Discovery of exercise-related genes and pathway analysis based on comparative genomes of Mongolian originated Abaga and Wushen horse.

The Mongolian horses have excellent endurance and stress resistance to adapt to the cold and harsh plateau conditions. Intraspecific genetic diversity is mainly embodied in various genetic advantages of different branches of the Mongolian horse. Since people pay progressive attention to the athletic performance of horse, we expect to guide the exercise-oriented breeding of horses through genomics research. We obtained the clean data of 630,535,376,400 bp through the entire genome second-generation sequencing for the whole blood of four Abaga horses and ten Wushen horses. Based on the data analysis of single nucleotide polymorphism, we severally detected that 479 and 943 positively selected genes, particularly exercise related, were mainly enriched on equine chromosome 4 in Abaga horses and Wushen horses, which implied that chromosome 4 may be associated with the evolution of the Mongolian horse and athletic performance. Four hundred and forty genes of positive selection were enriched in 12 exercise-related pathways and narrowed in 21 exercise-related genes in Abaga horse, which were distinguished from Wushen horse. So, we speculated that the Abaga horse may have oriented genes for the motorial mechanism and 21 exercise-related genes also provided a molecular genetic basis for exercise-directed breeding of the Mongolian horse.

[Construction of Fat-1 eukaryotic expression vector of excision markers and the establishment of transgenic sheep cell lines].

In order to establish marker-free transgenic cell lines, we cloned Fat-1 gene, attB and Loxp sequences by PCR. Then we inserted these sequences to pN1-EGFP vector and got pEGFP-N1-Fat-1 expression vector. PhiC31 integrase mRNA which was generated by in vitro transcription and a pEGFP-N1-Fat-1 expression vector co-electroporated into sheep fetal fibroblasts, and then we got transgenic cell lines expressing green fluorescence. Prokaryotic expression and purification of Cre recombinant protein was performed. Cre recombinant protein was transducted into stably-transfected cell colonies. We identified cell colonies by sequencing and established marker-free transgenic cell lines and eventually- established marker-free transgenic cell lines which were building more safely basic for producing Fat-1 transgenic animals.