Identification of WRKY gene family in Dioscorea opposita Thunb. reveals that DoWRKY71 enhanced the tolerance to cold and ABA stress.

WRKY transcription factors constitute one of the largest plant-specific gene families, regulating various aspects of plant growth, development, physiological processes, and responses to abiotic stresses. This study aimed to comprehensively analyze the WRKY gene family of yam (Dioscorea opposita Thunb.), to understand their expression patterns during the growth and development process and their response to different treatments of yam and analyze the function of DoWRKY71 in detail. A total of 25 DoWRKY genes were identified from the transcriptome of yam, which were divided into six clades (I, IIa, IIc, IId, IIe, III) based on phylogenetic analysis. The analysis of conserved motifs revealed 10 motifs, varying in length from 16 to 50 amino acids. Based on real-time quantitative PCR (qRT-PCR) analysis, DoWRKY genes were expressed at different stages of growth and development and responded differentially to various abiotic stresses. The expression level of DoWRKY71 genes was up-regulated in the early stage and then down-regulated in tuber enlargement. This gene showed responsiveness to cold and abiotic stresses, such as abscisic acid (ABA) and methyl jasmonate (MeJA). Therefore, further study was conducted on this gene. Subcellular localization analysis revealed that the DoWRKY71 protein was localized in the nucleus. Moreover, the overexpression of DoWRKY71 enhanced the cold tolerance of transgenic tobacco and promoted ABA mediated stomatal closure. This study presents the first systematic analysis of the WRKY gene family in yam, offering new insights for studying WRKY transcription factors in yam. The functional study of DoWRKY71 lays theoretical foundation for further exploring the regulatory function of the DoWRKY71 gene in the growth and development related signaling pathway of yam.

Isolation of endophytes from Dioscorea nipponica Makino for stimulating diosgenin production and plant growth.

KEY MESSAGE: Both bacterial and fungal endophytes exhibited one or more plant growth-promoting (PGP) traits. Among these strains, the Paenibacillus peoriae SYbr421 strain demonstrated the greatest activity in the direct biotransformation of tuber powder from D. nipponica into diosgenin. Endophytes play crucial roles in shaping active metabolites within plants, significantly influencing both the quality and yield of host plants. Dioscorea nipponica Makino accumulates abundant steroidal saponins, which can be hydrolyzed to produce diosgenin. However, our understanding of the associated endophytes and their contributions to plant growth and diosgenin production is limited. The present study aimed to assess the PGP ability and potential of diosgenin biotransformation by endophytes isolates associated with D. nipponica for the efficient improvement of plant growth and development of a clean and effective approach for producing the valuable drug diosgenin. Eighteen bacterial endophytes were classified into six genera through sequencing and phylogenetic analysis of the 16S rDNA gene. Similarly, 12 fungal endophytes were categorized into 5 genera based on sequencing and phylogenetic analysis of the ITS rDNA gene. Pure culture experiments revealed that 30 isolated endophytic strains exhibited one or more PGP traits, such as nitrogen fixation, phosphate solubilization, siderophore synthesis, and IAA production. One strain of endophytic bacteria, P. peoriae SYbr421, effectively directly biotransformed the saponin components in D. nipponica. Moreover, a high yield of diosgenin (3.50%) was obtained at an inoculum size of 4% after 6 days of fermentation. Thus, SYbr421 could be used for a cleaner and more eco-friendly diosgenin production process. In addition, based on the assessment of growth-promoting isolates and seed germination results, the strains SYbr421, SYfr1321, and SYfl221 were selected for greenhouse experiments. The results revealed that the inoculation of these promising isolates significantly increased the plant height and fresh weight of the leaves and roots compared to the control plants. These findings underscore the importance of preparing PGP bioinoculants from selected isolates as an additional option for sustainable diosgenin production.

Whole-genome sequencing and comparative genomics reveal candidate genes associated with quality traits in Dioscorea alata.

