DNA extraction
DNA extraction from fresh or dried leaf material should be carried out using the Qiagen (Venlo, NL) DNeasy Plant Mini Kit, according to the manufacturers instructions.
PCR amplification
PCR amplification of the Agt1 marker region can be performed with MyTaq DNA-polymerase (Bioline, London, UK) using 10-20 ng DNA template, 5x MyTaq reaction buffer (5 mM dNTPs and 15 mM MgCl), 10 pmol of each primer:
AGT1_SP6_Fw: 5‘-ATTTAGGTGACACTATAGATTGATGTCGCATTAACCGGC-3‘
AGT1_M13_Rev: 5’-AACAGCTATGACCATGGCAGTTCTTCAGTCCCCATG-3’
The following cycling conditions may be used:
- 95°C 3 min.
- [30 cycles: 95°C 30 sec.; 56°C 20 sec.; 72°C 20 sec.]
- 72°C 5 min.
Before designing bromeliad specific primers, we also used the canonical Agt1 primers that were used in previous studies [18, 23].
18. Li M, Wunder J, Bissoli G, Scarponi E, Gazzani S, Barbaro E, et al. Development of COS genes as universally amplifiable markers for phylogenetic reconstructions of closely related plant species. Cladistics. 2008.
23. Leme EMC, Heller S, Zizka G, Halbritter H. New circumscription of Cryptanthus and new cryptanthoid genera and subgenera (Bromeliaceae: Bromelioideae) based on neglected morphological traits and molecular phylogeny. Phytotaxa. 2017.
34. Schulte K, Horres R, Zizka G. Molecular phylogeny of Bromelioideae and its implications on biogeography and the evolution of CAM in the family (Poales, Bromeliaceae). Senckengergiana biologica 2005:113–125.
PCR amplification and sequencing of matK can be performed using the primer combination:
MatK 5F 5‘-ATACCCTGTTCTGACCATATTG-3‘
trnK2r 5‘-AACTAGTCGGATGGAGTAG-3‘
Internal sequencing of matK should be done using the primer TOmatK:
480F 5‘-CATCTKGAAATCTTGGTTC-3‘ [34].
Sanger sequencing
Prior to sequencing, PCR reactions need to be cleaned-up by combined treatment with Exonuclease I (New England Biolabs, Ipswich, MA, USA) and Shrimp Alkaline Phosphatase (Thermo Fischer Scientific, Waltham, MA, USA) according to the supplier recommendations.
Sanger Sequencing should be performed using standard sequencing primers:
SP6_Fw 5‘-ATTTAGGTGACACTATAG-3‘
M13_Rev 5‘-AACAGCTATGACCATG-3‘
Plasmid cloning
Agt1 PCR fragment cloning was done using the CloneJET PCR Cloning Kit (Thermo Fischer Scientific, Waltham, MA, USA) according to the supplier recommendations. Four to five plasmids per cloning assay were then sequenced at Eurofins Genomics (Eurofins Scientific, Luxemburg, LU) using universal plasmid sequencing primers M13 forward and SP6 reverse.
