DNA methylation changes can occur in some loci during callus culture, resulting in somaclonal variations (SVs). In the present study, we applied whole genome bisulfite sequencing to analyze context-specific DNA methylation changes in the pineapple genome between the cutting seedings and 5 SV plants. In general, SV plants exhibited methylation patterns analogous to those of cutting seedlings (CK). A total of 27.98% of the genomic cytosines of CK were methylcytosines, which was higher than that of 5 SV plants. Moreover, mCG and mCHG was hypermethylated, whereas mCHH was hypomethylated among the 5 SV plants genomic when compared with the CK. Most of the variation of DNA methylation was distributed in gene bodies, thus suggesting that phenotypic differences are probably perturbed by genes methylated from callus culture. In addition, the methylated genes were highly enriched for the Gene Ontology (GO) categories of binding and catalytic activity, cell part and organelle, cellular process, abiotic stimulus, and DNA modification. These results suggest that methylation mediates these pathways in the callus culture of pineapple. The results also suggested that the callus culture induced DNA methylation may result in the SV.

DNA methylation plays an important role in somaclonal variation. Cytosine-5 DNA methyltransferases (C5-MTases) are crucial enzymes for maintaining the de novo genome DNA methylation status, and the gene family has been identified and characterized in several species. However, limited information is available about the role of C5-MTases in the somaclonal variation of pineapple. Herein, the characteristic of DNA methylation in the leaf variants of pineapple showed that the methylation rate of variants decreased and the demethylation was mainly. Six AcC5-MTases were identified from pineapple genome. Phylogenetic analysis indicated that the six members could be classified into four subgroups, namely, AcMET, AcCMT, AcDRM, and AcDNMT, demonstrating evolutionary conservation in the C5-MTases across pineapple and other plant species. Cis-elements were presented in AcC5-MTase promoters, including light-responsive, phytohormone-responsive, and plant growth and development elements. Furthermore, we investigated the expression profiles of AcC5-MTases in different tissues and their transcript abundance in the leaves of somaclonal variation. Interestingly, most AcC5-MTase genes were highly expressed in leaf and carpopodium. AcMET, AcCMT2, and AcDRM2 were upregulated in pineapple somaclonal variants, indicating that AcC5-MTases might play an important role in the somaclonal variation of pineapple. Our results will aid in understanding the complex roles of methylation in plants and provide a basis for analyzing the role of methylation modification in the somaclonal variation of pineapple.

The gibberellic acid-stimulated Arabidopsis (GASA) gene family plays a crucial role in growth, development, and stress response, and it is specific to plants. This gene family has been extensively studied in various plant species, and its functional role in pineapple has yet to be characterized. In this study, 15 AcGASA genes were identified in pineapple through a genome-wide scan and categorized into three major branches based on a phylogenetic tree. All AcGASA proteins share a common structural domain with 12 cysteine residues, but they exhibit slight variations in their physicochemical properties and motif composition. Predictions regarding subcellular localization suggest that AcGASA proteins are present in the cell membrane, Golgi apparatus, nucleus, and cell wall. An analysis of gene synteny indicated that both tandem and segmental repeats have a significant impact on the expansion of the AcGASA gene family. Our findings demonstrate the differing regulatory effects of these hormones (GA, NAA, IAA, MeJA, and ABA) on the AcGASA genes. We analyzed the expression profiles of GASA genes in different pineapple tissue parts, and the results indicated that AcGASA genes exhibit diverse expression patterns during the development of different plant tissues, particularly in the regulation of floral organ development. This study provides a comprehensive understanding of GASA family genes in pineapple. It serves as a valuable reference for future studies on the functional characterization of GASA genes in other perennial herbaceous plants.

