Single-molecule Real-time Technology Research Articles

Integrating Full-Length and Second-Generation Transcriptomics to Reveal Differentially Expressed Genes Associated with the Development of Corydalis yanhusuo Tuber.

Corydalis yanhusuo is a medicinal herb in China that has been widely used to treat various kinds of pain. The tuber is the main organ of C. yanhusuo used for medicinal purposes, but changes in related genes during the development of the tuber have rarely been reported. To identify the differentially expressed genes during tuber development, C. yanhusuo full-length transcriptomic sequencing was performed using single-molecule real-time technology, and tubers at three development stages were selected for comparative transcriptome analysis. A total of 90,496 full-length non-chimeric transcripts were obtained, and 19,341 transcripts were annotated in at least one public database. A total of 9221 differentially expressed genes were identified during the swelling process of C. yanhusuo tuber. A Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis revealed that differentially expressed genes associated with a "starch and sucrose metabolism pathway", "phenylpropanoid biosynthesis pathway", "isoquinoline alkaloid biosynthesis pathway", "zeatin biosynthesis pathway", and "brassinosteroid biosynthesis pathway" were predominantly enriched. In addition, the genes involved in cell wall metabolism were potentially associated with tuber swelling. These processes regulated and were involved in C. yanhusuo tuber development. The results provide a foundation for further research on tuber formation in medicinal plants.

The comprehensive analysis of thalassemia alleles (CATSA) based on single-molecule real-time technology (SMRT) is a more powerful strategy in the diagnosis of thalassemia caused by rare variants

Thalassemia is one of the most widely distributed monogenic disorders in the world and affects the largest number of people. It can manifest a wide spectrum of phenotypes from asymptomatic to fatal, which is associated with the degree of imbalance between α- and β-globin chains. Therefore, individuals with different genotypes could present with a similar phenotype. Genetic analysis is always needed to make a correct diagnosis. However, routine genetic analysis of thalassemia used in the Chinese population identifies only 23 common variants, resulting in many cases undiagnosed or being misdiagnosed. In this study, we applied a long-read sequencing-based approach termed comprehensive analysis of thalassemia alleles (CATSA) to 30 subjects whose hematologic screening results could not be explained by the routine genetic test results. The identification of additional variants and the correction of genotypes allowed the interpretation of the clinical phenotype in 24 subjects, which have been confirmed to be correct by independent experiments. Moreover, we identified a novel 8.4-kb deletion containing the entire HBB and HBD genes as well as part of the HBBP1 gene, expanding the genotype spectrum of β-thalassemia. CATSA showed a great advantage over other genetic tests in the diagnosis of thalassemia caused by rare variants.

Identification of a novel 91.5 kb-deletion (αα)FJ in the α-globin gene cluster using single-molecule real-time (SMRT) sequencing

Objectives To present a novel 91.5-kb deletion of the α-globin gene cluster (αα)FJ identified by genetic assay and prenatal diagnosis in a Chinese family. Subjects and Methods The proband was a 34-year-old G3P1 (Gravida 3, Para 1) female at the gestational age of 21+ weeks with a history of an edematous fetus. A routine genetic assay (reverse dot blot hybridization, RDB) was performed to detect common thalassemia mutations. Multiplex ligation-dependent probe amplification (MLPA) and single-molecule real-time technology (SMRT) were used to detect rare thalassemia mutations. Results The hematological phenotypes of the proband, her mother, elder sister, husband, daughter, and nephew were consistent with the phenotype of α-thalassemia trait. No mutations were found in these family members by RDB, except for the proband’s husband who carried an α-globin gene deletion --SEA/αα. MLPA results showed that the proband and other α-thalassemia-suspected relatives had heterozygous deletions around the POLR3K-3-463nt, HS40-178nt, and HBA-HS40-382nt probes. The 5′-breakpoint was out of probe scope and could not be determined. SMRT was performed and a 91.5-kb deletion (NC_000016.10: g.39268_130758del) in the α-globin gene cluster (αα)FJ was identified in the proband and other suspected relatives, which could explain their phenotypes. At the proband’s gestational age of 22+ weeks, an amniotic fluid sample was collected and analyzed. As only the 91.5-kb deletion (αα)FJ was identified in the fetus with RDB, MLPA, and SMRT. The proband was suggested to continue the pregnancy. Conclusion We first reported a 91.5-kb deletion (NC_000016.10: g.hg38-chr16:39268-_130758del) of the HS-40 region in the α-globin gene cluster (αα)FJ identified in a Chinese family. Since the HS-40 loss of heterozygosity in combination with the heterozygous deletion --SEA might result in Hb Bart’s hydrops fetalis, routine genetic assay, and SMRT were recommended to individuals at risk for prenatal diagnosis.

