A comparative assessment of molecular marker assays (AFLP, RAPD and SSR) for white yam (Dioscorea rotundata) germplasm characterization

Several DNA‐based marker systems are available for genetic fingerprinting of plants but information on their relative usefulness for yam germplasm characterisation is lacking. The efficiency of RAPD, AFLP and SSR markers for the assessment of genetic relationships, and for cultivar identification and discrimination among 45 West and Central African white yam cultivars belonging to 22 morphotypes/cultivar groups was investigated. Dendrograms were produced based on band pattern scores using the UPGMA method. Results showed that each of the three techniques could unequivocably identify each cultivar, but that techniques differed in the mean number of profiles generated per primer (or primer pair) per cultivar, referred to as genotype index (GI). The order of merit based on this criterion in this study was AFLPs (GI = 2.56), SSRs (GI = 0.39) and RAPDs (GI = 0.35). Yam genotypes classified in the same cultivar group based on morphology were often genetically different, emphasising the need for molecular fingerprinting in yam germplasm characterisation. AFLPs showed the highest efficiency in detecting polymorphism and revealed genetic relationships that most closely reflected morphological classification.     

Phases of dormancy in Yam tubers (Dioscorea rotundata)

BACKGROUND AND AIMS: The control of dormancy in yam (Disocorea spp.) tubers is poorly understood and attempts to shorten the long dormant period (i.e. cause tubers to sprout or germinate much earlier) have been unsuccessful. The aim of this study was to identify and define the phases of dormancy in Dioscorea rotundata tubers, and to produce a framework within which dormancy can be more effectively studied. METHODS: Plants of 'TDr 131' derived from tissue culture were grown in a glasshouse simulating temperature and photoperiod at Ibadan (7 degrees N), Nigeria to produce tubers. Tubers were sampled on four occasions: 30 d before shoot senescence (149 days after planting, DAP), at shoot senescence (179 DAP), and twice during storage at a constant 25 degrees C (269 and 326 DAP). The development of the apical shoot bud was described from tissue sections. In addition, the responsiveness of shoot apical bud development to plant growth regulators (gibberellic acid, 2-chloroethanol and thiourea) applied to excised tuber sections was also examined 6 and 12 d after treatment. KEY RESULTS AND CONCLUSIONS: Three phases of tuber dormancy are proposed: Phase I, from tuber initiation to the appearance of the tuber germinating meristem; Phase II, from the tuber germinating meristem to initiation of foliar primordium; and Phase III, from foliar primordium to appearance of the shoot bud on the surface of the tuber. Phase I is the longest phase (approx. 220 d in 'TDr 131'), is not affected by PGRs and is proposed to be an endo-dormant phase. Phases II and III are shorter (<70 d in total), are influenced by PGRs and environmental conditions, and are therefore endo-/eco-dormant phases. To manipulate dormancy to allow off-season planting and more than one generation per year requires that the duration of Phase I is shortened.     

Inheritance of resistance to Yam mosaic virus, genus Potyvirus, in white yam (Dioscorea rotundata)

Yam mosaic virus (YMV) causes the most-widespread and economically important viral disease affecting white yam (Dioscorea rotundata) in West Africa. The genetic basis of resistance in white yam to a Nigerian isolate of YMV was investigated in three tetraploid D. rotundata genotypes: TDr 93–1, TDr 93–2 and TDr 89/01444. F₁ progeny were produced using TDr 87/00571 and TDr 87/00211 as the susceptible parents. Segregation ratios indicated that a single dominant gene in a simplex condition governs the resistance in TDr 89/01444, while the resistance in TDr 93–2 is associated with the presence of a major recessive gene in duplex configuration. Segregation of progeny of the cross TDr 93–1×TDr 87/00211 fitted a genetic ratio of 2.48:1 resistant:susceptible, which can be expected when two simplex heterozygotes are crossed, indicating the possible modifying effect of the susceptible parent. A triple antibody immunosorbent assay (TAS-ELISA) was used for virus detection in inoculated plants. Slight mosaic symptoms appeared on most resistant individuals, while asymptomatic resistant genotypes with high ELISA (A₄₀₅) values were observed in all crosses. Such a heterogeneous response suggests the influence of additional modifier genes that segregate in the progeny. The finding that resistance can be inherited as a dominant or recessive character has important implications for YMV resistance breeding. Received: 15 August 2000 / Accepted: 12 April 2001     

