Nr Database Research Articles

Mining and expression analysis of color related genes in Bougainvillea glabra bracts based on transcriptome sequencing.

Bract coloration is one of the key ornamental traits in Bougainvillea, yet research has predominantly focused on phenotypic color traits and pigment composition, with limited understanding of the molecular mechanisms underlying color formation. This gap hinders the improvement and innovation in bract coloration. To elucidate the regulatory mechanisms of bract coloration in Bougainvillea and to enhance the utilization of its germplasm resources, this study employed the Illumina Novaseq 6000 sequencing platform to conduct transcriptomic sequencing on 21 samples of bracts exhibiting seven distinct phenotypes. Comparative analysis against Nr, Pfam, EggNOG, GO, and KEGG databases annotated 90,279 unigenes. The highest annotation rates were achieved with the Nr (40.13%), GO (30.44%), and EggNOG (25.64%) databases. Among the species annotated, Beta vulgaris (20.08%) and Chenopodium quinoa (14.58%) shared the highest homology with Bougainvillea bract transcriptomes. WGCNA analysis identified 12 positively correlated tissue-specific modules, of which 2 are related to bract color formation. By comparing transcriptome data and genes within these specific modules against the KEGG database, a total of 321 unigenes associated with bract color formation in Bougainvillea were discovered. Among these, 220 unigenes are involved in anthocyanin synthesis, 43 unigenes are involved in betalain synthesis, 23 unigenes are annotated as Chlorophyll a-b binding protein genes, and 35 unigenes participate in carotenoid synthesis. Quantitative real-time PCR (qRT-PCR) validation of 16 differentially expressed genes (DEGs) including PAL2, CHS1, ANS, BZ1, 6GT, CDOPA5GT, ANR, CHS2, and DOPA, revealed significant expression differences among magenta, yellow, white, and cherry-colored bracts, suggesting their potential as candidate genes for bract color development. This study not only enriches the transcriptomic data of Bougainvillea but also identifies genes associated with bract coloration, providing a valuable theoretical basis for future gene cloning, genetic engineering, and breeding efforts in Bougainvillea.

Identification and phylogenetic of cysteine peptidase genes from the bamboo pest Hippotiscus dorsalis, based on RNA-seq and PacBio Iso-Seq

One of the insects that cause the most damage to Moso bamboos in the southern regions of China is Hippotiscus dorsalis (Hemiptera: Pentatomidae). However, the study of molecular and growth development analysis is limited because its genetic resources are still in the blank stage. In order to improve our knowledge of the molecular traits of this insect pest, we first generated the transcriptome from H dorsalis using PacBio Iso-Seq. With an average length of 1495 bp, 207,663 nonredundant full-length reads were produced. Using five databases, we annotated 36,238 transcripts: Uniprot (24,388), GO (1160), NR (24,110), Pfam (9333), and KEGG (14,497). According to aligning of each transcript in the NR database, most of them (85.147 %) were annotated into the functional genes from Halyomorpha halys. In the meantime, we discovered 33,689 long noncoding RNAs (lncRNAs), 4408 transcription factors (TFs), and 586 alternative splicing (AS) events, which involved in transcriptional regulation. Additionally, the candidate cathepsin genes from the bamboo pest were examined in order to comprehend how they adapted to their host. To sum up, our findings offer a fresh resource for comprehensive transcriptional data and insights into the genetics and gene expression of H dorsalis.

In-depth comparative pathogenome, virulome, and resistome analysis of an extensive drug resistant Ralstonia mannitolilytica strain isolated from blood

IntroductionRalstonia mannitolilytica is an global opportunistic pathogen responsible for various diseases. In this study, we reported the genome of a R. mannitolilytica isolate responsible for bacteremia in an acute exacerbation of chronic obstructive pulmonary disease (AECOPD). MethodsBacterial identification was performed with a Vitek2™ Automated System and 16S rRNA sequencing with BLASTn against the Non-Redundant Protein Sequence (Nr) database. Genome sequencing and analysis were performed using PacBio RS II sequencer, Hierarchical Genome Assembly Process assembly, as well as multiple annotation databases to better understand the innate features. Antibiotic resistance genes and virulence factors were specifically identified through Antibiotic Resistance Genes database and Virulence Factors of Pathogenic Bacteria databases. ResultsThe complete genome sequence was assembled into two chromosomes with 3,495,817 bp and 1,342,871 bp in length and GC% of 65.37 % and 66.43 %, respectively. The two chromosomes were fully annotated. In chromosome 1 and 2, 19 and 14 antibiotic resistant genes and 48 and 55 virulence factors were predicted, respectively. Specifically, beta-lactam resistance genes blaOXA-443, blaOXA-444 were acquired. ConclusionsThis study aids in the understanding of the innate features of R. mannitolilytica in AECOPD.

