Genome-Enabled Analysis of Population Dynamics and Virulence-Associated Loci in the Oat Crown Rust Fungus Puccinia coronata f. sp. avenae.

Puccinia coronata f. sp. avenae (Pca) is an important fungal pathogen causing crown rust that impacts oat production worldwide. Genetic resistance for crop protection against Pca is often overcome by the rapid virulence evolution of the pathogen. This study investigated the factors shaping adaptive evolution of Pca using pathogen populations from distinct geographic regions within the United States and South Africa. Phenotypic and genome-wide sequencing data of these diverse Pca collections, including 217 isolates, uncovered phylogenetic relationships and established distinct genetic composition between populations from northern and southern regions from the United States and South Africa. The population dynamics of Pca involve a bidirectional movement of inoculum between northern and southern regions of the United States and contributions from clonality and sexuality. The population from South Africa is solely clonal. A genome-wide association study (GWAS) employing a haplotype-resolved Pca reference genome was used to define 11 virulence-associated loci corresponding to 25 oat differential lines. These regions were screened to determine candidate Avr effector genes. Overall, the GWAS results allowed us to identify the underlying genetic factors controlling pathogen recognition in an oat differential set used in the United States to assign pathogen races (pathotypes). Key GWAS findings support complex genetic interactions in several oat lines, suggesting allelism among resistance genes or redundancy of genes included in the differential set, multiple resistance genes recognizing genetically linked Avr effector genes, or potentially epistatic relationships. A careful evaluation of the composition of the oat differential set accompanied by the development or implementation of molecular markers is recommended. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Genetic Analysis and Physical Mapping of Oat Adult Plant Resistance Loci Against Puccinia coronata f. sp. avenae.

Six quantitative trait loci (QTLs) for adult plant resistance against oat crown rust (Puccinia coronata f. sp. avenae) were identified from mapping three recombinant inbred populations. Using genotyping-by-sequencing with markers called against the OT3098 v1 reference genome, the QTLs were mapped on six different chromosomes: Chr1D, Chr4D, Chr5A, Chr5D, Chr7A, and Chr7C. Composite interval mapping with marker cofactor selection showed that the phenotypic variance explained by all identified QTLs for coefficient of infection range from 12.2 to 46.9%, whereas heritability estimates ranged from 0.11 to 0.38. The significant regions were narrowed down to intervals of 3.9 to 25 cM, equivalent to physical distances of 11 to 133 Mb. At least two flanking single-nucleotide polymorphism markers were identified within 10 cM of each QTL that could be used in marker-assisted introgression, pyramiding, and selection. The additive effects of the QTLs in each population were determined using single-nucleotide polymorphism haplotype data, which showed a significantly lower coefficient of infection in lines homozygous for the resistant alleles. Analysis of pairwise linkage disequilibrium also revealed high correlation of markers and presence of linkage blocks in the significant regions. To further facilitate marker-assisted breeding, polymerase chain reaction allelic competitive extension (PACE) markers for the adult plant resistance loci were developed. Putative candidate genes were also identified in each of the significant regions, which include resistance gene analogs that encode for kinases, ligases, and predicted receptors of avirulence proteins from pathogens.

Increased virulence of Puccinia coronata f. sp.avenae populations through allele frequency changes at multiple putative Avr loci.

Pathogen populations are expected to evolve virulence traits in response to resistance deployed in agricultural settings. However, few temporal datasets have been available to characterize this process at the population level. Here, we examined two temporally separated populations of Puccinia coronata f. sp. avenae (Pca), which causes crown rust disease in oat (Avena sativa) sampled from 1990 to 2015. We show that a substantial increase in virulence occurred from 1990 to 2015 and this was associated with a genetic differentiation between populations detected by genome-wide sequencing. We found strong evidence for genetic recombination in these populations, showing the importance of the alternate host in generating genotypic variation through sexual reproduction. However, asexual expansion of some clonal lineages was also observed within years. Genome-wide association analysis identified seven Avr loci associated with virulence towards fifteen Pc resistance genes in oat and suggests that some groups of Pc genes recognize the same pathogen effectors. The temporal shift in virulence patterns in the Pca populations between 1990 and 2015 is associated with changes in allele frequency in these genomic regions. Nucleotide diversity patterns at a single Avr locus corresponding to Pc38, Pc39, Pc55, Pc63, Pc70, and Pc71 showed evidence of a selective sweep associated with the shift to virulence towards these resistance genes in all 2015 collected isolates.

