A genetic linkage map of the cultivated strawberry (Fragaria × ananassa) and its comparison to the diploid Fragaria reference map

The cultivated strawberry, Fragaria × ananassa, is the most economically-important soft-fruit species, but few practical molecular tools for the purpose of marker assisted selection currently exist. As a precursor to the development of such tools, a genetic linkage map was developed from a F₁ population comprising 174 seedlings derived from a cross between two F. × ananassa cultivars, ‘Redgauntlet’ × ‘Hapil’. The resultant map is composed of 315 molecular markers—218 microsatellites, 11 gene-specific markers and 86 AFLP and RAPD markers—and spans 3,116 cM. In total, 69 linkage group fragments were recovered, more than the 56 linkage groups expected for the cultivated strawberry, however, all fragments contained a transferable marker that could be associated with one of 56 linkage group scaffolds. The female (Redgauntlet) and male (Hapil) linkage maps are composed, respectively of 170 loci in 32 linkage groups covering 1,675.3 cM and 182 loci in 37 linkage groups covering 1,440.7 cM, with 37 markers common to both maps. The maximum number of markers in one linkage group was 15, the minimum was two. All linkage groups resolved contained at least one transferable marker (SSR or gene-specific) that had been mapped on the diploid Fragaria reference map (FV × FB), and therefore all linkage groups could be identified as homologous to one of the seven diploid Fragaria linkage groups. When marker order was compared to the diploid Fragaria reference map, effectively complete colinearity was observed. However, the occurrence of duplicated loci on homologues of linkage groups FG1 and FG6 provided evidence of a putative chromosomal duplication or translocation event in Fragaria. The development of this linkage map will facilitate the study and dissection of QTL associated with traits of economic importance such as disease resistance and fruit quality, and provides a foundation for the development of markers for the purpose of marker assisted breeding and selection in the cultivated strawberry, F. × ananassa.     

Thermal imaging and carbon isotope composition indicate variation amongst strawberry (Fragaria × ananassa) cultivars in stomatal conductance and water use efficiency

Cultivars with low stomatal conductance (g_s) may show high water use efficiency (WUE) under drought conditions, but under optimal conditions low g_s may result in low vigour. A combination of thermal imaging and carbon isotope composition (δ¹³C) analysis offers potential for screening simultaneously for both high g_s and high WUE. Ten cultivars of strawberry (Fragaria × ananassa Duch.) were grown in well watered or water limited conditions. Thermal images were taken of the plants, with various approaches to determine the optimal protocol for detecting variation in g_s, including use of reference leaves, grids to maintain leaves horizontal, and collection of meteorological data in synchrony with thermal images. δ¹³C of leaves, fruit, and crowns was determined. An index of g_s derived from the temperature of horizontal leaves and the temperature of wet and dry references showed significant differences between cultivars and between well watered and water limited plants, as did g_s estimated from leaf temperature, the temperature of a dry reference, and humidity. Thermal imaging indicated low g_s in ‘Elsanta’ and ‘Totem’ and relatively high g_s in well watered ‘Elvira’, ‘Florence’ and ‘Cambridge Favourite’. δ¹³C of all plant material was higher in water limited than well watered plants and showed significant differences between cultivars. In one experiment leaf δ¹³C indicated lowest WUE in ‘Elvira’ and highest WUE in ‘Totem’. δ¹³C was inversely correlated with an index of g_s derived from thermal imaging. Although the results indicate substantial variation in g_s and WUE between cultivars, generally all cultivars responded to water deficit by lowering g_s and hence increasing WUE.     

In vitro assessment of strawberry (Fragaria × ananassa Duch.) plant responses to water shortage stress under nano-iron application

In Vitro Cellular & Developmental Biology - Plant - Strawberry is a susceptible plant to water stress, and its growth is severely declined under water shortage. Therefore, the aim of this study...     

Jasmonic acid improved in vitro strawberry (Fragaria × ananassa Duch.) resistance to PEG-induced water stress

Plant Cell, Tissue and Organ Culture (PCTOC) - To investigate the influence of jasmonic acid (JA) on morpho-physiological and biochemical characteristics of strawberry cv. Queen Elisa, under in...     

