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Over 100 years of studies in Drosophila melanogaster and related species in the genus Drosophila have facilitated key discoveries in genetics, genomics, and evolution. While high-quality genome assemblies exist for several species in this group, they only encompass a small fraction of the genus. Recent advances in long-read sequencing allow high-quality genome assemblies for tens or even hundreds of species to be efficiently generated. Here, we utilize Oxford Nanopore sequencing to build an open community resource of genome assemblies for 101 lines of 93 drosophilid species encompassing 14 species groups and 35 sub-groups. The genomes are highly contiguous and complete, with an average contig N50 of 10.5 Mb and greater than 97% BUSCO completeness in 97/101 assemblies. We show that Nanopore-based assemblies are highly accurate in coding regions, particularly with respect to coding insertions and deletions. These assemblies, along with a detailed laboratory protocol and assembly pipelines, are released as a public resource and will serve as a starting point for addressing broad questions of genetics, ecology, and evolution at the scale of hundreds of species.

Accurate and comprehensive characterization of genetic variation is essential for deciphering the genetic basis of diseases and other phenotypes. A vast amount of genetic variation stems from large-scale sequence changes arising from the duplication, deletion, inversion, and translocation of sequences. In the past 10 years, high-throughput short reads have greatly expanded our ability to assay sequence variation due to single nucleotide polymorphisms. However, a recent de novo assembly of a second Drosophila melanogaster reference genome has revealed that short read genotyping methods miss hundreds of structural variants, including those affecting phenotypes. While genomes assembled using high-coverage long reads can achieve high levels of contiguity and completeness, concerns about cost, errors, and low yield have limited widespread adoption of such sequencing approaches. Here we resequenced the reference strain of D. melanogaster (ISO1) on a single Oxford Nanopore MinION flow cell run for 24 hr. Using only reads longer than 1 kb or with at least 30x coverage, we assembled a highly contiguous de novo genome. The addition of inexpensive paired reads and subsequent scaffolding using an optical map technology achieved an assembly with completeness and contiguity comparable to the D. melanogaster reference assembly. Comparison of our assembly to the reference assembly of ISO1 uncovered a number of structural variants (SVs), including novel LTR transposable element insertions and duplications affecting genes with developmental, behavioral, and metabolic functions. Collectively, these SVs provide a snapshot of the dynamics of genome evolution. Furthermore, our assembly and comparison to the D. melanogaster reference genome demonstrates that high-quality de novo assembly of reference genomes and comprehensive variant discovery using such assemblies are now possible by a single lab for under $1,000 (USD).

Simple Summary: Paramutation is an epigenetic phenomenon in which one allele triggers an epigenetic conversion of another allele. How paramutation occurs is poorly understood, but it frequently involves small RNAs that can silence an alternate allele in trans. In one well-known case in Drosophila, small RNAs known as piRNAs mediate paramutation. In this paper, we describe a novel system of epigenetic conversion in Drosophila virilis that demonstrates paramutation-like behavior. This occurs at the telomere, where retrotransposon arrays are known to be regulated by piRNAs. This paramutation-like behavior demonstrates that the unique properties of the Drosophila telomere may play a role in triggering epigenetic conversion in trans. This system promises to reveal new mechanisms that underlie paramutation-like behavior. First discovered in maize, paramutation is a phenomenon in which one allele can trigger an epigenetic conversion of an alternate allele. This conversion causes a genetically heterozygous individual to transmit alleles that are functionally the same, in apparent violation of Mendelian segregation. Studies over the past several decades have revealed a strong connection between mechanisms of genome defense against transposable elements by small RNA and the phenomenon of paramutation. For example, a system of paramutation in Drosophila melanogaster has been shown to be mediated by piRNAs, whose primary function is to silence transposable elements in the germline. In this paper, we characterize a second system of piRNA-mediated paramutation-like behavior at the telomere of Drosophila virilis. In Drosophila, telomeres are maintained by arrays of retrotransposons that are regulated by piRNAs. As a result, the telomere and sub-telomeric regions of the chromosome have unique regulatory and chromatin properties. Previous studies have shown that maternally deposited piRNAs derived from a sub-telomeric piRNA cluster can silence the sub-telomeric center divider gene of Drosophila virilis in trans. In this paper, we show that this silencing can also be maintained in the absence of the original silencing allele in a subsequent generation. The precise mechanism of this paramutation-like behavior may be explained by either the production of retrotransposon piRNAs that differ across strains or structural differences in the telomere. Altogether, these results show that the capacity for piRNAs to mediate paramutation in trans may depend on the local chromatin environment and proximity to the uniquely structured telomere regulated by piRNAs. This system promises to provide significant insights into the mechanisms of paramutation. [ABSTRACT FROM AUTHOR]