A reference-anchored oat linkage map reveals quantitative trait loci conferring adult plant resistance to crown rust (Puccinia coronata f. sp. avenae).

KEY MESSAGE: We mapped three adult plant resistance (APR) loci on oat chromosomes 4D and 6C and developed flanking KASP/PACE markers for marker-assisted selection and gene pyramiding. Using sequence orthology search and the available oat genomic and transcriptomic data, we surveyed these genomic regions for genes that may control disease resistance. Sources of durable disease resistance are needed to minimize yield losses in cultivated oat caused by crown rust (Puccinia coronata f. sp. avenae). In this study, we developed five oat recombinant inbred line mapping populations to identify sources of adult plant resistance from crosses between five APR donors and Otana, a susceptible variety. The preliminary bulk segregant mapping based on allele frequencies showed two regions in linkage group Mrg21 (Chr4D) that are associated with the APR phenotype in all five populations. Six markers from these regions in Chr4D were converted to high-throughput allele specific PCR assays and were used to genotype all individuals in each population. Simple interval mapping showed two peaks in Chr4D, named QPc.APR-4D.1 and QPc.APR-4D.2, which were detected in the OtanaA/CI4706-2 and OtanaA/CI9416-2 and in the Otana/PI189733, OtanaD/PI260616, and OtanaA/CI8000-4 populations, respectively. These results were validated by mapping two entire populations, Otana/PI189733 and OtanaA/CI9416, genotyped using Illumina HiSeq, in which polymorphisms were called against the OT3098 oat reference genome. Composite interval mapping results confirmed the presence of the two quantitative trait loci (QTL) located on oat chromosome 4D and an additional QTL with a smaller effect located on chromosome 6C. This mapping approach also narrowed down the physical intervals to between 5 and 19 Mb, and indicated that QPc.APR-4D.1, QPc.APR-4D.2, and QPc.APR-6C explained 43.4%, 38.5%, and 21.5% of the phenotypic variation, respectively. In a survey of the gene content of each QTL, several clusters of disease resistance genes that may contribute to APR were found. The allele specific PCR markers developed for these QTL regions would be beneficial for marker-assisted breeding, gene pyramiding, and future cloning of resistance genes from oat.

Identification, introgression, and molecular marker genetic analysis and selection of a highly effective novel oat crown rust resistance from diploid oat, Avena strigosa.

KEY MESSAGE: Oat crown rust is one of the most damaging diseases of oat. We identified a new source of resistance and developed KASP and TaqMan markers for selection in breeding programs. A new highly effective resistance to oat crown rust (Puccinia coronata f. sp. avenae) was identified in the diploid oat Avena strigosa PI 258731 and introgressed into hexaploid cultivated oat. Young plants with this resistance show moderate susceptibility, whereas older plant tissues and adult plants are resistant with no virulent isolates encountered in over 8 years of testing. Resistance was incorporated into hexaploid oat by embryo rescue, colchicine chromosome doubling followed by backcrosses with a hexaploid parent, and selection for stable transmission of resistance. To mitigate flag leaf and panicle chlorosis/necrosis associated with the resistance, crosses were made with derived resistant lines to breeding lines of divergent parentage followed by selection. Subsequently, two F2 sister lines, termed MNBT1020-1 and MNBT1021-1, were identified in which the chlorosis/necrosis was reduced. These two lines performed well in replicated multi-location state trials in 2015 and 2016 out-yielding all cultivar entries. Segregating F2:3 plants resulting from crosses of MNBT lines to susceptible parents were genotyped with the oat 6K SNP array, and SNP loci with close linkage to the resistance were identified. KASP assays generated from linked SNPs showed accurate discrimination of the resistance in derivatives of the resistant MNBT lines crossed to susceptible breeding lines. A TaqMan marker was developed and correctly identified homozygous resistance in over 95% of 379 F4 plants when rust was scored in F4:5 plants in the field. Thus, a novel highly effective resistance and associated molecular markers are available for use in breeding, genetic analysis, and functional studies.

Detection of Race-Specific Resistance Against Puccinia coronata f. sp. avenae in Brachypodium Species.

