Thesis Abstracts

DNA fingerprinting study of neotropical parrot wild populations (Psittaciformes, Aves) and conservation

(Estudo de populações naturais de psitacídeos neotropicais (Psittaciformes, Aves) por técnica de identificação individual pelo DNA ("DNA fingerprinting"): enfoque em conservação)

Renato Caparroz*

*1998. Departamento de Biologia, Instituto de Biociências, Universidade de São Paulo, São Paulo, SP, Brasil. Master´s thesis. Orienting Professor: Dr. Anita Wajntal.

We estimated the genetic variability of natural populations of eight species of neotropical parrots (five Amazon and three macaws) by using the human minisatellite probes 33.6 and 33.15 developed by Jeffreys et al. (Nature 314: 67-73, 1985). We also estimated the sex ratio and studied the reproductive behavior of macaws with the same procedure. The species studied and their risk of extinction as determined by the International Union for Conservation of Nature (IUCN) were: Amazona a. aestiva (low risk/least concern or LR/lc); Amazona a. amazonica (LR/lc); A. pretrei (vulnerable or VU); A. brasiliensis (endangered); A. kawalli (recently described, probably VU); Ara ararauna (LR/lc); A. chloroptera (LR/lc), and Cyanopsitta spixii (critically endangered). Samples from different localities were available for A. brasiliensis, A. a. amazonica and A. pretrei; however, our analysis did not detect any significant differences between those samples. This result indicates that they probably belong to the same population. In C. spixii the similarity indexes between individuals, with previously unknown familial relationships, were in the range expected for first degree consanguinity. Decreased genetic variability was also present, as expected, in A. brasiliensis and populations of A. ararauna and A. chloroptera. Specific DNA fragments present in all individuals were detected in A. ararauna, A. chloroptera, C. spixii and A. kawalli. This is suggestive of allelic loss, and expected for small isolated populations. We attributed this finding to habitat fragmentation.

The sex-ratios in the macaw populations were close to 1. In one out of five nests of A. chloroptera with two chicks, the similarity index between the fledglings was suggestive of intraspecific brood parasitism or extra-pair copulation.

Our results show that it is possible to detect genetically vulnerable populations, even in non-threatened species. Since habitat destruction and fragmentation is considered to be the main cause of threat for wild populations, the ability to identify vulnerable populations might lead to the involvement of human communities in the preservation of their local ecosystem, using the vulnerable birds as a symbol.

Research supported by FAPESP, CNPq and CAPES.

Publication supported by FAPESP.

Genotoxic action of the sesquiterpene lactone centratherin on mammalian cells in vitro and in vivo

(Avaliação da ação genotóxica da lactona sesquiterpênica centraterina em células de mamíferos in vitro e in vivo)

Regislaine Valéria Burim*

*1998. Departamento de Genética, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brasil. Master's thesis. Orienting Professor: Dr. Catarina Satie Takahashi.

Centratherin (CTN) is a sesquiterpene lactone, extracted from plants of the family Vernonieae (Asteraceae), known for its bacteriostatic activity. Since the acceptance of a substance for medical use requires data about its toxicity, the aim of the present study was to determine the clastogenic and cytotoxic potential of CTN. Tests were made with human lymphocytes in culture and Swiss mouse bone marrow cells in vivo, respectively.

Human lymphocytes in culture were submitted to either continuous treatment or treatment during the G₂ phase of the cell cycle. Chomosomal aberrations (CA), mitotic index (MI), sister chromatid exchanges (SCE) and proliferation index (PI) were analyzed in continuous treatment, whereas in the G₂ treatment only CA and MI were analyzed. Three concentrations of CTN were tested for continuous treatment (0.05, 0.10 and 0.20 µg/ml) and for G₂ treatment (0.10, 0.30 and 0.50 µg/ml). At continuous treatment the 0.20 µg/ml concentration induced a significant increase in the total number of CA and SCE compared to the control, and it reduced the MI. However, CTN did not change PI values. CTN was strongly cytotoxic at concentrations above 0.50 µg/ml in continuous treatment. In the treatment during the G₂ phase, CTN induced a significant increase in the frequency of CA for all concentrations tested, but did not change the MI.

