QTL Analysis Of Oil Content And Related Quality Traits In Brassica Napus, And Cloning And Molecular Evolution Of Oleic Acid Desaturase Gene (FAD2)

Brassica napus is one of the most important oil crops in China, its planting area and total production are at the top of the world. The seed oil content and fatty acid composition both are very important constitute of rapeseed oil quality. The improvement of rapeseed oil quality began in the1980s, the process was from low (no) erucic acid breeding to the fine fatty acid composition breeding in China. The development of process reflected the improvement of people's living standard and health requirements for edible oil. Therefore, on the basis of double-low and high yield, increasing the content of oleic acid and decreasing the linolenic acid content are important goals of China's rapeseed quality breeding, and which also can increase the added value of rapeseed production, improve the enthusiasm of the people to plant rape and meet the improved required of people on oil quality.In this study, the recombinant inbred lines and F2populations of Brassica napus were used for QTL mapping of seed oil content and fatty acid composition; extreme oleic acid and linoleic acid content phenotype of materials in F2population and their parents were used to clone and sequence analysis of FAD2gene. And thus a number of genes internal-specific PCR primers, CAPS markers and absolute quantification PCR primers were developed. Bio-information science and molecular evolution were studied on FAD2gene in different species. All of this work was to provide a certain theoretical basis for high seed oil content and excellent fatty acid composition in rapeseed breeding research. The finished work and the main results are as follow:(1) In this study, the RIL (183lines) which were developed through successive selling up to eight generations from a cross between yellow-seeded high erucic female parent GH06and black-seeded male parent double-low P174by single seed descent to constitute the mapping population, and using the SSR, SRAP, AFLP and TRAP to construct genetic maps SWU-1, traits analysis and QTL mapping were analysis in two consecutive years.The seed oil content and protein content of the SWU-1groups showed a continuous distribution, which was controlled by multiple genes. The content of palmitic acid, oleic acid and linoleic acid, linolenic acid showed a positive deviation from the normal distribution, dodecenoic acid content and erucic acid content showed a negative deviation from the normal distribution. The correlation of seed oil content, protein content was significantly negatively. The correlation of seed oil content and fatty acid composition was almost same in two years.On the basis of our lab's work and increased molecular markers, a total of595polymorphic loci were used toconstruct SWU-1map on LOD=10.0. A total of451markers on the final linkage groups, which contained200SSR,199SRAP,106AFLP,7TRAP, the length of map was1587cM with an average interval of3.51cM between adjacent loci. By comparative mapping analysis,16linkage groups were identified and named as N1～N18. There are three linkage groups (N9, N14and N19) can not be determined the number of linkage groups because of no common markers, N2(N18) and N1(N11) contained corresponding common markers of the two linkage groups. A total of8oil content and40fatty acid composition QTL were analysis by CIM with LOD>2.5in two years, mainly cluster on N1, N8and N13. The seed oil content QTL located on the N1, N3, N4, N5, N7, N8and N13, and single locus explained variation of5.19%-13.57%; only a protein QTL on N8and explained phenotypic variation of12.73%.Major QTL of palmitic acid content were located on N8and N13, a single locus explained phenotypic variation of10.13%-34.79%, another minor QTL was located on N18, explained4.71%of the phenotypic variation. Major QTL of oleic acid content was located on N8and N13, a single locus explained variation of7.95%-25.42%, and other two minor QTL were located on N13and N10, a single QTL explained7.68%and5.42%of the phenotypic variation. Major QTL of linoleic acid content was located on the N8and N13, a single locus explained12.1%-22.92%of variation, three minor QTL were located o N5, N13and N12, a single QTL explained7.8%,5.63%and4.28%of phenotypic variation, respectively.6linolenic acid QTL were located on N4, N5, N7, N8, N10and N15, a single QTL explain11.69%,5.63%,9.72%,8.61%,6.17%,4.95%and8.55%of the phenotypic variation, respectively. Dodecenoic acid content QTL were located on the N8, N11, N13(?)d N18, a single QTL could explain4.36%-13.47%of the phenotypic variation; major erucic acid content QTL were located on N8and N13, a single QTL could explain30.52%-43.76%of phenotypic variation, and the additive effects were from GH06.(2) F2population was constructed by male parent10L422with black high-oleic, low-linolenic acid (C18:1=87.22%, C18:3=1.94%) and female parent10L421with yellow low-oleic acid, high-linolenic acid (C18:1=47.47%, C18:3=16.58%), using SSR markers to build the A01group. Only one linolenic content acid QTL was detected with explained15.03%of the phenotypic variation by CIM, LOD score was3.08and the additive effect was1.36from10L422.In F2population, the palmitic acid, oleic acid, linoleic acid and linolenic acid content showed a continuous distribution, which was controlled by multiple genes. Dodecenoic carbon acid content and stearic acid content showed a negative deviation from the normal distribution. Oleic acid have significant positively correlationship with other fatty acid composition.(3) FAD2gene were sequenced in parents of F2population and two types (BnFAD2-a and BnFAD2-b) of copy of FAD2gene were founded, the length was1155bp and1141bp, respectively. BnFAD2-a is the normal expression of the FAD2gene; there were exist14bp absence of base in the164bp-238bp interruptly and seven early termination codon in ORFs of BnFAD2-b.According to the difference of two types sequences, gene internal-specific PCR primers were designed and amplified in extreme phynotype of the F2population. The result found that some of the primers were specific amplified in some materials with oleic acid content more than85%and linolenic acid content less than4%. There were GCGC restriction sites at735bp site in partial sequence of BnFAD2-a, which can be enzyme digested by Hha I restriction enzyme and generated polymorphic fragments in the expected position, which can be used to identify the different types in BnFAD2-a. The number of copies of (BnFAD2-a and BnFAD2-b) were analysis by absolute quantification PCR sequence in the parents and extreme phenotypes materials. The number of BnFAD2-a was more than BnFAD2-b in most of the materials. The copy number of BnFAD2-b in the high oleic acid parent was much more than in the low oleic acid parent. So we speculated that maybe one of the functional copy (1155bp) in high oleic acid materials replaced by non-functional copy (1141bp) due to homologous recombination, which resulted in decreasing enzyme activity of FAD2gene.(4) The full-length mRNA of49FAD2gene nucleotide sequence and amino acid sequence from32species were analysis, found that the FAD2gene sequences has a high similarity in different species; FAD2gene sequences in all species were divided into three categories, more than10sub-categories from the phylogenetic tree; there were no obvious codon bias in FAD2genes; Ka/Ks detection also showed FAD2gene was conserved.