Complete genome sequence analysis of Pestalotiopsis microspora, a fungal pathogen causing kiwifruit postharvest rots

BackgroundThe postharvest rot of kiwifruit is one of the most devastating diseases affecting kiwifruit quality worldwide. However, the genomic basis and pathogenicity mechanisms of kiwifruit rot pathogens are lacking. Here we report the first whole genome sequence of Pestalotiopsis microspora, one of the main pathogens causing postharvest kiwifruit rot in China. The genome of strain KFRD-2 was sequenced, de novo assembled, and analyzed.ResultsThe genome of KFRD-2 was estimated to be approximately 50.31 Mb in size, with an overall GC content of 50.25%. Among 14,711 predicted genes, 14,423 (98.04%) exhibited significant matches to genes in the NCBI nr database. A phylogenetic analysis of 26 known pathogenic fungi, including P. microspora KFRD-2, based on conserved orthologous genes, revealed that KFRD-2’s closest evolutionary relationships were to Neopestalotiopsis spp. Among KFRD-2’s coding genes, 870 putative CAZy genes spanned six classes of CAZys, which play roles in degrading plant cell walls. Out of the 25 other plant pathogenic fungi, P. microspora possessed a greater number of CAZy genes than 22 and was especially enriched in GH and AA genes. A total of 845 transcription factors and 86 secondary metabolism gene clusters were predicted, representing various types. Furthermore, 28 effectors and 109 virulence-enhanced factors were identified using the PHI (pathogen host-interacting) database.ConclusionThis complete genome sequence analysis of the kiwifruit postharvest rot pathogen P. microspora enriches our understanding its disease pathogenesis and virulence. This study establishes a theoretical foundation for future investigations into the pathogenic mechanisms of P. microspora and the development of enhanced strategies for the efficient management of kiwifruit postharvest rots.

Full-Length Transcriptome Construction and Systematic Characterization of Virulence Factor-Associated Isoforms in Vairimorpha (Nosema) Ceranae.

Vairimorpha (Nosema) ceranae is a single-cellular fungus that obligately infects the midgut epithelial cells of adult honeybees, causing bee microsporidiosis and jeopardizing bee health and production. This work aims to construct the full-length transcriptome of V. ceranae and conduct a relevant investigation using PacBio single-molecule real-time (SMRT) sequencing technology. Following PacBio SMRT sequencing, 41,950 circular consensus (CCS) were generated, and 25,068 full-length non-chimeric (FLNC) reads were then detected. After polishing, 4387 high-quality, full-length transcripts were gained. There are 778, 2083, 1202, 1559, 1457, 1232, 1702, and 3896 full-length transcripts that could be annotated to COG, GO, KEGG, KOG, Pfam, Swiss-Prot, eggNOG, and Nr databases, respectively. Additionally, 11 alternative splicing (AS) events occurred in 6 genes were identified, including 1 alternative 5' splice-site and 10 intron retention. The structures of 225 annotated genes in the V. ceranae reference genome were optimized, of which 29 genes were extended at both 5' UTR and 3' UTR, while 90 and 106 genes were, respectively, extended at the 5' UTR as well as 3' UTR. Furthermore, a total of 29 high-confidence lncRNAs were obtained, including 12 sense-lncRNAs, 10 lincRNAs, and 7 antisense-lncRNAs. Taken together, the high-quality, full-length transcriptome of V. ceranae was constructed and annotated, the structures of annotated genes in the V. ceranae reference genome were improved, and abundant new genes, transcripts, and lncRNAs were discovered. Findings from this current work offer a valuable resource and a crucial foundation for molecular and omics research on V. ceranae.

Sex Differences in Antennal Transcriptome of Hyphantria cunea and Analysis of Odorant Receptor Expression Profiles.

Insects rely on olfaction for mating, finding oviposition sites, and locating hosts. Hyphantria cunea is a serious pest that severely damages forests. Differential expression analysis of olfactory-related genes between males and females is the basis for elucidating the functions of olfactory-related proteins in H. cunea. In this study, Illumina HiSeqTM 4000 high-throughput sequencing technology was used to perform transcriptome sequencing of the antennal tissues of adult male and female H. cunea. Functional annotation was conducted using the NR, Swiss-Prot, KOG, KEGG, and GO databases, and the results showed that the antennal transcriptome of adult H. cunea contained 50,158 unigenes. Differential expression analysis identified 3923 genes that were significantly differentially expressed between male and female antennae. A total of 221 olfactory-related genes were annotated, and 96 sex-biased genes were identified, including 13 odorant receptors (ORs), 48 odorant binding proteins (OBPs), 7 chemosensory proteins (CSPs), 10 ionotropic receptors (IRs), 10 sensory neuron membrane proteins (SNMPs), 2 gustatory receptors (GRs), and 6 odorant-degrading enzymes (ODEs), indicating that there were differences in olfaction between male and female H. cunea. Quantitative real-time PCR was used to verify the expression levels of 21 putative general odorant receptor genes in male and female antennae. HcunOR4 and HcunOR5 showed female-biased expression; HcunOR48, HcunOR49 and HcunOR50 showed male-biased expression. The results were consistent with the transcriptome differential analysis. The screening of male-biased odorant receptor genes might provide a theoretical basis for the functional characterization of odorant receptors for recognizing sex pheromones in H. cunea.

