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Genetics: Published Articles Ahead of Print, published on January 25, 2010 as 10.1534/genetics.109.112557
An interspecific plant hybrid shows novel changes
in parental splice forms of genes for splicing factors
Moira Scascitelli, Marie Cognet1, and Keith L. Adams
UBC Botanical Garden and Centre for Plant Research, and Department of Botany,
University of British Columbia, Vancouver, BC, V6T 1Z4, Canada
1
Present address: AgroParisTech, 16 rue claude Bernard, 75231 Paris cedex 05, France
Running title: Alternative splicing in hybrids
Key words: alternative splicing, interspecific hybrids, allelic expression, SR splicing factors,
plant evolution
Abbreviations: AS - alternative splicing; SR - serine/arginine
Corresponding author:
Keith Adams
2357 Main Mall
Vancouver, BC, V6T 1Z4, Canada
Phone: 604-822-2355
Fax: 604-822-6089
E-mail: [email protected]
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Abstract
Interspecific hybridization plays an important role in plant adaptive evolution and speciation, and
the process often results in phenotypic novelty. Hybrids can show changes in genome structure
and gene expression compared with their parents including chromosomal rearrangments, changes
in cytosine methylation, up- and down-regulation of gene expression, and gene silencing.
Alternative splicing is a fundamental aspect of the expression of many genes. However
alternative splicing patterns have not been examined in multiple genes in an interspecific plant
hybrid compared with its parents. Here we studied alternative splicing patterns in an interspecific
Populus hybrid and its parents by assaying 40 genes using reverse transcription PCR. Most of the
genes showed identical alternative splicing patterns between the parents and the hybrid. We
found new alternative splicing variants present in the hybrid in two SR genes, involved in the
regulation of splicing and alternative splicing. The novel alternative splicing patterns included
changes in donor and acceptor sites to create a new exon in one allele of PtRSZ22 in the hybrid,
and retention of an intron in both alleles of PtSR34a.1 in the hybrid, with effects on the function
of the corresponding truncated proteins, if present. Our results suggest that novel alternative
splicing patterns are present in a small percentage of genes in hybrids, but they could make a
considerable impact on the expression of some genes. Changes in alternative splicing are likely
to be an important component of the genetic changes that occur upon interspecific hybridization.
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Introduction
Hybridization between different species has been a common occurrence during plant
evolution and it is thought to play an important role in adaptive evolution, speciation, the transfer
of genetic adaptations, and ecological transitions (ARNOLD, 1997; RIESEBERG, 1997; RIESEBERG
et al. 2003; HEGARTY and HISCOCK, 2005; SOLTIS and SOLTIS, 2009). Hybridization can
generate novel phenotypes including a diverse variety of new and sometimes transgressive
phenotypes observed in hybrids (e.g., RIESEBERG et al. 1999; RIESEBERG et al. 2003).
Interspecific hybridization provides a large number of new alleles for gene evolution. Allelic
variation resulting from interspecific hybridization can potentially contribute to phenotypic
variation. The complementation and interaction of different alleles in hybrids are hypothesized to
be a component of the genetic basis for hybrid vigor (BIRCHLER et al. 2003; BIRCHLER et al.
2006; SPRINGER and STUPAR, 2007a). Hybridization can occur between two diploid species to
create a homoploid hybrid, it can be followed by chromosome doubling to create an
allopolyploid hybrid, or alternatively two polyploid species can hybridize to create a new
allopolyploid.
Interspecific hybridization can have considerable effects on the genome, including
chromosome rearrangements (e.g., RIESEBERG et al. 1996; SHAKED et al. 2001; LAI et al. 2005),
transposable element mobilization (LIU and WENDEL, 2000; SHAN et al. 2005), and DNA
methylation changes (e.g., SALMON et al. 2005; BEAULIEU et al. 2009), although not all of those
changes are observed in all hybrids (LIU et al. 2001). Thus hybrids generally have dynamic
genomes resulting from the merger of two divergent genomes in a common nucleus.
