Strong Purifying Selection Research Articles

Genome-wide analysis of the R2R3-MYB gene family and identification of candidate genes that regulate isoflavone biosynthesis in red clover (Trifolium pratense)

Red clover (Trifolium pratense) is a perennial legume with high feeding and medicinal value attributed to its abundant isoflavone content. Previous studies reported that R2R3-MYB transcription factors are involved in the biosynthesis of isoflavones; however, their specific role in red clover remains poorly understood. Through comprehensive genome-wide and transcriptome analyses, a total of 138 TpR2R3-MYB genes were identified and classified into 30 distinct subgroups within a phylogenetic tree. Importantly, six of these subgroups showed associations with isoflavone biosynthesis in red clover. The majority of segmental duplication events (Ka/Ks < 1) indicated that the TpR2R3-MYB gene underwent strong purifying selection during evolution. The qRT-PCR analysis demonstrated high expression levels of TpMYB79 and TpMYB53 in Minshan red clover at full flowering stage, consistent with the trend for isoflavone content determination, suggesting that TpMYB79 and TpMYB53 might be important regulators of isoflavone biosynthesis in red clover. Additionally, we observed nucleus and vacuole membrane localization of TpMYB53 and TpMYB79, with TpMYB53 primarily exerting transcriptional activation through its C-terminal activation motifs while TpMYB79 exhibited no transcriptional activity. These findings provided a foundation for the study of the biological function of R2R3-MYB transcription factors in red clover.

The Complete Mitochondrial Genome of Nephropsis grandis: Insights into the Phylogeny of Nephropidae Mitochondrial Genome.

The systematic phylogeny of Pleocyemata species, particularly within the family Nephropidae, remains incomplete. In order to enhance the taxonomy and systematics of Nephropidae within the evolutionary context of Pleocyemata, we embarked upon a comprehensive study aiming to elucidate the phylogenetic position of Nephropsis grandis. Consequently, we determined the complete mitochondrial DNA sequence for N. grandis. The circular genome spans a length of 15,344bp and exhibits a gene composition analogous to that observed in other metazoans, encompassing a comprehensive set of 37 genes. Additionally, the genome features an AT-rich region. The rRNAs exhibited the highest AT content among the 37 genes (70.41%), followed by tRNAs (67.42%) and protein-coding genes (PCGs) (62.76%). The absence of a dihydrouracil arm in trnS1 prevented the formation of the canonical cloverleaf secondary structure. Selective pressure analysis indicated that the PCGs underwent purifying selection. The Ka/Ks ratios for cox1, cox2, cox3, and cob were considerably lower compared to other PCGs, implying strong purifying selection acting upon these particular genes. The mitochondrial gene order in N. grandis was consistent with the reported order in ancestral Pleocyemata. Phylogenetic revealed that N. grandis forms a cluster with the genus Metanephrops, and this cluster further groups with Homarus and the genus Nephrops within the Nephropidae family. These findings provide robust support for N. grandis as an ancestral member of the Nephropidae family. This study highlights the significance of employing complete mitochondrial genomes in phylogenetic analysis and deepens our understanding of the evolution of the Nephropidae family.

Characterization, Codon Usage Pattern and Phylogenetic Implications of the Waterlily Aphid Rhopalosiphum nymphaeae (Hemiptera: Aphididae) Mitochondrial Genome

The water lily aphid, Rhopalosiphum nymphaeae, is the only known aphid that can live in both terrestrial and aquatic conditions. In this study, the complete mitochondrial genome of R. nymphaeae was generated using Illumina sequencing technology. The typical circular DNA mitochondrial genome of R. nymphaeae is 15,772 bp in length, with a high A+T content (84.34%). It contains 37 coding genes (13 protein-coding genes, 22 transport RNAs, and two ribosomal RNAs) and two non-coding regions (one control region and one repeat region). Enc-plot, PR2-bias, and neutrality plot analysis indicated that the codon usage of the protein-coding genes is mainly affected by natural selection. The evolution rate analysis (the ratio of nonsynonymous to synonymous, Ka/Ks) indicated that all the PCGs in R. nymphaeae are under a strong purifying selection. The control region has conserved structure elements, and two types of tandem repeat units exist. The length and sequence of the aphid-unique repeat region has high similarity with closely related species. Phylogenetic analyses determined by both maximum likelihood and Bayesian inference support the monophyly of Aphidinae, Aphidini, Aphidina, and Rhopalosiphina. However, the monophyly of the genera in Rhopalosiphina, such as Rhopalosiphum, is still not resolved. This study may help us to understand the phylogenetic relationship of aphids, and much more aphid data are needed in future studies.

