Long read sequencing to reveal the full complexity of a plant transcriptome by targeting both standard and long workflows.

BACKGROUND: Long read sequencing allows the analysis of full-length transcripts in plants without the challenges of reliable transcriptome assembly. Long read sequencing of transcripts from plant genomes has often utilized sized transcript libraries. However, the value of including libraries of differing sizes has not been established. METHODS: A comprehensive transcriptome of the leaves of Jojoba (Simmondsia chinensis) was generated from two different PacBio library preparations: standard workflow (SW) and long workflow (LW). RESULTS: The importance of using both transcript groups in the analysis was demonstrated by the high proportion of unique sequences (74.6%) that were not shared between the groups. A total of 37.8% longer transcripts were only detected in the long dataset. The completeness of the combined transcriptome was indicated by the presence of 98.7% of genes predicted in the jojoba male reference genome. The high coverage of the transcriptome was further confirmed by BUSCO analysis showing the presence of 96.9% of the genes from the core viridiplantae_odb10 lineage. The high-quality isoforms post Cd-Hit merged dataset of the two workflows had a total of 167,866 isoforms. Most of the transcript isoforms were protein-coding sequences (71.7%) containing open reading frames (ORFs) ≥ 100 amino acids (aa). Alternative splicing and intron retention were the basis of most transcript diversity when analysed at the whole genome level and by specific analysis of the apetala2 gene families. CONCLUSION: This suggests the need to specifically target the capture of longer transcripts to provide more comprehensive genome coverage in plant transcriptome analysis and reveal the high level of alternative splicing.

Highly sex specific gene expression in Jojoba.

BACKGROUND: Dioecious plants have male and female flowers on separate plants. Jojoba is a dioecious plant that is drought-tolerant and native to arid areas. The genome sequence of male and female plants was recently reported and revealed an X and Y chromosome system, with two large male-specific insertions in the Y chromosome. RESULTS: A total of 16,923 differentially expressed genes (DEG) were identified between the flowers of the male and female jojoba plants. This represented 40% of the annotated genes in the genome. Many genes, including those responsible for plant environmental responses and those encoding transcription factors (TFs), were specific to male or female reproductive organs. Genes involved in plant hormone metabolism were also found to be associated with flower and pollen development. A total of 8938 up-regulated and 7985 down-regulated genes were identified in comparison between male and female flowers, including many novel genes specific to the jojoba plant. The most differentially expressed genes were associated with reproductive organ development. The highest number of DEG were linked with the Y chromosome in male plants. The male specific parts of the Y chromosome encoded 12 very highly expressed genes including 9 novel genes and 3 known genes associated with TFs and a plant hormone which may play an important role in flower development. CONCLUSION: Many genes, largely with unknown functions, may explain the sexual dimorphisms in jojoba plants and the differentiation of male and female flowers.

The jojoba genome reveals wide divergence of the sex chromosomes in a dioecious plant.

Most flowering plants are hermaphrodites, but around 6% of species are dioecious, having separate male and female plants. Sex chromosomes and some sex-specific genes have been reported in plants, but the genome sequences have not been compared. We now report the genome sequence of male and female jojoba (Simmondsia chinensis) plants, revealing a very large difference in the sex chromosomes. The male genome assembly was 832 Mb and the female 822 Mb. This was explained by the large size differences in the Y chromosome (37.6 Mb) compared with the X chromosome (26.9 Mb). Relative to the X chromosome, the Y chromosome had two large insertions each of more than 5 Mb containing more than 400 genes. Many of the genes in the chromosome-specific regions were novel. These male-specific regions included many flowering-related and stress response genes. Smaller insertions found only in the X chromosome totalled 877 kb. The wide divergence of the sex chromosomes suggests a long period of adaptation to diverging sex-specific roles. Male and female plants may have evolved to accommodate factors such as differing reproductive resource allocation requirements under the stress of the desert environment in which the plants are found. The sex-determining regions accumulate genes beneficial to each sex. This has required the evolution of many more novel sex-specific genes than has been reported for other organisms. This suggest that dioecious plants provide a novel source of genes for manipulation of reproductive performance and environmental adaptation in crops.

Improvements in the sequencing and assembly of plant genomes.

