Search Results (19)

Phytoplasmas are linked to diseases in hundreds of economically important crops, including carrots. In carrots, phytoplasmosis is associated with leaf chlorosis and necrosis, coupled with inhibited root system development, ultimately leading to significant economic losses. During a field study conducted in Baden-Württemberg (Germany), two strains of the provisional taxon ‘Candidatus Phytoplasma asteris’ were identified within a carrot plot. For further analysis, strains M8 and M33 underwent shotgun sequencing, utilising single-molecule-real-time (SMRT) long-read sequencing and sequencing-by-synthesis (SBS) paired-end short-read sequencing techniques. Hybrid assemblies resulted in complete de novo assemblies of two genomes harboring circular chromosomes and two plasmids. Analyses, including average nucleotide identity and sequence comparisons of established marker genes, confirmed the phylogenetic divergence of ‘Ca. P. asteris’ and a different assignment of strains to the 16S rRNA subgroup I-A for M33 and I-B for M8. These groups exhibited unique features, encompassing virulence factors and genes, associated with the mobilome. In contrast, pan-genome analysis revealed a highly conserved gene set related to metabolism across these strains. This analysis of the Aster Yellows (AY) group reaffirms the perception of phytoplasmas as bacteria that have undergone extensive genome reduction during their co-evolution with the host and an increase of genome size by mobilome. Full article

Third-generation sequencing technology has found widespread application in the genomic, transcriptomic, and epigenetic research of both human and livestock genetics. This technology offers significant advantages in the sequencing of complex genomic regions, the identification of intricate structural variations, and the production of high-quality genomes. Its attributes, including long sequencing reads, obviation of PCR amplification, and direct determination of DNA/RNA, contribute to its efficacy. This review presents a comprehensive overview of third-generation sequencing technologies, exemplified by single-molecule real-time sequencing (SMRT) and Oxford Nanopore Technology (ONT). Emphasizing the research advancements in livestock genomics, the review delves into genome assembly, structural variation detection, transcriptome sequencing, and epigenetic investigations enabled by third-generation sequencing. A comprehensive analysis is conducted on the application and potential challenges of third-generation sequencing technology for genome detection in livestock. Beyond providing valuable insights into genome structure analysis and the identification of rare genes in livestock, the review ventures into an exploration of the genetic mechanisms underpinning exemplary traits. This review not only contributes to our understanding of the genomic landscape in livestock but also provides fresh perspectives for the advancement of research in this domain. Full article

Batocera horsfieldi (Hope) (Coleoptera: Cerambycidae) is an important forest pest in China that mainly infests timber and economic forests. This pest primarily causes plant tissue to necrotize, rot, and eventually die by feeding on the woody parts of tree trunks. To gain a deeper understanding of the genetic mechanism of B. horsfieldi, this study employed single-molecule real-time sequencing (SMRT) and Illumina RNA-seq technologies to conduct full-length transcriptome sequencing of the insect. Total RNA extracted from male and female adults was mixed and subjected to SMRT sequencing, generating a complete transcriptome. Transcriptome analysis, prediction of long non-coding RNA (lncRNA), coding sequences (CDs), analysis of simple sequence repeats (SSR), prediction of transcription factors, and functional annotation of transcripts were performed in this study. The collective 20,356,793 subreads (38.26 G, clean reads) were generated, including 432,091 circular consensus sequences and 395,851 full-length non-chimera reads. The full-length non-chimera reads (FLNC) were clustered and redundancies were removed, resulting in 39,912 consensus reads. SSR and ANGEL software v3.0 were used for predicting SSR and CDs. In addition, four tools were used for annotating 6058 lncRNAs, identifying 636 transcription factors. Furthermore, a total of 84,650 transcripts were functionally annotated in seven different databases. This is the first time that the full-length transcriptome of B. horsfieldi has been obtained using SMRT sequencing. This provides an important foundation for investigating the gene regulation underlying the interaction between B. horsfieldi and its host plants through gene editing in the future and provides a scientific basis for the prevention and control of B. horsfieldi. Full article

