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Bio Med Central Page 1 of 10(page number not for citation purposes) Plant Methods Open Access MethodologyAn inbred line of the diploid strawberry Fragaria vesca f. semperflorens for genomic and molecular genetic studies in the RosaceaeJanetPSlovin*1, KyleSchmitt2 and KevinMFolta2Address: 1Genetic Improvement of Fruits and Vegetables Laboratory, U.S. De partment of Agriculture Agricultural Research Service, Henry A Wallace Beltsville Agricultural Research Center, 10300 Baltimore Avenue, Beltsville, MD 20705, USA and 2Horticultural Sciences Department and the Graduate Program in Plant Molecular an d Cellular Biolo gy, 1301 Fifield Hall, Un iversity of Florida, Gainesville, FL 32611, USA Email: JanetPSlovin*-janet.slovin@ars.usda.gov; KyleSchm itt-pelican8@ufl.edu; KevinM Folta-kfolta@ifas.ufl.edu Corresponding author AbstractBackground: The diploid woodland strawberry ( Fragaria vesca ) is an attractive system for functional genomics studies. Its small stature, fast regeneration time, efficient transformabi lity and small genome size, together with substantia l EST and genomic sequence resources ma ke it an ideal reference plant for Fragaria and other herbaceous perennials. Mo st importantly, this species shar es gene sequence similarity and genomic microcolinearity with other members of the Ro saceae family, including large-statured tree crops (such as apple, peach and cherry), and brambles and rose s as well as with the cultivated octoploid strawberry, F ananassa F. vesca may be used to quickly address questions of gene function relevant to these valuable crop sp ecies. Although some F. vesca lines have been shown to be substantially homozygous, in our hands plants in purportedly homozygous po pulations exhibited a rang e of morphological and physiological variation, confoundin g phenotypic analyses. We also foun d the genotype of a named variety, thought to be well-characterized and even sold comme rcially, to be in question. An easy to grow, standardized, inbred diploid Fragaria line with documented genotype that is available to all members of the research community will fa cilitate comparison of results among laboratories and provide the research community with a necessary tool for f unctionally testing the large amount of sequence data that will soon be available for peach, apple, and strawberry. Results: A highly inbred line, YW5AF7 of a diploid strawberry Fragaria vesca f. semperflorens line called "Yellow Wonder" (Y2) was develo ped and examined. Botanical de scriptors were assessed for morphological characterization of th is genotype. The plant line was found to be rapidly transformable using established techniques and media formulations. Conclusion: The development of the documented YW5AF7 li ne provides an important tool for Rosaceae functional genomic analyses These day-neutral pl ants have a small genome, a seed to seed cycle of 3.0 3.5 months, and produce fruit in 7.5 cm pots in a gr owth chamber. YW5AF7 is runnerless and therefore easy to maintain in the greenhouse, forms abundant branch crowns for vege tative propagation, and produces highly aromatic yellow fruit throughout the year in the greenhouse. F. vesca can be transformed with Agrobacterium tumefaciens making these plants suitable for insertional mutagenesis, RNAi and overexpression studies that can be compared against a stable baseline of phenotypic descriptors and can be readily genetically substantiated.Published: 31 October 2009 Plant Methods 2009, 5 :15doi:10.1186/1746-4811-5-15 Received: 12 September 2008 Accepted: 31 October 2009 This article is available from: http ://www.plantmethods.com/content/5/1/15 2009 Slovin et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons. org/licenses/by/2.0 ), which permits unrestricted use, distribution, and reproduction in any medium, provided the orig inal work is properly cited.

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Plant Methods 2009, 5 :15http://www.plantmethods.com/content/5/1/15 Page 2 of 10(page number not for citation purposes)BackgroundThe family Rosaceae is comprised of diverse fruit, nut and ornamental plants. At this time, resources that will accelerate research efforts in this important crop family are being developed [1]. Genomes from three family members (peach, apple, and strawberry) are currently being sequenced and a massive number of transcribed sequences are being catalogued. While the amount of structural genomics information is increasing, the ability to put this information to work in a functional genomics context has not significantly advanced across rosaceous species. The growing wealth of genomics-level information requires development of agile transformation systems to enable direct tests of gene function. Unfortunately, the majority of the valuable crops in this family are large-architectured tree crops with long juvenility periods and substantial space requirements. Brambles and roses are challenging in culture, and require substantial time for regeneration. These characteristics slow the speed of discovery and greatly decrease the practicality of gene function studies in these systems. However, analysis of genome structure and content indicate remarkable similarities in protein sequence and colinearity between the studied members of the family [2], suggesting that gene regulation and function may be highly translatable between species. An excellent candidate system that circumvents many of these problems is the diploid strawberry ( Fragaria vesca L.; 2n = 14). F. vesca possesses many attributes that make it ideal for genomics, either as a reverse or forward genetic system or as a rapid means for direct tests of gene function [3-5]. It is a small plant commonly found along the edges of woodlands, with a wide distribution throughout Europe, Asia, and the United States [6]. The wild everbearing, or day-neutral form, F. vesca f. semperflorens is native to Europe [6], and produces fruit throughout the year in the greenhouse. Forms exist that reproduce by seed or branch crowns only, and there exist forms that are capable of reproducing vegetatively by runners as well [7]. The runnerless type, called Bush Alpine or Gaillon strawberry, tends to bear larger fruit than the runnered type [6]. White or pink flowered forms, single and double flowered forms, white or red fruited forms, as well as forms with three leaflets or one leaflet, have been described by Richardson [8-11]. Fruit aromas of lab-grown genotypes range from grape-like to sweet overripe banana and pineapple (Slovin, personal observation). Importantly, substantial evidence indicates that F. vesca shares a common ancestor with at least one of the subgenomes within the commercial octoploid strawberry, F ananassa [12,13], making findings in the reference species relevant to the cultivated germplasm. There are additional attributes that make F. vesca an attractive system to answer basic biological questions as well as solve agriculturally important problems. F. vesca is rapidly regenerable from tissue culture and can be transformed using Agrobacterium tumefaciens [14-17]. Each plant produces many achenes, making it suitable for genetic studies. The seed to seed cycle of F. vesca is complete in less than 4 months, and the plant can be grown to seed in a 7.5-10 cm pot in a small greenhouse, or even on a lighted laboratory shelf. Approximately 1% of the genome sequence appears in public databases (GenBank numbers EU024823 -EU024872 ), the most of any Roscaceae family member [1]. The genome of these plants is small, approximately 200 Mb [18-20]. The genome of F. vesca line 'Hawaii-4' (accession PI551572) is currently being sequenced and a substantial body of sequences from transcribed genes of octoploid and diploid Fragaria species is available. One issue that made F. vesca less than optimal for genome function studies is that, although being self-fertile and therefore likely to be substantially inbred, individual plants in populations of various lines growing under essentially uniform conditions in the greenhouse or growth chamber exhibited substantial phenotypic variability for certain traits. An example of this is given in Figure 1, which illustrates the differences observed in young plants grown from seeds of a single self-pollinated plant derived from accession Hawaii 4. These natural variations suggest an underlying level of heterozygosity, and have the potential to add complexity to downstream genetic analyses or assessment of gene function, making it difficult to assess if phenotypes arise from a transgene, genetic lesion, or genetic variation. The variability we have seen in this line and in our parental Yellow Wonder line also complicated interpretation of physiological studies. For A sample of phenotypic variation observed among F. vesca lines Figure 1 A sample of phenotypic variation observed among F. vesca lines Thirty-seven F4 inbred plants of accession Hawaii-4 were grown under uni form greenhouse conditions. While most plants maintained similar appearance (center) extreme phenotypes were still observed (left and right). These plants exhibited substant ial differences in runnering and flowering time.

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Plant Methods 2009, 5 :15http://www.plantmethods.com/content/5/1/15 Page 3 of 10(page number not for citation purposes)this reason, large numbers of plants were needed to achieve statistical significance when measuring parameters such as crown number, seedling root length and branching, or flowering time and number (J. Slovin, unpublished). A solution to these problems would be to develop an easily grown, genetically uniform line with a documented homozygous genotype. Inbred lines can serve as standard genotypes for studies of gene action and biochemical pathways [21]. In our own laboratories, assessment of traits such as tolerance to abiotic stresses using measurements of root growth for example, would benefit from such a genetically and phenotypically homogeneous starting population. Documented inbred Fragaria lines will facilitate comparisons between experiments and among laboratories using T-DNA insertion mutants or overexpression studies to test gene function in strawberry, and perhaps more widely in the family Rosaceae. The inbred line of F. vesca f. semperflorens var. Yellow Wonder described herein, YW5AF7, was developed at Beltsville, MD to facilitate such gene function studies in the genus Fragaria We chose to start with commercially available seed called "Yellow Wonder" because these plants are day neutral, do not runner, and have yellow fruit color. These three traits have been analyzed genetically in F. vesca and shown to be encoded by recessive genes [22-24], and efforts were being made to clone the responsible genes [23,24]. Seed designated as "Yellow Wonder" is available from several commercial sources and is listed by the United States National Clonal Germplasm Repository http://www.ars.usda.gov/Main/docs.htm?docid=11324 as PI 551827. PI 551827 is listed as being of uncertain pedigree and not available commercially in the United States. "Yellow Wonder" obtained from the Burpee Seed Company was used in a study to identify the yellow fruit color locus [25]. The Yellow Wonder seed used in this project for generating YW5AF7 was part of the seed collection at the USDA in Beltsville, MD. The need for a standardized line that can be used by all laboratories for gene function studies with confidence in its genotype can clearly be seen in Figure 2, which shows a comparison of two lines designated "Yellow Wonder" from different sources. PCR products from the Burpee "Yellow Wonder" line (YW1) used by Deng and Davis [25] are clearly different from the product obtained with DNA from the "Yellow Wonder" line (YW2) used for generating YW5AF7. Also shown for comparison in this figure are the products obtained with DNA from a different F. vesca subspecies (Pawt), and a different diploid Fragaria species, F. iinumae (J-17). The region amplified is the intron in a mitochondrial low molecular weight heat shock protein identified from our heat-treated "Yellow Wonder" seedling cDNA library. With the resolution of one molecular character it is apparent that not all "Yellow Wonder" accessions are equivalent. The advanced inbred diploid genotype, YW5AF7, provides a tool for direct tests of gene function in strawberry and other members of the Rosaceae family that can be used with confidence by all members of the research community. In this report we present information about the background of the accession, assessment of horticultural traits, and protocols for transformation and regeneration.ResultsTechnical Description PlantsAt 23C, YW5AF7 seedlings in 10 cm. pots will flower by 8 weeks after sowing. Four weeks later the achenes can be harvested and sown to start the next generation, even though the berry they grew on may not be completely ripe and the achenes may be slightly green. By twenty weeks, numerous berries are present and ripe (Figure 3). Sixmonth-old greenhouse grown plants in 15 cm pots average 25 cm in height and can be 35 cm across. Plants produce large numbers of branch crowns and can fill a 15 cm pot within 8 months when supplied with fertilizer biweekly. F. vesca lines called "Yellow Wonder may not have the same genotype Figure 2 F. vesca lines called "Yellow Wonder" may not have the same genotype Amplification of a region of a gene encoding a mitochondrial low molecular weight heat shock protein shows differences betw een "Yellow Wonder" plants from two different sources (Y1 and Y2). Primers were designed to amplify a region co ntaining an intron, and reveal polymorphisms between Y1 and Y2, as well as between subspecies of F. vesca (Y1, Y2 and Pawt) and between different diploid Fragaria species, F. vesca and F. iinumae (J-17). The same primers were used to amplify this region from a heat treated "Yellow Wonder" (Y2) seedling cDNA library (cDNA). bp: size markers in ba se pairs. M: size ladder.

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Plant Methods 2009, 5 :15http://www.plantmethods.com/content/5/1/15 Page 4 of 10(page number not for citation purposes)The leaves of YW5AF7 are thin, and show typical morphology for F. vesca The leaf is light medium green in color, with both sides pubescent. Stomates are found on the abaxial side only. The first two true leaves are unifoliate, round and serrated. Later leaves are trifoliate, although very small ectopic highly serrated unifoliate leaves are also sometimes found at the base of the plant. Under greenhouse conditions in a 15 cm pot, the largest leaves can reach 13 cm in width and 8 cm in length. The terminal leaflet is ovate and more rounded than found on F. vesca var. Ruegen grown under the same conditions. It is serrated, with an average of 19 serrations on the largest terminal leaflets, whereas the margins of the same size terminal leaflet of the octoploid F ananassa var. Chandler has about 25 serrations. Lateral leaflets of the largest leaves of YW5AF7 average 17 serrations. Serrations begin about half way up the inner edge of the lateral leaflets. The interveinal lamina are crinkled. Petioles are long and have a distinct adaxial groove. They are red in low light and tend to be greener toward the leaf. Petioles are pubescent, with straight, unbranched hairs. Stipules are red. Flowers and fruit are borne within the leaf canopy as well as on inflorescences that extend above the canopy. Occasionally these extend down over the sides of the pots because of the weight of the fruit. In 15 cm pots, pedicels of the first inflorescence can be 15-20 cm long. Pedicels are round, pubescent, and tend to be greener than the petioles. Inflorescences usually have 4 to 5 flowers, however, under some conditions the cymous inflorescence continues to branch and form additional flowers. Root initials sometimes form at the nodes and these will form roots if pegged to the soil. On very old, pot-bound plants, inflorescences with only one or two flowers become common. The flower is also typical of F. vesca It usually has five petals, a calyx consisting of 2 whorls of five sepals, and 20 stamens. In the center of the flower is a rounded receptacle, bearing yellow pistils, that extends well beyond the stamens when the flower bud opens and the anthers dehisce. Occasionally flowers have extra flower parts, the most obvious of these occurrences being 6 petals per flower. In addition, petaloid anthers have been observed. Both conditions are also seen in the parental generations, and the appearance is correlated with larger flowers. Primary flowers are usually 1.5-2 cm in diameter, depending on growth conditions. Secondary and later flowers tend to be smaller.FruitLike its progenitor, Yellow Wonder, the berry of YW5AF7 is soft and pale yellow in color with tan achenes when ripe, and pale green to white during development. Ripe berries are highly aromatic with sweet banana and pineapple overtones. When all achenes are fertilized, the berry shape is long conic, with some primary berries being necked long conic and reaching 27 mm in length and 20 mm in width in plants growing in 15 cm pots. The average fresh weight of a primary berry was 1.67 0.26 S.E. g., with the largest berry being 2.45 g. Achenes are borne on the surface of the berry, with an average 193 17 S.E. achenes per primary berry (225 on the largest). In comparison, an average of 518 achenes per primary fruit was reported for a commercial octoploid variety [26]. In the absence of insects or human interventions, only about half of the achenes are fertilized and enlarge, and the fruit tend to be smaller and of varied shapes. Pollination can be aided by transferring pollen from a flower with dehiscing anthers to a just opened flower using a small camel hair brush. The extent of fertilized ovules per fruit can be approximated over time by examining the expansion of developing achenes and the subtending receptacle tissue.PerformanceSeeds of YW5AF7 will germinate in soil in the greenhouse in one week. However, more uniform germination can be achieved by cold treatment of moist seed. Seeds of YW5AF7 in moistened soil did not achieve maximum germination [87%, n = 100 (10 pots with 10 seed each)] until 21 days after sowing. Following treatment of moist seed for 3 weeks in the dark at 5C, 74% of YW5AF7 seeds germinated in 7 days after being brought into the greenhouse, and by 14 days, maximum germination, 91%, was achieved. Seeds that have been disinfested using ethanol and bleach treatment will germinate in Petri dishes on 0.5 MS media [27] solidified with 0.8% Phytagar (Invitrogen, Carlsbad, CA). The resulting seedlings can be used The YW5AF7 plant Figure 3 The YW5AF7 plant The image shows a mature YW5AF7 plant with flowers and fruit in a 10 cm pot.

