A near-complete genome assembly of the bearded dragon Pogona vitticeps provides insights into the origin of Pogona sex chromosomes

  1. Qunfei Guo
  2. Youliang Pan
  3. Wei Dai
  4. Fei Guo
  5. Tao Zeng
  6. Wanyi Chen
  7. Yaping Mi
  8. Yanshu Zhang
  9. Shuaizhen Shi
  10. Wei Jiang
  11. Huimin Cai
  12. Beiying Wu
  13. Yang Zhou
  14. Ying Wang
  15. Chentao Yang
  16. Xiao Shi
  17. Xu Yan
  18. Junyi Chen
  19. Chongyang Cai
  20. Jingnan Yang
  21. Xun Xu
  22. Ying Gu
  23. Yuliang Dong
  24. Qiye Li

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Abstract

Background

The agamid dragon lizard Pogona vitticeps is one of the most popular domesticated reptiles to be kept as pets worldwide. The capacity of breeding in captivity also makes it emerging as a model species for a range of scientific research, especially for the studies of sex chromosome origin and sex determination mechanisms.

Results

By leveraging the CycloneSEQ and DNBSEQ sequencing technologies, we conducted whole genome and long-range sequencing for a captive-bred ZZ male to construct a chromosome-scale reference genome for P. vitticeps . The new reference genome is ∼1.8 Gb in length, with a contig N50 of 202.5 Mb and all contigs anchored onto 16 chromosomes. Genome annotation assisted by long-read RNA sequencing greatly expanded the P. vitticeps lncRNA catalog. With the chromosome-scale genome, we were able to characterize the whole Z sex chromosome for the first time. We found that over 80% of the Z chromosome remains as pseudo-autosomal region (PAR) where recombination is not suppressed. The sexually differentiated region (SDR) is small and occupied mostly by transposons, yet it aggregates genes involved in male development, such as AMH , AMHR2 and BMPR1A . Finally, by tracking the evolutionary origin and developmental expression of the SDR genes, we proposed a model for the origin of P. vitticeps sex chromosomes which considered the Z-linked AMH as the master sex-determining gene.

Conclusions

Our study provides novel insights into the sex chromosome origin and sex determination of this model lizard. The near-complete P. vitticeps reference genome will also benefit future study of amniote evolution and may facilitate genome-assisted breeding.

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  1. GigaScience

    AbstractBackground The agamid dragon lizard Pogona vitticeps is one of the most popular domesticated reptiles to be kept as pets worldwide. The capacity of breeding in captivity also makes it emerging as a model species for a range of scientific research, especially for the studies of sex chromosome origin and sex determination mechanisms.Results By leveraging the CycloneSEQ and DNBSEQ sequencing technologies, we conducted whole genome and long-range sequencing for a captive-bred ZZ male to construct a chromosome-scale reference genome for P. vitticeps. The new reference genome is ∼1.8 Gb in length, with a contig N50 of 202.5 Mb and all contigs anchored onto 16 chromosomes. Genome annotation assisted by long-read RNA sequencing greatly expanded the P. vitticeps lncRNA catalog. With the chromosome-scale genome, we were able to characterize the whole Z sex chromosome for the first time. We found that over 80% of the Z chromosome remains as pseudo-autosomal region (PAR) where recombination is not suppressed. The sexually differentiated region (SDR) is small and occupied mostly by transposons, yet it aggregates genes involved in male development, such as AMH, AMHR2 and BMPR1A. Finally, by tracking the evolutionary origin and developmental expression of the SDR genes, we proposed a model for the origin of P. vitticeps sex chromosomes which considered the Z-linked AMH as the master sex-determining gene.Conclusions Our study provides novel insights into the sex chromosome origin and sex determination of this model lizard. The near-complete P. vitticeps reference genome will also benefit future study of amniote evolution and may facilitate genome-assisted breeding.Competing Interest StatementThe authors have declared no competing interest.

    This work has been peer reviewed in GigaScience (see https://doi.org/10.1093/gigascience/giaf079), which carries out open, named peer-review. These reviews are published under a CC-BY 4.0 license and were as follows:

    Reviewer 2: Nazila Koochekian

    Impressive work but needs major revision to be accepted. The authors compressed everything in the result section and did not put enough effort into the other sections. Introduction and discussion need major changes and more details regarding many aspects of the study that comes in the results. Methods need rearrangement. It's common to keep the order of methods such as first DNA extraction, then sequencing and so on. The data availability needs to be completed. Biosamples for each sequenced tissue, all the reads, and even intermediate assemblies need to be submitted to the database and reported in the manuscript. More specific comments are on the copy of the manuscript attached for the authors.