BACKGROUND: Quality traits are essential determinants of consumer preferences. Dioscorea alata (Greater Yam), is a starchy tuber crop in tropical regions. However, a comprehensive understanding of the genetic basis underlying yam tuber quality remains elusive. To address this knowledge gap, we employed population genomics and candidate gene association approaches to unravel the genetic factors influencing the quality attributes of boiled yam. METHODS AND RESULTS: Comparative genomics analysis of 45 plant species revealed numerous novel genes absent in the existing D. alata gene annotation. This approach, adding 48% more genes, significantly enhanced the functional annotation of three crucial metabolic pathways associated with boiled yam quality traits: pentose and glucuronate interconversions, starch and sucrose metabolism, and flavonoid biosynthesis. In addition, the whole-genome sequencing of 127 genotypes identified 27 genes under selection and 22 genes linked to texture, starch content, and color through a candidate gene association analysis. Notably, five genes involved in starch content and cell wall composition, including 1,3-beta Glucan synthase, ß-amylase, and Pectin methyl esterase, were common to both approaches and their expression levels were assessed by transcriptomic data. CONCLUSIONS: The analysis of the whole-genome of 127 genotypes of D. alata and the study of three specific pathways allowed the identification of important genes for tuber quality. Our findings provide insights into the genetic basis of yam quality traits and will help the enhancement of yam tuber quality through breeding programs.

Unraveling the Molecular Basis of Color Variation in Dioscorea alata Tubers: Integrated Transcriptome and Metabolomics Analysis.

Dioscorea alata L. (Dioscoreaceae) is a widely cultivated tuber crop with variations in tuber color, offering potential value as health-promoting foods. This study focused on the comparison of D. alata tubers possessing two distinct colors, white and purple, to explore the underlying mechanisms of color variation. Flavonoids, a group of polyphenols known to influence plant color and exhibit antioxidant properties, were of particular interest. The total phenol and total flavonoid analyses revealed that purple tubers (PTs) have a significantly higher content of these metabolites than white tubers (WTs) and a higher antioxidant activity than WTs, suggesting potential health benefits of PT D. alata. The transcriptome analysis identified 108 differentially expressed genes associated with the flavonoid synthesis pathway, with 57 genes up-regulated in PTs, including CHS, CHI, DFR, FLS, F3H, F3'5'H, LAR, ANS, and ANR. The metabolomics analysis demonstrated that 424 metabolites, including 104 flavonoids and 8 tannins, accumulated differentially in PTs and WTs. Notably, five of the top ten up-regulated metabolites were flavonoids, including 6-hydroxykaempferol-7-O-glucoside, pinocembrin-7-O-(6″-O-malonyl)glucoside, 6-hydroxykaempferol-3,7,6-O-triglycoside, 6-hydroxykaempferol-7-O-triglycoside, and cyanidin-3-O-(6″-O-feruloyl)sophoroside-5-O-glucoside, with the latter being a precursor to anthocyanin synthesis. Integrating transcriptome and metabolomics data revealed that the 57 genes regulated 20 metabolites within the flavonoid synthesis pathway, potentially influencing the tubers' color variation. The high polyphenol content and antioxidant activity of PTs indicate their suitability as nutritious and health-promoting food sources. Taken together, the findings of this study provide insights into the molecular basis of tuber color variation in D. alata and underscore the potential applications of purple tubers in the food industry and human health promotion. The findings contribute to the understanding of flavonoid biosynthesis and pigment accumulation in D. alata tubers, opening avenues for future research on enhancing the nutritional quality of D. alata cultivars.

Diosgenin biosynthesis pathway and its regulation in Dioscorea cirrhosa L.

