坛紫菜“申福2号”的SSR分子标记

Research on SSR marker of the SF-2 strain of Porphyra haitanensis Chang et Zheng (Bangiales, Rhodophyta)

利用微卫星(SSR)分子标记技术,对坛紫菜(Porphyra haitanensis)优良品系"申福2号"的丝状体和叶状体分别进行了特异性分子标记鉴定,结果发现:用9#引物(序列为F:TCACAATGGGTGATATGGC;R:CCACATTTAAGTCCGACTCTG)对"申福2号"丝状体的DNA进行扩增,出现了能区别于其他14个品系(6个优良品系,3个杂交品系,5个野生品系)的特异性条带。用该引物对室内培养的"申福2号"叶状体的DNA进行扩增,也均获得了与丝状体相同的特异性条带。此外,用该引物分别对栽培在不同海区和不同时期采收的"申福2号"叶状体进行验证,结果均出现了与丝状体相同的特异性条带。该特异性条带的DNA测序结果证实,9#引物产生的SSR标记反映了微卫星DNA重复序列的变化。通过SSR Hunter软件搜索到了设计引物时的核心序列,所得产物大小在预期长度范围内,是特异性扩增。通过BLAST比对得知,该序列在核酸数据库中没有同源序列,是一个新序列。通过DNAMAN软件分析得知,"申福2号"、"申福1号"之间序列差异较小,与坛紫菜霞浦野生种差异较大。这证实该标记反映的是种内品系间的差异,可以用于种内品系鉴定。上述结果表明,由9#引物扩增出的这一特异性条带可以认为是"申福2号"丝状体和叶状体的特异性标记,可用于该品系的种质鉴定。

Microsatellite DNA （SSR） Marker was used to identify both conchocelis and gametophytic blades of the SF-2 strain of Porphyra haitanensis. Results showed that： A specific band was amplified by 9# primer-pair in conchocelis DNA of the SF-2 strain, which was significantly different from those in other 14 strains of P. haitanensis and also presented in the gametophytic blades of the SF-2 strain cultured in the laboratory. When the primer-pair was applied to discriminate SF-2 blades from the other cultivars in the Porphyra cultivation ground, the SSR marker showed the same results whatever the blades cultured at different cultivation grounds or harvested at different times. Analysis on DNA sequences of the specific bands showed that the SSR markers amplified by 9# primer-pair reflected the changes of simple sequence repeats. Respected motif sequences had been searched out by the software of SSR Hunter and the products were within the prospective lengths when the primer-pair was designed. The sequence turned out to be a new sequence because no similar sequence was found in the NCBI nucleic acid database by BLAST. Analysis of sequences by DNAMAN software showed that there were fewer differences between ＂SF-2＂ and ＂SF-I＂ but more differences compared to ＂WT-xp＂ cultivated in Xiapu, Fujian Province. Therefore, it was confirmed that the SSR marker reflected the differences between strains and could be used to identify intra-specific strains. All the above showed that the specific DNA band amplified by 9# primer-pair was the specific SSR marker in both conchocelis and gametophytic blades of the SF-2 strain and could be used to identify the SF-2 strain from the current cultivars ofP. haitanensis.