Genetic and Molecular Analyses of Barley for Seedling and Adult Plant Resistance Against Rust Diseases

Genetic and Molecular Analyses of Barley for Seedling and Adult Plant Resistance against Rust Diseases

Karanjeet Singh Sandhu

B.Sc. Agri. (Hons.), M.Sc. Agri. (Entomology)

A thesis submitted in fulfilment of the requirements for the degree of DOCTOR OF PHILOSOPHY

Plant Breeding Institute Cobbitty October 2011

Certificate of authorship

This thesis contains no material which has been accepted for the award of any other degree or diploma in any university, unless stated. To the best of my knowledge the text of this thesis is original and contains no material previously published by any other person, except where due references are made in the text.

Karanjeet Singh Sandhu

Acknowledgement

Above all, I am sincerely thankful to my supervisor Professor Robert F. Park, the Director of Cereal Rust Research, Plant Breeding Institute, the University of Sydney, Cobbitty, for his inspiring mentorship and constructive criticism throughout my research work and in writing this dissertation.

I am highly grateful to my associate supervisor Dr. Davinder Singh for his valuable suggestions during my research and in writing this manuscript. Mapping of Rph21 could have not been completed without the support of Dr. Urmil K. Bansal and I am profoundly thankful to her. I am extremely thankful to Dr. Haydar Karaoglu for his guidance in the molecular studies of Puccinia hordei Otth.

I am also thankful to Mathew Williams, Paul Kavanagh, Keshab Kendel, Kanwaljit Kaur, Hanif Miah, Mohamad B. Gill, Raul Rodriguez, Gary Standen, Sami Hoxha, James Hull, Kate Vincent, Catherine Cupitt and Beate Wildner for technical assistance and James Bell, Kate Rudd, Pradip Sen and Pradhan Dayaram for administration support at PBI. The financial support provided by the Grains Research and Development Corporation (GRDC) through cereal rust research scholarship is gratefully acknowledged. The provision of barley germplasm for testing by the University of Western Australia and International Centre for Agricultural Research in the Dry Areas (ICARDA) is highly acknowledged.

My special thanks go to Emeritus Professor R. A. McIntosh, who never curtailed answering my questions related to cereal rust diseases. I extend my gratitude to Associate Professor Harbans S. Bariana for encouraging me to start my doctoral studies. I owe my profound respect to my mother Mrs. Manjit Kaur Sandhu and father Mr. Gurdev Singh Sandhu, who have been instrumental in building up my educational carrier and taught me never to be afraid of doing new and progressive things in life.

I would also like to thank Associate Professor Colin R. Wellings for his guidance in stripe rust studies. I would like to acknowledge the field staff for helping in my field experiments. Further I am thankful to my charming fellow students Angela Dennett, Arvinder Toor, Jordan

Bailey, Lislé Snyman, Lida Derevnina, Philip Davies and Kapfuchira Tawanda for their friendship and support during the studies.

I am highly indebted to my daughters Puneet, Kirtveer and son Master Himmat Sandhu, who suffered a lot due to my late home comings from laboratory work and busy weekends. I must conclude with special thanks indeed to my beloved wife for her patience and unwavering support for the last four years.

"I am but one member of a vast team made up of many organizations, officials, thousands of scientists and millions of farmers - mostly small and humble - who for many years have been fighting a quiet, oftentimes losing war on the food production front."

Norman Borlaug Nobel laureate

Dedicated to

All the hardworking and marginal farmers of the world

Summary

Genetic studies were carried out to determine the inheritance of unknown seedling resistance (USR) to leaf rust (caused by Puccinia hordei Otth.) in the barley cultivar Ricardo. In the greenhouse Ricardo/Gus F3 (187 lines) and BC1F2 (130 lines) populations based studies using an array of P. hordei pathotypes (pts), revealed that the USR in Ricardo was conferred by a single dominant gene, which was tentatively named RphRic. Bulk segregant analysis (BSA) of the F3 population using a multiplex-ready PCR technique mapped RphRic on chromosome 4H flanked by markers GBM1220 and GBM1003 at distances of 17.4 cM and 20.4 cM, respectively. Being the first gene for leaf rust resistance mapped on chromosome 4H, RphRic was catalogued as Rph21. Phenotyping of Ricardo/Peruvian (Rph2) F3 populations and genotyping of both parents using the Rph2-linked marker ITS1 confirmed the presence of

Rph2 in Ricardo. The Ricardo/Gus F3 and BC1F2 populations segregated for the presence of an additional gene when tested under field conditions using the same pathotype (pt), 5457P+ (used in greenhouse). This uncharacterised adult plant resistance (APR) against P. hordei, found in Ricardo, appeared to be distinct from Rph20 when genotyped using a closely linked marker bPb-0837.

Responses of 113 advanced breeding lines and cultivars of barley (Hordeum vulgare L. subsp. vulgare), along with the susceptible control genotype Gus, were assessed against P. hordei pts in the greenhouse at seedling and field at adult plant growth stages. The tests revealed the presence of APR in 68 lines, USR in 23 lines and the seedling resistance gene Rph3 in three lines. Marker bPb-0837 was present in 35 of the 68 lines carrying APR, which suggested that these 35 lines carry APR gene Rph20. The remaining 33 lines, which lacked the Rph20 linked marker, likely carry new sources of APR. Pedigree analysis of the 68 lines found to carry APR revealed that 32 were related to cultivar (cv.) Gull and to H. laevigatum, two were related to cv. Bavaria and one related to cvv. Manchuria and Taganrog. Ancestral pedigree analysis also revealed the common presence of cv. Diamant (X-ray mutant) in the parentage of lines likely carrying Rph20. The remaining 19 lines lacked detectable seedling resistance and were susceptible in the field at adult plant growth stages.

Summary

Four international barley nurseries comprising 820 lines with 579 unique pedigrees were sourced from the International Centre for Agricultural Research in the Dry Areas (ICARDA) and analysed for resistance against isolates of P. hordei, P. graminis f. sp. tritici (Pgt) and barley grass stripe rust (BGYR). Overall analyses of the responses of 783 lines (excluding 37 missing lines) to P. hordei showed that 728 (93%) carried the major seedling resistance gene Rph3, five (0.65%) carried USR, six (0.75%) carried uncharacterised APR and 44 (5.6%) lines were susceptible at all growth stages. Of the six lines identified with uncharacterised APR, three likely carried Rph20 based on the presence of the Rph20-linked marker bPb- 0837. Based on tests with several control genotypes, marker bPb-0837 was found to be more reliable than Ebmag0833 in detecting the presence of Rph20. All lines were resistant to Pgt pt 98-1,2,3,5,6 when tested as seedlings in the greenhouse. Out of the 783 lines tested, 164 produced immune responses, 284 produced resistant (1= to 3) responses and 335 produced mesothetic (X type) responses against pt 98-1,2,3,5,6. All but two 783 lines were highly resistant to BGYR in greenhouse tests, showing immune responses.

The usefulness of 148 simple sequence repeats (SSRs) in revealing variability among Australian isolates of P. hordei were assessed. The markers comprised 71 developed for Pgt, 40 developed for P. triticina (Pt) and 37 developed for P. coronata f. sp. avenae (Pca). SSRs were tested across 22 pts of P. hordei from Australasia including one isolate of each of the control pathogens [Pt, Pgt, P. striformis f. sp. tritici (Pst), BGYR and P. graminis f. sp. avenae (Pga)]. Genotyping of P. hordei was also conducted with the PCR-fingerprinting primers M13 and (GACA)4. The SSRs developed from Pgt and Pt showed 100% cross amplification in P. hordei, while only nine Pca SSRs showed amplification in P. hordei. Of the 148 markers tested, only two Pgt SSRs (F4-15 and F7-22) were polymorphic. Both PCR- fingerprinting primers revealed polymorphisms among the isolates, with (GACA)4 generating the most informative fragments. Both SSRs and PCR fingerprinting markers out grouped the control pathogens Pt, Pgt, Pst, BGYR and Pga from the P. hordei pts. Polymorphic information content (PIC) values of SSR markers F4-15 and F7-22 were calculated as 0.50 and 0.55 respectively. Molecular analyses revealed evidence of clonal lineages among the P. hordei pts, supporting the hypothesis that some of the pts arose from mutational changes in the virulence of a founder pt. Sexual recombination within P. hordei in Australia on the alternate host Ornithogalum umbellatum may have resulted in some new pts with different

Summary

virulence against Rph genes. This is the first study of Australasian pts of P. hordei using PCR-fingerprinting technique and SSR genotyping.

III

Table of contents

Summary...... I List of Tables...... IX List of Figures...... XV List of Appendices...... XIX List of Abbreviations...... XXI Chapter I Introduction...... 1 Chapter II Review of literature...... 4 Origin of barley...... 4 Distribution of barley...... 5 Importance and usage of barley...... 8 Nutrition in barley...... 9 Geographical distribution of barley production…………………………………...... 10 Barley taxonomy...... 11 The barley genome...... 13 Rust diseases of barley...... 14 Barley leaf rust...... 15 Taxonomy of P. hordei...... 15 Host range of P. hordei...... 16 Life cycle of P. hordei...... 17 Pathogenic variation in P. hordei...... 19 Identifying pathotypes of P. hordei in Australia……………………………..21 Pathotype nomenclature in Australia...... 24 Pathotypes of P. hordei in Australia...... 25 Stem rust caused by P. graminis...... 26 Economic importance...... 26 Stripe rust...... 27 Genetic markers...... 27 Morphological markers...... 27 Biochemical markers...... 27 DNA markers...... 28 IV

Table of contents

Use of molecular markers in studying the genetic diversity of rust pathogens………29 Sources of rust resistance in barley...... 30 Genetic analyses for mapping the genes of disease resistance...... 32 Steps involved in the genetic analyses and mapping of genes for disease resistance...... 34 Development of genetic/mapping populations…...... 34 Phenotyping of populations...... 34 Genotyping of populations...... 35 Mapping of gene/s...... 35 Chapter III Genetic analyses and molecular mapping of an uncharacterised seedling gene conferring resistance to Puccinia hordei in the barley cultivar Ricardo...... 37 Abstract...... 37 Introduction...... 37 Materials and methods...... 40 Plant material...... 40 Pathogen material...... 40 Genetic populations...... 40 Greenhouse testing...... 42 Determining conditions for the optimal expression of seedling resistance in Ricardo...... 42 Inheritance of seedling resistance in Ricardo...... 43 Field tests...... 43 Evaluation of adult plant resistance in Ricardo...... 43 Molecular analyses and mapping of seedling resistance in Ricardo...... 44 DNA extraction...... 44 Bulk segregant analysis (BSA)...... 45 Linkage analyses and construction of consensus map...... 45 Assaying the presence of Rph2 in Ricardo with marker ITS1...... 46 PCR reaction and profiles...... 46 Restriction of PCR product...... 46 Marker genotyping of Rph20...... 46 PCR reaction and profiles...... 47

Table of contents

Separation of amplified PCR products...... 47 Chi squared analyses...... 48 Results...... 48 Expression of seedling resistance in Ricardo...... 48 Multipathotype testing for seedling resistance...... 50 Inheritance of seedling resistance...... 53 Detection of Rph2 in Ricardo...... 55 Genotyping of Ricardo with Rph2 linked marker ITS1...... 55 Molecular mapping of RphRic...... 57 Inheritance of adult plant resistance to leaf rust in Ricardo...... 59 Genotyping with marker bPb-0837 linked to APR gene Rph20...... 63 Discussion...... 64 Environment sensitivity of RphRic...... 64 Inheritance of seedling resistance...... 65 Presence of Rph2 in Ricardo...... 65 Inheritance of adult plant resistance in Ricardo...... 66 Genotyping of Ricardo with bPb-0837 marker linked to Rph20...... 68 Chapter IV Characterising resistance to Puccinia hordei in selected barley germplasm….. 69 Abstract...... 69 Introduction...... 69 Materials and methods...... 71 Plant material...... 71 Pathogen material...... 75 Greenhouse screening...... 75 Field screening...... 76 Extraction of genomic DNA...... 77 Genotyping with Rph20 linked marker bPb-0837...... 78 PCR reaction and profile...... 78 Results...... 79 Seedling resistance...... 79

Table of contents

Cultivars/lines lacking detectable seedling resistance...... 79 Cultivars/lines characterised with known seedling resistance……….79 Cultivars/lines with unknown seedling resistance...... 80 Resistance under field conditions...... 89 Field response of lines carrying uncharacterised seedling resistance...... 89 Field response of lines lacking seedling resistance effective in the field...... 89 Discussion...... 101 Chapter V Characterisation of rust resistance in four international barley nurseries...... 105 Abstract...... 105 Introduction...... 105 Materials and methods...... 107 Plant material...... 107 Pathogen material...... 107 Greenhouse screening...... 108 Field screening...... 109 Extraction of genomic DNA...... 110 Molecular markers...... 110 PCR reaction and profiles...... 111 Results...... 112 Leaf rust evaluation...... 112 Multipathotype testing...... 113 Stem rust evaluation...... 118 BGYR evaluation...... 119 Molecular marker analysis...... 120 Discussion...... 122 Chapter VI Short simple sequence repeats and PCR-fingerprinting based analyses of genetic diversity in Puccinia hordei Otth. in Australasia...... 127 Abstract...... 127 Introduction...... 127 Materials and methods...... 131

VII

Table of contents

Rust pathotypes...... 131 Extraction of genomic DNA from urediniospores...... 131 Molecular markers...... 135 PCR amplifications and profiles...... 135 SSR Primers...... ………………135 PCR-fingerprinting primers...... 136 Data analyses...... 136 Results...... 137 Discussion...... 147 Chapter VII General discussion...... 151 References...... 156 Appendices...... 176

VIII

List of tables

Table 2.1 Geographical distribution of diploid (2n = 14) barley...... 6

Table 2.2 Chemical compositions (percentage of grain dry matter) of barley grain compared with other important cereals...... 10

Table 2.3 Taxonomic classification of cultivated barley...... 12

Table 2.4 Taxonomic classification of Puccinia hordei...... 16

Table 2.5 Differential set used in Australia to identify pathotypes of Puccinia hordei...... 22

Table 2.6 System used in Australia to name pathotypes of Puccinia hordei, as exemplified by four recently isolated pathotypes...... 24

Table 2.7 Catalogued genes for resistance to Puccinia hordei...... 32

Table 3.1 Details of Puccinia hordei pathotypes used in the present study...... 41

Table 3.2 Details of molecular markers used to genotype Ricardo and control cultivars (Rph2 and Rph20)...... 47

Table 3.3 Infection types produced by Ricardo, Gus and Peruvian with different pathotypes of Puccinia hordei at three post-inoculation temperatures in the greenhouse under natural lighting...... 49

Table 3.4 Responses of Ricardo, Peruvian, Gus and differential lines in greenhouse tests with 10 pathotypes of Puccinia hordei...... 51

List of tables

Table 3.5 Observed number of segregating and homozygous susceptible lines in a BC1F2 population derived from the cross Ricardo/Gus//Gus, when inoculated with the Rph2-virulent Puccinia hordei pathotype 5457P+ at seedling stage in the greenhouse...... 54

Table 3.6 Observed number of homozygous resistant, segregating and homozygous susceptible lines in an F3 population derived from the cross Ricardo/Gus, when inoculated with the Rph2-virulent Puccinia hordei pathotype 5457P+ at seedling stage in the greenhouse...... 54

Table 3.7 Observed number of homozygous resistant, segregating and homozygous susceptible lines in F3 populations derived from the cross Ricardo/Peruvian (Rph2), when inoculated with the Rph2-avirulent Puccinia hordei pathotype 200P- at seedling stage...... 56

Table 3.8 Observed segregation among 15 randomly selected F3 lines derived from the cross Ricardo/Peruvian when inoculated with the Rph2-virulent Puccinia hordei pathotype 5457P+ at seedling stage...... 56

Table 3.9 Details of polymorphic SSRs published for chromosome 4H of barley used to genotype 187 F3 lines derived from the cross Ricardo/Gus...... 58

Table 3.10 Observed number of homozygous resistant, moderately susceptible, segregating and homozygous susceptible lines in an F3 population derived from the cross Ricardo/Gus when inoculated with Puccina hordei pathotype 5457P+ under field conditions...... 61

Table 3.11 Number of moderately susceptible, segregating and homozygous susceptible lines observed in an F3 population derived from the cross Ricardo/Gus that were scored as homozygous susceptible in seedling greenhouse tests when inoculated with Puccina hordei pathotype 5457P+ under field conditions...... 61

List of tables

Table 3.12 Observed number of segregating and homozygous susceptible lines in a BC1F2 population derived from the cross Ricardo/Gus//Gus, when inoculated with Puccina hordei pathotype 5457P+ under field conditions...... 62

Table 3.13 Observed number of segregating and homozygous susceptible lines in a BC1F2 population derived from the cross Ricardo/Gus//Gus that were scored as homozygous susceptible in greenhouse seedling tests when inoculated with Puccinia hordei pathotype 5457P+ under field conditions...... 62

Table 3.14 Assays of the marker bPb-0837, linked to the adult plant resistance gene Rph20, on Ricardo, Gus and three control genotypes...... 63

Table 4.1 Details of barley cultivars and lines assessed for response to Puccinia hordei at seedling (greenhouse) and adult plant (field) growth stages...... 71

Table 4.2 Pathogenicity details of Puccinia hordei pathotypes used in the studies to characterize resistance in barley germplasm...... 75

Table 4.3 Details of marker bPb-0837, linked to adult plant resistance gene Rph20 in barley...... 78

Table 4.4 Greenhouse infection types produced by differential/control genotypes against six Puccinia hordei pathotypes...... 81

Table 4.5 Responses of 87 barley genotypes susceptible to six Puccinia hordei pathotypes at seedling growth stages in the greenhouse tests...... 83

Table 4.6 Greenhouse multipathotype testing confirmed the presence of seedling resistance gene Rph3 in three barley genotypes...... 87

List of tables

Table 4.7 Responses of 23 barley genotypes postulated to carry unidentified seedling resistance to six pathotypes of Puccina hordei...... 87

Table 4.8 Field responses of cultivars/lines with uncharacterised seedling resistance effective against six pathotypes of Puccinia hordei...... 90

Table 4.9 Field responses of barley genotypes with seedling resistance ineffective against Puccinia hordei pathotype 5457P+...... 91

Table 4.10 Field responses of 19 barley genotypes that were susceptible to Puccinia hordei at both seedling and adult plant growth stages...... 94

Table 4.14 Pedigree analyses of barley cultivars/lines predicted to carry uncharacterised adult plant resistance against P. hordei...... 100

Table 5.1 Details of the four International Barley Observation Nurseries introduced from ICARDA...... 107

XII

List of tables

Table 5.2 Detail of rust isolates used for greenhouse and field tests of four international barley nurseries...... 108

Table 5.3 Detail of molecular markers used to genotype barley lines carrying adult plant resistance to leaf rust...... 111

Table 5.4 Classification of ICARDA germplasm with respect to resistance based on field and greenhouse tests with Puccinia hordei pathotypes 5652P+ and 5457P+...... 112

Table 5.5 Seedling responses of selected lines and control differential genotypes against six Puccinia hordei pathotypes...... 116

Table 5.6 Summary of greenhouse results of four international nurseries tested with Puccinia graminis f. sp. tritici pathotype 98-1,2,3,5,6...... 119

Table 5.7 Summary of infection types produced by four international nurseries when tested with Puccinia graminis f. sp. tritici pathotype 98-1,2,3,5,6...... 119

Table 5.8 Response of ICARDA germplasm against a standard isolate of the Barley Grass Stripe Rust (BGYR) pathogen...... 119

Table 5.9 Validation of markers bPb-0837 and Ebmag0833 on ICARDA barley lines carrying adult plant resistance to Puccinia hordei, plus controls...... 121

Table 6.2 SSRs and PCR-fingerprinting primers tested for utility in Puccinia hordei isolates from Australasia...... 135

XIII

List of tables

Table 6.3 Details of amplifications and polymorphisms generated with the 150 primers used to genotype selected Puccinia hordei pathotypes and control rust isolates...... 138

Table 6.4 Polymorphic SSRs and PCR-fingerprinting markers, including repeat motifs, primer sequences, PCR size, annealing temperatures, allele frequencies and polymorphism information content (PIC) values...... 139

Table 6.5 Cluster groupings of Puccinia hordei isolates based on the cluster analyses of SSR markers F4-15 and F7-22 and PCR-fingerprinting primers M13 and (GACA)4…………...145

Table 6.6 Groups of Puccinia hordei pathotypes based on the cluster analyses of PCR- fingerprinting primer (GACA)4 , showing their virulence against Rph genes………….…..146

XIV

List of figures

Fig. 2.1 Distribution Barley and other major crops, Source: (Leff et al. 2004)...... 9

Fig. 2.2 Complete life cycle of Puccinia hordei, the leaf rust pathogen of barley involving five different stages of spores (aeciospores, urediniospores, teliospores, basidiospores and pycniospores)...... 19

Fig. 3.1 Greenhouse infection types of Gus and Ricardo (in three sets) at three different post- inoculation temperatures against Puccinia hordei pt 5457P+...... 50

Fig. 3.2 DNA fragments generated by primers ITS5 and ITS2 (ITS1). Lanes 1 to 6: Gus (unrestricted, 350 bp), Gus (restricted), Ricardo (unrestricted, 300 bp), Ricardo (restricted), Peruvian (unrestricted, 300 bp) and Peruvian (restricted)...... 57

Fig. 3.3 Genetic map for RphRic and polymorphic microsatellite markers on chromosome 4H of barley, constructed using 187 F3 lines. Kosambi map distances (cM) shown on the left side...... 59

Fig. 3.4 Field responses of F3 lines derived from the cross Ricardo/Gus. L to R: R, MS and S, leading to leaf death as showed in last two leaves (Categories are described in Appendix 2)...... 62

Fig. 4.1 Barley genotypes likely to carry Rph20 produced a 245 bp band when genotyped with marker bPb-0837, while 33 lines with APR produced no band. One gel with 29 lines of the 68 with APR is presented only (numbers 1 to 29 represent cultivars/lines; ICB83-0157- 10AP-0TR-0AP-7AP-1APH-0AP, Glacier/Titan, Esperance, Belfor, Maris Mink, Hassan, Corvette, 74043, Galleon, WI-2553, 115-9505-B, SE627.02, SB01675, Nord GS1749, HOR 2410, UWA selection 4686, UWA late selection 8861, UWA thin seed selection 8861, M-Q- 54, Giza 127, Giza 128, GSHO 1452, Brenda, Chieftain, Decanter, Pewter, Chalice, Astoria and Expres respectively). M is one kb DNA ladder HyperLadder™ IV (Bioline)...... 93

List of figures

Fig. 4.2 Barley cultivars/lines with adult plant resistance to Puccinia hordei and lacking the Rph20-linked marker bPb-0837, showed three levels of APR; High, Moderate and Low, based on average AUDPC values from two field sites in 2009...... 102

Fig. 5.1 Percentage of leaf rust resistance among 783 barley lines from four international barley observation nurseries distributed by ICARDA. Categories (C1 to C4) of resistance as described in Table 5.4...... 113

Fig. 5.2 Infection type responses of control lines against Puccinia hordei pathotype 5457P+ L to R: Gus (3+), Ricardo (11+2C), Estate (Rph3, 3+), Egypt 4 (Rph8, 1++CN+), Cebada Capa (Rph7, ;N), 81882/BS1 (Rph17, ;1-C) and 38P18/8/1/10 (Rph18, 0;=)...... 114

Fig. 5.3 Field responses of barley lines with USR and UAPR, L to R: Immune (0), TR, MR, MS, S and leaf death due to high infection of leaf rust. Categories of resistance are described in Appendix 2...... 115

Fig. 5.4 L to R: Wheat cultivar Morocco showing susceptibility and barley line showing resistance against Puccinia graminis f. sp. tritici pathotype 98-1,2,3,5,6 under greenhouse conditions...... 118

Fig. 5.5 L to R: Wheat cultivar Chinese 166 showing susceptibility and barley line showing immunity against Barley Grass Stripe Rust (BGYR) pathogen under greenhouse conditions...... 120

Fig. 5.6 Marker bPb-0837 amplifications at 245 bp in (1 to 13): Lines 183, 202 (IBON 32) and 54, 110, 8, 41 (IBON 34) and controls Stirling, Flagship, Ricardo, Pompadour, Baronesse, WI 3407 and Gus...... 122

Fig. 5.7 Marker Ebmag0833 amplifications at 218 to 228 bp in (1 to 13): Lines 183, 202 (IBON 32) and 54, 110, 8, 41 (IBON 34) and controls Stirling, Flagship, Ricardo, Pompadour, Baronesse, WI 3407 and Gus...... 122

XVI

List of figures

Fig. 6.1 Amplifications of SSR F4-15 from 22 Puccinia hordei isolates (lanes 1–22) and controls (lanes 23–27) at 58˚C (TD). Numbers on top of the gel indicate the rust isolates corresponding to Table 6.1. M is the 1 kb DNA marker (Gibco®, Australia)...... 138

Fig. 6.2 Amplifications of the SSR F7-22 with 22 Puccinia hordei isolates (lanes 1–22) and control rust isolates (lanes 23–27) at 49˚C (TD). Ladder; 1 kb DNA marker (HyperLadder™ IV, Bioline)...... 140

Fig. 6.3 Non-polymorphic SSR F9-1 produced PCR amplifications among different pathotypes of Puccina hordei (lanes 1–22) and control rust pathotype P. triticina (lane 23). Ladder; 1 kb DNA marker (Gibco®, Australia)...... 140

Fig. 6.4 Genetic similarity dendrogram of 22 Puccinia hordei isolates and five control isolates (Pt, Pgt, Pst, BGYR and Pga) based on UPGMA composite cluster analyses using Dice algorithm (Bootstrap analyses) calculated from SSR markers F4-15 and F7-22. Similarity percentages are shown on the left hand side and bootstrap values on the right hand side of the group nodes. For the database construction in GelCompar II, BLR01 to 22 series was assigned to P. hordei pathotypes and 23 to 27 to the control cereal rust pathogen isolates. Pathotype names are shown on the right hand side...... 141

Fig. 6.5 Genetic similarity dendrogram of 22 Puccinia hordei isolates and five control isolates (Pt, Pgt, Pst, BGYR and Pga) based on UPGMA cluster analyses using Dice algorithm (CPCC) calculated from the PCR-fingerprinting marker M13. Similarity percentage values are shown on the left hand side of the group nodes. For the database construction in GelCompar II, BLR01 to 22 series was assigned to P. hordei pathotypes and 23 to 27 to control rust pathogen isolates. Pathotype names are shown on the right hand side...... 142

Fig. 6.6 PCR-fingerprinting amplification with primer M13 of 22 isolates of Puccinia hordei (1–22) and five control rust isolates (Pt, Pgt, Pst, BGYR and Pga) (23–27). Numbers on top of the gel indicate the isolate names corresponding to Table 6.1 and M is the 5 kb DNA marker (HyperLadder™ III, Bioline)...... 143

XVII

List of figures

Fig. 6.7 Genetic similarity dendrogram of 22 Puccinia hordei isolates and five control isolates (Pt, Pgt, Pst, BGYR and Pga) based on UPGMA cluster analyses using Dice algorithm (CPCC) calculated from PCR-fingerprinting marker (GACA)4. Similarity percentage values are shown on the left hand side of the group nodes. For the database construction in GelCompar II, BLR01 to 22 series was assigned to P. hordei pathotypes and 23 to 27 to control rust pathogen isolates. Pathotype names are shown on the right hand side...... 144

Fig. 6.8 Amplification profiles generated by the PCR-fingerprinting primer (GACA)4 for 22 isolates of Puccinia hordei (lanes 1–22) and five control rust isolates (Pt, Pgt, Pst, BGYR and Pga) (lanes 23–27). Numbers on top of the gel indicate the isolate names corresponding to Table 6.1 and M is the 5 kb DNA marker (HyperLadder™ III, Bioline)...... 145

XVIII

List of appendices

Appendix D1: Differential genotypes used in the greenhouse tests to assess pathogenicity of Puccinia hordei pathotypes...... 176

Appendix D2: Differential genotypes used in the greenhouse tests to assess pathogenicity of Puccinia graminis f. sp. tritici pathotypes...... 176

Appendix D3: Differential genotypes used in the greenhouse tests to assess pathogenicity of Puccinia striiformis...... 178

Appendix 1: Description of scale used in the greenhouse for scoring rust infections at seedling stage...... 179

Appendix 2: The modified Cobb’s scale used to score rust infections in the field...... 180

Appendix 3: Range of infection types produced on barley leaves in the greenhouse when inoculated with Puccinia hordei pathotypes. (;C denotes characteristic genetic black flecks)...... 181

Appendix 3.1 Observed number of segregating and homozygous susceptible BC1F2 lines developed from Ricardo by Gus cross, when tested against Puccinia hordei pathotype 5457P+ under greenhouse conditions...... 182

Appendix 3.2 Observed number of homozygous resistant, segregating and homozygous susceptible F3 lines developed from Ricardo by Gus cross, when tested against Puccinia hordei pathotype 5457P+ under greenhouse conditions...... 185

Appendix 4.1 Greenhouse infection types (ITs) data of all four International Barley Observation Nurseries (IBONs) and differential genotypes tested with Puccinia graminis f. sp. tritici (Pgt) pathotype 98-1,2,3,5,6...... 190

XIX

List of appendices

Appendix 4.2 Greenhouse infection types (ITs) data of all four International Barley Observation Nurseries (IBONs) and differential genotypes tested with BGYR pathotype...201

Appendix 5 Details of primers developed for P. graminis f.sp. tritici, P. triticina and P. coronata f.sp. avenae and tested on P. hordei including control pathotypes (P. triticina, P. graminis f.sp. tritici, P. striiformis f.sp. tritici, BGYR and P. graminis f.sp. avenae) to study the genetic variability among the P. hordei isolates from Australasia...... 213

List of abbreviations

@ At the rate of °C Degrees Celsius s Second min Minute hr Hour hrs Hours cM Centimorgan bp Base pairs kb Kilo base pairs (1,000 bp) Mb Mega base pairs (1,000,000 bp) mm Millimetre cm Centimetre m Metre µL Micro litre mL Millilitre L Litre mM Millimolar ng Nano gram μg Micro gram g Gram DNA Deoxyribonucleic acid PCR Polymerase chain reaction dNTPs Deoxynucleotide Triphosphates ddH2O Doubled distilled autoclaved water rpm Revolutions per minute V Volt BSA Bulk segregant analysis DArT Diversity array technology QTL Quantitative Trait Loci

XXI

List of abbreviations

AFLP Amplified Fragment Length Polymorphism RAPD Random Amplified Polymorphic DNA SAM Selectively Amplified Microsatellites SNPs Single Nucleotide Polymorphisms S-SAP Sequence-Specific Amplification Polymorphism SSR Simple sequence repeat BC Backcross DH Doubled Haploid RILs Recombinant Inbred Lines P Probability pt Pathotype pts Pathotypes APR Adult plant resistance ITs Infection Types Susc. Susceptible cv. Cultivar cvv. Cultivars Pst Puccinia striformis f. sp. tritici Pt Puccinia triticina Pgt Puccinia graminis f. sp. tritici Pca Puccinia coronata f. sp. avenae Pga Puccinia graminis f. sp. avenae BGYR Barley Grass Yellow Rust PBI Plant Breeding Institute UWA University of Western Australia NSW New South Wales QLD Queensland SA South Australia USA United States of America WA Western Australia BBA Barley Breeding Australia

XXII

Introduction

CHAPTER I Introduction

Cereals have remained an important part of human food since they were domesticated nearly 10,000 years ago. The ancient Romans worshipped the Goddess Ceres as a provider of grain and upon harvesting, offerings of barley were made to Ceres. These barley offerings were called ‘The Cerealia Munera’, from which the English word ‘cereal’ is derived (Hill 1937).

In the past, barley was a standard of measurement and a form of currency. Ancient civilisations used it as a medicine and even today barley leaves are considered a rich source of amino acids. Yu et al. (2002) reported the health benefits of barley grass and recently CSIRO in Australia released BARLEYmax®, variety with high levels of fibre, which can be a valuable content of health foods (CSIRO 2009). Barley is known as poor man’s food (Chandola 1999) because it is cheaper than other cereals such as wheat (Zohary and Hopf 1988). The Egyptians considered barley to be a nutritious food, providing enduring strength and energy (Weaver 1950). These days barley is also gaining popularity as a part of the human diet, as a breakfast cereal, as barley malt (an alternative to coffee) and as part of multi-grain breads etc.

Cultivated barley (Hordeum vulgare L. subsp. vulgare) is the world’s fourth most important cereal crop (FAO 2006; FAO 2008; Schulte et al. 2009) with its biggest market in Saudi Arabia (FAO 2011b). Barley can be grown on less fertile soils and has superior production per unit of moisture compared to wheat. Barley performs better than other cereal crops under drought, cold, saline and alkaline conditions (Bothmer et al. 1995). It is grown worldwide, normally at 350 to 1500 m above sea level, but in the Himalayan region, it is grown successfully at 2800 to 4050 m above sea level (Shao et al. 1982) indicating the high adaptability of barley to a range of environmental conditions.

Barley is an important multi-billion dollar industry in Australia. Recently, a national barley breeding program, Barley Breeding Australia (BBA), was formed with the aim to double Australia's 6.6 million tonne barley production by 2020, by increasing average yields from 2.0 t/ha to 2.6 t/ha and expanding the growing area from 3.3 million hectares to more than 5 million hectares (GRDC 2005). During 2009, 152.2 million tonnes of barley was produced 1

Introduction worldwide, with Australia being the sixth largest producer with 8 million tonnes of barley production (FAO 2011a). In several countries, barley is grown and used for animal feed and malt production and there is growing interest in its use for food and industrial purposes (Lea et al. 1992).

Throughout the world, cultivated barley is affected by at least 30 diseases. Barley is grown widely in Australia, where it is affected by 23 diseases, the most important of which are leaf rust, scald and the net blotches (Wallwork 2000; Williams 2003). In Australia, these important barley diseases cause an estimated average annual loss of A$ 252 million (Murray and Brennan 2010) to the barley industry, because of significant yield losses. Barley leaf rust, an economically important disease caused by the fungus Puccinia hordei Otth., affects barley production in many parts of the world (Clifford 1985). Barley leaf rust epidemics have caused significant yield losses in Australia (Cotterill et al. 1995; Cotterill et al. 1992; Waterhouse 1927), New Zealand (Arnst et al. 1979), Europe and the USA (Griffey et al. 1994; Melville et al. 1976). In addition to barley leaf rust, stem rust caused by either P. graminis Pers. f. sp. tritici Eriks. & E. Henn., P. graminis Pers. f. sp. secalis Eriks. & E. Henn. or the scabrum rust (Park et al. 2003) and a variant of P. striiformis called barley grass stripe rust (BGYR) (Wellings et al. 2000), can also affect barley in Australia. Barley stripe rust (P. striiformis f. sp. hordei) does not occur in Australia (McIntosh et al. 2001; Park 2008).

Australia has a long history of breeding rust resistant cereal cultivars (McIntosh 2007). Seedling resistance genes (Rph1 to Rph19) (Weerasena et al. 2004) were deployed against P. hordei in Australia and other parts of the world and majority of these genes have been overcome by the pathogen. In Australia only Rph7, Rph11, Rph14, Rph15 and Rph18 (Park 2003; Park 2010) and adult plant resistance (APR) gene Rph20 (Hickey et al. 2011) remained effective (Park 2010 unpublished) leaving breeders with limited choice of resistance genes for germplasm enhancement. The present study was aimed to characterise and/or to map the new sources of seedling resistance and APR against barley rust diseases and to find the genetic variation among the P. hordei pathotypes found in Australasia.

Introduction

Research projects:

1. Genetic analyses and molecular mapping of an uncharacterised seedling gene conferring resistance to P. hordei in barley cultivar Ricardo.

2. Characterisation of APR against P. hordei in barley germplasm containing a set of 113 advanced breeding lines and cultivars.

3. Evaluation of rust resistance against P. hordei, P. graminis Pers. f. sp. tritici and BGYR among four international barley nurseries comprising 820 barley lines, sourced from the International Centre for Agricultural Research in the Dry Areas (ICARDA).

4. Studying the usefulness of SSRs developed for P. graminis Pers. f. sp. tritici, P. triticina and P. coronata f. sp. avenae in detecting polymorphism among isolates of P. hordei and to detect the genetic variability in P. hordei isolates using polymorphic

SSR markers and PCR-fingerprinting primers M13 and (GACA)4.

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CHAPTER II Review of literature

Origin of barley As each cereal crop is considered to be derived from a single common ancestor, so different crops should likely carry the same genes derived from a common ancestor (Moore 1995; Pourkheirandish and Komatsuda 2007). It is estimated that the grass family emerged approximately 60 million years ago and that the divergence of the Triticeae occurred approximately 12 million years ago (Devos 2005; Pourkheirandish and Komatsuda 2007).

According to Molina-Cano et al. (1999), the origin of a crop can be described as the area of its first domestication and coexistence of its wild ancestors and developed cultivated species. The present day cultivated barley developed from a wild grass that originated in the Near East Fertile Crescent (Badr et al. 2000), though some researchers believe China was the place of origin (Xu 1987) and others consider it was Ethiopia (Bekele 1983). Remnants of wild barley (H. vulgare subsp. spontaneum) have been discovered from many sites across the belt that stretches from North Africa to Turkey, Iran and Afghanistan. H. vulgare subsp. spontaneum has no crossing barriers to the cultivated barley (H. vulgare subsp. vulgare) (Asfaw and Bothmer 1990) and is regarded as the wild ancestor of cultivated barley (Harlan and Zohary 1966).

Shao (1982) studied wild ancestors of H. vulgare found in the Tibetan region and concluded that two row wild barley is the oldest ancestor of cultivated barley. Cultivated barley (subsp. vulgare) has evolved through different stages as the two row barley evolved first, bottle necked wild barley second, six row barley third, followed by the fourth and final stage of cultivated barley (Shao 1982). Based on evolutionary studies, Shao (1982) claimed that barley was first domesticated in the Himalayan region. Other studies (Corke and Atsmon 1990; Shao 1987; Yang and Yen 1985) supported this hypothesis by showing that the wild ancestor (subsp. spontaneum) of cultivated barley is present in Tibet, Nepal, India, Pakistan and Afghanistan. In contrast, some researchers believe that before cultivation, the early forms of barley were six row as the original six row barley appears on many ancient coins and wall paintings. Xu (1987) also concluded that the six row naked forms of cultivated Chinese barleys might have first evolved from wild genotypes in Tibet. 4

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Phenotypic characterisation and an allelic study of 57 cultivated and 317 wild barley lines collected from different parts of the world showed that cultivated barley (H. vulgare subsp. vulgare) is monophyletic and that wild populations from Jordan and Israel are genetically more similar to the cultivated barley genome in comparison to populations from other parts of the world. This characterisation supports the hypothesis that cultivated barley originated from the fertile crescent of the Middle East (Badr et al. 2000). The prevalence of the diagnostic allele IIIa of gene BKn-3 in Himalayan landraces indicates that allelic substitution took place with the migration of barley from east to south Asia (Badr et al. 2000) and that diversification of domesticated barley occurred in the Himalaya.

Distribution of barley Barley is grown worldwide constituting an important crop across North America, Australia, Africa, South to East Asia and Europe. Barley was introduced to South America by European settlers during 17th century and later it spread to North America (Poehlman 1959). The geographical distribution of major crops as presented by Leff et al. (2004) shows that barley is concentrated around Europe, Central Asia, North Africa, Middle East, North America and Australasia (Fig. 2.1). At the end of the 19th Century, two row barley was introduced to Australia and Japan (Seko 1987). According to the ecogeographical distribution of the genus Hordeum (Bothmer et al. 1995), the worldwide distribution of the diploid species of barley including H. vulgare subsp. vulgare is given in Table 2.1.

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Table 2.1 Geographical distribution of diploid (2n = 14) barley Barley species Geographical distribution H. vulgare L. subsp. vulgare All temperate zones of the world. H. vulgare L. subsp. spontaneum (C. Koch.) Thell. Afghanistan, Egypt, Greece, Iran, southern Tajikistan and western Pakistan. H. vulgare L. subsp. agriochriton (Åberg) Bowd. From Middle East to 4500 m high in Himalayas and Mediterranean region. H. bulbosum L. Greece and western Egypt. H. murinum L. subsp. glaucum (Steudel) Tzvelev From south of Mediterranean region to east Iran, Afghanistan and Kashmir in India. H. pusillum Nuttal U.S.A. and few parts of Canada and Mexico. H. intercedens Nevski Parts of California near Santa Barbara Islands and northwest Baja California of Mexico. H. euclaston Steudel Central parts of Argentina, Uruguay and southern Brazil. H. flexuosum Steudel Buenos Aires, some parts of Argentina and Uruguay. H. muticum Prsl Argentina, Bolivia, Chile, Colombia, Ecuador and Peru. H. chilense Roemer & Schultes Rio Negro province of Argentina and Chile. H. cordobense Bothmer, Jacobsen & Nicora Central and northern parts of Argentina. H. stenostachys Gordon Central and northern parts of Argentina, Brazil and Uruguay. H. pubiflorum Hooker f. Santa Cruz in Argentina and Nubie in Chile. H. halophilum Grisebach Argentina, Chile, some parts of Bolivia and Peru. H. comosum Presl Mendoza province of Argentina and parts of Chile. H. marinum Hudson subsp. marinum Western parts of Mediterranean region. H. marinum Hudson subsp. gussoneaum (Parlatore) Thellung South west Asia to eastern parts of Mediterranean region.

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H. bogdanii Wilensky India, Iran, Afghanistan, Pakistan, China and Mongolia. H. roshevitzil Bowden Mongolia, southern parts of Siberia and north to central China. H. brachyantherum Nevski subsp. californicum (Covas & Stebbins) Bothmer, South-western California. Jacobsen & Seberg H. erectifolium Bothmer, Jacobsen & Jørgensen Part of Buenos Aires province of Argentina. H. patagonicum (Haumann) Covas and ssps. Provinces of Argentina like Chubut, Santa Cruz, Patagonia and inland parts of Tierra del Fuego. H. brevisubulatum (Trinius) Link subsp. brevisubulatum North of China, Mongolia and south-eastern Siberia. H. brevisubulatum (Trinius) Link subsp. nevskianum (Bowden) Tzvelev North of Afghanistan and Kashmir, Nepal, west of China and western Siberia. H. brevisubulatum (Trinius) Link subsp. turkestanicum (Nevski) Tzvelov Afghanistan, Pakistan, south of Tadzjikistan and west of China. H. brevisubulatum (Trinius) Link subsp. violaceum (Boissier & Hohenacker) Alborz mountains in Iran, Caucasus region and central Turkey. Tzvelov Table source: (Singh 2006)

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Fig. 2.1 Distribution of barley and other major crops. Source: (Leff et al. 2004)

Importance and usage of barley A significant amount of barley grain is used for animal feed and malting. Nevertheless more than five percent of global barley production is consumed as food (Edney 1996) with the highest consumption occurring in North African countries at a rate of 65 kg/person/annum (Bhatty 1993). Barley is a major staple food in some areas of Africa and the Near East, the highlands of Central Asia, the Horn of Africa, the Andean countries and the Baltic States. Food barley is generally found in regions where other cereals grow poorly due to low rainfall, altitude, or soil salinity. It remains the most viable option in dry areas (< 300 mm of rainfall) and in production systems where alternative food crops are limited, such as in the highlands and mountains.

In addition to food, feed and malt, transgenic barley has been used to produce enzymes, oral vaccines, antibodies, pharmaceuticals and vitamins etc. Xylanase, an enzyme used as a food additive to improve quality (Trogh et al. 2004) and in the manufacture of many products from biomasses (García-Aparicio et al. 2007), is

Review of literature produced on a large scale in barley grains (Patel et al. 2000). Special starches for industrial purposes could also be produced in barley. The bioplastic properties of potato, corn, wheat and rice starches have been studied (Soest et al. 1996) andhighlighting potential for similar uses of barley starch.

Nutrition in barley From a nutritional point of view, nitrogen, minerals, starch and fibre are the components of most interest in barley. Starch is an important component of many diets and is the principal source of energy in our diets and makes it possible to utilise protein efficiently. Barley is an efficient source of protein, rich in glutamic acid, proline and leucine amino acids (Grando et al. 2005). It is also a very good source of minerals such as iron, phosphorus, zinc and potassium. The phosphorus provided by hull-less barley varieties is absorbed better in the intestine, due to a lower phytic acid content (Grando et al. 2005).