Genome-wide identification of AP2/ERF gene family in Coptis Chinensis Franch reveals its role in tissue-specific accumulation of benzylisoquinoline alkaloids

BackgroundThe Plant-specific AP2/ERF gene family encodes proteins involved in various biological and physiological processes. Although the genome of Coptis chinensis Franch, a plant producing benzylisoquinoline alkaloids (BIAs), has been sequenced at the chromosome level, studies on the AP2/ERF gene family in C. chinensis are lacking. Thus, a genome-wide identification of AP2/ERF gene family in C. chinensis was conducted to explore its role in BIAs biosynthesis.ResultsA total of 96 CcAP2/ERF genes were identified and categorized into five subfamilies, including 43 ERFs, 32 DREBs, 17 AP2s, 3 RAVs, and 1 Soloist, based on their structural domains. These CcAP2/ERF genes were unevenly distributed across nine chromosomes. Analysis of gene duplication events identified 17 CcAP2/ERF gene pairs in the genome, with 7 involved in tandem duplication events and 10 involved in segmental duplicate events, indicating that both types of duplications contributed to the expansion of the AP2/ERF gene family. The Ka/Ks ratio analysis suggested that the CcAP2/ERF gene family underwent strong purifying selection. Two phytohormones, methyl jasmonate and abscisic acid, were identified as potential key inducers of BIAs biosynthesis due to the cis-acting element prediction. Analysis of the spatial transcriptomic data revealed that 28 differentially expressed AP2/ERF genes had the highest or relatively higher expression levels in the rhizome, 17 of which positively correlated with the tissue-specific accumulation of BIAs. Further real-time PCR verification and protein-protein interaction analysis indicated that DREB1B might be one of the central regulators in a highly complex BIAs biosynthesis network.ConclusionThese findings provide significant insight into the function of AP2/ERF genes in C. chinensis, particularly in the regulatory network of BIAs biosynthesis in C. chinensis. This study also identifies candidate genes for metabolic engineering to increase BIAs content in C. chinensis.

Sperm competition intensity shapes divergence in both sperm morphology and reproductive genes across murine rodents.

It remains unclear how variation in the intensity of sperm competition shapes phenotypic and molecular evolution across clades. Mice and rats in the subfamily Murinae are a rapid radiation exhibiting incredible diversity in sperm morphology and production. We combined phenotypic and genomic data to perform phylogenetic comparisons of male reproductive traits and genes across 78 murine species. We identified several shifts towards smaller relative testes mass, presumably reflecting reduced sperm competition. Several sperm traits were associated with relative testes mass, suggesting that mating system evolution selects for convergent suites of traits related to sperm competitive ability. We predicted that sperm competition would also drive more rapid molecular divergence in species with large testes. Contrary to this, we found that many spermatogenesis genes evolved more rapidly in species with smaller relative testes mass due to relaxed purifying selection. While some reproductive genes evolved rapidly under recurrent positive selection, relaxed selection played a greater role in underlying rapid evolution in small testes species. Our work demonstrates that postcopulatory sexual selection can impose strong purifying selection shaping the evolution of male reproduction, and that broad patterns of molecular evolution may help identify genes that contribute to male fertility.

Sperm competition intensity shapes divergence in both sperm morphology and reproductive genes across murine rodents.

