Complementing model species with model clades.

Model species continue to underpin groundbreaking plant science research. At the same time, the phylogenetic resolution of the land plant Tree of Life continues to improve. The intersection of these two research paths creates a unique opportunity to further extend the usefulness of model species across larger taxonomic groups. Here we promote the utility of the Arabidopsis thaliana model species, especially the ability to connect its genetic and functional resources, to species across the entire Brassicales order. We focus on the utility of using genomics and phylogenomics to bridge the evolution and diversification of several traits across the Brassicales to the resources in Arabidopsis, thereby extending scope from a model species by establishing a "model clade". These Brassicales-wide traits are discussed in the context of both the model species Arabidopsis thaliana and the family Brassicaceae. We promote the utility of such a "model clade" and make suggestions for building global networks to support future studies in the model order Brassicales.

Genome balance and dosage effect drive allopolyploid formation in Brassica.

Polyploidy is a major evolutionary force that has shaped plant diversity. However, the various pathways toward polyploid formation and interploidy gene flow remain poorly understood. Here, we demonstrated that the immediate progeny of allotriploid AAC Brassica (obtained by crossing allotetraploid Brassica napus and diploid Brassica rapa) was predominantly aneuploids with ploidal levels ranging from near-triploidy to near-hexaploidy, and their chromosome numbers deviated from the theoretical distribution toward increasing chromosome numbers, suggesting that they underwent selection. Karyotype and phenotype analyses showed that aneuploid individuals containing fewer imbalanced chromosomes had higher viability and fertility. Within three generations of self-fertilization, allotriploids mainly developed into near or complete allotetraploids similar to B. napus via gradually increasing chromosome numbers and fertility, suggesting that allotriploids could act as a bridge in polyploid formation, with aneuploids as intermediates. Self-fertilized interploidy hybrids ultimately generated new allopolyploids carrying different chromosome combinations, which may create a reproductive barrier preventing allotetraploidy back to diploidy and promote gene flow from diploids to allotetraploids. These results suggest that the maintenance of a proper genome balance and dosage drove the recurrent conversion of allotriploids to allotetraploids, which may contribute to the formation and evolution of polyploids.

The final piece of the Triangle of U: Evolution of the tetraploid Brassica carinata genome.

Ethiopian mustard (Brassica carinata) is an ancient crop with remarkable stress resilience and a desirable seed fatty acid profile for biofuel uses. Brassica carinata is one of six Brassica species that share three major genomes from three diploid species (AA, BB, and CC) that spontaneously hybridized in a pairwise manner to form three allotetraploid species (AABB, AACC, and BBCC). Of the genomes of these species, that of B. carinata is the least understood. Here, we report a chromosome scale 1.31-Gbp genome assembly with 156.9-fold sequencing coverage for B. carinata, completing the reference genomes comprising the classic Triangle of U, a classical theory of the evolutionary relationships among these six species. Our assembly provides insights into the hybridization event that led to the current B. carinata genome and the genomic features that gave rise to the superior agronomic traits of B. carinata. Notably, we identified an expansion of transcription factor networks and agronomically important gene families. Completion of the Triangle of U comparative genomics platform has allowed us to examine the dynamics of polyploid evolution and the role of subgenome dominance in the domestication and continuing agronomic improvement of B. carinata and other Brassica species.

The contributions from the progenitor genomes of the mesopolyploid Brassiceae are evolutionarily distinct but functionally compatible.

The members of the tribe Brassiceae share a whole-genome triplication (WGT), and one proposed model for its formation is a two-step pair of hybridizations producing hexaploid descendants. However, evidence for this model is incomplete, and the evolutionary and functional constraints that drove evolution after the hexaploidy are even less understood. Here, we report a new genome sequence of Crambe hispanica, a species sister to most sequenced Brassiceae. Using this new genome and three others that share the hexaploidy, we traced the history of gene loss after the WGT using the Polyploidy Orthology Inference Tool (POInT). We confirm the two-step formation model and infer that there was a significant temporal gap between those two allopolyploidizations, with about a third of the gene losses from the first two subgenomes occurring before the arrival of the third. We also, for the 90,000 individual genes in our study, make parental subgenome assignments, inferring, with measured uncertainty, from which of the progenitor genomes of the allohexaploidy each gene derives. We further show that each subgenome has a statistically distinguishable rate of homoeolog losses. There is little indication of functional distinction between the three subgenomes: the individual subgenomes show no patterns of functional enrichment, no excess of shared protein-protein or metabolic interactions between their members, and no biases in their likelihood of having experienced a recent selective sweep. We propose a "mix and match" model of allopolyploidy, in which subgenome origin drives homoeolog loss propensities but where genes from different subgenomes function together without difficulty.

