Dynamics of accessible chromatin regions and subgenome dominance in octoploid strawberry.

Subgenome dominance has been reported in diverse allopolyploid species, where genes from one subgenome are preferentially retained and are more highly expressed than those from other subgenome(s). However, the molecular mechanisms responsible for subgenome dominance remain poorly understood. Here, we develop genome-wide map of accessible chromatin regions (ACRs) in cultivated strawberry (2n = 8x = 56, with A, B, C, D subgenomes). Each ACR is identified as an MNase hypersensitive site (MHS). We discover that the dominant subgenome A contains a greater number of total MHSs and MHS per gene than the submissive B/C/D subgenomes. Subgenome A suffers fewer losses of MHS-related DNA sequences and fewer MHS fragmentations caused by insertions of transposable elements. We also discover that genes and MHSs related to stress response have been preferentially retained in subgenome A. We conclude that preservation of genes and their cognate ACRs, especially those related to stress responses, play a major role in the establishment of subgenome dominance in octoploid strawberry.

FvMYB108, a MYB Gene from Fragaria vesca, Positively Regulates Cold and Salt Tolerance of Arabidopsis.

MYB (myoblast) protein comes in large quantities and a wide variety of types and plays a role in most eukaryotes in the form of transcription factors (TFs). One of its important functions is to regulate plant responses to various stresses. However, the role of MYB TFs in regulating stress tolerance in strawberries is not yet well understood. Therefore, in order to investigate the response of MYB family members to abiotic stress in strawberries, a new MYB TF gene was cloned from Fragaria vesca (a diploid strawberry) and named FvMYB108 based on its structural characteristics and evolutionary relationships. After a bioinformatics analysis, it was determined that the gene belongs to the R2R3-MYB subfamily, and its conserved domain, phylogenetic relationships, predicted protein structure and physicochemical properties, subcellular localization, etc. were analyzed. After qPCR analysis of the expression level of FvMYB108 in organs, such as the roots, stems, and leaves of strawberries, it was found that this gene is more easily expressed in young leaves and roots. After multiple stress treatments, it was found that the target gene in young leaves and roots is more sensitive to low temperatures and salt stimulation. After these two stress treatments, various physiological and biochemical indicators related to stress in transgenic Arabidopsis showed corresponding changes, indicating that FvMYB108 may be involved in regulating the plant's ability to cope with cold and high-salt stress. Further research has found that the overexpression of this gene can upregulate the expression of AtCBF1, AtCOR47, AtERD10, and AtDREB1A related to low-temperature stress, as well as AtCCA1, AtRD29a, AtP5CS1, and AtSnRK2.4 related to salt stress, enhancing the ability of overexpressed plants to cope with stress.

[Identification and expression analysis of the YABBY gene family in strawberry].

YABBY proteins are important transcription factors that regulate morphogenesis and organ development in plants. In order to study the YABBY of strawberry, bioinformatic technique were used to identify the YABBY gene families in Fragaria vesca (diploid) and Fragaria×ananassa (octoploid), and then analyze the sequence characters, phylogeny and collinearity of the family members. The RNA-seq data and the quantitative reverse transcription-polymerase chain reaction (qRT-PCR) technique were used to assay the expression patterns of the family members. A green fluorescent protein (GFP) was fused with FvYABBYs and transiently expressed in tobacco leaf cells for the subcellular localization. As the results, six FvYABBY genes and 26 FxaYABBY genes were identified from F. vesca and F.×ananassa, respectively. The FvYABBY genes were grouped into five clades, and five family members were orthologous with AtYABBY genes of Arabidopsis. In F. vesca, all of the FvYABBYs were basically not expressed not expressed in root and receptacle, while FvYABBY1, FvYABBY2, FvYABBY5 and FvYABBY6 were highly expressed in leaf, shoot, flower and achene. In F.×ananassa, FxaYABBY1, FxaYABBY2, FxaYABBY5 and FxaYABBY6 were expressed in achene, and all FxaYABBY were poorly or not expressed in receptacle. Additionally, under the abiotic stresses of low temperature, high salt and drought, the expression of FvYABBY1, FvYABBY3, FvYABBY4 and FvYABBY6 were down-regulated, FvYABBY5 was up-regulated, and FvYABBY2 was up-regulated and then down-regulated. In tobacco leaf cells, the subcellular localization of FvYABBY proteins were in the nucleus. These results provides a foundation for the functional researches of YABBY gene in strawberry.

