Construction Of A High-density Genetic Linkage Map And QTL Mapping Of Growth Traits And Muscle Nutritive Components In Grass Carp(Ctenopharyngodon Idella)

Grass carp(Ctenopharyngodon idella),one of the most important aquaculture fish,has been cultured for thousands of years and its output has been ranking at the first place in freshwater aquaculture for several years in China.As the cost of next-generation sequencing decreases,QTL(quantitative trait locus)mapping by using high-density genetic linkage map is a fast and effective way to develop traits-related markers,while this work has not been reported in grass carp.In this study,a high-density genetic linkage map was constructed based on the previous consensus linkage map and draft genome of grass carp.Subsequently,several growth and muscle nutritive components of 10 month-old grass carp were located by the above linkage map.In addition,the effectiveness and practicability of several insert/deletion mutations were verified,and their correlations with growth traits of juvenile grass carp were studied.Last but not least,the bulked segregant analysis by RNA-seq(BSR-seq)was used for primary localization of body weight trait.This study aimed at detecting growth traits and muscle nutritive components related markers and genes,in order to provide reference for grass carp MAS(marker assisted selection)breeding.Main research contents were as follows: 1.Construction of a high-density genetic linkage map of grass carp2 parents and 185 progencies,whose parentage assignment was identified by twelve SSR(simple sequence repeats)markers,were used as mapping family to construct a high-density genetic linkage map of grass carp.Firstly,samples were sequenced and 2,629,210,652 raw reads were obtained.After data filtering,2,608,276,786 clean reads were retained,and the average clean reads,Q30 and GC content of all samples were 13,948,004,95.68% and 46.51%,respectively.By sequence alignment with the published female reference genome of grass carp,Samtools and GATK were used for SNP genotyping,and 21,733 polymorphic SNP markers were screened initially in the intersection results.After further strict filtration,3979 SNPs were finally reserved and used to construct the high-density genetic linkage map of grass carp.Number of linkage groups in this high-density genetic linkage map was 24,same with the previous consensus linkage map.Both total length(1,752.742 cM)and number of markers(3979)of this genetic linkage map were much improved,with an average resolution(0.44 cM per locus)nearly ten times of previous study(4.2 cM per locus).In present genetic linkage map,length of each linkage group ranged from 46.773(LG18)to 127.050 cM(LG2)with an average of 73.031 cM,and the number of markers varied from 46(LG9)to 232(LG2)with an average of 165.79,while the average resolution of all linkage groups changed from 0.263 to 2.106 cM per locus.Further more,the largest gaps in each linkage group ranged from 2.161(LG23)to 35.133 cM(LG2),and the ratios of gap<5 cM were 93.48 to 100%.In summary,the resolution of this genetic linkage map was much improved,and this map may lay the foundation for QTL fine mapping of economically traits in grass carp.2.QTL mapping for growth traits of 10 month-old grass carpGrowth traits are important economic traits of aquaculture fish,and QTL mapping of these traits may provide an important basis for MAS breeding.The high-density genetic linkage map was used for QTL mapping of growth-related traits in 10 month-old grass carp.Totally 35 linkage group-wide significant QTLs were detected relevant with growth-related traits: 13,8,10,2 and 2 QTLs were related with body wight(BWE),body length(BL),body height(BH),body width(BWE)and fatness(FN),respectively.BWE related QTLs were detected onto LG7(1),LG11(1),LG19(9),LG20(1)and LG23(1),respectively;and