| Spinibarbus hollandi(Cypriniformes: Cyprinidae)is widely distributed in southern Asia.It is an important commercial aquaculture species in the south of China,due to its dense succulent,delicate taste and rich nutrition.However,the breeding scale is limited by the low growth rate of S.hollandi.The most previous researches on S.hollandi focused on genetic resources,rearing conditions and morphology,whereas little is reported about the effect of feeding strategy on growth rate and screening of molecular markers associated with growth traits.This research will focus on the response to starvation and compensatory growth in S.hollandi,clone of genes related with growth and screen of SNPs related with growth traits.These experiments will provide the basic data for the improvement of growth traits in S.hollandi.The study mainly included four parts.The main results of these experiments are as follows:1.Feed deprivation is associated with reduced growth rates owing to its effect on cell division,proliferation and metabolism.And some fish exhibit compensatory growth during refeeding through increasing feeding intensity compared with continuously fed fish.This study was designed to investigate the effects of fasting and refeeding based on physiological responses,transcriptome,and changes in body weight in juvenile S.holland.The body weights of S.hollandi under fasting slightly decreased,and were insignificantly lower than continuously fed fish;after refeeding33 days,there was no significant difference between control group and test group.After fasting for 7 days,the diameters of cross-sectional muscle fibers were significantly smaller than continuously fed fish.There was no significant difference between controls and fasting-refeeding group.A total of 4499 differentially expressed genes were found among the three groups.Many genes associated with fat metabolism,protein metabolism,carbohydrate metabolism were significantly up-regulated,and genes related with cell cycle,DNA replication,and phagosome were down-regulated.In addition,some genes were significantly up-regulated in fasting-refeeding group than control group,these genes may be associated with compensatory growth.2.Brain plays an important role in energy regulation during fasting.This study investigated the response to fasting and refeeding in S.holland brain.In the RNA-seq analysis,936 differentially expressed genes were found among the three groups,these genes were divided into four parts based on the Hcluster.Genes in cluster 1 exhibited significantly up-regulated during fasting,and the majority of these genes were related with energy metabolism.Genes in cluster 2 exhibited significant difference between control group and fasting-refeeding group,many heat stock protein genes were involved in this cluster;the cluster 3 included genes highly expressed during fasting,these genes were associated with cell cycle,DNA replication,and steroid biosynthesis.Genes included in cluster 4 exhibited that the expression were significantly lower both in fasting group and refeeding group than control groups.In addition,results of RNA-seq were validated using Q-RTPCR.3.The melanocortin-4 receptor(MC4R)gene is a member of the central melanocortin pathway,which influences development,growth,metabolism,and appetite.In this study,MC4 R gene was cloned and characterized in S.hollandi.MC4 R sequence is composed of 3068 nucleotides with a 978 nucleotides open reading frame encoding a protein of 326 amino acids.Quantitative real-time PCR analysis revealed that MC4 R m RNA was ubiquitously expressed in tissues,with the most expression abundance in brain and gonad,and the expression levels of the MC4 R gene were significantly higher in the lightest group than those in the heaviest group of S.hollandi.A primary assessment of sequence variability revealed six single nucleotide polymorphisms(SNPs),among which,four were in the promoter(-698 A >G,-679 C > T,-326 C > T,and-202 A > G)and two were in the exon(36A > G and234 C >T).Three SNPs,-698 A > G,-679 C>T,and 234 C >T,were significantly associated with growth traits,two of which,-698 A > G and-679 C > T,were in complete linkage.The other three SNPs showed no significant associations with any of the growth traits.Our data indicated a significant association between genetic variations in the S.hollandi MC4 R gene and growth traits.Accordingly,the SNPs identified in the MC4 R gene could be used as potential genetic markers in future breeding programs for S.hollandi and other fish.4.Myostatin(MSTN)is a negative regulator of skeletal muscle development and growth in fish.In this study,the S.hollandi MSTN gene was cloned and characterized.MSTN is composed of three exons and two introns,with an open reading frame containing 1128 nucleotides encoding a protein of 375 amino acids.The deduced amino acid sequence of MSTN in S.hollandi showed 66.6～98.4% homology with MSTN of avian,mammalian,and teleostean species.MSTN was expressed in various tissues,with the highest expression observed in brain.Our result showed that the expression levels of MSTN in the skeletal muscle were higher in S.hollandi with considerably lower growth performance than those with extremely high growth,but this pattern was not observed in the brain.Five SNPs(including one SNP(1196T > A)in exon 2,and four SNPs(1428C > G,1430 T > G,1483 A > G,and 1562 A > C)in intron 2)in MSTN were detected and verified.Four SNPs,1196 T > A,1428 C > G,1430 T > G,and 1483 A > G,were significantly associated with growth traits.Our data indicated a significant association between genetic variations in MSTN and growth traits.The SNPs identified in MSTN could be used as genetic markers in future breeding programs for S.hollandi. |
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