_31365_0_55.7_LINEAR_1887 45.97 1887 LG1 68 scaffold3739_Locus_1301_0_57.5_COMPLEX_2028 45.97 2028 LG1 69 scaffold21828_Locus_31365_0_55.7_LINEAR_1305 45.97 1305 LG1

200

70 scaffold21828_Locus_31365_0_55.7_LINEAR_2139 45.97 2139 LG1 71 scaffold21828_Locus_31365_0_55.7_LINEAR_2247 45.97 2247 LG1 72 scaffold21828_Locus_31365_0_55.7_LINEAR_2653 45.97 2653 LG1 73 scaffold21828_Locus_31365_0_55.7_LINEAR_4200 45.97 4200 LG1 74 scaffold21828_Locus_31365_0_55.7_LINEAR_4788 45.97 4788 LG1 75 scaffold21828_Locus_31365_0_55.7_LINEAR_5106 45.97 5106 LG1 76 scaffold21828_Locus_31365_0_55.7_LINEAR_5140 45.97 5140 LG1 77 scaffold21828_Locus_31365_0_55.7_LINEAR_5514 45.97 5514 LG1 78 scaffold21828_Locus_31365_0_55.7_LINEAR_5817 45.97 5817 LG1 79 C201838_57.0_593 45.97 593 LG1 80 C215996_60.0_1159 47.84 1159 LG1 81 C215996_60.0_2174 47.84 2174 LG1 82 C202786_60.0_623 47.84 623 LG1 83 C213618_60.0_840 48.45 840 LG1 84 C214826_61.0_1282 49.64 1282 LG1 85 C208458_55.0_467 49.64 467 LG1 86 scaffold24697_Locus_41020_0_56.6_LINEAR_913 50.25 913 LG1 87 scaffold24697_Locus_41020_0_56.6_LINEAR_1560 50.25 1560 LG1 88 scaffold24697_Locus_41020_0_56.6_LINEAR_1921 50.25 1921 LG1 89 scaffold24697_Locus_41020_0_56.6_LINEAR_1969 50.25 1969 LG1 90 scaffold24697_Locus_41020_0_56.6_LINEAR_2048 50.25 2048 LG1 91 C195214_59.0_787 50.85 787 LG1 92 C195214_59.0_597 50.85 597 LG1 93 C195214_59.0_602 50.85 602 LG1 94 C195214_59.0_687 50.85 687 LG1 95 C195214_59.0_747 50.85 747 LG1 96 C195214_59.0_867 50.85 867 LG1 97 C195214_59.0_1149 50.85 1149 LG1 98 C215146_51.0_288 50.85 288 LG1 99 C215146_51.0_352 50.85 352 LG1 100 C215146_51.0_486 50.85 486 LG1 101 C215146_51.0_1008 50.85 1008 LG1 102 C215146_51.0_1628 50.85 1628 LG1 103 C175826_53.0_529 50.85 529 LG1 104 C205490_55.0_303 50.85 303 LG1 105 C179884_58.0_602 51.47 602 LG1 106 Locus_2331_Contig1_2442 51.47 2442 LG1 107 C212016_57.0_337 52.69 337 LG1

201

108 C213630_58.0_1480 52.69 1480 LG1 109 scaffold12446_Locus_10920_0_63.0_COMPLEX_1032 52.69 1032 LG1 110 scaffold12446_Locus_10920_0_63.0_COMPLEX_1058 52.69 1058 LG1 111 C212016_57.0_338 52.69 338 LG1 112 C212016_57.0_1627 52.69 1627 LG1 113 scaffold5774_Locus_2153_0_55.3_COMPLEX_65 53.29 65 LG1 114 scaffold22230_Locus_32345_0_62.2_COMPLEX_571 55.21 571 LG1 115 scaffold25380_Locus_46519_0_61.0_LINEAR_2168 55.83 2168 LG1 116 C193928_57.0_923 55.83 923 LG1 117 scaffold6679_Locus_2585_1_41.8_COMPLEX_1380 55.83 1380 LG1 118 C192680_50.0_804 57.08 804 LG1 119 C192680_50.0_861 57.08 861 LG1 120 C180352_52.0_473 57.70 473 LG1 121 C175966_53.0_483 57.70 483 LG1 122 scaffold22977_Locus_34540_0_56.2_LINEAR_448 57.70 448 LG1 123 C180352_52.0_672 57.70 672 LG1 124 C188628_60.0_536 58.93 536 LG1 125 Locus_144_Contig1_1741 60.11 1741 LG1 126 Locus_144_Contig1_1119 60.11 1119 LG1 127 Locus_144_Contig1_1165 60.11 1165 LG1 128 Locus_144_Contig1_2170 60.11 2170 LG1 129 Locus_144_Contig1_2173 60.11 2173 LG1 130 Locus_144_Contig1_2176 60.11 2176 LG1 131 Locus_144_Contig1_2321 60.11 2321 LG1 132 Locus_144_Contig1_2425 60.11 2425 LG1 133 Locus_144_Contig1_2515 60.11 2515 LG1 134 Locus_144_Contig1_2519 60.11 2519 LG1 135 Locus_144_Contig1_2531 60.11 2531 LG1 136 Locus_144_Contig1_2611 60.11 2611 LG1 137 Locus_144_Contig1_2618 60.11 2618 LG1 138 scaffold5554_Locus_2054_1_53.6_COMPLEX_388 60.11 388 LG1 139 C204764_60.0_206 60.11 206 LG1 140 C204764_60.0_212 60.11 212 LG1 141 scaffold5564_Locus_2059_1_56.4_COMPLEX_3284 60.11 3284 LG1 142 scaffold5553_Locus_2054_0_47.2_COMPLEX_832 60.11 832 LG1 143 scaffold5553_Locus_2054_0_47.2_COMPLEX_934 60.11 934 LG1 144 C215320_54.0_3244 62.49 3244 LG1 145 scaffold24275_Locus_39169_0_56.2_LINEAR_364 62.49 364 LG1

202

146 scaffold24275_Locus_39169_0_56.2_LINEAR_922 62.49 922 LG1 147 scaffold24275_Locus_39169_0_56.2_LINEAR_1714 62.49 1714 LG1 148 C210992_54.0_980 62.49 980 LG1 149 scaffold24275_Locus_39169_0_56.2_LINEAR_904 62.49 904 LG1 150 Locus_765_Contig1_2868 66.93 2868 LG1 151 C172230_63.0_340 67.51 340 LG1 152 C179988_62.0_439 67.51 439 LG1 153 C179988_62.0_506 67.51 506 LG1 154 C191240_57.0_649 67.51 649 LG1 155 C107934_34.0_22 67.51 22 LG1 156 Locus_9520_Contig1_1039 68.73 1039 LG1 157 C178924_58.0_198 68.73 198 LG1 158 C213884_57.0_606 68.73 606 LG1 159 Locus_9520_Contig1_1189 68.73 1189 LG1 160 Locus_9520_Contig1_1221 68.73 1221 LG1 161 C202072_60.0_291 70.68 291 LG1 162 scaffold25524_Locus_50156_0_60.1_LINEAR_450 70.68 450 LG1 163 scaffold25048_Locus_43142_0_57.6_LINEAR_1162 70.68 1162 LG1 164 scaffold25048_Locus_43142_0_57.6_LINEAR_1465 70.68 1465 LG1 165 scaffold25048_Locus_43142_0_57.6_LINEAR_1489 70.68 1489 LG1 166 scaffold25048_Locus_43142_0_57.6_LINEAR_1744 70.68 1744 LG1 167 C209926_58.0_694 72.55 694 LG1 168 C209926_58.0_923 72.55 923 LG1 169 C209926_58.0_1043 72.55 1043 LG1 170 C209926_58.0_1144 72.55 1144 LG1 171 scaffold10158_Locus_7178_1_54.0_BUBBLE_989 73.76 989 LG1 172 Locus_28453_Contig1_1773 83.74 1773 LG1 173 Locus_28453_Contig1_1683 83.74 1683 LG1 174 scaffold3969_Locus_1382_1_47.9_COMPLEX_1060 84.94 1060 LG1 175 scaffold3969_Locus_1382_1_47.9_COMPLEX_1114 84.94 1114 LG1 176 scaffold3969_Locus_1382_1_47.9_COMPLEX_1141 84.94 1141 LG1 177 scaffold3969_Locus_1382_1_47.9_COMPLEX_1294 84.94 1294 LG1 178 Locus_9179_Contig1_1351 85.52 1351 LG1 179 Locus_9179_Contig1_1144 85.52 1144 LG1 180 Locus_9179_Contig1_1747 85.52 1747 LG1 181 Locus_9179_Contig1_1768 85.52 1768 LG1 182 Locus_9179_Contig1_1990 85.52 1990 LG1 183 C187748_58.0_824 85.52 824 LG1

203

184 C187748_58.0_920 85.52 920 LG1 185 C186050_57.0_695 86.10 695 LG1 186 C186050_57.0_725 86.10 725 LG1 187 C186050_57.0_773 86.10 773 LG1 188 C186050_57.0_783 86.10 783 LG1 189 C186050_57.0_809 86.10 809 LG1 190 C178574_57.0_212 89.23 212 LG1 191 scaffold15250_Locus_16245_0_55.5_LINEAR_535 89.23 535 LG1 192 C179202_58.0_194 96.13 194 LG1 193 C179202_58.0_203 96.13 203 LG1 194 C179202_58.0_243 96.13 243 LG1 195 C179202_58.0_113 96.13 113 LG1 196 C179202_58.0_520 96.13 520 LG1 197 C179202_58.0_527 96.13 527 LG1 198 C179202_58.0_545 96.13 545 LG1 199 C179202_58.0_608 96.13 608 LG1 200 C179202_58.0_449 96.13 449 LG1 201 C179202_58.0_497 96.13 497 LG1 202 C179202_58.0_734 96.13 734 LG1 203 C179202_58.0_704 96.13 704 LG1 204 C203124_60.0_162 96.13 162 LG1 205 C179202_58.0_886 96.13 886 LG1 206 C203124_60.0_291 96.13 291 LG1 207 scaffold25315_Locus_45625_0_59.3_LINEAR_3517 96.74 3517 LG1 208 C215390_61.0_595 98.61 595 LG1 209 C215390_61.0_430 98.61 430 LG1 210 C215390_61.0_778 98.61 778 LG1 211 C215390_61.0_1019 98.61 1019 LG1 212 C215390_61.0_1726 98.61 1726 LG1 213 C215390_61.0_1786 98.61 1786 LG1 214 C215390_61.0_1976 98.61 1976 LG1 215 C212206_57.0_2033 101.66 2033 LG1 216 C212206_57.0_1914 101.66 1914 LG1 217 C212206_57.0_2118 101.66 2118 LG1 218 scaffold18260_Locus_22806_0_60.3_FORK_567 0.00 567 LG2 219 scaffold18260_Locus_22806_0_60.3_FORK_654 0.00 654 LG2 220 scaffold18169_Locus_22582_0_59.2_LINEAR_2280 12.77 2280 LG2 221 C214200_59.0_677 12.77 677 LG2

204

222 C167520_58.0_133 12.77 133 LG2 223 scaffold24924_Locus_42372_0_54.5_LINEAR_853 12.77 853 LG2 224 C215308_60.0_3306 12.77 3306 LG2 225 Locus_1283_Contig1_1875 12.77 1875 LG2 226 C197916_51.0_295 12.77 295 LG2 227 C197916_51.0_400 12.77 400 LG2 228 C197916_51.0_577 12.77 577 LG2 229 C197916_51.0_598 12.77 598 LG2 230 scaffold10619_Locus_7851_0_55.5_COMPLEX_1639 15.27 1639 LG2 231 scaffold10619_Locus_7851_0_55.5_COMPLEX_2874 15.27 2874 LG2 232 Locus_2281_Contig1_270 15.27 270 LG2 233 C196906_58.0_850 15.27 850 LG2 234 C196906_58.0_1083 15.27 1083 LG2 235 scaffold4679_Locus_1673_0_56.7_COMPLEX_2387 17.10 2387 LG2 236 scaffold4679_Locus_1673_0_56.7_COMPLEX_2414 17.10 2414 LG2 237 C207348_58.0_1188 17.10 1188 LG2 238 C176398_57.0_557 17.10 557 LG2 239 scaffold4679_Locus_1673_0_56.7_COMPLEX_4015 17.10 4015 LG2 240 scaffold21347_Locus_30137_0_55.6_LINEAR_300 17.10 300 LG2 241 scaffold21347_Locus_30137_0_55.6_LINEAR_463 17.10 463 LG2 242 C176398_57.0_524 17.10 524 LG2 243 C214166_53.0_1276 17.10 1276 LG2 244 scaffold4679_Locus_1673_0_56.7_COMPLEX_4790 17.10 4790 LG2 245 C214166_53.0_1760 17.10 1760 LG2 246 C214166_53.0_2611 17.10 2611 LG2 247 C212882_60.0_1400 23.21 1400 LG2 248 C212882_60.0_1390 23.21 1390 LG2 249 scaffold7265_Locus_2943_0_46.9_LINEAR_499 23.83 499 LG2 250 scaffold8377_Locus_4511_0_57.9_COMPLEX_195 26.40 195 LG2 251 Locus_1697_Contig1_419 27.59 419 LG2 252 Locus_1697_Contig1_437 27.59 437 LG2 253 Locus_1697_Contig1_692 27.59 692 LG2 254 Locus_1697_Contig1_1804 27.59 1804 LG2 255 scaffold24145_Locus_38546_0_60.5_LINEAR_1814 28.17 1814 LG2 256 scaffold24145_Locus_38546_0_60.5_LINEAR_1841 28.17 1841 LG2 257 scaffold24145_Locus_38546_0_60.5_LINEAR_2632 28.17 2632 LG2 258 scaffold24145_Locus_38546_0_60.5_LINEAR_2771 28.17 2771 LG2 259 scaffold24145_Locus_38546_0_60.5_LINEAR_3016 28.17 3016 LG2

205

260 scaffold24145_Locus_38546_0_60.5_LINEAR_3328 28.17 3328 LG2 261 scaffold3109_Locus_1074_0_55.8_FORK_541 28.17 541 LG2 262 scaffold14516_Locus_14816_0_59.0_LINEAR_2333 29.35 2333 LG2 263 scaffold9159_Locus_5608_0_56.0_COMPLEX_502 29.35 502 LG2 264 scaffold9159_Locus_5608_0_56.0_COMPLEX_748 29.35 748 LG2 265 scaffold9159_Locus_5608_0_56.0_COMPLEX_1210 29.35 1210 LG2 266 scaffold9159_Locus_5608_0_56.0_COMPLEX_1954 29.35 1954 LG2 267 scaffold14516_Locus_14816_0_59.0_LINEAR_817 29.35 817 LG2 268 scaffold14516_Locus_14816_0_59.0_LINEAR_1329 29.35 1329 LG2 269 scaffold14516_Locus_14816_0_59.0_LINEAR_1669 29.35 1669 LG2 270 scaffold14516_Locus_14816_0_59.0_LINEAR_3948 29.35 3948 LG2 271 C213592_59.0_2097 30.57 2097 LG2 272 C198066_60.0_998 31.79 998 LG2 273 C198066_60.0_719 31.79 719 LG2 274 C198066_60.0_728 31.79 728 LG2 275 C198066_60.0_977 31.79 977 LG2 276 C198066_60.0_1041 31.79 1041 LG2 277 scaffold24903_Locus_42256_0_60.4_LINEAR_1977 31.79 1977 LG2 278 scaffold24903_Locus_42256_0_60.4_LINEAR_1134 31.79 1134 LG2 279 C209576_58.0_541 32.96 541 LG2 280 C209576_58.0_191 32.96 191 LG2 281 C209576_58.0_1303 32.96 1303 LG2 282 scaffold1313_Locus_440_0_65.6_COMPLEX_3122 36.05 3122 LG2 283 scaffold1313_Locus_440_0_65.6_COMPLEX_3065 36.05 3065 LG2 284 scaffold1313_Locus_440_0_65.6_COMPLEX_3176 36.05 3176 LG2 285 scaffold1313_Locus_440_0_65.6_COMPLEX_3215 36.05 3215 LG2 286 scaffold1313_Locus_440_0_65.6_COMPLEX_3284 36.05 3284 LG2 287 scaffold1313_Locus_440_0_65.6_COMPLEX_3354 36.05 3354 LG2 288 C212238_61.0_1412 37.27 1412 LG2 289 C212238_61.0_2046 37.27 2046 LG2 290 C212238_61.0_1954 37.27 1954 LG2 291 C212238_61.0_1969 37.27 1969 LG2 292 C216242_61.0_3801 37.85 3801 LG2 293 C199808_61.0_711 37.85 711 LG2 294 C216242_61.0_1516 37.85 1516 LG2 295 Locus_23238_Contig1_972 40.33 972 LG2 296 Locus_23238_Contig1_681 40.33 681 LG2 297 Locus_23238_Contig1_882 40.33 882 LG2

