Applied Genomics to Coffee Breeding

Towards GWAS and Genomic Prediction in Coffee:

Development and Validation of a 26K SNP Chip for

Coffea Canephora.

Alan C. Andrade

[email protected]

Cultivated Coffea Species:

2n=2x=22

Allogamous species

2n=4x=44

Autogamous species

World Coffee Consumption

Challenges

Growers and Industry

•

Climate changes

•

Competition with other commodities (Bioenergy)

•

Sustainable production systems

•

Preservation of natural resources (minerals, water, etc.)

•

Quality (Competition with other beverages)

•

Socio-economic aspects

Applied Genomics to

Coffee Breeding

Coffea canephora

Embrapa´s Research on Coffea canephora

• Population of 3500 individuals (March 2008)

• Pool of seeds from an INCAPER field, containing 48

parents

• Introducing CIRAD Hybrids

Current Status

Statistical analysis

Physiological evaluations (



am)

Field evaluations (Productivity)

Lab evaluations (Morphological parameters)

Mapping sequences( Diversity groups, parentals)

SNP mining (GATK pipeline)

Genotyping (SNPsaurus/

25K Axiom Chip

)

Selection of individuals (1300 )

DNA extraction

Plant Material

Phenotyping

Genotyping

Association Studies



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

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

Coffea canephora

Phenotype Diversity

Affymetrix Parameters

Coverage parameters (> 30%); quality;

Strand bias.

Axiom Chip Design

Mapping reads in the reference genome

Local realignment; quality recalibration

(estimated PHRED scores)

Sequencing production

Sequencing

Mapping

Refinement

SNPs mining

SNPs selection

Genotype Diversity

• 11 SSRs

• 48 Parents Incaper

• 250 individuals

Parents INCAPER

Population EMBRAPA

SG1 Group

SG2 Group

Re-sequencing

• Paired end

Bulk of individuals

Conilons

Diversity

Cubry et al, 2013

Bulk

Generated sequences

Mapped

Conilon

704.388.334

568.553.344

Diversity

591.003.832

521.767.804

Pool

Group

Nº ind

Seq (Mi) Mapped

C1

Conilon

5

51,93

49,10

C2

Conilon

1

52,85

48,71

C3

Conilon

5

59,72

54,20

C4

Conilon

4

52,13

48,00

C5

Conilon

4

60,63

55,73

C6

Conilon

5

63,53

59,25

C7

Conilon

6

40,55

36,54

C8

Conilon

2

47,57

41,65

C9

Conilon

3

53,43

48,41

C10

Conilon

4

40,81

36,56

C11

Conilon

2

42,18

37,61

C12

Conilon

3

58,68

52,87

Pool

Sub-Group

Group

Nº ind

Seq (Mi) Mapped

D1

Luki

SG1

10

64,86

60,87

D2

Niaouli

SG1

9

69,43

58,27

D3

Nana

C

10

43,45

35,45

D4

Libengue

B

9

56,22

51,30

D5

INEAC

SG2

4

59,47

49,37

D6

Erect

SG2

2

67,32

62,14

D7

Nganda

SG2

2

61,84

58,36

D8

Wild

SG2

3

70,83

65,24

D9

Pelezi

G

1

50,71

39,26

D10

Mouniandougou

G

1

46,88

41,50

Genotyping in a Genomic Scale

Axiom_EMBR5SNP

25.456 SNPs

Chromosomes Size (Mb) of chromosomes

(%) of SNPs mapped

Chr0

278.51

2,260 (8.9)

Chr1

37.88

2,181 (8.6)

Chr2

58.38

4,483 (17.6)

Chr3

32.44

1,469 (5.8)

Chr4

33.84

1,915 (7.5)

Chr5

32.78

1,577 (6,2)

Chr6

40.51

2,697 (10.6)

Chr7

34.36

2,167 (8.5)

Chr8

31.93

1,759 (6.9)

Chr9

26.13

1,400 (5.5)

Chr10

29.42

1,872 (7.4)

Chr11

33.24

1,676 (6.6)

Genotyping at a Genomic Scale

Coding regions

Intergenic

SNPs Localization

Linkage disequilibrium (LD)