Hybridization between two species can result in extensive changes in gene expression, and that
has been a topic of considerable interest with numerous studies having been published in the last
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few years. Both up- and down-regulation of gene expression levels compared with their parents
have been shown in various diploid, triploid, allotetraploid, and allohexaploid hybrids (e.g.,
HEGARTY et al. 2006; WANG et al. 2006a; HEGARTY et al. 2009; RAPP et al. 2009). Other studies
that assayed expression levels of individual alleles in a diploid hybrid or homeologs in an
allopolyploid revealed allelic expression biases and silencing of one allele (e.g., SPRINGER and
STUPAR, 2007b; FLAGEL et al. 2008; HOVAV et al. 2008; ZHANG and BOREVITZ 2009). Current
evidence indicates that interspecific hybridization has a greater effect on gene expression than
chromosome doubling in allopolyploid hybrids (HEGARTY et al. 2006), as discussed in ADAMS
(2007) and DOYLE et al. (2008).
A fundamental aspect of the expression of many genes is alternative splicing (AS). AS
creates multiple mature mRNAs from a single precursor mRNA by using different 5’ and/or 3’
splice sites. There are several types of AS, including exon skipping where an exon is excluded
from the mature mRNA, intron retention in which a complete intron remains in the transcripts,
and AS at the 5’ end of an intron (alternative donor) or the 3’ end (alternative acceptor),
reviewed in (REDDY, 2007). Transcripts from the same gene can be altered by different
combinations of AS to create multiple mature mRNAs with different nucleotide sequences in the
regions of AS. AS is relatively common in plants, with current estimates of the frequency at
about 20-25% of genes in Arabidopsis and rice (WANG and BRENDEL, 2006). There is one
reported case of changes in AS pattern of a gene in an interspecific hybrid compared with its
parents: The SRK gene shows retention of all introns in an Arabidopsis thaliana x A. lyrata
hybrid but not in the A. lyrata parent, and the expression level of the major splice form varies
between the parent and the hybrid (NASRALLAH et al. 2007). No previous studies though, to our
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knowledge, have examined alternative splicing changes of multiple genes in plant hybrids in a
systematic way.
In this study we examined alternative splicing patterns of a sizable number of genes in an
interspecific hybrid and its parents. We used an F1 hybrid between Populus trichocarpa and
Populus deltoides and clones of its parents. The genome of P. trichocarpa is mostly sequenced
(TUSKAN et al. 2008), facilitating this study. We assayed the AS patterns of 40 genes, with a
focus on genes for serine/arginine rich splicing factors (SR genes), using RT-PCR. Our results
showed that two of the SR genes had novel AS forms in the hybrids compared with their parents.
The new splicing patterns likely were caused by interspecific hybridization, suggesting that AS
can be a component of the “transcriptome shock” (HEGARTY et al., 2006) commonly experienced
by hybrids.
MATERIALS AND METHODS
Sequence retrieval and analysis: We compared AS patterns between a P. trichocarpa x
P. deltoides F1 hybrid and its parents using 40 genes divided into three main groups: 26 SR
splicing factors; 6 genes associated with biotic or abiotic stress responses, with AS detected in
the homolog from Arabidopsis thaliana and, when possible, also in other plant species to
increase the likelihood that their homologs show AS also in poplars (Table 1; Table S1); and 8
genes with AS previously detected in Populus trichocarpa (BAEK et al. 2008). We retrieved P.
trichocarpa gene sequences, gene models, plus their genomic location from the National Center
for Biotechnology Information and from the whole-genome assembly and the Jamboree gene
model database from the DOE Joint Genome Institute (http://genome.jgi-
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psf.org/Poptr1_1/Poptr1_1.home.html). When gene models were not available, we analyzed
putative gene exon/intron structures and protein sequence predictions with gene finding
programs from Softberry (http://linux1.softberry.com/berry.phtml).
We performed a phylogenetic analysis of the 26 SR-genes found in poplars together with
the 19 known SR-genes from A. thaliana to categorize them into each of the seven SR
subfamilies. We made a multiple alignment of the coding sequences using transAlign (BININDAEMONDS, 2005). We manually checked the resulting alignment and retained only the conserved
regions in all SR-genes, roughly corresponding to the RRM domain. We used the aligned
sequenced region to construct an unrooted consensus tree after 100 bootstrap replicates using a
protein maximum likelihood estimation from PROML, with default settings including the JonesTaylor-Thornton probability model of change between amino acids, in the Phylip3.68 package
(FELSENSTEIN, 1989).