Oat crown rust caused by Puccinia coronata f. sp. avenae is the most destructive foliar disease of cultivated oat. Characterization of genetic factors controlling resistance responses to Puccinia coronata f. sp. avenae in nonhost species could provide new resources for developing disease protection strategies in oat. We examined symptom development and fungal colonization levels of a collection of Brachypodium distachyon and B. hybridum accessions infected with three North American P. coronata f. sp. avenae isolates. Our results demonstrated that colonization phenotypes are dependent on both host and pathogen genotypes, indicating a role for race-specific responses in these interactions. These responses were independent of the accumulation of reactive oxygen species. Expression analysis of several defense-related genes suggested that salicylic acid and ethylene-mediated signaling but not jasmonic acid are components of resistance reaction to P. coronata f. sp. avenae. Our findings provide the basis to conduct a genetic inheritance study to examine whether effector-triggered immunity contributes to nonhost resistance to P. coronata f. sp. avenae in Brachypodium spp.

A Role for CHH Methylation in the Parent-of-Origin Effect on Altered Circadian Rhythms and Biomass Heterosis in Arabidopsis Intraspecific Hybrids.

Hybrid plants and animals often show increased levels of growth and fitness, a phenomenon known as hybrid vigor or heterosis. Circadian rhythms optimize physiology and metabolism in plants and animals. In plant hybrids and polyploids, expression changes of the genes within the circadian regulatory network, such as CIRCADIAN CLOCK ASSOCIATED1 (CCA1), lead to heterosis. However, the relationship between allelic CCA1 expression and heterosis has remained elusive. Here, we show a parent-of-origin effect on altered circadian rhythms and heterosis in Arabidopsis thaliana F1 hybrids. This parent-of-origin effect on biomass heterosis correlates with altered CCA1 expression amplitudes, which are associated with methylation levels of CHH (where H = A, T, or C) sites in the promoter region. The direction of rhythmic expression and hybrid vigor is reversed in reciprocal F1 crosses involving mutants that are defective in the RNA-directed DNA methylation pathway (argonaute4 and nuclear RNA polymerase D1a) but not in the maintenance methylation pathway (methyltransferase1 and decrease in DNA methylation1). This parent-of-origin effect on circadian regulation and heterosis is established during early embryogenesis and maintained throughout growth and development.

cis- and trans-Regulation of miR163 and target genes confers natural variation of secondary metabolites in two Arabidopsis species and their allopolyploids.

MicroRNAs (miRNAs) play essential roles in plant and animal development, but the cause and effect of miRNA expression divergence between closely related species and in interspecific hybrids or allopolyploids are unknown. Here, we show differential regulation of a miR163-mediated pathway in allotetraploids and their progenitors, Arabidopsis thaliana and Arabidopsis arenosa. miR163 is a recently evolved miRNA in A. thaliana and highly expressed in A. thaliana, but its expression was undetectable in A. arenosa and repressed in resynthesized allotetraploids. Repression of A. arenosa MIR163 (Aa MIR163) is caused by a weak cis-acting promoter and putative trans-acting repressor(s) present in A. arenosa and allotetraploids. Moreover, ectopic Aa MIR163 precursors were processed more efficiently in A. thaliana than in resynthesized allotetraploids, suggesting a role of posttranscriptional regulation in mature miR163 abundance. Target genes of miR163 encode a family of small molecule methyltransferases involved in secondary metabolite biosynthetic pathways that are inducible by a fungal elicitor, alamethicin. Loss of miR163 or overexpression of miR163 in mir163 mutant plants alters target transcript and secondary metabolite profiles. We suggest that cis- and trans-regulation of miRNA and other genes provides a molecular basis for natural variation of biochemical and metabolic pathways that are important to growth vigor and stress responses in Arabidopsis-related species and allopolyploids.

Identification of Candidate Susceptibility Genes to Puccinia graminis f. sp. tritici in Wheat.

Wheat stem rust disease caused by Puccinia graminis f. sp. tritici (Pgt) is a global threat to wheat production. Fast evolving populations of Pgt limit the efficacy of plant genetic resistance and constrain disease management strategies. Understanding molecular mechanisms that lead to rust infection and disease susceptibility could deliver novel strategies to deploy crop resistance through genetic loss of disease susceptibility. We used comparative transcriptome-based and orthology-guided approaches to characterize gene expression changes associated with Pgt infection in susceptible and resistant Triticum aestivum genotypes as well as the non-host Brachypodium distachyon. We targeted our analysis to genes with differential expression in T. aestivum and genes suppressed or not affected in B. distachyon and report several processes potentially linked to susceptibility to Pgt, such as cell death suppression and impairment of photosynthesis. We complemented our approach with a gene co-expression network analysis to identify wheat targets to deliver resistance to Pgt through removal or modification of putative susceptibility genes.

