Model SNP development for complex genomes based on hexaploid oat using high-throughput 454 sequencing technology

Rebekah E. Oliver; Gerard R. Lazo; Joseph D. Lutz; Marc J. Rubenfield; Nicholas A. Tinker; Joseph M. Anderson; Nicole H. Wisniewski Morehead; Dinesh Adhikary; Eric N. Jellen; P. Jeffrey Maughan; Gina L. Brown Guedira; Shiaoman Chao; Aaron D. Beattie; Martin L. Carson; Howard W. Rines; Donald E. Obert; J. Michael Bonman; Eric W. Jackson

doi:10.1186/1471-2164-12-77

Model SNP development for complex genomes based on hexaploid oat using high-throughput 454 sequencing technology

Rebekah E. Oliver, Gerard R. Lazo, Joseph D. Lutz, Marc J. Rubenfield, Nicholas A. Tinker, Joseph M. Anderson, Nicole H. Wisniewski Morehead, Dinesh Adhikary, Eric N. Jellen, P. Jeffrey Maughan, Gina L. Brown Guedira, Shiaoman Chao, Aaron D. Beattie, Martin L. Carson, Howard W. Rines, Donald E. Obert, J. Michael Bonman, Eric W. Jackson

Plant Pathology

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Abstract

Background: Genetic markers are pivotal to modern genomics research; however, discovery and genotyping of molecular markers in oat has been hindered by the size and complexity of the genome, and by a scarcity of sequence data. The purpose of this study was to generate oat expressed sequence tag (EST) information, develop a bioinformatics pipeline for SNP discovery, and establish a method for rapid, cost-effective, and straightforward genotyping of SNP markers in complex polyploid genomes such as oat.Results: Based on cDNA libraries of four cultivated oat genotypes, approximately 127,000 contigs were assembled from approximately one million Roche 454 sequence reads. Contigs were filtered through a novel bioinformatics pipeline to eliminate ambiguous polymorphism caused by subgenome homology, and 96 in silico SNPs were selected from 9,448 candidate loci for validation using high-resolution melting (HRM) analysis. Of these, 52 (54%) were polymorphic between parents of the Ogle1040 × TAM O-301 (OT) mapping population, with 48 segregating as single Mendelian loci, and 44 being placed on the existing OT linkage map. Ogle and TAM amplicons from 12 primers were sequenced for SNP validation, revealing complex polymorphism in seven amplicons but general sequence conservation within SNP loci. Whole-amplicon interrogation with HRM revealed insertions, deletions, and heterozygotes in secondary oat germplasm pools, generating multiple alleles at some primer targets. To validate marker utility, 36 SNP assays were used to evaluate the genetic diversity of 34 diverse oat genotypes. Dendrogram clusters corresponded generally to known genome composition and genetic ancestry.Conclusions: The high-throughput SNP discovery pipeline presented here is a rapid and effective method for identification of polymorphic SNP alleles in the oat genome. The current-generation HRM system is a simple and highly-informative platform for SNP genotyping. These techniques provide a model for SNP discovery and genotyping in other species with complex and poorly-characterized genomes.

Original language	English (US)
Article number	77
Journal	BMC Genomics
Volume	12
DOIs	https://doi.org/10.1186/1471-2164-12-77
State	Published - Jan 27 2011

Bibliographical note

Funding Information:
The authors thank Irene Shackelford and Robert Campbell for excellent technical assistance. This project, in part, was funded by the USDA National Institute of Food and Agriculture, the USDA ARS National Program 301, and General Mills, Inc. The authors dedicate this work to the memory of Dr. Deon Stuthman. His contributions to this work and oat science in general played a pivotal role in the achievements of this project.

