STRAWBERRY

Flow Cytometry determination using DAPI with internal standard = iceberg lettuce ; performed by Plant Cytometry Services, The Netherlands, 7 August 2007.

Flow Cytometry determination using DAPI with internal standard = iceberg lettuce ; performed by Plant Cytometry Services, The Netherlands, 15 July 2008.

Flow Cytometry determination using DAPI with internal standard = Ilex crenata `Fastigiata?; performed by Plant Cytometry Services, The Netherlands, 13 October 2009.

Flow Cytometry determination using DAPI with internal standard = Ilex crenata `Fastigiata?; performed by Plant Cytometry Services, The Netherlands, May 2010.

Flow Cytometry determination using DAPI with internal standard = Ilex crenata `Fastigiata?; performed by Plant Cytometry Services, The Netherlands, June 2010.

Flow Cytometry determination using DAPI with internal standard = Ilex crenata `Fastigiata?; performed by Plant Cytometry Services, The Netherlands, July 2010.

Flow Cytometry determination using DAPI with internal standard = Ilex crenata `Fastigiata?; performed by Plant Cytometry Services, The Netherlands, November 2010.

Flow Cytometry determination using DAPI with internal standard = Ilex crenata `Fastigiata?; performed by Plant Cytometry Services, The Netherlands, 2011.

Curatorial selection of core accessions

Experiment Type: Field FIELD Study Year: 1985

Experiment Type: FILED FIELD Study Year: 1986

Experiment Type: Field FIELD Study Year: 1987

Study Name: Fragaria Fruit Observations Experiment Type: Field FIELD Study Year: 1988

Study Name: Fragaria Fruit Observations Experiment Type: Field FIELD Study Year: 1989 Year started: 06//1989

Study Name: Fragaria Bloom Data 1991 Experiment Type: Field FIELD Study Year: 1991 Exp. Location: NCGR-Corvall

Observed Dayneutral Fragaria during July, August and September 2002

Crown rot evaluation for Phythophthora cactorum on strawberry cultivars. Research was conducted in Norway by Eikemo et al., 2003. Resistance to crown rot (Phytophthora cactorum) in strawberry cultivars and in offspring from crosses between cultivars differing in susceptibility to the disease Ann. Appl. Bio. 142:83-89.

Determination of a diverse subset of the octoploid strawberry collection in 2001

data entered under FRAGARIA.Hummer.etal.StrawberryPhenotypeEval.2019

data entered under FRAGARIA.Hummer.etal.StrawberryPhenotypeEval.2020

In September 2018, 288 cultivars were planted in a field on the North Farm of the USDA, 33707 Peoria Road, Corvallis, Oregon, 97333-2521. For this field study, three replicates of each cultivar were planted in a randomized complete block design (RCBD). The blocks are defined based on irrigation proximity and soil compaction. Guard rows of strawberries were planted around the perimeters of the plot and central water wheel row. Plant spacing is 0.762m (2.5’) x 0.762m (2.5’) so that the plants can be tilled across rows and within rows using existing tractors and tillers. This prevents runners from contaminating adjacent clones. Irrigation is provided by a center waterwheel that travels the length of the plot. Traits evaluated in spring-summer 2019 and 2020 Plant Height measured early May First Flower Date taken weekly from 1 March through Mid-June Average number of runners per crown collected during the week of 1 July Average number of plants per runner Average runner length per crown Mean harvest date Average fruit weight of 5 berries per crown Average individual fruit weight per accession Average petiole length per crown pH of fruit Soluble solid content Titratable Acidity

In September 2018, 288 cultivars were planted in a field on the North Farm of the USDA, 33707 Peoria Road, Corvallis, Oregon, 97333-2521. For this field study, three replicates of each cultivar were planted in a randomized complete block design (RCBD). The blocks are defined based on irrigation proximity and soil compaction. Guard rows of strawberries were planted around the perimeters of the plot and central water wheel row. Plant spacing is 0.762m (2.5’) x 0.762m (2.5’) so that the plants can be tilled across rows and within rows using existing tractors and tillers. This prevents runners from contaminating adjacent clones. Irrigation is provided by a center waterwheel that travels the length of the plot. Traits evaluated in spring-summer 2019 and 2020 Plant Height measured early May First Flower Date taken weekly from 1 March through Mid-June Average number of runners per crown collected during the week of 1 July Average number of plants per runner Average runner length per crown Mean harvest date Average fruit weight of 5 berries per crown Average individual fruit weight per accession Average petiole length per crown pH of fruit Soluble solid content Titratable Acidity

