Characterization and screening for Cassava mosaic disease

(CMD)resistance

Paul. A. Asare (Ph.D)

Department of Crop Science

U. C. C

Outline of presentation

• Introduction

• Objective

• Materials and method

• Results

• Conclusion

• Some breeding work at UCC

Introduction

• Cassava (Manihot esculenta)

– Is one of the world’s most important tropical plants

– The fifth source of carbohydrate in the tropics

• Contributes 22% of the total Agriculture Gross

Domestic Product (AgGDP) (Parkes 2009)

• It is also a source of income for most rural dwellers.

CMD is an important constraint to cassava production in Africa (Geddes, 1990; Zhou et al., 1997; Huiping et al., 2010).

Estimated total crop yield losses due to CMD on the continent amounts to about US $440 million per annum (Thresh et al., 1997).

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Objectives of the study

• The objectives of the study were:

– to characterize the various accessions using morphological descriptors and molecular markers.

– to screen for mosaic resistant accessions.

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Materials and methods

• Forty three (43) different cassava plants (accessions) were used

• Land preparation – A portion of land, at the School of Agriculture Teaching and

Research farm, was cleared, ploughed and harrowed

• Planting of cassava accessions – Single row planting method of 1m x 1m length was used.

– Ten cuttings for each accession.

• .

.

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Morphological Characterization

• The accessions were first characterized base on morphological descriptors (IITA, 1990).

• Both qualitative and quantitative data were taken on shoot and root morphology

• Data collection started six weeks after planting through to 12 months after planting (MAP).

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Molecular Characterization

–Molecular characterization • DNA extraction was done using the CTAB

protocol I (Murray and Thompson 1990).

• DNA quality and quantitation was done using spectrophotometer.

• DNA was stored at -20oC.

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Molecular Characterization cont’d • PCR Amplification

– PCR amplification was carried out using 36 pairs of cassava SSR primers

– hPAGE was used

– Documentation

– 20 SSR primers that produced clear bands were used for the analysis

June 7, 2011 9

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Fig. 1 Distribution of first leaf colour in cassava germplasm

0

5

10

15

20

25

30

35

40

45

Light green Dark green Green purple Purple

Perc

en

tage

Fre

qu

ency

Colour of first leaf

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Results

Figure 2: Distribution of petiole colour in cassava germplasm.

Light green Dark green Green purple Purple

Petiole colour

0

10

20

30

40

50

60

70 P

erc

en

tage

Fre

qu

en

cy

June 7, 2011 11

Figure 3: Distribution of petiole length in cassava germplasm.

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0

10

20

30

40

50

60

5-14 15-24 >25

Perc

en

tage

Fre

qu

ency

Petiole lenght (cm)

Figure 4: Distribution of stem colour in cassava germplasm.

0

5

10

15

20

25

30

35

40

45

50

Silver green Light brown/orange Dark brown

Pe

rce

nta

ge F

req

ue

ncy

Stem colour

June 7, 2011 13

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Figure 5: Distribution of root surface colour in cassava germplasm.

0

10

20

30

40

50

60

White/Cream Light brown Dark brown

Perc

en

tage

Fre

qu

ency

Root surface colour

June 7, 2011 14 PAA

Figure 6: Distribution of storage root pulp colour in cassava germplasm.