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Plant Methods 2009, 5 :15http://www.plantmethods.com/content/5/1/15 Page 5 of 10(page number not for citation purposes)as aseptic explants for tissue culture. Under these conditions we have found that longer cold treatment (>3 weeks) results in more uniform germination, which can be critical for evaluating developmental or physiological parameters of transformed plants in gene function tests. Mildew susceptibility in F. vesca was found to be due to two dominant genes [28]. Both genes had to be absent to obtain a resistant plant, and cytoplasmic effects were noted. Seedlings of YW5AF7 are susceptible to powdery mildew at early stages in development, particularly in a growth chamber environment. In the greenhouse, YW5AF7 has been found to be susceptible to powdery mildew, thrips, two spotted mites, and aphids.Transformation and RegenerationWhile YW5AF7 is a strong candidate for genomics studies, it was important to test if it could be successfully transformed and regenerated. While diploid strawberry is routinely transformed, transformation and regeneration efficiency are highly genotype specific [reviewed in [29]]. To test the YW5AF7 line for the ability to produce shoots after gene insertion, explants from greenhouse-grown YW5AF7 plants were co-cultivated with Agrobacterium tumefaciens carrying a visible GFP reporter as described in Materials and Methods. Several published media formulations were evaluated for regeneration and are detailed in Table 1. Callusing was observed on all media types tested, but tissue vigor and regeneration were best supported by the formulation presented in Zhao et al. [30] (Figure 4, triangles). On this formulation, 20% of explants possessed shoots by six weeks whereas explants on other media formulations exhibited little or no organogenesis at this time point (Figure 4, black or white circles). At nine weeks, over 80 percent of explants had shoots on the Zhao formula, whereas shoot initiation on other media types was less frequent. Figure 5 shows that the medium producing the highest percentage of explants exhibiting organogenesis also resulted in a higher number of shoots per explant by nine weeks in culture than other formulations. These data indicate that the formulation by Zhao et al. [30] results in the highest number of explants exhibiting shoots, which is important for maximizing the number of independent transformation events in gene function experiments. Shoots were generated by direct organogenesis and were produced most quickly and abundantly on the basipetal end of petiole segments (as shown in Figure 6A). Evaluation of GFP fluorescence in emerging shoots revealed that about 40% of shoots were transformed, indicating that escapes can be present using 4 mg/L hygromycin for selection. Once differentiated, explants were moved to a media without TDZ to enhance shoot elongation. Although not formally quantified, at least one insertion event was observed on each explant, as evidenced by GFP fluorescence. Figure 6 shows germinating seed and two resulting seedlings from one such plant. In this random sample of seeds, the ratio of GFP positive to GFP negative (wild type) seeds was essentially 1:1, indicating that there was most likely a single insertion event in this transformant.DiscussionF. vesca has great potential as a system to study the genetic basis of agriculturally important biological questions in the Rosaceae family. Its small size, rapid growth, generous seed set, small genome, and sequence availability make it an excellent resource for development of genomics tools. Its genomic similarity to other valuable crops underlies its potential utility as a surrogate to test gene function relevant to many rosaceous species. A primary concern about the system has been the observation of variability among individuals in lines that have not been single seed propagated in the lab through several generations. Although typically self fertilizing and therefore expected to be largely homozygous, we observed clear variability in a number of horticultural traits among F. vesca Yellow Wonder and Hawaii-4 plants generated from achenes from a single fruit suggestive of some degree of residual heterozygosity. These phenotypic variations are potentially problematic in a seminal line proposed as a genomics-friendly genotype. This is an important consideration as several current efforts are developing populations of T-DNA insertion, activation tagged, overexpression, or RNAi lines using F. vesca and it could become difficult to discriminate between a phenotype resulting from an engineered genotypic variation and natural genetic variation in the line. Interpretations from a genetically noisy background may preclude, or at least delay, identification of gene-specific effects on morphol-Table 1: The media formulations used in regeneration experiments.MediumAuxinConcentrationCytokininConcentrationReference AIBA0.98 uMBA13.20 uM[32] B2,4-D0.45 uMTDZ4.54 uM[36] CIBA1.50 uMTDZ10.00 uM[30]

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Plant Methods 2009, 5 :15http://www.plantmethods.com/content/5/1/15 Page 6 of 10(page number not for citation purposes)ogy and physiology. High variability in results from physiological studies drastically increases the number of plants that must be used to obtain statistical significance. These populations may be suboptimal for quantitative studies of gene expression, as the variation in the baseline may lead to errors in interpreting microarray, digital, or qRTPCR gene expression profiles. Results must be able to be repeated in other laboratories, so a system based on a known genetic background will supplement these efforts and be of benefit to the wider research community. Our PCR analysis of two different "Yellow Wonder" lines (Figure 2) indicated that even a well-established and commercially available line of F. vesca may consist of different genotypes. Because "Yellow Wonder" is both non-red and non-runnering it would appear likely, given that these two loci must be homozygous recessive, that these plants are already substantially homozygous. However, clearly Y1 is not the same as Y2. There are no data from any of the suppliers to show that their "Yellow Wonder", the color of which would be expected to breed true from seed, is the same as a competitor's, which also would breed true from seed (at least for color and non-runnering), and no data to show that any of these are the same as others described in the literature. For these reasons, the pre-emptive development of a stable, highly inbred, prolific and documented genotype was considered useful, as it would provide a stable genotype for evaluation of gene function that could be shared among users. Botanical descriptors of YW5AF7 have been carefully evaluated and define a reproducible and firm foundation for later comparisons. Even subtle phenotypes induced by a transgene should be able to be reliably scored in this stable background. Many of the techniques used for studying Arabidopsis can be used with F. vesca YW5AF7. Seedling variations are almost indiscernible in populations of Arabidopsis seedlings, and their small stature makes in vitro assessment of phenotypes possible. Tests of early development in response to environmental conditions, growth regulators or nutrient status are also possible in F. vesca much like in Arabidopsis For YW5AF7 to have utility as a functional genomics system it must be transformable. As observed by many groups, transformation efficiency of various strawberry genotypes is highly genotype dependent and in some cases impossible [29]. The transformation capacity of YW5AF7 was tested with a GFP reporter gene. Many GFP foci were observed in co-cultivated tissues and GFP-posiYW5AF7 regeneration freque ncy on three published media formulations Figure 4 YW5AF7 regeneration frequency on three published media formulations Various explants from YW5AF7, including mature leaves, young leaves and petioles, were grown on three different media (Table 1) to test regeneration frequency. Medium A, whit e circles; Medium B, black circles and Medium C, triangles. The data are the means of two independent experiments. The number of shoots per YW5AF7 explant on three media formulations Figure 5 The number of shoots per YW5AF7 explant on three media formulations The mean number of shoots per explant was determined for three different media: Medium A, white circles, Medium B, blac k circles and Medium C, triangles. The data refle ct the mean of two independent experiments.

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Plant Methods 2009, 5 :15http://www.plantmethods.com/content/5/1/15 Page 7 of 10(page number not for citation purposes)tive plantlets were regenerated on media containing selective antibiotics. Three published (yet diverse) media formulations were evaluated for regeneration-inducing ability. In all cases shoots appeared via organogenesis with the best results arising from the media formulation presented in Zhao et al. [30]. A number of shoots were clearly initiated by 30 days and plantlets could be excised after 60 days and rooted in rooting media. This time course is reasonable yet could likely be optimized to improve the utility of the YW5AF7 system. The high frequency of shoot formation on independent explants ensures propagation of independent transformants. Antibiotic sensitivity was generally consistent with previouslypublished reports in strawberry [31,32] but subculture to progressively higher amounts of antibiotics may be advisable as regeneration of non-transformed shoots was observed using hygromycin at 4 ug/ml. The most prolific explants were leaf-adjacent petiole segments, with the first shoots appearing on the basipetal end of these explants. The most productive formulation contained thidiazuron, TDZ, as a principle growth regulator, a compound shown to be effective in inducing regeneration in a number of other studies. However, consistent with previous reports [33] growth on TDZ severely stunted shoot elongation, and increased somaclonal variation has been observed when this regulator has been employed [see, [34]]. Once clearly formed, the shooting explants were transferred to a TDZ-free media formulation that had been used to regenerate F. vesca accession Hawaii-4. Within one week the shoots elongated vigorously and could be transferred to rooting media. Other media formulations also induced shoots, but at a much slower rate. The YW5AF7 line is runnerless and this has distinct advantages to its adoption as a functional genomics model. YW5AF7 transformants Figure 6 YW5AF7 transformants A. A cluster of shoots emerging from the basi petal end of a petiole. Both transformed (GFP+; green) and non-transformed shoots (red in color) are present. B. Imbibed seed from one line of GFP expressing transformed 5AF7 plants. GFP positive and GFP negative seeds are present in a 19:20 ratio, indicating that a single insertion is likely. C. A wild-type seedling (left) and a GFP posi tive transgenic seedling (right) gr own from seed of the plant in (B).

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Plant Methods 2009, 5 :15http://www.plantmethods.com/content/5/1/15 Page 8 of 10(page number not for citation purposes)Runnerless plants are much easier to maintain in a greenhouse as plants may be located in close proximity without having to continually remove a tangle of runners or daughter plants that invade neighboring pots, such as is our experience with F. vesca Hawaii 4 and F. vesca Pawtuckaway. In large populations this can be a source of genotype contamination and requires dedication to constant manual management. On the other hand, one of the advantages of F. vesca as a functional genomics system is that plants can be propagated by branch crown divisions or runners or branch crown division, as well as by seed, making it possible to vegetatively propagate mutants that affect flowering or seed set. Although runnerless, the YW5AF7 line does produce abundant branch crowns. Seeds of YW5AF7 are available for research purposes from Dr. J. P. Slovin, USDA-ARS Genetic Improvement of Fruits and Vegetables Laboratory, Bldg. 010A, 10300 Baltimore Avenue, Beltsville, MD 20705 (phone: 301/504-5629; email: slovinj@ars.usda.gov). The seed are produced in the greenhouse under ambient conditions from F7 plants that are manually self-pollinated. Seed of this line have been deposited with the USDA, ARS National Clonal Germplasm Repository, Corvallis, OR (PI 641092).ConclusionThe highly inbred selection YW5AF7 has been generated and characterized. A set of botanical descriptors defines a baseline that may be compared to phenotypes of forward or reverse genetic mutants as well as overexpression and RNAi lines. This genotype, associated scored metrics, and transformation protocol permit the deployment of this system as a useful tool for the Rosaceae research community in the elucidation of gene function in a stable and consistent genetic background that complements existing systems.MethodsPlant Origin and Seedling SelectionThe inbred line, designated YW5AF7, was obtained by manually self-pollinating F. vesca var. "Yellow Wonder" plants grown from seeds in the Beltsville collection maintained originally by S. Hokanson. Seeds were planted in Metro Mix 510 (Scotts-Sierra Horticultural Products, Marysville, OH) supplemented with dolomitic lime. Plants were grown in the greenhouse with supplemental lighting from sodium halide lamps to give a daylength of at least 12 h. At least one hundred seed were planted at each generation. Flowers were self-pollinated by gentle brushing starting when their flowers just opened, and every day thereafter as their anthers dehisced until the flower petals fell. A new small camel hair artist's brush was used for each pollination. Flowers were tagged for the day of pollination. The plant to be used for the next generation was randomly chosen from ten plants that met a basic requirement of early flowering, general robust appearance, fruit set (as based on number of achenes that enlarge following pollination), high number of achenes on the primary fruit, and short number of days to mature fruit. Although germination rate and number of days to flowering varied, the other selecting parameters usually showed very little difference. However, some seedlings germinated from F3 seed showed morphological abnormalities such as dwarfness or a single cotyledon. Only seedlings with normal phenotypes were selected for further selfing. The selfing process was continued for seven generations to generate YW5AF7.Media PreparationThree previously defined media formulations were investigated to determine which would lead to optimal regeneration of YW5AF7 explants. Media included 1 Murashige and Skoog medium with vitamins, 2% sucrose and the growth regulators presented in Table 1. Media were prepared with deionized water, the pH adjusted to 5.6-5.8, and then autoclaved for 20 min at 121C and 15 psi. Growth regulators were co-autoclaved with the media. In all three media types, the selection agent used was 4 mg/L hygromycin B, which was added to the media after it was cooled to ~50C.Agrobacterium-mediat ed transformationLeaf, stem, and petiole segments were sterilized in 70% EtOH for 30 seconds and 1% sodium hypochlorite (20% bleach) for 10 min. A single transformed Agrobacterium colony carrying a 35S::GFP construct was grown overnight in Luria Broth with 10 mg/L rifampicin, 50 mg/L gentamicin, and 50 mg/L spectinomycin to an OD600 of 0.5, then pelleted at 1,000 g. The bacterial pellet was resuspended to 0.1 OD600 in co-cultivation medium consisting of 1 MS pH 5.8 with 2% sucrose, supplemented with 50 uM acetosyringone. Explants were added to the co-cultivation medium and incubated 20 min at room temperature, then blotted dry with sterile filter paper and transferred to media without selection for 2 d at 25C in darkness. After 2 d the explants were washed twice in co-cultivation medium liquid supplemented with 500 mg/L carbenicillin, followed by 30 min of incubation in fresh wash media. Explants were again blotted dry and transferred to solid media with selection. Transgenic shoots were selected on media containing 4 mg/L hygromycin B. The explants were regenerated under a 16 h light, 8 h dark photoperiod under cool-white fluorescent lighting. Explants were checked daily for contamination, and subcultured every 2 weeks. When distinct clumps of shoots were formed the entire clump was transferred to hormone-free rooting media consisting of 0.5 MS media (pH 5.8), 1% glucose, and 1% phytoagar. Roots

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Plant Methods 2009, 5 :15http://www.plantmethods.com/content/5/1/15 Page 9 of 10(page number not for citation purposes)formed within days to one month and individual plants could then be dissected from the groups of shoots.PCRPCR was performed using the touchdown protocol described by Sargent et al. [35] in a 20 l reaction containing HotStart Taq Master Mix (Qiagen, Valencia, CA), 0.4 M each primer, and 1.0 ng genomic DNA. PCR products were separated by electrophoresis through a 1.5% TAE agarose gel and visualized by ethidium bromide staining. Primers were designed to span the intron in the N-terminal domain of a low molecular weight heat shock protein gene identified as an EST (GenBank accession number CX661743.1 ) from a "Yellow Wonder" (Y2) heat-treated seedling cDNA library. Template DNA for Y1, Y2, F. iinumae J-17, and F. vesca subsp. americana Pawtuckaway was obtained from 50-100 mg young leaf tissue using a DNeasy Plant Mini kit (Qiagen). Y1 and F. vesca subsp. americana Pawtuckaway plants were obtained from T. Davis (University of New Hampshire) and F. iinumae J-17 plants were obtained from the US National Plant Germplasm collection.Competing interestsThe authors declare that they have no competing interests.Authors' contributionsJPS conceived of the project, performed all crosses, plant measurements and PCR, and participated in preparation of the manuscript. KS performed the co-cultivation and subculturing of strawberry tissues to test transformation efficiency. KMF supervised transformation and regeneration and participated in preparation of the manuscript.AcknowledgementsThe authors thank Andrea Murphy and Jeremy Goetz for greenhouse assistance, Sasha Drost, an Eleanor R oosevelt High School science intern for technical assistance, Todd Cooke for his help with botanical description, and anonymous reviewers for comments. This work was performed as part of NSF project 0701488 (KMF). Kyle Schmitt was supported with funding from the Howard Hughes Medical Inst itute "Science for Life" program at the University of Florida (KMF).References1.Shulaev V, Korban SS, Sosinski B, Abbott AG, Aldwinckle HS, Folta KM, Iezzoni A, Main D, Arus P, Dandekar AM, et al. : Multiple models for Rosaceae genomics. Plant Physiol 2008, 147(3): 985-1003. 2.Rousseau-Gueutin M, Lerceteau-Khler E, Barrot L, Sargent DJ, Monfort A, Simpson D, Ars P, Gurin G, Denoyes-Rothan B: Comparative genetic mapping betw een octoploid and diploid Fragaria species reveals a high level of colinearity between their genomes and the essential disomic behavior of the cultivated octoploid strawberry. Genetics 2008, 179(4): 2045-2060. 3.Davis TM, Pollard JE: Fragaria vesca chlorophyll mutants. HortScience 1991, 26(3): 311. 4.Albani M, Taylor S, Rodriquez Lo pez C, Cekic C, Al-Sheikh M, Greenland A, Wetten A, Wilkinson M, Battey NH: Fragaria vesca one way to understand flowering in perennials. Flowering Newsletter 2001, 31: 44-48. 5.Slovin J, Rabinowicz PD: Fragaria vesca a useful tool for Rosaceae genomics. In 6th North American Strawberry Symposium: 2007 Ventura, CA: American Soci ety for Horticultural Science; 2007:112-117. 6.Darrow GM: The Strawberry. New York: Holt, Rinehart and Winston; 1966. 7.Brown T, Waring PF: The genetical control of flowering and runnering in varieties of Fragaria vesca Heredity 1965, 20: 651. 8.Richardson CW: A preliminary note on the genetics of Fragaria Journal of Genetics 1914, 3: 171-178. 9.Richardson CW: A further note on the genetics of Fragaria Journal of Genetics 1918, 7: 167-170. 10.Richardson CW: Some notes on Fragaria Journal of Genetics 1920, 10: 39-46. 11.Richardson CW: Notes on Fragaria Journal of Genetics 1923, 13: 147-152. 12.Rousseau-Gueutin M, Gaston A, Anouche A, Anouche ML, Olbricht K, Staudt G, Richard L, Denoyes-Rothan B: Tracking the evolutionary history of polyploidy in Fragaria L. (strawberry): New insights from phylogenetic an alyses of low-copy nuclear genes. Molecular Phylogenet ics and Evolution 2009, 51(3): 515-530. 13.Hancock JF: Strawberries. New York: CABI Publishing; 1999. 14.Alsheikh MK, Suso H-P, Robson M, Battey NH, Wetten A: Appropriate choice of antibiotic and Agrobacterium strain improves transformation of antibiotic-sensitive Fragaria vesca and F. v. semperflorens Plant Cell Reports 2002, 20(12): 1173-1180. 15.El Mansouri I, Mercado J, Valpuest a V, Lopez-Aranda J, Pliego-Alfaro F, Quesada M: Shoot regeneration and Agrobacterium -mediated transformation of Fragaria vesca L. Plant Cell Reports 1996, 15: 642-646. 16.Haymes KM, Davis TM: Agrobacterium -mediated tr ansformation of 'Alpine' Fragaria vesca and transmission of transgenes to R1 progeny. Plant Cell Reports 1998, 17: 279-283. 17.Zhao Y, Liu Q, Davis RE: Transgene expression in strawberries driven by a heterologous phloem-specific promoter. Plant Cell Reports 2004, 23(4): 224-230. 18.Akiyama Y, Yamamoto Y, Ohmido N, Oshima M, Fukui K: Estimation of the nuclear DNA co ntent of strawberries ( Fragaria spp .) compared with Arabidopsis thaliana by using dual-stem flow cytometry. Cytologia 2001, 66: 431-436. 19.Bennett MD, Leitch IJ: Angiosperm DNA C-values Database (Release 6.0, Oct 2005). 2005 [http://www.kew.org/cvalues/ ]. 20.Bennett MD, Leitch IJ Price HJ, Johnston JS: Comparisons with Caenorhabditis (approximately 100 Mb) and Drosophila (approximately 175 Mb) using flow cytometry show genome size in Arabidopsis to be approximately 157 Mb and thus approximately 25% larger than the Arabidopsis genome initiative estimate of approximately 125 Mb. Annals of Botany 2003, 91(5): 547-557. 21.Tam YY, Slovin JP, Cohen JD: Selection and characterization of-methyltryptophan resistant lines of Lemna gibba showing a rapid rate of indole-3 -acetic acid turnover. Plant Physiol 1995, 107: 77-85. 22.Brown T, Wareing PF: The genetic control of the everbearing habit and three other char acters in varieties of Fragaria vesca Euphytica 1965, 14: 97-112. 23.Battey NH, LeMiere P, Tehranifar A, Cekic C, Taylor S, Shrives KJ, Hadley P, Greenland AJ, Darby J, Wilkinson MJ: Genetic and environmental control of flowering in strawberry. In Genetic and Environmental Manipulation of Horticultural Crops Edited by: Cockshull KE, Gray D, Seymour GB, Thomas B. New York: CABI Publishing; 1998:111-131. 24.Williamson SC, Yu H, Davis TM: Shikamate dehydrogenase allozymes: inheritance and close linkage to fruit color in the diploid strawberry. Journal of Heredity 1995, 86(1): 74-76. 25.Deng C, Davis TM: Molecular identification of the yellow fruit color (c) locus in diploid strawberry: a candidate gene approach. Theor Appl Genet 2001, 103: 316-322. 26.Gardner VR: Studies on the nutritio n of the strawberry. Mo Agr Exp Sta Res Bul 1923, 57: 31. 27.Murashige T, Skoog F: A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum 1962, 72: 473-497. 28.Harland SC, King EE: Inheritance of mild ew resistance in Fragaria with special reference to cytoplasmic effects. Heredity 1957, 11: 287.

