浅蛤属(Macridiscus)物种特异性线粒体DNA分子标记及其长度多态性和个体内异质性

浅蛤属(Macridiscus)包括3个形态非常相似的物种,即黑浅蛤(M .melanaegis)、等边浅蛤(M .multifarius)和半步目浅蛤(M .semicancellata),其中前二者为同域分布.根据形态学特征,浅蛤属物种较难区分.本研究以改进的CTAB法提取浅蛤个体基因组DNA 后,用引物Macr_CO1_F16834和Cytb_Macr_R2PCR 配对进行聚合酶链式反应(PCR),扩增线粒体DNA控制区至细胞色素b基因(Cytb)的DNA 片段并进行序列测定,结果发现在黑浅蛤控制区偏3′端,存在该种特有的长度为363bp的数目可变串联重复序列(variablenumberoftandemrepeat,VNTR),种内因该VNTR出现1~3次不等的串联拷贝,而表现出广泛的长度多态性和个体内线粒体异质性现象,这是目前在动物线粒体控制区中发现的最大的VNTR;在半步目浅蛤的控制区的偏3′端,则同时存在14和17bp的2个较短的VNTRs,该物种也因这2个VNTRs的拷贝数目不同,而存在广泛的长度多态性;而等边浅蛤的该段序列长度保守,没有与其他物种共享的VNTR.以该段序列作为分子标记,运用PCR技术能够快速准确地识别浅蛤属物种. 

Species-specific mitochondrial DNA marker,length polymorphism and heteroplasmy in the clam Macridiscus

In Genus Macridiscus there are three morphologically very similar species: M. melanaegis, M. multifarious and M. semicancellata, the first 2 are sympatric. It is difficult to distinguish these species solely by morphological characters. In this article,genomic DNA was extracted from ethanol preserved foot tissues by aCTAB extraction. A DNA fragment from mitochondrial control region of cytochrome b gene (Cyt b) was used for polymerase chain reaction (PCR) amplification with primers Macr_C01_F16834 and Cytb_Macr_R2. Sequencing of PCR products revealed a 363 bp variable number of tandem repeat (VNTR) close to 3' end of the control region leading to length polymorphism in 44％ heteroplasmic individuals in M. melanaegis. While in the control region of M. semicancellata,two short VNTRs, 14 bp and 17 bp each, were found close to 3' end with length polymorphism between individuals. M. multifarious demonstrated a length conserved control region. These data proved that the three species of Macridiscus could be efficiently and accurately identified via PCR with primer pair Macr_C01_F16834 and Cytb_Macr_R2.