Genetic and QTL mapping of Fusarium wilt (FOV) race 1 & race 4 resistance in cotton for breeding 

Mauricio Ulloa, USDA‐ARS, WICS Shafter 

Bob Hutmacher, Dept. Plant Science, UC‐Davis, Shafter 

Mike Davis, Dept. of Plant Pathology, UC Davis

Steve Wright, UC Cooperative Extension, Tulare 

Phil Roberts and Congli Wang,  Dept. Nematology, UC Riverside 

• FOV Plant Symptoms:• Wilting, yellowing, and necrosis of leaves.

• Internally, the xylem is dark brown [vascular root stain (VRS)].

Plant Material & Tests

Used two Mapping Populations:• F2 Pima‐S7 x ‘Acala NemX’  • RIL Upland TM‐1  x Pima 3‐79 

RIL Population evaluated in:• Three greenhouse artificial inoculated tests ( 4 –

5 plants in a CRD)• One known infested race 1 FOV field test ( 5 

plants, two reps, RCBD) 

FOV race 1 assays

• Used disease severity index (DSI) of leaves, and vascular stem and root staining [VRS (Scale 0 ‐5)].

• Disease was monitored on plants at different days after inoculation (dai) 9, 12, 14, 16, 19, 23, and 24. 

• QTL used mean phenotypic scores of dai from the various experiments.

Greenhouse TestSeeded RIL Population

race 1 FOV infection days after Inoculation (dai)

race 1 FOV Seedling Inoculation

Plant InfectionDisease severity index (DSI) of leaves, (scale 0 – 5)

Vascular stem and root staining (VRS)

DSI = 0 DSI = 2 ‐ 3 DSI = 5

VRS = 0 VRS = 2 ‐ 3 VRS =5

Field Assay

Field Assay

2006 Day 9      UC Riverside 139 2.84 1.372006 Day 14 139 3.68 1.232006 Day 23 139 4.56 0.88

2007 Day 12    UC Riverside 138 1.24 0.992007 Day 14 138 2.68 1.142007 Day 16 138 3.48 1.002007 Day 19 138 3.89 0.952007 Day 23 138 4.24 0.90

2008  Day 24  UC Kearney 137 2.52 1.08

Field Test  Shafter CA RIL No Mean SD

2007 Day 67 after planting 113 1.61 0.71

2007 Day 67 % Survival 113 85.74 13.37

2007 Day 146 % Survival 113 27.39 19.36

0

10

20

30

40

50

60

70

80

12 Days 14 Days 16 Days 19 Days 23 Days

Nu

mb

er o

f RIL

s

Days after Inoculation (dai)

Disease Severity Index (DSI)

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0

3-79

TM-1 3-79 TM-1

3-79 TM-1

F1s ranged from 2.3 to 4.3 (Pima S-7 x Acala NemX)

