Multicopy Gene Research Articles

Evaluation of Genetic Diversity and Parasite-Mediated Selection of MHC Class I Genes in Emberiza godlewskii (Passeriformes: Emberizidae)

The major histocompatibility complex (MHC) is a multi-copy immune gene family in vertebrates. Its genes are highly variable and code for antigen-presenting molecules. Characterization of MHC genes in different species and investigating the mechanisms that shape MHC diversity is an important goal in understanding the evolution of biological diversity. Here we developed a next-generation sequencing (NGS) protocol to genotype the MHC class I genes of 326 Godlewski’s buntings (Emberiza godlewskii) sampled in the Western mountain area of Beijing from 2014 to 2016. A total of 184 functional alleles were identified, including both non-classical and classical alleles, clustering into nine supertypes. Compared with other passerine birds, the number of MHC class I alleles per individual in Godlewski’s buntings is high (mean 16.1 ± 3.3, median 16). In addition, we demonstrated signatures of historical and contemporary selection on MHC genes. Reflecting historical selection, ten amino acid sites in the antigen-binding domain showed signatures of balancing selection, eight of which exhibit high amino acid polymorphism. In terms of contemporary selection, we found that specific MHC supertypes were nominally associated with the infection of two malaria parasite lineages. These findings indicate the action of historical and possibly also contemporary balancing selection and suggest negative frequency-dependent or fluctuating selection as possible selection mechanisms.

Genetic engineering and raising temperature enhance recombinant protein production with the cdna1 promoter in Trichoderma reesei

The fungus Trichoderma reesei is a powerful host for secreted production of proteins. The promoter of cdna1 gene, which encodes a small basic protein of unknown function and high expression, is commonly used for constitutive protein production in T. reesei. Nevertheless, the production level of proteins driven by this promoter still needs to be improved. Here, we identified that the region 600- to 700-bp upstream of the start codon is critical for the efficiency of the cdna1 promoter. Increasing the copy number of this region to three improved the production of a heterologous β-mannanase by 37.5%. Screening of several stressful conditions revealed that the cdna1 promoter is heat inducible. Cultivation at 37 °C significantly enhanced the production of β-mannanase as well as a polygalacturonase with the cdna1 promoter compared with those at 30 °C. Combing the strategies of promoter engineering, multi-copy gene insertion, and control of cultivation temperature, β-mannanase of 199.85 U/mL and relatively high purity was produced in shake flask, which was 6.6 times higher than that before optimization. Taken together, the results advance the understanding of the widely used cdna1 promoter and provide effective strategies for enhancing the production of recombinant proteins in T. reesei.Graphical

Sequencing our way to more accurate community abundance.

Over the last two decades, there has been a huge increase in our understanding of microbial diversity, structure and composition enabled by high-throughput sequencing technologies. Yet, it is unclear how the number of sequences translates to the number of cells or species within the community. In some cases, additional observational data may be required to ensure relative abundance patterns from sequence reads are biologically meaningful. The goal of DNA-based methods for biodiversity assessments is to obtain robust community abundance data, simultaneously, from environmental samples. In this issue of Molecular Ecology Resources, Pierella Karlusich et al.(2022) describe a new method for quantifying phytoplankton cell abundance. Using Tara Oceans data sets, the authors propose the photosynthetic gene psbO for reporting accurate relative abundance of the entire phytoplankton community from metagenomic data. The authors demonstrate higher correlations with traditional optical methods (including microscopy and flow cytometry), using their new method, improving upon molecular abundance assessments using multicopy marker genes. Furthermore, to facilitate application of their approach, the authors curated a psbO gene database for accessible taxonomic queries. This is an important step towards improving species abundance estimates from molecular data and eventually reporting of absolute species abundance, enhancing our understanding of community dynamics.

Accessing the Variability of Multicopy Genes in Complex Genomes using Unassembled Next-Generation Sequencing Reads: The Case of Trypanosoma cruzi Multigene Families.

