分子标记技术在草鱼个体识别中可行性探究

发布时间：2018-05-12 03:17

本文选题：总DNA提取 + 个体识别　；参考：《南昌大学》2017年硕士论文

【摘要】：本研究采用基于COI基因的DNA条形码技术和微卫星分子标记技术对草鱼个体识别进行研究,为后续草鱼育种养殖提供理论基础。传统酚-氯仿法是提取DNA的一种常用方法。该法的优势是DNA提取量大,但质量偏低,且传统法提取DNA通常选取样本肌肉或血液,对样本伤害极大。本研究在传统酚-氯仿法基础上进行改良,结合Tris饱和酚不含醌等氧化物、Chelex 100法提取DNA时间短的优势,优化获得一种以鱼鳞为原材料的,改良DNA提取法。此法所提取DNA效果明显优于传统法,并通过PCR实验显示,所提取的核基因和线粒体基因都可用于后续各类研究。COI又称细胞色素C氧化酶亚基I(cytochrome c oxidase subunit I),COI基因(cytochrome oxidase I)即为线粒体DNA上编码该亚基的基因,长度大约600bp,主要用于物种鉴定或生物系统发育研究。通过使用Dnaman等生物软件,对草鱼COI基因序列比对分析,了解草鱼COI基因之间相互关系,进一步探讨基于COI基因的DNA条形码技术在草鱼个体识别方面的可行性。结果显示,草鱼COI基因片段大小约为1440bp,碱基T、A、C、G的平均含量分别为30.2%、27.1%、25.0%,17.7%呈依次递减趋势,其中G的含量明显低于其它的含量,表现出很强的碱基组成偏向性。9尾草鱼COI基因序列共有24处碱基突变,在序列第280-440bp之间和第1300-1410bp之间分别有7个和6个突变位点,属于突变频率较高区段,可考虑作为DNA微型条形码。各草鱼之间的遗传距离为0.1-0.6%,平均遗传距离0.39%,小于同一物种内遗传距离的临界值2%,因此线粒体COI基因作为DNA条形码技术目的基因,不适用于草鱼的个体识别。微卫星又称简单重复序列(simple sequence repeats,SSR),存在于绝大多数真核生物的基因组中。利用微卫星突变率高、中性DNA、存在广泛且序列两端的保守性较好等特点,微卫星分子标记技术被广泛运用于各种生物领域。本研究利用该技术将少量多态性丰富的微卫星位点排列组合,从85对微卫星引物进行筛选出8对多态性较好的引物(HLJC115、HLJC104、HLJC107、13118、HLJC2、HLJC111、HLJC9和HLJC126),分别用于11尾草鱼样本和63尾草鱼样本的个体识别,11尾草鱼样本共扩增出19对等位基因,平均每个位点含4.75对等位基因,等位基因数目范围2-7之间。63尾草鱼样本共扩增出38对等位基因,平均每个位点含6.33对等位基因,等位基因数目范围4-9之间。研究结果显示微卫星分子标记技术对小范围样本(11尾)或大范围样本(63尾)的草鱼,个体识别率均可达100%。
[Abstract]:In this study, DNA barcode based on COI gene and microsatellite molecular marker were used to study the individual identification of grass carp, which provided a theoretical basis for the subsequent breeding and breeding of grass carp. Traditional phenol-chloroform method is a common method for extracting DNA. The advantage of this method is that the quantity of DNA is large, but the quality is low, and the traditional method of DNA extraction usually selects the sample muscle or blood, which is very harmful to the sample. Based on the traditional phenol-chloroform method and combined with the advantage of short extraction time of Tris saturated phenol without quinone, an improved DNA extraction method with fish scales as raw material was obtained. The DNA extracted by this method is obviously superior to that of the traditional method, and the results of PCR experiments show that, The extracted nuclear genes and mitochondrial genes can be used in all kinds of further studies. The cytochrome oxidase I gene of cytochrome C oxidase subunit I(cytochrome c oxidase subunit is the gene encoding this subunit on mitochondrial DNA. About 600 BP in length, mainly for species identification or phylogenetic research. By using Dnaman and other biological software to analyze the sequence alignment of COI gene of grass carp, to understand the relationship between COI gene of grass carp, and to explore the feasibility of DNA barcode based on COI gene in individual identification of grass carp. The results showed that the COI gene fragment size of grass carp was about 1440 BP, and the average content of the base TGG was 30.2% 27.1% and 25.0%, respectively, and the content of G was significantly lower than that of the other ones. The results showed that there were 24 base mutations in the COI gene sequence of grass carp with strong base composition bias. There were 7 and 6 mutation sites between 280-440bp and 1300-1410bp, respectively, which belonged to the higher mutation frequency region. Can be considered as a DNA minibar code. The genetic distance between grass carp is 0.1-0.6, and the average genetic distance is 0.39, which is smaller than the critical value of genetic distance within the same species. Therefore, mitochondrial COI gene is not suitable for individual identification of grass carp as the target gene of DNA barcode technology. Microsatellites, also known as simple sequence repeats, are present in the genomes of most eukaryotes. Microsatellite molecular marker technology has been widely used in various biological fields because of its high mutation rate, neutral DNA, wide range and good conservation at both ends of the sequence. In this study, a small number of polymorphic microsatellite loci were arranged and combined using this technique. Eight pairs of polymorphic primers HLJC115, HLJC104, HLJC107, HLJC2HJC111HLJC9 and HLJC126N were screened from 85 pairs of microsatellite primers. The alleles were amplified by 19 pairs of alleles in 11 samples of grass carp and 63 samples of grass carp, respectively. There were 4.75 alleles per locus, 38 alleles were amplified from 2-7 to 63.The average alleles were 6.33 alleles and 4-9 alleles per locus. The results show that the individual recognition rate of grass carp with microsatellite molecular marker technique can reach 100% for small samples (11 tails) or large scale samples (63 tails).
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S917.4

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