Establishment of Novel Simple Sequence Repeat (SSR) Markers from Chimonanthus praecox Transcriptome Data and Their Application in the Identification of Varieties.

Chimonanthus praecox, a member of the Calycanthaceae family, is a unique, traditional, and famous flowering economic tree species in China. Despite the existence of several varieties, only a few cultivars have been formally named. Currently, expression sequence tag-simple sequence repeat (EST-SSR) markers are extensively used to identify different species and varieties; a large number of microsatellites can be identified from transcriptome databases. A total of 162,638 unigenes were assembled using RNA-seq; 82,778 unigenes were annotated using the Nr, Nt, Swiss-Prot, Pfam, GO, KOG, and KEGG databases. In total, 13,556 SSR loci were detected from 11,691 unigenes, with trinucleotide repeat motifs being the most abundant among the six repeat motifs. To develop the markers, 64,440 pairs of SSR primers with polymorphism potential were designed, and 75 pairs of primers were randomly selected for amplification. Among these markers, seven pairs produced amplified fragments of the expected size with high polymorphism. Using these markers, 12 C. praecox varieties were clustered into two monophyletic clades. Microsatellites in the transcriptome of C. praecox exhibit rich types, strong specificity, and great polymorphism potential. These EST-SSR markers serve as molecular technical methods for identifying different varieties of C. praecox and facilitate the exploration of a large number of candidate genes associated with important traits.

Chromosome-Scale Genome Assembly Provides Insights into Fresh Pine Wood Decay Strategies of the Wolfiporia hoelen

The sclerotia of Wolfiporia hoelen (Fr.) Y.C. Dai & V. Papp is an important traditional Chinese medicine with diverse pharmacological properties. This study utilized a combination of PacBio Long-Read Sequencing, Illumina Short-Read Sequencing, and Hi-C Sequencing to generate a high-quality chromosome-level genome assembly of a W. hoelen strain Minling A5. There were 112 contigs in the genome, with 62.95 Mb in total length and 4.21 Mb in length for the contig N50. The average GC content was 51.89%. Based on Hi-C data, we corrected the CCS data and scaffolded them into 14 pseudo-chromosomes. The genome contained 44.37% repetitive sequences and 12,670 protein-coding genes, 86.53% (10,963) of which could be functionally annotated in at least one of the KOG, GO, Pfam, Swissprot, TrEMBL, NR, and KEGG databases. In addition, 240 transfer RNAs, 97 ribosomal RNAs, and 103 other non-coding RNAs were identified in the W. hoelen genome. A total of 755 pseudogenes were also identified, with an average length of 2665.51 bp. Further, there were 398, 100, 2837, 519, and 2068 genes annotated by CAZymes, TCDB, PHI, P450, and DFVF databases, respectively. One notable attribute of W. hoelen is its capacity to thrive in a substrate of fresh pine sawdust. Through an analysis of the growth on various pure wood sawdust culture media, we found that the growth of W. hoelen and Sparassis latifolia on pine sawdust was similar to that on broad-leaved wood sawdust, while the growth of Pleurotus ostreatus, P. eryngii, and Cyclocybe aegerita was slower than that on broad-leaved wood sawdust. By the functional annotation analysis of orthogroups in these five mushroom-forming fungi, it was determined that 645 orthogroups were specifically common in W. hoelen and S. latifolia. The genes in these specific orthogroups were significantly enriched in 12 pathways, including steroid biosynthesis, biosynthesis of antibiotics, and tyrosine metabolism. The high-quality genome and comparative genome analysis results significantly contribute to advancing our foundational knowledge of W. hoelen biology, while also offering valuable insights for the development of innovative biotechnological approaches aimed at enhancing the efficient and sustainable utilization of Pinus.

Genome- and Toxicology-Based Safety Assessment of Probiotic Akkermansia muciniphila ONE Isolated from Humans.