These days, barley is becoming a popular health food due to its health promoting highly soluble dietary fibre and β-glucan content. β-glucan is an important dietary fibre that has significant blood cholesterol lowering effects (Foster 1987; Martinez et al. 1992). It has been determined that barley grain comprises about 20% dietary fibre, of which 3–7% is β-glucan (Åman et al. 1985; Oscarsson et al. 1996; Ullrich et al. 1986). The β-glucan content of barley increases the viscosity of digestion in the intestine by slowing down the rate of digestion and absorption of starch (Anderson et al. 1990), which is highly beneficial for diabetics (Gosain 1996; Pick 1994). Chemical composition varies largely between different types of barley. According to Andersson et al. ( 1999) and Oscarsson et al. (1996), hull-less barley contains less ash and dietary fibre and more starch, protein and fat than covered barley. Fibre content in the kernel ranges from 6–10% with higher concentration in the basal or germinal region and lower in the distal region. Part of the aleurone layer and endosperm are detached during the pearling and some of the nutritive value is lost. Recently, CSIRO Australia released BARLEYmax® a two-row barley cultivar with unique grains containing twice the amount of fibre (soluble, insoluble and resistant starch) as found in wheat or oats (CSIRO 2009). The nutritional value of barley in comparison to other major cereals is described in Table 2.2. 9

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Table 2.2 Chemical compositions (percentage of grain dry matter) of barley grain compared with other important cereals Cereal Protein Fat Carbohydrates Fibre Ash

Covered Barley 12.2 1.9 75.9 6.8 3.1

Hull-less Barley 13.3 2.6 80.0 1.9 2.0

Pearled Barley 12.0 1.5 84.3 1.0 1.2

Wheat 13.4 2.4 79.9 2.4 1.9

Maize 10.3 4.5 81.5 2.3 1.4

Polished Rice 10.1 2.1 86.4 1.0 1.4

Polished Oat 14.7 8.0 72.0 4.0 2.0

Table source: (Grando et al. 2005)

Apart from grains, the green matter from young leaves of barley is full of nutrition as it contains vitamin C, antioxidants, amino acids, glucose and fructose (Paulickova et al. 2007). Barley grass shots (small quantity of juice made from young green leaves of barley) are becoming very popular with the awareness of its high nutritional qualities. According to Yu et al. (2002), barley grass was beneficial in lowering cholesterol levels in patients with type 2 diabetes. Supplementation with young barley leaf extract at the rate of 15 g/day can decrease plasma lipids and inhibit LDL oxidation, which may protect against atherosclerosis in hyperlipidemic smokers and/or non-smokers (Yu et al. 2003).

Geographical distribution of barley production Barley has been an important cereal crop since 8000 BC and remains a vital crop for poor people in dry and marginal areas. It was the first domesticated cereal crop, being cultivated around 10,000 years ago in the Fertile Crescent and was the foundation of old world agriculture (Badr et al. 2000; Pickering and Johnston 2005). Today, barley is cultivated all over the world from temperate to tropical regions, on about 57 million hectares (Grando et al. 2005) and by the year 2009 the world area under barley cultivation had risen to 59 million hectares (FAO 2011a). Most of the world's barley comes from Australia, Canada, France, Germany, Poland, Spain, Turkey, United 10

Review of literature

Kingdom, USA, Ukraine and the biggest producer Russia with 17.8 million tones of production during 2009 from 7.7 million hectares (FAO 2011a).

Barley is the second most important cereal crop in Australia (Murray and Brennan 2010). South Australia (SA), Western Australia (WA), New South Wales (NSW) and Victoria are the major barley producing states. Australia produced 5.9 million tonnes of barley during 2007 (FAO 2008) against a prediction of 5.5 million tonnes for 2007/08 season (ABARE 2007). Because of drought, barley production in 2006/07 in Australia was poor with 55% less production than in previous years (ABS 2008). In Australia, by the year 2009 the area under barley cultivation went to four million hectares with eight million tonnes of production. Average yields are low in Australia (1.9 t/ha) compared to Russia (2.3 t/ha) and the overall world average (2.8 t/ha) and in comparison with the top yield of 8.6 t/ha in Saudi Arabia (FAO 2011a). World barley production for 2011 is forecasted to be 133 million tonnes (FAO 2011b).

Barley taxonomy Barley is a monocotyledonous plant and is classified based on spike characters and growth habit. Cultivated barley is of two types based on head morphology, known by two row and six row barley. In six row barley, at each node of the head, three kernels are formed and only one kernel forms in the case of two row barley. At each node of the head, all three florets are fertile in six row barley and only one is fertile in two row barley. Some intermediate forms are also found in which the middle floret at each node is not fully developed. According to Harlan et al. (1940), progeny from crosses between two row and six row barley are very inferior, showing fewer yields compared to progeny from same row crosses. Lambert and Liang (1952), however, found that selections from two row by six row crosses out-yielded two by two row and six by six row crosses, which was further supported by the development of improved varieties from crosses between two row and six row barley by Aikasalo (1988). The six row phenotype is controlled by a recessive gene (vrs1) and the two row character is controlled by the dominant (Vrs1) gene located on 2HL (Lundqvist et al. 1996).

Barley is also classified as winter barley and spring barley based on climatic requirements; winter barley requires exposure to low temperatures for head initiation, 11

Review of literature where spring barley does not have such requirement. Spring barley can be grown successfully as a winter crop (Anderson et al. 2002).

The genus Hordeum consists of diploid (2n = 2x = 14), tetraploid (2n = 4x = 28) and hexaploid (2n = 6x = 42) cytotypes. According to Pourkheirandish and Komatsuda (2007), the German botanist Carl Koch was the first to discover the immediate ancestor of cultivated barley in Turkey and he described it as a separate species, H. spontaneum. Later, based on the biological species concept (Bothmer et al. 1995), it was regarded as a subspecies [subsp. spontaneum (C. Koch) Thell.] of Hordeum vulgare. According to Bothmer et al. (1995), cultivated barley (H. vulgare subsp. vulgare) is one of 32 species in the genus Hordeum that are divided into four sections: Hordeum, Anisolepis, Critesion and Stenostachys. The section Hordeum includes H. vulgare, H. bulbosum and H. murinum, all distributed widely throughout the world. Most Hordeum species are perennial and differ in their reproductive systems (Bothmer et al. 2003). Cultivated barley (H. vulgare L subsp. vulgare) is a self pollinated diploid with seven pairs of chromosomes (2n = 2x = 14). The scientific classification of cultivated barley is given in Table 2.3.

Table 2.3 Taxonomic classification of cultivated barley Kingdom Plantae Super Division Spermatophyta Class Angiospermae Sub Class Monocotyledonae Order Poales Family Poaceae (Gramineae) Genus Hordeum L. Species vulgare L. Sub species vulgare Scientific Name H. vulgare L. subsp. vulgare Common Name Cultivated Barley Source: (Bothmer et al. 1995; Singh 2006)

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The barley genome According to Bennett et al. (1995), the genome of diploid barley is very large at 5,300 million base pairs (Mb) and consists of 80% repetitive DNA (Flavell et al. 1974). The average distance between genes in barley is 240 kb (Dubcovsky et al. 2001). After hexaploid bread wheat (Triticum aestivum L.), the barley genome is one of the largest in the cereal crops (Bennett and Leitch 1995), being 12 times that of the rice genome and nearly twice the size of the human genome. The barley genome is the base model for species within the tribe Triticeae. The recombination rate differs between chromosomes. An observed high correlation between marker density and recombination frequency implies that most recombination takes place in gene rich areas of chromosomes (Künzel et al. 2000).

Due to the economic importance of barley and its suitability as a genomic model for other Triticeae genomes, numerous studies have been conducted on the genetics, cytogenetics, biosystematics, mutation, population genetics, molecular cytogenetics, cell culture, tissue culture and genetic transformation in barley (Singh 2006) and many genomic resources have been developed. A large number of genetic stocks and mutants, several well utilised genetic mapping populations (Caldwell et al. 2004; Lundqvist et al. 1996; Varshney et al. 2004), several bacterial artificial chromosome (BAC) libraries (Isidore et al. 2005; Yu et al. 2000) and the Affymetrix Gene Chip featuring 22,000 genes (IBSC 2006), have been developed. While these resources are important in advancing barley genetic research, a full genomic sequence is required to understand all important agricultural traits. In August 2006, during the meeting of International Triticeae Mapping Initiative (ITMI) held at Adelaide, a major international initiative to sequence the whole barley genome was established and to achieve this goal, the International Barley Genome Sequencing Consortium (IBSC) (http://barleygenome.org/) was formed.

Analyses of chromosome pairing in hybrids at metaphase 1 (M1) revealed the presence of four genomes in the genus Hordeum (Bothmer et al. 1986; Bothmer et al. 1995) and on this basis, the genus was divided into four basic genomes (H, I, X and Y). Subsequent molecular cytogenetic studies using genomic in situ hybridisation found three polyploid species, H. secalinum, H. capense and H. brachyantherum 13

Review of literature subsp. brachyantherum 6x, which are allopolyploids having combinations of the I and X genomes. Thus the genus is now divided into five genomes viz. H, I, X, Y and XI (Taketa et al. 1999; Taketa et al. 2001; Taketa et al. 2005).

According to the old system given by Burnham and Hagberg (1956), barley chromosomes were designated as 5, 2, 3, 4, 7, 6 and 1 (Singh and Tsuchiya 1982). A new designation was agreed to at the 7th International Barley Genetics Symposium (Linde-Laursen et al. 1997):

 The seven barley chromosomes were designated from 1 to 7 according to their homoeologous relationships with chromosomes of other Triticeae species. The chromosome number is followed by the letter H; e.g., 2H.  The genomes of H. vulgare and H. bulbosum are symbolised by the letter H.  The chromosome arms are designated by the letters S (small) and L (large).  The genome of the barley variety 'Betzes' was nominated as the reference genome in the Triticeae, to which definitions of translocations, short arm/long arm reversals, etc. are standardised in all species.  It was recommended that the seven barley chromosomes should be designated as 1H, 2H, 3H, 4H, 5H, 6H and 7H and that arm designation should follow the chromosome and genome designation without a space, e.g. 6HL.

Rust diseases of barley Rusts have undoubtedly been present throughout the evolution and development of cereals. Archaeological studies have found wheat lemma fragments with stem rust pustules dated between 1400 and 1200 BC (Kislev 1982). Cultivated barley is affected by many diseases in the different parts of the world and including Australia where it is commonly affected by leaf rust, scald and net blotches (Wallwork 2000; Williams 2003). According to Park et al. (2003), fungi causing rust diseases like leaf rust, stem rust and stripe rust have hindered the barley production in Australia for many years as many cereal rust epidemics occurred in the past.

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Barley leaf rust Also known as brown rust of barley, this disease is very wide spread and is found almost wherever barley is grown (Clifford 1985). Leaf rust of barley is caused by an obligate fungus, Puccinia hordei Otth. (syn. P. anomala Rostr., P. simplex Eriks. et Henn.) (Clifford 1985).

Leaf rust is an important disease and affects barley production in many parts of the world (Clifford 1985). A severe epidemic of leaf rust can reduce the yield of a susceptible cultivar by up to 62%, as was observed in Australia (Cotterill et al. 1992) and up to 20%, as observed in New Zealand (Arnst et al. 1979). Leaf rust is present in all barley growing regions of Australia (Park et al. 2003) and epidemics occurred in Queensland during 1978, 1983, 1984 and 1988 (Cotterill et al. 1995). In Europe, high yield losses were reported in trials of a susceptible cultivar, with a 17–31% reduction in yield due to reduced 1000-grain weight (Melville et al. 1976). According to Griffey et al. (1994), leaf rust epidemics resulted in 32% yield reductions in Virginia, USA.

Taxonomy of P. hordei P. hordei belongs to the genus Puccinia, the largest genus of the order Uredinales with 3000 to 4000 species (Littlefield 1981). Efforts to describe rust fungi started back in 1712. Giovanni Targioni Tozzetti described rust as the terrible scourge in his 1767 publication following a severe rust epidemic in Italy in 1766 (Bushnell and Roelfs 1984). This cereal rust epidemic motivated Felice Fontana to state in his pamphlet in 1767 that "On the 10th of June last year, I discovered that the rust, which had devastated the lands of Tuscany, is a grove of plant parasites that nourish themselves at the expense of the grain" (Bushnell and Roelfs 1984). In 1794 the Dutch researcher Christiaan Hendrick Persoon made significant efforts in fungal classification to provide detailed information on different fungi. Research work on rust taxonomy was continued by the Tulasne brothers, J.C. Arthur, G.B. Cummins, D.B. Savile and J. Eriksson. The taxonomic classification of P. hordei is described in Table 2.4.

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Table 2.4 Taxonomic classification of Puccinia hordei Taxon Eukaryota Kingdom Fungi Phylum Basidiomycota Order Uredinales Family Pucciniaceae Genus Puccinia Species hordei Otth. Scientific name Puccinia hordei Otth. Source: (Clifford 1985)

Host range of P. hordie P. hordei is a macrocyclic and heteroecious rust that can survive on several plant species. Uredinial and telial forms of the rust can survive on cultivated barley H. vulgare subsp. vulgare and the wild barleys H. spontaneum and H. bulbosum (Anikster and Wahl 1979). The gametophytic aecial stage is mainly formed on Ornithogalum and under highly favourable conditions on Leopoldia and Dipcadi species in the family Liliaceae (Clifford 1985).

Arthur (1929) reported that Anton de Bary (1831–1888), the writer of famous monograph "Brandpilze" was the first person to discover the alternation of hosts in life cycle of a rust fungus. Ornithogalum spp. as an alternate host to P. hordei was first considered by Tranzschel in 1914, with later support from other workers (Clifford 1985). Anikster (1982) successfully infected different species of Ornithogalum with basidiospores derived from leaf rust infected H. vulgare, H. spontaneum, H. bulbosum and H. murinum and found pycnial fertilisations on all alternate host species. In Europe, the alternate host does not play an important role as germination of telia does not synchronise with the season and growth of Ornithogalum (Clifford 1985). Star of Bethlehem (Ornithogalum umbellatum) is a widespread weed on the Yorke Peninsula in SA (Wallwork et al. 1992) and in the Murrumbidgee catchment areas including Henty and Junee in NSW (Murrumbidgee 2008). Occurrence of O. umbellatum beyond SA was indicated in the earlier study of pathogenic specialization of P. hordei in Australia (Park 2003).

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Life cycle of P. hordei P. hordei is macrocyclic rust (Cummins and Hiratsuka 1983) with five different spore stages (Clifford 1985; Petersen 1974), are described in Fig. 2.2.

Under favourable conditions, large amounts of rust inoculum are produced due to repeated infection and production of uredinia and urediniospores. Estimates have shown that 0.4 ha of susceptible wheat with a 10% stem rust severity can produce 1012 urediniospores (Rowell and Roelfs 1971), whilst one hectare of susceptible wheat at a 1% infection of leaf rust could produce 1011 urediniospores in a day (Parlevliet and Zadoks 1977). Agents such as human beings, animals, insects and wind, transport urediniospores to neighboring plants, fields, states and even to different countries (Kolmer 2005). For example pathotypes (pts) of P. hordei have been transported from Western Australia to eastern Australia, presumably by wind (Park 2003).

Initial P. hordei inoculum comes in the form of urediniospores from over-wintering or over-summering volunteer barley plants, cultivated barley plants or in the form of aeciospores from the alternate host if present. In many cases self sown volunteer barley plants on road sides, bridges, railway lines, around silos and fields act as a green bridge for the perpetuation of urediniospores for the next season. The aeciospore is globoid or ellipsoid in shape (18–26 x 16–33 µm) and urediniospore is subgloboid or ellipsoid (21–34 x 15–24 µm) in shape (Clifford 1985). Upon landing on a leaf, a urediniospore or aeciospore hydrates absorbing low molecular weight ionic material and is exposed to moisture during the formation of dew at night (Simkin and Wheeler 1974). Upon hydration and at suitable temperatures (5ºC to 25ºC; (Polly and Clarkson 1978), the urediniospores will then produce a germ tube. The germ tube is elongated and flattens at the end to produce a dome like appressorium (Clifford 1985). A penetration peg is formed at the end of appressorium, which penetrates through the stomatal guard cells (Clifford 1985). Maximum penetration by the appressorium takes place at 10 to 20ºC (Joshi et al. 1959; Simkin and Wheeler 1974). A substomatal vesicle is formed from the appressorium, which produces dilateral hyphae from either end (Clifford 1972). A mycelium is then formed by the hyphae, with haustoria on the end of each hypha. The pathogen derives nutrition from the host cell through haustoria whilst host cell remains alive. According 17

Review of literature to Polly and Clarkson (1978), under optimal conditions sporulation starts within 6 to 8 days of infection, but it can take up to 60 days at 5ºC (Simkin and Wheeler 1974). Uredinia rupture the epidermis to form orange-brown rust pustule. Mature urediniospores are easily detached and dispersed by wind and under cold and cloudy conditions, can survive up to 38 days, but lose viability quickly in hot and sunny weather (Teng and Close 1980).

At the end of the vegetative growth of maturing plants, smooth surfaced and black coloured telia form on the leaves and leaf sheath. Telia are covered by the epidermis and contain dark brown to black coloured teliospores. Teliospores have two cells, each of which carries two nuclei (Mendgen 1983). The two nuclei fuse to produce a single diploid nucleus by a process is known as karyogamy. With the formation of diploid cells, the teliospore wall is thickened and becomes dark in colour. Teliospores may germinate immediately or remain dormant. Dormant teliospores, the resting spore stage, remain intact within telia and perpetuate through winter in the infected straw or stubble. In spring, under favourable conditions, teliospores germinate and each cell produces a basidium. In the basidium, diploid cells undergo meiosis to start reduction division of the nucleus and four haploid unicellular basidiospores are produced, of both + (male) and - (receptive, female) type from each diploid cell. Mature basidiospores are ejected forcibly and carried away by the wind. Basidiospores cannot infect Hordeum species and do not play any role when the alternate host is absent.

Where the alternate host is present, both + and - basidiospores can germinate and penetrate leaves to produce pycnia or spermogonia. Pycniospores of + and - type are formed (Clifford 1985) and ooze out in the form of honey dew from separate + and - type pycnia (Anikster et al. 1999). Insects are attracted to the honey dew and help in the transportation of both types of pycniospores in both ways. On contact, mating takes place between the receptive hypha of - type pycniospores (female gamete) with the + type pycniospores (male gamete). The possibility of self fertilisation is prevented in P. hordei because only + pycniospores can fuse with the - type receptive hypha and vice versa. Fertilisation of a + pycniospore and a - receptive hypha (pycniospore) produces dikaryotic aecium, in which chains of aeciospores are 18

Review of literature produced by a process called plasmogamy (the cytoplasm of + pycniospore and - pycniospore fuse together without the fusion of nuclei). Mature aeciospores are dispersed by wind and can infect wild and or cultivated Hordeum spp. to produce a dikaryotic mycelium and eventually uredinia is produced and urediniospores released to start a new disease cycle. The combination of two genetically different haploid + pycniospore and - pycniospore can result in aeciospores with different virulence against host plants.

Fig. 2.2 Complete life cycle of Puccinia hordei, the leaf rust pathogen of barley involving five different stages of spores (aeciospores, urediniospores, teliospores, basidiospores and pycniospores)

Pathogenic variation in P. hordei Different isolates of P. hordei can vary in their capacity to infect different genotypes of Hordeum. These genetically different rust isolates are known as pts, first noted by Stakman and Piemeisel (1917) while studying P. graminis f. sp. tritici on wheat.

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New pts are formed through sexual or asexual (somatic) hybridisation of genetically different rust isolates and through random mutation (Park 2007b). The process of sexual hybridisation has resulted in new pts of P. hordei in Israel (Manisterski 1989), Greece (Critopoulos 1956) and in Australia (Wallwork et al. 1992). Golan et al. (1978) isolated new pts from alternate hosts that were virulent on the resistance genes Rph1, Rph2, Rph3, Rph4, Rph5, Rph6, Rph7, singly and in combination. P. hordei is the only cereal rust pathogen that undergoes sexual recombination in Australia (Park 2008). Uredinial isolates derived from aeciospores collected from infected plants of O. umbellatum in the Yorke Peninsula of SA, were identified as six different pts of P. hordei (Wallwork et al. 1992). More recent surveys of P. hordei in Australia found a high diversity of pts from samples collected in SA (12 pts from six samples), presumably due to initial inoculum coming from the alternate host O. umbellatum, which is present in the Yorke Peninsula (Park 2010).

Mutation is also believed to be an important source of pathogenic variation in P. hordei in Australia. Pathotypes 201, 210 and 220 virulent on Rph1, Rph4 and Rph5 respectively are believed to be the result of single step mutations from the founder pt 200 (Cotterill et al. 1995). Widespread cultivation of Rph12 carrying barley cultivars including Franklin, Tallon, Lindwall and Fitzgerald resulted in new pts like 4610P+, 5610P+ and 5453P- all virulent on Rph12 (Park 2003; Park 2008). Pathotype 5453P- is presumed to be mutated into a new pt 5453P+ with added virulence for Rph19 and both pts were detected from WA (Park 2006). Following the releases of cultivars Fitzroy, Yarra and Starmalt carrying Rph3, virulence for this gene was detected for the first time in Australia during 2009 rust survey, in pt 5457P+, which was considered to be mutated from pt 5453P+ (Park 2010).

Although not yet documented for P. hordei, new pts could emerge through somatic hybridisation between rust isolates, as has been observed in several other rust pathogens. A new pt of P. striiformis Westend resulted from an infection of intentionally mixed urediniospores of two different pts of P. striiformis f. sp. tritici (Wright and Lennard 1980). Park et al. (1999) provided evidence of somatic hybridisation in nature in P. triticina. According to Luig and Watson (1977),

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“scabrum” stem rust isolates are the product of somatic hybridisation between P. graminis f. sp. tritici and P. graminis f. sp. secalis.

Identifying pathotypes of P. hordei in Australia To identify pts, rust samples are tested on a set of barley genotypes (differentials) comprising barley varieties and lines with known resistance genes. Each differential tester carries one or more resistance gene(s), which are used to identify the virulence and avirulence of an isolate. Many workers have used local sets of differentials to identify pts. By using a set of nine barley varieties, Levine and Cherewick (1952) were able to differentiate 52 pts of P. hordei from isolates collected from Australia, Europe and North America.

The use of local differential sets created problems in comparing pathogenicity data at the international level. To overcome this problem, Clifford (1977) suggested a two tier system comprising a standard international differential set and a national level set according to local needs. Annual rust surveys of P. hordei in Australia are carried out under the Australian Cereal Rust Control Program (ACRCP) at the University of Sydney, Plant Breeding Institute (PBI), Cobbitty. The set of P. hordei differentials used at PBI includes 30 different genotypes representing most catalogued Rph genes and several uncharacterised genes (Table 2.5).

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Table 2.5 Differential set used in Australia to identify pathotypes of Puccinia hordei Resistance gene Barley Genotype Reference Susceptible Gus Rph1 Sudan (Unknown) Rph1 Berg (Park, unpublished) Rph2 Peruvian (Levine and Cherewick 1952; Starling 1956; Steffenson and Jin 1997) Rph2+ Rph5 Quinn (Roane and Starling 1967; Starling 1956) Rph2+ Rph6 Bolivia (Henderson 1945; Roane and Starling 1967; Starling 1956) Rph2+19 Reka 1 (Levine and Cherewick 1952; Moseman and Greeley 1965; Starling 1956) Rph2+? Ricardo (Henderson 1945; Moseman and Roan 1959; Zloten 1952) Rph3 Estate (Henderson 1945; Roane and Starling 1967) Rph4 Gold (Moseman and Reid 1961; Roane 1962) Rph5 Magnif 104 (Frecha 1970; Roane and Starling 1967; Starling 1956; Yahyaoui and Sharp 1987) Rph7 Cebada Capa (Johnson 1968; Nover and Lehmann 1974; Parlevliet 1976; Starling 1956) Rph8 Egypt 4 (Levine and Cherewick 1952; Tan 1977) Rph9 Hor 2596 (Tan 1977) Rph10 Clipper BC8 (Feuerstein et al. 1990) Rph11 Clipper BC67 (Feuerstein et al. 1990) Rph12 Triumph (Jin et al. 1993; Walther 1987) Rph13 PI 531849 (Jin et al. 1996; Sun 2007) Rph14 PI 584760 (Jin et al. 1996) Rph15 Bowman + Rph15 (Chicaiza et al. 1996)

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Rph17 81882/BS1 (Pickering et al. 1998) Rph18 38P18/8/1/10 (Pickering et al. 2000) Rph19 Prior (Park and Karakousis 2002) RphGat Gatam (Park, unpublished) RphB37 B37 (Park, unpublished) RphC Cantala (Park, unpublished) RphQ Q21861 (Borovkova et al. 1997) Rph? 36l50/3/5/1 (Unknown) Rph? 169P15/8 (Unknown)

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Pathotype nomenclature in Australia Different systems of nomenclature have been used by researchers to identify and name pts of rust pathogens. The nomenclature system developed by Stakman and Piemeisel (1917) did not permit the addition of new differential genotypes. To overcome this problem, Gilmour (1973) developed a nomenclature system using binary numbers. This flexible and open system of nomenclature was proposed by Clifford (1992) as an international system for naming pts of P. hordei.

A modified version of this system is in use at PBI. In this system, the barley variety Gus is included as a universal susceptible (but does not contribute to the pt code) and other differentials carrying genes from Rph1 to Rph12 are divided into four groups of three each. Rph1 to Rph12 are given octal values of ones, tens, hundreds and thousands in the same order. The pt code is based on virulence and avirulence against Rph1 to Rph12 and cultivar Prior (Rph19) as exemplified in Table 2.6. For example pathotype 5653P-, is virulent against Rph genes corresponding octal values 5000 (4000 + 1000; Rph12 and Rph10), 600 (400 + 200; Rph9 and Rph8), 50 (40 + 10; Rph2+6 and Rph4), 3 (2 + 1; Rph2 and Rph1) and is avirulent on Prior (“P-”).

Table 2.6 System used in Australia to name pathotypes of Puccinia hordei, as exemplified by four recently isolated pathotypes Differential Resistant gene Octal value P. hordei pathotypes Genotypes 5653P- 5610P+ 5453P- 5457P+ Gus Susc. V V V V Sudan Rph1 1 V A V V Peruvian Rph2 2 V A V V Estate Rph3 4 A A A V Gold Rph4 10 V V V V Magnif 104 Rph5 20 A A A A Bolivia Rph2+6 40 V A V V Cebada Capa Rph7 100 A A A A Egypt 4 Rph8 200 V V A A Abyssinian Rph9 400 V V V V Clipper BC8 Rph10 1000 V V V V

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Clipper BC67 Rph11 2000 A A A A Triumph Rph12 4000 V V V V Berg Rph1 V A V V Reka I Rph2+Rph19 A V A V Ricardo Rph2+? A A A A Quinn Rph2+Rph5 A A A A PI 531849 Rph13 A A A A PI 584760 Rph14 A A A A Prior (P) Rph19 A V A V Q21861 RphQ V A V V Cantala RphC V V A A PI366444 Rph? V V V V Gatam RphGatam V A V V A = avirulent, V = virulent and Susc. = susceptible

This system of nomenclature is incomplete because the pt code developed with it does not provide any information on pathogenicity of a pt against genes additional to Rph1–Rph12 except Rph19 (Prior).

Pathotypes of P. hordei in Australia Monitoring of rust pathogens via regular surveys for pathogenic variability has helped to understand the structure of rust pathogen populations in Australia. The results of these surveys have shown that rust populations vary according to the introduction of exotic pts, asexual or in the case of P. hordei sexual hybridisation, mutation and spread to the other parts of Australia (Park 2008; Park et al. 1995; Wellings and McIntosh 1990). Sporadic monitoring of the pathogenicity of P. hordei in Australia commenced in the 1920s (Waterhouse 1927). Early surveys found a pt similar to a European pt and another one that differed from one found in the North America (Waterhouse 1952; Watson and Butler 1947). Pathotypes UN14 and UN16 (Levine and Cherewick 1952) predominated in Australia prior to 1985 (Luig 1985). Cotterill et al. (1995) pathotyped 154 isolates of P. hordei collected in Australia from 1966 to 1990 and found that pts 243P+ and 243P- predominated from 1966 to 1979 and pts 210P+ and 200P+ were common respectively, in Queensland (Qld) and Northern NSW and SA. Virulence was also detected for Rph1, Rph2, Rph4, Rph5, Rph6, Rph8 and Rph9 and the resistance genes Rph3 and Rph7 were effective against all isolates examined (Cotterill et al.

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1995). Subsequent surveys detected the new pts 4610P+, 5610P+ and 5453P-, all virulent on Rph12 and no virulence was detected on the genes Rph3, Rph7, Rph11, Rph14, Rp15 and Rph18 (Park 2003), even though virulence for some of these genes has been reported in other parts of the world (Griffey et al. 1994). Pathotype 5453P- was first detected in WA (Park 2006) and in the 2005/06 survey another pt, a presumed mutant of 5453P- with added virulence for Rph19 (5453P+) was also found from WA. During the 2009 survey, a third new pt 5457P+ was detected from the Northern NSW (Park 2010). This pt is believed to have arisen via mutation to virulence for Rph3 in pt 5453P+.

+ - + + The PBI currently maintains a collection of 30 P. hordei pts [20P , 200P , 200P , 201, 201P , 210P+, 211, 211P+, 220P+, 220P+ (+Rph13), 222P+, 231P+, 232P+ (+Reka1), 242P+, 243P-, 243P+, 243P+ Yellow, 243 (+Reka1), 243 (+Ricardo), 253P-, 4610P+, 5610P+, 4673P+, 5453P-, 5653P+ -Rph13, 5453P+ +Rph13, 5652P+, 5653P+, 5653P+ +Rph13 and 5457P+] as a working collection and has more than 600 additional isolates preserved in liquid nitrogen.

Stem rust caused by P. graminis According to the 1906 publication ‘The Rusts of Australia’, by McAlpine, stem rust is the most destructive cereal rust in Australia (Park 2007b). P. graminis is a macrocyclic and heteroecious rust pathogen. In Australia, more than four formae speciales of P. graminis are known (Park 2007b), of which P. graminis f. sp. tritici, P. graminis f. sp. secalis and the hybrid “scabrum” stem rust infect barley (McIntosh et al. 1995b). Stem rust occurs on barley only in the presence of heavy inoculum of one or more stem rust pathogen (Park et al. 2003).

Economic importance Severe infection of stems caused by stem rust interrupts nutrient flow to the developing heads, resulting in shrivelled grain. In addition, stems weakened by rust infection are prone to lodging, causing further loss of grain (Roelfs et al. 1992). During 1982 and 1983, stem rust was severe in barley crops in some parts of Qld and farmers had to spray fungicides (Dill- Macky et al. 1990). Since then, few samples of the “scabrum” rust and P. graminis f. sp. tritici pt 34-1,2,7 +Sr38 were identified from stem rusted barley samples in annual stem rust surveys conducted at the PBI (Park 2007a).

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In recent years, little emphasis has been given to breeding barley for stem rust resistance in Australia and the disease still has the potential to become a serious problem.

Stripe rust The barley stripe rust pathogen P. striiformis f. sp. hordei (Psh) has not been reported in Australia (McIntosh et al. 2001), though it is prevalent in many other parts of the world (Brown et al. 2001; Dubin and Stubbs 1986; Stubbs 1985). Stripe rust caused by a variant of P. striiformis colloquially referred Barley Grass Stripe Rust (BGYR) (Wellings et al. 2000) is however occasionally seen on barley in Australia. BGYR differs from P. striiformis f. sp. tritici in its host range and in its DNA fingerprint (Keiper et al. 2003). It is believed to have been introduced to Australia sometime either during or before 1998 (Wellings et al. 2000).

Genetic markers Genetic markers reveal the genetic variability among the individual organisms and act as the sign posts of the genes controlling different traits. They are closely located to the genes but do not affect the traits controlled by different genes. A specific genomic position of a genetic marker in the chromosome is called locus. The genetic markers are classified into three major types:

1. Morphological makers: also known as classical or visible markers are the phenotypic characters of the organisms like height, shape colour etc. Classical example of a morphological marker for stem rust resistance gene Sr2 in wheat is the development of dark brown pigmentation (pseudo black chaff) close to the stem nodes and/or on the head. Though morphological markers are limited in number and can be affected by the environment, they have been very useful for the plant breeders.

2. Biochemical markers: are the allelic forms of the enzymes called allozymes or isozymes. Enzymes are the proteins consisting of amino acids and specific change in an amino acid may be due to DNA mutation leads to the change in the shape and charge of a molecule. These allelic variations can be detected by gel electrophoresis and subsequent enzyme-specific stains. Allozyme analysis is quick and easy to use as it does not require DNA extraction, sequence information and primers. The major disadvantage of biochemical markers is the low

Review of literature level of polymorphism and less abundance and these may be affected by the environmental conditions.

3. DNA markers: or molecular markers represent the sites of variation in the DNA of individual organisms. These markers are usually located in the non-coding region of DNA and are selectively neutral. DNA markers are not affected by the environmental conditions and/or the developmental stage of the plant (Winter and Kahl, 1995). DNA markers are present in abundance across the genome and are the most widely used form of the genetic markers.

DNA markers can be hybridisation, PCR and whole genome sequence-based. A range of DNA based molecular techniques are available to detect polymorphism at the DNA level and the choice is based on the type of study to be undertaken and the cost involved. DNA markers technology became practical in the 1980s with the first time development of the technique, Restriction Fragment Length Polymorphism (RFLP, Botstein et al., 1980). RFLPs are non- PCR based genetic markers and are simply inherited naturally occurring Mendelian characters. RFLPs are randomly distributed across the genome and present in abundance. RFLPs are codominant markers and highly reproducible but requires large quantities of high quality DNA. The invention of versatile PCR technique in the mid 1980s, lead to the development of many other molecular markers like; Amplified Fragment Length Polymorphism (AFLP, Vos et al., 1995), Random Amplified Polymorphic DNA (RAPD, Williams et al., 1990), Simple Sequence Repeats (SSRs, Tautz and Renz, 1984) and Single Nucleotide Polymorphisms (SNPs, Brookes, 1999). AFLPs and SSRs analysis require low quantities of moderate quality DNA and markers are highly reproducible. SSRs or microsatellites are codominant markers that generate maximum genetic information and are inherited according to the Mendelian laws (Liu et al. 1999). According to Chelkowski et al., 2003, AFLP and SSR markers have been frequently used in the mapping of loci conferring disease resistance in barley. Other molecular markers like Selectively Amplified Microsatellites (SAM) and Sequence-Specific Amplification Polymorphism (S-SAP) have been used to study the genetic variation among the cereal rust pathogens (Keiper et al. 2003).

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DNA markers have enormous potential in marker assisted selections (MAS) in plant breeding programs (Collard et al. 2005). Collard and Mackill (2008) has reported five different considerations in the use of molecular makers for MAS:

1. Reliability; a marker should be tightly linked, preferably < 5 cM from the target gene. 2. DNA quantity and quality; ideally, a marker that requires less DNA of normal quality should be used. 3. Technical procedure; quick robust methods are desirable. 4. Level of polymorphism; a marker should be polymorphic and discriminate different genotypes. 5. Cost; a marker assay must be cost effective.

Use of molecular markers in studying the genetic diversity of rust pathogens Recently developed molecular techniques can be used to understand genetic variation within rust pathogens. PCR based molecular tools like AFLPs, SAMs and S-SAPs can be helpful in understanding the genetic structure of cereal rust pathogen populations (Keiper et al. 2003). Using AFLPs and RAPDs, no polymorphism was detected among and between Australia and New Zealand isolates of P.striiformis f. sp. tritici but the same primers showed 5-15% polymorphic fragments between an isolate from UK, an isolate from Denmark and one from Colombia (Steele et al. 2001). Sun et al. (2007) examined genetic diversity of P. hordei using AFLP and found that isolates that grouped according to their virulence patterns and common regions of collection were not related based on AFLP genotypes.

Population studies of rust pathogens have been carried out using microsatellites. Recent studies of isolates of P. triticina collected from the Middle East and Central Asia using 23 SSRs showed that isolates from each region were different, indicating a lack of migration of P. triticina between the regions (Kolmer et al. 2011). In another recent study, four different pts of P. gramanis f. sp. tritici within the “Ug99” (TTKSK) lineage (TTKSF, TTKSP and PTKST) were evaluated using SSR markers, where these all four pts shared only 31% similarity with other South African pts and it was concluded that pts TTKSP and PTKST arose in South Africa as a result of exotic introduction (Visser et al. 2011).

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PCR-fingerprinting technique involving microsatellites (GACA)4 and (GTG)5 and minisatellite M13 derived from the core sequence of the wild type phase M13 bacteria was successfully used to study variability in fungal pathogens (Meyer et al. 2001). Gomez et al. (2006) studied polymorphism among 44 (25 Australasian and 19 European) isolates of Phragmidium violaceum, (rust fungus introduced to Australia for the biological control of European blackberry) using selective amplification of microsatellite polymorphic loci

(SAMPL) with primer pairs (GACA)4 + H-G and R1 + H-G and found European isolates more diverse than Australasian isolates, with 37 and 22% polymorphic loci respectively.

Sources of rust resistance in barley Some of the major or seedling resistance genes Rph1 to Rph19 have been deployed to control leaf rust in barley. Virulence is however known for many of these genes in different parts of the world (Griffey et al. 1994; Park et al. 2002). The seedling resistance genes Rph7, Rph11, Rph14, Rph15 and Rph18 remain effective in Australia (Park 2003; Park 2010). Out of 19 catalogued Rph genes (Table 2.7), three have been reported to be alleles of other genes. Seedling gene Rph5 is allelic to Rph6 (Zhong et al. 2003), Rph9 is allelic to Rph12 (Borovkova et al. 1998) and Rph15 is allelic to Rph16 (Weerasena et al. 2004). The seedling genes Rph1-9, Rph12, Rph19 and the adult plant resistance (APR) gene Rph20 were all described from H. vulgare, genes Rph10, Rph11, Rph13, Rph14, Rph15 and Rph16 from H. spontaneum and Rph17 and Rph18 from H. bulbosum.

As major seedling resistance genes can be easily overcome by new pts of P. hordei, APR was suggested by Park et al. (2003) and Golegaonkar et al. (2009b) as an alternative approach to develop leaf rust resistant barley cultivars. Golegaonkar et al. (2009b) identified a range of sources of APR to P. hordei in barley and further characterisation of these resistances is now needed to assist their use in breeding leaf rust resistant barley varieties. Recently in Australia, the APR gene Rph20 in barley cultivar Flagship and a QTL (quantitative trait loci) qRphND in line ND24260 were mapped to chromosomes 5HS and 6HL, respectively (Hickey et al. 2011). Molecular markers EBmag0833 and bPb-0837 linked to Rph20 were reported and the latter proposed for marker assisted selection of this gene (Liu et al. 2010).

Partial resistance causes slow rusting in the form of reduced infection frequency, increased latent period and reduced sporulation (Van der Plank 1963). This has been reported as 30

Review of literature conferring resistance to P. hordei in barley (Parlevliet 1976; Parlevliet 1979; Parlevliet and Ommerson 1975). Partial resistance to P. hordei in the barley cultivar Vada was mapped as seven QTLs, three of which were effective at seedling growth stages (Rphq1, Rphq2, Rphq3), and four of which were effective at adult plant growth stages (Rphq2, Rphq3, Rphq4 and Rphq5) (Qi et al. 1998). Under field conditions partial resistance is often difficult to select as all genotypes produce a susceptible reaction type (Parlevliet and Ommerson 1975). The relationship of this resistance to the APR identified by Golegaonkar et al. (2009b) remains unclear.

Stem rust was a serious problem on barley in the United States and Canada before the deployment of the stem rust resistance gene Rpg1 in 1942 (Brueggeman et al. 2002). Rpg1 remained effective and protected North American barley crops for more than 65 years (Mirlohi et al. 2008). The stem rust resistance genes Rpg1, Rpg2, Rpg3 and rpg4 are effective against P. graminis f. sp. tritici and RpgU, RpgBH and RpgQ (Rpg5) against P. graminis f. sp. secalis (Sun and Steffenson 2005). In Australia, Rpg1 cannot be detected with isolates of P. graminis and the effectiveness of this gene in providing field resistance is unclear (Park et al. 2009). Virulence for Rpg1 is present in North America in race QCCJ of P. graminis f. sp. tritici (Sun and Steffenson 2005) and stem rust pt “Ug99”, which threatens barley production in Eastern Africa, is also virulent for Rpg1 (Steffenson et al. 2009; Steffenson et al. 2007). This has led to the efforts to transfer a new stem rust resistance gene Rpg6 to cultivated barley (H. vulgare subsp. vulgare) from the wild relative H. bulbosum (Fetch et al. 2009).

Apart from cultivars Clipper, Maritime, Skiff and Tantangara, most Australian barley cultivars possess seedling resistance against BGYR, however, the genetic basis of this resistance is unknown (Park et al. 2009). Most Australian barley cultivars were susceptible to race 24 of Psh when tested in Mexico (Wellings et al. 2000), indicating that Psh is a serious exotic threat to the Australian barley industry.

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Table 2.7 Catalogued genes for resistance to Puccinia hordei Gene Chromosomal Reference Location Rph1 2H (Tan 1978; Tuleen and McDaniel 1971) Rph2 5HS (Borovkova et al. 1997; Franckowiak et al. 1997) Rph3 7HL (Jin et al. 1993) Rph4 1HS (Tan 1978; Tuleen and McDaniel 1971) Rph5 3HS (Mammadov et al. 2003; Tan 1978; Tuleen and McDaniel 1971) Rph6 3HS (Zhong et al. 2003) Rph7 3HS (Brunner et al. 2000; Graner et al. 2000) Rph8 ? Rph9 5HL (Borovkova et al. 1998) Rph10 3HL (Feuerstein et al. 1990) Rph11 6HL (Feuerstein et al. 1990) Rph12 5HL (Borovkova et al. 1998; Jin et al. 1993) Rph13* 3H (Jin et al. 1996; Sun 2007) Rph14 2HS (Golegaonkar et al. 2009a; Jin et al. 1996) Rph15 2HS (Weerasena et al. 2004) Rph16 2HS (Ivandic et al. 1998) Rph17 2HS (Pickering et al. 1998) Rph18 2HL (Pickering et al. 2000) Rph19 7HL (Park and Karakousis 2002) Rph20 5HS (Hickey et al. 2011) Source: (Franckowiak et al. 1997). *Previously mapped to 5HL (Jin et al. 1996)

Genetic analyses for mapping the genes of disease resistance Prior to the development of molecular markers, cytogenetic analysis was often used in the genetic analyses of important genes. According to McIntosh et al. (1995a), the success of this procedure depends upon the availability of suitable aneuploid stocks. Aneuploid plants have an abnormal number of chromosomes instead of normal diploid (2n) number. Sears (1954) produced complete sets of monosomics, nullisomics, trisomics and tetrasomics of the wheat cultivar Chinese Spring. Monosomics are diploids lacking a single chromosome (2n - 1) and nullisomic diploids lack a complete set of a chromosome (2n - 2). Trisomic diploids have an additional chromosome (2n + 1), while tetrasomics have a complete set of an additional chromosome (2n + 2). Aneuploids are very useful in locating genes for rust resistance as all 32

Review of literature the genes located on additional and missing chromosome/s are revealed by the phenotype of the particular aneuploid stock when compared to a normal diploid stock.

Various methods have been used to characterise genes providing resistance to leaf rust in barley. The genes Rph1, Rph4 and Rph5 (Tan 1978; Tuleen and McDaniel 1971) were located on chromosomes 2H, 1H and 3H, respectively, through trisomic analyses. Using linked morphological markers, Jin et al. (1993) mapped leaf rust resistance genes Rph3 and Rph12 on the long arms of chromosomes 7H and 5H, respectively. Seedling genes Rph10 and Rph11 were positioned on chromosomes 3H and 6H, respectively, using linkage with isozyme markers (Feuerstein et al. 1990). The use of aneuploids for genetic analyses has drawbacks that include sterility, poor germination, abnormal agronomic characters and the time involved in the development. Morphological and biochemical markers can be affected by environmental factors and plant growth stage (Winter and Kahl 1995).

Conventional methods of genetic analyses of rust resistance have now been largely replaced by DNA-based molecular techniques. Molecular markers that are located close to the gene of interest are identified and genetic maps are constructed based on marker data. The PCR based AFLP and SSR molecular marker systems have been used commonly in mapping disease resistance genes in barley (Williams 2003). For example, the seedling gene Rph15 was positioned on chromosome 2HS using these markers (Weerasena et al. 2004). Restriction Fragment Length Polymorphism (RFLP) markers were used to map the seedling gene Rph2 on chromosome 5H (Borovkova et al. 1997), Rph5 on 3HS (Mammadov et al. 2003), Rph6 on 3HS (Zhong et al. 2003), Rph7 on 3HS (Brunner et al. 2000; Graner et al. 2000), Rph16 (Ivandic et al. 1998) and Rph17 (Pickering et al. 1998) both on chromosome 2HS and Rph18 (Pickering et al. 2000) on chromosome 2HL. RAPD markers were used to map the alleles Rph9 and Rph12 on chromosome 5HL (Borovkova et al. 1998). Gene Rph19 was mapped to chromosome 7HL using SSR markers (Park and Karakousis 2002). Recently, Diversity Array Technology® (DArT) (Wenzel et al. 2007) markers were used to position Rph13 on chromosome 3H (Sun 2007), Rph14 on chromosome 2HS (Golegaonkar et al. 2009a) and the APR gene Rph20 on chromosome 5HS (Hickey et al. 2011). Previously, Rph13 was proposed to be present at chromosome 5HL based on the linkage analysis with Rph9 with a 30.4 ± 4.5 % of recombination fraction (Jin et al. 1996).

Review of literature

Steps involved in the genetic analyses and mapping of genes for disease resistance

To find the chromosomal location of gene/s for resistance, conventional genetic analysis involving intercrossing of a resistant and a susceptible genotype is followed by the phenotypic and genotypic study of individual plants and families in the following generations. Molecular markers are applied to the genetic/mapping populations and data is used to create genetic map/s of the chromosome/s locating the gene/s of interest. The following four major steps are involved in the genetic analyses of seedling resistance.

