Genetic diversity of chloroquine-resistant Plasmodium vivax parasites from the western Brazilian Amazon

The molecular basis of Plasmodium vivax chloroquine (CQ) resistance is still unknown. Elucidating the molecular background of parasites that are sensitive or resistant to CQ will help to identify and monitor the spread of resistance. By genotyping a panel of molecular markers, we demonstrate a similar genetic variability between in vitro CQ-resistant and sensitive phenotypes of P. vivax parasites. However, our studies identified two loci (MS8 and MSP1-B10) that could be used to discriminate between both CQ-susceptible phenotypes among P. vivax isolates in vitro. These preliminary data suggest that microsatellites may be used to identify and to monitor the spread of P. vivax-resistance around the world.

Genetic diversity of Cryptosporidium identified in clinical samples from cities in Brazil and Argentina

The identification and characterisation of Cryptosporidiumgenotypes and subtypes are fundamental to the study of cryptosporidiosis epidemiology, aiding in prevention and control strategies. The objective was to determine the genetic diversity ofCryptosporidium in samples obtained from hospitals of Rio de Janeiro, Brazil, and Buenos Aires, Argentina. Samples were analysed by microscopy and TaqMan polymerase chain reaction (PCR) assays forCryptosporidium detection, genotyped by nested-PCR-restriction fragment length polymorphism (RFLP) analysis of the 18S rRNA gene and subtyped by DNA sequencing of the gp60 gene. Among the 89 samples from Rio de Janeiro, Cryptosporidium spp were detected in 26 by microscopy/TaqMan PCR. In samples from Buenos Aires,Cryptosporidium was diagnosed in 15 patients of the 132 studied. The TaqMan PCR and the nested-PCR-RFLP detected Cryptosporidium parvum, Cryptosporidium hominis, and co-infections of both species. In Brazilian samples, the subtypes IbA10G2 and IIcA5G3 were observed. The subtypes found in Argentinean samples were IbA10G2, IaA10G1R4, IaA11G1R4, and IeA11G3T3, and mixed subtypes of Ia and IIa families were detected in the co-infections. C. hominis was the species more frequently detected, and subtype family Ib was reported in both countries. Subtype diversity was higher in Buenos Aires than in Rio de Janeiro and two new subtypes were described for the first time.

Genetic diversity of Brazilian Pantaneiro horse and relationships among horse breeds

The objective of this work was to evaluate the genetic diversity of Brazilian Pantaneiro horse by microsatellite markers, investigate the effect of genetic bottlenecks and estimate genetic differentiation among four horse breeds. Genetic variation was estimated through allele frequencies and mean breed heterozygosity. Nei's genetic distances among the breeds Pantaneiro, Thoroughbred, Arabian, Spanish Pure Breed (Andalusian), and Uruguay Creole were calculated, and it was used to construct an UPGMA dendrogram. Clustering at different K values was calculated to infer population structure and assign individuals to populations. Nei's distances showed a minimum distance between Pantaneiro horse and Spanish Pure Breed (0.228), and similar distances from Spanish Pure Breed to Thoroughbred and to Arabian (0.355 and 0.332). It was observed a great level of diversity, clear distance from Pantaneiro horse to other breeds, and genetic uniformity within breed. It was verified a certain level of substructure of Pantaneiro horse showing no influences from the other studied breeds.

Genetic diversity of cultivated accessions and wild species of rubber tree using EST‑SSR markers

The objective of this work was to evaluate the efficiency of EST‑SSR markers in the assessment of the genetic diversity of rubber tree genotypes (Hevea brasiliensis) and to verify the transferability of these markers for wild species of Hevea. Forty‑five rubber tree accessions from the Instituto Agronômico (Campinas, SP, Brazil) and six wild species were used. Information provided by modified Roger's genetic distance were used to analyze EST‑SSR data. UPGMA clustering divided the samples into two major groups with high genetic differentiation, while the software Structure distributed the 51 clones into eight groups. A parallel could be established between both clustering analyses. The 30 polymorphic EST‑SSRs showed from two to ten alleles and were efficient in amplifying the six wild species. Functional EST‑SSR microsatellites are efficient in evaluating the genetic diversity among rubber tree clones and can be used to translate the genetic differences among cultivars and to fingerprint closely related materials. The accessions from the Instituto Agronômico show high genetic diversity. The EST‑SSR markers, developed from Hevea brasiliensis, show transferability and are able to amplify other species of Hevea.

