Genetic Mechanism Of Black Coloration In Oujiang Color Common Carp Based On Genome Resequencing And CRISPR/Cas9 System

Pigmentation pattern(body color)is one of the most obvious traits of animals and serves a variety of functions.As a result,this trait is a major target of natural and artificial selection.Vertebrates possess the most variant pigmentation types and patterns,among which bony fishes have the most abundant pigment patterns.Since all the pigment cells in vertebrates originate from the neural crest,which is deeply involved in the development of many organs,understanding the formation mechanism and evolutionary process of fish pigment pattern can provide novel insights into the evolution of vertebrates.The formation of fish pigment pattern involves the synthesis and transfer of pigments,and the differentiation and migration of pigment cells.These biological activities involve many genes and regulatory pathways,which can also be affected by the environment.Carp have abundant body color types.Our research group has established 4 pure lines of pigment patterns of Oujiang color common carp,including "Dahua"(red-bottom with black spot RB),"Fenhua"(white-bottom with black spot WB),"Quanhong"(whole red WR)and "Fenyu"(whole white WW).These 4 pigment patterns can be consistently inherited by the next generation,which lays a foundation for the study of body color inheritance of Oujiang color common carp.In order to preliminarily explore the evolutionary relationship of different strains of carp,the selection of candidate genes related to pigment patterns and the regulatory function of body color related genes,this paper used whole genome resequencing to examine these traits.The functions of the selected body color related genes were verified through gene knockout technology.Relevant research results are presented as follows.1.Whole genome resequencing of 7 carp strainsA preliminary analysis of the evolutionary relationship of different strains of carp was conducted to select candidate genes related to pigment patterns.A total of 64 samples,including the Oujiang color common carp(RB,WB,WR and WW),red carp(Xingguo and Hebao),and wild common carp were analyzed by whole genome resequencing.A total of 17,638,455 SNP markers,1,931,463 indel markers,and 2,600,335 SV markers were obtained.To investigate the genetic and evolutionary relationship between carp with various pigment patterns,a PCA(principal component analysis),phylogenetic tree,LD analysis(linkage disequilibrium),and population genetic structure analyses were conducted on the 7 carp strains.In the PCA,the wild common carp and Xingguo red carp clustered together,the Hebao red carp was classified as its own group,and the Oujiang color common carp strains clustered together.The phylogenetic tree analysis suggested that there was no difference in evolutionary time among 4 colors of common carp.These fish differentiated from the wild carp,and the Xingguo carp ultimately differentiated from the wild common carp.The LD analysis revealed that the Oujiang color common carp and Hebao red carp differentiated at approximately the same time,with the Xingguo red carp evolving most recently.Among the 4 species of Oujiang color common carp,the genetic diversity of the WW population was the lowest.The population genetic structure analysis also indicated that the Xingguo red carp was closely related to wild carp,followed by the Hebao red carp.In addition,by comparing the SNPs among the 4 pigment patterns of the Oujiang color common carp,a large number of candidate genes related to the body color formation of the Oujiang color common carp were screened out.In order to verify the screening results of the whole genome sequencing,the following genes were selected as candidate genes for functional verification: ASIP(agouti-signaling protein),TYRP1(5,6-dihydroxyindole-2-carboxylic acid oxidase;Tyrosinase-related protein 1)and Mlpha(Melanophilin a).From these,ASIP is located upstream of the melanin pathway,and high expression levels of this gene can inhibit the synthesis of eumelanin.Furthermore,ASIP transcriptional control is complex;the gene uses at least two alternative promoters in different mammalian species.The TYRP1 gene is a member of the TRP gene family,which is the core of the melanin synthesis pathway.Located within the brown loci,TYRP1 mutants result in albinism type ? in humans.There are 2 copies of TYRP1 gene in zebrafish,TYRP1a(Tyrosinase related protein 1a)and TYRP1b(Tyrosinase related protein 1b).The Mlpha gene is associated with melanosome transfer in melanin cells,and it is located on the dilute loci.The mutation of Mlpha leads to melanosome transport disorders,which causes light coat color