206

298 Locus_23238_Contig1_2462 40.33 2462 LG2 299 Locus_23238_Contig1_2807 40.33 2807 LG2 300 Locus_23238_Contig1_3767 40.33 3767 LG2 301 Locus_23238_Contig1_4367 40.33 4367 LG2 302 Locus_23238_Contig1_4573 40.33 4573 LG2 303 Locus_23238_Contig1_4659 40.33 4659 LG2 304 Locus_23238_Contig1_4850 40.33 4850 LG2 305 scaffold15888_Locus_17561_0_61.5_BUBBLE_2757 42.16 2757 LG2 306 scaffold24064_Locus_38293_0_59.0_LINEAR_723 43.36 723 LG2 307 scaffold22926_Locus_34390_1_50.8_FORK_581 43.36 581 LG2 308 scaffold22926_Locus_34390_1_50.8_FORK_1173 43.36 1173 LG2 309 scaffold25459_Locus_48226_0_57.6_LINEAR_427 43.36 427 LG2 310 scaffold24064_Locus_38293_0_59.0_LINEAR_214 43.36 214 LG2 311 scaffold24064_Locus_38293_0_59.0_LINEAR_957 43.36 957 LG2 312 scaffold24064_Locus_38293_0_59.0_LINEAR_1551 43.36 1551 LG2 313 scaffold24064_Locus_38293_0_59.0_LINEAR_2537 43.36 2537 LG2 314 C212610_61.0_1598 44.58 1598 LG2 315 C214790_58.0_1345 49.39 1345 LG2 316 C207760_53.0_702 56.58 702 LG2 317 scaffold21717_Locus_31075_0_56.4_LINEAR_275 60.54 275 LG2 318 C183806_58.0_610 61.77 610 LG2 319 C183806_58.0_613 61.77 613 LG2 320 C213576_61.0_425 64.31 425 LG2 321 Locus_11549_Contig1_2630 65.59 2630 LG2 322 C213398_57.0_890 65.59 890 LG2 323 C213398_57.0_1361 65.59 1361 LG2 324 C213398_57.0_1385 65.59 1385 LG2 325 C213398_57.0_1388 65.59 1388 LG2 326 C213398_57.0_1445 65.59 1445 LG2 327 Locus_14496_Contig1_208 65.59 208 LG2 328 scaffold843_Locus_283_0_56.4_COMPLEX_1447 65.59 1447 LG2 329 scaffold843_Locus_283_0_56.4_COMPLEX_1025 65.59 1025 LG2 330 scaffold843_Locus_283_0_56.4_COMPLEX_1200 65.59 1200 LG2 331 scaffold843_Locus_283_0_56.4_COMPLEX_1466 65.59 1466 LG2 332 scaffold843_Locus_283_0_56.4_COMPLEX_1486 65.59 1486 LG2 333 Locus_617_Contig1_1714 66.22 1714 LG2 334 Locus_617_Contig1_1924 66.22 1924 LG2 335 Locus_617_Contig1_1231 66.22 1231 LG2

207

336 scaffold8902_Locus_5288_1_58.4_COMPLEX_1359 66.82 1359 LG2 337 scaffold8901_Locus_5288_0_59.5_COMPLEX_1853 66.82 1853 LG2 338 C210296_58.0_1699 0.00 1699 LG3 339 scaffold12990_Locus_11893_0_59.6_COMPLEX_1312 1.81 1312 LG3 340 C213294_58.0_986 2.41 986 LG3 341 C199880_58.0_1138 4.29 1138 LG3 342 scaffold9387_Locus_5964_0_57.8_COMPLEX_1835 4.29 1835 LG3 343 C191170_60.0_351 4.90 351 LG3 344 C201984_62.0_536 4.90 536 LG3 345 scaffold24805_Locus_41690_0_59.0_LINEAR_652 4.90 652 LG3 346 C207676_47.0_1585 4.90 1585 LG3 347 scaffold16298_Locus_18433_1_48.8_BUBBLE_483 5.51 483 LG3 348 C215940_56.0_2848 5.51 2848 LG3 349 C215940_56.0_2938 5.51 2938 LG3 350 C213302_58.0_229 5.51 229 LG3 351 Locus_1808_Contig2_3546 5.51 3546 LG3 352 Locus_2314_Contig1_854 5.51 854 LG3 353 C214198_61.0_2381 6.09 2381 LG3 354 C213320_61.0_2259 6.09 2259 LG3 355 C213210_55.0_1672 6.09 1672 LG3 356 C210768_60.0_1560 6.09 1560 LG3 357 scaffold16159_Locus_18164_0_49.9_COMPLEX_2624 6.09 2624 LG3 358 scaffold20838_Locus_28834_0_52.0_BUBBLE_274 6.09 274 LG3 359 Locus_19878_Contig1_304 7.33 304 LG3 360 Locus_19878_Contig1_1374 7.33 1374 LG3 361 scaffold22550_Locus_33313_0_54.0_LINEAR_529 8.63 529 LG3 362 scaffold22550_Locus_33313_0_54.0_LINEAR_785 8.63 785 LG3 363 Locus_15558_Contig1_342 8.63 342 LG3 364 scaffold7360_Locus_3076_1_50.4_COMPLEX_2116 12.53 2116 LG3 365 scaffold7360_Locus_3076_1_50.4_COMPLEX_2423 12.53 2423 LG3 366 scaffold25340_Locus_46072_0_53.5_LINEAR_2311 13.77 2311 LG3 367 C194214_55.0_517 14.35 517 LG3 368 scaffold25291_Locus_45336_0_56.4_LINEAR_481 14.35 481 LG3 369 C194214_55.0_501 14.35 501 LG3 370 C212660_55.0_1529 14.92 1529 LG3 371 C212660_55.0_1097 14.92 1097 LG3 372 Locus_2038_Contig1_1909 15.50 1909 LG3 373 Locus_2038_Contig1_1867 15.50 1867 LG3

208

374 Locus_2038_Contig1_2115 15.50 2115 LG3 375 Locus_2038_Contig1_2171 15.50 2171 LG3 376 C213600_58.0_1285 20.06 1285 LG3 377 C213600_58.0_312 20.06 312 LG3 378 C213600_58.0_316 20.06 316 LG3 379 C213600_58.0_1333 20.06 1333 LG3 380 C213600_58.0_1345 20.06 1345 LG3 381 C213600_58.0_1534 20.06 1534 LG3 382 C213600_58.0_1565 20.06 1565 LG3 383 C213600_58.0_1720 20.06 1720 LG3 384 C213600_58.0_1969 20.06 1969 LG3 385 C194226_40.0_462 20.06 462 LG3 386 scaffold24473_Locus_39958_0_56.1_LINEAR_2198 24.68 2198 LG3 387 C198726_57.0_479 27.28 479 LG3 388 C198726_57.0_769 27.28 769 LG3 389 C182858_54.0_457 27.28 457 LG3 390 scaffold11294_Locus_8960_0_26.3_LINEAR_703 27.87 703 LG3 391 scaffold11294_Locus_8960_0_26.3_LINEAR_976 27.87 976 LG3 392 scaffold11294_Locus_8960_0_26.3_LINEAR_1553 27.87 1553 LG3 393 C186430_51.0_620 30.92 620 LG3 394 scaffold4937_Locus_1772_0_61.5_COMPLEX_1297 30.92 1297 LG3 395 C186430_51.0_645 30.92 645 LG3 396 C186430_51.0_825 30.92 825 LG3 397 C186430_51.0_905 30.92 905 LG3 398 C186430_51.0_939 30.92 939 LG3 399 C191990_56.0_880 31.50 880 LG3 400 C202520_53.0_205 32.70 205 LG3 401 C165636_44.0_372 35.27 372 LG3 402 C165636_44.0_444 35.27 444 LG3 403 C210452_55.0_1651 38.44 1651 LG3 404 scaffold5618_Locus_2084_0_58.1_COMPLEX_601 38.44 601 LG3 405 scaffold5618_Locus_2084_0_58.1_COMPLEX_1870 38.44 1870 LG3 406 scaffold12100_Locus_10264_0_57.5_LINEAR_2155 38.44 2155 LG3 407 scaffold12100_Locus_10264_0_57.5_LINEAR_2608 38.44 2608 LG3 408 C215634_55.0_838 39.03 838 LG3 409 C215634_55.0_2509 39.03 2509 LG3 410 C215634_55.0_2734 39.03 2734 LG3 411 C188880_55.0_201 39.63 201 LG3

209

412 C188880_55.0_487 39.63 487 LG3 413 C188880_55.0_231 39.63 231 LG3 414 C212108_54.0_940 39.63 940 LG3 415 C212108_54.0_853 39.63 853 LG3 416 scaffold17470_Locus_20924_0_61.1_COMPLEX_1310 40.22 1310 LG3 417 scaffold17470_Locus_20924_0_61.1_COMPLEX_728 40.22 728 LG3 418 scaffold17470_Locus_20924_0_61.1_COMPLEX_953 40.22 953 LG3 419 scaffold17470_Locus_20924_0_61.1_COMPLEX_1070 40.22 1070 LG3 420 scaffold17470_Locus_20924_0_61.1_COMPLEX_1076 40.22 1076 LG3 421 scaffold17470_Locus_20924_0_61.1_COMPLEX_1145 40.22 1145 LG3 422 scaffold17470_Locus_20924_0_61.1_COMPLEX_1331 40.22 1331 LG3 423 C192336_56.0_848 40.22 848 LG3 424 C183868_56.0_617 40.22 617 LG3 425 C183868_56.0_661 40.22 661 LG3 426 C183868_56.0_729 40.22 729 LG3 427 C188880_55.0_708 40.22 708 LG3 428 scaffold2608_Locus_885_1_56.5_COMPLEX_204 40.22 204 LG3 429 scaffold4423_Locus_1574_0_62.6_COMPLEX_303 40.22 303 LG3 430 scaffold7950_Locus_3907_0_54.4_LINEAR_462 44.12 462 LG3 431 C211704_59.0_739 48.13 739 LG3 432 C213718_59.0_1611 0.00 1611 LG4 433 C213718_59.0_1356 0.00 1356 LG4 434 Locus_506_Contig1_3928 0.61 3928 LG4 435 C171954_58.0_17 0.61 17 LG4 436 C210180_54.0_1614 9.39 1614 LG4 437 C210180_54.0_1794 9.39 1794 LG4 438 C195454_60.0_694 10.63 694 LG4 439 scaffold6580_Locus_2547_1_57.1_BUBBLE_915 15.44 915 LG4 440 C185360_56.0_271 17.94 271 LG4 441 C185360_56.0_285 17.94 285 LG4 442 scaffold15870_Locus_17521_0_60.5_LINEAR_1042 19.79 1042 LG4 443 scaffold19598_Locus_25862_0_60.2_LINEAR_812 24.29 812 LG4 444 scaffold19598_Locus_25862_0_60.2_LINEAR_941 24.29 941 LG4 445 scaffold102_Locus_31_4_43.3_COMPLEX_187 24.29 187 LG4 446 C201160_56.0_982 24.29 982 LG4 447 C204826_56.0_475 24.89 475 LG4 448 Locus_2876_Contig1_3093 26.12 3093 LG4 449 Locus_2876_Contig1_3390 26.12 3390 LG4

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450 Locus_2876_Contig1_3576 26.12 3576 LG4 451 Locus_10297_Contig1_734 28.69 734 LG4 452 Locus_10297_Contig1_1249 28.69 1249 LG4 453 Locus_7003_Contig1_794 28.69 794 LG4 454 Locus_7003_Contig1_836 28.69 836 LG4 455 C195476_53.0_68 29.31 68 LG4 456 scaffold16251_Locus_18335_0_52.9_COMPLEX_280 29.31 280 LG4 457 C196640_52.0_442 29.94 442 LG4 458 Locus_8710_Contig1_1366 29.94 1366 LG4 459 scaffold5244_Locus_1903_0_52.6_COMPLEX_281 31.19 281 LG4 460 C202882_62.0_387 32.42 387 LG4 461 C202882_62.0_1344 32.42 1344 LG4 462 C213428_57.0_2513 33.06 2513 LG4 463 C213428_57.0_2561 33.06 2561 LG4 464 Locus_5114_Contig1_1392 33.72 1392 LG4 465 C215324_58.0_3472 33.72 3472 LG4 466 scaffold16480_Locus_18831_1_61.1_FORK_2621 33.72 2621 LG4 467 scaffold16479_Locus_18831_0_40.6_FORK_470 33.72 470 LG4 468 scaffold16092_Locus_18029_0_50.8_COMPLEX_951 33.72 951 LG4 469 scaffold16092_Locus_18029_0_50.8_COMPLEX_981 33.72 981 LG4 470 scaffold16092_Locus_18029_0_50.8_COMPLEX_1110 33.72 1110 LG4 471 scaffold16092_Locus_18029_0_50.8_COMPLEX_1178 33.72 1178 LG4 472 C213762_58.0_1693 33.72 1693 LG4 473 C207914_61.0_1066 34.33 1066 LG4 474 C207914_61.0_1351 34.33 1351 LG4 475 C207914_61.0_1468 34.33 1468 LG4 476 C207914_61.0_1513 34.33 1513 LG4 477 C207914_61.0_1625 34.33 1625 LG4 478 scaffold16733_Locus_19387_0_57.9_COMPLEX_3815 35.55 3815 LG4 479 Locus_32748_Contig1_2947 36.82 2947 LG4 480 C191772_31.0_487 40.88 487 LG4 481 scaffold25535_Locus_50938_0_58.0_LINEAR_2024 44.84 2024 LG4 482 scaffold25535_Locus_50938_0_58.0_LINEAR_4754 44.84 4754 LG4 483 scaffold24080_Locus_38331_1_60.2_FORK_271 46.71 271 LG4 484 scaffold24080_Locus_38331_1_60.2_FORK_301 46.71 301 LG4 485 scaffold24080_Locus_38331_1_60.2_FORK_343 46.71 343 LG4 486 scaffold24080_Locus_38331_1_60.2_FORK_512 46.71 512 LG4 487 scaffold24080_Locus_38331_1_60.2_FORK_539 46.71 539 LG4

