The genus Fragaria encompasses species at ploidy levels ranging from diploid to decaploid. The cultivated strawberry, Fragaria×ananassa, and its two immediate progenitors, F. chiloensis and F. virginiana, are octoploids. To elucidate the ancestries of these octoploid species, we performed a phylogenetic analysis using intron-containing sequences of the nuclear ADH-1 gene from 39 germplasm accessions representing nineteen Fragaria species and one outgroup species, Dasiphora fruticosa. All trees from Maximum Parsimony and Maximum Likelihood analyses showed two major clades, Clade A and Clade B. Each of the sampled octoploids contributed alleles to both major clades. All octoploid-derived alleles in Clade A clustered with alleles of diploid F. vesca, with the exception of one octoploid allele that clustered with the alleles of diploid F. mandshurica. All octoploid-derived alleles in clade B clustered with the alleles of only one diploid species, F. iinumae. When gaps encoded as binary characters were included in the Maximum Parsimony analysis, tree resolution was improved with the addition of six nodes, and the bootstrap support was generally higher, rising above the 50% threshold for an additional nine branches. These results, coupled with the congruence of the sequence data and the coded gap data, validate and encourage the employment of sequence sets containing gaps for phylogenetic analysis. Our phylogenetic conclusions, based upon sequence data from the ADH-1 gene located on F. vesca linkage group II, complement and generally agree with those obtained from analyses of protein-encoding genes GBSSI-2 and DHAR located on F. vesca linkage groups V and VII, respectively, but differ from a previous study that utilized rDNA sequences and did not detect the ancestral role of F. iinumae.
BACKGROUND: The diploid (2n = 2x = 14) strawberry model plant Fragaria vesca ssp. vesca 'Hawaii 4' was employed for functional analysis of expressed DNA sequences initially identified as being unique to Fragaria and of unknown or poorly understood function. 'Hawaii 4' is prominent in strawberry research due to its ease of Agrobacterium-mediated transformation and regenerability, and its status as the source of the first complete strawberry genomic sequence. Our studies of a set of transformants have documented intriguing, construct-associated effects on leaf morphology, and provide important and unexpected insights into the performance of the 'Hawaii 4' transformation and regeneration system.
RESULTS: Following Agrobacterium-mediated transformation of leaf explants with gene constructs carried by Gateway® vectors, plants were regenerated using a modified version of an established 'Hawaii 4' protocol. Expanding upon the findings of prior studies, we documented that plantlet regeneration was occurring via a somatic embryogenic rather than an organogenic developmental pathway. Among transformants, several variations in leaf morphology were observed. Unexpectedly, a particular leaf variant type, occurring in ~17% of all regenerants independent of construct type, was found to be attributable to tetraploidy. The tetraploidy-associated alteration in leaf morphology could be differentiated from the leaf morphology of diploid regenerants on the basis of a quantitative ratio of leaf dimensions: B/A, where B is the width of the central leaflet and A is the overall width of the trifoliate leaf. Variant effects on leaf morphology of four different transgenic constructs were also documented, and were in all cases distinguishable from the effects of tetraploidy.
CONCLUSIONS: These results define opportunities to optimize the existing 'Hawaii 4' protocol by focusing on treatments that specifically promote somatic embryogenesis. The reported morphological metric and descriptions will guide future transgenic studies using the 'Hawaii 4' model system by alerting researchers to the potential occurrence of polyploid regenerants, and to differentiating the effects on leaf morphology due to polyploidy versus transgenic manipulations. Finally, an intriguing spectrum of leaf morphology alterations resulting from manipulation of expressed sequences of uncertain function is documented, providing a foundation for detailed studies of the respective genes and their functional roles.
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. The phenotypic data for widely used founder germplasm that has contributed to current cultivars is available through the “Breeders Toolbox” at the Genome Database for Rosaceae (http://www.rosaceae.org/breeders_toolbox).
