The CRISPR/Cas9 genome-editing system serves as a pivotal tool for enhancing crop genetics.Within this system,single guide RNAs(sgRNAs)are instrumental in the precise cleavage of DNA double strands.However,the efficie...The CRISPR/Cas9 genome-editing system serves as a pivotal tool for enhancing crop genetics.Within this system,single guide RNAs(sgRNAs)are instrumental in the precise cleavage of DNA double strands.However,the efficiency of gene editing varies among sgRNAs,emphasizing the need to meticulously select target sites,especially in the context of Citrullus lanatus,a species notorious for its challenging and inefficient generation of transgenic plants through stable transformation.This study employed an Agrobacterium rhizogenes-mediated hairy root method to assess effective target sites for gene editing of ClCIPK17 across various Citrullus species.Hairy roots were successfully induced in different plant tissues at diverse growth stages of Citrullus lanatus,Citrullus mucosospermus,and Citrullus amarus.Employing a vector with two sgRNAs(sgRNA1 and sgRNA5)positioned within conserved regions of exon 1 and exon 5 of ClCIPK17 in the CRISPR/Cas9 system,targeted mutations were detected in 90.9%of accessions across the four Citrullus species.Notably,73.94%of all examined hairy roots exhibited mutations at the sgRNA1 site,while the sgRNA5 site showed no mutations.Among the 31 different mutation types identified at the sgRNA1 site,base deletion was the most prevalent.Using the sgRNA1 site of ClCIPK17,stable transgenic watermelon buds were obtained from explants,and the targeted mutations of the sgRNA1 site were confirmed.These findings underscore the viability of the hairy root transformation system in assessing the editing efficiency of sgRNA targets in diverse Citrullus species.展开更多
Watermelon is a highly cultivated fruit crop renowned for its quality properties of fruit flesh.Among various quality factors,fruit flesh firmness is a crucial quality parameter influencing the fruit texture,shelf lif...Watermelon is a highly cultivated fruit crop renowned for its quality properties of fruit flesh.Among various quality factors,fruit flesh firmness is a crucial quality parameter influencing the fruit texture,shelf life and its commercial value.The auxin/indole-3-acetic acid(Aux/IAA)plays a significant role in fruit development and ripening of non-climacteric fruits.However,the regulatory mechanism of Aux/IAA in controlling fruit flesh firmness and ripening in watermelon remains unknown.In this study,we employed an integrative approach combining genome-wide association study(GWAS)and bulked segregant RNA-Seq analysis(BSR-Seq)to identify an overlapping candidate region between 12776310 and 12968331 bp on chromosome 6,underlying an auxin-responsive gene(Aux/IAA)associated with flesh firmness in watermelon.Transcriptome analysis,followed by real-time quantitative reverse transcription PCR(qRT-PCR),confirmed that the expression of Aux/IAA was consistently higher in fruits with high flesh firmness.The sequence alignment revealed a single base mutation in the coding region of Aux/IAA.Furthermore,the concomitant Kompetitive/Competitive allele-specific PCR(KASP)genotyping data sets for F2 population and germplasm accessions identified Aux/IAA as a strong candidate gene associated with flesh firmness.Aux/IAA was enriched in the plant hormone signal transduction pathway,involving cell enlargement and leading to low flesh firmness.We determined the higher accumulation of abscisic acid(ABA)in fruits with low flesh firmness than hard flesh.Moreover,overexpression of Aux/IAA induced higher flesh firmness with an increased number of fruit flesh cells while reducing ABA content and flesh cell sizes.Additionally,the allelic variation in Aux/IAA for soft flesh firmness was found to exist in Citrullus mucosospermus and gradually fixed into Citrullus lanatus during domestication,indicating that soft flesh firmness was a domesticated trait.These findings significantly enhanced our understanding of watermelon fruit flesh firmness and consequently the watermelon fruit quality.展开更多
The organoleptic qualities of watermelon fruit are defined by the sugar and organic acid contents,which undergo considerable variations during development and maturation.The molecular mechanisms underlying these varia...The organoleptic qualities of watermelon fruit are defined by the sugar and organic acid contents,which undergo considerable variations during development and maturation.The molecular mechanisms underlying these variations remain unclear.In this study,we used transcriptome profiles to investigate the coexpression patterns of gene networks associated with sugar and organic acid metabolism.We identified 3 gene networks/modules containing 2443 genes highly correlated with sugars and organic acids.Within these modules,based on intramodular significance and Reverse Transcription Quantitative