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Summary Genome editing via artificial nucleases such as CRISPR/Cas9 has become popular in plants now. However, small insertions or deletions are major mutations and nucleotide substitutions rarely occur when DNA cleavage is induced. To induce nucleotide substitutions, a base editor utilizing dead or nickase‐type Cas9 fused with deaminase have been developed. However, the direction and position of practical substitution are still limited. In this context, homologous recombination (HR)‐mediated gene targeting (GT) has advantages because any mutations existing on the donor DNA are copied and passed onto the endogenous DNA. As HR‐mediated GT is extremely rare in higher plants, positive–negative selection has been used to isolate cells in which GT has occurred. After successful selection, positive selection marker is no longer needed and should ideally be eliminated. In a previous study, we reported a seamless piggyBac‐transposon‐mediated marker elimination system. Precision marker elimination efficiency in this system is very high. The piggyBac transposon integrates into the host genome at TTAA elements and excises without leaving a footprint at the excised site, so a TTAA sequence is necessary at the location of a positive selection marker. To compensate for this limitation, we have developed a novel marker elimination system using an I‐SceI break and subsequent single‐strand annealing (SSA)‐mediated DNA repair system.

African indigenous cattle harbor rich genetic diversity shaped by long-term adaptation to tropical climates and endemic diseases, notably trypanosomiasis. To investigate the genetic basis of natural disease resistance, we analyzed 33,147 SNPs in 1,047 individuals from 17 breeds using complementary selection scans (iHS, XP-EHH, and Rsb) in a multi-cohort framework. Fifteen pairwise comparisons across six breed groups enabled robust detection of recent positive selection, capturing both within-breed and breed-specific adaptive signals. We identified 229 selective regions, with 47 outlier SNPs consistently detected by multiple methods. Selection signals were enriched on chromosomes 5, 7, and 29, containing candidate genes such as ATF4, PKNOX2, DNAJB7, TEF, NFE2, and several SPINK and HOXC family members, many associated with immune function and trypanosome challenge response. These genomic regions represent promising targets for the development of breeding strategies aimed at enhancing disease resilience. By identifying genomic regions under selection, this study provides interesting insights for sustainable livestock improvement and conservation efforts in West and Central Africa. Our findings support the integration of indigenous genetic resources into breeding programs designed to optimize productivity while maintaining resilience to environmental and disease pressures.

The CONSTANS-like (COL) transcription factors integrate photoperiod cues with developmental regulation in plants, yet the evolutionary forces shaping their structural diversity remain poorly understood. Here, the evolutionary history of COL5 was reconstructed across 31 Brassicaceae genomes using a curated set of 284 high-confidence orthologs validated for domain architecture, alignment quality, and absence of substitution saturation. Branch-specific codon models identified a single episodically selected lineage within Arabidopsis thaliana , and site-level analyses mapped two non-synonymous amino-acid replacements uniquely acquired along this branch. Ancestral sequence reconstruction recovered the historical residues at both positions with posterior probability 1.0, enabling controlled reverse-evolution mutagenesis. Reintroduction of these ancestral states into the modern COL5 protein revealed a profound biophysical impact, Rosetta ΔΔG values indicated strong destabilization, and 100-ns molecular dynamics simulations showed large increases in structural deviation, compaction, loss of flexibility, and significantly elevated potential energy. These results demonstrate that the derived residues stabilize the contemporary COL5 fold, whereas the ancestral residues are incompatible with the evolved structural background. The findings provide direct mechanistic evidence that episodic positive selection on COL5 produced a lasting shift in protein stability and conformational dynamics, illustrating how adaptive molecular evolution can reshape protein energy landscapes and entrench derived states through historical contingency.

Human skin color diversity is considered an adaptation to environmental conditions such as UV radiation. Investigations into the genetic bases of such adaptation have identified a group of pigmentation genes contributing to skin color diversity in African and non-African populations. Here, we present a population analysis of the pigmentation gene KITLG with previously reported signal of Darwinian positive selection in both European and East Asian populations. We demonstrated that there had been recurrent selective events in the upstream and the downstream regions of KITLG in Eurasian populations. More importantly, besides the expected selection on the KITLG variants favoring light skin in coping with the weak UV radiation at high latitude, we observed a KITLG variant showing adaptation to winter temperature. In particular, compared with UV radiation, winter temperature showed a much stronger correlation with the prevalence of the presumably adaptive KITLG allele in Asian populations. This observation was further supported by the in vitro functional test at low temperature. Consequently, the pleiotropic effects of KITLG, that is, pigmentation and thermogenesis were both targeted by natural selection that acted on different KITLG sequence variants, contributing to the adaptation of Eurasians to both UV radiation and winter temperature at high latitude areas.

