商城肥鲵二碱基重复和四碱基重复微卫星DNA的结构特征及对筛选效率的影响

doi:10.3724/SP.J.1003.2012.08168

生物多样性 ›› 2012, Vol. 20 ›› Issue (1): 51-58. DOI: 10.3724/SP.J.1003.2012.08168 cstr: 32101.14.SP.J.1003.2012.08168

商城肥鲵二碱基重复和四碱基重复微卫星DNA的结构特征及对筛选效率的影响

王慧¹, 张保卫¹^,^*(), 史文博¹, 骆侠¹, 周立志¹, 韩德民¹, 常青²

1 安徽大学生命科学学院, 合肥 230039
2 南京师范大学生命科学学院, 南京 210046

收稿日期:2011-09-02 接受日期:2011-10-16 出版日期:2012-01-20 发布日期:2012-02-14
通讯作者: 张保卫
作者简介:*E-mail: zhangbw@ahu.edu.cn
基金资助:
国家自然科学基金(30800116);生物多样性与生物技术江苏省重点实验室开放基金(B200712-01);安徽大学青年骨干教师计划

Structural characteristics of di-nucleotide/tetra-nucleotide repeat microsatellite DNA in Pachyhynobius shangchengensisgenomes and its effect on isolation

Hui Wang¹, Baowei Zhang¹^,^*(), Wenbo Shi¹, Xia Luo¹, Lizhi Zhou¹, Demin Han¹, Qing Chang²

1 School of Life Sciences, Anhui University, Hefei 230039
2 School of Life Sciences, Nanjing Normal University, Nanjing 210046

Received:2011-09-02 Accepted:2011-10-16 Online:2012-01-20 Published:2012-02-14
Contact: Baowei Zhang

摘要/Abstract

摘要：

两栖类有尾目物种的微卫星分离中的筛选成功率常常较低。为探索微卫星结构对筛选效率的影响, 本研究通过AFLP快速分离法(fast isolation by AFLP of sequences containing repeats, FIASCO)对商城肥鲵(Pachyhynobius shangchengensis)二碱基重复类型和四碱基重复类型微卫星进行分离, 并对微卫星序列进行了分析。研究中发现二碱基微卫星位点多以微卫星DNA家族形式存在, 并因此导致了微卫星位点分离较低的筛选率; 在四碱基重复的微卫星位点中未发现微卫星DNA家族的存在。对研究中得到的3个微卫星DNA家族的分析发现, 同一家族的上、下游侧翼序列变异程度存在差异; 毗邻微卫星重复单元区的侧翼序列碱基变异程度较高, 而较远处的区段则相对保守。这些结构特征可能反映出微卫星DNA家族在演化中的复杂性。本文的研究结果提示在两栖动物的一些类群中, 微卫星的筛选必须考虑微卫星DNA家族的影响, 选取适宜的碱基重复类型将是决定筛选效率的关键。

关键词: 微卫星结构, 微卫星DNA家族, Pachyhynobius shangchengensis, 筛选效率

Abstract

A low success ratio is often encountered in the microsatellite isolation from the species of Caudata. In the present study, di-nucleotide/tetra-nucleotide repeat microsatellite loci were isolated following the FLASCO (fast isolation by AFLP of sequences containing repeats) approach and the sequences were analyzed for the purpose of learning how microsatellite structure impacted on isolation efficiency. We found that most di-nucleotide repeat microsatellite loci were involved in the microsatellite DNA family, which resulted in low isolation efficiency. In contrast, no microsatellite DNA family members were found in the tetra-nucleotide repeat microsatellite loci. On the basis of analysis of the three microsatellite DNA families found in the present study, there were significant differences in the extent of variation between the upstream and downstream flanking sequences in same microsatellite DNA family. This variation was the highest in the sequence sections adjacent to the core repeated units, but more conservative in other regions, which might reflect the complexity of the microsatellite DNA family evolution. These results suggest that in the process of microsatellite development for Caudata, we must pay attention to interference from microsatellite DNA family evolution. The choice of appropriate nucleotide repeats is key to obtaining a high isolation efficiency.

