Genome-wide characterization, functional analysis, and expression profiling of the Aux/IAA gene family in spinach.

BACKGROUND: The auxin/indole-3-acetic acid (Aux/IAA) gene family is a crucial element of the auxin signaling pathway, significantly influencing plant growth and development. Hence, we conducted a comprehensive investigation of Aux/IAAs gene family using the Sp75 and Monoe-Viroflay genomes in spinach. RESULTS: A total of 24 definitive Aux/IAA genes were identified, exhibiting diverse attributes in terms of amino acid length, molecular weight, and isoelectric points. This diversity underscores potential specific roles within the family, such as growth regulation and stress response. Structural analysis revealed significant variations in gene length and molecular weight. These variations indicate distinct roles within the Aux/IAA gene family. Chromosomal distribution analysis exhibited a dispersed pattern, with chromosomes 4 and 1 hosting the highest and lowest numbers of Aux/IAA genes, respectively. Phylogenetic analysis grouped the identified genes into distinct clades, revealing potential evolutionary relationships. Notably, the phylogenetic tree highlighted specific gene clusters suggesting shared genetic ancestry and potential functional synergies within spinach. Expression analysis under NAA treatment unveiled gene-specific and time-dependent responses, with certain genes exhibiting distinct temporal expression patterns. Specifically, SpoIAA5 displayed a substantial increase at 2 h post-NAA treatment, while SpoIAA7 and SpoIAA9 demonstrated continuous rises, peaking at the 4-hour time point. CONCLUSIONS: These observations indicate a complex interplay of gene-specific and temporal regulation in response to auxin. Moreover, the comparison with other plant species emphasized both shared characteristics and unique features in Aux/IAA gene numbers, providing insights into the evolutionary dynamics of this gene family. This comprehensive characterization of Aux/IAA genes in spinach not only establishes the foundation for understanding their specific functions in spinach development but also provides a valuable resource for experimental validation and further exploration of their roles in the intricate network of auxin signaling pathways.

Genome-wide screening and characterization of long noncoding RNAs involved in flowering/bolting of Lactuca sativa.

BACKGROUND: Lettuce (Lactuca sativa L.) is considered the most important vegetable in the leafy vegetable group. However, bolting affects quality, gives it a bitter taste, and as a result makes it inedible. Bolting is an event induced by the coordinated effects of various environmental factors and endogenous genetic components. Although bolting/flowering responsive genes have been identified in most sensitive and non-sensitive species, non-coding RNA molecules like long non-coding RNAs (lncRNAs) have not been investigated in lettuce. Hence, in this study, potential long non-coding RNAs that regulate flowering /bolting were investigated in two lettuce strains S24 (resistant strain) and S39 (susceptible strain) in different flowering times to better understand the regulation of lettuce bolting mechanism. For this purpose, we used two RNA-seq datasets to discover the lncRNA transcriptome profile during the transition from vegetative to reproductive phase. RESULTS: For identifying unannotated transcripts in these datasets, a 7-step pipeline was employed to filter out these transcripts and terminate with 293 novel lncRNAs predicted by PLncPRO and CREMA. These transcripts were then utilized to predict cis and trans flowering-associated targets and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Computational predictions of target gene function showed the involvement of putative flowering-related genes and enrichment of the floral regulators FLC, CO, FT, and SOC1 in both datasets. Finally, 17 and 18 lncRNAs were proposed as competing endogenous target mimics (eTMs) for novel and known lncRNA miRNAs, respectively. CONCLUSION: Overall, this study provides new insights into lncRNAs that control the flowering time of plants known for bolting, such as lettuce, and opens new windows for further study.

Global identification of long non-coding RNAs involved in the induction of spinach flowering.

