RESUMO
Transposable elements (TEs) make up a large portion of plant genomes and play a vital role in genome structure, function, and evolution. Cultivated strawberry (Fragaria x ananassa) is one of the most important fruit crops, and its octoploid genome was formed through several rounds of genome duplications from diploid ancestors. Here, we built a pan-genome TE library for the Fragaria genus using ten published strawberry genomes at different ploidy levels, including seven diploids, one tetraploid, and two octoploids, and performed comparative analysis of TE content in these genomes. The TEs comprise 51.83% (F. viridis) to 60.07% (F. nilgerrensis) of the genomes. Long terminal repeat retrotransposons (LTR-RTs) are the predominant TE type in the Fragaria genomes (20.16% to 34.94%), particularly in F. iinumae (34.94%). Estimating TE content and LTR-RT insertion times revealed that species-specific TEs have shaped each strawberry genome. Additionally, the copy number of different LTR-RT families inserted in the last one million years reflects the genetic distance between Fragaria species. Comparing cultivated strawberry subgenomes to extant diploid ancestors showed that F. vesca and F. iinumae are likely the diploid ancestors of the cultivated strawberry, but not F. viridis. These findings provide new insights into the TE variations in the strawberry genomes and their roles in strawberry genome evolution.
Assuntos
Fragaria , Humanos , Fragaria/genética , Elementos de DNA Transponíveis/genética , Poliploidia , Ploidias , Genoma de PlantaRESUMO
Studies on many plants have shown that mitogen-activated protein kinases (MAPKs) are key proteins involved in regulating plant responses to biotic and abiotic stresses. However, their involvement in cultivated strawberry development and ripening remains unclear. In this study, 43 FaMAPK gene family members were identified in the genome of cultivated strawberry (Fragaria × ananassa), phylogenetic analysis indicated that FaMAPKs could be classified into four groups. Systematic analysis of the conserved motif, exon-intron structure showed that there were significant varieties between different groups in structure, but in the same group they were similar. Multiple cis-regulatory elements associated with phytohormone response, and abiotic and biotic stresses were predicted in the promoter regions of FaMAPK genes. Transcriptional analysis showed that all FaMAPK genes were expressed at all developmental stages. Meanwhile, the effect of exogenous ABA and sucrose on the expression profile of FaMAPKs was investigated. Exogenous ABA, sucrose, and ABA plus sucrose treatments upregulated the expression of FaMAPK genes and increased the content of endogenous ABA, sucrose, and anthocyanin in strawberry fruits, suggesting that ABA and sucrose might be involved in the FaMAPK-mediated regulation of strawberry fruit ripening. Based on the obtained results, MAPK genes closely related to the ripening of strawberries were screened to provide a theoretical basis and support for future research on strawberries.
Assuntos
Fragaria , Ácido Abscísico/metabolismo , Ácido Abscísico/farmacologia , Fragaria/metabolismo , Frutas/metabolismo , Regulação da Expressão Gênica de Plantas , Filogenia , Proteínas de Plantas/metabolismo , Sacarose/metabolismoRESUMO
Fruit colour affects consumer preference and is an important trait for breeding in strawberry. Previously, we isolated the Reduced Anthocyanins in Petioles (RAP) gene encoding a glutathione S-transferase (GST) that binds anthocyanins to facilitate their transport from cytosol to vacuole in the diploid strawberry Fragaria vesca. The parent of rap was the F. vesca variety 'Yellow Wonder' that develops white fruit due to a natural mutation in the FveMYB10 gene. Here, we investigated the application potential of RAP in modulating fruit colours by overexpression of RAP in F. vesca and knockout of RAP in the cultivated strawberry Fragaria × ananassa. Unexpectedly, the RAP overexpression in Yellow Wonder background caused formation of red fruit. In addition, the red coloration occurs precociously at floral stage 10 and continues in the receptacle during early fruit development. Transcriptome analysis revealed that the anthocyanin biosynthesis genes were not up-regulated in RAP-ox; rap myb10 flowers at anthesis and largely inhibited at the turning stage in fruit, suggesting a coloration mechanism independent of FveMYB10. Moreover, we used CRISPR/Cas9 to knockout RAP in cultivated strawberry which is octoploid. Six copies of RAP were simultaneously knocked out in the T0 generation leading to the green stem and white-fruited phenotype. Several T1 progeny have segregated away the CRISPR/Cas9 transgene but maintain the green stem trait. Our results indicate that enhancing the anthocyanin transport could redirect the metabolic flux from proanthocyanidin to anthocyanin production at early developmental stages of fruit and that RAP is one promising candidate gene in fruit colour breeding of strawberry.
