Molecular characterization of Lebanese olive germplasm

Lebanon, an East Mediterranean country, does not have a comprehensive reference national olive collection. A report published 30 years ago indicated the presence of four Lebanese varieties, but the confusion regarding these varieties was discussed then and is still prevalent today hindering prospects for conserving and exploring the national germplasm. This study assessed the existing in situ diversity of old Lebanese olive groves using simple sequence repeat (SSR) and amplified fragment length polymorphisms (AFLP). Trees (140) were collected from 14 groves located in the four traditional olive areas. Analysis was based on 22 polymorphic alleles produced from six SSR primers, and on 135 polymorphic AFLP fragments. SSR and AFLP did not yield consistent results in terms of level of polymorphism, with SSR detecting higher variation than AFLP. On the other hand, both clustered trees based on geographic provenance. AFLP coefficient of similarity between trees ranged between 0.70 and 0.99 indicating the possible presence of varieties since some values fall within reported intervarietal ranges of 0.65 to 0.84. SSR unveiled the presence of nine different genotypes: four corresponding each to a provenance and five consisting of single trees characterized by the presence of less frequent alleles with an average of 0.33. Results also revealed a high incidence of clones greater than 90% in three of the four provenances. The findings of this study point for the need to plan for a sampling strategy that takes into consideration geographic provenances.     

Molecular characterization of Buffalograss germplasm using sequence-related amplified polymorphism markers

Buffalograss [Buchloe dactyloides (Nutt.) Englem] germplasm has a broad resource of genetic diversity that can be used for turfgrass, forage and conservation. Buffalograss is the only native grass that is presently used as a turfgrass in the Great Plains region of North America. Its low growth habit, drought tolerance and reduced requirement for fertilizer and pesticides contribute to interest in its use. The objectives of this study were to use sequence-related amplified polymorphism (SRAP) markers in the evaluation of genetic diversity and phenetic relationships in a diverse collection of 53 buffalograss germplasms, and to identify buffalograss ploidy levels using flow cytometry. Based on their DNA contents, buffalograss genotypes were grouped into four sets, corresponding to their ploidy levels. Thirty-four SRAP primer combinations were used. This is the first report of the detection of differentiating diploid, tetraploid, pentaploid and hexaploid buffalograss genotypes, representing diverse locations of origin, using SRAP markers. Cluster analysis by the unweighted pair-group method with arithmetic averages based on genetic similarity matrices indicated that there were eight clusters. The coefficients of genetic distance among the genotypes ranged from 0.33 up to 0.99 and averaged D=0.66. The genetic diversity estimate, He, averaged 0.35. These results demonstrated that genotypes with potential traits for turfgrass improvement could readily be distinguished, based on SRAP. The use of PCR-based technologies such as SRAP is an effective tool for estimating genetic diversity, identifying unique genotypes as new sources of alleles for enhancing turf characteristics, and for analyzing the evolutionary and historical development of cultivars at the genomic level in a buffalograss breeding program.Communicated by B. Friebe     

Molecular,morphological and agronomic characterization of the sweet potato (Ipomoea batatas L.) germplasm collection from Mozambique: Genotype selection for drought prone regions

Sweet potato (Ipomoea batatas (L.) Lam) is one of the most important root crops in Mozambique, ranking in the 3rd position, after cassava and maize. Within the scope of the national and regional strategies/initiatives, we have used a multi-analysis approach to characterize the national sweet potato germplasm collection at two different levels: i) genetic, morphological and agronomic diversity; and ii) agronomic potential (storage root yield, vine weight, biomass, harvest index and dry matter content) toward drought tolerance. This collection, composed by 44 accessions, comprises 28 genotypes cultivated in three different provinces of Mozambique (Gaza, Inhambane and Zambezia), nine from other African countries (Kenya, South Africa, Uganda and Zimbabwe), one from the United States of America, and six from CGIAR research centers (IITA and CIP). According to our results, the Mozambican germplasm bank presents a high level of diversity, comparable to those from the collections of the primary centers of origin and South Africa, therefore constituting of a good source of agronomic traits for breeding. Regarding drought tolerance, six Mozambican genotypes (Admarc, Chingova, Nhacoongo-1, Xihetamakote, Nwanatuyo, and Chissicuana-2), one from Uganda (NASPOT-5), one from Zimbabwe (Moz_white), one from Kenya (SPK 004), and one from the USA (Resisto) seem to have the highest potential to be used in regions with frequent drought seasons and in future breeding programs. The results showed that such integrated analysis can be used to successfully characterize the genetic material in terms of suitability to drought-prone regions, therefore helping sweet potato crop management, with economic and food security impacts.     

