Biotechnology and Crop Genetics is one of five research themes at Crops For the Future (CFF). The Biotechnology and Crop Genetics Theme is dedicated to applying biotechnology for the genetic improvement of underutilised crops, and works with all research programmes at CFF to identify the needs for each crop. Due to the lack of research on underutilised legumes in general, we employ methodology translation from model and major crops to underutilised crops to understand the underlying genetics for further improvement of these crops. The research carried out under this theme for underutilised legumes uses bambara groundnut (Vigna subterrana (L.) Verdc.) as the exemplar species, and winged bean (Phosphocarpus tetraglobulus) as our first test species.
Generating resources to assist breeding in underutilised crops
A major focus of the research theme is to access germplasm resources for the diversification of agricultural systems and the improvement of resilience traits. This happens alongside translation of information from major and model crops to underutilised crops to allow a better understanding of the traits of importance in these species and their modes of action in better studied species. Genome sequences currently exist for few underutilised crops, so one of our approaches is to focus on generating resources from transcriptomes and genome-wide genotyping using the Genotype-by-Sequencing (GbS) technique.
Note: Lablab sequences were generated in collaboration with Kirkhouse Trust (www.kirkhousetrust.org)
Developing SSR markers
The availability of functional microsatellite markers allows quality control and genetic diversity applications. For example, SSR with large size differences between parental genotypes can be used to evaluate the hybrid status of controlled crosses (Fig. 1). Analysis of 123 individual plant accessions from an Africa-wide collection of material using 12 SSR markers suggested an average residual observed heterozygosity (Ho) of 0.01, suggesting that bambara groundnut is a highly inbreeding species.
Fig. 1. Use of SSR markers to distinguish genuine bambara groundnut F1 hybrid seed (H) on a simple agarose gel system. P: parental alleles
Conversion of multi-locus SNP markers to a single locus format
The information rich multi-locus marker systems need to be converted to a simple and cheap format for use in breeding programmes, in this case, by using the specificity of a thermostable ligase and allele-specific tagged primers. Image shows conversion from the 64bp sequence tag for DArT Seq (Fig. 2).
Fig. 2. With the addition of known dNTPs, amplification (arrow) revealed (a) presence of the ‘T’ allele and (b) presence of the ‘G’ allele.
Translating resources to assist breeding in underutilised crops
Without having species-specific genome resources, we have attempted to utilise resources and information from major crops such as Phaseolus vulgaris, Glycine max and Cajun cajunus. These are estimated to have diverged 5, 20 and 20 million years ago from the ancestor of bambara groundnut, respectively (Canon et al., 2009). Using the 64bp sequence tag associated with DArT Seq markers, we have developed a number of approaches to map bambara groundnut genetic markers onto the physical genomes of sequenced major legume species (Fig. 3)
Figure 3: The alignment of DArTseq markers to different well annotated nearest available genomes
Generating a pseudo-physical map
Approximately 36% of the DArTseq tags detected a good match in the common bean sequence (>80% identity), allowing us to generate a comparative genetic (bambara groundnut) vs physical map (common bean) alignment, shown below. In general, bambara groundnut shows good correspondence to one or two physical chromosome segments in common bean, at least at the macrosynteny level.
Fig. 4. The linkage between the bambara groundnut genetic map and the Phaseolus vulgaris genome sequence