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1.  Using genetic diversity information to establish core collections of Stylosanthes capitata and Stylosanthes macrocephala 
Genetics and Molecular Biology  2012;35(4):847-861.
Stylosanthes species are important forage legumes in tropical and subtropical areas. S. macrocephala and S. capitata germplasm collections that consist of 134 and 192 accessions, respectively, are maintained at the Brazilian Agricultural Research Corporation Cerrados (Embrapa-Cerrados). Polymorphic microsatellite markers were used to assess genetic diversity and population structure with the aim to assemble a core collection. The mean values of HO and HE for S. macrocephala were 0.08 and 0.36, respectively, whereas the means for S. capitata were 0.48 and 0.50, respectively. Roger’s genetic distance varied from 0 to 0.83 for S. macrocephala and from 0 to 0.85 for S. capitata. Analysis with STRUCTURE software distinguished five groups among the S. macrocephala accessions and four groups among those of S. capitata. Nei’s genetic diversity was 27% in S. macrocephala and 11% in S. capitata. Core collections were assembled for both species. For S. macrocephala, all of the allelic diversity was represented by 23 accessions, whereas only 13 accessions were necessary to represent all allelic diversity for S. capitata. The data presented herein evidence the population structure present in the Embrapa-Cerrados germplasm collections of S. macrocephala and S. capitata, which may be useful for breeding programs and germplasm conservation.
doi:10.1590/S1415-47572012005000076
PMCID: PMC3526094  PMID: 23271947
Stylosanthes; tropical forage; microsatellites; genetic diversity; core collection
2.  Genetic variation in polyploid forage grass: Assessing the molecular genetic variability in the Paspalum genus 
BMC Genetics  2013;14:50.
Background
Paspalum (Poaceae) is an important genus of the tribe Paniceae, which includes several species of economic importance for foraging, turf and ornamental purposes, and has a complex taxonomical classification. Because of the widespread interest in several species of this genus, many accessions have been conserved in germplasm banks and distributed throughout various countries around the world, mainly for the purposes of cultivar development and cytogenetic studies. Correct identification of germplasms and quantification of their variability are necessary for the proper development of conservation and breeding programs. Evaluation of microsatellite markers in different species of Paspalum conserved in a germplasm bank allowed assessment of the genetic differences among them and assisted in their proper botanical classification.
Results
Seventeen new polymorphic microsatellites were developed for Paspalum atratum Swallen and Paspalum notatum Flüggé, twelve of which were transferred to 35 Paspalum species and used to evaluate their variability. Variable degrees of polymorphism were observed within the species. Based on distance-based methods and a Bayesian clustering approach, the accessions were divided into three main species groups, two of which corresponded to the previously described Plicatula and Notata Paspalum groups. In more accurate analyses of P. notatum accessions, the genetic variation that was evaluated used thirty simple sequence repeat (SSR) loci and revealed seven distinct genetic groups and a correspondence of these groups to the three botanical varieties of the species (P. notatum var. notatum, P. notatum var. saurae and P. notatum var. latiflorum).
Conclusions
The molecular genetic approach employed in this study was able to distinguish many of the different taxa examined, except for species that belong to the Plicatula group, which has historically been recognized as a highly complex group. Our molecular genetic approach represents a valuable tool for species identification in the initial assessment of germplasm as well as for characterization, conservation and successful species hybridization.
doi:10.1186/1471-2156-14-50
PMCID: PMC3682885  PMID: 23759066
Cross-species amplification; Genetic diversity; Germplasm evaluation; Microsatellite markers; Paspalum botanical varieties
3.  High-throughput transcriptome sequencing and preliminary functional analysis in four Neotropical tree species 
BMC Genomics  2014;15:238.
Background
The Amazonian rainforest is predicted to suffer from ongoing environmental changes. Despite the need to evaluate the impact of such changes on tree genetic diversity, we almost entirely lack genomic resources.
Results
In this study, we analysed the transcriptome of four tropical tree species (Carapa guianensis, Eperua falcata, Symphonia globulifera and Virola michelii) with contrasting ecological features, belonging to four widespread botanical families (respectively Meliaceae, Fabaceae, Clusiaceae and Myristicaceae). We sequenced cDNA libraries from three organs (leaves, stems, and roots) using 454 pyrosequencing. We have developed an R and bioperl-based bioinformatic procedure for de novo assembly, gene functional annotation and marker discovery. Mismatch identification takes into account single-base quality values as well as the likelihood of false variants as a function of contig depth and number of sequenced chromosomes. Between 17103 (for Symphonia globulifera) and 23390 (for Eperua falcata) contigs were assembled. Organs varied in the numbers of unigenes they apparently express, with higher number in roots. Patterns of gene expression were similar across species, with metabolism of aromatic compounds standing out as an overrepresented gene function. Transcripts corresponding to several gene functions were found to be over- or underrepresented in each organ. We identified between 4434 (for Symphonia globulifera) and 9076 (for Virola surinamensis) well-supported mismatches. The resulting overall mismatch density was comprised between 0.89 (S. globulifera) and 1.05 (V. surinamensis) mismatches/100 bp in variation-containing contigs.
Conclusion
The relative representation of gene functions in the four transcriptomes suggests that secondary metabolism may be particularly important in tropical trees. The differential representation of transcripts among tissues suggests differential gene expression, which opens the way to functional studies in these non-model, ecologically important species. We found substantial amounts of mismatches in the four species. These newly identified putative variants are a first step towards acquiring much needed genomic resources for tropical tree species.
