A highly mutagenised barley (cv. Golden Promise) TILLING population coupled with strategies for screening-by-sequencing

Miriam Schreiber, Abdellah Barakate, Nicola Uzrek, Malcolm Macaulay, Adeline Sourdille, Jenny Morris, Pete E. Hedley, Luke Ramsay, Robbie Waugh (Lead / Corresponding author)

Research output: Contribution to journalArticle

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Abstract

Background: We developed and characterised a highly mutagenised TILLING population of the barley (Hordeum vulgare) cultivar Golden Promise. Golden Promise is the 'reference' genotype for barley transformation and a primary objective of using this cultivar was to be able to genetically complement observed mutations directly in order to prove gene function. Importantly, a reference genome assembly of Golden Promise has also recently been developed. As our primary interest was to identify mutations in genes involved in meiosis and recombination, to characterise the population we focused on a set of 46 genes from the literature that are possible meiosis gene candidates.

Results: Sequencing 20 plants from the population using whole exome capture revealed that the mutation density in this population is high (one mutation every 154 kb), and consequently even in this small number of plants we identified several interesting mutations. We also recorded some issues with seed availability and germination. We subsequently designed and applied a simple two-dimensional pooling strategy to identify mutations in varying numbers of specific target genes by Illumina short read pooled-amplicon sequencing and subsequent deconvolution. In parallel we assembled a collection of semi-sterile mutants from the population and used a custom exome capture array targeting the 46 candidate meiotic genes to identify potentially causal mutations.

Conclusions: We developed a highly mutagenised barley TILLING population in the transformation competent cultivar Golden Promise. We used novel and cost-efficient screening approaches to successfully identify a broad range of potentially deleterious variants that were subsequently validated by Sanger sequencing. These resources combined with a high-quality genome reference sequence opens new possibilities for efficient functional gene validation.

Original languageEnglish
Article number99
Pages (from-to)1-14
Number of pages14
JournalPlant Methods
Volume15
DOIs
Publication statusPublished - 24 Aug 2019

Fingerprint

Hordeum
barley
screening
mutation
Mutation
Population
Genes
genes
Exome
Meiosis
meiosis
cultivars
Genome
genome assembly
Germination
Population Density
Hordeum vulgare
Genetic Recombination
Seeds
complement

Keywords

  • Amplicon sequencing
  • Barley
  • Exome capture
  • Functional genomics
  • Meiosis
  • Recombination

Cite this

Schreiber, Miriam ; Barakate, Abdellah ; Uzrek, Nicola ; Macaulay, Malcolm ; Sourdille, Adeline ; Morris, Jenny ; Hedley, Pete E. ; Ramsay, Luke ; Waugh, Robbie. / A highly mutagenised barley (cv. Golden Promise) TILLING population coupled with strategies for screening-by-sequencing. In: Plant Methods. 2019 ; Vol. 15. pp. 1-14.
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abstract = "Background: We developed and characterised a highly mutagenised TILLING population of the barley (Hordeum vulgare) cultivar Golden Promise. Golden Promise is the 'reference' genotype for barley transformation and a primary objective of using this cultivar was to be able to genetically complement observed mutations directly in order to prove gene function. Importantly, a reference genome assembly of Golden Promise has also recently been developed. As our primary interest was to identify mutations in genes involved in meiosis and recombination, to characterise the population we focused on a set of 46 genes from the literature that are possible meiosis gene candidates.Results: Sequencing 20 plants from the population using whole exome capture revealed that the mutation density in this population is high (one mutation every 154 kb), and consequently even in this small number of plants we identified several interesting mutations. We also recorded some issues with seed availability and germination. We subsequently designed and applied a simple two-dimensional pooling strategy to identify mutations in varying numbers of specific target genes by Illumina short read pooled-amplicon sequencing and subsequent deconvolution. In parallel we assembled a collection of semi-sterile mutants from the population and used a custom exome capture array targeting the 46 candidate meiotic genes to identify potentially causal mutations.Conclusions: We developed a highly mutagenised barley TILLING population in the transformation competent cultivar Golden Promise. We used novel and cost-efficient screening approaches to successfully identify a broad range of potentially deleterious variants that were subsequently validated by Sanger sequencing. These resources combined with a high-quality genome reference sequence opens new possibilities for efficient functional gene validation.",
keywords = "Amplicon sequencing, Barley, Exome capture, Functional genomics, Meiosis, Recombination",
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A highly mutagenised barley (cv. Golden Promise) TILLING population coupled with strategies for screening-by-sequencing. / Schreiber, Miriam; Barakate, Abdellah; Uzrek, Nicola; Macaulay, Malcolm; Sourdille, Adeline; Morris, Jenny; Hedley, Pete E.; Ramsay, Luke; Waugh, Robbie (Lead / Corresponding author).

