Identification of candidate genes and regulatory factors underlying intramuscular fat content through longissimus dorsi transcriptome analyses in heavy Iberian pigs

One of the most important determinants of meat quality is the intramuscular fat content (IMF). The development of high-throughput techniques as RNA-seq allows identifying gene pathways and networks with a differential expression (DE) between groups of animals divergent for a particular trait. The Iberian pig is characterized by having an excellent meat quality and a high content of intramuscular fat. The objectives of the current study were to analyze the longissimus dorsi transcriptome of purebred Iberian pigs divergent for IMF breeding value to identify differential expressed genes and regulatory factors affecting gene expression. RNA-seq technique allowed identifying in the samples analyzed around 10,000 of the 25,878 annotated genes. In addition to this, the 42.46% of the identified transcripts corresponded to newly predicted isoforms. The differential expression analyses revealed a total of 221 DE annotated genes and 116 DE newly isoforms. Functional analyses identified an enrichment of overexpressed genes in animals with a higher content of IMF involved in lipid metabolism (FASN, SCD, ELOVL6, DGAT2, PLIN1, CIDEC and ADIPOQ) and an enrichment of overexpressed genes related with myogenesis and adipogenesis (EGR1, EGR2, EGR3, JUNB, FOSB and SEMA4D) in the groups of animals with a lower content of IMF. In addition to this, potential regulatory elements of these DE genes were identified. On the one hand, co-expression networks analyses revealed six lncRNAs (ALDBSSCG0000002079, ALDBSSCG0000002093, ALDBSSCG0000003455, ALDBSSCG0000004244, ALDBSSCG0000005525, ALDBSSCG0000006849) co-expressed with SEMA4D and FOSB genes and one (ALDBSSCG0000004790) with SCD, ELOVL6, DGAT2, PLIN1 and CIDEC. The computation of the regulatory impact factors (RIFs) revealed a total of 301 transcriptionally regulatory factors involved in the expression differences, some of them have a role in adipogenesis (ARID5B, CREB1, VDR, ATF6 and SP1) and other ones take part of myogenesis and development of skeletal muscle (ATF3, KLF11 and MYF6). The results obtained provide a relevant insight about the knowledge of the genetics mechanisms underlying IMF content in purebred Iberian pigs and a set of candidate genes and regulatory factors is proposed for a further identification of polymorphisms susceptible of integrate in a selection program. Overall design: Twelve Iberian individuals with divergent breeding values for intramuscular fat content were selected (six per group), samples of Longissimus dorsi were collected after the slaughter, RNA from these samples was extracted, pair-end libraries we

肌内脂肪含量（intramuscular fat content, IMF）是影响肉质的最重要决定因素之一。以RNA测序（RNA-seq）为代表的高通量技术的发展，使得研究者能够鉴定出在特定性状上存在差异表达（differential expression, DE）的动物群体间的基因通路与调控网络。伊比利亚猪以优异的肉质与高肌内脂肪含量为典型特征。本研究旨在分析肌内脂肪育种值存在差异的纯种伊比利亚猪的背最长肌转录组，以鉴定影响基因表达的差异表达基因及调控因子。

RNA-seq技术在本次分析的样本中，成功鉴定出25878个注释基因中的约10000个。此外，所鉴定的转录本中有42.46%属于新预测的剪接异构体。差异表达分析共鉴定出221个差异表达注释基因与116个差异表达新异构体。功能富集分析显示，肌内脂肪含量较高的猪群体中，过表达基因富集于脂质代谢通路（包含FASN、SCD、ELOVL6、DGAT2、PLIN1、CIDEC及ADIPOQ）；而肌内脂肪含量较低的猪群体中，过表达基因则与肌发生和脂肪生成相关（包含EGR1、EGR2、EGR3、JUNB、FOSB及SEMA4D）。

此外，本研究还鉴定了上述差异表达基因的潜在调控元件。一方面，共表达网络分析显示，6个长链非编码RNA（long non-coding RNA, lncRNA）（ALDBSSCG0000002079、ALDBSSCG0000002093、ALDBSSCG0000003455、ALDBSSCG0000004244、ALDBSSCG0000005525、ALDBSSCG0000006849）可与SEMA4D及FOSB基因共表达，另有1个lncRNA（ALDBSSCG0000004790）可与SCD、ELOVL6、DGAT2、PLIN1及CIDEC共表达。调控影响因子（regulatory impact factors, RIFs）的计算结果显示，共有301个转录调控因子参与了表达差异调控，其中部分因子参与脂肪生成过程（包括ARID5B、CREB1、VDR、ATF6及SP1），其余则参与肌发生与骨骼肌发育过程（包括ATF3、KLF11及MYF6）。

本研究结果为阐明纯种伊比利亚猪肌内脂肪含量相关的遗传机制提供了重要见解，并提出了一系列候选基因与调控因子，可供后续筛选可整合进育种程序的多态性位点。整体实验设计：选取12个肌内脂肪含量育种值存在差异的伊比利亚猪个体（每组6只），屠宰后采集背最长肌样本，提取样本总RNA，构建双末端测序文库，我们