1. Development of genetic/mapping populations For genetic studies, segregating plant populations are developed by crossing two different parents with different traits. For pleminary genetic studies population size varies from 50 to 250 individuals per population (Mohan et al. 1997). Resistant by susceptible and resistant by resistant crosses are made to develop F1 hybrids to study the inheritance of resistance and allelic relationships among resistance genes. In the back cross (BC) approach, the F1

(resistant x susceptible) is again crossed with the susceptible parent. Individual F1s and

BC1F1s are harvested to develop the subsequent F2 population. These F2s are the simplest form of the mapping populations and are easy to develop. Self pollination of F2 individuals allows the construction of recombinanat inbred lines (RILs) viz. F2s, F3s and upto F8s. Usually six to eight generations are required to construct RILs and longer time involved in the process is a major disadvantage (Collard et al. 2005). Self pollinating F2 populations are preferred over BC populations as more labour and time is required to produce the maximum number of BC1F1 seeds. The BC1F2 generation lacks homozygous resistant (HR) genotypes as only heterozygotes (SEG) and homozygous susceptible (HS) lines are produced. The advantage of RILs is having homozygous types (HR and HS) of individuals each containing a unique combination of chromosomal segments from the original parents. In Doubled Haploid populations (DHs), homozygosity is acquired in the first generation through the induction of chromosomal doubling. The DH approach however requires more resources and is only possible in the species which are amenable to tissues culture.

2. Phenotyping of populations

F1s are tested to determine if the gene being investigated has a dominant or recessive mode of action. Individual F2 or F3 or BC1F2 lines are phenotyped using a suitable rust pt. Observed 34

Review of literature segregation ratios of phenotypes (number of HR, SEG and HS) in a population are used, according to the "Mendel's Law of Segregation", to establish the number/s of gene/s contributed by resistant parent. The F2 segregation ratios can be used to estimate the number of segregating loci, but F3 segregation ratios are preferred over F2 segregation data as they provide simultaneous genotypic classifications of multiple plants and a determination of the genotype of each F2 plant (Roelfs et al. 1992). The minimum number of individuals per population required to resolve segregating ratios for increasing number of genes can be calculated from basic genetic models (Hanson 1959).

3. Genotyping of populations The parents are genotyped using polymorphic molecular markers and the markers that differentiate the parents are used to genotype the mapping population(s). Bulked segregant analysis (BSA) (Michelmore et al. 1991) is commonly performed to find molecular markers linked to the loci/genes responsible for disease resistance, including PCR based molecular markers such as SSRs, STS, RFLPs and AFLPs. In BSA, genomic DNA of both parents, homozygous resistant and homozygous susceptible lines from a segregating population are pooled separately and screened for polymorphism using molecular markers. The markers that are polymorphic between the parents and between resistant and susceptible bulks are then applied across the whole population and data is generated in the form of presence or absence of markers. Hybridisation based DArT markers (Wenzel et al. 2007) and SSRs in a multiplex-ready PCR technique (Hayden et al. 2008a) were used in BSA to identify a number of markers associated with target rust resistance loci (Bansal et al. 2010; Golegaonkar 2007; Mammadov et al. 2003; Zhong et al. 2003).

4. Mapping of gene/s Based on the data generated from polymorphic markers, a partial map/s of a chromosome/s is constructed to locate the gene of resistance. A linkage analysis is performed to calculate recombination frequency between markers linked to the resistance gene. Linkage between marker loci is estimated by converting recombination frequency into genetic distance. The unit of genetic distance is called the centi-Morgan (cM), named after Professor T. H. Morgan. Kosambi (1944) mapping functions are commonly used to measure the genetic distance in cM. Based on different types of DNA markers, several genetic maps of barley chromosomes are available in the public domain (Varshney et al. 2007; Varshney et al. 2004). Computer 35

Review of literature programmes like Map Manager QTXb20 version 0.30 (Manly et al. 2001) can be used to perform a marker linkage analysis. Based on the recombination fractions calculated with Map Manager, another computer programme Map Chart 2.2 can be used to draw a consensus map of barley chromosome/s locating the gene/s of interest and the flanking markers.

P. hordei resistance in cultivar Ricardo

CHAPTER III Genetic analyses and molecular mapping of an uncharacterised seedling gene conferring resistance to Puccinia hordei in the barley cultivar Ricardo

Abstract

Ricardo/Gus F3 and BC1F2 populations were studied to determine the inheritance of unknown seedling resistance (USR) to leaf rust in the barley cultivar Ricardo. Genetic studies revealed that the USR in Ricardo was conferred by a single dominant gene, which was tentatively designated RphRic. Bulk segregant analysis (BSA) of the F3 population using a multiplex- ready PCR technique mapped RphRic on chromosome 4H. Given that this is the first gene for leaf rust resistance mapped on chromosome 4H, it was catalogued as Rph21. The presence of an additional gene, Rph2, in Ricardo, was confirmed by test of allelism and the Rph2-linked marker ITS1. An uncharacterised adult plant resistance (UAPR) against P. hordei, also found in Ricardo, appeared to be distinct from Rph20 based on marker analysis. Both Rph21 and the UAPR identified represent new useful sources of resistance to P. hordei.

Keywords: Barley leaf rust, Rph21, Rph2, Hordeum vulgare, Ricardo, adult plant resistance

Introduction Cultivated barley (Hordeum vulgare L. subsp. vulgare) is an important cereal crop worldwide (Schulte et al. 2009) and a multi-billion dollar grain industry in Australia. Leaf rust can be one of the most devastating diseases to affectbarley growth and productivity (Park 2003). Leaf rust is caused by the fungus Puccinia hordei Otth. and affects barley production in many parts of the world (Clifford 1985). Disease epidemics have caused significant yield losses globally (Arnst et al. 1979; Cotterill et al. 1992; Griffey et al. 1994; Melville et al. 1976), including in Australia (Murray and Brennan 2010).

Cereal rusts are often managed by developing and growing resistant cultivars (Steffenson et al. 2007) and Australia has a strong history of breeding cereal cultivars to manage rust diseases (McIntosh 2007). A total of 19 major seedling genes (Rph1 to Rph19) conferring resistance to P. hordei have been characterised in barley (Weerasena et al. 2004). Many of these major genes have been overcome by new pathotypes (pts) of P. hordei in Australia and elsewhere (Park 2003). Because only the seedling resistance genes Rph7, Rph11, Rph14, 37

P. hordei resistance in cultivar Ricardo

Rph15 and Rph18 (Park 2003; Park 2010) and APR gene Rph20 remain effective in Australia (Park 2010 unpublished), there is a dire need to find new sources of resistance to P. hordei.

Genetic loci conferring seedling resistance to P. hordei have been characterised using molecular, isozyme and morphological markers and also through trisomic analysis. Designated Rph genes are located on all H. vulgare chromosomes except 4H (detailed in Chapter II Table 2.7). Of the designated loci Rph1 to Rph19, six are reported to involve alleles; Rph5 and Rph6 (Zhong et al. 2003), Rph9 and Rph12 (Borovkova et al. 1998) and Rph15 and Rph16 (Weerasena et al. 2004). The seedling gene Rph2 was mapped on chromosome 5HS (Borovkova et al. 1997; Franckowiak et al. 1997). According to Franckowiak et al. (1997), Rph2 is a complex locus comprising many alleles. In addition to Rph2, the barley cultivar ‘Reka 1’ was reported to carry a second leaf rust resistance gene (Tan 1977), which was later characterised, mapped and designated as Rph19 (Park and Karakousis 2002). Gene Rph2 is reported to be allelic to RphQ, as no segregation was observed in F2 populations derived from the crosses between barley line Q21861 (RphQ) and sources of Rph2 (Peruvian, PI531840 and PI531841) when inoculated with an Rph2 avirulent P. hordei pt ND8702 (Borovkova et al. 1997). Pathotypes of P. hordei with different pathogenicities to Rph genes have been used to postulate new sources of resistance in barley germplasm (Cotterill et al. 1992; Golegaonkar et al. 2009b; Park and Karakousis 2002; Tan 1977). In other studies, recombinant inbred lines (RILs) and DNA markers were used to locate loci conferring resistance to leaf rust to chromosomes in H. vulgare, for example Rph2 (Borovkova et al. 1997; Franckowiak et al. 1997), Rph5 (Mammadov et al. 2003), Rph7 (Brunner et al. 2000; Graner et al. 2000) and Rph19 (Park and Karakousis 2002).

The use of molecular markers has fast tracked breeding programs by permitting marker assisted selection (Langridge and Barr 2003). Microsatellites or simple sequence repeats (SSRs) are the preferred molecular markers in cereal research due to their highly polymorphic and co-dominant characteristics (Gupta and Varshney 2000). Michelmore et al. (1991) developed the “bulk segregant analysis” (BSA) approach for the rapid detection of markers linked to specific genes. Golegaonkar et al. (2009a) performed BSA on parents and non segregating resistant and susceptible F3 bulks using Diversity Array Technology (DArT) markers, to map Rph14 on chromosome 2H. Based on BSA, a sequence tagged site (STS)

P. hordei resistance in cultivar Ricardo marker ITS1 (derived from Rrn2) was found to be closely linked (1.6 centi-Morgans (cM)) to RphQ, an allele of Rph2 (Borovkova et al. 1997).

Hayden et al. (2008a) developed an SSR-based multiplex-ready PCR technique for genotyping barley and wheat. Under standardised PCR conditions, the multiplex-ready PCR technique can achieve a success rate of up to 92% for the amplification of published sequences. The technique is highly suited for marker assisted selection in plant breeding programs (Hayden et al. 2008a; Hayden et al. 2008b). High density SSR-based linkage maps of barley are available (Ramsay et al. 2000; Varshney et al. 2007), which have increased the probability of finding polymorphic markers for specific chromosomal locations. The multiplex-ready PCR technique has been used widely to perform BSA on stripe rust resistance in bread wheat (Bansal et al. 2010) and of leaf rust resistance in durum wheat (Singh et al. 2010).

Adult plant resistance (APR) conferred by minor gene(s) and or by quantitative trait loci (QTL) is considered to be more durable than major (seedling) gene or qualitative resistance. Sources of APR to leaf rust in barley were characterised by Golegaonkar et al. (2009b) and the first gene conferring APR to leaf rust in barley, Rph20, was mapped to chromosome 5H (Hickey et al. 2011) and linked markers were reported for its selection in breeding programs (Liu et al. 2010).

Ricardo, a land race believed to have originated from Uruguay, carries Rph2 (Pa2) (Henderson 1945; Moseman and Roan 1959; Zloten 1952) and an uncharacterised seedling gene (Park unpublished; Stöcker 1983; Wallwork et al. 1992; Yahyaoui et al. 1988). Under different environmental conditions, Ricardo produced variable low infection types (ITs) against different Australian pts of P. hordei in the greenhouse (Park unpublished). Ricardo is reported to be highly resistant against P. hordei under field conditions and showed environmental sensitivity in the expression of seedling resistance under greenhouse conditions (Golegaonkar 2007). In the present study, the inheritance of seedling resistance and APR to leaf rust in Ricardo was investigated and the uncharacterised seedling resistance reported by previous workers was mapped. Allelic studies were then carried out to confirm the presence of Rph2 in Ricardo.

P. hordei resistance in cultivar Ricardo

Materials and methods

Plant material Seed of Ricardo, Gus (leaf rust susceptible cultivar) and control lines with known Rph genes was obtained from the germplasm collection held at the Plant Breeding Institute (PBI) of the University of Sydney. Originally, Ricardo (Variety: C.I. 6306; Pedigree: unknown) was sourced from the College of Agriculture, Davis California by Dr E. P. Baker at the University of Sydney. The lines Sudan (Rph1), Peruvian (Rph2), Estate (Rph3), Gold (Rph4), Magnif 104 (Rph5), Bolivia (Rph2 + 6), Cebada Capa (Rph7), Egypt 4 (Rph8), Abyssinian (Rph9), ClipperBC8 (Rph10), ClipperBC67 (Rph11), Triumph (Rph12), PI 531849 (Rph13), PI 584760 (Rph14), Bowman (Rph15), 81882/BS1 (Rph17), 38P18/8/1/10 (Rph18) and Prior (Rph19) were used to develop genetic populations. Differential lines used as controls are listed in Appendix D1.

Pathogen material Ten pts of P. hordei were selected for testing from the cereal rust collection maintained at the PBI. The pathogenicities and passport information for these pts is detailed in Table 3.1.

Genetic populations A field nursery of all parents including Ricardo, Gus and sources of Rph1 to Rph19, was raised at Karalee, PBI Cobbitty. Three replications were sown at 10-day intervals starting from mid June 2007. All lines were hand-sown as half meter rows using 30 cm spacing (30– 40 seeds/line). Plots were hand weeded 2–3 times during the season. Four weeks after sowing, plots were fertilised using granular urea (Incitec Pivot® w/w 46% nitrogen) @ 100 kg/hectare followed by irrigation. Plots were irrigated once a week or as required, using fixed sprinklers.

Table 3.1 Details of Puccinia hordei pathotypes used in the present study Pathotype Culture no. Virulence* 243P- 487 Rph1, Rph2, Rph6, Rph8 253P- 490 Rph1, Rph2, Rph4, Rph6, Rph8 200P- 518 Rph8 5610P+ 520 Rph4, Rph8, Rph9, Rph10, Rph12, Rph19

P. hordei resistance in cultivar Ricardo

5653P+ +Rph13 542 Rph1, Rph2, Rph4, Rph6, Rph8, Rph9, Rph10, Rph12, Rph13, Rph19 5453P- 560 Rph1, Rph2, Rph4, Rph6, Rph9, Rph10, Rph12 5652P+ 561 Rph2, Rph4, Rph6, Rph8, Rph9, Rph10, Rph12, Rph19 4673P+ 562 Rph1, Rph2, Rph4, Rph5, Rph6, Rph8, Rph9, Rph12, Rph19 5653P+ 584 Rph1, Rph2, Rph4, Rph6, Rph8, Rph9, Rph10, Rph12, Rph19 5457P+ 612 Rph1, Rph2, Rph3, Rph4, Rph6, Rph9, Rph10, Rph12, Rph19 *with respect to the resistance genes listed in Appendix D1

Genetic populations were developed by crossing Ricardo with all sources of Rph1 to Rph19 and the susceptible cultivar Gus. Inflorescences, just emerging out of boots, were emasculated using fine tweezers and covered immediately using labelled white glossy paper bags (15 cm x 5 cm) that were sealed around the rachis using paper clips. Pollinations were carried out two days after emasculation during the morning, followed by a second dose of pollen on the following morning. Three to four single heads were used for each cross using the maximum number of florets in each head. All equipment was sterilised using 70% ethanol before and after each emasculation and pollination. On maturity, individual crosses were harvested. Ten to 15 bulked heads of each parent were also harvested in paper bags, kept in a dehumidified (30-35% RH) and temperature (12°C) controlled seed store for two weeks and then threshed using a small motorised thresher. Crosses were hand threshed to avoid the loss of F1 seeds. Depending upon amount, seeds were stored in either 9 x 5 cm or 13 x 8 cm paper packets in a seed room fitted with a dehumidifier and maintained at 12°C until needed.

Sixteen F1 seeds from the cross Ricardo/Gus and 5–9 seeds from all other crosses were sown in the greenhouse in 9 cm-diameter pots filled with a mixture of fine bark and coarse sand and fertilised using Aquasol® (100 gm per 10 litre of water per 200 pots) prior to sowing. Following sowing, pots were kept in a growth room at 20 ± 2°C for germination. Seven-day old seedlings were fertilised with granular urea (Incitec Pivot® w/w 46% nitrogen; 50 gm per 10 litre of water per 200 pots). At the three and a half leaf stage, single plants were transplanted to medium sized (18 cm diameter) pots and maintained at 17 ± 2oC to promote tillering. An extra dose of urea was applied 7 days after transplanting. After 7 weeks, the pots were transferred to a naturally well lit tunnel/polythene house designed with an automatic irrigation system.

P. hordei resistance in cultivar Ricardo

Eight Ricardo/Gus F1 plants were back crossed to Gus with the intention of producing as many BC1F1 seeds as possible. Upon maturity, single plants were harvested and threshed individually. The progeny from each cross were space (30 cm) planted in long rows at

Karalee field site of PBI. Field plots were maintained as stated previously and individual F2 and BC1F1 plants were harvested to raise F3 and BC1F2 populations, respectively.

Greenhouse testing

Determining conditions for the optimal expression of seedling resistance in Ricardo Previous studies showed that the low ITs produced by the USR (referred to hereafter as RphRic) in Ricardo varied with environmental conditions and the P. hordei pt used (Park unpublished). It was therefore decided to determine what conditions permitted optimal expression of RphRic. Four sets of Ricardo, Peruvian and Gus, along with all differential lines (Appendix D1), were sown in the greenhouse in 9 cm diameter pots. Four clumps (parents and controls) and five clumps (differentials) per pot (8–10 seeds per clump) were sown according to the methods described earlier. The experiment was replicated three times with four sets per replicate. Four pts (viz. 5457P+, 5652P+, 4673P+ and 200P-) of P. hordei were used.

Nine to 10 day old seedlings at the one and a half leaf growth stage were inoculated in the greenhouse. The seedlings were moved to an enclosed chamber and urediniospores (10 to 12 mg/10 ml/200 pots) were suspended in a light mineral oil (Shellsol®, Mobil Oil) and sprayed over seedlings using an aerosol hydrocarbon propellant pressure pack. The chamber door was kept closed for 5 minutes to allow urediniospores to settle on the leaves completely. Spray nozzle fittings were stored in 70% ethanol and rinsed thoroughly with tap water before each inoculation to prevent cross contamination. In addition, the inoculation chamber was washed thoroughly with pressurised tap water following each inoculation. Leaf rust-inoculated seedlings were incubated for 24 hours at room temperature in a dark chamber where continuous mist was created by an ultrasonic humidifier. After incubation, seedlings were moved to naturally lit microclimate rooms maintained at 17 ± 2oC, 23 ± 2oC or 27 ± 2oC. Infection type responses were scored 10–12 days after inoculation according to the 0–4 scale used by Park and Karakousis (2002), described in Appendix 1.

P. hordei resistance in cultivar Ricardo

Ricardo, Peruvian and Gus, along with all differential lines (Appendix D1), were also tested in the greenhouse against 10 pts of P. hordei (Table 3.1) according to the methods described earlier.

Inheritance of seedling resistance in Ricardo

A total of 200 F3 lines (Ricardo/Gus), two populations comprising of 79 and 68 F3 lines

(Ricardo/Peruvian), 130 BC1F2 lines (Ricardo/Gus//Gus), parents Ricardo, Gus and Peruvian and all differential lines (Appendix D1), were sown in the greenhouse in 9-cm diameter pots with 30 to 35 seeds per F3 and BC1F2 line. Four clumps (parents and controls) and five clumps (differentials) per 9 cm diameter pot using 8–10 seeds per clump were sown according to the methods described earlier. Seedlings were inoculated with P. hordei pt 5457P+ at the one and half leaf stage (9–10 day old) and incubated as described earlier. After incubation, seedlings were transferred to naturally well lit microclimate rooms maintained at 23 ± 2oC. Disease infections were scored 10–12 days after inoculation according to the 0–4 scale.

Field tests

Evaluation of adult plant resistance in Ricardo + 130 BC1F2 lines (Ricardo/Gus//Gus) were tested with pt 5457P in the greenhouse using the methods described above. Susceptible plants from segregating lines were tagged with red wire rings. All lines were then transferred to a stand out area for two weeks, after which they st nd were transplanted to the Karalee field site on 1 and 2 June 2010. All 200 F3 lines from the cross Ricardo/Gus were hand sown (30–40 seeds space planted per 3.5 m row) directly into the field at Karalee on 7th to 9th June 2010, along with the parents Ricardo and Gus, which were sown as one meter rows. A susceptible rust spreader (Gus) row was sown after every two F3 lines. Plots were maintained as described earlier.

P. hordei resistance in cultivar Ricardo second inoculations, hot spots were established by watering and covering small areas of the rust spreader lines with plastic hoods overnight to ensure adequate dew formation in case natural dew formation did not occur. Rust responses were scored at regular intervals (seven days) from the onset of flag leaf emergence on the basis of disease severity and host response using a modified Cobb’s scale (Peterson et al. 1948), as described in Appendix 2. Three readings were taken from F3 and BC1F2 lines and the parents Ricardo and Gus and data were analysed based on the final reading to avoid any error if epidemic wasn't homogenous during first two readings.

Molecular analyses and mapping of seedling resistance in Ricardo

DNA extraction Genomic DNA was extracted from the leaf tissues of seedlings of Ricardo, Gus, Peruvian,

Pompadour, Flagship, Stirling and all lines of Ricardo/Gus F3 population. Parental and control lines were sown as four clumps per pot in the greenhouse as described earlier. All F3 lines were pruned after testing with pt 5457P+ and allowed to regrow in the greenhouse. Leaf tissue was collected from the regrowth and from actively growing seedlings 8–10 days after sowing. A 15 to 20 mm sample of leaf tissue was collected into 2 ml Eppendorf tubes from at least 10 to 15 plants per F3 line, parents and controls. The tubes were kept for 72 hours above silica beads to dry the leaf tissue. Two small stainless steel ball bearings were added per tube and dried leaves were crushed to powder using a Retsch MM300 Mixer Mill (Retsch, Germany) for 3 min at 25 rpm. Pre warmed (65°C) 700 µl of extraction buffer [50 mM Tris- pH 8.0, 10 mM EDTA-pH 8.0, 100 mM NaCl, SDS 1% (w/v), 10 mM ß-mercaptoethanol] was added per tube. Samples were incubated for 10 min at 65°C and 150 µl of 3 M K-acetate (pH 5.2) was added per tube. Tubes were shaken vigorously and kept in freezer (-20°C) for 15 min. Samples were centrifuged for 15 min at 12,000 rpm and 650 µl of supernatant was transferred to new 1.5 ml tubes. An equal volume (750 µl) of chilled (-20°C) isopropanol was added per tube and the supernatant was mixed thoroughly by inverting tubes several times. The tubes were placed in a freezer (-20°C) for 10 min to precipitate DNA and then centrifuged at 10,000 rpm for 10 min to pellet the DNA. The supernatant was discarded and the pellet was washed with 500 µl of 70% ethanol. DNA pellet was air dried and re- suspended in 200 µl of 10 mM Tris-HCl (pH 8.0). Rnase A @ 20 µl per 40 ml of 10 mM Tris-HCl was added before the re-suspension of DNA pellet. Tubes were kept in an oven 44

P. hordei resistance in cultivar Ricardo

(37°C) for 2 hrs to dissolve the DNA pellet properly. DNA was quantified using a Nanodrop ND-1000 spectrophotometer (Nanodrop® Technologies). Working dilutions of all samples

(50 ng/µl) using doubled distilled autoclaved water (ddH2O) were prepared from these stocks and stock DNA was stored in the freezer (-20°C).

Bulk segregant analysis (BSA)

The Ricardo/Gus F3 population, comprising 200 lines, was used for BSA analysis as described by Michelmore et al. (1991). Equal amounts of genomic DNA were pooled from

10 homozygous resistant and 10 homozygous susceptible F3 lines, to constitute resistant and susceptible bulks, respectively. Similar concentrations of genomic DNA were taken from both resistant and susceptible parents, Ricardo and Gus respectively.

The multiplex-ready PCR technique developed by Hayden et al. (2008a) was used to perform the BSA and genetic mapping of the locus conferring seedling resistance in Ricardo. The whole genome scan kit I, consisting of 488 published microsatellites (SSRs), was used to find marker-trait associations. BSA revealed 12 polymorphic markers on chromosome 4H, differentiating resistant and susceptible parents and bulks. Sequences of the linked markers EBmac0635, GBM1220, GBM1003, GBM1015, GBM1028 and GBM1044 (Varshney et al. 2007), HvBTAI0003 and HvHVO0003 (Hearnden et al. 2007), HVMLOE (Hayden et al. 2008a), EBmac0701 (Ramsay et al. 2000), HvPEPD1PR (Pillen et al. 2000) and Bmy1_INDEL6 (Hayden 2010, personal communication) are available in http://wheat.pw.usda.gov/cgi-bin/graingenes (GrainGenes 2010). These markers were genotyped on the whole Ricardo/Gus F3 population. PCR products were resolved on a GelScan2000 (Corbett Research) and ABI3730 DNA fragment analyser (Applied Biosystems). Using GeneMapper v4.0 (Applied Biosystems), SSR allele sizes were calculated for ABI3730 analysis (Hayden et al. 2008a).

Linkage analyses and construction of consensus map Map Manager QTXb20 version 0.30 (Manly et al. 2001) was used to perform the linkage analysis between the resistance gene and markers. Mapping functions as described by Kosambi (1944) were used to convert the recombination fraction percentages into map distances in cM. Based on the recombination fractions calculated with Map Manager,

P. hordei resistance in cultivar Ricardo software Map Chart 2.2 was used to predict the relationship between the gene and the markers.

Assaying the presence of Rph2 in Ricardo with marker ITS1 The STS marker ITS1, which shows close linkage (1.6 cM) to Rph2 (Borovkova et al. 1997), was used to genotype Gus, Ricardo and Peruvian. The sequences of the forward primer ITS5 and the reverse primer ITS2 are given in Table 3.2.

PCR reaction and profiles PCR was performed using 15 µl of reaction containing 2.0 µl of genomic DNA (50 ng), 1.0

µl of dNTPs (0.2 mM), 1.0 µl of 10x PCR buffer (Immobuffer, including 15 mM MgCl2), 2.25 µl of each forward and reverse primer (10 uM), 0.04 µl (5 u/µl) of Taq DNA (Immolase

DNA polymerase from Bioline) and 6.46 µl of ddH2O. PCR amplification profile comprised of an initial denaturation step at 95°C for 10 min, followed by 32 cycles of 60 s denaturation at 94°C, 60 s annealing at 55°C, 90 s extension at 72°C and a final extension step of 5 min at 72°C. Reaction was performed in a 96-well DNA theromocycler (Eppendorf Mastercycler, Germany).

Restriction of PCR product The restriction mixture contained 1.5 µl of 1x Buffer TaqI (10 mM Tris-HCl-pH 8.0, 5 mM

MgCl2, 100 mM NaCl and 0.1 mg/ml BSA), 0.5 µl (5 units) of restriction enzyme TaqI (5 x

3000 u, four base cutter 5’…T↓CGA…3’ and 3’…AGC↑T…5’) and 3.0 µl of ddH2O per 10 µl of PCR reaction. PCR products including restriction mix were incubated for 60 min at 65°C in DNA theromocycler (Eppendorf Mastercycler, Germany). Restriction enzyme and 1x Buffer TaqI were supplied by Fermantas Life Sciences.

Marker genotyping of Rph20 The recently published marker bPb-0837, linked to Rph20 at a distance of 0.7cM (Liu et al. 2010), was used to genotype the cultivars Ricardo, Pompadour, Stirling and Gus. The primer was synthesized and supplied by SIGMA (Sigma-Aldrich Australia) and sequence information is detailed in Table 3.2.

P. hordei resistance in cultivar Ricardo

PCR reaction and profiles Ten micro litres of PCR reaction contained 2.0 µl of genomic DNA (50 ng), 1.0 µl of dNTPs

(0.2 mM), 1.0 µl of 10x PCR buffer (Immobuffer, including 15 mM MgCl2), 0.25 µl of each forward and reverse primer (10 uM), 0.04 µl of Taq DNA (500U Immolase DNA polymerase from Bioline) and 5.46 µl of ddH2O. PCR amplification profile comprised of an initial denaturation step at 95°C for 10 min, followed by 35 cycles of 30 s denaturation at 94°C, 60 s annealing at 55°C, 60 s extension at 72°C and a final extension step of 5 min at 72°C.

Table 3.2 Details of molecular markers used to genotype Ricardo and control cultivars (Rph2 and Rph20) Size Name Sequence Marker Reference (bp) F 5’GGAAGTTAAAAGTCGTAACAAGG3’ 200, (Borovkova et al. ITS1 R 5’GCATCAATGAAGAACGCAGC 3’ STS 300 1997) bPb- F 5’GACACTTCGTGCCAGTTTGA 3’ DArT 245 (Liu et al. 2010) 0837 R 5’CCTCCCTCCCTCTTCTCAAC 3’

Separation of amplified PCR products

PCR products were mixed with 3.0 µl of formamide loading buffer [98% formamide, 10 mM EDTA (pH 8.0), 0.05% (wt/vol) Bromophenol blue and 0.05% xylene cyanol]. Two percent agarose gels were prepared by adding 2.0 gm agarose (Bioline) per 100 ml of 1x Tris-borate EDTA (TBE) buffer (90 mM Tris-borate + 2 mM EDTA-pH 8.0). For staining, 1.0 µl of ethidium bromide was added per 100 ml of gel solution. The gel solution was poured into moulds and allowed to cool for 40 min at room temperature. Eight to 10.0 µl of PCR product including loading buffer was loaded per well. One kb DNA ladder HyperLadder™ IV (Bioline) was used as reference. Electrophoresis was carried out at 80 to 110 V for 1 to 4 hrs depending upon the PCR product size. The separated bands were visualised under an ultra violet light unit fitted with a GelDoc-IT UVP Camera.

P. hordei resistance in cultivar Ricardo

Amplified PCR products were resolved on a GelScan2000 (Corbett Research). The 6% gel solution was prepared using 30 ml of acrylamide/bis-acrylamide (40% Amresco) and 60 ml of 2x TBE (Amresco) and the final volume was adjusted to 200 ml using ddH2O. In 18 ml of gel mix, 120 µl of APS (10% w/v) and 20 µl of TEMED was added and the solution mixed thoroughly. The gel solution was poured between two glass plates inserted with 0.2 mm thick comb and allowed to polymerise for 45 min, after which plates were washed with tap water to remove excess gel and wiped with lint free tissue. Dried plates were assembled into the gel scanner. Gels were pre run on 1200 V at 40°C for 30 min. To separate the PCR products using the GelScan2000, an equal volume of gel loading dye (98% formamide, 10 mM EDTA pH 8.0 and 0.25% bromophenol blue and xylene cyanol tracking dyes) was added to the PCR products and the mixture denatured for 5 min at 95°C on an Eppendorf Mastercycler followed by quick chill on ice. Electrophoresis was carried out at a constant power using 0.6x TBE buffer in the unit.

Chi squared analyses Goodness-of-fit of observed segregation ratios with the expected genetic ratios of phenotypic 2 data from F3 and BC1F2 populations was tested using Chi-squared (χ ) analysis. The two- tailed P values were computed from χ2 values using online calculator “QuickCalcs” by GraphPad software (http://www.graphpad.com/quickcalcs/pvalue1.cfm).

Results

Expression of seedling resistance in Ricardo To determine the optimal temperature for expression of RphRic, Ricardo was inoculated with three pathotypes of P. hordei, all of which were virulent for Rph2 and plants were then incubated at three post-inoculation temperatures. Ricardo expressed low ITs (11++C++ to 1++2C) against pt 5457P+ and slightly higher ITs against pts 4673P+ (1++2++ to 2++3C) and 5652P+ (12++C to 2++3-C) over a range of temperatures (17 ±2oC, 23 ±2oC and 27 ±2oC) under greenhouse conditions (Table 3.3). The lowest ITs of 11++C++ were noted against pt 5457P+ (Fig. 3.1) compared to 2++3C against 4673P+ and 2++3-C against pt 5652P+ at 23 ± 2oC. Ricardo produced a higher level of chlorosis at 23 ± 2oC in comparison to 17 ± 2oC and 27 ± 2oC when inoculated with pt 5457P+ (Fig. 3.1).

P. hordei resistance in cultivar Ricardo

Table 3.3 Infection types produced by Ricardo, Gus and Peruvian with different pathotypes of Puccinia hordei at three post-inoculation temperatures in the greenhouse under natural lighting Pathotype Temperature Ricardo Peruvian Gus 5457P+ 17 ± 2oC 1++2C 3+ 3+ 23 ± 2oC 11++C++ 3+ 3+ 27 ± 2oC 1++2C 3+ 3+ 5652P+ 17 ± 2oC 12++C 3+ 3+ 23 ± 2oC 2++3-C 3+ 3+ 27 ± 2oC 2++C 33+ 3+ 4673P+ 17 ± 2oC 1++2++ 33+ 3+ 23 ± 2oC 2++3C 3+ 3+ 27 ± 2oC 2++3C 3+ 3+ 200P- 17 ± 2oC ;N 0; 3+ 23 ± 2oC ;1=CN ;1=CN+ 3+ 27 ± 2oC ;C ;CN 3+ Pathotypes 5457P+, 5652P+ and 4673P+, while virulent on Rph2 (Peruvian), were avirulent on RphRic present in Ricardo and pt 200P- was avirulent on Rph2 and RphRic

P. hordei resistance in cultivar Ricardo

Fig. 3.1 Greenhouse infection types of Gus and Ricardo (in three sets) at three different post- inoculation temperatures against Puccinia hordei pathotype 5457P+

Multipathotype testing for seedling resistance To postulate the seedling resistance genes present in the parents Ricardo, Peruvian and Gus, each was tested against 10 different pts of P. hordei at 23 ±2oC in the greenhouse. Ricardo was resistant and Gus was susceptible to all 10 pts. Peruvian was susceptible to all pts except 200P- and 5610P+ (avirulent for Rph2). The results obtained were consistent with the presence of seedling resistance genes RphRic and Rph2 in Ricardo and Rph2 in Peruvian, where all differential lines produced the expected ITs against all used pts (Table 3.4).

P. hordei resistance in cultivar Ricardo

Table 3.4 Responses of Ricardo, Peruvian, Gus and differential lines in greenhouse tests with 10 pathotypes of Puccinia hordei 5653P+ Postulated Genotypes 243P- 253P- 200P- 5610P+ 5453P- 5652P+ 4673P+ 5653P+ 5457P+ +Rph13 Rph gene Ricardo 2+3-C 2+3-C ;1CN 12+CN 22+C 3-C 2++3-C 2++3C 122+C 11++C++ Rph2+RphRic Peruvian 33+ 33+C ;1CN ;1CN 33+ 33+ 3+ 33+ 33+ 3+ Rph2 DifferentialsD Gus/Susc. 3+ 3+ 3+ 3+ 3+ 3+ 3+ 3+ 3+ 3+ Nil Sudan/Rph1 3+ 3+ 0; 0;- 3+ 3+ ;N 3+ 3+ 3+ Rh1 Berg/Rph1 3+ 3+ 0;C 0;- 3+ 3+ ;N 3+ 3 3+ Rph1 Peruvian/Rph2 3+ 33+ ;1CN ;1CN 33+ 33+ 3+ 33+ 33+ 3+ Rph2 Gatam/RphGat 33+ 33+ 0;C 0;- 3+ ;11++C 33+ 3+ 33+ 3+ RphGat Reka 1/Rph2+RphP ;1+ ;1+N ;1=C ;C 3+ 3+ 3+ 3+ ;C 3+ Rph2+RphP Ricardo/Rph2+? 2+3-C 2+3-C ;1CN 12+C 22++C 3=C 2++3-C 12++3C 122+C 11++C++ Rph2+? Estate/Rph3 0; 0;- 0;= 0;-C 0;= 0;1=C ;-C ;- ;C 3+ Rph3 Gold/Rph4 ;1+ 3+ ;11- 3+ 3+ 3+ 3+ 3+ 33+ 3+ Rph4 PI 531849/Rph13 0;N 0;- 0;= 0;= 3+ ;1=C ;-N ;1N ;CN ;CN Rph13 Quinn/Rph2+Rph5 ;N ;N 0;C 0;- 0;- 0;= ;-N 3+ 0;- 0;C Rph2+Rph5 Magnif 104/Rph5 0;N ;-N 0;= 0;- ; 0; ;N 3+ 0;- 0;C Rph5 Bolivia/Rph2+Rph6 3+ 33+ ;C ;CN 3+ 3+ 33+ 33+ 3+ 3+ Rph2+Rph6 Cebada Capa/Rph7 0; 0;N 0;= 0;= ;CN+ ;N 0;N 0;N 0;= ;1=CN Rph7 PI 584760/Rph14 ;12- ;12+C 11++2+C 3 ;CN+ ;1-N ;12-N ;12N ;CN ;1-CN Rph14 Egypt 4/Rph8 3+ 3+ 3+ 3+ 3+ ;1CN 3+ 33+ 3+ 12++C Rph8

P. hordei resistance in cultivar Ricardo

Abyssinian/Rph9 233+ 233+ ;CN 3+ 33+ 3+ 3+ 3+ 3+ 3+ Rph9 Clipper BC8/Rph10 ;12+ ;12+ ;1=C 33+ 33+ 3+ 3+ 3 3+ 3+ Rph10 Clipper BC67/Rph11 ;12C ;1+2+C ;11+C 1++C ;1C 12+C 12+C 12+C 2++3 2++3C Rph11 Triumph/Rph12 22+ 12+C 0;CN 3+ 33+ 3+ 3+ 3+ 3+ 3+ Rph12 Prior/Rph19 ;1-N ;1-N 0;= 3+ 3+ 11-C 3+ 3+ 3+ 3+ Rph19 Cutter/Rph19 ;1N ;1N ;CN 0;- 33+ 11-N,3+ 3+ 3+ 3+ 3+ Rph19 Q21861/RphQ 33+ 3+ 0; ;C 3+ 3+ 3+ 3+ 33+ 3+ RphQ Cantala/RphC 12-C ;1-C 3+ 3+ 33+ 3+ 3+ 3+ 3+ 3+ RphC PI366444/RphB37 3 2++3C 3+ 3+ 3+ 3+ 3+ 3+ 3+ 3+ RphB37 Bowman+Rph15/Rph15 ;N ;N 0;C ;CN ;CN+ ;CN ;1-N ;N ;CN ;CN Rph15 81882/BS1/Rph17 ;1N ;1N ;C ;11+C ;1-CN 0;C ;N ;1+N ;C 0;C Rph17 36l50/3/5/1/Rph? ;1CN ;1-N ;C ;CN ;N 0;CN ;1CN ;1-N 0;C 0;CN Rph? 38P18/8/1/10/Rph18 0;N 0;N 0;= 0;= 0;= 0;= 0;N 0;N 0;- 0;= Rph18 169P15/8/ Rph? 0;N 0;N 0;= 0;= 0;= 0;= 0;N 0;N 0;- 0;= Rph? DGenotypes with known Rph genes. Susc.: susceptible, RphP: Prior/Rph19

P. hordei resistance in cultivar Ricardo

Inheritance of seedling resistance

Populations (F1, F3 and BC1F2) derived from intercrossing Ricardo and Gus were tested with + pt 5457P in the greenhouse. All F1s produced low ITs (11++C++), indicating a dominant inheritance of RphRic.

+ The 130 BC1F2 lines tested in greenhouse against 5457P were classified as 70 segregating and 60 homozygous susceptible (Appendix 4.1). Chi-squared analysis to determine the goodness-of-fit of these data to that expected for a single gene segregation among BC1F2 lines (1 Segregating : 1 Homozygous susceptible) supported the presence of a single gene (χ 2 = 0.76 P = 0.38) in Ricardo (Table 3.5).

P. hordei resistance in cultivar Ricardo

Cross No. of BC1F2 Lines Predicted ratio No. Segregating Homozygous susceptible Total χ2 P of genes

Ricardo/Gus//Gus 70 60 130 1:1 0.76 0.38 1 2 20 to 25 plants assessed per BC1F2 line, χ table value at P = 0.05 is 3.84 (1 d.f.) and at P = 0.01 is 6.64 (1 d.f.)

Cross No. of F3 Lines Predicted ratio No. Homozygous Segregating Homozygous Total χ2 P of genes resistant susceptible Ricardo/Gus 37 104 46 187 1:2:1 3.22 0.19 1 2 20 to 25 plants assessed per F3 line, χ table value at P = 0.05 is 5.99 (2 d.f.) and at P = 0.01 is 9.21 (2 d.f.)

P. hordei resistance in cultivar Ricardo

Of the 200 F3 lines derived from the cross Ricardo/Gus, 13 showed very poor germination and were excluded. The remaining 187 F3 lines were scored as 37 Homozygous resistant, 104 Segregating and 46 Homozygous susceptible when tested with pt 5457P+ in the greenhouse (Appendix 3.2). Chi-squared analyses confirmed the goodness-of-fit to a 1:2:1 ratio (χ 2 = 3.22, P = 0.19), expected for monogenic inheritance of RphRic (Table 3.6).

Detection of Rph2 in Ricardo

Populations derived from the cross Ricardo/Peruvian, consisting of 79 and 68 F3 lines, showed no segregation when tested with the Rph2-avirulent pt 200P-, consistent with the presence of Rph2 in both Ricardo and Peruvian (Table 3.7).

To ensure that the Ricardo/Peruvian crosses did not involve selfing, 15 F3 lines from both populations were selected randomly and tested with pt 5457P+ (virulent to Rph2 and avirulent to Ricardo). Segregation was observed in both cases (Table 3.8), indicating that selfing was not involved and the observed segregation pattern conformed to that expected from a single gene (P = 0.99).

Genotyping of Ricardo with Rph2 linked marker ITS1 The presence of Rph2 in the cultivars Ricardo and Peruvian was further tested using the STS marker ITS1, linked closely with Rph2 (Borovkova et al. 1997). The marker amplified a 300 bp product in Ricardo and Peruvian and 350 bp product in the cultivar Gus (lacking Rph2) and upon restriction, Ricardo and Peruvian produced similar bands whereas Gus showed an additional 175 bp band (Fig. 3.2). The amplification of similar bands in Ricardo and Peruvian before and after restriction supported the presence of Rph2 in both of these cultivars.

P. hordei resistance in cultivar Ricardo

Cross No. of F3 Lines Gene Ricardo/ Homozygous resistant Segregating Homozygous Total Peruvian susceptible P1 79 0 0 79 No Segregation Rph2 P2 68 0 0 68 No Segregation Rph2

20 to 25 plants assessed per F3 line for P1 (Population 1) and P2 (Population 2)

Table 3.8 Observed segregation among 15 randomly selected F3 lines derived from the cross Ricardo/Peruvian when inoculated with the Rph2- virulent Puccinia hordei pathotype 5457P+ at seedling stage

Cross No. of F3 Lines Predicted No. Ricardo/ Homozygous Segregating Homozygous Total ratio χ2 P of genes Peruvian resistant susceptible P1 4 7 4 15 1:2:1 0.02 0.99 1 P2 3 5 7 15 1:2:1 3.75 0.15 1

20 to 25 plants assessed per F3 line for P1 (Population 1) and P2 (Population 2) χ 2 table value at P = 0.05 is 5.99 (2 d.f.) and at P = 0.01 is 9.21 (2 d.f.)

P. hordei resistance in cultivar Ricardo

Molecular mapping of RphRic A total of 488 SSR markers (whole genome scan kit I) were used to identify markers linked closely to the uncharacterised locus responsible for seedling resistance to P. hordei in cultivar Ricardo. Twelve markers (EBmac0635, EBmac0701, HvBTAI0003, HvHVO0003, HVMLOE, HvPEPD1PR, GBM1220, GBM1003, GBM1015, GBM1028, GBM1044 and Bmy1_INDEL6) were polymorphic between the parents and displayed linkage with resistant and susceptible bulks. The PCR products produced by each primer are given in Table 3.9. All 12 markers are located on chromosome 4H, thereby providing strong evidence for the presence of the RphRic in this chromosome.

The 12 linked SSRs were used to genotype 187 F3 lines derived from the cross Ricardo/Gus. Based on marker data, a linkage analyses was performed using Map Manager. Linkage analyses grouped seven markers HvHVO0003, HVMLOE, HvPEPD1PR, GBM1220, GBM1003, GBM1015 and GBM1044 into a single linkage group of 85.1 cM, on chromosome 4H. Five markers (viz. EBmac0635, EBmac0701, HvBTAI0003, GBM1028 and Bmy1_INDEL6) were found unlinked. The locus RphRic was flanked by GBM1003 on the proximal end and by GBM1220 on the distal end, at distances of 20.4 cM and 17.4 cM, respectively. A genetic linkage map of chromosome 4H was drawn using Map Chart 2.2 (Fig. 3.3).

