Isolation of pollen S-determinant gene in Brassica species by using transformation and allelic polymorphysim

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Self-incompatibility (SI) in Brassica campestris is controlled by a single locus, termed S, with multiple alleles. To date, two polymorphyic genes have been identified at the S locus. We determined that the S receptor kinase (SRK) determines the S haplotype specificity of stigma, and the S locus glycoprotein (SLG) gene enhances the SI response. However, pollen S determinant was not identified, when we started this project. In our previous study, we isolated 76-kb contiguous genomic fragment containing SLG^9 and SRK^9, and determined the nucleotide sequence and expressed genes in the fragments. In the fragment, we identified two anther-specific genes, SAE1 and SP11. However, we did not determine which genes, SAE1 and SP11, were real pollen S determinant from their sequence information. Thus, we started to determine the real pollen S determinant by using transformation technique and allelic polymorphism.In the case of SAE1, we could not find allelic polymorphism among S alleles. In contr … More ast, we could isolate SP11-like cDNA clone from S^<52> allele. Furthermore, SP11-like cDNA clones from S^8 and S^<12> alleles were also isolated by co-operative research with Prof. Isogai's group, NAIST. These four clones encode the novel cysteine-rich pollen coat protein (PCP). They were located at the flanking region of SLG/SRK gene in each S allele, indicating that these four SP11-like genes were allelic. Their nucleotide sequences were highly diverted among S alleles. Furthermore, because this gene expressed in tapetum cells, we could explain that SI in Brassica is sporophytically controlled. Therefore, we selected this SP11 gene as a candidate gene for pollen S determinant. In order to determine that this SP11 gene is real pollen S determinant, SP11-9 gene were transformed into S^<52>S^<60> heterozygote. The pollen of transformant was rejected on S^9 stigma, and no phenotype change was observed in stigma side, indicating that the SP11 gene is real pollen S determinant. By using the combination of RT-PCR and CHEF analysis, 16 novel SP11 genes were isolated, and were located at the S locus, whose size was from 60 to 104 kb.When aligned the estimated amino acid sequences of SP11, six cysteine residues were completely conserved in all SP11, indicating that these cysteine residues are important for formation of tertiary conformation.When constructed the phylogenetic trees in SLG, SRK, and SP11, the pattern of the trees were similar among these three genes, indicating that these three genes co-evolved to make a new S allele.Furthermore, we isolated SP11 gene classified into class II S haplotype, and observed a similar trend to class I S haplotypes as described above. Less