Co-segregation analysis

For verification of genetic linkage and estimation of RF values between the sym11 gene and other genetic loci, we conducted a co-segregation analysis on both the mapping populations F₂(NGB1238 × N24) and F₂(SGE × N24). Genomic DNA was individually extracted from frozen leaves of F₂-plants using CTAB buffer. Based on the identified SNVs between an outcross line Sparkle (N24) and NGB1238 in pea transcripts (see Table 1) we developed CAPS and dCAPS markers applicable for genotyping (see Table 2). PCR primers were designed using the sequences of the transcripts from PNTA and similar pea transcriptome sequences publically available in the Nucleotide Collection of the NCBI database (https://www.ncbi.nlm.nih.gov/nuccore/), with the help of the online tool Primer-BLAST (Ye et al., 2012). The exon-intron structures of the pea transcripts were modeled by aligning their sequences with the sequences of orthologous genes of Medicago truncatula Gaertn. (version Mt4.0, http://www.phytozome.org) and were taken into account for primer design. The restriction endonucleases were selected by screening the polymorphic sequences by an in-house script. Fragments were amplified using the ready-to-use solution for PCR ScreenMix-HS (Evrogen, Moscow, Russia) and then digested by the selected restriction endonuclease: MboII, FastDigest Tru1I (Thermo Fisher Scientific, Waltham, MA, USA), BtsCI, Hpy166II (New England Biolabs, Ipswich, MA, USA), BssECI (SibEnzyme, Novosibirsk, Russia). Digestion patterns were analyzed with use of the microchip electrophoresis system MultiNA (Shimadzu, Kyoto, Japan).

Recombination rates between the gene-based markers and sym11 were calculated with the F2breed program (Zhernakov et al., 2017). Also, using the F2breed all genes were arranged into a linear genetic map.

For sequencing the candidate gene Sym33 the genome region including 99% of the pea IPD3 (GenBank: EF569222.1) open reading frame was PCR-amplified with Encyclo polymerase (Evrogen, Moscow, Russia). The target sequences for primer annealing used for amplification and sequencing are framed in Fig. S2.