doi:10.1186/1471-2164-15-238
PMCID: PMC3986928  PMID: 24673733
454-Pyrosequencing; Tropical rainforest tree species; Polymorphism discovery
4.  Behaviour of Sotalia guianensis (van Bénéden, 1864) (Cetacea, Delphinidae) and ethnoecological knowledge of artisanal fishermen from Canavieiras, Bahia, Brazil 
Artisanal fishermen, because of their direct and frequent contact with the aquatic environment, possess a wealth of knowledge about the natural history of the fauna of the region in which they live. This knowledge, both practical and theoretical, has been frequently utilized and integrated into academic research. Taking this into consideration, this study discusses the ethnoecological knowledge of artisanal fishermen from a community in Canavieiras, state of Bahia, Brazil regarding the Guiana dolphin (Sotalia guianensis), a typically costal member of the family Delphinidae that is little studied in this region. To this end, the behaviour of S. guianensis in Canavieiras was recorded over one year and the data obtained were compared with fishermen’s reports. A total of 609 hours of behavioural observations of S. guianensis was conducted from a fixed point in alternate morning and afternoon sessions between October 2009 and September 2010. Observations were conducted from a pier (15°40’59”S and 38°56’38”W) situated on the banks of the Pardo River estuary - the region’s main river - at 5.5 m above water level. For ethnoecological data collection, semi-structured interviews were carried out with 26 fishermen in May, June and September 2010 and January 2011 in the fishing community of Atalaia. Occasional boat expeditions were made with the fishermen to compare their reports with direct observations of the behaviour of S. guianensis. The results demonstrate that fishermen possess a body of knowledge about S. guianensis that describes in detail the main behavioural aspects of the species. They reported the presence of S. guianensis in the Pardo River estuary throughout the year and its gregarious behaviour. They cited a relationship between the movement of dolphins and tidal cycles, and their presence in the estuary associated with the search for food. In addition, the fishermen reported that numbers of infants in groups were proportional to group size. Behaviours described were compatible with the observations made in situ and with data found in the scientific literature, confirming the importance of traditional knowledge in complementing scientific data. One behaviour mentioned by the fishermen that had no equivalence in the scientific literature was confirmed in situ and, therefore, constitutes the first record for this species.
doi:10.1186/1746-4269-8-18
PMCID: PMC3480909  PMID: 22584063
Guiana dolphin; Behaviour patterns; Ethnobiology; Traditional knowledge
5.  European Phaseolus coccineus L. landraces: Population Structure and Adaptation, as Revealed by cpSSRs and Phenotypic Analyses 
PLoS ONE  2013;8(2):e57337.
Relatively few studies have extensively analysed the genetic diversity of the runner bean through molecular markers. Here, we used six chloroplast microsatellites (cpSSRs) to investigate the cytoplasmic diversity of 331 European domesticated accessions of the scarlet runner bean (Phaseolus coccineus L.), including the botanical varieties albiflorus, bicolor and coccineus, and a sample of 49 domesticated and wild accessions from Mesoamerica. We further explored the pattern of diversity of the European landraces using 12 phenotypic traits on 262 individuals. For 158 European accessions, we studied the relationships between cpSSR polymorphisms and phenotypic traits. Additionally, to gain insights into the role of gene flow and migration, for a subset of 115 accessions, we compared and contrasted the results obtained by cpSSRs and phenotypic traits with those obtained in a previous study with 12 nuclear microsatellites (nuSSRs). Our results suggest that both demographic and selective factors have roles in the shaping of the population genetic structure of the European runner bean. In particular, we infer the existence of a moderate-to-strong cytoplasmic bottleneck that followed the expansion of the crop into Europe, and we deduce multiple domestication events for this species. We also observe an adaptive population differentiation in the phenology across a latitudinal gradient, which suggests that selection led to the diversification of the runner bean in Europe. The botanical varieties albiflorus, bicolor and coccineus, which are based solely on flower colour, cannot be distinguished based on these cpSSRs and nuSSRs, nor according to the 12 quantitative traits.
doi:10.1371/journal.pone.0057337
PMCID: PMC3579852  PMID: 23451209
6.  Genetic diversity of peanut (Arachis hypogaea L.) and its wild relatives based on the analysis of hypervariable regions of the genome 
BMC Plant Biology  2004;4:11.
Background
The genus Arachis is native to a region that includes Central Brazil and neighboring countries. Little is known about the genetic variability of the Brazilian cultivated peanut (Arachis hypogaea, genome AABB) germplasm collection at the DNA level. The understanding of the genetic diversity of cultivated and wild species of peanut (Arachis spp.) is essential to develop strategies of collection, conservation and use of the germplasm in variety development. The identity of the ancestor progenitor species of cultivated peanut has also been of great interest. Several species have been suggested as putative AA and BB genome donors to allotetraploid A. hypogaea. Microsatellite or SSR (Simple Sequence Repeat) markers are co-dominant, multiallelic, and highly polymorphic genetic markers, appropriate for genetic diversity studies. Microsatellite markers may also, to some extent, support phylogenetic inferences. Here we report the use of a set of microsatellite markers, including newly developed ones, for phylogenetic inferences and the analysis of genetic variation of accessions of A. hypogea and its wild relatives.
Results
A total of 67 new microsatellite markers (mainly TTG motif) were developed for Arachis. Only three of these markers, however, were polymorphic in cultivated peanut. These three new markers plus five other markers characterized previously were evaluated for number of alleles per locus and gene diversity using 60 accessions of A. hypogaea. Genetic relationships among these 60 accessions and a sample of 36 wild accessions representative of section Arachis were estimated using allelic variation observed in a selected set of 12 SSR markers. Results showed that the Brazilian peanut germplasm collection has considerable levels of genetic diversity detected by SSR markers. Similarity groups for A. hypogaea accessions were established, which is a useful criteria for selecting parental plants for crop improvement. Microsatellite marker transferability was up to 76% for species of the section Arachis, but only 45% for species from the other eight Arachis sections tested. A new marker (Ah-041) presented a 100% transferability and could be used to classify the peanut accessions in AA and non-AA genome carriers.
Conclusion
The level of polymorphism observed among accessions of A. hypogaea analyzed with newly developed microsatellite markers was low, corroborating the accumulated data which show that cultivated peanut presents a relatively reduced variation at the DNA level. A selected panel of SSR markers allowed the classification of A. hypogaea accessions into two major groups. The identification of similarity groups will be useful for the selection of parental plants to be used in breeding programs. Marker transferability is relatively high between accessions of section Arachis. The possibility of using microsatellite markers developed for one species in genetic evaluation of other species greatly reduces the cost of the analysis, since the development of microsatellite markers is still expensive and time consuming. The SSR markers developed in this study could be very useful for genetic analysis of wild species of Arachis, including comparative genome mapping, population genetic structure and phylogenetic inferences among species.
doi:10.1186/1471-2229-4-11
PMCID: PMC491793  PMID: 15253775
7.  Genetic structure and differentiation in cultivated fig (Ficus carica L.) 
Genetica  2010;138(6):681-694.