BACKGROUND: Spinach is a beneficial annual vegetable species and sensitive to the bolting or early flowering, which causes a large reduction in quality and productivity. Indeed, bolting is an event induced by the coordinated effects of various environmental factors and endogenous genetic components. Although some key flowering responsive genes have been identified in spinach, non-coding RNA molecules like long non-coding RNAs (lncRNAs) were not investigated yet. Herein, we used bioinformatic approaches to analyze the transcriptome datasets from two different accessions Viroflay and Kashan at two vegetative and reproductive stages to reveal novel lncRNAs and the construction of the lncRNA-mRNA co-expression network. Additionally, correlations among gene expression modules and phenotypic traits were investigated; day to flowering was chosen as our interesting trait. RESULTS: In the present study, we identified a total of 1141 lncRNAs, of which 111 were differentially expressed between vegetative and reproductive stages. The GO and KEGG analyses carried out on the cis target gene of lncRNAs showed that the lncRNAs play an important role in the regulation of flowering spinach. Network analysis pinpointed several well-known flowering-related genes such as ELF, COL1, FLT, and FPF1 and also some putative TFs like MYB, WRKY, GATA, and MADS-box that are important regulators of flowering in spinach and could be potential targets for lncRNAs. CONCLUSIONS: This study is the first report on identifying bolting and flowering-related lncRNAs based on transcriptome sequencing in spinach, which provides a useful resource for future functional genomics studies, genes expression researches, evaluating genes regulatory networks and molecular breeding programs in the regulation of the genetic mechanisms related to bolting in spinach.

Transcriptome architecture reveals genetic networks of bolting regulation in spinach.

BACKGROUND: Bolting refers to the early flowering stem production on agricultural and horticultural crops before harvesting. Indeed, bolting is an event induced by the coordinated effects of various environmental factors and endogenous genetic components, which cause a large reduction in the quality and productivity of vegetable crops like spinach. However, little is known about the signaling pathways and molecular functions involved in bolting mechanisms in spinach. The genetic information regarding the transition from vegetative growth to the reproductive stage in spinach would represent an advantage to regulate bolting time and improvement of resistant cultivars to minimize performance loss. RESULTS: To investigate the key genes and their genetic networks controlling spinach bolting, we performed RNA-seq analysis on early bolting accession Kashan and late-bolting accession Viroflay at both vegetative and reproductive stages and found a significant number of differentially expressed genes (DEGs) ranging from 195 to 1230 in different comparisons. These genes were mainly associated with the signaling pathways of vernalization, photoperiod/circadian clock, gibberellin, autonomous, and aging pathways. Gene ontology analysis uncovered terms associated with carbohydrate metabolism, and detailed analysis of expression patterns for genes of Fructose-1, 6-bisphosphate aldolase, TREHALOSE-6-PHOSPHATE SYNTHASE 1, FLOWERING PROMOTING FACTOR 1, EARLY FLOWERING, GIGANTEA, and MADS-box proteins revealed their potential roles in the initiating or delaying of bolting. CONCLUSION: This study is the first report on identifying bolting and flowering-related genes based on transcriptome sequencing in spinach, which provides insight into bolting control and can be useful for molecular breeding programs and further study in the regulation of the genetic mechanisms related to bolting in other vegetable crops.

Organelle_PBA, a pipeline for assembling chloroplast and mitochondrial genomes from PacBio DNA sequencing data.

BACKGROUND: The development of long-read sequencing technologies, such as single-molecule real-time (SMRT) sequencing by PacBio, has produced a revolution in the sequencing of small genomes. Sequencing organelle genomes using PacBio long-read data is a cost effective, straightforward approach. Nevertheless, the availability of simple-to-use software to perform the assembly from raw reads is limited at present. RESULTS: We present Organelle-PBA, a Perl program designed specifically for the assembly of chloroplast and mitochondrial genomes. For chloroplast genomes, the program selects the chloroplast reads from a whole genome sequencing pool, maps the reads to a reference sequence from a closely related species, and then performs read correction and de novo assembly using Sprai. Organelle-PBA completes the assembly process with the additional step of scaffolding by SSPACE-LongRead. The program then detects the chloroplast inverted repeats and reassembles and re-orients the assembly based on the organelle origin of the reference. We have evaluated the performance of the software using PacBio reads from different species, read coverage, and reference genomes. Finally, we present the assembly of two novel chloroplast genomes from the species Picea glauca (Pinaceae) and Sinningia speciosa (Gesneriaceae). CONCLUSION: Organelle-PBA is an easy-to-use Perl-based software pipeline that was written specifically to assemble mitochondrial and chloroplast genomes from whole genome PacBio reads. The program is available at https://github.com/aubombarely/Organelle_PBA .

Comprehensive genetic discrimination of Leonurus cardiaca populations by AFLP, ISSR, RAPD and IRAP molecular markers.