Repetitive elements cause assembly fragmentation in complex eukaryotic genomes, limiting the study of their variability. The genome of Trypanosoma cruzi, the parasite that causes Chagas disease, has a high repetitive content, including multigene families. Although many T. cruzi multigene families encode surface proteins that play pivotal roles in host-parasite interactions, their variability is currently underestimated, as their high repetitive content results in collapsed gene variants. To estimate sequence variability and copy number variation of multigene families, we developed a read-based approach that is independent of gene-specific read mapping and de novo assembly. This methodology was used to estimate the copy number and variability of MASP, TcMUC, and Trans-Sialidase (TS), the three largest T. cruzi multigene families, in 36 strains, including members of all six parasite discrete typing units (DTUs). We found that these three families present a specific pattern of variability and copy number among the distinct parasite DTUs. Inter-DTU hybrid strains presented a higher variability of these families, suggesting that maintaining a larger content of their members could be advantageous. In addition, in a chronic murine model and chronic Chagasic human patients, the immune response was focused on TS antigens, suggesting that targeting TS conserved sequences could be a potential avenue to improve diagnosis and vaccine design against Chagas disease. Finally, the proposed approach can be applied to study multicopy genes in any organism, opening new avenues to access sequence variability in complex genomes. IMPORTANCE Sequences that have several copies in a genome, such as multicopy-gene families, mobile elements, and microsatellites, are among the most challenging genomic segments to study. They are frequently underestimated in genome assemblies, hampering the correct assessment of these important players in genome evolution and adaptation. Here, we developed a new methodology to estimate variability and copy numbers of repetitive genomic regions and employed it to characterize the T. cruzi multigene families MASP, TcMUC, and transsialidase (TS), which are important virulence factors in this parasite. We showed that multigene families vary in sequence and content among the parasite's lineages, whereas hybrid strains have a higher sequence variability that could be advantageous to the parasite's survivability. By identifying conserved sequences within multigene families, we showed that the mammalian host immune response toward these multigene families is usually focused on the TS multigene family. These TS conserved and immunogenic peptides can be explored in future works as diagnostic targets or vaccine candidates for Chagas disease. Finally, this methodology can be easily applied to any organism of interest, which will aid in our understanding of complex genomic regions.

Copy number variation of horse Y chromosome genes in normal equine populations and in horses with abnormal sex development and subfertility: relationship of copy number variations with Y haplogroups.

Structural rearrangements like copy number variations in the male-specific Y chromosome have been associated with male fertility phenotypes in human and mouse but have been sparsely studied in other mammalian species. Here, we designed digital droplet PCR assays for 7 horse male-specific Y chromosome multicopy genes and SRY and evaluated their absolute copy numbers in 209 normal male horses of 22 breeds, 73 XY horses with disorders of sex development and/or infertility, 5 Przewalski's horses and 2 kulans. This established baseline copy number for these genes in horses. The TSPY gene showed the highest copy number and was the most copy number variable between individuals and breeds. SRY was a single-copy gene in most horses but had 2-3 copies in some indigenous breeds. Since SRY is flanked by 2 copies of RBMY, their copy number variations were interrelated and may lead to SRY-negative XY disorders of sex development. The Przewalski's horse and kulan had 1 copy of SRY and RBMY. TSPY and ETSTY2 showed significant copy number variations between cryptorchid and normal males (P < 0.05). No significant copy number variations were observed in subfertile/infertile males. Notably, copy number of TSPY and ETSTY5 differed between successive male generations and between cloned horses, indicating germline and somatic mechanisms for copy number variations. We observed no correlation between male-specific Y chromosome gene copy number variations and male-specific Y chromosome haplotypes. We conclude that the ampliconic male-specific Y chromosome reference assembly has deficiencies and further studies with an improved male-specific Y chromosome assembly are needed to determine selective constraints over horse male-specific Y chromosome gene copy number and their relation to stallion reproduction and male biology.

Efficient Suppression of Natural Plasmid-Borne Gene Expression in Carbapenem-Resistant Klebsiella pneumoniae Using a Compact CRISPR Interference System.

