Homoeologous crossovers are distally biased and underlie genomic instability in first-generation neo-allopolyploid Arabidopsis suecica.

First-generation polyploids often suffer from more meiotic errors and lower fertility than established wild polyploid populations. One such example is the allopolyploid model species Arabidopsis suecica which originated c. 16 000 generations ago. We present here a comparison of meiosis and its outcomes in naturally evolved and first-generation 'synthetic' A. suecica using a combination of cytological and genomic approaches. We show that while meiosis in natural lines is largely diploid-like, synthetic lines have high levels of meiotic errors including incomplete synapsis and nonhomologous crossover formation. Whole-genome re-sequencing of progeny revealed 20-fold higher levels of homoeologous exchange and eightfold higher aneuploidy originating from synthetic parents. Homoeologous exchanges showed a strong distal bias and occurred predominantly in genes, regularly generating novel protein variants. We also observed that homoeologous exchanges can generate megabase scale INDELs when occurring in regions of inverted synteny. Finally, we observed evidence of sex-specific differences in adaptation to polyploidy with higher success in reciprocal crosses to natural lines when synthetic plants were used as the female parent. Our results directly link cytological phenotypes in A. suecica with their genomic outcomes, demonstrating that homoeologous crossovers underlie genomic instability in neo-allopolyploids and are more distally biased than homologous crossovers.

Little evidence for homoeologous gene conversion and homoeologous exchange events in Gossypium allopolyploids.

PREMISE: A complicating factor in analyzing allopolyploid genomes is the possibility of physical interactions between homoeologous chromosomes during meiosis, resulting in either crossover (homoeologous exchanges) or non-crossover products (homoeologous gene conversion). Homoeologous gene conversion was first described in cotton by comparing SNP patterns in sequences from two diploid progenitors with those from the allopolyploid subgenomes. These analyses, however, did not explicitly consider other evolutionary scenarios that may give rise to similar SNP patterns as homoeologous gene conversion, creating uncertainties about the reality of the inferred gene conversion events. METHODS: Here, we use an expanded phylogenetic sampling of high-quality genome assemblies from seven allopolyploid Gossypium species (all derived from the same polyploidy event), four diploid species (two closely related to each subgenome), and a diploid outgroup to derive a robust method for identifying potential genomic regions of gene conversion and homoeologous exchange. RESULTS: We found little evidence for homoeologous gene conversion in allopolyploid cottons, and that only two of the 40 best-supported events were shared by more than one species. We did, however, reveal a single, shared homoeologous exchange event at one end of chromosome 1, which occurred shortly after allopolyploidization but prior to divergence of the descendant species. CONCLUSIONS: Overall, our analyses demonstrated that homoeologous gene conversion and homoeologous exchanges are uncommon in Gossypium, affecting between zero and 24 genes per subgenome (0.0-0.065%) across the seven species. More generally, we highlighted the potential problems of using simple four-taxon tests to investigate patterns of homoeologous gene conversion in established allopolyploids.

Homoeologous exchanges contribute to branch angle variations in rapeseed: Insights from transcriptome, QTL-seq and gene functional analysis.

Branch angle (BA) is a critical morphological trait that significantly influences planting density, light interception and ultimately yield in plants. Despite its importance, the regulatory mechanism governing BA in rapeseed remains poorly understood. In this study, we generated 109 transcriptome data sets for 37 rapeseed accessions with divergent BA phenotypes. Relative to adaxial branch segments, abaxial segments accumulated higher levels of auxin and exhibited lower expression of six TCP1 homologues and one GA20ox3. A co-expression network analysis identified two modules highly correlated with BA. The modules contained homologues to known BA control genes, such as FUL, YUCCA6, TCP1 and SGR3. Notably, a homoeologous exchange (HE), occurring at the telomeres of A09, was prevalent in large BA accessions, while an A02-C02 HE was common in small BA accessions. In their corresponding regions, these HEs explained the formation of hub gene hotspots in the two modules. QTL-seq analysis confirmed that the presence of a large A07-C06 HE (~8.1 Mb) was also associated with a small BA phenotype, and BnaA07.WRKY40.b within it was predicted as candidate gene. Overexpressing BnaA07.WRKY40.b in rapeseed increased BA by up to 20°, while RNAi- and CRISPR-mediated mutants (BnaA07.WRKY40.b and BnaC06.WRKY40.b) exhibited decreased BA by up to 11.4°. BnaA07.WRKY40.b was exclusively localized to the nucleus and exhibited strong expression correlations with many genes related to gravitropism and plant architecture. Taken together, our study highlights the influence of HEs on rapeseed plant architecture and confirms the role of WRKY40 homologues as novel regulators of BA.

