Development and application of EST-SSR molecular markers in Pinus bungeana
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摘要:目的 白皮松作为我国濒危的乡土树种，具有重要的经济和园林观赏价值，为进一步开发和利用白皮松种质资源，本研究基于EST-SSR标记对北京地区3个不同种源地的白皮松群体遗传多样性开展评价。方法 以白皮松转录组数据为依据，对其微卫星位点进行筛选并设计合成了96对引物，并对北京温泉苗圃栽培收集的3个不同种源地（北京、山东、山西）的白皮松群体，共60个植株开展遗传多样性分析，对其群体内与群体间的遗传结构进行评价。结果 96对引物中共筛选获得了5对多态性引物，其观察杂合度、期望杂合度、多态性信息含量和等位基因位点个数变化范围分别为0.203 ~ 0.433、0.211 ~ 0.530、0.187 ~ 0.484和2 ~ 5。3个白皮松样本群体中等位基因数量、有效等位基因数量、香农信息指数、观察杂合度和固定指数变化范围为2.400 ~ 3.000、1.516 ~ 1.761、0.484 ~ 0.606、0.295 ~ 0.362和−0.075 ~ 0.081，平均值分别为2.677、1.632、0.560、0.333和−0.007。遗传分化系数和基因流变化范围分别为0.021 6 ~ 0.115 3和1.399 6 ~ 11.340 0，平均值分别为0.090 2和2.521 2。AMOVA分析显示遗传变异主要来自于群体内，群体间差异较小，仅占据11%。结论 本研究共获得了5对多态性的白皮松EST-SSR引物，可用于后续白皮松群体遗传多样性分析和分子标记辅助育种工作；北京温泉苗圃栽培收集的3个不同种源地的白皮松群体分析结果表明，当地现有收集的白皮松种源群体遗传相似度较高，在未来种质资源保存和苗木繁育工作中应考虑增加其他种源地的白皮松群体。Abstract:Objective As an endangered native tree species in China, Pinus bungeana has important economic and garden ornamental values. In order to further develop and utilize the species germplasm resources, the study evaluated the genetic diversity of three populations from different provenances in P. bungeana.Method Simple sequence repeats were selected from the transcriptome data in P. bungeana, and then 96 pairs of primers were developed, and the genetic diversity of 60 individuals in Beijing Hot Spring Nursery collected from three different provenances (Beijing, Shandong, and Shanxi) was analyzed, and genetic diversity parameters between intra- and inter-population were analyzed.Result The variation ranges of the observed heterozygosity, expected heterozygosity, polymorphism information content and the number of allele loci for 5 pairs of polymorphic primers were 0.203−0.433, 0.211−0.530, 0.187−0.484 and 2−5, respectively. The number of alleles, effective alleles, Shannon diversity index, observed heterozygosity and fixed index of the three populations ranged in 2.400−3.000, 1.516−1.761, 0.484−0.606, 0.295−0.362 and −0.075−0.081, the average values were 2.677, 1.632, 0.560, 0.333 and −0.007, respectively. The ranges of genetic differentiation coefficient and gene flows were 0.021 6−0.115 3 and 1.399 6−11.340 0, and the average values were 0.090 2 and 2.521 2, respectively. AMOVA analysis showed that genetic variation mainly comed from within the population, with small differences between populations, accounting for only 11%.Conclusion This study obtained 5 pairs of polymorphic EST-SSR primers in P. bungeana, which can be used for subsequent analysis of the population genetic diversity and molecular marker-assisted breeding. Genetic diversity analysis about the three populations of P. bungeana shows that the existing populations in Beijing Hot Spring Nursery have high genetic similarity, and the populations from other provenances should be considered for the future germplasm resource preservation and seedling breeding work.
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图 1 白皮松不同类型的EST-SSR重复序列的分布频率
Mono-. 单碱基重复基元；Di-. 二碱基重复基元；Tri-. 三碱基重复基元；Tetra-. 四碱基重复基元；Penta-. 五碱基重复基元；Hexa-. 六碱基重复基元。Mono-, one-base-repeat motif; Di-, two-base-repeat motif; Tri-, three-base-repeat motif; Tetra-, four-base-repeat motif; Penta-, five-base-repeat motif; Hexa-, six-base-repeat motif.