P. hordei resistance in cultivar Ricardo

Table 3.9 Details of polymorphic SSRs published for chromosome 4H of barley used to genotype 187 F3 lines derived from the cross Ricardo/Gus Marker Size (bp) Resistant bulk Ricardo Gus Susceptible bulk HvHVO0003 269 269 266 266 HVMLOE 269 269 266 266 GBM1220 197 197 195 195 GBM1003 229;235 235 229 229 GBM1044 265 265 262 262 HvPEPD1PR 211;218 211 218 218 GBM1015 283;295 283 295 283;295 EBmac0635 141 141 Na Na EBmac0701 175 175 Na Na HvBTAI0003 263 263 Na Na GBM1028 314 314 316 314;316 Bmy1_INDEL6 262 262 262;272 262;272 Figures separated by; indicates recombination shown by marker. Na: no amplification

P. hordei resistance in cultivar Ricardo

4H cM

0.0 GBM1044 2.2 GBM1003 0.0

20.4 GBM1003

RphRic2.2

RphRic 17.4 22.6 3.2 GBM1220 HvPEPD1PRn*hv0740 6.1 HvHVO000340.0

n*hv0667 20.0 43.2

n*hv0616GBM1015 15.8 49.3 HVMLOEn*hv0885

69.3

n*hv0666

Fig. 3.3 Genetic map for RphRic and polymorphic85.1 microsatellite markers on chromosome 4H of barley, constructed using 187 F3 lines. Kosambi4H map distances (cM) shown on the left side

GBM1044 Inheritance of adult plant resistance to leaf rust in Ricardo

The parents Ricardo and Gus, along with 200 F3 lines from the cross Ricardo/Gus, were + tested under field conditions using P. hordei pt 5457P . Twelve F3 lines showed very poor germination in the field and were excluded from the studies. The same Ricardo/Gus F3 population was tested under greenhouse conditions for the inheritance of seedling resistance using the same pathotype. At the adult plant stage under field conditions, Ricardo was highly resistant (0 to 5R) and Gus was highly susceptible (90 to 100S). In the field, the population showed a two gene segregation [7:8:1 (13 Homozygous resistant (HR) + 62 Moderately susceptible (MS) : 99 Segregating (SEG) : 14 Homozygous susceptible (HS))] (Table 3.10).

Goodness of fit for a two gene model was satisfactory at P = 0.51. Because the same F3 population showed single gene segregation with the same pt 5457P+ when tested at seedling growth stages in the greenhouse, the two gene segregation observed in the field indicated the presence of an APR gene in addition to RphRic. In the field segregating lines produced HR,

MS and HS type plants. Two resistance classes were observed among the F3 lines in the field; 59

P. hordei resistance in cultivar Ricardo lines displaying a high level of resistance may carry both RphRic and the unknown APR gene (AABB) and lines displaying a moderate level of resistance may carry the RphRic (AABb) or the APR gene (AaBB) only (Fig. 3.4). This was supported by the presence of only 13 lines (close to expected 11.75 lines) giving a high level of resistance (like Ricardo); one genotype (AABB) homozygous resistant for both genes is expected out of 16 (1AABB:2AABb:1AAbb:2AaBB:4AaBb:2Aabb:1aaBB:2aabB:1aabb) segregating for two genes. The 44 F3 lines that were scored as homozygous susceptible under greenhouse conditions segregated when tested under field conditions with the same pathotype (pt 5457P+). Chi-squared (χ2) analyses confirmed the goodness-of-fit to a 1:2:1 ratio among these lines, (11 Moderately susceptible : 19 Segregating : 14 Homozygous susceptible), expected for monogenic inheritance at P = 0.54 calculated from χ 2 value of 1.22 (Table 3.11).

Further studies of the APR present in Ricardo were undertaken using the 130 BC1F2 lines that displayed single gene inheritance under greenhouse conditions (Table 3.5). After greenhouse testing, these lines were transplanted to the field where they were tested using the same pathotype (pt 5457P+). Two lines did not survive transplantation and the remaining 128 lines showed a two gene segregation ratio when scored for leaf rust response post flag leaf emergence [3:1 (101 segregating : 27 Homozygous susceptible)] at P = 0.59 calculated from χ 2 value of 1.04 (Table 3.12). The BC1F2 lines that were homozygous susceptible in seedling greenhouse tests were analysed separately for the potential presence of an APR gene. A total of 58 BC1F2 lines followed the pattern of segregation expected for a single gene, [1:1 (33 Segregating : 25 Homozygous susceptible)] expected for monogenic inheritance at P = 0.29 and χ 2 value 1.1 (Table 3.13). The segregating lines produced moderately susceptible and susceptible phenotypes in the field.

P. hordei resistance in cultivar Ricardo

Cross No. of F3 Lines Predicted No. Homozygous Moderately Segregating Homozygous Total ratio χ2 P of genes resistant susceptible susceptible Ricardo/Gus 13 62 99 14 188 7:8:1a 1.33 0.51 2 2 20 to 25 plants assessed per F3 line, χ table value at P = 0.05 is 5.99 (2 d.f.) and at P = 0.01 is 9.21 (2 d.f.) aHomozygous resistant + Moderately susceptible : Segregating : Homozygous susceptible

Cross No. of F3 Lines Predicted ratio No. Moderately Segregating Homozygous Total χ2 P of genes susceptible (MS/S) susceptible Ricardo/Gus 11 19 14 44 1:2:1 1.22 0.54 1 2 20 to 25 plants assessed per F3 line. χ table value at P = 0.05 is 5.99 (2 d.f.) and at P = 0.01 is 9.21 (2 d.f.) MS: Moderately susceptible, S: Susceptible

P. hordei resistance in cultivar Ricardo

Cross No. of BC1F2 Lines Predicted No. Segregating Homozygous Total ratio χ2 P of genes susceptible Ricardo/Gus//Gus 101 27 128 3:1 1.04 0.59 2

20 to 25 plants assessed per BC1F2 line χ 2 table value at P = 0.05 is 3.84 (1 d.f.) and at P = 0.01 is 6.64 (1 d.f.)

Cross No. of BC1F2 Lines Predicted No. Segregating Homozygous susceptible Total ratio χ2 P of genes (MS/S) Ricardo/Gus//Gus 33 25 58 1:1 1.1 0.29 1 2 20 to 25 plants assessed per BC1F2 line. χ table value at P = 0.05 is 3.84 (1 d.f.) and at P = 0.01 is 6.64 (1 d.f.) MS: Moderately susceptible, S: Susceptible

P. hordei resistance in cultivar Ricardo

Genotyping with marker bPb-0837 linked to APR gene Rph20 The molecular marker bPb-0837 (Liu et al. 2010), closely linked to the APR gene Rph20, was used to genotype Ricardo, Gus and the control genotypes Pompadour (Rph20), Flagship (Rph20) and Stirling. The cultivars Ricardo, Gus and Stirling produced no band while Pompadour and Flagship amplified 245 bp bands (Table 3.14), suggesting that the APR in Ricardo differs from Rph20.

Fig. 3.4 Field responses of F3 lines derived from the cross Ricardo/Gus. L to R: R, MS and S, leading to leaf death as showed in last two leaves (Categories are described in Appendix 2)

P. hordei resistance in cultivar Ricardo

Table 3.14 Assays of the marker bPb-0837, linked to the adult plant resistance gene Rph20, on Ricardo, Gus and three control genotypes Genotypes Field score bPb-0837 Ricardo 0 – 5 R - Gus 70 – 100 S - Pompadour R – 10 R + Flagship R – 10 R + Stirling 70 – 90 S - + and - indicates presence and absence of band respectively

Discussion The present study aimed to characterise new sources of resistance to P. hordei to assist barley breeders by diversifying the resistance sources currently available to control this disease. The cultivar Ricardo was reported to carry the leaf rust resistance gene Rph2 (Pa2) (Henderson 1945; Moseman and Roan 1959; Zloten 1952) plus additional uncharacterised seedling resistance (Park unpublished; Stöcker 1983; Wallwork et al. 1992; Yahyaoui et al. 1988), referred to here as RphRic. Studies were therefore conducted to characterise RphRic, to prove the presence of Rph2 in Ricardo and to assess the protective properties of RphRic at adult plant growth stages under field conditions. To ensure accurate phenotyping of RphRic, studies were first conducted to identify the most congenial environment for optimal expression of this gene.

Environment sensitivity of RphRic The expression of rust resistance genes can differ with environmental conditions. In previous greenhouse studies, Ricardo produced ITs ranging from “1+” to “3” against different P. hordei pts (Park unpublished; Golegaonkar 2007). Ricardo, Gus and Peruvian seedlings were therefore inoculated with four different pts of P. hordei, all virulent for Rph2 except 200P- and incubated at a range of post-inoculation temperatures. The expression of the Ricardo resistance varied with post-inoculation temperature and was found to be most strongly expressed at temperatures of 23 ±2oC (Table 3.3). Temperature sensitivity of rust resistance genes has been reported earlier in both barley and other cereals. For example, seedlings of barley genotypes carrying the stem rust resistance gene rpg4 produced different ITs against pt QCCJ of P. graminis f. sp. tritici when incubated at 18–19oC and at 27–28oC (Sun and

P. hordei resistance in cultivar Ricardo

Steffenson 1997). Similarly, wheat seedlings carrying Yr17 expressed higher levels of resistance at 15–20oC and were susceptible at 12–15oC to P. striiformis f. sp tritici (Qamar et al. 2008). At 18oC and below, low ITs were produced by wheat seedlings carrying Sr15, while high ITs were produced at 26oC and above when inoculated with P. graminis f. sp. tritici (Gousseau et al. 1985).

Inheritance of seedling resistance To confirm the presence of RphRic in addition to the seedling gene Rph2 in Ricardo (Henderson 1945; Moseman and Roan 1959; Zloten 1952), multipathotype tests were carried out in the greenhouse. In addition to Rph2, the detection of RphRic in Ricardo (Table 3.4) was in accordance with earlier studies (Park unpublished; Stöcker 1983; Wallwork et al.

1992; Yahyaoui et al. 1988). Inheritance studies and Chi-squared analyses of F3

(Ricardo/Gus) and BC1F2 (Ricardo/Gus//Gus) families confirmed the dominant monogenic inheritance of RphRic. The pt 5457P+ was virulent on Rph2, therefore observed segregations were for RphRic only. Because virulence for this gene has not yet been detected in Australia (Park, unpublished), it is potentially a useful new source of resistance to P. hordei.

A BSA approach was adopted for the molecular mapping of RphRic. The multiplex-ready PCR technique (Hayden et al. 2008a) detected 12 markers polymorphic between resistant and susceptible bulks and between the resistant and susceptible parents. Of these 12 polymorphic markers, seven were linked to RphRic (Table 3.9). Genotyping of the Ricardo/Gus F3 population with these linked markers mapped RphRic on chromosome 4H, flanked by the markers GBM1003 and GBM1220 (Fig. 3.3). As no catalouged seedling leaf rust resistance gene has been located in chromosome 4H, the locus symbol Rph21 is proposed for RphRic.

Presence of Rph2 in Ricardo Many genotypes of barley are reported to carry the seedling gene Rph2 alone and in combination with other leaf rust resistance genes. For example, the barley cultivar Peruvian carries Rph2 (Levine and Cherewick 1952; Starling 1956; Steffenson and Jin 1997), Reka 1 carries Rph2 and Rph19 (Park and Karakousis 2002), Quinn carries Rph2 and Rph5 (Roane and Starling 1967; Starling 1956) and Bolivia carries Rph2 and Rph6 (Henderson 1945; Roane and Starling 1967; Starling 1956). Previous reports of the presence of Rph2 in Ricardo are based solely on gene postulation. In the present study, two sets of F3 populations derived 65

P. hordei resistance in cultivar Ricardo from the cross Ricardo/Peruvian (Rph2) were tested with the Rph2-avirulent pt 200P- in the greenhouse. No segregation was observed in both F3 populations (Table 3.7), providing convincing genetic evidence that Ricardo carries Rph2. Considering this, it was necessary to use an Rph2-virulent pathotype in studies that led to the characterisation of Rph21. When 15 randomly selected F3 lines from each population derived from the cross Ricardo/Peruvian were tested with the Rph2-virulent pt 5457P+, single gene segregation at the RphRic locus was observed, further supporting the presence of Rph2 in both parents. In similar studies,

Borovkova et al. (1997) reported no segregation in F2 populations derived from an intercross between RphQ (Q21861) and Rph2 (Peruvian, PI531840 and PI531841), when inoculated with a pathotype of P. hordei avirulent on Rph2, indicating the presence of same gene, Rph2, in both parents.

Results with the Rph2-linked marker ITS1 (Borovkova et al. 1997) in tests with DNA from Ricardo and the control cultivar Peruvian (Fig. 3.2) provided additional evidence for the presence of Rph2 in Ricardo. This is the first report of the validation of Rph2 marker ITS1 using Ricardo. The present study therefore established the presence of Rph2 and Rph21 in Ricardo.

Inheritance of adult plant resistance in Ricardo In earlier studies, Ricardo was found to be highly resistant under field conditions and was reported to carry APR (Golegaonkar et al. 2009b). The presence of slow rusting genes providing APR in barley was reported previously (Smit and Parlevliet 1990). Reductions in yield losses were reported in the wheat cultivars Banks, Bass, Cook, Kite and Suneca carrying APR against P. striiformis f. sp. tritici (Park et al. 1988). APR is often conditioned by the additive effects of more than one gene and is considered to be often durable (McIntosh 1992; Pretorius et al. 2007). Resistance that stays effective with prolonged use is considered durable and has been used successfully in wheat rust resistance breeding programs (Johnson 1984; McIntosh 1992).

When F3 lines (Ricardo/Gus) and BC1F2 lines (Ricardo/Gus//Gus) were tested against pt 5457P+ under field conditions, both populations segregated for rust response in the manner expected for two genes. The monogenic inheritance observed in the greenhouse and the

P. hordei resistance in cultivar Ricardo digenic inheritance observed under field conditions in both populations with the same pathotype confirmed the presence of a gene conferring APR to leaf rust in Ricardo.

Two resistant phenotypes, highly resistant and moderately susceptible were observed in the field among the F3 lines (Table 3.10). The highly resistant phenotype observed was similar to that of the parent Ricardo and is thought to result from the combination of Rph21 and the uncharacterised APR gene because only 13 out of 188 F3 lines were highly resistant in the field. This approximates to one line out of 16 (expected 11.75 of 188) being highly resistant, which conforms well to a 7:8:1 (1 HR + 6 MS : 8 SEG : 1 HS) model expected for two gene inheritance. Similarly, moderately susceptible phenotypes are expected from either Rph21 or UAPR singly, if both provide low levels of resistance in the field. The observed 62 moderately susceptible F3 lines out of 188, to the expected 70.5 lines with either Rph21 or UAPR gene in homozygous (1AAbb, 1aaBB) or in heterozygous (2Aabb, 2aaBb) form, fits well into 7:8:1 model of two gene inheritance. If only seedling gene Rph21 was highly effective under field conditions, then approximately 47 (25% of the total 188) homozygous resistant lines would have been expected and the same result would be expected if it was UAPR in place of Rph21. It is concluded that both genes are highly effective in combination under field conditions and that they display additivity. There are reports of APR genes in wheat like Sr2 that provide high levels of resistance when present in combination with seedling resistance genes such as Sr13, Sr24 and Sr30 (Bariana et al. 2007). Different levels of APR have also been reported in barley cultivars: for example, Ricardo, Derkado and Tweed showed high levels of APR; Egmont and Universe showed moderate levels of APR; and Atem, Belfore, Gilbert, Klimek, Optic and Uta showed low levels of APR to P. hordei under field conditions (Golegaonkar et al. 2009b). Recently, Hickey et al. (2011) mapped the APR gene Rph20 and a second QTL (qRphND) with smaller effect, on chromosome 6HL. In the absence of Rph20, qRphND confers a low level of resistance only.

Ricardo/Gus F3 and BC1F2 lines that were identified as homozygous susceptible in greenhouse seedling tests were analysed in the field at adult plant growth stages for APR. In both cases, single gene segregations were observed under field conditions, with 1:2:1 ratio of moderately susceptible : segregating : homozygous susceptible lines in case of F3 lines and

1:1 ratio of segregating : homozygous susceptible lines in case of BC1F2. Because these lines were homozygous susceptible in the greenhouse, the moderate resistance seen in the field 67

P. hordei resistance in cultivar Ricardo must be due the presence of the single APR gene, providing further evidence for additivity of this gene with Rph21.

Genotyping of Ricardo with bPb-0837 marker linked to Rph20 The APR gene Rph20 was recently mapped on chromosome 5H (Hickey et al. 2011) and the closely (0.7 cM) linked marker bPb-0837 was reported by Liu et. al. (2010). When the marker bPb-0837 was applied to Ricardo and the susceptible controls Stirling and Gus, it failed to amplify a product, but did so in the Rph20 carrying cultivars Pompadour and Flagship. The absence of this marker in Ricardo suggests that the APR present in Ricardo may be distinct from Rph20. Because Rph20 is considered to be present in a range of barley cultivars (Golegaonkar et al. 2009b; Hickey et al. 2011) globally and is the only catalogued gene for APR to leaf rust in barley, the distinct APR identified in Ricardo is potentially important, providing an additional source of APR that can be used in combination with Rph20. Pyramiding of four to five slow rusting minor genes with additive effects can provide high levels of resistance close to immunity (Singh et al. 2004). Further genetic analyses and mapping of the locus responsible for APR in Ricardo are highly recommended.

APR against P. hordei

CHAPTER IV Characterising resistance to Puccinia hordei in selected barley germplasm

Abstract A set of 113 advanced breeding lines and cultivars of barley (Hordeum vulgare L. subsp. vulgare), along with the susceptible control genotype Gus, was tested for response to the barley leaf rust pathogen Puccinia hordei Otth. in the greenhouse (as seedlings) and field (as adult plants). Tests revealed the presence of adult plant resistance (APR) in 68 lines, uncharacterised seedling resistance (USR) in 23 lines and the seedling resistance gene Rph3 in three lines. Nineteen lines lacked detectable seedling resistance and were susceptible in the field at adult plant growth stages. The presence of marker bPb-0837, linked to the APR gene Rph20, in 35 of the 68 lines carrying APR, suggested they carry this gene. The remaining 33 lines, which lacked the Rph20 linked marker, are likely sources of new uncharacterised APR. Pedigree analysis of the 68 lines found to carry APR revealed that 32 were related to cv. Gull and to H. laevigatum, two were related to cv. Bavaria and one related to cvv. Manchuria and Taganrog, suggesting that these genotypes may be the ancestrsal sources of the APR carried by each.

Keywords: Barley leaf rust, Adult plant resistance, Hordeum vulgare, AUDPC, Rph20

Introduction Cultivated barley (Hordeum vulgare L. subsp. vulgare) is the world’s most important cereal crop after wheat, maize and rice (FAO 2008; Poehlamn 1985). It is the second most important cereal crop in Australia (Murray and Brennan 2010), where it contributes billions of dollars to the national economy (GRDC 2005). In Australia, barley diseases cause estimated average annual losses of A$ 252 million (Murray and Brennan 2010). Leaf rust (caused by Puccinia hordei Otth.) is a serious disease of barley in many parts of the world (Clifford 1985). Epidemics of barley leaf rust have been reported in Australia (Cotterill et al. 1995; Cotterill et al. 1992; Waterhouse 1927), New Zealand (Arnst et al. 1979) and the USA (Griffey et al. 1994). Pathotypes (pts) of P. hordei with virulence matching important seedling genes conferring resistance have arisen in various regions. Following the releases of barley cultivars carrying Rph12 (Fitzgerald, Franklin, Lindwall and Tallon), eight new pts with virulence matching Rph12 emerged in Australia during the years 1992 to 2001 (Park 69

APR against P. hordei

2008). Similarly, the releases of cultivars Fitzroy, Yarra and Starmalt, each carrying the single major gene Rph3, were followed by the development of a new pt (5457P+) with virulence for this gene in 2009 (Park 2010). At present, only five seedling resistance genes, viz. Rph7, Rph11, Rph14, Rph15 and Rph18 ((Park 2003; Park 2010) and one adult plant resistance (APR) gene, Rph20 (Park 2010, unpublished), are effective in Australia, underscoring the narrow and limited genetic base of leaf rust resistance available in barley and highlighting a need to identify new sources of resistance (Golegaonkar et al. 2009b; Park 2003; Park 2008). Previous studies of rust resistance in wheat have shown that APR, especially in combination with major resistance genes, can provide durable resistance. For example, the APR gene Sr2 has been used effectively in combination with the seedling resistance genes Sr13, Sr24 and Sr30 (Bariana et al. 2007) and with several other Sr genes (Park 2007b), in controlling P. graminis f. sp. tritici. Similarly, APR genes like Lr34/Yr18 and Lr46/Yr29 have imparted durable resistance in wheat for more than 30 years (Park 2008) despite their extensive deployment. Recently, the first gene conferring APR to leaf rust in barley was characterised by Golegaonkar et al. (2010) and was subsequently mapped onto chromosome 5HS and given the designation Rph20 (Hickey et al. 2011). The molecular markers EBmag0833 and bPb-0837 linked to Rph20 were identified by Liu et al. (2010), the latter being recommended for marker assisted selection based on its closer (0.7cM) association with Rph20.

In studies of the field response of barley germplasm to leaf rust, Golegaonkar et al. (2009b) calculated an average coefficient of infection (ACI) by multiplying predetermined values with disease scores. In earlier studies, the barley cultivars Shyri, Clipper and Terán were assessed for different levels of partial resistance based on the area under the disease progress curve (AUDPC) under field conditions (Ochoa and Parlevliet 2007). In different studies, quantitative assessments of APR in cereal crops have been carried out by means of AUDPC and/or by means of ACI values (Broers et al. 1996; Parlevliet 1979; Pathan and Park 2006; Steffenson and Webster 1992). Based on disease scoring at periodic intervals, Shaner and Finney (1977) developed a formula to calculate AUDPC from qualitative disease ratings that was also used in subsequent studies (Haynes and Weingartner 2004; Jeger and Viljanen- Rollinson 2001; Mardi et al. 2005; Rahman et al. 2010).

APR against P. hordei

The characterisation of new sources of leaf rust resistance in barley will provide more options for breeders to enhance genetic resistance against P. hordei. The present study was conducted with the objective of finding new sources of resistance against P. hordei. Because only one gene conferring APR to leaf rust has been named in barley to date (i.e. Rph20), particular emphasis was placed on identifying new sources of APR.

Materials and methods

Plant material Barley germplasm comprising advanced breeding lines and cultivars was tested in the greenhouse and in artificially inoculated field rust nurseries for three years (2007–2009). One hundred and thirteen barley genotypes, plus the susceptible control genotype Gus, were screened for rust response in the greenhouse and field. A total of 95 barley lines were sourced from a barley germplasm importation program at the University of Western Australia (UWA) and the remaining 18 and Gus were obtained from a seed collection maintained at the Plant Breeding Institute (PBI) Cobbitty (Table 4.1). Differential lines used as controls are described in Appendix D1.

Table 4.1 Details of barley cultivars and lines assessed for response to Puccinia hordei at seedling (greenhouse) and adult plant (field) growth stages Cultivar/Line Source Pedigree 63081 UWA Prior/Lenta/Noyep/Lenta 74043 UWA Kenia/Erectoides 16 115-9505-Ba UWA Unknown AB30 UWA H. spontaneum///Clipper Andapi 3a UWA Unknown Argentine 2a UWA Unknown Astoria UWA Nevada/STA 13817 Atem PBI [(L 92/Minerva)/Emir]/Zephyr Baronesse PBI (Mentor/Minerva)/(Vada mutant////Carlsberg/ Union) (Opavsky/Salle///Ricardo/////Oriol/6153P40) Beecher UWA Atlas/Vaughn Belfor PBI Minerva/(Heine 4808/Piroline) Birte UWA Goldie/Cork 71

APR against P. hordei

BM9647-69 UWA TR251/CDC Kendall Bracken UWA Woodvale//Primus/SD67-297 Brenda UWA Nebi/11827-80//Gimpel Canada 110a UWA Unknown Caravela UWA Ribeka/Union Carboa UWA Unknown Casino PBI [(H. deficiens/Sergeant)/Georgie]/Regent Chalice UWA Cooper/NFC514-5/2/Chariot Chieftain UWA Brittania/Prisma CI 9819 UWA Ethiopian landrace CI 4976a UWA Unknown Corniche PBI (Diamant/14029/64)/F2[Emir/(HOR 3270/46132/68)] Corvette UWA Bonus/CI 3576 CPI 36396 Aa UWA Unknown Decanter UWA Heron/Dallas Derkado PBI Lada/Salome Egmont PBI (Maris Yak/W 1001)/Vada Emir UWA Delta/[Agio//(Kenia)/Arabian Variety] Esperance UWA (S)LV-Northern-Africa Expres UWA SK 3455/Akcent Farmington UWA WA-7190-86/Maresi Felicie PBI Patty/Nadir Galleon UWA Clipper/Hiproly///(Proctor/CI 3576) Gilbert PBI Koru reselection Giza 127a UWA Unknown Giza 128a UWA Unknown Glacier/Titan UWA Glacier/Titan GSHO 1436a UWA Unknown GSHO 1452a UWA Unknown Harriot UWA Hanka/Nordus//Annabell Hassan UWA [(Arabische/(Kenia)///Agio]/Delta HB369 UWA CSBA2209-1/CDC McGwire HOR 2410a UWA Unknown HOR 3877a UWA Unknown HOR 9696a UWA Unknown

APR against P. hordei

Hydrogen UWA Alis/Digger//Derkado I97-336R UWA Bowman/7/DWS1240 OB165 SLD ICB82-0114-6AP-0APa UWA Unknown ICB83-0157-10AP-0TR-0AP- UWA Unknown 7AP-1APH-0APa ICB88-1560-11AP-1APH- UWA Unknown 1APH-2AP-0TR-0APa ICB94-0705-AP-4APa UWA Unknown Ketch UWA Noyep/Lenta Klimek PBI Diva/Trumpf/Georgia/Diamant/ Pallidum/Orkisz/Nadja Laura UWA Pallas/Herta Mackay UWA Cameo/Koru Maris Mink UWA (Emir/Swallow)/Deba Abed M-Q-54a UWA Unknown Nord GS1749a UWA Unknown Nordus UWA 845/Krona Olbrana UWA Unknown Optic PBI Chad/(Corniche/Force) Orge 403-9a UWA Unknown Orzo-Nudo-Portignanoa UWA Unknown Pewter UWA NFC94-20/NFC94-11 Pompadour PBI FDO192/Patty Pusa 20a UWA Unknown Pusa 7a UWA Unknown Quasara UWA Unknown Ricardoa PBI Unknown Roland PBI Lud/Tellus M1D SB01675 UWA 4176n/CDC McGwire SB02420 UWA TR251/TR970 Scald and Powdery Mildew UWA Unknown Res. Selectiona SE627.02a UWA Unknown SE632.02a UWA Unknown SE644.02a UWA Unknown

APR against P. hordei

Shenmai 3 UWA Gobernadora/Humai 10 (BAR89-3-1) SHN094 UWA Shenmai 3/ND19119 SHN193 UWA Shenmai 3/ND19119 Tennessee Winter Coast UWA Unknown Selectiona Tifanga UWA Unknown TR03273 UWA TR251/TR253 TR03274 UWA TR253/BM9216-4 Trait d'Union UWA Firlbecks Union seln Tweed PBI (Akka/Maris Mink)/Maris Mink UC 711 74-2a UWA Unknown Universe PBI Abed 3371/Vada Ursa UWA Thuringa/Hanka//Annabell USA 10274a UWA Unknown USA 8279-1640a UWA Unknown Uta PBI Emir/Quantum UWA Selection 4685 UWA Selection from 91BYT-LRA 11 UWA Selection 8939 UWA Selection from SLB 66-050 UWA Selection 4900 UWA Selection from 91HBSN 4 UWA Selection 3849 UWA Selection from WA3849 UWA Selection 4887 UWA Selection from 91NBGP 51 UWA Late Selection 8861 UWA Selection from Tipper//WI-2291/WI-2269 UWA Selection 8951 UWA Selection from CI5791/Ca1607//Shyri/5 UWA Selection 1901 UWA Selection from WI-2615 UWA Selection 1968 UWA Selection from Nudideficiens UWA Selection 4884 UWA Selection from 91NGBP 47 UWA Selection 4886 UWA Selection from 91NBGP 50 UWA Thin Seed Selection UWA Selection from Tipper//WI-2291/WI-2269 8861 VB9935 UWA Chebec/VIC9104//Sloop WAU 4633a UWA Unknown WI 3407 PBI (Chieftain/Barque)/(Manley/VB9104) WI-2553 UWA CD28/WI-2231 Xi-An 2007a UWA Unknown Xi-An 91-2a UWA Unknown

APR against P. hordei

Yalea UWA Unknown Zhepi 2a UWA Unknown Gus (Susceptible control)a PBI Unknown aPedigree information not available PBI: Plant Breeding Institute Cobbitty; UWA: University of Western Australia

Pathogen material For seedling tests, three pts of P. hordei (5457P+, 5653P+ and 5652P+) were used. The P. hordei pts 5652P+ and 5653P+ were used at field sites “Karalee” and “Horse Unit”, respectively, during the years 2007 and 2008. Pathotype 5457P+ was used at both field sites during 2009. For multipathotype greenhouse seedling tests, three additional pts (200P-, 253P- and 4673P+) were used. The virulence/avirulence patterns of these pts on known Rph genes are provided in Table 4.2.

Table 4.2 Pathogenicity details of Puccinia hordei pathotypes used in the studies to characterise resistance in barley germplasm Pathotype Culture no. Virulence* 253P- 490 Rph1, Rph2, Rph4, Rph6, Rph8 200P- 518 Rph8 5652P+ 561 Rph2, Rph4, Rph6, Rph8, Rph9, Rph10, Rph12, Rph19 4673P+ 562 Rph1, Rph2, Rph4, Rph5, Rph6, Rph8, Rph9, Rph12, Rph19 5653P+ 584 Rph1, Rph2, Rph4, Rph6, Rph8, Rph9, Rph10, Rph12, Rph19 5457P+ 612 Rph1, Rph2, Rph3, Rph4, Rph6, Rph9, Rph10, Rph12, Rph19 *with respect to the resistance genes listed in Appendix D1

Greenhouse screening Pots were filled with a mixture of fine bark and coarse sand and fertilised using Aquasol® (100 gm per 10 litre of water per 200 pots) prior to sowing. Seedlings of differentials and barley lines were raised in 9 cm diameter pots by sowing four clumps (parents and controls) or five clumps (differentials) of each genotype using 8–10 seeds per clump. Following sowing, pots were kept in growth room at 20 ± 2°C for germination. Seven-day old seedlings were fertilised with granular urea using Incitec Pivot w/w 46% nitrogen (50 gm per 10 litre of water per 200 pots). Greenhouse inoculations were carried out on seedlings at the one and a half leaf growth stage (9–10 day old seedlings). The seedlings were moved to an enclosed 75

APR against P. hordei chamber and urediniospores suspended in a light mineral oil (Shellsol®, Mobil Oil) were atomized over seedlings (10–12 mg/10 ml/200 pots) using an aerosol hydrocarbon propellant pressure pack. The chamber door was kept closed for 5 minutes to allow urediniospores to settle on the leaves completely. Spray nozzle fittings were kept in 70% ethanol and rinsed thoroughly with tap water before each inoculation to prevent cross contamination between successive inoculations. In addition, the inoculation chamber was washed thoroughly with pressurised tap water between successive inoculations.

Leaf rust-inoculated seedlings were incubated for 24 hours at an ambient temperature in a dark chamber where continuous mist was created by an ultrasonic humidifier. After incubation, seedlings were moved to naturally lit microclimate rooms maintained at 23 ± 2oC. Infection types (ITs) were scored 10–12 days post inoculation according to the 0–4 scale used by Park and Karakousis (2002) and outlined in Appendix 1.

Field screening In the field, all genotypes were tested as single short rows (0.5 m, 30–40 seeds per row) at 30 cm spacing at the field sites “Karalee” and “Horse Unit” in 2007, 2008 and 2009. Two replications were sown at each field site. A row of the susceptible cultivar Gus was sown as a rust spreader after every five test genotypes to allow the build-up and uniform distribution of inoculum. Four weeks after sowing, plots were fertilised using granular urea Incitec Pivot w/w 46% nitrogen @ 100 kg/ha followed by irrigation. Plots were irrigated once a week or as required, using fixed sprinklers.

Field leaf rust epidemics were created following the procedures described by McIntosh et al. (1995b). Urediniospores (30–40 mg) were suspended in 1.5 litre of light mineral oil (Shellsol®, Mobil Oil) and sprayed over buffer/spreader lines with an ultra-low-volume applicator (Microfit®, Micron Sprayer Ltd., UK). Four to five inoculations were performed during late evening on days that had a strong forecast of overnight dew. During first and second inoculations, hot spots of disease were established by watering and covering small areas of the rust spreader with plastic hoods overnight to ensure adequate dew formation in case natural dew formation did not occur.

APR against P. hordei

Leaf rust was scored at weekly intervals on three occasions per year, starting from the flag leaf growth stage using a modified Cobb scale (Peterson et al. 1948) for disease severity and host response as described in Appendix 2. Disease severity and host response were converted into a quantitative score by calculating a coefficient of infection (CI) as described by Golegaonkar et al. (2009b) with slight modifications. CI was obtained by multiplying the field scores of disease severity and host responses of 0, R, TR, MR, MR–MS, MS, MS–S and S with predetermined values of 0, 0.01, 0.15, 0.30, 0.45, 0.60, 0.75 and 1.0 respectively.

The AUDPC was calculated according to the formula given by Shaner and Finney (1977)

{AUDPC = ∑i=1 to n [(Ri + Ri-1) ⁄ 2] [ti - ti-1]}, where Ri is the CI calculated from the ith observation, ti is the day of ith observation and n indicates the total number of observations. AUDPC was calculated for each genotype based on three disease scores per replicate per year (2007–2009). To avoid errors due to disease escape, replications with higher disease score were used to calculate the AUDPC values. Based on the AUDPC values calculated, lines were assessed for different levels of resistance in the field, with lines having AUDPC values of 500 and above considered susceptible and lines with AUDPC values of 0–150, 151–300 and 301–500, were considered as having high, moderate and low levels of resistance, respectively. Because the same pt was used at both field sites in 2009, an average AUDPC value from both field sites was used to assess resistance.

Extraction of genomic DNA Barley lines were sown in the greenhouse as described earlier. A 15 to 20 mm sample of leaf tissue representing at least 10–15 plants per line was collected into 2 ml Eppendorf tubes from actively growing seedlings 8–12 days after sowing. The tubes were kept for 72 hours above silica beads to dry the leaf tissue. Two small stainless steel ball bearings were added per tube and dried leaves were crushed to powder using a Retsch MM300 Mixer Mill (Retsch, Germany) for 3 min at 25 rpm. Pre warmed (65°C) 700 µl of extraction buffer [50 mM Tris-pH 8.0, 10 mM EDTA-pH 8.0, 100 mM NaCl, SDS 1% (w/v), 10 mM ß- mercaptoethanol] was added per tube. Samples were incubated for 10 min at 65°C and 150 µl of 3 M K-acetate (pH 5.2) was added per tube. Tubes were shaken vigorously and kept in a freezer (-20°C) for 15 min. Samples were centrifuged for 15 min at 12,000 rpm and 650 µl of supernatant was transferred to new 1.5 ml tubes. An equal volume (750 µl) of chilled (-20°C) isopropanol was added per tube and the supernatant was mixed thoroughly by inverting tubes 77

APR against P. hordei several times. The tubes were once again placed in a freezer (-20°C) for 10 min to precipitate DNA and were then centrifuged at 10,000 rpm for 10 min to make a pellet of the DNA. The supernatant was discarded, the pellet was washed with 500 µl of 70% ethanol and then air dried and re-suspended in 200 µl of 10 mM Tris-HCl (pH 8.0). Rnase A (1 μg/μl) @ 20 µl per 40 ml of 10 mM Tris-HCl was added before re-suspending the DNA pellet. Tubes were kept in an oven (37°C) for 2 hrs to dissolve the DNA pellet properly. DNA was quantified using a Nanodrop ND-1000 spectrophotometer (Nanodrop® Technologies). All DNA samples were diluted uniformly to 50 ng/µl using doubled distilled autoclaved water (ddH2O) and the stock DNA was stored in a freezer (-20°C) until needed.

Genotyping with Rph20 linked marker bPb-0837 Marker bPb-0837, closely linked (0.7 cM) to the APR gene Rph20 (Liu et al. 2010), was screened across the barley lines identified as carrying APR to leaf rust based on greenhouse and field rust testing. The primer was synthesized and supplied by SIGMA (Sigma-Aldrich Pty. Ltd. Australia) and sequence information is detailed in Table 4.3.

Table 4.3 Details of marker bPb-0837, linked to adult plant resistance gene Rph20 in barley Name Sequence Marker Size Reference (bp)

bPb-0837 F 5’GACACTTCGTGCCAGTTTGA 3’ DArT 245 (Liu et al. 2010) R 5’CCTCCCTCCCTCTTCTCAAC 3’

PCR reaction and profile Ten micro litres of PCR reaction contained 2.0 µl of genomic DNA (50 ng), 1.0 µl of dNTPs

APR against P. hordei formamide loading buffer [98% formamide, 10 mM EDTA (pH 8.0), 0.05% (w/v) Bromophenol blue and 0.05% xylene cyanol]. Agarose gels were prepared by adding 2.0 g agarose (Bioline) per 100 ml of 1x Tris-borate EDTA (TBE) buffer (90 mM Tris-borate + 2 mM EDTA-pH 8.0). For staining, 1.0 µl of ethidium bromide was added per 100 ml of gel solution. The gel solution was poured into moulds and allowed to cool for 40 min at room temperature. Eight to 10.0 µl of PCR product including loading buffer was loaded per well. One kb DNA ladder HyperLadder™ IV (Bioline) was used as a size reference. Electrophoresis was carried out at 110 V for 1.5 h. The separated bands were visualised under ultra violet light unit fitted with a GelDoc-IT UVP Camera.

Results The susceptible control genotype Gus expressed high ITs in the greenhouse (3+) and high terminal disease ratings in all field experiments (70–90 S), as expected.

Seedling resistance All differential lines showed the expected ITs against all the pathotypes used (Table 4.4). Based on the greenhouse testing against six P. hordei pts, the barley cultivars/lines were categorised into three different groups: (1) Cultivars/lines lacking detectable seedling resistance, (2) Cultivars/lines with known seedling resistance and, (3) Cultivars/lines with unidentified seedling resistance.

1. Cultivars/lines lacking detectable seedling resistance A total of 87 cultivars/lines produced high ITs against the six P. hordei pathotypes in the greenhouse (Table 4.5). While it is possible that these genotypes may carry seedling resistance genes, no such resistance could be detected with the pathotype array used, which permitted detection on all currently catalogued seedling Rph genes (Rph1 through Rph19).

2. Cultivars/lines characterised with known seedling resistance Three of the genotypes examined, Birte, Roland and line UWA selection 4884, were highly resistant against P. hordei pts 200P-, 253P-, 4673P+, 5652P+ and 5653P+ and susceptible to the Rph3 virulent pathotype 5457P+ (Table 4.6), supporting the presence Rph3 in all three.

APR against P. hordei

3. Cultivars/lines with unknown seedling resistance Twenty three genotypes displayed a response pattern to the six pathotypes used that did not match any known Rph gene and were therefore considered to carry unidentified seedling resistance. Most of the lines were resistant to all six pathotypes, except for Scald and powdery mildew resistance selection, WAU 4633 and VB 9935, all of which produced high ITs (3+) against pt 5457P+ (Table 4.7). While this pattern resembled that of Rph3, the ITs displayed against the Rph3-avirulent pathotypes were higher than those on the Rph3 control genotype Estate (Table 4.4).

APR against P. hordei

Table 4.4 Greenhouse infection types produced by differential/control genotypes against six Puccinia hordei pathotypes Differential/Rph gene 253P- 200P- 5653P+ 5652P+ 4673P+ 5457P+ Gus/Nil 3+ 3+ 3+ 3+ 3+ 3+ Sudan/Rph1 3+ 0; 3+ ;N 3+ 3+ Berg/Rph1 3+ 0;C 3 ;N 3+ 3++ Peruvian/Rph2 X++,33+ ;CN 33+ X++,3+ 33+ 3+ Gatam/RphGat 33+ 33+ X 33+ 3+ 3+ Reka 1/Rph2+RphP ;1+N ;1+ ;C 3+ 3+ 3+ Ricardo/Rph2+? ;12++C X++ ;C 2++3C 2++ 1++C+ Estate/Rph3 0; 0; ;C ;C ;- 3+ Gold/Rph4 3+ ;1+ 33+ 3+ 3+ 3+ PI 531849/Rph13 0; 0;N ;CN ;-N ;1N ;CN Quinn/Rph2+Rph5 ;N ;N 0;- ;-N 3+ 0;C Magnif 104/Rph5 ;-N 0;N 0;- ;N 3+ 0;C Bolivia/Rph2+Rph6 X++3 X+ 3+ X+C X++3C 3+ Cebada Capa/Rph7 0;N 0; 0;N 0;N 0;N ;CN PI 584760/Rph14 ;12+C ;12- ;CN ;12-N ;12N ;1-CN Egypt 4/Rph8 33+ 3+ 3+ 3CN 3CN 12++C Abyssinian/Rph9 X++3 XX+ 3+ X++3 3+ 3+ Clipper BC8/Rph10 ;12+ ;12+ 3+ 3+ 3- 3+ Clipper BC67/Rph11 ;1+2+C ;12C 2++ 12+C 12+C 2++3C Triumph/Rph12 12+C 22+ 3+ 3+ 3+ 3+

APR against P. hordei

Prior/Rph19 ;1-N ;1-N 22+ 3+ 3+ 3+ Cutter/Rph19 ;1N ;1N 3+ 3+ 3+ 3+ Q21861/RphQ 3+ 33+ 33+ 3+ 3+ 3+ Cantala/RphC ;1-C 12-C 3+ 3+ 3+ 3+ PI366444/RphB37 2++3C 3 XC 3+ 3+ 3+ Bowman+Rph15/Rph15 ;N ;N ;CN ;1-N ;N ;CN 81882/BS1/Rph17 ;1N ;1N ;C ;N ;1N 0;C 36l50/3/5/1/Rph? ;1-N ;1CN 0;C ;1CN ;1-N 0;CN

APR against P. hordei

Table 4.5 Responses of 87 barley genotypes susceptible to six Puccinia hordei pathotypes at seedling growth stages in the greenhouse tests Postulated Cultivar/Line 253P- 200P- 4673P+ 5652P+ 5653P+ 5457P+ resistanceR 63081 3+ 3+ 3+ 3+ 3+ 3+ Nil 74043 3+ 3+ 3+ 33+ 33+ 3+ Nil 115-9505-B 3+ 3+ 3+ 33+ 33+ 3+ Nil AB30 3+ 3+ 3+ 3+ 3+ 3+ Nil Andapi 3 3+ 3+ 3+ 3+ 3+ 3+ Nil Astoria 3+ 3+ 3+ 33+ 33+ 3+ Nil Atem 3 3 33+ 33+ 33+ 3+ Nil Baronesse 3+ 3+ 3+ 3+ 3+ 3+ Nil Belfor 3 33+ 33+ 33+ 33+ 3+ Nil BM9647-69 3+ 3+ 3+ 3+ 3+ 3+ Nil Bracken 3+ 3+ 3+ 3+ 3+ 3+ Nil Brenda 3+ 3+ 3+ 3 3 33+ Nil Canada 110 3+ 3+ 3+ 3+ 3+ 3+ Nil Caravela 3 3 33+ 3C 3C 3+ Nil Chalice 3+ 3+ 3+ 33+ 33+ 3+ Nil Chieftain 3+ 3+ 3+ 33+ 33+ 3+ Nil CI 9819 3+ 3+ 3+ 3+ 3+ 3+ Nil Corniche 3+ 3+ 3+ 3+ 3+ 3+ Nil Corvette 3+ 3+ 3+ 33+ 33+ 3+ Nil

APR against P. hordei

CPI 36396 A 33+ 33+ 3+ 3+ 3+ 3+ Nil Decanter 3+ 3+ 3+ 3 3 33+ Nil Derkado 3+ 3+ 3+ 33+C 3+ 3+ Nil Egmont 33+ 33+ 3+ 3+ 3+ 3+ Nil Emir 3+ 3+ 3+ 3+ 3+ 3+ Nil Esperance 3+ 3+ 3+ 3+ 3+ 3+ Nil Expres 3+ 3+ 3+ 3 3 3+ Nil Farmington 33+ 33+ 3+ 3+ 3+ 3+ Nil Galleon 3+ 3+ 3+ 3+ 3+ 3+ Nil Gilbert 3+ 3+ 3+ 3+ 3+ 3+ Nil Giza 127 3+ 3+ 3+ 33+ 33+ 3+ Nil Giza 128 3+ 3+ 3+ 33+ 33+ 3+ Nil Glacier/Titan 3+ 3+ 3+ 3+ 3+ 3+ Nil GSHO 1436 3 3 33+ 3 33+ 33+ Nil GSHO 1452 3 3 33+ 3 3 3+ Nil Harriot 3+ 3+ 3+ 33+ 33+ 3+ Nil Hassan 3+ 3+ 3+ 33+ 33+ 3+ Nil HOR 2410 3+ 3+ 3+ 3+ 3+ 3+ Nil HOR 9696 3 3 33+ 3 3 3+ Nil I97-336R 3+ 3+ 3+ 3+ 3+ 3+ Nil ICB82-0114-6AP-0AP 3 3 3 3 3 3+ Nil ICB83-0157-10AP-0TR-0AP-7AP-1APH-0AP 3+ 3+ 3+ 3+ 3+ 3+ Nil

APR against P. hordei

ICB88-1560-11AP-1APH-1APH-2AP-0TR-0AP 3+ 3+ 3+ 3+ 3+ 3+ Nil ICB94-0705-AP-4AP 33+ 33+ 33+ 33+ 33+ 3+ Nil Ketch 3+ 3+ 3+ 3+ 3+ 3+ Nil Klimek 3C 3C 3+ 3+ 33+ 3+ Nil Laura 33+ 3+ 3+ 3+ 3+ 3+ Nil Maris Mink 3+ 3+ 3+ 33+ 33+ 3+ Nil M-Q-54 3+ 3+ 3+ 3+ 3+ 3+ Nil Nord GS1749 3+ 3+ 3+ 33+ 33+ 3+ Nil Nordus 3+ 3+ 3+ 33+ 33+ 3+ Nil Olbran 3+ 3+ 3+ 3+ 3+ 3+ Nil Optic 33+ 33+ 3+ 3+ 3 3+ Nil Orge 403-9 3 3C 3+ 33+ 33+ 3+ Nil Pewter 3+ 3+ 3+ 33+ 33+ 3+ Nil Pompadour 3+ 3+ 3+ 33+ 3C 3+ Nil Pusa 20 3+ 3+ 3+ 3+ 3+ 3+ Nil SB01675 3+ 3+ 3+ 3+ 3+ 3+ Nil SB02420 3+ 3+ 3+ 3+ 3+ 3+ Nil SE627.02 3+ 3+ 3+ 3 3 33+ Nil Shenmai 3 3+ 3+ 3+ 3+ 3+ 3+ Nil SHN094 3+ 3+ 3+ 3+ 3+ 3+ Nil SHN193 3+ 3+ 3+ 3+ 3+ 3+ Nil Tennessee Winter Coast Selection 33+ 33+ 3+ 3+ 3+ 3+ Nil

APR against P. hordei

TR03273 3 3 3+ 33+ 33+ 3+ Nil TR03274 3 3 33+ 3 3 3+ Nil Trait d'Union 3+ 3+ 3+ 3+ 3+ 3+ Nil Tweed 3+ 3+ 3+ 33+ 33+ 3+ Nil UC 711 74-2 3+ 3+ 3+ 3+ 3+ 3+ Nil Universe 33+ 33+ 3+ 33+ 33+ 3+ Nil Ursa 3+ 3+ 3+ 33+ 33+ 3+ Nil Uta 33+ 33+ 3+ 3+ 3C 3+ Nil UWA Late Selection 8861 3+ 3+ 3+ 3+ 3+ 3+ Nil UWA Selection 1901 3+ 3+ 3+ 3+ 3+ 3+ Nil UWA Selection 1968 3+ 3+ 3+ 3+ 3+ 3+ Nil UWA Selection 3849 3 33+ 3+ 3+ 33+ 3+ Nil UWA Selection 4886 3+ 3+ 3+ 3+ 3+ 3+ Nil UWA Selection 4887 33+ 33+ 33+ 33+ 33+ 3+ Nil UWA Selection 4900 33+ 33+ 3+ 3+ 33+ 3+ Nil UWA Selection 8939 33+ 33+ 3+ 3+ 33+ 3+ Nil UWA Selection 8951 2++3 2++3 33+ 3 3 3+ Nil UWA Thin Seed Selection 8861 3+ 3+ 3+ 3+ 3+ 3+ Nil WI 3407 3+ 3+ 3+ 3+ 3+ 3+ Nil WI-2553 3+ 3+ 3+ 3+ 3+ 3+ Nil Xi-An 2007 3+ 3+ 3+ 3+ 3+ 3+ Nil Xi-An 91-2 3+ 3+ 3+ 3+ 3+ 3+ Nil

APR against P. hordei

Yale 2++3 2++3 3 3 3 3+ Nil Zhepi 2 3+ 3+ 3+ 3+ 3+ 3+ Nil Nil: No detectable seedling resistance, RPostulated seedling resistance gene/s

Table 4.6 Greenhouse multipathotype testing confirmed the presence of seedling resistance gene Rph3 in three barley genotypes Cultivar/Line 253P- 200P- 5653P+ 5652P+ 4673P+ 5457P+ Postulated gene(s) Birte 0;- 0;- 0;- 0;- 0;- 3+ Rph3 Roland 0;= 0;= 0;= 0;N 0;N 3+ Rph3 UWA Selection 4884 0;- 0;- 0;- 0;- 0;- 3+ Rph3 Estate 0; 0; ;C ;C ;- 3+ Rph3

Table 4.7 Responses of 23 barley genotypes postulated to carry unidentified seedling resistance to six pathotypes of Puccina hordei Postulated Cultivar/Line 253P- 200P- 5653P+ 5652P+ 4673P+ 5457P+ gene(s) Argentine 2 0;CN 0;CN ;1=CN ;1=CN ;1=CN 2++C USR Beecher 0; 0; 2++C 2++C 2++C 3C USR Carbo 0; 0; ;1+ ;1+ ;1+ ;1+ USR Casino 2CN 2CN 2++CN 1+2+CN 2++CN 3=C USR CI4976 0;1- 0;1- ;1C ;1C ;1+ 2++ USR Felicie 1+CN 2CN 2CN 2CN 2++CN 2++C USR HB369 0;1- 0;1- ;1+ ;1+ ;1+ 2++ USR

APR against P. hordei

HOR 3877 0;C 0;C ;1C ;1C ;1C ;1-C USR Hydrogen 0;1+ 0;1++ 2++ 2++ 2++ 2++ USR Mackay 0;C 0; ;1- ;1- ;1- 2++3 USR Orzo-Nudo-Portignano 0;C 0;C ;1+C ;1+C ;1+C ;1+2C USR Pusa 7 0; 0; 0; 0; 0; 2++3 USR Quasar 0; 0;C 1- 1- 1- 2++3 USR Ricardo ;12++C X++ ;C 2++3C 2++ 1++C+ USR Scald and Powdery Mildew Res. Selection ;1 ;1 11+ 11+ 2++ 3+ USR SE632.02 0;C 0;C 1C 1C 1C 2++C USR SE644.02 0;1=C 0; ;1- ;1- ;1- 2++ USR Tifang 3- 3= 3- 3 3 3 USR USA 10274 0;C 0;C ;1+C ;1+C ;1+C ;1++C USR USA 8279-1640 0;CN 0;CN ;1-CN ;1-CN ;1-CN 1++2C USR UWA Selection 4685 0;1 0;1 11+C 11+C 11++ 2++ USR VB9935 ;1- ;1- ;11-C ;11-C ;1++ 3+ USR WAU 4633 ;1 ;1 1++ 1++ 12++ 3+ USR USR: Uncharacterised seedling resistance

APR against P. hordei

Resistance under field conditions All 113 barley genotypes were tested against P. hordei pathotypes under field conditions at two different field sites (“Karalee” and “Horse Unit”) for three years (2007–2009). To assess critically the effectiveness of the seedling resistance detected in the field and the potential presence of APR, the genotypes were divided into two groups: firstly, those that were seedling resistant to all pathotypes used in the field; secondly, those that were seedling susceptible to at least one pathotype used in the field.

