Here we respond to Zhou (BMC Genomics 21:734, 2020) "Combined Transcriptome and Metabolome analysis of Pitaya fruit unveiled the mechanisms underlying peel and pulp color formation" published in BMC Genomics. Given the evolutionary conserved anthocyanin biosynthesis pathway in betalain-pigmented species, we are open to the idea that species with both anthocyanins and betalains might exist. However, in absence of LC-MS/MS spectra, apparent lack of biological replicates, and no comparison to authentic standards, the findings of Zhou (BMC Genomics 21:734, 2020) are not a strong basis to propose the presence of anthocyanins in betalain-pigmented pitaya. In addition, our re-analysis of the datasets indicates the misidentification of important genes and the omission of key flavonoid and anthocyanin synthesis genes ANS and DFR. Finally, our re-analysis of the RNA-Seq dataset reveals no correlation between anthocyanin biosynthesis gene expression and pigment status.

Comment on BMC Genomics. 2020 Oct 22;21(1):734.

Pantoea agglomerans DAPP-PG 734 was isolated as endophyte from knots (tumors) caused by Pseudomonas savastanoi pv. savastanoi DAPP-PG 722 in olive trees. To understand the plant pathogen-endophyte interaction on a genomic level, the whole genome of P. agglomerans DAPP-PG 734 was sequenced and annotated. The complete genome had a total size of 5'396'424 bp, containing one circular chromosome and four large circular plasmids. The aim of this study was to identify genomic features that could play a potential role in the interaction between P. agglomerans DAPP-PG 734 and P. savastanoi pv. savastanoi DAPP-PG 722. For this purpose, a comparative genomic analysis between the genome of P. agglomerans DAPP-PG 734 and those of related Pantoea spp. was carried out. In P. agglomerans DAPP-PG 734, gene clusters for the synthesis of the Hrp-1 type III secretion system (T3SS), type VI secretion systems (T6SS) and autoinducer, which could play an important role in a plant-pathogenic community enhancing knot formation in olive trees, were identified. Additional gene clusters for the biosynthesis of two different antibiotics, namely dapdiamide E and antibiotic B025670, which were found in regions between integrative conjugative elements (ICE), were observed. The in-depth analysis of the whole genome suggested a characterization of the P. agglomerans DAPP-PG 734 isolate as endophytic bacterium with biocontrol activity rather than as a plant pathogen.

BACKGROUND: Alternative splicing (AS) is an important channel for gene expression regulation and protein diversification, in addition to a major reason for the considerable differences in the number of genes and proteins in eukaryotes. In plants, U2 small nuclear ribonucleoprotein B″ (U2B″), a component of splicing complex U2 snRNP, plays an important role in AS. Currently, few studies have investigated plant U2B″, and its mechanism remains unclear. RESULT: Phylogenetic analysis, including gene and protein structures, revealed that U2B″ is highly conserved in plants and typically contains two RNA recognition motifs. Subcellular localisation showed that OsU2B″ is located in the nucleus and cytoplasm, indicating that it has broad functions throughout the cell. Elemental analysis of the promoter region showed that it responded to numerous external stimuli, including hormones, stress, and light. Subsequent qPCR experiments examining response to stress (cold, salt, drought, and heavy metal cadmium) corroborated the findings. The prediction results of protein-protein interactions showed that its function is largely through a single pathway, mainly through interaction with snRNP proteins. CONCLUSION: U2B″ is highly conserved in the plant kingdom, functions in the nucleus and cytoplasm, and participates in a wide range of processes in plant growth and development.

BACKGROUND: Plums are one of the most important economic crops of the Rosaceae family and are produced all over the world. China has many local varieties, but the genomic information is limited for genetic studies. Here, we first sequenced, assembled, and analyzed the plastomes of twelve plum cultivars and developed molecular markers to distinguish them. RESULTS: The twelve plastomes of plum cultivars have a circular structure of 157,863-157,952 bp containing a large single-copy region (LSC) of 86,109-86,287 bp, a small copy region (SSC) of 18,927-19,031 bp, and two inverted repeats (IR) of 26,353-26,387 bp each. The plastomes of plum cultivars encode 131 genes, including 86 protein-coding genes, 37 tRNA genes, and 8 rRNA genes. We detected 50, 54, 54, 53, 53, 50, 54, 54, 54, 49, 50, 54 SSRs in the twelve analyzed varieties, respectively. For repeat sequences, we identified 553 tandem repeats, 204 direct repeats, and 270 palindromic repeats. We also analyzed the expansion/contraction of IR regions. The genes rpl22, rps19, rpl2, ycf1, ndhF, and the trnH span on or near the boundary of IR and single-copy regions. Phylogenetic analysis showed that the twelve cultivars were clustered with the P. salicina and P. domestica. We developed eight markers LZ01 to LZ08 based on whole plastomes and nuclear genes and validated them successfully with six repetitions. CONCLUSIONS: The results obtained here could fill in the blanks of the plastomes of these twelve plum cultivars and provide a wider perspective based on the basis of the plastomes of Prunus to the molecular identification and phylogenetic construction accurately. The analysis from this study provides an important and valuable resource for studying the genetic basis for agronomic and adaptive differentiation of the Prunus species.

BACKGROUND: The bZIP gene family has important roles in various biological processes, including development and stress responses. However, little information about this gene family is available for Wheel Wingnut (Cyclocarya paliurus).  RESULTS: In this study, we identified 58 bZIP genes in the C. paliurus genome and analyzed phylogenetic relationships, chromosomal locations, gene structure, collinearity, and gene expression profiles. The 58 bZIP genes could be divided into 11 groups and were unevenly distributed among 16 C. paliurus chromosomes. An analysis of cis-regulatory elements indicated that bZIP promoters were associated with phytohormones and stress responses. The expression patterns of bZIP genes in leaves differed among developmental stages. In addition, several bZIP members were differentially expressed under drought stress. These expression patterns were verified by RT-qPCR. CONCLUSIONS: Our results provide insights into the evolutionary history of the bZIP gene family in C. paliurus and the function of these genes during leaf development and in the response to drought stress. In addition to basic genomic information, our results provide a theoretical basis for further studies aimed at improving growth and stress resistance in C. paliurus, an important medicinal plant.

BACKGROUND: Nematodes are a major group of soil inhabiting organisms. Heterorhabditis nematodes are insect-pathogenic nematodes and live in a close symbiotic association with Photorhabdus bacteria. Heterorhabditis-Photorhabdus pair offers a powerful and genetically tractable model to study animal-microbe symbiosis. It is possible to generate symbiont bacteria free (axenic) stages in Heterorhabditis. Here, we compared the transcriptome of symbiotic early-adult stage Heterorhabditis nematodes with axenic early-adult nematodes to determine the nematode genes and pathways involved in symbiosis with Photorhabdus bacteria. RESULTS: A de-novo reference transcriptome assembly of 95.7 Mb was created for H. bacteriophora by using all the reads. The assembly contained 46,599 transcripts with N50 value of 2,681 bp and the average transcript length was 2,054 bp. The differentially expressed transcripts were identified by mapping reads from symbiotic and axenic nematodes to the reference assembly. A total of 754 differentially expressed transcripts were identified in symbiotic nematodes as compared to the axenic nematodes. The ribosomal pathway was identified as the most affected among the differentially expressed transcripts. Additionally, 12,151 transcripts were unique to symbiotic nematodes. Endocytosis, cAMP signalling and focal adhesion were the top three enriched pathways in symbiotic nematodes, while a large number of transcripts coding for various responses against bacteria, such as bacterial recognition, canonical immune signalling pathways, and antimicrobial effectors could also be identified. CONCLUSIONS: The symbiotic Heterorhabditis nematodes respond to the presence of symbiotic bacteria by expressing various transcripts involved in a multi-layered immune response which might represent non-systemic and evolved localized responses to maintain mutualistic bacteria at non-threatening levels. Subject to further functional validation of the identified transcripts, our findings suggest that Heterorhabditis nematode immune system plays a critical role in maintenance of symbiosis with Photorhabdus bacteria.

BACKGROUND: Inferring the demographic history of a population is essential in population genetic studies. Though the inference methods based on the sequentially Markov coalescent can present the population history in detail, these methods assume that the population size remains unchanged in each time interval during discretizing the hidden state in the hidden Markov model. Therefore, these methods fail to uncover the detailed population history in each time interval. RESULTS: We present a new method called Beta-PSMC, which introduces the probability density function of a beta distribution with a broad variety of shapes into the Pairwise Sequentially Markovian Coalescent (PSMC) model to refine the population history in each discretized time interval in place of the assumption that the population size is unchanged. Using simulation, we demonstrate that Beta-PSMC can uncover more detailed population history, and improve the accuracy and resolution of the recent population history inference. We also apply Beta-PSMC to infer the population history of Adélie penguin and find that the fluctuation in population size is contrary to the temperature change 15-27 thousand years ago. CONCLUSIONS: Beta-PSMC extends PSMC by allowing more detailed fluctuation of population size in each discretized time interval with the probability density function of beta distribution and will serve as a useful tool for population genetics.

