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鲢(Hypophthal michthys molitrix)和鳙(Aristichys nobilis)是中国特有的四大家鱼中2个重要成员,主要分布于黑龙江、长江和珠江水系。传统人工养殖依靠天然鱼苗。但是,人口增长和人类经济活动加剧使其天然产卵和孵化场消失或遭到破坏。人工育苗技术虽然解决了鱼苗供应问题,但由于遗传资源管理和使用方法的不完善,使两种鱼的生长表现、抗病抗逆性和遗传多样性等都有明显的降低。另外,因洪水导致的养殖个体逃逸也使天然群体的遗传多样性受到干扰。近年来,比较大规模的人工鱼苗放流实践也加剧了对天然群体遗传多样性的扰动。对这些问题的深入研究迫切需要一套适用的分子标记。为评价鲢和鳙的遗传多样性、确定它们的遗传分化和地理分化、科学合理地管理和开发利用遗传资源,本研究构建了富集GT微卫星序列的基因组短片段文库。随机选择并测序的97个克隆中有87个含有微卫星序列。根据其中的21条序列,设计了22对微卫星标记引物并用来分析了在长江荆州段捕获的32尾野生鲢和7尾野生鳙的遗传多样性。所有标记引物在两种鱼中通用。在全部样品中共发现129个等位基因。每位点等位基因数在3~10个,平均5.9个。不同标记揭示的遗传多样性指数在0.33~2.00,平均1.22。由于使用的鱼个体数少,如鳙,只有7个个体,样品也只来源于长江荆州江段。本研究无法基于两种鱼的天然分布,对两种鱼的遗传分化、地理种群多样性比较、养殖群体和天然群体差异等问题进行深入分析。但是,这组标记的研制将有助于这2个中国特有经济鱼种的遗传多样性分析、遗传资源的管理及开发利用等相关研究。本研究中,微卫星DNA标记的研制使用了固定有微卫星核心序列的磁珠。这样的磁珠与两端接有已知序列的DNA片段杂交能富集出含有微卫星核心序列的片段。通过扩增和连接转化,可方便地获得大量含微卫星核心序列的片段。与已有的方法不同的是,本研究用AFLP方法的某些步骤使片段两端加上已知引物序列,方便易行。迄今,这两种鱼还没有微卫星标记连锁图谱。构建这样的图谱是本项目研究的长远目标。
Hypophthal michthys molitrix and Aristichys nobilis are two important members of the four major domestic fish species in China, mainly distributed in the Heilongjiang, Yangtze and Pearl River systems. Traditional artificial farming relies on natural fry. However, population growth and increased economic activities of man made their natural spawning and hatchery disappear or were destroyed. Although the artificial seedling raising technology solves the problem of fry supply, the growth performance, disease resistance and genetic diversity of the two species of fish have been significantly reduced due to the imperfect management and utilization of genetic resources. In addition, the individual’s escape due to flooding also disrupts the genetic diversity of the natural population. In recent years, the practice of releasing large-scale artificial fish fry has also aggravated the disturbance to the genetic diversity of natural populations. An in-depth study of these issues urgently needs a set of applicable molecular markers. In order to evaluate the genetic diversity of silver carp and bighead carp, determine their genetic differentiation and geographical differentiation, and manage and exploit genetic resources scientifically and reasonably, this study constructed a genomic short fragment library enriched in GT microsatellite sequences. Of the 97 clones randomly selected and sequenced, 87 contained microsatellite sequences. Based on the 21 sequences, 22 pairs of microsatellite markers were designed and used to analyze the genetic diversity of 32 wild and 7 wild silver carp captured in the Jingzhou section of the Yangtze River. All marker primers are common to both species of fish. A total of 129 alleles were found in all samples. The number of alleles per locus ranged from 3 to 10, with an average of 5.9. The genetic diversity index revealed by different markers ranged from 0.33 to 2.00 with an average of 1.22. Due to the small number of fish used, such as 鳙, only seven individuals, the sample only from the Yangtze River Jingzhou section. This study can not be based on the natural distribution of two species of fish, the genetic differentiation of two species of fish, the diversity of geographical populations, aquaculture populations and natural populations differences and other issues in-depth analysis. However, the development of this set of markers will contribute to the research on the genetic diversity analysis, genetic resources management, development and utilization of the two endemic economically species in China. In this study, microsatellite DNA markers were developed using magnetic beads immobilized with microsatellite core sequences. Such a magnetic bead hybridizes with a DNA fragment having a known sequence at both ends to enrich for a fragment containing a microsatellite core sequence. Through the amplification and ligation transformation, a large number of fragments containing microsatellite core sequences can be conveniently obtained. Different from the existing methods, some steps of the AFLP method in this study make it convenient to add known primer sequences to both ends of the fragment. So far, there is no microsatellite marker linkage map between the two species of fish. Building such a map is the long-term goal of this project.