Identification Of Differentially Expressed Genes In Different Wool Density Of Rex Rabbit And Association Of CCNA2 Gene Polymorphisms With Wool Density

Rex rabbit is a typical rabbit for fur. Wool density, hair length, coarse rate, villus diameter and area of hide are main indices of fur quality. In china, the current problem of rabbit fur quality is mainly focusing on the wool density and area of hide. How to improve the quality of our rex rabbit's fur in order to enhance market competitiveness is a critical problem. In this study, the gene expression patterns in different wool density of rex rabbit are presented and analyzed with cDNA microarray and statistical analysis, then verify the result of selected genes from microarray experiments by qRT-PCR. In order to give the effective molecular markers for the marker-assisted selection (MAS) on early production performance of rex rabbits, SNPs polymorphism of CCNA2 gene were conducted on rex rabbit by PCR-RFLP and DNA sequencing method. The relationship between SNPs polymorphism and fur traits had also been analyzed.The results were as follows:⑴The 2657 differentially expressed genes were identified in different wool density of rex rabbit. Among them, 1103 genes were functionally known genes. 687 genes were up-regulated and 419 down-regulated. GO analysis indicated that most differently expressed genes were monitored to be relative with cell signal transduction, cell development and cell proliferation, cell division, apoptosis, which were mainly belong to nucleotide binding, protein binding, DNA binding and ion binding molecule. And these genes were usually located at cell envelope. KEGG analysis showed that 95 signal pathways associated with up-regulated genes had changed significantly (P<0.05), these pathways were involved in focal adhesion, TGF-beta signaling pathway, cell adhesion molecules, MAPK signaling pathway, Wnt signaling pathway and so on; 87 signal pathways associated with down-regulated genes had changed significantly (P<0.05), this pathways were involved in cell cycle, oxidative phosphorylation, regulation of actin cytoskeleton, p53 signaling pathway and so on. The alteration of gene expression from array data was confirmed by quantitative real-time PCR.⑵Some important differentially expressed genes in different wool density of rex rabbit were identified, such as MMP2, TGF-β1, TGF-β2, IGF-1, BMP2, ActRⅡB, CDK2 and CCNA2. The abnormal expression of this genes may play an important role in the hair follicles initiation and development, finally lead to the distinction in wool density of rex rabbit. After bioinformatics analysis for all differently expressed genes, CCNA2 gene was selected as candidate gene in future studies.⑶Sequences of CCNA2 gene were amplified from the genome RNA samples extracted from blood of rex rabbits by RT-PCR. The polymorphisms of CCNA2 gene were detected by DNA sequencing and PCR-RFLP methods. The 129G>A SNP is a synonymous mutation of alanine, which creates the ScaⅠrestriction site. The 1140G>C SNP is a missense mutation from lysine to asparagine, which destroies the AfIⅡrestriction site.⑷At the 129G>A and 1140G>C SNP marker, the GG genotypes were dominance in population of rex rabbit, respectively; the G alleles were dominant allele, respectively. The two polymorphism sites showed moderate polymorphism(0.250.05).⑸The association between the CCNA2 polymorphism and fur traits (body weight, wool density, wool fineness and hair length) were analyzed in Rex rabbit. At the 129G>A SNP marker, the wool density of the GG genotype are significantly higher than those of GA and AA genotype; At the 1140G>C SNP marker, the wool density of the GG genotype are significantly higher than those of GC and CC genotype. The results indicated that, at the 129G>A and 1140G>C SNP marker, the GG genotype have high wool density, and have no adverse effect on the other fur indices(hair length, wool fineness and body weight). Therefore, the GG genotypes of"129G>A"and"1140G>C"were the most favorable at the CCNA2 gene for wool density trait in Rex rabbit.