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.