CA and MI were analyzed in the in vivo test system. CTN injected intraperitoneally in mice caused death at concentrations equal to or above 16.7 mg/kg body weight. All three concentrations tested in mice (3.3, 6.7 and 13.3 mg/kg body weight) induced a significant increase in CA compared to the negative control, but did not change MI values. On the basis of these results, we may state that, under the experimental conditions of the present study, CTN was clastogenic and cytotoxic for in vitro and in vivo mammalian systems.

Publicatin supported by FAPESP.

Assessment of the cytotoxic and clastogenic activities of the sesquiterpene lactone lychnopholide in in vitro and in vivo mammalian cells

(Avaliação da atividade citotóxica e clastogênica da lactona sesquiterpênica licnofolido em células de mamíferos in vitro e in vivo)

Renata Canalle*

*1998. Departamento de Genética, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brasil. Master's thesis. Orienting Professor: Dr. Catarina Satie Takahashi.

Lychnopholide (LNP) is a sesquiterpene lactone extracted from Vanillosmopsis erythropappa (Asteraceae) which is known for its antitumoral, antimicrobial and Trypanosoma cruzi-inhibiting activity. In view of the biological properties of this LNP compound, we evaluated the cytotoxic and clastogenic potential of this agent in mammalian cells in vitro and in vivo. The parameters for evaluation were chromosomal aberrations (600 cells/treatment) and mitotic index (6000 cells/treatment) in in vitro and in vivo systems, and sister chromatid exchanges (200 cells/treatment) and proliferation index (400 cells/treatment) in the in vitro assay only.

LNP at concentrations higher than 0.4 mg/ml culture medium was cytotoxic for human lymphocyte cultures, fully inhibiting cell growth in the continuous treatment. The concentrations tested (0.05, 0.1 and 0.2 mg/ml) did not cause a significant increase in the total number of chromosome aberrations or in the mean frequency of sister chromatid exchanges. However, at the highest concentration (0.2 mg/ml) LNF was cytotoxic to the cultures, inducing a significant decrease in mitotic index. In the G₂ phase, concentrations above 0.6 mg/ml interfered with chromosome condensation and fixation, preventing metaphase analysis. In this phase LNF induced a significant increase in the frequency of chromosome aberrations and in the number of altered cells in all treated cultures (0.1, 0.2 and 0.4 mg/ml) but did not change mitotic index, thus not interfering with cell division.

In the in vivo investigation of Swiss mouse bone marrow cells, LNP proved to be quite toxic when applied intraperitoneally at concentrations of 50 and 66.67 mg/kg body weight, causing the death of all treated animals, whereas 33.3 mg/kg caused the death of 50% of the animals. At the lowest LNP concentrations tested (6.67 and 13.33 mg/kg body weight) there was no alteration of any of the parameters analyzed, whereas the next highest concentration (26.67 mg/kg body weight) caused a significant increase in total number of chromosomal aberrations but did not interfere with cell division. Based on these results, we may state that, under these conditions, LNP has a clastogenic effect on both test systems and a cytotoxic effect in the continuous in vitro treatment.

Publication supported by FAPESP.

Morphological, agronomical, isoenzymatic and RAPD characterization of sweet orange varieties - Citrus sinensis (L.) Osbeck

(Caracterização morfológica, agronômica, isoenzimática e por RAPD de variedades de laranja doce - Citrus sinensis (L.) Osbeck)

Edson Tobias Domingues*

* 1998. Departamento de Genética, Escola Superior de Agricultura Luiz de Queiroz, USP, Piracicaba, SP, Brasil. Doctoral thesis. Orienting professor: Dr. Augusto Tulmann Neto.

