1. ABSTRACT Cassava mosaic disease (CMD) is the most important disease of cassava in Africa and can result in yield loss as high as 100%. The first deployed resistance to CMD was from Manihot glaziovii, which is thought to be polygenic. However, cassava varieties developed using this source of resistance has been observed to show increasing susceptibility to the disease. The discovery of the CMD2 dominant gene has improved breeding to cassava mosaic disease resistance. The use of monogenic or single gene is generally non-durable. Initiatives were therefore taken to identify new additional sources of resistance to build stable and durable resistance in cassava cultivars being developed at National Root Crops Research Institute (NRCRI) Umudike, Nigeria. Recently, International Institute of Tropical Agriculture (IITA) identified new varieties with very high resistance close to immunity that may likely represent new sources of resistance to the disease. Two of the varieties TMS 98/1089A and TMS 97/2205 are being used in crosses with CMD2 varieties to develop multigenic resistance to the disease. Seedling nursery evaluation of progenies developed were done at 3, 6, 12 months after planting on a scoring scale of 1 (highly resistant) to 5 (highly susceptible). In the last three years, genotypes within the range of moderate to high resistance were selected at 12 MAP and advanced into the next evaluation stage (Clonal trial) in the breeding scheme. Results from a four year evaluation study at the clonal trial stage in the NRCRI breeding programme indicate an average of 76% were highly resistant genotypes with severity score 1; 14.7% were resistant (severity score 2) and 0.034% were moderately resistant (severity score 3). The high proportion of the highly resistant genotypes compared with the other classes indicate that cross combination of resistant parents resulted in progenies of better and higher resistance to the disease and thus the likely possibility of enhancing durability. Initiatives are underway to map loci associated with disease resistance in the parent materials using SNPs. INTRODUCTION Cassava accounts for approximately one-third of the total staple food production and provides over 50% of the energy for more than 200 million people in sub-Saharan Africa (IITA 1992). In Africa alone, cassava is estimated to provide over 1000 Kcal per day to million people (Cock, 1985). CMD is one of the globally damaging plant virus disease with an annual economic loss of USD 1.9-2.7 billion (Legg and Fauquet, 2004). Cassava mosaic disease (CMD) is the most important disease of cassava in Africa and can result to yield loss as high as 100%. Host plant resistance is the most environmental friendly strategy to tackle the disease. The first source of resistance to the disease was deployed from Manihot glaziovii which is believed to be polygenic with a recessive component. Cassava breeding is complicated by the biology of the crop and its heterozygosity, which in the absence of inbred lines has imposed limitations to the successful use of this multigenic source of resistance in cassava genetic improvement. However, cassava varieties developed using this source of resistance have been observed to show increasing susceptibility to the disease. The second major source of resistance was identified in several Nigerian cassava landraces that have consistently shown strong resistance to CMD. High levels of resistance to CMD have been described in a group of closely related Nigerian cassava landraces (Akano et al., 2002). Since the 1990s, IITA and African NARs have been exploiting this resistance derived from TME 3 (Akano et al., 2002). The use of monogenic or single gene is generally non durable. Initiatives were therefore taken to identify new additional sources of resistance to build stable and durable resistance in cassava cultivars being developed at NRCRI. Under the CMD resistance project of IITA, resistance profile of cassava cultivars were tested in multilocational trials in Nigeria revealing top highly CMD resistant lines that are being considered as possible additional sources of CMD resistance. Two of these varieties include TMS 96/1089A and TMS 97/2205. We report here results of breeding activities to fully explore existing sources of CMD resistance to build durable and stable CMD resistance in new generation of cassava