B4FA 2013 Ghana: Cassava mosaic disease resistance - Paul Asare

1. Characterization and screening for Cassava mosaic disease (CMD)resistance Paul. A. Asare (Ph.D) Department of Crop Science U. C. C

2. Outline of presentation • Introduction • Objective • Materials and method • Results • Conclusion • Some breeding work at UCC

3. 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.

4. 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).

5. 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.

6. 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.

7. 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).

8. 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.

9. 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

10. 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 PercentageFrequency Colour of first leaf Results

11. 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 PercentageFrequency

12. Figure 3: Distribution of petiole length in cassava germplasm. 0 10 20 30 40 50 60 5-14 15-24 >25 PercentageFrequency Petiole lenght (cm)

13. 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 PercentageFrequency Stem colour

14. Figure 5: Distribution of root surface colour in cassava germplasm. 0 10 20 30 40 50 60 White/Cream Light brown Dark brown PercentageFrequency Root surface colour

15. Figure 6: Distribution of storage root pulp colour in cassava germplasm. 0 20 40 60 80 100 120 White Yellow PercentageFrequency Storage root pulp colour

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

21. IOHYEOKA KW148 KW001 AFS041 AFS048 OFF063 II ADEHYE CAPEVARS III UCC517 UCC506 IV NKABOM UCC153 V 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 VIIIAFS027 IXOFF058 0.000.050.100.150.200.25 Figure 2: Dendrogram of cassava accessions based on SSR data

22. IOHYEOKA KW148 KW001 AFS041 AFS048 OFF063 II ADEHYE CAPEVARS III UCC517 UCC506 IV NKABOM UCC153 V 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 VIIIAFS027 IXOFF058 0.000.050.100.150.200.25 Figure 2 Dendrogram of cassava accessions based on SSR data

26. IOHYEOKA KW148 KW001 AFS041 AFS048 OFF063 II ADEHYE CAPEVARS III UCC517 UCC506 IV NKABOM UCC153 V 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 VIIIAFS027 IXOFF058 0.000.050.100.150.200.25 Figure 2: Dendrogram of cassava accessions based on SSR data 26

31. 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 IOHYEOKA KW148 KW001 AFS041 AFS048 OFF063 II ADEHYE CAPEVARS III UCC517 UCC506 IV NKABOM UCC153 V 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 VIIIAFS027 IXOFF058 0.000.050.100.150.200.25 SSR markerMorphological markers

32. 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.

33. 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 AGAAAG ACA TTC TTG GC CCT GCAATT ATA TAG TGG CC GCG GAA TCC CTAACA TAA TC GCT CGT ATG TAT CCT CTA AGG CCT GCTC AAC TGG AGA CAC ACT TG CCT GCAACA 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

34. NoneCAPEVARS P4ADEHYE P1NKABOM ADW053 OFF063 P1 & P4 AFS041 OFF023 P1 & P3 P1, P2 & P4DMA002 AFS136 KW181 P2 & P4 P2, P3 & P4UCC470 UCC517 ADW051 ADW063 KW085 OFF058 OFF093 OFF146 P1 P2 & P4 OFF086 OFF136 P1, 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)

35. 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.

36. Conclusion cont’d This study identified only one genotype (Capevars) as resistant to the cassava mosaic disease CMD.

37. 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

B4FA 2013 Ghana: Cassava mosaic disease resistance - Paul Asare

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B4FA 2013 Ghana: Cassava mosaic disease resistance - Paul Asare