龙须菜(Gracilaria lemaneiformis)选育品系及其野生型的ISSR指纹分析

ISSR ANALYSIS OF WILD AND SELECTED STRAINS OF GRACILARIA LEMANEIFORMIS (RHODOPHYTA)

采用ISSR标记对龙须菜一个野生型群体和选育品系两个不同年代的栽培群体,进行了亲缘关系分析,构建了龙须菜选育品系的指纹图谱。通过实验从22个ISSR引物中筛选出16条引物,可以产生清晰稳定及可重复的带,共扩增出118个位点;野生型和选育品系两个群体的遗传相似率分别为0.7301、0.7189,选育品系的两养殖种群间的遗传相似率为0.9375;遗传距离聚类分析证明,龙须菜选育品系与其青岛野生型亲缘关系很近。7条引物产生的12条特异片段可用于龙须菜选育品系的种质鉴定,每条引物都能将龙须菜选育品系和其野生型分开;根据引物S848扩增的位点构建了指纹图谱,可用于区分龙须菜选育种群及野生种群。

Gracilaria lemaneiformis is edible for human being and a feed for shellfish (abalone). It is also a raw material from which phycocolloid agar is extracted. This algal material can be used as bioremediation and is important in coastal ecosystem. Usually, it was harvested from natural stocks, but over-harvesting reduced its populations and resulted in the shortage of a constant and reliable supply. Therefore, cultivation became an alternative source of supply. Currently, G. lemaneiformis is one of the main commercial red seaweeds in China, and was widely cultivated along coasts of Shandong, Zhejiang, Jiangsu, Fujian and Guangdong provinces driven by fast-growing agar-agar industry. Since fully artificial cultivation techniques were applied, strain's selection demanded by large-scale cultivation in China became the main task for scientists in recent years. Through over ten years of efforts, a strain of G. lemaneiformis was selected successfully with promising application. The objective characters of selected strain are fast growing and comparatively high agar yielding, it originally habited in Qingdao with wild populations, but now widely applied in large-scale cultivation in Shandong, Zhejiang, Jiangsu, Fujian and Guangdong provinces. Several-year successive cultivation proved that its objective characters were stable. It is however hard to distinguish the selected strain from wild type morphologically. Effective method for the distinguishing is suggested to search on genetic level. So far, genetic information in hands was very limited. Fingerprinting analysis could be a good alternative to reveal the differences between the selected strains and wild types in further genetic study of G. lemaneiformis. In this study, ISSR (Inter Simple Sequence Repeats) markers were applied to analyzing the relations between a wild population in Qingdao of Shandong and two cultivated populations in Lianyungang of Jiangsu, the latter was from our lab-selected strain to confirm the genetic background of the cultivated populations. An ISSR fingerprint of selected strains was constructed to offer an available tool for G. lemaneiformis germplasm verification. According to the ISSR data, 118 loci were obtained with 16 selected primers after amplification. The genetic identities for the wild and cultivated populations were 0.7301 and 0.7189 respectively; and 0.9375 between two cultivated populations. The genetic distance between the wild and cultivated were 0.3146 and 0.3300 respectively; and the one between two cultivated populations was 0.0645. Cluster analysis based on ISSR genetic distances was conducted by UPGMA method using TFPGA software, both wild and cultivated populations of G. lemaneiformis fall into one group which separated from Gracilaria tenuistipitata var. liui (outgroup); in G. lemaneiformis group, two cultivated populations were clustered together into one group and separated from the wild population. These results indicated that relationship between wild and cultivated G. lemaneiformis is very close, the selected strains of G. lemaneiformis original from its wild populations. Twelve specific loci of wild G. lemaneiformis yielded by seven ISSR primers (S858, S848, S873, S857, SP3, S884 and S864) could be used for its germplasm identification. The specific loci yielded by each of the seven ISSR primers can distinguish the selected strain from the wild population. A DNA fingerprint of G. lemaneiformis based on ISSR data with primer S848 was constructed. It is valid for identification of wild and selected populations of G. lemaneiformis, especially for those cultivated for many years.