Chromosome-level, nanopore-only genome and allele-specific DNA methylation of Pallas's cat, Otocolobus manul

Abstract

Pallas's cat, or the manul cat (Otocolobus manul), is a small felid native to the grasslands and steppes of central Asia. Population strongholds in Mongolia and China face growing challenges from climate change, habitat fragmentation, poaching, and other sources. These threats, combined with O. manul's zoo collection popularity and value in evolutionary biology, necessitate improvement of species genomic resources. We used standalone nanopore sequencing to assemble a 2.5 Gb, 61-contig nuclear assembly and 17097 bp mitogenome for O. manul. The primary nuclear assembly had 56× sequencing coverage, a contig N50 of 118 Mb, and a 94.7% BUSCO completeness score for Carnivora-specific genes. High genome collinearity within Felidae permitted alignment-based scaffolding onto the fishing cat (Prionailurus viverrinus) reference genome. Manul contigs spanned all 19 felid chromosomes with an inferred total gap length of less than 400 kilobases. Modified basecalling and variant phasing produced an alternate pseudohaplotype assembly and allele-specific DNA methylation calls; 61 differentially methylated regions were identified between haplotypes. Nearest features included classical imprinted genes, non-coding RNAs, and putative novel imprinted loci. The assembled mitogenome successfully resolved existing discordance between Felinae nuclear and mtDNA phylogenies. All assembly drafts were generated from 158 Gb of sequence using seven minION flow cells.

Figure 1.

Otocolobus manul holotype photo depicting Tater, the 5-year-old male manul cat sampled for genome assembly.

Figure 2.

OtoMan1.0 is a near-complete and highly contiguous genome assembly for Pallas's cat (Otocolobus manul). (A) Felid reference assembly completeness was compared to our O. manul assembly by locally searching Carnivora-specific genes (carnivora_odb10) with BUSCO v5.3.2. OtoMan1.0, a nanopore-only assembly, ranked within 1% of the highest felid assembly BUSCO scores. (B) Contig N50 distribution for O. manul and felid reference assemblies. The O. manul assembly exhibits high contiguity (N50 = 118.2 Mb) when compared to other Felidae genomes, which differ minimally (∼3 Mb) in size.

Figure 3.

OtoMan1.0 cross-genus alignment and scaffolding. (A) Genome sequence alignment of the fishing cat (P. viverrinus) reference genome (GCF_022837055.1) and the contig-level nanopore assembly of O. manul, OtoMan1.0, was generated using Nucmer (70) and Dot (https://github.com/marianattestad/dot). The alignment quality reflects gross genome collinearity present across Felidae (80,83). (B) Ideogram of OtoMan1.0 scaffolds generated via alignment to the fishing cat (P. viverrinus) reference assembly (GCF_022837055.1). Color transitions represent breakpoints between contigs on the scaffold. All 19 felid chromosomes were covered by the OtoMan1.0 assembly, and eight autosomes (A3, B2, B3, B4, C2, E2, E3 and F2) were captured by single contigs. Manul contigs were also aligned to two BUSCO-containing fishing cat unplaced scaffolds while two of the 61 O. manul contigs were unplaced. Correlations between the fishing cat chromosome lengths and O. manul scaffold lengths are provided in Supplemental Figure 5.

Figure 4.

Repetitive element content of the assembled O. manul genome and 17 other felid species assessed with RepeatMasker (47). Manul cat repeat content was highly similar to other members of Felidae and dominated by retroelements.

Figure 5.

Allele-specific DNA methylation at differentially located loci (DMRs) called between O. manul assembly pseudohaplotypes assembled from a whole blood sample. DMRs were visualized with the package Methylartist (61). Annotation of the 61 manually validated DMRs was accomplished by lifting gene coordinates from the Felis catus reference assembly (F.catus_Fca126_mat1.0) onto OtoMan1.0 and finding the nearest feature. The results revealed (A, B) singleton DMRs and (C, D) multi-DMR clusters within and near genes. Genes overlapping DMRs included previously described imprinted loci such as (A) potassium voltage-gated channel subfamily Q member 1 (KCNQ1) and (B) the guanine nucleotide-binding protein G(S) subunit alpha (GNAS) complex locus (aliases LOC101098453, LOC102900772). DMRs also overlapped genes not previously reported as imprinted, including (C) von Willebrand factor D and EGF domains (VWDE), a protein with predicted involvement in anatomical development (96). (D) The most notable locus was a 33.2 kb region containing 12 DMRs all hypomethylated on pseudohaplotype 2. The DMRS overlapped the 5’ end of a classical imprinted gene, zinc finger DBF-type containing 2 (ZDBF2), on the negative strand and the 5’ end of chemerin chemokine-like receptor 2 (CMKLR2) on the positive strand. The CMKLR2 antisense RNA (CMKLR2AS; alias GPR1AS), which is not annotated in F. catus, exhibits imprinted expression in the human placenta (97).

Figure 5.

Allele-specific DNA methylation at differentially located loci (DMRs) called between O. manul assembly pseudohaplotypes assembled from a whole blood sample. DMRs were visualized with the package Methylartist (61). Annotation of the 61 manually validated DMRs was accomplished by lifting gene coordinates from the Felis catus reference assembly (F.catus_Fca126_mat1.0) onto OtoMan1.0 and finding the nearest feature. The results revealed (A, B) singleton DMRs and (C, D) multi-DMR clusters within and near genes. Genes overlapping DMRs included previously described imprinted loci such as (A) potassium voltage-gated channel subfamily Q member 1 (KCNQ1) and (B) the guanine nucleotide-binding protein G(S) subunit alpha (GNAS) complex locus (aliases LOC101098453, LOC102900772). DMRs also overlapped genes not previously reported as imprinted, including (C) von Willebrand factor D and EGF domains (VWDE), a protein with predicted involvement in anatomical development (96). (D) The most notable locus was a 33.2 kb region containing 12 DMRs all hypomethylated on pseudohaplotype 2. The DMRS overlapped the 5’ end of a classical imprinted gene, zinc finger DBF-type containing 2 (ZDBF2), on the negative strand and the 5’ end of chemerin chemokine-like receptor 2 (CMKLR2) on the positive strand. The CMKLR2 antisense RNA (CMKLR2AS; alias GPR1AS), which is not annotated in F. catus, exhibits imprinted expression in the human placenta (97).

Figure 6.

Nanopore mitochondrial genome assembly of O. manul and felid mtDNA phylogeny. (A) The circular length of the built O. manul mitogenome assembly was 17 097 bp; read coverage was 800×. (B) Phylogenetic relationships of O. manul were derived through bootstrapped maximum likelihood comparison of the assembled mitogenome to existing Felidae references. In contrast to previously published mtDNA for O. manul (98), the current tree matches those generated through analysis of nuclear DNA (14).