Bansal UK

Bariana HS

Hayden MJ

Lagudah, E

Sambasivam PK

English

Sydney eScholarship

 1Identification of markers linked with stem rust resistance genes Sr33 and Sr45  Sambasivam PK1 , Bansal UK1, Hayden MJ 2, Dvorak J3, Lagudah ES4, Bariana HS1 1Faculty of Agriculture, Food and Natural Resources, University of Sydney, Plant Breeding Institute-Cobbitty, PMB11, Camden, NSW 2570, Australia. 2Plant Genomics Center, University of Adelaide, Hartley grove, Urrbrae, SA, 5064, Australia.3University of California Davis, USA 4CSIRO Plant Industry, GPO Box 1600,  Canberra, 2601 ACT, Australia.  INTRODUCTION  Stem rust caused by Puccinia graminis f. sp. tritici (Pgt) has been and still remains a major threat for wheat production world wide1,2. Following “Green Revolution”, the disease was brought under control by the use of stem rust resistant semi dwarf spring wheats and least attention was given to stem rust improvement for several decades3. Recently, evolution of a new Pgt pathotype, Ug99, in Africa has threatened the global wheat industry. This pathotype was initially detected in Uganda during 1999 and later spread to neighbouring countries like Kenya and Ethiopia and now has reached Iran2,4. This pathotype is virulent on many of the current stem rust resistance genes used in different wheat growing regions5. Some of the effective stem rust resistance are derived from the wild relatives of wheat. The genes Sr33 and Sr45, derived from Aegilops tauschii, were among the genes effective against Ug992. The present study was planned to identify molecular markers closely linked with stem rust resistance genes Sr33 and Sr45.   MATERIALS AND METHODS  Plant materials Substitutions lines for chromosome 1D in Chinese Spring carrying Sr33 (CS1D5405) and Sr45 (CS1D5406) were crossed with the susceptible parent Chinese Spring (CS) to generate single chromosome recombinant inbred line populations.   Stem rust evaluation The CS1D5405/CS and CS1D5406/CS RIL populations were screened for stem rust response segregation at the seedling stage under greenhouse conditions using Pgt pathotype 34-1,2,3,4,5,6,7,11 (culture no. 171), following the procedures described in Bariana and McIntosh (1993)6. These populations were sown at two locations, Karalee and Breakwell, at the University of Sydney, Plant Breeding Institute, Cobbitty. Each 50 row experimental block was surrounded by susceptible wheat lines serving as infector rows to create rust epidemic. Adult plant stem rust response assessments were made according to a 1-9 scale described by Bariana et al. (2007)7.  Molecular mapping Genomic DNA was extracted from the leaf tissue of CS1D5405/CS and CS1D5406/CS RIL populations using modified CTAB method of Kleinhoffs et al. (1993)8. Bulk segregant analysis (BSA) was performed to identify marker-trait associations. BSA was performed using multiplex ready PCR method9 with 80 markers located on chromosome 1D. Rust response-linked markers were mapped on RIL populations.  Data analyses and genetic mapping Chi-squared tests were used to determine the goodness of fit of observed RIL segregation to that of the expected genetic ratio. Recombination fractions were calculated with MAP MANAGER Version QTXb2010 using the Kosambi (1944)11 map function.   RESULTS  Genetic analysis The CS1D5405 (Sr33) and CS1D5406 (Sr45) produced low infection types (IT) IT2= and IT;;1-, respectively, when tested against Pgt pathotype 34-1,2,3,4,5,6,7,11 at the seedling stage. The susceptible parent Chinese spring displayed a susceptible response (IT3+). At the adult plant stage resistant parents produced rust response of 4 (CS1D5405) and 5 to 6 (CS1D5406), whereas susceptible parent produced high field response of 8. Both RIL populations showed 1:1 segregation both at the seedling and adult stages indicating the presence of single gene for resistance in each population (Table 1).   