Comparative genomics and DNA methylation analysis of Pseudomonas aeruginosa clinical isolate PA3 by single-molecule real-time sequencing reveals new targets for antimicrobials.

Pseudomonas aeruginosa (P.aeruginosa) is an important opportunistic pathogen with broad environmental adaptability and complex drug resistance. Single-molecule real-time (SMRT) sequencing technique has longer read-length sequences, more accuracy, and the ability to identify epigenetic DNA alterations. This study applied SMRT technology to sequence a clinical strain P. aeruginosa PA3 to obtain its genome sequence and methylation modification information. Genomic, comparative, pan-genomic, and epigenetic analyses of PA3 were conducted. General genome annotations of PA3 were discovered, as well as information about virulence factors, regulatory proteins (RPs), secreted proteins, type II toxin-antitoxin (TA) pairs, and genomic islands. A genome-wide comparison revealed that PA3 was comparable to other P. aeruginosa strains in terms of identity, but varied in areas of horizontal gene transfer (HGT). Phylogenetic analysis showed that PA3 was closely related to P. aeruginosa 60503 and P. aeruginosa 8380. P. aeruginosa's pan-genome consists of a core genome of roughly 4,300 genes and an accessory genome of at least 5,500 genes. The results of the epigenetic analysis identified one main methylation sites, N6-methyladenosine (m6A) and 1 motif (CATNNNNNNNTCCT/AGGANNNNNNNATG). 16 meaningful methylated sites were picked. Among these, purH, phaZ, and lexA are of great significance playing an important role in the drug resistance and biological environment adaptability of PA3, and the targeting of these genes may benefit further antibacterial studies. This study provided a detailed visualization and DNA methylation information of the PA3 genome and set a foundation for subsequent research into the molecular mechanism of DNA methyltransferase-controlled P. aeruginosa pathogenicity.

The frequency of HKαα allele in silent deletional α-thalassemia carriers in the Yulin region of southern China using the third-generation sequencing

Objectivesα-thalassemia is relatively prevalent in Yulin Region in southern China. In order to accurately detect α-globin gene aberrations for genetic counseling, the prevalence of HKαα (Hong Kong αα) allele in this subpopulation of silent deletional α-thalassemia were examined. Materials and methodsA total of 1845 subjects were selected in Yulin Region from January 2021 to March 2021. Peripheral blood was collected from each participant for routine genetic analysis of thalassemia. The HKαα allele was determined using the Single-molecule real-time (SMRT) technology for samples with -α3.7/αα, βN/βN genotype. ResultsTwo samples were identified with HKαα allele from 100 samples with -α3.7/αα, βN/βN genotype. The frequency of HKαα allele was 2.0 % (2/100) in -α3.7/αα, βN/βN carriers in Yulin Region. One sample was identified with a novel variant of the α-globin gene cluster named αHKαα by SMRT technology. One rare HBA2 variant and six HBB variants were found by SMRT technology, including -α3.7/HBA2:c.300 + 34G > A, HBB:c.316-45G > C/βN, HBB:c.315 + 180 T > C/βN, HBB:c.316-179A > C/βN. ConclusionA certain proportion of HKαα allele had been detected in Yulin Region. SMRT technology plays a crucial role for improving the diagnostic accuracy and positive detection rate of thalassemia. The completion of this study has great meaning for strengthening the prevention and control of thalassemia in Yulin Region.