Experiments performed in different genetic backgrounds occasionally exhibit failure in experimental reproducibility. This is a serious issue in Drosophila where there are no standard control stocks. Here, we illustrate the importance of controlling genetic background by showing that the timing of a major meiotic event, the breakdown of the synaptonemal complex (SC), varies in different genetic backgrounds. We assessed SC breakdown in three different control stocks and found that in one control stock, y w; svspa-pol, the SC broke down earlier than in Oregon-R and w1118 stocks. We further examined SC breakdown in these three control backgrounds with flies heterozygous for a null mutation in c(3)G, which encodes a key structural component of the SC. Flies heterozygous for c(3)G displayed differences in the timing of SC breakdown in different control backgrounds, providing evidence of a sensitizing effect of this mutation. These observations suggest that SC maintenance is associated with the dosage of c(3)G in some backgrounds. Lastly, chromosome segregation was not affected by premature SC breakdown in mid-prophase, consistent with previous findings that chromosome segregation is not dependent on full-length SC in mid-prophase. Thus, genetic background is an important variable to consider with respect to SC behavior during Drosophila meiosis. [ABSTRACT FROM AUTHOR]

Balancer chromosomes are multiply inverted and rearranged chromosomes that are widely used in Drosophila genetics. First described nearly 100 years ago, balancers are used extensively in stock maintenance and complex crosses. Recently, the complete molecular structures of several commonly used balancers were determined by whole-genome sequencing. This revealed a surprising amount of variation among balancers derived from a common progenitor, identified genes directly affected by inversion breakpoints, and cataloged mutations shared by balancers. These studies emphasized that it is important to choose the optimal balancer, because different inversions suppress meiotic recombination in different chromosomal regions. In this review, we provide a brief history of balancers in Drosophila, discuss how they are used today, and provide examples of unexpected recombination events involving balancers that can lead to stock breakdown. [ABSTRACT FROM AUTHOR]

The synaptonemal complex (SC) is a conserved meiotic structure that regulates the repair of double-strand breaks (DSBs) into crossovers or gene conversions. The removal of any central-region SC component, such as the Drosophila melanogaster transverse filament protein C(3)G, causes a complete loss of SC structure and crossovers. To better understand the role of the SC in meiosis, we used CRISPR/Cas9 to construct 3 in-frame deletions within the predicted coiledcoil region of the C(3)G protein. Since these 3 deletion mutations disrupt SC maintenance at different times during pachytene and exhibit distinct defects in key meiotic processes, they allow us to define the stages of pachytene when the SC is necessary for homolog pairing and recombination during pachytene. Our studies demonstrate that the X chromosome and the autosomes display substantially different defects in pairing and recombination when SC structure is disrupted, suggesting that the X chromosome is potentially regulated differently from the autosomes. [ABSTRACT FROM AUTHOR]

During early meiotic prophase, homologous chromosomes are connected along their entire lengths by a proteinaceous tripartite structure known as the synaptonemal complex (SC). Although the components that comprise the SC are predominantly studied in this canonical ribbon-like structure, they can also polymerize into repeated structures known as polycomplexes. We find that in Drosophila oocytes, the ability of SC components to assemble into canonical tripartite SC requires the E3 ubiquitin ligase Seven in absentia (Sina). In sina mutant oocytes, SC components assemble into large rod-like polycomplexes instead of proper SC. Thus, the wild-type Sina protein inhibits the polymerization of SC components, including those of the lateral element, into polycomplexes. These polycomplexes persist into meiotic stages when canonical SC has been disassembled, indicating that Sina also plays a role in controlling SC disassembly. Polycomplexes induced by loss-of-function sina mutations associate with centromeres, sites of double-strand breaks, and cohesins. Perhaps as a consequence of these associations, centromere clustering is defective and crossing over is reduced. These results suggest that while features of the polycomplexes can be recognized as SC by other components of the meiotic nucleus, polycomplexes nonetheless fail to execute core functions of canonical SC. [ABSTRACT FROM AUTHOR]

Meiotic recombination, which is necessary to ensure that homologous chromosomes segregate properly, begins with the induction of meiotic DNA double-strand breaks (DSBs) and ends with the repair of a subset of those breaks into crossovers. Here we investigate the roles of two paralogous genes, CG12200 and CG31053, which we have named Narya and Nenya, respectively, due to their relationship with a structurally similar protein named Vilya. We find that narya recently evolved from nenya by a gene duplication event, and we show that these two RING finger domain-containing proteins are functionally redundant with respect to a critical role in DSB formation. Narya colocalizes with Vilya foci, which are known to define recombination nodules, or sites of crossover formation. A separation-of-function allele of narya retains the capacity for DSB formation but cannot mature those DSBs into crossovers. We further provide data on the physical interaction of Narya, Nenya and Vilya, as assayed by the yeast two-hybrid system. Together these data support the view that all three RING finger domain-containing proteins function in the formation of meiotic DNA DSBs and in the process of crossing over. [ABSTRACT FROM AUTHOR]

