The Agassiz's desert tortoise genome provides a resource for the conservation of a threatened species

Abstract

Agassiz's desert tortoise (Gopherus agassizii) is a long-lived species native to the Mojave Desert and is listed as threatened under the US Endangered Species Act. To aid conservation efforts for preserving the genetic diversity of this species, we generated a whole genome reference sequence with an annotation based on deep transcriptome sequences of adult skeletal muscle, lung, brain, and blood. The draft genome assembly for G. agassizii has a scaffold N50 length of 252 kbp and a total length of 2.4 Gbp. Genome annotation reveals 20,172 protein-coding genes in the G. agassizii assembly, and that gene structure is more similar to chicken than other turtles. We provide a series of comparative analyses demonstrating (1) that turtles are among the slowest-evolving genome-enabled reptiles, (2) amino acid changes in genes controlling desert tortoise traits such as shell development, longevity and osmoregulation, and (3) fixed variants across the Gopherus species complex in genes related to desert adaptations, including circadian rhythm and innate immune response. This G. agassizii genome reference and annotation is the first such resource for any tortoise, and will serve as a foundation for future analysis of the genetic basis of adaptations to the desert environment, allow for investigation into genomic factors affecting tortoise health, disease and longevity, and serve as a valuable resource for additional studies in this species complex.

Fig 1. Assembly statistics for the genome of Gopherus agassizii and comparison with Python bivittatus and Alligator mississippiensis.

(A) Two k-mer frequency peaks exist for G. agassizii: a peak at 35X coverage, representing heterozygous regions and a peak at 71X coverage, representing homozygous regions. (B) Gene-based scaffolding greatly increased scaffold N50 from 169.2 kbp to 251.6 kbp, which places it within the range of recently published genomes of reptiles including the Burmese python [71] and the American alligator [72].

Fig 2. Frequency distribution (proportion) of GC content calculated in 5 kbp non-overlapping windows for Gopherus agassizii and other reptilian genomes.

GC content of G. agassizii closely matches that of the turtles Chrysemys picta bellii and Pelodiscus sinensis [17,18], and overlaps the American alligator (Alligator mississippiensis) [72], the green anole (Anolis carolinensis) [73], and the chicken (Gallus gallus) [70].

Fig 3. Gene structure of Gopherus agassizii compared to other reptiles.

Frequency distribution of genes or exons relative to (A) coding DNA sequence (CDS) lengths, (B) exon length and (C) intron length of Agassiz’s desert tortoise, chicken [74], western painted turtle [18], and Chinese softshell turtle [17]. CDS is for genes less than 10 kb.

Fig 4. Phylogeny of 10 sauropsids including Gopherus agassizii.

Phylogeny shows the relationships of sauropsids, including four chelonians, with complete genome sequences. Branch lengths are given in terms of DNA substitutions per site derived from fourfold degenerate sites.

Fig 5. Comparative genomics of chelonians.

Representation of Gene Ontology terms for biological processes shared by genes with pairwise Ka/Ks > 1 in comparisons of Gopherus agassizii to (A) Chrysemys picta bellii and (B) Chelonia mydas. Treemap boxes are both sized and colored by uniqueness. Abbreviations in (A): RB, receptor biosynthesis, LCFACB, long chain fatty acyl-CoA biosynthesis, MTMD, membrane to membrane docking.