0

20

40

60

80

100

120

White Yellow

Pe

rce

nta

ge F

req

ue

ncy

Storage root pulp colour

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BANKYEBOTAN

ADW051

UCC517

KW085

UCC506

I

AFS001

DMA002

ADW063

OFF146

KW070

OFF029

UCC470

KW181

AFS131

OFF025

II

KW001

NN43

AFS027

ADW004

OFF145

ADEHYE

CAPEVARS

OFF019

ADW053

DMA066

III

AFS126

OFF086

KW161

OFF023

ASAMAN

OFF058

NN42

AFS136

KW148

OHYEOKA

OFF093

BESEREBEMA

NKABOM

OFF063

OFF136

UCC153

AFS041

AFS048

IV

0.000.050.100.150.200.250.300.350.40

Figure 7: Dendrogram of cassava accessions based on morphological data

16

BANKYEBOTAN

ADW051

UCC517

KW085

UCC506

I

AFS001

DMA002

ADW063

OFF146

KW070

OFF029

UCC470

KW181

AFS131

OFF025

II

KW001

NN43

AFS027

ADW004

OFF145

ADEHYE

CAPEVARS

OFF019

ADW053

DMA066

III

AFS126

OFF086

KW161

OFF023

ASAMAN

OFF058

NN42

AFS136

KW148

OHYEOKA

OFF093

BESEREBEMA

NKABOM

OFF063

OFF136

UCC153

AFS041

AFS048

IV

0.000.050.100.150.200.250.300.350.40

Figure 7: Dendrogram of cassava accessions based on morphological data 17

BANKYEBOTAN

ADW051

UCC517

KW085

UCC506

I

AFS001

DMA002

ADW063

OFF146

KW070

OFF029

UCC470

KW181

AFS131

OFF025

II

KW001

NN43

AFS027

ADW004

OFF145

ADEHYE

CAPEVARS

OFF019

ADW053

DMA066

III

AFS126

OFF086

KW161

OFF023

ASAMAN

OFF058

NN42

AFS136

KW148

OHYEOKA

OFF093

BESEREBEMA

NKABOM

OFF063

OFF136

UCC153

AFS041

AFS048

IV

0.000.050.100.150.200.250.300.350.40

Figure 7: Dendrogram of cassava accessions based on morphological data

18

BANKYEBOTAN

ADW051

UCC517

KW085

UCC506

I

AFS001

DMA002

ADW063

OFF146

KW070

OFF029

UCC470

KW181

AFS131

OFF025

II

KW001

NN43

AFS027

ADW004

OFF145

ADEHYE

CAPEVARS

OFF019

ADW053

DMA066

III

AFS126

OFF086

KW161

OFF023

ASAMAN

OFF058

NN42

AFS136

KW148

OHYEOKA

OFF093

BESEREBEMA

NKABOM

OFF063

OFF136

UCC153

AFS041

AFS048

IV

0.000.050.100.150.200.250.300.350.40

Figure 7: Dendrogram of cassava accessions based on morphological data

19

BANKYEBOTAN

ADW051

UCC517

KW085

UCC506

I

AFS001

DMA002

ADW063

OFF146

KW070

OFF029

UCC470

KW181

AFS131

OFF025

II

KW001

NN43

AFS027

ADW004

OFF145

ADEHYE

CAPEVARS

OFF019

ADW053

DMA066

III

AFS126

OFF086

KW161

OFF023

ASAMAN

OFF058

NN42

AFS136

KW148

OHYEOKA

OFF093

BESEREBEMA

NKABOM

OFF063

OFF136

UCC153

AFS041

AFS048

IV

0.000.050.100.150.200.250.300.350.40

Figure 7: Dendrogram of cassava accessions based on morphological data

20

I OHYEOKA

KW148

KW001

AFS041

AFS048

OFF063

II

ADEHYE

CAPEVARSIII

UCC517

UCC506IV

NKABOM

UCC153V

B.BOTAN

OFF136

UCC470

KW161

OFF086

OFF023

OFF093

KW085

ADW051

KW181

OFF146

AFS126

OFF145

VI

ADW053

OFF025

AFS131

DMA002

DMA066

ADW004

ASAMAN

BESEREBEMA

ADW063

AFS136

OFF019

NN42

NN43

AFS001

KW070

OFF029

VII

VIII AFS027

IX OFF058

0.000.050.100.150.200.25

Figure 2: Dendrogram of cassava accessions based on SSR data 21

I OHYEOKA

KW148

KW001

AFS041

AFS048

OFF063

II

ADEHYE

CAPEVARSIII

UCC517

UCC506IV

NKABOM

UCC153V

B.BOTAN

OFF136

UCC470

KW161

OFF086

OFF023

OFF093

KW085

ADW051

KW181

OFF146

AFS126

OFF145

VI

ADW053

OFF025

AFS131

DMA002

DMA066

ADW004

ASAMAN

BESEREBEMA

ADW063

AFS136

OFF019

NN42

NN43

AFS001

KW070

OFF029

VII

VIII AFS027

IX OFF058

0.000.050.100.150.200.25

Figure 2 Dendrogram of cassava accessions based on SSR data

22

I OHYEOKA

KW148

KW001

AFS041

AFS048

OFF063

II

ADEHYE

CAPEVARSIII

UCC517

UCC506IV

NKABOM

UCC153V

B.BOTAN

OFF136

UCC470

KW161

OFF086

OFF023

OFF093

KW085

ADW051

KW181