There is an urgent need for efficient tools for genetic manipulation to assess plasmid function in clinical drug-resistant bacterial strains. To address this need, we developed an all-in-one CRISPR interference (CRISPRi) system that easily inhibited the gene expression of a natural multidrug-resistant plasmid in an sequence type 23 (ST23) Klebsiella pneumoniae isolate. We established an integrative CRISPRi system plasmid, pdCas9gRNA, harboring a dcas9 gene and a single guide RNA (sgRNA) unit under the control of anhydrotetracycline-induced and J23119 promoters, respectively, using a one-step cloning method. This system can repress the single resistance gene blaNDM-1, with a >1,000-fold reduction in the meropenem MIC, or simultaneously silence the resistance genes blaNDM-1 and blaSHV-12, with a 16-fold and 8-fold respective reduction in the meropenem and aztreonam MIC on a large natural multidrug-resistant pNK01067-NDM-1 plasmid in an ST23 K. pneumoniae isolate. Furthermore, an sgRNA targeting the blaNDM-1 promoter region can silence the entire blaNDM-1-bleMBL-trpF operon, confirming the existence of the operon. We also used this tool to knock down the multicopy resistance gene blaKPC-2 in pathogenic Escherichia coli, increasing the susceptibility to meropenem. In a word, the all-in-one CRISPRi system can be used for efficient interrogation of indigenous plasmid-borne gene functions, providing a rapid, easy genetic manipulation tool for clinical K. pneumoniae isolates.

Uncovering Pseudogenes and Intergenic Protein-coding Sequences in TriTryps' Genomes.

Trypanosomatids belong to a remarkable group of unicellular, parasitic organisms of the order Kinetoplastida, an early diverging branch of the phylogenetic tree of eukaryotes, exhibiting intriguing biological characteristics affecting gene expression (intronless polycistronic transcription, trans-splicing, and RNA editing), metabolism, surface molecules, and organelles (compartmentalization of glycolysis, variation of the surface molecules, and unique mitochondrial DNA), cell biology and life cycle (phagocytic vacuoles evasion and intricate patterns of cell morphogenesis). With numerous genomic-scale data of several trypanosomatids becoming available since 2005 (genomes, transcriptomes, and proteomes), the scientific community can further investigate the mechanisms underlying these unusual features and address other unexplored phenomena possibly revealing biological aspects of the early evolution of eukaryotes. One fundamental aspect comprises the processes and mechanisms involved in the acquisition and loss of genes throughout the evolutionary history of these primitive microorganisms. Here, we present a comprehensive in silico analysis of pseudogenes in three major representatives of this group: Leishmania major, Trypanosoma brucei, and Trypanosoma cruzi. Pseudogenes, DNA segments originating from altered genes that lost their original function, are genomic relics that can offer an essential record of the evolutionary history of functional genes, as well as clues about the dynamics and evolution of hosting genomes. Scanning these genomes with functional proteins as proxies to reveal intergenic regions with protein-coding features, relying on a customized threshold to distinguish statistically and biologically significant sequence similarities, and reassembling remnant sequences from their debris, we found thousands of pseudogenes and hundreds of open reading frames, with particular characteristics in each trypanosomatid: mutation profile, number, content, density, codon bias, average size, single- or multi-copy gene origin, number and type of mutations, putative primitive function, and transcriptional activity. These features suggest a common process of pseudogene formation, different patterns of pseudogene evolution and extant biological functions, and/or distinct genome organization undertaken by those parasites during evolution, as well as different evolutionary and/or selective pressures acting on distinct lineages.

Cistanche Species Mitogenomes Suggest Diversity and Complexity in Lamiales-Order Mitogenomes.

The extreme diversity and complexity of angiosperms is well known. Despite the fact that parasitic plants are angiosperms, little is known about parasitic plant mitogenomic diversity, complexity, and evolution. In this study, we obtained and characterized the mitogenomes of three Cistanche species (holoparasitic plants) from China to compare the repeats, segment duplication and multi-copy protein-coding genes (PCGs), to clarify the phylogenetic and evolution relationship within the Lamiales order, and to identify the mitochondrial plastid insertions (MTPT) in Cistanche mitogenomes. The results showed that the mitogenome sizes of the three Cistanche species ranged from 1,708,661 to 3,978,341 bp. The Cistanche species genome encodes 75–126 genes, including 37–65 PCGs, 31–58 tRNA genes and 3–5 rRNA genes. Compared with other Lamiales and parasitic species, the Cistanche species showed extremely high rates of multi-copy PCGs, ranging from 0.13 to 0.58 percent of the total number of PCGs. In addition, 37–133 Simple Sequence Repeat (SSRs) were found in these three mitogenomes, the majority of which were the mononucleotides Adenine/Thymine. The interspersed repeats contained forward and palindromic repeats. Furthermore, the segment-duplication sequence size ranged from 199,584 to 2,142,551 bp, accounting for 24.9%, 11.7% and 53.9% of the Cistanche deserticola, Cistanche salsa and Cistanche tubulosa mitogenome, respectively. Furthermore, the Ka/Ks analysis suggested that the atp4, ccmB, ccmFc and matR were probably positively selected during Lamiales evolution. The Cistanche plastome suggested the presence of MTPT. Moreover, 6–12 tRNA, 9–15 PCGs fragments and 3 rRNA gene fragments in the Cistanche mitogenomes were found in the MTPT regions. This work reports the Cistanche species mitogenome for the first time, which will be invaluable for study the mitogenome evolution of Orobanchaceae family.

