Single nucleotide polymorphism (SNP) genotypes and phenotypes for marker-trait association analysis of three pine species grown in a glasshouse

A common garden glasshouse trial was established in 2010 using seed from natural populations from three species of pine: Pinus sylvestris (SY), Pinus mugo (MU), Pinus uncinata (UN). Open-pollinated seeds were collected from three to five trees per population from twenty-eight natural populations in Europe covering the geographic range of each species. The collection consisted of thirteen populations of SY, nine MU, and six UN. The trial was divided into 25 randomized blocks with up to five families per population, of which the first 18 blocks were analysed by Wachowiak et al. (2018a).

Phenotypes (height, bud set and budburst) were assessed in the glasshouse during spring (budburst), autumn (bud set) and winter (height) between 2010 and 2013.

A subset of trees were selected to be genotyped using a multi-species SNP array. Trees were genotyped using a multi-species SNP array (Perry et al., 2020). DNA was extracted from needles using a Qiagen DNeasy 96 kit following the manufacturer’s instructions. Needles were dried on silica gel prior to extraction and DNA was quantified using a Qubit spectrophotometer to ensure a minimum standardized concentration of 35ng/µl. The quality of genomic DNA was also checked visually for fragmentation on 1 % agarose gel.

Genotyping was done at Edinburgh Genomics following DNA amplification, fragmentation, chip hybridisation, single-base extension through DNA ligation and signal amplification performed according to the Affymetrix Axiom® Assay protocol. Genotyping was performed in 384-well format on a GeneTitan according to the manufacturer’s procedure. Genotype calls were performed using Axiom Analysis Suite software as recommended by the manufacturer (ThermoFisher). Samples were assigned to an analysis group based on their call rate (CR) and dish QC (DQC: a metric provided by ThermoFisher which is generated by measuring signals at multiple sites in the genome known not to vary among individuals), using the following thresholds: DQC ‘high’ ≥ 0.82; DQC ‘low’ < 0.82; CR ‘high’ ≥ 96; CR ‘low’ < 96. Analyses: 1) DQC high + CR high; 2) DQC high + CR low; 3) DQC low + CR low. High quality samples (N=529), with high CR and DQC, were used to set thresholds for allele calls. Posteriors for allele calls were subsequently used as priors for analyses 2 (N = 753) and 3 (N = 251).

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Scots pine single nucleotide polymorphisms (SNP) for Axiom array