Waking up too early: TaMKK3-A underlies the major 4AL sprouting resistance locus, Phs-A1.

Oluwaseyi Shorinola

Monogram, 2017

@Oluwheat

"How does it happen there's no good bread?" asked Mrs.

Henchard

“Oh, 'tis the corn-factor—he's the man that our millers and bakers all

deal wi', and he has sold 'emgrowed wheat, … so that the

loaves be as flat as toads, and like suet pudden inside. I've been a

wife, and I've been a mother, and I never see such unprincipled bread

in Casterbridge as this before”

Thomas Hardy (1886)

“…Norwich’s growed wheat, …unprincipled bread”

Pre-harvest Sprouting (PHS)

The 4AL Phs-A1 locus confers resistance to sprouting

-Phs-A1 +Phs-A1

• Highly multi-genic trait

• Phs-A1 is major locus identified in numerous diverse global germplasm

• Phs-A1 delays dormancy loss during seed after-ripening.

***

0Days after Harvest Maturity

14

• Near isogenic lines (NILs) and recombinant lines (RILs) were developed for fine-mapping of Phs-A1 from two bi-parental populations.

0 20 40 60 80 100

% sproutingwmc420barc170wmc707wmc760wmc313

Robigus

Alchemy

NIL Group 1

NIL Group 2

NIL Group 3

NIL Group 4

NIL Group 5

NIL Group 6

NIL Group 7

5 cM Centromere

Chromosome 4A

Fine-mapping of Phs-A1 in bi-parental populations

Shorinola et al (2016) JxB 67: 4169-4178

Robigus (Non-dormant)

x Alchemy (Dormant)

Phs-A1 is further delimited to a sub-cM interval

Claire (Non-dormant)

xOption

(Dormant)

Chromosome 4A

barc170 wms894 xhbe03

0.5cM

RIL Group 1 (7)

RIL Group 2 (9)

RIL Group 3 (6)

RIL Group 4 (5)

Claire Control RIL

Option Control RIL

Centromere 0 10 20 30 40 50 60

% Sproutingwmc420

Shorinola et al (2016) JxB 67: 4169-4178

Brachypodium

Wheat

3 kb

5 kb

Synteny reveals gene content of the critical interval

• Good co-linearity between wheat and Brachypodium in the Phs-A1 interval.

• Synteny sometimes gives an incomplete picture.

Shorinola et al (2016) JxB 67: 4169-4178

Physical mapping of Phs-A1 interval

Shorinola et al. Manuscript in preparation

1

scaffold92430-3Chromosome 4A

250,000595,750,000596,750,000 596,500,000 596,250,000 596,000,000 595,500,000Wild-Emmer Physical Map

CS BAC Cluster 285 CS BAC Cluster 16421

Wheat Physical Map

High resolution fine-mapping of the Phs-A1 locus

Shorinola et al (2016) JxB doi: 10.1093/jxb/erw194

Stw17 PM19-A2

wms894

PP1-Like –LRR Kinase 2 ACC Oxidase-1

0.05 cM

xhbe03

Centromere

OTU Cysteine Protease

Robigus

Alchemy

RIL Group 21 (2)

RIL Group 22 (9)

RIL Group 23 (1)

RIL Group 25 (6)

RIL Group 24 (2)

Robigus (Non-dormant)

x Alchemy (Dormant)

0 20 40 60 80 100

% Sprouting

Claire

Option

Option Control RIL

RIL Group 11 (1)

RIL Group 12 (4)

RIL Group 13 (1)

RIL Group 14 (4)

RIL Group 15 (1)

Claire Control RIL

0 20 40 60 80 100

% Sprouting

High resolution fine-mapping of the Phs-A1 locus

Shorinola et al (2016) JxB 67: 4169-4178

Claire (susceptible)

xOption

(Resistant)

Stw17 PM19-A2

wms894

PP1-Like –LRR Kinase 2 ACC Oxidase-1

0.05 cM

xhbe03

Centromere

OTU Cysteine Protease

Robigus

Alchemy

RIL Group 21 (2)

RIL Group 22 (9)

RIL Group 23 (1)

RIL Group 25 (6)

RIL Group 24 (2)

Robigus (Non-dormant)

x Alchemy (Dormant)

0 20 40 60 80 100

% Sprouting

BAC Cluster 285 BAC Cluster 16421

Finding the causal gene for the Phs-A1 locus

Shorinola et al Manuscript in preparation

Phs-A1

Amino acid substitution in the catalytic domain caused by a C>A mutation results is associated with Phs-A1 effect.

