EST-SSR analysis of genetic diversity of Robinia pseudoacacia clones in Jixian County, Shanxi Province of northern China
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摘要:目的了解各对EST-SSR引物检出的等位简单序列重复片段数量，解析吉县良种基地刺槐无性系种质的多态性状况与种质间亲缘关系，为引物利用和刺槐种质遗传管理提供参考。方法利用课题组开发的45对EST-SSR引物进行PCR扩增，使用毛细管电泳仪对产物进行检测，用Popgene 1.32版软件对群体遗传参数进行评估，利用NTsys 2.10e进行聚类分析。结果45对EST-SSR引物中，除了引物RP-24均表现出良好的多态性。44对引物共检测到等位重复序列298个，每对引物检测到2 ~ 15个，平均6.77个。群体观测杂合度（Ho）的变化范围为0.022 7 ~ 0.842 1，平均为0.412 6。期望杂合度（He）的变化范围为0.088 7 ~ 0.822 3，平均为0.484 0。引物多态性信息量（PIC）变化范围为0.085 2 ~ 0.796 6，平均为0.444 4。结论该基地的刺槐无性系种质具有较高的遗传多样性。由聚类结果可知，在遗传距离为0.81时，可将96份刺槐无性系种质聚成5类。聚类图在分子水平上显示了刺槐无性系种质间的亲缘关系，研究结果对于利用这些引物用于刺槐的种质评价、资源管理和辅助育种有积极的参考价值。Abstract:Objective This paper aims to understand the number of alleles of simple sequence repeats detected by EST-SSR primers, and to analyze the polymorphisms of germplasm of Robinia pseudoacacia clones in Ji xian County of Shanxi Province,northern China and the genetic relationship between germplasms, and provide reference for primer utilization and genetic management of Robinia pseudoacacia germplasm.Method 45 pairs of EST-SSR primers developed by our laboratory were used for PCR amplification, and the products were detected by capillary electrophoresis, the genetic parameters of population were evaluated by Popgene 1.32 software, and cluster analysis was performed by NTsys 2.10e.Result Except for primer RP-24, 45 pairs of EST-SSR primers showed good polymorphism. A total of 298 allelic repeat sequences were detected in 44 pairs of primers, and 2 to 15 were detected in each pair, with an average of 6.77. The observed heterozygosity (Ho) of the population ranged from 0.022 7 to 0.842 1, with an average of 0.412 6. The expected heterozygosity (He) varies from 0.088 7 to 0.822 3, averaging 0.484 0. Primer polymorphism information (PIC) ranged from 0.085 2 to 0.796 6, with an average of 0.444 4.Conclusion Robinia pseudoacacia clonal germplasm in this base has high genetic diversity. It can be seen from the clustering results that 96 genetic clones of the hedgehog clones can be clustered into five classes when the genetic distance is 0.81. Cluster maps showed the genetic relationship among the clones of Robinia pseudoacacia at the molecular level, and the results of the study have positive reference value for the use of these primers for germplasm evaluation, resource management and assisted breeding of Robinia pseudoacacia.
