草鱼野生和养殖群体间遗传结构比较研究
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摘要
草鱼(Ctenopharyngodon idella)属于鲤形目(Cypriniformes)，鲤科(Cyprinidae)，雅罗鱼亚科(Leuciscinae)，因其草食性、生长快、肉质鲜美等特点，是优良的淡水养殖品种。由于人工繁殖的成功应用，改善了种苗供应不足的状况，也使很多地方引种后建立了独自的繁殖小群体。受生产条件和生产规模的限制，生产单位保存的亲鱼数量不多，且大部分选自自繁后代，如此经过多年的生殖隔离和多代近交，引起了养殖性状不同程度的退化，如性早熟、成熟个体趋小、抗病力降低等。
在群体遗传学的研究中，利用不同DNA标记在不同群体出现的频率，预测群体间的遗传分化已成为一个热门研究课题。但在草鱼的研究中，尚未见用不同分子标记各等位基因出现频率的差异，研究野生和养殖草鱼种质资源的报道。由于养殖性状的退化和稀有等位基因的丢失以及杂合度的降低密切相关，因此应用大量DNA标记研究人工繁殖条件下，草鱼由于近交产生的杂合度下降和不利突变的积累是一条可行的途径。
本文利用磁珠富集法对草鱼微卫星进行了筛选，然后运用微卫星(microsatellite or simple sequence length polymorphism)重复序列长度多态、TRAP(target region amplified polymorphism)靶位区域扩增多态、SRAP(sequence-related amplified polymorphism)序列相关扩增多态等标记，从基因组重复序列、表达序列标签以及开放阅读框等不同角度，通过比较草鱼野生群体和养殖群体间等位基因频率的变化，从分子水平揭示养殖群体遗传物质的变化，从而为解决养殖性状的退化提供理论依据，也为将来草鱼遗传图谱构建提供更多的遗传标记。具体研究内容如下：
1 草鱼基因组提取及CA重复微卫星富集文库的构建
利用传统酚-氯仿法及试剂盒法对草鱼血液及鳍条基因组提取效果进行比较，结果发现：以血液为试验材料，传统方法抽提效果好于试剂盒法，可能是由于购买的试剂盒适合人血液基因组的缘故；采用传统方法提取草鱼基因组时，血液提取效果好于鳍条。根据生物素(biotin)与链亲和素(strcptavidin)的强亲和性原理，用平端限制性内切酶Mbo Ⅰ消化草鱼基因组DNA，回收纯化300-1000bp的片段，连接上接头，与生物素标记的微卫星寡核苷酸探针(CA)16退火结合，用链亲和素磁珠(streptavidin magnetic beads)亲和捕捉，获得含有微卫星序列的单链目的片段，经PCR扩增形成双链，然后克隆到pMD18-T载体上，转化至DH5α中，首次成功构建草鱼基因组微卫星富集文库。抽样检测阳性率为69.23％，说明构建的富集文库质量较高，适合大规模测
Grass carp, Ctenopharyngodon idella, which belongs to Cypriniformes, Cyprinidae, Leuciscinae, is a widely cultured freshwater fish because of its herbivorous, fast growth and delicious flesh . The success of its artificial propagation improved the status of its fry in short supply and accelerated the foundation of many small breeding populations. In limited of conditions and scales, many seed farm kept few parents and apart of them were offspring of their inbreeding. Its germ plasm, however, such as prematurity, the decline of hatching ratio, and the ability of disease resistance and spawning has been seriously degenerating every year in recent years.
On population genetic study, it became a hot research subject that using different DNA marker to forecast genetic differentiation among different populations. However, we haven't seen such report in fishery research. Because many degenerating character related to loss of rare allele and decrease of heterozygosis, it is a useful and feasible method to use lots of DNA marker to predict decrease of heterozygosis and disbennifit mutation cased by inbreeding among different grasss carp populations.
In this paper, we constructed grass carp microsatellite-enriched library and then compared the allele frequency variation among wild and cultured stock of grass carp by using microsatellite or simple sequence length polymorphism, TRAP (target region amplified polymorphism) , SRAP (sequence-related amplified polymorphism) from different part of its genomic and made known the change of inbreeding population's germ plasm. This work supplied the theoretic basis to solve the problem of degenerating and offered many useful genetic markers for the construction of its genetic linkage map in the future.
