Characterisation of germplasm accessions of Napier grass (pennisetum purpureum and P. purpureum×P. glaucum Hybrids) and comparison with farm clones using RAPD

Fifty six germplasm accessions of the important East African fodder crop Napier grass, Pennisetum purpureum, and its hybrids with P. glaucum, were characterised using 67 random amplified polymorphic DNA (RAPD) fragments. No or very low intra-accession variation was found for 49 of the accessions examined, confirming field observations that this species is predominantly clonally propagated. Comparison of intra and inter-accession variation identified several groups of identical/similar accessions that could be targeted if the collection is to be rationalised, and also highlighted two misplantings of germplasm material during transfer to a field trial site. A neighbour joining dendrogram of Jaccard's similarity estimates, clearly separated 50 accessions of P. purpureum from three P. glaucum individuals, and placed six hybrid accessions in an intermediate position. These groupings were well supported by a nested AMOVA (P<0.001; 29.5% of total variance due to taxonomic delineation). The main group of P. purpureum individuals could be further differentiated into five sub-groups (designated East Africa, Southern Africa, USA1, USA2 and Miscellaneous, to reflect the majority membership of sub-groups) and examination of the within P. purpureum component of the nested AMOVA, found them to be significantly different (P<0.001; 18.8% of variance). Genetic diversity across all accessions was found to be fairly high (Shannon's diversity index 0.306) and thus the collection probably represents a wide genetic base for this species. In addition to germplasm accessions, 25 Kenyan farm clones were also analysed. A principal coordinate analysis found that all but one of the clones clustered with the main P. purpureum group of accessions, indicating that the majority are probably not interspecific hybrids. The origin and pedigree of clones is discussed based on genetic similarity amongst clones and to germplasm accessions.     

Isozyme Banding Pattern and Estimation of Genetic Diversity among Guinea Grass Germplasm

Isozyme banding pattern was studied in Guinea grass (Panicum maximum Jacq.), a widely cultivated grass having good fodder value. Similarity among 63 accessions collected from diverse sources was worked out using five enzyme systems (SOD, GOT, ACP, Esterase and Peroxidase) following horizontal starch gel electrophoresis. Biochemical markers such as isozymes are useful supplements in identifying the genetic variation present in any crop. A total of 35 clear and unambiguous bands were used for analysis of which 8 bands were monomorphic. Polymorphism exhibited by 27 bands from all five enzyme systems indicate presence of considerable diversity in this species. The dendrogram generated after UPGMA and SAHN cluster analysis using Jaccard genetic distance showed that 63 accessions from diverse geographical locations could be grouped in three main clusters, of which two could further be divided into sub-clusters. Although the clusters comprised members from different locations, most of the accessions from similar geographical locations tended to cluster in same group.     

Isozyme analysis of indigenous cassava germplasm for identification of duplicates

Isozyme analysis was utilized for identification of duplicates in indigenous collection of cassava germplasm. 786 indigenous accessions of cassava were screened for 11 key morphological characters and the morphological duplicates were identified. Two hundred and eighteen accessions of cassava consisting of ninety one sets of morphological duplicates were analysed for esterase isozyme polymorphism. Thirty seven polymorphic bands were obtained, showing high polymorphism for this enzyme. Thirty seven double sets and 17 multiple sets were found to be similar at isozyme level. These isozyme duplicates will be further analysed for DNA similarity before eliminating them from the field germplasm. The dissimilar sets showed variation in minor morphological characters, thus confirming the relation between isozyme polymorphism and phenotypic variation.     

Changes in the diversity and geographic distribution of cultivated millet (Pennisetum glaucum (L.) R. Br.) and sorghum (Sorghum bicolor (L.) Moench) varieties in Niger between 1976 and 2003

Changes in the diversity of landraces in centres of diversity of cultivated plants need to be assessed in order to monitor and conserve agrobioversity—a key-element of sustainable agriculture. This notably applies in tropical areas where factors such as increased populations, climate change and shifts in cropping systems are hypothesized to cause varietal erosion. To assess varietal erosion of staple crops in a country subjected to various anthropogenic and natural environmental changes, we carried out a study based on a comparison of the diversity of pearl millet and sorghum varieties collected in 79 villages spanning the entire cereal-growing zone of Niger over a 26 year period (1976–2003). For these two crops, the number, name and type of varieties according to important traits for farmers were considered at different spatial scales (country, region, village) at the two collection dates. The results confirmed the high diversity of millet and sorghum varieties in Niger. No erosion of varietal diversity was noted on a national scale during the period covered. Some changes were observed but were limited to the geographical distribution of certain varieties. This highlights that farmers’ management can preserve the diversity of millet and sorghum varieties in Niger despite recurrent and severe drought periods and major social changes. It also indicates that rainfed cereal cropping systems in Niger should remain to be based on millet and sorghum, while reinforcing farmers’ seed systems.     

