[논문]Isolation and characterization of EST-SSR markers for Astilboides tabularis (Saxifragaceae), endangered species in Korea

JUNG, Eui-Kwon; KANG, Dae-Hyun; YOO, Ki-Oug; KWAK, Myounghai; KIM, Young-Dong; KIM, Bo-Yun

doi:10.11110/kjpt.2018.48.3.195

Isolation and characterization of EST-SSR markers for Astilboides tabularis (Saxifragaceae), endangered species in Korea 원문보기

식물분류학회지 = Korean journal of plant taxonomy, v.48 no.3, 2018년, pp.195 - 200

JUNG, Eui-Kwon (Department of Life Science, Hallym University) , KANG, Dae-Hyun (Multidisciplinary Genome Institute, Hallym University) , YOO, Ki-Oug (Department of Biological Sciences, Kangwon National University) , KWAK, Myounghai (Plant Resources Division, National Institute of Biological Resources) , KIM, Young-Dong (Department of Life Science, Hallym University) , KIM, Bo-Yun (Multidisciplinary Genome Institute, Hallym University)

Abstract ▼ AI-Helper

Genetic assessments of rare and endangered species are among the first steps necessary to establish the proper management of natural populations. Transcriptome-derived single-sequence repeat markers were developed for the Korean endangered species Astilboides tabularis (Saxifragaceae) to assess its genetic diversity. A total of 96 candidate microsatellite loci were isolated based on transcriptome data using Illumina pair end sequencing. Of these, 26 were polymorphic, with one to five alleles per locus in 60 individuals from three populations of A. tabularis. The observed and expected heterozygosity per locus ranged from 0.000 to 0.950 and from 0.000 to 0.741, respectively. These polymorphic transcriptome-derived simple sequence repeat markers would be invaluable for future studies of population genetics and for ecological conservation of the endangered species A. tabularis.

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문제 정의

This study describes the first assembly and characterization of the leaf transcriptome of A. tabularis using the Illumina paired-end sequencing method. Twenty-six polymorphic markers were successfully amplified, revealing polymorphisms in A.

제안 방법

All PCRs were performed in a GeneAmp PCR System 9700 thermocycler (Applied Biosystems, Carlsbad, CA, USA) using the following program: initial denaturation at 98°C for 5 min followed by 30 cycles of denaturation at 95°C for 1 min, annealing at annealing at 59°C for 1 min with an extension at 72°C for 1.5 min, and a final extension step at 72°C for 10 min.
Finally, we identified 26 polymorphicmarkers based on genotyping data, and functional annotations for these markers were performed on a subset of ESTs with BLASTX scores (E-value < 1 × 10-10) using the gene ontology database (Table 2).
Fluorescently labeled (HEX, FAM) PCR products were analyzed by an automated sequencer (ABI 3730XL) 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. Finally, we identified 26 polymorphicmarkers based on genotyping data, and functional annotations for these markers were performed on a subset of ESTs with BLASTX scores (E-value < 1 × 10^-10) using the gene ontology database (Table 2).

대상 데이터

For the construction of the RNA library, the total RNA was extracted from leaves of individuals representing A. tabularis from three populations (Voucher No. NIBRVP0000655607) (Table 1). RNA was extracted using RNeasy kits, version 2.
2 (Illumina, San Diego, CA, USA) following the manufacturer’s instructions, and was subsequently used for TruSeq cDNA library preparation and high-throughput Illumina HiSeq 100 bp paired-end de novo transcriptome sequencing. The analysis results reads were obtained and assembled into 102,884 unigenes with 7,476,378,742 read numbers. The de novo transcriptome assembly of these reads was performed using the short read assembling program Trinity r20140717 (Haas et al.
To avoid collecting clones, we specified a distance of at least 2 m among individuals within each population. The voucher specimens were deposited in the National Institute of Biological Resources Herbarium (KB) and in the Herbarium of Hallym University (HHU) in Korea (Table 1). The locations of the sites have been withheld to prevent illegal collection.
Voucher information for the Astilboides tabularis populations sampled in this study. Vouchers were deposited in the Herbarium of the National Institute of Biological Resources (KB) and in the Herbarium of Hallym University (HHU), Republic of Korea. To prevent illegal collection, we did not disclose the exact locations of the sites.

후속연구

tabularis. This work can serve as a basis for further studies on the genetic diversity and structure of A. tabularis and can provide essential information for devising effective conservation strategies.

참고문헌 (16)

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