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利用ISSR和SRAP标记分析白及种质资源遗传多样性
doi: 10.11833/j.issn.2095-0756.20210690
Analysis of genetic diversity of Bletilla striata germplasm by ISSR and SRAP markers
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摘要:
目的 采用简单重复序列间扩增(ISSR)和相关序列扩增多态性(SRAP)等2种标记技术分析不同种源白及Bletilla striata样本的遗传多样性水平和遗传关系，为白及种质的鉴定、分类、保护和开发提供理论依据。 方法 从100个ISSR引物和238对SRAP引物组合中筛选出多态性高、扩增条带清晰、重复性好的引物进行聚合酶链式反应(PCR)扩增，用Popgene 32.0计算来自浙江、云南、贵州和四川等省32个不同种源白及的遗传多样性参数和遗传距离，用NTSYS-pc 2.10e进行聚类分析。 结果 从100个ISSR引物中筛选出11个多态性较好的引物，共扩增出188个条带，平均每个引物扩增出17.09个条带，其中多态性位点174个，占总扩增片段的92.20%；从238对SRAP引物组合中筛选出11对多态性较好的引物组合，共扩增出216个条带，平均每个引物扩增出19.64个条带，其中多态性位点202个，占总扩增片段的93.52%。综合ISSR和SRAP的标记结果发现：四川白及种源的遗传多样性水平最高，贵州最低；非加权组平均法(UPGMA聚类)和主坐标分析(PCoA分析)结果显示：聚为一类的白及种源大多来自同一省份，云南省与四川省白及种源的遗传距离较近，浙江省和贵州省白及种源的遗传距离较近，说明遗传距离与地理距离存在一定的重合，但并不呈正相关。 结论 本研究所选白及种源间具有较高的遗传多样性，ISSR和SRAP标记技术均可有效揭示白及的遗传多样性和亲缘关系。图4表4参25 -
关键词:
- 白及 /
- 遗传多样性 /
- 简单重复序列间扩增(ISSR)标记 /
- 相关序列扩增多态性(SRAP)标记
Abstract:Objective The objective is to analyze the genetic diversity and genetic relationship of 32 Bletilla striata samples from different provenances by ISSR and SRAP markers, so as to provide theoretical basis for identification, classification, conservation and development of B. striata germplasm. Method Primers with high polymorphism, clear amplification bands and good repeatability were selected from 100 ISSR primers and 238 pairs of SRAP primers for polymerase chain reaction (PCR) amplification. Genetic diversity parameters and genetic distance of B. striata from 32 different provenances in Zhejiang, Yunnan, Guizhou and Sichuan were calculated by Popgene 32.0, and cluster analysis was performed by NTSYS-PC 2.10e. Result 11 highly polymorphic primers were screened from 100 ISSR primers, and a total of 188 bands were amplified, with an average of 17.09 bands per primer, among which 174 were polymorphic loci, accounting for 92.20% of the total amplified fragments. 11 pairs of highly polymorphic primer pairs were screened from 238 pairs of SRAP primer pairs, and a total of 216 bands were amplified, with an average of 19.64 bands per primer, including 202 polymorphic loci, accounting for 93.52% of the total amplified fragments. Based on ISSR and SRAP markers, the genetic diversity level of B. striata population in Sichuan Province was the highest, while that in Guizhou Province was the lowest. UPGMA and PCoA analysis showed that the clustered B. striata samples were mostly from the same province. The genetic distance of B. striata population between Yunnan Province and Sichuan Province was relatively close, and that between Zhejiang Province and Guizhou Province was relatively close, indicating that there was a certain overlap between genetic distance and geographical distance, but there was no positive correlation. Conclusion B. striata provenances selected in this study have high genetic diversity. Both ISSR and SRAP markers can effectively reveal the genetic diversity and genetic relationship of B. striata. [Ch, 4 fig. 4 tab. 25 ref.] -