One hundred ninety-four germplasm accessions of fig representing the four fig types, Common, Smyrna, San Pedro, and Caprifig were analyzed for genetic diversity, structure, and differentiation using genetic polymorphism at 15 microsatellite loci. The collection showed considerable polymorphism with observed number of alleles per locus ranging from four for five different loci, MFC4, LMFC14, LMFC22, LMFC31 and LMFC35 to nine for LMFC30 with an average of 4.9 alleles per locus. Seven of the 15 loci included in the genetic structure analyses exhibited significant deviation from panmixia, of which two showed excess and five showed deficiency of heterozygote. The cluster analysis (CA) revealed ten groups with 32 instances of synonymy among cultivars and groups differed significantly for frequency and composition of alleles for different loci. The principal components analysis (PCA) confirmed the results of CA with some groups more differentiated than the others. Further, the model based Bayesian approach clustering suggested a subtle population structure with mixed ancestry for most figs. The gene diversity analysis indicated that much of the total variation is found within groups (HG/HT = 0.853; 85.3%) and the among groups within total component (GGT = 0.147) accounted for the remaining 14.7%, of which ~64% accounted for among groups within clusters (GGC = 0.094) and ~36% among clusters (GCT = 0.053). The analysis of molecular variance (AMOVA) showed approximately similar results with nearly 87% of variation within groups and ~10% among groups within clusters, and ~3% among clusters. Overall, the gene pool of cultivated fig analyzed possesses substantial genetic polymorphism but exhibits narrow differentiation. It is evident that fig accessions from Turkmenistan are somewhat genetically different from the rest of the Mediterranean and the Caucasus figs. The long history of domestication and cultivation with widespread dispersal of cultivars with many synonyms has resulted in a great deal of confusion in the identification and classification of cultivars in fig.
doi:10.1007/s10709-010-9442-3
PMCID: PMC2860561  PMID: 20217187
Ficus carica; Genetic polymorphism; Microsatellite; Genetic diversity; Population structure
8.  Development of microsatellite markers from an enriched genomic library for genetic analysis of melon (Cucumis melo L.) 
BMC Plant Biology  2004;4:9.
Background
Despite the great advances in genomic technology observed in several crop species, the availability of molecular tools such as microsatellite markers has been limited in melon (Cucumis melo L.) and cucurbit species. The development of microsatellite markers will have a major impact on genetic analysis and breeding of melon, especially on the generation of marker saturated genetic maps and implementation of marker assisted breeding programs. Genomic microsatellite enriched libraries can be an efficient alternative for marker development in such species.
Results
Seven hundred clones containing microsatellite sequences from a Tsp-AG/TC microsatellite enriched library were identified and one-hundred and forty-four primer pairs designed and synthesized. When 67 microsatellite markers were tested on a panel of melon and other cucurbit accessions, 65 revealed DNA polymorphisms among the melon accessions. For some cucurbit species, such as Cucumis sativus, up to 50% of the melon microsatellite markers could be readily used for DNA polymophism assessment, representing a significant reduction of marker development costs. A random sample of 25 microsatellite markers was extracted from the new microsatellite marker set and characterized on 40 accessions of melon, generating an allelic frequency database for the species. The average expected heterozygosity was 0.52, varying from 0.45 to 0.70, indicating that a small set of selected markers should be sufficient to solve questions regarding genotype identity and variety protection. Genetic distances based on microsatellite polymorphism were congruent with data obtained from RAPD marker analysis. Mapping analysis was initiated with 55 newly developed markers and most primers showed segregation according to Mendelian expectations. Linkage analysis detected linkage between 56% of the markers, distributed in nine linkage groups.
Conclusions
Genomic library microsatellite enrichment is an efficient procedure for marker development in melon. One-hundred and forty-four new markers were developed from Tsp-AG/TC genomic library. This is the first reported attempt of successfully using enriched library for microsatellite marker development in the species. A sample of the microsatellite markers tested proved efficient for genetic analysis of melon, including genetic distance estimates and identity tests. Linkage analysis indicated that the markers developed are dispersed throughout the genome and should be very useful for genetic analysis of melon.
doi:10.1186/1471-2229-4-9
PMCID: PMC419974  PMID: 15149552
9.  Analysis of molecular genetic diversity in a representative collection of foxtail millet [Setaria italica (L.) P. Beauv.] from different agro-ecological regions of India 
Foxtail millet [Setaria italica (L.) P. Beauv.], an important crop of East Asia is known for its drought tolerance and was once an indispensible crop of vast rainfed areas in semi-arid regions in India. In India it is cultivated in Andhra Pradesh, Karnataka, Maharashtra, Tamil Nadu, Rajasthan, Madhya Pradesh, Uttar Pradesh and north eastern states. The grain finds use in several local recipes such as roti (bread), jaula, singal, sirol. Foxtail millet grain contains 12.3 % protein, 4.7 % fat, 60.6 % carbohydrates, and 3.2 % ash. The present study was conducted to analyse the genetic diversity among foxtail accessions from different states of India and a few exotic accessions using RAPD and ISSR techniques and identify diverse accessions for use in variety improvement programmes. A set of 125 foxtail millet accessions selected from 11 different agro-ecological regions of India were analyzed using random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) marker techniques. A total of 146 (115 RAPD and 31 ISSR) scoreable markers were generated with 16 RAPD and four ISSR primers. The dendrogram generated using Nei’s genetic distances and principal component analyses revealed presence of two clusters and two subclusters in group I. The accessions from Andhra Pradesh, Karnataka, Maharashtra and Uttarakhand were more diverse since they were distributed in both the clusters. There was no clear geographical differentiation observable. The bootstrap support for the major groups identified was strong (above 80 %) indicating good statistical support. The average value of Nei and Li’s genetic distance was lowest (0.081) for accessions from West Bengal while the collections from Karnataka showed highest dissimilarity (average genetic distance = 0.239). The average genetic distance for all 125 accessions together was 0.177 indicating presence of only moderate genetic diversity in the collections. The analysis of molecular variance indicated that only 2.76 % variation was explained by variations among the groups and 11.55 % among populations within groups. However the percentage of variation observed within populations was high (85.68). The value of Fst was observed to be very low (0.028) indicating low differentiation of the accessions analysed. The population genetic analysis carried out indicates that highest number of alleles per locus (1.745 ± 0.438) was observed for Andhra Pradesh with 35 accessions. When four eco-geographic regions were considered, the southern region comprising AP, Karnataka and TN showed the highest number of alleles per locus (1.787 ± 0.411). The value of Gst was lowest for south (0.123) and highest for central west (0.455). This indicated that all the landraces from south share common alleles. The gene flow between the accessions from different regions was also observed to be high with the highest migration (3.557) recorded for south.