Genetic mapping:  Sr33 Fifteen chromosome 1D markers showed associations with resistant and susceptible bulks in CS1D5405/CS RIL population. These linked markers were mapped on entire RIL population consisting of 87 lines. Sr33 was flanked by Xbarc152 and Xcfd15 at a distance of 1.8 cM on either side. The LOD value for flanking markers was 20.5. The locus order and genetic distances between Sr33 and markers are presented in Fig 1a.  2 Table 1: Segregation for stem rust response in CS1D 5405/CS and CS1D 5406/CS RIL populations   Plant stage RIL Population Rust response χ2 1:1 Resistant SusceptibleSeedling stage (75L9=171) CS1D5405/CS 47 38 0.953CS1D5406/CS 20 26 0.783Adult plant stage (Karalee) CS1D5405/CS 47 38 0.953CS1D5406/CS 20 26 0.783Adult plant stage  (Breakwell) CS1D5405/CS 47 38 0.953CS1D5406/CS 20 26 0.783The table values ofχ2 at P=0.05 and P=0.01 and 1d.f. are 3.84 and 6.64, respectively.   Sr45 For Sr45, 30 markers showed linkage with bulks. The linked markers were genotyped on CS1D5406/CS RIL population consisting of 48 lines. The cluster of markers Xwmc222, Xgwm106, Xstm694tgag, Xcfa2158 and Xcfd21 mapped 3.3 cM distal to Sr45 and Xbarc229 was 3.7 cM proximal (LOD=8.5) to the gene (Fig 1b).   DISCUSSION  The D genome of hexaploid wheat, derived from Aegilops tauschii, contributed many important rust resistance genes12,13. Two stem rust resistance genes Sr33 and Sr45 genes were also transferred from chromosome 1D of Aegilops tauschii14,15. The substitution lines CS1D5405 and CS1D5406 showed low stem rust responses at the seedling and adult plant stages against Australian Pgt pathotypes. These genes were also effective against the Pgt pathotype Ug995. The seedling stem rust response segregation among CS1D5405/CS and CS1D5406/CS RIL populations conformed to monogenic inheritance of resistance in each population. The resistance gene Sr33 was flanked by markers barc152 (1.8cM) and cfd15 (1.8cM). The marker cluster wmc222/gwm106/stm694tgag/ cfa2158/cfd21 and the marker barc229 flanked Sr45 distally (3.3 cM) and proximally (3.7 cM). Marker order was consistent with the consensus map of Somers et al. 200416. Sr33 was previously reported to be tightly linked with Gli-D117. These additional Sr33 and Sr45 linked markers will be validated on backcross-derived germplasm in several different genetic backgrounds. The low recombination values suggest that these markers can be used in marker assisted selection of Sr33 and Sr45, where parental polymorphism exists. However the cluster of markers distal to Sr45 may indicate low resolution based on the small population size. Both these genes provide protection under field conditions and their pyramiding with other effective stem rust resistance would enable achievement of durability.      Figure 1: Genetic map of 1DS with respect to the location of Sr33 (a) and Sr45 (b).  Xbarc152 cMSr331.8Xcfd151.8Xwmc4321.8Xwmc2226.9Xcwem127.1  (a)Xbarc152cMXcfd154.6Xksm1172.4stm657tgag1.2Xwmc222 Xgwm106Xstm694tgag Xcfa2158Xcfd211.1Sr453.3Xbarc2293.7Xcfd72 Xcfd595.0(b) 1DS1DS 3REFERENCE  1. Fish S (1970). The history of plant pathology in Australia. Ann Rev Phytopathol 8: 13-36 2. 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Use of double-ditelosomic and normal chromosome 1D recombinant substitution lines to map Sr33 on chromosome arm 1DS in wheat. Genome 34:505-508                 

Identification of markers linked with stem rust resistance genes Sr33 and Sr45

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Identification of markers linked with stem rust resistance genes Sr33 and Sr45

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