Leonurus cardiaca is well known for its medicinal importance. In this investigation, genotypic characterization of this species from six eco-geographical regions of Iran was evaluated by four molecular techniques (AFLP, RAPD, ISSR and IRAP). A total of 899 polymorphic fragments were detected by used molecular markers (AFLP = 356, RAPD = 325, ISSR = 113 and IRAP = 105) with an overall average polymorphism of 81.24%. Genetic variation calculated using Shannon's Information index (I) and Nei's gene diversity index (H) showed high genetic diversity in studied germplasm. Also, analysis of molecular variance showed high genetic variation among (55%) and within populations (45%). UPGMA dendrogram constructed from combined data of molecular markers distinguished studied populations in accordance with the results obtained by each marker which all individuals were clearly differentiated into two major clusters. The correlation coefficients were statistically significant for all marker systems with the highest correlation between similarity matrixes of RAPD and ISSR markers (r = 0.82). The present results have an important implication for L. cardiaca germplasm characterization, improvement, and conservation. Furthermore, the characterized individuals exhibited a great deal of molecular variation and they seem to have a rich gene pool for breeding programs.

Transcriptome-guided selection of stable reference genes for expression analysis in spinach.

Accurate measurement of gene expression levels is vital for advancing plant biology research. This study explores the identification and validation of stable reference genes (RGs) for gene expression analysis in Spinacia oleracea. Leveraging transcriptome data from various developmental stages, we employed rigorous statistical analyses to identify potential RGs. A total of 1196 candidate genes were initially screened based on expression variability, with subsequent refinement using criteria such as low variance and stability. Among 12 commonly used candidate RGs, EF1α and H3 emerged as the most stable across diverse experimental conditions, while GRP and PPR exhibited lower stability. These findings were further validated through qRT-PCR assays and comprehensive statistical analyses, including geNorm, NormFinder, BestKeeper, and RefFinder. Our study underscores the importance of systematic RG selection to ensure accurate normalization in gene expression studies, particularly in the context of S. oleracea developmental stages and physiological processes like flowering. These validated RGs provide a robust foundation for future gene expression analysis in S. oleracea and contribute to the advancement of molecular research in plant biology.

Exploring genotypic variation and gene expression associated to cadmium accumulation in bread wheat.

Cadmium (Cd) contamination poses significant risks to agricultural productivity and human health, particularly through its accumulation in staple crops such as bread wheat (Triticum aestivum L.). This study evaluated Cd accumulation and tolerance among six bread wheat cultivars exposed to six Cd concentrations (0, 2.5, 5, 10, 15, 20, and 25 mg kg-1 soil). Phenotypic assessments and quantitative real-time PCR (qRT-PCR) were conducted to analyze the expression patterns of TaNRAMP and TaZIP genes in various tissues and developmental stages of wheat, which play crucial roles in Cd uptake and transport. Results demonstrated significant variability in Cd accumulation. The Barat cultivar exhibited the lowest accumulation in grain (ranging from 0.21 to 8.8 mg kg-1) and the highest tolerance. In contrast, Kavir and Pishtaz displayed elevated Cd levels in both grain and straw, while Parsi accumulated more Cd in straw at lower concentrations (56.9 mg kg-1 in Cd concentration of 10 mg kg-1 soil). The gene expression analysis revealed that most cultivars showed increased expression of TaNRAMP genes, particularly TaNRAMP2 in Cd concentration of 10 mg kg-1 soil, which facilitates Cd uptake from the soil, and TaZIP genes, such as TaZIP4 and TaZIP7, involved in transporting Cd within the plant. Notably, the expression of TaZIP1 was significantly lower in cultivars with high Cd accumulation, suggesting a potential regulatory mechanism for Cd tolerance. Furthermore, cultivars exhibiting higher Cd levels correlated with increased expression of stress-responsive genes, indicating a broader response to Cd stress. These findings highlight Barat's potential for bread-making applications due to its low Cd accumulation, while Morvarid and Pishtaz which show reduced Cd content in the straw even under high Cd exposure are better suited for animal feed. This research underscores the genetic variability of wheat cultivars in response to Cd stress and provides essential insights into the molecular mechanisms underlying Cd accumulation, offering valuable information for breeding programs aimed at developing Cd-tolerant varieties to ensure food security in contaminated regions.

Transcriptome and phytochemical analyses provide insights into the organic sulfur pathway in Allium hirtifolium.