Collectively, 435 F. × ananassa, 18 F. chiloensis, and 22 F. virginiana individuals (asexually propagated genetic resources) were phenotyped for resistance to PhCR in our study. The F. × ananassa individuals included 64 cultivars developed at University of California–Davis (UCD), 282 UCD hybrids (offspring from crosses between noninbred parents), 75 non-UCD cultivars, and 14 non-UCD hybrids. Thirty-eight individuals were phenotyped in 1 yr, whereas 437 individuals were phenotyped in both years of our studies. The latter were used as the “training” population for genomic prediction of breeding values and genetic variances. The training population included 321 UCD and 116 non-UCD individuals. Of the latter, 40 were wild ecotypes. The F. chiloensis and F. virginiana ecotypes were originally collected from habitats across their natural ranges in North and South America. Other than a single F. virginiana subsp. platypetala ecotype (15X001P001) collected near the Trout Creek Campground, CA (41.5° N, −121.9° W), the non-UCD genetic resources were originally acquired as single mother plants from the USDA National Plant Germplasm System National Clonal Germplasm Repository, Corvallis, OR. These individuals were multiplied from stolons in Winters, CA, and maintained in the UCD Strawberry Germplasm Collection. To produce bare-root clones (“daughter” plants) for replicated testing, bare-root “mother” plants were harvested in January, temporarily stored in the dark at −3.5 °C, and transplanted to a high-elevation (1,294 m asl) nursery in April 2017 and 2018 (Cedar Point Nursery, Dorris, CA). Clones (bare-root plants) of each individual were harvested in mid-October of each year and stored in the dark at 3.5 °C for 2–3 wk before pathogen inoculation and planting. ESTIMATED MARGINAL MEAN calculation.

Collectively, 435 F. × ananassa, 18 F. chiloensis, and 22 F. virginiana individuals (asexually propagated genetic resources) were phenotyped for resistance to PhCR in our study. The F. × ananassa individuals included 64 cultivars developed at University of California–Davis (UCD), 282 UCD hybrids (offspring from crosses between noninbred parents), 75 non-UCD cultivars, and 14 non-UCD hybrids. Thirty-eight individuals were phenotyped in 1 yr, whereas 437 individuals were phenotyped in both years of our studies. The latter were used as the “training” population for genomic prediction of breeding values and genetic variances. The training population included 321 UCD and 116 non-UCD individuals. Of the latter, 40 were wild ecotypes. The F. chiloensis and F. virginiana ecotypes were originally collected from habitats across their natural ranges in North and South America. Other than a single F. virginiana subsp. platypetala ecotype (15X001P001) collected near the Trout Creek Campground, CA (41.5° N, −121.9° W), the non-UCD genetic resources were originally acquired as single mother plants from the USDA National Plant Germplasm System National Clonal Germplasm Repository, Corvallis, OR. These individuals were multiplied from stolons in Winters, CA, and maintained in the UCD Strawberry Germplasm Collection. To produce bare-root clones (“daughter” plants) for replicated testing, bare-root “mother” plants were harvested in January, temporarily stored in the dark at −3.5 °C, and transplanted to a high-elevation (1,294 m asl) nursery in April 2017 and 2018 (Cedar Point Nursery, Dorris, CA). Clones (bare-root plants) of each individual were harvested in mid-October of each year and stored in the dark at 3.5 °C for 2–3 wk before pathogen inoculation and planting. GENOMIC-ESTIMATED BREEDING VALUE calculation.

Collectively, 435 F. × ananassa, 18 F. chiloensis, and 22 F. virginiana individuals (asexually propagated genetic resources) were phenotyped for resistance to PhCR in our study. The F. × ananassa individuals included 64 cultivars developed at University of California–Davis (UCD), 282 UCD hybrids (offspring from crosses between noninbred parents), 75 non-UCD cultivars, and 14 non-UCD hybrids. Thirty-eight individuals were phenotyped in 1 yr, whereas 437 individuals were phenotyped in both years of our studies. The latter were used as the “training” population for genomic prediction of breeding values and genetic variances. The training population included 321 UCD and 116 non-UCD individuals. Of the latter, 40 were wild ecotypes. The F. chiloensis and F. virginiana ecotypes were originally collected from habitats across their natural ranges in North and South America. Other than a single F. virginiana subsp. platypetala ecotype (15X001P001) collected near the Trout Creek Campground, CA (41.5° N, −121.9° W), the non-UCD genetic resources were originally acquired as single mother plants from the USDA National Plant Germplasm System National Clonal Germplasm Repository, Corvallis, OR. These individuals were multiplied from stolons in Winters, CA, and maintained in the UCD Strawberry Germplasm Collection. To produce bare-root clones (“daughter” plants) for replicated testing, bare-root “mother” plants were harvested in January, temporarily stored in the dark at −3.5 °C, and transplanted to a high-elevation (1,294 m asl) nursery in April 2017 and 2018 (Cedar Point Nursery, Dorris, CA). Clones (bare-root plants) of each individual were harvested in mid-October of each year and stored in the dark at 3.5 °C for 2–3 wk before pathogen inoculation and planting. GENOMIC-ESTIMATED BREEDING VALUE + presence of RPc2-associated SNP calculation.

