refseqR: an R package for common computational operations with records on RefSeq collection.

Summary: We introduce refseqR, an R package that offers a user-friendly solution, enabling common computational operations on RefSeq entries (GenBank, NCBI). The package is specifically designed to interact with records curated from the RefSeq database. Most importantly, the interoperability and integration with several Bioconductor objects allow connections to be applied to other projects. Availability and implementation: The package refseqR is implemented in R and published under the MIT open-source license. The source code, documentation, and usage instructions are available on CRAN (https://CRAN.R-project.org/package=refseqR).

Phenotypic and genetic characterization of a near-isogenic line pair: insights into flowering time in chickpea.

BACKGROUND: Cicer arietinum is a significant legume crop cultivated mainly in short-season environments, where early-flowering is a desirable trait to overcome terminal constraints. Despite its agricultural significance, the genetic control of flowering time in chickpea is not fully understood. In this study, we developed, phenotyped, re-sequenced and genetically characterized a pair of near-isogenic lines (NILs) with contrasting days to flowering to identify candidate gene variants potentially associated with flowering time. RESULTS: In addition to days to flowering, noticeable differences in multiple shoot architecture traits were observed between the NILs. The resequencing data confirms that the NILs developed in this study serve as appropriate plant materials, effectively constraining genetic variation to specific regions and thereby establishing a valuable resource for future genetic and functional investigations in chickpea research. Leveraging bioinformatics tools and public genomic datasets, we identified homologs of flowering-related genes from Arabidopsis thaliana, including ELF3 and, for the first time in chickpea, MED16 and STO/BBX24, with variants among the NILs. Analysis of the allelic distribution of these genes revealed their preservation within chickpea diversity and their potential association with flowering time. Variants were also identified in members of the ERF and ARF gene families. Furthermore, in silico expression analysis was conducted elucidating their putative roles in flowering. CONCLUSIONS: While the gene CaELF3a is identified as a prominent candidate, this study also exposes new targets in chickpea, such as CaMED16b and LOC101499101 (BBX24-like), homologs of flowering-related genes in Arabidopsis, as well as ERF12 and ARF2. The in silico expression characterization and genetic variability analysis performed could contribute to their use as specific markers for chickpea breeding programs. This study lays the groundwork for future investigations utilizing this plant material, promising further insights into the complex mechanisms governing flowering time in chickpea.

Efficient Single Nucleotide Polymorphism Marker-Assisted Selection to Fusarium Wilt in Chickpea.

Fusarium wilt is one of the most destructive chickpea diseases worldwide. Race 5 (Foc5) is the most harmful in the Mediterranean basin. The primary objective of this study is to validate a block of six SNP markers previously mapped in Ca2 in a diverse panel of cultivars, advanced and inbred lines phenotyped for resistance to fusarium wilt. Additionally, we aim to assess the effectiveness of using these markers in the selection of resistant Foc5 lines in an ongoing breeding program. The results showed a 100% coincidence between phenotype and expected haplotype in plant material evaluated for Foc5. We also analyzed 67 inbred lines previously phenotyped by different authors for fusarium wilt reaction, though the specific race was not specified. In these accessions, 65.8% of the analyzed lines exhibited complete correspondence between the phenotype and haplotype. Our results suggest that in early generations it is possible to select resistant materials with reliability, leading to the removal of a significant number of lines, thereby reducing costs and facilitating the handling of materials for additional trait evaluations. Functional annotation of genes delimited by the SNP block revealed several genes in the "response to stimulus" category with potential roles in the resistance reaction.

Genomic data of two chickpea populations sharing a potential Ascochyta blight resistance region.