varieties being developed at NRCRI. MATERIALS AND METHODS Parent materials In the breeding programme at NRCRI, genotypes with attributes for key breeding traits including resistance are selected for use in hybridization in the crossing block. TME 3, TMS 97/2205 (Fig. 1), TMS 98/1089A and genotypes developed from backcross derivatives of M. glaziovii representing four sources of CMD resistance were used as parents for CMD resistance breeding. Improved genotypes or varieties having TME 3, TMS 98/1089A and TMS 97/2205 in their pedigrees have also been frequently used as parents for breeding for CMD resistance. Crosses Given the importance of CMD as primary criterion for the release of improved varieties, each cross made was designed to have at least one parent with CMD resistance. The F 1 progenies are then used in further crosses with other sources of resistance to pyramid CMD resistance genes to improve durability. Often times, two parents developed using different sources of CMD resistance were used in crosses. Since 2009, in addition to the M. glaziovii and TME 3 sources, the breeding programme has increasingly used TMS 98/1089A and TMS 97/2205 as new sources of CMD resistance in crosses. Crosses have been made to increase the chances of pyramiding the four sources of CMD resistance in F 1 progenies developed. Apart from controlled hybridization, a polycross design to allow for possible wide crosses through open pollination among parents to increase CMD resistance was also explored. Selfing of parent genotypes to explore the multigenic source of resistance was also done. Field establishment The breeding crossing block was established at the National Root Crop Research Institute (NRCRI) headquarters, Umudike (with bimodal rainfall; mean annual precipitation of 2200 mm; altitude 120 m; mean temperature of 22- 31 o C; latitude 05 o 29'N; longitude 07 o N 33'E; luvisol soils; humid forest environment). Umudike is a high pressure disease zone for CMD. The parent materials were planted in single row fields using the polycross design with over 50 parents. The materials were planted at a spacing interval of 1m x 1m using matured planting stakes of 25-30 cm. The crossing block was established in April/May of each year between 2008 and 2011. Crosses were made between August and November each year with fruits harvested and seeds processed between January and March of the following year. Seedlings were raised from the seeds either in the screenhouse before transplanting to field or sowed directly on the field. The seedlings were then tagged for CMD resistance, harvested at 12 months after planting (MAP) and then advanced into single row clonal evaluation trial for CMD evaluation. CMD evaluation Each year in the breeding programme at NRCRI, over 20,000 seedlings raised were tagged for CMD resistance and advanced into the clonal trial for further evaluation for CMD resistance as shown in the breeding scheme in Fig 2. Symptom severity index of CMD was used solely to classify cassava genotypes for their reaction to CMD. The CMD symptom severity was rated on a scale of 1 (highly resistant) to 5 (highly susceptible) (Fig. 3). Plants with no symptoms were scored as 1. Symptom severity index of 2 (SSI 2) was assigned for mild symptoms, SSI 3 for moderate symptoms, SSI 4 for severe symptoms and SSI 5 for highly severe symptoms. In the clonal trial, the genotypes were scored five times at 1, 3, 6, 9 and 12 month(s) after planting (MAP). The highest severity score at the peak of the disease was used to assess the CMD resistance potential of each genotype. NR 8082, and TMS 30555 were used as susceptible check cultivars. RESULTS AND DISCUSSION The aim of breeding for resistance is to produce cultivars with improved resistance that persists under a wide range of environmental conditions. The continued expansion of the CMD pandemic in Africa is a threat to production gains achieved in recent decades. Durability of resistance is therefore a priority in Africa. The principal selection criterion at early breeding stage is resistance to CMD. Results from a four year evaluation study at the clonal trial stage in the NRCRI breeding programme indicate an average of 76% were highly resistant genotypes with severity score of 1; 14.7% were resistant (severity score 2) and 0.034% were moderately resistant (severity score 3). The highly resistant