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[Preprint]. 2025 Jul 25:2025.07.18.665411. [Version 1] doi: 10.1101/2025.07.18.665411

Gene model for the ortholog of Glyp in Drosophila ananassae

Gabriella N Bicanovsky 1, Evan N Bennett 2, Zachary Lill 3, Rebecca M McSweeney 4, Geoffrey D Findlay 4, Amy T Hark 3, Chinmay P Rele 1, Shallee T Page 2,*
PMCID: PMC12330513  PMID: 40777245

Abstract

We present a gene model for the ortholog of Glycogen phosphorylase (Glyp) in the D. ananassae May 2011 (Agencourt dana_caf1/DanaCAF1) genome assembly of Drosophila ananassae (GenBank Accession: GCA_000005115.1). This ortholog was characterized as part of a developing dataset to study the evolution of the insulin/insulin-like growth factor signaling pathway (IIS) across the genus Drosophila using the Genomics Education Partnership gene annotation protocol for Course-based Undergraduate Research Experiences.

Introduction

This article reports a predicted gene model generated by undergraduates using a structured gene model annotation protocol defined by the Genomics Education Partnership (GEP; thegep.org) for Course-based Undergraduate Research Experiences (CUREs).

“In this GEP CURE protocol, students use web-based tools to manually annotate genes in non-model Drosophila species based on orthology to genes in the well-annotated model organism fruitfly Drosophila melanogaster. The GEP uses web-based tools to allow undergraduates to participate in course-based research by generating manual annotations of genes in non-model species (Rele et al., 2023). Computational-based gene predictions in any organism are often improved by careful manual annotation and curation, allowing for more accurate analyses of gene and genome evolution (Mudge & Harrow, 2016; Tello-Ruiz et al., 2019). These models of orthologous genes across species then provide a reliable basis for further evolutionary genomic analyses when made available to the scientific community in public databases.” (Myers et al., 2024).

The D.ananassae Glyp ortholog described here was characterized “…as part of a developing dataset to study the evolution of the insulin/insulin-like growth factor signaling pathway (IIS) across the genus Drosophila. The IIS pathway is a highly conserved signaling pathway in animals and is central to mediating organismal responses to nutrients (Grewal, 2009; Hietakangas & Cohen, 2009).” (Myers et al., 2024). “Glycogen phosphorylase (Glyp; also known as GP, Glp1, DGPH, FBgn0004507), part of the insulin signaling pathway, has a high degree of amino acid similarity to mammalian enzymes (Tick et al., 1999), and contains repeats that are similar to repeats in several transmembrane proteins including Notch in D. melanogaster and Lin-28 in Caenorhabditis elegans (LaMarco et al., 1991). GLYP plays a major role in glycolytic activity in skeletal muscle in association with glycogen metabolism and body homeostasis (Yamada et al., 2019). The importance of Glyp is evident in that null homozygotes are lethal as full knockouts at the larval stage (Eanes et al., 2006; Yamada et al., 2019). The [Glyp gene product] has also shown to affect development, adult fitness, and aging in Drosophila (Bai et al., 2013; Yamada et al., 2019).” (Laskowski L.F. et al., 2025).

“D. ananassae (NCBI:txid7217) is part of the melanogaster species group within the subgenus Sophophora of the genus Drosophila (Sturtevant, 1939). First described by Doeschall (Doleschall, C.L., 1858), D. ananassae is circumtropical (Markow & O’Grady, 2006), and is often associated with human settlement (Singh, 2010). It has been extensively studied as a model for its cytogenetic and genetic characteristics, and in experimental evolution (Kent et al., 2002).” (Lawson et al., 2024).