these QTLs explained 8.3%-30.8% of phenotypic variance explained(PVE).BL related QTLs were detected onto LG11(2),LG13(1),LG19(4)and LG23(1),respectively;and PVE of these QTLs ranged from 8.6% to 16.9%.BH related QTLs were detected onto LG19(9)and LG20(1),respectively;and PVE of these QTLs varied from 8.0% to 32.8%.BWI related QTLs were detected onto LG7(1)and LG16(1)with the PVE of 13.0% and 10.1%,respectively.FN related QTLs were detected onto LG18 and the PVE ranged from 11.1 to 13.1%.6 BL related QTLs were overlapped with BWE related QTLs,they were qBL11-1,qBL19-4,qBL19-5,qBL19-6,qBL19-7 and qBL23-8,respectively.While 9 BH related QTLs(expect qBH19-7)were overlapped with BWE related QTLs;at the meantime,4 were overlapped with BL related QTLs,they were qBH19-2,qBH19-3,qBH19-6 and qBH19-9,respectively.Between BWI and BWE traits,only the qBWI7-1 was overlapped with qBWE7-1.It is worth mentioning that the PVE of qBWE19-3,qBWE20-12 and qBH20-10 were 20.5%,30.8% and 32.8%,respectively.In summary,these 35 growth related QTLs,especially the qBWE19-3,qBWE20-12 and qBH20-10,lay the foundation for futher study and MAS breeding of growth traits in grass carp.3.QTL mapping for muscle nutritive components of 10 month-old grass carpMuscle nutritive components related QTLs were screened based on the high-density genetic linkage map,in order to provide a basis for genetic improvement of flesh traits in grass carp.13 muscle nutritive components were measured and used for QTL mapping,6 of these components had no significant correlated QTLs.While 47 linkage group-wide significant QTLs,including 71 SNPs,were detected relevant with the other 7 components.14 crude fat(CF)-related QTLs were detected onto LG4(1),LG7(2),LG14(6),LG16(2)and LG21(3),whose PVE ranged from 8.3% to 21.5%;qCF4-1 and qCF14-5 had high PVEs of 21.5% and 21.1%,respectively.One crude protein(CP)-related QTL onto LG5,with the PVE of 9.7%;the other 6 CP-related QTLs were all onto LG22,with PVE of 8.8-21.8%;the qCP22-3 had the highest PVE of 14.9-21.8%.15 Arg-related QTLs were detected onto LG1(1),LG4(2),LG12(11)and LG13(1),whose PVE varied from 8.6% to 45.9%;qArg4-3 and qArg13-15 had high PVEs of 45.2-45.9% and 24.0%,respectively.His-related QTLs were detected onto LG8 and LG11,with the PVE of 30.0%(qHis8-1)and 11.1-11.3%,respectively.2 Lys-related QTLs were detected onto LG12 with PVE of 9.9-12.4%,the other QTL were detected onto LG13(qLys13-3)with the highest PVE of 28.7%.2 Pro-related QTLs were detected onto LG13 and LG19,whose PVE were 15.9% and 9.1%,respectively.4 Val-related QTLs were detected onto LG4(PVE: 8.5%),LG12(PVE: 19.3%),LG14(PVE: 10.5%)and LG24(PVE: 8.7%),respectively.In addition,qLys12-2 overlapped with qArg12-11,qVal4-1 overlapped with qArg4-2,qVal12-2 overlapped with qArg12-12,respectively.In summary,these 47 muscle nutritive components-related QTLs,especially the qCF4-1,qCF14-5,qCP22-3,qArg4-3,qArg13-15,qHis8-1 and qLys13-3,could provide basis for futher study and MAS breeding of flesh traits in grass carp.4.Characterization and linkage mapping of growth-related insertion/deletion markers in 10 month-old grass carpIn previous work,a body weight-related QTL of grass carp was found in LG15 by our research group(unpublished).In this study,we attempt to apply the STR genotyping method in characterization of insert/deletion mutations on three scaffolds in the LG15,according to the previous consensus linkage map and draft genome of grass carp.Moreover,the relevance between polymorphic mutations and growth traits of breeding populations was conducted.At last,polymorphic mutations were mapped on the above linkage map.Preliminary results found that 41 of the 85 selected insert/deletion mutations were SSRs,and 8 loci were failed to design primers.Therefore,36 pairs of primer were successfully designed,and PCR amplifications