团头鲂（Megalobrama amblycephala）低氧相关分子标记的开发及应用

发布时间：2018-01-09 13:04

本文关键词：团头鲂（Megalobrama amblycephala）低氧相关分子标记的开发及应用　出处：《华中农业大学》2016年博士论文　论文类型：学位论文

【摘要】：团头鲂(Megalobrama amblycephala,Yih),俗称武昌鱼、鳊鱼,是我国重要的草食性经济鱼类之一,2015年全国养殖总产量超过70万吨。与草鱼、鲤、鲫等大宗淡水养殖鱼类品种相比,团头鲂不耐低氧,对水体溶氧的变化比较敏感。随着市场需求持续增大以及养殖水域环境不断恶化,培育耐低氧团头鲂新品种(系)对团头鲂养殖产业的健康发展具有重要意义。耐低氧性状是受微效多基因控制的复杂数量性状,随着现代分子生物学的发展,分子标记辅助选择育种具有良好的应用前景。目前鱼类耐低氧性状的遗传研究基础薄弱,相关的基因和分子标记资源有限。本论文利用常规选育技术构建团头鲂低氧耐受和低氧敏感基础群体,分析低氧耐受性状的遗传特征,在肌肉组织转录组高通量测序基础上筛选、验证和开发低氧相关的SNP分子标记,并在子代群体中进行应用,验证分子标记的效果,为团头鲂耐低氧性状研究和新品种选育积累相关的理论和技术基础。本研究的主要结果和结论如下:1、团头鲂耐低氧性状的遗传分析通过低氧环境压力筛选,构建了团头鲂低氧耐受和低氧敏感育种基础群体,发现低氧耐受和低氧敏感群体间鱼体体长、体重、体高等形态学指标存在显著性差异(p0.05),而头长的差异不显著(p0.05),低氧耐受能力不同的群体间存在生长差异,大规格团头鲂相比小规格的低氧耐受能力要弱;采用组内半同胞方差分析估计,团头鲂低氧耐受性状的遗传力为0.31,属于中度遗传力水平,检验达到了显著水平(p0.05)。2、低氧胁迫后团头鲂肌肉转录组特征分析采用IlluminaHiSeq TM 2500高通量测序平台,对3种不同低氧处理团头鲂肌肉样本进行深度测序,总共产生了96,934,854个Raw Reads,88,200,889个clean reads,GC含量约为50%;拼接后共获得57,303个Unigenes(平均长度为807 bp,范围为201 bp~21643 bp),N50和N90值分别为1465 bp和298 bp;分子生物信息分析显示426个基因差异表达,整体上看,在低氧胁迫条件下下调差异基因的数目远大于上调的差异基因,表明肌肉组织主要是通过抑制基因表达的方式来应对低氧胁迫。下调差异基因富集的生物学功能集中在蛋白与大分子复合物的组装和组织过程,上调差异基因主要涉及肌肉收缩和血液循环。根据不同处理间差异表达基因的GO功能富集分析结果,可以初步推断出团头鲂肌肉组织应对低氧胁迫动用生物学过程的机制,肌肉组织应对低氧胁迫的主要目的在于保持低能量水平,新陈代谢能量消耗的控制能力不同是团头鲂转录组差异的潜在功能结果。3、团头鲂低氧相关SNPs标记的筛选和开发转录组测序共拼接64,797,236个碱基,发现了52623个可能的SNPs位点,其中碱基转换型30192个,颠换型位点16802个,转换与颠换比为1.80;同义SNPs占编码区总SNPs的99.7%,错义SNPs约0.3%;挑选28个非同义SNPs位点进行检测分析,利用聚合酶链式反应-限制性片段长度多态性(PCR-RFLP)分型方法,筛选出5个多态性SNPs。POPGENE软件分析显示5个SNPs的多态信息含量(PIC值)范围为0.1436-0.3469,平均值为0.2669,属于中度多态,除了Nr4a1-C2188T外,其他4个SNPs在低氧耐受和低氧敏感群体中均符合哈-温平衡(p0.05)。卡方检验结果表明Plin2-A1157G和Hif-3a-2917AG位点在低氧耐受和低氧敏感群体中基因和基因型差异显著(p㩳0.05),关联分析显示这2个SNPs与低氧耐受性状显著相关。4、子代群体的低氧性能测试和低氧相关SNPs标记的验证不同低氧耐受亲本杂交组合的子代群体间的受精率和孵化率差异不显著(p0.05)。低氧耐受能力比较显示耐受亲本子代敏感亲本子代,体重比较显示耐受亲本子代敏感亲本子代。关联分析结果表明在子代群体中2个低氧相关SNPs(Plin2-A1157G和Hif-3a-2917AG)与低氧耐受性能的关系均不显著(p0.05),相关研究有待进一步改进和深入开展。
[Abstract]:Bream (Megalobrama amblycephala, Yih), commonly known as Wuchang fish, bream, is one of China's important economic herbivorous fish farming, in 2015 the national total output of more than 700 thousand tons. Compared with grass carp, carp, crucian carp and other bulk freshwater aquaculture fish species, megalobramaamblycephala not resistant to hypoxia, is sensitive to the change of dissolved oxygen in the water. With the market demand continues to increase as well as the aquaculture water environment worsening, the cultivation of new varieties (lines) megalobramaamblycephala hypoxia has important significance for the healthy development of the aquaculture industry. Megalobramaamblycephala hypoxia tolerance trait is controlled by some genes of complex quantitative traits, with the development of modern molecular biology, molecular marker assisted selection has a good application prospect the genetic research foundation in fish hypoxia tolerance traits of weak related genes and molecular markers of limited resources. This paper constructs a bream hypoxia tolerance using conventional breeding techniques And the oxygen sensitivity of basic population, genetic characteristics of hypoxia tolerance traits, screening in high-throughput transcriptome sequencing of muscle tissue based on the validation and development of hypoxia related SNP molecular markers and application in progeny populations, molecular marker verification results, the accumulation of theoretical and technical basis for the related traits of megalobramaamblycephala hypoxia and the breeding of new varieties resistant. The main results and conclusions of this study are as follows: 1. Genetic analysis of hypoxia tolerance traits of Megalobrama amblycephala through hypoxia environmental stress screening, constructed bream hypoxia tolerance and hypoxia sensitive breeding base, found that hypoxia tolerance and hypoxia sensitive groups of body length, body weight, there was a significant difference higher body