Measuring Genome Sizes Using Read-Depth, k-mers, and Flow Cytometry: Methodological Comparisons in Beetles (Coleoptera).

Measuring genome size across different species can yield important insights into evolution of the genome and allow for more informed decisions when designing next-generation genomic sequencing projects. New techniques for estimating genome size using shallow genomic sequence data have emerged which have the potential to augment our knowledge of genome sizes, yet these methods have only been used in a limited number of empirical studies. In this project, we compare estimation methods using next-generation sequencing (k-mer methods and average read depth of single-copy genes) to measurements from flow cytometry, a standard method for genome size measures, using ground beetles (Carabidae) and other members of the beetle suborder Adephaga as our test system. We also present a new protocol for using read-depth of single-copy genes to estimate genome size. Additionally, we report flow cytometry measurements for five previously unmeasured carabid species, as well as 21 new draft genomes and six new draft transcriptomes across eight species of adephagan beetles. No single sequence-based method performed well on all species, and all tended to underestimate the genome sizes, although only slightly in most samples. For one species, Bembidion sp. nr. transversale, most sequence-based methods yielded estimates half the size suggested by flow cytometry.

Thoracic underreplication in Drosophila species estimates a minimum genome size and the dynamics of added DNA.

Many cells in the thorax of Drosophila were found to stall during replication, a phenomenon known as underreplication. Unlike underreplication in nuclei of salivary and follicle cells, this stall occurs with less than one complete round of replication. This stall point allows precise estimations of early-replicating euchromatin and late-replicating heterochromatin regions, providing a powerful tool to investigate the dynamics of structural change across the genome. We measure underreplication in 132 species across the Drosophila genus and leverage these data to propose a model for estimating the rate at which additional DNA is accumulated as heterochromatin and euchromatin and also predict the minimum genome size for Drosophila. According to comparative phylogenetic approaches, the rates of change of heterochromatin differ strikingly between Drosophila subgenera. Although these subgenera differ in karyotype, there were no differences by chromosome number, suggesting other structural changes may influence accumulation of heterochromatin. Measurements were taken for both sexes, allowing the visualization of genome size and heterochromatin changes for the hypothetical path of XY sex chromosome differentiation. Additionally, the model presented here estimates a minimum genome size in Sophophora remarkably close to the smallest insect genome measured to date, in a species over 200 million years diverged from Drosophila.

Comparative cytogenetics and derived phylogenic relationship among Sitophilus grain weevils (Coleoptera, Curculionidae, Dryophthorinae).

Cytogenetic characteristics and genome size are powerful tools for species characterization and identification of cryptic species, providing critical insights into phylogenetic and evolutionary relationships. Sitophilus Linnaeus, 1758 grain weevils can benefit from such tools as key pest species of stored products and also as sources of archeological information on human history and past urban environments. Moreover, the phylogenetic relationship among these weevil species remains controversial and is largely based on single DNA fragment analyses. Therefore, cytogenetic analyses and genome size determinations were performed for four Sitophilus grain weevil species, namely the granary weevil Sitophilus granarius (Linnaeus, 1758), the tamarind weevil S. linearis (Herbst, 1797), the rice weevil S. oryzae (Linnaeus, 1763), and the maize weevil S. zeamais Motschulsky, 1855. Both maize and rice weevils exhibited the same chromosome number (2n=22; 10 A + Xyp). In contrast, the granary and tamarind weevils exhibited higher chromosome number (2n=24; 11 A + Xyp and 11 A + neo-XY, respectively). The nuclear DNA content of these species was not proportionally related to either chromosome number or heterochromatin amount. Maize and rice weevils exhibited similar and larger genome sizes (0.730±0.003 pg and 0.786±0.003 pg, respectively), followed by the granary weevil (0.553±0.003 pg), and the tamarind weevil (0.440±0.001 pg). Parsimony phylogenetic analysis of the insect karyotypes indicate that S. zeamais and S. oryzae were phylogenetically closer than S. granarius and S. linearis, which were more closely related and share a more recent ancestral relationship.