We report the first whole genome sequence (WGS) assembly and annotation of a dwarf coconut variety, 'Catigan Green Dwarf' (CATD). The genome sequence was generated using the PacBio SMRT sequencing ...platform at 15X coverage of the expected genome size of 2.15 Gbp, which was corrected with assembled 50X Illumina paired-end MiSeq reads of the same genome. The draft genome was improved through Chicago sequencing to generate a scaffold assembly that results in a total genome size of 2.1 Gbp consisting of 7,998 scaffolds with N50 of 570,487 bp. The final assembly covers around 97.6% of the estimated genome size of coconut 'CATD' based on homozygous k-mer peak analysis. A total of 34,958 high-confidence gene models were predicted and functionally associated to various economically important traits, such as pest/disease resistance, drought tolerance, coconut oil biosynthesis, and putative transcription factors. The assembled genome was used to infer the evolutionary relationship within the palm family based on genomic variations and synteny of coding gene sequences. Data show that at least three (3) rounds of whole genome duplication occurred and are commonly shared by these members of the
family. A total of 7,139 unique SSR markers were designed to be used as a resource in marker-based breeding. In addition, we discovered 58,503 variants in coconut by aligning the Hainan Tall (HAT) WGS reads to the non-repetitive regions of the assembled CATD genome. The gene markers and genome-wide SSR markers established here will facilitate the development of varieties with resilience to climate change, resistance to pests and diseases, and improved oil yield and quality.
•Obtain a high-quality Ginkgo seed development-related transcript data set.•Reveals the diversity of TTLs regulation modes in ginkgo seed development.•Further optimized the structure and function ...annotations of the ginkgo genome.
Full-length transcriptome sequencing based on the PacBio sequencing platform could significantly optimize the annotation of gene structures. As an ancient relic gymnosperm in the monotypic order Ginkgoales, Ginkgo biloba L. contains rich terpenoids that are medicinally valuable. The seeds have abundant edible endosperm, which is delicious and of high nutritional value. However, existing molecular studies on the developmental process of ginkgo seeds are relatively weak, and the biosynthesis of terpenoids in seeds has received little attention. Therefore, single-molecule real-time (SMRT) technology and Illumina sequencing were combined to sequence six tissues related to the reproductive growth and development of ginkgo in order to generate a high-quality full-length transcription database. In total, 20.98 Gb of clean reads containing 178,548 full-length non-chimeric (FLNC) sequences were obtained. From these data, 4019 novel genes and 22,845 novel isoforms were predicted, 52.32 % of the novel genes were annotated, and three novel isoforms were annotated in terpene synthesis related pathways. The enrichment analysis of differentially expressed genes (DEGs) showed that, 95 genes were enriched into 21 categories related to seed development, and 47 DEGs were enriched in the skeletal pathway of terpene synthesis. Combined with the real-time quantitative reverse transcription PCR (qRT-PCR), the phosphosynthase family members synthesizing terpene precursors have diverse and complex expression trends during seed development. Our findings confirm the advantages of SMRT, which facilitated the construction a rich transcript data-set for research on the development of ginkgo seeds, enriching the annotation of the ginkgo genome, and enhancing our understanding of gene regulation of terpene biosynthesis in ginkgo seeds.
Introduction
The α‐globin fusion gene between the HBA2 and HBAP1 genes becomes clinically important in thalassemia screening because this fusion gene can cause severe hemoglobin (Hb) H disease when ...combining with α0‐thalassemia (α0‐thal). Due to its uncommon rearrangement in the α gene cluster without dosage changes, this fusion gene is undetectable by common molecular testing approaches used for α‐thal diagnosis.
Methods
In this study, we used the single‐molecule real‐time (SMRT) sequencing technique to detect this fusion gene in 23 carriers identified by next‐generation sequencing (NGS) among 16,504 screened individuals. Five primers for α and β thalassemia were utilized.