Rickett’s big-footed bat, Myotis pilosus, which belongs to the family Vespertilionida, is the only known piscivorous bat in East Asia. Accurate whole genome and transcriptome annotations are essential for the study of bat biological evolution. The lack of a whole genome for M. pilosus has limited our understanding of the molecular mechanisms underlying the species’ evolution, echolocation, and immune response. In the present work, we sequenced the entire transcriptome using error-corrected PacBio single-molecule real-time (SMRT) data. Then, a total of 40 GB of subreads were generated, including 29,991 full-length non-chimeric (FLNC) sequences. After correction by Illumina short reads and de-redundancy, we obtained 26,717 error-corrected isoforms with an average length of 3018.91 bp and an N50 length of 3447 bp. A total of 1528 alternative splicing (AS) events were detected by transcriptome structural analysis. Furthermore, 1032 putative transcription factors (TFs) were identified, with additional identification of several long non-coding RNAs (lncRNAs) with high confidence. Moreover, several key genes, including PRL-2, DPP4, Glul, and ND1 were also identified as being associated with metabolism, immunity, nervous system processes, and auditory perception. A multitude of pattern recognition receptors was identified, including NLR, RLR, SRCR, the antiviral molecule IRF3, and the IFN receptor subunit IFNAR1. High-quality reference genomes at the transcriptome level may be used to quantify gene or transcript expression, evaluate alternative splicing levels, identify novel transcripts, and enhance genome annotation in bats. Full article

As a unique tree species in the Salicaceae family, Chosenia arbutifolia is used primarily for construction materials and landscape planting in China. Compared with other Salicaceae species members, the genomic resources of C. arbutifolia are extremely scarce. Thus, in the present study, the full-length transcriptome of C. arbutifolia was sequenced by single-molecular real-time sequencing (SMRT) technology based on the PacBio platform. Then, it was compared against those of other Salicaceae species. We generated 17,397,064 subreads and 95,940 polished reads with an average length of 1812 bp, which were acquired through calibration, clustering, and polishing. In total, 50,073 genes were reconstructed, of which 48,174 open reading frames, 4281 long non-coding RNAs, and 3121 transcription factors were discovered. Functional annotation revealed that 47,717 genes had a hit in at least one of five reference databases. Moreover, a set of 12,332 putative SSR markers were screened among the reconstructed genes. Single-copy and special orthogroups, and divergent and conserved genes, were identified and analyzed to find divergence among C. arbutifolia and the five Salicaceae species. To reveal genes involved in a specific function and pathway, enrichment analyses for GO and KEGG were also performed. In conclusion, the present study empirically confirmed that SMRT sequencing realistically depicted the C. arbutifolia transcriptome and provided a comprehensive reference for functional genomic research on Salicaceae species. Full article

The highly versatile group of Herpesviruses cause disease in a wide range of hosts. In invertebrates, only two herpesviruses are known: the malacoherpesviruses HaHV-1 and OsHV-1 infecting gastropods and bivalves, respectively. To understand viral transcript architecture and diversity we first reconstructed full-length viral genomes of HaHV-1 infecting Haliotis diversicolor supertexta and OsHV-1 infecting Scapharca broughtonii by DNA-seq. We then used RNA-seq over the time-course of experimental infections to establish viral transcriptional dynamics, followed by PacBio long-read sequencing of full-length transcripts to untangle viral transcript architectures at two selected time points. Despite similarities in genome structure, in the number of genes and in the diverse transcriptomic architectures, we measured a ten-fold higher transcript variability in HaHV-1, with more extended antisense gene transcription. Transcriptional dynamics also appeared different, both in timing and expression trends. Both viruses were heavily affected by post-transcriptional modifications performed by ADAR1 affecting sense-antisense gene pairs forming dsRNAs. However, OsHV-1 concentrated these modifications in a few genomic hotspots, whereas HaHV-1 diluted ADAR1 impact by elongated and polycistronic transcripts distributed over its whole genome. These transcriptional strategies might thus provide alternative potential roles for sense-antisense transcription in viral transcriptomes to evade the host’s immune response in different virus–host combinations. Full article

