Discovery, PCR Markers And Genetic Analysis For Genes Controlling Adult-Plant Leaf Rust Resistance In Barley

Rust disease is an important disease in Triticeae crops. The development of cultivars with durable resistance is an efficient means of controlling rust disease. Due to it's race-non-specific and considered more durable for controlling rust disease, slow rusting is increasingly paid more attention from plant breeders. Adult-plant resistance varieties are considered as durable sources in both wheat and barley breeding programs for the reduction of infection frequency, pustule size and spore production. Leaf rust caused by a fungus Puccinia hordei is prevalent on cultivated barley. It is currently the most important rust disease affecting barley. In barley, adult plant resistance (APR) Rphq genes are considered potentially more durably effective for controlling barley leaf rust as compared to race-specific Rph-genes. Most annotated Rph-genes are only expressed in seedling development stage. Mining and utilisation of effective adult-plant leaf rust resistnace genes and development of durable resistance cultivars are economically and ecologically sustainable approaches to control barley leaf rust. In present study, two adult plant resistance cultivars Pompadour and WI3407 were used as the genetic material. We focuses on fine mapping an adult-plant leaf rust resistance gene on Pompadour chromosome 5HS by using 292 DH (Doubled haploid) lines derived from Pompadour×Stirling with comparative genomics approach among barley, wheat and rice detecting corresponding syntenic region. Sequencing and SSCP analysis were also implemented for mining polymorphic markers in the population . In addition, a new DH population generated by WI3407/Baudin used to analyze an adult plant resistance gene in WI3407 chromosome 5HS with the same markers as Pompadour。We expect developing new molecular markers for marker-assisted selection of the leaf rust resistance genes and providing a basis for utilizing Pompadour and WI3407.The results are as follows:1. The result of barley chromosome 5HS Bin1, Bin2 blasting mapped wheat ESTs displayed that among expected syntenic region located in wheat 5A, 5B and 5D, wheat deletion bins 5AS3-0.75-0.98, 5BS6-0.81-1.00 and 5DS2-0.78-1.00 were most likely the syntenic regions to the Pompadour adult plant leaf rust resistance gene.2. Blast completed between barley chromosome 5HS Bin1, Bin2 and Brachypodium distachyon genome revealed the syntenic relationship between chromosome 5HS Bin1, Bin2 and Brachypodium distachyon chromosome 4 -- Bd4. Since 54% Bin2 sequences found the corresponding Brachypodium distachyon genome sequences are located in Bd4:725573…779015 region, the postulated syntenic region of barley chromosome 5HS Bin2 was the most likely located in Bd4:725573…779015.3. Defence gene homologous (DGH) mapping strategy was carried out for fining mapping Pompadour chromosome 5HS APR gene. Linkage analysis performed on 292 DH plants from cross Pompadour/ Stirling showed among 115 primer pairs detected in the study seventeen PCR-based markers were mapped to the short arm of chromosome 5H. The markers covered 36.3cM with an average marker of distance 2.1 cM. There were 8 SNPs, 4 INDELs, 4 dominant and 1 SSR markers. ALL 4 DGH mapped in the 292 DH population were mined from wheat deletion bin 5AS3-0.75-0.98 including a multiple copy EST BE490728. The adult plant leaf rust resistance gene (LR-APR) was flanked by markers EBmag0833 and bPb-0837 which were mapped at 1.3 cM and 0.7 cM to the gene respectively. The LR-APR gene explained for 82% phenotype variation. For characterizing Pompadour chromosome 5HS LR-APR gene, 457 F₂ plants were generated from cross WABAR2482×Pompadour. According to the phenotypic results based on the modified Cobb's scale, 328 F₂ plants were classified as resistant and 129 as susceptible which showed 3:1 model for a major dominant gene.4. The updated integrated map for barley chromososme 5HS Bin1, Bin2 and Bin3 was constructed based on the 2009 Integrated-5H. The integrated map added 8 new markers mapped in the current 292 DH population including 4 wheat DGH (BM134523,BE490728,BE498794, BE403373) and 4 barley markers (bPb-2872,Mact426,K08520,K02762). The comparison to the mapped Rphq4 which originated from Vada and Pompadour LR-APR gene is performed. As a result, the two leaf rust resistance genes were postulated as different genes mapped on chromosome 5HS.5. 164 DH lines derived from cross WI3407/Baudin were used to detecting the adult plant resistance gene in WI3407. The DH lines phenotypic analysis revealed one major gene controls adult plant resistance of leaf rust in this population. The linkage analysis shows 10 markers are polymorphic in the DH population. The major gene in WI3407 flanked by markers Mact426 and GBS0412, mapped 3.7cM to the nearest marker Mact426 with LOD score 37.9. This adult plant resistance gene explained 97% of the phenotype variation. The additive effectiveness is about 39. Marker regression results showed the nearest marker Mact426 explained 88% of the phenotype variance (P<0.0001). The results found that the adult plant resistance gene in WI3407 could be a new gene located on chromosome 5HS Bin2 region.6. Association analysis between the markers detected in the present study and 457 F₂ plants phenotypic data was displayed a high correlation between EST-SNP marker K08520 and the adult plant resistance (R²=0.82). Genotype data demonstrated the co-dominant EST-SNP marker K08520 tested 109 resistant homozygous, 115 susceptible homozygous and 233 heterozygous in 457 F₂ population. Together with the association analysis data and phenotype data, the results showed that EST-SNP K08520 could be used in the molecular assisted selection (MAS) for the adult plant resistance to barley leaf rust in Pompadour. Besides, 2000 F₃ plants were developed for preparing the recombinant inbred lines (RIL). It will be the basis for dissection the LR-APR gene mapped in Pompadour.7. According to the mapping results of the LR-APR genes from Vada, Pompadour and WI3407 together with the total mapped DGHs, we assume that the barley chromosome 5HS Bin1, Bin2, Bin3 could be a resistance genes and DGHs rich region.