| The Chinese shrimp(Fenneropenaeus chinensis),belonging to Arthropoda,Crustacea,Decapoda,natantia,denderobranchiata,Penaeidae and Fenneropenaeus.At present,the breeding method of Fenneropenaeus chinensis mainly relies on traditional artificial breeding,which is based on the response of Fenneropenaeus chinensis to the environment and the phenotypic value of tolerance.Traditional breeding requires a large number of parents,but there are many uncertain factors,which are time-consuming and laborious.It can be combined with molecular marker assisted selection(MAS),and has been widely used in the breeding of new high-quality varieties.SNP,as the third generation of molecular marker technology,has the advantages of large number of loci,rich polymorphism,stable inheritance,and simple and fast detection method.At present,it has become one of the important research directions of molecular breeding to screen SNP loci and analyze SNP marker association with related traits by combining the results of transcriptome sequencing.In this study,on the basis of De novo sequencing of Fenneropenaeus chinensis,967 SNP loci were obtained by preliminary screening,and then sensitive and resistant groups were constructed for BSA analysis.After 237 potential SNP loci were obtained by direct sequencing,121 SNP loci were successfully typed by fluorescence quantitative PCR.Chi square test showed that 17 SNP markers were significantly related to the high pH tolerance(P<0.05).Through function prediction,10 SNP loci were screened The markers were matched to the functional genes with high sequence similarity.Among them,the gene of C3073-1338TC is N-acetyl-β-D-glucosaminidase(NAGase),which successfully amplified the full length of the open reading frame(ORF)DNA sequence of FcNAGase.The specific research contents are as follows:1. Transcriptome analysis of Fenneropenaeus chinensis and high-throughput development of SNP markersThe hepatopancreas of Fenneropenaeus chinensis was sampled at high pH(pH=9.2)stress for 3 hours.The transcriptional sequencing and analysis were carried out by using Illumina Hiseq 4000 sequencing technology.A total of 89.69GB data were measured,and 62,772 Unigenes were obtained after assembly and de redundancy.The total length,average length,N50 and GC content were 73,694,288bp,1,173bp,2,392bp and 44.17%,respectively.After data filtering,clean reads were assembled by Trinity software.In the transcripts assembled,there were 55,982 and 45,027 transcripts in the control group and the experimental group,respectively;42,668 and 35,263 transcripts in the Unigene assembly control group and the experimental group,respectively.The average length of the Unigene sequences in the control group and the experimental group was 992bp and 895bp.Unigene was compared to seven functional databases for annotation.Finally,33,015(NR:52.60%),28,011(Swssprot:44.62%),15,175(COG:24.17%),26,128(KEGG:41.62%),19,557(Interpro:31.16%),4,113(GO:6.55%),29426(NT:46.88%)Unigene obtained functional annotation,and 10 annotation organisms were obtained in the first five databases,421(16.6%)Unigene.Among the21,787 unigenes,10,630(48.8%)were classified into biological processes,6,122(28.1%)into cell components and 5,035(23.1%)into molecular functions.These results make up for the relevant information of the high pH stress genome of Fenneropenaeus chinensis,and provide a large number of data support for the study of the mechanism of high pH stress.Among the SNP sites predicted by transcriptome,49,215 and 65,112were detected in the experimental group and the control group respectively;for the same site,the results of the experimental group were the same three times,and the results of the control group were the same three times,but the two results were inconsistent,that is to say,to change the site to a high mutation rate,967 SNP sites related to high pH tolerance were initially obtained,laying the foundation for the next work.2. SNP loci development and polymorphism analysis of Fenneropenaeus chinensisAfter high pH(pH=9.3±0.05)stress,48 early dead and 48 late dead prawns were selected to form sensitive group and resistant group for BSA analysis.The primers were designed with 967 selected loci,and 68 were successfully amplified by direct sequencing,A total of 237 potential SNP sites were screened from 625 bp DNA fragments,and the SNP distribution frequency was 0.35 SNPs/100bp,62.44%(A/G,30.80%,C/T,31.64%),37.56%(A/C,A/T,C/G and G/T,10.12%,11.39%,3.38%and 12.67%,respectively);then according to the potential 237 SNP loci,the specific primers were designed,and the SNP loci were typed by fluorescence quantitative method.121 SNP loci were successfully typed Through the polymorphism analysis,we found that the observed heterozygosity(H_O)range of SNP was 0.2737～0.9149,the expected heterozygosity(He)range was 0.1119～0.5033,the pic value range was0.1050～0.3750,the low polymorphism(PIC<0.25)was 26,the moderate polymorphism(0.025<PIC<0.50)was 95,and there was no high polymorphism.Among them,31 SNP loci did not conform to hardy Weinberg equilibrium.The range of MAF was from 0.0591 to 0.5000,which indicated that the locus had high polymorphism and was suitable for association analysis.3. Correlation analysis between SNP locus and high pH tolerance121 SNP loci obtained were analyzed for association with high pH tolerance,and the SNP loci with significant correlation were used for functional prediction and gene mapping.A total of 17 SNP loci were found to be significantly correlated with high pH tolerance(P<0.05),such as C1111-1063GA,C3073-1338TC,C883-940GA,C944-390GA,C3784-997CT,C1042-1516GT,C1089-277AC,C1339-173GT,C2705-995AG,C2794-354CA,C2862-4787CT,C3239-1000TA,C3381-262GA,C3776-1023AG,C4032-495GA,C4848-698AC,C5216-510CT,among C1111-1063GA,C1042-1516GT,C3239-1000TA were significantly correlated with high pH tolerance(P<0.01).Through functional prediction,10 markers were selected to match the functional genes with high sequence similarity,namely,beta-N-acetylglucosaminidase,CTP synthase,major promoter super family domain containing protein 1-like protein1-like,5-phosphohydroxy-l-lysine phospho lyase like,protein rrp5 like homolog,small integrated membrane protein 8-like,von Willebrand factor a domain containing protein7-like),hepatic nuclear factor 4-gamma-like,cytochrome C-type heme lyse like,39s ribosomal protein L51.4. Amplification of fcnagase,development of SNP locus and its correlation with high pH toleranceIn this study,C3073-1338TC,a SNP locus related to high pH tolerance,was selected as the research object.beta-N-acetylglucosaminidase gene,the gene length of fcnagase was 2544 bp,which was composed of 5 exons and 4 introns.The boundary between exons and introns was in line with GT-AG principle,and 14 SNP loci were successfully developed.There are two missense mutations in the exon region,which are P2 mutation from ser(serine)to Ala(alanine),P9 mutation from Phe(phenylalanine)to Leu(leucine),and other exons are synonymous mutations.The results showed that the heterozygosity(H_O)was 0.1981～0.939,the expected heterozygosity(He)was0.0595～0.499,the polymorphism information content(PIC)was 0.0574～0.3731,the polymorphisms of p9,P14 and P18 were low,and the other 11 SNP loci were moderate.Chi-square test showed that P12,P27 were related to the high pH tolerance of Fenneropenaeus chinensis(P<0.05).The results of haplotypes showed that 14 SNPs consisted of 3 linkage disequilibrium haplotypes and 7 haplotypes,among which TCC haplotypes were significantly correlated with the high pH tolerance of Penaeus chinensis(P<0.05). |
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