Construction of SSR fingerprint and research of genetic structure in relative Quercus species
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摘要:目的对栎属近缘种质进行指纹图谱构建，并分析其遗传结构，为栎属近缘种鉴定及分类提供了重要工具。方法以23份栎属近缘种质为研究对象，利用遗传背景差异大的4份种质进行SSR引物筛选，选出9对扩增条带清晰、具有多态性且重复性好的引物对其进行扩增，利用毛细管电泳技术对PCR荧光产物进行检测，采用引物-分子量组合法构建23份种质的指纹图谱，利用NTsys和STRUCTURE软件进行聚类分析和群体遗传结构分析。结果9对SSR引物共扩增出78个条带，每个位点的等位标记数4~14个，平均每对引物为8.67个，多态信息含量变幅为0.58~0.82，平均为0.73。聚类分析显示辽东栎、蒙古栎分别聚为两个类群，猩红栎和北美红栎聚为一个类群，群体遗传结构分析显示23份种质为3个亚群体，遗传结构分析与聚类分析结果一致。结论23份栎属近缘种质可以划分为辽东栎组、蒙古栎组、猩红栎-北美红栎混合组，辽东栎与蒙古栎是两个独立的分类单位，上述结果为栎属分类、品种鉴定和知识产权保护提供了理论依据。Abstract:ObjectiveThe fingerprint of the relative Quercus species was constructed and the genetic structure was analyzed, it provides an important tool for the identification and classification in relative Quercus species in order to classify and identify the relative Quercus species.Method23 germplasms of relative Quercus were used as the materials in this study, SSR primers were selected by 4 accessions of genetically distant germplasms, 9 pairs of primers with clear amplification bands, high polymorphism and stable repeatability were selected and used for PCR amplification of the 23 germplasms, PCR products labeled fluorescent were detected using capillary electrophoresis technology, a strategy of combining primer pair with molecular weight for fingerprint construction was developed and applied to the 23 germplasms, the software of NTsys and STRUCTURE were used in clustering analysis and population genetic structure analysis.ResultThe 78 fragments were amplified by 9 pairs of SSR primers, the number of alleles at each locus was between 4 and 14, with an average of 8.67 alleles for each pair of primers, polymorphism information content values for the primer pairs ranged from 0.58 to 0.82, with an average of 0.73. Clustering analysis showed that Quercus liaotungensis and Quercus monglica were respectively divided into two groups, Quercus coccinea and Quercus rubra were clustered into a group, population genetic structure analysis showed that 23 germplasms for the three groups, genetic structure analysis and clustering analysis results were consistent.ConclusionThe 23 germplasms are divided into Quercus liaotungensis group, Quercus monglica group, Quercus coccinea-Quercus rubra mixed group, Quercus liaotungensis and Quercus monglica are two separate taxonomies, the results provide a theoretical basis for Quercus classification, variety identification and protection of intellectual property centers.
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Keywords:
- Quercus liaotungensis /
- Quercus monglica /
- fingerprint /
- genetic structure
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图 1 引物ssrQrZAG 112在辽东栎和蒙古栎中的扩增图谱
Figure 1. Polymorphic fingerprint detected by ssrQrZAG112 for Quercus liaotungensis and Quercus monglica
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图 3 利用STRUCTURE软件检测23份种质最佳亚群体数目(K)
Figure 3. Estimation of the optimal number (K) of subpopulations for 23 germplasms based on STRUCTURE
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图 4 23份种质的群体结构(K=3)
23份栎属近缘种质被划分为3个亚群体，每一个颜色代表一个亚群体，蓝色为辽东栎亚群；红色为蒙古栎亚群；绿色为猩红栎-北美红栎亚群。
Figure 4. Population structure for 23 germplasms(K=3)
The 23 germplasms were clustered into 3 sub-population, each color represents a sub-population, blue color represents Quercus liaotungensis sub-population, red color represents Q. monglica sub-population, green color represents Q. coccinea-Q. rubra sub-population.