Asian germplasm has significantly contributed to berry crops in America in several ways. The American wild octoploid species [Fragaria chiloensis (L.) Mill. and F. virginiana Mill.], and subsequently, the cultivated strawberry (F. ×ananassa Duch. ex Rozier), have benefitted from Asian heritage in the evolutionary time scale. Second, breeders have combined Asian germplasm in crosses for improved fruit cultivars. Third, Asian temperate fruit species have been collected from wild stands in their native ranges, imported, and in some cases improved and are now cultivated in the West or throughout the world. The objectives of this article were to 1) describe evolutionary contributions of Asian species to the American strawberry genome; 2) present examples of breeding Asian species (Rubus L. subgenus Idaeobatus) into cultivated raspberries; and 3) give examples of two Asian fruit species that have been recently introduced and cultivated or that could be developed for cultivation in the United States.
The genome sequence of the woodland strawberry (Fragaria vesca L.) is an important resource providing a reference for comparative genomics studies and future sequenced rosaceous species and has great utility as a model for the development of markers for mapping in the cultivated strawberry Fragaria ×ananassa Duchesne ex Rozier. A set of 152 microsatellite simple sequence repeat (SSR) primer pairs was developed and mapped, along with 42 previously published but unmapped SSRs, permitting the precise assignment of 28.2 Mbp of previously unanchored genome sequence scaffolds (13% of the F. vesca genome sequence). The original ordering of F. vesca sequence scaffolds was performed without a physical map, using predominantly SSR markers to order scaffolds via anchoring to a comprehensive linkage map. This report complements and expands resolution of the Fragaria spp. reference map and refines the scaffold ordering of the F. vesca genome sequence using newly devised tools. The results of this study provide two significant resources: (i) the concurrent validation of a substantial set of SSRs associated with these previously unmapped regions of the Fragaria spp. genome and (ii) the precise placement of previously orphaned genomic sequence. Together, these resources improve the resolution and completeness of the strawberry genome sequence, making it a better resource for downstream studies in Fragaria spp. and the family Rosaceae.
BACKGROUND: The genus Fragaria comprises species at ploidy levels ranging from diploid (2n = 2x = 14) to decaploid (2n = 10x = 70). Fluorescence in situ hybridization with 5S and 25S rDNA probes was performed to gather cytogenetic information that illuminates genomic divergence among different taxa at multiple ploidy levels, as well as to explore the evolution of ribosomal RNA genes during polyploidization in Fragaria.
RESULTS: Root tip cells of diploid taxa were typified by two 5S and six 25S rDNA hybridization signals of varying intensities, providing a baseline for comparisons within the genus. In three exceptional diploid genotypes, F. nilgerrensis (CFRA 1358 and CFRA 1825) and F. vesca 'Yellow Wonder', two 5S but only four 25S rDNA sites were found but with differing site losses. The numbers of 5S and 25S rDNA signals, respectively were three and nine in a triploid F. ×bifera accession, and were four and twelve in three tetraploids, thus occurring in proportional 1.5× and 2× multiples of the typical diploid pattern. In hexaploid F. moschata, a proportional multiple of six 5S rDNA sites was observed, but the number of 25S rDNA sites was one or two less than the proportionate prediction of eighteen. This apparent tendency toward rDNA site loss at higher ploidy was markedly expanded in octoploids, which displayed only two 5S and ten 25S rDNA sites. In the two decaploids examined, the numbers of 5S and 25S rDNA signals, respectively, were four and fifteen in F. virginiana subsp. platypetala, and six and twelve in F. iturupensis.
CONCLUSIONS: Among diploid Fragaria species, a general consistency of rDNA site numbers implies conserved genomic organization, but highly variable 25S signal sizes and intensities and two instances of site loss suggest concurrent high dynamics of rDNA copy numbers among both homologs and non-homologs. General conservation of rDNA site numbers in lower ploidy, but marked site number reductions at higher ploidy levels, suggest complex evolution of rDNA sites during polyploidization and/or independent evolutionary pathways for 6x versus higher ploidy strawberries. Site number comparisons suggest common genomic composition among natural octoploids, and independent origins of the two divergent decaploid accessions.