~ 400 ind./

~ 20.000 SNPs

Chr 0 Chr 1 Chr 2 Chr 3 Chr 4 Chr 5 Chr 6 Chr 7 Chr 8 Chr 9 Chr 10 Chr 11 0.2146 0.19 0.1884 0.1876 0.1878 0.1844 0.1858 0.1565 0.1862 0.1743 0.1838 0.1667 0.1324 0.1546 0.134 0.1757 0.1443 0.1663 0.1323 0.1544 0.15 0.1284 0.1744 0.1603 0.1628 0.1531 0.1213 0.1194 0.1663 0.1668 0.1623 0.1519 0.1471 0.0916 0.1635 0.1654 0.1576 0.1089 0.1513 0.1495 0.0569 0.0501 0.1608 0.1492 0.15 0.1469 0.1411 0.1471 0.0549 0.0449 0.148 0.1448 0.1491 0.1492 0.1413 0.1355 0.0948 0.1488 0.1458 0.1453 0.1314 0.1466 0.1484 0.1433 0.1407 0.1219 0.1049 0.1461 0.1452 0.1443 0.1445 0.1081 0.0665 0.1024 0.146 0.1425 0.1424 0.142 0.1424 0.1392 0.1053 0.1025 0.0662 0.0912 0.1009 0.145 0.1446 0.1336 0.1349 0.1048 0.104 0.1005 0.0934 0.0975 0.1437 0.1431 0.1405 0.1414 0.1045 0.1036 0.101 0.0987 0.0964 0.1403 0.1415 0.1372 0.104 0.1041 0.1026 0.1022 0.0964 0.0809 0.0941 0.1384 0.1398 0.1364 0.1364 0.1039 0.082 0.1038 0.1013 0.1004 0.0892 0.1015 0.0558 0.0546 0.0544 0.137 0.1368 0.1084 0.1031 0.053 0.0568 0.102 0.1032 0.0549 0.0518 0.096 0.0978 0.0985 0.0517 0.0544 0.0544 0.1327 0.1347 0.132 0.1332 0.1018 0.053 0.0567 0.1013 0.1009 0.1013 0.1027 0.0982 0.0516 0.0544 0.1017 0.1015 0.0567 0.0996 0.0826 0.0985 0.0894 0.0955 0.096 0.1005 0.1007 0.101 0.0993 0.0982 0.0985 0.0969 0.0939 0.0991 0.0821 0.0997 0.0991 0.0998 0.092 0.094 0.0961 0.0924 0.0761 0.0785 0.0982 0.0483 0.0917 0.0793 0.0973 0.0859 0.0918 0.0517 0.0508 0.0817 0.0535 0.0964 0.0537 0.0797 0.0784 0.0522 0.0855 0.0951 0.0803 0.045 0.0517 0.0507 0.0745 0.0534 0.0948 0.091 0.0728 0.0548 0.0522 0.0853 0.0833 0.0785 0.0507 0.0733 0.0463 0.0908 0.0492 0.0774 0.0756 0.0435 0.0723 0.0426 PAR66 302P4 CCG117 RD23 RD19 BRS 12 CCINC33 BRS7 RD34 108P5 CCI NC2-A1 CCINC748 RD37 RD1 6 CCG133 CCG56 RD33 RD2 4 RD29 RD26 CCG79 CCG73 CCG114_SG2+G RD25 RD32 CCG17 CCG80 RD27 RD35 RD31 20 7P2 203P2 CCINC6 71 C1028_B Libengue PAR333 RD30 RD28 CCG100 CCG4 CCG50 CCG107 CCG65 C1010_B Libengue C1038_B Libengue PAR1111 PAR222 109P2 CCINCPRES UL 61 CCG 14 CCG25 CCG22 CCINC6 04 CC E4P2 CCE 11P2 CCG52 CCG45 CCG72 CCG75 CCG51 CCG106 1637-1_SG1 Luki CCG62 CCG5 5 136A03(2) CCG 94 CCG108 CCG47 CCG 37 CC G104 CCG1 31 CCG30 CCG2 0 C3007_SG 1 Niaouli 13 6A03(3) 136 A03(5) 1635-2_SG1 Luki 1635-3_ SG1 Luki CCG1 3 CC G129 CCG28 CC G113 PAR147 C3008_SG 1 Niaouli 1640-1_SG1 Luk i 16 45-1_SG1 L uki 1647-1_ SG1 Luki CCG4 6 CCG1 27 CCG1 10 CCG102 CCG132 C3005_SG1 Ni aouli 1648-1_SG1 Luki 1649-1_SG1 Luki CCG49 CCG8 6 PAR 70 209P2 C3002_SG1 Niaouli C3003_SG 1 Nia ouli CCG15 CCG67 CCG26 CC G122 CCG40 107P2 PAR10 PAR14 308P2 308P 2-2 PAR80 CCINC2 V CCG 115 CC G105 CCINC3 R CCINC143 PAR2 201P3 CCINC24 BRS9 RD17 PAR20 208P4 PAR23 BRS1 BRS15 PAR130 305P3 CCINC2 -5V 205P 3 CC G7 CC G140 CCG125 CCG2 CC G120 CCG111 CC G119 CCG1 09 PAR 24 306P2 CCINC2 -03V PAR8 309P2 BRS2 204P3 CCINC6V CCINC2-6V PAR74 CCINC 22V CCI NC2 R CCINC1 20-6 R BRS6 RD20 BRS11 303P3 304P2 301P2 CCINC2-5+3 PAR13 CCINC 104 RD21 RD22 RD38 BRS13 PAR35 PAR5 BRS5 CCE2 P2 PAR7 PAR26 PAR30 PAR3 PAR22 206P2 CCE7 P2 PAR 152 PAR78 202P4 CCINC 2-18V PAR16 CCINC2-8V CCINC2 -12V PAR6 CCINC7V PAR4 PAR15 CCE6P2 CC E3P2 CCINC2-4V CC E5P3 101P 2 PAR25 PAR1 7 PAR18 PAR1 51 102P2 CCINC12 V PAR160 CCINC2-10V PAR 32 BRS8 RD18 CCE13P2 PA R148 CCINC10V PAR59 CCINC4V CCE9P1 CCE8P2 PAR19 105P2 PAR1 103P2 104P2 PAR9 CCINC462 PAR12 BRS3 CCINC9 V

