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Abstract Fertilization is a fundamental process that triggers seed and fruit development, but the molecular mechanisms underlying fertilization-induced seed development are poorly understood. Previous research has established AGamous-Like62 (AGL62) activation and auxin biosynthesis in the endosperm as key events following fertilization in Arabidopsis (Arabidopsis thaliana) and wild strawberry (Fragaria vesca). To test the hypothesis that epigenetic mechanisms are critical in mediating the effect of fertilization on the activation of AGL62 and auxin biosynthesis in the endosperm, we first identified and analyzed imprinted genes from the endosperm of wild strawberries. We isolated endosperm tissues from F1 seeds of 2 wild strawberry F. vesca subspecies, generated endosperm-enriched transcriptomes, and identified candidate Maternally Expressed and Paternally Expressed Genes (MEGs and PEGs). Through bioinformatic analyses, we identified 4 imprinted genes that may be involved in regulating the expression of FveAGL62 and auxin biosynthesis genes. We conducted functional analysis of a maternally expressed gene FveMYB98 through CRISPR-knockout and over-expression in transgenic strawberries as well as analysis in heterologous systems. FveMYB98 directly repressed FveAGL62 at stage 3 endosperm, which likely serves to limit auxin synthesis and endosperm proliferation. These results provide an inroad into the regulation of early-stage seed development by imprinted genes in strawberries, suggest the potential function of imprinted genes in parental conflict, and identify FveMYB98 as a regulator of a key transition point in endosperm development. 

Abstract Ancient whole-genome duplications (WGDs) are believed to facilitate novelty and adaptation by providing the raw fuel for new genes. However, it is unclear how recent WGDs may contribute to evolvability within recent polyploids. Hybridization accompanying some WGDs may combine divergent gene content among diploid species. Some theory and evidence suggest that polyploids have a greater accumulation and tolerance of gene presence-absence and genomic structural variation, but it is unclear to what extent either is true. To test how recent polyploidy may influence pangenomic variation, we sequenced, assembled, and annotated twelve complete, chromosome-scale genomes of Camelina sativa, an allohexaploid biofuel crop with three distinct subgenomes. Using pangenomic comparative analyses, we characterized gene presence-absence and genomic structural variation both within and between the subgenomes. We found over 75% of ortholog gene clusters are core in Camelina sativa and <10% of sequence space was affected by genomic structural rearrangements. In contrast, 19% of gene clusters were unique to one subgenome, and the majority of these were Camelina-specific (no ortholog in Arabidopsis). We identified an inversion that may contribute to vernalization requirements in winter-type Camelina, and an enrichment of Camelina-specific genes with enzymatic processes related to seed oil quality and Camelina’s unique glucosinolate profile. Genes related to these traits exhibited little presence-absence variation. Our results reveal minimal pangenomic variation in this species, and instead show how hybridization accompanied by WGD may benefit polyploids by merging diverged gene content of different species. 

Abstract Camelina (Camelina sativa), an allohexaploid species, is an emerging aviation biofuel crop that has been the focus of resurgent interest in recent decades. To guide future breeding and crop improvement efforts, the community requires a deeper comprehension of subgenome dominance, often noted in allopolyploid species, “alongside an understanding of the genetic diversity” and population structure of material present within breeding programs. We conducted population genetic analyses of a C. sativa diversity panel, leveraging a new genome, to estimate nucleotide diversity and population structure, and analyzed for patterns of subgenome expression dominance among different organs. Our analyses confirm that C. sativa has relatively low genetic diversity and show that the SG3 subgenome has substantially lower genetic diversity compared to the other two subgenomes. Despite the low genetic diversity, our analyses identified 13 distinct subpopulations including two distinct wild populations and others putatively representing founders in existing breeding populations. When analyzing for subgenome composition of long non-coding RNAs, which are known to play important roles in (a)biotic stress tolerance, we found that the SG3 subgenome contained significantly more lincRNAs compared to other subgenomes. Similarly, transcriptome analyses revealed that expression dominance of SG3 is not as strong as previously reported and may not be universal across all organ types. From a global analysis, SG3 “was only significant higher expressed” in flower, flower bud, and fruit organs, which is an important discovery given that the crop yield is associated with these organs. Collectively, these results will be valuable for guiding future breeding efforts in camelina. 

