Whole transcriptome analyses of six thoroughbred horses before and after exercising using RNA-Seq

We sequenced the whole mRNA of six thoroughbred horse (Equus caballus) blood and muscle tissues before and after exercising, generating a total of 1.3 billion short reads with 90-bp pair-end sequences from 24 samples. Comparing with current genome annotation, we identified 32,361 unigene clusters spanning 51.83 Mb that contained 11,933 (36.87%) annotated genes. More than 60% (20,428) unigene clusters did not match any current equine gene model. We identified 189,973 single nucleotide variations (SNVs) from the aligned sequences against the horse reference. Most SNVs (171,558 SNVs; 90.31%) were novel compared with over 1.1 million equine SNPs from two databases. Some genes have significantly different expression levels under different environment. We define those identical genes which have different expression levels are ‘differentially expressed’ and carried out differentially expressed gene analysis before and after exercise conditions. We discovered, 62 up- and 80 down-regulated genes in the blood and 878 up- and 285 down-regulated genes in the muscle from the 24 samples. Six out of 28 previously exercise-related known genes, HIF1A, ADRB2, PPARD, VEGF, TNC, and BDNF, were highly expressed in the muscle after exercise. We discovered 56 functionally unknown transcription factors that are probably associated with an early regulatory exercise mechanism from 91 differentially expressed transcription factors. We found interesting RNA expression patterns where different alternative splicing forms of the same gene showed reversed expressions before and after exercising. whole mRNA sequencing profiles of six thoroughbred horse (Equus caballus) blood and muscle tissues before and after exercising

本研究对6匹纯种马（Equus caballus）在运动前后的血液与肌肉组织的全长mRNA进行测序，从24个样本中共获得13亿条90 bp双端测序短读段。与现有基因组注释信息比对后，本研究共鉴定得到32361个单基因簇（unigene clusters），总跨度达51.83 Mb，其中包含11933个已注释基因，占比36.87%。超过60%（20428个）的单基因簇未匹配到当前任何马属基因模型。基于马参考基因组的比对序列，本研究共鉴定得到189973个单核苷酸变异（single nucleotide variations, SNVs）；相较于两个数据库中超过110万个马属单核苷酸多态性（single nucleotide polymorphism, SNPs）位点，其中171558个SNVs（占比90.31%）为全新发现的变异。部分基因在不同环境条件下的表达水平存在显著差异。本研究将表达水平存在差异的基因定义为"差异表达基因"，并针对运动前后的实验条件开展了差异表达基因分析。基于24个样本，本研究在血液中鉴定得到62个上调表达基因与80个下调表达基因，在肌肉中鉴定得到878个上调表达基因与285个下调表达基因。在28个已报道的运动相关已知基因中，HIF1A、ADRB2、PPARD、VEGF、TNC与BDNF这6个基因在运动后肌肉组织中呈高表达状态。在91个差异表达转录因子中，本研究发现了56个功能未知的转录因子，它们可能与运动早期调控机制相关。本研究还观察到了有趣的RNA表达模式：同一基因的不同可变剪接变体在运动前后呈现出相反的表达趋势。本数据集涵盖6匹纯种马（Equus caballus）在运动前后的血液与肌肉组织的全长mRNA测序表达谱。