Next-generation sequencing （NGS） technologies allow the cost-effective sequencing of whole genomes and have expanded the scope of genomics to novel applications, such as the genome-wide characterization of intraspec...Next-generation sequencing （NGS） technologies allow the cost-effective sequencing of whole genomes and have expanded the scope of genomics to novel applications, such as the genome-wide characterization of intraspecific polymorphisms and the rapid mapping and identification of point mutations. Next-generation sequencing platforms, such as the Illumina HiSeq2ooo platform, are now commercially available at affordable prices and routinely produce an enormous amount of sequence data, but their wide use is often hindered by a lack of knowledge on how to manipulateand process the information produced. In this review, we focus on the strategies that are available to geneticists who wish to incorporate these novel approaches into their research but who are not familiar with the necessary bioinformatic concepts and computational tools. In particular, we comprehensively summarize case studies where the use of NGS technologies has led to the identification of point mutations, a strategy that has been dubbed ＂mapping-by-sequencing＇, and review examples from plants and other model species such as Caenorhabditis elegans, Saccharomyces cerevisiae, and Drosophila mela- nogaster. As these technologies are becoming cheaper and more powerful, their use is also expanding to allow mutation identification in species with larger genomes, such as many crop plants.展开更多
Barley(Hordeum vulgare L.)is one of the earliest domesticated crop species and ranked as the fourth largest cereal production worldwide.Forward genetic studies in barley have greatly advanced plant genetics during the...Barley(Hordeum vulgare L.)is one of the earliest domesticated crop species and ranked as the fourth largest cereal production worldwide.Forward genetic studies in barley have greatly advanced plant genetics during the last century;however,most genes are identified by the conventional mapping method.Array genotyping and exome-capture sequencing have also been successfully used to target the causal mutation in barley populations,but these techniques are not widely adopted because of associated costs and partly due to the huge genome size of barley.This review summarizes three mapping cases of barley cuticle mutants in our laboratory with the help of RNA-sequencing.The causal mutations have been successfully identified for two of them and the target genes are located in the pericentromeric regions.Detailed information on the mapping-by-sequencing,mapping-and-sequencing,and RNA-sequencing assisted linkage mapping are presented and some limitations and challenges on the mapping assisted by RNA sequencing are also discussed.The alternative and elegant methods presented in this review may greatly accelerate forward genetics of barley mapping,especially for laboratories without large funding.展开更多
Despite the large number of genomic and transcriptomic resources in maize, there is still much to learn about the function of genes in developmental and biochemical processes. Some maize mutants that were generated by...Despite the large number of genomic and transcriptomic resources in maize, there is still much to learn about the function of genes in developmental and biochemical processes. Some maize mutants that were generated by gamma-irradiation showed clear segregation for the kernel phenotypes in B73 ? Mo17 F2 ears. To better understand the functional genomics of kernel development,we developed a mapping and gene identi?cation pipeline, bulked segregant exome sequencing(BSEx-seq), to map mutants with kernel phenotypes including opaque endosperm and reduced kernel size. BSEx-seq generates and compares the sequence of the exon fraction from mutant and normal plant F2 DNA pools. The comparison can derive mapping peaks, identify deletions within the mapping peak, and suggest candidate genes within the deleted regions. We then used the public kernel-speci?c expression data to narrow down the list of candidate genes/mutations and identi?ed deletions ranging from several kb to more than 1 Mb. A full deletion allele of the Opaque-2 gene was identi?ed in mutant 531, which occurs within a $200-kb deletion. Opaque mutant 1486 has a 6248-bp deletion in the mapping interval containing two candidate genes encoding RNA-directed DNA methylation 4(RdDM4) and AMP-binding protein, respectively. This study demonstrates the ef?-ciency and cost-effectiveness of BSEx-seq for causal mutation mapping and candidate gene selection,providing a new option in mapping-by-sequencing for maize functional genomics studies.展开更多