Vaccinium as a comparative system for understanding of complex flavonoid accumulation profiles and regulation in fruit.

The genus Vaccinium L. (Ericaceae) contains premium berryfruit crops, including blueberry, cranberry, bilberry, and lingonberry. Consumption of Vaccinium berries is strongly associated with various potential health benefits, many of which are attributed to the relatively high concentrations of flavonoids, including the anthocyanins that provide the attractive red and blue berry colors. Because these phytochemicals are increasingly appealing to consumers, they have become a crop breeding target. There has been substantial recent progress in Vaccinium genomics and genetics together with new functional data on the transcriptional regulation of flavonoids. This is helping to unravel the developmental control of flavonoids and identify genetic regions and genes that can be selected for to further improve Vaccinium crops and advance our understanding of flavonoid regulation and biosynthesis across a broader range of fruit crops. In this update we consider the recent progress in understanding flavonoid regulation in fruit crops, using Vaccinium as an example and highlighting the significant gains in both genomic tools and functional analysis.

Evolutionary history and pan-genome dynamics of strawberry (Fragaria spp.).

Strawberry (Fragaria spp.) has emerged as a model system for various fundamental and applied research in recent years. In total, the genomes of five different species have been sequenced over the past 10 y. Here, we report chromosome-scale reference genomes for five strawberry species, including three newly sequenced species' genomes, and genome resequencing data for 128 additional accessions to estimate the genetic diversity, structure, and demographic history of key Fragaria species. Our analyses obtained fully resolved and strongly supported phylogenies and divergence times for most diploid strawberry species. These analyses also uncovered a new diploid species (Fragaria emeiensis Jia J. Lei). Finally, we constructed a pan-genome for Fragaria and examined the evolutionary dynamics of gene families. Notably, we identified multiple independent single base mutations of the MYB10 gene associated with white pigmented fruit shared by different strawberry species. These reference genomes and datasets, combined with our phylogenetic estimates, should serve as a powerful comparative genomic platform and resource for future studies in strawberry.

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.

Autopolyploid inheritance and a heterozygous reciprocal translocation shape chromosome genetic behavior in tetraploid blueberry (Vaccinium corymbosum).

Understanding chromosome recombination behavior in polyploidy species is key to advancing genetic discoveries. In blueberry, a tetraploid species, the line of evidences about its genetic behavior still remain poorly understood, owing to the inter-specific, and inter-ploidy admixture of its genome and lack of in depth genome-wide inheritance and comparative structural studies. Here we describe a new high-quality, phased, chromosome-scale genome of a diploid blueberry, clone W85. The genome was integrated with cytogenetics and high-density, genetic maps representing six tetraploid blueberry cultivars, harboring different levels of wild genome admixture, to uncover recombination behavior and structural genome divergence across tetraploid and wild diploid species. Analysis of chromosome inheritance and pairing demonstrated that tetraploid blueberry behaves as an autotetraploid with tetrasomic inheritance. Comparative analysis demonstrated the presence of a reciprocal, heterozygous, translocation spanning one homolog of chr-6 and one of chr-10 in the cultivar Draper. The translocation affects pairing and recombination of chromosomes 6 and 10. Besides the translocation detected in Draper, no other structural genomic divergences were detected across tetraploid cultivars and highly inter-crossable wild diploid species. These findings and resources will facilitate new genetic and comparative genomic studies in Vaccinium and the development of genomic assisted selection strategy for this crop.

Downregulation of the expression of subgenomic chromosome A7 genes promotes plant height in resynthesized allopolyploid Brassica napus.

