Testing the Drosophila maternal haploid gene for functional divergence and a role in hybrid incompatibility

Testing the Drosophila maternal haploid gene for functional divergence and a role in hybrid incompatibility | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Testing the Drosophila maternal haploid gene for functional divergence and a role in hybrid incompatibility Dean M Castillo , Connor M Kean , Benjamin McCormick , Sahana Natesan , Daniel A Barbash doi: https://doi.org/10.1101/2022.03.23.485453 Dean M Castillo 1 Department of Biology, University of Nebraska at Omaha , Omaha, Nebraska 2 Department of Molecular Biology and Genetics, Cornell University , Ithaca, New York Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: deancastillo{at}unomaha.edu barbash{at}cornell.edu Connor M Kean 2 Department of Molecular Biology and Genetics, Cornell University , Ithaca, New York Find this author on Google Scholar Find this author on PubMed Search for this author on this site Benjamin McCormick 2 Department of Molecular Biology and Genetics, Cornell University , Ithaca, New York Find this author on Google Scholar Find this author on PubMed Search for this author on this site Sahana Natesan 2 Department of Molecular Biology and Genetics, Cornell University , Ithaca, New York Find this author on Google Scholar Find this author on PubMed Search for this author on this site Daniel A Barbash 2 Department of Molecular Biology and Genetics, Cornell University , Ithaca, New York Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: deancastillo{at}unomaha.edu barbash{at}cornell.edu Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Crosses between D. simulans females and D. melanogaster males produce viable F1 sons and poorly viable F1 daughters. Unlike most hybrid incompatibilities, this hybrid incompatibility violates Haldane’s rule, the observation that incompatibilities preferentially affect the heterogametic sex. Further, it is important to understand fully the genetic basis of this incompatibility because the causal allele in D. melanogaster is a large species-specific block of complex satellite DNA on its X chromosome known as the 359-bp satellite, rather than a protein-coding locus. The causal allele(s) in D. simulans are unknown but likely involve maternally expressed genes or factors since the F1 females die during early embryogenesis. The maternal haploid ( mh ) gene is an intriguing candidate because it is expressed maternally and its protein product localizes to the 359-bp repeat. We found that this gene has diverged extensively between D. melanogaster and D. simulans . This observation led to the hypothesis that D. melanogaster mh may have co-evolved with the 359-bp repeat, and that hybrid incompatibility thus results from the absence of a co-evolved mh allele in D. simulans . We tested for functional divergence of mh by creating matched transformants of D. melanogaster and D. simulans orthologs in both D. melanogaster and D. simulans strains. Surprisingly, we find that D. simulans mh fully complements the female sterile phenotype of D. melanogaster mh mutations. Contrary to our hypothesis, we find no evidence that adding a D. melanogaster mh gene to D. simulans increases hybrid viability. Introduction The evolution of reproductive isolation via hybrid incompatibilities can be complex, with multiple incompatibilities contributing to isolation within a single species pair. Two genetically distinct lethal hybrid incompatibilities exist between the sister species D. melanogaster and D. simulans ( Sawamura et al . 1993b ; Barbash 2010 ). When D. melanogaster females are crossed to D. simulans males, the F1 hybrid sons are invariably lethal, while the F1 daughters are generally fully viable, at least at lower temperatures (~<25°) ( Sturtevant 1920 ; Barbash et al . 2000 ). Importantly, this pattern of lethality is not sex-specific but rather caused by the presence of the D. melanogaster X chromosome. Experiments that can detect products of non-disjunction or use attached-X chromosomes demonstrate that daughters inheriting both X chromosomes from their D. melanogaster mother are lethal while sons inheriting their X from their D. simulans father are viable ( Barbash 2010 ). The lethality is caused by an incompatibility between the D. melanogaster allele of the gene Hmr on the D. melanogaster X, interacting with the D. simulans alleles of the autosomal genes Lhr and GFZF ( Watanabe 1979 ; Hutter and Ashburner 1987 ; Brideau et al . 2006 ; Phadnis et al . 