cDNA cloning of nine S alleles and establishment of a PCR-RFLP system for genotyping European pear cultivars

Abstract

Nine full-length cDNAs of S ribonucleases (S-RNases) were cloned from stylar RNA of European pear cultivars by RT-PCR and 3' and 5' RACE. Comparison of the nucleotide sequences between the nine S-RNases cloned and 13 putative S alleles previously amplified by genomic PCRs revealed that seven corresponded to Sa, Sb, Sd, Se, Sh, Sk and Sl alleles, and the other two were new S alleles (designated as Sq and Sr alleles). Genomic PCR with a set of a8FTQQYQa9 and a8EP-anti-IIWPNVa9 primers was used to amplify nine S alleles; 1,414 bp (Sl), ca. 1.3 kb (Sk and Sq), 998 bp (Se), 440 bp (Sb) and ca. 350 bp (Sa, Sd, Sh and Sr). Among these, S alleles of similar size were discriminated by digestion with BaeI, BglII, BssHII, HindIII, EcoO109I and SphI. The PCR amplification of S allele following digestion with the restriction enzymes provided a PCR-RFLP system for rapid S-genotyping European pear cultivars harboring nine S alleles. The PCR-RFLP system assigned a total of 63 European pear cultivars to 25 genotypes. Among these, 14 genotypes were shared by two or more cultivars, which were cross-incompatible. These results suggested that the genes cloned represented the S-RNases from European pear, and that there were many cross-incompatible combinations among European pear varieties.

Genomic characterization of self-incompatibility ribonucleases (S-RNases) in European pear cultivars and development of PCR detection for 20 alleles

“…Since the first report characterizing S-RNase sequences in this species (Zuccherelli et al 2002), 19 alleles have been published (Zuccherelli et al 2002;Zisovich et al 2004;Takasaki et al 2006;Moriya et al 2007;Sanzol and Robbins 2008). Takasaki et al (2006) and Moriya et al (2007) provided full-length cDNA and genomic sequences for 17 S-RNases, confirming that they have all the structural features found in the Maloideae S-RNase. This gene has three main characteristics that make it suitable for molecular S-genotyping: First, five conserved consensus regions (C1-C3, RC4, and C5) and the conserved sequence encoding "IIWPNV" located just downstream of the hyper-variable region (HVR), can be used to simultaneously amplify a large number of alleles using consensus PCR.…”

“…2a). Moreover, the restriction site utilized for the recognition of S r (Takasaki et al 2006) does not exist in S 9 (Fig. 2a).…”

“…Takasaki and co-workers developed a cleaved-amplified polymorphic sequences (CAPS) marker system to discriminate between 17 alleles in European pear (Takasaki et al 2006;Moriya et al 2007). This approach combines consensus PCR (using primers based on the C1 conserved region and the hexapeptide region "IIWPNV") and restriction analysis to type amplified products from consensus PCR.…”

S-genotype Assignments of Local Cultivars in Japanese Pear ‘Senryo’, ‘Kuroki’, and ‘Hogyoku’

“…The other products, which were different in size from the S 1 -to S 9 -RNases in Japanese pear, were cloned into pCR 2.1 vector and sequenced. Homology searching by BLASTN showed that PCR products of 998 bp from 'Senryo', 536 bp from 'Kuroki', and 309 bp from 'Hogyoku' were 100% homologous at the DNA level to the S e -RNase in European pear (Takasaki et al, 2006), the S 12 -RNase in Chinese pear (accession no. EU117115), and S 30 -RNase in Chinese pear (accession no.…”

“…In European pear, 17 S-alleles are found from 95 cultivars (Moriya et al, 2007;Takasaki et al, 2006). In Chinese pear, 32 S-RNase alleles have been cloned from 38 cultivars, and deposited in the DDBJ/EMBL/GenBank database (Tan et al, unpublished; Zhang et al, unpublished).…”