Genotype/Population

Expected Ratios R:H:S & R:S 

Observed ratio R:S χ2 P‐value

Parents

Pima S7  All R  38:5 

Acala   NemX  All S 5:39

F1 All S 6:60

F2 33:100 (1:3) 31:102 0.203 NS

Parents 

Upland TM1  All S 0:5

Pima 3‐79  All R 4:1

RIL (2007) Day 14 41:97 (1:3) 35:103 1.379 NS

(1:1) 35:103 18.03 0.001

Upland TM1 All S 0:4

Pima 3‐79  All R 1:3

RIL (2008) Day 24 52:78 (1:3) 34:103 17.83 0.001

52:78 (1:1) 68:69 4.94 0.05

Race 1 FOV Inheritance in Pima by Acala and Upland by Pima Cottons

LocusTM1 allele 

Mean3‐79 allele 

Mean K*† P value

24 SSRs  Identified 2.88 ‐ 3.10 1.70 – 2.35 8.2 – 22.5  0.01  

SSR Markers and QTLs for FOV race 1 Resistance based on Vascular Stem and Root 

Staining

Nine Chromosomes: 01, 06, 08, 09, 11, 12, 15, 16, and 19 

†Kruskal‐Wallis analysis (K*) test  ‐ nonparametric 1‐way ANOVA 

C I R 2 0 3 _ 2 4 0 b0 . 0G h 0 3 2 _ 1 1 6 / 9 74 . 5

B N L 2 5 6 9 _ 1 6 4 / 1 6 91 0 . 9G h 0 3 9 _ 1 3 1 / 1 1 91 5 . 3

N A U 5 2 6 9 _ 1 5 0 / 1 5 52 5 . 8

M U S B 0 7 5 4 _ 3 5 9 h4 5 . 4

B N L 3 8 1 2 _ 2 4 7 / 2 9 56 3 . 2

M U S S 2 7 5 _ 2 2 3 b7 8 . 1

B N L 2 8 8 4 _ 1 7 7 / 1 9 38 9 . 2

M U S B 0 7 8 0 - 1 3 5 / 1 3 20 . 0N A U 1 3 3 6 _ 1 6 3 / 1 5 94 . 3M U S S 1 3 0 _ 2 5 7 - 2 5 14 . 9N A U 1 1 6 4 _ 1 7 4 / 1 6 95 . 7

N A U 3 6 0 5 _ 2 2 2 / 2 2 71 3 . 8N A U 1 0 3 7 _ 2 1 3 / 2 2 11 4 . 9N A U 4 9 0 0 _ 1 4 8 / 1 4 21 7 . 0B N L 3 2 5 51 9 . 4M U S B 0 4 2 2 _ 3 3 02 2 . 0M U S B 0 1 0 0 - 1 0 4 / 9 42 4 . 2M U S S 2 5 0 b2 4 . 6N A U 3 7 0 8 _ 2 3 7 / 2 2 83 0 . 0

N A U 0 8 9 13 7 . 8N A U 0 9 5 4 _ 2 0 8 / 2 0 03 9 . 1N A U 1 3 0 2 _ 2 1 6 / 2 0 34 3 . 7M U C S 1 1 34 6 . 4N A U 3 4 9 9 _ 2 7 0 / 2 8 54 7 . 5

M U S S 0 2 20 . 0

B N L 2 5 9 08 . 8

M U S S 0 8 31 4 . 3

B N L 1 4 1 42 1 . 4M U S S 1 8 1 _ 1 8 3 / 1 8 42 2 . 9

N A U 1 0 4 6 _ 2 3 1 / 2 2 62 9 . 1M U C S 0 3 1 _ 2 6 . 0 h3 3 . 1G h 2 4 73 5 . 2B N L 1 3 1 73 6 . 9G h 4 8 44 1 . 4N A U 1 0 0 94 4 . 1M U C S 0 8 04 5 . 8M U S S 2 6 64 8 . 7G h 1 1 85 2 . 3N A U 3 9 6 7 _ 2 6 2 b5 3 . 1G h 4 9 55 4 . 8M U S S 5 7 9 _ 1 7 3 / 1 7 15 7 . 3N A U 1 3 6 0 _ 2 2 3 / 2 1 56 1 . 2M U S S 5 4 76 4 . 4B N L 1 6 7 26 6 . 8M U S S 1 1 0 _ 2 7 5 / 2 7 76 9 . 0M U S S 3 1 67 2 . 6M U C S 3 2 57 6 . 4

M U S B 0 4 0 4 _ 3 6 5 h0 . 0

C I R 0 0 3 _ 1 3 2 / 1 3 51 3 . 3

M U S B 0 8 4 9 _ 2 4 8 b1 8 . 6M U S B 0 8 2 7 _ 3 2 6 / 3 2 22 2 . 8B N L 3 5 9 2 _ 2 0 7 / 1 9 92 6 . 5B N L 1 4 0 8 _ 2 0 7 / 1 9 72 9 . 4N A U 1 0 1 4 _ 2 7 6 b3 1 . 9M U S B 1 2 7 8 _ 3 9 9 b3 3 . 5M U S B 0 8 2 3 _ 2 2 7 / 2 3 53 6 . 7M U S B 1 1 6 3 _ 2 8 8 b3 8 . 3M U S B 1 0 3 5 _ 4 2 5 h4 0 . 2M U S S 0 9 2 _ 3 1 4 / 3 1 84 4 . 5