KEY MESSAGE: Homoeolog expression bias and the gene dosage effect induce downregulation of genes on chromosome A7, causing a significant increase in the plant height of resynthesized allopolyploid Brassica napus. Gene expression levels in allopolyploid plants are not equivalent to the simple average of the expression levels in the parents and are associated with several non-additive expression phenomena, including homoeolog expression bias. However, hardly any information is available on the effect of homoeolog expression bias on traits. Here, we studied the effects of gene expression-related characteristics on agronomic traits using six isogenic resynthesized Brassica napus lines across the first ten generations. We found a group of genes located on chromosome A7 whose expression levels were significantly negatively correlated with plant height. They were expressed at significantly lower levels than their homoeologous genes, owing to allopolyploidy rather than inheritance from parents. Homoeolog expression bias resulted in resynthesized allopolyploids with a plant height similar to their female Brassica oleracea parent, but significantly higher than that of the male Brassica rapa parent. Notably, aneuploid lines carrying monosomic and trisomic chromosome A7 had the highest and lowest plant heights, respectively, due to changes in the expression bias of homoeologous genes because of alterations in the gene dosage. These findings suggest that the downregulation of the expression of homoeologous genes on a single chromosome can result in the partial improvement of traits to a significant extent in the nascent allopolyploid B. napus.

Evolution of Conserved Noncoding Sequences in Arabidopsis thaliana.

Recent pangenome studies have revealed a large fraction of the gene content within a species exhibits presence-absence variation (PAV). However, coding regions alone provide an incomplete assessment of functional genomic sequence variation at the species level. Little to no attention has been paid to noncoding regulatory regions in pangenome studies, though these sequences directly modulate gene expression and phenotype. To uncover regulatory genetic variation, we generated chromosome-scale genome assemblies for thirty Arabidopsis thaliana accessions from multiple distinct habitats and characterized species level variation in Conserved Noncoding Sequences (CNS). Our analyses uncovered not only PAV and positional variation (PosV) but that diversity in CNS is nonrandom, with variants shared across different accessions. Using evolutionary analyses and chromatin accessibility data, we provide further evidence supporting roles for conserved and variable CNS in gene regulation. Additionally, our data suggests that transposable elements contribute to CNS variation. Characterizing species-level diversity in all functional genomic sequences may later uncover previously unknown mechanistic links between genotype and phenotype.

Unraveling the Complex Hybrid Ancestry and Domestication History of Cultivated Strawberry.

Cultivated strawberry (Fragaria × ananassa) is one of our youngest domesticates, originating in early eighteenth-century Europe from spontaneous hybrids between wild allo-octoploid species (Fragaria chiloensis and Fragaria virginiana). The improvement of horticultural traits by 300 years of breeding has enabled the global expansion of strawberry production. Here, we describe the genomic history of strawberry domestication from the earliest hybrids to modern cultivars. We observed a significant increase in heterozygosity among interspecific hybrids and a decrease in heterozygosity among domesticated descendants of those hybrids. Selective sweeps were found across the genome in early and modern phases of domestication-59-76% of the selectively swept genes originated in the three less dominant ancestral subgenomes. Contrary to the tenet that genetic diversity is limited in cultivated strawberry, we found that the octoploid species harbor massive allelic diversity and that F. × ananassa harbors as much allelic diversity as either wild founder. We identified 41.8 M subgenome-specific DNA variants among resequenced wild and domesticated individuals. Strikingly, 98% of common alleles and 73% of total alleles were shared between wild and domesticated populations. Moreover, genome-wide estimates of nucleotide diversity were virtually identical in F. chiloensis,F. virginiana, and F. × ananassa (π = 0.0059-0.0060). We found, however, that nucleotide diversity and heterozygosity were significantly lower in modern F. × ananassa populations that have experienced significant genetic gains and have produced numerous agriculturally important cultivars.

The Evolutionary History of Wild, Domesticated, and Feral Brassica oleracea (Brassicaceae).

Understanding the evolutionary history of crops, including identifying wild relatives, helps to provide insight for conservation and crop breeding efforts. Cultivated Brassica oleracea has intrigued researchers for centuries due to its wide diversity in forms, which include cabbage, broccoli, cauliflower, kale, kohlrabi, and Brussels sprouts. Yet, the evolutionary history of this species remains understudied. With such different vegetables produced from a single species, B. oleracea is a model organism for understanding the power of artificial selection. Persistent challenges in the study of B. oleracea include conflicting hypotheses regarding domestication and the identity of the closest living wild relative. Using newly generated RNA-seq data for a diversity panel of 224 accessions, which represents 14 different B. oleracea crop types and nine potential wild progenitor species, we integrate phylogenetic and population genetic techniques with ecological niche modeling, archaeological, and literary evidence to examine relationships among cultivars and wild relatives to clarify the origin of this horticulturally important species. Our analyses point to the Aegean endemic B. cretica as the closest living relative of cultivated B. oleracea, supporting an origin of cultivation in the Eastern Mediterranean region. Additionally, we identify several feral lineages, suggesting that cultivated plants of this species can revert to a wild-like state with relative ease. By expanding our understanding of the evolutionary history in B. oleracea, these results contribute to a growing body of knowledge on crop domestication that will facilitate continued breeding efforts including adaptation to changing environmental conditions.