In this study, the genome of Akkermansia muciniphila ONE (designated AKK ONE) was sequenced, assembled, and analyzed. In addition, the safety of this strain was further evaluated by toxicological studies. The results showed that the AKK ONE genome is contained on a single chromosome with a total length of 2,817,524 bp and an average GC content of 55.48%. In total, 2411, 1131, 1168, 1745, and 1402 genes were annotated to the NR, GO, KEGG, COG, and SwissProt database, respectively. Potential resistance genes, adeF, tetW, ANT(3″)-IIa, and aadA1 were detected. AKK ONE was sensitive to ampicillin, ceftriaxone, cefotaxime, meropenem, tetracycline, and chloramphenicol and resistant to moxifloxacin. No potential virulence-related genes were detected. The PathogenFinder database analysis showed that AKK ONE was a non-potential human pathogen. This strain had good gastroenteric fluid tolerance and a weak ability to colonize the gut. No test item-related adverse effects were observed in the acute and subchronic toxicity test. AKK ONE did not display mutagenic activity either. This strain did not change the hematological and clinical biochemical parameters of mice. The weights of the organs were not affected by AKK ONE treatment. These results support that AKK ONE is safe for use as a probiotic at a dose of 8.28 × 109 CFU/kg bw/day.

Investigation of endogenous phytohormones and critical flowering genes during flower development in Iris germanica

With showy display and multi-color, Iris germanica (Bearded iris) is one of the most popular ornamental perennials, of which molecular mechanisms in regulation of flowering are poorly understood. The objectives of this study were to reveal profiles of endogenous phytohormones and identify critical genes in floral regulation during flower development. The development process of flowering from initiation of inflorescence to flower stem elongation was observed. Based on the observation results, plant materials at four critical development stages were collected to identify the endogenous phytochromes and genes regulating flowering by using high performance liquid chromatography (HPLC) and high-throughput RNA sequencing. The concentration of different endogenous phytohormones showed various trends at four stages. Among identified phytohormones, the concentrations of Indole-3-acetic acid (IAA) and abscisic acid (ABA) were relatively high when compared with the active forms of other phytohormones. Besides, the concentration of IAA was low at pistil and petal primordium stage under winter low temperature conditions and increased at petal and stem elongation stages, while the concentration changes of ABA showed opposite trend. With comprehensive analysis of the DEGs annotated with the GO, KEGG and NR databases, homologs of critical genes in flowering regulatory pathway, such as IgAP2, IgFRI, IgSVP, IgVIN3-1, IgVIN3-2, IgGI et al. were identified. Correlation analysis between DEGs and phytohormones showed that IgFLC, IgFRI, IgVIN3-1 had closely negative relations with IAA and positive relations with ABA. The result indicates that IgFLC, IgFRI and IgVIN3-1 might be key genes in regulation of the flowering process during the flower bud development. The profiles of endogenous phytohormones and critical flowering genes identified in this study could provide references for further research on regulation of flower time in I. germanica or other species in this genus.

Functional Analysis of Stress Resistance of Bacillus cereus SCL10 Strain Based on Whole-Genome Sequencing.

A Gram-positive, rod-shaped, aerobic, motile, and spore-forming bacterium, designated SCL10, was isolated from Acaudina molpadioides exposure to Co-60 radiation. In this study, whole-genome sequencing was performed to identify the strain as Bacillus cereus and functional characterization, with a focus on stress resistance. The genome of the B. cereus SCL10 strain was sequenced and assembled, revealing a size of 4,979,182 bp and 5167 coding genes. The genes involved in biological functions were annotated by using the GO, COG, KEGG, NR, and Swiss-Prot databases. The results showed that genes related to alkyl hydroperoxide reductase (ahpC, ahpF), DNA-binding proteins from starved cells (dps), spore and biofilm formation (spoVG, spo0A, gerP), cold shock-like protein (cspC, cspE), ATP-dependent chaperone (clpB), and photolyase, small, acid-soluble spore protein (SASP) and DNA repair protein (recA, radD) could explain the stress resistance. These findings suggest that antioxidant activity, sporulation, biofilm formation, and DNA protection may be considered as the main resistance mechanisms under exposure to radiation in the B. cereus SCL10 strain.

Construction of a Full-Length Transcriptome of Western Honeybee Midgut Tissue and Improved Genome Annotation.

