Bulked segregant analysis(BSA)is a rapid,cost-effective method for mapping mutations and quantitative trait loci(QTLs)in animals and plants based on high-throughput sequencing.However,the algorithms currently used for...Bulked segregant analysis(BSA)is a rapid,cost-effective method for mapping mutations and quantitative trait loci(QTLs)in animals and plants based on high-throughput sequencing.However,the algorithms currently used for BSA have not been systematically evaluated and are complex and fallible to operate.We developed a BSA method driven by deep learning,DeepBSA,for QTL mapping and functional gene cloning.DeepBSA is compatible with a variable number of bulked pools and performed well with various simulated and real datasets in both animals and plants.DeepBSA outperformed all other algorithms when comparing absolute bias and signal-to-noise ratio.Moreover,we applied DeepBSA to an F2 segregating maize population of 7160 individuals and uncovered five candidate QTLs,including three well-known plant-height genes.Finally,we developed a user-friendly graphical user interface for DeepBSA,by integrating five widely used BSA algorithms and our two newly developed algorithms,that is easy to operate and can quickly map QTLs and functional genes.The DeepBSA software is freely available to noncommercial users at http://zeasystemsbio.hzau.edu.cn/tools.html and https://github.com/lizhao007/DeepBSA.展开更多
Foxtail millet(Setaria italica)is an important C4 model crop;however,due to its high-density planting and high stature,lodging at the filling stage resulted in a serious reduction in yield and quality.Therefore,it is ...Foxtail millet(Setaria italica)is an important C4 model crop;however,due to its high-density planting and high stature,lodging at the filling stage resulted in a serious reduction in yield and quality.Therefore,it is imperative to identify and deploy the genes controlling foxtail millet plant height.In this study,we used a semi-dwarf line 263A and an elite high-stalk breeding variety,Chuang 29 to construct an F2 population to identify dwarf genes.We performed transcriptome analysis(RNA-seq)using internode tissues sampled at three jointing stages of 263A and Chuang 29,as well as bulk segregant analysis(BSA)on their F2 population.A total of 8918 differentially expressed genes(DEGs)were obtained from RNA-seq analysis,and GO analysis showed that DEGs were enriched in functions such as‘‘gibberellin metabolic process”and‘‘oxidoreductase activity”,which have previously been shown to be associated with plant height.A total 593 mutated genes were screened by BSA-seq method.One hundred and seventy-six out of the 593 mutated genes showed differential expression levels between the two parental lines,and seven genes not only showed differential expression in two or three internode tissues but also showed high genomic variation in coding regions,which indicated they play a crucial role in plant height determination.Among them,we found a gibberellin biosynthesis related GA20 oxidase gene(Seita.5G404900),which had a single-base at the third exon,leading to the frameshift mutation at 263A.Cleaved amplified polymorphic sequence assay and association analysis proved the single-base in Seita.5G404900 co-segregated with dwarf phenotype in two independent F2 populations planted in entirely different environments.Taken together,the candidate genes identified in this study will help to elucidate the genetic basis of foxtail millet plant height,and the molecular marker will be useful for marker-assisted dwarf breeding.展开更多
Peanut(Arachis hypogaea L.)is a globally important oil crop.Web blotch is one of the most important foliar diseases affecting peanut,which results in serious yield losses worldwide.Breeding web blotch-resistant peanut...Peanut(Arachis hypogaea L.)is a globally important oil crop.Web blotch is one of the most important foliar diseases affecting peanut,which results in serious yield losses worldwide.Breeding web blotch-resistant peanut varieties is the most effective and economically viable method for minimizing yield losses due to web blotch.In the current study,a bulked segregant analysis with next-generation sequencing was used to analyze an F2:3 segregating population and identify candidate loci related to web blotch resistance.Based on the fine-mapping of the candidate genomic interval using kompetitive allele-specific PCR(KASP)markers,we identified a novel web blotch resistance-related locus spanning approximately 169 kb on chromosome 16.This region included four annotated genes,of which only Arahy.35VVQ3 had a non-synonymous single nucleotide polymorphism in the coding region between the two parents.Two markers(Chr.16.12872635 and Chr.16.12966357)linked to this gene were shown to be co-segregated with the resistance of peanut web blotch by 72 randomly selected recombinant inbred lines(RIL),which could be used in marker-assisted breeding of resistant peanut varieties.展开更多
