Phytophthora sojae, the agent responsible for stem and root rot, is one of the most damaging plant pathogens of soybean. To establish a compatible-interaction, P. sojae secretes a wide array of effector proteins into the host cell. These effectors have been shown to act either in the apoplastic area or the cytoplasm of the cell to manipulate the host cellular processes in favor of the development of the pathogen. Deciphering effector-plant interactions is important for understanding the role of P. sojae effectors in disease progression and developing approaches to prevent infection. Here, we review the subcellular localization, the host proteins, and the processes associated with P. sojae effectors. We also discuss the emerging topic of effectors in the context of effector-resistance genes interaction, as well as model systems and recent developments in resources and techniques that may provide a better understanding of the soybean-P. sojae interaction. Phytophthora sojae Kaufm. & Gerd., a hemibiotrophic oomycete, is one of the most destructive pathogens of soybean . Bo. BoP. so soybean  47]. Us. UsP. soRps1-b and Rps1-k loci. Later, it was found that GmPUB1-1 interacts with a variety of other RxLR effectors, including PsAvr1d, whereas PsAvr1b and PsAvr1d interacted with a variety of other infection-induced GmPUB proteins, indicating that the pathogen modulates host immunity via a multiplex interaction of RxLR effectors with GmPUB proteins . Th. Th42]. proteins .Plants must have strict regulation over the defense-related gene expression to mount effective defenses . Reprogramming host gene expression appears to be a common virulence strategy used by microbial pathogens ,36,38,72Phytophthora spp. The PsAvr3c effector interacts with and stabilizes the soybean serine/lysine/arginine-rich proteins (GmSKRPs), which are components of the plant spliceosome and regulate host pre-mRNA splicing in order to modulate plant immunity . PI. PIPhytonfection . The PsAnfection . Interesnfection . This isessfully .Exploring the structure of effector proteins is critical to understand the molecular mechanisms behind the pathogenic and symbiotic interaction processes. For this purpose, genome-wide characterization of effector protein genes and proteins provides a great insight into their functional roles, conserved sequence motifs, and evolutionary patterns . MoreoveP. sojae that have been resolved experimentally using X-ray crystallography methods [Phytophthora species [So far, PsAvh240, PsPSR2, and PsAvr1d are the only three effector structures in  methods ,54,79. TMagnaporthe oryzae, Fusarium oxysporum, Puccinia graminis, and others have identified effector families possessing similar fold to known protein families having a role in plant-pathogen interactions or essential for pathogen virulence [In support of structural studies of effector proteins, recent breakthroughs in the development of tools such as AlphaFold and RoseTTaFold have revolutionized the analysis of effectors, as these can predict the structure of unannotated candidate effector proteins ,81. Seveirulence ,83,84. WP. sojae effectors. Therefore, in this review we have tried the AlphaFold program for modelling of the structure of P. sojae effector proteins in order to assess the efficacy of AlphaFold to the effector field  based DNA repair mechanism. Van West et al. (1999) successfully silenced inf1 in P. infestans through internuclear gene silencing. Silencing Avr4/6 using RNAi resulted in a gain of virulence of P. sojae isolates against Rps4 or Rps6 [Avr3a in P. sojae by transgenerational gene silencing [Homologous recombination based targeted gene knockout has been widely used for functional characterization of different genes in a number of plant pathogens . This ap or Rps6 ,87. Qutoilencing . HoweverP. sojae [P. infestans [CRISPR-Cas9 technology has been successfully applied in various pathogens and plant species . Using tP. sojae  and P. infestans . Once thnfestans ,93 and tP. sojae. Fang and Tyler (2016) overcame several technical hurdles to successfully implement CRISPR-Cas9 based genome editing in P. sojae [P. sojae [P. sojae were generated using the plasmids developed by Tyler’s lab [As a result of CRISPR-based gene editing methodology, functional characterization of effectors is gradually increasing in P. sojae . They opP. sojae ,54,94,95er’s lab .P. sojae effector PsAvh23 suppresses histone acetylation resulting in misregulation of defense-related genes in soybean. Using CRISPR, PsAvh23 gene was knocked out and replaced with NPTII, which resulted in upregulation of selected defense related genes [PsAvh23 was overexpressed in the soybean hairy root system, defense-related genes were downregulated favoring pathogen virulence [Avr1d promoted virulence by suppressing the self-ubiquitination of soybean E3 ligase GmPUB13 [P. sojae infection [Avr3a in proximity. Functional analyses using CRISPR-Cas9 knockout and constitutive expression demonstrated that Avr3a interacted with Rps8. This new finding shows that Avr3a interacts with Rps3a and Rps8 to trigger host immunity [P. sojae, the pathogen does harbor two chitin synthase (CHS) genes (PsCHS1 and PsCHS2) that are differentially regulated during the infection process. Knocking out PsCHS1 and PsCHS2 independently or simultaneously using CRISPR showed that only PsCHS1, is required for zoospore germination and hyphal branch formation during early infection [P. sojae would not have been possible without CRIPSR-Cas9 technology. Targeting a family of genes or multiple unrelated genes is possible by multiplexing sgRNA. In Fusarium fujikuroi, three different genes were targeted simultaneously using the same Pol III promoter [U. maydis, five members of eff1 effector genes were knocked out simultaneously using a multiplexed sgRNA expression cassette [P. sojae is still in its early stage as it requires development of multiple Pol III promoter containing a sgRNA cassette. To develop such multiplex cassettes, several factors need to be optimized. First and foremost, multiple RNA Pol III promoters in P. sojae need to be identified, and so far, the promoter RLP41 from P. sojae has been successfully used to express single sgRNA [ed genes . Interesirulence . When ovirulence . In addi GmPUB13 . Using t GmPUB13 . Knockounfection . The comimmunity . Even thnfection . It is ipromoter . Similarcassette . TargetiP. sojae interaction [Agrobacterium rhizogenes. Upon infection, A. rhizogenes transfers T-DNA from an Ri plasmid into plant cells, which results in the generation of excessive transformed roots. Hairy root technology has been utilized to produce phytochemicals and recombinant proteins, to identify novel genes, and to study host-pathogen interactions [P. sojae, the soybean cyst nematode, and Fusarium solani [Phytophthora infection [Avr1d promoted P. sojae infection and silencing of GmPUB13 by RNAi decreased infection. GmPUB13 was shown to act as a susceptibility gene with P. sojae [PsAvh181 of P. sojae in soybean hairy roots resulted in disease promotion and silencing of its interacting partner GmSNAPs, which also resulted in disease development [PsAvh23 or RNAi silencing of GmADA2/GmGCN5 in hairy roots enhanced P. sojae infection in soybean [The hairy root technique has been exploited to study the soybean-eraction ,100,101.ractions ,103,104.m solani ,106. Recnfection . Using sP. sojae . Similarelopment . Express soybean . Hairy rPhytophthora sojae-soybean research community should come together and agree on unified phenotypic method for effector and host protein functional characterization. Whether effector perception and corresponding downstream signalling pathway in root and leaf need not be conserved. In that case inoculating a root pathogen on leaf to validate the function of a candidate effector may not be an actual outcome.Knock out or knock down of a candidate gene followed by phenotyping assays is an essential prerequisite for functional characterization. Currently, there are different phenotyping approaches that are being exploited such as hypocotyl inoculation, root and detached leaf inoculation, and hydroponic assays . PhytophP. sojae infection, genetic resistance has been a key method [Rps genes are introduced into elite germplasm, the P. sojae rapidly evolves by adaptive changes to their Avr genes to overcome resistance genes thereby limiting the effectiveness of an Rps gene [Rps genes that can effectively limit the disease. Soybean cultivars resistant to distinct P. sojae pathotypes, i.e., those bearing specific effector (s) that could be recognised in a gene for gene interaction, have been thoroughly studied [Rps genes/alleles have been discovered in soybean and about 12 Avr genes have been identified and characterized in P. sojae  genes encode RxLR effectors and the only cloned Rps gene, Rps11, encodes a coiled-coil-nucleotide-binding leucine-rich repeat (CC-NB-LRR) [Avr genes code for CRNs. It would be interesting to see if the necrosis phenotype generated by some CRNs is linked to the plant defense response, and if so, whether NLR proteins activate the cell death response.To prevent y method . HoweverRps gene ,110. The studied ,111. So P. sojae  [87,112,-NB-LRR) . It is wRps genes have been integrated into breeding efforts to provide resistance against P. sojae infection [P. sojae under selection pressure to overcome Rps-mediated resistance. Gain of virulence is frequently associated with mutations that result in the Avr gene deletion, amino acids changes, frame shifts mutations and pseudogenization of specific effector genes are also used by P. sojae to evade host detection [P. sojae Avr genes [P. sojae also include gene copy number variation and epialleles, in addition to sequence diversification [Avr genes, which is favourable to a pathogen population since it allows effectors to be reused in the absence of Rps genes [In commercial soybean cultivars, nfection . This aletection ,120,122.vr genes ,124. Efffication ,125. EpiP. sojae has become one of the most devastating pathogens of this crop worldwide [P. sojae effectors and those of other pathogens. Since, the proteomic-based subcellular localization information is becoming more popular, the need of computationally predicted localization of unknown proteins is becoming less important. We believe that computational methods should become even more important for elucidating protein structures, the composition of protein complexes and interacting surfaces, dynamics, and interactions between proteins. Despite the report of more than 33 Rps genes/alleles in soybean [Rps genes has diversified the virulence profile of P. sojae through selection pressure [P. sojae and their corresponding Rps genes from soybean will contribute to a better inclusion of resisatnce genes in soybean breeding programs. The recent advancements and reduced costs in sequencing technologies should contribute to a better characterization of effector genes and identification of Rps genes. Efficient tools for the functional validation of these newly identified candidate genes are becoming more accessible. For instance, CRISPR-Cas systems have been developed to knock out candidate genes both in P. sojae and soybean and should be optimized in the future. One can envision that knock-out of multiple candidates of the same gene family through CRISPR-Cas could be exploited to accelerate functional studies of effector and Rps genes. Development of a P. sojae effector-R protein interacting network is largely understudied. Protein-protein interacting maps will help in identifying new and novel resistance proteins. Validating the interaction between newly identified P. sojae effectors and an R protein in soybean is challenging and time-consuming process and will benefit from faster transformation methods such as the hairy root technique that is amenable to root pathogens. In conclusion, a better understanding of P. sojae effectors and their targets in soybean is critical for the development of durable disease resistance.With the substantial increase in soybean acreage in many countries and particularly in Canada, orldwide . As revi soybean , only a  soybean . As a repressure ,127. Howpressure . Integra


