Temporal dynamics of chloroplast biogenesis revealed initiation of photosynthesis-related gene expression and protein complexes during alfalfa seed germination.

The chloroplast biogenesis occurs in cotyledon during alfalfa seed germination before true leaf formation, and is extremely important for the followed plant development and growth. In this study, we conducted a simulation of alfalfa seed germination in the soil by using tin foil and focused on 10 pivotal time points of chloroplast biogenesis in cotyledons before and after light exposure, which showed significant differences in multispectral images, and covered the whole process of chloroplast biogenesis from proplastid, etioplast to mature chloroplast. We revealed three phases that referred to the programmed involvements of photosynthesis promotion, ultrastructure maturity, transcriptomic expression, and protein complex construction, and observed distinct transcriptional expressions of genes from nuclear and chloroplast genomes. In phase I at dark germination before light exposure, chloroplast-encoded genes showed up-regulated expressions together with the importation of chloroplast proteins. In phase II for the first day after light exposure, nuclear-encoded genes' expressions were initiated at 2 h after light exposure (E2h), followed by swift assembly of chloroplast thylakoid membrane protein complexes, and roaring Fv/Fm and contents of chlorophyll a, chlorophyll b and carotenoid. The initiation at E2h was pronounced by the observation of gradual accumulation of single lamella, and facilitated the formation of granum stacks (thylakoid) at E8h in phase II. In phase III from the second day after light exposure, chloroplast became gradually complete with the fully established photosynthetic capacity. Altogether, our results layed a theoretical foundation for enhancing potential photosynthetic efficiency in alfalfa and related species.

Different microbial functional traits drive bulk and rhizosphere soil phosphorus mobilization in an alpine meadow after nitrogen input.

Enhanced nitrogen (N) input is expected to influence the soil phosphorus (P) cycling through biotic and abiotic factors. Among these factors, soil microorganisms play a vital role in regulating soil P availability. However, the divergent contribution of functional microorganisms to soil P availability in the rhizosphere and bulk soil under N addition remains unclear. We conducted an N addition experiment with four N input rates (0, 5, 10, and 15 g N m-2 year-1) in an alpine meadow over three years. Metagenomics was employed to investigate the functional microbial traits in the rhizosphere and bulk soil. We showed that N addition had positive effects on microbial functional traits related to P-cycling in the bulk and rhizosphere soil. Specifically, high N addition significantly increased the abundance of most microbial genes in the bulk soil but only enhanced the abundance of five genes in the rhizosphere soil. The soil compartment, rather than the N addition treatment, was the dominant factor explaining the changes in the diversity and network of functional microorganisms. Furthermore, the abundance of functional microbial genes had a profound effect on soil available P, particularly in bulk soil P availability driven by the ppa and ppx genes, as well as rhizosphere soil P availability driven by the ugpE gene. Our results highlight that N addition stimulates the microbial potential for soil P mobilization in alpine meadows. Distinct microbial genes play vital roles in soil P availability in bulk and rhizosphere soil respectively. This indicates the necessity for models to further our knowledge of P mobilization processes from the bulk soil to the rhizosphere soil, allowing for more precise predictions of the effects of N enrichment on soil P cycling.

Dysbiosis of Gut Microbiome Aggravated Male Infertility in Captivity of Plateau Pika.

Captivity is an important and efficient technique for rescuing endangered species. However, it induces infertility, and the underlying mechanism remains obscure. This study used the plateau pika (Ochotona curzoniae) as a model to integrate physiological, metagenomic, metabolomic, and transcriptome analyses and explore whether dysbiosis of the gut microbiota induced by artificial food exacerbates infertility in captive wild animals. Results revealed that captivity significantly decreased testosterone levels and the testicle weight/body weight ratio. RNA sequencing revealed abnormal gene expression profiles in the testicles of captive animals. The microbial α-diversity and Firmicutes/Bacteroidetes ratio were drastically decreased in the captivity group. Bacteroidetes and Muribaculaceae abundance notably increased in captive pikas. Metagenomic analysis revealed that the alteration of flora increased the capacity for carbohydrate degradation in captivity. The levels of microbe metabolites' short-chain fatty acids (SCFAs) were significantly high in the captive group. Increasing SCFAs influenced the immune response of captivity plateau pikas; pro-inflammatory cytokines were upregulated in captivity. The inflammation ultimately contributed to male infertility. In addition, a positive correlation was observed between Gastranaerophilales family abundance and testosterone concentration. Our results provide evidence for the interactions between artificial food, the gut microbiota, and male infertility in pikas and benefit the application of gut microbiota interference in threatened and endangered species.