The sweet orange, Citrus sinensis (L.) Osbeck, is the most important Citrus species in Brazil and in the world; however, few cultivars are employed in Brazilian orchards and little use has been made of germplasm collections to increase the genetic base for this crop. We characterized 61 sweet orange accessions of the germplasm collection at "Centro de Citricultura Sylvio Moreira/IAC". Eleven of them were 'Pêra' clones and six others were similar varieties; 44 genotypes represented the main groups of sweet orange (with high or low acidity, navel and blood fruits). Evaluations of fruit quality and harvesting period, determined by fruit morphology and ripening curves, were made fortnightly from June through December, 1995 to 1997. Intensity of polyembryony in seeds, germinated in the greenhouse and in vitro, and pollen fertility were determined. The reproductive system predominant in sweet orange was evaluated by fruit set analysis after self crossing, open or no pollination. Morphological and production polymorphism, observed by 29 qualitative and 47 quantitative data, were compared with polymorphism of isozymes and RAPD. The rate of polyembryony in seeds was greater than that observed after seed germination in vitro, and this was greater than that obtained in the greenhouse. The variety 'Pêra de Abril' proved to be monoembryonic; the others presented medium to high polyembryony. Viable pollen varied from 12 to 88%, depending on the variety. The main Brazilian sweet orange cultivars, 'Pêra', 'Valência' and 'Natal', had low percentages of viable pollen. Navel oranges had no pollen. Fruit settings were greater in those varieties that presented more evident stigmas and after cross and open pollination, indicating predominance of alogamy in this species. Univariate and multivariate analysis indicated considerable phenotypic variability. The characters responsible for most of the variance were determined by principal component analysis and genotypes were consistently orderer by cluster analysis, grouping, side by side, somatic mutants known to be of common origin. Molecular evaluations using isozymes and RAPDs detected low intra-specific variability, generally originating monomorphic bands. The elevated morphological and agronomic polymorphism compared with low isoenzymatic and molecular polymorphism indicates predominance of somatic mutation as the main source of intra-specific variability in Citrus sinensis (L.) Osbeck.

Publication supported by FAPESP.

Genetic studies of the resistance of Lycopersicon esculentum x L. hirsutum f. glabratum to the South American tomato pinworm (Tuta absoluta)

(Estudos genéticos sobre a resistência de Lycopersicon esculena x L. hirsutum f. glabratum ao oxiúro do tomate sul americano (Tuta absoluta))

Norma Eliane Pereira*

* 1998. Laboratório de Melhoramento Genético Vegetal, Centro de Ciências e Tecnologias Agropecuárias, Universidade Estadual do Norte Fluminense, Campos dos Goytacazes, Rio de Janeiro, RJ, Brasil. Doctoral thesis. Orienting Professor: Dr. Nilton Rocha Leal.

The South American tomato pinworm (Tuta absoluta) is one of the main Brazilian tomato pests and causes large crop losses. The use of pesticides has been the most common method of controlling this insect. However, chemical control is very expensive and causes environmental pollution. Genetic resistance to this insect is not found in cultivated tomato species, but it is known to occur in wild undomesticated species which are related to the cultivated tomato. This research concerned the genetic resistance to Tuta absoluta as well as the mechanisms of resistance such as density of foliar trichomes (type VI adaxial/abaxial) and levels of methyl-ketones (2-tridecanone and 2-undecanone) present in the leaves. The genetic material used was F₁ and F₂ generations Lycopersicon esculentum cv. IPA-6 x L. hirsutum f. glabratum PI 134418. Artificial infestations with T. absoluta were made by placing 1st instar larvae on the adaxial surface of the leaflets, which were then placed in dialysis tubing. The degree of insect foliage feeding and density of type VI trichomes were expressed as areas. The level of methyl-ketones in the foliage was determined by gas chromatography. Differences in resistance to T. absoluta and trichome density were found between genotypes. Genotype x environment interaction for T. absoluta resistance was seen under greenhouse and plastic tunnel conditions. The generation means analysis showed that the results cannot be explained by a simple additive-dominance model, except in the case of the abaxial trichome density trait, suggesting that the segregation distortion could be due to incongruity problems of interspecific crosses or epistatic gene action. The simple correlation coefficients showed a low and negative relationship between resistance to T. absoluta and methyl-ketone content. No associations were found between abaxial type VI trichome density and adaxial type VI trichome density. The heritability analysis showed that the ANOVA estimate was more accurate than the generations variance estimate. Although data of the means of the genotypes grown under three different environmental conditions showed low selection gain, it was responsible for the high coincidence of the best genotypes from each specific environment.