BACKGROUND: Long noncoding RNAs (lncRNAs) are a diverse class of RNAs that are critical for gene regulation, DNA repair, and splicing, and have been implicated in development, stress response, and cancer. However, the functions of many lncRNAs remain unknown. In Drosophila melanogaster, U snoRNA host gene 4 (Uhg4) encodes an antisense long noncoding RNA that is host to seven small nucleolar RNAs (snoRNAs). Uhg4 is expressed ubiquitously during development and in all adult tissues, with maximal expression in ovaries; however, it has no annotated function(s). RESULTS: We used CRISPR-Cas9 germline gene editing to generate multiple deletions spanning the promoter region and first exon of Uhg4. Females showed arrested egg development and both males and females were sterile. In addition, Uhg4 deletion mutants showed delayed development and decreased viability, and changes in sleep and responses to stress. Whole-genome RNA sequencing of Uhg4 deletion flies and their controls identified co-regulated genes and genetic interaction networks associated with Uhg4. Gene ontology analyses highlighted a broad spectrum of biological processes, including regulation of transcription and translation, morphogenesis, and stress response. CONCLUSION: Uhg4 is a lncRNA essential for reproduction with pleiotropic effects on multiple fitness traits.

BACKGROUND: Important regulation occurs at the level of transcription in Plasmodium falciparum and growing evidence suggests that these apicomplexan parasites have complex regulatory networks. Recent studies implicate long noncoding RNAs (lncRNAs) as transcriptional regulators in P. falciparum. However, due to limited research and the lack of necessary experimental tools, our understanding of their role in the malaria-causing parasite remains largely unelucidated. In this work, we address one of these limitations, the lack of an updated and improved lncRNA annotation in P. falciparum. RESULTS: We generated long-read RNA sequencing data and integrated information extracted and curated from multiple sources to manually annotate lncRNAs. We identified 1119 novel lncRNAs and validated and refined 1250 existing annotations. Utilising the collated datasets, we generated evidence-based ranking scores for each annotation and characterised the distinct genomic contexts and features of P. falciparum lncRNAs. Certain features indicated subsets with potential biological significance such as 25 lncRNAs containing multiple introns, 335 lncRNAs lacking mutations in piggyBac mutagenic studies and lncRNAs associated with specific biologic processes including two new types of lncRNAs found proximal to var genes. CONCLUSIONS: The insights and the annotation presented in this study will serve as valuable tools for researchers seeking to understand the role of lncRNAs in parasite biology through both bioinformatics and experimental approaches.

BACKGROUND: Sitting at the interface of gene expression and host-pathogen interaction, polymerase associated factor 1 complex (PAF1C) is a rising player in the innate immune response. The complex localizes to the nucleus and associates with chromatin to modulate RNA polymerase II (RNAPII) elongation of gene transcripts. Performing this function at both proximal and distal regulatory elements, PAF1C interacts with many host factors across such sites, along with several microbial proteins during infection. Therefore, translating the ubiquity of PAF1C into specific impacts on immune gene expression remains especially relevant. RESULTS: Advancing past work, we treat PAF1 knockout cells with a slate of immune stimuli to identify key trends in PAF1-dependent gene expression with broad analytical depth. From our transcriptomic data, we confirm PAF1 is an activator of traditional immune response pathways as well as other cellular pathways correlated with pathogen defense. With this model, we employ computational approaches to refine how PAF1 may contribute to both gene activation and suppression. Specifically focusing on transcriptional motifs and regulons, we predict gene regulatory elements strongly associated with PAF1, including those implicated in an immune response. Overall, our results suggest PAF1 is involved in innate immunity at several distinct axes of regulation. CONCLUSIONS: By identifying PAF1-dependent gene expression across several pathogenic contexts, we confirm PAF1C to be a key mediator of innate immunity. Combining these transcriptomic profiles with potential regulatory networks corroborates the previously identified functions of PAF1C. With this, we foster new avenues for its study as a regulator of innate immunity, and our results will serve as a basis for targeted study of PAF1C in future validation studies.

BACKGROUND: The identification of gene regulatory networks (GRNs) facilitates the understanding of the underlying molecular mechanism of various biological processes and complex diseases. With the availability of single-cell RNA sequencing data, it is essential to infer GRNs from single-cell expression. Although some GRN methods originally developed for bulk expression data can be applicable to single-cell data and several single-cell specific GRN algorithms were developed, recent benchmarking studies have emphasized the need of developing more accurate and robust GRN modeling methods that are compatible for single-cell expression data. RESULTS: We present SRGS, SPLS (sparse partial least squares)-based recursive gene selection, to infer GRNs from bulk or single-cell expression data. SRGS recursively selects and scores the genes which may have regulations on the considered target gene based on SPLS. When dealing with gene expression data with dropouts, we randomly scramble samples, set some values in the expression matrix to zeroes, and generate multiple copies of data through multiple iterations to make SRGS more robust. We test SRGS on different kinds of expression data, including simulated bulk data, simulated single-cell data without and with dropouts, and experimental single-cell data, and also compared with the existing GRN methods, including the ones originally developed for bulk data, the ones developed specifically for single-cell data, and even the ones recommended by recent benchmarking studies. CONCLUSIONS: It has been shown that SRGS is competitive with the existing GRN methods and effective in the gene regulatory network inference from bulk or single-cell gene expression data. SRGS is available at: https://github.com/JGuan-lab/SRGS .

17 Y-chromosomal STRs which are part of the Yfiler Amplification Kit were investigated in 493 unrelated Pakistani individuals belonging to the Punjabi, Sindhi, Baloch, and Pathan ethnic groups. We have assessed the forensic parameters and population genetic structure for each group. Among the 493 unrelated individuals from four ethnic groups (128 Baloch, 122 Pathan, 108 Punjabi, and 135 Sindhi), 82 haplotypes were observed with haplotype diversity (HD) of 0.9906 in Baloch, 102 haplotypes with HD value of 0.9957 in Pathans, 80 haplotypes with HD value of 0.9924 in Punjabi, and 105 haplotypes with HD value of 0.9945 in the Sindhi population. The overall gene diversity for Baloch, Pathan, Punjabi, and Sindhi populations was 0.6367, 0.6479, 0.6657, and 0.6112, respectively. The results had shown us that Pakistani populations do not have a unique set of genes but share the genetic affinity with regional (Central Asia and Northern India) populations. The observed low gene diversity (heterozygosity) values may be because of endogamy trends and this observation is equally supported by the results of forensic parameters which are mostly static across 4 combinations (minimal STRs, extended 11 Y-STRs, Powerplex 12 Y System, and Yfiler 17 Y-STRs) of STRs in these four populations.

BACKGROUND: Average backfat thickness (BFT) is a critical complex trait in pig and an important indicator for fat deposition and lean rate. Usually, genome-wide association study (GWAS) was used to discover quantitative trait loci (QTLs) of BFT in a single population. However, the power of GWAS is limited by sample size in a single population. Alternatively, meta-analysis of GWAS (metaGWAS) is an attractive method to increase the statistical power by integrating data from multiple breeds and populations. The aim of this study is to identify shared genetic characterization of BFT across breeds in pigs via metaGWAS.  RESULTS: In this study, we performed metaGWAS on BFT using 15,353 pigs (5,143 Duroc, 7,275 Yorkshire, and 2,935 Landrace) from 19 populations. We detected 40 genome-wide significant SNPs (Bonferroni corrected P < 0.05) and defined five breed-shared QTLs in across-breed metaGWAS. Markers within the five QTL regions explained 7 ~ 9% additive genetic variance and showed strong heritability enrichment. Furthermore, by integrating information from multiple bioinformatics databases, we annotated 46 candidate genes located in the five QTLs. Among them, three important (MC4R, PPARD, and SLC27A1) and seven suggestive candidate genes (PHLPP1, NUDT3, ILRUN, RELCH, KCNQ5, ITPR3, and U3) were identified. CONCLUSION: QTLs and candidate genes underlying BFT across breeds were identified via metaGWAS from multiple populations. Our findings contribute to the understanding of the genetic architecture of BFT and the regulating mechanism underlying fat deposition in pigs.

BACKGROUND: Climate change will result in more frequent droughts that can impact soil-inhabiting microbiomes (rhizobiomes) in the agriculturally vital North American perennial grasslands. Rhizobiomes have contributed to enhancing drought resilience and stress resistance properties in plant hosts. In the predicted events of more future droughts, how the changing rhizobiome under environmental stress can impact the plant host resilience needs to be deciphered. There is also an urgent need to identify and recover candidate microorganisms along with their functions, involved in enhancing plant resilience, enabling the successful development of synthetic communities. RESULTS: In this study, we used the combination of cultivation and high-resolution genomic sequencing of bacterial communities recovered from the rhizosphere of a tallgrass prairie foundation grass, Andropogon gerardii. We cultivated the plant host-associated microbes under artificial drought-induced conditions and identified the microbe(s) that might play a significant role in the rhizobiome of Andropogon gerardii under drought conditions. Phylogenetic analysis of the non-redundant metagenome-assembled genomes (MAGs) identified a bacterial genome of interest - MAG-Pseudomonas. Further metabolic pathway and pangenome analyses recovered genes and pathways related to stress responses including ACC deaminase; nitrogen transformation including assimilatory nitrate reductase in MAG-Pseudomonas, which might be associated with enhanced drought tolerance and growth for Andropogon gerardii. CONCLUSIONS: Our data indicated that the metagenome-assembled MAG-Pseudomonas has the functional potential to contribute to the plant host's growth during stressful conditions. Our study also suggested the nitrogen transformation potential of MAG-Pseudomonas that could impact Andropogon gerardii growth in a positive way. The cultivation of MAG-Pseudomonas sets the foundation to construct a successful synthetic community for Andropogon gerardii. To conclude, stress resilience mediated through genes ACC deaminase, nitrogen transformation potential through assimilatory nitrate reductase in MAG-Pseudomonas could place this microorganism as an important candidate of the rhizobiome aiding the plant host resilience under environmental stress. This study, therefore, provided insights into the MAG-Pseudomonas and its potential to optimize plant productivity under ever-changing climatic patterns, especially in frequent drought conditions.

