帚状江蓠质体UPA和线粒体COⅠ基因的序列比较及其系统进化分析

Sequence comparison and phylogenetic analysis of plastid UPA and mtDNA COⅠgene in Gracilaria edulis

为研究海南省帚状江蓠(Gracilaria edulis)野生群体的种质资源、遗传多样性状况及红藻门物种间亲缘关系,采集海南省文昌和莺歌海潮间带的野生帚状江蓠,采用PCR扩增获得帚状江蓠质体UPA基因片段(质体23S rRNA V区域)和线粒体COⅠ基因片段,分别对其进行序列比较及系统进化分析。结果表明,UPA基因片段T、C、A、G的含量分别为26.2%、16.2%、30.3%、27.3%;COⅠ基因片段T、C、A、G的含量分别为39.2%、15.1%、27.6%、18.1%;UPA和COⅠ基因片段的A+T的平均含量分别为56.5%和66.8%。UPA基因片段长度为370bp,共检测出1种单倍型,无多态性位点;COⅠ基因片段长度为664bp,共检测出2种单倍型,3个多态性位点,均为简约信息位点。与UPA基因相比,COⅠ基因具有更高的变异度,更适合用于帚状江蓠遗传多样性的分析。基于UPA和COⅠ基因片段对帚状江蓠进行系统进化分析,两者结果一致,结合形态学分析表明,帚状江蓠应是江蓠属内独立的分支。

Gracilaria edulis is a kind of marine macroalgae distributed in the coastal areas of Hainan and Guangdong provinces.Besides the function of improving water environment and repairing ecosystem,G.edulis is also rich in seaweed polysaccharides,alginate,as well as vitamins and trace elements.It has been used as an important raw material for production of agar and aquatic animal feed.The latest research indicates that the extracts of G.edulis play a certain role in inhibition of tumor cell proliferation in pharmaceutical industry.Currently,the way of obtaining G.edulis mainly relies on wild sources.Considering the huge potential economic value and the needs for sustainable development,it is necessary to carry out research on germplasm resource identification and genetic diversity analysis of G.edulis.DNA sequence analysis has been developed as a new tool to distinguish organisms that are difficult to be identified by traditional morphologic analysis,and it is widely used in the inference of phylogenetic relationships.The plastid UPA gene has served as a candidate marker in molecular identification of large algae.The mitochondrial COⅠgene has potential value as a DNA barcode of marine red algae,which can distinguish species with similar genetic relationships and biogeographic subgroups within species.The objective of this study is to figure out the genetic diversity and interspecies relationship of wild populations of G.edulis by applying molecular identification methods of UPA and COⅠgene sequences.Thirty wild G.edulis were sampled from the intertidal zone of Wenchang and Yinggehai in Hainan Province.The young stems and leaves of algae were stored in 95%ethanol and kept at-20℃until analysis.About 20 mg algae were sampled for DNA extract,then the plastid DNA UPA and mitochondrial DNA COⅠgene segment sequences were amplified by PCR method.The complete sequences of UPA and COⅠgene were obtained using solftware program of SeqMan and MEGA 11.Sequence nucleic acid diversity and haplotype polymorphism were calculated using DNAsp program.Phylogenetic tree was constructed by neighbor joining method.The results showed that T,C,A,G base contents of UPA gene segment sequences were 26.2%,16.2%,30.3%and 27.3%,while those of COⅠgene segment sequences were 39.2%,15.1%,27.6%and 18.1%,respectively.A+T base contents of UPA and COⅠgene segment sequences were 56.5%and 66.8%,respectively.One haplotype was detected in 370 bases of UPA gene segment sequences,and no polymorphic site was found.Moreover,2 haplotypes and 3 polymorphic sites were detected from 664 bases of COⅠgene segment sequences.Compared with UPA gene sequences,COⅠgene sequences had higher variability and were more suitable for analysis of genetic diversity of G.edulis.Results of the phylogenetic analysis of G.edulis based on UPA gene were consistent with that based on COⅠgene.Combined with the results of morphological analysis,it was shown that G.edulis was an independent branch in the genus Gracilaria.Both molecular phylogenetic tree based on UPA gene and COⅠgene could distinguish different genera of red algae.The results of this study were consistent with other studies showing that UPA gene had a lower evolution rate and was more conserved than COⅠgene.UPA gene was not suitable for the analysis of genetic diversity within species,but it could be used for species identification within the genus.Compared with UPA gene,COⅠdisplayed some advantages of distinguishing closely related species and was preferred for the analysis of genetic diversity within species of Gracilaria,but it was difficult to obtain COⅠgene sequence in some red algae.In conclusion,both UPA and COⅠgenes have high applicability in the molecular identification of G.edulis and have the potential to serve as DNA barcodes in marine algae.G.edulis resources are relatively simple in the South China Sea and the conservation of G.edulis resources shall be carried out in the future.