Identification of Y chromosome markers in the eastern three-lined skink (Bassiana duperreyi) using in silico whole genome subtraction

- Identification of Y chromosome markers in the eastern three-lined skink ( Bassiana.
- Background: Homologous sex chromosomes can differentiate over time because recombination is suppressed in the region of the sex determining locus, leading to the accumulation of repeats, progressive loss of genes that lack differential influence on the sexes and sequence divergence on the hemizygous homolog.
- Divergence in the non- recombining regions leads to the accumulation of Y or W specific sequence useful for developing sex-linked markers.
- Here we use in silico whole-genome subtraction to identify putative sex-linked sequences in the scincid lizard Bassiana duperreyi which has heteromorphic XY sex chromosomes..
- This study greatly improves our knowledge of the Y chromosome in B.
- In the absence of differential investment by the parents in male and female offspring, this system yields an evolution- arily stable 1:1 primary offspring sex ratio [1–3]..
- What follows over time is a chain of mutational events on the hemizygous member of the.
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- In humans, for example, the non- recombining region of the Y chromosome contains 78 protein coding genes encoding 27 proteins [7] compared with the 699 protein-coding genes with known function on the X [8].
- Such heterogeneity is brought about by variation in the evolu- tionary age of lineages with independently evolved sex chromosomes [11, 14].
- Suppression of re- combination along all or part of the sex chromosome length allows homologous sequences to diverge over time [19].
- Ran- dom amplified polymorphic DNA fingerprinting (RAPD) [27–29] and amplified fragment length polymorphisms (AFLP) [30–32] are PCR-based DNA fingerprinting techniques that sample only a fraction of the whole genome.
- Having no knowledge of the genomic context of the typically short markers can also render interpret- ation difficult..
- These RADseq and reduced representational approaches assess only a limited portion of the genome, and may miss many markers, particularly in species with small sex- specific domains or those with micro-sex chromosomes [47]..
- The species has heteromorphic XY sex chromosomes [57].
- duperreyi to apply an in silico whole genome subtraction approach, and de- velop new practical markers, useful in ongoing studies of this species in the laboratory and the wild..
- In silico whole genome subtraction.
- This equates to approximately 8x coverage of the genome estimated from the k-mer analysis.
- As expected, all 92 contigs passed the subtraction validation test where a product of the ex- pected size successfully amplified in the focal male and did not amplify in the focal female.
- Of these, 52 contigs yielded putative Y-chromosome markers when screened against the panel of 4 male and 4 female individuals, however, only 7 of these putative markers (Table 1) ran- ging in length from 628 bp to 824 bp, were validated as sex-specific when tested in the full panel of an additional 20 males and 20 females (Fig.
- The sequenced PCR products were aligned to the rele- vant full-length subtraction contig for each of the seven Y loci.
- When Piccadilly Circus and Anglesea populations were compared, alignment results showed a small number of discrepancies in the nucleotide composition obtained from five of the seven amplicons (Additional File 1: Figs S8 to S14.
- Of those that varied, sequence divergence ranged from 1.7% in the bdM27_.
- 0.3% in the bdM27_23_X5_798 amplicon (Additional File 1: Table S1).
- One of the seven Y-chromosome specific contigs, bdM27_23_X5_798, bears the partial sequence of an exon from the gene UBE2H, a member of a syntenic block conserved among jawed vertebrates [60].
- That said, the amplified sex specific region revealed some divergence between the Anglesea popula- tion and the Piccadilly Circus populations, suggesting that mutations could occur in the primer sites of some populations/taxa, limiting the generality of the sex-.
- relying on the divergence of the X and Y homologues in the region of recombination suppression.
- [79] in exploring variation among mammalian species in the Y chromosome, and recently applied to the yellow-bellied water skink, Eulamprus heatwolei [80], is to examine read copy number across the genome and identify the half copy number in the XY individuals compared to the XX individuals after screening out repetitive sequence.
- This technique identifies regions that have been lost from the non-recombining region of the Y chromosome but,.
- Here we used as an alternative complementary ap- proach, in silico whole genome subtraction to identify male-specific markers in the skink B.
- Our technique is useful for identifying novel sequences, often repetitive elements, gained by the non-recombining region of the Y chromo- some, or lost from the X chromosome.
- Lower read depth can be a challenge because it reduces the efficiency of the subtraction approach by increasing the number of false positives.
- Thus, PCR validation is effect- ive at eliminating the false positives resulting from auto- somal polymorphisms and differential coverage in the male and female..
- Our technique decomposes a set of reads from the genome to yield a unique, but highly redundant, repre- sentation of the genome as overlapping k-mers.
- We then select the k-mers found only in the XY (or ZW) individ- ual and reassemble the k-mers to yield Y (or W) enriched contigs that can be validated using PCR on a panel of individuals whose sex is known.