Dioscorea cirrhosa L. (D. cirrhosa) tuber is a traditional medicinal plant that is abundant in various pharmacological substances. Although diosgenin is commonly found in many Dioscoreaceae plants, its presence in D. cirrhosa remained uncertain. To address this, HPLC-MS/MS analysis was conducted and 13 diosgenin metabolites were identified in D. cirrhosa tuber. Furthermore, we utilized transcriptome data to identify 21 key enzymes and 43 unigenes that are involved in diosgenin biosynthesis, leading to a proposed pathway for diosgenin biosynthesis in D. cirrhosa. A total of 3,365 unigenes belonging to 82 transcription factor (TF) families were annotated, including MYB, AP2/ERF, bZIP, bHLH, WRKY, NAC, C2H2, C3H, SNF2 and Aux/IAA. Correlation analysis revealed that 22 TFs are strongly associated with diosgenin biosynthesis genes (-r2- > 0.9, P < 0.05). Moreover, our analysis of the CYP450 gene family identified 206 CYP450 genes (CYP450s), with 40 being potential CYP450s. Gene phylogenetic analysis revealed that these CYP450s were associated with sterol C-22 hydroxylase, sterol-14-demethylase and amyrin oxidase in diosgenin biosynthesis. Our findings lay a foundation for future genetic engineering studies aimed at improving the biosynthesis of diosgenin compounds in plants.

Identification of Dioscorea opposite Thunb. CDPK gene family reveals that DoCDPK20 is related to heat resistance.

Temperature affects the growth and yield of yam (Dioscorea opposite Thunb.), and calcium-dependent protein kinases (CDPKs) play an important role in the plant stress response. However, there has been a lack of system analyses of yam's CDPK gene family. In this study, 29 CDPK transcriptome sequences with complete open reading frames (ORFs) were identified from yam RNA sequencing data. The sequences were classified into four groups (I-VI) using phylogenetic analysis. Two DoCDPK genes were randomly selected from each group and the gene patterns of yam leaves were determined using quantitative real-time PCR (qRT-PCR) under high and low temperature stress in order to show their unique functions in mediating specific responses. Among them, DoCDPK20 was significantly induced in high temperatures. The pPZP221-DoCDPK20 was transformed into tobacco leaves using an agrobacterium-mediated method. Under high temperature stress, DoCDPK20 overexpression reduced photosynthesis and improved heat tolerance in transgenic tobacco. Our research offers meaningful perspectives into CDPK genes and new avenues for the genetic engineering and molecular breeding of yam.

Association mapping in multiple yam species (Dioscorea spp.) of quantitative trait loci for yield-related traits.

BACKGROUND: Yam (Dioscorea spp.) is multiple species with various ploidy level and considered as cash crop in many producing areas. Selection based phenotyping for yield and its related traits such as mosaic virus and anthracnose diseases resistance and plant vigor in multiple species of yam is lengthy however, marker information has proven to enhance selection efficiency. METHODOLOGY: In this study, a panel of 182 yam accessions distributed across six yam species were assessed for diversity and marker-traits association study using SNP markers generated from Diversity Array Technology platform. For the traits association analysis, the relation matrix alongside the population structure were used as co-factor to avoid false discovery using Multiple random Mixed Linear Model (MrMLM) followed by gene annotation. RESULTS: Accessions performance were significantly different (p < 0.001) across all the traits with high broad-sense heritability (H2). Phenotypic and genotypic correlations showed positive relationships between yield and vigor but negative for yield and yam mosaic disease severity. Population structure revealed k = 6 as optimal clusters-based species. A total of 22 SNP markers were identified to be associated with yield, vigor, mosaic and anthracnose diseases resistance. Gene annotation for the significant SNP loci identified some putative genes associated with primary metabolism, pest and resistance to anthracnose disease, maintenance of NADPH in biosynthetic reaction especially those involving nitro-oxidative stress for resistance to mosaic virus, and seed development, photosynthesis, nutrition use efficiency, stress tolerance, vegetative and reproductive development for tuber yield. CONCLUSION: This study provides valuable insights into the genetic control of plant vigor, anthracnose, mosaic virus resistance, and tuber yield in yam and thus, opens an avenue for developing additional genomic resources for markers-assisted selection focusing on multiple yam species.