It remains unclear how variation in the intensity of sperm competition shapes phenotypic and molecular evolution across clades. Mice and rats in the subfamily Murinae are a rapid radiation exhibiting incredible diversity in sperm morphology and production. We combined phenotypic and genomic data to perform phylogenetic comparisons of male reproductive traits and genes across 78 murine species. We identified several shifts towards smaller relative testes mass, presumably reflecting reduced sperm competition. Several sperm traits were associated with relative testes mass, suggesting that mating system evolution selects for convergent suites of traits related to sperm competitive ability. We predicted that sperm competition would also drive more rapid molecular divergence in species with large testes. Contrary to this, we found that many spermatogenesis genes evolved more rapidly in species with smaller relative testes mass due to relaxed purifying selection. While some reproductive genes evolved rapidly under recurrent positive selection, relaxed selection played a greater role in underlying rapid evolution in small testes species. Our work demonstrates that postcopulatory sexual selection can impose strong purifying selection shaping the evolution of male reproduction, and that broad patterns of molecular evolution may help identify genes that contribute to male fertility.

Duplication and sub-functionalization of flavonoid biosynthesis genes plays important role in Leguminosae root nodule symbiosis evolution.

Gene innovation plays an essential role in trait evolution. Rhizobial symbioses, the most important N2-fixing agent in agricultural systems that exists mainly in Leguminosae, is one of the most attractive evolution events. However, the gene innovations underlying Leguminosae root nodule symbiosis (RNS) remain largely unknown. Here, we investigated the gene gain event in Leguminosae RNS evolution through comprehensive phylogenomic analyses. We revealed that Leguminosae-gain genes were acquired by gene duplication and underwent a strong purifying selection. Kyoto Encyclopedia of Genes and Genomes analyses showed that the innovated genes were enriched in flavonoid biosynthesis pathways, particular downstream of chalcone synthase (CHS). Among them, Leguminosae-gain type Ⅱ chalcone isomerase (CHI) could be further divided into CHI1A and CHI1B clades, which resulted from the products of tandem duplication. Furthermore, the duplicated CHI genes exhibited exon-intron structural divergences evolved through exon/intron gain/loss and insertion/deletion. Knocking down CHI1B significantly reduced nodulation in Glycine max (soybean) and Medicago truncatula; whereas, knocking down its duplication gene CHI1A had no effect on nodulation. Therefore, Leguminosae-gain type Ⅱ CHI participated in RNS and the duplicated CHI1A and CHI1B genes exhibited RNS functional divergence. This study provides functional insights into Leguminosae-gain genetic innovation and sub-functionalization after gene duplication that contribute to the evolution and adaptation of RNS in Leguminosae.

Comparative analyses of plastomes in Allaeanthus and Malaisia: structure, evolution, and phylogeny.

The small genera Allaeanthus and Malaisia within the Moraceae have important edible, medicinal, and economic value. However, complete plastome blueprints and a well-resolved evolutionary history of these two genera are still lack, thereby limiting their conservation and application. The recent discovery of a new distribution of Allaeanthus kurzii in Hainan, China, marked by the collection of two unique samples, alongside three samples of Malaisia scandens, has opened new avenues for research. This study aimed to compare the Allaeanthus and Malaisia plastomes of Hainan Province samples with those of samples from other regions, focusing on plastome structure, codon usage bias, natural selection, and the evolutionary history of A. kurzii and M. scandens. The results showed that both species had a quadripartite plastome structure, with sizes ranging from 162,134 to 162,170bp for A. kurzii and 161,235 to 162,134bp for M. scandens. Both species displayed loss of the infA gene and reduction of the rpl22 gene. Two highly variable regions (petD-trnD-GUC and rpl20-clpP) and three highly variable genes (rpl20, petB, and rpl16) were identified in A. kurzii, while two highly variable regions (ycf2-ndhB and ccsA-ndhE) and three highly variable genes (psbT, rpl36, and ycf2) were found in M. scandens. The protein-coding sequences (CDSs) of the Allaeanthus and Malaisia plastomes exhibited similar patterns of adaptive indices and codon usage frequencies. The genes associated with photosynthesis underwent strong purifying selection. Phylogenetic analysis revealed that Allaeanthus, Broussonetia, and Malaisia constituted a monophyletic group, with Malaisia being more closely related to Broussonetia. Broussonetia diversified approximately 19.78 million years ago, Malaisia approximately 4.74 million years ago, and Allaeanthus approximately 16.18 million years ago. These new plastome-based discoveries will guide conservation planners and medicinal plant breeders and genetic resource development for these species in the region.

