Yam breeding at IITA: achievements, challenges, and prospects

Antonio Lopez-Montes (a.lopez-montes@cgiar.org), Ranjana Bhattacharjee, and Gezahegn Tessema
A. Lopez-Montes, Yam Breeder; R. Bhattacharjee, Molecular Geneticist; G. Tessema, Associate Professional Officer, IITA, Ibadan, Nigeria

Yam is an impotant staple food in West Africa. Photo by IITA.
Yam is an impotant staple food in West Africa. Photo by IITA.
Yam—an integral part of the West African food system
Yam (Dioscorea spp.) is a multi-species, clonally propagated crop cultivated for its starchy tubers. About 10 species are widely cultivated around the world, but only D. rotundata, D. alata, and D. cayenensis are the most widely cultivated species in West Africa, accounting for 93% of the global yam production. Since its inception, IITA R4D efforts have focused on developing new varieties of yam with desired agronomic and quality traits and to improve yam-based cropping systems.

Largest collection of yam genetic resources
IITA maintains the largest world collection of yam, accounting for over 3,000 accessions mainly of West African origin. The collection represents eight species: D. rotundata (67%), D. alata (25%), D. dumetorum (1.6%), D. cayenensis (2%), D. bulbifera (2%), D. mangenotiana (0.25%), D. esculenta (0.7%), and D. praehensilis (0.3%). The passport data and characterization information on these accessions are maintained in databases accessible at http://genebank.iita.org/. On request, these germplasm accessions are distributed following Standard Material Transfer Agreements (SMTA). As in many other crops, the request for gene bank accessions has been low for use in national and international yam improvement programs. Of a total of 3170 accessions, only 1077 accessions have been distributed in the last 10 years.

To increase the use of yam germplasm, which are a wealth of rare alleles for target traits, a core collection (391 accessions) was established in 2006 representing 75% of genetic diversity of the entire collection using data on 99 morphological descriptors and country of origin. The germplasm collection is being genotyped using 18 DNA-based markers. Presently, research efforts are under way in collaboration with CIRAD for cryopreservation, using liquid nitrogen, to reduce the cost of maintenance of such a large collection. Efforts to improve yam germplasm conservation and use will be continued under the framework of the CGIAR Research Program (CRP) on Roots, Tubers and Bananas (RTB) for Food Security and Income. As part of this program efforts will be made to (a) optimize ex situ and in situ yam conservation methodologies; (b) increase coverage of yam gene pools; (c) evaluate, genotype, and phenotype yam collections for important traits; (d) enrich databases with information on yam collections and make it freely accessible to users; and (e) improve procedures for safe exchange of RTB genetic resources.

Making the difference
IITA’s yam breeding program has mainly focused on clonal selection from landraces and hybridization of elite clones of D. alata and D. rotundata. Conventional breeding efforts in yam have resulted in substantial achievements leading to release of high-yielding and disease-resistant cultivars. For instance, through collaborative evaluation of IITA-derived breeding lines with national research institutes (National Root Crop Research Institute, Umudike, Nigeria, and the Crops Research Institute, Ghana), 10 varieties of D. rotundata (10 during 2001–2009 in Nigeria and 1 in 2007 in Ghana) and 5 varieties of D. alata (during 2008–2009 in Nigeria) were released. More lines are in the pipeline to be released by these institutions in Nigeria and Ghana, and also in Benin, Burkina Faso, Côte d’Ivoire, Sierra Leone, Togo, and Liberia. The released varieties have multiple pest and disease resistance, wide adaptability, and good organoleptic attributes.

Novel vertical sacs method for seed yam production using vine cuttings. Photo by L. Kumar.
Novel vertical sacs method for seed yam production using vine cuttings. Photo by L. Kumar.
Some work has also been carried out in interspecific hybridization, but it is faced with a lot of challenges, including cross-compatibility and synchronization of flowering. For instance, D. rotundata can be crossed to D. cayenensis, but crossing either of the two to D. alata has not been successful. Research effort in interspecific hybridization has been geared towards the genetic improvement of yam, primarily on D. rotundata, D. cayenensis, and D. alata by transferring complementary traits from one to the other, e.g., higher carotenoid in D. cayenensis transferred to D. rotundata by interspecific hybridization.

Besides success in hybridization, efforts of the breeding program resulted in identification of resistance to nematodes (D. dumetorum), fungi and viruses (D. alata and D. rotundata); selection of germplasm for their response to soil nutrients and nutrients use efficiency; physicochemical characterization of D. alata for food quality, sensory evaluation of ‘amala’ (yam flour paste) and pasting characteristics of fresh yam as indicators of textural quality in major food products. Studies are ongoing to determine the variation in nutrient retention during processing of yam into food products; characterization of tuber micronutrient density, specifically for iron, zinc, total carotenoids, ascorbic acid (vitamin C), phytate, and tannin content. Traits, such as photoperiod response, flowering, and dormancy are also being studied in D. rotundata.