Results
According to the NGS results, the 23 carriers include 14 pure heterozygotes, eight compound heterozygotes with common α‐thal alleles, and one homozygote. By using SMRT, the fusion mutant was successfully detected in all 23 carriers. Furthermore, SMRT corrected the diagnosis in two “pure” heterozygotes: one was compound heterozygote with anti‐3.7 triplication, and the other was homozygote.
Conclusion
Our results indicate that SMRT is a superior method compared to NGS in detecting the α fusion gene, attributing to its efficient, accurate, and one‐step properties.
Nanopore sequencing from Oxford Nanopore Technologies (ONT) and Pacific BioSciences (PacBio) single-molecule real-time (SMRT) long-read isoform sequencing (Iso-Seq) are revolutionizing the way ...transcriptomes are analyzed. These methods offer many advantages over most widely used high-throughput short-read RNA sequencing (RNA-Seq) approaches and allow a comprehensive analysis of transcriptomes in identifying full-length splice isoforms and several other post-transcriptional events. In addition, direct RNA-Seq provides valuable information about RNA modifications, which are lost during the PCR amplification step in other methods. Here, we present a comprehensive summary of important applications of these technologies in plants, including identification of complex alternative splicing (AS), full-length splice variants, fusion transcripts, and alternative polyadenylation (APA) events. Furthermore, we discuss the impact of the newly developed nanopore direct RNA-Seq in advancing epitranscriptome research in plants. Additionally, we summarize computational tools for identifying and quantifying full-length isoforms and other co/post-transcriptional events and discussed some of the limitations with these methods. Sequencing of transcriptomes using these new single-molecule long-read methods will unravel many aspects of transcriptome complexity in unprecedented ways as compared to previous short-read sequencing approaches. Analysis of plant transcriptomes with these new powerful methods that require minimum sample processing is likely to become the norm and is expected to uncover novel co/post-transcriptional gene regulatory mechanisms that control biological outcomes during plant development and in response to various stresses.
Posttranscriptional processing of precursor mRNAs contributes to transcriptome and protein diversity and gene regulatory mechanisms in eukaryotes. However, this posttranscriptional mechanism has not ...been studied in the marine macroalgae Gracilariopsis lemaneiformis, which is the most cultivated red seaweed species in China.
In the present study, third-generation sequencing (Pacific Biosciences single-molecule real-time long-read sequencing, SMRT-Seq) was used to sequence the full-length transcriptome of G. lemaneiformis to identify alternatively spliced transcripts and alternative polyadenylation (APA) sites in this species. RNAs were isolated from G. lemaneiformis under various treatments including abiotic stresses and exogenous phytohormones, and then equally pooled for SMRT-Seq. In summary, 346,544 full-length nonchimeric reads were generated, from which 13,630 unique full-length transcripts were obtained in G. lemaneiformis. Compared with the known splicing events in the gene models, more than 3000 new alternative splicing (AS) events were identified in the SMRT-Seq reads. Additionally, 810 genes were found to have poly (A) sites and 91 microRNAs (miRNAs), 961 long noncoding RNAs and 1721 novel genes were identified in G. lemaneiformis. Moreover, validation experiments showed that abiotic stresses and phytohormones could induce some specific AS events, especially intron retain isoforms, cause some alterations to the relative ratios of transcripts annotated to the same gene, and generate novel 3' ends because of differential APA. The growth of G. lemaneiformis was inhibited by Cu stress, while this inhibition was alleviated by ACC treatment. RNA-Seq analysis further revealed that 211 differential alternative splicing (DAS) events and 142 DAS events was obtained in CK vs Cu and Cu vs Cu + ACC, respectively, suggesting that AS of functional genes could be regulated by Cu stress and ACC. Compared with Cu stress, the expression of transcripts with DAS events mainly involved in the carbon fixation in photosynthetic organisms and oxidative phosphorylation pathway was upregulated in Cu + ACC treatment, revealing that ACC alleviated the growth inhibition by Cu stress by increasing carbon fixation and oxidative phosphorylation.
Our results provide the first comprehensive picture of the full-length transcriptome and posttranscriptional mechanism in red macroalgae, including transcripts that appeared in the presence of common abiotic stresses and phytohormones, which will improve the gene annotations of Gracilariopsis and contribute to the study of gene regulation in this important cultivated seaweed.
