Pinus taeda and Pinus lambertiana using high-performance computing resources. The quality of draft genomes and the genome annotation have significant consequences for the investigation of TEs and these aspects are discussed. Several TE families frequently inserted into genes or their flanks were identified in both species’ genomes. Potentially important sequence motifs were identified in TEs that could bind additional regulatory factors, promoting gene network formation with faster or enhanced transcription initiation. Node genes that contain many TEs were observed in multiple potential transposable element-associated networks. This study demonstrated the increased accumulation of TEs in the introns of stress-responsive genes of pines and suggests the possibility of rewiring them into responsive networks and sub-networks interconnected with node genes containing multiple TEs. Many such regulatory influences could lead to the adaptive environmental response clines that are characteristic of naturally spread pine populations.Sequencing the giga-genomes of several pine species has enabled comparative genomic analyses of these outcrossing tree species. Previous studies have revealed the wide distribution and extraordinary diversity of transposable elements (TEs) that occupy the large intergenic spaces in conifer genomes. In this study, we analyzed the distribution of TEs in gene regions of the assembled genomes of  The functional role of transposable element (TE) insertions distributed throughout plant genomes is less studied and phenotypic changes are less obvious compared with protein-coding regions ,2,3. Tracopia, Ty3-gypsy, etc.), families, and subfamilies. Up to 20% nucleotide sequence variation is permitted between family members [FRetro129 [Sukkula, [Grande, [Ogre, [TEs are organized into classes (retrotransposons and DNA transposons), superfamilies , while Sukkula, ,35, Gran[Grande, , and Ogr, [Ogre, ). Howeve, [Ogre, ,39. The , [Ogre, ,41,42. R, [Ogre, . Non-hom, [Ogre, . Some ge, [Ogre, . During , [Ogre, ,47,48. D, [Ogre, ,55,56,57Pinus taeda) genome contains a high proportion of TEs; there are approximately 1500 families with the prevalence of LTR RLXs (42% of genome sequence [Pinaceae and even in more distant gymnosperms [Pinus sylvestris) revealed a rapid expression of TE-containing sequences in response to stress conditions [P. taeda and P. lambertiana). We also explored the possibility of transferring this information to P. sylvestris genome studies. The obtained results were used to evaluate if the distribution of TEs in gene regions is random regarding different gene regions , species or TE families; if genes containing similar TE families are involved in similar processes; and if the investigated TEs contain potential gene regulatory motifs. Conifer genomes are greater than 15 Gbp in size and contain extended regions of non-coding DNA, much of which are derived from TEs. The genomes of pines represent one extreme in plants, with stable diploid genomes that are expanded by the proliferation of TEs, in contrast to frequent polyploidisation events in angiosperms ,59,60. Tsequence ,66. In cnosperms ,68. Our nditions . The comnditions . TherefoP. taeda v.1.01 and v.2.0 and P. lambertiana v.1.01 were downloaded from https://treegenesdb.org. The PIER v.2.0 (Pine Interspersed Element Resource) [UPPMAX  resource was initially used for analyses of genomic sequences. The Riga Technical University High Performance Computing (HPC) Centre was used for further analyses in Latvia. The publicly available PIER 2.0 database contains conifer TEs and nested repeats that were recognized by automatic genome annotation from the P. taeda v.1.01 genome. A large portion of the database entries contain nested repeats with 2–4 pairs of direct LTRs rather than full-length TE sequences, which could negatively influence the analyses in this study. One TE insertion within a particular gene will have hits to hundreds of different database entries containing high sequence similarity to this TE; therefore, all full-length elements were clustered with CD-Hit v.4.6.4 [PIER database, resulting in 24,591 repeats. The new data set of extracted repeats contained sequences from 79 bp to 13,295 bp in length, with an average length of 579 bp and a median length of 417 bp. The presence of nested repeats could lead to the extraction of direct repeats of the same TE family or sequences that represent a body of nested TEs. This was verified by individual sequence inspection and all-to-all alignment. To reduce such errors, extracted TE-derived repeats were CD-Hit clustered, resulting in 9659 entries. Sequences of only 0.1–2 kb in length were used for further analyses, resulting in a final database of 9107 entries (5.7% reduction). This was the TE-derived repeat database.Reference genomes for esource)  database v.4.6.4  utilizinBedtools v.2.27.1 [BLASTn v.2.2.26 [Samtools [BEDOPS v.2.4.35 [BLAST with the following parameters: percent identity ≥80%, alignment length ≥100 bp, Query Coverage Per High-scoring Segment Pair (HSP) ≥90%. For P. lambertiana analyses, the Query Coverage Per HSP parameter was lowered to ≥80% as the previous parameter set produced no hits. A t-test was calculated for the evaluation of TE enrichment with significance to flanking regions in the vicinity of genes compared with other regions. The standard deviation of the distribution of differences between independent sample means was estimated for each TE-derived repeat . Enrichment significance of p = 0.001 was considered if t was greater than 5.04 (df = 8) and p = 0.05 if t > 2.31 (df = 8).v.2.27.1  were usev.2.2.26 . SamtoolSamtools  were usev.2.4.35  used forP. taeda v.2.0 true genomic coordinates were evaluated for 15,534 transcripts annotated with any Gene Ontology term . Correct genomic coordinates of transcripts were identified by running a computationally intense local NCBI short-blast algorithm with parameters of sequence identity >98% and 100% query coverage per HSP. The resulting genomic coordinates were further sorted and corrected as follows: scaffolds containing identical hits and gene coordinates were deleted, with genes larger than 20 kb manually verified and repetitions of partial exon structures or gene repeats on one scaffold deleted, leaving only one conventional structure with all exons present. Further gene transcripts were screened against the PIER database, and genes matching TEs with more than 50% query coverage and more than 80% sequence identity were filtered out. All extractions and analyses were repeated with the new gene set. After all steps, genes containing two or more types of repeats (filling less than 50% of template) were still present in the dataset and were later filtered from the results. Gene genomic sequences from the transcription start to termination site were extracted from the reference genomes into separate databases.To overcome inconsistencies found in genome files, REPFIND repeat prediction tool [Repbase [Softberry tools , and PLE v.30.0  were useb server  were usehttp://www.geneontology.org/), and the P. taeda v.1.01 gene functional annotation was downloaded from PLAZA [P. taeda v.2.0 was generated by considering gene transcript homology to P. taeda v.1.01, which enabled comparison between differing numberings of genome versions. Fewer than 50% of the genes were categorized to any GO term for P. taeda v.2.0 . For P. lambertiana v.1.01, a functional annotation file was downloaded from the Treegenes data repository (https://treegenesdb.org/). In total, 8943 genes from 13,637 were assigned with any GO term for P. lambertiana v.1.01. Homology of the evaluated gene set in each network was tested by sequence comparison of transcripts (whole set) between two species .The Gene Ontology (GO) classification file was obtained from the Gene Ontology Consortium  were considered in the final networking analysis. BINGO v. 3.0.3 [Cytoscape v.3.3.0 [BINGO was used for the GO term overrepresentation test in one particular network compared to custom GO annotation of all pine genes. Comparative networks between two species or different TE locations (flanking regions or introns) were constructed using DyNet [CLC Genomics Workbench Blast2GO analysis workflow tools. The GenAlEx 6.05 software package [STRING v.11 [Gene networking analysis was performed only to assess potential functional significance of the evaluated gene groups. Each gene network was established by the presence of high-quality matches to similar TEs (members of one TE family), present in a particular location (5′ and 3′ 0–2 kb gene flanks or introns). High-quality sequence matches  than in the P. taeda v.2.0 genome assembly (the top-ranked TE had matches to only 39 genes). A similar situation was observed when analyzing the 5-kb flanking regions using the full-length TE database. However, when the TE-derived repeat database was used for the analyses, the number of genes with repeats in the introns or flanking regions was similar in both P. taeda genome versions. The main reason for these differences may be because the full-length TE elements identified from v.1.01 did not align over their entire length with the v.2.0 genomic sequence assembled from longer reads. Additionally, observation of particular gene introns with similarity to interspersed repeats revealed that incomplete TE sequences in v.2.0 were frequently present with masked sequences nearby. This suggests persistent problems with TE-containing read assembly and indicates the need for validation of observed matches to confirm the presence of full-length or partial TEs. Due to the diversity and repetitive nature of conifer TEs, the differentiation of full-length elements from partial, chimeric, or nested copies is often not possible without resequencing. The presence of any part of a TE could be indicative of the occurred insertion, and the partial or full-length structure could be further confirmed in additional experiments. Therefore, utilizing shorter repeats as representatives was more suitable at this point for evaluation of prevalent TEs in or near genes. For P. lambertiana, only one genome version is currently available, and using the full-length TE database, the most frequent families were found in the vicinity of 272 genes, which was comparable only with results from the P. taeda v.1.01 genome. However, utilizing the short TE-derived repeat database, approximately 500 matching genes were found for the most frequent TE family in both P. taeda v.1.01 and v.2.0, and 532 genes were identified in the P. lambertiana genome, which was comparable across all versions. Evaluated amounts of TEs are in accordance with whole-genome TE content data, where the larger genome size of the pine subgenus Strobus (P. lambertiana) correlates with a higher gene and repetitive element content [The quality of reference genomes and the repeat database used  play a major role when analyzing the presence of TEs in gene regions. Analysis of the first version of the  content .IFG family were found in gene introns of P. lambertiana, with more internal sequences present than LTRs . After in-depth analysis of the P. taeda v.2.0 genome, global errors in gene coordinates were revealed such that many protein-coding gene transcript