Development Of Gene-specific Markers For RGMS And High Oleic And Low Linolenic Acid Sterile Line Through Marker-assisted Selection In Brassica Napus L.

Oilseed rape is one of the important oil crops around the world, and rapeseed oil is also the first source of homegrown edible oil in China. Heterosis utilization can effectively increase yield as male sterility is the most significant approach to produce rapeseed hybrids. Nowadays, due to the complete stable sterility and not being affected by cytoplasm, genic male sterility (GMS) plays an increasingly large proportion in using heterosis for Brassica napus. At present, the molecular markers for recessive genic sterility in rapeseed were tightly linked with the target genes. However, there are relatively few studies for development of functional markers based on gene sequence polymorphisms. The objectives of this study were to develop specific markers for recessive genic male sterility in Brassica napus and apply the markers together with gene-specific markers for high oleic and low linolenic acid developed previously to marker-assisted selection (MAS) breeding of high oleic and low linolenic acid sterile line. Through such a combination of heterosis utilization and quality breeding by means of MAS, we expect to create elite resource for producing new hybrid varieties with high oleic and low linolenic acid trait. The main results are as follows:1. Based on SNP loci of BnMsl and fragment length difference of BnMs2, four allele-specific primers and two gene-specific primers were designed. According to the effect of PCR amplification, two gene-specific markers with polymorphysm between sterile and fertile lines, named ZLMS1and ZLMS2, were developed in Brassica napus L.2. The amplified effect for allele specific primer show that allele specific primers with single base difference could not reveal the polymorphic SNP. However, introducing mismatched base pairs of A/T at the third site of3’-end in the primers could obtain ideal polymorphic amplifications, which was also superior to mismatched base pairs of C/T. Besides, the application mismatched technology that improved extensive use of SNP markers has certain reference significance.3. Specific markers developed in this study were applied to identifying the genotype of backcross progenies in male sterility breeding. In each generation of backcross, the results showed that the separation ratio of genotypes was consistent with theoretical value. In addition,10sterile individuals with the trait of high oleic and low linolenic acid were selected from3078plants in BC1F2population. Furthermore, we investigated the phenotype of these ten plants in flowering period and confirmed the accordance between genotype and phenotype.4. With the help of the allele-specific markers for oleic and linolenic, we analyzed the content of fatty acid in self-fertilized rapeseed. Results showed that the content of oleic was more than73%, while linolenic acid reduced to less than5%compared with wildtype. The results demonstrated that the MAS at eary stage could be used effectively to identify desirable genotypes.5. In BC1F2generation, we have selected the recessive genic male sterile plants with high oleic and low linolenic acid locus and its maintainers through the molecular marker-assisted selection. The highest recovering ratio of recipient genome was88.67%and91.67%, respectively. Furthermore, we could take full advantage of these new materials to develop rape hybrids with high oleic and low linolenic.