Figure 1. Distribution frequency of EST-SSR repeats in different types of P. bungeana
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图 2 白皮松96对EST-SSR引物预试验扩增情况
Figure 2. Pre-experimental amplification of 96 pairs of EST-SSR primers in P. bungeana
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图 3 5个微卫星位点在3个群体中的等位基因分布模式
Na. 等位基因数量；Na freq. ≥5%. 基因分布频率大于等于5%的等位基因数量；Ne. 有效等位基因数量；Ⅰ. 香浓多样性指数；No. private alleles. 单个群体中特有的等位基因数量。Na, number of alleles; Na freq. ≥5%, number of alleles whose gene distribution frequency is greater than or equal to 5%; Ne, number of effective alleles; Ⅰ, Shannon’s diversity index; No. private alleles, number of alleles unique to a single population.
Figure 3. Allelic distribution patterns of five microsatellite loci in the three populations
表 1 白皮松3个群体的地理位置及主要生态因子
Table 1 Geographical location and main ecological factors of three populations in Pinus bungeana
林分代号
Abbreviation of
stand样本数量
Sample size林分来源
Stand source经度
Longitude纬度
Latitude海拔
Altitude/m年降水量
Annual
precipitation/mm林分起源
Stand originSDTA 20 山东泰安 Taian, Shandong 116°17′E 36°07′N 61 600.0 天然林 Natural forest SXXY 20 山西孝义 Xiaoyi, Shanxi 111°48′E 37°12′N 950 493.0 天然林 Natural forest BJMS 20 北京蟒山 Mangshan, Beijing 116°16′E 40°15′N 330 631.0 人工林 Plantation 
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表 2 白皮松EST-SSR引物
Table 2 Primers used for EST-SSR analysis of P. bungeana
引物编号 Primer No. 前引物序列 Forward primer sequence 后引物序列 Reverse primer sequence 元件 Motif BPS-SSR-01 CATAACACAGCAGCAACCGG ACACTGCTGCAAAAATAACCCT A BPS-SSR-02 GCCGAAACGACCTTTCACAC CTAAAGCATAAACCGCCGCC AAAT BPS-SSR-03 TCCAAGAAAGCGCGTTCTCT TGTTATTTTATTAGCGCGGCCG GAG BPS-SSR-04 ACAAGGGGAGAGCAATGTGG GCCATTTCCAACAGCTTAGCA GA BPS-SSR-05 TGAGAAGAAGGCCGAGGAGA GCTTGAGCACCTTGAAGATGT AAG BPS-SSR-06 AGCCCTAGTCCGAGTCCAAT GAGGCCCAGGAGGAGAAAAC CTG BPS-SSR-07 TGCCAGAGACAATGCAGACA GGAAATACCTGTCGGCCACA TGA BPS-SSR-08 TCTGGTGTTGTGGATGCCTC ACGCTGCTGTTACTTCCGAA GAAAG BPS-SSR-09 CGGCCTCAACAACCTCTTCT TCCTCCGGCTGTTCGTAGTA CTCTC BPS-SSR-10 AGCCGAAATCAAAAGCGCAG GGGGACAACGCGGATGAA CCTGAC BPS-SSR-11 GGCGTGGTGGTGAATTGAAC GAGCTTGGGGGCAATCTTCT CTC BPS-SSR-12 TTCACCCCCTGCAGATTTCC GCAAGCCTCCCCTTCAGAAT ATTCA BPS-SSR-13 AAGGGCAAGAGTAAACGGGG GTTGGCGGCATTGCAGAATC GAGGAA BPS-SSR-14 CTGGGTGTTCCTACCAGCAG ACCCTCAGTTTGTCAGGGGA TGATAA BPS-SSR-15 TACAGTAGGAAGCAGTGCCA GAGGGAGTGGCGTTATCGAG GGCAAC BPS-SSR-16 CTGCAGATGAGGAGGCTGAC AGCAGCAACCTAGAGGCAAT GAA BPS-SSR-17 ACAGAAAGTGGGAGGAAGCG TCTCCTCTCCCCTCCTCTCA GAAGA BPS-SSR-18 CTCCTGGCCATCCCAAACAA