Field response of lines carrying uncharacterised seedling resistance While uncharacterised seedling resistance was identified in 23 lines, three of these were seedling susceptible to pt 5457P+ (viz. Scald and powdery mildew resistance selection, WAU 4633 and VB 9935). These three lines were therefore included in the second group, along with the three lines postulated to carry Rph3, also ineffective against pt 5457P+ (see below).

The susceptible control Gus produced the highest AUDPC range of 665–1155 (Table 4.8). The 20 lines that were seedling resistant against all pathotypes used in the field all showed high levels of resistance (AUDPC 0.0–94.5) in the field, except Hydrogen and Tifang, which had moderate to low levels of resistance. The seedling gene/s present in these genotypes therefore represents potentially new and useful sources of resistance against P. hordei.

Field response of lines lacking seedling resistance effective in the field Of the 93 genotypes that were susceptible to at least one of the six pathotypes used in seedling tests, six were susceptible only to pt 5457P+. The potential presence of APR in these six genotypes could therefore only be assessed critically based on data from 2009, when pt 5457P+ was used in both field nurseries (Table 4.9). The three genotypes postulated to carry Rph3 were all highly resistant at adult plant growth stages in 2007 and 2008, most likely due to the effect of Rph3 in 2009 when this gene was ineffective, were all susceptible with AUDPC values of over 500 (Table 4.9). The three genotypes carrying unknown seedling resistance were less resistant in 2007 and 2008, indicating that the seedling resistance present was less effective when challenged with avirulent pathotypes. While all displayed moderate to low levels of resistance at adult plant stage in 2009 (Table 4.9), further testing is required to assess the value of this resistance.

APR against P. hordei

Table 4.8 Field responses of cultivars/lines with uncharacterised seedling resistance effective against six pathotypes of Puccinia hordei AUDPC Horse Unit AUDPC Karalee Cultivar/Line 2007 a 2008 a 2009 c 2007 b 2008 b 2009 c Argentine 2 0.6 0.1 0.6 0.1 0.6 0.6 Beecher 0.1 2.1 53.9 10.6 2.1 21.0 Carbo 0.0 2.1 0.0 5.3 22.6 0.5 Casino 0.3 0.1 1.1 0.1 0.1 0.4 CI4976 0.0 0.1 0.0 0.0 0.1 0.0 Felicie 0.1 0.1 0.4 0.1 0.1 0.1 HB369 0.6 6.5 6.5 10.6 54.1 6.5 HOR 3877 0.6 2.1 0.0 10.6 21.1 0.4 Hydrogen 47.4 178.5 204.8 78.9 273.0 178.5 Mackay 84.0 17.3 31.5 84.0 12.1 1.6 Orzo-Nudo-Portignano 0.6 0.1 0.1 10.6 32.6 0.1 Pusa 7 0.0 0.1 63.0 42.0 2.1 94.5 Quasar 0.6 43.1 1.1 21.1 94.5 1.1 Ricardo 0.0 0.0 0.0 0.0 0.0 0.0 SE632.02 0.0 2.1 0.0 21.0 22.6 0.0 SE644.02 0.5 12.1 0.0 10.5 43.6 0.2 Tifang 199.5 336.0 483.0 115.5 336.0 438.0 USA 10274 0.0 0.1 0.0 0.0 0.1 0.0 USA 8279-1640 0.3 0.6 2.1 6.3 22.6 0.0

APR against P. hordei

UWA Selection 4685 42.5 64.6 22.6 53.0 53.6 1.9 Gus (susceptible control) 665.0 1085.0 1190.0 665.0 1155.0 910.0 a Pathotype 5653P+, b Pathotype 5652P+, c Pathotype 5457P+

Table 4.9 Field responses of barley genotypes with seedling resistance ineffective against Puccinia hordei pathotype 5457P+ AUDPC Horse Unit AUDPC Karalee Characterised Cultivar/Line 2007 a 2008 a 2009 c 2007 b 2008 b 2009 c Resistance Birte 0.0 0.0 595.5 0.0 0.0 595.5 Rph3 Roland 0.1 0.1 752.5 0.1 0.1 812.0 Rph3 UWA Selection 4884 21.1 22.1 840.5 21.1 2.1 840.5 Rph3 Scald and Powdery Mildew Res. Selection 31.5 2.1 199.5 78.8 22.6 215.3 ? WAU 4633 242.0 178.5 252.5 126.5 441.0 127.1 ? VB9935 409.5 483.0 472.5 304.5 483.0 273.5 ? Gus 665.0 1085.0 1190.0 665.0 1155.0 910.0 Susc. a Pathotype 5653P+, b Pathotype 5652P+, c Pathotype 5457P+, Susc.: susceptible

APR against P. hordei

Of the remaining 87 genotypes, 19 were susceptible in the field in all three years (AUDPC > 500; Table 4.10) and hence were concluded to be of no use as potential sources of resistance to P. hordei. The remaining 68 genotypes carried different levels of APR based on susceptibility in seedling greenhouse tests and resistance in adult plant field tests with one or more pathotype (Table 4.11 and 4.12).

To assess for the likely presence or absence of the APR gene Rph20, the 68 genotypes found to carry APR were genotyped using marker bPb-0837. The marker amplified a 245 bp bands (Fig 4.1) in 35 lines, indicating the likely presence of Rph20 (Table 4.11). With the exception of Emir, all of these genotypes displayed very high levels of APR in all three years (Table 4.11). Although the AUDPC values for Emir were higher than the other genotypes in this group in 2007 and 2008, it still displayed high levels of APR. In 2009, however, Emir displayed AUDPC values of 483.5 and 472.5, regarded as very minor APR (Table 4.11). This indicates that these 34 lines expressing high levels of APR may carry additional gene(s) compared to Emir.

The remaining 33 lines did not amplify a product when tested with marker bPb-0837, indicating that they may carry uncharacterised, new APR to leaf rust (Table 4.12). These genotypes displayed a range of APR levels, with Egmont, GSHO 1452 and Universe showing consistently high levels of APR over the three years, with AUDPC ranges of 0.1–12.1, 0.6– 33.1 and 1.6–21.0, respectively (Table 4.12). Because disease severity was more consistent during 2009 at both field sites, average of AUDPC values from this year from the two field sites were used to classify each genotype as carrying high (AUDPC 0.1–141.7; Egmont, Universe, GSHO 1452, Farmington, UWA Selection 4887, HOR 2410, Belfor and UWA Selection 4886), medium (AUDPC 152.2–298.5; GSHO 1436, Atem, SB02420, UWA Selection 8939, Gilbert, Glacier/Titan, Optic, UWA Selection 3849, HOR 9696, Caravela and TR03274), or low (AUDPC 317.6–475.3; Uta, UWA Selection 8951, Tennessee Winter Coast Selection, UWA Selection 4900, ICB94-0705-AP-4AP, Klimek, Laura, ICB82-0114- 6AP-0AP, Orge 403-9, Yale, TR03273, CPI 36396 A, ICB88-1560-11AP-1APH-1APH- 2AP-0TR-0AP and Andapi 3) levels of APR (Fig. 4.2).

APR against P. hordei

Table 4.10 Field responses of 19 barley genotypes that were susceptible to Puccinia hordei at both seedling and adult plant growth stages AUDPC Horse Unit AUDPC Karalee Cultivar/Line 2007 a 2008 a 2009 c 2007 b 2008 b 2009 c 63081 532.0 875.0 717.5 525.0 875.0 805.4 AB30 525.0 588.0 665.5 519.8 840.0 528.5 BM9647-69 506.6 574.0 700.5 540.8 595.0 700.5 Bracken 566.1 980.0 901.3 567.0 514.5 735.4 Canada 110 1015.0 805.0 805.0 910.0 805.0 805.0 CI 9819 722.8 805.0 805.0 682.5 805.0 805.0 I97-336R 875.0 945.0 507.5 805.0 980.0 560.5 Ketch 784.0 875.0 805.5 679.0 875.0 840.5 Pusa 20 770.0 980.0 805.5 638.8 1120.0 577.5 Shenmai 3 743.8 805.0 770.5 603.8 805.0 665.0 SHN094 743.8 945.0 782.3 540.8 840.0 735.0 SHN193 742.0 1050.0 883.8 525.0 1085.0 763.0 Trait d'Union 591.5 735.0 700.5 507.5 539.0 770.0 UC 711 74-2 805.0 1015.0 906.5 735.0 910.0 887.3 UWA Selection 1901 556.5 945.0 535.5 591.5 910.0 560.0 UWA Selection 1968 521.5 805.0 638.8 528.5 735.0 533.8 Xi-An 2007 700.0 1155.0 805.5 630.0 1155.0 770.5 Xi-An 91-2 805.0 945.0 735.5 665.0 1050.0 770.0 Zhepi 2 609.0 1050.0 817.3 609.0 1085.0 883.8

APR against P. hordei

Gus (Susceptible control) 665.0 1085.0 1190.0 665.0 1155.0 910.0 a Pathotype 5653P+, b Pathotype 5652P+, c Pathotype 5457P+

Table 4.11 Field responses of barley genotypes likely to carry adult plant resistance gene Rph20 based on the AUDPC calculated from the results of three years (2007–2009) testing at two field sites and greenhouse testing against Puccinia hordei and genotyping with Rph20 linked marker bPb-0837 AUDPC Horse Unit AUDPC Karalee Marker Cultivar/Line 2007 a 2008 a 2009 c 2007 b 2008 b 2009 c bPb-0837 74043 0.1 33.1 53.6 31.6 53.0 43.1 + 115-9505-B 0.1 0.9 0.0 5.4 22.1 0.1 + Astoria 0.0 2.1 0.0 0.5 17.3 0.0 + Baronesse 2.1 2.1 0.7 2.1 2.1 0.2 + Brenda 10.6 6.8 0.0 21.1 22.6 0.0 + Chalice 5.4 6.8 2.5 21.1 22.6 0.7 + Chieftain 10.6 2.1 0.0 10.6 22.6 0.0 + Corniche 0.1 0.1 0.0 0.6 0.1 0.0 + Corvette 1.6 2.1 0.3 2.1 33.1 0.1 + Decanter 0.1 2.1 0.0 10.6 27.8 0.0 + Derkado 0.6 2.1 0.0 0.6 6.8 0.4 + Emir 84.1 85.6 483.5 105.1 106.6 472.5 + Esperance 0.8 2.6 1.1 5.4 21.9 0.1 +

APR against P. hordei

Expres 0.1 6.8 1.6 5.4 22.6 1.6 + Galleon 1.9 2.3 1.6 6.8 22.6 1.6 + Giza 127 0.0 2.1 0.5 0.5 12.1 0.5 + Giza 128 0.1 2.1 2.1 0.6 17.3 0.6 + Harriot 10.5 0.1 0.6 21.0 10.6 0.0 + Hassan 0.1 1.6 0.5 0.6 22.1 0.2 + ICB83-0157-10AP-0TR-0AP-7AP-1APH-0AP 0.6 1.6 0.6 0.6 22.6 0.2 + Maris Mink 0.1 3.0 1.2 10.6 12.1 0.6 + M-Q-54 0.5 12.1 0.0 10.5 17.3 0.2 + Nord GS1749 0.6 2.1 1.1 21.1 6.8 0.3 + Nordus 0.1 22.6 2.1 10.6 33.1 0.0 + Olbran 0.6 12.1 1.1 28.1 22.6 0.2 + Pewter 0.1 2.1 1.1 0.6 2.1 0.5 + Pompadour 0.1 0.1 0.5 0.1 0.1 0.1 + SB01675 22.1 3.7 6.8 22.6 21.5 0.6 + SE627.02 0.0 0.0 0.0 10.5 2.1 0.1 + Tweed 0.1 0.1 0.5 0.6 0.1 0.1 + Ursa 0.0 2.1 0.6 10.5 22.6 0.1 + UWA Late Selection 8861 2.8 4.9 2.1 21.5 4.9 2.5 + UWA Thin Seed Selection 8861 63.6 37.5 64.4 0.6 2.1 53.2 + WI 3407 2.1 2.1 0.0 2.1 2.1 0.0 + WI-2553 5.4 12.1 1.6 5.4 53.0 0.2 +

APR against P. hordei

Gus (Susceptible control) 665.0 1085.0 1190.0 665.0 1155.0 910.0 - a Pathotype 5653P+, b Pathotype 5652P+, c Pathotype 5457P+, +: Marker present, -: Marker absent

Table 4.12 Field responses of barley genotypes identified as likely carrying uncharacterised adult plant resistance to leaf rust based on the AUDPC calculated from the results of three years (2007–2009) testing at two field sites and in the greenhouse against Pucccinia hordei and lacking the Rph20 linked marker bPb-0837 AUDPC Horse Unit AUDPC Karalee Marker Cultivar/Line 2007 a 2008 a 2009 c 2007 b 2008 b 2009 c bPb-0837 BelforH 105.1 367.5 189.0 126.1 168.0 33.1 - EgmontH 0.1 2.1 0.1 10.6 12.1 0.1 - FarmingtonH 42.1 64.6 84.5 42.1 186.4 32.6 - GSHO 1452H 5.4 2.1 0.6 10.6 17.3 33.1 - HOR 2410H 105.1 0.1 63.1 47.4 1.6 157.5 - UniverseH 10.5 12.1 2.1 21.0 12.1 1.6 - UWA Selection 4886H 84.1 54.1 94.5 84.1 392 189 - UWA Selection 4887H 10.5 94.5 48.8 131.3 378 148.1 - AtemM 105.1 199.5 210.5 84.1 451.5 111.5 - CaravelaM 173.3 215.3 126.5 236.3 483.0 441.0 - GilbertM 278.3 283.5 330.8 315.0 199.5 63.7 - Glacier/TitanM 105.0 115.5 283.5 105.0 168.0 147.0 - GSHO 1436M 241.5 147.0 210.0 278.3 357.0 94.5 - HOR 9696M 353.5 189.5 127.6 388.5 406.0 336.5 -

APR against P. hordei

OpticM 178.5 388.5 304.5 157.5 210.0 147.5 - SB02420M 175.0 168.0 176.6 175.0 469.0 210.0 - TR03274M 210.1 388.5 211.6 175.1 441.0 385.5 - UWA Selection 3849M 10.6 399 245 245.1 451.5 210.1 - UWA Selection 8939M 210 147.5 176.1 245 490 211.1 - Andapi 3L 157.5 446.3 488.3 53.0 490.0 462.4 - CPI 36396 AL 413.0 257.3 499.3 308.0 446.3 446.3 - ICB82-0114-6AP-0APL 418.3 443.6 399.5 348.3 469.0 441.0 - ICB88-1560-11AP-1APH-1APH-2AP-0TR-0APL 64.6 483.0 476.0 80.3 371.0 473.0 - ICB94-0705-AP-4APL 21.0 498.8 368.0 175.0 399.0 374.5 - KlimekL 63.1 63.0 435.8 105.1 241.5 336.5 - LauraL 237.3 441.0 322.0 245.1 441.0 472.5 - Orge 403-9L 371.0 402.5 438.0 267.8 241.5 403.0 - Tennessee Winter Coast SelectionL 477.8 315.0 348.3 446.3 477.8 336.0 - TR03273L 175.1 399.0 420.5 245.1 234.5 462.5 - UtaL 84.1 147.0 367.5 126.1 220.5 267.8 - UWA Selection 4900L 459.4 472.5 406.5 294 141.8 287 - UWA Selection 8951L 105.1 294 367.5 175.1 94.5 315 - YaleL 385.0 488.3 423.5 147.0 483.0 420.5 - Gus (susceptible control) 665.0 1085.0 1190.0 665.0 1155.0 910.0 - a Pathotype 5653P+, b Pathotype 5652P+, c Pathotype 5457P+, H High APR, M Moderate APR and L Low APR, -: Marker absent

APR against P. hordei

Table 4.13 Pedigree analyses of barley cultivars/lines characterised with adult plant resistance against P. hordei pathotypes and tested positive for the presence of marker bPb-0837 linked to the adult plant resistance Rph20 Possible APR derivative Cultivar/Line based on ancestry Gull Astoria, Galleon Gull, Erectoides 16 (Maja 74043 X-ray Mutant) Gull, Diamant Chieftain, WI 3407 Gull, H. laevigatum Casino, Corvette, Emir, Felicie, Hassan, Maris Mink, Pompadour Gull, H. laevigatum, Brenda, Chalice, Corniche, Decanter, Derkado, Harriot, Nordus, Tweed,Ursa Diamant Gull, H. laevigatum, Baronesse Vada Mutant Bavaria, Diamant Expres Unknown 115-9505-B, Esperance, Giza 127, Giza 128, ICB83-0157-10AP-0TR-0AP-7AP-1APH-0AP, M-Q-54, Olbran, SE627.02, Pewter, SB01675, UWA Late Selection 8861, UWA Thin Seed Selection 8861, WI-2553, Nord GS1749 Ancestral pedigree information sourced from: (BBSRC 2002) and http://genbank.vurv.cz/barley/pedigree/pedigree.asp

APR against P. hordei

Table 4.14 Pedigree analyses of barley cultivars/lines predicted to carry uncharacterised adult plant resistance against P. hordei Possible APR derivative based on ancestry Cultivar/Line Bavaria Caravela Manchuria, Taganrog Glacier/Titan Gull, H. laevigatum Atem, Belfor, Egmont, Farmington, Gilbert, Universe, Uta Gull, H. laevigatum, Diamant Klimek, Optic Gull, Bonus X-ray Mutant Laura Ancestral pedigree information sourced from: (BBSRC 2002) and http://genbank.vurv.cz/barley/pedigree/pedigree.asp

100

APR against P. hordei

Discussion The recent detection of virulence for the seedling resistance gene Rph3 (Park 2010), along with previously detected virulences for Rph4 carried by cultivars like Grimmett (Cotterill et al. 1995) and Rph12 carried by cultivars like Franklin, Tallon, Lindwall and Fitzgerald (Park 2008) and the effectivness of only six resistance genes (viz. Rph7, Rph11, Rph14, Rph15, Rph18 and Rph20) in Australia, together highlight the importance of finding and deploying durable resistance to leaf rust. Accordingly, the present study was undertaken to search for new sources of resistance to leaf rust for use in barley improvement. Considerable effort was made to characterise the genotypes for APR to leaf rust in an attempt to identify potentially new sources for use in combination with the only known gene conferring APR to leaf rust in barley, Rph20.

Most of the 23 barley cultivars/lines characterised with unidentified seedling resistance were highly resistant in the field (Table 4.7). This indicates that the seedling resistance gene/s present in these genotypes are highly effective under field conditions. Both Casino and Felicie were also reported to be resistant under greenhouse conditions against a range of P. hordei pathotypes by Golegaonkar et al. (2009b) and shown to likely carry Rph20 based on the presence of the linked marker bPb-0837 (Singh D., personal communication). Combining major seedling resistance genes with APR has been proposed as a strategy in breeding for durable rust resistance in wheat (Park 2008) and so both of these cultivars may be useful donors of durable resistance to leaf rust. Genetic analyses of the seedling resistances along with assessments of potential APR in Casino and Felicie are needed to fully assess their value in resistance breeding.

Other cultivars/lines characterised with unidentified seedling resistance, including Hydrogen, Powdery mildew resistance selection, Tifang, WAU4633 and VB 9935, displayed moderate to low levels of resistance under field conditions (Table 4.7). Pedigree information was not available for most of these lines and so it was not possible to explore the origin of the resistance present in each and their relationships with each other. Cultivar Beecher was found to be derived from the cross Atlas/Vaughn, similar to Glacier (Atlas/Vaughn), a parent in the line Glacier/Titan. All include Breustedt, Schladener, Taganrog (South Russian selection) and Club Mariout (Egyptian variety selection) from the parent Vaughn. In view of the distinctiveness of the seedling resistance in many of these lines, plus its highly effective 101

APR against P. hordei nature under field conditions, genetic analyses of these potentially new sources of seedling resistance seem warranted.

Cultivars Birte, Roland and line UWA selection 4884 were resistant against pts 5652P+ and 5653P+ in the greenhouse and in the field (2007 to 2008) and susceptible against the Rph3 virulent pt 5457P+ both in the greenhouse and field (2009). These lines were considered to carry seedling gene Rph3 only. The presence of Rph3 in cultivar Roland is consistent with earlier multipathotype gene postulation studies by Golegaonkar et al. (2009b), which were undertaken in the absence of a pathotype virulent for Rph3. None of these cultivars were found to carry residual APR when field tested with pt 5457P+ in 2009.

2,048.00 1,024.00 512.00 256.00 128.00 A 64.00 U 32.00 16.00 High APR Moderate APR Low APR D 8.00 4.00 P 2.00 1.00 C 0.50 0.25 0.13 0.06

Barley Genotypes With Unknown APR

With the exception of Emir, all 35 barley genotypes that were found to carry APR and marker bPb-0837 linked to Rph20 displayed very high levels of APR in all three years (Table 4.11). While Emir displayed high levels of APR in 2007 and 2008, it became more heavily rusted in

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2009 and displayed low APR only. The reasons for the higher AUDPC values in 2009 are not clear and could be due to either a sowing error or a combination of higher disease pressure and the aggressiveness of pt 5457P+ used in 2009. This cultivar has been reported in previous studies to carry partial resistance to P. hordei (Parlevliet 1979; Parlevliet 1983).

Although the pedigrees of most cultivars carrying the marker bPb-0837 (Astoria, Baronesse, Brenda, Casino, Chalice, Chieftain, Corniche, Corvette, Decanter, Derkado, Emir, Felicie, Galleon, Harriot, Hassan, Maris Mink, Nordus, Pewter, Pompadour, Tweed, Ursa, SB01675, 74043, WI 2553 and WI 3407) are different (Table 4.1), a detailed analysis revealed that all include Gull and H. laevigatum (Table 4.13). The pedigree of Expres includes Bavaria (a German selection) and Diamant (an X-ray mutant of Valticky barley). While the cultivar Caravela lacking the Rph20-linked marker bPb-0837, was also found to trace back to Bavaria (Table 4.14), it showed lower levels of APR (AUDPC values 126.5–483) than did Expres (AUDPC values 0.1 to 22.6), suggesting that the gene(s) conferring APR in these cultivars differ and that the high levels of APR in cultivar Expres may have originated from the X-ray mutant Diamant, which was also found to be present in the pedigrees of Brenda, Chalice, Chieftain, Corniche, Decanter, Derkado, Expres, Harriot, Nordus, Tweed, Ursa and WI 3407. Given that Gull was reported as susceptible to P. hordei (Golegaonkar et al. 2009b), these results suggest that the APR present in many of these genotypes may have originated from H. laevigatum. The presence of Diamant in many of the other gneotypes that were positive for marker bPb-0837 is consistent with this X-ray mutant also being a common source of Rph20.

Field tests of leaf rust response over three years, in combination with seedling leaf rust tests, revealed varying levels of APR in 33 genotypes that lacked the marker bPb-0837 (Fig. 4.2). Given that the linkage between marker bPb-0837 and Rph20 is not complete, it is possible that some or all of these genotypes carry Rph20 in the absence of the marker. It is also possible that these genotypes carry new and uncharacterised APR to leaf rust. Like many of the cultivars that carried both the marker and APR, the genotypes Belfor, Egmont, Farmington and Universe all displayed high levels of APR and were found to have Gull and H. laevigatum in their pedigrees. This further suggests that the APR in all of these genotypes, only some of which carry bPb-0837 could be the same. Clearly, genetic studies including tests of allelism using stocks known to carry Rph20 [e.g. Vada, Nagrad, Patty, Pompadour (Golegaonkar et al. 2010); Flagship (Hickey et al. 2011)], are needed to resolve this. 103

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The genetic bases of the APR identified in the present study are unknown. Several studies of APR to stripe rust in wheat have revealed that high levels of APR can be conferred by the presence of multiple, additive genes (Pathan et al. 2007; Singh et al. 2000). Consistent differences in the level of APR across both years and sites was observed in the present study among barley genotypes lacking marker bPb-0837 (e.g. Egmont (high APR), Optic (moderate APR) and Yale (low APR), suggesting the presence of different APR genes and possibly additivity among these genes. Recently, the QTL qRphND present in barley line ND24260 was mapped to chromosome 6HL and reported to be highly effective in combination with APR gene Rph20 against P. hordei at adult plant stages (Hickey et al. 2011). Targeted genetic studies are now needed to understand the genetic control of these resistances, not only the number of genes present but also potential additivity among them and other sources of effective resistance, both seedling (Rph7, Rph11, Rph14, Rph15, Rph18) and APR (Rph20).

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CHAPTER V Characterisation of rust resistance in four international barley nurseries

Abstract Barley nurseries comprising 820 lines with 579 unique pedigrees were sourced from the International Centre for Agricultural Research in the Dry Areas (ICARDA) and screened for resistance against Australian isolates of Puccinia hordei, P. graminis f. sp. tritici (Pgt) and barley grass stripe rust (BGYR). Ninty three percent of the lines were postulated to carry the seedling leaf rust resistance gene Rph3 based on their susceptibility in the greenhouse and field against Rph3 virulent pathotype (pt) and resistance against Rph3 avirulent pathotypes (pts). Remaining 0.65% lines showed uncharacterised seedling resistance (USR), 0.75% carried uncharacterised adult plant resistance (UAPR) and 5.6% were susceptible at both growth stages. Of the six lines identified to carry UAPR, three likely carried Rph20 based on the presence of the Rph20-linked marker bPb-0837. Based on tests with several control genotypes, marker bPb-0837 was more reliable than Ebmag0833 in detecting the presence of Rph20. All lines were resistant against Pgt pt 98-1,2,3,5,6 and BGYR when tested as seedlings in the greenhouse. The results suggested that most of the ICARDA germplasm tested is not suitable for leaf rust resistance under Australian conditions due to the presence of virulence for Rph3. Further genetic analysis of the lines carrying UAPR and USR is proposed.

Keywords Hordeum vulgare, barley leaf rust, stem rust, BGYR, adult plant resistance, Rph3

Introduction Cultivated barley (Hordeum vulgare L. subsp. vulgare) ranks fourth in the world’s production after wheat, maize and rice (Schulte et al. 2009). It is grown widely in Australia, where it is an important multi-billion dollar industry (GRDC 2005). The gross value of barley production in Australia is, however, hampered by many constraints, of which diseases alone account for an estimated average annual loss of $252 million (Murray and Brennan 2010). Of the diseases that afflict barley, leaf rust (caused by Puccinia hordei Otth.) is considered to be most destructive in many parts of the world (Clifford 1985). Significant losses due to leaf rust epidemics have been reported in Australia, New Zealand, Europe and USA (Arnst et al. 1979; Cotterill et al. 1992; Griffey et al. 1994; Melville et al. 1976). Many of the known 105

Resistance among IBONs seedling leaf rust resistance genes have been rendered ineffective by the emergence of new pts of P. hordei with matching virulence (Park 2003). During the years 1992 to 2001, eight new pts, each virulent for Rph12, were detected in Australia (Park 2008) and recently, following the release of several barley cultivars with Rph3, a new pt with virulence matching Rph3 (5457P+) was detected (Park 2010). Currently, only five seedling resistance genes (Rph7, Rph11, Rph14, Rph15 and Rph18) are effective in Australia. In this context, several previous studies (Golegaonkar et al. 2009b; Park 2003; Park 2008) stressed the need to identify new sources of resistance to leaf rust in barley, including adult plant resistance (APR). Recently, the first gene conferring APR to leaf rust in barley, Rph20, was mapped on chromosome 5HS (Hickey et al. 2011). Two markers linked to Rph20, EBmag0833 and bPb- 0837, were reported by Liu et al. (2010), who proposed the use of bPb-0837 in marker assisted selection for APR against P. hordei.

In addition to barley leaf rust, in the presence of heavy inoculum, stem rust caused by either P. graminis Pers. f. sp. tritici Eriks. & E. Henn. (Pgt), P. graminis Pers. f. sp. secalis Eriks. & E. Henn. (Pgs), or the scabrum rust (Park 2008), can affect barley in Australia. Stem rust of barley in North America was managed by using the resistance genes Rpg1, Rpg2, Rpg3 and rpg4 against Pgt and RpgU, RpgBH and RpgQ (Rpg5) against Pgs (Sun and Steffenson 2005). In the U.S.A. and Canada, the stem rust resistance gene Rpg1 was deployed in 1942 (Brueggeman et al. 2002) and remained effective for more than 65 years (Mirlohi et al. 2008). The role of stem rust resistance gene Rpg1 in Australia is not clear as it cannot be detected in the greenhouse or field (Park et al. 2009).

The barley stripe rust pathogen P. striiformis f. sp. hordei (Psh) does not occur in Australia (McIntosh et al. 2001; Park 2008), but poses a serious threat to the Australian barley industry as vast majority of Australia barleys were susceptible when tested in Mexico with local Psh pts (Wellings et al. 2000). Stripe rust caused by a new variant of P. striiformis (barley grass stripe rust; BGYR) (Wellings et al. 2000), can also infect some barley genotypes and wild barley grass in Australia.

The rust resistance of entries in several recent International Barley Observation Nurseries (IBONs) developed at the International Centre for Agricultural Research in the Dry Areas (ICARDA) was examined in an attempt to identify potentially new sources of seedling 106

Resistance among IBONs resistance and APR to leaf rust. Tests were also conducted to determine the responses of entries to stem rust and BGYR. Four different IBONs (31st to 34th), released from 2003 to 2006, were examined.

Materials and methods

Plant material Eight hundred and twenty lines representing four IBONs were introduced from ICARDA (Table 5.1) and screened for rust response in the greenhouse and field. The original nurseries were provided by the international nurseries program at ICARDA, Aleppo, Syria. Sets of differential lines for each rust pathogen, as described in Appendices D1, D2 and D3, were included in each experiment.

Pathogen material For seedling tests, two pts of P. hordei (5457P+ and 5652P+), one standard isolate of BGYR and one pt of Pgt (98-1,2,3,5,6) were used. In field testing, the predominant P. hordei pts were 5652P+ (2007 and 2008) and 5457P+ (2009). For multipathotype tests, four additional pts (200P-, 253P-, 5610P+ and 5653P+ +Rph13) were used. The virulence of the pts against seedling resistance (Rph) genes is detailed in Table 5.2.

Table 5.1 Details of the four International Barley Observation Nurseries introduced from ICARDA used in this study

IBON Year of release No. of lines No. of unique pedigrees

31st 2003 205 117

32nd 2004 266 190

33rd 2005 205 160

34th 2006 144 112

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Table 5.2 Detail of rust isolates used for greenhouse and field tests of four international barley nurseries Pathogen Pathotype Culture Virulence* no. P. hordei 200P- 518 Rph8 P. hordei 253P- 490 Rph1, Rph2, Rph4, Rph6, Rph8 P. hordei 5610P+ 520 Rph4, Rph8, Rph9, Rph10, Rph12, Rph19 P. hordei 5653P+ +Rph13 542 Rph1, Rph2, Rph4, Rph6, Rph8, Rph9, Rph10, Rph12, Rph13, Rph19 P. hordei 5652P+ 561 Rph2, Rph4, Rph6, Rph8, Rph9, Rph10, Rph12, Rph19 P. hordei 5457P+ 612 Rph1, Rph2, Rph3, Rph4, Rph6, Rph9, Rph10, Rph12, Rph19 BGYR 589 Yr1 P. graminis f. sp. tritici 98-1,2,3,5,6 279 Sr5, Sr6, Sr8a, Sr9b, Sr9g, Sr11, Sr17 *with respect to the resistance genes listed in Appendices D1, D2 and D3

Greenhouse screening For greenhouse tests, all lines along with differential sets were planted in pots filled with a mixture of fine bark and coarse sand and fertilised using “Aquasol®” (100 gm per 10 litre of water per 200 pots) prior to sowing. Seedlings of differentials and barley lines were raised in 9 cm diameter pots by sowing four clumps (test lines) or five clumps (differentials) of each genotype using 8–10 seeds per clump. Following sowing, pots were kept in a growth room at 20 ± 2°C for germination. Seven-day old seedlings were fertilised with granular urea using “Incitec Pivot” w/w 46% nitrogen (50 gm per 10 litre of water per 200 pots). Greenhouse inoculations were carried out on seedlings at the one and a half leaf growth stage (9–10 day old seedlings). The seedlings were moved to an enclosed chamber and urediniospores (10 to 12 mg/10 ml/200 pots) suspended in a light mineral oil (Shellsol®, Mobil Oil) were sprayed over seedlings using an aerosol hydrocarbon propellant pressure pack. The chamber door was kept closed for 5 minutes to allow urediniospores to settle on the leaves completely. Spray nozzle fittings were stored in 70% ethanol and rinsed thoroughly with tap water before each inoculation to prevent cross contamination. In addition, the inoculation chamber was washed thoroughly with pressurised tap water between successive inoculations.

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Leaf rust-inoculated seedlings were incubated for 24 hours at room temperature in a dark chamber in which continuous mist was created by an ultrasonic humidifier. Seedlings inoculated with BGYR were incubated under polythene hoods kept on stainless steel trays filled with de-chlorinated water to create a sealed environment, in darkness at 13oC for 24 hours. The inside of the hoods were sprayed with distilled water to ensure high humidity for dew formation. Stem rust-inoculated seedlings were incubated in the same manner as BGYR inoculated seedlings but using hoods under natural light at 18oC for 48 hours. The seedlings were then transferred to naturally well lit microclimate rooms maintained at 23 ± 2oC (leaf rust), 17 ± 2oC (BGYR) and 27 ± 2oC (stem rust), respectively. The infected seedlings were scored 10–12 (leaf rust) and 12–16 (stem rust and BGYR) days after inoculation using 0–4 scale as described in Appendix 1.

Field screening All lines were tested at the field site Karalee. The lines were hand-sown as hill plots (30–40 seeds/plot) using 30 cm spacing during mid to late June in 2007, 2008 and 2009. A row of the susceptible cultivar Gus was sown as a rust spreader after every five hill plots of barley lines to allow the build-up and uniform distribution of inoculum. Plots were hand weeded two to three times each season. Four weeks after sowing, plots were fertilised using granular urea “Incitec Pivot” w/w 46% nitrogen @ 100 kg/hectare followed by irrigation. Plots were irrigated once a week or as required, using fixed sprinklers.

Field epidemics of leaf rust were created following the procedures described by McIntosh et al. (1995b). Urediniospores (30–40 mg) were suspended in 1.5 L of light mineral oil (Shellsol®, Mobil Oil) and sprayed over buffer/spreader lines with an ultra-low-volume applicator (Microfit®, Micron Sprayer Ltd., UK). Four to five inoculations were performed during late evening on days that had a strong forecast of overnight dew. On the first and second inoculations, hot spots of disease were established by watering and covering small areas of the rust spreader with plastic hoods overnight to ensure adequate dew formation in case natural dew formation did not occur. Leaf rust was scored at the flag leaf growth stage in all three seasons using a modified Cobb’s scale for disease severity and host response as described in Appendix 2.

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Extraction of genomic DNA Genomic DNA was extracted from the leaf tissues of selected lines. Barley lines were sown in the greenhouse as described earlier. Leaf tissues were collected from actively growing seedlings 8–12 days after sowing. A 15 to 20 mm sample of leaf tissue from at least 10 to 15 plants per line of each genotype and controls was collected into a 2 ml Eppendorf tube. The tubes were kept for 72 hours above silica beads to desiccate leaf tissue. Two small stainless steel ball bearings were added per tube and dried leaves were crushed to powder using a Retsch MM300 Mixer Mill (Retsch, Germany) for 3 min at 25 rpm. Pre warmed (65°C) 700 µl of extraction buffer (50 mM Tris-pH 8.0, 10 mM EDTA-pH 8.0, 100 mM NaCl, SDS 1% (w/v), 10 mM ß-mercaptoethanol ) was added per tube. Samples were incubated for 10 min at 65°C and 150 µl of 3 M K-acetate (pH 5.2) was added per tube. Tubes were shaken vigorously and kept in freezer (-20°C) for 15 min. Samples were centrifuged for 15 min at 12,000 rpm and 650 µl of supernatant was transferred to new 1.5 ml tubes. An equal volume (750 µl) of chilled (-20°C) isopropanol was added per tube and the supernatant was mixed thoroughly by inverting tubes several times. Again tubes were placed in a freezer (-20°C) for 10 min for precipitation of DNA and then centrifuged at 10,000 rpm for 10 min to make a pellet of the DNA. The supernatant was discarded and the pellet was washed with 500 µl of 70% ethanol. DNA pellet was air dried and re-suspended in 200 µl of 10 mM Tris-HCl (pH 8.0). Rnase A @ 20 µl per 40 ml of 10 mM Tris-HCl was added before the re-suspension of DNA pellet. Tubes were kept in an oven (37°C) for 2 hrs to dissolve the DNA pellet properly. DNA was quantified using a Nanodrop ND-1000 spectrophotometer (Nanodrop® Technologies). All DNA samples were uniformly diluted to 50 ng/µl using doubled distilled autoclaved water (ddH2O) and stock DNA was stored in the freezer (-20°C).

Molecular markers The molecular markers bPb-0837 and EBmag0833, closely linked to the APR leaf rust resistance gene Rph20, located on chromosome 5HS, were reported by Liu et al. (2010). Molecular marker bPb-0837 amplified 245 bp fragments in barley cultivars Pompadour and null allele in Stirling and similarly, the marker Ebmag0833 amplified a 218–228 bp fragment in Pompadour and null allele in Stirling (Liu et al. 2010). Both markers were used to genotype lines selected from different nurseries and the controls. Sequence information of both primers is detailed in Table 5.3 and primers were synthesised and supplied by SIGMA (Sigma-Aldrich Pty. Ltd. Australia). 110

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Table 5.3 Detail of molecular markers used to genotype barley lines carrying adult plant resistance to leaf rust

Name Sequence Marker Size (bp) Reference

EBmag0833 F5’AAGCAATAAGTAAGGTACTCCC 3’ SSR 218 – 228 (Liu et al. R 5’ATTCACGCATCTTATGTCC 3’ 2010)

F 5’GACACTTCGTGCCAGTTTGA 3’ bPb-0837 DArT 245 (Liu et al. R 5’CCTCCCTCCCTCTTCTCAAC 3’ 2010)

PCR reaction and profiles Ten micro litres of PCR reaction contained 2.0 µl of genomic DNA (50 ng), 1.0 µl of dNTPs

(0.2 mM), 1.0 µl of 10x PCR buffer (Immobuffer, including 15 mM MgCl2), 0.25 µl of each forward and reverse primer (10 µM), 0.04 µl of Taq DNA (500 U Immolase DNA polymerase from Bioline) and 5.46 µl of ddH2O. The PCR amplification profile comprised an initial denaturation step at 95°C for 10 min, followed by 35 cycles of 30 s denaturation at 94°C, 60 s annealing at 55°C, 60 s extension at 72°C and a final extension step of 5 min at 72°C. Reaction was performed in a 96-well DNA theromocycler (Eppendorf Mastercycler, Germany). PCR products were mixed with 3.0 µl of formamide loading buffer [98% formamide, 10 mM EDTA (pH 8.0), 0.05% (wt/vol) Bromophenol blue and 0.05% xylene cyanol]. Two percent agarose gels were prepared by adding 2.0 gm agarose (Bioline) per 100 ml of 1x Tris-borate EDTA (TBE) buffer (90 mM Tris-borate + 2 mM EDTA-pH 8.0). For staining, 1.0 µl of ethidium bromide was added per 100 ml of gel solution. The gel solution was poured into moulds and allowed to cool for 40 min at room temperature. Eight to 10.0 µl of PCR product including loading buffer was loaded per well. One kb DNA marker HyperLadder™ IV (Bioline) was used as reference. Electrophoresis was carried out at 110 V for 1.5 hrs. The separated bands were visualised under ultra violet light unit fitted with a GelDoc-IT UVP Camera.

