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Class III peroxidases (PODs) are plant-specific glycoproteins, that play essential roles in various plant physiological processes and defence responses. To date, scarce information is available about the POD gene family in soybean. Hence, the present study is the first comprehensive report about the genome-wide characterization of GmPOD gene family in soybean (Glycine max L.). Here, we identified a total of 124 GmPOD genes in soybean, that are unevenly distributed across the genome. Phylogenetic analysis classified them into six distinct sub-groups (A-F), with one soybean specific subgroup. Exon-intron and motif analysis suggested the existence of structural and functional diversity among the sub-groups. Duplication analysis identified 58 paralogous gene pairs; segmental duplication and positive/Darwinian selection were observed as the major factors involved in the evolution of GmPODs. Furthermore, RNA-seq analysis revealed that 23 out of a total 124 GmPODs showed differential expression between drought-tolerant (DTL and DTP) and drought-sensitive (DSL and DSP) genotypes under stress conditions; however, two of them (GmPOD40 and GmPOD42) revealed the maximum deregulation in all contrasting genotypes. Overexpression (OE) lines of GsPOD40 showed considerably higher drought tolerance compared to wild type (WT) plants under stress treatment. Moreover, the OE lines showed enhanced photosynthesis and enzymatic antioxidant activities under drought stress, resulting in alleviation of ROS induced oxidative damage. Hence, the GsPOD40 enhanced drought tolerance in soybean by regulating the key physiological and biochemical pathways involved in the defence response. Lastly, the results of our study will greatly assist in further functional characterization of GsPODs in plant growth and stress tolerance in soybean.

Vertebrate photoreceptors are categorized into two broad classes, rods and cones, responsible for dim- and bright-light vision, respectively. While many molecular features that distinguish rods and cones are known, gene expression differences among cone subtypes remain poorly understood. Teleost fishes are renowned for the diversity of their photoreceptor systems. Here, we used single-cell RNA-seq to profile adult photoreceptors in zebrafish, a teleost. We found that in addition to the four canonical zebrafish cone types, there exist subpopulations of green and red cones (previously shown to be located in the ventral retina) that express red-shifted opsin paralogs (opn1mw4 or opn1lw1) as well as a unique combination of cone phototransduction genes. Furthermore, the expression of many paralogous phototransduction genes is partitioned among cone subtypes, analogous to the partitioning of the phototransduction paralogs between rods and cones seen across vertebrates. The partitioned cone-gene pairs arose via the teleost-specific whole-genome duplication or later clade-specific gene duplications. We also discovered that cone subtypes express distinct transcriptional regulators, including many factors not previously implicated in photoreceptor development or differentiation. Overall, our work suggests that partitioning of paralogous gene expression via the action of differentially expressed transcriptional regulators enables diversification of cone subtypes in teleosts.

Target enrichment (such as Hyb-Seq) is a well-established high throughput sequencing method that has been increasingly used for phylogenomic studies. Unfortunately, current widely used pipelines for analysis of target enrichment data do not have a vigorous procedure to remove paralogs in target enrichment data. In this study, we develop a pipeline we call Putative Paralogs Detection (PPD) to better address putative paralogs from enrichment data. The new pipeline is an add-on to the existing HybPiper pipeline, and the entire pipeline applies criteria in both sequence similarity and heterozygous sites at each locus in the identification of paralogs. Users may adjust the thresholds of sequence identity and heterozygous sites to identify and remove paralogs according to the level of phylogenetic divergence of their group of interest. The new pipeline also removes highly polymorphic sites attributed to errors in sequence assembly and gappy regions in the alignment. We demonstrated the value of the new pipeline using empirical data generated from Hyb-Seq and the Angiosperms353 kit for two woody genera Castanea (Fagaceae, Fagales) and Hamamelis (Hamamelidaceae, Saxifragales). Comparisons of data sets showed that the PPD identified many more putative paralogs than the popular method HybPiper. Comparisons of tree topologies and divergence times showed evident differences between data from HybPiper and data from our new PPD pipeline. We further evaluated the accuracy and error rates of PPD by BLAST mapping of putative paralogous and orthologous sequences to a reference genome sequence of Castanea mollissima. Compared to HybPiper alone, PPD identified substantially more paralogous gene sequences that mapped to multiple regions of the reference genome (31 genes for PPD compared with 4 genes for HybPiper alone). In conjunction with HybPiper, paralogous genes identified by both pipelines can be removed resulting in the construction of more robust orthologous gene data sets for phylogenomic and divergence time analyses. Our study demonstrates the value of Hyb-Seq with data derived from the Angiosperms353 probe set for elucidating species relationships within a genus, and argues for the importance of additional steps to filter paralogous genes and poorly aligned regions (e.g., as occur through assembly errors), such as our new PPD pipeline described in this study. [Angiosperms353; Castanea; divergence time; Hamamelis; Hyb-Seq, paralogs, phylogenomics.]