AFLP Fingerprinting in Pigeonpea (Cajanus cajan (L.) Millsp.) and its Wild Relatives

Detection of DNA polymorphism in cultivated pigeonpea (Cajanus cajan) and two of its wild relatives Cajanus volubilis and Rhynchosia bracteata is reported here for the first time using amplified fragment length polymorphism (AFLP) fingerprinting. For this purpose, two EcoRI (three selective nucleotides) and 14 MseI (three selective nucleotides) primers were used. The two wild species shared only 7.15% bands with the pigeonpea cultivars, whereas 86.71% common bands were seen among cultivars. Similarly, 62.08% bands were polymorphic between C. volubilis and pigeonpea cultivars in comparison to 63.33% polymorphic bands between R. bracteata and pigeonpea cultivars, and 13.28% polymorphic bands among pigeonpea cultivars. The cluster analysis revealed low polymorphism among pigeonpea cultivars and very high polymorphism between cultivated pigeonpea and its wild relatives. The AFLP analysis also indicated that only one primer combination (EcoRI + ACT and MseI + CTG), at the most any four primer pair combinations, are sufficient for obtaining reliable estimation of genetic diversity in closely related cultivars like pigeonpea material analyzed herein. AFLP analysis may prove to be a useful tool for molecular characterization of pigeonpea cultivars and its wild relatives and for possible use in genome mapping.     

Germplasm Management of Faba Bean (Vicia faba L.): Monitoring Intercrossing between Accessions with Inter-plot Barriers

Knowledge of gene flow is essential for designing strategies of germplasm multiplication. Inter and intra-plot gene flow and pollen dispersion between small plots in a field of Vicia faba germplasm multiplication were measured under four isolation zones at two locations. The four isolation zones were: a barren zone, a nucleocytoplasmic Vicia faba male sterile line, a Vicia faba tetraploid genotype and a Vicia narbonensis L. population. Genotypes fixed for alternative isozyme and allozyme alleles allowed the identification of inter and intra-plot hybrids through progeny testing. A set of log-likelihood ratio (LLR) G tests were used to analyze the effects of geographical location, isolation zone and genotype on the different components of gene flow. Considerable heterogeneity among the genotypes in all the components, inter and intra-plot gene flow and pollen dispersion, of gene flow has been found. It has been shown that gene flow is location, isolation zone and genotype dependent. Planting a barrier surrounding the plots rather than using a non cultivated area between plots seemed more or less efficient for preventing inter-plot gene flow depending on the genotype and the location. Specific associations between genotype and male sterile or tetraploid border rows for every location can bring about gene flow as high, or even higher, than those produced by a barren zone. Differences among the genotypes and borders in flowering overlapping appear not to be the major factor influencing the pattern of gene flow. Implications of the pattern of gene flow observed are discussed in relation to crop diversity managed by farmers.     

Allelic Size Standards and Reference Genotypes to Unify International Cocoa (Theobroma cacao L.) Microsatellite Data

Standardisation of microsatellite allele profiles between laboratories is of fundamental importance to the transferability of genetic fingerprint data and the identification of clonal individuals held at multiple sites. Here we describe two methods of standardisation applied to the microsatellite fingerprinting of 429 Theobroma cacao L. trees representing 345 accessions held in the worlds largest Cocoa Intermediate Quarantine facility: the use of a partial allelic ladder through the production of 46 cloned and sequenced allelic standards (AJ748464 to AJ48509), and the use of standard genotypes selected to display a diverse allelic range. Until now a lack of accurate and transferable identification information has impeded efforts to genetically improve the cocoa crop. To address this need, a global initiative to fingerprint all international cocoa germplasm collections using a common set of 15 microsatellite markers is in progress. Data reported here have been deposited with the International Cocoa Germplasm Database and form the basis of a searchable resource for clonal identification. To our knowledge, this is the first quarantine facility to be completely genotyped using microsatellite markers for the purpose of quality control and clonal identification. Implications of the results for retrospective tracking of labelling errors are briefly explored.     

