| The Aral barbel（Luciobarbus brachycephalus）belongs to Cyprinidae,Barbinae,and Barbus;it is native to the Aral Sea of Uzbekistan.To promote the development of inland saline-alkalinity fisheries in China,the Heilongjiang River Fisheries Research Institute of Chinese Academy of Fishery Sciences introduced more than 100 wild fish species through the"948"project of the Ministry of Agriculture in 2003.In 2012,Aral barbel was successfully carried out the wholly artificial propagation and prompted to be cultured in more than twenty provinces and municipalities across China,achieving good economic and social benefits.The wild L.brachycephalus is gray-black in the dorsal and silvery-white in the abdomen;however,a few individuals were mutated to yellow during the culture.The yellow Aral barbel is an excellent germplasm with the advantages of stable genetic body color,good domestication cluster,strong resistance to low oxygen,disease resistance,and high ornamental value.However,due to the scarcity of L.brachycephalus genetic resources,the deposited genetic resources are not sufficient to support the analysis of the genetic mechanism of economic traits and molecular breeding of new varieties;there is an urgent need to develop its genetic resources to promote the selection and breeding process of its varieties.In this study,we developed the genomic resources of L.brachycephalus by assembling the genome,constructing a genetic linkage map,and QTL mapping for growth-related traits.We also screened genes associated with the yellow body color by differential expression analysis of the transcriptome and bulked segregant RNA-Seq（BSR-Seq）and revealed the role of candidate genes in body color formation and the genetic mechanism of yellow body color variation in L.brachycephalus.We provide an important theoretical basis for the selective breeding of it and other fish species with diverse body colors.（1）The draft genome of L.brachycephalus was assembled using Pac Bio long reads,totaling1,698.3 Mb,consisting of 653 contigs with an N50 size of 4.5 Mb,which covered 94.01%of the genome estimated by Kmers with a length of 1,806.6 Mb.In the genome,54,600 protein-coding genes were predicted.The protein-coding genes were more than that of the diploid relatives,zebrafish（Danio rerio）and grass carp（Ctenopharyngodon idellus）.In contrast,they were similar to the tetraploid carp（Cyprinus carpio）and schizothorax（Schizothorax o’connori）.Functional annotations were available for 92.9%of the genes（50,727）.Comparative genomics analysis showed that Aral barbel was divergent from its related species,Labeo rohita,around 42.6 million years ago（Ma）.In addition,there were 6,306 expanded gene families and 338 contractive famil ies;the expanded gene families were involved in ion transport,osmotic pressure regulation,and pigment synthesis,among other functions.（2）To construct the high-density linkage map of L.brachycephalus,146 individuals and their parents from a full sibling family were sequenced by loci specific amplification fragment（SLAF-Seq）.A total of 66,771 single nucleotide polymorphisms（SNP）were obtained,of which 31,019SNPs were assigned to 50 linkage groups.The linkage map contained 4,304 unique loci with a total span of 2,419.2 c M and an average length of 48.4 c M.The length of each linkage group ranged from 5.2 c M to 76.2 c M,with an average interval gap of 0.4 c M to 0.8 c M.In addition,the QTLs of four traits,head width（HB）,total length（TL）,body height（BH）,and body color（BC）of L.brachycephalus,were further precisely mapped.The QTL mapping showed that no significant QTLs were obtained for the BC trait,while 72 significant QTLs were obtained for the other three traits,including 3 QTLs for HB,38 QTLs for BH,and 48 QTLs for BH.Moreover,186,140,and 5 genes were characterized to be associated with BH,TL,and HB in these QTL regions,respectively.