Genomics for transforming yam breeding

Ranjana Bhattacharjee (, 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.

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.
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.

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.

Leveraging “agrigenomics” for crop improvement

Melaku Gedil ( and Ismail Rabbi
M. Gedil, Head, Bioscience Center; I. Rabbi, Postdoctoral Fellow (Molecular Genetics), IITA, Ibadan, Nigeria

Harnessing state-of-the art genomics technologies
The potential application of “Omics” technology, as demonstrated by the steadily growing impact of biosciences, in alleviating the multitude of constraints in agricultural production is rapidly becoming a reality with the advent of next-generation DNA sequencing and genotyping technologies, high throughput (HTP) metabolomics and transcriptomics, informatics, and decision-making tools. These technologies, together with rapidly evolving bio-computational tools, are accelerating the discovery of genes and closely linked molecular markers underlying important traits, leading to the rapid accumulation of genomic resources necessary for devising an efficient and effective breeding strategy geared toward the faster development of varieties of choice.

Researchers in IITA's Bioscience Center. Photo by L. Kumar.
Researchers in IITA's Bioscience Center. Photo by L. Kumar.
The state-of-the-art technologies including the next-generation sequencing (NGS) for genome and transcriptome analysis, as well as genotyping-by-sequencing (GBS) are being adopted in R4D programs at IITA. For instance, the NGS through outsourcing and multi-partner collaboration; the RNAseq for HTP expression study in cassava; the Illumina’s Golden Gate Assay for HTP single nucleotide polymorphism (SNP) genotyping in cassava, soybean, and maize as well as GBS in maize and cassava. Data generated by these techniques are being applied for marker-assisted recurrent selection (MARS) of drought-tolerant maize, and genome selection (GS) for high-yielding, disease-resistant cassava.

Development of an integrated molecular breeding platform
The new technologies, however, are very data-intensive and demand advanced computational and communication technologies and infrastructure for data acquisition, analysis, and management. For the effective integration of genomics technologies in our breeding schemes, we are building capacity (connectivity to the internet, the necessary hardware/software, and skilled personpower) to acquire, store, and analyze terabytes of data.

The Generation Challenge Program (GCP) of the CGIAR is developing an integrated breeding platform (IBP) to build a comprehensive and integrated crop information system enabling linkages among molecular, phenotypic, and pedigree data. The maize version of International Crop Information System (ICIS), dubbed International Maize Information System (IMIS), has been expanded to include all pedigrees of IITA maize under the Drought Tolerant Maize for Africa (DTMA) project. It has some functionality in terms of molecular data storage but this is limited and we are now generating data sets of hundreds of thousands of markers per line that require different storage solutions. The GCP is consulting with other initiatives such as iPlant and DArT and is working on collaboratively creating solutions for the needs of several user-cases including DTMA, Tropical Legumes (TL)-I, and TL-II projects. In the IBP initiative, IITA is the leading crop center to host the main web-accessible databases of cassava, cowpea, yam, and soybean. The form and functionality of the databases are still a work in progress although activities are ongoing in the application of current versions of ICIS to cassava, yam, and cowpea.

In view of the IBP initiative, we are developing a bioinformatics capacity to (a) manage the newly generated genomic resources of IITA’s research crops, particularly those clonally propagated, (b) use the genomic resources in the public sector for soybean and maize, (c) use comparative genomics techniques for other African orphan crops of high importance, such as cassava, yam, and cowpea, and (d) create a bioinformatics center of excellence to train and provide access for African research scientists.

HTP by genotyping and informatics support tools
The increasing affordability of the NGS technologies has shifted critical consideration from genotyping to phenotyping. According to leading experts, it is now cheaper to genotype than to phenotype a plant. Quality phenotypic data are essential for the interpretation and use of the deluge of genomic data to identify the changes in DNA sequences that influence important traits. The fact that priority agronomic traits are complex and polygenic and interact with the environment necessitates conducting extensive and precise multi-environment evaluations of candidate breeding materials (over several years and in several locations). Therefore, there is a need to invest in precision phenotyping of traits and data capture (from electronic sample tracking to non-invasive HTP) through the use of hand-held devices such as barcode readers and near-infrared spectroscopy. Efforts are being made to develop rapid and accurate phenotyping protocols to integrate with genomic tools in establishing breeding schemes at IITA.

A wide array of techniques and tools is being deployed to associate molecular markers with desirable phenotypic traits. Associated markers can be used to accelerate germplasm enhancement via MARS, marker-assisted backcrossing for the introgression of disease resistance and other simple traits, hence bypassing the necessity of evaluating breeding materials in the field; MARS for rapid cycle population improvement in bi-parental crosses based on genomic estimated breeding value; and GS based on a model developed with a training population to select untested samples.

Our efforts to harness the unparalleled scientific progress in the fields of genomics and bioinformatics are expected to find solutions to the recalcitrant problems confronting small-holder farmers in sub-Saharan Africa.