Genomic analyses provide insights into sex differentiation of tetraploid strawberry (Fragaria moupinensis).

The strawberry genus, Fragaria, exhibits a wide range of sexual systems and natural ploidy variation. Nearly, all polyploid strawberry species exhibit separate sexes (dioecy). Research has identified the sex-determining sequences as roughly conserved but with repeatedly changed genomic locations across octoploid strawberries. However, it remains unclear whether tetraploid wild strawberries evolved dioecy independently or shared a common origin with octoploid strawberries. In this study, we investigated the sex determinants of F. moupinensis, a dioecious plant with heterogametic females (ZW). Utilizing a combination of haplotype-resolved genome sequencing of the female F. moupinensis, k-mer-based and coverage-based genome-wide association studies (GWAS), and transcriptomic analysis, we discovered a non-recombining, approximately 33.6 kb W-specific region on chromosome 2a. Within this region, only one candidate sex-determining gene (FmoAFT) was identified. Furthermore, an extensive resequencing of the entire Fragaria genus indicated that the W-specific region displays conservative female specificity across all tetraploid species. This observation suggests that dioecy evolved independently in tetraploid and octoploid strawberries. Moreover, employing virus-induced gene silencing (VIGS), we knocked down the expression of the FmoAFT homologue transcript in cultivated strawberries, revealing its potential role in promoting female functions during early carpel development. We also applied DNA affinity purification sequencing (DAP-seq) and yeast one-hybrid assays to identify potential direct targets of FmoAFT. These insights shed new light on the genetic basis and evolutionary history of sex determination in strawberries, thereby facilitating the formulation of strategies to manipulate sex determination in breeding programs.

Comparative Analysis of Transposable Elements in Strawberry Genomes of Different Ploidy Levels.

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.

Haplotype-resolved genomes of wild octoploid progenitors illuminate genomic diversifications from wild relatives to cultivated strawberry.

Strawberry is an emerging model for studying polyploid genome evolution and rapid domestication of fruit crops. Here we report haplotype-resolved genomes of two wild octoploids (Fragaria chiloensis and Fragaria virginiana), the progenitor species of cultivated strawberry. Substantial variation is identified between species and between haplotypes. We redefine the four subgenomes and track the genetic contributions of diploid species by additional sequencing of the diploid F. nipponica genome. We provide multiple lines of evidence that F. vesca and F. iinumae, rather than other described extant species, are the closest living relatives of these wild and cultivated octoploids. In response to coexistence with quadruplicate gene copies, the octoploid strawberries have experienced subgenome dominance, homoeologous exchanges and coordinated expression of homoeologous genes. However, some homoeologues have substantially altered expression bias after speciation and during domestication. These findings enhance our understanding of the origin, genome evolution and domestication of strawberries.

A point mutation in the gene encoding magnesium chelatase I subunit influences strawberry leaf color and metabolism.

Magnesium chelatase (MgCh) catalyzes the insertion of magnesium into protoporphyrin IX, a vital step in chlorophyll (Chl) biogenesis. The enzyme consists of 3 subunits, MgCh I subunit (CHLI), MgCh D subunit (CHLD), and MgCh H subunit (CHLH). The CHLI subunit is an ATPase that mediates catalysis. Previous studies on CHLI have mainly focused on model plant species, and its functions in other species have not been well described, especially with regard to leaf coloration and metabolism. In this study, we identified and characterized a CHLI mutant in strawberry species Fragaria pentaphylla. The mutant, noted as p240, exhibits yellow-green leaves and a low Chl level. RNA-Seq identified a mutation in the 186th amino acid of the CHLI subunit, a base conserved in most photosynthetic organisms. Transient transformation of wild-type CHLI into p240 leaves complemented the mutant phenotype. Further mutants generated from RNA-interference (RNAi) and CRISPR/Cas9 gene editing recapitulated the mutant phenotype. Notably, heterozygous chli mutants accumulated more Chl under low light conditions compared with high light conditions. Metabolite analysis of null mutants under high light conditions revealed substantial changes in both nitrogen and carbon metabolism. Further analysis indicated that mutation in Glu186 of CHLI does not affect its subcellular localization nor the interaction between CHLI and CHLD. However, intramolecular interactions were impaired, leading to reduced ATPase and MgCh activity. These findings demonstrate that Glu186 plays a key role in enzyme function, affecting leaf coloration via the formation of the hexameric ring itself, and that manipulation of CHLI may be a means to improve strawberry plant fitness and photosynthetic efficiency under low light conditions.

