Isolation and characterization of novel microsatellites for Abies koreana and A. nephrolepis (Pinaceae)

Abstract

ABSTRACT.Abies koreana is an endemic and rare species from Korea and is classified as endangered by the International Union for Conservation of Nature. Although the genetic diversity assessment for current population of A. koreana needs to be performed urgently, no microsatellite markers have been developed for this species. In the present study, we developed 22 novel polymorphic microsatellite loci and the characteristics of these loci were determined in A. koreana as well as in Abies nephrolepis, the most closely related species, and these loci were compared with previously reported microsatellite markers developed for the Abies genus. Genomic sequence (161 Mbp; 325,776 reads) was obtained from one individual of A. koreana using Roche 454 GS-FLX Titanium sequencing and 19,258 repeat motifs were identified from it. A total of 288 primer pairs with high copy numbers of direpeat motifs were evaluated for amplification in A. koreana and A. nephrolepis. A total of 71 primer pairs successfully amplified fragments, of which 22 showed polymorphisms in A. koreana and A. nephrolepis. The average expected diversity was 0.767 and 0.717 in A. koreana and A. nephrolepis, respectively; these heterozygosity levels were moderate compared to the previously reported microsatellite loci from Abies species. This is the first set of microsatellite markers developed for A. koreana as well as A. nephrolepis and further population genetic studies of both species and genetic delimitation can be carried out for the species conservation and management.

Spatial genetic structure and seed quality of a southernmost Abies nephrolepis population

“…For genotyping, nine microsatellite markers, AK87, AK171, AK173, AK176, AK246, AK247, AK252 57 , As13, and As20 58 (Supplementary Table S4 ), were selected based on their polymorphism and amplification pattern. PCR amplifications were performed with a total volume of 12 μl containing 20 ng DNA, 1 X A-Star Taq Reaction Buffer, 2.5 mM MgCl 2 , 0.2 mM dNTP Mix, 0.5 U A-Star Taq DNA polymerase (BioFact, Daejeon, Korea), 0.04 μM HEX/FAM M13(-19) primer, and 0.2 μM of each reverse and forward primer.…”

Selection of Abies nephrolepis Materials for Restoration of Genetic Diversity in Mt. Gariwangsan Degraded Area

“…Nuclear simple-sequence repeat (nSSR) markers are located in the nuclear genome and are commonly used to analyze genetic variation in populations due to their co-dominant characteristics [32][33][34]. For this study, ten markers (AK087, AK240, AK246, AK252, As13, As20, NFF7, NFH15, Aat04, and Aat12) that were stably amplified in A. nephrolepis were selected by referring to A. koreana [35], A. sachalinensis [36], A. nordmanniana [37], and A. alba [38]. The results were checked for genotyping errors using a Micro-Checker program [39].…”

Spatial Genetic Structure and Seed Quality of a Southernmost Abies nephrolepis Population

“…For genotyping, nine microsatellite markers, AK87, AK171, AK173, AK176, AK246, AK247, AK252 (Hong et al 2016), As13, and As20 (Lian et al 2007) (Table S1), were selected based on their polymorphism and ampli cation pattern. PCR ampli cations were performed with a total volume of 12 µL containing 20 ng DNA, 1 X A-Star Taq Reaction Buffer, 2.5 mM MgCl 2 , 0.2 mM dNTP Mix, 0.5 U A-Star Taq DNA polymerase (BioFact, Daejeon, Korea), 0.04 µM HEX/FAM M13(-19) primer, and 0.2 µM of each reverse and forward primer.…”