龙须菜（Gracilaria lemaneiformis）微卫星标记的筛选及品系间多态性分析
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摘要
龙须菜(Gracilaria lemaneiformis)已经成为我国继海带和紫菜之后的第三大栽培海藻,不仅是江蓠属的一个重要的产琼胶物种,而且还是遗传学研究的好材料。因此,进行龙须菜的基础研究,对更好地开发其经济效益具有重要意义和推动作用。微卫星(SSR)是一种广泛用于遗传多样性、系统发生和种质鉴定等领域的分子标记技术。本论文从三个方面对龙须菜微卫星进行了初步的开发,并对开发出的微卫星进行了多态性检测及种间转移扩增。
1.利用生物信息学方法,从公开发表的龙须菜公共核苷酸数据库中寻找多态信息含量丰富的微卫星位点,在筛选出的微卫星位点中选取序列,成功设计合成了5对引物,并在青岛野生龙须菜群体中进行多态性检测。结果显示,5对引物均有扩增产物,但却未检测到多态性。
2.从江蓠属其它种的EST数据库中查找微卫星位点,根据Gracilaria changii的EST序列设计合成了10对微卫星引物。通过对青岛野生龙须菜群体中的多态性检测,其中1对引物的扩增产物具有多态性。
3.为了获得更多的微卫星引物,通过查找相关文献获得江蓠属其他种(细江蓠Gracilaria gracilis)的4对已知微卫星引物,用在龙须菜上进行引物种间转移扩增研究,有2对引物在龙须菜中有具多态性的扩增产物。实验获得的微卫星引物可用来进行龙须菜种群遗传多样性研究、遗传图谱构建。
此外,为比较龙须菜不同品系及江蓠属不同物种在微卫星位点的差异,我们将从龙须菜核酸公共数据库中查找到的微卫星引物在相关品系和相关种间做了转移扩增研究。根据计算得到的各样品间Nei氏遗传相似系数进行聚类分析,结果显示青岛野生型龙须菜样品QD与养殖选育品系样品981、石岛的龙须菜样品SD与龙须岛的龙须菜样品LD之间的遗传相似系数最高;LV与龙须菜之间的差异远远高于种内水平的差异,而成为一种种间水平的差异。
Gracilaria lemaneiformis has becoming the third largest cultivable species in China following Laminaria and Porphyra, which is not only one of the important agarophytic species of Gracilaria genus but also an ideal material for studies of genetics and molecular biology. Therefore, carrying out basic research on Gracilaria lemaneiformis is both crucial and propulsive for the commercial usage of the species. SSR technique has been widely applied to various fields, such as genetic diversity, phylogenies and identification of variants. In this study, we are trying to develop microsatellite loci in Gracilaria lemaneiformis. Based on the microsatellite loci, polymorphic was characterized and the transferring amplifications of interspecies were carried out in other relative breeds and species.
1. By making use of bioinformatic mining method in searching of microsatellites from public nucleotide resources of Gracilaria lemaneiformis, we have designed 5 selective primer pairs from the identified microsatellite loci. With the polymorphic detection in the wild population of G. lemaneiformis from Qingdao, both the primer pairs have amplification products but monomorphism.
2. By searching the EST sequences of Gracilaria genus, 10 primer pairs were designed and synthesized based on the EST sequences of Gracilaria changii. Among the 10 primer pairs, 1 pairs have amplification products with polymorphism. In order to find more microsatellite loci, the transferring amplifications of interspecies were carried out in wild population of G. lemaneiformis by using 4 primer pairs of microsatellite from other available Gracilaria genus (Gracilaria gracilis). Among the 4 primer pairs, 2 pairs have amplification products with polymorphism. Therefore the microsatellite primers derived from the experiment can be used in researching the diversity of population genetics and in DNA fingerprint mapping.
