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为获得与尼罗罗非鱼(Oreochromis niloticus)链球菌病抗性相关的单核苷酸多态性(single nucleotide polymophisms,SNPs)标记,本研究克隆了尼罗罗非鱼补体C9基因(On C9)的基因组DNA序列,通过直接测序法从20个家系的39条尼罗罗非鱼On C9的测序序列中分析得到22个SNPs位点,其中S1(C483T)、S3(G954A)、S4(T1180C)、S5(G1539A)、S6(G1627T)、S7(A2590T)、S10(T2876G)、S11(C2915G)、S12(T2939G)、S13(A2942C)、S14(A3544C)、S15(T3604C)、S16(T3670C)、S17(T3841C)、S18(T4339C)、S19(G4494C)、S20(A4678G)、S21(T5790A)、S22(T5816G)等19个SNPs位点在内含子中,S2(C644T)、S8(C2660T)和S9(A2751T)等3个SNPs位点位于外显子中,其中S2、S9属于非同义突变,S8为同义突变。此外,本研究获得的On C9 SNPs位点中有一半为碱基转换产生,另一半为碱基颠换产生。通过snapshot分型法对易感群体(susceptible group,SG)(84尾)和抗病群体(resistance group,RG)(81尾)进行了分型,并利用Popgen32软件统计分析了尼罗罗非鱼On C9各个SNPs位点在两个群体的多态性和遗传参数,结果显示多态信息含量(polymorphism information content,PIC)值为0.09~0.37,表明所有SNPs位点均为中度或低度多态。采用SPSS 20软件分析了22个SNPs位点在两个群体中的基因型频率、等位基因频率,并采用卡方检验其显著性,分析其与抗病性状之间的相关性,发现S8和S21两个位点与抗病性状显著相关。通过Haploview 4.2软件分析了On C9中的22个SNPs位点所形成的单倍块和连锁不平衡状态。结果表明,22个位点可构成4个单倍块和11个单倍型。其中GTTAG和ATGA两个单倍型与尼罗罗非鱼链球菌病易感性显著相关(P<0.05)。本研究显示,筛选到的尼罗罗非鱼On C9的两个SNPs位点和两个单倍型具有用于尼罗罗非鱼抗链球菌(Streptococcus agalactiae)病性状的分子辅助育种的潜力。
In order to obtain single nucleotide polymorphisms (SNPs) markers associated with streptococcal disease in Oreochromis niloticus, this study cloned the C9 gene of Onychomycosis Nilai (On C9) ) Were sequenced and 22 SNPs loci were obtained from the sequencing of 39 Cynomolgus nigra tilapia On C9 from 20 families by direct sequencing. Among them, S1 (C483T), S3 (G954A), S4 (T1180C ), S5 (G1539A), S6 (G1627T), S7 (A2590T), S10T2876G, S11C2915G, S12T2939G, S13A2942C, S14A3544C, S15T3604C, S16T3670C 19 (S64), S17 (T3341C), S19 (G4494C), S20 (A4678G), S21 (T5790A) and S22 (T5816G) C2660T) and S9 (A2751T) were located in exon, of which S2 and S9 belonged to non-synonymous mutation and S8 was synonymous mutation. In addition, half of the On C9 SNPs obtained in this study were base-conversion and the other half were base-transversion. The susceptible group (SG) (84) and resistance group (RG) (81) were classified by using the snapshot typing method. Populus was used to analyze the distribution of Nile tilapia Onc9 SNP loci in two populations of polymorphisms and genetic parameters, the results showed that the polymorphism information content (PIC) value of 0.09 ~ 0.37, indicating that all SNPs loci were moderate or low state. SPSS 20 software was used to analyze the genotype frequency and allele frequency of 22 SNPs loci in two populations and the significance was tested by chi-square test. The correlation between them and disease resistance traits was analyzed. It was found that S8 and Two sites of S21 were significantly associated with disease resistance. Haploview 4.2 software was used to analyze the haplotypes and linkage disequilibrium of 22 SNPs in On C9. The results showed that 22 loci could constitute 4 haplotypes and 11 haplotypes. The GTTAG and ATGA haplotypes were significantly associated with susceptibility to Nile tilapia (P <0.05). The present study shows that the two SNPs and two haplotypes of On C9 of the selected Nile tilapia have the potential of molecular-assisted breeding for the trait of Nile tilapia.