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.     

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.     

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 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...     

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.     

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.     

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.     

Physiological and growth responses to water deficits in cultivated strawberry (Fragaria × ananassa) and in one of its progenitors,Fragaria chiloensis

Cultivation of strawberry (Fragaria × ananassa) requires irrigation. Improving crop water use efficiency (WUE) is important for future production. Fragaria chiloensis, a progenitor of cultivated strawberry, grows in sandy soils, and may prove useful in breeding for improved WUE. Little, however, is known about variation in drought tolerance within this species. This research explores drought tolerance in a range of F. chiloensis and F. × ananassa genotypes. Four cultivars of F. × ananassa and four accessions of F. chiloensis were compared when well watered, and when subjected to a water deficit (65% of evapotranspiration). New leaf production, stomatal conductance, and photosynthetic rate were significantly reduced under water deficit, and also significantly differed between genotypes. A significant interaction of genotype and irrigation was found for transpiration rate, leaf area and dry mass, production of runners, predawn water potential, a measure of transpiration efficiency (shoot biomass produced per litre water transpired), and carbon isotope composition, indicating that some genotypes were more severely affected by water deficit than others. The South American F. chiloensis accession ‘Manzanar Alto’ had a similar rate of transpiration to the commercial cultivars, but the remaining (North American) F. chiloensis accessions used far less water than the F. × ananassa. Well-watered F. chiloensis plants used less water than water-limited plants of the F. × ananassa cultivar ‘Florence’. Transpiration efficiency of the F. chiloensis accession ‘BSP14’ was improved by water deficit: this was the only genotype not to show a reduction in leaf area and dry mass under water deficit. Greater drought resistance in three F. chiloensis accessions compared to F. × ananassa results from a conservative vegetative growth strategy, reducing loss of water.     

Induced expression of the Fragaria × ananassa Rapid alkalinization factor-33-like gene decreases anthracnose ontogenic resistance of unripe strawberry fruit stages

Rapid alkalinization factor (RALF) genes encode for ubiquitous small peptides that stimulate apoplastic alkalinization through interaction with malectin-like receptor kinase. RALF peptides may act as negative regulators of plant immune response, inhibiting the formation of the signal receptor complex for immune activation. Recently RALF homologues were identified in different fungal pathogen genomes contributing to host infection ability. Here, FaRALF-33-like gene expression was evaluated in strawberry fruits inoculated with Colletotrichum acutatum, Botrytis cinerea, or Penicillium expansum after 24 and 48 h post-infection. To investigate the role of FaRALF-33-like in strawberry susceptibility, transient transformation was used to overexpress it in white unripe fruits and silence it in red ripe fruits. Agroinfiltrated fruits were inoculated with C. acutatum and expression, and histological analysis of infection were performed. Silencing of FaRALF-33-like expression in C. acutatum-inoculated red fruits led to a delay in fruit colonization by the fungal pathogen, and infected tissues showed less penetrated infective hyphae than in wild-type fruits. In contrast, C. acutatum-inoculated white unripe fruits overexpressing the FaRALF-33-like gene decreased the ontogenic resistance of these fruits, leading to the appearance of disease symptoms and penetrated subcuticular hyphae, normally absent in white unripe fruits. The different response of transfected strawberry fruits to C. acutatum supports the hypothesis that the FaRALF-33-like gene plays an important role in the susceptibility of fruits to the fungal pathogen C. acutatum.     

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.     

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.     

Detachment-accelerated ripening and senescence of strawberry (Fragaria × ananassa Duch. cv. Akihime) fruit and the regulation role of multiple phytohormones

To study the detachment stress on the ripeness of strawberry fruit, physiological characteristics of strawberry fruit on and off plant during ripeness and senescence processes were investigated. The results indicated that the ripeness of strawberry fruit upon detachment was accelerated, in terms of firmness, soluble solid content and especially color development. The color of fruit off plant changed rapidly from white to full red in 1–2 days. The respiratory rate in fruit off plant was strengthened, higher than that on plant. Abscisic acid level and ethylene production in fruit off plant were also higher than those on plant and auxin degradation was exacerbated by detachment. Expression levels of FaMYB1, FabHLH3 and FaTTG1 were generally reduced with phenotypes of redder color and more anthocyanin accumulation in fruit off plant. Results also suggested that the detachment initially stimulated ethylene and abscisic acid production and auxin degradation, which modulated ripening-related gene expression and at last enhanced fruit pigmentation.     