Assuntos
Fragaria , Antocianinas , Flores/genética , Fragaria/genética , Frutas/genética , Perfilação da Expressão GênicaRESUMO
Anthocyanins are responsible for the red color of strawberry, they are a subclass of flavonoids synthesized in cytosol and transferred to vacuole to form the visible color. Previous studies in model and ornamental plants indicated members of the glutathione S-transferase (GST) gene family were involved in vacuolar accumulation of anthocyanins. In the present study, a total of 130 FaGST genes were identified in the genome of cultivated strawberry (Fragaria × ananassa), which were unevenly distributed across the 28 chromosomes from the four subgenomes. Evolutionary analysis revealed the expansion of FaGST family was under stable selection and mainly drove by WGD/segmental duplication event. Classification and phylogenetic analysis indicated that all the FaGST genes were clarified into seven subclasses, among which FaGST1, FaGST37, and FaGST97 belonging to Phi class were closely related to FvRAP, an anthocyanin-related GST of wildwood strawberry, and this clade was clustered with other known anthocyanin-related GSTs. RNAseq-based expression analysis at different developmental stages of strawberry revealed that the expression of FaGST1, FaGST37, FaGST39, FaGST73, and FaGST97 was gradually increased during the fruit ripening, consistent with the anthocyanins accumulation. These expression patterns of those five FaGST genes were also significantly correlated with those of other anthocyanin biosynthetic genes such as FaCHI, FaCHS, and FaANS, as well as anthocyanin regulatory gene FaMYB10. These results indicated FaGST1, FaGST37, FaGST39, FaGST73, and FaGST97 may function in vacuolar anthocyanin accumulation in cultivated strawberry.
Assuntos
Antocianinas/metabolismo , Fragaria/genética , Frutas/genética , Genoma de Planta/genética , Glutationa Transferase/genética , Proteínas de Plantas/genética , Fragaria/metabolismo , Frutas/metabolismo , Perfilação da Expressão Gênica/métodos , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Ontologia Genética , Glutationa Transferase/classificação , Glutationa Transferase/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Filogenia , Proteínas de Plantas/classificação , Proteínas de Plantas/metabolismoRESUMO
KEY MESSAGE: Fra a 1 protein in strawberry causes oral allergic syndrome. Over 39 Fra a 1 paralogs have been identified in strawberry genome. Fra a 1.01 is major accumulating protein in edible organs. Strawberry fruits contain allergenic proteins that cause oral allergic syndrome. The hypothesized major allergen is Fra a 1, an ortholog of the birch pollen allergen protein Bet v 1. We organized Fra a 1 genes and analyzed their localizations at the transcriptional and translational levels. In total, 15 new Fra a 1 proteins were identified from the genomic database, increasing the total number of Fra a 1 to 30 proteins encoded by 39 genes. Fra a 1.02 was mostly expressed in receptacles, and Fra a 1.01 in achenes, when analyzed by RNA sequencing. Immunoblotting showed that the Fra a 1.01 protein was broadly accumulated in strawberry organs, while the Fra a 1.02 protein was mostly expressed in receptacles. Recombinant Fra a 1.01 strongly reacted with human IgE. The mRNA and protein expression levels of Fra a 1 did not correlate, indicating the importance of protein levels when evaluating the abundance of allergens in strawberry. Based on the localizations, accumulation levels and reactivity to human IgE, we determined that Fra a 1.01 was the most important allergen, followed by Fra a 1.02, and then other Fra a 1 proteins. The information obtained here will be useful for selecting the target Fra a 1 paralogs when breeding hypoallergenic strawberry.