M U C S 3 9 9 _ 2 1 9 / 2 2 44 9 . 1

M U S B 1 0 1 5 _ 2 9 7 b5 5 . 0

M U S B 0 6 4 1 _ 3 0 2 h6 7 . 2M U S B 1 0 0 0 _ 3 5 6 / 3 0 86 9 . 3

B N L 0 5 8 0 _ 2 7 4 h0 . 0

B N L 2 9 8 6 _ 1 6 5 / 1 7 11 0 . 2

B N L 3 0 0 8 _ 1 4 4 / 1 5 51 5 . 3G h 2 9 5 _ 9 6 / 8 41 7 . 8

M U C S 5 9 4 _ 2 4 0 b2 6 . 8G h 3 4 5 _ 1 2 2 / 1 1 33 0 . 3

M U C S 6 1 6 _ 0 / 2 3 74 0 . 8

M U S B 0 8 1 2 _ 4 3 4 / 3 9 74 7 . 0

N A U 0 9 2 20 . 0

C I R 3 7 36 . 1N A U 3 6 5 0 _ 2 5 6 h9 . 9

N A U 5 2 7 3 _ 2 5 8 b1 6 . 0

N A U 2 5 0 3 _ 2 4 0 / 2 1 72 5 . 3N A U 1 2 2 1 _ 2 3 2 / 2 3 82 6 . 9G h 1 0 9 _ 1 1 9 / 1 0 83 1 . 5N A U 2 8 1 6 _ 1 7 0 / 1 7 83 1 . 9B N L 3 3 4 7 _ 1 7 7 / 1 7 03 3 . 7B N L 8 5 2 b3 7 . 8B N L 0 8 2 7 _ 1 8 6 / 1 9 03 9 . 3B N L 1 6 7 1 _ 1 8 7 / 1 6 14 3 . 2B N L 1 6 7 1 _ 1 8 2 / 1 7 34 7 . 9

N A U 2 2 3 2 _ 1 8 1 h5 4 . 1

Chromosome 6 Chromosome 9 Chromosome 16

Chromosome 8 Chromosome 11 Chromosome 19

QTL =

MGG 2011

MUCS594_269b0.0MUSB0812_452h0.6

MUCS616_256b10.7

M1E731.1BNLl3008_150/13933.3Gh295_86/10034.3M-CAA_E-AGC-16935.5

M-CAA_E-AAC-8540.2

Gh345_116/12642.8

Fov148.5

Fov1-C1652.0MUSB0812_412/40152.3

BNL0580_274h0.0

BNL2986_165/17110.2

BNL3008_144/15515.3Gh295_96/8417.8

MUCS594_240b26.8

Gh345_122/11330.3

MUCS616_0/23740.8

Fov1-C1646.9MUSB0812_434/39747.0

Chromosome 16

F2 (Pima‐S7 x NemX) Population

RIL (TM1 x Pima 3‐79) Population

Major QTLs were detected : QTL on chromosome 6 explained from 8.0 to 22.3% of variation in DSI with additive effect ranging from 0.33 to 0.51. QTL on chromosome 8 explained from 8.8 to 10.6% of DSI variation with additive effect ranging from 0.31 to 0.33. QTLs on chromosome 11 (two) explained from 9.0 to 14.2% of DSI variation with an additive effect averaging 0.39 − 0.43. QTL on Chromosome 19explained 13% of DSI variation with an additive effect of 0.46.

QTL explained 27% of DSI variation with an additive 

effect of 0.77.  

QTL explained 31% of VRS variation with an additive 

effect of 0.72.

LOD > 6

Mapping Fusarium wilt (FOV) race 1 resistance  summary

A set of 24 SSR markers indicated gene interactions and inheritance from nine cotton chromosomes, with major QTLs detected on five. Breeding targets.

The single recessive locus (Fov1), previously identified in F2 (Pima‐S7x NemX) mapped to chromosome 16 and co‐located with the Fov1 and QTL in the TM1 x 3‐79 RIL. Breeding target. 

Breeding for Race 4 Fusarium wilt (FOV) Disease Resistance

Fusarium wilt (FOV) Symptoms

Plant InfectionResistant < 2.0 & Susceptible > 2.0

Disease severity index (DSI) of leaves, (scale 0 – 5)

Vascular stem and root staining [VRS (scale 0 ‐ 5)]

DSI = 0 DSI = 2 ‐ 3 DSI = 5

VRS = 0 VRS = 2 ‐ 3 VRS = 5

Race 4 FOV Disease ResistanceResistant & Susceptible

Resistant & F1 Progeny

0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

4.00

NMSI

1601

8810

DP-74

4Ph

yto-81

0RPh

yto-80

0OA

-353

PS-7

A1_9

A1_1

8A1

_52

A2_3

1A2

_72

A2_8

0A2

_241

D_65

D_70

F_DamageV_Stain

Identification of Resistant Sources from Early EvaluationsPima Cottons and Accessions

