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Development of Polymorphic SSR Markers from Pinus densiflora (Pinaceae) Natural Population in Korea

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,*
Plant Breeding and Biotechnology 2019;7(1):67-71.
Published online: March 1, 2019

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

*Yi Lee, leeyi22@hanmail.net, Tel: +82-43-261-3373, Fax: +82-43-271-0413

These authors contributed equally.

• Received: February 15, 2019   • Revised: February 17, 2019   • Accepted: February 17, 2019

Copyright © 2019 The Korean Society of Breeding Science

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.

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  • 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.
  • Key words: Genetic diversity, Next-generation sequencing, Pinaceae, Pinus densiflora, Simple sequence repeat
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.
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).
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.
PBB-7-067_suppl.docx
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.
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.
Table 1
Characteristics of 17 SSRs developed in Pinus densiflora.
Table 1
Locus Primer sequences (5′-3′) Repeat motif Allele size range (bp) Tm (°C) GenBank accession no.
CPDE 0002 F;GAGGGTGCGAGGATTTAAGA (TA)9 171–239 54 KY826503
R;CAGCTGCTAGGAGTTGTTGATG
CPDE 0032 F;ACCGCTGATCGATGTGTT (AT)16 174–214 58 KY826504
R;CATGAGGAGCATGTACCGA
CPDE 0039 F;TTCCAAGAACTCCTGGCTCT (AT)15 169–209 58 KY826505
R;GGGAACAGGTCCTCATTTCT
CPDE 0048 F;GCTTCTGCCCTTTGTTCTCT (TA)9 184–222 55 KY826506
R;GGGGCACTACGTATTTTACCAC
CPDE 0057 F;CAGTGACCAACATACCTACCTC (AAT)18 138–189 55 KY826507
R;CGGTAGTGACGAAAGTAGAACG
CPDE 0058 F;CAGTGGTCCACCACACTAACT (TA)10 172–200 57 KY826508
R;GTGTGGACCATGTAAGGTATGC
CPDE 0060 F;ATTGATGCATGGCACCTG (GT)16 145–163 53 KY826509
R;ACAGGAGTTCCGATGAGGTT
CPDE 0076 F;CTCAACTGGCCACTGTAGAACT (GA)9 188–206 57 KY826510
R;AAGGTTCAGGTTGGCATC
CPDE 0077 F;ACTACAAGAGGGTCGGTCTTC (TA)15 180–216 54 KY826511
R;GTGCGATATCGTTGCATGTC
CPDE 0079 F;CAACAGCAAGTTGAGAACCC (AC)12 169–191 54 KY826512
R;GTTGGTTCCTTCTTGGCAC
CPDE 0093 F;GGCAAAGGGGATGAGAAT (TG)12 170–182 52 KY826513
R;GCAAGGTCTTCTCACATCCA
CPDE 0106 F;CAGATGTTAATCTGGTAGCCCC (AT)11 160–190 55 KY826514
R;CACCTAAGTTGCCACAATGC
CPDE 0110 F;ACCTCATTCTCCACCATGC (TTA)7 172–190 55 KY826515
R;CCCTTGAGATAAGCCTTAGTGG
CPDE 0115 F;GGCAGCTTGAAATGGGTACT (AAAC)5 158–186 55 KY826517
