【作者】 聂竹兰；

【导师】 王卫民；

【作者基本信息】 华中农业大学 ， 水产养殖， 2014， 博士

【摘要】 三角鲂(Megalobrama terminalis),属鲤科(Cyprinidae)、鲂属(Megalobrama),主要分布在东南沿海,长江水系、黄河水系、黑龙江水系以及俄罗斯远东地区,是鲂属中分布最广泛的鱼类。但野生三角鲂资源量逐年减少,为了保护三角鲂的野生资源,同时建立三角鲂良种选育平台,本实验室以钱塘江三角鲂原种为亲本,建立了50个全同胞家系。F1代5月龄时,随机在1个家系中选取200尾个体,测量体长体重后,分别选取体重的前5%(10尾)个体的肌肉、肝脏和脑组织提RNA,各组织的RNA等量混合成1个样品L,同样分别选取体重的后5%(10尾)个体的肌肉、肝脏和脑组织提RNA,各组织的RNA等量混合成1个样品S。L和S的总RNA提取mRNA后,本实验室按照Ion Torrent测序仪200bp读长的转录组测序指南,构建出L和S的cDNA文库；制备318芯片后,经本实验室的Ion Torrent测序仪测序。采用生物信息学方法比较分析三角鲂F1代中L和S两个转录组；利用微卫星搜索软件搜索三角鲂F1代转录组中的SSR序列,筛选出三角鲂的分子标记,用于三角鲂不同地理种群的遗传多样性研究。具体研究结果如下：(1)依据Ion Torrent测序仪200bp读长的转录组测序指南和相关试剂盒,构建了三角鲂F1代L和S的cDNA文库。经检测,L和S cDNA文库浓度分别为5.06ng/μL和34.8ng/μL,均大于5ng/μL,符合Ion Torrent RNA-Seq测序要求,文库长度均约200bp,表明三角鲂F1代L和S的cDNA文库构建成功。(2)利用318芯片,在Ion Torrent PGMTM测序仪上测序,获得了三角鲂F1代L和S的转录组数据。其中,L和S分别获得2,163,601个raw reads和3,893,877个raw reads。下机序列两次去除引物、接头及短序列后,L获得1,987,905cleaned reads;S得到3,795,177cleaned reads。L所有cleaned reads成功拼接出27,673contigs,其中N50为156bp,平均长度为164bp (101bp-2,824bp); S所有cleaned reads成功拼接出78,370contigs,其中N50为152bp,平均长度为162bp(101bp-3,376bp)。与NR蛋白数据库比对,L和S分别比对上15,786和38,923contigs,鉴定出10,800和21,718个unique protein。 GO注释中,L和S分别有3486个contigs、7134个contigs注释上,这些contigs被分为三大生物类别。KEGG分析中,L有28个生长相关的转录本,S有117个生长相关的转录本。三角鲂F1代转录组序列经微卫星搜索软件搜索,L有118个微卫星序列,S有383个微卫星序列,L和S中可用于设计微卫星引物的序列分别为46和143个。利用三角鲂的SSR序列开发了81对微卫星引物,最终筛选出2个可以稳定扩增出目的条带的微卫星位点。(3)利用本实验室开发的30个多态性较高的团头鲂微卫星位点,在三角鲂中跨种扩增,筛选出能稳定扩增出目的条带的位点13个,其中有11个位点在三角鲂中具有多态性。基于三角鲂转录组开发的2个微卫星位点及跨种扩增筛选出的11个微卫星位点的观测杂合度(Ho)与期望杂合度(He)的分布范围分别为0.7000-0.9667和0.6480-0.8264;多态信息含量(PIC)为0.5710-0.7885,均大于0.5,表明这13个位点可用于三角鲂遗传多样性研究。(4)利用13个位点对三角鲂钱塘江、北江、金沙河水库及黑龙江等4个群体分析遗传多样性。三角鲂4个群体的平均有效等位基因数在4.0196与4.4093之间,观测杂合度分布于0.4至0.9667之间,期望杂合度分布在0.6073到0.8661范围内。固定指数Fst的遗传分化研究指出,北江群体与钱塘江群体、北江群体与金沙河群体间均存在中等程度的遗传分化(0.05<Fst<0.25)。 UPGMA聚类显示钱塘江群体和黑龙江群体先聚为一类,再与金沙河水库群体聚为一类,最后与北江群体聚为一类。分子方差显示,三角鲂大部分的差异来自于个体间而非群体间或群体内。以上研究结果为三角鲂后期选育个体大、生长快的良种工作奠定基础。更多还原

