【作者】 于海洋；

【导师】 张全启； 郭希明；

【作者基本信息】 中国海洋大学 ， 遗传学， 2010， 博士

【摘要】 Ⅰ.牙鲆微卫星分子标记的筛选和鉴定本研究利用基因组文库—菌落原位杂交法、构建微卫星富集文库法和利用公共数据库EST序列分析法筛选牙鲆微卫星标记。1.文库构建法筛选微卫星构建了牙鲆基因组文库,通过菌落原位杂交和测序获得牙鲆微卫星序列20个筛选出具有多态性的微卫星标记8个。通过构建微卫星富集文库和菌落原位杂交法获得阳性克隆350个,测序结果显示其中253个克隆含299个微卫星序列,其中设计了173对引物,有131对引物能扩增出预期产物,有60对引物的扩增产物呈现多态性。选取从基因组文库中获得的8个多态性微卫星标记和从富集文库获得的38个多态性微卫星标记,利用30个野生牙鲆个体对其进行多态性评价,结果显示,46个微卫星的扩增等位基因数目从4到22个不等,平均等位基因数目为14.3,观测杂合度和期望杂合度的范围分别为0.3285～0.9655和0.3854～0.9732,多态信息含量PIC平均值为0.79。2.数据库分析法筛选微卫星利用公共数据库分析从牙鲆EST库筛查含有微卫星的序列,分析了GenBank数据库中包含的牙鲆EST序列(截止2010年2月达11111个),其中751EST序列含有微卫星序列882个,占分析EST总数的6.76%,其中,二碱基、三碱基、四碱基、五碱基、和六碱基重复序列分别为444条、254条、53条、95条和40条。选择了10条含有二碱基重复且侧翼序列合适的微卫星序列设计引物,并进行引物的优化和多态性的检测,有8个位点具有多态性,其等位基因数目为6-12,平均为8.88,观测和期望杂合度分别为0.3103-0.8346和0.7272-0.8736,多态信息含量PIC平均值为0.8。在这微卫星筛选方法中,EST库筛查法是最简便,而且最省人力物力的,另外,构建微卫星富集文库法由于其富集率较高达到80%,相比较基因组文库法其效率有很大的提高,是大量标记中实现高效低耗的较好选择。Ⅱ.美洲牡蛎抗病相关候选基因的单核苷酸多态性分析选用美洲牡蛎免疫相关的大防御素基因(CvBD)和丝氨酸蛋白酶抑制因子1(CvSerpinl),通过重测序的方法,在两个基因中探寻单核苷酸多态(SNP)位点,同时选取部分SNP位点,利用高分辨率溶解曲线技术在美洲牡蛎不同地理群体和感染疾病死亡前后选育家系中进行检测,分析CvBD, CvSerpinl基因的SNP位点与美洲牡蛎感染帕金虫和尼氏单孢子虫的死亡相关性。1.CvSerpinl基因的单核苷酸多态性分析为鉴定牡蛎CvSerpinl基因(丝氨酸蛋白酶抑制因子1基因)与美洲牡蛎感染疾病死亡相关的SNP标记,我们使用来自不同地理群体的30个牡蛎样本,PCR扩增基因全长并重测序其cDNA外显子,利用软件对其序列进行分析,获得了14个SNP位点,均位于编码区,其中同义突变5个,非同义突变9个。同时利用不同地理群体和死亡前后选育家系样本,选择3个SNP位点,使用高分辨率溶解曲线技术进行SNP检测和分型,结果显示只有一个SNP位点CvSPI224(A-C)在群体和家系内均具多态性,且符合哈迪温伯格平衡。检测结果在美国北方(马塞诸塞州、新泽西州)和南方群体(佛罗里达州、德克萨斯州)之间具有显著差异,推测差异来源于地理隔离。同时在德克萨斯州和佛罗里达州群体之间检测结果也存在明显差异,推测存在差异的原因可能与两地疾病严重程度不同有关。家系间的分析结果表明此位点在死亡前后家系间存在显著差异,这意味着CvSPI224位点多态性可能同美洲牡蛎抵抗夏季MSX、Dermo病原感染有着一定的相关性。此结果同La Peyre等人利用CvSerpinl基因进行的基因表达和感染实验相一致。2. CvBD基因的单核苷酸多态性分析为鉴定牡蛎CvBD基因(大防御素基因)与美洲牡蛎感染疾病死亡有关的SNP标记,本实验使用来自不同地理群体的25个牡蛎样本,PCR扩增基因全长并重测序其CDNA外显子,对其序列进行分析,获得了25个SNP位点,除一个一个位于5’非翻译区外,其余均均位于编码区。在所有SNP位点中,同义突变只有2个,非同义突变达到了23个。SNP位点中极高的NS/S比率揭示此基因可能在进化中受到了强烈的阳性选择。利用与前文一致的样本,同样使用高分辨率溶解曲线技术对其中的3个非同义突变的SNP位点进行SNP检测和分型,其中2个位点结果在群体和家系内均具有多态性,并有一定的差异。不同地理群体结果中SNP CvBD378(T-G)仅在南方与北方群体中显示出显著的差异,而SNP CvBD90(T-G)位点显示佛罗里达群体与其他群体存在明显差异,同时两位点在群体中对于哈迪温伯格平衡存在一定的偏离(P<0.05)。在感染前后家系分析中没有观察到明显的差异,但在XG3家系中两位点均发生了偏离孟德尔遗传比的情况。推测原因可能与该基因中存在拷贝数变异(Copy Numbers Variant,CNV)有关,该变异可能影响邻近的SNP检测结果,并使出现连锁不平衡现象。对于该现象的验证,需要通过测序和更多的SNP分析来进行。本论文对于海洋生物的分子标记筛选以及其在抗病育种中的作用进行了初步研究,在牙鲆中利用不同方法获得了较多的分子标记,并对其多态性进行了检测,为下一步构建连锁图谱奠定了基础。对美洲牡蛎疾病候选基因SNP分子标记的研究表明,美洲牡蛎丝氨酸蛋白酶抑制因子1基因中的SNP位点CvSPI224 C基因型与美洲牡蛎对夏季大规模死亡相关疾病的抗性具有相关性,提示其可作为与美洲牡蛎抗病相关的候选分子标记应用于其抗病育种中,同时为贝类的标记辅助育种提供参考。而大防御素基因的抗病相关分子标记需要进一步研究验证。更多还原

【Abstract】 I.Isolation and characterization of SSR markers in Paralichthys olivaceusIn this study, microsatellite (or simple sequence repeat,SSR)markers of Japanese flounder (Paralichthys olivaceus Temminck et Schlegel)were isolated and characterized through construction of genomic genome library and colony hybridization, construction of microsatellite-enriched library, and mining of EST sequences in GenBank database.1.Library construction and colony hybridizationA genomic library of selected fragment sizes was constructed. Through colony hybridization using (AC)10 and (AG)10 as probes 20 SSRs were identified, of which 8 SSRs were detected to be polymorphic.A SSR-enriched library was constructed using (GA)20和(CA) 2o as probes.A total of 350 positive clones were screened by colony hybridization, and 299 