Isolation,characterization and cross‐species amplification of microsatellite DNA loci in the tropical American yam Dioscorea trifida

We developed microsatellite markers in American yam (Dioscorea trifida). A microsatellite sequence‐enriched genomic library was screened, and after sequencing, primers were designed for 20 microsatellites. Among these, eight primer pairs yielded amplification products that were both interpretable and polymorphic in 24 yam cultivars. The number of alleles per locus ranged from two to 13 and the overall expected heterozygosity was around 0.5. Six of the eight Dioscorea trifida microsatellite loci gave amplification products in other Dioscorea species.     

Effect of mound size and fertilizer on white Guinea yam (Dioscorea rotundata) in Southern Nigeria

Summary Field trials were conducted in the forest zone of southern Nigeria on three soil series, gravelly loamy sand Ibadan soil (Oxic paleustalf), gravelly sandy loam Egbeda soil (Oxic paleustalf) and sandy loam Alagba soil (Oxic paleustalf). The trials were carried out to study the effects of planting on flatversus various mound sizes and NPK fertilizer on performance of white Guinea yam (Dioscorea rotundata) cultivar Laoko.Mound size appeared to have a more pronounced effect on tuber yield than fertilizer even on land which was in the second and third year of cropping after bush fallow. The average tuber yield for the three locations without fertilizers was 7.83 tons/ha on the flat compared with 9.44 tons/ha on large mound (about 30 cm height). With fertilizer application, tuber yields were 7.43 tons/ha on the flat and 11.30 tons/ha on large mound respectively. Total yield reduction on flat may in part be related to physical soil impedence. Planting on large mounds also resulted in longer tubers and shorter harvesting time.     

Isolation, characterisation and identification of a potyvirus from Dioscorea alata L. (water yam) in Nigeria

A yam potyvirus was isolated from Dioscorea alata samples collected in Nigeria. The virus was not transmissible mechanically but was transmitted by Aphis craccivora to four cowpea cultivars (Ife Brown, IT84S-2114, IT82E-10 and TVu2657), and from which it could be mechanically transmitted between the cowpea cultivars. In infectivity- tests using cowpea extracts, the virus had a dilution end point of 10^-4, a thermal inactivation point of 60–65°C and longevity in vitro of 2 days at room temperature. The virus coat protein had an estimated molecular weight of 32 100 daltons. The virus was identified as an isolate of Dioscorea alata virus (DAV; syn. yam virus 1) due to its biological characteristics and its serological reaction with antiserum raised against DAV. The virus is not related to yam mosaic virus, but distantly related to blackeye cowpea mosaic virus and cowpea aphid-borne mosaic virus.     

Effects of jasmonic acid and its methylester on in vitro microtuberisation of three food yam (Dioscorea) species

The effects of jasmonic acid (JA) applied in the medium and its methylester (MeJA) applied either in the medium or as a vapour, on shoot growth and microtuber formation were evaluated in three important food yam species (Dioscorea alata, D. cayenensis, and D. rotundata). Single nodes with leaves, derived from in vitro-multiplied material, were used as explants. When delivered at higher concentrations (10 or 50 μm) both JA and MeJA suppressed node formation. Microtuberisation was supported in all three species by adding either JA or MeJA to the medium. Significant promotory effects were observed only when photoperiod, salt compositions and sucrose concentrations known to favour microtuberisation processes in yams were used. MeJA applied as a vapour strongly inhibited microtuber differentiation in D. alata on all media tested but in D. rotundata and D. cayenensis yams MeJA, also applied in the vapour phase, exhibited slight promotory effects on microtuberisation. Received: 28 January 1998 / Revision received: 7 October 1999 / Accepted: 7 January 2000     

Segregation patterns of isozyme loci and microsatellite markers show the diploidy of African yam Dioscorea rotundata (2n=40)

The cultivated yam species Dioscorea rotundata (2n=40) has been considered by most authors as a tetraploid species with a basic chromosome number of ten. In this paper, we analysed the segregation of two isozyme loci and six microsatellite markers in the progeny of a self-fertilised monoecious plant. For the eight markers, segregation patterns could be explained by only two genetic models: diploidy or tetraploidy with two null alleles. Given the nature of studied markers, the most parsimonious hypothesis was that the parental plant was diploid. These results, data from a diversity survey and results of other authors led to the conclusion that D. rotundata is a diploid species.     

