【作者】 曾献存；

【导师】 贾斌； 赵宗胜；

【作者基本信息】 石河子大学 ， 动物遗传育种与繁殖， 2010， 博士

【摘要】 一.新疆部分绵羊品种遗传多样性分析利用10个微卫星位点对新疆地区7个地方绵羊品种和1个育成品种进行了遗传学检测,初步探讨了新疆8个绵羊品种的遗传多样性和遗传分化关系。结果表明:10个微卫星位点在8个绵羊群体中的平均PIC为0.492,平均He为0.536,其基因多态性和遗传多样性相对贫乏;各个位点GST在0.017~0.182之间,平均为0.08;8个绵羊群体FIS在0.255~0.556之间,处于较高水平,说明这些绵羊群体内存在一定程度的近交。以中国美利奴(新疆军垦型)羊为外群,UPGMA聚类分析表明,阿勒泰羊、哈萨克羊和巴什拜羊遗传距离较近,首先聚在一起,然后和巴音布鲁克羊聚为一大类;和田羊和策勒黑羊先聚在一起,然后和多浪羊聚为另一大类,这与各绵羊群体的地理分布、起源及育成史基本一致。二.绵羊主要经济性状候选基因的研究1、利用SSCP或RFLP方法,以具有毛品质、生长和繁殖性状差异的中国美利奴羊、德国美利奴羊和德国美利奴羊公羊与中国美利奴羊母羊杂交F1代羊为研究对象,对绵羊羊毛性状(KAP8.2、KAP7、KAP5.4、KAP8.1、KAP1.3和Ⅰ型IF基因)、生长性状(PROP1、POU1F1、IGFBP3和MC4R基因)和繁殖性状(PRLR、RBP4、PGR、FSHβ、GnRHR、BMP4和BMP2基因)候选基因的部分序列进行了多态性检测。结果发现KAP8.1基因、KAP1.3基因、Ⅰ型IF基因、PROP1基因PROP1-1～PROP1-4引物、MC4R基因MC4R-3和MC4R-4引物、PRLR基因PRLR-1和PRLR-2引物和GnRHR基因扩增片段存在多态。2、利用荧光实时定量PCR技术,研究了中国美利奴羊KAP8.1基因、KAP1.3基因、PROP1基因和PRLR基因的表达情况。结果表明中国美利奴羊不同生长阶段(0、7、14、30、60和90日龄)皮肤组织中KAP8.1和KAP1.3基因的发育表达模式基本一致,在0日龄时最高,随后下降。KAP8.1基因表达量在0日龄时高于其他日龄(P<0.01),在14到90日龄间基本趋于平稳状态。KAP1.3基因表达量在0日龄时高于14到90日龄(P<0.01),在7日龄时高于14到90日龄(P<0.05)。PROP1基因仅在绵羊垂体组织中表达,而PRLR基因在绵羊各种组织中广泛表达,且在子宫和下丘脑组织中的表达量最高。垂体组织中的PROP1基因表达量较低,在7日龄时高于30(P<0.01)、14和60日龄(P<0.05),PRLR基因表达量呈现先波动上升后下降的趋势,各日龄间无差异(P>0.05)。PRLR基因表达量在卵巢组织中从7日龄起呈先下降后上升的趋势,90日龄高于0~30日龄(P<0.05);在睾丸组织中呈波动上升趋势,各日龄间无差异(P>0.05);在皮肤组织中0~14日龄高于30～90日龄(P<0.01)。3、对KAP8.1、KAP1.3和Ⅰ型IF基因不同基因型与中国美利奴羊羊毛性状进行关联分析,结果显示,KAP1.3基因Bsr I酶切多态性与羊毛细度极显著相关(P<0.01),AA基因型个体羊毛细度低于AB(P<0.05)和BB(P<0.01)基因型个体;KAP8.1基因SSCP多态性与羊毛细度显著相关(P<0.05),BC基因型个体羊毛细度低于AB和BB基因型个体(P<0.05)。KAP8.1和KAP1.3基因组合合并基因型对羊毛细度有显著影响,BC-AA为羊毛细度有利基因型,其个体羊毛细度低于BB-BB、BB-AB、AB-BB和AB-AB基因型个体(P<0.05)。4、PROP1和MC4R基因多态性与中国美利奴羊生长性状的关联分析结果显示: PROP1基因PROP1-4引物扩增片段多态性存在显著的基因型效应,BB基因型为生长性状有利基因型,其个体胸围和胸宽显著高于AA基因型个体(P<0.05),尻宽高于AB基因型个体(P<0.05),体斜长显著高于AA和AB基因型个体(P<0.05);MC4R基因MC4R-4引物扩增片段多态性与体斜长显著相关(P<0.05),BB基因型个体体斜长最大,高于AB、AC和BC基因型个体(P<0.05);PROP1-4和MC4R-4基因组合合并基因型与中国美利奴羊体斜长显著相关(P<0.05),两基因合并效应>单基因效应,合并基因型的效应不是各自基因型效应的简单相加,要高于单个基因型效应。5、利用χ~2检验进行中国美利奴羊单胎和多胎个体PRLR-1、PRLR-2和GnRHR引物扩增片段的基因型差异显著性检验,结果表明PRLR-2引物扩增片段AC(P<0.01)和CC(P<0.05)基因型在多胎个体中的频率高于单胎;GnRHR引物扩增片段BB基因型在多胎个体中的频率高于单胎(P<0.01),而AA基因型在多胎个体中的频率低于单胎(P<0.05),推测PRLR-2引物扩增片段AC和CC基因型、GnRHR基因扩增片段BB基因型可能为中国美利奴羊产羔数的有利基因型。6、在中国美利奴羊群体中,对羊毛性状和生长性状候选基因进行合并基因型分析,发现PROP1-4和KAP1.3基因组合合并基因型BB-AA个体胸围、体斜长和体重均最大,且胸围高于合并基因型AA-AA、AB-AA和AA-AB个体(P<0.05)。BB-AA合并基因型对体斜长、胸围和体重的有大的正向遗传贡献率,对羊毛细度的遗传贡献率较小。更多还原

【Abstract】 一. Studies on the Genetic Diversity of Xinjiang Sheep BreedsTo reveal the genetic diversity and systemic relationship of main sheep breeds in Xingjiang, the genetic polymorphisms of 10 microsatellites in 7 local and 1 improved sheep breeds were studied. The results showed that the average PIC (0.492) and He (0.536) of the whole population were all lower than those of other sheep breeds reported in the documents, which showed the gene polymorphisms and genetic diversity in these sheep breeds were relatively rare. The unbiased coefficients of gene differentiation (GST) of the loci varied from 0.017 to 0.182, with an average of 0.08. The high positive FIS value (ranging