Mycobacterium abscessus (M. abscessus) is a highly antimicrobial-resistant pathogen that causes refractory pulmonary disease. Recently, the possibility of M. abscessus cross-transmission among cystic fibrosis (CF) patients has been reported. CF is rare in Asia, but M. abscessus pulmonary disease is common. Therefore, we investigated the possibility of M. abscessus cross-transmission in a Japanese hospital setting. Of 104 M. abscessus isolates, 25 isolates from 24 patients were classified into four clusters based on their variable number of tandem repeat profiles and were subjected to whole-genome sequencing (WGS). The epidemiological linkages among our patients were investigated by integrating the WGS data of previously reported nosocomial outbreak-related M. abscessus clinical isolates in the United Kingdom and the United States. Eight transmissible clusters (TCs) were identified. The United Kingdom and United States isolates were assigned to four clusters  and one cluster (TC3), respectively. A total of 12 isolates from our hospital belonged to 4 clusters . Epidemiological linkage analysis inferred direct or indirect transmission between patients in our hospital in TC4 and TC5 but not in TC6 and TC7. In TC5, the single nucleotide polymorphism distance between isolates from Japanese and United Kingdom patients was less than 21; however, there was no contact. This study revealed that genetically closely related isolates exist, even in non-CF patients. However, the transmission route remains unclear, and further research is warranted to clarify whether cross-transmission is involved.IMPORTANCE Although the possibility of Mycobacterium abscessus (M. abscessus) cross-transmission in cystic fibrosis (CF) patients has often been reported, it is not clear whether similar events have occurred in Asian non-CF patients. Whole-genome sequencing analysis of M. abscessus isolates from Fukujuji Hospital in Japan indicated that genetically closely related M. abscessus isolates exist. In addition, according to epidemiological linkage analysis, some clusters were suspected of direct or indirect transmission between patients within our hospital. However, the transmission route of M. abscessus remains unclear, because interestingly, one cluster showed a single nucleotide polymorphism distance of less than 21 from the United Kingdom isolates, but no epidemiological linkage was identified. Mycobacterium abscessus (M. abscessus) is a highly antimicrobial-resistant NTM that causes refractory disease  patients has recently been reported  pulmonary disease has been increasing worldwide in recent decades , 2. Myco disease . Two syseported 68, indicaM. abscessus in a CF center in the United Kingdom by analyzing pairwise single nucleotide polymorphism (SNP) distances , and multilocus sequence typing, have been utilized , 9–11. R9–istances . They deistances , 13. HowM. abscessus clinical isolates from 517 CF patients in Europe, the United States, and Australia revealed that approximately 74% of CF patients worldwide were infected with two M. abscessus subsp. abscessus clones or one M.abscessus subsp. massiliense clone . The most common radiological finding was noncavitary nodular bronchiectatic , followed by cavitary nodular bronchiectatic , fibrocavitary , and unclassified type . Colony morphotypes were classified as follows: rough, 40% (10 isolates); smooth, 44% (11); mixed, 8% (2); and intermediate, 8% (2). Regarding susceptibility to clarithromycin, all M. abscessus subsp. abscessus isolates had inducible resistance, while all but one M. abscessus subsp. massiliense isolate was susceptible. All isolates were amikacin-susceptible or intermediate.A total of 25 isolates were obtained from the 24 patients enrolled in the present study. Of these, 23 patients met the diagnostic criteria of NTM pulmonary disease established by the American Thoracic Society/European Respiratory Society/European Society of Clinical Microbiology and Infectious Diseases/Infectious Diseases Society of America (ATS/ERS/ESCMID/IDSA), but 1 did not . The chaM. abscessus subsp. abscessus and M. abscessus subsp. massiliense with low genetic diversity were identified, all of which were consistent with each of the four VNTR clusters classified in a previous study (M. abscessus subsp. massiliense isolate (RGM-96_post) obtained serially from the patient from whom RGM-96_pre was isolated at a different time point was assigned to the same PC IV as RGM-96_pre.The phylogenetic tree of the 25 isolates generated using the WGS data is shown in us study . One M. M. abscessus subsp. massiliense isolates collected from the same individuals in three hospitals  was 25 in our analysis (M. abscessus subsp. massiliense clusters 1 and 2), and the remaining nonclustered isolates were assigned to TC5 and TC8 ., 11.M. aanalysis . The UniThe total numbers of overlapping days when the patients were in the outpatient department, hospital ward, and outpatient department-hospital ward on the same date were 6, 8, and 4 days for TC4 and 21, 3, and 13 days for TC5, respectively . When hoM. abscessus among non-CF patients. Using our analysis pipeline, WGS analysis of publicly available isolates from the United Kingdom and the United States showed that the maximum SNP distance within the same individuals was 25, consistent with a previous report  located in northwestern Tokyo, Japan, between September 2004 and December 2014. Basically, the first available positive culture from each patient was stored. Of the 104 M. abscessus isolates, 57 isolates were identified as subsp. abscessus, 45 as subsp. massiliense, and 2 as subsp. bolletii using multiplex PCR and rpoB gene sequencing as previously described  and de novo assembled using the Shovill pipeline. To assess the assembly quality of the 109 isolates, we calculated the genome fraction (%) of each isolate, which is the total number of bases aligned with the reference genome (M. abscessus subsp. massiliense JCM 15300), using QUAST software . To identify possible cross-transmission events, we used rPinecone software to define sublineages from clonal expansions , and admission periods and wards from the first visit to Fukujuji Hospital through the end of the study. We considered that opportunities for nosocomial cross-transmission could occur if a patient who once had a positive sputum acid-fast bacilli (AFB) culture shared the same space at the same time with another patient in the ward or at the outpatient clinic. We counted those days and investigated the possibility of contact. Radiological findings based on computed tomography were classified into the following disease patterns: fibrocavitary type (FC type), cavitary nodular bronchiectatic type (C-NB type), noncavitary nodular bronchiectatic type (NC-NB type), and unclassified type . RegardiThe study protocol was approved by the Fukujuji Hospital Institutional Review Board (protocol number 21024), and informed consent was waived because of the retrospective nature of the analysis.DRA012694.The raw read data of whole-genome sequencing were deposited in the DNA Data Bank of Japan (DDBJ) under BioProject accession number


Transposable elements (TEs) play key roles in crucial biological pathways. Therefore, several tools enabling the quantification of their expression were recently developed. However, many of the existing tools lack the capability to distinguish between the transcription of autonomously expressed TEs and TE fragments embedded in canonical coding/non-coding non-TE transcripts. Consequently, an apparent change in the expression of a given TE may simply reflect the variation in the expression of the transcripts containing TE-derived sequences. To overcome this issue, we have developed TEspeX, a pipeline for the quantification of TE expression at the consensus level. TEspeX uses Illumina RNA-seq short reads to quantify TE expression avoiding counting reads deriving from inactive TE fragments embedded in canonical transcripts.https://github.com/fansalon/TEspeX (Zenodo URL: https://doi.org/10.5281/zenodo.6800331).The tool is implemented in python3, distributed under the GNU General Public License (GPL) and available on Github at Bioinformatics online. TEspeX is a flexible tool, developed to measure the expression of any TE, regardless of its classification.Transposable elements (TEs) are repetitive and mobile DNA sequences that occupy large portions of the eukaryotic genomes . Therefohttps://broadinstitute.github.io/picard/). The pipeline rationale is to select for the TE expression quantification of only the RNA-seq reads deriving from the transcription of autonomously expressed TEs. Therefore, all the reads possibly transcribed from TE fragments embedded in canonical coding/non-coding non-TE transcripts are discarded. To this end, first, the reference transcriptome composed by TE consensus sequences and annotated coding/non-coding transcripts (introns not included) is generated and indexed, then RNA-seq reads are mapped to the reference transcriptome. Best scoring alignments are selected and all the reads mapping to any annotated non-TE transcript are discarded. Finally, the selected reads are counted (TEspeX is developed in python3 and takes advantage of STAR (v2.6.0c)  and Pica counted . Comprehin silico RNA-seq reads from the Repbase TE consensus sequences , supporting the evidence of TEspeX properly working on a broad set of species (D.melanogaster (dm6), M.musculus (mm39) and H.sapiens (hg38). TE expression was then quantified at the consensus sequence level using TEspeX, SalmonTE   (Having assessed the proper functioning of TEspeX on 2.2e−16) . Next, tarisons) . Moreovearisons) .H.sapiens dataset where TE transcriptional activation was observed in human neural progenitor cells (hNPC) upon DNMT1 knock-out (KO) by P-value = 1.56e−06) , the correlation coefficient was low (rho = 0.27), with several TEs identified as expressed by the custom pipeline used by P < 2.2e−16), which is the only tool, in addition to TEspeX, implemented to specifically distinguish between autonomous and passive TE transcription  . Althougcription . We thencription .An additional source of TE passive expression is represented by the presence of introns in the mRNA preps. It is now clear that the total RNA RNA-seq libraries and, to a lesser extent, the polyA+ ones might contain intronic sequences deriving from immature transcripts  PhD fellowship and Istituto Italiano di Tecnologia (IIT) postdoctoral fellowship to F.A. and the International School for Advanced Studies (SISSA) PhD fellowships to N.G. and M.E.Conflict of Interest: none declared.btac526_Supplementary_DataClick here for additional data file.


