BAC Library Clones for Lupinus Genome

Single copy BAC clones from the L. angustifolius nuclear genome BAC library [33 (link)] identified as Lang06-specific in the previous study [17 (link)] were used. Due to the dispersed mapping pattern of 067H16 BAC clone in wild lupins, one additional probe (059F07) specific to Lang06 [34 (link)] was used instead. Moreover, BAC clone 127N17 was also not included in FISH because of its overlapping sequence with the BAC 051D03. The complete list of used BAC clones and their alignment to pseudochromosomes is included in the Supplementary Materials (Supplementary Table S1). DNA isolation from BAC clones was performed using miniprep kits (QIAprep Spin; Qiagen, Hilden, Germany). BAC DNA thus obtained was labeled by nick-translation (Sigma–Aldrich, St. Louis, MI, USA), using either digoxigenin-11-dUTP (Sigma–Aldrich) or tetramethylrhodamine-5-dUTP (Sigma–Aldrich). BAC clone DNA isolation and labeling were done as described by Susek et al. [35 (link)]. To obtain information on localization of BAC clones in the genome assembly, nucleotide sequences of inserts were downloaded from the NCBI database (accession numbers provided in Table S1) and aligned to the L. angustifolius pseudomolecules and/or scaffolds [18 (link)] using Basic Local Alignment Search Tool (BLAST) implemented in Geneious 9.1.8 program (Biomatters, Ltd., Auckland, New Zealand).