表 1 试验材料
Table 1 Experimental materials
编号Code 种质Germplasm 种源Provenance Q1 晋辽东栎1号Querecus liaotungensis ‘Jinliaodong1’ 山西省中条山国有林管理局横河林场，良种编号：晋R-SC-QL-001-2016
Shanxi Province Zhongtiao Mountain State Forest Administration, Henghe Forest Farm, the code of improved variety: Jin R-SC-QL-001-2016Q2 晋辽东栎2号Querecus liaotungensis ‘Jinliaodong2’ 山西省太岳山国有林管理局灵空山林场，良种编号：晋R-SC-QL-002-2016
Shanxi Province Taiyue Mountain State Forest Administration, Lingkongshan Forest Farm, the code of improved variety: Jin R-SC-QL-002-2016Q3 晋辽东栎3号Querecus liaotungensis ‘Jinliaodong3’ 山西省吕梁山国有林管理局康城林场，良种编号：晋R-SC-QL-003-2016
Shanxi Province Lüliang Mountain State Forest Administration, Kangcheng Forest Farm, the code of improved variety: Jin R-SC-QL-003-2016Q4 晋辽东栎4号Querecus liaotungensis ‘Jinliaodong4’ 山西省吕梁山国有林管理局东山林场，良种编号：晋R-SC-QL-004-2016
Shanxi Province Lüliang Mountain State Forest Administration, Dongshan Forest Farm, the code of improved variety: Jin R-SC-QL-004-2016Q5 晋辽东栎5号Querecus liaotungensis ‘Jinliaodong5’ 山西省太行山国有林管理局坪松林场，良种编号：晋S-SS-QL-005-2014
Shanxi Province Taihang Mountain State Forest Administration, Pinsong Forest Farm, the code of improved variety: Jin S-SS-QL-005-2014Q6 晋辽东栎6号Querecus liaotungensis ‘Jinliaodong6’ 山西省关帝山国有林管理局真武山林场，良种编号：晋S-SS-QL-006-2014
Shanxi Province Guandi Mountain State Forest Administration, Pinsong Forest Farm, the code of improved variety: Jin S-SS-QL-006-2014Q7 蒙古栎1号Quercus monglica 1 吉林省引种种质The introduction of germplasm in Jilin Province Q8 蒙古栎2号Quercus monglica 2 吉林省引种种质The introduction of germplasm in Jilin Province Q9 蒙古栎3号Quercus monglica 3 吉林省引种种质The introduction of germplasm in Jilin Province Q10 蒙古栎4号Quercus monglica 4 吉林省引种种质The introduction of germplasm in Jilin Province Q11 蒙古栎5号Quercus monglica 5 吉林省引种种质The introduction of germplasm in Jilin Province Q12 猩红栎1号Quercus coccinea 1 美国北卡罗来纳州引种种质The introduction of germplasm in the United States North Carolina Q13 猩红栎2号Quercus coccinea 2 美国北卡罗来纳州引种种质The introduction of germplasm in the United States North Carolina Q14 猩红栎3号Quercus coccinea 3 美国北卡罗来纳州引种种质The introduction of germplasm in the United States North Carolina Q15 猩红栎4号Quercus coccinea 4 美国北卡罗来纳州引种种质The introduction of germplasm in the United States North Carolina Q16 猩红栎5号Quercus coccinea 5 美国北卡罗来纳州引种种质The introduction of germplasm in the United States North Carolina Q17 猩红栎6号Quercus coccinea 6 美国北卡罗来纳州引种种质The introduction of germplasm in the United States North Carolina Q18 北美红栎1号Quercus rubra 1 美国北卡罗来纳州引种种质The introduction of germplasm in the United States North Carolina Q19 北美红栎2号Quercus rubra 2 美国北卡罗来纳州引种种质The introduction of germplasm in the United States North Carolina Q20 北美红栎3号Quercus rubra 3 美国北卡罗来纳州引种种质The introduction of germplasm in the United States North Carolina Q21 北美红栎4号Quercus rubra 4 美国北卡罗来纳州引种种质The introduction of germplasm in the United States North Carolina Q22 北美红栎5号Quercus rubra 5 美国北卡罗来纳州引种种质The introduction of germplasm in the United States North Carolina Q23 北美红栎6号Quercus rubra 6 美国北卡罗来纳州引种种质The introduction of germplasm in the United States North Carolina 
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表 2 SSR引物信息表
Table 2 Table of SSR primer information
引物名称
Primer name正向引物
Forward primer (5′→3′)反向引物
Reverse primer(5′→3′)ssrQrZAG 96 CCCAGTCACATCCACTACTGTCC GGTTGGGAAAAGGAGATCAGA ssrQrZAG 102 GCCTACACTCTTCAATCTACATGA GACTTGTAACACCTTAAGCATTATCT ssrQrZAG 112 TTCTTGCTTTGGTGCGCG GTGGTCAGAGACTCGGTAAGTATTC ssrQrZAG 7 CAACTTGGTGTTCGGATCAA GTGCATTTCTTTTATAGCATTCAC Qden 03011 AACCC AACCTTCCCTTCATC GCAGTGGTGCCTAATGTAGAC Qden 03021 ACAGCAAACCAGACTCCAC CCCCAAAGTTTCGGCTAATAC Qden 03032 AGTTGTGGTCCTGCTCGC GAAAAGTGCGATGACGGTTG Qden 05011 CCCACTCCCTGTCCATTGT CACTGTGTGCTGCGACTTG Qden 05031 CCCCGATTCGCCATCATTGT GTAACGCCGTTTTTCTCCACC