OFF146

AFS126

OFF145

VI

ADW053

OFF025

AFS131

DMA002

DMA066

ADW004

ASAMAN

BESEREBEMA

ADW063

AFS136

OFF019

NN42

NN43

AFS001

KW070

OFF029

VII

VIII AFS027

IX OFF058

0.000.050.100.150.200.25

Figure 2: Dendrogram of cassava accessions based on SSR data

23

I OHYEOKA

KW148

KW001

AFS041

AFS048

OFF063

II

ADEHYE

CAPEVARSIII

UCC517

UCC506IV

NKABOM

UCC153V

B.BOTAN

OFF136

UCC470

KW161

OFF086

OFF023

OFF093

KW085

ADW051

KW181

OFF146

AFS126

OFF145

VI

ADW053

OFF025

AFS131

DMA002

DMA066

ADW004

ASAMAN

BESEREBEMA

ADW063

AFS136

OFF019

NN42

NN43

AFS001

KW070

OFF029

VII

VIII AFS027

IX OFF058

0.000.050.100.150.200.25

Figure 2: Dendrogram of cassava accessions based on SSR data

24

I OHYEOKA

KW148

KW001

AFS041

AFS048

OFF063

II

ADEHYE

CAPEVARSIII

UCC517

UCC506IV

NKABOM

UCC153V

B.BOTAN

OFF136

UCC470

KW161

OFF086

OFF023

OFF093

KW085

ADW051

KW181

OFF146

AFS126

OFF145

VI

ADW053

OFF025

AFS131

DMA002

DMA066

ADW004

ASAMAN

BESEREBEMA

ADW063

AFS136

OFF019

NN42

NN43

AFS001

KW070

OFF029

VII

VIII AFS027

IX OFF058

0.000.050.100.150.200.25

Figure 2: Dendrogram of cassava accessions based on SSR data

25

I OHYEOKA

KW148

KW001

AFS041

AFS048

OFF063

II

ADEHYE

CAPEVARSIII

UCC517

UCC506IV

NKABOM

UCC153V

B.BOTAN

OFF136

UCC470

KW161

OFF086

OFF023

OFF093

KW085

ADW051

KW181

OFF146

AFS126

OFF145

VI

ADW053

OFF025

AFS131

DMA002

DMA066

ADW004

ASAMAN

BESEREBEMA

ADW063

AFS136

OFF019

NN42

NN43

AFS001

KW070

OFF029

VII

VIII AFS027

IX OFF058

0.000.050.100.150.200.25

Figure 2: Dendrogram of cassava accessions based on SSR data 26

I OHYEOKA

KW148

KW001

AFS041

AFS048

OFF063

II

ADEHYE

CAPEVARSIII

UCC517

UCC506IV

NKABOM

UCC153V

B.BOTAN

OFF136

UCC470

KW161

OFF086

OFF023

OFF093

KW085

ADW051

KW181

OFF146

AFS126

OFF145

VI

ADW053

OFF025

AFS131

DMA002

DMA066

ADW004

ASAMAN

BESEREBEMA

ADW063

AFS136

OFF019

NN42

NN43

AFS001

KW070

OFF029

VII

VIII AFS027

IX OFF058

0.000.050.100.150.200.25

Figure 2: Dendrogram of cassava accessions based on SSR data

27

I OHYEOKA

KW148

KW001

AFS041

AFS048

OFF063

II

ADEHYE

CAPEVARSIII

UCC517

UCC506IV

NKABOM

UCC153V

B.BOTAN

OFF136

UCC470

KW161

OFF086

OFF023

OFF093

KW085

ADW051

KW181

OFF146

AFS126

OFF145

VI

ADW053

OFF025

AFS131

DMA002

DMA066

ADW004

ASAMAN

BESEREBEMA

ADW063

AFS136

OFF019

NN42

NN43

AFS001

KW070

OFF029

VII

VIII AFS027

IX OFF058

0.000.050.100.150.200.25

Figure 2: Dendrogram of cassava accessions based on SSR data

28

I OHYEOKA

KW148

KW001

AFS041

AFS048

OFF063

II

ADEHYE

CAPEVARSIII

UCC517

UCC506IV

NKABOM

UCC153V

B.BOTAN

OFF136

UCC470

KW161

OFF086

OFF023

OFF093

KW085

ADW051

KW181

OFF146

AFS126

OFF145

VI

ADW053

OFF025

AFS131

DMA002

DMA066

ADW004

ASAMAN

BESEREBEMA

ADW063

AFS136

OFF019

NN42

NN43

AFS001

KW070

OFF029

VII

VIII AFS027

IX OFF058

0.000.050.100.150.200.25

Figure 2: Dendrogram of cassava accessions based on SSR data

29

I OHYEOKA

KW148

KW001

AFS041

AFS048

OFF063

II

ADEHYE

CAPEVARSIII

UCC517

UCC506IV

NKABOM

UCC153V

B.BOTAN

OFF136

UCC470

KW161

OFF086

OFF023

OFF093

KW085

ADW051

KW181

OFF146

AFS126

OFF145

VI

ADW053

OFF025

AFS131

DMA002

DMA066

ADW004

ASAMAN

BESEREBEMA

ADW063

AFS136

OFF019

NN42

NN43

AFS001

KW070

OFF029

VII

VIII AFS027

IX OFF058

0.000.050.100.150.200.25

Figure 2: Dendrogram of cassava accessions based on SSR data

June 7, 2011 30

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Figure 13: Dendrogram of cassava accessions based on SSR data