Endemic Radiation of African Moonfish, Selene dorsalis (Gill 1863), in the Eastern Atlantic: Mitogenomic Characterization and Phylogenetic Implications of Carangids (Teleostei: Carangiformes).

This study offers an in-depth analysis of the mitochondrial genome of Selene dorsalis (Gill 1863), a species native to the Eastern Atlantic Ocean. The circular mitochondrial DNA molecule measures 16,541 base pairs and comprises 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, two ribosomal RNA genes, and a control region (CR). The nucleotide composition exhibits a notable adenine-thymine (AT) bias, accounting for 53.13%, which aligns with other species in the Carangidae family. Most PCGs initiate with the ATG codon, with the exception of Cytochrome C oxidase subunit I, which starts with GTG. Analysis of relative synonymous codon usage reveals that leucine and serine are the most prevalent amino acids in the mitochondrial genome of S. dorsalis and its congeners (S. vomer and S. setapinnis). All tRNAs display the typical cloverleaf structure, though tRNA Serine (S1) lacks a dihydrouracil arm. Pairwise comparisons of synonymous and nonsynonymous substitutions for all PCGs yielded values below '1', indicating strong purifying selection. The CR spans 847 bp, representing 5.12% of the mitochondrial genome, and is characterized by high AT content (62.81%). It is situated between tRNA-Pro (TGG) and tRNA-Phe (GAA). The CR contains conserved sequence blocks, with CSB-1 being the longest at 22 bp and CSB-D the shortest at 18 bp. Phylogenetic analysis, using Bayesian and Maximum-likelihood trees constructed from concatenated PCGs across 72 species, successfully differentiates S. dorsalis from other carangids. This study also explores how ocean currents and gyres might influence lineage diversification and parapatric speciation of Selene species between the Atlantic and Pacific Oceans. These results highlight the importance of the mitochondrial genome in elucidating the structural organization and evolutionary dynamics of S. dorsalis and its relatives within marine ecosystems.

Genome-Wide Identification and Expression Analysis of MYB Transcription Factor Family in Response to Various Abiotic Stresses in Coconut (Cocos nucifera L.).

Abiotic stresses such as nitrogen deficiency, drought, and salinity significantly impact coconut production, yet the molecular mechanisms underlying coconut's response to these stresses are poorly understood. MYB proteins, a large and diverse family of transcription factors (TF), play crucial roles in plant responses to various abiotic stresses, but their genome-wide characterization and functional roles in coconut have not been comprehensively explored. This study identified 214 CnMYB genes (39 1R-MYB, 171 R2R3-MYB, 2 3R-MYB, and 2 4R-MYB) in the coconut genome. Phylogenetic analysis revealed that these genes are unevenly distributed across the 16 chromosomes, with conserved consensus sequences, motifs, and gene structures within the same subgroups. Synteny analysis indicated that segmental duplication primarily drove CnMYB evolution in coconut, with low nonsynonymous/synonymous ratios suggesting strong purifying selection. The gene ontology (GO) annotation of protein sequences provided insights into the biological functions of the CnMYB gene family. CnMYB47/70/83/119/186 and CnMYB2/45/85/158/195 were identified as homologous genes linked to nitrogen deficiency, drought, and salinity stress through BLAST, highlighting the key role of CnMYB genes in abiotic stress tolerance. Quantitative analysis of PCR showed 10 CnMYB genes in leaves and petioles and found that the expression of CnMYB45/47/70/83/85/119/186 was higher in 3-month-old than one-year-old coconut, whereas CnMYB2/158/195 was higher in one-year-old coconut. Moreover, the expression of CnMYB70, CnMYB2, and CnMYB2/158 was high under nitrogen deficiency, drought, and salinity stress, respectively. The predicted secondary and tertiary structures of three key CnMYB proteins involved in abiotic stress revealed distinct inter-proteomic features. The predicted interaction between CnMYB2/158 and Hsp70 supports its role in coconut's drought and salinity stress responses. These results expand our understanding of the relationships between the evolution and function of MYB genes, and provide valuable insights into the MYB gene family's role in abiotic stress in coconut.

