| The mandarin fish(Siniperca chuatsi)is an important freshwater economic fish in China.However,with years of artificial breeding,the natural germplasm resources and breeding quality have declined,the growth rate has decreased and the disease resistance have been weakened,which seriously affected the breeding effect and the quality safety of the products.Therefore,it is necessary to develop germplasm resources protection and breeding of fine varieties in order to support the healthy development of S.chuatsi aquaculture.In this study,based on the growth trait related QTLs obtained in a linkage map of S.chuatsi constructed previously by our group,the molecular markers related to the growth of S.chuatsi were excavated.SSR markers were selected from the growth differential transcriptome sequencing and used to analyze the genetic diversity of the selection population,as well as for the paternity identification of F2 population.The main contents are as follows:1.Minning of the growth related QTLs in S.chuatsi—Correlation analysis of mandarin fish GH gene polymorphisms with growth traitsScreening of trait-associated molecular markers can be used to enhance the efficiency of selective breeding.Previously,we produced the first high-density genetic linkage map for the mandarin fish and identified 11 quantitative trait loci(QTLs)significantly associated with growth,one of which was located within the growth hormone(GH)gene.To investigate the correlation between mandarin fish GH gene polymorphism and growth,we cloned the complete GH gene sequence and examined its polymorphism.We identified 32 single-nucleotide polymorphisms(SNPs)and one simple sequence repeat(SSR)in a small population.Eight SNPs(G1–G8)and the SSR(GH-AG)were selected for genotyping and correlation analysis with growth traits in a large population.Four loci,G1–G3 and GH-AG,were found to be significantly correlated with growth traits(P < 0.05).Of them,G1,G3,and GH-AG showed highly significant correlations with multiple growth traits(P < 0.01).G1–G3 formed four effective diplotypes(D1–D4)which mainly manifests as D1>D3>D2>D4 in important growth traits such as total length,weight,etc.,and D1 was highly significantly greater than D4(P < 0.01).Our results show that the four polymorphic loci G1–G3 and GH-AG within the mandarin fish GH gene are significantly correlated with growth traits and can be used as candidate molecular markers for selective breeding of superior varieties of mandarin fish.It also would provide more reliable guidelines for breeding selection of other fish species.2.Sequencing of growth transcriptome and development of new SSR markers for Siniperca chuatsiThe transcriptome of musculature in three fast growing and three slow growing individuals of Siniperca chuatsi was sequenced by Illumina Hiseq 2500.The transcriptome data was assembled,and SSRs were searched in the obtained unigenes.41 533 736 high-quality reads were obtained,and 138 728 non-redundant unigenes were assembled with an average length of 808.94 bp.Among them,57 009 unigenes(41.09%)were at least annotated in one of Nr,Swissprot,KEGG or COG database,and 7125 unigenes(5.14%)were annotated in all four databases.138 728 unigenes was retrieved by using Misa,mreps and trf software to search for their common SSRs.There were 4692 SSR loci distributed in 4662 unigenes,which were retrieved,and the frequency of occurrence was 3.36%.Among all SSR loci,the mononucleotide repeat,dinucleotide and trinucleotide were the main types,which respectively accounted for 62.66%,24.87% and 10.83%.AC(CA)/GT(TG)was the dominant repeat motifs in the dinucleotide repeat motifs.AGG(GGA,GAG)/CCT(CTC,TCC)was the dominant repeat motifs in trinucleotide repeat motifs.Primer 5 was used to design primers for the 4692 SSR sequences,and 80 pairs of them were randomly selected for test of PCR validity and SSR polymorphism.The results showed that 41 pairs of them could get clear and stable target bands with an effective amplification rate at 51.25%,and 23 of them can obtain stable and repeatable polymorphic products by PCR.Finally,the genetic parameters of 24 samples of S.chuatsi were calculated and analyzed by the 23 SSR loci.The expected heterozygosity(He)and polymorphic information content(PIC)were 0.198~0.888 and 0.175~0.857,respectively.The results of this study indicated that,the unigene information generated by transcriptome sequencing of S.chuatsi could be used to mine functional genes in batches,and also be an effective source for the development of new SSR markers.These SSR loci developed by transcriptme could be used in genetic diversity analysis,germplasm conservation and genetic breeding of S.chuatsi.3.Genetic diversity of the selection population of F1 and F2 of S.chuatsi,and the paternity identification of F2In this study,9 SSR markers with high polymorphism obtained above were selected and used to analyze the genetic diversity of F1 and F2 generation,and also used to identify paternity relationship of the 630 individuals in the eight mixed families of F2 generation.The result showed that these SSR loci were all in high polymorphism(PIC>0.5),except for one being moderately polymorphic locus.A total of 64 alleles were detected in all the 820 individuals in F1 and F2 generation,with an average 0.6604 of PIC.The genetic diversity of parents and offsprings were analyzed.It was found that the genetic diversity of the two generation F1 and F2 was high(He >0.5).Among the 8 mixed families in the F2,the highest genetic diversity were found in family 3 and family 8(He at 0.614 and 0.618,respectively).The above 9 SSR markers were used for paternity identification of the 8 mixed families in F2 generation.The result showed that the success rate of paternity identification were 89%?61%?90%?91%?86%?73%?73% and 83% corresponding to family 1,3,4,5,6,7,8 and 10,with confidence level of 80%.The genetic analysis of F1 and F2 and paternity identification of the F2 generations would provide reliable genetic data and pedigree relationship for further selected breeding of S.chuatsi. |
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