genotypes ranged from 66 to 82% for the four year period. The high proportion of the highly resistant genotypes compared with the other classes indicate that cross combination of resistant parents over years resulted in progeny of better and high resistant to the disease and thus the likely possibility of enhancing durability over times. Year*CMD scores 12345 200882.513.70.0400 200986.313.40.00200 201071.60.240.0400 201166.429.80.0400 Table 1. CMD evaluation in the Clonal Evaluation trial at NRCRI Umudike from 2008 - 2011 *CMD severity scores on a scale of 1(highly resistant) to 5 (highly susceptible). The number of individuals per CMD score in the Table is expressed in percentage of the total number of individuals evaluated each year. Fig. 1. TMS 97/2205 at early development stage on the field Fig. 2 Breeding scheme for cassava as used in Africa. Source: IITA 13 4 5 Fig. 3: Cassava Mosaic Disease (CMD) severity scores REFERENCES Akano, A.O., Dixon, A.G.O, Mba, C. & Barrera, E. 2002. Genetic mapping of dominant gene conferring resistance to cassava mosaic disease. Theoretical and Applied Genetics. 105: 521-525. IITA. 1992. Cassava in tropical Africa. A reference manual. Chayce Publication Services, UK. Kawano, K. 1980. Cassava. P. 225-233. In: W. R. Fehr and H. H. Hadley (ed.) Hybridization of crop plants. ASA, Madison, WI. Legg, J.P., and Thresh, J.M.. 2000. Cassava mosaic virus disease in East Africa: A dynamic disease in a changing environment. Virus Res. 71:135–149. Okogbenin, E., Porto, M.C.M, Egesi, C., Mba, C., Ospinosa, E., Santos, L. G., Ospina, C., Marin, J., Barera, E.,Gutierrez, J., Ekanayake, I., Iglesias C. and Fregene, M. 2007. Marker aided introgression of CMD resistance in Latin American germplasm for genetic improvement of cassava in Africa. Crops Science 47:1895-1904 Overtime, record has revealed that the number of the families selected were dominated by progeny from TME 3, TMS 96/1089A and TMS 97/2205. For example, in the 2011 selection, 22 families of the 38 families were largely from those having either TMS 96/1089A or TMS 97/2205 CMD resistant parents. In the families having TMS 97/2205 as a parent, 59% of the progeny were highly resistant to the disease while 36% were resistant. For TMS 96/1089A families, 51% were highly resistant with 49% being resistant. The strategy adapted in selfing some of the resistant genotypes was largely to take advantage of the multigenic resistance for the M. glaziovii source. Cassava is highly heterozygous and cassava breeding has until recent years not explored selfing due to inbreeding depression. Good vigor and field performance have been reported for advanced selfed generation showing that selfing may be explored for genetic gain in cassava. Evaluation of CMD resistance showed that the average CMD score in the last four years in selected germplasm have been consistently low with severity scores ranging from 1.1 to 1.4 (Fig. 4). This indicates that the currently deployed sources of variation are very effective and could prove yet useful in combating the threat posed by the strong evolutionary capacity of the virus. The current scheme is largely envisaged to permit the successful pyramiding of CMD resistance genes in the new varieties developed. Genetic mapping of CMD loci have been done for TME 3 and partially for M. glaziovii source. Current initiative are underway at NRCRI to map genes with SNP markers in TMS 97/2205 and TMS 96/1089A Recently released varieties by NRCRI have been demonstrated to show good resistance indicating that the present breeding strategy for durable resistance to CMD is effective and is expected to rapidly result in improved cassava production in Nigeria and Africa. Fig. 4 Average CMD score in the clonal evaluation for four years at NRCRI Umudike, Nigeria Kenneth Eluwa 1, Emmanuel Okogbenin 1 *, Chiedozie Egesi 1, Olalekan Akinbo 1, Eunice Ekaette 1, Favour Ewa 1, Ezenwaka Lydia 1, M. Okoro 1 and Martin Fregene 2 1 National Root Crops Research Institute (NRCRI) Umudike, Nigeria; 2 CIAT, Cali, Colombia corresponding email address: eokogbenin@yahoo.com ACKNOWLEDGEMENT We acknowledge National Root Crops Research Institute (NRCRI), Umudike, Nigeria for providing the fund and conducive environment for this study. The fund for attending this conference was provided by Bill and Melinda Gates Foundation through the organizers of GCP21.