Genetic and functional analysis of plasmids pRJ6 and pRJ9 and comparison to other bacteriocinogenic plasmids of Staphylococcus aureus

(Análise genética e funcional dos plasmídios pRJ6 e pRJ9 e comparação com outros plasmídios bacteriocinogênicos de Staphylococcus aureus)

Selma Soares de Oliveira*

* 1998. Laboratório de Genética Molecular, Instituto de Microbiologia Prof. Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brasil. PhD thesis. Orienting Professor: Dr. Maria do Carmo de Freire Bastos.

pRJ6 and pRJ9 are small Staphylococcus aureus plasmids which code for bacteriocin (Bac) production and immunity (Imm) to Bac action. Bacteriocins encoded by both plasmids exhibited a bactericidal activity against several lactic acid bacteria and strains of Listeria monocytogenes, an important food-borne pathogen. Filter-mating experiments using plasmid derivatives tagged with either Tn551 or Tn917-lac showed that pRJ6, but not pRJ9, could be mobilized by staphylococcal conjugative plasmids. Using transposons as insertional mutagens, we generated a number of inserts that allowed us to locate some genetic functions present on both plasmids. The Bac and Imm structural genes of pRJ6 are part of the same operon, which is located in the HindIII-A fragment, around coordinate 4.0, being transcribed from right to left. However, gene cloning experiments using a staphylococcal vector showed some evidence for the involvement of additional functions of pRJ6 in Bac expression. At least one function involved in pRJ6 mobilization also mapped in the fragment HindIII-A, around coordinate 5.2, and it appears to be transcribed from left to right. The bactericidal action exerted by strains harboring pRJ9 appears to reflect the activity of two Bac genes, whose gene clusters are located at least in part in the HindIII-A and HindIII-C fragments, respectively, and whose combined action results in a broader spectrum of activity and in a higher antagonistic activity.

The 2.5-kb plasmid, pRJ5, which has been used as a vector for cloning of Bac and Imm structural genes, confers constitutive resistance to macrolide-lincosamide-streptogramin B (MLS) antibiotics. It presents a physical map very similar to that of pT48, which encodes MLS-induced resistance. DNA sequencing showed that pRJ5 contains a 28-bp direct tandem duplication in the leader/attenuator region of ermC, the gene that codes for the methylase responsible for MLS resistance. This duplication probably changes the secondary structure of the methylase mRNA, allowing constitutive expression of ermC.

Antimicrobial substance (AMS) production was detected in four strains of S. aureus isolated from cattle in the State of Paraíba, Brazil. The biochemical properties presented by all AMS showed that they are bacteriocins, with a broad spectrum of activity. These strains were shown to harbor plasmids of about 8.0 kb (pRJ34) or 50 kb (pRJ35, pRJ61 and pRJ65). Experiments of plasmid elimination associated the AMS production with the single plasmid present in strain 215FN (pRJ35). Plasmid pRJ34 was found to generate restriction fragment patterns very similar to those of pRJ6. A strong homology between pRJ34 and pRJ6 was detected in the region that would correspond to the structural genes for Bac and Imm. It was observed that pRJ34 codes for a Bac with chemical properties (sensitivity to proteases, heat resistance, activity under anaerobiosis and molecular weight) similar to those specified by pRJ6 and pRJ29, conferring cross immunity to them. The Bac produced by strain 215FN presented a broad spectum of action, which was different from all other bacteriocins tested. No cross immunity was observed between 215FN and other bacteriocin-producing strains, suggesting that the antimicrobial substance produced by 215FN is a new Bac.

