applying next-generation sequencing to enable marker-assisted...

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

Long-term impact aims

Establish molecular breeding capability for adapting Phaseolus bean to the UK climate

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

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

Haricot bean is not currently grown in the UK

Crop ideotype

Disease resistance (bacterial, viral, fungal)

Crop ideotype

Disease resistance (bacterial, viral, fungal)

Cold tolerance

Crop ideotype

Disease resistance (bacterial, viral, fungal)

Cold tolerance

Early maturity

Crop ideotype

Disease resistance (bacterial, viral, fungal)

Cold tolerance

Early maturity

Root architecture

Crop ideotype

Disease resistance (bacterial, viral, fungal)

Cold tolerance

Early maturity

Root architecture

Nutrient acquisition efficiency

Crop ideotype

Disease resistance (bacterial, viral, fungal)

Cold tolerance

Early maturity

Root architecture

Nutrient acquisition efficiency

Plant architecture and growth habit

Crop ideotype

Disease resistance (bacterial, viral, fungal)

Cold tolerance

Early maturity

Root architecture

Nutrient acquisition efficiency

Plant architecture and growth habit

Human nutritional qualities

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

F6 and F7 recombinant inbred populations

National Vegetable Research Station cultivar

Multiple-disease-resistant

Haricot characteristics

(Conway et al., 1982)

“Edmund”

F6 and F7 recombinant inbred populations

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

Experimental approach Pathology & physiology

Identify and characterise contrasting adaptive-trait phenotypes in the parental lines

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

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

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

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

Variant-calling pipeline

(Bolser, 2014)

Candidate-gene SNP and INDEL markers

Fragment analysis of INDELs CAPS assay of SNPs

Genotyping-by-sequencing (GBS)

Reduced genome representation

Genotyping-by-sequencing (GBS)

Reduced genome representation

Genome-wide simultaneous SNP/INDEL discovery and genotyping

Genotyping-by-sequencing (GBS)

Reduced genome representation

Genome-wide simultaneous SNP/INDEL discovery and genotyping

Reference-anchored or pairwise alignment of reads (tags)

The plant immune system

(Dangl et al., 2013)

Halo blight Caused by the Gram-negative bacterium Pseudomonas syringae pv. phaseolicola (Psph)

Halo blight Caused by the Gram-negative bacterium Pseudomonas syringae pv. phaseolicola (Psph)

Type III secretion system

Halo blight Caused by the Gram-negative bacterium Pseudomonas syringae pv. phaseolicola (Psph)

Type III secretion system

Seed-borne

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

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

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

Causes yield losses of up to 45% (Singh and Shwartz, 2010)

Halo blight

(CABI, 2012)

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)

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)

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

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?

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?

Race 6 resistance maps to one major-effect locus

P < 0.00001

Race 1 resistance maps to the same locus

P < 0.00001

Pse-3 maps to the I gene (BCMV) locus P < 0.00001

P > 0.1

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)

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

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

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

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

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