A chromosome-level genome assembly for Onobrychis viciifolia reveals gene copy number gain underlying enhanced proanthocyanidin biosynthesis.

Sainfoin (Onobrychis viciifolia), which belongs to subfamily Papilionoideae of Leguminosae, is a vital perennial forage known as "holy hay" due to its high contents of crude proteins and proanthocyanidins (PAs, also called condensed tannins) that have various pharmacological properties in animal feed, such as alleviating rumen tympanic disease in ruminants. In this study, we select an autotetraploid common sainfoin (2n = 4x = 28) and report its high-quality chromosome-level genome assembly with 28 pseudochromosomes and four haplotypes (~1950.14 Mb, contig N50 = 10.91 Mb). The copy numbers of genes involved in PA biosynthesis in sainfoin are significantly greater than those in four selected Fabales species, namely, autotetraploid Medicago sativa and three other diploid species, Lotus japonicus, Medicago truncatula, and Glycine max. Furthermore, gene expansion is confirmed to be the key contributor to the increased expression of these genes and subsequent PA enhancement in sainfoin. Transcriptomic analyses reveal that the expression of genes involved in the PA biosynthesis pathway is significantly increased in the lines with high PA content compared to the lines with medium and low PA content. The sainfoin genome assembly will improve our understanding of leguminous genome evolution and biosynthesis of secondary metabolites in sainfoin.

Characterization of phyllosphere endophytic lactic acid bacteria reveals a potential novel route to enhance silage fermentation quality.

The naturally attached phyllosphere microbiota play a crucial role in plant-derived fermentation, but the structure and function of phyllosphere endophytes remain largely unidentified. Here, we reveal the diversity, specificity, and functionality of phyllosphere endophytes in alfalfa (Medicago sativa L.) through combining typical microbial culture, high-throughput sequencing, and genomic comparative analysis. In comparison to phyllosphere bacteria (PB), the fermentation of alfalfa solely with endophytes (EN) enhances the fermentation characteristics, primarily due to the dominance of specific lactic acid bacteria (LAB) such as Lactiplantibacillus, Weissella, and Pediococcus. The inoculant with selected endophytic LAB strains also enhances the fermentation quality compared to epiphytic LAB treatment. Especially, one key endophytic LAB named Pediococcus pentosaceus EN5 shows enrichment of genes related to the mannose phosphotransferase system (Man-PTS) and carbohydrate-metabolizing enzymes and higher utilization of carbohydrates. Representing phyllosphere, endophytic LAB shows great potential of promoting ensiling and provides a novel direction for developing microbial inoculant.

Structural Reorganization in Two Alfalfa Mitochondrial Genome Assemblies and Mitochondrial Evolution in Medicago Species.

Plant mitochondria are crucial for species evolution, phylogenetics, classification, and identification as maternal genetic material. However, the presence of numerous repetitive sequences, complex structures, and a low number of genes in the mitochondrial genome has hindered its complete assembly and related research endeavors. In this study, we assembled two mitochondrial genomes of alfalfa varieties of Zhongmu No.1 (299,123 bp) and Zhongmu No.4 (306,983 bp), based on a combination of PacBio, Illumina, and Hi-C sequences. The comparison of genome assemblies revealed that the same number of mitochondrial genes, including thirty-three protein-coding genes, sixteen tRNA genes, and three rRNA genes existed in the two varieties. Additionally, large fragments of repetitive sequences were found underlying frequent mitochondrial recombination events. We observed extensive transfer of mitochondrial fragments into the nuclear genome of Zhongmu No.4. Analysis of the cox1 and rrn18s genes in 35 Medicago accessions revealed the presence of population-level deletions and substitutions in the rrn18s gene. We propose that mitochondrial structural reorganizations may contribute to alfalfa evolution.

Single-cell transcriptome and metagenome profiling reveals the genetic basis of rumen functions and convergent developmental patterns in ruminants.

