A telomere to telomere phased genome assembly and annotation for the Australian central bearded dragon Pogona vitticeps

  1. Hardip R. Patel
  2. Kirat Alreja
  3. Andre L.M. Reis
  4. J King Chang
  5. Zahra A. Chew
  6. Hyungtaek Jung
  7. Jillian M. Hammond
  8. Ira W. Deveson
  9. Aurora Ruiz-Herrera
  10. Laia Marin-Gual
  11. Clare E. Holleley
  12. Xiuwen Zhang
  13. Nicholas C. Lister
  14. Sarah Whiteley
  15. Lei Xiong
  16. Duminda S.B. Dissanayake
  17. Paul D. Waters
  18. Arthur Georges

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Abstract

Background

The central bearded dragon ( Pogona vitticeps) is widely distributed in central eastern Australia and adapts readily to captivity. Among other attributes, it is distinctive because it undergoes sex reversal from ZZ genotypic males to phenotypic females at high incubation temperatures. Here, we report an annotated telomere to telomere phased assembly of the genome of a female ZW central bearded dragon.

Results

Genome assembly length is 1.75 Gbp with a scaffold N50 of 266.2 Mbp, N90 of 28.1 Mbp, 26 gaps and 42.2% GC content. Most (99.6%) of the reference assembly is scaffolded into 6 macrochromosomes and 10 microchromosomes, including the Z and W microchromosomes, corresponding to the karyotype. The genome assembly exceeds standard recommended by the Earth Biogenome Project (6CQ40): 0.003% collapsed sequence, 0.03% false expansions, 99.8% k-mer completeness, 97.9% complete single copy BUSCO genes and an average of 93.5% of transcriptome data mappable back to the genome assembly. The mitochondrial genome (16,731 bp) and the model rDNA repeat unit (length 9.5 Kbp) were assembled. Male vertebrate sex genes Amh and Amhr2 were discovered as copies in the small non-recombining region of the Z chromosome, absent from the W chromosome.

This, coupled with the prior discovery of differential Z and W transcriptional isoform composition arising from pseudoautosomal sex gene Nr5a1 , suggests that complex interactions between these genes, their autosomal copies and their resultant transcription factors and intermediaries, determines sex in the bearded dragon.

Conclusion

This high-quality assembly will serve as a resource to enable and accelerate research into the unusual reproductive attributes of this species and for comparative studies across the Agamidae and reptiles more generally.

Species Taxonomy

Eukaryota; Animalia; Chordata; Reptilia; Squamata; Iguania; Agamidae; Amphibolurinae; Pogona ; Pogona vitticeps (Ahl, 1926) (NCBI:txid103695).

Graphical Abstract

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  1. GigaScience

    AbstractBackground The central bearded dragon (Pogona vitticeps) is widely distributed in central eastern Australia and adapts readily to captivity. Among other attributes, it is distinctive because it undergoes sex reversal from ZZ genotypic males to phenotypic females at high incubation temperatures. Here, we report an annotated telomere to telomere phased assembly of the genome of a female ZW central bearded dragon.Results Genome assembly length is 1.75 Gbp with a scaffold N50 of 266.2 Mbp, N90 of 28.1 Mbp, 26 gaps and 42.2% GC content. Most (99.6%) of the reference assembly is scaffolded into 6 macrochromosomes and 10 microchromosomes, including the Z and W microchromosomes, corresponding to the karyotype. The genome assembly exceeds standard recommended by the Earth Biogenome Project (6CQ40): 0.003% collapsed sequence, 0.03% false expansions, 99.8% k-mer completeness, 97.9% complete single copy BUSCO genes and an average of 93.5% of transcriptome data mappable back to the genome assembly. The mitochondrial genome (16,731 bp) and the model rDNA repeat unit (length 9.5 Kbp) were assembled. Male vertebrate sex genes Amh and Amhr2 were discovered as copies in the small non-recombining region of the Z chromosome, absent from the W chromosome.This, coupled with the prior discovery of differential Z and W transcriptional isoform composition arising from pseudoautosomal sex gene Nr5a1, suggests that complex interactions between these genes, their autosomal copies and their resultant transcription factors and intermediaries, determines sex in the bearded dragon.Conclusion This high-quality assembly will serve as a resource to enable and accelerate research into the unusual reproductive attributes of this species and for comparative studies across the Agamidae and reptiles more generally.Species Taxonomy Eukaryota; Animalia; Chordata; Reptilia; Squamata; Iguania; Agamidae; Amphibolurinae; Pogona; Pogona vitticeps

