Desenvolvimento de marcadores microssatélites para Ololygon centralis (Anura: Hylidae) por sequenciamento de segunda geração com baixa cobertura
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2018-03-12
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Universidade Federal de Goiás
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The ease access to Next Generation Sequence methodologies has improved the development
of several projects, once it makes possible the de novo assembly of DNA sequences,
creating a great amount of data and giving access to unknown genomes of different
organisms. The anuran species Ololygon centralis belong to Hylidae family. It is considered
as endemic of Cerrado Biome and little is known about its genomics. The aim of this work
was to realize a draft of the genome of O. centralis in order to identify, to characterize, to
quantify repetitive elements and to predict possible genes. Also, we aimed to develop
microsatellite markers for the species. The genome draft assembled in 13989 scaffolds,
which correspond to 4926069 bp with N50 = 336. The repetitive elements found in the O.
centralis genome (1795 transposons, 957, microsatellite, one satellite DNA and 25 small
nuclear RNAs) comprised 531337 bp. Between the retrotransposons, the LINE element
(49,83%) was the most abundant, followed by LTR (48,66%) and SINE (1,56%). It was
possible to identify coding genes for the 5S, 18S and 28S subunits of ribosomes. Also, 18
types of coding regions for tRNA and 26 putative genes were found in the O. centralis
genome. From the microsatellite regions, it was possible to design 87 pairs of primers for
the flanking sites. There were found 47 regions composed of tetranucleotides, 39
dinucleotides and on trinucleotide on the microsatellite sites. From them, 31 pairs of primers
were selected for amplification and 18 showed good results. The annealing temperatures
varied from 50° e 60° C. Seven markers showed polymorphism. From them, only 5 markers
(PCE05, OCE11, PCE20, OCE21, OCE26) were used to characterize the genotype of 30
individuals. The medium number of alleles was 10, the allelic amplitude varied from 148 bp
(OCE20) to 318 bp (OCE21). The Expected Heterozygosity was 0,7 and the observed was
0,5. The probability of parentage exclusion (Q) was 0,993 and the probability of combined
identity (I) was 1,13x10-6. The global value of the fixation index (FST) was significant and
equal to 0,2 (p<0.05). The data obtained from the NGS Illumina platform represent one
important step for the knowledge of genomics of this species and of the anuran in general.
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CASTRO, Andrezza Arantes. Desenvolvimento de marcadores microssatélites para Ololygon centralis (Anura: Hylidae) por sequenciamento de segunda geração com baixa cobertura. 2018. 97 f. Dissertação (Mestrado em Genética e Biologia Molecular) - Universidade Federal de Goiás, Goiânia, 2018.