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The recent development and affordable accessibility of the next-generation highthroughput sequencing technology and artificial intelligence have propelled more researchers to get involved in genomics and to the threshold of a new beginning in understanding, utilizing, and conserving the biodiversity. However, one of the biggest challenges for the analysis of high-throughput sequencing reads is the whole genome assembly and annotation. Availability of user-friendly free software that manages all types of sequenced DNA to be used in a local environment is lacking. Hence, the Genomic Analysis Made Easy (GAME v1) software has been developed using Python to provide a user-friendly, fast, free, and automated GUI-based solution for plant genome assembly and annotation. The software performs on a Linux-based operating environment with a minimum of 16 GB RAM and 100 GB disc space, fully automated from the installation to execution, thus requiring minimal bioinformatics expertise for the execution. The GAME v1 generates detailed quality reports of the raw reads, GenomeScope heterozygosity report, QUAST contigs and scaffolds results, BUSCO summary plot, COG functional annotation chart, GO chart, NCBI and UniProt annotations, KEGG pathway distribution graph, and RepeatMasker plots. The nuclear genome of Chenopodium pallidicaule retrieved from NCBI was assembled and annotated successfully using GAME v1, revealing preliminary genome size estimate of around 419.54 Mb based on GenomeScope analysis prior to assembly. The final assembly, as assessed by QUAST, unveiled a total length of 285.85 Mb (0.29 Gb) containing 23,806 genes. This automated solution will facilitate plant genomics research by revealing the underlying insights of draft nuclear genomes. The software is available at https://arraygen.com/game [ABSTRACT FROM AUTHOR]

Heavy and costly use of phosphorus (P) fertiliser is often needed to achieve high crop yields, but only a small amount of applied P fertiliser is available to most crop plants. Hakea prostrata (Proteaceae) is endemic to the P‐impoverished landscape of southwest Australia and has several P‐saving traits. We identified 16 members of the Phosphate Transporter 1 (PHT1) gene family (HpPHT1;1‐HpPHT1;12d) in a long‐read genome assembly of H. prostrata. Based on phylogenetics, sequence structure and expression patterns, we classified HpPHT1;1 as potentially involved in Pi uptake from soil and HpPHT1;8 and HpPHT1;9 as potentially involved in Pi uptake and root‐to‐shoot translocation. Three genes, HpPHT1;4, HpPHT1;6 and HpPHT1;8, lacked regulatory PHR1‐binding sites (P1BS) in the promoter regions. Available expression data for HpPHT1;6 and HpPHT1;8 indicated they are not responsive to changes in P supply, potentially contributing to the high P sensitivity of H. prostrata. We also discovered a Proteaceae‐specific clade of closely‐spaced PHT1 genes that lacked conserved genetic architecture among genera, indicating an evolutionary hot spot within the genome. Overall, the genome assembly of H. prostrata provides a much‐needed foundation for understanding the genetic mechanisms of novel adaptations to low P soils in southwest Australian plants. [ABSTRACT FROM AUTHOR]

Abstract Background The short-read whole-genome sequencing (WGS) approach has been widely applied to investigate the genomic variation in the natural populations of many plant species. With the rapid advancements in long-read sequencing and genome assembly technologies, high-quality genome sequences are available for a group of varieties for many plant species. These genome sequences are expected to help researchers comprehensively investigate any type of genomic variants that are missed by the WGS technology. However, multiple genome alignment (MGA) tools designed by the human genome research community might be unsuitable for plant genomes. Results To fill this gap, we developed the AnchorWave-Cactus Multiple Genome Alignment (ACMGA) pipeline, which improved the alignment of repeat elements and could identify long (> 50 bp) deletions or insertions (INDELs). We conducted MGA using ACMGA and Cactus for 8 Arabidopsis (Arabidopsis thaliana) and 26 Maize (Zea mays) de novo assembled genome sequences and compared them with the previously published short-read variant calling results. MGA identified more single nucleotide variants (SNVs) and long INDELs than did previously published WGS variant callings. Additionally, ACMGA detected significantly more SNVs and long INDELs in repetitive regions and the whole genome than did Cactus. Compared with the results of Cactus, the results of ACMGA were more similar to the previously published variants called using short-read. These two MGA pipelines identified numerous multi-allelic variants that were missed by the WGS variant calling pipeline. Conclusions Aligning d e novo assembled genome sequences could identify more SNVs and INDELs than mapping short-read. ACMGA combines the advantages of AnchorWave and Cactus and offers a practical solution for plant MGA by integrating global alignment, a 2-piece-affine-gap cost strategy, and the progressive MGA algorithm.