Molecular characterization of an anther-preferential gene from rice

Expression levels of anther-expressed genes in rice were estimated by plaque hybridization. A total of 33 cDNAs, isolated randomly from an anther-enriched cDNA library, were used as probes to hybridize both anther and leaf cDNAs. The expression level of individual cDNA clones was then estimated by counting the number of plaques hybridized to each probe. Based on abundance patterns that appeared in both anther and leaf cDNA libraries, the clones were classified into three groups. This classification showed that the majority of the clones (one group) exhibited expression in both cDNA libraries at almost equal frequency. The other two groups showed either low or no expression in the leaf cDNA library. Among the cDNA clones,RA1003 (detected only in the anther cDNA library) was selected and further characterized at the molecular level. Consistent with the results of the plaque hybridization experiment, northern blot analysis also revealed no gene expression in vegetative organs, leaves, or roots. However, expression was high in the flowers, especially in the anthers. Detailed molecular studies of the gene are also described and discussed here.     

Molecular characterization of an earliest cacao (Theobroma cacao L.) collection from Upper Amazon using microsatellite DNA markers

Cacao (Theobroma cacao L.) is indigenous to the Amazon region of South America. The river basins in the Upper Amazon harbor a large number of diverse cacao populations. Since the 1930s, several numbers of populations have been collected from the present-day Peruvian Amazon and maintained as ex situ germplasm repositories in various countries, with the largest held in the International Cacao Genebank in Trinidad. The lack of information on population structure and pedigree relationship and the incorrect labeling of accessions are major concerns for efficient conservation and use of cacao germplasm. In the present study, we assessed the individual identity, sibship, and population structure in cacao populations collected from the present-day Loreto Region, Peru in the 1930–1940s. Using a capillary electrophoresis genotyping system, we analyzed the simple sequence repeat variation of 612 cacao accessions collected from the Marañon, Nanay, and Ucayali river systems. A total of 180 cases of mislabeling were identified using a Bayesian clustering method for admixture detection. Using maximum likelihood-based methods, we reconstructed 78 full-sib families nested in 48 half-sib families, indicating that the pods collected in the 1930s were from 48 mother trees, maximum. Likelihood simulation also identified eight probable parents that are responsible for 117 pairs of mother–offspring relationships in this collection. Principal coordinate analysis (PCoA) and the Bayesian clustering method cohesively demonstrated a pronounced structure of genetic diversity, stratified by the river systems of the Peruvian Amazon. Our results also show that, in spite of the high level of allelic diversity in this collection, it was composed of a large number of related family members collected from a relatively small area, including a couple of sites in the Ucayali and Nanay rivers, as well as the lower Marañon river near Iquitos. The vast majority of the Peruvian Amazon, especially the upper Marañon River and its tributaries, have not been sampled by collecting expeditions. The improved understanding of the individual identities, genealogical relationships, and geographical origin of cacao germplasm in this collection will contribute to more efficient conservation and utilization of these germplasm. Additionally, this study also provides more baseline information to help guide future collecting expeditions in the Peruvian Amazon.     

