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Key message More than 40 QTLs associated with 14 stress-related, quality and agro-morphological traits were identified. A catalogue of favourable SNP markers for MAS and a list of candidate genes are provided. Abstract Cassava (Manihot esculenta) is one of the most important starchy root crops in the tropics due to its adaptation to marginal environments. Genetic progress in this clonally propagated crop can be accelerated through the discovery of markers and candidate genes that could be used in cassava breeding programs. We carried out a genome-wide association study (GWAS) using a panel of 5130 clones developed at the International Institute of Tropical Agriculture—Nigeria. The population was genotyped at more than 100,000 SNP markers via genotyping-by-sequencing (GBS). Genomic regions underlying genetic variation for 14 traits classified broadly into four categories: biotic stress (cassava mosaic disease and cassava green mite severity); quality (dry matter content and carotenoid content) and plant agronomy (harvest index and plant type) were investigated. We also included several agro-morphological traits related to leaves, stems and roots with high heritability. In total, 41 significant associations were uncovered. While some of the identified loci matched with those previously reported, we present additional association signals for the traits. We provide a catalogue of favourable alleles at the most significant SNP for each trait-locus combination and candidate genes occurring within the GWAS hits. These resources provide a foundation for the development of markers that could be used in cassava breeding programs and candidate genes for functional validation.

Cassava (Manihot esculenta L.) is one of the most important food crops in the tropics, but yields are far below their potential. The gene pool of cas- sava contains natural genetic diversity relevant to many important breeding goals, but breeding progress has been slow, partly because of insuf- ficient genomic resources. As a first step toward implementing genomewide genetic studies that will facilitate rapid genetic gain through breed- ing, we genotyped-by-sequencing a set of 182 full-sibs population of cassava that segregated in several traits: resistance to the cassava mosaic disease (CMD) and yield under CMD pressure; increased carotenoid content in storage roots; color of stem exterior and anthocyanin pigmen- tation in the petioles, inner root skin, and api- cal leaves. Employing a rare-cutting restriction enzyme, PstI, in a genotyping-by-sequencing (GBS) library preparation, we obtained 2478 segregating single nucleotide polymorphisms (SNPs), of which 1257 passed standard filter - ing for missing genotypes and deviation from expected genotypic frequencies. We mapped 772 SNPs across 19 linkage groups and anchored 313 unique scaffolds from the version 4.1 of the cassava genome assembly. Most of the stud- ied morphological traits as well as resistance to CMD and root carotenoid content showed quali- tative inheritance. As expected, quantitative trait loci analysis for these traits revealed single loci surrounded by small confidence intervals. Yield under CMD was associated with the CMD resis- tance locus. We show that GBS is a powerful genotyping tool that provides a sufficient number of markers for unraveling the genetic architecture of Mendelian traits in cassava in addition to the development of a robust genetic map that can help anchor unassembled genomic scaffolds.

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 (R2=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.

Cross contamination arising from random natural processes and breeding errors is one of the prominent challenges to breeding in many crops including cassava ( Manihot esculenta ). This study attempts to identify molecular marker tools suitable for verifying the genetic purity of putative progenies of populations of cassava. It also proposes a rapid, high-throughput genomic DNA extraction protocol which is a modification of an existing extraction protocol adapted to the pace required for the DNA-based verification of often large population sizes. Three polymorphic simple sequence repeat (SSR) markers were selected from a total of 125 and used for genotyping three populations. The petiole color trait was also used to verify TMS 96/1089A X TME117 where the pink color of the male parent was dominant over the female’s green color. The pace of genomic analysis of populations used in the study was enhanced using a modified , quicker DNA isolation protocol which slashed extraction time by 60%. SSRY153 identified six false progenies in the selfed line, 1M18. Segregation patterns of In combination, both markers identified 2 selfed individuals out of a total of 207. NS890 identified two false hprogenies resulting from selfing in the female parent of the cross TMS 30001 X TMS 96/1089A, out of a total of 93, The principles described for verification using each of the SSR markers applied in this study can be extended to other SSR markers with corresponding nature of polymorphism in any population of the crop. Increased attention would go to the development of more efficient markers such as single nucleotide polymorphisms (SNPs) and other gene-based markers using new and advanced genomics techniques for routine integration of such quality control step in the breeding scheme. Key words: Cassava, simple sequence repeat (SSR), morphological trait, molecular markers, genomic DNA.