Chickpea (Cicer arietinum L.) is one of the most important crops worldwide and a valuable nutritional source. The availability of data from different genotypes and populations is important for the comprehension of the biology and trait control of chickpea. Tissue from young leaves was collected from adult plants and sequenced using an Illumina HiSeq X platform, which provided sequencing data for a total of 169 individuals from two different populations. Furthermore, functional annotation was performed with BLAST2GO software in a candidate region for resistance to Ascochyta blight, a devastating disease that produces huge yield reductions if the growth conditions are optimal for the fungus. A total of 273 different genes in a region spanning ∼4.67 Mb in chromosome 4 were fully annotated. The raw DNA sequences and functional annotation data can be reused by the scientific community for the analysis of different agronomic traits of interest in chickpea.

Aldehyde Dehydrogenase 3 Is an Expanded Gene Family with Potential Adaptive Roles in Chickpea.

Legumes play an important role in ensuring food security, improving nutrition and enhancing ecosystem resilience. Chickpea is a globally important grain legume adapted to semi-arid regions under rain-fed conditions. A growing body of research shows that aldehyde dehydrogenases (ALDHs) represent a gene class with promising potential for plant adaptation improvement. Aldehyde dehydrogenases constitute a superfamily of proteins with important functions as 'aldehyde scavengers' by detoxifying aldehydes molecules, and thus play important roles in stress responses. We performed a comprehensive study of the ALDH superfamily in the chickpea genome and identified 27 unique ALDH loci. Most chickpea ALDHs originated from duplication events and the ALDH3 gene family was noticeably expanded. Based on the physical locations of genes and sequence similarities, our results suggest that segmental duplication is a major driving force in the expansion of the ALDH family. Supported by expression data, the findings of this study offer new potential target genes for improving stress tolerance in chickpea that will be useful for breeding programs.

Transcriptome analysis identifies genes related to the waxy coating on blueberry fruit in two northern-adapted rabbiteye breeding populations.

BACKGROUND: Blueberry is of high economic value. Most blueberry varieties selected for the fresh market have an appealing light blue coating or "bloom" on the fruit due to the presence of a visible heavy epicuticular wax layer. This waxy layer also serves as natural defense against fruit desiccation and deterioration. RESULTS: In this study, we attempted to identify gene(s) whose expression is related to the protective waxy coating on blueberry fruit utilizing two unique germplasm populations that segregate for the waxy layer. We bulked RNA from waxy and non-waxy blueberry progenies from the two northern-adapted rabbiteye hybrid breeding populations ('Nocturne' x T 300 and 'Nocturne' x US 1212), and generated 316.85 million RNA-seq reads. We de novo assembled this data set integrated with other publicly available RNA-seq data and trimmed the assembly into a 91,861 blueberry unigene collection. All unigenes were functionally annotated, resulting in 79 genes potentially related to wax accumulation. We compared the expression pattern of waxy and non-waxy progenies using edgeR and identified overall 1125 genes in the T 300 population and 2864 genes in the US 1212 population with at least a two-fold expression difference. After validating differential expression of several genes by RT-qPCR experiments, a candidate gene, FatB, which encodes acyl-[acyl-carrier-protein] hydrolase, emerged whose expression was closely linked to the segregation of the waxy coating in our populations. This gene was expressed at more than a five-fold higher level in waxy than non-waxy plants of both populations. We amplified and sequenced the cDNA for this gene from three waxy plants of each population, but were unable to amplify the cDNA from three non-waxy plants that were tested from each population. We aligned the Vaccinium deduced FATB protein sequence to FATB protein sequences from other plant species. Within the PF01643 domain, which gives FATB its catalytic function, 80.08% of the amino acids were identical or had conservative replacements between the blueberry and the Cucumis melo sequence (XP_008467164). We then amplified and sequenced a large portion of the FatB gene itself from waxy and non-waxy individuals of both populations. Alignment of the cDNA and gDNA sequences revealed that the blueberry FatB gene consists of six exons and five introns. Although we did not sequence through two very large introns, a comparison of the exon sequences found no significant sequence differences between the waxy and non-waxy plants. This suggests that another gene, which regulates or somehow affects FatB expression, must be segregating in the populations. CONCLUSIONS: This study is helping to achieve a greater understanding of epicuticular wax biosynthesis in blueberry. In addition, the blueberry unigene collection should facilitate functional annotation of the coming chromosomal level blueberry genome.