Replaying the evolutionary tape to investigate subgenome dominance in allopolyploid Brassica napus.

Allopolyploidisation merges evolutionarily distinct parental genomes (subgenomes) into a single nucleus. A frequent observation is that one subgenome is 'dominant' over the other subgenome, often being more highly expressed. Here, we 'replayed the evolutionary tape' with six isogenic resynthesised Brassica napus allopolyploid lines and investigated subgenome dominance patterns over the first 10 generations postpolyploidisation. We found that the same subgenome was consistently more dominantly expressed in all lines and generations and that >70% of biased gene pairs showed the same dominance patterns across all lines and an in silico hybrid of the parents. Gene network analyses indicated an enrichment for network interactions and several biological functions for the Brassica oleracea subgenome biased pairs, but no enrichment was identified for Brassica rapa subgenome biased pairs. Furthermore, DNA methylation differences between subgenomes mirrored the observed gene expression bias towards the dominant subgenome in all lines and generations. Many of these differences in gene expression and methylation were also found when comparing the progenitor genomes, suggesting that subgenome dominance is partly related to parental genome differences rather than just a byproduct of allopolyploidisation. These findings demonstrate that 'replaying the evolutionary tape' in an allopolyploid results in largely repeatable and predictable subgenome expression dominance patterns.

Diversification, spread, and admixture of octoploid strawberry in the Western Hemisphere.

PREMISE: Polyploid species often have complex evolutionary histories that have, until recently, been intractable due to limitations of genomic resources. While recent work has further uncovered the evolutionary history of the octoploid strawberry (Fragaria L.), there are still open questions. Much is unknown about the evolutionary relationship of the wild octoploid species, Fragaria virginiana and Fragaria chiloensis, and gene flow within and among species after the formation of the octoploid genome. METHODS: We leveraged a collection of wild octoploid ecotypes of strawberry representing the recognized subspecies and ranging from Alaska to southern Chile, and a high-density SNP array to investigate wild octoploid strawberry evolution. Evolutionary relationships were interrogated with phylogenetic analysis and genetic clustering algorithms. Additionally, admixture among and within species is assessed with model-based and tree-based approaches. RESULTS: Phylogenetic analysis revealed that the two octoploid strawberry species are monophyletic sister lineages. The genetic clustering results show substructure between North and South American F. chiloensis populations. Additionally, model-based and tree-based methods support gene flow within and among the two octoploid species, including newly identified admixture in the Hawaiian F. chiloensis subsp. sandwicensis population. CONCLUSIONS: F. virginiana and F. chiloensis are supported as monophyletic and sister lineages. All but one of the subspecies show extensive paraphyly. Furthermore, phylogenetic relationships among F. chiloensis populations supports a single population range expansion southward from North America. The inter- and intraspecific relationships of octoploid strawberry are complex and suggest substantial gene flow between sympatric populations among and within species.

Single-molecule sequencing of the desiccation-tolerant grass Oropetium thomaeum.

Plant genomes, and eukaryotic genomes in general, are typically repetitive, polyploid and heterozygous, which complicates genome assembly. The short read lengths of early Sanger and current next-generation sequencing platforms hinder assembly through complex repeat regions, and many draft and reference genomes are fragmented, lacking skewed GC and repetitive intergenic sequences, which are gaining importance due to projects like the Encyclopedia of DNA Elements (ENCODE). Here we report the whole-genome sequencing and assembly of the desiccation-tolerant grass Oropetium thomaeum. Using only single-molecule real-time sequencing, which generates long (>16 kilobases) reads with random errors, we assembled 99% (244 megabases) of the Oropetium genome into 625 contigs with an N50 length of 2.4 megabases. Oropetium is an example of a 'near-complete' draft genome which includes gapless coverage over gene space as well as intergenic sequences such as centromeres, telomeres, transposable elements and rRNA clusters that are typically unassembled in draft genomes. Oropetium has 28,466 protein-coding genes and 43% repeat sequences, yet with 30% more compact euchromatic regions it is the smallest known grass genome. The Oropetium genome demonstrates the utility of single-molecule real-time sequencing for assembling high-quality plant and other eukaryotic genomes, and serves as a valuable resource for the plant comparative genomics community.