Eudicots are the most diverse group of flowering plants that compromise five well-defined lineages: core eudicots, Ranunculales, Proteales, Trochodendrales, and Buxales. However, the phylogenetic relationships between these five lineages and their chromosomal evolutions remain unclear, and a lack of high-quality genome analyses for Buxales has hindered many efforts to address this knowledge gap.Buxus austro-yunnanensis . Our phylogenomic analyses revealed that Buxales and Trochodendrales are genetically similar and classified as sisters. Additionally, both are sisters to the core eudicots, while Ranunculales was found to be the first lineage to diverge from these groups. Incomplete lineage sorting and hybridization were identified as the main contributors to phylogenetic discordance (34.33%) between the lineages. In fact, B. austro-yunnanensis underwent only one whole-genome duplication event, and collinear gene phylogeny analyses suggested that separate independent polyploidizations occurred in the five eudicot lineages. Using representative genomes from these five lineages, we reconstructed the ancestral eudicot karyotype (AEK) and generated a nearly gapless karyotype projection for each eudicot species. Within core eudicots, we recovered one common chromosome fusion event in asterids and malvids, respectively. Further, we also found that the previously reported fused AEKs in Aquilegia  and Vitis (core eudicots) have different fusion positions, which indicates that these two species have different karyotype evolution histories.Here, we present a high-quality chromosome-level genome of Based on our phylogenomic and karyotype evolution analyses, we revealed the likely relationships and evolutionary histories of early eudicots. Ultimately, our study expands genomic resources for early-diverging eudicots.The online version contains supplementary material available at 10.1186/s12915-022-01420-1. Eudicots are the most diverse and abundant group of flowering plants on earth and contain more than 280,000 species from approximately 44 orders. Together, they make up over 75% of all flowering plants –3 and ocNelumbo nucifera  [Tetracentron sinense  [Aquilegia  and Vitis  revealed a similar genomic fusion that appears to have occurred in the ancestral eudicot karyotype [Polyploidization events, such as tetraploidization  and hexaploidization , occur frequently in plants and are a major source of evolutionary change that enables rapid adaptation to different environments –15. Prevoteales) , 20 or Tndrales) , 22. Intaryotype . From tharyotype . A recenaryotype . Still, aryotype .In addition to the uncertainty concerning this WGT, the phylogenetic relationships between the well-defined core eudicot lineage , 27, 28 Buxus austro-yunnanensis (2n = 28) [B. austro-yunnanensis and representative species of the other four lineages. Using high-quality early-diverging and core eudicot genomes, we reconstructed an accurate ancestral eudicot karyotype (AEK) and generated nearly gapless karyotype projections for the chosen eudicot species. Specifically, we observed one chromosome fusion within asterids and another in malvids and also confirmed that Aquilegia  and Vitis (eudicots) have completely different karyotype evolution histories with no shared chromosome fusions. Thus, our results provide considerably new insights on the genomic and karyotype evolution of early-diverging eudicots.Here, we present a high-quality chromosome-level reference genome for 2n = 28)  that wasB. austro-yunnanensis genome, which revealed that over 98.72% of the Illumina short reads could be mapped to the assembly. The GC content followed a Poisson distribution  and other early-diverging eudicot species , which were used to construct the contig assembly with NextDenovo  of the Aristolochia fimbriata and Liriodendron chinense as outgroups) were selected for inclusion in our phylogenetic analyses  . Only one obvious polyploidization event was detected in B. austro-yunnanensis , even though no polyploidization was detected in this species in an earlier study [Carthamus tinctorius  was a WGT rather than a WGD event [Olea europaea  that occurred between a previously reported WGD [The intragenomic syntenic analysis of nts Fig. . Intergeer study . Second,GD event . Finallyrted WGD  and the Vitis vinifera  and Coffea canephora (asterids) were selected to represent core eudicots because neither of them showed evidence of any polyploidization event other than the γ event. We found that most gene trees supported independent WGD events that occur within each early-diverging eudicot order and with the core eudicots. Specifically, Vitis vinifera and Coffea canephora shared the γ event and two species of Trochodendrales  shared two rounds of WGDs , we observed many chromosome-scaled conserved synteny relationships . The dot plots between AEK and PAEK showed that most of the chromosome variants were intra-chromosome variants, which supports high accuracy of conserved gene clustering. Only one inter-chromosome variant was detected where a piece of the PAEK 6 (corresponding to AEK 7) region was translocated into the head of AEK 6 (corresponding to PAEK 7) , but the fusion position differs from that in the asterids. Specially, the collinearity near the dash line was discontinuous, which indicates that the occurrence of the fusion in these two clades is an example of parallel evolution  revealed a complete absence of this fusion. A fusion of B1 and C1 was also detected in Betula, but the fusion position was different, which indicates that this fusion event was independent compared to the events in rosid clades I and III. It should also be noted that species in rosid clades I and III were previously considered to cluster together in the malvids clade [After the γ event, there were three copies of each of the seven AEKs in the ancestor of the core eudicots. These ancestral chromosomes are designated A1-A7, B1-B7, and C1-C7, where the letters A, B, and C represent the duplicates. We observed one common chromosome fusion that occurred in all asterids where A3 fused with A7 via end-end joining (EEJ) Fig. . This wads clade , 28, butds clade , 67.Aquilegia and Vitis based on the occurrence of a similar fusion event in both orders [Vitis and Cercidiphyllum). Our karyotype analysis revealed a similar fusion of AEK 3 (green) and 5 (purple) in Aquilegia Chr 5 and Vitis Chr 7 that is absent in other early-diverging eudicots  and core eudicot Cercidiphyllum  event between AEK 4 and 7  that was formed by the two RTA events involving AEK 3, 5, and 7, but without the subsequent EEJ  and different fusion positions. This data strongly supports the occurrence of independent WGDs in early-diverging eudicots and core eudicots. Overall, our results show that only considering the phenotype of ancestral chromosome fusions without investigating the details of the associated chromosome rearrangements may lead to unreliable results and conclusions.Although our collinear gene tree analyses suggested that independent WGD events occurred in the early-diverging eudicot lineages and core eudicots, an earlier study suggested a shared WGD between h orders . To direum Figs. . Howevernts Fig. . This cod 7 Fig. . A subser 5 Fig. . The conEEJ Fig. . Thus, ABuxus austro-yunnanensis genome displayed high quality of continuity, completeness, and accuracy when compared to many other previous studies on early-diverging eudicot species [Our assembled  species , 54, 68.Ks distribution of synteny genes, which is one of the most common ways. The peak of a Ks distribution within one species usually indicates one polyploidization event, but this characteristic is not able to distinguish how many times the genome multiplies in an event. Further, it is also difficult to determine whether a duplication event is shared or not between two species as evolutionary rates vary and cause the Ks to differ greatly [Ks analysis in regards with determining ploidy. However, it often depends on the well assembled genomes, especially at the chromosome-level, to get accurate results [S. chinensis, C. tinctorius, and O. europaea  . To circquilegia . In our quilegia  or in plAquilegia and Vitis . Since further karyotype construction of more species can be carried out, it will be more advantageous and more reliable to analyze phylogeny considering evidence from both sequences and karyotype features.Buxus austro-yunnanensis were obtained that enrich the genomic resources available for the early-diverging eudicot order Buxales. By analyzing this genome together with representative genomes of early-diverging eudicots and other core eudicot lineages, we inferred their phylogenetic relationships and showed the relative contribution of gene tree estimation errors, ILS, and hybridization to observed phylogenetic discordance. Further, we reconstructed a better ancient eudicot karyotype (AEK) using high-quality genomic information on early-diverging eudicots and other core eudicots, and clarified the karyotype evolution of various eudicot orders, which revealed one common fusion in asterids and another in malvids. We also reconstructed and confirmed the different karyotype evolution histories of Aquilegia and Vitis based on the AEK. Together, this information suggests that high resolution reconstruction of ancestral chromosomes provides significant insights into karyotype evolutionary processes that are not accessible by other means. When judging common chromosome variants, it is essential to fully consider the karyotype evolution process to avoid misinterpreting outcomes that result from parallel evolution. These findings provide new insights into the evolution of eudicots and will inspire more karyotype analysis and genomic research into these species, as well as others in the future.In this work, high-quality chromosome-level genome sequences of B. austro-yunnanensis individual was planted in the Xishuangbanna Tropical Botanical Garden at the Chinese Academy of Sciences, Mengla 666303, Yunnan, China; stored samples were assigned the accession number 0020010744. Fresh leaves were collected, immediately frozen in liquid nitrogen, and sent to Grandomics  for genome