Recovering individual haplotypes and a contiguous genome assembly from pooled long read sequencing of the diamondback moth (Lepidoptera: Plutellidae)

BackgroundRecent advances in genomics have addressed the challenge that divergent haplotypes pose to the reconstruction of haploid genomes. However for many organisms, the sequencing of either field-caught individuals or a pool of heterogeneous individuals is still the only practical option. Here we present methodological approaches to achieve three outcomes from pooled long read sequencing: the generation of a contiguous haploid reference sequence, the sequences of heterozygous haplotypes; and reconstructed genomic sequences of individuals related to the pooled material.ResultsPacBio long read sequencing, Dovetail Hi-C scaffolding and linkage map integration yielded a haploid chromosome-level assembly for the diamondback moth (Plutella xylostella), a global pest of Brassica crops, from a pool of related individuals. The final assembly consisted of 573 scaffolds, with a total assembly size of 343.6Mbp a scaffold N50 value of 11.3Mbp (limited by chromosome size) and a maximum scaffold size of 14.4Mbp. This assembly was then integrated with an existing RAD-seq linkage map, anchoring 95% of the assembled sequence to defined chromosomal positions.ConclusionsWe describe an approach to resolve divergent haplotype sequences and describe multiple validation approaches. We also reconstruct individual genomes from pooled long-reads, by applying a recently developed k-mer binning method.

Alternative haplotypes of antigen processing genes in zebrafish diverged early in vertebrate evolution

Antigen processing and presentation genes found within the MHC are among the most highly polymorphic genes of vertebrate genomes, providing populations with diverse immune responses to a wide array of pathogens. Here, we describe transcriptome, exome, and whole-genome sequencing of clonal zebrafish, uncovering the most extensive diversity within the antigen processing and presentation genes of any species yet examined. Our CG2 clonal zebrafish assembly provides genomic context within a remarkably divergent haplotype of the core MHC region on chromosome 19 for six expressed genes not found in the zebrafish reference genome: mhc1uga, proteasome-β 9b (psmb9b), psmb8f, and previously unknown genes psmb13b, tap2d, and tap2e. We identify ancient lineages for Psmb13 within a proteasome branch previously thought to be monomorphic and provide evidence of substantial lineage diversity within each of three major trifurcations of catalytic-type proteasome subunits in vertebrates: Psmb5/Psmb8/Psmb11, Psmb6/Psmb9/Psmb12, and Psmb7/Psmb10/Psmb13. Strikingly, nearby tap2 and MHC class I genes also retain ancient sequence lineages, indicating that alternative lineages may have been preserved throughout the entire MHC pathway since early diversification of the adaptive immune system ∼500 Mya. Furthermore, polymorphisms within the three MHC pathway steps (antigen cleavage, transport, and presentation) are each predicted to alter peptide specificity. Lastly, comparative analysis shows that antigen processing gene diversity is far more extensive than previously realized (with ancient coelacanth psmb8 lineages, shark psmb13, and tap2t and psmb10 outside the teleost MHC), implying distinct immune functions and conserved roles in shaping MHC pathway evolution throughout vertebrates.

Archaic adaptive introgression in TBX15/WARS2

A recent study conducted the first genome-wide scan for selection in Inuit from Greenland using SNP chip data. Here, we report that selection in the region with the second most extreme signal of positive selection in Greenlandic Inuit favored a deeply divergent haplotype that is closely related to the sequence in the Denisovan genome, and was likely introgressed from an archaic population. The region contains two genes, WARS2 and TBX15, and has previously been associated with adipose tissue differentiation and body-fat distribution in humans. We show that the adaptively introgressed allele has been under selection in a much larger geographic region than just Greenland. Furthermore, it is associated with changes in expression of WARS2 and TBX15 in multiple tissues including the adrenal gland and subcutaneous adipose tissue, and with regional DNA methylation changes in TBX15.

Genetic divergence and phylogeography of the alpine plant taxon Onobrychis transsilvanica (Fabaceae)

Although the Carpathians represent one of the main elements of the European Alpine System and an important area of endemism, only a few phylogeographic studies concerning this region have focused on the detailed intraspecific variation in alpine plant species. Using two molecular marker systems, we (1) aimed to elucidate the controversy concerning the status of the endemic Carpathian taxon Onobrychis transsilvanica Simonk. in relation to the more widespread Onobrychis montana DC. and (2) determined the phylogeographic structure of O. transsilvanica within the Carpathians. For O. transsilvanica, our data suggest either a recent postglacial speciation with incomplete lineage sorting or genetic divergence followed by subsequent continuous gene flow during the glacial period. The genetic structure of the complex does not support O. transsilvanica as a distinct species from O. montana. Within the Carpathians, the extant populations of O. transsilvanica comprise two major allopatric lineages, which have been isolated from each other for a long period of time. Unexpectedly, the major genetic break was not in line with a classical biogeographical boundary in the Carpathians but rather separated a group from the southwestern edge of the mountains. We also discovered an additional divergent haplotype lineage, with weaker genetic support, within the O. transsilvanica populations.