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.

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.

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.

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.

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.

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.

Genome-wide genotyping elucidates the geographical diversification and dispersal of the polyploid and clonally propagated yam (Dioscorea alata).

BACKGROUND AND AIMS: Inferring the diffusion history of many human-dispersed species is still not straightforward due to unresolved past human migrations. The centre of diversification and routes of migration of the autopolyploid and clonally propagated greater yam, Dioscorea alata, one of the oldest edible tubers, remain unclear. Here, we address yam demographic and dispersal history using a worldwide sample. METHODS: We characterized genome-wide patterns of genetic variation using genotyping by sequencing 643 greater yam accessions spanning four continents. First, we disentangled the polyploid and clonal components of yam diversity using allele frequency distribution and identity by descent approaches. We then addressed yam geographical origin and diffusion history with a model-based coalescent inferential approach. KEY RESULTS: Diploid genotypes were more frequent than triploids and tetraploids worldwide. Genetic diversity was generally low and clonality appeared to be a main factor of diversification. The most likely evolutionary scenario supported an early divergence of mainland Southeast Asian and Pacific gene pools with continuous migration between them. The genetic make-up of triploids and tetraploids suggests that they have originated from these two regions before westward yam migration. The Indian Peninsula gene pool gave origin to the African gene pool, which was later introduced to the Caribbean region. CONCLUSIONS: Our results are congruent with the hypothesis of independent domestication origins of the two main Asian and Pacific gene pools. The low genetic diversity and high clonality observed suggest a strong domestication bottleneck followed by thousands of years of widespread vegetative propagation and polyploidization. Both processes reduced the extent of diversity available for breeding, and this is likely to threaten future adaptation.

[Genetic stability study on autotetraploid plant of Dioscorea zingiberensis].

OBJECTIVE: To study the genetic stability of autotetraploid plant of Dioscorea zingiberensis. METHODS: The chromosome of root-tip was determined by photomicroscope, and the agronomic characters were observed in the period of stable growth. The protein content was determined and the experiment of protein polyacrylamide gel electrophoresis was carried out. Furthemore, the diosgenin content was determined and compared. RESULTS: The chromosome number of autotetraploid plantlet was 2n = 4x = 40. The agronomic characters showed typical autotetraploid characteristics. The contents of diosgenin and protein of autotetraploid were higher than that of the diploid. The protein electrophoresis bands of all the lines were similar. CONCLUSION: The experiment confirmed that the autotetraploid plant of Dioscorea zingiberensis, which was artificially induced, had good genetic stability. It lays the foundation for the polyploid breeding to develop superior varieties of Dioscorea zingiberensis.

Molecular cloning and differential expression analysis of a squalene synthase gene from Dioscorea zingiberensis, an important pharmaceutical plant.

Diosgenin is a steroid derived from cholesterol in plants and used as a typical initial intermediate for synthesis of numerous steroidal drugs in the world. Commercially, this compound is extracted mainly from the rhizomes or tubers of some Dioscorea species. Squalene synthase (SQS: EC 2.5.1.21) catalyzes the condensation of two molecules of farnesyl diphosphate to form squalene, the first committed step for biosynthesis of plant sterols including cholesterol, and is thought to play an important role in diosgenin biosynthesis. A full-length cDNA of a putative squalene synthase gene was cloned from D. zingiberensis and designated as DzSQS (Genbank Accession Number KC960673). DzSQS was contained an open reading frame of 1,230 bp encoding a polypeptide of 409 amino acids with a predicted molecular weight of 46 kDa and an isoelectric point of 6.2. The deduced amino acid sequence of DzSQS shared over 70 % sequence identity with those of SQSs from other plants. The truncated DzSQS in which 24 amino acids were deleted from the carboxy terminus was expressed in Escherichia coli, and the resultant bacterial crude extract was incubated with farnesyl diphosphate and NADPH. GC-MS analysis showed that squalene was detected in the in vitro reaction mixture. Quantitative real-time PCR analysis revealed that DzSQS was expressed from highest to lowest order in mature leaves, newly-formed rhizomes, young leaves, young stems, and two-year-old rhizomes of D. zingiberensis.