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Results

Leaf rust evaluation Eighty nine per cent (728 out of 820) of the lines tested from the four nurseries were resistant to pt 5652P+ in seedling greenhouse tests and in adult plant field tests during 2007 and 2008. All 728 lines were susceptible in the greenhouse and in the field when tested with the Rph3 virulent pt 5457P+ in 2009, indicating the very probable presence of seedling gene Rph3 only in all of these lines (Table 5.4).

Five lines (IBON 32.34, IBON 32.126, IBON 34.88, IBON 34.95 and IBON 34.126) displayed seedling resistance in greenhouse tests to all pts and were also resistant in the field against the two P. hordei pts used. Six lines (IBON 32.183, IBON 32.202, IBON 34.8, IBON 34.41, IBON 34.54 and IBON 34.110) were resistant only in the field (during all three years), indicating the presence of APR. Line numbers IBON 32.202, IBON 34.41, IBON 34.54 and IBON 34.110 showed resistant responses of TR to 10R at adult plant growth stages and line numbers IBON 32.183 and IBON 34.8 showed MR to MR–MS responses under field conditions (Table 5.9). Categories of resistance are described in Appendix 2. Adult plant responses against P. hordei pt 5457P+ under field conditions are shown in Fig. 5.3.

Table 5.4 Classification of ICARDA germplasm with respect to resistance based on field and greenhouse tests with Puccinia hordei pathotypes 5652P+ and 5457P+ Category1 IBON 31 IBON 32 IBON 33 IBON 34 Total Postulation C1 197 233 180 118 728 Rph3 C2 0 2 0 3 5 USR2 C3 0 2 0 4 6 UAPR3 C4 3 21 4 16 44 Susceptible C5 5 8 21 3 37 Missing lines 1 C1 = Lines resistant to pathotype 5652P+ and susceptible to 5457P+ both in greenhouse and field; C2 = Lines resistant to pts 5652P+ and 5457P+ both in the greenhouse and the field; C3 = Lines susceptible in the greenhouse but resistant in the field to pts 5652P+ and 5457P+; C4 = Lines susceptible in the greenhouse and the field to pts 5652P+ and 5457P+; C5 = Missing; 2 USR = Uncharacterised seedling resistance; 3 UAPR = Uncharacterised adult plant resistance

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An overall analysis of the total 783 lines tested (excluding 37 missing lines) showed that 93% carried the major seedling resistance gene Rph3, 5.6% of the lines were susceptible at both growth stages, 0.65% of the lines were resistant to both P. hordei pts at both growth stages and 0.75% of the lines possessed UAPR (Fig. 5.1).

C3 (UAPR) 6 0.75%

C1 (Rph3) C2 (USR) 728 5 93% 0.65%

C4 (Susc.) 44 5.6%

Rph3 APR Totally Resistant Totally Susceptible

Multipathotype testing The five lines in category C2 and six lines in category C3 (described in Table 5.4) were subjected to multipathotype tests in the greenhouse using six pts of P. hordei. Sets of differential lines included in these tests showed the expected infection types (ITs) against all six pts (Table 5.5).

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Barley lines IBON 34.8, IBON 34.41, IBON 34.54, IBON 34.110, IBON 32.183 and IBON 32.202, which were resistant in the field, displayed high ITs against all six pts when tested in the greenhouse, indicating that they lacked detectable seedling resistance genes and confirming the presence of UAPR in all six lines. In contrast, lines IBON 32.34, IBON 32.126, IBON 34.88, IBON 34.95 and IBON 34.126, resistant in the field, also showed resistance in the greenhouse against all six P. hordei pts (Table 5.5). Lines 88 and 95 from IBON 34 had the same pedigree and are hence sib-lines. Based on the resistance of these lines against the six pts used, they could carry either Rph5, Rph7, Rph11, Rph14, Rph15, Rph18 or another uncharacterised seedling resistance gene. However, differences in the ITs produced by these lines in comparison with control genotypes carrying these genes suggested that the gene(s) present in each may be different.

Fig. 5.2 Infection type responses of control lines against Puccinia hordei pt 5457P+ L to R: Gus (3+), Ricardo (11+2C), Estate (Rph3, 3+), Egypt 4 (Rph8, 1++CN+), Cebada Capa (Rph7, ;N), 81882/BS1 (Rph17, ;1-C) and 38P18/8/1/10 (Rph18, 0;=)

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Fig. 5.3 Field responses of barley lines with USR and UAPR, L to R: Immune (0), TR, MR, MS, S and leaf death due to high infection of leaf rust. Categories of resistance are described in Appendix 2

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Table 5.5 Seedling responses of selected lines and control differential genotypes against six Puccinia hordei pathotypes

Nursery Line No. 253P- 200P- 5610P+ 5653P++Rph13 5652P+ 5457P+ Postulated Pedigrees Resistance Gene IBON 32 34 0;= 0;= 0;= 0;= 0;- 0; USR Petunia 1/Winchester//Ciru IBON 32 126 0;= 0;= 0;= 0;= ;C 0; USR M9878/Cardo//Quina/3/Petunia 1/4/Ciru IBON 34 88 0;= 0;= 0;= 0;= 0;= 0;= USR Br2/l.p//Azaf IBON 34 95 0;= 0;= 0;= 0;= 0;= 0;= USR Br2/l.p//Azaf IBON 34 126 0;= 0;= 0;= 0;= 0;= 0;= USR Canela/pfc9201//Msel IBON 32 183 33+ 33+ 33+ 33+ 3+ 3+ UAPR Atah92/Gob//f101.78/3/Arupo/k8755//Mora IBON 32 202 33+ 33+ 3+ 33+ 3+ 3+ UAPR Triumph-bar/Tyra//Arupo*2/abn- b/3/Canela/4/Canela/Zhedar#2 IBON 34 8 33+ 33+ 33+ 33+ 3+ 3+ UAPR Cheng du 105/Cabuya//Petunia 1 IBON 34 41 33+ 33+ 33+ 33+ 3+ 3+ UAPR Scotia1/wa1356.70//wa1245.68/Boyer/ 3/mja/brb2//Quina/4/La Molina 94 IBON 34 54 33+ 33+ 33+ 33+ 3+ 3+ UAPR Acuario t95/br2//Msel IBON 34 110 33+ 3+ 33+ 33+ 3+ 3+ UAPR Atah92/Gob//f101.78/3/Arupo/k8755//Mora Differential/Rph gene Gus/Nil 3+ 3+ 3+ 3+ 3+ 3+ Nil Sudan/Rph1 3+ 0; 0;- 3+ 0;- 3+ Rh1 Berg/Rph1 3+ 0;C 0;- 3+ 0;- 3+ Rph1 Peruvian/Rph2 33+C ;1CN ;1CN 33+ 3+ 3+ Rph2 Gatam/RphGat 11+C 0;C 0;- 3+ 33+ 3+ RphGat Reka 1/Rph2+RphP ;1=C ;1=C ;C 3+ 3+ 3+ Rph2+RphP

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Ricardo/Rph2+? 2++3 ;1CN 12+CN 1+2-CN 2++3-C 1++2C+ Rph2+? Estate/Rph3 ;-C 0;- ;-C 0;= ;-C 3+ Rph3 Gold/Rph4 2++3 ;11- 3+ 3+ 3+ 3+ Rph4 PI 531849/Rph13 0;= 0;= 0;= 3+ 0;-C ;CN Rph13 Quinn/Rph2+Rph5 0;= 0;C 0;- 0;- 0;= 0;C Rph2+Rph5 Magnif 104/Rph5 0;= 0;= 0;- ; 0;= 0;C Rph5 Bolivia/Rph2+Rph6 1++,3+ ;C ;CN 3+ 3+ 3+ Rph2+Rph6 Cebada Capa/Rph7 0;-N 0;N 0;CN ;CN+ 0;N ;1=CN Rph7 PI 584760/Rph14 11+C 11++2+C 33+ ;CN+ ;1=C ;1-CN Rph14 Egypt 4/Rph8 0;- 3+ 3+ 3+ 3+ 1++CN+ Rph8 Abyssinian/Rph9 ;CN ;CN 3+ 33+ 3+ 3+ Rph9 Clipper BC8/Rph10 ;1=C ;1=C 33+ 33+ 3+ 3+ Rph10 Clipper BC67/Rph11 ;1-C ;11+C 1++C ;1C ;11++ 2++3C Rph11 Triumph/Rph12 0;C 0;CN 3+ 33+ 3+ 3+ Rph12 Prior/Rph19 0;- 0;= 3+ 3+ 3+ 3+ Rph19 Cutter/Rph19 0;- ;CN 0;- 33+ 3+ 3+ Rph19 Q21861/RphQ 0;- 0; ;C 3+ 3+ 3+ RphQ Cantala/RphC ;1C 3+ 3+ 33+ 3+ 3+ RphC PI366444/RphB37 ;1=C 3+ 3+ 3+ 3+ 3+ RphB37 Bowman+Rph15/Rph15 ;CN+ 0;C ;CN ;CN+ ;CN ;CN+ Rph15 81882/BS1/Rph17 ;1-C ;C ;11+C ;1-CN ;1=C ;1-C Rph17 36l50/3/5/1/Rph? 0; ;C ;CN ;N ;CN ;CN Rph? 38P18/8/1/10/Rph18 0;= 0;= 0;= 0;= 0;= 0;= Rph18 169P15/8/ Rph? 0;= 0;= 0;= 0;= 0;= 0;= Rph? USR = uncharacterised seedling resistance, UAPR = uncharacterised adult plant resistance

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Stem rust evaluation All lines from the four nurseries were resistant to Pgt pt 98-1,2,3,5,6 when tested as seedlings in the greenhouse (Table 5.6). While there was some variation in the ITs displayed by the lines, all were low and a majority displayed mesothetic ITs. Different ITs displayed by entries in the four IBONs are summarised in Table 5.7. Out of the 783 lines tested, 164 produced immune (0;= to 0;) responses, 284 produced resistant (1= to 3) responses and 335 produced mesothetic resistant (X types) responses under greenhouse conditions. All differential lines showed the expected ITs when inoculated with the same pt of Pgt (Fig. 5.4). Raw data of greenhouse stem rust testing of IBONs 31 to 34 is provided in the Appendix 4.1.

Fig. 5.4 L to R: Wheat cultivar Morocco showing susceptibility and barley line showing intermediate resistance against Puccinia graminis f. sp. tritici pt 98-1,2,3,5,6 under greenhouse conditions

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Table 5.6 Summary of greenhouse results of four international nurseries tested with Puccinia graminis f. sp. tritici pathotype 98-1,2,3,5,6 31st IBON 32nd IBON 33rd IBON 34rth IBON Total Resistant 200 258 184 141 783 Susceptible 0 0 0 0 0 Missing lines 5 8 21 3 37 Total Lines 205 266 205 144 820

Table 5.7 Summary of infection types produced by four international nurseries when tested with Puccinia graminis f. sp. tritici pathotype 98-1,2,3,5,6 31st IBON 32nd IBON 33rd IBON 34rth IBON Total Immune 28 56 52 28 164 Resistant 83 95 64 42 284 Mesothetic (X types) 89 107 68 71 335 Missing lines 5 8 21 3 37 Total Lines 205 266 205 144 820

BGYR evaluation All 783 lines showed high levels of resistance against BGYR in the greenhouse tests (Table 5.8). More than 99% of the lines were immune to BGYR (ITs of 0;-) when tested under greenhouse conditions. Only two lines, IBON 34.5 and IBON 32.173 showed admixtures with resistant ITs (1 to 2++). All differential lines except Chinese 166 were resistant to BGYR, as expected (Fig. 5.5). BGYR testing data of all the IBON entries is compiled in the Appendix 4.2.

Table 5.8 Response of ICARDA germplasm against a standard isolate of the Barley Grass Stripe Rust (BGYR) pathogen 31st IBON 32nd IBON 33rd IBON 34th IBON Resistant 200 258 184 141 Susceptible 0 0 0 0 Missing 5 8 21 3 Total Lines 205 266 205 144

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Fig. 5.5 L to R: Wheat cultivar Chinese 166 showing susceptibility and barley line showing immunity against Barley Grass Stripe Rust (BGYR) pathogen under greenhouse conditions

Molecular marker analysis Barley lines IBON 34.8, IBON 34.41, IBON 34.54, IBON 34.110, IBON 32.183 and IBON 32.202, which were resistant at adult plant stage in the field and displayed high ITs against all six pts at the seedling growth stage (Table 5.5), were selected for genotyping with the markers closely linked to Rph20 (Liu et al. 2010).

Barley lines IBON 32.183, IBON 34.54 and IBON 34.110 amplified a 245 bp band, whereas no amplification occurred in lines IBON 32.202, IBON 34.8 and IBON 34.41 with marker bPb-0837. The control cultivars Pompadour, Baronesse, WI 3407 and Flagship amplified 245 bp bands, while no band was produced in tests with Stirling, Gus and Ricardo (Fig. 5.6 and Table 5.9). The microsatellite marker Ebmag0833 amplified a 218–228 bp band in barley lines IBON 32.183, IBON 32.202 and IBON 34.8 and in the control genotypes Baronesse and Stirling. Lines IBON 34.41, IBON 34.54, IBON 34.110 and the controls Pompadour, WI

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3407, Flagship, Ricardo and Gus failed to amplify a fragment with marker Ebmag0833 (Fig. 5.7 and Table 5.9). The cultivar Ricardo did not amplify any PCR product with either marker. PCR reactions were repeated for DNA of all barley lines and controls using the microsatellite marker Ebmag0833 and the same results were obtained. The results of marker bPb-0837 are more reliable as it produced the expected bands in Rph20 carrying controls while marker Ebmag0833 produced conflicting results (Table 5.9).

Table 5.9 Validation of markers bPb-0837 and Ebmag0833 on ICARDA barley lines carrying adult plant resistance to Puccinia hordei, plus controls IBON No. Line No. Field score bPb-0837 Ebmag0833 32 183 40 MR + + 202 TR - + 34 8 60 MR-MS - + 41 TR - - 54 TR + - 110 10 R + - Controls Pompadour 10 R + - Baronesse 10 R + + WI 3407 5 R + - Flagship 10 R + - Stirling 70 S - + Ricardo 10 R - - Gus 90 S - - + and - indicates presence and absence of band respectively

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Fig. 5.6 Marker bPb-0837 amplifications at 245 bp in lanes 1 to 13: Lines 183, 202 (IBON 32) and 54, 110, 8, 41 (IBON 34) and controls Stirling, Flagship, Ricardo, Pompadour, Baronesse, WI 3407 and Gus

Fig. 5.7 Marker Ebmag0833 amplifications at 218 to 228 bp in lanes 1 to 13: Lines 183, 202 (IBON 32) and 54, 110, 8, 41 (IBON 34) and controls Stirling, Flagship, Ricardo, Pompadour, Baronesse, WI 3407 and Gus

Discussion Of the designated major seedling genes that confer resistance to P. hordei in barley (Rph1 to Rph19), only Rph7, Rph11, Rph14, Rph15, Rph18 (Park 2003; Park 2010) and Rph20 (Park 2010 unpublished) are effective in Australia. It is well known that major genes can be easily overcome by new pts of P. hordei. This situation has occurred in Australia, with the frequency of virulence for Rph4 increasing following the widespread use of cultivar

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Grimmett carrying Rph4 (Cotterill et al. 1995), for Rph12 following the releases and widespread cultivation of barley cultivars including Franklin, Tallon, Lindwall and Fitzgerald, all of which carry Rph12 (Park 2008) and more recently for Rph3, following the releases of cultivars Fitzroy, Yarra and Starmalt carrying Rph3 (Park 2010). Plant breeders therefore have a limited choice in terms of resistance sources against P. hordei. In view of this, the present study sought new sources of resistance to P. hordei in four of the International Barley Observation Nurseries, which are distributed annually by ICARDA. The nursery entries were also assessed for response to stem rust and BGYR.

Tests of leaf rust response indicated that 44 lines were susceptible at both seedling and adult plant growth stages to all of the pts tested. During 2007 and 2008, more than 93% of the entries tested showed high levels of resistance to leaf rust in both seedling greenhouse and adult plant field tests. In early 2009, for the first time in Australia, virulence for the seedling resistance gene Rph3 was detected with the identification of a pt 5457P+ from the northern NSW (Park 2010). When the nursery entries were tested with this new pt, 93% were susceptible in both the greenhouse and the field, strongly indicating that the resistance detected in previous tests was due to a single major gene, Rph3 and that no additional resistance was present in these lines. The occurrence of Rph3 only, in 93% of this germplasm indicated that it is highly vulnerable to leaf rust.

Five entries showed resistance to leaf rust that was effective in the field against pts 5652P+ and 5457P+ as well as to a range of pts in the greenhouse (Table 5.5). Based on the ITs generated against a range of pts, it was postulated that these lines carry one or more unknown seedling resistance genes. While it is possible that these lines may carry one of the resistance genes (Rph5, Rph7, Rph11, Rph14, Rph15 and or Rph18) that is effective against all of the pts used, this was considered unlikely because all five entries showed ITs that differed from those shown by all of the known effective genes except Rph18. It was considered unlikely that these nursery entries carried Rph18 because this resistance gene is derived from Hordeum bulbosum (Pickering et al. 2000) and has not yet been deployed in breeding programs. Genetic studies are therefore needed to characterise the seedling resistance identified in these five lines.

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On the basis of multipathotype testing in the greenhouse and field testing for three consecutive seasons, six lines that carry APR to leaf rust were identified. It is known that many European cultivars carry APR to P. hordei (Golegaonkar et al. 2009b; Park 2008). Positive validation of the molecular marker bPb-0837 amplified 245 bp bands in lines IBON 32.183, IBON 34.54 and IBON 34.110 indicated the likely presence of the APR gene Rph20, reported on chromosome 5H and closely linked to this marker (Liu et al. 2010). Similar amplification of 245 bp bands from DNA of reference stock; Pompadour, Baronesse, WI 3407 and Flagship by marker bPb-0837 (Liu et al. 2010) also supported the likely presence of Rph20 in genotypes IBON 32.183, IBON 34.54and IBON 34.110. The marker bPb-0837 failed to amplify a product in lines IBON 32.202, IBON 34.8 and IBON 34.41, indicating the likely presence of uncharacterised APR in each. These lines were resistant in the field and susceptible in the greenhouse to a range of P. hordei pts. The lines IBON 32.202 and IBON 34.41 showed identical field responses and it is possible that they might have a gene in common. Further genetic analysis of these lines will provide useful information regarding the APR present in each.

The SSR marker Ebmag0833 amplified a positive product in the susceptible control Stirling and no band was amplified in resistant control Pompadour. These results are not in agreement with the results published by Liu et al. (2010). Amplification of 245 and 218 bp products with markers bPb-0837 and Ebmag0833 respectively showed the presence of Rph20 in IBON 32.183. Expected PCR products were amplified in controls with marker bPb-0837, although marker Ebmag0833 did not amplify in the control Pompadour but amplified in Stirling. All genotyping was repeated with marker Ebmag0833 to verify this and similar results were obtained. The results produced with marker bPb-0837 were similar and consistent to those published by Liu et al. (2010), hence this marker proved to be more reliable. This is likely due to the closer linkage between bPb-0837 (0.7cM) with Rph20 compared to Ebmag0833 (1.3 cM). Marker bPb-0837 should therefore be helpful in marker assisted selection and pyramiding of APR against P. hordei.

The cultivar Ricardo was resistant in the field but failed to produce a PCR product when genotyped using these two markers, indicating the likely presence of an unknown resistance under field conditions. It will be useful therefore to undertake genetic analysis and allelic studies of unknown APR present in lines IBON 32.202, IBON 34.8 and IBON 34.41, along 124

Resistance among IBONs with Ricardo, to determine the mode of inheritance and their genetic relationship with the only other named APR gene for leaf rust in barley, Rph20 on chromosome 5H.

All lines were resistant to stem rust when tested in the greenhouse. The development of stem rust epidemics in field nurseries was unfortunately not sufficient enough to assess the effectiveness of the seedling resistances under field conditions. In barley, stem rust resistance genes Rpg1, Rpg2, Rpg3 and rpg4 are reported to be effective against Pgt (Sun and Steffenson 2005). In the U.S.A. and Canada, stem rust resistance gene Rpg1 was used in barley breeding programs (Brueggeman et al. 2002) and remained effective for more than six decades (Mirlohi et al. 2008). Stem rust resistance among the IBON entries may be due to either any one or combination of these stem rust resistance genes, or due to uncharacterised resistance genes. All 164 lines from four nurseries showing immunity to P. graminis may carry a single major gene for resistance to stem rust. Further genetic analyses of the resistance identified among the nursery entries may be very helpful in breeding stem rust resistant barley, however, a critical analyses of the level of protection afforded by these resistance sources in the field is needed to ensure the resistance is useful in protecting barley from stem rust. It would be worthwhile to test this germplasm with North American Rpg1 virulent and avirulent pts, as attempts to detect this gene in seedling tests using Australian isolates of P. graminis have not been successful (Park et al. 2009). The molecular marker pM13 (Kilian et al. 1997) could also be used to assess the presence Rpg1in these lines.

The germplasm tested was highly resistant in seedling tests with BGYR and there was no variation in ITs other than two possible admixtures. The results were not unexpected as this pathogen has only a limited tendency to infect barley genotypes (Park 2008). The BGYR pathogen is distinct from the true barley stripe rust pathogen P. striiformis f. sp. hordei, which up to now has not been detected in Australia (Keiper et al. 2003; Wellings et al. 2000). While the results obtained for BGYR cannot be directly related to Psh, they nonetheless provide useful information on resistance of ICARDA nurseries with respect to BGYR as this germplasm has not been tested against this pathogen before. The resistance identified to BGYR in the lines identified as also carrying effective leaf rust resistance is important if any of the leaf rust resistant entries are to be used by Australian breeders, to prevent the inadvertent introduction of susceptibility to BGYR into breeding populations. It will also be useful to test this germplasm with Psh to assist their utilisation in breeding programs. 125

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Given that virulence for seedling resistance gene Rph3 is now present in eastern Australia, 93% of the germplasm tested here carries Rph3 only is of limited value for leaf rust resistance. The diversity of leaf rust resistance among these four ICARDA nurseries is very narrow as only 11 lines (6 with unknown APR and 5 with unknown seedling resistance) were identified in the study. Out of six lines with APR, three likely carry Rph20. Eight lines (three with APR and five with seedling resistance) were identified that carry potentially uncharacterised resistance to leaf rust and are therefore potentially valuable as new sources of resistance. It is recommended to undertake genetic analysis of these eight lines to understand their inheritance and genetic relationship with other known genes for their effective utilisation in breeding programs. The present studies again stress the importance of identifying new sources of leaf rust resistance to diversify the genetic base of resistance and for additional and better choice of resistance in breeding programs. At the same time, deployment of single major known effective genes (Rph7, Rph11, Rph14, Rph15 and Rph18) should be conducted wisely by avoiding the release of cultivars with single major genes only. Efforts should be made to pyramid the resistance genes available to reduce the chance of matching virulence developing in pathogens. Given that only one gene conferring APR to leaf rust in barley has been characterised to date, the identification of one or more potentially new sources of APR could provide a means of achieving durable resistance via APR gene pyramiding.

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CHAPTER VI Short simple sequence repeats and PCR-fingerprinting based analyses of genetic diversity in Puccinia hordei Otth. in Australasia

Abstract A total of 148 microsatellite markers, comprising 71 developed for P. graminis f. sp. tritici, 40 developed for P. triticina and 37 developed for P. coronata f. sp. avenae, were tested across 22 pathotypes of Puccinia hordei Otth. from Australasia to assess their usefulness in revealing genetic variability in this pathogen. Genotyping of P. hordei was also conducted with the PCR-fingerprinting primers M13 and (GACA)4. The SSRs developed from P. graminis f. sp. tritici and P. triticina showed 100% cross amplification in P. hordei, while only nine P. coronata f. sp. avenae SSRs showed amplification in P. hordei. Of the 148 markers tested, only two P. graminis f. sp. tritici SSRs were polymorphic. Both PCR- fingerprinting primers revealed polymorphisms among the isolates, with (GACA)4 generating the most informative fragments. Molecular analyses revealed evidence of clonal lineages among the P. hordei pathotypes, supporting the hypothesis that some of the pathotypes arose from mutational changes in the virulence of a founder pathotype. Evidence was also obtained for sexual recombination within P. hordei in Australia on the alternate host Ornithogalum umbellatum. This is the first study of Australasian pathotypes of P. hordei using PCR- fingerprinting technique and SSR genotyping. Given that only two polymorphic SSR markers were identified in these studies, further work is recommended to identify additional polymorphic SSR markers for this rust species.

Key words: Barley leaf rust, molecular analysis, M13, (GACA)4, SSRs

Introduction

Annual surveys of pathogenic variability in the rust pathogens that infect cereal crops in Australia have provided evidence that variation in these fungi arises via either the introduction of exotic genotypes, simple mutations, asexual hybridisation and in the case of Puccinia hordei Otth. only, sexual hybridisation (Park 2008; Park et al. 1995; Wellings and McIntosh 1990).

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Different barley genotypes, known collectively as a differential set and comprising barley varieties and lines with resistance genes, were used by Cherewick (1952) and Clifford (1977) to characterise pathotypes (pts) among different isolates of P. hordei. The differential set used to characterise pts of P. hordei at the University of Sydney Plant Breeding Institute (PBI) comprises 30 different barley genotypes with one or more resistance (Rph) gene (Appendix D1). The first assessment of pathogenic variability in P. hordei in Australia was made in 1920 by Waterhouse (1927), who detected two pts, one similar to an European pt and another that differed in virulence on some genotypes compared to a pt found in North America (Waterhouse 1952; Watson and Butler 1947). In a later Australian study, Cotterill et al. (1995) found substantial pathogenic variation among P. hordei isolates collected between 1966 and 1990. This study identified 11 different pathotypes among 154 isolates, of which pt 210P+ was the most common. Up to 1995, virulence was detected for the leaf rust resistance genes Rph1, Rph2, Rph4, Rph5, Rph6, Rph8, Rph9 and Rph12 and the genes Rph3 and Rph7 remained effective (Cotterill et al. 1995). Pathotype 4610P+ virulent on Rph12 was detected first time in 1991 from Tasmania. Later on (1996 to 2002), more pathogenic variation was detected in P. hordei including the identification of two new Rph12 virulent pts with an added virulence for the resistance gene Rph10 (viz. 5610P+ and 5453P-) (Park 2003). While no virulence was detected in these studies for genes Rph3, Rph7, Rph11, Rph14, Rph15 and Rph18 (Park 2003), virulence for Rph3 was detected in 2009 in pt 5457P+, isolated from northern NSW (Park 2010). This pathotype is believed to have arisen from pt 5453P-, first detected in Western Australia in 2001 (Park 2006), via sequential single step mutations for virulence to Rph19 (pt 5453P+) and then Rph3 (5457P+) (Park 2010).

P. hordei is a macrocyclic and heteroecious rust pathogen that forms its aecial stage on various species of Ornithogalum, Leopoldia and Dipcadi in the family Liliaceae (Clifford 1985). The alternate host Ornithogalum umbellatum occurs in Australia, where it is present on the Yorke Peninsula of South Australia (SA) (Wallwork et al. 1992) and in the Murrumbidgee catchment areas including Henty and Junee in NSW (Murrumbidgee 2008). In an earlier study of pathogenic specialization of P. hordei in Australia (Park 2003), it was suspected that O. umbellatum may be also present in other parts of Australia beyond the Yorke Peninsula. While six pts of P. hordei were identified among uredinial isolates derived from aeciospores collected from infected plants of O. umbellatum from the Yorke Peninsula

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(Wallwork et al. 1992), the overall role of sexual recombination in generating genetic variability in P. hordei in Australia is largely unknown.

Although information on variability obtained from pathogenicity on differential genotypes is important in the genetic control of rusts, it is of limited use in assessing genetic variation in these pathogens. Both biochemical and molecular markers have been applied to evaluate genetic diversity among various plant pathogens (McDermott and McDonald 1993). Amplified fragment length polymorphism (AFLP) analyses were used to study genetic diversity among isolates of P. hordei in relation to their virulence (Sun et al. 2007). This study revealed an association between molecular diversity and virulence patterns in P. hordei isolates collected from different geographical regions of the world. Keiper et al. (2003) studied the genetic structure of several cereal rust pathogens using various polymerase chain reaction (PCR) based tools like AFLP, selectively amplified microsatellites (SAM) and sequence-specific amplification polymorphisms (S-SAP). This study was able to discriminate five rust taxa [P. triticina (Pt), P. graminis f. sp. tritici (Pgt), P. striiformis f. sp. tritici (Pst), barley grass stripe rust (BGYR) and P. graminis f. sp. avenae (Pga)], although the level of polymorphism observed within individual taxa was low. In a separate study that used AFLPs and random amplified polymorphic DNA (RAPDs), Steele et al. (2001) found no polymorphism among Australian and New Zealand isolates of Pst. However, the same AFLP primers showed five to 15% polymorphic fragments among isolates of Pst from the UK, Denmark and Colombia. These results were consistent with clonality in Australian populations of Pst.

Microsatellites, or simple sequence repeats (SSRs), are tandemly repeated DNA sequences composed of 1–6 base pair (bp) arrays that are highly polymorphic and evenly distributed in abundance across genomes. SSRs are co-dominant, generate maximum genetic information and are inherited according to Mendelian laws (Liu et al. 1999). Due to their informative power, high throughput and PCR reproducibility, SSRs are the preferred choice of markers for a variety of studies including discrimination, kinship, population genetics and mapping (Jarne and Lagoda 1996).

To date, SSRs have been developed and applied to study different rust pathogens. SSRs developed specifically for the crown rust pathogen P. coronata f. sp. avenae (Pca) were 129

Genetic diversity in P. hordei highly polymorphic among 35 Pca isolates, with an allelic diversity of two to 16 alleles per locus (Dambroski and Carson 2008). Similarly, SSR markers developed from a urediniospore derived expressed sequence tag (EST) resource were used to study genetic diversity among the Australian and New Zealand isolates of P. coronata f. sp. lolli, causing crown rust on rye grass (Dracatos et al. 2009). Out of 72 single pustule samples collected from both the North and South Islands of New Zealand and from south-eastern Australia, 59 were found to be distinct genotypes. Furthermore, high levels of genetic diversity were observed within and between the isolates collected from different locations, with Victorian isolates showing high population differentiation from New Zealand populations (Dracatos et al. 2009).

More recently, 118 isolates of Pt collected from the Middle East and Central Asia were genotyped using 23 SSRs (Kolmer et al. 2011). All Middle Eastern isolates differed from the Central Asian isolates, suggesting a lack of pathogen migration between the two regions. In another study that compared North American and South American Pt isolates using SSRs, a high degree of similarity was found, suggesting that Pt was introduced to America from a common origin (Ordoñez et al. 2010). In the SSR genotyping of Italian Pt isolates, virulence phenotypes and molecular genotypes were found to be highly correlated (Mantovani et al. 2010).

SSRs have also been developed and used to genotype Pgt isolates. Keiper et al. (2006) used 110 SSRs to genotype 10 pathogenically diverse isolates of Pgt and demonstrated that some of these SSRs were also useful in revealing polymorphism among isolates of the oat stem rust pathogen Pga. Recently, the Pgt pts TTKSF, TTKSP and PTKST, all believed to belong to a clonal lineage typified by pt TTKSK (“Ug99”) and selected South African isolates of Pgt, were genotyped using SSR markers. The four “Ug99” pts shared only 31% similarity with other South African pts and it was concluded that pts TTKSP and PTKST arose in South Africa as a result of exotic introduction (Visser et al. 2011).

PCR-fingerprinting is a technique that involves using microsatellites (GACA)4 and (GTG)5 and the minisatellite M13 derived from the core sequence of the wild type phase M13 bacterium, as single primers in PCR to amplify hypervariable DNA sequences (Meyer et al. 2001). The PCR-fingerprinting technique has been used successfully to reveal polymorphism among various fungal and bacterial pathogens. For example, Vuyst et al. (2008) used (GTG)5 130

Genetic diversity in P. hordei to identify acetic acid bacteria in cocoa beans and the primers GTG, GACA and M13 were used to study population dynamics in several human pathogens (Cogliati et al. 2007; Delhaes et al. 2008; Meyer et al. 2001; Roque et al. 2006; Trilles et al. 2008). Selective amplification of the microsatellite polymorphic loci (SAMPL) markers (GACA)4 + H-G and R1 + H-G were used to study polymorphism among 44 (25 Australasian and 19 European) isolates of Phragmidium violaceum (causal agent of blackberry rust), revealing more diversity in European isolates than in Australasian isolates, with 37 and 22% polymorphic loci respectively (Gomez et al. 2006). In all of these studies, the primers GACA and M13 generated the most discriminating and informative DNA profiles.

To date no attempt has been made to study genetic variation in Australian populations of P. hordei. The present study attempted to develop SSR markers for P. hordei, by assessing the utility of SSR primers developed for Pgt, Pt and Pca in revealing polymorphism among isolates of P. hordei. Studies were then made to investigate genetic variability among Australasian isolates of P. hordei using the polymorphic SSR markers found and also by

PCR-fingerprinting profiles with primers M13 and (GACA)4.

Materials and methods

Rust pathotypes A total of 22 pts of P. hordei, comprising 20 from Australia and two from New Zealand, along with five other rust pathogen controls (single reference isolates of Pt, Pgt, Pst, BGYR and Pga), were included in this study (Table 6.1). The P. hordei pts used were selected to represent those identified in different regions within Australia and New Zealand in annual pathogenicity surveys conducted from 1980 to 2009.

Extraction of genomic DNA from urediniospores Freshly collected urediniospores were desiccated over silica for 12 hrs. A sample of 25–30 mg of urediniospores of each rust isolate was put in labelled Lysing Matrix C tubes (Impact resistant tubes with 1.0 mm silica spheres, Mp Biomedical, Ohio, USA). One ml of 2x CTAB extraction buffer [(CTAB 2% (w/v), 20 mM EDTA (pH 8.0), 1.4 M NaCl, Polyvinylpyrrolidone (PVP; 40000 MW) 1% (w/v), 100 mM Tris-HCl (pH 8.0) and dH2O] was added to each sample, mixed well by inversion and tubes were submerged in ice for 2 131

Genetic diversity in P. hordei min. Tubes were then shaken for 15 s on a FastPrep® Cell Distrupter (Qbiogene, USA) at speed 6, returned to ice for 3 min and shaken again for 20 s at the same speed. Tubes were kept in a pre-warmed water bath at 65˚C for 30 min and inverted every 10 min, after which they were removed, mixed well by inversion and the solution in each tube/sample was divided (~ 500 µl in each tube) into two new 1.5 ml Eppendorf tubes to generate duplicate extractions. DNA extraction was carried in a fume hood by adding ~ 250 µl of cold phenol, followed by ~ 250 µl of cold chloroform: isoamyl alcohol, to each tube. Samples were mixed gently by inverting (~ 100 times) the tubes until a thick emulsion formed. Tubes were centrifuged at 13,000 rpm for 15 min and the supernatant was transferred into sterile 1.5 ml Eppendorf tubes. The process of phenol and chloroform: isoamyl alcohol extraction was repeated. About 50 µl of 3 M NaOAc and ~ 500 µl of cold isopropanol were added to each tube and tubes were then stored at -20˚C. The following day, the tubes were centrifuged at 13,000 rpm for 30 min and the DNA pellet thus formed was drained carefully. The pellets were washed with 500 µl of ethanol, centrifuged at 13,000 rpm for 15 min, drained carefully and allowed to air dry. The dried pellet was re-suspended in 100 µl double distilled autoclaved water (ddH2O) and stored overnight at 4˚C. The following day, 5 µl of Rnase-A (10 μg/μl) was added to each tube and incubated at 37˚C for 2 hr. All DNA samples were quantified using a Nanodrop ND-1000 spectrophotometer (Nanodrop® Technologies) and diluted to working dilution of 10 ng/µl using ddH2O.

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Table 6.1 Details of Puccinia hordei pathotypes and control isolates of P. triticina, P. graminis f. sp. tritici, P. striiformis f. sp. tritici, P. graminis f. sp. avenae and “BGYR” analysed using SSR and PCR-fingerprinting primers Isolate Pathogen Pathotype Culture No. Origin Host Year Number 1 P. hordei 211P+ 484 Coonamble, NSW O’Connor 1992 2 P. hordei 220P+ 485 Yanco, NSW Nigrinudum 1992 3 P. hordei 253P- 490 Grafton, NSW Barley 1992 4 P. hordei 243 (+Reka1) 537 Grafton, NSW Barley 1999 5 P. hordei 200P+ 570 Yanco, NSW Gus 2002 6 P. hordei 232 (+Reka1) 506 Balaclava, SA Galleon 1994 7 P. hordei 201 480 St Leonards, VIC Barley 1992 8 P. hordei 201P+ 481 Rochester, VIC Barley 1992 9 P. hordei 242P+ 531 Borung, VIC Barley 1998 10 P. hordei 5653P- -Rph13 569 Byaduk, VIC Franklin 2002 11 P. hordei 243P+ Yellow 489 Monto, QLD NA 1992 12 P. hordei 243 (+Ricardo) 507 Toowoomba, QLD Dampier 1994 13 P. hordei 5453P- 560 Esperance, WA Schooner 2002 14 P. hordei 5653P+ 584 Wongan Hills, WA Barley 2004 15 P. hordei 4610P+ 491 Cressy, TAS Franklin 1992 16 P. hordei 5653P+ +Rph13 542 Glen Esk, TAS Gairdner 2000 17 P. hordei 211 483 Aorangi, NZ Barley 1992 18 P. hordei 231P+ 486 Aorangi, NZ Barley 1992

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19 P. hordei 5610P+ 520 Ravensthorpe, WA Barley 1997 20 P. hordei 220P+ +Rph13 577 SA Star of Bethlehem 2003 21 P. hordei 200P- 518 NA Barley 1995 22 P. hordei 5457P+ 612 Legume, QLD Barley 2009 23 P. triticina 104-2,3,(6),(7),11 423 Mt Derimut, VIC Nebraska 1984 24 P. graminis f. sp. tritici 194-2,3,7,8,9 344 Hermitage, QLD Wheat 1980 25 P. striiformis f. sp. tritici 110 E143 A+ 444 Richmond, TAS Hartog 1987 26 Barley Grass Stripe Rust 981549 589 Turretfield, SA Barley 1998 (“BGYR”) 27 P. graminis f. sp. avenae 41+Pg9 496 Rutherglen, VIC NA 1993 Pathotypes details are sourced from the Cereal Rust Collection, maintained at the University of Sydney, PBI, Cobbitty

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Molecular markers A total of 148 SSR primers and two PCR-based fingerprinting markers (Appendix 5) were tested for their information content among the 22 P. hordei pts and five control rust isolates. The SSR primers used in the study included 52 developed for Pgt by Karaoglu et al. (unpublished), 19 developed for Pgt by Szabo (2007), 19 developed for Pt by Szabo and Kolmer (2007), 21 developed for Pt by Wang et al. (2010) and 37 developed for Pca by Dambroski and Carson (2008). Two oligonucleotide primers were used in the PCR- fingerprinting of the rust pts: the microsatellite-specific (GACA)4 and M13 minisatellite DNA sequence markers derived from the core unit of M13 vector (Meyer et al. 2001) (Table 6.2).

Table 6.2 SSRs and PCR-fingerprinting primers tested for utility in Puccinia hordei isolates from Australasia No. of primers Loci name Primer reference tested F1-1 to F9-47 (Pgt) 52 (Karaoglu et al., unpublished) PgtSSR 53 – 71 (Pgt) 19 (Szabo 2007) PtSSR 72 – 90 (Pt) 19 (Szabo and Kolmer 2007) PtSSR 91 – 111 (Pt) 21 (Wang et al. 2010) PcaSSR 112 – 148 (Pca) 37 (Dambroski and Carson 2008)

M13, (GACA)4 (PCR-fingerprinting) 2 (Meyer et al. 2001) Total 150 Locus details are given in Appendix 5

PCR amplifications and profiles

SSR Primers PCR was performed using 15 µl of reaction containing 2.0 µl of genomic DNA (10 ng/µl), 1.5 µl of dNTPs (0.2 mM), 1.5 µl of 10x PCR buffer (NH4 Reaction buffer, Bioline), 0.9 µl of 50 mM MgCl2 (Bioline), 0.9 µl of each forward and reverse primer (2 mM), 0.15 µl (5 u/µl) of Taq DNA (Bioline, Australia) and 7.15 µl of ddH2O. PCR was carried out for 33 cycles with denaturing at 94°C for 30 s, annealing and extension at 58°C for 3 min, with a final extension of 7 min. When this condition was not applicable, or when the PCR products contained spurious priming, a touchdown step was included to the normal PCR profile. After 135

Genetic diversity in P. hordei the first denaturing for 4 min at 94°C, the touchdown PCR was carried out for 20 cycles with denaturation at 94°C for 30 s, annealing at 68–58°C for 30 s (F4-15) and 59–49°C (F7-22) for 30 s by reducing 1/2°C at each cycle and extension at 72°C for 7 min. An addition of 20– 30 cycles were done with denaturing at 94°C for 30 s, annealing at 49–58 °C (locus specific) and extension at 72°C for 30 s, with a final extension of 72°C for 7 min. Reactions were performed in a 96-well DNA thermocycler (Eppendorf Mastercycler, Germany). PCR products were resolved on 2–3% high resolution agarose (MetaPhor® Agarose, Lonza, USA) gels at 120 V electrophoresis for 2 hrs. One kb DNA markers Gibco® and HyperLadder™ IV (Bioline) were used as ladders. The separated bands were visualised under an ultra violet light unit fitted with a GelDoc-IT UVP Camera.

PCR-fingerprinting primers 50 µl of PCR reactions contained 3.0 µl of genomic DNA (10 ng/µl), 5.0 µl of dNTPs (0.2 mM), 5.0 µl of 10x PCR buffer (NH4 Reaction buffer, Bioline), 3.0 µl of 50 mM MgCl2 (Bioline), 5.0 µl of primer (2 mM), 0.5 µl (5 u/µl) of Taq DNA (Immolase DNA polymerase from Bioline) and 28.5 µl of ddH2O. PCR amplification profile comprised of an initial denaturation step at 94°C for 5 min, followed by 35 cycles of 30 s denaturation at 94°C, 60 s annealing at 47°C (M13) and 40°C (GACA)4, 30 s extension at 72°C and a final extension of 7 min at 72°C. Reactions were performed in a 96-well DNA theromocycler (Eppendorf Mastercycler, Germany). PCR products were concentrated to 30 µl by placing in fan forced oven for 45 min at 65°C and resolved on 2% high resolution agarose (MetaPhor® Agarose, Lonza, Rockland Inc.USA) gels at 80 V electrophoresis for 6 hrs. Five kb DNA marker HyperLadder™ III (Bioline) was used as reference. The separated bands were visualised as described earlier.

Data analyses Gel images were analysed using the software GelCompar II (6th edition, Applied Maths, Belgium). Bands were scored as either “1” (present) or “0” (absent) and unclear bands were deselected manually. Based on the standard DNA ladders used, molecular weights of selected bands were assigned automatically. Band scoring for SSRs ranged from 100–300 bp and for PCR-fingerprinting primers from 500–2000 bp. Genetic diversity among the P. hordei pts examined was evaluated using UPGMA (Unweighted pair group method for arithmetic averages) cluster analyses based on a distance matrix calculated using the Dice algorithm. 136

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Band position optimisation and tolerance was set to 1% and 1.5% respectively. The quality of similarity clusters was calculated using the cluster validity index Cophenetic correlation coefficient (CPCC). The CPCC was used to test the efficiency of the clusters/similarities that resulted from the individual analyses of markers M13 and (GACA)4. The CPCC is a simple correlation coefficient between the original dissimilarity matrix and the final dissimilarity matrix (Cophenetic matrix) produced after the clustering algorithm recalculates the dissimilarities (Lessig 1972). With GelCompar II, Bootstrap (Efron 1979) analysis was carried out in order to test the reliability of the similarity patterns obtained from the composite analyses of SSRs. Bootstrap analysis is often used in molecular genetics as it yields probability values for the clusters that are assessments of the uncertainty in clustering and these probabilities can be used to identify reliable clusters through their statistical significance (Efron et al. 1996). Moreover, bootstrap analysis is an appropriate way to test the reliability or quality of results and clusters, with a bootstrap value of ≥ 95% being considered to reflect correct relationship, though 70% may be acceptable as a cut-off point (Kumar and Filipski 2008). Dendrograms were constructed for individual and composite analyses of primers. Based on similarity clusters of primer (GACA)4, the P. hordei pts were grouped into different (GACA) groups.

