*2.5. Plant Material*

A total of 107 forms and cultivars of spring triticale were used, including selection forms obtained at the Department of Genetics, Russian State Agrarian University, and accessions from the VIR collection (Supplementary Table S1).

### *2.6. DNA Isolation and PCR Analysis*

PCR amplification was conducted using specific primers (Table 1) designed using Primer 3.0 software (http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi/, [30]). The PCR conditions for custom primers were 94 ◦C for 1 min, 35 cycles: 94 ◦C for 1 min; 58 ◦C for 1 min; 72 ◦C for 1 min; and final elongation: 72 ◦C for 3 min. For NWP primers [11], the annealing temperature was 55 ◦C.


**Table 1.** Primers designed in this study and the expected length of the PCR product.

For verification, PCR products of three selected lines were purified and sequenced using ABI 3130×l Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's instructions.