    This work has been peer reviewed in GigaScience (see https://doi.org/10.1093/gigascience/giaf085), 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

    Patel et al. present a genome assembly of the bearded dragon Pogona vitticeps a lizard species that is widely distributed as a pet and known for its interesting sex-determination, which may switch from genetic sex-determination (ZW) to temperature dependent sex-reversal. The methods chosen to assemble the genome are very state-of-the-art including HIFI and ONT long reads, Hi-C and suitable bioinformatic tools.

    I have to admit that I have recently been reviewing a similar manuscript for Gigascience (https://www.biorxiv.org/content/10.1101/2024.09.05.611321v1), where a female ZZ P. vitticeps had been sequenced/assembled from long read data of a different nanopore technology and analyses of the ZW-chromosome was done by short read coverage analysis. One of my major comments was that this approach lacked a true assembly of the W-chromosome. Thus, I am happy to see that the assembly of the W-specific region has been achieved here and the sequencing technologies used might even improve the assembly quality over the ZZ assembly in terms of phasing, consensus accuracy etc. The two manuscripts are highly complementary and I think they should be published, if possible, in the very same issue of Gigascience. Surely both groups have invested a lot of efforts. (Reading L. 685, I just have realized that this seems to be the intention of the journal and I very much support this idea.)

    Still there are some minor points that need improvement for the current manuscript:

    Why do you leave the Z and W splitted into PAR, Z- and W-specific scaffolds and do not assemble the full-length chromosomes (L. 676)? Would the Hi-C data not support that?

    Mitochondrial assembly: from ONT only (L. 307), please do a consensus correction with illumina data, or at least show that the MT assembly has a high consensus accuracy (Q40-Q50).

    Genome annotation: show BUSCO scores for annotated proteins (do they fit to BUSCO performed on the whole genome?). If possible, compare to results of the NCBI RefSeq annotation (is it already available?). In this regard please explain the relatively low mapping rates (L. 647) of RNAseq to the annotated sequences.

    Could you provide some expression data for the Z-specific Amh and AmhR2? Is it differentially expressed in testis/ovary (after correction for copy number)?

    Table1, could you show results for the two different ONT library types (ligation vs. ultralong kit). It seems the overall yield was low (5 cells -> 100Gb), any speculation why?

    I think assembly statistics (Table2) should also contain contig N50 length as an additional value to show the high continuity of the assembly.

    L. 488: "48.36 (1 error in 146kb)", I think something is wrong here. Q48.36 would be 1 error in 68.5kb. I would suggest to re-check these values and incorporate them in Table2. The high consensus accuracy is one selling point compared to the competitor's assembly.

    L. 490: "Individual haplotypes were 85.5% complete…". Explain why you are confident that the haplotypes are more complete than the Merqury results suggest (just one sentence).