R;GACGGTGTGAGGTAGGTGAATA
CPDE 0122 F;GCCTTTAGGGGACGTTATAGTG (TA)5 171–215 55 KY826518
R;CCTTTCCCCACAAAACACCT
CPDE 0137 F;AAACCCTAGCTGCAGAGGAT (TG)8 160–178 55 KY826519
R;ACCATTGACACTTGGTGCTC
CPDE 0372 F;GGTCCAAAACCATAAGGCTC (TTTA)5 156–180 57 KY826520
R;AAGGAGGCTTGGGAAAGA
Table 2
Genetic properties of 17 polymorphic SSR markers in four Pinus densiflora populations.
Table 2
Locus Population 1 (N=35) Population 2 (N=25) Population 3 (N=33) Population 4 (N=28) Total
Az) Hey) Hox) HWEw) A He Ho HWE A He Ho HWE A He Ho HWE A PICv)
CPDE 0002 3 0.5174 0.0000 0.000* 8 0.7888 0.3200 0.000* 4 0.6747 0.2143 0.000* 4 0.6111 0.000* 0.000* 9 0.6604
CPDE 0032 3 0.2669 0.1724 0.101 6 0.5584 0.2000 0.000* 6 0.3609 0.2414 0.010 4 0.2013 0.0435 0.000* 10 0.3394
CPDE 0039 13 0.7845 0.1714 0.000* 10 0.7624 0.2800 0.000* 9 0.8194 0.4667 0.000* 13 0.8731 0.2222 0.000* 17 0.8094
CPDE 0048 6 0.4453 0.2286 0.000* 3 0.1844 0.2000 1.000 4 0.2928 0.0667 0.000* 5 0.5242 0.2143 0.000* 8 0.3700
CPDE 0057 11 0.8278 0.7143 0.016 11 0.8712 0.6000 0.015 11 0.8646 0.6061 0.000* 11 0.8457 0.5714 0.001 12 0.8488
CPDE 0058 8 0.8098 0.6857 0.019 6 0.7313 0.5500 0.001 6 0.7415 0.3636 0.000* 6 0.7679 0.4286 0.000* 9 0.7666
CPDE 0060 7 0.7143 0.3143 0.000* 7 0.7174 0.2609 0.000* 8 0.7158 0.4545 0.000* 6 0.7462 0.6071 0.098 10 0.7185
CPDE 0076 5 0.5882 0.4857 0.010 6 0.6784 0.5600 0.109 4 0.6717 0.4545 0.000* 5 0.6626 0.5357 0.044 6 0.6676
CPDE 0077 10 0.7543 0.2000 0.000* 9 0.8507 0.0833 0.000* 9 0.7555 0.0968 0.000* 10 0.8306 0.2308 0.000* 15 0.8119
CPDE 0079 10 0.7943 0.7714 0.003 5 0.6953 0.9167 0.080 7 0.7153 0.4375 0.000* 7 0.6435 0.6429 0.087 10 0.7151
CPDE 0093 3 0.2152 0.0800 0.005 2 0.3403 0.2609 0.253 2 0.4537 0.3478 0.354 3 0.2675 0.1304 0.007 3 0.2789
CPDE 0106 9 0.8032 0.5000 0.001 7 0.7996 0.5652 0.006 9 0.7966 0.6364 0.001 10 0.8482 0.9286 0.014 12 0.8067
CPDE 0110 5 0.5986 0.1765 0.000* 4 0.5425 0.2000 0.000* 5 0.4458 0.2593 0.001 4 0.5302 0.3333 0.000* 6 0.4694
CPDE 0115 3 0.4503 0.5588 0.349 2 0.3848 0.5200 0.143 3 0.3802 0.4848 0.346 4 0.4681 0.5000 0.387 4 0.3688
CPDE 0122 6 0.7163 0.9714 0.003 2 0.2778 0.3333 1.000 7 0.6769 0.8387 0.131 7 0.7309 1.0000 0.032 8 0.6616
CPDE 0137 5 0.5913 0.5588 0.334 6 0.6112 0.6000 0.350 6 0.6782 0.4375 0.005 6 0.6301 0.5357 0.351 7 0.5814
CPDE 0372 7 0.7739 0.5313 0.000* 6 0.7117 0.3043 0.000* 7 0.7640 0.5172 0.000* 7 0.7655 0.3077 0.000* 8 0.7426
Mean 6.7 0.6266 0.4189 5.9 0.6180 0.3973 6.3 0.6357 0.4073 6.6 0.6439 0.4254 9.1 0.6245

