Development of DNA microsatellite markers in tropical yam (Dioscorea sp.)

We developed new simple sequence repeat (SSR) markers in different species of yam (Dioscorea sp.). A microsatellite‐enriched bank was created from Dioscorea alata, Dioscorea abyssinica and Dioscorea praehensilis. Sixteen polymorphic loci were characterized. Several of these markers are transferable to species of other Dioscorea sections.     

Complementarity between Geographical and social patterns in the preservation of yam (Dioscorea sp.) diversity in Northern Benin

This study was undertaken to contribute to the sustainable management of yam (Dioscorea sp.) diversity in Benin. We surveyed 220 farms from eight different villages in the north part of Benin. We hypothesized that the richness of yam cultivars is more related to farmers’ ethnic group than to their geographical location. Cultivar diversity may differ from one ethnic group to another within a given area, but remains constant for a given ethnic group independent of its geographical location. This study identified 182 yam cultivars in the region; however, only a few farmers on a very small scale marginally produce more than 50% of that total richness. Cultivation practices as well as historical and socio-cultural determinants played important roles in the creation and maintenance of yam diversity within each ethnic group. The Bariba and the Gando ethnic groups, having developed yam cultivation since time immemorial, still hold the most diverse collection of yam varieties. The access of farmers to main roads and the availability of arable and in a given village greatly influenced the overall yam diversity in the region. This study highlighted the need to combine social determinants and geographical patterns in the conservation of agricultural diversity in Benin.     

Rapid assessment of resistance of tissue-cultured water yam (Dioscorea alata) and white guinea yam (Dioscorea rotundata) to anthracnose (Colletotrichum gloeosporioides Penz.)

Yam anthracnose is caused by the pathogen Colletotrichum gloeosporioides Penz. and has been identified as the most important biotic constraint to yam production worldwide. Rapid assessment of the disease is vital to its effective diagnosis and management. In this study, tissue-cultured yam plantlets of five lines of Dioscorea alata and nine of D. rotundata were rapidly assessed for their reactions to two isolates of yam anthracnose. The plantlets, obtained from meristem of the nodal cuttings, were grown for 8?weeks on Murashige and Skoog (MS) basal medium, acclimatised for 3?weeks, hardened for an additional 3?weeks, arranged in screen house in completely randomised design and sprayed with spore inocula prepared from 7?day-old culture of the two strains of Colletotrichum gloeosporioidies Penz. The relative resistance of the different Dioscorea spp. was evaluated using three disease indices – severity at seventh day after inoculation, SD7; area under disease progress curve, AUDPC; and disease severity rate, Rd. A modified rank-sum classification method put TDa 1425 and TDr 2040, with rank sum of 2.0 each, as resistant. TDr 2121, TDr 2287 and TDr 2048 were susceptible with rank sum of 27.50, 25.50 and 24.50, respectively. Dioscorea alata TDa 1425 and Dioscorea rotundata TDr 2040 were recommended in areas endemic with yam anthracnose, and also as parent lines while breeding for resistance to anthracnose.     

Genomic variation and relationships in aerial yam (Dioscorea bulbifera L.) detected by random amplified polymorphic DNA.

Random amplified polymorphic DNA (RAPD) markers were used to assess intraspecific variability and relationships in aerial yam (Dioscorea bulbifera L.). A total of 23 accessions from different geographic locations in Africa, Asia, and Polynesia were analyzed by 10 arbitrarily chosen GC-rich decamer primers. Using cesium chloride purified genomic template DNA, highly reproducible polymorphic fingerprints were generated by all 10 primers, resulting in a total of 375 informative characters. Only eight bands were monomorphic among all investigated accessions. A binary character matrix was generated by scoring for presence/absence of a band at a particular position, transformed into a matrix of pairwise distances using either the Jaccard or a simple matching coefficient, and analyzed by neighbour joining, UPGMA (unweighted pair group method with arithmetic averaging) cluster analysis, or split decomposition. All methods of data evaluation resulted in similar groupings that reflected the geographical origin of the samples. The African accessions formed a distinct isolated group, whereas Asian and Polynesian accessions proved to be more heterogeneous. With two exceptions (var. suavior and var. sativa), the RAPD data supported previous varietal classification based on morphological characters. Stepwise reduction of the number of evaluated characters did not affect branching patterns of the trees above a minimum threshold of 150. Key words : Dioscorea bulbifera, random amplified polymorphic DNA (RAPD), genetic variation, genetic relatedness.     

Severity of spoilage storage rots of white yam (Dioscorea rotundata Poir.)