Meiosis is a defining characteristic of eukaryotes, believed to have evolved only once, over one billion years ago. While the general progression of meiotic events is conserved across multiple diverse organisms, the specific pathways and proteins involved can be highly divergent, even within species from the same genus. Here we investigate the rapid evolution of Matrimony (Mtrm), a female meiosis-specific regulator of Polo kinase (Polo) in Drosophila. Mtrm physically interacts with Polo and is required to restrict the activity of Polo during meiosis. Despite Mtrm's critical role in meiosis, sequence conservation within the genus Drosophila is poor. To explore the functional significance of this rapid divergence, we expressed Mtrm proteins from 12 different Drosophila species in the Drosophila melanogaster female germline. Distantly related Mtrm homologs are able to both physically interact with D. melanogaster Polo and rescue the meiotic defects seen in mtrm mutants. However, these distant homologs are not properly degraded after the completion of meiosis. Rather, they continue to inhibit Polo function in the early embryo, resulting in dominant maternal-effect lethality. We show that the ability of Mtrm to be properly degraded, and thus release Polo, is partially due to residues or motifs found within Mtrm's least-conserved regions. We hypothesize that, while Mtrm regions critical for its meiotic function are under strong purifying selection, changes that occurred in its unconserved regions may have been advantageous, potentially by affecting the timing or duration of meiosis and/or the early embryonic divisions. [ABSTRACT FROM AUTHOR]

The authors presents a comment to the article "Condensin II drives large-scale folding and spatial partitioning of interphase chromosomes in Drosophila nuclei" by Eric F. Joyce and colleagues. They discuss three contributions by Joyce and colleagues to the understanding of genome organization in Drosophila. The authors also relate the results of the study which found that condensin II depletion leads to increased intermixing of chromosomes.

Selfish genetic elements use a myriad of mechanisms to drive their inheritance and ensure their survival into the next generation, often at a fitness cost to its host. 1,2 Although the catalog of selfish genetic elements is rapidly growing, our understanding of host drive suppression systems that counteract self-seeking behavior is lacking. Here, we demonstrate that the biased transmission of the non-essential, non-driving B chromosomes in Drosophila melanogaster can be achieved in a specific genetic background. Combining a null mutant of matrimony , a gene that encodes a female-specific meiotic regulator of Polo kinase, 3,4 with the TM3 balancer chromosome creates a driving genotype that is permissive for the biased transmission of the B chromosomes. This drive is female-specific, and both genetic components are necessary, but not individually sufficient, for permitting a strong drive of the B chromosomes. Examination of metaphase I oocytes reveals that B chromosome localization within the DNA mass is mostly abnormal when drive is the strongest, indicating a failure of the mechanism(s) responsible for the proper distribution of B chromosomes. We propose that some proteins important for proper chromosome segregation during meiosis, like Matrimony, may have an essential role as part of a meiotic drive suppression system that modulates chromosome segregation to prevent genetic elements from exploiting the inherent asymmetry of female meiosis. [Display omitted] • Female meiotic drive promotes biased B chromosome inheritance in D. melanogaster • A genetic reduction of Mtrm and the TM3 balancer chromosome are necessary for drive • Strong drive correlates with aberrant B chromosome arrangements during metaphase I • The distributive segregation system may act to suppress drive during female meiosis B chromosomes are genetic renegades that can rapidly spread through a population when left unchecked. Hanlon and Hawley show that the B chromosomes in D. melanogaster are subject to female meiotic drive in a specific mutant, leading to the identification of a drive suppression system that may protect against invasion by selfish genetic elements. [ABSTRACT FROM AUTHOR]

: During the oocyte-to-embryo transition in Drosophila, degradation of the Polo kinase inhibitor, Matrimony, depends on Cortex, a meiosis-specific form of the Anaphase Promoting Complex/Cyclosome that is required for the oocyte's normal transition from meiosis to mitosis. [ABSTRACT FROM AUTHOR]

The structure of the meiosis-specific synaptonemal complex, which is perhaps the central visible characteristic of meiotic prophase, has been a matter of intense interest for decades. Although a general picture of the interactions between the transverse filament proteins that create this structure has emerged from studies in a variety of organisms, a recent analysis of synaptonemal complex structure in Caenorhabditis elegans by Schild-Prüfert et al. (2011) has provided the clearest picture of the structure of the architecture of a synaptonemal complex to date. Although the transverse filaments of the worm synaptonemal complex are assembled differently then those observed in yeast, mammalian, and Drosophila synaptonemal complexes, a comparison of the four assemblies shows that achieving the overall basic structure of the synaptonemal complex is far more crucial than conserving the structures of the individual transverse filaments. [ABSTRACT FROM AUTHOR]