Background: The short-read whole-genome sequencing (WGS) approach has been widely applied to investigate the genomic variation in the natural populations of many plant species. With the rapid advancements in long-read sequencing and genome assembly technologies, high-quality genome sequences are available for a group of varieties for many plant species. These genome sequences are expected to help researchers comprehensively investigate any type of genomic variants that are missed by the WGS technology. However, multiple genome alignment (MGA) tools designed by the human genome research community might be unsuitable for plant genomes. Results: To fill this gap, we developed the AnchorWave-Cactus Multiple Genome Alignment (ACMGA) pipeline, which improved the alignment of repeat elements and could identify long (> 50 bp) deletions or insertions (INDELs). We conducted MGA using ACMGA and Cactus for 8 Arabidopsis (Arabidopsis thaliana) and 26 Maize (Zea mays) de novo assembled genome sequences and compared them with the previously published short-read variant calling results. MGA identified more single nucleotide variants (SNVs) and long INDELs than did previously published WGS variant callings. Additionally, ACMGA detected significantly more SNVs and long INDELs in repetitive regions and the whole genome than did Cactus. Compared with the results of Cactus, the results of ACMGA were more similar to the previously published variants called using short-read. These two MGA pipelines identified numerous multi-allelic variants that were missed by the WGS variant calling pipeline. Conclusions: Aligning denovo assembled genome sequences could identify more SNVs and INDELs than mapping short-read. ACMGA combines the advantages of AnchorWave and Cactus and offers a practical solution for plant MGA by integrating global alignment, a 2-piece-affine-gap cost strategy, and the progressive MGA algorithm. [ABSTRACT FROM AUTHOR]

Abstract Crop pangenomes made from individual cultivar assemblies promise easy access to conserved genes, but genome content variability and inconsistent identifiers hamper their exploration. To address this, we define pangenes, which summarize a species coding potential and link back to original annotations. The protocol get_pangenes performs whole genome alignments (WGA) to call syntenic gene models based on coordinate overlaps. A benchmark with small and large plant genomes shows that pangenes recapitulate phylogeny-based orthologies and produce complete soft-core gene sets. Moreover, WGAs support lift-over and help confirm gene presence-absence variation. Source code and documentation: https://github.com/Ensembl/plant-scripts .

The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is a protein encoded by the PRKDC gene in humans and plays a crucial role in repairing DNA double-strand breaks (DSBs). Recent studies have revealed that DNA-PKcs has additional functions in the cell beyond DSB repair, including transcriptional regulation, telomere protection and capping, preserving chromosomal integrity, and regulating senescence, apoptosis, and autophagy. Moreover, DNA-PKcs has also been implicated in regulating the innate immune response, and dysregulation of DNA-PKcs has been commonly observed in various types of cancers. Until recently it was believed that DNA-PKcs is not present in plants in general. However, DNA-PKcs is conserved in green plants ranging from microscopic green algae such as Ostreococcus of the chlorophytes to the tallest living trees on earth, Sequoia of the gymnosperms. Interestingly, DNAPKcs has not been detected in angiosperms, or in basal angiosperms which are considered sister groups to all other flowering plants. The long polypeptide and gene length of DNA-PKcs coupled with errors in genome assembly, annotation, and gene prediction, have contributed to the challenges in detecting and extracting DNA-PKcs sequences in plant lineages. Sequence alignment showed that several amino acids throughout the length of DNA-PKcs are conserved between plants and human, and all the typical domains identified in human DNA-PKcs are also found in DNA-PKcs from green plants suggesting possible structural and functional conservation. Given the highly conserved nature of DNA repair pathways between mammals and plants further highlights the potential significance of DNA-PKcs in plant biology. [ABSTRACT FROM AUTHOR]

SUMMARY: By contrast to their conserved mammalian counterparts, plant long interspersed nuclear elements (LINEs) are highly variable, splitting into many low‐copy families. Curiously, LINE families from the retrotransposable element (RTE) clade retain a stronger sequence conservation and hence reach higher copy numbers. The cause of this RTE‐typical property is not yet understood, but would help clarify why some transposable elements are removed quickly, whereas others persist in plant genomes. Here, we bring forward a detailed study of RTE LINE structure, diversity and evolution in plants. For this, we argue that the nightshade family is the ideal taxon to follow the evolutionary trajectories of RTE LINEs, given their high abundance, recent activity and partnership to non‐autonomous elements. Using bioinformatic, cytogenetic and molecular approaches, we detect 4029 full‐length RTE LINEs across the Solanaceae. We finely characterize and manually curate a core group of 458 full‐length LINEs in allotetraploid tobacco, show an integration event after polyploidization and trace hybridization by RTE LINE composition of parental genomes. Finally, we reveal the role of the untranslated regions (UTRs) as causes for the unique RTE LINE amplification and evolution pattern in plants. On the one hand, we detected a highly conserved motif at the 3′ UTR, suggesting strong selective constraints acting on the RTE terminus. On the other hand, we observed successive rounds of 5′ UTR cycling, constantly rejuvenating the promoter sequences. This interplay between exchangeable promoters and conserved LINE bodies and 3′ UTR likely allows RTE LINEs to persist and thrive in plant genomes. Significance Statement: Here, we bring forward a detailed study of retrotransposable element (RTE) long interspersed nuclear element (LINE) structure, diversity and evolution in plants and find that the sequence of the evolutionarily optimized RTE LINE body can persist across time and species because it is paired with a constantly renewed 5′ untranslated region (UTR). In this way, RTE LINEs may escape from epigenetic silencing. Hence, 5′ UTR renewal likely works as a regular cure for aging. [ABSTRACT FROM AUTHOR]