Puccinia striiformis Westend. f. sp. tritici(Pst) pathotype CYR34 is widely virulent and prevalent in China.Here, we report identification of a strpie rust resistance(Yr) gene, designated Yr041133, in winter wheat lin...Puccinia striiformis Westend. f. sp. tritici(Pst) pathotype CYR34 is widely virulent and prevalent in China.Here, we report identification of a strpie rust resistance(Yr) gene, designated Yr041133, in winter wheat line 041133. This line produced a hypersensitive reaction to CYR34 and conferred resistance to 13 other pathotypes. Resistance to CYR34 in line 041133 was controlled by a single dominant gene. Bulked segregant RNA sequencing(BSR-Seq) was performed on a pair of RNA bulks generated by pooling resistant and susceptible recombinant inbred lines. Yr041133 was mapped to a 1.7 c M genetic interval on the chromosome arm 7 BL that corresponded to a 0.8 Mb physical interval(608.9–609.7 Mb) in the Chinese Spring reference genome. Based on its unique physical location Yr041133 differred from the other Yr genes on this chromosome arm.展开更多
Fruit wart is an important appearance trait influencing consumer preferences of bitter gourd(Momordica charantia L.).The molecular genetic mechanisms underlying fruit wart formation in bitter gourd are largely unknown...Fruit wart is an important appearance trait influencing consumer preferences of bitter gourd(Momordica charantia L.).The molecular genetic mechanisms underlying fruit wart formation in bitter gourd are largely unknown.In this study,genetic analysis based on four generations showed that fruit wart formation in bitter gourd was controlled by a single dominant locus named as Fwa.The Fwa locus was initially mapped into a 4.82 Mb region on pseudochromosome 4 by BSA-seq analysis and subsequently narrowed down to a 286.30 kb region by linkage analysis.A large F2population consisting of 2360 individuals was used to screen recombinants,and the Fwa locus was finally fine mapped into a 22.70 kb region harboring four protein-coding genes through recombination analysis.MC04g1399,encoding an epidermal patterning factor 2-like protein,was proposed as the best candidate gene for Fwa via sequence variation and expression analysis.In addition,a 1-bp insertion and deletion(InDel)variation within MC04g1399 was converted to a cleaved amplified polymorphic sequence(CAPS)marker that could precisely distinguish between the warty and non-warty types with an accuracy rate of 100%among a wide panel of 126 bitter gourd germplasm resources.Our results not only provide a scientific basis for deciphering the molecular mechanisms underlying fruit wart formation but also provide a powerful tool for efficient genetic improvement of fruit wart via marker-assisted selection.展开更多
Leaf senescence is normally the last stage of plant development. Early senescence of functional leaves significantly reduces the photosynthetic time and efficiency, seriously affecting grain yield and quality in wheat...Leaf senescence is normally the last stage of plant development. Early senescence of functional leaves significantly reduces the photosynthetic time and efficiency, seriously affecting grain yield and quality in wheat. Discovering genes responsible for early leaf senescence(els) are necessary for developing novel germplasms and cultivars with delayed leaf-senescence through molecular manipulation and marker assisted selection. In this study, we identified an early leaf senescence line M114 in a derivative of a wheat breeding population. Genetic analysis indicated that early leaf senescence in M114 is controlled by a single recessive gene, provisionally designated els1. By applying bulked segregant analysis and RNA-Seq(BSR-Seq), seven polymorphic markers linked to els1 were developed and the gene was located on chromosome arm 2 BS in a 1.5 c M genetic interval between markers WGGB303 and WGGB305. A co-segregating marker, WGGB302, provide a starting point for fine mapping and map-based cloning of els1.展开更多
Hypsizygus marmoreus is one of the most important edible fungi in Basidiomycete division and includes white and gray strains.However,very limited knowledge is known about the genomic structures and the genetic basis f...Hypsizygus marmoreus is one of the most important edible fungi in Basidiomycete division and includes white and gray strains.However,very limited knowledge is known about the genomic structures and the genetic basis for the white/gray diversity of this mushroom.Here,we report the near-complete high-quality H.marmoreus genome at the chromosomal level.Comparative genomics analysis indicates that chromosome structures were relatively conserved,and variations in collinearity and chromosome number were mainly attributed by chromosome split/fusion events in Aragicales,whereas the fungi genome experienced many genomic chromosome fracture,fusion,and genomic replication events after the split of Aragicales from Basidiomycetes.Resequencing of 57 strains allows us to classify the population into four major groups and associate genetic variations with morphological features,indicating that white strains were not originated independently.We further generated genetic populations and identified a cytochrome P450 as the candidate causal gene for the melanogenesis in H.marmoreus based on bulked segregant analysis (BSA)and comparative transcriptome analysis.The high-quality H.marmoreus genome and diversity data compiled in this study provide new knowledge and resources for the molecular breeding of H.marmoreus as well as the evolution of Basidiomycete.展开更多