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Publish with Bio Med Central and every scientist can read your work free of charge"BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright Submit your manuscript here:http://www.biomedcentral.com/info/publishing_adv.asp Bio Med central Plant Methods 2009, 5 :15http://www.plantmethods.com/content/5/1/15 Page 10 of 10(page number not for citation purposes)29.Folta KM, Dhingra A: Transformation of st rawberry: The basis for translational genomics in Rosaceae. In Vitro Cell Dev-Pl 2006, 42(6): 482-490. 30.Zhao Y, Liu QZ, Davis RE: Transgene expression in strawberries driven by a heterologo us phloem-specific promoter. Plant Cell Reports 2004, 23(4): 224-230. 31.Debnath SC, Teixeira da Silva JA: Strawberry culture in vitro : applications in genetic tran sformation and biotechnology. Fruit, Vegetable, and Cereal Science Biotechnology 2007, 1: 1-2. 32.Oosumi T, Gruszewski HA, Blischak LA, Baxter AJ, Wadl PA, Shuman JL, Veilleux RE, Shulaev V: High-efficiency tran sformation of the diploid strawberry ( Fragaria vesca ) for functional genomics. Planta 2006, 223(6): 1219-1230. 33.Debnath SC: Zeatin overcomes thidia zuron-induced inhibition of shoot elongation and promote s rooting in strawberry culture in vitro. J Hortic Sci Biotech 2006, 81(3): 349-354. 34.Arnold D, Flegmann A, Clarkson J: Somaclonal variation in watercress for resistan ce to crook root disease. Plant Cell Reports 1995, 14(4): 241-244. 35.Sargent DJ, Hadonou AM, Simpson DW: Development and characterization of polymorphic microsatellite markers from Fragaria viridis a wild diploid strawberry. Mol Ecol Notes 2003, 3(4): 550-552. 36.Schaart JG, Krens FA, Pelgrom KTB, Mendes O, Rouwendal GJA: Effective production of mark er-free transgenic strawberry plants using inducible site-specific recombination and a bifunctional selectable marker gene. Plant Biotechnol J 2004, 2(3): 233-240.