Evolutionary force of AT‐rich repeats to trap genomic and episomal DNAs into the rice genome: lessons from endogenous pararetrovirus

In plant genomes, the incorporation of DNA segments is not a common method of artificial gene transfer. Nevertheless, various segments of pararetroviruses have been found in plant genomes in recent decades. The rice genome contains a number of segments of endogenous rice tungro bacilliform virus‐like sequences (ERTBVs), many of which are present between AT dinucleotide repeats (ATrs). Comparison of genomic sequences between two closely related rice subspecies, japonica and indica, allowed us to verify the preferential insertion of ERTBVs into ATrs. In addition to ERTBVs, the comparative analyses showed that ATrs occasionally incorporate repeat sequences including transposable elements, and a wide range of other sequences. Besides the known genomic sequences, the insertion sequences also represented DNAs of unclear origins together with ERTBVs, suggesting that ATrs have integrated episomal DNAs that would have been suspended in the nucleus. Such insertion DNAs might be trapped by ATrs in the genome in a host‐dependent manner. Conversely, other simple mono‐ and dinucleotide sequence repeats (SSR) were less frequently involved in insertion events relative to ATrs. Therefore, ATrs could be regarded as hot spots of double‐strand breaks that induce non‐homologous end joining. The insertions within ATrs occasionally generated new gene‐related sequences or involved structural modifications of existing genes. Likewise, in a comparison between Arabidopsis thaliana and Arabidopsis lyrata, the insertions preferred ATrs to other SSRs. Therefore ATrs in plant genomes could be considered as genomic dumping sites that have trapped various DNA molecules and may have exerted a powerful evolutionary force.     

Plastid phylogenetics of Oceania yams (Dioscorea spp., Dioscoreaceae) reveals natural interspecific hybridization of the greater yam (D. alata)

Phylogenetic relationships of Oceanian staple yams (species of Dioscorea section Enantiophyllum) were investigated using plastid trnL‐F and rpl32‐trnL^(UAG) sequences and nine nuclear co‐dominant microsatellites. Analysis of herbarium specimens, used as taxonomic references, allowed the comparison with samples collected in the field. It appears that D. alata, D. transversa and D. hastifolia are closely related species. This study does not support a direct ancestry from D. nummularia to D. alata as previously hypothesized. The dichotomy in D. nummularia previously described by farmers in semi‐perennial and annual types was reflected by molecular markers, but the genetic structure of D. nummularia appears more complex. Dioscorea nummularia displayed two haplotypes, each corresponding to a different genetic group. One, including a D. nummularia voucher from New Guinea, is closer to D. tranversa, D. alata and D. hastifolia and encompasses only semi‐perennial types. The second group is composed of semi‐perennial and annual yams. However, some of these annual yams also displayed D. alata haplotypes. Nuclear markers revealed that some annual yams shared alleles with D. alata and semi‐perennial D. nummularia, suggesting a hybrid origin, which may explain their intermediate morphotypes and the difficulty met in classifying them.     

紫乌头复合体nrDNA的ITS序列与系统发育分析

对乌头属 Aconitum L. 27个类群的nrDNA ITS序列进行了简约法与邻接法分析,两种方法得到的系统发育树基本一致。乌头亚属subgen. Aconitum 的蔓乌头系 ser. Volubilia 不是一个单系类群,显柱乌头系 ser. Stylosa 与兴安乌头系ser. Ambigua 各自作为单系类群也没有得到支持。特产于云南西北部横断山区的一些种之间存在非常近的系统发育关系,说明这些种可能是近期物种形成的产物。紫乌头 A. delavayi 复合体的不同类群在系统发育树上位     

The application of strand invasion phenomenon,directed by peptide nucleic acid (PNA) and single‐stranded DNA binding protein (SSB) for the recognition of specific sequences of human endogenous retroviral HERV‐W family

