Chromosome evolution and the genetic basis of agronomically important traits in greater yam

Yams (*genus Dioscorea*) are a crucial food and income source in tropical and subtropical regions, providing over 200 dietary calories daily for approximately 300 million people. Their nutritional richness, including carbohydrates, protein, and vitamin C, coupled with months-long storability, ensures year-round availability. Global yam production, estimated at 72.6 million tons in 2018, is heavily concentrated in West Africa, where the crop holds significant cultural and religious importance. While primarily dioecious outcrossers, yams are vegetatively propagated, facilitating the preservation of desirable traits across planting seasons. Greater yam (*Dioscorea alata* L.) stands out due to its wide global distribution, believed to originate in Southeast Asia or Melanesia, spreading to East and West Africa over centuries. Its adaptability, high yield, ease of propagation, and nutritional value make it a prime candidate for systematic improvement. Despite a doubling of global yam production in the last two decades, this increase is largely attributed to expanded cultivated areas, not enhanced productivity. This necessitates the urgent development of improved yam varieties to meet growing demands and mitigate production constraints. Conventional breeding for greater yam faces challenges due to its long growth cycle, erratic flowering, and polyploidy. Breeders are actively developing varieties with improved yield, pest and disease resistance, and desirable tuber quality. Anthracnose, a devastating fungal disease caused by *Colletotrichum gloeosporioides*, poses a major threat, causing significant production losses. However, some resistance exists within greater yam landraces and breeding lines. The development of high-quality genomic resources is crucial for accelerating breeding methods and improving yam production.

Previous research on greater yam has focused on understanding its genetic diversity, ploidy status, and resistance to diseases like anthracnose. Studies using AFLP markers and microsatellite analysis have investigated genetic diversity and relationships with related species. Cytogenetic and microsatellite analyses have clarified the polyploidy nature of many *Dioscorea* species. Efforts have also been made to construct genetic linkage maps for QTL mapping of anthracnose resistance and sex determination. However, a high-quality, chromosome-scale genome assembly has been lacking until now, hindering the application of modern genomic tools to yam improvement. Existing genomic resources, including EST-SSRs and GBS-SNPs, have been used to map QTLs for anthracnose resistance; however, the mapping resolution was limited, and the identified QTL did not always align between studies, suggesting challenges associated with phenotypic plasticity or other technical considerations.

This study generated a high-quality reference genome sequence for *D. alata* using a combination of long-read (PacBio) and short-read (Illumina) sequencing technologies. PacBio sequencing provided long reads (234x coverage, 15.1 kb N50 read length) for assembling the genome, while Illumina sequencing was used for polishing and mate-pair linkage. Hi-C data and a composite meiotic linkage map were utilized to organize contigs into chromosome-scale sequences. The genome assembly spans 479.5 Mb, aligning well with previous estimates. To corroborate the assembly and provide tools for genetic analysis, ten genetic linkage maps were constructed from eleven mapping populations using DArTseq technology. A total of 13,584 biallelic markers were generated, which were integrated into a single, high-resolution composite linkage map (1817.9 cM) with 10,448 markers. This composite map represents a significant improvement in resolution compared to previous efforts. The genome annotation identified an estimated 25,189 protein-coding genes. Extensive transcriptome data (short-read RNA-seq and long, single-molecule direct-RNA sequencing) were incorporated into the annotation process. Comparative genomics was performed using the *D. alata* genome sequence, along with those of *D. rotundata*, *D. dumetorum*, and *D. zingiberensis* to assess genome-wide synteny, collinearity, and structural rearrangements. This provided insights into the evolution of the *Dioscorea* genome. To evaluate the utility of the generated resources, a QTL mapping analysis was conducted for anthracnose resistance and tuber quality traits using multiple mapping populations. Phenotyping involved field trials and detached leaf assays for anthracnose, and measurements of dry matter, oxidation, tuber color, and corm characteristics. Whole-genome resequencing of eight breeding lines was also conducted to investigate genetic variation and relationships between the lines. Analyses involved estimating identity-by-descent (IBD) to assess relationships among the breeding lines. The SNV data was used to assess relationships among lines, quantify genomic regions of homozygosity (ROH) and heterozygosity, and to infer potential interspecific introgression. Phylogenetic analysis of mitochondrial and plastid sequences provided further insights into the evolutionary relationships of different *Dioscorea* species. Statistical tests were implemented to assess the evidence of autotetraploidy vs. allotetraploidy following genome duplication events. The statistical methods to do this were explicitly detailed within the methods section. The whole process is described in extensive detail in the supplementary information.

