An Integrated Approach To Stabilising HFN In Wheat ... · How the HFN Project Emerged • Breeder...

WGIN Stakeholders Meeting 22/11/05

An Integrated Approach To Stabilising HFN In Wheat: Screens, Genes And Understanding

= HFN LINK

Peter Jack, RAGT


HFN LINK Presentation

• Project overview & Industry Role – Peter Jack (RAGT)• WP1 – Analysis of Dormancy – Mike Holdsworth (University

of Nottingham)• WP2 – Pre-maturity amylase (PMA) smart screen – Peter

Kettlewell (Harper Adams)• WP3 – PMA molecular characterisation – Andy Phillips

(Rothamsted)• WP4 – Candidate Genes - Mike Holdsworth – (University of

Nottingham)• WP5 – QTL identification & relation to candidate genes –

John Flintham (John Innes)• Questions – please hold till end


The HFN Problem – Value/Cost• HFN (Hagberg Falling Number) is Industry standard for Starch

integrity.

• Starch degradation > loss of quality - bread with a dark crust and sticky crumb.

• HFN is key parameter in meeting Group1/2 specification. Failure:– Lost premium to grower in bad years > volatility of income– Wasted N input to meet protein specification– Importation to replace (Shipping energy cost; Balance of payments;

additional cost to Miller)– Exports reduced

• Preventative Costs which could be reduced– Grain drying – energy cost– Glyphosate to promote ripening– Quality control & blending costs by millers/end users


How the HFN Project Emerged• Breeder concern that Public projects not matching Industry needs

• Traits Meeting organised by MH/NP on 10/6/04 > Key Priorities

• HFN instability at top of list for breeders & very high for end users

• RAGT meeting October 2004– Breeders & Academics. Agree Problem; Brainstorm Options > Overall Plan

• Utilised WGIN gatherings to progress concept/submissions– Nov 04; Jan 05; May 05

• Breeders Meetings March & July 2005– Finalise field work & genotyping & Final Commitment to project.

• Project Submissions– DEFRA (8/3/05 & 15/6/05); BBSRC (15/4/05); HGCA (14/5/05)– Project Defense at DTI – 7/7/05 – project approved by DEFRA

• This project is strongly endorsed by the entire UK wheat breeding industry– Very substantial project involvement (materials, work and management time)


ParticipantsINDUSTRY

RAGT AdvantaCPB TwyfordSvalof Weibull NickersonElsoms(Totals >95% UK wheat)

Biogemma (Biotech)

HGCA (Growers)NABIM (Millers)CCFRA (Bakers)SWRI (Distillers)

PUBLIC SCIENCE

Rothamsted ResearchUniversity of NottinghamJohn Innes Centre (NIAB)Harper Adams

CASH FUNDING

BBSRCDEFRAHGCA

ADVISERS

Dr. John Lenton (ex LARS)Prof. Daryl Mares (U. Adelaide)


Project Components

Develop controlled conditions to reliably induce PHS & PMA

Apply to segregating populations if not triggered in field trials over 3 years

Use to test involvement of candidate genes & identification of additional ones (incl. TILLING, RNAi)

QTLs

Field trial: 6 Populations over 3 seasons on 2 sites

Genotype populations

Fully validated Marker System for Direct use in Breeding Programme

Validate Markers (ideally Gene-based) in Breeding lines

Position candidate genes:1. By chromosome arm2. Map those aligning with QTLs

Identify set candidate Genes

Complex project – Good Communication & Management is Critical


Key Features

• Success requires large multidisciplinary project– Impossible to achieve other than through Public-

Private Partnership such as LINK – Complementary skill set – Breeders & Public Sector

• Addresses Fundamental Science issues as well as major commercial problem.

• Focus on elite germplasm to ensure rapid exploitation

• Addresses key Environmental & Economic Sustainability Issues

• Industrial leadership & focus throughout


Industry Participation

• Breeders & Biotechnology Company– Provision & Genotyping of 4 new Populations– Phenotyping 6 populations x 3 years– Bioinformatics & Candidate Gene Characterisation– Marker Validation– Route to Market (Varietal Development)

– Project Management

• Dissemination of Information– HGCA, NABIM, CCFRA, SWRI


HFN LINKWork Package 1; Physiological analysis of

wheat seed dormancy and pre harvest

sprouting.