Targeted Mutagenesis of the Multicopy Myrosinase Gene Family in Allotetraploid Brassica juncea Reduces Pungency in Fresh Leaves across Environments.

Recent breeding efforts in Brassica have focused on the development of new oilseed feedstock crop for biofuels (e.g., ethanol, biodiesel, bio-jet fuel), bio-industrial uses (e.g., bio-plastics, lubricants), specialty fatty acids (e.g., erucic acid), and producing low glucosinolates levels for oilseed and feed meal production for animal consumption. We identified a novel opportunity to enhance the availability of nutritious, fresh leafy greens for human consumption. Here, we demonstrated the efficacy of disarming the ‘mustard bomb’ reaction in reducing pungency upon the mastication of fresh tissue—a major source of unpleasant flavor and/or odor in leafy Brassica. Using gene-specific mutagenesis via CRISPR-Cas12a, we created knockouts of all functional copies of the type-I myrosinase multigene family in tetraploid Brassica juncea. Our greenhouse and field trials demonstrate, via sensory and biochemical analyses, a stable reduction in pungency in edited plants across multiple environments. Collectively, these efforts provide a compelling path toward boosting the human consumption of nutrient-dense, fresh, leafy green vegetables.

Multi-level metabolic engineering of Escherichia coli for high-titer biosynthesis of 2'-fucosyllactose and 3-fucosyllactose.

Fucosyllactoses (FL), including 2'-fucosyllactose (2'-FL) and 3-fucosyllactose (3-FL), have garnered considerable interest for their value in newborn formula and pharmaceuticals. In this study, an engineered Escherichia coli was developed for high-titer FL biosynthesis by introducing multi-level metabolic engineering strategies, including (1) individual construction of the 2'/3-FL-producing strains through gene combination optimization of the GDP-L-fucose module; (2) screening of rate-limiting enzymes (α-1,2-fucosyltransferase and α-1,3-fucosyltransferase); (3) analysis of critical intermediates and inactivation of competing pathways to redirect carbon fluxes to FL biosynthesis; (4) enhancement of the catalytic performance of rate-limiting enzymes by the RBS screening, fusion peptides and multi-copy gene cloning. The final strains EC49 and EM47 produced 9.36 g/L for 2'-FL and 6.28 g/L for 3-FL in shake flasks with a modified-M9CA medium. Fed-batch cultivations of the two strains generated 64.62 g/L of 2'-FL and 40.68 g/L of 3-FL in the 3-L bioreactors, with yields of 0.65 mol 2'-FL/mol lactose and 0.67 mol 3-FL/mol lactose, respectively. This research provides a viable platform for other high-value-added compounds production in microbial cell factories.

S1.4d Cryptococcus qPCR assays: the future for routine mycology labs and clinical trials dealing with cryptococcosis

