Insights into phylogeny,sex function and age of Fragaria based on whole chloroplast genome sequencing

The cultivated strawberry is one of the youngest domesticated plants, developed in France in the 1700s from chance hybridization between two western hemisphere octoploid species. However, little is known about the evolution of the species that gave rise to this important fruit crop. Phylogenetic analysis of chloroplast genome sequences of 21 Fragaria species and subspecies resolves the western North American diploid F. vesca subsp. bracteata as sister to the clade of octoploid/decaploid species. No extant tetraploids or hexaploids are directly involved in the maternal ancestry of the octoploids.There is strong geographic segregation of chloroplast haplotypes in subsp. bracteata, and the gynodioecious Pacific Coast populations are implicated as both the maternal lineage and the source of male-sterility in the octoploid strawberries. Analysis of sexual system evolution in Fragaria provides evidence that the loss of male and female function can follow polyploidization, but does not seem to be associated with loss of self-incompatibility following genome doubling. Character-state mapping provided insight into sexual system evolution and its association with loss of self-incompatibility and genome doubling/merger. Fragaria attained its circumboreal and amphitropical distribution within the past one to four million years and the rise of the octoploid clade is dated at 0.372–2.05 million years ago.     

A Phylogenetic Analysis of the Genus Fragaria (Strawberry) Using Intron-Containing Sequence from the ADH-1 Gene

The genus Fragaria encompasses species at ploidy levels ranging from diploid to decaploid. The cultivated strawberry, Fragaria×ananassa, and its two immediate progenitors, F. chiloensis and F. virginiana, are octoploids. To elucidate the ancestries of these octoploid species, we performed a phylogenetic analysis using intron-containing sequences of the nuclear ADH-1 gene from 39 germplasm accessions representing nineteen Fragaria species and one outgroup species, Dasiphora fruticosa. All trees from Maximum Parsimony and Maximum Likelihood analyses showed two major clades, Clade A and Clade B. Each of the sampled octoploids contributed alleles to both major clades. All octoploid-derived alleles in Clade A clustered with alleles of diploid F. vesca, with the exception of one octoploid allele that clustered with the alleles of diploid F. mandshurica. All octoploid-derived alleles in clade B clustered with the alleles of only one diploid species, F. iinumae. When gaps encoded as binary characters were included in the Maximum Parsimony analysis, tree resolution was improved with the addition of six nodes, and the bootstrap support was generally higher, rising above the 50% threshold for an additional nine branches. These results, coupled with the congruence of the sequence data and the coded gap data, validate and encourage the employment of sequence sets containing gaps for phylogenetic analysis. Our phylogenetic conclusions, based upon sequence data from the ADH-1 gene located on F. vesca linkage group II, complement and generally agree with those obtained from analyses of protein-encoding genes GBSSI-2 and DHAR located on F. vesca linkage groups V and VII, respectively, but differ from a previous study that utilized rDNA sequences and did not detect the ancestral role of F. iinumae.     

Editor's choice: Dissection of the Octoploid Strawberry Genome by Deep Sequencing of the Genomes of Fragaria Species

Cultivated strawberry (Fragaria x ananassa) is octoploid and shows allogamous behaviour. The present study aims at dissecting this octoploid genome through comparison with its wild relatives, F. iinumae, F. nipponica, F. nubicola, and F. orientalis by de novo whole-genome sequencing on an Illumina and Roche 454 platforms. The total length of the assembled Illumina genome sequences obtained was 698 Mb for F. x ananassa, and ∼200 Mb each for the four wild species. Subsequently, a virtual reference genome termed FANhybrid_r1.2 was constructed by integrating the sequences of the four homoeologous subgenomes of F. x ananassa, from which heterozygous regions in the Roche 454 and Illumina genome sequences were eliminated. The total length of FANhybrid_r1.2 thus created was 173.2 Mb with the N50 length of 5137 bp. The Illumina-assembled genome sequences of F. x ananassa and the four wild species were then mapped onto the reference genome, along with the previously published F. vesca genome sequence to establish the subgenomic structure of F. x ananassa. The strategy adopted in this study has turned out to be successful in dissecting the genome of octoploid F. x ananassa and appears promising when applied to the analysis of other polyploid plant species.     

