Abstract
Pinus koraiensis has significant economic and ecological value in Northeast China. However, due to the lack of suitable molecular markers, only a few available microsatellite markers were developed for further population genetics studies. In this study, for the first time we developed expressed sequence tag–simple sequence repeat (EST-SSR) markers from the cold-stressed transcriptome of P. koraiensis using Illumina Sequencing. We identified a total of 7,235 EST-SSRs from 97,376 sequences, and we tested their transferability among seven related Pinus species. The results showed that trinucleotides were the most abundant type of repeat (1287, 18.74%) excluding mononucleotides, followed by dinucleotides (1284, 18.7%) and tetranucleotides (72, 1.05%). The most dominant dinucleotides and trinucleotide repeat motifs were AT/AT (535, 7.79%) and AAT/ATT (103, 1.5%). The observed heterozygosity (Ho) and expected heterozygosity (He) ranged from 0.002 to 0.986 and 0.017 to 0.743, respectively, and the polymorphism information content (PIC) values and number of alleles (Na) varied from 0.029 to 0.794 and 2 to 23, respectively. A total of 8 natural P. koraiensis populations were divided into two main genetic clusters. Furthermore, nine of twenty polymorphic primer pairs were successfully amplified in seven Pinus species, and at least 80% of the successful P. koraiensis EST-SSR primers could be amplified in more than four species (16, 80%). Combined results for the development of EST-SSR markers in P. koraiensis and transferability among related species would contribute to improved studies on the genetic diversity and population structure in P. koraiensis and phylogenetic relationships among Pinus species. They would also provide a significant source for quantitative trait locus analysis.
Highlights
Pinus koraiensis, an evergreen conifer species, is an important native and precious forest tree in Northeastern China, as well as in the far eastern region of Russia and the Korean peninsula [1,2].P. koraiensis has great ecological and economic value, and plays a significant role in ecological environmental construction and economic development in China
The main reasons for these cases were as follows—(1) the transferability of expressed sequence tag–simple sequence repeat (EST-simple sequence repeats (SSRs)) primers is related to the genetic relationship between two species; (2) Pinus sibirica, Pinus pumila, and Pinus wallichiana belong to the five-needle group and are widely assumed to have a closer genetic relationship than two- and three-needle species. These results revealed high genome homology among seven Pinus species, and nine of 20 newly developed EST-SSRs from cold-stressed transcriptome of P. koraiensis could be used as universal markers in the Pinus species for further population genetics studies in related species and the identification of functional genes with significant economic traits
These results indicated that most molecular variance was within populations, which was similar to the results for analyses of molecular variance (AMOVA) analysis in the P. koraiensis (97.65% within populations), based on the nine EST-SSR markers [12]
Summary
An evergreen conifer species, is an important native and precious forest tree in Northeastern China, as well as in the far eastern region of Russia and the Korean peninsula [1,2]. P. koraiensis has great ecological and economic value, and plays a significant role in ecological environmental construction and economic development in China. P. koraiensis has been extensively used as a major source of excellent timber, natural remedies and edible pine nuts, due to its excellent wood properties and nutritional value [3,4,5]. Pine nuts have a high nutritional value, and they include a variety of essential nutrients, such as vitamins, proteins, and unsaturated fatty acids, among others [6,7]. P. koraiensis is considered a main ecological and pioneer tree species in
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