BACKGROUND: DBA/1 mice have a higher susceptibility to generalized audiogenic seizures (AGSz) and seizure-induced respiratory arrest (S-IRA) than C57/BL6 mice. The gene expression profile might be potentially related to this difference. This study aimed to investigate the susceptibility difference in AGSz and S-IRA between DBA/1 and C57BL/6 mice by profiling long noncoding RNAs (lncRNAs) and mRNA expression. METHODS: We compared lncRNAs and mRNAs from the brainstem of the two strains with Arraystar Mouse lncRNA Microarray V3.0 (Arraystar, Rockville, MD). Gene Ontology (GO) and pathway analyses were performed to determine the potentially related biological functions and pathways based on differentially expressed mRNAs. qRT-PCR was carried out to validate the results. RESULTS: A total of 897 lncRNAs and 438 mRNAs were differentially expressed (fold change ≥2, P < 0.05), of which 192 lncRNAs were upregulated and 705 lncRNAs were downregulated. A total of 138 mRNAs were upregulated, and 300 mRNAs were downregulated. In terms of specific mRNAs, Htr5b, Gabra2, Hspa1b and Gfra1 may be related to AGSz or S-IRA. Additionally, lncRNA Neat1 may participate in the difference in susceptibility. GO and pathway analyses suggested that TGF-β signaling, metabolic process and MHC protein complex could be involved in these differences. Coexpression analysis identified 9 differentially expressed antisense lncRNAs and 115 long intergenic noncoding RNAs (lincRNAs), and 2010012P19Rik and its adjacent RNA Tnfsf12-Tnfsf13 may have participated in S-IRA by regulating sympathetic neuron function. The results of the qRT-PCR of five selected lncRNAs (AK038711, Gm11762, 1500004A13Rik, AA388235 and Neat1) and four selected mRNAs (Hspa1b, Htr5b, Gabra2 and Gfra1) were consistent with those obtained by microarray. CONCLUSION: We concluded that TGF-β signaling and metabolic process may contribute to the differential sensitivity to AGSz and S-IRA. Among mRNAs, Htr5b, Gabra2, Hspa1b and Gfra1 could potentially influence the susceptibility. LncRNA Neat1 and 2010012P19Rik may also contribute to the different response to sound stimulation. Further studies should be carried out to explore the underlying functions and mechanisms of differentially expressed RNAs.

BACKGROUND: Plant mitogenomes vary widely in size and genomic architecture. Although hundreds of plant mitogenomes of angiosperm species have already been sequence-characterized, only a few mitogenomes are available from gymnosperms. Silver fir (Abies alba) is an economically important gymnosperm species that is widely distributed in Europe and occupies a large range of environmental conditions. Reference sequences of the nuclear and chloroplast genome of A. alba are available, however, the mitogenome has not yet been assembled and studied. RESULTS: Here, we used paired-end Illumina short reads generated from a single haploid megagametophyte in combination with PacBio long reads from high molecular weight DNA of needles to assemble the first mitogenome sequence of A. alba. Assembly and scaffolding resulted in 11 mitogenome scaffolds, with the largest scaffold being 0.25 Mbp long. Two of the scaffolds displayed a potential circular structure supported by PCR. The total size of the A. alba mitogenome was estimated at 1.43 Mbp, similar to the size (1.33 Mbp) of a draft assembly of the Abies firma mitogenome. In total, 53 distinct genes of known function were annotated in the A. alba mitogenome, comprising 41 protein-coding genes, nine tRNA, and three rRNA genes. The proportion of highly repetitive elements (REs) was 0.168. The mitogenome seems to have a complex and dynamic structure featured by high combinatorial variation, which was specifically confirmed by PCR for the contig with the highest mapping coverage. Comparative analysis of all sequenced mitogenomes of gymnosperms revealed a moderate, but significant positive correlation between mitogenome size and proportion of REs. CONCLUSIONS: The A. alba mitogenome provides a basis for new comparative studies and will allow to answer important structural, phylogenetic and other evolutionary questions. Future long-read sequencing with higher coverage of the A. alba mitogenome will be the key to further resolve its physical structure. The observed positive correlation between mitogenome size and proportion of REs will be further validated once available mitogenomes of gymnosperms would become more numerous. To test whether a higher proportion of REs in a mitogenome leads to an increased recombination and higher structural complexity and variability is a prospective avenue for future research.

BACKGROUND: Bacteriophages are widely considered to be highly abundant and genetically diverse, with their role in the evolution and virulence of many pathogens becoming increasingly clear. Less attention has been paid on phages preying on Bacillus, despite the potential for some of its members, such as Bacillus anthracis, to cause serious human disease. RESULTS: We have isolated five phages infecting the causative agent of anthrax, Bacillus anthracis. Using modern phylogenetic approaches we place these five new Bacillus phages, as well as 21 similar phage genomes retrieved from publicly available databases and metagenomic datasets into the Tyrovirus group, a newly proposed group named so due to the conservation of three distinct tyrosine recombinases. Genomic analysis of these large phages (~ 160-170 kb) reveals their DNA packaging mechanism and genomic features contributing to virion morphogenesis, host cell lysis and phage DNA replication processes. Analysis of the three tyrosine recombinases suggest Tyroviruses undergo a prophage lifecycle that may involve both host integration and plasmid stages. Further we show that Tyroviruses rely on divergent invasion mechanisms, with a subset requiring host S-layer for infection. CONCLUSIONS: Ultimately, we expand upon our understanding on the classification, phylogeny, and genomic organisation of a new and substantial phage group that prey on critically relevant Bacillus species. In an era characterised by a rapidly evolving landscape of phage genomics the deposition of future Tyroviruses will allow the further unravelling of the global spread and evolutionary history of these Bacillus phages.

Cadmium (Cd) is a highly toxic pollutant in soil and water that severely hampers the growth and reproduction of plants. Phytoremediation has been presented as a cost-effective and eco-friendly method for addressing heavy metal pollution. However, phytoremediation is restricted by the limited number of accumulators and the unknown mechanisms underlying heavy metal tolerance. In this study, we demonstrated that Erigeron canadensis (Asteraceae), with its strong adaptability, is tolerant to intense Cd stress (2 mmol/L CdCl2 solution). Moreover, E. canadensis exhibited a strong ability to accumulate Cd2+ when treated with CdCl2 solution. The activity of some antioxidant enzymes, as well as the malondialdehyde (MDA) level, was significantly increased when E. canadensis was treated with different CdCl2 solutions (0.5, 1, 2 mmol/L CdCl2). We found high levels of superoxide dismutase (SOD) and ascorbate peroxidase (APX) activities under 1 mmol/L CdCl2 treatment. Comparative transcriptomic analysis identified 5,284 differentially expressed genes (DEGs) in the roots and 3,815 DEGs in the shoots after E. canadensis plants were exposed to 0.5 mM Cd. Functional annotation of key DEGs indicated that signal transduction, hormone response, and reactive oxygen species (ROS) metabolism responded significantly to Cd. In particular, the DEGs involved in auxin (IAA) and ethylene (ETH) signal transduction were overrepresented in shoots, indicating that these genes are mainly involved in regulating plant growth and thus likely responsible for the Cd tolerance. Overall, these results not only determined that E. canadensis can be used as a potential accumulator of Cd but also provided some clues regarding the mechanisms underlying heavy metal tolerance.

BACKGROUND: Infectious Salmon Anaemia virus (ISAV) is an orthomyxovirus responsible for large losses in Atlantic salmon (Salmo salar) aquaculture. Current available treatments and vaccines are not fully effective, and therefore selective breeding to produce ISAV-resistant strains of Atlantic salmon is a high priority for the industry. Genomic selection and potentially genome editing can be applied to enhance the disease resistance of aquaculture stocks, and both approaches can benefit from increased knowledge on the genomic mechanisms of resistance to ISAV. To improve our understanding of the mechanisms underlying resistance to ISAV in Atlantic salmon we performed a transcriptomic study in ISAV-infected salmon with contrasting levels of resistance to this virus. RESULTS: Three different tissues (gills, head kidney and spleen) were collected on 12 resistant and 12 susceptible fish at three timepoints (pre-challenge, 7 and 14 days post challenge) and RNA sequenced. The transcriptomes of infected and non-infected fish and of resistant and susceptible fish were compared at each timepoint. The results show that the responses to ISAV are organ-specific; an important response to the infection was observed in the head kidney, with up-regulation of immune processes such as interferon and NLR pathways, while in gills and spleen the response was more moderate. In addition to immune related genes, our results suggest that other processes such as ubiquitination and ribosomal processing are important during early infection with ISAV. Moreover, the comparison between resistant and susceptible fish has also highlighted some interesting genes related to ubiquitination, intracellular transport and the inflammasome. CONCLUSIONS: Atlantic salmon infection by ISAV revealed an organ-specific response, implying differential function during the infection. An immune response was observed in the head kidney in these early timepoints, while gills and spleen showed modest responses in comparison. Comparison between resistance and susceptible samples have highlighted genes of interest for further studies, for instance those related to ubiquitination or the inflammasome.

