B4FA 2013 Ghana: Cassava mosaic disease resistance - Paul Asare
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Characterization and screening for Cassava mosaic disease (CMD)resistance Paul. A. Asare (Ph.D) Department of Crop Science U. C. C
Transcript of B4FA 2013 Ghana: Cassava mosaic disease resistance - Paul Asare
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
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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
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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
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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
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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
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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 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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