AACTACGAAAGCTCCAGGCC GGTGAT BPS-SSR-19 CAGCTCAACTCGCATTACGC ACCACCTGTGTTTGGCAAGA GCA BPS-SSR-20 TGGGATTGTCCAAGAGCAGG TCACTCTGATTGTGACTGCGA CAATCC BPS-SSR-21 GGATGCAGAGGACGAGAGG TCGGATCTCCTCTTCCCCTG AGGGGA BPS-SSR-22 TGCTCCACAGACAGAAGCTG CCCCTAGCATTGTTGTACCTTC GAT BPS-SSR-23 CATTCTTGGGGGCTCTACCG TAAACGGCCTGACTCCAACC AT BPS-SSR-24 TCCAGGTGGCTTTGGTTCTC GCAGGTCACTGGTATCGACC T BPS-SSR-25 CACAGGGAGTGGCGGTATAC AGCATCGATTCCTCCACTGC GGCGAT BPS-SSR-26 GGTGACCCTTTGGAGAGGTC AAAATCGCCACCATTGCCAC CCACA BPS-SSR-27 TTGTGTGAGCAGCCAACAGA GGGGAGATCAATGACAACCCA TTCCTC BPS-SSR-28 TACCTCCATGGGGGTGTTGA GCTCTGGACTGGATGGTCAG CAGAG BPS-SSR-29 ATTGGGAGGGCGGAGAAAAG GGCGGCATTGACAATGAGAC GGCAGA BPS-SSR-30 GCTTATTCCCTGGGCATGGT CTGTGGCCAAGCATTGCTTC CAGCGA BPS-SSR-31 TCTCTCACGCGTTTTGCAGA AGAAGTGGTCCACTCCCGTA TGATG BPS-SSR-32 GCGATTGATCAGGATGCACC TGCTGAATTTTTGCAGCAGAGA AT BPS-SSR-33 TCGCTCGTACTTGCACCATT TCGGACTCAGGCCAGTACTT TCTTC BPS-SSR-34 TCAAAACGGCACTGGCATTG CCGATCAATGGCCATGGCTA TGGCTT BPS-SSR-35 TCCCTGCACAGCTGAAAACA AATGACAATGCTTTGGCCGG ACTGCG BPS-SSR-36 CTCTGTTCCGTCGGTGATCC GCTCTTCCAACTTGCGCTTC GGACAT BPS-SSR-37 TTTGGAGCAATGTTTCGGCG ACACTGCATCCCACTGTCTG ATAGAG BPS-SSR-38 ACTTCGTGAGCAGGTTGAGG AATTCCTCTGCTTCGGGTGC AAG BPS-SSR-39 TTACTGACAGGTCGCCTTCG TATATGGGTGGCTGCCTCTG ATGTCG BPS-SSR-40 ACCACGTCATCATCGTCGTC AAACTGAGGCAGAGGCTTCC GAGGCA BPS-SSR-41 ATTATGGGCGGTGGCAGAAA CCCCGTTCCTACCATCCAAT GAA BPS-SSR-42 GGAATCTCAGCGCTTCCTGT AAGCTCTCACAAACGTGGCA CAG BPS-SSR-43 CTGTCGAGCGAAGGTTACCC AGGTCCCTTGCCCATCTCTA GCAGTT BPS-SSR-44 GGGTGAGCTAGACCGTCAAA TATTGACGGCGGCCTTTCTT CTCTG BPS-SSR-45 CCGCGAAGACAGAGTATCCC AGCTGGGAAAAAGATCGCCA TAT BPS-SSR-46 GAAAATTGCCCCCGTCGAAG GCAAAGACGCTTCCAGCAAA GAGGAA BPS-SSR-47 GCCTTATCTTCTTCCTTGTGTGA CACATAGAAGAGGCGGCAGT TCT BPS-SSR-48 GGAAGGTTGCCAGGTGGTAA ACATCACAACGAAGGGACCC TGGAGA BPS-SSR-49 CTCGAGTCTCTCACCCTTGC CCGTGTTTGTCCACCAGACT AGCCGC BPS-SSR-50 ACGCCGGATTTCAACAGACT GGGGAGAACGGCGATTGTTA AACGCC BPS-SSR-51 AAGGGCTCCATGATGTCTGC ACGTGGGCATAGGCTTGAAA CTTCAC BPS-SSR-52 ATCTTTGTCCAGCGCTTCGA GGAAGCACGGGAATCAGACA CCTCT BPS-SSR-53 ATCAGTACAACGCTGCCCAG CTGCCCGGAGTCTGAGAATC CAG BPS-SSR-54 GTCTTTCGGTGTGGGCTGTA CCGTCACACGATCGTCCAAT TTGT BPS-SSR-55 TGCAGGGAGTGTCTACAGGA GAGAAACGTGAAGCCCCTCA GAC BPS-SSR-56 CCTTTGCTCTGTACGAGGGG TCAGTCGTTTCAGTCGCAGT GCACGG BPS-SSR-57 TGGGGGAAAAGCTAGATCCA CTTTGTGTTTGGCCTCCAGC AGAGGC BPS-SSR-58 TGAGGGCAATGTTACTCGCT AACAATCGCCGCTATCAGCT AGG BPS-SSR-59 AGGGGCCTCAGCTGATATCA AACAACAGCAACAGCAGCAG TGC BPS-SSR-60 CTATCAGGGGATTGTGGGGC TCTGCTGCTGGAGACGTAAC