First Record of the Complete Mitochondrial Genome for the Genus Borbo (Lepidoptera, Hesperiidae): Characterization and Comparative Genomic Analysis

Butterflies of the genus Borbo are mainly distributed in the Oriental and Australian regions and are considered pests of important crops. However, no mitochondrial genomes have been reported for this genus until now, leaving the evolutionary characteristics and differentiation patterns of their mitogenomes unclear. In this study, we present the first complete mitochondrial genome sequence of the rice swift, Borbo cinnara. The circular double-stranded mitogenome was 15,508 bp in length, comprising 13 protein-coding genes (PCGs), 2 ribosomal RNA genes (rRNAs), 22 transfer RNA genes (tRNAs), and 1 non-coding control region (CR). Among the mitogenomes of Hesperiinae, the ND3 gene was found to be the most variable PCG, while COX1 was the most conserved. Selection pressure analysis revealed that ND3 was under relaxed purifying selection, whereas COX1 was subjected to strong purifying selection. Phylogenetic trees reconstructed using both the Bayesian inference (BI) and maximum likelihood (ML) methods yielded robust and identical topologies, confirming the sister relationship between B. cinnara and Pelopidas mathias at the mitogenome level. Methodologically, this research enriches novel molecular markers for the species identification of butterflies and enhances our understanding of mitogenomic evolution in Lepidoptera.

Genome-wide identification of the phenylalanine ammonia-lyase gene from Epimedium Pubescens Maxim. (Berberidaceae): novel insight into the evolution of the PAL gene family

BackgroundPhenylalanine ammonia-lyase (PAL) serves as a key gateway enzyme, bridging primary metabolism and the phenylpropanoid pathway, and thus playing an indispensable role in flavonoid, anthocyanin and lignin biosynthesis. PAL gene families have been extensively studied across species using public genomes. However, a comprehensive exploration of PAL genes in Epimedium species, especially those involved in prenylated flavonol glycoside, anthocyanin, or lignin biosynthesis, is still lacking. Moreover, an in-depth investigation into PAL gene family evolution is warranted.ResultsSeven PAL genes (EpPAL1-EpPAL7) were identified. EpPAL2 and EpPAL3 exhibit low sequence identity to other EpPALs (ranging from 61.09 to 64.38%) and contain two unique introns, indicating distinct evolutionary origins. They evolve at a rate ~ 10 to ~ 54 times slower compared to EpPAL1 and EpPAL4-7, suggesting strong purifying selection. EpPAL1 evolved independently and is another ancestral gene. EpPAL1 formed EpPAL4 through segmental duplication, which lead to EpPAL5 and EpPAL6 through tandem duplications, and EpPAL7 through transposed duplication, shaping modern EpPALs. Correlation analysis suggests EpPAL1, EpPAL2 and EpPAL3 play important roles in prenylated flavonol glycosides biosynthesis, with EpPAL2 and EpPAL3 strongly correlated with both Epimedin C and total prenylated flavonol glycosides. EpPAL1, EpPAL2 and EpPAL3 may play a role in anthocyanin biosynthesis in leaves. EpPAL2, EpPAL3, EpPAL6, and EpPAL7 might be engaged in anthocyanin production in petals, and EpPAL2 and EpPAL3 might also contribute to anthocyanin synthesis in sepals. Further experiments are needed to confirm these hypotheses. Novel insights into the evolution of PAL gene family suggest that it might have evolved from a monophyletic group in bryophytes to large-scale sequence differentiation in gymnosperms, basal angiosperms, and Magnoliidae. Ancestral gene duplications and vertical inheritance from gymnosperms to angiosperms likely occurred during PAL evolution. Most early-diverging eudicotyledons and monocotyledons have distinct histories, while modern angiosperm PAL gene families share similar patterns and lack distant gene types.ConclusionsEpPAL2 and EpPAL3 may play crucial roles in biosynthesis of prenylated flavonol glycosides and anthocyanins in leaves and flowers. This study provides novel insights into PAL gene family evolution. The findings on PAL genes in E. pubescens will aid in synthetic biology research on prenylated flavonol glycosides production.