The HERV‐W family of human endogenous retroviruses represents a group of numerous sequences that show close similarity in genetic composition. It has been documented that some members of HERV‐W–derived expression products are supposed to play significant role in humans' pathology, such as multiple sclerosis or schizophrenia. Other members of the family are necessary to orchestrate physiological processes (eg, ERVWE1 coding syncytin‐1 that is engaged in syncytiotrophoblast formation). Therefore, an assay that would allow the recognition of particular form of HERV‐W members is highly desirable. A peptide nucleic acid (PNA)–mediated technique for the discrimination between multiple sclerosis‐associated retrovirus and ERVWE1 sequence has been developed. The assay uses a PNA probe that, being fully complementary to the ERVWE1 but not to multiple sclerosis‐associated retrovirus (MSRV) template, shows high selective potential. Single‐stranded DNA binding protein facilitates the PNA‐mediated, sequence‐specific formation of strand invasion complex and, consequently, local DNA unwinding. The target DNA may be then excluded from further analysis in any downstream process such as single‐stranded DNA‐specific exonuclease action. Finally, the reaction conditions have been optimized, and several PNA probes that are targeted toward distinct loci along whole HERV‐W env sequences have been evaluated. We believe that PNA/single‐stranded DNA binding protein–based application has the potential to selectively discriminate particular HERV‐W molecules as they are at least suspected to play pathogenic role in a broad range of medical conditions, from psycho‐neurologic disorders (multiple sclerosis and schizophrenia) and cancers (breast cancer) to that of an auto‐immunologic background (psoriasis and lupus erythematosus).     

Multi‐gene phylogenetic analysis of south‐east Asian pest members of the Bactrocera dorsalis species complex (Diptera: Tephritidae) does not support current taxonomy

Bactrocera dorsalis sensu stricto, B. papayae, B. philippinensis and B. carambolae are serious pest fruit fly species of the B. dorsalis complex that predominantly occur in south‐east Asia and the Pacific. Identifying molecular diagnostics has proven problematic for these four taxa, a situation that cofounds biosecurity and quarantine efforts and which may be the result of at least some of these taxa representing the same biological species. We therefore conducted a phylogenetic study of these four species (and closely related outgroup taxa) based on the individuals collected from a wide geographic range; sequencing six loci (cox1, nad4‐3′, CAD, period, ITS1, ITS2) for approximately 20 individuals from each of 16 sample sites. Data were analysed within maximum likelihood and Bayesian phylogenetic frameworks for individual loci and concatenated data sets for which we applied multiple monophyly and species delimitation tests. Species monophyly was measured by clade support, posterior probability or bootstrap resampling for Bayesian and likelihood analyses respectively, Rosenberg's reciprocal monophyly measure, P(AB), Rodrigo's (P(RD)) and the genealogical sorting index, gsi. We specifically tested whether there was phylogenetic support for the four ‘ingroup’ pest species using a data set of multiple individuals sampled from a number of populations. Based on our combined data set, Bactrocera carambolae emerges as a distinct monophyletic clade, whereas B. dorsalis s.s., B. papayae and B. philippinensis are unresolved. These data add to the growing body of evidence that B. dorsalis s.s., B. papayae and B. philippinensis are the same biological species, which poses consequences for quarantine, trade and pest management.     

Descriptions and phylogenetic relationships of two new genera and four new species of Oligo‐Miocene waterfowl (Aves: Anatidae) from Australia

The Tertiary anatid fossils (Aves: Anatidae) from Oligocene and Miocene deposits in Australia are described. Most fossils derive from the Late Oligocene – Early Miocene (26–24 Mya) Etadunna and Namba Formations, respectively, in the Lake Eyre and Lake Frome Basins of South Australia. The local faunas from these two formations contain the same suite of anatid species. Two new genera, the oxyurine Pinpanetta, with three new species (Pi. tedfordi, 18 specimens; Pi. vickersrichae, 15 specimens; Pi. fromensis, 20 specimens), and the tadornine Australotadorna, for a large new species known from eight specimens, are established. Three anatid bones from the Waite Formation (c. 8 Mya) at Alcoota, Northern Territory reveal the presence of a tadornine that is neither Australotadorna nor an extant Tadorna species, and an indeterminate duck about the size of Malacorhynchus. Phylogenetic analyses establish Pinpanetta as a basal member of an oxyurine (stiff‐tailed duck) radiation. Oxyurines are found to include the Recent Stictonetta and Malacorhynchus as basal members, along with the fossil taxa Mionetta, Manuherikia, and Dunstanetta, and the traditionally included Recent Oxyura, Biziura, Thalassornis, and Nomonyx. © 2009 The Linnean Society of London, Zoological Journal of the Linnean Society, 2009, 156 , 411–454.     

