Critical Metabolic Pathways And Genes Cooperate For Epoxy Fatty Acid-Enriched Oil Production In Developing Seeds of Vernonia Galamensis, An Industrial Oleaginous Plant

Background: Vernonia galamensis, originated from Africa, is an annual oleaginous plant of Asteraceae family. As a newly-establishing industrial oil crop, this plant produces high level (> 70%) of vernolic acid (cis-12-epoxyoctadeca-cis-9-enoic acid), an unusual epoxy fatty acid (EFA) with multiple industrial applications. Here, transcriptome analysis and fatty acid profiling from developing V. galamensis seeds were integrated to uncover the critical metabolic pathways responsible for high EFA accumulation and identify the targets that could be used in assembly of EFA biosynthesis pathway in existing oilseed crops. Results: Based on oil accumulation dynamics of V. galamensis seeds, we harvested seed samples from three stages (17, 38, and 45 days after pollination, DAP) representing the initial, fast and final EFA accumulation phases, and one mixed sample from different tissues for RNA-sequencing, with three biological replicates at each sample. Using Illumina platform, we have generated a total of 265 million raw cDNA reads. After filtering process, de novo assembly of clean reads yielded 67,114 unigenes with an N50 length of 1,316 nt. Functional annotation resulted in the identification of almost all genes involved in diverse lipid-metabolic pathways, including the novel fatty acid desaturase/epoxygenase, diacylglycerol acyltransferase 1 and 2, and phospholipid:diacylglycerol acyltransferases. By comparison, we found that various genes associated with acyl editing, fatty acid β-oxidation, triacylglycerol assembly and oil-body formation had greater expression levels at middle developmental stage (38 DAP), which are consistent with the fast accumulation of EFA in V. galamensis developing seed, implying their fundamental roles in EFA production. Additionally, we isolated some transcription factors (such as WRI1, FUS3 and ABI4), which putatively regulated the production of V. galamensis seed oils. The transient assay of selected genes resulted in a synergistic increase of EFA-enriched TAG accumulation in tobacco leaves. Transcriptome data were further confirmed by quantitative real-time PCR for twelve key genes in EFA biosynthesis. Finally, a comprehensive network for high EFA accumulation in V. galamensis seed was established.Conclusions: Our results are of significance with regard to understanding the molecular mechanisms underlying the natural epoxy oil production in this plant and provide valuable reference for developing other oilseeds with high level of valued epoxy oil.

Different DGAT1s show different TAG synthesis abilities and a specific amino acid substitution enhances oil accumulation

Triacylglycerols (TAGs) are the major component of plant storage lipids. Acyl-CoA:diacylglycerol acyltransferase (DGAT) catalyzes the final step of the Kennedy pathway, and responsible for plant oil accumulation. We previously found DGAT activity of Vernonia galamensis DGAT1 was distinctively higher than that of Arabidopsis thaliana DGAT1 and soybean DGAT1 in a yeast microsome assay. In this study, the DGAT1 cDNAs of Arabidopsis, Vernonia, soybean, and castor were introduced into Arabidopsis (ecotype Col-0). All Vernonia DGAT1 expressing lines showed a significantly higher oil content (average 49% relative increase compared to the wild type) followed by soybean, and castor. Most Arabidopsis DGAT1 over-expressing lines did not show a significant increase. In addition to these four DGAT1s, sunflower, Jatropha and sesame DGAT1 genes were introduced into the TAG biosynthesis defective yeast mutant (H1246). In the yeast expression culture, DGAT1s from Arabidopsis, castor, and soybean only slightly increased TAG content, however, DGAT1s from Vernonia, sunflower, Jatropha, and sesame remarkably increased TAG content more than 10 times higher than the former three DGAT1s. Three amino acid residues were characteristically common in the latter four DGAT1s. Using soybean DGAT1, these amino acid substitutions by site-directed mutagenesis was performed and analyzed. These substitutions substantially increased the TAG content.HighlightDGAT1s from several plant species were tested their TAG accumulation promotion in Arabidopsis and yeast. They were divided into high and low function and single amino acid substitution enhanced function

Genetic diversity and population structure of Vernonia [Vernonia galamensis (Cass.) Less] populations from Ethiopia revealed by SSR markers

Background: Vernonia (Vernonia galamensis) is a potential novel industrial crop due to high demand for its natural epoxidized oil, which can be used for the manufacturing of oleochemicals such as paints, plastic formulations (polyvinyl chloride), and pharmaceutical products. Assessment of the extent of genetic diversity in V. galamensis was the first and the most step in cultivation, breeding and genetic resource conservation. Hence, this study was aimed to assess the genetic diversity and population structure of this plant from different agro-ecologies in Ethiopia using SSR markers. Results: Twenty SSR markers were used for genetic diversity analyses of 150 individual V. galamensis accessions representing 10 populations, from which a total of 79 bands were identified for the 20 loci. All the loci used showed high polymorphism that ranged from 0.50 to 0.96, while the mean observed heterozygosity (Ho) was 0.15 across all the 20 markers evaluated. The molecular variance analysis (AMOVA) showed significant variations among populations which accounted for 11% of the variations. Populations clustering showed that the dendrogram and principal coordinate’s analysis roughly classified the accessions into four groups. However, the Bayesian model-based clustering (STRUCTURE) grouped into 6 (K = 6) major gene pools. Since, the cluster and the STRUCTURE analyses did not group the populations into sharply distinct clusters, due to presence of gene flow and mode of reproduction of the plant. Conclusions: The SSR molecular markers used in this study are highly polymorphic. Among the ten populations, East Showa and East Hararghe revealed higher genetic diversity, signaled that these areas are the hotspots for in-situ conservation of V. galamensis. In addition, the values of SSR markers such as heterozygosity, Shannon‘s index, polymorphic information content, and population clusters are important baseline information for future V. galamensis cultivation, breeding and genetic resource conservation endeavors in Ethiopia.