!DOCTYPE art SYSTEM 'http:www.biomedcentral.comxmlarticle.dtd'
ui 1746-4811-5-15
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dochead Methodology
bibl
title
p An inbred line of the diploid strawberry it Fragaria vesca f. semperflorens for genomic and molecular genetic studies in the Rosaceae
aug
au id A1 ca yes
snm Slovin
mi P
fnm Janet
insr iid I1
email janet.slovin@ars.usda.gov
A2
Schmitt
Kyle
I2
pelican8@ufl.edu
A3
Folta
M
Kevin
kfolta@ifas.ufl.edu
insg
ins
Genetic Improvement of Fruits and Vegetables Laboratory, U.S. Department of Agriculture Agricultural Research Service, Henry A Wallace Beltsville Agricultural Research Center, 10300 Baltimore Avenue, Beltsville, MD 20705, USA
Horticultural Sciences Department and the Graduate Program in Plant Molecular and Cellular Biology, 1301 Fifield Hall, University of Florida, Gainesville, FL 32611, USA
source Plant Methods
issn 1746-4811
pubdate 2009
volume 5
issue 1
fpage 15
url http://www.plantmethods.com/content/5/1/15
xrefbib
pubidlist
pubid idtype pmpid 19878589
doi 10.1186/1746-4811-5-15
history
rec
date
day 12
month 9
year 2008
acc
31
10
2009
pub
31
10
2009
cpyrt
2009
collab Slovin et al; licensee BioMed Central Ltd.
note This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
abs
sec
st
Abstract
Background
The diploid woodland strawberry (Fragaria vesca) is an attractive system for functional genomics studies. Its small stature, fast regeneration time, efficient transformability and small genome size, together with substantial EST and genomic sequence resources make it an ideal reference plant for Fragaria and other herbaceous perennials. Most importantly, this species shares gene sequence similarity and genomic microcolinearity with other members of the Rosaceae family, including large-statured tree crops (such as apple, peach and cherry), and brambles and roses as well as with the cultivated octoploid strawberry, F. ×ananassa. F. vesca may be used to quickly address questions of gene function relevant to these valuable crop species. Although some F. vesca lines have been shown to be substantially homozygous, in our hands plants in purportedly homozygous populations exhibited a range of morphological and physiological variation, confounding phenotypic analyses. We also found the genotype of a named variety, thought to be well-characterized and even sold commercially, to be in question. An easy to grow, standardized, inbred diploid Fragaria line with documented genotype that is available to all members of the research community will facilitate comparison of results among laboratories and provide the research community with a necessary tool for functionally testing the large amount of sequence data that will soon be available for peach, apple, and strawberry.
Results
A highly inbred line, YW5AF7, of a diploid strawberry Fragaria vesca f. semperflorens line called "Yellow Wonder" (Y2) was developed and examined. Botanical descriptors were assessed for morphological characterization of this genotype. The plant line was found to be rapidly transformable using established techniques and media formulations.
Conclusion
The development of the documented YW5AF7 line provides an important tool for Rosaceae functional genomic analyses. These day-neutral plants have a small genome, a seed to seed cycle of 3.0 3.5 months, and produce fruit in 7.5 cm pots in a growth chamber. YW5AF7 is runnerless and therefore easy to maintain in the greenhouse, forms abundant branch crowns for vegetative propagation, and produces highly aromatic yellow fruit throughout the year in the greenhouse. F. vesca can be transformed with Agrobacterium tumefaciens, making these plants suitable for insertional mutagenesis, RNAi and overexpression studies that can be compared against a stable baseline of phenotypic descriptors and can be readily genetically substantiated.
meta
classifications
classification type bmc subtype user_supplied_xml endnote
bdy
Background
The family Rosaceae is comprised of diverse fruit, nut and ornamental plants. At this time, resources that will accelerate research efforts in this important crop family are being developed abbrgrp abbr bid B1 1. Genomes from three family members (peach, apple, and strawberry) are currently being sequenced and a massive number of transcribed sequences are being catalogued. While the amount of structural genomics information is increasing, the ability to put this information to work in a functional genomics context has not significantly advanced across rosaceous species. The growing wealth of genomics-level information requires development of agile transformation systems to enable direct tests of gene function. Unfortunately, the majority of the valuable crops in this family are large-architectured tree crops with long juvenility periods and substantial space requirements. Brambles and roses are challenging in culture, and require substantial time for regeneration. These characteristics slow the speed of discovery and greatly decrease the practicality of gene function studies in these systems. However, analysis of genome structure and content indicate remarkable similarities in protein sequence and colinearity between the studied members of the family B2 2, suggesting that gene regulation and function may be highly translatable between species.
An excellent candidate system that circumvents many of these problems is the diploid strawberry (Fragaria vesca L.; 2n = 14). F. vesca possesses many attributes that make it ideal for genomics, either as a reverse or forward genetic system or as a rapid means for direct tests of gene function B3 3B4 4B5 5. It is a small plant commonly found along the edges of woodlands, with a wide distribution throughout Europe, Asia, and the United States B6 6. The wild everbearing, or day-neutral form, F. vesca f. semperflorens, is native to Europe 6, and produces fruit throughout the year in the greenhouse. Forms exist that reproduce by seed or branch crowns only, and there exist forms that are capable of reproducing vegetatively by runners as well B7 7. The runnerless type, called Bush Alpine or Gaillon strawberry, tends to bear larger fruit than the runnered type 6. White or pink flowered forms, single and double flowered forms, white or red fruited forms, as well as forms with three leaflets or one leaflet, have been described by Richardson B8 8B9 9B10 10B11 11. Fruit aromas of lab-grown genotypes range from grape-like to sweet overripe banana and pineapple (Slovin, personal observation). Importantly, substantial evidence indicates that F. vesca shares a common ancestor with at least one of the subgenomes within the commercial octoploid strawberry, F. ×ananassa B12 12B13 13, making findings in the reference species relevant to the cultivated germplasm.
There are additional attributes that make F. vesca an attractive system to answer basic biological questions as well as solve agriculturally important problems. F. vesca is rapidly regenerable from tissue culture and can be transformed using Agrobacterium tumefaciens B14 14B15 15B16 16B17 17. Each plant produces many achenes, making it suitable for genetic studies. The seed to seed cycle of F. vesca is complete in less than 4 months, and the plant can be grown to seed in a 7.5-10 cm pot in a small greenhouse, or even on a lighted laboratory shelf. Approximately 1% of the genome sequence appears in public databases (GenBank numbers ext-link ext-link-type gen ext-link-id EU024823 EU024823-EU024872 EU024872), the most of any Roscaceae family member 1. The genome of these plants is small, approximately 200 Mb B18 18B19 19B20 20. The genome of F. vesca line 'Hawaii-4' (accession PI551572) is currently being sequenced and a substantial body of sequences from transcribed genes of octoploid and diploid Fragaria species is available.
One issue that made F. vesca less than optimal for genome function studies is that, although being self-fertile and therefore likely to be substantially inbred, individual plants in populations of various lines growing under essentially uniform conditions in the greenhouse or growth chamber exhibited substantial phenotypic variability for certain traits. An example of this is given in Figure figr fid F1 1, which illustrates the differences observed in young plants grown from seeds of a single self-pollinated plant derived from accession Hawaii 4. These natural variations suggest an underlying level of heterozygosity, and have the potential to add complexity to downstream genetic analyses or assessment of gene function, making it difficult to assess if phenotypes arise from a transgene, genetic lesion, or genetic variation. The variability we have seen in this line and in our parental Yellow Wonder line also complicated interpretation of physiological studies. For this reason, large numbers of plants were needed to achieve statistical significance when measuring parameters such as crown number, seedling root length and branching, or flowering time and number (J. Slovin, unpublished).
fig
Figure 1
caption
A sample of phenotypic variation observed among F. vesca lines
text
b A sample of phenotypic variation observed among F. vesca lines. Thirty-seven F4 inbred plants of accession Hawaii-4 were grown under uniform greenhouse conditions. While most plants maintained similar appearance (center) extreme phenotypes were still observed (left and right). These plants exhibited substantial differences in runnering and flowering time.
graphic file 1746-4811-5-15-1
A solution to these problems would be to develop an easily grown, genetically uniform line with a documented homozygous genotype. Inbred lines can serve as standard genotypes for studies of gene action and biochemical pathways B21 21. In our own laboratories, assessment of traits such as tolerance to abiotic stresses using measurements of root growth for example, would benefit from such a genetically and phenotypically homogeneous starting population. Documented inbred Fragaria lines will facilitate comparisons between experiments and among laboratories using T-DNA insertion mutants or overexpression studies to test gene function in strawberry, and perhaps more widely in the family Rosaceae.
The inbred line of F. vesca f. semperflorens var. Yellow Wonder described herein, YW5AF7, was developed at Beltsville, MD to facilitate such gene function studies in the genus Fragaria. We chose to start with commercially available seed called "Yellow Wonder" because these plants are day neutral, do not runner, and have yellow fruit color. These three traits have been analyzed genetically in F. vesca and shown to be encoded by recessive genes B22 22B23 23B24 24, and efforts were being made to clone the responsible genes 2324. Seed designated as "Yellow Wonder" is available from several commercial sources and is listed by the United States National Clonal Germplasm Repository http://www.ars.usda.gov/Main/docs.htm?docid=11324 as PI 551827. PI 551827 is listed as being of uncertain pedigree and not available commercially in the United States. "Yellow Wonder" obtained from the Burpee Seed Company was used in a study to identify the yellow fruit color locus B25 25. The Yellow Wonder seed used in this project for generating YW5AF7 was part of the seed collection at the USDA in Beltsville, MD.
The need for a standardized line that can be used by all laboratories for gene function studies with confidence in its genotype can clearly be seen in Figure F2 2, which shows a comparison of two lines designated "Yellow Wonder" from different sources. PCR products from the Burpee "Yellow Wonder" line (YW1) used by Deng and Davis 25 are clearly different from the product obtained with DNA from the "Yellow Wonder" line (YW2) used for generating YW5AF7. Also shown for comparison in this figure are the products obtained with DNA from a different F. vesca subspecies (Pawt), and a different diploid Fragaria species, F. iinumae (J-17). The region amplified is the intron in a mitochondrial low molecular weight heat shock protein identified from our heat-treated "Yellow Wonder" seedling cDNA library. With the resolution of one molecular character it is apparent that not all "Yellow Wonder" accessions are equivalent.
Figure 2
F. vesca lines called "Yellow Wonder" may not have the same genotype
F. vesca lines called "Yellow Wonder" may not have the same genotype. Amplification of a region of a gene encoding a mitochondrial low molecular weight heat shock protein shows differences between "Yellow Wonder" plants from two different sources (Y1 and Y2). Primers were designed to amplify a region containing an intron, and reveal polymorphisms between Y1 and Y2, as well as between subspecies of F. vesca (Y1, Y2 and Pawt) and between different diploid Fragaria species, F. vesca and F. iinumae (J-17). The same primers were used to amplify this region from a heat treated "Yellow Wonder" (Y2) seedling cDNA library (cDNA). bp: size markers in base pairs. M: size ladder.
1746-4811-5-15-2
The advanced inbred diploid genotype, YW5AF7, provides a tool for direct tests of gene function in strawberry and other members of the Rosaceae family that can be used with confidence by all members of the research community. In this report we present information about the background of the accession, assessment of horticultural traits, and protocols for transformation and regeneration.
Results
Technical Description
Plants
At 23°C, YW5AF7 seedlings in 10 cm. pots will flower by 8 weeks after sowing. Four weeks later the achenes can be harvested and sown to start the next generation, even though the berry they grew on may not be completely ripe and the achenes may be slightly green. By twenty weeks, numerous berries are present and ripe (Figure F3 3). Six-month-old greenhouse grown plants in 15 cm pots average 25 cm in height and can be 35 cm across. Plants produce large numbers of branch crowns and can fill a 15 cm pot within 8 months when supplied with fertilizer biweekly.
Figure 3
The YW5AF7 plant
The YW5AF7 plant. The image shows a mature YW5AF7 plant with flowers and fruit in a 10 cm pot.
1746-4811-5-15-3
The leaves of YW5AF7 are thin, and show typical morphology for F. vesca. The leaf is light medium green in color, with both sides pubescent. Stomates are found on the abaxial side only. The first two true leaves are unifoliate, round and serrated. Later leaves are trifoliate, although very small ectopic highly serrated unifoliate leaves are also sometimes found at the base of the plant. Under greenhouse conditions in a 15 cm pot, the largest leaves can reach 13 cm in width and 8 cm in length. The terminal leaflet is ovate and more rounded than found on F. vesca var. Ruegen grown under the same conditions. It is serrated, with an average of 19 serrations on the largest terminal leaflets, whereas the margins of the same size terminal leaflet of the octoploid F. ×ananassa var. Chandler has about 25 serrations. Lateral leaflets of the largest leaves of YW5AF7 average 17 serrations. Serrations begin about half way up the inner edge of the lateral leaflets. The interveinal lamina are crinkled. Petioles are long and have a distinct adaxial groove. They are red in low light and tend to be greener toward the leaf. Petioles are pubescent, with straight, unbranched hairs. Stipules are red.
Flowers and fruit are borne within the leaf canopy as well as on inflorescences that extend above the canopy. Occasionally these extend down over the sides of the pots because of the weight of the fruit. In 15 cm pots, pedicels of the first inflorescence can be 15-20 cm long. Pedicels are round, pubescent, and tend to be greener than the petioles. Inflorescences usually have 4 to 5 flowers, however, under some conditions the cymous inflorescence continues to branch and form additional flowers. Root initials sometimes form at the nodes and these will form roots if pegged to the soil. On very old, pot-bound plants, inflorescences with only one or two flowers become common.
The flower is also typical of F. vesca. It usually has five petals, a calyx consisting of 2 whorls of five sepals, and 20 stamens. In the center of the flower is a rounded receptacle, bearing yellow pistils, that extends well beyond the stamens when the flower bud opens and the anthers dehisce. Occasionally flowers have extra flower parts, the most obvious of these occurrences being 6 petals per flower. In addition, petaloid anthers have been observed. Both conditions are also seen in the parental generations, and the appearance is correlated with larger flowers. Primary flowers are usually 1.5-2 cm in diameter, depending on growth conditions. Secondary and later flowers tend to be smaller.
Fruit
Like its progenitor, Yellow Wonder, the berry of YW5AF7 is soft and pale yellow in color with tan achenes when ripe, and pale green to white during development. Ripe berries are highly aromatic with sweet banana and pineapple overtones. When all achenes are fertilized, the berry shape is long conic, with some primary berries being necked long conic and reaching 27 mm in length and 20 mm in width in plants growing in 15 cm pots. The average fresh weight of a primary berry was 1.67 ± 0.26 S.E. g., with the largest berry being 2.45 g. Achenes are borne on the surface of the berry, with an average 193 ± 17 S.E. achenes per primary berry (225 on the largest). In comparison, an average of 518 achenes per primary fruit was reported for a commercial octoploid variety B26 26. In the absence of insects or human interventions, only about half of the achenes are fertilized and enlarge, and the fruit tend to be smaller and of varied shapes. Pollination can be aided by transferring pollen from a flower with dehiscing anthers to a just opened flower using a small camel hair brush. The extent of fertilized ovules per fruit can be approximated over time by examining the expansion of developing achenes and the subtending receptacle tissue.
Performance
Seeds of YW5AF7 will germinate in soil in the greenhouse in one week. However, more uniform germination can be achieved by cold treatment of moist seed. Seeds of YW5AF7 in moistened soil did not achieve maximum germination [87%, n = 100 (10 pots with 10 seed each)] until 21 days after sowing. Following treatment of moist seed for 3 weeks in the dark at 5°C, 74% of YW5AF7 seeds germinated in 7 days after being brought into the greenhouse, and by 14 days, maximum germination, 91%, was achieved. Seeds that have been disinfested using ethanol and bleach treatment will germinate in Petri dishes on 0.5× MS media B27 27 solidified with 0.8% Phytagar (Invitrogen, Carlsbad, CA). The resulting seedlings can be used as aseptic explants for tissue culture. Under these conditions we have found that longer cold treatment (3 weeks) results in more uniform germination, which can be critical for evaluating developmental or physiological parameters of transformed plants in gene function tests./p
pMildew susceptibility in itF. vesca /itwas found to be due to two dominant genes abbrgrpabbr bid="B28"28/abbr/abbrgrp. Both genes had to be absent to obtain a resistant plant, and cytoplasmic effects were noted. Seedlings of YW5AF7 are susceptible to powdery mildew at early stages in development, particularly in a growth chamber environment. In the greenhouse, YW5AF7 has been found to be susceptible to powdery mildew, thrips, two spotted mites, and aphids./p
/sec
sec
st
pTransformation and Regeneration/p
/st
pWhile YW5AF7 is a strong candidate for genomics studies, it was important to test if it could be successfully transformed and regenerated. While diploid strawberry is routinely transformed, transformation and regeneration efficiency are highly genotype specific [reviewed in abbrgrpabbr bid="B29"29/abbr/abbrgrp]. To test the YW5AF7 line for the ability to produce shoots after gene insertion, explants from greenhouse-grown YW5AF7 plants were co-cultivated with itAgrobacterium tumefaciens /itcarrying a visible GFP reporter as described in Materials and Methods. Several published media formulations were evaluated for regeneration and are detailed in Table tblr tid="T1"1/tblr./p
tbl id="T1"
title
pTable 1/p
/title
caption
pThe media formulations used in regeneration experiments./p
/caption
tblbdy cols="6"
r
c ca="left"
p
bMedium/b
/p
/c
c ca="left"
p
bAuxin/b
/p
/c
c ca="left"
p
bConcentration/b
/p
/c
c ca="left"
p
bCytokinin/b
/p
/c
c ca="left"
p
bConcentration/b
/p
/c
c ca="center"
p
bReference/b
/p
/c
/r
r
c cspan="6"
hr/
/c
/r
r
c ca="left"
pA/p
/c
c ca="left"
pIBA/p
/c
c ca="left"
p0.98 uM/p
/c
c ca="left"
pBA/p
/c
c ca="left"
p13.20 uM/p
/c
c ca="center"
p
abbrgrp
abbr bid="B32"32/abbr
/abbrgrp
/p
/c
/r
r
c ca="left"
pB/p
/c
c ca="left"
p2,4-D/p
/c
c ca="left"
p0.45 uM/p
/c
c ca="left"
pTDZ/p
/c
c ca="left"
p4.54 uM/p
/c
c ca="center"
p
abbrgrp
abbr bid="B36"36/abbr
/abbrgrp
/p
/c
/r
r
c ca="left"
pC/p
/c
c ca="left"
pIBA/p
/c
c ca="left"
p1.50 uM/p
/c
c ca="left"
pTDZ/p
/c
c ca="left"
p10.00 uM/p
/c
c ca="center"
p
abbrgrp
abbr bid="B30"30/abbr
/abbrgrp
/p
/c
/r
/tblbdy
/tbl
pCallusing was observed on all media types tested, but tissue vigor and regeneration were best supported by the formulation presented in Zhao et al. abbrgrpabbr bid="B30"30/abbr/abbrgrp (Figure figr fid="F4"4/figr, triangles). On this formulation, 20% of explants possessed shoots by six weeks whereas explants on other media formulations exhibited little or no organogenesis at this time point (Figure figr fid="F4"4/figr, black or white circles). At nine weeks, over 80 percent of explants had shoots on the Zhao formula, whereas shoot initiation on other media types was less frequent. Figure figr fid="F5"5/figr shows that the medium producing the highest percentage of explants exhibiting organogenesis also resulted in a higher number of shoots per explant by nine weeks in culture than other formulations./p
fig id="F4"
title
pFigure 4/p
/title
caption
pYW5AF7 regeneration frequency on three published media formulations/p
/caption
text
pbYW5AF7 regeneration frequency on three published media formulations/b. Various explants from YW5AF7, including mature leaves, young leaves and petioles, were grown on three different media (Table 1) to test regeneration frequency. Medium A, white circles; Medium B, black circles and Medium C, triangles. The data are the means of two independent experiments./p
/text
graphic file="1746-4811-5-15-4"/
/fig
fig id="F5"
title
pFigure 5/p
/title
caption
pThe number of shoots per YW5AF7 explant on three media formulations/p
/caption
text
pbThe number of shoots per YW5AF7 explant on three media formulations/b. The mean number of shoots per explant was determined for three different media: Medium A, white circles, Medium B, black circles and Medium C, triangles. The data reflect the mean of two independent experiments./p
/text
graphic file="1746-4811-5-15-5"/
/fig
pThese data indicate that the formulation by Zhao et al. abbrgrpabbr bid="B30"30/abbr/abbrgrp results in the highest number of explants exhibiting shoots, which is important for maximizing the number of independent transformation events in gene function experiments. Shoots were generated by direct organogenesis and were produced most quickly and abundantly on the basipetal end of petiole segments (as shown in Figure figr fid="F6"6A/figr). Evaluation of GFP fluorescence in emerging shoots revealed that about 40% of shoots were transformed, indicating that escapes can be present using 4 mgL hygromycin for selection./p
fig id="F6"
title
pFigure 6/p
/title
caption
pYW5AF7 transformants/p
/caption
text
pbYW5AF7 transformants/b. A. A cluster of shoots emerging from the basipetal end of a petiole. Both transformed (GFP+; green) and non-transformed shoots (red in color) are present. B. Imbibed seed from one line of GFP expressing transformed 5AF7 plants. GFP positive and GFP negative seeds are present in a 19:20 ratio, indicating that a single insertion is likely. C. A wild-type seedling (left) and a GFP positive transgenic seedling (right) grown from seed of the plant in (B)./p
/text
graphic file="1746-4811-5-15-6"/
/fig
pOnce differentiated, explants were moved to a media without TDZ to enhance shoot elongation. Although not formally quantified, at least one insertion event was observed on each explant, as evidenced by GFP fluorescence. Figure figr fid="F6"6/figr shows germinating seed and two resulting seedlings from one such plant. In this random sample of seeds, the ratio of GFP positive to GFP negative (wild type) seeds was essentially 1:1, indicating that there was most likely a single insertion event in this transformant./p
/sec
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pDiscussion/p
/st
pitF. vesca /ithas great potential as a system to study the genetic basis of agriculturally important biological questions in the Rosaceae family. Its small size, rapid growth, generous seed set, small genome, and sequence availability make it an excellent resource for development of genomics tools. Its genomic similarity to other valuable crops underlies its potential utility as a surrogate to test gene function relevant to many rosaceous species./p
pA primary concern about the system has been the observation of variability among individuals in lines that have not been single seed propagated in the lab through several generations. Although typically self fertilizing and therefore expected to be largely homozygous, we observed clear variability in a number of horticultural traits among itF. vesca /itYellow Wonder and Hawaii-4 plants generated from achenes from a single fruit suggestive of some degree of residual heterozygosity. These phenotypic variations are potentially problematic in a seminal line proposed as a genomics-friendly genotype. This is an important consideration as several current efforts are developing populations of T-DNA insertion, activation tagged, overexpression, or RNAi lines using itF. vesca/it, and it could become difficult to discriminate between a phenotype resulting from an engineered genotypic variation and natural genetic variation in the line. Interpretations from a genetically noisy background may preclude, or at least delay, identification of gene-specific effects on morphology and physiology. High variability in results from physiological studies drastically increases the number of plants that must be used to obtain statistical significance. These populations may be suboptimal for quantitative studies of gene expression, as the variation in the baseline may lead to errors in interpreting microarray, digital, or qRT-PCR gene expression profiles. Results must be able to be repeated in other laboratories, so a system based on a known genetic background will supplement these efforts and be of benefit to the wider research community./p
pOur PCR analysis of two different "Yellow Wonder" lines (Figure figr fid="F2"2/figr) indicated that even a well-established and commercially available line of itF. vesca /itmay consist of different genotypes. Because "Yellow Wonder" is both non-red and non-runnering it would appear likely, given that these two loci must be homozygous recessive, that these plants are already substantially homozygous. However, clearly Y1 is not the same as Y2. There are no data from any of the suppliers to show that their "Yellow Wonder", the color of which would be expected to breed true from seed, is the same as a competitor's, which also would breed true from seed (at least for color and non-runnering), and no data to show that any of these are the same as others described in the literature. For these reasons, the pre-emptive development of a stable, highly inbred, prolific and documented genotype was considered useful, as it would provide a stable genotype for evaluation of gene function that could be shared among users./p
pBotanical descriptors of YW5AF7 have been carefully evaluated and define a reproducible and firm foundation for later comparisons. Even subtle phenotypes induced by a transgene should be able to be reliably scored in this stable background./p
pMany of the techniques used for studying itArabidopsis /itcan be used with itF. vesca /itYW5AF7. Seedling variations are almost indiscernible in populations of Arabidopsis seedlings, and their small stature makes itin vitro /itassessment of phenotypes possible. Tests of early development in response to environmental conditions, growth regulators or nutrient status are also possible in itF. vesca/it, much like in itArabidopsis/it./p
