Medaka (Oryzias spp.) is an important fish species in ecotoxicology and 31 considered as a model species due to its biological features including small body size and 32 short generation time. Since Japanese medaka Oryzias latipes is a freshwater species with 33 access to an excellent genome resources, the marine medaka Oryzias melastigma is also 34 applicable for marine ecotoxicology. In genome era, a high-density genetic linkage map is a 35 very useful resource in genomic research, providing a means for comparative genomic 36 analysis and verification of de novo genome assembly. In this study, we developed a high-37 density genetic linkage map for O. melastigma using restriction-site associated DNA 38 sequencing (RAD-seq). The genetic map consisted of 24 linkage groups with 2,481 RAD-tag 39 markers. The total map length was 1,784 cM with an average marker space of 0.72 cM. The 40 genetic map was integrated with the reference-assisted chromosome assembly (RACA) of O. 41 melastigma, which anchored 90.7% of the assembled sequence onto the linkage map. The 42 values of complete Benchmarking Universal Single-Copy Orthologs (BUSCO) were similar 43 to RACA assembly but N50 (23.74 Mb; total genome length 779.4 Mb; gap 5.29%) increased 44 to 29.99 Mb (total genome length 778.7 Mb; gap 5.2%). Using MapQTL analysis with a 45 single nucleotide polymorphism markers, we identified a major quantitative trait locus for sex 46 traits on the Om10. The integration of the genetic map with the reference genome of marine 47 medaka will serve as a good resource for studies in molecular toxicology, genomics, 48 CRISPR/Cas9, and epigenetics. 49 50 KEYWORDS Marine medaka, Oryzias melastigma, linkage map, reference genome, sex, 51 quantitative trait locus 52 3 Many fish species are useful for ecotoxicological research, as they indicate an early 53 warning of environmental contamination caused by various aquatic pollutants (Arellano-54 Aguilar et al. 2009). Recently, an increase in the contamination levels in estuaries and coastal 55 water due to anthropogenic pollutants emphasizes the needs for marine sentinel model fish 56 species. Medaka (Oryzias spp.) is an important fish species in ecotoxicology and considered a 57 model species, as its biological features include certain advantages such as small body size 58 and short generation time (Kim et al. 2016). Japanese medaka Oryzias latipes is important 59 and widely used model species for the studies of genetics, evolution, and ecotoxicological 60 study, with abundant genomic resources. Since O. latipes is a freshwater species, their 61 responses to environmental toxicants can be different in those of marine fish (Shi et al. 2008; 62