Key words: microsatellite, structure characteristics, microsatellite DNA family, Pachyhynobius shangchengensis, isolation efficiency

王慧, 张保卫, 史文博, 骆侠, 周立志, 韩德民, 常青 (2012) 商城肥鲵二碱基重复和四碱基重复微卫星DNA的结构特征及对筛选效率的影响. 生物多样性, 20, 51-58. DOI: 10.3724/SP.J.1003.2012.08168.

Hui Wang, Baowei Zhang, Wenbo Shi, Xia Luo, Lizhi Zhou, Demin Han, Qing Chang (2012) Structural characteristics of di-nucleotide/tetra-nucleotide repeat microsatellite DNA in Pachyhynobius shangchengensisgenomes and its effect on isolation. Biodiversity Science, 20, 51-58. DOI: 10.3724/SP.J.1003.2012.08168.

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链接本文: https://www.biodiversity-science.net/CN/10.3724/SP.J.1003.2012.08168

https://www.biodiversity-science.net/CN/Y2012/V20/I1/51

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参考文献 28

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序列组 Sequence set	包含的序列数目 Sequence number	长度范围 Size range	序列间的遗传距离范围 Range of genetic distance among sequence	序列间平均的遗传距离 Average genetic distance among sequence
Psmf1上游侧翼序列 Upstream flanking sequence ofPsmf1	11	165-438	0.016-0.084	0.042
Psmf1下游侧翼序列 Downstream flanking sequence ofPsmf1	11	52-482	-	-
Psmf2上游侧翼序列 Upstream flanking sequence ofPsmf2	4	277-297	0.109-0.253	0.174
Psmf2下游侧翼序列 Downstream flanking sequence ofPsmf2	4	79-140	0.090-0.295	0.159
Psmf3上游侧翼序列 Upstream flanking sequence ofPsmf3	26	314-500	0.021-0.416	0.168
Psmf3下游侧翼序列 Downstream flanking sequence ofPsmf3	26	60-85	0-0.210	0.091

序列组 Sequence set	包含的序列数目 Sequence number	长度范围 Size range	序列间的遗传距离范围 Range of genetic distance among sequence	序列间平均的遗传距离 Average genetic distance among sequence
Psmf1上游侧翼序列 Upstream flanking sequence ofPsmf1	11	165-438	0.016-0.084	0.042
Psmf1下游侧翼序列 Downstream flanking sequence ofPsmf1	11	52-482	-	-
Psmf2上游侧翼序列 Upstream flanking sequence ofPsmf2	4	277-297	0.109-0.253	0.174
Psmf2下游侧翼序列 Downstream flanking sequence ofPsmf2	4	79-140	0.090-0.295	0.159
Psmf3上游侧翼序列 Upstream flanking sequence ofPsmf3	26	314-500	0.021-0.416	0.168
Psmf3下游侧翼序列 Downstream flanking sequence ofPsmf3	26	60-85	0-0.210	0.091