Mike HoldsworthJohn Foulkes

John Lenton

1 postdoc 4 yr(with WP4)1 technician 3.5 yr


WP 1 – University of Nottingham, Physiological analysis of seed

development

Mike Holdsworth; Molecular analysis of wheat development, physiology of PHS,genetics of germination.

John Foulkes; Physiological analysis of wheat Development

John Lenton; Physiological/molecular analysis of wheat development, the role ofphytohormones in seed development


WP1 Objectives & HypothesesHypotheses:1. That environmental conditions can be defined that allow

consistent induction of PHS in susceptible genotypes. 2. That the relative contribution of true embryo dormancy (and ABA

responsiveness) and surrounding maternal tissues to 'in ear' PHSin the field can be established.

3. That the defined environmental conditions used to develop the 'smart screen' can be validated against field-based PHS scores and used successfully for identification of QTLs for PHS susceptibility and resistance in key parents of mapping populations.

Objective: To develop a physiologically relevant ‘smart screen’ to Expose susceptibility to PHS


WP1 – Experimental ApproachEXPERIMENT 1: Initial analysis of dormancy levels in harvest ripe grain from

field-grown material. Output: Initial definition of range of phenotypes, initial development of assay protocols.

EXPERIMENT 2: Analysis of dormancy and PHS susceptibility of whole seeds and isolated ears at harvest ripeness Output: Further definition of phenotype range, and development of assay.

EXPERIMENT 3: Analysis of the effect of assay temperature & ABA-responsiveness on germination potential of whole grains. Output: Determination of temperature influence on germination potential and the relationship between embryo ABA responsiveness and germination potential, further development of assay.

EXPERIMENT 4: Analysis of dormancy and PHS susceptibility of whole seeds and isolated ears from beginning of net grain water loss to harvest ripeness: Output: Determination of characteristics of dormancy induction x genotype, further development of assay.

EXPERIMENT 5: Use of defined environmental conditions to analyse predictability of assay conditions and behaviour of specific wheat lines. Comparison with field assay (36-48 months). Output: Use of developed assay with test materials, definition of comparability with field assay results.


WP1 – Benefit/Linkage to Other WPs

WP1

WP3

WP2 WP4

WP5

PMA smart screen Candidate genes

Molecular characterisation of PMA

QTL Identification, validation & relation to candidate genes.

- screen conditions- screen conditions- materials for molecular

analyses

- Validation of smart screen- germplasm for molecular

marker analyses

- materials for molecularanalyses


WP1 – Timelines & Milestones


HFN LINKWork Package 2

PMA Smart Screen development

Peter Kettlewell & Aidan Farrell – Harper AdamsAndy Phillips – Rothamsted John Snape & John Flintham – JICPeter Jack (coordinating breeders)– RAGT Daryl Mares – Adelaide


WP 2 – Harper Adams

• Shown how agronomy and climate affects HFN and PMA

• Quantified contribution of PHS and PMA and other mechanisms to low HFN

• Explored environmental stimuli for PMA


WP2 Objectives & Hypotheses

• Objectives– To define a reproducible screen for PMA– To phenotype mapping populations for

PMA• Hypotheses– PMA can be induced by environmental

stimuli– PMA can be induced by increased

assimilate supply


WP2 – Experimental Approach

• Controlled environment experiments• Compare all published environmental

stimuli for PMA• Most involve transition from hot to cool• Degraining is a possible alternative• Validate with field material• Scale-up to phenotype mapping

populations



WP2 WP1

WP3 WP4 WP5

Parallel approach

Screenconditions

Improved understanding & techniques Phenotyping

Validation


Phenotyping

Validation

Assess field material

Fine-tuning screen

CE testing

3rd2nd1st4th3rd2nd1st4th3rd2nd1st4th

2008200720062005Task

WP2 – Timelines




Andy Phillips - GA biosynthesis in wheat

Alison Huttly - a-Amylase; Grain development

Peter Hedden - GA biosynthesis & quantification

Peter Shewry - Grain development & quality

Peter Kettlewell - PMA induction (WP2)

1 post-doc 3 yrs (RRes)


HFN LINKWP3: Background

PMA (Pre-maturity amylase):

is due to α-amylase accumulation in the grain during maturation, which affects grain quality (HFN).