pFor YW5AF7 to have utility as a functional genomics system it must be transformable. As observed by many groups, transformation efficiency of various strawberry genotypes is highly genotype dependent and in some cases impossible abbrgrpabbr bid="B29"29/abbr/abbrgrp. The transformation capacity of YW5AF7 was tested with a GFP reporter gene. Many GFP foci were observed in co-cultivated tissues and GFP-positive plantlets were regenerated on media containing selective antibiotics. Three published (yet diverse) media formulations were evaluated for regeneration-inducing ability. In all cases shoots appeared via organogenesis with the best results arising from the media formulation presented in Zhao et al. abbrgrpabbr bid="B30"30/abbr/abbrgrp. A number of shoots were clearly initiated by 30 days and plantlets could be excised after 60 days and rooted in rooting media. This time course is reasonable yet could likely be optimized to improve the utility of the YW5AF7 system. The high frequency of shoot formation on independent explants ensures propagation of independent transformants. Antibiotic sensitivity was generally consistent with previously-published reports in strawberry abbrgrpabbr bid="B31"31/abbrabbr bid="B32"32/abbr/abbrgrp but subculture to progressively higher amounts of antibiotics may be advisable as regeneration of non-transformed shoots was observed using hygromycin at 4 ugml./p
pThe most prolific explants were leaf-adjacent petiole segments, with the first shoots appearing on the basipetal end of these explants. The most productive formulation contained thidiazuron, TDZ, as a principle growth regulator, a compound shown to be effective in inducing regeneration in a number of other studies. However, consistent with previous reports abbrgrpabbr bid="B33"33/abbr/abbrgrp growth on TDZ severely stunted shoot elongation, and increased somaclonal variation has been observed when this regulator has been employed [see, abbrgrpabbr bid="B34"34/abbr/abbrgrp]. Once clearly formed, the shooting explants were transferred to a TDZ-free media formulation that had been used to regenerate itF. vesca /itaccession Hawaii-4. Within one week the shoots elongated vigorously and could be transferred to rooting media. Other media formulations also induced shoots, but at a much slower rate./p
pThe YW5AF7 line is runnerless and this has distinct advantages to its adoption as a functional genomics model. Runnerless plants are much easier to maintain in a greenhouse as plants may be located in close proximity without having to continually remove a tangle of runners or daughter plants that invade neighboring pots, such as is our experience with itF. vesca /itHawaii 4 and itF. vesca /itPawtuckaway. In large populations this can be a source of genotype contamination and requires dedication to constant manual management. On the other hand, one of the advantages of itF. vesca /itas a functional genomics system is that plants can be propagated by branch crown divisions or runners or branch crown division, as well as by seed, making it possible to vegetatively propagate mutants that affect flowering or seed set. Although runnerless, the YW5AF7 line does produce abundant branch crowns./p
pSeeds of YW5AF7 are available for research purposes from Dr. J. P. Slovin, USDA-ARS Genetic Improvement of Fruits and Vegetables Laboratory, Bldg. 010A, 10300 Baltimore Avenue, Beltsville, MD 20705 (phone: 301504-5629; e-mail: emailslovinj@ars.usda.gov/email). The seed are produced in the greenhouse under ambient conditions from F7 plants that are manually self-pollinated. Seed of this line have been deposited with the USDA, ARS National Clonal Germplasm Repository, Corvallis, OR (PI 641092)./p
/sec
sec
st
pConclusion/p
/st
pThe highly inbred selection YW5AF7 has been generated and characterized. A set of botanical descriptors defines a baseline that may be compared to phenotypes of forward or reverse genetic mutants as well as overexpression and RNAi lines. This genotype, associated scored metrics, and transformation protocol permit the deployment of this system as a useful tool for the Rosaceae research community in the elucidation of gene function in a stable and consistent genetic background that complements existing systems./p
/sec
sec
st
pMethods/p
/st
sec
st
pPlant Origin and Seedling Selection/p
/st
pThe inbred line, designated YW5AF7, was obtained by manually self-pollinating itF. vesca /itvar. "Yellow Wonder" plants grown from seeds in the Beltsville collection maintained originally by S. Hokanson. Seeds were planted in Metro Mix 510 (Scotts-Sierra Horticultural Products, Marysville, OH) supplemented with dolomitic lime. Plants were grown in the greenhouse with supplemental lighting from sodium halide lamps to give a daylength of at least 12 h. At least one hundred seed were planted at each generation. Flowers were self-pollinated by gentle brushing starting when their flowers just opened, and every day thereafter as their anthers dehisced until the flower petals fell. A new small camel hair artist's brush was used for each pollination. Flowers were tagged for the day of pollination. The plant to be used for the next generation was randomly chosen from ten plants that met a basic requirement of early flowering, general robust appearance, fruit set (as based on number of achenes that enlarge following pollination), high number of achenes on the primary fruit, and short number of days to mature fruit. Although germination rate and number of days to flowering varied, the other selecting parameters usually showed very little difference. However, some seedlings germinated from F3 seed showed morphological abnormalities such as dwarfness or a single cotyledon. Only seedlings with normal phenotypes were selected for further selfing. The selfing process was continued for seven generations to generate YW5AF7./p
/sec
sec
st
pMedia Preparation/p
/st
pThree previously defined media formulations were investigated to determine which would lead to optimal regeneration of YW5AF7 explants. Media included 1× Murashige and Skoog medium with vitamins, 2% sucrose and the growth regulators presented in Table tblr tid="T1"1/tblr. Media were prepared with deionized water, the pH adjusted to 5.6-5.8, and then autoclaved for 20 min at 121°C and 15 psi. Growth regulators were co-autoclaved with the media. In all three media types, the selection agent used was 4 mgL hygromycin B, which was added to the media after it was cooled to ~50°C./p
/sec
sec
st
pAgrobacterium-mediated transformation/p
/st
pLeaf, stem, and petiole segments were sterilized in 70% EtOH for 30 seconds and 1% sodium hypochlorite (20% bleach) for 10 min. A single transformed itAgrobacterium /itcolony carrying a 35S::GFP construct was grown overnight in Luria Broth with 10 mgL rifampicin, 50 mgL gentamicin, and 50 mgL spectinomycin to an ODsub600 /subof 0.5, then pelleted at 1,000 × g. The bacterial pellet was resuspended to 0.1 ODsub600 /subin co-cultivation medium consisting of 1× MS pH 5.8 with 2% sucrose, supplemented with 50 uM acetosyringone. Explants were added to the co-cultivation medium and incubated 20 min at room temperature, then blotted dry with sterile filter paper and transferred to media without selection for 2 d at 25°C in darkness. After 2 d the explants were washed twice in co-cultivation medium liquid supplemented with 500 mgL carbenicillin, followed by 30 min of incubation in fresh wash media. Explants were again blotted dry and transferred to solid media with selection./p
pTransgenic shoots were selected on media containing 4 mgL hygromycin B. The explants were regenerated under a 16 h light, 8 h dark photoperiod under cool-white fluorescent lighting. Explants were checked daily for contamination, and subcultured every 2 weeks. When distinct clumps of shoots were formed the entire clump was transferred to hormone-free rooting media consisting of 0.5× MS media (pH 5.8), 1% glucose, and 1% phytoagar. Roots formed within days to one month and individual plants could then be dissected from the groups of shoots./p
/sec
sec
st
pPCR/p
/st
pPCR was performed using the touchdown protocol described by Sargent et al. abbrgrpabbr bid="B35"35/abbr/abbrgrp in a 20 μl reaction containing HotStart Taq Master Mix (Qiagen, Valencia, CA), 0.4 μM each primer, and 1.0 ng genomic DNA. PCR products were separated by electrophoresis through a 1.5% TAE agarose gel and visualized by ethidium bromide staining. Primers were designed to span the intron in the N-terminal domain of a low molecular weight heat shock protein gene identified as an EST (GenBank accession number ext-link ext-link-type="gen" ext-link-id="CX661743.1"CX661743.1/ext-link) from a "Yellow Wonder" (Y2) heat-treated seedling cDNA library. Template DNA for Y1, Y2, itF. iinumae /itJ-17, and itF. vesca /itsubsp. itamericana /itPawtuckaway was obtained from 50-100 mg young leaf tissue using a DNeasy Plant Mini kit (Qiagen). Y1 and itF. vesca /itsubsp. itamericana /itPawtuckaway plants were obtained from T. Davis (University of New Hampshire) and itF. iinumae /itJ-17 plants were obtained from the US National Plant Germplasm collection./p
/sec
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sec
st
pCompeting interests/p
/st
pThe authors declare that they have no competing interests./p
/sec
sec
st
pAuthors' contributions/p
/st
pJPS conceived of the project, performed all crosses, plant measurements and PCR, and participated in preparation of the manuscript. KS performed the co-cultivation and subculturing of strawberry tissues to test transformation efficiency. KMF supervised transformation and regeneration and participated in preparation of the manuscript./p
/sec
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bm
ack
sec
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pAcknowledgements/p
/st
pThe authors thank Andrea Murphy and Jeremy Goetz for greenhouse assistance, Sasha Drost, an Eleanor Roosevelt High School science intern for technical assistance, Todd Cooke for his help with botanical description, and anonymous reviewers for comments. This work was performed as part of NSF project 0701488 (KMF). Kyle Schmitt was supported with funding from the Howard Hughes Medical Institute "Science for Life" program at the University of Florida (KMF)./p
/sec
/ack
refgrp
bibl id="B1"
title
pMultiple models for Rosaceae genomics/p
/title
aug
au
snmShulaev/snm
fnmV/fnm
/au
au
snmKorban/snm
fnmSS/fnm
/au
au
snmSosinski/snm
fnmB/fnm
/au
au
snmAbbott/snm
fnmAG/fnm
/au
au
snmAldwinckle/snm
fnmHS/fnm
/au
au
snmFolta/snm
fnmKM/fnm
/au
au
snmIezzoni/snm
fnmA/fnm
/au
au
snmMain/snm
fnmD/fnm
/au
au
snmArus/snm
fnmP/fnm
/au
au
snmDandekar/snm
fnmAM/fnm
/au
etal/
/aug
sourcePlant Physiol/source
pubdate2008/pubdate
volume147/volume
issue3/issue
fpage985/fpage
lpage1003/lpage
xrefbib
pubidlist
pubid idtype="pmpid" link="fulltext"18487361/pubid
pubid idtype="doi"10.1104pp.107.115618/pubid
pubid idtype="pmcid"2442536/pubid
/pubidlist
/xrefbib
/bibl
bibl id="B2"
title
pComparative genetic mapping between octoploid and diploid Fragaria species reveals a high level of colinearity between their genomes and the essential disomic behavior of the cultivated octoploid strawberry/p
/title
aug
au
snmRousseau-Gueutin/snm
fnmM/fnm
/au
au
snmLerceteau-Köhler/snm
fnmE/fnm
/au
au
snmBarrot/snm
fnmL/fnm
/au
au
snmSargent/snm
fnmDJ/fnm
/au
au
snmMonfort/snm
fnmA/fnm
/au
au
snmSimpson/snm
fnmD/fnm
/au
au
snmArús/snm
fnmP/fnm
/au
au
snmGuérin/snm
fnmG/fnm
/au
au
snmDenoyes-Rothan/snm
fnmB/fnm
/au
/aug
sourceGenetics/source
pubdate2008/pubdate
volume179/volume
issue4/issue
fpage2045/fpage
lpage2060/lpage
xrefbib
pubidlist
pubid idtype="pmpid" link="fulltext"18660542/pubid
pubid idtype="doi"10.1534genetics.107.083840/pubid
pubid idtype="pmcid"2516079/pubid
/pubidlist
/xrefbib
/bibl
bibl id="B3"
title
pitFragaria vesca /itchlorophyll mutants/p
/title
aug
au
snmDavis/snm
fnmTM/fnm
/au
au
snmPollard/snm
fnmJE/fnm
/au
/aug
sourceHortScience/source
pubdate1991/pubdate
volume26/volume
issue3/issue
fpage311/fpage
/bibl
bibl id="B4"
title
pitFragaria vesca/it, one way to understand flowering in perennials/p
/title
aug
au
snmAlbani/snm
fnmM/fnm
/au
au
snmTaylor/snm
fnmS/fnm
/au
au
snmRodriquez Lopez/snm
fnmC/fnm
/au
au
snmCekic/snm
fnmC/fnm
/au
au
snmAl-Sheikh/snm
fnmM/fnm
/au
au
snmGreenland/snm
fnmA/fnm
/au
au
snmWetten/snm
fnmA/fnm
/au
au
snmWilkinson/snm
fnmM/fnm
/au
au
snmBattey/snm
fnmNH/fnm
/au
/aug
sourceFlowering Newsletter/source
pubdate2001/pubdate
volume31/volume
fpage44/fpage
lpage48/lpage
/bibl
bibl id="B5"
title
pitFragaria vesca/it, a useful tool for Rosaceae genomics/p
/title
aug
au
snmSlovin/snm
fnmJ/fnm
/au
au
snmRabinowicz/snm
fnmPD/fnm
/au
/aug
source6th North American Strawberry Symposium: 2007/source
publisherVentura, CA: American Society for Horticultural Science/publisher
pubdate2007/pubdate
fpage112/fpage
lpage117/lpage
/bibl
bibl id="B6"
title
pThe Strawberry/p
/title
aug
au
snmDarrow/snm
fnmGM/fnm
/au
/aug
publisherNew York: Holt, Rinehart and Winston/publisher
pubdate1966/pubdate
/bibl
bibl id="B7"
title
pThe genetical control of flowering and runnering in varieties of itFragaria vesca/it/p
/title
aug
au
snmBrown/snm
fnmT/fnm
/au
au
snmWaring/snm
fnmPF/fnm
/au
/aug
sourceHeredity/source
pubdate1965/pubdate
volume20/volume
fpage651/fpage
xrefbib
pubid idtype="doi"10.1038hdy.1965.35/pubid
/xrefbib
/bibl
bibl id="B8"
title
pA preliminary note on the genetics of itFragaria/it/p
/title
aug
au
snmRichardson/snm
fnmCW/fnm
/au
/aug
sourceJournal of Genetics/source
pubdate1914/pubdate
volume3/volume
fpage171/fpage
lpage178/lpage
xrefbib
pubid idtype="doi"10.1007BF02981712/pubid
/xrefbib
/bibl
bibl id="B9"
title
pA further note on the genetics of itFragaria/it/p
/title
aug
au
snmRichardson/snm
fnmCW/fnm
/au
/aug
sourceJournal of Genetics/source
pubdate1918/pubdate
volume7/volume
fpage167/fpage
lpage170/lpage
xrefbib
pubid idtype="doi"10.1007BF02983543/pubid
/xrefbib
/bibl
bibl id="B10"
title
pSome notes on itFragaria/it/p
/title
aug
au
snmRichardson/snm
fnmCW/fnm
/au
/aug
sourceJournal of Genetics/source
pubdate1920/pubdate
volume10/volume
fpage39/fpage
lpage46/lpage
xrefbib
pubid idtype="doi"10.1007BF02983521/pubid
/xrefbib
/bibl
bibl id="B11"
title
pNotes on itFragaria/it/p
/title
aug
au
snmRichardson/snm
fnmCW/fnm
/au
/aug
sourceJournal of Genetics/source
pubdate1923/pubdate
volume13/volume
fpage147/fpage
lpage152/lpage
xrefbib
pubid idtype="doi"10.1007BF02983050/pubid
/xrefbib
/bibl
bibl id="B12"
title
pTracking the evolutionary history of polyploidy in itFragaria /itL. (strawberry): New insights from phylogenetic analyses of low-copy nuclear genes/p
/title
aug
au
snmRousseau-Gueutin/snm
fnmM/fnm
/au
au
snmGaston/snm
fnmA/fnm
/au
au
snmAïnouche/snm
fnmA/fnm
/au
au
snmAïnouche/snm
fnmML/fnm
/au
au
snmOlbricht/snm
fnmK/fnm
/au
au
snmStaudt/snm
fnmG/fnm
/au
au
snmRichard/snm
fnmL/fnm
/au
au
snmDenoyes-Rothan/snm
fnmB/fnm
/au
/aug
sourceMolecular Phylogenetics and Evolution/source
pubdate2009/pubdate
volume51/volume
issue3/issue
fpage515/fpage
lpage530/lpage
xrefbib
pubid idtype="doi"10.1016j.ympev.2008.12.024/pubid
/xrefbib
/bibl
bibl id="B13"
title
pStrawberries/p
/title
aug
au
snmHancock/snm
fnmJF/fnm
/au
/aug
publisherNew York: CABI Publishing/publisher
pubdate1999/pubdate
/bibl
bibl id="B14"
title
pAppropriate choice of antibiotic and itAgrobacterium /itstrain improves transformation of antibiotic-sensitive itFragaria vesca /itand itF. v. semperflorens/it/p
/title
aug
au
snmAlsheikh/snm
fnmMK/fnm
/au
au
snmSuso/snm
fnmH-P/fnm
/au
au
snmRobson/snm
fnmM/fnm
/au
au
snmBattey/snm
fnmNH/fnm
/au
au
snmWetten/snm
fnmA/fnm
/au
/aug
sourcePlant Cell Reports/source
pubdate2002/pubdate
volume20/volume
issue12/issue
fpage1173/fpage
lpage1180/lpage
xrefbib
pubid idtype="doi"10.1007s00299-002-0453-0/pubid
/xrefbib
/bibl
bibl id="B15"
title
pShoot regeneration and itAgrobacterium/it-mediated transformation of itFragaria vesca /itL/p
/title
aug
au
snmEl Mansouri/snm
fnmI/fnm
/au
au
snmMercado/snm
fnmJ/fnm
/au
au
snmValpuesta/snm
fnmV/fnm
/au
au
snmLopez-Aranda/snm
fnmJ/fnm
/au
au
snmPliego-Alfaro/snm
fnmF/fnm
/au
au
snmQuesada/snm
fnmM/fnm
/au
/aug
sourcePlant Cell Reports/source
pubdate1996/pubdate
volume15/volume
fpage642/fpage
lpage646/lpage
xrefbib
pubid idtype="doi"10.1007BF00232469/pubid
/xrefbib
/bibl
bibl id="B16"
title
pitAgrobacterium/it-mediated transformation of 'Alpine' itFragaria vesca/it, and transmission of transgenes to R1 progeny/p
/title
aug
au
snmHaymes/snm
fnmKM/fnm
/au
au
snmDavis/snm
fnmTM/fnm
/au
/aug
sourcePlant Cell Reports/source
pubdate1998/pubdate
volume17/volume
fpage279/fpage
lpage283/lpage
xrefbib
pubid idtype="doi"10.1007s002990050392/pubid
/xrefbib
/bibl
bibl id="B17"
title
pTransgene expression in strawberries driven by a heterologous phloem-specific promoter/p
/title
aug
au
snmZhao/snm
fnmY/fnm
/au
au
snmLiu/snm
fnmQ/fnm
/au
au
snmDavis/snm
fnmRE/fnm
/au
/aug
sourcePlant Cell Reports/source
pubdate2004/pubdate
volume23/volume
issue4/issue
fpage224/fpage
lpage230/lpage
xrefbib
pubidlist
pubid idtype="pmpid" link="fulltext"15235813/pubid
pubid idtype="doi"10.1007s00299-004-0812-0/pubid
/pubidlist
/xrefbib
/bibl
bibl id="B18"
title
pEstimation of the nuclear DNA content of strawberries (itFragaria spp/it.) compared with Arabidopsis thaliana by using dual-stem flow cytometry/p
/title
aug
au
snmAkiyama/snm
fnmY/fnm
/au
au
snmYamamoto/snm
fnmY/fnm
/au
au
snmOhmido/snm
fnmN/fnm
/au
au
snmOshima/snm
fnmM/fnm
/au
au
snmFukui/snm
fnmK/fnm
/au
/aug
sourceCytologia/source
pubdate2001/pubdate
volume66/volume
fpage431/fpage
lpage436/lpage
/bibl
bibl id="B19"
title
pAngiosperm DNA C-values Database (Release 6.0, Oct 2005)/p
/title
aug
au
snmBennett/snm
fnmMD/fnm
/au
au
snmLeitch/snm
fnmIJ/fnm
/au
/aug
pubdate2005/pubdate
urlhttp:www.kew.orgcvalues/url
/bibl
bibl id="B20"
title
pComparisons with itCaenorhabditis /it(approximately 100 Mb) and itDrosophila /it(approximately 175 Mb) using flow cytometry show genome size in itArabidopsis /itto be approximately 157 Mb and thus approximately 25% larger than the itArabidopsis /itgenome initiative estimate of approximately 125 Mb/p
/title
aug
au
snmBennett/snm
fnmMD/fnm
/au
au
snmLeitch/snm
fnmIJ/fnm
/au
au
snmPrice/snm
fnmHJ/fnm
/au
au
snmJohnston/snm
fnmJS/fnm
/au
/aug
sourceAnnals of Botany/source
pubdate2003/pubdate
volume91/volume
issue5/issue
fpage547/fpage
lpage557/lpage
xrefbib
pubidlist
pubid idtype="pmpid"12646499/pubid
pubid idtype="doi"10.1093aobmcg057/pubid
/pubidlist
/xrefbib
/bibl
bibl id="B21"
title
pSelection and characterization of itα/it-methyltryptophan resistant lines of Lemna gibba showing a rapid rate of indole-3-acetic acid turnover/p
/title
aug
au
snmTam/snm
fnmYY/fnm
/au
au
snmSlovin/snm
fnmJP/fnm
/au
au
snmCohen/snm
fnmJD/fnm
/au
/aug
sourcePlant Physiol/source
pubdate1995/pubdate
volume107/volume
fpage77/fpage
lpage85/lpage
xrefbib
pubidlist
pubid idtype="pmcid"161170/pubid
pubid idtype="pmpid" link="fulltext"12228344/pubid
/pubidlist
/xrefbib
/bibl
bibl id="B22"
title
pThe genetic control of the everbearing habit and three other characters in varieties of itFragaria vesca/it/p
/title
aug
au
snmBrown/snm
fnmT/fnm
/au
au
snmWareing/snm
fnmPF/fnm
/au
/aug
sourceEuphytica/source
pubdate1965/pubdate
volume14/volume
fpage97/fpage
lpage112/lpage
/bibl
bibl id="B23"
title
pGenetic and environmental control of flowering in strawberry/p
/title
aug
au
snmBattey/snm
fnmNH/fnm
/au
au
snmLeMiere/snm
fnmP/fnm
/au
au
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/au
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/au
au
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fnmS/fnm
/au
au
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fnmKJ/fnm
/au
au
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fnmP/fnm
/au
au
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fnmAJ/fnm
/au
au
snmDarby/snm
fnmJ/fnm
/au
au
snmWilkinson/snm
fnmMJ/fnm
/au
/aug
sourceGenetic and Environmental Manipulation of Horticultural Crops/source
publisherNew York: CABI Publishing/publisher
editorCockshull KE, Gray D, Seymour GB, Thomas B/editor
pubdate1998/pubdate
fpage111/fpage
lpage131/lpage
/bibl
bibl id="B24"
title
pShikamate dehydrogenase allozymes: inheritance and close linkage to fruit color in the diploid strawberry/p
/title
aug
au
snmWilliamson/snm
fnmSC/fnm
/au
au
snmYu/snm
fnmH/fnm
/au
au
snmDavis/snm
fnmTM/fnm
/au
/aug
sourceJournal of Heredity/source
pubdate1995/pubdate
volume86/volume
issue1/issue
fpage74/fpage
lpage76/lpage
/bibl
bibl id="B25"
title
pMolecular identification of the yellow fruit color (c) locus in diploid strawberry: a candidate gene approach/p
/title
aug
au
snmDeng/snm
fnmC/fnm
/au
au
snmDavis/snm
fnmTM/fnm
/au
/aug
sourceTheor Appl Genet/source
pubdate2001/pubdate
volume103/volume
fpage316/fpage
lpage322/lpage
xrefbib
pubid idtype="doi"10.1007s001220100648/pubid
/xrefbib
/bibl
bibl id="B26"
title
pStudies on the nutrition of the strawberry/p
/title
aug
au
snmGardner/snm
fnmVR/fnm
/au
/aug
sourceMo Agr Exp Sta Res Bul/source
pubdate1923/pubdate
volume57/volume
fpage31/fpage
/bibl
bibl id="B27"
title
pA revised medium for rapid growth and bioassays with tobacco tissue cultures/p
/title
aug
au
snmMurashige/snm
fnmT/fnm
/au
au
snmSkoog/snm
fnmF/fnm
/au
/aug
sourcePhysiologia Plantarum/source
pubdate1962/pubdate
volume72/volume
fpage473/fpage
lpage497/lpage
xrefbib
pubid idtype="doi"10.1111j.1399-3054.1962.tb08052.x/pubid
/xrefbib
/bibl
bibl id="B28"
title
pInheritance of mildew resistance in itFragaria /itwith special reference to cytoplasmic effects/p
/title
aug
au
snmHarland/snm
fnmSC/fnm
/au
au
snmKing/snm
fnmEE/fnm
/au
/aug
sourceHeredity/source
pubdate1957/pubdate
volume11/volume
fpage287/fpage
/bibl
bibl id="B29"
title
pTransformation of strawberry: The basis for translational genomics in Rosaceae/p
/title
aug
au
snmFolta/snm
fnmKM/fnm
/au
au
snmDhingra/snm
fnmA/fnm
/au
/aug
sourceIn Vitro Cell Dev-Pl/source
pubdate2006/pubdate
volume42/volume
issue6/issue
fpage482/fpage
lpage490/lpage
xrefbib
pubid idtype="doi"10.1079IVP2006807/pubid
/xrefbib
/bibl
bibl id="B30"
title
pTransgene expression in strawberries driven by a heterologous phloem-specific promoter/p
/title
aug
au
snmZhao/snm
fnmY/fnm
/au
au
snmLiu/snm
fnmQZ/fnm
/au
au
snmDavis/snm
fnmRE/fnm
/au
/aug
sourcePlant Cell Reports/source
pubdate2004/pubdate
volume23/volume
issue4/issue
fpage224/fpage
lpage230/lpage
xrefbib
pubidlist
pubid idtype="pmpid" link="fulltext"15235813/pubid
pubid idtype="doi"10.1007s00299-004-0812-0/pubid
/pubidlist
/xrefbib
/bibl
bibl id="B31"
title
pStrawberry culture itin vitro/it: applications in genetic transformation and biotechnology/p
/title
aug
au
snmDebnath/snm
fnmSC/fnm
/au
au
snmTeixeira da Silva/snm
fnmJA/fnm
/au
/aug
sourceFruit, Vegetable, and Cereal Science Biotechnology/source
pubdate2007/pubdate
volume1/volume
fpage1/fpage
lpage2/lpage
/bibl
bibl id="B32"
title
pHigh-efficiency transformation of the diploid strawberry (itFragaria vesca/it) for functional genomics/p
/title
aug
au
snmOosumi/snm
fnmT/fnm
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/au
/aug
sourcePlanta/source
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xrefbib
pubidlist
pubid idtype="pmpid" link="fulltext"16320068/pubid
pubid idtype="doi"10.1007s00425-005-0170-3/pubid
/pubidlist
/xrefbib
/bibl
bibl id="B33"
title
pZeatin overcomes thidiazuron-induced inhibition of shoot elongation and promotes rooting in strawberry culture in vitro/p
/title
aug
au
snmDebnath/snm
fnmSC/fnm
/au
/aug
sourceJ Hortic Sci Biotech/source
pubdate2006/pubdate
volume81/volume
issue3/issue
fpage349/fpage
lpage354/lpage
/bibl
bibl id="B34"
title
pSomaclonal variation in watercress for resistance to crook root disease/p
/title
aug
au
snmArnold/snm
fnmD/fnm
/au
au
snmFlegmann/snm
fnmA/fnm
/au
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snmClarkson/snm
fnmJ/fnm
/au
/aug
sourcePlant Cell Reports/source
pubdate1995/pubdate
volume14/volume
issue4/issue
fpage241/fpage
lpage244/lpage
xrefbib
pubid idtype="doi"10.1007BF00233641/pubid
/xrefbib
/bibl
bibl id="B35"
title
pDevelopment and characterization of polymorphic microsatellite markers from itFragaria viridis/it, a wild diploid strawberry/p
/title
aug
au
snmSargent/snm
fnmDJ/fnm
/au
au
snmHadonou/snm
fnmAM/fnm
/au
au
snmSimpson/snm
fnmDW/fnm
/au
/aug
sourceMol Ecol Notes/source
pubdate2003/pubdate
volume3/volume
issue4/issue
fpage550/fpage
lpage552/lpage
xrefbib
pubid idtype="doi"10.1046j.1471-8286.2003.00507.x/pubid
/xrefbib
/bibl
bibl id="B36"
title
pEffective production of marker-free transgenic strawberry plants using inducible site-specific recombination and a bifunctional selectable marker gene/p
/title
aug
au
snmSchaart/snm
fnmJG/fnm
/au
au
snmKrens/snm
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/au
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snmMendes/snm
fnmO/fnm
/au
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snmRouwendal/snm
fnmGJA/fnm
/au
/aug
sourcePlant Biotechnol J/source
pubdate2004/pubdate
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fpage233/fpage
lpage240/lpage
xrefbib
pubidlist
pubid idtype="pmpid"17147614/pubid
pubid idtype="doi"10.1111j.1467-7652.2004.00067.x/pubid
/pubidlist
/xrefbib
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Abstract
Background
The diploid woodland strawberry (Fragaria vesca) is an attractive system for functional genomics studies. Its small stature, fast regeneration time, efficient transformability and small genome size, together with substantial EST and genomic sequence resources make it an ideal reference plant for Fragaria and other herbaceous perennials. Most importantly, this species shares gene sequence similarity and genomic microcolinearity with other members of the Rosaceae family, including large-statured tree crops (such as apple, peach and cherry), and brambles and roses as well as with the cultivated octoploid strawberry, F. ×ananassa. F. vesca may be used to quickly address questions of gene function relevant to these valuable crop species. Although some F. vesca lines have been shown to be substantially homozygous, in our hands plants in purportedly homozygous populations exhibited a range of morphological and physiological variation, confounding phenotypic analyses. We also found the genotype of a named variety, thought to be well-characterized and even sold commercially, to be in question. An easy to grow, standardized, inbred diploid Fragaria line with documented genotype that is available to all members of the research community will facilitate comparison of results among laboratories and provide the research community with a necessary tool for functionally testing the large amount of sequence data that will soon be available for peach, apple, and strawberry.
Results
A highly inbred line, YW5AF7, of a diploid strawberry Fragaria vesca f. semperflorens line called "Yellow Wonder" (Y2) was developed and examined. Botanical descriptors were assessed for morphological characterization of this genotype. The plant line was found to be rapidly transformable using established techniques and media formulations.
Conclusion
The development of the documented YW5AF7 line provides an important tool for Rosaceae functional genomic analyses. These day-neutral plants have a small genome, a seed to seed cycle of 3.0 3.5 months, and produce fruit in 7.5 cm pots in a growth chamber. YW5AF7 is runnerless and therefore easy to maintain in the greenhouse, forms abundant branch crowns for vegetative propagation, and produces highly aromatic yellow fruit throughout the year in the greenhouse. F. vesca can be transformed with Agrobacterium tumefaciens, making these plants suitable for insertional mutagenesis, RNAi and overexpression studies that can be compared against a stable baseline of phenotypic descriptors and can be readily genetically substantiated.
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Slovin, Janet P
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Title: An inbred line of the diploid strawberry Fragaria vesca f. semperflorens for genomic and molecular genetic studies in the Rosaceae
Series Title: Plant Methods 2009, 5:15
Physical Description: Mixed Material
Creator: Slovin JP
Schmitt K
Folta KM
Publication Date: 40117
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Plant methods
Plant Methods g.oMed Central


