【作者】 张恩平；

【导师】 袁志发； 陈玉林；

【作者基本信息】 西北农林科技大学 ， 动物遗传育种与繁殖， 2005， 博士

【摘要】 本研究旨在运用RAPD、微卫星和候选基因SNP分子标记技术,对102只南江黄羊基因组DNA进行标记分析,研究影响南江黄羊生长性状的QTL遗传标记,建立分了标记数据分析统计数学模型,分析标记与生长性能之间的相关关系,为南江黄羊的选种选育提供分子遗传学依据,同时也为其它家畜分子标记研究提供统计学处理方法。论文研究结果如下: 1.RAPD标记与体尺体性状的相关分析 (1) 从10条随机引物中筛选出多态性良好的4条引物,对102只南江黄羊组基因DNA进行RAPD分析,共扩增出明显清晰的标记条带33条,其中多态性条带21条,多态频率58.86%。每个引物扩增条带数在7～9条之间,扩增片断长度在180～2000bp之间,条带分布均匀,分离良好。说明所选引物适于对南江黄羊基因组进行RAPD多态性分析。 (2) RAPD标记分析结果表明,与体重性状显著相关的标记有:A05引物扩增多态条带B和BF条带组合;F09引物扩增多态条带C、F及CF组合;CY0817引物扩增多态条带A和条带F;CY0818引物扩增多态条带A和条带F。上述标记中,A05引物扩增标记条带B、BF组合及F09引物扩增标记条带CF对体重和体尺性状均有显著影响,其它标记条带对体重性状作用显著,但对体尺性状影响不显著。 (3) 各标记条带替代效应对体重性状的贡献率在6.72%～19.26%之间,其中A05-B的替代效应贡献率最高,为19.26%,F09-CF次之,为13.53%,CY0817-A和CY0818-A最低,为6.72%。各标记条带替代效应对体尺性状的贡献率在1.14%～5.81%之间,部分标记与体尺性状呈负相关,但差异不显著。 2.微卫星标记与体重性状的相关分析 (1) 对南江黄羊群体10微卫星位点的遗传参数分析结果表明,微卫星位点的等位基因数在4～16之间,平均等位基因数为8.1个;有效等位基因数在3.84～11.47之间,平均有效等位基因数为6.14个;多态信息含量在0.4611～0.7738之间,平均值为0.5934;微卫星位点杂合度在0.7397～0.9128之间,平均值为0.8203。在10个微卫星位点中,INRA063位点等位基因数最少,为4个;BM1227位点等位基因数最多,为16个。南江黄羊群体含有丰富的微卫星多态信息,适合于微卫星标记分析。 (2) 建立了微卫星与性状间的相关系数的统计学模型,该模型在r_j显著的情况下有以下结论:若r_j>0,说明所研究的性状与该标记的出现与否呈正相关,相关系数越大,说明性状与标记的一致性越强,可用该标记作为所研究性状的辅助选择标记;若r_j<0,说明所研究的性状与更多还原