The rumen undergoes developmental changes during maturation. To characterize this understudied dynamic process, we profiled single-cell transcriptomes of about 308,000 cells from the rumen tissues of sheep and goats at 17 time points. We built comprehensive transcriptome and metagenome atlases from early embryonic to rumination stages, and recapitulated histomorphometric and transcriptional features of the rumen, revealing key transitional signatures associated with the development of ruminal cells, microbiota, and core transcriptional regulatory networks. In addition, we identified and validated potential cross-talk between host cells and microbiomes and revealed their roles in modulating the spatiotemporal expression of key genes in ruminal cells. Cross-species analyses revealed convergent developmental patterns of cellular heterogeneity, gene expression, and cell-cell and microbiome-cell interactions. Finally, we uncovered how the interactions can act upon the symbiotic rumen system to modify the processes of fermentation, fiber digestion, and immune defense. These results significantly enhance understanding of the genetic basis of the unique roles of rumen.

Complete chloroplast genomes of three wild perennial Hordeum species from Central Asia: genome structure, mutation hotspot, phylogenetic relationships, and comparative analysis.

Hordeum L. is widely distributed in mountain or plateau of subtropical and warm temperate regions around the world. Three wild perennial Hordeum species, including H. bogdanii, H. brevisubulatum, and H. violaceum, have been used as forage and for grassland ecological restoration in high-altitude areas in recent years. To date, the degree of interspecies sequence variation in the three Hordeum species within existing gene pools is still not well-defined. Herein, we sequenced and assembled chloroplast (cp) genomes of the three species. The results revealed that the cp genome of H. bogdanii showed certain sequence variations compared with the cp genomes of the other two species (H. brevisubulatum and H. violaceum), and the latter two were characterized by a higher relative affinity. Parity rule 2 plot (PR2) analysis illuminated that most genes of all ten Hordeum species were concentrated in nucleotide T and G. Numerous single nucleotide polymorphism (SNP) and insertion/deletion (In/Del) events were detected in the three Hordeum species. A series of hotspots regions (tRNA-GGU ~ tRNA-GCA, tRNA-UGU ~ ndhJ, psbE ~ rps18, ndhF ~ tRNA-UAG, etc.) were identified by mVISTA procedures, and the five highly polymorphic genes (tRNA-UGC, tRNA-UAA, tRNA-UUU, tRNA-UAC, and ndhA) were proved by the nucleotide diversity (Pi). Although the distribution and existence of cp simple sequence repeats (cpSSRs) were predicted in the three Hordeum cp genomes, no rearrangement was found between them. A similar phenomenon has been found in the cp genome of the other seven Hordeum species, which has been published so far. In addition, evolutionary relationships were reappraised based on the currently reported cp genome of Hordeum L. This study offers a framework for gaining a better understanding of the evolutionary history of Hordeum species through the re-examination of their cp genomes, and by identifying highly polymorphic genes and hotspot regions that could provide important insights into the genetic diversity and differentiation of these species.

Integrating optical imaging techniques for a novel approach to evaluate Siberian wild rye seed maturity.

Advances in optical imaging technology using rapid and non-destructive methods have led to improvements in the efficiency of seed quality detection. Accurately timing the harvest is crucial for maximizing the yield of higher-quality Siberian wild rye seeds by minimizing excessive shattering during harvesting. This research applied integrated optical imaging techniques and machine learning algorithms to develop different models for classifying Siberian wild rye seeds based on different maturity stages and grain positions. The multi-source fusion of morphological, multispectral, and autofluorescence data provided more comprehensive information but also increases the performance requirements of the equipment. Therefore, we employed three filtering algorithms, namely minimal joint mutual information maximization (JMIM), information gain, and Gini impurity, and set up two control methods (feature union and no-filtering) to assess the impact of retaining only 20% of the features on the model performance. Both JMIM and information gain revealed autofluorescence and morphological features (CIELab A, CIELab B, hue and saturation), with these two filtering algorithms showing shorter run times. Furthermore, a strong correlation was observed between shoot length and morphological and autofluorescence spectral features. Machine learning models based on linear discriminant analysis (LDA), random forests (RF) and support vector machines (SVM) showed high performance (>0.78 accuracies) in classifying seeds at different maturity stages. Furthermore, it was found that there was considerable variation in the different grain positions at the maturity stage, and the K-means approach was used to improve the model performance by 5.8%-9.24%. In conclusion, our study demonstrated that feature filtering algorithms combined with machine learning algorithms offer high performance and low cost in identifying seed maturity stages and that the application of k-means techniques for inconsistent maturity improves classification accuracy. Therefore, this technique could be employed classification of seed maturity and superior physiological quality for Siberian wild rye seeds.

