【作者】 申雪艳；

【导师】 杨官品；

【作者基本信息】 中国海洋大学 ， 生物化学与分子生物学， 2007， 博士

【摘要】 为建立孔雀鱼(Poecilia reticulata)微卫星DNA序列快速分离方法,并利用微卫星DNA标记结合AFLP标记构建孔雀鱼的遗传连锁图谱,我们进行了两部分实验:(1)用磁珠富集法从孔雀鱼基因组DNA中分离富含微卫星的DNA序列;(2)利用SSR和AFLP分子标记结合拟测交策略,以孔雀鱼的单对杂交亲本及其F1代为作图群体,构建了孔雀鱼雌、雄两张遗传连锁图谱。主要内容如下:1、将孔雀鱼(P. reticulata)的一个个体的基因组DNA用Mse I单酶切并与AFLP分析所用的Mse I接头序列相连,通过AFLP体系扩增获得短片段;将这些片断与生物素标记的(AC)20寡核苷酸杂交,再通过生物素与包被在磁珠上的链亲和素结合;分离磁珠,洗脱,获得富集含微卫星DNA的单链短基因组DNA片段;再经过PCR扩增获得包含微卫星DNA的双链短基因组DNA片段;将这些片段与pMD18-T载体连接,电转化E. coli JM109,构建富集微卫星序列的基因组DNA短片段文库。随机挑取450个菌落,选用3个单独的PCR反应(第一个反应用载体上的正向测序引物M13(-47)和反向测序引物M13(-48);第二个反应用正向测序引物M13(-47)和SSR-1寡核苷酸序列;第三个反应用反向测序引物M13(-48)和SSR-1寡核苷酸序列(所用引物序列见论文第二章))筛选到阳性克隆287个(阳性克隆率约为63.78%);测序后得到262个含有微卫星序列的克隆(91.2%),利用Tandem Repeats Finder软件共找到281个微卫星重复序列。对所有重复类型进行统计:除两碱基重复类型(AC/TG)数目最多外(242个),还观察到GCTTT、AAAAG、ATGG、TGCG、TATG、GTAT、CAAG、CACG、CGCA、TCC、TAA、AGG、ACA、AAC、GAA、GAG、CAA、CT、GA、A/T等微卫星重复序列。此外,有一个克隆含有重复类型为7个核苷酸的串联重复序列(CACACAC),即小卫星序列。在得到的281个微卫星完整序列中,完美型微卫星共有76个,占27.05%:非完美型微卫星173个,占61.57% ;复合型微卫星32个,占11.39%。设计并合成189对微卫星引物,经过优化反应条件,共有156对引物在孔雀鱼基因组DNA上扩增出特异性条带。利用30尾孔雀鱼个体对其中51对引物进行多态性检测。结果表明:51对引物在孔雀鱼群体中均呈多态性,每个位点的等位基因数量从2到16个不等,揭示的观测杂合度值在0.10和0.63之间,揭示的期望杂合度值在0.23和0.77之间,有四个位点(G95、G278、G307和G401)显著偏离了哈温伯格平衡(P<0.05)。所有51个位点中都没有出现非扩增等位基因。利用上述51对孔雀鱼的微卫星引物对玛丽鱼(Poecilia latipinna),剑尾鱼(Xiphophorus hellerii)和月光鱼(Xiphophorus maculates)的基因组DNA进行PCR扩增,检测孔雀鱼微卫星引物的通用性。PCR结果表明,51对孔雀鱼微卫星引物中,有27对能在玛丽鱼上稳定扩增,16对和19对分别能在剑尾鱼和月光鱼基因组DNA上扩增。这些引物在玛丽鱼(花鳉属)基因组中扩增的成功率远高于在剑尾鱼属的两个物种(剑尾鱼和月光鱼)。2、利用微卫星和AFLP标记结合拟测交策略构建孔雀鱼的遗传连锁图谱。221个微卫星位点中,有101个符合1:1或1:1:1:1分离;此外,91对AFLP引物组合共产生符合1:1分离的标记336个,对这些分离标记进行连锁分析,得到了雌、雄两张遗传连锁图谱。在雌性连锁图谱中,135个标记(含38个微卫星和97个AFLP标记)构成了22个连锁群(4个或4个以上标记),每个连锁群含4-13个标记,连锁群长度在19.6-202.3cM之间,所有标记平均间隔为11.2cM,图谱长度为1267.7cM。计算产生20个小连锁群,包括2个三联体和18个连锁对。加上三联体和连锁对,图谱总长度为1469.7cM。在雄性连锁图谱中,有172个标记(含49个微卫星、122 AFLP和1个性别标记)定位在20个连锁群(4个或4个以上标记),每个连锁群含4-14个标记,连锁群长度在31.5-175.6cM之间,图谱总标记平均间隔为11.7cM,图谱长度为1771.2cM。另外有16个标记构成了4个三联体和2个连锁对。加上三联体和连锁对图谱总长度为1886.2cM。雌雄图谱框架图的长度分别为1267.7cM和1771.2cM,相应覆盖率分别为72.5%和79.3%;若把三联体和连锁对考虑在内,雌雄图谱的覆盖率则分别为84.0%和84.5%。作图群体所有个体的性别均作为标记整合到雌雄分离信息中,在61个F1个体中,30尾雌鱼,31尾雄鱼。在雄性图谱第2连锁群上,性别与13个标记(包括4个微卫星和9个AFLP)紧密连锁,4个微卫星标记与性别位点的图距分别为114. cM,23.3cM,12.1cM和9.2cM。本研究的结果表明:磁珠富集法可作为分离孔雀鱼微卫星DNA标记的有效手段,并可为今后进一步开发大量的孔雀鱼基因组微卫星DNA标记奠定基础;通用SSR引物在孔雀鱼、玛丽鱼、剑尾鱼和月光鱼中的应用,将促进这四种鱼基因组比较研究的顺利开展,有利于遗传信息在不同物种间的相互利用;利用SSR和AFLP分子标记构建的孔雀鱼遗传连锁图谱是国内孔雀鱼分子遗传学研究的新探索,并为经济鱼类的遗传学研究提供了借鉴。更多还原