Next-generation sequencing based genotyping, cytometry and phenotyping for understanding diversity and evolution of Guinea yams.

KEY MESSAGE: Genotyping by sequencing (GBS) is used to understand the origin and domestication of guinea yams, including the contribution of wild relatives and polyploidy events to the cultivated guinea yams. Patterns of genetic diversity within and between two cultivated guinea yams (Dioscorea rotundata and D. cayenensis) and five wild relatives (D. praehensilis, D. mangenotiana, D. abyssinica, D. togoensis and D. burkilliana) were investigated using next-generation sequencing (genotyping by sequencing, GBS). Additionally, the two cultivated species were assessed for intra-specific morphological and ploidy variation. In guinea yams, ploidy level is correlated with species identity. Using flow cytometry a single ploidy level was inferred across D. cayenensis (3x, N = 21), D. praehensilis (2x, N = 7), and D. mangenotiana (3x, N = 5) accessions, whereas both diploid and triploid (or aneuploid) accessions were present in D. rotundata (N = 11 and N = 32, respectively). Multi-dimensional scaling and maximum parsimony analyses of 2,215 SNPs revealed that wild guinea yam populations form discrete genetic groupings according to species. D. togoensis and D. burkilliana were most distant from the two cultivated yam species, whereas D. abyssinica, D. mangenotiana, and D. praehensilis were closest to cultivated yams. In contrast, cultivated species were genetically less clearly defined at the intra-specific level. While D. cayenensis formed a single genetic group, D. rotundata comprised three separate groups consisting of; (1) a set of diploid individuals genetically similar to D. praehensilis, (2) a set of diploid individuals genetically similar to D. cayenensis, and (3) a set of triploid individuals. The current study demonstrates the utility of GBS for assessing yam genomic diversity. Combined with morphological and biological data, GBS provides a powerful tool for testing hypotheses regarding the evolution, domestication and breeding of guinea yams.

Efficiency of RAPD, ISSR, AFLP and ISTR markers for the detection of polymorphisms and genetic relationships in camote de cerro (Dioscorea spp. )

Background At present, species known as camote de cerro (Dioscorea spp.) are found only in the wilderness in Mexico, but their populations are extremely depleted because they are indiscriminately collected, it is urgent to evaluate the conservation status of these plants in order to design conservation genetics programs. In this study, genetic diversity parameters along with cluster analysis based on Jaccard's coefficient were estimated with the objective to assess the efficiency of Random Amplified Polymorphic DNA (RAPD), Inter Simple Sequence Repeat (ISSR), Amplified Fragment Length Polymorphism (AFLP) and Inverse Sequence Tagged Repeat (ISTR) molecular DNA markers in the Dioscorea genus. Results The polymorphic information contents were quite similar for all markers (≈0.48). Genetic variation of Dioscorea spp., in terms of average heterozygosity was lower with ISTR (0.36), and higher when other markers were used (RAPD = 0.43; ISSR = 0.45 and AFLP = 0.47). Conclusion This indicates an important level of genetic differences despite the fact that the plant is asexually propagated. Based on the diversity statistics, any marker tested in present work can be recommended for use in large-scale genetic studies of populations. However, the low correlations among different molecular marker systems show the importance of the complementarity of the information that is generated by different markers for genetic studies involving estimation of polymorphism and relationships.

