Molecular Marker-assisted Selection Of Blast And Bacterial Blight Resistance In Hybrid Rice

Developing disease-resistant rice cultivars through molecular marker-assisted selection (MAS) is not only an economic, powerful strategy to fight against various rice diseases, but also an important component of the comprehensive rice breeding target to obtain high-yield, high-quality and multifaceted resistant varieties, playing a significant role in rice production practice.Based on the reported mapping result of the rice blast resistance gene, Pi25(t), 14 additional SSR markers identified in the interval harboring Pi25(t) was detected for parental polymorphism and four of them, RM3330, RM564, RM3628 and RM6298 were added to chromosome 6 of the new genetic map consisting of 181 markers.G19, G36 and G114, chosen from the 304 recombinant inbred lines (RILs) of indica rice cross Zhong 156/Gumei 2, as female parents, were crossed to high-yield restorers, 3-11 and 9308, respectively at China National Rice Research Institute (CNRRI), Hangzhou, China. From a single F₁ plant, seeds were collected and advanced by single seed decent (SSD). In 2004, six RIL populations (FS6001-FS6006) at the F₆ generation were obtained. Three molecular markers, RM3330, A7 and RM7193 were employed to survey the six populations and 109 lines homozygous for Pi25(t) using the SSR makers RM3330 and A7 were obtained. In addition, 20 lines homozygous for combination of Pi25(t) and fertility restoring genes were obtained through second-round screening of the selected 109 lines using markers linked to fertility restoring genes. Aiming to analyze the efficiency of MAS, three populations were selected to be artificially inoculated with M. grisea isolate 05-20-1 prevalent in Zhejiang Province. The result manifested that different MAS efficiency values were observed for the markers in different populations and MAS efficiency of each individual marker was around 70% to 80% and the MAS efficiency of some marker in a specific population was merely 44.3%. The MAS efficiency value was significantly increased with the maximum value amounting to 96.3% and the minimum value exceeding 77%, as two markers flanking the target gene on both sides were used for the assay, implying that simultaneous selection of markers flanking the gene on both sides can increase the MAS efficiency tremendously. Elementary study concerning the fertility restoring ability of selected lines homozygous for fertility restoring genes concluded that seed-setting rate of crosses between cytoplasmic male sterility (CMS) line, Zhong 9A and those lines reached the normal level.High-yield restorer, 3-11, as female parent, was crossed to IRBB58, IRBB59 and IRBB60 carrying bacterial blight (BB) resistance genes, respectively at CNRRI, Hangzhou, China. In 2004, three populations at the F₆ generation were obtained through SSD. STS marker pTA248 and SSR markers RM433, RM153 and RM264 flanking BB resistance genes, Xa21, xa13, xa5 and Xa4, respectively were chosen to screen the three F₆ populations for lines homozygous for individual genes and those having different combinations. For each population, first-round screening was carried out to obtain lines homozygous for Xa21 and the second-round screening for the other BB resistance genes, xa13, xa5 and Xa4. As the results showed, 41 lines carrying multiple combinations of BB resistance genes were acquired from the three F₆ populations comprising of 203 lines, including 2 lines having combination ofBB resistance genes Xa21+xal3+xa5+Xa4, 7 lines for combination of Xa21+xal3+xa5, 1 line for Xa21+xal3+Xa4, 23 lines for Xa21+xal3, 25 lines for Xa21+xa5 and 5 lines for Xa21+Xa4. Inoculation with six isolates of the pathogen indicated that combinations of three resistance genes were more effective against the pathogen than combinations of two resistance genes and combinations of resistance genes Xa21/xa13 and Xa21/xa5 demonstrated a higher-level of resistance to the pathogen than the combinations of Xa21/Xa4, suggesting that xal3 and xa5 are two valuable BB resistance genes with great potential for utilization. All the lines of the two populations were also inoculated with isolate 05-10 widely prevalent in Zhejiang Province to appraise the MAS efficiency of the selected markers, pTA248, RM433 and RM153. The results suggested that average MAS efficiency of pTA248 for FS6008 and FS6009 was 61.1% and 68.4%, respectively and MAS efficiency of markers in combinations was a little bit higher. In the two populations of FS6008 and FS6009, average MAS efficiency of pTA248+RM433 and pTA248+RM153 was67.5%, 79.2% and 68.3%, 81.7%, respectively. In line with the target of selecting lines homozygous for resistance genes, it can be inferred that MAS efficiency of RM153 was the highest (83.3%), followed by RM433 (60.0%) and pTA248 (57.9%) was the least effective for MAS assay.From the lines homozygous for multiple resistance genes obtained through the above-mentioned MAS, five lines carrying genes Xa21, xal3 and xa5 in various combinations and another five lines homozygous for Pi25(t) were selected for sexual hybridizations and 25 crosses were achieved. Screening of the crosses via MAS concluded that only one cross, DH146/TM487, proved to be suitable for MAS analysis. The F2 population of the cross DH146/TM487 was firstly inoculated with M. grisea isolate 05-20-1 to eliminate those susceptible plants and followed by MAS analysis by markers linked to various resistance genes. Lines homozygous for Pi25(t) were screened through linkage markers RM3330 and A7 and markers pTA248, RM433 and RM153 for BB resistance genes Xa21, xal3 and xa5, respectively. Lines carrying multiple resistance genes in various combinations were obtained and inoculation with Xoo isolate C23 demonstrated that the selected lines were similar to the parents TM487 and IRBB60 regarding resistance level to the pathogen isolates. The results suggested pyramiding BB and rice blast resistance genes through markers pTA248, RM433 and RM153 was effective and reliable.The lines carrying multiple disease resistance genes in combinations through pyramiding both BB and blast resistance gene could be introduced to practical rice breeding projects, and also as resistance gene donors for variety improvement programs.