1

TL1 meeting Progress in Objective 3:

Common beans

Bodo Raatz, CIAT

Addis Ababa, May 2012

2

Road map

• Ethiopian program

• SNPs

– QTLs in the making in MAGIC, MARS, association mapping

• Markers in application MAS

• Phenotyping

3

Andean

Gene Pool

Cultivated common

bean:

• Two major gene

pools

• Specific grain

classes

…that define the

structure of breeding

programs

Mesoamerican

Gene Pool

4

Activity 2: SNP assay

• Collaboration with USDA-Beltsville, mediated by Xavier

• 1500 SNPs converted to Kaspar (Kbio)

• SNPs chosen that are polymorphic within gene pools and grain classes

• 94 lines genotyped (Milestone 2)

5

Mesoamerican

Andean

Phaseolus coccineus

Phaseolus acutifolius

Phylogeny analysis …

Activity 2: SNP assay genotypes

ma

rke

rs

6

Activity 5.1: MARS PhD thesis of Fitsum Alemayehu SARI, Ethiopia

Sister lines SABs 626, 628, 659, 686

– Drought

– Improved remobilization?

CAL 143

– Released in 6 countries in southern Africa

– Vegetative vigor

– Tolerant of poor soils

X

• 165 markers selected for genotyping

• Results in analysis

7

Activity 5.1: MARS genotyping

sc00244ln360837_151643_G_T_157sc00244ln360837_80567_T_C_1578sc00334ln300981_254308_G_A_187sc00334ln300981_202658_C_T_187sc00964ln122791_59538_A_C_3065

sc00429ln249479_88921_A_G_2138

sc00477ln232797_55899_C_T_2254

sc00050ln749641_133318_T_C_569

sc00022ln1003704_490507_A_C_32

sc00022ln1003704_88768_G_A_325

sc00003ln2130026_318965_G_A_46sc00003ln2130026_249989_T_G_45sc00003ln2130026_221589_T_C_45sc00618ln186586_8943_T_C_25469sc01409ln79819_3642_A_C_350551sc01409ln79819_56167_T_C_35060

0.41.00.7

10.1

41.8

14.5

18.6

5.9

15.3

0.41.14.00.2

1

sc00831ln140879_35627_G_A_2890

BARC-PV-0005808

sc00458ln240490_238835_A_C_221

sc00025ln963649_165119_T_G_356sc00025ln963649_327459_T_C_357

sc00038ln842375_5033_G_A_47257

sc00038ln842375_820428_T_C_480

sc00027ln946161_647185_A_G_380sc00027ln946161_730836_A_C_381

66.8

13.9

55.1

0.7

11.6

4.1

11.2

0.5

2

sc00377ln276023_97213_T_G_2002

sc00426ln251751_240727_C_T_213

sc00079ln659676_266197_T_C_776sc00880ln134596_94488_T_C_2958sc00134ln519503_497021_C_T_110sc00023ln985577_737723_A_G_342sc00023ln985577_404408_A_G_339

sc00061ln715360_52902_C_T_6499

sc00009ln1640450_1304280_C_T_1

62.3

17.5

3.42.14.02.2

12.7

24.0

3

BARC-PV-0000055sc01018ln115966_70654_C_T_3130sc01018ln115966_84234_C_T_3130sc00518ln214431_82881_A_G_2346sc00518ln214431_38754_C_T_2345sc00112ln569344_270381_C_T_979sc00112ln569344_446130_C_T_981sc00112ln569344_538657_G_A_982sc00835ln140787_95095_C_T_2896sc01131ln102720_84539_A_G_3253sc00283ln338214_136440_C_T_171sc00283ln338214_153572_G_T_171sc00283ln338214_233599_A_C_171sc00283ln338214_239137_G_A_171sc00283ln338214_293493_T_C_171sc01149ln100495_6599_A_G_32715sc01149ln100495_19761_C_T_3271sc01149ln100495_32702_T_C_3271sc00043ln806145_763080_A_G_521sc00043ln806145_731182_A_G_521sc00043ln806145_170363_C_A_516sc00043ln806145_102278_G_A_515sc00043ln806145_27550_A_G_5145