Genetic Diversity in a Worldwide Collection of Cherimoya Cultivars

Genetic diversity of 206 cherimoya (Annona cherimola Mill.) cultivars representing the major regions of production was determined from an analysis of 23 isozyme loci. Gene diversity in cherimoya was high, although not equally distributed among countries. Peru and Ecuador, the countries of origin of this species, showed the greatest diversity, and Spain and Madeira (Portugal) showed the least, probably due to genetics bottlenecks resulting from limited introduction. Cultivars from California showed high values for several genetic diversity measures, likely due to their diverse origins. The expansion of cherimoya cultivation out the area of origin has produced a change in the apportionment of genetic diversity, with an increase in the inter-populational components, but likely without a general erosion overall.     

Genetic Diversity in Tef [<Emphasis Type="Italic">Eragrostis tef</Emphasis> (Zucc.) Trotter] Germplasm

One hundred and forty-four heterogeneous tef germplasm accessions collected from 10 major tef growing areas in Ethiopia were evaluated for 18 quantitative traits in a simple lattice design, at Holetta and Ginchi, in 2001 main growing season to study the genetic diversity in tef. The combined analysis of variance revealed the presence of significant variation among germplasm accessions for all traits studied. Cluster analysis revealed the overlapping of the germplasm accessions from different origin and the accessions were grouped into eight distinct clusters of 1–78. Generalized distance square confirmed the presence of significant genetic distance between clusters. In principal component (Prin) analysis, the first four principal components with latent root values above one accounted for about 80.6% of the variability existing among the germplasm accessions. Prin1 constituted about 55% of the variability mainly from almost equal contribution of 10 quantitative traits, indicating that most of the traits are equally important in tef diversity. In this study, the regions and altitudes of origin were not found to have a substantial effect on the genetic diversity in tef germplasm. Diversity within the regions was found to be significant and, hence, an opportunity for exploitation in tef improvement.     

Short-term measurements of uptake of nitrogen fixed in the rhizospheres of sorghum (Sorghum bicolor) and millet (Pennisetum americanum)

Summary In a series of short-term experiments root systems of young sorghum and millet plants inoculated with N₂-fixing bacteria were exposed to ¹⁵N₂-enriched atmospheres for 72 h. The plants were grown in a normal atmosphere for up to 22 days after the end of the exposure to allow them to take up the fixed N₂. Environmental conditions and genotypes of sorghum and millet were selected to maximise N₂-fixation in the rhizosphere. Detectable amounts of fixed N (> 16 g/plant) were rapidly incorporated into sorghum plants grown in a sand/farmyard manure medium, but measurable fixation was found on only one occasion in plants grown in soil. N₂ fixation was detectable in some experiments with soil-grown millet plants but the amounts were small (2–4 g/plant) and represented less than 1 % of plant N accumulated over the same period. In many cases there was no detectable ¹⁵N₂ incorporation despite measurable increases in ethylene concentration found during an acetylene reduction assay.Published as ICRISAT Journal Article No. JA 740     

Spatial and temporal dynamics in genetic diversity in upland rice and late millet (<Emphasis Type="Italic">Pennisetum glaucum</Emphasis> (L.) R. Br.) in The Gambia

Ambiguity exists about the level of genetic diversity represented by farmer crop varieties, how it develops over time and how it relates to the diversity comprised by formal varieties. As part of an interdisciplinary technological/sociological study on farmer management of gene flow, upland rice (Oryza sativa L.) and late millet (Pennisetum glaucum (L.) R.Br.) from The Gambia were investigated for morphological and molecular variation. The goal of these analyses was to obtain insight into the level of crop genetic diversity of farmer’s materials planted in several case study villages in The Gambia. For both crops, samples were collected from villages and various research institutes. Based on variety names, different rice and millet varieties were expected to be used in different villages. In fact, there was a large overlap in genetic diversity for both crops, masked by the use of synonyms. The considerable similarity in rice genetic diversity between villages most likely results from the exchange of varieties between farmers. For millet this seems the result of development of varieties from the same gene pool. Some farmer varieties of rice, however, are apparent hybrid forms between the species O. sativa and O. glaberrima Steud., and farmer varieties in general displayed higher levels of genetic diversity than formal varieties. This indicates that, for rice, genetic diversity develops in farmers’ fields and may have potential use in formal breeding programs.     

