基于3D基因组技术解析猪肌纤维类型标志基因受cis-eQTL调控的分子机制

Muscle fiber type composition are closely related with meat quality traits including meat color, pH value, tenderness and water-holding capacity, which can be regarded as sub-trait for genetic dissection of meat quality traits. We conducted eQTL mapping analysis for porcine Longissimus dorsi, which identified three cis-eQTL regulating myofiber type specific marker genes. The present study was going to do fine mapping to narrow the QTL region, and discover all mutations surrounding the three target genes of cis-eQTL by deep resequencing of haplotype-representative homozygote. Meanwhile, we will identify important transcriptional regulatory element of target genes and capture the DNA interaction events with 3D genome technologies. Furthermore, we will conduct functional annotation for all identified mutations according to multidimensional data, from which potential functional mutations will be selected for functional experiments validation in cell line. And genome-editing mouse model targeting important functional mutation will be generated to further evaluate their effect to myofiber characters. Moreover, we will do genotyping for the identified polymorphism sites in multiple populations to evaluate their effect to myofiber and meat quality traits. The present study will reveal the molecular mechanism regulating the expression of myofiber type marker genes, which will deep our understanding to the genetic mechanism of myofiber characters and meat quality traits. Meanwhile, the identification of functional variants will provide new molecular markers for breeding of porcine meat quality, which will effectively promote the research advance of pig breeding target meat quality. In conclusion, the present study has not only theoretical value but also practical significance for pig breeding.

肌纤维类型组成与肉色、pH值、嫩度和系水力等指标密切相关，可作为解析肉质性状遗传基础的精细组分性状。申请人基于猪背最长肌eQTL分析鉴定到3个调控肌纤维类型标志基因的cis-eQTL。本研究拟通过精细定位缩小QTL区域，对代表性单倍型纯合个体深度重测序挖掘cis-eQTL靶基因相关的各类突变；利用3D基因组技术鉴定靶基因附近重要转录调控区并捕获DNA互作事件；综合多种数据进行突变位点功能注释并通过细胞水平实验鉴定cis-eQTL功能突变；建立基因编辑小鼠模型评估功能突变对于肌纤维特性的影响，并在多群体中检验功能突变对肌纤维特性和肉质性状的效应。研究结果将揭示调控这些肌纤维类型标志基因表达的分子机制，深化人们对猪肌纤维和肉质性状遗传基础的认识；功能突变的鉴定将为肉质性状遗传改良增添新的分子标记，有力地促进猪的肉质育种研究，所以，本研究兼具重要的理论价值和育种实践意义。

肌纤维类型与肌肉颜色、pH值、嫩度和系水力等肉质指标密切相关，但是其遗传基础研究进展缓慢。本项目前期基于大群体猪背最长肌RNA-seq数据开展了eQTL分析研究，鉴定到MYLPF、TNNC2和TNNI1这3个与肌纤维直径显著相关且受cis-eQTL调控的肌纤维类型标志基因，本项目重点围绕这些基因开展cis-eQTL功能变异鉴定工作。针对MYLPF基因，本项目在其第一外显子区鉴定到一个同时影响剪接与转录活性的突变位点，并经双荧光素酶报告基因载体、mini-gene载体和EMSA实验证实该突变是导致cis-eQTL信号产生的功能突变。TNNC2基因不仅存在cis-eQTL信号，也存在ASE现象，在TNNC2-201转录本的第一外显子剪接供体区域鉴定到一个11bp的缺失，同时在其前面37bp处存在17bp的插入变异；通过荧光素酶报告基因载体和真核表达及RT-PCR实验证实这两个偶联的变异位点导致转录本结构发生变化，而且是导致其表达水平产生变化的功能变异，这两个偶联的变异位点多态性与肌纤维性状及肌内脂肪含量存在极显著关联。此外，在本项目支持下，我们还开展了群体水平的肌内脂肪含量性状相关、eQTL和GWAS联合分析，鉴定到SFXN4等212个肌内脂肪含量相关基因；鉴定到猪肉颜色候选基因NUDT7基因相关cis-eQTL功能变异。本项目鉴定到的功能变异位点为肉质性状的高效选育提供了精准分子标记，新发现的肉质性状候选基因也为肉质性状遗传解析提供了新的基因资源。