Abstract The transporters of the ATP-Binding Cassette (ABC) Superfamily involved in the Multidrug Resistance (MDR) phenomena are also known as ABC-Pleiotropic Drug Resistance (PDR) proteins. The homologs of the Saccharomyces cerevisiae SNQ2 and PDR18 genes were identified in 171 yeast genomes, representing 68 different hemiascomycetous species. All early-divergent yeast species analyzed in this work lack Snq2/Pdr18 homologs, suggesting that the origin of these ABC-PDR genes in hemiascomycete yeasts resulted from a horizontal transfer event. The evolutionary pathway of the Snq2/Pdr18 protein subfamily in pathogenic Candida species was also reconstructed, revealing a main gene lineage leading to the Candida albicans SNQ2 gene. The results indicate that, after the gene duplication event at the origin of the SNQ2/PDR18 paralogs, the PDR18 ortholog has been under strong diversifying selection and suggest that a small portion of the sequence of the SNQ2 ancestral ortholog might have been under mild positive selection. The results also showed that strong positive selection was exerted over one of the two paralogs generated by the Whole Genome Duplication (WGD) event, corresponding to the duplicate at the origin of a “short-lived” WGD sublineage. In this study, the transporters of the ATP-Binding Cassette (ABC) Superfamily involved in the multidrug resistance phenomena, also known as ABC- Pleiotropic Drug Resistance proteins, are analyzed from an evolutionary perspective.

The adaptive evolution of the glutathione S-transferase (GST) gene family in Salix lindleyana provides insights into the relationship between enzyme structure and function. In this study, 37 genes encoding the GST protein were cloned from S. lindleyana with no genomic data available, and their expression levels and enzyme activity were determined in vitro . The 22 genes encoding the Tau GST subfamily were divided into Clades A and B, with Clade A subjected to more relaxed selection pressure than Clade B. Clade A was split into two smaller branches, Clades a and b. Three genes under positive selection from Clade a were chosen for 36 site-directed mutations, with Trp162 and Pro202 crucially affecting variations in GST enzyme activity. Crystal structure analysis of SliGSTU7 complexed with GSH revealed that the Trp162 residue was located at the bottom of the hydrophobic cavity. Homology modeling and molecular docking revealed that the W162G/P202A mutation in SliGSTU7 significantly reduced the neighboring effect during the formation of GS-DNB. A study of the GST gene family of S. lindleyana identified Trp162 and Pro202 as key amino acids that regulate the release rate of GS-X.

Background Foreign DNA cloning is a critical step in genetic engineering and molecular biology for acquiring target genes. Efficient and accurate identification of positive clones from transformants is essential to this process. Results Here, we engineered an insertion-activated cloning vector that accepts PCR products directly. In this study, we verified that 60-bp NptII promoters can effectively drive the expression of the mScarlet-I gene. In contrast, the 45-bp NptII promoter can’t drive the expression of the mScarlet-I gene. The insertion-activated cloning vector harbored with the 45-bp truncated NptII promoter and ORF of mScarlet-I gene. This cloning vector contains an Eco RⅤ restriction site, which serves as the cloning site for PCR products. When amplifying exogenous DNA, the designed reverse primer is added with the 15-bp core sequence from − 60 to -46 of the NptII promoter. If the PCR product is cloned into the insertion-activated cloning vector with the correct orientation, the 15-bp core promoter and the 45-bp truncated NptII promoter can be connected and drives expression of the reporter gene, mScarlet-I , which allows positive recombinants to be identified with red fluorescence. Five PCR products with different sizes were tested using the one-step restriction enzyme digestion and ligation approach or in vivo cloning method. The. Conclusions The positive selection marker in this insertion-activated cloning vector can achieve higher screening efficiency.