Genome composition in Brassica interspecific hybrids affects chromosome inheritance and viability of progeny.

Interspecific hybridization is widespread in nature and can result in the formation of new hybrid species as well as the transfer of traits between species. However, the fate of newly formed hybrid lineages is relatively understudied. We undertook pairwise crossing between multiple genotypes of three Brassica allotetraploid species Brassica juncea (2n = AABB), Brassica carinata (2n = BBCC), and Brassica napus (2n = AACC) to generate AABC, BBAC, and CCAB interspecific hybrids and investigated chromosome inheritance and fertility in these hybrids and their self-pollinated progeny. Surprisingly, despite the presence of a complete diploid genome in all hybrids, hybrid fertility was very low. AABC and BBAC first generation (F1) hybrids both averaged ~16% pollen viability compared to 3.5% in CCAB hybrids: most CCAB hybrid flowers were male-sterile. AABC and CCAB F1 hybrid plants averaged 5.5 and 0.5 seeds per plant, respectively, and BBAC F1 hybrids ~56 seeds/plant. In the second generation (S1), all confirmed self-pollinated progeny resulting from CCAB hybrids were sterile, producing no self-pollinated seeds. Three AABC S1 hybrids putatively resulting from unreduced gametes produced 3, 14, and 182 seeds each, while other AABC S1 hybrids averaged 1.5 seeds/plant (0-8). BBAC S1 hybrids averaged 44 seeds/plant (range 0-403). We also observed strong bias towards retention rather than loss of the haploid genomes, suggesting that the subgenomes in the Brassica allotetraploids are already highly interdependent, such that loss of one subgenome is detrimental to fertility and viability. Our results suggest that relationships between subgenomes determine hybridization outcomes in these species.

Dosage-sensitivity shapes how genes transcriptionally respond to allopolyploidy and homoeologous exchange in resynthesized Brassica napus.

The gene balance hypothesis proposes that selection acts on the dosage (i.e. copy number) of genes within dosage-sensitive portions of networks, pathways, and protein complexes to maintain balanced stoichiometry of interacting proteins, because perturbations to stoichiometric balance can result in reduced fitness. This selection has been called dosage balance selection. Dosage balance selection is also hypothesized to constrain expression responses to dosage changes, making dosage-sensitive genes (those encoding members of interacting proteins) experience more similar expression changes. In allopolyploids, where whole-genome duplication involves hybridization of diverged lineages, organisms often experience homoeologous exchanges that recombine, duplicate, and delete homoeologous regions of the genome and alter the expression of homoeologous gene pairs. Although the gene balance hypothesis makes predictions about the expression response to homoeologous exchanges, they have not been empirically tested. We used genomic and transcriptomic data from 6 resynthesized, isogenic Brassica napus lines over 10 generations to identify homoeologous exchanges, analyzed expression responses, and tested for patterns of genomic imbalance. Groups of dosage-sensitive genes had less variable expression responses to homoeologous exchanges than dosage-insensitive genes, a sign that their relative dosage is constrained. This difference was absent for homoeologous pairs whose expression was biased toward the B. napus A subgenome. Finally, the expression response to homoeologous exchanges was more variable than the response to whole-genome duplication, suggesting homoeologous exchanges create genomic imbalance. These findings expand our knowledge of the impact of dosage balance selection on genome evolution and potentially connect patterns in polyploid genomes over time, from homoeolog expression bias to duplicate gene retention.

Demographic history inference and the polyploid continuum.

