4 DISCUSSION
We integrated whole genome resequencing data from a comprehensive
sampling of long-monitored populations of P. vulgaris with
knowledge of the recently assembled genomes of P. vulgaris andP. veris to explain the causes and consequences of the transition
from distyly to homostyly. We identified a novel loss-of-function
structural mutation in CYPT associated with the
transition to homostyly that had remained undetected using exonic Sanger
sequencing (Mora-Carrera et al., 2021). Importantly, we found no
evidence for a potential single origin of homostyly in P.
vulgaris via mutations in CYPT promoter region
or structural mutations involving CYPT exons,
thus the previously supported hypothesis of multiple transitions to
homostyly via independent loss-of-function mutations inCYPT exons stands (Mora-Carrera et al.,2021). Furthermore, population genetic analyses validated theoretical
expectations for the evolutionary consequences of hemizygosity on
S-locus genes and revealed differences of selective constraints among
S-locus genes. Finally, the genomic resources newly available in thePrimula system enabled, for the first time, the testing of
long-standing predictions on changing frequencies of S-locus genotypes
during intraspecific transitions from distyly to homostyly, partially
supporting the possible role of viability differences between homostyles
with haploid vs. diploid S-locus genotypes in preventing the fixation of
homostyly. Jointly, our study provides a detailed overview of the early
molecular- and population genetic causes and consequences of
mating-system transitions.
Genetic basis of transitions from distyly to homostyly in Primula
vulgaris
Shifts from outcrossing to selfing are common in flowering plants and
can be caused by loss-of-function mutations in the genes of interest,
structural rearrangements of their exons, or mutations in their
promoters (Shimizu & Tsuchimatsu, 2015). One question concerns whether
disruptive mutations in the alleles that determine outcrossing act
dominantly or recessively. In Brassicaceae, loss of self-incompatibility
often stems from mutations in dominant alleles of genes controlling this
trait (Busch et al ., 2011; Nasrallah et al. , 2017;
Tsuchimatsu et al., 2012; Bachmann et al., 2019), although
in Arabidopsis lyrata loss of self-incompatibility caused
by mutations in recessive alleles has also been discovered (Mableet al ., 2017). In Primula , the S-locus controlling distyly
is hemizygous in both P. vulgaris and P. veris (Huu et
al., 2016; 2020), but previous models assumed S-locus heterozygosity,
with thrum phenotype associated with the dominant S-locus allele
(Bateson & Gregory, 1905). Based on this model and greenhouse crossing
experiments, Crosby (1949) assumed that S-locus alleles associated with
homostyly should be recessive. Previously, we documented sevenCYPT haplotypes
(CYPT -2 to CYPT -7)
with putative loss-of-function mutations that are occurring exclusively
in P. vulgaris homostyles (Mora-Carrera et al., 2021). Two
of these haplotypes (CYPT -2 andCYPT -6) have an early stop codon causing
premature termination of translation. Our results indicate that, when
hemizygous or homozygous, these disrupted CYPT alleles lead to homostyly. However, CYPT -2
behaves recessively in the single heterozygous individual carrying one
functional and one disrupted copy of CYPT(i.e., CYPT-1/CYPT-2 ,
represented as S/S* in Figure 4), determining a thrum phenotype. This
finding aligns with results of crossing experiments between thrums and
homostyles of Primula oreodoxa showing that S* is recessive to S
when the two alleles co-occur (Yuan et al ., 2018), corroborating
Crosby’s prediction (1949). Therefore, our results indicate that the
presence of a disrupted CYPT allele does not
alter the thrum morph when paired with a functionalCYPT allele, hence the disrupted allele acts
recessively.
In addition to loss-of-function mutations in coding regions,
mating-system shifts can also stem from transcription silencing or
exonic rearrangements in pertinent genes (Chakraborty et al. ,
2023). For instance, down-regulation caused by transposon-like
insertions in the promoter regions of the male-determining
self-incompatibility genes MGST and BnSP11-1 trigger the
shift from self-incompatibility to self-compatibility in Prunus
avium and Brassica napus , respectively (Gao et al ., 2016;
Ono et al ., 2020). In Primula vulgaris , previous Sanger
sequencing of individual CYPT exons identified
homostyles with an apparently functional CYPTallele (CYPT-1 ; Figure 1B), suggesting that
homostyles might also arise through CYPTsilencing caused by a disruptive mutation in its promoter region or
exonic rearrangements in CYPT not detectable
via Sanger sequencing (Mora-Carrera et al ., 2021). However, 20 of
the 31 homostyles analyzed in the present study had a promoter region
identical to that of 37 thrums, implying that the shift to homostyly in
these plants was likely caused by loss-of-function mutations inCYPT exons rather than in its promoter. The
remaining 11 homostyles were characterized by a large 2150 bp deletion
that eliminated both CYPT exon 1 and its
promoter region (Figure 3). Thus, our current results do not support the
conclusion that mutations in the promoter region or exonic
rearrangements in CYPT can alone cause the
shift to homostyly in P. vulgaris .
The evidence above also has implications for determining whether
homostyly arose once or multiple times in P. vulgaris . The single
origin of homostyly, followed by independent mutations inCYPT , would have been supported if all studied
homostyles had shared the same promoter mutation or rearrangement inCYPT . However, this is not the case, favoring
the hypothesis of multiple origins of homostyly via independent
mutations in CYPT exons, as previously proposed
(Mora Carrera et al ., 2021). Nevertheless, a study of a single
homostyle from Chiltern Hills, England (population not included in our
analyses), found reduced expression of CYPTwhen compared to a thrum, suggesting epigenetic silencing might play a
role in the shift to homostyly (Huu et al., 2016). The mentioned
study however did not provide sequences of CYPTexons, thus it remains unknown whether they contained any potentially
disruptive mutations in the coding region. Therefore, transcriptome
analyses of homostylous flowers are necessary to conclusively discard
the possibility that disruptive promoter mutations causing reducedCYPT expression might also cause the shift to
homostyly.
Finally, it remains to be explained why the three homostyles previously
thought to have the functional CYPT -1 allele
based on Sanger sequencing of the five CYPTexons (Mora-Carrera et al ., 2021) were here found to contain the
2150 bp deletion including exon 1 (i.e., CYPT-8 haplotype: see Figures 1B, 2, and 3). A possible explanation is that
exon 1 was deleted from the S-locus (causingCYPT loss of function, hence homostyly) and
translocated to a highly repetitive genomic region. The translocation
could have allowed targeted amplification and subsequent Sanger
sequencing using exon-1-specific PCR primers, while preventing exon-1
detection via next generation sequencing due to biases arising, for
example, during genomic DNA sonication used to produce short DNA
fragments prior to short-read library preparation (Poptsova et
al. , 2014; Garafutdinov et al. , 2016; Jennings et al. ,
2017). Notably, a few low-quality sequencing reads did map toCYPT exon 1, suggesting this exon is indeed
present in the genome of these homostyles but was not successfully
sequenced using short-read sequencing methodology. Long-read sequencing
technologies capable of sequencing through repetitive regions would be
necessary to definitively resolve whether CYPTexon 1 was translocated to a highly repetitive genomic region in these
homostyles. To summarize, our findings indicate that the homostyles
previously identified as having a functionalCYPT allele in fact possess a disruptedCYPT allele due to exon 1 deletion (designatedCYPT -8 allele: Figure 3). Overall, these
results emphasize that not only non-synonymous mutations or small
deletions, but also structural rearrangements such as large deletions
and translocations can cause mating-system transitions.