Use of NAD tagSeq II to identify growth phase-dependent alterations in E. coli RNA NAD+ capping.

Recent findings regarding nicotinamide adenine dinucleotide (NAD+)-capped RNAs (NAD-RNAs) indicate that prokaryotes and eukaryotes employ noncanonical RNA capping to regulate gene expression. Two methods for transcriptome-wide analysis of NAD-RNAs, NAD captureSeq and NAD tagSeq, are based on copper-catalyzed azide-alkyne cycloaddition (CuAAC) click chemistry to label NAD-RNAs. However, copper ions can fragment/degrade RNA, interfering with the analyses. Here we report development of NAD tagSeq II, which uses copper-free, strain-promoted azide-alkyne cycloaddition (SPAAC) for labeling NAD-RNAs, followed by identification of tagged RNA by single-molecule direct RNA sequencing. We used this method to compare NAD-RNA and total transcript profiles of Escherichia coli cells in the exponential and stationary phases. We identified hundreds of NAD-RNA species in E. coli and revealed genome-wide alterations of NAD-RNA profiles in the different growth phases. Although no or few NAD-RNAs were detected from some of the most highly expressed genes, the transcripts of some genes were found to be primarily NAD-RNAs. Our study suggests that NAD-RNAs play roles in linking nutrient cues with gene regulation in E. coli.

AtHDA6 functions as an H3K18ac eraser to maintain pericentromeric CHG methylation in Arabidopsis thaliana.

Pericentromeric DNA, consisting of high-copy-number tandem repeats and transposable elements, is normally silenced through DNA methylation and histone modifications to maintain chromosomal integrity and stability. Although histone deacetylase 6 (HDA6) has been known to participate in pericentromeric silencing, the mechanism is still yet unclear. Here, using whole genome bisulfite sequencing (WGBS) and chromatin immunoprecipitation-sequencing (ChIP-Seq), we mapped the genome-wide patterns of differential DNA methylation and histone H3 lysine 18 acetylation (H3K18ac) in wild-type and hda6 mutant strains. Results show pericentromeric CHG hypomethylation in hda6 mutants was mediated by DNA demethylases, not by DNA methyltransferases as previously thought. DNA demethylases can recognize H3K18ac mark and then be recruited to the chromatin. Using biochemical assays, we found that HDA6 could function as an 'eraser' enzyme for H3K18ac mark to prevent DNA demethylation. Oxford Nanopore Technology Direct RNA Sequencing (ONT DRS) also revealed that hda6 mutants with H3K18ac accumulation and CHG hypomethylation were shown to have transcriptionally active pericentromeric DNA.

Arabidopsis EXTRA-LARGE G PROTEIN 1 (XLG1) functions together with XLG2 and XLG3 in PAMP-triggered MAPK activation and immunity.

Pattern-triggered immunity (PTI) is an essential strategy used by plants to deploy broad-spectrum resistance against pathogen attacks. Heterotrimeric G proteins have been reported to contribute to PTI. Of the three non-canonical EXTRA-LARGE G PROTEINs (XLGs) in Arabidopsis thaliana, XLG2 and XLG3 were shown to positively regulate immunity, but XLG1 was not considered to function in defense, based on the analysis of a weak xlg1 allele. In this study, we characterized the xlg1 xlg2 xlg3 triple knockout mutants generated from an xlg1 knockout allele. The strong xlg1 xlg2 xlg3 triple mutants compromised pathogen-associated molecular pattern (PAMP)-triggered activation of mitogen-activated protein kinases (MAPKs) and resistance to pathogen infection. The three XLGs interacted with MAPK cascade proteins involved in defense signaling, including the MAPK kinase kinases MAPKKK3 and MAPKKK5, the MAPK kinases MKK4 and MKK5, and the MAPKs MPK3 and MPK6. Expressing a constitutively active form of MKK4 restored MAPK activation and partially recovered the compromised disease resistance seen in the strong xlg1 xlg2 xlg3 triple mutant. Furthermore, mutations of all three XLGs largely restored the phenotype of the autoimmunity mutant bak1-interacting receptor-like kinase 1. Our study reveals that all three XLGs function redundantly in PAMP-triggered MAPK activation and plant immunity.