For each polymorphic marker, Polymorphic information content (PIC) value was calculated using the formula PIC = 1-Σ P¡ ², where P¡ (Frequency of particular allele/total genotypes) is the frequency of the ¡th allele among the selected rust pts (Anderson JA et al. 1993). PIC measures the usefulness and value of a marker in detecting polymorphism within a population and it depends upon the number of detectable alleles and the distributions of their frequency (Anderson JA et al. 1993; Botstein et al. 1980). Codominant marker loci with PIC > 0.50, 0.25 to 0.50 and < 0.25 are highly, reasonably and slightly informative, respectively and loci with many alleles and a PIC close to 1.0 are highly desirable (Botstein et al. 1980).

Results All 148 SSR primers amplified the DNA of all isolates, but only two (Table 6.3) were polymorphic among the P. hordei isolates (Figs. 6.1 and 6.2). Most of the non polymorphic SSRs produced strong monomorphic amplification products across the P. hordei pts (Fig.

6.3). The oligonucleotides M13 and (GACA)4 amplified all rust isolates and revealed polymorphism among all (Figs. 6.6 and 6.8). 137

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Table 6.3 Details of amplifications and polymorphisms generated with the 150 primers used to genotype selected Puccinia hordei pathotypes and control rust isolates Amplifying Polymorphic Primer origin Total primers tested primers primers P. graminis f. sp. tritici 71 71 2 P. triticina 40 40 0 P. coronata f. sp. avenae 37 9 0 PCR-fingerprinting 2 2 2 Total 150 122 4

The SSR marker F4-15 generated band sizes between 200–300 bp in P. hordei and the control isolates at an annealing temperature (Tm) of 58°C and a touchdown profile (TD) (Fig. 6.1). Similarly, the SSR marker F7-22 produced bands of approximately 130–160 bp in P. hordei and 100–200 bp in the control isolates at 49°C (Tm) with TD (Fig. 6.2). The PCR- fingerprinting primers M13 and (GACA)4 produced bands between 500 to 1500 bp (Fig. 6.6) and 500 to 2000 bp (Fig. 6.8), respectively, among the isolates examined at 47°C (Tm) and

40°C (Tm). The PIC values of SSR markers F4-15 and F7-22 were higher (0.65 and 0.69) when calculated including controls, compared to 0.50 and 0.55 when calculated without controls (Table 6.4).

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Locus Name Repeat Motif Primer sequence (5'–3') PCR Size Tm (˚C) Na PICnc PICc (bp)

F4-15 (CAA)11 F: CTCAAGCACCCTCAACATCC 200–290 58TD 2 0.50 0.65 R: CGTCGTCCCTCCATAGTCTT

F7-22 (ATG)14 F: TAACCGACCAACAACAACAA 130–160 49TD 3 0.55 0.69 R: TTCTCCCATCGCTCTCTCTC M13 NA GAGGGTGGCGGTTCT 500–1500 47

(GACA)4 NA GACAGACAGACAGACA 500–2000 40

Tm: Annealing temperature in ˚C, TD: Touchdown, Na: Numbers of alleles, PICnc: Polymorphism information content value without controls, PICc: Polymorphic information content value including controls

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Fig. 6.2 Amplifications of the SSR F7-22 with 22 Puccinia hordei isolates (lanes 1–22) and control rust isolates (lanes 23–27) at 49˚C (TD). Ladder; 1 kb DNA marker (HyperLadder™ IV, Bioline)

Fig. 6.3 Non-polymorphic SSR F9-1 produced PCR amplifications among different pathotypes of Puccina hordei (lanes 1–22) and control rust pathotype of P. triticina (lane 23). Ladder; 1 kb DNA marker (Gibco®, Australia)

Both SSR markers F4-15 and F7-22 (Fig. 6.4) and the PCR-fingerprinting primers M13 (Fig.

6.5) and (GACA)4 (Fig. 6.6) out grouped representative control isolates of Pt, Pgt, Pst, BGYR and Pga from the P. hordei isolates examined. The two SSRs grouped Pst and BGYR, Pt and Pgt, leaving Pga as a distinct group. The two clades formed by Pst and BGYR and Pt and Pgt, were supported by high bootstrap values of 92% and 82%, respectively. Bootstrap values among the clusters of P. hordei isolates ranged from 48 to 58%, indicating low support only for these (Fig. 6.4). Both fingerprinting primers grouped Pst and BGYR, with Pgt and Pga in another group, while Pt was in a standalone group.

Composite cluster analyses of the two SSR markers F4-15 and F7-22 produced seven groups among the P. hordei isolates, with 45% to 100% similarities (Fig. 6.4). Cluster analyses of the marker M13 produced seven groups among the P. hordei isolates with 75.9% to 100% similarities (Fig. 6.5), while marker (GACA)4 revealed higher variability among the P. hordei isolates and produced 10 different groups with 70.5% to 100% similarities (Fig. 6.7).

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100 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 5 23-Pt .104-2,3,(6),(7),11 54.5 82 16.0 24-Pgt .194-2,3,7,8,9 35

11.7 27-Pga .41+Pg9 60 25-Pst .110 E143A+ 72.7 92 26-BGYR .981549 BLR07 .201 57 BLR08 .201P+ 57 52BLR12 .243 (+Ricardo) + 52BLR16 .5653P +Rph13 BLR22 .5457P +

80.0 + 43 BLR05 .200P 51 BLR10 .5653P- -Rph13 48 0.9 - 46BLR13 .5453P 52.2 + 7 BLR20 .220P +Rph13 BLR15 .4610P + 44 BLR19 .5610P+ 44 80.0 - 40 BLR21 .200P 47.4 + 12 BLR11 .243P Yellow BLR06 .232 (+Reka1) 57 BLR09 .242P + BLR01 .211P + 80.0 86 58 + 45.0 BLR02 .220P 52 86 - 86BLR03 .253P 41BLR04 .243 (+Reka1) BLR14 .5653P + BLR17 .211 67 BLR18 .231P +

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100 30 35 40 45 50 55 60 65 70 75 80 85 90 95 25-Pst .110 E143A+ 57.1 26-BGYR .981549 34.3 24-Pgt .194 -2,3,7,8,9 66.7 57.9 27-Pga .41+Pg9 23-Pt .104 -2,3,(6),(7),11 BLR17 .211 BLR18 .231P + 93.3 + BLR08 .201P BLR12 .243 (+Ricardo) BLR16 .5653P + +Rph13 BLR01 .211P + BLR02 .220P + 26.4 BLR03 253P. - BLR04 .243 (+Reka1) BLR05 .200P+ 87.8 BLR06 .232 (+Reka1) BLR07 .201 BLR09 .242P + BLR11 .243P + Yellow + 85.4 BLR15 .4610P BLR19 .5610P + 95.9 BLR20 .220P + +Rph13 94.9 - 75.9 BLR21 .200P BLR14 .5653P + BLR10 .5653P - -Rph13 BLR13 .5453P - + BLR22 .5457P

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All SSR and PCR-fingerprinting markers clustered pts 211 and 231P+ together, both of which originated from New Zealand. The UPGMA similarity dendrograms produced from the cluster analyses based on markers F4-15 plus F7-22, M13 and (GACA)4, grouped all 22 P. hordei isolates (Table 6.5). Given that the marker (GACA)4 resolved the greatest genetic variation among the P. hordei isolates, different “GACA” groups were defined (Table 6.6).

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100 35 40 45 50 55 60 65 70 75 80 85 90 95 25-Pst .110 E143A+ 83.3 52.3 26-BGYR .981549

45.0 23-Pt .104 -2,3,6,7,11 24-Pgt .194 -2,3,7,8,9 66.7 27-Pga .41+Pg9 BLR01 .211P + BLR02 .220P + BLR03 .253P - BLR04 .243 (+Reka1) BLR05 .200P + BLR06 .232 (+Reka1) BLR07 .201 BLR08 .201P + 33.3 BLR09 242P. + 96.0 BLR12 .243 (+Ricardo) 88.9 BLR19 .5610P + BLR14 .5653P + BLR11 .243P + Yellow 87.1 + BLR16 .5653P +Rph13 90.9 BLR15 4610P. + 89.0 BLR20 .220P+ +Rph13 85.8 BLR10 .5653P - -Rph13 BLR17 .211 70.5 + 91.7 BLR18 .231P BLR21 .200P - BLR13 .5453P - 90.9 BLR22 .5457P +

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Table 6.5 Cluster groupings of Puccinia hordei isolates based on the cluster analyses of SSR markers F4-15 and F7-22 and PCR-fingerprinting primers M13 and (GACA)4

Gp F4-15 + F7-22 Gp M13 Gp (GACA)4 1 201 1 201P+ 1 211P+ 201P+ 243 (+Ricardo) 220P+ 243 (+Ricardo) 5653P+ +Rph13 253P- 5653P+ +Rph13 2 211P+ 243 (+Reka1) 5457P+ 220P+ 200P+ 2 200P+ 253P- 232 (+Reka1) 5653P- -Rph13 243 (+Reka1) 201 5453P- 200P+ 201P+ 220P+ +Rph13 232 (+Reka1) 242P+ 3 4610P+ 201 243 (+Ricardo) 5610P+ 242P+ 2 5610P+ 200P- 243P+ Yellow 3 5653P+ 4 243P+ Yellow 4610P+ 4 243P+ Yellow 5 232 (+Reka1) 5610P+ 5653P+ +Rph13 242P+ 220P+ +Rph13 4610P+ 6 211P+ 3 200P- 5 220P+ +Rph13 253P- 4 5653P+ 6 5653P- -Rph13 220P+ 5 5653P- -Rph13 7 211

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243 (+Reka1) 6 5453P- 231P+ 5653P+ 5457P+ 8 200P- 7 211 7 211 9 5453P- 231P+ 231P+ 10 5457P+

Gp: Similarity groups based on the cluster analyses of P. hordei pathotype carried out with GelCompar II software

Table 6.6 Groups of Puccinia hordei pathotypes based on the cluster analyses of PCR- fingerprinting primer (GACA)4 , showing their virulence against Rph genes GACA Isolate Pathotype Virulence to Rph genes*

Gp number** 1 1 211P+ Rph1, Rph4, Rph8, Rph19 2 220P+ Rph5, Rph8, Rph19 3 253P- Rph1, Rph2, Rph4, Rph6, Rph8 4 243 (+Reka1) Rph1, Rph2, Rph6, Rph8, Rph19 5 200P+ Rph8, Rph19 6 232 (+Reka1) Rph2, Rph4, Rph5, Rph8, Rph19 7 201 Rph1, Rph8 8 201P+ Rph1, Rph8, Rph19 9 242P+ Rph2, Rph6, Rph8, Rph19 12 243 (+Ricardo) Rph1, Rph2, Rph6, Rph8 2 19 5610P+ Rph4, Rph8, Rph9, Rph10, Rph12, Rph19 3 14 5653P+ Rph1, Rph2, Rph4, Rph6, Rph8, Rph9, Rph10, Rph12, Rph19 4 11 243P+ Yellow Rph1, Rph2, Rph6, Rph8, Rph19 16 5653P+ +Rph13 Rph1, Rph2, Rph4, Rph6, Rph8, Rph9, Rph10, , Rph12, Rph13, Rph19 15 4610P+ Rph4, Rph8, Rph9, Rph12, Rph19 5 20 220P+ +Rph13 Rph5, Rph8, Rph13, Rph19 6 10 5653P- -Rph13 Rph1, Rph2, Rph4, Rph6, Rph8, Rph9, Rph10 Rph12 7 17 211 Rph1, Rph4, Rph8 18 231P+ Rph1, , Rph4, Rph5, Rph8, Rph19 8 21 200P- Rph8 9 13 5453P- Rph1, Rph2, Rph4, Rph6, Rph9, Rph10, Rph12

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10 22 5457P+ Rph1, Rph2, Rph3, Rph4, Rph6, Rph9, Rph10, Rph12, Rph19

GACA Gp: GACA groups of P. hordei pathotypes, *with respect to the resistance genes listed in Appendix D1, ** From Table 6.1

Discussion The evolution of new virulent pts of P. hordei is a significant constraint in the economical production of barley in Australia and worldwide. Understanding genetic diversity in P. hordei is fundamental in the efforts to develop cultivars of barley with resistance to this pathogen. For example, genetically diverse fungal pathogens may have a greater potential to evolve new pts with the ability to overcome resistance. In earlier work, six pts of P. hordei were identified from aeciospores collected from infected plants of O. umbellatum in SA (Wallwork et al. 1992). Furthermore, high diversities of P. hordei pts have been reported in SA in pathogenicity surveys, suggesting that sexual recombination is contributing to pathogen diversity (Park 2010). Prior to the current study, no attempt had been made to study the genetic diversity of P. hordei in Australia.

The ability of SSRs developed based on Pt to amplify alleles in P. coronata and P. graminis was reported by Wang et al. (2010) and the usefulness of the PCR-fingerprinting primers M13 and GACA in discriminating fugal pathogens has been shown in several studies (Cogliati et al. 2007; Delhaes et al. 2008; Meyer et al. 2001; Roque et al. 2006; Trilles et al. 2008). In view of this, SSRs developed for Pt, Pgt and Pca, along with the PCR- fingerprinting primers M13 and (GACA)4, were assessed for their utility in P. hordei. All Pgt and Pt SSRs showed 100% cross amplification, producing PCR products in all 22 P. hordei isolates. In contrast, only nine of the 37 SSR primers developed for Pca amplified in P. hordei, consistent with previous reports of the specificity of these markers for Pca (Dambroski and Carson 2008). Overall, the usefulness of the SSRs tested in studying P. hordei was very limited as only two, both developed from Pgt sequence information, were polymorphic. The failure to find more polymorphic loci in P. hordei was surprising given the fact that all Pgt and Pt SSRs displayed flanking sequence conservation and excellent PCR amplification of all alleles. The monomorphism of these SSRs may be due to a number of factors such as genome size, low SSR mutation rates leading to shorter motif repeats, or the clonal nature of the Australian isolates of P. hordei used.

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Cluster analyses of all markers used revealed from three to 10 clusters among the 22 P. hordei isolates and control pathogen isolates. A high percentage of similarity was observed among the P. hordei clusters, whereas the control pathogen isolates were more diverse. The

SSR markers F4-15 and F7-22 and the PCR-fingerprinting primers M13 and (GACA)4 clearly differentiated Pt, Pgt, Pst, BGYR, Pga from each other and from the isolates of P. hordei

(Fig. 6.4, 6.5 and 6.7). The two SSRs, M13 and (GACA)4, revealed 0.9%, 26.4% and 33.3 % genetic similarities only, respectively, between P. hordei and the control rust pts. These findings are in accordance with earlier studies in which isolates of Pgt were clearly differentiated from isolates of P. hordei using AFLP (Sun et al. 2007).

In this study, the two polymorphic SSRs and the PCR-fingerprinting markers distinguished Pst and BGYR with 57% to 83.3% genetic similarities respectively, in accordance with an earlier study of these rust pathogens by Keiper et al. (2003) in which Pst and BGYR were distinct but more similar compared to other rust pathogen species. SSRs grouped together Pt and Pgt with a high level of confidence (bootstrap value 82%) and distinguished these from the other controls and the P. hordei isolates whereas Pga was stand alone and these results are in accordance with the results of SAMs and S-SAPs used to genotype the rust pathogens by Keiper et al. (2003). The significant separation of oat stem rust (Pga) from wheat rust pathogens (Pst and Pt) is in accordance with their different host. Both markers M13 and

(GACA)4 formed clades of Pga and Pgt which is consistent with earlier results of an AFLP study on these rust pathogens (Keiper et al. 2003). The current results support the informative value and usefulness of the SSRs and PCR-fingerprinting markers used in differentiating rust pathogens.

The SSR markers F4-15 and F7-22 and the PCR-fingerprinting primer M13 clustered the 22

P. hordei isolates into seven groups, while the marker (GACA)4 resolved 10 groups among the P. hordei isolates (Table 6.5) and detected more polymorphism. Interestingly, all markers + grouped P. hordei pts 211 and 231P with 100% similarity (Gp 7, Table 6.5) and differentiated them from all other P. hordei pts. Both originated from New Zealand and differ only in virulence on Rph2 and Rph19. It is therefore possible that these two pathotypes are simply related and their distinctiveness from the Australian isolates indicates that the two populations are distinct. This contrasts with results from long-term surveys of pathogenic 148

Genetic diversity in P. hordei variability in wheat rust pathogens across Australia and New Zealand, which have provided substantial evidence of rust migration between the two land masses (Luig 1985). These studies have also provided evidence that wheat rust movement is predominantly from west to east (Luig 1985; Wellings et al. 2003). In view of this, the distinctiveness of the two isolates of P. hordei from New Zealand from those in Australia suggests that they may have originated from a region outside Australasia and that they have remained localized to New Zealand.

Based on pathogenicity, Cotterill et al. (1995) suggested that the appearance of a group of pts distinct from pt 243 and typified by pt 200 and its subsequent single-step mutations in the form of pts 201, 210 and 220 in the 1980s, may have resulted from an exotic incursion. Present results also support this hypothesis that exotic incursions of P. hordei may have occurred and resulted in new pts in Australia.

Studies of pathogenic variability in all three wheat rust pathogens in Australia have provided strong evidence of clonality, with presumed clonal lineages comprising closely related pathotypes derived by sequential single-step mutations from a common ancestor (Keiper et al. 2006). In contrast, pathotypes of P. hordei detected in Australia between 1992 and 2001 did not appear to be so simply related (Park 2003). Of the pathotypes examined in the present study, pt 5457P+ is believed to have originated from pt 5453P- via step-wise mutation for virulence for Rph19 and then Rph3 (Park, unpublished). Surprisingly, while markers M13 and

(GACA)4 grouped these two pathotypes and separated them from all other pathotypes, they were not identical (Figs. 6.5 and 6.7, respectively). Furthermore, both differed with respect to their SSR genotypes (Fig. 6.4). These results show that the relationship between these two pathotypes is not simple and that pt 5457P+ may have arisen through a mechanism other than simple mutation or possibly through sexual recombination.

The molecular analyses in the present study did however provide some evidence of clonal lineages in P. hordei in Australasia. Both SSR markers and the marker (GACA)4 revealed pts 201P+ and 201 to be 100% genetically similar and given that pt 201P+ differs from 201 only in being virulent for Rph19, consistent with pt 201P+ arising via a single step mutation in pt 201 with an added virulence for Rph19. The lack of molecular variation among some of the pts studied support the hypothesis of single-step mutation being an important source of 149

Genetic diversity in P. hordei pathogenic variation in P. hordei, which is consistent with the results published by Steele et al. (2001) who found a similar situation among Australian isolates of Pst.

PCR-fingerprinting primers M13 and (GACA)4 revealed more informative fragments and this technique can be a very efficient and an effective tool to find genetic variations in P. hordei and other rust pts. The PIC value of SSRs F4-15 and F7-22 calculated > 0.50 is in accordance with the standard of high informativeness of SSRs (Botstein et al. 1980) and while these two markers were effective in differentiating the P. hordei pts examined, it is clear that more SSR markers are needed to gain a more detailed and better understanding of genetic variation in this pathogen in Australia.

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General discussion

CHAPTER VII General discussion

World population is expected to reach 9 billion by the middle of this century, increasing the demand for food, land, water and energy. This growth will affect our ability to grow crops in an environmently friendly manner (Godfray et al. 2010). Agricultural scientists face an immense challenge to achieve the target of 70% more food by 2050, a goal set by the recent world summit on food security (Tester and Langridge 2010). During 2011, the FAO food price index rose to 232 points and feed barley prices soared up to 50–100 % in different regions of the world (FAO 2011b). Globally, cultivated barley (Hordeum vulgare L. subsp. vulgare) is a major cereal crop that plays an important role as food, malt and feed in the food chain. To ensure that world's poorest people receive adequate nutrition, there is a need to meet the growing demand of food, with environmentally and socially sustainable production systems. In such systems, integrated pest management (IPM) will play an important role. The use of plant resistance against diseases is often the basis on which IPM is based. The adaptation of cereal rust pathogens to deployed resistance genes is likely to have most damaging impacts in terms of food security (Newton et al. 2010). The ability of the leaf rust pathogen of barley, Puccinia hordei, to overcome resistance genes in Australia has made it a disease of high economic importance (Park 2003; Park 2010) and necessitated the discovery, characterisation and deployment of new resistance genes. The present study was therefore conducted to discover and characterise new sources of seedling and adult plant resistance to P. hordei.

The barley cultivar Ricardo was previously reported to carry an uncharacterised seedling resistance (USR) gene (Park unpublished; Stöcker 1983; Wallwork et al. 1992; Yahyaoui et al. 1988) in conjunction with the characterised seedling resistance gene Rph2 (Pa2) (Henderson 1945; Moseman and Roan 1959; Zloten 1952). A study was conducted to confirm the presence of Rph2 in Ricardo, to characterise the USR present in this cultivar and to investigate the level of resistance afforded by the latter under field conditions, reported previously by Golegaonkar et al. (2009b). The USR in Ricardo was expressed optimally at 23 ± 2oC. Based on phenotyping of populations derived from intercrossing Ricardo with the leaf rust susceptible genotype Gus, the USR in Ricardo was found to be controlled by a single dominant gene, tentatively designated RphRic. Bulk segregant analysis (BSA) and molecular 151

General discussion

mapping of an F3 population derived from the cross Ricardo/Gus using a multiplex-ready PCR technique (Hayden et al. 2008a) located RphRic to chromosome 4H. Being the first gene mapped for leaf rust resistance on this chromosome, it was catalogued as Rph21. When tested with the same P. hordei pathotype (pt) in the field, this F3 population was found to segregate for two genes, indicating the presence of an unknown adult plant resistance (APR) gene in addition to the Rph21. Genotyping with Rph20 (Hickey et al. 2011) linked marker bPb-0837 (Liu et al. 2010) indicated that unknown APR found in Ricardo is new. Because only five characterised seedling resistance genes (Rph7, Rph11, Rph14, Rph15 and Rph18 (Park 2003; Park 2010) and one APR gene (Rph20 Park, unpublished) remain effective against P. hordei in Australia, the new seedling gene Rph21 and the unknown APR found in Ricardo are potentially valuable additional sources of resistance that can be deployed against P. hordei. The presence of Rph2 in Ricardo, reported earlier based solely on gene postulation (Henderson 1945; Moseman and Roan 1959; Zloten 1952), was confirmed by testing a set of

F3 populations derived from intercrossing Ricardo with Peruvian (Rph2) using a pt avirulent for Rph2 and by genotyping with Rph2 linked marker ITS1. This was the first time the marker ITS1 has been validated using Ricardo.

In wheat, APR genes such as Sr2 have been deployed in combination with various seedling resistance genes including Sr13, Sr24 and Sr30 (Bariana et al. 2007), as well as other APR genes including Yr18, Yr29 and Lr34 (Park 2008) to achieve durable resistance against rust pathogens. The role of APR as an important contributor in attaining durable resistance was stressed in several previous studies (Golegaonkar et al. 2009b; Park 2003; Park 2008). Because only one APR gene (Rph20) conferring resistance to leaf rust has been characterised in barley, a search was made for new sources of APR among a set of 113 barley cultivars/lines that were tested in the greenhouse and field over three years (2007–2009). Phenotyping in the greenhouse and the field with P. hordei and genotyping using the Rph20 linked marker bPb-0837 revealed that 23 barley genotypes carried USR, three carried Rph3, the likely presence of Rph20 in 35 barley cultivars/lines and potentially new APR in 33 genotypes. The genotypes found to carry USR, including the culvivars Casino and Felicie, expressed high levels of resistance under field conditions. Both Casino and Felicie may carry a combination of seedling and APR genes, supported by the present results and previous reports of their resistance under greenhouse conditions against a range of P. hordei pts (Golegaonkar et al. 2009b) and the likely presence of APR gene Rph20 based on genotyping 152

General discussion with linked marker bPb-0837 (Singh D., personal communication). Both cultivars appear to be potentially valuable sources of durable resistance against P. hordei, given that they carry a combination of a major seedling resistance gene and APR, as was proposed in breeding for durable rust resistance in wheat (Park 2008). Other genotypes, like Hydrogen, Powdery mildew resistance selection, Tifang, WAU 4633 and VB 9935, all with USR, showed moderate to low levels of resistance under field conditions, which suggests that the seedling resistance in many of these lines is distinct. Apart from Emir, all 35 barley genotypes that were found to carry APR and marker bPb-0837 linked to Rph20 displayed very high levels of APR. Partial resistance to P. hordei in the cultivar Emir was reported in earlier studies (Parlevliet 1979; Parlevliet 1983). Pedigree analysis of most cultivars identified to carry APR, which also carried marker bPb-0837, revealed that all include Gull, H. laevigatum and Diamant (X-ray mutant), whereas 33 genotypes characterised with unknown APR and lacked the marker bPb-0837, carried Gull and H. laevigatum. As the linkage between marker bPb- 0837 and Rph20 is not complete, the APR in all of these genotypes, some of which carry bPb-0837 and some of which do not, could be the same. Genetic analyses using Rph20 stocks (Golegaonkar et al. 2010; Hickey et al. 2011) are necessary to determine the genetic bases of the APR identified in the present study. Given that Gull was reported as susceptible to P. hordei (Golegaonkar et al. 2009b), these results suggest that the APR present in many of these genotypes originated from H. laevigatum. The presence of Diamant in many of the other gneotypes that were positive for marker bPb-0837 also qualifies this genotype as a candidate source of Rph20. Different levels of APR were observed consistently among barley genotypes lacking marker bPb-0837, suggesting the presence of different APR genes or different number of APR genes and possibly additivity among genes. Studies of APR to stripe rust in wheat have revealed that high levels of APR can be conferred by the presence of multiple, additive minor genes (Pathan et al. 2007; Singh et al. 2000). Recently, the QTL qRphND present in barley line ND24260 was reported to be highly effective in combination with APR gene Rph20 against P. hordei at adult plant stages (Hickey et al. 2011).

With the similar aim of finding new sources of resistance to rust, four international nurseries comprising 820 barley lines were sourced from the International Centre for Agricultural Research in the Dry Areas (ICARDA). The lines were tested for resistance to P. hordei, P. graminis f. sp. tritici (Pgt) and barley grass stripe rust (BGYR) in both the greenhouse and field. In the leaf rust studies, 93% of the germplasm was found to possess the seedling 153

General discussion resistance gene Rph3. Five lines were identified as carrying USR and three with unknown APR. While these eight lines represent new and potentially useful resistance, the susceptibility of 812 entries indicated that overall resistance to leaf rust was poor and that care is needed to prevent inadvertent introduction of leaf rust susceptibility should any of this material is used for barley improvement in Australia. Further genetic studies are needed to characterise the resistance present in the eight lines. All lines showed resistance against Pgt in seedling greenhouse tests, however, the effectiveness of this resistance in the field could not be assessed due to poor epidemic development. Given that markers are available for the stem rust resistance genes Rpg1 (Kilian et al. 1997) and rpg4 (Borovkova et al. 1995), it would be worth genotyping the lines with these linked markers. In tests with BGYR, with the exception of two lines, all were immune, which was not unexpected because this pathogen rarely infects barley genotypes (Park 2008). It would also be useful to screen the germplasm for response to true barley stripe rust, P. striiformis f. sp. hordei (Psh) for a prospective source of multiple-resistance against these pathogens.

The utility of simple sequence repeats (SSRs) developed for Pgt, P. triticina (Pt) and P. coronata f. sp. avenae (Pca) in understanding genetic variation in Australian isolates of P. hordei was investigated. To complement these studies, PCR-fingerprinting of the P. hordei isolates was carried out using the primers M13 and (GACA)4. Out of 148 SSRs tested, only two (developed for Pgt) were polymorphic. All markers differentiated the control rust pathogens Pt, Pgt, P. striiformis f. sp. tritici (Pst), BGYR and P. gramanis f. sp. avenae (Pga) from each other and from P. hordei. The molecular analyses revealed evidence of clonal lineages among the P. hordei pts, supporting the hypothesis that many arose from simple mutational changes in the virulence of a founder pt. The marker (GACA)4 was the most informative of those tested, distinguishing 10 clusters among the P. hordei pts tested. The genetic diversity observed among the P. hordei isolates examined provided evidence that some may have originated via sexual recombination on the alternate host Ornithogalum umbellatum. This is the first study on understanding genetic diversity of Australasian pts of P. hordei using PCR-fingerprinting technique and SSR genotyping. These are preliminary findings and further work is required to identify more polymorphic SSRs to increase our understanding of genetic diversity in this rust species.

154

General discussion

In summary, the present studies suggest the following future research recommendations for achieving durable resistance against P. hordei and understanding the genetics of P. hordei:

1. Characterisation of unknown APR possessed by Ricardo and development of markers closely linked to Rph21 for its marker assisted selection in breeding programs and its deployment in combination with other seedling genes and APR genes like Rph20.

2. Genetic analyses of barley cultivars/lines identified with unknown APR and USR to leaf rust and their additional testing with Psh and Pgt and molecular markers.

3. Development and application of more specific SSRs to improve our understanding of genetic diversity in P. hordei.

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Appendices

Appendix D1: Differential genotypes used in the greenhouse tests to assess pathogenicity of Puccinia hordei pathotypes Number Resistance Gene Genotype 1 Rph1 Sudan 2 Rph1 Berg 3 Rph2 Peruvian 4 Rph2 + Rph19 Reka 1 5 Rph2 + Rph5 Quinn 6 Rph2 + Rph6 Bolivia 7 Rph2+? Ricardo 8 Rph3 Estate 9 Rph4 Gold 10 Rph5 Magnif 104 11 Rph7 Cebada Capa 12 Rph8 Egypt 4 13 Rph9 Abyssinian 14 Rph10 Clipper BC8 15 Rph11 Clipper BC67 16 Rph12 Triumph 17 Rph13 PI 531849 18 Rph14 PI 584760 19 Rph15 Bowman + Rph15 20 Rph17 81882/BS1 21 Rph18 38P18/8/1/10 22 Rph19 Cutter 23 Rph19 Prior 24 RphB37 PI366444 25 RphCantala Cantala 26 RphGat Gatam 27 RphQ Q21861 28 Rph? 36l50/3/5/1 29 Rph? 169P15/8 30 Susceptible Gus

Appendix D2: Differential genotypes used in the greenhouse tests to assess pathogenicity of Puccinia graminis f. sp.tritici pathotypes Number Resistance Gene Genotype 1 Sr2, Sr5, Sr6, Sr8a, Sr12 Gatcher Res 2 Sr5, Sr16 Reliance 3 Sr5, Sr6, Sr8a, Sr36 Cook 4 Sr5, Sr8a, Sr9b, Sr12 Egret 176

Appendices

5 Sr5, Sr8a, Sr9b, Sr12, Sr30 Banks 6 Sr5b, Sr7b, Sr9b Combination X 7 Sr6 McMurachy 8 Sr7b, Sr17 Renown 9 Sr7b, Sr9b W2402 10 Sr7b, SrX+Sr18, Sr19, Sr20 Marquis 11 Sr8a Mentana 12 Sr8b Barleta Benvenuto 13 Sr9b Mokoan 14 Sr9e Emmer 15 Sr9g, SrX Acme 16 Sr11 Yalta 17 Sr11, Sr17, Sr36 Mendos 18 Sr13, Sr17 Line S 19 Sr15 Norka 20 Sr21 Einkorn 21 Sr22 W3534 22 Sr24 Agent 23 Sr26 Kite 24 Sr27 Coorong 25 Sr30 Festiguay 26 Sr31 Mildress 27 Sr32 W3531 28 Sr35 Sr35/3*77W549 29 Sr36 TD 30 Sr38 Trident 31 SrAgi TAF 2 32 SrNin Sr Nin 33 SrNorin40 Norin 40 34 SrSatu Satu 35 ? 51209 36 Susceptible Morocco

177

Appendices

Appendix D3: Differential genotypes used in the greenhouse tests to assess pathogenicity of Puccinia striiformis Number Resistance Gene Genotype 1 Yr1 Chinese 166

2 Yr2 Kalyansona 3 Yr2, + Heines VII 4 Yr2, Yr6, Yr25 Heines Kolben 5 Yr3 Vilmorin 27 6 Yr4 Hybrid 46 7 Yr5 Triticum spelta 8 Yr6, Yr2 Heins Peko 9 Yr7 Reichersberg 42 10 Yr7, Yr22, Yr23 Lee 11 Yr8, Yr19 Compair 12 Yr9 Fed 4/Kavkaz 13 Yr9, Yr2, +? Clement 14 Yr10 Moro 15 Yr15 Yr 15/6 Avocet S 16 Yr17 Trident 17 Yr17 Ellison 18 Yr17 Binnu 19 Yr25 Hugenoot 20 Yr27 Selkirk 21 Yr33 Gregory 22 YrA Avocet R 23 YrCV, Yr32 Carstens V 24 YrJ Breakwell/Jackie 25 YrND Nord Desprez 26 YrSD, Yr25 Strubes Dickkopf 27 YrSP Spalding Prolific 28 YrSU Suwon 92/ Omar 29 Susceptible Avocet S

178

Appendices

Appendix 1: Description of scale used in the greenhouse for scoring rust infections at seedling stage Its Rust Response Description 0;= Immune No visible uredinia or other macroscopic sign of infection. 0; to ; Resistant Necrotic flecks of varying sizes. ;N Resistant Necrotic areas, dead tissues without uredinia. 1 Resistant Restricted small uredinia surrounded by necrosis. 2 Resistant Small to medium uredinia often surrounded by chlorosis or necrosis, green island may be surrounded by chlorosis or necrosis. X Resistant Random distribution of variable sized uredinia on single leaf with a pure rust culture. Y Resistant Ordered distribution of variable sized uredinia, with larger uredinia at the leaf tip and smaller uredinia at the leaf base. Z Resistant Ordered distribution of variable sized uredinia, with larger uredinia at the leaf base and smaller uredinia at the leaf tip. 3 Resistant Medium sized uredinia that may be associated with chlorosis or rarely necrosis. 3+ to 4 Susceptible Large uredinia without chlorosis or necrosis/abundant sporulation. ITs: Infection types. An Infection type followed by letters C, N and CN denotes chlorosis, necrosis and chlorosis turning into necrosis respectively

179

Appendices

Appendix 2: The modified Cobb’s scale used to score rust infections in the field Rust Score Abbreviation Description Rust Response Description 0 Immune No symptoms R Resistant Few to more, light or dark flecks, no uredinia TR Resistant to Moderately Resistant Trace or minute uredinia with no sporulation MR Moderately Resistant Small uredinia with slight sporulation MR–MS Moderately Resistant to Moderately Small to medium uredinia with moderate sporulation Susceptible MS Moderately Susceptible Medium sized uredinia with moderate sporulation MS–S Moderately Susceptible to Susceptible Medium sized uredinia with moderate to heavy sporulation S Susceptible Large uredinia with abundant sporulation, lesions formed

Percentage of leaf area infected was followed by scale 0, R, TR, MR, MS and S

180

Appendices

Appendix 3: Range of infection types produced on barley leaves in the greenhouse when inoculated with P. hordei pathotypes. (;C denotes characteristic genetic black flecks)

181

Appendices

Appendix 3.1 Observed number of segregating and homozygous susceptible BC1F2 lines developed from Ricardo/Gus//Gus cross, when tested against Puccinia hordei pathotype 5457P+ under greenhouse conditions of 23oC temperature and natural light

BC1F2s Line No. Resistant Plants Susceptible Plants

1 17 6 2 17 6 3 HS 4 HS 5 HS 6 18 2 7 13 11 8 21 3 9 19 5 10 19 4 11 HS 12 HS 13 17 6 14 15 6 15 HS 16 21 4 17 18 3 18 HS 19 21 4 20 HS 21 HS 22 13 4 23 20 5 24 21 8 25 16 5 26 20 7 27 HS 28 HS 29 HS 30 24 6 31 13 5 32 14 5 33 HS 34 HS 35 HS 36 HS 37 HS 38 15 3 39 HS 182

Appendices

40 12 5 41 HS 42 17 9 43 HS 44 HS 45 HS 46 19 6 47 20 7 48 21 9 49 14 4 50 HS 51 16 7 52 18 8 53 HS 54 16 8 55 17 6 56 HS 57 13 4 58 HS 59 19 7 60 HS 61 HS 62 HS 63 HS 64 19 6 65 HS 66 19 6 67 HS 68 HS 69 15 5 70 19 7 71 HS 72 HS 73 6 20 74 HS 75 HS 76 17 6 77 HS 78 17 7 79 HS 80 19 7 81 20 6 82 HS 83 21 7 84 20 7

183

Appendices

85 HS 86 HS 87 HS 88 15 9 89 HS 90 23 4 91 17 6 92 HS 93 16 7 94 HS 95 18 7 96 10 2 97 18 5 98 HS 99 11 11 100 HS 101 14 7 102 19 7 103 18 2 104 17 8 105 HS 106 HS 107 19 6 108 HS 109 17 6 110 HS 111 HS 112 HS 113 18 6 114 HS 115 HS 116 8 3 117 10 16 118 HS 119 HS 120 15 5 121 17 6 122 20 4 123 HS 124 17 6 125 20 6 126 16 6 127 8 1 128 17 7 129 22 6

184

Appendices

130 HS TOTAL=130 SEG=70 HS=60 Note: Ricardo produced 11++C++ and Gus produced 3+ infection types. SEG denotes segregating lines and HS denotes non segregating susceptible lines

Appendix 3.2 Observed number of homozygous resistant, segregating and homozygous susceptible F3 lines developed from Ricardo/Gus cross, when tested against Puccinia hordei pathotype 5457P+ under greenhouse conditions of 23oC temperature and natural light

F3s Line No. Resistant Plants Susceptible Plants DNA Extraction

1 18 4 Yes 2 8 3 Yes 3 HS Yes 4 21 3 Yes 5 HR Yes 6 14 6 Yes 7 HR Yes 8 HS Yes 9 14 2 Yes 10 9 2 Yes 11 HS Yes 12 HS Yes 13 HR Yes 14 HR Yes 15 16 3 Yes 16 5 2 Yes 17 15 3 Yes 18 15 2 Yes 19 HR Yes 20 HS Yes 21 9 2 Yes 22 15 3 Yes 23 12 2 Yes 24 HS Yes 25 12 3 Yes 26 1P 3+ No 27 5 2 Yes 28 HS Yes 29 HR Yes 30 14 7 Yes 31 HR Yes 32 3 2 Yes 33 1 1 No 185

Appendices

34 16 3 Yes 35 HS Yes 36 HS Yes 37 12 6 Yes 38 10 10 Yes 39 4 4 No 40 5 1 Yes 41 5 3 Yes 42 HS Yes 43 13 3 Yes 44 1P No 45 No 46 HS Yes 47 10 5 Yes 48 HS Yes 49 18 7 Yes 50 HS Yes 51 10 9 Yes 52 HS Yes 53 17 6 Yes 54 9 3 Yes 55 10 6 Yes 56 HR Yes 57 7 2 Yes 58 17 5 Yes 59 HS Yes 60 13 4 Yes 61 3 3 No 62 14 4 Yes 63 6 3 Yes 64 HS Yes 65 10 2 Yes 66 9 4 Yes 67 2 1 No 68 10 9 Yes 69 HS Yes 70 13 3 Yes 71 15 3 Yes 72 15 8 Yes 73 12 9 Yes 74 HS Yes 75 5 3 Yes 76 17 5 Yes 77 3 4 No 78 18 5 Yes 186

Appendices

79 15 7 Yes 80 15 11 Yes 81 HR Yes 82 17 5 Yes 83 HR Yes 84 HS Yes 85 15 3 Yes 86 7 5 Yes 87 HR Yes 88 HR Yes 89 11 6 Yes 90 HR Yes 91 HS Yes 92 HS Yes 93 18 3 Yes 94 18 3 Yes 95 15 7 Yes 96 19 10 Yes 97 16 4 Yes 98 HR Yes 99 HR Yes 100 16 7 Yes 101 HR Yes 102 HR Yes 103 HR Yes 104 19 3 Yes 105 HR Yes 106 11 3 Yes 107 20 6 Yes 108 HR Yes 109 5 6 Yes 110 16 5 Yes 111 19 7 Yes 112 HR Yes 113 17 4 Yes 114 10 2 Yes 115 HR Yes 116 HR Yes 117 No 118 HR Yes 119 4 1 Yes 120 13 4 Yes 121 No 122 HS Yes 123 HR Yes 187

Appendices

124 17 6 Yes 125 10 3 Yes 126 17 4 Yes 127 HS Yes 128 HS Yes 129 18 4 Yes 130 19 8 Yes 131 HR Yes 132 HS Yes 133 HR Yes 134 HR Yes 135 10 5 Yes 136 HS Yes 137 HS Yes 138 10 3 Yes 139 HS Yes 140 11 4 Yes 141 HR Yes 142 No 143 21 6 Yes 144 11 5 Yes 145 HR Yes 146 HS Yes 147 No 148 HS Yes 149 15 6 Yes 150 HS Yes 151 17 7 Yes 152 HS Yes 153 8 4 Yes 154 HS Yes 155 HS Yes 156 12 6 Yes 157 17 3 Yes 158 12 5 Yes 159 HS Yes 160 HS Yes 161 21 4 Yes 162 HS Yes 163 18 6 Yes 164 HR Yes 165 12 2 Yes 166 16 2 Yes 167 HS Yes 168 15 4 Yes 188

Appendices

169 HS Yes 170 HS Yes 171 HS Yes 172 16 5 Yes 173 13 6 Yes 174 19 5 Yes 175 HR Yes 176 20 5 Yes 177 HS Yes 178 15 7 Yes 179 8 3 Yes 180 HS Yes 181 13 3 Yes 182 24 3 Yes 183 19 3 Yes 184 21 3 Yes 185 16 3 Yes 186 16 6 Yes 187 25 4 Yes 188 HR Yes 189 HS Yes 190 HR Yes 191 16 8 Yes 192 HR Yes 193 HS Yes 194 HS Yes 195 HR Yes 196 2 1 No 197 9 3 Yes 198 16 5 Yes 199 HR Yes 200 14 4 Yes Total 187 HR = 37 SEG = 104 HS = 46

Note: Ricardo produced 11++C++ and Gus produced 3+ infection types. F3 lines with fewer plants were discarded and total remained 187 after exclusions. HR denotes homozygous resistant, SEG denotes segregating and HS denotes non segregating susceptible

189

Appendices

IBON 31 Line Pgt Pgt IBON 33 Line Pgt IBON 34 Line Pgt IBON 32 Line No. No. ITs ITs No. ITs No. ITs 1 X-C 1 X=C 1 X=C 1 ;1-C 2 Missing 2 X=C 2 0; 2 XC 3 13C 3 XC 3 0; 3 X 4 X 4 ;1- 4 0; 4 X 5 X-C 5 X=C 5 ;1 5 X 6 X-C 6 ;1+ 6 ;1 6 1+3 7 X-C 7 X 7 ;1-C 7 X 8 X-C 8 X=C 8 0;- 8 X 9 X-C 9 ;1++ 9 ;1-C 9 XC 10 Missing 10 X 10 ;1++ 10 XC 11 X-C 11 X=C 11 0; 11 XC 12 X-C 12 0;- 12 0;- 12 XC 13 13 13 XC 13 0;- 13 Missing 14 X-C 14 XC 14 X-C 14 XC 15 X-C 15 XC 15 0; 15 XC 16 Missing 16 XC 16 1++ 16 XC 17 X-C 17 X-C 17 ;1+ 17 ;1C 18 X=C 18 X=C 18 0;- 18 XC 19 XC 19 X-C 19 X 19 XC 20 X-C 20 X=C 20 0;- 20 XC 21 11+ 21 Missing 21 ;1= 21 X

190

Appendices

22 13-C 22 X 22 ;1= 22 0; 23 13-C 23 X-C 23 0;- 23 0; 24 XC 24 X 24 XC 24 X=C 25 X 25 ;1-C 25 X 25 XC 26 13- 26 XC 26 X- 26 0; 27 X-C 27 X 27 X- 27 X=C 28 13 28 X 28 1+ 28 X=C 29 X-C 29 X 29 Missing 29 ;1+C 30 ;1 30 X 30 Missing 30 X=C 31 1++ 31 X- 31 Missing 31 X=C 32 0;- 32 X=C 32 Missing 32 ;1++C 33 X- 33 X 33 Missing 33 ;1++C 34 X=C 34 0;= 34 Missing 34 XC 35 X=C 35 0;- 35 Missing 35 XC 36 X=C 36 0; 36 Missing 36 XC 37 X=C 37 X 37 Missing 37 XC 38 X=C 38 ;1 38 Missing 38 Missing 39 1++ 39 ;1+ 39 Missing 39 XC 40 X-C 40 X 40 Missing 40 ;1=C 41 ;1++ 41 ;1- 41 ;1+ 41 ;1-C 42 1++ 42 0; 42 0; 42 ;1-C 43 X-C 43 X 43 X 43 XC 44 X-C 44 X=C 44 X=C 44 XC 45 X=C 45 ;1 45 X- 45 0;C 46 X 46 ;1 46 1+ 46 ;1+ 47 X=C 47 ;1C 47 X 47 ;1+ 48 X=C 48 X-C 48 ;1- 48 XC

