Outcrossing rate between 'Haden' and 'Tommy Atkins' mangoes estimated using microsatellite and AFLP markers

The objective of this work was to estimate outcrossing rates between Haden and Tommy Atkins mango cultivars, using AFLP and microsatellite markers. Progenies of an isolated 'Haden' plant, identified in a 'Tommy Atkins' commercial orchard, in Petrolina, PE, Brazil, were analyzed. Total DNA was isolated from the progeny leaves and used for AFLP and microsatellite reactions. Multilocus outcrossing rates (t m) were estimated by direct count of AFLP or microsatellite markers and by the mLTR software. Outcrossing rates ranged from 0.85 to 0.87 with the analysis based on seven AFLP markers, and from 0.83 to 0.91 based on three microsatellite primers. No unexpected band patterns were observed for 'Haden' and 'Tommy Atkins'. The estimates obtained with the mLTR software were close to those obtained by direct AFLP and microsatellite allele counting, which indicates that the multilocus model was appropriate for this kind of study. The microsatellites mMiCIR005, mMiCIR030, and mMiCIR036 can be used to elucidate the origin of 'Haden' and 'Tommy Atkins' seedlings.

Molecular characterization of papaya genotypes using AFLP markers

Due to the low genetic variability reported in the commercial plantations of papaya (Carica papaya L.), the objective of this study was analyze the genetic diversity of 32 genotypes including cultivars, landraces, inbred lines, and improved germplasm using the AFLP technique (Amplified Fragment Length Polymorphism). The genetic distance matrix was obtained using the Nei and Li genetic distance and clustering was performed using the unweighted pair-method with arithmetic mean (UPGMA). Using 11 combinations of EcoRI/MseI primers, 383 polymorphic bands were obtained. On average, 34.8 polymorphic bands were obtained per primer combination. Five clusters were formed. The traditional cultivar 'Sunrise' and the inbred line CMF-L30-08 were the closest genotypes, and the improved germplasm (CMF041) and landrace (CMF233) the most distant. The main papaya cultivars commercially grown in Brazil, as well as four inbred lines and three improved germplasm, were clustered together, however, were not grouped in the same branch. The genetic distance between the Sunrise and Golden cultivars was 0.329, and even arising from mutation and selection within the Sunrise variety, the Golden stores considerable genetic variability. Additional variability was observed in the inbred lines derived from papaya breeding program at Embrapa Cassava and Fruits.

AFLP markers differentiate isolates of Colletotrichum gossypii from C. gossypii var. cephalosporioides

Fungal diseases in cotton (Gossypium hirsutum), such as anthracnose caused by Colletotrichum gossypii and ramulose caused by C. gossypii var. cephalosporioides, are responsible for large yield losses. These pathogens are seed borne and morphologically similar although they induce different symptoms, which can lead to misdiagnosis using the blotter testing method. The present study was carried out to assess the viability of using Amplified Fragment Length Polymorphism (AFLP) markers to differentiate these pathogens. Five isolates, for each pathogen, were classified according to pathogenicity on cotton plants, and mycelial growth morphology. Conidial suspensions were sprayed on 30-day-old cotton plants and the symptoms assessed ten and 40 days after inoculation. For growth morphology 200 cottonseeds were inoculated with seven-day-old pure cultures, and the mycelial traits observed under a stereoscopic microscope seven days after inoculation. The DNA for AFLP analysis was obtained from seven-day-old fungal mycelia grown in liquid medium, using the Dneasy Qiagen protocol. Using the AFLP technique 318 polymorphic bands were selected to estimate similarities using Dice's Coefficient. The results clearly distinguished between ramulose and anthracnose isolates, which agreed with morphological and pathogenicity testing.

Assessment of silver-stained AFLP markers for studying DNA polymorphism in proso millet (Panicum miliaceum L.)

Proso millet (Panicum miliaceum L.) is a serious weed in North America. A high number of wild proso millet biotypes are known but the genetic basis of its phenotypic variation is poorly understood. In the present study, a non-radioactive silver staining method for PCR-Amplified Fragment Length Polymorphism (AFLP) was evaluated for studying genetic polymorphism in American proso millet biotypes. Twelve biotypes and eight primer combinations with two/three and three/three selective nucleotides were used. Pair of primers with two/three selective nucleotides produced the highest number of amplified DNA fragments, while pair of primers with three/three selective nucleotides were more effective for revealing more polymorphic DNA fragments. The two better primer combinations were EcoR-AAC/Mse-CTT and EcoR-ACT/Mse-CAA with seven and eleven polymorphic DNA fragments, respectively. In a total of 450 amplified fragments, at least 339 appeared well separated in a silver stained acrylamide gel and 39 polymorphic DNA bands were scored. The level of polymorphic DNA (11.5%) using only eight pairs of primers were effective for grouping proso millet biotypes in two clusters but insufficient for separating hybrid biotypes from wild and crop. Nevertheless, the present result indicates that silver stained AFLP markers could be a cheap and important tool for studying genetic relationships in proso millet.