A Novel 3-O-rhamnoside: 2″-O-xylosyltransferase Responsible for Terminal Modification of Prenylflavonol Glycosides in Epimedium pubescens Maxim.

Prenylated flavonol glycosides in Epimedium plants, as key medicinal components, are known to have great pharmaceutical activities for human health. Among the main prenylated flavonol glycosides, the modification mechanism of different sugar moieties is still not well understood. In the current study, a novel prenylated flavonol rhamnoside xylosyltransferase gene (EpF3R2″XylT) was cloned from E. pubescens, and the enzymatic activity of its decoding proteins was examined in vitro with different prenylated flavonol rhamnoside substrates and different 3-O-monosaccharide moieties. Furthermore, the functional and structural domains of EpF3R2″XylT were analyzed by bioinformatic approaches and 3-D protein structure remodeling. In summary, EpF3R2″XylT was shown to cluster with GGT (glycosyltransferase that glycosylates sugar moieties of glycosides) through phylogenetic analysis. In enzymatic analysis, EpF3R2″XylT was proven to transfer xylose moiety from UDP-xylose to prenylated flavonol rhamnoside at the 2″-OH position of rhamnose. The analysis of enzymatic kinetics showed that EpF3R2″XylT had the highest substrate affinity toward icariin with the lowest Km value of 75.96 ± 11.91 mM. Transient expression of EpF3R2″XylT in tobacco leaf showed functional production of EpF3R2″XylT proteins in planta. EpF3R2″XylT was preferably expressed in the leaves of E. pubescens, which is consistent with the accumulation levels of major prenylflavonol 3-O-triglycoside. The discovery of EpF3R2″XylT will provide an economical and efficient alternative way to produce prenylated flavonol trisaccharides through the biosynthetic approach.

Single Seed Identification in Three Medicago Species via Multispectral Imaging Combined with Stacking Ensemble Learning.

Multispectral imaging (MSI) has become a new fast and non-destructive detection method in seed identification. Previous research has usually focused on single models in MSI data analysis, which always employed all features and increased the risk to efficiency and that of system cost. In this study, we developed a stacking ensemble learning (SEL) model for successfully identifying a single seed of sickle alfalfa (Medicago falcata), hybrid alfalfa (M. varia), and alfalfa (M. sativa). SEL adopted a three-layer structure, i.e., level 0 with principal component analysis (PCA), linear discriminant analysis (LDA), and quadratic discriminant analysis (QDA) as models of dimensionality reduction and feature extraction (DRFE); level 1 with support vector machine (SVM), multiple logistic regression (MLR), generalized linear models with elastic net regularization (GLMNET), and eXtreme Gradient Boosting (XGBoost) as basic learners; and level 3 with XGBoost as meta-learner. We confirmed that the values of overall accuracy, kappa, precision, sensitivity, specificity, and sensitivity in the SEL model were all significantly higher than those in basic models alone, based on both spectral features and a combination of morphological and spectral features. Furthermore, we also developed a feature filtering process and successfully selected 5 optimal features out of 33 ones, which corresponded to the contents of chlorophyll, anthocyanin, fat, and moisture in seeds. Our SEL model in MSI data analysis provided a new way for seed identification, and the feature filter process potentially could be used widely for development of a low-cost and narrow-channel sensor.

Genome-Wide Analysis and Expression Profiling of Glutathione Reductase Gene Family in Oat (Avena sativa) Indicate Their Responses to Abiotic Stress during Seed Imbibition.

