以2b-RAD技术构建凡纳滨对虾遗传连锁图谱及生长性状QTL定位

Genetic linkage map construction by 2b-RAD technology and QTL mapping of Litopenaeus vannamei growth traits

  • 摘要: 【目的】构建凡纳滨对虾高密度遗传连锁图谱,并对生长相关性状进行QTL定位,筛选出生长性状相关候选基因,为后续开展凡纳滨对虾分子标记辅助育种、生长相关功能基因精细定位研究等提供理论依据。【方法】以耐低盐选育凡纳滨对虾为父本,厄瓜多尔野生凡纳滨对虾为母本,单尾交配,以2个亲本及150个F1代个体为作图群体,通过2b-RAD测序挖掘SNP分子标记并构建遗传连锁图谱;结合生长性状表型数据,使用MapQTL 6.0在构建的遗传连锁图谱上对体质量、全长、体长、头胸甲长、头胸甲宽、头胸甲高等13个生长相关性状进行QTL定位。筛选QTL区间SNP分子标记附近的基因,经GO功能注释及KEGG信号通路富集分析,挖掘生长相关候选基因;并采用实时荧光定量PCR检测候选基因在凡纳滨对虾不同组织及不同群体间的表达情况。【结果】构建的凡纳滨对虾遗传连锁图谱包括3136个SNPs标记,分布在44个连锁群上;总图谱全长为5430.54 cM,平均图距为1.73 cM。生长性状QTL定位共产生79个生长性状相关QTLs,LOD范围为3.00~11.04,可解释的表型变异范围为9.0%~28.8%。根据GO功能注释及KEGG信号通路富集分析结果,最终筛选出4个生长相关候选基因(TOB2CRATCCT6KLF4)。4个候选基因在凡纳滨对虾各组织中均普遍表达,且CCT6KLF4TOB2基因在耐低盐选育家系群体中的相对表达量均高于常规的凡纳滨对虾群体,其中CCT6基因表达差异达显著水平(P<0.05)。【结论】基于2b-RAD技术构建的凡纳滨对虾遗传连锁图谱鉴定出79个与生长性状相关的QTLs,并筛选出4个与凡纳滨对虾生长性状相关的候选基因(CCT6KLF4TOB2CRAT)。可见,以2b-RAD技术结合QTL定位能高效、快捷挖掘出凡纳滨对虾生长性状相关候选基因,为开展分子标记辅助育种、生长相关功能基因精细定位研究等提供技术支持。

     

    Abstract: 【Objective】 To construct high-density genetic linkage map of Litopenaeus vannamei, to conduct quantitative trait loci(QTL) mapping for growth-related traits, and to screen candidate genes related to growth traits, so as to provide theoretical basis for molecular marker-assisted L. vannamei breeding, fine mapping of growth-related functional genes and other studies.【Method】 In this study, a low salt-tolerant breeding L. vannamei was used as the male parent, and a wild L. vannamei from Ecuador the female parent. With 2 parents and 150 F1 individuals as the mapping population, a large number of SNP molecular markers were found through 2b-RAD sequencing to construct a genetic linkage map. According to phenotypic data of growth-related traits, QTLs of 13 growth traits including body mass, total length, body length, carapace length, carapace width and carapace height were mapped on the genetic linkage map constructed through MapQTL 6.0. The genes near SNPs in the QTL interval were screened out, and growth-related candidate genes were mined through GO functional annotation and KEGG signal pathway enrichment analysis to get candidate genes related to growth. The expression of candidate genes in different tissues and populations of L. vannamei was detected by quantitative realtime PCR(qRT-PCR).【Result】The genetic linkage map of L. vannamei had 3136 SNPs, distributed in 44 linkage groups. The total map length was 5430.54 cM, and the average map distance was 1.73 cM. A total of 79 growth trait-related QTLs were mapped, with LOD ranging from 3.00 to 11.04, and the range of phenotypic variation that could be explained was 9.0%-28.8%. According to GO function annotation and KEGG signal pathway enrichment analysis results, 4 growth-related candidate genes(TOB2, CRAT, CCT6, and KLF4) were screened. The 4 candidate genes were generally expressed in all tissues of L. vannamei, and the relative expression of CCT6, KLF4, and TOB2 genes in low salt-tolerant breeding families was higher than conventional L. vannamei populations, of which the expression difference of CCT6 gene reached a significant level(P<0.05).【Conclusion】 The genetic linkage map of L. vannamei based on 2b-RAD technology identifies 79 QTLs related to growth traits, and screens 4 candidate genes related to growth traits of L. vannamei(CCT6, KLF4, TOB2 and CRAT), indication 2b-RAD technology and QTL mapping together can efficiently and quickly discover candidate genes related to growth traits of L. vannamei, thus providing technical support for molecular marker-assisted breeding, and fine mapping of growth-related functional genes.

     

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