A ddRAD Based Linkage Map of the Cultivated Strawberry, Fragaria xananassa.
ABSTRACT: The cultivated strawberry (Fragaria ×ananassa Duch.) is an allo-octoploid considered difficult to disentangle genetically due to its four relatively similar sub-genomic chromosome sets. This has been alleviated by the recent release of the strawberry IStraw90 whole genome genotyping array. However, array resolution relies on the genotypes used in the array construction and may be of limited general use. SNP detection based on reduced genomic sequencing approaches has the potential of providing better coverage in cases where the studied genotypes are only distantly related from the SNP array's construction foundation. Here we have used double digest restriction-associated DNA sequencing (ddRAD) to identify SNPs in a 145 seedling F1 hybrid population raised from the cross between the cultivars Sonata (?) and Babette (?). A linkage map containing 907 markers which spanned 1,581.5 cM across 31 linkage groups representing the 28 chromosomes of the species. Comparing the physical span of the SNP markers with the F. vesca genome sequence, the linkage groups resolved covered 79% of the estimated 830 Mb of the F. × ananassa genome. Here, we have developed the first linkage map for F. × ananassa using ddRAD and show that this technique and other related techniques are useful tools for linkage map development and downstream genetic studies in the octoploid strawberry.
Project description: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.
Project description:The strawberry, Fragaria?×?ananassa, is an allo-octoploid (2n?=?8x?=?56) and outcrossing species. Although it is the most widely consumed berry crop in the world, its complex genome structure has hindered its genetic and genomic analysis, and thus discrimination of subgenome-specific loci among the homoeologous chromosomes is needed. In the present study, we identified candidate subgenome-specific single nucleotide polymorphism (SNP) and simple sequence repeat (SSR) loci, and constructed a linkage map using an S1 mapping population of the cultivar 'Reikou' with an IStraw90 Axiom® SNP array and previously published SSR markers.The 'Reikou' linkage map consisted of 11,574 loci (11,002 SNPs and 572 SSR loci) spanning 2816.5 cM of 31 linkage groups. The 11,574 loci were located on 4738 unique positions (bin) on the linkage map. Of the mapped loci, 8999 (8588 SNPs and 411 SSR loci) showed a 1:2:1 segregation ratio of AA:AB:BB allele, which suggested the possibility of deriving loci from candidate subgenome-specific sequences. In addition, 2575 loci (2414 SNPs and 161 SSR loci) showed a 3:1 segregation of AB:BB allele, indicating they were derived from homoeologous genomic sequences. Comparative analysis of the homoeologous linkage groups revealed differences in genome structure among the subgenomes.Our results suggest that candidate subgenome-specific loci are randomly located across the genomes, and that there are small- to large-scale structural variations among the subgenomes. The mapped SNPs and SSR loci on the linkage map are expected to be seed points for the construction of pseudomolecules in the octoploid strawberry.
Project description: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.
Project description:The cultivated strawberry (Fragaria×ananassa) is consumed worldwide for its flavor and nutritional benefits. Genetic analysis of commercially important traits in strawberry are important for the development of breeding methods and tools for this species. Although several quantitative trait loci (QTL) have been previously detected for fruit quality and flowering traits using low-density genetic maps, clarity on the sub-genomic locations of these QTLs was missing. Recent discoveries in allo-octoploid strawberry genomics led to the development of the IStraw90 single-nucleotide polymorphism (SNP) array, enabling high-density genetic maps and finer resolution QTL analysis. In this study, breeder-specified traits were evaluated in the Eastern (Michigan) and Western (Oregon) United States for a common set of breeding populations during 2 years. Several QTLs were validated for soluble solids content (SSC), fruit weight (FWT), pH and titratable acidity (TA) using a pedigree-based QTL analysis approach. For fruit quality, a QTL for SSC on linkage group (LG) 6A, a QTL for FWT on LG 2BII, a QTL for pH on LG 4CII and two QTLs for TA on LGs 2A and 5B were detected. In addition, a large-effect QTL for flowering was detected at the distal end of LG 4A, coinciding with the FaPFRU locus. Marker haplotype analysis in the FaPFRU region indicated that the homozygous recessive genotype was highly predictive of seasonal flowering. SNP probes in the FaPFRU region may help facilitate marker-assisted selection for this trait.
Project description:The cultivated strawberry (Fragaria × ananassa) is an octoploid (2n = 8x = 56) of the Rosaceae family whose genomic architecture is still controversial. Several recent studies support the AAA'A'BBB'B' model, but its complexity has hindered genetic and genomic analysis of this important crop. To overcome this difficulty and to assist genome-wide analysis of F. × ananassa, we constructed an integrated linkage map by organizing a total of 4474 of simple sequence repeat (SSR) markers collected from published Fragaria sequences, including 3746 SSR markers [Fragaria vesca expressed sequence tag (EST)-derived SSR markers] derived from F. vesca ESTs, 603 markers (F. × ananassa EST-derived SSR markers) from F. × ananassa ESTs, and 125 markers (F. × ananassa transcriptome-derived SSR markers) from F. × ananassa transcripts. Along with the previously published SSR markers, these markers were mapped onto five parent-specific linkage maps derived from three mapping populations, which were then assembled into an integrated linkage map. The constructed map consists of 1856 loci in 28 linkage groups (LGs) that total 2364.1 cM in length. Macrosynteny at the chromosome level was observed between the LGs of F. × ananassa and the genome of F. vesca. Variety distinction on 129 F. × ananassa lines was demonstrated using 45 selected SSR markers.
