An Integrated Approach to Stabilising HFN in Wheat: Screens, Genes & Understanding

A four-year LINK programme to target pre-harvest sprouting and pre-maturity amylase in wheat

Andy PhillipsRothamsted Research

The Defra Wheat Genetic Improvement Network (WGIN)

Started July 2003

Aim: To Underpin Wheat Improvement by Plant Breeders

Approaches:1. Characterisation and provision of genetic resources2. Genetic mapping and marker development3. Trait identification eg. NUE, disease4. Identification and generation of novel variation in key traits

using non-GM approaches (mutagenesis and TILLING)6. Liaison and communication

WGIN Traits MeetingHeld June 10th, 2004, RRes

Breeders’ target traits (in order of priority):

Hagberg Falling NumberSeptoria resistance

Second wheat syndromeOrange blossom midge

Lodging resistanceBarley Yellow Dwarf Virus

Other insect pestsNitrogen use efficiency

HFN AND BREADMAKING QUALITY

HFN variation by cultivar and year

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An Integrated Approach to Stabilising HFN in Wheat: Screens, Genes & Understanding

PHS (Pre-harvest sprouting)

- is the result of premature germination of grain in the ear, promoted in susceptible varieties by warm weather followed by rain in the period between maturity and harvest.

- Germination results in induction of α-amylase expression in the scutellum and peripheral aleurone layer, leading to loss of breadmaking quality.

An Integrated Approach to Stabilising HFN in Wheat: Screens, Genes & Understanding

PMA (Pre-maturity amylase):

­ is due to α-amylase accumulation in the grain during maturation.

­ expressed in the distal (non-embryo) part of the grain.

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

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

An Integrated Approach to Stabilising HFN in Wheat: Screens, Genes & Understanding

Partners:

RAGTAdvantaCPB TwyfordsSvalof WeibullNickersonsBiogemmaElsomsNABIMCCFRASWRI

Rothamsted ResearchUniversity of NottinghamJICHarper Adams University CollegeNIAB

Funding

BBSRCDEFRAHGCA

Coordinators: Peter Jack (RAGT), Andy Phillips (RRes)

HFN Workpackages

1. Physiological analysis of wheat seed dormancy and pre harvest sprouting.

2. PMA Smart Screen development

3. Molecular characterisation of PMA

4. Identifying candidate genes using a post-genomics comparative approach.

5. Field- and smart screen-based QTL identification and correlation with candidate genes.

HFN LINKWork Package 1: Physiological analysis of wheat

seed dormancy and pre harvest sprouting.

Mike HoldsworthJohn FoulkesTanja GerjetsDuncan SchofieldJohn Lenton

Aim: To develop a physiologically relevant ‘smart screen’ to expose susceptibility to PHS

WP1 Objectives & Hypotheses

Hypotheses:

1. That controlled environmental conditions can be defined that allow consistent induction of PHS in susceptible genotypes.

2. 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.

Germination assays for analysing grain dormancy levels

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WP1: Physiology of PHS

HFN LINKWork Package 2

PMA Smart Screen development

Peter Kettlewell & Aidan Farrell – Harper AdamsAndy Phillips & Peter Hedden – Rothamsted Peter Jack (coordinating breeders) – RAGT Daryl Mares – U. Adelaide

Objective: To develop smart screens for PMA under controlled conditions that reflect the phenomenon observed in the field

PMA Smart Screen developmentDaryl Mares (Adelaide)

Plants in potsat 18C (night) 28C (day)

25 dpa: Cool shock7 days at 12/18C

Mature at 18/25C

HFN LINKWork Package 3

Molecular characterisation of PMA

Andy PhillipsAlison HuttlyPeter HeddenPeter ShewryYongfang Wan

Peter Kettlewell

Aims: To characterize the PMA syndrome at a molecular level, including identification of the genes and mechanisms involved in their induction.

HFN LINKWP3: Experimental approach

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

3. Transcript profiling of specific tissues during induction of PMA to identify pathways involved

2. Does a single, common syndrome exist in all susceptible varieties and

inductive conditions?

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

Candidates genes from the GA pathway?

Rht dwarfing genes suppress PMA

OVER-EXPRESSION OF GA20ox IN ENDOSPERMRESULTS IN LARGER GRAIN

50 GRAINSCADENZACONTROL

50 GRAINSCADENZAHMWGlu::GA20ox

1000-grain weight: 48g

1000-grain weight: 63g

ENHANCED GA BIOSYNTHSIS IN ENDOSPERMINCREASES GRAIN SIZE AND WEIGHT

400μmsections

HMWGlu::GA20ox

Control

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IN DEVELOPING GRAIN AFFECTS AMYLASE LEVELS AT HARVEST

Detection of a-Amy1 protein in the aleuroneof HMWGlu::GA20ox grain

Tissue print onto nitrocellulose-coated slide, probed withanti-a-Amy1 antibody

Mike HoldsworthSean May (NASC)

Andy PhillipsPeter Hedden

John FlinthamJohn SnapeSimon Griffiths

Biogemma

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

post-genomics comparative approach.

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

WP4 Objectives & Hypotheses

• Hypotheses:• 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.

• 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.

HFN LINK Work Package 5

QTL Identification &Relation to Candidate Genes

John FlinthamJohn SnapeJames Simmonds

• RAGT• Nickerson-Advanta• CPB Twyford

• Elsoms• Svalöf Weibull• NIAB

Aims: To identify robust QTLs for PHS and PMA, validated across multiple field sites and multiple populations, and to develop linked markers.

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.

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y = 0.7497x + 4.2273R2 = 0.5185

Avalon x Cadenza DHL’s: PHS in JIC irrigated plots, 2006 v 2005

Arcsine percent visibly sprouted grains 2005

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An Integrated Approach to Stabilising HFN in Wheat: Screens, Genes & Understanding

Candidategenes

Allelemining

Fieldexperiments

QTLmapping

Understandingthe physiology

Smartscreens

Validation of QTLs&

Candidate genesMarkers

Breedingprogrammes