[논문]Development of EST-SSR markers for the Korean endemic species Chrysosplenium aureobracteatum (Saxifragaceae)

SHIN, Jae-Seo; KIM, Bo-Yun; KIM, Yong-In; LEE, Jung-Hoon; KIM, Young-Dong

doi:10.11110/kjpt.2020.50.1.22

Development of EST-SSR markers for the Korean endemic species Chrysosplenium aureobracteatum (Saxifragaceae) 원문보기

식물분류학회지 = Korean journal of plant taxonomy, v.50 no.1, 2020년, pp.22 - 26

SHIN, Jae-Seo (Department of Life Science, Multidisciplinary Genome Institute, Hallym University) , KIM, Bo-Yun (National Institute of Biological Resources) , KIM, Yong-In (Multidisciplinary Genome Institute, Hallym University) , LEE, Jung-Hoon (Multidisciplinary Genome Institute, Hallym University) , KIM, Young-Dong (Department of Life Science, Multidisciplinary Genome Institute, Hallym University)

Abstract ▼ AI-Helper

Chrysosplenium aureobracteatum Y. I. Kim & Y. D. Kim (Saxifragaceae) is a recently described endemic species growing in the central part of the Korean peninsula. It requires constant monitoring for conservation due to its limited distributions. There is also a need for molecular markers for proper assessments of the genetic differentiation of C. aureobracteatum from species morphologically similar to it. In this study, we developed microsatellite markers that can be used to evaluate the genetic diversity of this species, representing fundamental data with which to conserve the natural populations of the species. A total of 17 expressed sequence tag-simple sequence repeat (EST-SSR) markers were developed by the Illumina pair-end sequencing of the transcriptomes of C. aureobracteatum. These markers were successfully applied to populations of C. aureobracteatum and to its most closely related species, C. barbatum, revealing high polymorphism in both species. The EST-SSR markers developed in this study were proven to be useful not only to monitor the population genetic structure of C. aureobracteatum for conservation purposes but also to study the genetic delimitation of the species from species closely related to it.

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문제 정의

, 2019). In this study, EST-SSR markers based on the transcriptome data of C. aureobracteatum were developed to evaluate the genetic diversity of this newly described species.

제안 방법

Alleles were identified with an ABI 3730XL (automated sequencer) equipped with the GeneScan 500 LIZ Size Standard (Applied Biosystems). Genotyping was performed using GeneMapper 3.7 (Applied Biosystems), and peaks were scored manually by visual inspections. The PCR products with sizes deviating from the expected size ranges (as estimated by the Primer3 program) were eliminated from the genotyping process.
PCR for the selected loci was conducted using a mixture with a total volume of 25 µL consisting of 10× Ex Taq buffer at 2.5 µL (TaKaRa Bio Inc., Otsu, Japan), 2.5 mM dNTP at 2 µL, 10 pmol forward and reverse primer labeled with fluorescent dye (FAM, HEX, NED) each at 0.5 µL, template DNA (5–10 ng) at 2 µL, Ex Taq DNA polymerase at 0.12 µL and SIGMA free water using a GeneAmp PCR System 9700 thermocycler (Applied Biosystems Inc., San Diego, CA, USA).

대상 데이터

barbatum for the marker evaluation and cross-specific amplification test. The voucher specimens were deposited in the HHU (Herbarium of Hallym University) in Korea (Table 1).

성능/효과

As a result of applying the 17 EST-SSR markers on a population (with 20 individuals) of C. aureobracteatum, polymorphisms were detected from 11 markers, and a total of 33 alleles were identified (2 to 7 alleles depending on the polymorphic markers). The observed heterozygosity (H_o) and the expected heterozygosity (H_e) ranged from 0.
From the identified SSR motifs, 96 candidate markers were selected in consideration of the number of repetitions, the coverage depth, the annealing temperature, and the PCR product size. Excluding nonspecific bands with sizes deviating from the expected sizes, a total of 17 EST-SSR loci (markers) showing polymorphisms for Chrysosplenium aureobracteatum and C. barbatum were finally developed (Table 3).
It is important to note that the six monomorphic markers in C. aureobracteatum (CA_005, CA_006, CA_010, CA_052, CA_066, CA_104) were highly polymorphic in C. barbatum. In contrast, the monomorphic markers for C.
The candidate EST-SSR markers developed from C. aureobracteatum were applied to its most closely related species, C. barbatum, to confirm and obtain markers exhibiting polymorphism in both species. We examined 20 individuals of Chrysosplenium aureobracteatum and C.
A total of 15,547 SSR motifs were identified from the trimmed data. The identified SSR motifs consisted of 40% mono-nucleotides, 36% dinucleotides, 23% tri-nucleotides, and 1% tetra-, penta-, and hexa-nucleotides (Table 2).
aureobracteatum (Table 4). These results suggest that the EST-SSR markers developed for C. aureobracteatum are useful not only to examine the population genetic structure but also for species delimitation among related taxa.

참고문헌 (13)

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원문보기 상세보기
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