In oil crops, triacylglycerol biosynthesis is an important metabolic pathway in which glycerol-3-phosphate acyltransferase (GPAT) performs the first acylation step. Mass spectrometry analysis of developing sunflower (Helianthus annuus) seed membrane fractions identified an abundant GPAT, HaGPAT9 isoform 1, with a N-terminal peptide that possessed two phosphorylated residues with possible regulatory function. HaGPAT9-1 belongs to a broad eukaryotic GPAT family, similar to mammalian GPAT3, and it represents one of the two sunflower GPAT9 isoforms, sharing 90% identity with HaGPAT9-2. Both sunflower genes are expressed during seed development and in vegetative tissues, with HaGPAT9-1 transcripts accumulating at relatively higher levels than those for HaGPAT9-2. Green fluorescent protein tagging of HaGPAT9-1 confirmed its subcellular accumulation in the endoplasmic reticulum. Despite their overall sequence similarities, the two sunflower isoforms displayed significant differences in their enzymatic activities. For instance, HaGPAT9-1 possesses in vivo GPAT activity that rescues the lethal phenotype of the cmy228 yeast strain, while in vitro assays revealed a preference of HaGPAT9-1 for palmitoyl-, oleoyl- and linoleoyl-CoAs of one order of magnitude, with the highest increase in yield for oleoyl- and linoleoyl-CoAs. By contrast, no enzymatic activity could be detected for HaGPAT9-2, even though its over-expression modified the TAG profile of yeast.

Genome-wide maps of histone modifications have been obtained for several plant species. However, most studies focus on model systems and do not enforce FAIR data management principles. Here we study the H3K27me3 epigenome and associated transcriptome of Brassica rapa, an important vegetable cultivated worldwide. We performed H3K27me3 chromatin immunoprecipitation followed by high-throughput sequencing and transcriptomic analysis by 3′-end RNA sequencing from B. rapa leaves and inflorescences. To analyze these data we developed a Reproducible Epigenomic Analysis pipeline using Galaxy and Jupyter, packaged into Docker images to facilitate transparency and reuse. We found that H3K27me3 covers roughly one-third of all B. rapa protein-coding genes and its presence correlates with low transcript levels. The comparative analysis between leaves and inflorescences suggested that the expression of various floral regulatory genes during development depends on H3K27me3. To demonstrate the importance of H3K27me3 for B. rapa development, we characterized a mutant line deficient in the H3K27 methyltransferase activity. We found that braA.clf mutant plants presented pleiotropic alterations, e.g., curly leaves due to increased expression and reduced H3K27me3 levels at AGAMOUS-like loci. We characterized the epigenetic mark H3K27me3 at genome-wide levels and provide genetic evidence for its relevance in B. rapa development. Our work reveals the epigenomic landscape of H3K27me3 in B. rapa and provides novel genomics datasets and bioinformatics analytical resources. We anticipate that this work will lead the way to further epigenomic studies in the complex genome of Brassica crops.

The usual analysis of RNA sequencing (RNA-seq) reads is based on an existing reference genome and annotated gene models. However, when a reference for the sequenced species is not available, alternatives include using a reference genome from a related species or reconstructing transcript sequences with de novo assembly. In addition, researchers are faced with many options for RNA-seq data processing and limited information on how their decisions will impact the final outcome. Using both a diploid and polyploid species with a distant reference genome, we have tested the influence of different tools at various steps of a typical RNA-seq analysis workflow on the recovery of useful processed data available for downstream analysis. At the preprocessing step, we found error correction has a strong influence on de novo assembly but not on mapping results. After trimming, a greater percentage of reads could be used in downstream analysis by selecting gentle quality trimming performed with Skewer instead of strict quality trimming with Trimmomatic. This availability of reads correlated with size, quality, and completeness of de novo assemblies and with number of mapped reads. When selecting a reference genome from a related species to map reads, outcome was significantly improved when using mapping software tolerant of greater sequence divergence, such as Stampy or GSNAP. The selection of bioinformatic software tools for RNA-seq data analysis can maximize quality parameters on de novo assemblies and availability of reads in downstream analysis.

Taraxacum kok-saghyz (TKS) carries great potential as alternative natural rubber source. To better inform future breeding efforts with TKS and gain a deeper understanding of its genetic diversity, we utilized de novo sequencing to generate novel genomic simple sequence repeats markers (gSSRs). We utilized 25 gSSRs on a collection of genomic DNA (gDNA) samples from germplasm bank, and two gDNA samples from historical herbarium specimens. PCR coupled with capillary electrophoresis and an array of population genetics tools were employed to analyze the dataset of our study as well as a dataset of the recently published genic SSRs (eSSRs) generated on the same germplasm. Our results using both gSSRs and eSSRs revealed that TKS has low- to- moderate genetic diversity with most of it partitioned to the individuals and individuals within populations, whereas the species lacked population structure. Nineteen of the 25 gSSR markers cross-amplified to other Taraxacum spp. collected from Southeastern United States and identified as T. officinale by ITS sequencing. We used a subset of 14 gSSRs to estimate the genetic diversity of the T. officinale gDNA collection. In contrast to the obligatory outcrossing TKS, T. officinale presented evidence for population structure and clonal reproduction, which agreed with the species biology. We mapped the molecular markers sequences from this study and several others to the well-annotated sunflower genome. Our gSSRs present a functional tool for the biodiversity analyses in Taraxacum, but also in the related genera, as well as in the closely related tribes of the Asteraceae.