Description
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This article reports a predicted gene model generated by undergraduate work using a structured gene model annotation protocol defined by the Genomics Education Partnership (GEP;
thegep.org
) for Course-based Undergraduate Research Experience (CURE). The following information in quotes may be repeated in other articles submitted by participants using the same GEP CURE protocol for annotating Drosophila species orthologs of Drosophila melanogaster detoxification genes.
“Within insects, the process of detoxifying xenobiotics and host secondary metabolites is a three-phase process that involves functionalization, conjugation, and excretion of these compounds. Expansions of known detoxification gene families (
e.g.
, cytochrome P450s) is associated with diet breadth and insecticide resistance (Ranson et al., 2002; Després et al., 2007; Rane et al., 2016). With the increasing availability of high-quality genomes for non-model organisms, including
Drosophila
species beyond
D. melanogaster
, it is now possible to perform large scale comparative studies (Robinson et al., 2011; Kim et al., 2021; Threfall and Baxter, 2021). Careful manual annotation and curation of gene models can improve upon computational gene predictions in non-model species, which aids the accuracy of studies on gene and genome evolution (Mudge and Harrow, 2016; Tello-Ruiz et al., 2019). To aid in these annotations, the Genomics Education Partnership (thegep.org) developed a curriculum involving web-based tools that allow undergraduates to engage in authentic course-based research focused on manually annotating genes in non-model species (Rele et al., 2023). The orthologous gene models, including the one presented here, then provide a reliable basis for further evolutionary genomic analyses when made available to the scientific community. The gene ortholog described here in
D. funebris
for
Peptidoglycan recognition protein SB1
(
PGRP-SB1
), a member of the peptidoglycan recognition protein family that contributes to the innate immune system of insects, was characterized as part of a developing dataset for a comparative study of detoxification gene families in the
immigrans
-
tripunctata
radiation of the genus
Drosophila
.” (Williams et al., 2026)
Drosophila funebris
(Fabricius, 1787) is a member of the
funebris
species group, which occurs in the
immigrans-tripunctata
radiation of the
Drosophila
subgenus (Bächli, 2005; ICZN, 2010). It is also the type species of the genus
Drosophila
. This species is a globally distributed human commensal (Grimaldi, 2022). While
D. funebris
feeds on shelf fungi and fruit (Kimura et al., 1977; Prigent et al., 2003), it does not tolerate the mushroom toxin α-amanitin (Stump et al., 2011; Erlenbach et al., 2023).
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When considering the detoxification of xenobiotics, the focus is often directed towards genes that produce detoxification enzymes. A growing body of work is beginning to highlight the role of genes in the insect's innate immune system in metabolizing these toxic compounds (Bartling et al., 2021; Jiang et al., 2021; Rahmat et al., 2024). One component of the innate immune system, peptidoglycan recognition proteins (PGRP), are pattern recognition molecules that detect molecular patterns found in pathogens but absent in the host organism (Akira et al., 2006; Lemaitre and Hoffman, 2007). Within
Drosophila melanogaster
, Werner et al. (2000) identified
12 PGRPs. These proteins are split into two subgroups (long and short) based on their transcript size, cellular location, and function (Werner et al., 2000).
Peptidoglycan recognition protein SB1
(
PGRP-SB1
)
is a short PGRP (Werner et al., 2000). It is an amidase that exhibits antibacterial activity against peptidoglycans that possess a diaminopimelic acid residue (Mellroth and Steiner, 2006). When
Drosophila
flies are exposed to wasp, mite, or nematode parasites, the expression of this gene is increased (Benoit et al., 2020). In addition, exposure to sublethal doses of six insecticides also increased the expression of
PGRP-SB1
(Gao et al., 2021).
We propose a gene model for the
D. funebris
ortholog of the
D. melanogaster
Peptidoglycan recognition protein SB1
(
PGRP-SB1
) gene. The genomic region of the ortholog corresponds to the Augustus gene prediction
JAEIFK010000688
.g512.t1 in the ASM1890182v1 Genome Assembly of
D. funebris
(GCA_018901825.1- Kim et al. 2021). This model is based on mixed sex, adult RNA-Seq data from
D. funebris
(Erlenbach et al. 2023;
https://doi.org/10.5061/dryad.hdr7sqvq2
) and
PGRP-SB1
in
D. melanogaster
using FlyBase release FB2024_02 (
GCA_000001215.4
; Gramates et al., 2022; Jenkins et al., 2022; Larkin et al.,
2021).