BACKGROUND: The brown adipose tissue (BAT) is a target for treating obesity. BAT losses thermogenic capacity and gains a "white adipose tissue-like" phenotype ("BAT whitening") under thermoneutral environments, which is a potential factor causing a low curative effect in BAT-related obesity treatments. Circular RNAs (circRNAs) and long non-coding RNAs (lncRNAs) can act as competing endogenous RNAs (ceRNA) to mRNAs and function in various processes by sponging shared microRNAs (miRNAs). However, the roles of circRNA- and lncRNA-related ceRNA networks in regulating BAT whitening remain litter known. RESULTS: In this study, BATs were collected from rabbits at day0 (D0), D15, D85, and 2 years (Y2). MiRNA-seq was performed to investigate miRNA changes during BAT whitening. Then, a combined analysis of circRNA-seq and whole-transcriptome sequencing was used for circRNA assembly and quantification during BAT whitening. Our data showed that 1187 miRNAs and 6204 circRNAs were expressed in the samples, and many of which were identified as significantly changed during BAT whitening. Target prediction showed that D0-selective miRNAs were significantly enriched in the Ras, MAPK, and PI3K-Akt signaling pathways, and Y2-selective miRNAs were predicted to be involved in cell proliferation. The cyclization of several circRNAs could form novel response elements of key thermogenesis miRNAs at the back-splicing junction (BSJ) sites, and in combination with a dual-luciferase reporter assay confirmed the binding between the BSJ site of novel_circ_0013792 and ocu-miR-378-5p. CircRNAs and lncRNAs have high cooperativity in sponging miRNAs during BAT whitening. Both circRNA-miRNA-mRNA and lncRNA-miRNA-mRNA triple networks were significantly involved in immune response-associated biological processes. The D15-selective circRNA might promote BAT whitening by increasing the expression of IDH2. The Y2-selective circRNA-related ceRNA network and lncRNA-related ceRNA network might regulate the formation of the WAT-like phenotype of BAT via MAPK and Ras signaling pathways, respectively. CONCLUSIONS: Our work systematically revealed ceRNA networks during BAT whitening in rabbits and might provide new insight into BAT-based obesity treatments.

BACKGROUND: Potential functional variants (PFVs) can be defined as genetic variants responsible for a given phenotype. Ultimately, these are the best DNA markers for animal breeding and selection, especially for polygenic and complex phenotypes. Herein, we described the identification of PFVs for complex phenotypes (in this case, Feed Efficiency in beef cattle) using a systems-biology driven approach based on RNA-seq data from physiologically relevant organs. RESULTS: The systems-biology coupled with deep molecular phenotyping by RNA-seq of liver, muscle, hypothalamus, pituitary, and adrenal glands of animals with high and low feed efficiency (FE) measured by residual feed intake (RFI) identified 2,000,936 uniquely variants. Among them, 9986 variants were significantly associated with FE and only 78 had a high impact on protein expression and were considered as PFVs. A set of 169 significant uniquely variants were expressed in all five organs, however, only 27 variants had a moderate impact and none of them a had high impact on protein expression. These results provide evidence of tissue-specific effects of high-impact PFVs. The PFVs were enriched (FDR < 0.05) for processing and presentation of MHC Class I and II mediated antigens, which are an important part of the adaptive immune response. The experimental validation of these PFVs was demonstrated by the increased prediction accuracy for RFI using the weighted G matrix (ssGBLUP+wG; Acc = 0.10 and b = 0.48) obtained in the ssGWAS in comparison to the unweighted G matrix (ssGBLUP; Acc = 0.29 and b = 1.10). CONCLUSION: Here we identified PFVs for FE in beef cattle using a strategy based on systems-biology and deep molecular phenotyping. This approach has great potential to be used in genetic prediction programs, especially for polygenic phenotypes.

BACKGROUND: Squamous promoter binding protein-like (SPL) proteins are a class of transcription factors that play essential roles in plant growth and development, signal transduction, and responses to biotic and abiotic stresses. The rapid development of whole genome sequencing has enabled the identification and characterization of SPL gene families in many plant species, but to date this has not been performed in quinoa (Chenopodium quinoa). RESULTS: This study identified 23 SPL genes in quinoa, which were unevenly distributed on 18 quinoa chromosomes. Quinoa SPL genes were then classified into eight subfamilies based on homology to Arabidopsis thaliana SPL genes. We selected three dicotyledonous and monocotyledonous representative species, each associated with C. quinoa, for comparative sympatric mapping to better understand the evolution of the developmental mechanisms of the CqSPL family. Furthermore, we also used 15 representative genes from eight subfamilies to characterize CqSPLs gene expression in different tissues and at different fruit developmental stages under six different abiotic stress conditions. CONCLUSIONS: This study, the first to identify and characterize SPL genes in quinoa, reported that CqSPL genes, especially CqSPL1, play a critical role in quinoa development and in its response to various abiotic stresses.

BACKGROUND: Most susceptible loci of hepatocellular carcinoma (HCC) identified by genome-wide association studies (GWAS) are located in non-coding regions, and the mechanism of action remains unclear. The objective of this study was to explore the association of single nucleotide polymorphisms (SNPs) on long non-coding RNAs (lncRNAs) that affect competing endogenous RNAs (ceRNA) regulation mechanism with the risk and prognosis of HCC. METHODS: Based on a set of bioinformatics strategies, eight lncRNA genes that affect HCC through the mechanism of lncRNA-mediated ceRNA were systematically screened, and 15 SNPs that affect microRNA (miRNA) binding in these lncRNA genes were annotated. Genotyping was performed in 800 HCC cases and 801 healthy controls to examine associations of these SNPs with HCC in a northeastern Chinese Han population. RESULTS: The GG, GC and GG + GC genotypes of HOTAIR rs7958904 were associated with a 0.65, 0.59 and 0.63-fold decreased HCC risk, respectively. In addition, HCC patients with PVT1 rs3931282 AA + GA genotypes were less prone to develop late-stage cancers in a stratified analysis of clinical characteristics. When stratified by clinical biochemical indexes, rs1134492 and rs10589312 in PVT1 and rs84557 in EGFR-AS1 showed significant associations with aspartate aminotransferase (AST), alanine aminotransferase (ALT) or AST/ALT ratio in HCC patients. Furthermore, we constructed potential ceRNA regulatory axes that might be affected by five positive SNPs to explain the causes of these genetic associations. CONCLUSIONS: HOTAIR rs7958904, PVT1 rs3931282, rs1134492 and rs10589312, and EGFR-AS1 rs84557 might be predictors for HCC risk or prognosis. Our results provide new insights into how SNPs on lncRNA-mediated ceRNAs confer interindividual differences to occurrence and progression of HCC.

BACKGROUND: Pre-harvest sprouting (PHS) is one of the most serious rice production constraints in areas where prolonged rainfall occurs during harvest. However, the molecular mechanisms of transcriptional regulation underlying PHS remain largely unknown. RESULTS: In the current study, comparative transcriptome analyses were performed to characterize the similarities and differences between two rice varieties: PHS-sensitive Jiuxiangzhan (JXZ) and PHS-resistant Meixiangxinzhan (MXXZ). The physiological experimental results indicated that PHS causes a significant decrease in starch content and, in contrast, a significant increase in soluble sugar content and amylase activity. The extent of change in these physiological parameters in the sensitive variety JXZ was greater than that in the resistant variety MXXZ. A total of 9,602 DEGs were obtained from the transcriptome sequencing data, and 5,581 and 4,021 DEGs were identified in JXZ and MXXZ under high humidity conditions, respectively. The KEGG pathway enrichment analysis indicated that many DEGs under high humidity treatment were mainly linked to plant hormone signal transduction, carbon metabolism, starch and sucrose metabolism, and phenylpropanoid biosynthesis. Furthermore, the number of upregulated genes involved in these pathways was much higher in JXZ than in MXXZ, while the number of downregulated genes was higher in MXXZ than in JXZ. These results suggest that the physiological and biochemical processes of these pathways are more active in the PHS-sensitive JXZ than in the PHS-resistant MXXZ. CONCLUSION: Based on these results, we inferred that PHS in rice results from altered phytohormone regulation, more active carbon metabolism and energy production, and enhanced phenylpropanoid biosynthesis. Our study provides a theoretical foundation for further elucidation of the complex regulatory mechanism of PHS in rice and the molecular breeding of PHS-resistant rice varieties.

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of paratuberculosis. As a potential zoonotic pathogen, MAP also seriously threatens human health and social security. At present, long non-coding RNA (lncRNA) has attracted wide attention as an useful biomarker in various diseases. Therefore, our study analyzed the lncRNA expression profiles and lncRNA-mRNA regulatory network of MAP infected bovine monocytes-macrophages and uninfected bovine cells by high-throughput sequencing. A total of 4641 differentially expressed lncRNAs genes were identified, including 3111 up-regulated genes and 1530 down-regulated genes. In addition, lncRNA-mRNA interaction analysis was performed to predict the target genes of lncRNA. Among them, after MAP infection, 86 lncRNAs targeted to mRNA, of which only 6 genes were significantly different. The results of Gene Ontology (GO) enrichment analysis showed that the differentially expressed genes significantly enriched in functional groups were related to immune regulation. Multiple signal pathways including NF-κB, NOD-like receptor, Cytokine-cytokine receptor, Toll-like receptor signaling pathway, Chemokine signaling pathway, and other important biochemical, metabolic and signal transduction pathways were enriched in Kyoto Encyclopedia of Genes and Genomes (KEGG). In this study, analysis of macrophage transcriptomes in response to MAP infection is expected to provide key information to deeply understand role of the pathogen in initiating an inappropriate and persistent infection in susceptible hosts and molecular mechanisms that might underlie the early phases of paratuberculosis.

