Morphological and molecular diversity among cassava genotypes

The objective of this work was to characterize morphologically and molecularly the genetic diversity of cassava accessions, collected from different regions in Brazil. A descriptive analysis was made for 12 morphological traits in 419 accessions. Data was transformed into binary data for cluster analysis and analysis of molecular variance. A higher proportion of white or cream (71%) root cortex color was found, while flesh colors were predominantly white (49%) and cream (42%). Four accession groups were classified by the cluster analysis, but they were not grouped according to their origin, which indicates that diversity is not structured in space. The variation was greater within regions (95.6%). Sixty genotypes were also evaluated using 14 polymorphic microsatellite markers. Molecular results corroborated the morphological ones, showing the same random distribution of genotypes, with no grouping according to origin. Diversity indices were high for each region, and a greater diversity was found within regions, with: a mean number of alleles per locus of 3.530; observed and expected heterozygosity of 0.499 and 0.642, respectively; and Shannon index of 1.03. The absence of spatial structure among cassava genotypes according to their origins shows the anthropic influence in the distribution and movement of germplasm, both within and among regions.

Genetic associations in the detection of QTLs for wheat spike-related traits

The objective of this work was to assess the genetic diversity and population structure of wheat genotypes, to detect significant and stable genetic associations, as well as to evaluate the efficiency of statistical models to identify chromosome regions responsible for the expression of spike-related traits. Eight important spike characteristics were measured during five growing seasons in Serbia. A set of 30 microsatellite markers positioned near important agronomic loci was used to evaluate genetic diversity, resulting in a total of 349 alleles. The marker-trait associations were analyzed using the general linear and mixed linear models. The results obtained for number of allelic variants per locus (11.5), average polymorphic information content value (0.68), and average gene diversity (0.722) showed that the exceptional level of polymorphism in the genotypes is the main requirement for association studies. The population structure estimated by model-based clustering distributed the genotypes into six subpopulations according to log probability of data. Significant and stable associations were detected on chromosomes 1B, 2A, 2B, 2D, and 6D, which explained from 4.7 to 40.7% of total phenotypic variations. The general linear model identified a significantly larger number of marker-trait associations (192) than the mixed linear model (76). The mixed linear model identified nine markers associated to six traits.

Genetic variations among passion fruit species using rapd markers

It has been evaluated the genetic variability through the use of RAPD molecular markers on the following passionflower species: Passiflora amethystina, P. caerulea, P. cincinnata, P. coccinea, P. serrato digitata, P. foetida, P. maliformis, P. alata, P. giberti, P. laurifolia, P. macrocarpa, P. nitida, P. setacea, P. suberosa, P. ligularis, P. capsularis, P. edulis Sims and its botanical variety P. edulis Sims f. flavicarpa Deg. In this research work, the analyses of the random amplified polymorphic DNA products (RAPD) were employed to estimate the genetic diversity and the taxonomic linkage within the species above. The total of 21 primers were used in this study which generated 270 different polymorphic products. It was possible to detect that the Passiflora species had shown a similarity of 17,3%, and between Passiflora edulis Sims and Passiflora edulis Sims f. flavicarpa a similarity of 34,35% has been found. The rate of similarity within edulis specie is low, making it clear that a large variability between the yellow and the purple forms exists.

Diversity and genetic struture in cajá tree (spondias mombin l.) populations in northeastern Brazil

Spondias mombin L. is a fruit tree from the American continent from the Anacardiaceae family. In Brazil it is common in different vegetation types but is more frequent in the Atlantic and Amazonian rainforests. It is economically important because of its fruits, which are widely consumed raw or processed as fruit jellies, juices and ice creams. The leaves have great importance in the pharmaceutical industry because of their antibacterial properties. In the state of Pernambuco, cajá tree is widely distributed in the Zona da Mata region and less frequently in the Agreste and Sertão areas. In this work diversity and genetic structure were studied in four populations of cajá tree from Pernambuco's Zona da Mata, Northeast Brazil, using isozymes polymorphism analyses from electrophoreses. The result showed 100% of polymorphism (P) for nine alleles and the average of alleles per locus s was 2.4. The expected heterozygosity ranged from 0.530 to 0.574 and the observed heterozygosity , from 0.572 to 0.735. It was not observed inbreeding and the average F IT was -0.175, whereas within population inbreeding (f) varied from -0.08 to- 0.37. The genetic divergence among the populations (F ST) ranged from 0.006 to 0.028 and the average was 0.026. The average of estimated gene flow (Nm) was high (5.27). The CG-IPA population, corresponding to the germplasm collection of IPA, showed more than 96% of genetic similarity with other populations; therefore, it is a good representative of the existent genetic diversity in the Zona da Mata region.