Genomika porównawcza i biologia systemowa oferują niespotykane możliwości testowania głównych zasad biologii ewolucyjnej sformułowanych przez Darwina w O powstawaniu gatunków w 1859 roku i rozszerzonych sto lat później w ramach Nowoczesnej Syntezy. Badania w dziedzinie genomiki ewolucyjnej pokazują, że dobór naturalny stanowi tylko jedną z sił kształtujących ewolucję genomu i wcale nie występującą najczęściej, natomiast procesy nieadaptacyjne mają znacznie większe znaczenie niż wcześniej przypuszczano. Duży wkład horyzontalnego transferu genów i różnych samolubnych elementów genetycznych w ewolucję genomu podważa koncepcję drzewa życia. Adekwatny opis ewolucji wymaga bardziej złożonej koncepcji sieci lub „lasu” życia. Nie istnieje spójny trend ewolucji w kierunku większej złożoności genomowej, a kiedy złożoność wzrasta, wydaje się, że jest to raczej nieadaptacyjna konsekwencja ewolucji drogą słabego doboru oczyszczającego niż adaptacji. Odkryto rozmaite powszechniki ewolucji genomu, w tym niezmiennicze rozkłady tempa ewolucji pośród genów ortologowych z różnych genomów oraz rozmiarów rodzin genów paralogowych. Dostrzeżono też negatywną korelację między poziomem ekspresji genów a tempem ewolucji sekwencji. Niektóre z tych powszechników uzyskują wyjaśnienie dzięki zastosowaniu prostych, nieadaptacjonistycznych modeli ewolucji, co sugeruje, że w dość nieodległej przyszłości powstać może nowa synteza biologii ewolucyjnej.

Microorganisms harbor bulks of functionally similar or undefined genes, which belong to paralogous gene family. There is a necessity of exploring combinatorial or interactive functions of these genes, but conventional loss-of-function strategy with one-by-one rounds suffers extremely low efficiency for generating mutant library with all genes permutations. Here, taking histidine kinases (HKs) in Clostridium acetobutylicum as a proof-of-concept, we developed a multi-plasmid cotransformation strategy for generating all theoretical HKs combinations in one round. For five HKs with 31 theoretical combinations, the library containing 22 mutants within all the possible HKs-inactivated combinations was constructed with 11 days compared to 242 days by conventional strategy, while the other 9 combinations cannot survive. Six mutants with the enhanced butanol production and tolerance were obtained with changes of cell development during fermentation, one of which could produce 54.2% more butanol (56.4% more solvents), while the butanol production of other mutants was unchanged or decreased. The cotransformation strategy demonstrated potentials for fast exploring pleiotropic function of paralogous family genes in cell survival, cell development, and target product metabolism. This article is protected by copyright. All rights reserved.

To improve livestock and aquaculture-raised fish as food, targeted mutagenesis using genome editing technologies is becoming more realizable. Myostatin (Mstn), which functions as the negative regulator of skeletal muscle growth, is one of the major targets to improve the edible ratio of livestock and farmed fish. We previously reported that the deficiency of Pm-mstn, one of Myostatin paralogs, improves muscle growth and changes body shape in a finfish species, red seabream (Pagrus major), as a result of editing the gene by means of CRISPR/Cas9. In this study, we established Pm-mstnb-deficient red seabream, which is a null-allelic mutant of another paralogous gene of Myostatin in the species, and analyzed their phenotype in terms of growth traits and body shape. A comparison of all growth traits between Pm-mstnbwt/wt and Pm-mstnb-5/-5 revealed no significant differences. In addition, all metrics for body shape, defined as the ratios of body depth, body width, and depth of the caudal peduncle to body length, respectively, were also similar in Pm-mstnbwt/wt and Pm-mstnb-5/-5. Therefore, we concluded that Pm-mstnb does not function as a negative regulator of skeletal muscle growth in red seabream.

Plants regularly experience multiple abiotic and biotic pressures affecting their normal development. The 90-kDa heat shock protein (HSP90) plays a dynamic role in countering abiotic and biotic stresses via a plethora of functional mechanisms. The HSP90 has been investigated in many plant species. However, there is little information available about this gene family in the cultivated Mediterranean olive tree, Olea europaea subsp. europaea var. europaea. In the current study, we systematically performed genome-wide identification and characterization of the HSP90 gene family in O. europaea var. europaea (OeHSP90s). Twelve regular OeHSP90s were identified, which were phylogenetically grouped into two major clusters and four sub-clusters, showing five paralogous gene pairs evolving under purifying selection. All of the 12 proteins contained a Histidine kinase-like ATPase (HATPase_c) domain, justifying the role played by HSP90 proteins in ATP binding and hydrolysis. The predicted 3D structure of OeHSP90 proteins provided information to understand their functions at the biochemical level. Consistent with their phylogenetic relationships, OeHSP90 members were predicted to be localized in different cellular compartments, suggesting their involvement in various subcellular processes. In consonance with their spatial organization, olive HSP90 family members were found to share similar motif arrangements and similar number of exons. We found that OeHSP90 promoters contained various cis-acting elements associated with light responsiveness, hormone signaling pathways and reaction to various stress conditions. In addition, expression sequence tags (ESTs) analysis offered a view of OeHSP90 tissue- and developmental stage specific pattern of expression. Proteins interacting with OeHSP90s were predicted and their potential roles were discussed. Overall, our results offer premises for further investigation of the implication of HSP90 genes in the physiological processes of the olive and its adaptation to stresses.

Few ophiostomatoid fungi have been reported from the margin of the Tibetan Plateau and none have been found in the central portion of the region. In a survey of ophiostomatoid fungi associated with spruce bark beetles in Tibetan subalpine forests, numerous strains of Leptographium s. l. (Ophiostomataceae) were isolated from Orthotomicus sp. (Coleoptera: Scolytinae) and its galleries infesting Picea likiangensis var. balfouriana. Morphological characters and phylogenetic analysis based on multiple DNA sequence data (ITS2-partial LSU rDNA region, beta-tubulin and transcription elongation factor-1α genes) revealed a new species in the “Grosmannia penicillata complex”, which is proposed as G. tibetensis. The species is characterized by both Leptographium and Pesotum asexual states, which is unique in the “G. penicillata complex”. Additionally, sequences of the tubC paralogue gene were found combining with tub2 sequences in many species of the “G. penicillata complex”, resulting in incongruent trees. This is the first report of tubulin paralogue genes in ophiostomatoid fungi. Gene duplication and losses make beta-tubulin a potentially challenging locus for use as a molecular marker for tracing speciation.