Hb Q-Thailand heterozygosity unlinked with the (–α4.2/) α+-thalassemia deletion allele identified by long-read SMRT sequencing: hematological and molecular analyses

ABSTRACTObjectiveIn the present study, two unrelated cases of Hb Q-Thailand heterozygosity unlinked with the (–α4.2/) α+-thalassemia deletion allele were identified by long-read single molecule real-time (SMRT) sequencing in southern China. The aim of this study was to report the hematological and molecular features as well as diagnostic aspects of the rare manifestation.MethodsHematological parameters and hemoglobin analysis results were recorded. A suspension array system for routine thalassemia genetic analysis and long-read SMRT sequencing were applied in parallel for thalassemia genotyping. Traditional methods, including Sanger sequencing, multiplex gap-polymerase chain reaction (gap-PCR) and multiplex ligation-dependent probe amplification (MLPA), were used together to confirm the thalassemia variants.ResultsLong-read SMRT sequencing was used to diagnose two Hb Q-Thailand heterozygous patients for whom the hemoglobin variant was unlinked to the (–α4.2/) allele for the first time. The hitherto undescribed genotypes were verified by traditional methods. Hematological parameters were compared with those of Hb Q-Thailand heterozygosity linked with the (–α4.2/) deletion allele in our study. For the positive control samples, long-read SMRT sequencing revealed a linkage relationship between the Hb Q-Thailand allele and the (–α4.2/) deletion allele.ConclusionsIdentification of the two patients confirms that the linkage relationship between the Hb Q-Thailand allele and the (−α4.2/) deletion allele is a common possibility but not a certainty. Remarkably, as it is superior to traditional methods, SMRT technology may eventually serve as a more comprehensive and precise method that holds promising prospects in clinical practice, especially for rare variants.

Characterization and Analysis of the Full-Length Transcriptome Provide Insights into Fruit Quality Formation in Kiwifruit Cultivar Actinidia arguta cv. Qinziyu

Kiwifruit is an economically important horticultural crop with extremely high values in nutrition and health care. However, the molecular mechanisms underlying fruit quality formation remain largely limited for most kiwifruit varieties. Recently, a new kiwifruit cultivar with a high level of soluble solids, Actinidia arguta cv. Qinziyu (full-red flesh) was discovered through the introduction and propagation test. To provide new insights into fruit quality formation in a typical kiwifruit cultivar, we integrated full-length transcriptome surveys based on PacBio single-molecule real-time (SMRT) sequencing, key enzyme genes expression involved in carbohydrate and amino acids metabolism pathways, and bHLH gene family analysis to enhance the understanding of soluble sugar, organic acid, and anthocyanin biosynthesis in A. arguta cv. Qinziyu. A total of 175,913 CCSs were generated, of which 124,789 were identified as FLNC transcripts. In total, 45,923 (86.99%) transcripts were successfully annotated, and more than 76.05% of the transcripts were longer than 1 Kb. KEGG pathway analysis showed that 630 candidate genes encoding 55 enzymes were mainly involved in carbohydrate and amino acid biosynthesis pathways. Further analysis verified the expression of 12 key enzyme genes (e.g., pyruvate kinase (PK), enolase (ENO), hexokinase (HK), and phosphoglycerate kinase (PGK)) in flowers using quantitative real-time PCR. Furthermore, we also screened 10 AabHLH proteins’ function in anthocyanin biosynthesis and characterized the AabHLH gene family in A. arguta cv. Qinziyu. Overall, our research data generated by SMRT technology provide the first set of gene isoforms from a full-length transcriptome in A. arguta cv. Qinziyu and more comprehensive insights into the molecular mechanism of fruit quality formation.

Strain-level resolution and pneumococcal carriage dynamics by single-molecule real-time (SMRT) sequencing of the plyNCR marker: a longitudinal study in Swiss infants

BackgroundPneumococcal carriage has often been studied from a serotype perspective; however, little is known about the strain-specific carriage and inter-strain interactions. Here, we examined the strain-level carriage and co-colonization dynamics of Streptococcus pneumoniae in a Swiss birth cohort by PacBio single-molecule real-time (SMRT) sequencing of the plyNCR marker.MethodsA total of 872 nasal swab (NS) samples were included from 47 healthy infants during the first year of life. Pneumococcal carriage was determined based on the quantitative real-time polymerase chain reaction (qPCR) targeting the lytA gene. The plyNCR marker was amplified from 214 samples having lytA-based carriage for pneumococcal strain resolution. Amplicons were sequenced using SMRT technology, and sequences were analyzed with the DADA2 pipeline. In addition, pneumococcal serotypes were determined using conventional, multiplex PCR (cPCR).ResultsPCR-based plyNCR amplification demonstrated a 94.2% sensitivity and 100% specificity for Streptococcus pneumoniae if compared to lytA qPCR. The overall carriage prevalence was 63.8%, and pneumococcal co-colonization (≥ 2 plyNCR amplicon sequence variants (ASVs)) was detected in 38/213 (17.8%) sequenced samples with the relative proportion of the least abundant strain(s) ranging from 1.1 to 48.8% (median, 17.2%; IQR, 5.8–33.4%). The median age to first acquisition was 147 days, and having ≥ 2 siblings increased the risk of acquisition.ConclusionThe plyNCR amplicon sequencing is species-specific and enables pneumococcal strain resolution. We therefore recommend its application for longitudinal strain-level carriage studies of Streptococcus pneumoniae.7kNn5E5Dxp8AY6Abk1AWrtVideo