In many animal species the meiosis I spindle in oocytes is anastral and lacks centrosomes. Previous studies of Drosophila oocytes failed to detect the native form of the germline-specific γ-tubulin (γTub37C) in meiosis I spindles, and genetic studies have yielded conflicting data regarding the role of γTub37C in the formation of bipolar spindles at meiosis I. Our examination of living and fixed oocytes carrying either a null allele or strong missense mutation in the γtub37C gene demonstrates a role for γTub37C in the positioning of the oocyte nucleus during late prophase, as well as in the formation and maintenance of bipolar spindles in Drosophila oocytes. Prometaphase I spindles in γtub37C mutant oocytes showed wide, non-tapered spindle poles and disrupted positioning. Additionally, chromosomes failed to align properly on the spindle and showed morphological defects. The kinetochores failed to properly co-orient and often lacked proper attachments to the microtubule bundles, suggesting that γTub37C is required to stabilize kinetochore microtubule attachments in anastral spindles. Although spindle bipolarity was sometimes achieved by metaphase I in both γtub37C mutants, the resulting chromosome masses displayed highly disrupted chromosome alignment. Therefore, our data conclusively demonstrate a role for γTub37C in both the formation of the anastral meiosis I spindle and in the proper attachment of kinetochore microtubules. Finally, multispectral imaging demonstrates the presences of native γTub37C along the length of wild-type meiosis I spindles. [ABSTRACT FROM AUTHOR]

Abstract: Ca2+ signaling pathways play important roles to complete meiosis from metaphase II arrest in vertebrate oocytes. However, less is known about the molecular mechanism of completion of meiosis in Drosophila females. Here, we provide direct evidence that calcineurin, a Ca2+/calmodulin (CaM)-dependent phosphatase, is essential for meiotic progression beyond metaphase I in Drosophila oocytes. Oocytes from germline clones lacking CanB2, a calcineurin regulatory subunit B, failed to complete meiosis after egg activation, and laid eggs exhibited a meiotic arrested anaphase I chromosome configuration. Genetic analyses suggest that calcineurin activity is regulated by Sarah (Sra), a family member of regulators of calcineurin (RCANs), through a Sra phosphorylation-dependent mechanism. Our results support a view in which the phosphorylation of Sra not only acts to relieve the inhibitory effects of Sra, but also acts to activate calcineurin, thus explaining the role of RCAN proteins as positive regulators of calcineurin. [ABSTRACT FROM AUTHOR]

The Drosophila gene ald encodes the fly ortholog of mps1, a conserved kinetochore-associated protein kinase required for the meiotic and mitotic spindle assembly checkpoints. Using live imaging, we demonstrate that oocytes lacking Ald/Mps1 (hereafter referred to as Ald) protein enter anaphase I immediately upon completing spindle formation, in a fashion that does not allow sufficient time for nonexchange homologs to complete their normal partitioning to opposite half spindles. This observation can explain the heightened sensitivity of nonexchange chromosomes to the meiotic effects of hypomorphic ald alleles. In one of the first studies of the female meiotic kinetochore, we show that Ald localizes to the outer edge of meiotic kinetochores after germinal vesicle breakdown, where it is often observed to be extended well away from the chromosomes. Ald also localizes to numerous filaments throughout the oocyte. These filaments, which are not observed in mitotic cells, also contain the outer kinetochore protein kinase Polo, but not the inner kinetochore proteins Incenp or Aurora-B. These filaments polymerize during early germinal vesicle breakdown, perhaps as a means of storing excess outer kinetochore kinases during early embryonic development. [ABSTRACT FROM AUTHOR]

Nod is a chromokinesin-like protein that plays a critical role in segregating achiasmate chromosomes during female meiosis. The C-terminal half of the Nod protein contains two putative DNA-binding domains. The first of these domains, known as the HMGN domain, consists of three tandemly repeated high-mobility group N motifs. This domain was previously shown to be both necessary and sufficient for binding of the C-terminal half of Nod to mitotic chromosomes in embryos. The second putative DNA-binding domain, denoted HhH(2)/NDD, is a helix-hairpin-helix(2)/Nod-like DNA-binding domain. Although the HhH(2)/NDD domain is not required or sufficient for chromosome binding in embryos, several well-characterized nod mutations have been mapped in this domain. To characterize the role of the HhH(2)/NDD domain in mediating Nod function, we created a series of UAS-driven transgene constructs capable of expressing either a wild-type Nod-GFP fusion protein or proteins in which the HhH(2)/NDD domain had been altered by site-directed mutagenesis. Although wild-type Nod-GFP localizes to the oocyte chromosomes and rescues the segregation defect in nod mutant oocytes, two of three proteins carrying mutants in the HhH(2)/NDD domain fail to either rescue the nod mutant phenotype or bind to oocyte chromosomes. However, these mutant proteins do bind to the polytene chromosomes in nurse-cell nuclei and enter the oocyte nucleus. Thus, even though the HhH(2)/NDD domain is not essential for chromosome binding in other cell types, it is required for chromosome binding in the oocyte. These HhH(2)/NDD mutants also block the localization of Nod to the posterior pole of stage 9–10A oocytes, a process that is thought to facilitate the interaction of Nod with the plus ends of microtubules (CUI et al. 2005). This observation suggests that the Nod HhH2/NDD domain may play other roles in addition to binding Nod to meiotic chromosomes. [ABSTRACT FROM AUTHOR]

