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R&D Center ToursR&D Center Tours

Use of DNA markers in cocoa to manage collection, traceability and

breeding program.

PRIVAT I., BELLANGER L., CROUZILLAT D.

Nestlé Centre R&D, Tours, France.

SOESILO AW., PRIYONO P.

Indonesian Coffee and Cocoa Research Institute

R&D Center Tours2

Establish a core cacao collection in producing countries for evaluation of genetic & environment interactions

Traceability of cocoa bean to end-product

Identify genetic markers for traits of interest (Agro, technologic,…)

Main key research areas of biology molecular for cocoa improvement at R&D Nestlé

R&D Center Tours3

Cocoa Genetic Diversity analysis using SSRs- 18 SSR

- 1137 cocoa clones from various local and international collections

This result indicate that the cocoa genetic diversity is linked to the geographic origin

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-3

-2

-1

0

1

2

3

-2,5 -2 -1,5 -1 -0,5 0 0,5 1 1,5

EcAr

Ec54 Ec52

Ecuador

Ve30

Ve28

Ve29

Ve51

Venezuela

Br25

Br50

Brazil

In26

Ni21

Ni64

Ni22

Ni20

Nigeria

Ghana

Ivory Coast

Gh18Gh55

Gh57

Gh17

Gh19

Gh58Gh56

IC63

IC13

IC14 IC59

IC62IC64

IC61 IC16

21,7

%

58,5%

The 2 axis explained 80.2% of the variance

Identification of the geographic origins of cocoa liquors used in

Nestlé supply chain

DNA traceability to assess the geographic

origin of cocoa liquorsFrom the cocoa tree to the end-product

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Genetics

Chemistry Sensory

C1680

At5g6686012CIR11814At4g3728017At4g3751020At3g4990022CIR1523

C6630

At4g3966034

At5g0872044

CIR27048At2g4270049C75At2g3259053At2g3290055At2g0484257

At4g1276062

At4g0268072

CIR9777At1g0291079At3g5286081

At3g6258085At4g0206086C138At1g0109090

At4g00560101At3g61080102CIR275At1g48050104At5g13700At5g52820111At3g63520113C86114

1At4g163400At1g807701CIR732

At1g26810CIR119

At1g2323030

CIR22435At1g2456038

At1g2735051At2g3545053

At4g0340059CA2161CIR17662At4g1518064

C21180

At2g1994084

CIR10089

CIR19101At5g49960103

2CIR490

At1g490405

CIR2115

C6526At5g5032029At5g63290At3g4850030At1g2932031At1g0535033C20835At5g1991036

At4g3341045CIR7848At5g2430050At4g2770051At4g1712055CIR167At4g2111056

At5g4119062

At4g2384066CIR20269

3C1040At3g55250At2g046901

At3g017807

C20915

CIR17At3g0230020

CA0626

C4836CIR1237

At1g0469044At3g0605048CIR9549At4g1474651At3g2349054CIR6757At5g18570C7058At3g1681060At3g1704061

4CIR1220

CIR27913

At3g1006020

CA2429

At5g1628033CIR19736At5g0508037

CIR25646

At1g4831055CIR24557

At4g2760061At5g3597063

At1g1625069At1g0883071At3g0426072C16476

At1g0915089

At1g0934094CIR27496

5CIR60At4g209301

CIR20812

At1g1608017

CIR23542

At4g1826047

CIR2551

At4g2171057

CIR29173

6CIR1770

At3g14770CIR557

CIR714

ccg114126

CIR11346

CIR9350

7CIR750

CIR225At1g20410At1g42430

11

CIR200CCG207524

CIR2629

At4g0828033At1g2164035At5g2580038At3g01790CIR18942

CIR10349

8At2g24395At4g311500At5g199401At4g321402CIR2666

C4920

CIR3030At4g2940032At4g2906033At5g1995036CIR2437At4g2013041

CIR16046

CIR12452

At5g1962062

At2g2693068At1g0505571At2g2849072

CIR64At5g2262081

At2g2890094C106At2g2709098

CA16106

9C2060

At5g059309At3g5552012

CIR15517

CIR91At3g1067034

At2g3873042

CIR22358

10

Improve raw material by Integrative research

Set up specific crosses to analyze the

progenies

0

5

10 Sweet Acid

Bitter

CacaoFruity

FlowerySpicy

Roasted

Nut

GreenDefault

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Nacional

(Arriba Flavour)