【Abstract】 Megalobrama terminalis, belonging to Family Cyprinidae, Genus Megalobrama, is the most widely distributed of bream genus which mainly distrubuted in the southeast coast area, Yangtze River, Yellow River, Heilongjiang River and the Russian Far East. However, due to the decreasing of the wild resource of M. terminalis, thus, in order to protect the wild resources and establish the breeding program of this species. In our laboratory,50full-sib families were bred using the broodstock original from Qiantang River. After,200five-month-old fishes from a family of F1generation were randomly selected for measuring body weight and length, of which,10(5%) fishes have highest growth rate (L) and10(5%) fishes have lowest growth rate (S) were sampled. The tissues of muscles, liver and brain were collected, extracted RNA and equally pooled into two separate samples of L and S, respectively. The Ion Torrent sequencing with the read length of200bp was used in this present study following to manufacture’s instruction. Briefly, after mRNA was isolated from total RNA of L and S, cDNA libraries of L and S were synthesized,318chip preparation, and sequencing using the Ion Torrent sequencer. Bioinformatics analysis was used to compare the differences between two transcriptomes of M. terminalis L and S in F1generation; using software for microsatellite searching to find out the SSRs in M. terminalis transcriptomes, discovery molecular markers in M. terminalis, study on genetic diversity of M. terminalis by using different geographic populations. The present study results were revealed following:(1) Based on the Ion Torrent sequencing with read length of200bp instruction and related kits, two cDNA libraries of L and S in F1generation of M. terminalis were constructed. After measuring, the concentration of cDNA library of L and S was5.06ng/μL and34.8ng/μL, respectively, which were higher than5ng/μL, accordance with the requirements of Ion Torrent RNA-Seq sequencing; and the length of the libraries was about200bp in average, which showed that the cDNA libraries of L and S in F1generation of M. terminalis were successfully constructed.(2) Using318chips, the transcriptomes of M. terminalis L and S in Fi generation were achieved from the Ion Torrent PGMTM sequencing. The L and S obtained2,163,601and3,893,877raw reads, respectively. After removal twice of the primer sequences, linkers and short sequences, the L obtained1,987,905cleaned reads and the S obtained3,795,177cleaned reads. The all cleaned reads of L were successfully assembled to27,673contigs, where N50was156bp and the average length was164(from101to2,824) bp. The all cleaned reads of S were successfully assembled to78,370contigs, where N50was152bp and the average length was162(101-3,376) bp. Compared with NR protein database, the L and S was hit to15,786and38,923contigs, and identification of10,800and21,718unique protein, respectively. In GO annotation, a number of3486contigs (in the L) and7134contigs (in the S) were annotated and divided into three biological categories. In KEGG analysis, the L had28growth-related transcripts, and the S had117growth-related transcripts. Through microsatellite searching software in the transcriptomes of M. terminalis L and S in F1generation,118and383microsatellite sequences were identified in the L and S, respectively. Among obtained microsatellites,46(in the L) and143(in the S) could be used to design microsatellite primer sequences. Using the M. terminalis SSR sequences,81microsatellite primer pairs were developed, and only two stable microsatellites were selected to amplify the target band of microsatellite loci.(3) Using of30previously developed high polymorphic microsatellite loci from blunt snout bream, a total of13microsatellite loci were amplified target band in M. terminalis by cross-species amplification. Of which these microsatellite loci,11were polymorphism. Based on two microsatellite loci from transcriptome profile and11microsatellite loci from cross-species amplification, the observed heterozygosity (Ho), expected heterozygosity (He) was calculated as in range of0.7000-0.9667and0.6480-0.8264, respectively; the polymorphism information content (PIC) was estimated in ranging of0.5710-0.7885(were higher than0.5). These13microsatellite loci may be used in studying on bream genetic diversity.(4) The13developed microsatellite loci were used to analyze the genetic diversity of four populations distributing in Qiantang River, the Beijiang River, Jinsha Reservoir and Heilongjiang. The average number of effective alleles of the four M. terminalis populations was in ranging of4.0196-4.4093. The observed heterozygosity and expected heterozygosity distributed between0.4-0.9667and0.8661-0.6073, respectively. The genetic differentiation fixation index Fst in the North River and Qiantang River population, the Beijiang River and Jinshahe Reservoir population was0.05<Fst<0.25. The UPGMA cluster results displayed that the Qiantang population and Heilongjiang population grouped together, and then with the Jinshahe Reservoir clustered together and finally with the Beijiang River populations clustered together. The results of AMOVA analysis showed that the most difference of genetic diversity was from different individuals rather than between populations or within population of M. terminalis.The paper provides basic information for further selection of large body and fast-growing of M. terminalis broodstock.更多还原