The multiply inverted X chromosome balancer FM7 strongly suppresses, or eliminates, the occurrence of crossing over when heterozygous with a normal sequence homolog. We have utilized the LacI-GFP: lacO system to visualize the effects of FM7 on meiotic pairing, synapsis, and double-strand break formation in Drosophila oocytes. Surprisingly, the analysis of meiotic pairing and synapsis for three lacO reporter couplets in FM7/X heterozygotes revealed they are paired and synapsed during zygotene/pachytene in 70%-80% of oocytes. Moreover, the regions defined by these lacO couplets undergo double-strand break formation at normal frequency. Thus, even complex aberration heterozygotes usually allow high frequencies of meiotic pairing, synapsis, and double-strand break formation in Drosophila oocytes. However, the frequencies of failed pairing and synapsis were still 1.5- to 2-fold higher than were observed for corresponding regions in oocytes with two normal sequence X chromosomes, and this effect was greatest near a breakpoint. We propose that heterozygosity for breakpoints creates a local alteration in synaptonemal complex structure that is propagated across long regions of the bivalent in a fashion analogous to chiasma interference, which also acts to suppress crossing over. [ABSTRACT FROM AUTHOR]

The semisterile meiotic mutant mei-352 alters the distribution of meiotic exchanges without greatly affecting their total frequency. We show that the mei-352 mutation is an allele of the klp3A gene, which encodes a kinesin-like protein of the Kinesin-4 family. The semisterility observed in mei-352 females results from a known defect of klp3A oocytes in mediating pronuclear fusion. Interestingly, other klp3A alleles also exhibit defects in meiotic recombination similar to those of mei-352. Finally, we show that the Klp3A protein localizes within the oocyte nucleus during meiotic prophase, the time at which exchange distribution is established, and extensively colocalizes with DNA. The parallel of the klp3A phenotype with a meiotic defect observed for kar3 mutants in yeast suggests a role for kinesins in early meiosis and might reflect a previously suggested role for this class of kinesins in chromosome condensation. [ABSTRACT FROM AUTHOR]

The synaptonemal complex (SC) is intimately involved in the process of meiotic recombination in most organisms, but its exact role remains enigmatic. One reason for this uncertainty is that the overall structure of the SC is evolutionarily conserved, but many SC proteins are not. Two putative SC proteins have been identified in Drosophila: C(3)G and C(2)M. Mutations in either gene cause defects in SC structure and meiotic recombination. Although neither gene is well conserved at the amino acid level, the predicted secondary structure of C(3)G is similar to that of transverse-filament proteins, and C(2)M is a distantly related member of the α-kleisin family that includes Rec8, a meiosis-specific cohesin protein. Here, we use immunogold labeling of SCs in Drosophila ovaries to localize C(3)G and C(2)M at the EM level. We show that both C(3)G and C(2)M are components of the SC, that the orientation of C(3)G within the SC is similar to other transverse-filament proteins, and that the N terminus of C(2)M is located in the central region adjacent to the lateral elements (LEs). Based on our data and the known phenotypes of C(2)M and C(3)G mutants, we propose a model of SC structure in which C(2)M links C(3)G to the LEs. [ABSTRACT FROM AUTHOR]

Previous data suggested that anastral spindles, morphologically similar to those found in oocytes, can assemble in a centrosome-independent manner in cells that contain centrosomes. It is assumed that the microtubules that build these acentrosomal spindles originate over the chromatin. However, the actual processes of centrosome-independent microtubule nucleation, polymerisation, and sorting have not been documented in centrosome-containing cells. We have identified two experimental conditions in which centrosomes are kept close to the plasma membrane, away from the nuclear region, throughout meiosis I in Drosophila spermatocytes. Time-lapse confocal microscopy of these cells labelled with fluorescent chimeras reveals centrosome-independent microtubule nucleation, growth, and sorting into a bipolar spindle array over the nuclear region, away from the asters. The onset of noncentrosomal microtubule nucleation is significantly delayed with respect to nuclear envelope breakdown and coincides with the end of chromosome condensation. It takes place in foci that are close to the membranes that ensheath the nuclear region, not over the condensed chromosomes. Metaphase plates are formed in these spindles, and, in a fraction of them, some degree of polewards chromosome segregation takes place. In these cells that contain both membrane-bound asters and an anastral spindle, the orientation of the cytokinesis furrow correlates with the position of the asters and is independent of the orientation of the spindle. We conclude that the fenestrated nuclear envelope may significantly contribute to the normal process of spindle assembly in Drosophila spermatocytes. We also conclude that the anastral spindles that we have observed are not likely to provide a robust back-up able to ensure successful cell division. We propose that these anastral microtubule arrays could be a constitutive component of wild-type spindles, normally masked by the abundance of centrosome-derived... [ABSTRACT FROM AUTHOR]