sc02743ln31009_27860_A_G_41677sc00721ln160814_116732_A_G_272sc00736ln157270_32680_T_C_2748sc00036ln871953_816000_T_G_463sc00036ln871953_623598_A_C_461sc00036ln871953_260922_C_A_457sc00716ln161188_139140_T_G_271

sc00018ln1167623_926274_C_T_29sc00018ln1167623_515133_C_A_28sc00018ln1167623_30331_G_T_281sc01035ln113702_42942_G_A_3149sc00175ln461633_78259_G_T_1298sc00175ln461633_42284_G_A_1298sc00914ln129176_38656_G_A_3003

1.41.22.11.00.30.40.40.70.21.90.6

10.10.30.8

56.9

0.83.42.01.62.53.9

17.9

3.56.71.32.60.43.9

4

8

sc00516ln215718_103432_C_T_234

sc00169ln473620_158171_C_T_127

sc00004ln1947458_369015_C_T_68sc00004ln1947458_475808_A_G_69sc00004ln1947458_609051_G_A_70sc00004ln1947458_651581_A_G_71sc00004ln1947458_666119_G_A_71sc00004ln1947458_704961_G_A_71sc00004ln1947458_901631_G_A_73sc00004ln1947458_1019379_A_G_7sc00004ln1947458_1047835_C_A_7sc00004ln1947458_1101740_A_C_7sc00004ln1947458_968323_T_C_74sc00004ln1947458_1211738_C_T_7sc00004ln1947458_1308111_T_C_7sc00004ln1947458_1320715_T_C_7sc00004ln1947458_1733897_T_C_8

43.3

32.4

1.90.31.77.70.60.30.44.7

5

sc01815ln56854_15487_C_T_37774

sc00162ln482802_338752_C_T_123sc00162ln482802_86819_T_G_1236

30.7

6

sc00431ln249067_99164_A_G_2143

sc00014ln1397360_934650_A_G_23sc00502ln219883_127438_T_C_231

sc00067ln694271_486308_T_C_696

sc00052ln745181_210054_A_G_585sc00052ln745181_283027_C_T_586sc00064ln701495_548353_A_G_676sc00583ln198651_1233_G_A_24795

sc00153ln494563_454394_G_A_119sc00158ln489092_253333_G_A_121

sc00262ln351368_101277_A_G_164

BARC-PV-0001413sc00002ln2152649_1120369_C_T_3BARC-PV-0001385sc00002ln2152649_1550949_T_G_3

9.7

3.9

5.4

8.8

6.11.7

9.8

2.7

30.9

50.4

1.50.92.1

7

sc00089ln640327_255418_C_T_841

sc01794ln57480_20276_G_A_37654

sc00035ln877458_767887_C_T_454

sc00187ln435150_76559_G_A_1352

67.2

59.6

11.3

8

Activity 5.1: MARS genotyping

9

QTL / association mapping for drought tolerance

Andean genepool Mesoamerican genepool

CAL143 NATAL

SUGAR

PAN127 R.C.