sequencing. Total genomic DNA was extracted using the CTAB method and purified with the QIAGEN® Genomic kit . For the ONT long reads, a library of large DNA fragments (> 20 kb) was constructed with the Ligation Sequencing Kit 1D (SQK-LSK108) and then sequenced on the PromethION platform. Raw long reads were further filtered by removing adaptors and low-quality nucleotides . For Illumina short reads, a paired sequencing library with an insertion size of 350 bp was constructed and sequenced using the Illumina HiSeq 4000 platform. The raw Illumina reads were then filtered with fastp v0.20.1 [The sampled  v0.20.1  using thhttps://github.com/Nextomics/NextDenovo) was then used to correct the ONT long reads and construct the preliminary contig assembly with the following parameters “read_cutoff=7k, seed_cutoff=37072, blocksize=8g.” The assembly was then subjected to two rounds of polishing with Nextpolish v1.2.4 [The genome size was first estimated using GCE v1.0.0  with K-mh v1.2.4  using thh v1.2.4   and then used RepeatMasker to search the repeat sequences against the combined library. The insertion times of complete LTRs were inferred using LTR_retriever. To predict protein coding genes, we first used GeMoMa v1.6.1 [Nymphaea colorata, Nymphaea thermarum, Aquilegia coerulea, Tetracentron sinense, Trochodendron aralioides, Vitis vinifera, and Arabidopsis thaliana. Three programs  assembled with trinity [Repetitive elements were first annotated using a combination of evidence-based and RF v4.09  was usedRF v4.09  and RepeRF v4.09  and Repea v1.6.1 , 90 to pS v3.3.3 , GENSCANS v3.3.3 , GlimmerS v3.3.3 ) were us trinity  was also SRR93044 assemble trinity  was used trinity . Metabol trinity , using aAristolochia fimbriata and Liriodendron chinense) as outgroups together with 26 species representing 25 eudicot orders , Asterales (Carthamus tinctorius), Brassicales (Bretschneidera sinensis), Buxales (B. austro-yunnanensis), Caryophyllales (Simmondsia chinensis), Celastrales (Tripterygium wilfordii), Cornales (Davidia involucrata), Cucurbitales (Cucumis hystrix), Ericales (Rhododendron simsii), Fabales (Phaseolus vulgaris), Fagales (Betula pendula), Gentianales (Coffea canephora), Lamiales (Olea europaea), Malpighiales (Populus trichocarpa), Malvales (Theobroma cacao), Myrtales , Oxalidales (Averrhoa carambola), Proteales (Nelumbo nucifera), Ranunculales (Aquilegia coerulea), Rosales , Sapindales (Acer yangbiense), Saxifragales (Cercidiphyllum japonicum), Solanales (Ipomoea trifida), Trochodendrales , and Vitales (Vitis vinifera). A total of 1208 single copy gene families were identified among the 28 species by SonicParanoid v1.0 [http://www.timetree.org), and 94 Mya as the lower boundary for the Vitis-Eurosid split [Phylogenetic analysis was performed using two magnoliids  to assess the contributions of three factors to gene tree variations: gene tree estimation error, incomplete lineage sorting (ILS), and hybridization (gene flow). In brief, we first calculated bootstrap values for the species tree with all gene trees and used these values to represent the gene tree variation; higher bootstrap values represent lower gene tree variation. For the gene tree estimation error, because there were two species with different topologies in the concatenated and coalescent trees, we first reassessed the branch length with the fixed coalescent topology using IQ-TREE. Next, we used Seq-Gen v1.3.4 [Aristolochia, Aquilegia, Nelumbo, Buxus, Trochodendron, Simmondsia, Davidia, Cercidiphyllum and Vitis) were selected with Aristolochia as the outgroup of the total analysis and the default parameters of QuIBL were used. To distinguish an ILS-only model and a hybridization model, we used the Bayesian Information Criterion (BIC) test with a strict cutoff of ΔBIC > 10.Many factors could give rise to incongruent tree topologies among nuclear genes or between nuclear and plastome genes. Here, we used a recently published method   between collinear genes were estimated using the Nei–Gojobori approach as implemented in the PAML package v.4.9h [Ks values were used to represent each syntenic block, and Ks peak fitting was performed with WGDI using “-pf” option. Dot plots of collinear genes and synteny blocks were used to determine syntenic ratios between different species to confirm their polyploidy levels. We also used the collinear genes to perform phylogenetic analyses to determine whether WGD had occurred independently within early-diverging eudicots and core eudicots. Collinear genes between all species pairs from the seven selected species  were extracted using WGDI with the “-at” option, and IQ-TREE was used to construct gene trees as described above. For each gene tree, we randomly rooted a collinear gene from one species using nw_reroot from the Newick utilities v1.6 [All of the previously mentioned 26 eudicot species were included in the WGD analyses. Synteny blocks and collinear genes within each species and between species were identified using WGDI  with thee v.4.9h . The medies v1.6  and thenVitis, Trochodendron, Tetracentron, Buxus, Nelumbo and Aquilegia) were selected to represent early-diverging and core eudicots to construct the AEK. WGDI was used to detect collinear genes/blocks between all chromosome pairs among all six species. We firstly generated the dot plots between the all chromosomes and detected many chromosomes showed a nearly intact synteny relationships with others. Then, we used the following method to group these chromosomes and complete the ancestral chromosomes reconstructing. For a pair of chromosomes i and j, we defined the collinearity ratio as Z-transformation: μi and σi represent the average values and standard deviation of the collinearity ratios between chromosome i and the other chromosomes. The normalized collinearity ratios were used to cluster the chromosomes, which were then displayed using Pheatmap in R. Seven clusters were generated, and we picked the most complete chromosome from each cluster  as a reference. Each reference chromosome was then augmented as follows: if there were no more than five genes in another chromosome between two adjacent collinear genes (located in a single collinear block), we inserted these genes into the reference chromosome between the two collinear genes. After adding all the specific genes from other chromosomes in this way, we obtained the final AEK.Six species , gene tree estimation error (Est. error), and hybridization in generating gene tree variation. Fig. S14. Collinear gene dot plots between Buxus austro-yunnanensis and Aristolochia, Aquilegia, Nelumbo, Tetracentron, Cercidiphyllum, Vitis. Fig. S15. Collinear gene dot plots between Cercidiphyllum, Vitis and Simmondsia, Carthamus, Olea. Fig. S16. Merged dotplot of Aquilegia, Buxus, Nelumbo, Trochodendron, Tetracentron and Vitis. Fig. S17. Heatmap of cluster of collinearity relationships in Aquilegia (aco), Buxus austro-yunnanensis (byu), Nelumbo (nnu), Trochodendron (tar), Tetracentron (tsi) and Vitis (vvi). Fig. S18. Demonstration of pieces of AEK 1 in Aquilegia (aco), Buxus austro-yunnanensis (byu), Nelumbo (nnu), Trochodendron (tar), Tetracentron (tsi) and Vitis (vvi). Fig. S19. Demonstration of pieces of AEK 2 in Aquilegia (aco), Buxus austro-yunnanensis (byu), Nelumbo (nnu), Trochodendron (tar), Tetracentron (tsi) and Vitis (vvi). Fig. S20. Demonstration of pieces of AEK 3 in Aquilegia (aco), Buxus austro-yunnanensis (byu), Nelumbo (nnu), Trochodendron (tar), Tetracentron (tsi) and Vitis (vvi). Fig. S21. Demonstration of pieces of AEK 4 in Aquilegia (aco), Buxus austro-yunnanensis (byu), Nelumbo (nnu), Trochodendron (tar), Tetracentron (tsi) and Vitis (vvi). Fig. S22. Demonstration of pieces of AEK 5 in Aquilegia (aco), Buxus austro-yunnanensis (byu), Nelumbo (nnu), Trochodendron (tar), Tetracentron (tsi) and Vitis (vvi). Fig. S23. Demonstration of pieces of AEK 6 in Aquilegia (aco), Buxus austro-yunnanensis (byu), Nelumbo (nnu), Trochodendron (tar), Tetracentron (tsi) and Vitis (vvi). Fig. S24. Demonstration of pieces of AEK 7 in Aquilegia (aco), Buxus austro-yunnanensis (byu), Nelumbo (nnu), Trochodendron (tar), Tetracentron (tsi) and Vitis (vvi). Fig. S25. Comparison between the previous AEK (PAEK) and our constructed AEK. Fig. S26. Karyotype projection of Aquilegia, Nelumbo, Buxus austro-yunnanensis, Tetracentron, Trochodendron and Coffea genome based on PAEK. Fig. S27. Karyotype projection of Vitis and Cercidiphyllum based on PAEK and AEK. Fig. S28. Karyotype projection of Aquilegia, Nelumbo, Buxus austro-yunnanensis, Tetracentron, Trochodendron and Coffea genome based on AEK. Fig. S29. Collinear gene dot plots and karyotype projection between Cercidiphyllum and Bretschneidera, Theobroma, Acer, Averrhoa, Populus, Tripterygium. Fig. S30. Collinear gene dot plots and karyotype projection between Cercidiphyllum and Cucumis, Phaseolus, Malus, Betula, Eucalyptus, Vitis. Fig. S31. Collinear gene dot plots and karyotype projection between Cercidiphyllum and Coffea, Ipomoea, Olea, Apium, Carthamus, Davidia, Rhododendron, Simmondsia. Fig. S32. Collinear gene dot plots and karyotype projection between Coptis and Aquilegia, Cercidiphyllum and Muscadinia, Vitis and Muscadinia. Fig. S33. Collinear gene dot plots and karyotype projection between Tetracentron and Trochodendron. Fig. S34. Synteny comparison among asterids species with Davidia as reference. Fig. S35. Synteny comparison among rosids species with Tripterygium as reference. Table S1. The detail sequencing information of Buxus austro-yunnanensis. Table S2. Summary of genome assembly. Table S3. Contig N50 values of the early-diverging eudicots. Table S4. Summary of chromosome level assembly. Table S5. Statistic of repetitive elements in the assembled genome. Table S6. Comparison of gene space of Buxus austro-yunnanensis with other genomes. Table S7. Assessment of the predicted genes by BUSCO (database: eudicotyledons_odb10). Table S8. Functional annotation of the predicted genes. Table S9. Summary of the 28 species genome that used in this study. Table S10. Average total introgression proportion per species pair in QuIBL analysis. Table S11. The QuIBL analysis result.


