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Background: Whole genome duplication (WGD) events are common in the evolutionary history of many living organisms. For decades, researchers have been trying to understand the genetic and epigenetic impact of WGD and its underlying molecular mechanisms. Particular attention was given to allopolyploid study systems, species resulting from an hybridization event accompanied by WGD. Investigating the mechanisms behind the survival of a newly formed allopolyploid highlighted the key role of DNA methylation. With the improvement of high-throughput methods, such as whole genome bisulfite sequencing (WGBS), an opportunity opened to further understand the role of DNA methylation at a larger scale and higher resolution. However, only a few studies have applied WGBS to allopolyploids, which might be due to lack of genomic resources combined with a burdensome data analysis process. To overcome these problems, we developed the Automated Reproducible Polyploid EpiGenetic GuIdance workflOw (ARPEGGIO): the first workflow for the analysis of epigenetic data in polyploids. This workflow analyzes WGBS data from allopolyploid species via the genome assemblies of the allopolyploid’s parent species. ARPEGGIO utilizes an updated read classification algorithm (EAGLE-RC), to tackle the challenge of sequence similarity amongst parental genomes. ARPEGGIO offers automation, but more importantly, a complete set of analyses including spot checks starting from raw WGBS data: quality checks, trimming, alignment, methylation extraction, statistical analyses and downstream analyses. A full run of ARPEGGIO outputs a list of genes showing differential methylation. ARPEGGIO was made simple to set up, run and interpret, and its implementation ensures reproducibility by including both package management and containerization. Results: We evaluated ARPEGGIO in two ways. First, we tested EAGLE-RC’s performance with publicly available datasets given a ground truth, and we show that EAGLE-RC decreases the error rate by 3 to 4 times compared to standard approaches. Second, using the same initial dataset, we show agreement between ARPEGGIO’s output and published results. Compared to other similar workflows, ARPEGGIO is the only one supporting polyploid data. Conclusions: The goal of ARPEGGIO is to promote, support and improve polyploid research with a reproducible and automated set of analyses in a convenient implementation. ARPEGGIO is available at https://github.com/supermaxiste/ARPEGGIO., MC genomics, 22 (1), ISSN:1471-2164

Summary Polyploidization is pervasive in plants, but little is known about the niche divergence of wild allopolyploids (species that harbor polyploid genomes originating from different diploid species) relative to their diploid progenitor species and the gene expression patterns that may underlie such ecological divergence. We conducted a fine‐scale empirical study on habitat and gene expression of an allopolyploid and its diploid progenitors.We quantified soil properties and light availability of habitats of an allotetraploid Cardamine flexuosa and its diploid progenitors Cardamine amara and Cardamine hirsuta in two seasons. We analyzed expression patterns of genes and homeologs (homeologous gene copies in allopolyploids) using RNA sequencing.We detected niche divergence between the allopolyploid and its diploid progenitors along water availability gradient at a fine scale: the diploids in opposite extremes and the allopolyploid in a broader range between diploids, with limited overlap with diploids at both ends. Most of the genes whose homeolog expression ratio changed among habitats in C. flexuosa varied spatially and temporally.These findings provide empirical evidence for niche divergence between an allopolyploid and its diploid progenitor species at a fine scale and suggest that divergent expression patterns of homeologs in an allopolyploid may underlie its persistence in diverse habitats.

Contemporary speciation provides a unique opportunity to directly observe the traits and environmental responses of a new species. Cardamine insueta is an allotriploid species that appeared within the past 150 years in a Swiss village, Urnerboden. In contrast to its two progenitor species, C. amara and C. rivularis that live in wet and open habitats, respectively, C. insueta is found in-between their habitats with temporal water level fluctuation. This triploid species propagates clonally and serves as a triploid bridge to form higher ploidy species. Although niche separation is observed in field studies, the mechanisms underlying the environmental robustness of C. insueta are not clear. To characterize responses to a fluctuating environment, we performed a time-course analysis of homeolog gene expression in C. insueta in response to submergence treatment. For this purpose, the two parental (C. amara and C. rivularis) genome sequences were assembled with a reference-guided approach, and homeolog-specific gene expression was quantified by using HomeoRoq software. We found that C. insueta and C. rivularis initiated vegetative propagation by forming ectopic meristems on leaves, while C. amara did not. We examined homeolog-specific gene expression of three species at nine time points during the treatment. The genome-wide expression ratio of homeolog pairs was 2:1 over the time-course, consistent with the ploidy number. By searching the genes with high coefficient of variation of expression over time-course transcriptome data, we found many known key transcriptional factors related to meristem development and formation upregulated in both C. rivularis and rivularis-homeolog of C. insueta, but not in C. amara. Moreover, some amara-homeologs of these genes were also upregulated in the triploid, suggesting trans-regulation. In turn, Gene Ontology analysis suggested that the expression pattern of submergence tolerant genes in the triploid was inherited from C. amara. These results suggest that the triploid C. insueta combined advantageous patterns of parental transcriptomes to contribute to its establishment in a new niche along a water-usage gradient.

