Linkage analysis of scaffolds
To clarify the linkages between scaffolds, we adapted the genetic approach. First, we obtained backcross generation 1 (BC1) individuals between S. ricini and S. c. pryeri , a closely related species to S. ricini. Crossing scheme was (Samia cynthia pryeri x S. ricini ) x S. ricini. Since meiotic recombination does not occur in ovaries of lepidopteran species, chromosomes in BC1 individuals should be S. ricini -S. c. pryeri heterozygotic or S. ricini -S. ricini homozygotic.
We designed thirty-five genetic markers which can specifically detect thirty-five scaffolds longer than 1 Mbp and can molecularly distinguishS. ricini and S. c. pryeri (Fig. S1A) and then perform genomic PCR. Genomic DNA was extracted from the legs of eight BC1 larvae using the DNeasy Blood and Tissue Kit (QIAGEN). The genomic PCR program was as follows: 40 cycles of 10 s at 98°C, 5 s at 60°C and 5 s at 68°C. KOD OneTM PCR Master Mix (TOYOBO) was used to perform genomic PCR. Then, the allele combinations of scaffolds in 8 BC1 individuals were examined. According to the result, we designated the identified linkage groups according to Yoshido et al. (2011). Electrophoresis was conducted using 2.0% agarose gel or MultiNA microchip electrophoresis system (Shimadzu, Kyoto, Tokyo). Table S4 listed all primers used for linkage analysis.