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The Rosaceae crop family (including almond, apple, apricot, blackberry, peach, pear, plum, raspberry, rose, strawberry, sweet cherry, and sour cherry) provides vital contributions to human well-being and is economically significant across the U.S. In 2003, industry stakeholder initiatives prioritized the utilization of genomics, genetics, and breeding to develop new cultivars exhibiting both disease resistance and superior horticultural quality. However, rosaceous crop breeders lacked certain knowledge and tools to fully implement DNA-informed breeding—a “chasm” existed between existing genomics and genetic information and the application of this knowledge in breeding. The RosBREED project (“Ros” signifying a Rosaceae genomics, genetics, and breeding community initiative, and “BREED”, indicating the core focus on breeding programs), addressed this challenge through a comprehensive and coordinated 10-year effort funded by the USDA-NIFA Specialty Crop Research Initiative. RosBREED was designed to enable the routine application of modern genomics and genetics technologies in U.S. rosaceous crop breeding programs, thereby enhancing their efficiency and effectiveness in delivering cultivars with producer-required disease resistances and market-essential horticultural quality. This review presents a synopsis of the approach, deliverables, and impacts of RosBREED, highlighting synergistic global collaborations and future needs. Enabling technologies and tools developed are described, including genome-wide scanning platforms and DNA diagnostic tests. Examples of DNA-informed breeding use by project participants are presented for all breeding stages, including pre-breeding for disease resistance, parental and seedling selection, and elite selection advancement. The chasm is now bridged, accelerating rosaceous crop genetic improvement.

The first high-resolution genetic linkage map of the ancestral octoploid (2n = 8x = 56) strawberry species, Fragaria virginiana, was constructed using segregation data obtained from a pentaploid progeny population. This novel mapping population of size 178 was generated by crossing highly heterozygous F. virginiana hybrid “LB48” as a paternal parent with diploid (2n = 2x = 14) Fragaria vesca “Hawaii 4”. The LB48 linkage map comprises 6055 markers genotyped on the Axiom® IStraw90 strawberry SNP array. The map consists of 28 linkage groups (LGs) organized into seven homoeology groups of four LGs each, and excludes a small 29th LG of undefined homoeology. One member of each homoeology group was assignable to an “A” subgenome associated with ancestral diploid Fragaria vesca, while no other subgenomes were defined. Despite an intriguing discrepancy within homoeology group VI, synteny comparisons with the previously published Fragaria ×ananassa DA × MO linkage map revealed substantial agreement. Following initial map construction, examination of crossover distributions revealed that six of the total 5162 (=29 chromosomes/individual × 178 individuals) chromosomes making up the data set exhibited abnormally high crossover counts, ranging from 15 to 48 crossovers per chromosome, as compared with the overall mean of 0.66 crossovers per chromosome. Each of these six hyper-recombinant (HypR) chromosomes occurred in a different LG and in a different individual. When calculated upon exclusion of the six HypR chromosomes, the canonical (i.e., broadly representative) LB48 map had 1851 loci distributed over a total map length of 1873 cM, while their inclusion increased the number of loci by 130, and the overall map length by 91 cM. Discovery of these hyper-recombinant chromosomes points to the existence of a sporadically acting mechanism that, if identified and manipulable, could be usefully harnessed for multiple purposes by geneticists and breeders.

BACKGROUND: The fungal disease verticillium wilt has been recognized as an obstacle to strawberry production since its initial description in 1931. The full potential of genetic resistance as a solution to this problem has yet to be determined or realized. OBJECTIVE: Our investigations are concerned with defining new sources of resistance to verticillium wilt disease in cultivated and wild strawberry germplasm, and with advancing genetic studies on the basis of resistance/susceptibility. METHODS: We screened 23 diploid, 1 decaploid, and 26 octoploid Fragaria (strawberry) germplasm accessions and cultigens for response to root-dip inoculation with Verticillium dahliae isolate V1. Pedigree relationships of 10 studied cultigens were examined. Crosses were performed between resistant and susceptible accessions. RESULTS: Variability in inoculation response existed within and between species at diploid and octoploid levels. Very or moderately resistant accessions were found within each of three diploid and three octoploid species. Moderately or very susceptible accessions were documented within F. vesca and each octoploid species. Segregation for resistance/susceptibility was evident in progeny populations. CONCLUSIONS: The verticillium wilt resistance ratings reported here and discussed in relation to prior studies adds to the body of publically available knowledge about sources of wilt resistance and susceptibility in Fragaria germplasm.

