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High Resolution Mapping of RphMBR1012 Conferring Resistance to Puccinia hordei in Barley (Hordeum vulgare L.) Dissertation zur Erlangung des Doktorgrades der Agrarwissenschaften (Dr. agr.) der Naturwissenschaftlichen Fakultät III Agrar‐ und Ernährungswissenschaften, Geowissenschaften und Informatik der Martin‐Luther‐Universität Halle‐Wittenberg vorgelegt von Frau Leila Fazlikhani Geb. am 04. September 1983 in Tehran (Iran) 1. Gutachter: Prof. Dr. Frank Ordon 2. Gutachter: Prof. Dr. Holger B. Deising 3. Gutachter: Prof. Dr. Jens Leon Verteidigung am: 27. January 2020 Table of Contents 1 General introduction ............................................................................................................................................... 1 1.1 Barley history and importance ................................................................................................................. 1 1.2 World production and uses ........................................................................................................................ 2 1.3 Barley Leaf Rust and economic importance ........................................................................................ 3 1.4 Resistance sources and mapping of leaf rust resistance genes in barley ................................ 5 1.5 Histology of race-specific resistance ...................................................................................................... 9 1.6 The barley genome ...................................................................................................................................... 10 1.7 Barley genomic resources ........................................................................................................................ 10 1.8 Novel sequencing technology ................................................................................................................. 11 1.9 Genetic maps and molecular markers ................................................................................................. 13 1.10 Gene identification and positional cloning in barley .................................................................... 14 1.11 Physical mapping ......................................................................................................................................... 15 1.12 Objectives ........................................................................................................................................................ 16 2 Material and methods .......................................................................................................................................... 17 2.1 Plant material and construction of high resolution mapping population ............................ 17 2.2 Disease assessment ..................................................................................................................................... 18 2.2.1 Propagation of urediniospores of Puccinia hordei .................................................................... 18 2.2.2 Macroscopic Screening ......................................................................................................................... 18 2.2.3 Microscopic investigation of fungal development .................................................................... 19 2.3 DNA extraction .............................................................................................................................................. 20 2.4 Polymerase chain reaction (PCR) ......................................................................................................... 21 2.5 Agarose gel electrophoresis analysis .................................................................................................. 21 2.6 Marker saturation ........................................................................................................................................ 22 2.6.1 Markers derived from the genome zipper (GZ) and the 9K iSelect Illumina array ..... 22 2.6.1.1 Primer design....................................................................................................................................... 23 2.6.1.2 Cleaved Amplified Polymorphic Sequences (CAPS) marker analysis .......................... 24 2.6.2 Marker development from 50K and genotyping-by-sequencing (GBS) ........................... 24 2.6.2.1 50K iSelect genotyping .................................................................................................................... 24 2.6.2.2 Genotyping-by-sequencing (GBS) ............................................................................................... 25 2.6.2.3 KASP markers analysis .................................................................................................................... 27 2.7 Linkage analysis ........................................................................................................................................... 30 2.8 Testing the diagnostic value of newly developed markers ........................................................ 30 2.9 Anchoring RphMBR1012 fine map to the barley reference genome sequence .......................... 32 2.10 Allele specific re-sequencing of candidate genes ........................................................................... 33 3 Results ........................................................................................................................................................................ 34 3.1 High-resolution genetic mapping and marker saturation of the leaf rust resistance gene RphMBR1012 ....................................................................................................................................................................... 34 3.1.1 50K iSelect genotyping ......................................................................................................................... 35 3.1.2 Genotyping-by-sequencing (GBS) .................................................................................................... 36 3.2 Phenotyping of homozygous recombinants inbreed lines (RILs) in the RphMBR1012 region ………………………………………………………………………………………………………………………………38 3.2.1 Macroscopic assessment ...................................................................................................................... 38 3.2.2 Microscopic investigation .................................................................................................................... 38 3.3 Physical mapping and anchoring leaf rust resistance locus to the physical map of barley ………………………………………………………………………………………………………………………………40 3.4 Allele specific re-sequencing of candidate genes from the interval ....................................... 44 3.5 Development of diagnostic markers for the RphMBR1012 gene ..................................................... 50 4 Discussion ................................................................................................................................................................. 54 4.1 Advanced genomic resources greatly facilitate gene isolation in barley.............................. 54 4.2 Barley physical map is a fundamental platform for gene identification ............................... 56 4.3 Map-based cloning approach as a method of choice in gene isolation .................................. 58 4.4 Identification of candidate genes .......................................................................................................... 59 4.5 Plant-pathogen interaction ...................................................................................................................... 61 5 Outlook ...................................................................................................................................................................... 64 6 Summary ................................................................................................................................................................... 65 7 Zusammenfassung ................................................................................................................................................ 67 8 References ................................................................................................................................................................ 69 9 Appendix ................................................................................................................................................................... 86 10 Abbreviations ....................................................................................................................................................... 108 11 List of figures ........................................................................................................................................................ 112 12 List of tables .......................................................................................................................................................... 114 Acknowledgements………….…………..…………………………………..………………………………..…………………………………115 Curriculum vitae……………………………………………………………………………………………………………116 Declaration under oath………………………………………………………..…………………………….………….118 G e n e r a l i ntroduction |1 1 General introduction 1.1 Barley history and importance Barley (Hordeum vulgare L ssp. vulgare) is a cereal crop that belongs to the family of grasses (Poaceae), which is of prime economic importance in
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