The complete mitochondrial genome of the imperiled Bullnose ray Myliobatis freminvillei (Myliobatiformes: Myliobatidae) with comments on its phylogenetic position and claims of diversifying selection affecting protein coding genes in a closely related species

The Bullnose ray Myliobatis freminvillei is a bentho-pelagic eagle ray that inhabits the north Gulf of Mexico and the Western Atlantic Ocean Coast, discontinuously, from Massachusetts, USA to Buenos Aires, Argentina. Myliobatis freminvillei is currently listed as vulnerable by the 2019 IUCN Red List of Threatened Species given that it is often captured as bycatch by artisanal and commercial fisheries, along the coasts of Argentina, Brazil, and Venezuela. This study, for the first time, assembled and characterized the mitochondrial genome of M. fremnvillei. The A+T rich mitochondrial genome of M. fremnvillei is 18,356 bp long and encodes 22 transfer RNA genes (tRNA), 2 ribosomal RNA genes (12S ribosomal RNA and 16S ribosomal RNA), 13 protein coding genes (PCGs), and also contains a non coding control region 2,617 bp long. Nonsynonomous codon usage with a preference for A+T rich codons was observed in all 13 PCGs. Leu (CTA), Ile (ATC), Phe (TTC), Thr (ACA), and Ala (GCC) were the most frequently used codons. Ka/Ks ratios estimated for all 13 PCGs exhibited values < 1, indicating strong purifying selection affecting all these genes. In contrast to the results of a previous study that claimed diversifying selective pressure in two mitochondrial PCGs of Mobula tarapacana, reanalysis of the Ka/Ks values for the same species indicated purifying selection in all 13 PCGs. Of the 22 tRNA genes, all have a cloverleaf secondary structure except tRNA-Ser1 which has a truncated dihydrouridine arm. In the control region, A+T rich microsatellites (n = 42) and short tandem repeats (n = 6) were identified, and the secondary structure of the same region contained numerous hairpin loops. Phylomitogenomic analyses supported the monophyletic status of the order Myliobatiformes and family Myliobatidae. The assembled mitochondrial genome will assist with conservation efforts in Myliobatis fremnvillei.

Comparative genomics and evolutionary analysis of dengue virus strains circulating in Pakistan.

Dengue fever virus (DENV) poses a significant public health risk in tropical and subtropical regions across the world. Although the dengue fever virus (DENV) exhibits significant genetic diversity and has the potential to evolve, there is a lack of comprehensive research on the comparative genomics and evolutionary dynamics of the virus in Pakistan. Phylogenetic analysis demonstrated the circulation of all four dengue virus serotypes (DENV-1, -2, -3, and -4) with prevalent genotypes III and V within DENV-1, cosmopolitan genotype within DENV-2, genotype III within DENV-3, and genotype I within DENV-4 during 2006-2014. Based on the complete envelope region, genome-wide residue signature and genetic diversity indicate that there is a high level of genetic diversity among DENV-1 strains, while DENV-3 strains exhibit the least genetic diversity. Comparative analysis of all four DENV serotypes revealed that certain codons in DENV-2 and -4 were subject to strong purifying selection, while a few codon sites in the envelope region showed evidence of positive selection. These findings provided valuable insights into the comparative genomics and evolutionary pattern of DENV strains reported from Pakistan. Whether those characteristics conferred a fitness advantage to DENV-1 genotypes within a specific geography and time interval warrants further investigations. The findings of the current study will contribute to tracking disease dynamics, understanding virus transmission and evolution, and formulating effective disease control strategies.

Genome-Wide Analyses of CCHC Family Genes and Their Expression Profiles under Drought Stress in Rose (Rosa chinensis).