S1.4 Fungal infections in Asia, bringing it out of the dark, September 21, 2022, 11:00 AM - 12:30 PMBackground: Routine laboratory testing for cryptococcal meningitis currently consists of Cryptococcal antigen (CrAg) testing in blood and cerebrospinal fluid (CSF), CSF India ink, and CSF fungal culture. Quantitative cryptococcal culture (QCC) is labor intensive and not feasible in most settings.ObjectivesWe evaluated quantitative (qPCR) and reverse transcriptase qPCR (RT-qPCR) assays to quantify cryptococcal load in CSF, plasma, and blood. We also investigated the dynamics of fungal DNA and RNA detection during antifungal treatment.MethodsWe developed a qPCR assay that can differentiate serotypes A, D, and B/C of Cryptococcus neoformans and C. gattii based on the amplification of a unique nuclear Quorum sensing protein 1 (QSP1) and a multicopy 28S rRNA gene and evaluated the assays on 205-patient samples from the AMBITION-cm trial in Botswana and Malawi (2018-2021). CSF, plasma, and whole blood samples were stored per patient and were sampled at day 0 (baseline), day 7 and 14 for CSF and at day 1, 3 and 7 for plasma and whole blood post antifungal treatment initiation. A Roche LightCycler480 and Graph pad prism were used for data analysis.ResultsA total of 205/209 stored patient samples (85 from Botswana; 124 from Malawi), were used. For QSP1 qPCR tested in CSF at D0, 138 (81.7%) were serotype A, 28 (16.6%) were serotype B/C and 3 (1.8%) were a mixed infection of serotype A and B/C. There was no amplification with 36 (17.6%) samples. There was no difference in fungal loads at D0, D7, and D14 between serotype A and B/C with the QSP1 qPCR assay, and QCC. QCC showed a good correlation with qPCR quantification with QSP1 qPCR (slope = 0.797, R2 = 0.73) and with 28S rRNA qPCR (Slope = 0.771, R2 = 0.778) assays. The fungal load at D0 was significantly higher in patients who died at week 2 (w2) and at week 10 (w10) as compared with patients who survived post-week 10 (P <.01), with no significant difference in initial fungal load in both treatment regimens (P >.05). Detection of Cryptococcus DNA (28S rRNA qPCR) in plasma or whole blood within the first 24 h of treatment was significantly associated with early mortality at w2 and mortality at w10 (P <.01). QSP1 RT-qPCR showed that detection of DNA was due to viable fungal cells as the quantification of QSP1 whole nucleic acids was systematically higher (X2 to 5) than that of DNA.ConclusionQuantification of C. neoformans and C. gattii load in CSF and plasma at D0 is useful in identifying patients at risk of death and may be a promising tool for monitoring treatment response in the future.

P498 Pneumocystis Jirovecii pneumonia: Clinical profile and diagnostic modalities

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PM ObjectivesIn developing countries like India, the diagnosis of Pneumocystis jirovecii infection is often made either by conventional staining or clinically. This study was planned to know the utility of polymerase chain reaction (PCR) in diagnosing Pneumocystis jirovecii pneumonia (PJP), to compare the PCR results with that of the staining technique, and also to correlate the results with the radiological condition of patients from a tertiary care center in India.MethodA retrospective study included 3500 adult in-patients with symptoms of lower respiratory tract infection. Sputum and bronchoalveolar lavage samples were proceeded for both staining and PCR for multicopy mtLSU gene and singlecopy DHPS fas gene of P. jirovecii.ResultsThe present study was carried out in the Department of Microbiology, Max, Hospital, Saket, New Delhi, India. Between the years 2019 and 2022, the patients with clinically suspected pneumocystis pneumonia who attended outpatient or admitted to the Departments of Medicine, Gastroenterology, and Neurology, etc. of our hospital were retrospectively analyzed. The details of these patients were analyzed as per a well-structured proforma that included the detailed clinical history and laboratory data from the hospital records. A total of 3500 patients were included in the present study. Of these 3500 patients, 1.42% (50/3500) patients of pneumocystis pneumonia were confirmed, based on positivity by at least any two of the above-mentioned techniques. The age of the patients ranged from 29 to 80 years (mean 32 years). Among these 50 patients, 34 (68%) were males and 16 (32%) were females and the male-to-female ratio was 2:1. A total of 10 patients had human immunodeficiency virus (HIV) infection, while the other 25 were HIV negative. The immune status of five patients was unknown. Fever and dyspnea (n = 44, 88%), non-productive cough and abnormal auscultation sound (n = 30, 60%), and also chills and weight loss (n = 10, 20%) were the documented clinical symptoms of PJP. Radiological diagnosis of Pneumocystis pneumonia was noticed in 34 cases. Foamy alveolar casts were present in 19 cases. Silver methenamine stain enhanced the rounded, helmet, or cleft forms of sporozoites. Inflammatory infiltrate was mainly polymorphonuclear. All the cases were confirmed as P. jiroveci by PCR amplification.ConclusionClinicians often face the diagnostic dilemmas presented in the study. Individual modalities of the diagnosis are available, but all have drawbacks with varied sensitivity and specificity. Combining the available clinical, radiological, and microbiological modalities to reach early diagnosis can go a long way to avoid misdiagnosis and unnecessary delays in treatment.