In this study, a novel beta-cypermethrin (beta-cyp)-degrading strain Lactobacillus pentosus 3–27 (LP3–27) was screened from beta-cyp-contaminated silage. The strain could degrade 96% of beta-cyp ...(50 mg/L) in MSM medium after 4 d of culture, while the strain lost its degradation ability when the beta-cyp concentration reached 250 mg/L. The effects of LP 3–27 on fermentation, bacterial community, and bioremediation of contaminated alfalfa silage at two dry matter (DM) contents were studied. The results showed that inoculation with LP3–27 not only degraded beta-cyp, but also improved the fermentation quality of alfalfa silage after 60 d of ensiling. Meanwhile, L. pentosus dominated the bacterial community during ensiling in LP3–27 inoculated silages, whereas Pediococcus acidilactici was the dominant species in the control silage. LP3–27 inoculation also simplified the bacterial interaction networks of ensiled alfalfa. Beta-cyp degradation was positively correlated with L. pentosus in LP- inoculated silages, which confirmed the function of beta-cyp degradation by L. pentosus. In addition, higher beta-cyp degradation was observed in silage with 35% versus 43% DM. In summary, strain LP3–27 could be used as a candidate inoculum for bioremediation of beta-cyp-contaminated silage and to produce safe silage for animal production.
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•A novel beta-cypermethrin-degrading L. pentosus 3-27 was screened from silage.•The LP3-27 degraded 96% of beta-cyp (50 mg/L) in MSM medium after 4 d culturing.•LP3-27 effectively degraded beta-cyp and improved fermentation of alfalfa silage.•LP3–27 inoculation simplified bacterial interaction networks in alfalfa silage.•A higher beta-cyp degradation was observed in silage with 35% DM versus 43% DM.
Carbohydrate responsive element-binding protein (ChREBP) has been identified as a primary transcription factor that maintains energy homeostasis through transcriptional regulation of glycolytic, ...lipogenic, and gluconeogenic enzymes in response to a high-carbohydrate diet. Amino acids are important substrates for gluconeogenesis, but nevertheless, knowledge is lacking about whether this transcription factor regulates genes involved in the transport or use of these metabolites. Here, we demonstrate that ChREBP represses the expression of the amino acid transporter sodium-coupled neutral amino acid transporter 2 (SNAT2) in response to a high-sucrose diet in rats by binding to a carbohydrate response element (ChoRE) site located -160 bp upstream of the transcriptional start site in the SNAT2 promoter region. Additionally, immunoprecipitation assays revealed that ChREBP and silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) interact with each other, as part of the complex that repress SNAT2 expression. The interaction between these proteins was confirmed by an in vivo chromatin immunoprecipitation assay. These findings suggest that glucogenic amino acid uptake by the liver is controlled by ChREBP through the repression of SNAT2 expression in rats consuming a high-carbohydrate diet.
This study highlights the key role of carbohydrate responsive element-binding protein (ChREBP) in the fine-tuned regulation between glucose and amino acid metabolism in the liver via regulation of the amino acid transporter sodium-coupled neutral amino acid transporter 2 (SNAT2) expression after the consumption of a high-carbohydrate diet. ChREBP binds to a carbohydrate response element (ChoRE) site in the SNAT2 promoter region and recruits silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) corepressor to reduce SNAT2 transcription. This study revealed that ChREBP prevents the uptake of glucogenic amino acids upon the consumption of a high-carbohydrate diet.
•The conventional methods and Next-generation sequencing to diagnose thalassemia have limitations.•Long-read SMRT sequencing has been demonstrated to be more effective and accurate than conventional ...methods, especially for rare and complicated thalassemia variants.•A 762 bp deletion and a 342 bp insertion in α-globin gene cluster were identified by SMRT sequencing and reported for the first time.•Subjects with other rare mutations including α Fusion mutation, α-triplicates, α-quadruplicates and conversion of HBA2 to HBA1 was precisely identified by SMRT sequencing.
