S-RNASE BASED PCR-RFLP SYSTEM FOR S-GENOTYPE ASSIGNMENT IN JAPANESE PEAR

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.

International Symposium on Asian Pears, Commemorating the 100th Anniversary of Nijisseiki Pear

C. Castillo, T. Nakanishi, T. Ishimizu, T. Takasaki, S. Norioka, T. Saito

Pyrus pyrifolia, self-incompatibility, S₁ to S9-alleles, endonucleases, specificity

https://doi.org/10.17660/ActaHortic.2002.587.59