showed they were effective.Afterwards,PCR products were sequenced to validate the STR genotyping results.Subsequent sequencing results showed STR genotyping method could not only identify these insertion/deletion mutantions accurately,but also reduce the genotyping costs.The mean effective number of alleles(Ne),observed heterozygosity(Ho),expected heterozygosity(He)and polymorphic information content(PIC)of 36 mutations in wild individuals were 1.4976,0.3190,0.2970 and 0.2349,respectively.However,only 7 of 36 mutations were found polymorphic in breeding populations.Then,the general linear model was used to analyze the relevance between those 7 insert/deletion mutations and growth traits of 323 juvenile grass carp.Correlation results found that 2 loci(ID-10 H and ID-41F)were not related with growth traits(P>0.05);while the other 5 loci were significantly related with one or more growth traits of juvenile grass carp(P<0.05).Among the 5 loci,Locus ID-6H was significantly correlated with fatness traits of juvenile grass carp(P<0.05);while locus ID-11 F,ID-15 F,ID-32 F and ID-39 F were significantly correlated with body weight or length or width or height(P<0.05).Finally,these 7 polymorphic mutations were mapped on LG15 of the high-density genetic linkage map.To summarise,the inexpensive STR genotyping method could be used in development of insert/deletion mutations,and these growth-related mutations might be used in molecular marker assisted breeding of grass carp.5.Excavation of markers and genes linked to body weight traits of 10 month-old grass carp by BSR-seq technologyBSR-seq technology was used to excavate body weight-related molecular markers and genes in 10 month-old grass carp.In a fullsib F1 family,individuals with extreme body weight traits were average divided into six groups.Firstly,the quality and quantity of total RNA in six groups were assessed.The OD280/260,RIN,28S:18S and total quantity were all achieved the inspection standard,which ranging from 1.85 to 1.94,6.8 to 7.4(>6.5),1.1 to1.9(>1)and 5.24 to 7.75 ng(>2 ng),respectively.Six cDNA libraries were sequenced by Illumina HiSeq X sequencing machine,and totally 45.18 G data was obtained.Raw data,GC content and Q30 of six groups were varied from 6.9560 to 8.0913 G,47.02 to 48.73%,and 90.30 to 91.34%,respectively.After strict filtration and sequence alignment with the published female genome,the mapped rate of clean data ranged from 86.60 to 89.23%.Compared with the three smaller-BW groups,334 genes up-regulated and 191 genes down-regulated in the three bigger-BW groups.Then GO and KEGG enrichment analysis were performed for these 525 DEGs.GO enrichment analysis found most of these DEGs were significantly involved in 31 GO terms(p-classicFisher<0.05),including skeletal muscle tissue,skeletal muscle organ,striated muscle tissue,muscle tissue,muscle organ,muscle structure development,and so on.While KEGG enrichment analysis showed most of these DEGs were significantly involved in 12 KEGG pathways(qvalue<0.05),including regulation of actin cytoskeleton,mTOR signaling pathway,and so on.In addition,strict filtrated SNPs were used for BSR-seq analysis,and 440-540 SNPs involved in 171 candidate intervals were detected.Sequences in candidate intervals were extracted and analyzed.164 candidate genes,including 8 DEGs,were screened out,and 6 of these differential expression genes had functional annotation.These annotated DEGs were Proline-rich AKT1 substrate 1(AKT1S1),Supervillin(SVIL),Cardiomyopathy-associated protein 5(CMYA5),Eukaryotic translation initiation factor 4 gamma 1(EIF4G1),PDZ and LIM domain protein 5(Pdlim5)and myosin heavy chain,respectively.Make a summary,according to the BSR-seq technology,440-540 SNPs involved in 171 candidate intervals and 8 candidate DEGs were linked to body weight trait of 10 month-old grass carp.