Sequence of ScGAMYB
The 769 bp fragment of ScGAMYB amplified from DNA of Ot1–3 and 541 parental lines showed 95–97% identity with orthologous genes of wheat and barley deposited in NCBI database (Table 1).The E value between rye and wheat or barley sequences was 0.0.
Comparative analysis of ScGAMYB chromatograms representing gene fragment of 769 bp length revealed 4 SNPs differentiating Ot1–3 and 541 lines (C-T, A-G transitions at positions 470 and 476, respectively and G-C transversions at 485 and 495 positions of the ScGAMYB fragment sequence). All identified point mutations (ESM1) were used to design primers allowing to generate allele-specific products (Tab. 2). AS-PCR markers uncovered polymorphisms not only between parental 541/Ot1–3 lines but also between S32N/RXL10 lines and within mapping populations. In total, 10 pairs of primers were designed (Table 2). All of them amplified stable and repeatable products specific to the alleles tested.
Geneious software, version 10.2.4. was used to align the fragment of ScGAMYB sequence to the whole genome shotgun sequence assembly of rye cultivar Lo7 [20]. This approach allowed to identify homologous gene in the scaffold no. Sc170168 being a DNA fragment containing entire sequence of ScGAMYB located on chromosome 3R in position of 92.15706326 cM. The identity coefficients between the analyzed sequences and that found within the scaffold were 97.46% and 96.56% for Ot1–3 and 541 lines, respectively. Moreover, the sequence alignment of the ScGAMYB gene fragments to sequences of rye transcriptome (Góralska et al. unpublished) revealed their high level of identity to contig no. c81081_g3_i1 i.e. 98.23% for line Ot1–3 and 97.33% for line 541.
Additionally, bioinformatics analysis of the raw sequences data deposited in Sequence Read Archive (SRA), in GeneBank (NCBI) for DS2, RXL10, M12 and L35 rye inbred lines allowed to disclose the complete mRNA sequence of the ScGAMYB geneaccessions SRX2636904—SRX2636920). The alignment of the obtained rye sequences of GAMYB gave a total gene length of 3,700 bp for M12 and DS2 lines. Sequences for RXL10 and L35 rye inbred lines were incomplete within exon 1. All sequences contained the entire coding sequence of ScGAMYB.Comparative analysis of these four sequences revealed SNPs in 22 positions (ESM1).
The structure of ScGAMYB gene was derived by comparing sequencing data reported in this paper with rye DNA sequences presented by Bauer et al. [20] and those deposited in NCBI database. It has a total length of 3,700bp and contains 4 exons and 3 introns, similarly as coding sequences of orthologous genes in wheat and barley. The particular exons of ScGAMYB gene sequence contain 248bp, 387bp, 1009bp and 597bp being interspaced by three introns of 618bp, 829bp and 82bp lengths, with the coding sequence spanning from exon 2 to exon 4 (Fig. 1).
The coding sequence of ScGAMYB gene having 1659bp was translated to protein sequence containing 552 amino acids (ESM2), using Geneious software. This analysis revealed that particular SNPs resulted in change of amino acid sequences at positions: 76 (F/N), 87 (A/I), 233 (P/Q), 367 (S/C) and 459 (S/P) (ESM2). In addition, the protein structure prediction conducted by EMBOSS 6.5.7 plug showed that these polymorphisms affected on secondary structure of ScGAMYB (Fig. 2). Moreover, two polymorphisms resulted in change of protein sequence were observed in region (44–93 AA) coding functional MYB domain, responsible for DNA-binding. The second region of DNA-binding domain (97–144 AA) was highly conservative.
The similarity relationships between rye inbred lines and related species established based on GAMYB sequences are shown on the Figure 3.
Mapping of the ScGAMYB on chromosome 3R
Polymorphic AS-PCR markers of ScGAMYB segregated within the rye mapping populations in a ratio not significantly deviated from the 1 : 1 and 3 : 1 expectations (population RIL and F2,respectively), thus allowing for successful mapping. ScGAMYB was mapped on proximal part of the long arm of chromosome 3R in tight linkage with the DArT marker XrPt401390 on the 541×Ot1–3 (RIL-K) map and between DArTseq-silico 3589123 and 3357900 on the S32N/07×RXL10 (BSR-F2) map (Fig. 4, ESM 3).
Associations of ScGAMYB polymorphisms with selected phenotypic traits
ScGAMYB locus showed significant relationship with trait variation for leaf rolling (RL), α-amylase activity in the grain (AMY), flowering date (FD), plant height (PH), spike length (SL), grain number per spike (GNPS), grain weight per spike (GWPS) and thousand grain weight (TGW) (Table 3). While relationship with leaf rolling, amylase activity, grain number per spike and grain weight per spike were detected in one mapping population and in one year of study, the remaining traits showed significant relationship across years and populations (spike length, plant height) or across years of study (thousand-grain weight and flowering date).