Silver nanoparticles improved explant disinfection,in vitro growth,runner formation and limited ethylene accumulation during micropropagation of strawberry (Fragaria × ananassa)

Plant Cell, Tissue and Organ Culture (PCTOC) - One of the common problems in strawberry (Fragaria?×?ananassa) micropropagation is the vitrification phenomenon (succulent plantlets,...     

Effect of soil cadmium application and pH on growth and cadmium accumulation in roots, leaves and fruit of strawberry plants (Fragaria × ananassa Duch.)

Three strawberry (Fragaria × ananassa Duch.) cultivars Rainier, Totem and Selva were grown under greenhouse conditions in a Parkhill sandy loam soil with a background DTPA-extractable Cd concentration of 0.18 mg kg^-1 and a pH of 5.1. Experimental treatments included combinations of 4 Cd applications (0, 15, 30 and 60 mg Cd kg^-1 soil) applied as CdSO₄ and 2 soil pH values 5.1 and 6.8. Both the application of Cd and pH of the soil significantly affected plant growth, yield and Cd accumulation in plant tissue anf fruit. Although roots accumulated the highest concentrations of Cd of all plant parts investigated, increased soil Cd application reduced leaf weight more than root weight. In general, yield of strawberries was decreased by an increase in amount of soil-applied Cd, however the yield response varied among cultivars. At 60 mg Cd kg^-1 soil, yield of Rainier cultivar was reduced to 17.6% of control plants. Over 90% of total Cd taken up by plants grown in Cd-treated soil accumulated in roots, regardless of the Cd level in the soil. Root Cd concentrations ranged from 2.6 mg kg^-1 (control plants) to 505.7 mg kg^-1 (Totem plants grown in soil at highest Cd and a soil pH 5.1) and were directly related to soil Cd concentrations. Cd translocation from roots to leaves and fruit was very limited, resulting in a maximum Cd concentration in root leaf tissue of 10.2 mg kg^-1. Accumulation of Cd in fruit was found to correlate well with leaf Cd, although even at the highest amount of applied Cd, fruit Cd concentration did not exceed 700 g kg^-1 of fresh weight.Contribution no. 951     

Identification of gibberellins in leaf tissues of day-neutral strawberry (Fragaria × ananassa Duch.) cultivars

The endogenous gibberellins (GAs) in leaf tissues of two day-neutral cultivars (Rapella and Selva) of strawberry (Fragaria × ananassa Duch.) were analysed using combined gas chromatography -- mass spectrometry (GC-MS). Seven of the later members of the 13-hydroxylation GA biosynthetic pathway were identified, by comparison of Kovats retention indices and mass spectral data obtained for methyl ester trimethylsilyl ether derivatives, either with data obtained from authentic compounds or literature values. GA₁, GA₃, GA₈, GA₁₇, GA₁₉, GA₂₀ and GA₂₉ were detected in extracts of both cultivars.     

Tissue-specific expression of the β-glucuronidase reporter gene in transgenic strawberry (Fragaria × ananassa) plants

The strawberry ( Fragaria spp) is regarded as a false fruit because it originates from the receptacle, which is a non-ovarian tissue. For this reason, fruit-specific promoters isolated from plant species in which the fruit is derived from the ovary wall might not be suited to control gene expression in a fruit-specific way in strawberry. In order to achieve (false) fruit-specific expression in strawberry, we tested the petunia FBP7 (floral binding protein7) promoter, which proved to be active in the receptacles of petunia flowers, in transgenic strawberry fruits. In strawberry plants containing the FBP7 promoter fused to the ß-glucuronidase (GUS) reporter gene ( gus), GUS activity was found in floral and fruit tissues of all developmental stages tested but not in leaf, petiole and root tissue . Surprisingly, Northern blot analysis showed the presence of gus-derived mRNAs in root (strong) and petiole (weak) tissue of fbp7- gus plants in addition to the floral and fruit tissues. Therefore, it is concluded that the histological GUS phenotype does not necessarily correspond with expression at the mRNA level. mRNA quantification using the TaqMan polymerase chain reaction technology confirmed the Northern results and showed that in red strawberry tissue the cauliflower mosaic virus 35S promoter is at least sixfold stronger than the FBP7 promoter.     