Wounding induces suberin deposition, relevant gene expressions and changes of endogenous phytohormones in Chinese yam (Dioscorea opposita) tubers.

Wounds on Chinese yam (Dioscorea opposita ) tubers can ocurr during harvest and handling, and rapid suberisation of the wound is required to prevent pathogenic infection and desiccation. However, little is known about the causal relationship among suberin deposition, relevant gene expressions and endogenous phytohormones levels in response to wounding. In this study, the effect of wounding on phytohormones levels and the expression profiles of specific genes involved in wound-induced suberisation were determined. Wounding rapidly increased the expression levels of genes, including PAL , C4H , 4CL , POD , KCSs , FARs , CYP86A1 , CYP86B1 , GPATs , ABCGs and GELPs , which likely involved in the biosynthesis, transport and polymerisation of suberin monomers, ultimately leading to suberin deposition. Wounding induced phenolics biosynthesis and being polymerised into suberin poly(phenolics) (SPP) in advance of suberin poly(aliphatics) (SPA) accumulation. Specifically, rapid expression of genes (e.g. PAL , C4H , 4CL , POD ) associated with the biosynthesis and polymerisation of phenolics, in consistent with SPP accumulation 3days after wounding, followed by the massive accumulation of SPA and relevant gene expressions (e.g. KCSs , FARs , CYP86A1 /B1 , GPATs , ABCGs , GELPs ). Additionally, wound-induced abscisic acid (ABA) and jasmonic acid (JA) consistently correlated with suberin deposition and relevant gene expressions indicating that they might play a central role in regulating wound suberisation in yam tubers.

Dioscorea composita WRKY5 positively regulates AtSOD1 and AtABF2 to enhance drought and salt tolerances.

KEY MESSAGE: DcWRKY5 increases the antioxidant enzyme activity and proline accumulation, oppositely, reduces the accumulation of ROS and MDA, through directly activating the genes expression, finally enhances the salt and drought tolerance. Drought and salinity are two main environmental factors that limit the large-scale cultivation of the medicinal plant Dioscorea composita (D. composita). WRKY transcription factors (TFs) play vital roles in regulating drought and salt tolerance in plants. Nevertheless, the molecular mechanism of WRKY TF mediates drought and salt resistance of D. composita remains largely unknown. Here, we isolated and characterized a WRKY TF from D. composita, namely DcWRKY5, which was localized to the nucleus and bound to the W-box cis-acting elements. Expression pattern analysis showed that it was highly expressed in root and significantly up-regulated in the presence of salt, polyethylene glycol-6000 (PEG-6000) and abscisic acid (ABA). Heterologous expression of DcWRKY5 increased salt and drought tolerance in Arabidopsis, but was insensitive to ABA. In addition, compared with the wild type, the DcWRKY5 overexpressing transgenic lines had more proline, higher antioxidant enzyme (POD, SOD, and CAT) activities, less reactive oxygen species (ROS) and malondialdehyde (MDA). Correspondingly, the overexpression of DcWRKY5 modulated the expression of genes related to salt and drought stresses, such as AtSS1, AtP5CS1, AtCAT, AtSOD1, AtRD22, and AtABF2. Dual luciferase assay and Y1H were further confirmed that DcWRKY5 activate the promoter of AtSOD1 and AtABF2 through directly binding to the enrichment region of the W-box cis-acting elements. These results suggest that DcWRKY5 is a positive regulator of the drought and salt tolerance in D. composita and has potential applications in transgenic breeding.

Identification of BBX gene family and its function in the regulation of microtuber formation in yam.

