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Development of Polymorphic SSR Markers from Pinus densiflora (Pinaceae) Natural Population in Korea
Plant Breeding and Biotechnology 2019;7:67-71
Published online March 30, 2019
© 2019 Korean Society of Breeding Science.

Hee Chung1,†, Jaebok Lee1,†, Jinsu Gil1, Yurry Um2, Ji Hyun Kim1, Min Yeong Hwang1, Ho Bang Kim3, Chang Pyo Hong4, Shin Gi Park4, Donghwan Shim5, Yi Lee1,*

1Department of Industrial Plant Science & Technology, Chungbuk National University, Cheongju 28644, Korea, 2Forest Medicinal Resources Research Center, National Institute of Forest Science, Yeongju 36040, Korea, 3Life Sciences Research Institute, Biomedic Co., Ltd., Bucheon 14548, Korea, 4Theragen Etex Bio Institute, Theragen Etex, Suwon 16229, Korea, 5Department of Forest Genetic Resources, National Institute of Forest Science, Suwon 16631, Korea
Corresponding author: *Yi Lee, leeyi22@hanmail.net, Tel: +82-43-261-3373, Fax: +82-43-271-0413
Received February 15, 2019; Revised February 17, 2019; Accepted February 17, 2019.
This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract

Simple sequence repeat (SSR) markers were developed from Pinus densiflora, a species native to Asia, to investigate its genetic diversity and population structure in order to provide information for the management and breeding of this species. Using next-generation sequencing, a total of 1,008 putatively polymorphic SSR primer sets were designed. Seventeen polymorphic SSR markers in 121 individuals belonging to four natural populations of P. densiflora were identified and characterized, with three to seventeen alleles per locus. The expected heterozygosity ranged from 0.1844 to 0.8731 in four populations, and the average of the PIC values ranged from 0.2789 to 0.8488. Cross amplification of these markers was performed among the related species P. rigida, P. koraiensis, P. parviflora, and P. bungeana. The developed novel SSR markers are promising tools for studying the genetic diversity or population structure of P. densiflora and its related species.

Keywords : Genetic diversity, Next-generation sequencing, Pinaceae, Pinus densiflora, Simple sequence repeat
INTRODUCTION

Pinus densiflora is widely distributed in East Asian countries including Korea, Japan, China, and Russia. Especially in the Korean Peninsula, the distribution extends from Jeju Island (33° 29′ N), the southernmost island in Korea, to the northern part of Baekdu Mt. in North Korea (41° 59′ N) (Kim et al. 1981). Although members of the Pinaceae family are among the most important plants ecologically and economically (Liu et al. 2015), there were only a few reports of genetic relationships or population diversity among pine trees because of their very large genome. Molecular tools are necessary to understand the distribution of pine trees, to identify genetic resources, and to improve systematic breeding of pine trees. Several types of molecular markers have been reported from genus Pinus. Kim et al. (1995) reported the investigation of the genetic structure and the degree of genetic variation in natural P. densiflora populations using random amplified polymorphic DNA (RAPD); they classified subjects into two groups dependent on geographic patterning. Ahn et al. (2015) estimated the genetic diversity and differentiation of thirteen P. densiflora populations in South Korea using expressed sequence tag polymorphism (ESTP) markers. Hong et al. (2014) defined phylogenetic relationships within 17 taxa of Pinus in Korea using two gene loci in chloroplast DNA; they classified them into diploxylon and haploxylon.

Simple sequence repeats (SSRs) are excellent molecular markers for many eukaryotes, and are widely applied to examine genetic diversity, molecular-assisted breeding, and germplasm resources for identification in a variety of species (Neale et al. 2017). Lian et al. (2000) developed SSR markers from P. densiflora to investigate reproductive behavior and tested for amplification in 12 Pinus species. Watanabe et al. (2006) developed seven SSR loci from P. densiflora using a dual PCR technique and they investigated Mendelian inheritance in pine wood nematode-resistant trees using the developed markers. In related species, EST-SSR markers from P. sylvestris (Fang et al. 2014) and SSR markers from P. armandii (Dong et al. 2016) and P. kesiya (Cai et al. 2017) were developed. However, there is still limited genomic sequence information for the study of Pinus species. In this study, we developed novel SSR markers for P. densiflora by using next-generation sequencing to analyze the genetic diversity and population structure of this species.

MATERIALS AND METHODS

Needle samples of 121 individuals from four P. densiflora natural populations in different areas of South Korea and 20 individuals from related species (P. rigida, P. koraiensis, P. parviflora, and P. bungeana) were collected (Supplementary Table S1). All needle samples were ground into a fine powder using liquid nitrogen, and total genomic DNA was isolated from each sample using Biomedic ® Plant gDNA Extraction Kit (Biomedic, Bucheon, Gyeonggi, Korea). Paired-end libraries were constructed on two samples and sequenced by a customer sequencing service (Theragen Etex, Suwon, Gyeonggi, Korea) using Illumina HiSeq 2500 platform (Illumina, San Diego, California, USA). All the produced reads from the two representative plants were trimmed and assembled using CLC Genomics Workbench (CLC bio, Aarhus, Denmark) version 7.5.