Overexpression of a Fragaria vesca 1R-MYB Transcription Factor Gene (FvMYB114) Increases Salt and Cold Tolerance in Arabidopsis thaliana.

The MYB (v-MYB avian myeloblastosis viral oncogene homolog) transcription factor (TF) family has numerous members with complex and diverse functions, which play an indispensable role in regulating the response of plants to stress. In this study, a new 1R-MYB TF gene was obtained from Fragaria vesca (a diploid strawberry) by cloning technology and given a new name, FvMYB114. According to the subcellular localization results, FvMYB114 protein was a nuclear localization protein. Overexpression of FvMYB114 greatly enhanced the adaptability and tolerance of Arabidopsis thaliana to salt and low temperature. Under salt and cold stress, the transgenic plants had greater proline and chlorophyll contents and higher activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) than the wild-type (WT) and unloaded-line (UL) A. thaliana. However, malondialdehyde (MDA) was higher in the WT and UL lines. These results suggested that FvMYB114 may be involved in regulating the response of A. thaliana to salt stress and cold stress. FvMYB114 can also promote the expression of genes, such as the genes AtSOS1/3, AtNHX1 and AtLEA3 related to salt stress and the genes AtCCA1, AtCOR4 and AtCBF1/3 related to cold stress, further improving the tolerance of transgenic plants to salt and cold stress.

Comprehensive profiling of endogenous phytohormones and expression analysis of 1-aminocyclopropane-1-carboxylic acid synthase gene family during fruit development and ripening in octoploid strawberry (Fragaria× ananassa).

The non-climacteric octoploid strawberry (Fragaria × ananassa Duchesne ex Rozier) was used as a model to study its regulation during fruit ripening. High performance liquid chromatography electrospray tandem-mass spectrometry (HPLC-ESI-MS/MS) was employed to profile 28 different endogenous phytohormones in strawberry. These include auxins, cytokinins (CKs), abscisic acid (ABA), ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC), jasmonates, and phenolic compounds salicylic acid (SA), benzoic acid (BzA) and phenylacetic acid (PAA) together with their various metabolic forms that have remained largely unexplored thus far. ABA, ACC and CK N6-(Δ2-isopentenyl)adenine (iP) were found to be associated with ripening while ABA catabolites 9-hydroxy-ABA and phaseic acid mimicked the pattern of climacteric decline at the turning phase of strawberry ripening. The content of other CK forms except iP decreased as fruit ripened, as also that of auxins indole-3-acetic acid (IAA) and oxo-IAA, and of jasmonates. Data presented here also suggest that both the transition and progression of strawberry fruit ripening are associated with N6-(Δ2-isopentenyl)adenosine-5'-monophosphate (iPRMP) → N6-(Δ2-isopentenyl)adenosine (iPR) → iP as the preferred CK metabolic pathway. In contrast, the ethylene precursor ACC was present at higher levels, with its abundance increasing from the onset of ripening to the red ripe stage. Further investigation of ripening-specific ACC accumulation revealed the presence of a large ACC synthase (ACS) encoding gene family in octoploid strawberry that was previously unknown. Seventeen ACS genes were found differentially expressed in fruit tissues, while six of them showed induced expression during strawberry fruit ripening. These data suggest a possible role(s) of ACC, ABA, and iP in strawberry fruit ripening. These data add new dimension to the existing knowledge of the interplay of different endogenous phytohormones in octoploid strawberry, paving the way for further investigation of their individual role(s) in fruit ripening.

Proanthocyanidins Delay Fruit Coloring and Softening by Repressing Related Gene Expression during Strawberry (Fragaria × ananassa Duch.) Ripening.

Proanthocyanidins (PAs), also known as condensed tannins, are widespread throughout the plant kingdom, presenting diverse biological and biochemical activities. Being one of the most abundant groups of natural polyphenolic antioxidant, PAs are applied to improve plant tolerance to (a)biotic stresses and delay the senescence of fruit by scavenging the reactive oxygen species (ROS) and enhancing antioxidant responses. The effects of PAs on coloring and softening of strawberries (Fragaria × ananassa Duch.), a worldwide demanded edible fruit and typical material for studying non-climacteric fruit ripening, were firstly assessed in this work. The results showed that exogenous PAs delayed the decrease in fruit firmness and anthocyanins accumulation but improved the fruit skin brightness. Strawberries treated with PAs had similar total soluble solids, total phenolics, and total flavonoids, but lower titratable acidity content. Moreover, the contents of endogenous PAs, abscisic acid and sucrose, were somehow increased by PA treatment, while no obvious change was found in fructose and glucose content. In addition, the anthocyanin- and firmness-related genes were significantly repressed, while the PA biosynthetic gene (anthocyanin reductase, ANR) was highly up-regulated by PA treatment at the key point for fruit softening and coloring. In summary, the results presented in this study suggest that PAs slow down strawberry coloration and softening by inhibiting the expression of related genes, which could be helpful for a better understanding of the biological role of PAs and provide a new strategy to regulate strawberry ripening.