Ethylene treatment improves diosgenin accumulation in in vitro cultures of Dioscorea zingiberensis via up-regulation of CAS and HMGR gene expression

Background: The perennial medicinal herb Dioscorea zingiberensis is a very important plant used for steroid drug manufacturing for its high level of diosgenin in rhizome. Although the stimulation of diosgenin accumulation by ethylene has been reported in a few of plant species, its regulation is not yet characterized at the molecular level, the underlying molecular mechanism remains elusive. Results: In this study, the effects of ethylene on diosgenin biosynthesis in in vitro cultures of D. zingiberensis were described. The results showed that, in samples treated with ethylene at concentration E3 (10(4) dilution of 40% ethephon), the diosgenin biosynthesis was significantly promoted in comparison with the control samples. Treatment with high concentrations of ethylene had inhibitory effect, whereas with low concentration of the gas elicitor brought about no detectable deleterious effect on the growth rate and diosgenin content of the cultures. The considerable increase of diosgenin level in in vitro cultured Dioscorea zingiberensis by ethylene application is accompanied by the concomitant increase of soluble proteins and chlorophyll content. The gene expressions of cycloartenol synthase and 3-hydroxy-3-methylglutaryl-CoA reductase but not of squalene synthase or farnesyl pyrophosphate synthase were up-regulated by applied ethylene. Conclusions: Our results suggest that ethylene treatment enhanced diosgenin accumulation via up-regulation of the gene expressions of cycloartenol synthase and 3-hydroxy-3-methylglutaryl-CoA reductase.

Microsatellite and flow cytometry analysis to help understand the origin of Dioscorea alata polyploids.

BACKGROUND AND AIMS: Dioscorea alata is a polyploid species with a ploidy level ranging from diploid (2n = 2x = 40) to tetraploid (2n = 4x = 80). Ploidy increase is correlated with better agronomic performance. The lack of knowledge about the origin of D. alata spontaneous polyploids (triploids and tetraploids) limits the efficiency of polyploid breeding. The objective of the present study was to use flow cytometry and microsatellite markers to understand the origin of D. alata polyploids. METHODS: Different progeny generated by intracytotype crosses (2x × 2x) and intercytotype crosses (2x × 4x and 3x × 2x) were analysed in order to understand endosperm incompatibility phenomena and gamete origins via the heterozygosity rate transmitted to progeny. RESULTS: This work shows that in a 2x × 2x cross, triploids with viable seeds are obtained only via a phenomenon of diploid female non-gametic reduction. The study of the transmission of heterozygosity made it possible to exclude polyspermy and polyembryony as the mechanisms at the origin of triploids. The fact that no seedlings were obtained by a 3x × 2x cross made it possible to confirm the sterility of triploid females. Flow cytometry analyses carried out on the endosperm of seeds resulting from 2x × 4x crosses revealed endosperm incompatibility phenomena. CONCLUSIONS: The major conclusion is that the polyploids of D. alata would have appeared through the formation of unreduced gametes. The triploid pool would have been built and diversified through the formation of 2n gametes in diploid females as the result of the non-viability of seeds resulting from the formation of 2n sperm and of the non-viability of intercytotype crosses. The tetraploids would have appeared through bilateral sexual polyploidization via the union of two unreduced gametes due to the sterility of triploids.

Molecular cloning, structural analysis and mass spectrometric identification of native dioscorins of various yam species.

BACKGROUND: Dioscorins are the major storage proteins of yam tubers. However, the molecular nature of their heterogeneity in tubers has not been fully elucidated. In this study the authors isolated the dioscorin gene families of Dioscorea japonica and Dioscorea pseudojaponica, performed matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) and elucidated which dioscorin isoforms are the major constituents in tubers. RESULTS: The dioscorin gene families of D. japonica (Dj-dioA1-Dj-dioA4, Dj-dioB1 and Dj-dioB2) and D. pseudojaponica (Dp-dioA1-Dp-dioA5 and Dp-dioB1) were cloned from cDNA libraries of yam tubers. The dioscorins isolated from Dioscorea alata (Da-dioscorins), D. japonica (Dj-dioscorins) and D. pseudojaponica (Dp-dioscorins) were mainly monomers, with a few dimers. The monomers contained one intramolecular disulfide bond (Cys(28)-Cys(187)) and belonged to Class A dioscorins with two cysteine residues. The dimers consisted of Class B dioscorins with one intermolecular disulfide bond (Cys(40)-Cys(40)). Results of MALDI-TOF-MS revealed that the Da-dioscorins were mainly encoded by Da-dioA2, Da-dioA3 and Da-dioA4. The majority of the Dj-dioscorins were encoded by Dj-dioA1, Dj-dioA2, Dj-dioA3 and Dj-dioB2. The Dp-dioscorins mainly comprised proteins encoded by Dp-dioA1, Dp-dioA3, Dp-dioA4, Dp-dioB1 and Dp-dioB2. CONCLUSION: Determination of the constituents of dioscorin isoforms in yam tubers provides a basis for future studies of their physiological and biomedical functions.