Adsorption kinetics of a basic dye from aqueous solutions onto apricot stone activated carbon

The preparation of activated carbon from apricot stone with H₂SO₄ activation and its ability to remove a basic dye, astrazon yellow 7GL, from aqueous solutions were reported in this study. The adsorbent was characterized by FTIR, BET and SEM, respectively. The effects of various experimental parameters, such as initial dye concentration, pH, adsorbent dosage and temperature were investigated in a batch-adsorption technique. The optimum conditions for removal of the basic dye were found to be pH 10, 6 g/l of adsorbent dosage and equilibrium time of 35 min, respectively. A comparison of three kinetic models, the pseudo first-order, second-order and diffusion controlled kinetic models, on the basic dye-adsorbent system showed that the removal rate was heavily dependent on diffusion controlled kinetic models. The adsorption isotherm data were fitted well to Langmuir and Freundlich isotherms. The adsorption capacity was calculated as 221.23 mg/g at 50 °C. Thermodynamics parameters were also evaluated. The values of enthalpy and entropy were 49.87 kJ/mol and 31.93 J/mol K, respectively, indicating that this process was spontaneous and endothermic. The experimental studies were indicated that ASC had the potential to act as an alternative adsorbent to remove the basic dye from aqueous solutions.     

Assessing genetic diversity in a sugarcane germplasm collection using an automated AFLP analysis

An assessment of genetic diversity within and between Saccharum, Old World Erianthus sect. Ripidium, and North American E.giganteus (S.giganteum) was conducted using Amplified Fragment Length Polymorphism (AFLP^TM) markers. An automated gel scoring system (GelCompar^TM) was successfully used to analyse the complex AFLP patterns obtained in sugarcane and its relatives. Similarity coefficient calculations and clustering revealed a genetic structure for Saccharum and Erianthus sect. Ripidium that was identical to the one previously obtained using other molecular marker types, showing the appropriateness of AFLP markers and the associated automated analysis in assessing genetic diversity in sugarcane. A genetic structure that correlated with cytotype (2n=30, 60, 90) was revealed within the North American species, E. giganteus (S.giganteum). Complex relationships among Saccharum, Erianthus sect. Ripidium, and North American E.giganteus were revealed and are discussed in the light of a similar study which involved RAPD markers. This revised version was published online in July 2006 with corrections to the Cover Date.     

Molecular characterization and genetic structure of Quercus acutissima germplasm in China using microsatellites

Quercus acutissima is native to eastern Asia. It has a wide distribution in China and China is an important component in understanding the ecology and genetic structure of this species. Q. acutissima attained high economic value for hardwood product and can be managed as an energy tree species. To investigate the genetic variation of Q. acutissima provenances, 12 microsatellite primer pairs were used to analyze 672 trees sampled from 28 provenances of Q. acutissima in China. All of the tested microsatellite loci proved to be effective for the studied Q. acutissima provenances. The results revealed that allele numbers varied from 5 to 13 per locus, with an average of 8 alleles per locus. The mean observed heterozygosity and expected heterozygosity were 0.4927 and 0.7023, respectively. The relatedness of the provenances was studied using the arithmetic mean algorithm based on Nei’s genetic distance and principal coordinates analysis. Interestingly, both approaches revealed two main groups: one consisted of the eastern Chinese provenances, and the other comprised of the western Chinese provenances. An analysis of molecular variance indicated that most genetic variation was contained within populations (84 %). The two microsatellite markers developed in this study may be employed for genetic characterization of other oak species. Considering the management or breeding programs of Q. acutissima provenances in China, we should treat each main group as a single gene resource.     

Molecular characterization of global maize breeding germplasm based on genome-wide single nucleotide polymorphisms

Characterization of genetic diversity is of great value to assist breeders in parental line selection and breeding system design. We screened 770 maize inbred lines with 1,034 single nucleotide polymorphism (SNP) markers and identified 449 high-quality markers with no germplasm-specific biasing effects. Pairwise comparisons across three distinct sets of germplasm, CIMMYT (394), China (282), and Brazil (94), showed that the elite lines from these diverse breeding pools have been developed with only limited utilization of genetic diversity existing in the center of origin. Temperate and tropical/subtropical germplasm clearly clustered into two separate groups. The temperate germplasm could be further divided into six groups consistent with known heterotic patterns. The greatest genetic divergence was observed between temperate and tropical/subtropical lines, followed by the divergence between yellow and white kernel lines, whereas the least divergence was observed between dent and flint lines. Long-term selection for hybrid performance has contributed to significant allele differentiation between heterotic groups at 20% of the SNP loci. There appeared to be substantial levels of genetic variation between different breeding pools as revealed by missing and unique alleles. Two SNPs developed from the same candidate gene were associated with the divergence between two opposite Chinese heterotic groups. Associated allele frequency change at two SNPs and their allele missing in Brazilian germplasm indicated a linkage disequilibrium block of 142 kb. These results confirm the power of SNP markers for diversity analysis and provide a feasible approach to unique allele discovery and use in maize breeding programs.     