coordinates were mapped to TE genomic coordinates without any sequence similarity. This increased the number of LTRs found in the 0–1 kb flanks of false genes. After creating our own genome annotation (see methods), similar tendencies were revealed in the TE distribution in all studied genomes, namely, an increase in hits to TE-derived repeats with increasing distance from genes.An overview and comparison of the number of extracted gene-flanking regions and total number of hits to TE-derived repeats in 5′ and 3′ gene-flanking regions between the two pine species are presented in P. taeda v.2.0 genome currently has the best scaffold quality regarding TE location. To analyze the distribution of TEs relative to distance from genes, the 5-kb flanking regions of filtered P. taeda v.2.0 genes were divided into 1-kb regions ; the distance and the position (5′ or 3′) relative to each gene were therefore retained. For each repeat, t-statistics were calculated comparing average unique hit numbers to the gene-flanking regions. In total, 135 different TE-derived repeats were identified in gene-flanking regions. Significantly increased occupancy in the 0–1 kb gene-flanking regions was determined for nine (p = 0.001) and seven TEs (p = 0.05) compared to the 2–5 kb flanking regions (p = 0.001) was also significantly increased in the 0–2 kb flanking regions. The number of genes where a particular TE was enriched in the 5′ and 3′ 0–1 kb flanks ranged from 27 to 48 genes. False genes were identified and evaluated in subsequent detailed sequence analyses. These false gene sequences consisted of parts of several different TEs that were not present in databases and therefore could not be filtered out in previous stages. Therefore, fewer protein-coding genes actually contained TEs in their vicinity than initially identified, and all gene groups were repeatedly screened against the NCBI database using a blastx search to confirm that they were protein coding. The  regions . Of thesPlater and describe in further detail below. In addition, we identified evidence of the distribution of a Copia RLX RYX6 LTR family in the 0–2 kb flanks of P. taeda genes. This element had 91% similarity to the PpRT6 partial RNaseH-like gene (EF102091) previously reported for P. pinaster [RYX6 due to masked regions prevented determination of a consensus sequence and verification of the results, indicating that sequence scaffolding problems persist in the case of longer repetitive elements. Another enriched repeat identified belonged to the 5767-bp RLX Copia-2602 with 160-bp LTRs. After filtering of false-positives, the hit count to the newly identified RLX increased with distance from the gene. The relatively short LTR of this RLX contains three TATA box motifs and three CAAT boxes on the negative strand; therefore, insertion of this element could promote antisense transcription. Only two P. lambertiana genes possessed a Copia-2602 insertion in 0–1 kb flanks; these were a probable histone H2B.3 gene and a Piwi-like domain containing argonaute family protein-coding gene. In the P. taeda 2.0 genome, two gene flanks contained hits to Copia-2602 (serine/threonine protein kinase PEPKR2 and carnitine transporter 4). Insertions of the Copia-2602 LTRs into 3′ flanks of a TMV resistance protein N-like gene and one unknown gene were found. Copia-2602 was also identified in the 1-2 kb flanks of flavonoid 3′,5′-hydroxylase 2, probable disease resistance protein At4g33300, chitinase 2-like, and cytochrome P450 (CYP736A12) genes.Analysis of enriched repeats in gene-flanking regions allowed identification of a MITE element in both species’ genomes, which we have named pinaster . The fulP. taeda and P. lambertiana genomes, all gene sets contained almost no highly frequent TE insertions in the 0–1 kb gene-flanking regions (with the exception of MITE). Therefore, nearby gene regions were characterized with moderate TE distribution and diverse TE insertions. Furthermore, the number of most TE-derived repeats gradually increased with distance from genes, suggesting a slight elimination of TEs from the gene vicinity. Most TE insertions accumulated in the gene introns  were considered for further networking analyses  analysis was performed for the extracted gene groups. However, approximately 50% of all pine genes were not annotated to any GO category and therefore these genes were automatically excluded from the networking analysis. For flanking regions, GO analysis was performed for genes with a statistically higher frequency of TE-derived repeats . Some meDyNet comparative trees, common GO categories shared between the two species are indicated in white (A higher diversity of TEs were identified within gene introns. Gene networks were built for genes having similar TE insertions in their introns without taking into account the exact location of TEs regarding intron number and position . GO catein white . Genes fin white . PtRXX_3321 was significantly enriched in the P. taeda v.1.01 gene flanks (0–2 kb) and was also frequently found in gene introns . The stem sequence was flanked with 40-bp direct repeats which were identified in the first intron of the probable pectinesterase/pectinesterase inhibitor gene. A consensus sequence of the MITE transposon Plater was built for each species genome. Compared with P. taeda, the P. lambertiana Plater MITE contained a 10-bp insertion in the 3′ stem sequence and was 265 bp in length with 28-bp TIRs. The P. taeda Plater element (sharing more than 80% sequence similarity) was found in 74 gene introns, of which 58 unique gene introns contained Plater MITE insertions with 99–100% nucleotide identity . Plater MITE insertions were identified in the vicinity of 191 P. taeda genes and of 65 P. lambertiana genes , pigment metabolic processes, plastid organization, maturation of ribosomal proteins, potassium ion transmembrane transport, and proline biosynthetic processes  that disrupted a site (TTTGACC) identical to several WRKY TFBS, but gained a site identical to co-dependent regulatory elements responsible for pollen-specific activation , which is involved in plant systemic acquired resistance and the salicylic acid-mediated signaling pathway. NPR1 contained several TE-derived repeats in the second intron and all repeats were tested with additional searches, but only Irbe-related repeat was distributed and found in an additional 200 gene introns from the non-filtered P. taeda v.2.0 genes and formed a network of stress-responsive genes , hAT family C-terminal dimerization region (pfam05699), and BED zinc finger domain (pfam02892), indicating that this element belongs to the DNA TE class. The FindMiRNA tool (http://www.softberry.com/) predicted seven probable pre-microRNAs in the Irbe TE with free energies ranging from −53.75 to −45.82 kcal/mol. A search of MiRBase revealed homology to the mature microRNA sly-miR9472-3p from a drought-tolerant tomato line [P. lambertiana gene introns contained more hits to the newly isolated Irbe element; 143 genes contained hits longer than 1 kb with sequence identity >82%. Irbe consensus sequences from both species were compared, but no species-specific structural polymorphisms were identified, suggesting ancient transposition events and probable distribution in genes of other pine species. Gene transcripts from P. taeda and P. lambertiana containing the Irbe TE were compared and one homologous gene was identified with more than 95% sequence identity and 100% query coverage. This gene was annotated as 26S proteasome non-ATPase regulatory subunit 4. The Irbe DNA TE was not identified in the 0–1 kb gene flanks of both species.A conserved domain search of the in pfam022, indicaato line . The P. IFG RLX is a remarkable TE family, as it is highly distributed in conifer genomes and is far more ancient than other RLXs, but sequence homology is still maintained [IFG sequences to gene introns were considered in gene network analyses, resulting in 99 genes from the filtered P. taeda v.2.0 dataset and 317 genes from high quality (HQ) genes in P. lambertiana , and 7987 bp in total from intron II was masked. In the P. taeda v.2.0 tyrosine protein kinase gene, IFG was inserted into the first intron, which is 106,013 bp long, and only 1639 bp was masked. Both protein kinase genes shared an identical exon-intron structure and 97% cDNA similarity. While IFG LTRs were 82% similar, the IFG body was interrupted by sequence masking. A receptor-like protein kinase gene contained exon duplication events in both species, with both introns containing one full-length IFG insertion on the minus strand. These insertions were 87% similar in the IFG body and 79–83% similar in their LTRs. The P. taeda v.2.0 gene contained an additional IFG sequence with one attached LTR on the positive strand, two IFGs on the positive strand, and four nested LTRs with surrounding masked regions. The P. lambertiana receptor-like protein kinase gene contained one full-length IFG and one single LTR on the positive strand.The intained ,68. Highbertiana . The folIFG insertions were also found in gene flanks (IFG LTR insertion was in close vicinity to 14 filtered P. taeda genes (0–1 kb) in the 5′ flanks. Genes encoding sugar transport protein 7 contained only the IFG body in the 5′ 0–1 kb flank; 12 genes’ 3′gene flanks contained IFG insertions  in the P. lambertiana or P. taeda v.2.0 genomes.e flanks , but RLXsertions , but onlto genes . No geneDaugava was found frequently distributed within gene introns in the P. lambertiana and P. taeda genomes. The RLX isolated from a BAC clone was 6884 bp long with two large 1386 bp LTRs, with conventional TG and CA sites. The RLX internal region was weakly similar at the protein level to the Retrovirus-related Pol polyprotein from tobacco tnt1 . The PPT (AAGAGGGAG) site was identical for elements from P. taeda and P. lambertiana. Some inserts in other locations probably have shorter LTRs, but due to multiple masked regions, it was not possible to extract more copies for the consensus structure. The P. lambertiana photosystem II stability/assembly factor HCF136-coding gene contained a Daugava RLX in the first intron in the positive orientation; the LTR was similar to an RLX from P. taeda with 34% query coverage and 82% sequence identity, and the RLX body was 89% similar with 62% coverage. However, some parts of this RLX were masked. The P. taeda Daugava LTR contained the following two AG-rich tracts: (AGNN)3(AG)3(NNAG)2 and (AGNN)2(AGN)4. The P. lambertiana Daugava LTR also contained polypurine-rich motifs 25 bp apart: AA(AGG)2A3(AGG)2GA3AGG and GAG(AGG)3AGA(AG)3. The P. taeda filtered gene set contained 80 strong hits to genes with Daugava RLX in the introns  were analyzed .High TE diversity within gene introns was observed in both pine species, and the presence of intronic TE insertions could form gene networks with similar expression and response patterns. If each interspersed repeat introduced additional gene regulation signals in gene introns, then genes containing a single interspersed repeat might show specificity regarding their function or expression pattern. To test this assumption, genes that contained an insertion of the analyzed . Five ofDaugava RLX network genes were further analyzed regarding combinations