Genomic SSR regions for P. densiflora were detected using the SSRIT program (Temnykh et al. 2001), and 1,008 pairs of primer sequences were successfully designed for the putatively polymorphic loci. A total of 192 primer sets with di, tri, and tetra-nucleotide SSR motifs were randomly selected to screen using the two representative individuals.

The reaction mixtures for SSR amplification included 50 ng of DNA template, 1 × HS™ Taq DNA polymerase buffer, 1.5 mM MgCl2, 0.2 mM of each dNTP, 0.2 mM of each primer, and 1.25 units HS™ Taq DNA polymerase (Dongsheng Biotech, Guangzhou, Guangdong, China). The conditions for PCR amplification were as follows: 5 minutes for initial denaturation at 95°C, 35 cycles of 30 seconds at 94°C, 30 seconds at 56°C, 30 seconds at 72°C, concluding with 1 cycle of 10 minutes at 72°C. The PCR products were inspected with the Fragment Analyzer Automated CE system (Advanced Analytical Technologies [AATI], Ames, Iowa, USA) with the Quant-iT PicoGreen dsDNA reagent kit, 1–500 bp (Invitrogen, Carlsbad, California, USA). The raw data were analyzed by using PROSize version 2.0 software (AATI).

Statistical parameters such as number of alleles per locus, observed (Ho) and expected heterozygosities (He), Hardy-Weinberg equilibrium (HWE), and polymorphism information content (PIC) of each locus were calculated with PowerMarker version 3.25 (Liu and Muse 2005).

RESULTS AND DISCUSSION

A total of 147 from 192 primer pairs showed at least one band, and 77 primer pairs showed monomorphisms (data not shown). The remaining 70 primer pairs showed polymorphic bands between the two P. densiflora plants. Finally, 17 primer pairs showing clear polymorphic bands were selected as novel genomic SSR markers (Table 1). These markers were applied to 121 tree samples that were collected from four regions in South Korea. Among the 121 genotyped individuals, the number of alleles per locus varied from three to seventeen. The averages of the expected heterozygosity (He) and observed heterozygosity (Ho) were highest in population 4, which was 0.6439 and 0.4254, respectively. The average PIC value of the markers ranged from 0.2789 to 0.8488, and twelve markers showed more than 0.5 (Table 2). Also, transferability was tested from four Pinus species; two markers, CPDE 0039 and CPDE 0076, amplified clear bands from the four Pinus species (Table 3).

In this study, we developed 17 novel polymorphic SSR markers from P. densiflora. These markers would be helpful for genetic studies and diversity analyses and could be applicable to four related species of Pinus. Also, these results would provide valuable information for molecular marker-assisted breeding, germplasm identification, and gene flow in pine trees.

Supplementary Information
ACKNOWLEDGEMENTS

This work was carried out with the support of “Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ01344302)” Rural Development Administration, Republic of Korea.

Tables

Characteristics of 17 SSRs developed in Pinus densiflora.

LocusPrimer sequences (5′-3′)Repeat motifAllele size range (bp)Tm (°C)GenBank accession no.
CPDE 0002F;GAGGGTGCGAGGATTTAAGA(TA)9171–23954KY826503
R;CAGCTGCTAGGAGTTGTTGATG
CPDE 0032F;ACCGCTGATCGATGTGTT(AT)16174–21458KY826504
R;CATGAGGAGCATGTACCGA
CPDE 0039F;TTCCAAGAACTCCTGGCTCT(AT)15169–20958KY826505
R;GGGAACAGGTCCTCATTTCT
CPDE 0048F;GCTTCTGCCCTTTGTTCTCT(TA)9184–22255KY826506
R;GGGGCACTACGTATTTTACCAC
CPDE 0057F;CAGTGACCAACATACCTACCTC(AAT)18138–18955KY826507
R;CGGTAGTGACGAAAGTAGAACG
CPDE 0058F;CAGTGGTCCACCACACTAACT(TA)10172–20057KY826508
R;GTGTGGACCATGTAAGGTATGC
CPDE 0060F;ATTGATGCATGGCACCTG(GT)16145–16353KY826509
R;ACAGGAGTTCCGATGAGGTT
CPDE 0076F;CTCAACTGGCCACTGTAGAACT(GA)9188–20657KY826510
R;AAGGTTCAGGTTGGCATC
CPDE 0077F;ACTACAAGAGGGTCGGTCTTC(TA)15180–21654KY826511
R;GTGCGATATCGTTGCATGTC
CPDE 0079F;CAACAGCAAGTTGAGAACCC(AC)12169–19154KY826512
R;GTTGGTTCCTTCTTGGCAC
CPDE 0093F;GGCAAAGGGGATGAGAAT(TG)12170–18252KY826513
R;GCAAGGTCTTCTCACATCCA
CPDE 0106F;CAGATGTTAATCTGGTAGCCCC(AT)11160–19055KY826514
R;CACCTAAGTTGCCACAATGC
CPDE 0110F;ACCTCATTCTCCACCATGC(TTA)7172–19055KY826515
R;CCCTTGAGATAAGCCTTAGTGG
CPDE 0115F;GGCAGCTTGAAATGGGTACT(AAAC)5158–18655KY826517
R;GACGGTGTGAGGTAGGTGAATA
CPDE 0122F;GCCTTTAGGGGACGTTATAGTG(TA)5171–21555KY826518
R;CCTTTCCCCACAAAACACCT
CPDE 0137F;AAACCCTAGCTGCAGAGGAT(TG)8160–17855KY826519
R;ACCATTGACACTTGGTGCTC
CPDE 0372F;GGTCCAAAACCATAAGGCTC(TTTA)5156–18057KY826520
R;AAGGAGGCTTGGGAAAGA