The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is a protein encoded by the PRKDC gene in humans and plays a crucial role in repairing DNA double-strand breaks (DSBs). Recent studies have revealed that DNA-PKcs has additional functions in the cell beyond DSB repair, including transcriptional regulation, telomere protection and capping, preserving chromosomal integrity, and regulating senescence, apoptosis, and autophagy. Moreover, DNA-PKcs has also been implicated in regulating the innate immune response, and dysregulation of DNA-PKcs has been commonly observed in various types of cancers. Until recently it was believed that DNA-PKcs is not present in plants in general. However, DNA-PKcs is conserved in green plants ranging from microscopic green algae such as Ostreococcus of the chlorophytes to the tallest living trees on earth, Sequoia of the gymnosperms. Interestingly, DNA-PKcs has not been detected in angiosperms, or in basal angiosperms which are considered sister groups to all other flowering plants. The long polypeptide and gene length of DNA-PKcs coupled with errors in genome assembly, annotation, and gene prediction, have contributed to the challenges in detecting and extracting DNA-PKcs sequences in plant lineages. Sequence alignment showed that several amino acids throughout the length of DNA-PKcs are conserved between plants and human, and all the typical domains identified in human DNA-PKcs are also found in DNA-PKcs from green plants suggesting possible structural and functional conservation. Given the highly conserved nature of DNA repair pathways between mammals and plants further highlights the potential significance of DNA-PKcs in plant biology.

IntroductionClimate change is already affecting ecosystems around the world and forcing us to adapt to meet societal needs. The speed with which climate change is progressing necessitates a massive scaling up of the number of species with understood genotype-environment-phenotype (G×E×P) dynamics in order to increase ecosystem and agriculture resilience. An important part of predicting phenotype is understanding the complex gene regulatory networks present in organisms. Previous work has demonstrated that knowledge about one species can be applied to another using ontologically-supported knowledge bases that exploit homologous structures and homologous genes. These types of structures that can apply knowledge about one species to another have the potential to enable the massive scaling up that is needed through in silico experimentation.MethodsWe developed one such structure, a knowledge graph (KG) using information from Planteome and the EMBL-EBI Expression Atlas that connects gene expression, molecular interactions, functions, and pathways to homology-based gene annotations. Our preliminary analysis uses data from gene expression studies in Arabidopsis thaliana and Populus trichocarpa plants exposed to drought conditions.ResultsA graph query identified 16 pairs of homologous genes in these two taxa, some of which show opposite patterns of gene expression in response to drought. As expected, analysis of the upstream cis-regulatory region of these genes revealed that homologs with similar expression behavior had conserved cis-regulatory regions and potential interaction with similar trans-elements, unlike homologs that changed their expression in opposite ways.DiscussionThis suggests that even though the homologous pairs share common ancestry and functional roles, predicting expression and phenotype through homology inference needs careful consideration of integrating cis and trans-regulatory components in the curated and inferred knowledge graph.

Chromosomal inversions are theorized to play an important role in adaptation by preventing recombination, but testing this hypothesis requires an understanding of the rate of inversion fixation. Here, we use chromosome-level whole-genome assemblies for 32 genera of plants to ask how fast inversions accumulate and what factors affect this rate. We find that on average species accumulate 4–25 inversions per million generations, but this rate is highly variable, and we find no correlation between sequence divergence or repeat content and the number of inversions or the proportion of genome that was inverted and only a small correlation with chromosome size. We also find that inversion regions are depleted for genes and enriched for TEs compared to the genomic background. This suggests that idiosyncratic forces, like natural selection and demography, are controlling how fast inversions fix. [ABSTRACT FROM AUTHOR]

The nucleotide-binding and leucine-rich repeat (NB-LRR) genes, also known as resistance (R)-genes, play an important role in the activation of immune responses. In recent years, large-scale studies have been performed to highlight the diversification of plant NB-LRR repertories. It is well known that, to provide new functionalities, NB-LRR sequences are subject to duplication, domain fusions and acquisition and other kinds of mutations. Although some mechanisms that govern NB-LRR protein domain adaptations have been uncovered, to retrace the plant-lineage-specific evolution routes of R protein structure, a multi-genome comparative analysis was performed. This study allowed us to define groups of genes sharing homology relationships across different species. It is worth noting that the most populated groups contained well-characterized R proteins. The arsenal profile of such groups was investigated in five botanical families, including important crop species, to underline specific adaptation signatures. In addition, the dissection of 70 NB domains of well-characterized R-genes revealed the NB core motifs from which the three main R protein classes have been diversified. The structural remodeling of domain segments shaped the specific NB-LRR repertoires observed in each plant species. This analysis provided new evolutionary and functional insights on NB protein domain shuffling. Taken together, such findings improved our understanding of the molecular adaptive selection mechanisms occurring at plant R loci. [ABSTRACT FROM AUTHOR]