2015 ). In contrast, the reciprocal cross of D. simulans females to D. melanogaster males produces viable F1 sons and poorly viable F1 daughters that die as early embryos ( Sturtevant 1920 ). While the D. melanogaster X is again implicated in causing this embryonic lethality, Sawamura et al. (1993c) showed that Hmr is not responsible for this F1 lethality. Instead, the lethal effect of the D. melanogaster X maps to the pericentromeric heterochromatin in a region called Zhr ( Sawamura and Yamamoto 1993 ). The lethal effect of Zhr + appears to be caused by missegregation during early embryogenesis of a multi-million base pair block of complex satellite DNA sequences ( Ferree and Barbash 2009 ). These satellite sequences are known alternatively as the 359-bp or the 1.688 g/cm 3 satellites ( Sawamura and Yamamoto 1993 ; Ferree and Barbash 2009 ) ( Lohe and Brutlag 1986 ). While D. simulans contains some dispersed 359-bp repeats, it does not have the large X-linked block found in D. melanogaster ( Lohe et al . 1993 ; Sproul et al . 2020 ; de Lima et al . 2020 ). This extensive difference in abundance of the 359-bp satellite between the species suggests that D. melanogaster may contain allele(s) that have coevolved with the X-linked 359-bp satellite block to help promote its proper mitotic segregation. This logic further implies that the lack of the X-linked 359-bp satellite block in D. simulans would cause it to be unable to regulate this satellite block when inherited from a D. melanogaster parent, leading to hybrid incompatibility. Because the F1 hybrid female lethality occurs during early embryonic development, the allele(s) hypothesized to be missing from D. simulans are likely to be maternally expressed ( Sturtevant 1920 ; Ferree and Barbash 2009 ). The penetrance of F1 hybrid female lethality is highly variable across strains, which has complicated efforts to identify the genes causing this lethality. Sawamura et al. identified a strain of D. simulans called maternal hybrid rescue ( mhr ) that produces high viability of F1 hybrid daughters and mapped the effect to the second chromosome ( Sawamura et al . 1993a ). Orr also implicated the D. simulans second chromosome in contributing to hybrid lethality in this cross ( Orr 1996 ). Another study directly tested the satellite-binding protein D1 as a candidate but found no evidence for a role in the incompatibility ( Ferree and Barbash 2009 ). Further attempts to identify the genetic basis of the D. simulans maternal effect on hybrid viability led to the plausible conclusion that it is a polygenic effect ( Gérard and Presgraves 2012 ). Others have suggested that the incompatibility may be caused by the absence in D. simulans of maternally deposited small RNAs homologous to the 359-bp repeat, rather than by protein-coding genes ( Ferree and Barbash 2007 ). Several studies have shown that such RNAs are produced by heterochromatic satellites including the X-linked 359-bp satellite block ( Usakin et al . 2007 ; Wei et al . 2021 ), though their potential role in hybrid lethality remains untested. The X-linked gene maternal haploid ( mh ) is an intriguing candidate for contributing to this interspecific incompatibility. Mutations in mh were first identified based on its female sterility phenotype ( Gans et al . 1975 ). Embryos from mh mutant mothers typically arrest within the first few nuclear cycles with condensation defects specific to the paternally inherited chromosomes, with a minority reaching late embryogenesis as lethal gynogenetic haploids ( Zalokar et al . 1975 ; Loppin et al . 2001 ). The mh gene encodes a predicted metalloprotease, homologs of which are involved in DNA damage repair ( Delabaere et al . 2014 ; Tang et al . 2017 ). Most relevant to this study, the mh protein localizes to the 359-bp satellite during embryogenesis and the satellite shows aberrant segregation in the embryonic progeny of mh mutant mothers ( Tang et al . 