Cloning and characterization of cDNAs encoding S-RNases from almond (Prunus dulcis): primary structural features and sequence diversity of the S-RNases in Rosaceae

Abstract

cDNAs encoding three S-RNases of almond (Prunus dulcis), which belongs to the family Rosaceae, were cloned and sequenced. The comparison of amino acid sequences between the S-RNases of almond and those of other rosaceous species showed that the amino acid sequences of the rosaceous S-RNases are highly divergent, and intra-subfamilial similarities are higher than inter-subfamilial similarities. Twelve amino acid sequences of the rosaceous S-RNases were aligned to characterize their primary structural features. In spite of their high level of diversification, the rosaceous S-RNases were found to have five conserved regions, C1, C2, C3, C5, and RC4 which is Rosaceae-specific conserved region. Many variable sites fall into one region, named RHV. RHV is located at a similar position to that of the hypervariable region a (HVa) of the solanaceous S-RNases, and is assumed to be involved in recognizing S-specificity of pollen. On the other hand, the region corresponding to another solanaceous hypervariable region (HVb) was not variable in the rosaceous S-RNases. In the phylogenetic tree of the T2/S type RNase, the rosaceous S-RNase fall into two subfamily-specific groups (Amygdaloideae and Maloideae). The results of sequence comparisons and phylogenetic analysis imply that the present S-RNases of Rosaceae have diverged again relatively recently, after the divergence of subfamilies.

Allelic diversity of S-RNase at the self-incompatibility locus in natural flowering cherry populations (Prunus lannesiana var. speciosa)

“…The putative introns differed considerably in size (Table 1). The nucleotide sequences of the exon regions showed high homology (80-90%) to those of the S-RNases previously reported in P. dulcis (Ushijima et al, 1998) and P. avium (Sonneveld et al, 2001;Wiersma et al, 2001). The alignment of the deduced amino-acid sequences is shown in Figure 3.…”

“…In the phylogenetic tree, the S-RNases of flowering cherry and Prunus cultivars (P. dulcis and P. avium) clustered together, but they did not form species-specific subgroups. This suggests that the S-RNase of flowering cherry and Prunus cultivars diverged before the divergence of species, as suggested by Ushijima et al (1998).…”

“…PCR amplification of S-RNase genes from genomic DNA To amplify S-RNases from genomic DNA, primers C1-sense (5 0 -TATTTTCAATTTGTGCAACA-3 0 ) and C5-antisense (5 0 -CAAAATACCACTTCATGTAA-3 0 ) were designed from conserved domains (C1 and C5) of the P. avium (Sonneveld et al, 2001;Wiersma et al, 2001) and P. dulcis (Ushijima et al, 1998) S-RNases. PCR was performed with 35 cycles at 951C for 30 s, 501C for 30 s, 721C for 1 min with initial denaturing at 951C for 5 min, and a final extension at 721C for 5 min.…”

“…The alignment of the deduced amino-acid sequences is shown in Figure 3. These regions of sequences included the three conserved domains (C2, C3 and RC4) in addition to the C1 and C5 domains used as the PCR primers and a region that is a hyper-variable region (RHV), previously reported in the S-RNases of Rosaceae (Ushijima et al, 1998). The intron position was always in the RHV region, and the RHV was highly variable in the flowering cherry (Figure 3).…”

“…The numbers of synonymous (Ks) and nonsynonymous (Ka) substitutions per site were calculated from pairwise comparisons within and between the seven sequences of flowering cherry, and the nine sequences from Prunus cultivars, S 1 , S 2 , S 3 , S 4 , S 5 and S 6 of P. avium (Sonneveld et al, 2001;Wiersma et al, 2001) and S b , S c and S d of P. dulcis (Ushijima et al, 1998). Table 2 shows the results.…”