Advances in DNA sequencing have made it easier to sequence and assemble plant genomes. Here, we extend an earlier study, and compare recent methods for long read sequencing and assembly. Updated Oxford Nanopore Technology software improved assemblies. Using more accurate sequences produced by repeated sequencing of the same molecule (Pacific Biosciences HiFi) resulted in less fragmented assembly of sequencing reads. Using data for increased genome coverage resulted in longer contigs, but reduced total assembly length and improved genome completeness. The original model species, Macadamia jansenii, was also compared with three other Macadamia species, as well as avocado (Persea americana) and jojoba (Simmondsia chinensis). In these angiosperms, increasing sequence data volumes caused a linear increase in contig size, decreased assembly length and further improved already high completeness. Differences in genome size and sequence complexity influenced the success of assembly. Advances in long read sequencing technology continue to improve plant genome sequencing and assembly. However, results were improved by greater genome coverage, with the amount needed to achieve a particular level of assembly being species dependent.

Salt-Tolerant Phenomena, Sequencing and Characterization of a Glyoxalase I (Jojo-Gly I) Gene from Jojoba in Comparison with Other Glyoxalase I Genes.

Plant response to salt stress and the mechanism of salt tolerance have received major focus by plant biology researchers. Biotic stresses cause extensive losses in agricultural production globally, but abiotic stress causes significant increase in the methylglyoxal (MG) level of GlyoxalaseI (Gly I). Identification of salt-tolerant genes when characterizing their phenotypes will help to identify novel genes using polymerase chain reaction (PCR) to amplify the DNA coding region for glyoxalase I. This method is specific, requiring only genomic DNA and two pairs of PCR primers, and involving two successive PCR reactions. This method was used rapidly and easily identified glyoxalase I sequences as salt-tolerant genes from Jojoba (Simmondsia chinensis (Link) Schneider). In the present study, the glyoxalase I gene was isolated, amplified by PCR using gene-specific primers and sequenced from the jojoba plant, then compared with other glyoxalase I sequences in other plants and glyoxalase I genes like in Brassica napus, ID: KT720495.1; Brassica juncea ID: Y13239.1, Arachis hypogaea; ID: DQ989209.2; and Arabidopsis thaliana L, ID: AAL84986. The structural gene of glyoxalase I, when sequenced and analyzed, revealed that the uninterrupted open reading frame (ORF) of jojoba Gly I (Jojo-Gly I) spans 775 bp, corresponding to 185 amino acid residues, and shares 45.2% amino acid sequence identity to jojoba (Jojo-Gly I). The cloned ORF, in a multicopy constitutive expression plasmid, complemented the Jojo-Gly I, confirming that the encoded Jojo-Gly I in jojoba showed some homology with other known glyoxalase I sequences of plants.

Profiling microRNA expression during multi-staged date palm (Phoenix dactylifera L.) fruit development.

MicroRNAs (miRNAs) play crucial roles in multiple stages of plant development and regulate gene expression at posttranscriptional and translational levels. In this study, we first identified 238 conserved miRNAs in date palm (Phoenix dactylifera) based on a high-quality genome assembly and defined 78 fruit-development-associated (FDA) miRNAs, whose expression profiles are variable at different fruit development stages. Using experimental data, we subsequently detected 276 novel P. dactylifera-specific FDA miRNAs and predicted their targets. We also revealed that FDA miRNAs function mainly in regulating genes involved in starch/sucrose metabolisms and other carbon metabolic pathways; among them, 221 FDA miRNAs exhibit negative correlation with their corresponding targets, which suggests their direct regulatory roles on mRNA targets. Our data define a comprehensive set of conserved and novel FDA miRNAs along with their expression profiles, which provide a basis for further experimentation in assigning discrete functions of these miRNAs in P. dactylifera fruit development.

Transcriptomic study of the red palm weevil Rhynchophorus ferrugineus embryogenesis.

