Identification Of Molecular Markers Of Restorer Gene In Nsa CMS And Stem Rot Resistance Of Restorer Lines In B.napus

CMS is the main approach for heterosis utilization. As a major pollination control system of hybrid production, it plays a decisive role in improving the output of rapeseed. At present, several CMS systems have been reported, but only a few of them are used in rapeseed production. Pol CMS is widely used in China presently, but the singularity of rapeseed hybrid cytoplasm increases the risk of epidemic disease and affects the sustainable development of rapeseed production. So creating and exploring of new CMS systems is imperative. Nsa CMS is a new cytoplasmic male sterility system established through protoplast fusion. The sterility of this CMS is stable, rarely produces trace pollen under low temperature conditions. However, because the fertility restoration source of alloplasmic male sterility is narrow, it was difficult to identify restoration materials through test cross in the cultivated Brassica species. The only resource found so far with restoration was from the offspring of the somatic hybrids derived from the same fusion combination as the male sterile line. The breeding and improvement of restorer lines all rely on crossing to transfer the restoration. Good restorer lines is the necessary precondition to produce high purity hybrid using Nsa CMS.To speed up the breeding process of restorers for Nsa CMS system, studies related to the restorer gene and stem rot resistance of the existing restorer lines had been carried out. The main results are as follows:1. DNA samples from ten sterile and ten fertile F₂ single plants of a cross between the sterile line and the restorer were mixed respectively to make sterile bulk and fertile bulk. AFLP, SRAP, RAPD, SSR analysis were used to identify polymorphic bands between the two bulks. Results showed that seventeen among 180 AFLP primer combinations, ten among 94 RAPD primers, two among 96 pairs of SSR primers, and 30 among 170 pairs of SRAP primers had polymorphic bands between sterile bulk and fertile bulk.2. Primers (or primer pairs) which could amplify polymorphic bands between the two bulks had been used to determine the genetic distance of markers from the restorer gene. Four SRAP molecular markers (m1e8, m6e10, m9e10 and m3e9) which linked to restorer gene had been identified, and the genetic distance of these markers from the restorer gene were 28 cM, 23 cM, 3.1 cM and 13.5 cM. With other three marker systems, no marker had been found closely linked with the restorer gene.3. Through reclaiming of fragment, sequencing and primer design, the SRAP marker amplified by primer pair m3e9 had been successfully converted into a SCAR marker. This marker was 3.1 cM from the restorer gene, and had been defined as SCAR₂₈₁.4. In vitro inoculation, toothpicks inoculation, natural infection in the field, and other methods had been used to assess the stem rot resistance of the restorer line. The results of variance analysis showed that, after inoculating, there was a significant difference between restorer line 7330 and the controls, indicating the resistance of Nsa restorer lines were higher than controls Zhongshuang 4, Zhongshuang 9 and Zhongyou 821.5. The changes of phenylalanine ammonialyase (PAL), polyphenol oxidase (PPO), peroxidase (POD) activity were used to analyze the mechanism of stem rot resistance. Activity of three kinds of enzymes in the resistant restorer line was higher than controls (Zhongshuang 4, Zhongshuang 9 and Zhongyou 821), indicating these three kinds of enzymes played a certain role on this disease resistance.