BBX proteins play important roles in all of the major light-regulated developmental processes. However, no systematic analysis of BBX gene family regarding the regulation of photoperiodic microtuber formation has been previously performed in yam. In this study, a systematic analysis on the BBX gene family was conducted in three yam species, with the results, indicating that this gene plays a role in regulating photoperiodic microtuber formation. These analyses included identification the BBX gene family in three yam species, their evolutionary relationships, conserved domains, motifs, gene structure, cis-acting elements, and expressional patterns. Based on these analyses, DoBBX2/DoCOL5 and DoBBX8/DoCOL8 showing the most opposite pattern of expression during microtuber formation were selected as candidate genes for further investigation. Gene expression analysis showed DoBBX2/DoCOL5 and DoBBX8/DoCOL8 were highest expressed in leaves and exhibited photoperiod responsive expression patterns. Besides, the overexpression of DoBBX2/DoCOL5 and DoBBX8/DoCOL8 in potato accelerated tuber formation under short-day (SD) conditions, whereas only the overexpression of DoBBX8/DoCOL8 enhanced the accelerating effect of dark conditions on tuber induction. Tuber number was increased in DoBBX8/DoCOL8 overexpressing plants under dark, as well as in DoBBX2/DoCOL5 overexpressing plants under SD. Overall, the data generated in this study may form the basis of future functional characterizations of BBX genes in yam, especially regarding their regulation of microtuber formation via the photoperiodic response pathway.

Characterization of the cholesterol biosynthetic pathway in Dioscorea transversa.

Cholesterol is the precursor of bioactive plant metabolites such as steroidal saponins. An Australian plant, Dioscorea transversa, produces only two steroidal saponins: 1ß-hydroxyprotoneogracillin and protoneogracillin. Here, we used D. transversa as a model in which to elucidate the biosynthetic pathway to cholesterol, a precursor to these compounds. Preliminary transcriptomes of D. transversa rhizome and leaves were constructed, annotated, and analyzed. We identified a novel sterol side-chain reductase as a key initiator of cholesterol biosynthesis in this plant. By complementation in yeast, we determine that this sterol side-chain reductase reduces Δ24,28 double bonds required for phytosterol biogenesis as well as Δ24,25 double bonds. The latter function is believed to initiate cholesterogenesis by reducing cycloartenol to cycloartanol. Through heterologous expression, purification, and enzymatic reconstitution, we also demonstrate that the D. transversa sterol demethylase (CYP51) effectively demethylates obtusifoliol, an intermediate of phytosterol biosynthesis and 4-desmethyl-24,25-dihydrolanosterol, a postulated downstream intermediate of cholesterol biosynthesis. In summary, we investigated specific steps of the cholesterol biosynthetic pathway, providing further insight into the downstream production of bioactive steroidal saponin metabolites.

Unravelling the genetic diversity of water yam (Dioscorea alata L.) accessions from Tanzania using simple sequence repeat (SSR) markers.

Water yam (Dioscorea alata L.) is among the most cultivated species used as a source of food and income for small-scale farmers in Tanzania. However, little is documented about Dioscorea species available in Tanzania, including their genetic diversity. This study used ten polymorphic microsatellite markers to determine the genetic diversity and relationship of 63 D. alata accessions from six major producing regions. Results revealed a polymorphic information content (PIC) of 0.63, while the number of alleles per locus ranged from 4 to 12 with a mean of 7.60. The expected heterozygosity ranged from 0.20to 0.76, with a mean of 0.53, which suggests moderate genetic diversity of D. alata accessions. Kagera region had the highest mean number of (1.5) private alleles. Analysis of molecular variance revealed that 54% of the variation was attributed to within individual, 39% was attributed to among individual while among population contributed 7% of the total variation. The highest Nei's genetic distance (0.43) was for accessions sampled from Kilimanjaro and Mtwara regions. Principal coordinate analysis and cluster analysis using Unweighted Paired Group Method using Arithmetic (UPGMA) grouped D. alata accessions into two major clusters regardless of geographical origin and local names. The Bayesian structure analysis confirmed the two clusters obtained in UPGMA and revealed an admixture of D. alata accessions in all six regions suggesting farmers' extensive exchange of planting materials. These results are helpful in the selection of D. alata accessions for breeding programs in Tanzania.

