Hochauflösende Kartierung der Virusresistenzgene <i>rym11</i> und <i>Ryd3</i> der Gerste (<i>Hordeum vulgare</i> L.)

Abstract

Barley yellow mosaic virus disease, caused by Barley yellow mosaic virus (BaYMV) and Barley mild mosaic virus (BaMMV), as well as Barley yellow dwarf disease which is caused by Barley yellow dwarf virus (BYDV) and Cereal yellow dwarf virus (CYDV) lead to severe yield losses in susceptible cultivars. For both diseases chemical control is neither economically nor ecologically reasonable, so rendering the cultivation of tolerant/resistant varieties is the most effective way to prevent yield losses. In barley several resistance genes are available for resistance breeding against both virus diseases, with Ryd3 explaining 75 % of the phenotypic variance regarding tolerance against BYDV-PAV and BYDV-MAV, while rym11 confers resistance against all European agents of Barley yellow mosaic virus disease.
In order to get detailed information on the structure and the underlying resistance mechanism of both genes as well as providing a better targeted introgression of the resistance genes into elite material, the aim of this study was to isolate these genes. Since no information about the gene product was available, a mapped based cloning strategy was applied. Because of the gene´s proximity to the cenromere of barley chromosome 4H and 6H, respectively, the genes had to be mapped at high resolution.
For Ryd3 3,210 F2-plants of a cross between the susceptible line `L94-QTL3´ with the resistant line `L94´ were analysed with the co-dominant flanking markers GMS006 and GBM1063. Out of a set of 31 public markers, 10 markers mapped in the 7.31 cM interval of GMS006 – GBM1063 and 7 markers co-segregated with Ryd3. For the shortened marker-interval GBS0655 – WBE201 121 RILs showed a recombination event and the phenotypical analysis for tolerance (t) vs. susceptibility (s) against BYDV was consistent with the expected 1r:1s ratio for a major gene (58t vs. 63s; Chi²_(1t:1s) = 0.207).
A sequence comparison with the rice genome identified a syntenic region of 19 Mb on rice chromosome Os02 and further marker were developed by exploitation of a widely existing macrocollinearity to rice, Brachypodium and sorghum from public EST sequences and from the Genome Zipper. Out of 15 barley ESTs the unigenes U35_02578_540-712 and U35_16315_345-563 co-segregated with Ryd3. With DArT-marker bPb-3722 another co-segregating marker was identified by means of a bulked segregant analysis (BSA).
A test of the diagnostic value of the developed markers on a panel of 33 barley genotypes consisting of known Ryd2 and Ryd3 donors as well as susceptible cultivars showed that no single marker alone could consistently discriminate Ryd3 from non-Ryd3 genotypes, but the combination of the markers GBMS0107 and GBMS0135 could.
For rym11 5,102 F2-plants of a cross between the susceptible cv. `Naturel´ with the resistant lines `W757/612´ and `W757/112´ were analysed with the co-dominant flanking markers HVM03 and HVM68. Out of a set of 82 public markers 18 markers mapped in the 10.72 cM interval of HVM03 – HVM68. For the shortened marker interval GBS0887 – GBS0506 100 RILs had a recombination event and the phenotypical analysis for resistance (r) vs. susceptibility (s) against BaMMV was consistent with the expected 1r:1s ratio (52r vs. 48s; Chi<sup>2</sup>( <sub>(1t:1s)</sub> = 0.163).
A well preserved collinearity with rice chromosome Os03 was exploited for a synteny based marker saturation approach and could be automated after anchoring the genetic map of rym11 with the Genome Zipper.
Besides marker development out of 17 informative unigenes additional 70 sequence-tagged-site (STS) markers were developed out of sequence information from the Genome Zipper and the markers U32_3699A and sel.1167 could be mapped. In the last step of marker saturation markers were developed out of 8 informative contigs of the cultivar `Morex´ and the markers C_1030750_B and C_1012894_B shortened the gene carrying interval to 0,074 cM, whereas the markers C_205243_B and C_205243_E1f&4730r were co-segregating with rym11. The polymorphism of marker C_205243_E1f&4730r is based on a 1,375 bp large deletion which causes in the resistant parents `W757-112´ und `W757-612´ a loss of function of the barley gene HvPDIL5-1 so denying BaYMV and BaMMV a host factor.