polymerase chain reaction(RT-qPCR),we identified 7 genes involved in the metabolism of sugars and organic acids.Among these genes,Cla97C01G000640,Cla97C05G087120 and Cla97C01G018840(r^(2)=0.83 with glucose content)were identified as sugar transporters(SWEET,EDR6 and STP)and Cla97C03G064990(r^(2)=0.92 with sucrose content)was identified as a sucrose synthase from information available for other crops.Similarly,Cla97C07G128420,Cla97C03G068240 and Cla97C01G008870,having strong correlations with malic(r^(2)=0.75)and citric acid(r^(2)=0.85),were annotated as malate and citrate transporters(ALMT7,CS,and ICDH).The expression profiles of these 7 genes in diverse watermelon genotypes revealed consistent patterns of expression variation in various types of watermelon.These findings add significantly to our existing knowledge of sugar and organic acid metabolism in watermelon.展开更多
Objective: To report presence of Leishmania major in Khyber Pakhtunkhwa of Pakistan, where cutaneous leishmaniasis(CL) is endemic and was thought to be caused by Leishmania tropica only. Methods: Biopsy samples from 4...Objective: To report presence of Leishmania major in Khyber Pakhtunkhwa of Pakistan, where cutaneous leishmaniasis(CL) is endemic and was thought to be caused by Leishmania tropica only. Methods: Biopsy samples from 432 CL suspected patients were collected from 3 southern districts of Khyber Pakhtunkhwa during years 2011–2016. Microscopy on Giemsa stained slides were done followed by amplification of the ribosomal internal transcribed spacer 1 gene. Results: Leishmania amastigotes were detected by microscopy in 308 of 432 samples(71.3%) while 374 out of 432 samples(86.6%) were positive by ribosomal internal transcribed spacer 1 PCR. Subsequent restriction fragment length polymorphism confirmed Leishmania tropica in 351 and Leishmania major in 6 biopsy samples. Conclusions: This study is the first molecular characterization of Leishmania species in southern Khyber Pakhtunkhwa. It confirmed the previous assumptions that anthroponotic CL is the major CL form present in Khyber Pakhtunkhwa province. Furthermore, this is the first report of Leishmania major from a classical anthroponotic CL endemic focus identified in rural areas of Kohat district in southern Khyber Pakhtunkhwa.展开更多
基金supported by the Basic Research Funds of Chinese Academy of Agricultural Sciences(Grant No.1610192023201)the National Key Research and Development Program of China(Grant No.SQ2023YFE0201239)+3 种基金China Agriculture Research System of MOF and MARA(Grant No.CARS-25-03)the Major Science and Technology Projects of Henan Province(Grant No.221100110400)the Funding of Joint Research on Agricultural Variety Improvement of Henan Province(Grant No.2022010503)the Agricultural Science and Technology Innovation Program(Grant No.CAAS-ASTIP-2021-ZFRI)。
文摘The CRISPR/Cas9 genome-editing system serves as a pivotal tool for enhancing crop genetics.Within this system,single guide RNAs(sgRNAs)are instrumental in the precise cleavage of DNA double strands.However,the efficiency of gene editing varies among sgRNAs,emphasizing the need to meticulously select target sites,especially in the context of Citrullus lanatus,a species notorious for its challenging and inefficient generation of transgenic plants through stable transformation.This study employed an Agrobacterium rhizogenes-mediated hairy root method to assess effective target sites for gene editing of ClCIPK17 across various Citrullus species.Hairy roots were successfully induced in different plant tissues at diverse growth stages of Citrullus lanatus,Citrullus mucosospermus,and Citrullus amarus.Employing a vector with two sgRNAs(sgRNA1 and sgRNA5)positioned within conserved regions of exon 1 and exon 5 of ClCIPK17 in the CRISPR/Cas9 system,targeted mutations were detected in 90.9%of accessions across the four Citrullus species.Notably,73.94%of all examined hairy roots exhibited mutations at the sgRNA1 site,while the sgRNA5 site showed no mutations.Among the 31 different mutation types identified at the sgRNA1 site,base deletion was the most prevalent.Using the sgRNA1 site of ClCIPK17,stable transgenic watermelon buds were obtained from explants,and the targeted mutations of the sgRNA1 site were confirmed.These findings underscore the viability of the hairy root transformation system in assessing the editing efficiency of sgRNA targets in diverse Citrullus species.
基金the Agricultural Science and Technology Innovation Program(Grant No.CAAS-ASTIP-2021-ZFRI)China Agriculture Research System of MOF and MARA(Grant No.CARS-25-03)+3 种基金National Nature Science Foundation of China(Grant Nos.31672178 and 31471893)the Natural Science Foundation of Henan Province(Grant No.212300410312)the scientific and technological research in Henan Province(Grant No.202102110398)the key project of the Action of“Rejuvenating Mongolia with Science and Technology”(Grant No.NMKJXM202114).
