PAG Australia 2023: Exploring Dingo Ecology and Evolution with Chromosome-Level Canid Genomes

Richard J Edwards, Matt F Field and J William O Ballard - PAG Australia 2023

Dogs are uniquely associated with human dispersal and bring novel insight into human migration and the domestication process. Dingoes represent an intriguing case within canine evolution being geographically isolated for thousands of years. The exact origin(s) and people(s) who transported the canines that became dingoes to Australia is debated, but it has been suggested they arrived by boat ~5,000-8,000 BP. Published morphological and genetic evidence has established the presence of at least two dingo lineages. The Alpine dingo is commonly found in south-eastern Australia while the Desert ecotype is found in the north, central and western Australia. The relationship of dingoes to modern dogs, and the ecotypes to each other, has important implications management and protection of this top predator, as well as providing interesting perspectives on human colonisation and canine domestication.

We have generated chromosome-level assemblies of both dingo ecotypes, along with domesticated dogs representing both ancient (Basenji) and derived (German Shepherd) breeds. In each case, long-read sequencing and Hi-C scaffolding have been combined to produce genome assemblies with high contiguity and structural completeness. Comparison of these assemblies with additional dog breeds, using the Greenland wolf as an outgroup, places the dingo as an early offshoot of modern dogs, situated between the grey wolf and the domesticated dogs of today. This is supported by patterns of genetic variation, and chromosome structure. Furthermore, we confirm that dingoes have not experienced the expansion of the AMY2B pancreatic amylase gene that occurred during domestication of modern dogs. This has important implications for dingo ecology and behaviour, and raises the prospect of using AMY2B copy number as a novel and reliable in-field discriminator between dingoes and feral dogs.

The Australasian dingo archetype: De novo chromosome-length genome assembly, DNA methylome, and cranial morphology

Ballard JWO, Field MA, Edwards RJ, Wilson LAB, Koungoulos LG, Rosen BD, Chernoff B, Dudchenko O, Omer A, Keilwagen J, Skvortsova K, Bogdanovic O, Chan E, Zammit R, Hayes V & Aiden EL (2023): The Australasian dingo archetype: De novo chromosome-length genome assembly, DNA methylome, and cranial morphology. Gigascience 12:giad018. [Gigascience] [PubMed]

Background

One difficulty in testing the hypothesis that the Australasian dingo is a functional intermediate between wild wolves and domesticated breed dogs is that there is no reference specimen. Here we link a high-quality de novo long-read chromosomal assembly with epigenetic footprints and morphology to describe the Alpine dingo female named Cooinda. It was critical to establish an Alpine dingo reference because this ecotype occurs throughout coastal eastern Australia where the first drawings and descriptions were completed.

Findings

We generated a high-quality chromosome-level reference genome assembly (Canfam_ADS) using a combination of Pacific Bioscience, Oxford Nanopore, 10X Genomics, Bionano, and Hi-C technologies. Compared to the previously published Desert dingo assembly, there are large structural rearrangements on chromosomes 11, 16, 25, and 26. Phylogenetic analyses of chromosomal data from Cooinda the Alpine dingo and 9 previously published de novo canine assemblies show dingoes are monophyletic and basal to domestic dogs. Network analyses show that the mitochondrial DNA genome clusters within the southeastern lineage, as expected for an Alpine dingo. Comparison of regulatory regions identified 2 differentially methylated regions within glucagon receptor GCGR and histone deacetylase HDAC4 genes that are unmethylated in the Alpine dingo genome but hypermethylated in the Desert dingo. Morphologic data, comprising geometric morphometric assessment of cranial morphology, place dingo Cooinda within population-level variation for Alpine dingoes. Magnetic resonance imaging of brain tissue shows she had a larger cranial capacity than a similar-sized domestic dog.

Conclusions

These combined data support the hypothesis that the dingo Cooinda fits the spectrum of genetic and morphologic characteristics typical of the Alpine ecotype. We propose that she be considered the archetype specimen for future research investigating the evolutionary history, morphology, physiology, and ecology of dingoes. The female has been taxidermically prepared and is now at the Australian Museum, Sydney.