of all identified intronic TE insertions to determine if genes with specific TE combinations had similar functions. The presence of unique, short TE-derived repeats (95 loci) and unique >1 kb hits (22 loci) were used to form TE insertion patterns present in introns . While these analyses provide some initial clues about the formation of gene networks characterized by specific patterns of insertions of multiple TEs, further investigations are required using improved genomic sequences, gene annotations, and expression data for pine species.The evaluated  introns . The preDaugava RLX network was evaluated . This could be explained by the lower GC content of LTRs of some RLXs distributed in the gene introns. The mean GC content of the gene transcripts (involved in the Daugava RLX network) was 44% for P. lambertiana and P. taeda, which was higher than any average estimate for introns and published estimates for whole BAC clones and whole genome sequences of gymnosperms ..Daugava P. taeda v.2.0 genome, 75 genes were identified containing more than eight different unique TE-derived repeats in their introns, with the two-pore potassium channel coding gene containing the maximum of 65 different repeats. According to the PLAZA Gymnosperm database, P. taeda contains 14 members of the two-pore potassium channel gene family, which contains 659 co-occurring terms, indicating the involvement of these gene products in many plant-cell processes [P. taeda v.2.0 were chloroplastic/amyloplastic 1,4-alpha-glucan-branching enzyme coding gene, GTP binding protein Der, S-formylglutathione hydrolase, cytochrome P450, B3 domain-containing transcription repressor VAL2, serine/threonine protein kinase GRIK1, ribosome production factor 1, jasmonic acid-amido synthetase JAR1, COP9 signalosome complex subunit, chaperone protein ClpB3, and others  and ubiquitin-like fold (cl28922) conserved domains. A search of the Uniprot database  with this gene identified an HD domain-containing protein with 63% identity and 75% positives  that is involved in guanosine tetraphosphate metabolic processes (GO:0015969) with 107 co-occurring terms. A gene containing the second largest number of TEs in P. lambertiana was also an unknown protein containing the AMN1 domain (cl2816) and F-box domain (pfam12937), annotated only with the parent term “protein binding”. Other genes with a high amount of TEs in P. lambertiana were DNA repair helicase XPD, mitochondrial substrate carrier family protein, translation initiation factor elF-2B, syntaxin-81, ADB-ribosylation factor, acyl-CoA dehydrogenase IBR3, and others  contains 329 co-occurring terms, and 12 TEs were similar in gene homologs. SrmB conserved domains were present in ATP-dependent RNA helicases, but these gene transcripts were not identical. ATP-dependent helicase activity (GO:0003724) contained 929 co-occurring terms. A similar situation was found with proteins containing conserved domains of chromosome segregation protein SMC (cl37069) from nuclear pore complex protein NUP62, WD40 domain from protein WRAP73, RAE1 and actin-related proteins (found in a number of eukaryotic proteins that cover a wide variety of functions), alpha-tubulin suppressor (ATS1) domain-containing proteins (cl34932), and mitochondrial carrier protein domain .Interestingly, eight homologous genes were identified in both bertiana , from whde novo evaluation of repeated elements, analysis of the presence of specific structural features, and combined techniques [P. taeda v.2.0 genome currently has the highest quality conifer genome regarding scaffold length. Version 2.0 was improved by merging small contigs; while the first version of the P. taeda genome contained 16.5 million contigs, the second version contains only 2.9 million [PacBio read length used to reconstruct genome v.2.0 was 9665 bp with 12x coverage, which is shorter than many RLXs. The mean TE length in the PIER database is 6273 bp (median 5383 bp) but 3046 TEs are longer than 10 kb with the availability of better quality or longer-read genome assemblies, TE identification should be repeated de novo. Genome annotation of conifers is an ongoing process. Indeed, our study revealed errors in annotation files and datasets, where gene IDs of transcripts did not coincide with genomic sequences bearing identical gene IDs, and genes had noticeably varying intron lengths in different genome versions. Some additional errors involve automated annotation, nested repeat annotation, pools of identical reverse transcriptase domains defined as different genes, and intense masking of ambiguous regions, which are not discussed sufficiently in associated publications. Consequently, comparison of genomes assembled with different approaches and of varying quality should be performed with caution to avoid errors when interpreting the results. Additionally, widespread gene capture by TEs has often been described for large plant genomes [P. lambertiana high-quality gene set [Automated TE detection relies on several strategies, such as searches for sequences with homology to known elements, chniques . In the chniques ,96. Othechniques ,98. Typichniques ,99. Sevechniques ,66,100;  million . However genomes ,102,103 gene set . EvaluatVeju [BARE-2 [Cassandra elements in pear contain three LTRs [Diverse conifer RLXs sharing high partial sequence similarities may not be classified in one family by full-length alignments used in bioinformatic studies, resulting in inflated family counts . Due to Veju  and BARE [BARE-2 . TE struree LTRs . ConsideIrbe DNA TE could provide microRNA target sites or produce microRNA, or both. However, further analysis is required to determine if these sequences represent microRNA precursors or, alternatively target sites [P. taeda NPR1 gene, a key regulator of systemic acquired resistance [Irbe-related repeats, approximately 200 genes could be involved in this network in the P. taeda genome. In P. lambertiana, insertions of Irbe TE within 143 high-quality gene introns were found, many of which are important genes coding for transcription factors, chromatin modification enzymes, protein kinases, and receptors. The short Plater MITE family identified in proximal gene-flanking regions and introns could provide TATA boxes and several ARR1, DOF, W-box, GT, and MYB-binding sites, which are important signals in plant transcription activation and stress-response regulation [P. taeda Plater element. The transcription factor-type response regulator ARR1 directs transcriptional activation of the ARR6 gene, which responds to cytokinins without de novo protein synthesis [4A motif is one of the most common TFBS for plant promoters [Daugava identified in this study contains longer AG-rich tracts; this RLX family is distributed in pine gene introns and several flanks and might form an important responsive gene network. Genome-wide approaches in mammalian genomes demonstrate that TEs contribute to rewiring and selection of gene networks [P. taeda and P. lambertiana, extensive transcriptome data are not currently available; therefore, the role of TE insertions within introns in regulation of gene expression requires additional investigations. Using the Daugava RLX gene network identified in this study, the formation of specific patterns or intronic genotypes was tested. TE patterns could influence gene availability, responsiveness, stability, or higher order organization structures in the nucleus. Several revealed genes with unique Daugava RLX insertions are involved in the process of pre-mRNA maturation and splicing, while other genes with identical combinations of TE insertions are linked to protein metabolic processes and Golgi body homeostasis, which are related processes. Further investigation will enable more thorough analyses of relevance. The function of genes where multiple TEs were identified within introns  suggests involvement in the maintenance of cell homeostasis under stress conditions. These genes were found to have many co-occurring GO terms, indicating that gene products are involved in many cellular processes and these genes may be expressed or retain stability under a broad range of conditions. We suggest that genes with many different types of TEs could act as node genes that are functional or stable across a range of conditions and could be important in early defense responses and rapid metabolome switching. This is also supported by the discovery of several homologous genes with large introns in both pine species. Cases of independent transpositions of different RLX families into homologous genes in varieties of differing origin resulting in similar phenotypes have been reported [P. taeda and P. lambertiana was comparable (44%) and higher than the reported genome average of 38% [In the current study, we identified several TE families that unite many pine stress-responsive genes and contain potentially important gene regulatory signals. The et sites . This DNsistance , the PSMgulation ,112,113.ynthesis . Cytokinynthesis ,116,117,ynthesis . The , a distance over which linkage equilibrium extends in conifers [Plater was also frequently inserted in the introns of genes of the studied pine species. However, in this study, no genes with several Plater insertions in different non-coding regions  were identified. This distribution suggests the formation of differentially regulated gene sub-networks, depending on the location of MITE insertions. For example, it was determined that many genes containing Plater in flanking regions are involved in the regulation of developmental processes and cell division, but genes having Plater in their introns are associated with immune responses and cell-wall biosynthesis, among other activities. Therefore, we could hypothesize about the mechanisms of action of gene-TE associated networks. If Plater cis-acting elements in the more accessible introns help to activate a specific network of stress-responsive genes, then non-coding RNA products of splicing from these genes could be involved in feedback loop mechanisms for blocking transcription of genes with flanking Plater insertions, near genes involved in developmental processes. In this example, identical TEs initiate transcription of defense genes under unfavorable conditions, while at the same time have a down-regulatory effect on developmental gene transcription, switching to an energy-saving mode. Similar strategies could be highly advantageous for the rapid activation of defense responses and switching of metabolic functions. However, it is only one explanation among the other possibilities and this could be a subject of further studies. Additionally, in the current study, Plater MITE insertions were found in both analyzed pine species, belonging to separate subgenera, suggesting similar distributions also in other pine species. Therefore, Plater could be a useful molecular marker for genotyping of pine species, as shown for MITEs in other plant species [It remains unclear if TE sequences have insertional preferences, but it has been reported that a relaxed chromatin configuration promotes TE insertions and leads to an increased mutation probability in tissue- and stage-specific genes ,135. In conifers . MITEs aconifers ,138,139.conifers . Plater  species ,141,142.Strobus (P. lambertiana) and Pinus (P. taeda) and speciation [Irbe DNA TE in homologous genes of both species was found in PSMD4, a 26S proteasome non-ATPase regulatory subunit gene . Insertion of the ancient LTR RLX IFG [P. taeda and P. lambertiana gene pairs, surprisingly, all encoding different protein kinases. However, some of the IFG insertions were located in different introns of kinase genes and therefore could also represent independent transpositions. Analyzed IFG sequences contained masked regions and therefore the age of the insertions was not evaluated. Notably, insertions of other TEs were also frequently found in different receptor-like protein kinases and tyrosine protein kinases in our study. Protein kinase genes belong to one of the most proliferated gene superfamilies in plants, whose members are linked with a range of key metabolic and various plant-specific adaptation processes [TE distribution is linked with speciation ,144. In eciation ,68,145.  RLX IFG  was founrocesses ,148,149.P. sylvestris, which is an important species in northern Europe. In the two analyzed pine species, TE insertions were more often found in gene introns but less commonly in gene-flanking regions, similar to other plant species [In conclusion, the source of TE sequences expressed in response to stress conditions could be the transcription of introns of many stress-responsive genes, which could explain the observed highly correlated expression levels of RLX families within individuals . Transfe species ,152,153. species . Cultiva species . A recen species . Gymnosp species ,158,159. species , hence s species ,162,163.