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.

Table 3
Transferability of the 17 SSR markers developed in Pinus densiflora across four Pinus species.
Table 3
Locus P. densiflora P. rigida P. koraiensis P. parviflora P. bungeana
CPDE 0002 171–239 218–222 - - -
CPDE 0032 174–214 -z) - - -
CPDE 0039 169–209 190–206 162–170 162 170–176
CPDE 0048 184–222 - - - -
CPDE 0057 138–189 141 - - -
CPDE 0058 172–200 174–192 - - -
CPDE 0060 145–163 - - - -
CPDE 0076 188–206 190–218 212–216 212 216
CPDE 0077 180–216 - - - -
CPDE 0079 169–191 - - - -
CPDE 0093 170–182 - - - -
CPDE 0106 160–190 168–186 - - -
CPDE 0110 172–190 - - - -
CPDE 0115 158–186 162–190 - - -
CPDE 0122 171–215 - - - -
CPDE 0137 160–178 - - - -
CPDE 0372 156–180 - - - -

z)- indicates unsuccessful amplification.

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Development of Polymorphic SSR Markers from Pinus densiflora (Pinaceae) Natural Population in Korea
Plant Breed. Biotech.. 2019;7(1):67-71.   Published online March 1, 2019
DOI: https://doi.org/10.9787/PBB.2019.7.1.67
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Development of Polymorphic SSR Markers from Pinus densiflora (Pinaceae) Natural Population in Korea
Plant Breed. Biotech.. 2019;7(1):67-71.   Published online March 1, 2019
DOI: https://doi.org/10.9787/PBB.2019.7.1.67
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Development of Polymorphic SSR Markers from Pinus densiflora (Pinaceae) Natural Population in Korea
Development of Polymorphic SSR Markers from Pinus densiflora (Pinaceae) Natural Population in Korea

Characteristics of 17 SSRs developed in Pinus densiflora.

Locus Primer sequences (5′-3′) Repeat motif Allele size range (bp) Tm (°C) GenBank accession no.
CPDE 0002 F;GAGGGTGCGAGGATTTAAGA (TA)9 171–239 54 KY826503
R;CAGCTGCTAGGAGTTGTTGATG
CPDE 0032 F;ACCGCTGATCGATGTGTT (AT)16 174–214 58 KY826504
R;CATGAGGAGCATGTACCGA
CPDE 0039 F;TTCCAAGAACTCCTGGCTCT (AT)15 169–209 58 KY826505
R;GGGAACAGGTCCTCATTTCT
CPDE 0048 F;GCTTCTGCCCTTTGTTCTCT (TA)9 184–222 55 KY826506
R;GGGGCACTACGTATTTTACCAC
CPDE 0057 F;CAGTGACCAACATACCTACCTC (AAT)18 138–189 55 KY826507
R;CGGTAGTGACGAAAGTAGAACG
CPDE 0058 F;CAGTGGTCCACCACACTAACT (TA)10 172–200 57 KY826508
R;GTGTGGACCATGTAAGGTATGC
CPDE 0060 F;ATTGATGCATGGCACCTG (GT)16 145–163 53 KY826509
R;ACAGGAGTTCCGATGAGGTT
CPDE 0076 F;CTCAACTGGCCACTGTAGAACT (GA)9 188–206 57 KY826510
R;AAGGTTCAGGTTGGCATC
CPDE 0077 F;ACTACAAGAGGGTCGGTCTTC (TA)15 180–216 54 KY826511
R;GTGCGATATCGTTGCATGTC
CPDE 0079 F;CAACAGCAAGTTGAGAACCC (AC)12 169–191 54 KY826512
R;GTTGGTTCCTTCTTGGCAC
CPDE 0093 F;GGCAAAGGGGATGAGAAT (TG)12 170–182 52 KY826513
R;GCAAGGTCTTCTCACATCCA
CPDE 0106 F;CAGATGTTAATCTGGTAGCCCC (AT)11 160–190 55 KY826514
R;CACCTAAGTTGCCACAATGC
CPDE 0110 F;ACCTCATTCTCCACCATGC (TTA)7 172–190 55 KY826515
R;CCCTTGAGATAAGCCTTAGTGG
CPDE 0115 F;GGCAGCTTGAAATGGGTACT (AAAC)5 158–186 55 KY826517
R;GACGGTGTGAGGTAGGTGAATA
CPDE 0122 F;GCCTTTAGGGGACGTTATAGTG (TA)5 171–215 55 KY826518
R;CCTTTCCCCACAAAACACCT
CPDE 0137 F;AAACCCTAGCTGCAGAGGAT (TG)8 160–178 55 KY826519
R;ACCATTGACACTTGGTGCTC
CPDE 0372 F;GGTCCAAAACCATAAGGCTC (TTTA)5 156–180 57 KY826520
R;AAGGAGGCTTGGGAAAGA

