Characterization of new Schistosoma mansoni microsatellite loci in sequences obtained from public DNA databases and microsatellite enriched genomic libraries

In the last decade microsatellites have become one of the most useful genetic markers used in a large number of organisms due to their abundance and high level of polymorphism. Microsatellites have been used for individual identification, paternity tests, forensic studies and population genetics. Data on microsatellite abundance comes preferentially from microsatellite enriched libraries and DNA sequence databases. We have conducted a search in GenBank of more than 16,000 Schistosoma mansoni ESTs and 42,000 BAC sequences. In addition, we obtained 300 sequences from CA and AT microsatellite enriched genomic libraries. The sequences were searched for simple repeats using the RepeatMasker software. Of 16,022 ESTs, we detected 481 (3%) sequences that contained 622 microsatellites (434 perfect, 164 imperfect and 24 compounds). Of the 481 ESTs, 194 were grouped in 63 clusters containing 2 to 15 ESTs per cluster. Polymorphisms were observed in 16 clusters. The 287 remaining ESTs were orphan sequences. Of the 42,017 BAC end sequences, 1,598 (3.8%) contained microsatellites (2,335 perfect, 287 imperfect and 79 compounds). The 1,598 BAC end sequences 80 were grouped into 17 clusters containing 3 to 17 BAC end sequences per cluster. Microsatellites were present in 67 out of 300 sequences from microsatellite enriched libraries (55 perfect, 38 imperfect and 15 compounds). From all of the observed loci 55 were selected for having the longest perfect repeats and flanking regions that allowed the design of primers for PCR amplification. Additionally we describe two new polymorphic microsatellite loci.

Genotyping of the MTL loci and susceptibility to two antifungal agents of Candida glabrata clinical isolates

The opportunistic fungal pathogen Candida glabrata is the second most common isolate from bloodstream infections worldwide and is naturally less susceptible to the antifungal drug fluconazole than other Candida species. C. glabrata is a haploid yeast that contains three mating-type like loci (MTL), although no sexual cycle has been described. Strains containing both types of mating information at the MTL1 locus are found in clinical isolates, but it is thought that strains containing type a information are more common. Here we investigated if a particular combination of mating type information at each MTLlocus is more prevalent in clinical isolates from hospitalized patients in Mexico and if there is a correlation between mating information and resistance to fluconazole and 5-fluorocytosine. We found that while both types of information at MTL1 are equally represented in a collection of 64 clinical isolates, the vast majority of isolates contain a-type information at MTL2 and α-type at MTL3. We also found no correlation of the particular combination of mating type information at the three MTL loci and resistance to fluconazole.

Mapping of quantitative trait loci for thermosensitive genic male sterility in indica rice

The objective of this work was to select and use microsatellite markers, to map genomic regions associated with the genetic control of thermosensitive genic male sterility (TGMS) in rice. An F2 population, derived from the cross between fertile and TGMS indica lines, was used to construct a microsatellite-based genetic map of rice. The TGMS phenotype showed a continuous variation in the segregant population. A low level of segregation distortion was detected in the F2 (14.65%), whose cause was found to be zygotic selection. There was no evidence suggesting a cause-effect relationship between zygotic selection and the control of TGMS in this cross. A linkage map comprising 1,213.3 cM was constructed based on the segregation data of the F2 population. Ninety-five out of 116 microsatellite polymorphic markers were assembled into 11 linkage groups, with an average of 12.77 cM between two adjacent marker loci. The phenotypic and genotypic data allowed for the identification of three new quantitative trait loci (QTL) for thermosensitive genic male sterility in indica rice. Two of the QTL were mapped on chromosomes that, so far, have not been associated with the genetic control of the TGMS trait (chromosomes 1 and 12). The third QTL was mapped on chromosome 7, where a TGMS locus (tms2) has recently been mapped. Allelic tests will have to be developed, in order to clarify if the two regions are the same or not.