New insights into Arabidopsis transcriptome complexity revealed by direct sequencing of native RNAs.

Arabidopsis thaliana transcriptomes have been extensively studied and characterized under different conditions. However, most of the current 'RNA-sequencing' technologies produce a relatively short read length and demand a reverse-transcription step, preventing effective characterization of transcriptome complexity. Here, we performed Direct RNA Sequencing (DRS) using the latest Oxford Nanopore Technology (ONT) with exceptional read length. We demonstrate that the complexity of the A. thaliana transcriptomes has been substantially under-estimated. The ONT direct RNA sequencing identified novel transcript isoforms at both the vegetative (14-day old seedlings, stage 1.04) and reproductive stages (stage 6.00-6.10) of development. Using in-house software called TrackCluster, we determined alternative transcription initiation (ATI), alternative polyadenylation (APA), alternative splicing (AS), and fusion transcripts. More than 38 500 novel transcript isoforms were identified, including six categories of fusion-transcripts that may result from differential RNA processing mechanisms. Aided by the Tombo algorithm, we found an enrichment of m5C modifications in the mobile mRNAs, consistent with a recent finding that m5C modification in mRNAs is crucial for their long-distance movement. In summary, ONT DRS offers an advantage in the identification and functional characterization of novel RNA isoforms and RNA base modifications, significantly improving annotation of the A. thaliana genome.

NAD tagSeq reveals that NAD+-capped RNAs are mostly produced from a large number of protein-coding genes in Arabidopsis.

The 5' end of a eukaryotic mRNA transcript generally has a 7-methylguanosine (m7G) cap that protects mRNA from degradation and mediates almost all other aspects of gene expression. Some RNAs in Escherichia coli, yeast, and mammals were recently found to contain an NAD+ cap. Here, we report the development of the method NAD tagSeq for transcriptome-wide identification and quantification of NAD+-capped RNAs (NAD-RNAs). The method uses an enzymatic reaction and then a click chemistry reaction to label NAD-RNAs with a synthetic RNA tag. The tagged RNA molecules can be enriched and directly sequenced using the Oxford Nanopore sequencing technology. NAD tagSeq can allow more accurate identification and quantification of NAD-RNAs, as well as reveal the sequences of whole NAD-RNA transcripts using single-molecule RNA sequencing. Using NAD tagSeq, we found that NAD-RNAs in Arabidopsis were produced by at least several thousand genes, most of which are protein-coding genes, with the majority of these transcripts coming from <200 genes. For some Arabidopsis genes, over 5% of their transcripts were NAD capped. Gene ontology terms overrepresented in the 2,000 genes that produced the highest numbers of NAD-RNAs are related to photosynthesis, protein synthesis, and responses to cytokinin and stresses. The NAD-RNAs in Arabidopsis generally have the same overall sequence structures as the canonical m7G-capped mRNAs, although most of them appear to have a shorter 5' untranslated region (5' UTR). The identification and quantification of NAD-RNAs and revelation of their sequence features can provide essential steps toward understanding the functions of NAD-RNAs.

Membrane Proteomic Profiling of Soybean Leaf and Root Tissues Uncovers Salt-Stress-Responsive Membrane Proteins.