The woodland strawberry, Fragaria vesca (2n = 2x = 14), is a versatile experimental plant system. This diminutive herbaceous perennial has a small genome (240 Mb), is amenable to genetic transformation and shares substantial sequence identity with the cultivated strawberry (Fragaria × ananassa) and other economically important rosaceous plants. Here we report the draft F. vesca genome, which was sequenced to ×39 coverage using second-generation technology, assembled de novo and then anchored to the genetic linkage map into seven pseudochromosomes. This diploid strawberry sequence lacks the large genome duplications seen in other rosids. Gene prediction modeling identified 34,809 genes, with most being supported by transcriptome mapping. Genes critical to valuable horticultural traits including flavor, nutritional value and flowering time were identified. Macrosyntenic relationships between Fragaria and Prunus predict a hypothetical ancestral Rosaceae genome that had nine chromosomes. New phylogenetic analysis of 154 protein-coding genes suggests that assignment of Populus to Malvidae, rather than Fabidae, is warranted.
The KNOTTED-LIKE HOMEODOMAIN (KNOX) genes play a central role in maintenance of the shoot apical meristem. They also contribute to the morphology of simple and compound leaves. In this report we characterize the FaKNOX1 gene from strawberry (Fragaria spp.) and demonstrate its function in trasgenic plants. The FaKNOX1 cDNA was isolated from a cultivated strawberry (F.×ananassa) flower EST library. The sequence is most similar to Class I KNOX genes, and was mapped to linkage group VI of the diploid strawberry genome. Unlike most KNOX genes studied, steady-state transcript levels were highest in flowers and fruits. Transcripts were also detected in emerging leaf primordia and the apical dome. Transgenic strawberry plants suppressing or overexpressing FaKNOX1 exhibited conspicuous changes in plant form. The FaKNOX1 RNAi plants presented a dwarfed phenotype with deeply serrated leaflets and exaggerated petiolules. They also exhibited a high level of cellular disorganization of the shoot apical meristem and leaves. Overexpression of FaKNOX1 caused dwarfed stature with wrinkled leaves. These gain- and loss-of-function assays in strawberry functionally demonstrate the contributions of a KNOX domain protein in a rosaceous species.
In the emerging eraof Next Generation (NextGen) technologies, molecular markers will continue to play important roles in research requiring targeted genotyping rather than global sequencing. Using chloroplast DNA (cpDNA) markers, we generated the first evidence confirming that cpDNA is maternally inherited in Fragaria, and defined likely diploid sources of the chloroplast genomes harbored by the Fragaria hexaploid, octoploid, and decaploid allopolyploid species. As a resource for future research on Fragaria cpDNA, we used a NextGen sequencing approach to assemble a draft sequence of the Fragaria vesca subsp. americana ‘Pawtuckaway’ chloroplast genome.
BACKGROUND: Although technological advances allow for the economical acquisition of whole genome sequences, many organisms' genomes remain unsequenced, and fully sequenced genomes may contain gaps. Researchers reliant upon partial genomic or heterologous sequence information require methods for obtaining unknown sequences from loci of interest. Various PCR based techniques are available for sequence walking - i.e., the acquisition of unknown DNA sequence adjacent to known sequence. Many such methods require rigid, elaborate protocols and/or impose narrowly confined options in the choice of restriction enzymes for necessary genomic digests. We describe a new method, TOPO(R) Vector-Ligation PCR (or TVL-PCR) that innovatively integrates available tools and familiar concepts to offer advantages as a means of both targeted sequence walking and paralog mining.
FINDINGS: TVL-PCR exploits the ligation efficiency of the pCR(R)4-TOPO(R) (Invitrogen, Carlsbad, California) vector system to capture fragments of unknown sequence by creating chimeric molecules containing defined priming sites at both ends. Initially, restriction enzyme-digested genomic DNA is end-repaired to create 3' adenosine overhangs and is then ligated to pCR4-TOPO vectors. The ligation product pool is used directly as a template for nested PCR, using specific primers to target orthologous sequences, or degenerate primers to enable capture of paralogous gene family members. We demonstrated the efficacy of this method by capturing entire coding and partial promoter sequences of several strawberry Superman-like genes.