morphology index (P0.05), but there was no significant difference in head length (P0.05), there are differences in growth of hypoxia tolerance of different groups, large size megalobramaamblycephala compared to the small size of the hypoxia tolerance The ability to use weak; half sib group analysis of variance estimation of heritability, bream hypoxia tolerance characteristics was 0.31, belonging to moderate heritability level test, reached a significant level (.2, P0.05) after hypoxic stress megalobramaamblycephala muscle transcription group characteristics analysis using IlluminaHiSeq TM 2500 high-throughput sequencing platform, deep sequencing of 3 different hypoxia megalobramaamblycephala muscle samples, produced a total of 96934854 Raw Reads, 88200889 clean reads, GC was about 50%; 57303 Unigenes were obtained after stitching (average length of 807 BP, range 201 bp~ 21643 BP), N50 and N90 were 1465 BP and 298 BP; molecular biology information analysis showed that 426 genes were differentially expressed, on the whole, the number of differentially expressed genes down regulated genes in hypoxia stress conditions is far greater than the increase, suggests that the muscle tissue is mainly through the inhibition of gene expression patterns The biological function of hypoxic stress. By differential gene enrichment in the assembly and the concentration of tissue protein and macromolecular complexes, upregulation of these genes mainly involved in muscle contraction and blood circulation. According to the results of GO gene function enrichment analysis expression differences between different treatments, the preliminary inference group bream muscle tissue hypoxia stress coping mechanism use the biological process, the main purpose of muscle tissue hypoxia stress response is to maintain a low energy level, the ability to control the energy consumption of different The new supersedes the old. potential function results in.3 megalobramaamblycephala transcriptome differences, screening and development of transcription SNPs related markers megalobramaamblycephala hypoxia group were confirmed mosaic of 64797236 bases, found 52623 possible SNPs the base site, conversion type 30192, type 16802 transversion sites, transition and transversion ratio of 1.80; the total synonymous SNPs SNPs encoding region 9 9.7% missense SNPs is about 0.3%; selected 28 nonsynonymous SNPs loci was detected by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) genotyping method, screened polymorphic information content of 5 polymorphic SNPs.POPGENE analysis showed that 5 SNPs (PIC) is in the range of 0.1436-0.3469, the average value is 0.2669, the moderate polymorphism, except Nr4a1-C2188T, the other 4 SNPs in hypoxia tolerance and hypoxia sensitive groups were consistent with Hardy Weinberg equilibrium (P0.05). The chi square test results showed that Plin2-A1157G and Hif-3a-2917AG genes in hypoxia tolerance and hypoxia sensitive group and genotype had significant difference (P? 0.05), correlation analysis the 2 SNPs and hypoxia tolerance traits were significantly related to.4, hypoxia performance test and hypoxia SNPs markers related to the offspring population of hypoxia tolerance verification of different hybrid combinations of the offspring groups between the fertilization rate and hatching The rate of no significant difference (P0.05). Hypoxia tolerance compared to parent offspring tolerance sensitive parent offspring, parent offspring showed weight tolerance sensitive parent offspring. Correlation analysis showed that in the progeny of 2 hypoxia related SNPs (Plin2-A1157G and Hif-3a-2917AG) and hypoxia tolerance performance were not significant (P0.05), the related research needs further improvement and development.

【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S917.4

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