from 0.255 to 0.556) indicated inbreeding to be one of the main causes in all breeds. Using Chinese Merino as the out-group, UPGMA cluster analysis showed that Aletai, Kazak and Bashibai were clustered first due to a relatively small genetic distance among them,and then they clustered with the Bayinbuluke sheep; and Hetian and Qira black were clustered first and then they clustered with the Duolang sheep, which indicated that the relationship among breeds displayed some degree of consistency with their geographical distribution, origin and breeding history.二. Studies on the Candidate Genes of Major Economic Traits1、Using SSCP or RFLP methods, the polymorphisms of partial sequence of candiate genes of wool traits (KAP8.2, KAP7, KAP5.4, KAP8.1, KAP1.3 and TypeⅠIF genes), growth traits (PROP1, POU1F1, IGFBP3 and MC4R genes) and reproductive traits (PRLR, RBP4, PGR, FSHβ, GnRHR , BMP4 and BMP2 genes) were detected in Chinese Merino, German Merino and hybrid F1 generation of Germany Merino rams and Chinese Merino ewes (with different wool, growth and reproduction traits). The results showed that the PCR fragment of KAP8.1 gene, KAP1.3 gene, TypeⅠIF gene, PROP1-1~PROP1-4 primers of PROP1 gene, MC4R-3 and MC4R-4 primers of MC4R gene, PRLR-1 and PRLR-2 primers of PRLR gene and GnRHR gene have polymorphisms.2、The real-time PCR technique was used to study the expression level of KAP8.1, KAP1.3, PROP1 and PRLR genes in Chinese Merino sheep. Results showed that: KAP8.1 and KAP1.3 genes showed the same developmental expression patterns at different growth stages (0, 7, 14, 30, 60 and 90 days) in skin tissue in Chinese Merino, the highest at 0 days of age, then decreased. KAP8.1 gene expression level at 0 days was higher than the other days (P<0.01), within 14 to 90 days tendency to stability state. KAP1.3 gene expression level at 0 day was higher than the 14-90 days of age (P<0.01), at 7 days of age is higher than the 14-90 days of age (P<0.05). PROP1 gene expressed only in sheep pituitary, while PRLR gene expressed widely in various tissues, and showed a high expression in uterus and pituitary gland tissues. A relatively lower expression occurred in pituitary of PROP1 gene, on the age of 7 days, the expression level of PROP1 gene was higher than that of 30 (P<0.01), 14 and 60 days (P<0.05); the PRLR gene expression level showed a rising trend with fluctuation, then downward, no significant difference between the ages (P>0.05). In ovarian tissue, the PRLR gene expression level from the 7 days of age showed a decline after the rise, at 90 days of age higher than the 0 to 30 days (P <0.05); fluctuations in the testis showed an upward trend among all age no difference (P>0.05); in skin tissue from 0 to 14 days of age higher than 30 to 90 days (P<0.01).3、The relationships between different genotypes of KAP8.1, KAP1.3 and TypeⅠIF genes and wool traits were analysed in Chinese Merino. Results showed that the Bsr I restriction polymorphism of KAP1.3 gene was