Assessing the Genetic Diversity in the International Cocoa Genebank,Trinidad (ICG,T) using Isozyme Electrophoresis and RAPD

Random amplified polymorphic DNA (RAPD) and isozyme electrophoresis (IE) techniques were used to estimate the level of genetic diversity in a sample of cacao germplasm existing at the International Cocoa Genebank, Trinidad. Twenty-six cocoa populations represented by 459 cocoa genotypes were analysed using IE and 22 populations represented by 353 cocoa genotypes were analysed using RAPD. Despite few differences in the classification of the populations, both techniques revealed three major groups: the indigenous trees, the cultivated Trinitario and the cultivated trees from Ecuador. Two-thirds of the partitioned diversity were found within populations and one-third between the populations, with both techniques.     

Characterization of Seashore Paspalum (Paspalum vaginatum Swartz) Germplasm by Transferred SSRs from Wheat, Maize and Sorghum

One hundred and thirty SSR markers from wheat, maize and sorghum were screened for the transferability to Paspalum. The transfer rate was 67.5, 49.0 and 66.8% respectively. This would be a very efficient approach for DNA marker development for species which are not well studied molecularly. The polymorphism level for transferred SSR markers was 51.5% within species (Paspalum vaginatum) and 87.1% among Paspalum species. The high level of polymorphism is directly related to the high degree of heterozygosity maintained by its way of reproduction, i.e. self-incompatibility. Forty transferred polymorphic SSR markers were selected and used for characterization and evaluation of seventy-three Paspalum accessions. In total, 209 polymorphic bands were detected from these 40 SSR markers, with an average of five polymorphic bands per marker. The Paspalum accessions clustered into three major groups. Two very similar dendrograms can be generated from either 109 or 209 polymorphic bands. This led us to determine that 18 of the transferred SSR markers were sufficient for genetically differentiating the investigated germplasm accessions. The number of SSR markers required for germplasm characterization and evaluation is discussed. This is the first report of the transfer of SSR markers from major field crops to newly emerged environmental turfgrasses.     

Variability Among Maize (Zea mays L.) Inbred Lines for Seed Longevity

Genotypes conserved in active collections may suffer genetic erosion and modifications. The objective of this work was to investigate changes in germination and vigour in maize (Zea mays L.) inbred lines during cold storage in an active collection. Seeds of 16 maize inbreds produced along 16 years were evaluated for emergence and seedling vigour in a growth chamber. Linear and quadratic regressions of vigour and viability-related traits over seed age were calculated and tested for homogeneity. The seed of five production years of five inbreds that behaved differently in the regression analysis was multiplied in 1998, and original and renewed seed were evaluated in a growth chamber in 1999. Viability and vigour decreased linearly with age for most inbreds, particularly for B84 and EP10, varied at random for a few inbreds, and remained high for EP56 and A295. Aging caused reduction of vigour and loss of viability in most inbreds. There was variability for seed longevity among inbred lines; longevity was highest for inbred EP56. During storage, some seeds of each inbred died, while enduring seeds, when multiplied, produced new seed with enhanced viability and vigour compared to the average seed of the inbred, suggesting natural selection for viability and vigour during storage within inbred lines.     

Geographic variation for isozymes in cherimoya (<Emphasis Type="Italic">Annona cherimola</Emphasis> Mill.)

Cherimoya (Anonna cherimola Mill.) is a fruit tree which originated in Peru and Ecuador and is now cultivated in several subtropical areas of the world. The characterization of cherimoya cultivars at allozyme level has been previously reported, but the geographic distribution and organization of this variation have not been fully characterized. In this study, we assessed the relationships among 206 cherimoya and four atemoya (A. cherimola ×A. squamosa) cultivars based on allozyme polymorphism. We have confirmed the genetic differences between atemoya and cherimoya cultivars, and showed that cherimoya accessions from Madeira, Bolivia and Spain form homogeneous groups of cultivars. Accessions from Chile and California form heterogeneous groups, probably due to their mixed origins. Cultivars from Peru and Ecuador showed a wide range of allelic variation, as is expected for accessions from the center of origin of this species.     

