Overexpression of a Fragaria vesca NAM, ATAF, and CUC (NAC) Transcription Factor Gene (FvNAC29) Increases Salt and Cold Tolerance in Arabidopsis thaliana.

The NAC (NAM, ATAF1/2, CUC2) family of transcription factors (TFs) is a vital transcription factor family of plants. It controls multiple parts of plant development, tissue formation, and abiotic stress response. We cloned the FvNAC29 gene from Fragaria vesca (a diploid strawberry) for this research. There is a conserved NAM structural domain in the FvNAC29 protein. The highest homology between FvNAC29 and PaNAC1 was found by phylogenetic tree analysis. Subcellular localization revealed that FvNAC29 is localized onto the nucleus. Compared to other tissues, the expression level of FvNAC29 was higher in young leaves and roots. In addition, Arabidopsis plants overexpressing FvNAC29 had higher cold and high-salinity tolerance than the wild type (WT) and unloaded line with empty vector (UL). The proline and chlorophyll contents of transgenic Arabidopsis plants, along with the activities of the antioxidant enzymes like catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD) under 200 mM NaCl treatment or -8 °C treatment, were higher than those activities of the control. Meanwhile, malondialdehyde (MDA) and the reactive oxygen species (ROS) content were higher in the WT and UL lines. FvNAC29 improves transgenic plant resistance to cold and salt stress by regulating the expression levels of AtRD29a, AtCCA1, AtP5CS1, and AtSnRK2.4. It also improves the potential to tolerate cold stress by positively regulating the expression levels of AtCBF1, AtCBF4, AtCOR15a, and AtCOR47. These findings suggest that FvNAC29 may be related to the processes and the molecular mechanisms of F. vesca response to high-salinity stress and LT stress, providing a comprehensive understanding of the NAC TFs.

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.

Overexpression of transcription factor FaMYB63 enhances salt tolerance by directly binding to the SOS1 promoter in Arabidopsis thaliana.

Salinity is a pivotal abiotic stress factor with far-reaching consequences on global crop growth, yield, and quality and which includes strawberries. R2R3-MYB transcription factors encompass a range of roles in plant development and responses to abiotic stress. In this study, we identified that strawberry transcription factor FaMYB63 exhibited a significant upregulation in its expression under salt stress conditions. An analysis using yeast assay demonstrated that FaMYB63 exhibited the ability to activate transcriptional activity. Compared with those in the wild-type (WT) plants, the seed germination rate, root length, contents of chlorophyll and proline, and antioxidant activities (SOD, CAT, and POD) were significantly higher in FaMYB63-overexpressing Arabidopsis plants exposed to salt stress. Conversely, the levels of malondialdehyde (MDA) were considerably lower. Additionally, the FaMYB63-overexpressed Arabidopsis plants displayed a substantially improved capacity to scavenge active oxygen. Furthermore, the activation of stress-related genes by FaMYB63 bolstered the tolerance of transgenic Arabidopsis to salt stress. It was also established that FaMYB63 binds directly to the promoter of the salt overly sensitive gene SOS1, thereby activating its expression. These findings identified FaMYB63 as a possible and important regulator of salt stress tolerance in strawberries.

Genetic gains underpinning a little-known strawberry Green Revolution.

The annual production of strawberry has increased by one million tonnes in the US and 8.4 million tonnes worldwide since 1960. Here we show that the US expansion was driven by genetic gains from Green Revolution breeding and production advances that increased yields by 2,755%. Using a California population with a century-long breeding history and phenotypes of hybrids observed in coastal California environments, we estimate that breeding has increased fruit yields by 2,974-6,636%, counts by 1,454-3,940%, weights by 228-504%, and firmness by 239-769%. Using genomic prediction approaches, we pinpoint the origin of the Green Revolution to the early 1950s and uncover significant increases in additive genetic variation caused by transgressive segregation and phenotypic diversification. Lastly, we show that the most consequential Green Revolution breeding breakthrough was the introduction of photoperiod-insensitive, PERPETUAL FLOWERING hybrids in the 1970s that doubled yields and drove the dramatic expansion of strawberry production in California.

Changes in Phytohormones and Transcriptomic Reprogramming in Strawberry Leaves under Different Light Qualities.