Arsenophonus nasoniae is the son-killer microbe of the parasitic wasp Nasonia vitripennis and exists with the wasp throughout its life cycle. We completed its genome with the aid of recently developed long-read technology. This assembly contained over 50 chromosomal regions of phage origin and 17 extrachromosomal elements within the genome, encoding many important traits at the host-microbe interface. Thus, the biology of this symbiont is enabled by a complex array of mobile elements.The biology of many bacteria is critically dependent on genes carried on plasmid and phage mobile elements. These elements shuttle between microbial species, thus providing an important source of biological innovation across taxa. It has recently been recognized that mobile elements are also important in symbiotic bacteria, which form long-lasting interactions with their host. In this study, we report a bacterial symbiont genome that carries a highly complex array of these elements.  Arsenophonus nasoniae, the son-killer symbiont of the parasitic wasp Nasonia vitripennis. This microbe’s genome has the highest prophage complement reported to date, with over 50 genomic regions that represent either intact or degraded phage material. Moreover, the genome is predicted to include 17 extrachromosomal genetic elements, which carry many genes predicted to be important at the microbe-host interface, derived from a diverse assemblage of insect-associated gammaproteobacteria. In our system, this diversity was previously masked by repetitive mobile elements that broke the assembly derived from short reads. These findings suggest that other complex bacterial genomes will be revealed in the era of long-read sequencing.Mobile elements—plasmids and phages—are important components of microbial function and evolution via traits that they encode and their capacity to shuttle genetic material between species. We here report the unusually rich array of mobile elements within the genome of  Buchnera and the reproductive parasitic phenotype of male-killing Spiroplasma in Drosophila melanogaster are encoded on plasmids  allow closure of prophage-rich bacterial genomes whose repetitive nature prevented them from being completed with previous technologies. Our own attempts to complete the genome of id wasps , represeipositor . The micA. nasoniae (strain FIN). Hybrid assembly using PacBio  and Nanopore long reads  with subsequent Illumina polishing resulted in a closed circular genome .Our first attempt to sequence this genome used standard and paired-end libraries with 454 sequencing, resulting in a fragmented assembly with 665 contigs (143 scaffolds and 261 sequencing gaps) . We havehttps://doi.org/10.6084/m9.figshare.11845134). This density of phage-derived elements (6.9/Mb) is almost double that of Lactococcus lactis subsp. cremoris MG1363, the most prophage-rich genome recorded in previous systematic analyses . Flanking attachment sites were observed in all cases. However, these putative prophage elements varied in their ranges of core phage functions identified through homology. Two of 18 putative prophage elements lacked genes predicted to function in lysis, 4 lacked predicted packaging genes, and 3 lacked structural genes. From these data, we conclude either (i) that some of these elements are not autonomous (excision would require complementation by other phage-derived elements) or (ii) that the missing components are novel and within the unannotated portion of the putative prophage element.Phage-derived elements classed as potentially complete prophage by PHASTER were examined in more detail , combinations of long-read pairs that overlap and form a circle, and the presence of open reading frames (ORFs) predicted to function in plasmid maintenance (retention systems and/or replication initiation proteins) support their status as plasmids (https://doi.org/10.6084/m9.figshare.11845197 and https://doi.org/10.6084/m9.figshare.11845305). The predicted plasmid diversity exceeds the 11 recorded in Marinovum algicola, which the authors considered “unprecedented for proteobacteria” (Borrelia burgdorferi (A. nasoniae (https://doi.org/10.6084/m9.figshare.11857836). ORFs present are predicted to encode diverse functions, including the capacity to induce apoptosis in host cells, toxin elements and transporters, type III secreted effectors that manipulate eukaryotic cell physiology, and proteins that allow microbes to adhere to and invade eukaryotic cells.The assembly also predicted a complex complement of extrachromosomal DNA, with at least 17 extrachromosomal DNA elements  . Patternacteria” . Outsidehttps://doi.org/10.6084/m9.figshare.11845305) and were classified as plasmids on the basis of their Rep-encoding genes or addiction systems. Synteny mapping indicates that extrachromosomal elements share many repetitive elements with each other, and these regions are primarily of phage origin (https://doi.org/10.6084/m9.figshare.11857794).The extrachromosomal DNA is predicted to harbor 28 further phage-derived elements . Four of the extrachromosomal elements consisted entirely of circularized prophage DNA . However, they do not contain ORFs of obvious eukaryotic origin, as typified in the eukaryote association modules of Wolbachia phage . Of the genes in A. nasoniae phage-derived regions, the best matches were in the genus Arsenophonus, other closely related genera, and other insect-associated gammaproteobacteria. In contrast, with regard to the genes not present within the predicted phage-derived regions, the best matches were most commonly to genes in the Arsenophonus symbiont from Nilaparvata lugens.The ORFs present in phage-derived regions highlight the potentially rich capacity for phages to drive lateral transfer of important genes between bacterial lineages. Previous work indicated shared phage-derived elements between  defensa . We examhttps://doi.org/10.6084/m9.figshare.11857869). Predicted capsid proteins and predicted phage portal proteins from A. nasoniae prophage-derived elements were most closely related to the respective gene in prophage elements from Xenorhabdus, Proteus, Providencia, and Morganella, genera closely related to Arsenophonus. In contrast, predicted lysozyme proteins from A. nasoniae prophage-derived regions had a distinct pattern, with most closely related genes being from other Arsenophonus strains, and this cluster then allied to ones derived from prophages in the aphid symbiont Regiella insecticola.We complemented this approach with phylogenetic analysis of three prophage-encoded proteins: portal protein, lysozyme, and capsid protein  (https://doi.org/10.6084/m9.figshare.11857887). A notable difference is that in A. nasoniae, the cas2-cas3 fusion gene, a typical characteristic of the type I-F CRISPR-Cas system . These long-range scaffolding tools make assemblies relatively robust for long repetitive elements, such as prophages. Conversely, closed bacterial chromosomes completed using short-read sequencing methods tend not to contain large numbers of phage-derived elements. Short-read sequencing methods may have thus led to an underrepresentation of closed bacterial genomes with complex architectures. Because the causes of assembly failure , in which cosmid- or fosmid-based scaffolding processes are employed (e phages ) are bioSRR8797496, SRR8797497, and SRR8797498 for PacBio, Illumina, and Nanopore data, respectively. Data can be found under BioProject number PRJNA529362. Supplementary material may be downloaded from https://doi.org/10.6084/m9.figshare.c.4853367.v1.Raw sequences have been submitted to the NCBI SRA under accession numbers


Paris polyphylla var. chinensis is a species of flowering herb of the family Liliaceae and widely distributed in 12 provinces in China. It has been used in Chinese traditional medicine for centuries. The chloroplast (cp) genome of P. polyphylla var. chinensis, sequenced based on next-generation platform (NEOSAT), is 164,429 bp in size. The cp genome encodes 133 genes, including eight rRNA genes, 87 protein-coding genes (PCGs), and 38 tRNA genes. Phylogenetic relationship analysis based on complete cp genome sequences exhibited that P. polyphylla var. chinensis was most related to Daiswa forrestii. Paris polyphylla var. chinensis, an endemic to China, is a species of flowering herb of the family Liliaceae and widely distributed in 12 provinces in China . It was frozen in a dry ice-ethanol bath and deposited in a deep freezer at the herbarium of Hunan Academy of Forestry. An Illumina Hiseq 2500 system was employed to carry out paired-end sequencing. After trimming, around 7.2 Gb clean data were assembled against the online published cp genome of P. rugosa  was sampled from a P. polyphylla var. chinensis (GenBank no. MN528722) is a circular DNA molecule with 164,429 bp in size having 36.94% of total GC content. It contains two inverted repeat regions  with GC content 39.62%, a small single-copy region  with GC content 32.16% and a large single-copy region  with GC content 35.56%. There are a total of 133 genes annotated in the cp genome, comprising eight rRNA genes, 87 PCGs and 38 tRNA genes. Eleven tRNA genes, nine PCGs, and four rRNA genes were duplicated in the IRs. There are six intron-bearing PCGs. Two of them (ycf3 and clpP) bear two introns each, while four of them , bear one intron each.The complete cp genome of P. polyphylla var. chinensis was most related to Daiswa forrestii (For phylogenetic maximum likelihood (ML) analysis, multiple alignment was completed by MAFFT v7.2 with cp genomes downloaded from Genbank with default parameters (Katoh and Standley orrestii .