doi:10.1007/s12298-011-0085-3
PMCID: PMC3550584  PMID: 23573030
Foxtail; Genetic diversity; RAPD; ISSR markers; Spatial distribution
10.  Molecular evaluation of orphan Afghan common wheat (Triticum aestivum L.) landraces collected by Dr. Kihara using single nucleotide polymorphic markers 
BMC Plant Biology  2014;14(1):320.
Background
Landraces are an important source of genetic diversity in common wheat, but archival collections of Afghan wheat landraces remain poorly characterised. The recent development of array based marker systems, particularly single nucleotide polymorphism (SNP) markers, provide an excellent tool for examining the genetic diversity of local populations. Here we used SNP analysis to demonstrate the importance of Afghan wheat landraces and found tremendous genetic diversity and province-specific characteristics unique to this geographic region.
Results
A total of 446 Afghan wheat landraces were analysed using genotype by sequencing (GBS) arrays containing ~10 K unique markers. Pair-wise genetic distance analyses revealed significant genetic distances between landraces, particularly among those collected from distanced provinces. From these analyses, we were able to divide the landraces into 14 major classes, with the greatest degree of diversity evident among landraces isolated from Badakhshan province. Population-based analyses revealed an additional 15 sub-populations within our germplasm, and significant correlations were evident in both the provincial and botanical varieties. Genetic distance analysis was used to identify differences among provinces, with the strongest correlations seen between landraces from Herat and Ghor province, followed closely by those between Badakhshan and Takhar provinces. This result closely resembles existing agro-climatic zones within Afghanistan, as well as the wheat varieties commonly cultivated within these regions. Molecular variance analysis showed a higher proportion of intra-province variation among landraces compared with variation among all landraces as a whole.
Conclusion
The SNP analyses presented here highlight the importance and genetic diversity of Afghan wheat landraces. Furthermore, these data strongly refute a previous analysis that suggested low genetic diverse within this germplasm. Ongoing analyses include phenotypic characterisation of these landraces to identify functional traits associated with individual genotypes. Taken together, these analyses can be used to help improve wheat cultivation in Afghanistan, while providing insights into the evolution and selective pressures underlying these distinct landraces.
Electronic supplementary material
The online version of this article (doi:10.1186/s12870-014-0320-5) contains supplementary material, which is available to authorized users.
doi:10.1186/s12870-014-0320-5
PMCID: PMC4255927  PMID: 25432399
Afghan wheat landraces; Botanical varieties; Genetic diversity; Population structure; Single nucleotide polymorphism
11.  Antimicrobial activity of amazonian medicinal plants 
SpringerPlus  2013;2:371.
Objectives
The aqueous extracts of currently utilized Amazonian medicinal plants were assayed in vitro searching for antimicrobial activity against human and animal pathogenic microorganisms.
Methods
Medium resuspended lyophilized aqueous extracts of different organs of Amazonian medicinal plants were assayed by in vitro screening for antimicrobial activity. ATCC and standardized microorganisms obtained from Oswaldo Cruz Foundation/Brazil were individually and homogeneously grown in agar plate, and holes previously perforated in the gel were filled with diluted plant aqueous extracts. Inhibition halos were evaluated and controlled by the use of the fluoroquinolone ciprofloxacin.
Results
The Amazonian medicinal plants, Hymenelobium petraeum showed inhibitory activity over Staphylococcus aureus, Enterococcus faecalis, Salmonella enterica serovar Typhi, Acinetobacter baumannii and Candida albicans, while Vatairea guianensis and Symphonia globulifera presented inhibitory activity exclusively for Staphylococcus aureus. Also, Ptychopetalum olacoides and Pentaclethra macroloba inhibited the growth of Klebsiella ozaenae and Acinetobacter baumannii.
Conclusion
The aqueous botanic extracts that showed activity against microroganisms of ATTC and Osvaldo Cruz strains had at least 40% of antimicrobial activity when compared to halo inhibition produced by the commercial antibiotic ciprofloxacin utilized as a control. Of all plants extracts assayed, the Hymenelobium petraeum had the best performance, sometimes exhibiting higher activity than ciprofloxacin. It is not well-defined by the physicians the exact indication of the majority of medicinal plants in the Amazon area in Brazil. Natives utilize the plants according to their symptoms, based on the traditional knowledge transmitted orally from generation to generation, among Amerindians, Afrodescendents and ethnic mixed populations. A significant number of Amazonian medicinal plants are totally unknown related to their medicinal properties including mechanism of action and therapeutic effects, as very few information is reported in the scientific literature. A tiny amount of data is presented, as the preliminary antimicrobial properties of the medicinal plants here accessed, under the urgent necessity of new antibiotics in the market and in face of the increased resistance of infectious microorganisms to antimicrobials.
doi:10.1186/2193-1801-2-371
PMCID: PMC3738913  PMID: 23961431
Antimicrobials; Environment; Medicinal plants; Metabolites; Amazon
12.  Comparison of glucosamine sulfate and a polyherbal supplement for the relief of osteoarthritis of the knee: a randomized controlled trial [ISRCTN25438351] 
Background
The efficacy and safety of a dietary supplement derived from South American botanicals was compared to glucosamine sulfate in osteoarthritis subjects in a Mumbai-based multi-center, randomized, double-blind study.