SSRs were identified from 253 clones.Primers were designed for 173 SSRs,from which 131 pairs of primers could amplify fragments of the expected sizes.All the 8 polymorphic SSRs from the genomic library and 38 of the 131 polymorphic SSRs from the enriched library were selected for polymorphism analysis in 30 wild individuals.The results should that the amplified number of alleles of the 46 SSRs varied from 4 to 22, with average allele number of 14.3. The observed (Ho) and the expected heterozygocity(He) was 0.3285～0.9655 and 0.3854～0.9732, respectively,and the average polymorphism information content PIC was 0.79.2. Data mining from GenBank EST sequences EST data mining was also used for searching SSRs in Japanese flounder. There is a large amount of sequences in EST database of Japanese flounder. A total of 11111 EST sequences were found in the database, from which 882 SSRs were identified in 751 EST sequences that accounted for 6.76% of all sequences analyzed.Among these,dinucleotide SSRs,trinucleotide SSRs, tetranucleotide SSRs,pentanucleotide SSRs and hexanucleotide SSRs were 444, 25,53,95 and 40, respectively. Ten dinucleotide sequences were selected to design primers and to assay the polymorphism, of which 8 loci were proved to be polymorphic.The number of alleles ranged from 6 to 12, and averaged 8.88.The values of He and Ho ranged from 0.3285 to 0.9655 and 0.3854 to 0.9732, respectively, and the average PIC value was 0.79. Among these different methods identification, EST database mining is proven to be an efficient and low-cost method to obtaining new microsatellite markers.Furthermore, prior to the genome library method, the construction of microsatellite-enriched library was an optimal choice for its high efficiency and relative low cost.II Analysis of the SNPs in candidate genes associated with resistance of Crassostrea virginicaIn this study, the Big defensin gene (CvBD) and Serpin 1 gene in C. virginica(cvSerpin 1) were chosen as disease resistant genes for analysis of single nucleotide polymorphisms (SNP) and detection of their associations with the susceptibility or resistance to MSX (caused by the parasite Haplosporidium nelsoni) and Dermo(caused by the parasite Perkinsus marinus). Not only different geographic populations but also different families before and after mortality were used in the analysis.1.Single nucleotide polymorphism analysis in the CvSerpinl geneIn an attempt to identify useful markers that could predict susceptibility to the disease and mortality of the American oyster(C. virginica),we resequenced all 24 exons from 3 different geographical populations (MA, DB and AL).Fourteen SNPs were identified, all in the coding exons.Of these, nine were non-synonymous SNPs,while the remaining five were synonymous.SNPs genotyping were used for screening by Real-time PCR with High Resolution Melting curve analysis in four wild populations ((Massachusetts (MA),New Jersey (NJ),Florida (FL) and Texas (TX))from north to south of the USA and three cultured families before and after mortality. Only one synonymous SNP CvSPI224(A-C)was well genotyped and polymorphic in all the populations and families detected. This SNP showed significant difference