Development of TEX-QTL mapping strategy
For each pooled tissue sample, genomic DNA was isolated using Qiagen® plant genomic DNA maxi prep kit. To remove polysaccharide contamination, the isolated genomic DNA was re-extracted with the CTAB method (Maniatis et al. 1983). For each sample, 3 µg of genomic DNA was fragmented with a Covaris® S220 Focused-ultrasonicator. The library preparation and probe hybridization were conducted following the protocol of Agilent® SureSelect target enrichment system (2013). Briefly, fragmented genomic DNA was end-repaired with a single adenine nucleotide overhang, and ligated to sequencing adaptors with a single thymine nucleotide overhang. The successfully ligated DNA fragments were amplified by PCR and hybridized with biotin-labeled RNA probes. The RNA/DNA hybrids were isolated by incubation with streptavidin-coated magnetic beads, which bound the biotin. The enriched genomic DNA fragments were released, bar-coded, and sequenced in Illumina’s HiSeq™ with 100 base and paired-end sequencing.