A population genetics study of Anopheles darlingi (Diptera: Culicidae) from Colombia based on random amplified polymorphic DNA-polymerase chain reaction and amplified fragment lenght polymorphism markers

The genetic variation and population structure of three populations of Anopheles darlingi from Colombia were studied using random amplified polymorphic markers (RAPDs) and amplified fragment length polymorphism markers (AFLPs). Six RAPD primers produced 46 polymorphic fragments, while two AFLP primer combinations produced 197 polymorphic fragments from 71 DNA samples. Both of the evaluated genetic markers showed the presence of gene flow, suggesting that Colombian An. darlingi populations are in panmixia. Average genetic diversity, estimated from observed heterozygosity, was 0.374 (RAPD) and 0.309 (AFLP). RAPD and AFLP markers showed little evidence of geographic separation between eastern and western populations; however, the F ST values showed high gene flow between the two western populations (RAPD: F ST = 0.029; Nm: 8.5; AFLP: F ST = 0.051; Nm: 4.7). According to molecular variance analysis (AMOVA), the genetic distance between populations was significant (RAPD:phiST = 0.084; AFLP:phiST = 0.229, P < 0.001). The F ST distances and AMOVAs using AFLP loci support the differentiation of the Guyana biogeographic province population from those of the Chocó-Magdalena. In this last region, Chocó and Córdoba populations showed the highest genetic flow.

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.

Variability and Genetic Structure in Populations of Arrabidaea bilabiata (Sprague) Sandwith in the Amazonas State

Weeds in pastures can intoxicate animals, and Arrabidaea bilabiata is the most important species for herbivores in floodplain areas in the Amazon Basin. Genetic diversity studies in natural populations may contribute to the better understanding of the range of toxicity and the genetic variability organization in this species. The objective of this study was to assess the variability and genetic structure in six populations of A. bilabiata sampled in floodplain areas in three municipalities of the Amazonas State, from the AFLP markers analysis. AFLP markers were efficient to characterize the genetic variability of the 65 individuals analyzed. From four combinations of oligonucleotides, a total of 309 AFLP fragments was obtained, where 304 (98.38%) were polymorphic. By the dendrogram and Bayesian cluster analysis, there was a formation of two isolated groups, the first one comprising individuals from Autazes municipality and the second one comprising individuals from Itacoatiara and Parintins. However, depending on the method to define the most probable cluster number, there was a separation of the six populations, according to their geographical origin. Mantel test confirmed that geographically closer populations are more akin, although low gene flow (0.538) is observed among the sampled populations. The molecular analysis of variance found that 49.29% of the genetic variability are among individuals inside populations and 50.71% among the populations analyzed. The results indicate the possibility that isolated A. bilabiata populations contain plants with different toxicity levels and suggest a strong adaptability of the species.

Genetic diversity and relationship in American and African oil palm as revealed by RFLP and AFLP molecular markers

The objective of this work was to evaluate the genetic diversity, its organization and the genetic relationships within oil palm (Elaeis oleifera (Kunth) Cortés, from America, and E. guineensis (Jacq.), from Africa) germplasm using Restriction Fragment Length Polymorphism (RFLP) and Amplified Fragment Length Polymorphism (AFLP). In complement to a previous RFLP study on 241 E. oleifera accessions, 38 E. guineensis accessions were analyzed using the same 37 cDNA probes. These accessions covered a large part of the geographical distribution areas of these species in America and Africa. In addition, AFLP analysis was performed on a sub-set of 40 accessions of E. oleifera and 22 of E. guineensis using three pairs of enzyme/primer combinations. Data were subjected to Factorial Analysis of Correspondence (FAC) and cluster analysis, with parameters of genetic diversity being also studied. Results appeared congruent between RFLP and AFLP. In the E. oleifera, AFLP confirmed the strong structure of genetic diversity revealed by RFLP, according to geographical origin of the studied material, with the identification of the same four distinct genetic groups: Brazil, French Guyana/Surinam, Peru, north of Colombia/Central America. Both markers revealed that genetic divergence between the two species is of the same magnitude as that among provenances of E. oleifera. This finding is in discrepancy with the supposed early tertiary separation of the two species.

Genetic diversity of sweet sorghum germplasm in Mexico using AFLP and SSR markers

The objective of this work was to evaluate the diversity and genetic relationships between lines and varieties of the sweet sorghum (Sorghum bicolor) germplasm bank of the National Institute for Forestry, Agriculture and Livestock Research, Mexico, using AFLP and SSR markers. The molecular markers revealed robust amplification profiles and were able to differentiate the 41 genotypes of sweet sorghum evaluated. Analysis of the frequency and distribution of polymorphic fragments allowed for the detection of unique (AFLP) and rare (SSR) alleles in several genotypes (RBSS‑8, RBSS‑9, RBSS‑25, RBSS‑32, and RBSS‑37), indicating that these markers may be associated with a feature that has not yet been determined or may be useful for the identification of these genotypes. The genetic relationships indicated the presence of at least two types of sweet sorghum: a group of modern genotypes used for sugar and biofuel production, and another group consisting of historic and modern genotypes used for the production of syrups. Sweet sorghum genotypes may be used to develop new varieties with higher sugar and juice contents.