SUMMARY Acer truncatum (purpleblow maple) is a woody tree species that produces seeds with high levels of valuable fatty acids (especially nervonic acid). However, the lack of a complete genome sequence has limited both basic and applied research on A. truncatum. We describe a high‐quality draft genome assembly comprising 633.28 Mb (contig N50 = 773.17 kb; scaffold N50 = 46.36 Mb) with at least 28 438 predicted genes. The genome underwent an ancient triplication, similar to the core eudicots, but there have been no recent whole‐genome duplication events. Acer yangbiense and A. truncatum are estimated to have diverged about 9.4 million years ago. A combined genomic, transcriptomic, metabonomic, and cell ultrastructural analysis provided new insights into the biosynthesis of very long‐chain monounsaturated fatty acids. In addition, three KCS genes were found that may contribute to regulating nervonic acid biosynthesis. The KCS paralogous gene family expanded to 28 members, with 10 genes clustered together and distributed in the 0.27‐Mb region of pseudochromosome 4. Our chromosome‐scale genomic characterization may facilitate the discovery of agronomically important genes and stimulate functional genetic research on A. truncatum. Furthermore, the data presented also offer important foundations from which to study the molecular mechanisms influencing the production of nervonic acids., Significance Statement Here, we provide the first report on the Acer truncatum genome. Our work provides extensive genetic resources necessary for very long‐chain monounsaturated fatty acid biosynthesis.

International audience; In human, as in the other mammals, the neuroendocrine control of reproduction is ensured by the brain-pituitary gonadotropic axis. Multiple internal and environmental cues are integrated via brain neuronal networks, ultimately leading to the modulation of the activity of gonadotropin-releasing hormone (GnRH) neurons. The decapeptide GnRH is released into the hypothalamic-hypophyseal portal blood system, and stimulates the production of pituitary glycoprotein hormones, the two gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH). A novel actor, the neuropeptide Kiss, acting upstream of GnRH, has attracted increasing attention in recent years. Other neuropeptides, such as gonadotropin-inhibiting hormone (GnIH)/ RF-amide related peptide (RFRP), and other members of the RF-amide peptide superfamily, as well as various non-peptidic neuromediators such has dopamine, serotonin also provide a large panel of stimulatory or inhibitory regulators. This paper addresses the origin and evolution of the vertebrate gonadotropic axis. Brain-pituitary neuroendocrine axes are typical of vertebrates, the pituitary gland, mediator and amplifier of brain control on peripheral organs, being a vertebrate innovation. The paper reviews, from molecular and functional perspectives, the evolution across vertebrate radiation of some key-actors of the vertebrate neuroendocrine control of reproduction, and traces back their origin along the vertebrate lineage and in other metazoa before the emergence of vertebrates. A focus is given on how gene duplications, resulting from either local events or from whole genome duplication events (WGD), and followed by paralogous gene loss or conservation, might have shaped the evolutionary scenarios of current families of key-actors of the gonadotropic axis.

Graphical abstract, Crocus sativus, containing remarkably amounts of crocin, picrocrocin and safranal, is the source of saffron with tremendous medicinal, economic and cultural importance. Here, we present a high-quality full-length transcriptome of the sterile triploid C. sativus, using the PacBio SMRT sequencing technology. This yields 31,755 high-confidence predictions of protein-coding genes, with 50.1% forming paralogous gene pairs. Analysis on distribution of Ks values suggests that the current genome of C. sativus is probably a product resulting from at least two rounds of whole-genome duplication (WGD) events occurred at ~28 and ~114 million years ago (Mya), respectively. We provide evidence demonstrating that the recent β WGD event confers a major impact on family expansion of secondary metabolite genes, possibly leading to an enhanced accumulation of three distinct compounds: crocin, picrocrocin and safranal. Phylogenetic analysis unravels that the founding member (CCD2) of CCD enzymes necessary for the biosynthesis of apocarotenoids in C. sativus might be evolved from the CCD1 family via the β WGD event. Based on the gene expression profiling, CCD2 is found to be expressed at an extremely high level in the stigma. These findings may shed lights on further genomic refinement of the characteristic biosynthesis pathways and promote germplasm utilization for the improvement of saffron quality.

The Lateral Organ Boundaries Domain (LBD) genes encode highly conserved plant-specific LOB domain proteins which regulate growth and development in various species. However, members of the LBD gene family have yet to be identified in Brassica rapa var. rapa. In the present study, fifty-nine LBD genes were identified and distributed on 10 chromosomes. The BrrLBD proteins are predicted to encode hydrophobic polypeptides between 118 and 394 amino acids in length and with molecular weights ranging from 13.31 to 44.24 kDa; the theoretical pI for these proteins varies from 4.83 to 9.68. There were 17 paralogous gene pairs in the BrrLBD family, suggesting that the amplification of the BrrLBD gene family involved large-scale gene duplication events. Members of the BrrLBD family were divided into 7 subclades (class I a to e, class II a and b). Analysis of gene structure and conserved domains revealed that most BrrLBD genes of the same subclade had similar gene structures and protein motifs. The expression profiles of 59 BrrLBD genes were determined through Quantitative Real-time fluorescent PCR (qRT-PCR). Most BrrLBD genes in the same subclade had similar gene expression profiles. However, the expression patterns of 7 genes differed from their duplicates, indicating that although the gene function of most BrrLBD genes has been conserved, some BrrLBD genes may have undergone evolutionary change. Keywords: LBD, Transcription factors, LBD gene sequence analysis, Expression profiles, Brassica rapa var. rapa