CONCLUSIONS: TVL-PCR is a convenient and efficient method for DNA sequence walking and paralog mining that is applicable to any organism for which relevant DNA sequence is available as a basis for primer design.
BACKGROUND: Strawberry (Fragaria ssp.) is the familiar name of a group of economically important crop plants and wild relatives that also represent an emerging system for the study of gene and genome evolution. Its small stature, rapid seed-to-seed cycle, transformability and miniscule basic genome make strawberry an attractive system to study processes related to plant physiology, development and crop production; yet it lacks substantial genomics-level resources. This report addresses this deficiency by characterizing 0.71 Mbp of gene space from a diploid species (F. vesca). The twenty large genomic tracks (30-52 kb) captured as fosmid inserts comprise gene regions with roles in flowering, disease resistance, and metabolism.
RESULTS: A detailed description of the studied regions reveals 131 Blastx-supported gene sites and eight additional EST-supported gene sites. Only 15 genes have complete EST coverage, enabling gene modelling, while 76 lack EST support. Instances of microcolinearity with Arabidopsis thaliana were identified in twelve inserts. A relatively high portion (25%) of targeted genes were found in unanticipated tandem duplications. The effectiveness of six FGENESH training models was assessed via comparisons among ab initio predictions and homology-based gene and start/stop codon identifications. Fourteen transposable-element-related sequences and 158 simple sequence repeat loci were delineated.
CONCLUSIONS: This report details the structure and content of targeted regions of the strawberry genome. The data indicate that the strawberry genome is gene-dense, with an average of one protein-encoding gene or pseudogene per 5.9 kb. Current overall EST coverage is sparse. The unexpected gene duplications and their differential patterns of EST support suggest possible subfunctionalization or pseudogenization of these sequences. This report provides a high-resolution depiction of targeted gene neighborhoods that will aid whole-genome sequence assembly, provide valuable tools for plant breeders and advance the understanding of strawberry genome evolution.
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.
Strawberry (Fragaria spp.) is a valuable fruit crop as well as an outstanding system for studying functional genomics in plants. The goal of this study was to substantially increase and analyze the available expressed sequence information in the genus by examining the transcriptome of the cultivated strawberry (Fragaria × ananassa Duchesne). To maximize transcript diversity and discovery, plants representing an octoploid strawberry cultivar were subjected to a broad range of treatments. Plant materials were pooled by tissue type. cDNA pools were sequenced by the Roche-454 GS-FLX system and assembled into over 32,000 contigs. Predictions of cellular localization and function were made by associating assembled contigs to annotated homologs, and the tissue pool tags provided a means to assess the overall expression pattern for any given transcript. Contigs comprised of reads originating from only one organ type and those present equally in all plant organs were both identified. Bacterial and fungal sequences found in the strawberry samples provide a metagenomic survey of the microbial community of a greenhouse strawberry plant. This study utilized an innovative assembly strategy on pooled tissues, thus providing a foundation for developing tissue-specific tools, an opportunity to identify alleles for marker-assisted selection, a reference of strawberry gene annotations, and a basis for comparative transcriptomics between cultivated strawberry, its diploid ancestors, and the wider Rosaceae family.
The United States Department of Agriculture (USDA) - Agricultural Research Service (ARS) - National Clonal Germplasm Repository (NCGR) in Corvallis, Oregon, is a genebank that preserves strawberry genetic resources. The Fragaria L. collection consists of more than 1700 accessions of 17 species from 37 countries. In 2004, Asian diploid species, F. iinumae Makino and F. nipponica Makino, were collected during an expedition to Hokkaido, Japan. An ancestor of F. iinumae may be a progenitor of the “B” genome for octoploid strawberry species. The objective of this study was to evaluate the diversity of these two species using microsatellite markers. We report preliminary results obtained from SSR analysis of 139 accessions based on 20 SSRs. A higher genetic diversity was observed in F. nipponica, which tends to be cross pollinated, than in F. iinumae, which tends to be self-pollinated. This study identified putative hybrids between F. iinumae and F. nipponica as well as an unexpected octoploid accession from Hokkaido, Japan. The hybrid nature of these accessions and the possible source of this octoploid accession will be further evaluated using chloroplast and nuclear markers as well as morphological traits.
