Genetic Diversity in Chestnut Germplasm Assessed by Nuclear and Chloroplast Simple Sequence Repeat Markers

To assess the genetic diversity in chestnut germplasm (Castanea spp.), we used 15 nuclear simple sequence repeats markers (ncSSR markers) from Castanea mollissima (Inoue et al., 2009) and 10 chloroplast simple sequence repeats markers (cpSSR markers) from C. sativa (Sebastiani et al., 2003; Inoue et al., 2007).  80 genotypes from 9 chestnut species containing C. sativa, C. mollisshima, C. crenata, C. seguinii, C. henryi, C. dentata, C. alnifolia, C. ozarkensis and C. pumila in chestnut germplasm were used in this research.  Total DNA were isolated from the mature leaves of each genotype and used for SSR polymerase chain reaction (PCR) with ncSSR and cpSSR primers.  The PCR products were separated using an ABI3130xl sequencer with the Genemapper software and a GS500LIZ ladder.  Pairwise genetic distances were determined on the basis of the proportion of shared alleles for all the genotypes and the dendrogram were generated using the unweighted pair group method with arithmetic mean (UPGMA) cluster analysis method.  By the 15 ncSSR primers, 22-129 putative alleles per locus were detected and 3-60 species specific alleles were identified.  The 9 species used in this study were identified to different cluster by using the allelic information obtained from the 15 ncSSR loci.  C. crenata, C. seguinii, and C. dentata were belonged to a single clade in a dendrogram.  Another 6 Castanea species also belonged to another single clade.  On the other hand, by the 10 cpSSR primers, 2-7 putative alleles per locus were detected.  All the genotypes were classified into 16 kinds of haplo type by the genotypes of cpSSR loci.  The 9 species used in this study were also identified to different haplo type by using the allelic information obtained from the 10 cpSSR loci.  The 8 Castanea species without C. crenata were belonged to a single clade in a dendrogram.  The difference in a result of classification between ncSSR and cpSSR genotypes is discussed in this presentation.