Methods
Subjects (n = 95) were screened and randomized to receive glucosamine sulfate (n = 47, 1500 mg/day) or reparagen (n = 48, 1800 mg/day), a polyherbal consisting of 300 mg of vincaria (Uncaria guianensis) and 1500 mg of RNI 249 (Lepidium meyenii) administered orally, twice daily. Primary efficacy variable was response rate based on a 20% improvement in WOMAC pain scores. Additional outcomes were WOMAC scores for pain, stiffness and function, visual analog score (VAS) for pain, with assessments at 1, 2, 4, 6 and 8 weeks. Tolerability, investigator and subject global assessments and rescue medication consumption (paracetamol) were measured together with safety assessments including vital signs and laboratory based assays.
Results
Subject randomization was effective: age, gender and disease status distribution was similar in both groups. The response rates (20% reduction in WOMAC pain) were substantial for both glucosamine (89%) and reparagen (94%) and supported by investigator and subject assessments. Using related criteria response rates to reparagen were favorable when compared to glucosamine. Compared to baseline both treatments showed significant benefits in WOMAC and VAS outcomes within one week (P < 0.05), with a similar, progressive improvement over the course of the 8 week treatment protocol (45–62% reduction in WOMAC or VAS scores). Tolerability was excellent, no serious adverse events were noted and safety parameters were unchanged. Rescue medication use was significantly lower in the reparagen group (p < 0.01) at each assessment period. Serum IGF-1 levels were unaltered by treatments.
Conclusion
Both reparagen and glucosamine sulfate produced substantial improvements in pain, stiffness and function in subjects with osteoarthritis. Response rates were high and the safety profile was excellent, with significantly less rescue medication use with reparagen. Reparagen represents a new natural productive alternative in the management of joint health.
Trial registration
Current Controlled Trials ISRCTN25438351.
doi:10.1186/1472-6882-7-34
PMCID: PMC2131759  PMID: 17974032
13.  Genetic Diversity and Population Structure of Miscanthus sinensis Germplasm in China 
PLoS ONE  2013;8(10):e75672.
Miscanthus is a perennial rhizomatous C4 grass native to East Asia. Endowed with great biomass yield, high ligno-cellulose composition, efficient use of radiation, nutrient and water, as well as tolerance to stress, Miscanthus has great potential as an excellent bioenergy crop. Despite of the high potential for biomass production of the allotriploid hybrid M. ×giganteus, derived from M. sacchariflorus and M. sinensis, other options need to be explored to improve the narrow genetic base of M. ×giganteus, and also to exploit other Miscanthus species, including M. sinensis (2n = 2x = 38), as bioenergy crops. In the present study, a large number of 459 M. sinensis accessions, collected from the wide geographical distribution regions in China, were genotyped using 23 SSR markers transferable from Brachypodium distachyon. Genetic diversity and population structure were assessed. High genetic diversity and differentiation of the germplasm were observed, with 115 alleles in total, a polymorphic rate of 0.77, Nei’s genetic diversity index (He) of 0.32 and polymorphism information content (PIC) of 0.26. Clustering of germplasm accessions was primarily in agreement with the natural geographic distribution. AMOVA and genetic distance analyses confirmed the genetic differentiation in the M. sinensis germplasm and it was grouped into five clusters or subpopulations. Significant genetic variation among subpopulations indicated obvious genetic differentiation in the collections, but within-subpopulation variation (83%) was substantially greater than the between-subpopulation variation (17%). Considerable phenotypic variation was observed for multiple traits among 300 M. sinensis accessions. Nine SSR markers were found to be associated with heading date and biomass yield. The diverse Chinese M. sinensis germplasm and newly identified SSR markers were proved to be valuable for breeding Miscanthus varieties with desired bioenergy traits.
doi:10.1371/journal.pone.0075672
PMCID: PMC3792140  PMID: 24116066
14.  Carcass Characteristics and Meat Quality of Swamp Buffaloes (Bubalus bubalis) Fattened at Different Feeding Intensities 
Twenty-four male 1-year old swamp buffaloes (Bubalus bubalis) were randomly allocated to 4 groups. One group grazed on guinea grass (GG) and another on guinea grass and the legume Stylosanthes guianensis (GL). The other two groups were kept in pens and fed freshly cut guinea grass and concentrate at an amount of 1.5% (GC1.5) and 2.0% (GC2.0) of body weight, respectively. The effect of the different feeding intensities on carcass characteristics and meat quality were assessed. The mean body weight at slaughter was 398 (±16) kg. Average daily gain was higher in concentrate-supplemented groups (570 and 540 g/d in GC1.5 and GC2.0, respectively) when compared to GG (316 g/d) and GL (354 g/d) (p<0.01). Likewise, the warm carcass weight was higher in GC1.5 and GC2.0 compared to GG and GL. Dressing percentage was 48.1% and 49.5% in GC1.5 and GC2.0 in comparison to 42.9% and 44.8% observed in GG and GL, respectively. Meat of Longissimus throracis from GC1.5 and GC2.0 was redder in color (p<0.01), while water holding capacity (drip and thawing loss) was improved in pasture-fed groups (p<0.05). Protein and fat content of Longissimus thoracis was higher in animals supplemented with concentrate (p<0.01), as was cholesterol content (p<0.05), whereas PUFA:SFA ratio was higher and n-6/n-3 ratio lower (p<0.01) in pasture-fed buffaloes. Results of the present study showed that the supplementation of pasture with concentrate enhances the growth and carcass characteristics of swamp buffaloes expressed in superior dressing percentage, better muscling, and redder meat with a higher content of protein and fat, whereas animals grazing only on pasture had a more favorable fatty acid profile and water holding capacity. In conclusion, the supplementation of concentrate at a rate of about 1.5% of body weight is recommended to improve the performance and carcass quality of buffaloes.
doi:10.5713/ajas.2013.13555
PMCID: PMC4093524  PMID: 25049987
Swamp Buffaloes; Guinea Grass; Stylosanthes guianensis; Meat Quality; Carcass Composition; Fatty Acids
15.  Genetic Biodiversity of Italian Olives (Olea europaea) Germplasm Analyzed by SSR Markers 
The Scientific World Journal  2014;2014:296590.