ASTRAL-Pro 2: ultrafast species tree reconstruction from multi-copy gene family trees.

Species tree inference from multi-copy gene trees has long been a challenge in phylogenomics. The recent method ASTRAL-Pro has made strides by enabling multi-copy gene family trees as input and has been quickly adopted. Yet, its scalability, especially memory usage, needs to improve to accommodate the ever-growing dataset size. We present ASTRAL-Pro 2, an ultrafast and memory efficient version of ASTRAL-Pro that adopts a placement-based optimization algorithm for significantly better scalability without sacrificing accuracy. The source code and binary files are publicly available at https://github.com/chaoszhang/ASTER; data are available at https://github.com/chaoszhang/A-Pro2_data. Supplementary data are available at Bioinformatics online.

PRAMEY: A Bovid-Specific Y-Chromosome Multicopy Gene Is Highly Related to Postnatal Testicular Growth in Hu Sheep.

Simple SummaryPRAMEY (preferentially expressed antigen in melanoma, Y-linked) is a cancer-testis antigen and is believed to play an important role in testicular development and gametogenesis. However, the structure, expression and function of PRAMEY in sheep remain unknown. In this paper, we found that PRAMEY was extremely expressed in the testis and significantly upregulated during testicular growth. The mRNA abundance of PRAMEY was significantly correlated with testicular size traits in Hu sheep at 6 months. These results imply that PRAMEY may play an important role in testicular growth.PRAMEY (preferentially expressed antigen in melanoma, Y-linked) belongs to the cancer-testis antigens (CTAs) gene family and is predominantly expressed in testis, playing important roles in spermatogenesis and testicular development. This study cloned the full-length cDNA sequence of ovine PRAMEY using the rapid amplification of cDNA ends (RACE) method and analyzed the expression profile and copy number variation (CNV) of PRAMEY using quantitative real-time PCR (qPCR). The results revealed that the PRAMEY cDNA was 2099 bp in length with an open reading frame (ORF) of 1536 bp encoding 511 amino acids. PRAMEY was predominantly expressed in the testis and significantly upregulated during postnatal testicular development. The median copy number (MCN) of PRAMEY was 4, varying from 2 to 25 in 710 rams across eight sheep breeds. There was no significant correlation between the CNV of PRAMEY and testicular size, while a significant positive correlation was observed between the mRNA expression and testicular size in Hu sheep. The current study suggests that the expression levels of PRAMEY were closely associated with testicular size, indicating that PRAMEY may play an important role in testicular growth.

The genome of Orychophragmus violaceus provides genomic insights into the evolution of Brassicaceae polyploidization and its distinct traits

Orychophragmus violaceus, referred to as “eryuelan” (February orchid) in China, is an early-flowering ornamental plant. The high oil content and abundance of unsaturated fatty acids in O. violaceus seeds make it a potential high-quality oilseed crop. Here, we generated a whole-genome assembly for O. violaceus using Nanopore and Hi-C sequencing technologies. The assembled genome of O. violaceus was ∼1.3 Gb in size, with 12 pairs of chromosomes. Through investigation of ancestral genome evolution, we determined that the genome of O. violaceus experienced a tetraploidization event from a diploid progenitor with the translocated proto-Calepineae karyotype. Comparisons between the reconstructed subgenomes of O. violaceus identified indicators of subgenome dominance, indicating that subgenomes likely originated via allotetraploidy. O. violaceus was phylogenetically close to the Brassica genus, and tetraploidy in O. violaceus occurred approximately 8.57 million years ago, close in time to the whole-genome triplication of Brassica that likely arose via an intermediate tetraploid lineage. However, the tetraploidization in Orychophragmus was independent of the hexaploidization in Brassica, as evidenced by the results from detailed phylogenetic analyses and comparisons of the break and fusion points of ancestral genomic blocks. Moreover, identification of multi-copy genes regulating the production of high-quality oil highlighted the contributions of both tetraploidization and tandem duplication to functional innovation in O. violaceus. These findings provide novel insights into the polyploidization evolution of plant species and will promote both functional genomic studies and domestication/breeding efforts in O. violaceus.