Exogenous ethylene is commonly utilized to initiate flower induction in pineapple (Ananas comosus (L.) Merr.). However, the molecular mechanisms and metabolic changes involved are not well understood. In this study, we explored the genetic network and metabolic shifts in the ‘Comte de Paris’ pineapple variety during ethylene-induced flowering. This was achieved through an integrative analysis of metabolome and transcriptome profiles at vegetative shoot apexes (0 d after ethephon treatment named BL_0d), the stage of bract primordia (8 d after ethephon treatment named BL_8d), stage of flower primordia (18 d after ethephon treatment named BL_18d), and the stage of stopped floret differentiation (34 d after ethephon treatment named BL_34d). We isolated and identified 804 metabolites in the pineapple shoot apex and inflorescence, categorized into 24 classes. Notably, 29, 31, and 46 metabolites showed significant changes from BL_0d to BL_8d, BL_8d to BL_18d, and BL_18d to BL_34d, respectively. A marked decrease in indole was observed, suggesting its role as a characteristic metabolite during flower induction. Transcriptomic analysis revealed 956, 1768, and 4483 differentially expressed genes (DEGs) for BL_0d vs. BL_8d, BL_8d vs. BL_18d, and BL_18d vs. BL_34d, respectively. These DEGs were significantly enriched in carbohydrate metabolism and hormone signaling pathways, indicating their potential involvement in flower induction. Integrating metabolomic and transcriptomic data, we identified several candidate genes, such as Agamous-Like9 (AGL9), Ethylene Insensitive 3-like (ETIL3), Apetala2 (AP2), AP2-like ethylene-responsive transcription factor ANT (ANT), and Sucrose synthase 2 (SS2), that play potentially crucial roles in ethylene-induced flower induction in pineapple. We also established a regulatory network for pineapple flower induction, correlating metabolites and DEGs, based on the Arabidopsis thaliana pathway as a reference. Overall, our findings offer a deeper understanding of the metabolomic and molecular mechanisms driving pineapple flowering.

Nursery trays are essential agricultural tools in rice production. Plastic nursery trays pose problems such as resource waste and environmental pollution. Biomass seedling trays are an effective way to achieve sustainable agricultural development. Previous research has been conducted on biomass seedling tray molding equipment and molding process, but the impact of raw material ratio on seedling tray molding quality and seedling growth is still unclear, and the ratio combination still needs further optimization. In this study, we used slurry concentration, pulp content, adhesive content, and the ratio of straw to cow manure as variables. We selected the bowl hole molding rate and the strong seedling index as evaluation indicators, and carried out biomass seedling tray forming experiments and seedling cultivation experiments. The response surface analysis method was used to optimize the raw material ratio of biomass seedling trays from the perspectives of forming effect and seedling quality. The results show that when the slurry concentration is 30%, the pulp content is 20%, the adhesive content is 530 g, and the mass ratio of straw to cow manure is 2:1; the bowl hole molding rate is 91.03%, and the strong seedling index is 0.22, indicating good molding effect and seedling growth. The verification test results indicate that the theoretical analysis results are accurate, and the model fitting is good. These research results provide a theoretical basis for the preparation of biomass seedling trays and technical support for achieving green development in agriculture.

Breeding is an effective method for the varietal development of pineapple. However, due to open pollination, it is necessary to conduct authentic identification of the hybrid offspring. In this study, we identified the authenticity of offspring and analyzed the genetic diversity within the offspring F₁ hybrids resulting from crosses between ‘Josapine’ and ‘MD2’ by single nucleotide polymorphism (SNP) markers. From the resequencing data, 26 homozygous loci that differentiate between the parents have been identified. Then, genotyping was performed on both the parents and 36 offspring to select SNP markers that are suitable for authentic identification. The genotyping results revealed that 2 sets of SNP primers, namely SNP4010 and SNP22550, successfully identified 395 authentic hybrids out of 451 hybrid offspring. We randomly selected two true hybrids and four pseudohybrids for sequencing validation, and the results have shown that two true hybrids had double peaks with A/G, while pseudohybrids had single peaks with base A or G. Further study showed that the identification based on SNP molecular markers remained consistent with the morphological identification results in the field, with a true hybridization rate of 87.58%. K-means clustering and UPGMA tree analysis revealed that the hybrid offspring could be categorized into two groups. Among them, 68.5% of offspring aggregated with MD2, while 31.95% were grouped with Josapine. The successful application of SNP marker to identify pineapple F₁ hybrid populations provides a theoretical foundation and practical reference for the future development of rapid SNP marker-based methods for pineapple hybrid authenticity and purity testing.