基于simc1基因筛选3种尖塘鳢特异性SNP标记

Screening of the Specific SNP Markers Based on Simc1 Gene in Three Species of Oxyeleotris

为了开发云斑尖塘鳢、线纹尖塘鳢及其杂交尖塘鳢种间特异性分子标记,选择泛素化相互作用基序包含蛋白1(sumo-interacting motif-containing protein 1,simc1)基因作为候选基因。利用该基因mRNA序列设计引物对P1针对3种尖塘鳢基因组进行扩增,均扩增到893 bp的外显子序列。该序列在3种尖塘鳢种内个体间均未获得单核苷酸多态性(singe nucleotide polymorphisms,SNP)位点,但种间共存在21个SNP位点,其中有7个同义突变,14个错义突变。选择较容易设计引物的SNP15位点设计等位基因特异性PCR(allele specific PCR,AS-PCR)引物对P2和P3。利用这两对引物针对待测样品基因组进行扩增,若P2扩增出222 bp条带,而P3无扩增条带,则样品为线纹尖塘鳢;若P2无扩增条带,而P3扩增出566 bp条带,则为云斑尖塘鳢;若P2和P3同时扩增出222 bp和566 bp条带,则为杂交尖塘鳢。通过对模板进行梯度稀释,检测P2和P3引物对灵敏度,结果表明,P2引物对检测的基因组最低浓度为5×10^(-4)mg/L,P3引物对检测的基因组最低浓度为5×10^(-2)mg/L。3种尖塘鳢特异性SNP标记的开发及快速检测方法的建立,可为尖塘鳢种质资源鉴定及新品种培育等工作提供技术支撑。

In order to screen for the specific molecular markers among Oxyeleotris marmoratus,Oxyeleotris lineolatus and their hybrids,the candidate gene of sumo-interacting motif-containing protein 1(simc1)was selected to distinguish the three fish species.Using the mRNA sequence of hybrids simc1 gene,P1 primer pair was designed to amplify the genome DNA of the three species of Oxyeleotris,and the 893 bp fragment was amplified.This sequence had no SNP within intraspecific individuals of the three Oxyeleotris species,but 21 SNP sites were identified among interspecific individuals of the three species of Oxyeleotris,and all of them were in exon regions,including 7 synonymous mutations and 14 nonsynonymous mutations.Then P2 and P3 primer pairs were designed for allele-specific PCR(AS-PCR)according to the genotypes of the SNP15 in the three species of Oxyeleotris.After PCR amplification and electrophoresis,O.lineolatus could be identified by a 222 bp fragment with P2 and no fragment with P3 primer pair,and O.marmoratus could be identified by no fragment with P2 and a 566 bp fragment with P3.And the hybrids could be identified by both 222 bp and 566 bp fragments with P2 and P3.The sensitivity of P2 and P3 was detected by the gradient dilution of DNA templates.The results showed that the minimum detection concentrations of genomic DNA were 5×10^(-4)mg/L and 5×10^(-2)mg/L for P2 and P3 primer pairs,respectively.The screening for specific molecular markers of the three species of Oxyeleotris and the establishment of rapid detection methods can provide technical supports for the study of germplasm resources and the breeding of new varieties.