The level of resistance to radiation and the developmental and molecular responses can vary between species, and even between developmental stages of one species. For flies (order: Diptera), prior studies concluded that the fungus gnat Bradysia (Sciara) coprophila (sub-order: Nematocera) is more resistant to irradiation-induced mutations that cause visible phenotypes than the fruit fly Drosophila melanogaster (sub-order: Brachycera). Therefore, we characterized the effects of and level of resistance to ionizing radiation on B. coprophila throughout its life cycle. Our data show that B. coprophila embryos are highly sensitive to even low doses of gamma-irradiation, whereas late-stage larvae can tolerate up to 80 Gy (compared to 40 Gy for D. melanogaster) and still retain their ability to develop to adulthood, though with a developmental delay. To survey the genes involved in the early transcriptional response to irradiation of B. coprophila larvae, we compared larval RNA-seq profiles with and without radiation treatment. The up-regulated genes were enriched for DNA damage response genes, including those involved in DNA repair, cell cycle arrest, and apoptosis, whereas the down-regulated genes were enriched for developmental regulators, consistent with the developmental delay of irradiated larvae. Interestingly, members of the PARP and AGO families were highly up-regulated in the B. coprophila radiation response. We compared the transcriptome responses in B. coprophila to the transcriptome responses in D. melanogaster from 3 previous studies: whereas pathway responses are highly conserved, specific gene responses are less so. Our study lays the groundwork for future work on the radiation responses in Diptera.

Homoeologous relationships between the arms of wheat group 2 chromosomes were analysed by studying homoeologous pairing in wheat—rye hybrids. Chromosomes 2A and 2D were identified using telocentrics 2AS, 2DS or 2DL. The remaining wheat chromosomes and their arms were identified by C-banding. Wheat chromosomes in the hybrids were derived from plants which were heterozygous(Ph1/ph1b) and monotelocentric for 2AS, 2DS or 2DL. In these plants, chromosome 2B and the telocentric showed regular bivalent pairing. From the results of homoeologous pairing in wheat—rye hybrids, normal homoeologous relationships between 2AS, 2BS and 2DS, as well as between 2AL, 2BL and 2DL, were deduced. There was no evidence that wheat group 2 chromosomes were involved in evolutionary chromosome rearrangements. The existence of a pairing promoter gene on chromosome arm 2DS was supported.

The lower Dipteran fungus fly, Sciara coprophila , has many unique biological features. For example, Sciara undergoes paternal chromosome elimination and maternal X chromosome nondisjunction during spermatogenesis, paternal X elimination during embryogenesis, intrachromosomal DNA amplification of DNA puff loci during larval development, and germline-limited chromosome elimination from all somatic cells. Paternal chromosome elimination in Sciara was the first observation of imprinting, though the mechanism remains a mystery. Here, we present the first draft genome sequence for Sciara coprophila to take a large step forward in aiding these studies. We approached assembling the Sciara genome using multiple sequencing technologies: PacBio, Oxford Nanopore MinION, and Illumina. To find an optimal assembly using these datasets, we generated 44 Illumina assemblies using 7 short-read assemblers and 50 long-read assemblies of PacBio and MinION sequence data using 6 long-read assemblers. We ranked assemblies using a battery of reference-free metrics, and scaffolded a subset of the highest-ranking assemblies using BioNano Genomics optical maps. RNA-seq datasets from multiple life stages and both sexes facilitated genome annotation. Moreover, we anchored nearly half of the Sciara genome sequence into chromosomes. Finally, we used the signal level of both the PacBio and Oxford Nanopore data to explore the presence or absence of DNA modifications in the Sciara genome since DNA modifications may play a role in imprinting in Sciara , as they do in mammals. These data serve as the foundation for future research by the growing community studying the unique features of this emerging model system.

