Leopard coral grouper(Plectropomus leopardus)is a commercially important marine fish species.It is important to study how to prevent it from infecting with various viruses.In this study,we established and characterize...Leopard coral grouper(Plectropomus leopardus)is a commercially important marine fish species.It is important to study how to prevent it from infecting with various viruses.In this study,we established and characterized a new cell line derived from the fin tissue of leopard coral grouper(PLF).The PLF cells were cultured for more than 55 passages.Cytochrome B gene sequencing confirmed the origin of the PLF cells is P.leopardus.Immunostaining against cytokeratin indicated that the PLF cells predominantly consist of epithelial cells.The chromosome number of PLF was 48.The cells grew well in Dulbecco's modified Eagle's medium(DMEM)supplemented with 10%–20%fetal bovine serum(FBS)at temperature between 20–28℃,with the highest growth rate at28℃.Transfection with pEGFP-N3 plasmid showed the transfection efficiency was about 35%.Virus susceptibility tests revealed that PLF cells are susceptible to red-spotted grouper nervous necrosis virus(NNV)and viral hemorrhagic septicemia virus(VHSV),and viral proliferation was confirmed by qRT-PCR and western blot.The altered expressions of immune-related genes TBK1,IRF3,and Mx after NNV and VHSV infections suggested that PLF cells can mount an immune response to fish viruses.Thus the PLF cells can be employed for studying virus-host interactions and developing antiviral strategies.展开更多
Two morphotypes of Plectropomus leopardus have been identified; morphometric and meristic analyses show that there is no diagnostic difference between them. A difference in color pattern was the most ap- propriate phe...Two morphotypes of Plectropomus leopardus have been identified; morphometric and meristic analyses show that there is no diagnostic difference between them. A difference in color pattern was the most ap- propriate phenotypic character with which to distinguish between the two morphotypes. Complete mito- chondrial DNA sequencing, however, indicated a clear difference between the two morphotypes. Barcoding analysis revealed no significant difference （P〉0.05） in CO1 or ND2 divergence among intramorphotypic individuals, even between geographically separated populations, whereas the intermorphotypic CO1 and ND2 divergences were large enough （averaging 0.95% for CO1 and 1.37% for ND2） to clearly discriminate between the two morphotypes. The color pattern difference, geographical distribution, together with the mtDNA and barcode sequencing data, suggest that the two morphotypes should be of two subspecies or even two species.展开更多
The leopard coral grouper(Plectropomus leopardus)is a species of significant economic importance.Although artificial cultivation of P.leopardus has thrived in recent decades,the advancement of selective breeding has b...The leopard coral grouper(Plectropomus leopardus)is a species of significant economic importance.Although artificial cultivation of P.leopardus has thrived in recent decades,the advancement of selective breeding has been hindered by the lack of comprehensive population genomic data.In this study,we identified over 8.73 million single nucleotide polymorphisms(SNPs)through whole-genome resequencing of 326 individuals spanning six distinct groups.Furthermore,we categorized 226 individuals with high-coverage sequencing depth(≥14×)into eight clusters based on their genetic profiles and phylogenetic relationships.Notably,four of these clusters exhibited pronounced genetic differentiation compared with the other populations.To identify potentially advantageous loci for P.leopardus,we examined genomic regions exhibiting selective sweeps by analyzing the nucleotide diversity(θπ)and fixation index(FST)in these four clusters.Using these high-coverage resequencing data,we successfully constructed the first haplotype reference panel specific to P.leopardus.This achievement holds promise for enabling high-quality,cost-effectiveimputationmethods.Additionally,we combined low-coverage sequencing data with imputation techniques for a genome-wide association study,aiming to identify candidate SNP loci and genes associated with growth traits.A significant concentration of these genes was observed on chromosome 17,which is primarily involved in skeletal muscle and embryonic development and cell proliferation.Notably,our detailed investigation of growth-related SNPs across the eight clusters revealed that cluster 5 harbored the most promising candidate SNPs,showing potential for genetic selective breeding efforts.These findings provide a robust toolkit and valuable insights into the management of germplasm resources and genome-driven breeding initiatives targeting P.leopardus.展开更多