Preferential retention of genes from one parental genome after polyploidy illustrates the nature and scope of the genomic conflicts induced by hybridization.

Polyploidy is increasingly seen as a driver of both evolutionary innovation and ecological success. One source of polyploid organisms' successes may be their origins in the merging and mixing of genomes from two different species (e.g., allopolyploidy). Using POInT (the Polyploid Orthology Inference Tool), we model the resolution of three allopolyploidy events, one from the bakers' yeast (Saccharomyces cerevisiae), one from the thale cress (Arabidopsis thaliana) and one from grasses including Sorghum bicolor. Analyzing a total of 21 genomes, we assign to every gene a probability for having come from each parental subgenome (i.e., derived from the diploid progenitor species), yielding orthologous segments across all genomes. Our model detects statistically robust evidence for the existence of biased fractionation in all three lineages, whereby genes from one of the two subgenomes were more likely to be lost than those from the other subgenome. We further find that a driver of this pattern of biased losses is the co-retention of genes from the same parental genome that share functional interactions. The pattern of biased fractionation after the Arabidopsis and grass allopolyploid events was surprisingly constant in time, with the same parental genome favored throughout the lineages' history. In strong contrast, the yeast allopolyploid event shows evidence of biased fractionation only immediately after the event, with balanced gene losses more recently. The rapid loss of functionally associated genes from a single subgenome is difficult to reconcile with the action of genetic drift and suggests that selection may favor the removal of specific duplicates. Coupled to the evidence for continuing, functionally-associated biased fractionation after the A. thaliana At-α event, we suggest that, after allopolyploidy, there are functional conflicts between interacting genes encoded in different subgenomes that are ultimately resolved through preferential duplicate loss.

Genome-wide association of volatiles reveals candidate loci for blueberry flavor.

Plants produce a range of volatile organic compounds (VOCs), some of which are perceived by the human olfactory system, contributing to a myriad flavors. Despite the importance of flavor for consumer preference, most plant breeding programs have neglected it, mainly because of the costs of phenotyping and the complexity of disentangling the role of VOCs in human perception. To develop molecular breeding tools aimed at improving fruit flavor, we carried out target genotyping of and VOC extraction from a blueberry population. Metabolite genome-wide association analysis was used to elucidate the genetic architecture, while predictive models were tested to prove that VOCs can be accurately predicted using genomic information. A historical sensory panel was considered to assess how the volatiles influenced consumers. By gathering genomics, metabolomics, and the sensory panel, we demonstrated that VOCs are controlled by a few major genomic regions, some of which harbor biosynthetic enzyme-coding genes; can be accurately predicted using molecular markers; and can enhance or decrease consumers' overall liking. Here we emphasized how the understanding of the genetic basis and the role of VOCs in consumer preference can assist breeders in developing more flavorful cultivars at a more inexpensive and accelerated pace.

Subgenome Dominance in an Interspecific Hybrid, Synthetic Allopolyploid, and a 140-Year-Old Naturally Established Neo-Allopolyploid Monkeyflower.

Recent studies have shown that one of the parental subgenomes in ancient polyploids is generally more dominant, having retained more genes and being more highly expressed, a phenomenon termed subgenome dominance. The genomic features that determine how quickly and which subgenome dominates within a newly formed polyploid remain poorly understood. To investigate the rate of emergence of subgenome dominance, we examined gene expression, gene methylation, and transposable element (TE) methylation in a natural, <140-year-old allopolyploid (Mimulus peregrinus), a resynthesized interspecies triploid hybrid (M. robertsii), a resynthesized allopolyploid (M. peregrinus), and progenitor species (M. guttatus and M. luteus). We show that subgenome expression dominance occurs instantly following the hybridization of divergent genomes and significantly increases over generations. Additionally, CHH methylation levels are reduced in regions near genes and within TEs in the first-generation hybrid, intermediate in the resynthesized allopolyploid, and are repatterned differently between the dominant and recessive subgenomes in the natural allopolyploid. Subgenome differences in levels of TE methylation mirror the increase in expression bias observed over the generations following hybridization. These findings provide important insights into genomic and epigenomic shock that occurs following hybridization and polyploid events and may also contribute to uncovering the mechanistic basis of heterosis and subgenome dominance.

