Vol. 75 No: 5

Title:
Determining the S-genotypes of several sweet cherry cultivars based on PCR-RFLP analysis

Authors:
HISAYO YAMANEA, RYUTARO TAO, HIDEKI MURAYAMA and AKIRA SUGIURA

pp: 562-567

Abstract:
Based on the cDNA sequences encoding sweet cherry self-incompatibility associated ribonucleases (S-RNases), a PCR-based S-allele typing system for sweet cherry cultivars has been recently developed. Using this technique, we determined S-genotypes of the three newly released Japanese cvs Kouka-Nishiki, Beni-Sayaka and Beni-Shuho and one British cv Merton Glory that was classified as a Universal Donor, which is able to be used as a pollen donor for all cultivars in pollen incompatibility groups I to XIII. Furthermore, we also determined the partial sequences of the S-RNase genes of 'Rainier' (S¹S⁴)' and 'Sato-Nishiki (S³S⁶)', which leads to the development of a more reliable S- allele identification method of PCR-RFLP for sweet cherry cultivars. Total DNA isolated from leaves of the four cultivars along with those from ten cultivars with known S-genotypes were PCR amplified with two sets of primers that were designed from DNA sequences encoding the signal peptide (Pru-T²) and two conserved domains (Pru-C₂ and Pru-C⁴R) of sweet cherry S-RNases. By comparing the size of PCR products on agarose gel, the S-genotypes of 'Kouka-Nishiki', 'Beni-Sayaka', 'Beni-Shuho' and 'Merton Glory' were suggested to be S¹S³, S¹S⁶,S⁴S⁶, and S⁴S⁶,respectively. Two of these three S-genotypes (S¹S⁶ and S⁴S⁶) were found for the first time. DNA sequencing of PCR products from S-alleles of 'Rainier' and 'Sato-Nishiki' revealed that Ban II, Nru I, Apa LI and Ava I sites, respectively, were unique in the S¹-, S³-, S⁴- and S⁶- sequences flanked by Pru-T² and Pru-C⁴R primers. RFLP analysis of the PCR products using these enzymes confirmed that S¹-, S³-, S⁴- and S⁶-alleles of the four cultivars contained the respective restriction enzyme recognition sites.

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