CCHC-type zinc finger proteins (CCHC-ZFPs), ubiquitous across plant species, are integral to their growth, development, hormonal regulation, and stress adaptation. Roses (Rosa sp.), as one of the most significant and extensively cultivated ornamentals, account for more than 30% of the global cut-flower market. Despite its significance, the CCHC gene family in roses (Rosa sp.) remains unexplored. This investigation identified and categorized 41 CCHC gene members located on seven chromosomes of rose into 14 subfamilies through motif distribution and phylogenetic analyses involving ten additional plant species, including Ginkgo biloba, Ostreococcus lucimarinus, Arabidopsis thaliana, and others. This study revealed that dispersed duplication likely plays a crucial role in the diversification of the CCHC genes, with the Ka/Ks ratio suggesting a history of strong purifying selection. Promoter analysis highlighted a rich presence of cis-acting regulatory elements linked to both abiotic and biotic stress responses. Differential expression analysis under drought conditions grouped the 41 CCHC gene members into five distinct clusters, with those in group 4 exhibiting pronounced regulation in roots and leaves under severe drought. Furthermore, virus-induced gene silencing (VIGS) of the RcCCHC25 member from group 4 compromised drought resilience in rose foliage. This comprehensive analysis lays the groundwork for further investigations into the functional dynamics of the CCHC gene family in rose physiology and stress responses.

Genome-Wide Identification, Expression, and Protein Analysis of CKX and IPT Gene Families in Radish (Raphanus sativus L.) Reveal Their Involvement in Clubroot Resistance.

Cytokinins (CKs) are a group of phytohormones that are involved in plant growth, development, and disease resistance. The isopentenyl transferase (IPT) and cytokinin oxidase/dehydrogenase (CKX) families comprise key enzymes controlling CK biosynthesis and degradation. However, an integrated analysis of these two gene families in radish has not yet been explored. In this study, 13 RsIPT and 12 RsCKX genes were identified and characterized, most of which had four copies in Brassica napus and two copies in radish and other diploid Brassica species. Promoter analysis indicated that the genes contained at least one phytohormone or defense and stress responsiveness cis-acting element. RsIPTs and RsCKXs were expanded through segmental duplication. Moreover, strong purifying selection drove the evolution of the two gene families. The expression of the RsIPT and RsCKX genes distinctly showed diversity in different tissues and developmental stages of the root. Expression profiling showed that RsCKX1-1/1-2/1-3 was significantly upregulated in club-resistant materials during primary infection, suggesting their vital function in clubroot resistance. The interaction network of CKX proteins with similar 3D structures also reflected the important role of RsCKX genes in disease resistance. This study provides a foundation for further functional study on the IPT and CKX genes for clubroot resistance improvement in Raphanus.

Large-scale analysis of the PAC domain structure of arogenate dehydratases reveals their evolutionary patterns in angiosperms

Arogenate dehydratase (ADT) is the key limiting enzyme of plant phenylalanine biosynthesis, but some ADTs display a prephenate decarboxylase/dehydratase activity-conferring (PAC) domain. The genome resources of 70 species were employed to identify genes and outline their characteristics, especially the number and type of PAC domain structures. We obtained 522 ADTs, and their size, exon number, amino acid number and putative protein isoelectric point greatly varied from 306 to 2520 bp, 1 to 15, 101 to 839 and 4.37 to 11.18, respectively. We classified the ADTs into Class α (without a PAC domain) (115, 22.0 %), β (with a type I PAC domain) (244, 46.7 %) and γ (with a type II PAC domain) (163, 31.2 %), and their distribution frequencies exhibited large differences among various branches of angiosperms. We found that Class γ members are more conserved than Class β members, although they commonly experienced multiple duplication events and strong purifying selection, which resulted in a small number, and the putative origin order was from Class α to β and then to γ. In addition, the co-occurrence of both Class β and γ members could ensure the survival of angiosperms, while their optimized composition and strategically intertwined regulation may facilitate core eudicot success.

Genome-wide identification and analysis of the EIN3/EIL gene family in broomcorn millet (Panicum miliaceum L.).