- Specifically, our in silico subtrac- tion method surveys the entire available genome, assuming adequate read depth, to identify sex specific differences and does not rely on a highly reduced repre- sentation of the genome as with RAD and ddRAD ap- proaches, that may miss many putative markers.
- Although various members of the ubi- quitin conjugating enzyme family are involved in testes specific processes (e.g.
- testis-specific UBC4-testis in the rat, [82] and an ascidian, [83]) we make no suggestion that UBE2H plays a role in sex determination in these skinks, merely that it is a gene on the sex chromosomes..
- Investigating the occurrence of temperature sex reversal will increase our understanding of sex rever- sal as a driver of sex-chromosome turn-over in the wild [75] and establish links between environmental extremes and reptile sex determining modes [84].
- The success rate of future Y-marker discovery via gen- ome subtraction could be improved by implementing ef- forts to reduce false positives caused by autosomal insertion/deletion polymorphisms in the focal sequenced individuals.
- S E, 1246 m a.s.l.) in Namadgi National Park, 40 km west of Canberra in the Australian Capital Territory, and from Anglesea S, 144°12′.
- 150 μg/g body weight), dissected, and phenotypic sex con- firmed by examination of the gonads.
- DNA was extracted from fresh liver samples of the two focal animals and from the tail snips of the 60 validation.
- Reads from the focal male and the focal female were analysed independently as follows (Fig.
- K-mers in the subtraction with a count less than 2 for males and 5 for females were considered to rep- resent sequencing errors and were removed from the ana- lysis.
- This decision was based on examination of the k- mer spectra, identifying the minima immediately to the right of the peak arising from presumed read errors.
- Briefly, the assembler ini- tially took a focal k-mer at random and searched for other k-mers that matched exactly k-1 bp of the focal k-mer.
- To validate the sex specificity of each of the contigs and remove false positives derived from autosomal and X chromosome polymorphisms, we designed primers for each contig using Primer 3 [89] implemented in Geneious [90] (version R8).
- PCR tests in the validation animals using the following conditions.
- To confirm that the subtraction pipeline had suc- cessfully identified a presence/absence polymorphism in the two focal individuals, we first screened those two indi- viduals to confirm presence of an amplified fragment in the male and the absence of an amplified fragment in the fe- male.
- 3 Schematic diagram showing methodology of the genome subtraction pipeline a A hypothetical schematic of the B.
- e Female k-mers are subtracted from the male k-mers.
- At each of the stages, the loci that did not appear as sex specific were eliminated as candidate sex markers.
- The probability of an autosomal or X chromosome polymorphism being present in the focal male, 4 males and 20 additional males, and absent in the focal female, 4 females and 20 additional females, is suffi- ciently low.
- To confirm the amplification of the desired sequence, PCR products for all 7 putative Y-loci were visually assessed using gel electrophoresis and then Sanger se- quenced in a single direction, using the forward primer, on an AB 3730xl DNA Analyzer at the Biomolecular Re- search Facility, Australian National University, Canberra, Australia.
- The phenotypic sex of each of the karyotyped animals was confirmed by gross examination of gonads followed by histological examination.
- To discover homologies of the male-specific contigs and identify any partial gene sequences that may exist, we used BLASTN to search each contig against representa- tive reptilian and avian genomes available in Ensembl, Release 99 (Anolis carolinensis, Crocodylus porosus, Gallus gallus, Pelodiscus sinensis, Podarcis muralis, Pogona vitticeps, Pseudonaja textilis, Notechis scutatus, Varanus komodoensis, Sphenodon punctatus) with a minimum E-value of 0.000001 for reported alignments and a filter for low complexity regions.
- Hits to known repeats in the Dfam database.
- Additional file 2: Original gel images to accompany Figure 1 of the manuscript.
- Detailed specimen list available in the Additional file 1: Table S4..
- We are grateful to Lasanthika Thewarage for assistance in the field and Wendy Ruscoe and Jacqui Richardson of the animal facility at the University of Canberra for animal husbandry..
- DSBD led the writing of the manuscript with contributions from JED and all other authors.
- Funding agency played no role in the study design, data analysis and interpretation, or in writing the manuscript..
- Data and materials are presented in the main paper and additional files, and in public repositories.
- Illumina reads used as the basis of the k-mer analysis and subtraction are available from NCBI SRA re- pository (Accession Numbers SAMN .
- 3 Present Address: Centre for Gene Therapy, Beckman Research Institute of the City of Hope, Duarte, CA, USA..
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- Development of sexing primers in Glyptemys insculpta and Apalone spinifera turtles uncovers an XX/XY sex-determining system in the critically- endangered bog turtle Glyptemys muhlenbergii.
- Screening and characterization of sex-linked DNA markers and marker-assisted selection in the southern catfish (Silurus meridionalis).
- Sex determination mode does not affect body or genital development of the central bearded dragon (Pogona vitticeps).
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- Differentiation and development of testis in the oviparous lizard, Calotes versicolor (Daud

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