The genome sequence of any organism is key to understanding the biology and utility of that organism. Plants have diverse, complex and sometimes very large nuclear genomes, mitochondrial genomes and much smaller and more highly conserved chloroplast genomes. Plant genome sequences underpin our understanding of plant biology and serve as a key platform for the genetic selection and improvement of crop plants to achieve food security. The development of technology that can capture large volumes of sequence data at low costs and with high accuracy has driven the acceleration of plant genome sequencing advancements. More recently, the development of long read sequencing technology has been a key advance for supporting the accurate sequencing and assembly of chromosome-level plant genomes. This review explored the progress in the sequencing and assembly of plant genomes and the outcomes of plant genome sequencing to date. The outcomes support the conservation of biodiversity, adaptations to climate change and improvements in the sustainability of agriculture, which support food and nutritional security. [ABSTRACT FROM AUTHOR]

Summary: Genome phasing is a recently developed assembly method that separates heterozygous eukaryotic genomic regions and builds haplotype‐resolved assemblies. Because differences between haplotypes are ignored in most published de novo genomes, assemblies are available as consensus genomes consisting of haplotype mixtures, thus increasing the need for genome phasing. Here, we review the operating principles and characteristics of several freely available and widely used phasing tools (TrioCanu, FALCON‐Phase, and ALLHiC). An examination of downstream analyses using haplotype‐resolved genome assemblies in plants indicated significant differences among haplotypes regarding chromosomal rearrangements, sequence insertions, and expression of specific alleles that contribute to the acquisition of the biological characteristics of plant species. Finally, we suggest directions to solve addressing limitations of current genome‐phasing methods. This review provides insights into the current progress, limitations, and future directions of de novo genome phasing, which will enable researchers to easily access and utilize genome‐phasing in studies involving highly heterozygous complex plant genomes. [ABSTRACT FROM AUTHOR]

Long-read DNA sequencing technologies require high molecular weight (HMW) DNA of adequate purity and integrity, which can be difficult to isolate from plant material. Plant leaves usually contain high levels of carbohydrates and secondary metabolites that can impact DNA purity, affecting downstream applications. Several protocols and kits are available for HMW DNA extraction, but they usually require a high amount of input material and often lead to substantial DNA fragmentation, making sequencing suboptimal in terms of read length and data yield. We here describe a protocol for plant HMW DNA extraction from low input material (0.1 g) which is easy to follow and quick (2.5 h). This method successfully enabled us to extract HMW from four species from different families (Orchidaceae, Poaceae, Brassicaceae, Asteraceae). In the case of recalcitrant species, we show that an additional purification step is sufficient to deliver a clean DNA sample. We demonstrate the suitability of our protocol for long-read sequencing on the Oxford Nanopore Technologies PromethION® platform, with and without the use of a short fragment depletion kit. [ABSTRACT FROM AUTHOR]

Long-read DNA sequencing technologies require high molecular weight (HMW) DNA of adequate purity and integrity, which can be difficult to isolate from plant material. Plant leaves usually contain high levels of carbohydrates and secondary metabolites that can impact DNA purity, affecting downstream applications. Several protocols and kits are available for HMW DNA extraction, but they usually require a high amount of input material and often lead to substantial DNA fragmentation, making sequencing suboptimal in terms of read length and data yield. We here describe a protocol for plant HMW DNA extraction from low input material (0.1 g) which is easy to follow and quick (2.5 h). This method successfully enabled us to extract HMW from four species from different families (Orchidaceae, Poaceae, Brassicaceae, Asteraceae). In the case of recalcitrant species, we show that an additional purification step is sufficient to deliver a clean DNA sample. We demonstrate the suitability of our protocol for long-read sequencing on the Oxford Nanopore Technologies PromethION® platform, with and without the use of a short fragment depletion kit.

