一种基于线粒体COI PCR-RFLP单酶切快速鉴定4种巨蛎属牡蛎的方法

A method for rapid identification of four Crassostrea oyster species based on the PCR-RFLP analysis of single digestion of mitochondrial COI

本文报道了一种基于线粒体细胞色素氧化酶亚基I(mt COI)基因序列PCR-RFLP单酶切的牡蛎物种鉴定方法。本方法可快速鉴定福建牡蛎(Crassostrea angulata)、熊本牡蛎(Crassostrea sikamea)、香港牡蛎(Crassostrea hongkongensis)和近江牡蛎(Crassostrea ariakensis)等中国沿海常见的4种巨蛎属(Crassostrea)牡蛎。该方法以甲基转移酶(Msp I)作为限制性内切酶,对4种巨蛎属牡蛎的线粒体DNA COI扩增序列进行酶切,以得到的特异性条带为依据进行物种鉴定。本方法的鉴定结果与COI测序方法的鉴定结果一致,并且筛选出的单一的限制性内切酶Msp I在4种牡蛎的COI序列中不存在酶切位点的突变,准确率达到100%,能够为巨蛎属牡蛎的物种鉴别提供简便可靠的技术支撑。

Four species of Crassostrea oysters,Crassostrea angulata,C.sikamea,C.hongkongensis,and C.ariakensis,are found along the coast of China.Oysters have strong appearance plasticity,which decreases the reliability of traditional mor-phological methods for species identification.Herein,polymerase chain reaction-restriction fragment length polymorphism was used to identify four Crassostrea oyster species at the molecular level.Mitochondrial cytochrome oxidase subunit I(COI)fragments of C.angulata,C.sikamea,C.hongkongensis,and C.ariakensis were amplified using universal primers and then sequenced.Restriction sites were analyzed according to the sequencing results.After multiple sequence alignment,the restriction endonuclease Msp I was selected to digest the COI fragments of the four oysters;digestion products were detected via 2%agarose gel electrophoresis.The COI sequences of the four Fujian coastal Crassostrea oysters were digested with Msp I to produce different numbers and lengths of fragments,which can quickly and reliably identify these oysters.The accuracy rate reached 100%after a large number of samples were tested,demonstrating high reliability using a simple and fast method for the identification of these four Crassostrea oyster species from Chinese coastal waters.