| 张小芳1,2 杨淑慧1 马跃1,2 徐艳春1,2△ 宋伟杰1.微卫星复杂结构对分型的影响：以熊UamD116 和UamB1 为例[J].,2012,12(20):3812-3816 |
| 微卫星复杂结构对分型的影响：以熊UamD116 和UamB1 为例 |
| The Impacts of Complex Allele Structure on Genotyping Accuracy ofMicrosatellite: a Lesson from Bear Microsatellites UamD116 and UamB1 |
| |
| DOI： |
| 中文关键词: 微卫星 分型 等位基因结构 亚洲黑熊 Ursus thibetanus |
| 英文关键词: Microsatellite Genotyping Allele structure Asiatic black bear Ursus thibetanus |
| 基金项目:国家林业局野生动物保护与繁育项目（2008）；中央高校基本科研业务费专项资金项目（04004）；
黑龙江省青年科学基金项目（QC05C72） |
|
| 摘要点击次数: 885 |
| 全文下载次数: 1002 |
| 中文摘要: |
| 目的：微卫星是基因组上的短串联重复序列，具有高度多态性，表现为核心序列中重复单位的重复次数的变化，这种变化造
成不同等位基因核心序列的长度不同。因此，其基因型主要依靠PCR 扩增片段长度来判定。在各类研究中，人们更倾向于使用4
碱基重复的微卫星以减少2 碱基微卫星的stutter 等问题的影响。但是4 碱基微卫星核心序列结构复杂时，就会对分型的正确性
产生影响，从而影响到下游分析的正确性。在很多野生动物的研究中，这一问题常常被忽略。本文以亚洲黑熊（Ursus thibetanus）的
2 个四碱基微卫星位点UamD116 和UamB1 为例，揭示内部结构对分型的影响。方法：我们选用96 份亚洲黑熊样品（包括血液、
肌肉组织和毛发等样品）进行微卫星分型研究，通过荧光标记的PCR 扩增和毛细管电泳分型，比较了基于扩增片段长度的分型和
基于序列核心结构的分型效果的差异。结果：UamD116 核心序列结构除了含有多种不同的重复单位外，还在重复单位之间有碱
基插入，出现单碱基T、二碱基TC 和三碱基AAG 插入；并在一类等位基因下游侧翼序列有1 个GA 缺失。基于序列结构的分型
中可以将不同的等位基因分开，而在基于片段长度的分型中，容易将不同的等位基因合并为1 个等位基因。在位点UamB1 共发
现两种类型的等位基因，在一类等位基因中出现一个3bp 的插入，使等位基因之间的差异不再是4bp，而是1bp。在仅依据片段长
度分型时，相差1bp 的等位基因被认定为1 个。此外，还有不同等位基因核心序列不同，但是二者长度完全一致。依据片段长度分
型共发现8 个等位基因，而经过序列分型确定的等位基因数为12 个，相应地基因频率及其他遗传学参数都发生相应的改变。结
论：对于核心序列结构复杂的微卫星必须通过等位基因测序来矫正片段长度分型的结果，才能得到可靠的群体遗传学结论。 |
| 英文摘要: |
| Objective: Microsatellite refers to a kind of polymorphic DNA fragments on the genome which contains variable
number of tandem repeats of a short specific sequence of nucleotides (motif). The polymorphism of micorsatellite is reflected by variation
of repeating number of motifs resulting in variation of fragment length among alleles. Therefore, routine genotyping approach is based on
the fragment length of amplified alleles using polymerase chain reaction (PCR) method. Tetra-nucleotide microsatellites are preferable than
di-nucleotide ones because of the influence of stutters on genotyping of the later. However, tetra-nucleotide microsatellites often contain
complex motif structure potentially impacting genotyping and such problem is usually ignored in studies. To address the impacts of complex
allele structure on the genotyping, we genotyped two tetra-nucleotide loci of the Asiatic black bear (Ursus thibetanus), UamD116
and UamB1, using fragment length based approach and sequence based approach, and compared the results. Methods：A total of 96 samples
including muscle, blood and hair were used in the experiments. PCR using fluorescently labeled upper primers were performed for
each sample. PCR products were isolated using capillary electrophoresis on an automated DNA sequencer. Results: UamD116 contained
multiple types of motifs, and mono-, di- tri-nucloetide insertions between motifs. Meanwhile, a 1-bp deletion occurred in the downstream
flanking region of some alleles. Fragment length based genotyping was not able to discriminate the differences and sorted different alleles
with similar length as a single one. UamB1 contained 2 types of alleles, one type contained a 3-bp insertion resulting in the shifts of size
difference between the two types of alleles from 4 bp to 1 bp. In addition, alleles with different motifs had similar size. Fragment length
based genotyping identified 8 alleles, while sequence based approach identified 12 ones. Conclusion: For complex tetra-nucleotide microsatellites,
structural analysis of alleles by sequencing is necessary for correcting errors occurring in fragment based genotyping so as to
access population genetic features accurately. |
|
查看全文
查看/发表评论 下载PDF阅读器 |
| 关闭 |
|
|
|