Evidence of diploidy in the wild Amerindian yam, a putative progenitor of the endangered species Dioscorea trifida (Dioscoreaceae).

The fundamental question about Dioscorea trifida (Dioscoreaceae), the most important Amerindian yam, that remains unresolved concerns its evolutionary origin, since no wild relative has been reported. In this paper we report the existence of D. trifida's wild relative for the first time. The diploidy of wild D. trifida (2n = 40) is clearly demonstrated by flow cytometry, chromosome counts, and microsatellite pattern analysis, whereas the cultivated form was previously shown to be autotetraploid (2n = 80). In the coastal region where the wild and cultivated forms are sympatric, tetraploid and triploid cytotypes coexist within the same populations. In the sites where the wild and cultivated forms are allopatric, the wild diploid cytotype predominates. AFLP (amplified fragment length polymorphism) analyses gave an initial idea of the position of the wild forms in relation to the cultivated forms. All the wild and cultivated types form a monophyletic group structured into two major subgroups corresponding to the tetraploid cytotype of the cultivated form and the diploid cytotype of the wild form. The triploid cytotypes of the wild form are in an intermediary position. Wild accessions are grouped on the basis of their geographic origin. The data presented in this paper are significant for the effective breeding and conservation of D. trifida and to assess its genetic diversity and population structure for the general understanding of the evolution and domestication of the species.

Evaluación en campo de plantas de ñame (Dioscorea alata L) obtenidas de los microtubérculos formados en sistema de inmersión temporal / Field evaluation of yam plants (Dioscorea alata L) obtained from microtubers produced in a temporary immersion system

Los microtubérculos en algunas especies de plantas constituyen una importante alternativa como material vegetal de plantación. Se definió como objetivo de trabajo evaluar en campo la respuesta morfoagronómica de las plantas obtenidas de los microtubérculos de ñame formados en Sistema de Inmersión Temporal (SIT). Como variantes experimentales se plantaron tres categorías de microtubérculos, clasificados según su masa fresca (I. de 0,5 a 0,9 g; II. de 1,0 a 2,9 g; III. igual o mayor de 3,0 g), plantas in vitro previamente aclimatadas y corona de tubérculo. Se evaluó el efecto de la masa fresca de los microtubérculos sobre su brote, supervivencia y posterior desarrollo de las plantas derivadas de ellos en campo. Con los microtubérculos de ñame, con una masa fresca igual o superior a 3,0 g, se alcanzó el más alto porcentaje de brotación (91,30%) y supervivencia de las plantas (96,50%), así como las mejores respuestas en los caracteres cuantitativos que se evaluaron en campo. Estos resultados confirmaron la importancia de la masa fresca de los microtubérculos para ser empleados como material vegetal de plantación directo en campo.

Microtubers in some plant species represent an important alternative crop-planting material. The presentwork involved field work for evaluating the morphoagronomic response of plants obtained from yam microtubersproduced in a temporary immersion system (TIS). Three categories of microtuber were planted asexperimental variants; they were classified by fresh mass (1 - 0.5 to 0.9 g, 2 - from 1.0 to 2.9 g and 3 - equal to or greater than 3.0 g), previously in vitro-acclimated plants and tuber crowns. The effect of microtuber freshweight on their sprouting, survival and later development of the plants derived from them in the field were evaluated. The highest sprouting (91.30%) and plant survival percentages (96.50%) and the best response in quantitative traits evaluated in the field were obtained with yam microtubers having a fresh mass equal to or greater than 3.0 g. These results confirmed the importance of microtubers’ fresh weight for using them as plant material in direct planting in the field.