Identification of quantitative trait loci for resistance against soybean sudden death syndrome caused by Fusarium tucumaniae

The objective of this work was to identify genomic regions that underlie resistance to Fusarium tucumaniae sp. nov., the causing agent of sudden death syndrome (SDS) in soybean in South America, using a population with a genetic background different from that previously reported for Fusarium virguliforme sp. nov. (F. solani f. sp. glycines), also responsible for SDS in soybean. Although major genes and quantitative trait loci (QTL) for SDS resistance have been identified, little is known about the same disease caused by Fusarium tucumaniae sp. nov., in South America. To identify genetic factors related to resistance to F. tucumaniae and DNA markers associated with them, a QTL analysis was performed using recombinant inbred lines. The map locations of the four loci, here identified, differed from those SDS resistance QTL previously described. It was screened a residual heterozygous line (RHL), which was heterozygous around the most effective QTL, RSDS1, and homozygous for the other genomic regions. The genetic effect of RSDS1 was confirmed using near-isogenic lines (NIL) derived from the RHL. The line which was homozygous for the Misuzudaizu genotype showed resistance levels comparable with that of the line homozygous for the Moshidou Gong 503 genotype.

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.

Variabilidade genética de Eugenia uniflora L. em remanescentes florestais em diferentes estádios sucessionais

A compreensão da diversidade genética fornece elementos básicos sobre a dinâmica e funcionamento de populações, auxiliando na conservação e uso sustentável das espécies. Supõe-se que populações sucessionais precoces poderiam ser geneticamente mais diferenciadas do que populações sucessionais mais tardias. Visando testar esta hipótese, o presente trabalho teve como objetivo analisar a variabilidade genética de populações de Eugenia uniflora L. em manchas florestais em diferentes estádios sucessionais. Foram selecionadas duas áreas em diferentes estádios de sucessão, sendo a primeira em estádio inicial e a segunda em estádio avançado. A área de estudo apresenta um remanescente florestal em transição de Floresta Ombrófila Mista e Floresta Estacional Semidecídua. Por meio da técnica de RAPD (Random Amplified Polymorphic DNA) e análise multivariada, a diversidade gênica esperada e a porcentagem de loci polimórficos foram estimadas, além da similaridade genética entre as populações de cada mancha florestal e a diversidade de cada área por meio do índice de diversidade de Simpson. Os resultados indicaram 79% de loci polimórficos para a área em estádio avançado e 70% para a área em estádio inicial de sucessão. A similaridade genética entre pares de indivíduos variou entre 0,55 e 0,86 na área em estádio inicial de sucessão e entre 0,45 e 0,78 para a área em estádio avançado. Não houve diferenças significativas entre a diversidade das duas áreas (P = 89). Um escalonamento multidimensional não-métrico indicou menor distância genética entre os indivíduos da área em estádio inicial. Da mesma forma, uma análise de similaridade - ANOSIM indicou separação entre os indivíduos das duas áreas.

Diagnosis of plague and identification of virulence markers in Yersinia pestis by multiplex-PCR

We have developed a procedure for the rapid diagnosis of plague that also allows the identification of prominent virulence markers of Y. pestis strains. This procedure is based upon the use of a single polymerase chain reaction with multiple pairs of primers directed at genes present in the three virulence plasmids as well as in the chromosomal pathogenicity island of the bacterium. The technique allowed the discrimination of strains which lacked one or more of the known pathogenic loci, using as template total DNA obtained from bacterial cultures and from simulated blood cultures containing diluted concentration of bacteria. It also proved effective in confirming the disease in a blood culture from a plague suspected patient. As the results are obtained in a few hours this technique will be useful in the methodology of the Plague Control Program.

MOLECULAR CHARACTERIZATION AND SEQUENCE PHYLOGENETIC ANALYSIS OF SURFACE ANTIGEN 3 (SAG3) GENE OF LOCAL INDIAN ISOLATES (CHENNAI AND IZATNAGAR) OF Toxoplasma gondii

Context and objective:The molecular characterization of local isolates of Toxoplasma gondii is considered significant so as to assess the homologous variations between the different loci of various strains of parasites.Design and setting:The present communication deals with the molecular cloning and sequence analysis of the 1158 bp entire open reading frame (ORF) of surface antigen 3 (SAG3) of two Indian T. gondii isolates (Chennai and Izatnagar) being maintained as cryostock at the IVRI.Method:The surface antigen 3 (SAG3) of two local Indian isolates were cloned and sequenced before being compared with the available published sequences.Results:The sequence comparison analysis revealed 99.9% homology with the standard published RH strain sequence of T. gondii. The strains were also compared with other established published sequences and found to be most related to the P-Br strain and CEP strain (both 99.3%), and least with PRU strain (98.4%). However, the two Indian isolates had 100% homology between them.Conclusion:Finally, it was concluded that the Indian isolates were closer to the RH strain than to the P-Br strain (Brazilian strain), the CEP strain and the PRU strains (USA), with respect to nucleotide homology. The two Indian isolates used in the present study are known to vary between themselves, as far as homologies related to other genes are concerned, but they were found to be 100% homologous as far as SAG3 locus is concerned. This could be attributed to the fact that this SAG3 might be a conserved locus and thereby, further detailed studies are thereby warranted to exploit the use of this particular molecule in diagnostics and immunoprophylactics. The findings are important from the point of view of molecular phylogeny.

