【作者】 梁祚仁；

【导师】 刘超；

【作者基本信息】 广州医学院 ， 生物化学与分子生物学， 2009， 硕士

【摘要】 人类Y染色体属于性染色体,为正常男性所特有,除拟常染区外,在遗传过程中不发生重组,其序列结构除突变外由父代不变的传递给子代。由于此特性,通过对Y染色体第二代遗传标记――短串联重复序列（STR,short tandem repeats）的研究,可以对不同男性个体之间的种族,民族鉴定有重要的参考作用;还可根据其STR分型进行家系识别,父权鉴定;在常染色体STR比较难发挥作用的领域,如男女混合斑中男性成份的鉴定等方面能快速,有效的检出其中男性成份。基于Y-STR分析技术有此特性的前提下,为了解我国广西壮族自治区少数民族男性Y染色体STR基因座的遗传多态信息,本研究对广西的10个少数民族共1149无关男性的17个Y-STR基因座的等位基因频率分布、基因多态性、单倍型多态性进行了统计,并与其它包括广东汉族、福建汉族、南克罗地亚人、奥大利亚人的各基因座基因频率分布之间的差异作对比,观察不同人群在这些STR基因座上的差别。对10个民族作基于单倍型的分子方差分析（AMOVA）,以研究10个民族间的遗传变异关系,并进一步应用DA遗传距离、N-J系统发生树、多维尺度分析（MDS）等分析方法在17个Y-STR基因座基因频率分布的基础上与上述的4个其它人群之间的进化关系作比较研究。通过这些让我们充分认识了Y-STR复合扩增技术在法医学和遗传学上的重要价值。本研究在广西壮族自治区各民族聚集地对相应民族的无关男性采集口腔拭子,其中侗族101例,苗族101例,壮族107例,瑶族105例、水族150例、仡佬族130例、仫佬族146例、京族100例、彝族101例、毛南族108例,共计1149例。利用DNA自动提取仪通过磁珠法对口腔拭子提取DNA,然后选用包括ISFH认定的最小单倍型（MHL）的9个基因座和SWGDAM推荐的基因座以及另外几个多态性较高基因座（DYS437, DYS448,DYS456, DYS458, DYS635 （Y GATA C4）, and Y_GATA_H4）在内,共计17个Y-STR基因座的美国ABI公司的AmpFISTR? filerTM复合扩增试剂盒进行PCR扩增,扩增产物在3100XL型遗传分析仪进行毛细管电泳,用软件Foundation data collection收集原始扫描数据,GeneMapper ID进行Y-STR基因分型。得到Y-STR基因分型数据后,用直接计数法计算各等位基因频率。基因多样性（gene diversity,GD）及单倍型多样性（haplotype diversity,HD）,按公式h=n（1-∑Pi²）/（n-1）计算,n为样本例数,Pi为等位基因频率。并计算累积的GD值（TGD,相当于常染色体STR评估指标中的累积非父排除率CPE）按公式TGD=1-（1-GD₁）（1-GD₂）（1-GD₃）（1-GD₄）…（1-GD_n）计算,GD_n为各基因座的GD值。用arlequin 3.11软件对10个少数民族基于单倍型进行分子方差分析（AMOVA）,比较10个少数民族与其它包括广东汉族、福建汉族,南克罗地亚人,澳大利亚人4个人群在内的共计14个人群的17个Y-STR基因座等位基因频率分布的差异性。用DISPAN软件根据14个人群的17个Y-STR基因座等位基因频率计算Nei提出的DA遗传距离,再利用MEGA4软件根据DA遗传距离矩阵绘制N-J进化树以及利用SPSS12.0软件的多维尺度分析（MDS）对14个人群的遗传进化关系进行分析。通过上述处理,本研究获得10个少数民族1149名男性17个Y-STR基因座的等位基因频率分布资料,在10个民族的17个基因座中共检测出个98等位基因（不包括DYS385a/b）和66个DYS385a/b单体型。频率从最低的0.0066（DYS385a/b）-最高的0.9307（彝族DYS438*10）。10个民族1149个样本观察到1129种单倍型,各民族间只观察到苗族和仡佬族有1种共享单倍型。侗族、京族、毛南族有1种单倍型出现2次,壮族、苗族,仡佬族有2种单倍型出现2次,水族和仫佬族有3种单倍型出现2次,彝族有4种单倍型出现2次。10个民族17个Y-STR基因座的GD值在0.1327-0.9663之间,单倍型多样性（HD）在0.99920-0.99983之间,累积GD值均接近1。10个少数民族的AMOVA分析表明各民族间的遗传变异较大,达到7.23%。遗传距离、N-J系统发生树以及MDS分析表明白种人和黄种人之间有明显差别,10个少数民族与汉族间有明显差别,10个少数民族互相之间也有明显差别。结论在不同民族,不同种族的Y染色体遗传多态性信息研究中,单个Y-STR的信息量是有限的,但从本研究中可以看到,选择多态性高的多个Y-STR基因座的联合扩增技术在对不同人群的STR多态信息进行调查分析后,显示无论在法医学还是人类遗传学上都有重要意义: 1、多个Y-STR基因座的多态性分析对男性个体所属种族,民族的区分方面具有重要的参考意义。2、多个Y-STR基因座的多态性分析在法医领域的个体识别,父权鉴定,男性家系的鉴别,混合斑中男性成分的鉴定中有显著效果。3、多个Y-STR基因座的多态性分析在人类遗传学中,在民族的迁徙、演变及其来源的研究中,是重要的研究手段之一。此外,对少数民族DNA数据库的完善也有重要补充作用。更多还原