α-Amylase production is not accompanied by germination and may be restricted to the cavity aleurone.

Inductive conditions are less clear, but may involve interaction between ambient temperature and water availability during grain filling & maturation (WP2).


HFN LINKWP3: Aims and Hypotheses

Aims:To characterize the PMA syndrome at a molecular level, including identification of the genes and mechanisms involved in their induction. This will inform strategies for improving PMA resistance in UK wheat varieties by targeting specific candidate genes.

Hypotheses:- That a single, common PMA syndrome exists in all susceptible varieties (British and Australian) under the different inductive conditions.

-That the PMA syndrome is confined to aleurone cells surrounding the endosperm cavity.

-That PMA thus represents an abnormal activation of cavity aleurone responses.

-That this involves gibberellin signalling, thus identifying potential candidate genes that determine susceptibility to PMA.


HFN LINKWP3: Experimental approach

1. In which cells are a-amylase genes expressed during PMA?

2. Transcript profiling of cavity aleurone cells during induction of PMA to identify pathways involved

3. Does a single, common syndrome exist in all susceptible varieties?

4. Assess the role of gibberellin hormones in the induction of PMA & inform selection of candidate genes


HFN LINKWP3: Links to other workpackages

WP3

WP2PMA smart screens

WP5Field screening & QTLs

WP4Candidate genes

Inductive conditions;Induced material

Plant materialCandidate genesfor mapping, allele discovery etc

Novel genetic material for assay

Novel screening protocols


HFN LINKWP3: Milestones

RRes/JIC/UofN/BiogemmaMap candidate PMA genesm3.7

Biogemma/RRes

Identify variation in PMA candidate genes using TILLING

m3.6

RResIdentify candidate genes by transcript profilingm3.5

RResHormone analysis of developing grainm3.4

RRes/HAUCMolecular characterisation of PMA: timing & location

m3.3

RResEstablishment of laser capture microdissectionm3.2

RResIntrogression of transgenes into characterised wheat lines

m3.1

3214321432143214

200920082007200605PartnerTask/Milestone

Year/Quarter


Mike HoldsworthSean May (NASC)

Andy PhillipsPeter Hedden

John FlinthamJohn Snape

Biogemma

1 postdoc 4 yr (with WP1)

HFN LINKWork Package 4; Identifying candidate genes for increased HFN stability in wheat using a

post-genomics comparative approach.


WP 4 – University of Nottingham/ Biogemma;

Identifying candidate genes

Andy PhillipsPeter Hedden;Rothamsted Research

John FlinthamJohn Snape;John Innes Centre

Molecular Genetic mechanisms regulatingGermination and PHS;Bioinformatics (through the EuropeanArabidposis Stock Centre)

Genetic mechanisms regulatingPHS and HFN

Hormonal mechanisms Controlling PMA/PHS

Mike HoldsworthSean May;University ofNottingham

Tina Barsby;Biogemma

Candidate genesTransgenic wheat


WP4 Objectives & HypothesesHypotheses:1. That information from previous molecular studies of model

systems can be used to define a set of candidate genes with possible roles in regulating PHS/PMA.

2. That the candidate gene list can be further refined using a variety of criteria related to functionality in wheat, to provide a core set.

3. That co-alignment between candidate gene polymorphisms and QTLs can be used to develop gene-based diagnostics for direct selection of elite germplasm with stable HFN.

Objective: To develop a set of developmentally informed relevantmolecular markers for PHS


WP4 – Experimental Approach• Three approaches will be used to define candidate genes:A. Definition through scrutiny of previous genetic analysis of model species B. Definition through comparative transcriptome analysis of germination C. Definition through scrutiny of previous molecular/biochemical studies

related to germination/seed dormancy



WP4

WP3

WP2 WP1

WP5

PMA smart screen PHS smart screen


QTL Identification, validation & relation to candidate genes.