This paper reports on a new role for mei-41 in cell cycle control during meiosis. This function is revealed by the requirement of mei-41 for the precocious anaphase observed in crossover-defective mutants. Normally in Drosophila oocytes, tension on the meiotic spindle causes a metaphase I arrest. This tension results because crossovers, and the resulting chiasmata, hold homologs together that are being pulled by kinetochore microtobules toward opposite spindle poles. In the absence of tension, such as in a recombination-defective mutant, metaphase arrest is not observed and meiosis proceeds through the two divisions. Here we show that in some recombination-defective mutants, the precocious anaphase requires the mei-41 gene product. For example, metaphase arrest is not observed in mei-218 mutants because of the severe reduction in crossing over. In mei-41 mei-218 double mutants, however, metaphase arrest was restored. The effect of mei-41 is dependent on double-strand break formation. Thus, in mutants that fail to initiate meiotic recombination the absence of mei-41 has no effect. [ABSTRACT FROM AUTHOR]

Determines the extent to which recombinatorial processes act to control recombinant DNA (rDNA) copy number in wild-type drosophila. Basic structure and genetics of the rDNA; rDNA-mediated recombination between X and Y chromosomes; Significance of rDNA magnification; Spontaneous rDNA recombination and separate maintenance of the two nucleolus organizers.

Our current knowledge of B chromosome biology has been augmented by an increase in the number and diversity of species observed to carry B chromosomes as well as the use of next-generation sequencing for B chromosome genomic analysis. Within the genus Drosophila, B chromosomes have been observed in a handful of species, but recently they were discovered in a single laboratory stock of Drosophila melanogaster. In this paper, we review the B chromosomes that have been identified within the Drosophila genus and pay special attention to those recently found in D. melanogaster. These newly-discovered B chromosomes have centromeres, telomeres, and a number of simple satellite repeats. They also appear to be entirely heterochromatic since next-generation sequencing of isolated B chromosomes did not detect sequences associated with known genic regions. We also summarize what effects the B chromosomes have been found to have on the A chromosomes. Lastly, we highlight some of the outstanding questions regarding B chromosome biology and discuss how studying B chromosomes in Drosophila melanogaster, which is a versatile model system with a wealth of genetic and genomic tools, may advance our understanding of the B chromosome's unique biology. [ABSTRACT FROM AUTHOR]

R. Scott Hawley is an Investigator at the Stowers Institute for Medical Research in Kansas City and a Professor of Molecular Biosciences at the University of Kansas. He is best known for his studies of meiotic chromosome behaviour in Drosophila females. Indeed, an anonymous reviewer of one of his research proposals once claimed that Hawley “had elevated the genetic analysis of meiosis in Drosophila to an art form”. He is also well-known for his efforts in the teaching of genetics. He has co-authored two books and is hard at work on the third. [Copyright &y& Elsevier]

Studies in Drosophila support the view that a failure of cohesion between sister chromatids may contribute to meiotic nondisjunction in humans. Moreover, the demonstration of a meiotic aneugen in mice provides important clues to the higher frequencies of nondisjunction observed in older women. [Copyright &y& Elsevier]

Summary: The conserved kinase Mps1 is necessary for the proper functioning of the mitotic and meiotic spindle checkpoints (MSCs), which monitor the integrity of the spindle apparatus and prevent cells from progressing into anaphase until chromosomes are properly aligned on the metaphase plate []. In Drosophila melanogaster, a null allele of the gene encoding Mps1 was recently shown to be required for the proper functioning of the MSC, but it did not appear to exhibit a defect in female meiosis []. We demonstrate here that the meiotic mutant ald1 is a hypomorphic allele of the mps1 gene. Both ald1 and a P-insertion allele of mps1 exhibit defects in female meiotic chromosome segregation. The observed segregational defects are substantially more severe for pairs of achiasmate homologs, which are normally segregated by the achiasmate (or distributive) segregation system, than they are for chiasmate bivalents. Furthermore, cytological analysis of ald1 mutant oocytes reveals both a failure in the coorientation of achiasmate homologs at metaphase I and a defect in the maintenance of the chiasmate homolog associations that are normally observed at metaphase I. We conclude that Mps1 plays an important role in Drosophila female meiosis by regulating processes that are especially critical for ensuring the proper segregation of nonexchange chromosomes. [Copyright &y& Elsevier]