Application of the Single-Molecule Real-Time Technology (SMRT) for Identification of HKαα Thalassemia Allele.

Single-molecule real-time technology (SMRT) is a sequencing technology using the DNA polymerases and fluorescently tagged nucleotides to accurately sequence DNA strands. The purpose of this study was to evaluate the detection accuracy of SMRT for identification of the Hong Kongαα (HKαα) thalassemia allele. We conducted a blinded study of 33 samples of known HKαα alleles. These alleles were detected using SMRT to evaluate accuracy. We conducted a blinded study of 33 known HKαα samples and found all HKαα variants detected by SMRT to be concordant with those independently assigned by gap-polymerase chain reaction (gap-PCR), reverse dot blot hybridization, and 2-round nested PCR. In addition, SMRT detected 2 β-thalassemia variants that were missed by conventional techniques. The results demonstrate that SMRT offers a higher detection accuracy of thalassemia rare and new loci. It is an efficient, reliable, and broad-spectrum test that can be widely used for thalassemia screening in the clinic.

Detection of four rare thalassemia variants using Single-molecule realtime sequencing.

Conventional methods for the diagnosis of thalassemia include gap polymerase chain reaction (Gap-PCR), reverse membrane hybridization (RDB), multiplex ligation-dependent probe amplification (MLPA) and Sanger sequencing. In this study, we used single molecule real-time technology (SMRT) sequencing and discovered four rare variants that have not been identified by conventional diagnostic methods for thalassemia. We also performed genotype and phenotype analyses on family members of thalassemia patients. The SMRT technology detected five cases in which the proband had abnormal results by conventional diagnostic methods or inconsistencies between the genotype and phenotype. The variants included two cases of an α-globin gene cluster 27,311 bp deletion, --27.3/αα (hg38 chr16:158664-185974), one case of an HS-40 region 16,079 bp deletion (hg38 chr16:100600-116678), one case of a rearrangement of -α3.7α1α2 on one allele and one case of a ß-globin gene cluster HBG1-HBG2 4,924 bp deletion (hg38 chr11:5249345-5254268). This study clarified the hematological phenotypes of four rare variants and indicated the application value of SMRT in the diagnosis of rare α-globin and ß-globin gene cluster deletions, gene recombination and deletion breakpoints. The SMRT method is a comprehensive one-step technology for the genetic diagnosis of thalassemia and is particularly suitable for the diagnosis of thalassemia with rare deletions or genetic recombination.

Full-Length Transcriptome Analysis of the Halophyte Nitraria sibirica Pall.

Background: Nitraria sibirica Pall. is one of the pioneer tree species in saline–alkali areas due to its extreme salt tolerance. However, the lack of information on its genome limits the further exploration of the molecular mechanisms in N. sibirica under salt stress. Methods: In this study, we used single-molecule real-time (SMRT) technology based on the PacBio Iso-Seq platform to obtain transcriptome data from N. sibirica under salt treatment for the first time, which is helpful for our in-depth analysis of the salt tolerance and molecular characteristics of N. sibirica. Results: Our results suggested that a total of 234,508 circular consensus sequences (CCSs) with a mean read length of 2121 bp were obtained from the 19.26 Gb raw data. Furthermore, based on transcript cluster analysis, 93,713 consensus isoforms were obtained, including 92,116 high-quality isoforms. After removing redundant sequences, 49,240 non-redundant transcripts were obtained from high-quality isoforms. A total of 37,261 SSRs, 1816 LncRNAs and 47,314 CDSs, of which 40,160 carried complete ORFs, were obtained. Based on our transcriptome data, we also analyzed the coding genes of H+-PPase, and the results of both bioinformatics and functional analyses indicated that the gene prediction via full-length transcripts obtained by SMRT technology is reliable and effective. In summary, our research data obtained by SMRT technology provides more reliable and accurate information for the further analysis of the regulatory network and molecular mechanism of N. sibirica under salt stress.