Key words:
- Bletilla striata /
- genetic diversity /
- ISSR marker /
- SRAP marker
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图 1 引物UBC876对所采集白及样本的ISSR-PCR扩增结果
Figure 1 Amplification results of B. striata with primer UBC876 of ISSR
图 2 引物组合Me9-Em13对所采集白及样本的SRAP-PCR扩增结果
Figure 2 Amplification results of B. striata with primer combination Me9-Em13 of SRAP
表 1 白及种源信息
Table 1. Source information of B. striata samples
编号 种源地 编号 种源地 编号 种源地 编号 种源地 1 浙江温州泰顺 9 云南丽江永胜 17 四川乐山沐川(2) 25 四川汶川水磨(2) 2 浙江台州天台 10 云南普洱思茅 18 四川雅安石棉 26 四川汶川水磨(3) 3 浙江衢州江山 11 云南曲靖会泽 19 四川内江东兴(1) 27 四川都江堰虹口(1) 4 浙江金华磐安 12 贵州遵义正安 20 四川内江东兴(2) 28 四川都江堰虹口(2) 5 浙江宁波象山 13 贵州黔西南安龙 21 四川内江东兴(3) 29 四川成都大邑 6 浙江杭州临安 14 贵州遵义赤水 22 四川内江东兴(4) 30 四川成都彭州(1) 7 云南红河蒙自(1) 15 贵州毕节大方 23 四川内江东兴(5) 31 四川成都彭州(2) 8 云南红河蒙自(2) 16 四川乐山沐川(1) 24 四川汶川水磨(1) 32 四川甘孜康定 
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表 2 ISSR引物信息及扩增结果
Table 2. ISSR primer information and amplification results
引物编号 ISSR引物 序列
(5′→3′)扩增条
带数/条多态性条
带数/条PPB/% 引物编号 ISSR引物 序列
(5′→3′)扩增条
带数/条多态性条
带数/条PPB/% 1 UBC823 (TC)8C 15 12 80.00 8 UBC876 (GATA)2(GACA)2 25 25 100.00 2 UBC826 (AC)8C 18 17 94.44 9 UBC879 C(TTCAC)3 15 15 100.00 3 UBC827 (AC)8G 16 16 100.00 10 UBC880 (GGAGA)3 17 11 64.71 4 UBC842 (GA)8(C/T)G 18 18 100.00 11 UBC881 (GGGTG)3 20 20 100.00 5 UBC855 (AC)8(C/T)T 9 9 100.00 合计 188 174 6 UBC866 (CTC)6 15 12 80.00 平均 17.09 15.82 92.20 7 UBC868 (GAA)6 20 19 95.00
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表 3 SRAP引物信息及扩增结果
Table 3. SRAP primer information and amplification results
引物
编号SRAP引物 正向引
物(5′→3′)反向引
物(5′→3′)扩增条
带数/条多态性条
带数/条PPB/% 引物
编号SRAP引物 正向引
物(5′→3′)反向引
物(5′→3′)扩增条
带数/条多态性条
带数/条PPB/% 1 Me1-Em12 BATA DCAT 19 17 89.47 8 Me11-Em12 BACG DCAT 18 15 83.33 2 Me3-Em2 BAAT DTGC 22 20 90.91 9 Me12-Em6 BAAA DGCA 16 16 100.00 3 Me5-Em5 BAAG DAAC 18 17 94.44 10 Me12-Em7 BAAA DCAA 17 16 94.12 4 Me6-Em2 BACT DTGC 19 16 84.21 11 Me13-Em9 BAAC DCGA 28 27 96.43 5 Me9-Em13 BACA DCTA 18 18 100.00 合计 216 202 6 Me9-Em14 BACA DCTC 19 18 94.74 平均 19.64 18.36 93.52 7 Me11-Em10 BACG DCAG 22 22 100.00 说明：B表示TGAGTCCAAACCGG；D表示GACTGCGTACGAATT
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表 4 白及的遗传多样性
Table 4. Genetic diversity of B. striata
研究方法 种源地 Na Ne H I PPB/% 浙江省 1.5000 1.3215 0.1841 0.2729 50.00 ISSR 云南省 1.6489 1.4042 0.2328 0.3473 64.89 贵州省 1.5000 1.3176 0.1824 0.2717 50.00 四川省 1.7926 1.4393 0.2607 0.3940 79.26 群体水平 1.6104 1.3707 0.2150 0.3215 61.04 物种水平 1.9255 1.4619 0.2819 0.4327 92.55 SRAP 浙江 1.5833 1.3753 0.2153 0.3191 58.33 云南 1.6713 1.4163 0.2432 0.3631 67.13 贵州 1.5000 1.3373 0.1923 0.2835 50.00 四川 1.8704 1.4891 0.2886 0.4357 87.04 群体水平 1.6563 1.4045 0.2349 0.3504 65.63 物种水平 1.9352 1.5309 0.3103 0.4667 93.52 ISSR+SRAP 浙江 1.5446 1.3503 0.2007 0.2976 54.46 云南 1.6609 1.4107 0.2384 0.3558 66.09 贵州 1.5000 1.3281 0.1877 0.2780 50.00 四川 1.8342 1.4660 0.2756 0.4163 83.42 群体水平 1.6349 1.3888 0.2256 0.3369 63.49 物种水平 1.9307 1.4988 0.2971 0.4509 93.07
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