基金supported by grants from the Ministerio de Economiay Competitividad of Spain(BFU2011-22825 and CSD2007-00057(TRANSPLANTA))the Generalitat Valenciana(PROMETEOII/2014/003)+2 种基金H.C.was a recipient of a Marie Curie International Reintegration Grant(PIRG03-GA-2008-231073)Research in the laboratory of H.C.was supported by a grant from the Ministerio de Economiay Competitividad of Spain(BFU2012-31719)R.C.S.held a fellowship from the Ministerio de Economfa y Competitividad of Spain(BES-2009-014106)
文摘Next-generation sequencing （NGS） technologies allow the cost-effective sequencing of whole genomes and have expanded the scope of genomics to novel applications, such as the genome-wide characterization of intraspecific polymorphisms and the rapid mapping and identification of point mutations. Next-generation sequencing platforms, such as the Illumina HiSeq2ooo platform, are now commercially available at affordable prices and routinely produce an enormous amount of sequence data, but their wide use is often hindered by a lack of knowledge on how to manipulateand process the information produced. In this review, we focus on the strategies that are available to geneticists who wish to incorporate these novel approaches into their research but who are not familiar with the necessary bioinformatic concepts and computational tools. In particular, we comprehensively summarize case studies where the use of NGS technologies has led to the identification of point mutations, a strategy that has been dubbed ＂mapping-by-sequencing＇, and review examples from plants and other model species such as Caenorhabditis elegans, Saccharomyces cerevisiae, and Drosophila mela- nogaster. As these technologies are becoming cheaper and more powerful, their use is also expanding to allow mutation identification in species with larger genomes, such as many crop plants.
基金grants from the National Natural Science Foundation of China(No.41621001,No.31870381,and No.31970352)by the Youth Innovation Promotion Association,CAS(2018463).
文摘Barley(Hordeum vulgare L.)is one of the earliest domesticated crop species and ranked as the fourth largest cereal production worldwide.Forward genetic studies in barley have greatly advanced plant genetics during the last century;however,most genes are identified by the conventional mapping method.Array genotyping and exome-capture sequencing have also been successfully used to target the causal mutation in barley populations,but these techniques are not widely adopted because of associated costs and partly due to the huge genome size of barley.This review summarizes three mapping cases of barley cuticle mutants in our laboratory with the help of RNA-sequencing.The causal mutations have been successfully identified for two of them and the target genes are located in the pericentromeric regions.Detailed information on the mapping-by-sequencing,mapping-and-sequencing,and RNA-sequencing assisted linkage mapping are presented and some limitations and challenges on the mapping assisted by RNA sequencing are also discussed.The alternative and elegant methods presented in this review may greatly accelerate forward genetics of barley mapping,especially for laboratories without large funding.
基金supported by the Agriculture and Food Research Initiative competitive grant (Grant No.2013-02278)the United States Department of Agriculture,National Institute of Food and Agriculture (USDA-NIFA)Center for Plant Science Innovation Program of Excellence and Department of Agronomy and Horticulture,University of NebraskaLincoln,United States
文摘Despite the large number of genomic and transcriptomic resources in maize, there is still much to learn about the function of genes in developmental and biochemical processes. Some maize mutants that were generated by gamma-irradiation showed clear segregation for the kernel phenotypes in B73 ? Mo17 F2 ears. To better understand the functional genomics of kernel development,we developed a mapping and gene identi?cation pipeline, bulked segregant exome sequencing(BSEx-seq), to map mutants with kernel phenotypes including opaque endosperm and reduced kernel size. BSEx-seq generates and compares the sequence of the exon fraction from mutant and normal plant F2 DNA pools. The comparison can derive mapping peaks, identify deletions within the mapping peak, and suggest candidate genes within the deleted regions. We then used the public kernel-speci?c expression data to narrow down the list of candidate genes/mutations and identi?ed deletions ranging from several kb to more than 1 Mb. A full deletion allele of the Opaque-2 gene was identi?ed in mutant 531, which occurs within a $200-kb deletion. Opaque mutant 1486 has a 6248-bp deletion in the mapping interval containing two candidate genes encoding RNA-directed DNA methylation 4(RdDM4) and AMP-binding protein, respectively. This study demonstrates the ef?-ciency and cost-effectiveness of BSEx-seq for causal mutation mapping and candidate gene selection,providing a new option in mapping-by-sequencing for maize functional genomics studies.