Assuntos
Alérgenos/genética , Antígenos de Plantas/genética , Fragaria/genética , Frutas/genética , Proteínas de Plantas/genética , Alérgenos/imunologia , Alérgenos/metabolismo , Sequência de Aminoácidos , Animais , Antígenos de Plantas/classificação , Antígenos de Plantas/metabolismo , Fragaria/metabolismo , Frutas/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Cobaias , Humanos , Soros Imunes/imunologia , Immunoblotting , Imunoglobulina E/imunologia , Especificidade de Órgãos/genética , Filogenia , Proteínas de Plantas/metabolismo , Análise de Sequência de RNA , Homologia de Sequência de AminoácidosRESUMO
BACKGROUND: The strawberry, Fragaria × ananassa, is an allo-octoploid (2n = 8x = 56) and outcrossing species. Although it is the most widely consumed berry crop in the world, its complex genome structure has hindered its genetic and genomic analysis, and thus discrimination of subgenome-specific loci among the homoeologous chromosomes is needed. In the present study, we identified candidate subgenome-specific single nucleotide polymorphism (SNP) and simple sequence repeat (SSR) loci, and constructed a linkage map using an S1 mapping population of the cultivar 'Reikou' with an IStraw90 Axiom® SNP array and previously published SSR markers. RESULTS: The 'Reikou' linkage map consisted of 11,574 loci (11,002 SNPs and 572 SSR loci) spanning 2816.5 cM of 31 linkage groups. The 11,574 loci were located on 4738 unique positions (bin) on the linkage map. Of the mapped loci, 8999 (8588 SNPs and 411 SSR loci) showed a 1:2:1 segregation ratio of AA:AB:BB allele, which suggested the possibility of deriving loci from candidate subgenome-specific sequences. In addition, 2575 loci (2414 SNPs and 161 SSR loci) showed a 3:1 segregation of AB:BB allele, indicating they were derived from homoeologous genomic sequences. Comparative analysis of the homoeologous linkage groups revealed differences in genome structure among the subgenomes. CONCLUSIONS: Our results suggest that candidate subgenome-specific loci are randomly located across the genomes, and that there are small- to large-scale structural variations among the subgenomes. The mapped SNPs and SSR loci on the linkage map are expected to be seed points for the construction of pseudomolecules in the octoploid strawberry.
Assuntos
Mapeamento Cromossômico , Fragaria/genética , Loci Gênicos/genética , Genômica , Poliploidia , Genoma de Planta/genética , Filogenia , Polimorfismo de Nucleotídeo ÚnicoRESUMO
Heat shock transcription factors (HSFs) are mainly involved in the activation of genes in response to heat stress as well as other abiotic and biotic stresses. The growth, development, reproduction, and yield of strawberry are strongly limited by extreme temperatures and droughts. In this study, we used Illumina sequencing and obtained transcriptome data set from Fragaria × ananassa Duchessne cv. Toyonoka. Six contigs and three unigenes were confirmed to encode HSF proteins (FaTHSFs). Subsequently, we characterized the biological functions of two particularly selected unigenes, FaTHSFA2a and FaTHSFB1a, which were classified into class A2 and B HSFs, respectively. Expression assays revealed that FaTHSFA2a and FaTHSFB1a expression was induced by heat shock and correlated well with elevated ambient temperatures. Overexpression of FaTHSFA2a and FaTHSFB1a resulted in the activation of their downstream stress-associated genes, and notably enhanced the thermotolerance of transgenic Arabidopsis plants. Besides, both FaTHSFA2a and FaTHSFB1a fusion proteins localized in the nucleus, indicating their similar subcellular distributions as transcription factors. Our yeast one-hybrid assay suggested that FaTHSFA2a has trans-activation activity, whereas FaTHSFB1a expresses trans-repression function. Altogether, our annotated transcriptome sequences provide a beneficial resource for identifying most genes expressed in octoploid strawberry. Furthermore, HSF studies revealed the possible insights into the molecular mechanisms of thermotolerance, thus rendering valuable molecular breeding to improve the tolerance of strawberry in response to high-temperature stress.