The red palm weevil (RPW), Rhynchophorus ferrugineus (Coleoptera: Curculionidae), is an invasive, concealed and destructive tissue borer, and it becomes a lethal pest of the palm family of plants and has been reported to attack 20 palm species around the globe. Here we report a systematic transcriptomic study on embryogenesis of RPW, where we analyze the transcriptomes across five developmental stages of RPW embryogenesis, involving four embryonic stages (E1, E2, E3 and E4) and one larval stage (L1). Using the RNA-seq and next-generation platforms, we generated 80 to 91 million reads for each library and assemble 22 532 genes that are expressed at different embryonic stages. Among the total transcripts from the five embryonic development stages, we found that 30.45 % are differentially expressed, 10.10 % show stage-specificity and even a larger fraction, 62.88 %, exhibit constitutive expression in all the stages. We also analyzes the expression dynamics of several conserved signaling pathways (such as Hedgehog, JAK-STAT, Notch, TGF-ß, Ras/MAPK and Wnt), as well as key developmental genes, including those related to apoptosis, axis formation, Hox complex, neurogenesis and segmentation. The datasets provide an essential resource for gene annotation and RPW functional genomics, including studies by using tools and concepts from multiple disciplines, such as development, physiology, biochemistry, molecular biology and genetics.

Seasonally variable intestinal metagenomes of the red palm weevil (Rhynchophorus ferrugineus).

The intestinal microbes residing in the red palm weevil (RPW, Rhynchophorus ferrugineus) larva consume tender interior fibrous tissues of date palm trunks. The understanding of such microbiota at molecular level provides vital clues for the biological control of this devastating pest. Using pyrosequencing and shotgun strategy, we first study taxonomic profiles of the microbiota sampled at different months (March, July and November), and then confirm the impact of high-temperature stress on the microbial populations based on data from 16S rRNA amplicons using both field and laboratory samples. We further identify Klebsiella pneumoniae in November and Lactococcus lactis in July as the dominant species of the microbiota. We find that the RPW gut microbiota degrades polysaccharides and sucrose with hydrolases and that different active bacterial species in November and July are responsible for the symbiotic relationship between the microbiota and the host. Our results provide vital information for pest control and cellulolytic bacterial species characterization.

A large-scale gene discovery for the red palm weevil Rhynchophorus ferrugineus (Coleoptera: Curculionidae).

The red palm weevil (RPW; Rhynchophorus ferrugineus) is a devastating pest of palms, prevalent in the Middle East as well as many other regions of the world. Here, we report a large-scale de novo complementary DNA (cDNA) sequencing effort that acquired ∼5 million reads and assembled them into 26 765 contigs from 12 libraries made from samples of different RPW developmental stages based on the Roche/454 GS FLX platform. We annotated these contigs based on the publically available known insect genes and the Tribolium castaneum genome assembly. We find that over 80% of coding sequences (CDS) from the RPW contigs have high-identity homologs to known proteins with complete CDS. Gene expression analysis shows that the pupa and larval stages have the highest and lowest expression levels, respectively. In addition, we also identified more than 60 000 single nucleotide polymorphisms and 1 200 simple sequence repeat markers. This study provides the first large-scale cDNA dataset for RPW, a much-needed resource for future molecular studies.

Genome sequence of the date palm Phoenix dactylifera L.

Date palm (Phoenix dactylifera L.) is a cultivated woody plant species with agricultural and economic importance. Here we report a genome assembly for an elite variety (Khalas), which is 605.4 Mb in size and covers >90% of the genome (~671 Mb) and >96% of its genes (~41,660 genes). Genomic sequence analysis demonstrates that P. dactylifera experienced a clear genome-wide duplication after either ancient whole genome duplications or massive segmental duplications. Genetic diversity analysis indicates that its stress resistance and sugar metabolism-related genes tend to be enriched in the chromosomal regions where the density of single-nucleotide polymorphisms is relatively low. Using transcriptomic data, we also illustrate the date palm's unique sugar metabolism that underlies fruit development and ripening. Our large-scale genomic and transcriptomic data pave the way for further genomic studies not only on P. dactylifera but also other Arecaceae plants.

The organelle genomes of Hassawi rice (Oryza sativa L.) and its hybrid in saudi arabia: genome variation, rearrangement, and origins.