- Candidate genesExpression patterns

- Definition of validity asMolecular markers

-Resources for molecular analyses

- Candidate genesExpression patterns


WP4 – Timelines & MilestonesYEAR:

Nottingham1 Define wheat orthologous sequences for model candidate genes (BBSRC) 2 Definition of genome position using synteny (BBSRC) 3 Further analysis of candidate gene expression patterns (BBSRC) 4 Analysis of relationships of candidate genes to defined QTLs for dormancy/PHS. Analysis of transgenic plants (BBSRC).

JIC1 Initiate candidate gene mapping on aneuploid/deletion stocks (BBSRC) 2 Further deletion and fine mapping of candidate genes (BBSRC). 3 BAC screening and sequencing of key candidate genes (BBSRC). 4 Completion of BAC screening and gene sequencing. Marker development and validation (BBSRC).

RRes123 Identify candidate genes through transcript profiling using microarrays (BBSRC). 4 Identify endogenous and novel variation in candidate genes through TILLING. Map candidate genes. (BBSRC).

Biogemma1 Bioinformatics, especially design homoeologous gene-specific assays. 2 Further characterisation of candidate genes and marker development. 3 Validation of candidate genes through map location and allelic distribution. Possible transgenic POC. 4 Further validation of candidate genes, including possible transgenic POC.



QTL Identification&

Relation to Candidate Genes

John FlinthamJohn SnapeJames Simmonds


WP 5 – Partners

• John Innes Centre - Crop Genetics DepartmentHFN research recordGenetic stocks, DNA markersQTL mapping expertiseStrong collaborations

• RAGT• Nickerson• CPB Twyford

• Elsoms• Advanta• Svalöf Weibull

• NIAB• Biogemma


WP5 Hypothesis & Objectives• Hypothesis:

Elite UK germplasm is segregating for genes (QTLs) controlling HFN that can be related to DNA markers and candidate genes.

• Objectives1) Genetic mapping of QTLs for PHS / PMA.2) Validation of QTLs in advanced breeding lines.3) Map alignments of QTLs with candidate genes.4) Deliver high-throughput, validated DNA markers for

genetically stable HFN.



• Core Germplasm• QTL Mapping Populations• Smart Screens• Genotyping• Comparative Mapping• Marker Validation



• Core Germplasm30 varieties sampling the range of genetic variation in modern UK winter wheats, plus PHS / PMA checks and key genetic models.

PHS and PMA trials at JIC (years 1-4)and at NIAB (year 1),replicate scores for HFN and visible sprouting.



• QTL Mapping Populations1) Core set of 6 populations (3 PHS, 3 PMA) in trials at JIC

and with breeders.Parents plus ~120 recombinant lines per population; > 50 population x year x location datasets.HFN on all, plus visible sprouting on weathered / irrigated plots (JIC / SW / Nickerson / Elsoms / CPB / Advanta).

2) Eight validation populations (JIC).



• Smart ScreensApplications of physiological screening protocols / controlled environments from WP1 & WP2 to selected mapping lines (JIC / HAAC / Nottingham).

• GenotypingConstruction of genetic linkage maps, based on DNA markers, for each experimental population (WGIN / JIC / RAGT / CPB / SW / Advanta).


WP5 – Experimental Approach• Comparative mapping

Candidate gene mapping – aligning QTL maps with hormone / dormancy loci from WP3 & WP4 – developing gene-specific DNA markers.

• Marker validationTesting DNA markers for QTL candidates in validation populations; association genetics in advanced breeders’lines.



• Provision of genetic stocks for field, glasshouse, controlled environment and laboratory experiments (WP1 – 4).

• WP1 & WP2: WP5 generates new genotypes for validation of smart screens, uses smart screens to extend QTL datasets.

• WP3 & WP4: WP5 identifies genomic regions for candidate gene mining.


WP5 – Timelines & Milestones• Years 1-4: QTL mapping of PHS & PMA.

• Year 1: Seed multiplication & distribution.

• Year 2: Deletion mapping of candidates.

• Year 3: Fine-scale gene mapping.

• Year 4: QTL / Candidate gene validation;

• Delivery of fully validated markers to breeders, for new QTL combinations in elite UK germplasm.


Questions for All Speakers

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