Generation and analysis of expressed sequence tags (ESTs) for marker development in yam (Dioscorea alata L.)

Background: Anthracnose (Colletotrichum gloeosporioides) is a major limiting factor in the production of yam (Dioscorea spp.) worldwide. Availability of high quality sequence information is necessary for designing molecular markers associated with resistance. However, very limited sequence information pertaining to yam is available at public genome databases. Therefore, this collaborative project was developed for genetic improvement and germplasm characterization of yams using molecular markers. The current investigation is focused on studying gene expression, by large scale generation of ESTs, from one susceptible (TDa 95-0310) and two resistant yam genotypes (TDa 87-01091, TDa 95-0328) challenged with the fungus. Total RNA was isolated from young leaves of resistant and susceptible genotypes and cDNA libraries were sequenced using Roche 454 technology. Results: A total of 44,757 EST sequences were generated from the cDNA libraries of the resistant and susceptible genotypes. Greater than 56% of ESTs were annotated using MapMan Mercator tool and Blast2GO search tools. Gene annotations were used to characterize the transcriptome in yam and also perform a differential gene expression analysis between the resistant and susceptible EST datasets. Mining for SSRs in the ESTs revealed 1702 unique sequences containing SSRs and 1705 SSR markers were designed using those sequences. Conclusion: We have developed a comprehensive annotated transcriptome data set in yam to enrich the EST information in public databases. cDNA libraries were constructed from anthracnose fungus challenged leaf tissues for transcriptome characterization, and differential gene expression analysis. Thus, it helped in identifying unique transcripts in each library for disease resistance. These EST resources provide the basis for future microarray development, marker validation, genetic linkage mapping and QTL analysis in Dioscorea species

Development of genomic simple sequence repeat markers for yam

Yam ( Dioscorea spp.) is a major staple crop widely cultivated for its starchy tubers. To date, very few marker resources are publicly avail - able as tools for genetic and genomic studies of this economically important crop. In this study, 90 simple sequence repeat (SSR) markers were developed from an enriched genomic library of yellow Guinea yam ( D. cayenensis Lam.). Cross- amplification revealed that 85 (94.4%) and 51 (56.7%) of these SSRs could be successfully transferred to the two major cultivated species of D. rotundata Poir. and D. alata L., respec - tively. Polymorphisms in 30 markers selected on the basis of reliability and reproducibility of DNA bands were evaluated using a panel of 12 D. cayenensis , 48 D. rotundata , and 48 D. alata accessions. Accordingly, number of alleles per locus ranged from 2 to 8 in D. cayenensis (mean = 3.9), 3 to 30 in D. rotundata (mean = 13.9), and 2 to 22 in D. alata (mean = 12.1). The average observed and expected heterozygosi - ties were 0.156 and 0.634 ( D. cayenensis ), 0.326 and 0.853 ( D. rotundata ), and 0.247 and 0.836 ( D. alata ), respectively. Clustering based on six SSRs that were polymorphic in at least four of the five cultivated Dioscorea species studied, including D. cayenensis , D. rotundata , D. alata , D. dumetorum (Kunth) Pax., and D. bulbifera L., detected groups consistent with the phyloge - netic relationships of the species except for D. dumetorum . These new SSR markers are invalu - able resources for applications such as genetic diversity analysis and marker-assisted breedingYam ( Dioscorea spp.) is a major staple crop widely cultivated for its starchy tubers. To date, very few marker resources are publicly avail - able as tools for genetic and genomic studies of this economically important crop. In this study, 90 simple sequence repeat (SSR) markers were developed from an enriched genomic library of yellow Guinea yam ( D. cayenensis Lam.). Cross- amplification revealed that 85 (94.4%) and 51 (56.7%) of these SSRs could be successfully transferred to the two major cultivated species of D. rotundata Poir. and D. alata L., respec - tively. Polymorphisms in 30 markers selected on the basis of reliability and reproducibility of DNA bands were evaluated using a panel of 12 D. cayenensis , 48 D. rotundata , and 48 D. alata accessions. Accordingly, number of alleles per locus ranged from 2 to 8 in D. cayenensis (mean = 3.9), 3 to 30 in D. rotundata (mean = 13.9), and 2 to 22 in D. alata (mean = 12.1). The average observed and expected heterozygosi - ties were 0.156 and 0.634 ( D. cayenensis ), 0.326 and 0.853 ( D. rotundata ), and 0.247 and 0.836 ( D. alata ), respectively. Clustering based on six SSRs that were polymorphic in at least four of the five cultivated Dioscorea species studied, including D. cayenensis , D. rotundata , D. alata , D. dumetorum (Kunth) Pax., and D. bulbifera L., detected groups consistent with the phyloge - netic relationships of the species except for D. dumetorum . These new SSR markers are invalu - able resources for applications such as genetic diversity analysis and marker-assisted breedin

