Characterization of S haplotype in a new self-compatible Brassica rapa cultivar Dahuangyoucai

The most important Brassica species, B. rapa, is naturally self-incompatible. Self-compatible mutants would be useful for dissecting the molecular mechanism of self-incompatibility (SI), a process that promotes outcrossing by recognizing and refusing self-pollens. The S haplotype in a new self-compatible B. rapa cultivar, Dahuangyoucai, was characterized for the first time in this study. Sequence analysis of the S-locus genes, SLG (S-locus glycoprotein), SRK (S-locus receptor kinase) and SCR (S-locus cysteine-rich protein) revealed that Dahuangyoucai contained S haplotype highly similar to S-f2, a non-functional class I S haplotype identified in another self-compatible B. rapa cultivar, Yellow Sarson. Mutations of MLPK (M-locus protein kinase) and non-transcription of the male determinant, SCR, were observed in this cultivar, which is similar to the situation reported in Yellow Sarson. With respect to the female determinant, SRK, no transcript was detected in Yellow Sarson but two fragments were detected in Dahuangyoucai. One fragment was highly similar to SRK-f2, but the other fragment was different from the signal factors previously identified in the SI reaction. The results suggest that Dahuangyoucai and Yellow Sarson have the same origin and a similar mechanism of self-compatibility, but diverge after mutations in SRK, SCR and MLPK. Further studying the self-compatibility of Dahuangyoucai might identify novel factors involved in the SI signalling cascade and provide new insights into the mechanisms of SI in Brassicaceae.

Genomic Organization of the S Locus: Identification and Characterization of Genes in SLG/SRK Region of S9 Haplotype of Brassica campestris (syn. rapa)

In Brassica, two self-incompatibility genes, encoding SLG (S locus glycoprotein) and SRK (S-receptor kinase), are located at the S locus and expressed in the stigma. Recent molecular analysis has revealed that the S locus is highly polymorphic and contains several genes, i.e., SLG, SRK, the as-yet-unidentified pollen S gene(s), and other linked genes. In the present study, we searched for expressed sequences in a 76-kb SLG/SRK region of the S9 haplotype of Brassica campestris (syn. rapa) and identified 10 genes in addition to the four previously identified (SLG9, SRK9, SAE1, and SLL2) in this haplotype. This gene density (1 gene/5.4 kb) suggests that the S locus is embedded in a gene-rich region of the genome. The average G + C content in this region is 32.6%. An En/Spm-type transposon-like element was found downstream of SLG9. Among the genes we identified that had not previously been found to be linked to the S locus were genes encoding a small cysteine-rich protein, a J-domain protein, and an antisilencing protein (ASF1) homologue. The small cysteine-rich protein was similar to a pollen coat protein, named PCP-A1, which had previously been shown to bind SLG.

Evolutionary aspects of the S-related genes of the Brassica self-incompatibility system: synonymous and nonsynonymous base substitutions.

In the Brassicaceae, self-vs. nonself-recognition in self-incompatibility is controlled by sporophytic S-alleles. Haplotypes specifying both SRK (S-receptor kinase) and SLG (S-locus glycoprotein) are considered to play an important role in the recognition reactions. We compared the nucleotide sequences of SRK9(Bc) and SRK6(Bo). The number of nonsynonymous substitutions per site (Pn) was lower, constrained, in the kinase than the receptor domain, while the numbers of synonymous substitutions (Ps) in the two domains were largely comparable. Pairwise values for Ps and Pn were calculated among 17 operational taxonomic units, including eight SLGs, the receptor domains of two SRKs, four SRAs (S-related A) and three SRBs (S-related B), which have high homologies with each other. The values of Ps and Pn of SLG were mostly comparable to those of the receptor domain of SRK. Dendrograms constructed on the basis of Pn and Ps indicated that SRA differentiated first, followed by SRB. The differentiation of SLG alleles is one of prerequisite factors for the establishment of self-incompatibility, and the allelic differentiation has occurred more than tens of million years ago.