The olive is an important fruit species cultivated for oil and table olives in Italy and the Mediterranean basin. The conservation of cultivated plants in ex situ collections is essential for the optimal management and use of their genetic resources. The largest ex situ olive germplasm collection consists of approximately 500 Italian olive varieties and corresponding to 85% of the total Italian olive germplasm is maintained at the Consiglio per la Ricerca e sperimentazione per l'Agricoltura, Centro di Ricerca per l'Olivicoltura e l'Industria Olearia (CRA-OLI), in Italy. In this work, eleven preselected nuclear microsatellite markers were used to assess genetic diversity, population structure, and gene flows with the aim of assembling a core collection. The dendrogram obtained utilizing the unweighted pair group method highlights the presence of homonymy and synonymy in olive tree datasets analyzed in this study. 439 different unique genotype profiles were obtained with this combination of 11 loci nSSR, representing 89.8% of the varieties analyzed. The remaining 10.2% comprises different variety pairs in which both accessions are genetically indistinguishable. Clustering analysis performed using BAPS software detected seven groups in Italian olive germplasm and gene flows were determined among identified clusters. We proposed an Italian core collection of 23 olive varieties capturing all detected alleles at microsatellites. The information collected in this study regarding the CRA-OLI ex situ collection can be used for breeding programs, for germplasm conservation, and for optimizing a strategy for the management of olive gene pools.
doi:10.1155/2014/296590
PMCID: PMC3958686  PMID: 24723801
16.  Next generation DNA sequencing technology delivers valuable genetic markers for the genomic orphan legume species, Bituminaria bituminosa 
BMC Genetics  2011;12:104.
Background
Bituminaria bituminosa is a perennial legume species from the Canary Islands and Mediterranean region that has potential as a drought-tolerant pasture species and as a source of pharmaceutical compounds. Three botanical varieties have previously been identified in this species: albomarginata, bituminosa and crassiuscula. B. bituminosa can be considered a genomic 'orphan' species with very few genomic resources available. New DNA sequencing technologies provide an opportunity to develop high quality molecular markers for such orphan species.
Results
432,306 mRNA molecules were sampled from a leaf transcriptome of a single B. bituminosa plant using Roche 454 pyrosequencing, resulting in an average read length of 345 bp (149.1 Mbp in total). Sequences were assembled into 3,838 isotigs/contigs representing putatively unique gene transcripts. Gene ontology descriptors were identified for 3,419 sequences. Raw sequence reads containing simple sequence repeat (SSR) motifs were identified, and 240 primer pairs flanking these motifs were designed. Of 87 primer pairs developed this way, 75 (86.2%) successfully amplified primarily single fragments by PCR. Fragment analysis using 20 primer pairs in 79 accessions of B. bituminosa detected 130 alleles at 21 SSR loci. Genetic diversity analyses confirmed that variation at these SSR loci accurately reflected known taxonomic relationships in original collections of B. bituminosa and provided additional evidence that a division of the botanical variety bituminosa into two according to geographical origin (Mediterranean region and Canary Islands) may be appropriate. Evidence of cross-pollination was also found between botanical varieties within a B. bituminosa breeding programme.
Conclusions
B. bituminosa can no longer be considered a genomic orphan species, having now a large (albeit incomplete) repertoire of expressed gene sequences that can serve as a resource for future genetic studies. This experimental approach was effective in developing codominant and polymorphic SSR markers for application in diverse genetic studies. These markers have already given new insight into genetic variation in B. bituminosa, providing evidence that a division of the botanical variety bituminosa may be appropriate. This approach is commended to those seeking to develop useful markers for genomic orphan species.
doi:10.1186/1471-2156-12-104
PMCID: PMC3265443  PMID: 22171578
17.  Stem and Root Nodulation in Aeschynomene spp 
Nodulation ability of 15 Rhizobium strains isolated from root and stem nodules of tropical Aeschynomene species was studied on 20 different Aeschynomene species and four other legumes—Arachis hypogaea, Stylosanthes guianensis, Macroptilium atropurpureum, and Sesbania rostrata. The results of this investigation showed that Aeschynomene species could be divided into three groups according to the cross-inoculation group concept.
PMCID: PMC238704  PMID: 16346895
18.  Genetic Characterization of a Core Set of a Tropical Maize Race Tuxpeño for Further Use in Maize Improvement 
PLoS ONE  2012;7(3):e32626.
The tropical maize race Tuxpeño is a well-known race of Mexican dent germplasm which has greatly contributed to the development of tropical and subtropical maize gene pools. In order to investigate how it could be exploited in future maize improvement, a panel of maize germplasm accessions was assembled and characterized using genome-wide Single Nucleotide Polymorphism (SNP) markers. This panel included 321 core accessions of Tuxpeño race from the International Maize and Wheat Improvement Center (CIMMYT) germplasm bank collection, 94 CIMMYT maize lines (CMLs) and 54 U.S. Germplasm Enhancement of Maize (GEM) lines. The panel also included other diverse sources of reference germplasm: 14 U.S. maize landrace accessions, 4 temperate inbred lines from the U.S. and China, and 11 CIMMYT populations (a total of 498 entries with 795 plants). Clustering analyses (CA) based on Modified Rogers Distance (MRD) clearly partitioned all 498 entries into their corresponding groups. No sub clusters were observed within the Tuxpeño core set. Various breeding strategies for using the Tuxpeño core set, based on grouping of the studied germplasm and genetic distance among them, were discussed. In order to facilitate sampling diversity within the Tuxpeño core, a minicore subset of 64 Tuxpeño accessions (20% of its usual size) representing the diversity of the core set was developed, using an approach combining phenotypic and molecular data. Untapped diversity represents further use of the Tuxpeño landrace for maize improvement through the core and/or minicore subset available to the maize community.
doi:10.1371/journal.pone.0032626
PMCID: PMC3296726  PMID: 22412898
19.  Genetic diversity in Capsicum baccatum is significantly influenced by its ecogeographical distribution 
BMC Genetics  2012;13:68.