Strawberry plants require light for growth, but the frequent occurrence of low-light weather in winter can lead to a decrease in the photosynthetic rate (Pn) of strawberry plants. Light-emitting diode (LED) systems could be used to increase Pn. However, the changes in the phytohormones and transcriptomic reprogramming in strawberry leaves under different light qualities are still unclear. In this study, we treated strawberry plants with sunlight, sunlight covered with a 50% sunshade net, no light, blue light (460 nm), red light (660 nm), and a 50% red/50% blue LED light combination for 3 days and 7 days. Our results revealed that the light quality has an effect on the contents of Chl a and Chl b, the minimal fluorescence (F0), and the Pn of strawberry plants. The light quality also affected the contents of abscisic acid (ABA), auxin (IAA), trans-zeatin-riboside (tZ), jasmonic acid (JA), and salicylic acid (SA). RNA sequencing (RNA-seq) revealed that differentially expressed genes (DEGs) are significantly enriched in photosynthesis antenna proteins, photosynthesis, carbon fixation in photosynthetic organisms, porphyrin and chlorophyll metabolisms, carotenoid biosynthesis, tryptophan metabolism, phenylalanine metabolism, zeatin biosynthesis, and linolenic acid metabolism. We then selected the key DEGs based on the results of a weighted gene co-expression network analysis (WGCNA) and drew nine metabolic heatmaps and protein-protein interaction networks to map light regulation.

Structural and transcriptional characterization of pyruvate decarboxylase (PDC) gene family during strawberry fruit ripening process.

Strawberry is one of the most popular fruits in the world, because their high fruit quality, especially with respect to the combination of aroma, flavor, color, and nutritional compounds. Pyruvate decarboxylase (PDC) is the first of two enzymes specifically required for ethanolic fermentation and catalyzes the decarboxylation of pyruvate to yield acetaldehyde and CO2. The ethanol, an important alcohol which acts as a precursor for the ester and other alcohols formation in strawberry, is produced by the PDC. The objective was found all different PDCs genes present in the strawberry genome and investigate PDC gene expression and ligand-protein interactions in strawberry fruit. Volatile organic compounds were evaluated during the development of the fruit. After this, eight FaPDC were identified with four genes that increase the relative expression during fruit ripening process. Molecular dynamics simulations were performed to analyze the behavior of Pyr and TPP ligands within the catalytic and regulatory sites of the PDC proteins. Results indicated that energy-restrained simulations exhibited minor fluctuations in ligand-protein interactions, while unrestrained simulations revealed crucial insights into ligand affinity. TPP consistently displayed strong interactions with the catalytic site, emphasizing its pivotal role in enzymatic activity. However, FaPDC6 and FaPDC9 exhibited decreased pyruvate affinity initially, suggesting unique binding characteristics requiring further investigation. Finally, the present study contributes significantly to understanding PDC gene expression and the intricate molecular dynamics underlying strawberry fruit ripening, shedding light on potential targets for further research in this critical biological pathway.

Leaf dissection and margin serration are independently regulated by two regulators converging on the CUC2-auxin module in strawberry.

The remarkable diversity of leaf forms allows plants to adapt to their living environment. In general, leaf diversity is shaped by leaf complexity (compound or simple) and leaf margin pattern (entire, serrated, or lobed). Prior studies in multiple species have uncovered a conserved module of CUC2-auxin that regulates both leaf complexity and margin serration. How this module is regulated in different species to contribute to the species-specific leaf form is unclear. Furthermore, the mechanistic connection between leaf complexity and leaf serration regulation is not well studied. Strawberry has trifoliate compound leaves with serrations at the margin. In the wild strawberry Fragaria vesca, a mutant named salad was isolated that showed deeper leaf serrations but normal leaf complexity. SALAD encodes a single-Myb domain protein and is expressed at the leaf margin. Genetic analysis showed that cuc2a is epistatic to salad, indicating that SALAD normally limits leaf serration depth by repressing CUC2a expression. When both Arabidopsis homologs of SALAD were knocked out, deeper serrations were observed in Arabidopsis rosette leaves, supporting a conserved function of SALAD in leaf serration regulation. We incorporated the analysis of a third strawberry mutant simple leaf 1 (sl1) with reduced leaf complexity but normal leaf serration. We showed that SL1 and SALAD independently regulate CUC2a at different stages of leaf development to, respectively, regulate leaf complexity and leaf serration. Our results provide a clear and simple mechanism of how leaf complexity and leaf serration are coordinately as well as independently regulated to achieve diverse leaf forms.

[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.