【Abstract】 Genome DNA marking and analysis of 102 Nanjiang Yellow Goats was carried out by molecular marking techniques of radom amplificated DNA(RAPD), microsatellite and single nucleotide polymorphism(SNP) in this study. The purpose was to seek quantity trait loci(QTL) genetic markers of growth trait, to establish mathematic model for molecular marker data analysis, to analyze dependence relation between markers and growth trait. As the result, it will offer molucular genetics base for breeding on Nanjiang Yellow Goat and analysis methed for molecular marker data on other animal. The result of study as follow:1. Corelation analysis of RAPD markers and Traits of weight and body size(1) 4 Of 10 primers was slected for RAPD analysis on 102 samples of Nanjiang yellow goat. 33 visibile and bananced distribution segments were amplificated at all, 21 polymorphic segments of which. The polymorphic ratio was 58.86%, the segment length in the region of 180-2000bp. The result indicted that slected primers fited to analyze on Nanjiang Yellow Goat genome DNA.(2) The result of RAPD analysis showed that several markers close linked with researched traitsas follow: A05-B and A05-BF;F09-C and F09-CF;CY0817-A and CY0817-F;CY0818-A and CY0818-F, these markers all have signifficently effect on weight trait, in which, except A05-B, A05-BF and F09-CF also have have signifficently effect on body size traits, the others make non-signifficent effect.(3) Contribution ratio of substitution effect on weight of each marker is in the region of 6.72%-19.26%, among them, A05-B is the biggest for 19.26%, F09-CF next to it for 13.53%, CY0817-A and CY0818-A is lowest for 6.72%. Contribution ratio of substitution effect on body size of each marker is in the region of 1.14%-5.18%. It is negative correlation between body size traits and some markers, but is not significance difference.2. The correlation analysis of weight trait and microsatellite markers(1) Genetic parameter analysis of 10 microsatellite sites in population of Nanjiang Yellow goat indicated that, allele number is at 4-16, average allele number is 8.1, effective allele number is at 3.84-11.47, average effective allele number is 6.14;Polymorphic information content(PIC) is at 0.4611-0.7738, average is 0.5934;heterozygosity is at 0.7397—0.9128, average is 0.8203. Among the 10 microsatellite sites, INRA063 have minimum number alleles for 4, BM1227 have maximum for 16.(2) Mathematical model of correlation analysis of trait and microsatellite markers was established. In the situation of r_j is significant, this model can induced conclusion as follow: if r_j>0, it means positive correlation between the trait and marker. The bigger of r_j the more concordance between the trait and marker, the marker can be used in molecular marker assistant slect(MMAS);if r_j<0, it means negative correlation, incompatibility between the trait and marker. The smaller of r, the more concordance between the trait and marker, the marker also can be used in MMAS as a negative select.(3) The result of correlation analysis showed that these alleles make very significant positivecorrelated with body weight, such as BM3413-186,190, MFA70-109, INRA063-178,184, MB066-110, MB3501-176, correlation coefficient is at 0.5180-0.7375. BM3413-182, MFA70-115, MB066-98, these alleles make significant negative relation with bodyweight, correlation coefficient is at -0.6539~-0.3880.(4) Inbreeding coefficient genetic model was established, and inbreeding coefficient of Nanjiang Yellow Goat populatin was estimated by this model. The inbreeding coefficient of 10 microsatellite sites was at the region of 0.2065~0.5400, the average was 0.3677. This result was fitting to the reality of heigh close breeding with in-line breeding and blocked breeding in the population of Nanjiang Yellow Goat. In addition, to establish inbreeding coefficient with microsatellite markeers can overcome the difficult of classical genetic analysis for lacking pedigree information in the condition of pasturage or semi-pasturage in Nanjiang Yellow Goat population.3. SNP analysis of candidate genes which related to growth trait(1) The amplified products including 5’-region and exonl and part of intronl of GH gene was digested by Haelll, the results of PAGE and sequencing showed that there was a SNP site at +355, some samples lose the restriction site for base replacement (C—T) , there were 3 genotype were obtained in this site: AA(420/420),AB(420/354+66)and BB(354+66/354+66). The population was in Hardy-Weinberg balanced state in this site tested by x2.(2) The amplified product of GHR promoter PI at 5’-region was digested by Haelll, and 3 fragments(540bp/280bp/260bp) were obtained, the results of sequencing indicated that in the site of Haelll, some samples lose the restriction site for base replacement (C—T) .(3) The sequencing result of exon41 and part of coding sequence of IGF-1 showed that it was even conservative in this site, there are no mutation body in 20 samples.(4) The correlation analysis of weight and body size traits and SNP of GH gene: Typing of product of GH gene5’-region and exonl by PCR-RFLP, there were 3 genome types were obtained, AA, AB and BB. The body weight of samples with AA was significant less than population average, and AB was significant greater than the average, BB have no significant difference with population average. Body length of AA type was very significant greater than that of BB and average. The height at withers and heart girth of AB type was significant greater than the other 2 types and average. The result demonstrated that heterozygote is advantageous on body weight.(5) In the sequence of GHR promoter PI at5’-region, there are no significant difference between different genome types or each type and average by variance analysis.4. The research on consistent degree of different molecular marking method in Nanjiang Yellow Goat population(1) consistent degree calculation formula of different molecular marking method was established,kN C =------------^^---------x 100%, and consistent degree of 3 marking methods were calculated inNA+NB+- + Nknanjiang yellow goat, the consistent degree of RAPD and microsaatellite was 86.67%, RAPD and SNP was 77.19%, microsaatellite and SNP was 71.70%. The consistent degree was 63.53% if selecting by three kinds of markings at the same time.(2) It is not total consistence that to select by different marking in Nanjiang Yellow Goat population. In breeding practice, which molecular marking method should be used as MAS must in accordance with cases of condition, cost and convenience of the method, for more important, it must make sure according to breeding actual effect. 5. About sampling and marking methodThe result of study showed that, to search markers correlated with weight trait in the extram population that have great phenotype value variation, and then analyzing random samples with the markers to confirm their validity, this process can increase the efficiency of research and it is operable.更多还原