Assuntos
Arabidopsis/genética , Proteínas de Ligação a DNA/genética , Fragaria/genética , Resposta ao Choque Térmico/genética , Proteínas Recombinantes de Fusão/metabolismo , Termotolerância/genética , Fatores de Transcrição/genética , Sequência de Aminoácidos , Fragaria/crescimento & desenvolvimento , Fragaria/metabolismo , Regulação da Expressão Gênica de Plantas , Fatores de Transcrição de Choque Térmico , Proteínas de Choque Térmico/metabolismo , Temperatura Alta , Plantas Geneticamente Modificadas/genética , Alinhamento de Sequência , Estresse Fisiológico/genéticaRESUMO
Fragaria nilgerrensis is a wild strawberry species widely distributed in southwest China and has strong ecological adaptability. Akihime (F. × ananassa Duch. cv. Akihime) is one of the main cultivated strawberry varieties in China and is prone to infection with a variety of diseases. In this study, high-throughput sequencing was used to analyze and compare the soil and root microbiomes of F. nilgerrensis and Akihime. Results indicate that the wild species F. nilgerrensis showed higher microbial diversity in nonrhizosphere soil and rhizosphere soil and possessed a more complex microbial network structure compared with the cultivated variety Akihime. Genera such as Bradyrhizobium and Anaeromyxobacter, which are associated with nitrogen fixation and ammonification, and Conexibacter, which is associated with ecological toxicity resistance, exhibited higher relative abundances in the rhizosphere and nonrhizosphere soil samples of F. nilgerrensis compared with those of Akihime. Meanwhile, the ammonia-oxidizing archaea Candidatus Nitrososphaera and Candidatus Nitrocosmicus showed the opposite tendencies. We also found that the relative abundances of potential pathogenic genera and biocontrol bacteria in the Akihime samples were higher than those in the F. nilgerrensis samples. The relative abundances of Blastococcus, Nocardioides, Solirubrobacter, and Gemmatimonas, which are related to pesticide degradation, and genus Variovorax, which is associated with root growth regulation, were also significantly higher in the Akihime samples than in the F. nilgerrensis samples. Moreover, the root endophytic microbiomes of both strawberry species, especially the wild F. nilgerrensis, were mainly composed of potential biocontrol and beneficial bacteria, making them important sources for the isolation of these bacteria. This study is the first to compare the differences in nonrhizosphere and rhizosphere soils and root endogenous microorganisms between wild and cultivated strawberries. The findings have great value for the research of microbiomes, disease control, and germplasm innovation of strawberry.
RESUMO
The diploid woodland strawberry (F. vesca) represents an important model for the genus Fragaria. Significant advances in the understanding of the molecular mechanisms regulating seasonal alternance of flower induction and vegetative reproduction has been made in this species. However, this research area has received little attention on the cultivated octoploid strawberry (F. × ananassa) despite its enormous agronomical and economic importance. To advance in the characterization of this intricated molecular network, expression analysis of key flowering time genes was performed both in short and long days and in cultivars with seasonal and perpetual flowering. Analysis of overexpression of FaCO and FaSOC1 in the seasonal flowering 'Camarosa' allowed functional validation of a number of responses already observed in F. vesca while uncovered differences related to the regulation of FaFTs expression and gibberellins (GAs) biosynthesis. While FvCO has been shown to promote flowering and inhibit runner development in the perpetual flowering H4 accession of F. vesca, our study showed that FaCO responds to LD photoperiods as in F. vesca but delayed flowering to some extent, possibly by induction of the strong FaTFL1 repressor in crowns. A contrasting effect on runnering was observed in FaCO transgenic plants, some lines showing reduced runner number whereas in others runnering was slightly accelerated. We demonstrate that the role of the MADS-box transcription factor FaSOC1 as a strong repressor of flowering and promoter of vegetative growth is conserved in woodland and cultivated strawberry. Our study further indicates an important role of FaSOC1 in the photoperiodic repression of FLOWERING LOCUS T (FT) genes FaFT2 and FaFT3 while FaTFL1 upregulation was less prominent than that observed in F. vesca. In our experimental conditions, FaSOC1 promotion of vegetative growth do not require induction of GA biosynthesis, despite GA biosynthesis genes showed a marked photoperiodic upregulation in response to long days, supporting GA requirement for the promotion of vegetative growth. Our results also provided insights into additional factors, such as FaTEM, associated with the vegetative developmental phase that deserve further characterization in the future.
RESUMO
Anthracnose caused by Colletotrichum spp. was widespread in recent years and resulted in great damage to strawberry production. Soil microbial communities were key contributors to host nutrition, development, and immunity; however, the difference between the microbial communities of healthy and anthracnose-infected strawberry rhizosphere soils remains unclear. In this study, the Illumina sequencing technique was used to comparatively study the prokaryotic and fungal community compositions and structures between healthy and anthracnose-infected strawberry rhizosphere soils in Yuxi, Yunnan Province. Both microbial community diversities and richness of anthracnose-infected strawberry rhizosphere soils were higher than those of healthy strawberry rhizosphere soils. A total of 2,518 prokaryotic and 556 fungal operational taxonomic units (OTUs) were obtained at the 97% similarity threshold. Proteobacteria, Thaumarchaeota, and Acidobacteria were the dominant prokaryotic phyla; Ascomycota, unclassified_k__Fungi, and Mortierellomycota were the dominant fungal phyla. The relative abundances of beneficial bacterial phyla Actinobacteria and Firmicutes, genera Streptomyces, Azospirillum, and Bacillus were significantly reduced in anthracnose-infected strawberry rhizosphere soils; the relative abundance of beneficial fungal species Trichoderma asperellum shows a similar tendency with bacterial abundance. Besides Colletotrichum, 15 other potential fungal pathogen genera and seven fungal pathogen species were identified; among the potential pathogen genera and species, eight pathogen genera and Fusarium oxysporum showed significant differences between healthy and anthracnose-infected strawberry rhizosphere soils. The results suggested that strawberry planted in this area may be infected by other fungal pathogens except for Colletotrichum spp. Our present research will provide theoretical basis and data reference for the isolation and identification of strawberry pathogens and potential probiotics in future works.