significantly associated with fiber diameter (P<0.01). Individuals with genotype AA were significantly lower than those of individuals with genotype AB (P<0.05) and BB (P<0.01) in fiber diameter. The SSCP genetic polymorphism of KAP8.1 gene was significantly associated with fiber diameter (P<0.05). Individuals with genotype BC were significantly lower than those of individuals with genotype AB and BB (P<0.05) in fiber diameter. Combination genotype BC-AA was the favorable genotype for fiber diameter, individuals with genotype BC-AA were significantly lower than those of individuals with genotype BB-BB, BB-AB, AB-BB and AB-AB (P<0.05).4、The relationships between different genotypes of PROP1 and MC4R genes and growth traits were analysed in Chinese Merino sheep. Results showed that the significant genotype effects existed in PROP1-4 amplified fragment polymorphism, BB genotype was the favorable genotype for growth traits; individuals with genotype BB were significantly higher than those of individuals with AA genotype in girth and chest width(P<0.05), AB genotype in rump width(P<0.05) and AA and AB genotype in body length (P<0.05); The genetic polymorphism of MC4R-4 amplified fragment was significantly associated with body length (P<0.05), individuals with genotype BB were significantly higher than those of individuals with AB, AC and BC genotypes (P<0.05); Combination genotypes of PROP1-4 and MC4R-4 genes was significantly associated with body length (P<0.05), two gene effect > single gene effect, genetic effect of combination genotypes was not the simple addition of different genotypic effects, may higher than that of a single genotype effect.5、The genotype frequency of PRLR-1、PRLR-2 and GnRHR genes with different little size in Chinese Merino were analyzed byχ~2 test. Results showed that genotype frequencies of AC (P<0.01) and CC (P<0.05) on the locus of PRLR-2 as well as genotype frequencies of BB on GnRHR gene were higher in prolific individuals than in singleton pregnancy individuals of in Chinese Merino sheep. Whereas, genotype frequencies of AA on GnRHR gene in prolific individuals was lower than that in singleton pregnancy individuals of Chinese Merino sheep. These results indicated that genotypes of AC and CC on PRLR-2 locus as well as genotype BB on GnRHR gene were favorable genotypes for the number of little size in Chinese Merino sheep.6、The effect of combined genotypes of the candidate genes for wool and growth traits was tested by least square means in Chinese Merino sheep. The results showed that the combined genotype BB-AA of PROP1-4 and KAP1.3 genes was the predominant genotype on chest girth, body length and body weight, which is more effective on chest girth than AA-AA, AB-A and AA-AB combined genotypes (P<0.05). The contribute percentage(CP)of chest girth, body length and body weight with BB-AA genotype were high(4.08%, 10.50% and 7.63%, respectively), and the contribute percentage(CP)of fiber diameter was low(0.85%).更多还原