DNA methylation in the wild: epigenetic transgenerational inheritance can mediate adaptation in clones of wild strawberry (Fragaria vesca).

Due to the accelerating climate change, it is crucial to understand how plants adapt to rapid environmental changes. Such adaptation may be mediated by epigenetic mechanisms like DNA methylation, which could heritably alter phenotypes without changing the DNA sequence, especially across clonal generations. However, we are still missing robust evidence of the adaptive potential of DNA methylation in wild clonal populations. Here, we studied genetic, epigenetic and transcriptomic variation of Fragaria vesca, a predominantly clonally reproducing herb. We examined samples from 21 natural populations across three climatically distinct geographic regions, as well as clones of the same individuals grown in a common garden. We found that epigenetic variation was partly associated with climate of origin, particularly in non-CG contexts. Importantly, a large proportion of this variation was heritable across clonal generations. Additionally, a subset of these epigenetic changes affected the expression of genes mainly involved in plant growth and responses to pathogen and abiotic stress. These findings highlight the potential influence of epigenetic changes on phenotypic traits. Our findings indicate that variation in DNA methylation, which can be environmentally inducible and heritable, may enable clonal plant populations to adjust to their environmental conditions even in the absence of genetic adaptation.

FvemiR160-FveARF18A-FveAP1/FveFUL module regulates flowering time in woodland strawberry.

Flowering is an indicator of plant transformation from vegetative to reproductive growth. miR160 has been shown to have a significant effect on the growth and development of fruits, leaves, and roots of plants or their stress response to environment, but the participation of miR160 in regulating flowering time in plants is unclear. In this study, we found that two FvemiR160s (FvemiR160a/FvemiR160b) mature sequences in strawberry (Fragaria vesca) were consistent. It was displayed that the miR160 mature sequence is highly conserved in various species, and the miR160 mature sequence formed by the 5' arm of the MIR160 precursor was more conserved. Three FveARFs in woodland strawberry were negatively regulated by FvemiR160a, among which FveARF18A was the most significant. Phylogenetic analysis indicated that FvemiR160 is closely related to apple (Malus domestica), grape (Vitis vinifera), and Arabidopsis thaliana, while FveARF18A is closely related to RcARF18. Subsequently, we demonstrated that FvemiR160a can target cutting FveARF18A to negatively regulate its expression by RLM-5' RACE, cleavage site mutation, and GFP fluorescence assay. Moreover, we observed that FveMIR160a overexpressed plants have advanced flowering, while mFveARF18A overexpressed plants have delayed flowering. We also verified that FveARF18A negatively regulates the expression of FveAP1 and FveFUL by binding their promoters by yeast one-hybrid, LUC, and GUS assay, and FveAP1 and FveFUL transgenic Arabidopsis showed early flowering phenotype. In addition, the expression level of FvemiR160a was decreased obviously while that of FveARF18A was increased obviously by MeJA, GA and IAA. In conclusion, our study reveals the important role of the FvemiR160-FveARF18A-FveAP1/FveFUL module in the flowering process of woodland strawberry and provides a new pathway for studying flowering.

FvDFR2 rather than FvDFR1 play key roles for anthocyanin synthesis in strawberry petioles.

The accumulation of anthocyanins can be found in both the fruit and petioles of strawberries, but the fruit appears red while the petioles appear purple-red. Additionally, in the white-fruited diploid strawberries, the petioles can accumulate anthocyanins normally, suggesting a different synthesis pattern between the petioles and fruits. We screened the EMS mutagenized population of a red-fruited diploid strawberry 'Ruegen' and discovered a mutant which showed no anthocyanin accumulation in the petioles but normal accumulation in the fruit. Through BSA sequencing and allelic test, it was found that a mutation in FvDFR2 was responsible for this phenotype. Furthermore, the complex formed by the interaction between the petiole-specific FvMYB10L and FvTT8 only binds the promoter of FvDFR2 but not FvDFR1, resulting in the expression of only FvDFR2 in the petiole. FvDFR2 can catalyze the conversion of DHQ and eventually the formation of cyanidin and peonidin, giving the petiole a purplish-red color. In the fruit, however, both FvDFR1 and FvDFR2 can be expressed, which can mediate the synthesis of cyanidin and pelargonidin. Our study clearly reveals different regulation of FvDFR1 and FvDFR2 in mediating anthocyanin synthesis in petioles and fruits.

