Variations And Interactions Between S-Locus Genes From Progenitors During The Formation Of Brassica Napus L.

Self-incompatibility(SI) is one of the evolved mechanisms to avoid inbreeding depression and to keep genetic variation. The study of SI will contribute to the development of biology and agriculture. SI in Brassica species was controlled by a single S-locus containing multiallelic genes, SRK(S-locus receptor kinase) determining the self-recognition specificity of the stigma, SP11/SCR(S-locus protein 11/S-locus cysteine rich protein) controlling the self-recognition specificity in the pollen. S alleles were divided into two groups, class I and class II, based on the sequence similarity between allelic genes of the three S-locus genes, SLG(S-locus glycoprotein), SRK and SP11.Allopolyploidy Brassica napus L.(2n=4x=38, AACC) was formed ~7500 years ago by hybridization between Brassica rapa L.(2n=2x=20, AA) and Brassica Oleracea L.(2n=2x=18, CC), followed by a series of evolution process. Although the cultivated B.napus is self-compatible(SC), B. rapa, B. oleracea and artificially synthesized B. napus are self-incompatible. Interestingly, the B. rapa restituted from B. napus by the total elimination of C-genome chromosomes in intertribal hybridizations regains self-incompatibility. The novel B. rapa genotypes provide unique opportunity for the study of the interaction between S-locus genes from ancestral species in the evolution of B. napus and for the elucidation of the conversion mechanism from SI to SC. In this study,the restituted B. rapa(OroA and ZS11A) from B. napus cultivars ‘Oro’ and ‘ZS11’ and artificially synthesized B. napus were used for phenotype characterization, gene cloning and expression analysis to reveal the differentiation and interaction between S-locus genes in the formation process of B. napus. The main results were given as follows:1. Structural change and expression of S-locus genes in synthesized B. napusThe parental S haplotype of B. rapa(AA) was similar to BoS-45 and designated as BrS-AA and of B. oleracea(CC) was BoS-15. Analysis of the S haplotypes of artificially synthesized B. napus at the generations S0-S6 showed that the S-locus genes of S0(AA.CC and CC.AA) were neither A nor C types from two parents. Particularly, in AA.CC and CC.AA, the exons 2-4 of their classⅠSRK showed similarities to BnSRK-1 and BnSRK-910, respectively, and their exon 1(coding the S domain) both shared perfect match with BcSRK-99, suggesting that different regions of SRK experienced different changes. Even more, the classⅠSLG of AA.CC showed only 88% identity to BrSLG-99,revealing the inconsistent variation between the S domain of SRK and SLG. Unlike in CC.AA, classⅠS-locus genes from female parent in AA.CC tended to alter to BnS-1 from synthesis. As for classⅡS-locus genes, the sequence of SRK from male parent was more similar with BnSRK-60 and BOLSRK-1 than with BnSRK-6, indicating that the transformation of classⅡS-locus gene from male parent was more complex than fromfemale parent. In generations from S3 to S6, the S haplotype became the common combination BnS-1/BnS-6. Expression analysis showed that not only nucleic sequence but also gene expression changed in synthesized B. napus. Although the sequence of classⅠSRK of CC.AA and S1-2 were highly similar, the expression products were diverse.Because both classⅠandⅡSP11 had normal expressions in the anthers of CC.AA and S1-2, we speculated that possible insertion or deletion in upstream or downstream of SP11 invalidated classⅡSP11.The parental S haplotype of B. rapa cv. Qinghai Dahuang and B. oleracea var.alboglabra(CJ) was BrS-f2 and BoS-5, respectively. The S haplotype combination in synthetic B. napus at generations S1 and S5 was BrS-f2/BoS-5. ClassⅠSRK normally expressed in S5 but not in S1 and classⅡSRK normally expressed in S1 but not in S5, but ClassⅡSP11 expressed in both. Therefore, even if the S haplotypes kept the same in synthetics, the expression of S-locus genes changed towards self-compatibility.2. Structural and expression of S-locus gene in restituted B. rapaThe S haplotypes of two B. napus donors were BnS-1/BnS-6, while the S haplotypes of their restituted B. rapa were not the one from parental A genome. The classⅠSRK sequence of OroA exhibited similarity with BnSRK-910 and its expression product revealed 99% identity to BrSRK-99, which inferred that the changed SRK led to the aberrance in SRK protein or the mismatch with other key factors and then broke the dominant relationship to classⅡSRK. In the above situation, the classⅡS-locus genes probably was the SI determinant for both SRK-60 and SP11-60 with normal expressions.In ZS11 A, the classⅠS haplotype was BrS-AA and classⅡSRK and SLG were deficient.ClassⅡSP11 and BcSLR-2 were separately expressed in the anther and stigma of ZS11 A,albeit classⅠSRK and SP11 showed no expression. It was probable that BcSLR-2interacted with SP11, resulting in the SI of ZS11 A.3. The change in S-locus gene in synthesized B. napus and restituted B.rapaThe comparison of the sequence of classⅠSRK and SLG between AA and ZS11 A and between CC.AA and OroA showed that the similarities of each pair were 99%, 97%and 99%, 93%, respectively. Accordingly, in the process of allopolyploidization and A-genome extraction, changes in the sequence of S-locus genes were reversible, or in other words, S-locus genes experienced certain evolutionary change before finalizing the common S haplotype combinations. At the same time, this reversible change process likely explained that the most common BnS-1/BnS-6 of B. napus in the nature evolved not necessarily from the parental S haplotypes BrS-47 and BoS-15 but also from more S haplotypes.