Gene Expression Variation And Epigenetic Modification Associated With Skin Color In Koi Carp(Cyprinus Carpio Var. Koi)

Cyprinus carpio var. Koi dubordinated to Cyprinidae, is varieties of common carp and a important worldwide ornamental fish. The variable colors and color patterns of Koi makes it a unique system for studying the genetics and epigenetics of pigmentation. In this study, red skin and white skin transcriptome were sequenced by RNA-Seq, the main conclusion as follows:Transcriptome sequencing yielded about 10.3 million pairs of reads for red skin and white skin, respectively. The clean reads were aligned to common carp genomes with Top Hat, respectively. Combining the merged alignments of two tissues with reference annotation, 85,823 transcripts were constructed with Cufflinks. Among the initial assembly, 81,959(95.3%) transcripts were covered in the reference gene regions. However, there were still 3,846 transcripts falling away from the reference genes. They were transcribed from 3,157 transcribed loci, of which were 437 cis-antisense gene or 2,720 intergenic region.Among 3,157 novel transcribed loci, coding potential prediction with Transdecode prediction and Blastx homolog search identified 2,295 novel protein-coding gene. The remaining 862 transcripts might be non-coding RNAs. Search against NONCODE database and teleost non-coding RNA dataset found that 143 loci were significantly homologous to known non-coding RNAs.To validate the accuracy of RNA-Seq result and the expression difference analysis, we used Cuffdiff, DESeq and edge R to identify DEGs between red skin and white skin. A total of 169, 453, 1453 genes were predicted to be significant differently expressed, respectively. Cross-over of these three results found that 62 DEGs were identified using all methods. Among the 62 DEGs, 52 genes were co-expressed in two skins. The other 10 genes were tissue-specific. All 52 DEGs were selected for q RT-PCR analysis in the two skin. Overall, except for six genes without significant differences, the expression patterns of the other 46 genes were significantly different and similar to those indicated by the RNA-Seq analysis.The BS-PCR methylation research were performed in Koi carp gpib and klf4 a genes, respectively. The methylation ratio of gpib in red skin is 83.98% and in white skin is 68.68%. klf4 a methylation ratio in red and white skin is 79.8% and 95.8%. The mathylation level and gene expression have an inverse relationship, and the results of this experiment are corresponding to the RNA-Seq and q RT-PCR expression patterns.The transcriptome sequences and annotations obtained in this study will be valuable resources for basic research of skin color mechanism.