Genomic DNA was extracted from needles using a modified protocol from Dellaporta et al.  for all marker analyses, but for the 1536 and 384 GoldenGate assays (Illumina Inc., San Diego, CA, USA), for which a commercial Invisorb DNA plants HTS
96kit (Invitek GmbH, Berlin, Germany) was used. Four types of molecular marker were used for genotyping the mapping populations: nSSRs, EST-Ps, SAMPLs and SNPs.
nSSRs: Forty seven primer pairs designed for amplification of nSSR loci in P. pinaster and P. taeda[60, 66, 67] were tested for segregation in the mapping populations. Thirteen loci were polymorphic, 27 were monomorphic, and seven resulted in muti-banding or non-clear patterns. Amplification of A6F03, A5B01, A5A11, A6F10, A6D04, A5B07 loci was performed as in Guevara et al. . Amplification of NZPR823, NZPR413,NZPR114, NZPR544, SsrPt_ctg64, SsrPt_ctg275 loci was performed as described by Chagné et al.  and the amplification of PtTX3116 followed the protocol described by Auckland et al.  with modified touchdown profile, using 55°C and 45°C as starting and final temperatures . A Perkin-Elmer GenAmp 9700 thermal cycler (Perkin Elmer Inc., Waltham, Massachusetts, USA) was used to carry out PCR reactions. Amplified products were separated in denaturing gels containing 6% acrylamide / bisacrylamide (19:3), 7 M urea and 1x TBE. Amplified products were visualized in a DNA Analyzer System (4300, LI-COR Biosciences, Lincoln, NE, USA). Fragments were scored visually as codominant markers.
EST-Ps: EST-P genotyping was carried out by Tilling (Targeting Induced Local Lesions in Genomes) as described by Till et al. . This technique allows detection of multiple SNP sites heterozygous in the same progenitor . A set of 14 EST-P primer pairs (PtIFG_893, PtIFG_9136, PtIFG_9034, PtIFG_1955, PtIFG_8429, PtIFG_8702, PtIFG_3C8E, PtIFG_22B8, PtIFG_1CA6C, PtIFG_9044, PtIFG_2253, PtIFG_8436, PtIFG_8887, PtIFG_C6H11) derived from cDNA sequences of P. taeda and P. pinaster[61, 72, 73] were tested, in order to identify the most informative markers. A total of 11 EST-P primer pairs generated 25 polymorphic markers. PCRs were performed in 10 μl containing 10 ng of DNA; 1x PCR reaction buffer (Fermentas, Ontario, Canada), 0.2 mM of each dNTP, 2 mM MgSO4, 0.25U Pfu DNA polymerase (Fermentas, Ontario, Canada), 0.2 μM of each primer (forward primers were labeled on its 5’ end with IRDye 700 and reverse primers with IRDye 800). A Perkin-Elmer GenAmp 9700 thermal cycler (Perkin Elmer Inc., Waltham, Massachusetts, USA) was used to carry out PCR reactions. Thermocycler parameters were: 94°C 2 min, 10 touchdown cycles of 94°C 20s, (Tm + 3)°C, 45 s (−0.8°C/cycle), 72°C 1 min; 45 cycles of 94°C 20s, (Tm-5)°C 45 s, 72°C 1 min and final extension step of 72°C for 7 min. Amplification products were visualized on 1% agarose gels to verify amplification. PCR products were digested with CEL I nuclease purified as described by Till et al. . Previously, the concentration of nuclease added, was screened to optimize the detection of heteroduplex between heterozygous sites. Partial DNA digestions were stopped by the addition of 5 μl of 0.5 M EDTA. The mixture were transferred to 96-well Sephadex G50 spin plates (GE HeathCare, Waukesha, WI, USA) for cleaning up by centrifugation into formamide solution and heated at 70°C to reduce the volume to 8 μl. DNA fragments were separated in denaturing gels containing 8% Long Ranger polyacrylamide (Cambrex, East Rutherford, NJ, USA), 7 M urea and 1x TBE. Fragments detection was carried out on a DNA Analyzer System (4300, LI-COR Biosciences, Lincoln, NE, USA). Fragments were scored as dominant markers. Polymorphism was inferred from the resulting fragment pattern and confirmed by sequencing independently undigested amplified products from four haploid megagametophyte DNAs for each progenitor.
SAMPLs: SAMPL genotyping was performed as indicated by Vos et al.  with several modifications . Preamplifications were carried out using three primer combinations (EcoRI + A/ MseI + G; EcoRI + A/ MseI + C; EcoRI + A/ MseI + T). For the selective amplification a SAMPL primer [CATA: (CA)8(TA)2; GATA: (GA)8(TA)2], was used in combination with an EcoRI + 3 primer. In order to select the most informative combinations (those with a higher level of polymorphism) different combinations were tested using template DNA from the parental lines and 9 offspring. Progenitor C14 revealed lower levels of polymorphisms than C15 (see Results section), thus primer combinations were chosen in order to equilibrate the number of markers segregating from each progenitor. A total of 31 CATA/EcoRI and 26 GATA/EcoRI primer combinations were used for the selective amplification. Selective PCR reaction were performed in 10 μl of 1x PCR Buffer (10 mM Tris–HCl, 50 mM KCl, pH 8.3), 0.1 mM of each dNTP, 2.5 mM MgCl2 (Roche, Basel, Switzerland), 3 ng IRDye 800 5’end labeled CATA or GATA primers, 15 ng EcoRI + 3 primer, 0.2U Taq DNA polymerase (Invitrogen, Grand Island, NY, USA) and 5 μl of 10-fold diluted pre-amplification DNA fragments using classical AFLP cycling parameters . Samples were loaded into denaturing gels containing 8% Long Ranger polyacrylamide (Cambrex, East Rutherford, NJ, USA), 7 M urea and 1x TBE. Fragments detection was carried out on a DNA Analyzer System (4300, LI-COR Biosciences, Lincoln, NE, USA). Fragments were scored visually as dominant markers.
SNPs: two SNP genotyping assays were used in this study; a 1,536 BeadArray™ and a 384 BeadXpress® Golden Gate assays (Illumina Inc., San Diego, CA, USA). SNPs selected for 1,536 Golden Gate assay corresponded to three different sets (see Chancerel et al.  for further details): in vitro polymorphisms from 35 candidate genes for cell wall formation and drought stress resistance; in silico SNPs from a maritime pine EST assembly; and in silico polymorphism from re-sequenced amplicons of the species. In this genotyping assay, 95 DNA samples of the mapping progenies were genotyped (73 for C14xC15 and 22 for C15xC14). In order to increase the number of genotyped individuals for a set of genes of interest, another genotyping assay was developed. This genotyping assay (384 SNPlex) consisted in a subsample of SNPs selected from the 1,536 genotyping assay and 14 additional SNPs from candidate genes for drought resistance . It was carried out at Center for Genomic Regulation (CRG, Barcelona, Spain) for a total of 119 DNA samples (79 for C14x15 and 40 for C15xC14). Both genotyping assays were realized according to the manufacturer’s instructions (Illumina Inc., San Diego, CA, USA) and SNPs clusters revised manually with Illumina Bead Studio v2.0 Software. When the same SNP was successfully genotyped in both assays priority was given for the 384 Vera Code data because of the higher number of DNA samples genotyped in this assay. Contig and gene sequences containing the polymorphic SNPs are presented in Additional file 1.