MAIN CONCLUSION Genome wide analysis, expression pattern analysis, and functional characterization of RAV genes highlight their roles in roots, stem development and hormonal response. RAV (Related to ABI3 and VP1) gene family members have been involved in tissues/organs growth and hormone signaling in various plant species. Here, we identified 247 RAVs from 12 different species with 33 RAV genes from G. hirsutum. Phylogenetic analysis classified RAV genes into four distinct groups. Analysis of gene structure showed that most GhRAVs lack introns. Motif distribution pattern and protein sequence logos indicated that GhRAV genes were highly conserved during the process of evolution. Promotor cis-acting elements revealed that promotor regions of GhRAV genes encode numerous elements related to plant growth, abiotic stresses and phytohormones. Chromosomal location information showed uneven distribution of 33 GhRAV genes on different chromosomes. Collinearity analysis identified 628 and 52 orthologous/ paralogous gene pairs in G. hirsutum and G. barbadense, respectively. Ka/Ks values indicated that GhRAV and GbRAV genes underwent strong purifying selection pressure. Selecton model and codon model selection revealed that GhRAV amino acids were under purifying selection and adaptive evolution exists among GhRAV proteins. Three dimensional structure of GhRAVs indicated the presence of numerous alpha helix and beta-barrels. Expression level revealed that some GhRAV genes exhibited high expression in roots (GhRAV3, GhRAV4, GhRAV11, GhRAV18, GhRAV20 and GhRAV30) and stem (GhRAV3 and GhRAV18), indicating their potential role in roots and stem development. GhRAV genes can be regulated by phytohormonal stresses (BL, JA and IAA). Our study provides a reference for future studies related to the functional analysis of GhRAVs in cotton.

Protein disulfide isomerases (PDI) and PDI-like proteins catalyze the formation and isomerization of protein disulfide bonds in the endoplasmic reticulum and prevent the buildup of misfolded proteins under abiotic stress conditions. In the present study, we conducted the first comprehensive genome-wide exploration of the PDI gene family in tomato (Solanum lycopersicum L.). We identified 19 tomato PDI genes that were unevenly distributed on 8 of the 12 tomato chromosomes, with segmental duplications detected for 3 paralogous gene pairs. Expression profiling of the PDI genes revealed that most of them were differentially expressed across different organs and developmental stages of the fruit. Furthermore, most of the PDI genes were highly induced by heat, salt, and abscisic acid (ABA) treatments, while relatively few of the genes were induced by cold and nutrient and water deficit (NWD) stresses. The predominant expression of SlPDI1-1, SlPDI1-3, SlPDI1-4, SlPDI2-1, SlPDI4-1, and SlPDI5-1 in response to abiotic stress and ABA treatment suggested they play regulatory roles in abiotic stress tolerance in tomato in an ABA-dependent manner. Our results provide new insight into the structure and function of PDI genes and will be helpful for the selection of candidate genes involved in fruit development and abiotic stress tolerance in tomato.

The heat shock protein 90 (Hsp90) is a protein produced in plants in response to stress. This study identified and analyzed Hsp90 gene family members in the perennial ryegrass genome. From the results, eight Hsp90 proteins were obtained and their MW, pI and number of amino acid bases varied. The amino acid bases ranged from 526 to 862. The CDS also ranged from 20 (LpHsp0-4) to 1 (LpHsp90-5). The least number of CDS regions was 1 (LpHsp90-5) with 528 kb amino acids, while the highest was 20 (LpHsp90-4) with 862 kb amino acids, which showed diversity among the protein sequences. The phylogenetic tree revealed that Hsp90 genes in Lolium perenne, Arabidopsis thaliana, Oryza sativa and Brachypodium distachyon could be divided into two groups with five paralogous gene pairs and three orthologous gene pairs. The expression analysis after perennial ryegrass was subjected to heat, salt, chromium (Cr), cadmium (Cd), polyethylene glycol (PEG) and abscisic acid (ABA) revealed that LpHsp90 genes were generally highly expressed under heat stress, but only two LpHsp90 proteins were expressed under Cr stresses. Additionally, the expression of the LpHsp90 proteins differed at each time point in all treatments. This study provides the basis for an understanding of the functions of LpHsp90 proteins in abiotic stress studies and in plant breeding.