Accurate and comprehensive characterization of genetic variation is essential for deciphering the genetic basis of diseases and other phenotypes. A vast amount of genetic variation stems from largescale sequence changes arising from the duplication, deletion, inversion, and translocation of sequences. In the past 10 years, high-throughput short reads have greatly expanded our ability to assay sequence variation due to single nucleotide polymorphisms. However, a recent de novo assembly of a second Drosophila melanogaster reference genome has revealed that short read genotyping methods miss hundreds of structural variants, including those affecting phenotypes. While genomes assembled using highcoverage long reads can achieve high levels of contiguity and completeness, concerns about cost, errors, and low yield have limited widespread adoption of such sequencing approaches. Here we resequenced the reference strain of D. melanogaster (ISO1) on a single Oxford Nanopore MinION flow cell run for 24 hr. Using only reads longer than 1 kb or with at least 30x coverage, we assembled a highly contiguous de novo genome. The addition of inexpensive paired reads and subsequent scaffolding using an optical map technology achieved an assembly with completeness and contiguity comparable to the D. melanogaster reference assembly. Comparison of our assembly to the reference assembly of ISO1 uncovered a number of structural variants (SVs), including novel LTR transposable element insertions and duplications affecting genes with developmental, behavioral, and metabolic functions. Collectively, these SVs provide a snapshot of the dynamics of genome evolution. Furthermore, our assembly and comparison to the D. melanogaster reference genome demonstrates that high-quality de novo assembly of reference genomes and comprehensive variant discovery using such assemblies are now possible by a single lab for under $1,000 (USD). [ABSTRACT FROM AUTHOR]

The Drosophila genus is a unique group containing a wide range of species that occupy diverse ecosystems. In addition to the most widely studied species, Drosophila melanogaster, many other members in this genus also possess a well-developed set of genetic tools. Indeed, high-quality genomes exist for several species within the genus, facilitating studies of the function and evolution of cis-regulatory regions and proteins by allowing comparisons across at least 50 million years of evolution. Yet, the available genomes still fail to capture much of the substantial genetic diversity within the Drosophila genus. We have therefore tested protocols to rapidly and inexpensively sequence and assemble the genome from any Drosophila species using single-molecule sequencing technology from Oxford Nanopore. Here, we use this technology to present highly contiguous genome assemblies of 15 Drosophila species: 10 of the 12 originally sequenced Drosophila species (ananassae, erecta, mojavensis, persimilis, pseudoobscura, sechellia, simulans, virilis, willistoni, and yakuba), four additional species that had previously reported assemblies (biarmipes, bipectinata, eugracilis, and mauritiana), and one novel assembly (triauraria). Genomes were generated from an average of 29x depth-of-coverage data that after assembly resulted in an average contig N50 of 4.4 Mb. Subsequent alignment of contigs from the published reference genomes demonstrates that our assemblies could be used to close over 60% of the gaps present in the currently published reference genomes. Importantly, the materials and reagents cost for each genome was approximately $1,000 (USD). This study demonstrates the power and cost-effectiveness of long-read sequencing for genome assembly in Drosophila and provides a framework for the affordable sequencing and assembly of additional Drosophila genomes. [ABSTRACT FROM AUTHOR]

Symbiotic interactions between microbes and their multicellular hosts have manifold biological consequences. To better understand how bacteria maintain symbiotic associations with animal hosts, we analyzed genome-wide gene expression for the endosymbiotic a-proteobacteria Wolbachia pipientis across the entire life cycle of Drosophila melanogaster. We found that the majority of Wolbachia genes are expressed stably across the D. melanogaster life cycle, but that 7.8% of Wolbachia genes exhibit robust stage- or sexspecific expression differences when studied in the whole-organism context. Differentially-expressed Wolbachia genes are typically up-regulated after Drosophila embryogenesis and include many bacterial membrane, secretion system, and ankyrin repeat-containing proteins. Sex-biased genes are often organized as small operons of uncharacterized genes and are mainly up-regulated in adult Drosophila males in an age-dependent manner. We also systematically investigated expression levels of previously-reported candidate genes thought to be involved in host-microbe interaction, including those in the WO-A and WO-B prophages and in the Octomom region, which has been implicated in regulating bacterial titer and pathogenicity. Our work provides comprehensive insight into the developmental dynamics of gene expression for a widespread endosymbiont in its natural host context, and shows that public gene expression data harbor rich resources to probe the functional basis of the Wolbachia-Drosophila symbiosis and annotate the transcriptional outputs of the Wolbachia genome. [ABSTRACT FROM AUTHOR]