Plant material and nucleic acid extraction: We used an F1 hybrid created by crossing
Populus trichocarpa Nisqually-1 accession (maternal parent) x P. deltoides ILL 101 accession
(paternal parent), and clones derived from cuttings of its parents, also used in ZHUANG and
ADAMS (2007). The hybrid was created by Dan Carson of Kruger Products in Harrison Mills,
British Columbia. Cuttings of the parents and hybrids were grown under the same greenhouse
conditions for several months before leaf collection. Total genomic DNA was extracted from
young leaves using the Qiagen (Valencia, CA) DNeasy plant minikit. Total RNA was extracted
from young leaves of about the same size from each genotype, with tissue samples from different
plants for each of three replicates, using a method previously described (ADAMS et al. 2003).
Tissue collection was done at the same time for all individuals and genotypes, and the samples
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were immediately frozen in liquid nitrogen. Extractions were repeated from new tissue samples
at a later date for additional replication of PtRSZ22 and PtSR34a.1 that showed novel AS forms
in the hybrid. The RNA quality was checked on an agarose gel, and residual DNA was removed
using DNaseI (New England Biolabs, Beverly, MA).
RT-PCR analysis of AS: DNase-treated RNA (500 ng) was used to synthesize firststrand cDNA in a final reaction volume of 20 μl, using M-MLV reverse transcriptase
(Invitrogen, Carlsbad, CA), following the manufacturer’s instructions. For each sample, we
performed a parallel reaction without the reverse transcriptase enzyme to test for genomic DNA
contamination.
We designed PCR primers with the oligo analyzer tool available from Integrated DNA
Technologies (IDT, Iowa City, IA). When the putative gene size exceeded about 2000 bp we
used multiple internal primers to cover the whole region. Locations of primers in the genes are
shown in Figure S1. We performed PCR reactions in a final volume of 20 μl, adding 20 ng of
genomic DNA or 1 μl of first-strand cDNA, 1 unit Paq5000TM DNA polymerase (Stratagene), 1x
Paq5000TM reaction buffer, 2.5 mM MgCl2, 0.25 mM of each dNTP, and 0.25 μM of each
primer. We amplified poplar cDNA using three replicates per species and genomic DNA using a
touch-down PCR method (DON et al. 1991) to reduce nonspecific fragment amplification, with
the following conditions: initial denaturing step of 3 min at 95ºC; 12 touch-down cycles at 95ºC
for 30 sec; T*anneal for 30 sec (where the annealing temperature T*ann started at 6ºC higher than
the final Tann and it was dropped by 0.5ºC each touch-down cycle); 72ºC for 1 min per kilobase;
28 cycles at 95ºC for 30 sec; Tanneal for 30 sec; 72ºC for 1 min/Kb), with a final extension period
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at 72ºC for 15 min. Amplified fragments were resolved with electrophoresis runs on 2-2.5%
agarose gels.
Sequencing of hybrid-specific AS variants: For the two genes with novel AS bands
visible only in the F1 hybrid but not in its parents, we purified PCR products with Qiagen
(Valencia, CA) Qiaquick PCR purification kit, cloned them with pCR2.1-TOPO vector
(Invitrogen), following the manufacturer’s protocol, and sequenced them using Big Dye
Terminator v3.1 (Applied Biosystems) sequencing chemistry. We aligned the obtained sequences
against the corresponding genomic DNA using SPIDEY
(http://www.ncbi.nlm.nih.gov/IEB/Research/Ostell/Spidey/). We performed direct sequencing of
parental cDNA for the same genes after PCR and purification using the Qiaquick PCR
purification kit (Qiagen, Valencia, CA) to determine if one or both alleles were present. We
inspected the quality of the chromatogram in the region of the exon-exon junction to verify if the
hybrid-specific AS band was present in the parents and, for the same purpose, we tried to
amplify parental and hybrid cDNA using a primer designed in the intronic region clearly retained
in the hybrids.