Background
The exotic pepper species Capsicum baccatum, also known as the aji or Peruvian hot pepper, is comprised of wild and domesticated botanical forms. The species is a valuable source of new genes useful for improving fruit quality and disease resistance in C. annuum sweet bell and hot chile pepper. However, relatively little research has been conducted to characterize the species, thus limiting its utilization. The structure of genetic diversity in a plant germplasm collection is significantly influenced by its ecogeographical distribution. Together with DNA fingerprints derived from AFLP markers, we evaluated variation in fruit and plant morphology of plants collected across the species native range in South America and evaluated these characters in combination with the unique geography, climate and ecology at different sites where plants originated.
Results
The present study mapped the ecogeographic distribution, analyzed the spatial genetic structure, and assessed the relationship between the spatial genetic pattern and the variation of morphological traits in a diverse C. baccatum germplasm collection spanning the species distribution. A combined diversity analysis was carried out on the USDA-ARS C. baccatum germplasm collection using data from GIS, morphological traits and AFLP markers. The results demonstrate that the C. baccatum collection covers wide geographic areas and is adapted to divergent ecological conditions in South America ranging from cool Andean highland to Amazonia rainforest. A high level of morphological diversity was evident in the collection, with fruit weight the leading variable. The fruit weight distribution pattern was compatible to AFLP-based clustering analysis for the collection. A significant spatial structure was observed in the C. baccatum gene pool. Division of the domesticated germplasm into two major regional groups (Western and Eastern) was further supported by the pattern of spatial population structure.
Conclusions
The results reported improve our understanding of the combined effects of geography, ecology and human intervention on organization of the C. baccatum genepool. The results will facilitate utilization of C. baccatum for crop improvement and species conservation by providing a framework for efficient germplasm collection management and guidance for future plant acquisitions.
doi:10.1186/1471-2156-13-68
PMCID: PMC3496591  PMID: 22866868
20.  Molecular Characterization of Cultivated Bromeliad Accessions with Inter-Simple Sequence Repeat (ISSR) Markers 
Bromeliads are of great economic importance in flower production; however little information is available with respect to genetic characterization of cultivated bromeliads thus far. In the present study, a selection of cultivated bromeliads was characterized via inter-simple sequence repeat (ISSR) markers with an emphasis on genetic diversity and population structure. Twelve ISSR primers produced 342 bands, of which 287 (~84%) were polymorphic, with polymorphic bands per primer ranging from 17 to 34. The Jaccard’s similarity ranged from 0.08 to 0.89 and averaged ~0.30 for the investigated bromeliads. The Bayesian-based approach, together with the un-weighted paired group method with arithmetic average (UPGMA)-based clustering and the principal coordinate analysis (PCoA), distinctly grouped the bromeliads from Neoregelia, Guzmania, and Vriesea into three separately clusters, well corresponding with their botanical classifications; whereas the bromeliads of Aechmea other than the recently selected hybrids were not well assigned to a cluster. Additionally, ISSR marker was proven efficient for the identification of hybrids and bud sports of cultivated bromeliads. The findings achieved herein will further our knowledge about the genetic variability within cultivated bromeliads and therefore facilitate breeding for new varieties of cultivated bromeliads in future as well.
doi:10.3390/ijms13056040
PMCID: PMC3382752  PMID: 22754348
cultivated bromeliads; genetic diversity; population structure; ISSR
21.  Diversification of indigenous gene- pool by using exotic germplasm in lentil (Lens culinaris Medikus subsp. culinaris) 
Genetic diversity was studied among 21 accessions of lentil using SSR markers and morphological traits in order to assess the diversification of Indian gene-pool of lentil through introgression of exotic genes and introduction of germplasm. Among these , 16 genotypes either had ‘Precoz’ gene, an Argentine line in their pedigree or genes from introduced lines from ICARDA. Sixty five SSR markers and eight phenotypic traits were used to analyse the level of genetic diversity in these genotypes. Forty three SSR markers (66 %) were polymorphic and generated a total of 177 alleles with an average of 4.1 alleles per SSR marker. Alleles per marker ranged from 2 to 6. The polymorphic information content ranged 0.33 to 0.80 with an average of 0.57, suggesting that SSR markers are highly polymorphic among the studied genotypes. Genetic dissimilarity based a dendrogram grouped these accessions into two main clusters (cluster I and cluster II) and it ranged 33 % to 71 %, suggesting high level of genetic diversity among the genotypes. First three components of PCA based morphological traits explained higher variance (95.6 %) compared to PCA components based on SSR markers (42.7 %) of total genetic variance. Thus, more diversity was observed for morphological traits and genotypes in each cluster and sub-cluster showed a range of variability for seed size, earliness, pods/plant and plant height. Molecular and phenotypic diversity analysis thus suggested that use of germplasm of exotic lines have diversified the genetic base of lentil germplasm in India. This diversified gene-pool will be very useful in the development of improved varieties of lentil in order to address the effect of climate change, to adapt in new cropping systems niches such as mixed cropping, relay cropping, etc. and to meet consumers’ preference.
doi:10.1007/s12298-013-0214-2
PMCID: PMC3925481  PMID: 24554846
Alien gene; Introgression; SSR marker; Morphological traits; Diversification; Lentil
22.  Genetic divergence of rubber tree estimated by multivariate techniques and microsatellite markers 
Genetics and Molecular Biology  2010;33(2):308-318.
Genetic diversity of 60 Hevea genotypes, consisting of Asiatic, Amazonian, African and IAC clones, and pertaining to the genetic breeding program of the Agronomic Institute (IAC), Brazil, was estimated. Analyses were based on phenotypic multivariate parameters and microsatellites. Five agronomic descriptors were employed in multivariate procedures, such as Standard Euclidian Distance, Tocher clustering and principal component analysis. Genetic variability among the genotypes was estimated with 68 selected polymorphic SSRs, by way of Modified Rogers Genetic Distance and UPGMA clustering. Structure software in a Bayesian approach was used in discriminating among groups. Genetic diversity was estimated through Nei's statistics. The genotypes were clustered into 12 groups according to the Tocher method, while the molecular analysis identified six groups. In the phenotypic and microsatellite analyses, the Amazonian and IAC genotypes were distributed in several groups, whereas the Asiatic were in only a few. Observed heterozygosity ranged from 0.05 to 0.96. Both high total diversity (HT' = 0.58) and high gene differentiation (G st' = 0.61) were observed, and indicated high genetic variation among the 60 genotypes, which may be useful for breeding programs. The analyzed agronomic parameters and SSRs markers were effective in assessing genetic diversity among Hevea genotypes, besides proving to be useful for characterizing genetic variability.
doi:10.1590/S1415-47572010005000039
PMCID: PMC3036869  PMID: 21637487
genealogy; genetic diversity; Hevea brasiliensis; multivariate analysis; SSRs
23.  Genome-wide characterization of genetic diversity and population structure in Secale 
BMC Plant Biology  2014;14:184.