The germplasm accessions (C0 population) phenotyped in our study were 853 asexually propagated F. ×ananassa, F. chiloensis, and F. virginiana individuals preserved in the UC Davis (UCD) Strawberry Germplasm Collection (SGC) and USDA National Plant Germplasm System (NPGS) collections. We acquired ‘mother’ plants for 265 of these individuals from the USDA NPGS National Clonal Germplasm Repository in Corvallis, Oregon. The other 588 were among holdings in the SCG as of February 1, 2015. C0 individuals were preserved and annually propagated from mother plant stolons at the Wolkskill Experiment Orchard (WEO), Winters, CA, over the course of our studies. UCD and USDA identification and plant introduction numbers, aliases, pedigrees, and passport information for C0 population individuals (n=853) are tabulated in Supplemental File S1). The C0 population was comprised of 788 F. ×ananassa cultivars and other hybrids, 39 F. chiloensis ecotypes, and 36 F. virginiana ecotypes (SupplementalFileS1). The daughter plants (bare-root clones) of C0 individuals were produced from mother plants grown at a low-elevation location (41m; Winters, CA) for the spring-planted 2016 study and a high-elevation location (1284m; Dorris, CA) for the fall-planted 2016 to 2017 study.

Large-Scale Standardized Phenotyping of Strawberry in RosBREED. Perception of sweetness and presence of off-flavors; Scored 1-9; 1 = not sweet, bad off-flavors; 9 = very sweet, no off-flavors. Rated in Oregon, Michigan, and New Hampshire.

Responses of strawberry species and cultivars to the root-lesion and northern root-knot nematodes

Study Name: Determination of TDS, SS, pH and TA in small fruits at NCGR Study Year: 1988

Perpetual-flowering (PF) is a highly desirable trait within cultivated strawberries (Fragaria texttimesananassa) for the commercial and home garden markets. The most widely used source of the PF trait was originally introgressed from a wild F. virginiana subsp. glauca accession collected in the Wasatch Mountains near Salt Lake City, UT in 1955. This source is conferred by a single dominant QTL, FaPFRU, and was recently identified in multiple bi-parental populations. Multiple markers have been proposed as diagnostic tests for marker-assisted selection (MAS). These markers were proposed after looking at a relatively small sample of germplasm. To identify the best diagnostic testing procedure for MAS, the markers were evaluated individually and in combination on a training set of cultivars with known genotypes and the best test was used to determine the distribution of the FaPFRU source of PF within a large sample of octoploid Fragaria germplasm. Of the tests evaluated, the microsatellite marker Bx215 alone was found to have the best diagnostic ability for MAS with an accuracy of 93.1% in controlled conditions. When utilizing the test on 390 F. texttimesananassa accessions, 164 accessions were identified to likely have the FaPFRU locus. Nine octoploid Fragaria accessions were PF and did not have this marker, indicating possible recombination events or potentially novel sources of the PF trait. Future work will be needed to dissect the PF trait in these nine individuals.

Plant and fungal materials. Information about the studied plant accessions, their geographic origins (wild-collected materials) or breeding program sources, and their identification numbers (local and/or Plant Introduction) is provided for diploids and polyploids, respectively, in Tables 1 and 2. Subspecies designations are not provided for California accessions of F. vesca due to uncertainty in differentiating the subspecies Fragaria vesca ssp. bracteata from F. vesca ssp. californica and their possible hybrids. Among diploid accessions (Table 1); CFRA364.002, CFRA333.001, and CFRA520.001 were obtained from the NCGR, accessions from Hokkaido, Japan were collected by Thomas M. Davis and Kim Hummer [32], and GS1J was collected by Gunter Staudt, while all other UNH-numbered accessions were collected by Davis.

