Allo-octoploid cultivated strawberry (Fragaria × ananassa) originated through a combination of polyploid and homoploid hybridization, domestication of an interspecific hybrid lineage, and continued admixture of wild species over the last 300 years. While genes appear to flow freely between the octoploid progenitors, the genome structures and diversity of the octoploid species remain poorly understood. The complexity and absence of an octoploid genome frustrated early efforts to study chromosome evolution, resolve subgenomic structure, and develop a single coherent linkage group nomenclature. Here, we show that octoploid Fragaria species harbor millions of subgenome-specific DNA variants. Their diversity was sufficient to distinguish duplicated (homoeologous and paralogous) DNA sequences and develop 50K and 850K SNP genotyping arrays populated with co-dominant, disomic SNP markers distributed throughout the octoploid genome. Whole-genome shotgun genotyping of an interspecific segregating population yielded 1.9M genetically mapped subgenome variants in 5,521 haploblocks spanning 3,394 cM in F. chiloensis subsp. lucida, and 1.6M genetically mapped subgenome variants in 3,179 haploblocks spanning 2,017 cM in F. × ananassa. These studies provide a dense genomic framework of subgenome-specific DNA markers for seamlessly cross-referencing genetic and physical mapping information and unifying existing chromosome nomenclatures. Using comparative genomics, we show that geographically diverse wild octoploids are effectively diploidized, nearly completely collinear, and retain strong macro-synteny with diploid progenitor species. The preservation of genome structure among allo-octoploid taxa is a critical factor in the unique history of garden strawberry, where unimpeded gene flow supported its origin and domestication through repeated cycles of interspecific hybridization.

Cultivated strawberry (Fragaria × ananassa) is one of our youngest domesticates, originating in early eighteenth-century Europe from spontaneous hybrids between wild allo-octoploid species (F. chiloensis and F. virginiana). The improvement of horticultural traits by 300 years of breeding has enabled the global expansion of strawberry production. Here, we describe the genomic history of strawberry domestication from the earliest hybrids to modern cultivars. We observed a significant increase in heterozygosity among interspecific hybrids and a decrease in heterozygosity among domesticated descendants of those hybrids. Selective sweeps were found across the genome in early and modern phases of domestication— 59-76% of the selectively swept genes originated in the three less dominant ancestral subgenomes. Contrary to the tenet that genetic diversity is limited in cultivated strawberry, we found that the octoploid species harbor massive allelic diversity and that Fragaria × ananassa harbors as much allelic diversity as either wild founder. We identified 41.8M subgenome-specific DNA variants among resequenced wild and domesticated individuals. Strikingly, 98% of common alleles and 73% of total alleles were shared between wild and domesticated populations. Moreover, genome-wide estimates of nucleotide diversity were virtually identical in F. chiloensis, F. virginiana, and Fragaria × ananassa (π = 0.0059-0.0060). We found, however, that nucleotide diversity and heterozygosity were significantly lower in modern Fragaria × ananassa populations that have experienced significant genetic gains and have produced numerous agriculturally important cultivars.