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488 scaffold24080_Locus_38331_1_60.2_FORK_432 46.71 432 LG4 489 C199490_57.0_914 46.71 914 LG4 490 scaffold6102_Locus_2313_0_54.2_COMPLEX_1224 47.33 1224 LG4 491 scaffold20971_Locus_29169_0_60.2_LINEAR_534 48.57 534 LG4 492 C177350_54.0_447 51.77 447 LG4 493 scaffold10263_Locus_7328_0_60.0_COMPLEX_1224 54.16 1224 LG4 494 C215956_61.0_3824 54.16 3824 LG4 495 scaffold10266_Locus_7328_3_65.3_COMPLEX_922 54.16 922 LG4 496 Locus_18070_Contig1_798 54.16 798 LG4 497 Locus_18070_Contig1_913 54.16 913 LG4 498 C215576_60.0_3597 54.16 3597 LG4 499 C196326_39.0_1072 55.38 1072 LG4 500 scaffold6804_Locus_2649_1_55.5_COMPLEX_1367 56.02 1367 LG4 501 C197294_54.0_908 57.32 908 LG4 502 C197294_54.0_881 57.32 881 LG4 503 C210024_58.0_1639 57.32 1639 LG4 504 Locus_221_Contig1_992 61.12 992 LG4 505 Locus_221_Contig1_1064 61.12 1064 LG4 506 Locus_221_Contig1_1157 61.12 1157 LG4 507 Locus_221_Contig1_1758 61.12 1758 LG4 508 C176868_60.0_377 61.12 377 LG4 509 C176868_60.0_571 61.12 571 LG4 510 C176868_60.0_594 61.12 594 LG4 511 C176868_60.0_782 61.12 782 LG4 512 scaffold7135_Locus_2851_1_57.7_COMPLEX_963 61.12 963 LG4 513 scaffold7135_Locus_2851_1_57.7_COMPLEX_1319 61.12 1319 LG4 514 scaffold7135_Locus_2851_1_57.7_COMPLEX_1344 61.12 1344 LG4 515 scaffold7135_Locus_2851_1_57.7_COMPLEX_1351 61.12 1351 LG4 516 scaffold7135_Locus_2851_1_57.7_COMPLEX_1352 61.12 1352 LG4 517 scaffold7135_Locus_2851_1_57.7_COMPLEX_1360 61.12 1360 LG4 518 scaffold7135_Locus_2851_1_57.7_COMPLEX_1378 61.12 1378 LG4 519 scaffold7135_Locus_2851_1_57.7_COMPLEX_1387 61.12 1387 LG4 520 scaffold7135_Locus_2851_1_57.7_COMPLEX_1389 61.12 1389 LG4 521 Locus_221_Contig1_1580 61.12 1580 LG4 522 C201820_50.0_281 62.29 281 LG4 523 C212052_58.0_815 62.29 815 LG4 524 scaffold25500_Locus_49384_0_60.0_LINEAR_3247 64.03 3247 LG4 525 scaffold25500_Locus_49384_0_60.0_LINEAR_1188 64.03 1188 LG4

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526 scaffold25500_Locus_49384_0_60.0_LINEAR_3397 64.03 3397 LG4 527 Locus_27651_Contig1_513 65.21 513 LG4 528 Locus_27651_Contig1_579 65.21 579 LG4 529 Locus_27651_Contig1_585 65.21 585 LG4 530 Locus_27651_Contig1_648 65.21 648 LG4 531 scaffold5827_Locus_2181_1_57.0_COMPLEX_1283 67.65 1283 LG4 532 scaffold5827_Locus_2181_1_57.0_COMPLEX_2045 67.65 2045 LG4 533 scaffold5827_Locus_2181_1_57.0_COMPLEX_2087 67.65 2087 LG4 534 scaffold5827_Locus_2181_1_57.0_COMPLEX_2810 67.65 2810 LG4 535 scaffold5827_Locus_2181_1_57.0_COMPLEX_2816 67.65 2816 LG4 536 scaffold5827_Locus_2181_1_57.0_COMPLEX_2936 67.65 2936 LG4 537 scaffold5827_Locus_2181_1_57.0_COMPLEX_2963 67.65 2963 LG4 538 scaffold5827_Locus_2181_1_57.0_COMPLEX_3006 67.65 3006 LG4 539 C181368_49.0_824 70.06 824 LG4 540 C174362_52.0_560 74.01 560 LG4 541 C200682_59.0_350 78.70 350 LG4 542 C200682_59.0_458 78.70 458 LG4 543 C200682_59.0_423 78.70 423 LG4 544 C209350_49.0_1525 78.70 1525 LG4 545 C198294_56.0_404 79.26 404 LG4 546 C198294_56.0_974 79.26 974 LG4 547 scaffold20945_Locus_29109_0_48.5_LINEAR_122 79.26 122 LG4 548 C210854_43.0_877 79.83 877 LG4 549 C210854_43.0_809 79.83 809 LG4 550 C210854_43.0_1082 79.83 1082 LG4 551 C212280_58.0_1970 79.83 1970 LG4 552 C212280_58.0_2078 79.83 2078 LG4 553 C210854_43.0_806 79.83 806 LG4 554 scaffold2640_Locus_898_0_65.2_COMPLEX_1724 83.12 1724 LG4 555 scaffold21987_Locus_31736_0_56.1_FORK_1306 83.12 1306 LG4 556 scaffold21987_Locus_31736_0_56.1_FORK_1411 83.12 1411 LG4 557 scaffold21987_Locus_31736_0_56.1_FORK_1603 83.12 1603 LG4 558 C182608_54.0_588 85.18 588 LG4 559 Locus_768_Contig1_260 87.18 260 LG4 560 Locus_768_Contig1_263 87.18 263 LG4 561 Locus_768_Contig1_557 87.18 557 LG4 562 scaffold12525_Locus_11052_0_54.3_COMPLEX_781 87.18 781 LG4 563 Locus_2093_Contig1_876 87.18 876 LG4

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564 scaffold12525_Locus_11052_0_54.3_COMPLEX_772 87.18 772 LG4 565 C208552_60.0_1022 87.18 1022 LG4 566 Locus_2093_Contig1_906 87.18 906 LG4 567 Locus_768_Contig1_677 87.18 677 LG4 568 C192644_57.0_305 0.00 305 LG5 569 C188808_45.0_683 2.67 683 LG5 570 C202154_57.0_756 13.27 756 LG5 571 C207678_45.0_1579 13.90 1579 LG5 572 C199104_60.0_377 16.46 377 LG5 573 C150490_28.0_298 17.70 298 LG5 574 Locus_7052_Contig1_2100 20.91 2100 LG5 575 Locus_7052_Contig1_913 20.91 913 LG5 576 C196518_55.0_277 20.91 277 LG5 577 C196518_55.0_573 20.91 573 LG5 578 Locus_27101_Contig1_355 22.10 355 LG5 579 Locus_27101_Contig1_508 22.10 508 LG5 580 Locus_27101_Contig1_784 22.10 784 LG5 581 Locus_27101_Contig1_827 22.10 827 LG5 582 Locus_27101_Contig1_835 22.10 835 LG5 583 Locus_27101_Contig1_913 22.10 913 LG5 584 Locus_27101_Contig1_971 22.10 971 LG5 585 Locus_27101_Contig1_979 22.10 979 LG5 586 Locus_27101_Contig1_1099 22.10 1099 LG5 587 Locus_27101_Contig1_1393 22.10 1393 LG5 588 Locus_27101_Contig1_1540 22.10 1540 LG5 589 Locus_27101_Contig1_1973 22.10 1973 LG5 590 Locus_27101_Contig1_2060 22.10 2060 LG5 591 Locus_27101_Contig1_2069 22.10 2069 LG5 592 C209140_57.0_451 23.95 451 LG5 593 C183474_62.0_367 25.85 367 LG5 594 C183474_62.0_376 25.85 376 LG5 595 C201382_51.0_797 31.51 797 LG5 596 C201382_51.0_1070 31.51 1070 LG5 597 scaffold14200_Locus_14227_0_59.0_FORK_1209 31.51 1209 LG5 598 C213264_54.0_1760 31.51 1760 LG5 599 C188916_57.0_104 31.51 104 LG5 600 C167014_64.0_495 31.51 495 LG5 601 scaffold14200_Locus_14227_0_59.0_FORK_762 31.51 762 LG5

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602 C205172_60.0_311 34.68 311 LG5 603 C210460_55.0_1836 35.25 1836 LG5 604 C199840_59.0_1368 35.25 1368 LG5 605 C196012_59.0_549 35.25 549 LG5 606 C196012_59.0_1181 35.25 1181 LG5 607 C197180_56.0_678 35.85 678 LG5 608 C207980_54.0_1302 35.85 1302 LG5 609 scaffold25376_Locus_46472_0_39.2_LINEAR_2694 35.85 2694 LG5 610 scaffold20706_Locus_28526_0_45.3_LINEAR_1458 36.44 1458 LG5 611 scaffold20706_Locus_28526_0_45.3_LINEAR_386 36.44 386 LG5 612 scaffold20706_Locus_28526_0_45.3_LINEAR_1268 36.44 1268 LG5 613 scaffold20706_Locus_28526_0_45.3_LINEAR_1817 36.44 1817 LG5 614 C214122_59.0_2028 36.44 2028 LG5 615 C212758_61.0_612 37.01 612 LG5 616 C212758_61.0_631 37.01 631 LG5 617 C206392_61.0_853 37.01 853 LG5 618 scaffold439_Locus_149_1_41.8_COMPLEX_347 37.01 347 LG5 619 scaffold7525_Locus_3275_0_57.6_COMPLEX_243 37.01 243 LG5 620 scaffold439_Locus_149_1_41.8_COMPLEX_348 37.01 348 LG5 621 scaffold439_Locus_149_1_41.8_COMPLEX_351 37.01 351 LG5 622 scaffold439_Locus_149_1_41.8_COMPLEX_386 37.01 386 LG5 623 C182060_59.0_563 37.01 563 LG5 624 C212880_59.0_1597 37.01 1597 LG5 625 C212880_59.0_2263 37.01 2263 LG5 626 C182060_59.0_292 37.01 292 LG5 627 C212758_61.0_1466 37.01 1466 LG5 628 C206392_61.0_1402 37.01 1402 LG5 629 C206392_61.0_1465 37.01 1465 LG5 630 C206392_61.0_1468 37.01 1468 LG5 631 Locus_2099_Contig1_2422 38.20 2422 LG5 632 scaffold15593_Locus_16960_0_63.7_FORK_1308 38.20 1308 LG5 633 C211428_58.0_619 38.20 619 LG5 634 Locus_1436_Contig1_1944 38.20 1944 LG5 635 Locus_1436_Contig1_1959 38.20 1959 LG5 636 Locus_1436_Contig1_2026 38.20 2026 LG5 637 Locus_1436_Contig1_2052 38.20 2052 LG5 638 Locus_3556_Contig1_571 38.78 571 LG5 639 Locus_3556_Contig1_580 38.78 580 LG5

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640 Locus_3556_Contig1_664 38.78 664 LG5 641 Locus_3556_Contig1_916 38.78 916 LG5 642 scaffold5149_Locus_1863_0_57.7_COMPLEX_2420 38.78 2420 LG5 643 C211532_60.0_169 38.78 169 LG5 644 scaffold20212_Locus_27295_1_56.2_FORK_1589 42.58 1589 LG5 645 C170458_59.0_692 45.79 692 LG5 646 C187658_58.0_673 46.39 673 LG5 647 scaffold7352_Locus_3071_0_40.7_COMPLEX_619 46.97 619 LG5 648 scaffold11802_Locus_9724_1_53.8_COMPLEX_1125 47.57 1125 LG5 649 C192842_59.0_538 49.57 538 LG5 650 Locus_23696_Contig1_1246 49.57 1246 LG5 651 C169752_44.0_176 49.57 176 LG5 652 C214224_57.0_653 50.82 653 LG5 653 scaffold24136_Locus_38522_0_60.8_COMPLEX_947 54.68 947 LG5 654 scaffold25475_Locus_48731_0_45.3_LINEAR_480 55.90 480 LG5 655 scaffold25475_Locus_48731_0_45.3_LINEAR_1451 55.90 1451 LG5 656 C203762_57.0_351 57.77 351 LG5 657 C203762_57.0_1159 57.77 1159 LG5 658 C204908_58.0_670 57.77 670 LG5 659 C203762_57.0_357 57.77 357 LG5 660 C203762_57.0_379 57.77 379 LG5 661 C203762_57.0_804 57.77 804 LG5 662 C207614_61.0_298 58.38 298 LG5 663 C203228_56.0_490 59.00 490 LG5 664 C210080_59.0_317 59.00 317 LG5 665 C214356_59.0_487 60.88 487 LG5 666 C214356_59.0_413 60.88 413 LG5 667 C214356_59.0_488 60.88 488 LG5 668 C214356_59.0_1931 60.88 1931 LG5 669 Locus_1335_Contig1_1199 73.44 1199 LG5 670 scaffold11722_Locus_9598_0_50.7_COMPLEX_250 79.55 250 LG5 671 scaffold11722_Locus_9598_0_50.7_COMPLEX_454 79.55 454 LG5 672 scaffold16910_Locus_19745_0_53.0_FORK_794 80.17 794 LG5 673 C173838_53.0_299 85.34 299 LG5 674 scaffold13195_Locus_12272_0_56.9_BUBBLE_507 90.91 507 LG5 675 C206904_60.0_532 92.15 532 LG5 676 C206904_60.0_746 92.15 746 LG5 677 C206904_60.0_686 92.15 686 LG5

216

678 C206904_60.0_752 92.15 752 LG5 679 C206904_60.0_704 92.15 704 LG5 680 C206904_60.0_842 92.15 842 LG5 681 C206904_60.0_996 92.15 996 LG5 682 C206904_60.0_1142 92.15 1142 LG5 683 C190330_56.0_219 92.75 219 LG5 684 C209334_62.0_457 92.75 457 LG5 685 C209334_62.0_499 92.75 499 LG5 686 C209334_62.0_523 92.75 523 LG5 687 C190330_56.0_135 92.75 135 LG5 688 C190330_56.0_209 92.75 209 LG5 689 C190330_56.0_360 92.75 360 LG5 690 C190330_56.0_603 92.75 603 LG5 691 C190330_56.0_606 92.75 606 LG5 692 scaffold10369_Locus_7473_0_54.8_LINEAR_1006 93.33 1006 LG5 693 scaffold20360_Locus_27621_0_62.3_COMPLEX_640 93.33 640 LG5 694 scaffold20360_Locus_27621_0_62.3_COMPLEX_928 93.33 928 LG5 695 scaffold20360_Locus_27621_0_62.3_COMPLEX_2810 93.33 2810 LG5 696 scaffold20360_Locus_27621_0_62.3_COMPLEX_1424 93.33 1424 LG5 697 scaffold20360_Locus_27621_0_62.3_COMPLEX_1611 93.33 1611 LG5 698 scaffold10369_Locus_7473_0_54.8_LINEAR_1411 93.33 1411 LG5 699 Locus_1575_Contig1_2679 0.00 2679 LG6 700 scaffold14079_Locus_13985_0_59.2_BUBBLE_697 1.27 697 LG6 701 scaffold18402_Locus_23200_0_53.0_FORK_733 1.90 733 LG6 702 C208262_41.0_851 1.90 851 LG6 703 C198700_61.0_852 3.82 852 LG6 704 C215306_60.0_1426 3.82 1426 LG6 705 C216256_61.0_3821 4.43 3821 LG6 706 scaffold25526_Locus_50243_0_58.6_LINEAR_1115 8.39 1115 LG6 707 scaffold25526_Locus_50243_0_58.6_LINEAR_2365 8.39 2365 LG6 708 C194956_58.0_454 8.39 454 LG6 709 C194956_58.0_926 8.39 926 LG6 710 scaffold2996_Locus_1032_0_60.7_COMPLEX_606 9.64 606 LG6 711 C206104_54.0_328 10.92 328 LG6 712 Locus_10160_Contig1_624 10.92 624 LG6 713 Locus_5504_Contig1_2262 14.13 2262 LG6 714 Locus_1049_Contig1_2062 14.13 2062 LG6 715 scaffold12435_Locus_10894_0_55.5_COMPLEX_716 14.13 716 LG6