The polyploid accessions of octoploids Fragaria virginiana and F. chiloensis, and decaploid F. cascadensis belong-ing to the Fragaria supercore collection were obtained from the NCGR. The studied F.×ananassa accessions were of interest to our breeding programs. Plants of ‘Sparkle’ and ‘Tristar’ were purchased from Nourse Farms, Whately MA, while the other cultivars and breeding lines were provided by Andrew R. Jamieson. F.×ananassa cultivars ‘Annapolis’, ‘Cavendish’, ‘Evangeline’, ‘Mira’, ‘Wendy’ (aka ‘AC Wendy’), and ‘Laurel’ (tested as K93-20), and numbered breeding clones K05-9 and M903 were developed at the Agri-Food Canada Kentville Research Station, Nova Scotia, Canada. Cultivar ‘Sparkle’ is a 1942 release from the New Jersey State University breeding program [18], while ‘Tristar’ is a 1981 release from the USDA Beltsville program.

Screening methods. A series of trials was conducted, in which from five to 20 genotypes were screened. In each trial, each genotype was represented by three orfour inoculated and two uninoculated (control) plants. Pots containing inoculated or uninoculated plants were maintained within separate containment trays, and plants were randomly distributed within the trays. Some genotypes were included in multiple trials.

Verticillium dahliae isolate V1 was obtained from Mansun Kong at Driscoll Strawberry Associates, Watsonville, CA, USA, and was originally isolated from an infected strawberry plant (M. Kong, personal communication). Plants were maintained in the UNH MacFarlane Greenhouse facility in Pro-Mix Mycorrhizae™ (Premier Tech Horticulture LTD, Canada), and were propagated by rooting stolons that were still attached to mother plants to produce runner plantlets. Runner nodes were pinned onto the surface of Metro-mix 360 (Hummert™ International, Missouri) medium in 4” standard round polypropylene pots (Dillen Products Company, Ltd., Middlefield, Ohio) using staples made from plastic-coated wire. Plantlets were allowed to root for two weeks prior to separation from the mother plant and inoculation. Ten plantlets were rooted from each mother plant, and eight were ultimately used in each trial (four inoculated plantlets and four uninoculated controls). Concurrent with plant propagation, fungal inoculum was cultured in an appropriate volume (∼10 ml for each plant to be inoculated) of autoclaved Difco™ Czapek-Dox broth (BD Biosciences). The broth, contained in 1L flasks, was inoculated in a laminar flow hood with two to four ∼1cm2 pieces of fungus-covered Czapek-Dox agar from fungal culture plates. The inoculated broth was then incubated for two weeks at room temperature on an orbital shaker at 200 RPM.

On the day of inoculation, the fungal culture was strained through two layers of cheesecloth and one layer of Miracloth (EMD Millipore, Billerica, Massachusetts), and then centrifuged at 10,000× g for 5 min. The culture medium was decanted and the conidial pellet was resuspended in a volume of sterile distilled water equivalent to that of the decanted culture medium. Conidia were quantified using a hemacytometer (Bright Light Counting Chamber Improved Neubauer, Hausser Scientific, Horsham Pa), and the suspension was diluted, if necessary, with sterile distilled water to ∼2 × 107 conidia/ml. Immediately prior to root dipping, rooted plantlets were separated from mother plants and trimmed to remove runners. Soil was shaken from roots prior to dipping. Root dipping was performed in 50-ml polyvinyl chloride pipet basins (Fisher Scientific, Pittsburgh, PA). Root systems were immersed, two at a time per basin, for 5 min in either 20 ml fungal spore suspension or 20 ml sterile distilled water (uninoculated control). After dipping, the plants were replanted in new 4” pots of sterile (autoclaved) Metromix medium, then moved to a greenhouse under ambient light and temperature conditions or to a 22◦ C temperature-controlled growth room under broad-spectrum (140 moles/m2/sec) fluorescent lights, then maintained in containment trays with minimal watering for a period of at least eight weeks.

Plant verticillium wilt disease ratings. At the end of the observational period, each individual plant was rated relative to controls according to the following rating scale, as exemplified by plants shown in Fig. 1. A rating of 1 (healthy) was given to plants closely resembling uninoculated controls. A rating of 1.5 (slightly symptomatic) indicated mild stunting and/or mild outer leaf necrosis and/or browning. A rating of 2.0 (mildly symptomatic) indicated distinct stunting and/or distorted growth, more leaf necrosis and browning, and perhaps one or two dead leaves. A rating of 2.5 (very symptomatic) was given to plants with severe stunting, leaf necrosis and browning, yet still having one to a few green leaves. A 3.0 (dead) rating indicated that all of the leaves were necrotic and the plant was considered to be nearly or completely dead.