Physalis L. is a genus of herbaceous plants of the family Solanaceae, which has important medicinal, edible, and ornamental values. The morphological characteristics of Physalis species are similar, and it is difficult to rapidly and accurately distinguish them based only on morphological characteristics. At present, the species classification and phylogeny of Physalis are still controversial. In this study, the complete chloroplast (cp) genomes of four Physalis species  were sequenced, and the first comprehensive cp genome analysis of Physalis was performed, which included the previously published cp genome sequence of Physalis peruviana.Physalis cp genomes exhibited typical quadripartite and circular structures, and were relatively conserved in their structure and gene synteny. However, the Physalis cp genomes showed obvious variations at four regional boundaries, especially those of the inverted repeat and the large single-copy regions. The cp genomes’ lengths ranged from 156,578 bp to 157,007 bp. A total of 114 different genes, 80 protein-coding genes, 30 tRNA genes, and 4 rRNA genes, were observed in four new sequenced Physalis cp genomes. Differences in repeat sequences and simple sequence repeats were detected among the Physalis cp genomes. Phylogenetic relationships among 36 species of 11 genera of Solanaceae based on their cp genomes placed Physalis in the middle and upper part of the phylogenetic tree, with a monophyletic evolution having a 100% bootstrap value.The Physalis. The availability of these cp genomes will provide abundant information for further species identification, increase the taxonomic and phylogenetic resolution of Physalis, and assist in the investigation and utilization of Physalis plants.Our results enrich the data on the cp genomes of the genus  Physalis L., consisting of 75–120 species, is a well-known genera of the family Solanaceae because of its significant economic value, owing to the medicinal, edible and ornamental uses of its members , 1.6 mM dNTPs, 0.5 μM of each primer pair  library construction, following the manufacturer’s protocol (Illumina) [Total genomic DNA was extracted from ~ 0.1 g of preserved leaves  according to a modified CTAB method . The modg et al. ) (Additillumina) . DNA libwww.nipgr.res.in/ngsqctoolkit.html). Second, the high quality pair-ended reads were assembled using CLC Genomics Workbench version 8  and SOAPdenovo (http://soap.genomics.org.cn/soapdenovo.html) with a k-mer length of 63. Third, highly similar genome sequences were identified using BLAST (http://blast.ncbi.nlm.nih.gov/) with default parameters. Output scaffolds/contigs larger than 1000 bps were mapped to the reference cp genome of P. peruviana (NC_026570). Finally, we determined the order of aligned scaffolds/contigs according to the reference genome and resolved any gaps that were present by mapping the raw reads to the assembly.De novo and reference-guided strategies were used to assemble cp genomes. First, Illumina short reads were assembled into contigs using NGS QC Toolkit v2.3.3 (http://dogma.ccbb.utexas.edu/) tool [Physalis cp genomes. Start and stop codons of protein-coding genes and intron positions were manually corrected based on the reference genome (NC_026570). DOGMA and tRNA scan-SE version 1.21 [Physalis species were performed using the mVISTA program (http://genome.lbl.gov/vista/mvista/about.shtml). MEGA 6 software was used to analyze GC content, codon usage and phylogenetic analyses as described below [The Dual Organellar GenoMe Annotator (DOGMA) (u/) tool  was usedion 1.21  were useion 1.21 . The cp ome NC_0270. DOGMAhttp://pgrc.ipk-gatersleben.de/misa/) [Physalis cp genomes. The parameters were set as follows: 10 repeat units for mononucleotide SSRs, 5 repeat units for dinucleotide SSRs, 4 repeat units for trinucleotide SSRs, and 3 repeat units for tetra-, penta- and hexanucleotide repeats. REPuter was used to identify forward (direct), reverse, and palindromic repeats, within the cp genome, with a minimum repeat size of 30 bp and 90% sequence identity (Hamming distance of 3) [The Perl script MISA (e/misa/)  was usedce of 3) .Physalis species within the Solanaceae family, multiple alignments were performed using the complete cp genome sequences of 36 Solanaceae species representing 11 genera  and S. insignis (NC_028533), as outgroups. The MAFFT7.017 and ClustalX alignment software were used to compare and analyze the complete cp genome sequences of all the species, manual adjustments were made where necessary [To elucidate the phylogenetic positions of ecessary . Maximumecessary , using tAdditional file 1: Table S1. Information on the four Physalis species used in the study. Table S2. Quality control of the Illumina sequencing of chloroplast genome of Physalis species. Table S3. Relative synonymous codon usage (RSCU) in five Physalis chloroplast genomes. Table S4. Evolutionary divergence among Physalis species based on complete chloroplast genome sequences. Table S5. Universal primers for amplifying complete chloroplast genomes. Table S6. The 36 studied species belonging to 11 genera of Solanaceae, and the corresponding chloroplast whole genome GenBank accession number.Additional file 2: Figure S1. Gene map of the P. alkekengi var. franchetii chloroplast genome. Genes shown outside the outer circle are transcribed clockwise, and those inside are transcribed counterclockwise. Genes belonging to different functional groups are color coded. The darker gray in the inner circle indicates the GC content, and the lighter gray indicates the AT content. The inner circle also indicates that the chloroplast genome contains two copies of the inverted repeat (IRA and IRB), a large single-copy region (LSC) and a small single-copy region (SSC). The map was constructed using OrganellarGenomeDRAW. Figure S2. Gene map of the P. minima chloroplast genome. Genes shown outside the outer circle are transcribed clockwise, and those inside are transcribed counterclockwise. Genes belonging to different functional groups are color coded. The darker gray in the inner circle indicates the GC content, and the lighter gray indicates the AT content. The inner circle also indicates that the chloroplast genome contains two copies of the inverted repeat (IRA and IRB), a large single-copy region (LSC) and a small single-copy region (SSC). The map was constructed using OrganellarGenomeDRAW. Figure S3. Gene map of the P. pubescens chloroplast genome. Genes shown outside the outer circle are transcribed clockwise, and those inside are transcribed counterclockwise. Genes belonging to different functional groups are color coded. The darker gray in the inner circle indicates the GC content, and the lighter gray indicates the AT content. The inner circle also indicates that the chloroplast genome contains two copies of the inverted repeat (IRA and IRB), a large single-copy region (LSC) and a small single-copy region (SSC). The map was constructed using OrganellarGenomeDRAW. Figure S4. Amino acid frequencies in the chloroplast genomes of five Physalis species. Figure S5. Neighbor-joining (NJ) tree based on the complete chloroplast genome sequences of 36 species of Solanaceae. Numbers above branches indicate bootstrap support, and circled by the red dotted lines are the five Physalis species.


