微卫星复杂结构对分型的影响：以熊UamD116 和UamB1 为例

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。

The Impacts of Complex Allele Structure on Genotyping Accuracy of Microsatellite: a Lesson from Bear Microsatellites UamD116 and UamB1

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.