Honeybees are an indispensable pollinator in nature with pivotal ecological, economic, and scientific value. However, a full-length transcriptome for Apis mellifera, assembled with the advanced third-generation nanopore sequencing technology, has yet to be reported. Here, nanopore sequencing of the midgut tissues of uninoculated and Nosema ceranae-inoculated A. mellifera workers was conducted, and the full-length transcriptome was then constructed and annotated based on high-quality long reads. Next followed improvement of sequences and annotations of the current reference genome of A. mellifera. A total of 5,942,745 and 6,664,923 raw reads were produced from midguts of workers at 7 days post-inoculation (dpi) with N. ceranae and 10 dpi, while 7,100,161 and 6,506,665 raw reads were generated from the midguts of corresponding uninoculated workers. After strict quality control, 6,928,170, 6,353,066, 5,745,048, and 6,416,987 clean reads were obtained, with a length distribution ranging from 1 kb to 10 kb. Additionally, 16,824, 17,708, 15,744, and 18,246 full-length transcripts were respectively detected, including 28,019 nonredundant ones. Among these, 43,666, 30,945, 41,771, 26,442, and 24,532 full-length transcripts could be annotated to the Nr, KOG, eggNOG, GO, and KEGG databases, respectively. Additionally, 501 novel genes (20,326 novel transcripts) were identified for the first time, among which 401 (20,255), 193 (13,365), 414 (19,186), 228 (12,093), and 202 (11,703) were respectively annotated to each of the aforementioned five databases. The expression and sequences of three randomly selected novel transcripts were confirmed by RT-PCR and Sanger sequencing. The 5' UTR of 2082 genes, the 3' UTR of 2029 genes, and both the 5' and 3' UTRs of 730 genes were extended. Moreover, 17,345 SSRs, 14,789 complete ORFs, 1224 long non-coding RNAs (lncRNAs), and 650 transcription factors (TFs) from 37 families were detected. Findings from this work not only refine the annotation of the A. mellifera reference genome, but also provide a valuable resource and basis for relevant molecular and -omics studies.

Transcriptome analysis and development of EST-SSR markers in the mushroom Auricularia heimuer

Auricularia heimuer, the third most frequently cultivated edible mushroom species worldwide, has high medicinal value. However, a shortage of molecular marker hinders the efficiency and accuracy of genetic breeding efforts for A. heimuer. High-throughput transcriptome sequencing data are essential for gene discovery and molecular markers development. This study aimed to clarify the distribution of SSR loci across the A. heimuer transcriptome and to develop highly informative EST-SSR markers. These tools can be used for phylogenetic analysis, functional gene mining, and molecular marker-assisted breeding of A. heimuer. This study used Illumina high-throughput sequencing technology to obtain A. heimuer transcriptome data. The results revealed 37,538 unigenes in the A. heimuer transcriptome. Of these unigenes, 24,777 (66.01%) were annotated via comparison with the COG, Pfam, and NR databases. Overall, 2510 SSRs were identified from the unigenes, including 6 types of SSRs. The most abundant type of repeats were trinucleotides (1425, 56.77%), followed by mononucleotides (391, 15.58%) and dinucleotides (456, 18.17%). Primer pairs for 102 SSR loci were randomly designed for validity confirmation and polymorphism identification; this process yielded 53 polymorphic EST-SSR markers. Finally, 13 pairs of highly polymorphic EST-SSR primers were used to analyze the genetic diversity and population structure of 52 wild A. heimuer germplasms, revealing that the 52 germplasms could be divided into three categories. These results indicated that SSR loci were abundant in types, numbers, and frequencies, providing a potential basis for germplasm resource identification, genetic diversity analysis, and molecular marker-assisted breeding of A. heimuer.

The genomic database of fruits: A comprehensive fruit information database for comparative and functional genomic studies

Fruit has an important role in human nutrition and health; therefore, the systematic study of fruit genomic data is essential. The Genomic Database of Fruits (TGDF, http://tgdf.bio2db.com/), established through whole-genome analyses of 44 fruit species, is a comprehensive, user-friendly fruit database. TGDF contains a wealth of functional genes, including 11,350 flowering genes, 3161 auxin signaling genes, 2164 anthocyanin synthesis genes, 1464 abscisic acid (ABA) synthesis genes, 10,931 ​cell division and expansion genes, 1786 starch synthesis genes, 294 fruit size genes, and 6311 sugar transporter genes. Additionally, TGDF contains 1,433,368 CRISPR guide sequences from various fruit genomes, along with information on homologous genes and duplication types for the 44 fruit species. TGDF contains 6,417,060 gene annotations sourced from TrEMBL, SwissProt, Nr, and Gene Ontology databases, along with tools such as Sequence Fetch, BLAST, Synteny, and JBrowse for bioinformatics analyses. Transcriptomic data were also collected and collated from fruits, including details on instruments, tissues, or growth stages. This comprehensive, user-friendly resource is the first collection of fruit genomic data. Users can easily download genomic sequences, gene annotations, and bioinformatics analysis results from TGDF, which will be updated continually. We anticipate that TGDF will become a primary resource for fruit comparative and functional genomic studies.

A Full-Length Transcriptome and Analysis of the NHL-1 Gene Family in Neocaridina denticulata sinensis.