3. In order to find out the conservation quality of microsatellite loci in different breeds of G. lemaneiformis and different species of Gracilaria genus, the transferring amplifications of interspecies were carried out based on the microsatellite loci identified form public nucleotide resources of G. lemaneiformis. The data was analyzed by means of Nei’s similarity coefficient, and a phenogram was constructed. Analyses of data showed that G. lemaneiformis from Qingdao (QD) and its selected strain (981), G. lemaneiformis from Shidao (SD) and G. lemaneiformis from Longxudao (LD) displayed the highest genetic similarity of all the samples; G. lemaneiformis from Venezuela should not be ascribed to species of G. lemaneiformis.
1.利用生物信息学方法,从公开发表的龙须菜公共核苷酸数据库中寻找多态信息含量丰富的微卫星位点,在筛选出的微卫星位点中选取序列,成功设计合成了5对引物,并在青岛野生龙须菜群体中进行多态性检测。结果显示,5对引物均有扩增产物,但却未检测到多态性。
2.从江蓠属其它种的EST数据库中查找微卫星位点,根据Gracilaria changii的EST序列设计合成了10对微卫星引物。通过对青岛野生龙须菜群体中的多态性检测,其中1对引物的扩增产物具有多态性。
3.为了获得更多的微卫星引物,通过查找相关文献获得江蓠属其他种(细江蓠Gracilaria gracilis)的4对已知微卫星引物,用在龙须菜上进行引物种间转移扩增研究,有2对引物在龙须菜中有具多态性的扩增产物。实验获得的微卫星引物可用来进行龙须菜种群遗传多样性研究、遗传图谱构建。
此外,为比较龙须菜不同品系及江蓠属不同物种在微卫星位点的差异,我们将从龙须菜核酸公共数据库中查找到的微卫星引物在相关品系和相关种间做了转移扩增研究。根据计算得到的各样品间Nei氏遗传相似系数进行聚类分析,结果显示青岛野生型龙须菜样品QD与养殖选育品系样品981、石岛的龙须菜样品SD与龙须岛的龙须菜样品LD之间的遗传相似系数最高;LV与龙须菜之间的差异远远高于种内水平的差异,而成为一种种间水平的差异。
Gracilaria lemaneiformis has becoming the third largest cultivable species in China following Laminaria and Porphyra, which is not only one of the important agarophytic species of Gracilaria genus but also an ideal material for studies of genetics and molecular biology. Therefore, carrying out basic research on Gracilaria lemaneiformis is both crucial and propulsive for the commercial usage of the species. SSR technique has been widely applied to various fields, such as genetic diversity, phylogenies and identification of variants. In this study, we are trying to develop microsatellite loci in Gracilaria lemaneiformis. Based on the microsatellite loci, polymorphic was characterized and the transferring amplifications of interspecies were carried out in other relative breeds and species.
1. By making use of bioinformatic mining method in searching of microsatellites from public nucleotide resources of Gracilaria lemaneiformis, we have designed 5 selective primer pairs from the identified microsatellite loci. With the polymorphic detection in the wild population of G. lemaneiformis from Qingdao, both the primer pairs have amplification products but monomorphism.
2. By searching the EST sequences of Gracilaria genus, 10 primer pairs were designed and synthesized based on the EST sequences of Gracilaria changii. Among the 10 primer pairs, 1 pairs have amplification products with polymorphism. In order to find more microsatellite loci, the transferring amplifications of interspecies were carried out in wild population of G. lemaneiformis by using 4 primer pairs of microsatellite from other available Gracilaria genus (Gracilaria gracilis). Among the 4 primer pairs, 2 pairs have amplification products with polymorphism. Therefore the microsatellite primers derived from the experiment can be used in researching the diversity of population genetics and in DNA fingerprint mapping.
3. In order to find out the conservation quality of microsatellite loci in different breeds of G. lemaneiformis and different species of Gracilaria genus, the transferring amplifications of interspecies were carried out based on the microsatellite loci identified form public nucleotide resources of G. lemaneiformis. The data was analyzed by means of Nei’s similarity coefficient, and a phenogram was constructed. Analyses of data showed that G. lemaneiformis from Qingdao (QD) and its selected strain (981), G. lemaneiformis from Shidao (SD) and G. lemaneiformis from Longxudao (LD) displayed the highest genetic similarity of all the samples; G. lemaneiformis from Venezuela should not be ascribed to species of G. lemaneiformis.
引文
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