These 19 wild species belong to 9 genome types , representing wide genomic diversity in the genus. Using the genotype information, we analyzed the genome diversity of Oryza species. Other features of OryzaGenome facilitate the use of information on single nucleotide polymorphisms (SNPs) between O. sativa and its wild progenitor O. rufipogon in rice research, including breeding as well as basic science. For example, we provide Variant Call Format (VCF) files for genome-wide SNPs of 33 O. rufipogon accessions against the O. sativa reference genome, IRGSP1.0. In addition, we provide a new SNP Effect Table function, allowing users to identify SNPs or small insertion/deletion polymorphisms in the 33 O. rufipogon accessions and to search for the effect of these polymorphisms on protein function if they reside in the coding region . Furthermore, the SNP Viewer for 446 O. rufipogon accessions was updated by implementing new tracks for possible selective sweep regions and highly mutated regions that were potentially exposed to selective pressures during the process of domestication.Here we release OryzaGenome2.1, the first major update of OryzaGenome. The main feature in this version is the inclusion of newly sequenced genotypes and their meta-information, giving a total of 217 accessions of 19 wild Oryza accessions collected around the world and on the development of resources to speed up the identification of critical trait-related genes, especially from O. rufipogon. It aims to promote the use of genotype information from wild accessions in rice breeding and potential future crop improvements. Diverse genotypes will be a key resource for evolutionary studies in Oryza, including polyploid biology.OryzaGenome2.1 focuses on comparative genomic analysis of diverse wild The online version contains supplementary material available at 10.1186/s12284-021-00468-x. Oryza species into cultivated rice species, with the aim of exploiting agronomically advantageous traits from diverse wild relatives.Rice is an essential food for humankind, playing a critical role in food security. In addition, rice is a well-established model monocot in plant science, enabling advanced genomic breeding approaches for rapid development of improved cultivars. One such approach is genomic introgression from either closely or distantly related wild Oryza sativa L.) genotypes have been made available to the public , a major update. This release features newly sequenced genotypes and their meta-information, for a total of 217 wild Oryza accessions from 19 Oryza species  as a reference genome sequence  following the instruction of the software   . Variants were called by HaplotypeCaller of the Genome Analysis Toolkit   files are accessible from the Downloads page Fig. . The anahttp://viewer.shigen.info/oryzagenome/mapview/VariantView.do?action=Downloads) was downloaded and re-imputed by beagle 4.0 software (Browning and Browning https://faculty.washington.edu/browning/beagle/b4_0.html). This file contains phased, non-missing genotypes for all non-reference positions and is available from the Downloads page.Imputed genome data were obtained as follows. Whole-genome information in OryzaGenome v. 1 Variant List , DataTables (https://www.datatables.net/), Magnific Popup (http://dimsemenov.com/plugins/magnific-popup/), Google chart tools (https://developers.google.com/chart/), Prototype (http://prototypejs.org/), and script.aculo.us (https://script.aculo.us/) were used. Other conventional utilities for UNIX computing were installed on the server as necessary. All the OryzaGenome2.1 resources are stored on the server and are available at http://viewer.shigen.info/oryzagenome21detail/index.xhtml.Java and C++ were used as server-side application languages. JavaScript was used to implement client-side rich applications. The JavaScript libraries jQuery (Oryza species (average coverage 18×) , and their variants against the japonica reference genome  217 Oryza Genotypes; (2) 33 O. rufipogon Genotypes and Variants; (3) SNP Viewer for 446 Imputed O. rufipogon Genotypess, with new information tracks; (4) SNP Effect Table function to search for the effects of variants in 33 O. rufipogon accessions against IRGSP1.0; and (5) Misc Downloads.OryzaGenome release 2.1 provides the genotypes of 217 highly diverse accessions from 19 wild Oryza species  are provided. The 19 wild species belong to 9 genome types , representing much more genomic divergence than in the previous version. Each accession has been genotyped by Illumina paired-end short-read DNA sequencing (100- to 150-bp read length for one end) of over 8× coverage (average 18× coverage). Users can browse the sequencing platform, total bases, coverage, and file size in a pop-up window by mouse-over action at the accession numbers that are hyperlinked to NCBI and DDBJ on the Downloads page -derived genotypes of 217 accessions from 19 wild age Fig.  describiOryza. K-mer analysis was conducted to estimate the size of genome, the content of repetitive sequences and the heterozygosity in the genome of each accession  for 33 accessions of O. rufipogon. The raw sequence data of these accessions, as well as VCF files containing information on variants against Os-Nipponbare-Reference-IRGSP-1.0, are provided on the Downloads page  is updated in this release. Chromosome coordinates, genome sequences, and gene annotations are based on the latest japonica reference genome, Os-Nipponbare-Reference-IRGSP-1.0 . The genomic coordinates show regions or loci that appear to have been exposed to particular selective pressures through the process of domestication. In addition to RAP-DB  was obtained by using the SnpEff tool  . BED-formatted annotations and Fst values for the 446 O. rufipogon accessions published in Huang et al.  as well as information on these 446 accessions including accession number, habitat/country of origin, ecotype, sequence coverage, and deposited IDs of NGS raw data.In OryzaGenome1, an initial set of imputation-derived variant information on 446 Oryza accessions. Most of the wild Oryza bioresources used for acquisition of genotype data in OryzaGenome are preserved at the National Institute of Genetics in Japan. These materials were collected during the 1950s to 1980s from around the world, and their biomaterials (such as seeds) are available from Oryzabase (https://shigen.nig.ac.jp/rice/oryzabase/) upon request. Thus, OryzaGenome will help to promote the use of wild Oryza bioresources in basic research and speed up the identification of critical agricultural-trait-related genes from these bioresources. OryzaGenome aims to promote rice breeding science as well as potential future crop improvement. OryzaGenome2.1 is a significant update, providing a genus-wide comprehensive genomic repository for wild Oryza species.The goal of OryzaGenome is to facilitate comparative and evolutionary genomic analysis of diverse wild O. rufipogon. For genes with known function, it is now possible to quickly narrow down SNPs linked to regions that may affect their function. Lists of SNPs in candidate regions can be quickly obtained on the web interface and downloaded as an Excel file.Improved web-based tools (the SNP Effect Table and SNP Viewer) allow visual inspection of SNPs of interest and the evolutionary background of the surrounding regions in Oryza genotypes, including newly sequenced genotypes of seven tetraploid Oryza species: O. minuta (BBCC), O. punctata (BBCC), O. alta (CCDD), O. grandiglumis (CCDD), O. latifolia (CCDD), O. longiglumis (HHJJ), and O. ridleyi (HHJJ). Compared with other comprehensive databases for Oryza genomics  and its wild progenitor (O. rufipogon) and to survey the effect of polymorphisms. These functions will contribute to the analysis of domestication processes and the identification of agriculturally important genes from wild progenitors.OryzaGenome2.1 provides access to a wide variety of Oryza genome information, such as OryzaGenome2.1, OMAP (http://www.omap.org/index.html), Gramene (http://www.gramene.org), Ensembl Plants (https://plants.ensembl.org/index.html), and Rice SNP seek (https://snp-seek.irri.org), will greatly facilitate evolutionary studies of Oryza and contribute to agricultural science. We also foresee that installing and integrating genome browsers for resequencing data, such as TASUKE+, in future versions of OryzaGenome will improve the user experience (Kumagai et al. We expect that cross-references between databases storing Additional file 1: Table S1. Accession list of OryzaGenome2.1.Additional file 2: Table S2. Summary of K-mer analysis using genotypes of 217 wild Oryza accessions.Additional file 3: Table S3 Number of homozygous small nucleotide polymorphisms in 33 O. rufipogon.Additional file 4: Fig. S1. Analysis of genome diversity among genus Oryza. A. A plot of estimated genome size and estimated repeat length among genus Oryza. B. A plot of estimated genome size and estimated repeat content among genus Oryza.