To study the genetic structure of clonal plant populations, genotyping and genet detection using genetic markers are necessary to assign ramets to corresponding genets. Assignment is difficult as it involves setting a robust threshold of genetic distance for genet distinction as neighbouring genets in a plant population are often genetically related. Here, we used restriction site-associated DNA sequencing (RAD-seq) for a rhizomatous clonal herb, Cardamine leucantha [Brassicaceae] to accurately determine genet structure in a natural population. We determined a draft genome sequence of this species for the first time, which resulted in 66 617 scaffolds with N50 = 6086 bp and an estimated genome size of approximately 253 Mbp. Using genetic distances based on the RAD-seq analysis, we successfully distinguished ramets that belonged to distinct genets even from a half-sib family. We applied these methods to 372 samples of C. leucantha collected at 1-m interval grids within a 20 × 20 m plot in a natural population in Hokkaido, Japan. From these samples, we identified 61 genets with high inequality in terms of genet size and patchy distribution. Spatial autocorrelation analyses indicated significant aggregation within 7 and 4 m at ramet and genet levels, respectively. An analysis of parallel DNA microsatellite loci (simple sequence repeats) suggested that RAD-seq can provide data that allows robust genet assignment. It remains unclear whether the large genets identified here became dominant stochastically or deterministically. Precise identification of genets will assist further study and characterization of dominant genets., Clonal plants produce genetically identical offspring through vegetative growth. A ‘ramet’ refers to a single physiological individual produced by clonal propagation; a ‘genet’ refers to a group of ramets that originate from a single seed. Here we investigated how ramets that belong to different genets distribute in a natural population of Carrdamine leucantha using a genome-wide SNP markers. We applied the method to 372 samples collected at 1-m interval grids within a 20 × 20 m plot, and identified 61 genets with high inequality in terms of genet size and patchy distribution.

Population genetics studies of allopolyploid species lag behind those of diploid species because of practical difficulties in analysis of homeologs-duplicated gene copies originating from hybridized parental species. Pool-Seq, i.e. massive parallel sequencing of pooled individuals, has high potential for detecting nucleotide polymorphisms within and among multiple populations; however, its use has been limited to diploid species. We applied Pool-Seq to an allopolyploid species by developing a bioinformatic pipeline that assigns reads to each homeolog as well as to each polymorphic allele within each homeolog. We simultaneously sequenced eight genes from twenty individuals from each of 24 populations, and found over 100 polymorphic sites in each homeolog. For two sites, we estimated allele frequencies using the number of reads and then validated these estimations by making individual-based estimations. Pool-Seq using our bioinformatic pipeline allows efficient evaluation of nucleotide polymorphisms in a large number of individuals, even in allopolyploid species.

Background:Genetic variation in plants alters insect abundance and community structure in the field; however, little is known about the importance of a single gene among diverse plant genotypes. In this context,Arabidopsistrichomes provide an excellent system to discern the roles of natural variation and a key gene,GLABRA1, in shaping insect communities. In this study, we transplanted two independent glabrous mutants (gl1-1andgl1-2) and 17 natural accessions ofArabidopsis thalianato two localities in Switzerland and Japan.Results:Fifteen insect species inhabited plant accessions, with 10–30% broad-sense heritability of community indices being detected, such as species richness and diversity. The total abundance of leaf-chewing herbivores was negatively correlated with trichome density at both the field sites, while glucosinolates had variable effects on leaf chewers between the two sites. Interestingly, there was a parallel tendency for the abundance of leaf chewers to be higher ongl1-1andgl1-2than for their different parental accessions, Ler-1 and Col-0, respectively. Furthermore, the loss of function in theGLABRA1gene significantly decreased the resistance of plants to the two predominant chewers, flea beetles and turnip sawflies.Conclusions:Overall, our results indicate that insect community composition onA. thalianais heritable across two distant field sites, withGLABRA1playing a key role in altering the abundance of leaf-chewing herbivores. Given that such a trichome variation is widely observed in Brassicaceae plants, the present study exemplifies the community-wide impact of a single plant gene on crucifer-feeding insects in the field.