217

716 Locus_5504_Contig1_2560 14.13 2560 LG6 717 C191584_45.0_343 15.96 343 LG6 718 C212920_53.0_1733 15.96 1733 LG6 719 C210504_61.0_167 17.84 167 LG6 720 C210504_61.0_182 17.84 182 LG6 721 C183372_58.0_699 18.45 699 LG6 722 C183372_58.0_769 18.45 769 LG6 723 scaffold10884_Locus_8235_0_58.7_COMPLEX_1641 22.51 1641 LG6 724 scaffold17841_Locus_21852_0_58.6_COMPLEX_122 23.12 122 LG6 725 scaffold17841_Locus_21852_0_58.6_COMPLEX_224 23.12 224 LG6 726 scaffold17841_Locus_21852_0_58.6_COMPLEX_260 23.12 260 LG6 727 C214086_58.0_304 25.69 304 LG6 728 C194748_61.0_881 29.07 881 LG6 729 scaffold22436_Locus_32972_0_56.9_LINEAR_262 30.30 262 LG6 730 scaffold22436_Locus_32972_0_56.9_LINEAR_298 30.30 298 LG6 731 C215236_61.0_1633 36.04 1633 LG6 732 Locus_11301_Contig1_2131 37.34 2131 LG6 733 Locus_121_Contig1_1915 38.58 1915 LG6 734 C134088_37.0_189 38.58 189 LG6 735 C215626_58.0_1134 38.58 1134 LG6 736 C215626_58.0_1245 38.58 1245 LG6 737 C215260_56.0_1072 41.18 1072 LG6 738 C191654_54.0_1034 0.00 1034 LG7 739 scaffold11589_Locus_9403_1_60.4_FORK_4958 0.00 4958 LG7 740 C191654_54.0_1057 0.00 1057 LG7 741 scaffold6391_Locus_2459_0_48.4_BUBBLE_1025 0.60 1025 LG7 742 C197728_60.0_581 3.90 581 LG7 743 C197728_60.0_616 3.90 616 LG7 744 C197728_60.0_626 3.90 626 LG7 745 scaffold7849_Locus_3750_0_54.8_LINEAR_518 3.90 518 LG7 746 scaffold17091_Locus_20175_0_58.4_FORK_2247 7.56 2247 LG7 747 C194514_59.0_359 7.56 359 LG7 748 C194514_59.0_717 7.56 717 LG7 749 scaffold17091_Locus_20175_0_58.4_FORK_2119 7.56 2119 LG7 750 scaffold6078_Locus_2301_0_52.0_FORK_2550 8.15 2550 LG7 751 scaffold6078_Locus_2301_0_52.0_FORK_2591 8.15 2591 LG7 752 scaffold6078_Locus_2301_0_52.0_FORK_2617 8.15 2617 LG7 753 scaffold6078_Locus_2301_0_52.0_FORK_2700 8.15 2700 LG7

218

754 C189760_56.0_500 8.15 500 LG7 755 C189760_56.0_335 8.15 335 LG7 756 C189760_56.0_797 8.15 797 LG7 757 Locus_15833_Contig1_724 9.38 724 LG7 758 C216150_61.0_480 9.98 480 LG7 759 C206434_59.0_1226 11.83 1226 LG7 760 Locus_341_Contig1_197 11.83 197 LG7 761 scaffold13391_Locus_12637_0_45.6_LINEAR_548 11.83 548 LG7 762 Locus_31413_Contig1_1002 15.04 1002 LG7 763 Locus_37392_Contig1_708 16.31 708 LG7 764 C196784_59.0_369 18.31 369 LG7 765 C196784_59.0_1041 18.31 1041 LG7 766 C212666_59.0_1282 23.89 1282 LG7 767 C212666_59.0_1291 23.89 1291 LG7 768 C212666_59.0_1342 23.89 1342 LG7 769 scaffold23672_Locus_36867_0_61.3_FORK_1517 27.95 1517 LG7 770 C168680_54.0_621 0.00 621 LG8 771 C209546_60.0_580 0.60 580 LG8 772 C209546_60.0_250 0.60 250 LG8 773 C209546_60.0_338 0.60 338 LG8 774 C209546_60.0_370 0.60 370 LG8 775 C209546_60.0_691 0.60 691 LG8 776 C209546_60.0_820 0.60 820 LG8 777 C209546_60.0_925 0.60 925 LG8 778 C209546_60.0_1162 0.60 1162 LG8 779 C209546_60.0_1627 0.60 1627 LG8 780 C209000_56.0_710 0.60 710 LG8 781 scaffold25216_Locus_44698_0_49.5_LINEAR_4195 1.79 4195 LG8 782 scaffold25216_Locus_44698_0_49.5_LINEAR_4196 1.79 4196 LG8 783 C184098_57.0_318 2.39 318 LG8 784 scaffold20070_Locus_26951_0_53.8_LINEAR_1237 3.00 1237 LG8 785 scaffold20070_Locus_26951_0_53.8_LINEAR_1240 3.00 1240 LG8 786 scaffold17569_Locus_21151_0_48.0_LINEAR_53 3.58 53 LG8 787 scaffold17569_Locus_21151_0_48.0_LINEAR_109 3.58 109 LG8 788 scaffold17569_Locus_21151_0_48.0_LINEAR_843 3.58 843 LG8 789 scaffold17569_Locus_21151_0_48.0_LINEAR_1245 3.58 1245 LG8 790 scaffold17569_Locus_21151_0_48.0_LINEAR_1806 3.58 1806 LG8 791 scaffold17569_Locus_21151_0_48.0_LINEAR_657 3.58 657 LG8

219

792 C210136_57.0_134 4.19 134 LG8 793 C210136_57.0_238 4.19 238 LG8 794 C210136_57.0_187 4.19 187 LG8 795 C210136_57.0_244 4.19 244 LG8 796 scaffold5252_Locus_1908_0_56.3_BUBBLE_720 10.75 720 LG8 797 C212246_60.0_1751 10.75 1751 LG8 798 C212246_60.0_2060 10.75 2060 LG8 799 C212246_60.0_2044 10.75 2044 LG8 800 C212246_60.0_2055 10.75 2055 LG8 801 C215472_56.0_2713 16.86 2713 LG8 802 C209312_53.0_402 18.09 402 LG8 803 C209312_53.0_1602 18.09 1602 LG8 804 C210352_51.0_693 18.70 693 LG8 805 C210352_51.0_912 18.70 912 LG8 806 C210352_51.0_1119 18.70 1119 LG8 807 C210352_51.0_1269 18.70 1269 LG8 808 scaffold3470_Locus_1208_0_57.5_COMPLEX_2296 18.70 2296 LG8 809 C213904_51.0_2253 20.56 2253 LG8 810 C213904_51.0_2295 20.56 2295 LG8 811 C213904_51.0_2361 20.56 2361 LG8 812 C214120_59.0_1530 21.17 1530 LG8 813 C214120_59.0_2192 21.17 2192 LG8 814 C214120_59.0_2430 21.17 2430 LG8 815 scaffold12394_Locus_10806_1_53.3_FORK_300 21.78 300 LG8

220

Appendix 7.2: BLASTx-based sequence analysis of contigs/scaffold sequences underlying SNP markers against protein sequences of Fabaceae family

Percent Alignment Query Subject identity length q.start q.end s.start s.end evalue bit_score scaffold20706_Locus_28526_0_45.3_LINEAR GAU48978.1 93.93 643 201 2123 2 635 0 1036 scaffold20706_Locus_28526_0_45.3_LINEAR XP_003601283.1 89.88 642 201 2123 2 630 0 1025 scaffold20706_Locus_28526_0_45.3_LINEAR XP_004501935.1 93.52 648 201 2123 2 640 0 1019 scaffold20706_Locus_28526_0_45.3_LINEAR PNY13944.1 93.68 554 201 1856 2 547 0 865 scaffold20706_Locus_28526_0_45.3_LINEAR XP_003522373.1 82.24 642 204 2123 1 632 0 844 scaffold20706_Locus_28526_0_45.3_LINEAR XP_016203196.1 84.19 658 195 2123 2 645 0 844 scaffold20706_Locus_28526_0_45.3_LINEAR XP_007138087.1 83.49 648 204 2123 1 645 0 843 scaffold20706_Locus_28526_0_45.3_LINEAR XP_015967725.1 84.6 656 195 2123 2 646 0 841 scaffold20706_Locus_28526_0_45.3_LINEAR XP_025701602.1 84.45 656 195 2123 2 646 0 838 scaffold20706_Locus_28526_0_45.3_LINEAR RDX80819.1 83.25 609 300 2123 381 981 0 836 scaffold20706_Locus_28526_0_45.3_LINEAR XP_020230293.1 80.56 648 201 2123 2 629 0 833 scaffold20706_Locus_28526_0_45.3_LINEAR XP_014501528.1 82.02 645 204 2123 1 636 0 827 scaffold20706_Locus_28526_0_45.3_LINEAR XP_017421979.1 81.89 646 204 2123 1 637 0 825 scaffold20706_Locus_28526_0_45.3_LINEAR XP_003528152.1 80.66 641 219 2123 3 634 0 817 scaffold20706_Locus_28526_0_45.3_LINEAR KYP52478.1 80.61 624 273 2123 346 949 0 816 scaffold20706_Locus_28526_0_45.3_LINEAR XP_019437163.1 81.83 644 195 2123 1 623 0 814 scaffold20706_Locus_28526_0_45.3_LINEAR XP_019437162.1 80.83 652 195 2123 1 633 0 812 scaffold20706_Locus_28526_0_45.3_LINEAR XP_015970464.1 85.69 594 351 2123 40 625 0 811 scaffold20706_Locus_28526_0_45.3_LINEAR XP_024633747.1 89.3 514 582 2123 1 503 0 809 scaffold20706_Locus_28526_0_45.3_LINEAR XP_025603684.1 85.52 594 351 2123 40 625 0 807 scaffold20706_Locus_28526_0_45.3_LINEAR XP_016191481.1 84.68 594 351 2123 40 622 0 790

scaffold20706_Locus_28526_0_45.3_LINEAR XP_019464075.1 80.2 611 303 2123 23 626 0 781 scaffold20706_Locus_28526_0_45.3_LINEAR OIW15392.1 78.34 591 477 2123 25 599 0 721

221

scaffold20706_Locus_28526_0_45.3_LINEAR XP_019464083.1 78.3 553 303 1949 23 568 0 672 scaffold20706_Locus_28526_0_45.3_LINEAR KRH54411.1 78.56 555 213 1859 1 546 0 664 scaffold20706_Locus_28526_0_45.3_LINEAR XP_007138088.1 80.44 450 204 1529 1 449 1.57E-172 533 scaffold20706_Locus_28526_0_45.3_LINEAR XP_004508896.1 69.11 586 369 2123 47 608 4.76E-170 532 scaffold20706_Locus_28526_0_45.3_LINEAR XP_003601282.1 84 250 4257 5000 48 296 6.08E-119 380 scaffold20706_Locus_28526_0_45.3_LINEAR XP_004501936.1 83.47 248 4257 5000 33 266 9.16E-119 379 scaffold20706_Locus_28526_0_45.3_LINEAR XP_003541783.2 56.27 638 267 2117 18 641 9.47E-104 351 scaffold20706_Locus_28526_0_45.3_LINEAR GAU48982.1 78.8 250 4257 5000 33 258 1.51E-103 335 scaffold20706_Locus_28526_0_45.3_LINEAR KHN19019.1 56.27 638 267 2117 11 634 1.72E-103 350 scaffold20706_Locus_28526_0_45.3_LINEAR XP_020221250.1 56.27 590 408 2117 80 640 2.64E-101 344 scaffold20706_Locus_28526_0_45.3_LINEAR KYP63079.1 56.27 590 408 2117 70 630 3.74E-101 343 scaffold20706_Locus_28526_0_45.3_LINEAR XP_003532602.2 58.23 589 405 2117 122 697 1.08E-99 341 scaffold20706_Locus_28526_0_45.3_LINEAR XP_019421614.1 57.6 599 420 2123 117 700 1.45E-97 335 scaffold20706_Locus_28526_0_45.3_LINEAR XP_020230327.1 73.81 252 4257 4985 27 269 1.50E-96 316 scaffold20706_Locus_28526_0_45.3_LINEAR GAU48981.1 84.41 186 4257 4808 33 215 1.43E-94 280 scaffold20706_Locus_28526_0_45.3_LINEAR GAU48981.1 70.83 96 4790 5077 211 301 1.43E-94 92.4 scaffold20706_Locus_28526_0_45.3_LINEAR KYP52479.1 71.65 261 4257 4985 27 278 3.57E-94 310 scaffold20706_Locus_28526_0_45.3_LINEAR XP_003523075.1 74.1 251 4245 4985 24 264 4.13E-94 309 scaffold20706_Locus_28526_0_45.3_LINEAR RDX80820.1 70.72 263 4257 4985 117 377 1.26E-93 312 scaffold20706_Locus_28526_0_45.3_LINEAR XP_014518827.1 57.24 601 366 2117 59 642 8.65E-93 320 scaffold20706_Locus_28526_0_45.3_LINEAR XP_020221255.1 55.74 549 408 1997 80 602 1.74E-91 315 scaffold20706_Locus_28526_0_45.3_LINEAR BAT86915.1 55.15 602 366 2117 58 641 6.91E-91 314 scaffold20706_Locus_28526_0_45.3_LINEAR XP_017420550.1 72.91 251 4257 4985 27 270 1.08E-89 297 scaffold20706_Locus_28526_0_45.3_LINEAR XP_007138086.1 71.95 246 4257 4985 27 270 4.78E-89 295 scaffold20706_Locus_28526_0_45.3_LINEAR XP_014499773.1 72.8 250 4257 4985 27 270 1.43E-88 293 scaffold20706_Locus_28526_0_45.3_LINEAR XP_019434905.1 71.15 253 4257 4991 21 269 1.16E-87 291 scaffold20706_Locus_28526_0_45.3_LINEAR XP_003527020.1 72.29 249 4257 4985 20 259 1.10E-86 288

222

scaffold20706_Locus_28526_0_45.3_LINEAR XP_017434847.1 53.46 621 366 2117 58 660 1.34E-86 302 scaffold20706_Locus_28526_0_45.3_LINEAR KOM53939.1 53.87 607 408 2117 6 597 2.12E-86 300 scaffold20706_Locus_28526_0_45.3_LINEAR XP_016203197.1 71.43 245 4257 4982 27 253 2.46E-85 283 scaffold20706_Locus_28526_0_45.3_LINEAR XP_015967726.1 71.84 245 4257 4982 27 253 1.87E-84 281 scaffold20706_Locus_28526_0_45.3_LINEAR XP_025701600.1 71.84 245 4257 4982 27 253 1.97E-84 281 scaffold20706_Locus_28526_0_45.3_LINEAR XP_004508862.1 71.26 254 4281 4991 36 288 4.87E-84 281 scaffold20706_Locus_28526_0_45.3_LINEAR XP_019456912.1 64.58 271 4260 4979 31 298 3.64E-81 273 scaffold20706_Locus_28526_0_45.3_LINEAR XP_019464810.1 68.22 258 4281 4979 43 296 1.01E-80 272 scaffold20706_Locus_28526_0_45.3_LINEAR RDX91239.1 52.24 647 300 2141 35 663 1.53E-79 282 scaffold20706_Locus_28526_0_45.3_LINEAR XP_017428724.1 70.49 244 4281 4982 31 265 3.11E-79 266 scaffold20706_Locus_28526_0_45.3_LINEAR OIW18348.1 61.54 286 4260 4979 31 313 5.78E-78 265 scaffold20706_Locus_28526_0_45.3_LINEAR AFK43639.1 70.63 252 4281 4991 36 287 6.17E-78 264 scaffold20706_Locus_28526_0_45.3_LINEAR OIW00333.1 64.84 273 4281 4979 43 311 7.10E-78 265 scaffold20706_Locus_28526_0_45.3_LINEAR XP_003608969.2 70.63 252 4281 4991 36 287 7.20E-78 263 scaffold20706_Locus_28526_0_45.3_LINEAR KOM32815.1 69.48 249 4281 4982 31 270 8.62E-78 263 scaffold20706_Locus_28526_0_45.3_LINEAR XP_007155418.1 66.94 248 4281 4982 31 265 2.26E-77 261 scaffold20706_Locus_28526_0_45.3_LINEAR XP_003550739.2 70.87 230 4317 4982 43 261 8.52E-77 259 scaffold20706_Locus_28526_0_45.3_LINEAR GAU19643.1 66.4 250 4281 4985 36 277 1.40E-75 256 scaffold20706_Locus_28526_0_45.3_LINEAR XP_020221256.1 54.27 503 408 1859 80 556 4.95E-74 262 scaffold20706_Locus_28526_0_45.3_LINEAR KRH42084.1 57.46 503 663 2117 6 495 5.42E-74 260 scaffold20706_Locus_28526_0_45.3_LINEAR XP_016191492.1 69.01 242 4281 4979 45 282 9.88E-73 249 scaffold20706_Locus_28526_0_45.3_LINEAR XP_015970473.1 69.01 242 4281 4979 45 282 7.07E-72 246 scaffold20706_Locus_28526_0_45.3_LINEAR RCW19386.1 68.35 218 4356 4985 39 244 3.65E-71 243 scaffold20706_Locus_28526_0_45.3_LINEAR XP_003525500.1 86.75 151 4533 4985 6 156 1.49E-70 237 scaffold20706_Locus_28526_0_45.3_LINEAR XP_020227401.1 66.27 249 4260 4982 22 255 3.53E-64 223 scaffold20706_Locus_28526_0_45.3_LINEAR KYP56305.1 64.84 256 4260 4982 22 262 2.74E-62 218 scaffold20706_Locus_28526_0_45.3_LINEAR KHN34308.1 78.15 151 4539 4985 1 151 1.26E-60 208

