## 3 Materials and Methods  
In Silico Analysis of B. napus, B. oleraca, B. rapa, C. sativa, and R. sativus RSH Genes and Proteins  
The _RSH1_, _RSH2_, _RSH3_, and _CRSH_ in the plant genomes selected for this study from _Brassicaceae_ family genes have been identified using _A. thaliana_ _RSH_ cDNA sequences (_AtRSH1_, _AtRSH2_, _AtRSH3_, and _AtCRSH_) as queries. A search was performed using BLASTN (Basic Local Alignment Search Tool) using the NCBI (ncbi.nlm.nih.gov, accessed on 10 May 2021) nucleotide database. The analysis of the intron-exon organisation was carried out using the CIWOG tool ([[http://peroxibase.toulouse.inra.fr/tools/ciwog_search_form](http://peroxibase.toulouse.inra.fr/tools/ciwog_search_form)]([http://peroxibase.toulouse.inra.fr/tools/ciwog_search_form](http://peroxibase.toulouse.inra.fr/tools/ciwog_search_form)), accessed on 15 May 2021) [74]. The putative amino acid sequences were then obtained from the NCBI protein database. For primary and secondary structure predictions of RSHproteins InterProScan ([[https://www.ebi.ac.uk/interpro/search/sequence/](https://www.ebi.ac.uk/interpro/search/sequence/)]([https://www.ebi.ac.uk/interpro/search/sequence/](https://www.ebi.ac.uk/interpro/search/sequence/)), accessed on 23 May 2021), Conserved Domain Search ([[https://www.ncbi.nlm.nih.gov/Structure/cdd/wrrpsb.cgi](https://www.ncbi.nlm.nih.gov/Structure/cdd/wrrpsb.cgi)]([https://www.ncbi.nlm.nih.gov/Structure/cdd/wrrpsb.cgi](https://www.ncbi.nlm.nih.gov/Structure/cdd/wrrpsb.cgi)), accessed on 23 May 2021), and PSIPRED ([[http://bioinf.cs.ucl.ac.uk/psipred/](http://bioinf.cs.ucl.ac.uk/psipred/)]([http://bioinf.cs.ucl.ac.uk/psipred/](http://bioinf.cs.ucl.ac.uk/psipred/)), accessed on 24 May 2021) were utilized. Clustal Omega was used for multiple sequence alignments ([[http://www.clustal.org/omega/](http://www.clustal.org/omega/)]([http://www.clustal.org/omega/](http://www.clustal.org/omega/)), accessed on 27 May 2021) [75]. For calculation of molecular mass and pI of putative RSH proteins the Compute pI/Mw tool ([[https://web.expasy.org/compute_pi/](https://web.expasy.org/compute_pi/)]([https://web.expasy.org/compute_pi/](https://web.expasy.org/compute_pi/)), accessed on 13 June 2021) was utilised. TargetP ([[http://www.cbs.dtu.dk/services/TargetP/](http://www.cbs.dtu.dk/services/TargetP/)]([http://www.cbs.dtu.dk/services/TargetP/](http://www.cbs.dtu.dk/services/TargetP/)), accessed on 14 June 2021) [76; 77] was used to predict subcellular localization of analysed RSH proteins. The phylogenetic analysis was carried out in MEGA7 software [78; 79] using the neighbour-joining method [80].  
The promoter regions of _ButRSH_ genes were analysed using the PlantCARE database ([[http://bioinformatics.psb.ugent.be/webtools/plantcare/html/](http://bioinformatics.psb.ugent.be/webtools/plantcare/html/)]([http://bioinformatics.psb.ugent.be/webtools/plantcare/html/](http://bioinformatics.psb.ugent.be/webtools/plantcare/html/)), accessed on 10 May 2021) [59]. For each _BnRSH_ gene a 1500-bp long fragment including promoter and 5'UTR of genomic DNA was retrieved from the NCBI GenBank ([[http://www.ncbi.nlm.nih.gov/genbank/](http://www.ncbi.nlm.nih.gov/genbank/)]([http://www.ncbi.nlm.nih.gov/genbank/](http://www.ncbi.nlm.nih.gov/genbank/)), accessed on 5 May 2021).