RESUMO
We genotyped strawberry cultivars by two newly selected and two previously reported SSR markers. All four markers produced interpretable electropherograms from 75 accessions consisting of 72 Fragaria × ananassa cultivars or lines and three octoploid Fragaria species accessions. These SSR markers were highly polymorphic; in particular, one of the newly developed markers, FxaHGA02P13, was capable of distinguishing all of the accessions except for a mutant strain that was derived from another accession in the set. When two markers were combined, all 48 full-sib individuals could be distinguished. Fingerprinting patterns were reproducible between multiple samples, including the leaves, sepals, and fruit flesh of the same accession. Principal-coordinate analysis of the 75 accessions detected several groups, which reflect taxon and breeding site. Together with other available markers, these SSR markers will contribute to the management of strawberry genetic resources and the protection of breeders' rights.
RESUMO
The cultivated strawberry, Fragaria×ananassa possesses a genetically complex allo-octoploid genome. Advances in genomics research in Fragaria, including the release of a genome sequence for F. vesca, have permitted the development of a high throughput whole genome genotyping array for strawberry, which promises to facilitate genetics and genomics research. In this investigation, we used the Axiom® IStraw90®)array for linkage map development, and produced a linkage map containing 8,407 SNP markers spanning 1,820cM. Whilst the linkage map provides good coverage of the genome of both parental genotypes, the map of 'Monterey' contained significantly fewer mapped markers than did that of 'Darselect'. The array contains a novel marker class known as haploSNPs, which exploit homoeologous sequence variants as probe destabilization sites to effectively reduce marker ploidy. We examined these sites as potential indicators of subgenomic identities by using comparisons to allele states in two ancestral diploids. On this basis, haploSNP loci could be inferred to be derived from F. vesca, F. iinumae, or from an unknown source. When the identity classifications of haploSNPs were considered in conjunction with their respective linkage map positions, it was possible to define two discrete subgenomes, while the remaining homoeologues of each chromosome could not be partitioned into two discrete subgenomic groupings. These findings suggested a novel hypothesis regarding octoploid strawberry subgenome structure and evolutionary origins.
Assuntos
Mapeamento Cromossômico/métodos , Fragaria/genética , Genoma de Planta/genética , Polimorfismo de Nucleotídeo Único , Poliploidia , Alelos , Sequência de Bases , Cromossomos de Plantas/genética , Diploide , Evolução Molecular , Ligação Genética , Genótipo , Haplótipos , Homologia de Sequência do Ácido NucleicoRESUMO
Flowering time control is important for fruit production in Fragaria × ananassa. The flowering inhibition pathway has been extensively elucidated in the woodland strawberry, Fragaria vesca, whereas the factors involved in its promotion remain unclear. In this study, we investigated the environmental responses of F. × ananassa FT and TFL1-like genes, which are considered key floral promoters and repressors in many plants, respectively. A putative floral promoter, FaFT3, was up-regulated in the shoot tip under short-day and/or low growth temperature, in accordance with the result that these treatments promoted flowering. FaFT3 mRNA accumulated before induction of a floral meristem identity gene, FaAP1. FaFT2, a counterpart of FvFT2, expressed in the flower bud of F. vesca, was not induced in the shoot tip differentiating sepal or stamen, suggesting that this gene works at a later stage than stamen formation. In F. vesca, FvFT1 transmits the long-day signal perceived in the leaves to the shoot tip, and induces the potent floral inhibitor FvTFL1. FaFT1 was expressed in the leaves under long-day conditions in F. × ananassa. Expression of FaTFL1 was higher in the shoot tip under long-day than short-day conditions. Independent of day-length, FaTFL1 expression was higher under high temperature than low temperature conditions. These results suggest that FaFT3 induction by short-day or low temperature stimuli is a key step for flowering initiation. As in F. vesca, F. × ananassa floral inhibition pathways depend on FaTFL1 regulation by day-length via FaFT1, and by temperature.