Leishmania major is the main causative agent of cutaneous leishmaniasis in humans. The Friedlin strain of this species (LmjF) was chosen when a multi-laboratory consortium undertook the objective of deciphering the first genome sequence for a parasite of the genus Leishmania. The objective was successfully attained in 2005, and this represented a milestone for Leishmania molecular biology studies around the world. Although the LmjF genome sequence was done following a shotgun strategy and using classical Sanger sequencing, the results were excellent, and this genome assembly served as the reference for subsequent genome assemblies in other Leishmania species. Here, we present a new assembly for the genome of this strain (named LMJFC for clarity), generated by the combination of two high throughput sequencing platforms, Illumina short-read sequencing and PacBio Single Molecular Real-Time (SMRT) sequencing, which provides long-read sequences. Apart from resolving uncertain nucleotide positions, several genomic regions were reorganized and a more precise composition of tandemly repeated gene loci was attained. Additionally, the genome annotation was improved by adding 542 genes and more accurate coding-sequences defined for around two hundred genes, based on the transcriptome delimitation also carried out in this work. As a result, we are providing gene models (including untranslated regions and introns) for 11,238 genes. Genomic information ultimately determines the biology of every organism; therefore, our understanding of molecular mechanisms will depend on the availability of precise genome sequences and accurate gene annotations. In this regard, this work is providing an improved genome sequence and updated transcriptome annotations for the reference L. major Friedlin strain. Full article

(1) Background: Short-read sequencing allows for the rapid and accurate analysis of the whole bacterial genome but does not usually enable complete genome assembly. Long-read sequencing greatly assists with the resolution of complex bacterial genomes, particularly when combined with short-read Illumina data. However, it is not clear how different assembly strategies affect genomic accuracy, completeness, and protein prediction. (2) Methods: we compare different assembly strategies for Haemophilus parasuis, which causes Glässer’s disease, characterized by fibrinous polyserositis and arthritis, in swine by using Illumina sequencing and long reads from the sequencing platforms of either Oxford Nanopore Technologies (ONT) or SMRT Pacific Biosciences (PacBio). (3) Results: Assembly with either PacBio or ONT reads, followed by polishing with Illumina reads, facilitated high-quality genome reconstruction and was superior to the long-read-only assembly and hybrid-assembly strategies when evaluated in terms of accuracy and completeness. An equally excellent method was correction with Homopolish after the ONT-only assembly, which had the advantage of avoiding hybrid sequencing with Illumina. Furthermore, by aligning transcripts to assembled genomes and their predicted CDSs, the sequencing errors of the ONT assembly were mainly indels that were generated when homopolymer regions were sequenced, thus critically affecting protein prediction. Polishing can fill indels and correct mistakes. (4) Conclusions: The assembly of bacterial genomes can be directly achieved by using long-read sequencing techniques. To maximize assembly accuracy, it is essential to polish the assembly with homologous sequences of related genomes or sequencing data from short-read technology. Full article

This study aimed to obtain the full-length transcriptome of purslane (Portulaca oleracea); assorted plant samples were used for single-molecule real-time (SMRT) sequencing. Based on SMRT, functional annotation of transcripts, transcript factors (TFs) analysis, simple sequence repeat analysis and long non-coding RNAs (LncRNAs) prediction were accomplished. Total 15.33-GB reads were produced; with 9,350,222 subreads and the average length of subreads, 1640 bp was counted. With 99.99% accuracy, after clustering, 132,536 transcripts and 78,559 genes were detected. All unique SMART transcripts were annotated in seven functional databases. 4180 TFs (including transcript regulators) and 7289 LncRNAs were predicted. The results of RNA-seq were confirmed with qRT–PCR analysis. Illumina sequencing of leaves and roots of two purslane genotypes was carried out. Amounts of differential expression genes and related KEGG pathways were found. The expression profiles of related genes in the biosynthesis of unsaturated fatty acids pathway in leaves and roots of two genotypes of purslane were analyzed. Differential expression of genes in this pathway built the foundation of ω-3 fatty acid accumulation in different organs and genotypes of purslane. The aforementioned results provide sequence information and may be a valuable resource for whole-genome sequencing of purslane in the future. Full article

The SLC6A4 gene has been implicated in psychiatric disorder susceptibility and antidepressant response variability. The SLC6A4 promoter is defined by a variable number of homologous 20–24 bp repeats (5-HTTLPR), and long (L) and short (S) alleles are associated with higher and lower expression, respectively. However, this insertion/deletion variant is most informative when considered as a haplotype with the rs25531 and rs25532 variants. Therefore, we developed a long-read single molecule real-time (SMRT) sequencing method to interrogate the SLC6A4 promoter region. A total of 120 samples were subjected to SLC6A4 long-read SMRT sequencing, primarily selected based on available short-read sequencing data. Short-read genome sequencing from the 1000 Genomes (1KG) Project (~5X) and the Genetic Testing Reference Material Coordination Program (~45X), as well as high-depth short-read capture-based sequencing (~330X), could not identify the 5-HTTLPR short (S) allele, nor could short-read sequencing phase any identified variants. In contrast, long-read SMRT sequencing unambiguously identified the 5-HTTLPR short (S) allele (frequency of 0.467) and phased SLC6A4 promoter haplotypes. Additionally, discordant rs25531 genotypes were reviewed and determined to be short-read errors. Taken together, long-read SMRT sequencing is an innovative and robust method for phased resolution of the SLC6A4 promoter, which could enable more accurate pharmacogenetic testing for both research and clinical applications. Full article