Theoretical and applied studies demonstrate the difficulty of detecting extremely over-dominant and smalleffect genes for quantitative traits via bulked segregant analysis(BSA)in an F_(2)population.To address this iss...Theoretical and applied studies demonstrate the difficulty of detecting extremely over-dominant and smalleffect genes for quantitative traits via bulked segregant analysis(BSA)in an F_(2)population.To address this issue,we proposed an integrated strategy for mapping various types of quantitative trait loci(QTLs)for quantitative traits via a combination of BSA and whole-genome sequencing.In this strategy,the numbers of read counts of marker alleles in two extreme pools were used to predict the numbers of read counts of marker genotypes.These observed and predicted numbers were used to construct a new statistic,G_(w),for detecting quantitative trait genes(QTGs),and the method was named dQTG-seq1.This method was significantly better than existing BSA methods.If the goal was to identify extremely over-dominant and smalleffect genes,another reserved DNA/RNA sample from each extreme phenotype F_(2)plant was sequenced,and the observed numbers of marker alleles and genotypes were used to calculate G_(w)to detect QTGs;this method was named dQTG-seq2.In simulated and real rice dataset analyses,dQTG-seq2 could identify many more extremely over-dominant and small-effect genes than BSA and QTL mapping methods.dQTGseq2 may be extended to other heterogeneous mapping populations.The significance threshold of G_(w)in this study was determined by permutation experiments.In addition,a handbook for the R software dQTG.seq,which is available at https://cran.r-project.org/web/packages/dQTG.seq/index.html,has been provided in the supplemental materials for the users’convenience.This study provides a new strategy for identifying all types of QTLs for quantitative traits in an F_(2)population.展开更多
基金supported by the National Natural Science Foundation of China(31922068)HainanYazhouBay Seed Lab(B21HJ8102)+2 种基金themajor Program of Hubei Hongshan Laboratory(2021hszd008)Huazhong 3 Agricultural University Scientific&Technological Self-innovation Foundation(2021ZKPY001)Fundamental Research Funds for the Central Universities of China(2662020LXQD002).
文摘Bulked segregant analysis(BSA)is a rapid,cost-effective method for mapping mutations and quantitative trait loci(QTLs)in animals and plants based on high-throughput sequencing.However,the algorithms currently used for BSA have not been systematically evaluated and are complex and fallible to operate.We developed a BSA method driven by deep learning,DeepBSA,for QTL mapping and functional gene cloning.DeepBSA is compatible with a variable number of bulked pools and performed well with various simulated and real datasets in both animals and plants.DeepBSA outperformed all other algorithms when comparing absolute bias and signal-to-noise ratio.Moreover,we applied DeepBSA to an F2 segregating maize population of 7160 individuals and uncovered five candidate QTLs,including three well-known plant-height genes.Finally,we developed a user-friendly graphical user interface for DeepBSA,by integrating five widely used BSA algorithms and our two newly developed algorithms,that is easy to operate and can quickly map QTLs and functional genes.The DeepBSA software is freely available to noncommercial users at http://zeasystemsbio.hzau.edu.cn/tools.html and https://github.com/lizhao007/DeepBSA.
基金supported by the National Key Research and Development Program of China (2018YFD1000702/ 2018YFD1000700)the Agricultural Science and Technology Innovation Program of Chinese Academy of Agricultural SciencesOperating Expenses for Basic Scientific Research of Institute of Crop Science, Chinese Academy of Agricultural Sciences
文摘Foxtail millet(Setaria italica)is an important C4 model crop;however,due to its high-density planting and high stature,lodging at the filling stage resulted in a serious reduction in yield and quality.Therefore,it is imperative to identify and deploy the genes controlling foxtail millet plant height.In this study,we used a semi-dwarf line 263A and an elite high-stalk breeding variety,Chuang 29 to construct an F2 population to identify dwarf genes.We performed transcriptome analysis(RNA-seq)using internode tissues sampled at three jointing stages of 263A and Chuang 29,as well as bulk segregant analysis(BSA)on their F2 population.A total of 8918 differentially expressed genes(DEGs)were obtained from RNA-seq analysis,and GO analysis showed that DEGs were enriched in functions such as‘‘gibberellin metabolic process”and‘‘oxidoreductase activity”,which have previously been shown to be associated with plant height.A total 593 mutated genes were screened by BSA-seq method.One hundred and seventy-six out of the 593 mutated genes showed differential expression levels between the two parental lines,and seven genes not only showed differential expression in two or three internode tissues but also showed high genomic variation in coding regions,which indicated they play a crucial role in plant height determination.Among them,we found a gibberellin biosynthesis related GA20 oxidase gene(Seita.5G404900),which had a single-base at the third exon,leading to the frameshift mutation at 263A.Cleaved amplified polymorphic sequence assay and association analysis proved the single-base in Seita.5G404900 co-segregated with dwarf phenotype in two independent F2 populations planted in entirely different environments.Taken together,the candidate genes identified in this study will help to elucidate the genetic basis of foxtail millet plant height,and the molecular marker will be useful for marker-assisted dwarf breeding.
