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.
The underlying high-temperature oxidation mechanisms of the heat-resistant austenitic stainless steel with gradient nanostructured surface layer is revealed through systematic analysis of the ...microstructure and composition. Nanoscale oxide grains and high-density grain boundaries promoted the formation of pronounced spinel oxides, which suppressed elemental diffusion and CrO3 volatilization. High level of residual stress in the GNS layer facilitated the formation of high-density precipitates at the oxide/matrix interface and grain boundaries, which hindered the growth of oxide scale and reactive elements diffusion. The enhanced high-temperature oxidation resistance resulted from the synergistic combination of spinel oxides, precipitates, and high-density grain boundaries.
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•Improved oxidation resistance was the synergy result of spinel oxides, precipitates, and grain boundaries.•Extremely fine oxide grains promoted outward diffusion of Mn and forming thick spinel oxide layer.•Gradient strain induced high-density precipitates formation in the gradient nanostructured layer.•Outward diffusion of reactive elements reduced the thermal stability of nanograins.
Tripsacum dactyloides (2n = 4x = 72) and Zea perennis (2n = 4x = 40) are tertiary gene pools of Zea mays L. and exhibit many abiotic adaptations absent in modern maize, especially salt tolerance. A ...previously reported allopolyploid (hereafter referred to as MTP, 2n = 74) synthesized using Zea mays, Tripsacum dactyloides, and Zea perennis has even stronger salt tolerance than Z. perennis and T. dactyloides. This allopolyploid will be a powerful genetic bridge for the genetic improvement of maize. However, the molecular mechanisms underlying its salt tolerance, as well as the key genes involved in regulating its salt tolerance, remain unclear.
Single-molecule real-time sequencing and RNA sequencing were used to identify the genes involved in salt tolerance and reveal the underlying molecular mechanisms. Based on the SMRT-seq results, we obtained 227,375 reference unigenes with an average length of 2300 bp; most of the unigenes were annotated to Z. mays sequences (76.5%) in the NR database. Moreover, a total of 484 and 1053 differentially expressed genes (DEGs) were identified in the leaves and roots, respectively. Functional enrichment analysis of DEGs revealed that multiple pathways responded to salt stress, including "Flavonoid biosynthesis," "Oxidoreductase activity," and "Plant hormone signal transduction" in the leaves and roots, and "Iron ion binding," "Acetyl-CoA carboxylase activity," and "Serine-type carboxypeptidase activity" in the roots. Transcription factors, such as those in the WRKY, B3-ARF, and bHLH families, and cytokinin negatively regulators negatively regulated the salt stress response. According to the results of the short time series-expression miner analysis, proteins involved in "Spliceosome" and "MAPK signal pathway" dynamically responded to salt stress as salinity changed. Protein-protein interaction analysis revealed that heat shock proteins play a role in the large interaction network regulating salt tolerance.
Our results reveal the molecular mechanism underlying the regulation of MTP in the response to salt stress and abundant salt-tolerance-related unigenes. These findings will aid the retrieval of lost alleles in modern maize and provide a new approach for using T. dactyloides and Z. perennis to improve maize.
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.
•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.
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 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 ability to generate long sequencing reads and access long-range linkage information is revolutionizing the quality and completeness of genome assemblies. Here we use a hybrid approach that ...combines data from four genome sequencing and mapping technologies to generate a new genome assembly of the honeybee Apis mellifera. We first generated contigs based on PacBio sequencing libraries, which were then merged with linked-read 10x Chromium data followed by scaffolding using a BioNano optical genome map and a Hi-C chromatin interaction map, complemented by a genetic linkage map.
Each of the assembly steps reduced the number of gaps and incorporated a substantial amount of additional sequence into scaffolds. The new assembly (Amel_HAv3) is significantly more contiguous and complete than the previous one (Amel_4.5), based mainly on Sanger sequencing reads. N50 of contigs is 120-fold higher (5.381 Mbp compared to 0.053 Mbp) and we anchor > 98% of the sequence to chromosomes. All of the 16 chromosomes are represented as single scaffolds with an average of three sequence gaps per chromosome. The improvements are largely due to the inclusion of repetitive sequence that was unplaced in previous assemblies. In particular, our assembly is highly contiguous across centromeres and telomeres and includes hundreds of AvaI and AluI repeats associated with these features.
The improved assembly will be of utility for refining gene models, studying genome function, mapping functional genetic variation, identification of structural variants, and comparative genomics.
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.
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