ISHS Acta Horticulturae 587: International Symposium on Asian Pears, Commemorating the 100th Anniversary of Nijisseiki Pear
S-RNASE BASED PCR-RFLP SYSTEM FOR S-GENOTYPE ASSIGNMENT IN JAPANESE PEAR |
| Authors: | C. Castillo, T. Nakanishi, T. Ishimizu, T. Takasaki, S. Norioka, T. Saito |
| Keywords: | Pyrus pyrifolia, self-incompatibility, S1 to S9-alleles, endonucleases, specificity |
| Abstract:
The PCR-RFLP (S1 to S7) is a high reliable system for S-genotype identification of Japanese pear cultivars carrying S1 to S7 allele. It was evidenced to the analysis of two undetermined S-genotype cultivars ‘Shinchuu’ and ‘Wasekouzou’ assigned as S3S5 and S2S5, respectively, and then corroborated by our pollination tests. The PCR-RFLP system (S1 to S7), however, was not adapted to other seven cultivars. An S-RNase fragment of unexpected size was amplified from ‘Ichiharawase’, ‘Meigetsu’ and ‘Heiwa’, whereas, only one S-RNase fragment from ‘Shinkou’, ‘Shinsei’, ‘Amanogawa’ and ‘Niitaka’. Using RT-PCR S8-RNase and S9-RNase cDNA fragments were cloned from pistils of ‘Ichiharawase’ and ‘Heiwa’, and from those of ‘Shinkou’ and ‘Shinsei’, respectively. These two S-RNases possessed all the characteristic features of Rosaceae S-RNases, including one quite different hypervariable region (HV) compared to S1 to S7-RNase. A reverse primer ‘anti-(I/T)IWPNV’ was designed to achieve the amplification of S1 to S9-RNase fragments from genomic DNA. Based on the intron sequences of S8- and S9-RNase, we selected NruI and BstBI as their allele specific restriction endonucleases. Finally, we developed a new PCR-RFLP system (S1 to S9) for discriminating S-genotypes of Japanese pear cultivars carrying S1 to S9 allele. ‘Ichiharawase’, ‘Meigetsu’ and ‘Heiwa’, were assigned as S1S8, S1S8, S4S8, and those of ‘Shinkou’, ‘Shinsei’, ‘Amanogawa’ and ‘Niitaka’ as S4S9, S4S9, S1S9 and S3S9, respectively. | |
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