Genetic diversity between improved banana diploids using canonical variables and the Ward-MLM method

The objective of this work was to estimate the genetic diversity of improved banana diploids using data from quantitative analysis and from simple sequence repeats (SSR) marker, simultaneously. The experiment was carried out with 33 diploids, in an augmented block design with 30 regular treatments and three common ones. Eighteen agronomic characteristics and 20 SSR primers were used. The agronomic characteristics and the SSR were analyzed simultaneously by the Ward-MLM, cluster, and IML procedures. The Ward clustering method considered the combined matrix obtained by the Gower algorithm. The Ward-MLM procedure identified three ideal groups (G1, G2, and G3) based on pseudo-F and pseudo-t² statistics. The dendrogram showed relative similarity between the G1 genotypes, justified by genealogy. In G2, 'Calcutta 4' appears in 62% of the genealogies. Similar behavior was observed in G3, in which the 028003-01 diploid is the male parent of the 086079-10 and 042079-06 genotypes. The method with canonical variables had greater discriminatory power than Ward-MLM. Although reduced, the genetic variability available is sufficient to be used in the development of new hybrids.

Evaluation of Xylella fastidiosa genetic diversity by fAFPL markers

The first phytopathogenic bacterium with its DNA entirely sequenced is being detected and isolated from different host plants in several geographic regions. Although it causes diseases in cultures of economic importance, such as citrus, coffee, and grapevine little is known about the genetic relationships among different strains. Actually, all strains are grouped as a single species, Xylella fastidiosa, despite colonizing different hosts, developing symptoms, and different physiological and microbiological observed conditions. The existence of genetic diversity among X. fastidiosa strains was detected by different methodological techniques, since cultural to molecular methods. However, little is know about the phylogenetic relationships developed by Brazilian strains obtained from coffee and citrus plants. In order to evaluate it, fAFLP markers were used to verify genetic diversity and phylogenetic relationships developed by Brazilian and strange strains. fAFLP is an efficient technique, with high reproducibility that is currently used for bacterial typing and classification. The obtained results showed that Brazilian strains present genetic diversity and that the strains from this study were grouped distinctly according host and geographical origin like citrus-coffee, temecula-grapevine-mulberry and plum-elm.

GENETIC DIVERSITY AND STRUCTURE OF Oenocarpus mapora GERMPLASM CONSERVED AT EASTERN AMAZON

ABSTRACT The aim of this study was to evaluate the genetic diversity and structure in the germoplasm of Oenocarpus mapora conserved at Eastern Amazon. Thus, 88 individuals were genotyped with five microsatellite loci. These individuals belong to 24 accessions that were sampled in eight sample places of three Brazilian Amazon states conserved at the Active Germplasm Bank (AGB) of Embrapa Eastern Amazon. All loci were polymorphic and they generated 85 alleles with an average of 17 alleles per loci. Total genetic diversity (HE) was 0.48. Sample places were considered genetically distinct, with ?p = 0.354. The analysis of molecular variance (AMOVA) identified that the genetic portion among areas was of 36.14% and within 63.86%. The Nei distances varied from 0.091 between Abaetetuba and Santo Antônio do Tauá, both in the state of Pará (PA), to 4.18, between Parintins, AM and Rio Branco, AC. By means of Bayesian analysis, it was identified nine clusters that compose the accessions of the germplasm bank, with different distributions among individuals. The study showed high fixation rates per sample area, which indicates that there may have been significant inbreeding or crossing among parental individuals. It suggests that future samples should be made of different plants in natural populations. Even though, it was verified that there is considerable genetic variation in the germplasm of O. mapora.