Subgenome phasing for complex allopolyploidy: case-based benchmarking and recommendations.

Accurate subgenome phasing is crucial for understanding the origin, evolution and adaptive potential of polyploid genomes. SubPhaser and WGDI software are two common methodologies for subgenome phasing in allopolyploids, particularly in scenarios lacking known diploid progenitors. Triggered by a recent debate over the subgenomic origins of the cultivated octoploid strawberry, we examined four well-documented complex allopolyploidy cases as benchmarks, to evaluate and compare the accuracy of the two software. Our analysis demonstrates that the subgenomic structure phased by both software is in line with prior research, effectively tracing complex allopolyploid evolutionary trajectories despite the limitations of each software. Furthermore, using these validated methodologies, we revisited the controversial issue regarding the progenitors of the octoploid strawberry. The results of both methodologies reaffirm Fragaria vesca and Fragaria iinumae as progenitors of the octoploid strawberry. Finally, we propose recommendations for enhancing the accuracy of subgenome phasing in future studies, recognizing the potential of integrated tools for advanced complex allopolyploidy research and offering a new roadmap for robust subgenome-based phylogenetic analysis.

A multi-omics framework reveals strawberry flavor genes and their regulatory elements.

Flavor is essential to consumer preference of foods and is an increasing focus of plant breeding programs. In fruit crops, identifying genes underlying volatile organic compounds has great promise to accelerate flavor improvement, but polyploidy and heterozygosity in many species have slowed progress. Here we use octoploid cultivated strawberry to demonstrate how genomic heterozygosity, transcriptomic intricacy and fruit metabolomic diversity can be treated as strengths and leveraged to uncover fruit flavor genes and their regulatory elements. Multi-omics datasets were generated including an expression quantitative trait loci map with 196 diverse breeding lines, haplotype-phased genomes of a highly-flavored breeding selection, a genome-wide structural variant map using five haplotypes, and volatile genome-wide association study (GWAS) with > 300 individuals. Overlaying regulatory elements, structural variants and GWAS-linked allele-specific expression of numerous genes to variation in volatile compounds important to flavor. In one example, the functional role of anthranilate synthase alpha subunit 1 in methyl anthranilate biosynthesis was supported via fruit transient gene expression assays. These results demonstrate a framework for flavor gene discovery in fruit crops and a pathway to molecular breeding of cultivars with complex and desirable flavor.

Chromosome-scale assembly with a phased sex-determining region resolves features of early Z and W chromosome differentiation in a wild octoploid strawberry.

When sex chromosomes stop recombining, they start to accumulate differences. The sex-limited chromosome (Y or W) especially is expected to degenerate via the loss of nucleotide sequence and the accumulation of repetitive sequences. However, how early signs of degeneration can be detected in a new sex chromosome is still unclear. The sex-determining region of the octoploid strawberries is young, small, and dynamic. Using PacBio HiFi reads, we obtained a chromosome-scale assembly of a female (ZW) Fragaria chiloensis plant carrying the youngest and largest of the known sex-determining region on the W in strawberries. We fully characterized the previously incomplete sex-determining region, confirming its gene content, genomic location, and evolutionary history. Resolution of gaps in the previous characterization of the sex-determining region added 10 kb of sequence including a noncanonical long terminal repeat-retrotransposon; whereas the Z sequence revealed a Harbinger transposable element adjoining the sex-determining region insertion site. Limited genetic differentiation of the sex chromosomes coupled with structural variation may indicate an early stage of W degeneration. The sex chromosomes have a similar percentage of repeats but differ in their repeat distribution. Differences in the pattern of repeats (transposable element polymorphism) apparently precede sex chromosome differentiation, thus potentially contributing to recombination cessation as opposed to being a consequence of it.