Diversity and genetic structure in natural populations of Hancornia speciosa var. speciosa Gomes in northeastern Brazil

Hancornia speciosa Gomes is a fruit tree native from Brazil that belongs to Apocinaceae family, and is popularly known as Mangabeira. Its fruits are widely consumed raw or processed as fruit jam, juices and ice creams, which have made it a target of intense exploitation. The extractive activities and intense human activity on the environment of natural occurrence of H. speciosa has caused genetic erosion in the species and little is known about the ecology or genetic structure of natural populations. The objective of this research was the evaluation of the genetic diversity and genetic structure of H. speciosa var. speciosa. The genetic variability was assessed using 11 allozyme loci with a sample of 164 individuals distributed in six natural populations located in the States of Pernambuco and Alagoas, Northeastern Brazil. The results showed a high level of genetic diversity within the species (e= 0.36) seeing that the most of the genetic variability of H. speciosa var. speciosa is within its natural populations with low difference among populations ( or = 0.081). The inbreeding values within ( = -0.555) and among populations ( =-0.428) were low showing lacking of endogamy and a surplus of heterozygotes. The estimated gene flow ( m ) was high, ranging from 2.20 to 13.18, indicating to be enough to prevent the effects of genetic drift and genetic differentiation among populations. The multivariate analyses indicated that there is a relationship between genetic and geographical distances, which was confirmed by a spatial pattern analysis using Mantel test (r = 0.3598; p = 0.0920) with 1000 random permutations. The high genetic diversity index in these populations indicates potential for in situ genetic conservation.

Diversity and genetic relatedness among genotypes of Vitis spp. using microsatellite molecular markers

The purpose of this research was to study the genetic diversity and genetic relatedness of 60 genotypes of grapevines derived from the Germplasm Bank of Embrapa Semiárido, Juazeiro, BA, Brazil. Seven previously characterized microsatellite markers were used: VVS2, VVMD5, VVMD7, VVMD27, VVMD3, ssrVrZAG79 and ssrVrZAG62. The expected heterozygosity (He) and polymorphic information content (PIC) were calculated, and the cluster analysis were processed to generate a dendrogram using the algorithm UPGMA. The He ranged from 81.8% to 88.1%, with a mean of 84.8%. The loci VrZAG79 and VVMD7 were the most informative, with a PIC of 87 and 86%, respectively, while VrZAG62 was the least informative, with a PIC value of 80%. Cluster analysis by UPGMA method allowed separation of the genotypes according to their genealogy and identification of possible parentage for the cultivars 'Dominga', 'Isaura', 'CG 26916', 'CG28467' and 'Roni Redi'.

Telomere and microsatellite primers reveal diversity among Sclerotinia sclerotiorum isolates from Brazil

Sclerotinia sclerotiorum, the causal agent of white mold, is a problem of winter bean (Phaseolus vulgaris) production in Brazil under center-pivot irrigation. Isolates of S. sclerotiorum were obtained from a center-pivot-irrigated field near Guaíra-SP, Brazil. Mycelial compatibility group (MCG) studies revealed the presence of only two MCG. PCR/RFLP analysis of the ITS1-5.8S-ITS2 ribosomal subunit regions of these field isolates of S. sclerotiorum failed to show any genetic differences between these two MCGs. DNA amplification with a chromosomal telomere sequence-based primer and one microsatellite primer revealed genetic polymorphisms among isolates within the same MCG. Isolates taken from beans and two other crops from another region of Brazil showed the same two MCG and had identical banding patterns for the telomere and microsatellite primers. These findings support the use of telomere sequence-based primers for revealing genotypic differences among S. sclerotiorum isolates.