【Abstract】 In order to establish an efficient method to clone microsatellite DNA sequences and use microsatellite DNA and AFLP markers to construct genetic linkage maps of guppy (Poecilia reticulata), two parts of experiments were done: (1) isolation microsatellite DNA by magnetic beads enrichment; (2) construction of guppy (P. reticulata) genetic linkage maps using microsatellite DNA and AFLP markers and pseudo-testcross mapping strategy. The main contents were as follow:A genomic DNA library enriched with AC/TG-microsatellite DNA was constructed using DNA extracted from a guppy individual using FIASCO (Fast Isolation by AFLP of Sequences Containing Repeats) method. A biotinylated (AC)20 oligonucleotide was used as probe. DNA fragments containing microsatellite DNA were ligated with pMD18-T vector and electroporated into E. coli JM109.In total, about 450 white colonies were screened, each colony was subjected to 3 individual PCR reactions. In the first reaction, universal forward and reverse sequencing primers (M13-47 and M13-48) were used. In the second reaction, the universal forward primer was used in combination with a (CA)20 oligonucleotide and in the third one the universal reverse primer was used in combination with a (CA)20 oligonucleotide. In the white colonies screened, 287 clones are positive. After sequencing, 262 contained microsatellite repeat sequences. Besides the motif of (CA/GT) contained in the oligoprobe, we also found GCTTT、AAAAG、ATGG、TGCG、TATG、GTAT、CAAG、CACG、CGCA、TCC、TAA、AGG、ACA、AAC、GAA、GAG、CAA、CT、GA、A/T repeats. In addition, 1 (CACACAC) minisatellite clone was obtained. According to Weber (1990),the microsatellite sequences could be categorized structurally as follows: perfect (27.05%), imperfect (61.57%), and compound (11.39%).One hundred and eighty-nine microsatellite DNA primers of guppy (P.reticulata) were developed. Fifty-one of the markers detected the diversity in a guppy population. The number of alleles each locus ranged from 2 to16. Observed and expected heterozygosities varied from 0.10 to 0.63 and from 0.23 to 0.77, respectively. Four loci (G95, G278, G307and G401) displayed significant deviations from the expectations of Hardy–Weinberg equalifiation ( P<0.05 ). There was no null allele detected at any of the loci.Cross-species amplification was investigated in 3 closely related species, Poecilia latipinna, Xiphophorus hellerii and Xiphophorus maculates. The cases of successful cross-species amplification were higher in P. latipinna than in two species of genus Xiphophorus.Genetic linkage maps of guppy (P.reticulata) were constructed using microsatellite DNA and AFLP markers and pseudo-testcross mapping strategy. Of 221 microsatellite DNA markers, 101 segregated at 1:1 or 1:1:1:1 ratios. In addition, 336 AFLP markers segregated also in F1 progenies at 1:1 ratio, which were produced using 91 primer combinations.Two linkage maps were constructed: one for male parent and the other for female parent. The female parent map consisted of 219 markers, of which 135 (97 AFLP and 38 microsatellite DNA) were assigned to 22 linkage groups, at least four markers each. The 135 markers covered 1267.7 cM in length with an average interval of 11.2 cM,. The length of the individual linkage group varied from 19.6 to 202.3 cM. The remaining 46 markers distributed as two triplets and eighteen doublets. The male parent map consisted of 227 markers, of which 172 (122 AFLP, 49 microsatellite DNA and the sex determinant) were assigned to 20 linkage group. The 172 markers covered 1771.2 cM in length with an average interval of 11.7 cM. The length of the linkage groups ranged from 31.5 to 175.6 cM, the number of markers per group varied from 4 to 14. The remaining 16 distributed as four triplets and two doublets. On the basis of the expected genome lengths, genome coverage of female and male maps was 72.5% and 79.3%, respectively. When considering all the triplets and doublets, the map coverage increased to 84.0% for the female and 84.5% for the male.The sex of the mapping progenies was treated as a marker for linkage analysis in the female and male (Li et al., 2003). Of 61 progenies, 30 were female and 31 were male. The sex marker was the only phenotypic trait placed on the map. In the male parent map, sex was mapped onto linkage group 2, which contained 13 markers including four microsatellite DNA and nine AFLP. Four microsatellite DNA markers separated from the sex locus at the distance of 114.3 cM, 23.3 cM, 12.1cM and 9.2 cM, respectively.The present study has shown that enrichment by magnetic beads is a simple and efficient method for rapid isolation of guppy genome microsatellite DNA and the (AC) -enriched library created in this study will be useful resource for developing additional genomic microsatellite DNA markers for guppy in the future. Moreover, the construction of guppy linkage maps will be important for genetic studies of economically important fish.更多还原