title: BsRADseq: screening DNA methylation in natural populations of non-model species.
abstract: Epigenetic modifications are expected to occur at a much faster rate than genetic mutations, potentially causing isolated populations to stochastically drift apart, or if they are subjected to different selective regimes, to directionally diverge. A high level of genome-wide epigenetic divergence between individuals occupying distinct habitats is therefore predicted. Here, we introduce bisulfite-converted restriction site associated DNA sequencing (bsRADseq), an approach to quantify the level of DNA methylation differentiation across multiple individuals. This reduced representation method is flexible in the extent of DNA sequence interrogated. We showcase its applicability in three natural systems, each comprising individuals adapted to divergent environments: a diploid plant (Heliosperma, Caryophyllaceae), a tetraploid plant (Dactylorhiza, Orchidaceae) and an animal (Gasterosteusaculeatus, Gasterosteidae). We present a robust bioinformatic pipeline, combining tools for RAD locus assembly, SNP calling, bisulfite-converted read mapping and DNA methylation calling to analyse bsRADseq data with or without a reference genome. Importantly, our approach accurately distinguishes between SNPs and methylation polymorphism (SMPs). Although DNA methylation frequency between different positions of a genome varies widely, we find a surprisingly high consistency in the methylation profile between individuals thriving in divergent ecological conditions, particularly in Heliosperma. This constitutive stability points to significant molecular or developmental constraints acting on DNA methylation variation. Altogether, by combining the flexibility of RADseq with the accuracy of bisulfite sequencing in quantifying DNA methylation, the bsRADseq methodology and our bioinformatic pipeline open up the opportunity for genome-wide epigenetic investigations of evolutionary and ecological relevance in non-model species, independent of their genomic features.
authors.affiliation: Department of Botany and Biodiversity Research, University of Vienna, Rennweg 14, 1030, Vienna, Austria.|Department of Biosciences, Centre for Ecological and Evolutionary Synthesis, University of Oslo, PO Box 1066 Blindern, N-0316, Oslo, Norway.
authors.forename: Emiliano
authors.initials: E
authors.lastname: Trucchi
authors.affiliation: Department of Biosciences, Centre for Ecological and Evolutionary Synthesis, University of Oslo, PO Box 1066 Blindern, N-0316, Oslo, Norway.
authors.forename: Anna B
authors.initials: AB
authors.lastname: Mazzarella
authors.affiliation: Department of Medical Genetics, Oslo University Hospital, PO Box 4590 Nydalen, N-0420, Oslo, Norway.
authors.forename: Gregor D
authors.initials: GD
authors.lastname: Gilfillan
authors.affiliation: Department of Botany and Biodiversity Research, University of Vienna, Rennweg 14, 1030, Vienna, Austria.
authors.forename: Maria T
authors.initials: MT
authors.lastname: Lorenzo
authors.affiliation: Institute of Botany, University of Innsbruck, Sternwartestrasse 15, A-6020, Innsbruck, Austria.
authors.forename: Peter
authors.initials: P
authors.lastname: Schönswetter
authors.affiliation: Department of Botany and Biodiversity Research, University of Vienna, Rennweg 14, 1030, Vienna, Austria.