Simple Summary: Transposable elements are dynamic components of plant genomes, and display a high diversity of lineages and distribution as the result of evolutionary driving forces and overlapping mechanisms of genetic and epigenetic regulation. They are now regarded as main contributors for genome evolution and function, and important regulators of endogenous gene expression. In this review, we survey recent progress and current challenges in the identification and classification of transposon lineages in complex plant genomes, highlighting the molecular specificities that may explain the expansion and diversification of mobile genetic elements in land plants. Transposable elements (TEs) are important components of most plant genomes. These mobile repetitive sequences are highly diverse in terms of abundance, structure, transposition mechanisms, activity and insertion specificities across plant species. This review will survey the different mechanisms that may explain the variability of TE patterns in land plants, highlighting the tight connection between TE dynamics and host genome specificities, and their co-evolution to face and adapt to a changing environment. We present the current TE classification in land plants, and describe the different levels of genetic and epigenetic controls originating from the plant, the TE itself, or external environmental factors. Such overlapping mechanisms of TE regulation might be responsible for the high diversity and dynamics of plant TEs observed in nature. [ABSTRACT FROM AUTHOR]

Abstract Background Plant genomes are rich in repetitive sequences, and transposable elements (TEs) are the most accumulated of them. This mobile fraction can be distinguished as Class I (retrotransposons) and Class II (transposons). Retrotransposons that are transposed using an intermediate RNA and that accumulate in a “copy-and-paste” manner were screened in three genomes of peppers (Solanaceae). The present study aimed to understand the genome relationships among Capsicum annuum, C. chinense, and C. baccatum, based on a comparative analysis of the function, diversity and chromosome distribution of TE lineages in the Capsicum karyotypes. Due to the great commercial importance of pepper in natura, as a spice or as an ornamental plant, these genomes have been widely sequenced, and all of the assemblies are available in the SolGenomics group. These sequences were used to compare all repetitive fractions from a cytogenomic point of view. Results The qualification and quantification of LTR-retrotransposons (LTR-RT) families were contrasted with molecular cytogenetic data, and the results showed a strong genome similarity between C. annuum and C. chinense as compared to C. baccatum. The Gypsy superfamily is more abundant than Copia, especially for Tekay/Del lineage members, including a high representation in C. annuum and C. chinense. On the other hand, C. baccatum accumulates more Athila/Tat sequences. The FISH results showed retrotransposons differentially scattered along chromosomes, except for CRM lineage sequences, which mainly have a proximal accumulation associated with heterochromatin bands. Conclusions The results confirm a close genomic relationship between C. annuum and C. chinense in comparison to C. baccatum. Centromeric GC-rich bands may be associated with the accumulation regions of CRM elements, whereas terminal and subterminal AT- and GC-rich bands do not correspond to the accumulation of the retrotransposons in the three Capsicum species tested.

Diclofenac (DCF) is a very common pharmaceutical that, due to its high use and low removal rate, is considered a prominent contaminant in surface and groundwater worldwide. In this study, Solanum lycopersicum L. cv. Micro-Tom (tomato) was used to disclose the role of glutathione (GSH)-related enzymes, as GSH conjugation with DCF is a well reported detoxification mechanism in mammals and some plant species. To achieve this, S. lycopersicum plants were exposed to 0.5 and 5 mg L−1 of DCF for 5 weeks under a semi-hydroponic experiment. The results here obtained point towards an efficient DCF detoxification mechanism that prevents DCF bioaccumulation in fruits, minimizing any concerns for human health. Although a systemic response seems to be present in response to DCF, the current data also shows that its detoxification is mostly a root-specific process. Furthermore, it appears that GSH-mediated DCF detoxification is the main mechanism activated, as glutathione-S-transferase (GST) activity was greatly enhanced in roots of tomato plants treated with 5 mg L−1 DCF, accompanied by increased glutathione reductase activity, responsible for GSH regeneration. By applying a targeted gene expression analysis, we provide evidence, for the first time, that SlGSTF4 and SlGSTF5 genes, coding for GSTs from phi class, were the main players driving the conjugation of this contaminant. In this sense, and even though tomato plants appear to be somewhat tolerant to DCF exposure, research on GST activity can prove to be instrumental in remediating DCF-contaminated environments and improving plant growth under such conditions. • Pharmaceutical contamination is considered a threat to the environment. • DCF detoxification was achieved through GST-mediated GSH-DCF conjugation. • A sufficient GSH pool was maintained through the regeneration of GSH from GSSG. • SlGSTF4 and SlGSTF5 were the main GST-encoding genes involved in this process. [ABSTRACT FROM AUTHOR]

Abstract Background MicroRNAs (miRNAs) play crucial roles in post-transcriptional regulation of eukaryotic gene expression and are involved in many aspects of plant development. Although several prediction tools are available for metazoan genomes, the number of tools dedicated to plants is relatively limited. Results Here, we present miRkwood, a user-friendly tool for the identification of miRNAs in plant genomes using small RNA sequencing data. Deep-sequencing data of Argonaute associated small RNAs showed that miRkwood is able to identify a large diversity of plant miRNAs and limits false positive predictions. Moreover, it outperforms current tools such as ShortStack and contrary to ShortStack, miRkwood provides a quality score allowing users to rank miRNA predictions. Conclusion miRkwood is a very efficient tool for the annotation of miRNAs in plant genomes. It is available as a web server, as a standalone version, as a docker image and as a Galaxy tool: http://bioinfo.cristal.univ-lille.fr/mirkwood