Genetic diversity among isolates of stemphylium solani from cotton

The fungus Stemphylium solani causes leaf blight of tomato (Lycopersicon esculentum) in Brazil. In recent years, severe epidemics of a new leaf blight of cotton (Gossipium hyrsutum) caused by S. solani occurred in three major cotton-growing Brazilian states (PR, MT and GO). Molecular analysis was performed to assess the genetic diversity among the S. solani isolates from cotton, and to verify their relationship with representative S. solani isolates from tomato. Random amplified polymorphic DNA (RAPD) markers and internal transcribed spacers of ribosomal DNA (rDNA) were used to compare 33 monosporic isolates of S. solani (28 from cotton and five from tomato). An isolate of Alternaria macrospora from cotton was also used for comparison. RAPD analysis showed the presence of polymorphism between the genera and the species. The A. macrospora and the S. solani isolates from cotton and tomato were distinct from each other, and fell into separate groups. Variation by geographic region was observed for the tomato isolates but not for the cotton isolates. Amplifications of the ITS region using the primer pair ITS4/ITS5 resulted in a single PCR product of approximately 600 bp for all the isolates. Similarly, when amplified fragments were digested with eight restriction enzymes, identical banding patterns were observed for all the isolates. Hence, rDNA analysis revealed no inter-generic or intra-specific variation. The genetic difference observed between the cotton and the tomato isolates provides evidence that S. solani attacking cotton in Brazil belongs to a distinct genotype.

Genetic diversity of begomovirus infecting tomato and associated weeds in Southeastern Brazil

The genetic diversity of begomovirus isolates from tomato (Lycopersicon esculentum) fields in the Southeastern region of Brazil was analyzed by direct sequencing of PCR fragments amplified by using universal oligonucleotides for the begomovirus DNA-A, and subsequent computer-aided phylogenetic analysis. Samples of tomato plants and associated weeds showing typical symptoms of virus infection were collected at seven locations in the states of Minas Gerais, Espírito Santo and Rio de Janeiro. A total of 137 out of 369 samples were infected with a begomovirus based on PCR analysis. Phylogenetic analysis indicated a high degree of genetic diversity among begomoviruses infecting tomatoes in the sampled area. One species (Tomato chlorotic mottle virus, TCMV) occurs predominantly in Minas Gerais, whereas in Rio de Janeiro and Espírito Santo a distinct species, not yet fully characterized, predominates. Phylogenetic analysis further indicates the presence of an additional four possible new species. This high degree of genetic diversity suggests a recent transfer of indigenous begomovirus from wild hosts into tomatoes. The close phylogenetic relationship verified between begomovirus infecting tomato and associated weeds favors this hypothesis.

Nuclear and mitochondrial DNA markers in traceability of retail beef samples

Several characteristics are important in a traceability system of animal products, such as age at slaughter, breed composition, besides information of the productive chain. In general, the certification agent records information about the animals and the system which it came from, although cannot guarantee that the slaughtering, meat processing and distribution are error proof. Besides, there is a differential price, at least at the international market, based on sex and breed composition of the animals. Genetic markers allow identification of characteristics controlled in the beef cattle traceability program, as sex and breed composition, in order to correctly identify and appraise the final product for the consumer. The hypothesis of this study was that the majority beef samples retailed in the local market originate from female with a great participation of zebu breeds. Therefore, the objective of this work was to characterize retail beef samples with DNA markers that identify cattle sex and breed composition. Within 10 beef shops localized in Pirassununga, SP, Brazil, 61 samples were collected, all were genotyped as harboring Bos taurus mitochondrial DNA and 18 were positive for the Y chromosome amplification (male). For the marker sat1711b-Msp I the frequency of the allele A was 0.278 and for the marker Lhr-Hha I the frequency of the allele T was 0.417. The results of sat1711b-Msp I and Lhr-Hha I allelic frequencies are suggestive that the proportion of indicus genome compared with the taurine genome in the market meat is smaller than the observed in the Nellore breed. The procedure described in this study identified sex and subspecies characteristics of beef meat samples, with potential application in meat products certification in special as an auxiliary tool in beef cattle traceability programs.