BACKGROUND: The transcriptional changes around zygotic genome activation (ZGA) in preimplantation embryos are critical for studying mechanisms of embryonic developmental arrest and searching for key transcription factors. However, studies on the transcription profile of porcine ZGA are limited. RESULTS: In this study, we performed RNA sequencing in porcine in vivo developed (IVV) and somatic cell nuclear transfer (SCNT) embryo at different stages and compared the transcriptional activity of porcine embryos with mouse, bovine and human embryos. The results showed that the transcriptome map of the early porcine embryos was significantly changed at the 4-cell stage, and 5821 differentially expressed genes (DEGs) in SCNT embryos failed to be reprogrammed or activated during ZGA, which mainly enrichment to metabolic pathways. c-MYC was identified as the highest expressed transcription factor during ZGA. By treating with 10,058-F4, an inhibitor of c-MYC, the cleavage rate (38.33 ± 3.4%) and blastocyst rate (23.33 ± 4.3%) of porcine embryos were significantly lower than those of the control group (50.82 ± 2.7% and 34.43 ± 1.9%). Cross-species analysis of transcriptome during ZGA showed that pigs and bovines had the highest similarity coefficient in biological processes. KEGG pathway analysis indicated that there were 10 co-shared pathways in the four species. CONCLUSIONS: Our results reveal that embryos with impaired developmental competence may be arrested at an early stage of development. c-MYC helps promote ZGA and preimplantation embryonic development in pigs. Pigs and bovines have the highest coefficient of similarity in biological processes during ZGA. This study provides an important reference for further studying the reprogramming regulatory mechanism of porcine embryos during ZGA.

BACKGROUND: Although knowledge of the sizes, contents, and forms of plant mitochondrial genomes (mitogenomes) is increasing, little is known about the mechanisms underlying their structural diversity. Evolutionary information on the mitogenomes of Primula, an important ornamental taxon, is more limited than the information on their nuclear and plastid counterparts, which has hindered the comprehensive understanding of Primula mitogenomic diversity and evolution. The present study reported and compared three Primula mitogenomes and discussed the size expansion of mitogenomes in Ericales. RESULTS: Mitogenome master circles were sequenced and successfully assembled for three Primula taxa and were compared with publicly available Ericales mitogenomes. The three mitogenomes contained similar gene contents and varied primarily in their structures. The Primula mitogenomes possessed relatively high nucleotide diversity among all examined plant lineages. In addition, high nucleotide diversity was found among Primula species between the Mediterranean and Himalaya-Hengduan Mountains. Most predicted RNA editing sites appeared in the second amino acid codon, increasing the hydrophobic character of the protein. An early stop in atp6 caused by RNA editing was conserved across all examined Ericales species. The interfamilial relationships within Ericales and interspecific relationships within Primula could be well resolved based on mitochondrial data. Transfer of the two longest mitochondrial plastid sequences (MTPTs) occurred before the divergence of Primula and its close relatives, and multiple independent transfers could also occur in a single MTPT sequence. Foreign sequence [MTPTs and mitochondrial nuclear DNA sequences (NUMTs)] uptake and repeats were to some extent associated with changes in Ericales mitogenome size, although none of these relationships were significant overall. CONCLUSIONS: The present study revealed relatively conserved gene contents, gene clusters, RNA editing, and MTPTs but considerable structural variation in Primula mitogenomes. Relatively high nucleotide diversity was found in the Primula mitogenomes. In addition, mitogenomic genes, collinear gene clusters, and locally collinear blocks (LCBs) all showed phylogenetic signals. The evolutionary history of MTPTs in Primula was complicated, even in a single MTPT sequence. Various reasons for the size variation observed in Ericales mitogenomes were found.

BACKGROUND: Elucidating the phylogenetic relationships within species-rich genera is essential but challenging, especially when lineages are assumed to have been going through radiation events. Mahonia Nutt. (Berberidaceae) is a genus with cosmopolitan distribution, comprising approximately 100 species, two of which are known as Caulis Mahoniae (M. bealei and M. fortunei) with crucial pharmacological significance in Chinese herbal medicine. Mahonia is a taxonomically challenging genus, and intrageneric phylogenetic relationships still need to be explored using genome data. Universal DNA barcodes and floral morphological attributes have limited discriminatory power in Mahonia. RESULTS: We sequenced 17 representative plastomes and integrated three published plastome data together to conduct comparative and phylogenetic analyses. We found that Mahonia and Berberis share a large IR expansion (~ 12 kb), which is recognized as a typical character of Berberideae. Repeated sequences are revealed in the species of Mahonia, which are valuable for further population genetic studies. Using a comparative plastome analysis, we determined eight hypervariable regions whose discriminative power is comparable to that of the whole plastid genomes. The incongruence of the ITS and the plastome tree topologies may be ascribed to ancestral hybridization events and/or to incomplete lineage sorting. In addition, we suggest that leaf epidermal characters could help to distinguish closely related species in Mahonia. CONCLUSIONS: We propose an integrative approach combining special barcodes and micromorphological traits to circumscribe Mahonia species. The results cast a new light on the development of an integrative method for accurate species circumscription and provide abundant genetic resources for further research on Mahonia.

BACKGROUND: Maize-infecting viruses are known to inflict significant agronomic yield loss throughout the world annually. Identification of known or novel causal agents of disease prior to outbreak is imperative to preserve food security via future crop protection efforts. Toward this goal, a large-scale metagenomic approach utilizing high throughput sequencing (HTS) was employed to identify novel viruses with the potential to contribute to yield loss of graminaceous species, particularly maize, in North America. RESULTS: Here we present four novel viruses discovered by HTS and individually validated by Sanger sequencing. Three of these viruses are RNA viruses belonging to either the Betaflexiviridae or Tombusviridae families. Additionally, a novel DNA virus belonging to the Geminiviridae family was discovered, the first Mastrevirus identified in North American maize. CONCLUSIONS: Metagenomic studies of crop and crop-related species such as this may be useful for the identification and surveillance of known and novel viral pathogens of crops. Monitoring related species may prove useful in identifying viruses capable of infecting crops due to overlapping insect vectors and viral host-range to protect food security.

BACKGROUND: The first metazoan genome sequenced, that of Caenorhabditis elegans, has motivated animal genome evolution studies. To date > 50 species from the genus Caenorhabditis have been sequenced, allowing research on genome variation. RESULTS: In the present study, we describe a new gonochoristic species, Caenorhabditis niphades n. sp., previously referred as C. sp. 36, isolated from adult weevils (Niphades variegatus), with whom they appear to be tightly associated during its life cycle. Along with a species description, we sequenced the genome of C. niphades n. sp. and produced a chromosome-level assembly. A genome comparison highlighted that C. niphades n. sp. has the smallest genome (59 Mbp) so far sequenced in the Elegans supergroup, despite being closely related to a species with an exceptionally large genome, C. japonica. CONCLUSIONS: The compact genome of C. niphades n. sp. can serve as a key resource for comparative evolutionary studies of genome and gene number expansions in Caenorhabditis species.

BACKGROUND: Rectal cancer (RC) is one of the most common malignant tumors. Ferroptosis is an iron-dependent form of cell death, which plays an important role in various cancers. However, the correlation between ferroptosis-related genes (FRGs) and prognosis in RC remains unclear. METHODS: Gene expression data from The Cancer Genome Atlas Rectum adenocarcinoma (TCGA-READ) and GSE87211 were downloaded. Clustering and functional enrichment were evaluated. A FRGs risk score was established based on the univariate Cox analysis and the Least absolute shrinkage and selection operator (LASSO) analysis. K-M analysis and ROC analysis were conducted to determine prognostic values. qRT-PCR was performed to validate levels of mRNA expression. Multivariate Cox analysis was used to build a prognostic prediction model based on the risk score. RESULTS: Based on FRGs, RC patients were grouped into two clusters. In the functional enrichment of differentially expressed genes between the two clusters, immune-related pathways dominated. A novel FRGs signature with 14 genes related to the overall survival (OS) of RC was established. qRT-PCR of the 14 genes identified TP63, ISCU, PLIN4, MAP3K5, OXSR, FANCD2 and ATM were overexpressed in RC tissue; HSPB1, MAPK1, ABCC1, PANX1, MAPK9 and ATG7 were underexpressed; TUBE1 had no difference. The high-risk group had a significantly lower OS than the low-risk group (P < 0.001), and ROC curve analysis confirmed the signature's predictive capacity. Multivariate analysis demonstrated that the risk score and age were independent prognostic factors. CONCLUSION: A novel FRGs model can be used to predict the prognosis in RC, as well as to guide individual treatment.

BACKGROUND: Protoporphyrin IX (Pp IX) is the primary pigment for brown eggshells. However, the regulatory mechanisms directing Pp IX synthesis, transport, and genetic regulation during eggshell calcification in chickens remain obscure. In this study, we investigated the mechanism of brown eggshell formation at different times following oviposition, using White Leghorn hens (WS group), Rhode Island Red light brown eggshell line hens (LBS group) and Rhode Island Red dark brown eggshell line hens (DBS group). RESULTS: At 4, 16 and 22 h following oviposition, Pp IX concentrations in LBS and DBS groups were significantly higher in shell glands than in liver (P < 0.05). Pp IX concentrations in shell glands of LBS and DBS groups at 16 and 22 h following oviposition were significantly higher than WS group (P < 0.05). In comparative transcriptome analysis, δ-aminolevulinate synthase 1 (ALAS1), solute carrier family 25 member 38 (SLC25A38), ATP binding cassette subfamily G member 2 (ABCG2) and feline leukemia virus subgroup C cellular receptor 1 (FLVCR1), which were associated with Pp IX synthesis, were identified as differentially expressed genes (DEGs). RT-qPCR results showed that the expression level of ALAS1 in shell glands was significantly higher in DBS group than in WS group at 16 and 22 h following oviposition (P < 0.05). In addition, four single nucleotide polymorphisms (SNPs) in ALAS1 gene that were significantly associated with eggshell brownness were identified. By identifying the differential metabolites in LBS and DBS groups, we found 11-hydroxy-E4-neuroprostane in shell glands and 15-dehydro-prostaglandin E1(1-) and prostaglandin G2 2-glyceryl ester in uterine fluid were related to eggshell pigment secretion. CONCLUSIONS: In this study, the regulatory mechanisms of eggshell brownness were studied comprehensively by different eggshell color and time following oviposition. Results show that Pp IX is synthesized de novo and stored in shell gland, and ALAS1 is a key gene regulating Pp IX synthesis in the shell gland. We found three transporters in Pp IX pathway and three metabolites in shell glands and uterine fluid that may influence eggshell browning.