Brassinosteroids (BRs), an efficient plant endogenous hormone, significantly promotes plant nutrient growth adapting to biological and abiotic adversities. BRs mainly promote plant cell elongation by regulating gene expression patterns. EXORDIUM (EXO) genes have been characterized as the indicators of BR response genes. Cotton, an ancient crop, is of great economic value and its fibers can be made into all kinds of fabrics. However, EXO gene family genes have not been full identified in cotton. 175 EXO genes were identified in nine plant species, of which 39 GhEXO genes in Gossypium hirsutum in our study. A phylogenetic analysis grouped all of the proteins encoded by the EXO genes into five major clades. Sequence identification of conserved amino acid residues among monocotyledonous and dicotyledonous species showed a high level of conservation across the N and C terminal regions. Only 25% the GhEXO genes contain introns besides conserved gene structure and protein motifs distribution. The 39 GhEXO genes were unevenly distributed on the 18 At and Dt sub-genome chromosomes. Most of the GhEXO genes were derived from gene duplication events, while only three genes showed evidence of tandem duplication. Homologous locus relationships showed that 15 GhEXO genes are located on collinear blocks and that all orthologous/paralogous gene pairs had Ka > Ks values, indicating purifying selection pressure. The GhEXO genes showed ubiquitous expression in all eight tested cotton tissues and following exposure to three phytohormones, IAA, GA, and BL. Furthermore, GhEXO7_At was mainly expressed in response to BL treatment, and was predominantly expressed in the fibers. GhEXO7_At was found to be a plasma membrane protein, and its ectopic expression in Arabidopsis mediated BR-regulated plant growth and development with altered expression of DWF4, CPD, KCS1, and EXP5. Additionally, the functions of GhEXO7_At were confirmed by virus-induced gene silencing (VIGS) in cotton. This study will provide important genetic resources for future cotton breeding programs.

The survival of viruses depends on their ability to resist host defenses and, of all animal virus families, the poxviruses have the most antidefense genes. Orthopoxviruses (ORPV), a genus within the subfamily Chordopoxvirinae, infect diverse mammals and include one of the most devastating human pathogens, the now eradicated smallpox virus. ORPV encode ∼200 genes, of which roughly half are directly involved in virus genome replication and expression as well as virion morphogenesis. The remaining ∼100 “accessory” genes are responsible for virus-host interactions, particularly counter-defense of innate immunity. Complete sequences are currently available for several hundred ORPV genomes isolated from a variety of mammalian hosts, providing a rich resource for comparative genomics and reconstruction of ORPV evolution. To identify the provenance and evolutionary trends of the ORPV accessory genes, we constructed clusters including the orthologs of these genes from all chordopoxviruses. Most of the accessory genes were captured in three major waves early in chordopoxvirus evolution, prior to the divergence of ORPV and the sister genus Centapoxvirus from their common ancestor. The capture of these genes from the host was followed by extensive gene duplication, yielding several paralogous gene families. In addition, nine genes were gained during the evolution of ORPV themselves. In contrast, nearly every accessory gene was lost, some on multiple, independent occasions in numerous lineages of ORPV, so that no ORPV retains them all. A variety of functional interactions could be inferred from examination of pairs of ORPV accessory genes that were either often or rarely lost concurrently. IMPORTANCE Orthopoxviruses (ORPV) include smallpox (variola) virus, one of the most devastating human pathogens, and vaccinia virus, comprising the vaccine used for smallpox eradication. Among roughly 200 ORPV genes, about half are essential for genome replication and expression as well as virion morphogenesis, whereas the remaining half consists of accessory genes counteracting the host immune response. We reannotated the accessory genes of ORPV, predicting the functions of uncharacterized genes, and reconstructed the history of their gain and loss during the evolution of ORPV. Most of the accessory genes were acquired in three major waves antedating the origin of ORPV from chordopoxviruses. The evolution of ORPV themselves was dominated by gene loss, with numerous genes lost at the base of each major group of ORPV. Examination of pairs of ORPV accessory genes that were either often or rarely lost concurrently during ORPV evolution allows prediction of different types of functional interactions.

The APETALA2 (AP2)/ethylene response factor plays vital functions in response to environmental stimulus. The ethylene response factor (ERF) subfamily B3 group belongs to the AP2/ERF superfamily and contains a single AP2/ERF domain. Phylogenetic analysis of the ERF subfamily B3 group genes from Arabdiposis thaliana, Gossypium arboreum, Gossypium hirsutum, and Gossypium raimondii made it possible to divide them into three groups and showed that the ERF subfamily B3 group genes are conserved in cotton. Collinearity analysis identified172 orthologous/paralogous gene pairs between G. arboreum and G. hirsutum; 178 between G. hirsutum and G. raimondii; and 1,392 in G. hirsutum. The GhERF subfamily B3 group gene family experienced massive gene family expansion through either segmental or whole genome duplication events, with most genes showing signature compatible with the action of purifying selection during evolution. Most G. hirsutum ERF subfamily B3 group genes are responsive to salt stress. GhERF13.12 transgenic Arabidopsis showed enhanced salt stress tolerance and exhibited regulation of related biochemical parameters and enhanced expression of genes participating in ABA signaling, proline biosynthesis, and ROS scavenging. In addition, the silencing of the GhERF13.12 gene leads to increased sensitivity to salt stress in cotton. These results indicate that the ERF subfamily B3 group had remained conserved during evolution and that GhERF13.12 induces salt stress tolerance in Arabidopsis and cotton.

Dendrobium is a semi-shade epiphytic Orchidaceae herb with important ornamental and medicinal value. Parts of the cultivation of Dendrobium germplasm resources, as well as the identification of medicinal components, are more studied, but the functional characterization of the flowering regulation in Dendrobium plants is less reported. Here, six PEBP family genes (DhFT3, DhFT1, DhMFT, DhTFL1b, DhFT2, and DhTFL1a) were identified from the Dendrobium huoshanense genome. The chromosome-level mapping showed that these genes were sequentially distributed on chromosomes 6, 9, 15, and 17. The paralogous gene DhTFL1b corresponded to DhTFL1a, which was determined through tandem duplication. The gene structure and conserved motif of DhPEBP indicated five PEBP genes apart from DhMFT contained four exons and three introns entirely. The phylogeny analysis showed that the PEBP gene family in A. thaliana, O. sativa, Z. mays, S. lycopersicum, and P. equestris were classified into three subclades, FT, TFL, and MFT, which maintained a high homology with D. huoshanense. The conserved domain of the amino acid demonstrated that two highly conserved short motifs (DPDXP and GXHR) embed in DhPEBPs, which may contribute to the conformation of the ligand binding bag. The 86th position of DhFTs was tyrosine (Y), while the 83th and 87th of DhTFL1s belonged to histidine (H), suggesting they should have distinct functions in flowering regulation. The promoter of six DhPEBPs contained several cis-elements related to hormone induction, light response, and abiotic stress, which indicated they could be regulated by the environmental stress and endogenous signaling pathways. The qRT-PCR analysis of DhPEBPs in short-term days induced by GA indicated the gene expressions of all DhFTs were gradually increased, whereas the expression of DhTFL1 was decreased. The results implied that DhPEBPs have various regulatory functions in modulating flowering, which will provide a scientific reference for the flowering regulation of Dendrobium plants.