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

Locus Population 1 (N=35) Population 2 (N=25) Population 3 (N=33) Population 4 (N=28) Total
Az) Hey) Hox) HWEw) A He Ho HWE A He Ho HWE A He Ho HWE A PICv)
CPDE 0002 3 0.5174 0.0000 0.000* 8 0.7888 0.3200 0.000* 4 0.6747 0.2143 0.000* 4 0.6111 0.000* 0.000* 9 0.6604
CPDE 0032 3 0.2669 0.1724 0.101 6 0.5584 0.2000 0.000* 6 0.3609 0.2414 0.010 4 0.2013 0.0435 0.000* 10 0.3394
CPDE 0039 13 0.7845 0.1714 0.000* 10 0.7624 0.2800 0.000* 9 0.8194 0.4667 0.000* 13 0.8731 0.2222 0.000* 17 0.8094
CPDE 0048 6 0.4453 0.2286 0.000* 3 0.1844 0.2000 1.000 4 0.2928 0.0667 0.000* 5 0.5242 0.2143 0.000* 8 0.3700
CPDE 0057 11 0.8278 0.7143 0.016 11 0.8712 0.6000 0.015 11 0.8646 0.6061 0.000* 11 0.8457 0.5714 0.001 12 0.8488
CPDE 0058 8 0.8098 0.6857 0.019 6 0.7313 0.5500 0.001 6 0.7415 0.3636 0.000* 6 0.7679 0.4286 0.000* 9 0.7666
CPDE 0060 7 0.7143 0.3143 0.000* 7 0.7174 0.2609 0.000* 8 0.7158 0.4545 0.000* 6 0.7462 0.6071 0.098 10 0.7185
CPDE 0076 5 0.5882 0.4857 0.010 6 0.6784 0.5600 0.109 4 0.6717 0.4545 0.000* 5 0.6626 0.5357 0.044 6 0.6676
CPDE 0077 10 0.7543 0.2000 0.000* 9 0.8507 0.0833 0.000* 9 0.7555 0.0968 0.000* 10 0.8306 0.2308 0.000* 15 0.8119
CPDE 0079 10 0.7943 0.7714 0.003 5 0.6953 0.9167 0.080 7 0.7153 0.4375 0.000* 7 0.6435 0.6429 0.087 10 0.7151
CPDE 0093 3 0.2152 0.0800 0.005 2 0.3403 0.2609 0.253 2 0.4537 0.3478 0.354 3 0.2675 0.1304 0.007 3 0.2789
CPDE 0106 9 0.8032 0.5000 0.001 7 0.7996 0.5652 0.006 9 0.7966 0.6364 0.001 10 0.8482 0.9286 0.014 12 0.8067
CPDE 0110 5 0.5986 0.1765 0.000* 4 0.5425 0.2000 0.000* 5 0.4458 0.2593 0.001 4 0.5302 0.3333 0.000* 6 0.4694
CPDE 0115 3 0.4503 0.5588 0.349 2 0.3848 0.5200 0.143 3 0.3802 0.4848 0.346 4 0.4681 0.5000 0.387 4 0.3688
CPDE 0122 6 0.7163 0.9714 0.003 2 0.2778 0.3333 1.000 7 0.6769 0.8387 0.131 7 0.7309 1.0000 0.032 8 0.6616
CPDE 0137 5 0.5913 0.5588 0.334 6 0.6112 0.6000 0.350 6 0.6782 0.4375 0.005 6 0.6301 0.5357 0.351 7 0.5814
CPDE 0372 7 0.7739 0.5313 0.000* 6 0.7117 0.3043 0.000* 7 0.7640 0.5172 0.000* 7 0.7655 0.3077 0.000* 8 0.7426
Mean 6.7 0.6266 0.4189 5.9 0.6180 0.3973 6.3 0.6357 0.4073 6.6 0.6439 0.4254 9.1 0.6245

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.

Locus P. densiflora P. rigida P. koraiensis P. parviflora P. bungeana
CPDE 0002 171–239 218–222 - - -
CPDE 0032 174–214 -z) - - -
CPDE 0039 169–209 190–206 162–170 162 170–176
CPDE 0048 184–222 - - - -
CPDE 0057 138–189 141 - - -
CPDE 0058 172–200 174–192 - - -
CPDE 0060 145–163 - - - -
CPDE 0076 188–206 190–218 212–216 212 216
CPDE 0077 180–216 - - - -
CPDE 0079 169–191 - - - -
CPDE 0093 170–182 - - - -
CPDE 0106 160–190 168–186 - - -
CPDE 0110 172–190 - - - -
CPDE 0115 158–186 162–190 - - -
CPDE 0122 171–215 - - - -
CPDE 0137 160–178 - - - -
CPDE 0372 156–180 - - - -

z)- indicates unsuccessful amplification.

Table 1 Characteristics of 17 SSRs developed in Pinus densiflora.
Table 2 Genetic properties of 17 polymorphic SSR markers in four Pinus densiflora populations.

A, number of alleles,

He, expected heterozygosity,

Ho, observed heterozygosity,

P-value of Hardy-Weinberg equilibrium test,

Significant at the 0.05 probability level,

polymorphism information content.

Table 3 Transferability of the 17 SSR markers developed in Pinus densiflora across four Pinus species.

- indicates unsuccessful amplification.

Table 1

Table 2

Table 3

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