Genetic properties of 17 polymorphic SSR markers in four Pinus densiflora populations.

LocusPopulation 1 (N=35)Population 2 (N=25)Population 3 (N=33)Population 4 (N=28)Total





Az)Hey)Hox)HWEw)AHeHoHWEAHeHoHWEAHeHoHWEAPICv)
CPDE 000230.51740.00000.000*80.78880.32000.000*40.67470.21430.000*40.61110.000*0.000*90.6604
CPDE 003230.26690.17240.10160.55840.20000.000*60.36090.24140.01040.20130.04350.000*100.3394
CPDE 0039130.78450.17140.000*100.76240.28000.000*90.81940.46670.000*130.87310.22220.000*170.8094
CPDE 004860.44530.22860.000*30.18440.20001.00040.29280.06670.000*50.52420.21430.000*80.3700
CPDE 0057110.82780.71430.016110.87120.60000.015110.86460.60610.000*110.84570.57140.001120.8488
CPDE 005880.80980.68570.01960.73130.55000.00160.74150.36360.000*60.76790.42860.000*90.7666
CPDE 006070.71430.31430.000*70.71740.26090.000*80.71580.45450.000*60.74620.60710.098100.7185
CPDE 007650.58820.48570.01060.67840.56000.10940.67170.45450.000*50.66260.53570.04460.6676
CPDE 0077100.75430.20000.000*90.85070.08330.000*90.75550.09680.000*100.83060.23080.000*150.8119
CPDE 0079100.79430.77140.00350.69530.91670.08070.71530.43750.000*70.64350.64290.087100.7151
CPDE 009330.21520.08000.00520.34030.26090.25320.45370.34780.35430.26750.13040.00730.2789
CPDE 010690.80320.50000.00170.79960.56520.00690.79660.63640.001100.84820.92860.014120.8067
CPDE 011050.59860.17650.000*40.54250.20000.000*50.44580.25930.00140.53020.33330.000*60.4694
CPDE 011530.45030.55880.34920.38480.52000.14330.38020.48480.34640.46810.50000.38740.3688
CPDE 012260.71630.97140.00320.27780.33331.00070.67690.83870.13170.73091.00000.03280.6616
CPDE 013750.59130.55880.33460.61120.60000.35060.67820.43750.00560.63010.53570.35170.5814
CPDE 037270.77390.53130.000*60.71170.30430.000*70.76400.51720.000*70.76550.30770.000*80.7426
Mean6.70.62660.41895.90.61800.39736.30.63570.40736.60.64390.42549.10.6245

z)A, number of alleles,

y)He, expected heterozygosity,

x)Ho, observed heterozygosity,

w)P-value of Hardy-Weinberg equilibrium test,

*Significant at the 0.05 probability level,

v)polymorphism information content.


Transferability of the 17 SSR markers developed in Pinus densiflora across four Pinus species.

LocusP. densifloraP. rigidaP. koraiensisP. parvifloraP. bungeana
CPDE 0002171–239218–222---
CPDE 0032174–214-z)---
CPDE 0039169–209190–206162–170162170–176
CPDE 0048184–222----
CPDE 0057138–189141---
CPDE 0058172–200174–192---
CPDE 0060145–163----
CPDE 0076188–206190–218212–216212216
CPDE 0077180–216----
CPDE 0079169–191----
CPDE 0093170–182----
CPDE 0106160–190168–186---
CPDE 0110172–190----
CPDE 0115158–186162–190---
CPDE 0122171–215----
CPDE 0137160–178----
CPDE 0372156–180----

z)- indicates unsuccessful amplification.


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