To gain more valuable genomic information about betalain biosynthesis, the full-length transcriptome of pitaya pulp from ‘Zihonglong’ (red pulp) and ‘Jinghonglong’ (white pulp) in four fruit developmental stages was analyzed using Single-Molecule Real-Time (SMRT) sequencing corrected by Illumina RNA-sequence (Illumina RNA-Seq). A total of 65,317 and 91,638 genes were identified in ‘Zihonglong’ and ‘Jinghonglong’, respectively. A total of 11,377 and 15,551 genes with more than two isoforms were investigated from ‘Zihonglong’ and ‘Jinghonglong’, respectively. In total, 156,955 genes were acquired after elimination of redundancy, of which, 120,604 genes (79.63%) were annotated, and 30,875 (20.37%) sequences without hits to reference database were probably novel genes in pitaya. A total of 31,169 and 53,024 simple sequence repeats (SSRs) were uncovered from the genes of ‘Zihonglong’ and ‘Jinghonglong’, and 11,650 long non-coding RNAs (lncRNAs) in ‘Zihonglong’ and 11,113 lncRNAs in ‘Jinghonglong’ were obtained herein. qRT-PCR was conducted on ten candidate genes, the expression level of six novel genes were consistent with the Fragments Per Kilobase of transcript per Million mapped reads (FPKM) values. In conclusion, we firstly undertook SMRT sequencing of the full-length transcriptome of pitaya, and the valuable resource that was acquired through this sequencing facilitated the identification of additional betalain-related genes. Notably, a list of novel putative genes related to the synthesis of betalain in pitaya fruits was assembled. This may provide new insights into betalain synthesis in pitaya. Full article

Shepherd’s purse (Capsella bursa-pastoris) is a cosmopolitan annual weed and a promising model plant for studying allopolyploidization in the evolution of angiosperms. Though plant mitochondrial genomes are a valuable source of genetic information, they are hard to assemble. At present, only the complete mitogenome of C. rubella is available out of all species of the genus Capsella. In this work, we have assembled the complete mitogenome of C. bursa-pastoris using high-precision PacBio SMRT third-generation sequencing technology. It is 287,799 bp long and contains 32 protein-coding genes, 3 rRNAs, 25 tRNAs corresponding to 15 amino acids, and 8 open reading frames (ORFs) supported by RNAseq data. Though many repeat regions have been found, none of them is longer than 1 kbp, and the most frequent structural variant originated from these repeats is present in only 4% of the mitogenome copies. The mitochondrial DNA sequence of C. bursa-pastoris differs from C. rubella, but not from C. orientalis, by two long inversions, suggesting that C. orientalis could be its maternal progenitor species. In total, 377 C to U RNA editing sites have been detected. All genes except cox1 and atp8 contain RNA editing sites, and most of them lead to non-synonymous changes of amino acids. Most of the identified RNA editing sites are identical to corresponding RNA editing sites in A. thaliana. Full article