The future thrust will be on reducing the breeding period required to develop improved varieties with consumer-preferred traits, as well as increased participation of stakeholders for improved efficiency and impact of the yam breeding program. Developing participatory value chain strategy will set priorities not only for research and development but also for a consistent value chain articulation and low risk models to link farmers to markets. Yam for food security, food industry (flour, pasta, noodles, pancakes etc.), and pharmacology (drugs, cosmetics) needs prioritized by stakeholders will drive the development of new varieties, that are high yielding, resistant to diseases and pests, and with good adaptability to specific production systems, low fertility soils, and dry environments. GIS-based characterization of yam production systems, yam growth models and genome sequencing will provide strategic knowledge for the success of the yam breeding program. Rapid and high-ratio seed yam propagation systems will support the variety development and dissemination efforts to breeders and other stakeholders. The implementation of the new scheme is expected to reduce the time to develop and recommend new varieties from 9 to 3.5 years and facilitate rapid release of consumer-preferred varieties by the national programs.

Genomic resources for yam improvement
Research on biotechnology of yam includes tissue culture, genetic transformation, and development and use of molecular markers. However, no genetically modified yam has been produced so far although this approach could be used to transfer resistance to virus and anthracnose diseases into popular commercial varieties. Progress on yam genomics and transformation is covered in Bhattacharjee et al.

Researchers in accelerated yam breeding trial plot. Photo by L. Kumar.
Researchers in accelerated yam breeding trial plot. Photo by L. Kumar.
Future prospects
Review of constraints in yam production in West Africa identified the high cost of planting material, high labor costs, poor soil fertility, low yield potential of local varieties, pests and diseases (on-farm and in storage), and shortage of quality seed yam of popular landraces and released varieties as major limitations. To overcome these challenges, in the next five years under the CRP-RTB framework, yam breeding efforts will focus on (a) development of new breeding tools and strategies, (b) trait capture and gene discovery, (c) pre-breeding for new traits, (d) development of new varieties incorporating consumer-preferred characters, and (e) aligning research with farmer and end-user priorities.

These efforts will be supported by the ongoing R4D programs on developing efficient phenotyping protocols for nutrient use efficiency, moisture stress tolerance and biotic stresses in different yam species; regeneration protocol for transformation of various species (D. rotundata, D. alata, and D. cayenensis); methods for efficient interspecific hybridization among D. alata, D. rotundata, D. bulbifera, D. cayenensis, and D. dumetorum; establishment of marker-assisted breeding platform; techniques for rapid propagation of high quality seed yam; protocol for double haploids from yam microspores; and adoption of stakeholder participatory approaches in development and release of new varieties. Ongoing efforts to strengthen seed yam systems for ensuring sustainable production and supply of quality seed yam in West Africa, and communication and promotional strategies for the dissemination of breeding materials and improved varieties underpin the success of these efforts.

Genomics for transforming yam breeding

Ranjana Bhattacharjee (r.bhattacharjee@cgiar.org), Melaku Gedil, and Antonio Lopez-Montes
R. Bhattacharjee, Molecular Geneticist; M. Gedil, Head, Bioscience Center; A. Lopez-Montes, Yam Breeder, IITA, Ibadan, Nigeria

Breeding challenges in yam
Yam (Dioscorea spp.), a multi-species, polyploidy, and vegetatively propagated crop, is an economically important staple food for more than 300 million people in West Africa, Asia, Oceania, and the Caribbean. The five major yam-producing countries in West Africa (Bénin, Côte d’Ivoire, Ghana, Nigeria, and Togo) account for 93% of worldwide production. Dioscorea rotundata and D. alata are the species most commonly cultivated in West Africa1.

Scientists strategizing genomics for precision breeding. Photo by L. Kumar.
Scientists strategizing genomics for precision breeding. Photo by L. Kumar.
The genetic improvement of yam is faced with several constraints, including the long growth cycle (about 8 months or more), dioecy, plants that flower poorly or not at all, polyploidy, vegetative propagation, heterozygous genetic background, and poor knowledge about the genetics of the crop2. Progress has been made in breeding to develop F1 full-sib mapping populations from crossing male and female parents of D. rotundata for traits such as multiple tuber production, improved cooking quality, and virus disease resistance; and of D. alata for resistance to anthracnose, improved cooking quality, and reduced tuber oxidation3. These are valuable sources of populations for genetic analysis in yam for its improvement.

Current status of yam genomics
There is no convenient model system for yam genomics. In recent years, some progress has been made in the development of molecular markers to assess their potential for germplasm characterization and phylogenetic studies in D. rotundata-cayenensis and their wild progenitors, such as D. abyssinica and D. prahensilis. Two framework linkage maps were constructed using D. alata that included 338 AFLP markers on 20 linkage groups with a total map length of 1055 cM; and D. rotundata in which 107 AFLP markers were mapped on 12 linkage groups (585 cM) for the male and 13 linkage groups (700 cM) for the female. Three quantitative trait loci (QTLs) on the male and one QTL on the female were identified for resistance to yam mosaic virus (YMV). Similarly, one AFLP marker was found to be associated with anthracnose resistance on linkage group 2, explaining about 10% of the total phenotypic variance.