文摘Watermelon is a highly cultivated fruit crop renowned for its quality properties of fruit flesh.Among various quality factors,fruit flesh firmness is a crucial quality parameter influencing the fruit texture,shelf life and its commercial value.The auxin/indole-3-acetic acid(Aux/IAA)plays a significant role in fruit development and ripening of non-climacteric fruits.However,the regulatory mechanism of Aux/IAA in controlling fruit flesh firmness and ripening in watermelon remains unknown.In this study,we employed an integrative approach combining genome-wide association study(GWAS)and bulked segregant RNA-Seq analysis(BSR-Seq)to identify an overlapping candidate region between 12776310 and 12968331 bp on chromosome 6,underlying an auxin-responsive gene(Aux/IAA)associated with flesh firmness in watermelon.Transcriptome analysis,followed by real-time quantitative reverse transcription PCR(qRT-PCR),confirmed that the expression of Aux/IAA was consistently higher in fruits with high flesh firmness.The sequence alignment revealed a single base mutation in the coding region of Aux/IAA.Furthermore,the concomitant Kompetitive/Competitive allele-specific PCR(KASP)genotyping data sets for F2 population and germplasm accessions identified Aux/IAA as a strong candidate gene associated with flesh firmness.Aux/IAA was enriched in the plant hormone signal transduction pathway,involving cell enlargement and leading to low flesh firmness.We determined the higher accumulation of abscisic acid(ABA)in fruits with low flesh firmness than hard flesh.Moreover,overexpression of Aux/IAA induced higher flesh firmness with an increased number of fruit flesh cells while reducing ABA content and flesh cell sizes.Additionally,the allelic variation in Aux/IAA for soft flesh firmness was found to exist in Citrullus mucosospermus and gradually fixed into Citrullus lanatus during domestication,indicating that soft flesh firmness was a domesticated trait.These findings significantly enhanced our understanding of watermelon fruit flesh firmness and consequently the watermelon fruit quality.
基金supported by the Agricultural Science and Technology Innovation Program(CAAS-ASTIP-2016-ZFRI)National Key R&D Program of China(2018YFD0100704)the China Agriculture Research System(CARS-25-03)+1 种基金National Natural Science Foundation of China[31672178&31471893]Scientific and Technological Project of Henan Province(202102110197).
文摘The organoleptic qualities of watermelon fruit are defined by the sugar and organic acid contents,which undergo considerable variations during development and maturation.The molecular mechanisms underlying these variations remain unclear.In this study,we used transcriptome profiles to investigate the coexpression patterns of gene networks associated with sugar and organic acid metabolism.We identified 3 gene networks/modules containing 2443 genes highly correlated with sugars and organic acids.Within these modules,based on intramodular significance and Reverse Transcription Quantitative polymerase chain reaction(RT-qPCR),we identified 7 genes involved in the metabolism of sugars and organic acids.Among these genes,Cla97C01G000640,Cla97C05G087120 and Cla97C01G018840(r^(2)=0.83 with glucose content)were identified as sugar transporters(SWEET,EDR6 and STP)and Cla97C03G064990(r^(2)=0.92 with sucrose content)was identified as a sucrose synthase from information available for other crops.Similarly,Cla97C07G128420,Cla97C03G068240 and Cla97C01G008870,having strong correlations with malic(r^(2)=0.75)and citric acid(r^(2)=0.85),were annotated as malate and citrate transporters(ALMT7,CS,and ICDH).The expression profiles of these 7 genes in diverse watermelon genotypes revealed consistent patterns of expression variation in various types of watermelon.These findings add significantly to our existing knowledge of sugar and organic acid metabolism in watermelon.
基金grateful to Higher Education Commission Government of Pakistan for providing fund Grant No: 1384 to Kohat university of Science and technology Kohat,Pakistangrateful to French Embassy,Islamabad for funding under their split Ph D fellowship programs,a 6 months Ph D fellowship to Dr. Mubbashir Hussain at ANSES,Animal Health Laboratory,Maisons-Alfort,France
文摘Objective: To report presence of Leishmania major in Khyber Pakhtunkhwa of Pakistan, where cutaneous leishmaniasis(CL) is endemic and was thought to be caused by Leishmania tropica only. Methods: Biopsy samples from 432 CL suspected patients were collected from 3 southern districts of Khyber Pakhtunkhwa during years 2011–2016. Microscopy on Giemsa stained slides were done followed by amplification of the ribosomal internal transcribed spacer 1 gene. Results: Leishmania amastigotes were detected by microscopy in 308 of 432 samples(71.3%) while 374 out of 432 samples(86.6%) were positive by ribosomal internal transcribed spacer 1 PCR. Subsequent restriction fragment length polymorphism confirmed Leishmania tropica in 351 and Leishmania major in 6 biopsy samples. Conclusions: This study is the first molecular characterization of Leishmania species in southern Khyber Pakhtunkhwa. It confirmed the previous assumptions that anthroponotic CL is the major CL form present in Khyber Pakhtunkhwa province. Furthermore, this is the first report of Leishmania major from a classical anthroponotic CL endemic focus identified in rural areas of Kohat district in southern Khyber Pakhtunkhwa.