Nocardia iowensis DSM 45197T. N. iowensis DSM 45197T is a spore-forming, mesophilic, Gram-positive bacterium that was isolated from garden soil in Osceola, Iowa, USA. This organism has been exploited for its production of glycocinnamolyspermidine antibiotics and biotransformation of xenobiotic substances. Other significant features of N. iowensis DSM 45197T include the first fully characterized carboxylic acid reductase (CAR) and the first bacterial nitric oxide synthase system. The genome sequence of N. iowensis DSM 45197T can facilitate further understanding of its function, as well as the pathogenesis of Nocardia spp. N. iowensis DSM 45197T has a genome size of 8.95 Mbp; about 46% of the coding sequences have no known homologues and were labeled hypothetical proteins. This finding implies further potential for biomedical and biotechnological research applications.We report the complete genome sequence and predicted functional profile of  Nocardia iowensis DSM 45197T, a versatile bacterium that was initially of interest as an antibiotic producer. Subsequent discoveries of versatile biocatalytic abilities have sustained interest because it biotransforms many natural and synthetic substrates into value-added products, including flavonoids, vanillic acid, and 4-vinylphenol  according to the manufacturer's instructions. DNA integrity was checked on a FemtoPulse system . A SMRTbell template library was prepared according to the instructions from Pacific Biosciences (PacBio)  . Conditions for annealing of sequencing primers and binding of polymerase to purified SMRTbell templates were assessed with the calculator in single-molecule real-time (SMRT) Link. Libraries were sequenced on the Sequel IIe platform (PacBio), taking one 15-h movie per SMRT cell. In total, one SMRT cell resulted in 109,275 reads, with a mean (filtered) length of 7,321 bp and an N50 value of 83,86 bp. Read quality control, error correction (preassembly), and adapter trimming were performed using the PacBio microbial assembly protocol.https://www.bioinformatics.babraham.ac.uk/projects/fastqc). Long-read genome assembly was performed using the microbial assembly protocol included in SMRT Link v10.1.0, with a target genome size of 8 Mbp. The microbial assembly process resulted in one circular chromosomal contig, which was adjusted to dnaA. Identification of dnaA was carried out using BLAST, and circularization and rotation were performed with the genomecirculator.py tool (https://github.com/boykebunk/genomefinish). Error correction was performed by mapping Illumina short reads onto the completed genome using BWA (https://github.com/JHartlich/AlternateReferenceMaker). The genome was annotated using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP)  with modifications . Samplessing BWA , with sue (PGAP) . DefaultNocardia iowensis DSM 45197T have been deposited in GenBank under BioProject accession number PRJNA743876, BioSample accession number SAMN20062579, SRA accession numbers SRX11360990 (PacBio reads) and SRX11360991 (Illumina reads), and GenBank nucleotide accession number CP078145.Complete genome sequences of


Prosopis cineraria is native to the Near East and Indian subcontinent, where it thrives in very hot desert environments. To develop a tool to better understand desert plant adaptation mechanisms, we sequenced the P. cineraria genome to near-chromosomal assembly, with a total sequence length of ~691 Mb. We predicted 77,579 gene models  from the assembled genome, among them 55,325 (~72%) protein-coding genes that were functionally annotated. This genome was found to consist of over 58% repeat sequences, primarily long terminal repeats (LTR-)-retrotransposons. We find an expansion of terpenoid metabolism genes in P. cineraria and its relative Prosopis alba, but not in other legumes. We also observed an amplification of NBS-LRR disease-resistance genes correlated with LTR-associated retrotransposition, and identified 410 retrogenes with an active burst of chimeric retrogene creation that approximately occurred at the same time of divergence of P. cineraria from a common lineage with P. alba~23 Mya. These retrogenes include many biotic defense responses and abiotic stress stimulus responses, as well as the early Nodulin 93 gene. Nodulin 93 gene amplification is consistent with an adaptive response of the species to the low nitrogen in arid desert soil. Consistent with these results, our differentially expressed genes show a tissue specific expression of isoprenoid pathways in shoots, but not in roots, as well as important genes involved in abiotic salt stress in both tissues. Overall, the genome sequence of P. cineraria enriches our understanding of the genomic mechanisms of its disease resistance and abiotic stress tolerance. Thus, it is a very important step in crop and legume improvement.The mimosoid legumes are a clade of ~40 genera in the Caesalpinioideae subfamily of the Fabaceae that grow in tropical and subtropical regions. Unlike the better studied Papilionoideae, there are few genomic resources within this legume group. The tree  Legumes (Fabaceae) are a key flowering plant family, associated with the unique ability to fix nitrogen in soil though interaction with microbes. Traditionally, Fabaceae is classified in three subfamilies, Papilionoideae, Mimosoideae, and Caesalpinioideae . A molecMedicago truncatula Gaertn. [Lotus japonicus L. [Glycine max (L.) Merr. [Cajanus cajan (L.) Millsp. [Cicer arietinum L. [Vigna radiata (L.) R. Wilczek [2, temperature, and humidity in the Pliocene [Due to their economic importance, most legume genomic resource development has focused on the Papilionoideae subfamily. These include six published genome sequences:  Gaertn. , Lotus jnicus L. , Glycine.) Merr. , Cajanus Millsp. , Cicer atinum L. ,9, and V Wilczek . Compara Wilczek . Previou Wilczek ,12,13, a Wilczek ,14. ThisPliocene .Acacia, Prosopis, and Mimosa. One genus in the Caesalpinioideae, Clade: Mimosoid, is Prosopis , which is particularly well adapted to severe desert environments, including via some of the world’s deepest and broadest root systems. Prosopis cineraria (L.) Druce (Arabic name Ghaf) thrives in harsh desert environments, including as the most abundant wild legume tree in the Arabian Peninsula, and is the national tree of the United Arab Emirates (UAE) . P. cineraria is also abundant throughout the middle east and arid regions of the Indian subcontinent [P. cineraria leaves, pods, trunk, and bark are used by humans for various purposes. The unripened and ripened pods of the plant are eaten as vegetables or fruit, respectively [In contrast to the well-studied Papilionoideae subfamily, little is known about the genome organization and evolution of Mimosoideae and Caesalpinioideae, which includes important economic and ecological members, such as in the clade mimosoids ontinent . P. cineectively . Moreoveectively . The truectively . This leP. cineraria is a long-lived phreatophyte, which means it produces a long tap root that will reach more than 30 m in search for underground aquifers [P. cineraria is drought and salt tolerant, and is able to survive heat extremes that span both high (>45 °C) and low (<10 °C) temperatures that occur seasonally in desert regions. Unusually, it has a flowering mechanism in which blossoms appear during the hot dry season, where water availability is relatively scarce [Prosopis glandulosa Torr., in which early flowering and fruiting were more abundant under water deficit and heat stress compared to well-watered plants [aquifers . P. ciney scarce ,18,22. Gd plants .P. cineraria to a near-chromosomal level assembly. We annotated and characterized repeats and genes, with a particular emphasis on stress resistance genes, in the genome of P. cineraria in a comparative approach to other legumes. In the P. cineraria genome, we identified a dramatic burst of new gene creation by retrotransposition, and studied the expression of these retrogenes. We also increased our understanding of the genes and pathways involved in salt stress response using a transcriptomic approach. The genome resource and analysis of P. cineraria developed in this study provide insights into the biology and evolution of ecologically important desert trees, and generate an important new foundation for advancing the genetic improvement of legume crops. In this study, we sequenced the genome of P. cineraria tree located in a desert environment in the United Arab Emirates  terms and Interpro signatures were identified through InterProScan search. In total, 9132 transcription factors belonging to 58 plant transcription factor families were annotated from the gene models . MYB-relP. cineraria and Prosopis alba Griseb.  belonging to the mimosoid clade were compared to five members of the Papilionoideae subfamily within the legumes  Heynh. and Oryza sativa L. as outgroup species. The comparison of the nine species in this study led to the identification of 33,123 gene families in total  are enriched for defense response loci such as disease resistance (NBS-LRR), for terpenoid and isoprenoid metabolism, as well as reverse transcriptase genes (p < 10−2) contraction of gene families enriched for PFAM genes related to plant organ developmental regulation and water response , as well as flowering time and circadian rhythm coordination genes such as AP2, were contracted. The contraction of water response gene families is surprising, given the arid conditions where P. cineraria grows.We observed in the pansions A. P. cinse genes B,C. In cresponse B,C. For P. cineraria; we also found specific gene families for pathogen resistance that were legume-specific. Our analysis found an intriguing connection between some of these disease-resistance genes and LTR-retrotransposon sequences. We observed this by mining the genome of P. cineraria for all types of repeats, and found that the genome has ~58.2% repeats, of which ~51.3% are LTR-retrotransposons , followed by G. max (35.1%), Trifolium pratense L. (30.4%), Phaseolus vulgaris L. (26.6%) and the two outgroups O. sativa and A. thaliana with 26.7% and 8.5%, respectively. It should be noted that Arachis species have the highest LTR-retrotransposon contents fraction in these studied legumes (~60%). Our analysis indicates a significant expansion of disease-resistance gene families in nsposons A. As expP. cineraria and P. alba , but only a few in others of these species (p = 0.0003), a standard observation for this category of gene that has LRR regions under diversifying selection. Interestingly, we observed a frequent co-localization of disease-resistance genes (NBS-LRR) and LTR-retrotransposons. To test for co-localization, we calculated the average distance between LTR-retrotransposons and disease-resistance genes (NBS-LRR), and compared this to the overlap distance of LTR-retrotransposons to all genes in the genome. We observe that LTR-retrotransposons are in closer proximity to NBS-LRR genes compared to the rest of the genome (p < 0.05) using the R package regionR, which confirms that the co-localization of LTR-retrotransposons with NBS-LRR genes is not due to chance.An abundant and important category of plant disease-resistance genes, the NBS-LRR genes, were mined in the same manner for each of these species in a