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Male-specific molecular genetic markers in the Japanese subterranean termite Reticulitermes speratus

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Abstract

Sex-specific genetic markers are often required for studying sex-associated phenomena. Restriction site-associated DNA sequencing (RAD-seq) allows detection of a huge number of genetic polymorphisms and is particularly useful for identifying sex-specific DNA-based (or molecular) genetic markers. Although sex determination in the Japanese subterranean termite Reticulitermes speratus depends on an XX/XY chromosome system, in which male-specific molecular genetic markers can be developed, no such marker has been developed. In this study, we developed male-specific genetic markers for R. speratus using RAD-seq with two restriction enzymes (i.e. double digest [dd] RAD-seq). We subjected 58 field-collected individuals per sex to ddRAD-seq and obtained around 210 million paired-end reads. Our in silico analysis of ddRAD-seq data detected 25 male-specific loci but no female-specific loci, thus confirming an XX/XY system. To verify the male specificity of those loci, we conducted PCR with primers designed for male-specific loci. Accordingly, we obtained male-specific PCR amplifications in six of the 25 loci. Furthermore, we examined whether these six male-specific markers were also applicable to individuals derived from four Japanese populations of R. speratus, including from the northern and southern ends of the distribution range in Japan. Of the six markers, five exhibited male-specific PCR amplifications in all four populations; the sixth was applicable to three populations. PCR amplification tests for cross-species applicability in R. kanmonensis found that none of these markers were cross-species applicable. The six male-specific markers would be useful for sex and sex chromosome identification in R. speratus individuals at all life stages.

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Acknowledgements

We thank D. Watanabe for his assistance with termite collection in the field and H. Asao for her kind instruction regarding RAD-seq library construction. Computational resources were provided by the Data Integration and Analysis Facility, National Institute for Basic Biology (NIBB). This study was conducted as part of the Model Organism Development Collaborative Research Projects of the NIBB. This study was partly supported by a Grant for Basic Science Research Projects from the Sumitomo Foundation (No. 150189 to YH) and JSPS KAKENHI Grants (Nos. 16K18591 and 25251041 to YH and TM, respectively).

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    Authors and Affiliations

    1. Graduate School of Environmental Science, Hokkaido University, Sapporo, 060-0810, Japan

      Y. Hayashi, K. Oguchi & T. Miura

    2. NIBB Core Research Facilities, National Institute for Basic Biology, National Institutes of Natural Sciences, Okazaki, 444-8585, Japan

      K. Yamaguchi & S. Shigenobu

    3. College of Science, Ibaraki University, Mito, 310-8512, Japan

      O. Kitade

    4. Graduate School of Science and Engineering, University of Toyama, Toyama, 930-8555, Japan

      K. Maekawa

    5. Department of Basic Biology, School of Life Science, Graduate University for Advanced Studies, Okazaki, 444-8585, Japan

      S. Shigenobu

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    1. Y. Hayashi
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    Correspondence to Y. Hayashi.

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    Yoshinobu Hayashi and Kohei Oguchi have contributed equally to this work.

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    Hayashi, Y., Oguchi, K., Yamaguchi, K. et al. Male-specific molecular genetic markers in the Japanese subterranean termite Reticulitermes speratus . Insect. Soc. 64, 357–364 (2017). https://doi.org/10.1007/s00040-017-0553-z

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