| Walnut is one of the world’s four major nuts, with rich nutritional value, and is an important nut crop in China. Annamocarya sinensis (Dode) Leroy is a tertiary relict tree, and is listed as second class state protection in Chinese Red Date Book. The expressed sequence tags in public database represent a potential valuable resource for the development of simple sequence repeats. EST-SSR primers reveal polymorphisms not only within a genus but also among genus. In our study, we newly developed EST-SSRs from walnut ERTs, and tested the primers’universality and polymorphism among J. regia, J. nigra, C. cathayensis, C. dabieshanensis and A. sinensis. The main results are as follows:(1) A total of7,262unigenes, including1,991contigs and5,351singletons, were obtained from the13,559walnut ESTs. After screening,805SSR loci were got from706EST-SSR unigenes.309EST-SSRs were initially developed, and the number (percentage) of effective PCR amplification in J. regia, J. nigra, C. cathayensis, C. dabieshanensis and A. sinensis were191(61.8%),233(75.4%),152(49.2%),130(42.1%),120(38.8%), respectively. And77EST-SSRs performed well in all five species. After polymorphisms analysis, the number (percentage) of polymorphic primers in J. regia, J. nigra, C. cathayensis, C. dabieshanensis and A. sinensis were33(42.9%),32(41.6%),10(13.0%),12(15.6%),21(27.3%), respectively. And13polymorphic primers were further used for genetic analysis in the above five species.(2) The genetic diversity and population structure of A. sinensis were investigated by70individuals from three populations using12polymorphic EST-SSRs. The results revealed high genetic diversity and low genetic differentiation: mean observed number of allele=4.667±0.436, effective number of alleles=2.913±0.249, observed heterozygosity=0.586±0.039, expected heterozygosity0.582±0.029, fixation index=-0.028±0.057, genetic differentiation=0.066±0.011, and87%of the variance within populations, and13%among populations. So the protection strategy should aim at protect as many individuals and populations as possible. It is also advisable for A. sinensis that promoting sexual production among various genotypes and establishing an outcrossing program. |
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