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Keywords:
- Robinia pseudoacacia /
- EST-SSR /
- genetic diversity /
- cluster analysis
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图 1 基于EST-SSR数据的96份刺槐资源聚类图
Figure 1. Dendrogram of 96 Robinia pseudoacacia resources based on EST-SSR data
表 1 试验所用EST-SSR引物信息
Table 1 Information of EST-SSR primers used in the experiment
位点 Locus 引物序列 Primer sequence (5′−3′) 重复基元 Repeat motif 大小 Size/bp 退火温度 Ta/℃ Rp-01 F: TGCAGAAAGAGAAAGCAGAGG （TGTGAA）4 140 58 R: CCGAACCCTTTCTGGTTAGTC Rp-02 F: GCTGCGTTTAATTTTGTCAGG （GAAT）4 170 58 R: TCAATCCATCAAAGAGGAAACA Rp-03 F: GTGAGAAGTGGTTAGGGTTTT （CTC）7 186 55 R: TCAAGATCACCAACGTACAA Rp-04 F: CTCGTGATGATGGTGTTGATG （AATGGT）4 146 58 R: AATGGTCCAAACAACACGAAG Rp-05 F: CCTTGCACATTTATCCCAGAA （TCTGGC）3 158 57 R: CGACCTCGATCTTTTCTTGTG Rp-06 F: TGGACAAAACATCATCGTGTG （TGAGTT）4 147 59 R: CTCTCTTCTTTCTGCCCCTCA Rp-07 F: TTTTTCTCCCAACGAAACAAA （CT）10 144 56 R: TGATGTGTTGTACGGAGGTGA Rp-08 F: TCAGGTGCATAAGCTCATTACTTC （AAAAT）4 152 56 R: GGTTGTCAGATGAAATGCACA Rp-09 F: CGTTTAGAAGCTGAGGCAGAA （CTTT）5 153 58 R: TGAGATATCTTAGTGCAGGAGCA Rp-10 F: GGCATGTGGCTATGAAGATGT （CCTTT）4 154 57 R: TCAGTGGGACTTGGTTTCTTG Rp-11 F: GAAGCTATCACCGCAAATGAA （AG）10 150 57 R: GTCGAAGTGCGTCCTAGATCA Rp-12 F: AAGAGTCATCACGGAGACCAA （AGCAGA）4 150 56 R: GGAGTCCAATTAAGTGCGAGA Rp-13 F: CATTTCCGATTTCCAATTCCT （CTCTTC）4 151 56 R: GCCGAGGACTCGGTAGAAGT Rp-14 F: TTAGCACGAACCTGGTTATGG （TGCAAC）4 151 56 R: CACTTCATTTGGTTCCTTGAGA Rp-15 F: TTAACTAATGCGGCGAGAAGA （TCAC）5 119 56 R: GAGAGGAAGTGTGCGAAACAA Rp-16 F: TATGAGACAGTGTTGGTTGGT （TTCAGT）4 175 56 R: CGTGCCAGAAGAGTATAACAG Rp-17 F: GTAAGTCTGCAAAGAAGACCA （AACCA）4 150 56 R: GCTTTTCACCTATCAACTCAA Rp-18 F: GGATGAACTTTGGCAATCCTT （GGTCAG）4 158 55 R: AATTTGTTGGGAATGCTGTTG Rp-19 F: CAGGAGTGGCAGCATTAGTGT （AGGCTG）4 123 56 R: CACAACAAGCACATTTTGCAC Rp-20 F: TTTCTTGGCTTGCTTTTGCTA （GCAGCT）3 145 56 R: TCTTGGATACGCAAGGTTGTC Rp-21 F: TATGATCACGTCCCCTAATGC （CCA）7 146 57 R: AAGTGGAAAGAAATGGGATGG Rp-22 F: GGTAAGGTGAAGGAGGTGGAG （AGGGTT）4 150 56 R: AGCTTGGTCTCCTAGGTCGTC Rp-23 F: GGAGGAGCAACCATCTGTGTA （AGAAGT）4 146 56 R: CTCCCTCTTCATCCTCACCTC Rp-24 F: TGCACATATTTGCCTGGTTTA （AATA）4 160 56 R: AAAATGAGCATGACACAACCA Rp-25 F: CGGCAACAAGTTGAGAAGAAC （AAAG）5 139 56 R: GGCTCACAAACCAACCTATGA Rp-26 F: GCTGCAAGCAAAGGATCTTAC （ATGATA）4 139 56 R: CCTCATCATCCTCGTCATCAT Rp-27 F: TGGACAAAACATCATCGTGTG （TGAGTT）4 147 57 R: CTCTCTTCTTTCTGCCCCTCA Rp-28 F: CTTGGTCTAGAAAGTCCTGCT （CAG）7 151 56 R: GGTCATCAAGGTTAGTTGGAT Rp-29 F: CCTGATGATCAAAACGACGAC （GATC）4 148 56 R: GGAGGTGACCCCTCTTATCCT Rp-30 F: TTGAACCAAAACTGGAAGAGC （GCT）8 151 56 R: GCACCGTACAGTTACCCTATCC Rp-31 F: GACCCCATTTTTCTCAAGGAC （ATT）7 140 56 R: TTGGATAAGTCGGTGAAGGTG Rp-32 F: CCACGTGGTTCTTCAAACATT （GTG）7 163 57 R: CAACAACAACCCACAAACACA Rp-33 F: CAAACAGTCTCATGGAAATGGA （ATC）7 141 56 R: GGGTTGGTATTGTTGGGAAAT Rp-34 F: AGGATATTAGCCAAGTCCATC （TGGTGA）4 164 57 R: AGTAACCATCACCACAATCAC Rp-35 F: TCAGACGTGGTAGAGCAGTGTT （CACAC）4 152 58 R: ATTTGTTTTTGGGGGAGATTG Rp-36 F: CGTTTCAGCCATTGATTTTGT （GAATC）5 141 57 R: GATCATCACCGTCCACCTTC Rp-37 F: TGTCGTCATTTTATTTTACCC （GAACGA）4 152 56 R: CTCACCCTTTTTATTTCCATT Rp-38 F: TCCATTCCCTGGTTTCTTCTT （TC）10 150 56 R: AGCACAATTTCCTCAGTGCAG Rp-39 F: TTAAAGAATGTTCCGTTCAGA （AAGAGG）3 152 56 R: GAGAAGATAGCCTCCTAGCTG Rp-40 F: TCATTGGACATCCCTCCATAA （TAA）8 139 56 R: GGCTCGACATGGTTGATTTT Rp-41 F: AACTCACCCAATTGCACACTC （CCA）7 143 56 R: GAGCAAGAGCTAAAGCAGCAA Rp-42 F: CTTCGCAATCCTCACTCTTTG （AATC）4 169 55 R: CTTACCCAGAAGCCAACAATG Rp-43 F: CAAAGCAGAGAGAATGTATGG （CAAAAT）4 155 57 R: ATCCCTTGCTCCTTGTAATAG Rp-44 F: TATCTGGGAGAATCGAGAGCA （ATCA）5 145 57 R: CCACCATGGTTGTCCTTCTAA Rp-45 F: GGGTTGAGGAAGAGAGGAGAA （TTC）7 156 57 R: AAAAATCGAATCGTGTTGGTG 
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表 2 44对引物扩增结果及多态性信息
Table 2 Amplification results and polymorphic information of 44 pairs of primers
引物 Primer 观测杂合度 Ho 期望杂合度 He Nei氏指数 Nei 等位重复序列 Na 有效等位重复序列 Ne Shannon信息指数 I 引物多态性信息量 PIC RP-01 0.623 7 0.629 4 0.626 0 6 2.674 0 1.152 8 0.557 1 RP-02 0.106 7 0.125 3 0.124 4 2 1.142 1 0.244 9 0.116 8 RP-03 0.641 3 0.628 1 0.624 6 11 2.664 1 1.329 8 0.571 7 RP-04 0.781 6 0.790 8 0.786 2 8 4.678 0 1.638 4 0.752 4 RP-05 0.540 2 0.673 0 0.669 2 15 3.022 8 1.680 0 0.652 5 RP-06 0.734 0 0.738 4 0.734 4 7 3.765 6 1.445 5 0.690 5 RP-07 0.842 1 0.816 8 0.811 5 11 5.303 9 1.892 0 0.788 2 RP-08 0.505 4 0.726 6 0.722 7 7 