Candidate genes expression profiling during wilting in chickpea caused by Fusarium oxysporum f. sp. ciceris race 5.

Chickpea production may be seriously threatened by Fusarium wilt, a disease caused by the soil-borne fungus Fusarium oxysporum f. sp. ciceris. F. oxysporum race 5 is the most important race in the Mediterranean basin. Recently, the region responsible for resistance race 5 has been delimited within a region on chromosome 2 that spans 820 kb. To gain a better understanding of this genomic region, we used a transcriptomic approach based on quantitative real-time PCR to analyze the expression profiles of 22 selected candidate genes. We used a pair of near-isogenic lines (NILs) differing in their sensitivity to Fusarium race 5 (resistant vs susceptible) to monitor the transcriptional changes over a time-course experiment (24, 48, and 72 hours post inoculation, hpi). Qualitative differences occurred during the timing of regulation. A cluster of 12 genes were induced by the resistant NIL at 24 hpi, whereas a second cluster contained 9 genes induced by the susceptible NIL at 48 hpi. Their possible functions in the molecular defence of chickpea is discussed. Our study provides new insight into the molecular defence against Fusarium race 5 and demonstrates that development of NILs is a rich resource to facilitate the detection of candidate genes. The new genes regulated here may be useful against other Fusarium races.

geneHummus: an R package to define gene families and their expression in legumes and beyond.

BACKGROUND: During the last decade, plant biotechnological laboratories have sparked a monumental revolution with the rapid development of next sequencing technologies at affordable prices. Soon, these sequencing technologies and assembling of whole genomes will extend beyond the plant computational biologists and become commonplace within the plant biology disciplines. The current availability of large-scale genomic resources for non-traditional plant model systems (the so-called 'orphan crops') is enabling the construction of high-density integrated physical and genetic linkage maps with potential applications in plant breeding. The newly available fully sequenced plant genomes represent an incredible opportunity for comparative analyses that may reveal new aspects of genome biology and evolution. The analysis of the expansion and evolution of gene families across species is a common approach to infer biological functions. To date, the extent and role of gene families in plants has only been partially addressed and many gene families remain to be investigated. Manual identification of gene families is highly time-consuming and laborious, requiring an iterative process of manual and computational analysis to identify members of a given family, typically combining numerous BLAST searches and manually cleaning data. Due to the increasing abundance of genome sequences and the agronomical interest in plant gene families, the field needs a clear, automated annotation tool. RESULTS: Here, we present the geneHummus package, an R-based pipeline for the identification and characterization of plant gene families. The impact of this pipeline comes from a reduction in hands-on annotation time combined with high specificity and sensitivity in extracting only proteins from the RefSeq database and providing the conserved domain architectures based on SPARCLE. As a case study we focused on the auxin receptor factors gene (ARF) family in Cicer arietinum (chickpea) and other legumes. CONCLUSION: We anticipate that our pipeline should be suitable for any taxonomic plant family, and likely other gene families, vastly improving the speed and ease of genomic data processing.

Segmental and Tandem Duplications Driving the Recent NBS-LRR Gene Expansion in the Asparagus Genome.

Garden asparagus is an important horticultural plant worldwide. It is, however, susceptible to a variety of diseases, which can affect the potential yield, spear quality, and lifespan of production fields. Screening studies have identified resistant germplasm. The genetic resistance is usually complex, and the genes underlying that resistance are still unknown. Most often, disease resistance is determined by resistance genes (R). The most predominant R-genes contain nucleotide binding site and leucine-rich repeat (NBS-LRR) domains. Using bioinformatics and data mining approaches, we identified and characterized 68 NBS predicted proteins encoded by 49 different loci in the asparagus genome. The NBS-encoding genes were grouped into seven distinct classes based on their domain architecture. The NBS genes are unevenly distributed through the genome and nearly 50% of the genes are present in clusters. Chromosome 6 is significantly NBS-enriched and one single cluster hosts 10% of the genes. Phylogenetic analysis points to their diversification into three families during their evolution. Recent duplications are likely to have dominated the NBS expansion with both tandem genes and duplication events across multiple chromosomes. Transcriptome sequencing data provided evidence for their transcription and tissue-specific expression. The total number of cis-regulatory elements as well as their relative positions within the NBS promoters suggests a complex transcriptional network regulating defense responses. Our study provides a strong groundwork for the isolation of candidate R-genes in garden asparagus.