位点名称 Loci	核心序列重复单元 Repeated core units	引物序列(上游/下游) Primer sequence (Upper primer/down primer)	扩增结果 PCR result
Psh110	AC₁₆	5′-CACCGCTGACCTGGACACTT-3′ ∕ 5′-GTGACTGACAGCTAGGGAAG-3′	单带 Single band
Psh116	AC₂₁	5′-CAAGAGGCAATGAATGTGAG-3′ ∕ 5′-TACTATGTCCCATCTTC-3′	单带 Single band
Psh118	AC₁₂	5′-GGTGGTTCTCTGGAGTGTTG-3′ ∕ 5′-TGGTCCTCCAGTCTCACAGC-3′	单带 Single band
Psh124	AC₁₅	5′-CACGGATCTCGGAAGCT-3′ ∕ 5′-AGAATAAAGTAACCTCAATG-3′	单带 Single band
Psh131	AC₁₆	5′-TATTCCCTTTCCTACTCCT-3′ ∕ 5′-AACAGACCCATGATAAGAGC-3′	单带 Single band
Psh135	AC₂₂	5′-AGGAAAGAGCTCCACCAAG-3′ ∕ 5′-GTGAAAGAAGAGAAGCAGAG-3′	单带 Single band
Psh138	AC₂₉	5′-CTCCTATTTCCTCCTCAGAC-3′ ∕ 5′-AAGGGTGTAGGCTGAGAG-3′	弥散带 Smear
Psh143	AC₂₁	5′-TGTGTGGGCTCTGCATTTAC-3′ ∕ 5′-CAGGATGGGGGAAAATATAC-3′	单带 Single band
Psh209	AG₁₉	5′-CACATTTATACGGACACCAC-3′ ∕ 5′-TCCACATTTTTACAGCCAGG-3′	单带 Single band
Psh211	AG₂₈	5′-TGAGGAGTTCCCCTAAGCAC-3′ ∕ 5′-GGTGAACGGAAAAGGATTAC-3′	多带 Multi-band
Psh216	AG₂₇	5′-AACAGAGGCAATGTCCACAC-3′ ∕ 5′-CAACAGTCTGCATGTGAAAG-3′	单带 Single band
Psh229	AG₄₃	5′-TTTTTGGGACTCTGCCTCTC-3′ ∕ 5′-CTATAGGGCTATGAAG-3′	无法扩增 No band
Psh230	AG₂₇	5′-ACAGGGTAGGAAGGCAGTAG-3′ ∕ 5′-AGTAAGGGAGAGAAAAAAGC-3′	弥散带 Smear
Psh238	AG₂₈	5′-TCGCTCGCTCACCCATTCAC-3′ ∕ 5′-GCAGGGGATTGAGATAGTTG-3′	多带 Single band
Psh245	AG₅₃	5′-CTGTTTCTTTAGAGTGAGTG-3′ ∕ 5′-GGTAGCAAGTTTGATAAGTG-3′	单带 Single band
Psh246	AG₃₄	5′-TAGGCATCAACTAAGGGGAC-3′ ∕ 5′-TGTTCCATATTTGGGACTGG-3′	单带 Single band
Psh248	AG₃₇	5′-AGGTATTGTGCGGAACGATC-3′ ∕ 5′-TGGACTGTTTTTGCCACCAG-3′	单带 Single band
Psh228	AG₄₀	5′-GAAAATGGAGAGGGATGAAC-3′ ∕ 5′-ACAGTCTGCATGAGAAAGGC-3′	单带 Single band
Psh150	AC₃₀	5′-TAAATGTGAGTCCAGCAAAC-3′ ∕ 5′-CGATTTGAGTATTTGGGTAG-3′	单带 Single band
Psh274	AG₅₂	5′-GGTGAGCCCTTGAGTTTGAG-3′ ∕ 5′-GCCGCTTCACATTAGTTTTC-3′	无法扩增 No band
Psh207	AG₂₄	5′-TCTTTGTTGAGACTCGGGAC-3′ ∕ 5′-AAAGCAAAAGGCAAACC-3′	弥散带 Smear