A new set of polymorphic simple sequence repeat (SSR) markers from a wild strawberry (Fragaria vesca) are transferable to other diploid Fragaria species and to Fragaria × ananassa

A set of 41 polymorphic microsatellite markers were developed using a CT/AG‐enriched genomic library of Fragaria vesca cv. Reine des Vallées. Thirty‐five of them were polymorphic in F. vesca and were tested in one accession each of six additional diploid Fragaria species and the octoploid Fragaria× ananassa. A mean of 5.3 alleles per locus and a low level of observed heterozygosity were generally detected in the 32 single‐locus simple sequence repeats of F. vesca. Most of these loci amplify in the other diploid species and in F. × ananassa.     

Relationship between ploidy level and genome size in strawberries

This study investigated the pattern of variation in nuclear DNA content at different ploidy levels in Fragaria (Strawberry, Rosaceae) using flow cytometry based on mean fluorescent intensity (MFI) reflected by propidium-iodide-stained nuclei. On average, MFI values were 237 for diploids F. vesca, F. viridis, and F. nubicola, 416.5 for tetraploid F. orientalis, 621.5 for hexaploid F. moschata, and 798 for octoploids F. × ananassa, F. virginiana, and F. chiloensis. Within diploids MFI ranged from 225.9 in F. vesca ssp. vesca to 255.4 in F. nubicola, and within octoploids varied from 766 in F. × ananassa to 808 in F. virginiana. The nuclear DNA variation was significant among diploid species (N = 21, P < 0.008), but not across octoploid species (N = 17, P>0.386). MFI values were also variable among different genotypes of a given species though not significant. The values of mean basic genome DNA (MFI divided by ploidy level) were 118.5, 104, 103.5, and 99.8, respectively, for diploids, tetraploid, hexaploid, and octoploid species. This indicates that relative genomic size decreases by increasing ploidy level, and that there is no direct proportional relationship between DNA content and ploidy levels in Fragaria, supporting the idea of genome downsizing during polyploidization in plants.     

EST‐SSR markers from Fragaria vesca L. cv. Yellow Wonder

Fourteen microsatellite primer pairs were developed from a cDNA library of heat‐treated seedlings of Fragaria vesca cv. yellow wonder. Transferability to 13 species of Fragaria ranged from 71% in diploid species F. gracilis Losinsk., F. iinumae Makino, F. nilgerrensis Schltdl. ex J. Gay and F. nipponica Makino, to 100% in octoploid domestic strawberry and its progenitors. Polymorphism was high in polyploid Fragaria species. However, polymorphism and heterozygosity of eight EST‐SSRs (expressed sequence tag–simple sequence repeats) was low in 14 F. vesca genotypes.     

Plastid genomes reveal recurrent formation of allopolyploid Fragaria

Premise of the Study

Recurrent formation of polyploid taxa is a common observation in many plant groups. Haploid, cytoplasmic genomes like the plastid genome can be used to overcome the problem of homeologous genes and recombination in polyploid taxa. Fragaria (Rosaceae) contains several octo‐ and decaploid species. We use plastome sequences to infer the plastid ancestry of these taxa with special focus on the decaploid Fragaria cascadensis.

Methods

We used genome skimming of 96 polyploid Fragaria samples on a single Illumina HiSeq 3000 lane to obtain whole plastome sequences. These sequences were used for phylogenetic reconstructions and dating analyses. Ploidy of all samples was inferred with flow cytometry, and plastid inheritance was examined in a controlled cross of F. cascadensis.

Key Results

The plastid genome phylogeny shows that only the octoploid F. chiloensis is monophyletic, all other polyploid taxa were supported to be para‐ or polyphyletic. The decaploid Fragaria cascadensis has biparental plastid inheritance and four different plastid donors. Diversification of the F. cascadensis clades occurred in the last 230,000 years. The southern part of its distribution range harbors considerably higher genetic diversity, suggestive of a potential refugium.

Conclusions

Fragaria cascadensis had at least four independent origins from parents with different plastomes. In contrast, para‐ and polyphyletic taxa of the octoploid Fragaria species are best explained by incomplete lineage sorting and/or hybridization. Biogeographic patterns in F. cascadensis are probably a result of range shift during the last glacial maximum.     