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Oliver, R. E., Lazo, G. R., Lutz, J. D., Rubenfield, M. J., Tinker, N. A., Anderson, J. M., Wisniewski Morehead, N. H., Adhikary, D., Jellen, E. N., Maughan, P. J., Brown Guedira, G. L., Chao, S., Beattie, A. D., Carson, M. L., Rines, H. W., Obert, D. E., Bonman, J. M., & Jackson, E. W. (2011). Model SNP development for complex genomes based on hexaploid oat using high-throughput 454 sequencing technology. BMC Genomics, 12, Article 77. https://doi.org/10.1186/1471-2164-12-77

Oliver, RE, Lazo, GR, Lutz, JD, Rubenfield, MJ, Tinker, NA, Anderson, JM, Wisniewski Morehead, NH, Adhikary, D, Jellen, EN, Maughan, PJ, Brown Guedira, GL, Chao, S, Beattie, AD, Carson, ML, Rines, HW, Obert, DE, Bonman, JM & Jackson, EW 2011, 'Model SNP development for complex genomes based on hexaploid oat using high-throughput 454 sequencing technology', BMC Genomics, vol. 12, 77. https://doi.org/10.1186/1471-2164-12-77

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abstract = "Background: Genetic markers are pivotal to modern genomics research; however, discovery and genotyping of molecular markers in oat has been hindered by the size and complexity of the genome, and by a scarcity of sequence data. The purpose of this study was to generate oat expressed sequence tag (EST) information, develop a bioinformatics pipeline for SNP discovery, and establish a method for rapid, cost-effective, and straightforward genotyping of SNP markers in complex polyploid genomes such as oat.Results: Based on cDNA libraries of four cultivated oat genotypes, approximately 127,000 contigs were assembled from approximately one million Roche 454 sequence reads. Contigs were filtered through a novel bioinformatics pipeline to eliminate ambiguous polymorphism caused by subgenome homology, and 96 in silico SNPs were selected from 9,448 candidate loci for validation using high-resolution melting (HRM) analysis. Of these, 52 (54%) were polymorphic between parents of the Ogle1040 × TAM O-301 (OT) mapping population, with 48 segregating as single Mendelian loci, and 44 being placed on the existing OT linkage map. Ogle and TAM amplicons from 12 primers were sequenced for SNP validation, revealing complex polymorphism in seven amplicons but general sequence conservation within SNP loci. Whole-amplicon interrogation with HRM revealed insertions, deletions, and heterozygotes in secondary oat germplasm pools, generating multiple alleles at some primer targets. To validate marker utility, 36 SNP assays were used to evaluate the genetic diversity of 34 diverse oat genotypes. Dendrogram clusters corresponded generally to known genome composition and genetic ancestry.Conclusions: The high-throughput SNP discovery pipeline presented here is a rapid and effective method for identification of polymorphic SNP alleles in the oat genome. The current-generation HRM system is a simple and highly-informative platform for SNP genotyping. These techniques provide a model for SNP discovery and genotyping in other species with complex and poorly-characterized genomes.",

author = "Oliver, {Rebekah E.} and Lazo, {Gerard R.} and Lutz, {Joseph D.} and Rubenfield, {Marc J.} and Tinker, {Nicholas A.} and Anderson, {Joseph M.} and {Wisniewski Morehead}, {Nicole H.} and Dinesh Adhikary and Jellen, {Eric N.} and Maughan, {P. Jeffrey} and {Brown Guedira}, {Gina L.} and Shiaoman Chao and Beattie, {Aaron D.} and Carson, {Martin L.} and Rines, {Howard W.} and Obert, {Donald E.} and Bonman, {J. Michael} and Jackson, {Eric W.}",

note = "Funding Information: The authors thank Irene Shackelford and Robert Campbell for excellent technical assistance. This project, in part, was funded by the USDA National Institute of Food and Agriculture, the USDA ARS National Program 301, and General Mills, Inc. The authors dedicate this work to the memory of Dr. Deon Stuthman. His contributions to this work and oat science in general played a pivotal role in the achievements of this project.",

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T1 - Model SNP development for complex genomes based on hexaploid oat using high-throughput 454 sequencing technology

AU - Oliver, Rebekah E.

AU - Lazo, Gerard R.

AU - Lutz, Joseph D.

AU - Rubenfield, Marc J.

AU - Tinker, Nicholas A.

AU - Anderson, Joseph M.

AU - Wisniewski Morehead, Nicole H.

AU - Adhikary, Dinesh

AU - Jellen, Eric N.

AU - Maughan, P. Jeffrey

AU - Brown Guedira, Gina L.

AU - Chao, Shiaoman

AU - Beattie, Aaron D.

AU - Carson, Martin L.

AU - Rines, Howard W.

AU - Obert, Donald E.

