Denise Aparecida Andrade de Oliveira

Deep barcode divergence in Brazilian freshwater fishes: the case of the São Francisco River basin

Background and aims. The application of DNA barcoding as a global standard for fish identification is probing diverse worldwide realms (Nearctic, Australian and the Neotropics) and environments (e.g. marine and freshwater). Comparing the patterns of sequence divergence among conspecific and congeneric taxa between realms can provide valuable information on recent evolutionary histories of lineages as barcode data accumulates. Materials and methods. Herein, we have analyzed over 100 species (around 50%) of the Neotropical fish fauna from the São Francisco River, in southeast Brazil. Our aims were to test the performance of DNA barcoding in this biodiversity-rich region, and to compare patterns of genetic divergence with previous studies. Results. The mean Kimura two-parameter distances within species, genera, families, orders, and classes were 0.5, 10.6, 21.0, 22.7, and 24.4%, respectively, with 100% of the species examined successfully differentiated by barcoding. With the exception of Astyanax bimaculatus lacustris, Piabina argentea, and Bryconamericus stramineus, all other species yield a single, cohesive cluster of barcode sequences. The average ‘nearest-neighbor distance’ was 11.12%, 21-fold higher than the mean within species distance of around 0.54%. In a few instances, deep lineage divergences among conspecifics (up to 10%) and congenerics (up to 22.9%) taxa were revealed. Conclusions. Reflecting possible cases of cryptic speciation and the deeper phylogeographic history of São Francisco fish fauna, with some higher clades extending back into the late Cretaceous and Cenozoic (90 mya), when much of the diversification of the Neotropical region apparently took place. In addition, barcodes also highlighted misidentifications and helped to document range extensions for known species.

DNA barcoding unveils a high rate of mislabeling in a commercial freshwater catfish from Brazil

Background and Aims. Molecular markers have contributed to species authentication by flagging mislabeling and the misidentification of commercial landings. Such tools are of great value since the market substitution of fish of lower value for highly commercialized species is expected to become more pronounced due to a shortage of natural stocks. Materials and Methods. Here we report on the molecular identification 4results from processed fish products (i.e. fillets) and whole fishes sold in Brazilian markets under the common name surubim (Pseudoplatystoma spp.). Results. DNA barcoding revealed the incorrect labeling of around 80% of all samples analyzed, with mislabeling being more pronounced within fillets rather than whole fish. Conclusion. To our knowledge, this is the first report correlating the rate of fraud with processed fish products. The establishment of an official list of acceptable common names for freshwater fish and seafood is urgently needed in Brazil for further trade regulations to take place.

Caracterização genética de seis plantéis comerciais de tilápia (Oreochromis) utilizando marcadores microssatélites

Two hundred and thirty five individuals from six commercial stocks of tilapias (Ceara, Chitralada, Israel, Nilotica, Taiwan and Red) from the Southeastern region of the country were genetically characterized using five microsatellite loci. The results suggest large genetic difference among the stocks, estimated through the fixation allele index (Fst = 0.3263), and a considerable loss of heterozigosity accurs in most of the stocks, according to the population inbreeding coefficient (Fis=0.0486). The Israel and Nilotica stocks were genetically similar (Ig=0.6663), while Chitralada and Taiwan showed less genes in common (Ig=0.2463). The Red stock was the most distinct stock.

Association of the bovine major histocompatibility complex (BoLA) BoLA-DRB3 gene with fat and protein production and somatic cell score in Brazilian Gyr dairy cattle (Bos indicus)

The effect of the bovine major histocompatibility complex (BoLA) locus on animal health may be due to a direct action of its alleles on immune functions, whereas its indirect effect on production traits might be explained by the better general health conditions of more productive animals. In the present study, the BoLA-DRB3 gene was investigated in 1058 cows belonging to seven Brazilian Gyr Dairy herds (Bos indicus, Zebu cattle). A total of 37 alleles were identified, 15 of them described for the first time in a Zebu breed. A highly significant association (p < 0.02) was observed between allele *54 and a decrease (-26.1 kg) in milk protein yield and there was a significant association (p < 0.05) between this allele and lower (-26.07 kg) milk fat yield. There was also a significant association (p < 0.05) between allele *6 and decreased (-12.47 kg) milk protein and allele *7 and increased (12.72 kg) milk protein. There were also indications of association (p < 0.10) between somatic cell score (SCS) and alleles *3 (SCS increased by 0.54 units) and *31 (SCS increased by 0.46 units). The highly significant association of allele *54 with lower protein yield suggests the possible use of this allele in marker-assisted selection programs.

Frequency of benzimidazole resistance in Haemonchus contortus populations isolated from buffalo, goat and sheep herds

Anthelmintic resistance is an increasing problem that threatens livestock production worldwide. Understanding of the genetic basis of benzimidazole resistance recently allowed the development of promising molecular diagnostic tools. In this study, isolates of Haemonchus contortus obtained from goats, sheep and buffaloes raised in Brazil were screened for presence of the polymorphism Phe200Tyr in the β-tubulin 1 gene, which confers resistance to benzimidazole. The allelic frequency of the mutation conferring resistance ranged from 7% to 43%, and indicated that resistance to benzimidazole could be found in nematodes isolated from all the ruminant species surveyed. Although significant variation in the frequency of the F200Y mutation was observed between different herds or host species, no significant variation could be found in populations isolated from animals within the same herd. These findings suggest that screening of samples from a few animals has the potential to provide information about the benzimidazole resistance status of the entire herd, which would enable a considerable reduction in the costs of diagnosis for the producer. Molecular diagnosis has practical advantages, since it can guide the choice of anthelmintic drug that will be used, before its application in the herd, thus reducing the economic losses driven by anthelmintic resistance.

