Transcriptome Characterization Involved In Growth And High-density Genetic Linkage Map Construction In Chinese Perch

The Chinese perch Siniperca chuatsi(Basilewsky)is one of the most commercially important freshwater fishes for aquaculture in China.Due to its excellent taste,fast growth,and high market price,nowadays it has been massively cultured in China,with total production of 298,057 tons in 2015.However,the cultured population of this species gradually exhibits growth depression,mainly in slow growth rate and large differences in individual body size.In addition,fish growth traits are governed by interactions of multiple genetic loci,which are hard to be identified using traditional quantitative genetics.These have led commercial breeders to incorporate significant selection for increased growth rate in breeding programs by marker-assisted selection(MAS).This paper conducted the transcriptome characterization involved in body size,species-specific microsatellite markers development,correlation analysis between single nucleotide polymorphisms(SNPs)and growth traits,and a genetic linkage map construction for S.chuatsi by next generation sequencing.The main results are as followed:To identify the major genes regulating Chinese perch body size at different development stage,we compared transcriptomic profiles between different size at 30 days post-hatch(dph)and 180 dph.Firstly,transcriptome sequencing was performed in S.chuatsi with differential body size at 180 dph using the Illumina Hiseq^TM 2500 system.By RNA-Seq,44,666,047 and 38,771,920 clean reads were obtained in small-size and big-size group,respectively.After assembling high quality reads,a total of 4,317,338contigs,292,265 transcripts,and 115,576 unigenes(mean length:865 bp,N50:1,896 bp)were obtained,respectively.Of 115,576 unigenes,29,465 unigenes were aligned function by BLASTx against databases including COG,KEGG,Swiss-Prot and NCBI.Then,we identified 2,014 differentially expressed genes(DEGs)in S.chuatsi between small-size group and big-size group at 180 dph(FDR<0.01,Fold Change?2),of which 675 genes were up-regulated and 1,339 genes were down-regulated in big-size when compared with genes in small-size group.A total of 1,319 DEGs were assigned with one or more GO terms,509 genes were assigned to COG terms,and 719 genes were assigned to 156known KEGG pathways.In our previous study,2,171 DEGs have been identified between small-size and big-size fish at 30 dph.Finally,we made a comparative analysis of these differentially expressed genes and their enrichment pathways at two different stage groups in Chinese perch.These genes were involved in several signaling pathways,including growth hormone–insulin-like growth factor(GH–IGF)axis,cell proliferation and differentiation,appetite control,glucose metabolism,reproduction and sexual size dimorphism pathways.RT quantitative PCR(q PCR)analysis showed that the 10 selected DEGs in Chinese perch that went through the same training procedure.This study will provide basic information for further functional analysis and selection breeding.Here,a total of 8,923 simple sequence repeats(SSRs)were obtained from 23,923transcriptome sequences longer than 1,000 bp.All the identified SSRs were detected in7,135 unigenes,5,083 of which were annotated in all SSRs.Then,we used these SSR-containing sequences to develop EST microsatellite markers and species-specific microsatellite markers among S.chuatsi,Siniperca kneri,and Siniperca scherzeri.A total of 991 SSR primer pairs were identified in 8,923 SSR-containing sequences.566 loci were randomly selected for primer synthesis and 394 of which were successfully amplified in S.chuatsi.All the non-polymorphic loci(308)were evaluated among three Siniperca species,and 18 species-specific microsatellite markers were finally validated among three Siniperca species.These species-specific microsatellite markers would not only easily discriminate these three Siniperca species at juvenile stage,but also effectively monitor the statuses of hybridization and introgression for germplasm resources protection.Growth hormone(GH)has been considered as a candidate gene for growth traits in fish.In this study,polymorphisms of the GH gene were evaluated for associations with growth traits in 282 S.chuatsi individuals.Using directly sequencing,four SNPs were identified in GH gene,with two mutations in intron 4(g.4940A>C,g.4948A>T),one mutation in exon 5(g.5045T>C),and one in intron 5(g.5234T>G).The effective allele numbers ranged from 1.68 to 1.74,with a mean of 3.8 alleles per locus.The observed heterozygosities(Ho)and expected heterozygosities(He)values ranged from 0.3475 to0.4255,and from 0.4056 to 0.4274,respectively.According to the classification of polymorphism information content(PIC),the mixed pedigrees of S.chuatsi population belongs to the median polymorphism level(PIC=0.33).Notably,three of them(g.4940A>C,g.4948A>T,and g.5045T>C)were significantly associated with growth performance,particularly for g.4940A>C which was highly correlated with all the four growth traits.In conclusion,our results demonstrated that these SNPs in GH gene could influence growth performance of S.chuatsi and could be used for MAS in this species.A high-density genetic linkage map of Chinese perch was constructed with 2,437SNPs using genotyping-by-sequencing(GBS)technique in an F1 mapping panel containing 190 progenies.To identify SNP markers for map estimation,Ape KI reduced representation libraries from the mapping family were constructed and sequenced on the Illumina Hi Seq^TM4000 platform.After filtering,a total of 974.48 M reads were obtained,with a mean of approximately 10.55 and 5.02 million reads for parents and offspring,respectively.A total of 12,466 were informative GBS markers were discovered,of which,2,767 were successfully genotyped in both parents and offspring.After getting rid of the markers showing segregation distortion significantly,there were 24 linkage groups(LGs)in the final linkage maps,comprising 1,001 markers in the male map,1,003 markers in the female map,and 2,437 markers in the sex-averaged map.The male,female,and sex-averaged maps covered a total genetic distance of 1,764.05 c M,1,469.29 c M,and1,895.76 c M,respectively,with an average inter-location space of 1.47 c M(male map),1.89 c M(female map),and 0.72 c M(sex-averaged map).Females had an overall increase in recombination rate relative to the males in S.chuatsi,with the ratio of 1.30:1.This high-density genetic linkage map solidified the basis for identifying QTLs associated with economically important traits and implementing MAS toward genetic improvement in S.chuatsi farming.