Allele-specific analysis of AS: We designed primers to directly sequence the hybridspecific AS variants in order to check whether the gain of AS involved one or both parental
alleles. One of the two primers spanned the retained portion of the intron to amplify exclusively
cDNA with AS but not the fully spliced cDNA from the same gene; primers also surrounded one
or more sites where the two parental sequences were divergent. In the case of biallelic AS, a
polymorphic site in the hybrids, represented by a double peak in the chromatogram, was
expected whereas the two parents had a different nucleotide. In the case of monoallelic AS, a
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single peak corresponding to either P. trichocarpa or P. deltoides specific nucleotide along the
sequence, was expected in the hybrids. Cases of uniparental loss of AS were not detectable with
agarose gels, therefore we sequenced a total of 13 AS variants, visible in the hybrid and in both
parents, after gel extraction and purification with the Qiaquick Gel extraction kit (Qiagen,
Valencia, CA). We analyzed the sequences obtained with Sequencer 4.8 and MEGA v4 (KUMAR
et al. 2008).
RESULTS
Identification and phylogenetic analysis of SR genes in Populus: We focused on
genes for serine/arginine-rich splicing factors (SR genes) because those genes are known to have
multiple AS forms in Arabidopsis thaliana (REDDY, 2004; PALUSA et al. 2007) and they are
regulators of constitutive and alternative splicing that are involved in the process of spliceosome
assembly and splice site recognition (reviewed in LORKOVIĆ et al. 2000; SANFORD et al. 2005;
BARTA et al. 2008). SR genes have conserved domains for pre-mRNA recognition (RRM) and
domains rich in serine and arginine residues (SR-rich domains) involved mainly in proteinprotein interactions. We identified Populus homologs of SR genes from A. thaliana using
BLAST searches of Populus sequence databases. Genes were assigned names based on their
homologs in A. thaliana, with “Pt” for Populus trichocarpa preceding each name. Our analysis
revealed that there are at least 26 SR-genes in Populus, with all seven subfamilies represented
(Figure 1), and that the family is larger compared with A. thaliana that has 19 SR genes
(KALYNA and BARTA, 2004). In addition we included in this study six alternatively spliced genes
associated with biotic and abiotic stress responses whose homologs in Arabidopsis have AS, and
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eight other genes with AS in P. trichocarpa (BAEK et al. 2008) for a total of 40 genes (Table 1;
Table S1).
Novel AS forms in the hybrid compared with its parents: We assayed AS patterns in
all 40 genes by reverse-transcription PCR (RT-PCR) and gel electrophoresis to identify gains of
AS bands in one or both parental alleles of the hybrid, and losses of parental AS variants,
checking for different numbers of AS bands in the hybrid relative to its parents. We found at
least one intron with AS in all genes except PtSCL28, PtSCL28.1 and PtRSZ21 in all genotypes,
and PtRSZ22 in the parents. Most of the 40 genes analyzed (Table 1) had identical splicing
patterns among P. trichocarpa, P. deltoides, and the hybrid; examples are shown in Figure 2.
The AS patterns in the two diploid parental species were identical in all 40 genes examined in
this study. Little is known about the conservation of AS patterns between plant species within a
genus, so it is impossible to say if this level of conservation between P. trichocarpa and P.
deltoides is typical.
In contrast to most of the genes, two genes belonging to the SR-gene family, PtRSZ22
and PtSR34a.1, showed an extra band in the hybrid (Figure 3; Figure S2). Sequencing confirmed
that the hybrid-specific extra bands represented new variants due to AS in both genes. The use of
alternative donor and acceptor splicing sites within the third intron of PtRSZ22 caused the
inclusion of a 66 bp fragment (Figure 3A), while the retention of the fourth intron in PtSR34a.1
added 76 bp to the full-length coding sequence (Figure 3B). PtRSZ22 did not show any AS in the
two parental species, similar to what was found in the homologous gene in A. thaliana (PALUSA
et al. 2007). In contrast to PtRSZ22, PtSR34a.1 has at least three AS isoforms in the two parents
that are conserved in the F1 hybrid (Figure 2). The AS events in PtSR34a.1 are different from
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those in A. thaliana (Table S3; Palusa et al. 2007). The hybrid-specific extra splice variant in
PtSR34a.1 is not associated with any of the three AS isoforms common between the two parents
and the hybrid. The conceptual translation of the hybrid-specific AS forms revealed a premature
termination codon in both cases (Figure 3). If the transcripts are translated they would result in
truncated proteins where only the domain for recognition and binding of RNA (RRM, Figure 3)
is retained, while the domain for interaction with other SR proteins and with the spliceosome
machinery (SR domain, Figure 3) is lost, with likely consequences on function. RSZ22 interacts
with the U1 small nuclear ribonucleoprotein particle (snRNP) 70K protein (GOLOVKIN and
REDDY, 1998) that is involved in the recognition and choice of the donor splice site. An
alternative fate for the alternatively spliced transcripts is that they could be degraded instead of
being translated, potentially lowering the total level of expression of the gene, as discussed in
REDDY (2007) and BARBAZUK et al. (2008). It has been predicted that transcripts with premature
stop codons that are located greater than 50 bp upstream of the last exon-exon junction are
targets for nonsense mediated decay (LEWIS et al. 2003; WANG and BRENDEL, 2006).