Leopard coral groupers(Plectropomus leopardus),commercially bred in South China,are a significant economical fish species.In this study,by means of quantitative real-time PCR(qRT-PCR)technology,we assessed the stabili...Leopard coral groupers(Plectropomus leopardus),commercially bred in South China,are a significant economical fish species.In this study,by means of quantitative real-time PCR(qRT-PCR)technology,we assessed the stability of six common reference genes expression and selected the appropriate reference genes in leopard coral groupers with or without Vibrio harveyi stimulation at different time points.These data produced by qRT-PCR was handled via the geNorm,NormFinder,and BestKeeper software.The results revealed all the examined reference genes had manifest tissue-specific expression in different tissues.Prior to V.harveyi stimulation,RPL13 gene was the appropriate reference gene among eleven tissue types(blood,spleen,hepatopancreas,kidney,stomach,gill,heart,skin,muscle,intestine,brain)in leopard coral groupers.Under V.harveyi stimulation,the most reliable reference genes varied from tissue to tissue and were closely hinged upon different time points.At 6-h post-bacterial injection,the appropriate reference genes in hepatopancreas,spleen,kidney,and gill were Actin,B2M,UBCE,and Actin,respectively.At 9-and 12-h post-bacterial injection,the appropriate reference genes in hepatopancreas,spleen,kidney,and gill were RPL13,Actin,Actin,and Actin,respectively.If one reference gene was preferable,RPL13,Actin,Actin,and Actin could be selected as the reference gene in hepatopancreas,spleen,kidney,and gill of leopard coral groupers after V.harveyi infection,respectively.Expression profiles of two target genes(IL-6 and NK-lysin)were used to further validate reliability of these selected appropriate candidates.This study will lay a solid foundation for the future research on qRTPCR analysis of gene expression in leopard coral groupers.展开更多
基金the National Natural Science Foundation of China(Nos.32473189,32173001,32273115)the Guangdong Province Special Support Plan Youth Top Talent Project(No.NIQN2024002)+2 种基金the Scientific and Technological Planning Project of Guangzhou City(No.2023B03J1267)the Natural Science Foundation of Guangxi(No.2021GXNSFDA075015)the Natural Science Foundation of Guangdong Province(Nos.2023B1515120074,2024A1515010880)。
文摘Leopard coral grouper(Plectropomus leopardus)is a commercially important marine fish species.It is important to study how to prevent it from infecting with various viruses.In this study,we established and characterized a new cell line derived from the fin tissue of leopard coral grouper(PLF).The PLF cells were cultured for more than 55 passages.Cytochrome B gene sequencing confirmed the origin of the PLF cells is P.leopardus.Immunostaining against cytokeratin indicated that the PLF cells predominantly consist of epithelial cells.The chromosome number of PLF was 48.The cells grew well in Dulbecco's modified Eagle's medium(DMEM)supplemented with 10%–20%fetal bovine serum(FBS)at temperature between 20–28℃,with the highest growth rate at28℃.Transfection with pEGFP-N3 plasmid showed the transfection efficiency was about 35%.Virus susceptibility tests revealed that PLF cells are susceptible to red-spotted grouper nervous necrosis virus(NNV)and viral hemorrhagic septicemia virus(VHSV),and viral proliferation was confirmed by qRT-PCR and western blot.The altered expressions of immune-related genes TBK1,IRF3,and Mx after NNV and VHSV infections suggested that PLF cells can mount an immune response to fish viruses.Thus the PLF cells can be employed for studying virus-host interactions and developing antiviral strategies.
基金Key Program of the Science and Technology Department Foundation of Fujian Province under contract No.2007N0050the Seed Industry Innovation and Industrialization Project of Fujian Province,China(on the subtopic of groupers)
文摘Two morphotypes of Plectropomus leopardus have been identified; morphometric and meristic analyses show that there is no diagnostic difference between them. A difference in color pattern was the most ap- propriate phenotypic character with which to distinguish between the two morphotypes. Complete mito- chondrial DNA sequencing, however, indicated a clear difference between the two morphotypes. Barcoding analysis revealed no significant difference （P〉0.05） in CO1 or ND2 divergence among intramorphotypic individuals, even between geographically separated populations, whereas the intermorphotypic CO1 and ND2 divergences were large enough （averaging 0.95% for CO1 and 1.37% for ND2） to clearly discriminate between the two morphotypes. The color pattern difference, geographical distribution, together with the mtDNA and barcode sequencing data, suggest that the two morphotypes should be of two subspecies or even two species.
