The genus Orthotrichum is a widespread moss group, which includes approximately 159 species [18], and is the second largest genera in the family Orthotrichaceae. Taxa belonging to this genus are found throughout the world from the Arctic to the Antarctic, except in deserts and wet tropical forests. Species of the genus Orthotrichum grow on trees and rocks to an elevation of ca. 5000 m above sea level [19]. The subdivision within this genus has been a matter of a continuing debate since the end of the 19th century. Certain taxa have been alternately included in and excluded from the genus Orthotrichum in the attempt to divide it into lower taxonomic units, subgenera and sections. The basis for the classification of the genus Orthotrichum in a historical perspective has been described in detail by Lewinsky [19] and Lewinsky-Haapasaari and Hedenäs [20].

Orthotrichum species have a wide geographical range and are usually characterized by high genetic diversity, in some cases pointing to the occurrence of cryptic species [17,18]. Several widespread species, including O. speciosum of the subgenus Gymnoporus, showed a very low level of genetic variation in the analyzed regions. O. speciosum is a common representative of the genus, found across the entire Holarctic ecozone. The species is well defined morphologically [19,21] and genetically [17], and it is characterized by a low level of genetic variation in nuclear and chloroplast sequences [17,22], which is why it has been selected as a model species for the present study. The developed markers will also be used in ecological studies, to replace less polymorphic ones. A population of O. speciosum from the Czech Republic was used for DNA isolation (Kouty nad Desnou, Hruby Jesenik Mts).

Total genomic DNA was extracted from 30 fresh stems. The stems were ground with silica beads in a MiniBead-Beater tissue disruptor for 50 seconds, and were subsequently processed using the DNeasy^® Plant Mini Kit (Qiagen, Hilden, Germany) following the manufacturer’s protocol. DNA quantity was estimated with the Qubit fluorometer system (Invitrogen, Carlsbad, NM, USA), using the Quant-IT ds-DNA BR Assay kit (Invitrogen).

Eight hundred nanograms of DNA was sheared by nebulization, purified with the MinElute PCR Purification Kit (Qiagen), and subsequently processed according to the GS Rapid Library Preparation Kit Method Manual (Roche/454 Life Sciences). The quality of DNA library was assessed by gel electrophoresis in the FlashGel System (Lonza). DNA fragments were clonally amplified using the GS Junior Titanium emPCR Lib-L Kit (Roche/454 Life Sciences). Sequencing was performed using the GS Junior pyrosequencing system according to the Sequencing Method Manual (Roche/454 Life Sciences).

Pyrosequencing data were assembled using GS Reference Mapper software (Roche/454 Life Sciences). A two-step assembly was performed. First, the obtained sequences were assembled using the chloroplast genome data of Syntrichia ruralis (GenBank: NC_012052.1) to separate chloroplast reads from nuclear reads. Syntrichia ruralis is one of the two moss species with sequenced cpDNA genomes and is closer related to Orthotrichum than the Physcomitriella patens. The remaining reads were assembled using the GS Newbler de novo assembler (Roche/454 Life Sciences).

The obtained contigs were searched for microsatellite motifs using msatcommander with default settings [23]. This program was also used for primer design. To avoid designing primers for any potential SSR locus twice, the contigs containing the same motif were compared in Bioedit 7.0.5 [24].

We tested PCR amplification and the level of polymorphism of the designed primer pairs. The sequences used in genotyping test were deposited in GenBank (accession numbers from JX154169 to JX154203). The polymorphism of SSR markers was tested in two O. speciosum populations of 20 specimens each, and in the material used in our previous studies [17,22]. The cross-species amplification of SSR loci was tested in both closely related O. affine and O. striatum, and more phylogenetically distant O. diaphanum and O. pallens [25,26].

SSR-PCR reactions were performed in 20 μL of a reaction mixture containing 40 ng genomic DNA, 1.0 μM of each primer, 1.5 mM MgCl₂, 200 μL M dNTP (dATP, dGTP, dCTP, dTTP), 1× PCR buffer, 1 μL BSA and 1 U Genomic RedTaq polymerase (Sigma, St. Louis, USA). SSR marker reactions were performed under the following thermal conditions: (1) initial denaturation—5 min at a temperature of 94 °C; (2) denaturation—1 min at 94 °C; (3) annealing—1 min at 53 °C; (4) elongation—1 min at 72 °C, final elongation7 min at 72 °C. Stages 2–4 were repeated 34 times. The products of the PCR reaction were separated on the QIAxcel capillary electrophoresis system, which is a cost-effective system suitable for SSR marker electrophoresis [27]. Electrophoresis was performed using the Qiaxcel High Resolution Kit with the alignment marker 15–500 bp (Qiagen) and the DNA size marker 25–500 bp (Qiagen). Standard OM700 settings were used as the electrophoresis program.

To check consistency of designed primers, randomly selected 24 amplicons were resequenced using amplification primers. Purified PCR products were sequenced in both directions using the ABI BigDye 1.1 Terminator Cycle Kit (Applied Biosystems, Foster City, USA), and were visualized using an ABI Prism 3130 Automated DNA Sequencer (Applied Biosystems).

An analysis of genetic diversity was performed using PopGen 1.32 software [28]. The linkage disequilibrium (LD) was tested using FSTAT v.2.9.3 [29].