The genus Fragaria has a basic chromosome number of seven (x=7) (Ichijima, 1926), and four main fertility groups are recognized: the diploids (2n=2x=14) which include the model species for the genus, F. vesca, (Oosumi et al., 2006) amongst the 14 described species; the tetraploids (2n=4x=28) including F. orientalis; the single hexaploid species F. moschata (2n=6x=42); and four octoploid species (2n=8x=56): F. chiloensis, F. iturupensis, F. virginiana and the hybrid cultivated strawberry, F. ×ananassa.
Thirty fosmids were randomly selected from a library of Fragaria vesca subsp. americana (cv. Pawtuckaway) DNA. These fosmid clones were individually sheared, and ∼4- to 5-kb fragments were subcloned. Subclones on a single 384-well plate were sequenced bidirectionally for each fosmid. Assembly of these data yielded 12 fosmid inserts completely sequenced, 14 inserts as 2 to 3 contiguous sequences (contigs), and 4 inserts with 5 to 9 contigs. In most cases, a single unambiguous contig order and orientation was determined, so no further finishing was required to identify genes and their relative arrangement. One hundred fifty-eight genes were identified in the ∼1.0 Mb of nuclear genomic DNA that was assembled. Because these fosmids were randomly chosen, this allowed prediction of the genetic content of the entire ∼200 Mb F. vesca genome as about 30,500 protein-encoding genes, plus >4700 truncated gene fragments. The genes are mostly arranged in gene-rich regions, to a variable degree intermixed with transposable elements (TEs). The most abundant TEs in F. vesca were found to be long terminal repeat (LTR) retrotransposons, and these comprised about 13% of the DNA analyzed. Over 30 new repeat families were discovered, mostly TEs, and the total TE content of F. vesca is predicted to be at least 16%.
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 F1 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.
As a step toward greater understanding of the genetics of verticillium wilt resistance in plants, we report the sequencing of a candidate wilt resistance gene, mVe1, from the mint diploid model species, Mentha longifolia (Lamiaceae). mVe1 is a putative homolog of tomato (Solanum lycopersicum L.) verticillium wilt (Ve) resistance genes. The mVe1 gene has a coding region of 3,051 bp. The predicted mVe1 protein contains a leucine-rich repeat domain, a common feature of plant disease resistance proteins. We compared 13 mVe1 alleles from three mint species. These alleles shared 96.2-99.6% nucleotide identity. We analyzed four M. longifolia populations segregating with respect to mVe1 alleles and wilt resistance versus susceptibility and found one association between mVe1 genotype and wilt phenotype. We conclude that mVe1 may play a role in mint verticillium wilt resistance, but variation for resistance in our segregating progenies is likely polygenic. Therefore, further investigations of mVe1 and identification of additional candidate genes are both warranted.
The genome composition of the octoploid, cultivated strawberry awaits rigorous delineation. Initial insight into the organization and content of strawberry gene space comes from analysis of 50 genomic (fosmid) library inserts totaling ~1.75 Mb from the ~200 Mb genome of the diploid model species Fragaria vesca. Intergenic distances were generally low (0.5 to 5 kb), allowing for amplification of PCR products spanning highly polymorphic intergenic regions. Allele mining at such “gene pair” loci offers a new window onto allelic diversity and genome compositions of octoploids F. ×ananassa, F. chiloensis and F. virginiana. Three distinct haplotype classes were detected at the gRGA1-Subtilase gene pair locus in F. virginiana, providing molecular evidence that its genome is derived from at least three distinct diploid ancestors. Considerable variation was also present within haplotype classes, supporting the expectation that up to eight haplotypes can be differentiated at a single locus in an octoploid individual. A diploid (F. vesca) × octoploid (F. virginiana) cross has yielded a segregating pentaploid population, which is being used to assess patterns of haplotype transmission from the octoploid parent.