To rule out the possibility that AS bands detected by RT-PCR only in the hybrid were
present also in the parents but at too low concentration to be visible on agarose gels, we
performed direct sequencing of parental mRNAs after RT-PCR. The nucleotide region where
alternative splicing occurred in the hybrid, corresponding to exon boundaries in the parents,
showed clear chromatograms (Figure 4) whereas background noise would be expected if there
were low levels of alternatively spliced products in the parents. To further confirm these results
we did RT-PCR using one primer in the intron region and the other primer in the flanking exon
to try to amplify the retained intron region in the parents. RT-PCR amplification was successful
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only in the hybrid (Figure 4C). These results together confirmed that the AS isoforms discovered
in the hybrid were novel and not present in either parent.
One or both alleles can display novel AS in the hybrid: For the two cases of AS gain
in PtRSZ22 and PtSR34a.1, we determined if one or both parental alleles in the hybrid showed
the novel AS form by identifying polymorphic sites between the two parental species and
checking the nucleotide composition at those sites in the hybrid-specific AS isoforms. The
hybrid-specific AS isoform of PtRSZ22 showed a chromatogram with clear single peaks in
which each peak represents a nucleotide corresponding only to the P. deltoides allele (Figure 5a).
The comparison of the two parental genomic sequences in the region where hybrid-specific AS
occurred revealed an interesting difference. The acceptor splicing site ‘ag’ within the fourth
intron, active in the hybrid-specific AS isoform, is present only in P. deltoides sequence while it
is replaced by ‘at’ in P. trichocarpa (Figure 5a). This implies that the presence of a splicing site
in the P. deltoides allele is sensed as a signal of intron termination from the hybrid spliceosome
machinery, so that the adjacent fragment is retained as a cryptic exon. The same acceptor site is
not used by the spliceosome machinery in the P. deltoides parent, at least not in the tissue
analyzed. One possible explanation is that this cryptic splicing site is maintained in a silent state
in P. deltoides through coevolution of species-specific interactions between cis- and transsplicing factors, but the process of hybridization disrupted the parental-specific regulation
causing activation of the site. Alternatively, it could represent a weak splice site active in the
parent P. deltoides only in particular tissues, developmental stages, or after stresses.
In contrast to PtRSZ22, the allelic analysis of the hybrid-specific AS band of PtSR34a.1
showed that both parental alleles in the hybrid retain intron 4 (Figure 5b). Thus the splicing
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machinery in the hybrid is missing splice sites that were detected in the parents. The two AS
isoforms gained by the F1 hybrid, which result in either skipping of parental splicing sites or a
use of new sites relative to the parents, indicate that multiple types of changes in AS patterns can
occur in a hybrid.
While losses of AS forms in both parental alleles and AS gains in one or both parental
alleles were visible on agarose gels as differential numbers of bands in the hybrid compared to its
parents, possible cases of AS losses in only one of the two parental alleles were expected to give
the same number of bands in the hybrid and its parents. Therefore, to monitor the possible
occurrence of uni-parental losses of AS in the hybrid we sequenced 13 bands corresponding to
AS forms after extraction from agarose gels, checking the chromatograms in genomic regions
where the two parents had a divergent sequence. Both parental alleles were present in all cases
(Table S3) and thus there were no analyzed cases where AS was lost in one of the two parental
alleles after interspecific hybridization.