1 Extraction of grass carp genomic DNA and construction the grass carp genomic microsatellite-enriched library.
Grass carp genomic DNA was extracted from fin and blood by using traditional method and reagent-box. The result show: the quality of genomic DNA extracted from
在群体遗传学的研究中，利用不同DNA标记在不同群体出现的频率，预测群体间的遗传分化已成为一个热门研究课题。但在草鱼的研究中，尚未见用不同分子标记各等位基因出现频率的差异，研究野生和养殖草鱼种质资源的报道。由于养殖性状的退化和稀有等位基因的丢失以及杂合度的降低密切相关，因此应用大量DNA标记研究人工繁殖条件下，草鱼由于近交产生的杂合度下降和不利突变的积累是一条可行的途径。
本文利用磁珠富集法对草鱼微卫星进行了筛选，然后运用微卫星(microsatellite or simple sequence length polymorphism)重复序列长度多态、TRAP(target region amplified polymorphism)靶位区域扩增多态、SRAP(sequence-related amplified polymorphism)序列相关扩增多态等标记，从基因组重复序列、表达序列标签以及开放阅读框等不同角度，通过比较草鱼野生群体和养殖群体间等位基因频率的变化，从分子水平揭示养殖群体遗传物质的变化，从而为解决养殖性状的退化提供理论依据，也为将来草鱼遗传图谱构建提供更多的遗传标记。具体研究内容如下：
1 草鱼基因组提取及CA重复微卫星富集文库的构建
利用传统酚-氯仿法及试剂盒法对草鱼血液及鳍条基因组提取效果进行比较，结果发现：以血液为试验材料，传统方法抽提效果好于试剂盒法，可能是由于购买的试剂盒适合人血液基因组的缘故；采用传统方法提取草鱼基因组时，血液提取效果好于鳍条。根据生物素(biotin)与链亲和素(strcptavidin)的强亲和性原理，用平端限制性内切酶Mbo Ⅰ消化草鱼基因组DNA，回收纯化300-1000bp的片段，连接上接头，与生物素标记的微卫星寡核苷酸探针(CA)16退火结合，用链亲和素磁珠(streptavidin magnetic beads)亲和捕捉，获得含有微卫星序列的单链目的片段，经PCR扩增形成双链，然后克隆到pMD18-T载体上，转化至DH5α中，首次成功构建草鱼基因组微卫星富集文库。抽样检测阳性率为69.23％，说明构建的富集文库质量较高，适合大规模测
Grass carp, Ctenopharyngodon idella, which belongs to Cypriniformes, Cyprinidae, Leuciscinae, is a widely cultured freshwater fish because of its herbivorous, fast growth and delicious flesh . The success of its artificial propagation improved the status of its fry in short supply and accelerated the foundation of many small breeding populations. In limited of conditions and scales, many seed farm kept few parents and apart of them were offspring of their inbreeding. Its germ plasm, however, such as prematurity, the decline of hatching ratio, and the ability of disease resistance and spawning has been seriously degenerating every year in recent years.
On population genetic study, it became a hot research subject that using different DNA marker to forecast genetic differentiation among different populations. However, we haven't seen such report in fishery research. Because many degenerating character related to loss of rare allele and decrease of heterozygosis, it is a useful and feasible method to use lots of DNA marker to predict decrease of heterozygosis and disbennifit mutation cased by inbreeding among different grasss carp populations.
In this paper, we constructed grass carp microsatellite-enriched library and then compared the allele frequency variation among wild and cultured stock of grass carp by using microsatellite or simple sequence length polymorphism, TRAP (target region amplified polymorphism) , SRAP (sequence-related amplified polymorphism) from different part of its genomic and made known the change of inbreeding population's germ plasm. This work supplied the theoretic basis to solve the problem of degenerating and offered many useful genetic markers for the construction of its genetic linkage map in the future.
1 Extraction of grass carp genomic DNA and construction the grass carp genomic microsatellite-enriched library.
Grass carp genomic DNA was extracted from fin and blood by using traditional method and reagent-box. The result show: the quality of genomic DNA extracted from
引文
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