CGG BPS-SSR-61 TATTCGCCACTCTGCACGTT CCTCCATGGGGAAGGGAATG TTGCAG BPS-SSR-62 ATTGATCAGGCGAGGCAGAC GCAGGTTTTCGTTGTGGGTG GGGCGA BPS-SSR-63 ATCGAACCCGTAGTCTCCCA AAGTCAAGAAAGTGGCCGCT TGGGCT BPS-SSR-64 CCGCGGTTTTGATGTGAACC CCCTGTCCAGTTCGTTGTCA GACAAC BPS-SSR-65 GGCTAGCGAGTGGTGATTGT GAGTTGCTTCTCCTCCGCAT CCTGGC BPS-SSR-66 TTGCGAAGGAGGTCCATAGC GGGGTAGATGGAGAATGGCC AAAGGC BPS-SSR-67 GCTTCGTTGGTAAGGGCCTA AGTTCTTGCACTGCCCAACT GATGGA BPS-SSR-68 GCCTTGTAGTTTTGGCCTGC CTGCAGGATGGGGCTCTTTT AGA BPS-SSR-69 AAGTCCTCGGCTTCGCATTC GAGCCCAGTGCCTCAATCTT CTT BPS-SSR-70 CGGGCAACAGTCTGTTAGGT GCCTGAAACAGCCTCCTCAT ATGGGC BPS-SSR-71 TAAGAAGCCTGCGTGGACTG GCATATTTGAATGCCTTTCTGGG AT BPS-SSR-72 CTCACCCGACAACTACAACA TGAGGGCTCTTGTTGACAGT CCCCTG BPS-SSR-73 CGTACCAAGTGCGCCTCTAT TAGTGCCAACTCTGCGCTAC GAG BPS-SSR-74 ACTGGGGGCAACAATATCAGA GAGTGGTGGGTGTGGAAGAG AGTGA BPS-SSR-75 CCTGCAACTGGATGACGACT ACCGGCACACAGAAAAGCTA CCACGA BPS-SSR-76 GCAGTCCCCTCGCTATAACC TCAGAAACCCAGCGGTGTAC AAATAA BPS-SSR-77 GAAGTAGAAGCAGGCGGAGG ATAACGCTGCCAGTGCTTCT AGCAGA BPS-SSR-78 GCTGATCCTCTTGACTCCGG GGCAATGGTGAGAGTGTGGA TGACCC BPS-SSR-79 GCCTAGGCACTGGAATTCCT GGTGGGCATGACAGATGGAA TTCCTC BPS-SSR-80 TGAGCTTTCACAACACCCGA GGCCCAATTCTCCTCCTTCC TGAT BPS-SSR-81 CCTCAGCTCATCCTACAGCG TCTCTTCCAGTTCGACTTCCA AAGGTG BPS-SSR-82 ACGGGTCATTGCGCCATTAT CCACGCCAACATTGACCTTG ATGGCG BPS-SSR-83 TTGTTCAATCCCTCCACCGC GGAATCTTCCCGTCCTGCAA TTGTT BPS-SSR-84 GGCCCTATGAGTGCACTGTT ACAGCCCCTACTATTGCGAA CAGGG BPS-SSR-85 GGGGATTCTTCAATCTGTAACGC ACCCCACGTAACCCTTATGC GATCCT BPS-SSR-86 TAGATACTCCGGTGCCAACG ATTTCAGCAGCCCATCCGAG CAG BPS-SSR-87 AAGAAAATGGTGGGCCGGAT CTTCCTCCAGCTCCCCAAAG AGCAGT BPS-SSR-88 TCTACACGGTCCAAGCGATG TTCCCTTTCGATGACCTGCA GAGCAG BPS-SSR-89 TGTCGAGAAGGCTATTGCTGA TGACATCTGCAACGTCTCCT ATA BPS-SSR-90 GGAAGCTTTGCATGTGCCAA GCAACCAGCTCTTCCGACTT TTTCA BPS-SSR-91 ATGGACATGCGTTGTGGAGT CTGGATACACATGGCCTCCC AGAGTG BPS-SSR-92 CGAACGTCTCCGGATCGAAT AGGGGAAGGCATGATTGTGG TCCTCT BPS-SSR-93 TCCGCCTGAGTTCCAAGTTC ACGGTGATGAAGGGAAAACCA A BPS-SSR-94 GATCTTCAGAGAGCTCCGGC ACCACTGGAAACACTTCGCA AAT BPS-SSR-95 GAGCATCGTCCGAATGTGGA TTGCCCCTCCTCTTCTTCCT AGA BPS-SSR-96 AAGCTGCTGAATTGGGCTCT ACACATGATGACAGGGCAGG TCTTGT
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表 3 PCR反应体系
Table 3 PCR reaction system
μL 类别
Category预试验反应体系
Pre-experiment
reaction systemSSR-PCR反应体系
SSR-PCR reaction
system2 × PCR MIX 10.0 10.0 DNA模板 DNA template 2.0 2.0 前引物 Forward prime 3.2 0.8 后引物 Reverse prime 3.2 3.2 M13引物 M13 primer 3.2 ddH2O 1.6 0.8 总计 Total 20.0 20.0