基金supported by the Key Research Project of the Shennong Laboratory,China(SN01-2022-03)the Henan Provincial Science and Technology R&D Program Joint Fund(Superiority Discipline Cultivation)Project,China(222301420100)+4 种基金the Major Science and Technology Projects of Henan Province,China(221100110300)the China Agriculture Research System of MOF and MARA(CARS-13)the Henan Provincial Agriculture Research System,China(S2012-5)the Outstanding Young Scientists of Henan Academy of Agricultural Sciences,China(2022YQ16)the Independent Innovation Project of the Henan Academy of Agricultural Sciences,China(2023ZC093)。
文摘Peanut(Arachis hypogaea L.)is a globally important oil crop.Web blotch is one of the most important foliar diseases affecting peanut,which results in serious yield losses worldwide.Breeding web blotch-resistant peanut varieties is the most effective and economically viable method for minimizing yield losses due to web blotch.In the current study,a bulked segregant analysis with next-generation sequencing was used to analyze an F2:3 segregating population and identify candidate loci related to web blotch resistance.Based on the fine-mapping of the candidate genomic interval using kompetitive allele-specific PCR(KASP)markers,we identified a novel web blotch resistance-related locus spanning approximately 169 kb on chromosome 16.This region included four annotated genes,of which only Arahy.35VVQ3 had a non-synonymous single nucleotide polymorphism in the coding region between the two parents.Two markers(Chr.16.12872635 and Chr.16.12966357)linked to this gene were shown to be co-segregated with the resistance of peanut web blotch by 72 randomly selected recombinant inbred lines(RIL),which could be used in marker-assisted breeding of resistant peanut varieties.
基金Financial support of this research by the National Key Research and Development Program of China(2017YFD0101000)the Agricultural Science and Technology Innovation Program of CAAS(CAAS-ZDRW202002)。
文摘Puccinia striiformis Westend. f. sp. tritici(Pst) pathotype CYR34 is widely virulent and prevalent in China.Here, we report identification of a strpie rust resistance(Yr) gene, designated Yr041133, in winter wheat line 041133. This line produced a hypersensitive reaction to CYR34 and conferred resistance to 13 other pathotypes. Resistance to CYR34 in line 041133 was controlled by a single dominant gene. Bulked segregant RNA sequencing(BSR-Seq) was performed on a pair of RNA bulks generated by pooling resistant and susceptible recombinant inbred lines. Yr041133 was mapped to a 1.7 c M genetic interval on the chromosome arm 7 BL that corresponded to a 0.8 Mb physical interval(608.9–609.7 Mb) in the Chinese Spring reference genome. Based on its unique physical location Yr041133 differred from the other Yr genes on this chromosome arm.
基金supported by the Science and Technology Planning Project of Guangdong Province(Grants Nos.2022 B0202160015 and 2019A050520002)the Seed Industry Revitalization Project of Special Funds for Provincial Rural Revitalization Strategy(Grant No.2022-NPY-00-027)+1 种基金the Guangzhou Science and Technology Plan Projects(Grants Nos.202002020086,202102020800 and 202206010170)the Guangzhou Basic and Applied Basic Research Project(Grant No.SL2023A04J01673)。
文摘Fruit wart is an important appearance trait influencing consumer preferences of bitter gourd(Momordica charantia L.).The molecular genetic mechanisms underlying fruit wart formation in bitter gourd are largely unknown.In this study,genetic analysis based on four generations showed that fruit wart formation in bitter gourd was controlled by a single dominant locus named as Fwa.The Fwa locus was initially mapped into a 4.82 Mb region on pseudochromosome 4 by BSA-seq analysis and subsequently narrowed down to a 286.30 kb region by linkage analysis.A large F2population consisting of 2360 individuals was used to screen recombinants,and the Fwa locus was finally fine mapped into a 22.70 kb region harboring four protein-coding genes through recombination analysis.MC04g1399,encoding an epidermal patterning factor 2-like protein,was proposed as the best candidate gene for Fwa via sequence variation and expression analysis.In addition,a 1-bp insertion and deletion(InDel)variation within MC04g1399 was converted to a cleaved amplified polymorphic sequence(CAPS)marker that could precisely distinguish between the warty and non-warty types with an accuracy rate of 100%among a wide panel of 126 bitter gourd germplasm resources.Our results not only provide a scientific basis for deciphering the molecular mechanisms underlying fruit wart formation but also provide a powerful tool for efficient genetic improvement of fruit wart via marker-assisted selection.