Escherichia coli are one of the commonest bacteria causing bloodstream infection (BSI). The aim of the research was to identify the serotypes, MLST (Multi Locus Sequence Type), virulence genes, and antimicrobial resistance of E. coli isolated from bloodstream infection hospitalized patients in Cipto Mangunkusumo National Hospital Jakarta. We used whole genome sequencing methods rather than the conventional one, to characterized the serotypes, MLST (Multi Locus Sequence Type), virulence genes, and antimicrobial resistance (AMR) of E. coli. The composition of E. coli sequence types (ST) was as follows: ST131 (n = 5), ST38 (n = 3), ST405 (n = 3), ST69 (n = 3), and other STs . Enteroaggregative E. coli (EAEC) and Extra-intestinal pathogenic E. coli (ExPEC) groups were found dominant in our samples. Twenty isolates carried virulence genes for host cells adherence and 15 for genes that encourage E. coli immune evasion by enhancing survival in serum. ESBL-genes were present in 17 E. coli isolates. Other AMR genes also encoded resistance against aminoglycosides, quinolones, chloramphenicol, macrolides and trimethoprim. The phylogeny analysis showed that phylogroup D is dominated and followed by phylogroup B2. The E. coli isolated from 22 patients in Cipto Mangunkusumo National Hospital Jakarta showed high diversity in serotypes, sequence types, virulence genes, and AMR genes. Based on this finding, routinely screening all bacterial isolates in health care facilities can improve clinical significance. By using Whole Genome Sequencing for laboratory-based surveillance can be a valuable early warning system for emerging pathogens and resistance mechanisms. There is currently enlarge in the prevalence of infections worldwide due to multidrug-resistant (MDR). Due to their correlation with a high level of mortality and morbidity which are triggered by the insufficient of potent antibiotics, Gram-negative bacteria became a critical threat to global public health –3. The pEscherichia coli naturally inhabits the human gastrointestinal tract (GIT) and classified as Gram-negative commensals bacterium. Two main classification of pathogenic E. coli—diarrheagenic E. coli (DEC) and extra-intestinal pathogenic E. coli (ExPEC)—are perceived, varying in their associated clinical syndromes and virulence genes. Extraintestinal pathogenic E. coli (ExPEC) can cause a diversity of infections, including sepsis, neonatal meningitis, and urinary tract infections (UTI). The urinary tract is the most commonly infected by these bacteria, which then, is a common source for bloodstream infections , enteropathogenic E. coli [EPEC], enteroaggregative E. coli [EAEC], Shiga toxin (Stx)-producing E. coli [STEC], enteroinvasive E. coli [EIEC], and diffusely adherent E. coli [DAEC], are identified as the leading etiological agents of childhood and travellers’ diarrhoea. EAEC has long been considered as an intestinal pathogen and thereof unlikely to cause disease in normal patients outside of the intestinal tract blaTEM-1Bhe Korea  have consistance .E. coli isolates. Based on our results, we found the specific virulence genes in ExPEC and EAEC groups. Adherence protein (iha) was found dominating in ExPEC adherence virulence genes. We also found several toxins which belong to ExPEC group, such as cnf1, ireA, sat, vat, senB, pic, and tsh. Another prevalent group of virulence gene were liable for E. coli immune evasion by increasing serum survival (iss). The iss gene recognized for its role in ExPEC for enhanced survival of bacteria in the serum. The iss gene is located on plasmid ColV, a huge virulence plasmid typical of avian pathogenic E. coli strains, which reveal that a replacement of plasmids and, as the result, replacement of those virulence genes is feasible between human and avian pathogenic E. coli strains [This current research also characterized virulence genes in all  strains .aar; dispersin, required for proper dispersal of AAFs on the bacterial surface; and the aat transporter system, which mediates dispersin secretion [aggR gene was found in ST38 (H30) sample and both aggR and aar, was found in ST69 (O15:H18) sample. Strains harbouring the aggR regulon or its constituents have been termed typical EAEC. We also found two samples that harbouring EAEC toxin, i.e. astA, which both of them belong to ST38 (O86:H18). These virulence genes have been defined as suggestive of virulent serotypes and may be used as reliable markers for the detection of pathogenic E. coli [EAEC-specific virulence genes present in our 12 isolates included aggregative adherence fimbria (AAF) variant I; AggR, a global regulator of EAEC virulence; AggR-activated regulator, ecretion . The agg E. coli .E. coli pathotypes, such extraintestinal pathogenic E. coli (ExPEC). ExPEC genes found among the EAEC strains included i) increased serum survival-encoding gene, iss (63.6%); ii) Secreted autotransporter toxin, sat (18.2%); iii) Plasmid encoded enterotoxin, senB (9.1%); iv) Temperature-sensitive hemagglutinin, tsh (9.1%), and Salmochelin receptor, iroN (18.2%). The similar result was also found in another study by Boisen et. al. 2020 [Based on our results, the existence of a substantial number of genes characteristically correlated with another al. 2020 , that shet al [aatA and aar. This research underscore that E. coli should not be considered as non-pathogenic until the determinants of pathogenic and antimicrobial resistance have been verified to be missing. In addition, the WGS-based result suggested that there are nosocomial transmissions in the hospital, thus encouraging the formulation of pragmatic antimicrobial management, control initiatives and infection prevention.Potential clinical implications for the results received are that caution must be taken when explaining and utilizing microbiology outcomes. A study from Havt et al , showed E. coli isolates, which may limit generalization of the results. With a limited number of the isolates analyzed, it is essential to figure out that another limitation that this work may suffer is the insufficiency of deeper statistical analysis to associate the isolates resistance and virulence results with patient characteristics such as room, age, ward, and gender. Also, the presence of genes that encode various resistance and virulence genes only shows the genes that are in the isolates.We understand and acknowledge the limitations of this study. The assessment was carried out on a limited number of E. coli isolated from 22 patients in Cipto Mangunkusumo National Hospital Jakarta showed high diversity in serotypes, sequence types, virulence genes, and AMR genes. Based on this finding, routinely screening all bacterial isolates in health care facilities can improve clinical significance. By using Whole Genome Sequencing for laboratory-based surveillance can be a valuable early warning system for emerging pathogens and resistance mechanisms.The S1 Table(XLSX)Click here for additional data file.


Streptococcus anginosus group (SAG) is a subgroup of viridans streptococci and can be found ubiquitously in normal human flora. SAG is known to form invasive pyogenic infection when it becomes pathogenic. Yet, SAG is a very rare cause of endocarditis, and there is a dearth of case reports on this topic. We present a rare case of native bicuspid aortic valve endocarditis secondary to S. anginosus that caused aortic insufficiency and ascending aortic aneurysm. To our knowledge, this is the first well-documented case report of community-acquired S. anginosus endocarditis on a bicuspid aortic valve in an immunocompetent patient. The patient first presented with cough that was likely due to bronchus irritation from a 5.5 x 5.2 cm ascending aortic aneurysm. He underwent aortic valve replacement with bovine bioprosthesis and ascending aortic aneurysm repairment and was treated with a two-week regimen of IV ceftriaxone and gentamicin followed by another four weeks of IV ceftriaxone. He was eventually discharged to a rehabilitation facility. SAG is usually susceptible to beta-lactam antibiotics. The prognosis of SAG infection is usually good, but progression to bacteremia carries a poor outcome. It was first isolated by Guthof in 1956 from dental abscesses  = 0.047 μg/mL) and ceftriaxone (MIC = 0.25 μg/mL). Kirby-Bauer disc-diffusion antibiotic susceptibility testing revealed that it was also susceptible to clindamycin, erythromycin, vancomycin, and levofloxacin. Therefore, it was confirmed that he had aortic valve IE secondary to S. anginosus bacteremia. Yet, the dental consultation did not find an oral source of IE. The patient was started on IV ceftriaxone 2 grams every 24 hours and IV gentamicin 220 mg every 24 hours.Aerobic and anaerobic blood culture bottles obtained in the emergency room grew after 16 hours of incubation, with Gram-positive cocci in pairs noticed on microscopy. Intra-operative transesophageal echocardiogram showed that the patient had a bicuspid aortic valve. He underwent surgery for excision of infected bicuspid aortic valve and aortic valve replacement utilizing a 23-mm bovine pericardial Carpentier-Edwards bioprosthesis. The aortic valve leaflets consisted of two partially calcified, tan-yellow, valvular tissue measuring 3.0 x 3.0 x 1.2 cm in aggregate. High-power microscopic view demonstrated bacterial colonies on the aortic valve Figure . No obviS. anginosus, which was susceptible to penicillin and ceftriaxone. He underwent upper endoscopy and colonoscopy, which failed to show any gastrointestinal (GI) malignancy. His recovery from surgery was optimal. He completed the antibiotics course of IV ceftriaxone and gentamicin for two weeks after the surgery in the hospital. Even though the pathology report did not show bacterial involvement of the aortic aneurysm, the infectious diseases team made the decision to extend the patient’s ceftriaxone regimen to six weeks to avoid any risk of developing untreated mycotic aneurysm. He was subsequently discharged to a rehabilitation facility on another four weeks of IV ceftriaxone. He was followed closely in our Infectious Disease Clinic. Blood culture showed no growth of bacterium two months after discharge.The surgical culture from the aortic vegetations also grew S. anginosus, S. constellatus, and S. intermedius, are gram-positive, catalase-negative cocci and nonmotile facultative anaerobes that have typically small colonies (≤0.5 mm of diameter) [S. anginosus can further classify as S. anginosus subsp. anginosus, S. anginosus subsp. whileyi, S. anginosusgenomosubsp. AJ1, and a potentially new strain called S. anginosusgenomosubsp. vellorensis [S. constellatus has three subspecies, which are S. constellatus subsp. constellatus, S. constellatus subsp. pharyngis, and S. constellatussubsp. viborgensis [S. intermedius can form abscesses of the brain and liver, whereas both S. anginosus and S. constellatus can be isolated from a wider range of sites  and infections [S. anginosus [SAG, including iameter) . They caiameter) . Those tiameter) . Howeveriameter) . Nucleiclorensis . On the orgensis . When thfections . Anginosfections . Howeverfections . In a stnginosus .The HANDOC score is a sensitive tool to determine if an echocardiogram is indicated in patients with non-β-hemolytic streptococci bacteremia . There wBeta-lactams are the treatment of choice for SAG infections. Almost all isolates of SAG are susceptible to penicillin; however, intermediate susceptibility was seen in 15% of isolates . In a JaSAG has a propensity in forming abscesses and invasive pyogenic infections, but it is a rare cause of endocarditis. Our case report showed that SAG endocarditis can occur on a bicuspid aortic valve in an immunocompetent host. Most species of SAG are penicillin-sensitive, but the treatment outcomes can be compromised by bacteremia or endocarditis.


