Appendix S1. SNP marker extraction
RNA-extraction
Moss shoots and sporophytes were collected during the end of February 2011. Total RNA
was extracted using the RNeasy Plant Mini Kit (Qiagen) with liquid nitrogen. RNA samples
were treated with RNase-free DNase Set (Qiagen) to remove DNA. To get a high number of
expressed genes, three different samples were pooled for construction of cDNA libraries: 1)
Three individuals from each of three different populations (Arrie ponds, Käglinge ponds and
Gafvelsbjer, all located within a couple of km from each other in south western Scania), being
placed in plastic tubes in a cold greenhouse for 24 hrs before RNA extraction. 2) Three
individuals from three different populations (same as in sample one), being placed in plastic
tubes in room temperature for 24 hrs (surrounded by a plastic bag). 3) Approximately 20
sporophytes from two populations (Arrie ponds and Käglinge ponds), a mixture from the cold
and warm treatment (according to library 1 and 2).
cDNA library preparations and 454 sequencing
Sequencing was conducted at Lund University Sequencing Facility (Faculty of Science, Lund,
Sweden, October 2011). Total RNA from the three different samples were chemically
fragmentized using ZnCl2, then reduced for short fragments by using Agencourt RNA Clean
(Beckman Coulter, Brea, California, USA) and inspected on a RNA 6000 Pico kit on a 2100
Bioanalyzer (Agilent Technologies, Santa Clara, California, USA). Double-stranded cDNA
was constructed using the cDNA Synthesis System Kit (Roche/454 life sciences, Branford,
Connecticut, USA) and by following the 454 cDNA Rapid Library Preparation Manual
(Roche, October 2009, Rev. Jan 2010) including attachment of individual standard MIDs to
each library (sample 1 = C, sample 2 = W and sample 3 = SC; Roche). Libraries were reduced
for short fragments by using Agencourt AMPure XP (Beckman Coulter) and again inspected
using a High Sensitivity DNA kit (Agilent Technologies). cDNA was quantified using the
Quant-iT dsDNA assay kit (Invitrogen, Carlsbad, California, USA) and a Quantiflour
fluorometer (Promega), and pooled in equal molar amounts. The pool was diluted to obtain a
total of 1×107 copies ul‑ 1. Titration and library production (aiming at 10-15 % enrichment)
was performed using emulsion PCR and the Lib-L kit (Roche). DNA positive beads were
enriched, counted on an Innovatis CASY particle counter (Roche), processed using XLR70
sequencing kit (Roche), and loaded onto a picotiter plate for pyrosequencing on a 454 Life
Sciences Genome Sequencer Titanium FLX machine (Roche).
rRNA removal
A BLAST (Altschul et. al. 1990) homology search (using e-value 1e-5) against a custom made
rRNA database (using sequences from SILVA, Quast et al. 2013, and the 5S ribosomal RNA
database, Maciej et al. 2002) revealed that 60 % of the read sequences originated from
ribosomal RNAs. Therefore, a second 454 sequencing run was conducted with RNA from the
same original samples. Prior to this second run, total RNA from the three different samples
was enriched by 2 rounds mRNA purification using the PolyATract kit (Promega, Fitchburg,
Wisconsin, USA) before ZnCl2 fragmentation. A subsequent BLAST homology search
revealed that now only 8 % of the read sequences originated from ribosomal rRNAs.
Transcriptome assembly
After rRNA filtration 594,000 of the originally 1,040,000 reads remained from the two runs.
Sequencing reads were assembled using GS De Novo Assembler (version 2.6, 454 Life
Sciences; often called Newbler). Default settings were used except for that the -cdna option
was added. 387,000 read sequences (65 %) were successfully assembled. The output from
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runAssembly consists of (in Life Science vocabulary) isotigs or transcript variants. All isotigs
with sequence lengths less than 30 nucleotides were removed. Since runAssembly produces
flow based alignments, the read sequences were remapped to their isotig sequences with the
alignment software Exonerate (version 2.2.0, Slate & Birney 2005) using the est2genome
option. After these steps, a final number of 15,040 isotigs representing 369,075 reads (C: 138
013, W: 176 193, SC: 54 869) remained to be analysed. The average isotig length was 995
nucleotides. All isotigs are stored in an annotated database available at http://mioserv2.mbioekol.lu.se/Mosses/.
SNP calling and filtering
In our approach to study population genetics of H. lutescens we have chosen to use only
single nucleotide polymorphisms (SNPs); in other words, insertions or deletions of
nucleotides were deliberately avoided. The SNP calling was performed by a custom made
Perl-program (available on request). In total 170,496 SNP positions were predicted. The SNPs
were filtered using the following criteria: I: The less common allele (out of two alternative
alleles in each SNP position) had a frequency of 19 % or higher. II: The read coverage of the
SNP position was at least 26. III: There was zero (or at most one) adjacent SNP position
within 60 nucleotides upstream or downstream of the targeted SNP position. The filtering
resulted in 125 high quality SNPs.
References
Slater GS, Birney E (2005). Automated generation of heuristics for biological sequence
comparison. BMC Bioinform 6: 31.
Szymanski M, Barciszewska MZ, Erdmann VA, Barciszewski J (2002). 5S ribosomal RNA
database. Nucleic Acids Res 30: 176-178.
Quast C, Pruesse E, Yilmaz P, Gerken J, Schweer T, Yarza P, Peplies J, Glöckner FO (2013).
The SILVA ribosomal RNA gene database project: improved data processing and webbased tools. Nucleic Acids Res 41: D590-D596.
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