Polyploidy is an important generator of evolutionary novelty across diverse groups in the Tree of Life, including many crops. However, the impact of whole-genome duplication depends on the mode of formation: doubling within a single lineage (autopolyploidy) versus doubling after hybridization between two different lineages (allopolyploidy). Researchers have historically treated these two scenarios as completely separate cases based on patterns of chromosome pairing, but these cases represent ideals on a continuum of chromosomal interactions among duplicated genomes. Understanding the history of polyploid species thus demands quantitative inferences of demographic history and rates of exchange between subgenomes. To meet this need, we developed diffusion models for genetic variation in polyploids with subgenomes that cannot be bioinformatically separated and with potentially variable inheritance patterns, implementing them in the dadi software. We validated our models using forward SLiM simulations and found that our inference approach is able to accurately infer evolutionary parameters (timing, bottleneck size) involved with the formation of auto- and allotetraploids, as well as exchange rates in segmental allotetraploids. We then applied our models to empirical data for allotetraploid shepherd's purse (Capsella bursa-pastoris), finding evidence for allelic exchange between the subgenomes. Taken together, our model provides a foundation for demographic modeling in polyploids using diffusion equations, which will help increase our understanding of the impact of demography and selection in polyploid lineages.

Patterns of Chromosomal Variation, Homoeologous Exchange, and Their Relationship with Genomic Features in Early Generations of a Synthetic Rice Segmental Allotetraploid.

Polyploidization is a driving force in plant evolution. Chromosomal variation often occurs at early generations following polyploid formation due to meiotic pairing irregularity that may compromise segregation fidelity and cause homoeologous exchange (HE). The trends of chromosomal variation and especially factors affecting HE remain to be fully deciphered. Here, by whole-genome resequencing, we performed nuanced analyses of patterns of chromosomal number variation and explored genomic features that affect HE in two early generations of a synthetic rice segmental allotetraploid. We found a wide occurrence of whole-chromosome aneuploidy and, to a lesser extent, also large segment gains/losses in both generations (S2 and S4) of the tetraploids. However, while the number of chromosome gains was similar between S2 and S4, that of losses in S4 was lower than in S2. HEs were abundant across all chromosomes in both generations and showed variable correlations with different genomic features at chromosomal and/or local scales. Contents of genes and transposable elements (TEs) were positively and negatively correlated with HE frequencies, respectively. By dissecting TEs into different classes, retrotransposons were found to be negatively correlated with HE frequency to a stronger extent than DNA transposons, whereas miniature terminal inverted elements (MITEs) showed a strong positive correlation. Local HE frequencies in the tetraploids and homologous recombination (HR) rates in diploids within 1 Mb sliding windows were significantly correlated with each other and showed similar overall distribution profiles. Nonetheless, non-concordant trends between HE and HR rates were found at distal regions in some chromosomes. At local scale, both shared and polymorphic retrotransposons between parents were negatively correlated with HE frequency; in contrast, both shared and polymorphic MITEs showed positive correlations with HE frequency. Our results shed new light on the patterns of chromosomal number variation and reveal genomic features influencing HE frequency in early generations following plant polyploidization.

Patterns, mechanisms, and consequences of homoeologous exchange in allopolyploid angiosperms: a genomic and epigenomic perspective.

Allopolyploids result from hybridization between different evolutionary lineages coupled with genome doubling. Homoeologous chromosomes (chromosomes with common shared ancestry) may undergo recombination immediately after allopolyploid formation and continue over successive generations. The outcome of this meiotic pairing behavior is dynamic and complex. Homoeologous exchanges (HEs) may lead to the formation of unbalanced gametes, reduced fertility, and selective disadvantage. By contrast, HEs could act as sources of novel evolutionary substrates, shifting the relative dosage of parental gene copies, generating novel phenotypic diversity, and helping the establishment of neo-allopolyploids. However, HE patterns vary among lineages, across generations, and even within individual genomes and chromosomes. The causes and consequences of this variation are not fully understood, though interest in this evolutionary phenomenon has increased in the last decade. Recent technological advances show promise in uncovering the mechanistic basis of HEs. Here, we describe recent observations of the common patterns among allopolyploid angiosperm lineages, underlying genomic and epigenomic features, and consequences of HEs. We identify critical research gaps and discuss future directions with far-reaching implications in understanding allopolyploid evolution and applying them to the development of important phenotypic traits of polyploid crops.