Complete Chloroplast Genome of Four Thai Native Dioscorea Species: Structural, Comparative and Phylogenetic Analyses.

The chloroplast genomes of Dioscorea brevipetiolata, D. depauperata, D. glabra, and D. pyrifolia are 153,370-153,503 bp in size. A total of 113 genes were predicted, including 79 protein-coding sequences (CDS), 30 tRNA, and four rRNA genes. The overall GC content for all four species was 37%. Only mono-, di-, and trinucleotides were present in the genome. Genes adjacent to the junction borders were similar in all species analyzed. Eight distinct indel variations were detected in the chloroplast genome alignment of 24 Dioscorea species. At a cut-off point of Pi = 0.03, a sliding window analysis based on 25 chloroplast genome sequences of Dioscorea species revealed three highly variable regions, which included three CDS (trnC, ycf1, and rpl32), as well as an intergenic spacer region, ndhF-rpl32. A phylogenetic tree based on the complete chloroplast genome sequence displayed an almost fully resolved relationship in Dioscorea. However, D. brevipetiolata, D. depauperata, and D. glabra were clustered together with D. alata, while D. pyrifolia was closely related to D. aspersa. As Dioscorea is a diverse genus, genome data generated in this study may contribute to a better understanding of the genetic identity of these species, which would be useful for future taxonomic work of Dioscorea.

Biosynthetic Gene Expression and Tissue Distribution of Diosgenin in Dioscorea japonica.

Diosgenin is an aglycone of dioscin, a major bioactive steroidal saponin found in plants, including Himalayan Paris (Paris polyphylla), fenugreek (Trigonella foenum-graecum), and yam (Dioscorea spp.). We have previously demonstrated that a species of natural yam, Dioscorea japonica, contains a promising bioactive compound diosgenin, which induces anti-carcinogenic and anti-hypertriacylglycerolemic activities. Here, we found for the first time that Japanese yam (D. japonica) bulbils are richer in diosgenin than the edible tubers (rhizomes) and leaves. LC-MS and imaging-MS analyses revealed that diosgenin accumulated in the peripheral region of D. japonica bulbils. Additionally, we performed RNA-seq analysis of D. japonica, and multiple sequence alignment identified D. japonica CYP90 (DjCYP90), the orthologous gene of CYP90G4 in P. polyphylla, CYP90B50 in T. foenum-graecum, CYP90G6 in Dioscorea zingiberensis, and CYP90G in Dioscorea villosa, which encodes a diosgenin biosynthetic rate-limiting enzyme. The expression levels of DjCYP90 were significantly upregulated in D. japonica bulbils than in its rhizomes and leaves. Since diosgenin is one of the most promising functional food factors executing several favorable bioactivities, D. japonica bulbils rich in diosgenin would be a beneficial natural resource.

Genetic diversity and population structure of Uganda's yam (Dioscorea spp.) genetic resource based on DArTseq.

Assessing the genetic diversity of yam germplasm from different geographical origins for cultivation and breeding purposes is an essential step for crop genetic resource conservation and genetic improvement, especially where the crop faces minimal attention. This study aimed to classify the population structure, and assess the extent of genetic diversity in 207 Dioscorea rotundata genotypes sourced from three different geographical origins. A total of 4,957 (16.2%) single nucleotide polymorphism markers were used to assess genetic diversity. The SNP markers were informative, with polymorphic information content ranging from 0.238 to 0.288 and a mean of 0.260 across all the genotypes. The observed and expected heterozygosity was 0.12 and 0.23, respectively while the minor allele frequency ranged from 0.093 to 0.124 with a mean of 0.109. The principal coordinate analysis, model-based structure and discriminant analysis of principal components, and the Euclidean distance matrix method grouped 207 yam genotypes into three main clusters. Genotypes from West Africa (Ghana and Nigeria) had significant similarities with those from Uganda. Analysis of molecular variance revealed that within-population variation across three different geographical origins accounted for 93% of the observed variation. This study, therefore, showed that yam improvement in Uganda is possible, and the outcome will constitute a foundation for the genetic improvement of yams in Uganda.

