Computational protocol: A Polynucleotide Repeat Expansion Causing Temperature-Sensitivity Persists in Wild Irish Accessions of Arabidopsis thaliana

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Protocol publication

[…] The Bur-0 × Col-0 recombinant inbred lines (RILs) have been described previously (Simon et al., ). The collection of wild accessions from Ireland is described below. Plants were grown either in long days (LD, 16 h light/8 h dark) at 23°C for seed collection and in short days (SD, 8 h light/16 h dark) at 23°C or 27°C for iil phenotyping, QTL analysis and temperature assays as described previously (Sanchez-Bermejo et al., ). For Diversity Arrays Technology sequencing (DArT-seq), DNA from the plant from which the seeds were bulked was extracted as described previously (Balasubramanian et al., ). GAA/TTC repeat expansion was analyzed through Polymerase chain reaction (PCR) using primers described in Table . For sequencing the microsatellite repeats, purified PCR products were sequenced (Micromon, Monash University) and analyzed through Seqman (DNAStar-Lasergene). [...] To analyze germination and flowering time, six bulked seeds per accession were sown in a fully randomized design in individually numbered positions of multiple-well soil flats. For phenotyping, eight plants of each of the 344 RILs were grown in a growth room at 27°C in short days. Plants were grown as single individuals in 30-well trays in a completely randomized design as described previously (Lempe et al., ). The trays were repositioned systematically on a weekly basis to minimize the effect of micro-environmental variation. The iil phenotype was first assessed against the parental Bur-0 line. We first photographed all the variant phenotypes and generated a scale of 1–10 (Figure ). Using this scale, every plant was scored and a quantitative score for the iil phenotype was obtained (Table ). Flowering time was measured as Days To Flower (DTF), as described previously (Sanchez-Bermejo et al., ). QTL mapping was done using R/qtl package through simple interval mapping or two-dimensional scans (Broman et al., ). [...] Population structure was analyzed using the model-based Bayesian clustering algorithm implemented by STRUCTURE v.2.3.4 (Pritchard et al., ) and the kinship procedure of Hardy (Hardy, ) using SPAGeDi (Hardy and Vekemans, ), respectively. STRUCTURE analyses were performed assuming an admixture model with default settings (no informative priors were used). STRUCTURE was run from 1 to 10 inferred clusters (K) with 4 independent runs for each K, each run starting with a burn-in period of 100,000 steps followed by 100,000 Markov Chain Monte Carlo iterations. The most probable value of K was selected according to the Evanno method (Evanno et al., ), implemented through the Structure Harvester web tool (Earl and vonHoldt, ). To assess population structure at the chromosomal level we calculated genetic distances to construct genome-wide and chromosome unrooted neighbor-joining trees from maximum-likelihood distances inferred with a TN93 substitution model constructed for all using Perl scripts and the package Ape v3.3 (Paradis et al., ) in RStudio v0.00.467 for 4556 uniquely mapping non-duplicate sites. […]

Pipeline specifications

Software tools DNASTAR Lasergene, R/qtl
Applications WGS analysis, Sanger sequencing
Organisms Homo sapiens, Arabidopsis thaliana
Diseases Friedreich Ataxia, Huntington Disease, Genetic Diseases, Inborn