Rapid large-scale genomic introgression in Arabidopsis suecica via an autoallohexaploid bridge.

Gene flow between species in the genus Arabidopsis occurs in significant amounts, but how exactly gene flow is achieved is not well understood. Polyploidization may be one avenue to explain gene flow between species. One problem, however, with polyploidization as a satisfying explanation is the occurrence of lethal genomic instabilities in neopolyploids as a result of genomic exchange, erratic meiotic behavior, and genomic shock. We have created an autoallohexaploid by pollinating naturally co-occurring diploid Arabidopsis thaliana with allotetraploid Arabidopsis suecica (an allotetraploid composed of A. thaliana and Arabidopsis arenosa). Its triploid offspring underwent spontaneous genome duplication and was used to generate a multigenerational pedigree. Using genome resequencing, we show that 2 major mechanisms promote stable genomic exchange in this population. Legitimate meiotic recombination and chromosome segregation between the autopolyploid chromosomes of the 2 A. thaliana genomes occur without any obvious bias for the parental origin and combine the A. thaliana haplotypes from the A. thaliana parent with the A. thaliana haplotypes from A. suecica similar to purely autopolyploid plants. In addition, we repeatedly observed that occasional exchanges between regions of the homoeologous chromosomes are tolerated. The combination of these mechanisms may result in gene flow leading to stable introgression in natural populations. Unlike the previously reported resynthesized neoallotetraploid A. suecica, this population of autoallohexaploids contains mostly vigorous, and genetically, cytotypically, and phenotypically variable individuals. We propose that naturally formed autoallohexaploid populations might serve as an intermediate bridge between diploid and polyploid species, which can facilitate gene flow rapidly and efficiently.

Effects of homoeologous exchange on gene expression and alternative splicing in a newly formed allotetraploid wheat.

Homoeologous exchange (HE) is a major mechanism generating post-polyploidization genetic variation with important evolutionary consequences. However, the direct impacts of HE on gene expression and transcript diversity in allopolyploids without the intertwined evolutionary processes remain to be fully understood. Here, we analyzed high-throughput RNA-seq data of young leaves from plant groups of a synthetic allotetraploid wheat (AADD), which contained variable numbers of HEs. We aimed to investigate if and to which extent HE directly impacts gene expression and alternative splicing (AS). We found that HE impacts expression of genes located within HE regions primarily via a cis-acting dosage effect, which led to significant changes in the total expression level of homoeologous gene pairs, especially for homoeologs whose original expression was biased. In parallel, HE also influences expression of a large number of genes residing in non-HE regions by trans-regulation leading to convergent expression of homoeologs. Intriguingly, when taking the original relative homoeolog expression states into account, homoeolog pairs under trans-effect are more prone to manifesting a convergent response to the HEs whereas those under cis-regulation tended to show further exacerbated subgenome-biased expression. Moreover, HE-induced quantitative, largely individual-specific, changes of AS events were detected. Similar to homoeologous expression, homoeo-AS events under trans-effect were more responsive to HE. HE therefore exerts multifaceted immediate effects on gene expression and, to a less extent, on individualized transcript diversity in nascent allopolyploidy.

Homoeologous chromosome exchange explains the creation of a QTL affecting soil-borne pathogen resistance in tobacco.

Crop plant partial resistance to plant pathogens controlled by quantitative trait loci (QTL) is desirable in cultivar development programmes because of its increased durability. Mechanisms underlying such resistance are difficult to study. We performed RNA-seq analyses for tobacco (Nicotiana tabacum) nearly isogenic lines (NILs) with and without favourable allele(s) at Phn7.1, a major QTL influencing partial resistance to the soil-borne pathogens Phytophthora nicotianae and Ralstonia solanacearum. Based upon combined analyses of transcriptome-based sequence variation and gene expression profiles, we concluded that allelic variability at the Phn7.1 locus was likely generated from homoeologous exchange, which led to deletion of low-expressing members of the SAR8.2 gene family and duplication of high-expressing SAR8.2 genes from a different subgenome of allotetraploid tobacco. The high expression of endogenous Phn7.1-associated SAR8.2 genes was correlated with observed resistance to P. nicotianae. Our findings suggest a role for genomic rearrangements in the generation of favourable genetic variability affecting resistance to pathogens in plants.