Dioscorea composita WRKY12 is involved in the regulation of salt tolerance by directly activating the promoter of AtRCI2A.

Dioscorea composita (D. composita) is an important medicinal plant worldwide with high economic value. However, its large-scale cultivation was limited by soil salinization. Identification of genes and their mechanisms of action in response to salt stress are critically important. In the present study, we isolated a classical WRKY transcription factor from D. composita, namely DcWRKY12, and analyzed its function in salt tolerance. Expression pattern analysis showed DcWRKY12 is mainly expressed in roots and significantly induced by NaCl, polyethylene glycol-6000 (PEG-6000), and abscisic acid (ABA). Phenotypic and physiological analyses revealed that heterologous expression of DcWRKY12 enhanced salt and osmotic stress tolerance by increasing antioxidant enzyme activity, osmoregulatory substance content, maintaining relative water content and ion homeostasis, decreasing reactive oxygen species and malondialdehyde content. Correspondingly, the overexpression of DcWRKY12 modulated the expression of salt stress-responsive and ion transport-related genes. Dual luciferase assay and Y1H were further confirmed that DcWRKY12 activates the promoter of AtRCI2A through directly binding to the specific W-box cis-acting elements. These results suggest that DcWRKY12 is a positive regulator of salt tolerance in D. composita and has potential applications in salt stress.

Genomic, spatial and morphometric data for discrimination of four species in the Mediterranean Tamus clade of yams (Dioscorea, Dioscoreaceae).

BACKGROUND AND AIMS: Among the numerous pantropical species of the yam genus, Dioscorea, only a small group occurs in the Mediterranean basin, including two narrow Pyrenean endemics (Borderea clade) and two Mediterranean-wide species (D. communis and D. orientalis, Tamus clade). However, several currently unrecognized species and infraspecific taxa have been described in the Tamus clade due to significant morphological variation associated with D. communis. Our overarching aim was to investigate taxon delimitation in the Tamus clade using an integrative approach combining phylogenomic, spatial and morphological data. METHODS: We analysed 76 herbarium samples using Hyb-Seq genomic capture to sequence 260 low-copy nuclear genes and plastomes, together with morphometric and environmental modelling approaches. KEY RESULTS: Phylogenomic reconstructions confirmed that the two previously accepted species of the Tamus clade, D. communis and D. orientalis, are monophyletic and form sister clades. Three subclades showing distinctive geographic patterns were identified within D. communis. These subclades were also identifiable from morphometric and climatic data, and introgression patterns were inferred between subclades in the eastern part of the distribution of D. communis. CONCLUSIONS: We propose a taxonomy that maintains D. orientalis, endemic to the eastern Mediterranean region, and splits D. communis sensu lato into three species: D. edulis, endemic to Macaronesia (Canary Islands and Madeira); D. cretica, endemic to the eastern Mediterranean region; and D. communis sensu stricto, widespread across western and central Europe. Introgression inferred between D. communis s.s. and D. cretica is likely to be explained by their relatively recent speciation at the end of the Miocene, disjunct isolation in eastern and western Mediterranean glacial refugia and a subsequent westward recolonization of D. communis s.s. Our study shows that the use of integrated genomic, spatial and morphological approaches allows a more robust definition of species boundaries and the identification of species that previous systematic studies failed to uncover.

Probiotic Characterization of Indigenous Kocuria flava Y4 Strain Isolated from Dioscorea villosa Leaves.