  2. GigaScience

    AbstractBackground The agamid dragon lizard Pogona vitticeps is one of the most popular domesticated reptiles to be kept as pets worldwide. The capacity of breeding in captivity also makes it emerging as a model species for a range of scientific research, especially for the studies of sex chromosome origin and sex determination mechanisms.Results By leveraging the CycloneSEQ and DNBSEQ sequencing technologies, we conducted whole genome and long-range sequencing for a captive-bred ZZ male to construct a chromosome-scale reference genome for P. vitticeps. The new reference genome is ∼1.8 Gb in length, with a contig N50 of 202.5 Mb and all contigs anchored onto 16 chromosomes. Genome annotation assisted by long-read RNA sequencing greatly expanded the P. vitticeps lncRNA catalog. With the chromosome-scale genome, we were able to characterize the whole Z sex chromosome for the first time. We found that over 80% of the Z chromosome remains as pseudo-autosomal region (PAR) where recombination is not suppressed. The sexually differentiated region (SDR) is small and occupied mostly by transposons, yet it aggregates genes involved in male development, such as AMH, AMHR2 and BMPR1A. Finally, by tracking the evolutionary origin and developmental expression of the SDR genes, we proposed a model for the origin of P. vitticeps sex chromosomes which considered the Z-linked AMH as the master sex-determining gene.Conclusions Our study provides novel insights into the sex chromosome origin and sex determination of this model lizard. The near-complete P. vitticeps reference genome will also benefit future study of amniote evolution and may facilitate genome-assisted breeding.Competing Interest StatementThe authors have declared no competing interest.

    This work has been peer reviewed in GigaScience (see https://doi.org/10.1093/gigascience/giaf079), which carries out open, named peer-review. These reviews are published under a CC-BY 4.0 license and were as follows:

    Reviewer 1: Heiner Kuhl

    Guo et al. present a new reference genome for Pogona vitticeps, a widespread reptile model organism that is also common as a domestic animal worldwide. The genome assembly shows much improvement over an older assembly from 2017. There are two points that make this manuscript outstanding from common genome assembly papers:

    1. The authors find a new sex determination locus in this species.
    2. the authors use a new nanopore sequencing technology ("CycloneSEQ"), which has so far only described in a preprint (https://www.biorxiv.org/content/10.1101/2024.08.19.608720v1).

    In my opinion this deserves a publication in Gigascience, but both points must be focused more in a revised manuscript.

    Major comments:

    1. The authors have sequenced a male individual (ZZ), which means the long-read reference assembly is missing the W-chromosome. PAR and SDR regions are deduced from the Z sequence, by analysis of sequencing coverage of only a few sexed samples (2 females and 4 males). It is unclear if these individuals are from the same family, which could mean that the newly found SD-region could just be a family specific variation. To make the whole story more intriguing and statistical sound the authors should at least test 15 males and 15 females from different P. vitticeps populations for W-specific markers near the proposed AMH deletion. The authors should also show that the prior proposed SD locus (nr5a1) does not carry W-specific mutations in these 15+15 individuals. Furthermore, a phased assembly of a female (ZW) Pogona vitticeps individual, could enable the assembly of the missing W-chr and should be included, it would even improve analysis of W-specific sequences in the proposed additional individuals.

    2. A technology aware reader would like to see more information on the specifics of the CycloneSEQ data quality and handling and maybe a comparison to competing technologies. Which enzymes and buffers were used to prepare the library? In the sections on the methods, there are only superficial descriptions such as (DNA repair buffer/enzyme, DNA clean beads, wash buffer for long fragments). Is it a kit or were the enzymes and buffers purchased individually? I cannot find the procedure for preparation and sequencing of the long-read cDNA libraries. How many flowcells were needed to generate the different datasets? How do the read-length distributions look like (statistics over all reads not only selected 40Kb+)? How was the variability between those runs, especially culmulative output over time? What hardware was needed to run the basecalling and what was the runtime? How is the Q-Value distribution of the reads? Why is the consensus accuracy of the assembly low (Q36.4)? can it be improved? Typically reference quality genomes should have Q40+. Which regions of the genome display lower consensus accuracies (is it random or sequence specific)?

    Minor comments:

    L.900: PRJNAxxxxxx looks like a placeholder, insert the true number,please.

  3. Version published to 10.1101/2024.09.05.611321 on bioRxiv