位点名称 Loci	核心序列重复单元 Repeated core units	引物序列(上游/下游) Primer sequence (Upper primer/down primer)	扩增结果 PCR result
Psh110	AC₁₆	5′-CACCGCTGACCTGGACACTT-3′ ∕ 5′-GTGACTGACAGCTAGGGAAG-3′	单带 Single band
Psh116	AC₂₁	5′-CAAGAGGCAATGAATGTGAG-3′ ∕ 5′-TACTATGTCCCATCTTC-3′	单带 Single band
Psh118	AC₁₂	5′-GGTGGTTCTCTGGAGTGTTG-3′ ∕ 5′-TGGTCCTCCAGTCTCACAGC-3′	单带 Single band
Psh124	AC₁₅	5′-CACGGATCTCGGAAGCT-3′ ∕ 5′-AGAATAAAGTAACCTCAATG-3′	单带 Single band
Psh131	AC₁₆	5′-TATTCCCTTTCCTACTCCT-3′ ∕ 5′-AACAGACCCATGATAAGAGC-3′	单带 Single band
Psh135	AC₂₂	5′-AGGAAAGAGCTCCACCAAG-3′ ∕ 5′-GTGAAAGAAGAGAAGCAGAG-3′	单带 Single band
Psh138	AC₂₉	5′-CTCCTATTTCCTCCTCAGAC-3′ ∕ 5′-AAGGGTGTAGGCTGAGAG-3′	弥散带 Smear
Psh143	AC₂₁	5′-TGTGTGGGCTCTGCATTTAC-3′ ∕ 5′-CAGGATGGGGGAAAATATAC-3′	单带 Single band
Psh209	AG₁₉	5′-CACATTTATACGGACACCAC-3′ ∕ 5′-TCCACATTTTTACAGCCAGG-3′	单带 Single band
Psh211	AG₂₈	5′-TGAGGAGTTCCCCTAAGCAC-3′ ∕ 5′-GGTGAACGGAAAAGGATTAC-3′	多带 Multi-band
Psh216	AG₂₇	5′-AACAGAGGCAATGTCCACAC-3′ ∕ 5′-CAACAGTCTGCATGTGAAAG-3′	单带 Single band
Psh229	AG₄₃	5′-TTTTTGGGACTCTGCCTCTC-3′ ∕ 5′-CTATAGGGCTATGAAG-3′	无法扩增 No band
Psh230	AG₂₇	5′-ACAGGGTAGGAAGGCAGTAG-3′ ∕ 5′-AGTAAGGGAGAGAAAAAAGC-3′	弥散带 Smear
Psh238	AG₂₈	5′-TCGCTCGCTCACCCATTCAC-3′ ∕ 5′-GCAGGGGATTGAGATAGTTG-3′	多带 Single band
Psh245	AG₅₃	5′-CTGTTTCTTTAGAGTGAGTG-3′ ∕ 5′-GGTAGCAAGTTTGATAAGTG-3′	单带 Single band
Psh246	AG₃₄	5′-TAGGCATCAACTAAGGGGAC-3′ ∕ 5′-TGTTCCATATTTGGGACTGG-3′	单带 Single band
Psh248	AG₃₇	5′-AGGTATTGTGCGGAACGATC-3′ ∕ 5′-TGGACTGTTTTTGCCACCAG-3′	单带 Single band
Psh228	AG₄₀	5′-GAAAATGGAGAGGGATGAAC-3′ ∕ 5′-ACAGTCTGCATGAGAAAGGC-3′	单带 Single band
Psh150	AC₃₀	5′-TAAATGTGAGTCCAGCAAAC-3′ ∕ 5′-CGATTTGAGTATTTGGGTAG-3′	单带 Single band
Psh274	AG₅₂	5′-GGTGAGCCCTTGAGTTTGAG-3′ ∕ 5′-GCCGCTTCACATTAGTTTTC-3′	无法扩增 No band
Psh207	AG₂₄	5′-TCTTTGTTGAGACTCGGGAC-3′ ∕ 5′-AAAGCAAAAGGCAAACC-3′	弥散带 Smear

商城肥鲵二碱基重复和四碱基重复微卫星DNA的结构特征及对筛选效率的影响

Structural characteristics of di-nucleotide/tetra-nucleotide repeat microsatellite DNA in Pachyhynobius shangchengensisgenomes and its effect on isolation

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