The influence of photoperiodic growth condition on isolation of RNA from strawberry (Fragaria × ananassa duch.) tissue

Purification of high-quality RNA from different strawberry tissues is often affected by the presence of high levels of contamination by polysaccharides and phenolic compounds. With the protocol detailed here we describe for the first time total RNA purification from petiole tissue. Treating the plants used as source of material with short-day light regime prior the extraction we are able to obtain RNA suitable for further applications such as in vitro translation, RT-PCR, and RNA blot analysis. The yield of total RNA extraction is significantly enhanced when tissue from plants grown under short-day photoperiodic condition is used compared with that taken from plants grown under long day photoperiod.     

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

Background

A high-throughput genotyping platform is needed to enable marker-assisted breeding in the allo-octoploid cultivated strawberry Fragaria × ananassa. Short-read sequences from one diploid and 19 octoploid accessions were aligned to the diploid Fragaria vesca ‘Hawaii 4’ reference genome to identify single nucleotide polymorphisms (SNPs) and indels for incorporation into a 90 K Affymetrix® Axiom® array. We report the development and preliminary evaluation of this array.

Results

About 36 million sequence variants were identified in a 19 member, octoploid germplasm panel. Strategies and filtering pipelines were developed to identify and incorporate markers of several types: di-allelic SNPs (66.6%), multi-allelic SNPs (1.8%), indels (10.1%), and ploidy-reducing “haploSNPs” (11.7%). The remaining SNPs included those discovered in the diploid progenitor F. iinumae (3.9%), and speculative “codon-based” SNPs (5.9%). In genotyping 306 octoploid accessions, SNPs were assigned to six classes with Affymetrix’s “SNPolisher” R package. The highest quality classes, PolyHigh Resolution (PHR), No Minor Homozygote (NMH), and Off-Target Variant (OTV) comprised 25%, 38%, and 1% of array markers, respectively. These markers were suitable for genetic studies as demonstrated in the full-sib family ‘Holiday’ × ‘Korona’ with the generation of a genetic linkage map consisting of 6,594 PHR SNPs evenly distributed across 28 chromosomes with an average density of approximately one marker per 0.5 cM, thus exceeding our goal of one marker per cM.

Conclusions

The Affymetrix IStraw90 Axiom array is the first high-throughput genotyping platform for cultivated strawberry and is commercially available to the worldwide scientific community. The array’s high success rate is likely driven by the presence of naturally occurring variation in ploidy level within the nominally octoploid genome, and by effectiveness of the employed array design and ploidy-reducing strategies. This array enables genetic analyses including generation of high-density linkage maps, identification of quantitative trait loci for economically important traits, and genome-wide association studies, thus providing a basis for marker-assisted breeding in this high value crop.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1310-1) contains supplementary material, which is available to authorized users.     

Identification of gibberellins in leaf tissues of strawberry (Fragaria × ananassa Duch.) grown under different photoperiods

In studies of the effect of long or short-day photoperiod treatments on the qualitative gibberellin (GA) content of mature leaves of a facultative short-day (SD) strawberry cultivar (Fragaria × ananassa Duch. cv. Elsanta), GA₁, GA₈, GA₁₇, GA₁₉, GA₂₀, GA₂₉ and GA₄₄ were identified by full-scan gas chromatography - mass spectrometry (GC-MS) in extracts from plants grown under long-day (LD) conditions, and GA₁, GA₅, GA₈, GA₁₉, GA₂₀ and GA₂₉ in similar extracts from plants subjected to eight SD cycles after growth under LD conditions. The early 13-hydroxylation GA biosynthetic pathway thus appeared to predominate, with the apparent absence of GA₅ in LD and of GA₁₇ and GA₄₄ in SD extracts providing evidence of modulation of this pathway by photoperiod. A search, including GC-MS with selected ion monitoring, failed to detect GA₃, or the polyhydroxylated GA₈₅, GA₈₆, GA₈₇ or GA₃₂ for which some extracts were specifically purified.This paper is respectfully dedicated to the memory of Gordon Browning, who died suddenly on the 1st July, 1993. He will be sorely missed, both as a friend and colleague.     

