Identification of resistance genes against powdery mildew in wild barley PI284752 by DNA markers
2008
A newly detected accession of wild barley (Hordeum vulgare ssp. spontaneum) resistant to powdery mildew caused by Blumeria graminis f. sp. hordei was studied with the aim of finding the number and identity of genes conferring the resistance. Therefore, F2 population derived from a cross between the winter variety Tiffany and the wild barley accession PI284752 consisted of 449 plants was established. Use of DNA markers was focused on the identification of individual resistance genes by means of their chromosomal locations. The number of plants in the two phenotypic categories (427 resistant and 22 susceptible) found in F2 population was compared with a theoretical Mendelian segregation ratio by a chi-square test and a two-locus model of resistance was shown by this genetic analysis (chi-square=1.37). In PI284752, dominant alleles of two independent genes were present; the allelism test indicated that one gene was located at the Mla locus. All parental plants of PI284752 showed the highest reaction type RT0; the F1 generation with RTs ranging between 0 and 1-2 reflected that RT of the other gene was 1-2 and, in parental plants, was overridden by the effect of the first gene. The linkage between a microsatellite DNA marker and particular resistance gene was revealed by means of bulk segregant analysis (BSA) when resistance was dominant over susceptibility. Bmac0213 on chromosome 1HS and Bmag0134 on chromosome 2HS were indicated for linkage with the resistance genes. Linkage analysis with the polymorphic markers for which a linkage with individual resistance genes was traced by BSA was carried out with 112 F2 plants. The RGH1a gene sequence from the Mla locus was used as source data for development of the cleaved amplified polymorphic sequence (CAPS) marker RGH1aE2I2 after AluI digestion. An expected position of one resistance gene of the cross Tiffany x PI284752 was established by interval mapping on chromosome 1HS between the markers Bmac0213 and RGH1aE2I2, 6 cM and 1 cM, respectively. The location of the other R gene was determined on chromosome 2HS in association with Bmac0134. The LRS score between the RGH1aE2I2 marker and the resistance gene was 166.3. A high significance threshold of 15.7 was determined by a permutation test. Co-segregation between tightly linked markers and the powdery mildew resistance was analyzed by specific DNA fragments associated with each allele of both genes amplified in 112 F2 plants. For the co-dominant CAPS marker RGH1aE2I2, 440 bp fragments were amplified from F2 plants that exhibited the highest resistance reaction type 0 to powdery mildew; whereas, 395 bp fragments were amplified in susceptible plants. In F2 plants with RT1 to RT2-3, the resistance was conferred by the R gene tightly linked with Bmac0134 in homozygous or heterozygous constitution.
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