WONDER

SER8 SEQ11 SEC16 SUG131 SER22 SER16

F1 F1 F1 F1 F1 F1 F1 F1 F1 F1 F1 F1 F1 F1 F1 F1 F1 F1 F1 F1 F1 F1 F1 F1 F1

F2

F3

F4

F2

F3

F4

F2

F4:6 F4:6

160 plants harvested

400 lines

QTL/Association analysis

10

SNP set: Regenotyping for improved QTL map

BAT 881 x G21212 330 polymorphic SNPs

SER16 X (SER16 X G35346) G35346 = Phaseolus coccineus

400 polymorphic SNPs

Pinto Saltillo X Pinto Villa 170 polymorphic SNPs

11

Cut seeds

DNA extraction from seeds

Amplification Test

BM 185 Tissue Lyser

seeds macerated with

stainless steel beads

DNA

extraction

12

Seed storage to planting

Germination test

In field

Cut seeds germinate in the field

13

Activity 5.2: Advanced backcross

• Five Adv. BC populations BC2-F4

• Most lines badly adapted

• 2012: - few lines in breeding program

- QTL appropach abandoned

SOURCE

SER 16 SER 48

CAL 143 126 78

CAL 96 156 128

CIM 9314-36 39 -

14

Mesoamerican donor SER 16 Andean recurrent parent CAL 96

Activity 5.2: Advanced backcross

15

Activity 5.2: Advanced backcross

16

SXB 412 INB 827 ALB 213 SEN 56 SCR 2 MIB 778 SCR 9 INB 841

79 F1 5 F1 38 F1 99 F1

323 F1 272 F1

728 F1

500 selected

499 F2

996 F3 families

996 F3.5 families

F5:7 families for drought phenotyping

MAGIC population in common bean (mesoamerican gene pool)

Genotype in F5??

Genotype individuals

17

Activity 3: MAS for biotic traits

Storage insects:

Bruchids (Zabrotes

subfasciatus)

18

SSR marker for bruchid resistance

PV-ATCT001 – Arc

Cortesia: H.F. Buendia

•20 crosses

Agarose based marker:

RA

Z4

4

RA

Z105

RA

Z106

G18264

CA

L143

SE

Q1006

SE

Q1027

Bla

nco

19

RI =

lo

g{(

Em

erg

ed a

dults/h

atc

hed e

ggs)

+ 1

} /

DA

E *

100

Yield (Kg/Ha)

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29 30 31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

51

52

53

87 88

89

90

91

93

94

95 96

97

98

99

100

102

104

105

106

108

111

112

113

114

115

116

RAZ103

RAZ104 RAZ168

54

55

56

57

117

58 59

60

61 62

63

64

65

66

67

68

70

71

72

73

74

75 76

77

78

125

126 127 128

129

CAL143

KATB9

79 80

81

82

83

84

85

86 119

120

121 122

123 124

0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

1000 1200 1400 1600 1800 2000 2200 2400 2600 2800

R

RI

S

positive

heterozygote

negative

No Amplification

PV-ATCT001 marker

Activity 3: MAS for bruchid resistance

20

Activity 3: SNPs for bruchid resistance

BAC wild meso

BAT93 meso

G19833 andean

kb

APA: Arcelin-Phytohemagglutinin-amylase Inhibitor family

BAC: 71F18, Kami et al, 2006

21

Precision introgression

• Bruchid resistance (arcelin gene) originates from mesoamerican pool

• 4 markers easily available between genepools

The perfect marker is four markers

R locus

Positive

marker

Positive

marker

negative

marker

negative

marker

1cM 1cM

22

Activity 3: MAS for biotic traits

BGYMV (bean golden yellow

mosaic virus):

SCAR for bgm-1, but occasional

recombinants (14 crosses)

Recessive bc-3 resistance for BCMNV

(bean common mosaic necrotic virus) (64 crosses)

23

C-A

bc-3 candidate gene: eIF4E NADERPOUR et al. MOLECULAR PLANT PATHOLOGY (2010)

180 bp

820 bp

r r r r r r r r s s

1000 bp

24

Common Bacterial Blight (CBB)

MAS with

• SU91 on Chr8 (Pedraza et al. 1997)

• SCAR’s : BC420 on Chr6 (Yu et al. 1998)

• SAP6 on Chr10 (Miklas et al. 2006) .