Methodology


An inbred line of the diploid strawberry Fragaria vesca f.
semperflorens for genomic and molecular genetic studies in the
Rosaceae
Janet P Slovin*1, Kyle Schmitt2 and Kevin M Folta2


Address: 'Genetic Improvement of Fruits and Vegetables Laboratory, U.S. Department of Agriculture Agricultural Research Service, Henry A
Wallace Beltsville Agricultural Research Center, 10300 Baltimore Avenue, Beltsville, MD 20705, USA and 2Horticultural Sciences Department and
the Graduate Program in Plant Molecular and Cellular Biology, 1301 Fifield Hall, University of Florida, Gainesville, FL 32611, USA
Email: Janet P Slovin* janet.slovin@ars.usda.gov; Kyle Schmitt pelican8@ufl.edu; Kevin M Folta kfolta@ifas.ufl.edu
* Corresponding author



Published: 31 October 2009 Received: 12 September 2008
Plant Methods 2009, 5:15 doi:10. 1186/1746-4811-5-15 Accepted: 31 October 2009
This article is available from: http://www.plantmethods.com/content/5/1/15
2009 Slovin et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.



Abstract
Background: The diploid woodland strawberry (Fragaria vesca) is an attractive system for functional
genomics studies. Its small stature, fast regeneration time, efficient transformability and small genome size,
together with substantial EST and genomic sequence resources make it an ideal reference plant for Fragaria
and other herbaceous perennials. Most importantly, this species shares gene sequence similarity and
genomic microcolinearity with other members of the Rosaceae family, including large-statured tree crops
(such as apple, peach and cherry), and brambles and roses as well as with the cultivated octoploid
strawberry, F. xananassa. F. vesca may be used to quickly address questions of gene function relevant to
these valuable crop species. Although some F. vesca lines have been shown to be substantially homozygous,
in our hands plants in purportedly homozygous populations exhibited a range of morphological and
physiological variation, confounding phenotypic analyses. We also found the genotype of a named variety,
thought to be well-characterized and even sold commercially, to be in question. An easy to grow,
standardized, inbred diploid Fragaria line with documented genotype that is available to all members of the
research community will facilitate comparison of results among laboratories and provide the research
community with a necessary tool for functionally testing the large amount of sequence data that will soon
be available for peach, apple, and strawberry.
Results: A highly inbred line, YW5AF7, of a diploid strawberry Fragaria vesca f. semperflorens line called
"Yellow Wonder" (Y2) was developed and examined. Botanical descriptors were assessed for
morphological characterization of this genotype. The plant line was found to be rapidly transformable using
established techniques and media formulations.
Conclusion: The development of the documented YW5AF7 line provides an important tool for Rosaceae
functional genomic analyses. These day-neutral plants have a small genome, a seed to seed cycle of 3.0 -
3.5 months, and produce fruit in 7.5 cm pots in a growth chamber. YW5AF7 is runnerless and therefore
easy to maintain in the greenhouse, forms abundant branch crowns for vegetative propagation, and
produces highly aromatic yellow fruit throughout the year in the greenhouse. F. vesca can be transformed
with Agrobacterium tumefaciens, making these plants suitable for insertional mutagenesis, RNAi and
overexpression studies that can be compared against a stable baseline of phenotypic descriptors and can
be readily genetically substantiated.


Page 1 of 10
(page number not for citation purposes)


Open Access







http://www.plantmethods.com/content/5 /1/15


Background
The family Rosaceae is comprised of diverse fruit, nut and
ornamental plants. At this time, resources that will accel-
erate research efforts in this important crop family are
being developed [1]. Genomes from three family mem-
bers (peach, apple, and strawberry) are currently being
sequenced and a massive number of transcribed
sequences are being catalogued. While the amount of
structural genomics information is increasing, the ability
to put this information to work in a functional genomics
context has not significantly advanced across rosaceous
species. The growing wealth of genomics-level informa-
tion requires development of agile transformation sys-
tems to enable direct tests of gene function.
Unfortunately, the majority of the valuable crops in this
family are large-architectured tree crops with long juvenil-
ity periods and substantial space requirements. Brambles
and roses are challenging in culture, and require substan-
tial time for regeneration. These characteristics slow the
speed of discovery and greatly decrease the practicality of
gene function studies in these systems. However, analysis
of genome structure and content indicate remarkable sim-
ilarities in protein sequence and colinearity between the
studied members of the family [2], suggesting that gene
regulation and function may be highly translatable
between species.

An excellent candidate system that circumvents many of
these problems is the diploid strawberry (Fragaria vesca L.;
2n = 14). F. vesca possesses many attributes that make it
ideal for genomics, either as a reverse or forward genetic
system or as a rapid means for direct tests of gene function
[3-5]. It is a small plant commonly found along the edges
of woodlands, with a wide distribution throughout
Europe, Asia, and the United States [6]. The wild everbear-
ing, or day-neutral form, F. vesca f. semperflorens, is native
to Europe [6], and produces fruit throughout the year in
the greenhouse. Forms exist that reproduce by seed or
branch crowns only, and there exist forms that are capable
of reproducing vegetatively by runners as well [7]. The
runnerless type, called Bush Alpine or Gaillon strawberry,
tends to bear larger fruit than the runnered type [6]. White
or pink flowered forms, single and double flowered
forms, white or red fruited forms, as well as forms with
three leaflets or one leaflet, have been described by Rich-
ardson [8-11]. Fruit aromas of lab-grown genotypes range
from grape-like to sweet overripe banana and pineapple
(Slovin, personal observation). Importantly, substantial
evidence indicates that F. vesca shares a common ancestor
with at least one of the subgenomes within the commer-
cial octoploid strawberry, F. xananassa [12,13], making
findings in the reference species relevant to the cultivated
germplasm.