BACKGROUND: Retained placenta (RP) is a prevalent disorder in cattle with many health-related and economic costs for the farm owners. Its etiology has not been clarified yet and there is no definite therapy for this disorder. In this study we conducted RNA-seq, hematologic and histologic experiments to survey the causes of RP development. METHODS: Blood samples were collected from 4 RP and 3 healthy cows during periparturtion period for hematological assessments followed by placentome sampling within 30 min after parturition. Cows were grouped as RP and control in case the placenta was retained or otherwise expelled, respectively. Total RNA was extracted from placentome samples followed by RNA-sequencing. RESULTS: We showed 240 differentially expressed genes (DEGs) between the RP and control groups. Enrichment analyzes indicated immune system and lipid metabolism as prominent over- and under-represented pathways in RP cows, respectively. Hormonal assessments showed that estradiol-17β (E2) was lower and cortisol tended to be higher in RP cows compared to controls at the day of parturition. Furthermore, histologic experiment showed that villi-crypt junctions remain tighter in RP cows compared to controls and the crypts layer seemed thicker in the placentome of RP cows. Complete blood cell (CBC) parameters were not significantly different between the two groups. CONCLUSION: Overall, DEGs derived from expression profiling and these genes contributed to enrichment of immune and lipid metabolism pathways. We suggested that E2 could be involved in development of RP and the concentrations of P4 and CBC counts periparturition might not be a determining factor.

BACKGROUND: The Kurds as an ethnic group are believed to be a combination of earlier Indo-European tribes who migrated and inhabited a mountainous area thousands of years ago. However, as it is difficult to describe the precise history of their origin, it is necessary to investigate their population relationship with other geographical and ethnic groups. RESULTS: Seventeen Short Tandem Repeat markers on the Y chromosome (Y-STR) included in the AmpFLSTR™ Yfiler™ PCR Amplification Kit (Thermo Fisher Scientific, USA) were used to type DNA samples from the Sorani (Central) Kurdish population in Sulaymaniyah province. One hundred fifty-seven haplotypes were obtained from 162 unrelated male individuals. The highest and lowest gene diversities were DYS385a/b (GD = 0.848) and DYS392 (GD = 0.392), respectively. The haplotypes were used to predict the most likely haplogroups in the Sulaymaniyah population. CONCLUSION: Haplogroup prediction indicated predominance (28%) of subclade J2 (44/157) in the Sorani Kurds, northeast of Iraq. The pairwise genetic distance results showed that the Kurdish group clustered along with Asian populations, whereas the furthest countries were Europeans and Africans.

BACKGROUND: Older adults are more prone to develop systemic dehydration. Systemic dehydration has implications for vocal fold biology by affecting gene and protein expression. The objective of this study was to quantify vocal fold protein changes between two age groups and hydration status, and to investigate the interaction of age and hydration status on protein expression, which has not been investigated in the context of vocal folds before. Comparative proteomics was used to analyze the vocal fold proteome of 6.5-month-old and > 3-year-old rabbits subjected to water ad libitum or water volume restriction protocol. RESULTS: Young and older adult rabbits (n = 22) were either euhydrated (water ad libitum) or dehydrated by water volume restriction. Dehydration was confirmed by body weight loss of - 5.4% and - 4.6% in young and older groups, respectively, and a 1.7-fold increase of kidney renin gene expression in the young rabbits. LC-MS/MS identified 2286 proteins in the rabbit vocal folds of young and older adult rabbits combined. Of these, 177, 169, and 81 proteins were significantly (p ≤ 0.05) affected by age, hydration status, or the interaction of both factors, respectively. Analysis of the interaction effect revealed 32 proteins with opposite change patterns after dehydration between older and young rabbit vocal folds, while 31 proteins were differentially regulated only in the older adult rabbits and ten only in the young rabbits in response to systemic dehydration. The magnitude of changes for either up or downregulated proteins was higher in the older rabbits. These proteins are predominantly related to structural components of the extracellular matrix and muscle layer, suggesting a disturbance in the viscoelastic properties of aging vocal fold tissue, especially when subjected to systemic dehydration. CONCLUSIONS: Water restriction is a laboratory protocol to assess systemic dehydration-related changes in the vocal fold tissue that is translatable to human subjects. Our findings showed a higher number of proteins differentially regulated with a greater magnitude of change in the vocal folds of older adult rabbits in the presence of systemic dehydration compared to younger rabbits. The association of these proteins with vocal fold structure and biomechanical properties suggests that older human subjects may be more vulnerable to the effects of systemic dehydration on vocal function. The clinical implications of these protein changes warrant more investigation, but age should be taken into consideration when evaluating vocal treatment recommendations that interfere with body fluid balance.

BACKGROUND: Mytilidae, also known as marine mussels, are widely distributed in the oceans worldwide. Members of Mytilidae show a tremendous range of ecological adaptions, from the species distributed in freshwater to those that inhabit in deep-sea. Mitochondria play an important role in energy metabolism, which might contribute to the adaptation of Mytilidae to different environments. In addition, some bivalve species are thought to lack the mitochondrial protein-coding gene ATP synthase F0 subunit 8. Increasing studies indicated that the absence of atp8 may be caused by annotation difficulties for atp8 gene is characterized by highly divergent, variable length. RESULTS: In this study, the complete mitochondrial genomes of three marine mussels (Xenostrobus securis, Bathymodiolus puteoserpentis, Gigantidas vrijenhoeki) were newly assembled, with the lengths of 14,972 bp, 20,482, and 17,786 bp, respectively. We annotated atp8 in the sequences that we assembled and the sequences lacking atp8. The newly annotated atp8 sequences all have one predicted transmembrane domain, a similar hydropathy profile, as well as the C-terminal region with positively charged amino acids. Furthermore, we reconstructed the phylogenetic trees and performed positive selection analysis. The results showed that the deep-sea bathymodiolines experienced more relaxed evolutionary constraints. And signatures of positive selection were detected in nad4 of Limnoperna fortunei, which may contribute to the survival and/or thriving of this species in freshwater. CONCLUSIONS: Our analysis supported that atp8 may not be missing in the Mytilidae. And our results provided evidence that the mitochondrial genes may contribute to the adaptation of Mytilidae to different environments.

BACKGROUND: The cold pressor test (CPT) is a widely used pain provocation test to investigate both pain tolerance and cardiovascular responses. We hypothesize, that performing multi-omic analyses during CPT gives the opportunity to home in on molecular mechanisms involved. Twenty-two females were phenotypically assessed before and after a CPT, and blood samples were taken. RNA-Sequencing, steroid profiling and untargeted metabolomics were performed. Each 'omic level was analyzed separately at both single-feature and systems-level (principal component [PCA] and partial least squares [PLS] regression analysis) and all 'omic levels were combined using an integrative multi-omics approach, all using the paired-sample design. RESULTS: We showed that PCA was not able to discriminate time points, while PLS did significantly distinguish time points using metabolomics and/or transcriptomic data, but not using conventional physiological measures. Transcriptomic and metabolomic data revealed at feature-, systems- and integrative- level biologically relevant processes involved during CPT, e.g. lipid metabolism and stress response. CONCLUSION: Multi-omics strategies have a great potential in pain research, both at feature- and systems- level. Therefore, they should be exploited in intervention studies, such as pain provocation tests, to gain knowledge on the biological mechanisms involved in complex traits.

The effect of DNA methylation on the regulation of gene expression has been extensively discussed in the literature. However, the potential association between DNA methylation and alternative splicing is not understood well. In this study, we integrated multiple omics data types from The Cancer Genome Atlas (TCGA) and systematically examined the relationship between DNA methylation and alternative splicing. Using the methylation data and exon expression data, we identified many CpG sites significantly associated with exon expression in various types of cancers. We further observed that the direction and strength of significant CpG-exon correlation tended to be consistent across different cancer contexts, indicating that some CpG-exon correlation patterns reflect fundamental biological mechanisms that transcend tissue- and cancer- types. We also discovered that CpG sites correlated with exon expressions were more likely to be associated with patient survival outcomes compared to CpG sites that did not correlate with exon expressions. Furthermore, we found that CpG sites were more strongly correlated with exon expression than expression of isoforms harboring the corresponding exons. This observation suggests that a major effect of CpG methylation on alternative splicing may be related to the inclusion or exclusion of exons, which subsequently impacts the relative usage of various isoforms. Overall, our study revealed correlation patterns between DNA methylation and alternative splicing, which provides new insights into the role of methylation in the transcriptional process.