Synteny
The reference gene,
PGRP-SB1
,
occurs on
chromosome 3L in
D. melanogaster
and is flanked upstream by
Dead box protein 73D
(
Dbp73D
) and
CG9701
and downstream by
Peptidoglycan recognition protein SB2
(
PGRP-SB2
) and
CG13026
. The
tblastn
search of
D. melanogaster
PGRP-SB1-PA (query) against the
D. funebris
(GenBank Accession:
GCA_018901825.1
Genome Assembly database (subject) placed the putative ortholog of
PGRP-SB1
within contig_693 (
JAEIFK010000688
) which corresponds to the Augustus gene prediction
JAEIFK010000688
.g512.t1 (E-value: 3.00e-113; percent identity: 78.2%; query coverage: 98% as determined by
blastp
). The putative ortholog is flanked upstream by Augustus gene predictions
JAEIFK010000688
.g513.t1 and
JAEIFK010000688
.g514.t1, which correspond to
Dbp73D
and
CG9701
in
D. melanogaster
(E-value: 0.0 and 0.0; identity: 66.47% and 88.70%, respectively, as determined by
blastp
;
Figure 1A
; Altschul et al., 1990). The putative ortholog of
PGRP-SB1
is flanked downstream by
JAEIFK010000688
.g511.t1 and
JAEIFK010000688
.g510.t1, which correspond to
CG13026
and
Disabled
(
Dab
) in
D. melanogaster
(E-value: 1.00e-13 and 0.0 identity: 52.75% and 68.16%, respectively, as determined by
blastp
).
Dab
is the third gene downstream of the target gene,
PGRP-SB1
, in
D. melanogaster
. The putative ortholog assignment for
PGRP-SB1
in
D. funebris
is supported by the following evidence: The gene predictions surrounding the
PGRP-SB1
ortholog are all orthologous to the genes at the same locus in
D. melanogaster
except for the loss of the
PGRP-SB2
gene, gene expression data corresponds with each prediction, and local synteny is almost entirely conserved, supported by E-values and percent identities, so we conclude that
JAEIFK010000688
.g512.t1 is an ortholog of
PGRP-SB1
in
D. funebris
(
Figure 1A
).
Protein Model
PGRP-SB1
in
D. funebris
has 2 coding sequences (CDS) within the genome sequence. The only unique protein sequence (PGRP-SB1-PA) is translated from 1 mRNA isoform (PGRP-SB1-RA;
Figure 1B
). Relative to the ortholog in
D. melanogaster
, the CDS number and protein isoform count are conserved
.
The sequence of
PGRP-SB1-PA
in
D. funebris
has 78.1% identity (89.1% similarity) with the
protein-coding isoform
PGRP-SB1-PA
in
D. melanogaster
,
as determined by
blastp
(
Figure 1C
). This level of divergence is not surprising given that
D. funebris
and
D. melanogaster
belong to two separate subgenera (
Drosophila
and
Sophophora
respectively) that diverged approximately 45-60 MYA (Russo et al., 1995; Tamura et al., 2004; Obbard et al., 2012). Coordinates of this curated gene model are archived in the CaltechDATA repository (see “Extended Data” section below).
Methods
The annotation methods used in this project are adapted from those described in Rele et al. (2023), which includes algorithms, database versions, and citations for the complete annotation process developed for the Pathways Project. The methods for the current project are detailed in brief below with notes on significant differences between this protocol and the one described in Rele et al. (2023). The students use the GEP instance of the UCSC Genome Browser v.435 (
https://gander.wustl.edu
;
Kent et al., 2002; Raney et al., 2024) to examine the genomic neighborhood of their reference detoxification gene in the
D. melanogaster
genome assembly (Aug. 2014; BDGP Release 6 + ISO1 MT/dm6). Students obtain the protein sequence for the
D. melanogaster
target gene for a given isoform and use a
tblastn
search of the sequence against their target
Drosophila
species genome assembly (
D. funebris
(
GCA_018901825.1
– Kim et al., 2013)) on the NCBI BLAST server (
https://blast.ncbi.nlm.nih.gov/Blast.cgi
, Altschul et al., 1990) to identify the putative ortholog location. Students compare the genomic neighborhood of the putative ortholog to that of the reference gene in
D. melanogaster
. This local synteny analysis includes a minimum of two upstream and downstream genes relative to the potential ortholog. As no RefSeq protein data is available for these species, comparisons are based on gene predictions that correlate with gene expression data in the putative ortholog neighborhood. Using the multiple alignment tracks feature in the Genome Browser, students examine other sets of genomic evidence, including Spaln alignment of
D. melanogaster
proteins, multiple gene prediction tracks (e.g., GeMoMa, Augustus, NSCAN PASA-EST), and mixed sex RNA-Seq adult expression data from the target species generated by Erlenbach et al. (2023;
https://doi.org/10.5061/dryad.hdr7sqvq2
). Information on the genomic structure information (e.g., CDSs, intron-exon number, number of isoforms) for the reference gene in
D. melanogaster
is retrieved using Gene Record Finder (
https://gander.wustl.edu/~wilson/dmelgenerecord/index.html
; Rele et al
.,
2023). To determine approximate splice sites within the target gene, a
tblastn
search using the CDSs from the
D. melanogaste
r reference gene against the putative ortholog location (10kb up- and downstream of the target gene prediction). Coordinates of the CDS(s) are refined by examining aligned RNA-Seq data, identifying canonical splice site sequences, and ensuring the maintenance of an open reading frame. Students confirm the biological validity of their target gene model using the FlySeq Gene Model Checker (
https://gander2.wustl.edu/~wilson/genechecker-flyseq/
), which compares the hypothesized target gene model's structure and translated sequence against the
D. melanogaster
reference
gene. At least two independent models for this gene are generated. These models are reconciled by a third independent researcher to produce the final model presented here. Note: comparison of 5' and 3' UTR sequence information is not included in this GEP CURE protocol.