DNA Methyltransferase HsdM Induce Drug Resistance on Mycobacterium tuberculosis via Multiple Effects.

Besides the genomic variants, epigenetic mechanisms such as DNA methylation also have an effect on drug resistance. This study aimed to investigate the methylomes of totally/extensively drug-resistant M. tuberculosis clinical isolates using the PacBio single-molecule real-time technology. The results showed they were almost the same as the pan-susceptible ones. Genetics and bioinformatics analysis confirmed three DNA methyltransferases—MamA, MamB, and HsdM. Moreover, anti-tuberculosis drug treatment did not change the methylomes. In addition, the knockout of the DNA methyltransferase hsdM gene in the extensively drug-resistant clinical isolate 11826 revealed that the motifs of GTAYN4ATC modified by HsdM were completely demethylated. Furthermore, the results of the methylated DNA target analysis found that HsdM was mainly involved in redox-related pathways, especially the prodrug isoniazid active protein KatG. HsdM also targeted three drug-targeted genes, eis, embB, and gyrA, and three drug transporters (Rv0194, Rv1410, and Rv1877), which mildly affected the drug susceptibility. The overexpression of HsdM in M. smegmatis increased the basal mutation rate. Our results suggested that DNA methyltransferase HsdM affected the drug resistance of M. tuberculosis by modulating the gene expression of redox, drug targets and transporters, and gene mutation.

Correction to: The value of single-molecule real-time technology in the diagnosis of rare thalassemia variants and analysis of phenotype–genotype correlation

Correction to: The value of single-molecule real-time technology in the diagnosis of rare thalassemia variants and analysis of phenotype–genotype correlation

The value of single-molecule real-time technology in the diagnosis of rare thalassemia variants and analysis of phenotype\u2013genotype correlation

To compare single-molecule real-time technology (SMRT) and conventional genetic diagnostic technology of rare types of thalassemia mutations, and to analyze the molecular characteristics and phenotypes of rare thalassemia gene variants, we used 434 cases with positive hematology screening as the cohort, then used SMRT technology and conventional gene diagnosis technology [(Gap-PCR, multiple ligation probe amplification technology (MLPA), PCR-reverse dot blot (RDB)] for thalassemia gene screening. Among the 434 enrolled cases, conventional technology identified 318 patients with variants (73.27%) and 116 patients without variants (26.73%), SMRT identified 361 patients with variants (83.18%), and 73 patients without variants (16.82%). The positive detection rate of SMRT was 9.91% higher than conventional technology. Combination of the two methods identified 485 positive alleles among 49 types of variant. The genotypes of 354 cases were concordant between the two methods, while 80 cases were discordant. Among the 80 cases, 76 cases had variants only identified in SMRT method, 3 cases had variants only identified in conventional method, and 1 false positive result by the traditional PCR detection technology. Except the three variants in HS40 and HBG1-HBG2 loci, which was beyond the design of SMRT method in this study, all the other discordant variants identified by SMRT were validated by further Sanger sequencing or MLPA. The hematological phenotypic parameters of 80 discordant cases were also analyzed. SMRT technology increased the positive detection rate of thalassemia genes, and detected rare thalassemia cases with variable phenotypes, which had great significance for clinical thalassemia gene screening.

SARS-CoV-2 Quasispecies Provides an Advantage Mutation Pool for the Epidemic Variants.

