大口黑鲈转录组SNPs筛选及其与生长的关联分析

摘要

为开发人工饲料代替冰鲜杂鱼养殖大口黑鲈的分子标记,以食用冰鲜鱼和配合饲料的同批大口黑鲈为研究材料,利用RNA-Seq (RNA sequencing)技术挖掘SNPs (Single nucleotide polymorphisms)标记,并以关联分析筛选可用于育种的候选标记。转录组进行测序共获得174 M数据,8681个SNPs位点。挑选其中具有表达差异的50个SNPs位点进行SNaPshot分型,结果39个分型成功,其中有4个为假阳性,通过转录组技术开发出SNPs标记35个,成功率为70.0%。为进一步检验这些标记是否可用于评估驯食饲料的大口黑鲈选育研究,研究以327尾摄食人工配合饲料的大口黑鲈为试验材料, SPSS软件进行一般线性模型分析SNPs的不同基因型与生长性状的相关性,结果显示有2个SNPs位点与体质量、全长和体高等生长性状存在显著相关性(P<0.05),可作为候选标记用于大口黑鲈的分子辅助育种。由于转录组数据直接反应基因的表达情况,从中挖掘与性状相关的优势基因型与分子标记的成功率高,效果较好。同时也为解决大口黑鲈选育研究中标记缺乏提供了有效途径,为选育提供遗传依据、加速育种进程。%It is a hot research area to substitute frozen trash fish (FF) with artificial compound diet (CD) in feeding the carnivorous economic fish. Largemouth bass (Micropterus salmoides) is one of the important aquaculture species with an annual output of about 340000 tons in fresh water in China. Effect of artificial compound diet at the transcriptome level of largemouth bass is rare reported. The current study utilized RNA-Seq (RNA sequencing) technology on the Il-lumina sequencing platform (Illumina HiSeqTM 2000) to screen polymorphic SNP (single nucleotide polymorphism) markers of largemouth bass in two diets and analyzed the association of SNPs with growth trait. As a result, a total of 174M reads were assembled to generate 95024 unigenes. We used BWA (http://sourceforge.net/projects/bio-bwa/) and SamTools (http://source forge.net/projects/samtools/) to detect SNPs. Several filtering steps were performed to reduce the false positive of SNPs, 8681 SNPs met the selection criteria. 50 SNPs sequences in different genes were validated by PCR amplification and genotyped by the SNaPshot assay. 39 out of 50 SNPs were confirmed. Of these, 4 SNPs were monomorphic for all individuals and 35 SNPs were polymorphic. Validation of selected SNPs was 70%. 327 large-mouth bass with marketable sizes fed by CD were used to evaluate the correlation between these validated SNPs and growth traits including body mass, total length, head length, body width, withers height and caudal peduncle length. Genotyping data were computed and analyzed by SPSS 19.0 statistical software package. Two SNPs were associated with body mass, total length and withers height, which could be used as important candidate molecular markers for breeding selection of largemouth bass. This study shows that RNA-Seq is an efficient approach to uncover gene-asso-ciated SNPs, which could facilitate genetic and functional genomics research in the artificial domestication of formu-lated feeding of largemouth bass.