Cultivated soybean (Glycine max (L.)), the world's most important legume crop, has high-to-moderate salt sensitivity. Being the frontier for sensing and controlling solute transport, membrane proteins could be involved in cell signaling, osmoregulation, and stress-sensing mechanisms, but their roles in abiotic stresses are still largely unknown. By analyzing salt-induced membrane proteomic changes in the roots and leaves of salt-sensitive soybean cultivar (C08) seedlings germinated under NaCl, we detected 972 membrane proteins, with those present in both leaves and roots annotated as receptor kinases, calcium-sensing proteins, abscisic acid receptors, cation and anion channel proteins, proton pumps, amide and peptide transporters, and vesicle transport-related proteins etc. Endocytosis, linoleic acid metabolism, and fatty acid biosynthesis pathway-related proteins were enriched in roots whereas phagosome, spliceosome and soluble NSF attachment protein receptor (SNARE) interaction-related proteins were enriched in leaves. Using label-free quantitation, 129 differentially expressed membrane proteins were found in both tissues upon NaCl treatment. Additionally, the 140 NaCl-induced proteins identified in roots and 57 in leaves are vesicle-, mitochondrial-, and chloroplast-associated membrane proteins and those with functions related to ion transport, protein transport, ATP hydrolysis, protein folding, and receptor kinases, etc. Our proteomic results were verified against corresponding gene expression patterns from published C08 RNA-seq data, demonstrating the importance of solute transport and sensing in salt stress responses.

Effect of Adding L-carnitine to High-Fat/Low-Protein Diets of Common Carp (Cyprinus carpio) and the Mechanism of Regulation of Fat and Protein Metabolism.

L-carnitine is a low molecular weight amino acid that plays an essential role in the oxidation of long-chain fatty acids. The regulatory effects and molecular mechanisms of L-carnitine on fat and protein metabolism in common carp (Cyprinus carpio) were investigated in this study. Common carp (n = 270) were randomly divided into three groups and fed either (1) common carp diet, (2) high-fat/low-protein diet, or (3) L-carnitine-high-fat/low-protein diet. Growth performance, plasma biochemistry, muscle composition, and ammonia excretion rate were all examined after 8 weeks. Additionally, each group's hepatopancreas was subjected to transcriptome analysis. The results showed that decreasing the feed protein/fat ratio resulted in a considerable increase in feed conversion ratio and a significant decrease in common carp-specific growth rate to 1.19 ± 0.02 (P < 0.05). Similarly, total plasma cholesterol sharply increased to 10.15 ± 2.07, while plasma urea nitrogen, muscle protein, and ammonia excretion levels dropped (P < 0.05). After adding L-carnitine to the high-fat/low-protein diet, it was found that the specific growth rate and protein content of the dorsal muscle increased significantly (P < 0.05). In contrast, the plasma total cholesterol and ammonia excretion rate decreased considerably at most time points after feeding (P < 0.05). The expression of genes in the hepatopancreas differed substantially between the different groups. Through GO analysis, it was demonstrated that L-carnitine increased the ability of fat decomposition by up-regulating the expression of cpt1 in the hepatopancreas and decreased the expression of fasn and elovl6 to reduce the production and extension of lipids. Simultaneously, mtor was more abundant in the hepatopancreas, implying that L-carnitine can increase protein synthesis. According to the findings, adding L-carnitine to high-fat/low-protein diets can stimulate growth by enhancing lipolysis and protein synthesis.

PlaMoM: a comprehensive database compiles plant mobile macromolecules.

In plants, various phloem-mobile macromolecules including noncoding RNAs, mRNAs and proteins are suggested to act as important long-distance signals in regulating crucial physiological and morphological transition processes such as flowering, plant growth and stress responses. Given recent advances in high-throughput sequencing technologies, numerous mobile macromolecules have been identified in diverse plant species from different plant families. However, most of the identified mobile macromolecules are not annotated in current versions of species-specific databases and are only available as non-searchable datasheets. To facilitate study of the mobile signaling macromolecules, we compiled the PlaMoM (Plant Mobile Macromolecules) database, a resource that provides convenient and interactive search tools allowing users to retrieve, to analyze and also to predict mobile RNAs/proteins. Each entry in the PlaMoM contains detailed information such as nucleotide/amino acid sequences, ortholog partners, related experiments, gene functions and literature. For the model plant Arabidopsis thaliana, protein-protein interactions of mobile transcripts are presented as interactive molecular networks. Furthermore, PlaMoM provides a built-in tool to identify potential RNA mobility signals such as tRNA-like structures. The current version of PlaMoM compiles a total of 17 991 mobile macromolecules from 14 plant species/ecotypes from published data and literature. PlaMoM is available at http://www.systembioinfo.org/plamom/.