This aim of the study was to isolate and screen potential probiotics from Dioscorea villosa leaves. The potential isolate Y4 was obtained from the Dioscorea villosa leaves, and its ability to grow in a medium containing high NaCl concentrations (2-10%) indicated its negative hemolytic activity. Furthermore, Y4 demonstrated inhibitory activity against human pathogens, such as Klebsiella pneumonia, Staphylococcus aureus, Citrobacter koseri, and Vibrio cholerae, as well as towards a plant pathogen isolate OR-2 (obtained from Citrus sinensis). Some biologically important functional groups of Y4 metabolites, such as sulfoxide; aliphatic ether; 1, 2, 3-trisubstituted, tertiary alcohol: vinyl ether; aromatic amine; carboxylic acid; nitro compound; alkene mono-substituted; and alcohol, were identified through FTIR analysis. The 16S rRNA sequencing and subsequent phylogenetic tree analysis indicated that Y4 and OR-2 are the closest neighbors to Kocuria flava (GenBank accession no. MT773277) and Pantoea dispersa (GenBank accession no. MT766308), respectively. The potential isolate Y4 was found to exhibit adhesion, auto-aggregation, co-aggregation, and weak biofilm activity. It also exhibited a high level of antimicrobial activity and antibiotic susceptibility. The safety of K. flava Y4 isolate, which is proposed to be a probiotic, was evaluated through acute oral toxicity test and biogenic amine production test. Moreover, the preservation potential of isolate Y4 was assessed through application on fruits under different temperatures. Thus, our results confirmed that Kocuria flava Y4 is a prospective probiotic and could also be used for the preservation of fruits.

Genome Sequence Resource for Colletotrichum gloeosporioides, an Important Pathogenic Fungus Causing Anthracnose of Dioscorea alata.

Anthracnose disease is one of the most important diseases of Dioscorea alata and many other food yams, which is caused by Colletotrichum gloeosporioides fungus from the Glomerellaceae family of the Sordariomycetes class. In the present study, a C. gloeosporioides starin named CgDa01 was isolated from D. alata, and its genome was sequenced based on Oxford Nanopore technology (ONT) and the Illumina sequencing platform. The high-quality genome of CgDa01 was assembled with a 62.78 Mb genome size and 15,845 predicted protein-coding genes. The proteins of predicted genes were annotated using multiple public databases, including the nonredundant protein database, the InterProScan databases, and Kyoto Encyclopedia of Genes and Genomes. Among the annotated protein-coding genes, 55 were predicted as potential virulence genes by the fungal virulence factor database. The C. gloeosporioides CgDa01 genome assembly described in this study can serve as a resource for better understanding the pathogenic mechanism of C. gloeosporioides on yam hosts.

Overexpression of DoBAM1 from Yam (Dioscorea opposita Thunb.) Enhances Cold Tolerance in Transgenic Tobacco.

ß-amylase (BAM) plays an important role in plant development and response to abiotic stresses. In this study, 5 DoBAM members were identified in yam (Dioscorea opposita Thunb.). A novel ß-amylase gene BAM1, (named DoBAM1), was isolated from yam varieties Bikeqi and Dahechangyu. The open reading frame (ORF) of DoBAM1 is 2806 bp and encodes 543 amino acids. Subcellular localization analysis indicates that DoBAM1 localizes to the cell membrane and cytoplasm. In the yam variety Dahechangyu, the starch content, ß-amylase activity, and expression of DoBAM1 were characterized and found to all be higher than in Bikeqi. DoBAM1 overexpression in tobacco is shown to promote the accumulation of soluble sugar and chlorophyll content and to increase the activities of peroxidase (POD), superoxide dismutase (SOD), catalase (CAT), and ß-amylase. Under cold treatment, we observed the induced upregulation of DoBAM1 and lower starch content and malondialdehyde (MDA) accumulation than in WT plants. In conclusion, these results demonstrate that DoBAM1 overexpression plays an advanced role in cold tolerance, at least in part by raising the levels of soluble sugars that are capable of acting as osmolytes or antioxidants.