An XY sex chromosome system and a boa and python

17 Gamble T.

Zarkower D. Identification of sex-specific molecular markers using restriction site-associated DNA sequencing. 41 Felip A.

Young W.P.

Wheeler P.A.

Thorgaard G.H. An AFLP-based approach for the identification of sex-linked markers in rainbow trout (Oncorhynchus mykiss). 42 Devlin R.H.

Biagi C.A.

Smailus D.E. Genetic mapping of Y-chromosomal DNA markers in Pacific salmon. 43 Baird N.A.

Etter P.D.

Atwood T.S.

Currey M.C.

Shiver A.L.

Lewis Z.A.

Selker E.U.

Cresko W.A.

Johnson E.A. Rapid SNP discovery and genetic mapping using sequenced RAD markers. 17 Gamble T.

Zarkower D. Identification of sex-specific molecular markers using restriction site-associated DNA sequencing. 17 Gamble T.

Zarkower D. Identification of sex-specific molecular markers using restriction site-associated DNA sequencing. 21 Gamble T.

Coryell J.

Ezaz T.

Lynch J.

Scantlebury D.P.

Zarkower D. Restriction site-associated DNA sequencing (RAD-seq) reveals an extraordinary number of transitions among gecko sex-determining systems. 22 Gamble T. Using RAD-seq to recognize sex-specific markers and sex chromosome systems. The identification of sex-specific genetic markers can be used to infer a species’ sex chromosome system []. Here we identified sex-specific markers from RAD-seq data. RAD-seq uses Illumina sequencing to produce paired-end reads from libraries made from restriction digested DNA []. The process involves sequencing thousands of RAD markers from multiple confidently sexed males and females to identify the sex-specific markers, that is, RAD markers found in one sex but not the other []. These sex-specific RAD markers are presumed to be on the Y or W. Thus, species with an excess of male-specific markers have an XY system while species with an excess of female-specific markers have a ZW system [].

44 Peterson B.K.

Weber J.N.

Kay E.H.

Fisher H.S.

Hoekstra H.E. Double digest RADseq: an inexpensive method for de novo SNP discovery and genotyping in model and non-model species. 34 Card D.C.

Schield D.R.

Adams R.H.

Corbin A.B.

Perry B.W.

Andrew A.L.

Pasquesi G.I.

Smith E.N.

Jezkova T.

Boback S.M.

et al. Phylogeographic and population genetic analyses reveal multiple species of Boa and independent origins of insular dwarfism. 44 Peterson B.K.

Weber J.N.

Kay E.H.

Fisher H.S.

Hoekstra H.E. Double digest RADseq: an inexpensive method for de novo SNP discovery and genotyping in model and non-model species. 45 Schield D.R.

Card D.C.

Adams R.H.

Jezkova T.

Reyes-Velasco J.

Proctor F.N.

Spencer C.L.

Herrmann H.-W.

Mackessy S.P.

Castoe T.A. Incipient speciation with biased gene flow between two lineages of the western diamondback rattlesnake (Crotalus atrox). 21 Gamble T.

Coryell J.

Ezaz T.

Lynch J.

Scantlebury D.P.

Zarkower D. Restriction site-associated DNA sequencing (RAD-seq) reveals an extraordinary number of transitions among gecko sex-determining systems. 46 Etter P.D.

Bassham S.

Hohenlohe P.A.

Johnson E.A.

Cresko W.A. SNP discovery and genotyping for evolutionary genetics using RAD sequencing. We produced four groups of multiplexed RAD-seq libraries that each included multiple male and females samples. These were (1) double digest or ddRAD libraries for Western Diamondback Rattlesnake (Crotalus atrox); (2) ddRAD libraries for Burmese python (Python bivittatus); (3) ddRAD libraries for the Central American Boa constrictor (Boa imperator); and (4) single digest RAD libraries for the Central American Boa constrictor (Boa imperator) ( Tables 1 and S4 ). Double-digest RAD-seq (ddRAD) libraries for boas (six males and nine females) and pythons (three males and four females) were constructed following the protocol of Peterson et al. [] with minor modifications following Card et al. []. We used enzymes PstI and Sau3AI and a size selection of 570 to 690 bp (including adapters) for boa ddRAD libraries. For pythons, we used enzymes SpeI and Sau3AI and size selected 300 to 625 bp. Libraries were sequenced on an Illumina HiSeq2500 using 100 bp paired-end reads. Rattlesnake ddRAD libraries (seven males and seven females) also followed Peterson et al. [] and used enzymes SbfI and Sau3AI and a size selection of 590 to 640 bp []. Libraries were sequenced on an Illumina HiSeq2500. We also made a single digest RAD library for additional boa samples (six males and five females), all siblings from a single litter, using the SbfI enzyme and size selection of 300 to 550 bp [] and sequenced these on an Illumina HiSeq2500 using 125 bp paired-end reads. Sequencing reads are available at the NCBI Short Read Archive (Boa, NCBI SRA: PRJNA382366, PRJNA387612; Python, NCBI SRA: PRJNA382347; Crotalus, NCBI SRA: PRJNA269607).