comparative approach. We observed a great abundance of NBS-LRR gene candidates in  species A. There  species B, compare genome . We ran p = 10−5), as well as genome size and predicted NBS-LRR gene numbers . In addition, NBS-LRR gene numbers are also significantly correlated with total repeat sequence numbers  and LTR-retrotransposons sequences   C.P. cineraria predicted genes encoding protein sequences were mapped against the near-chromosomal level genome assembly using RetroScan. We identified 785 candidate retrocopies that originated from 410 parental genes  to synonymous (Ks) substitutions, comparing the Ka/Ks ratio between retrogenes and their parental genes. We observed that the distribution of this ratio for chimerical retrogenes shows a peak at Ka/Ks < 0.2, and intact retrogenes have lower Ka/Ks than pseudoretrogenes  to validate the activity of retrogenes. We observed 401 expressed retrocopies (FPKM > 0) and if we consider the salt treatment versus control (FPKM > 0), the number is 355. However, the number of robustly expressed retrocopies (FPKM > 1) in control and salt environments was found to be 77 . More chP. cineraria genome sequence  and shoot (control and treatment) tissues . After asequence . For eacsequence . We carrIn shoot samples, we found 2065 up-regulated and 2108 down-regulated genes that differentiated control and salt stressed tissues  A. ImportIn root samples, the DGE analysis revealed 2024 up-regulated genes and 2701 down-regulated genes in control vs salt stressed B. Genes Prosopis cineraria by combining different NGS sequencing technologies . We present a near-chromosomal level assembly with 14 pseudochromosomes and a high N50 of ~41 Mb compared to the reported genome assembly of a related South American species, P. alba, with N50 of ~248 Kb (NCBI genome data source).In this study, we generated a high-quality genome assembly of P. cineraria and P. alba in the mimosoid clade compared to other Papilionoideae legumes. The expanded terpene synthase gene family in this clade is consistent with the fact that terpenes are important bioactive substances in Prosopis spp. [P. cineraria terpenoid (isoprenoid) bioactivity is diverse may partly explain why human exploitation of this species is widespread in the Near East and Indian subcontinent [Our orthologous gene group analysis highlights enrichment of genes involved in terpenoid metabolism that are specific to pis spp. . As a gapis spp. ,27,28. Ipis spp. ,30,31. Tontinent ,32.P. cineraria and P. alba have amplified NBS-LRR gene numbers compared to other legumes, and this is significantly correlated with both the abundance and location of LTR-containing retroelements. Our evidence for retro-amplification of NBS-LRR genes is consistent to what is known in some other plants [P. cineraria suggests balancing selection acting on these genes. One possible explanation is that the woody nature and long lifespan of P. cineraria may lead to greater exposure to pathogens and less meiotic recombination events to generate novelty. Thus, more NBS-LRR genes could provide a broader array of pathogen sensors as well as promote frequent unequal recombination to amplify and diverge these genes [The innate immune system in plants uses a repertoire of receptors that sense pathogens and trigger an immune response. A big fraction of these receptors is from the NBS-LRR gene family. r plants ,34,35. Ise genes ,37,38.P. cineraria genome. In general, nuclear sequences are assumed to have a mutation rate on the order of 10−8 to 10−9 substitutions/site/generation [P. cineraria, we use an estimate of ~10−9 from woody long-lived trees such as conifers [P. cineraria peaks between 0.02–0.05, which is equivalent to ~10–25 Mya divergence, suggesting that a large fraction of these retrogenes originated in this evolutionary time period.It has been surmised that by recruiting new proteins, chimerical retrogenes are likely to drive genetic innovation and adaptive evolution . Our ananeration . Since wconifers . The Ks P. cineraria from shared lineage with P. alba, as well as a similar estimate by Cardoso and coworkers [ [P. cineraria suggest that retrotransposition contributed to the origin of new genes that have played a role in evolutionary adaptation.Interestingly, this burst of chimerical retrogene generation coincides with our estimate of the divergence of oworkers . Our resoworkers . In addioworkers ,44. For oworkers ,34,35. Coworkers ,46. A se G. max) . Moreove G. max) . Moreove2; FDR value: 8.49 × 10−25) in salt stressed samples. In rice, PPR gene function was validated to enhance both drought and salt stress tolerance [p-value: 1.14 × 10−20) and root  samples during the salt stress. In maize, this gene was reported to be correlated with cell-wall extensibility under salt stress [The transcriptome analysis performed under control and salt stress conditions in both root and leaf samples revealed key genes that are possibly related to salt stress response. In leaf samples, a pentatricopeptide repeat-containing  protein gene was up-regulated almost 13-fold  and plant-pathogen defense in shoots, but not found in roots. This suggest that biosynthesis of these compounds is restricted to the shoots, which could be tightly linked to internal and external stimuli regulation to fine-tune terpenoid development to mediate proper interaction with the environment ,53,54.P. cineraria were collected from one tree (~15–20 years old) growing in the desert of Sweihan area (w/v) agarose gel.For the reference genome assembly, fresh leaves of han area . The quaTo assist gene annotation, RNA was extracted from leaves, roots, or flowers using a modification of a previously published method  the det. The isoP. cineraria DNA. Illumina compatible libraries (insert size 300–600 bp) were generated and sequenced (150 bp PE chemistry) on the Illumina NextSeq 2000 platform following Illumina chemistry and instructions. The raw Illumina data were trimmed  using Trimmomatic v.3 [We generated shot-gun sequencing reads (Illumina-based) and long sequencing reads (PacBio-based) data from the isolated atic v.3  and furtatic v.3 . ContinuP. cineraria was estimated using GenomeScope v.1 online tool [The Illumina shotgun PE reads generated were used for genome size estimation using a k-mer-based method. We generated k-mer count  as well as k-mer histogram files from trimmed and cleaned Illumina reads using Jellyfish v.2.3.0 . Based oine tool .Initial genome assembly was carried out by CANU (v.1.6)  softwareFor scaffolding the genome into a near-chromosomal level, we used Omini-C libraries (Dovetail) generated using the NEBNext Ultra enzymes kit and sequenced on the Illumina HiSeqX platform (target to obtain ~30X coverage). We used HiRise  for scafP. cineraria for training gene models  from the mimosoid clade in comparison with other legumes, such as G. max (GCA_000004515), T. pratense (GCA_020283565), M. truncatula (GCA_003473485), V. radiata (GCA_000741045), and P. vulgaris (GCA_000499845). We used A. thaliana and O. sativa as outgroups. All these genomes are available from the NCBI genome database. Orthogroup presence and absence were summarized in an UpsetR [P. cineraria and P. alba, were extracted and GO enrichment was summarized using REVIGO [P. cineraria and P. alba but not shared with other legumes.Orthofinder2 v.2.3.12  was usedn UpsetR  plot. Org REVIGO . A similhttps://www.ncbi.nlm.nih.gov, accessed on 2 March 2022), including the outgroups A. thaliana and O. sativa, Orthofinder2 was used on all protein-coding genes. Single copy Orthogroups mined by Orthofinder2 were used to construct a phylogenetic tree. R8s [A. thaliana and O. sativa median divergence time from TimeTree , which is ~152 Mya, and the number of sites  that went into generating the species tree.To estimate the evolutionary relationships among 18 legumes species  to extract the top significant expansions, contractions and under selection at p < 10−2. A Venn diagram for the intersection of expanded, contracted. and under selection gene families was generated as well as GO term enrichment. Barplots for gene families expanded, contracted, and under selection were generated using R package GOplot [All expansions, contractions, and rapidly evolving gene families from the 18 legumes with the two outgroups were determined using Orthofinder2. Species-specific gene family’s expansion and contractions were identified using CAFÉ v.5  programse GOplot .P. cineraria, we used ab initio predictions of three available programs, RepeatModeler v.2.0.1 [P. cineraria genome was carried out using RepeatModeler tool. From the predicted repeat dataset, possible proteins and transcripts that were related to repeats were removed from the assembled genome prior to gene prediction using default parameters to run RepeatMasker v.4.1 tool [To build a de novo repeat library for  v.2.0.1 , for all v.2.0.1 ,93. LTR 4.1 tool . IdenticNBS-LRR  disease-resistance genes were mined using NLgenomesweeper . This toP. cineraria, we used the RetroScan [https://github.com/Vicky123wzy/RetroScan, accessed on 20 January 2022) for visualization of the results and for generation of retrocopy distribution, localization, and expression under salt stress as well as the selective force acting upon retrogenes in the genome. GO enrichment analysis for retrogenes was summarized using REVIGO. Filtered raw reads from the salt experiment in triplicates was used for retrogenes activity analysis.To identify retrogenes in the genome of etroScan  pipelineP. cineraria seed were collected from the same tree used for genome sequencing. Collected seed were sterilized with 30% (v/v) bleach (Clorox) for 15 min and washed three times with sterile Milli-Q water. They were placed on 1/2 strength MS agar (0.7% w/v) medium and allowed to grow at 25 °C in a growth chamber. Five-week-old seedlings were transferred to hydroponic trays and acclimatized for five weeks. Later, the seedlings were subjected to salt stress (48 h) by replacing the media in the trays with fresh medium with and without 250 mM salt stress for test and control, respectively. Three replicates of roots and shoots were collected from the control and treated samples and were snap-frozen for transcriptome expression analysis (For the salt stress experiment, analysis . The samThe quality of raw transcriptome reads was assayed using the FastQC program. Adapters and low quality regions found in the reads were trimmed using Trimmomatic tool. Here, we followed a reference-based transcriptome approach for the gene expression analysis . The refFrom the up-regulated and down-regulated genes that were differentially expressed, GO enrichment was predicted using dcGO  and the Prosopis cineraria is among a small number of native trees that thrives in arid environments. It is used extensively in desert societies for its social, economic, ecological, and medicinal values. Given that the rate of desertification has been increasing in the past many decades, understanding desert plants’ survival strategies may help support translational agronomics, breeding, genetics, and genomics for crop improvement. The released P. cineraria genome will provide a key genomic resource and assist identification of the adaptive responses of genes that might be used in the enhancement of crop legumes.


