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

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.