A mean disease rating was then calculated for each germplasm accession or cultigen on the basis of the respective total number of rated plants. As guided by previous literature [9, 31, 33] and to facilitate comparison with prior studies, we then assigned a qualitative classification to each accession or cultigen based upon its mean disease rat-ing. For this purpose, the scale of mean disease ratings was partitioned into five ordered categories. The category ranges and corresponding classifications (in parentheses) were: 1.0 to 1.3 (very resistant = VR), 1.4 to 1.7 (moder-ately resistant = MR), 1.8 to 2.2 (intermediate = I), 2.3 to 2.6 (moderately susceptible = MS), and 2.7 to 3.0 (very susceptible = VS). For example, an accession with a mean disease rating of 1.5 would have been classified as MR.

Abstract. Thirty-two wild strawberry genotypes and two commercial cultivars were obtained from the US Department of Agriculture (USDA), Agricultural Research Service (ARS), National Clonal Germplasm Repository-Corvallis (NCGR) and planted in the field to test cold hardiness and foliar disease resistance at the University of Minnesota North Central Research and Outreach Center at Grand Rapids, MN. This station is located in USDA plant hardiness zone 3b. Among the 34 genotypes tested, Fragaria iinumae was sensitive to powdery mildew, leaf scorch, frost and mid-winter injury while F. nipponica was resistant to powdery mildew and leaf scorch. Fragaria iinumae suffered severe injury after winter 2009-10, while F. nipponica and F. orientalis survived that winter well, producing dense and vigorous plants in 2010. These species could be potentially useful diploid or tetraploid parents in breeding programs developing strawberries for the mid-western United States. Fragaria chiloensis PI 637983, was similar to the standard cultivars for winter hardiness and disease resistance. Wild strawberries bloomed earlier and had softer, smaller fruit than did the commercial F ×ananassa 'Jewel' and 'Mesabi'. Fragaria nipponica had the softest fruit. Fragaria orientalis PI 637933 and PI 637939 were notable for their pleasing taste and aroma, while most Fragaria nipponica, F itrupensis, and F. virginiana were very sour. The feral F. ×ananassa PI 641087 and PI 641088 may have the greatest immediate utility, with a combination of winter hardiness and excellent disease resistance, for octoploid breeding programs.

Material and methods
The evaluation was conducted at the University of Minnesota North Central Research and Outreach Center (NCROC) at Grand Rapids, MN, in USDA plant hardiness zone 3b. Field trials at this location have been useful in evaluating strawberry winter injury over the past three decades and select­ing cultivars for northern climates based on winter hardiness and other important traits in a collaborative breeding program between the USDA Agriculture Research Service (ARS) and the University of Minnesota. The evaluation included 34 genotypes of Fragaria iinumae, F. orientalis, F. nipponica, F. iturupensis, F vesca, F. chiloensis, F. virginiana, and wild accessions and standard cultivars ('Jewel' and 'Mesabi') of F ×ananassa Duchesne ex Rozier. 'Mesabi' was selected at Grand Rapids, MN, and is well adapted there for winter survival and fruiting. 'Jewel,' selected in New York, is widely grown in the eastern and mid-western US but" is not reliably winter hardy for com­mercial production at Grand Rapids.

Wild strawberries were shipped as runners from NCGR-Corvallis and propagated in the greenhouse and in the field at NCROC in 2008. In mid-October 2008, plants were dug and potted to 10 cm pots and grown in a heated greenhouse until January 2009, when they were moved to a cellar to spend their dormancy. Potted plants were moved out of the cellar on May 10, 2009, and stayed outdoors until planting.
On 28 May 2009, two, two-plant plots of each genotype were established in each of four blocks in a split block design. In each block, there were two sub-blocks with identical plant­ing plans. One was overwintered with straw mulch, and the adjacent sub-block was over­wintered without mulch. fn addition to the 34 replicated entries, two other wild genotypes, PI 637954 and Pl 641089, with insufficient plants for complete replication, were planted in a border row for observation. Starter fertilizer (11-52-0), monoammonium phosphate at rate of 80 g·114 L-1 rate and 500 ml solution per plant was used at planting and no additional fertilizer was applied after planting. Drip irrigation was installed as one T-tape per row (emitters spaced at 305 mm, 1.7 L·min-1, 102 L·h-1 for 30 mat 55.6 k Pa, John Deere Water, San Marcos, CA) and the field was irrigated once or twice per week as a supplement to precipitation. Weeds were manually removed and the space between rows was tilled as necessary to control weeds and runners. Straw mulch of 10-15 cm was applied on mulched plots in early November 2009, and removed to between rows in early April 2010. Straw was also added between the rows of the non-mulched plots in April so that all plots had surrounding straw during the 2010 growing season.