ABSTRACTThe dynamics of quasispecies afford RNA viruses a great fitness on cell tropism and host range. To study the quasispecies features and the intra-host evolution of SARS-CoV-2, we collected nine confirmed patients and sequenced the haplotypes of spike gene using a single-molecule real-time platform. Fourteen samples were extracted from sputum, nasopharyngeal swabs, or stool, which in total produced 283,655 high-quality circular consensus sequences. We observed a stable quasispecies structure that one master mutant (mean abundance ∼0.70), followed by numerous minor mutants (mean abundance ∼1.21 × 10−3). Under high selective pressure, minor mutants may obtain a fitness advantage and become the master ones. The later predominant substitution D614G existed in the minor mutants of more than one early patient. An epidemic variant had a possibility to be independently originated from multiple hosts. The mutant spectrums covered ∼85% amino acid variations of public genomes (GISAID; frequency ≥ 0.1) and likely provided an advantage mutation pool for the current/future epidemic variants. Notably, 32 of 35 collected antibody escape substitutions were preexistent in the early quasispecies. Virus populations in different tissues/organs revealed potentially independent replications. The quasispecies complexity of sputum samples was significantly lower than that of nasopharyngeal swabs (P = 0.02). Evolution analysis revealed that three continuous S2 domains (HR1, CH, and CD) had undergone a positive selection. Cell fusion-related domains may play a crucial role in adapting to the intrahost immune system. Our findings suggested that future epidemiologic investigations and clinical interventions should consider the quasispecies information that has missed by routine single consensus genome.IMPORTANCE RNA virus population in a host does not consist of a consensus single haplotype but rather an ensemble of related sequences termed quasispecies. The dynamics of quasispecies afford SARS-CoV-2 a great ability on genetic fitness during intrahost evolution. The process is likely achieved by changing the genetic characteristics of key functional genes, such as the spike glycoprotein. Previous studies have applied the next-generation sequencing (NGS) technology to evaluate the quasispecies of SARS-CoV-2, and results indicated a low genetic diversity of the spike gene. However, the NGS platform cannot directly obtain the full haplotypes without assembling, and it is also difficult to predict the extremely low-frequency variations. Therefore, we introduced a single-molecule real-time technology to directly obtain the haplotypes of the RNA population and further study the quasispecies features and intrahost evolution of the spike gene.

Complete Genome Resources for Ralstonia Bacterial Wilt Strains UW763 (Phylotype I); Rs5 and UW700 (Phylotype II); and UW386, RUN2474, and RUN2279 (Phylotype III).

We share whole genome sequences of six strains from the Ralstonia solanacearum species complex, a diverse group of Betaproteobacteria that cause plant vascular wilt diseases. Using single-molecule real-time technology, we sequenced and assembled full genomes of Rs5 and UW700, two phylotype IA-sequevar 7 (IIA-7) strains from the southeastern United States that are closely related to the R. solanacearum species type strain, K60, but were isolated >50 years later. Four sequenced strains from Africa include a soil isolate from Nigeria (UW386, III-23), a tomato isolate from Senegal (UW763, I-14), and two potato isolates from the Madagascar highlands (RUN2474, III-19 and RUN2279, III-60). This resource will support studies of the genetic diversity, ecology, virulence, and microevolution of this globally distributed group of high-impact plant pathogens.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Integrated SMRT Technology with UMI RNA-Seq Reveals the Hub Genes in Stamen Petalody in Camellia oleifera

Male sterility caused by stamen petalody is a key factor for a low fruit set rate and a low yield of Camellia oleifera but can serve as a useful genetic tool because it eliminates the need for artificial emasculation. However, its molecular regulation mechanism still remains unclear. In this study, transcriptome was sequenced and analyzed on two types of bud materials, stamen petalody mutants and normal materials, at six stages of stamen development based on integrated single-molecule real-time (SMRT) technology with unique molecular identifiers (UMI) and RNA-seq technology to identify the hub genes responsible for stamen petalody in C. oleifera. The results show that a large number of alternative splicing events were identified in the transcriptome. A co-expression network analysis of MADSs and all the differentially expressed genes between the mutant stamens and the normal materials showed that four MADS transcription factor genes, CoSEP3.1, CoAGL6, CoSEP3.2, and CoAP3, were predicted to be the hub genes responsible for stamen petalody. Among these four, the expression patterns of CoAGL6 and CoSEP3.2 were consistently high in the mutant samples, but relatively low in the normal samples at six stages, while the patterns of CoSEP3.1 and CoAP3 were initially low in mutants and then were upregulated during development but remained relatively high in the normal materials. Furthermore, the genes with high connectivity to the hub genes showed significantly different expression patterns between the mutant stamens and the normal materials at different stages. qRT-PCR results showed a similar expression pattern of the hub genes in the RNA-seq. These results lay a solid foundation for the directive breeding of C. oleifera varieties and provide references for the genetic breeding of ornamental Camellia varieties.

