Characterization of the First W-Specific Protein-Coding Gene for Sex Identification inHelicoverpa armigera

Helicoverpa armigerais a globally-important crop pest with a WZ (female)/ZZ (male) sex chromosome system. The absence of discernible sexual dimorphism in its egg and larval stages makes it impossible to address any sex-related theoretical and applied questions before pupation unless a W-specific sequence marker is available for sex diagnosis. To this end, we used one pair of morphologically pre-sexed pupae to PCR-screen 17 non-transposon transcripts selected from 4855 W-linked candidate reads identified by mapping a publicly available egg transcriptome of both sexes to the male genome of this species and detected the read SRR1015458.67499 only in the female pupa. Subsequent PCR screenings of this read and the previously reported female-specific RAPD (random amplified polymorphic DNA) markerAF18with ten more pairs of pre-sexed pupae and different annealing positions and/or temperatures as well as its co-occurrence with the female-specific transcript splicing isoforms ofdoublesexgene ofH. armigera (Hadsx)and amplification and sequencing of their 5 ' unknown flanking sequences in three additional pairs of pre-sexed pupae verified that SRR1015458.67499 is a single copy protein-coding gene unique to W chromosome (namedGUW1) whileAF18is a multicopy MITE transposon located on various chromosomes. Test application ofGUW1as a marker to sex 30 neonates ofH. armigerayielded a female/male ratio of 1.14: 1.00. BothGUW1andHadsxsplicing isoforms assays revealed that theH. armigeraembryo cell line QB-Ha-E-1 is a male cell line. Taken together,GUW1is not only a reliable DNA marker for sexing all stages ofH. armigeraand its cell lines, but also represents the first W-specific protein-coding gene in lepidopterans.