Strawberry (Fragaria × ananassa cv. Romina) methanolic extract attenuates Alzheimer's beta amyloid production and oxidative stress by SKN-1/NRF and DAF-16/FOXO mediated mechanisms in C. elegans.

Bioactive compounds from strawberries have been associated with multiple healthy benefits. The present study aimed to assess chemical characterization of a methanolic extract of the Romina strawberry variety in terms of antioxidant capacity, polyphenols profile and chemical elements content. Additionally, potential toxicity, the effect on amyloid-ß production and oxidative stress of the extract was in vivo evaluated in the experimental model Caenorhabditis elegans. Results revealed an important content in phenolic compounds (mainly ellagic acid and pelargonidin-3-glucoside) and minerals (K, Mg, P and Ca). The treatment with 100, 500 or 1000 µg/mL of strawberry extract did not show toxicity. On the contrary, the extract was able to delay amyloid ß-protein induced paralysis, reduced amyloid-ß aggregation and prevented oxidative stress. The potential molecular mechanisms present behind the observed results explored by RNAi technology revealed that DAF-16/FOXO and SKN-1/NRF2 signaling pathways were, at least partially, involved.

Identification of MYB Transcription Factors Involving in Fruit Quality Regulation of Fragaria × ananassa Duch.

The cultivated strawberry (Fragaria × ananassa Duch.) is an important horticultural crop. The economic values of strawberry cultivars are decided by their fruit qualities including taste, color and aroma. The important role of MYB transcription factors in fruit quality regulation is recognized increasingly with the identification of MYB genes involved in metabolism. A total of 407 MYB genes of F. × ananassa (FaMYBs) were identified in the genome-wide scale and named according to subgenome locations. The 407 FaMYBs were clustered into 36 groups based on phylogenetic analysis. According to synteny analysis, whole genome duplication and segmental duplication contributed over 90% of the expansion of the FaMYBs family. A total of 101 FaMYB loci with 1-6 alleles were identified by the homologous gene groups on homologous chromosomes. The differentially expressed FaMYB profiles of three cultivars with different fruit quality and fruit ripe processes provided the 8 candidate loci involved in fruit quality regulation. In this experiment, 7, 5, and 4 FaMYBs were screeded as candidate genes involved in the regulation of metabolism/transportation of anthocyanins, sugars or organic acids and 4-hydroxy-2, 5-dimethyl-3(2H)-furanone, respectively. These results pointed out the key FaMYBs for further functional analysis of gene regulation of strawberry fruit quality and would be helpful in the clarification on ofe roles of MYBs in the metabolism of fruit crops.

Genome-Wide Analysis of MYB10 Transcription Factor in Fragaria and Identification of QTLs Associated with Fruit Color in Octoploid Strawberry.

The genus Fragaria encompass fruits with diverse colors influenced by the distribution and accumulation of anthocyanin. Particularly, the fruit colors of strawberries with different ploidy levels are determined by expression and natural variations in the vital structural and regulatory genes involved in the anthocyanin pathway. Among the regulatory genes, MYB10 transcription factor is crucial for the expression of structural genes in the anthocyanin pathway. In the present study, we performed a genome wide investigation of MYB10 in the diploid and octoploid Fragaria species. Further, we identified seven quantitative trait loci (QTLs) associated with fruit color in octoploid strawberry. In addition, we predicted 20 candidate genes primarily influencing the fruit color based on the QTL results and transcriptome analysis of fruit skin and flesh tissues of light pink, red, and dark red strawberries. Moreover, the computational and transcriptome analysis of MYB10 in octoploid strawberry suggests that the difference in fruit colors could be predominantly influenced by the expression of MYB10 from the F. iinumae subgenome. The outcomes of the present endeavor will provide a platform for the understanding and tailoring of anthocyanin pathway in strawberry for the production of fruits with aesthetic colors.

Diversification, spread, and admixture of octoploid strawberry in the Western Hemisphere.