基金supported by the National Key Research and Development Program of China (2022YFD2400501)Key R&D Project of Hainan Province (ZDYF2021XDNY133)+2 种基金Project of Sanya Yazhouwan Science and Technology City Management Foundation (SKJC-2020-02-009)PhD Scientific Research and Innovation Foundation of Sanya Yazhou Bay Science and Technology City (HSPHDSRF-2022-02-007)Young Elite Scientists Sponsorship Program by CAST (2023QNRC001)。
文摘The leopard coral grouper(Plectropomus leopardus)is a species of significant economic importance.Although artificial cultivation of P.leopardus has thrived in recent decades,the advancement of selective breeding has been hindered by the lack of comprehensive population genomic data.In this study,we identified over 8.73 million single nucleotide polymorphisms(SNPs)through whole-genome resequencing of 326 individuals spanning six distinct groups.Furthermore,we categorized 226 individuals with high-coverage sequencing depth(≥14×)into eight clusters based on their genetic profiles and phylogenetic relationships.Notably,four of these clusters exhibited pronounced genetic differentiation compared with the other populations.To identify potentially advantageous loci for P.leopardus,we examined genomic regions exhibiting selective sweeps by analyzing the nucleotide diversity(θπ)and fixation index(FST)in these four clusters.Using these high-coverage resequencing data,we successfully constructed the first haplotype reference panel specific to P.leopardus.This achievement holds promise for enabling high-quality,cost-effectiveimputationmethods.Additionally,we combined low-coverage sequencing data with imputation techniques for a genome-wide association study,aiming to identify candidate SNP loci and genes associated with growth traits.A significant concentration of these genes was observed on chromosome 17,which is primarily involved in skeletal muscle and embryonic development and cell proliferation.Notably,our detailed investigation of growth-related SNPs across the eight clusters revealed that cluster 5 harbored the most promising candidate SNPs,showing potential for genetic selective breeding efforts.These findings provide a robust toolkit and valuable insights into the management of germplasm resources and genome-driven breeding initiatives targeting P.leopardus.
基金Supported by the Natural Science Foundation of Hainan Province (Nos. 320QN212, 2019RC078)the Key Research Project of Hainan Province (No. ZDKJ2019011)
文摘Leopard coral groupers(Plectropomus leopardus),commercially bred in South China,are a significant economical fish species.In this study,by means of quantitative real-time PCR(qRT-PCR)technology,we assessed the stability of six common reference genes expression and selected the appropriate reference genes in leopard coral groupers with or without Vibrio harveyi stimulation at different time points.These data produced by qRT-PCR was handled via the geNorm,NormFinder,and BestKeeper software.The results revealed all the examined reference genes had manifest tissue-specific expression in different tissues.Prior to V.harveyi stimulation,RPL13 gene was the appropriate reference gene among eleven tissue types(blood,spleen,hepatopancreas,kidney,stomach,gill,heart,skin,muscle,intestine,brain)in leopard coral groupers.Under V.harveyi stimulation,the most reliable reference genes varied from tissue to tissue and were closely hinged upon different time points.At 6-h post-bacterial injection,the appropriate reference genes in hepatopancreas,spleen,kidney,and gill were Actin,B2M,UBCE,and Actin,respectively.At 9-and 12-h post-bacterial injection,the appropriate reference genes in hepatopancreas,spleen,kidney,and gill were RPL13,Actin,Actin,and Actin,respectively.If one reference gene was preferable,RPL13,Actin,Actin,and Actin could be selected as the reference gene in hepatopancreas,spleen,kidney,and gill of leopard coral groupers after V.harveyi infection,respectively.Expression profiles of two target genes(IL-6 and NK-lysin)were used to further validate reliability of these selected appropriate candidates.This study will lay a solid foundation for the future research on qRTPCR analysis of gene expression in leopard coral groupers.