2009 Evaluations
Plants in each plot were initially spaced 0.6 m apart in rows 1.3 m apart. Plants were allowed to runner in 2010 to form short matted row plots. Some genotypes with excessive runners were trimmed manually to maintain them within their plot. Runners per plot were rated on 6 Aug. 2010 from 1 = 1-5 runners per plot; 2 = 6-10; 3 = 11-20; 4 = 21-30; and 5 => 30. Powdery mildew (Podosphaera aphanis (Wallr.) U. Braun & S. Takamatsu) and fungal leaf spot (Mycosphaerella fragariae (Tul.) Lindau) and leaf scorch (Diplocarpon earliana Ell. et Ev. (Wolf)) infections were rated on 6 Aug. and 25 September 2009, from 1 = no disease to 9 = severe infection. Frost resistance was evaluated on 13 October 2009, after several hard frosts from 9 to 13 October 2009, on a scale from I = no damage to 9 = all leaves fully desiccated.

2010 Evaluations
In 2010, genotypes were evaluated for several plant and fruit traits. The stand (% coverage of the plot) was estimated on 15 May during flowering and again at the early stage fruiting on 18 June. Winter injury was rated on 4 June from 1 (= all plants surviving , and vigorously growing) to 9 (= all plants dead) based on visual estimation of survival of the plants and the health and regrowth of the surviving plants. Plant vigor was rated on 18 June from 0 (= dead) to 9 (= highly vigorous) based primarily on the number and size of leaves produced. Growth habit was rated on 28 June from 1 (= prostrate) to 5 (= erect). Productivity was rated from 0 (no fruit) to 9 (heavily fruiting) when approximately 50% of the fruit appeared to be ripe. Using the same rating scale as in 2009, powdery mildew, fungal leaf spotting (leaf scorch/blight/spot) severity were rated on 7 July and 27 July. Fungal leaf spotting diseases appeared to include leaf scorch, leaf blight and leaf spot in 2010. As all three could be observed on one genotype, and necrotic lesions often coincided, a single fungal leaf disease score was given for each plot.
Berry weight was estimated based on random samples of 20 berries from a midseason harvest date (approximately 50% ripe fruit) from plots that fruited. Fruit shape was described as oblate, globose, globose conic, conic, long conic, necked, long wedge or short wedge according to the University of Florida key. External and internal fruit colors were described. Skin toughness was rated from 1 (very tender) to 9 (very tough) based on resistance to thumb abrasion when rubbed between thumb and forefinger. Firmness was rated from 1 (very soft) to 9 (very firm) when squeezed between thumb and forefinger. Flavor was characterized with descriptors and rated hedonically by JJL from 1 (very poor) to 9 (excellent).
Ratings were performed by SY and JJL in 2009 and 2010, respectively. Data for plant and fruit traits in each year were analyzed, where appropriate, using ANOVA with Statistix Software (Analytical Software, Tallahassee, FL). Mean separations were based on Fisher's protected LSD (P<=0.05).