Another linkage map was generated for D. alata based on 508 AFLP markers that covered a total length of 1233 cM on 20 linkage groups, accounting for about 65% of the entire genome. Genes conferring resistance to YMV have been identified in D. rotundata and to anthracnose in D. alata by the successful use of bulked segregant analysis (BSA). Two RAPD markers, OPW18850 and OPX15850, closely linked in coupling phase with the dominant YMV-resistance locus Ymv-1 were identified. Similarly, two RAPD markers, OPI171700 and OPE6950, closely linked in coupling phase with anthracnose resistance gene, Dcg-1, were identified2.

Designing molecular markers using a bioinformatics platform. Photo by A. Alonge, IITA.
Designing molecular markers using a bioinformatics platform. Photo by A. Alonge, IITA.
Enriching the repertoire of molecular markers
In an effort to develop additional genomics resources, IITA was involved in sequencing ESTs from a cDNA library constructed from floral tissue. However, the first several hundred sequences were predominantly housekeeping genes. Recently, in a collaborative project with University of Virginia through USAID-Linkage funds, several thousand ESTs were generated using cDNA libraries from yam leaf tissues challenged with Colletotrichum gloeosporioides, the fungal pathogen responsible for yam anthracnose disease. This resulted in the identification of >800,000 EST sequences, from which about 1152 EST-SSRs were generated in D. alata for use in a yam improvement program. Although AFLP markers have been used for generating linkage maps so far, efforts are under way to saturate the maps with these EST-SSRs to identify the genomic regions associated with resistance to anthracnose disease.

DNA barcoding
Species identification in the genus Dioscorea has remained a challenge when active domestication is continuing in several parts of West Africa. Research on DNA barcoding is under way using chloroplast markers (rbcL, matK, and trnH-psbA) to understand the phylogenetic relationship between different species and also to get an insight into the ongoing domestication process.

Whole genome sequencing
Important considerations for the whole genome sequencing of yam include the genome size, ploidy level, and availability of homozygous clones. Estimation of the genome sizes of various Dioscorea species showed widely variable figures: D. alata and D. rotundata have genome sizes of about 800 mega base pairs (Mbp). Recently, an initiative was launched at IITA in collaboration with the Japan International Research Center for Agricultural Sciences (JIRCAS) to complete the whole genome sequencing of D. rotundata. Preliminary data yielded reasonable sequences. Further work is in progress to generate additional sequence data from the BAC library to facilitate the assembly of the genome which will culminate in producing the first draft genome sequence of Dioscorea species. Additional genomic information produced by resequencing several breeding materials and a parallel project in transcriptome analysis are poised to result in the discovery of a large number of molecular markers and help in the annotation of the genome.

Transcriptome analysis
In contrast to the genome sequence, which is fixed and uniform in all cells of a particular organism, transcriptome refers to the study of the total set of transcripts (expressed genes) in a given cell/tissue at a particular developmental stage or external environmental condition that could influence the physiology of the cell/tissue. IITA, in collaboration with USDA-Agricultural Research Service, Stoneville, embarked on RNAseq, the latest revolutionary tool for transcriptome profiling, based on differential gene expression for anthracnose disease. One of the expected outcomes of this project is to enrich the genomic resources available for yam improvement, including the discovery of SNPs. The latest informatics and statistical methods will be applied to saturate the available linkage map and high resolution mapping of the QTL(s) for anthracnose resistance in different genetic backgrounds.

Genotyping-by-sequencing
With advances in the next generation technologies, the costs of DNA sequencing have come down to such an extent that genotyping-by-sequencing (GBS) is now possible in almost all crops. IITA has recognized the potential of such innovative techniques in accelerating the breeding of clonally propagated crops, such as yam. Hence, in an ongoing USAID-Linkage project, a diverse panel of D. alata genotypes, including parents of available mapping population progenies segregating for anthracnose disease will be genotyped by sequencing to identify a large set of SNPs and determine the divergence among the parents.

Yam roots. Photo by IITA.
Yam roots. Photo by IITA.
Conclusions
To meet the steadily increasing demand, the viable approach is to adopt the innovative plant breeding strategies for yam that integrate the latest innovations in molecular technologies with conventional breeding practices. As efforts are under way to obtain the complete genome sequences and the development of additional genomic resources, the groundwork for deploying yam molecular breeding has been laid. With the availability of new genomic markers and GBS, it would be possible to fingerprint yam germplasm to identify duplicates/mislabeled accessions, to conduct diversity analysis and association mapping. As the genus Dioscorea contains several other useful species, comparative genomic tools can be used to transfer or deduce genetic and genomic information in other species.

References
1 Gedil, M. and A. Sartie. 2010. Aspects of Applied Biology 96:123–135.
2 Mignouna, H.D., M.M. Abang, and R. Asiedu. 2007. Yams. Pages 271–296 in: Genome mapping and molecular breeding Vol. 3: Pulses, Sugar and Tuber Crops, edited by C. Kole. Springer, Heidelberg, Berlin, New York, and Tokyo.
3 Sartie, A. and R. Asiedu. 2011. Development of mapping populations for genetic analysis in yams (Dioscorea rotundata Poir. and Dioscorea alata L.). African Journal of Biotechnology 10: 3040–3050.