Genetic diversity and differentiation in natural Plasmodium falciparum populations inferred by molecular typing of the merozoite surface proteins 1 and 2

Genetic diversity and differentiation, inferred by typing the polymorphic genes coding for the merozoite surface proteins 1 (Msp-1) and 2 (Msp-2), were compared for 345 isolates belonging to seven Plasmodium falciparum populations from three continents. Both loci yielded similar estimates of genetic diversity for each population, but rather different patterns of between-population differentiation, suggesting that natural selection on these loci, rather than the transmission dynamics of P. falciparum, determines the variation in allele frequencies among populations.

Genetic variability among Anopheles species belonging to the Nyssorhynchus and Anopheles subgenera in the Amazon region

INTRODUCTION: Isoenzymatic analyses were performed involving species of the Nyssorhynchus and Anopheles subgenera in order to estimate the intra and interspecies genetic variability. METHODS: Mosquitoes were caught at different localities in the Amazon region. The collection and rearing of mosquitoes in the laboratory followed specific protocols. For the genetic variability analyses, the technique of horizontal electrophoresis on starch and starch-agarose gel with appropriate buffer systems was used. The alloenzyme variation was estimated using the Biosys-1 software. RESULTS: Out of the 13 loci, eight were polymorphic. Anopheles nuneztovari presented the largest number of alleles per locus, while the smallest number was detected in Anopheles marajoara from Macapá. The largest number of polymorphic loci was found for Anopheles marajoara from Maruanum and the smallest for Anopheles benarrochi (Guayará Mirim). Anopheles darlingi (Macapá) presented the greatest heterozygosity (Ho = 0.167 ± 0.071), while the lowest heterozygosity (Ho = 0.045 ± 0.019) was observed in Anopheles intermedius (Pacoval) of the subgenus Anopheles. Wright's F coefficient revealed considerable genetic structuring between the populations of Anopheles darlingi (Fst = 0.110) and between the populations of Anopheles marajoara (Fst = 0.082). CONCLUSIONS: Considering all the species studied, the genetic distance ranged from 0.008 to 1.114. The greatest distance was between Anopheles mattogrossensis and Anopheles oswaldoi, while the smallest was between the Anopheles benarrochi populations.

Biologia de Anofelinos Amazônicos. VI. Variabilidade Enzimática em ANOPHELES DARLINGI Root. (DIPTERA, CULICIDAE)

São relatados resultados preliminares sobre o polimorfismo protético de populuções naturais de Anopheles darlingi, procedentes de duas localidades da região amazônica-Ariquemes (Rondônia) e Rodovia PA-422 (Pará). Pata as esterases, foi detectada variação para o locus Est-2na população de Ariquemes, enquanto foi monomórfico na população da PA-422, sendo verificados nesta última, apenas indivíduos com o genótipo Est2*F/Est2*F. A freqüência do alelo Est2*F foi estimada em 0,80 na população de Ariquemes, valor este muito maior do que o observado na população da BR-174 (Manaus/Boa Vista), em estudos anteriores, que foi de 0,48. Os resultados para o sistema da leucina aminopeptidase (LAP) sugerem que a enzima é controlada por três loci, aparentemente monomórficos em ambas as populações.

BIOLOGIA DE ANOFELINOS AMAZÔNICOS. XIV. ISOENZIMAS DE ESTERASE EM ANOPHELES TRIANNULATUS (NEIVA & PINTO, 1922)

Foram estudadas populações de Anopheles triannulatusprocedentes da hidrelétrica de Balbina (AM), quanto ao sistema das esterases, usando-se gel de poliacrilamida e como substrato α-naftil propionato A análise eletroforética possibilitou identificar seis zonas de atividade e o mecanismo de herança foi estudado para cinco locique foram polimórficos. Foram detectados dois alelos para as esterases 1, 2 e 4 e três para as esterases 3 e 5, sendo todos de ação codominante. Os dados de frequências gênicas mostraram-se diferentes para os alelos em cada locus.Os valores de qui-quadrados para as frequências genotípicas evidenciaram que a população não está em equilíbrio para todos os loci,sendo as frequências observadas dos homozigotos superiores as esperadas. Esses resultados foram interpretados como decorrentes de alterações ambientais que estão ocorrendo na área em estudo.