Venezolano Amarillo

(Trinitario)

EET 95

Selfing (F2)

In average, for each locus the segregating progenies have the following ratios:

¼ Nacional homozygote, ½ Hybrid, ¼ Venezolano homozygote

Reference cross EET95 progeny

(R&D – Tours

& Nestlé Ecuador)

R&D Center Tours

C1680


C6630

At4g3966034

At5g0872044CIR27048At2g4270049C75At2g3259053At2g3290055At2g0484257

At4g1276062

At4g0268072

CIR9777At1g0291079At3g5286081

At3g6258085At4g0206086C138At1g0109090


1At4g163400At1g807701CIR732

At1g26810CIR119

At1g2323030

CIR22435At1g2456038

At1g2735051At2g3545053

At4g0340059CA2161CIR17662At4g1518064

C21180

At2g1994084

CIR10089

CIR19101At5g49960103

2CIR490

At1g490405

CIR2115



At5g4119062

At4g2384066CIR20269

3C1040At3g55250At2g046901

At3g017807

C20915

CIR17At3g0230020

CA0626

C4836CIR1237


4CIR1220

CIR27913

At3g1006020

CA2429

At5g1628033CIR19736At5g0508037

CIR25646

At1g4831055CIR24557

At4g2760061At5g3597063

At1g1625069At1g0883071At3g0426072

C16476

At1g0915089

At1g0934094CIR27496

5CIR60At4g209301

CIR20812

At1g1608017

CIR23542

At4g1826047

CIR2551

At4g2171057

CIR29173

6CIR1770

At3g14770CIR557

CIR714

ccg114126

CIR11346

CIR9350

7CIR750

CIR225At1g20410At1g42430

11

CIR200CCG207524

CIR2629


CIR10349


C4920


CIR16046

CIR12452

At5g1962062

At2g2693068At1g0505571At2g2849072

CIR64At5g2262081

At2g2890094C106At2g2709098

CA16106

9C2060

At5g059309At3g5552012

CIR15517

CIR91At3g1067034

At2g3873042

CIR22358

10

Building genetic map based on SSR or SNP markers

R&D Center Tours8

C1680


C6630

At4g3966034

At5g0872044CIR27048At2g4270049C75At2g3259053At2g3290055At2g0484257

At4g1276062

At4g0268072

CIR9777At1g0291079At3g5286081

At3g6258085At4g0206086C138At1g0109090


1At4g163400At1g807701CIR732

At1g26810CIR119

At1g2323030

CIR22435At1g2456038

At1g2735051At2g3545053

At4g0340059CA2161CIR17662At4g1518064

C21180

At2g1994084

CIR10089

CIR19101At5g49960103

2CIR490

At1g490405

CIR2115



At5g4119062

At4g2384066CIR20269

3C1040At3g55250At2g046901

At3g017807

C20915

CIR17At3g0230020

CA0626

C4836CIR1237


4CIR1220

CIR27913

At3g1006020

CA2429

At5g1628033CIR19736At5g0508037

CIR25646

At1g4831055CIR24557

At4g2760061At5g3597063

At1g1625069At1g0883071At3g0426072

C16476

At1g0915089

At1g0934094CIR27496

5CIR60At4g209301

CIR20812

At1g1608017

CIR23542

At4g1826047

CIR2551

At4g2171057

CIR29173

6CIR1770

At3g14770CIR557

CIR714

ccg114126

CIR11346

CIR9350

7CIR750

CIR225At1g20410At1g42430

11

CIR200CCG207524

CIR2629


CIR10349


C4920


CIR16046

CIR12452

At5g1962062

At2g2693068At1g0505571At2g2849072

CIR64At5g2262081

At2g2890094C106At2g2709098

CA16106

9C2060

At5g059309At3g5552012

CIR15517

CIR91At3g1067034

At2g3873042

CIR22358

10

Spicy

Cocoa and 3 volatile

compounds

Organic &

amino acids

Caffeine

2 non-volatile compounds

related to quality

Acidity +2 non-volatile

markers

Identify QTLs and select the most promising ones

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Progenies from EET95 cross Getting dry beans and liquors from each tree