Abiotic stress disturbs plant cellular redox homeostasis, inhibiting seed germination and plant growth. This is a crucial limitation to crop yield. Glutathione reductase (GR) is an important component of the ascorbate-glutathione (AsA-GSH) cycle which is involved in multiple plant metabolic processes. In the present study, GRs in A. sativa (AsGRs) were selected to explore their molecular characterization, phylogenetic relationship, and RNA expression changes during seed imbibition under abiotic stress. Seven AsGR genes were identified and mapped on six chromosomes of A, C, and D subgenomes. Phylogenetic analysis and subcellular localization of AsGR proteins divided them into two sub-families, AsGR1 and AsGR2, which were predicted to be mainly located in cytoplasm, mitochondrion, and chloroplast. Cis-elements relevant to stress and hormone responses are distributed in promoter regions of AsGRs. Tissue-specific expression profiling showed that AsGR1 genes were highly expressed in roots, leaves, and seeds, while AsGR2 genes were highly expressed in leaves and seeds. Both AsGR1 and AsGR2 genes showed a decreasing-increasing expression trend during seed germination under non-stress conditions. In addition, their responses to drought, salt, cold, copper, H2O2, and ageing treatments were quite different during seed imbibition. Among the seven AsGR genes, AsGR1-A, AsGR1-C, AsGR2-A, and AsGR2-D responded more significantly, especially under drought, ageing, and H2O2 stress. This study has laid the ground for the functional characterization of GR and the improvement of oat stress tolerance and seed vigor.

Spatial variations of root-associated bacterial communities of alpine plants in the Qinghai-Tibet Plateau.

Exploring the geospatial variation of root-associated microbiomes is critical for understanding plant-microbe-environment interactions and plant environmental adaptability. Root-associated bacterial communities from the three compartments [rhizosphere surrounding soil (RSS), rhizosphere soil (rhizosphere), and root endosphere (endophytic)] are influenced by multiple factors, including plant species and geographical locations. Nonetheless, these communities remain poorly understood under harsh conditions. In this study, we selected four dominant alpine plants on the Qinghai-Tibet Plateau (i.e., Elymus nutans, Festuca sinensis, Kobresia pygmaea, and Kobresia humilis) to investigate their root-associated bacterial communities across 11 geographical locations and determine the factors driving spatial variation. The results showed that the microbiota of the three compartments had significantly different community compositions, with more Pseudomonadaceae and Enterobacteriaceae present in the endosphere. Spatial variations in root endophytic microbiota were mainly governed by stochastic processes, which were different from the deterministic processes in the other two compartments. Meanwhile, the geographical location had greater effects on bacterial communities than plant species, and the spatial variation of α-diversity in the endosphere was much higher than that in the RSS and rhizosphere. We further found that the differentiation of bacterial diversity in the endosphere among sympatric plant species was enhanced by higher annual precipitation, lower soil nutrients (carbon and nitrogen), and pH. For example, the coefficient of variation of endosphere Pseudomonadaceae abundance was positively correlated with annual mean precipitation, whereas that of Enterobacteriaceae abundance was negatively correlated with soil pH. The co-occurrence network analysis identified a higher proportion of bacterial coexistence in the endosphere (70.9%) than in the RSS (49.5%) and rhizosphere soil (50.9%). Finally, we revealed the relative convergence of endophytic communities among sympatric plant species in the alpine grasslands.

Physiological and Molecular Responses of Zoysia japonica to Rust Infection.

Zoysiagrass (Zoysia japonica) is a popular turfgrass species and is widely used for sport turf and urban landscape. Zoysiagrass is often infected by Puccinia zoysiae, which causes a loss in turf quality. The physiological and molecular mechanisms of rust resistance are poorly understood in this species. In this study, the rust-resistant and susceptible lines of zoysiagrass were inoculated with P. zoysiae, and alterations of leaf cell structure, physiological indicators and transcriptomic response were investigated at the various stages of inoculation. After inoculation, the cell membranes, nucleus, mitochondria, and chloroplast were all impaired, followed by abnormal physiological metabolism. The damage occurred earlier and more severely in the susceptible line. Changes in electrolyte leakage and chlorophyll content varied with the genotype and the inoculation stages. The transcriptome analysis showed that plant hormones, MAPK signal transduction pathway, photosynthesis and energy generation pathways were significantly enriched in the early response, in both the resistant and susceptible lines. The results provided insights into the physiological and molecular mechanisms of rust disease resistance and would benefit the breeding of rust-resistant varieties in zoysiagrass and related turfgrass species.