Recently, increasing attempts have been made to understand how plant genes function in natura. In this context, transcriptional profiles represent plant physiological status in response to environmental stimuli. Herein, we combined high-throughput RNA-Seq with insect survey data on 19 accessions of Arabidopsis thaliana grown at a field site in Switzerland. We found that genes with the gene ontology (GO) annotations of “glucosinolate biosynthetic process” and “response to insects” were most significantly enriched, and the expression of these genes was highly variable among plant accessions. Nearly half of the total expression variation in the glucosinolate biosynthetic genes (AOPs, ESM1, ESP, and TGG1) was explained by among-accession variation. Of these genes, the expression level of AOP3 differed among Col-0 accession individuals depending on the abundance of the mustard aphid (Lipaphis erysimi). We also found that the expression of the major cis-jasmone activated gene CYP81D11 was positively correlated with the number of flea beetles (Phyllotreta striolata and Phyllotreta atra). Combined with the field RNA-Seq data, bioassays confirmed that AOP3 was up-regulated in response to attack by mustard aphids. The combined results from RNA-Seq and our ecological survey illustrate the feasibility of using field transcriptomics to detect an inducible defense, providing a first step towards an in natura understanding of biotic interactions involving phenotypic plasticity.

The habitats of polyploid species are generally distinct from their parental species. Stebbins described polyploids as 'general purpose genotypes', which can tolerate a wide range of environmental conditions. However, little is known about its molecular basis because of the complexity of polyploid genomes. We hypothesized that allopolyploid species might utilize the expression patterns of both parents depending on environments (polyploid plasticity hypothesis). We focused on hydrological niche segregation along fine-scale soil moisture and waterlogging gradients. Two diploid species, Cardamine amara and Cardamine hirsuta, grew best in submerged and unsubmerged conditions, respectively, consistent with their natural habitats. Interestingly, the allotetraploid Cardamine flexuosa derived from them grew similarly in fluctuating as well as submerged and unsubmerged conditions, consistent with its wide environmental tolerance. A similar pattern was found in another species trio: allotetraploid Cardamine scutata and its parents. Using the close relatedness of Cardamine and Arabidopsis, we quantified genomewide expression patterns following dry and wet treatments using an Arabidopsis microarray. Hierarchical clustering analysis revealed that the expression pattern of C. flexuosa clustered with C. hirsuta in the dry condition and with C. amara in the wet condition, supporting our hypothesis. Furthermore, the induction levels of most genes in the allopolyploid were lower than in a specialist diploid species. This reflects a disadvantage of being allopolyploid arising from fixed heterozygosity. We propose that recurrent allopolyploid speciation along soil moisture and waterlogging gradients confers niche differentiation and reproductive isolation simultaneously and serves as a model for studying the molecular basis of ecological speciation and adaptive radiation.

Whole genome sequences, which can be provided even for non-model organisms owing to high-throughput sequencers, are valuable in enhancing the understanding of adaptive evolution. Metrosideros polymorpha, a tree species endemic to the Hawaiian Islands, occupies a wide range of ecological habitats and shows remarkable polymorphism in phenotypes among/within populations. The biological functions of genetic variations observed within this species could provide significant insights into the adaptive radiation found in a single species. Here de novo assembled genome sequences of M. polymorpha are presented to reveal basic genomic parameters about this species and to develop our knowledge of ecological divergences. The assembly yielded 304-Mbp genome sequences, half of which were covered by 19 scaffolds with >5 Mbp, and contained 30 K protein-coding genes. Demographic history inferred from the genome-wide heterozygosity indicated that this species experienced a dramatic rise and fall in the effective population size, possibly owing to past geographic or climatic changes in the Hawaiian Islands. This M. polymorpha genome assembly represents a high-quality genome resource useful for future functional analyses of both intra- and interspecies genetic variations or comparative genomics.