An endangered new species of edible yam (<Emphasis Type="Italic">Dioscorea</Emphasis>, <Emphasis Type="Italic">Dioscoreaceae</Emphasis>) from Western Madagascar and its conservation

Summary  A new species of Dioscorea from Morondava prefecture in Western Madagascar is described. Dioscorea bako Wilkin differs from D. alatipes Burkill & H. Perr. by its usually shallowly and irregularly lobed leaf margins, broader leaf blade (grey-green below), longer petiole and the broadly cuneate area where the petiole is inserted onto the leaf blade in the basal sinus. It is pubescent, especially densely on young shoots and inflorescences. It is endemic to Morondava Préfecture and is endangered under IUCN Red List category criteria (IUCN 2001). The unusual morphological features of its inflorescences, some of which are shared with D. alatipes are discussed. Dioscorea bako is a favoured food source for people of the Menabe region and is reported by them to be increasingly hard to find. Immediate conservation measures are necessary to ensure that it remains extant and, in the medium to long term, research should be undertaken to guide its sustainable utilisation.     

Cryptic diversity of small‐sized species of Phalacroma (Dinophysales,Dinophyceae) from Denmark Strait (Eastern Arctic Greenland)

Phalacroma currently comprises 69 species of mostly marine heterotrophic dinophysoids. With a round cell body and short sulcal and cingular lists, Phalacroma spp. have a simple morphology compared to other dinophysoid genera. Therefore, species identification is not always a trivial matter. A few Arctic species have been described and, with the exception of Ph. rotundatum, they are not only infrequently recorded but also occur in low cell abundances. Here, we studied a Lugol fixed sample from Denmark Strait. From this sample 13 cells with a Phalacroma‐like morphology were isolated under a stereo microscope, photo documented, and used for single‐cell PCR determination of nuclear‐encoded LSU rDNA sequences. The sequences fell in three groups defined by their ribotype. A single ribotype sequence representing each group was added to an alignment containing a diverse assemblage of dinophysoids and analyzed using Bayesian analysis and maximum likelihood. Based on comparative light microscopy one of the ribotypes (ribotype 1) was similar but not identical to Ph. ruudi and ribotype 2 could not be matched to a known species of Phalacroma. In the phylogeny both clustered with other species of Phalacroma including the type species. The morphology of ribotype 3 was similar but not identical to Ph. braarudii. In the phylogeny ribotype 3 clustered outside the core group of Phalacroma species. Hence, ribotype 3 was designated ‘Phalacroma’ sp. 3 and has to be described as a new genus pending additional data. This study has revealed the existence of cryptic species diversity within the Phalacroma morphotype boundary. Two additional Phalacroma species (Ph. apicatum and Ph. cf. argus) also clustered outside Phalacroma sensu stricto further emphasizing the presence of cryptic species within the genus. Future studies with a polyphasic approach are needed to better address the taxonomy of Phalacroma and Phalacroma‐like dinophysoids.     

Identification of microsatellite loci from Epimedium koreanum and cross‐species amplification in four species of Epimedium (Berberidaceae)

Nineteen polymorphic microsatellite markers were isolated and characterized from a trinucleotide enriched partial genomic library of Epimedium koreanum. The average allele number of these microsatellites was 5.9 per locus, ranging from two to 11. The observed heterozygosity (H_O) and expected heterozygosity (H_E) at the population level were 0.00–0.90 and 0.12–0.90, respectively. In addition, the results of cross‐species amplification of this set of microsatellite markers in four closely related Epimedium species, E. brevicornum, E. sagittatum, E. pubescens and E. wushanense, revealed that these microsatellite markers were useful for population genetic structure evaluation and genotype analysis of major Epimedium species that have been used as traditional Chinese medicines.