Construction of a strawberry breeding core collection to capture and exploit genetic variation.

BACKGROUND: Genetic diversity is crucial for the success of plant breeding programs and core collections are important resources to capture this diversity. Many core collections have already been constructed by gene banks, whose main goal is to obtain a panel of a limited number of genotypes to simplify management practices and to improve shareability while retaining as much diversity as possible. However, as gene banks have a different composition and goal than plant breeding programs, constructing a core collection for a plant breeding program should consider different aspects. RESULTS: In this study, we present a novel approach for constructing a core collection by integrating both genomic and pedigree information to maximize the representation of the breeding germplasm in a minimum subset of genotypes while accounting for future genetic variation within a strawberry breeding program. Our stepwise approach starts with selecting the most important crossing parents of advanced selections and genotypes included for specific traits, to represent also future genetic variation. We then use pedigree-genomic-based relationship coefficients combined with the 'accession to nearest entry' criterion to complement the core collection and maximize its representativeness of the current breeding program. Combined pedigree-genomic-based relationship coefficients allow for accurate relationship estimation without the need to genotype every individual in the breeding program. CONCLUSIONS: This stepwise construction of a core collection in a strawberry breeding program can be applied in other plant breeding programs to construct core collections for various purposes.

A Novel R2R3-MYB Transcription Factor FaMYB10-like Promotes Light-Induced Anthocyanin Accumulation in Cultivated Strawberry.

Anthocyanins widely accumulate in the vegetative and reproductive tissues of strawberries and play an important role in stress resistance and fruit quality. Compared with other fruits, little is known about the molecular mechanisms regulating anthocyanin accumulation in strawberry vegetative tissues. In this study, we revealed an R2R3-MYB transcription factor, FaMYB10-like (FaMYB10L), which positively regulated anthocyanin accumulation and was induced by light in the petiole and runner of cultivated strawberry. FaMYB10L is a homologue of FveMYB10-like and a nuclear localization protein. Transient overexpression of FaMYB10L in a white fruit strawberry variety (myb10 mutant) rescued fruit pigmentation, and further qR-PCR analysis revealed that FaMYB10L upregulated the expression levels of anthocyanin biosynthesis-related genes and transport gene. A dual luciferase assay showed that FaMYB10L could activate the anthocyanin transport gene FaRAP. Anthocyanin accumulation was observed in FaMYB10L-overexpressing strawberry calli, and light treatment enhanced anthocyanin accumulation. Furthermore, transcriptomic profiling indicated that the DEGs involved in the flavonoid biosynthesis pathway and induced by light were enriched in FaMYB10L-overexpressing strawberry calli. In addition, yeast two-hybrid assays and luciferase complementation assays indicated that FaMYB10L could interact with bHLH3. These findings enriched the light-involved regulatory network of anthocyanin metabolism in cultivated strawberries.

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.

A medium-density genotyping platform for cultivated strawberry using DArTag technology.

Genomic prediction in breeding populations containing hundreds to thousands of parents and seedlings is prohibitively expensive with current high-density genetic marker platforms designed for strawberry. We developed mid-density panels of molecular inversion probes (MIPs) to be deployed with the "DArTag" marker platform to provide a low-cost, high-throughput genotyping solution for strawberry genomic prediction. In total, 7742 target single nucleotide polymorphism (SNP) regions were used to generate MIP assays that were tested with a screening panel of 376 octoploid Fragaria accessions. We evaluated the performance of DArTag assays based on genotype segregation, amplicon coverage, and their ability to produce subgenome-specific amplicon alignments to the FaRR1 assembly and subsequent alignment-based variant calls with strong concordance to DArT's alignment-free, count-based genotype reports. We used a combination of marker performance metrics and physical distribution in the FaRR1 assembly to select 3K and 5K production panels for genotyping of large strawberry populations. We show that the 3K and 5K DArTag panels are able to target and amplify homologous alleles within subgenomic sequences with low-amplification bias between reference and alternate alleles, supporting accurate genotype calling while producing marker genotypes that can be treated as functionally diploid for quantitative genetic analysis. The 3K and 5K target SNPs show high levels of polymorphism in diverse F. × ananassa germplasm and UC Davis cultivars, with mean pairwise diversity (π) estimates of 0.40 and 0.32 and mean heterozygous genotype frequencies of 0.35 and 0.33, respectively.

4mC-CGRU: Identification of N4-Methylcytosine (4mC) sites using convolution gated recurrent unit in Rosaceae genome.