Genome-Wide Identification and Expression Analysis of eIF Family Genes from Brassica rapa in Response to TuMV Resistance.

Brassica rapa is one of the most important leafy vegetables worldwide, and has a long history of cultivation. However, it has not been possible to completely control the damage of turnip mosaic virus (TuMV), a serious virus in B. rapa, to production. In this study, the genome-wide identification and expression detection of eIF family genes from B. rapa in response to TuMV resistance were analyzed, including the identification of eIF family genes, chromosomal distribution, three-dimensional (3D) structure and sequence logo analyses, and the expression characterization as well as differential metabolite analysis of eIF family genes in resistant/susceptible lines, which may further prove the whole-genome tripling (WGT) event in B. rapa evolution and provide evidence for the functional redundancy and functional loss of multicopy eIF genes in evolution. A qRT-PCR analysis revealed that the relative expressions of eIF genes in a susceptible line (80461) were higher than those in a resistant line (80124), which may prove that, when TuMV infects host plants, the eIF genes can combine with the virus mRNA 5′ end cap structure and promote the initiation of virus mRNA translation in the susceptible B. rapa line. In addition, the metabolite substances were detected, the differences in metabolites between disease-resistant and disease-susceptible plants were mainly manifested by altered compounds such as flavonoids, jasmonic acid, salicylic acid, ketones, esters, etc., which inferred that the different metabolite regulations of eIF family genes and reveal the resistance mechanisms of eIF genes against TuMV in brassica crops. This study may lay a new theoretical foundation for revealing eIF family gene resistance to TuMV in B. rapa, as well as advancing our understanding of virus–host interactions.

High prevalence of Toxoplasma gondii in Nigerian wild rats by molecular detection

Toxoplasmosis has been reported in Nigeria using several diagnostic tools with high prevalence in humans and some food animals. Rodents have been recognised as vital intermediate hosts of Toxoplasma gondii. However, there is paucity of information on the occurrence of T. gondii in wild rats found in Nigeria. This study aimed at molecular detection of T. gondii in Zyzomys pedunculatus and to evaluate its involvement in the epidemiology of toxoplasmosis in Nigeria. A total of 84 rats were sampled across three states of the North Central Nigeria, and DNA was extracted from the brain, lungs, kidney and intestine of the rats for the detection of T. gondii DNA by nested PCR to amplify the multicopy B1 gene. Sixty-four of the 84 samples (76.2%) were positive for T. gondii out of which 5 samples were sequenced and had an identity score of between 97.73% and 99.35% with the reference B1 gene of T. gondii in GenBank. This study suggests Nigerian wild rats may be an important intermediate hosts of T. gondii and may play a role in the epidemiology and maintenance of T. gondii circulation in Nigeria.

Phylogenomics Resolves the Phylogeny of Theaceae by Using Low-Copy and Multi-Copy Nuclear Gene Makers and Uncovers a Fast Radiation Event Contributing to Tea Plants Diversity

