5.8S rDNA-ITS区片段的序列分析在坛紫菜种质鉴定中的应用

Sequence analysis and application of 5.8S rDNA-ITS regions of Porphyra haitanensis

为探讨DNA序列标记技术在坛紫菜种质鉴定中的应用,对10个野生坛紫菜种质材料的5.8S rDNA-ITS区进行PCR扩增和序列分析,结果发现扩增的片段长度在1208～1219bp之间,可以分为ITS1区,5.8S区和ITS2区3个部分,其中5.8S区片段的长度完全一致,均为160bp;ITS1区和ITS2区片段的长度也非常接近,只有几个碱基的差异。多重序列比对发现10个种质材料的ITS区(包括ITS1和ITS2)序列都存在一定差异,序列同源性在95.82%～99.73%之间,而5.8S区序列则完全一致,但与其它种紫菜的5.8S区序列有很大差异,序列同源性在79.7%～95.0%之间。由此认为5.8S rDNA-ITS区这种高度保守区和高变区交替排列的形式可以成为坛紫菜种质鉴定及系统进化分析的强有力工具。

Porphyra haitanensis is an important economic marine crop,and has been widely cultivated along the coasts of South China. The correct identification of species or forma of Porphyra is necessary for ensuring the well-bred cultivation and the quality of production. However,as the gametophytic blade of Porphyra is morphologically simple and marked variations occur as environmental conditions change,it is very difficult to identify the species or forma of Porphyra based only on their morphological characteristics. In order to find a new way to discriminate the forma of P. haitanensis,the internal transcribed spacer （ITS） regions of nuclear ribosomal DNA （including 5.8S rDNA） of 10 germplasm materials of wild-type P. haitanensis were amplified and sequence analyzed by NCBI blast. The sequence length of the amplified fragments that ranged from 1 208 bp to 1 219 bp,can be divided into ITS1,5.8S and ITS2 three regions. The 5.8S rDNA region of the 10 materials was of an identical size,160 bp. The ITS1 and ITS2 regions varied slightly by two or three bps. By multiple sequences alignment,the result showed that the sequences of ITS region （including ITS1 and ITS2） of the 10 materials are different from each other and identical sequence pair was not found among these strains,sequence homology was 95.82%-99.73%,so it is easy to discriminate 10 germplasm materials of P. haitanensis by comparing the sequences of their ITS regions. And the sequences of 5.8S region of the 10 materials are identical,but difference with other Porphyra species,sequences homology was 79.7%-95.0%,so the sequences of 5.8S rDNA can be used to discriminate the species of Porphyra. In other words,the sequence of 5.8S rDNA -ITS region of P. haitanensis was alternate array by conservative region and high variational region,and the mode of sequence array of 5.8S rDNA-ITS regions can be a powerful tool in variety identification of P. haitanensis. The phylogenetic analyses based on these data also support the results