Ten seedlings from 36 crosses representing eastern and western North American short day and remontant genotypes were evaluated in 2011 and 2012 in California, Michigan, New Hampshire and Oregon, for phenology, flower related traits, plant characteristics, fruit characteristics and fruit chemistry traits. There was significant variability among genotypes, locations and evaluation year for most of the characteristics; however, few genotype × location and genotype × year interactions were detected. General combining ability variance components were significant for all traits and greater than SCA variance components for peduncle length, total flowering weeks, flowering cycles, truss size, growing degree days for harvest data, remontancy, achene position, ease of capping, fruit weight, percent soluble solids, titratable acidity and soluble solids/titratable acidity. ‘Sarian’ was identified as the best contributing parent for remontancy. Narrow-sense heritability estimates were moderate to high (0.33–0.78) for total flowering weeks, flowering cycle, truss size, remontancy, number of runners, fruit weight, pH, and titratable acidity. Having a better understanding of these attributes will provide breeders guidance on the most effective breeding strategies for incorporating superior traits from this germplasm into their programs.

UMR 1332 - Equipe OrFE; The Axiom® IStraw90 SNP (single nucleotide polymorphism) array was developed to enable high-throughput genotyping in allo-octoploid cultivated strawberry (Fragaria ×ananassa). However, high cost ($ 80-105 sample-1) limits throughput for certain applications. On average the IStraw90 has yielded 50 to 60% usable data points, defined as PHR (Poly High Resolution) and NMH (No Minor Homozygote) marker classes. Thus, an array is needed with a higher percentage of usable data points at a lower cost. We initiated an effort to identify IStraw90 SNP markers that were genetically mapped in one or more strawberry populations from research programs around the world. Seven programs participated in this endeavor. A total of 41,183 SNP probes were submitted to Affymetrix for quality check, 38,506 of which were accommodated on the Axiom® IStraw35 384HT design. In order to assess the performance of the Axiom® IStraw35 384HT array, 384 DNA samples from the University of Florida strawberry breeding program were assayed at a cost of $ 50 per sample, all inclusive. The performance of the array met expectations. More than 87% of markers belonged to the PHR and NMH categories. This array is expected to provide high-quality genome scanning at a more affordable price for strawberry researchers worldwide.

Makino is recognized as an ancestor of the octoploid strawberry species, which includes the cultivated strawberry, × Duchesne ex Rozier. Here we report the construction of the first high-density linkage map for . The linkage map (Fii map) is based on two high-throughput techniques of single nucleotide polymorphism (SNP) genotyping: the IStraw90 Array (hereafter “Array”), and genotyping by sequencing (GBS). The F generation mapping population was derived by selfing hybrid F1D, the product of a cross between two divergent accessions collected from Hokkaido, Japan. The Fii map consists of seven linkage groups (LGs) and has an overall length of 451.7 cM as defined by 496 loci populated by 4173 markers: 3280 from the Array and 893 from GBS. Comparisons with two versions of the ssp. L. ‘Hawaii 4’ pseudo-chromosome (PC) assemblies reveal substantial conservation of synteny and colinearity, yet identified differences that point to possible genomic divergences between and , and/or to genomic assembly errors. The Fii map provides a basis for anchoring a genome assembly as a prerequisite for constructing a second diploid reference genome for .

Of the three plant genomic components (nuclear, plastid, and mitochondrial) the mitochondrial DNA (mtDNA) has received the least research attention. We describe a phylogenetically informative sequence in the matR gene region of the Fragaria (strawberry) mtDNA, and the first mtDNA marker developed for this economically important genus. The sequence was obtained from a fosmid genomic library clone of F. vesca subsp. americana 'Pawtuckaway'. New mtDNA PCR primer sets work on members of the Rosaceae family, and have potential for broad applicability in the angiosperms. An informative mtDNA CAPS (cleaved amplified polymorphic site) marker provided evidence that mtDNA was transmitted maternally in two interspecific crosses, and that diploid F. iinumae is the likely mtDNA donor to the octoploid species F. chiloensis and F. virginiana, but not to decaploid F. iturupensis. We expanded the database of Fragaria mtDNA sequence by assembling a 67 kb mtDNA contig from Illumina 36 bp paired-end reads from 'Pawtuckaway', providing a basis to develop additional mtDNA markers.

