Improved genome assembly of American alligator genome reveals conserved architecture of estrogen signaling

Abstract

The American alligator, , like all crocodilians, has temperature-dependent sex determination, in which the sex of an embryo is determined by the incubation temperature of the egg during a critical period of development. The lack of genetic differences between male and female alligators leaves open the question of how the genes responsible for sex determination and differentiation are regulated. Insight into this question comes from the fact that exposing an embryo incubated at male-producing temperature to estrogen causes it to develop ovaries. Because estrogen response elements are known to regulate genes over long distances, a contiguous genome assembly is crucial for predicting and understanding their impact. We present an improved assembly of the American alligator genome, scaffolded with in vitro proximity ligation (Chicago) data. We use this assembly to scaffold two other crocodilian genomes based on synteny. We perform RNA sequencing of tissues from American alligator embryos to find genes that are differentially expressed between embryos incubated at male- versus female-producing temperature. Finally, we use the improved contiguity of our assembly along with the current model of CTCF-mediated chromatin looping to predict regions of the genome likely to contain estrogen-responsive genes. We find that these regions are significantly enriched for genes with female-biased expression in developing gonads after the critical period during which sex is determined by incubation temperature. We thus conclude that estrogen signaling is a major driver of female-biased gene expression in the post-temperature sensitive period gonads.

New advances in sequence assembly

“…This data can be used to group contigs by chromosome, reconstruct chromosome-length scaffolds, and phase haplotypes (Burton et al 2013;Kaplan and Dekker 2013;Selvaraj et al 2013). In this issue, Rice et al (2017) demonstrates a related approach, using in vitro reconstituted chromatin and Illumina sequencing to assemble the American alligator genome. Another approach to boosting short reads uses highthroughput barcoding to tag groups of "linked reads" that all originate from a larger, single molecule of DNA.…”

Improving Illumina assemblies with Hi‐C and long reads: An example with the North African dromedary

“…Other genome assembly projects that began with Illumina short‐ and long‐insert libraries have also taken advantage of chromosome conformation capture and/ or long‐read technologies to improve assemblies. For example, the AllMis1 assembly (American alligator, Alligator mississippiensis ) was assembled with Illumina short‐insert libraries and scaffolded with mate‐pair and BAC libraries (Green et al, ) and then subsequently improved with Dovetail Chicago libraries resulting in the AllMis2 assembly (Rice et al, ). Further, the sooty manageby ( Cercocebus atys ) genome assembly was de novo assembled with Illumina short‐insert and mate‐pair libraries, and gaps were filled in with ~12X coverage of PacBio RS I and II reads (Palesch et al, ).…”

A high-quality reference genome assembly of the saltwater crocodile, Crocodylus porosus, reveals patterns of selection in Crocodylidae

“…The data were assembled with AllPaths-LG (Gnerre, et al 2011). More recently, a highly-contiguous assembly from Alligator mississippiensis (Rice, et al 2017) was used to re-scaffold the All-Paths assembly (Green, et al 2014) using Ragout (Kolmogorov, et al 2014). Here, we prepared and re-assembled using our Chicago libraries (see Library preparation and de novo shotgun assembly, above).…”

“…Next, we used BUSCO v 3.0.2 (Simao, et al 2015) to obtain quantitative measures of gene content, using 3,950 single-copy orthologous genes from the tetrapod lineage database, tetrapoda_odb9, and setting chicken as the Augustus species gene finding parameter. We then used the JupiterPlot (https://github.com/JustinChu/JupiterPlot) pipeline to visually compare the assembly from (Rice, et al 2017) (set as the reference) to our assembly, setting the minimum size of a contiguous region to render to 100 bp, considering all reference chromosomes larger than 100 bp, and using the largest reference scaffolds that are equal to 96.4% of our genome, to the full-length of the reference genome. Finally, MUMmer v. 4.0.0 (Kurtz, et al 2004) was used to align and draw a dot plot to evaluate synteny between assemblies.…”

“…Although CroPor_comp1 has high contiguity, this is based on assumed orthologous contiguity. Thus, a high-quality, well-annotated de novo genome assembly of C. porosus similar in quality to the most recently released version of the A. mississippensis (Rice, et al 2017) will allow a more comprehensive assessment of the species' genome in terms of contiguity, gene space and annotations. This work attempts to bridge that gap by presenting an annotated and highly contiguous draft genome of C. porosus.…”