Emergence of the Ug99 lineage of the wheat stem rust pathogen through somatic hybridisation.

Parasexuality contributes to diversity and adaptive evolution of haploid (monokaryotic) fungi. However, non-sexual genetic exchange mechanisms are not defined in dikaryotic fungi (containing two distinct haploid nuclei). Newly emerged strains of the wheat stem rust pathogen, Puccinia graminis f. sp. tritici (Pgt), such as Ug99, are a major threat to global food security. Here, we provide genomics-based evidence supporting that Ug99 arose by somatic hybridisation and nuclear exchange between dikaryons. Fully haplotype-resolved genome assembly and DNA proximity analysis reveal that Ug99 shares one haploid nucleus genotype with a much older African lineage of Pgt, with no recombination or chromosome reassortment. These findings indicate that nuclear exchange between dikaryotes can generate genetic diversity and facilitate the emergence of new lineages in asexual fungal populations.

A Novel Approach to Assess Salt Stress Tolerance in Wheat Using Hyperspectral Imaging.

Salinity stress has significant adverse effects on crop productivity and yield. The primary goal of this study was to quantitatively rank salt tolerance in wheat using hyperspectral imaging. Four wheat lines were assayed in a hydroponic system with control and salt treatments (0 and 200 mM NaCl). Hyperspectral images were captured one day after salt application when there were no visual symptoms. Subsequent to necessary preprocessing tasks, two endmembers, each representing one of the treatment, were identified in each image using successive volume maximization. To simplify image analysis and interpretation, similarity of all pixels to the salt endmember was calculated by a technique proposed in this study, referred to as vector-wise similarity measurement. Using this approach allowed high-dimensional hyperspectral images to be reduced to one-dimensional gray-scale images while retaining all relevant information. Two methods were then utilized to analyze the gray-scale images: minimum difference of pair assignments and Bayesian method. The rankings of both methods were similar and consistent with the expected ranking obtained by conventional phenotyping experiments and historical evidence of salt tolerance. This research highlights the application of machine learning in hyperspectral image analysis for phenotyping of plants in a quantitative, interpretable, and non-invasive manner.

A Near-Complete Haplotype-Phased Genome of the Dikaryotic Wheat Stripe Rust Fungus Puccinia striiformis f. sp. tritici Reveals High Interhaplotype Diversity.

A long-standing biological question is how evolution has shaped the genomic architecture of dikaryotic fungi. To answer this, high-quality genomic resources that enable haplotype comparisons are essential. Short-read genome assemblies for dikaryotic fungi are highly fragmented and lack haplotype-specific information due to the high heterozygosity and repeat content of these genomes. Here, we present a diploid-aware assembly of the wheat stripe rust fungus Puccinia striiformis f. sp. tritici based on long reads using the FALCON-Unzip assembler. Transcriptome sequencing data sets were used to infer high-quality gene models and identify virulence genes involved in plant infection referred to as effectors. This represents the most complete Puccinia striiformis f. sp. tritici genome assembly to date (83 Mb, 156 contigs, N50 of 1.5 Mb) and provides phased haplotype information for over 92% of the genome. Comparisons of the phase blocks revealed high interhaplotype diversity of over 6%. More than 25% of all genes lack a clear allelic counterpart. When we investigated genome features that potentially promote the rapid evolution of virulence, we found that candidate effector genes are spatially associated with conserved genes commonly found in basidiomycetes. Yet, candidate effectors that lack an allelic counterpart are more distant from conserved genes than allelic candidate effectors and are less likely to be evolutionarily conserved within the P. striiformis species complex and Pucciniales In summary, this haplotype-phased assembly enabled us to discover novel genome features of a dikaryotic plant-pathogenic fungus previously hidden in collapsed and fragmented genome assemblies.IMPORTANCE Current representations of eukaryotic microbial genomes are haploid, hiding the genomic diversity intrinsic to diploid and polyploid life forms. This hidden diversity contributes to the organism's evolutionary potential and ability to adapt to stress conditions. Yet, it is challenging to provide haplotype-specific information at a whole-genome level. Here, we take advantage of long-read DNA sequencing technology and a tailored-assembly algorithm to disentangle the two haploid genomes of a dikaryotic pathogenic wheat rust fungus. The two genomes display high levels of nucleotide and structural variations, which lead to allelic variation and the presence of genes lacking allelic counterparts. Nonallelic candidate effector genes, which likely encode important pathogenicity factors, display distinct genome localization patterns and are less likely to be evolutionary conserved than those which are present as allelic pairs. This genomic diversity may promote rapid host adaptation and/or be related to the age of the sequenced isolate since last meiosis.

