Characterization of the first W-unique protein-coding gene for sex determination in Helicoverpa armigera

Authors: DENG, ZHONGYUAN - Zhengzhou University; ZHANG, YAKUN - Chinese Academy Of Agricultural Sciences; ZHANG, MIN - Zhengzhou University; HUANG, JINYONG - Zhengzhou University; Ni, Xinzhi; LI, XIANCHUN - University Of Arizona

Submitted to: Frontiers in Genetics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/28/2020
Publication Date: 6/19/2020
Citation: Deng, Z., Zhang, Y., Zhang, M., Huang, J., Ni, X., Li, X. 2020. Characterization of the first W-unique protein-coding gene for sex determination in Helicoverpa armigera. Frontiers in Genetics. 11:649. https://doi.org/10.3389/fgene.2020.00649.
DOI: https://doi.org/10.3389/fgene.2020.00649

Interpretive Summary: Sex identification at the early life stage of a pest can be a useful tool for integrated pest management. However, most of morphological differences between males and females are only visible in adult and/or pupal stages. This is because most insects do not exhibit conspicuous sexual dimorphism before pupation, and thus their eggs and larvae are often morphologically indistinguishable. Yet, characterization of the upstream diverged primary signal, master binary switch gene and autoregulatory gene of the sex determination pathway in various insects necessitates gender determination of eggs and young larvae/nymphs. Having a reliable method to sex eggs and larvae/nymphs of insects is also required for many other theoretical and applied research, such as molecular mechanisms of sexual trait development and genetic control of insect pests. The Old World bollworm is one of the most destructive polyphagous pest with hundreds of host plants belong to over 40 plant families. The bollworm has a unique sex determination system, in which males are homogametic and females are heterogametic sex. Male and female adults can be reliably sexed by their morphological dimorphism in pupal and adult stages. However, there are no morphological characters that can differentiate male from female eggs and larvae younger than the 3rd instar. Therefore, we conducted a study to develop a polymerase chain reaction-based sexing method using chromosome specific unique DNA sequence for identification of male and female eggs, larvae and even pupae of the bollworm. These experiments demonstrated that a transposon located in multiple loci on both sex-determination chromosome and other chromosomes, whereas a single copy protein-coding gene unique to sex chromosome (called GUW1) can be used as a reliable DNA marker for sexing all growth stages of the Old World bollworm.

Technical Abstract: Helicoverpa armigera is 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-unique sequence marker is available for sex determination. 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 publically available egg transcriptome of both sexes to the male genome of this species and detected the read SRR1015458.67499 only in the female pupae. Subsequent PCR screenings of this read and the previously reported female-specific RAPD (random amplified polymorphic DNA) marker AF18 with 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 of doublesex gene of H. armigera 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 (named GUW1) while AF18 is a multicopy MITE transposon located on various chromosomes. Test application of GUW1 as a marker to sex 30 neonate larvae of H. armigera yielded a female/male ratio of 1.14 : 1.0. Taken together, GUW1 is not only a reliable DNA marker for sexing all stages of H. armigera, but also represents the first W-unique protein-coding gene in lepidopterans.