SNP genotyping
DNA was extracted by Diversity Arrays Technologies (DArT Pty Ltd, Canberra, Australia, www.diversityarrays.com) using a NucleoMag 96 Tissue Kit (Macherey-Nagel, Düren, Germany) coupled with NucleoMag SEP to allow automated separation of high-quality DNA on a Freedom Evo robotic liquid handler (TERAN Pty Ltd, Port Melbourne, Australia).
Sequencing for SNP genotyping was done using DArTseq™ (DArT Pty Ltd) which uses a combination of complexity reduction using restriction enzymes, implicit fragment size selection and next generation sequencing (Sansaloni et al., 2011), as described in detail by Kilian et al. (2012). The technique is similar to double-digest restriction associated DNA sequencing (ddRAD) (Peterson et al., 2012), but has the advantages of accepting lower quantities of DNA, greater tolerance of lower quality DNA and higher call rates (Sansaloni et al., 2011). The restriction enzyme combination of PstI (recognition sequence 5′-CTGCA|G-3′) and SphI (5’-GCATG|C-3’) was used for the double digestion.
The PstI-compatible adapter included the Illumina flowcell attachment sequence, a sequencing primer sequence, a barcode region of variable length (see Elshire et al., 2011) and the PstI-compatible overhang sequence. The reverse adapter contained flowcell attachment sequence and SphI-compatible overhang sequence. Only fragments generated by the PstI-SphI double digest were effectively amplified by polymerase chain reaction (PCR) (Georges et al., 2018).
Sequences generated from each lane were processed using proprietary DArT analytical pipelines as outlined by Georges et al. (2018) to yield repeatable SNP markers. In addition, DArT processes approximately one third of the samples twice from DNA to allelic calls as technical replicates and scoring consistency (repeatability) was used as the main selection criteria for high quality/low error rate markers.