Computational protocol: From QTL to candidate gene: Genetical genomics of simple and complex traits in potato using a pooling strategy

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Protocol publication

[…] Gene specific primers for candidate genes were designed using the beacon designer™. cDNA synthesis of genotypes and RNA bulks was carried out using iScript One-Step RT-PCR kit (Bio-Rad). qRT-PCR using SYBR green was carried out in duplicates on the iCycler (Bio-Rad) and quantified with the Bio-Rad iQ5 optical system software. As a reference a eukaryotic translation initiation factor 3E-like gene from potato was used. All primer sequences are listed in Additional file . [...] Based on an earlier version of the C × E genetic map [], a simplified map was made using mapping software Joinmap 4.0® [], based on 94 individuals with few additional markers (Kumari, unpublished results). Genetic markers for candidate genes targeted for mapping were either PCR-based (StTLRP [gb|GU233535], micro.17361.c1) http://pgrc.ipk-gatersleben.de/poci or CAPS-based (bch [gb|GU233534]), and integrated using the same linkage mapping software. PCR primers and conditions and the restriction enzymes used for marker scoring, are listed in Additional file .QTL analysis of quantitative data was performed using the software package MapQTL® Version 5.0 []. The phenotypic data were tested for normality. QTL analysis was initially done using the interval mapping method []. Detection of a significant QTL was done using a genome-wide LOD threshold calculated with the permutation test option provided in the program. Gene expression data obtained with qRT-PCR were normalized against the reference gene and the relative expression levels were treated as a quantitative trait. […]

Pipeline specifications

Software tools Beacon Designer, JoinMap, MapQTL
Applications WGS analysis, qPCR
Organisms Solanum tuberosum
Chemicals beta Carotene