Genome-wide identification of the auxin response factor gene family in Cicer arietinum.

BACKGROUND: Auxin Response Factors act as critical components of the auxin-signaling pathway by regulating the transcription of auxin-responsive genes. The release of the chickpea reference genome provides an opportunity to identify and characterize the ARF gene family in this important legume by a data mining coupled by comparative genomics approaches. RESULTS: We performed a comprehensive characterization and analysis of 24 ARF genes in the chickpea reference genome. Comparative phylogenetic analysis of the ARF from chickpea, Medicago and Arabidopsis suggests that recent duplications have played a very limited role in the expansion of the ARF chickpea family. Gene structure analysis based on exon-intron organization provides additional evidence to support the evolutionary relationship among the ARF members. Conserved motif analysis shows that most of the proteins fit into the canonical ARF structure model, but 9 proteins lack or have a truncated dimerization domain. The mechanisms underlying the diversification of the ARF gene family are based on duplications, variations in domain organization and alternative splicing. Concerning duplications, segmental, but not tandem duplications, have contributed to the expansion of the gene family. Moreover, the duplicated pair genes have evolved mainly under the influence of purifying selection pressure with restricted functional divergence. Expression profiles responding to various environmental stimuli show a close relationship between tissue and expression patterns. Promoter sequence analysis reveals an enrichment of several cis-regulatory elements related to symbiosis, and modulation of plant gene expression during the interaction with microbes. CONCLUSIONS: In conclusion, this study provides a comprehensive overview of the ARF gene family in chickpea. Globally, our data supports that auxin signaling pathway regulates a wide range of physiological processes and stress responses. Our findings could further provide new insights into the complexity of the regulation of ARF at the transcription level that may be useful to develop rational chickpea breeding strategies to improve development or stress responses. Our study also provides a foundation for comparative genomic analyses and a framework to trace the dynamic evolution of ARF genes on a large time-scale within the legume family.

Genome-scale examination of NBS-encoding genes in blueberry.

Blueberry is an important crop worldwide. It is, however, susceptible to a variety of diseases, which can lead to losses in yield and fruit quality. Although screening studies have identified resistant germplasm for some important diseases, still little is known about the molecular basis underlying that resistance. The most predominant type of resistance (R) genes contains nucleotide binding site and leucine rich repeat (NBS-LRR) domains. The identification and characterization of such a gene family in blueberry would enhance the foundation of knowledge needed for its genetic improvement. In this study, we searched for and found a total of 106 NBS-encoding genes (including 97 NBS-LRR) in the current blueberry genome. The NBS genes were grouped into eleven distinct classes based on their domain architecture. More than 22% of the NBS genes are present in clusters. Ten genes were mapped onto seven linkage groups. Phylogenetic analysis grouped these genes into two major clusters based on their structural variation, the first cluster having toll and interleukin-1 like receptor (TIR) domains and most of the second cluster containing a coiled-coil domain. Our study provides new insight into the NBS gene family in blueberry and is an important resource for the identification of functional R-genes.

Proteome dynamics of cold-acclimating Rhododendron species contrasting in their freezing tolerance and thermonasty behavior.