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表 4 白皮松中25个微卫星位点的特征
Table 4 Characteristics of 25 microsatellite loci developed in P. bungeana
引物编号
Primer No.等位基因数
Number of alleles (Na)元件
Motif观察杂合度
Observed
heterozygosity (Ho)期望杂合度
Expected
heterozygosity (He)多态性信息含量
Polymorphism information
content (PIC)BPS-SSR-01 2 A 0.203 0.211 0.187 BPS-SSR-09 4 CTCTC 0.364 0.530 0.484 BPS-SSR-14 3 TGATAA 0.433 0.487 0.374 BPS-SSR-52 5 CCTCT 0.417 0.423 0.361 BPS-SSR-83 2 TTGTT 0.265 0.232 0.204 BPS-SSR-62 2 GGGCGA 0.033 0.033 0.032 BPS-SSR-96 2 TCTTGT 0.000 0.130 0.120 BPS-SSR-04 1 GA BPS-SSR-08 1 GAAAG BPS-SSR-12 1 ATTCA BPS-SSR-16 1 GAA BPS-SSR-30 1 CAGCGA BPS-SSR-50 1 AACGCC BPS-SSR-54 1 TTGT BPS-SSR-63 1 TGGGCT BPS-SSR-64 1 GACAAC BPS-SSR-75 1 CCACGA BPS-SSR-79 1 TTCCTC BPS-SSR-82 1 ATGGCG BPS-SSR-90 1 TTTCA BPS-SSR-91 1 AGAGTG BPS-SSR-93 1 A BPS-SSR-95 1 AGA BPS-SSR-80 2 TGAT BPS-SSR-13 2 GAGGAA
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表 5 3个白皮松群体遗传多样性参数
Table 5 Genetic diversity parameters of the three populations in P. bungeana
群体
PopulationNa 有效等位基因数量
Number of effective allele (Ne)香农多样性指数
Shannon’s diversity index (I)Ho 固定指数
Fixation index (Fix)SDTA 2.400 1.516 0.484 0.295 −0.026 SXXY 3.000 1.620 0.589 0.362 −0.075 BJMS 2.600 1.761 0.606 0.343 0.081 均值 Mean 2.667 1.632 0.560 0.333 −0.007
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表 6 白皮松中不同SSR位点的F-统计量
Table 6 F-statistic of SSR locus in P. bungeana
位点 Locus 亚群内 Within subgroup (Fis) 群体内 Within group (Fit) 亚群间 Between subgroups (Fst) 基因流 Gene flow (Nm) BPS-SSR-01 −0.021 4 0.028 6 0.048 9 4.859 4 BPS-SSR-09 0.189 6 0.312 4 0.151 6 1.399 6 BPS-SSR-14 0.082 9 0.102 7 0.021 6 11.340 0 BPS-SSR-52 −0.123 6 0.006 0 0.115 3 1.918 1 BPS-SSR-83 −0.257 6 −0.149 3 0.086 1 2.652 0 均值 Mean 0.011 4 0.100 6 0.090 2 2.521 2
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表 7 群体间遗传相似度GI（对角线之上）和遗传距离GD（对角线之下）
Table 7 Nei’s genetic identity (GI, above diagonal) and genetic distance (GD, below diagonal) among populations
群体 Population SDTA SXXY BJMS SDTA 0.935 5 0.905 9 SXXY 0.066 7 0.934 7 BJMS 0.098 8 0.067 5
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表 8 3个白皮松群体方差分析结果
Table 8 ANOVA of the three populations in P. bungeana
变异来源
Source of variation自由度
Degree of freedom (df)离差平方和
Sum of squares (SS)方差成分
Variance component变异所占百分比
Percentage of variation/%群体间 Among populations 2 17.617 0.310 11 群体内 Within population 57 148.450 2.604 89 合计 Total 59 166.067 2.915 100
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