Fusarium graminearum sensu stricto (F.g.). This species is responsible for cereal diseases and negatively impacts grain production worldwide. The fungus belongs to the monophyletic fungal complex referred to as F. graminearum species complex including at least sixteen cryptic species, a few among them may be involved in cereal diseases in certain agricultural areas. We analyzed WGS data from a collection of 99 F.g. strains and 33 strains representing all known cryptic species belonging to the FGSC complex. As a first step, we performed a phylogenomic analysis to reveal species-specific clustering. A RAxML maximum likelihood tree grouped all analyzed strains of F.g. into a single clade, supporting the clustering-based identification approach. Although, phylogenetic reconstructions are essential in detecting cryptic species, a phylogenomic tree does not fulfill the criteria for rapid and cost-effective approach for identification of fungi, due to the time-consuming nature of the analysis. As an alternative, analysis of WGS information by mapping sequence data from individual strains against reference genomes may provide useful markers for the rapid identification of fungi. We provide a robust framework for typing F.g. through the web-based PhaME workflow available at EDGE bioinformatics. The method was validated through multiple comparisons of assembly genomes to F.g. reference strain PH-1. We showed that the difference between intra- and interspecies variability was at least two times higher than intraspecific variation facilitating successful typing of F.g. This is the first study which employs WGS data for typing plant pathogenic fusaria.Recent improvements in microbiology and molecular epidemiology were largely stimulated by whole- genome sequencing (WGS), which provides an unprecedented resolution in discriminating highly related genetic backgrounds. WGS is becoming the method of choice in epidemiology of fungal diseases, but its application is still in a pioneer stage, mainly due to the limited number of available genomes. Fungal pathogens often belong to complexes composed of numerous cryptic species. Detecting cryptic diversity is fundamental to understand the dynamics and the evolutionary relationships underlying disease outbreaks. In this study, we explore the value of whole-genome SNP analyses in identification of the pandemic pathogen  Fungi are among the most diverse groups of eukaryotic organisms on earth and include a wide range of taxonomic groups with various morphological and phenotypic characters, ecologies and life cycles . With thUndoubtedly, taxonomic information is crucial for understanding fungal diversity and evolution. Taxonomy provides a common language for scientists and the regulatory community ,b, and iAlternaria, Botryosphaeria, Cercospora, Diaporthe, and Fusarium ITS sequence alone cannot be used to identify most species  which are amplified and sequenced with the use of universal primers. Today, the internal transcribed spacer (ITS) region in the ribosomal RNA gene cluster has been validated as the best DNA barcode marker for fungi. However, its reliability as taxonomic marker for fungi has been criticized, because of: (i) the presence of divergent intragenomic ITS sequences in several fungal lineages  and (ii) species .A number of fungi previously proved to be a single species proved species complexes composed of multiple cryptic species. Identification of these diverged species is usually achieved through multilocus sequence typing (MLST). This approach involves the amplification of housekeeping genes by PCR, followed by DNA sequencing of amplicons and defining an allelic profile corresponding to a Sequence Type (ST) . The useRecent advances in genomic sequence analysis and Single Nucleotide Polymorphism (SNP) discovery have empowered researchers to explore microbial diversity in more detail. The increasing amount of sequenced fungal genomes opens up the possibility to better understand the tremendous diversity of fungi and their evolution. Fungal genomes are relatively small in comparison to plant and animal genomes, but vary from several to nearly 1,000 megabases (Mb) . GenomesFusarium graminearum sensu stricto (F.g.) (F.g. belongs to the F. graminearum species complex (FGSC), which includes at least sixteen cryptic species  . This cro (F.g.) . F.g. be species , among t species . Determi species . We haveequences .F.g. strains and 33 strains representing all known cryptic species from the FGSC complex. We constructed a phylogenomic tree to demonstrate species-specific clustering for species recognition. We also show that the web-based calculation of the number of SNPs via comparison to reference PH-1 strain of F.g. provides an easy method of typing F.g. This is the first report on typing plant pathogenic fusaria through WGS data.In this paper, sequence data from previous WGS project were first used to assemble genomes from a total of 99 F.g. strains and 33 strains representing all known cryptic species from the FGSC complex: Fusarium acaciae-mearnsii (three strains), Fusarium aethiopicum (one strain), Fusarium asiaticum (n = 3), Fusarium austroamericanum (n = 2), Fusarium boothii (n = 4), Fusarium brasilicum (n = 1), Fusarium cortadariae (n = 3), Fusarium gerlachii (n = 2), Fusarium louisianense (n = 2), Fusarium meridionale (n = 2), Fusarium mesoamericanum (n = 1), Fusarium nepalense (n = 2), Fusarium ussurianum (n = 4), Fusarium vorosii (n = 2), and strain CBS 123663 (NRRL34461), which lacks a Latin binomial. In addition, four single strains from the closely related morphospecies F. culmorum, F. pseudograminearum, F. sambucinum and F. venenatum were incorporated into SNP analysis. Detailed information on all strains used in this study are included in In total, 136 strains were analyzed in this study, with 99 via FastQC (ver. 0.11.9) (via SPAdes (v.3.13.2) . Libraries were prepared using the KAPA HyperPlus Kit . An Illumina HiSeq X Ten was used to sequence the genomes using a paired-end read length of 2 × 150 bp with an insert size of 350 bp. For 12 strains: ar1 (CBS_139514), ar3 (119-12), us2 (CBS_119173), po12 (CBS138561), po13 (CBS138562), po14 , fas1 (CBS110258), fam3 (CBS123662), fau1 (CBS110244), fbo3 (CBS119170), fme2 (CBS110260) and sa2 (CBS119800), whole genome libraries were prepared using a Nextera XT kit  and sequenced on the Illumina Miseq platform with the 250 bp paired-end read, version 2. The sequencing quality was assessed  0.11.9) . Low-qua 0.11.9)  and the .3.13.2)  with k-min reads . The comin reads . Genome in reads . The proF. pseudograminearum strain CS3096 was used as an outgroup.Locally installed PhaME  was used1 (F.g. (GenBank assembly accession: GCA_900044135.1). The total number of SNPs is counted as the number of positions that are variable between two genomes.To extract core SNPs, we used the whole-genome SNP-based phylogeny tool PhaME, which is integrated into the EDGE bioinformatics platform1 . PhaME eF. culmorum (GenBank assembly accession: GCA_019055245.1) and F. cerealis (GenBank assembly accession: GCA_019055205.1) was used as an outgroup. Variation within each sequence was identified as a SNP and counted with the use of an in-house Python script. Nucleotide diversity values (π) for were calculated with TASSEL software  of whole-genome sequences were generated in this study. Details of genome assembly quality is given in We analyzed genomes of 99 F. aethiopicum, F. acaciae-mearnsii), Asia  and Australia (F. acaciae-mearnsii). The second larger clade was more diverse and included species endemic to North  and South America  and New Zealand (F. cortadariae). F. boothii and F. meridionale reported from diverse regions of Asia, Africa, and Latin America . Its basal position in the F.g. clade may suggest that subclade I includes genotypes with the highest genetic relationship to ancestors of F.g. Interestingly, subclade I included the oldest known strains of F.g.: un1 (CBS 185.32) and un2 (CBS 104.09), which were isolated/deposited in the collection of the Westerdijk Fungal Biodiversity Institute in 1932 and 1904, respectively. Unfortunately, associated metadata of these two strains do not include information on their geographic origin. The first three subclades  diverging from subclade I were represented by single strains, among which two: ar4 (114-2) and ar1 (CBS_139514) come from South America and one  from Australia. Most European strains were grouped into clusters VI-XIII. Interestingly, subclades VI (n = 7), VII (n = 1), VIII (n = 10) and IX (n = 2) grouped mostly Polish strains. A small subclade X clustered only two strains: ne5 (79E1) originating from Netherlands and po11 (16-390-z) originating from Poland. This small clade was located between Polish subclade IX and west European subclade XI grouping strains (n = 34) mostly from West Europe. The observed high degree of geographic clustering of the strains was not observed in subclade XIII (n = 27), which diverged from west European subclade XI. It grouped strains from diverse geographic locations such Argentina, Brazil, Italy, Germany, the Netherland, Poland, Russia and Serbia, pointing out evidence of their most recent spread to new geographic locations. Geographic expansion in accessible directions may be also indicated by clustering two European strains ge3 (CS10007) (from Germany) and ru13 (70725) (from Russia) into subclade I. Other examples come from subclade XI, which included a single strain from South Africa , and subclade VI, which included strain ir1 (CBS 110263) from Iran.To minimize the impact of geographic variation that may interfere with the results, we incorporated a large set of geographically diverse strains. Such a strategy is especially critical for pandemic species, which are often subdivided into genetically distinct populations . F. gramF.g., which displaced of F. culmorum, which had been the major FHB agent of wheat since the 1840s  unexpectedly clustered outside the F.g. clade and was grouped in the F. boothii clade. This strain is held in the fungal collection Westerdijk Fungal Biodiversity Institute  as F. graminearum and according to available metadata it was isolated from maize in South Africa where F. boothii has been frequently reported (F.g. strain CBS 104.09 (NCBI accession no. KR011238). Partial (659 nt) tef gene sequence of CBS 119800 (NCBI accession no. KT855180) shows 100% identity to F.g. (NCBI accession no. KP267345), indicating that tef alone could not definitively confirm its taxonomic affiliation. Blast search against NCBI non-redundant (nr) nucleotide database with an e-value cutoff of ≤ e-0.0, 100% identity and 100% coverage also yielded nearly 30 hits to F. boothii. To further clarify its taxonomic status, we retrieved the complete sequence of the topoisomerase 1 (top1) and phosphoglycerate kinase (pgk) genes, which have become widely used taxonomic markers for Fusaria  yielded two hits to F. boothii (NCBI accession no. KY952952 and KY952951) with 100% sequence identity. However, blast searching with the pgk gene as a query did not produce hits with 100% sequence identity, presumably due to the lack of pgk sequences from F. boothii in GenBank database. To determine sequence similarity of pgk between CBS 119800 and other strains of F. boothii, we retrieved its sequence from genome assembly of three strains of F. boothii: CBS 316.73, CBS 110251 and CBS 119170. Subsequent sequence comparison (data not shown) revealed that all four strains shared 100% identity in the pgk gene, thus, supporting its identity as F. boothii.Among the reported . Howeverreported . The com Fusaria . Blast sF. asiaticum or F. meridionale or a hybrid strain  is in range of intraspecific variability found for F.g. (discussed in later sections), which is indicative of its taxonomic assignment as F. meridionale.Our results of phylogenomic analysis may also be helpful in resolving the uncertain taxonomic status of CBS 110260. This strain was isolated from maize in Nepal and has been assigned to either d strain . Most re49 fme1, , which sanalyses  confirmeF.g.. The method was validated through multiple comparisons of assembly genomes to reference PH-1 strain. We estimated intra and interspecific differences in the number of SNPs facilitating species recognition , with one major exception. In subclade I grouping most of the strains from either North or South America had high bootstrap support (92). In addition, phylogenetic tree supported higher genetic divergence of CBS 119173 (us2) by clustering of this strain at the basal position of the F.g. clade. Grouping of cryptic species of FGSC corresponded to phylogenomic clustering and confirmed clarification of taxonomic issues of CBS 119800 (sa2) and CBS 110260 (fme2) as F. boothii and F. meridionale, respectively.Phylogenetic analyses of different housekeeping genes  help to Fusarium graminearum sensu stricto (F.g.) based on intra- and interspecific differences in the number of SNPs. Analysis of genome comparison does not require specialized software and can be rapidly performed in a user-friendly web-based workflows. Our results seem to be sufficiently robust to the effects that the quality of the assembly can have on SNPs calling  can be found in the article/TK conceived the idea, acquired funding, designed the study, and prepared the draft manuscript. TM reconstructed phylogeny of the strains. GF performed assessment of genome-wide SNP counts. AD, AS, JO, KB, MB, MP, and SS provided fungal strains for analysis. KT, KB, and GF provided the technical support during the study. All authors read the manuscript, critically revised, agreed to the content of the manuscript, read and approved the final manuscript.