Phylogeny and multiple independent whole-genome duplication events in the Brassicales.

PREMISE: Whole-genome duplications (WGDs) are prevalent throughout the evolutionary history of plants. For example, dozens of WGDs have been phylogenetically localized across the order Brassicales, specifically, within the family Brassicaceae. A WGD event has also been identified in the Cleomaceae, the sister family to Brassicaceae, yet its placement, as well as that of WGDs in other families in the order, remains unclear. METHODS: Phylo-transcriptomic data were generated and used to infer a nuclear phylogeny for 74 Brassicales taxa. Genome survey sequencing was also performed on 66 of those taxa to infer a chloroplast phylogeny. These phylogenies were used to assess and confirm relationships among the major families of the Brassicales and within Brassicaceae. Multiple WGD inference methods were then used to assess the placement of WGDs on the nuclear phylogeny. RESULTS: Well-supported chloroplast and nuclear phylogenies for the Brassicales and the putative placement of the Cleomaceae-specific WGD event Th-ɑ are presented. This work also provides evidence for previously hypothesized WGDs, including a well-supported event shared by at least two members of the Resedaceae family, and a possible event within the Capparaceae. CONCLUSIONS: Phylogenetics and the placement of WGDs within highly polyploid lineages continues to be a major challenge. This study adds to the conversation on WGD inference difficulties by demonstrating that sampling is especially important for WGD identification and phylogenetic placement. Given its economic importance and genomic resources, the Brassicales continues to be an ideal group for assessing WGD inference methods.

Usability of reference-free transcriptome assemblies for detection of differential expression: a case study on Aethionema arabicum dimorphic seeds.

BACKGROUND: RNA-sequencing analysis is increasingly utilized to study gene expression in non-model organisms without sequenced genomes. Aethionema arabicum (Brassicaceae) exhibits seed dimorphism as a bet-hedging strategy - producing both a less dormant mucilaginous (M+) seed morph and a more dormant non-mucilaginous (NM) seed morph. Here, we compared de novo and reference-genome based transcriptome assemblies to investigate Ae. arabicum seed dimorphism and to evaluate the reference-free versus -dependent approach for identifying differentially expressed genes (DEGs). RESULTS: A de novo transcriptome assembly was generated using sequences from M+ and NM Ae. arabicum dry seed morphs. The transcripts of the de novo assembly contained 63.1% complete Benchmarking Universal Single-Copy Orthologs (BUSCO) compared to 90.9% for the transcripts of the reference genome. DEG detection used the strict consensus of three methods (DESeq2, edgeR and NOISeq). Only 37% of 1533 differentially expressed de novo assembled transcripts paired with 1876 genome-derived DEGs. Gene Ontology (GO) terms distinguished the seed morphs: the terms translation and nucleosome assembly were overrepresented in DEGs higher in abundance in M+ dry seeds, whereas terms related to mRNA processing and transcription were overrepresented in DEGs higher in abundance in NM dry seeds. DEGs amongst these GO terms included ribosomal proteins and histones (higher in M+), RNA polymerase II subunits and related transcription and elongation factors (higher in NM). Expression of the inferred DEGs and other genes associated with seed maturation (e.g. those encoding late embryogenesis abundant proteins and transcription factors regulating seed development and maturation such as ABI3, FUS3, LEC1 and WRI1 homologs) were put in context with Arabidopsis thaliana seed maturation and indicated that M+ seeds may desiccate and mature faster than NM. The 1901 transcriptomic DEG set GO-terms had almost 90% overlap with the 2191 genome-derived DEG GO-terms. CONCLUSIONS: Whilst there was only modest overlap of DEGs identified in reference-free versus -dependent approaches, the resulting GO analysis was concordant in both approaches. The identified differences in dry seed transcriptomes suggest mechanisms underpinning previously identified contrasts between morphology and germination behaviour of M+ and NM seeds.