A new economical storage technique for strawberry (Fragaria × ananassa Duch.) in vitro

In Vitro Cellular & Developmental Biology - Plant - Strawberry plants grown in vitro are typically stored and maintained on agar containing Murashige and Skoog (MS) media and sucrose as a...     

In vitro establishment of nitrogen-fixing strawberry (Fragaria × ananassa) via artificial symbiosis with Azomonas insignis

Summary Artificial symbiosis was established between diazotrophic Azomonas insignis and strawberry (Fragaria × ananassa). The partnership was created by in vitro techniques through callus induction and organogenesis. Suitable micropropagation [M3=Murashige and Skoog (1962) (MS) basal medium supplemented with 2.5 μM N⁶-benzyladenine (BA), 0.3 μM gibberellic acid (GA₃), 2.2 μM indole-3-butyric acid (IBA), and 3% sucrose] and plant regeneration [R3=MS mineral salts+555 μM myo-inositol, 1.2 μM thiamine HCl, 4.4 μM BA, 0.5 μM IBA, 0.3 μM α-naphthaleneacetic acid (NAA), 0.5 μM 2,4-dichlorophenoxyacetic acid (2,4-D)] media were developed for the test cultivar Fert?di F5. New shoots containing bacteria were rooted, acclimatized, and planted outdoors. The basis of the partnership during the in vitro phase is the bacterial dependence on the plant metabolic activity, using maltose in the medium as carbon and energy source that can be utilized by the plant cells only. The presence of bacteria in the intercellular spaces of the callus tissues and regenerated plants was proved by re-isolation and microscopic techniques. Nitrogenase activity was also detected in the plant tissues.     

Dynamics of phytohormone and DNA methylation patterns changes during dormancy induction in strawberry (Fragaria?×?ananassa Duch.)

Changes in endogenous phytohormone levels, DNA methylation patterns, and expression levels of related genes during induction of dormancy in two strawberry cultivars, Darselect and All Star, were studied under controlled environmental conditions. At 12°C, regardless of day length, potted, runner-derived plants of both cultivars gradually exhibited morphological traits typical of dormancy after treatment for 8 weeks. These morphological changes were accompanied by a synchronous significant decline in indole-3-acetic acid (IAA) level and increases in abscisic acid (ABA) content and global genomic DNA methylation in young leaves. Exposed at 15°C and a short-day photoperiod, the changes in morphology, phytohormone levels and DNA methylation of both cultivars were similar to those observed at 12°C. Slight but non-significant changes in IAA and ABA levels and genomic DNA methylation occurred in young leaves at both 15°C with long days and 18°C with short days. These results indicated that temperature alone was sufficient to induce strawberry to enter the typical dormant phase, and day length had no impact at 12°C. The higher temperature permissible for dormancy induction in strawberry was 15°C, but at this temperature dormancy induction was modified by day length. The expression patterns of FaPIN1, FaNCED1, FaDRM and FaROS1 were coincident with the changes in phytohormone levels and DNA methylation. Although the two tested cultivars have different temporal responses with the different degree of cold tolerance and depth of dormancy, both the endogenous phytohormone and DNA methylation were changed when induced by external environmental factors.     

Responses of Strawberry (Fragaria × ananassa) to Supplemental UV-B Radiation and Selenium Under Field Conditions

In greenhouse experiments, selenium (Se) has been shown to defend plants against detrimental effects of heavy UV-B radiation stress. The aim of this study was to investigate whether this positive effect can be found in open-field conditions with enhancement of UV-B radiation. In the experiment, conducted with strawberry (Fragaria×ananassa, cultivars “Jonsok” and “Polka”) over two growing seasons, plants were exposed to UV-B radiation (including UV-A) and cultivated without Se or supplied with Se added at two levels (0.1 and 1.0 mg kg⁻¹). The plants were monitored for growth, flavonoids, chlorophyll fluorescence, net photosynthesis as well as tissue and cell structure. Photosystem II was observed to be sensitive to UV-B stress under field conditions. In the leaves, a decrease in F_v/F_m was seen at the end of the growing season, implying a cumulative effect of UV-B stress. Several parameters, especially cell and tissue structures, were affected by UV-B and UV-A treatments, which proves the need for UV-A control in outdoor UV-B supplementation studies. Addition of Se did not ameliorate the harmful effects of UV-B but the lower Se-increment level increased leaf growth. The effects of UV-B and Se differed during the two experimental years, indicating the need to repeat experiments during several growing seasons.     