The Australian dingo is an early offshoot of modern breed dogs

Field MA, Yadav S, Dudchenko O, Esvaran M, Rosen BD, Skvortsova K, Edwards RJ, Keilwagen J, Cochran BJ, Manandhar B, Bustamante S, Rasmussen JA, Melvin RG, Chernoffl B, Omer A, Colaric Z, Chan EKF, Minoche AE, Smith TPL, Gilbert MTP, Bogdanovic O, Zammit RA, Thomas T, Aiden EL & Ballard JWO (2022): The Australian dingo is an early offshoot of modern breed dogs. Science Advances 8(16):abm5944; DOI: 10.1126/sciadv.abm5944. [Sci Adv] [PubMed] [PDF]

Dogs are uniquely associated with human dispersal and bring transformational insight into the domestication process. Dingoes represent an intriguing case within canine evolution being geographically isolated for thousands of years. Here, we present a high-quality de novo assembly of a pure dingo (CanFam_DDS). We identified large chromosomal differences relative to the current dog reference (CanFam3.1) and confirmed no expanded pancreatic amylase gene as found in breed dogs. Phylogenetic analyses using variant pairwise matrices show that the dingo is distinct from five breed dogs with 100% bootstrap support when using Greenland wolf as the outgroup. Functionally, we observe differences in methylation patterns between the dingo and German shepherd dog genomes and differences in serum biochemistry and microbiome makeup. Our results suggest that distinct demographic and environmental conditions have shaped the dingo genome. In contrast, artificial human selection has likely shaped the genomes of domestic breed dogs after divergence from the dingo.

Chromosome-length genome assembly and structural variations of the primal Basenji dog (Canis lupus familiaris) genome

Our latest genome paper is now out at BMC Genomics. This was a second collaboration in the team behind the German Shepherd Dog genome last year, led by Bill Ballard. This time, we used a combination of BGI short reads, ONT long reads, and Hi-C scaffolding to generate a chromosome-length assembly. This is one of the most intact and complete dog genomes generated to date, and joins only a handful of published breed-specific chromosome-length assemblies.

The Basenji is particularly interesting as it sits at the base of the dog breed family tree, making it a good unbiased reference for future comparisons between breeds.

The paper also has a few nice nuggets for those interesting in genome assembly. Of particular interest, our initial assembly had an artefact where the entire mitochondrial genome got assembled (in two copies) into the middle of one of the nuclear chromosomes. It is not entirely clear why this happened, but it was inserted into a NUMT (nuclear mitochondrial DNA insertion) fragment at that location. To make finding such things easier, we’ve released a new NUMT finding tool, NUMTFinder.

As with our previous dog genome, the German Shepherd Dog, we also observe that the tandem repeat of Amy2B (Amylase Alpha 2B) genes, was assembled intact but with fewer copies than are present in the actual genome. (This gene is of interest for dog domestication and adaptations to a starch-rich diet.) Crucially, without looking at the raw sequencing data, it would not have been clear that the assembly under-represents Amy2B copy number. This kind of analysis can be repeated using the regcheck or regcnv run modes of Diploidocus, which estimates the copy number of a region based on its read depth versus the single-copy read depth determined from BUSCO single-copy complete genes.

Overall, this presents a nice case study of the need for a bit of TLC and manual curation, even when you have some very impressive completeness and contiguity statistics.

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Edwards RJ, Field MA, Ferguson JM, Dudchenko O, Keilwagen K, Rosen BD, Johnson GS, Rice ES, Hillier L, Hammond JM, Towarnicki SG, Omer A, Khan R, Skvortsova K, Bogdanovic O, Zammit RA, Lieberman Aiden E, Warren WC & Ballard JWO (2021): Chromosome-length genome assembly and structural variations of the primal Basenji dog (Canis lupus familiaris) genome. BMC Genomics 22:188

Abstract

Background: Basenjis are considered an ancient dog breed of central African origins that still live and hunt with tribesmen in the African Congo. Nicknamed the barkless dog, Basenjis possess unique phylogeny, geographical origins and traits, making their genome structure of great interest. The increasing number of available canid reference genomes allows us to examine the impact the choice of reference genome makes with regard to reference genome quality and breed relatedness.