There are additional attributes that make F. vesca an attrac-
tive system to answer basic biological questions as well as
solve agriculturally important problems. F. vesca is rapidly
regenerable from tissue culture and can be transformed
using Agrobacterium tumefaciens [14-17]. Each plant pro-
duces many achenes, making it suitable for genetic stud-
ies. The seed to seed cycle of F. vesca is complete in less
than 4 months, and the plant can be grown to seed in a
7.5-10 cm pot in a small greenhouse, or even on a lighted
laboratory shelf. Approximately 1% of the genome
sequence appears in public databases (GenBank numbers
EIU024823-EU024872), the most of any Roscaceae family
member [1]. The genome of these plants is small, approx-
imately 200 Mb [18-20]. The genome of F. vesca line
'Hawaii-4' (accession PI551572) is currently being
sequenced and a substantial body of sequences from tran-
scribed genes of octoploid and diploid Fragaria species is
available.

One issue that made F. vesca less than optimal for genome
function studies is that, although being self-fertile and
therefore likely to be substantially inbred, individual
plants in populations of various lines growing under
essentially uniform conditions in the greenhouse or
growth chamber exhibited substantial phenotypic varia-
bility for certain traits. An example of this is given in Fig-
ure 1, which illustrates the differences observed in young
plants grown from seeds of a single self-pollinated plant
derived from accession Hawaii 4. These natural variations
suggest an underlying level of heterozygosity, and have
the potential to add complexity to downstream genetic
analyses or assessment of gene function, making it diffi-
cult to assess if phenotypes arise from a transgene, genetic
lesion, or genetic variation. The variability we have seen in
this line and in our parental Yellow Wonder line also
complicated interpretation of physiological studies. For


Figure I
A sample of phenotypic variation observed among F.
vesca lines. Thirty-seven F4 inbred plants of accession
Hawaii-4 were grown under uniform greenhouse conditions.
While most plants maintained similar appearance (center)
extreme phenotypes were still observed (left and right).
These plants exhibited substantial differences in runnering
and flowering time.



Page 2 of 10
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Plant Methods 2009, 5:15







http://www.plantmethods.com/content/5 /1/15


this reason, large numbers of plants were needed to
achieve statistical significance when measuring parame-
ters such as crown number, seedling root length and
branching, or flowering time and number (J. Slovin,
unpublished).

A solution to these problems would be to develop an eas-
ily grown, genetically uniform line with a documented
homozygous genotype. Inbred lines can serve as standard
genotypes for studies of gene action and biochemical
pathways [21]. In our own laboratories, assessment of
traits such as tolerance to abiotic stresses using measure-
ments of root growth for example, would benefit from
such a genetically and phenotypically homogeneous start-
ing population. Documented inbred Fragaria lines will
facilitate comparisons between experiments and among
laboratories using T-DNA insertion mutants or overex-
pression studies to test gene function in strawberry, and
perhaps more widely in the family Rosaceae.

The inbred line ofF. vesca f. semperflorens var. Yellow Won-
der described herein, YW5AF7, was developed at Belts-
ville, MD to facilitate such gene function studies in the
genus Fragaria. We chose to start with commercially avail-
able seed called "Yellow Wonder" because these plants are
day neutral, do not runner, and have yellow fruit color.
These three traits have been analyzed genetically in F.
vesca and shown to be encoded by recessive genes [22-24],
and efforts were being made to clone the responsible
genes [23,24]. Seed designated as "Yellow Wonder" is
available from several commercial sources and is listed by
the United States National Clonal Germplasm Repository
http://www.ars.usda.gov/Main/docs.htm?docid= 11324
as PI 551827. PI 551827 is listed as being of uncertain
pedigree and not available commercially in the United
States. "Yellow Wonder" obtained from the Burpee Seed
Company was used in a study to identify the yellow fruit
color locus [25]. The Yellow Wonder seed used in this
project for generating YW5AF7 was part of the seed collec-
tion at the USDA in Beltsville, MD.

The need for a standardized line that can be used by all
laboratories for gene function studies with confidence in
its genotype can clearly be seen in Figure 2, which shows
a comparison of two lines designated "Yellow Wonder"
from different sources. PCR products from the Burpee
"Yellow Wonder" line (YW1) used by Deng and Davis
[25] are clearly different from the product obtained with
DNA from the "Yellow Wonder" line (YW2) used for gen-
erating YW5AF7. Also shown for comparison in this figure
are the products obtained with DNA from a different F.
vesca subspecies (Pawt), and a different diploid Fragaria
species, F. iinumae (J-17). The region amplified is the
intron in a mitochondrial low molecular weight heat
shock protein identified from our heat-treated "Yellow


M -OV I


Figure 2
F. vesca lines called "Yellow Wonder" may not have
the same genotype. Amplification of a region of a gene
encoding a mitochondrial low molecular weight heat shock
protein shows differences between "Yellow Wonder" plants
from two different sources (YI and Y2). Primers were
designed to amplify a region containing an intron, and reveal
polymorphisms between YI and Y2, as well as between sub-
species of F. vesca (Y I, Y2 and Pawt) and between different
diploid Fragaria species, F. vesca and F. iinumae (J- 17). The
same primers were used to amplify this region from a heat
treated "Yellow Wonder" (Y2) seedling cDNA library
(cDNA). bp: size markers in base pairs. M: size ladder.


Wonder" seedling cDNA library. With the resolution of
one molecular character it is apparent that not all "Yellow
Wonder" accessions are equivalent.

The advanced inbred diploid genotype, YW5AF7, pro-
vides a tool for direct tests of gene function in strawberry
and other members of the Rosaceae family that can be
used with confidence by all members of the research com-
munity. In this report we present information about the
background of the accession, assessment of horticultural
traits, and protocols for transformation and regeneration.

Results
Technical Description
Plants
At 23 C, YW5AF7 seedlings in 10 cm. pots will flower by
8 weeks after sowing. Four weeks later the achenes can be
harvested and sown to start the next generation, even
though the berry they grew on may not be completely ripe
and the achenes may be slightly green. By twenty weeks,
numerous berries are present and ripe (Figure 3). Six-
month-old greenhouse grown plants in 15 cm pots aver-
age 25 cm in height and can be 35 cm across. Plants pro-
duce large numbers of branch crowns and can fill a 15 cm
pot within 8 months when supplied with fertilizer
biweekly.


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Figure 3
The YWSAF7 plant. The image shows a mature YW5AF7
plant with flowers and fruit in a 10 cm pot.



The leaves of YW5AF7 are thin, and show typical mor-
phology for F. vesca. The leaf is light medium green in
color, with both sides pubescent. Stomates are found on
the abaxial side only. The first two true leaves are unifoli-
ate, round and serrated. Later leaves are trifoliate,
although very small ectopic highly serrated unifoliate
leaves are also sometimes found at the base of the plant.
Under greenhouse conditions in a 15 cm pot, the largest
leaves can reach 13 cm in width and 8 cm in length. The
terminal leaflet is ovate and more rounded than found on
F. vesca var. Ruegen grown under the same conditions. It
is serrated, with an average of 19 serrations on the largest
terminal leaflets, whereas the margins of the same size ter-
minal leaflet of the octoploid F. xananassa var. Chandler
has about 25 serrations. Lateral leaflets of the largest
leaves of YW5AF7 average 17 serrations. Serrations begin
about halfway up the inner edge of the lateral leaflets. The
interveinal lamina are crinkled. Petioles are long and have
a distinct adaxial groove. They are red in low light and
tend to be greener toward the leaf. Petioles are pubescent,
with straight, unbranched hairs. Stipules are red.

Flowers and fruit are borne within the leaf canopy as well
as on inflorescences that extend above the canopy. Occa-
sionally these extend down over the sides of the pots
because of the weight of the fruit. In 15 cm pots, pedicels
of the first inflorescence can be 15-20 cm long. Pedicels
are round, pubescent, and tend to be greener than the pet-
ioles. Inflorescences usually have 4 to 5 flowers, however,
under some conditions the cymous inflorescence contin-
ues to branch and form additional flowers. Root initials
sometimes form at the nodes and these will form roots if


pegged to the soil. On very old, pot-bound plants, inflo-
rescences with only one or two flowers become common.

The flower is also typical ofF. vesca. It usually has five pet-
als, a calyx consisting of 2 whorls of five sepals, and 20 sta-
mens. In the center of the flower is a rounded receptacle,
bearing yellow pistils, that extends well beyond the sta-
mens when the flower bud opens and the anthers dehisce.
Occasionally flowers have extra flower parts, the most
obvious of these occurrences being 6 petals per flower. In
addition, petaloid anthers have been observed. Both con-
ditions are also seen in the parental generations, and the
appearance is correlated with larger flowers. Primary flow-
ers are usually 1.5-2 cm in diameter, depending on growth
conditions. Secondary and later flowers tend to be
smaller.

Fruit
Like its progenitor, Yellow Wonder, the berry of YW5AF7
is soft and pale yellow in color with tan achenes when
ripe, and pale green to white during development. Ripe
berries are highly aromatic with sweet banana and pine-
apple overtones. When all achenes are fertilized, the berry
shape is long conic, with some primary berries being
necked long conic and reaching 27 mm in length and 20
mm in width in plants growing in 15 cm pots. The average
fresh weight of a primary berry was 1.67 + 0.26 S.E. g.,
with the largest berry being 2.45 g. Achenes are borne on
the surface of the berry, with an average 193 17 S.E.
achenes per primary berry (225 on the largest). In com-
parison, an average of 518 achenes per primary fruit was
reported for a commercial octoploid variety [26]. In the
absence of insects or human interventions, only about
half of the achenes are fertilized and enlarge, and the fruit
tend to be smaller and of varied shapes. Pollination can be
aided by transferring pollen from a flower with dehiscing
anthers to a just opened flower using a small camel hair
brush. The extent of fertilized ovules per fruit can be
approximated over time by examining the expansion of
developing achenes and the subtending receptacle tissue.

Performance
Seeds of YW5AF7 will germinate in soil in the greenhouse
in one week. However, more uniform germination can be
achieved by cold treatment of moist seed. Seeds of
YW5AF7 in moistened soil did not achieve maximum ger-
mination [87%, n = 100 (10 pots with 10 seed each)] until
21 days after sowing. Following treatment of moist seed
for 3 weeks in the dark at 5 C, 74% of YW5AF7 seeds ger-
minated in 7 days after being brought into the green-
house, and by 14 days, maximum germination, 91%, was
achieved. Seeds that have been disinfested using ethanol
and bleach treatment will germinate in Petri dishes on
0.5x MS media [27] solidified with 0.8% Phytagar (Invit-
rogen, Carlsbad, CA). The resulting seedlings can be used


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as aseptic explants for tissue culture. Under these condi-
tions we have found that longer cold treatment (>3
weeks) results in more uniform germination, which can
be critical for evaluating developmental or physiological
parameters of transformed plants in gene function tests.

Mildew susceptibility in F. vesca was found to be due to
two dominant genes [28]. Both genes had to be absent to
obtain a resistant plant, and cytoplasmic effects were
noted. Seedlings of YW5AF7 are susceptible to powdery
mildew at early stages in development, particularly in a
growth chamber environment. In the greenhouse,
YW5AF7 has been found to be susceptible to powdery
mildew, thrips, two spotted mites, and aphids.

Transformation and Regeneration
While YW5AF7 is a strong candidate for genomics studies,
it was important to test if it could be successfully trans-
formed and regenerated. While diploid strawberry is rou-
tinely transformed, transformation and regeneration
efficiency are highly genotype specific [reviewed in [29]].
To test the YW5AF7 line for the ability to produce shoots
after gene insertion, explants from greenhouse-grown
YW5AF7 plants were co-cultivated with Agrobacterium
tumefaciens carrying a visible GFP reporter as described in
Materials and Methods. Several published media formula-
tions were evaluated for regeneration and are detailed in
Table 1.

Callusing was observed on all media types tested, but tis-
sue vigor and regeneration were best supported by the for-
mulation presented in Zhao et al. [30] (Figure 4,
triangles). On this formulation, 20% of explants pos-
sessed shoots by six weeks whereas explants on other
media formulations exhibited little or no organogenesis at
this time point (Figure 4, black or white circles). At nine
weeks, over 80 percent of explants had shoots on the Zhao
formula, whereas shoot initiation on other media types
was less frequent. Figure 5 shows that the medium pro-
ducing the highest percentage of explants exhibiting orga-
nogenesis also resulted in a higher number of shoots per
explant by nine weeks in culture than other formulations.

These data indicate that the formulation by Zhao et al.
[30] results in the highest number of explants exhibiting
shoots, which is important for maximizing the number of
independent transformation events in gene function

Table I: The media formulations used in regeneration experiments.


experiments. Shoots were generated by direct organogen-
esis and were produced most quickly and abundantly on
the basipetal end of petiole segments (as shown in Figure
6A). Evaluation of GFP fluorescence in emerging shoots
revealed that about 40% of shoots were transformed, indi-
cating that escapes can be present using 4 mg/L hygromy-
cin for selection.

Once differentiated, explants were moved to a media
without TDZ to enhance shoot elongation. Although not
formally quantified, at least one insertion event was
observed on each explant, as evidenced by GFP fluores-
cence. Figure 6 shows germinating seed and two resulting
seedlings from one such plant. In this random sample of
seeds, the ratio of GFP positive to GFP negative (wild
type) seeds was essentially 1:1, indicating that there was
most likely a single insertion event in this transformant.

Discussion
F. vesca has great potential as a system to study the genetic
basis of agriculturally important biological questions in
the Rosaceae family. Its small size, rapid growth, generous
seed set, small genome, and sequence availability make it
an excellent resource for development of genomics tools.
Its genomic similarity to other valuable crops underlies its
potential utility as a surrogate to test gene function rele-
vant to many rosaceous species.

A primary concern about the system has been the observa-
tion of variability among individuals in lines that have
not been single seed propagated in the lab through several
generations. Although typically self fertilizing and there-
fore expected to be largely homozygous, we observed clear
variability in a number of horticultural traits among F.
vesca Yellow Wonder and Hawaii-4 plants generated from
achenes from a single fruit suggestive of some degree of
residual heterozygosity. These phenotypic variations are
potentially problematic in a seminal line proposed as a
genomics-friendly genotype. This is an important consid-
eration as several current efforts are developing popula-
tions of T-DNA insertion, activation tagged,
overexpression, or RNAi lines using F. vesca, and it could
become difficult to discriminate between a phenotype
resulting from an engineered genotypic variation and nat-
ural genetic variation in the line. Interpretations from a
genetically noisy background may preclude, or at least
delay, identification of gene-specific effects on morphol-


Concentration


Cytokinin


0.98 uM
0.45 uM
1.50 uM


Concentration


Reference


13.20 uM
4.54 uM
10.00 uM


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Medium


Auxin

IBA
2,4-D
IBA


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90
S80
i 70
S60

40
0
S30


10


35 42 48 57 62
Age (Days)


Figure 4
YW5AF7 regeneration frequency on three published
media formulations. Various explants from YW5AF7,
including mature leaves, young leaves and petioles, were
grown on three different media (Table I) to test regenera-
tion frequency. Medium A, white circles; Medium B, black cir-
cles and Medium C, triangles. The data are the means of two
independent experiments.


ogy and physiology. High variability in results from phys-
iological studies drastically increases the number of plants
that must be used to obtain statistical significance. These
populations may be suboptimal for quantitative studies
of gene expression, as the variation in the baseline may
lead to errors in interpreting microarray, digital, or qRT-
PCR gene expression profiles. Results must be able to be
repeated in other laboratories, so a system based on a
known genetic background will supplement these efforts
and be of benefit to the wider research community.

Our PCR analysis of two different "Yellow Wonder" lines
(Figure 2) indicated that even a well-established and com-
mercially available line ofF. vesca may consist of different
genotypes. Because "Yellow Wonder" is both non-red and
non-runnering it would appear likely, given that these two
loci must be homozygous recessive, that these plants are
already substantially homozygous. However, clearly Y1 is
not the same as Y2. There are no data from any of the sup-
pliers to show that their "Yellow Wonder", the color of
which would be expected to breed true from seed, is the
same as a competitor's, which also would breed true from
seed (at least for color and non-runnering), and no data
to show that any of these are the same as others described
in the literature. For these reasons, the pre-emptive devel-
opment of a stable, highly inbred, prolific and docu-
mented genotype was considered useful, as it would


A


42 48 57 62
Age (Days)

Figure 5
The number of shoots per YW5AF7 explant on three
media formulations. The mean number of shoots per
explant was determined for three different media: Medium A,
white circles, Medium B, black circles and Medium C, trian-
gles. The data reflect the mean of two independent experi-
ments.


provide a stable genotype for evaluation of gene function
that could be shared among users.

Botanical descriptors of YW5AF7 have been carefully eval-
uated and define a reproducible and firm foundation for
later comparisons. Even subtle phenotypes induced by a
transgene should be able to be reliably scored in this sta-
ble background.

Many of the techniques used for studying Arabidopsis can
be used with F. vesca YW5AF7. Seedling variations are
almost indiscernible in populations of Arabidopsis seed-
lings, and their small stature makes in vitro assessment of
phenotypes possible. Tests of early development in
response to environmental conditions, growth regulators
or nutrient status are also possible in F. vesca, much like in
Arabidopsis.