The genus Rhododendron (Ericaceae), which includes horticulturally important plants such as azaleas, is a highly diverse and widely distributed genus of >1,000 species. Here, we report the chromosome-scale de novo assembly and genome annotation of Rhododendron williamsianum as a basis for continued study of this large genus. We created multiple short fragment genomic libraries, which were assembled using ALLPATHS-LG. This was followed by contiguity preserving transposase sequencing (CPT-seq) and fragScaff scaffolding of a large fragment library, which improved the assembly by decreasing the number of scaffolds and increasing scaffold length. Chromosome-scale scaffolding was performed by proximity-guided assembly (LACHESIS) using chromatin conformation capture (Hi-C) data. Chromosome-scale scaffolding was further refined and linkage groups defined by restriction-site associated DNA (RAD) sequencing of the parents and progeny of a genetic cross. The resulting linkage map confirmed the LACHESIS clustering and ordering of scaffolds onto chromosomes and rectified large-scale inversions. Assessments of the R. williamsianum genome assembly and gene annotation estimate them to be 89% and 79% complete, respectively. Predicted coding sequences from genome annotation were used in syntenic analyses and for generating age distributions of synonymous substitutions/site between paralgous gene pairs, which identified whole-genome duplications (WGDs) in R. williamsianum. We then analyzed other publicly available Ericaceae genomes for shared WGDs. Based on our spatial and temporal analyses of paralogous gene pairs, we find evidence for two shared, ancient WGDs in Rhododendron and Vaccinium (cranberry/blueberry) members that predate the Ericaceae family and, in one case, the Ericales order.

Background Chlamydia are ancient intracellular pathogens with reduced, though strikingly conserved genome. Despite their parasitic lifestyle and isolated intracellular environment, these bacteria managed to avoid accumulation of deleterious mutations leading to subsequent genome degradation characteristic for many parasitic bacteria. Results We report pan-genomic analysis of sixteen species from genus Chlamydia including identification and functional annotation of orthologous genes, and characterization of gene gains, losses, and rearrangements. We demonstrate the overall genome stability of these bacteria as indicated by a large fraction of common genes with conserved genomic locations. On the other hand, extreme evolvability is confined to several paralogous gene families such as polymorphic membrane proteins and phospholipase D, and likely is caused by the pressure from the host immune system. Conclusions This combination of a large, conserved core genome and a small, evolvable periphery likely reflect the balance between the selective pressure towards genome reduction and the need to adapt to escape from the host immunity.

Background INDETERMINATE DOMAIN (IDD) transcription factors form one of the largest and most conserved gene families in plant kingdom and play important roles in various processes of plant growth and development, such as flower induction in term of flowering control. Till date, systematic and functional analysis of IDD genes remained infancy in cotton. Results In this study, we identified total of 162 IDD genes from eight different plant species including 65 IDD genes in Gossypium hirsutum. Phylogenetic analysis divided IDDs genes into seven well distinct groups. The gene structures and conserved motifs of GhIDD genes depicted highly conserved exon-intron and protein motif distribution patterns. Gene duplication analysis revealed that among 142 orthologous gene pairs, 54 pairs have been derived by segmental duplication events and four pairs by tandem duplication events. Further, Ka/Ks values of most of orthologous/paralogous gene pairs were less than one suggested the purifying selection pressure during evolution. Spatiotemporal expression pattern by qRT-PCR revealed that most of the investigated GhIDD genes showed higher transcript levels in ovule of seven days post anthesis, and upregulated response under the treatments of multiple abiotic stresses. Conclusions Evolutionary analysis revealed that IDD gene family was highly conserved in plant during the rapid phase of evolution. Whole genome duplication, segmental as well as tandem duplication significantly contributed to the expansion of IDD gene family in upland cotton. Some distinct genes evolved into special subfamily and indicated potential role in the allotetraploidy Gossypium hisutum evolution and development. High transcript levels of GhIDD genes in ovules illustrated their potential roles in seed and fiber development. Further, upregulated responses of GhIDD genes under the treatments of various abiotic stresses suggested them as important genetic regulators to improve stress resistance in cotton breeding.