223

scaffold20706_Locus_28526_0_45.3_LINEAR KRH49836.1 61.92 302 1233 2123 101 393 1.03E-57 209 scaffold20706_Locus_28526_0_45.3_LINEAR XP_004504849.1 62.42 298 1236 2117 405 694 1.23E-57 218 scaffold20706_Locus_28526_0_45.3_LINEAR XP_004504849.1 68.18 154 408 869 103 256 3.65E-42 171 scaffold20706_Locus_28526_0_45.3_LINEAR KHN19312.1 77.18 149 4608 4985 1 149 1.39E-57 200 scaffold20706_Locus_28526_0_45.3_LINEAR XP_003608391.2 64.24 302 1233 2123 416 708 1.42E-57 218 scaffold20706_Locus_28526_0_45.3_LINEAR XP_003608391.2 66.45 155 405 869 106 260 7.96E-40 164 scaffold20706_Locus_28526_0_45.3_LINEAR RDX66722.1 63.29 286 1269 2117 175 451 1.18E-56 208 scaffold20706_Locus_28526_0_45.3_LINEAR XP_017420551.1 66.67 195 4254 4823 29 216 1.29E-56 201 scaffold20706_Locus_28526_0_45.3_LINEAR KRH49835.1 62 300 1233 2117 231 521 1.30E-56 210 scaffold20706_Locus_28526_0_45.3_LINEAR KRH49834.1 61.92 302 1233 2123 288 580 6.64E-56 209 scaffold20706_Locus_28526_0_45.3_LINEAR KRH49834.1 64.34 129 483 869 3 131 2.19E-29 130 scaffold20706_Locus_28526_0_45.3_LINEAR XP_020222330.1 61.3 323 1164 2117 364 676 8.36E-56 211 scaffold20706_Locus_28526_0_45.3_LINEAR XP_020222330.1 65.09 169 366 869 67 235 3.35E-40 164 scaffold20706_Locus_28526_0_45.3_LINEAR KRH54409.1 66.14 189 4257 4805 20 199 1.18E-55 197 scaffold20706_Locus_28526_0_45.3_LINEAR XP_006581928.1 66.14 189 4257 4805 20 199 1.20E-55 197 scaffold20706_Locus_28526_0_45.3_LINEAR KOM31247.1 61.75 285 1269 2117 145 421 1.27E-55 204 scaffold20706_Locus_28526_0_45.3_LINEAR XP_003528395.1 62 300 1233 2117 407 697 1.73E-55 211 scaffold20706_Locus_28526_0_45.3_LINEAR XP_003528395.1 64 150 420 869 101 250 1.03E-35 150 scaffold20706_Locus_28526_0_45.3_LINEAR PNX81692.1 74.03 181 4170 4706 3 180 4.20E-55 194 scaffold20706_Locus_28526_0_45.3_LINEAR KHN18461.1 77.7 148 4539 4982 2 127 5.83E-55 191 scaffold20706_Locus_28526_0_45.3_LINEAR XP_025638096.1 62.13 301 1233 2117 423 713 1.05E-54 209 scaffold20706_Locus_28526_0_45.3_LINEAR XP_025638096.1 68.15 157 408 878 112 268 1.49E-40 166 scaffold20706_Locus_28526_0_45.3_LINEAR XP_019423479.1 63.67 289 1269 2123 418 696 1.31E-54 208 scaffold20706_Locus_28526_0_45.3_LINEAR XP_019423479.1 66.67 150 420 869 110 259 3.24E-37 155 scaffold20706_Locus_28526_0_45.3_LINEAR OIV93133.1 63.67 289 1269 2123 425 703 1.38E-54 208 scaffold20706_Locus_28526_0_45.3_LINEAR OIV93133.1 63.69 157 420 869 110 266 5.45E-35 149 scaffold20706_Locus_28526_0_45.3_LINEAR XP_007148360.1 59.94 327 1164 2117 188 508 4.27E-54 202

224

scaffold20706_Locus_28526_0_45.3_LINEAR XP_015956104.1 61.79 301 1233 2117 415 705 1.57E-53 205 scaffold20706_Locus_28526_0_45.3_LINEAR XP_015956104.1 68.15 157 408 878 112 268 4.99E-41 167 scaffold20706_Locus_28526_0_45.3_LINEAR XP_017438756.1 61.75 285 1269 2117 410 686 2.82E-53 204 scaffold20706_Locus_28526_0_45.3_LINEAR XP_017438756.1 68.39 155 405 869 88 242 4.16E-41 167 scaffold20706_Locus_28526_0_45.3_LINEAR XP_007159151.1 61.62 284 1272 2117 407 682 3.19E-53 204 scaffold20706_Locus_28526_0_45.3_LINEAR XP_007159151.1 67.1 155 405 869 88 242 6.37E-41 167 scaffold20706_Locus_28526_0_45.3_LINEAR XP_007148361.1 59.94 327 1164 2117 307 627 4.40E-53 202 scaffold20706_Locus_28526_0_45.3_LINEAR XP_007148361.1 59.51 205 279 887 37 234 8.89E-40 162 scaffold20706_Locus_28526_0_45.3_LINEAR XP_025655308.1 64.06 192 4257 4823 27 200 1.77E-52 189 C197180_56.0 CAA74889.1 89 291 181 1041 1 288 6.66E-152 436 C197180_56.0 XP_013460981.1 79.93 304 181 1041 1 288 2.38E-129 379 C197180_56.0 AFK44430.1 79.93 304 181 1041 1 288 1.36E-128 377 C197180_56.0 XP_004501850.1 77.6 308 181 1041 1 294 2.78E-118 351 C197180_56.0 KEH35016.1 78.67 286 181 987 1 270 1.58E-116 346 C197180_56.0 PNY02779.1 75.25 303 181 1041 1 276 1.88E-115 343 C197180_56.0 XP_020235774.1 74.15 294 181 1041 1 286 7.67E-112 334 C197180_56.0 NP_001348128.1 72.17 309 181 1041 1 302 3.92E-110 331 C197180_56.0 GAU40419.1 77.9 276 238 1041 18 271 6.48E-107 321 C197180_56.0 RDX74639.1 71.88 313 139 1038 30 327 2.20E-106 322 C197180_56.0 XP_007137988.1 72.9 310 181 1041 1 297 2.39E-106 321 C197180_56.0 NP_001239665.1 73.38 293 181 1041 1 287 4.43E-105 317 C197180_56.0 KHN07969.1 73.04 293 181 1041 1 287 2.31E-104 315 C197180_56.0 XP_014502417.1 72.49 309 181 1041 1 299 7.87E-103 312 C197180_56.0 XP_017419777.1 69.16 308 181 1041 1 299 1.68E-102 311 C197180_56.0 XP_025688583.1 65.82 316 208 1041 7 316 1.69E-99 304 C197180_56.0 XP_015954522.1 65.82 316 208 1041 7 316 3.04E-99 303 C197180_56.0 KRH63663.1 69.87 302 181 1020 1 295 7.84E-98 300

225

C197180_56.0 XP_016187623.1 65.61 314 208 1041 7 314 1.70E-95 294 C197180_56.0 XP_014510072.1 69.08 304 172 1041 2 302 3.95E-95 292 C197180_56.0 XP_020218344.1 71.18 288 184 1041 6 289 1.61E-94 290 C197180_56.0 KYP65342.1 72.2 277 217 1041 7 279 3.71E-94 289 C197180_56.0 XP_025641509.1 64 325 175 1041 41 359 1.66E-93 290 C197180_56.0 XP_016189637.1 66.56 317 193 1041 2 313 1.89E-93 288 C197180_56.0 XP_019464184.1 79 219 409 1041 95 308 4.06E-93 287 C197180_56.0 OIW00293.1 79 219 409 1041 90 303 5.74E-93 287 C197180_56.0 XP_004515855.1 67.43 307 181 1041 1 299 5.93E-93 286 C197180_56.0 XP_017420438.1 68.42 304 172 1041 2 302 3.90E-92 285 C197180_56.0 XP_015964618.1 85.71 189 475 1041 130 313 6.76E-92 285 C197180_56.0 XP_025703283.1 85.71 189 475 1041 130 313 9.06E-92 284 C197180_56.0 XP_019457367.1 66.46 316 181 1041 1 302 2.13E-91 283 C197180_56.0 KOM32736.1 70.11 281 217 1041 7 285 2.55E-91 282 C197180_56.0 NP_001341807.1 68.44 301 175 1041 1 296 7.22E-91 281 C197180_56.0 XP_013457715.1 67.75 307 181 1041 1 298 1.27E-90 281 C197180_56.0 XP_019433522.1 69.15 295 235 1041 22 311 1.75E-90 281 C197180_56.0 OIW21658.1 69.15 295 235 1041 15 304 2.20E-90 280 C197180_56.0 XP_007155499.1 67.76 304 172 1041 2 302 2.64E-90 280 C197180_56.0 AFK42498.1 67.1 307 181 1041 1 298 7.71E-89 276 C197180_56.0 GAU50596.1 67.43 307 181 1041 1 299 1.03E-88 276 C197180_56.0 XP_019415396.1 85.16 182 496 1041 134 310 6.31E-87 271 C197180_56.0 PNY03038.1 74.89 223 379 1041 5 225 5.98E-85 263 C197180_56.0 NP_001242356.1 64.9 302 181 1038 1 296 7.25E-85 266 C197180_56.0 ACF22879.1 64.8 304 175 1038 1 298 8.16E-85 266 C197180_56.0 RDX94712.1 64.29 294 238 1041 14 298 5.92E-83 261 C197180_56.0 KHN05811.1 87.2 164 550 1041 1 161 2.28E-79 247

226

C197180_56.0 OIW18313.1 64.77 298 181 987 1 284 3.29E-79 251 C197180_56.0 ACU18482.1 66.9 281 175 981 1 276 4.87E-78 248 C197180_56.0 ACJ84773.1 74.58 236 181 837 1 220 5.35E-78 246 C197180_56.0 AFK47384.1 65.35 303 181 1032 1 300 4.72E-76 243 C197180_56.0 RDX75942.1 65.53 206 448 1044 69 274 1.35E-61 205 C197180_56.0 XP_015951587.1 67.5 200 448 1041 67 266 2.61E-61 204 C197180_56.0 XP_016184868.1 67.5 200 448 1041 67 266 3.10E-61 204 C197180_56.0 XP_020239889.1 67.02 191 493 1044 89 279 1.76E-60 202 C197180_56.0 NP_001348127.1 65.05 206 448 1044 70 275 3.48E-60 201 C197180_56.0 KHN48247.1 64.73 207 448 1044 73 279 4.09E-60 201 C197180_56.0 NP_001240946.1 64.25 207 448 1044 73 279 1.29E-59 200 C197180_56.0 PNX81175.1 77.48 151 589 1041 1 150 6.13E-59 194 C197180_56.0 XP_007144372.1 68.78 189 493 1041 83 271 2.04E-58 197 C197180_56.0 XP_004497514.1 63.78 196 499 1041 87 282 3.83E-57 194 C197180_56.0 XP_019421698.1 64.68 201 499 1041 100 300 4.54E-54 186 C197180_56.0 XP_014514950.1 64.62 195 493 1044 83 277 6.67E-54 185 C197180_56.0 XP_014510849.1 64.77 193 493 1044 83 275 8.84E-54 184 C197180_56.0 XP_017412587.1 64.4 191 493 1038 83 273 1.52E-51 179 C197180_56.0 XP_017413347.1 65.96 188 493 1038 83 270 1.72E-51 179 C197180_56.0 OIW01591.1 61.88 202 499 1041 99 300 7.24E-51 179 C197180_56.0 XP_019461671.1 61.88 202 499 1041 99 300 8.13E-51 178 C197180_56.0 GAU48474.1 65.1 192 499 1041 85 276 2.99E-48 170 C197180_56.0 XP_003590481.1 62.63 198 493 1041 83 278 7.74E-47 166 C197180_56.0 AFK37653.1 57.21 215 493 1041 86 300 8.02E-47 167 C197180_56.0 PNX73157.1 61.35 207 493 1041 83 278 2.06E-46 166 C197180_56.0 ACJ85252.1 62.63 198 493 1041 83 278 4.81E-46 164 C197180_56.0 XP_016176842.1 58.22 213 418 1032 86 282 7.67E-46 164

227

C197180_56.0 RDX80558.1 60.2 196 484 1032 100 279 2.39E-45 162 C197180_56.0 GAU39640.1 62.37 186 499 1032 105 274 4.42E-45 162 C197180_56.0 XP_019414814.1 62.3 183 493 1032 82 253 5.42E-45 161 C197180_56.0 XP_025617871.1 57.75 213 418 1032 86 282 6.17E-45 162 C197180_56.0 XP_017413348.1 57.21 229 364 1032 69 277 7.40E-45 161 C197180_56.0 XP_020227068.1 61.83 186 499 1032 110 279 1.02E-44 161 C197180_56.0 XP_003590174.2 62.9 186 499 1032 114 283 2.16E-44 160 C197180_56.0 ACJ85270.1 62.9 186 499 1032 114 283 2.37E-44 160 C197180_56.0 XP_014514849.1 60.64 188 493 1032 102 273 4.37E-44 159 C197180_56.0 AFK38283.1 60.64 188 493 1032 100 271 6.10E-44 159 C197180_56.0 NP_001347344.1 61.83 186 499 1032 111 280 6.10E-44 159 C197180_56.0 XP_007145349.1 61.96 184 499 1032 114 283 1.67E-43 158 C197180_56.0 XP_019437964.1 53.65 233 343 1032 66 276 2.18E-43 157 C197180_56.0 PNX97045.1 63.13 179 499 1032 98 267 1.22E-42 155 C197180_56.0 AFK34861.1 61.83 186 499 1032 114 283 4.25E-42 154 C197180_56.0 KOM34828.1 64.71 170 493 984 83 252 5.22E-42 155 C197180_56.0 CAA66479.1 62.01 179 499 1032 118 287 9.09E-42 153 C197180_56.0 AHA84143.1 60.87 184 499 1032 114 283 1.85E-41 152 C197180_56.0 KOM34502.1 56.13 212 364 981 69 260 8.06E-40 148 C197180_56.0 NP_001241528.1 61.83 186 499 1032 118 287 8.16E-39 145 C197180_56.0 KHN41542.1 60.95 169 499 981 111 263 9.15E-39 145 C197180_56.0 XP_019444198.1 58.91 202 472 1032 97 298 2.35E-38 145 C197180_56.0 XP_004494258.1 55.98 234 346 1032 63 290 2.46E-38 145 C197180_56.0 XP_013450228.1 58.05 205 433 1032 87 291 5.96E-38 144 C197180_56.0 KRG90317.1 59.14 186 499 1032 118 286 7.35E-38 143 C197180_56.0 XP_025678792.1 77.19 114 700 1041 57 165 1.01E-37 139 C197180_56.0 XP_020967644.1 76.07 117 691 1041 41 152 1.02E-37 138