Genetic and phenotypic characterization of isolates of Pyricularia grisea from the rice cultivars epagri 108 and 109 in the State of Tocantins

An epidemic of rice (Oryza sativa) blast occurred on cultivars Epagri 108 and 109 in the municipalities of Lagoa da Confusão and Duerê in the State of Tocantins, during the rice-growing season 1998-99. DNA fingerprinting and virulence phenotype analysis were utilized to determine the diversity of Pyricularia grisea isolates collected from these cultivars in one epidemic year. Rep-PCR analysis of isolates was done by using two primer sequences from Pot2. Two distinct fingerprint groups or lineages were identified among 53 isolates collected from nine different commercial fields. The virulence pattern of isolates retrieved from these two cultivars was analyzed in artificial inoculation tests utilizing 32 genotypes in the greenhouse. A dendrogram constructed from virulence phenotype data showed a single group considering 77% similarity level. The predominant pathotype IB-45 was represented by 47 of the 53 isolates corresponding to 83%. Four other pathotypes (IB-1, IB-9, IB-13 and IB-41) were identified at random among the isolates from these cultivars. There was no relation between rep-PCR grouping and pathotypes. The results showed that the isolates of P. grisea recovered from cultivars Epagri108 and 109 in farmers' fields had narrow phenotypic and genetic diversity. The blast outbreak on these two cultivars one year after their introduction could be attributed to the new pathotype IB-45 or its increase, which was hitherto existing in low frequency.

Genotypic diversity among brazilian isolates of Macrophomina phaseolina revealed by RAPD

Macrophomina phaseolina has been considered one of the most prevalent soybean (Glycine max) pathogens in Brazil. No genetic resistance has been determined in soybean and very little is known about the genetic diversity of this pathogen in tropical and sub-tropical regions. Fifty-five isolates from soybean roots were collected in different regions and analyzed through RAPD for genetic diversity. The UPGMA cluster analysis for 74 loci scored permitted identification of three divergent groups with an average similarity of 99%, 92% and 88%, respectively. The three groups corresponded to 5.45%, 59.95% and 34.6%, respectively of all isolates used. A single plant had three different haplotypes, while 10.9% of the analyzed plants had two different haplotypes. In another study the genetic similarity was evaluated among isolates from different hosts [soybean, sorghum (Sorghum bicolor), sunflower (Helianthus annuus), cowpea (Vigna unguiculata), corn (Zea mays) and wheat (Triticum aestivum)] as well as two soil samples from native areas. Results showed that more divergent isolates originated from areas with a single crop. Isolates from areas with crop rotation were less divergent, showing high similarity values and consequently formed the largest group. Amplification of the ITS region using primers ITS1 and ITS4 produced only one DNA fragment of 620 bp. None of the isolates were differentiated through PCR-RFLP. Our results demonstrated genetic variability among Brazilian isolates of M. phaseolina and showed that one single root can harbor more than one haplotype. Moreover, cultivation with crop rotation tends to induce less specialization of the pathogen isolates. Knowledge of this variation may be useful in screening soybean genotypes for resistance to charcoal rot.

Variability in ribosomal DNA genic and spacer regions in Verticillium dahliae isolates from different hosts

Using PCR-based assays with specific primers for amplification of the ribosomal DNA intergenic spacer region (IGS) and a portion of the mitochondrial DNA small subunit ribosomal RNA gene (mtDNA SSU rRNA), the genetic variability among Verticillium dahliae isolates from olive (Olea europaea) and other host species from Argentina and Brazil was estimated. The derived UPGMA-generated phenograms based upon the restriction fingerprinting data of rDNA IGS products revealed genetic differences, correlating with the host of origin. Isolates infecting olive genetically distinct from those from cocoa (Theobroma cacao) and sunflower (Helianthus annuus). Digestion of mitochondrial DNA SSU rRNA PCR products revealed less variability, distinguishing only one isolate from sunflower. Ribosomal DNA ITS restriction patterns were identical for all isolates of V. dahliae, irrespective of host of origin. These preliminary results may have relevance for Verticillium wilt control practices, possibly reflecting a different evolutionary origin, or reproductive isolation of the pathogen in olive, distinct from populations of other hosts.

Genetic and antigenic analysis of Babesia bigemina isolates from five geographical regions of Brazil

