| Grass carp(Ctenopharyngodon idella)has the highest production in China’s freshwater aquaculture industry,however,no good varieties of grass carp have been bred in the breeding industry.In recently years,the individuals with close relationship,small size and early sexual maturity were often selected for the breeding in many grass carp breeding farms,which caused a germplasm degradation of grass carp breeding and restricted the healthy development of the breeding industry.The sexual maturity age of grass carp is generally 4-5 years old which leads to a long breeding cycle,and it is necessary to explore the molecular marker assisted breeding technology to promote the progress of grass carp breeding.Gene aggregation breeding technology is widely used in the breed improvement of crop and livestock,but rarely reported in fish.Currently,the researches about the molecular mechanism of grass carp growth regulation and genetic breeding are still weak,it is necessary to explore the genes or molecular markers linked with growth characters to provide a basis for multi gene polymerization breeding in grass carp.In this study,RNA-Seq technology was used to screen genes related to growth traits of grass carp,then SNP markers related to growth traits were identified from candidate genes.Further,the dominant genotype of growth-related markers were aggregated to analyze the correlation between the number of aggregated dominant genotypes and growth traits,to provide basic infromation for grass carp gene aggregation breeding.The studies are as follows:1.Screening of candidate genes related to growth traits in grass carpRNA-Seq technology was used to analyze the transcriptome of liver,muscle and brain tissue in the fast and slow growing groups of grass carp.A total of 319.53 million raw reads were obtained,and 314.65 million high-quality clean reads were obtained after filtration.After assembly,34147 unigenes were obtained,with anaverage length of 1060 bp.Through the BLASTX method,the unigenes were annotated to the NR,KEGG and Swiss-prot databases respectively,and 30751 unigenes obtained the functional annotation.Edge R software was used to analyze the differences in gene expression among these samples,1013,552 and 372 differentially expressed genes were screened from liver,muscle and brain tissues,respectively,which mainly involved in GH-IGF growth axis,PI3K/Akt signaling pathway,MAPK signaling pathway,m TOR signaling pathway,TGF-β signaling pathway,glycolysis pathway and JAK-STAT pathway.These genes could be used as candidate genes for the genes related to the growth traits in grass carp.2.Screening of SNP markers in candidate genes related to growth traits in grass carpA total of 4580 SNP markers were screened from the candidate growth-related genes in the grass carp transcriptomes,and after detected by the SNa Pshot method,30 of 34 were the correct polymorphic markers.Besides,the c DNA full length of GHRH was amplified by RACE technology,including 120 bp for 5′ UTR,41 bp for 3′ UTR and 441 bp for ORF,encoding 142 amino acids.The genome sequence length of GHRH is 5244 bp,and 12 SNP markers were screened from the genome sequence by direct sequencing.3.Correlation analysis between SNP markers in candidate genes and growth traitsThe genotypes of each SNP marker were detected by using the method of snapshot in 300 individuals of grass carp.The results of correlation analysis showed that unigene00810126-8014 and unigene00810126-2903 in Somatolactin alpha,unigene00870394-525 in early growth response protein-1,unigene02938762-11628 in myosin heavy chain,C+1798T,T+2340C,A+2782T and T+3925C in GHRH were significantly correlated with growth traits of grass carp(P<0.05),which could be used as molecular markers in assisted breeding of grass carp.4.The effects of pyramiding growth-related genotypes in grass carpThree growth-related SNP markers obtained in this study and seven growth-related SNP markers obtained in the previously researches were used to detect the genotypes of each marker in 48 parent populations.The parents with the most numbers of aggregatable dominant genotypes were selected to construct a family,and the effects of aggregating the dominant genotypes of the family offspring was analyzed until 8-month-old.The results showed that the numbers of related dominant genotypes was 0-7 in the offspring,correspond to the number of corresponding individuals was 44,67,83,85,44,38,15 and 6,and the corresponding average bodyweight was 129.66,144.45,151.33,153.53,154.77,160.50,167.50 and 176.67 g.These results revealed that the numbers of aggregation of growth-related dominant genotypes were positively correlated with the growth traits in grass carp.The average number of dominant genotypes in the offspring population is 2.58,which is significantly higher than the average number of dominant genotypes of the parent population(1.00).In conclusion,aggregating the dominant genes related to growth traits in grass carp could obtain the individuals with good growth characters in the offspring,which provides a theoretical basis for the application of multi gene aggregation breeding in grass carp. |
