These results highlight the power and accuracy of diploid Mendelian exclusion analysis methods for pedigree authentication , intellectual property protection, and quality control monitoring of germplasm and nursery stock collections in octoploid strawberry using subgenome-specific DNA markers. The application of these approaches was straightforward because of the simplicity and accuracy of subgenome-specific genotyping approaches in octoploid strawberry populations . The development and robustness of SNP genotyping platforms has facilitated the application of standard diploid genetic theory and methods in octoploid strawberry, including the exclusion analysis methods applied in the present study . The power and accuracy of these methods were rigorously tested and affirmed in a court of law where DNA forensic evidence was pivotal in proving the theft of University of California intellectual property by the defendants in a 2017 case in US District Court for the Northern District of California captioned The Regents of the University of California v California Berry Cultivars, LLC, Shaw, and Larson . The DNA forensic approaches and evidence applied in that case are documented in a publicly available expert report identified by case number 3:16- cv-02477 .We estimated that 80-90% of the individuals in the California and Cosmopolitan pedigree networks were extinct . Using SNP array-genotyped individuals preserved in public germplasm collections as anchor points, we searched for evidence that the allelic diversity transmitted by extinct founders had been “lost.” This is a difficult question to answer with certainty; however, the findings reported here,drainage pot combined with the findings of Hardigan et al. , suggest that genetic diversity has been exceptionally well preserved in domesticated populations.
Using SNA and PCAs of H, we did not observe structural features in sociograms or PCA plots that were indicative of the loss of novel ancestral genetic diversity . The kinship or numerator relationship matrix was estimated for the entire pedigree of genotyped and ungenotyped individuals . For the present study, 1,495 historically important and geographically diverse UCD and USDA F. ananassa individuals were genotyped with high-density SNP arrays . The genomic relationship matrix was estimated for the genotyped individuals and combined with the A matrix to estimate the H matrix for the entire pedigree . The global H matrix was partitioned as needed for subsequent analyses . PCAs of the H matrices yielded two-dimensional visualizations of genetic relationships that were remarkably similar in shape and structure to sociograms for the California and Cosmopolitan populations . We observed distinctive differences in the shapes and structures of the sociograms and PCA plots between the populations . The pattern in the Cosmopolitan population was a characteristic of pervasive admixture among individuals across geographies . We observed a strong chronological trend in the California population but not in Cosmopolitan population . We observed a mid-twentieth-century bottleneck in the California population , in addition to a bottleneck pinpointed to approximately 1987-1993 when the California population became closed. We discovered that 48 founders contributed 100% of the allelic diversity to the California population from 1987 onward . Hardigan et al. showed that even though nucleotide diversity had been progressively reduced by bottlenecks and selection, a significant nucleotide diversity has persisted in the California population but was found to be unevenly distributed across the genome.Our genealogy search did not uncover pedigree records for F. ananassa cultivars developed between 1714 and 1775, the 61-year period following the initial migration of F. chiloensis ecotypes from Chile to Europe .
The scarcity of pedigree records from the eighteenth century was anticipated because the interspecific hybrid origin of F. ananassa was not discovered until the mid-1700s . “Madame Moutot” was the only cultivar in the database with ancestry that could bedirectly traced to one of the putative original wild octoploid progenitors of the earliest F. ananassa hybrids that emerged in France in the early 1700s . Although the genealogy primarily covers the past 200 years of domestication and breeding , ascendants in the pedigree of the cultivar “Madame Moutot” traced to “Chili de Plougastel” , a putative clone of one of the original F. chiloensis subsp. chiloensis plants imported from Chile to France by the explorer Ame´de´e-Franc¸ois Fre´zier . These plants were carried aboard the French frigate “St. Joseph,” delivered by Fre´zier to Brest, France , and shared with Antoine Laurent de Jussieu, a botanist at the Jardin des plantes de Paris. According to de Lambertye , the Fre´zier clone was widely disseminated and cultivated in Plougastel near Brest and interplanted with F. virginiana . Hence, some of the earliest spontaneous hybrids between F. chiloensis and F. virginiana undoubtedly arose in the strawberry fields of Brittany in the early 1700s . The French naturalist Bernard de Jussieu, the brother of Antoine Laurent de Jussieu and a mentor of Antoine Duchesne—“the father of the modern strawberry”—brought clones of the original Fre´zier F. chiloensis plants to the Jardins du Chaˆ teau de Versailles where Duchesne unraveled the interspecific hybrid origin of F. ananassa . The next earliest F. chiloensis founders appear to be a California ecotype identified in German breeding records from the mid-1800s and an anonymous ecotype in the pedigree of the French cultivar “La Constante” from 1855 . The origins and identities of the earliest F. virginiana founders of F. ananassa remain a mystery because their migrations from North America to Europe in the early 1600s and subsequent intracontinental migrations were not well documented . The oldest F. virginiana individuals identified in historic documents and pedigree records were “Large Early Scarlet” , “Old Scarlet” , and “Hudson Bay” , all extinct .