基金supported by the National Key Research and Development Program of China(2017YFD0101004)Science and Technology Service Network Initiative of Chinese Academy of Sciences(KFJ-STSZDTP-024)
文摘Leaf senescence is normally the last stage of plant development. Early senescence of functional leaves significantly reduces the photosynthetic time and efficiency, seriously affecting grain yield and quality in wheat. Discovering genes responsible for early leaf senescence(els) are necessary for developing novel germplasms and cultivars with delayed leaf-senescence through molecular manipulation and marker assisted selection. In this study, we identified an early leaf senescence line M114 in a derivative of a wheat breeding population. Genetic analysis indicated that early leaf senescence in M114 is controlled by a single recessive gene, provisionally designated els1. By applying bulked segregant analysis and RNA-Seq(BSR-Seq), seven polymorphic markers linked to els1 were developed and the gene was located on chromosome arm 2 BS in a 1.5 c M genetic interval between markers WGGB303 and WGGB305. A co-segregating marker, WGGB302, provide a starting point for fine mapping and map-based cloning of els1.
基金supported by program for Seed Innovation and Industrialization in Fujian Province-Breeding and Industrialization of Major Edible Fungithe Science and Technology Major Project of Fujian Province (2016NZ0001)+1 种基金the Program for New Century Excellent Talents in Fujian Province (KLa17073A)agricultural technology extension service system for Edible fungus industry in Fujian, China (KNJ-153011-1)。
文摘Hypsizygus marmoreus is one of the most important edible fungi in Basidiomycete division and includes white and gray strains.However,very limited knowledge is known about the genomic structures and the genetic basis for the white/gray diversity of this mushroom.Here,we report the near-complete high-quality H.marmoreus genome at the chromosomal level.Comparative genomics analysis indicates that chromosome structures were relatively conserved,and variations in collinearity and chromosome number were mainly attributed by chromosome split/fusion events in Aragicales,whereas the fungi genome experienced many genomic chromosome fracture,fusion,and genomic replication events after the split of Aragicales from Basidiomycetes.Resequencing of 57 strains allows us to classify the population into four major groups and associate genetic variations with morphological features,indicating that white strains were not originated independently.We further generated genetic populations and identified a cytochrome P450 as the candidate causal gene for the melanogenesis in H.marmoreus based on bulked segregant analysis (BSA)and comparative transcriptome analysis.The high-quality H.marmoreus genome and diversity data compiled in this study provide new knowledge and resources for the molecular breeding of H.marmoreus as well as the evolution of Basidiomycete.
基金This work was supported by the National Natural Science Foundation of China(32070557 and 31871242)the Fundamental Research Funds for the Central Universities(2662020ZKPY017)the Huazhong Agricul-tural University Scientific and Technological Self-Innovation Foundation(2014RC020).
文摘Theoretical and applied studies demonstrate the difficulty of detecting extremely over-dominant and smalleffect genes for quantitative traits via bulked segregant analysis(BSA)in an F_(2)population.To address this issue,we proposed an integrated strategy for mapping various types of quantitative trait loci(QTLs)for quantitative traits via a combination of BSA and whole-genome sequencing.In this strategy,the numbers of read counts of marker alleles in two extreme pools were used to predict the numbers of read counts of marker genotypes.These observed and predicted numbers were used to construct a new statistic,G_(w),for detecting quantitative trait genes(QTGs),and the method was named dQTG-seq1.This method was significantly better than existing BSA methods.If the goal was to identify extremely over-dominant and smalleffect genes,another reserved DNA/RNA sample from each extreme phenotype F_(2)plant was sequenced,and the observed numbers of marker alleles and genotypes were used to calculate G_(w)to detect QTGs;this method was named dQTG-seq2.In simulated and real rice dataset analyses,dQTG-seq2 could identify many more extremely over-dominant and small-effect genes than BSA and QTL mapping methods.dQTGseq2 may be extended to other heterogeneous mapping populations.The significance threshold of G_(w)in this study was determined by permutation experiments.In addition,a handbook for the R software dQTG.seq,which is available at https://cran.r-project.org/web/packages/dQTG.seq/index.html,has been provided in the supplemental materials for the users’convenience.This study provides a new strategy for identifying all types of QTLs for quantitative traits in an F_(2)population.