Hassawi rice (Oryza sativa L.) is a landrace adapted to the climate of Saudi Arabia, characterized by its strong resistance to soil salinity and drought. Using high quality sequencing reads extracted from raw data of a whole genome sequencing project, we assembled both chloroplast (cp) and mitochondrial (mt) genomes of the wild-type Hassawi rice (Hassawi-1) and its dwarf hybrid (Hassawi-2). We discovered 16 InDels (insertions and deletions) but no SNP (single nucleotide polymorphism) is present between the two Hassawi cp genomes. We identified 48 InDels and 26 SNPs in the two Hassawi mt genomes and a new type of sequence variation, termed reverse complementary variation (RCV) in the rice cp genomes. There are two and four RCVs identified in Hassawi-1 when compared to 93-11 (indica) and Nipponbare (japonica), respectively. Microsatellite sequence analysis showed there are more SSRs in the genic regions of both cp and mt genomes in the Hassawi rice than in the other rice varieties. There are also large repeats in the Hassawi mt genomes, with the longest length of 96,168 bp and 96,165 bp in Hassawi-1 and Hassawi-2, respectively. We believe that frequent DNA rearrangement in the Hassawi mt and cp genomes indicate ongoing dynamic processes to reach genetic stability under strong environmental pressures. Based on sequence variation analysis and the breeding history, we suggest that both Hassawi-1 and Hassawi-2 originated from the Indonesian variety Peta since genetic diversity between the two Hassawi cultivars is very low albeit an unknown historic origin of the wild-type Hassawi rice.

Large-scale collection and annotation of gene models for date palm (Phoenix dactylifera, L.).

The date palm (Phoenix dactylifera L.), famed for its sugar-rich fruits (dates) and cultivated by humans since 4,000 B.C., is an economically important crop in the Middle East, Northern Africa, and increasingly other places where climates are suitable. Despite a long history of human cultivation, the understanding of P. dactylifera genetics and molecular biology are rather limited, hindered by lack of basic data in high quality from genomics and transcriptomics. Here we report a large-scale effort in generating gene models (assembled expressed sequence tags or ESTs and mapped to a genome assembly) for P. dactylifera, using the long-read pyrosequencing platform (Roche/454 GS FLX Titanium) in high coverage. We built fourteen cDNA libraries from different P. dactylifera tissues (cultivar Khalas) and acquired 15,778,993 raw sequencing reads-about one million sequencing reads per library-and the pooled sequences were assembled into 67,651 non-redundant contigs and 301,978 singletons. We annotated 52,725 contigs based on the plant databases and 45 contigs based on functional domains referencing to the Pfam database. From the annotated contigs, we assigned GO (Gene Ontology) terms to 36,086 contigs and KEGG pathways to 7,032 contigs. Our comparative analysis showed that 70.6 % (47,930), 69.4 % (47,089), 68.4 % (46,441), and 69.3 % (47,048) of the P. dactylifera gene models are shared with rice, sorghum, Arabidopsis, and grapevine, respectively. We also assigned our gene models into house-keeping and tissue-specific genes based on their tissue specificity.

A complete sequence and transcriptomic analyses of date palm (Phoenix dactylifera L.) mitochondrial genome.

Based on next-generation sequencing data, we assembled the mitochondrial (mt) genome of date palm (Phoenix dactylifera L.) into a circular molecule of 715,001 bp in length. The mt genome of P. dactylifera encodes 38 proteins, 30 tRNAs, and 3 ribosomal RNAs, which constitute a gene content of 6.5% (46,770 bp) over the full length. The rest, 93.5% of the genome sequence, is comprised of cp (chloroplast)-derived (10.3% with respect to the whole genome length) and non-coding sequences. In the non-coding regions, there are 0.33% tandem and 2.3% long repeats. Our transcriptomic data from eight tissues (root, seed, bud, fruit, green leaf, yellow leaf, female flower, and male flower) showed higher gene expression levels in male flower, root, bud, and female flower, as compared to four other tissues. We identified 120 potential SNPs among three date palm cultivars (Khalas, Fahal, and Sukry), and successfully found seven SNPs in the coding sequences. A phylogenetic analysis, based on 22 conserved genes of 15 representative plant mitochondria, showed that P. dactylifera positions at the root of all sequenced monocot mt genomes. In addition, consistent with previous discoveries, there are three co-transcribed gene clusters-18S-5S rRNA, rps3-rpl16 and nad3-rps12-in P. dactylifera, which are highly conserved among all known mitochondrial genomes of angiosperms.

High-throughput sequencing-based gene profiling on multi-staged fruit development of date palm (Phoenix dactylifera, L.).