The activity of different Bac genes produced by S. aureus strains (MB92, A70, 146L, 215FN and A53) was evaluated in comparison to strains that are pathogenic for cattle. Strain A53 showed a broader spectrum of action, being active against Moraxella bovis, the agent of pink eye in cattle, and all 65 Staphylococcus sp. strains involved in bovine mastitis tested, suggesting that its Bac can be used for treating infections caused by these bacteria.

The molecular genotyping of Bac⁺ strains of S. aureus was determined by the use of the AP-PCR and rep-PCR techniques. Strains containing small Bac plasmids (A70, 146L, MB91, MB92 and MB93) were classified into different clones, although a good similarity among them was detected (78%). Strains MB92 and MB93 seem to be more closely related (similarity of 97%) than the others. Among the Bac⁺ strains tested, only 215FN and A53 presented a coefficient of similarity of 100%. The PCR techniques used also showed that RN7242 belongs to a clone different from that of RN450, RN451 and RN8411. These four strains have been used as recipients of Bac plasmids in gene transfer experiments. The differences exhibited by RN7242 could be related to an increased Bac expression in this strain. According to this study, the AP-PCR and rep-PCR techniques allow a good discrimination among bacteriocin-producing strains of S. aureus.

This study was supported by grants from CNPq, FAPERJ, FUJB, FINEP, and PRONEX (BRAZIL) and from TWAS (RGA No. 96-273).

Race identification and genetic diversity of Uromyces appendiculatus var. appendiculatus and resistance inheritance in the common bean

(Identificação de raças, diversidade genética de Uromyces appendiculatus var. appendiculatus e herança da resistência no feijoeiro)

Fábio Gelape Faleiro*

*1997. Núcleo de Biotecnologia Aplicada à Agropecuária - BIOAGRO, Departamento de Biologia Geral, Universidade Federal de Viçosa, Viçosa, MG, Brasil. Master's thesis. Orienting professor: Dr. Everaldo Gonçalves de Barros.

Fifteen monopustular isolates collected in four counties of the State of Minas Gerais, Brazil, were grouped into 13 races. To facilitate exchange of information and the classification of isolates into races, we proposed a simplified classification scale and a new system for race designation. Even with this simplified scale no similarities were observed between the races identified in this work and those identified previously in Brazil and in the United States. We established a protocol for DNA extraction from uredospores in order to study the genetic diversity of the pathogen based on RAPD markers. Each DNA sample was amplified with 20 primers, yielding a total of 262 fragments, 208 of which were polymorphic. The genetic distances among the isolates based on these data varied between 9.6 and 83.12%. Cluster analyses based on the genetic distance matrix confirmed the high variability of U. appendiculatus and demonstrated that isolates belonging to the same race or collected in the same region tend to cluster in the same groups. Fourteen cultivars were initially evaluated for resistance inheritance. As cultivar Ouro Negro showed no pustules when inoculated with a mixture of U. appendiculatus spores, it was used as a resistant parent. Populations F₁, F₂ and BC from the crosses Pinto 111 x Ouro Negro and Rudá (A285) x Ouro Negro were tested for resistance to U. appendiculatus race 4. Symptoms of the plants were scored 10 and 20 days after inoculation and the data were analyzed by the chi-square test. The data collected 10 days after inoculation suggest that resistance is governed by more than one gene. However, resistance determined 20 days after inoculation was determined to be monogenic. Our data confirm the extensive variability of U. appendiculatus, demonstrate that RAPD markers can be used to aid the studies of genetic variability of the pathogen, and also that resistance of the common bean to rust is controlled by a major gene and several minor genes.

Publication supported by CAPES.

Genetic mapping of quantitative trait loci associated with morphology, photosynthesis and yield in the bean (Phaseolus vulgaris L.)

(Mapeamento de locos de características quantitativas associados com a morfologia, a fotossíntese e o rendimento do feijão (Phaseolus vulgaris L.))

Ricardo Bressan-Smith^*

*1998. Laboratório de Melhoramento Genético Vegetal, Centro de Ciências e Tecnologias Agropecuárias, Universidade Estadual do Norte Fluminense, Rio de Janeiro, RJ, Brasil. Doctoral thesis. Orienting Professor: Dr. Messias Gonzaga Pereira.

