SSR Marker Of Restorer Gene Of Cytoplasmic Male Sterile L04-05A And Fertility Analysis In Brassica Napus

This thesis based on the cytoplasmic male sterile line L04-05A of brassica napus which bred from agricultural college of Gansu agricultural university and F1, F2 segregating population which prepared from restoring material L04-05C1 and L04-05C2, and the Bulked sergeant analysis(BSA) was adopted to screen molecular marker and to linkage analysis of the restorer gene of cytoplasmic male sterile. At the same time, the exploration on the application of the SSR (Simple sequence repeat) technology on molecular breeding in brassica napus has been made. And the results are as follows:1. The research on the floral organ morphology of the sterile material PLCMS(L04-05A) and its maintainer Pol CMS(Pol 5A)and Ogu CMS showed that about PLCMS(L04-05A), the diameter of corolla is large, the petal is big and petal flattening; the sterility is completely, steadily, and there's no fertile pollen by microscopic examination anther in full flowering stage. Compare with PLCMS, Pol CMS has significant difference, and Ogu CMS has some difference, and PLCMS is a new type of sterile cytoplasm which different from Pol CMS and Ogura CMS.2. Based on the F₂ segregating population, it proved that the fertility restoring of L04-05C₁ and L04-05C₂ to L04-05A is controlled by one dominant gene through fertility segregation analysis in florescence.3. Take the brassica napus genome DNA as the template, and to optimize the brassica napus PCR reaction system from DNA, Taq enzyme, dNTPs, primers and template, so the SSR optimized reaction system suit for brassica napus has been established: the reaction volume is 25ul, Mg²⁺ is 2.0ul(concentration is 2.5mM),dNTPs is 2.0ul(concentration is 2.5mM), 10×buffer is 2.5ul(concentration is 7.5mM), Taq enzyme 0.4ul(concentration is 2.5U/ul), primer is 4ul(concentration is 10uM), template DNA is1ul(concentration is 15-40 ng/L).PCR reaction program is: pre-denature at 94℃for 3min;denature at 94℃for 30s, annealing at 51℃for 30s, extention at 72℃for 1min, cycling 30 times, last extention at 72℃for 5min。4. To screen 60 primers by using fertile DNA mixing pool and sterile DNA mixing pool which established from number 6 F₂ population and number 9 F₂ population respectively; and it showed that there's two primers of differential band between the two DNA mixing pool, then to further prove by the single plant that used for establishing mixing pool, and it was found that the molecular weight of 150bp band which amplified by primer Ol13-E08 is the differential marker for fertile plant, and it can't be amplified this band in sterile plant, with good reproducibility. Then further amplified the F2 population of number 6 and 9 respectively by primer Ol13-E08 make sure that Ol13-E08-150bp is the SSR marker linked to the fertility restorer gene of PLCMS ( L04-05A ) , and the genetic distance between Ol13-E08-150bp and fertility restorer gene is 5.13 cM.