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表 3 9对SSR引物的多态检测
Table 3 Polymorphism detection of 9 pairs of SSR primer
引物名称
Primer name等位标记数
Allele number多态信息含量
PICssrQrZAG 96 8 0.76 ssrQrZAG 102 14 0.82 ssrQrZAG 112 8 0.77 ssrQrZAG 7 10 0.71 Qden 03011 7 0.65 Qden 03021 4 0.58 Qden 03032 7 0.78 Qden 05011 12 0.79 Qden 05031 8 0.72 总计Total 78 平均Mean 8.67 0.73
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表 4 23份种质的指纹图谱
Table 4 Fingerprint of 23 germplasms based on SSR markers
编号Code 指纹图谱Fingerprint Q1 ssrQrZAG96, 152, 152 ssrQrZAG112, 78, 90 ssrQrZAG7, 124, 140 Qden03011, 142, 144 Qden03021, 252, 254 Qden 05011, 174, 180 Q2 ssrQrZAG96, 154, 166 ssrQrZAG112, 81, 81 ssrQrZAG7, 134, 140 Qden03011, 140, 150 Qden03021, 252, 252 Qden 05011, 180, 188 Q3 ssrQrZAG96, 145, 150 ssrQrZAG112, 96, 98 ssrQrZAG7, 128, 138 Qden03011, 140, 140 Qden03021, 252, 256 Qden 05011, 176, 178 Q4 ssrQrZAG96, 150, 168 ssrQrZAG112, 90, 90 ssrQrZAG7, 126, 128 Qden03011, 140, 140 Qden03021, 252, 260 Qden 05011, 172, 180 Q5 ssrQrZAG96, 160, 186 ssrQrZAG112, 88, 90 ssrQrZAG7, 124, 124 Qden03011, 140, 142 Qden03021, 254, 256 Qden 05011, 176, 192 Q6 ssrQrZAG96, 154, 166 ssrQrZAG112, 90, 94 ssrQrZAG7, 126, 126 Qden03011, 138, 140 Qden03021, 252, 260 Qden 05011, 180, 186 Q7 ssrQrZAG96, 147, 149 ssrQrZAG112, 88, 94 ssrQrZAG7, 124, 134 Qden03011, 137, 137 Qden03021, 254, 254 Qden 05011, 175, 183 Q8 ssrQrZAG96, 147, 151 ssrQrZAG112, 88, 88 ssrQrZAG7, 124, 124 Qden03011, 137, 137 Qden03021, 254, 254 Qden 05011, 175, 183 Q9 ssrQrZAG96, 147, 149 ssrQrZAG112, 88, 88 ssrQrZAG7, 124, 134 Qden03011, 137, 139 Qden03021, 252, 252 Qden 05011, 175, 175 Q10 ssrQrZAG96, 149, 149 ssrQrZAG112, 88, 98 ssrQrZAG7, 124, 134 Qden03011, 137, 139 Qden03021, 252, 254 Qden 05011, 177, 183 Q11 ssrQrZAG96, 149, 151 ssrQrZAG112, 88, 88 ssrQrZAG7, 124, 134 Qden03011, 137, 139 Qden03021, 252, 252 Qden 05011, 175, 183 Q12 ssrQrZAG96, 149, 151 ssrQrZAG112, 92, 92 ssrQrZAG7, 134, 146 Qden03011, 138, 140 Qden03021, 252, 252 Qden 05011, 183, 185 Q13 ssrQrZAG96, 151, 151 ssrQrZAG112, 92, 92 ssrQrZAG7, 134, 146 Qden03011, 140, 142 Qden03021, 252, 252 Qden 05011, 183, 185 Q14 ssrQrZAG96, 149, 149 ssrQrZAG112, 92, 94 ssrQrZAG7, 130, 134 Qden03011, 138, 142 Qden03021, 252, 254 Qden 05011, 183, 185 Q15 ssrQrZAG96, 151, 151 ssrQrZAG112, 92, 94 ssrQrZAG7, 130, 130 Qden03011, 138, 140 Qden03021, 254, 254 Qden 05011, 185, 185 Q16 ssrQrZAG96, 149, 151 ssrQrZAG112, 92, 94 ssrQrZAG7, 130, 138 Qden03011, 140, 155 Qden03021, 256, 256 Qden 05011, 183, 185 Q17 ssrQrZAG96, 149, 151 ssrQrZAG112, 92, 96 ssrQrZAG7, 134, 138 Qden03011, 138, 138 Qden03021, 252, 252 Qden 05011, 183, 183 Q18 ssrQrZAG96, 149, 151 ssrQrZAG112, 92, 94 ssrQrZAG7, 129, 131 Qden03011, 140, 142 Qden03021, 252, 252 Qden 05011, 183, 183 Q19 ssrQrZAG96, 149, 151 ssrQrZAG112, 92, 92 ssrQrZAG7, 127, 129 Qden03011, 136, 140 Qden03021, 252, 254 Qden 05011, 185, 185 Q20 ssrQrZAG96, 149, 151 ssrQrZAG112, 92, 92 ssrQrZAG7, 134, 144 Qden03011, 138, 140 Qden03021, 252, 252 Qden 05011, 183, 183 Q21 ssrQrZAG96, 151, 151 ssrQrZAG112, 92, 92 ssrQrZAG7, 130, 134 Qden03011, 136, 136 Qden03021, 252, 254 Qden 05011, 183, 183 Q22 ssrQrZAG96, 149, 151 ssrQrZAG112, 92, 92 ssrQrZAG7, 130, 134 Qden03011, 138, 142 Qden03021, 254, 254 Qden 05011, 174, 183 Q23 ssrQrZAG96, 149, 151 ssrQrZAG112, 92, 92 ssrQrZAG7, 124, 130 Qden03011, 142, 151 Qden03021, 254, 254 Qden 05011, 180, 183
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