Population Structure and Diversity Analysis All analyses of diversity and structure were conducted using R v 4.0.3 (ref. 31). Accessions were stratified into 13 geographical regions based on their passport information on GRIN Global. The regions within North America included: Alaska, U.S., Northwest U.S. (Idaho, Oregon, Washington, and Wyoming), California, U.S., Midwest U.S. (Illinois, Indiana, Iowa, Michigan, Minnesota, Missouri, and Wisconsin), Southeastern U.S. (Arkansas, Florida, Louisiana, Mississippi, North Carolina, Tennessee, Texas, and South Carolina), Mid-Atlantic U.S. (Maryland and Delaware), New England U.S. (Connecticut, Maine, Massachusetts, New Hampshire, New Jersey, and New York), Western Canada (British Columbia and Alberta), and Eastern Canada (Ontario and Nova Scotia). Europe was divided into Western Europe (Belgium, Denmark, France, Germany, Italy, Ireland, the Netherlands, Norway, Sweden, and the United Kingdom) and Eastern Europe (Belarus, Lithuania, Poland, and Western Russia). The Asia geographic region included accessions from Eastern Russia, Japan, and Taiwan. Finally, a single accession that originated from South Africa made up the final geographic region. Three methods were used to evaluate population structure. The first was via principal component analysis (PCA) followed by k-means clustering as implemented in adegenet v. 2.1.3 (refs. 32 & 33). When conducting k-means clustering the maximum number of principal components were retained and the optimum number of clusters was selected using the minimum Bayesian information criterion. The sparse non-negative matrix factorization (sNMF) algorithm as implemented in the R package LEA 34, 35 was used to evaluate population structure and admixture between populations. The number of K subpopulations evaluated ranged from 2 to 14 and each analysis was repeated 10 times. The elbow method was used to identify K clusters for the sNMF algorithm. Sample orders were calculated using CLUMPP v. 1.1.2 (ref. 37) and results were visualized using the barchart function from LEA 34. Finally, population structure and admixture between populations was assessed using STRUCTURE v. 2.3.4 (ref. 36) for 2 to 14 subpopulations. Parameters were set to 25,000 burn-in steps and 50,000 Markov-Chain Monte Carlo (MCMC) steps with 10 replications per k subpopulation. All remaining parameters were set to default. The optimal number of k subpopulations for the STRUCTURE results was identified using STRUCTURE HARVESTER v. 0.6.94 (refs. 38 & 39). Sample orders were calculated using CLUMPP v. 1.1.2 (ref. 37) and results were visualized using Structure Plot v2.0 (ref. 40). The geographic sub-populations, except for the South African accession, were evaluated for population richness, intra group diversity, expected heterozygosity, and evenness. Intra group diversity was evaluated using Simpson’s index 36 and expected heterozygosity was evaluated using Nei’s expected heterozygosity 37. Richness, Simpson’s index, Nei’s expected heterozygosity, and evenness were calculated using the R package poppr v 2.8.7 (refs. 41 & 42). The pairwise fixation index (FST) was also calculated for each geographic sub-population, excluding South Africa, using hierfstat v 0.5-7 (ref. 43) to assess the amount of interbreeding/sharing of germplasm between breeding programs in these regions. Core Collection Creation Two 100 individual core collections were created using the R package corehunter (v. 3.2.1 (ref. 6). The first core collection was a type 1 core collection (also known as a CC-I collection) designed to evenly represent the diversity of the collection. The second was a type 2 core collection (also known as a CC-X collection) designed to represent the extremes of the entire collection. The type 1 collection used the average distance between each accession and the nearest entry (A-NE) criterion and works to minimize this value 44. The type 2 collection used the average distance between each entry and the nearest neighboring entry (E-NE) criterion and works to maximize this value 7. For each collection, a set of 13 accessions were pre-selected as “seeds”. These individuals were selected based on their geographical origin and because they are positive controls for various DNA tests, were sequenced or a parent of a major mapping population, or have been known to be notable cultivars from their geographic region 12, 45, 46. These 13 accessions were as follows: ‘Camarosa’ (PI 670238), ‘Charm’ (PI 664911), ‘Deutsch Evern’ (PI 551626), ‘Holiday’ (PI 551653), ‘Korona’ – Netherlands (PI 666636), ‘Mara des Bois’ (PI 687353), ‘Ooishi shikinari 2’ (PI 641185), ‘Senga Sengana’ (PI 264680), ‘Strawberry Festival’ (PI 664337), ‘Tochiotome’ (PI 617008), ‘Totem’ (PI 551501), ‘Tribute’ (PI 551953), and US 4809 (PI 637938). Prevosti’s absolute genetic distance was used in construction of each core collection 47. The corehunter package was run 2,000 times when constructing each core collection, retaining the core collection with minimum A-NE or maximum E-NE criterion depending on the collection type, due to the stochastic algorithms used in the package. Pedigree Confirmation Percent identity by state (IBS) was calculated between each pair of individuals for all individuals. Individuals with greater than or equal to 98% were considered to be synonyms. The software COLONY v 2.0.6.6 (ref. 48) was used for parentage inference. The parameters polygamy for both males and female, inbreeding mating, without clones, monoecious, and diploid were used to describe hybridization within strawberry. The full-likelihood estimates algorithm with precision set to high was used. All remaining parameters were set to the default. Potential parents with a pairwise likelihood under 90% were excluded as parental candidates unless a full-likelihood estimate was provided. Trait Associated Haplotype Prevalence For haplotype identification, markers from the curated dataset that had been mapped to the F. ×ananassa ‘Camarosa’ v. 1.0 assembly were used 14. Data were imputed and phased using Beagle v 5.2 (refs. 49 & 50). Pairwise linkage disequilibrium was calculated using VCFtools v 0.1.16 (ref. 51) to assess linkage disequilibrium decay. Haploblocks for each region of interest were defined as N nucleotides proximally and distally from markers associated with each gene or QTL, where N is the genome-wide distance required to reach an r2 of 0.20 when estimating linkage disequilibrium. The genetic regions for the remontancy gene FaPFRU 20 and disease resistance genes FaRCa1 (anthracnose fruit rot; ref. 26), FaRCg1 (Colletotrichum crown rot; ref. 22), FaRMp1 (charcoal rot; ref. 24), FaRMp2 (charcoal rot; ref. 24), FaRPc2 (Phytophthora crown rot; ref. 23), and Fw1 (Fusarium wilt; ref. 25) were investigated. Haplotypes associated with perpetual flowering and disease resistance were identified using previously reported favorable alleles in known positive accessions within the collection. The prevalence of these haplotypes within the collection and their geographical distributions were assessed. Haplotypes with identical sequences were arbitrarily named except for those that have been previously identified. Previously identified haplotypes were named using the gene name followed by any signifying haplotype in previous research.

