3 RESULTS
3.1 Mutations putatively disrupting CYPTfunction in P. vulgaris
Disruptive mutations in CYPT coding region - Consistent with expectations (Huu et al ., 2016; Gilmartin et al ., 2016; Potente et al ., 2022) and previous findings by Mora-Carrera et al . (2021), we detected putatively disruptive CYPTmutations exclusively in homostyles (Figure 2). Specifically, we identified a non-synonymous mutation [Serine to Stop codon] in exon 2 (referred to as allele CYPT- 2 in Mora-Carrera et al.,2021) and an 8bp deletion in exon 1 that shifts the open reading frame of CYPT (referred to as alleleCYPT- 6 in Mora-Carrera et al., 2021; Figure 1B). Both mutations in CYPT- 2 andCYPT-6 introduce a premature stop codon, likely causing incomplete CYPT translation. In our dataset, CYPT-2 occurred in two homostyles of population EN4-T and all ten homostyles of EN5-T, whileCYPT-6 occurred in eight homostyles of EN4-T.
Additionally, we discovered four non-synonymousCYPT mutations in thrums never reported before. These mutations were observed in different exons and individuals. Specifically, in the 37 thrums analyzed, two non-synonymous mutations were detected in exon 1 of four out of five individuals from SK-D; one non-synonymous mutation was found in exon 3 of two thrums from EN5-T; and one non-synonymous mutation was identified in exon 5 of one individual from CH-D. In contrast to the non-synonymous mutations discovered in homostyles (see above), none of the above mentioned mutations found in thrums introduced a premature stop codon.
Structural rearrangements in CYPT- A drop in sequencing coverage compared to the rest of the genome revealed for the first time a large deletion (ca. 2150 bps) encompassingCYPT exon 1 and its upstream and downstream regions (Figure 3), which was exclusively detected in the 11 homostyles from population EN6-M (Figures 2 and 3). Additional analyses of WGR data in IGV showed that a small number of reads within this region had paired-mates mapping to different chromosomes (not shown), suggesting a local deletion of exon 1 and its potential translocation to another location in the genome. Notably, the remaining fourCYPT exons in these homostyles showed sequencing read coverage values comparable to average sequence read coverage of the genome and did not exhibit any additional disruptive mutations. Apart from the local deletion of exon 1, we did not identify additional inversions or translocations involving the remainingCYPT exons. We designated this previously unreported allele as CYPT -8 (Figure 1B).
Mutations in putative promoter region of CYPT- We identified 20 conserved motifs in the region upstream ofCYPT that are potential binding sites for seven different transcription-factor families, as summarized in Table 2. Among these motifs, ten were associated with various subfamilies of the DREB (Dehydration-Responsive Element-Binding protein) group within the ERF/AP2 transcription-factor family, four were related to the MYB (Myeloblastosis viral oncogene) transcription factor, two were associated with the basic leucine-zipper transcription factor, and one each were linked to the telomere-repeat binding factor, basic pentacysteine, CHC (Clathrin Heavy Chain) protein, and a FAR1 (Fatty Acyl-CoA Reductase 1) related protein. All these conserved motifs were found within a range of 1361 to 15 base pairs upstream ofCYPT exon 1. Since CYPT is a brassinosteroid-degrading enzyme expressed exclusively in the style in P. vulgaris (Huu et al., 2016), we focused on eight conserved motifs involved in brassinosteroid regulation and associated with genes expressed in the carpel of Arabidopsis thaliana , based on the TAIR database (Table 2). Inspection of the aligned sequences upstream of CYPT indicated that the 3kb sequence upstream of CYPT in 20 out of the 31 homostyles were identical to those of the 37 thrums with a functional S-locus (Supplementary Data). However, in the 11 homostyles with the 2150 bp deletion affecting CYPT exon 1 and surrounding upstream and downstream regions (i.e.,CYPT -8 allele: see above), all eight conserved motifs were absent from the promoter region. In summary,CYPT promoter regions are conserved and likely functional in most analyzed homostyles, while the 11 homostyles with a deleted CYPT exon 1 (CYPT -8 allele) also appear to lack the promoter region.