The caseinolytic protease complex component CLPC1 in Arabidopsis maintains proteome and RNA homeostasis in chloroplasts.

BACKGROUND: Homeostasis of the proteome is critical to the development of chloroplasts and also affects the expression of certain nuclear genes. CLPC1 facilitates the translocation of chloroplast pre-proteins and mediates protein degradation. RESULTS: We found that proteins involved in photosynthesis are dramatically decreased in their abundance in the clpc1 mutant, whereas many proteins involved in chloroplast transcription and translation were increased in the mutant. Expression of the full-length CLPC1 protein, but not of the N-terminus-deleted CLPC1 (ΔN), in the clpc1 mutant background restored the normal levels of most of these proteins. Interestingly, the ΔN complementation line could also restore some proteins affected by the mutation to normal levels. We also found that that the clpc1 mutation profoundly affects transcript levels of chloroplast genes. Sense transcripts of many chloroplast genes are up-regulated in the clpc1 mutant. The level of SVR7, a PPR protein, was affected by the clpc1 mutation. We showed that SVR7 might be a target of CLPC1 as CLPC1-SVR7 interaction was detected through co-immunoprecipitation. CONCLUSION: Our study indicates that in addition to its role in maintaining proteome homeostasis, CLPC1 and likely the CLP proteasome complex also play a role in transcriptome homeostasis through its functions in maintaining proteome homeostasis.

An RNA polymerase II- and AGO4-associated protein acts in RNA-directed DNA methylation.

DNA methylation is an important epigenetic mark in many eukaryotes. In plants, 24-nucleotide small interfering RNAs (siRNAs) bound to the effector protein, Argonaute 4 (AGO4), can direct de novo DNA methylation by the methyltransferase DRM2 (refs 2, 4-6). Here we report a new regulator of RNA-directed DNA methylation (RdDM) in Arabidopsis: RDM1. Loss-of-function mutations in the RDM1 gene impair the accumulation of 24-nucleotide siRNAs, reduce DNA methylation, and release transcriptional gene silencing at RdDM target loci. RDM1 encodes a small protein that seems to bind single-stranded methyl DNA, and associates and co-localizes with RNA polymerase II (Pol II, also known as NRPB), AGO4 and DRM2 in the nucleus. Our results indicate that RDM1 is a component of the RdDM effector complex and may have a role in linking siRNA production with pre-existing or de novo cytosine methylation. Our results also indicate that, although RDM1 and Pol V (also known as NRPE) may function together at some RdDM target sites in the peri-nucleolar siRNA processing centre, Pol II rather than Pol V is associated with the RdDM effector complex at target sites in the nucleoplasm.

A KH-domain RNA-binding protein interacts with FIERY2/CTD phosphatase-like 1 and splicing factors and is important for pre-mRNA splicing in Arabidopsis.

Eukaryotic genomes encode hundreds of RNA-binding proteins, yet the functions of most of these proteins are unknown. In a genetic study of stress signal transduction in Arabidopsis, we identified a K homology (KH)-domain RNA-binding protein, HOS5 (High Osmotic Stress Gene Expression 5), as required for stress gene regulation and stress tolerance. HOS5 was found to interact with FIERY2/RNA polymerase II (RNAP II) carboxyl terminal domain (CTD) phosphatase-like 1 (FRY2/CPL1) both in vitro and in vivo. This interaction is mediated by the first double-stranded RNA-binding domain of FRY2/CPL1 and the KH domains of HOS5. Interestingly, both HOS5 and FRY2/CPL1 also interact with two novel serine-arginine (SR)-rich splicing factors, RS40 and RS41, in nuclear speckles. Importantly, FRY2/CPL1 is required for the recruitment of HOS5. In fry2 mutants, HOS5 failed to be localized in nuclear speckles but was found mainly in the nucleoplasm. hos5 mutants were impaired in mRNA export and accumulated a significant amount of mRNA in the nuclei, particularly under salt stress conditions. Arabidopsis mutants of all these genes exhibit similar stress-sensitive phenotypes. RNA-seq analyses of these mutants detected significant intron retention in many stress-related genes under salt stress but not under normal conditions. Our study not only identified several novel regulators of pre-mRNA processing as important for plant stress response but also suggested that, in addition to RNAP II CTD that is a well-recognized platform for the recruitment of mRNA processing factors, FRY2/CPL1 may also recruit specific factors to regulate the co-transcriptional processing of certain transcripts to deal with environmental challenges.

