Identification and validation of simple sequence repeats (SSRs) Markers for Androctonus crassicauda scorpion using RNA-Seq data

Document Type : Research Paper

Authors

1 Ph. D student in Department of Animal Science, Faculty of Animal science and Food Technology, Agricultural Science and Natural Resources University of Khuzestan, Mollasani, Iran.

2 Department of Animal Science, Faculty of Animal science and Food Technology, Agricultural Science and Natural Resources University of Khuzestan, Mollasani, Iran.

3 Department of Animal Science, Faculty of Animal Science and Food Technology, Agricultural Science and Natural Resources University of Khuzestan

4 Assistant Professor, Department of Venomous Animals and Anti-venom Production, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Ahvaz, Iran.

10.22103/jab.2025.24286.1625

Abstract

Objective
Simple sequence repeats (SSRs) markers or microsatellites are one of the best and most complete molecular tools in the study of genetic diversity due to the appropriate genomic coverage and high repeatability. Simple sequence repeats markers for the scorpion Andractonus crassicouda have not been reported so far. Therefore, this study was conducted with the aim of identifying and validating simple repeat sequence markers of the scorpion Andractonus crassicouda using RNA-Seq data.
Materials and methods
The samples were evaluated qualitatively and quantitatively after RNA extraction from the scorpion venom gland. Then the samples were sequenced with the Illumina HiSeq 2000 platform. Reconstruction of transcripts was done by De novo assembly method using Trinity software. Next, in order to identify new SSR markers, the transcriptome of the scorpion Andrachtonus crassicouda was analyzed using FullSSR software (version 1.5). To validate the identified SSR markers, 8 pairs of primers were designed by PRIMER 3 software and evaluated by polymerase chain reaction method on 60 DNA samples extracted from scorpion tissue.
Results
Assembling transcripts by the Trinity program generated a total of 744,804 transcripts and 563,526 unigenes. In this study, the examination of 952,725 sequences by FullSSR software led to the identification of 315,395 SSR markers. Among SSRs, two and three nucleotide repeats accounted for the highest number of repetitions with 71.85% and 22.36%, respectively. Among the 8 studied loci, only 2 loci showed polymorphism. The expected and observed heterozygosity for the RK1354 locus was calculated as 0.765 and 0.683 respectively, and for the NK1362 locus as 0.768 and 0.633 respectively. The fixation index statistic (Fis) for both loci was almost 0.1, which indicates low inbreeding in the population. In addition, the chi-square test showed that the population was not in Hardy-Weinberg equilibrium for both loci.
Conclusions
The different criteria of genetic diversity all indicate that the population is highly diverse, but some factors reduce the diversity among the studied population. This means that the population is out of balance and the number of observed heterozygotes is less than the expected heterozygotes. It is necessary to investigate the factors that have led to the reduction of diversity to prevent its further reduction. In general, the results of this research showed that new SSRs can be useful for understanding the population structure and investigating the genetic diversity of the scorpion Andractonus crassicouda.

Keywords

References

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Agricultural Biotechnology Journal
Volume 17, Issue 2
May 2025
Pages 31-54

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