The Full-Length Transcriptome Sequencing and Identification of Na+/H+ Antiporter Genes in Halophyte Nitraria tangutorum Bobrov.

Nitraria tangutorum Bobrov is a halophyte that is resistant to salt and alkali and is widely distributed in northwestern China. However, its genome has not been sequenced, thereby limiting studies on this particular species. For species without a reference genome, the full-length transcriptome is a convenient and rapid way to obtain reference gene information. To better study N. tangutorum, we used PacBio single-molecule real-time technology to perform full-length transcriptome analysis of this halophyte. In this study, a total of 21.83 Gb of data were obtained, and 198,300 transcripts, 51,875 SSRs (simple sequence repeats), 55,574 CDS (coding sequence), and 74,913 lncRNAs (long non-coding RNA) were identified. In addition, using this full-length transcriptome, we identified the key Na+/H+ antiporter (NHX) genes that maintain ion balance in plants and found that these are induced to express under salt stress. The results indicate that the full-length transcriptome of N. tangutorum can be used as a database and be utilized in elucidating the salt tolerance mechanism of N. tangutorum.

The use of PacBio SMRT technology to explore the microbial network and fermentation characteristics of woody silage prepared with exogenous carbohydrate additives.

To effectively use woody plant resources to prepare silage for ruminants, Pacific Biosciences (PacBio) single-molecule real-time (SMRT) sequencing was applied to study the microbial network and fermentation characteristics of paper mulberry (PM) silage prepared with corn meal (CM) and rice bran (RB) as exogenous additives. PM is rich in nutrients and contains more than 26% crude protein in dry matter. After ensiling, the microbial diversity and abundance in PM, CM and RB decreased due to the anaerobic environment and acidic conditions. The CM-treated PM silage accelerated the conversion of the dominant microbial community from harmful bacteria to lactic acid bacteria and promoted lactic acid fermentation. When RB was used to treat PM silage, Enterobacter and Clostridium species became the main bacterial community during ensiling, leading to butyric acid fermentation and protein decomposition. Compared with RB, CM increased the amount of fermentation substrates, changed the microbial community structure and affected metabolic pathways (global metabolism, carbohydrate metabolism and amino acid metabolism), which improved the flavour and quality of the PM silage. The CM addition of improved the fermentation quality of PM silage, with PM + CM being the ideal combination. The SMRT sequencing technology could accurately obtain specific details of the microbial networks and fermentation characteristics. Our results indicate that PM can be used as a potential high-protein silage in animal production. In tropics, the effective use of abundant natural biomass resources such as woody plants to prepare silage for feed preservation can solve the problem of restricting livestock production due to the shortage of feed in the dry season. SMRT sequencing technology was used to accurately analyze the microbial network and fermentation characteristics of woody silage prepared with CM as an exogenous additive to improve the fermentation quality of silage.

Mitochondrial Genome of Fagopyrum esculentum and the Genetic Diversity of Extranuclear Genomes in Buckwheat.

Fagopyrum esculentum (common buckwheat) is an important agricultural non-cereal grain plant. Despite extensive genetic studies, the information on its mitochondrial genome is still lacking. Using long reads generated by single-molecule real-time technology coupled with circular consensus sequencing (CCS) protocol, we assembled the buckwheat mitochondrial genome and detected that its prevalent form consists of 10 circular chromosomes with a total length of 404 Kb. In order to confirm the presence of a multipartite structure, we developed a new targeted assembly tool capable of processing long reads. The mitogenome contains all genes typical for plant mitochondrial genomes and long inserts of plastid origin (~6.4% of the total mitogenome length). Using this new information, we characterized the genetic diversity of mitochondrial and plastid genomes in 11 buckwheat cultivars compared with the ancestral subspecies, F. esculentum ssp. ancestrale. We found it to be surprisingly low within cultivars: Only three to six variations in the mitogenome and one to two in the plastid genome. In contrast, the divergence with F. esculentum ssp. ancestrale is much higher: 220 positions differ in the mitochondrial genome and 159 in the plastid genome. The SNPs in the plastid genome are enriched in non-synonymous substitutions, in particular in the genes involved in photosynthesis: psbA, psbC, and psbH. This presumably reflects the selection for the increased photosynthesis efficiency as a part of the buckwheat breeding program.