Camellia oleifera Abel. is a critical oil tree species. Camellia oil, which is extracted from the seeds, is widely regarded as a premium cooking oil, with the content of oleic acid being over 80%. Light is thought to be one of the largest essential natural components in the regulation of plant developmental processes, and different light qualities can considerably influence plant physiological and phenotypic traits. In this research, we examined the growth and physiological responses of C. oleifera “MIN 43” cultivar plantlets to three different wavelengths of light, containing white, red, and blue light, and we utilized the combination of the PacBio single-molecule real-time (SMRT) and Illumina HiSeq RNA sequencing to obtain the mRNA expression profiles. The results showed that plantlets growing under blue light conditions displayed superior growth performance, including stimulated enhancement of the leaf area, increased leaf number, increased chlorophyll synthesis, and improved photosynthesis. Furthermore, SMAT sequencing created 429,955 reads of inserts, where 406,722 of them were full-length non-chimeric reads, and 131,357 non-redundant isoforms were produced. Abundant differentially expressed genes were found in leaves under different light qualities by RNA-sequencing. Gene expression profiles of actin, dynein, tubulin, defectively organized tributaries 3 (DOT3), and ADP ribosylation factor 5 (ARF5) were associated with the greatest leaf performance occurring under blue light conditions. Moreover, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis identified hundreds of pathways involved in different light conditions. The pathways of the plant circadian rhythm and hormone signal transduction were associated with different light quality responses in C. oleifera. Phytochrome B (PHYB), constitutively photomorphogenic 1 (COP1), long hypocotyl 5 (HY5), auxin/indole-3-acetic acid (AUX/IAA), Gretchen Hagen 3 (GH3), and small auxin-up RNA (SAUR), which were differentially expressed genes involved in these two pathways, play a vital role in responses to different wavelengths of light in C. oleifera. In addition, blue light significantly promotes flavonoid biosynthesis via changing expression of related genes. Full article

The genus Gnetum includes pantropical trees, shrubs and lianas, with unresolved phylogenetic relationships with other seed plant groups. Despite the reference genome for this genus being recently published, the molecular mechanisms that regulate the reproductive organ development of Gnetum remain unclear. A previous study showed that indole-3-acetic acid is involved in the regulation of female strobili of Gnetum, while the diversity and evolution of indole-3-acetic acid-related genes—the Aux/IAA genes—have never been investigated in Gnetales. Thus, a pooled sample from different developmental stages of female strobili in Gnetum luofuense C.Y. Cheng was sequenced using PacBio single-molecular long-read technology (SMRT) sequencing. PacBio SMRT sequencing generated a total of 53,057 full-length transcripts, including 2043 novel genes. Besides this, 10,454 alternative splicing (AS) events were detected with intron retention constituting the largest proportion (46%). Moreover, 1196 lncRNAs were identified, and 8128 genes were found to possess at least one poly (A) site. A total of 3179 regulatory proteins, including 1413 transcription factors (e.g., MADS-box and bHLHs), 477 transcription regulators (e.g., SNF2), and 1289 protein kinases (e.g., RLK/Pelles) were detected, and these protein regulators probably participated in the female strobili development of G. luofuense. In addition, this is the first study of the Aux/IAA genes of the Gnetales, and we identified 6, 7 and 12 Aux/IAA genes from Gnetum luofuense, Welwitschia mirabilis, and Ephedra equistina, respectively. Our phylogenetic analysis reveals that Aux/IAA genes from the gymnosperms tended to cluster and possessed gene structures as diverse as those in angiosperms. Moreover, the Aux/IAA genes of the Gnetales might possess higher molecular evolutionary rates than those in other gymnosperms. The sequencing of the full-length transcriptome paves the way to uncovering molecular mechanisms that regulate reproductive organ development in gymnosperms. Full article

DNA barcoding has been used for decades, although it has mostly been applied to some single-species. Traditional Chinese medicine (TCM), which is mainly used in the form of combination-one type of the multi-species, identification is crucial for clinical usage. Next-generation Sequencing (NGS) has been used to address this authentication issue for the past few years, but conventional NGS technology is hampered in application due to its short sequencing reads and systematic errors. Here, a novel method, Full-length multi-barcoding (FLMB) via long-read sequencing, is employed for the identification of biological compositions in herbal compound formulas in adequate and well controlled studies. By directly sequencing the full-length amplicons of ITS2 and psbA-trnH through single-molecule real-time (SMRT) technology, the biological composition of a classical prescription Sheng-Mai-San (SMS) was analyzed. At the same time, clone-dependent Sanger sequencing was carried out as a parallel control. Further, another formula—Sanwei-Jili-San (SJS)—was analyzed with genes of ITS2 and CO1. All the ingredients in the samples of SMS and SJS were successfully authenticated at the species level, and 11 exogenous species were also checked, some of which were considered as common contaminations in these products. Methodology analysis demonstrated that this method was sensitive, accurate and reliable. FLMB, a superior but feasible approach for the identification of biological complex mixture, was established and elucidated, which shows perfect interpretation for DNA barcoding that could lead its application in multi-species mixtures. Full article