Perfil proteico de tilápia nilótica chitralada (Oreochromis niloticus), submetida ao estresse crônico por hipóxia

This study evaluated the secundary response variation to hypoxia stress in a strain of chitralada nilotic tilapia (Oreochromis niloticus) during 18 days, in a recirculation system. The effects were measured by the proteic electrophoretic profile and the difference between the genders. One hundred twenty-six fish were used, 60 males and 66 females, both averaging 800g. The chronic hypoxia stress altered (P<0.05) the relative values of albumin, α+β-globulins, and γ-globulin; modified (P<0.05) the levels of total protein due to an increase in the male group; significantly decreased of the absolute mean values of albumin due to a decrease in the female group; and decreased the g-globulin values in males. The proteic profile, albumin, and γ-globulin were influenced by gender.

Nucleotide excision repair in Trypanosoma brucei: specialization of transcription-coupled repair due to multigenic transcription

Nucleotide excision repair (NER) is a highly conserved genome repair pathway acting on helix distorting DNA lesions. NER is divided into two subpathways: global genome NER (GG‐NER), which is responsible for repair throughout genomes, and transcription‐coupled NER (TC‐NER), which acts on lesions that impede transcription. The extent of the Trypanosoma brucei genome that is transcribed is highly unusual, since most genes are organized in multigene transcription units, each transcribed from a single promoter. Given this transcription organization, we have addressed the importance of NER to T. brucei genome maintenance by performing RNAi against all predicted contributing factors. Our results indicate that TC‐NER is the main pathway of NER repair, but only CSB, XPBz and XPG contribute. Moreover, we show that UV lesions are inefficiently repaired in T. brucei, perhaps due to preferential use of RNA polymerase translesion synthesis. RNAi of XPC and DDB was found to be lethal, and we show that these factors act in inter‐strand cross‐link repair. XPD and XPB appear only to act in transcription, not repair. This work indicates that the predominance of multigenic transcription in T. brucei has resulted in pronounced adaptation of NER relative to the host and may be an attractive drug target.

Microsatellite markers for the Amazon peacock bass (Cichla piquiti)

A set of primers to amplify 10 microsatellite DNA loci was developed for the Neotropical fish Cichla piquiti, one of the largest sized cichlids in the Amazon Basin. These loci were used to genotype individuals from two populations, one native population from the Tocantins River, the other an introduced population in southeast Brazil, Upper Paraná River. Cross‐amplification was also successful for another species of peacock bass, C. kelberi. An average of 4.4 alleles per locus (2–9 alleles) was detected. These markers will be useful for the characterization of genetic structure of native populations, and also for invasive biology studies since Cichla species have been introduced in many river basins outside their native ranges.

Comparação entre métodos de estocagem de DNA extraído de amostras de sangue, sêmen e pêlos e entre técnicas de extração

DNA samples of six bovines obtained from three tissues (blood, semen and hair) were extracted using two different techniques. After the extraction procedures the samples were divided in six fractions. Three were stored at -20° C and three at 4° C. Every three months one sample of each tissue/extraction procedure was analyzed in spectrophotometer, to determine the quantity of the DNA and the extract was amplified using the primer RM 29. No differences in the DNA quantity or in the level of protein contamination among the three periods of analyses were observed. All the DNA extracted by quick extraction technique showed good amplification patterns during the nine months, meaning that this technique can be used in laboratory routine instead of the permanent extraction technique. The extract obtained from blood, using the permanent extraction technique, showed the higher quantity of DNA with the smaller index of protein contamination. The high quantity of protein contamination found in the semen samples preserved in egg yolk demanded modifications in both extraction techniques. After that the results were positive, showing good amplification patterns.

Design and validation of a 90K SNP genotyping assay for the water buffalo (Bubalus bubalis).

Background The availability of the bovine genome sequence and SNP panels has improved various genomic analyses, from exploring genetic diversity to aiding genetic selection. However, few of the SNP on the bovine chips are polymorphic in buffalo, therefore a panel of single nucleotide DNA markers exclusive for buffalo was necessary for molecular genetic analyses and to develop genomic selection approaches for water buffalo. The creation of a 90K SNP panel for river buffalo and testing in a genome wide association study for milk production is described here. Methods The genomes of 73 buffaloes of 4 different breeds were sequenced and aligned against the bovine genome, which facilitated the identification of 22 million of sequence variants among the buffalo genomes. Based on frequencies of variants within and among buffalo breeds, and their distribution across the genome, inferred from the bovine genome sequence, 90,000 putative single nucleotide polymorphisms were selected to create an Axiom® Buffalo Genotyping Array 90K. Results This 90K “SNP-Chip” was tested in several river buffalo populations and found to have ∼70% high quality and polymorphic SNPs. Of the 90K SNPs about 24K were also found to be polymorphic in swamp buffalo. The SNP chip was used to investigate the structure of buffalo populations, and could distinguish buffalo from different farms. A Genome Wide Association Study identified genomic regions on 5 chromosomes putatively involved in milk production. Conclusion The 90K buffalo SNP chip described here is suitable for the analysis of the genomes of river buffalo breeds, and could be used for genetic diversity studies and potentially as a starting point for genome-assisted selection programmes. This SNP Chip could also be used to analyse swamp buffalo, but many loci are not informative and creation of a revised SNP set specific for swamp buffalo would be advised.