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
4.6 Putative candidate genes for Rrs2 ..............................................................121
4.7 Outlook ..........................................................................................................126
5 SUMMARY 128
6 ZUSAMMENFASSUNG 129
7 REFERENCES 131
8 SUPPLEMENTARY MATERIAL 157LIST OF FIGURES IV
List of Figures
Fig. 1.1: Zigzag model of the plant immune system by JONES and DANGL (2006)
(modified). ...................................................................................................................3
Fig. 1.2: Classes of plant disease resistance proteins and examples of cloned R
proteins for each class (modified from MCDOWELL and WOFFENDEN (2003) with
information from DANGL and JONES (2001) and CHISHOLM et al. (2006))......................5
Fig. 1.3: Receptor-ligand model (A) and guard hypothesis (B,C) based on figures in
DANGL and JONES (2001) and DEYOUNG and INNES (2006)..........................................7
Fig. 1.4: Locations of mapped resistance genes against Rhynchosporium secalis on
the barley bin map (http://www.barleyworld.org, status of August 11, 2006)..............13
Fig. 1.5: Effectiveness of the Rrs2 mediated resistance in the field observed at the
Bayerische Landesanstalt für Landwirtschaft (LfL-Bavaria) in Freising in the year
2005. .........................................................................................................................16
Fig. 1.6: Rrs2 region on barley chromosome 7HS with flanking markers and in blue
the corresponding homologous region on rice chromosome Os6. ............................18
Fig. 1.7: Schematical representation of the establishment of the distal barley BAC
contig by COSSU et al. (unpublished).........................................................................19
Fig. 1.8: Schematical representation of the establishment of the proximal barley BAC
contig by COSSU et al. (unpublished).........................................................................20
Fig. 1.9: Graphical representation of the annotation of the proximal BAC contig
MO134N7_MO246J13_MO524N3_MO288D11 performed by Thomas Wicker
(University of Zürich). ................................................................................................22
Fig. 2.1: Overview of the establishment of the F -mapping population. ....................252
Fig. 2.2: Schematic representation of the allocation of BAC clone DNA into pools for
the Morex BAC library using BAC library plate 12 as example..................................27
Fig. 2.3: Section of the physical map of the Rrs2 locus on barley chromosome 7HS
depicting the name and location of six PCR fragments analyzed in the association
study..........................................................................................................................35
Fig. 3.1: Genetic map of the Rrs2 region on chromosome 7HS................................40
Fig. 3.2: Distal barley BAC contig for the Rrs2 locus on barley chromosome 7HS
OSSUestablished by C et al. (unpublished), status at the beginning of the presented
PhD work...................................................................................................................41
Fig. 3.3: Schematical representation of the work flow for the identification of barley
BAC clone MO668A17 and its anchoring in the existing distal barley BAC contig. ...43
Fig. 3.4: Schematical representation of the work flow for the identification of barley
BAC clones MO348I22 and MO3M16 and their anchoring in the distal BAC contig..45
Fig. 3.5: Prolonged distal barley BAC contig of the Rrs2 locus after screening of
several different BAC libraries. ..................................................................................46
Fig. 3.6: Proximal barley BAC contig for the Rrs2 locus on barley chromosome 7HS
established by COSSU et al. (unpublished), status at the beginning of the presented
PhD work...................................................................................................................47LIST OF FIGURES V
Fig. 3.7: Schematical representation of work flow for the identification of BAC clone
MO16J08 and its intended anchoring in the proximal BAC contig.............................48
Fig. 3.8: Summary of the genetic and physical map (minimal BAC clone tiling path) of
the Rrs2 locus on barley chromosome 7HS. .............................................................50
Fig. 3.9: Section of the physical map of the Rrs2 locus on barley chromosome 7HS
indicating the available sequence information of the co-segregating area of Rrs2....52
Fig. 3.10: Graphical representation of the annotated Rrs2 co-segregating region of
the distal BAC contig. ................................................................................................57
Fig. 3.11: Section of the physical map of the Rrs2 locus on barley chromosome 7HS
indicating the genes identified in the co-segregating area of Rrs2. ...........................62
Fig. 3.12: Graphical representation of the syntenic relationships between the Rrs2
locus on barley chromosome 7HS and rice (Oryza sativa)........................................65
Fig. 3.13: Graphical representation of the syntenic relationships between the Rrs2
locus on barley chromosome 7HS and Brachypodium distachyon............................68
Fig. 3.14: Overview of a section of the physical map of the Rrs2 locus on barley
chromosome 7HS indicating the genomic location and names of six PCR fragments
which were analyzed in the association study. ..........................................................72
Fig. 3.15: Distribution of haplotypes at six genomic regions across the Rrs2 locus on
barley chromosome 7HS based on data of 72 barley accessions representing 58
different genotypes....................................................................................................74
Fig. 3.16: Cluster analysis of 72 barley accessions. .................................................76
Fig. 3.17: Linkage Disequilibrium (LD) matrix showing the correlation of haplotypes of
six PCR fragments and Rrs2 mediated resistance. ...................................................78
2Fig. 3.18: LD measurement R of pairwise comparisons of all haplotypes across the
analyzed genomic area with the Rrs2 phenotype......................................................79
Fig. 3.19: CAPS marker assays for PCR fragment Put_acri_res_gene_7H with
Bsp68I (A) and Eco32I (B), white numbered samples do not carry Rrs2, yellow
labelled samples carry Rrs2. .....................................................................................87
Fig. 3.20: CAPS marker assays for PCR fragment 668A17_g1-3 with BncI (A) and