DISCUSSION
Novel splice forms of SR genes in the hybrid compared with its parents: Our
comparison of AS banding patterns of a first generation hybrid between Populus trichocarpa and
Populus deltoides with the corresponding parents revealed the presence of new AS isoforms,
unique to the hybrids, in two genes for SR-rich proteins that function in intron splicing. The
novel AS forms were most likely caused by the process of interspecific hybridization. The two
hybrid-specific AS variants are the result of both gains and losses of splicing sites, indicating that
the splicing machinery in the hybrid can recognize new splicing sites and skip other sites. We
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showed two different types of novel splice forms: intron retention and new cryptic exon creation
from a region of an intron. The novel splice forms affected one allele of PtRSZ22 and both
alleles of PtSR34a.1. The new AS forms create premature stop codons that would truncate the
proteins if translation occurs. The truncated proteins both would be missing the SR domain and
the glycine-rich domain, and PtRSZ22 also would be missing the zinc knuckle domain while
PtSR34.1a would be missing the ΨRRM and P domains (Figure 3). It has been speculated that
truncated SR proteins might interact with some spliceosomal proteins and function as dominant
negative regulators (REDDY, 2007). Thus if the transcripts are translated they might affect
splicing functions. It has been hypothesized that changes in splicing patterns of genes whose
products are involved in splicing, like the SR genes studied here, could lead to alterations in
splicing patterns of other genes (PALUSA et al. 2007). Thus the splicing of other genes may be
affected in the Populus hybrid from novel AS forms of PtRSZ22 and PtSR34a.1.
The presence of a premature termination codon in the alternatively spliced transcripts
might instead cause degradation of those transcripts through the nonsense mediated decay
pathway (BELOSTOTSKY, 2008) or by other transcript decay mechanisms, with possible effects on
gene expression by lowering the total level of steady state transcripts produced from the gene, as
discussed in REDDY (2007) and BARBAZUK et al. (2008). Many of the SR genes in Arabidopsis
thaliana have alternatively spliced forms that have been shown to be present at higher levels in a
mutant for one of the genes involved in nonsense-mediated decay (upf3), indicating that some of
the alternatively spliced transcripts are degraded (PALUSA and REDDY, 2009). The SR protein
RSZ33 in A. thaliana has been shown to autoregulate its own AS, with the AS form containing a
premature stop codon that results in transcript degradation and down-regulation of expression
(KALYNA et al. 2003). Another example of alternatively spliced transcripts that contain
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premature stop codons, where the AS has important functional consequences, is the flowering
time gene FCA in Arabidopsis thaliana that controls the transition from the vegetative to the
reproductive phase. Production of three AS forms of FCA mRNAs with premature stop codons
limits the amount of FCA protein, both spatially and temporally, to prevent precocious flowering
(MACKNIGHT et al. 2002).
Changes in alternative splicing patterns of some genes in hybrids could create new
protein isoforms or regulate gene expression in novel ways with phenotypic consequences. An
intriguing case in this regard is the SRK gene in Arabidopsis lyrata that shows different transcript
levels of the main functional form in hybrids of A. thaliana x A. lyrata compared with the
parental A. lyrata. The changes in AS are associated with the loss of self-incompatibility in the
hybrid (NASRALLAH et al. 2007). Altered allelic AS patterns might be one of the factors involved
in generating novel and transgressive phenotypes that often appear in F1 hybrids including
characteristics of hybrid vigor. Future research including transformation experiments with
alternatively spliced gene products might be useful in testing that hypothesis.
Alternative splicing contributes to genetic novelty in interspecific hybrids: AS
changes in interspecific hybrids are one of the molecular processes, including up- and downregulation of gene expression, gene silencing, chromosomal rearrangements, and cytosine
methylation changes, that can take place after the merger of genomes from two species in a
common nucleus during interspecific hybridization (outlined in the Introduction section).
Alterations in AS are a further indication of the dynamic nature of genomes and gene expression
patterns in hybrids. What molecular mechanisms might cause novel AS patterns in hybrids? One
possibility, first proposed in the context of altered gene expression levels in allopolyploids
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(OSBORN et al. 2003; RIDDLE and BIRCHLER, 2003), is that the combination of diverged
regulatory factors from both parents, AS factors in this case, could lead to suboptimal
interactions of AS factors with their target sequences and thus changes in AS. Another
possibility is that changes in chromatin structure and histone modifications that are seen in
allopolyploid hybrids (WANG et al. 2006b; NI et al. 2009) might cause changes in AS patterns,
because chromatin structure and histone modifications are known to affect AS patterns
(BATSCHÉ et al. 2006; ALLÓ et al. 2009).
