A new hybrid approach for MHC genotyping: high-throughput NGS and long read MinION nanopore sequencing, with application to the non-model vertebrate Alpine chamois (Rupicapra rupicapra).

The major histocompatibility complex (MHC) acts as an interface between the immune system and infectious diseases. Accurate characterization and genotyping of the extremely variable MHC loci are challenging especially without a reference sequence. We designed a combination of long-range PCR, Illumina short-reads, and Oxford Nanopore MinION long-reads approaches to capture the genetic variation of the MHC II DRB locus in an Italian population of the Alpine chamois (Rupicapra rupicapra). We utilized long-range PCR to generate a 9 Kb fragment of the DRB locus. Amplicons from six different individuals were fragmented, tagged, and simultaneously sequenced with Illumina MiSeq. One of these amplicons was sequenced with the MinION device, which produced long reads covering the entire amplified fragment. A pipeline that combines short and long reads resolved several short tandem repeats and homopolymers and produced a de novo reference, which was then used to map and genotype the short reads from all individuals. The assembled DRB locus showed a high level of polymorphism and the presence of a recombination breakpoint. Our results suggest that an amplicon-based NGS approach coupled with single-molecule MinION nanopore sequencing can efficiently achieve both the assembly and the genotyping of complex genomic regions in multiple individuals in the absence of a reference sequence.

Relaxed selective constraints drove functional modifications in peripheral photoreception of the cavefish P. andruzzii and provide insight into the time of cave colonization.

The genetic basis of phenotypic changes in extreme environments is a key but rather unexplored topic in animal evolution. Here we provide an exemplar case of evolution by relaxed selection in the Somalian cavefish Phreatichthys andruzzii that has evolved in the complete absence of light for at least 2.8 million years. This has resulted in extreme degenerative phenotypes, including complete eye loss and partial degeneration of the circadian clock. We have investigated the molecular evolution of the nonvisual photoreceptor melanopsin opn4m2, whose mutation contributes to the inability of peripheral clocks to respond to light. Our intra- and inter-species analyses suggest that the 'blind' clock in P. andruzzii evolved because of the loss of selective constraints on a trait that was no longer adaptive. Based on this change in selective regime, we estimate that the functional constraint on cavefish opn4m2 was relaxed at ∼5.3 Myr. This implies a long subterranean history, about half in complete isolation from the surface. The visual photoreceptor rhodopsin, expressed in the brain and implicated in photophobic behavior, shows similar evolutionary patterns, suggesting that extreme isolation in darkness led to a general weakening of evolutionary constraints on light-responsive mechanisms. Conversely, the same genes are still conserved in Garra barreimiae, a cavefish from Oman, that independently and more recently colonized subterranean waters and evolved troglomorphic traits. Our results contribute substantially to the open debate on the genetic bases of regressive evolution.