applying next-generation sequencing to enable marker-assisted...
TRANSCRIPT
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Applying next-generation sequencing to enable marker-assisted breeding for adaptive traits in a home-
grown haricot bean (Phaseolus vulgaris L.)
Andrew Tock Prof Eric Holub & Dr Guy Barker
University of Warwick, UK
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Long-term impact aims
Establish molecular breeding capability for adapting Phaseolus bean to the UK climate
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Long-term impact aims
Establish molecular breeding capability for adapting Phaseolus bean to the UK climate Provide UK farmers with a novel, short-season legume break crop that would promote soil renewal and aid black-grass control
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Long-term impact aims
Establish molecular breeding capability for adapting Phaseolus bean to the UK climate Provide UK farmers with a novel, short-season legume break crop that would promote soil renewal and aid black-grass control
Establish a food production and supply chain for haricot beans in the UK, providing consumers with a nutritious source of vegetable protein
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Haricot bean is not currently grown in the UK
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Crop ideotype
Disease resistance (bacterial, viral, fungal)
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Crop ideotype
Disease resistance (bacterial, viral, fungal)
Cold tolerance
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Crop ideotype
Disease resistance (bacterial, viral, fungal)
Cold tolerance
Early maturity
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Crop ideotype
Disease resistance (bacterial, viral, fungal)
Cold tolerance
Early maturity
Root architecture
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Crop ideotype
Disease resistance (bacterial, viral, fungal)
Cold tolerance
Early maturity
Root architecture
Nutrient acquisition efficiency
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Crop ideotype
Disease resistance (bacterial, viral, fungal)
Cold tolerance
Early maturity
Root architecture
Nutrient acquisition efficiency
Plant architecture and growth habit
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Crop ideotype
Disease resistance (bacterial, viral, fungal)
Cold tolerance
Early maturity
Root architecture
Nutrient acquisition efficiency
Plant architecture and growth habit
Human nutritional qualities
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Crop ideotype
Disease resistance (bacterial, viral, fungal)
Cold tolerance
Early maturity
Root architecture
Nutrient acquisition efficiency
Plant architecture and growth habit
Human nutritional qualities
Seed colour, size and shape
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F6 and F7 recombinant inbred populations
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National Vegetable Research Station cultivar
Multiple-disease-resistant
Haricot characteristics
(Conway et al., 1982)
“Edmund”
F6 and F7 recombinant inbred populations
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National Vegetable Research Station cultivar
Multiple-disease-resistant
Haricot characteristics
(Conway et al., 1982)
Early maturing
Cold-tolerant
Drought-tolerant
(Dodd and Taylor, 1991, unpublished data)
“Edmund” “SOA-BN”
F6 and F7 recombinant inbred populations
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Experimental approach Pathology & physiology
Identify and characterise contrasting adaptive-trait phenotypes in the parental lines
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Experimental approach Pathology & physiology
Identify and characterise contrasting adaptive-trait phenotypes in the parental lines Derive models of inheritance by characterising and interpreting phenotypic variation within RIL populations:
• in response to infection with economically important diseases; and
• with regard to physiological resilience traits
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Experimental approach Pathology & physiology
Identify and characterise contrasting adaptive-trait phenotypes in the parental lines Derive models of inheritance by characterising and interpreting phenotypic variation within RIL populations:
• in response to infection with economically important diseases; and
• with regard to physiological resilience traits
Computational, molecular & statistical genetics Identify sequence variations between parental lines and develop polymorphic markers using RNA-seq and genotyping-by-sequencing (GBS) data
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Experimental approach Pathology & physiology
Identify and characterise contrasting adaptive-trait phenotypes in the parental lines Derive models of inheritance by characterising and interpreting phenotypic variation within RIL populations:
• in response to infection with economically important diseases; and
• with regard to physiological resilience traits
Computational, molecular & statistical genetics Identify sequence variations between parental lines and develop polymorphic markers using RNA-seq and genotyping-by-sequencing (GBS) data Derive a high-resolution genetic map for a bi-parental RIL population
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Experimental approach Pathology & physiology
Identify and characterise contrasting adaptive-trait phenotypes in the parental lines Derive models of inheritance by characterising and interpreting phenotypic variation within RIL populations:
• in response to infection with economically important diseases; and
• with regard to physiological resilience traits
Computational, molecular & statistical genetics Identify sequence variations between parental lines and develop polymorphic markers using RNA-seq and genotyping-by-sequencing (GBS) data Derive a high-resolution genetic map for a bi-parental RIL population Define a mapping interval for potentially durable race-nonspecific halo blight resistance
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Variant-calling pipeline
(Bolser, 2014)
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Candidate-gene SNP and INDEL markers
Fragment analysis of INDELs CAPS assay of SNPs
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Genotyping-by-sequencing (GBS)
Reduced genome representation
