【作者】 张建勇；

【导师】 张全启；

【作者基本信息】 中国海洋大学 ， 海洋生物学， 2011， 博士

【摘要】 单核苷酸多态性（single nucleotide polymorphisms, SNPs）被认为是很有应用前景的分子标记技术。本研究在中国对虾（Fenneropenaeus chinensis）转录组454高通量GS FLX系统大规模测序的基础上,依据预测的SNP位点设计引物,利用四引物扩增受阻突变体系PCR（tetra-primer amplification refractory mutation system PCR, Tetra-primer ARMS-PCR）,对单核苷酸突变进行检测。利用开发的SNP标记,以F₁家系为作图群体,构建了中国对虾遗传连锁图谱并对相关生长性状体长和体重进行了QTL分析。本研究的主要结果如下:1、完成了中国对虾转录组454高通量测序、contig的拼装和SNP位点的预测,该项在国家人类基因组南方研究中心帮助下完成。首先设计了80组ARMS-PCR引物,优化了四引物ARMS-PCR的扩增条件,为大规模引物设计筛选奠定了基础。其中有20组引物得到良好的分型效果。利用20组tetra-primer ARMS–PCR SNP引物对中国对虾6个家系共180个中国对虾个体基因组DNA进行扩增,均表现出多态性并分型成功。有效等位基因数分布范围1.127¹.993,平均1.600。期望杂合度和观测杂合度分布范围分别为0.505⁰.609和0.373⁰.487,多态信息含量分析显示20个位点的PIC值范围为0.145⁰.373,低于标准值0.5,对于一个只有两个等位片段的标记或基因来说,多态信息含量较高,可以作为分子标记。通过哈迪-温博格平衡检验,结果显示只有8个群体位点明显偏离（P<0.05）,93.3%群体位点符合哈迪-温博格平衡。表明四引物扩增受阻突变体系聚合酶链式反应是SNP基因分型的一种可行方法,可在中国对虾SNP遗传图谱构建、重要性状QTL定位以及分子标记辅助育种等研究中应用。2、构建了中国对虾父母本的遗传连锁图谱。共设计了800组tetra-primerARMS-PCR引物,能扩增出目的产物的引物中,经过PCR扩增、琼脂糖凝胶电泳检测,最终筛选出能扩增出不同基因型PCR产物的tetra-primerARMS-PCR引物310组。对作图群体的筛选表明,310组引物中符合中国对虾遗传连锁图谱构建的tetra-primerARMS-PCR引物200组。其中母本标记为119组,父本标记为115组。分别构建了中国对虾父母本的遗传连锁图谱,覆盖率分别为51.94%和53.77%。整合后的中国对虾连锁图谱包含16个连锁群,每个连锁群标记数6～24,共有180个标记,平均每个连锁群上有11.5个。连锁群长度范围36.3～106.8cM,图谱总长度是899.3cM,图谱平均间隔是5.2cM。对180个SNP标记在作图群体中的分型结果进行分析,有效等位基因数分布范围1.041¹.993,期望杂合度和观测杂合度分布范围分别为0.067⁰.772和0.169⁰.657,最小等位基因频率分布范围为0.021⁰.492,多态信息含量分布范围为0.145⁰.481,通过哈迪-温博格平衡检验,显示171个位点（95%）符合哈迪-温博格平衡。3、中国对虾家系体长、体重性状的测量值都显示出连续变异的特点,显示这些与生长相关的性状都是典型的数量性状或者多基因遗传。利用所构建的家系进行相关性状的QTL定位,在图谱中仅初步定位了1个与体重性状相关的QTL区间,分布在LG16连锁群上。利用最小二乘法对标记座位与中国对虾体重、体长性状进行连锁显著性检验,在180个SNP座位中,与体重显著相关的SNP位点有2个,分别是C2904-168和C12871-235。在中国对虾转录组454高通量测序GS FLX系统大规模测序的基础上,依据预测的SNP位点设计引物,通过四引物扩增受阻突变体系PCR技术（Tetra-primer ARMS-PCR）进行SNP位点多态性的验证,建立简便快捷的中国对虾SNP分型方法。验证了SNP分子标记构建中国对虾遗传连锁图谱的可行性,并构建了包含一定数目SNP分子标记的遗传连锁图谱。为中国对虾重要经济性状的QTL定位,早期鉴定具有优良性状的个体,筛选优良亲本,从而加快育种进程,缩短育种周期,进行分子标记辅助育种奠定基础。更多还原