Allium is one of the well-known genera of the Amaryllidaceae family, which contains over 780 species. Onions, garlic, leeks, and shallots are the most important species of this genus. Allium hirtifolium (shallot) is a rich source of proteins, carbohydrates, lipids, amino acids, and bioactive compounds such as organic sulfur compounds with an expansive range of biological activities and medicinal attributes. To identify the putative compounds and genes involved in the organic sulfur pathway, we applied GC-MS and RNA-seq techniques for the bulb, stem, and flower tissues of A. hirtifolium. The essential oil analysis revealed the maximum amount of sulfur compounds in stem against flower and bulb tissues. Transcriptome profiling showed 6155, 6494, and 4259 DEGs for bulb vs. flower, bulb vs. stem, and flower vs. stem, respectively. Overall, more genes were identified as being up-regulated rather than down-regulated in flower tissue compared to the stem and bulb tissues. Our findings in accordance with other results from different papers, suggest that carbohydrates are vital to bulb formation and development because a high number of identified DEGs (586 genes) were mapped to carbohydrate metabolism. This study has detected the genes in the organic sulfur pathway and indicated that the alliinase gene shows a high variability among different tissues. In general, this study formed a useful genomic resource data to explore tissue-specific sulfur pathway in A. hirtifolium, which is helpful for functional breeding.

Identification of the Complex Interplay Between Nematode-Related lncRNAs and Their Target Genes in Glycine max L.

Soybean (Glycine max) is a major plant protein source and oilseed crop. However, plant-parasitic nematodes (PPNs) affect its annual yield. In the current study, in order to better understand the regulation of defense mechanism against PPNs in soybean, we investigated the role of long non-coding RNAs (lncRNAs) in response to two nematode species, Heterodera glycines (SCN: soybean cyst nematode) and Rotylenchulus reniformis (reniform). To this end, two publicly available RNA-seq data sets (SCN data set and RAD: reniform-associated data set) were employed to discover the lncRNAome profile of soybean under SCN and reniform infection, respectively. Upon identification of unannotated transcripts in these data sets, a seven-step pipeline was utilized to sieve these transcripts, which ended up in 384 and 283 potential lncRNAs in SCN data set and RAD, respectively. These transcripts were then used to predict cis and trans nematode-related targets in soybean genome. Computational prediction of target genes function, some of which were also among differentially expressed genes, revealed the involvement of putative nematode-responsive genes as well as enrichment of multiple stress responses in both data sets. Finally, 15 and six lncRNAs were proposed to be involved in microRNA-mediated regulation of gene expression in soybean in response to SNC and reniform infection, respectively. Collectively, this study provides a novel insight into the signaling and regulatory network of soybean-pathogen interactions and opens a new window for further research.

Comparative transcriptome provides molecular insight into defense-associated mechanisms against spider mite in resistant and susceptible common bean cultivars.

Common bean (Phaseolus vulgaris L.) is a major source of proteins and one of the most important edible foods for more than three hundred million people in the world. The common bean plants are frequently attacked by spider mite (Tetranychus urticae Koch), leading to a significant decrease in plant growth and economic performance. The use of resistant cultivars and the identification of the genes involved in plant-mite resistance are practical solutions to this problem. Hence, a comprehensive study of the molecular interactions between resistant and susceptible common bean cultivars and spider mite can shed light into the understanding of mechanisms and biological pathways of resistance. In this study, one resistant (Naz) and one susceptible (Akhtar) cultivars were selected for a transcriptome comparison at different time points (0, 1 and 5 days) after spider mite feeding. The comparison of cultivars in different time points revealed several key genes, which showed a change increase in transcript abundance via spider mite infestation. These included genes involved in flavonoid biosynthesis process; a conserved MYB-bHLH-WD40 (MBW) regulatory complex; transcription factors (TFs) TT2, TT8, TCP, Cys2/His2-type and C2H2-type zinc finger proteins; the ethylene response factors (ERFs) ERF1 and ERF9; genes related to metabolism of auxin and jasmonic acid (JA); pathogenesis-related (PR) proteins and heat shock proteins.

Genome-Wide SNP discovery and genomic characterization in avocado (Persea americana Mill.).

Modern crop breeding is based on the use of genetically and phenotypically diverse plant material and, consequently, a proper understanding of population structure and genetic diversity is essential for the effective development of breeding programs. An example is avocado, a woody perennial fruit crop native to Mesoamerica with an increasing popularity worldwide. Despite its commercial success, there are important gaps in the molecular tools available to support on-going avocado breeding programs. In order to fill this gap, in this study, an avocado 'Hass' draft assembly was developed and used as reference to study 71 avocado accessions which represent the three traditionally recognized avocado horticultural races or subspecies (Mexican, Guatemalan and West Indian). An average of 5.72 M reads per individual and a total of 7,108 single nucleotide polymorphism (SNP) markers were produced for the 71 accessions analyzed. These molecular markers were used in a study of genetic diversity and population structure. The results broadly separate the accessions studied according to their botanical race in four main groups: Mexican, Guatemalan, West Indian and an additional group of Guatemalan × Mexican hybrids. The high number of SNP markers developed in this study will be a useful genomic resource for the avocado community.