Results: Here, we report two high quality de novo Basenji genome assemblies: a female, China (CanFam_Bas), and a male, Wags. We conduct pairwise comparisons and report structural variations between assembled genomes of three dog breeds: Basenji (CanFam_Bas), Boxer (CanFam3.1) and German Shepherd Dog (GSD) (CanFam_GSD). CanFam_Bas is superior to CanFam3.1 in terms of genome contiguity and comparable overall to the high quality CanFam_GSD assembly. By aligning short read data from 58 representative dog breeds to three reference genomes, we demonstrate how the choice of reference genome significantly impacts both read mapping and variant detection.

Conclusions: The growing number of high-quality canid reference genomes means the choice of reference genome is an increasingly critical decision in subsequent canid variant analyses. The basal position of the Basenji makes it suitable for variant analysis for targeted applications of specific dog breeds. However, we believe more comprehensive analyses across the entire family of canids is more suited to a pangenome approach. Collectively this work highlights the importance the choice of reference genome makes in all variation studies.

Canfam_GSD: De novo chromosome-length genome assembly of the German Shepherd Dog (Canis lupus familiaris) using a combination of long reads, optical mapping, and Hi-C

Our latest paper is out! This one is a bit more photogenic than the cane toad - a German Shepherd Dog called Nala. This was a big international effort in a collaboration led by Bill Ballard at UNSW that included a dozen institutions across four continents. We threw all the main sequencing technologies at this one and achieved a chromosome-level assembly of better quality than the current “CanFam” reference genome.

You can find out more in the UNSW press release.

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Field MA, Rosen BD, Dudchenko O, Chan EKF, Minoche AM, Edwards RJ, Barton K, Lyons RJ, Enosi Tuipulotu D, Hayes VM, Omer AD, Colaric Z, Keilwagen J, Skvortsova K, Bogdanovic O, Smith MA, Lieberman Aiden E, Smith TPL, Zammit RA & Ballard JWO (2020): Canfam_GSD: De novo chromosome-length genome assembly of the German Shepherd Dog (Canis lupus familiaris) using a combination of long reads, optical mapping, and Hi-C. GigaScience 9(4):giaa027. [GigaScience]

Abstract

Background

The German Shepherd Dog (GSD) is one of the most common breeds on earth and has been bred for its utility and intelligence. It is often first choice for police and military work, as well as protection, disability assistance, and search-and-rescue. Yet, GSDs are well known to be susceptible to a range of genetic diseases that can interfere with their training. Such diseases are of particular concern when they occur later in life, and fully trained animals are not able to continue their duties.

Findings

Here, we provide the draft genome sequence of a healthy German Shepherd female as a reference for future disease and evolutionary studies. We generated this improved canid reference genome (CanFam_GSD) utilizing a combination of Pacific Bioscience, Oxford Nanopore, 10X Genomics, Bionano, and Hi-C technologies. The GSD assembly is ∼80 times as contiguous as the current canid reference genome (20.9 vs 0.267 Mb contig N50), containing far fewer gaps (306 vs 23,876) and fewer scaffolds (429 vs 3,310) than the current canid reference genome CanFamv3.1. Two chromosomes (4 and 35) are assembled into single scaffolds with no gaps. BUSCO analyses of the genome assembly results show that 93.0% of the conserved single-copy genes are complete in the GSD assembly compared with 92.2% for CanFam v3.1. Homology-based gene annotation increases this value to ∼99%. Detailed examination of the evolutionarily important pancreatic amylase region reveals that there are most likely 7 copies of the gene, indicative of a duplication of 4 ancestral copies and the disruption of 1 copy.

Conclusions

GSD genome assembly and annotation were produced with major improvement in completeness, continuity, and quality over the existing canid reference. This resource will enable further research related to canine diseases, the evolutionary relationships of canids, and other aspects of canid biology.

Photo credit: Outdoor Action Photography.