Inheritance, Heterosis, Molecular Markers And Key Gene Cloning And Characterization For High Erucic Acid Content In The Especially High Erucic Acid Industrial Specialty Rapeseed In Brassica Napus L.

Erucic acid is a special fatty acid with long carbon chain in the Brassica seeds. As a food oil, erucic acid is difficult to be digested. But as an industrial material, erucic acid has very wide applications in oilfield chemistry, petrochemical industry, daily chemicals, pharmaceutical Chemicals, and so on. High erucic acid Brassica napus L. is the most important source of erucic acid in present time. In this study, three especially high erucic acid content lines, two high erucic acid content lines, two middle erucic acid content lines,and two low erucic acid content lines were selected as the study materials to explore the inheritance of especially high erucic acid content.in Brassica napus L. (1) An especially high erucic acid content, low oleic acid content genetic male sterile line 703AB-4 (Brassica napus L.) was crossed to a low erucic acid content and high oleic acid content variety Zhongshuang11(B. napus L). and six genetic populations (P1, P2, F1, B1, B2 and F2) from this cross were prepared for analyses of genetic models for erucic acid and oleic acid contents in the especially high erucic acid rapeseed lines and correlation between erucic acid content and other main fatty acids. (2) Nine parental lines (B. napus L) with varified erucic acid contents and genetic backgrounds were used to make 72 reciprocal hybrid combinations, according to the Complete Diallel Cross Design. Heterosis, combining ability and environment effects in erucic acid content were studied with a randomized block design in three different ecological regions in Sichuan province (Chendu, Mianyang,Yibin). (3)SSR markers were analyzed with an F2 population deviated from a cross between an especially high erucic acid line,703AB-4, and a low erucic acid line, Zhongshuang 11. SSR markers distinguishing high and low erucic acid contents were screened in order to be applied to Marker-assisted Selection in high erucic acid rapeseed breeding.(4) Two key regulating genes, FAE1 and FAD2, in the synthesis pathway of erucic acid were cloned and campared with the especially high erucic acid content line 703AB-4, middle erucic acid content line L155 and low erucic acid content line Zhongshuang 11. The main results of the study were the fowllowing:1. The optimum genetic model for erucic acid content was E-0, i.e. two major additive-dominant-epistatic genes+additive-dominant-epistatic polygenes. Erucic acid was mainly controlled by major gene and polygenes were quite weak. The additive effects of the two major genes were equal and amounted to 13.21, significantly higher than those reported in earlier studies. The inheritability of the major genes in the Bl, B2 and F2 populations was 98.5%,94.3% and 98.2%, respectively. The optimum genetic model for oleic acid content was E-1, i.e. two major additive-dominant-epistatic genes+ additive-dominant polygenes. The inheritability of major genes in B1 and F2 populations were 98.00% and 95.53%, respectively. The inheritability of major genes in B2 population was only 73.71%. It was revealed that oleic acid content was also mainly controlled by two major genes and the effects of polygenes were weak.2. The erucic acid content was highly significantly and negatively correlated with oleic content and linoleic acid content, respectively. The oleic acid content changed greatly, amounted to55 percentage points. The change in linoleic acid content was little, less than10 percentage points. The erucic acid content was not significantly correlated with linolenic acid content. There was complex relationship between the erucic acid content and arachidonic acid content. When erucic acid content was under 15%, erucic acid content was positively correlated with arachidonic acid content. Otherwise, erucic acid content was negatively correlated with arachidonic acid content. When erucic acid content reached to 50%, library of arachidonic acid content got small to promote erucic acid content continued to rise.3. Most cross combinations showed significant mid-parent heterosis in erucic acid content, and a few combinations showed over-parent heterosis. With greater differences in erucic acid content between the two parents, the mid-parent heterosis was stronger, while over-parent heterosis was only shown between especially high erucic acid content parents. It was indicated that heterosos in erucic acid content was mainly mid-parent heterosis. The effects of environments on erucic acid content were observed in parents and hybrids, however, the effects of environment on parents were smaller than hybrids. The performance of heterosis in erucic acid content was consistent in different environments, and was mainly shown as mid-parent heterosis. The heterosis of erucic acid content is variable among different test locations, but the combinations with higher heterosis appeared consistently, it was easier to get the hybrids with heterosis of erucic acid content in Yibin. Because of the importance of parents in raising the erucic acid content of the hybrids, it is necessary in the breeding process to consider use of high erucic acid material as parents, simultaneously taking into account environmental factors.4. It was shown that erucic acid contents in the hybrid combinations were mainly controlled by general combining ability (GCA). To obtain a hybrid of high erucic acid content, firstly, we should choose parents with high erucic acid content and high GCA, simultaneously taking into account the reciprocal effects..The erucic acid content was mainly controlled by genetic, but environmental factors showed certain level of influence on it, according to the stability analysis for combining ability. The effects of material could also play a role in cross combinations performance for high erucic acid content in Brassica napus L.. There were significant differences in erucic acid content between reciprocal crosses when maternal effects were positive. Furthermore, non-maternal effects that were only observed in some specific combinations may have a great influence on erucic acid content in the hybrid combinations.5. Two SSR markers, CB,0364 and BRMS-017, were identified to be tightly linked to the erucic acid content genes. All individual plants bearing both CB]0364-a and BRMS-017-a markers showed an especially high erucic acid content of over 57%. The segregation ratios of the two SSR markers in the F2 population well fit to Mendelian laws based on X2 test. The two SSR markers could be used to reliably separate the especially high erucic acid plants in the hybrid population.6. Low erucic acid content materials and middle erucic acid content materials had two FAE1 gene copy located in the chromosomes A8 and C3 of B. napus L.. The especially high erucic acid content materials had only one FAE1 gene copy. The results of comparisons in the encoded amino acid sequences offrom the low erucic acid materials (FAE1-1.1), the middle erucic acid materials(FAE1-2.1) and the especially high erucic acid materials 9FAE1-30, showed that F (phenylalanine) was present in the 282th amino acid site for low erucic acid materials FAE1-1.1, while S (serine) was present for middle erucic acid materials FAE1-2.1 and the especially high erucic acid materials FAE1-3. In 286,323, 395 and 406 amino acid sites, the especially high erucic acid materials FAE1-3 encoded the amino acid sequences of R (arginine), T (threonine), K (lysine) and G (glycine) respectively, while the amino acid sequences in low erucic acid and moderate erucic acid genes were successively G (glycine), I (isoleucine), R (arginine) and A (alanine).7. There were four different FAD2 gene sequences in the low erucic acid content and middle erucic acid content materials, and three various FAD2 sequences in the especially high erucic acid content materials. Homologous analysis showed that, as a class, FAD2-1.1, FAD2-2.1 and FAD2-3.1 encoded exactly the same amino acid sequence; FAD2-1.2, FAD2-2.2, FAD2-3.2 encoded exactly the same amino acid sequence; FAD2-1.3, FAD2-2.3 encoded a same class, But a termination codon appeared in advance in FAD2-1.4, FAD2-2.4, FAD2-3.3 as a class, homology in their encoded amino acid sequence was up to 99.91%, only varying in the 20 amino acid sites, T (threonine) for middle erucic acid materials (material No.2), while N-aspartic acid for low erucic acid materials (material No. 1) and especially high erucic acid materials (material No.3), namely, the amino acid sequences in low erucic acid and the especially high erucic acid materials were completely consistent.