Genome-wide analysis of alternative splicing of pre-mRNA under salt stress in Arabidopsis.

BACKGROUND: Alternative splicing (AS) of precursor mRNA (pre-mRNA) is an important gene regulation process that potentially regulates many physiological processes in plants, including the response to abiotic stresses such as salt stress. RESULTS: To analyze global changes in AS under salt stress, we obtained high-coverage (~200 times) RNA sequencing data from Arabidopsis thaliana seedlings that were treated with different concentrations of NaCl. We detected that ~49% of all intron-containing genes were alternatively spliced under salt stress, 10% of which experienced significant differential alternative splicing (DAS). Furthermore, AS increased significantly under salt stress compared with under unstressed conditions. We demonstrated that most DAS genes were not differentially regulated by salt stress, suggesting that AS may represent an independent layer of gene regulation in response to stress. Our analysis of functional categories suggested that DAS genes were associated with specific functional pathways, such as the pathways for the responses to stresses and RNA splicing. We revealed that serine/arginine-rich (SR) splicing factors were frequently and specifically regulated in AS under salt stresses, suggesting a complex loop in AS regulation for stress adaptation. We also showed that alternative splicing site selection (SS) occurred most frequently at 4 nucleotides upstream or downstream of the dominant sites and that exon skipping tended to link with alternative SS. CONCLUSIONS: Our study provided a comprehensive view of AS under salt stress and revealed novel insights into the potential roles of AS in plant response to salt stress.

SNP calling using genotype model selection on high-throughput sequencing data.

MOTIVATION: A review of the available single nucleotide polymorphism (SNP) calling procedures for Illumina high-throughput sequencing (HTS) platform data reveals that most rely mainly on base-calling and mapping qualities as sources of error when calling SNPs. Thus, errors not involved in base-calling or alignment, such as those in genomic sample preparation, are not accounted for. RESULTS: A novel method of consensus and SNP calling, Genotype Model Selection (GeMS), is given which accounts for the errors that occur during the preparation of the genomic sample. Simulations and real data analyses indicate that GeMS has the best performance balance of sensitivity and positive predictive value among the tested SNP callers. AVAILABILITY: The GeMS package can be downloaded from https://sites.google.com/a/bioinformatics.ucr.edu/xinping-cui/home/software or http://computationalbioenergy.org/software.html. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Seasonal and Soil Microbiota Effects on the Adaptive Strategies of Wild Goitered Gazelles Based on the Gut Microbiota.