To gain a better understanding of cold acclimation in rhododendron and in woody perennials in general, we used the 2D-DIGE technique to analyze the rhododendron proteome during the seasonal development of freezing tolerance. We selected two species varying in their cold acclimation ability as well as their thermonasty response (folding of leaves in response to low temperature). Proteins were extracted from leaves of non-acclimated (NA) and cold acclimated (CA) plants of the hardier thermonastic species, R. catawbiense (Cata.), and from leaves of cold acclimated plants of the less hardy, non-thermonastic R. ponticum (Pont.). All three protein samples (Cata.NA, Cata.CA, and Pont.CA) were labeled with different CyDyes and separated together on a single gel. Triplicate gels were run and protein profiles were compared resulting in the identification of 72 protein spots that consistently had different abundances in at least one pair-wise comparison. From the 72 differential spots, we chose 56 spots to excise and characterize further by mass spectrometry (MS). Changes in the proteome associated with the seasonal development of cold acclimation were identified from the Cata.CA-Cata.NA comparisons. Differentially abundant proteins associated with the acquisition of superior freezing tolerance and with the thermonastic response were identified from the Cata.CA-Pont.CA comparisons. Our results indicate that cold acclimation in rhododendron involves increases in abundance of several proteins related to stress (freezing/desiccation tolerance), energy and carbohydrate metabolism, regulation/signaling, secondary metabolism (possibly involving cell wall remodeling), and permeability of the cell membrane. Cold acclimation also involves decreases in abundance of several proteins involved in photosynthesis. Differences in freezing tolerance between genotypes can probably be attributed to observed differences in levels of proteins involved in these functions. Also differences in freezing tolerance may be attributed to higher levels of some constitutive protective proteins in Cata. than in Pont. that may be required to overcome freeze damage, such as glutathione peroxidase, glutamine synthetase, and a plastid-lipid-associated protein.

Selection of internal reference genes for normalization of reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis in the rumen epithelium.

The rumen is lined on the luminal side by a stratified squamous epithelium that is responsible for not only absorption, but also transport, extensive short-chain fatty acid (SCFA) metabolism and protection. Butyrate has been demonstrated to initiate the differentiation of the tissue following introduction of solid feed to the weaning neonate as well as affecting the metabolism of other nutrients and absorption of nutrients in in vitro experiments. The objective of the present study was to validate expression stability of eight putative reference genes bovine rumen, considering the intrinsic heterogeneity of bovine rumen with regard to different luminal characteristics due to direct infusion of butyrate to double the intra-ruminal content of the rumen liquor. Our focus was on identifying stable reference genes which are suitable to normalize real-time RT-qPCR experiments from rumen samples collected from clinical assays, irrespective of localization within the organ and the across physiological state. The most stably expressed genes included: ACTB, UXT, DBNDD2, RPS9, DDX54 and HMBS. Their high stability values suggest these reference genes will facilitate better evaluation of variation of across an array of conditions including: localization within the rumen, differences among cattle fed an array of rations, as well as response to development in the weaning animal. Moreover, we anticipate these reference genes may be useful for expression studies in other ruminants.

Design and Sampling Plan Optimization for RT-qPCR Experiments in Plants: A Case Study in Blueberry.

The qPCR assay has become a routine technology in plant biotechnology and agricultural research. It is unlikely to be technically improved, but there are still challenges which center around minimizing the variability in results and transparency when reporting technical data in support of the conclusions of a study. There are a number of aspects of the pre- and post-assay workflow that contribute to variability of results. Here, through the study of the introduction of error in qPCR measurements at different stages of the workflow, we describe the most important causes of technical variability in a case study using blueberry. In this study, we found that the stage for which increasing the number of replicates would be the most beneficial depends on the tissue used. For example, we would recommend the use of more RT replicates when working with leaf tissue, while the use of more sampling (RNA extraction) replicates would be recommended when working with stems or fruits to obtain the most optimal results. The use of more qPCR replicates provides the least benefit as it is the most reproducible step. By knowing the distribution of error over an entire experiment and the costs at each step, we have developed a script to identify the optimal sampling plan within the limits of a given budget. These findings should help plant scientists improve the design of qPCR experiments and refine their laboratory practices in order to conduct qPCR assays in a more reliable-manner to produce more consistent and reproducible data.