17 Gamble T.

Zarkower D. Identification of sex-specific molecular markers using restriction site-associated DNA sequencing. 21 Gamble T.

Coryell J.

Ezaz T.

Lynch J.

Scantlebury D.P.

Zarkower D. Restriction site-associated DNA sequencing (RAD-seq) reveals an extraordinary number of transitions among gecko sex-determining systems. 35 Catchen J.M.

Amores A.

Hohenlohe P.

Cresko W.

Postlethwait J.H. Stacks: building and genotyping loci de novo from short-read sequences. 36 Baxter S.W.

Davey J.W.

Johnston J.S.

Shelton A.M.

Heckel D.G.

Jiggins C.D.

Blaxter M.L. Linkage mapping and comparative genomics using next-generation RAD sequencing of a non-model organism. 21 Gamble T.

Coryell J.

Ezaz T.

Lynch J.

Scantlebury D.P.

Zarkower D. Restriction site-associated DNA sequencing (RAD-seq) reveals an extraordinary number of transitions among gecko sex-determining systems. 17 Gamble T.

Zarkower D. Identification of sex-specific molecular markers using restriction site-associated DNA sequencing. 47 Kircher M.

Sawyer S.

Meyer M. Double indexing overcomes inaccuracies in multiplex sequencing on the Illumina platform. 37 Kearse M.

Moir R.

Wilson A.

Stones-Havas S.

Cheung M.

Sturrock S.

Buxton S.

Cooper A.

Markowitz S.

Duran C.

et al. Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Sex-specific markers were identified using a previously published bioinformatic pipeline []. We used the “process radtags” script from Stacks-1.41 [] to demultiplex, filter, and trim raw Illumina reads. RADtools 1.2.4 [] was used to generate candidate alleles for each individual and candidate loci across all individuals from the forward reads. All species were analyzed separately. Settings for the RADtags script included a cluster distance of 10, minimum quality score of 20, and read threshold of 5. Settings for the RADmarkers script, which generates candidate loci and alleles across individuals using output from the RADtags script, included a tag count threshold of 4 and the maximum number of mismatches set at 2. The RADtools output includes the presence/absence of each locus and allele for every sampled individual, enabling the identification of sex-specific RAD markers. We used a python script [] to identify putative sex-specific markers from the RADtools output. This script also produces a second list, a subset of the initial set of sex-specific RAD markers, that excludes from further consideration any sex-specific markers that also appear in the original reads files from the opposite sex, we call these “confirmed sex-specific RAD markers” following Gamble et al. []. This removes rare, but potentially inaccurate RAD markers that may arise due to problems with multiplex sequencing on the Illumina platform []. Forward and reverse reads from the confirmed sex-specific markers are subsequently assembled into contigs using Geneious R9 [].

17 Gamble T.

Zarkower D. Identification of sex-specific molecular markers using restriction site-associated DNA sequencing. 21 Gamble T.

Coryell J.

Ezaz T.

Lynch J.

Scantlebury D.P.

Zarkower D. Restriction site-associated DNA sequencing (RAD-seq) reveals an extraordinary number of transitions among gecko sex-determining systems. 17 Gamble T.

Zarkower D. Identification of sex-specific molecular markers using restriction site-associated DNA sequencing. Using the preceding methods, species with an excess of male-specific RAD markers have an XY sex chromosome system while species with an excess of female-specific RAD markers have a ZW sex chromosome system. However, we cannot rule out that some number of sex-specific markers may be identified by chance, particularly when sample size is small, e.g., our python dataset with only three males and four females. False positives may be present because there exists some probability that a RAD marker could exhibit a sex-specific pattern simply by chance. This chance is higher when the number of sampled individuals is small and decreases as the number of individuals increases. The chance of false positives should also increase as the number of RAD markers increases. We addressed this by permuting the sex labels among sampled individuals for each dataset to create a null distribution of the number of sex-specific markers that could be expected solely by chance. We then determined whether the observed number of sex-specific markers is a plausible sample of this null distribution, e.g., contained within the 95% confidence interval of the null distribution, or whether the observed number of sex-specific RAD markers is larger than expected by chance alone. We did this for each species, calculating null distributions using the same number of males and females as our original dataset ( Table 1 ) using 100 permutations. We performed these permutations using the total number of sex-specific RAD markers identified in each dataset not the number of “confirmed sex-specific RAD markers.” Evaluating the number of confirmed sex-specific RAD markers would have also involved permuting the raw read data, which was computationally burdensome. However, since the number of sex-specific markers in each dataset is proportional to the number of confirmed sex-specific markers ( Table 1 ) [] we feel that this is an acceptable means of assessing the significance of our RAD-seq results. It should be noted that previous work has shown that sex-specific markers can still be identified when sample sizes are small []. However, the true sex-specific markers will be contained within an increasingly larger sample of false positives as sample size decreases.