Bean morphology, photosynthesis and yield (Phaseolus vulgaris L.) were investigated by means of quantitative genetics based on DNA markers. Random amplified polymorphic DNA markers (RAPD) were used to construct a partial genetic linkage map for the bean (N = 11) and to map quantitative trait loci (QTL) which control photosynthesis, plant morphology and yield. Eighty-eight plants from an F₂ population derived from a cross between a snap bean (cv HAB-52) and a common bean (cv BAC-6) were used. Among 160 random primers (10 bases) tested for the two parents, 73% showed at least one polymorphic band, yielding 312 polymorphic markers (2.66 markers/primer). The partial linkage map consisted of 72 RAPD loci and one morphologic locus distributed along 13 linkage groups, covering a distance of 1032.1 cM, with an average of 14 cM between adjacent loci. Twenty-eight markers showed distorted segregation, revealed by means of the chi-square test, and 16 were mapped. Linkage group B had six distorted markers, suggesting the presence of a gametophytic factor affecting the segregation proportions. Interval mapping identified 36 QTL associated with morphology, photosynthesis and yield, described as follows: one for plant height, three for canopy diameter, two for leaf width, one for leaf length, two for leaf width/length ratio, three for pod weight, two for pod length, one for total seed weight, two for number of seeds/pod, one for carbon dioxide assimilation rate, two for leaf temperature, five for stomatal conductance, one for internal CO₂ concentration, two for stomatal resistance, one for leaf chlorophyll a content, one for leaf chlorophyll b content, two for chlorophyll a/b ratio, one for total leaf chlorophyll content, one for carotenoid content, one for chlorophyll/carotenoid ratio and two for total soluble leaf protein content. For each QTL identified, the most likely position on the linkage map, the magnitude of effects, the gene action, and the source (which parents) of the favorable allele increasing the mean were evaluated. The phenotypic traits were evaluated based on means and variances of 193 F₂ plants in order to obtain heritability, gene effects and gene actions. Heritability was medium to high for most of the traits. Gene action and effect on phenotypic traits were calculated and then compared with gene action and effect on the correspondently identified QTL, showing similarity in some cases. Most of the phenotypic distributions did not deviate significantly from a normal distribution. The photosynthetic assimilation of CO₂ did not correlate with yield components; however, positive correlation was observed with stomatal conductance and leaf temperature. The combination of the characteristics cited above could allow the generation of lines, initiated from individuals of an F₂ population, better adapted to environments which suffer from water deficits. A high correlation was found among chlorophyll a, chlorophyll b and carotenoids, further demonstrated by the identification of closely linked or pleiotropic QTL on linkage group B. These results demonstrate that gene polymorphism affecting photosynthesis and yield can be identified and utilized in breeding programs.

X-chromosomal inactivation studies in 49,XXXXY and 49,XXXXX polysomies

(Estudo da inativação do cromossomo X nas polissomias 49,XXXXY e 49,XXXXX)

Lúcia de Fátima Marques de Moraes*

1997. Programa de Biologia Molecular, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, UFRJ, Rio de Janeiro, RJ, Brasil. Master's thesis. Orienting Professor: Dr. Juan Clinton Llerena Jr.

Cytogenetic and molecular analyses were made on five patients with 49,XXXXY and 49,XXXXX to study the X-inactivation process. 49,XXXXY and 49,XXXXX are rare numerical sex chromosomal aberrations with an incidence of 1:85,000 livebirths. Among the clinical abnormalities observed, skeletal and mental retardation are considered the main features. Clinical similarities with 47,XXY and 48,XXXY cases have been reported, however a greater decrease in mental capabilites has been observed among the 49,XXXXY/49,XXXXX patients. Two hypotheses have been put forward to explain these clinical observations: a) an excess of active X chromosome segments corresponding to the excess of chromosomes and/or b) an asynchronous pattern of X-replication with an overall active product different from that expected by Lyon's law. In our investigation we characterized the cytogenetic X chromosome inactivation pattern with 5-BrdU in peripheral lymphocyte cultures of five patients with 49,XXXXY (#4) and 49,XXXXX (#1) karyotype. An obvious asynchronic replication pattern was present in all cases. FISH investigation using a digoxigenin-XIST probe revealed the integrity of locus XIST (Xq13) in all X chromosomes. Dosage assays of the glucose-6-phosphate dehydrogenase (G-6-PD) in these patients turned out to be compatible with the presence of only one active X chromosome per cell. Parental origin of the extra X chromosome set was investigated using the polymorphic pattern of the first exon for the human androgen receptor. All four cases investigated showed a maternal derived set of extra X chromosomes, most likely as a consequence of first and second meiotic non-disjunction errors. Molecular analysis of the methylation of X chromosomes in three non-mosaic patients (49,XXXXY/49,XXXXX) and their mothers revealed a skewed inactivation pattern compatible with non-random inactivation.

