DOI: 10.7280/d1rh56
title: Understanding the Early Evolutionary Stages of a Tandem Drosophila melanogaster - Specific Gene Family: A Structural and Functional Population Study

description: Gene families underlie genetic innovation and phenotypic diversification.
 However, our understanding of the early genomic and functional evolution
 of tandemly arranged gene families remains incomplete as paralog sequence
 similarity hinders their accurate characterization.  The D.
 melanogaster-specific gene family Sdic is tandemly repeated and impacts
 sperm competition.  We scrutinized Sdic in 20 geographically
 diverse populations using reference-quality genome assemblies, read-depth
 methodologies, and qPCR, finding that ~90% of the individuals harbor 3-7
 copies as well as evidence of population differentiation. In strains with
 reliable gene annotations, copy number variation (CNV) and differential
 transposable element insertions distinguish one structurally distinct
 version of the Sdic region per strain.  All 31 annotated copies
 featured protein-coding potential and, based on the protein variant
 encoded, were categorized into 13 paratypes differing in their 3’ ends,
 with 3-5 paratypes coexisting in any strain examined. Despite widespread
 gene conversion, the only copy present in all strains has functionally
 diverged at both coding and regulatory levels under positive selection.
 Contrary to artificial tandem duplications of the Sdic region that
 resulted in increased male expression, CNV in cosmopolitan strains did not
 correlate with expression levels, likely as a result of differential
 genome modifier composition.  Duplicating the region did not
 enhance sperm competitiveness, suggesting a fitness cost at high
 expression levels or a plateau effect. Beyond facilitating a minimally
 optimal expression level, Sdic CNV acts as a catalyst of protein and
 regulatory diversity, showcasing a possible evolutionary path recently
 formed tandem multigene families can follow toward long-term consolidation
 in eukaryotic genomes.
description: <strong>Progeny contribution scores in offense
 double-matings of genotypes carrying the duplication of the Sdic
 region</strong> Offense double-mating experiments
 for duplication-bearing males were performed as reported (Yeh, et al.
 2013), and concomitantly with those for other male genotypes whose results
 were already published (Yeh, et al. 2012). Briefly, sperm competitive
 ability for any given male genotype was calculated with the P2 metric,
 which measures the relative contribution of the second male to mate to the
 total progeny of doubly-mated females. The angular transformation was
 applied to the P2 values (Sokal and Rohlf 1994).
 <strong>Original Ct values in qRT-PCR Sdic expression
 surveys across males of 10 D. melanogaster
 strains</strong> Experiments were done using four
 replicates of total RNA extractions from whole-body males with a CFX-96
 1000 touch real-time instrument (BioRad) using the PowerUP SYBR Green
 Master Mix (Applied Biosystems) with 1 µl cDNA in a 20 µl reaction. Total
 RNA was extracted from 10 strains (Fig. 5) using TRIzol reagent (Thermo
 Fisher) following manufacturer instructions. Fifty naive males per
 replicate per strain were systematically sacrificed at 3 pm to control for
 circadian rhythms and extracted on separate days to avoid strain
 cross-contamination. DNA traces were subsequently eliminated using the
 RNeasy mini kit with DNase I (Qiagen). RNA integrity, purity, and
 concentration were assessed using gel electrophoresis, Nanodrop, and a
 Qubit RNA BR assay kit, respectively. Each sample was converted to cDNA
 using 1.5 µg total RNA and the SuperScript IV first-strand synthesis
 system with an RNase inhibitor (Invitrogen). Effective reverse
 transcriptase reactions were confirmed through successful RT-PCR of the
 gene Gapdh2. The gene clot was used as the reference gene and males from
 ISO-1 were used for calibration. Expression estimates were obtained
 accounting for variable primer efficiencies for the gene of interest
 (Sdic) and the reference gene (Pfaffl 2001).
 <strong>Original Ct values in qPCR Sdic CNV surveys across
 24 genotypes</strong> Real-time PCR experiments
 were performed in 20µl reactions using PowerUP SYBR Green Master Mix
 (Applied Biosystems), 5µM of each primer, and ~30ng of purified genomic
 DNA in 96-well plates on a Bio-Rad CFX-96 1000 touch real-time PCR
 instrument. For each interrogated genotype, three biological replicates
 were used. In each extraction, 20 entire whole bodies from less than 10
 days post-eclosion individuals were homogenized with motorized pestles in
 1.5ml tubes. Genomic DNA was extracted using the Qiagen’s Puregene Core
 Kit B, and further purified using Zymo Research’s Genomic DNA Clean
 & Concentrator-10 kit following manufacturer’s instructions. DNA
 purity was confirmed with a NanoDrop 8000 spectrophotometer (Thermo
 Fisher), and the specificity of expected amplicons by agarose gel
 electrophoresis of the qPCR products and the analysis of the melting
 curves from the qPCR instrument. DNA concentrations were measured using a
 Qubit 2.0 fluorometer with either Qubit dsDNA BR Assay Kit or Qubit dsDNA
 HS Assay Kit reagents when appropriate.