Allopolyploids possess complete sets of genomes derived from different parental species and exhibit a range of variation in various traits. Reproductive traits may play a key role in the reproductive isolation between allopolyploids and their parental species, thus affecting the thriving of allopolyploids. However, empirical data, especially in natural habitats, comparing reproductive trait variation between allopolyploids and their parental species remain rare. Here, we documented the flowering phenology and floral morphology of the allopolyploid wild plantCardamine flexuosaand its diploid parentsC. amaraandC. hirsutain their native range in Switzerland. The flowering ofC. flexuosastarted at an intermediate time compared with those of the parents and the flowering period ofC. flexuosaoverlapped with those of the parents.Cardamine flexuosaresembledC. hirsutain the size of flowers and petals and the length/width ratio of petals, while it resembledC. amarain the length/width ratio of flowers. These results provide empirical evidence of the trait-dependent variation of allopolyploid phenotypes in natural habitats at the local scale. They also suggest that the variation in some reproductive traits inC. flexuosais associated with self-fertilization. Therefore, it is helpful to consider the mating system in furthering the understanding of the processes that may have shaped trait variation in polyploids in nature.

Genome duplication is widespread in wild and crop plants. However, little is known about genome-wide selection in polyploids due to the complexity of duplicated genomes. In polyploids, the patterns of purifying selection and adaptive substitutions may be affected by masking owing to duplicated genes or homeologs as well as effective population size. Here, we resequence 25 accessions of the allotetraploid Arabidopsis kamchatica, which is derived from the diploid species A. halleri and A. lyrata. We observe a reduction in purifying selection compared with the parental species. Interestingly, proportions of adaptive non-synonymous substitutions are significantly positive in contrast to most plant species. A recurrent pattern observed in both frequency and divergence–diversity neutrality tests is that the genome-wide distributions of both subgenomes are similar, but the correlation between homeologous pairs is low. This may increase the opportunity of different evolutionary trajectories such as in the HMA4 gene involved in heavy metal hyperaccumulation., Nature Communications, 9, ISSN:2041-1723

Finger millet (Eleusine coracana (L.) Gaertn) is an important crop for food security because of its tolerance to drought, which is expected to be exacerbated by global climate changes. Nevertheless, it is often classified as an orphan/underutilized crop because of the paucity of scientific attention. Among several small millets, finger millet is considered as an excellent source of essential nutrient elements, such as iron and zinc; hence, it has potential as an alternate coarse cereal. However, high-quality genome sequence data of finger millet are currently not available. One of the major problems encountered in the genome assembly of this species was its polyploidy, which hampers genome assembly compared with a diploid genome. To overcome this problem, we sequenced its genome using diverse technologies with sufficient coverage and assembled it via a novel multiple hybrid assembly workflow that combines next-generation with single-molecule sequencing, followed by whole-genome optical mapping using the Bionano Irys® system. The total number of scaffolds was 1,897 with an N50 length >2.6 Mb and detection of 96% of the universal single-copy orthologs. The majority of the homeologs were assembled separately. This indicates that the proposed workflow is applicable to the assembly of other allotetraploid genomes., DNA Research, 25 (1), ISSN:1340-2838, ISSN:1756-1663

Pitchers are modified leaves used by carnivorous plants for trapping prey. Their fluids contain digestive enzymes from the plant and they harbor abundant microbes. In this study, the diversity of bacterial communities was assessed in Nepenthes pitcher fluids and the composition of the bacterial community was compared to that in other environments, including the phyllosphere of Arabidopsis, animal guts and another pitcher plant, Sarracenia. Diversity was measured by 454 pyrosequencing of 16S rRNA gene amplicons. A total of 232,823 sequences were obtained after chimera and singleton removal that clustered into 3260 distinct operational taxonomic units (OTUs) (3% dissimilarity), which were taxonomically distributed over 17 phyla, 25 classes, 45 orders, 100 families, and 195 genera. Pyrosequencing and fluorescence in situ hybridization yielded similar estimates of community composition. Most pitchers contained high proportions of unique OTUs, and only 22 OTUs (

Background and Aims The coexistence of hermaphrodites and female-sterile individuals, or androdioecy, has been documented in only a handful of plants and animals. This study reports its existence in the plant species Cardamine amara (Brassicaceae), in which female-sterile individuals have shorter pistils than seed-producing hermaphrodites. Methods Morphological analysis, in situ manual pollination, microsatellite genotyping and differential gene expression analysis using Arabidopsis microarrays were used to delimit variation between female-sterile individuals and hermaphrodites. Key Results Female sterility in C. amara appears to be caused by disrupted ovule development. It was associated with a 2.4- to 2.9-fold increase in clonal propagation. This made the pollen number of female-sterile genets more than double that of hermaphrodite genets, which fulfils a condition of co-existence predicted by simple androdioecy theories. When female-sterile individuals were observed in wild androdioecious populations, their ramet frequencies ranged from 5 to 54 %; however, their genet frequencies ranged from 11 to 29 %, which is consistent with the theoretically predicted upper limit of 50 %. Conclusions The results suggest that a combination of sexual reproduction and increased asexual proliferation by female-sterile individuals probably explains the invasion and maintenance of female sterility in otherwise hermaphroditic populations. To our knowledge, this is the first report of the coexistence of female sterility and hermaphrodites in the Brassicaceae.