228

C197180_56.0 XP_003554474.1 55.93 236 340 1032 59 290 1.68E-37 143 C210460_55.0 XP_013460694.1 84.1 327 1756 779 2 324 1.41E-156 462 C210460_55.0 XP_004503159.1 82.78 331 1762 779 2 305 2.87E-148 440 C210460_55.0 XP_007163425.1 79.76 331 1762 779 2 305 4.18E-141 422 C210460_55.0 XP_014496376.1 79.15 331 1762 779 2 305 2.07E-139 418 C210460_55.0 GAU12743.1 84.34 332 1762 779 1 314 5.56E-139 417 C210460_55.0 XP_017415877.1 78.85 331 1762 779 2 305 3.08E-138 415 C210460_55.0 XP_025982507.1 78.48 330 1762 785 3 300 5.48E-138 414 C210460_55.0 XP_003537459.1 77.71 332 1762 785 3 305 9.43E-136 409 C210460_55.0 RDX95109.1 80.06 331 1762 779 1 305 8.40E-135 406 C210460_55.0 PNX96810.1 84.52 407 1744 542 10 397 3.10E-134 405 C210460_55.0 XP_019419778.1 75.98 333 1762 779 1 309 3.11E-128 389 C210460_55.0 XP_019439458.1 76.13 331 1762 779 1 306 1.34E-125 382 C210460_55.0 XP_016190165.1 76.99 339 1762 779 1 323 1.92E-124 379 C210460_55.0 XP_015956524.1 76.99 339 1762 779 1 323 3.33E-124 379 C210460_55.0 KRG97885.1 73.94 330 1762 785 1 280 5.61E-122 372 C210460_55.0 XP_019422227.1 73.27 333 1762 779 1 313 2.44E-113 351 C210460_55.0 XP_020237400.1 67.06 337 1762 779 2 263 8.81E-111 342 C210460_55.0 XP_020233938.1 66.06 330 1744 779 3 294 1.25E-93 300 C210460_55.0 RDX99426.1 63.89 324 1750 779 9 283 2.38E-91 293 C210460_55.0 XP_007132046.1 62.58 326 1750 779 9 285 1.03E-90 291 C210460_55.0 XP_007141397.1 64.94 328 1744 794 3 289 1.64E-90 291 C210460_55.0 XP_017406217.1 61.96 326 1750 779 9 285 2.70E-90 290 C210460_55.0 XP_014494451.1 61.66 326 1750 779 9 285 4.21E-90 289 C210460_55.0 XP_003537982.1 61.01 336 1750 779 9 291 8.66E-90 288 C210460_55.0 RDX60843.1 65.34 326 1744 791 3 294 2.58E-89 288 C210460_55.0 XP_015935453.1 62.54 315 1750 806 10 278 4.04E-88 285

229

C210460_55.0 XP_016170215.1 63.17 315 1750 806 10 278 4.08E-88 285 C210460_55.0 AES99400.1 65.77 336 1744 779 3 304 2.58E-87 283 C210460_55.0 PNY00934.1 60 325 1753 779 8 287 2.74E-87 282 C210460_55.0 XP_014503008.1 65.74 324 1744 794 3 289 4.39E-87 282 C210460_55.0 XP_003606705.1 61.8 322 1759 794 6 286 4.43E-87 282 C210460_55.0 GAU49337.1 60.31 325 1753 779 9 290 6.12E-87 281 C210460_55.0 XP_004486987.1 62.8 328 1762 791 1 281 1.31E-86 279 C210460_55.0 XP_020214787.1 63.27 324 1750 779 9 287 2.33E-86 279 C210460_55.0 XP_003545254.1 64.72 326 1744 794 3 292 4.44E-86 280 C210460_55.0 XP_019448615.1 63.27 324 1762 791 1 273 2.85E-85 276 C210460_55.0 XP_025982216.1 65.35 329 1744 794 3 297 3.71E-85 278 C210460_55.0 XP_003539637.1 62.77 325 1750 779 9 289 3.83E-85 276 C210460_55.0 XP_003597390.1 63.04 322 1756 791 9 294 2.32E-84 274 C210460_55.0 XP_016187260.1 62.88 326 1750 779 9 284 2.94E-84 274 C210460_55.0 XP_004490878.1 61.88 341 1744 779 3 312 1.15E-83 274 C210460_55.0 XP_015952231.1 62.39 327 1750 779 9 285 1.66E-83 271 C210460_55.0 RDX66728.1 60.19 324 1762 791 1 266 3.06E-83 271 C210460_55.0 XP_025686208.1 63 327 1750 779 9 285 3.49E-83 271 C210460_55.0 GAU47130.1 62.31 337 1744 779 3 303 4.30E-83 272 C210460_55.0 XP_025609322.1 62.11 351 1744 806 3 339 7.65E-83 274 C210460_55.0 XP_020983121.1 61.65 352 1744 806 3 340 8.47E-83 274 C210460_55.0 XP_004507169.1 60.62 325 1750 779 10 287 9.75E-83 270 C210460_55.0 XP_019447967.1 66.36 321 1750 791 9 283 1.07E-82 270 C210460_55.0 XP_020962982.1 60.85 355 1744 806 3 344 2.76E-82 273 C210460_55.0 XP_014496469.1 58.33 324 1762 791 1 264 4.30E-82 267 C210460_55.0 XP_017423871.1 59.26 324 1762 791 1 264 1.01E-81 266 C210460_55.0 KYP48506.1 60.06 328 1744 779 3 267 1.10E-81 267

230

C210460_55.0 XP_003543521.1 58.41 327 1762 788 1 256 2.46E-81 265 C210460_55.0 XP_007150348.1 57.41 324 1762 791 1 264 5.32E-81 264 C210460_55.0 XP_020223918.1 57.72 324 1762 791 1 260 2.30E-80 262 C210460_55.0 XP_017428556.1 64.2 324 1744 794 3 289 3.66E-80 264 C210460_55.0 XP_003547238.1 58.95 324 1762 791 1 253 3.67E-80 261 C210460_55.0 XP_003616442.2 65.3 317 1687 779 3 285 1.26E-78 259 C210460_55.0 KYP58518.1 57.1 324 1762 791 1 260 1.74E-78 258 C210460_55.0 PNY02711.1 62.04 353 1744 779 3 326 2.01E-78 261 C210460_55.0 KHN17914.1 85.71 182 1330 785 1 179 4.13E-77 252 C210460_55.0 KHN07574.1 85.71 182 1330 785 1 169 6.86E-77 251 C210460_55.0 XP_019455796.1 54.57 416 1744 542 3 382 1.60E-75 252 C210460_55.0 PNY09352.1 63.41 328 1762 791 6 296 3.81E-74 248 C210460_55.0 KYP68217.1 65.98 241 1750 1028 9 225 6.66E-74 245 C210460_55.0 KYP44759.1 80.95 189 1330 779 1 169 6.00E-73 241 C210460_55.0 GAU17308.1 62.5 328 1762 791 6 292 1.01E-72 242 C210460_55.0 GAU17309.1 62.5 328 1762 791 6 292 2.23E-72 243 C210460_55.0 XP_019454083.1 57.94 321 1753 791 7 274 3.00E-70 236 C210460_55.0 AGV54862.1 58.18 318 1726 779 17 286 5.31E-69 234 C210460_55.0 OIW04064.1 55.03 398 1690 542 2 363 5.63E-65 223 C210460_55.0 XP_003529105.1 54.46 325 1744 794 9 250 1.63E-63 217 C210460_55.0 XP_019459456.1 55.69 325 1744 806 8 260 4.85E-63 217 C210460_55.0 XP_017417312.1 53.35 328 1744 794 9 256 1.78E-60 210 C210460_55.0 XP_006602601.2 54.71 329 1762 794 4 255 1.41E-59 207 C210460_55.0 XP_014497251.1 53.66 328 1744 794 9 256 3.52E-59 206 C210460_55.0 XP_007140509.1 53.54 325 1741 794 11 259 5.84E-54 192 C210460_55.0 KHN41107.1 56.62 302 1666 794 83 326 1.20E-50 186 C210460_55.0 XP_020215697.1 52.73 256 1759 1019 10 228 7.73E-41 155

231

C210460_55.0 RDY11870.1 52.27 264 1759 1019 10 228 5.41E-40 153 C210460_55.0 XP_019424034.1 52.4 250 1741 1019 28 235 3.88E-39 151 C210460_55.0 XP_019421645.1 51.41 249 1741 1019 27 238 1.38E-38 150 C210460_55.0 XP_017413323.1 52.27 264 1762 1001 1 242 3.61E-38 148 C210460_55.0 KHN21932.1 69.66 145 1210 779 1 124 5.14E-38 144 C210460_55.0 RDX96378.1 52.83 265 1762 1001 68 310 7.09E-38 149 C210460_55.0 XP_016183514.1 50.56 267 1762 1004 1 238 1.07E-37 147 C210460_55.0 AFK44009.1 49.81 269 1762 1001 1 239 1.08E-37 146 C210460_55.0 XP_020223740.1 51.52 264 1762 1001 1 242 1.14E-37 146 C210460_55.0 XP_014514318.1 52.27 264 1762 1001 1 242 1.23E-37 146 C210460_55.0 XP_015949647.1 50.19 267 1762 1004 1 238 1.24E-37 146 C210460_55.0 XP_003556948.1 49.09 275 1762 1001 1 243 5.88E-37 144 C210460_55.0 XP_019434714.1 51.38 253 1741 1019 18 223 6.90E-37 144 C210460_55.0 ACU24187.1 49.09 275 1762 1001 1 243 7.22E-37 144 C210460_55.0 XP_007145499.1 51.89 264 1762 1001 1 242 7.25E-37 144 C210460_55.0 XP_003518992.1 49.08 271 1762 1001 1 239 2.49E-35 140 C210460_55.0 OIV90031.1 50.74 270 1762 1019 1 233 5.06E-35 139 C210460_55.0 XP_025620857.1 51.38 253 1741 1019 14 256 7.03E-35 139 C210460_55.0 XP_015941905.1 51.38 253 1741 1019 14 256 7.68E-35 139 C210460_55.0 XP_019428949.1 50.74 270 1762 1019 35 267 1.26E-34 139 scaffold14200_Locus_14227_0_59.0_FORK XP_003601037.1 98.55 276 864 37 114 389 0 539 scaffold14200_Locus_14227_0_59.0_FORK XP_003601037.1 84.96 113 1192 866 1 113 0 177 scaffold14200_Locus_14227_0_59.0_FORK GAU49179.1 97.46 276 864 37 103 378 0 531 scaffold14200_Locus_14227_0_59.0_FORK GAU49179.1 76.32 114 1192 866 1 102 2.23E-35 138 scaffold14200_Locus_14227_0_59.0_FORK OIW02747.1 96.74 276 864 37 671 946 0 530 scaffold14200_Locus_14227_0_59.0_FORK OIW02747.1 73.55 121 1195 866 551 670 0 149 scaffold14200_Locus_14227_0_59.0_FORK KHN04809.1 97.46 276 864 37 125 400 0 529

232

scaffold14200_Locus_14227_0_59.0_FORK KHN04809.1 69.05 126 1192 866 1 124 1.32E-36 142 scaffold14200_Locus_14227_0_59.0_FORK XP_003538500.1 97.46 276 864 37 112 387 0 528 scaffold14200_Locus_14227_0_59.0_FORK XP_003538500.1 77.88 113 1192 866 1 111 0 159 scaffold14200_Locus_14227_0_59.0_FORK KHN31331.1 97.1 276 864 37 113 388 0 526 scaffold14200_Locus_14227_0_59.0_FORK KHN31331.1 75.44 114 1192 866 1 112 2.16E-38 146 scaffold14200_Locus_14227_0_59.0_FORK NP_001241559.1 97.1 276 864 37 112 387 0 526 scaffold14200_Locus_14227_0_59.0_FORK NP_001241559.1 79.65 113 1192 866 1 111 0 157 scaffold14200_Locus_14227_0_59.0_FORK AFK49509.1 97.83 276 864 37 115 390 0 526 scaffold14200_Locus_14227_0_59.0_FORK AFK49509.1 78.45 116 1192 866 1 114 0 157 scaffold14200_Locus_14227_0_59.0_FORK XP_007163609.1 97.1 276 864 37 112 387 0 521 scaffold14200_Locus_14227_0_59.0_FORK XP_007163609.1 77.88 113 1192 866 1 111 0 156 scaffold14200_Locus_14227_0_59.0_FORK XP_025609062.1 97.1 276 864 37 122 397 0 521 scaffold14200_Locus_14227_0_59.0_FORK XP_025609062.1 95.38 65 1060 866 57 121 2.92E-33 132 scaffold14200_Locus_14227_0_59.0_FORK XP_020221475.1 96.38 276 864 37 110 385 0 521 scaffold14200_Locus_14227_0_59.0_FORK XP_020221475.1 77.48 111 1192 866 1 109 0 152 scaffold14200_Locus_14227_0_59.0_FORK XP_020221467.1 96.38 276 864 37 112 387 0 521 scaffold14200_Locus_14227_0_59.0_FORK XP_020221467.1 80.53 113 1192 866 1 111 0 160 scaffold14200_Locus_14227_0_59.0_FORK KYP75811.1 96.38 276 864 37 115 390 0 521 scaffold14200_Locus_14227_0_59.0_FORK KYP75811.1 70.59 119 1192 866 1 114 3.22E-32 130 scaffold14200_Locus_14227_0_59.0_FORK RDY07010.1 96.74 276 864 37 100 375 0 521 scaffold14200_Locus_14227_0_59.0_FORK RDY07010.1 67.26 113 1192 866 1 99 8.41E-30 122 scaffold14200_Locus_14227_0_59.0_FORK XP_019460966.1 96.74 276 864 37 115 390 0 521 scaffold14200_Locus_14227_0_59.0_FORK XP_019460966.1 90.12 81 1105 866 34 114 1.59E-38 147 scaffold14200_Locus_14227_0_59.0_FORK XP_025664178.1 96.74 276 864 37 122 397 0 518 scaffold14200_Locus_14227_0_59.0_FORK XP_025664178.1 95.38 65 1060 866 57 121 2.95E-33 132 scaffold14200_Locus_14227_0_59.0_FORK XP_025664177.1 96.74 276 864 37 124 399 0 518 scaffold14200_Locus_14227_0_59.0_FORK XP_025664177.1 95.45 66 1063 866 58 123 7.22E-34 134

233

scaffold14200_Locus_14227_0_59.0_FORK XP_016166192.1 96.74 276 864 37 124 399 0 518 scaffold14200_Locus_14227_0_59.0_FORK XP_016166192.1 93.94 66 1063 866 58 123 9.93E-33 131 scaffold14200_Locus_14227_0_59.0_FORK XP_017418615.1 96.38 276 864 37 112 387 0 517 scaffold14200_Locus_14227_0_59.0_FORK XP_017418615.1 80.53 113 1192 866 1 111 0 155 scaffold14200_Locus_14227_0_59.0_FORK KOM39645.1 96.38 276 864 37 94 369 0 516 scaffold14200_Locus_14227_0_59.0_FORK KOM39645.1 65.49 113 1192 866 1 93 2.33E-24 107 scaffold14200_Locus_14227_0_59.0_FORK XP_014494576.1 96.01 276 864 37 112 387 0 515 scaffold14200_Locus_14227_0_59.0_FORK XP_014494576.1 77.88 113 1192 866 1 111 0 156 scaffold14200_Locus_14227_0_59.0_FORK XP_015973764.2 88.41 276 864 37 122 374 2.03E-162 465 scaffold14200_Locus_14227_0_59.0_FORK XP_015973764.2 95.38 65 1060 866 57 121 2.03E-162 133 scaffold14200_Locus_14227_0_59.0_FORK GAU46728.1 72.83 276 864 37 120 394 1.00E-106 303 scaffold14200_Locus_14227_0_59.0_FORK GAU46728.1 79.45 73 1084 866 47 119 1.00E-106 109 scaffold14200_Locus_14227_0_59.0_FORK PNX75430.1 97.48 159 513 37 1 159 3.08E-105 311 scaffold14200_Locus_14227_0_59.0_FORK AFK34407.1 98.74 159 513 37 1 159 4.61E-105 311 scaffold14200_Locus_14227_0_59.0_FORK XP_020231368.1 73.19 276 864 37 118 392 5.90E-105 300 scaffold14200_Locus_14227_0_59.0_FORK XP_020231368.1 78.08 73 1084 866 45 117 5.90E-105 107 scaffold14200_Locus_14227_0_59.0_FORK KYP51357.1 73.19 276 864 37 109 383 6.35E-105 300 scaffold14200_Locus_14227_0_59.0_FORK KYP51357.1 78.08 73 1084 866 36 108 6.35E-105 107 scaffold14200_Locus_14227_0_59.0_FORK XP_019443876.1 72.1 276 864 37 120 394 1.10E-104 298 scaffold14200_Locus_14227_0_59.0_FORK XP_019443876.1 78.67 75 1090 866 45 119 1.10E-104 108 scaffold14200_Locus_14227_0_59.0_FORK XP_007149382.1 72.46 276 864 37 118 392 1.37E-104 298 scaffold14200_Locus_14227_0_59.0_FORK XP_007149382.1 79.45 73 1084 866 45 117 1.37E-104 107 scaffold14200_Locus_14227_0_59.0_FORK RDX68454.1 73.19 276 864 37 119 393 1.55E-104 299 scaffold14200_Locus_14227_0_59.0_FORK RDX68454.1 79.45 73 1084 866 46 118 1.55E-104 106 scaffold14200_Locus_14227_0_59.0_FORK AFK39152.1 97.48 159 513 37 1 159 2.63E-104 309 scaffold14200_Locus_14227_0_59.0_FORK XP_017425855.1 73.19 276 864 37 117 391 3.32E-104 298 scaffold14200_Locus_14227_0_59.0_FORK XP_017425855.1 79.45 73 1084 866 44 116 3.32E-104 106