（3）The transcriptome analysis of the black and yellow skin of L.brachycephalus was performed.After removing duplicate and low-quality reads,119.0 M and 131.6 M cleaned reads were obtained,respectively.A total of 163,700 transcripts were assembled,with length s ranging from201 bp to 14,389 bp and an N50 length of 1,943 bp.A total of 855 differentially expressed genes were identified.Of these,659 genes were significantly down-regulated in yellow skin,and 196genes were significantly up-regulated in yellow skin.KEGG pathway enrichment results showed that the differential genes were significantly enriched in 238 pathways,including melanin generation,c AMP signaling pathway,tyrosine metabolism pathway,cysteine and methionine metabolism,and other pathways related to pigmentation.Six genes（ywhae,cdk4,rhoa,mt-ATP6,tuba1,c3）related to melanin production and transfer were screened in these pathways.In addition,five genes were related to tyrosine metabolism（hexa,trinity＿dn34596＿c0＿g1,met,trinity＿dn16612＿c0＿g2,hpd）,and two genes related to xanthin expression（hpd,pvalb7）.RT-q PCR verified the expression pattern and was consistent with transcriptome analysis.（4）In order to obtain genes related to body color,BSR-Seq of the skin in black and yellow L.brachycephalus was performed.The sequencing pools were composed of black parent skin（T1）,yellow parent skin（T2）,30 yellow skin（T3）,and 30 black skin（T4）from a full sibling family F1population.A total of 496.11 M cleaned reads were generated by BSR-Seq,with four pool reads of 43.18 M（T1）,44.12 M（T2）,205.50 M（T3）,and 203.31 M（T4）,respectively.As a result,1,824,915 SNPs were detected in two parents and two mixed pools using GATK software,among which 8,978 high-quality SNPs were used for subsequentΔ（SNP-index）analysis.One hundred and eight differentially significant SNPs were screened byΔ（SNP-index）?0.4.These SNPs were located in 67 candidate genes,including tyrosinase,cysteine-type peptidase,zinc ion binding protein binding,and cysteine and histidine-rich domain-containing protein,which could be used as candidates associated with skin pigmentation.（5）The tyr gene is a key gene for melanin synthesis.To verify whether it is related to the mutation of the yellow body color in L.brachycephalus,CRISPR/Cas9 gene editing technology was used to knockout tyr in Aral barbel.A total of 697 F0 individuals were obtained,including 331yellow-spotted mutants with a mutation rate of 47.49%.Mutants were classified into four categories based on spotted area:55（16.62%）mutants with a spotted area greater than 75%（Y75）,83（25.08%）mutants with a spotted area less than 75%and greater than 50%（Y50）,56（16.92%）mutants with a spotted area less than 50%and greater than 25%（Y25）,and 137（41.39%）mutants with a spotted area less than 25%（Y25-）.Most of the skin in the Y75 mutant is yellow,with a small,scattered distribution of black skin on the trunk,dendritic melanocytes,no massive melanocytes,and the melanin does not entirely cover the surrounding yellow pigment cells.TA clone sequencing results showed nine mutant alleles were characterized in the mutants,includi ng 13 frameshift mutations in3 target loci,and 66.67%（6/9）of the alleles were stop-codon gained and pre-matured.Most Y25-mutant skins appeared black,with a small,scattered distribution of yellow skin on the head and trunk.The melanocytes were blocky and dendritic in shape and could obscure the surrounding yellow pigment cells completely.There were four mutant alleles,including seven frameshift mutations,and 25%（1/5）of alleles were stop-codon gained and pre-matured.The Y50 and Y25mutants are intermediate in phenotype,with a mosaic of black and yellow skin on the body.The expression levels of tyr and the downstream genes,tyrp1 and dct,in the skin of the four mutants were decreased,the melanin content was significantly reduced,whereas the carotenoid content was significantly increased.These results confirm that tyr might have a role in the body color mutations in L.brachycephalus.In summary,this study was the first report on the genome of L.brachycephalus and obtained a high-quality genetic linkage map and 72 QTLs associated with growth traits,including BH,TL,and HB.This study also characterized 855 significantly differentially expressed genes and four candidate genes（tyrosinase,cysteine-type peptidase,zinc ion binding protein,and cysteine and histidine-rich domain-containing protein）related to body color by transcriptome and BSR-Seq analysis.Moreover,this study knocked out tyr in the black L.brachycephalus using CRISPR/Cas9gene editing technology and obtained yellow spotted mutants in which melanocytes in the mutant were significantly reduced and replaced by yellow pigment cell patches,confirming that tyr has an important role in the yellow body color mutation of L.brachycephalus. |
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