The objective of this study was to evaluate 29 diverse octoploid and 1 decaploid strawberries for soilborne pathogen mortality.

Germplasm. The NCGR in Corvallis, OR provided 30 accessions from the Fragaria Supercore including 15 F. chiloensis accessions, 14 F. virginiana accessions, and one F. cascadensis accession. A total of 21 F. ×ananassa accessions were also evaluated. These accessions consisted of 10 selections and cultivars from the MSU strawberry breeding program in East Lansing, MI and 11 from the HCRU breeding program in Corvallis, OR. The public cultivars Monterey, Portola, and Albion were used as susceptible controls for Fusarium, Verticillium, and Macrophomina, respectively. Plant propagation was conducted to produce daughter plants for replicated inoculation trials for the Supercore and F. ×ananassa accessions. Runners for the F. ×ananassa accessions were produced by the University of California, Davis in a high elevation nursery in Dorris, CA and sent to the California Polytechnic State University (Cal Poly) in San Luis Obispo, CA.

Runners for the Supercore accessions were produced at Cal Poly. The Supercore mother plants were maintained in 7.6-L containers and fertilized with slow-release Osmocote Plus (15N-3.9P-4.8K). Plants were irrigated as needed throughout the trial using a showertype sprayer nozzle directed at the base of the crown. To establish daughter plants for inoculation, runners of each accession were rooted in plug cells (L × W × D of 8.00 cm × 3.94 cm × 5.92 cm; Greenhouse Megastore, Sacramento, CA) containing a substrate consisting of 33% coconut coir, 33% sphagnum peat moss, and 33% perlite. The runners were kept under intermittent mist for seven to 10 days until roots colonized the substrate. Once rooted, plants were allowed to grow for another four to six weeks before host resistance screens were initiated.

Phenotyping.Trials were conducted in the strawberry greenhouse at the Cal Poly Crops Unit. Host resistance screens were conducted for each of the three pathogens (F. oxysporum f. sp. fragariae, V. dahliae, and M. phaseolina). Seven plants of each selection were used per pathogen screen, inoculating six and leaving one as a non-inoculated control. Local isolates of each pathogen originating from diseased strawberry crowns were used for each inoculation. Inoculum for each trial consisted of 1 × 106 conidia per mL of water for F. oxysporum f. sp. fragariae (isolate GLI080), 5 × 106 conidia per mL of water for V. dahliae (isolates Vd1, Vd3, Vd7, and Vd20), or a slurry of M. phaseolina microsclerotia at 2,500 colony-forming-units per mL of slurry (isolates Mp8, Mp21, and Mp22). Plants were inoculated by soaking the roots in the inoculum for 5 min.

The noninoculated controls were soaked in water for 5 min. Plants were then transplanted into 0.5 L pots (10.2 cm diameter) with the same potting mix as previously described and arrayed randomly on a greenhouse bench. Plant mortality was assessed after the susceptible control cultivars had a mortality of greater than 83.3 % for their respective diseases. Susceptible control cultivars were evaluated only in trials in which they were the susceptible control. Fusarium and Verticillium trials were conducted twice for all accessions. Macrophomina trials were conducted once for Supercore accessions and twice for breeding accessions. Average percent mortality was calculated and accessions with less than 33.3 % mortality were considered resistant.