【作者】 叶华；

【导师】 王志勇；

【作者基本信息】 湖南农业大学 ， 动物遗传育种与繁殖， 2010， 博士

【摘要】 采用FIASCO方法构建大黄鱼(AC)n微卫星富集文库,阳性克隆率为66.4%,总共得到631个含有微卫星的序列,设计了微卫星引物413对。用其中11对引物分析了官井洋野生群体(N=30)和宁波养殖群体(N=38)的遗传多样性,野生群体的平均观测杂合度为0.648,平均期望杂合度0.803,平均等位基因数为8.5,平均多态信息含量值为0.764。养殖群体中平均观测杂合度为0.708,平均期望杂合度为0.693,平均等位基因数为4.5。结果表明大黄鱼野生群体和养殖群体的遗传多样性都较低。从NCBI数据库中获得1205条大黄鱼EST序列,其中48条含有微卫星,利用30个官井洋野生大黄鱼个体检测48个位点的多态性,从中筛选出16个多态性的微卫星标记,这些位点在官井洋野群体中的等位基因数为3-11,多态信息含量为0.115～0.866。16个EST-SSRs位点在小黄鱼、黑鳃梅童鱼、棘头梅童鱼、白姑鱼和皮氏叫姑鱼中能够通用的引物对分别为14、12、11、7和6对。大黄鱼微卫星引物在近缘物种的通用性及扩增出的多态性位点比例随着物种间遗传距离的增加而呈下降。以来自不同群体、遗传差异较大4尾雌鱼与4尾雄鱼为亲本,构建了1个4♀×4♂混合繁育群体,养殖到成鱼阶段,借助微卫星标记进行亲子鉴定重建群体的系谱关系,从中选择2个同父异母的半同胞家系为作图群体,利用LINKMFEX软件分别构建了2个家系大黄鱼微卫星标记连锁图谱,分别含有115个和224个标记。借助LINKMFEX软件的MERGE程序进行图谱整合,整合的大黄鱼遗传连锁图谱上共有251个微卫星标记,其中包括47个EST-SSR标记。雌性整合图谱含有206个标记,覆盖24个连锁群,每个连锁群的平均标记数为8.6个,图谱总长度为827.9 cM,标记间的平均间隔为4.5 cM,图谱的覆盖率为76.5%；雄性整合图谱含有179个标记,覆盖24个连锁群,每个连锁群的平均标记数为7.5个,图谱总长度为1000.4 cM,标记间的平均间隔为6.5 cM,图谱的覆盖率71.5%。同一连锁群上相同位点间的重组率存在雌雄不一现象,总体上是雌性重组率高于雄性,雌性亲本与雄性亲本重组率的比值从0.1到3.6,平均为1.3：1。利用构建的微卫星标记连锁图谱,采用MapQTL 5.0软件对1个作图家系(CC家系)的全长、体长、体高和体重性状进行了QTL定位分析,这4个性状的表型相关均达到极显著水平(P<0.001),Pearson相关系数从0.955至0.991。总共定位了26个QTL,包括7个全长、6个体长、7个体高和6个体重QTL,成簇分布于LG1、LG2、LG7、LG8、LG10、LG11、LG17、LG23上。这些QTL在大黄鱼整合图谱上存在成簇分布现象,说明这些生长相关的性状可能具有共同的遗传基础。单个QTL可解释的表型变异从6.5%到73.0%。用ANOVA分析QTL临近标记不同基因型个体表型值的差异,发现了11个位点各基因型个体间表型值存在显著或极显著差异。更多还原

【Abstract】 We constructed an (AC)n-microsatellite-enriched library for large yellow croaker Pseudosciaena crocea (Richardson 1846) using the method of FIASCO. The rate of positive clones reached 66.4%,631 sequences containing microsatellite were obtained, and 413 primer pairs were designed. Eleven primer pairs were genotyped on 30 P. crocea individuals from Guangjingyang wild population (WP) in Fujian province and 38 individuals from Ningbo cultured population in Zhejiang province (CP). In WP, the average observed and expected heterozygosoties was 0.648 and 0.803, respectively, the average number of allele and polymorphism information content was 8.5 and 0.764, respectively. In CP, the average observed heterozygosoty, expected heterozygosoty and number of allele was 0.708,0.693 and 4.5, respectively. These results indicated that both WP and CP have low genetic diversity.Simple sequence repeat (SSR) markers were also obtained for the large yellow croaker Pseudosciaena crocea using 1205 expressed sequences tags (ESTs) from the NCBI database. The loci were screened in 30 specimens from a wild population in China. Sixteen of 48 loci were polymorphic, and they were amplified with the number of alleles per locus ranging from 3 to 11. Polymorphic information content ranged from 0.115 to 0.866. The number of successfully amplified loci in Pseudosciaena polyactis, Collichthys niveatus, Collichthys lucidus, Argyrosomus argentatus and Johnius grypotus was 14,12,11,7, and 6, respectively. These results showed that the amplification probability should correlate with their phylogenetic proximity to P. crocea.A mixed group of large yellow croaker Pseudosciaena crocea was constructed, whose parents (4♀×4♂) came from different populations. After they grew up to adults, parental allocation was conducted by using microsatellite markers. Two half-sib families were selected as mapping families to construct the genetic linkage maps of microsatellite markers using LINKMFEX software. The number of segregation makers was 115 and 224 in WC family and in CC family, respectively. Using the module MERGE of LINMFEX software, new linkage group orders were built for preexisting linkage group orders that were obtained from the two full-sib families. A total of 251 microsatellite markers were integrated into linkage maps, in which contained 47 EST-SSR markers. The composite female map contained 206 markers in 24 linkage groups, with an average of 8.6 markers per group, spanning 827.9 cM with an average interval 4.5 cM. The coverage of female map was 76.5%. For the composite male map,179 markers were assigned in 24 linkage groups, with an average of 7.5 markers per group. The total length of male map was 1000.4 cM with an average interval of 6.5 cM. The coverage of male map was 71.5%. The recombination frequency was greater in the female than that in the male with a female to male recombination frequency ratio of 0.1 to 3.6, with the average of 1.3:1.The location and effects of QTLs were estimated for total length, body length, body weight, body height trait using MapQTL 5.0 based on the composite maps of large yellow croaker. A total of 26 putative QTLs were located, including 7 for total length,6 for body length,7 for body height, and 6 for body weight. These QTLs were mapped in LG2, LG7, LGI0, LG11, LG17, and LG23. They were clustered on the composite maps of large yellow croaker, which means these growth-related traits shared the common genetic elements. The phenotypic variation explained by the QTLs for growth-related traits ranged from 6.5% to 73.0%. The association between phenotypic traits and genotypes of four allelic combinations from markers nearest to QTL was analyzed by ANOVA method, and 11 significant QTLs were found.更多还原