BANKYEBOTAN

ADW051

UCC517

KW085

UCC506

I

AFS001

DMA002

ADW063

OFF146

KW070

OFF029

UCC470

KW181

AFS131

OFF025

II

KW001

NN43

AFS027

ADW004

OFF145

ADEHYE

CAPEVARS

OFF019

ADW053

DMA066

III

AFS126

OFF086

KW161

OFF023

ASAMAN

OFF058

NN42

AFS136

KW148

OHYEOKA

OFF093

BESEREBEMA

NKABOM

OFF063

OFF136

UCC153

AFS041

AFS048

IV

0.000.050.100.150.200.250.300.350.40

I OHYEOKA

KW148

KW001

AFS041

AFS048

OFF063

II

ADEHYE

CAPEVARSIII

UCC517

UCC506IV

NKABOM

UCC153V

B.BOTAN

OFF136

UCC470

KW161

OFF086

OFF023

OFF093

KW085

ADW051

KW181

OFF146

AFS126

OFF145

VI

ADW053

OFF025

AFS131

DMA002

DMA066

ADW004

ASAMAN

BESEREBEMA

ADW063

AFS136

OFF019

NN42

NN43

AFS001

KW070

OFF029

VII

VIII AFS027

IX OFF058

0.000.050.100.150.200.25

SSR marker Morphological markers

31

Marker Assisted Selection

• Laboratory screening.

– Six (6) pairs of CMD diagnostic primers were used to check for the various strains of the virus

– Four (4) pairs of primers associated with resistant (CMD2 gene) to the virus were used.

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Lab. Screening…cont’d • PCR amplification

Table 2: Nucleotide sequences of DNA primers used in polymerase chain reaction for the detection of cassava mosaic begomovirus

Harrison et al. (1997) and that of Zhou et al. (1997)

CMD2 markers were SSRY28, NS156, NS169 and RME1

(Akano et al., 2002; Fregene et al., 2001 )

Virus Name of primer Sequence (5’ to 3’)

ACMV ACMV-F1 (P1) ACMV-R1

ACMV-F2 (P2) ACMV-R2

ACMV-AL1/F (P3) ACMV-ARO/R

ACMV-1 (P3) ACMV-2

TTC AGT TAT CAG GGC TCG TAA

GAG TG AAG TTG ACT CAT GA

GTG AGA AAG ACA TTC TTG GC

CCT GCA ATT ATA TAG TGG CC

GCG GAA TCC CTA ACA TAA TC

GCT CGT ATG TAT CCT CTA AGG CCT

GCTC AAC TGG AGA CAC ACT TG

CCT GCA ACA TAC TTA CGC TT

EACMV/EACMV UV-AL3/F (P5) UV-AL1/F1

TAC ACA TGC CTC RAA TCC TG

CTC CGC CAC AAA CTT ACG TT

EACMV-Ug UV-AL1/F1 (P6) ACMV-CP/R3

TGT CTT CTG GGA CTT GTG TG TGC CTC CTG ATG ATT ATA TGTC

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None CAPEVARS

P4 ADEHYE

P1 NKABOM

ADW053

OFF063P1 & P4

AFS041

OFF023P1 & P3

P1, P2 & P4 DMA002

AFS136

KW181P2 & P4

P2, P3 & P4 UCC470

UCC517

ADW051

ADW063

KW085

OFF058

OFF093

OFF146

P1 P2 & P4

OFF086

OFF136P1, P3 & P4

NN42

NN43

OFF019

OFF025

OFF029

OFF145

OHYEOKA

UCC153

UCC506

ADW004

AFS001

AFS027

AFS048

AFS126

AFS131

ASAMAN

B.BOTAN

BESEREBEMA

DMA066

KW001

KW070

KW148

KW161

P1, P2, P3 & P4

0.000.050.100.150.200.250.30

Figure 3: Dendrogram showing 43 cassava accessions reaction patterns with four mosaic primers as determined by the unweighted pair

group method with arithmetic averages of binary character matrices using the similarity coefficient index (Nei, 1983)

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Conclusion

• The 43 cassava accessions used were genetically variable and clustered into groups not necessarily based on the source of collection.

• Molecular markers were more efficient in distinguishing the 43 accessions into 9 clusters, compared to morphological markers, which grouped them into 4 clusters

• The only CMG strain responsible for the disease symptoms in the study area was ACMV.

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Conclusion cont’d

This study identified only one genotype (Capevars) as resistant to the cassava mosaic disease CMD.

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Some plant breeding work at UCC

• Cassava germplasm collection – Central & Western Regions – 516 accessions from 23 districts – Through conventional breeding 2 varieties were released

to farmers

• Characterization of water yam accessions using both morphological and molecular techniques.

• Molecular characterization of Ghanaian Avocado pear

• Screening work – Striga resistance in cowpea – Drought resistance in cowpea – Drought resistance and ‘stay green’ trait in sorghum – Blight tolerance/resistance in taro