Protocol for preparing Drosophila genomic DNA to create chromosome-level de novo genome assemblies.

De novo genome assemblies are common tools for examining novel biological phenomena in non-model organisms. Here, we present a protocol for preparing Drosophila genomic DNA to create chromosome-level de novo genome assemblies. We describe steps for high-molecular-weight DNA preparation with phenol or Genomic-tips, quality control, long-read nanopore sequencing, short-read DNA library preparation, and sequencing. We then detail procedures of genome assembly, annotation, and assessment that can be used for downstream comparison and functional analysis. For complete details on the use and execution of this protocol, please refer to Sperling et al.1.

Whole mount multiplexed visualization of DNA, mRNA, and protein in plant-parasitic nematodes.

BACKGROUND: Plant-parasitic nematodes compromise the agriculture of a wide variety of the most common crops worldwide. Obtaining information on the fundamental biology of these organisms and how they infect the plant has been restricted by the ability to visualize intact nematodes using small molecule stains, antibodies, or in situ hybridization. Consequently, there is limited information available about the internal composition of the nematodes or the biology of the effector molecules they use to reprogram their host plant. RESULTS: We present the Sperling prep - a whole mount method for nematode preparation that enables staining with small molecules, antibodies, or in situ hybridization chain reaction. This method does not require specialized apparatus and utilizes typical laboratory equipment and materials. By dissociating the strong cuticle and interior muscle layers, we enabled entry of the small molecule stains into the tissue. After permeabilization, small molecule stains can be used to visualize the nuclei with the DNA stain DAPI and the internal structures of the digestive tract and longitudinal musculature with the filamentous actin stain phalloidin. The permeabilization even allows entry of larger antibodies, albeit with lower efficiency. Finally, this method works exceptionally well with in situ HCR. Using this method, we have visualized effector transcripts specific to the dorsal gland and the subventral grand of the sugar beet cyst nematode, Heterodera schachtii, multiplexed in the same nematode. CONCLUSION: We were able to visualize the internal structures of the nematode as well as key effector transcripts that are used during plant infection and parasitism. Therefore, this method provides an important toolkit for studying the biology of plant-parasitic nematodes.

Protocol for screening facultative parthenogenesis in Drosophila.

Most species of sexually reproducing Drosophila are capable of some degree of facultative parthenogenesis, which involves the initiation of development in an unfertilized egg. Here, we present an optimized protocol to screen facultative parthenogenesis in Drosophila. We describe steps for the collection and maintenance of virgin flies. We then detail offspring screening for the analysis of parthenogenesis. This protocol can be applied to different Drosophila strains and can be adapted for the analysis of parthenogenesis in other animals. For complete details on the use and execution of this protocol, please refer to Sperling et al.1.

A genetic basis for facultative parthenogenesis in Drosophila.

Facultative parthenogenesis enables sexually reproducing organisms to switch between sexual and asexual parthenogenetic reproduction. To gain insights into this phenomenon, we sequenced the genomes of sexually reproducing and parthenogenetic strains of Drosophila mercatorum and identified differences in the gene expression in their eggs. We then tested whether manipulating the expression of candidate gene homologs identified in Drosophila mercatorum could lead to facultative parthenogenesis in the non-parthenogenetic species Drosophila melanogaster. This identified a polygenic system whereby increased expression of the mitotic protein kinase polo and decreased expression of a desaturase, Desat2, caused facultative parthenogenesis in the non-parthenogenetic species that was enhanced by increased expression of Myc. The genetically induced parthenogenetic Drosophila melanogaster eggs exhibit de novo centrosome formation, fusion of the meiotic products, and the onset of development to generate predominantly triploid offspring. Thus, we demonstrate a genetic basis for sporadic facultative parthenogenesis in an animal.