2017 ). These findings, as well as the evolutionary patterns described below, motivated us to test for functional divergence of mh orthologs between D. melanogaster and D. simulans and for possible effects on hybrid lethality. In particular, we tested the hypothesis that hybrid female embryonic lethality results from the inability of the maternally expressed D. simulans Mh protein to properly interact with the paternally inherited X-linked D. melanogaster 359-bp satellite block. Under this hypothesis, we predict that adding mel-mh to D. simulans mothers would suppress hybrid lethality. Materials and Methods Nomenclature We use the abbreviations mel-mh and sim-mh to refer to the mh ortholog in D. melanogaster and D. simulans , respectively. We use phi{mel-mh-Gfp} and phi{sim-mh-Gfp} to designate PhiC31-mediated transgenes containing Gfp fusions of mel-mh and sim-mh , respectively. As described in the Results, D. simulans has a tandem duplication of mh . The mh-p copy is more similar than the mh-d copy in sequence and structure to D. melanogaster mh . We therefore consider sim-mh-p to be the ortholog of D. melanogaster mh , and for simplicity refer to it as sim-mh . Drosophila stocks w mh 6 / FM7a, P{sChRFP}1 and w, mh 31 /FM7, GFP + stocks were kindly provided by Xiaona Tang and Yikang Rong. D. simulans stocks containing attP landing sites were kindly provided by David Stern ( Stern et al . 2017 ). Sequence analysis The sequences for both duplicates of mh from the D. simulans genome were taken from the genome sequence and aligned with the D. melanogaster ortholog to determine the consensus coding sequence (see Results). We then calculated D N /D S between the D. melanogaster mh and both D. simulans orthologs using MEGA 11 ( Tamura et al . 2021 ). We compared these ratios to a genome-wide sample of D N /D S ratios from a previously published study to determine their percentile and relative rate of evolutionary change ( Stanley and Kulathinal 2016 ). These data all consist of two-by-two (i.e. pairwise) sequence comparisons between D. melanogaster and D. simulans orthologs. mh transgene constructs The mel-mh-gfp transgene was kindly provided by Xiaona Tang and Yikang Rong and previously described as gfp-mh-pTV2gw ( Tang et al . 2017 ). We added an attB site to this to create the plasmid gfp-mh-pTV2gw-attB by PCR-amplifying using oligos 502/503 from a plasmid with an attB site that derived from the pTA-attB plasmid ( Groth et al . 2004 ). The PCR product was digested using NotI and inserted into gfp-mh-pTV2gw at its NotI site. All oligonucleotide sequences are listed in Table S1 . View this table: View inline View popup Download powerpoint Table S1. Oligonucleotide sequences The w+-attB-sim-mh-eGFP (p834) construct was designed to be parallel in structure to gfp-mh-pTV2gw-attB ( Fig 1 and Sup File 1) and was made in the following steps: w+-attB-sim-mh . The D. simulans mh genomic region covering the coding region and ~3000 bp upstream was PCR amplified from the strain w 501 using oligos 2099/2100 and cloned into pCR-Blunt II-TOPO (Invitrogen). The sequence of sim-mh was confirmed by Sanger sequencing (using oligos 788, 823, 2078, 2079, 2080, 2081, 2082, 2083, 2084 and 2101). The insert was then released by XbaI digestion and ligated into the XbaI site of the plasmid w + -attB , a gift from Jeff Sekelsky (Addgene plasmid # 30326; http://n2t.net/addgene:30326 ; RRID:Addgene_30326). The resulting clone w + -attB-sim-mh was confirmed by checking the pattern of restriction enzyme digestion. pCR-Blunt II-TOPO_sim-mh(partial)-eGFP . The coding sequence of eGFP was inserted into the coding sequence of sim-mh immediately after the start codon by Gibson assembly of Dsmh1, eGFP and Dsmh2. Dsmh1 and Dsmh2 were PCR amplified from D. simulans (strain w 501 ) using oligo pairs 2016/2017and 2020/2021, respectively, while eGFP was amplified using oligos 2018/2019 from pEGFP-attB (Drosophila Genomics Resource Center). A fusion product of Dsmh1-eGFP -Dsmh2 was PCR-amplified from the Gibson Assembly reaction mixture, using oligos 2080/2082 and cloned into pCR-Blunt II-TOPO. The sequence of the insert, i.e. the fusion of eGFP CDS and Dsmh (partial), was checked by Sanger sequencing using oligos 498, 788, 823, 2081. w+-attB-Dsmh-eGFP . The Dsmh(partial)_eGFP fragment was released from the pCR-Blunt II TOPO vector and ligated into w+-attB-sim-mh using double digestion (BsiWI-BstEII). The final construct w+attB-sim_mh-eGFP was confirmed by checking the restriction pattern of the construct. Download figure Open in new tab Fig 1. Map of mh region in D. simulans demonstrating the duplication event. The region in D. melanogaster has the same structure as the mh-p and Alg14 region. The dashed line shows the region duplicated to form mh-d and Alg14-d . Black arrows show translation start sites; the gray arrow in mh-d shows the alternative translation start site proposed by ( Chakraborty et al . 