Association of Azospirillum brasilense with pearl millet (Pennisetum americanum (L.) Leeke)

Summary Microscopic observations of the root system of pearl millet (Pennisetum americanum (L.) Leeke) var. BJ 104 after surface sterilization and incubation in phosphate malate triphenyl tetrazolium chloride (TTC) revealed extensive colonization by Azospirillum spp. when plants were grown in sterile, partially sterile and field conditions as evidenced by the TTC-reducing property of active cells of the bacterium. Quantitative studies showed the need to standardize the techniques further to ensure more precise monitoring of the bacteria in the rhizosphere, as large numbers of soil bacteria were found capable of growth on specific media, thus interfering with the plate counts. Seed inoculation with A. brasilense increased the mean grain yield of pearl millet under different agroclimatic conditions in India. The mean increase in grain yield due to inoculation over uninoculated controls was also noticed with graded levels of fertilizer nitrogen (urea). Inoculation alone contributed to increased nitrogen uptake of plants with varying levels of fertilizer nitrogen application under sandy loam soil conditions (pH 7.3). The effects of inoculation were more prominent under lower levels of nitrogen than at the higher levels. The root biomass under field conditions was increased with Azospirillum spp. inoculation at 10 and 20 kg N/ha than their corresponding uninoculated controls.     

Searching for New Sources of Pink Stem Borer Resistance in Maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.)

The pink stem borer (Sesamia nonagrioides Lef.) is the main corn (Zea mays L.) pest in the Mediterranean area. Although, screening for resistance to this pest has been successful, the level of resistance shown by the most resistant varieties is not high. The objectives of the present work were: (i) the evaluation for pink stem borer resistance of the nontested inbred lines, field and popcorn materials, from the collection maintained at the Misión Biológica de Galicia and (ii) the study of the performance, under pink stem borer infestation, of inbreds selected for resistance to pink stem borer in hybrid combination. Forty four inbred lines nontested yet for resistant to pink stem borer along with five inbred lines previously reported as resistant to stem and ear attack, were evaluated in 2002 and 2003. In adjacent experiments, hybrids EP79× W552, EP77× B93, and A661× EP42 were tested along with a resistant hybrid, MEB531-Bt, and a susceptible hybrid, INRA 260. New sources of resistance to pink stem borer have been detected among inbred lines improving the level of resistance presented by previously tested inbreds. Some of these lines were successfully developed by pedigree selection for resistance to pink stem borer.     

Physico-chemical characterisation of yam (Dioscorea alata L.) tubers from Vanuatu

The objectives of this study were: (1) to analyse the physico-chemical characteristics of 48 Dioscorea alata varieties representing a core sample of the Vanuatu national germplasm collection; (2) to relate those characteristics with the varieties eating quality; and (3) to assess the possibility of selecting varieties according to their chemotype. Overall, 331 accessions were collected from 15 different islands of Vanuatu, planted in an ex situ germplasm collection and described during 3 years. The 48 varieties included in the core sample were selected according to their island of origin, eating quality, tuber shape, tuber flesh colour and morphotype. Analyses of their tubers were made for percentage dry matter, starch, amylose, lipids, minerals, proteins, sugars and gelatinisation temperature range. Significant variation exists for each of these characteristics except for gelatinisation temperature. Varieties with good eating quality are characterised with high dry matter, starch and amylose contents. Chemotypes appear to be genetically controlled and further screening of germplasm and/or breeding will have to take into consideration these characteristics, important for farmers’ adoption.     

Allozyme Variation Among Some Pearl Millet (<Emphasis Type="Italic">Pennisetum glaucum</Emphasis> L.) Cultivars Collected from Tunisia and West Africa

Allozyme variation was assessed in a collection of pearl millet cultivars derived from three different regions of traditional cultivation of this cereal in Tunisia and West African countries. Using starch gel electrophoresis, 11 loci corresponding to seven enzyme staining systems were identified. Important within-cultivar genetic diversity was detected. Genetic diversity within Tunisian cultivars was similar and was more pronounced than for West African ones. This result could be explained by the low level of the human selection characterizing the Tunisian cultivars. Most of the total genetic diversity is maintained within subregions suggesting little genetic differentiation. Cluster analysis did not reveal separate clusters for the various subregion cultivars. However the cluster analysis should help in the selection of interesting genotypes for future breeding programs.