CAPS markers improved by cluster-specific amplification for identification of octoploid strawberry (Fragaria × ananassa Duch.) cultivars, and their disomic inheritance

Cleavage amplified polymorphic sequence (CAPS) markers of strawberry (Fragaria × ananassa Duch.) can be useful for identifying mislabeled or patent-infringing cultivars in the marketplace. However, CAPS markers in octoploid strawberry tend to give unclear bands because multiple homologous sites are simultaneously amplified by the non-selective PCR. To overcome this problem, we used cluster-specific amplification based on the nucleotide sequences of PCR products and were able to improve the band clarity of 18 CAPS markers. By analyzing the marker segregation ratio, we demonstrated that 13 clarified markers were derived from single diploid loci that were transmitted to progeny in a manner consistent with Mendelian inheritance. We discuss the genomic structure of octoploid strawberry from the viewpoint of cluster and segregation analysis and suggest that it comprises independent genomes. We tested the utility of all of the markers we developed for cultivar identification and confirmed their ability to distinguish among 64 strawberry cultivars.     

Reduced clonal reproduction indicates low potential for establishment of hybrids between wild and cultivated strawberries (Fragaria vesca × F. × ananassa)

The genus Fragaria (Rosaceae) contains 24 species, including hybrid species such as the garden strawberry (Fragaria × ananassa Duch.). Natural hybridization between Fragaria species has repeatedly been reported, and studies on the hybridization potential between F. × ananassa and its wild relatives have become increasingly important with the outlook for genetically modified garden strawberries. In Europe, a candidate species for hybridization with garden strawberries is the common woodland strawberry (Fragaria vesca L.). Although a previous field survey indicated that the potential for hybridization between F. vesca and F. × ananassa is low, it is not clear whether the lack of natural hybrids is caused by known pre- and postzygotic barriers, or whether hybrid plants lack the fitness to establish in natural F. vesca populations. We grew different F. vesca and F. vesca × F. × ananassa hybrid clones with and without competition in a greenhouse and assessed biomass production, clonal reproduction, and sexual reproduction of plants. While some hybrid clones exceeded F. vesca in biomass production, general clonal reproduction was much lower and delayed in hybrids. Furthermore, hybrids were sterile. These results demonstrate a mechanism by which the general lack of F. vesca × F. × ananassa hybrids in natural habitats can be explained, in addition to the known low hybridization potential between garden and woodland strawberries. We conclude that hybrids have a competitive disadvantage against co-occurring F. vesca plants due to inferior and delayed clonal reproduction, and that the potential for hybrid establishment under natural conditions is low.     

Seasonal changes in adenine phosphoribosyltransferase and adenosine kinase activities as markers of the dormancy and the growth of Fragaria × ananassa

Because of the potential involvement of adenosine in the winter re-acquisition of nucleotide synthesis capability of strawberry plants (Fragaria × ananassa Duch., Elsanta), the properties and the time-course activity of an adenosine kinase (EC 2.7.1.20) and an adenine phosphoribosyltransferase (APRTase, EC 2.4.2.7) of the adenosine recycling pathway were characterized. The results showed an increase in APRTase activity during winter re-acquisition of nucleotide synthesis capability and an increase in adenosine kinase activity during spring growth of strawberry plants. Western blot analysis, performed with polyclonal rabbit antibodies raised against peach bud adenosine kinase, showed a concomitant rise in the strawberry enzyme. These results suggest that APRTase activity could be a good marker of the break of strawberry plant dormancy and adenosine kinase a marker of subsequent spring growth.     

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     

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...     

Lateral root formation in the strawberry Fragaria × ananassa Duch. revealed by histone H4 in situ hybridisation

Abstract

Our study was carried out in bench rhizotrons using the Camarosa variety of strawberry (Fragaria × ananassa Duch.), by exciding the apex of fast-growing primary roots at two distances (1 or 8 cm) from the apex. It was demonstrated that new lateral meristems were rapidly induced by excision of the root apex at either distance; after 24 h, histone H4 in situ hybridisation detected groups of cell-organising root primordia just a few millimetres below the cut. After a further 24 h, new lateral roots were about to protrude from the original root. Results show that lateral roots can be formed anywhere along the primary roots of strawberry plants from a few stem cells distributed along the pericycle close to the protoxylem arches.