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Genotyping-by-sequencing (GBS)
Reduced genome representation
Genome-wide simultaneous SNP/INDEL discovery and genotyping
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Genotyping-by-sequencing (GBS)
Reduced genome representation
Genome-wide simultaneous SNP/INDEL discovery and genotyping
Reference-anchored or pairwise alignment of reads (tags)
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The plant immune system
(Dangl et al., 2013)
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Halo blight Caused by the Gram-negative bacterium Pseudomonas syringae pv. phaseolicola (Psph)
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Halo blight Caused by the Gram-negative bacterium Pseudomonas syringae pv. phaseolicola (Psph)
Type III secretion system
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Halo blight Caused by the Gram-negative bacterium Pseudomonas syringae pv. phaseolicola (Psph)
Type III secretion system
Seed-borne
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Halo blight Caused by the Gram-negative bacterium Pseudomonas syringae pv. phaseolicola (Psph)
Type III secretion system
Seed-borne
Spread by inoculum-splash and wind during rainfall
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Halo blight Caused by the Gram-negative bacterium Pseudomonas syringae pv. phaseolicola (Psph)
Type III secretion system
Seed-borne
Spread by inoculum-splash and wind during rainfall
Bacteria enter through leaf stomata and grow in intercellular spaces
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Halo blight Caused by the Gram-negative bacterium Pseudomonas syringae pv. phaseolicola (Psph)
Type III secretion system
Seed-borne
Spread by inoculum-splash and wind during rainfall
Bacteria enter through leaf stomata and grow in intercellular spaces
Cultivated and weedy alternative hosts include most Phaseoleae tribe members
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Causes yield losses of up to 45% (Singh and Shwartz, 2010)
Halo blight
(CABI, 2012)
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Causes yield losses of up to 45% (Singh and Shwartz, 2010)
Annual losses of 181.3 thousand tonnes in sub-Saharan Africa (Wortmann et al., 1998)
Halo blight
(CABI, 2012)
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Causes yield losses of up to 45% (Singh and Shwartz, 2010)
Annual losses of 181.3 thousand tonnes in sub-Saharan Africa (Wortmann et al., 1998)
Disease prevention Genetic resistance conferred by regionally appropriate R genes
Halo blight
(CABI, 2012)
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Table 1. Host reactions of differential cultivars and type accessions when inoculated with isolates of the nine identified races of Pseudomonas syringae pv. phaseolicola, with putative resistance (R) genes indicated
Adapted from Teverson (1991: 60) and Taylor et al. (1996a,b: 474, 482), as modified by Miklas et al. (2011: 2440). +, apparent susceptible (compatible) reaction; –, apparent resistant (incompatible) reaction; –a, apparent resistant reaction with severe hypersensitive response.
Psph race 6 was undetected by known R-genes
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Error bars: standard deviation of infection scores recorded following separate inoculations with Psph race 6 isolate 716B (2–4 replicates) and race 1 isolate 725A (2–4 replicates).
F6 S×E and F7 E×S RILs; a quantitative trait?
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Error bars: standard deviation of infection scores recorded following separate inoculations with Psph race 6 isolate 716B (1 replicate) and race 1 isolate 725A (1 replicate).
F7 and F13 J. D. Taylor lines; a quantitative trait?
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Race 6 resistance maps to one major-effect locus
P < 0.00001
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Race 1 resistance maps to the same locus
P < 0.00001
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Pse-3 maps to the I gene (BCMV) locus P < 0.00001
P > 0.1
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LG 1 LG 2 LG 5 LG 6 LG 7
| fin (growth habit) | P
(potentiates pigment in seed coat, flower & hypocotyl, & pod speckling)
| V (black / violet seed coat, hypocotyl colour)
| Growth habit
| Pse-3 (HB) & I (BCMV)
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Preliminary conclusions and further work Potentially durable race-nonspecific resistance to halo blight is governed by one major-effect locus within a ~600-kbp mapping interval
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Preliminary conclusions and further work Potentially durable race-nonspecific resistance to halo blight is governed by one major-effect locus within a ~600-kbp mapping interval
Key recombinants to be genotyped at parental polymorphisms within noncoding regions to reduce interval size
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Preliminary conclusions and further work Potentially durable race-nonspecific resistance to halo blight is governed by one major-effect locus within a ~600-kbp mapping interval
Key recombinants to be genotyped at parental polymorphisms within noncoding regions to reduce interval size
Local Resistance gene enrichment and Sequencing (RenSeq) to be pursued to reveal parental polymorphisms with regard to nucleotide-binding site–leucine-rich repeat (NBS–LRR) genes located within the mapping interval
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Preliminary conclusions and further work Potentially durable race-nonspecific resistance to halo blight is governed by one major-effect locus within a ~600-kbp mapping interval
Key recombinants to be genotyped at parental polymorphisms within noncoding regions to reduce interval size
Local Resistance gene enrichment and Sequencing (RenSeq) to be pursued to reveal parental polymorphisms with regard to nucleotide-binding site–leucine-rich repeat (NBS–LRR) genes located within the mapping interval
Genome-wide association genetics to identify type-III-secreted virulence effectors conserved amongst all races of the pathovar, as candidate targets for race-nonspecific resistance
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Acknowledgements
Eric Holub Guy Barker Joana Vicente John Taylor Siva Samavedam Peter Walley Laura Baxter Sajjad Awan Vegetable Research Trust Medical and Life Sciences Research Fund