191

Appendices

49 0;- 49 0; 49 0; 49 ;1C 50 0;-,1P1 50 0; 50 ;1= 50 X=C 51 0;-,1P1 51 0;- 51 ;1-C 51 X-C 52 0;- 52 0;- 52 0; 52 ;1C 53 X-C 53 0; 53 XC 53 ;1=C 54 X-C 54 0;- 54 0; 54 XC 55 X-C 55 0;- 55 X- 55 XC 56 X-C 56 X 56 XC 56 X 57 0;- 57 X 57 X- 57 X-C 58 0;- 58 0; 58 0; 58 X-C 59 13 59 X- 59 X- 59 0;,1 60 13- 60 X- 60 1++ 60 X 61 X=C 61 ;1 61 X 61 X-C 62 ;1C 62 ;1+ 62 ;1+C 62 X-C 63 ;1- 63 ;1+ 63 X 63 ;1C 64 0;- 64 ;1 64 Missing 64 X-C 65 ;1 65 X 65 XC 65 X-C 66 XC 66 ;1 66 X-C 66 0; 67 X-C 67 Missing 67 X 67 X=C 68 0;- 68 ;11+ 68 ;1-C 68 X=C 69 0;- 69 X 69 X- 69 ;1C 70 1+ 70 0;- 70 ;1=C 70 X=C 71 X=C 71 ;1C 71 X=C 71 0; 72 1++ 72 0;- 72 X-C 72 ;1=C 73 ;1+ 73 ;1= 73 X 73 XC 74 X=C 74 0;- 74 X- 74 XC 75 X+ 75 ;1- 75 Missing 75 0;

192

Appendices

76 1+ 76 ;1- 76 ;1= 76 ;1++C 77 1++ 77 0; 77 ;1C 77 ;1++C 78 X 78 ;1++C 78 ;1C 78 ;1++C 79 X 79 ;13 79 ;1C 79 ;1+C 80 X 80 0; 80 0; 80 XC 81 1+ 81 ;1= 81 X 81 X-C 82 1+ 82 0;- 82 ;1C 82 X=C 83 1+ 83 0;- 83 XC 83 0; 84 X 84 0;- 84 X=C 84 1++ 85 X+ 85 0;- 85 XC 85 X 86 0;- 86 Missing 86 0; 86 0; 87 1+3- 87 0;- 87 ;1-C 87 0;- 88 0;- 88 0;- 88 0; 88 ;-C 89 X-C 89 0;- 89 ;1-C 89 ;,C 90 13-C 90 0;- 90 XC 90 X=C 91 X 91 Missing 91 Missing 91 X 92 X 92 0;- 92 ;1C 92 0;- 93 ;1+ 93 X 93 X=C 93 0; 94 13=C 94 1+ 94 ;1C 94 X=C 95 1+ 95 ;1- 95 X-C 95 0; 96 11+C 96 X=C 96 X=C 96 XC 97 X 97 ;1C 97 13 97 XC 98 13 98 0;- 98 X- 98 0; 99 13 99 X=C 99 X 99 ;-C 100 0; 100 X 100 11+ 100 0; 101 0;- 101 ;1- 101 ; 101 1++ 102 11- 102 ;1= 102 X=C 102 X

193

Appendices

103 0;-,1P1 103 ;1= 103 1 103 ;C 104 ;1-C 104 0;1= 104 0;- 104 11+C 105 ;1-C 105 X=C 105 0;C 105 ;1-C 106 ;1=C 106 ;1-C 106 0;- 106 X 107 ;1+C 107 1=C 107 ;1=C 107 0; 108 X 108 ;1=C 108 0; 108 XC 109 ;11-C 109 X 109 ;1+ 109 X 110 ;11- 110 0;-3 110 ;1+ 110 3= 111 ;1=C 111 X 111 Missing 111 X=C 112 ;1-C 112 0; 112 0; 112 ;1++ 113 X 113 X 113 ;1+C 113 ;13 114 0;-,1 114 1++ 114 XC 114 XC 115 ;1- 115 1 115 0;- 115 X-C 116 ;1- 116 X 116 ;1+ 116 Missing 117 X= 117 ;1 117 X 117 ;1++ 118 X- 118 1+ 118 X 118 X 119 X 119 X=C 119 ;1++ 119 X=C 120 X 120 ;1C 120 3 120 X 121 ;1=C 121 ;1 121 X-C 121 0;1+ 122 ;1C 122 0; 122 ;1C 122 0;1+ 123 ;11+C 123 XC 123 X=C 123 0;1+ 124 X 124 X=C 124 X-C 124 0;1+ 125 0;-,1P3 125 X 125 0;- 125 0;- 126 ;11+3 126 X 126 XC 126 0;- 127 ;1C 127 ;1 127 XC 127 ;1+ 128 X-C 128 ;1 128 ;1++ 128 ;C 129 ;1+ 129 X=C 129 0; 129 ;1+

194

Appendices

130 ;1+3 130 X-C 130 0;C 130 0; 131 X 131 X- 131 0; 131 ;1 132 X-C 132 1++3X 132 0;C 132 0;1- 133 X- 133 ;1- 133 0; 133 ;13- 134 X 134 13 134 X=C 134 0;1- 135 X-C 135 13 135 X=C 135 ;1=C 136 ;1 136 X-C 136 XC 136 X 137 X-C 137 ;1- 137 X=C 137 X 138 X 138 ; 138 X=C 138 X 139 0;- 139 X-C 139 X=C 139 ;1C 140 X+ 140 X=C 140 X-C 140 ;1C 141 ;1C 141 ;1++ 141 X 141 ;1+C 142 1++3 142 X=C 142 ;1+ 142 0; 143 ;1 143 X 143 X-C 143 X=C 144 0; 144 X-C 144 Missing 144 0;- 145 0;- 145 XC 145 0;-

146 X 146 X 146 0;-

147 1+3 147 X= 147 0;

148 X 148 0; 148 X

149 ;1+ 149 0; 149 X

150 X-C 150 X 150 ;1-

151 X 151 XC 151 0;-

152 X-C 152 X-C 152 0;-

153 X 153 0;- 153 ;1-C

154 X=C 154 0;- 154 0;-

155 X 155 0; 155 X-C

156 ;1 156 Missing 156 0;

195

Appendices

157 X-C 157 ;13 157 XC

158 13- 158 X 158 ;1C

159 ;1 159 0; 159 X

160 X=C 160 0;1= 160 X1-C

161 X 161 0;- 161 0;

162 X 162 0;1= 162 ;1+

163 X=C 163 0;1= 163 ;1+

164 X-C 164 0;1= 164 ;1+

165 1+3 165 X 165 0;

166 X 166 ;1+ 166 0;

167 X=C 167 ;1 167 X

168 1++ 168 ;1 168 ;1-C

169 11- 169 11+ 169 ;1=C

170 0;- 170 0; 170 0;

171 11+ 171 ;1 171 Missing

172 X=C 172 ;1 172 0;

173 13= 173 X 173 0;

174 0;- 174 ;1- 174 0;

175 ;11+ 175 X- 175 ;C

176 ;11+ 176 X-C 176 X

177 X-C 177 0;- 177 ;1C

178 0;- 178 X=C 178 ;1+C

179 ;1C 179 X=C 179 X

180 X 180 X=C 180 ;1+

181 X 181 X- 181 X

182 X=C 182 ;1+ 182 0;

183 ;1- 183 X 183 0;1

196

Appendices

184 0;- 184 X+ 184 0;'1

185 ;1- 185 ;1 185 0;

186 11- 186 XC 186 0;

187 11+ 187 ;C 187 ;1=C

188 1++ 188 ;1C 188 ;1-

189 ;1+ 189 X=C 189 0;

190 ;1+ 190 ;1=C 190 XC

191 0;- 191 ;1=C 191 X-C

192 ;1-C 192 X-C 192 X

193 ;1 193 X 193 ;1+

194 13 194 X=C 194 X

195 X 195 ;1+C 195 Missing

196 13 196 0; 196 11+

197 ;1 197 X-C 197 11+

198 1 198 0; 198 ;1C

199 0;- 199 0;- 199 ;1+C

200 0;- 200 ;1-C 200 ;1C

201 ;1,13 201 X 201 ;1C

202 Missing 202 ;1 202 Missing

203 0;-,1P1- 203 X 203 0;

204 Missing 204 ;1=C 204 ;1=C

205 ;1+ 205 0;= 205 Missing

206 1++

207 0;-

208 0;=.1P1++

209 ;1

210 ;1+

197

Appendices

211 X-

212 0;-

213 X-C

214 ;1+

215 X

216 Missing

217 X-C

218 ;1+

219 ;1++

220 Missing

221 ;1

222 ;1

223 X

224 X

225 Missing

226 X

227 ;1-

228 0;-

229 X

230 ;1++3

231 X

232 X

233 ;1-C

234 X

235 X

236 ;1+

237 XC

198

Appendices

238 13

239 ;1

240 ;1-

241 0;-

242 ;1=

243 ;1++

244 ;1-C

245 ;1

246 X-C

247 X=C

248 X

249 X

250 X=C

251 X-C

252 X=C

253 0;-

254 ;1=

255 ;1-

256 X

257 X

258 0;-

259 X

260 X=C

261 ;1

262 0;-

263 ;1-C

264 ;1-C

199

Appendices

265 ;1-C

266 ;1-C

No. Differential*

1 Reliance 3+

2 Marquis 3+

3 Acme 3+

4 Emmer 3+

5 Einkorn ;CN

6 Line S 0;-

7 McMurachy 3+

8 Yalta 3+

9 W2402 33+

10 TD 33+

11 Renown 33+

12 Mentana 0;-,3-

13 Norka 0;C

14 Festiguay ;1-C

15 TAF 2 ;-CN

16 51209 ;1C

17 Barleta ;1++3

18 Coorong 0;

19 Satu 0;

20 Sr Nin ;

21 Gatcher 0;-

22 Comb X 3+

23 Kite 0;-

24 Agent 0;-

200

Appendices

25 Norin 40 ;1+C

26 Cook 0;-

27 Banks 2++C

28 Egret 3+

29 Mendos 0;=

30 Mildress ;1-C

31 Mokoan 3+

32 W3534 23-C

33 Sr32 ;1=

34 Sr35 0;-

35 Trident 0;-

36 Morocco 3+

*Sr genes explained in appendix D2

Appendix 4.2 Greenhouse infection types (ITs) data of all four International Barley Observation Nurseries (IBONs) and differential genotypes tested with BGYR pathotype

IBON 31 Line BGYR IBON 32 Line BGYR IBON 33 Line BGYR IBON 34 Line BGYR No. ITs No. ITs No. ITs No. ITs 1 0;- 1 0;- 1 0;- 1 0;- 2 Missing 2 0;- 2 0;- 2 0;- 3 0;- 3 0;- 3 0;- 3 0;- 4 0;- 4 0;- 4 0;- 4 0;- 5 0;- 5 0;- 5 0;- 5 ;C,2++ 6 0;- 6 0;- 6 0;- 6 0;- 7 0;C 7 0;- 7 0;- 7 0;- 8 0;- 8 0;- 8 0;- 8 0;- 201

Appendices

9 0;- 9 0;- 9 0;- 9 0;- 10 Missing 10 0;- 10 0;- 10 0;- 11 0;- 11 0;- 11 0;- 11 0;- 12 0;- 12 0;- 12 0;- 12 0;- 13 0;- 13 0;- 13 0;- 13 Missing 14 0;- 14 0;- 14 0;- 14 0;- 15 0;- 15 0;- 15 0;- 15 0;- 16 Missing 16 0;- 16 0;- 16 0;- 17 0;C 17 0;- 17 0;- 17 0;- 18 0;- 18 0;- 18 0;- 18 0;- 19 0;- 19 0;- 19 0;- 19 0;- 20 0;C 20 0;- 20 0;- 20 0;- 21 0;- 21 Missing 21 0;- 21 0;- 22 0;- 22 0;- 22 0;- 22 0;- 23 0;- 23 0;- 23 0;- 23 0;- 24 0;- 24 0;- 24 0;- 24 0;- 25 0;- 25 0;- 25 0;- 25 0;- 26 0;- 26 0;- 26 0;- 26 0;- 27 0;- 27 0;- 27 0;- 27 0;- 28 0;- 28 0;- 28 0;- 28 0;- 29 0;- 29 0;- 29 Missing 29 0;- 30 0;- 30 0;- 30 Missing 30 0;- 31 0;- 31 0;- 31 Missing 31 0;- 32 0;- 32 0;- 32 Missing 32 0;- 33 0;- 33 0;- 33 Missing 33 0;- 34 0;- 34 0;- 34 Missing 34 0;- 35 0;- 35 0;- 35 Missing 35 0;-

202

Appendices

36 0;- 36 0;- 36 Missing 36 0;- 37 0;- 37 0;- 37 Missing 37 0;- 38 0;C 38 0;- 38 Missing 38 Missing 39 0;- 39 0;- 39 Missing 39 0;- 40 0;- 40 0;- 40 Missing 40 0;- 41 0;- 41 0;- 41 0;- 41 0;- 42 0;- 42 0;- 42 0;- 42 0;- 43 0;C 43 0;- 43 0;- 43 0;- 44 0;C 44 0;- 44 0;- 44 0;- 45 0;- 45 0;- 45 0;- 45 0;- 46 0;- 46 0;- 46 0;- 46 0;- 47 0;- 47 0;- 47 0;- 47 0;- 48 0;- 48 0;- 48 0;- 48 0;- 49 0;- 49 0;- 49 0;- 49 0;- 50 0;- 50 0;- 50 0;- 50 0;- 51 0;- 51 0;- 51 0;- 51 0;- 52 0;- 52 0;- 52 0;- 52 0;- 53 0;- 53 0;- 53 0;- 53 0;- 54 0;- 54 0;- 54 0;- 54 0;- 55 0;- 55 0;- 55 0;- 55 0;- 56 0;- 56 0;- 56 0;- 56 0;- 57 0;- 57 0;- 57 0;- 57 0;- 58 0;- 58 0;- 58 0;- 58 0;- 59 0;- 59 0;- 59 0;- 59 0;- 60 0;- 60 0;- 60 0;- 60 0;- 61 0;- 61 0;- 61 0;- 61 0;- 62 0;- 62 0;- 62 0;- 62 0;-

203

Appendices

63 0;- 63 0;- 63 0;- 63 0;- 64 0;- 64 0;- 64 Missing 64 0;- 65 0;- 65 0;- 65 0;- 65 0;- 66 0;- 66 0;- 66 0;- 66 0;- 67 0;- 67 Missing 67 0;- 67 0;- 68 0;- 68 0;- 68 0;- 68 0;- 69 0;- 69 0;- 69 0;- 69 0;- 70 0;- 70 0;- 70 0;- 70 0;- 71 0;- 71 0;- 71 0;- 71 0;- 72 0;C 72 0;- 72 0;- 72 0;- 73 0;- 73 0;- 73 0;- 73 0;- 74 0;- 74 0;- 74 0;- 74 0;- 75 0;- 75 0;- 75 Missing 75 0;- 76 0;- 76 0;- 76 0;- 76 0;- 77 0;- 77 0;- 77 0;- 77 0;- 78 0;- 78 0;- 78 0;- 78 0;- 79 0;- 79 0;- 79 0;- 79 0;- 80 0;- 80 0;- 80 0;- 80 0;- 81 0;- 81 0;- 81 0;- 81 0;- 82 0;- 82 0;- 82 0;- 82 0;- 83 0;- 83 0;- 83 0;- 83 0;- 84 0;- 84 0;- 84 0;- 84 0;- 85 0;- 85 0;- 85 0;- 85 0;- 86 0;- 86 Missing 86 0;- 86 0;- 87 0;- 87 0;- 87 0;- 87 0;- 88 0;- 88 0;- 88 0;- 88 0;- 89 0;- 89 0;- 89 0;- 89 0;-

204

Appendices

90 0;- 90 0;- 90 0;- 90 0;- 91 0;- 91 Missing 91 Missing 91 0;- 92 0;- 92 0;- 92 0;- 92 0;- 93 0;- 93 0;- 93 0;- 93 0;- 94 0;- 94 0;- 94 0;- 94 0;- 95 0;- 95 0;- 95 0;- 95 0;- 96 0;- 96 0;- 96 0;- 96 0;- 97 0;- 97 0;- 97 0;- 97 0;- 98 0;- 98 0;- 98 0;- 98 0;- 99 0;- 99 0;- 99 0;- 99 0;- 100 0;- 100 0;- 100 0;- 100 0;- 101 0;- 101 0;- 101 0;- 101 0;- 102 0;- 102 0;- 102 0;- 102 0;- 103 0;- 103 0;- 103 0;- 103 0;- 104 0;- 104 0;- 104 0;- 104 0;- 105 0;- 105 0;- 105 0;- 105 0;- 106 0;- 106 0;- 106 0;- 106 0;- 107 0;- 107 0;- 107 0;- 107 0;- 108 0;- 108 0;- 108 0;- 108 0;- 109 0;- 109 0;- 109 0;- 109 0;- 110 0;- 110 0;- 110 0;- 110 0;- 111 0;- 111 0;- 111 Missing 111 0;- 112 0;- 112 0;- 112 0;- 112 0;- 113 0;- 113 0;- 113 0;- 113 0;- 114 0;- 114 0;- 114 0;- 114 0;- 115 0;- 115 0;- 115 0;- 115 0;- 116 0;- 116 0;- 116 0;- 116 Missing

205

Appendices

117 0;- 117 0;- 117 0;- 117 0;- 118 0;- 118 0;- 118 0;- 118 0;- 119 0;- 119 0;- 119 0;- 119 0;- 120 0;- 120 0;- 120 0;- 120 0;- 121 0;- 121 0;- 121 0;- 121 0;- 122 0;- 122 0;- 122 0;- 122 0;- 123 0;- 123 0;- 123 0;- 123 0;- 124 0;- 124 0;- 124 0;- 124 0;- 125 0;- 125 0;- 125 0;- 125 0;- 126 0;- 126 0;- 126 0;- 126 0;- 127 0;- 127 0;- 127 0;- 127 0;- 128 0;- 128 0;- 128 0;- 128 0;- 129 0;- 129 0;- 129 0;- 129 0;- 130 0;- 130 0;- 130 0;- 130 0;- 131 0;- 131 0;- 131 0;- 131 0;- 132 0;- 132 0;- 132 0;- 132 0;- 133 0;- 133 0;- 133 0;- 133 0;- 134 0;- 134 0;- 134 0;- 134 0;- 135 0;- 135 0;- 135 0;- 135 0;- 136 0;- 136 0;- 136 0;- 136 0;- 137 0;- 137 0;- 137 0;- 137 0;- 138 0;- 138 0;- 138 0;- 138 0;= 139 0;- 139 0;- 139 0;- 139 0;- 140 0;- 140 0;- 140 0;- 140 0;- 141 0;C 141 0;- 141 0;- 141 0;- 142 0;- 142 0;- 142 0;- 142 0;- 143 0;- 143 0;- 143 0;- 143 0;-

206

Appendices

144 0;- 144 0;- 144 Missing 144 0;- 145 0;- 145 0;- 145 0;-

146 0;- 146 0;- 146 0;-

147 0;- 147 0;- 147 0;-

148 0;- 148 0;- 148 0;-

149 0;- 149 0;- 149 0;-

150 0;- 150 0;- 150 0;-

151 0;- 151 0;- 151 0;-

152 0;- 152 0;- 152 0;-

153 0;- 153 0;- 153 0;-

154 0;- 154 0;- 154 0;-

155 0;- 155 0;- 155 0;-

156 0;- 156 Missing 156 0;

157 0;- 157 0;- 157 0;-

158 0;- 158 0;- 158 0;-

159 0;- 159 0;- 159 0;-

160 0;- 160 0;- 160 0;-

161 0;- 161 0;- 161 0;-

162 0;- 162 0;- 162 0;-

163 0;- 163 0;- 163 0;-

164 0;- 164 0;- 164 0;-

165 0;- 165 0;- 165 0;-

166 0;- 166 0;- 166 0;-

167 0;- 167 0;- 167 0;-

168 0;- 168 0;- 168 0;-

169 0;- 169 0;- 169 0;-

170 0;- 170 0;- 170 0;-

207

Appendices

171 0;- 171 0;- 171 Missing

172 0;- 172 0;- 172 0;-

173 0;- 173 0;=,1P1 173 0;-

174 0;- 174 0;- 174 0;-

175 0;- 175 0;- 175 0;-

176 0;- 176 0;- 176 0;-

177 0;- 177 0;- 177 0;-

178 0;- 178 0;- 178 0;-

179 0;- 179 0;- 179 0;-

180 0;- 180 0;- 180 0;-

181 0;- 181 0;- 181 0;-

182 0;- 182 0;- 182 0;-

183 0;- 183 0;- 183 0;-

184 0;- 184 0;- 184 0;-

185 0;- 185 0;- 185 0;-

186 0;- 186 0;- 186 0;-

187 0;- 187 0;- 187 0;-

188 0;- 188 0;- 188 0;-

189 0;- 189 0;- 189 0;-

190 0;- 190 0;- 190 0;-

191 0;- 191 0;- 191 0;-

192 0;- 192 0;- 192 0;-

193 0;- 193 0;- 193 0;-

194 0;- 194 0;- 194 0;-

195 0;- 195 0;- 195 Missing

196 0;- 196 0;- 196 0;-

197 0;- 197 0;- 197 0;-

208

Appendices

198 0;- 198 0;- 198 0;-

199 0;- 199 0;- 199 0;-

200 0;- 200 0;- 200 0;-

201 0;- 201 0;- 201 0;-

202 Missing 202 0;- 202 Missing

203 0;- 203 0;- 203 0;-

204 Missing 204 0;- 204 0;-

205 0;- 205 0;- 205 Missing

206 0;-

207 0;-

208 0;-

209 0;-

210 0;-

211 0;-

212 0;-

213 0;-

214 0;-

215 0;-

216 Missing

217 0;-

218 0;-

219 0;-

220 Missing

221 0;-

222 0;-

223 0;-

224 0;-

209

Appendices

225 Missing

226 0;-

227 0;-

228 0;-

229 0;-

230 0;-

231 0;-

232 0;-

233 0;-

234 0;-

235 0;-

236 0;-

237 0;-

238 0;-

239 0;-

240 0;-

241 0;-

242 0;-

243 0;-

244 0;-

245 0;-

246 0;-

247 0;-

248 0;-

249 0;-

250 0;-

251 0;-

210

Appendices

252 0;-

253 0;-

254 0;-

255 0;-

256 0;-

257 0;-

258 0;-

259 0;-

260 0;-

261 0;-

262 0;-

263 0;-

264 0;-

265 0;-

266 0;-

No. Differentials*

1 Chinese 166 3+

2 Lee 0;-

3 Heines Kolben 0;-

4 Vilmorin 27 0;-

5 Moro 0;-

6 Strubes Dickkopf 0;-

7 Suwon 92/ Omar 0;-

8 Clement 0;-

9 Triticum spelta 0;-

10 Hybrid 46 0;-

11 Reichersberg 42 0;-

211

Appendices

12 Heins Peko 0;-

13 Nord Desprez 0;-

14 Compair 0;-

15 Carstens V 0;-

16 Spalding Prolific 0;-

17 Heines VII 0;-

18 Avocet R 0;C

19 Kalyansona 0;-

20 Trident 0;-

21 Yr 15/6AvS 0;-

22 Hugenoot 0;-

23 Selkirk 0;-

24 Fed 4/Kavkaz 0;-

25 Avocet S 0;-

26 Gregory 0;-

27 Ellison 0;-

28 Binnu 0;-

29 Breakwell/ 0;- Jackie *Yr genes explained in appendix D3

212

Appendices

No. Locus Repeat Motif Primer sequence F: (5'–3') Primer sequence R: (5'–3') Size (bp) Ply

1 F1-1 (GA)12 CGGGAGTAGCTGAAGTAACC CCGCTACGTGATTGCTTATC 393 No

2 F1-10 (CT)30 TATCGCAGACGGTGTCAATG CGCGAGAGTTGTAGACGAG 208 No

3 F1-19 (CTT)18 AAAGATCCTGTTTGCCTACC CGGAATTAGATGGGACCATA 327 No

4 F1-22 (AAAG)22 GAAGCGAAAAACAGGGTAAG GGGCAAGATCAGACCTAGTG 307 No

5 F2-7 (TC)29 GCAGCGGAAGTAGGGCGATG GGCCGGGCAGGATCGTTTAT 402 No

6 F2-14 (GAA)22 GGCGGAATTAGATGGGACCA TCCTCCCCCTCTTCCTCTTC 216 No

7 F2-22 (ACACA)28 CATCACATCCCTAACTAC CTTGGTCCTAAGACATCT 273 No

8 F3-5 (CT)13 AGTCGTTTCGTCGTCGGTTCAT GGAAGGAGAAGCGGAGTCA 306 No

9 F3-9 (CT)15 ATTGCCTCTGCTTCTAGTGT ACCCAGCCACATCATAGTTT 243 No

10 F3-14 (ACT)12 CATTTGCTCTGCGCTCAG TGGGCTCTTCCGACCTTT 236 No

11 F3-20 (ACA)14 CCATCCACCCCAACCACAAC CGGGAGAGGAGGGATTGAT 187 No

12 F4-9 (GGA)8 CGGCCTCTTCTTCGTCTCA ATCGGCAGTGGTCCCTTAC 244 No

13 F4-11 (AAC)10 TTTATTCCCTCCTGTGATTGC GATGAGTTGGGTGCTGATTGAG 338 No

14 F4-15 (CAA)11 CTCAAGCACCCTCAACATCC CGTCGTCCCTCCATAGTCTT 200 - 300 Yes

15 F4-17 (GTT)11 GATGAGGGATGGATGTGATT CGAGACGCAGTAGATCAAG 257 No

16 F4-18 (TTG)11 GGGTCGTTGTGTGGCATTAT CTCAGGAACGCACCTATCTT 250 No

17 F4-20 (GAAA)8 GAAGGCTCAGCGAAGGAT CTCCAGCAGTCTTTCCGATA 294 No

18 F4-23 (AAAAC)13 GTTTGCCTTATTCGTCTCA GGGGTATAAAGTGTATGAAG 438 No

19 F5-9 (AG)13b TTCACGGCCAAATAACAACT ATCGCCATCGTCTCGTCCTC 180 No

20 F5-19 (CT)20b GGTGAGAGTGGCTATTGAGA ATTTTATTCCTTTCAACCAG 274 No

213

Appendices

21 F5-22 (GA)22 GGCAATAAATCAACCTCAACT GCAGGCCATCTAATATCAC 211 No

22 F6-30 (ATATC)8 CGTGAAAAGGTGCTACATCT GGTGAGAAGAGGCATACTAA 247 No

23 F6-32 (ACATT)10 CTATTTTTATCCCTATTACT TCCTTTCCTTACCTAATAGA 152 No

24 F6-36 (TTTGG)12 GGGGGGTGACTGAAAAATA CCCTTTTCTACCTCCTTACTT 312 No

25 F6-31 (TTTGT)9 CGGGGTGACTGAAGATAGAG CCCTTTCTACCTCCTTACTT 306 No

26 F7-7 (TC)21 CCTTCCGCGTTCTCTCAAAG GCCCCGCTTAGTCATTTCGT 286 No

27 F7-9 (AG)24 TAGGGAGGAGAACAAGAGAG ACAGCCTCCAAACTCAAACA 219 No

28 F7-19 (TTG)11 TTTCGCTTGGTTTTTCTGTT CCCCTCACCCCCACCCCTCT 271 No

29 F7-22 (ATG)14 TAACCGACCAACAACAACAA TTCTCCCATCGCTCTCTCTC 100 - 200 Yes

30 F7-26 (AAC)30 TCCCCCATCATCACCCAGCC ATGGGGTTTTATGTTTTTAC 266 No

31 F7-29 (T)5(TTGT)5(T)6(A)5 CGCAATCTCAGTATAAGCAT TGCGGGTAGGTCTGGGTGGC 295 No

32 F8-2 (TTCC)7 CGTTTTGTTTCCTTTTCTCA TCGACCAATCTCCAAAACCA 222 No

33 F8-6 (TTGG)8 ATCGGTTGTGTCGTAGTCTG AAGCGGTCAACAAGAGAAA 225 No

34 F9-1 (CAAAA)7 TCATCACATCCCTAACTACA GTTGGTTTATTTCACTCTTG 163 No

35 F9-2 (CGCTA)7 ACGCTGGGCCGCTACGCTAC AGCGGGTTTTTTGCCTTTTT 259 No

36 F9-4 (TTTTG)7 TACCCAGTAGACAGTCTTG ATCACATCCCTAACTACACA 214 No

37 F9-5 (TTTTA)7 ATCCCCTCCCCCAACTTATC GAGGAAATAAGGGCAGATGA 321 No

38 F9-9 (GTTTT)9++ TCTTGATTATTTTGGGTTGT TCATCACATCCCTAACTACA 267 No

39 F9-12 (TTTTG)10 TCTCAGGATGTTGGTTTAT AAAAACAAGACCACACTACA 156 No

40 F9-14 (AAAAC)11 TCACATCCCTAACTACAACA TCACCTCAAATCCCCACTCA 470 No

41 F9-15 (CACAA)22 ATCACATCCCTAACTACAAC ATCACCTCAATCCCCACTCA 445 No

42 F9-17 (AAAAG)13++ ACGGGGCGAATGGAAATGCT TTCCATGGGCCCTGAAGTTG 286 No

43 F9-19 (AACAC)15 CATCAAATCCCTAACTACAA TGTGCCGTCCTGTATCGTCC 187 No

44 F9-22 (AGAAC)17 ACAAAAACAAGCCCACACTA TGGTTTCCTCATTTTATTAT 389 No

45 F9-23 (AACAA)18+ ATCACATCCCTAACTACAAC GTTTAATTTTCTTGGTCCTA 272 No

214

Appendices

46 F9-27 (ACACA)22 CACATCCCTAACTACAACA TTAATTTTCTTGGTCCTAAG 268 No

47 F9-28 (ACAGA)23 ACATCCCTAACGACAACACA TGTTGGTTTATTTCACTCTT 244 No

48 F9-29 (AAAAC)24 GCACGGCTCAACACAACACA ACCTCAATCCCCACTCAGTC 389 No

49 F9-43 (TTTGTG)7 TGTGGTGGGTGGAACTTGAA CAATCCTGCCATCACTACTC 273 No

50 F9-44 (TGAAGA)8+ AGAGGGGACATCAAGACTAT GGCGGGTTGGGGGGTTATTC 379 No

51 F9-46 (TGTTGG)9 CGGTGAGTATGGGTGTGAAA CCAGCCGGTTCATCCCCTCA 310 No

52 F9-47 (CAACAT)10 AGCACCAGTAACACCAACAC ACGGTGAGTATGAGTGTGAA 211 No

53 PgtSSR6 (TC)9 CCAGCCAAGGAATGGTTAGA AATGCCACTACCCAACTTCG 157-177 No

54 PgtSSR11 (TC)10+(TC)11+(TGG)3 AGTTCGGCATAGGGAATCCT GATTTGCTGGCTTCGGTTAG 161-171 No

55 PgtSSR12 (TC)15 GGACTACTTCATCAGCATTACCA TTCCTCTGTTTTCTCTCTCTCTCTC 155-169 No

56 PgtSSR13 (AC)7TC(AC)4C(AC)2 TGAGTTTGACATGTTGCCGTA CAGTTCCCTTTTCCCCATTT 215-245 No

57 PgtSSR14 (TC)12+(ATT)6 TTCCACATTTCGAACAACGA GCTTGTGTCCCAAGAGCTTC 196-218 No

58 PgtSSR20 (TC)11 CTAGATGAGGGGCAGCGAAT TTCTCTCTCCTTCATCCTAA 145-153 No 59 PgtSSR21 TC rich AAAATGATGGTCTCCTTGGCTA CGTCGCCGACCTTATCTAAT 164-170 No

60 PgtSSR47 (TC)14 GACTACTGGTGGCGGTCCT AATCAGGTTGACCAGGATGG 186-202 No

61 PgtSSR68 (TC)16+(TC)4 AACCAGGGAACCAAAGGTCT GATTGACTCGGCAGTTGGAG 161-179 No

62 PgtSSR90 (TC)4T(TC)3+(TC)2 GTCGTCCACCATCCTCAACT TCAAGAGCAATTGAAATGGAA 274-282 No

63 PgtSSR119 (TC)7+(TC)6+(TC)29 AGAGATCATGCTCATTGATGGA TCCACTCACCATGTTCTTGC 306-320 No

64 PgtSSR129 (TC)9+(TC)7+(TC)3+(TTG)4+(TC)4 CGTGACAGTTCTCACCAAAAA CTGGCACAAAACCTACAGCA 354-358 No

65 PgtSSR134 (TC)11 ATCGGGCTCCCTTTTGTATC TTGGTCTGTTCGATTGCTTG 355-365 No

66 PgtSSR140 (TC)16 TTTGGAATCTATGCGGTTATTT CCTTCCGCTCTTCCTTTCAC 237-265 No

67 PgtSSR147 (TC)2TT(TC)13 GGATTCCGAGTGAGAATTGG CTCACCTCTCGCACAGTCAA 206-218 No

68 PgtSSR149 (AC)15TC(AC)5TC(AC)1 CAGTTCCCTTTTCACCCATT GACTACCGATGAGTTAGACATGTTG 227-245 No

69 PgtSSR151 (TC)11 CTTTCCCCCACACCATTCC AATTTGGTTGTGGAAAGAGAAC 248-260 No

70 PgtSSR164 (TC)14+(TC)3+(TC)4+(TC)3 GCTCTTTATCGCGTTCGTA AGTTAGTGGGCGGACAATTT 107-153 No

215

Appendices

71 PgtSSR180 (TC)10 CGACTAGCTTGAACGGGAAC CTAGTCCCACCCAAACTTCG 200-210 No 72 PtSSR3 (TC)5,6,6,6,6,7 TTCAATTTGCCCCTTGACTC AGGTAGCATTGCCAGTGGCA 271-301 No

73 PtSSR13 (TC)9 CGAATTCGCGTTTTATGTCC TGATCCAATCGAACCTAGCC 128-130 No

74 PtSSR50 (TC)16 CATCGGAATGGTCTGTCTCC CCAAATGCTATGAGTGGAAAA 360-366 No

75 PtSSR55 (TC)10 AGCTTACGGTCCTCAATCG AGTGAAAGGGGCTGGGAGT 302-304 No

76 PtSSR61 (TG)9 CGAACTGGTACAACGCACTG CGCAAAAAGGCTGATCTCTG 297-303 No

77 PtSSR68 (TG)16 GACTCAGCCCACTGCTAACC GATGGCGACGTATTTGGTCT 305-327 No

78 PtSSR76 (TG)14 GGCGTCGTATTTCTCGTAGC TTCGGACTACTGGGTAAGCA 393-402 No

79 PtSSR91 (TG)9 ATCTTGCGTCTCAGCCATCT CGCCGCTCTTCATCTCTTAC 378-380 No

80 PtSSR92 (TG)13 CCAAGGAACAGTCCACCAAG GAGTCGGGTAAGCCATCTGA 242-252 No

81 PtSSR151A (AAC)12 TCATCGCACTCCACTCAGAC ATGCTGCCCAACCTGCTC 456-476 No

82 PtSSR152 (TG)7,3,3 CTCCGTTCCTCTTTCTGTCG CCATCGCAACCAACAAACA 384-388 No

83 PtSSR154 (AAC)21 + (GAC)6 ACGGTCAACAGCCAACTACC CCTCGTCATCCTGGTTGAGT 242-272 No

84 PtSSR158 (CAA)13 GACGACTTCGTCACTGCTGA GAGGAGAAGCCGTTCTGTTG 227-232 No

85 PtSSR161 (TC)13 ACTGCCTCCTGTGCCTTCT TAGTCCGAGGGTGACGAAGT 213-215 No

86 PtSSR164 (TC)13 GTGGAAGTGAGCGGAAGAAG GGAGATGGGCAGATGAGGTA 214-222 No

87 PtSSR173 (TC)3,12 CTCAGCGACCTCAAAGAACC GAGACGACGGATGTCAACAA 211-219 No

88 PtSSR184 (TC)5,5,6,6,2 GGTCTGGCGAATCTTTCCTT CATTTTTAGTTGTGAGCCCTTG 373-569 No

89 PtSSR186 (AAC)9 GCCACGAGAAATACATAGAAATAAAA GGTTGTTGATGGGCTTGAGT 335-347 No

90 PtSSR187 (CAA)15 TTCAGTCGAAAACAGGCAAA TTGGAGCTGAAGGTCGAGTT 268-271 No 91 PtSSR0083 (GA)n ATGGATTTGGAGACCAGTCG GTTGAAAGATCTGGGGGTGA 292-298 No 92 PtSSR6981 (ATC)n ACGTGGTGAGGTTTCTGCTC TTCCGTTTTTGAAAGCAAGC 163-171 No 93 PtSSR0019 (ATC)n GTTCGGATACCCCGTTTCTC TTTGGAGCATGTTGTTTTGG 148-153 No 94 PtSSR5649 (CT)n CAGACGACCATCAACATTCG CATGAACCAAACAAACAGCTTC 189-195 No 95 PtSSR0085 (TTT)n CCAAAATTATCCCGCCCTAT GCGAGGGGGTAGGAAGTAAT 289-295 No

216

Appendices

96 PtSSR6259 (TCA)n GTTCAACACATTGCGCTGTT ATGGGTTGTGCAGATCGAGT 238-244 No 97 PtSSR2948 (GAT) CACACACCACACAAAACCAA CCCAACAAGCTCGTGTCTTT 117-121 No 98 PtSSR0536 (AAA)n TGTTGCGAATTGATGGTACG GAAGTTCTGCTCTGCTGTCG 180-186 No 99 PtSSR3233 (GA)n GTAAGCTCGCTTTGGCTACG TTTGGAGCATGTTGTTTCCA 165-167 No 100 PtSSR5594 (GAT)n CGGACCAAACACAAAGGAAA CCCTGCGTTTAACACCTTGT 205-217 No 101 PtSSR0189 (AT)n TCTCAACCAAAAATCAATCTACG CTTCCACGAAGACGAAGCAC 102-118 No 102 PtSSR0801 (TG)n CAATGGTAGTGGCAAGCAAA GCACCTCTCACGCTCTTAGC 201-204 No 103 PtSSR6863 (CT)n TAGATGGGCACACAACCAAA AAGCAAAGTGCAAGGAGCAT 212-248 No 104 PtSSR0243 (CA)n+(AT)n CTCACTCGCTCGCTTGTTCT GACGAAAAGATCGGGTTTGA 201-211 No 105 PtSSR0125 (GAT)n ATCGTGTCATGCAACCAAAA AGAGAGGGACGTGAGGGATA 177-183 No 106 PtSSR0481 (TTT)n CCACAATCCTCCGTTCTGAT CGAAAGCAAAACACATGAGG 192-199 No 107 PtSSR0639 (GAA)n TCTCCGCCTACCAACACTG AAAGGAGGGAGAGGGGAGG 204-210 No 108 PtSSR3145 (TCTT) TAGGTGCGTGGTTTTCATCA CAAATGAGAGCGACGAACAA 181-189 No 109 PtSSR6542 (CT)n TGTGATCTCGCCCGTACATA TGGGAATGATGGACACACAC 142-162 No 110 PtSSR0182 (CT)n CGAATCCCTTGTCTTTTGCT TGTAGAGAGCGGGAGAAGAAA 172-174 No 111 PtSSR6386 (CAT)n AATGAGGTGACTCGGATGGA GAAGAAGGCGAAGTTGTTGC 193-199 No

112 PcaSSRA03 (CA)14 GCCGTACGATCCTAGTCCAG TGGTTCATATGGTGAGGATGG 148-154 No

113 PcaSSRA04 (GA)18 CGGATTGGAGAGTCGGTAGA CCTTCAAAGAGGGCACTCAC 223-241 No

114 PcaSSRA10 (CT)16 CATTCGCCCATTGAATTACC ACTGTTTCGGAACGTGGAAG 223-229 No

115 PcaSSRA18 (CA)14 GCGTAACCATAGGACCCAGA AGACATGCGACAGAATGGTG 233-237 No

116 PcaSSRA26 (AC)9T(CA)10 TTCGAGAAGCGCTTGATCTT ATCACCACAGTCCACACGAA 155-162 No

117 PcaSSRA35 (CT)9 AACCTCCCTTCCCCCTCT GGGGCAAGATACTCGAAACA 150-156 No

118 PcaSSRA36 (CT)14 CCTGATCGAGTCCAGCTACC AGTCCAGAATATCATCCACGA 178-182 No

119 PcaSSRA41 (CT)3TT(CT)9 ATCGAAGTCACCGAGAAGGA CCAAACTGACCCTTCGTGTT 169-173 No

120 PcaSSRA52 (GA)11 GAAAGGGAGCCTGAAGGAGT AGACCTCCAATGTCGGTACG 150-158 No

217

Appendices

121 PcaSSRA59 (CA)15 TTCACAGTCACCCATCAACAA TCGAATACTCGTCCGTTTACC 143-151 No

122 PcaSSRA66 (CT)18 TGCTGCTGTACATCCATCGT GACCCAGACTGCAAGAGGAA 167-191 No

123 PcaSSRA67 (GT)12 CCACAAGGAGATGAAAGGCTA CGAGGGAAGCCGAATACATA 188-196 No

124 PcaSSRA73 (TC)9TGA(TAA)4 CTTTCAACTCTGTCCGACCA TCGAATCGATCGAAAAGGAC 150-152 No

125 PcaSSRA75 (AG)16 CTGGAGGAAAGATTGGTGGA CGACTGGGTCAAGGTCAAAT 182-192 No

126 PcaSSRA92 (CA)13 GGGAATCTCGTCCTCCATTT GGGAATCTCGTCCTCCATTT 169-175 No

127 PcaSSRB02 (CT)14TT(CT)6 AGAATGCGAGCCAGGACTAA ATTGGAGATCGGGCATGATA 160-164 No

128 PcaSSRB09 (CT)18 CCTCTCGTTGTTAGGTTACACTG CCCATCATGAACCACTTCCT 145-163 No

129 PcaSSRB13 (GT)10 ATGCAACTCGGTCACTCACA TGACTCCCACCACAATGGTA 233-235 No

130 PcaSSRB14 (CA)10 GTGATCGTGCTCGTCGTCTA ATGCCTATGTTCCCCATGAA 190-205 No

131 PcaSSRB15 (CT)17 CGCGGGTTGTAGTACACCTT CTGGGGAGCCGAATTATACA 190-194 No

132 PcaSSRB16 (CT)12 GGTCCCCGGAAACTTTAATC GAAATTCCGTGGTCATAGCC 222-240 No

133 PcaSSRB17 (CT)11 GGTTAAACAGGCAGAGGGAGA ACTCTTCACCAGGAATAGTGATCT 141-149 No

134 PcaSSRB25 (GT)13 CCCCTCCCGAGTCTCTATCT ACGGATCATGGAAAATCCAA 196-214 No

135 PcaSSRB32 (CT)9 ATGCGTGTTCTGCTGTTGAC GGTGAAAGATTCCTGGCTGA 170-172 No

136 PcaSSRB33 (GT)19 GTACACCTCGCCGTTGATCT GAGGGATGAGCCATCAGGTA 189-226 No

137 PcaSSRB37 (GA)8 CAGCGCAAGACATAGAGAGG CTAGATGTCTGGGCCGATCA 153-157 No

138 PcaSSRB54 (CA)3TT(CA)5TT(CA)5TT(CA)3 CATCGACAGCACCACCAG TTCGAGGAGACGAAGGAGAA 214-249 No

139 PcaSSRB66 (GA)9 AGATGTCTCCGCAAAGAGGA CTGCCTCAACTCGTCATCAA 168-174 No

140 PcaSSRB78 (GA)11 TCTACCGACGATCAATGCTG ACCATCAATCTCCTCGTTGC 241-243 No

141 PcaSSRB81 (GA)20 AAGTAGGCAGCCAAGTCAAAA TCATCTTCCCTCCATCCTTG 213-240 No

142 PcaSSRB84 (AG)6T(GA)9 CTTGCTGGATAAGGGAACGA TACTCAACTGGCACCCCATC 169-183 No

143 PcaSSRB87 (GT)17TT(GT)3 GCCGAGGCCAGAAAGGAT CAGACGGAGATGATCCCAAA 146-152 No

144 PcaSSRC26 (CT)10 CCGAAAACGCTCTCAAGAAG AAGGCGAGAGGGAAGAATGT 164-196 No

145 PcaSSRC33 (GA)10 TGAAGTGTTCGACCTGAGGA GTACCATTCGCTGTGCATGA 142-152 No

218

Appendices

146 PcaSSRC52 (CT)13 CACCGCTTTCTTGTTCCAGT CCCTGGAGTACCGACAGAGA 197-207 No

147 PcaSSRC76 (CA)17 CGCAAGTCTCTGTGTCGTCT ACCATCGATGAGTCCAGAAA 153-193 No

148 PcaSSRC77 (CT)11 CTGGTAGTTGTGGGCTAGGG GAATGGCCTTCTCGGATGTA 164-166 No

149 Olign M13 GAGGGTGGCGGTTCT 500 - 1500 Yes

150 Olign (GACA)4 GACAGACAGACAGACA 500 - 2000 Yes

Olign: oligonucleotide, F: forward primer, R: reverse primer, Ply: polymorphic on P. hordei isolates

219