Neocaridina denticulata sinensis has emerged as a promising model organism for basic studies in Decapod. However, the current transcriptome information on this species is based on next-generation sequencing technologies, which are limited by a short read length. Therefore, the present study aimed to generate a full-length transcriptome assembly of N. denticulata sinensis utilizing the PacBio Sequel Ⅱ platform. The resulting transcriptome assembly comprised 5831 transcripts with an N50 value of 3697 bp. Remarkably, 90.5% of these transcripts represented novel isoforms of known genes. The transcripts were further searched against the NR, SwissProt, KEGG, KOG, GO, NT, and Pfam databases. A total of 24.8% of the transcripts can be annotated across all seven databases. Additionally, 1236 alternative splicing events, 344 transcription factors, and 124 long non-coding RNAs (LncRNAs) were predicted. Based on the alternative splicing annotation results, a RING finger protein NHL-1 gene from N. denticulata sinensis (NdNHL-1) was identified. There are 15 transcripts in NdNHL-1. The longest transcript is 4995 bp in length and encodes a putative protein of 1665 amino acids. A phylogenetic analysis showed its close relationship with NHL-1 from other crustacean species. This report represents the full-length transcriptome of N. denticulata sinensis and will facilitate research on functional genomics and environmental adaptation in this species.

First report of pepper chlorosis-associated virus infecting tobacco (Nicotiana tabacum) plants in Sichuan Province in China.

Tobacco (Nicotiana tabacum) is an economically important crop in China, and more than 30 viruses have been reported to infect tobacco (Yin et al. 2022). In July 2022, we observed interveinal necrosis on tobacco leaves in fields in Sichuan Province (N 27.9172, E 105.6662) (Fig. 1). Total RNA was isolated from multiple leaves of one plant using an RNAprep Pure Polysaccharide Polyphenol Plant Total RNA Extraction Kit (TIANGEN, Beijing, China). Total RNAs were pooled, and a TruSeq Stranded Total RNA with RiboZero Gold Kit (Illumina, San Diego, CA, USA) was used to eliminate ribosomal RNA. An RNA-Seq library was constructed using VAHTS Universal V6 RNA-seq Library Prep (Nanjing Vazyme, China). High-throughput sequencing was performed on the Illumina DNBseq platform (BGI-ShenZhen, China), which yielded 20,102,087 reads with an average length of 150 nt (total size >6 Gb). Unaligned reads were assembled de novo using SPAdes (Bankevich et al. 2012). Contigs with length ≥200 nt were subjected to local BLASTn and BLASTx analyses against the GenBank nt and nr databases, respectively (Wang et al. 2022). A total of 23 contigs were identified through BLASTx (e-value cut-off = 10 -3), ranging from 631 to 1555 bp long, with 82% to 96% coverage to partial genomic sequences of pepper chlorosis-associated virus (PepCaV-Higashitsuno_2021; Accessions: LC719619 to LC719621) and one contig (6459 bp) with 99% similarity to tobacco mosaic virus (Accession: OP525281) isolate DSMZ PV-0109 from Germany. The complete genome sequence of PepCaV was obtained using primers based on the assembled contigs. The 5'- and 3'-terminal regions of the RNA genome were obtained by 5'- and 3'-rapid amplification of cDNA ends. These amplicons were cloned using the pEASY-Blunt Zero Cloning Kit (TRANSGEN, Nangjing, China) and sequenced by Sanger sequencing. Complete genome sequences of tripartite PepCaV from tobacco samples were 7697, 1808, and 1557 nucleotides long (Accession: OR451987 to OR451989) and showed genome organization typical of the genus Ophiovirus in the family Aspiviridae. The complete sequences of RNA1, RNA2 and RNA3 genome segments shared 92.36%, 90.43%, and 95.24%, nucleotide sequence identities, respectively, with the isolate PepCaV-Higashitsuno_2021 pepper isolate (Accession: LC719619 to LC719621) (Shimomoto et al. 2023), but PepCaV has not been reported to infect N. tabacum. In June 2023, 10 plants collected from each place of Macheng (N 27.9094, E 105.6740), Xiangyang (N 28.0936, E 105.6249) and Moni (N 27.8899, E 105.5936) showing interveinal necrosis symptoms were tested using RT-PCR using PepCaV-MP610-F (5'-TGTTCTCTGCTATGCGGTTG -3') and PepCaV-MP610-R (5'-AGCAATCTCGCACCTGAAGT-3') to product 610bp amplicon. Twenty-five tobacco plants were positive for PepCaV. Single sequence from each location was submitted to GenBank (Accession: PP728631 to PP728633). Sap extracts from the original field leaf samples collected from Sichuan Province were used to mechanically inoculate tobacco plants (10 plants) at the four-leaf stage. After 7 days, leaf samples were tested using RT-PCR assay specific to PepCaV and TMV while samples were positive only for TMV but failed to transmit PepCaV by mechanical inoculation. According to previous literature, ophioviruses may be transmitted though soilborne fungus (Jeong et al. 2014). Further research is needed to understand the transmission, epidemiology, and pathological properties of the PepCaV. To our knowledge, this is the first report documenting natural PepCaV infection of tobacco plants in China, providing a scientific basis for PepCaV infection control in tobacco plantations.