A molecular epidemiological study was performed with Babesia bigemina isolates from five geographical regions of Brazil. The genetic analysis was done with random amplification of polymorphic DNA (RAPD), repetitive extragenic palindromic elements-polymerase chain reaction (REP-PCR) and enterobacterial repetitive intergenic consensus sequences-polymerase chain reaction (ERIC-PCR) that showed genetic polymorphism between these isolates and generated fingerprinting. In RAPD, ILO872 and ILO876 primers were able to detect at least one fingerprinting for each B. bigemina isolate. The amplification of B. bigemina DNA fragments by REP-PCR and ERIC-PCR gave evidence for the presence in this haemoprotozoan of the sequences described previously in microorganisms of the bacterial kingdom. For the first time it was demonstrated that both techniques can be used for genetic analysis of a protozoan parasite, although the ERIC-PCR was more discriminatory than REP-PCR. The dendogram with similarity coefficient among isolates showed two clusters and one subcluster. The Northeastern and Mid-Western isolates showed the greatest genetic diversity, while the Southeastern and Southern isolates were the closest. The antigenic analysis was done through indirect fluorescent antibody technique and Western blotting using a panel of monoclonal antibodies directed against epitopes on the merozoite membrane surface, rhoptries and membrane of infected erythrocytes. As expected, the merozoite variable surface antigens, major surface antigen (MSA)-1 and MSA-2 showed antigenic diversity. However, B cell epitopes on rhoptries and infected erythrocytes were conserved among all isolates studied. In this study it was possible to identify variable and conserved antigens, which had already been described as potential immunogens. Considering that an attenuated Babesia clone used as immunogen selected populations capable of evading the immunity induced by this vaccine, it is necessary to evaluate more deeply the cross-protection conferred by genetically more distant Brazilian B. bigemina isolates and make an evaluation of the polymorphism degree of variable antigens such as MSA-1 and MSA-2.

Genotype characterization of Haematobia irritans from different Brazilian geographic regions based on randomly amplified polymorphic DNA (RAPD) analysis

Blood-sucking diptera are important parasites in bovine production systems, especially regarding confinement conditions. Haematobia irritans, the horn fly, is one of the most troublesome species within bovine production systems, due to the intense stress imposed to the animals. An important aspect while studying the variability within a species is the study of the geographic structure of its populations and, attempting to find out the genetic flow of Brazilian populations of horn fly, the RAPD technique, which is suited for this purpose, has been used. The use of molecular markers generated from RAPD made it possible to identify the geographic origin of samples from different Brazilian geographic regions, as well as to estimate the genotypic flow among the different Brazilian populations of the horn fly.

Genetic polymorphism of fifteen microsatellite loci in Brazilian (blue-egg Caipira) chickens

The purpose of this study was to investigate the genetic polymorphism of fifteen microsatellites loci in Brazilian (blue-egg Caipira) chickens. Samples were collected from 100 blue eggs of Caipira chickens from rural properties in the city of Dois Lajeados, RS. After DNA extraction, the fragments related to molecular markers LEI0248, LEI0221, LEI0214, LEI0192, LEI0217, LEI0254, LEI0194, LEI0212, MCW0371, ADL0278, LEI0234, MCW0183, MCW0216, MCW0330 and MCW0081 were obtained by polymerase chain reaction (PCR). The statistical analysis were carried out with the softwares ARLEQUIN 3.5 version and CERVUS 3.0.3 version. The allelic and genotypic frequencies, deviations from Hardy-Weinberg equilibrium, estimates of observed (HO) and expected (HE) heterozygosity and polymorphic information content (PIC) were obtained for each marker locus. A total of 186 alleles from 15 loci were obtained, with sizes ranging of 83 to 490 base pairs. The medium number of alleles was 12.4, the HE was 0.76±0.14 and HO was 0.49±0.21 and PIC was 0.706. The first conclusion is that the microsatellites used are polymorphic and can be used to genetic studies in chickens. The second is that the "Caipira" chicken (blue eggs) population investigated has a great genic variability, which makes than an important source of genetic resources for future animal breeding programs.

Genetic variability among Commelina weed species from the states of Paraná and São Paulo, Brazil

This work aims to carry out a comparative analysis using RAPD molecular markers in four Commelina weed species from the state of Paraná and C. benghalensis populations from the states of Paraná and São Paulo, Brazil. The genomic plant DNA sample was extracted from the leaves, separated, randomly fragmented and amplified by PCR. Random amplified polymorphic DNA fragments (RAPD markers) were analyzed by using POPGENE statistical program. Eighty-five primer sequences were tested but only three were suitable as molecular markers producing 37 DNA polymorphic fragments for comparisons among four Commelina species and 22 polymorphic fragments for comparisons among C. benghalensis populations. The results showed that there were inter-specific and intra-specific genetic variabilities among Commelina plant genera. Genetic diversity analysis between species indicated four mono-specific clusters and it was suggested to keep C. villosa as one species. Regarding the intra-specific genetic variability of C. benghalensis alone, three groups were verified, although there were 13 populations from two geographical areas. However, these clusters do not correspond to the distinct characteristics verified.

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.