Background
Numerous rye accessions are stored in ex situ genebanks worldwide. Little is known about the extent of genetic diversity contained in any of them and its relation to contemporary varieties, since to date rye genetic diversity studies had a very limited scope, analyzing few loci and/ or few accessions. Development of high throughput genotyping methods for rye opened the possibility for genome wide characterizations of large accessions sets. In this study we used 1054 Diversity Array Technology (DArT) markers with defined chromosomal location to characterize genetic diversity and population structure in a collection of 379 rye accessions including wild species, landraces, cultivated materials, historical and contemporary rye varieties.
Results
Average genetic similarity (GS) coefficients and average polymorphic information content (PIC) values varied among chromosomes. Comparison of chromosome specific average GS within and between germplasm sub-groups indicated regions of chromosomes 1R and 4R as being targeted by selection in current breeding programs. Bayesian clustering, principal coordinate analysis and Neighbor Joining clustering demonstrated that source and improvement status contributed significantly to the structure observed in the analyzed set of Secale germplasm. We revealed a relatively limited diversity in improved rye accessions, both historical and contemporary, as well as lack of correlation between clustering of improved accessions and geographic origin, suggesting common genetic background of rye accessions from diverse geographic regions and extensive germplasm exchange. Moreover, contemporary varieties were distinct from the remaining accessions.
Conclusions
Our results point to an influence of reproduction methods on the observed diversity patterns and indicate potential of ex situ collections for broadening the genetic diversity in rye breeding programs. Obtained data show that DArT markers provide a realistic picture of the genetic diversity and population structure present in the collection of 379 rye accessions and are an effective platform for rye germplasm characterization and association mapping studies.
doi:10.1186/1471-2229-14-184
PMCID: PMC4236688  PMID: 25085433
24.  Genetic diversity among INERA maize inbred lines with single nucleotide polymorphism (SNP) markers and their relationship with CIMMYT, IITA, and temperate lines 
BMC Genetics  2014;15(1):127.
Background
Genetic diversity provides the capacity for plants to meet changing environments. It is fundamentally important in crop improvement. Fifty-nine local maize lines developed at INERA and 41 exotic (temperate and tropical) inbred lines were characterized using 1057 SNP markers to (1) analyse the genetic diversity in a diverse set of maize inbred lines; (2) determine the level of genetic diversity in INERA inbred lines and patterns of relationships of these inbred lines developed from two sources; and (3) examine the genetic differences between local and exotic germplasms.
Results
Roger’s genetic distance for about 64% of the pairs of lines fell between 0.300 and 0.400. Sixty one per cent of the pairs of lines also showed relative kinship values of zero. Model-based population structure analysis and principal component analysis revealed the presence of 5 groups that agree, to some extent, with the origin of the germplasm. There was genetic diversity among INERA inbred lines, which were genetically less closely related and showed a low level of heterozygosity. These lines could be divided into 3 major distinct groups and a mixed group consistent with the source population of the lines. Pairwise comparisons between local and exotic germplasms showed that the temperate and some IITA lines were differentiated from INERA lines. There appeared to be substantial levels of genetic variation between local and exotic germplasms as revealed by missing and unique alleles.
Conclusions
Allelic frequency differences observed between the germplasms, together with unique alleles identified within each germplasm, shows the potential for a mutual improvement between the sets of germplasm. The results from this study will be useful to breeders in designing inbred-hybrid breeding programs, association mapping population studies and marker assisted breeding.
Electronic supplementary material
The online version of this article (doi:10.1186/s12863-014-0127-2) contains supplementary material, which is available to authorized users.
doi:10.1186/s12863-014-0127-2
PMCID: PMC4253617  PMID: 25421948
Maize inbred line; Diversity; Allele frequency
25.  Processes Underpinning Development and Maintenance of Diversity in Rice in West Africa: Evidence from Combining Morphological and Molecular Markers 
PLoS ONE  2014;9(1):e85953.
We assessed the interplay of artificial and natural selection in rice adaptation in low-input farming systems in West Africa. Using 20 morphological traits and 176 molecular markers, 182 farmer varieties of rice (Oryza spp.) from 6 West African countries were characterized. Principal component analysis showed that the four botanical groups (Oryza sativa ssp. indica, O. sativa ssp. japonica, O. glaberrima, and interspecific farmer hybrids) exhibited different patterns of morphological diversity. Regarding O. glaberrima, morphological and molecular data were in greater conformity than for the other botanical groups. A clear difference in morphological features was observed between O. glaberrima rices from the Togo hills and those from the Upper Guinea Coast, and among O. glaberrima rices from the Upper Guinea Coast. For the other three groups such clear patterns were not observed. We argue that this is because genetic diversity is shaped by different environmental and socio-cultural selection pressures. For O. glaberrima, recent socio-cultural selection pressures seemed to restrict genetic diversity while this was not observed for the other botanical groups. We also show that O. glaberrima still plays an important role in the selection practices of farmers and resulting variety development pathways. This is particularly apparent in the case of interspecific farmer hybrids where a relationship was found between pericarp colour, panicle attitude and genetic diversity. Farmer varieties are the product of long and complex trajectories of selection governed by local human agency. In effect, rice varieties have emerged that are adapted to West African farming conditions through genotype × environment × society interactions. The diversity farmers maintain in their rice varieties is understood to be part of a risk-spreading strategy that also facilitates successful and often serendipitous variety innovations. We advocate, therefore, that farmers and farmer varieties should be more effectively involved in crop development.
doi:10.1371/journal.pone.0085953
PMCID: PMC3896413  PMID: 24465809

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