Gap- polymerase chain reaction (PCR), reverse dot-blot assay (RDB), real-time PCR based multicolor melting curve analysis (MMCA assay), multiplex ligation-dependent probe amplification (MLPA) and Sanger sequencing are conventional methods to diagnose thalassemia but all of them have limitations. In this study, we applied single-molecule real-time (SMRT) sequencing following multiplex long-range PCR to uncover rare mutations in nine patients and their family members. The patients with different results between Gap-PCR and MMCA assay or with phenotype not matching genotype were included. Using SMRT sequencing, we first identified the carriers with αααanti3.7/HKαα, -α762bpα/αα (chr16:172,648–173,409), ααfusion/αQSα (in a trans configuration), two cases with novel gene rearrangements and another case with a novel 341 bp insertion in α-globin gene cluster, respectively. One carrier with --SEA/αααanti4.2, and two carriers with the coexistence of globin variant and an α-globin gene duplication were also found. Most importantly, we could determine two defects in α-globin gene cluster being a cis or trans configuration in a single test. Our results showed that SMRT has great advantages in detection of α-globin gene triplications, rare deletions and determination of a cis or trans configuration. SMRT is a comprehensive and one-step method for thalassemia screening and diagnosis, especially for detection of rare thalassemia mutations.
In order to effectively use locally available woody forage and hay resources in tropics, paper mulberry (PM) silages were prepared using Napier grass (NG) and rice straw (RS) hays. The mixing ...proportion of hays were 10%, 20%, and 30% on fresh matter basis. After 60 days of ensiling, single molecule, real-time (SMRT) sequencing technology was used to analyse the dynamic changes of microbial community and silage fermentation. When PM silages were prepared with both hays, the moisture of PM were adjusted below 67.6%. The dominant microbial community rapidly shifted from Gram-negative to Gram-positive bacteria during ensiling. Before ensiling, aerobic bacteria were dominant in materials, but after ensiling, Lactobacillus plantarum became the main bacterial community and dominated the fermentation process. The result of Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis shown that hay-treated PM silages increased the proportion of Global and overview maps and carbohydrate metabolic pathways, while decreasing the proportion of amino acid metabolic pathway. Fatty acid metabolism, glycolysis and proteolysis categories may improve the flavour and quality of the silage. PM silage prepared with 10% hay effectively improved better fermentation quality than PM alone silage. The result confirmed that PM and hay are the ideal combination, they can prepare good-quality silage for ruminant feed to alleviate the shortage of feed in tropics.
•Napier grass and rice straw improve fermentation suitability of paper mulberry.•Paper mulberry with 10% hay are the ideal combination for silage preparation.•Microbial metabolic pathways improve the flavour and quality of the silage.•Hay addition increases the proportion of carbohydrate metabolic pathway in silage.•SMRT technology accurately revealed specific microbial information of silage.
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is the causal agent of the COVID-19 pandemic that emerged in late 2019. The outbreak of variants with mutations in the region ...encoding the spike protein S1 sub-unit that can make them more resistant to neutralizing or monoclonal antibodies is the main point of the current monitoring. This study examines the feasibility of predicting the variant lineage and monitoring the appearance of reported mutations by sequencing only the region encoding the S1 domain by Pacific Bioscience Single Molecule Real-Time sequencing (PacBio SMRT). Using the PacBio SMRT system, we successfully sequenced 186 of the 200 samples previously sequenced with the Illumina COVIDSeq (whole genome) system. PacBio SMRT detected mutations in the S1 domain that were missed by the COVIDseq system in 27/186 samples (14.5%), due to amplification failure. These missing positions included mutations that are decisive for lineage assignation, such as G142D (
= 11), N501Y (
= 6), or E484K (
= 2). The lineage of 172/186 (92.5%) samples was accurately determined by analyzing the region encoding the S1 domain with a pipeline that uses key positions in S1. Thus, the PacBio SMRT protocol is appropriate for determining virus lineages and detecting key mutations.