We extracted genomic DNA from quill bases of feathers, blood, muscle and other tissue material either with a GeneMole® automated nucleic acid extraction instrument (Mole Genetics), the DNeasy Blood and Tissue Kit (QIAGEN) or with Chelex. Standard procedures were applied except for the quill bases, which were lysated with 1% DTT. Feather material was sampled from a European green woodpecker Picus viridis killed by traffic. Live animals were not sampled for this study. Other tissue material was borrowed from museum collections and from the collections of colleagues, the National Veterinary Institute SVA in Uppsala and Uppsala City Council. We performed mtDNA barcoding with COI, following the Stockholm protocol outlined in [68 (link)], to confirm labelling of selected tissue samples and to identify species Ramphastos tucanus from an unspecified toucan tissue sample.
Using the degenerate primers SU80a [69 (link)], SU149a, SU161a, SU193a [42 (link)] or SU200Ca, combined with SU304b [15 (link)] or SU306b [42 (link)] we amplified a gene fragment coding for residues from aa sites 81–94, located in the 2nd α-helical transmembrane region of the SWS1 opsin. We conducted PCR on an Eppendorf MasterCycler Gradient or a PE Applied Biosystems Geneamp® PCR System 9700 with reactions containing 0.5-2.5 ng/μl DNA extracts, 1 unit Taq-polymerase (Applied Biosystems) plus reaction buffer, 0.4 pmol of forward and reverse primers, 0.2 mM of each dNTP, and 2 mM MgCl2. Each PCR reaction contained 0.5–2.5 ng/μl total DNA extracts, 1 unit Taq-polymerase (Applied Biosystems) with reaction buffer, 0.4 pmol of forward and reverse primers, 0.2 mM of each dNTP, and 2 mM MgCl2. For some reactions, PuReTaq™ Ready-To-Go™ PCR beads (GE Healthcare) replaced separate volumes of Taq-polymerase, dNTP’s and MgCl2. Initially, the reaction conditions followed [42 (link)] (i.e. 90 s at 94°C, 5 × (30 s at 94°C, 30 s at 54°C and 1 s at 72°C), 38 × (15 s at 94°C, 30 s at 54°C and 5 s at 72°C) and 10 min at 72°C) but were later optimized to exclude the extension phase in order to minimize nonspecific amplification of longer fragments. The final version of thermocycling started with 90 s at 94°C, was followed by 48 × (5 s at 94°C, 15 s at 54°C) and ended with 1 s at 72°C. We used a different protocol for the primer pair SU80a/SU306b, namely 2 min 30 s at 95°C, 40 × (30 s at 95°C, 30 s at 54°C and 10 s at 72°C) and 1 min at 72°C. Two percent agarose gel electrophoresis for 90 min at 80 V confirmed amplification and expected fragment length. When there were extra fragments present we sometimes performed a second PCR on the products using internal primers.
The PCR products were purified with EXOsap-IT (USB). Macrogen Inc. (South Korea) then performed double-stranded sequencing using the same primers as above plus SU200a [15 (link)], SU200Ga [60 (link)], and SU296b 5′-AAG AYR AAG TAD CCS YGS G-3′, which we designed for this study with the help of Primer3 online software (http://frodo.wi.mit.edu/ ) [70 ].
We translated our DNA sequences into aa’s to identify the spectral tuning sites 86, 90, and 93 [5 (link),10 (link)]. From the aa residues presents at these sites we estimated λmax values following Wilkie et al. [5 (link)], Yokoyama et al. [10 (link)] and Carvalho et al. [11 (link)] as outlined in [15 (link)].
Using the degenerate primers SU80a [69 (link)], SU149a, SU161a, SU193a [42 (link)] or SU200Ca, combined with SU304b [15 (link)] or SU306b [42 (link)] we amplified a gene fragment coding for residues from aa sites 81–94, located in the 2nd α-helical transmembrane region of the SWS1 opsin. We conducted PCR on an Eppendorf MasterCycler Gradient or a PE Applied Biosystems Geneamp® PCR System 9700 with reactions containing 0.5-2.5 ng/μl DNA extracts, 1 unit Taq-polymerase (Applied Biosystems) plus reaction buffer, 0.4 pmol of forward and reverse primers, 0.2 mM of each dNTP, and 2 mM MgCl2. Each PCR reaction contained 0.5–2.5 ng/μl total DNA extracts, 1 unit Taq-polymerase (Applied Biosystems) with reaction buffer, 0.4 pmol of forward and reverse primers, 0.2 mM of each dNTP, and 2 mM MgCl2. For some reactions, PuReTaq™ Ready-To-Go™ PCR beads (GE Healthcare) replaced separate volumes of Taq-polymerase, dNTP’s and MgCl2. Initially, the reaction conditions followed [42 (link)] (i.e. 90 s at 94°C, 5 × (30 s at 94°C, 30 s at 54°C and 1 s at 72°C), 38 × (15 s at 94°C, 30 s at 54°C and 5 s at 72°C) and 10 min at 72°C) but were later optimized to exclude the extension phase in order to minimize nonspecific amplification of longer fragments. The final version of thermocycling started with 90 s at 94°C, was followed by 48 × (5 s at 94°C, 15 s at 54°C) and ended with 1 s at 72°C. We used a different protocol for the primer pair SU80a/SU306b, namely 2 min 30 s at 95°C, 40 × (30 s at 95°C, 30 s at 54°C and 10 s at 72°C) and 1 min at 72°C. Two percent agarose gel electrophoresis for 90 min at 80 V confirmed amplification and expected fragment length. When there were extra fragments present we sometimes performed a second PCR on the products using internal primers.
The PCR products were purified with EXOsap-IT (USB). Macrogen Inc. (South Korea) then performed double-stranded sequencing using the same primers as above plus SU200a [15 (link)], SU200Ga [60 (link)], and SU296b 5′-AAG AYR AAG TAD CCS YGS G-3′, which we designed for this study with the help of Primer3 online software (
We translated our DNA sequences into aa’s to identify the spectral tuning sites 86, 90, and 93 [5 (link),10 (link)]. From the aa residues presents at these sites we estimated λmax values following Wilkie et al. [5 (link)], Yokoyama et al. [10 (link)] and Carvalho et al. [11 (link)] as outlined in [15 (link)].
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