3.606 8 1.426 1 0.679 1 RP-09 0.648 9 0.660 8 0.657 3 15 2.918 1 1.529 9 0.615 7 RP-10 0.433 0.422 9 0.420 6 5 1.725 8 0.798 4 0.375 7 RP-11 0.719 1 0.822 3 0.817 7 14 5.485 5 2.018 3 0.796 6 RP-12 0.750 0 0.776 8 0.772 7 10 4.400 1 1.646 2 0.737 7 RP-13 0.315 8 0.362 2 0.360 3 9 1.563 2 0.878 1 0.350 0 RP-14 0.395 6 0.708 7 0.704 8 10 3.387 6 1.556 5 0.671 7 RP-15 0.416 7 0.371 6 0.369 6 8 1.586 4 0.802 4 0.345 2 RP-16 0.162 8 0.173 2 0.172 2 4 1.208 0 0.374 9 0.163 6 RP-17 0.250 0 0.247 7 0.246 4 7 1.327 0 0.572 3 0.236 1 RP-18 0.434 2 0.599 3 0.595 4 7 2.471 5 1.085 8 0.512 3 RP-19 0.446 8 0.610 6 0.607 4 7 2.547 1 1.118 6 0.528 9 RP-20 0.046 0 0.088 7 0.088 2 3 1.096 7 0.203 8 0.085 2 RP-21 0.246 8 0.280 6 0.278 8 6 1.386 6 0.624 5 0.266 4 RP-22 0.602 2 0.595 9 0.592 7 5 2.455 0 1.141 4 0.546 7 RP-23 0.341 2 0.502 1 0.499 1 6 1.996 4 0.957 2 0.446 5 RP-25 0.104 2 0.139 2 0.138 5 5 1.160 7 0.345 2 0.134 9 RP-26 0.212 8 0.214 0 0.212 8 6 1.270 4 0.472 0 0.201 0 RP-27 0.118 3 0.112 5 0.111 9 3 1.126 0 0.242 6 0.106 6 RP-28 0.651 7 0.719 5 0.715 4 7 3.514 2 1.436 0 0.671 6 RP-29 0.022 7 0.107 8 0.107 2 2 1.120 0 0.218 1 0.101 4 RP-30 0.268 8 0.416 6 0.414 3 5 1.707 4 0.775 8 0.367 3 RP-31 0.376 6 0.561 2 0.557 6 10 2.260 4 1.269 3 0.532 9 RP-32 0.511 9 0.635 9 0.632 1 7 2.718 0 1.246 2 0.578 3 RP-33 0.557 9 0.444 1 0.441 7 4 1.791 2 0.696 4 0.357 3 RP-34 0.500 0 0.523 5 0.520 6 8 2.086 0 1.106 7 0.485 3 RP-35 0.375 0 0.565 1 0.561 9 5 2.282 7 0.987 2 0.486 1 RP-36 0.512 2 0.661 5 0.657 4 4 2.919 0 1.167 6 0.590 9 RP-37 0.612 9 0.665 1 0.661 5 6 2.954 4 1.353 5 0.628 0 RP-38 0.556 8 0.774 9 0.770 5 15 4.357 9 1.895 0 0.741 3 RP-39 0.392 4 0.592 3 0.588 5 5 2.430 3 1.021 3 0.507 8 RP-40 0.178 9 0.182 0 0.181 0 3 1.221 0 0.345 7 0.166 4 RP-41 0.315 8 0.331 3 0.329 5 6 1.491 5 0.706 5 0.311 7 RP-42 0.281 7 0.459 5 0.456 3 2 1.839 1 0.648 7 0.352 2 RP-43 0.221 1 0.305 3 0.303 7 4 1.436 1 0.613 7 0.285 1 RP-44 0.081 4 0.133 3 0.132 6 4 1.152 8 0.317 1 0.129 0 RP-45 0.315 2 0.398 4 0.396 2 4 1.656 2 0.661 0 0.333 4 总计 Total 298 104.907 6 均值 Mean 0.412 6 0.484 0 0.481 2 6.772 7 2.384 3 0.991 9 0.444 4
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