Transcriptome Landscape Variation in the Genus Thymus.

Among the Lamiaceae family, the genus Thymus is an economically important genera due to its medicinal and aromatic properties. Most Thymus molecular research has focused on the determining the phylogenetic relationships between different species, but no published work has focused on the evolution of the transcriptome across the genus to elucidate genes involved in terpenoid biosynthesis. Hence, in this study, the transcriptomes of five different Thymus species were generated and analyzed to mine putative genes involved in thymol and carvacrol biosynthesis. High-throughput sequencing produced ~43 million high-quality reads per sample, which were assembled de novo using several tools, then further subjected to a quality evaluation. The best assembly for each species was used as queries to search within the UniProt, KEGG (Kyoto Encyclopedia of Genes and Genomes), COG (Clusters of Orthologous Groups) and TF (Transcription Factors) databases. Mining the transcriptomes resulted in the identification of 592 single-copy orthogroups used for phylogenetic analysis. The data showed strongly support a close genetic relationship between Thymus vulgaris and Thymus daenensis. Additionally, this study dates the speciation events between 1.5-2.1 and 9-10.2 MYA according to different methodologies. Our study provides a global overview of genes related to the terpenoid pathway in Thymus, and can help establish an understanding of the relationship that exists among Thymus species.

Comparative analysis of the root and leaf transcriptomes in Chelidonium majus L.

Chelidonium majus is a traditional medicinal plant, which commonly known as a rich resource for the major benzylisoquinoline alkaloids (BIAs), including morphine, sanguinarine, and berberine. To understand the biosynthesis of C. majus BIAs, we performed de novo transcriptome sequencing of its leaf and root tissues using Illumina technology. Following comprehensive evaluation of de novo transcriptome assemblies produced with five programs including Trinity, Bridger, BinPacker, IDBA-tran, and Velvet/Oases using a series of k-mer sizes (from 25 to 91), BinPacker was found to produce the best assembly using a k-mer of 25. This study reports the results of differential gene expression (DGE), functional annotation, gene ontology (GO) analysis, classification of transcription factor (TF)s, and SSR and miRNA discovery. Our DGE analysis identified 6,028 transcripts that were up-regulated in the leaf, and 4,722 transcripts that were up-regulated in the root. Further investigations showed that most of the genes involved in the BIA biosynthetic pathway are significantly expressed in the root compared to the leaf. GO analysis showed that the predominant GO domain is "cellular component", while TF analysis found bHLH to be the most highly represented TF family. Our study further identified 10 SSRs, out of a total of 39,841, that showed linkage to five unigenes encoding enzymes in the BIA pathway, and 10 conserved miRNAs that were previously not detected in this plant. The comprehensive transcriptome information presented herein provides a foundation for further explorations on study of the molecular mechanisms of BIA synthesis in C. majus.

Assessment of Genetic Diversity and Population Structure in Iranian Cannabis Germplasm.

Cannabis sativa has a complex history reflected in both selection on naturally occurring compounds and historical trade routes among humans. Iran is a rich resource of natural populationswhich hold the promise to characterize historical patterns of population structure and genetic diversity within Cannabis. Recent advances in high-throughput DNA sequencing technologies have dramatically increased our ability to produce information to the point that it is now feasible to inexpensively obtain population level genotype information at a large scale. In the present investigation, we have explored the use of Genotyping-By-Sequencing (GBS) in Iranian cannabis. We genotyped 98 cannabis samples 36 from Iranian locations and 26 accessions from two germplasm collections. In total, 24,710 high-quality Single Nucleotide Polymorphisms (SNP) were identified. Clustering analysis by Principal Component Analysis (PCA) identified two genetic clusters among Iranian populations and fineSTRUCTURE analysis identified 19 populations with some geographic partitioning. We defined Iranian cannabis in two main groups using the results of the PCA and discovered some strong signal to define some locations as population according to fineSTRUCTURE analyses. However, single nucleotide variant analysis uncovered a relatively moderate level of variation among Iranian cannabis.