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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Sparassis latifolia, a highly valued edible fungus, is a crucial medicinal and food resource owing to its rich active ingredients and pharmacological effects. Excessive oxalic acid secreted on a pine-sawdust-dominated substrate inhibits its mycelial growth, and severely restricts the wider development of its cultivation. However, the mechanism underlying the relationship between oxalic acid and slow mycelial growth remains unclear. The present study reported the transcriptome-based response of S. latifolia induced by different oxalic acid concentrations. In total, 9206 differentially expressed genes were identified through comparisons of three groups; 4587 genes were down-regulated and 5109 were up-regulated. Transcriptome analysis revealed that excessive oxalic acid mainly down-regulates the expression of genes related to carbohydrate utilization pathways, energy metabolism, amino acid metabolism, protein synthesis metabolism, glycan biosynthesis, and signal transduction pathways. Moreover, genes encoding for wood-degrading enzymes were predominantly down-regulated in the mycelia treated with excessive oxalic acid. Taken together, the study results provide a speculative mechanism underlying the inhibition of saprophytic growth by excessive oxalic acid and a foundation for further research on the growth of S. latifolia mycelia. Sparassis latifolia is a major commercial mushroom widely cultivated and consumed in Asia [S. latifolia. in Asia . It is a in Asia . A subst in Asia . HoweverS. latifolia is the key to directly understanding its mycelial growth. Unlike most saprophytic edible fungi, S. latifolia possesses two different nutritional modes: saprophytic and parasitic [S. latifolia mycelia to oxalic acid during the process of saprophytic growth has not been deeply elucidated.The utilization of a substrate by arasitic . In prevarasitic . NeverthS. latifolia.Oxalic acid is an important pathogenic or toxic factor in the interaction between pathogenic fungi and their hosts. This organic acid can attack defense substances on the plant surface, making it easier for pathogens to infect plants. Oxalic acid accumulates continuously in the infected site, directly inducing toxin action and leading to host cell necrosis ,7. OxaliAbortiporus biennis revealed that the addition of oxalic acid significantly promoted the expression of cellulolytic enzyme genes, but significantly inhibited the expression of lignolytic enzyme genes [Polyporus umbellatus in the presence of oxalic acid [Gene expression profiles of edible fungi have recently been considered effective tools for understanding molecular mechanisms. RNA-seq is a practicable method for determining differential transcript levels of edible fungi under different treatments . Transcrme genes . Xing etlic acid . These tS. latifolia and the inhibitory role of oxalic acid in its slow mycelial growth rate. Illumina sequencing technology was used here to detect the differential properties of S. latifolia at different oxalic acid concentrations and thus establish a critical basis for elucidating the mechanism underpinning the slow mycelial growth rate during the saprophytic process.No researchers have currently focused on the reason for the slow saprophytic growth rate of S. latifolia CCMJ 1100 was obtained from the Culture Collection Center of Mycology of Jilin Agriculture University. For the pure culture of S. latifolia, a mycelial disc (diameter: 5 mm) was cultured on the center of potato dextrose peptone agar (PDPA) at 24 °C [S. latifolia were inoculated into pine sawdust medium  in a bag (17 × 35 cm) and incubated at 24 °C [at 24 °C . To growat 24 °C .Total RNA was isolated using the Trizol Reagent  according to the manufacturer’s protocol. The Nanodrop 2000 spectrophotometer  and the Agilent 2100 Bioanalyzer and 2100 RNA nano 6000 assay kit  were used to evaluate the concentration and purity and the integrity of RNA samples, respectively.After the quality check procedure, RNA with poly-A in eukaryotic total RNA samples was enriched using the TurboCapture 96 mRNA Kit . Then, transcriptome sequencing libraries were constructed with the TruSeq RNA Library Preparation Kit V2 . Briefly, library construction involved capture of mRNA fragmentation, mRNA disruption and synthesis of double-stranded cDNA, end repair, A-tailing, ligation of sequencing adapters, size selection, and library PCR amplification. Qubit 2.0 fluorometer  was used to quantified library concentration. Subsequently, the concentration was diluted to 1 ng/µL. The insert size was checked by an Agilent 2100 Bioanalyzer and quantified to greater accuracy using quantitative PCR (q-PCR). The clustering of the index-coded samples was performed on a cBot Cluster Generation System by using TruSeq PE Cluster Kit v3-cBot-HS  . After chttp://www.usadellab.org/cms/index.php?page=trimmomatic, version 0.39, accessed on 28 May 2022) [S. latifolia reference genome at JGI genome portal  by using the TopHat software tool  with a default option [https://pypi.org/project/HTSeq/, version 2.0.2, accessed on 28 May 2022) was used to count the read numbers mapped to each gene. Then, fragments per kilobase of exon per million mapped reads (FPKM) of each gene was used to quantify the abundance of mRNA. The DESeq2 R package  was used for differential expression analysis of two groups [p values. To identify genes responding to different oxalic acid concentrations, a gene with an adjusted p value < 0.05 and an absolute value of log2 fold change > 1 was considered as a DEG.The raw data generated through high-throughput sequencing were provided in the fastq format sequences. Clean data (clean reads) were obtained by removing reads containing adapter and trimming low-quality bases with Trimmomatic ay 2022) . Simultat option . Htseq-c22 . Simup value of ≤ 0.05 were considered significantly enriched by DEGs. Genome annotation was based on Ensembl and Kyoto Encyclopedia of Genes and Genomes (KEGG) [Gene Ontology (GO) enrichment analysis of DGEs was performed using the topGO R package . GO terms (KEGG) . The stas (KEGG) .18S rbs) was used as the internal control gene. qRT-PCR primers were designed with Primer 6 and are listed in TM kit  was used according to the manufacturer’s protocol. Three biological replicates were performed, and the relative RNA expression level was calculated using the 2−ΔΔCt method.According to the biological role of genes clarified through GO and KEGG analyses and the levels and differences in gene expression between samples, 6 DEGs were randomly selected for validation through qRT-PCR on a Bio-Rad CFX96 System . The 18S ribosomal RNA gene (p ≤ 0.05) were used to determine the statistically significant difference. The statistical analysis was applied through SPSS version 20.0 software .All data are presented as the mean ± standard deviation (SD). Analysis of variance (ANOVA) and Duncan’s multiple range tests , S. latifolia mycelial growth was significantly inhibited, and the mycelial growth rate was the lowest , DEGs were identified and annotated in three comparisons , and the normalized read counts  of the nine samples were statistically compared. Compared with the CK group, 4478 DEGs were identified in the OA group (2248 up-regulated and 2030 down-regulated) , and 133 samples B–D. The  samples . Taken tTo investigate the DEGs’ function, the identified genes were annotated using GO and KEGG databases. In the OA vs. CK comparison, 231 terms were enriched from 4478 DEGs, including 56 molecular functions (MFs), 136 biological processes (BPs), and 39 cellular components (CCs) . Among tIn the IOA vs. CK comparison, 117 terms were enriched from 1331 DEGs, including 47 MFs, 55 BPs, and 15 CCs . Among tS. latifolia mycelial growth, we conjecture that the negative effect of high oxalic acid concentrations may be exerted through these processes.In the OA vs. IOA comparison, 200 terms were enriched from 3397 DEGs, including 43 MFs, 139 BPs, and 18 CCs . Among tS. latifolia grown in the presence of different oxalic acid concentrations were analyzed. KEGG functional annotation was performed, and the results of KEGG pathway enrichment analysis from the OA vs. CK, IOA vs. CK, and OA vs. IOA comparisons were provided , exo-glucanase (mRNA_8946), glycoside hydrolase , exo-β-1,3-glucosidase (mRNA_6544), and laccases (mRNA_3922), were found in the criptome . Comparecriptome .qRT-PCR was used to confirm the accuracy of gene expression profiles obtained through RNA-seq. Three up-regulated and three down-regulated DEGs were randomly selected to verify the relative expression levels of RNA-seq data. As shown in S. latifolia mycelial growth, especially for identifying candidate genes associated with slow saprophytic growth caused by high concentrations of oxalic acid.Oxalic acid is a common low-molecular-weight organic acid commonly produced by the majority of white and brown rot fungi and has been suggested to play multiple roles during the wood degradation process . This orS. latifolia significantly reduced under high oxalic acid concentration [S. latifolia to lignocellulose under high oxalic acid concentration will inevitably reduce its growth rate. Moreover, transcriptomic profiling also revealed that the expression level of many carbohydrate utilization pathways, such as galactose metabolism, starch and sucrose metabolism, and fructose and mannose metabolism, were significantly down-regulated at high oxalic acid concentrations (S. latifolia by excessive oxalic acid.The most direct effect of oxalic acid on fungal mycelia is its growth rate . In our ntration . Therefotrations . CarbohyS. latifolia mycelium.The cell membrane is a thin semi-permeable membrane that plays the function of transmembrane transport of certain substances . AccordiS. latifolia mycelia.Oxidative phosphorylation is a critical coupling reaction wherein energy is released during mitochondria oxidation of substances . MitochoS. latifolia mycelia may be due to the decreased ability of the fungus to synthesize glycans at high oxalic acid concentrations.The fungal mycelium growth is accompanied by continuous synthesis and assembly of cell wall polysaccharides that mainly include glucans, chitin, mannans, and glycoproteins . All livS. latifolia mycelial growth. Furthermore, many genes associated with amino acid metabolism and vitamins were enriched in the group of down-regulated DEGs at high oxalic acid concentrations, including the synthesis and catabolism of various amino acids (S. latifolia mycelia. However, the mechanism of biosynthesis regulated by excessive oxalic acid needs to be revealed by in-depth research.Mycelial growth is also accompanied by the synthesis of numerous proteins and their assembly and post-translational modifications. Ribosomes are ribonucleoprotein particles, and act as molecular machinery for protein synthesis in the cells ,42. Ribono acids . Amino aS. latifolia mycelia. Moreover, our previous study also revealed that the MAPK, PI3K–Akt, and mTOR signaling pathways play important roles in primordium formation and fruiting body development [S. latifolia, leading to the prolongation of the cultivation cycle.Previous studies have reported that many signal transduction pathways are involved in fungal mycelial growth, such as cAMP/PKA pathways , MAPK sielopment . TherefoS. latifolia growing in PSM with different oxalic acid concentrations. In summary, the comparison of gene expression profiles showed that high oxalic acid concentrations inhibited many S. latifolia pathways related to the growth and development of mycelia, such as lignocellulose degradation metabolism, carbohydrate metabolism, and signal transduction processes. The inhibition of these metabolic pathways may be the reason for the slow saprophytic growth and long cultivation cycle of S. latifolia. On the one hand, observed differential expression patterns of DEGs suggest a seemingly complex regulation of oxalic acid in S. latifolia. On the other hand, transcriptome analysis identified some valuable candidate genes for further studies. However, the mechanism of oxalic acid in the degradation of pine sawdust by S. latifolia must be complicated and needs to be further explored. In conclusion, our results reveal the new mechanisms underlying the inhibitory effect of excessive oxalic acid on S. latifolia mycelia. Results of the present work will be beneficial for future work seeking to investigate in depth the initiation of saprophytic growth of S. latifolia.Our work presents the first of comparative transcriptome analysis of