2021 ). The two boxes shown for Alg14-d represent a frameshift relative to Alg14 . A full annotation of this region is shown in Supp File 1. mh transgenic lines D. melanogaster transgenic lines were made by phiC31-mediated integration into the strain y w P{nos-phiC31\int.NLS}X; P{CaryP}attP40 . D. simulans transformant lines were made by phiC31-mediated integration into the strains y w; pBac{3XP3::EYFP,attP}1048-2R and y w; pBac{3XP3::EYFP,attP}1029-3R ( Stern et al . 2017 ). Microinjections were done by Rainbow Transgenic Flies, Inc. Fertility tests The mel-mh-Gfp and sim-mh-Gfp transgenes transformed into D. melanogaster were compared for their ability to complement the female sterility of mh null mutations. A smallscale pilot experiment was initially done at room temperature by crossing single virgin w mh 6 / w mh 31 ; {mh-Gfp, w + }attP40/+ females to two DGRP-882 (wild type) males, where mh-Gfp represents either of the two transgenes. Two sets were done, the first with females aged 3-4 days-old before mating and the second aged 9-10 days. Vials were cleared after 5 days; if either the female or both males were dead, then the vial was discarded To generate F1 females to assay in a large-scale experiment, a parental cross was set up of w mh 6 ; {mh-Gfp, w + }attP40 females and mh 31 /Y males, where mh-Gfp represents either of the two transgenes. Virgin F1 daughters of genotype w mh 6 / w mh 31 ; {mh-Gfp, w + }attP40/+ were collected and aged for 3-5 days, followed by test crosses containing one virgin female and two Canton-S (wild type) males. After 4-5 days, parents were flipped to new vials, and flipped again after another 4-5 days. At each flip, if either the female or both males were dead, then the vial was discarded. Otherwise, vials were kept for 16 (27°) or 18 (25°) days and all progeny counted. Three flips were performed at 27° and four at 25°; however very few parents survived until the fourth flip at 25° and thus we only report the first three flips in Fig 3B . The overall time period across all three flips was 12 days at 25° and 12-13 days at 27°. Progeny per day are reported in order to normalize between flips that were 4 or 5 days. Hybrid viability tests The mel-mh-Gfp and sim-mh-Gfp transgenes transformed into D. simulans were assessed for their ability to rescue female viability in F1 D. simulans/D. melanogaster hybrids as compared to a matched control group lacking the transgenes. To generate F1 hybrids either carrying or not carrying a mh-Gfp transgene, for each transgenic D. simulans line, the following parental crosses were set; first, w 501 virgin females were crossed to y w/Y; {mh-Gfp, w+} males, where mh-Gfp represents either of the two transgenes. Virgin daughters of the genotype w 501 /y w; {mh-Gfp, w+}/+ collected from this cross were subsequently mated to w 501 / Y males. For each set, 30-40 y? w/w 501 ; {mh-Gfp, w+}/+ and y? w/w 501 virgin daughters were separately collected, aged 0-1 days, and mated to 40-50 3-5 day old D. melanogaster Canton-S virgin males. The crosses were kept at room temperature (19.4-22.0°) and flipped every 2-4 days until they stopped producing progeny; the adult F1 hybrids were scored for sex. Western blots Young female virgin flies were fed yeast paste 2-3 days prior to dissection. Ovaries were dissected in 0.7% NaCl with 2 pairs of tweezers after flies were anesthetized by carbon dioxide. Ovaries were collected into 1.7 ml microcentrifuge tubes and flash-frozen in liquid nitrogen, then stored at −80°C until all dissections were completed. To extract proteins, ovaries were ground in SDS sample buffer (62.5 mM Tris pH6.8, 2% SDS, 10% glycerol, 1% ß-mercaptoethanol, 0.05% bromophenol blue), boiled for 3 minutes, and centrifuged at 10,000 rpm for 5 minutes. Proteins were separated via 7.5% SDS-PAGE using a BioRad Mini-PROTEAN Vertical Electro Cell and transferred to PVDF (polyvinylidene difluoride) membrane using BioRad Mini Trans-Blot. The protein standard was Thermo Scientific™ PageRuler™ Prestained Protein Ladder, 10 to 180 kDa. The protein-bound membrane was blocked in 5% skim milk in TBST (150mM NaCl, 20 mM Tris pH7.5, 0.1% Tween-20) for 1 hour at room temperature, followed by primary antibody incubation at 4°C for 16 hours, and secondary antibody incubation at room temperature for 1 hour. The membrane was washed 3 x 10 minutes in TBST after each antibody incubation. Primary antibodies used were: Anti-GFP Rabbit Polyclonal Antibody (1/5,000, Rockland Immunochemical 600-401-215S) and Monoclonal Mouse Anti-α-Tubulin antibody (1/20,000, Sigma T9026), secondary antibodies: HRP-Goat Anti-Rabbit IgG (H+L) (1/4,000, Jackson 111-035-003) and HRP-Goat Anti-Mouse IgG (H+L) (1/8,000, Jackson 115-035-003). Antibodies were diluted in