Severity of storage rots in different sections of white yam tubers (Dioscorea rotundata Poir.) was investigated. Yam samples with rots were collected from a yam barn and from selected markets in Accra, Ghana, to identify the most predominant pathogens associated with the rots. Nine fungal spoilage microorganisms, including Aspergillus flavus, Aspergillus niger, Botryodiplodia theobromae, Fusarium culmorum, Fusarium oxysporium, Fusarium sp., Penicillium brevi‐compactum, Penicillium sp. and Rhizopus stolonifer and a bacterium species Erwinia carotovora were identified. The mean incidence of occurrence of the organisms on rotten tissues ranged from 1.2% to 28.5%. Of the 10 microorganisms isolated, B. theobromae, F. oxysporium and R. stolonifer were the most frequently encountered spoilage microorganisms in the markets. E. carotovora, Fusarium solani and Penicillium sp. were relatively sparse (incidence not exceeding 3%) compared to the other yam spoilage microorganisms. The surface area and weight of necrotic tissue induced by the spoilage fungi in the various zones of the tubers over a 28‐day storage period were assessed. All the spoilage microorganisms produced rots in the yams, although to different degrees. The severity of the rots increased in weight and area over the period when the tubers were in store but were normally not significantly different in the zones of tubers. There was, however, a linear progression of rots in the various zones of the yam tubers. Although there was generally no significant (P ≥ 0.05) difference in the severity of rots induced by the different microorganisms in the tubers, R. stolonifer commonly induced more rot in the zones of the tubers compared to B. theobromae and F. oxysporium.     

A genetic linkage map of Guinea yam ( Dioscorea rotundata Poir.) based on AFLP markers

A genetic linkage map of the tetraploid white yam (Dioscorea rotundata Poir.) was constructed based on 341 co-dominantly scored amplified fragment length polymorphism (AFLP) markers segregating in an intraspecific F₁ cross. The F₁ mapping population was produced by crossing a landrace cultivar TDr 93-1 as female parent to a breeding line TDr 87/00211 as the male parent. The marker segregation data were split into maternal and paternal data sets, and separate genetic linkage maps were constructed since the mapping population was an F₁ cross between two presumed heterozygous parents. The markers segregated like a diploid cross-pollinator population suggesting that the D. rotundata genome is an allo-tetraploid (2n = 4x = 40). The maternal map comprised 155 markers mapped on 12 linkage groups with a total map length of 891 cM. Three linkage groups consisted of maternal parent markers only. The paternal map consisted of 157 markers mapped on 13 linkage groups with a total map length of 852 cM. Three and one quantitative trait loci (QTLs) with effects on resistance to Yam Mosaic Virus (YMV) were identified on the maternal and paternal linkage maps, respectively. Prospects for detecting more QTLs and using marker-assisted selection in white yam breeding appear good, but this is subject to the identification of additional molecular markers to cover more of the genome.     

New starches from traditional Chinese medicine (TCM)--Chinese yam (Dioscorea opposita Thunb.) cultivars

The starches separated from two different Dioscorea opposita Thunb. cultivars were investigated for morphological, thermal and crystal properties. The shape of starch granules separated from different D. opposita Thunb. cultivars varied from round to oval or irregular. The surface of the starch granules appeared to be smooth without any fissures. The average particle diameter of starches from different D. opposita Thunb. cultivars was 40.3 and 38.7mum for D. 47 and D. SXY starch, respectively. The transition temperatures (T(o), T(p) and T(c)) and enthalpy of gelatinization (DeltaH(gel)) were determined using differential scanning calorimetry (DSC). The D. SXY starch showed the lower T(o) (74.2 degrees C) and the broader R (12.4). T(p) and T(c) of starch from D. 47 were higher than that of D. SXY starch. DeltaH(gel) values (11.37J/g) of D. 47 was higher than that of D. SXY starch (10.78J/g). The crystal type of starches separated from two different D. opposita cultivars was a typical C-type pattern. The degree of crystallinity of two D. opposita cultivars starches was about 45.9% and 31.5%, respectively.     

Genetic screening for prey in the gut contents from a giant squid (Architeuthis sp.)

Giant squids (Architeuthis sp.) remain mysterious; they have evaded observation and are rarely taken from their deep sea habitat. Information on the diet of Architeuthis is scarce due to the limited number of specimens with morphologically recognizable remains in their digestive tracts. We explored the use of polymerase chain reaction (PCR)-based methods for detection of DNA in the prey remains and amorphous slurry from an Architeuthis gut sample. The DNA region amplified varied in size, allowing separation of fish and squid components. Sequence comparisons identified fish prey as Macruronus novaezelandiae. Isolation of Architeuthis DNA from an ingested tentacle and the presence of chitin fragments indicate cannibalism occurs in giant squid. Denaturing gradient gel electrophoresis was used to screen for less common DNA types, revealing a high frequency of PCR-generated false alleles, but no additional prey species.     