Metabolite profiling reveals novel multi-level cold responses in the diploid model Fragaria vesca (woodland strawberry)

Winter freezing damage is a crucial factor in overwintering crops such as the octoploid strawberry (Fragaria × ananassa Duch.) when grown in a perennial cultivation system. Our study aimed at assessing metabolic processes and regulatory mechanisms in the close-related diploid model woodland strawberry (Fragaria vesca L.) during a 10-days cold acclimation experiment. Based on gas chromatography/time-of-flight-mass spectrometry (GC/TOF-MS) metabolite profiling of three F. vesca genotypes, clear distinctions could be made between leaves and non-photosynthesizing roots, underscoring the evolvement of organ-dependent cold acclimation strategies. Carbohydrate and amino acid metabolism, photosynthetic acclimation, and antioxidant and detoxification systems (ascorbate pathway) were strongly affected. Metabolic changes in F. vesca included the strong modulation of central metabolism, and induction of osmotically-active sugars (fructose, glucose), amino acids (aspartic acid), and amines (putrescine). In contrast, a distinct impact on the amino acid proline, known to be cold-induced in other plant systems, was conspicuously absent. Levels of galactinol and raffinose, key metabolites of the cold-inducible raffinose pathway, were drastically enhanced in both leaves and roots throughout the cold acclimation period of 10 days. Furthermore, initial freezing tests and multifaceted GC/TOF-MS data processing (Venn diagrams, independent component analysis, hierarchical clustering) showed that changes in metabolite pools of cold-acclimated F. vesca were clearly influenced by genotype.     

Development and characterization of polymorphic microsatellite markers from Fragaria viridis,a wild diploid strawberry

To date, the development of microsatellite (SSR) markers in the genus Fragaria has focused on F. vesca. However, further species are thought to have contributed to the complex allo‐octoploid genome of the cultivated strawberry, F.×ananassa. Here, we present 22 new SSR markers developed from the diploid species F. viridis. Twenty‐one of the primer pairs amplified polymorphisms in six F. viridis accessions, with an average of 4.95 alleles per primer pair and an average expected heterozygosity of 0.68. Fourteen of these primer pairs, and a locus monomorphic in F. viridis, amplified polymorphic alleles in the parents of a F. vesca mapping population.     

TDZ, auxin and genotype effects on leaf organogenesis in Fragaria

The different types of organogenic (roots and adventitious shoots) and callus formation responses of leaves from 30-day-old proliferating shoots of different Fragaria spp. genotypes were studied in response to MS medium supplemented with 4.54 μM 1-phenyl-3-(1,2,3-thiadiazol-5-yl) urea (thidiazuron; TDZ) alone and in combination with 0.98 μM indole-3-butyric acid (IBA), 0.84 μM3-benzo[b]selenienyl acetic acid (BSAA) or 0.90 μM2,4-dichlorophenoxy acetic acid (2,4-D). The study included: nine octoploid Fragaria x ananassa cultivars and breeding selections; two octoploid breeding selections from F. virginiana glauca inter-species crosses; two diploid F. vesca cultivars; and one diploid clone of F. nubicola Lindl. TDZ plus IBA promoted the highest shoot regeneration efficiencies from leaves of nearly all of the genotypes, while the TDZ/BSAA and TDZ/2,4-D combinations promoted high regeneration efficiencies for only some of the genotypes (Alpina W.O., Sveva, AN 91.371.53, Onda, Paros and FO93.143.5). For the more efficient regenerating genotypes, IBA induced the highest frequency of regenerating leaves, while BSAA induced the highest number of regenerated shoots from leaves and more callus production for most of the genotypes.     

Solitary bees – Potential vectors for gene flow from cultivated to wild strawberries

The genus Fragaria (Rosaceae) contains 24 plant species, including hybrid species such as the octoploid garden strawberry (F. × ananassa). Natural hybridization between Fragaria species has repeatedly been reported, and the potential future cultivation of genetically modified strawberries has made the study of hybridization potential between F. × ananassa and its wild relatives increasingly important. In Europe, F. × ananassa is the only octoploid species present, and the most likely candidate for hybridization is the common diploid woodland strawberry (F. vesca). To date, it is unknown whether pollinator spectra of the two Fragaria species overlap and thus might promote interspecific gene flow. We carried out a survey of flower visitors in northwestern Switzerland to identify major flower visitors of F. vesca and F. × ananassa. This survey indicated that wild bees are the most important shared flower visitors of F. × ananassa and F. vesca. Therefore, we studied flower choice behavior of the common wild bee Osmia bicornis in a greenhouse experiment. Osmia bicornis did not discriminate between F. × ananassa and F. vesca flowers. We conclude that wild bees are important shared flower visitors of both F. × ananassa and F. vesca and are potential vectors for gene flow between cultivated and wild strawberries.     