In: Plant Methods, Vol. 15, 99, 24.08.2019, p. 1-14.

Research output: Contribution to journalArticle

TY - JOUR

T1 - A highly mutagenised barley (cv. Golden Promise) TILLING population coupled with strategies for screening-by-sequencing

AU - Schreiber, Miriam

AU - Barakate, Abdellah

AU - Uzrek, Nicola

AU - Macaulay, Malcolm

AU - Sourdille, Adeline

AU - Morris, Jenny

AU - Hedley, Pete E.

AU - Ramsay, Luke

AU - Waugh, Robbie

N1 - The research leading to these results was funded by the H2020 European Research Council (ERC Shuffle, Project ID: 66918) to RW. We acknowledge the Scottish Government RESAS Strategic research program for supporting this research.

PY - 2019/8/24

Y1 - 2019/8/24

N2 - Background: We developed and characterised a highly mutagenised TILLING population of the barley (Hordeum vulgare) cultivar Golden Promise. Golden Promise is the 'reference' genotype for barley transformation and a primary objective of using this cultivar was to be able to genetically complement observed mutations directly in order to prove gene function. Importantly, a reference genome assembly of Golden Promise has also recently been developed. As our primary interest was to identify mutations in genes involved in meiosis and recombination, to characterise the population we focused on a set of 46 genes from the literature that are possible meiosis gene candidates.Results: Sequencing 20 plants from the population using whole exome capture revealed that the mutation density in this population is high (one mutation every 154 kb), and consequently even in this small number of plants we identified several interesting mutations. We also recorded some issues with seed availability and germination. We subsequently designed and applied a simple two-dimensional pooling strategy to identify mutations in varying numbers of specific target genes by Illumina short read pooled-amplicon sequencing and subsequent deconvolution. In parallel we assembled a collection of semi-sterile mutants from the population and used a custom exome capture array targeting the 46 candidate meiotic genes to identify potentially causal mutations.Conclusions: We developed a highly mutagenised barley TILLING population in the transformation competent cultivar Golden Promise. We used novel and cost-efficient screening approaches to successfully identify a broad range of potentially deleterious variants that were subsequently validated by Sanger sequencing. These resources combined with a high-quality genome reference sequence opens new possibilities for efficient functional gene validation.

AB - Background: We developed and characterised a highly mutagenised TILLING population of the barley (Hordeum vulgare) cultivar Golden Promise. Golden Promise is the 'reference' genotype for barley transformation and a primary objective of using this cultivar was to be able to genetically complement observed mutations directly in order to prove gene function. Importantly, a reference genome assembly of Golden Promise has also recently been developed. As our primary interest was to identify mutations in genes involved in meiosis and recombination, to characterise the population we focused on a set of 46 genes from the literature that are possible meiosis gene candidates.Results: Sequencing 20 plants from the population using whole exome capture revealed that the mutation density in this population is high (one mutation every 154 kb), and consequently even in this small number of plants we identified several interesting mutations. We also recorded some issues with seed availability and germination. We subsequently designed and applied a simple two-dimensional pooling strategy to identify mutations in varying numbers of specific target genes by Illumina short read pooled-amplicon sequencing and subsequent deconvolution. In parallel we assembled a collection of semi-sterile mutants from the population and used a custom exome capture array targeting the 46 candidate meiotic genes to identify potentially causal mutations.Conclusions: We developed a highly mutagenised barley TILLING population in the transformation competent cultivar Golden Promise. We used novel and cost-efficient screening approaches to successfully identify a broad range of potentially deleterious variants that were subsequently validated by Sanger sequencing. These resources combined with a high-quality genome reference sequence opens new possibilities for efficient functional gene validation.

KW - Amplicon sequencing

KW - Barley

KW - Exome capture

KW - Functional genomics

KW - Meiosis

KW - Recombination

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U2 - 10.1186/s13007-019-0486-9

DO - 10.1186/s13007-019-0486-9

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SN - 1746-4811

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