Delimitation of physiological regions in yam tubers (Dioscorea sp.) and distribution pattern of saccharide degrading enzymes,cell sap pH and protein in these regions

Physiological regions of yam tubers were morphologically defined in different specie into ‘Head’, ‘Middle’ and ‘Tail’, while the limits of these regions were studied using phosphorylase activity. Variation in enzyme activity, pH and protein concentration was found in different regions of the tubers. Old yam tubers had significantly higher activities of saccharide degrading enzymes, hexokinase, phosphorylase, glucose-6-phosphate dehydrogenase, phosphofructokinase and pyruvate kinase, than the new tubers. However, activity of phosphofructokinase in newD. rotundata was higher than that of old tuber. The high activity of phosphorylase in different regions of all the yam tubers examined indicates a very important role of this enzyme in starch degradation inDioscorea species. The measured pH and protein concentration were also higher in old yam tubers. Except for phosphorylase, these enzymes had alkaline pH optima.     

Direct plantlet regeneration from male inflorescences of medicinal yam (Dioscorea floribunda Mart. & Gal.)

Summary Segments of male inflorescences of medicinal yam (Dioscorea floribunda) cultured on Murashige and Skoog (MS) medium supplemented with 13.94 μM kinetin (Kn) resulted in the conversion of floral buds into vegetative buds and these later developed into plantlets. Growth and multiplication of shoots could be obtained by culturing individual shoots in MS modified basal medium, replacing the MS standard three vitamins with 10.0 mgl⁻¹ thiamine in addition to 13.94 μM Kn. Root induction was also obtained simultaneously from the base of the shoots in the same medium. Such plantlets have been successfully transferred to potted soil, where they grew normally. Plantlets were also made to develop tubers on MS medium with 18.91 μM abscisic acid (ABA) and also with 2.68 μM α-naphthaleneacetic acid (NAA) and 40–50 gl⁻¹ sucrose.     

Antifungal properties of yam (Dioscorea alata) peel extract

The extraction of natural antifungal compounds from the peels of yam (Dioscorea alata) and the effect of these compounds on both the vegetative and reproductive structures of some yam rot pathogens were studied. Four prominent antifungal components were obtained; one of the components was fully characterized and identified as β-sitosterol. The antifungal activity of the compounds toward the germination of spores of two yam pathogens showed an inhibition of less than 57% at a concentration of 50 mg/L while inhibition on the elongation of germ-tubes ofFusarium moniliforme was as high as 82% at the same concentration. However, the ED₅₀ for inhibition of germ-tube elongation in the yam compounds for the same organism was below 32 mg/L. The role of the yam compounds at high concentrations in disease resistance is discussed.     

Physical map of chloroplast DNA of aerial yam,Dioscorea bulbifera L.

Summary A physical map of chloroplast DNA (ctDNA) of aerial yam, Dioscorea bulbifera L. was constructed using three restriction endonucleases, PstI, SalI, and SmaI. In addition, a clone bank of the BamHI-digested fragments were generated, and the locations of most BamHI fragments on the map were also determined. The ctDNA of D. bulbifera was found to be a circular molecule with a total size of ca. 152 kb involving two inverted repeats of ca. 25.5 kb, and small and large single copy regions of ca. 18.5 and 83.4 kb, respectively. The genes for the large subunit of the ribulose 1,5-bisphosphate carboxylase (rbcL) and the ATP-synthase subunits and (atpB/atpE) were mapped.Contribution from the Plant Germ-plasm Institute and the Laboratory of Genetics (No. 504), Faculty of Agriculture, Kyoto University, Japan. The work was supported in part by a Grant-in-Aid (No. 60400005) from the Ministry of Education, Science and Culture, Japan     

An elicitor-induced cDNA from aerial yam (Dioscorea bulbifera L.) encodes a pathogenesis-related type 4 protein

We established Dioscorea bulbifera (aerial yam) cell suspension cultures to study the expression of defense-related genes upon elicitation with the yam pathogenic ascomycete Colletotrichum gloeosporioides. The induction of phenylalanine-ammonia lyase (PAL) mRNA, coding for a key enzyme involved in phytoalexin biosynthesis, was observed upon elicitation. Using RT-PCR, we isolated an elicited cDNA clone with an open reading frame of 453 nucleotides which showed high homology to cDNA sequences of pathogenesis-related proteins belonging to the PRP-4 group. Yam PRP-4 expression is increased in elicited cell cultures as well as in elicited leaves, and is encoded by a small multigene family. This is the first example for the cloning of a cDNA that might be involved in defense reactions of yam plants. Received: December 1997 / Revision received: June 1998 / Accepted: November 1998     