Seasonal variation in extreme environments is a threat to endangered species. The gut microbiota is important in the adaptive strategies of wild herbivores, and herbivores will contact the soil microbiota when they are feeding. However, there are no studies about the effects of soil microbiota on the gut microbiota of wild herbivores. Understanding the seasonal adaptive strategies of wild herbivores based on their gut microbiota and the effects of soil microbiota on the herbivorous gut microbiota is indispensable for making optimal conservation recommendations. To address those issues, we compared the diversity and functions of gut microbiota in goitered gazelles between winter and summer with a non-invasive fecal sampling method from the Qaidam Basin based on 16S rRNA V3-V4 regions. The data showed that seasonal variations caused the significant changes in gut microbiota at α-and ß-diversity levels. The main gut microbial function was "Metabolism." It showed significant seasonal changes. The goitered gazelles adapted to the seasonal changes by increasing the relative abundance of Firmicutes, Christensenellaceae, Bacteroides and the function about "Metabolism" in the winter to improve the adaptability. We also compared the effects of soil microbiota on the gut microbiota between winter and summer, covering source tracking analysis and the seasonal differences in ecological assembly processes. The contribution of soil microbiota on the gut microbiota of goitered gazelles was 5.3095% and 15.6347% in winter and summer, respectively, which was greater than on species of animals living underground. Seasonal variation also influenced the ecological processes of microbiota both in the gut and soil. Due to the differences in environments, the ecological processes between fecal microbiota and soil microbiota showed significant differences, and they were dominated by stochastic processes and deterministic processes, respectively. The soil microbiota has contributed to the gut microbiota, but not a decisive factor. Our research laid the foundation on the seasonal and soil microbiota effects on the adaptive strategies of goitered gazelles, and is the first study to explain the soil microbiota influence on the gut microbiota of wild herbivores.

Main Factors Influencing the Gut Microbiota of Datong Yaks in Mixed Group.

The Datong yak (Bos grunniens) is the first artificial breed of yaks in the world and has played an important role in the improvement of domestic yak quality on the Qinghai-Tibet Plateau. The Datong yak breeding farm in the Qinghai province of China is the main place for the breeding and feeding of Datong yaks. It hosts domestic Datong yaks and wild male yaks, mainly in mixed groups. Different managements have different effects on livestock. The gut microbiota is closely related to the health and immunity of Datong yaks, and mixed grouping can affect the composition and diversity of the gut microbiota of Datong yaks. To reveal the effects of mixed grouping on the gut microbiota of Datong yaks and wild yaks and identify the main dominant factors, we compared the gut microbial diversities of domestic males and females and wild males based on 16S rRNA V3-V4 regions using fresh fecal samples. The data showed significant differences in the gut microbial diversity of these three groups, and the α-diversity was the highest in wild males. Different factors influence the gut microbiota, and the main influencing factors were different in different groups, including sex differences, host genetics, and physical interactions. We also compared ecological assembly processes in the three groups. The results showed that mixed grouping contributed to the improvement of gut microbial diversity in domestic females. Our study provides effective and feasible suggestions for the feeding and management of the Datong yaks.

Digestive Tract Morphology and Gut Microbiota Jointly Determine an Efficient Digestive Strategy in Subterranean Rodents: Plateau Zokor.

Rodents' lifestyles vary in different environments, and to adapt to various lifestyles specific digestion strategies have been developed. Among these strategies, the morphology of the digestive tracts and the gut microbiota are considered to play the most important roles in such adaptations. However, how subterranean rodents adapt to extreme environments through regulating gut microbial diversity and morphology of the digestive tract has yet to be fully studied. Here, we conducted the comparisons of the gastrointestinal morphology, food intake, food assimilation, food digestibility and gut microbiota of plateau zokor Eospalax baileyi in Qinghai-Tibet Plateau and laboratory rats Rattus norvegicus to further understand the survival strategy in a typical subterranean rodent species endemic to the Qinghai-Tibet Plateau. Our results revealed that plateau zokor evolved an efficient foraging strategy with low food intake, high food digestibility, and ultimately achieved a similar amount of food assimilation to laboratory rats. The length and weight of the digestive tract of the plateau zokor was significantly higher than the laboratory rat. Particularly, the weight and length of the large intestine and cecum in plateau zokor is three times greater than that of the laboratory rat. Microbiome analysis showed that genus (i.e., Prevotella, Oscillospira, CF231, Ruminococcus and Bacteroides), which are usually associated with cellulose degradation, were significantly enriched in laboratory rats, compared to plateau zokor. However, prediction of metagenomic function revealed that both plateau zokor and laboratory rats shared the same functions in carbohydrate metabolism and energy metabolism. The higher digestibility of crude fiber in plateau zokor was mainly driven by the sizes of cecum and cecum tract, as well as those gut microbiota which associated with cellulose degradation. Altogether, our results highlight that both gut microbiota and the morphology of the digestive tract are vital to the digestion in wild rodents.