Dihydroflavonol 4-Reductase Genes Encode Enzymes with Contrasting Substrate Specificity and Show Divergent Gene Expression Profiles in Fragaria Species

During fruit ripening, strawberries show distinct changes in the flavonoid classes that accumulate, switching from the formation of flavan 3-ols and flavonols in unripe fruits to the accumulation of anthocyanins in the ripe fruits. In the common garden strawberry (Fragaria×ananassa) this is accompanied by a distinct switch in the pattern of hydroxylation demonstrated by the almost exclusive accumulation of pelargonidin based pigments. In Fragaria vesca the proportion of anthocyanins showing one (pelargonidin) and two (cyanidin) hydroxyl groups within the B-ring is almost equal. We isolated two dihydroflavonol 4-reductase (DFR) cDNA clones from strawberry fruits, which show 82% sequence similarity. The encoded enzymes revealed a high variability in substrate specificity. One enzyme variant did not accept DHK (with one hydroxyl group present in the B-ring), whereas the other strongly preferred DHK as a substrate. This appears to be an uncharacterized DFR variant with novel substrate specificity. Both DFRs were expressed in the receptacle and the achenes of both Fragaria species and the DFR2 expression profile showed a pronounced dependence on fruit development, whereas DFR1 expression remained relatively stable. There were, however, significant differences in their relative rates of expression. The DFR1/DFR2 expression ratio was much higher in the Fragaria×ananassa and enzyme preparations from F.×ananassa receptacles showed higher capability to convert DHK than preparations from F. vesca. Anthocyanin concentrations in the F.×ananassa cultivar were more than twofold higher and the cyanidin:pelargonidin ratio was only 0.05 compared to 0.51 in the F. vesca cultivar. The differences in the fruit colour of the two Fragaria species can be explained by the higher expression of DFR1 in F.×ananassa as compared to F. vesca, a higher enzyme efficiency (K _cat/K _m values) of DFR1 combined with the loss of F3’H activity late in fruit development of F.×ananassa.     

A reliable multiplexed microsatellite set for genotyping <Emphasis Type="Italic">Fragaria</Emphasis> and its use in a survey of 60 <Emphasis Type="Italic">F</Emphasis>. × <Emphasis Type="Italic">ananassa</Emphasis> cultivars

We have identified a reliable set of multiplexed microsatellite (SSR) markers for the genotyping of strawberry cultivars and their octoploid progenitors. Over 100 SSRs were screened in two F. × ananassa genotypes and from these, 32 that showed promise for genotyping were selected for further analysis. These SSRs were used to screen a set of 16 strawberry cultivars and a set of fingerprints were produced. Those SSRs that produced reliable, reproducible and easy to interpret fingerprints, that could also distinguish readily between the 16 strawberry cultivars screened, and which could be conveniently included in three multiplex reactions, were selected to form the genotyping set. The genotyping set, consisting of 10 previously-reported SSRs was used to fingerprint a total of 56 cultivated strawberry, and four octoploid Fragaria species accessions. The SSRs used could reliably distinguish between all 60 genotypes surveyed, including sibling cultivars derived from the same parental lines. The primers could be combined for multiplex PCR and represent a useful and convenient genotyping set for Fragaria that will permit fingerprinting data to be shared between laboratories.     

Expression of genes involved in the anthocyanin biosynthesis pathway in white and red fruits of Fragaria pentaphylla and genetic variation in the dihydroflavonol-4-reductase gene

Structural and regulatory genes control fruit colors in plants. Real-time quantitative PCR results showed significantly higher expression levels of structural genes (FpCHS, FpDFR, FpANS, and FpUFGT) as well as of the regulatory gene MYB10 in red fruits of Fragaria pentaphylla compared to white fruits. These genes were strongly associated with anthocyanin accumulation within fruits. The full-length sequence of the FpDFR gene in red fruits of F. pentaphylla had a length of 2080 bp, was separated by five introns, and shared 95% homology with the F. vesca DFR sequence. Twenty-seven SNPs were detected in the FpDFR gDNA sequences between red and white fruits. Among these, transition substitutions were more frequent than transversions (66.7% vs. 33.3%), and a larger number of nucleotide variants existed in introns compared to exons (70.4% vs. 29.6%). A Chi-square test showed only three SNPs significantly associated with fruit color. Combined with structural analyses of the FpDFR protein and an expression analysis of the anthocyanin pathway genes, these results indicate that trans-regulation might contribute to color control in F. pentaphylla.     

