Striking sequence similarity in inter- and intra-specific comparisons of class I SLG alleles from Brassica oleracea and Brassica campestris: implications for the evolution and recognition mechanism

Abstract

Self-incompatibility in Brassica is controlled by a single multi-allelic locus (S locus), which contains at least two highly polymorphic genes expressed in the stigma: an S glycoprotein gene (SLG) and an S receptor kinase gene (SRK). The putative ligand-binding domain of SRK exhibits high homology to the secretory protein SLG, and it is believed that SLG and SRK form an active receptor kinase complex with a self-pollen ligand, which leads to the rejection of self-pollen. Here, we report 31 novel SLG sequences of Brassica oleracea and Brassica campestris. Sequence comparisons of a large number of SLG alleles and SLG-related genes revealed the following points. (i) The striking sequence similarity observed in an inter-specific comparison (95.6% identity between SLG14 of B. oleracea and SLG25 of B. campestris in deduced amino acid sequence) suggests that SLG diversification predates speciation. (ii) A perfect match of the sequences in hypervariable regions, which are thought to determine S specificity in an intra-specific comparison (SLG8 and SLG46 of B. campestris) and the observation that the hypervariable regions of SLG and SRK of the same S haplotype were not necessarily highly similar suggests that SLG and SRK bind different sites of the pollen ligand and that they together determine S specificity. (iii) Comparison of the hypervariable regions of SLG alleles suggests that intragenic recombination, together with point mutations, has contributed to the generation of the high level of sequence variation in SLG alleles. Models for the evolution of SLG/SRK are presented.

Figure 1

Structure of the SLG in B. oleracea and B. campestris. As an example, the deduced amino acid sequence of SLG⁶Bol is shown. Shaded boxes represent the hypervariable regions I, II, and III. Filled, stippled, and hatched boxes show signal sequence, the deletable region, and the C-terminal variable region, respectively. ∗, Indicates perfectly conserved potential N-linked glycosylation sites. Boldface bars are the 12 conservative cysteine residues. Variable regions are boxed.

Figure 2

Comparisons of deduced amino acid sequence between markedly similar SLG pairs. Shaded “N” and filled circles show the positions of potential N-linked glycosylation sites and conserved cysteine residues, respectively. Boxed regions represent the deletable region, the hypervariable regions, and the C-terminal variable region. Dots indicate identical amino acid residues. (a) SLG¹⁴Bol and SLG²⁵Bca. (b) SLG⁸Bca and SLG⁴⁶Bca.

Figure 3

Neighbor joining tree of SLG and SLG-related sequences. #, Indicates SLG sequences reported in this paper. ##, Indicates sequences for which our data show some differences from the sequences reported previously (17, 28). Our data were used in the analysis. Amino acid sequences of the putative mature protein regions of SLG, SLR1, SLR2, and S domain of SRK were used for the analysis. SLR1 group was used as an outgroup. The numbers besides the branches are the bootstrap values (%) with 1000 repeats. The scale shows amino acid substitutions/site.

Figure 4

(a) Pairwise distribution of the number of substitutions in two different regions of class I SLG alleles in B. oleracea. All pairwise comparisons are shown. Open boxes and open circles indicate pairs of sequences showing a significant deviation from the expected binomial distribution (P < 0.05 and P < 0.01, respectively). (b) Neighbor-joining trees using nucleotide sequences of the hypervariable regions I, II, III, and the C-terminal variable region of SLG⁷, SLG¹², SLG¹⁴, SLG³², and SLG⁶³. SLG² was used as an outgroup (not shown in Fig. 4b). The numbers besides the branches are the bootstrap values (%) with 1,000 repeats.

Figure 5

Two models for the evolution process of SLG and SRK alleles. Arrows indicate gene conversion between SLG and SRK. Broken lines indicate possible stages at which the S haplotypes acquired the function of self-incompatibility (see text).