De Novo Assembly and Phasing of Dikaryotic Genomes from Two Isolates of Puccinia coronata f. sp. avenae, the Causal Agent of Oat Crown Rust.

Oat crown rust, caused by the fungus Pucinnia coronata f. sp. avenae, is a devastating disease that impacts worldwide oat production. For much of its life cycle, P. coronata f. sp. avenae is dikaryotic, with two separate haploid nuclei that may vary in virulence genotype, highlighting the importance of understanding haplotype diversity in this species. We generated highly contiguous de novo genome assemblies of two P. coronata f. sp. avenae isolates, 12SD80 and 12NC29, from long-read sequences. In total, we assembled 603 primary contigs for 12SD80, for a total assembly length of 99.16 Mbp, and 777 primary contigs for 12NC29, for a total length of 105.25 Mbp; approximately 52% of each genome was assembled into alternate haplotypes. This revealed structural variation between haplotypes in each isolate equivalent to more than 2% of the genome size, in addition to about 260,000 and 380,000 heterozygous single-nucleotide polymorphisms in 12SD80 and 12NC29, respectively. Transcript-based annotation identified 26,796 and 28,801 coding sequences for isolates 12SD80 and 12NC29, respectively, including about 7,000 allele pairs in haplotype-phased regions. Furthermore, expression profiling revealed clusters of coexpressed secreted effector candidates, and the majority of orthologous effectors between isolates showed conservation of expression patterns. However, a small subset of orthologs showed divergence in expression, which may contribute to differences in virulence between 12SD80 and 12NC29. This study provides the first haplotype-phased reference genome for a dikaryotic rust fungus as a foundation for future studies into virulence mechanisms in P. coronata f. sp. avenaeIMPORTANCE Disease management strategies for oat crown rust are challenged by the rapid evolution of Puccinia coronata f. sp. avenae, which renders resistance genes in oat varieties ineffective. Despite the economic importance of understanding P. coronata f. sp. avenae, resources to study the molecular mechanisms underpinning pathogenicity and the emergence of new virulence traits are lacking. Such limitations are partly due to the obligate biotrophic lifestyle of P. coronata f. sp. avenae as well as the dikaryotic nature of the genome, features that are also shared with other important rust pathogens. This study reports the first release of a haplotype-phased genome assembly for a dikaryotic fungal species and demonstrates the amenability of using emerging technologies to investigate genetic diversity in populations of P. coronata f. sp. avenae.

Optimization and Comparative Analysis of Plant Organellar DNA Enrichment Methods Suitable for Next-generation Sequencing.

Plant organellar genomes contain large, repetitive elements that may undergo pairing or recombination to form complex structures and/or sub-genomic fragments. Organellar genomes also exist in admixtures within a given cell or tissue type (heteroplasmy), and an abundance of subtypes may change throughout development or when under stress (sub-stoichiometric shifting). Next-generation sequencing (NGS) technologies are required to obtain deeper understanding of organellar genome structure and function. Traditional sequencing studies use several methods to obtain organellar DNA: (1) If a large amount of starting tissue is used, it is homogenized and subjected to differential centrifugation and/or gradient purification. (2) If a smaller amount of tissue is used (i.e., if seeds, material, or space is limited), the same process is performed as in (1), followed by whole-genome amplification to obtain sufficient DNA. (3) Bioinformatics analysis can be used to sequence the total genomic DNA and to parse out organellar reads. All these methods have inherent challenges and tradeoffs. In (1), it may be difficult to obtain such a large amount of starting tissue; in (2), whole-genome amplification could introduce a sequencing bias; and in (3), homology between nuclear and organellar genomes could interfere with assembly and analysis. In plants with large nuclear genomes, it is advantageous to enrich for organellar DNA to reduce sequencing costs and sequence complexity for bioinformatics analyses. Here, we compare a traditional differential centrifugation method with a fourth method, an adapted CpG-methyl pulldown approach, to separate the total genomic DNA into nuclear and organellar fractions. Both methods yield sufficient DNA for NGS, DNA that is highly enriched for organellar sequences, albeit at different ratios in mitochondria and chloroplasts. We present the optimization of these methods for wheat leaf tissue and discuss major advantages and disadvantages of each approach in the context of sample input, protocol ease, and downstream application.