either 5% skim milk or 5% BSA, in TBST. Signals were detected by applying ECL2 Western Blotting Substrate (Thermo Scientific 80197) to the membrane and exposing it to autoradiography film (VWR 490001-930). Results mh is duplicated in D. simulans When attempting to identify the mh ortholog in D. simulans , we noticed two different regions with homology to mel-mh . The first region was a contig on the X with high similarity but that appeared to be incomplete and possibly contain partial duplications. While pursuing this analysis a PacBio assembly of the D. simulans genome reported that mh is tandemly duplicated on the X along with the flanking gene alg14 ( Chakraborty et al . 2021 ). Following Chakraborty et al. (2021) , we refer to the D. simulans duplicates as mh-p (mh-proximal) and mh-d (mh-distal), though as noted below we suggest that mh-p can also be considered the parental copy and mh-d the daughter copy. We confirmed the duplication structure of D. simulans mh in an independent assembly of D. simulans created using Nanopore sequencing ( Miller et al . 2018 ). The Nanopore and PacBio assemblies fully agree in structure, with the exception of a ~25 bp insertion in the PacBio assembly immediately distal to the proximal copy (Supp File 1). The two genome assemblies also contain no differences in the coding sequences of either mh-p or mh-d . We used Artemis and the Artemis Comparison Tool to compare and annotate the structures of the D. melanogaster and D. simulans mh regions (cite) (Sup file 1). We estimate that the duplicated region corresponds to 3160 bp of sequence (relative to D. melanogaster ). This leaves D. simulans mh-d having a duplication of its 3’ region as its 5’ region, which may be responsible for its novel testis-enriched expression pattern reported by Chakraborty et al (2021) . There is a 16 bp deletion in the coding region of exon 1 of mh-d , relative to mh-p and D. melanogaster mh . We confirmed that mh-d has this deletion in five D. simulans strains that were sequenced using Sanger sequencing ( Begun et al . 2007 ). This deletion changes the coding potential of exon 1, but it could potentially splice to exon 2 and restore the same reading frame as in mh-p . We have annotated mh-d to have the same length of its N-terminal region as mh-p and D. melanogaster mh ( Fig. 1 ; Supp file 1). Charkrobarty et al. (2021) instead annotated the mh-d CDS as beginning in what would be its third exon relative to the mh-p gene structure, based on the reduced RNA-Seq reads mapping to exons 1 and 2. As there are some RNA-Seq reads that appear to map to the (potential) exons 1 and 2 of mh-d , further work is warranted to determine the protein product(s) made by mh-d . Regardless of this uncertainty regarding the N-terminal structure of mh-d, mh-p has greater similarity D. melanogaster mh in its structure, primary sequence, and expression pattern, suggesting that mh-p can be considered to be the parental copy and mh-d the daughter copy of the duplication. We thus define D. simulans mh-p as the ortholog of D. melanogaster mh . The second region of lesser homology mapped to an intron of the tkv gene on chr 2. This region of chr 2 is annotated as the pseudogene CR14033, and was identified as producing siRNAs in testis ( Czech et al . 2008 ; Okamura et al . 2008 ) that were noted as homologous to mh ( Czech et al . 2008 ). mh coding sequences are rapidly evolving We calculated pairwise divergence among the mh genes in D. melanogaster and D. simulans ( Table 1 ). The D N /D S ratios are relatively high compared to a genome-wide sample of loci, indicating substantial non-synonymous divergence. The comparison between mel-mh and sim-mh-p had a D N /D S ratio of 0.373, placing it in the top 10% of the genome-wide distribution. The comparison between mel-mh and sim-mh-d had a D N /D S ratio of 0.541 which is in the top 5% of the genome distribution. For reference, the hybrid incompatibility loci Hmr and Lhr are in the top 3% of the distribution. Both D N and D N /D S are higher between the paralogs sim-mh-p and sim-mh-d than between the orthologs mel-mh and sim-mh-p , though we reiterate that there are uncertainties in the annotation of sim-mh-d noted in the previous section. View this table: View inline View popup Download powerpoint Table 1. Divergence of mh genes between D. melanogaster and D. simulans D. simulans mh complements D. melanogaster mh mutants We constructed a transgene of D. simulans mh tagged with Gfp (called sim-mh-Gfp ) to precisely match in structure the