Simple SummaryThe Theaceae includes more than 300 species of great morphological diversity and has immense economic, cultural, and ornamental values. However, the evolutionary history of this family remains elusive. We integrated 91 genomes and transcriptome datasets of Theaceae and successfully resolved the phylogeny of Theaceae including relatives of cultivated tea plants from both extensive low-copy and multi-copy nuclear gene markers. Bayes-based molecular dating revealed that the ancestor of the tea family originated slightly earlier than the K-Pg boundary (Mass extinction events including the extinction of dinosaurs) with early diversification of three tribes associated with the Early Eocene Climatic Optimum. Further speciation analysis suggested a sole significant diversification shift rate in the common ancestor of Camellia associated with the Mid-Miocene Climatic Optimum. Collectively, polyploidy events, and key morphological innovation characters, such as pericarp with seed coat hardening, could possibly contribute to the Theaceae species diversity.Tea is one of the three most popular nonalcoholic beverages globally and has extremely high economic and cultural value. Currently, the classification, taxonomy, and evolutionary history of the tea family are largely elusive, including phylogeny, divergence, speciation, and diversity. For understanding the evolutionary history and dynamics of species diversity in Theaceae, a robust phylogenetic framework based on 1785 low-copy and 79,103 multi-copy nuclear genes from 91 tea plant genomes and transcriptome datasets had been reconstructed. Our results maximumly supported that the tribes Stewartieae and Gordonieae are successive sister groups to the tribe Theeae from both coalescent and super matrix ML tree analyses. Moreover, in the most evolved tribe, Theeae, the monophyletic genera Pyrenaria, Apterosperma, and Polyspora are the successive sister groups of Camellia. We also yield a well-resolved relationship of Camellia, which contains the vast majority of Theaceae species richness. Molecular dating suggests that Theaceae originated in the late L-Cretaceous, with subsequent early radiation under the Early Eocene Climatic Optimal (EECO) for the three tribes. A diversification rate shift was detected in the common ancestors of Camellia with subsequent acceleration in speciation rate under the climate optimum in the early Miocene. These results provide a phylogenetic framework and new insights into factors that likely have contributed to the survival of Theaceae, especially a successful radiation event of genus Camellia members to subtropic/tropic regions. These novel findings will facilitate the efficient conservation and utilization of germplasm resources for breeding cultivated tea and oil-tea. Collectively, these results provide a foundation for further morphological and functional evolutionary analyses across Theaceae.

Histone editing elucidates the functional roles of H3K27 methylation and acetylation in mammals.

Posttranslational modifications of histones (PTMs) are associated with specific chromatin and gene expression states1,2. Although studies in Drosophila melanogaster have revealed phenotypic associations between chromatin-modifying enzymes and their histone substrates, comparable studies in mammalian models do not exist3-5. Here, we use CRISPR base editing in mouse embryonic stem cells (mESCs) to address the regulatory role of lysine 27 of histone H3 (H3K27), a substrate for Polycomb repressive complex 2 (PRC2)-mediated methylation and CBP/EP300-mediated acetylation6,7. By generating pan-H3K27R (pK27R) mutant mESCs, where all 28 alleles of H3.1, H3.2 and H3.3 have been mutated, we demonstrate similarity in transcription patterns of genes and differentiation to PRC2-null mutants. Moreover, H3K27 acetylation is not essential for gene derepression linked to loss of H3K27 methylation, or de novo activation of genes during cell-fate transition to epiblast-like cells (EpiLCs). In conclusion, our results show that H3K27 is an essential substrate for PRC2 in mESCs, whereas other PTMs in addition to H3K27 acetylation are likely involved in mediating CBP/EP300 function. Our work demonstrates the feasibility of large-scale multicopy gene editing to interrogate histone PTM function in mammalian cells.

A phenome-wide association study identifies effects of copy-number variation of VNTRs and multicopy genes on multiple human traits

The human genome contains tens of thousands of large tandem repeats and hundreds of genes that show common and highly variable copy-number changes. Due to their large size and repetitive nature, these variable number tandem repeats (VNTRs) and multicopy genes are generally recalcitrant to standard genotyping approaches and, as a result, this class of variation is poorly characterized. However, several recent studies have demonstrated that copy-number variation of VNTRs can modify local gene expression, epigenetics, and human traits, indicating that many have a functional role. Here, using read depth from whole-genome sequencing to profile copy number, we report results of a phenome-wide association study (PheWAS) of VNTRs and multicopy genes in a discovery cohort of ∼35,000 samples, identifying 32 traits associated with copy number of 38 VNTRs and multicopy genes at 1% FDR. We replicated many of these signals in an independent cohort and observed that VNTRs showing trait associations were significantly enriched for expression QTLs with nearby genes, providing strong support for our results. Fine-mapping studies indicated that in the majority (∼90%) of cases, the VNTRs and multicopy genes we identified represent the causal variants underlying the observed associations. Furthermore, several lie in regions where prior SNV-based GWASs have failed to identify any significant associations with these traits. Our study indicates that copy number of VNTRs and multicopy genes contributes to diverse human traits and suggests that complex structural variants potentially explain some of the so-called "missing heritability" of SNV-based GWASs.