Transcriptomic Analysis of Green Leaf Plants and White-Green Leaf Mutants in Haworthia cooperi var. pilifera.

Haworthia cooperi var. pilifera is a succulent plant with ornamental value. The white-green leaf mutant (wl) showed a significant difference in leaf color from the wild-type plant (WT). In this study, we integrated the transcriptomes of wl and WT plants to screen differentially expressed genes related to leaf color variation. The results of transcriptome analysis showed that 84,163 unigenes were obtained after de novo assembly and the NR database annotated the largest number of unigenes, which accounted for 57.13%, followed by NT (43.02%), GO (39.84%), Swiss-Prot (39.25%), KEGG (36.06%), and COG (24.88%). Our finding showed that 2586 genes were differentially expressed in the two samples, including 1996 down-regulated genes and 590 up-regulated genes. GO analysis predicted that these differentially expressed genes (DEGs) participate in 12 cellular components, 20 biological processes, and 13 molecular function terms and KEGG analysis showed that metabolic pathways, plant-pathogen interaction, glycerophospholipid metabolism, endocytosis, plant hormone signal transduction, and ether lipid metabolism were enriched among all identified pathways. Through functional enrichment analysis of DEGs, we found that they were involved in chloroplast division and the biosynthesis of plant pigments, including chlorophyll, carotenoids, anthocyanin, and transcription factor families, which might be related to the formation mechanism of leaf color. Taken together, these results present insights into the difference in gene expression characteristics in leaves between WT and wl mutants and provide a new insight for breeding colorful leaf phenotypes in succulent plants.

Metagenomic and flavoromic profiling reveals the correlation between the microorganisms and volatile flavor compounds in Monascus-fermented cheese

The Monascus-fermented cheese (MC) is a unique cheese product that undergoes multi-strain fermentation, imparting it with distinct flavor qualities. To clarify the role of microorganisms in the formation of flavor in MC, this study employed SPME (arrow)-GC–MS, GC-O integrated with PLS-DA to investigate variations in cheese flavors represented by volatile flavor compounds across 90-day ripening periods. Metagenomic datasets were utilized to identify taxonomic and functional changes in the microorganisms. The results showed a total of 26 characteristic flavor compounds in MC at different ripening periods (VIP＞1, p < 0.05), including butanoic acid, hexanoic acid, butanoic acid ethyl ester, hexanoic acid butyl ester, 2-heptanone and 2-octanone. According to NR database annotation, the genera Monascus, Lactococcus, Aspergillus, Lactiplantibacillus, Staphylococcus, Flavobacterium, Bacillus, Clostridium, Meyerozyma, and Enterobacter were closely associated with flavor formation in MC. Ester compounds were linked to Monascus, Meyerozyma, Staphylococcus, Lactiplantibacillus, and Bacillus. Acid compounds were linked to Lactococcus, Lactobacillus, Staphylococcus, and Bacillus. The production of methyl ketones was closely related to the genera Monascus, Staphylococcus, Lactiplantibacillus, Lactococcus, Bacillus, and Flavobacterium. This study offers insights into the microorganisms of MC and its contribution to flavor development, thereby enriching our understanding of this fascinating dairy product.

Insights into the mechanism of color formation of the freshwater prawn (Macrobrachium rosenbergii) revealed by de novo assembly transcriptome analysis

Body color is an important visual indicator of crustacean quality and plays a major role in consumer acceptability, perceived quality, and the market price of crustaceans. The freshwater prawn (Macrobrachium rosenbergii) has two distinct phenotypic variations, characterized by dark blue and light yellow body colors. However, the underlying mechanisms regulating the body color of M. rosenbergii remain unclear. In this study, the composition of shell color parameters and pigment cells of raw and cooked dark blue and light yellow M. rosenbergii was investigated and the mechanisms associated with body color were elucidated by transcriptome analysis. The results showed significant differences in the raw shells of the dark blue and light yellow M. rosenbergii (L: 26.20 ± 0.53 vs. 29.25 ± 0.45; a: −0.88 ± 0.19 vs. 0.35 ± 0.18; b: 1.73 ± 0.20 vs. 3.46 ± 0.37; dE: 70.33 ± 0.53 vs. 67.34 ± 0.45, respectively, p = 0.000) as well as the cooked shells (L: 58.14 ± 0.81 vs. 55.78 ± 0.55; a: 19.30 ± 0.56 vs. 16.42 ± 0.40; b: 23.60 ± 0.66 vs. 20.30 ± 0.40, respectively, p < 0.05). Transcriptome differential gene analysis obtained 39.02 Gb of raw data and 158,026 unigenes. Comprehensive searches of the SwissProt, Nr, KEGG, Pfam, and KOG databases resulted in successful annotations of 23,902 (33 %), 40,436 (25.59 %), 32,015 (20.26 %), 26,139 (16.54 %), and 22,155 (14.02 %) proteins, respectively. By KEGG pathway analysis, numerous differentially expressed genes were related to pigmentation-related pathways (MAPK signaling pathway, Wnt signaling pathway, melanin production, tyrosine metabolism, and cell-cell communication process). Candidate DEGs that may be involved in body color included apolipoprotein D, crustacyanin, cytochrome P450, and tyrosinase, as verified by quantitative real-time PCR. The results of this study provide useful references to further elucidate the molecular mechanisms of color formation of M. rosenbergii and other crustaceans.