Building sensory profiles of liquors

Correlate sensory traits to chemical/genetic profiles

05

101520253035

Perform QTL analysis for traits of interest

Selecting cocoa material having the right markers

Muliplying elite cocoa trees by somatic embryogenesis

Improve raw material by Integrative research

Parent Parent éélite A: 2 QTL A et Blite A: 2 QTL A et B

A

B


A

B

A

B

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Collaboration between Nestlé and ICCRI

Genotyping• Involved seven cocoa clones selected by ICCRI: TSH

858, SUL1, SUL2, NIC7, ICS13, KW165 (BAL209) and KEE2

• 115 SSR makers have been tested according to the genetic diversity among the seven cocoa clones. Only 83 markers showed correct DNA amplification on those plants.

• In parallel, 18 other SSR markers were used to assess genetic diversity of these clones in regards to 1137 cocoa genotypes from various cocoa collections from different countries

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• 83 SSR markers used for this analysis show a high level of genetic diversity among the 7 cocoa parental genotypes.

• 79 markers are polymorphic among the 7 genotypes.– The PIC values of the polymorphic SSRs are ranking from

0.13 to 0.83.– A total of 237 alleles were detected for the 83 SSRs and

the average allele number per locus is 2.9.• Level of heterozygosity for each clone was estimated based

on the use of these 83 SSR markers.– The value is ranking from 26 % for KEE2 to 65 % for

ICS13.– This data fit well with the genetic diversity study since

ICS13 (Trinitario type, hybrid clone) has the highest value and KEE2 (Amelonado) has the lowest value.


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• Cocoa clones ICS13 and NIC7 are located closed to Trinitario genotypes.

• On the opposite, KEE2 genotype matched with cocoa clones from Africa (Ivory Coast) such as Amelonado cluster.

• The last four genotypes SUL1, SUL2, TSH858, KW165 (BAL209) are closely related and seems to be Forastero hybrids such as BAL209 (PA35 X NA32) resulting from a cross between Maranon (PA35) and Nanay (NA32) cluster genotypes


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3,00 2,25 1,50 0,75 0,00

Dissimilarity

TSH858

KEE2

SUL1

BAL209

SUL2

NIC7

ICS13


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ICS13

NIC7 SUL2

TSH858BAL209

SUL1

KEE2

- 18 SSR

- 1137 cocoa clones from various local and international collections


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Génotype ♂

TSH858 (S) SUL1 (R) KEE2 (R) NIC7 (S) ICS13 (S)

Gén

otyp

e ♀

TSH858 (S) HKW2 HKW1 HKW3 HKW4

SUL1 (R) HKW10 HKW9 HKW11 HKW12KEE2 (R) HKW5 HKW7 HKW8NIC7 (S) HKW6

SUL2 (MR) HKW13

BAL209 (R) HKW14

Traits of interest YieldFat contentVSD resistance

Factorial plan / Connected populations


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A

B


A

B

A

B

CD

CD

CD

CA

B

DCA

B

DCA

B

D

Parent 1 Parent 2

QTL A and B QTL C and D

MAS

Combine QTL A, B, C and D in elite clones

Deploy SAM (Marker Assisted Selection) for cocoa

Large bi-parental population

(Nestlé Ecudador)

Factorial plan

Connected populations

(ICCRI/VSD)

R&D Center Tours17

Use of DNA markers in cocoa to manage collection, traceability and breeding program.

• Define the genetic diversity from different cocoa collections or producing countries and help to identify genetic clusters to orientate parents selection for breeding programs

• Allow to trace the raw material all along the supply chain from the field to the end product

• Establish correlation between traits of interest and specific zones of the genome

• Develop MAS strategy for cacao and combine QTLs of key interest for polygenic traits

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Acknowledgments

Nestlé R&D TOURS

Dr. Jwanro Husson

Dr. Pierre Broun

ICCRI

Dr. Teguh Wahyudi

Dr. Abdoellah Soetanto

NRC Lausanne

Dr. Jean-Claude Spadone

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