of mammals.2.Effect of ASIP on skin color of Oujiang color common carpThe ASIP gene is located upstream of the melanin pathway,and high expression of this gene can inhibit the synthesis of eumelanin.The expression is regulated by several promoters,which have spatial specificity and influences the formation of animal patterns.By comparing genomic data,there were 2 ASIP genes in the Oujiang color common carp genome,of which ASIP1 is located in LG9,and ASIP2 is located in LG11.After cloning and assembly of the gene,a total length of 2140 bp of ASIP1 DNA and 2304 bp of ASIP2 DNA were obtained.Both ASIP1 and ASIP2 have a cds of 378 bp,and the two genes share 96% nucleotide identity.In this study,two ASIP genes(ASIP1 and ASIP2)were expressed at higher levels in the white belly than in the dark dorsal skin were observed.These observations are in agreement with the typical dorsal-ventral expression pattern as in wild common carp.Meanwhile,unusually high expression was observed in nonmelanophore side skin compared with melanophore side skin in the Oujiang color common carp.Using CRISPR/Cas9,the two ASIP genes were disrupted in the Oujiang color common carp of red with big black patches(RB)pattern.It was observed that the black patches disappeared,and melanophores dispersed along the dorsal skin in the F0 mosaic RB individuals.The present study indicated that the ASIP gene may play an essential role in the regulation of melanin aggregation and distribution during the formation of black patches in common carp.3.Effect of the TYRP1 gene on the skin color of Oujiang color common carpThere were 3 TYRP1 genes observed in the Oujiang color common carp genome,TYRP1 a,TYRP1b and TYRP1 c,respectively.TYRP1 a is located on scaffold 2034,and TYRP1 b,TYRP1c are located on LG1.After cloning of the genes,the total length and cds length of the TYRP1 a gene DNA were 9769 bp and 1575 bp,respectively.For the TYRP1 b gene DNA,the lengths were 5796 bp and 1581 bp respectively.For TYRP1 c,the DNA lengths were 5837 bp and 1581 bp,respectively.The similarity between TYRP1 a and TYRP1 b or TYRP1 c was 72%,and that between TYRP1 b and TYRP1 c was 97%.All 3 TYRP1 genes were highly expressed in the black tissues,and at low levels in the nonmelanin tissues.The expression patterns of TYRP1 b and TYRP1 c were very similar,and were expressed in more tissues than TYRP1 a.Therefore the TYRP1 b and TYRP1 c may have doubled from the same gene origin.The result of TYRP1 knock out in zebrafish indicated that disruption of one of the TYRP1 genes does not result in phenotype change,but knocking out the 2 TYRP1 genes simultaneously would silence melanin expression.By knocking out the 3 TYRP1 genes in WB,3 mutant phenotypes were obtained.The complete mutant showed melanin was absent in skin,and the mosaic mutant showed brown or gray patches instead of black patches.Taken together,these results suggest that the function of multiple copies of the TYRP1 gene is repetitive in zebrafish and Oujiang colored common carp.In addition,the melanin deleted WB mutant indicated that TYRP1 is responsible for the apoptosis of melanocytes,and the brown and gray mutants may be induced by the disruption of different functional domains of the TYRP1 b gene.Our results indicated that TYRP1 may induce the different phenotypes between WB and WW.4.Effect of the Mlpha gene on the skin color of the Oujiang color common carpThe Mlpha gene has been associated with the transport of melanosomes in melanocytes.In mammals,Mlpha mutations induce melanosome transport obstacles,resulting in light coat color.Two Mlpha genes(Mlpha1 and Mlpha2)were found by combining genomic analysis and gene cloning in this chapter.The total length of Mlpha1 DNA was 7565 bp,and the cds length was 1716 bp.The total length of Mlpha2 DNA was 13267 bp and the cds length was 1668 bp.Results of RT-PCR indicated that Mlpha expression showed no significant correlation with the black patches and the highest levels of expression in the skin of WR.It was speculated that Mlpha may be involved in other regulatory mechanisms in WR,such as pigment cell migration.When the Mlpha gene is knocked out,the melanocytes dramatically shrink and the hollow melanocytes with degenerate dendrites were found in the mutant RB and WB fish.It was also predicted that the different mutant phenotypes were caused by the absence of different functional domains of the Mlpha gene.The findings reported that a few mutants of RB showed the phenotype of localized red body color fading.However,this observation requires further verification that the phenotype is a result of the mutation.In addition,mutation of the Mlpha gene in this study may be related to the immunodeficiency of fish.