not only between north and south populations,which may due to the geographic isolation, but also significant difference between Texas and Florida population.This evolutionary selection between Texas and Florida may due to the different situation of infection by oyster disease such as MSX and Dermo. Also significant differentiation was detected in the main cultured families before and after mortality. This may suggest it has significant association between the SNP CvSPI224(A-C)CC genotype and the disease phenotype, which means this locus was related to the disease-resistance.These results were identified to the conclusion of La Peyre et al.(2009),which based on the CvSerpinl gene expression and plasma protease inhibitor activity in American oysters.2.Single nucleotide polymorphism analysis in the CvBD geneWe also resequenced all 24 exons of gene highly homologous to the big defensin gene of American oyster(CvBD) from three different geographical populations.Twenty-five SNPs were identified, twenty-four in the coding regions and one in the 5’Untranslated region. Of these, twenty-three were non-synonymous SNPs,two were synonymous.The surprisingly high NS/S ratio is one feature thought to be consistent with strong positive selection in evolutionary change of the gene.Two non-synonymous and polymorphic SNPs were used for screening by High Resolution Melting Curve analysis in four wild populations (MA,NJ, FL, TX) and three families before and after mortality. SNP CvBD378 (T-G)just showed significant difference between the north and south populations. But SNP CvBD90 (T-G)was interesting due to its significant differences between FL and other populations,which may suggest it has evolutionary selection between FL population and others.Screening on families before and after mortality with these SNPs was also addressed. No difference between families before and after mortality in both two SNPs could be concluded, which may not support the association between the genetic polymorphism of CvBDand the oyster mortality caused by MSX and Dermo in these families.However, the deviation from the mendelian inheritance ration in XG3 family and the deviation from Hardy-Weinberg equilibrium in both the two SNPs.These results indicated that the copy numbers variant (CNV) occuring in the CvBD gene may affect the assessment of SNPs adjacent to. More SNPs need to be developed and details of CNVs locations still need to be studied to convince their relationships to the innate defense mechanisms and susceptibility to disease.In the present study, we isolated and characterized a panel of microsatellite markers with three different methods,and also studied their polymorphism,laying a foundation for the mapping and MAS in Japanese flounder. SNPs of two candidate gene for disease resistance were isolated and screened.The-results suggested that,the SNP CvSPI224(A-C)CC genotype of CvSerpinl gene was significantly associated with the resistance of American oyster MSX, Dermo and other factors involved in the mortality, and could be potential marker applied in future selection of American oyster with enhanced disease resistance.These results would hopefully provide reference for MAS in mollusks.更多还原