AU - Bonman, J. Michael

AU - Jackson, Eric W.

N1 - Funding Information: The authors thank Irene Shackelford and Robert Campbell for excellent technical assistance. This project, in part, was funded by the USDA National Institute of Food and Agriculture, the USDA ARS National Program 301, and General Mills, Inc. The authors dedicate this work to the memory of Dr. Deon Stuthman. His contributions to this work and oat science in general played a pivotal role in the achievements of this project.

PY - 2011/1/27

Y1 - 2011/1/27

N2 - Background: Genetic markers are pivotal to modern genomics research; however, discovery and genotyping of molecular markers in oat has been hindered by the size and complexity of the genome, and by a scarcity of sequence data. The purpose of this study was to generate oat expressed sequence tag (EST) information, develop a bioinformatics pipeline for SNP discovery, and establish a method for rapid, cost-effective, and straightforward genotyping of SNP markers in complex polyploid genomes such as oat.Results: Based on cDNA libraries of four cultivated oat genotypes, approximately 127,000 contigs were assembled from approximately one million Roche 454 sequence reads. Contigs were filtered through a novel bioinformatics pipeline to eliminate ambiguous polymorphism caused by subgenome homology, and 96 in silico SNPs were selected from 9,448 candidate loci for validation using high-resolution melting (HRM) analysis. Of these, 52 (54%) were polymorphic between parents of the Ogle1040 × TAM O-301 (OT) mapping population, with 48 segregating as single Mendelian loci, and 44 being placed on the existing OT linkage map. Ogle and TAM amplicons from 12 primers were sequenced for SNP validation, revealing complex polymorphism in seven amplicons but general sequence conservation within SNP loci. Whole-amplicon interrogation with HRM revealed insertions, deletions, and heterozygotes in secondary oat germplasm pools, generating multiple alleles at some primer targets. To validate marker utility, 36 SNP assays were used to evaluate the genetic diversity of 34 diverse oat genotypes. Dendrogram clusters corresponded generally to known genome composition and genetic ancestry.Conclusions: The high-throughput SNP discovery pipeline presented here is a rapid and effective method for identification of polymorphic SNP alleles in the oat genome. The current-generation HRM system is a simple and highly-informative platform for SNP genotyping. These techniques provide a model for SNP discovery and genotyping in other species with complex and poorly-characterized genomes.

AB - Background: Genetic markers are pivotal to modern genomics research; however, discovery and genotyping of molecular markers in oat has been hindered by the size and complexity of the genome, and by a scarcity of sequence data. The purpose of this study was to generate oat expressed sequence tag (EST) information, develop a bioinformatics pipeline for SNP discovery, and establish a method for rapid, cost-effective, and straightforward genotyping of SNP markers in complex polyploid genomes such as oat.Results: Based on cDNA libraries of four cultivated oat genotypes, approximately 127,000 contigs were assembled from approximately one million Roche 454 sequence reads. Contigs were filtered through a novel bioinformatics pipeline to eliminate ambiguous polymorphism caused by subgenome homology, and 96 in silico SNPs were selected from 9,448 candidate loci for validation using high-resolution melting (HRM) analysis. Of these, 52 (54%) were polymorphic between parents of the Ogle1040 × TAM O-301 (OT) mapping population, with 48 segregating as single Mendelian loci, and 44 being placed on the existing OT linkage map. Ogle and TAM amplicons from 12 primers were sequenced for SNP validation, revealing complex polymorphism in seven amplicons but general sequence conservation within SNP loci. Whole-amplicon interrogation with HRM revealed insertions, deletions, and heterozygotes in secondary oat germplasm pools, generating multiple alleles at some primer targets. To validate marker utility, 36 SNP assays were used to evaluate the genetic diversity of 34 diverse oat genotypes. Dendrogram clusters corresponded generally to known genome composition and genetic ancestry.Conclusions: The high-throughput SNP discovery pipeline presented here is a rapid and effective method for identification of polymorphic SNP alleles in the oat genome. The current-generation HRM system is a simple and highly-informative platform for SNP genotyping. These techniques provide a model for SNP discovery and genotyping in other species with complex and poorly-characterized genomes.

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Model SNP development for complex genomes based on hexaploid oat using high-throughput 454 sequencing technology

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