To develop and characterize valid simple sequence repeat (SSR) markers in chestnut (Castanea spp.), we used a selective hybridization method to perform SSR sequence enrichment in the genomic library of chinese chestnut (Castanea mollissima). From 47 sequences of the enriched library, we designed 24 primer pairs for SSR polymerase chain reaction (PCR). SSR PCR was performed for 23 chinese chestnut cultivars. Among the 24 primers, 22 primers amplified their respective SSR loci; 21 primer pairs yielded polymorphic fragments across the cultivars. Among these 21 primer pairs, 17 primer pairs amplified single SSR loci with polymorphisms. Multilocus amplification patterns were observed in the other four primers. For the corresponding 17 SSR loci, the number of alleles per locus was two to 13, and the average number of alleles was 7.19. The observed heterozygosity (number of genotypes of the heterozygote/total number of genotypes scored at that locus) ranged from 0.00 to 0.87 (average = 0.68), and the expected heterozygosity ranged from 0.00 to 0.87 (average = 0.68). One of the SSR loci—ICMA014—was a unique locus, because the primer pair for this locus did not amplify any fragment in any of the cultivars, except in Qianchi, which was used to construct the SSR-enriched genomic library. Cross-species amplifications using the 17 primer pairs were also successful in the other species of genus Castanea. A dendrogram depicting the relationships among the genotypes on the basis of genetic distances was generated using the unweighted pair group method with arithmetic mean cluster analysis. The genotypes were clearly separated into three large groups, which reflected the three cultivated species, namely, C. mollissima, C. crenata, and C. sativa.
Effective DNA fragmentation technique for simple sequence repeat detection with a microsatellite-enriched library and high-throughput sequencing