Development of mapping populations for genetic analysis in yams (D. rotundata and D. alata)

Progress is being made at the International Institute of Tropical Agriculture to develop molecular tools for marker-assisted selection that would complement and expedite conventional breeding approaches for genetic improvement of yams (Dioscorea spp.). F mapping populations 1 were developed from crossing male and female parents of D. rotundata Poir. and D. alata Lam. that differ in specific traits of interest towards identification of molecular markers linked to those traits. Success in hybridization was validated based on DNA analysis with SSR markers on agarose gel. Traits for which the populations were developed included multiple tuber production, cooking quality and virus disease resistance in D. rotundata, and anthracnose disease, cooking quality and tuber oxidation in D. alata. Death of plants in the field and rotting of tubers in storage, possibly due to pests, diseases and other environmental factors were encountered that led to the reduction in sizes of the populations. Low seed multiplication ratio necessitates two to three cycles of tuber multiplication of mapping population genotypes to achieve adequate numbers of seed tubers for field experimentation. These mapping populations are valuable tools for genetic analysis and molecular marker development in yam improvement programmes

Development of mapping populations for genetic analysis in yams (Dioscorea rotundata and D. alata)

Progress is being made at the International Institute for Tropical Agriculture to develop molecular tools for marker-assisted selection that would complement and expedite conventional breeding approaches for genetic improvement of yams (Dioscorea spp.). F1 mapping populations were developed from crossing male and female parents of D. rotundata Poir. and D. alata Lam. that differ in specific traits of interest towards identification of molecular markers linked to those traits. Success in hybridization was validated based on DNA analysis with SSR markers on agarose gel. Traits for which the populations were developed included multiple tuber production and cooking quality in D. rotundata, and anthracnose disease, cooking quality and tuber oxidation in D. alata. Death of plants in the field and rotting of tubers in storage, possibly due to pests, diseases and other environmental factors were encountered that led to the reduction in size of the populations. Low seed multiplication ratio necessitates two to three cycles of tuber multiplication of mapping population genotypes to achieve adequate numbers of seed tubers for field experimentation. These mapping populations are valuable tools for genetic analysis and molecular marker development in yam improvement programmes

Phenotypic analysis of tuber yield and maturityrelated traits in white yam (Dioscorea rotundata)

Inadequate yield potential of available varieties and their long growth periods are two of the factors limiting yam (Dioscorea spp.) production. Identifying yield- and maturity-related traits and breeding for them will enhance production. Ten morphological/physiological traits: time of shoot emergence, time of tuber initiation, plant height, shoot dry weight, time of shoot senescence, tuber fresh weight (tuber yield), tuber number/plant, tuber parenchyma colour, tuber dry matter content and tuber dormancy period were assessed in eight accessions of D. rotundata (white Guinea yam) on the field in 2008 and 2009. Shoot dry weight and plant height were identified as the major tuber yield-related traits. Early field emergence and tuber initiation, short tuber dormancy period and low tuber dry matter content may also be related to tuber yield. High yielding accessions were low in tuber dry matter content. Moreover, early and late maturing accessions could be separated by time of attainment of uniform parenchyma colour within a tuber, length of tuber dormancy period and time of shoot senescence. Accessions were identified that may be used as parents for developing mapping populations for some of the traits assessed in the study

Genetic and phenotypic diversity in a germplasm working collection of cultivated tropical yams (Dioscorea spp.)