This article describes the use of cytogenomic and molecular approaches to explore the origin and evolution of Cardamine schulzii, a textbook example of a recent allopolyploid, in its ;110-year history of human-induced hybridization and allopolyploidy in the Swiss Alps. Triploids are typically viewed as bridges between diploids and tetraploids but rarely as parental genomes of high-level hybrids and polyploids. The genome of the triploid semifertile hybrid Cardamine 3 insueta (2n = 24, RRA) was shown to combine the parental genomes of two diploid (2n =2 x = 16) species, Cardamine amara (AA) and Cardamine rivularis (RR). These parental genomes have remained structurally stable within the triploid genome over the >100 years since its origin. Furthermore, we provide compelling evidence that the alleged recent polyploid C. schulzii is not an autohexaploid derivative of C. 3 insueta. Instead, at least two hybridization events involving C. 3 insueta and the hypotetraploid Cardamine pratensis (PPPP, 2n =4 x22 = 30) have resulted in the origin of the trigenomic hypopentaploid (2n =5 x22 = 38, PPRRA) and hypohexaploid (2n =6 x22 = 46, PPPPRA). These data show that the semifertile triploid hybrid can promote a merger of three different genomes and demonstrate how important it is to reexamine the routinely repeated textbook examples using modern techniques.

Allopolyploidization combines parental genomes and often confers broader species distribution. However, little is known about parentally transmitted gene expression underlying quantitative traits following allopolyploidization because of the complexity of polyploid genomes. The allopolyploid species Arabidopsis kamchatica is a natural hybrid of the zinc hyperaccumulator Arabidopsis halleri and of the nonaccumulator Arabidopsis lyrata. We found that A. kamchatica retained the ability to hyperaccumulate zinc from A. halleri and grows in soils with both low and high metal content. Hyperaccumulation of zinc by A. kamchatica was reduced to about half of A. halleri, but is 10-fold greater than A. lyrata. Homeologs derived from A. halleri had significantly higher levels of expression of genes such as HEAVY METAL ATPASE4 (HMA4), METAL TRANSPORTER PROTEIN1 and other metal ion transporters than those derived from A. lyrata, which suggests cis-regulatory differences. A. kamchatica has on average about half the expression of these genes compared with A. halleri due to fixed heterozygosity inherent in allopolyploids. Zinc treatment significantly changed the ratios of expression of 1% of homeologous pairs, including genes putatively involved in metal homeostasis. Resequencing data showed a significant reduction in genetic diversity over a large genomic region (290 kb) surrounding the HMA4 locus derived from the A. halleri parent compared with the syntenic A. lyrata-derived region, which suggests different evolutionary histories. We also estimated that three A. halleri-derived HMA4 copies are present in A. kamchatica. Our findings support a transcriptomic model in which environment-related transcriptional patterns of both parents are conserved but attenuated in the allopolyploids., Molecular Biology and Evolution, 33 (11), ISSN:0737-4038, ISSN:1537-1719

Self-incompatibility (SI) systems in flowering plants distinguish self- and non-self pollen to prevent inbreeding. While other SI systems rely on the self-recognition between specific male- and female-determinants, the Solanaceae family has a non-self recognition system resulting in the detoxification of female-determinants of S-ribonucleases (S-RNases), expressed in pistils, by multiple male-determinants of S-locus F-box proteins (SLFs), expressed in pollen. It is not known how many SLF components of this non-self recognition system there are in Solanaceae species, or how they evolved. We identified 16?20 SLFs in each S-haplotype in SI Petunia, from a total of 168 SLF sequences using large-scale next-generation sequencing and genomic polymerase chain reaction (PCR) techniques. We predicted the target S-RNases of SLFs by assuming that a particular S-allele must not have a conserved SLF that recognizes its own S-RNase, and validated these predictions by transformation experiments. A simple mathematical model confirmed that 16?20 SLF sequences would be adequate to recognize the vast majority of target S-RNases. We found evidence of gene conversion events, which we suggest are essential to the constitution of a non-self recognition system and also contribute to self-compatible mutations., [プレスリリース]植物が自分以外の花粉を選ぶための遺伝子セットを特定！ ～生物の非自己認識システムの進化の謎解明へ～, [Author's version title]Gene duplication and genetic exchange drive the evolution of S-RNase based self-incompatibility

