The Construction Of An EMS-Induced M2 Population For TILLING Analysis In Oilseed Rape (Brassica Napus L.) And The Investigation On Allelic Variation Of The Loci Associating With Seed Qualitative Traits

Brassica oilseeds are important sources of edible oil in many parts of the world. Molecular Marker-Assisted Plant Breeding (MMAPB) is highly efficient in improving crop agronomic and seed quality traits. The availability of various kinds of DNA molecular markers and the sequence data of genomes and ESTs facilitate genetic research and MMAPB programs. Knowledge about the genes implicated in lipid biosynthesis acquired from the model plant Arabidopsis is useful in understanding the formation of seed oil in Brassicas.Nevertheless, little is known about allelic variations of the genes regulating lipid biosynthesis and their association with a certain kind of fatty acid accumulation in Brassica oilseeds. The current study was envisaged to identify the DNA polymorphisms of functional genes that are preferably expressed in seeds and are putatively important to the fatty acid composition among EMS-induced M2 individual plants as well as a collection of 54 local Brassica oilseed cultivars. The process of our research and the results are as followed:1. The ethyl methan sulfonate (EMS) was applied to treat the pure seeds of the 'double low'cultivar "Zhe-Shuang 72" (Brassca napus L.). The mutants with abnormal appearance of leaf color, branch number, plant height, stem or flower color, floral fertility and life cycle were observed in M1 population, screened in the M2 population and partly verified in the M3 population. In general, the mutants accounts for 8.46% of the total population in the M2 generation. Correlation analysis among the fatty acids and between the fatty acids and total oil content has been conducted in the M3 seeds. The variation degrees of the fatty acid components and oil content of the M3 seeds are described. A DNA pool from 2558 individual M2 plants were constructed for the analysis of allelic variations of the major genetic loci regulating grain quality traits. Materials with particular mutated traits are available for breeding projects targeting genetic improvement of oilseed rape.2. The comparison of the transcription level of the FATTY ACID DESATURATION genes as FAD2, FAD6, FAD7 and FAD8 between leaf and seed tissues revealed that FAD2 and FAD8 transcribed with similar level in seeds and leaves, whereas FAD7 and FAD8 preferably expressed in leaves to seeds. Alignment among sequences of the FAD genes indicated a higher similarity among FAD3, FAD7 and FAD8 genes. On the other hand, the sequences of FAD2, FAD5 and FAD6 were more different from each other and also diverged significantly from the group including the FAD3, FAD7 and FAD8 genes. Three primer pairs covering the coding region of FAD2 were used for High Resolution Melting (HRM) PCR and SNP detection.3. A total of 12 SNPs that gave rise to non-synonymous amino acid changes were identified on the basis of one-thousand M2 individual plants. Six of the corresponding mutants were characterized with lower oleic acid content, and three of the mutants demonstrated higher oleic acid content, whereas the other three mutants was not significantly different from the control in terms of oleic acid content.4. Polymorphic markers at the loci putative for the seed oil formation between two geographically different genotypes:the Chinese cultivar Ningyou-7 and the European cultivar Tapidor were systemically screened. These primer pairs (150) were designed based on 75 Brassica genes that were Arabidopsis orthologues implicated in the oil formation. A total of 52 out of the 150 primer pairs associated with 47 of the 75 genes showed polymorphisms between the two genotypes. The type of polymorphisms that could be detected on capillary electrophoresis images and their respective visual futures are described. Further,34 polymorphic markers were selected to scan allelic variations and rich DNA polymorphisms among the 54 Brassica oilseed cultivars were found. On the average, each primer pair resulted in 5.6 alleles at the region that was covered. The correlation between the alleles and seed quality traits revealed that the alleles of BnFAD7 were related to the variation of linolenic acid (C18:3) contents among the cultivars. The allele FAD7-ics11170 (3/4)-b that was significantly correlated with high linolenic acid content can be used as an efficient marker for the selection of breeding materials with high linolenic acid content.Taken together, we described the allelic variation of some loci that regulate seed fatty acid composition among an EMS-induced M2 population as well as a collection of Brassica oilseeds germplasm. Our results could be an enriching addition to the body of work that attempts to understand the accumulation of fatty acids in Brassica oilseeds. The start-up work to screen for alleles correlating with high content unsaturated fatty acids laid a solid foundation for MMAPB project towards the improvement of the seed quality of Brassica crops.