Improved culture technique for strawberry (Fragaria × ananassa Duch.) protoplasts and the determination of DNA content in protoplast derived plants

A reproducible and efficient protoplast to plant-system has been developed for strawberry. By using young shoots growing on medium supplemented with cytokinin we were able to exclude the detrimental enzyme Pectolyase from our enzyme solution. The more gentle isolation procedure reported in this paper, together with the agarose-embedding system had a pronounced effect on protoplast viability and growth. Plant regeneration was enhanced by using thidiazuron instead of benzyladenine. Flow cytometric measurement of the DNA content in protoplast derived plants revealed that 27 out of 51 plants contained the amount of DNA corresponding to octoploid level. Fifteen plants exhibited chromosome doubling(16×), one was diplodecaploid(12×) and eight plants mixoploid.     

A microsatellite linkage map for the cultivated strawberry (Fragaria?×?ananassa) suggests extensive regions of homozygosity in the genome that may have resulted from breeding and selection

The linkage maps of the cultivated strawberry, Fragaria × ananassa (2n = 8x = 56) that have been reported to date have been developed predominantly from AFLPs, along with supplementation with transferrable microsatellite (SSR) markers. For the investigation of the inheritance of morphological characters in the cultivated strawberry and for the development of tools for marker-assisted breeding and selection, it is desirable to populate maps of the genome with an abundance of transferrable molecular markers such as microsatellites (SSRs) and gene-specific markers. Exploiting the recent release of the genome sequence of the diploid F. vesca, and the publication of an extensive number of polymorphic SSR markers for the genus Fragaria, we have extended the linkage map of the ‘Redgauntlet’ × ‘Hapil’ (RG × H) mapping population to include a further 330 loci, generated from 160 primer pairs, to create a linkage map for F. × ananassa containing 549 loci, 490 of which are transferrable SSR or gene-specific markers. The map covers 2140.3 cM in the expected 28 linkage groups for an integrated map (where one group is composed of two separate male and female maps), which represents an estimated 91% of the cultivated strawberry genome. Despite the relative saturation of the linkage map on the majority of linkage groups, regions of apparent extensive homozygosity were identified in the genomes of ‘Redgauntlet’ and ‘Hapil’ which may be indicative of allele fixation during the breeding and selection of modern F. × ananassa cultivars. The genomes of the octoploid and diploid Fragaria are largely collinear, but through comparison of mapped markers on the RG × H linkage map to their positions on the genome sequence of F. vesca, a number of inversions were identified that may have occurred before the polyploidisation event that led to the evolution of the modern octoploid strawberry species.     

Positive effects of cold pretreatment, iron source, and silver nitrate on anther culture of strawberry (Fragaria × ananassa Duch.)

Androgenesis by anther culture or isolated microspore culture is the most efficient method for haploid production. In this study, the effects of cold pretreatment (4 °C), silver nitrate, and iron source in the medium were investigated on anther culture of five strawberry cultivars (Camarosa, Selva, Pajaro, Paros, and Gaviota) in three independent experiments, and three traits including percentage of androgenic anthers, embryogenesis, and callogenesis were determined. In the first experiment, cold pretreatment of 4 °C for 2 and 3 days produced the highest percentage of androgenic anthers in three cultivars (Camarosa, Selva, and Pajaro) and cold pretreatment of 4 °C for 2 days produced the highest percentage of androgenic anthers in cultivar Paros. In the second experiment, the effect of Fe-EDDHA was more effective than Fe-EDTA, and increased the percentage of androgenic anthers and embryogenesis. The use of 57.5 mg l^?1 Fe-EDDHA produced the highest percentage of androgenic anthers in cultivar Camarosa and the best embryogenesis in cultivars Camarosa and Gaviota. In the third experiment, the use of 15 mg l^?1 silver nitrate in medium significantly increased the percentage of androgenic anthers and embryogenesis in cultivar Camarosa.