Superior cross-species reference genes: a blueberry case study.

The advent of affordable Next Generation Sequencing technologies has had major impact on studies of many crop species, where access to genomic technologies and genome-scale data sets has been extremely limited until now. The recent development of genomic resources in blueberry will enable the application of high throughput gene expression approaches that should relatively quickly increase our understanding of blueberry physiology. These studies, however, require a highly accurate and robust workflow and make necessary the identification of reference genes with high expression stability for correct target gene normalization. To create a set of superior reference genes for blueberry expression analyses, we mined a publicly available transcriptome data set from blueberry for orthologs to a set of Arabidopsis genes that showed the most stable expression in a developmental series. In total, the expression stability of 13 putative reference genes was evaluated by qPCR and a set of new references with high stability values across a developmental series in fruits and floral buds of blueberry were identified. We also demonstrated the need to use at least two, preferably three, reference genes to avoid inconsistencies in results, even when superior reference genes are used. The new references identified here provide a valuable resource for accurate normalization of gene expression in Vaccinium spp. and may be useful for other members of the Ericaceae family as well.

Carotenogenic gene expression and carotenoid accumulation in three varieties of Cucurbita pepo during fruit development.

The control of gene expression is a crucial regulatory mechanism in carotenoid accumulation of fruits and flowers. We investigated the role of transcriptional regulation of nine genes involved in the carotenoid biosynthesis pathway in three varieties of Cucurbita pepo with evident differences in fruit color. The transcriptional levels of the key genes involved in the carotenoid biosynthesis were higher in flower-, leaf-, and fruit skin tissues than flesh tissues. This correlated with higher concentration of carotenoid content in these tissues. The differential expression among the colored and white cultivars detected for some genes, such as LCYe, in combination with other regulatory mechanisms, could explain the large differences found in terms of carotenoid content among the three varieties. These results are a first step to elucidate carotenogenesis in C. pepo and demonstrate that, in general, regulation of the pathway genes is a critical factor that determines the accumulation of these compounds.

RNA quality assessment: a view from plant qPCR studies.

Reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) is probably the most common molecular technique used in transcriptome analyses today. The simplicity of the technology and associated protocols that generate results without the need to understand the underlying principles has made RT-qPCR the method of choice for RNA quantification. Rather than the 'gold standard technology' often used to describe it, the performance of RT-qPCR suffers from considerable pitfalls during general workflow. The inconsistency of conventional methods for the evaluation of RNA quality and its influence on qPCR performance as well as stability of reference genes is summarized and discussed here.

Characterization of the 3':5' ratio for reliable determination of RNA quality.

Determination of RNA quality is a critical first step in obtaining meaningful gene expression data. The PCR-based 3':5' assay is an RNA quality assessment tool. This assay is a simple, fast, and low-cost method of selecting samples for further analysis. However, its practical applications are unexploited primarily because of the absence of an experimental threshold. We show that, by anchoring the 5' assay a specific distance from the 3' end of the sequence and by spacing the 3' at a distance of a number of nucleotides, a cutoff determines whether a sample is suitable for downstream quantification studies.

Selection of reference genes for gene expression studies in zucchini (Cucurbita pepo) using qPCR.

The zucchini (Cucurbita pepo) is an important food crop, the transcriptomics of which are a fundamental tool to accelerate the development of new varieties by breeders. However, the suitability of reference genes for data normalization in zucchini has not yet been studied. The aim of this study was to assess the suitability of 13 genes for their potential use as reference genes in quantitative real-time PCR. Assays were performed on 34 cDNA samples representing plants under different stresses and at different developmental stages. The application of geNorm and NormFinder software revealed that the use of a combination of UFP, EF-1A, RPL36aA, PP2A, and CAC genes for the different experimental sets was the best strategy for reliable normalization. In contrast, 18S rRNA and TUA were less stable and unsuitable for use as internal controls. These results provide the possibility to allow more accurate use of qPCR in this horticultural crop.