authors.forename: Ovidiu
authors.initials: O
authors.lastname: Paun
mesh_terms: D000818:Animals; D029748:Caryophyllaceae / Q000145:classification / Q000235:genetics*; D019295:Computational Biology; D019175:DNA Methylation; D044127:Epigenesis, Genetic; D005828:Genetics, Population; D016678:Genome; D029595:Orchidaceae / Q000145:classification / Q000235:genetics*; D020641:Polymorphism, Single Nucleotide; D017422:Sequence Analysis, DNA; D023701:Smegmamorpha / Q000235:genetics*
keywords: DNA methylation; RADseq; bisulfite sequencing; bsRADseq; population epigenetics
references.citation: New Phytol. 2005 Oct;168(1):81-91
references.pmid: 16159323
references.citation: PLoS Biol. 2004 May;2(5):E109
references.pmid: 15069472
references.citation: Nucleic Acids Res. 2012 Feb;40(4):e32
references.pmid: 22156374
references.citation: PLoS One. 2010 Apr 26;5(4):e10326
references.pmid: 20436669
references.citation: Curr Opin Genet Dev. 2008 Apr;18(2):221-6
references.pmid: 18337082
references.citation: PLoS One. 2012;7(9):e41143
references.pmid: 22984395
references.citation: Nat Rev Genet. 2012 Jan 04;13(2):97-109
references.pmid: 22215131
references.citation: Nat Methods. 2012 Mar 04;9(4):357-9
references.pmid: 22388286
references.citation: Proc Natl Acad Sci U S A. 2010 May 11;107(19):8689-94
references.pmid: 20395551
references.citation: Epigenetics. 2010 Jan 1;5(1):47-9
references.pmid: 20081358
references.citation: Mol Biol Evol. 2010 Nov;27(11):2465-73
references.pmid: 20551043
references.citation: Nature. 2010 Jul 15;466(7304):388-92
references.pmid: 20512117
references.citation: Trends Ecol Evol. 2004 Sep;19(9):456-9
references.pmid: 16701305
references.citation: Genet Res Int. 2012;2012:698421
references.pmid: 22567398
references.citation: BMC Genomics. 2012 Jul 02;13:300
references.pmid: 22747568
references.citation: Nat Commun. 2013;4:2875
references.pmid: 24285012
references.citation: PLoS One. 2008;3(10):e3376
references.pmid: 18852878
references.citation: Proc Natl Acad Sci U S A. 2012 Aug 7;109(32):E2183-91
references.pmid: 22733782
references.citation: Bioinformatics. 2011 Jun 1;27(11):1571-2
references.pmid: 21493656
references.citation: Science. 2001 Aug 10;293(5532):1068-70
references.pmid: 11498573
references.citation: Elife. 2015 May 05;4:e05255
references.pmid: 25939354
references.citation: Genetics. 2010 Apr;184(4):1037-50
references.pmid: 20100941
references.citation: Mol Ecol. 2015 Sep;24(17):4406-18
references.pmid: 26206253
references.citation: Nat Rev Genet. 2010 Mar;11(3):191-203
references.pmid: 20125086
references.citation: Nat Rev Genet. 2010 Mar;11(3):204-20
references.pmid: 20142834
references.citation: Genome Res. 2009 Jun;19(6):959-66
references.pmid: 19273618
references.citation: Mol Ecol Resour. 2012 Mar;12(2):333-43
references.pmid: 21999839
references.citation: Mol Phylogenet Evol. 2007 Apr;43(1):140-55
references.pmid: 17188521
references.citation: Science. 2014 Mar 7;343(6175):1145-8
references.pmid: 24505129
references.citation: Nature. 1999 Sep 9;401(6749):157-61
references.pmid: 10490023
references.citation: Cell. 2013 Mar 28;153(1):193-205
references.pmid: 23540698
references.citation: Nature. 2008 Mar 13;452(7184):215-9
references.pmid: 18278030
references.citation: Genome Biol Evol. 2014 Dec 23;7(1):154-71
references.pmid: 25539727
references.citation: Am J Bot. 2014 Aug;101(8):1309-13
references.pmid: 25143467
references.citation: Methods Mol Biol. 2011;772:157-78
references.pmid: 22065437
references.citation: Cell. 2008 May 2;133(3):523-36
references.pmid: 18423832
references.citation: Curr Opin Plant Biol. 2011 Apr;14(2):130-6
references.pmid: 21470901
references.citation: Proc Natl Acad Sci U S A. 2003 Aug 5;100(16):9440-5
