Isis version 5

We used alpaca CHORI-246 genomic Bacterial Artificial Chromosome (BAC) library¹⁰ to obtain probes for FISH. BAC clones containing ASIP and TYRP1 were previously identified and mapped in the alpaca (Avila et al., 2014a (link)). To obtain BACs for MC1R, we screened CHORI-246 filters with MC1R-specific radioactively labeled ([³²P] dATP/dCTP) overgo primers (Table 1) as described by Avila et al. (2014b) (link). The final BACs containing MC1R were further verified by PCR with MC1R exon primers (Table 1). BAC DNA was isolated with Plasmid Mini Kit (Qiagen) according to the manufacturer’s protocol. Probe labeling, hybridization and signal detection were conducted according to standard protocols (Raudsepp and Chowdhary, 2008 (link)). Because of difficulties to unambiguously identify camelid chromosomes by conventional cytogenetic methods (Avila et al., 2014b (link)), BACs containing the three genes were co-hybridized with a differently labeled reference gene from the alpaca cytogenetic map (Avila et al., 2014a (link)). Composite information about the BACs used for comparative FISH mapping is presented in Table 3. Images for at least 10 metaphases for each experiment were captured and analyzed using a Zeiss Axioplan 2 fluorescence microscope, equipped with the Isis Version 5.2 (MetaSystems GmbH) software.

The DNA of individual BACs was labeled with Biotin or digoxigenin using Biotin or DIG Nick Translation Mix (Roche Diagnostics), respectively, and the manufacturer’s protocol. In situ hybridization and signal detection was done following standard protocols described elsewhere [25 (link),35 (link)]. Biotin-labeled probes were detected with avidin-FITC (Vector Laboratories) and dig-labeled probes with anti-DIG-rhodamine (Roche Applied Science). In order to precisely determine the cytogenetic location of the 35 new markers, each marker was co-hybridized with a differently labeled previously FISH-mapped reference marker [5 (link),23 (link)] (Table 1 and Table S3). Images for a minimum of 10 metaphase spreads and 10 interphase nuclei were captured for each experiment and analyzed using a Zeiss Axioplan 2 fluorescence microscope, equipped with the Isis Version 5.2 (MetaSystems GmbH, Altlussheim, Germany) software. Chomosomes were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) and identified according to the previously proposed nomenclature [22 (link),23 (link),35 (link)].

Painting probes generated from flow-sorted alpaca X and Y chromosomes (Avila et al., 2014b (link)) were used to test the presence and integrity of sex chromosomes. The origin of the autosomal translocation was investigated through series of dual-color FISH experiments with chromosome-specific markers (BAC clones from CHORI-246 BAC library¹ derived from the alpaca whole genome cytogenetic map (Avila et al., 2014a (link)). BAC DNA isolation, labeling and FISH were performed following standard protocols (Raudsepp and Chowdhary, 2008 (link); Avila et al., 2014b (link)). Images for a minimum of 10 metaphase spreads were captured for each experiment and analyzed with a Zeiss Axioplan2 fluorescence microscope equipped with Isis Version 5.2 (MetaSystems GmbH) software.