Dynamic Responses of Antioxidant and Glyoxalase Systems to Seed Aging Based on Full-Length Transcriptome in Oat (Avena sativa L.).

Seed aging is a major challenge for food security, agronomic production, and germplasm conservation, and reactive oxygen species (ROS) and methylglyoxal (MG) are highly involved in the aging process. However, the regulatory mechanisms controlling the abundance of ROS and MG are not well characterized. To characterize dynamic response of antioxidant and glyoxalase systems during seed aging, oat (Avena sativa L.) aged seeds with a range of germination percentages were used to explore physiological parameters, biochemical parameters and relevant gene expression. A reference transcriptome based on PacBio sequencing generated 67,184 non-redundant full-length transcripts, with 59,050 annotated. Subsequently, eleven seed samples were used to investigate the dynamic response of respiration, ROS and MG accumulation, antioxidant enzymes and glyoxalase activity, and associated genes expression. The 48 indicators with high correlation coefficients were divided into six major response patterns, and were used for placing eleven seed samples into four groups, i.e., non-aged (Group N), higher vigor (Group H), medium vigor (Group M), and lower vigor (Group L). Finally, we proposed a putative model for aging response and self-detoxification mechanisms based on the four groups representing different aging levels. In addition, the outcomes of the study suggested the dysfunction of antioxidant and glyoxalase system, and the accumulation of ROS and MG definitely contribute to oat seed aging.

Seasonal variations in the composition and functional profiles of gut microbiota reflect dietary changes in plateau pikas.

Seasonal variations in gut microbiota of small mammals and how they are influenced by environmental variables are relatively poorly understood. We sampled 162 wild plateau pikas (Ochotona curzoniae) in 4 seasons over 2 and a half years and recorded the air temperature, precipitation, and nutrient content in edible vegetation at the sampling site. After conducting 16S rRNA and shotgun metagenomic sequencing, we found that the highest alpha diversity, the relative abundance of Firmicutes, and the simplest co-occurrence network occurred in winter, whereas the highest relative abundance of Proteobacteria and the most complex network structure were observed in spring. The highest relative abundance of Verrucomicrobiota and Spirochaetota was seen in summer and autumn, respectively. Air temperature, precipitation, and the contents of crude protein, crude fiber, and polysaccharide in vegetation had significant effects on the seasonal changes in gut microbiota. Diet contributed more to microbial variation than climatic factors. Metagenomic analysis revealed that the amino acid metabolism pathway and axillary activity enzymes were most abundant in summer, while abundance of carbohydrate-binding modules and carbohydrate esterases was highest in spring. These microbial variations were related to the changes in dietary nutrition, indicating that gut microbiota of plateau pika contribute to the efficient use of food resources. This study provides new evidence of how external environmental factors affect the intestinal environment of small mammals.

InMut-finder: a software tool for insertion identification in mutagenesis using Nanopore long reads.

BACKGROUND: Biological mutagens (such as transposon) with sequences inserted, play a crucial role to link observed phenotype and genotype in reverse genetic studies. For this reason, accurate and efficient software tools for identifying insertion sites based on the analysis of sequencing reads are desired. RESULTS: We developed a bioinformatics tool, a Finder, to identify genome-wide Insertions in Mutagenesis (named as "InMut-Finder"), based on target sequences and flanking sequences from long reads, such as Oxford Nanopore Sequencing. InMut-Finder succeeded in identify > 100 insertion sites in Medicago truncatula and soybean mutants based on sequencing reads of whole-genome DNA or enriched insertion-site DNA fragments. Insertion sites discovered by InMut-Finder were validated by PCR experiments. CONCLUSION: InMut-Finder is a comprehensive and powerful tool for automated insertion detection from Nanopore long reads. The simplicity, efficiency, and flexibility of InMut-Finder make it a valuable tool for functional genomics and forward and reverse genetics. InMut-Finder was implemented with Perl, R, and Shell scripts, which are independent of the OS. The source code and instructions can be accessed at https://github.com/jsg200830/InMut-Finder .

Unveiling the Complexity of Red Clover (Trifolium pratense L.) Transcriptome and Transcriptional Regulation of Isoflavonoid Biosynthesis Using Integrated Long- and Short-Read RNAseq.