A mutation in TaMKK3-A is associated with Phs-A1 effect

Shorinola et al Manuscript in preparation

Torada et al (2016) Curr Biol 26 (6):782-787

TaMKK3-A C>A mutation

PM19-A1 promoter InDel

Robigus A Deletion

Alchemy C Insertion

Claire A Deletion

Option C Insertion

Baxter A Deletion

Chara A Deletion

Westonia A Deletion

Yitpi C Insertion

SW95-50213 A Deletion

Cunningham C Deletion

Münstertaler A Insertion

Kitamoe C Insertion

Haruyokoi A Insertion

OS21-5 C Insertion

A mutation in TaMKK3-A is associated with Phs-A1 effect

UK

Au

stra

liaJa

pan

Non-dormant

Dormant

TaMKK3-A allele distribution in global germplasm

Germplasm Description

Triticumurartu

Diploid progenitor of bread wheat

T. diccocoides

Tetraploid progenitor of bread wheat

Watkins Global landraces before 1930

Gediflux European germplasm 1945-2000

Shorinola et al. Manuscript in preparation

H_ 1 C C T C G G C C G C A T T T T G G A G A C G G C C T A C C G A T G C G A A T T

H_ 2 C C T C G G C C G C A T T T T G G A G A C G G C C T A C C G A T G G G A A T T

H_ 3 C C T C G G C C G C A T T T T G G A G A C G G T C T A C C G A T G G G A A T T

H_ 4 C C T C G G C C G C A T T T T G G C G A C G G C C T A C C G A T G G G A A T T

H_ 5 C C T C G G C C G C A T T T T G G C G A C G G C c T A C T T G C A G G G C C G

H_ 6 C C T C G G C C G C A T t T T G G C G A C G G C c T A C C G G C A G G A A T T

H_ 7 C C T C G G C C G C A T T T T G G C G A C G G C C T A C C G G C A G G G C C G

H_ 8 C C T C G G C C G C A T T T T G G A G A C G G T C T A A T T G C A G G G C C G

H_ 9 C C T C G G C C G C A T T T T G G C G A C G G C C T A C C G A T G G G A C C G

H_ 10 C T C C G T C T G C C C C T C G A C A G C A A C C A G C C G G C A G G G C C G

H_ 11 C T C C G T C T G C C C C T C G A c A G C A A C C A G C C G G C A G G A A T T

H_ 12 C T C C G T C T G C C C C T C G A C A G C A A C C A G C C G G C A G T G C C G

H_ 13 C T C C G T C T G C C C C T c G A C A G C A A C C A G C C G G C A G T G C T T

H_ 14 T C C G A T T T A T C C C C C A A C A G G A G C T A G A T T G C A G G G C C G

ERF-C

Fourteen haplotypes around the Phs-A1 locus in global germplasm

BAC Cluster 285 BAC Cluster 16421

PP1-Like TaMKK3-A ASC1-Like LRR1-Kinase 1

LRR1-Kinase 2 PM19-A2 PM19-A1

Shorinola et al. Manuscript in preparation

• Using newly available wheat haplotype resource: “A haplotype map of allohexaploid wheat ..”Jordan et al. (2015). Genome Biol 16:48.

Implication for PHS breeding

Non-Dormant

37%Dormant

63%H3H8

H1/H2

EU Unique

H12

H10

H14Others

Non-Dormant

56%

Dormant44%

H1/H2

H3 H8 Aus Unique

H5/H7H10

H11

H6

Others

UK (176 varieties)

Australia (200 varieties)

Helium:Shaw et al. (2014) BMC Bioinf 15: 259

TaMKK3-A and “unprincipled bread”

NABIM Group End-use

Group 1 Bread-making

Group 2 Bread-making potential

Group 3 Biscuits, cakes etc

Group 4 Animal feed

Summary

- Phs-A1 + Phs-A1

1

ZavitanScaffold92430-3

250,000

ZavitanChromosome 4A

595,750,000596,750,000 596,500,000 596,250,000 596,000,000 595,500,000

TaMKK3-A is the major causal gene underlying Phs-A1 in UK germplasm.

Phs-A1 confers major resistance to sprouting in diverse population.

TaMKK3-A mutation present in progenitors and increased in elite modern germplasm

TaMKK3-A marker is associated to grain end-use in the UK

John Flintham

Nicholas Bird

Cristobal Uauy

Jose Sanchez Barrero (CSIRO, Aus)Atshushi Torada (HIRA, Japan)Peter Jack (RAGT, UK)Simon Berry (Limagrain, UK)Tina Henriksson (Lantmannen)Peter Werner (KWS, UK)

Acknowledgements

Tanja Gergets (UoN, UK)Duncan Scholefield (UoN, UK)Michael Holdsworth (UoNt, UK)Miroslav Valarik (IEB, Czech Rep)Bara Klocova (IEB, Czech Rep)Assaf Distelfeld (TAU, Israel)

Uauy lab members