To evaluate crops generated by new breeding techniques, it is important to confirm the removal of recombinant DNAs (rDNAs) derived from foreign genes including unintentionally introduced short rDNA(s). We attempted to develop a sensitive detection method for such short rDNAs using Southern blot analysis and performed a model study targeting single-copy endogenous genes in plants. To increase the detection sensitivity, the general protocol for Southern blot analysis was modified. In the model study, we used endogenous-gene-targeting probes in which complementary sequences were serially replaced by dummy sequences, and detected complementary sequences as well as 30 bp. We further evaluated the sensitivity using short rDNAs derived from GM sequences as pseudoinsertions, and the results demonstrated that rDNA-insertions as small as 30 bp could be detected. The results suggested that unintentionally introduced rDNA-insertions were 30 bp or more in length could be detected by the Southern blot analysis. Short sequences, equal to or longer than 30 bp, could be detected by a modified Southern blot analysis. The method would be used for an evaluation of crops generated by new breeding techniques. [ABSTRACT FROM AUTHOR]

Background: Plant genomes are rich in repetitive sequences, and transposable elements (TEs) are the most accumulated of them. This mobile fraction can be distinguished as Class I (retrotransposons) and Class II (transposons). Retrotransposons that are transposed using an intermediate RNA and that accumulate in a "copy-and-paste" manner were screened in three genomes of peppers (Solanaceae). The present study aimed to understand the genome relationships among Capsicum annuum, C. chinense, and C. baccatum, based on a comparative analysis of the function, diversity and chromosome distribution of TE lineages in the Capsicum karyotypes. Due to the great commercial importance of pepper in natura, as a spice or as an ornamental plant, these genomes have been widely sequenced, and all of the assemblies are available in the SolGenomics group. These sequences were used to compare all repetitive fractions from a cytogenomic point of view. Results: The qualification and quantification of LTR-retrotransposons (LTR-RT) families were contrasted with molecular cytogenetic data, and the results showed a strong genome similarity between C. annuum and C. chinense as compared to C. baccatum. The Gypsy superfamily is more abundant than Copia, especially for Tekay/Del lineage members, including a high representation in C. annuum and C. chinense. On the other hand, C. baccatum accumulates more Athila/Tat sequences. The FISH results showed retrotransposons differentially scattered along chromosomes, except for CRM lineage sequences, which mainly have a proximal accumulation associated with heterochromatin bands. Conclusions: The results confirm a close genomic relationship between C. annuum and C. chinense in comparison to C. baccatum. Centromeric GC-rich bands may be associated with the accumulation regions of CRM elements, whereas terminal and subterminal AT- and GC-rich bands do not correspond to the accumulation of the retrotransposons in the three Capsicum species tested. [ABSTRACT FROM AUTHOR]

Retrotransposons (repeated DNA sequences capable of moving across the genome) contribute considerably to the evolution of plant genomes. They are involved in determining variation of genome size, in structuring the chromatin, in altering the regulatory patterns of gene regions, also changing the epigenetic setting of DNA. Sequencing of genomic DNA at low coverage (also called genome skimming) and consequent sequence assembling is a practice that can be used in structural genomics to identify and collect retrotransposon sequences in eukaryotic genomes and to gain insights into plant genome structure and evolution. The replicative mechanism of retrotransposons starts with their transcription. Therefore, it is important to have strategies that allow whole genome analysis of their expression. Besides reporting many studies that use genome skimming and assembling for structural genomics, here we point out the use of genome skimming to produce a whole genome library of these elements, to be used as a reference for analysis of retrotransposon expression in RNA-seq libraries, evidencing pros and cons of such a strategy. [ABSTRACT FROM AUTHOR]

Milkweeds (Asclepias) are used in wide-ranging studies including floral development, pollination biology, plant-insect interactions and co-evolution, secondary metabolite chemistry, and rapid diversification. We present a transcriptome and draft nuclear genome assembly of the common milkweed, Asclepias syriaca. This reconstruction of the nuclear genome is augmented by linkage group information, adding to existing chloroplast and mitochondrial genomic resources for this member of the Apocynaceae subfamily Asclepiadoideae. The genome was sequenced to 80.4× depth and the draft assembly contains 54,266 scaffolds ≥1 kbp, with N50 = 3,415 bp, representing 37% (156.6 Mbp) of the estimated 420 Mbp genome. A total of 14,474 protein-coding genes were identified based on transcript evidence, closely related proteins, and ab initio models, and 95% of genes were annotated. A large proportion of gene space is represented in the assembly, with 96.7% of Asclepias transcripts, 88.4% of transcripts from the related genus Calotropis, and 90.6% of proteins from Coffea mapping to the assembly. Scaffolds covering 75 Mbp of the Asclepias assembly formed 11 linkage groups. Comparisons of these groups with pseudochromosomes in Coffea found that six chromosomes show consistent stability in gene content, while one may have a long history of fragmentation and rearrangement. The progesterone 5β-reductase gene family, a key component of cardenolide production, is likely reduced in Asclepias relative to other Apocynaceae. The genome and transcriptome of common milkweed provide a rich resource for future studies of the ecology and evolution of a charismatic plant family. [ABSTRACT FROM AUTHOR]