The EIN3/EIL gene family holds a pivotal role as it encodes a crucial transcription factor in plants. During the process of polyploidization in broomcorn millet (Panicum miliaceum L.), there is an intriguing above-average amplification observed within the EIN3/EIL gene family. Nonetheless, our current knowledge of this gene family in broomcorn millet remains limited. Hence, in this study, we conducted a comprehensive analysis of the EIN3/EIL gene family in broomcorn millet, aiming to provide a deeper understanding of the potential evolutionary changes. Additionally, we analyzed the EIN3/EIL gene family of Panicum hallii L., a close relative of broomcorn millet, to enhance our characterization efforts. Within this study, we identified a total of 15 EIN3/EIL genes specific to broomcorn millet. Through covariance analysis, it was revealed that all PmEIL genes, except PmEIL1 and PmEIL15, had duplicate copies generated through genome-wide duplication events. Importantly, the Ka/Ks values of all duplicated genes were found to be less than 1, indicating strong purifying selection. Phylogenetic analysis showed that these genes could be categorized into four distinct evolutionary branches, showcasing similar characteristics among members within the same branch. However, there appeared to be an uneven distribution of cis-acting elements amid the EIN3/EIL genes. Further examination of transcriptomic data shed light on the diverse spatiotemporal and stress-related expression patterns exhibited by the EIN3/EIL genes in broomcorn millet. Notably, under cold stress, the expression of PmEIL3/4/8/14 was significantly up-regulated, while under drought stress, PmEIL4/5/6 displayed significant up-regulation. Intriguingly, the expression pattern of PmEIL15 showed an opposite pattern in resistant and sensitive cultivars. The findings of this study augment our understanding of the EIN3/EIL gene family in broomcorn millet and offer a valuable reference for future investigations into polyploid studies. Moreover, this study establishes a theoretical foundation for further exploration of the ethylene signaling pathway in broomcorn millet.

Maternal age enhances purifying selection on pathogenic mutations in complex I genes of mammalian mtDNA.

Mitochondrial diseases, caused mainly by pathogenic mitochondrial DNA (mtDNA) mutations, pose major challenges due to the lack of effective treatments. Investigating the patterns of maternal transmission of mitochondrial diseases could pave the way for preventive approaches. In this study, we used DddA-derived cytosine base editors (DdCBEs) to generate two mouse models, each haboring a single pathogenic mutation in complex I genes (ND1 and ND5), replicating those found in human patients. Our findings revealed that both mutations are under strong purifying selection during maternal transmission and occur predominantly during postnatal oocyte maturation, with increased protein synthesis playing a vital role. Interestingly, we discovered that maternal age intensifies the purifying selection, suggesting that older maternal age may offer a protective effect against the transmission of deleterious mtDNA mutations, contradicting the conventional notion that maternal age correlates with increased transmitted mtDNA mutations. As collecting comprehensive clinical data is needed to understand the relationship between maternal age and transmission patterns in humans, our findings may have profound implications for reproductive counseling of mitochondrial diseases, especially those involving complex I gene mutations.

Unique myoglobin adaptation to endothermy and flight since the origin of birds.

Myoglobin (Mb) mediates oxygen diffusion and storage in muscle tissue and thus is important for the energy utilization and activity of animals. Birds generally have a high body temperature, and most species also possess the capability of powered flight. Both of these require high levels of aerobic metabolism. Within endothermic mammals, bats also independently evolved flight. Although the functional evolution of myoglobins in deep-diving amniote vertebrates has been well-studied, the functional evolution of myoglobin since the origins of both birds and bats is unclear. Here, with Mb-coding sequences from >200 extant amniote species, we reconstructed ancestral sequences to estimate the functional properties of myoglobin through amniote evolution. A dramatic change in net surface charge on myoglobin occurred during the origin of Aves, which might have been driven by positively selected amino acid substitutions that occurred on the lineage leading to all birds. However, in bats, no change in net surface charge occurred and instead, the Mb genes show evidence of strong purifying selection. The increased net surface charge on bird myoglobins implies an adaptation to flight-related endothermic and higher body temperatures, possibly by reducing harmful protein aggregations. Different from the findings of net surface charge, myoglobins of extant birds show lower stability compared with other amniotes, which probably accelerates the rate of oxygen utilization in muscles. In bats and other mammals, higher stability of Mb may be an alternative pathway for adaptation to endothermy, indicating divergent evolution of myoglobin in birds and bats.