Homoeologous exchanges occur through intragenic recombination generating novel transcripts and proteins in wheat and other polyploids.

Recombination between homeologous chromosomes, also known as homeologous exchange (HE), plays a significant role in shaping genome structure and gene expression in interspecific hybrids and allopolyploids of several plant species. However, the molecular mechanisms that govern HEs are not well understood. Here, we studied HE events in the progeny of a nascent allotetraploid (genome AADD) derived from two diploid progenitors of hexaploid bread wheat using cytological and whole-genome sequence analyses. In total, 37 HEs were identified and HE junctions were mapped precisely. HEs exhibit typical patterns of homologous recombination hotspots, being biased toward low-copy, subtelomeric regions of chromosome arms and showing association with known recombination hotspot motifs. But, strikingly, while homologous recombination preferentially takes place upstream and downstream of coding regions, HEs are highly enriched within gene bodies, giving rise to novel recombinant transcripts, which in turn are predicted to generate new protein fusion variants. To test whether this is a widespread phenomenon, a dataset of high-resolution HE junctions was analyzed for allopolyploid Brassica, rice, Arabidopsis suecica, banana, and peanut. Intragenic recombination and formation of chimeric genes was detected in HEs of all species and was prominent in most of them. HE thus provides a mechanism for evolutionary novelty in transcript and protein sequences in nascent allopolyploids.

DNA methylation repatterning accompanying hybridization, whole genome doubling and homoeolog exchange in nascent segmental rice allotetraploids.

Allopolyploidization, which entails interspecific hybridization and whole genome duplication (WGD), is associated with emergent genetic and epigenetic instabilities that are thought to contribute to adaptation and evolution. One frequent genomic consequence of nascent allopolyploidization is homoeologous exchange (HE), which arises from compromised meiotic fidelity and generates genetically and phenotypically variable progenies. Here, we used a genetically tractable synthetic rice segmental allotetraploid system to interrogate genome-wide DNA methylation and gene expression responses and outcomes to the separate and combined effects of hybridization, WGD and HEs. Progenies of the tetraploid rice were genomically diverse due to genome-wide HEs that affected all chromosomes, yet they exhibited overall methylome stability. Nonetheless, regional variation of cytosine methylation states was widespread in the tetraploids. Transcriptome profiling revealed genome-wide alteration of gene expression, which at least in part associates with changes in DNA methylation. Intriguingly, changes of DNA methylation and gene expression could be decoupled from hybridity and sustained and amplified by HEs. Our results suggest that HEs, a prominent genetic consequence of nascent allopolyploidy, can exacerbate, diversify and perpetuate the effects of allopolyploidization on epigenetic and gene expression variation, and hence may contribute to allopolyploid evolution.

Mapping of homoeologous chromosome exchanges influencing quantitative trait variation in Brassica napus.

Genomic rearrangements arising during polyploidization are an important source of genetic and phenotypic variation in the recent allopolyploid crop Brassica napus. Exchanges among homoeologous chromosomes, due to interhomoeologue pairing, and deletions without compensating homoeologous duplications are observed in both natural B. napus and synthetic B. napus. Rearrangements of large or small chromosome segments induce gene copy number variation (CNV) and can potentially cause phenotypic changes. Unfortunately, complex genome restructuring is difficult to deal with in linkage mapping studies. Here, we demonstrate how high-density genetic mapping with codominant, physically anchored SNP markers can detect segmental homoeologous exchanges (HE) as well as deletions and accurately link these to QTL. We validated rearrangements detected in genetic mapping data by whole-genome resequencing of parental lines along with cytogenetic analysis using fluorescence in situ hybridization with bacterial artificial chromosome probes (BAC-FISH) coupled with PCR using primers specific to the rearranged region. Using a well-known QTL region influencing seed quality traits as an example, we confirmed that HE underlies the trait variation in a DH population involving a synthetic B. napus trait donor, and succeeded in narrowing the QTL to a small defined interval that enables delineation of key candidate genes.