Study On Genetic Basis Of Black Patches In Oujiang Color Common Carp Using BSA,MBD-Seq And CRISPR/Cas9 Technologies

Pigmentation is an important phenotype that widely exists in vertebrates,and molecular mechanism of pigmentation has been widely studied in animals.Oujiang color common carp?Cyprinus carpio var.color?is a variety of common carp with 5basic body color,in which the�WB�?White background with big black-patches?and�RB�?Red background with big black-patches?type of Oujiang color common carps keep phenotype of obvious black-patches.In order to uncover the molecular genetic basis of the black-patches in Oujiang color common carp,we conducted a number of studies as follow:Firstly,phenotype statistics and gene mapping based on next generation sequencing such as BSA were conducted to investigate the genetic basis of the phenotype of black-patches.Then,Methylated Cp G binding protein 2 enrichment sequencing?MBD-seq?was applied to profile the epigenetic basis under the black-patches phenotype of Oujiang color common carp.Finally,the effects of Dopachrome tautomerase?Dct?,one of key genes in melanin pathway,on black-patches of Oujiang color common carp were studied by using CRISPR/Cas9 gene editing technology.The results of these studies are as follows:1.Gene mapping of black-patches Oujiang color common carp by Bulked Segregant AnalysisIn this chapter,using the F₂populations established by crossing�WB�and�WW�?Whole White?types of Oujiang color common carp,the phenotypic characteristics were quantitated and the sizes of black-patches were measured,and gene mappings of black-patches phenotype were conducted through BSA method combined with selective sweep analysis based on Next Generation Sequencing.The results showed that,black-patches in�WB�was dominant compared to none-black-patches in�WW�type of Oujiang color common carp.The separation ratio of"WB"and"WW"in the F₂ population was about 4:1.The black area proportion in�WB�ranged from 2%to 35%,and the two distinct peaks between 9%-10%and 18%-19%were observed,which represented 31 and 15?2:1?individuals of the investigated Oujiang color common carp respectively.It can be inferred that the black-patches trait might be dominated by a major gene,which also had addictive effect on the size of black-patches in�WB�type of Oujiang color common carp.Through BSA mapping,a total of 86candidate regions were obtained,which mainly distributed on the chromosomes 11,chromosomes 33 and chromosomes 10.There were 191 candidate genes annotated in the candidate regions.Based on the analysis of the selective sweep regions of"WB"and"WW"types,205 genes were annotated in this regions,in which,25 genes coexisted in both of the BSA analysis and selective sweep analysis,especially,Cntn4displayed the most significant signal in the BSA analysis,implying this gene may play an important role in the formation of black-patches in Oujiang color common carp.2.DNA methylation basis of black-patches phenotype in Oujiang color common carpIn this chapter,MBD-seq was applied to investigate DNA methylation basis in 4different types of Oujiang color common carps from the same F₂ family.The results showed that,DNA methylation is widely existed on the genome of Oujiang color common carp.Compared to�WR�?Whole Red?and�WW�types with no black-patches phenotypes,the�RB�and�WB�with black-patches kept higher level of DNA methylation and the principal components of DMRs showed that�WR�and�WW�types shared more similarities in DNA methylation,which indicated that DNA methylation may play an important role in formation or maintain of the black-patches phenotype.The total of 316,132 differentially methylated regions?DMRs?were respectively captured between the�WB�and�WW�?�RB�and�WR�,and GO enrichment analysis of DMRs on the promoter and exon region of genes showed that DNA methylation regions were enriched in the biological processes such as nerve Cell differentiation,blood vessel diameter regulation,mucus secretion regulation,muscle cell differentiation and development.3.Effect of gene Dct on black patches of pigmentation pattern in Oujiang color common carpDopachrome tautomerase?Dct?is one of tyrosinase family members,playing an important role in the synthesis of the melanin.The effect that Dct on the pigment pattern of fish remains elusive to date.In this chapter,Dct genes in Oujiang color common carp were cloned and gene expression levels of skin tissues in�WB�type of Oujiang color common carps were obtained through RT-PCR.Furthermore,CRIPSPR/Cas9 gene editing technology was applied to construct the Dct highly mutated�WB�type of Oujiang color common carp.This study showed that 2 Dct genes were detected in Oujiang color common carp and the gene expression of Dct1 and Dct2 showed different expression pattern in black and white region of skin in�WB�type of Oujiang color commpn carp.For Dct1,the gene expression is significantly higher in the black-patches region than the white region of skin?P<0.05?.For Dct2,on the contrary,the white skin of the�WB�kept the significantly higher level of gene expression?P<0.05?.The observation of Dct mutated�WB�Oujiang color common carp?efficiency>50%?showed that the black-patches diminished and more non-dendritic like melanocytes appeared.