Background: MicroRNAs (miRNAs) play crucial roles in post-transcriptional regulation of eukaryotic gene expression and are involved in many aspects of plant development. Although several prediction tools are available for metazoan genomes, the number of tools dedicated to plants is relatively limited. Results: Here, we present miRkwood, a user-friendly tool for the identification of miRNAs in plant genomes using small RNA sequencing data. Deep-sequencing data of Argonaute associated small RNAs showed that miRkwood is able to identify a large diversity of plant miRNAs and limits false positive predictions. Moreover, it outperforms current tools such as ShortStack and contrary to ShortStack, miRkwood provides a quality score allowing users to rank miRNA predictions. Conclusion: miRkwood is a very efficient tool for the annotation of miRNAs in plant genomes. It is available as a web server, as a standalone version, as a docker image and as a Galaxy tool: http://bioinfo.cristal.univ-lille.fr/mirkwood [ABSTRACT FROM AUTHOR]

Abstract Computational analyses play crucial roles in characterizing splicing isoforms in plant genomes. In this review, we provide a survey of computational tools used in recently published, genome-scale splicing analyses in plants. We summarize the commonly used software and pipelines for read mapping, isoform reconstruction, isoform quantification, and differential expression analysis. We also discuss methods for analyzing long reads and the strategies to combine long and short reads in identifying splicing isoforms. We review several tools for characterizing local splicing events, splicing graphs, coding potential, and visualizing splicing isoforms. We further discuss the procedures for identifying conserved splicing isoforms across plant species. Finally, we discuss the outlook of integrating other genomic data with splicing analyses to identify regulatory mechanisms of AS on genome-wide scale. Highlights • Review recent progress and discovery in plant alternative splicing. • Summarize computational tools for plant splicing analysis and quantification. • Discuss methods for identification of conserved splicing events and isoforms. • Provide a guideline for computational analysis of splicing in plants. [ABSTRACT FROM AUTHOR]

Summary: The Orchidaceae is a diverse and ecologically important plant family. Approximately 69% of all orchid species are epiphytes, which provide diverse microhabitats for many small animals and fungi in the canopy of tropical rainforests. Moreover, many orchids are of economic importance as food flavourings or ornamental plants. Phalaenopsis aphrodite, an epiphytic orchid, is a major breeding parent of many commercial orchid hybrids. We provide a high‐quality chromosome‐scale assembly of the P. aphrodite genome. The total length of all scaffolds is 1025.1 Mb, with N50 scaffold size of 19.7 Mb. A total of 28 902 protein‐coding genes were identified. We constructed an orchid genetic linkage map, and then anchored and ordered the genomic scaffolds along the linkage groups. We also established a high‐resolution pachytene karyotype of P. aphrodite and completed the assignment of linkage groups to the 19 chromosomes using fluorescence in situ hybridization. We identified an expansion in the epiphytic orchid lineage of FRS5‐like subclade associated with adaptations to the life in the canopy. Phylogenetic analysis further provides new insights into the orchid lineage‐specific duplications of MADS‐box genes, which might have contributed to the variation in labellum and pollinium morphology and its accessory structure. To our knowledge, this is the first orchid genome to be integrated with a SNP‐based genetic linkage map and validated by physical mapping. The genome and genetic map not only offer unprecedented resources for increasing breeding efficiency in horticultural orchids but also provide an important foundation for future studies in adaptation genomics of epiphytes. [ABSTRACT FROM AUTHOR]

Background The growing wealth of knowledge on whole-plant genome sequences is highlighting the key role of transposable elements (TEs) in plant evolution, as a driver of drastic changes in genome size and as a source of an important number of new coding and regulatory sequences. Together with polyploidization events, TEs should thus be considered the major players in evolution of plants. Scope This review outlines the major mechanisms by which TEs impact plant genome evolution and how polyploidy events can affect these impacts, and vice versa. These include direct effects on genes, by providing them with new coding or regulatory sequences, an effect on the epigenetic status of the chromatin close to genes, and more subtle effects by imposing diverse evolutionary constraints to different chromosomal regions. These effects are particularly relevant after polyploidization events. Polyploidization often induces bursts of transposition probably due to a relaxation in their epigenetic control, and, in the short term, this can increase the rate of gene mutations and changes in gene regulation due to the insertion of TEs next to or into genes. Over longer times, TE bursts may induce global changes in genome structure due to inter-element recombination including losses of large genome regions and chromosomal rearrangements that reduce the genome size and the chromosome number as part of a process called diploidization. Conclusions TEs play an essential role in genome and gene evolution, in particular after polyploidization events. Polyploidization can induce TE activity that may explain part of the new phenotypes observed. TEs may also play a role in the diploidization that follows polyploidization events. However, the extent to which TEs contribute to diploidization and fractionation bias remains unclear. Investigating the multiple factors controlling TE dynamics and the nature of ancient and recent polyploid genomes may shed light on these processes. [ABSTRACT FROM AUTHOR]