Tetraploidization of diploid Dioscorea results in activation of the antioxidant defense system and increased heat tolerance.

Polyploidy is reported to show increased tolerance to environmental stress. In this work, tetraploid plants of Dioscorea zingiberensis were obtained by colchicine treatment of shoots propagated in vitro. The highest tetraploid induction rate was achieved by treatment with 0.15% colchicine for 24h. Diploid and tetraploid plants were exposed to normal (28 degrees C) and high temperature (42 degrees C) for 5d during which physiological indices were measured. Compared with diploid plants, relative electrolyte leakage and contents of malondialdehyde, superoxide anions and hydrogen peroxide were lower in tetraploids, while activities of antioxidant enzymes, such as superoxide dismutase, peroxidase, catalase, ascorbate peroxidase and glutathione reductase, were stimulated and antioxidants (ascorbic acid and glutathione) were maintained at high concentrations. These results indicate that tetraploid plants possess a stronger antioxidant defense system and increased heat tolerance.

[Analysis of biochemical indices and efficient ingredients in autotetraploid of Dioscorea zingibiernsis].

OBJECTIVE: To improve the yield and quality of Dioscrea zingibiernsis. METHODS: The Biochemical indices and the diosgenin's content were analysed in the autotetraploid lines. RESULTS: The results showed that the activities of APX, SOD and POD in most of autotetraploid lines were higher than that in diploid line or close to it, there was also difference between autotetraploid lines and control lines in the SDS-PAGE of soluble proteins. The content of diosgenin in most autotetraploid plantlets were higher than that in the control. CONCLUSION: There were difference between autotetraploid and control lines in the content of diosgenin and Biochemical indices, therefore, inducing autotetraploid could be an effective way to breeding the superior varieties.

Revision of ploidy status of Dioscorea alata L. (Dioscoreaceae) by cytogenetic and microsatellite segregation analysis.

Dioscorea alata is a polyploid species with several ploidy levels and its basic chromosome number has been considered by most authors to be x = 10. Standard chromosome counting and flow cytometry analysis were used to determine the chromosome number of 110 D. alata accessions of the CIRAD germplasm collection. The results revealed that 76% of accessions have 2n = 40 chromosomes, 7% have 2n = 60 chromosomes and 17% have 2n = 80 chromosomes. Progenies were produced from 2n = 40 types of D. alata and the segregation patterns of six microsatellite markers in four different progenies were analysed. The Bayesian method was used to test for diploid versus tetraploid (allo- and autotetraploid) modes of inheritance. The results provided the genetic evidence to establish the diploidy of plants with 2n = 40 chromosomes and to support the hypothesis that plants with 2n = 40, 60 and 80 chromosomes are diploids, triploids and tetraploids, respectively, and that the basic chromosome number of D. alata is x = 20. The findings obtained in the present study are significant for effective breeding programs, genetic diversity analysis and elucidation of the phylogeny and the species origin of D. alata.

Molecular cloning of a genomic DNA encoding yam class IV chitinase.

Genomic DNA for a class IV chitinase was cloned from yam (Dioscorea opposita Thunb) leaves and sequenced. The deduced amino acid sequence shows 50 to 59% identity to class IV chitinases from other plants. The yam chitinase, however, has an additional sequence of 8 amino acids (a C-terminal extension) following the cysteine that was reported as the last amino acid for other class IV chitinases; this extension is perhaps involved in subcellular localization. A homology model based on the structure of a class II chitinase from barley was used as an aid to interpreting the available data. The analysis suggests that the class IV enzyme recognizes an even shorter segment of the substrate than class I or II enzymes. This observation might help to explain why class IV enzymes are better suited to attack against pathogen cell walls.