Molecular and cellular characterization of an AT-hook protein from Leishmania

AT-rich DNA, and the proteins that bind it (AT-hook proteins), modulate chromosome structure and function in most eukaryotes. Unlike other trypanosomatids, the genome of Leishmania species is unusually GC-rich, and the regulation of Leishmania chromosome structure, replication, partitioning is not fully understood. Because AT-hook proteins modulate these functions in other eukaryotes, we examined whether AT-hook proteins are encoded in the Leishmania genome, to test their potential functions. Several Leishmania ORFs predicted to be AT-hook proteins were identified using in silico approaches based on sequences shared between eukaryotic AT-hook proteins. We have used biochemical, molecular and cellular techniques to characterize the L. amazonensis ortholog of the L. major protein LmjF06.0720, a potential AT-hook protein that is highly conserved in Leishmania species. Using a novel fusion between the AT-hook domain encoded by LmjF06.0720 and a herpesviral protein, we have demonstrated that LmjF06.0720 functions as an AT-hook protein in mammalian cells. Further, as observed for mammalian and viral AT-hook proteins, the AT-hook domains of LmjF06.0720 bind specific regions of condensed mammalian metaphase chromosomes, and support the licensed replication of DNA in mammalian cells. LmjF06.0720 is nuclear in Leishmania, and this localization is disrupted upon exposure to drugs that displace AT-hook proteins from AT-rich DNA. Coincidentally, these drugs dramatically alter the cellular physiology of Leishmania promastigotes. Finally, we have devised a novel peptido-mimetic agent derived from the sequence of LmjF06.0720 that blocks the proliferation of Leishmania promastigotes, and lowers amastigote parasitic burden in infected macrophages. Our results indicate that AT-hook proteins are critical for the normal biology of Leishmania. In addition, we have described a simple technique to examine the function of Leishmania chromatin-binding proteins in a eukaryotic context amenable to studying chromosome structure and function. Lastly, we demonstrate the therapeutic potential of compounds directed against AT-hook proteins in Leishmania.     

Molecular characterization and genetic diversity in an avocado collection of cultivars and local Spanish genotypes using SSRs

In this work, 75 avocado accessions maintained in an ex situ germplasm collection at the E.E. la Mayora in Málaga (Spain) were characterized with 16 microsatellites previously developed in this species. This avocado collection includes both local Spanish genotypes obtained through prospection and genotypes obtained by exchange with different countries. A total of 156 different amplification fragments were detected ranging from 4 to 16 per locus with an average of 9.75 alleles per locus. All the microsatellites were highly informative with an expected heterozygosity higher than 0.5 and a probability of identity below 0.36. The total probability of identity was 2.85x10(-14). Fifteen of the 16 loci studied showed a positive Wright's fixation index (F) indicating a deficit of heterozygotes with an average over all the SSRs of 0.18. A dendrogram was generated using UPGMA (unweighted pair group method with arithmetic averages) based on the Nei and Li similarity index. This dendrogram classified most of the genotypes analyzed into three major groups which mainly differed in racial origin although with low bootstrap support probably due to the presence of many interracial hybrids in the collection. All the genotypes studied could be unequivocally distinguished with the combination of SSRs used except some putative mutations of 'Hass' and an additional group of two cultivars. The results obtained indicate that the set of SSRs used is highly informative and are discussed in terms of their implications for avocado germplasm characterization and management.     

Molecular cloning and characterization of an inorganic pyrophosphatase from barley

A cDNA clone with sequence homology to soluble inorganic pyrophosphatase (IPPase) was isolated from a library of developing barley grains. The protein encoded by this clone was produced in transgenic Escherichia coli, and showed IPPase activity. In nondormant barley grains, the gene appeared to be expressed in metabolically active tissue such as root, shoot, embryo and aleurone. During imbibition, a continuous increase of the steady state mRNA level of IPPase was observed in embryos of non-dormant grains. In the embryos of dormant grains its production declined, after an initial increase. With isolated dormant and nondormant embryos, addition of recombinant IPPase, produced by E. coli, enhanced the germination rate. On the other hand, addition of pyrophosphate (PPi), substrate for this enzyme, appeared to reduce the germination rate. A role for this IPPase in germination is discussed.     