BACKGROUND: Hemiptera is one of the most speciose orders of insects, and the most speciose considering Hemimetabola. Through their evolutive history, hemipterans with different feeding habits have adapted to deal with different chemical challenges. Three major gene families are involved in xenobiotic detoxification in insects: the cytochromes P450 (CYPs), carboxyl/cholinesterases (CCEs), and glutathione transferases (GSTs). Here we perform a comparative analysis on the complement of these gene superfamilies across five hemipteran species; four heteropterans (the pentatomid plant feeders Nezara viridula and Halyomorpha halys; the hematophagous Cimex lectularius, Cimicidae, and Rhodnius prolixus, Reduviidae), and one Auchenorrhyncha plant feeder (Nilaparvata lugens). RESULTS: Our results point to an expansion of several enzyme families associated with xenobiotic detoxification in heteropterans with respect to other species and the existence of a dynamic evolution pattern including CYP3 clan, hormone and pheromone processing class in the CCE superfamily, and sigma class in GST superfamily. Other detoxification-related families are reduced in the hemipteran species analyzed here: reduction or even absence of epsilon class and reduced delta class in GST superfamily; absence of mitochondrial CYP12 family; absence of CYP9 family in CYP3 clan; and reduction or even absence of some dietary/detoxification groups of CCEs. Interestingly, the most polyphagous species analyzed here (H. halys) is also the one that presents the largest repertoire of detoxification enzymes. Gene cluster analysis suggests that this could be due to gene duplication events. CONCLUSIONS: The evolutionary analysis performed here reveals characteristics that are both common and particular for heteropterans. The composition and organization of detoxification-related gene families could shed light on evolutionary forces that shaped their divergence. These families are important for both the detoxification of diet products and for conferring tolerance or resistance to synthetic insecticides. Furthermore, we present the first comprehensive analysis of detoxification gene superfamilies in N. viridula, an understudied species in spite of its economic relevance as a crop pest. The information obtained is of interest for basic insect science as well as for the control of harmful species and the management of insecticide resistance.

BACKGROUND: In eukaryotes, cell-to-cell communication relies on the activity of small signaling peptides. In plant genomes, many hundreds of genes encode for such short peptide signals. However, only few of them are functionally characterized and due to the small gene size and high sequence variability, the comprehensive identification of such peptide-encoded genes is challenging. The CLAVATA3 (CLV3)/EMBRYO SURROUNDING REGION-RELATED (CLE) gene family encodes for short peptides that have a role in plant meristem maintenance, vascular patterning and responses to environment. The full repertoire of CLE genes and the role of CLE signaling in tomato (Solanum lycopersicum)- one of the most important crop plants- has not yet been fully studied. RESULTS: By using a combined approach, we performed a genome-wide identification of CLE genes using the current tomato genome version SL 4.0. We identified 52 SlCLE genes, including 37 new non annotated before. By analyzing publicly available RNAseq datasets we could confirm the expression of 28 new SlCLE genes. We found that SlCLEs are often expressed in a tissue-, organ- or condition-specific manner. Our analysis shows an interesting gene diversification within the SlCLE family that seems to be a result of gene duplication events. Finally, we could show a biological activity of selected SlCLE peptides in the root growth arrest that was SlCLV2-dependent. CONCLUSIONS: Our improved combined approach revealed 37 new SlCLE genes. These findings are crucial for better understanding of the CLE signaling in tomato. Our phylogenetic analysis pinpoints the closest homologs of Arabidopsis CLE genes in tomato genome and can give a hint about the function of newly identified SlCLEs. The strategy described here can be used to identify more precisely additional short genes in plant genomes. Finally, our work suggests that the mechanism of root-active CLE peptide perception is conserved between Arabidopsis and tomato. In conclusion, our work paves the way to further research on the CLE-dependent circuits modulating tomato development and physiological responses.

BACKGROUND: Although a number of imprinted genes are known to be highly expressed in the brain, and in certain brain regions in particular, whether they are truly over-represented in the brain has never been formally tested. Using thirteen single-cell RNA sequencing datasets we systematically investigated imprinted gene over-representation at the organ, brain region, and cell-specific levels. RESULTS: We established that imprinted genes are indeed over-represented in the adult brain, and in neurons particularly compared to other brain cell-types. We then examined brain-wide datasets to test enrichment within distinct brain regions and neuron subpopulations and demonstrated over-representation of imprinted genes in the hypothalamus, ventral midbrain, pons and medulla. Finally, using datasets focusing on these regions of enrichment, we identified hypothalamic neuroendocrine populations and the monoaminergic hindbrain neurons as specific hotspots of imprinted gene expression. CONCLUSIONS: These analyses provide the first robust assessment of the neural systems on which imprinted genes converge. Moreover, the unbiased approach, with each analysis informed by the findings of the previous level, permits highly informed inferences about the functions on which imprinted gene expression converges. Our findings indicate the neuronal regulation of motivated behaviours such as feeding and sleep, alongside the regulation of pituitary function, as functional hotspots for imprinting. This adds statistical rigour to prior assumptions and provides testable predictions for novel neural and behavioural phenotypes associated with specific genes and imprinted gene networks. In turn, this work sheds further light on the potential evolutionary drivers of genomic imprinting in the brain.

BACKGROUND: Since inception of the COVID-19 pandemic, early detection and isolation of positive cases is one of the key strategies to restrict disease transmission. Real time reverse transcription polymerase chain reaction (qRTPCR) has been the mainstay of diagnosis. Most of the qRTPCR kits were designed against the target genes of original strain of SARS-CoV-2. However, with the emergence of variant strains of SARS-CoV-2, sensitivity of the qRTPCR assays has reportedly reduced. In view of this, it is critical to continuously monitor the performance of the qRTPCR kits in the backdrop of variant strains of SARS-CoV-2. Real world monitoring of assay performance is challenging. Therefore, we developed a two-step in-silico screening process for evaluating the performance of various qRTPCR kits used in India. RESULTS: We analysed 73 qRT-PCR kits marketed in India, against the two SARS-CoV-2 VoCs. Sequences of both Delta (B.1.617.2) and Omicron (B.1.1.529) VoCs submitted to GISAID within a specific timeframe were downloaded, clustered to identify unique sequences and aligned with primer and probe sequences. Results were analysed following a two-step screening process. Out of 73 kits analysed, seven were unsatisfactory for detection of both Delta and Omicron VoCs, 10 were unsatisfactory for Delta VoC whereas 2 were unsatisfactory for only Omicron VoC. CONCLUSION: Overall, we have developed a useful screening process for evaluating the performance of qRTPCR assays against Delta and Omicron VoCs of SARS-CoV-2 which can be used for detecting SARS-CoV-2 VoCs that may emerge in future and can also be redeployed for other evolving pathogens of public health importance.

BACKGROUND: Continuous tilling and the lateral growth of rhizomes confer rhizomatous grasses with the unique ability to laterally expand, migrate and resist disturbances. They play key roles especially in degraded grasslands, deserts, sand dunes, and other fragile ecological system. The rhizomatous plant Leymus secalinus has both rhizome buds and tiller buds that grow horizontally and upward at the ends of rhizome differentiation and elongation, respectively. The mechanisms of rhizome formation and differentiation in L. secalinus have not yet been clarified. RESULTS: In this study, we found that the content of gibberellin A3 (GA3) and indole-3-acetic acid (IAA) were significantly higher in upward rhizome tips than in horizontal rhizome tips; by contrast, the content of methyl jasmonate and brassinolide were significantly higher in horizontal rhizome tips than in upward rhizome tips. GA3 and IAA could stimulate the formation and turning of rhizomes. An auxin efflux carrier gene, LsPIN1, was identified from L. secalinus based on previous transcriptome data. The conserved domains of LsPIN1 and the relationship of LsPIN1 with PIN1 genes from other plants were analyzed. Subcellular localization analysis revealed that LsPIN1 was localized to the plasma membrane. The length of the primary roots (PRs) and the number of lateral roots (LRs) were higher in Arabidopsis thaliana plants overexpressing LsPIN1 than in wild-type (Col-0) plants. Auxin transport was altered and the gravitropic response and phototropic response were stronger in 35S:LsPIN1 transgenic plants compared with Col-0 plants. It also promoted auxin accumulation in root tips. CONCLUSION: Our findings indicated that LsPIN1 plays key roles in auxin transport and root development. Generally, our results provide new insights into the regulatory mechanisms underlying rhizome development in L. secalinus.

BACKGROUND: Elucidation of the genetic mechanisms underlying treatment response to standard induction chemotherapy in AML patients is warranted, in order to aid in risk-adapted treatment decisions as novel treatments are emerging. In this pilot study, we explored the treatment-induced expression patterns in a small cohort of AML patients by analyzing differential gene expression (DGE) over the first 2 days of induction chemotherapy. METHODS: Blood samples were collected from ten AML patients at baseline (before treatment initiation) and during the first 2 days of treatment (Day 1; approximately 24 h, and Day 2; approximately 48 h after treatment initiation, respectively) and RNA was extracted for subsequent RNA sequencing. DGE between time points were assessed by pairwise analysis using the R package edgeR version 3.18.1 in all patients as well as in relation to treatment response (complete remission, CR, vs non-complete remission, nCR). Ingenuity Pathway Analysis (Qiagen) software was used for pathway analysis and visualization. RESULTS: After initial data quality control, two patients were excluded from further analysis, resulting in a final cohort of eight patients with data from all three timepoints. DGE analysis demonstrated activation of pathways with genes directly or indirectly associated with NF-κB signaling. Significant activation of the NF-κB pathway was seen in 50% of the patients 2 days after treatment start, while iNOS pathway effects could be identified already after 1 day. nCR patients displayed activation of pathways associated with cell cycle progression, oncogenesis and anti-apoptotic behavior, including the STAT3 pathway and Salvage pathways of pyrimidine ribonucleotides. Notably, a significant induction of cytidine deaminase, an enzyme responsible for the deamination of Ara-C, could be observed between baseline and Day 2 in the nCR patients but not in patients achieving CR. CONCLUSIONS: In conclusion, we show that time-course analysis of gene expression represents a feasible approach to identify relevant pathways affected by standard induction chemotherapy in AML patients. This poses as a potential method for elucidating new drug targets and biomarkers for categorizing disease aggressiveness and evaluating treatment response. However, more studies on larger cohorts are warranted to elucidate the transcriptional basis for drug response.