D. melanogaster mh-Gfp transgene of Tang et al. (2017) . ( Fig 1 ; Methods). We transformed and integrated both transgenes into D. melanogaster at the same autosomal position. Western blots indicate that both transgenes express at similar levels ( Fig 2 ). We then crossed the transgenes into a mh 6 mutant background. Stable stocks were established, indicating that both transgenes can complement the sterility of mh 6 . To quantitatively compare the activity of these transgenes, we performed fertility assays of females trans-heterozygous for two mh null alleles and heterozygous for a mh-Gfp transgene; that is w mh 6 / w mh 31 ; {mel-mh-Gfp, w + }/+ compared to w mh 6 / w mh 31 ; {sim-mh-Gfp, w + }/+ . A small-scale pilot experiment found greater fertility among females carrying mel-mh-Gfp , but only among younger females ( Fig 3A ). We then performed a more extensive experiment, examining fertility across a 12-13 day period at two different temperatures ( Fig 3B ). No significant difference between the transgenic genotypes was observed at any time point, except for the first flip at 25°, where females carrying the mel-mh-Gfp transgene had significantly fewer progeny than those carrying the sim-mh-Gfp transgene. We conclude that the D. melanogaster and D. simulans orthologs have not substantially diverged for the essential female fertility function of mh . Download figure Open in new tab Fig 2. Western blot comparing mh-Gfp protein accumulation in different transgenic genotypes. Western blot with an anti-GFP antibody recognizes the Mh-eGFP protein in ovary extracts from females (top panel). Signals on the same membrane detected by an anti-α-Tubulin antibody served as a loading control (lower panel). Protein sizes of the pre-stained protein ladder were hand-marked on the membrane. Lanes 2-6 are from D. simulans extracts and lanes 7-9 from D. melanogaster extracts. Two independent transformants of the transgenes in D. simulans were analyzed, at attP sites 1029 and 1048. The attP-1048 and attP-40 samples are negative controls because they are the untransformed strains that carry the attP sites. The predicted molecular weights of mel-Mh-Gfp and sim-Mh-Gfp proteins are 108.6 and 109 kD, respectively. Download figure Open in new tab Fig. 3. Comparison of fertility between mh mutant females carrying mel-mh-Gfp versus sim-mh-Gfp transgenes (single w mh6 / w mh31; {mel-mh-Gfp, w+}attP40/+ versus w mh6 / w mh31; {sim-mh-Gfp, w+}attP40/+). Statistical significance between genotypes was tested by a two-sample unpaired t-test: *** = 0.0001 < p ≤ 0.001; * = 0.01 0.05 (not significant). A) Pilot experiment at room temperature (~20°-22°). Females were collected as virgins and aged for either 3-4 days (left) or 9-10 days (right) prior to mating and progeny collection for 5 days. Progeny are reported as ‘per-day’ to normalize with Fig 3B . B) Two large-scale experiments, performed at 25° and 27°. Virgin females were aged for 3-5 days prior to mating. Crosses were flipped to fresh vials after 4 or 5 days, and again after an additional 4 or 5 days, for a total collection period of 12-13 days; the exact length of each flip was recorded and is accounted for in calculating the ‘progeny per day’. Note that vials were discarded if the parents died, which is why N goes down between flips (see Methods). D. melanogaster mh does not affect hybrid viability Having established that sim-mh-Gfp is functional within D. melanogaster , we turned to the primary question motivating this study, of whether mh is a hybrid incompatibility gene. We tested this by comparing the D. simulans and D. melanogaster mh transgenes for their ability to modulate F1 hybrid female viability. Specifically, we sought to determine whether the addition of a mh + transgene to the mothers of these hybrids would increase viability of their daughters. We transformed these transgenes into two different attP sites in D. simulans , with Western blots with an anti-Gfp antibody demonstrating similar protein levels for both transgenes ( Fig 2 ). We then generated F1 interspecific hybrids by crossing D. simulans females heterozygous for either mel-mh-Gfp or sim-mh-Gfp , along with sibling females not carrying a transgene as controls, to wild type D. melanogaster males ( Table 2 ). The viability of the control F1 daughters not carrying a transgene was quite high, ranging from ~47%-69% relative to F1 sons, which limited our ability to detect substantial increases in the progeny of interspecific hybrids from mothers carrying a transgene. This relatively