PS: R2 = 0.56 &

h2 = 0.61

F_Damage: R2 = 0.58 &

h2 = 0.64

VRS: R2 = 0.63 &

h2 = 0.70

VRS

VRS

Sca l e

Race 4 FOV Inheritance Ulloa et al (2006)

Pima by Pima Crosses

Root top Vascular Stain Evaluation

Vascular Root Staining Fusarium wilt (FOV)

0.000.501.001.502.002.503.003.504.004.505.00

Phyto

gen 7

2

Pima S

-7Ph

ytoge

n 800

SJ-07

P-FR0

1SJ

-07P-F

R02

SJ-07

P-FR0

3SJ

-07P-F

R04

Cultivars

Roo

t Sta

inin

g

S40 FOV race 1 & RKNKern FOV race R4Fresno FOV race R4

Plant Percentage Survival

Percentage Survival Rate Fusarium wilt (FOV)

0.010.020.030.040.050.060.070.080.090.0

100.0

Phyto

gen 7

2

Pima S

-7Ph

ytoge

n 800

SJ-07

P-FR

01SJ

-07P-

FR02

SJ-07

P-FR

03SJ

-07P-

FR04

Cultivars

Perc

enta

ge S40 FOV race 1 & RKNKern FOV race R4Fresno FOV race R4

Four Pima Germplasm Lines Jointly Released by USDA-ARS, Univ. of California, & NMSU

SJ-07P-FR01

SJ-07P-FR02

SJ-07P-FR03

SJ-07P-FR04

Continuing efforts for releasing additional germplasm with improved Yield, Fiber, and Pest Resistance

0.00

400.00

800.00

1,200.00

1,600.00

2,000.00

2,400.00

2,800.00

3,200.00

3,600.00

4,000.00

Seed Cotton Lint Yield

Pima New Releases USDA-ARS and Univ. of CA. (R. Percy, R. Hutmacher, S. Wright, and M. Ulloa)

LB per Acre

0.00

0.50

1.00

1.50

2.00

2.50

F_Damage

V_Stain

Identification of Resistant Sources from Early EvaluationsAcala and Upland Cottons

0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

4.00

4.50

5.00

AU‐1107

AU‐5367

AU‐5491

AU‐6103

Ark0001‐01

‐03

Ark0002‐03

‐02

Ark0008‐22

‐10

Ark0012‐03

‐08

Ark0015‐06

‐11

GA2

004089

GA2

004230

GA2

004303

GA2

006128

LA05307029

LA05307061

LA05307083

LA05307094

0149

‐17 ne

0147

‐22 ne

0020

‐31 ne

0028

‐16 ne

8921

‐2‐2‐14‐13

‐11

04PST‐246

04PST‐250

04‐PST‐275

Acala 1517

‐99

NM 03012

PD03011

PD03025

PD04012

PD99041

TAM B182‐34

DP 393

FM 958

Phytogen

 72

SG 105

Pima‐S7

Phytogen

800

SJ‐07P

‐FR0

1

SJ‐07P

‐FR0

2

SJ‐07P

‐FR0

3

SJ‐07P

‐FR0

4

Shafter FOV race 1 & RKN Kern FOV race 4 Fresno FOV race 4

Vascular Root Staining [scale 0 = no staining to 5 = plant death]

AL AR GA LA MS MS NM SC TX Checks CA

RBTN Entries in 2008

0

10

20

30

40

50

60

70

80

90

100

AU‐5491

AU‐5367

AU‐6103

AU‐1107

Ark0012‐03

‐08

Ark0015‐06

‐11

Ark0002‐03

‐02

Ark0001‐01

‐03

Ark0008‐22

‐10

GA2

004230

GA2

004089

GA2

004303

GA2

006128

LA05307029

LA05307061

LA05307083

LA05307094

0149

‐17 ne

0147

‐22 ne

0020

‐31 ne

0028

‐16 ne

8921

‐2‐2‐14‐13

‐11

04PST‐250

04‐PST‐275

04PST‐246

Acala 1517

‐99

NM 03012

PD99041

PD04012

PD03025

PD03011

TAM B182‐34

DP 393

FM 958

SG 105

Phytogen

 72

Pima‐S7

Phytogen

800

SJ‐07P

‐FR0

1

SJ‐07P

‐FR0

2

SJ‐07P

‐FR0

3

SJ‐07P

‐FR0

4

Shafter FOV race 1 & RKN Kern FOV race 4 Fresno FOV race 4

AL AR GA LA MS MS NM SC TX Checks CA

Percent of Plant Survival [scale 0 to 100 %]

