DEVELOPMENT OF GENOMIC RESOURCES AND IDENTIFICATION OF MARKER-TRAIT ASSOCIATIONS IN STRAWBERRY

Crosses were performed and progeny populations were developed in diploid and octoploid strawberry (Fragaria) species for the purposes of genetic analysis and breeding. A high throughput genotyping platform - the Affymetrix IStraw90® Axiom® strawberry SNP array - was evaluated and employed for these purposes. Phenotyping was conducted with respect to several traits of interest, including flower color, flower and fruit pigment compositions, and verticillium wilt resistance, for the ultimate purpose of identifying marker-trait associations useful in breeding. In the ancestral diploid species Fragaria iinumae, an F2 mapping population was developed from a cross between two accessions previously collected in Hokkaido, Japan. High-throughput genotyping platforms, the IStraw90 SNP Array and Genotyping-by-Sequencing (GBS), were utilized to develop high density linkage maps for F. iinumae. A linkage map based on 21 of the F2 generation plants genotyped on both platforms consists of 4,110 markers, while the second map based 85 F2 plants genotyped on only GBS consists of 895 markers. The linkage maps will used in a parallel research project to anchor an F. iinumae genomic sequence assembly, with the aim of providing an additional reference genome for Fragaria. A 455 member Closed Pedigree Set of octoploid strawberry plants was developed with diverse flower colors and progress was made on improvement of fruit quality through selection. The range of floral cyanidin and pelargonidin contents determined by HPLC in the progeny exceeded the range of contents of the founding parents, and thus exhibited transgressive segregation. The non-white (NW) flower trait was found to be a completely dominant to white, and no true-breeding, non-white flowered plants were found. The IStraw90 SNP Array was utilized to genotype 41 members of the Closed Pedigree Set and among a total of 5,674 segregating markers, 35 markers predictive of NW flower color were identified on chromosome 5. Additional genotyping will be required to further characterize the marker-trait association for color and to identify quantitative trait loci for hue. Fragaria diploid and polyploid species were phenotyped for verticillium wilt resistance, and disease resistance ratings of the octoploid cultivars were compared to the results of previous published studies and presented in a pedigree format to help identify sources of resistance. The results will be used to identify germplasm for developing populations for high throughput genotyping on the IStraw90 Array for the ultimate goal of identifying marker-trait associations for the purpose of marker-assisted breeding for resistance

A High-Density Linkage Map of the Ancestral Diploid Strawberry, Fragaria iinumae, Constructed with Single Nucleotide Polymorphism Markers from the IStraw90 Array and Genotyping by Sequencing

Fragaria iinumae Makino is recognized as an ancestor of the octoploid strawberry species, which includes the cultivated strawberry, Fragaria ×ananassa Duchesne ex Rozier. Here we report the construction of the first high-density linkage map for F. iinumae. The F. iinumae 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 F2 generation mapping population was derived by selfing F. iinumae hybrid F1D, the product of a cross between two divergent F. iinumae 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 Fragaria vesca ssp. vesca L. ‘Hawaii 4’ pseudo-chromosome (PC) assemblies reveal substantial conservation of synteny and colinearity, yet identified differences that point to possible genomic divergences between F. iinumae and F. vesca, and/or to F. vesca genomic assembly errors. The Fii map provides a basis for anchoring a F. iinumae genome assembly as a prerequisite for constructing a second diploid reference genome for Fragaria

Large-scale standardized phenotyping of strawberry in RosBREED

In an effort to implement marker-assisted breeding in Rosaceae, many traits need to be characterized in diverse germplasm. The USDA-NIFA Specialty Crop Research Initiative-funded RosBREED project includes breeding programs of four Rosaceae crops (apple, peach, cherry and strawberry). Phenotyping each crop for specific horticultural and commercial traits is an important process needed to translate genomic knowledge through Marker-assisted breeding into enhanced breeding efficiency. These data will directly aid in the identification of quantitative trait loci or marker-trait associations that will be used to assist breeding programs in the future. Large-scale, standardized phenotyping protocols have been set up for each crop. The standardized,phenotyping protocol for strawberries was agreed upon by the breeding teams in Oregon, Michigan, New Hampshire, California and Florida and includes four trait categories: phenology and other flower-related traits, plant characteristics, fruit characteristics, and fruit chemistry traits. We describe how each of the traits in the categories was evaluated. A summary of mean values for 37 traits of the genotypes planted at the RosBREED locations in 2011 and 2012 is provided

Large-scale standardized phenotyping of strawberry in RosBREED

In an effort to implement marker-assisted breeding in Rosaceae, many traits need to be characterized in diverse germplasm. The USDA-NIFA Specialty Crop Research Initiative-funded RosBREED project includes breeding programs of four Rosaceae crops (apple, peach, cherry and strawberry). Phenotyping each crop for specific horticultural and commercial traits is an important process needed to translate genomic knowledge through Marker-assisted breeding into enhanced breeding efficiency. These data will directly aid in the identification of quantitative trait loci or marker-trait associations that will be used to assist breeding programs in the future. Large-scale, standardized phenotyping protocols have been set up for each crop. The standardized,phenotyping protocol for strawberries was agreed upon by the breeding teams in Oregon, Michigan, New Hampshire, California and Florida and includes four trait categories: phenology and other flower-related traits, plant characteristics, fruit characteristics, and fruit chemistry traits. We describe how each of the traits in the categories was evaluated. A summary of mean values for 37 traits of the genotypes planted at the RosBREED locations in 2011 and 2012 is provided

Development and preliminary evaluation of a 90 K Axiom® SNP array for the allo-octoploid cultivated strawberry Fragaria × ananassa

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

RosBREED: bridging the chasm between discovery and application to enable DNA-informed breeding in rosaceous crops

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