Date palm provides both staple food and gardening for the Middle East and North African countries for thousands of years. Its fruits have diversified significantly, such as nutritional content, size, length, weight color, and ripping process. Dates palm represent an excellent model system for the study of fruit development and diversity of fruit-bearing palm species that produce the most versatile fruit types as compared to other plant families. Using Roche/454 GS FLX instrument, we acquired 7.6 million sequence tags from seven fruiting stages (F1-F7). Over 99% of the raw reads are assembled, and the numbers of isotigs (equivalent to transcription units or unigenes) range from 30,684 to 40,378 during different fruiting stages. We annotated isotigs using BLASTX and BLASTN, and mapped 74% of the isotigs to known functional sequences or genes. Based on gene ontology categorization and pathway analysis, we have identified 10 core cell division genes, 18 ripening related genes, and 7 starch metabolic enzymes, which are involved as nutrition storage and sugar/starch metabolisms. We noticed that many metabolic pathways vary significantly during fruit development, and carbohydrate metabolism (especially sugar synthesis) is particularly prominent during fruit ripening. Transcriptomics study on various fruiting stages of date palm shows complicated metabolic activities during fruit development, ripening, synthesis and accumulation of starch enzymes and other related sugars. Most Genes are highly expressed in early stages of development, while late developmental stages are critical for fruit ripening including most of the metabolism associated ones.

A pangenomic study of Bacillus thuringiensis.

Bacillus thuringiensis (B. thuringiensis) is a soil-dwelling Gram-positive bacterium and its plasmid-encoded toxins (Cry) are commonly used as biological alternatives to pesticides. In a pangenomic study, we sequenced seven B. thuringiensis isolates in both high coverage and base-quality using the next-generation sequencing platform. The B. thuringiensis pangenome was extrapolated to have 4196 core genes and an asymptotic value of 558 unique genes when a new genome is added. Compared to the pangenomes of its closely related species of the same genus, B. thuringiensis pangenome shows an open characteristic, similar to B. cereus but not to B. anthracis; the latter has a closed pangenome. We also found extensive divergence among the seven B. thuringiensis genome assemblies, which harbor ample repeats and single nucleotide polymorphisms (SNPs). The identities among orthologous genes are greater than 84.5% and the hotspots for the genome variations were discovered in genomic regions of 2.3-2.8Mb and 5.0-5.6Mb. We concluded that high-coverage sequence assemblies from multiple strains, before all the gaps are closed, are very useful for pangenomic studies.

The complete chloroplast genome sequence of date palm (Phoenix dactylifera L.).

BACKGROUND: Date palm (Phoenix dactylifera L.), a member of Arecaceae family, is one of the three major economically important woody palms--the two other palms being oil palm and coconut tree--and its fruit is a staple food among Middle East and North African nations, as well as many other tropical and subtropical regions. Here we report a complete sequence of the data palm chloroplast (cp) genome based on pyrosequencing. METHODOLOGY/PRINCIPAL FINDINGS: After extracting 369,022 cp sequencing reads from our whole-genome-shotgun data, we put together an assembly and validated it with intensive PCR-based verification, coupled with PCR product sequencing. The date palm cp genome is 158,462 bp in length and has a typical quadripartite structure of the large (LSC, 86,198 bp) and small single-copy (SSC, 17,712 bp) regions separated by a pair of inverted repeats (IRs, 27,276 bp). Similar to what has been found among most angiosperms, the date palm cp genome harbors 112 unique genes and 19 duplicated fragments in the IR regions. The junctions between LSC/IRs and SSC/IRs show different features of sequence expansion in evolution. We identified 78 SNPs as major intravarietal polymorphisms within the population of a specific cp genome, most of which were located in genes with vital functions. Based on RNA-sequencing data, we also found 18 polycistronic transcription units and three highly expression-biased genes--atpF, trnA-UGC, and rrn23. CONCLUSIONS: Unlike most monocots, date palm has a typical cp genome similar to that of tobacco--with little rearrangement and gene loss or gain. High-throughput sequencing technology facilitates the identification of intravarietal variations in cp genomes among different cultivars. Moreover, transcriptomic analysis of cp genes provides clues for uncovering regulatory mechanisms of transcription and translation in chloroplasts.