Bacterial blight: Xanthomonas

25

BMbS15 0 BMbS13 SUcat01 BMbS12

3

BMbS5 5 SU91 9 SUag01 gVQ

12

BMbS10 13 BMbS4 15

40

BMa27 60

BM151 78 BMb386 83 BMb277 86

PVBR173 94

BMc121 111

PVBR71 141

BMb1319w 167

Xf

0

2

4

6

8

10

12

14

16

18

20

22

24

26

28

30

Xc

Chr 8

Map size : 166.82 cM

BMb1196w

BM212 0

BMd42 21

PV158 38

PV181 47 PV185 51 SAP6 55

BMb96 93 BMc63 102 IAC54 109 BMb302 111 BMb167 113 BMc66 118

0

1

2

3

Xc

Xf

Xc

4

6

8

10

Chr 10

Map size : 117.59 cM

R2 =20%

R2 =49%

BMbS12

A

CBB resistance QTLs

26

• 36 advanced lines characterized (milestone 6)

• Drought trials in

– 2 saison in CIAT (2010, 2011), Palmira, ±drought

– 1 saison in Ethiopia, ±drought

• 3 visual scales for wilting, podfilling and degree of leaf loss analyzed (milestone 7)

Activity 4: Drought tolerant traits

27

Irrigated grain yield (kg ha-1)

1800 2000 2200 2400 2600 2800 3000 3200 3400 3600 3800

Rain

fed

gra

in y

ield

(kg

ha

-1)

200

400

600

800

1000

1200

CAL 143

CAL 96

DAB 295DAB 489

DAB 494DAB 528

DAB 441

DAB 520

DAB 318

DAB 530

DAB 302

DAB 261

DAB 395

DAB 557

DAB 545

DAB 353 DAB 317

DAB 344

DAB 380

DAB 564DAB 493

KATB 1 AFR 298

DAB 432

DAB 525DAB 541

DAB 555

Mean: 798LSD0.05: 383

Mean: 2934LSD0.05: 565

Relationship between irrigated and rainfed grain yield of 36 common bean genotypes grown under field conditions at

CIAT-Colombia

28

Activity 4: Visual scales

Resistance to Wilting

P.a.

29

Adapted from King et al. 2009 Crop Science 49:290-298;

0 = no wilting and 100 = plant death

Wilting score at 41 days after planting

5 10 15 20 25 30 35

Rain

fed

gra

in y

ield

(kg

ha

-1)

200

400

600

800

1000

1200

CAL 143

CAL 96

DAB 295

DAB 489 DAB 494 DAB 528

DAB 441

DAB 504

DAB 532

DAB 520

DAB 318

DAB 530

DAB 302

DAB 261DAB 395

DAB 326

DAB 557DAB 534

DAB 545

DAB 353 DAB 290

DAB 514

DAB 317

DAB 344DAB 380

DAB 555

DAB 564

DAB 493

KATB 1AFR 298

DAB 432

Mean: 798LSD0.05: 383

Mean: 18.9LSD0.05: 8.6

r = -0.36***

Relationship between leaf wilting and rainfed grain yield of 36 common bean genotypes grown under field conditions

at CIAT-Colombia

30

0 = well-filled grain; 10 = more than 50% of grain is not well-filled

Rainfed seed fill score2 3 4 5 6 7

Rain

fed

gra

in y

ield

(kg

ha

-1)

200

400

600

800

1000

1200

CAL 143

CAL 96

DAB 295

DAB 489DAB 494 DAB 528

DAB 441

DAB 504

DAB 520

DAB 318

DAB 530 DAB 302

DAB 261DAB 395

DAB 532DAB 557

DAB 534

DAB 545

DAB 353

DAB 290

DAB 317

DAB 344DAB 380

DAB 555

DAB 564

DAB 493KATB 1AFR 298

DAB 432

DAB 549DAB 525

DAB 286Mean: 798LSD0.05: 383

Mean: 4.78LSD0.05: 2.0

r = -0.47***

Relationship between seed filling and rainfed grain yield of 36 common bean genotypes grown under field conditions

at CIAT-Colombia

31

Conclusions

• SNPs used in QTL and association mapping

• MARS moving forward

– Seed DNA extraction permits flexibility

• MAS for biotic stress in implementation, moving toward routine

• Advanced lines show improved yield

• Visual scale of pod filling useful

32

Thank you for your attention!