cDNA cloning of nine S alleles and establishment of a PCR-RFLP system for genotyping European pear cultivars.
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.
Takasaki
T
T
Faculty of Agriculture, Kobe University, Kobe 657-8501, Japan. taka@kobe-u.ac.jp
Moriya
Y
Y
Okada
K
K
Yamamoto
K
K
Iwanami
H
H
Bessho
H
H
Nakanishi
T
T
D000483:Alleles; D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D017124:Conserved Sequence; D018076:DNA, Complementary / Q000235:genetics*; D018744:DNA, Plant / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification; D005060:Europe; D005838:Genotype; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D031989:Pyrus / Q000235:genetics*; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D018744:DNA, Plant