Dosage compensation is a fundamental mechanism in many species that corrects for the inherent imbalance in X-chromosome copy number between XY males and XX females. In Drosophila melanogaster , transcriptional output from the single male X-chromosome is equalized to that of XX females by recruitment of the Male Specific Lethal (MSL) complex to specific sequences along the length of the X-chromosome. The initial recruitment of MSL complex to the X-chromosome is dependent on a recently discovered zinc finger protein called Chromatin-Linked Adapter for MSL Proteins (CLAMP). However, further studies on the in vivo function of CLAMP remained difficult because the location of the gene in pericentric heterochromatin made it challenging to create null mutations or deficiencies. Using the CRISPR/Cas9 genome editing system, we generated the first null mutant in the clamp gene that eliminates expression of CLAMP protein. We show that CLAMP is necessary for both male and female viability. While females die at the third instar larval stage, males die earlier, likely due to the essential role of CLAMP in male dosage compensation. Moreover, we demonstrate that CLAMP promotes dosage compensation in males and represses key male-specific transcripts involved in sex-determination in females. Our results reveal that CLAMP is an essential protein with dual roles in males and females, which together assure that dosage compensation is a sex-specific process.

Dosage compensation is an essential process that equalizes transcript levels of X-linked genes between sexes by forming a domain of coordinated gene expression. Throughout the evolution of Diptera, many different X-chromosomes acquired the ability to be dosage compensated. Once each newly evolved X-chromosome is targeted for dosage compensation in XY males, its active genes are upregulated two-fold to equalize gene expression with XX females. In Drosophila melanogaster, the CLAMP zinc finger protein links the dosage compensation complex to the X-chromosome. However, the mechanism for X-chromosome identification has remained unknown. Here, we combine biochemical, genomic and evolutionary approaches to reveal that expansion of GA-dinucleotide repeats likely accumulated on the X-chromosome over evolutionary time to increase the density of CLAMP binding sites, thereby driving the evolution of dosage compensation. Overall, we present new insight into how subtle changes in genomic architecture, such as expansions of a simple sequence repeat, promote the evolution of coordinated gene expression.

Heterogametic species require chromosome-wide gene regulation to compensate for differences in sex chromosome gene dosage. In Drosophila melanogaster, transcriptional output from the single male X-chromosome is equalized to that of XX females by recruitment of the male-specific lethal (MSL) complex, which increases transcript levels of active genes 2-fold. The MSL complex contains several protein components and two non-coding RNA on the X ( roX) RNAs that are transcriptionally activated by the MSL complex. We previously discovered that targeting of the MSL complex to the X-chromosome is dependent on the chromatin-linked adapter for MSL proteins (CLAMP) zinc finger protein. To better understand CLAMP function, we used the CRISPR/Cas9 genome editing system to generate a frameshift mutation in the clamp gene that eliminates expression of the CLAMP protein. We found that clamp null females die at the third instar larval stage, while almost all clamp null males die at earlier developmental stages. Moreover, we found that in clamp null females roX gene expression is activated, whereas in clamp null males roX gene expression is reduced. Therefore, CLAMP regulates roX abundance in a sex-specific manner. Our results provide new insights into sex-specific gene regulation by an essential transcription factor.

Targeted gene insertion is a goal of genome editing and has been performed in cultured cells but only in a handful of whole organisms. The existing method to integrate foreign DNA using the homologous recombination pathway is inherently low efficiency, and many systems are refractory to this method. Several additional manipulations have been developed to gain greater efficiency by suppressing the competing dominant repair pathway of nonhomologous end-joining. However, this can be laborious and in practice limits the range of hosts where the method is applicable. Here, we use the preferred pathway of nonhomologous end-joining (used previously to create indels for gene inactivation) for precise integration of large DNA into the specified genomic target site of an intact animal. Our method uses site-specific cleavage, end-capture of cohesive ends, and obligate ligation-gated recombination. This approach is straight-forward and yields high efficiency without additional gene manipulations; therefore it is easily applicable to a much broader range of organisms. We demonstrate its application to the fungus fly Sciara coprophila where a transformation system has not existed before. We integrated a 6.5 kb transgene precisely at the desired genomic target site of Sciara using this method. This provides the foundation for future experiments to explore the unique genetic features of this organism. Similarly, the method described here will allow insertion of large pieces of DNA into a diverse group of organisms for studies of their genetic attributes.

Oxford Nanopore Technologies’ nanopore sequencing device, the MinION, holds the promise of sequencing ultra-long DNA fragments >100kb. An obstacle to realizing this promise is delivering ultra-long DNA molecules to the nanopores. We present our progress in developing cost-effective ways to overcome this obstacle and our resulting MinION data, including multiple reads >100kb.