Synteny

The reference gene, Glyp, occurs on chromosome 2L in D. melanogaster and is flanked upstream by Odorant receptor 22c (Or22c) and CG12674, nested by defective proboscis extension response 3 (dpr3), and downstream by tho2 and CG11723. The tblastn search of D. melanogaster Glyp-PA (query) against the D. ananassae (GenBank Accession: GCA_000005115.1) Genome Assembly database placed the putative ortholog of Glyp within scaffold_12916 (European Nucleotide Archive: CH902620.1) at locus LOC6497423 (XP_001962117.1)— with an E-value of 0.0 and a percent identity of 94.58%. Furthermore, the putative ortholog is flanked upstream by LOC6497421 (XP_001962120.1), LOC26513848 (XP_014762266.1) and LOC6498121 (XP_044571907.1), which correspond to Or22c, CG12674 and dpr3 in D. melanogaster (E-value: 0.0, 5e-109 and 0.0; identity: 85.21%, 50.31% and 72.63%, respectively, as determined by blastp; Figure 1A; Altschul et al., 1990). The putative ortholog of Glyp is flanked downstream by LOC6498120 (XP_032307372.1) and LOC6498119 (XP_001962115.2), which correspond to tho2 and CG11723 in D. melanogaster (E-value: 0.0 and 5e-129; identity: 86.37% and 53.97%, respectively, as determined by blastp). The putative ortholog assignment for Glyp in D. ananassae is supported by the following evidence: The genes surrounding the Glyp ortholog are orthologous to the genes at the same locus in D. melanogaster and local synteny is completely conserved, supported by results generated from tblastn and blastp, so we conclude that LOC6497423 is the correct ortholog of Glyp in D. ananassae (Figure 1A).

Figure 1: Glyp gene model comparison between D. melanogaster and D. ananassae: (A) Synteny of the genomic neighborhood of Glyp in D. melanogaster and D. ananassae.

Figure 1:

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The thin terminal arrow pointing to the right indicates that Glyp is on the (+) strand in D. melanogaster; the arrow pointing to the left indicates that Glyp is on the (−) strand in D. ananassae. Notations within the large white arrows in D. ananassae indicate the locus ID and the orthology to the corresponding gene in D. melanogaster. The wide gene arrows pointing in the same direction as Glyp are on the same strand relative to the thin underlying arrows, while wide gene arrows pointing in the opposite direction of Glyp are on the opposite strand. The embedded arrow indicates that the Dpr-3 gene contains a nested gene (CG12674). The gene names given in the D. ananassae gene arrows indicate the orthologous gene in D. melanogaster, while the locus identifiers are specific to D. ananassae. (B) The custom gene model in UCSC Track Hub (Raney et al., 2014). The gene model in D. ananassae is depicted in black with exons depicted with wide boxes, introns with narrow lines, and arrows indicating direction of transcription. Evidence tracks below include BLAT alignments of NCBI Ref-Seq Genes (blue, alignment of Ref-Seq genes for D. ananassae), Spaln of D. melanogaster Proteins (purple, alignment of Ref-Seq proteins from D. melanogaster), RNA-Seq (alignment of Illumina RNA-Seq reads from D. ananassae) from adult females (red), adult males (blue), and Wolbachia-cured embryos (pink; alignment of Illumina RNA-Seq reads from D. ananassae), transcripts (green) including coding regions predicted by TransDecoder and splice junctions predicted by regtools using D. ananassae RNA-Seq (SRP006203, SRP007906, PRJNA257286, PRJNA388952). Splice junctions shown have a minimum read-depth of 17761 with >1000 supporting the reads shown. (C) Dot plot of Glyp-PA in D. melanogaster (x-axis) vs. the orthologous polypeptide in D. ananassae (y-axis). Amino acid number is indicated along the left and bottom; exon number is indicated along the top and right, and exons are also highlighted with alternating colors.

Protein Model

Glyp in D. ananassae has four coding sequences (CDSs) within the genomic sequence. The unique protein sequence (Glyp-PA; Glyp-PB) is translated from two mRNA isoforms that differ in their UTRs (Glyp-RA; Glyp-RB; Figure 1B). Relative to the ortholog in D. melanogaster, the CDS number and protein isoform count are conserved. The sequence of Glyp-PA in D. ananassae has 98.46% identity (E-value: 0.0) with the protein-coding isoform Glyp-PA in D. melanogaster, as determined by blastp (Figure 1C). Coordinates of this curated gene model are stored by NCBI at GenBank/BankIt (accession BK063018 and BK063019). These data are also available at the TrackHub listed in “Extended Data” below.