Genetic diversity among proso millet (Panicum miliaceum) biotypes assessed by AFLP technique

The Amplified Fragment Length Polymorphism (AFLP) technique was used to access genetic diversity between three domestic and nine wild proso millet biotypes from the United States and Canada. Eight primer combinations detected 39 polymorphic DNA fragments, with the genetic distance estimates among biotypes ranging from 0.02 to 0.04. Colorado-Weld County black seeded and Wyoming-Platte County were the most distinct biotypes according to the dissimilarity level. A UPGMA cluster analysis revealed two distinct groups of proso millet without any geographic association. Six weed biotypes exhibiting some characters of cultivated plants were grouped together with domesticated biotypes of proso millet while the three typical wild phenotypes were clearly clustered into another group according to AFLP markers.

Characterization of six rat strains (Rattus norvegicus) by mitochondrial DNA restriction fragment length polymorphism

Restriction fragment length polymorphism (RFLP) was used to examine the extent of mtDNA polymorphism among six strains of rats (Rattus norvegicus) - Wistar, Wistar Munich, Brown Norway, Wistar Kyoto, SHR and SHR-SP. A survey of 26 restriction enzymes has revealed a low level of genetic divergence among strains. The sites of cleavage by EcoRI, NcoI and XmnI were shown to be polymorphic. The use of these three enzymes allows the 6 strains to be classified into 4 haplotypes and identifies specific markers for each one. The percentage of sequence divergence among all pairs of haplotypes ranged from 0.035 to 0.33%, which is the result of a severe population constriction undergone by the strains. These haplotypes are easily demonstrable and therefore RFLP analysis can be employed for genetic monitoring of rats within animal facilities or among different laboratories.

Genetic diversity in sugar apple (Annona squamosa L.) by using RAPD markers

Genetic diversity in a collection of 64 sugar apple accessions collected from different municipalities in northern Minas Gerais was assessed by RAPD analysis. Using 20 selected RAPD primers 167 fragments were generated, of which 48 were polymorphic (28.7%) producing an average of 2.4 polymorphic fragments per primer. Low percentage of polymorphism (< 29%) was observed by using the set of primers indicating low level of genetic variation among the 64 accessions evaluated. Genetic relationships were estimated using Jaccard's coefficient of similarity. Accessions from different municipalities clustered together indicating no correlation between molecular grouping and geographical origin. The dendrogram revealed five clusters. The first cluster grouped C19 and G29 accessions collected from the municipalities of Verdelândia and Monte Azul, respectively. The second cluster grouped G16 and B11 accessions collected from the municipalities of Monte Azul and Coração de Jesus, respectively. The remaining accessions were grouped in three clusters, with 8, 15 and 37 accessions, respectively. In summary, RAPD showed a low percentage of polymorphism in the germplasm collection.

Determination of human cytomegalovirus genetic diversity in different patient populations in Costa Rica

Seroprevalence of HCMV in Costa Rica is greater than 95% in adults; primary infections occur early in life and is the most frequent congenital infection in newborns. The objectives of this study were to determine the genetic variability and genotypes of HCMV gB gene in Costa Rica. Samples were collected from alcoholics, pregnant women, blood donors, AIDS patients, hematology-oncology (HO) children and HCMV isolates from neonates with cytomegalic inclusion disease. A semi-nested PCR system was used to obtain a product of 293-296 bp of the gB gene to be analyzed by Single Stranded Conformational Polymorphism (SSCP) and sequencing to determine the genetic polymorphic pattern and genotypes, respectively. AIDS patients showed the highest polymorphic diversity with 14 different patterns while fifty-six percent of HO children samples showed the same polymorphic pattern, suggesting in this group a possible nosocomial infection. In neonates three genotypes (gB1, gB2 and gB3), were determined while AIDS patients and blood donors only showed one (gB2). Of all samples analyzed only genotypes gB1, 2 and 3 were determined, genotype gB2 was the most frequent (73%) and mixed infections were not detected. The results of the study indicate that SSCP could be an important tool to detect HCMV intra-hospital infections and suggests a need to include additional study populations to better determine the genotype diversity and prevalence.