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

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-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) 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 (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 (named GUW1) while AF18 is a multicopy MITE transposon located on various chromosomes. Test application of GUW1 as a marker to sex 30 neonates of H. armigera yielded a female/male ratio of 1.14: 1.00. Both GUW1 and Hadsx splicing isoforms assays revealed that the H. armigera embryo cell line QB-Ha-E-1 is a male cell line. Taken together, GUW1 is not only a reliable DNA marker for sexing all stages of H. armigera and its cell lines, but also represents the first W-specific protein-coding gene in lepidopterans.

Identification and Characterization of the Masculinizing Function of the Helicoverpa armigera Masc Gene

“…This is because the femalespecific W chromosome in Lepidoptera is packed with transposons that can be transcribed into non-coding RNAs [6,[49][50][51]. Our recent characterization of the first W-specific proteincoding gene GUW1 from H. armigera [35] suggests that the W-specific GUW1 may function as the primary signal to minimize HaMasc expression in female cotton bollworms. Further experiments are required to examine these two possibilities.…”

“…Genomic PCR gel analysis showed that the band intensity of HaMasc amplified from male (AA + ZZ = 2 copies of each autosome + 2 copies of Z chromosome) pupa was roughly two times of that from female (AA + ZW = 2 copies of each autosome + 1 copy of Z chromosome/1 copy of W chromosome) pupa, whereas the band intensity of the autosomal gene EF-1α was equal between the male and female pupa (Figure 4A). Further genomic qPCR analysis revealed that the male copy/female copy ratio was 0.0023 for the W chromosome gene GUW1 [35], 1.00 for the autosomal gene EF-1α, 0.98 for the autosomal gene β-actin, and 2.32 for HaMasc (Figure 4B). These data confirm that the HaMasc gene is located on Z chromosome.…”

Chromosome‐level genome reference and genome editing of the tea geometrid

“…Another sex chromosome (W) was identified according to the criterion that mapped reads are completely absent in male but in theory drop to half in females. In fact, the mean sequencing coverage of W in males is not completely absent due to the high repeat content of Lepidoptera W chromosomes (Abe et al, 2005; Deng et al, 2020). Basic genomic features including sequencing coverage, coverage ratio between females and males, GC content, and gene density were displayed using circos version 0.69–8 (Krzywinski et al, 2009).…”

“…Thus, we defined this chromosome as the Z chromosome. However, the W chromosome was not properly assembled in this study, possibly due to the complex structure and high repetitive content widely found on lepidopteran W chromosomes (Abe et al, 2005;Dalíková et al, 2017;Deng et al, 2020). Alternatively, we characterized 40 scaffolds comprising ~3.9 Mb, which showed extreme sequencing coverage bias in females (ZW) as W candidate scaffolds (Figure S3, Table S4).…”

“…Another sex chromosome (W) was identified according to the criterion that mapped reads are completely absent in male but in theory drop to half in females. In fact, the mean sequencing coverage of W in males is not completely absent due to the high repeat content of Lepidoptera W chromosomes (Abe et al, 2005;Deng et al, 2020).…”