First report of Hibiscus soymovirus in Hibiscus rosa-sinensis in Colombia in mixed infection.

Hibiscus is native to southeast Asia but well suited to Colombia's arid soil and dry climates from the coast to the mountains of Bogotá. Viruses infecting hibiscus in Colombia are largely unexplored, with four viruses previously known: hibiscus chlorotic ringspot virus (HCRSV), hibiscus latent Fort Pierce virus (HLFPV), hibiscus latent Singapore virus (HLSV), and citrus leprosis virus C2 (CiLV-C2) (Padmanabhan et al., 2023). Mixed infections between these viruses were frequently detected. A recent virome analysis of a single hibiscus plant from Colombia revealed multiple viruses in mixed infection; : HCRSV, HLFPV, passion fruit green spot virus (PFGSV), a strain of physalis vein necrosis nepovirus, four novel carlavirus, one new potexvirus and a mitovirus. In addition, few smaller contigs of blunervirus and soymovirus were also identified in the high throughput sequencing (HTS) data, but their presence in the mixed infection could not be validated (A. Roy et al. 2023unpublish data). During Brevipalpus-transmitted virus (BTV) surveys, two asymptomatic and 15 hibiscus foliar samples showing green ringspots with central chlorotic spots in senescing areas, mosaic, and black or chlorotic spots were collected from six departments (states) in three geographical regions of Colombia: Tolima (n=4) and Cauca Valley (n=2) (Andean region), Meta (n=6) and Casanare (n=1) (Orinoquia region), and Quindío (n=1) and Risaralda (n=1) (coffee growing region). About 100 mg of 17 hibiscus leaf samples were separately processed for RNA isolation without DNase I treatment and tested for known BTVs, and for newly discovered hibiscus soymovirus (HSV; genus Soymovirus family Caulimoviridae) using PCR assays (Padmanabhan et al. 2023, Wang et al. 2023). To identify potential HSV infection in the samples, published SVF1/SVR1 and newly designed primer pairs (HSV-REP-F/-R and HSV-CPG-F/-R) were used to amplify the 430 nt transactivation (ORF-VI), 631 nt replicase (REP) and 401 nt coat protein gene (CPG), respectively (Supplementary 1). Of 17 samples tested, three from Tolima and one each from Meta and Quindío yielded all three expected size amplicons. Bi-directional sequencing followed by BLASTn analysis revealed 95-98% nt identity with the CPG, REP, and ORF-VI genes of HSV (OP757659). Ribo-depleted libraries were prepared using the RNA extracts of five HSV PCR positive samples. HTS yielded 11.6 to 50.3 million raw reads per sample library. Adapters were trimmed and filtered from the raw reads with Trimmomatic v0.39 and then assembled using SPAdes v3.15.5 (Padmanabhan et al., 2023). Contigs were blasted against the Arabidopsis proteome and a RefSeq-based viral protein database. Potential viral sequences were then blasted against the complete NCBI nr database. Assembled soymo contigs covered 99-100% of the HSV genome, with per-nucleotide read depths of 23.8 to 393. Contigs from the Tolima (Accessions; OR621030- OR621032 and Quindío samples (OR621033) covered 99-100% of the HSV genome and had >96-98% nt identity to Hawaiian isolate (OP757659) whereas the Meta sample contigs covered 78% of the genome with 9495% nt identity. HTS contigs shared >98-99% nt identities with their PCR amplicons. Along with HSV, other virus sequences (HCRSV, HLFPV, PFGSV, CiLV-C2, and mycoviruses) were variously detected from all five libraries. Due to mixed infection no symptom similarity was noticed among these 5 samples. The findings in hibiscus in Tolima, Meta and Quindío represent the first confirmed report of HSV infection in hibiscus in Colombia. The widespread distribution suggests the possibility of HSV dispersion via movement of planting material, and potential further spread to another hibiscus growing region.