Potato cultivar identification using molecular markers

The objective of this work was to evaluate a set of microsatellite markers for varietal identification and characterization of the most widespread potato cultivars in Brazil. The DNA from 14 potato cultivars was genotyped using microsatellite markers and the alleles were scored in silver-stained polyacrylamide gel. Twenty-four microsatellite markers were evaluated, and only one locus was monomorphic. Based on band patterns, a set of two microsatellites that were able to identify and differentiate all examined cultivars was obtained.

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.

Identification of 'Ubá' mango tree zygotic and nucellar seedlings using ISSR markers

Polyembryonic seeds are characterized by the development of over one embryo in the same seed, which can be zygotic and nucellar. The objective of this work was to identify the genetic origin, whether zygotic or nucellar, of seedlings of polyembryonic seeds of 'Ubá' mango tree using ISSR markers, and relating them with the vigor of the seedlings. Thus, mangos were harvested in Visconde do Rio Branco (accession 102) and Ubá (accessions 112, 138, 152 and 159), whose seeds were germinated in plastic trays filled with washed sand. Fifty days after sowing, seedlings from five seeds of each one of the accessions 102, 112, 138, 159 and from 10 seeds of the accession 152, were analyzed. These sseedlings were characterized and evaluated for plant height, stem circumference and mass of fresh aerial part and the most vigorous seedling was the one displaying at least two of these traits higher than the other seedlings from seed. Leaves were collected for genomic DNA extraction, which was amplified using seven ISSR primers previously selected based on the amplification profile and considering the number and resolution of fragments. Zygotic seedlings were found in 18 seeds, which were the most vigorous in six seeds. The results evidenced the existence of genetic variability in orchards using seedlings grown from seeds, because the farmer usually uses the most vigorous ones, assuming that this is of nucellar origin. These results also indicate that the most vigorous seedling are not always nucellar, inasmuch as of 20% of the total seeds evaluated, the zygotic seedling was the most vigorous.

Comparison of serum hepatitis B virus replication markers in patients with chronic hepatitis B: studies on HBeAg/Anti-HBe system, viral dna polymerase and HBV-DNA

The detection of HBV-DNA in serum by molecular hybridization is the most sensitive and specific marker of replication and infectivity of hepatitis B virus and currently is proposed as a routine diagnostic technique in the follow-up of HBV - related diseases. Comparing different techniques already described, we found that direct spotting of serum samples on nitrocellulose membranes under vacuum filtration, followed by denaturing and neutralizing washes is more practical, simple, sensible and reproducible. DNA polymerase assay using phosphonoformic acid as specific viral inhibitor has shown 86.8% of concordance with HBV-DNA detection, and so, it is an useful alternative in the follow-up of hepatitis B chronic patients. We found 19.2% HBeAg positive samples with no other markers of viral replication and no anti-HBe positive sample had detectable HBV-DNA. Discordance between the 2 systems have been extensively described, and we confirm this for the first time in our country. Molecular biological techniques are essential to determine the replication status of chronic hepatitis B patients.

Potential infectivity of blood from HBsAG asymptomatic carriers due to the presence of HBV-DNA and comparison with other markers of hbv infection

Serum samples from 356 HBsAg positive asymptomatic carriers, which were titrated by reverse passive hemagglutination, were analysed for the presence of HBV-DNA, HBsAg and IgM anti-HBc. The samples were divided in three classes, according to the titers of HBsAg and IgM anti-HBc and the distribution of HBV-DNA and HBsAg among these classes was studied. In the high titer class of HBsAg, 65% of samples have one or both markers against only 19% in the low titer class. From the total of 356 samples, 121 gave positive results for IgM anti-HBc (33.9%). From these, 38.9% of HBV-DNA and 47.9% of HBeAg were observed, whereas in samples with absence of IgM anti-HBc, 18.3% and 16.6% were respectively found. A higher frequency of agreement between all these markers was found in the class of high titers of HBsAg; however, HBV-DNA was detected in the low titer class of HBsAg and little or no IgM anti-HBc, showing potential blood infectivity even in HBsAg positive borderline samples.

Evaluation of phenotypic markers associated with pathogenicity in the genus Listeria

A total of 130 Listeria strains were tested in order to evaluate lecithinase production and capacity for Congo red adsorption as markers of pathogenicity. The strains were identified according to acid production from sugars and by the CAMP test and the data were correlated with the ability to produce keratoconjunctivitis in guinea pigs. L. monocytogenes cultures presented 51.8% and 88.8% positivity rates for Congo red adsorption and lecithinase production, respectively, whereas 80.8% and 100% for L. innocua cultures were negative for the two test, respectively