Project description: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.
Project description:Cultivated strawberry (Fragaria × ananassa) is a genetically complex allo-octoploid crop with 28 pairs of chromosomes (2n = 8x = 56) for which a genome sequence is not yet available. The diploid Fragaria vesca is considered the donor species of one of the octoploid sub-genomes and its available genome sequence can be used as a reference for genomic studies. A wide number of strawberry cultivars are stored in ex situ germplasm collections world-wide but a number of previous studies have addressed the genetic diversity present within a limited number of these collections. Here, we report the development and application of two platforms based on the implementation of Diversity Array Technology (DArT) markers for high-throughput genotyping in strawberry. The first DArT microarray was used to evaluate the genetic diversity of 62 strawberry cultivars that represent a wide range of variation based on phenotype, geographical and temporal origin and pedigrees. A total of 603 DArT markers were used to evaluate the diversity and structure of the population and their cluster analyses revealed that these markers were highly efficient in classifying the accessions in groups based on historical, geographical and pedigree-based cues. The second DArTseq platform took benefit of the complexity reduction method optimized for strawberry and the development of next generation sequencing technologies. The strawberry DArTseq was used to generate a total of 9,386 SNP markers in the previously developed '232' × '1392' mapping population, of which, 4,242 high quality markers were further selected to saturate this map after several filtering steps. The high-throughput platforms here developed for genotyping strawberry will facilitate genome-wide characterizations of large accessions sets and complement other available options.
Project description:Macrosynteny and colinearity between Fragaria (strawberry) species showing extreme levels of ploidy have been studied through comparative genetic mapping between the octoploid cultivated strawberry (F. xananassa) and its diploid relatives. A comprehensive map of the octoploid strawberry, in which almost all linkage groups are ranged into the seven expected homoeologous groups was obtained, thus providing the first reference map for the octoploid Fragaria. High levels of conserved macrosynteny and colinearity were observed between homo(eo)logous linkage groups and between the octoploid homoeologous groups and their corresponding diploid linkage groups. These results reveal that the polyploidization events that took place along the evolution of the Fragaria genus and the more recent juxtaposition of two octoploid strawberry genomes in the cultivated strawberry did not trigger any major chromosomal rearrangements in genomes involved in F. xananassa. They further suggest the existence of a close relationship between the diploid Fragaria genomes. In addition, despite the possible existence of residual levels of polysomic segregation suggested by the observation of large linkage groups in coupling phase only, the prevalence of linkage groups in coupling/repulsion phase clearly demonstrates that the meiotic behavior is mainly disomic in the cultivated strawberry.
Project description:Genotyping-by-sequencing (GBS) was used to survey genome-wide single-nucleotide polymorphisms (SNPs) in three biparental strawberry (Fragaria × ananassa) populations with the goal of evaluating this technique in a species with a complex octoploid genome. GBS sequence data were aligned to the F. vesca 'Fvb' reference genome in order to call SNPs. Numbers of polymorphic SNPs per population ranged from 1,163 to 3,190. Linkage maps consisting of 30-65 linkage groups were produced from the SNP sets derived from each parent. The linkage groups covered 99% of the Fvb reference genome, with three to seven linkage groups from a given parent aligned to any particular chromosome. A phylogenetic analysis performed using the POLiMAPS pipeline revealed linkage groups that were most similar to ancestral species F. vesca for each chromosome. Linkage groups that were most similar to a second ancestral species, F. iinumae, were only resolved for Fvb 4. The quantity of missing data and heterogeneity in genome coverage inherent in GBS complicated the analysis, but POLiMAPS resolved F. × ananassa chromosomal regions derived from diploid ancestor F. vesca.
Project description:Octoploid strawberry (Fragaria ×ananassa) is a valuable specialty crop, but profitable production and availability are threatened by many pathogens. Efforts to identify and introgress useful disease resistance genes (R-genes) in breeding programs are complicated by strawberry's complex octoploid genome. Recently-developed resources in strawberry, including a complete octoploid reference genome and high-resolution octoploid genotyping, enable new analyses in strawberry disease resistance genetics. This study characterizes the complete R-gene collection in the genomes of commercial octoploid strawberry and two diploid ancestral relatives, and introduces several new technological and data resources for strawberry disease resistance research. These include octoploid R-gene transcription profiling, dN/dS analysis, expression quantitative trait loci (eQTL) analysis and RenSeq analysis in cultivars. Octoploid fruit eQTL were identified for 76 putative R-genes. R-genes from the ancestral diploids Fragaria vesca and Fragaria iinumae were compared, revealing differential inheritance and retention of various octoploid R-gene subtypes. The mode and magnitude of natural selection of individual F. ×ananassa R-genes was also determined via dN/dS analysis. R-gene sequencing using enriched libraries (RenSeq) has been used recently for R-gene discovery in many crops, however this technique somewhat relies upon a priori knowledge of desired sequences. An octoploid strawberry capture-probe panel, derived from the results of this study, is validated in a RenSeq experiment and is presented for community use. These results give unprecedented insight into crop disease resistance genetics, and represent an advance toward exploiting variation for strawberry cultivar improvement.