The role of mitochondria in plant development and stress tolerance.

Eukaryotic cells require orchestrated communication between nuclear and organellar genomes, perturbations in which are linked to stress response and disease in both animals and plants. In addition to mitochondria, which are found across eukaryotes, plant cells contain a second organelle, the plastid. Signaling both among the organelles (cytoplasmic) and between the cytoplasm and the nucleus (i.e. nuclear-cytoplasmic interactions (NCI)) is essential for proper cellular function. A deeper understanding of NCI and its impact on development, stress response, and long-term health is needed in both animal and plant systems. Here we focus on the role of plant mitochondria in development and stress response. We compare and contrast features of plant and animal mitochondrial genomes (mtDNA), particularly highlighting the large and highly dynamic nature of plant mtDNA. Plant-based tools are powerful, yet underutilized, resources for enhancing our fundamental understanding of NCI. These tools also have great potential for improving crop production. Across taxa, mitochondria are most abundant in cells that have high energy or nutrient demands as well as at key developmental time points. Although plant mitochondria act as integrators of signals involved in both development and stress response pathways, little is known about plant mtDNA diversity and its impact on these processes. In humans, there are strong correlations between particular mitotypes (and mtDNA mutations) and developmental differences (or disease). We propose that future work in plants should focus on defining mitotypes more carefully and investigating their functional implications as well as improving techniques to facilitate this research.

Dissecting Plant Chromosomes by the Use of Ionizing Radiation.

Radiation treatment of genomes is used to generate chromosome breaks for numerous applications. This protocol describes the preparation of seeds and the determination of the optimal level of irradiation dosage for the creation of a radiation hybrid (RH) population. These RH lines can be used to generate high-resolution physical maps for the assembly of sequenced genomes as well as the fine mapping of genes. This procedure can also be used for mutation breeding and forward/reverse genetics.

Natural variation in timing of stress-responsive gene expression predicts heterosis in intraspecific hybrids of Arabidopsis.

The genetic distance between hybridizing parents affects heterosis; however, the mechanisms for this remain unclear. Here we report that this genetic distance correlates with natural variation and epigenetic regulation of circadian clock-mediated stress responses. In intraspecific hybrids of Arabidopsis thaliana, genome-wide expression of many biotic and abiotic stress-responsive genes is diurnally repressed and this correlates with biomass heterosis and biomass quantitative trait loci. Expression differences of selected stress-responsive genes among diverse ecotypes are predictive of heterosis in their hybrids. Stress-responsive genes are repressed in the hybrids under normal conditions but are induced to mid-parent or higher levels under stress at certain times of the day, potentially balancing the tradeoff between stress responses and growth. Consistent with this hypothesis, repression of two candidate stress-responsive genes increases growth vigour. Our findings may therefore provide new criteria for effectively selecting parents to produce high- or low-yield hybrids.

Ploidy and Hybridity Effects on Growth Vigor and Gene Expression in Arabidopsis thaliana Hybrids and Their Parents.

Both ploidy and hybridity affect cell size and growth vigor in plants and animals, but the relative effects of genome dosage and hybridization on biomass, fitness, and gene expression changes have not been systematically examined. Here we performed the first comparative analysis of seed, cell, and flower sizes, starch and chlorophyll content, biomass, and gene expression changes in diploid, triploid, and tetraploid hybrids and their respective parents in three Arabidopsis thaliana ecotypes: Columbia, C24, and Landsberg erecta (Ler). Ploidy affects many morphological and fitness traits, including stomatal size, flower size, and seed weight, whereas hybridization between the ecotypes leads to altered expression of central circadian clock genes and increased starch and chlorophyll content, biomass, and seed weight. However, varying ploidy levels has subtle effects on biomass, circadian clock gene expression, and chlorophyll and starch content. Interestingly, biomass, starch content, and seed weight are significantly different between the reciprocal hybrids at all ploidy levels tested, with the lowest and highest levels found in the reciprocal triploid hybrids, suggesting parent-of-origin effects on biomass, starch content, and seed weight. These findings provide new insights into molecular events of polyploidy and heterosis, as well as complex agronomic traits that are important to biomass and seed production in hybrid and polyploid crops.