references.pmid: 12883005
references.citation: Science. 2010 Oct 29;330(6004):622-7
references.pmid: 21030646
references.citation: Mol Ecol. 2010 Apr;19(7):1283-95
references.pmid: 20298470
references.citation: Proc Natl Acad Sci U S A. 1992 Mar 1;89(5):1827-31
references.pmid: 1542678
references.citation: Nature. 2012 Apr 04;484(7392):55-61
references.pmid: 22481358
references.citation: Nat Rev Genet. 2008 Jun;9(6):465-76
references.pmid: 18463664
references.citation: New Phytol. 2010 Mar;185(4):1108-18
references.pmid: 20003072
references.citation: Mol Genet Genomics. 2012 Aug;287(8):643-50
references.pmid: 22773086
references.citation: PLoS Genet. 2009 Jun;5(6):e1000530
references.pmid: 19557164
references.citation: Nucleic Acids Res. 2005 Oct 13;33(18):5868-77
references.pmid: 16224102
references.citation: Elife. 2015 Jan 28;4:e05290
references.pmid: 25629660
references.citation: Nature. 2015 Jul 9;523(7559):212-6
references.pmid: 26030523
references.citation: Cell. 2013 Jan 17;152(1-2):352-64
references.pmid: 23313553
references.citation: Nat Rev Genet. 2012 Jan 31;13(3):153-62
references.pmid: 22290458
references.citation: Mol Ecol. 2015 Aug;24(15):3823-30
references.pmid: 25951436
references.citation: Mol Ecol. 2012 Jun;21(12):2852-62
references.pmid: 22384978
references.citation: BMC Evol Biol. 2011 Apr 26;11:113
references.pmid: 21521507
references.citation: Nucleic Acids Res. 2015 Apr 30;43(8):3911-21
references.pmid: 25813042
references.citation: Mol Ecol. 2013 Jun;22(11):3124-40
references.pmid: 23701397
references.citation: Genes Dev. 2011 May 15;25(10):1010-22
references.pmid: 21576262
references.citation: Q Rev Biol. 2009 Jun;84(2):131-76
references.pmid: 19606595
references.citation: Science. 2005 Mar 25;307(5717):1928-33
references.pmid: 15790847
references.citation: Cytogenet Genome Res. 2013;140(2-4):270-85
references.pmid: 23751292
references.citation: Genome Biol. 2009;10(3):R25
references.pmid: 19261174
references.citation: Nature. 2011 Sep 20;480(7376):245-9
references.pmid: 22057020
references.citation: PLoS Genet. 2015 Jan 08;11(1):e1004920
references.pmid: 25569172
references.citation: Brief Funct Genomics. 2010 Dec;9(5-6):416-23
references.pmid: 21266344
references.citation: Nat Genet. 2006 Aug;38(8):948-52
references.pmid: 16832354
references.citation: Ecol Lett. 2008 Feb;11(2):106-15
references.pmid: 18021243
references.citation: Mol Genet Genomics. 2002 Dec;268(4):510-7
references.pmid: 12471448
references.citation: Nature. 2009 Nov 19;462(7271):315-22
references.pmid: 19829295
references.citation: Syst Biol. 2009 Jun;58(3):328-45
references.pmid: 20525587
references.citation: Nat Genet. 2005 Aug;37(8):853-62
references.pmid: 16007088
references.citation: Nat Struct Mol Biol. 2014 Jan;21(1):64-72
references.pmid: 24336224
references.citation: Biochem Soc Trans. 2013 Jun;41(3):727-40
references.pmid: 23697932
references.citation: PLoS Genet. 2013 Apr;9(4):e1003437
references.pmid: 23593031
references.citation: Integr Comp Biol. 2013 Aug;53(2):340-50
references.pmid: 23583961
references.citation: Genome Res. 2013 Sep;23(9):1363-72
references.pmid: 23908385
references.citation: Mol Biol Evol. 2015 Apr;32(4):888-95
references.pmid: 25534027
references.citation: Nat Genet. 2003 Mar;33 Suppl:245-54
references.pmid: 12610534
references.citation: Mol Ecol. 2014 Oct;23(20):4926-38
references.pmid: 25208110
references.citation: Mol Ecol. 2011 Apr;20(8):1572-4
references.pmid: 21612021
references.citation: Nature. 2013 Mar 14;495(7440):193-8
references.pmid: 23467092
references.citation: Mol Ecol. 2014 Jul;23(14):3523-37
references.pmid: 24943730
references.citation: Cold Spring Harb Perspect Biol. 2013 Dec 01;5(12):a017764
references.pmid: 24296171