PREMISE: Polyploid species often have complex evolutionary histories that have, until recently, been intractable due to limitations of genomic resources. While recent work has further uncovered the evolutionary history of the octoploid strawberry (Fragaria L.), there are still open questions. Much is unknown about the evolutionary relationship of the wild octoploid species, Fragaria virginiana and Fragaria chiloensis, and gene flow within and among species after the formation of the octoploid genome. METHODS: We leveraged a collection of wild octoploid ecotypes of strawberry representing the recognized subspecies and ranging from Alaska to southern Chile, and a high-density SNP array to investigate wild octoploid strawberry evolution. Evolutionary relationships were interrogated with phylogenetic analysis and genetic clustering algorithms. Additionally, admixture among and within species is assessed with model-based and tree-based approaches. RESULTS: Phylogenetic analysis revealed that the two octoploid strawberry species are monophyletic sister lineages. The genetic clustering results show substructure between North and South American F. chiloensis populations. Additionally, model-based and tree-based methods support gene flow within and among the two octoploid species, including newly identified admixture in the Hawaiian F. chiloensis subsp. sandwicensis population. CONCLUSIONS: F. virginiana and F. chiloensis are supported as monophyletic and sister lineages. All but one of the subspecies show extensive paraphyly. Furthermore, phylogenetic relationships among F. chiloensis populations supports a single population range expansion southward from North America. The inter- and intraspecific relationships of octoploid strawberry are complex and suggest substantial gene flow between sympatric populations among and within species.

Karyotypic stability of Fragaria (strawberry) species revealed by cross-species chromosome painting.

Chromosome karyotyping analysis is particularly useful in determining species relationships and the origin of polyploid species. Identification of individual chromosomes is the foundation for karyotype development. For Fragaria (strawberry) species, definitive identification of the individual chromosomes is extremely difficult because of their small size and similar shape. Here, we identified all chromosomes for 11 representative Fragaria species with different ploidy using a set of oligonucleotide-based probes developed in Fragaria vesca. Comprehensive molecular cytogenetic karyotypes were established based on the individually identified chromosomes. In addition, we used oligo probes to assign the 5S and 45S rDNA loci to specific chromosomes in 16 Fragaria species. We found that these Fragaria species maintained a remarkably conserved karyotype. No inter-chromosomal structural rearrangements at the cytological level were observed in any of the chromosomes among these species. Despite karyotypic stability and similarity, variations in the signal intensity of oligo probes were observed among the homologous chromosomes in several polyploid species. Moreover, most Fragaria species also showed differences in the distribution patterns of 45S and 5S rDNA. These data provide new insights into the origins of several polyploid Fragaria species.

N6-methyladenosine RNA modification regulates strawberry fruit ripening in an ABA-dependent manner.

BACKGROUND: Epigenetic mark such as DNA methylation plays pivotal roles in regulating ripening of both climacteric and non-climacteric fruits. However, it remains unclear whether mRNA m6A methylation, which has been shown to regulate ripening of the tomato, a typical climacteric fruit, is functionally conserved for ripening control among different types of fruits. RESULTS: Here we show that m6A methylation displays a dramatic change at ripening onset of strawberry, a classical non-climacteric fruit. The m6A modification in coding sequence (CDS) regions appears to be ripening-specific and tends to stabilize the mRNAs, whereas m6A around the stop codons and within the 3' untranslated regions is generally negatively correlated with the abundance of associated mRNAs. We identified thousands of transcripts with m6A hypermethylation in the CDS regions, including those of NCED5, ABAR, and AREB1 in the abscisic acid (ABA) biosynthesis and signaling pathway. We demonstrate that the methyltransferases MTA and MTB are indispensable for normal ripening of strawberry fruit, and MTA-mediated m6A modification promotes mRNA stability of NCED5 and AREB1, while facilitating translation of ABAR. CONCLUSION: Our findings uncover that m6A methylation regulates ripening of the non-climacteric strawberry fruit by targeting the ABA pathway, which is distinct from that in the climacteric tomato fruit.

Ethylene application at the immature stage of Fragaria chiloensis fruit represses the anthocyanin biosynthesis with a concomitant accumulation of lignin.

Ethylene seems to play a secondary role in non-climacteric strawberry ripening compared to abscisic acid. However, this does not exclude that ethylene can regulate some specific events related to the ripening process. Preliminary experiments of applications of ethylene or its inhibitor 1-MCP to strawberry fruits have reinforced this hypothesis. Here, we reveal some previously non-covered physiological effects of ethylene using an in vitro strawberry ripening system. Fruits of Fragaria chiloensis treated with ethephon at the large green developmental stage showed inhibition of anthocyanin biosynthesis and downregulation of essential anthocyanin biosynthesis genes during the ripening. At the same time, ethylene stimulated lignin biosynthesis and remarkably upregulated the expression of FcPOD27. Since contrasting results have been reported when ethylene was applied at late ripening developmental stages, our findings support the hypothesis of a temporal-specific ethylene role in the ripening of strawberry fruits.