Summary: The pairing of homologous chromosomes and the intimate synapsis of the paired homologs by the synaptonemal complex (SC) are essential for subsequent meiotic processes including recombination and chromosome segregation. Here we show that the centromere clustering plays an important role in initiating homolog synapsis during meiosis in Drosophila females. Although centromeres are not clustered prior to the onset of meiosis, all four pairs of centromeres are actively clustered into one or two masses during early meiotic prophase. Within the 16-cell cyst, centromeric clustering appears to define the first step in the initiation of synapsis. Clustering is restricted to the nuclei that form the SC and is dependent on all known SC proteins. Surprisingly, both centromeric clusters and the SC components associated with them persist long after the disassembly of the euchromatic SC at the end of pachytene. The initiation of homologous recombination through the formation of programmed double-strand breaks (DSBs) is not required for either the formation or the maintenance of the centromeric clusters. Our data support a view in which the SC-mediated clustering at the centromeres is the initiating event for meiotic synapsis. [ABSTRACT FROM AUTHOR]

Abstract: Chiasmata established by recombination are normally sufficient to ensure accurate chromosome segregation during meiosis by physically interlocking homologs until anaphase I. Drosophila melanogaster female meiosis is unusual in that it is both exceptionally tolerant of nonexchange chromosomes and competent in ensuring their proper segregation. As first noted by Puro and Nokkala [Puro, J., Nokkala, S., 1977. Meiotic segregation of chromosomes in Drosophila melanogaster oocytes. A cytological approach. Chromosoma 63, 273–286], nonexchange chromosomes move precociously towards the poles following formation of a bipolar spindle. Indeed, metaphase arrest has been previously defined as the stage at which nonexchange homologs are symmetrically positioned between the main chromosome mass and the poles of the spindle. Here we use studies of both fixed images and living oocytes to show that the stage in which achiasmate chromosomes are separated from the main mass does not in fact define metaphase arrest, but rather is a component of an extended prometaphase. At the end of prometaphase, the nonexchange chromosomes retract into the main chromosome mass, which is tightly repackaged with properly co-oriented centromeres. This repackaged state is the true metaphase arrest configuration in Drosophila female meiosis. [Copyright &y& Elsevier]

Members of the minichromosome maintenance (MCM) family have pivotal roles in many biological processes. Although originally studied for their role in DNA replication, it is becoming increasingly apparent that certain members of this family are multifunctional and also play roles in transcription, cohesion, condensation, and recombination. Here we provide a genetic dissection of the mcm5 gene in Drosophila that demonstrates an unexpected function for this protein. First, we show that homozygotes for a null allele of mcm5 die as third instar larvae, apparently as a result of blocking those replication events that lead to mitotic divisions without impairing endo-reduplication. However, we have also recovered a viable and fertile allele of mcm5 (denoted mcm5A7) that specifically impairs the meiotic recombination process. We demonstrate that the decrease in recombination observed in females homozygotis for mcm5A7 is not due to a failure to create or repair meiotically induced double strand breaks (DSBs), hut rather to a failure to resolve those DSBs into meiotic crossovers. Consistent with their ability to repair meiotically induced DSBs, flies homozygous for mcm5A7 are hilly proficient in somatic DNA repair. These results strengthen the observation that members of the prereplicative complex have multiple functions and provide evidence that mcm5 plays a critical role in the meiotic recombination pathway. [ABSTRACT FROM AUTHOR]

In a screen for new DNA repair mutants, we tested 6275 Drosophila strains bearing homozygous mutagenized autosomes (obtained from C. Zuker) for hypersensitivity to methyl methanesulfonate (MMS) and nitrogen mustard (HN2). Testing of 2585 second-chromosome lines resulted in the recovery of 18 mutants, 8 of which were alleles of known genes. The remaining 10 second-chromosome mutants were solely sensitive to MMS and define 8 new mutagen-sensitive genes (mus212-mus219). Testing of 3690 third chromosomes led to the identification of 60 third-chromosome mutants, 44 of which were alleles of known genes. The remaining 16 mutants define 14 new mutagen-sensitive genes (mus314-mus327). We have initiated efforts to identify these genes at the molecular level and report here the first two identified. The HN2-sensitive mus322 mutant defines the Drosophila ortholog of the yeast snm1 gene, and the MMS- and HN2-sensitive mus301 mutant defines the Drosophila ortholog of the human HEL308 gene. We have also identified a second-chromosome mutant, mus215ZIII-2059, that uniformly reduces the frequency of meiotic recombination to <3% of that observed in wild type and thus defines a function required for both DNA repair and meiotic recombination. At least one allele of each new gene identified in this study is available at the Bloomington Stock Center. [ABSTRACT FROM AUTHOR]