high rescue may reflect the fact that the transgenes were outcrossed to the D. simulans w 501 strain, which produced high hybrid viability in other crosses ( Gérard and Presgraves 2012 ). Regardless, the results are opposite to our hypothesis. Both sets of crosses with the sim-mh-Gfp transgene showed increased female viability from transgenic mothers compared to control mothers, though only set D (with the 1048 insertion) was statistically significant. In contrast, crosses with the mel-mh-Gfp transgene showed essentially no differences in the relative viability of daughters between the transgenic and control genotypes. Within the resolution of our assay, we find no evidence suggesting that hybrid lethality results from the absence of mel-mh alleles in D. simulans . View this table: View inline View popup Download powerpoint Table 2. Testing mh transgenes for modulation of interspecific F1 hybrid viability Discussion The mh gene is interesting based on its unusual mutant phenotype of producing gynogenetic haploid embryos. It also displays a relatively high rate of coding sequence evolution, and has experienced two duplications in Drosophila. Our major motivation in this study derived from the association of mel-Mh with the X-linked 359-bp satellite DNA block that is present in D. melanogaster but absent in D. simulans . We used transgenic constructs to compare the activity of mh orthologs from D. melanogaster and D. simulans in the background of both species. We designed the sim-mh-Gfp transgene to be parallel in structure to a mel-mh-Gfp transgene previously shown to be functional. Our sim-mh-Gfp transgene complements D. melanogaster mh mutations, as evidenced by the ability to maintain a fertile mh 6 ; sim-mh-Gfp stock. In the course of this study, Brand and Levine (2022) independently published a study of the evolution of mh between D. melanogaster and D. simulans . They compared mh ortholog function by replacing the endogenous D. melanogaster mh locus with the D. simulans ortholog. They found that females of this replacement line have reduced fertility compared to the D. melanogaster mh control line, and further found that this reduced fertility is dependent on an intact Zhr + locus, which contains a large block of the 359 bp satellite. Below we summarize our results and compare them to those of Brand and Levine where appropriate. Similar fertility function of mel-mh and sim-mh We observed some reduction of fertility in D. melanogaster mh mutants carrying sim-mh-Gfp compared to mel-mh-Gfp controls in one of two small scale initial experiments ( Fig 3A ). However, follow-up experiments at much larger scale failed to find any reduction (with one out of the six comparisons showing a modest but significant increase in fertility of sim-mh-Gfp relative to mel-mh-Gfp females; Fig 3B ). We conclude that D. melanogaster and D. simulans mh orthologs are largely interchangeable for female fertility. This contrasts with the result of Brand and Levine, who report a more than two-fold mean reduction in fertility of D. melanogaster females carrying sim-mh compared to mel-mh . One possible explanation for these differences is that the fertility of mh genotypes are inherently variable due to environmental (or other) variation, which is plausible for any genotype that may be sub-fertile but not completely sterile. Another possibility may be differences in experimental design of the fertility assays. We assayed females individually while Brand and Levine analyzed fertility in broods of four females. If a female (and/or the males they were mating with) died during the course of our experiment we could exclude that vial, while in the Brand and Levine design individual deaths may not have been recorded and would reduce the brood size and thus presumably the progeny count. We saw very high rates of death in one of our experiments (at 25°) that varied by genotype: mel-mh-Gfp crosses dropped from 118 to 71 from flip 1 to 2, while for sim-mh-Gfp the drop was much greater, from 112 to 47. There are also significant differences in experimental design of the gene replacements between the two studies. Here we have used transgenic constructs integrated into an autosomal site and crossed into mh null-allele backgrounds to ‘replace’ mh and compare mel-mh and sim-mh . We designed our sim-mh-Gfp transgene to match a previously described mel-mh-Gfp transgene that was shown to provide wild type mh function ( Tang et al . 