The fruit cyanidin (Cyn) and pelargonidin (Plr) contents in 73 Fragaria accessions were assayed using a modified HPLC method, and % Cyn (Cyn as % of Cyn-plus-Plr) values were calculated. Plr content increased during fruit ripening, while Cyn content held steady. Consistent with previous studies, % Cyn was comparatively low (5-20%) among twelve F. ×ananassa cultivars, but was >40% in diploid F. vesca. Importantly, three F. chiloensis accessions had greater than 50% Cyn, and ssp. lucida CFRA1691 had the overall highest Cyn content: 707 μg/g FDW. An F 1 population of 'Bountiful' × 'Pink Panda' segregated for both Cyn and Plr contents. Our investigation identifies useful germplasm, offering promise for increasing Cyn content and antioxidant potential through introgressive breeding.

This is Scientific Contribution Number 2565 from the New Hampshire Agricultural Experiment Station., [Background]: A high-throughput genotyping platform is needed to enable marker-assisted breeding in the allo-octoploid cultivated strawberry Fragaria × ananassa. Short-read sequences from one diploid and 19 octoploid accessions were aligned to the diploid Fragaria vesca ‘Hawaii 4’ reference genome to identify single nucleotide polymorphisms (SNPs) and indels for incorporation into a 90 K Affymetrix® Axiom® array. We report the development and preliminary evaluation of this array., [Results]: About 36 million sequence variants were identified in a 19 member, octoploid germplasm panel. Strategies and filtering pipelines were developed to identify and incorporate markers of several types: di-allelic SNPs (66.6%), multi-allelic SNPs (1.8%), indels (10.1%), and ploidy-reducing “haploSNPs” (11.7%). The remaining SNPs included those discovered in the diploid progenitor F. iinumae (3.9%), and speculative “codon-based” SNPs (5.9%). In genotyping 306 octoploid accessions, SNPs were assigned to six classes with Affymetrix’s “SNPolisher” R package. The highest quality classes, PolyHigh Resolution (PHR), No Minor Homozygote (NMH), and Off-Target Variant (OTV) comprised 25%, 38%, and 1% of array markers, respectively. These markers were suitable for genetic studies as demonstrated in the full-sib family ‘Holiday’ × ‘Korona’ with the generation of a genetic linkage map consisting of 6,594 PHR SNPs evenly distributed across 28 chromosomes with an average density of approximately one marker per 0.5 cM, thus exceeding our goal of one marker per cM., [Conclusions]: The Affymetrix IStraw90 Axiom array is the first high-throughput genotyping platform for cultivated strawberry and is commercially available to the worldwide scientific community. The array’s high success rate is likely driven by the presence of naturally occurring variation in ploidy level within the nominally octoploid genome, and by effectiveness of the employed array design and ploidy-reducing strategies. This array enables genetic analyses including generation of high-density linkage maps, identification of quantitative trait loci for economically important traits, and genome-wide association studies, thus providing a basis for marker-assisted breeding in this high value crop., This study was partially funded by the USDA’s National Institute of Food and Agriculture – Specialty Crop Research Initiative project, “RosBREED: Enabling marker-assisted breeding in Rosaceae” (2009-51181-05808). Partial funding was also provided by the New Hampshire Agricultural Experiment Station (Projects NH00433 and NH00588 - to TMD), and from USDA-CSREES National Research Initiative (NRI) Plant Genome Grant 2008-35300-04411 (to TMD). Technological Top Institute Green Genetics, project number 1C004RP, is acknowledged for financial support. Financial support was also provided by AGL2010-21414 from the Spanish Ministry of Science (to AM); from the USDA-ARS CRIS number 5358-21000-044-00D (to NVB).

Strawberry is one of the five fruit crops included in the USDA-funded multi-institutional and trans-disciplinary project, “RosBREED: Enabling Marker-Assisted Breeding in Rosaceae”. A Crop Reference Set (CRS) was developed of 900 genotypes and seedlings from 40 crosses representing the breadth of relevant diversity and encompassing founders used in breeding the domesticated strawberry. Individual native species and cultivar genotypes were included along with 10 progeny from 36 of the crosses of genotypes representing eastern and western North American and European short day and remontant cultivars. This CRS has been phenotyped in five U.S. states. Over 14 fruit quality traits have been studied, as well as remontancy, truss size, peduncle length, crop estimate, plant architecture, and disease resistance. The phenotyping conducted in the first growing season showed considerable variability amongst the genotypes and the locations for all of the characteristics. General and specific combining ability variance components were determined from the populations in order to provide breeders with guidance on the most effective breeding strategies for incorporating the superior traits from this germplasm into their programs.