Fine mapping and marker development for the resistance gene Rrs2 against Rhynchosporium secalis in barley [Elektronische Ressource] / Anja Hanemann

Sujets

• Biologie

Informations

• Publié par: technische_universitat_munchen
• Publié le: 01 janvier 2009
• Nombre de lectures: 17
• Langue: English
• Poids de l'ouvrage: 10 Mo

Extrait

TECHNISCHE UNIVERSITÄT MÜNCHEN
Lehrstuhl für Pflanzenzüchtung
Fine Mapping and Marker Development for
the Resistance Gene Rrs2 against
Rhynchosporium secalis in Barley
Anja Hanemann
Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan
für Ernährung, Landnutzung und Umwelt der Technischen Universität München zur
Erlangung des akademischen Grades eines
Doktors der Naturwissenschaften
genehmigten Dissertation.
Vorsitzende: Univ.-Prof. Dr. Chr.-C. Schön
Prüfer der Dissertation: 1. Univ.-Prof. Dr. G. Wenzel
2. Univ.-Prof. Dr. R. Hückelhoven
3. Priv.-Doz. Dr. V. Mohler
Die Dissertation wurde am 13.05.2009 bei der Technischen Universität München
eingereicht und durch die Fakultät Wissenschaftszentrum Weihenstephan für
Ernährung, Landnutzung und Umwelt der Technischen Universität München am
14.10.2009 angenommen.dedicated with many thanks to S. Mikolajewski" Ignorance more frequently begets confidence than does knowledge: it is those
who know little, not those who know much, who so positively assert that this or
that problem will never be solved by science. "
Charles DarwinTABLE OF CONTENTS I
Table of contents
1 INTRODUCTION 1
1.1 Disease resistance in plants – a short overview............................................2
1.2 Cloned disease resistance genes of cereals with focus on cloned barley R
genes .................................................................................................................7
1.3 Scald in barley caused by Rhynchosporium secalis...................................10
1.3.1 Epidemiology and genetic variability of Rhynchosporium secalis .................11
1.3.2 Development of Rhynchosporium secalis on barley and effects of its toxins 11
1.3.3 Known resistance genes against Rhynchosporium secalis ..........................13
1.3.4 Known functions of resistance genes against Rhynchosporium secalis with
focus on Rrs1 ...............................................................................................14
1.3.5 Effectiveness of Rrs2 resistance...................................................................15
1.4 Previous work on Rrs2 ...................................................................................17
1.4.1 Mapping of the Rrs2 gene ............................................................................17
1.4.2 Establishment of a physical BAC contig for the Rrs2 locus ..........................17
1.5 Aim of the present work .................................................................................23
2 MATERIAL AND METHODS 24
2.1 Establishment of the F -mapping population...............................................242
2.2 Scald resistance test ......................................................................................26
2.3 Physical map establishment..........................................................................26
2.3.1 BAC libraries and BAC library screening ......................................................26
2.3.2 BAC clone fingerprinting...............................................................................28
2.3.3 Subcloning of BAC clones ............................................................................28
2.4 Nucleic acid isolation and quantification .....................................................29
2.4.1 Genomic DNA...............................................................................................29
2.4.2 Plasmid DNA ................................................................................................29
2.4.3 RNA..............................................................................................................30
2.5 PCR and RT-PCR.............................................................................................30
2.5.1 Analysis of PCR products .............................................................................31
2.6 DNA sequencing .............................................................................................31
2.7 Sequence analysis and database mining .....................................................32
2.7.1 In-silico sequence analysis ...........................................................................32
2.7.2 Sequence annotation....................................................................................32
2.8 Molecular marker development .....................................................................33
2.8.1 CAPS markers..............................................................................................33
2.8.2 Pyrosequencing markers ..............................................................................34TABLE OF CONTENTS II
2.9 Association Study...........................................................................................34
2.9.1 Linkage disequilibrium ..................................................................................36
2.9.2 Cluster Analysis ............................................................................................36
3 RESULTS 37
3.1 Fine Mapping of the Rrs2 gene......................................................................37
3.2 Continued establishment of a physical BAC contig for the Rrs2 region ...41
3.2.1 Distal BAC contig..........................................................................................41
3.2.2 Proximal BAC contig.....................................................................................47
3.3 Summary of results for the map based cloning approach..........................49
3.4 Sequence annotation of the Rrs2 co-segregating region............................51
3.4.1 Sequence annotation of the distal BAC contig, BAC clones MO668A17 and
MO348I22, as well as gene information obtained from the HarvEST Barley
Integrated Map 04/16/08...............................................................................52
3.4.2 Sequence annotation of the proximal BAC contig and summary of identified
genes in the co-segregating region...............................................................61
3.5 Synteny of the Rrs2 region to other members of the Poacea family..........62
3.5.1 Synteny to rice (Oryza sativa L.)...................................................................62
3.5.2 Synteny to Brachypodium distachyon...........................................................66
3.6 Association study...........................................................................................69
3.6.1 SNP and haplotype patterns of six genomic regions located near or within the
co-segregating area of Rrs2 on barley chromosome 7HS............................71
3.6.2 Cluster analysis ............................................................................................75
3.6.3 Linkage disequilibrium (LD) analysis of haplotypes ......................................77
3.6.4 Association of SNPs and haplotypes of six PCR fragments with the Rrs2
phenotype.....................................................................................................79
3.7 Development of diagnostic markers for the Rrs2 gene...............................86
3.7.1 CAPS markers based on fragment Put_acri_res_gene_7H..........................87
3.7.2 CAPS markers based on fragment 668A17_g1-3.........................................88
3.7.3 CAPS marker based on fragment 668A17_e11-2.........................................89
3.7.4 Pyrosequencing marker based on PCR fragment Put_acri_res_gene_7H...90
3.7.5 Pyrosequencing marker based on fragment 668A17_g1-3...........................90
3.7.6 Pyrosequencing marker based on fragment 668A17_e11-2.........................91
3.8 Expression Analysis.......................................................................................92
3.8.1 PCR fragment RGH-3...................................................................................93
3.8.2 PCR fragment Put_acr_res_gene_7H ..........................................................93
3.8.3 PCR fragment FST-2 and primer combination PK95 ....................................94
3.8.4 PCR fragment 668A17_g1-3 and primer combinations PK37 and PK38......96
3.8.5 PCR fragment 668A17_e11-2.......................................................................97
3.8.6 PCR fragment 134N7_con5-3 and primer combination PK18 ......................97
3.8.7 Summary of the expression analysis ............................................................99
3.9 Summary of results.......................................................................................100TABLE OF CONTENTS III
4 DISCUSSION 102
4.1 High-resolution mapping of the Rrs2 region..............................................102
4.2 Possible reasons for suppressed recombination in the vicinity of the Rrs2
gene ...............................................................................................................105
4.3 The Rrs2 region coincides with a region which is poorly represented in
BAC libraries .................................................................................................110
4.4 Synteny to rice and Brachypodium............................................................. 111
4.5 Association study, development of diagnostic molecular markers for Rrs2,
and possible origin of the Rrs2 gene ..........................................................115