Efficient shoot organogenesis in petioles of yam (Dioscorea spp)

Shoot organogenesis was successfully achieved in petiole explants excised from 6 to 8-week old in vitro plantlets of yam Dioscorea rotundata P. cv. Kponan fissa, Dioscorea cayenensis L. cv. Krengle IB14 and cv. Krengle IB35, and Dioscorea alata L. cv. Bete bete. Only the basipetal portion of the petiole acquired competence, and plants regenerated within 21?days on MS medium supplemented with 2?% sucrose, 100?mg/l myo-inositol, 10???M kinetin and 1.5?mM putrescine referred to as yam regeneration medium (YRM). Plant regeneration was significantly (p?<?0.01) higher (42?%) with 2?% sucrose compared to 1, 3 and 4?% sucrose which produced 4, 25 and 15?% regeneration respectively. The age of the donor yam plantlet was critical to regeneration, and the highest regeneration efficiency was obtained with explants from 8-week old plantlets. Addition of the antioxidants lipoic acid (19.4???M), and l-cysteine (28.5???M) to the culture medium stimulated axillary branching in regenerated shoots. Among the four yam cultivars tested, cv. Kponan fissa, cv. Krengle IB14 and cv. Bete bete had similar response at 10???M kinetin, while the cv. Krengle IB35 regenerated best at a lower concentration of kinetin (0.5???M).     

Studies on tuber starches III. Impact of annealing on the molecular structure,composition and physicochemical properties of yam (Dioscorea sp.) starches grown in Sri Lanka

This study reports the impact of one step annealing on the composition, molecular structure, granule morphology and physicochemical properties of starches isolated from cultivars of Dioscorea esculenta (kukulala, java-ala-nattala) and Dioscorea alata (hingurala, raja-ala), yam tubers grown in Sri Lanka. In all starches, granule morphology (shape, size, surface appearance), birefringence patterns, acid hydrolysis profile and X-ray patterns remained unchanged on annealing. Crystallinity remained unchanged on annealing in hingurala, kukulala and java-ala. However, crystallinity of raja-ala and nattala increased and decreased, respectively, on annealing. In all starches, annealing decreased the gelatinization temperature range (kukulala ～ hingurala > nattala ～ raja-ala > java-ala), amylose leaching (raja-ala > nattala > hingurala > kukulala > java-ala), granular swelling (raja-ala ～ hingurala > kukulala > java-ala > nattala), peak viscosity (raja-ala > hingurala > kukulala > java-ala > nattala), enthalpy of retrogradation (kukulala ～ java-ala ～ nattala ～ hingurala ～ raja-ala) and susceptibility towards acid hydrolysis (java-ala > raja-ala > hingurala ～ nattala > kukulala). However, annealing increased gelatinization temperatures (kukulala ～ java-ala ～ nattala ～ raja-ala ～ hingurala) and the enthalpy of gelatinization (kukulala > hingurala > java-ala > nattala > raja-ala). Set-back viscosity increased in nattala, but decreased in the other starches (raja-ala > hingurala > kukulala > java-ala) on annealing. The study showed that the different responses shown by the cultivars of the Dioscorea starches towards annealing were to a large extent influenced by their composition and molecular structure.     

白刺花中3种生物碱的含量及HPLC指纹图谱

为建立白刺花高效液相指纹图谱,并测定白刺花不同部位(根、茎、叶)苦参碱、槐果碱和氧化苦参碱的含量。实验采用AgilentTC-C₁₈(2)色谱柱(250×4.6 mm,5μm),以乙腈-0.09%三乙胺水溶液(4∶96,磷酸调pH3.5)为流动相,流速为1.0 mL/min,紫外检测波长205 nm,柱温35℃。建立了白刺花的高效液相指纹图谱,确定了14个共有峰,样品的相似度在0.9以上。在该色谱条件下,各成分有较好的分离度,苦参碱、槐果碱和氧化苦参碱分别在6.34～101.5、6.47～103.5、8.38～100.5μg/mL范围内呈良好的线性关系(r>0.999)。用该方法测得白刺花根中苦参碱、槐果碱、氧化苦参碱的平均含量分别为8.204 8、1.237 1、66.147 6 mg/g;茎中分别为4.246 3、1.549 6、25.035 1 mg/g;叶中分别为9.314 8、6.947 4、1.573 4 mg/g。该方法简单、准确,可用于白刺花的质量控制。