Development of Methods for Improving Flowering and Seed Set of Diverse Germplasm in Cassava Breeding.

Cassava breeding faces obstacles due to late flowering and poor flower and seed set. The acceleration of breeding processes and the reduction in each cycle's duration hinge upon efficiently conducting crosses to yield ample progeny for subsequent cycles. Our primary objective was to identify methods that provide tools for cassava breeding programs, enabling them to consistently and rapidly generate offspring from a wide array of genotypes. In greenhouse trials, we examined the effects of the anti-ethylene silver thiosulfate (STS) and the cytokinin benzyladenine (BA). STS, administered via petiole infusion, and BA, applied as an apical spray, combined with the pruning of young branches, significantly augmented the number of flowers. Controls produced no flowers, whereas treatments with pruning plus either BA or STS alone produced an average maximum of 86 flowers per plant, and the combination of pruning, BA and STS yielded 168 flowers per plant. While STS had its primary effect on flower numbers, BA increased the fraction of female flowers from less than 20% to ≥87%, thus increasing the number of progeny from desired parents. Through field studies, we devised an optimal protocol that maintained acceptable levels of phytodamage ratings while substantially increasing seed production per plant compared to untreated plants. This protocol involves adjusting the dosage and timing of treatments to accommodate genotypic variations. As a result, cassava breeding programs can effectively leverage a diverse range of germplasm to develop cultivars with the desired traits.

Tracking crop varieties using genotyping-by-sequencing markers: a case study using cassava (Manihot esculenta Crantz).

BACKGROUND: Accurate identification of crop cultivars is crucial in assessing the impact of crop improvement research outputs. Two commonly used identification approaches, elicitation of variety names from farmer interviews and morphological plant descriptors, have inherent uncertainty levels. Genotyping-by-sequencing (GBS) was used in a case study as an alternative method to track released varieties in farmers' fields, using cassava, a clonally propagated root crop widely grown in the tropics, and often disseminated through extension services and informal seed systems. A total of 917 accessions collected from 495 farming households across Ghana were genotyped at 56,489 SNP loci along with a "reference library" of 64 accessions of released varieties and popular landraces. RESULTS: Accurate cultivar identification and ancestry estimation was accomplished through two complementary clustering methods: (i) distance-based hierarchical clustering; and (ii) model-based maximum likelihood admixture analysis. Subsequently, 30% of the identified accessions from farmers' fields were matched to specific released varieties represented in the reference library. ADMIXTURE analysis revealed that the optimum number of major varieties was 11 and matched the hierarchical clustering results. The majority of the accessions (69%) belonged purely to one of the 11 groups, while the remaining accessions showed two or more ancestries. Further analysis using subsets of SNP markers reproduced results obtained from the full-set of markers, suggesting that GBS can be done at higher DNA multiplexing, thereby reducing the costs of variety fingerprinting. A large proportion of discrepancy between genetically unique cultivars as identified by markers and variety names as elicited from farmers were observed. Clustering results from ADMIXTURE analysis was validated using the assumption-free Discriminant Analysis of Principal Components (DAPC) method. CONCLUSION: We show that genome-wide SNP markers from increasingly affordable GBS methods coupled with complementary cluster analysis is a powerful tool for fine-scale population structure analysis and variety identification. Moreover, the ancestry estimation provides a framework for quantifying the contribution of exotic germplasm or older improved varieties to the genetic background of contemporary improved cultivars.

High-resolution mapping of resistance to cassava mosaic geminiviruses in cassava using genotyping-by-sequencing and its implications for breeding.

Cassava mosaic disease (CMD), caused by different species of cassava mosaic geminiviruses (CMGs), is the most important disease of cassava in Africa and the Indian sub-continent. The cultivated cassava species is protected from CMD by polygenic resistance introgressed from the wild species Manihot glaziovii and a dominant monogenic type of resistance, named CMD2, discovered in African landraces. The ability of the monogenic resistance to confer high levels of resistance in different genetic backgrounds has led recently to its extensive usage in breeding across Africa as well as pre-emptive breeding in Latin America. However, most of the landraces carrying the monogenic resistance are morphologically very similar and come from a geographically restricted area of West Africa, raising the possibility that the diversity of the single-gene resistance could be very limited, or even located at a single locus. Several mapping studies, employing bulk segregant analysis, in different genetic backgrounds have reported additional molecular markers linked to supposedly new resistance genes. However, it is not possible to tell if these are indeed new genes in the absence adequate genetic map framework or allelism tests. To address this important question, a high-density single nucleotide polymorphism (SNP) map of cassava was developed through genotyping-by-sequencing a bi-parental mapping population (N=180) that segregates for the dominant monogenic resistance to CMD. Virus screening using PCR showed that CMD symptoms and presence of virus were strongly correlated (r=0.98). Genome-wide scan and high-resolution composite interval mapping using 6756 SNPs uncovered a single locus with large effect (R(2)=0.74). Projection of the previously published resistance-linked microsatellite markers showed that they co-occurred in the same chromosomal location surrounding the presently mapped resistance locus. Moreover, their relative distance to the mapped resistance locus correlated with the reported degree of linkage with the resistance phenotype. Cluster analysis of the landraces first shown to have this type of resistance revealed that they are very closely related, if not identical. These findings suggest that there is a single source of monogenic resistance in the crop's genepool tracing back to a common ancestral clone. In the absence of further resistance diversification, the long-term effectiveness of the single gene resistance is known to be precarious, given the potential to be overcome by CMGs due to their fast-paced evolutionary rate. However, combining the quantitative with the qualitative type of resistance may ensure that this resistance gene continues to offer protection to cassava, a crop that is depended upon by millions of people in Africa against the devastating onslaught of CMGs.