Red clover (Trifolium pratense L.) is used as forage and contains a high level of isoflavonoids. Although isoflavonoids in red clover were discovered a long time ago, the transcriptional regulation of isoflavonoid biosynthesis is virtually unknown because of the lack of accurate and comprehensive characterization of the transcriptome. Here, we used a combination of long-read (PacBio Iso-Seq) and short-read (Illumina) RNAseq sequencing to develop a more comprehensive full-length transcriptome in four tissues (root, stem, leaf, and flower) and to identify transcription factors possibly involved in isoflavonoid biosynthesis in red clover. Overall, we obtained 50,922 isoforms, including 19,860 known genes and 2817 novel isoforms based on the annotation of RefGen Tp_v2.0. We also found 1843 long non-coding RNAs, 1625 fusion genes, and 34,612 alternatively spliced events, with some transcript isoforms validated experimentally. A total of 16,734 differentially expressed genes were identified in the four tissues, including 43 isoflavonoid-biosynthesis-related genes, such as stem-specific expressed TpPAL, TpC4H, and Tp4CL and root-specific expressed TpCHS, TpCHI1, and TpIFS. Further, weighted gene co-expression network analysis and a targeted compound assay were combined to investigate the association between the isoflavonoid content and the transcription factors expression in the four tissues. Twelve transcription factors were identified as key genes for isoflavonoid biosynthesis. Among these transcription factors, the overexpression of TpMYB30 or TpRSM1-2 significantly increased the isoflavonoid content in tobacco. In particular, the glycitin was increased by 50-100 times in the plants overexpressing TpRSM1-2, in comparison to that in the WT plants. Our study provides a comprehensive and accurate annotation of the red clover transcriptome and candidate genes to improve isoflavonoid biosynthesis and accelerate research into molecular breeding in red clover or other crops.

Dietary Fiber Influences Bacterial Community Assembly Processes in the Gut Microbiota of Durco × Bamei Crossbred Pig.

Several studies have shown that dietary fiber can significantly alter the composition and structure of the gut bacterial community in humans and mammals. However, few researches have been conducted on the dynamics of the bacterial community assembly across different graded levels of dietary fiber in different gut regions. To address this, 24 Durco × Bamei crossbred pigs were randomly assigned to four experimental chows comprising graded levels of dietary fiber. Results showed that the α-and ß-diversity of the bacterial community was significantly different between the cecum and the jejunum. Adding fiber to the chow significantly increased the α-diversity of the bacterial community in the jejunum and cecum, while the ß-diversity decreased. The complexity of the bacterial network increased with the increase of dietary fiber in jejunal content samples, while it decreased in cecal content samples. Furthermore, we found that stochastic processes governed the bacterial community assembly of low and medium dietary fiber groups of jejunal content samples, while deterministic processes dominated the high fiber group. In addition, deterministic processes dominated all cecal content samples. Taken together, the variation of gut community composition and structure in response to dietary fiber was distinct in different gut regions, and the dynamics of bacterial community assembly across the graded levels of dietary fiber in different gut regions was also distinct. These findings enhanced our knowledge on the bacterial community assembly processes in gut ecosystems of livestock.

Chromosome-Scale Genome Assembly for Chinese Sour Jujube and Insights Into Its Genome Evolution and Domestication Signature.

Sour or wild jujube fruits and dried seeds are popular food all over the world. In this study, we reported a high-quality genome assembly of sour jujube (Ziziphus jujuba Mill. var. spinosa), with a size of 406 Mbp and scaffold N50 of 30.3 Mbp, which experienced only γ hexaploidization event, without recent genome duplication. Population structure analysis identified four jujube subgroups (two domesticated ones, i.e., D1 in West China and D2 in East/SouthEast China, semi-wild, and wild), which underwent an evolutionary history of a significant decline of effective population size during the Last Glacial Period. The respective selection signatures of three subgroups were discovered, such as strong peaks on chromosomes #3 in D1, #1 in D2, and #4 in wild. Genes under the most significant selection on chromosomes #4 in wild were confirmed to be involved in fruit variations among jujube accessions, in transcriptomic analysis. Our study offered novel insights into the jujube population structure and domestication and provided valuable genomic resources for jujube improvement in stress response and fruit flavor in the future.