We identified 30 anonymous F. virginiana and 76 anonymous F. chiloensis founders in the pedigree records. These individuals were assigned unique alphanumerical aliases to facilitate the reconstruction of the genealogy; e.g., FV22 is the alias for an anonymous F. virginiana founder and FC71 is the alias for an anonymous F. chiloensis founder in the pedigree of “Madame Moutot” .Once the interspecific hybrid origin of F. ananassa became widely known , domestication began in earnest with extensive intra- and interspecific hybridization, artificial selection, and intra- and intercontinental migration . These forces shaped the genetic structure of the F. ananassa populations that emerged in Europe and North America, and ultimately migrated around the globe . Over the next 250 years, horticulturalists and plant breeders repeatedly tapped into the wild reservoir of genetic diversity, especially wild octoploid taxa native to North America . There are numerous narrative accounts of what transpired, especially in Europe, North America, and California , but none have painted a holistic picture of the complicated wild ancestry and dynamic forces that shaped genetic diversity in F. ananassa. We identified 1,438 founders in the genealogy of cultivated strawberry . Here and elsewhere, “founders” are individuals with unknown parents, whereas “ancestors” are ascendants that may or may not be founders . The terminal nodes in the pedigree networks are either founders or the youngest descendants in a pedigree . Of the 1,438 founders, 267 were wild species and 1,171 were F. ananassa individuals . Because the F. ananassa founders are either interspecific hybrids or descendants of interspecific hybrids, the number of wild species founders could exceed 268. One of the challenges we had with estimating the number of wild species founders was the anonymity of ecotypes that were used as parents before breeders began carefully documenting pedigrees . We could not rule out that some of the anonymous wild species founders in the pedigree records might have been clones of the same individuals, drainage planter pot which means that the estimated number of wild species founders reported here could be inflated. As interspecific hybridization with wild founders became less important and intraspecific hybridization became more important in strawberry breeding, the proportional GC of wild founders to the gene pool of cultivated strawberry decreased . This seems paradoxical because 100% of the alleles found in F. ananassa were inherited from wild founders, but increasingly flowed through F. ananassa descendants over time—wild octoploids numerically only constituted 14% of the founders we identified . Several trends emerged from our analyses of genetic relationships and founder contributions. First, inbreeding has steadily increased over time as a consequence of population bottlenecks and directional selection . Second, the California population was significantly more inbred than the Cosmopolitan population . These results were consistent with the findings of Hardigan et al. from genome-wide analyses of DNA variants and population structure. They found selective sweeps on several chromosomes in the California population, which was shown to be unique and bottle necked. Finally, the relative number of founder equivalents has decreased over time, consistent with the increase in inbreeding over time .
The primary gene pool of cultivated strawberry is comprised of eight cross-compatible, interfertile octoploid taxa: F. chiloensis subsp. chiloensis, F. chiloensis subsp. lucida, F. chiloensis subsp. pacifica, F. chiloensis subsp. sandwicensis, F. virginiana subsp. virginiana, F. virginiana subsp. glauca, F. virginiana subsp. grayana, and F. virginiana subsp. platypetala , seven of which were found in pedigree records . The only primary gene pool taxon not found in the pedigree records was F. virginiana subsp. grayana. We identified 112 F. chiloensis, 65 F. virginiana, and 1,171 F. ananassa founders, which constituted 95% of the founders and were estimated to have contributed 99% of the allelic diversity to global, California, and Cosmopolitan F. ananassa populations . Even though wild species from the secondary gene pool constituted 6% of the founders and 30% of the wild species founders identified in pedigree records, they were estimated to have contributed < 0:1% of the allelic diversity in the global F. ananassa population . While the assignment of F. chiloensis and F. virginiana subspecies to the primary gene pool was unequivocal and uncontroversial, the assignment of non-octoploid Fragaria and Potentilla species to secondary or tertiary gene pools, as per the definitions of Harlan and de Wet , was tenuous because evidence for the inheritance of alleles from exotic donors among inter-ploidy hybrid offspring with cultivated strawberry was not always clear from genealogical and breeding records. We lumped the non-octoploid Fragaria and Potentilla into the secondary gene pool solely because they were recorded as ancestors of F. ananassa individuals , which implied that interspecific, intergeneric, and inter-ploidy hybrid descendants inherited alleles transmitted by secondary gene pool donors. However, the genetic proof was not always clear or available. One or more of the species assigned to the secondary gene pool might belong in the tertiary gene pool , a distinction of negligible practical importance. The secondary gene pool founders in the genealogy were nearly always parents of orphans or other isolated individuals in short dead-end pedigrees that have not materially contributed allelic diversity to important cultivated strawberry populations or cultivars. The exotic founders have included decaploid F. vescana and pentaploid F. bringhurstii individuals . Although cited as important genetic resources for strawberry breeding , the secondary gene pool species have had a limited utility because of the range of biological challenges one encounters when attempting to introgress alleles from exotic donors through interspecific, intergeneric, and inter-ploidy hybrids, e.g., reproductive and recombination barriers, ploidy differences, meiotic abnormalities, and hybrid sterility . Genetic variation in the secondary gene pool has not been needed to drive genetic gains or solve problems in strawberry breeding. As highlighted earlier, Hardigan et al. showed that genetic diversity is massive in the primary gene pool and has not been eroded by domestication and breeding on a global scale, even though it has been significantly reduced and restructured in certain populations, e.g., the California population. The profound changes and restructuring in the California population over time, as previously noted, were clearly evident in the sociograms and PCAs of the pedigree–genomic relationship matrices . Because the California population has been the source of numerous historically and commercially important cultivars, we hypothesize that intense selection and population bottlenecks have purged a high frequency of unfavorable alleles compared to many other populations, thereby yielding an elite population with lower genetic diversity than the highly admixed Cosmopolitan population .We used coancestry, betweenness-centrality , and out-degree statistics to estimate the GC of founders and non-founders to genetic variation within a population and identify the most prominent and important ancestors in the genealogy of cultivated strawberry . For founders, GC was estimated by the mean coancestry or MK between each founder and cultivars within a focal population .