Published Online: 7 February, 2012A working collection of yam (Dioscoreaspp.) comprising 53 landraces and seven improved cultivars of four species (D. alata L., D. cayenensis Lam., D. dumetorum (Kunth) and D. rotundata Poir.) was evaluated for phenotypic and genetic diversity. The evaluation involved field assessment of 24 morphological traits and DNA analysis with 32 Simple sequence repeat (SSR) polymorphic markers. Diversity was greater between species than within species; highest in D. rotundata and lowest in D. alata and D. cayenensis. Analysis based on combined phenotypic and SSR marker data sets revealed a close relationship between D. rotundata and D. cayenensis, but D. alata and D. dumetorum remained as distinct species. D. alata was related genetically to D. rotundata and D. cayenensis, but phenotypically to D. dumetorum. The study showed that cultivars obtained from different farmers may bear the same name but be genetically different. Polymorphic SSR markers were identified that may be used for genetic analysis across yam species. The working collection assessed in this study represents a good gene pool for intra- and inter-specific hybridization in yam genetic improvement

Colchicine-induced variations in survival rate and morphological characteristics of water yam (Dioscorea alata)

The effects of 0.2% aqueous solution of colchicine on the survival rate and morphological features of five accessions of Dioscorea alata were investigated. Sprouting buds of two month old plants were treated with 0.2% colchicine and their performances were monitored until maturity. Survival of buds was lower in all the treated plants (ranging between 6.25% and 8.75%) compared to controls (between 13.25% and 15.25%). However, colchicine treated vines had higher survival rates when exposed to drought (13.8% - 24%) compared to controls (7.2% - 16.69%). Higher number of leaves, larger leaf width and fewer numbers of stomata were observed among the treated plants. The survival of buds between the treated plants and control plants was signifi cantly different at P ≤ 0.05. The treated and non-treated plants were also signifi cantly different at P ≤ 0.05 for leaf width in accessions TDa02/00246, TDa98/00116 and TDa99/00240, and for stomata number in accessions TDa02/00151 and TDa02/00246. Our results suggest that colchicine can be used to induce mutagenic changes in yam which may be of agronomic importance in the production of the crop

An improved method of DNA extraction from plants for pathogen detection and genotyping by polymerase chain reaction

Polymerase chain reaction (PCR)-based applications in plant molecular biology and molecular diagnostics for plant pathogens require good quality DNA for reliable and reproducible results. Leaf tissue is often the choice for DNA extraction, but the use of other sources such as tubers, stems, or seeds, is not uncommon. The extraction of DNA from different tissue sources often requires different protocols. In this study, a simple protocol was established for the extraction of DNA from leaves, tubers, stems, seeds and even fungal mycelia. The protocol is simple and suitable for high-throughput DNA extraction using automated tissue grinders. It yielded large quantities of DNA (0.4 ?g to 2 mg DNA from 100 mg tissue) of high quality from seeds of maize, soybean, and cowpea, tubers of yam, tuberous roots of cassava, and leaf tissues of banana and plantain, yam, cassava, maize, okra, mango, and other species. DNA was successfully used for the detection of fungal and viral pathogens and the genotyping of yam and cassava by PCR

In vitro androgenesis in Dioscorea rotundata Poir

Conventional breeding takes several years to achieve homozygosity in yam. Anther culture technology is a rapid and viable alternative for the development of homozygous plants, therefore optimizing key parameters for the establishment of an efficient protocol for yam anther culture is indispensable. Influence of anther developmental stages, sterilization treatments, concentration and combination of growth hormones on in vitro androgenesis of Dioscorea rotundata was investigated. Anthers were grouped into five developmental stages from least to most matured, designated as Sml to Sm5. The criteria used for the grouping were anther size, color and location on the inflorescence. Three sterilization treatments were designed to produce contamination-free anther cultures. These are NaOCI (sodiumhypochlorite), NaOCI with 70% ethanol pretreatment and NaOCI with ethanol and Tween 20. Sterilization of spikes was carried out using 2%, 3%, 5%,10%, 15% and 20% NaOCI concentrations for 5, 10, 15 and 20 minutes. Anthers were cultured on MS media supplemented with 2, 4-D or NAA at three concentrations (1.0, 1.5 and 2.0 mg/l) combined with Kinetin or 6-BAP at two concentrations (0.5 1.0 mg/l). Viable and contamination-free anthers were recorded two weeks after culture while callus production was recorded four weeks after culture. Anthers of Sm2 had100% viabitity under dark condition. Fifteen percent NaOCI sterilization for 20 minutes with ethanol gave 100% contamination-free anthers. Optimum callus (15%) was produced when 1.5 mg/I 2, 4-D was combined with 1.0 mg/I 6-BAP. The optimal conditions derived for yam anther culture would facilitate the production of haploid genotypes