Molecular characterization of an avian astrovirus

Astroviruses are known to cause enteric disease in several animal species, including turkeys. However, only human astroviruses have been well characterized at the nucleotide level. Herein we report the nucleotide sequence, genomic organization, and predicted amino acid sequence of a turkey astrovirus isolated from poults with an emerging enteric disease.     

New evidence of old stone tools from the Mekong terraces,Cambodia

The study of prehistoric sites with lithic remains indicates that the occupation of continental Asia, notably India and China, seems to have taken place earlier than previously thought. However, this Early Pleistocene human dispersal out of Africa remains debatable for the Southeast of Asia, in spite of the discovery of original lithic assemblages on the Mekong terraces dated to the very beginning of the Middle Pleistocene in the centre of Cambodia, by Saurin and Carbonnel in the 1960–1970s. Although this fundamental lithic material has become a reference, it has not been subjected to renewed study of these artefacts over the past decades, and it is thus not possible, for the moment, to attribute it with certainty to a particular culture. In this paper, we present an analysis of the raw materials and a techno-typological study of a similar series of prehistoric tools gathered by one of us in order to bring to light new elements concerning the first Palaeolithic occupation of this region of the world.     

Genetic diversity in a world germplasm collection of tall fescue

Festuca arundinacea Schreb., commonly known as tall fescue, is a major forage crop in temperate regions. Recently, a molecular analysis of different accessions of a world germplasm collection of tall fescue has demonstrated that it contains different species from the genus Festuca and allowed their rapid classification into the three major morphotypes (Continental, Mediterranean and Rhizomatous). In this study, we explored the genetic diversity of 161 accessions of Festuca species from 29 countries, including 28 accessions of INTA (Argentina), by analyzing 15 polymorphic SSR markers by capillary electrophoresis. These molecular markers allowed us to detect a total of 214 alleles. The number of alleles per locus varied between 5 and 24, and the values of polymorphic information content ranged from 0.627 to 0.840. In addition, the accessions analyzed by flow cytometry showed different ploidy levels (diploid, tetraploid, hexaploid and octaploid), placing in evidence that the world germplasm collection consisted of multiple species, as previously suggested. Interestingly, almost all accessions of INTA germplasm collection were true hexaploid tall fescue, belonging to two eco-geographic races (Continental and Mediterranean). Finally, the data presented revealed an ample genetic diversity of tall fescue showing the importance of preserving the INTA collection for future breeding programs.     

Molecular and cytological characterization of the global <Emphasis Type=Italic>Musa</Emphasis> germplasm collection provides insights into the treasure of banana diversity

Bananas (Musa spp.) are one of the main fruit crops grown worldwide. With the annual production reaching 144 million tons, their production represents an important contribution to the economies of many countries in Asia, Africa, Latin-America and Pacific Islands. Most importantly, bananas are a staple food for millions of people living in the tropics. Unfortunately, sustainable banana production is endangered by various diseases and pests, and the breeding for resistant cultivars relies on a far too small base of genetic variation. Greater diversity needs to be incorporated in breeding, especially of wild species. Such work requires a large and thoroughly characterized germplasm collection, which also is a safe depository of genetic diversity. The largest ex situ Musa germplasm collection is kept at the International Transit Centre (ITC) in Leuven (Belgium) and currently comprises over 1500 accessions. This report summarizes the results of systematic cytological and molecular characterization of the Musa ITC collection. By December 2015, 630 accessions have been genotyped. The SSR markers confirmed the previous morphological based classification for 84% of ITC accessions analyzed. The remaining 16% of the genotyped entries may need field verification by taxonomist to decide if the unexpected classification by SSR genotyping was correct. The ploidy level estimation complements the molecular data. The genotyping continues for the entire ITC collection, including newly introduced accessions, to assure that the genotype of each accession is known in the largest global Musa gene bank.