Analysis of genetic variability in Pimelodidae and Rhamdiidae (Pisces - Siluriformes) from the Tibagi River basin

(Análise da variabilidade genética em Pimelodidae e Rhamdiidae (Pisces - Siluriformes) da bacia do rio Tibagi)

Fernanda Simões de Almeida

1998. Mestrado em Genética e Melhoramento, Centro de Ciências Biológicas, Departamento de Biologia Geral, Universidade Estadual de Londrina (UEL), Londrina, PR, Brasil. Master's thesis. Orienting Professor: Dr. Leda Maria Koelblinger Sodré.

Genetic variability expressed differently within and among populations does not only facilitate adaptation to a particular habitat, but also sets limits to colonization and distribution.

RAPD and isoenzyme techniques were used for genetic variability analyses of six fish species, Iheringichthys labrosus, Pimelodus maculatus, Pimelodus absconditus, Pimelodella aff. gracillis, Pimelodella sp. and Pinirampus pirinampu, collected from five locations in the Tibagi River basin: Sertaneja, Jataizinho and Londrina (belonging to lower Tibagi), Sapopema (medium Tibagi) and Tibagi (upper Tibagi). Quantification of the genetic variability was measured through the mean of the proportion of polymorphic loci (m)

I.labrosus, collected at four places, was the species that presented the largest genetic variability (m 53.7%). In the dendrogram obtained the individuals of each of these sites grouped together, corroborating the idea that this species is sedentary or has restricted displacement.

P. maculatus is the only one of these species which migrates great distances. It was collected at three places. For this species the m was of 30.2%. The analysis of the dendrogram shows that the individuals of a given site did not group separately from the others.

P. absconditus, collected from three sites, had a m of 47.4%. This species has an external morphology similar to I. labrosus, nevertheless it was possible to separate them with the RAPDs and isoenzymes.

P. aff. gracillis was only collected at Sertaneja. It had a low proportion of polymorphic loci (18.18%). Pimelodella sp. presented a m of 43.2%. It was collected at four sites. In the analysis of the dendrogram, the individuals of each of these places grouped separately.

P. pirinampu was also collected only at Sertaneja, and had a low proportion of polymorphic loci (27.2%).

The RAPD analysis indicates that for increased precision of the quantification of genetic variability, it would be best to analyze at least five individuals of the species or population. In the species collected in more than one place (I. labrosus, P. absconditus, P. maculatus and Pimelodella sp.), the low number of individuals analyzed in some places coincides with a low value of genetic variability. However, for the species P. aff. gracillis and P. pirinampu the low genetic variability found, in spite of 10 and 12 individuals analyzed respectively, is due to restricted mobility.

In the comparative analysis of the profiles of RAPD each of the six species presented different patterns. Those within the same genus were similar. The analysis of the dendrogram shows the formation of two groups, one constituted by the species P. maculatus, P. absconditus, I. labrosus and P. pirinampu (Pimelodidae) and the other by P. aff. gracillis and Pimelodella sp. (Rhamdiidae). The RAPD technique proved to be useful for the analysis of natural populations of fish, and was capable to identifying species.

Publication Dates

• Publication in this collection
  02 June 1999
• Date of issue
  Mar 1999