4,5-DOPA dioxygenase (DODA) is a crucial enzyme in the biosynthetic pathway of betalain. Previous studies have shown that DODA is present in plants, fungi and bacteria. Using updated data, here we show that DODA genes (BmDODA) in the domestic silkworm (Bombyx mori) and other lepidopteran insects are most likely to be horizontally transferred from fungi. A synteny analysis indicated that BmDODA1 is orthologous to other lepidopteran DODAs and that BmDODA2 is a paralogous gene. To explore the function of DODA in Lepidoptera, we first examined the expression patterns of BmDODA1. BmDODA1 showed high transcriptional and translational levels in the midgut and head. Then, we exogenously expressed the BmDODA1 gene, detected 4,5-DOPA ring-cleaving activity and calculated the kinetic parameters of the recombinant BmDODA1. We found that the transcription levels of BmDODA1 were significantly induced by the pathogens Bacillus bombyseptieus and Escherichia coli. Thus, the horizontal transfer of the BmDODA gene in the silkworm may be involved in dopa metabolism and contribute to antimicrobial activity in this species. Our results provide a documented example of functional horizontal gene transfer (HGT) between fungi and animals and expand our knowledge of HGT amongst eukaryotes.

The Asteraceae (Compositae), a large plant family of approximately 24 000–35 000 species, accounts for ∼10% of all angiosperm species and contributes a lot to plant diversity. The most representative members of the Asteraceae are the economically important chrysanthemums (Chrysanthemum L.) that diversified through reticulate evolution. Biodiversity is typically created by multiple evolutionary mechanisms such as whole-genome duplication (WGD) or polyploidization and locally repetitive genome expansion. However, the lack of genomic data from chrysanthemum species has prevented an in-depth analysis of the evolutionary mechanisms involved in their diversification. Here, we used Oxford Nanopore long-read technology to sequence the diploid Chrysanthemum nankingense genome, which represents one of the progenitor genomes of domesticated chrysanthemums. Our analysis revealed that the evolution of the C. nankingense genome was driven by bursts of repetitive element expansion and WGD events including a recent WGD that distinguishes chrysanthemum from sunflower, which diverged from chrysanthemum approximately 38.8 million years ago. Variations of ornamental and medicinal traits in chrysanthemums are linked to the expansion of candidate gene families by duplication events including paralogous gene duplication. Collectively, our study of the assembled reference genome offers new knowledge and resources to dissect the history and pattern of evolution and diversification of chrysanthemum plants, and also to accelerate their breeding and improvement.

The TIFY gene family, a key plant-specific transcription factor (TF) family, is involved in diverse biological processes including plant defense and growth regulation. Despite TIFY proteins being reported in some plant species, a genome-wide comparative and comprehensive analysis of TIFY genes in plant species can reveal more details. In the current study, the members of the TIFY gene family were significantly increased by the identification of 18 and six new members using maize and tomato reference genomes, respectively. Thus, a genome-wide comparative analysis of the TIFY gene family between 48 tomato (Solanum lycopersicum, a dicot plant) genes and 26 maize (Zea mays, a monocot plant) genes was performed in terms of sequence structure, phylogenetics, expression, regulatory systems, and protein interaction. The identified TIFYs were clustered into four subfamilies, namely, TIFY-S, JAZ, ZML, and PPD. The PPD subfamily was only detected in tomato. Within the context of the biological process, TIFY family genes in both studied plant species are predicted to be involved in various important processes, such as reproduction, metabolic processes, responses to stresses, and cell signaling. The Ka/Ks ratios of the duplicated paralogous gene pairs indicate that all of the duplicated pairs in the TIFY gene family of tomato have been influenced by an intense purifying selection, whereas in the maize genome, there are three duplicated blocks containing Ka/Ks > 1, which are implicated in evolution with positive selection. The amino acid residues present in the active site pocket of TIFY proteins partially differ in each subfamily, although the Mg or Ca ions exist heterogeneously in the centers of the active sites of all the predicted TIFY protein models. Based on the expression profiles of TIFY genes in both plant species, JAZ subfamily proteins are more associated with the response to abiotic and biotic stresses than other subfamilies. In conclusion, globally scrutinizing and comparing the maize and tomato TIFY genes showed that TIFY genes play a critical role in cell reproduction, plant growth, and responses to stress conditions, and the conserved regulatory mechanisms may control their expression.

Background E2F/DP proteins have been shown to regulate genes implicated in cell cycle control and DNA repair. However, to date, research into the potential role of the Moso bamboo E2F/DP family has been limited. Results Here, we identified 23 E2F/DPs in the Moso bamboo genome, including nine E2F genes, six DP genes, eight DEL genes and one gene with a partial E2F domain. An estimation of the divergence time of the paralogous gene pairs suggested that the E2F/DP family expansion primarily occurred through a whole-genome duplication event. A regulatory element and coexpression network analysis indicated that E2F/DP regulated the expression of cell cycle-related genes. A yeast two-hybrid assay and expression analysis based on transcriptome data and in situ hybridization indicated that the PheE2F-PheDP complex played important roles in winter Moso bamboo shoot growth. The qRT-PCR results showed that the PheE2F/DPs exhibited diverse expression patterns in response to drought and salt treatment and diurnal cycles. Conclusion Our findings provide novel insights into the Moso bamboo E2F/DP family and partial experimental evidence for further functional verification of the PheE2F/DPs.

Modules of interacting proteins regulate vesicle budding and fusion in eukaryotes. Distinct paralogous copies of these modules act at distinct sub-cellular locations. The processes by which such large gene modules are duplicated and retained remain unclear. Here we show that interspecies hybridization is a potent source of paralogous gene modules. We study the dynamics of paralog doublets derived from the 100-million-year-old hybridization event that gave rise to the whole genome duplication clade of budding yeast. We show that paralog doublets encoding vesicle traffic proteins are convergently retained across species. Vesicle coats and adaptors involved in secretory and early-endocytic pathways are retained as doublets, while tethers and other machinery involved in intra-Golgi traffic and later endocytic steps are reduced to singletons. These patterns reveal common selective pressures that have sculpted traffic pathways in diverse yeast species. They suggest that hybridization may have played a pivotal role in the expansion of the endomembrane system.