  2. GigaScience

    AbstractBackground The central bearded dragon (Pogona vitticeps) is widely distributed in central eastern Australia and adapts readily to captivity. Among other attributes, it is distinctive because it undergoes sex reversal from ZZ genotypic males to phenotypic females at high incubation temperatures. Here, we report an annotated telomere to telomere phased assembly of the genome of a female ZW central bearded dragon.Results Genome assembly length is 1.75 Gbp with a scaffold N50 of 266.2 Mbp, N90 of 28.1 Mbp, 26 gaps and 42.2% GC content. Most (99.6%) of the reference assembly is scaffolded into 6 macrochromosomes and 10 microchromosomes, including the Z and W microchromosomes, corresponding to the karyotype. The genome assembly exceeds standard recommended by the Earth Biogenome Project (6CQ40): 0.003% collapsed sequence, 0.03% false expansions, 99.8% k-mer completeness, 97.9% complete single copy BUSCO genes and an average of 93.5% of transcriptome data mappable back to the genome assembly. The mitochondrial genome (16,731 bp) and the model rDNA repeat unit (length 9.5 Kbp) were assembled. Male vertebrate sex genes Amh and Amhr2 were discovered as copies in the small non-recombining region of the Z chromosome, absent from the W chromosome.This, coupled with the prior discovery of differential Z and W transcriptional isoform composition arising from pseudoautosomal sex gene Nr5a1, suggests that complex interactions between these genes, their autosomal copies and their resultant transcription factors and intermediaries, determines sex in the bearded dragon.Conclusion This high-quality assembly will serve as a resource to enable and accelerate research into the unusual reproductive attributes of this species and for comparative studies across the Agamidae and reptiles more generally.Species Taxonomy Eukaryota; Animalia; Chordata; Reptilia; Squamata; Iguania; Agamidae; Amphibolurinae; Pogona; Pogona vitticeps (Ahl, 1926) (NCBI:txid103695).

    This work has been peer reviewed in GigaScience (see https://doi.org/10.1093/gigascience/giaf085), which carries out open, named peer-review. These reviews are published under a CC-BY 4.0 license and were as follows:

    Reviewer 2: Yuan Li

    The authors de novo assembled a telomere to telomere phased genome assembly of the Australian central bearded dragon Pogona vitticeps, using PacBio HiFi, ONT, HiC, and Illumina sequencing platforms. The assembly achieves remarkable contiguity (scaffold N50: 266.2 Mb) and completeness (97.9% BUSCO score), surpassing Earth Biogenome Project standards. The phased assembly of sex chromosomes (Z/W) and identification of candidate sex-determining genes (Amh, Amhr2, and Nr5a1) provide valuable insights into reptilian sex determination. Overall, the study is well-executed and provides a valuable resource for comparative genomics and reproductive biology.

    Major concern: 1.The description of read depth had errors at lines 401-402, such as 60.6x. In addition, "4 x promethION", "2x150 bp" were should be revised and please check and revise all the similar description in the manuscript. 2.There are errors in the citation format of the journal references, such as the absence of punctuation "."marks between the title name and the journal name at lines 1005-1009, mixing abbreviations (e.g., "PNAS" vs. "Proceedings of the National Academy of Sciences USA") (lines 988-990, 1005-1009). Please check carefully the format of all references. 3.The script "calculateGC.py and processtrftelo.py" (lines 242 and 245) are mentioned without code availability or parameter details. Provide effective links or repository access. 4.The inconsistent use of "Gb" and "Gbp" is observed; it is recommended to adopt a unified description. 5.Units were missing in the descriptions in multiple places in Table 1 and 2, such as the unit for "Total Bases" and "Assembly length"; please include them. 6.At lines 683-687, the conclusion that Amh/Amhr2 are sex-determining genes relies solely on positional evidence. Discuss the need for functional studies (e.g., CRISPR knockouts) to strengthen claims. 7.There were errors in "Vasimuddin et al. 2019" (line 238) and "Danecek et al. 2021" (line 239). Please check all the other formats of references. 8.At lines 476-481, BAC mappings are cited as validation but lack visual evidence (e.g., alignment plots in figures or supplements). Please verify the accuracy of Figure 7 at line 478, as it does not correspond with the description.

  3. Version published to 10.1101/2025.05.01.651798 on bioRxiv