234

scaffold14200_Locus_14227_0_59.0_FORK XP_006592935.1 73.55 276 864 37 121 395 7.02E-104 297 scaffold14200_Locus_14227_0_59.0_FORK XP_006592935.1 78.08 73 1084 866 48 120 7.02E-104 105 scaffold14200_Locus_14227_0_59.0_FORK XP_003540489.1 73.55 276 864 37 120 394 7.32E-104 297 scaffold14200_Locus_14227_0_59.0_FORK XP_003540489.1 78.08 73 1084 866 47 119 7.32E-104 105 scaffold14200_Locus_14227_0_59.0_FORK XP_006592936.1 73.55 276 864 37 119 393 8.22E-104 297 scaffold14200_Locus_14227_0_59.0_FORK XP_006592936.1 78.08 73 1084 866 46 118 8.22E-104 105 scaffold14200_Locus_14227_0_59.0_FORK XP_014521546.1 72.46 276 864 37 117 391 9.47E-104 296 scaffold14200_Locus_14227_0_59.0_FORK XP_014521546.1 79.45 73 1084 866 44 116 9.47E-104 106 scaffold14200_Locus_14227_0_59.0_FORK XP_014521545.1 72.46 276 864 37 118 392 1.02E-103 296 scaffold14200_Locus_14227_0_59.0_FORK XP_014521545.1 79.45 73 1084 866 45 117 1.02E-103 106 scaffold14200_Locus_14227_0_59.0_FORK XP_003596359.1 72.46 276 864 37 121 395 1.64E-103 296 scaffold14200_Locus_14227_0_59.0_FORK XP_003596359.1 80.3 66 1063 866 55 120 1.64E-103 105 scaffold14200_Locus_14227_0_59.0_FORK XP_006594732.1 73.55 276 864 37 119 393 2.84E-103 296 scaffold14200_Locus_14227_0_59.0_FORK XP_006594732.1 79.45 73 1084 866 46 118 2.84E-103 105 scaffold14200_Locus_14227_0_59.0_FORK XP_003543210.1 73.55 276 864 37 120 394 2.88E-103 296 scaffold14200_Locus_14227_0_59.0_FORK XP_003543210.1 79.45 73 1084 866 47 119 2.88E-103 105 scaffold14200_Locus_14227_0_59.0_FORK XP_006594731.1 73.55 276 864 37 121 395 2.96E-103 296 scaffold14200_Locus_14227_0_59.0_FORK XP_006594731.1 79.45 73 1084 866 48 120 2.96E-103 105 scaffold14200_Locus_14227_0_59.0_FORK XP_017442451.1 96.23 159 513 37 1 159 3.16E-103 306 scaffold14200_Locus_14227_0_59.0_FORK KOM43413.1 73.19 276 864 37 117 391 7.69E-103 297 scaffold14200_Locus_14227_0_59.0_FORK KOM43413.1 80.3 66 1063 866 51 116 7.69E-103 102 scaffold14200_Locus_14227_0_59.0_FORK XP_014620580.1 73.55 276 864 37 71 345 1.78E-102 297 scaffold14200_Locus_14227_0_59.0_FORK XP_014620580.1 78.79 66 1063 866 5 70 1.78E-102 101 scaffold14200_Locus_14227_0_59.0_FORK KOM58634.1 96.36 165 531 37 444 608 1.80E-102 320 scaffold14200_Locus_14227_0_59.0_FORK KHN36887.1 73.55 276 864 37 72 346 5.03E-102 296 scaffold14200_Locus_14227_0_59.0_FORK KHN36887.1 80.3 66 1063 866 6 71 5.03E-102 101 scaffold14200_Locus_14227_0_59.0_FORK OIW11581.1 72.1 276 864 37 48 322 4.39E-97 298

235

scaffold14200_Locus_14227_0_59.0_FORK XP_015933652.1 71.74 276 864 37 18 292 9.16E-97 296 scaffold14200_Locus_14227_0_59.0_FORK KRH21966.1 73.55 276 864 37 18 292 1.10E-96 295 scaffold14200_Locus_14227_0_59.0_FORK XP_015933651.1 71.74 276 864 37 48 322 1.18E-96 296 scaffold14200_Locus_14227_0_59.0_FORK XP_016171563.1 71.74 276 864 37 18 292 6.04E-96 294 scaffold14200_Locus_14227_0_59.0_FORK XP_016171562.1 71.74 276 864 37 48 322 7.10E-96 295 scaffold14200_Locus_14227_0_59.0_FORK XP_015933649.1 71.74 276 864 37 117 391 9.96E-96 296 scaffold14200_Locus_14227_0_59.0_FORK XP_015933649.1 58.21 134 1195 812 7 136 1.31E-22 103 scaffold14200_Locus_14227_0_59.0_FORK XP_016171561.1 71.74 276 864 37 117 391 4.82E-95 295 scaffold14200_Locus_14227_0_59.0_FORK XP_016171561.1 69.15 94 1078 812 46 136 1.45E-22 103 scaffold14200_Locus_14227_0_59.0_FORK AFK42086.1 70.71 280 864 25 117 395 1.26E-94 294 scaffold14200_Locus_14227_0_59.0_FORK AFK42086.1 69.79 96 1084 812 44 136 9.89E-25 109 scaffold14200_Locus_14227_0_59.0_FORK XP_004489026.1 72.22 216 864 217 120 334 4.34E-83 226 scaffold14200_Locus_14227_0_59.0_FORK XP_004489026.1 79.45 73 1084 866 47 119 4.34E-83 107 scaffold14200_Locus_14227_0_59.0_FORK ACJ85135.1 68.2 217 864 217 121 336 2.06E-75 203 scaffold14200_Locus_14227_0_59.0_FORK ACJ85135.1 80.3 66 1063 866 55 120 2.06E-75 105 scaffold14200_Locus_14227_0_59.0_FORK XP_020986158.1 69.38 209 864 238 122 329 5.27E-73 205 scaffold14200_Locus_14227_0_59.0_FORK XP_020986158.1 74.65 71 1078 866 51 121 5.27E-73 95.1 scaffold14200_Locus_14227_0_59.0_FORK XP_020986157.1 69.38 209 864 238 151 358 6.07E-73 205 scaffold14200_Locus_14227_0_59.0_FORK XP_020986157.1 74.65 71 1078 866 80 150 6.07E-73 95.1 scaffold14200_Locus_14227_0_59.0_FORK XP_015938931.2 69.38 209 864 238 109 316 9.75E-73 205 scaffold14200_Locus_14227_0_59.0_FORK XP_015938931.2 75 68 1069 866 41 108 9.75E-73 94.4 scaffold14200_Locus_14227_0_59.0_FORK XP_015938930.1 69.38 209 864 238 48 255 7.94E-67 205 scaffold14200_Locus_14227_0_59.0_FORK XP_015938930.1 80.85 47 1006 866 1 47 7.94E-67 74.7 scaffold14200_Locus_14227_0_59.0_FORK PNX86649.1 96.33 109 864 544 38 146 1.92E-65 196 scaffold14200_Locus_14227_0_59.0_FORK PNX86649.1 97.3 37 976 866 1 37 1.92E-65 79 scaffold14200_Locus_14227_0_59.0_FORK AFK37069.1 69.14 175 864 340 121 294 5.99E-64 165 scaffold14200_Locus_14227_0_59.0_FORK AFK37069.1 80.3 66 1063 866 55 120 5.99E-64 105

236

scaffold14200_Locus_14227_0_59.0_FORK XP_020979774.1 70.33 209 864 238 18 225 1.02E-62 206 scaffold14200_Locus_14227_0_59.0_FORK XP_025654685.1 70.33 209 864 238 40 247 1.81E-62 206 scaffold14200_Locus_14227_0_59.0_FORK XP_015938923.1 69.38 209 864 238 18 225 4.15E-62 205 scaffold14200_Locus_14227_0_59.0_FORK PNY02732.1 68.99 158 864 391 102 258 4.15E-60 149 scaffold14200_Locus_14227_0_59.0_FORK PNY02732.1 82.09 67 1066 866 35 101 4.15E-60 107 scaffold14200_Locus_14227_0_59.0_FORK XP_016178266.1 67.03 182 816 274 87 267 2.36E-43 156 scaffold14200_Locus_14227_0_59.0_FORK XP_025647603.1 63.12 160 639 160 53 203 1.36E-41 150 scaffold14200_Locus_14227_0_59.0_FORK XP_020978101.1 71.21 132 555 160 80 211 2.53E-41 149 scaffold14200_Locus_14227_0_59.0_FORK AFK46459.1 76.58 111 1192 881 1 109 3.37E-40 143 scaffold14200_Locus_14227_0_59.0_FORK XP_025665105.1 67.5 160 864 385 152 310 1.01E-37 142 scaffold14200_Locus_14227_0_59.0_FORK XP_020963516.1 72.17 115 504 160 44 158 8.46E-36 133 scaffold14200_Locus_14227_0_59.0_FORK PNX97929.1 78.41 88 300 37 4 91 3.65E-32 121 scaffold14200_Locus_14227_0_59.0_FORK GAU44874.1 73.77 61 1048 866 19 79 3.79E-25 85.9 scaffold14200_Locus_14227_0_59.0_FORK GAU44874.1 64.06 64 864 673 80 142 3.79E-25 54.7 scaffold14200_Locus_14227_0_59.0_FORK XP_020999660.1 67.03 91 1078 812 24 114 8.78E-23 99 scaffold14200_Locus_14227_0_59.0_FORK PNX58403.1 85.51 69 1183 986 5 73 1.50E-21 92 scaffold14200_Locus_14227_0_59.0_FORK XP_020973237.1 66.67 90 1066 812 9 95 2.15E-21 94 C196012_59.0 XP_013460594.1 93.56 264 1217 426 1 264 3.60E-174 491 C196012_59.0 XP_012572024.1 93.94 264 1217 426 1 264 1.76E-173 489 C196012_59.0 NP_001296625.1 93.56 264 1217 426 1 264 1.32E-172 487 C196012_59.0 NP_001296625.1 93.56 264 1217 426 1 264 1.32E-172 487 C196012_59.0 GAU50719.1 92.8 264 1214 426 3 266 6.49E-172 486 C196012_59.0 AAY52461.1 88.64 264 1217 426 1 261 4.87E-155 442 C196012_59.0 XP_020225060.1 87.64 267 1217 426 1 264 4.53E-154 440 C196012_59.0 NP_001236929.2 87.88 264 1217 426 1 261 1.31E-153 439 C196012_59.0 XP_019439483.1 86.57 268 1217 426 1 268 5.56E-152 435 C196012_59.0 AFK45329.1 91.21 239 1217 501 1 239 1.30E-149 428

237

C196012_59.0 AAY85184.1 86.74 264 1217 426 1 259 1.47E-148 426 C196012_59.0 XP_007163386.1 84.91 265 1217 426 1 262 1.69E-148 426 C196012_59.0 XP_003538394.1 86.74 264 1217 426 1 259 2.69E-148 426 C196012_59.0 XP_014494748.1 86.04 265 1217 426 1 262 7.03E-147 422 C196012_59.0 XP_017418984.1 85.28 265 1217 426 1 262 1.15E-146 421 C196012_59.0 XP_025690332.1 87.17 265 1217 426 1 263 3.19E-145 418 C196012_59.0 XP_025638534.1 86.42 265 1217 426 1 263 2.38E-144 416 C196012_59.0 RDX57856.1 85.02 267 1217 432 1 260 2.65E-141 414 C196012_59.0 XP_019459154.1 84.33 268 1217 426 1 263 6.54E-139 402 C196012_59.0 XP_015956745.1 91.32 219 1082 426 1 219 5.47E-135 390 C196012_59.0 KRH30941.1 86.86 236 1217 510 1 231 1.85E-130 379 C196012_59.0 OIW14166.1 86.25 240 1217 510 1 240 9.38E-129 385 C196012_59.0 KOM39219.1 83 247 1217 480 1 244 8.99E-127 376 C196012_59.0 ACJ86279.1 90.82 196 1217 630 1 196 2.78E-118 347 C196012_59.0 AKU47314.1 95.54 157 1052 582 1 157 1.97E-100 300 C196012_59.0 XP_020207160.1 78.37 208 1055 432 3 210 7.92E-94 285 C196012_59.0 XP_004497312.1 79.52 210 1055 426 3 212 4.91E-93 283 C196012_59.0 GAU12147.1 78.37 208 1055 432 3 210 4.16E-92 281 C196012_59.0 AFK38549.1 76.47 221 1085 426 25 245 5.56E-91 279 C196012_59.0 XP_014510876.1 77.51 209 1055 432 3 211 1.17E-90 277 C196012_59.0 XP_017412557.1 77.51 209 1055 432 3 211 1.39E-90 277 C196012_59.0 NP_001236937.2 77.99 209 1055 432 3 211 2.21E-90 276 C196012_59.0 XP_019452567.1 77.4 208 1055 432 3 210 2.40E-89 274 C196012_59.0 XP_013470460.1 78.1 210 1055 426 3 212 3.32E-89 273 C196012_59.0 ACU14052.1 77.51 209 1055 432 3 211 4.75E-89 273 C196012_59.0 XP_019427688.1 78.37 208 1055 432 3 210 7.65E-89 272 C196012_59.0 NP_001242242.2 77.03 209 1055 432 3 211 1.11E-88 272

238

C196012_59.0 PNX76279.1 91.95 149 1217 774 1 149 2.72E-88 269 C196012_59.0 PNX75228.1 91.95 149 1217 774 1 149 3.02E-88 268 C196012_59.0 ACJ84466.1 77.62 210 1055 426 3 212 3.85E-88 271 C196012_59.0 XP_015942302.1 73.81 210 1055 426 3 212 9.06E-88 270 C196012_59.0 XP_025620947.1 73.81 210 1055 426 3 212 1.35E-87 269 C196012_59.0 KHN37319.1 76.56 209 1055 432 3 211 3.33E-87 268 C196012_59.0 XP_016176231.1 73.33 210 1055 426 3 212 4.31E-87 268 C196012_59.0 GAU12146.1 75.96 208 1055 432 3 210 7.18E-87 267 C196012_59.0 XP_007142703.1 77 200 1022 426 14 213 3.94E-86 265 C196012_59.0 ACU20520.1 77.84 194 1055 474 3 196 2.32E-83 258 C196012_59.0 RDX87903.1 70.61 228 1055 432 3 230 1.79E-81 256 C196012_59.0 AFK39744.1 83.23 155 1217 753 1 152 5.11E-75 235 C196012_59.0 XP_020963912.1 86.3 146 1010 573 10 155 6.02E-75 234 C196012_59.0 ACU18720.1 79.04 167 1217 717 1 162 2.73E-72 228 C196012_59.0 OIW02668.1 77.27 154 1217 768 1 149 1.63E-60 197 C196012_59.0 XP_020997264.1 87.61 113 911 573 29 141 2.37E-56 186 C196012_59.0 XP_025692847.1 87.72 114 911 573 2 115 2.79E-56 185 C196012_59.0 XP_020210473.1 73.83 107 1055 735 3 109 9.93E-39 139 C196012_59.0 KYP75478.1 73.83 107 1055 735 47 153 2.04E-38 140 C196012_59.0 PNX55982.1 91.8 61 608 426 1 61 1.51E-29 113 C196012_59.0 KOM37906.1 83.12 77 878 648 241 317 2.80E-28 120 C196012_59.0 PNX66320.1 79.49 78 1001 768 1 78 4.19E-28 110 C196012_59.0 KYP30947.1 80 75 734 510 1 75 3.74E-23 98.6 C207980_54.0 GAU22443.1 92.97 498 1763 285 2 499 0 886 C207980_54.0 PNX76336.1 93.89 491 1748 288 1 491 0 881 C207980_54.0 PNX72448.1 93.88 490 1763 306 2 491 0 878 C207980_54.0 PNX83736.1 94.23 485 1730 288 1 485 0 874