CCINC2-9V PAR60 CCE1

2P2 CCE10P2 PA R28 CCINC19/86 CC E1P2 PAR31 307P3 0.05

Conilons

Diversity

Phenotyping

• Yield (2011/12; 2012/13; 2013/14)

• Morphology of fruits

• (Fruit size and Bean size/Sieve)

• Vigour

• Pre-dawn leaf water potential (



pd)

• Disease resistance (0-3)

• Biochemical composition NIRs

Phenotyping

0 100 200 300 400 500 600 0, 1 -3 ,0 7 3, 0 8-6 ,0 5 6, 0 6-9 ,0 3 9, 0 4-1 2, 02 12 ,0 3-15 15 ,0 1-17 ,9 8 17 ,9 8-20 ,9 6 N u mb e r o f in d iv id u al s Average Production (L)

Total: 1292 ind.

Yield

(Average of 3 harvests)

2011-2013

Phenotyping

Total: 404 ind.

Pre-dawn Leaf

Water Potential

Total: 1292 ind.

Fruit Morphology

Área 3,1942 3,0670 3,1608 2,0485 2,8882 2,8717 Perímetro 10,2846 10,0100 10,0965 8,3837 9,6723 9,6894 Menor eixo 0,8476 0,8548 0,8979 0,6393 0,8476 0,8174 Maior eixo 1,1996 1,1421 1,1206 1,0200 1,0846 1,1134 Área 2,7595 3,0292 2,9748 2,2486 2,4950 2,7014 Perímetro 9,5112 9,8257 9,7624 8,5690 9,0959 9,3528 Menor eixo 0,8045 0,9194 0,8907 0,7327 0,7470 0,8189 Maior eixo 1,0918 1,0487 1,0631 0,9769 1,0631 1,0487 Área 2,7543 2,7689 3,3083 2,4859 2,9070 2,8449 Perímetro 9,4084 9,4497 10,3036 8,9968 9,6568 9,5631 Menor eixo 0,8476 0,8404 0,9338 0,7758 0,8763 0,8548 Maior eixo 1,0344 1,0487 1,1277 1,0200 1,0559 1,0573 Área 3,5965 3,7512 3,4234 3,1398 3,5732 3,4968 Perímetro 10,8373 11,0463 10,5376 10,0058 10,7474 10,6349 Menor eixo 0,9266 0,9553 0,9194 0,9338 0,9482 0,9367 Maior eixo 1,2355 1,2499 1,1852 1,0703 1,1996 1,1881 Área 2,5131 2,3896 2,6324 2,6324 2,7232 2,5781 Perímetro 8,9727 8,7076 9,1905 9,1905 9,3274 9,0777 Menor eixo 0,8189 0,8404 0,8332 0,8332 0,8620 0,8375 Maior eixo 0,9769 0,9051 1,0056 1,0056 1,0056 0,9798 L1P65 L2P8 L4P51 E5Média Foto L1P15 L1P49 PlantaMedidas (cm) E1 E2 E3 E4

Phenotyping

Phenotyping

Total: 100 ind.

NIRs.

0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 0 20 40 60 80 100 120

sucrose

proteins

lipids

trigoneline

cafeine

total chlorogenic acids

Association Studies

~ 400 ind./~ 20000 SNPs

Tassel (MLM)

Yield

Drought

Fruit Morphology

Bean Biochemical

Association Studies

Drought



a.m.

Summary

•

Perennial crops like coffee may benefit most from GS

programs.

•

A cost-effective genotyping platform with high reproducibility

is an important tool for coffee researchers aiming at GS

programs.

•

Additional

phenotyping

for

other

traits

and

other

environmental conditions is under progress.

•

A new population derived from selected clones is already in

the field!

G.C . RODRIGUES Embrapa-CNPTIA A.F. GUERRA Embrapa-Café G.F . BARTHOLO Embrapa-Café P. MARRACCINI HDR / AGAP-Burst O.C . ROcha Embrapa-Cerrados M.A.G. FERRÃO Embrapa-Café D. Grattapaglia Embrapa-Cenargen M. A. F. Carvalho Embrapa Café A. Delly Veiga Embrapa Cerrados

Research Team:

Jwanro Husson

Nestlé R&D

Centre - Tours

Ray Ming University of Illinois USA Orzenil Silva Embrapa-Cenargen