2017 ). This introduces a potential position effect as mh is now in an autosomal location. It is also possible that sim-mh-Gfp does not express properly in a D. melanogaster background since it has its endogenous regulatory regions. We did, however, see robust protein expression from our transgenes. Brand and Levine (2022) used a CRISPR replacement strategy, replacing the endogenous mh locus in D. melanogaster with FLAG-tagged and codon-optimized mel-mh and sim-mh coding sequences. This means that the sim-mh allele is synthetic in the sense that the synonymous sites in the transgene are not native to D. simulans . But again, Western blots suggest that it is fully expressed. Among other differences in the two studies is that our fertility assays were done with one functional copy of mh in the mothers, while the Brand and Levine (2022) design has two functional copies. Brand and Levine (2022) found that D. melanogaster females with one mel-mh and one sim-mh allele are fully fertile, and other experiments support their conclusion that deleterious effects of sim-mh on fertility and ovarian morphology are dose-dependent. Lack of effect of mel-mh on hybrid viability We found no evidence that the absence of mel-mh contributes to hybrid female lethality. In crosses of D. simulans females to D. melanogaster males, D. simulans mothers carrying a mel-mh-Gfp transgene produced the same ratio of female hybrids compared to control crosses without the transgene. It remains possible that repeating our experiments in D. simulans genetic backgrounds that have a lower baseline of hybrid female viability might reveal more subtle effects that we could not detect. Surprisingly, we did observe a moderate effect of increased hybrid female viability produced by mothers that carried the sim-mh-Gfp transgene. This finding suggests that increased mh dosage may reduce mis-segregation of the 359-bp satellite in hybrids, but that such effects may not be dependent on the mel-mh ortholog that has co-evolved with the D. melanogaster 359-bp satellite block. Acknowledgements Plasmids were obtained from the Drosophila Genomics Resource Center (supported by NIH Grant 2P40OD010949). The w+attB plasmid was a gift from Jeff Sekelsky (Addgene plasmid # 30326). 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Erk , 1975 Developmental defects of female-sterile mutants of Drosophila melanogaster . Dev. Biol . 47 : 419 – 432 . OpenUrl CrossRef PubMed Web of Science Back to top Previous Next Posted March 23, 2022. Download PDF Supplementary Material Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following Testing the Drosophila maternal haploid gene for functional divergence and a role in hybrid incompatibility Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. Your Personal Message CAPTCHA This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Share Testing the Drosophila maternal haploid gene for functional divergence and a role in hybrid incompatibility Dean M Castillo , Connor M Kean , Benjamin McCormick , Sahana Natesan , Daniel A Barbash bioRxiv 2022.03.23.485453; doi: https://doi.org/10.1101/2022.03.23.485453 Share This Article: Copy Citation Tools Testing the Drosophila maternal haploid gene for functional divergence and a role in hybrid incompatibility Dean M Castillo , Connor M Kean , Benjamin McCormick , Sahana Natesan , Daniel A Barbash bioRxiv 2022.03.23.485453; doi: https://doi.org/10.1101/2022.03.23.485453 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Evolutionary Biology Subject Areas All Articles Animal Behavior and Cognition (8026) Biochemistry (18809) Bioengineering (14934) Bioinformatics (44541) Biophysics (22658) Cancer Biology (19787) Cell Biology (26969) Clinical Trials (138) Developmental Biology (14004) Ecology (21064) Epidemiology (2067) Evolutionary Biology (25507) Genetics (16191) Genomics (23547) Immunology (18750) Microbiology (42609) Molecular Biology (18122) Neuroscience (93684) Paleontology (701) Pathology (2989) Pharmacology and Toxicology (5106) Physiology (8135) Plant Biology (16038) Scientific Communication and Education (2098) Synthetic Biology (4575) Systems Biology (10259) Zoology (2394) window.__CF$cv$params={r:'a405e7488d14f48a',t:'MTc5MDI5NjA1MA==',u:'01a0d5f5d10b74018f083b7802bffb2f',ut:'dOKK8vSmStE94gA3fHOkrBIxgLutPv1Zdg4q.RvmrHg-1790296051-1.2.1.1-jH5J67W2h0.bQYk22VyKK37cbMxmCT_0uYN4e8EIY8CMnvA6OkBcjoEaixXSEVSyimTkavEptrYa3Qpzm_CxRxfgJomynroktEK5bOzkCD0',i:60};(function(){if(!document.body)return;var s=document.createElement('script');s.src='/cdn-cgi/challenge-platform/scripts/precursor/main.js';document.head.appendChild(s);})();