High variability and disomic segregation of microsatellites in the octoploid Fragaria virginiana Mill. (Rosaceae)

The objectives of the present study were to develop microsatellite markers for the wild strawberry, Fragaria virginiana, to evaluate segregation patterns of microsatellite alleles in this octoploid species, and assess genetic variability at microsatellite loci in a wild population. A genomic library was screened for microsatellite repeats and several PCR primers were designed and tested. We also tested the use of heterologous primers and found that F. virginiana primers amplified products in cultivated strawberry, Fragaria × ananassa Duch. and Fragaria chiloensis. Similarly, microsatellite loci developed from cultivated strawberry also successfully amplified F. virginiana loci. We investigated four microsatellite loci in detail, three developed from F. virginiana and one from cultivated strawberry. A survey of 100 individuals from a population of F. virginiana in Pennsylvania demonstrated high heterozygosities (H_e or gene diversity ranged from 0.80 to 0.88 per locus) and allelic diversity (12–17 alleles per locus), but individual plants had no more than two alleles per locus. Segregation patterns in parents and progeny of two controlled crosses at these four loci were consistent with disomic Mendelian inheritance. Together these findings suggest that the genome of F. virginiana is "highly diploidized" and at least a subset of microsatellite loci can be treated as codominant, diploid markers. Significant heterozygote deficiencies were found at three of the four loci for hermaphroditic individuals but for only one locus among females in this gynodioecious species.Communicated by J. Dvorak     

Isolation and characterization of microsatellite loci in Fragaria

This study reports the development and characterization of 20 microsatellite primer pairs in wild strawberry Fragaria vesca. One hundred primers were obtained from an AC‐enriched library developed in the cultivar ‘Ilaria’. A set of eight F. vesca genotypes was used to detect the polymorphism resulting in an average of 7.0 alleles, an average observed heterozygosity of 0.32 and an average expected heterozygosity of 0.73. Nineteen (95%) of the primers also amplified the cultivated octoploid strawberry Fragaria×ananassa.     

Tracking the evolutionary history of polyploidy in Fragaria L. (strawberry): New insights from phylogenetic analyses of low-copy nuclear genes

Phylogenetic utility of two nuclear genes (GBSSI-2 and DHAR) was explored in genus Fragaria in order to clarify phylogenetic relationships among taxa and to elucidate the origin of the polyploid species. Orthology of the amplified products was assessed by several methods. Our results strongly suggest the loss of one GBSSI duplicated copy (GBSSI-1) in the Fragariinae subtribe. Phylogenetic analyses provided new insights into the evolutionary history of Fragaria, such as evidence supporting the presence of three main diploid genomic pools in the genus and demonstrating the occurrence of independent events of polyploidisation. In addition, the data provide evidence supporting an allopolyploid origin of the hexaploid F. moschata, and the octoploids F. chiloensis, F. iturupensis and F. virginiana. Accordingly, a new pattern summarizing our present knowledge on the Fragaria evolutionary history is proposed. Additionally, sequence analyses also revealed relaxed constraints on homoeologous copies at high ploidy level, as demonstrated by deletion events within DHAR coding sequences of some allo-octoploid haplotypes.     

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.     

Differences between Fragaria clones by fourth derivative room temperature spectroscopy of intact leaves

Clones of Fragaria were characterized by the attenuance (absorption) spectra of their photosynthetic apparatus. This method uses fourth derivative analysis of spectra of intact lamina of detached leaves at room temperature (20–25 C). Twenty-one different species and hybrids of Fragaria were examined. F. chiloensis accessions frequently have characteristic fourth derivative spectra distinct from that of other Fragaria species. These spectra are characterized by a strong band that appears to be photosystem I component Ca 693; and is correlated with a higher chlorophyll b content per unit area of leaf, and a lower chlorophyll a/b ratio.Abbreviations Chl chlorophyll - chiloensis Fragaria chiloensis - 4th deriv fourth derivative - PSI photosystem I - PSH photosystem II