Abstract Subgenome dominance has been reported in diverse allopolyploid species, where genes from one subgenome are preferentially retained and are more highly expressed than those from other subgenome(s). However, the molecular mechanisms responsible for subgenome dominance remain poorly understood. Here, we develop genome-wide map of accessible chromatin regions (ACRs) in cultivated strawberry (2n = 8x = 56, with A, B, C, D subgenomes). Each ACR is identified as an MNase hypersensitive site (MHS). We discover that the dominant subgenome A contains a greater number of total MHSs and MHS per gene than the submissive B/C/D subgenomes. Subgenome A suffers fewer losses of MHS-related DNA sequences and fewer MHS fragmentations caused by insertions of transposable elements. We also discover that genes and MHSs related to stress response have been preferentially retained in subgenome A. We conclude that preservation of genes and their cognate ACRs, especially those related to stress responses, play a major role in the establishment of subgenome dominance in octoploid strawberry. 

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

Abstract The spatial organization of genes within plant genomes can drive evolution of specialized metabolic pathways. Terpenoids are important specialized metabolites in plants with diverse adaptive functions that enable environmental interactions. Here, we report the genome assemblies of Prunella vulgaris , Plectranthus barbatus , and Leonotis leonurus . We investigate the origin and subsequent evolution of a diterpenoid biosynthetic gene cluster (BGC) together with other seven species within the Lamiaceae (mint) family. Based on core genes found in the BGCs of all species examined across the Lamiaceae, we predict a simplified version of this cluster evolved in an early Lamiaceae ancestor. The current composition of the extant BGCs highlights the dynamic nature of its evolution. We elucidate the terpene backbones generated by the Callicarpa americana BGC enzymes, including miltiradiene and the terpene (+)-kaurene, and show oxidization activities of BGC cytochrome P450s. Our work reveals the fluid nature of BGC assembly and the importance of genome structure in contributing to the origin of metabolites. 

Abstract Background Transposable elements (TEs) are powerful creators of genotypic and phenotypic diversity due to their inherent mutagenic capabilities and in this way they serve as a deep reservoir of sequences for genomic variation. As agents of genetic disruption, a TE’s potential to impact phenotype is partially a factor of its location in the genome. Previous research has shown TEs’ ability to impact the expression of neighboring genes, however our understanding of this trend is hampered by the exceptional amount of diversity in the TE world, and a lack of publicly available computational methods that quantify the presence of TEs relative to genes. Results Here, we have developed a tool to more easily quantify TE presence relative to genes through the use of only a gene and TE annotation, yielding a new metric we call TE Density. Briefly defined as the proportion of TE-occupied base-pairs relative to a window-size of the genome. This new pipeline reports TE density for each gene in the genome, for each type descriptor of TE (order and superfamily), and for multiple positions and distances relative to the gene (upstream, intragenic, and downstream) over sliding, user-defined windows. In this way, we overcome previous limitations to the study of TE-gene relationships by focusing on all TE types present in the genome, utilizing flexible genomic distances for measurement, and reporting a TE presence metric for every gene in the genome. Conclusions Together, this new tool opens up new avenues for studying TE-gene relationships, genome architecture, comparative genomics, and the tremendous diversity present of the TE world. TE Density is open-source and freely available at: https://github.com/sjteresi/TE_Density . 

Abstract Anthracnose fruit rot (AFR), caused by the fungal pathogen Colletotrichum fioriniae, is among the most destructive and widespread fruit disease of blueberry, impacting both yield and overall fruit quality. Blueberry cultivars have highly variable resistance against AFR. To date, this pathogen is largely controlled by applying various fungicides; thus, a more cost-effective and environmentally conscious solution for AFR is needed. Here we report three quantitative trait loci associated with AFR resistance in northern highbush blueberry (Vaccinium corymbosum). Candidate genes within these genomic regions are associated with the biosynthesis of flavonoids (e.g. anthocyanins) and resistance against pathogens. Furthermore, we examined gene expression changes in fruits following inoculation with Colletotrichum in a resistant cultivar, which revealed an enrichment of significantly differentially expressed genes associated with certain specialized metabolic pathways (e.g. flavonol biosynthesis) and pathogen resistance. Using non-targeted metabolite profiling, we identified a flavonol glycoside with properties consistent with a quercetin rhamnoside as a compound exhibiting significant abundance differences among the most resistant and susceptible individuals from the genetic mapping population. Further analysis revealed that this compound exhibits significant abundance differences among the most resistant and susceptible individuals when analyzed as two groups. However, individuals within each group displayed considerable overlapping variation in this compound, suggesting that its abundance may only be partially associated with resistance against C. fioriniae. These findings should serve as a powerful resource that will enable breeding programs to more easily develop new cultivars with superior resistance to AFR and as the basis of future research studies.