【作者】 沈祖楠；

【导师】 李祥龙；

【作者基本信息】 河北农业大学 ， 动物遗传育种与繁殖， 2010， 硕士

【摘要】 利用GOATMAP DATABASE和NCBI生物学数据库查询牛、山羊和绵羊常染色体上的标记,然后用Phylogenetic Computer Tools与进化树软件Phylip进行牛、绵羊和山羊染色体同源性分析。研究结果表明山羊、牛和绵羊染色体具有较高的同源性,相似性系数在0.0000～1.0000之间。牛和山羊染色体同源性较绵羊和山羊、牛和绵羊高,进一步证实绵羊1、2和3号染色体分别由山羊1与3号、2与8号和5与11号染色体重组所致。应用比较基因组学和生物信息学方法比较了绵羊、山羊、牛、狗、马、兔子、猪、挪威鼠CLPG基因部分序列,并对该基因的遗传多样性和遗传分化进行分析。结果表明在被比对的12条基因序列中检测到176个多态位点,物种间及物种内的CLPG基因存在较丰富的遗传多样性。在12条CLPG基因序列中发现9种单倍型,表明物种间遗传变异丰富。物种间单倍型平均核苷酸差异数（k）为28.045,核苷酸多样性（π）和单倍型多样性（Hd）分别为0.15935和0.881～0.997。对不同物种CLPG基因部分序列进行聚类。绵羊先与鬣羊、岩羊首先聚在一起,再与牛聚在一起,再和山羊聚,总体来讲符合牛科系统分类。通过物种间生物信息分析将CLPG基因电子定位于山羊21号染色体。细胞遗传学分析表明山羊染色体以端部着丝粒染色体为主（2n=60）。以山羊CLPG基因部分DNA序列为探针利用荧光原位杂交技术（FISH）对CLPG基因进行了物理定位,进一步确认CLPG基因位于山羊21号染色体,丰富了山羊21号染色体物理图谱和整合图谱。同时将山羊皮毛颜色候选基因Myo5a、Brca1、Sox10、Zic2、kit、Snai2、Wnt3a和Tyrp1电子定位于山羊10、17、5、12、6、14、7和8号染色体。根据GenBank上发表的绵羊Callipyge基因（AF401294）和牛21号染色体基因组重叠群（NW₀01494068）进行引物设计。PCR扩增CLPG基因P0、P1、P2和P3片段,测序拼接得到2709bp序列。在辽宁绒山羊、济宁青山羊、唐山奶山羊、南江黄羊、雷州黑山羊和波尔山羊CLPG基因P2序列内进行突变位点筛选,发现A316G和C372G。对A316G位点进行PCR-RFLP分析,结果显示在这些群体中G等位基因频率都比较低,以辽宁绒山羊最低为0.2000,唐山奶山羊最高为0.5000。从基因型分布来看GG基因型以唐山奶山羊、济宁青山羊、雷州黑山羊和南江黄羊（快长系）较高。辽宁绒山羊和波尔山羊中没有发现GG基因型。Hardy-Weinberg平衡检验表明在A316G位点辽宁绒山羊、济宁青山羊、唐山奶山羊、南江黄羊、雷州黑山羊和波尔山羊六个品种均处于Hardy-Weinberg平衡状态（P>0.05）。通过一般线性模型分析发现:在波尔山羊群体中个体基因型对初生重影响不显著0.121（P>0.05）。更多还原

【Abstract】 The homology of chromosomes among cattle, sheep and goat was analyzed using Phylogenetic Computer Tools and Phylip software based on markers that exist in GOATMAP DATABASE and NCBI biological database located at their autosomes. The results indicated that similarity of homologous chromosomes among three species was between 0.0000～1.0000. The chromosomal homology of cattle and goat is higher than that of sheep and goat, and that of cattle and sheep, which further confirmed that sheep chromosome 1, 2 and 3 came from chromosomal reorganization of goat chromosome 1 and 3, goat chromosome 2 and 8, and goat chromosome 5 and 11, respectively.Part of CLPG gene sequences of Ovis aries, Capra hircus, Bos taurus, Canis familiaris, Equus caballus, Oryctolagus cuniculus, Sus scrofa and Rattus norvegicus was analyzed using the method of comparative genomics and bioinformatics and the Genetic diversity and genetic differentiation were analyzed. The results showed that a total of 176 polymorphic sites were detected from 12 sequences, more genetic diversity of the CLPG gene existed within and among species.9 haplotypes were derived from the 12 CLPG gene sequences, which indicated that rich genetic variation existed among species. The average number of nucleotide differences among species（k）, nucleotide diversity（π） and haplotype diversity（Hd） are 28.045, 0.15935 and from 0.881 to 0.997, respectively. The partial CLPG gene sequences from different species were clustered. Results showed that Ovis aries, Aoudad and Bharal were clustered together firstly, followed by cattle, then by goat, which was consistent with the biological classification of bovine.The goat CLPG gene was predicted at chromosome 21 by bioinformatics analysis among species. Most of goat chromosomes are telocentric in metaphase （2n=60）. Via fluorescent in situ hybridization （FISH）, physical location of CLPG gene was set, which showed that CLPG gene located at the 21th chromosome of goat, having enriched the physical map and integration map of goat chromosome 21. The coat color candidate genes of goat, Myo5a, Brca1, Sox10, Zic2, kit, Snai2, Wnt3a and Tyrp1, were predicted at chromosome 10, 17, 5, 12, 6, 14, 7 and 8.According to the reported sheep Callipyge gene sequence （AF401294） and cattle chromosome 21 genomic contig （NW₀01494068）, pairs of primers were designed and P0, P1, P2 and P3 fragments of CLPG gene were amplified. 2709bp sequence was got after connecting these fragments. Then we identified two SNPs （A316G and C372G） in the P2 fragment from Liaoning Cashmere Goat, Jining gray goat, Tangshan dairy goat, Nanjiang brown goat, Leizhou black goat and Boar goat. PCR-RFLP analysis of A316G showed that allele G had lower frequency in each of the analyzed populations, with Liaoning cashmere goat the lowest （0.2000） and Tangshan dairy goat the highest （0.5000）. GG genotype was mainly found in Tangshan diary goat, Jining gray goat, Leizhou black goat and Nanjiang brown goat （fast grow strain）, but not found in Liaoning cashmere goat and Boer goat. Hardy-Weinberg equilibrium test （chi-square） showed that all these involved populations were in equilibrium for A316G （P>0.05）. General linear model analysis showed that the individual genotype had no significant effect （P=0.121>0.05） on birth weight of Boer goat.更多还原