For YW5AF7 to have utility as a functional genomics sys-
tem it must be transformable. As observed by many
groups, transformation efficiency of various strawberry
genotypes is highly genotype dependent and in some
cases impossible [29]. The transformation capacity of
YW5AF7 was tested with a GFP reporter gene. Many GFP
foci were observed in co-cultivated tissues and GFP-posi-


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Figure 6
YW5AF7 transformants. A. A cluster of shoots emerging from the basipetal end of a petiole. Both transformed (GFP+;
green) and non-transformed shoots (red in color) are present. B. Imbibed seed from one line of GFP expressing transformed
5AF7 plants. GFP positive and GFP negative seeds are present in a 19:20 ratio, indicating that a single insertion is likely. C. A
wild-type seedling (left) and a GFP positive transgenic seedling (right) grown from seed of the plant in (B).


tive plantlets were regenerated on media containing selec-
tive antibiotics. Three published (yet diverse) media
formulations were evaluated for regeneration-inducing
ability. In all cases shoots appeared via organogenesis
with the best results arising from the media formulation
presented in Zhao et al. [30]. A number of shoots were
clearly initiated by 30 days and plantlets could be excised
after 60 days and rooted in rooting media. This time
course is reasonable yet could likely be optimized to
improve the utility of the YW5AF7 system. The high fre-
quency of shoot formation on independent explants
ensures propagation of independent transformants. Anti-
biotic sensitivity was generally consistent with previously-
published reports in strawberry [31,32] but subculture to
progressively higher amounts of antibiotics may be advis-
able as regeneration of non-transformed shoots was
observed using hygromycin at 4 ug/ml.


The most prolific explants were leaf-adjacent petiole seg-
ments, with the first shoots appearing on the basipetal
end of these explants. The most productive formulation
contained thidiazuron, TDZ, as a principle growth regula-
tor, a compound shown to be effective in inducing regen-
eration in a number of other studies. However, consistent
with previous reports [33] growth on TDZ severely
stunted shoot elongation, and increased somaclonal vari-
ation has been observed when this regulator has been
employed [see, [34]]. Once clearly formed, the shooting
explants were transferred to a TDZ-free media formulation
that had been used to regenerate F. vesca accession
Hawaii-4. Within one week the shoots elongated vigor-
ously and could be transferred to rooting media. Other
media formulations also induced shoots, but at a much
slower rate.

The YW5AF7 line is runnerless and this has distinct advan-
tages to its adoption as a functional genomics model.


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Runnerless plants are much easier to maintain in a green-
house as plants may be located in close proximity without
having to continually remove a tangle of runners or
daughter plants that invade neighboring pots, such as is
our experience with F. vesca Hawaii 4 and F. vesca Paw-
tuckaway. In large populations this can be a source of gen-
otype contamination and requires dedication to constant
manual management. On the other hand, one of the
advantages of F. vesca as a functional genomics system is
that plants can be propagated by branch crown divisions
or runners or branch crown division, as well as by seed,
making it possible to vegetatively propagate mutants that
affect flowering or seed set. Although runnerless, the
YW5AF7 line does produce abundant branch crowns.

Seeds of YW5AF7 are available for research purposes from
Dr. J. P. Slovin, USDA-ARS Genetic Improvement of Fruits
and Vegetables Laboratory, Bldg. 010A, 10300 Baltimore
Avenue, Beltsville, MD 20705 (phone: 301/504-5629; e-
mail: slovinj@ars.usda.gov). The seed are produced in the
greenhouse under ambient conditions from F7 plants that
are manually self-pollinated. Seed of this line have been
deposited with the USDA, ARS National Clonal Germ-
plasm Repository, Corvallis, OR (PI 641092).

Conclusion
The highly inbred selection YW5AF7 has been generated
and characterized. A set of botanical descriptors defines a
baseline that may be compared to phenotypes of forward
or reverse genetic mutants as well as overexpression and
RNAi lines. This genotype, associated scored metrics, and
transformation protocol permit the deployment of this
system as a useful tool for the Rosaceae research commu-
nity in the elucidation of gene function in a stable and
consistent genetic background that complements existing
systems.

Methods
Plant Origin and Seedling Selection
The inbred line, designated YW5AF7, was obtained by
manually self-pollinating F. vesca var. "Yellow Wonder"
plants grown from seeds in the Beltsville collection main-
tained originally by S. Hokanson. Seeds were planted in
Metro Mix 510 (Scotts-Sierra Horticultural Products, Mar-
ysville, OH) supplemented with dolomitic lime. Plants
were grown in the greenhouse with supplemental lighting
from sodium halide lamps to give a daylength of at least
12 h. At least one hundred seed were planted at each gen-
eration. Flowers were self-pollinated by gentle brushing
starting when their flowers just opened, and every day
thereafter as their anthers dehisced until the flower petals
fell. A new small camel hair artist's brush was used for
each pollination. Flowers were tagged for the day of polli-
nation. The plant to be used for the next generation was
randomly chosen from ten plants that met a basic require-


ment of early flowering, general robust appearance, fruit
set (as based on number of achenes that enlarge following
pollination), high number of achenes on the primary
fruit, and short number of days to mature fruit. Although
germination rate and number of days to flowering varied,
the other selecting parameters usually showed very little
difference. However, some seedlings germinated from F3
seed showed morphological abnormalities such as dwarf-
ness or a single cotyledon. Only seedlings with normal
phenotypes were selected for further selfing. The selfing
process was continued for seven generations to generate
YW5AF7.

Media Preparation
Three previously defined media formulations were inves-
tigated to determine which would lead to optimal regen-
eration of YW5AF7 explants. Media included lx
Murashige and Skoog medium with vitamins, 2% sucrose
and the growth regulators presented in Table 1. Media
were prepared with deionized water, the pH adjusted to
5.6-5.8, and then autoclaved for 20 min at 121 C and 15
psi. Growth regulators were co-autoclaved with the media.
In all three media types, the selection agent used was 4
mg/L hygromycin B, which was added to the media after
it was cooled to ~50C.

Agrobacterium-mediated transformation
Leaf, stem, and petiole segments were sterilized in 70%
EtOH for 30 seconds and 1% sodium hypochlorite (20%
bleach) for 10 min. A single transformed Agrobacterium
colony carrying a 35S::GFP construct was grown overnight
in Luria Broth with 10 mg/L rifampicin, 50 mg/L gen-
tamicin, and 50 mg/L spectinomycin to an OD600 of 0.5,
then pelleted at 1,000 x g. The bacterial pellet was resus-
pended to 0.1 OD600 in co-cultivation medium consisting
of 1x MS pH 5.8 with 2% sucrose, supplemented with 50
uM acetosyringone. Explants were added to the co-cultiva-
tion medium and incubated 20 min at room temperature,
then blotted dry with sterile filter paper and transferred to
media without selection for 2 d at 25 C in darkness. After
2 d the explants were washed twice in co-cultivation
medium liquid supplemented with 500 mg/L carbenicil-
lin, followed by 30 min of incubation in fresh wash
media. Explants were again blotted dry and transferred to
solid media with selection.

Transgenic shoots were selected on media containing 4
mg/L hygromycin B. The explants were regenerated under
a 16 h light, 8 h dark photoperiod under cool-white fluo-
rescent lighting. Explants were checked daily for contami-
nation, and subcultured every 2 weeks. When distinct
clumps of shoots were formed the entire clump was trans-
ferred to hormone-free rooting media consisting of 0.5
MS media (pH 5.8), 1% glucose, and 1% phytoagar. Roots



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formed within days to one month and individual plants
could then be dissected from the groups of shoots.

PCR
PCR was performed using the touchdown protocol
described by Sargent et al. [35] in a 20 |il reaction contain-
ing HotStart Taq Master Mix (Qiagen, Valencia, CA), 0.4
|iM each primer, and 1.0 ng genomic DNA. PCR products
were separated by electrophoresis through a 1.5% TAE
agarose gel and visualized by ethidium bromide staining.
Primers were designed to span the intron in the N-termi-
nal domain of a low molecular weight heat shock protein
gene identified as an EST (GenBank accession number
CX661743.1) from a "Yellow Wonder" (Y2) heat-treated
seedling cDNA library. Template DNA for Y1, Y2, F. iinu-
mae J-17, and F. vesca subsp. americana Pawtuckaway was
obtained from 50-100 mg young leaf tissue using a
DNeasy Plant Mini kit (Qiagen). Y1 and F. vesca subsp.
americana Pawtuckaway plants were obtained from T.
Davis (University of New Hampshire) and F. iinumae J-17
plants were obtained from the US National Plant Germ-
plasm collection.

Competing interests
The authors declare that they have no competing interests.

Authors' contributions
JPS conceived of the project, performed all crosses, plant
measurements and PCR, and participated in preparation
of the manuscript. KS performed the co-cultivation and
subculturing of strawberry tissues to test transformation
efficiency. KMF supervised transformation and regenera-
tion and participated in preparation of the manuscript.

Acknowledgements
The authors thank Andrea Murphy and Jeremy Goetz for greenhouse
assistance, Sasha Drost, an Eleanor Roosevelt High School science intern
for technical assistance, Todd Cooke for his help with botanical description,
and anonymous reviewers for comments. This work was performed as part
of NSF project 0701488 (KMF). Kyle Schmitt was supported with funding
from the Howard Hughes Medical Institute "Science for Life" program at
the University of Florida (KMF).

References
I. Shulaev V, Korban SS, Sosinski B, Abbott AG, Aldwinckle HS, Folta
KM, lezzoni A, Main D, Arus P, Dandekar AM, et ao: Multiple mod-
els for Rosaceae genomics. Plant Physiol 2008, 147(3):985-1003.
2. Rousseau-Gueutin M, Lerceteau-Kohler E, Barrot L, Sargent DJ, Mon-
fort A, Simpson D, Aris P, Guerin G, Denoyes-Rothan B: Compar-
ative genetic mapping between octoploid and diploid
Fragaria species reveals a high level of colinearity between
their genomes and the essential disomic behavior of the cul-
tivated octoploid strawberry. Genetics 2008, 179(4):2045-2060.
3. Davis TM, Pollard JE: Fragaria vesca chlorophyll mutants. Hort-
Science 1991, 26(3):31 I.
4. Albani M, Taylor S, Rodriquez Lopez C, Cekic C, AI-Sheikh M, Green-
land A, Wetten A, Wilkinson M, Battey NH: Fragaria vesca, one
way to understand flowering in perennials. Flowering Newsletter
2001, 31:44-48.
5. Slovin J, Rabinowicz PD: Fragaria vesca, a useful tool for
Rosaceae genomics. In 6th North American Strawberry Symposium:


2007 Ventura, CA: American Society for Horticultural Science;
2007:112-117.
6. Darrow GM: The Strawberry. New York: Holt, Rinehart and Win-
ston; 1966.
7. Brown T, Waring PF: The genetical control of flowering and
runnering in varieties of Fragaria vesca. Heredity 1965, 20:651.
8. Richardson CW: A preliminary note on the genetics of Fra-
garia. Journal of Genetics 1914, 3:171-178.
9. Richardson CW: A further note on the genetics of Fragaria.
journal of Genetics 1918, 7:167-170.
10. Richardson CW: Some notes on Fragaria. journal of Genetics 1920,
10:39-46.
II. Richardson CW: Notes on Fragaria. journal of Genetics 1923,
13:147-152.
12. Rousseau-Gueutin M, Gaston A, AYnouche A, AYnouche ML, Olbricht
K, Staudt G, Richard L, Denoyes-Rothan B: Tracking the evolu-
tionary history of polyploidy in Fragaria L. (strawberry): New
insights from phylogenetic analyses of low-copy nuclear
genes. Molecular Phylogenetics and Evolution 2009, 51(3):515-530.
13. Hancock JF: Strawberries. New York: CABI Publishing; 1999.
14. Alsheikh MK, Suso H-P, Robson M, Battey NH, Wetten A: Appro-
priate choice of antibiotic and Agrobacterium strain improves
transformation of antibiotic-sensitive Fragaria vesca and F. v.
semperflorens. Plant Cell Reports 2002, 20(1 2):1173-1180.
15. El Mansouri I, Mercado J, Valpuesta V, Lopez-Aranda J, Pliego-Alfaro
F, Quesada M: Shoot regeneration and Agrobacterium-medi-
ated transformation of Fragaria vesca L. Plant Cell Reports 1996,
15:642-646.
16. Haymes KM, Davis TM: Agrobacterium-mediated transforma-
tion of'Alpine' Fragaria vesca, and transmission of transgenes
to RI progeny. Plant Cell Reports 1998, 17:279-283.
17. Zhao Y, Liu Q, Davis RE: Transgene expression in strawberries
driven by a heterologous phloem-specific promoter. Plant Cell
Reports 2004, 23(4):224-230.
18. Akiyama Y, Yamamoto Y, Ohmido N, Oshima M, Fukui K: Estima-
tion of the nuclear DNA content of strawberries (Fragaria
spp.) compared with Arabidopsis thaliana by using dual-stem
flow cytometry. Cytologia 2001, 66:431-436.
19. Bennett MD, Leitch IJ: Angiosperm DNA C-values Database
(Release 6.0, Oct 2005). 2005 [http://www.kew.org/cvalues/].
20. Bennett MD, Leitch IJ, Price HJ, Johnston JS: Comparisons with
Caenorhabditis (approximately 100 Mb) and Drosophila
(approximately 175 Mb) using flow cytometry show genome
size in Arabidopsis to be approximately 157 Mb and thus
approximately 25% larger than the Arabidopsis genome initi-
ative estimate of approximately 1 25 Mb. Annals of Botany 2003,
91(5):547-557.
21. Tam YY, Slovin JP, Cohen JD: Selection and characterization of
a-methyltryptophan resistant lines of Lemna gibba showing
a rapid rate of indole-3-acetic acid turnover. Plant Physiol 1995,
107:77-85.
22. Brown T, Wareing PF: The genetic control of the everbearing
habit and three other characters in varieties of Fragaria
vesca. Euphytica 1965, 14:97-1 12.
23. Battey NH, LeMiere P, Tehranifar A, Cekic C, Taylor S, Shrives KJ,
Hadley P, Greenland AJ, Darby J, Wilkinson MJ: Genetic and envi-
ronmental control of flowering in strawberry. In Genetic and
Environmental Manipulation of Horticultural Crops Edited by: Cockshull
KE, Gray D, Seymour GB, Thomas B. New York: CABI Publishing;
1998:111-131.
24. Williamson SC, Yu H, Davis TM: Shikamate dehydrogenase
allozymes: inheritance and close linkage to fruit color in the
diploid strawberry. Journal of Heredity 1995, 86(1):74-76.
25. Deng C, Davis TM: Molecular identification of the yellow fruit
color (c) locus in diploid strawberry: a candidate gene
approach. Theor Appl Genet 2001, 103:316-322.
26. Gardner VR: Studies on the nutrition of the strawberry. Mo Agr
Exp StaRes Bul 1923, 57:31.
27. Murashige T, Skoog F: A revised medium for rapid growth and
bioassays with tobacco tissue cultures. Physiologia Plantarum
1962, 72:473-497.
28. Harland SC, King EE: Inheritance of mildew resistance in Fra-
garia with special reference to cytoplasmic effects. Heredity
1957, 11:287.





Page 9 of 10
(page number not for citation purposes)


Plant Methods 2009, 5:15








http://www.plantmethods.com/content/5 /1/15


29. Folta KM, Dhingra A: Transformation of strawberry: The basis
for translational genomics in Rosaceae. In Vitro Cell Dev-PI 2006,
42(6):482-490.
30. Zhao Y, Liu QZ, Davis RE: Transgene expression in strawber-
ries driven by a heterologous phloem-specific promoter.
Plant Cell Reports 2004, 23(4):224-230.
31. Debnath SC, Teixeira da Silva JA: Strawberry culture in vitro:
applications in genetic transformation and biotechnology.
Fruit, Vegetable, and Cereal Science Biotechnology 2007, 1:1-2.
32. Oosumi T, Gruszewski HA, Blischak LA, Baxter Aj, Wadl PA, Shuman
JL, Veilleux RE, Shulaev V: High-efficiency transformation of the
diploid strawberry (Fragaria vesca) for functional genomics.
Plant 2006, 223(6): 1219-1230.
33. Debnath SC: Zeatin overcomes thidiazuron-induced inhibition
of shoot elongation and promotes rooting in strawberry cul-
ture in vitro. j Hortic Sci Biotech 2006, 81(3):349-354.
34. Arnold D, Flegmann A, Clarkson J: Somaclonal variation in
watercress for resistance to crook root disease. Plant Cell
Reports 1995, 14(4):241-244.
35. Sargent DJ, Hadonou AM, Simpson DW: Development and char-
acterization of polymorphic microsatellite markers from
Fragaria viridis, a wild diploid strawberry. Mol Ecol Notes 2003,
3(4):550-552.
36. Schaart JG, Krens FA, Pelgrom KTB, Mendes O, Rouwendal GJA:
Effective production of marker-free transgenic strawberry
plants using inducible site-specific recombination and a
bifunctional selectable marker gene. Plant Biotechnol j 2004,
2(3):233-240.


Page 10 of 10
(page number not for citation purposes)


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