Histone deacetylases (HDACs) are known as erasers that remove acetyl groups from lysine residues in histones. Although plant HDACs play essential roles in physiological processes, including various stress responses, our knowledge concerning HDAC gene families and their evolutionary relationship remains limited. In Brassica rapa genome, we identified 20 HDAC genes, which are divided into three major groups: RPD3/HDA1, HD2, and SIR2 families. In addition, seven pairs of segmental duplicated paralogs and one pair of tandem duplicated paralogs were identified in the B. rapa HDAC (BraHDAC) family, indicating that segmental duplication is predominant for the expansion of the BraHDAC genes. The expression patterns of paralogous gene pairs suggest a divergence in the function of BraHDACs under various stress conditions. Furthermore, we suggested that BraHDA3 (homologous of Arabidopsis HDA14) encodes the functional HDAC enzyme, which can be inhibited by Class I/II HDAC inhibitor SAHA. As a first step toward understanding the epigenetic responses to environmental stresses in Chinese cabbage, our results provide a solid foundation for functional analysis of the BraHDAC family.

The family Apiaceae includes many important vegetables and medicinal plants. Auxin response factors (ARFs) play critical roles in regulating plant growth and development. Here, we performed a comprehensive analysis of the ARF gene family in three Apiaceae species, celery, coriander, and carrot, and compared the results with the ARF gene family of lettuce, Arabidopsis, and grape. We identified 156 ARF genes in all six species and 89 genes in the three Apiaceae species, including 28, 34, and 27 in celery, coriander, and carrot, respectively. The paralogous gene number in coriander was far greater than that in carrot and celery. Our analysis revealed that ARF genes of the three Apiaceae species in 34 branches of the phylogenetic tree underwent significant positive selection. Additionally, our findings indicated that whole-genome duplication played an important role in ARF gene family expansion. Coriander contained a greater number of ARF genes than celery and carrot because of more gene duplications and less gene losses. We also analyzed the expression of ARF genes in three tissues by RNA-seq and verified the results by quantitative real-time PCR. Furthermore, we found that several paralogous genes exhibited divergent expression patterns. Overall, this study provides a valuable resource for exploring how ARF family genes regulate plant growth and development in other plants. Since this is the first report of the ARF gene family in Apiaceae, our results will serve as a guide for comparative and functional analyses of ARF and other gene families in Apiaceae.

A cDNA encoding NPGL was first identified in the chicken hypothalamus. The orthologous NPGL gene was found throughout vertebrates. In chicks, the central and peripheral infusion of NPGL increases body mass gain. In particular, the chronic intracerebroventricular infusion of NPGL in chicks stimulates food intake and fat accumulation. NPGL induces food intake and increases the mass of white adipose tissue through de novo lipogenesis in rodents. NPGL is a novel neuronal regulator that drives food intake and fat accumulation through de novo lipogenesis and acts to maintain a steady-state fat level. On the other hand, the database search found the presence of the paralogous gene, NPGM, in vertebrates. These small proteins may take part in the regulation of energy homeostasis in vertebrates.

The COBRA-like (COBL) gene family has been associated with the regulation of cell wall expansion and cellulose deposition. COBL mutants result in reduced levels and disorganized deposition of cellulose causing defects in the cell wall and inhibiting plant development. In this study, we report the identification of 24 COBL genes (GmCOBL) in the soybean genome. Phylogenetic analysis revealed that the COBL proteins are divided into two groups, which differ by about 170 amino acids in the N-terminal region. The GmCOBL genes were heterogeneously distributed in 14 of the 20 soybean chromosomes. This study showed that segmental duplication has contributed significantly to the expansion of the COBL family in soybean during all Glycine-specific whole-genome duplication events. The expression profile revealed that the expression of the paralogous genes is highly variable between organs and tissues of the plant. Only 20% of the paralogous gene pairs showed similar expression patterns. The high expression levels of some GmCOBLs suggest they are likely essential for regulating cell expansion during the whole soybean life cycle. Our comprehensive overview of the COBL gene family in soybean provides useful information for further understanding the evolution and diversification of COBL genes in soybean.

CRISPR-based forward genetic screening represents a powerful approach for the systematic characterization of gene function. Recent efforts have been directed toward establishing CRISPR-based tools for the programmable delivery of combinatorial genetic perturbations, most of which are mediated by a single nuclease and the expression of structurally identical guide backbones from two promoters. In contrast, we have developed CHyMErA (Cas hybrid for multiplexed editing and screening applications), which is based on the co-expression of Cas9 and Cas12a nucleases in conjunction with a hybrid guide RNA (hgRNA) engineered by the fusion of Cas9 and Cas12a guides and expressed from a single U6 promoter. CHyMErA is suitable for the high-throughput deletion of genetic segments including the excision of individual exons. Furthermore, CHyMErA enables the concomitant targeting of two or more genes and can thus be used for the systematic mapping of genetic interactions in mammalian cells. CHyMErA can also be applied for the perturbation of paralogous gene pairs, thereby allowing the capturing of phenotypic roles that would otherwise be masked because of genetic redundancy. Here, we provide instructions for the cloning of hgRNA screening libraries and individual hgRNA constructs and offer guidelines for designing and performing combinatorial pooled genetic screens using CHyMErA. Starting with the generation of Cas9- and Cas12a-expressing cell lines, CHyMErA screening can be implemented within 15-20 weeks.