The challenge of allelic diversity for assembling haplotypes is exemplified in polyploid genomes containing homoeologous chromosomes of identical ancestry, and significant homologous variation within their ancestral subgenomes. Cultivated strawberry (Fragaria × ananassa) and its wild progenitors are outbred octoploids (2n = 8x = 56) in which up to eight homologous and homoeologous alleles are preserved. This introduces significant risk of haplotype collapse, switching, and chimeric fusions during assembly. Using third generation HiFi sequences from PacBio, we assembled the genome of the day-neutral octoploid F. × ananassa hybrid ‘Royal Royce’ from the University of California. Our goal was to produce subgenome-and haplotype-resolved assemblies of all 56 chromosomes, accurately reconstructing the parental haploid chromosome complements. Previous work has demonstrated that partitioning sequences by parental phase supports direct assembly of haplotypes in heterozygous diploid species. We leveraged the accuracy of HiFi sequence data with pedigree-informed sequencing to partition long read sequences by phase, and reduce the downstream risk of subgenomic chimeras during assembly. We were able to utilize an octoploid strawberry recombination breakpoint map containing 3.6 M variants to identify and break chimeric junctions, and perform scaffolding of the phase-1 and phase-2 octoploid assemblies. The N50 contiguity of the phase-1 and phase-2 assemblies prior to scaffolding and gap-filling was 11 Mb. The final haploid assembly represented seven of 28 chromosomes in a single contiguous sequence, and averaged fewer than three gaps per pseudomolecule. Additionally, we re-annotated the octoploid genome to produce a custom F. × ananassa repeat library and improved set of gene models based on IsoSeq transcript data and an expansive RNA-seq expression atlas. Here we present ‘FaRR1’, a gold-standard reference genome of F. × ananassa cultivar ‘Royal Royce’ to assist future genomic research and molecular breeding of allo-octoploid strawberry.

The widely recounted story of the origin of cultivated strawberry (Fragaria × ananassa) oversimplifies the complex interspecific hybrid ancestry of the highly admixed populations from which heirloom and modern cultivars have emerged. To develop deeper insights into the three century long domestication history of strawberry, we reconstructed the genealogy as deeply as possible—pedigree records were assembled for 8,851 individuals, including 2,656 cultivars developed since 1775. The parents of individuals with unverified or missing pedigree records were accurately identified by applying exclusion analysis to array-genotyped single nucleotide polymorphisms. We identified 187 wild octoploid and 1,171 F. × ananassa founders in the genealogy, from the earliest hybrids to modern cultivars. The pedigree networks for cultivated strawberry are exceedingly complex labyrinths of ancestral interconnections formed by diverse hybrid ancestry, directional selection, migration, admixture, bottlenecks, overlapping generations, and recurrent hybridization with common ancestors that have unequally contributed allelic diversity to heirloom and modern cultivars. Fifteen to 333 ancestors were predicted to have transmitted 90% of the alleles found in country-, region-, and continent-specific populations. Using parent-offspring edges in the global pedigree network, we found that selection cycle lengths over the last 200 years of breeding have been extraordinarily long (16.0-16.9 years/generation) but decreased to a present-day range of 6.0-10.0 years/generation. Our analyses uncovered conspicuous differences in the ancestry and structure of North American and European populations and shed light on forces that have shaped phenotypic diversity in F. × ananassa.

Background Shape is a critical element of the visual appeal of strawberry fruit and is influenced by both genetic and non-genetic determinants. Current fruit phenotyping approaches for external characteristics in strawberry often rely on the human eye to make categorical assessments. However, fruit shape is an inherently multi-dimensional, continuously variable trait and not adequately described by a single categorical or quantitative feature. Morphometric approaches enable the study of complex, multi-dimensional forms but are often abstract and difficult to interpret. In this study, we developed a mathematical approach for transforming fruit shape classifications from digital images onto an ordinal scale called the Principal Progression of k Clusters (PPKC). We use these human-recognizable shape categories to select quantitative features extracted from multiple morphometric analyses that are best fit for genetic dissection and analysis. Results We transformed images of strawberry fruit into human-recognizable categories using unsupervised machine learning, discovered 4 principal shape categories, and inferred progression using PPKC. We extracted 68 quantitative features from digital images of strawberries using a suite of morphometric analyses and multivariate statistical approaches. These analyses defined informative feature sets that effectively captured quantitative differences between shape classes. Classification accuracy ranged from 68% to 99% for the newly created phenotypic variables for describing a shape. Conclusions Our results demonstrated that strawberry fruit shapes could be robustly quantified, accurately classified, and empirically ordered using image analyses, machine learning, and PPKC. We generated a dictionary of quantitative traits for studying and predicting shape classes and identifying genetic factors underlying phenotypic variability for fruit shape in strawberry. The methods and approaches that we applied in strawberry should apply to other fruits, vegetables, and specialty crops.