Development and characterization of new microsatellites for walnut (Juglans regia).

The expressed sequence tag (EST) database represents a potentially valuable resource for the development of simple sequence repeat (SSR) markers for use in evolutionary studies. EST-SSRs reveal polymorphisms not only within the source taxon, but in related taxa as well. In this paper, we describe a case study in which the publicly available walnut (Juglans regia) EST database was used to develop SSR markers for use in the genetic analysis of the widespread Juglans nigra and Carya cathayensis and an endangered species Annamocarya sinensis. A total of 7262 unigenes, including 1911 contigs and 5351 singletons, were obtained from 13,559 ESTs retrieved from the NCBI database. The 7262 unigenes were further reduced to 706 EST-SSR sequences containing 805 SSR loci. Then, 309 EST-SSR primers were randomly designed, and 77 were identified with five high across-species transferability cross-species: namely, J. regia, J. nigra, C. cathayensis, Carya dabieshanensis, and A. sinensis. Thirteen highly polymorphic EST-SSRs were further used for genetic analyses in these above five species.

Conservation genetics of Annamocarya sinensis (Dode) Leroy, an endangered endemic species.

The endangered perennial plant Annamocarya sinensis (Dode) Leroy is a tertiary relict tree restricted to southeastern China and northern Vietnam. To explore endangerment mechanisms, develop protection strategies, and guide reintroduction efforts for this species, we investigated genetic diversity and population structure by surveying 70 individuals from three distinct populations using 12 polymorphic microsatellite markers. We found high genetic diversity for A. sinensis as indicated by high allelic diversity (allelic number = 4.667 ± 0.436, effective number of alleles = 2.913 ± 0.249), excess heterozygosity (observed heterozygosity = 0.586 ± 0.039, expected heterozygosity = 0.582 ± 0.029), and low fixation index (-0.028 ± 0.057). Our research revealed low genetic differentiation (FST = 0.066 ± 0.011) and no correlation between genetic distance and geographic distance. Analysis of molecular variance attributed 87% of the variance to differences within the population, whereas 13% was distributed among populations. The protection strategy should aim to protect as many populations as possible. Promoting sexual reproduction among various genotypes and establishing an outcrossing program are advisable for A. sinensis.