An epigenetic modification is DNA N4-methylcytosine (4mC) that affects several biological functions without altering the DNA nucleotides, including DNA conformation, cell development, replication, stability, and DNA structural changes. To prevent restriction enzyme from damaging self-DNA, 4mC performs a critical role in restriction-modification functions. Existing studies mainly focused on finding hand-crafted features to identify 4mC locations, but these methods are inefficient due to high time consuming and high costs. In our research work, we propose a 4mC-CGRU which is a deep learning-based computational model with a standard encoding method to identify the 4mC sites from DNA sequences that learned autonomous feature selection in the Rosaceae genome, particularly in Rosa chinensis (R. chinensis) and Fragaria vesca (F. vesca). The proposed model consists of a convolutional neural network (CNN) and a gated recurrent unit network (GRU)-based model for identifying 4mC sites from Fragaria vesca and Rosa chinensis in the genomes. The CNN model extracts useful features from the datasets and the GRU classifies the DNA sequences. Thus, our approach can automatically extract important features to detect relative sites from DNA sequence. The performance analysis shows that the proposed model consistently outperforms over the state-of-the-art works in detecting 4mC sites.

Effects of multiple N, P, and K fertilizer combinations on strawberry growth and the microbial community.

Nitrogen (N), phosphorus (P), and potassium (K) exert various effects on strawberry (Fragaria ananassa Duchesne) yields. In this study, we employed an orthogonal experimental design (T1-T9) with three fertilization treatments (N, P, and K) at three levels to identify an optimal fertilization scheme for strawberry cultivation. The effects of fertilizer combinations the rhizosphere soil microbial community were also explored by using bacterial full-length 16S rRNA and fungal ITS (internal transcribed spacer) sequencing (30 samples for each analysis). The results showed that the average plant height and leaf area of the fertilized groups were 24.6% and 41.6% higher than those of the non-fertilized group (T0). After 60 d of planting, the sucrase activity in the T6 group increased by 76.67% compared to the T0 group, with phosphate fertilizer exerting a more significant impact on sucrase activity. The T6 treatment group had the highest alpha diversity index among bacterial and fungal microorganisms, and had a different microbial community structure compared with the control group. The most abundant bacterial taxa in the strawberry rhizosphere soil were Proteobacteria, Bacteroidota, and Acidobacteriota, and the most abundant fungal phyla were Monoblepharomycota, Glomeromycota, and Mucoromycota. Application of the optimal combined fertilizer treatment (T6) significantly increased the abundance of Proteobacteria and altered the abundance of Gemmatimonas compared to other treatment groups. Notably, Gemmatimonas abundance positively correlated with strawberry plant height and soil N, P, and K levels. These findings indicated that the relative abundance of beneficial bacteria could be enhanced by the application of an optimal fertilizer ratio, ultimately improving strawberry agronomic traits.

Effect of graphene oxide nanoparticles on in vitro growth of Fragaria x ananassa (Cameron Highlands white Strawberry) and evaluation of genetic stability using DAMD and ISSR markers.

Graphene oxide (GO) is a novel nanomaterial with distinct physical properties and significant biological applications. The use of GO in plant tissue culture offers several new properties and potential applications. This research is vital due to the growing need for innovative techniques to promote plant growth, improve plant productivity and mitigate challenges posed by environmental stressors. This study focused on the rare Cameron Highlands white strawberry plants (Fragaria x ananassa) and addressed issues such as callus production during direct shoot induction and hyperhydricity. The research aimed to investigate the effects of GO on the regeneration process and genetic stability of white strawberry plants and to use molecular markers to ensure that plants propagated in vitro are true to type. For this purpose, shoot tip explants were used and different concentrations of GO (0, 2.5, 5.0, 7.5, 10 mg/L) were added to the Murashige and Skoog (MS) medium for six weeks. The results showed that the optimum concentration for promoting the development of white strawberry seedlings was 7.5 mg/L of GO. The study also revealed that the addition of 7.5 mg/L GO in combination with 8 µM TDZ to the MS medium facilitated the induction of multiple shoots. Moreover, the clonal fidelity of the in vitro plants treated with GO showed a genetic similarity of over 97%. These results confirm that lower GO concentrations improve plant development and stability. Consequently, this nanomaterial has a positive effect on the growth of strawberry plants and is therefore well suited for strawberry tissue culture.