High-density linkage mapping and genetic dissection of resistance to broomrape (Orobanche crenata Forsk.) in pea (Pisum sativum L.)

Leaf tissue samples were collected from three individuals of the parental lines and one individual of each RIL. DNA was isolated using the DNeasy Plant Mini Kit (Qiagen) according to the manufacturer’s protocol and checked for quality and concentration using agarose gel (0.8%) electrophoresis and the Qubit 3.0 fluorometer (Life Technologies). A multiplexed ApeKI-GBS library was prepared as described by Elshire et al. (2011) and sequenced by paired-end approach using the Illumina Novoseq 6000 sequencing system (Elshire Group Ltd.). After demultiplexing with the Axe algorithm (Murray and Borevitz, 2018), reads were trimmed for adapter and reverse-barcode sequences using the batch_trim.pl script from github (https://github.com/Lanilen/GBS-PreProcess). Alignment to the Pisum sativum v1.0 reference genome (Kreplak et al. 2019) was performed using bowtie2 (Langmead and Salzberg 2012). After pooling together alignments of the biological replicates of the parental lines, the Stacks pipeline (Catchen et al. 2013) with the biparental filtering mode was used for SNP calling. Further filtering was performed in TASSEL 5.2.31 (Bradbury et al. 2007) by selecting SNP loci showing polymorphism between the parental lines and associated with call rate >90%, minor allele frequency (MAF) >0.25 and heterozygous calls