Outcrossing rate of Couratari multiflora (J.Smith) Eyma (Lecythidaceae), a low-density tropical tree species from a central Amazonian rainforest

A multilocus mixed-mating model was used to evaluate the mating system of a population of Couratari multiflora, an emergent tree species found in low densities (1 individual/10 ha) in lowland forests of central Amazonia. We surveyed and observed phenologically 41 trees in an area of 400 ha. From these, only four mother trees were analyzed here because few of them set fruits, which also suffered high predation. No difference was observed between the population multilocus outcrossing rate (t mp = 0.953 ± 0.040) and the average single locus rate (t sp = 0.968 ± 0.132). The four mother trees were highly outcrossed (t m ~ 1). Two out of five loci showed departures from the Hardy-Weinberg Equilibrium (HWE) expectations, and the same results occurred with the mixed-mating model. Besides the low number of trees analyzed, the proportion of loci in HWE suggests random mating in the population. However, the pollen pool was heterogeneous among families, probably due to both the small sample number and the flowering of trees at different times of the flowering season. Reproductive phenology of the population and the results presented here suggest, at least for part of the population, a long-distance pollen movement, around 1,000 m.

Identification of duplicates of cassava accessions sampled on the North Region of Brazil using microsatellite markers

Duplicates are common in germplasm banks and their identification is needed to facilitate germplasm bank management and to reduce maintenance costs. The aim of this work was to identify duplicates of cassava from a germplasm bank in Eastern Amazon, which had been previously characterized both morphological and agronomically. In order to be genotyped with 15 microsatellite loci, 36 accessions were selected. These accessions were classified into 13 groups of similar morpho-agronomical characteristics. All loci were polymorphic, and 75 alleles were identified, with an average of five alleles per loci and H E = 0.66. There were determined 34 pairs of genotypes with identical multiloci profiles and the probability of genetic identity was 1.1x10-12 with probability of exclusion of 99.9999%. Among these duplicates, there are accessions sampled on different years and places, but with different names and accessions with the same name sampled in different places and years. The study identified genotypes that are grown in different places and that have been maintained over the years by local farmers.

Soldadura por uma das extremidades de dois cromossômios homólogos do tityus

In this paper the author describes a very interesting case of union of two homologous chromosomes of the scorpion Tityus bahiensis just by the opposite extremities. The two normal pairs of chromosomes behave as ordinarily, the members of each pair showing at times a slight disturbance in their regular parallelism. The complex chromosome, on the contrary, behaves itself as if it were devoid of kinetochores, that is, it does not orient like normal chromosomes nor reveal any kind of active movement. The fusion of the chromosomes has resulted from terminal breakage at the opposite ends, the correspondig fragments having been found unpaired in a cell in which two pairs of chromosomes were present. Consequently, the compound chromosome, like the normal ones, is provided with a kinetochore at each one of the free ends. Being thus a centric chromosome its behavior, or more exactly, its kinetic inactivity may be compared with that of the monovalents found elsewhere in meioses. It is due o the failure of a partner. The fusion of two homologous chromosomes has transformed them into a new chromosomal unit in whose corresponding parts the ability of pairing was entirely abolished. This result is in full contradiction with the theory of a point-to point attraction between homologous chromosomes attributed to particular power of the genes, since, if genes really exist, being placed in their original loci, they would promote the union side by side of the members of the compound chromosome. If an attraction loci-to-loci should prevail the compound chromosome would be bent as in Fig. 8, C or form a ring similar to the loops observed in the inverted segment of sailvary chromosomes of Drosophila, as represented in the Fig. 8, D and this, in accordance with the order of the loci resulting from an union of corresponding or opposite ends of the fused chromosomes, as indicated in the Fig, 8 A and B. The evidence in hand points to a fusion by non homologous extremities. The expected rings, however, have never been found in metaphase plates. From this fact the author concludes that there is no point-to-point attraction between chromosomes, a conclusion in full agreement with the behavior of Hemipteran chromosomes which, in spite of geing composed of two equivalent halves do not bend in order to adjust the corresponding loci. (Cf. the papers on Hemiptera published by the author in this volume).