RBTN Entries in 2008

QTL mapping & breeding resistance to FOV race 4

1200 genome-wide markers analyzed

Populations from Upland-by-Upland, Upland-by-Pima, and Pima-by-Pima cottons.

FBCX-2 Shorty PS 6 3-79 DP 744 Uzbekistan A1

Nem_ARS X X X X X

FBCX-2 X X X

Shorty X X X XPS 6 X X X XPS 7 X X X X X X

DP 744 X X X XTM 1 X X XA2 X

Parents, Crosses, & Progeny for FOV & Root-knot nematode Research

0

20

40

60

0 1 2 3 4 50

20

40

60

0 1 2 3 4 5

Num

ber o

f R

ILs

0

20

40

60

80

0 1 2 3 4 5

0

10

20

30

40

0 1 2 3 4 5

0

20

40

60

0 1 2 3 4 5

0

20

40

60

0 1 2 3 4 5

A

Num

ber o

f Pl

ants

Num

ber o

f R

ILs

Num

ber o

f R

ILs

Num

ber o

f Pl

ants

Num

ber o

f Pl

ants

TM-1

Pima-S6F1

Pima-S6

F1

B

C

Shorty

Pima-S6Shorty

D

E

F

TM-1Pima 3-79

Pima 3-79TM-1

Pima 3-79

TM-1

FOV 4 Screens for VSR in F2 and RILs

VRS Scale VRS Scale

VRS Scale

VRS Scale VRS Scale

VRS Scale

MUSB0255_228/2640.0Gh465_1161.9Gh272_134/1244.3GH237_102/1116.9

GH171_248/23411.2

MUSS354_3970.0

Fov4-C146.0

BNL0834_150/14326.9

CIR2921_49/19045.5

BNL3932_249/23652.0

CIR228_20165.7

A

BNL3347_172/1590.0

Gh109_107/9411.0

NAU2816_176/8414.9

NAU1221_233/24120.1

BNL1671_19329.1

NAU2232_159h38.4

BNL1690_128/14142.3

Gh537_174/1650.0

Gh224_111/1048.6

BNL3103_192/21012.9NAU3092_163/15815.1

BNL2691_304/29023.0

A

Chromosome 8

Chromosome 14

Chromosome 19

Chromosome 25

Fig 2

Major QTLs =Detected on three populations:RIL TM-1 x Pima 3-79 (A)F2 TM-1 x Pima-S6 (B)F2 Shorty x Pima-S6 (C)

Minor QTLs = Detected on at least two populations:

A B C

BC A B

C

A B

Major QTL for FOV1 QTL on chromosome 16 explained from 27 to 31% of variation in DSI  & VRSwith additive effect ranging from 0.72 to 0.77.QTL on chromosome 6 explained from 8.0 to 22.3% of variation in DSI with additive effect ranging from 0.33 to 0.51. QTL on chromosome 8 explained from 8.8 to 10.6% of DSI variation with additive effect ranging from 0.31 to 0.33. QTLs on chromosome 11 (two) explained from 9.0 to 14.2% of DSI variation with an additive effect averaging 0.39 − 0.43. QTL on Chromosome 19 explained 13% of DSI variation with an additive effect of 0.46.

Major QTL for FOV4 QTL on chromosome 8 explained from 5 to 19% of variation in DSI  & VRS.QTL on chromosome 14 explained from 18 to 80% of variation in DSI with additive effect ranging from 0.36 to 1.58; and 76% of variation for DSI and 1.10 additive effect. QTL on chromosome 19 explained 72% of VRS variation with dominant effect ranging of 1.80. QTLs on chromosome 25 explained from 15% of VRS variation and 1.80 additive effect . 

Different QTL for Resistance to Race 1 and Race 4