【Abstract】 Y chromatosome is sex-chromosome, it is presence on normal male .when the Y-chromosome is inheritance from father to son, the sequence of gene is Constant excluded the pseudoautosomal region unless variation is occurrence.Therefore,a men can be appreciationed which nation he is through the test of Y-chromosome short tandem repeats（STR） . Also the test of Y-STR is powerful for Judging Paternity and genealogy. In the mixed Stains from Sexual Assault Cases, the tested Y-chromosome STRs is better than the tested autosomal STRs.For researching the Polymorphism of Y-STR loci in the minority populations in Guangxi of China, we investigated the genic information of 17 Y-STR in don,miao,yi ,gelao, melao, maonan, jing,yao,shui and zhuang nationality of Guangxi of China,and then evaluate their application in forensic and genetics.as well as the information of polymorphism is significant to build DNA database.The oral swab came from1149 independence male of guangxi in china.dong is 101,miao is 101,zhuang is 107,yao is105,shui is 150,gelao is 130,melao is 146,jing is 100,yi is101,maonan is 108. the DNA was extracted from oral swab of minority population with magnetic beads method and then seventeen Y-STR loci were amplified with AmpFlSTR? Yfiler^TM PCR Amplification Kit System. The PCR products were detected with 3l30XL Genetic Analyzer.The allele frequencies was calculate directly,diversities and genetic distance were calculated by the equation: h=n（1-∑Pi²）/（n-1）.TGD was calculated by the equation:TGD=1-（1-GD₁）（1-GD₂）（1-GD₃）（1-GD₄）…（1-GD_n）. Nei’DA distance was calcu- lated by DISPAN soft. compared the allele distribution between 10 minority populations and han nationality by arlequin 3.11soft.and AMOVA base on allele frequencie is performed by arlequin. N-J tree base on DA distance was draw by MEGA 4.0 soft. MDS base on DA distance was performed by SPSS 12.0.The information of allele distribution and gene diversity of the 17 Y-STR loci were obtained from ten minority population. 96 allele exclude DYS385a/b and 66 haplotype of DYS385a/b was find , The allele frequencies is 0.0066-0.9307 .1129 haplotypes of 17 Y-STR were found in 10 minority population, only one haplotype are shared between gelao and miao population. One haplotype is twice in dong,jing, maonan population. two haplotype is twice in zhuang,miao,gelao population. three haplotype is twice in shui,mielao population. four haplotype is twice in yi.GD of the 17 Y-STR loci is 0.1327-0.9663 in ten minority population. HD is 0.99920-0.99983.TGD all is close to 1.AMOVA indicate that genetic variance between ten population is major,it is 7.23%. D_A distance, N-J tree and MDS indicate that there have significant difference between white people and yellow people.The significant difference is presence between han nation and ten minority population, also interior of ten minority population.Conclusion : Single Y-STRs is less capability of discrimination than the multiple-Y-STRs. From ours research,we know that :1.the haplotype diversity of 17 Y-STR loci is so effective in identity discrimination , paternity identification and test of mixing stains. 2.To estimate which nation or racial a man is ,the haplotype diversity of 17 Y-STR have significance. 3. haplotype diversity of 17 Y-STR is one of significant technique to research history of nation or migration of human population. also, It is significant to build DNA database of minority population.更多还原