The mitochondrion, which is an intracellular organelle responsible for most of the energy‐producing pathways, can have its genome targeted for climate‐driven selection. However, climate‐driven mitochondrial selection remains a sparsely studied area in reptiles. Here, we reported the complete mitochondrial genome sequence of a lacertid lizard (Takydromus intermedius) and used mitogenomes from 54 species of lacertid lizards to study their phylogenetic relationships and to identify the mitochondrial genes under positive selection by climate. The length of the complete mitochondrial genome sequence of T. intermedius was 17,713 bp, which was within the range of lengths (17,224–18,943) ever reported for Takydromus species. The arrangement of mitochondrial genes in T. intermedius was the same as in other congeneric species. The 54 lacertid species could be divided into three geographically and climatically different clades. We identified three mitochondrial genes (ATP6, ATP8, and ND3) under positive selection by climate, and found that isothermality, temperature seasonality, precipitation of wettest month, and precipitation seasonality were the most important climatic variables contributing to the gene selection. We investigated the mitogenoms of Takydromus intermedius. We found three genes (ATP6, ATP8, and ND3) positively selected by climate inlizards.

Key messagePositive selection genes are related to metabolism, while differentially expressed genes are related to photosynthesis, suggesting that genetic adaptation and expression regulation may play independent roles in different gene classes.Genome-wide investigation of the molecular mechanisms for high-altitude adaptation is an intriguing topic in evolutionary biology. The Qinghai-Tibet Plateau (QTP) with its extremely variable environments is an ideal site for studying high-altitude adaptation. Here, we used transcriptome data of 100 individuals from 20 populations collected from various altitudes on the QTP to investigate the adaptive mechanisms of the aquatic plant Batrachium bungei at both the genetic and transcriptional level. To explore genes and biological pathways that may contribute to QTP adaptation, we employed a two-step approach, in which we identified positively selected genes and differentially expressed genes using the landscape genomic and differential expression approaches. The positive selection analysis showed that genes involved in metabolic regulation played a crucial role in B. bungei adaptation to the extreme environments of the QTP, especially intense ultraviolet radiation. Altitude-based differential expression analysis suggested that B. bungei could increase the rate of energy dissipation or reduce the efficiency of light energy absorption by down regulating the expression of photosynthesis-related genes to adapt to the strong ultraviolet radiation. Weighted gene co-expression network analysis identified ribosomal genes as hubs of altitude adaptation in B. bungei. Only a small part of genes (about 10%) overlapped between positively selected genes and differentially expressed genes in B. bungei, suggesting that genetic adaptation and gene expression regulation might play relatively independent roles in different categories of functional genes. Taken together, this study enriches our understanding of the high-altitude adaptation mechanism of B. bungei on the QTP.

Aldama (Heliantheae, Asteraceae) is a diverse genus in the sunflower family. To date, nearly 200 Asteraceae chloroplast genomes have been sequenced, but the plastomes of Aldama remain undescribed. Plastomes in Asteraceae usually show little sequence divergence, consequently, our hypothesis is that species of Aldama will be overall conserved. In this study, we newly sequenced 36 plastomes of Aldama and of five species belonging to other Heliantheae genera selected as outgroups (i.e., Dimerostemma asperatum, Helianthus tuberosus, Iostephane heterophylla, Pappobolus lanatus var . lanatus, and Tithonia diversifolia) . We analyzed the structure and gene content of the assembled plastomes and performed comparative analyses within Aldama and with other closely related genera. As expected, Aldama plastomes are very conserved, with the overall gene content and orientation being similar in all studied species. The length of the plastome is also consistent and the junction between regions usually contain the same genes and have similar lengths. A large ∼20 kb and a small ∼3 kb inversion were detected in the Large Single Copy (LSC) regions of all assembled plastomes, similarly to other Asteraceae species. The nucleotide diversity is very low, with only 1,509 variable sites in 127,466 bp (i.e., 1.18% of the sites in the alignment of 36 Aldama plastomes, with one of the IRs removed, is variable). Only one gene, rbcL , shows signatures of positive selection. The plastomes of the selected outgroups feature a similar gene content and structure compared to Aldama and also present the two inversions in the LSC region. Deletions of different lengths were observed in the gene ycf2 . Multiple SSRs were identified for the sequenced Aldama and outgroups. The phylogenetic analysis shows that Aldama is not monophyletic due to the position of the Mexican species A. dentata . All Brazilian species form a strongly supported clade. Our results bring new understandings into the evolution and diversity of plastomes at the species level.