How common are changes in AS forms after interspecific hybridization? Data from this
study of 40 genes suggest that novel AS patterns occur at a relatively low but detectable
frequency, perhaps about 5% of genes with AS being affected. This frequency is roughly
comparable to the frequency of up- and down-regulation of gene expression seen in some diploid
and synthetic allopolyploid hybrids (KASHKUSH et al. 2002; ADAMS et al. 2004; SWANSONWAGNER et al. 2006; WANG et al. 2006a), although differing methodologies in the studies make
comparisons difficult. It is now estimated that AS affects about 42% of genes in the Arabidopsis
thaliana genome (FILICHKIN et al. 2009) and the frequency in other plants may well be
comparable. Considering that the Populus trichocarpa genome has about 45,000 genes (TUSKAN
et al. 2006), if about 42% have AS and 5% of those genes show novel AS patterns in an F1
hybrid, there would be about 950 genes showing novel AS patterns. Such a large number of
genes with novel AS patterns in the hybrid could have a significant impact on gene expression
and potentially on phenotypic variation between hybrids and their parents. However at this point
the actual number of genes showing novel AS patterns in an interspecific plant hybrid is
speculative, and it might vary by species. This study is likely to stimulate further research on
novel AS patterns in hybrids to determine the genomic extent of the phenomenon.
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Acknowledgements: We thank Noushin Moshgabadi for technical assistance, and Dan
Carson from Kruger Products in New Westminster, British Columbia, for letting us make
cuttings from his P. trichocarpa x P. deltoides F1 hybrids. Also thanks to Quentin Cronk,
Armando Geraldes, Loren Rieseberg, Jonathan Wendel, and the Adams lab for comments on an
earlier version of manuscript. This study was supported by a grant from the Natural Sciences and
Engineering Council of Canada, by start-up funds from the Faculty of Land and Food Systems at
the University of British Columbia, and by infrastructure funds from the Canadian Foundation
for Innovation.
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_____________________________________________
Table 1. Genes assayed for alternative splicing
_____________________________________________
RS-like subfamily
PtSR31.1, PtSR31.2, PtSR40.1, PtSR40.2, PtSR40.3,
PtSR40.4, PtSR40.5
SC35-like subfamily
PtSC35.1, PtSC35.2, PtSC35.3
SCL-like subfamily
PtSCL28.1, PtSCL28.2, PtSCL30.1, PtSCL30.2,
PtSCL30.3, PtSCL30.4, PtSCL30.5
ASF/SF2-like subfamily
PtSR34.1, PtSR34.2, PtSR34.3, PtSR34a.1, PtSR34a.2
9G8-like subfamily
PtSRZ21, PtSRZ22
SR2Z-like subfamily
PtSRZ32.1, PtSRZ32.2
SR45-like subfamily
PtSR45
Putative functions of other genes analyzed (number)
TIR-NBS-LRR class of disease resistance proteins (2)
Putative GRP, RNA binding activity (2)
MYB, DNA binding activity (2)
Putative phosphoinositide phosphatase activity (1)
Translation initiation factor (1)
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Ribosomal protein L35 family protein (1)
Ubiquitin conjugating enzyme (1)
Pre-mRNA splicing factor (1)
Predicted snRNP core protein (1)
Unknown function (2)
________________________________________________
See supplementary table 1 for sequence accession numbers.
29
Figure legends
FIGURE 1. - Phylogenetic analysis of SR-genes in Populus trichocarpa (Pt) and Arabidopsis
thaliana (At). We obtained an unrooted consensus tree after 100 bootstrap replicates with the
maximum likelihood method using Phylip v3.6 package. Seven subfamilies are distinguishable,
three of which (SF2/ASf-like, 9G8-like and SC35-like) are common among all eukaryotes, while
the remaining four (SR2Z-, SCL-, SR45- and RS-like) are plant-specific. Genes were named
based on their homologs in A. thaliana, with “Pt” for Populus trichocarpa preceding each name.
The two underlined genes in bold gained AS after interspecific hybridization.
FIGURE 2. - Examples of genes with conservation of AS patterns between the two parental
species Populus trichocarpa (Pt) and P. deltoides (Pd) and their F1 hybrid (F1). One out of three
replicates is shown for each gene. PtSCL28.2, PtSR34a.1 and PtRSZ32.2 belong to the SR-rich
protein gene family (see Figure 1 for details about gene subfamilies), whereas TC74948 has an
unknown function (Table 1). PtSCL28.2 shows absence of alternative splicing in all samples.