The genus Nepenthes, a carnivorous plant, has a pitcher to trap insects and digest them in the contained fluid to gain nutrient. A distinctive character of the pitcher fluid is the digestive enzyme activity that may be derived from plants and dwelling microbes. However, little is known about in situ digestive enzymes in the fluid. Here we examined the pitcher fluid from four species of Nepenthes. High bacterial density was observed within the fluids, ranging from 7×10(6) to 2.2×10(8) cells ml(-1). We measured the activity of three common enzymes in the fluid: acid phosphatases, β-D-glucosidases, and β-D-glucosaminidases. All the tested enzymes detected in the liquid of all the pitcher species showed activity that considerably exceeded that observed in aquatic environments such as freshwater, seawater, and sediment. Our results indicate that high enzyme activity within a pitcher could assist in the rapid decomposition of prey to maximize efficient nutrient use. In addition, we filtered the fluid to distinguish between dissolved enzyme activity and particle-bound activity. As a result, filtration treatment significantly decreased the activity in all enzymes, while pH value and Nepenthes species did not affect the enzyme activity. It suggested that enzymes bound to bacteria and other organic particles also would significantly contribute to the total enzyme activity of the fluid. Since organic particles are themselves usually colonized by attached and highly active bacteria, it is possible that microbe-derived enzymes also play an important role in nutrient recycling within the fluid and affect the metabolism of the Nepenthes pitcher plant.

Ever since Darwin's pioneering research, the evolution of self-fertilisation (selfing) has been regarded as one of the most prevalent evolutionary transitions in flowering plants. A major mechanism to prevent selfing is the self-incompatibility (SI) recognition system, which consists of male and female specificity genes at the S-locus and SI modifier genes. Under conditions that favour selfing, mutations disabling the male recognition component are predicted to enjoy a relative advantage over those disabling the female component, because male mutations would increase through both pollen and seeds whereas female mutations would increase only through seeds. Despite many studies on the genetic basis of loss of SI in the predominantly selfing plant Arabidopsis thaliana, it remains unknown whether selfing arose through mutations in the female specificity gene (S-receptor kinase, SRK), male specificity gene (S-locus cysteine-rich protein, SCR; also known as S-locus protein 11, SP11) or modifier genes, and whether any of them rose to high frequency across large geographic regions. Here we report that a disruptive 213-base-pair (bp) inversion in the SCR gene (or its derivative haplotypes with deletions encompassing the entire SCR-A and a large portion of SRK-A) is found in 95% of European accessions, which contrasts with the genome-wide pattern of polymorphism in European A. thaliana. Importantly, interspecific crossings using Arabidopsis halleri as a pollen donor reveal that some A. thaliana accessions, including Wei-1, retain the female SI reaction, suggesting that all female components including SRK are still functional. Moreover, when the 213-bp inversion in SCR was inverted and expressed in transgenic Wei-1 plants, the functional SCR restored the SI reaction. The inversion within SCR is the first mutation disrupting SI shown to be nearly fixed in geographically wide samples, and its prevalence is consistent with theoretical predictions regarding the evolutionary advantage of mutations in male components.

Genome duplication with hybridization, or allopolyploidization, occurs commonly in plants, and is considered to be a strong force for generating new species. However, genome-wide quantification of homeolog expression ratios was technically hindered because of the high homology between homeologous gene pairs. To quantify the homeolog expression ratio using RNA-seq obtained from polyploids, a new method named HomeoRoq was developed, in which the genomic origin of sequencing reads was estimated using mismatches between the read and each parental genome. To verify this method, we first assembled the two diploid parental genomes of Arabidopsis halleri subsp. gemmifera and Arabidopsis lyrata subsp. petraea (Arabidopsis petraea subsp. umbrosa), then generated a synthetic allotetraploid, mimicking the natural allopolyploid Arabidopsis kamchatica. The quantified ratios corresponded well to those obtained by Pyrosequencing. We found that the ratios of homeologs before and after cold stress treatment were highly correlated (r = 0.870). This highlights the presence of nonstochastic polyploid gene regulation despite previous research identifying stochastic variation in expression. Moreover, our new statistical test incorporating overdispersion identified 226 homeologs (1.11% of 20 369 expressed homeologs) with significant ratio changes, many of which were related to stress responses. HomeoRoq would contribute to the study of the genes responsible for polyploid-specific environmental responses.