239

C207980_54.0 PNX82250.1 94.02 485 1748 306 6 490 0 873 C207980_54.0 PNX72702.1 94.23 485 1730 288 1 485 0 873 C207980_54.0 XP_004503106.1 92.48 492 1748 288 1 492 0 858 C207980_54.0 XP_003600668.2 92.26 491 1748 282 1 486 0 838 C207980_54.0 XP_020228225.1 90.52 496 1766 288 4 496 0 826 C207980_54.0 XP_020228226.1 91.02 490 1748 288 1 490 0 826 C207980_54.0 XP_019460900.1 91.63 490 1748 288 1 490 0 826 C207980_54.0 XP_014515012.1 90.89 494 1766 288 4 494 0 824 C207980_54.0 XP_007135475.1 90.89 494 1766 288 4 494 0 823 C207980_54.0 XP_017407369.1 90.69 494 1766 288 4 494 0 822 C207980_54.0 XP_003538383.1 89.9 495 1766 288 4 488 0 810 C207980_54.0 KHN08788.1 89.29 495 1766 288 4 488 0 801 C207980_54.0 KRG97977.1 90.64 481 1766 330 4 481 0 800 C207980_54.0 XP_016190353.1 88.93 488 1739 288 2 489 0 786 C207980_54.0 XP_025690368.1 88.52 488 1739 288 2 489 0 779 C207980_54.0 XP_015956718.1 88.52 488 1739 288 2 489 0 777 C207980_54.0 XP_025638604.1 87.3 488 1739 288 2 483 0 764 C207980_54.0 RDX95125.1 92.24 451 1631 288 90 540 0 758 C207980_54.0 KRH03172.1 87.77 466 1748 357 1 456 0 742 C207980_54.0 XP_019460901.1 83.61 488 1748 288 1 445 0 738 C207980_54.0 XP_025649899.1 81.29 481 1700 282 34 500 0 654 C207980_54.0 XP_016196790.1 81.29 481 1700 282 3 469 0 654 C207980_54.0 XP_015962337.1 80.67 481 1700 282 3 469 0 650 C207980_54.0 XP_025696630.1 80.67 481 1700 282 3 469 0 649 C207980_54.0 RHN63526.1 79.29 478 1763 345 11 442 0 641 C207980_54.0 AES91174.2 77.41 478 1763 345 2 423 0 619 C207980_54.0 AET00425.1 73.59 409 1712 486 14 421 8.86E-162 476

240

C207980_54.0 PNY12294.1 66.92 399 1475 285 1 357 2.67E-130 391 C207980_54.0 GAU12385.1 68.98 374 1475 360 1 347 2.13E-128 388 C207980_54.0 RHN68089.1 95.5 200 1748 1149 1 200 1.38E-121 364 C207980_54.0 KHN18380.1 91.67 204 1766 1155 4 204 5.83E-112 339 C207980_54.0 KYP57101.1 66.67 297 1178 288 89 308 6.37E-106 327 C207980_54.0 KYP57101.1 59.83 117 1766 1416 4 80 1.88E-29 123 C207980_54.0 XP_024639438.1 71.2 250 1235 486 2 251 7.45E-94 295 C207980_54.0 XP_004491357.1 58 300 1259 375 10 260 1.07E-65 220 C207980_54.0 AFK43459.1 84.96 133 674 282 1 128 2.45E-58 196 C207980_54.0 XP_003617332.2 50.59 340 1256 285 23 329 1.49E-53 190 C207980_54.0 XP_004496834.1 83.2 125 674 300 1 122 1.85E-50 175 C207980_54.0 AET00252.2 52.1 286 1133 285 16 274 4.70E-50 179 C207980_54.0 XP_003617266.1 49.12 340 1277 345 5 316 1.46E-49 179 C207980_54.0 AFK42483.1 49.12 340 1277 345 5 316 1.59E-49 179 C207980_54.0 XP_003617402.1 48.38 339 1277 345 5 315 3.76E-48 176 C207980_54.0 XP_003617522.2 50.31 320 1256 366 15 306 4.54E-48 176 C207980_54.0 XP_003610784.1 48.41 347 1277 342 5 308 4.45E-46 170 C207980_54.0 XP_003610929.1 50.3 328 1277 369 5 289 1.26E-45 168 C207980_54.0 XP_003611210.2 47.16 352 1277 348 5 324 1.37E-44 166 C207980_54.0 AES94184.2 46.88 352 1277 348 5 324 2.34E-44 166 C207980_54.0 XP_003611226.3 46.88 352 1277 348 8 327 2.54E-44 166 C207980_54.0 AET00481.1 49.58 238 1019 366 8 225 8.07E-39 147 C207980_54.0 AET00390.1 46.25 320 1256 366 15 288 1.66E-38 149 C207980_54.0 GAU19655.1 72.36 123 818 468 48 170 1.37E-37 142 C207980_54.0 PNY12293.1 58.64 162 839 360 2 136 5.57E-34 131 C207980_54.0 KHN48121.1 89.33 75 905 681 6 80 1.59E-33 127 C207980_54.0 PNX54805.1 54.59 185 1361 822 1 179 1.60E-24 105

241

C207980_54.0 BAT98326.1 70.65 92 674 402 15 104 6.10E-22 95.1 C207980_54.0 KOM25825.1 83.33 54 1745 1584 8 61 2.57E-21 95.1 scaffold25376_Locus_46472_0_39.2_LINEAR GAU33703.1 92.88 365 2633 1542 45 409 0 536 scaffold25376_Locus_46472_0_39.2_LINEAR XP_003601245.1 95.89 341 2564 1542 54 394 1.09E-180 533 scaffold25376_Locus_46472_0_39.2_LINEAR XP_004501915.1 92.76 359 2621 1545 74 432 1.84E-174 518 scaffold25376_Locus_46472_0_39.2_LINEAR XP_020231197.1 93.13 364 2633 1542 31 394 3.37E-171 508 scaffold25376_Locus_46472_0_39.2_LINEAR AFK40049.1 93.02 344 2564 1533 59 402 1.26E-170 507 scaffold25376_Locus_46472_0_39.2_LINEAR XP_004501916.1 92.9 352 2621 1566 74 425 2.06E-170 508 scaffold25376_Locus_46472_0_39.2_LINEAR XP_020231196.1 93.77 337 2552 1542 65 401 8.35E-170 505 scaffold25376_Locus_46472_0_39.2_LINEAR XP_016179541.1 92.63 339 2564 1548 68 406 1.15E-167 499 scaffold25376_Locus_46472_0_39.2_LINEAR XP_015937793.1 92.04 339 2564 1548 68 406 4.98E-167 498 scaffold25376_Locus_46472_0_39.2_LINEAR XP_025616137.1 92.04 339 2564 1548 68 406 1.34E-166 497 scaffold25376_Locus_46472_0_39.2_LINEAR RDX70073.1 88.98 354 2564 1542 96 449 7.45E-165 494 scaffold25376_Locus_46472_0_39.2_LINEAR XP_017420960.1 91.67 360 2621 1542 40 399 4.17E-164 490 scaffold25376_Locus_46472_0_39.2_LINEAR XP_014500001.1 91.11 360 2621 1542 35 394 5.92E-163 487 scaffold25376_Locus_46472_0_39.2_LINEAR XP_007138060.1 90.83 360 2621 1542 40 399 9.64E-163 487 scaffold25376_Locus_46472_0_39.2_LINEAR XP_003523084.1 93.2 338 2564 1551 57 394 1.17E-161 484 scaffold25376_Locus_46472_0_39.2_LINEAR XP_014630278.1 92.92 339 2564 1551 57 395 6.34E-160 479 scaffold25376_Locus_46472_0_39.2_LINEAR XP_019417465.1 91.81 342 2564 1548 59 400 2.61E-153 462 scaffold25376_Locus_46472_0_39.2_LINEAR XP_019417464.1 92.84 335 2552 1548 67 401 3.01E-153 462 scaffold25376_Locus_46472_0_39.2_LINEAR KEH35038.1 95.74 282 2564 1719 54 335 1.48E-149 451 scaffold25376_Locus_46472_0_39.2_LINEAR OIV96958.1 91.96 336 2564 1566 59 394 2.24E-148 453 scaffold25376_Locus_46472_0_39.2_LINEAR OIV96958.1 92.31 91 1820 1548 396 486 1.56E-30 130 scaffold25376_Locus_46472_0_39.2_LINEAR XP_020985665.1 86.14 339 2564 1548 68 386 1.90E-146 444 scaffold25376_Locus_46472_0_39.2_LINEAR KRH63558.1 93.53 278 2384 1551 1 278 1.35E-130 399 scaffold25376_Locus_46472_0_39.2_LINEAR PNX77279.1 93.27 223 2633 1968 39 261 1.71E-96 310 scaffold25376_Locus_46472_0_39.2_LINEAR PNX83994.1 93.01 143 1970 1542 1 143 1.08E-67 226

242

scaffold25376_Locus_46472_0_39.2_LINEAR KYP51564.1 94.7 132 1961 1566 1 132 1.56E-61 210 scaffold25376_Locus_46472_0_39.2_LINEAR PNX67409.1 99.1 111 2390 2058 1 111 5.00E-41 150 scaffold25376_Locus_46472_0_39.2_LINEAR RHN68767.1 84.52 84 1720 1971 16 99 9.21E-35 133 scaffold25376_Locus_46472_0_39.2_LINEAR XP_019416428.1 85.58 104 1841 1548 22 125 2.84E-33 128 scaffold25376_Locus_46472_0_39.2_LINEAR KYP78379.1 55.07 207 2162 1557 2 208 1.44E-24 107 C205172_60.0 GAU51254.1 97.94 389 335 1495 1 389 0 763 C205172_60.0 XP_003600853.1 98.19 387 335 1495 1 387 0 760 C205172_60.0 XP_004503228.1 97.42 387 335 1495 1 387 0 720 C205172_60.0 XP_020211284.1 94.57 387 335 1495 1 387 0 716 C205172_60.0 XP_017415904.1 94.57 387 335 1495 1 387 0 716 C205172_60.0 XP_014496325.1 94.83 387 335 1495 1 387 0 716 C205172_60.0 XP_007163512.1 94.32 387 335 1495 1 387 0 716 C205172_60.0 XP_003552524.1 94.83 387 335 1495 1 387 0 712 C205172_60.0 ACU23254.1 94.83 387 335 1495 1 387 0 710 C205172_60.0 XP_003538446.1 94.57 387 335 1495 1 387 0 708 C205172_60.0 RDX94736.1 94.83 387 335 1495 1 387 0 707 C205172_60.0 XP_019439410.1 93.56 388 335 1495 1 388 0 702 C205172_60.0 PNY11097.1 98.02 354 434 1495 1 354 0 701 C205172_60.0 XP_016166720.1 92.54 389 335 1495 1 389 0 682 C205172_60.0 XP_015931861.1 92.29 389 335 1495 1 389 0 682 C205172_60.0 XP_025609645.1 90.93 397 335 1495 1 397 0 677 C205172_60.0 ACJ84871.1 84.75 387 335 1495 1 336 0 624 C205172_60.0 ACU18796.1 94.97 179 959 1495 1 179 6.44E-113 337 C205172_60.0 AFK49549.1 92.35 170 335 841 1 170 2.95E-96 294 C205172_60.0 KYP59805.1 62.56 219 803 1459 1 167 2.74E-65 214 C205172_60.0 KYP35032.1 70.69 174 962 1459 98 261 8.73E-57 196 C205172_60.0 KYP35027.1 65.27 167 959 1459 31 169 1.16E-47 168

243

C205172_60.0 KRH31099.1 45.42 262 656 1438 7 178 2.13E-38 143 C205172_60.0 XP_004485532.1 49.65 288 680 1471 67 341 1.35E-23 106 C205172_60.0 ADK60786.1 92 50 755 904 1 50 3.58E-23 96.7 C205172_60.0 XP_003531565.1 51.43 280 680 1462 67 338 1.14E-22 103 C205172_60.0 BAT86832.1 49.82 285 680 1471 67 341 8.51E-22 101 C205172_60.0 XP_019418322.1 48.96 288 680 1471 67 341 1.15E-21 100 C205172_60.0 XP_014518096.1 50.18 285 680 1462 67 338 2.08E-21 100 C205172_60.0 XP_003593121.1 49.65 282 680 1471 67 341 2.37E-21 99.8 C205172_60.0 XP_020220831.1 49.14 291 680 1456 67 336 5.38E-21 98.6 C205172_60.0 XP_020238099.1 48.3 265 680 1426 67 327 6.20E-21 98.6 C205172_60.0 KYP43947.1 48.33 269 671 1426 69 333 7.41E-21 98.6 C199840_59.0 XP_004516451.1 91.69 349 1298 276 1 347 0 558 C199840_59.0 XP_004516450.1 91.43 350 1298 276 1 348 0 557 C199840_59.0 GAU44355.1 91.35 347 1298 276 1 345 0 542 C199840_59.0 XP_013445560.1 92.94 326 1250 276 60 384 0 534 C199840_59.0 AAO16018.1 89.05 347 1298 276 1 345 0 520 C199840_59.0 XP_022634489.1 87.79 344 1298 276 1 344 0 517 C199840_59.0 XP_017431339.1 87.5 344 1298 276 1 344 0 514 C199840_59.0 AAO16020.1 90.8 326 1250 276 15 338 0 513 C199840_59.0 XP_014519571.1 87.5 344 1298 276 1 343 1.07E-180 513 C199840_59.0 XP_017431340.1 87.21 344 1298 276 1 343 9.92E-180 510 C199840_59.0 XP_020214909.1 87.79 344 1298 276 1 344 1.56E-179 510 C199840_59.0 KHN46954.1 88.25 349 1307 276 214 560 1.81E-179 518 C199840_59.0 XP_003527280.1 88.15 346 1298 276 1 344 3.35E-179 509 C199840_59.0 ACU23864.1 88.76 338 1274 276 4 340 2.07E-178 507 C199840_59.0 NP_001240262.1 86.67 345 1298 276 1 344 4.61E-178 506 C199840_59.0 KHN16011.1 86.67 345 1298 276 16 359 7.75E-178 506

244

C199840_59.0 XP_007133142.1 87.39 341 1298 285 1 341 1.16E-176 503 C199840_59.0 XP_019413479.1 86.67 345 1298 276 1 344 1.56E-175 500 C199840_59.0 OIV99148.1 87.54 337 1274 276 5 340 7.58E-175 498 C199840_59.0 XP_019413478.1 84.94 352 1298 276 1 351 4.57E-173 494 C199840_59.0 XP_025690595.1 85.92 348 1298 276 1 345 4.80E-172 491 C199840_59.0 XP_016190381.1 85.63 348 1298 276 1 345 5.84E-172 491 C199840_59.0 PNX94362.1 98.11 265 1067 276 1 265 1.61E-164 469 C199840_59.0 XP_015956820.2 95.27 275 1100 276 8 282 2.55E-161 461 C199840_59.0 RDY10688.1 84.08 333 1298 345 1 331 1.13E-155 451 C199840_59.0 KRH26036.1 85.62 306 1298 393 1 305 2.18E-149 432 C199840_59.0 XP_006591900.1 84.39 314 1298 369 1 313 3.93E-149 432 C199840_59.0 KOM49494.1 84.67 274 1298 486 1 274 4.88E-131 384 C199840_59.0 XP_025980325.1 85.87 276 1298 486 1 274 2.40E-128 377 C199840_59.0 PNX79793.1 84.55 123 1211 852 1 123 8.38E-58 191 C199840_59.0 PNX54748.1 95.18 83 1067 819 1 83 4.35E-44 154

245

Minerva Access is the Institutional Repository of The University of Melbourne

Author/s: Dadu, Rama Harinath Reddy

Title: Identification and characterisation of potential sources of resistance to Ascochyta blight within the exotic germplasm of lentil

Date: 2018

Persistent Link: http://hdl.handle.net/11343/221831

File Description: PhD thesis - Identification and characterisation of potential sources of resistance to Ascochyta blight within the exotic germplasm of lentil

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