The region of PtSR34a.1 amplified here is between exons 5-11; the region between exons 1-5 of
the same gene showed gain of AS in the F1 hybrid (Figure 3). All the bands in the figure have
been sequenced and the events of AS that generated these bands are reported in Table S3.
Controls include: a negative control of the reverse-transcription reaction (rt-) for each of the
three samples to exclude genomic DNA contamination; P. trichocarpa genomic DNA (g) to
show the size of the gene including introns; and a negative control (N) to test for contamination
during PCR reactions.
30
FIGURE 3. - Effects of interspecific hybridization on AS of two genes for SR-rich proteins,
PtRSZ22 (A) and PtSR34a.1 (B). Gel pictures on the left side of each panel show the presence of
a single, consitutively spliced band in both parents (Pt = P. trichocarpa, Pd = P. deltoides), and a
gain of an AS form in the F1 hybrid (F1). All bands were sequenced to confirm their identity.
One biological replicate of each genotype is shown here; two additional tissue replicates from
different plants (Figure S2), plus replicates from tissue samples harvested at a later date, were
used to check for reproducibility. There was no variability in band patterns among the replicates.
Controls include: a negative control of the reverse-transcription reaction (rt-) for each of the
three samples to exclude genomic DNA contamination; P. trichocarpa genomic DNA (g) to
show the size of the gene including introns; and a negative control (N) to test for contamination
during PCR reactions. The exon and intron structure is shown in the centre of each panel with
filled boxes and lines respectively, at the same level of the resulting band, while the portion of
the intron retained in the hybrid-specific AS variant is highlighted with a white box. Arrows
represent locations of the PCR primer sets used for the RT-PCR reactions shown here. Note that
a different set of primers (Table S2) for PtSr34a.1 was used here, amplifying exons 1-5, than for
the same gene in Figure 2, amplifying exons 5-11. The right side of each panel shows domains of
the predicted full length protein and the effect of AS on its sequence. Asterisks represent
premature termination codons, the box with oblique lines shows a change in the amino acid
sequence due to a reading frame shift after AS. Domains include: RRM, RNA recognition motif;
ΨRRM, domain exclusive of SF2/ASF gene subfamily, containing the SWQDLKD motif; SR,
serine/arginine-rich domain; G, glycine-rich domain; Z, Zinc knuckle domain; P, PSK
(proline/serine/lysine-rich) domain.
31
FIGURE 4. - Sequencing and RT-PCR analysis of AS forms of PtRSZ22 (A) and PtSR34a.1 (B).
Direct sequencing after PCR of P. trichocarpa and P. deltoides mRNAs, from Figure 3, showed
clear chromatograms (shown on the right side of the picture), where the sequences correspond
only to the junction between the exons (in capital letters). A schematic diagram of the region
under analysis is shown on the left side of the panel. We therefore excluded the presence of low
amounts of the AS product in the parental diploids, whose first and last four nucleotides are
represented with lower case letters. Di-nucleotide sites of splicing are bolded and italicized. For
PtRSZ22 (A), the alternative acceptor site used only in the hybrids is underlined also in the
parents to show the divergent sequence (see text for explanation). (C). RT-PCR using one primer
located in an exon and another primer located in the intron with AS. Amplification was
successful only in the hybrids, consistent with the sequencing results.
FIGURE 5. - Sequencing based analysis of the hybrid-specific AS bands from RT-PCR products
to check whether the alteration of AS involved one or both parental alleles. Shown are the
chromatograms of F1 hybrid AS isoforms in sites where the two parental species (P. trichocarpa
and P. deltoides) had polymorphisms in their nucleotide sequences, highlighted with black
boxes. The hybrid AS band from PtRSZ22 (A) showed single peaks corresponding only to the P.
deltoides allele. The presence of double peaks at polymorphic sites for PtSR34a.1 (B) indicated
that the gain of a novel AS isoform involved both parental alleles.
32
0.1 substitution/site
A
B
C
PtRSZ22
PtSR34a.1
Pt Pd F1 rt- g N
Pt Pd F1 rt- g N