【Abstract】 Fenneropenaeus chinensis is a commercially important farmed species in China since the 1970s. Since the development of molecular biology techniques in the early 1990s, DNA markers have been widely applied to the structural and functional analysis of important genes, the analysis of population genetic structure, development of genetic linkage map, marker-assisted selection and QTL analysis. The purposes of this study were to identify the facticity of putative SNPs with amplification refractory mutation system （ARMS）-PCR method. Tetra-primer amplification refractory mutation system PCR （Tetra-primer ARMS PCR） technique is one of the better methods for SNP genotyping because of the lower cost and convenience.1. Tetra-primer ARMA-PCR is introduced to investigate single nucleotide polymorphisms （SNPs） genotyping in Chinese shrimp （Fenneropenaeus chinensis） with 80 putative SNPs locus. For a SNP locus, 2 allele specific inner primers which their 3 ends matched to the two alleles and 2 flanking outer primers were designed, respectively. Primers were designed to amplify fragments of variying sizes for each allele band in order for them to be easily resolved using agarose gel electrophoresis. The SNP was successfully genotyped as PCR were conducted with Mg²⁺ 1.5mmol/L, dNTP 0.2mmol/L, Taq polymerase 0.5U, inner/outer primers 4/1 at annealing temperature in Ta and following touchdown profiles. 20 out of the 80 tetra-primer ARMA-PCR primer sets were validated and the outer and the expected inner bands were amplified. Homozygous and heterozygous were detected by agarose gel and the genotypes were obtained.Twenty SNP tetra-primer ARMA-PCR primer sets were validated and used to investigate the genetic structure of six selected families of marine shrimp F. chinensis. The effective number of alleles ranged from 1.127 to 1.993, with an average value of 1.600. The average values of expected and observed heterozygosities of the SNPs ranged from 0.505⁰.609 and 0.373⁰.487, respectively. Polymorphism information content （PIC） values ranged from 0.145 to 0.373. Among 120 population-locus cases （six populations×twenty loci）, only 8 （6.7%） showed significant deviation （P<0.05）, while the other 112 （93.3%） were in Hardy-Weinberg equilibrium （HWE） （P>0.05）. The frequencies of minor alleles ranged from 0.378 to 0.497.2. Three hundred and ten SNP tetra-primer ARMA-PCR primer sets were validated of eight hundred primers and the polymorphic SNP primers were two hundred sets between the parents. The polymorphic markers presented in this study provide a useful tool for population genetics, pedigree analysis, linkage map construction and marker-assisted selection （MAS） of Fenneropenaeus chinensis. The genetic linkage map of Fenneropenaeus chinensis was constructed using 200 SNP markers. Linkage map was performed using one F₁ family, and a composite linkage map was generated by incorporating map information from the male and female map. The composite linkage map contained 180 markers covering 899.3cM with an average spacing of 5.2cM. The number of linkage groups in the intergrated linkage map was 16.One hundred and eighty SNP tetra-primer ARMA-PCR primer sets were validated and used to investigate the genetic structure of F₁ family. The effective number of alleles ranged from 1.041 to 1.962. The values of expected and observed heterozygosities of the SNPs ranged from 0.067⁰.772 and 0.169⁰.657, respectively. Polymorphism information content （PIC） values ranged from 0.135 to 0.481. The MAF ranged from 0.021 to 0.492.3、A GLM procedure was used to analyze the correlation between the 180 SNPs and body weight. Results uncovered that C2904-168 and C12871-235 had a significant impact on body weight. The detection and location of QTL were performed based on map. One putative QTL （LOD≥2.0） associated with the body weight was detected and located in the male map LG16. The location of QTLs with the growth would be very useful for molecular-assisted selection and map-based cloning of functional genes.The results indicated that tetra-primer ARMA-PCR is a simple, rapid and efficient method for SNP genotyping which make it useful in a broad aspects of Fenneropenaeus chinensis genetic and breeding studies. The genetic linkage map of Fenneropenaeus chinensis was constructed using SNP markers. In summary, the SNPs study of Fenneropenaeus.chinensis is encouraging and suggesting that SNP markers have been useful for genetic and breeding studies in Fenneropenaeus. chinensis.更多还原