In our previous study, a methodology was established to predict transcriptional regulatory elements in promoter sequences using transcriptome data based on a frequency comparison of octamers. Some transcription factors, including the NAC family, cannot be covered by this method because their binding sequences have non-specific spacers in the middle of the two binding sites. In order to remove this blind spot in promoter prediction, we have extended our analysis by including bipartite octamers that are composed of '4 bases--a spacer with a flexible length--4 bases'. 8,044 pre-selected bipartite octamers, which had an overrepresentation of specific spacer lengths in promoter sequences and sequences related to core elements removed, were subjected to frequency comparison analysis. Prediction of ER stress-responsive elements in the BiP/BiPL promoter and an ANAC017 target sequence resulted in precise detection of true positives, judged by functional analyses of a reported article and our own in vitro protein-DNA binding assays. These results demonstrate that incorporation of bipartite octamers with continuous ones improves promoter prediction significantly. [ABSTRACT FROM AUTHOR]

Post Arabidopsis thaliana, 55 genomes comprising 49 different plant species have been decoded by use of clone-by-clone, whole genome shotgun and next generation sequencing approaches. The structural outcomes of these sequenced genomes shed light on their genomic constitution, particularly the way genes, transposable elements and genetic markers are organized within the genomes. The functional outcomes provide a brief account of specific phenotypic trait characteristics of crop genomes by digging deep into the genetic make-up of transcription factors, regulatory elements and gene families governing multiple agronomic traits in these crop plants. The comparative and evolutionary outcomes deduce the genetic basis of biological diversity and basic process of genome evolution by analysing the syntenic relationships among genes and genomes/chromosomes of the sequenced crop plants. Therefore, a revisit to published genome sequence landmarks in 30 major cultivated food crops constituting major groups (cereals, legumes, vegetables, fruits, oilseeds and fibres) would significantly assist us to gain a detailed insight into their genome organization and dissect the structural, functional, comparative and evolutionary intricacies for identifying species- and lineage-specific genes controlling multiple characteristics in crop plants. The essential inputs obtained will be helpful in devising efficient strategies to develop high-yielding climate ready crop varieties through translational genomics. [ABSTRACT FROM AUTHOR]

The introduction of the elite pineapple variety, MD-2, has caused a significant market shift in the pineapple industry. Better productivity, overall increased in fruit quality and taste, resilience to chilled storage and resistance to internal browning are among the key advantages of the MD-2 as compared with its previous predecessor, the Smooth Cayenne. Here, we present the genome sequence of the MD-2 pineapple (Ananas comosus (L.) Merr.) by using the hybrid sequencing technology from two highly reputable platforms, i.e. the PacBio long sequencing reads and the accurate Illumina short reads. Our draft genome achieved 99.6% genome coverage with 27,017 predicted protein-coding genes while 45.21% of the genome was identified as repetitive elements. Furthermore, differential expression of ripening RNASeq library of pineapple fruits revealed ethylene-related transcripts, believed to be involved in regulating the process of non-climacteric pineapple fruit ripening. The MD-2 pineapple draft genome serves as an example of how a complex heterozygous genome is amenable to whole genome sequencing by using a hybrid technology that is both economical and accurate. The genome will make genomic applications more feasible as a medium to understand complex biological processes specific to pineapple. [ABSTRACT FROM AUTHOR]

Environmental factors, such as drought, salinity, extreme temperature, ozone poisoning, metal toxicity etc., significantly affect crops. To study these factors and to design a possible remedy, biological experimental data concerning these crops requires the quantification of gene expression and comparative analyses at high throughput level. Development of microarrays is the platform to study the differential expression profiling of the targeted genes. This technology can be applied to gene expression studies, ranging from individual genes to whole genome level. It is now possible to perform the quantification of the differential expression of genes on a glass slide in a single experiment. This review documents recently published reports on the use of microarrays for the identification of genes in different plant species playing their role in different cellular networks under abiotic stresses. The regulation pattern of differentially-expressed genes, individually or in group form, may help us to study different pathways and functions at the cellular and molecular level. These studies can provide us with a lot of useful information to unravel the mystery of abiotic stresses in important crop plants. [ABSTRACT FROM AUTHOR]