Aegilops

The WGS sequence assembly of CDC Bethune (http://www.phytozome.net/flax, NCBI genome project #68161) [8 (link)] was used as reference for mapping of all sequence reads. Reads from all eight genotypes including CDC Bethune were aligned using Bowtie (version 0.12.8) and BWA (version 0.6.1) using default settings. An additional parameter to report only the uniquely mapped reads (m=1) was added in Bowtie. The software package SAMtools was used to convert the sequence alignment files from sequence alignment/map (SAM) to sorted binary alignment/map (BAM). The pileup files containing the SNPs were processed through an updated AGSNP pipeline [61 (link)] to filter SNPs that had a minimum of three occurrences within any accession. The method is outlined in Figure 1.
Sequence coverage expressed as genome equivalents (X) was calculated by dividing the total read length by the estimated size of the flax genome (~370 Mbp) [43 (link),44 (link)]. Mapping coverage percentage (MCP) and mapped read depth (MRD) were used to characterize the sequence coverage and average read mapping depth within a defined interval of the concatenated reference sequence referred to as bins. MCP represents the percentage of the reference sequence (318 Mbp) covered by reads of individual genotypes within a bin size of 0.5 Mbp. Similarly, MRD is the average number of mapped reads per mapped position within a bin size of 0.5 Mbp from the individual genotypes mapped separately onto the reference sequence. The heat maps showing MCP, MRD and SNP density were generated using an in-house program written in Java.
In the original AGSNP pipeline [61 (link)], the average mapped read depth ( $\overline{X}$ ) was used to identify single copy reference sequences and to set a maximum read depth for filtering paralogous genes or repetitive sequences. Average mapping read depth plus 2 times its standard deviation (s), $\overline{X}$ + 2s, was considered to be an optimal cut-off value in the self-pollinating species Aegilops tauschii. We used the same criteria for flax, applying it to each genotype to remove potentially false-positive SNPs due to highly repetitive sequences or mis-mapping. $\overline{X}$ and s were estimated for each genotype based on the mapping results using a pipeline program in the AGSNP package. The SNP filtering criteria are listed in Table 2.
To determine SNP location within genes, we used the gene prediction database available at http://www.phytozome.net/flax that was created using Augustus (version 2.5.5), a Hidden Markov Model-based gene finding program [84 (link)] and Glimmer HMM (version 3.0.1) [85 (link)].

FISH was carried out to study the mitotic chromosomes of root meristems. On the other hand, GISH was used to examine both the mitotic chromosomes of root meristemes and meiotic chromosomes of PMCs. Four probes were subjected to in situ hybridization on the same chromosome preparations. First FISH was made according to Książczyk et al. (2011 (link)) with minor modifications of Kwiatek et al. (2013 (link)), using 25S (used for detection of 25-5.8-18S rDNA loci) and 5S rDNA (pTa794). The hybridization mixture (40 μl per slide) contained 90 ng of each probe in the presence of salmon sperm DNA, 50 % formamide, 2 × SSC, 10 % dextran sulphate, and was denatured at 75 °C for 10 min and stored on ice for 10 min. Chromosomal DNA was denatured in the presence of the hybridization mixture at 75 °C for 5 min and allowed to hybridize overnight at 37 °C. For detection of the hybridization signals, anti-digoxigenin conjugated with FITC (Roche) was used. After documentation of the FISH sites, the slides were washed according to Heslop-Harrison (2000 (link)) (2 × 45 min in 4 × SSC Tween, 2 × 5 min in 2 × SSC, at room temperature).
Second FISH with pSc119.2 and pAs1 (labelled with digoxygenin-11-dUTP and tetramethyl-rhodamine-5-dUTP, respectively) was made with the same conditions after reprobing. After second reprobing, GISH was carried out according to Kwiatek et al. (2012 (link)) with modifications. Multicolour GISH was carried out using U-genome probe (from Ae. umbellulata), S^l-genome probe (from Ae. longissima) and unlabelled triticale genomic DNA which was used as specific blocker. The GISH mixture (40 μL per slide), containing 50 % formamide, 2 × SSC, 10 % dextran sulphate, 90 ng each of the genome probes, and 4.5 μg blocking DNA, was denatured at 75 °C for 10 min and stored on ice for 10 min. In case of initial GISH on triticale ‘Lamberto’ chromosomes, the hybridization mix contained the following: A-genome probe generated from genomic DNA of Triticum monococcum L., R-genome probe (rye, S. cereale L.) and blocking DNA from B-genome (Aegilops speltoides Tausch; 2n = 2x = 14; SS). The chromosomal DNA denaturation, hybridization and immunodetection conditions were the same as above-mentioned. Mitotic and meiotic (MI) cells were examined with an Olympus XM10 CCD camera attached to an Olympus BX 61 automatic epifluorescence microscope. Image processing was carried out using Olympus Cell-F (version 3.1; Olympus Soft Imaging Solutions GmbH: Münster, Germany) imaging software and PaintShop Pro X5 software (version 15.0.0.183; Corel Corporation, Ottawa, Canada). The identification of particular chromosomes were made by comparing the signal pattern of 5S rDNA, 25S rDNA, pSc119.2 and pAs1 probes according previous study (Kwiatek et al. 2013 (link)) and similar cytogenetic analysis (Cuadrado and Jouve 1994 (link); Schneider et al. 2003 (link), 2005 (link); Wiśniewska et al. 2013 (link)). Single-factor analysis of variance and Tukey’s Honest Significant Difference (HSD) test was used to examine the differences of means of chromosome configurations between plants from respective generations and the differences of means of chromosome configurations between plants from BC₂F₁ with comparison to their progeny in BC₂F₂ generation.