BACKGROUND: Evolutionary divergence and speciation often occur at a slower rate in the marine realm due to the higher potential for long-distance reproductive interaction through larval dispersal. One common evolutionary pattern in the Indo-Pacific, is divergence of populations and species at the peripheries of widely-distributed organisms. However, the evolutionary and demographic histories of such divergence are yet to be well understood. Here we address these issues by coupling genome-wide SNP data with mitochondrial DNA sequences to test the patterns of genetic divergence and possible secondary contact among geographically distant populations of the highly valuable spiny lobster Panulirus homarus species complex, distributed widely through the Indo-Pacific, from South Africa to the Marquesas Islands. RESULT: After stringent filtering, 2020 SNPs were used for population genetic and demographic analyses, revealing strong regional structure (FST = 0.148, P < 0001), superficially in accordance with previous analyses. However, detailed demographic analyses supported a much more complex evolutionary history of these populations, including a hybrid origin of a North-West Indian Ocean (NWIO) population, which has previously been discriminated morphologically, but not genetically. The best-supported demographic models suggested that the current genetic relationships among populations were due to a complex series of past divergences followed by asymmetric migration in more recent times. CONCLUSION: Overall, this study suggests that alternating periods of marine divergence and gene flow have driven the current genetic patterns observed in this lobster and may help explain the observed wider patterns of marine species diversity in the Indo-Pacific.

BACKGROUND: The dung beetle Phelotrupes auratus is a holometabolous insect belonging to the order Coleoptera, and it is widely distributed in Japan. The P. auratus habitat depends on herbivores. P. auratus eats the dung of the herbivores and carries it underground for its young. In this process, herbivore droppings disappear from the ground, not only keeping the ground hygienic but also maintaining good soil conditions for plant growth. In this way, a rich ecosystem is maintained. In recent years, the population of P. auratus has decreased, and the main cause has been the decrease in grazing land. It seems that Japanese dung beetles are mainly dependent on herbivores for nutrient sources. However, the physiological relationship between herbivores and P. auratus has not been well investigated. Here, we investigated the nutritional metabolism system of P. auratus by performing whole gene expression analysis of individuals collected from two areas where the ecosystem is occupied by different herbivores. RESULTS: We obtained 54,635 transcripts from P. auratus from Nara Park and Cape Toi and identified 2,592 differentially expressed genes in the fat bodies of the Nara Park and Cape Toi groups. We annotated P. auratus transcripts using Homo sapiens and Drosophila melanogaster genes as references; 50.5% of P. auratus transcripts were assigned to H. sapiens genes, and 54.0% of P. auratus transcripts were assigned to D. melanogaster genes. To perform gene set enrichment analysis, we chose H. sapiens genes for P. auratus transcript annotation. Principal component analysis and gene set enrichment analysis revealed that the nutritional metabolism of P. auratus from Cape Toi might differ from that of P. auratus from Nara Park. CONCLUSION: We analyzed the nutritional metabolism system of P. auratus from Cape Toi and Nara Park and found that the characteristics of the nutritional metabolism process might depend on the plants consumed by the herbivores. Our findings will contribute to elucidating the relationships among habitat plants, herbivores, and dung decomposers and may aid in the maintenance of sustainable land health cycles.

BACKGROUND: Shotgun metagenome analysis provides a robust and verifiable method for comprehensive microbiome analysis of fungal, viral, archaeal and bacterial taxonomy, particularly with regard to visualization of read mapping location, normalization options, growth dynamics and functional gene repertoires. Current read classification tools use non-standard output formats, or do not fully show information on mapping location. As reference datasets are not perfect, portrayal of mapping information is critical for judging results effectively. RESULTS: Our alignment-based pipeline, Wochenende, incorporates flexible quality control, trimming, mapping, various filters and normalization. Results are completely transparent and filters can be adjusted by the user. We observe stringent filtering of mismatches and use of mapping quality sharply reduces the number of false positives. Further modules allow genomic visualization and the calculation of growth rates, as well as integration and subsequent plotting of pipeline results as heatmaps or heat trees. Our novel normalization approach additionally allows calculation of absolute abundance profiles by comparison with reads assigned to the human host genome. CONCLUSION: Wochenende has the ability to find and filter alignments to all kingdoms of life using both short and long reads, and requires only good quality reference genomes. Wochenende automatically combines multiple available modules ranging from quality control and normalization to taxonomic visualization. Wochenende is available at https://github.com/MHH-RCUG/nf_wochenende .

Flowering time and active accumulated temperature (AAT) are two key factors that limit the expanded production especially for soybean across different regions. Wild soybean provides an important germplasm for functional genomics study in cultivar soybean. However, the studies on genetic basis underlying flowering time in response to AAT especially in wild soybean were rarely reported. In this study, we used 294 wild soybean accessions derived from major soybean production region characterized by different AAT in Northeast of China. Based on genome-wide association study (GWAS), we identified 96 SNPs corresponded to 342 candidate genes that significantly associated with flowering time recorded in two-year experiments. Gene Ontology enrichment analysis suggests that the pathways of photosynthesis light reaction and actin filament binding were significantly enriched. We found three lead SNPs with -log10(p-value) > 32 across the two-year experiments, i.e., Chr02:9490318, Chr04:8545910 and Chr09:49553555. Linkage disequilibrium block analysis shows 28 candidate genes within the genomic region centered on the lead SNPs. Among them, expression levels of three genes (aspartic peptidase 1, serine/threonine-protein kinase and protein SCAR2-like) were significantly differed between two subgroups possessing contrasting flowering time distributed at chromosome 2, 4 and 9, respectively. There are 6, 7 and 3 haplotypes classified on the coding regions of the three genes, respectively. Collectively, accessions with late flowering time phenotype are typically derived from AAT zone 1, which is associated with the haplotypic distribution and expression levels of the three genes. This study provides an insight into a potential mechanism responsible for flowering time in response to AAT in wild soybean, which could promote the understanding of genetic basis for other major crops.

BACKGROUND: Understanding the micro--evolutionary response of populations to demographic declines is a major goal in evolutionary and conservation biology. In small populations, genetic drift can lead to an accumulation of deleterious mutations, which will increase the risk of extinction. However, demographic recovery can still occur after extreme declines, suggesting that natural selection may purge deleterious mutations, even in extremely small populations. The Chatham Island black robin (Petroica traversi) is arguably the most inbred bird species in the world. It avoided imminent extinction in the early 1980s and after a remarkable recovery from a single pair, a second population was established and the two extant populations have evolved in complete isolation since then. Here, we analysed 52 modern and historical genomes to examine the genomic consequences of this extreme bottleneck and the subsequent translocation. RESULTS: We found evidence for two-fold decline in heterozygosity and three- to four-fold increase in inbreeding in modern genomes. Moreover, there was partial support for temporal reduction in total load for detrimental variation. In contrast, compared to historical genomes, modern genomes showed a significantly higher realised load, reflecting the temporal increase in inbreeding. Furthermore, the translocation induced only small changes in the frequency of deleterious alleles, with the majority of detrimental variation being shared between the two populations. CONCLUSION: Our results highlight the dynamics of mutational load in a species that recovered from the brink of extinction, and show rather limited temporal changes in mutational load. We hypothesise that ancestral purging may have been facilitated by population fragmentation and isolation on several islands for thousands of generations and may have already reduced much of the highly deleterious load well before human arrival and introduction of pests to the archipelago. The majority of fixed deleterious variation was shared between the modern populations, but translocation of individuals with low mutational load could possibly mitigate further fixation of high-frequency deleterious variation.

BACKGROUND: So far, a lot of binning approaches have been intensively developed for untangling metagenome-assembled genomes (MAGs) and evaluated by two main strategies. The strategy by comparison to known genomes prevails over the other strategy by using single-copy genes. However, there is still no dataset with all known genomes for a real (not simulated) bacterial consortium yet. RESULTS: Here, we continue investigating the real bacterial consortium F1RT enriched and sequenced by us previously, considering the high possibility to unearth all MAGs, due to its low complexity. The improved F1RT metagenome reassembled by metaSPAdes here utilizes about 98.62% of reads, and a series of analyses for the remaining reads suggests that the possibility of containing other low-abundance organisms in F1RT is greatly low, demonstrating that almost all MAGs are successfully assembled. Then, 4 isolates are obtained and individually sequenced. Based on the 4 isolate genomes and the entire metagenome, an elaborate pipeline is then in-house developed to construct all F1RT MAGs. A series of assessments extensively prove the high reliability of the herein reconstruction. Next, our findings further show that this dataset harbors several properties challenging for binning and thus is suitable to compare advanced binning tools available now or benchmark novel binners. Using this dataset, 8 advanced binning algorithms are assessed, giving useful insights for developing novel approaches. In addition, compared with our previous study, two novel MAGs termed FC8 and FC9 are discovered here, and 7 MAGs are solidly unearthed for species without any available genomes. CONCLUSION: To our knowledge, it is the first time to construct a dataset with almost all known MAGs for a not simulated consortium. We hope that this dataset will be used as a routine toolkit to complement mock datasets for evaluating binning methods to further facilitate binning and metagenomic studies in the future.