Applications and potentials of nanopore sequencing in the (epi)genome and (epi)transcriptome era.

The Human Genome Project opened an era of (epi)genomic research, and also provided a platform for the development of new sequencing technologies. During and after the project, several sequencing technologies continue to dominate nucleic acid sequencing markets. Currently, Illumina (short-read), PacBio (long-read), and Oxford Nanopore (long-read) are the most popular sequencing technologies. Unlike PacBio or the popular short-read sequencers before it, which, as examples of the second or so-called Next-Generation Sequencing platforms, need to synthesize when sequencing, nanopore technology directly sequences native DNA and RNA molecules. Nanopore sequencing, therefore, avoids converting mRNA into cDNA molecules, which not only allows for the sequencing of extremely long native DNA and full-length RNA molecules but also document modifications that have been made to those native DNA or RNA bases. In this review on direct DNA sequencing and direct RNA sequencing using Oxford Nanopore technology, we focus on their development and application achievements, discussing their challenges and future perspective. We also address the problems researchers may encounter applying these approaches in their research topics, and how to resolve them.

Potential Risks for Seahorse Stock Enhancement: Insight From the Declivity of Genetic Levels With Hatchery Management.

Stock enhancement is one of the potential management strategies for the fishery. To better understand the impaction of stock enhancement, we simulated an experiment for lined seahorse (Hippocampus erectus) and evaluated the genetic structure after stock enhancement. In this study, we found the numbers of alleles (N A ) and heterozygosity (H O ) of stock enhancement strains were lower than those of the wild collections, while the inbreeding coefficient (F IS ) and relatedness index were higher. Within the 3 generations of stock enhancement strain, the N A , H O and polymorphism information content (PIC) didn't change significantly. In addition, the F ST value indicated that the genetic differentiation between the stock enhancement strains and the first wild collection reached an intermediate level, which could lead to substructuring in wild populations. Overall, these findings revealed a potential genetic risk associated with the release of hatchery strains into wild populations.

Genome-wide DNA mutations in Arabidopsis plants after multigenerational exposure to high temperatures.

BACKGROUND: Elevated temperatures can cause physiological, biochemical, and molecular responses in plants that can greatly affect their growth and development. Mutations are the most fundamental force driving biological evolution. However, how long-term elevations in temperature influence the accumulation of mutations in plants remains unknown. RESULTS: Multigenerational exposure of Arabidopsis MA (mutation accumulation) lines and MA populations to extreme heat and moderate warming results in significantly increased mutation rates in single-nucleotide variants (SNVs) and small indels. We observe distinctive mutational spectra under extreme and moderately elevated temperatures, with significant increases in transition and transversion frequencies. Mutation occurs more frequently in intergenic regions, coding regions, and transposable elements in plants grown under elevated temperatures. At elevated temperatures, more mutations accumulate in genes associated with defense responses, DNA repair, and signaling. Notably, the distribution patterns of mutations among all progeny differ between MA populations and MA lines, suggesting that stronger selection effects occurred in populations. Methylation is observed more frequently at mutation sites, indicating its contribution to the mutation process at elevated temperatures. Mutations occurring within the same genome under elevated temperatures are significantly biased toward low gene density regions, special trinucleotides, tandem repeats, and adjacent simple repeats. Additionally, mutations found in all progeny overlap significantly with genetic variations reported in 1001 Genomes, suggesting non-uniform distribution of de novo mutations through the genome. CONCLUSION: Collectively, our results suggest that elevated temperatures can accelerate the accumulation, and alter the molecular profiles, of DNA mutations in plants, thus providing significant insight into how environmental temperatures fuel plant evolution.