The hidden Markov model (HMM) profile of the RCC1 gene family (PF00415) in PFAM (http://pfam.xfam.org/) was downloaded and used to identify the RCC1 genes in the local protein database of bread wheat, emmer wheat (Triticum dicoccoides, 2n = 4x = 28, AABB) and Aegilops tauschii (2n = 2x = 14, DD) (downloaded from Ensembl Plants, http://plants.ensembl.org/index.html) with the hmmsearch tool of HMMER3.1 software (HMMER 3.1; http://hmmer.org/). To avoid missing RCC1 family members, an aligned file of a high-quality protein set (E value < 1 × 10⁻²⁰) in MEGA X software (Kumar et al., 2018 (link)
) was used to reconstruct the new HMM profile, which was used as the query to search all the RCC1 members (E value < 0.01) in all bread wheat, emmer wheat, and Aegilops tauschii proteins, respectively. All the detected protein sequences were submitted to the PFAM (http://pfam.xfam.org/), SMART domain search (http://smart.embl.de/smart/batch.pl) (Letunic et al., 2021 (link)
), and NCBI Batch CD-search database (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi) to confirm the structural integrity of the RCC1 domain (Marchler-Bauer et al., 2005 (link)
). The non-redundant, verified genes encoding proteins with RCC1 domains were assigned as members of the RCC1 gene family.

Chromosome preparation was carried out according to Han et al. (2006 (link), 2009) (link) with minor modifications. In brief, the seed roots with the length of 1-2 cm were collected, pretreated with N₂O under 10 atm pressure for 2 hours and then fixed in 90% acetic acid for 8 min. The root tips were cut and digested with 2% cellulase and 1% pectinase before washed with 75% ethyl alcohol. The root tips were mashed using dissecting needle and diluted with 100% acetic acid. Finally, 10 μL mixture was dropped onto the center of a slide. The slides with good mitotic phases were used in sequential genomic in situ hybridization (GISH) and multi-color fluorescence in situ hybridization (mc-FISH).
The genomic DNA (gDNA) of Th. ponticum was labeled as a probe with Alexa Fluor-488-dUTP (green). While the gDNA of CS was used as a block. The probe and block with ratio of 1:200 were used in GISH analysis. After GISH, two repetitive probes, pAs1 (GenBank Accession number: D30736.1), isolated from Aegilops tauschii Coss. and labeled with Texas-red-5-dCTP (red), pSc119.2 (GenBank Accession number: KF719093), isolated from S. cereale and labeled with Alexa Fluor-488-dUTP (green), were used in the FISH analysis (Rayburn and Gill, 1986 (link); McIntyre et al., 1990 (link)). After hybridization, the slides were washed in 2× Saline Sodium Citrate buffer followed by counterstain with 4, 6-diamidino-2-phenylindole (DAPI). The cells with clear hybridization signals were photographed by a DP80 CCD camera attached to an Olympus BX53 and analyzed using the program CellSens Standard 1.12 (Olympus, Tokyo, Japan).