The study successfully generated a high-quality, chromosome-scale genome assembly for *D. alata*, representing a significant advance in genomic resources for this important crop. The genome assembly revealed an ancient allotetraploidization event in the *Dioscorea* lineage, followed by extensive genome reorganization. The genome encodes an estimated 25,189 protein-coding genes. A high-resolution genetic linkage map was constructed, which corroborated the genome assembly. Comparative genomic analysis indicated significant conservation of chromosome structure between *D. alata* and *D. rotundata*, but considerable rearrangement relative to *D. zingiberensis*. Analysis of the *D. alata* genome revealed evidence of two ancient paleotetraploidies, with the most recent one (designated 'delta') likely an allotetraploidy based on biased gene retention analysis. This delta duplication is estimated to have occurred approximately 64 million years ago. The older duplication event, ‘tau’, is inferred to be shared by other core monocots. QTL mapping identified eight significant QTLs: three for anthracnose resistance (chromosomes 5, 19, and 6) and five for tuber quality traits (dry matter, oxidation, tuber color, corm type, and tuber size/shape, chromosomes 18, 12, and 7). The chromosome 5 anthracnose resistance QTL explained 48.2% of phenotypic variance and showed an additive effect. Analysis of candidate genes within these QTL regions suggested potential roles of disease-resistance proteins and immune regulators in anthracnose response. Analysis of the eight resequenced breeding lines revealed extensive inbreeding, evidenced by long runs of homozygosity. However, it also revealed regions of high heterozygosity, suggesting potential interspecific introgression from a related yam species, possibly *D. nummularia*, during domestication.

The findings of this study directly address the lack of genomic resources for greater yam, a major constraint for improving this important crop. The high-quality genome assembly and dense genetic map provide powerful tools for marker-assisted selection (MAS), genome-wide association studies (GWAS), and genomic selection, enabling more efficient breeding strategies. The identified QTLs for anthracnose resistance and tuber quality traits represent valuable targets for MAS, facilitating the development of improved varieties with enhanced disease resistance and desirable tuber characteristics. The observed extensive inbreeding within the studied breeding lines highlights a need for broader germplasm utilization to prevent inbreeding depression and increase genetic diversity for selection. The identified regions of potential interspecific introgression highlight the potential benefits of using a wider range of species in breeding programs. The genome sequence will also enable gene editing techniques to be explored further to accelerate the improvement of this crop.

This study provides a comprehensive genomic resource for greater yam, including a chromosome-scale genome assembly, a high-resolution genetic map, and a catalog of genetic variation within breeding lines. The identified QTLs for anthracnose resistance and tuber quality traits are valuable targets for future breeding efforts. The discovery of extensive inbreeding and potential interspecific introgression highlights strategies to maximize genetic diversity for future selection. Future research should focus on validating the identified QTLs under diverse environmental conditions, further characterizing the potential interspecific introgression, and exploring the application of gene editing technologies. Further exploration of the implications of the detected genome duplication events will be important.

The study focused on a limited number of breeding lines and may not fully capture the genetic diversity of greater yam across its entire distribution range. The QTL analysis relies on field trials and detached leaf assays; while these methods are standard for anthracnose research, potential environmental factors could affect the findings, resulting in some differences between different studies of QTL for anthracnose resistance. Phenotyping in the field might confound genetic effects with environmental variations. The use of a limited number of yam lines could underestimate the full genetic diversity and relationships among greater yam germplasm. The identification of introgression is largely based on heterozygosity and requires confirmation by comparative genomics with related species.