Methods

“Detailed methods including algorithms, database versions, and citations for the complete annotation process can be found in Rele et al. (Rele et al., 2023). Students used the GEP instance of the UCSC Genome Browser v.435 (https://gander.wustl.edu) (Kent et al., 2002; Navarro Gonzalez et al., 2021) to examine the genomic neighborhood of Glyp gene in the D. melanogaster genome assembly (Aug. 2014; BDGP Release 6 + ISO1 MT/dm6). Students then retrieved the protein sequence for the D. melanogaster Glyp for a given isoform and ran it using tblastn against the D. ananassae genome assembly (GCA_000005115.1) (Drosophila 12 Genomes Consortium et al., 2007) on the NCBI BLAST server (https://blast.ncbi.nlm.nih.gov/Blast.cgi, (Altschul et al., 1990) to identify potential orthologs. To validate the potential ortholog, students compared the local genomic neighborhood of the D. ananassae ortholog with the genomic neighborhood of Glyp in D. melanogaster. This local synteny analysis included the two upstream and downstream genes relative to the putative D. ananassae ortholog. They also explored other sets of genomic evidence using multiple alignment tracks in the Genome Browser, including BLAT alignments of RefSeq Genes, Spaln alignment of D. melanogaster proteins, and multiple gene prediction tracks (e.g., GeMoMa, Geneid, Augustus), and modENCODE RNA-Seq from the target species. Genomic structure information (e.g., CDSs, intron-CDS number and boundaries, number of isoforms) for the D. melanogaster reference gene was retrieved through the Gene Record Finder (https://gander.wustl.edu/~wilson/dmelgenerecord/index.html) (Gruys et al., 2025; Rele et al., 2023). Approximate splice sites within the target gene were determined using Combined Splice Junction Predictions (Kim et al., 2015) and tblastn using the CDSs from the D. melanogaster reference gene. “Coordinates of CDSs were then refined by examining aligned modENCODE RNA-Seq data, and by applying paradigms of molecular biology such as identifying canonical splice site sequences and ensuring the maintenance of an open reading frame across hypothesized splice sites. Students then confirmed the biological validity of their target gene model using the Gene Model Checker (https://gander.wustl.edu/~wilson/dmelgenerecord/index.html) (Rele et al., 2023), which compares the structure and translated sequence from their hypothesized target gene model against the D. melanogaster reference gene model. Three independent models for this gene were generated by students under mentorship of their faculty course instructors. These models were then reconciled by a third independent researcher mentored by the project leaders to produce the final model presented here. Note: comparison of 5’ and 3’ UTR sequence information is not included in this GEP CURE analysis.” (Gruys et al., 2025)

Discussion

Here, we propose a gene model for the D. ananassae ortholog of the D. melanogaster Glycogen phosphorylase (Glyp) gene. The genomic region of the ortholog corresponds to the uncharacterized protein XP_001962117.1 (Locus ID LOC6497423) in the Agencourt danacaf1/Dana-CAF1 Genome Assembly of D. ananassae (GCA_000005115.1 (Graveley et al., 2011)). This model is based on RNA-Seq data from D. ananassae (SRP006203, SRP007906, PRJNA257286, PRJNA388952) and Glyp in D. melanogaster using FlyBase release FB2024_02 (GCA_000001215.4) (Gramates et al., 2022; Jenkins et al., 2022; Larkin et al., 2021).

Supplementary Material

Supplement 1
media-1.zip (9.2KB, zip)

Acknowledgements:

We would like to thank Laura K. Reed and Wilson Leung, who created and maintained the GEP technological infrastructure. Thank you to FlyBase for providing the definitive database for Drosophila melanogaster gene models.

Funding:

This material is based upon work supported by the National Science Foundation under Grant No. IUSE-1915544 and the National Institute of General Medical Sciences of the National Institutes of Health Award R25GM130517. Support was also received from New Hampshire-INBRE through an Institutional Development Award (IDeA), P20GM103506, from the National Institute of General Medical Sciences of the NIH to ENB and STP. The Genomics Education Partnership is fully financed by federal funding. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Footnotes

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Supplementary Materials

Supplement 1
media-1.zip (9.2KB, zip)

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