Candida species such as Candida auris have emerged recently as important invasive fungal diseases. We report a case of C. bovina bloodstream infection in a 94-year-old patient in France. The species led to identification issues because it was misidentified by phenotypic and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry methods.New  Streptococcus mitis/oralis and S. gordonii. Parenteral nutrition was stopped, the peripheral catheter was removed, and the patient received piperacillin/tazobactam. On day 56, a catheter culture was positive for S. mitis/oralis and for a yeast. We identified this yeast by using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry with the Vitek MS 3.2 system , which revealed Candida slooffiae with a maximal score of probability. A 94-year-old patient in France with no remarkable medical history was hospitalized because of a fall. During hospitalization, the patient had digestive disorders with postprandial regurgitations, leading to undernutrition. At hospital day 24, we performed an abdominal computed tomography scan, which indicated a dolicho-mega esophagus. Because of undernutrition, we introduced parenteral nutrition through peripheral venous catheter on day 36. We performed endoscopic pneumatic dilation to treat achalasia of the esophagus lower sphincter on day 51. One day later (day 52), the patient had a fever, triggering a bloodstream culture, which was positive after 24 hours (day 53) for C. slooffiae by using Vitek MS, and anaerobic culture was positive for S. anginosus. We initiated treatment with caspofungin . Three days later, the patient was nonpyretic, and the inflammatory syndrome regressed. Results of blood cultures performed on day 63 were negative. Caspofungin was continued for 14 days after the negative culture, and no valvular disease or cardiac failure was noted. Moreover, funduscopic examination revealed no abnormalities. We performed bloodstream cultures on day 57. Aerobic culture was positive on day 58 for a yeast again, which we identified as Candida 18S ribosomal DNA to confirm identification of this rare species. This sequencing identified the isolate as C. bovina (GenBank accession no. MN704805). Sequence analysis showed 100% similarity with ribosomal DNA sequences  of 2 C. bovina isolates from 2 different collection centers (GenBank nos. KY103626.1 and KP132306.1). After 90 days of monitoring, the patient showed improvement and had no relapse of infection.Although Vitek MS indicated a MALDI-TOF mass spectrometry high score of identification, we performed sequencing of Candida bloodstream infections has increased over recent decades in many countries, mainly because of use of immunosuppressive drugs and critical care therapies  telluris complex, which includes C. (Kazachstania) bovina, C. (Kazachstania) pintolopesii, C. (Kazachstania) sloofiae, K. heterogenica, and K.telluris  in 24 hours and forms small colonies. This yeast was inhibited by cycloheximide and was found to be white on a chromogenic CAN2 medium (bioMérieux). Attention must be paid to misidentification through use of MALDI-TOF mass spectrometry technology, which appears unable to distinguish yeasts inside the Kazachstania (Arxiozyma) telluris complex. Vitek MS 3.2 identified the organism as C. slooffiae; identification was K. telluris by using Bruker Biotyper MS 4.1.80 . Phenotypic identification using the Vitek2 YST card (bioMérieux) was unable to identify fungi. Sequencing of ITS1 and ITS4  must be performed and appears able to effectively differentiate species inside this complex.C. glabrata Candida is 2 mg/L. However, this MIC is higher than those of common Candida, such as C. albicans (MIC90<0.25 mg/L) (We evaluated MICs by using European Committee on Antimicrobial Susceptibility Testing broth microdilution . Among aCandida species about which laboratory staff are advised to be aware. These new species might lead to issues with identification and antifungal susceptibility. Clinicians should remain skeptical of infections with uncommon yeast species and consider confirmation through DNA sequencing.In conclusion, this case illustrates the emergence of an uncommon


Over the past few decades advances in modern medicine have resulted in a global increase in the prevalence of fungal infections. Particularly people undergoing organ transplants or cancer treatments with a compromised immune system are at an elevated risk for lethal fungal infections such as invasive candidiasis, aspergillosis, cryptococcosis, etc. The emergence of drug resistance in fungal pathogens poses a serious threat to mankind and it is critical to identify new targets for the development of antifungals. Calcineurin and TOR proteins are conserved across eukaryotes including pathogenic fungi. Two small molecules FK506 and rapamycin bind to FKBP12 immunophilin and the resulting complexes (FK506-FKBP12 and rapamycin-FKBP12) target calcineurin and TOR, respectively in both humans and fungi. However, due to their immunosuppressive nature these drugs in the current form cannot be used as an antifungal. To overcome this, it is important to identify key differences between human and fungal FKBP12, calcineurin, and TOR proteins which will facilitate the development of new small molecules with higher affinity toward fungal components. The current review highlights FK506/rapamycin-FKBP12 interactions with calcineurin/TOR kinase in human and fungi, and development of non-immunosuppressive analogs of FK506, rapamycin, and novel small molecules in inhibition of fungal calcineurin and TOR kinase. Aspergillus fumigatus, Cryptococcus neoformans, and Candida albicans, there has been a rising number of cases caused by other fungi such as those belonging to Mucorales  kinase. Rapamycin was further investigated as a possible antifungal agent with potent activity against C. albicans however, it was later discovered that rapamycin possessed potent immunosuppressive activity  was the binding partner for FK506. The AfFKBP12 shares 55% sequence similarity with HsFKBP12  comprises of five β sheets that wrap around the central α helix and also has three extended loops  surrounding the FK506 binding pocket  of 80s loop docks into the active site of adjacent subunit and vice-versa facilitating FKBP12-FKBP12 interaction in apostate  which is absent in HsFKBP12   and one regulatory subunit (CnbR)  . Mutantsnsition) . M. circt (CnbR) . We havet (CnbR) , 2015. St (CnbR) . The Cnat (CnbR) .Affkbp12 was replaced with Mcfkbp12 in A. fumigatus, FK506 was not able to inhibit calcineurin function. As mentioned above, in the presence of FK506 the F88 residue in the 80s loop of AfFKBP12 is critical in the FK506-AfFKBP12-calcineurin interaction. Interestingly, McFKBP12 possesses a tyrosine at the 88th residue instead of phenylalanine because of which McFKBP12-FK506 failed to inhibit calcineurin function in A. fumigatus  while retaining antifungal properties.C. neoformans at 37°C, however, its potency was 10–100 times lower than FK506 , 9-deoxo-prolyl-FK506 (9DP-FK506), 31-O-demethyl-FK506 (31OD-FK506), and 9-deoxo-31-O-demethyl-FK506 (9D31OD-FK506)  were gences spp. . These aan FK506 . All theconazole .C. albicans and A. fumigatus when compared to the previously described analog 9D31OD-FK506. This new analog also exhibited synergistic antifungal activity with fluconazole against C. neoformans  transporters in the genomes  also resulted in rapamycin and FK506 resistance (M. circinelloides FKBP12 was complemented in an S. cerevisiae mutant strain lacking FKBP12 resulting in the restoration of rapamycin sensitivity (M. circinelloides FKBP12 and Tor interacted in a rapamycin-dependent fashion (in vitro studies found that rapamycin exhibited potent growth inhibition against M. circinelloides followed by an in vivo study with Galleria mellonella improving survival by 50% post-rapamycin treatment (One major hurdle encountered by physicians treating mucormycosis patients is that Mucorales are resistant to most antifungal drugs . Thus, ihomologs . In thishomologs . Total dsistance . To furtsitivity . Further fashion . Finallyreatment . These fC. albicans, C. neoformans, and A. fumigatus (Aspergillus were tested, all of which were clinical isolates derived from patients with invasive aspergillosis (We have previously established that the TOR pathway in fungi can regulate various cell stress pathways . This maumigatus . Beauverumigatus . The preumigatus . This acumigatus . Furtherumigatus . Similarumigatus . In thisgillosis . The fungillosis . It was gillosis .C. albicans (C. albicans rbp1/rbp1 mutant strain lacking a homolog of the FKBP12 protein (C. albicans, C. neoformans, and S. cerevisiae, while analogs 18 and 19 were weakly toxic to C. neoformans and had no activity against the other fungi tested. In this study, analogs 2 and 23 were found to be the most toxic to C. albicans and C. neoformans while sharing a common mechanism of action with rapamycin involving an FKBP12-dependent inhibition of the TOR kinases (The synthesis of rapamycin analogs that present a lowered immunosuppressive activity in humans has been previously explored . One majalbicans . In thisalbicans  were stualbicans . Previoualbicans . This wa protein . It was  protein . With re kinases .Fungal calcineurin and TOR are key targets to develop new antifungals. In this review, we highlighted the structural differences between human and fungal FKBP12, calcineurin, and TOR components. FK506 targets both human and fungal calcineurin. However, several FK506 analogs, particularly which had modifications at C9, C21, C22, and C31 had increased affinity toward fungal FKBP12 and calcineurin resulting in lower immunosuppression while retaining antifungal activity. The sequences of FKBP12, calcineurin, and TOR components vary among fungal species. As a result, the antifungal efficacy of these analogs varies among the fungal pathogens tested. Nevertheless, these studies provide a proof of concept for developing non-immunosuppressive FK506 or rapamycin analogs with antifungal activity.SL and SV conceived the study. SV, AG, and SL wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.