GsuI (B), white numbered samples do not carry Rrs2, yellow labelled samples carry
Rrs2...........................................................................................................................88
Fig. 3.21: CAPS marker assay for PCR fragment 668A17_e11-2 with Hin1II...........89
Fig. 3.22: Results of the pyrosequencing marker assay for fragment
Put_acri_res_gene_7H of varieties Atlas (Rrs2+) and Steffi (Rrs2-)..........................90
Fig. 3.23: Results of the pyrosequencing marker assay for fragment 668A17_g1-3 for
the varieties Atlas (Rrs2+) and Steffi (Rrs2-). ............................................................91
Fig. 3.24: Results of the pyrosequencing marker assay for fragment 668A17_e11-2
for the varieties Atlas (Rrs2+) and Steffi (Rrs2-). .......................................................91
Fig. 3.25: PCR products separated on 2% agarose gel showing the amplification of
genomic DNA and cDNA of Atlas and Steffi with a primer combination amplifying part
of the barley actin gene (GenBank Acc. AY145451.1)...............................................92LIST OF FIGURES VI
Fig. 3.26: Alignment of PCR fragment RGH-3, EST Acc. CA004050 and HarvEST
unigene #41873 with the genomic region of RGH-3 (Acc. AY853252) and with
primers used for the expression analysis. .................................................................93
Fig. 3.27: Amplification of cDNA of Atlas and Steffi with primer combination PK95 and
actin...........................................................................................................................95
Fig. 3.28: Graphical representation of alignment of the genomic region of the
predicted HvFST-2 gene with EST consensus TC188981, EST Acc. BY842569,
HarvEST unigene #31645 and #7621 together with PCR fragment FST-2 and RT-
PCR fragment of PK95..............................................................................................95
Fig. 3.29: Amplification of cDNA of Atlas and Steffi with primer combinations PK37,
PK38 and Actin..........................................................................................................96
Fig. 3.30: Alignment of BAC subclone 668A17_plate3_g1-t3 with EST consensus hit
TC154956 and HarvEST unigene#2643, PCR fragment 668A17_g1-3 and with RT-
PCR fragments PK37 and PK38. ..............................................................................97
Fig. 3.31: Amplification of cDNA of Atlas and Steffi with primer combinations PK37,
PK38 and Actin..........................................................................................................98
Fig. 3.32: Alignment of PCR fragment 134N7_con5-3 with EST consensus hit
TC184695 and HarvEST unigenes #3681 and #3682 and with RT-PCR fragment
PK18. ........................................................................................................................98
Fig. 4.1: Geographical origin and common pedigree of barley varieties carrying Rrs2.
................................................................................................................................120LIST OF TABLES VII
List of Tables
Table 1.1: Cloned resistance genes of cereals (based on AYLIFFE and LAGUDAH
(2004) and DAI et al. (2007); updated).........................................................................8
Table 3.1: Summary of the different screening steps for establishment of the Rrs2
mapping population...................................................................................................39
Table 3.2: Overview of genetic distances and expected physical distances achieved
with the mapping population......................................................................................39
Table 3.3: Summary of the expected and actual physical distances of the marker
interval between markers AFLP14 and P1D23R .......................................................51
Table 3.4: List of putative genes identified in the Rrs2 co-segregating region of the
distal BAC contig and on BAC clones MO668A17 and ME194H14...........................55
Table 3.5: Comparison of the location of orthologous genes found in the Rrs2 locus
on barley chromosome 7HS, in rice and Brachypodium............................................69
Table 3.6: Distribution of country of origin, seasonal habit, row number, and
adaptation status among the 58 different genotypes studied in the association study.
..................................................................................................................................71
Table 3.7: Summary of the SNP and haplotype analysis of 58 barley genotypes
based on sequence data of six PCR fragments originating from the Rrs2 region on
barley chromosome 7HS...........................................................................................73
Table 3.8: LD analysis results of the pairwise comparisons of haplotypes H2 of PCR
fragments Put_acri_res_gene_7H, 668A17_g1-3, 668A17_e11-2, and 134N7_con5-3
with each other and with the Rrs2 gene. ...................................................................77
Table 3.9: Associations for SNPs and haplotypes of PCR fragment RGH-3 with the
Rrs2 resistance phenotype calculated for 66 barley accessions using the General
Linear Model (GLM). .................................................................................................80
Table 3.10: Associations for SNPs and haplotypes of PCR fragment
Put_acri_res_gene_7H with the Rrs2 resistance phenotype calculated for 72 barley
accessions using the General Linear Model (GLM)...................................................81
Table 3.11: Associations for SNPs and haplotypes of PCR fragment FST-2 with the
Rrs2 resistance phenotype calculated for 72 barley accessions using the General
Linear Model (GLM). .................................................................................................82
Table 3.12: Associations for SNPs and haplotypes of PCR fragment 668A17_g1-3
with the resistance phenotype calculated for 72 barley accessions using the General
Linear Model (GLM). .................................................................................................83
Table 3.13: Associations for SNPs and haplotypes of PCR fragment 668A17_e11-2
with the resistance phenotype calculated for 72 barley accessions using the General
Linear Model (GLM). .................................................................................................84
Table 3.14: Associations for SNPs and haplotypes of PCR fragment 134N7_con5-3
with the resistance phenotype calculated for 71 barley accessions using the General
Linear Model (GLM). .................................................................................................85