Genetic diversity and population structure of Vernonia [Vernonia galamensis (Cass.) Less] populations from Ethiopia revealed by SSR markers

Background: Vernonia (Vernonia galamensis) is a potential novel industrial crop due to high demand for its natural epoxidized oil, which can be used for the manufacturing of oleochemicals such as paints, plastic formulations (polyvinyl chloride), and pharmaceutical products. This study was initiated for the systematic and intensive genetic diversity assessment of V. galamensis accessions by SSR molecular markers to minimize the existing research gaps, provide a clue for germplasm conservation and further research. Results: Twenty SSR markers were used for genetic diversity analyses of 150 individual V. galamensis accessions representing 10 populations, from which a total of 79 bands were identified for the 20 loci. All the loci used showed high polymorphism that ranged from 0.50 to 0.96, while the mean observed heterozygosity (Ho) was 0.15 across all the 20 markers evaluated. The molecular variance analysis (AMOVA) showed significant variations among populations which accounted for 11% of the variations. Populations clustering showed that the dendrogram and principal coordinate’s analysis roughly classified the accessions into four groups. However, the Bayesian model-based clustering (STRUCTURE) grouped into 6 (K = 6) major gene pools. Since, the cluster and the STRUCTURE analyses did not group the populations into sharply distinct clusters, due to presence of gene flow and mode of reproduction of the plant. Conclusions: The SSR molecular markers used in this study are highly polymorphic. Among the ten populations, East Showa and East Hararghe revealed higher genetic diversity, signaled that these areas are the hotspots for in-situ conservation of V. galamensis. In addition, the values of SSR markers such as heterozygosity, Shannon‘s index, polymorphic information content, and population clusters are important baseline information for future V. galamensis cultivation, breeding and genetic resource conservation endeavors in Ethiopia.

Genetic diversity and population structure of Vernonia [Vernonia galamensis (Cass.) Less] populations from Ethiopia revealed by SSR markers

BackgroundVernonia (Vernonia galamensis) is a potential novel industrial crop due to high demand for its natural epoxidised oil, which can be used for the manufacturing of oleochemicals such as paints, plastic formulations (polyvinyl chloride), and pharmaceutical products. This study is initiated for the systematic and intensive genetic diversity assessment of V. galamensis accessions by SSR molecular markers to minimize the existing research gaps, provide a clue for germplasm conservation and further research. ResultsTwenty SSR markers were used for genetic diversity analyses of 150 individual V. galamensis accessions representing 10 populations, from which a total of 79 bands were identified across the entire loci. All the loci used showed high polymorphism that ranged from 0.50 to 0.96, while the mean observed heterozygosity (Ho) was 0.15 across all the 20 markers evaluated. The molecular variance analysis (AMOVA) showed significant variations despite low differentiation among populations which accounted for only 11% of the total variations. Populations clustering showed that the dendrogram and principal coordinate’s analysis roughly classified the 150 accessions into four groups. However, the Bayesian model-based clustering (STRUCTURE) grouped into 6 (K = 6) major gene pools. These analyses showed accessions collected from the same region of origin did not often grouped entirely together within a given major groups. ConclusionsThe result suggested that the markers applied to ten populations, in which East Showa and East Harerghe revealed higher genetic diversity, signaled that these areas are the hotspots for in-situ conservation of V. galamensis. In addition, the values of SSR markers such as heterozygosity, Shannon‘s index, polymorphic information content, and population clusters are important baseline information for future V. galamensis cultivation, breeding and genetic resource conservation endeavors in Ethiopia.

Genetic diversity and population structure of Vernonia [Vernonia galamensis (Cass.) Less] populations from Ethiopia revealed by SSR markers

Background Vernonia (Vernonia galamensis) is a potential novel industrial crop due to high demand to its natural epoxidised oil, which can be used for the manufacturing of polyvinyl chloride, adhesives, and petrochemicals, cosmetic and pharmaceutical products. This study is initiated for the systematic and intensive assessment of V. galamensis accessions genetic diversity through SSR molecular markers to minimize the existing research gaps, and provide a clue for germplasm conservation and further research. Results A total of 150 V. galamensis accessions were analyzed using 20 SSR markers. The markers detected a total of 79 with an average of 3.9 alleles per locus. The mean number of effective alleles was 3.06 and, the mean observed heterozygosity (Ho) was 0.15 across all the 20 markers evaluated. The marker also showed the highest percent of polymorphism that ranged from 0.50 to 0.96 with an average of 0.76. The analysis of molecular variance showed only 11% variation was among populations, 22% among individuals within populations and 67% within individuals. The largest number of migrants per generation was occurred between Derashie and Wollo (Nm=7.37) whereas the lowest values was between East Harerghe and West Harerghe (Nm =1.42). A factor analysis including dendrogram clusters and principal coordinates classified the 150 accessions into 4 groups. However, the Bayesian model based clustering (STRUCTURE) grouped into 3 (K = 3) major gene pools. These analyses showed accessions collected from the same region of origin did not often grouped entirely together within a given major groups. Conclusions Molecular genetic diversity analysis, using SSR markers was the first report in V. galamensis. All the markers used were polymorphic in the population studied. The markers detected the larger number of alleles, higher expected heterozygosity than observed heterozygosity. The markers applied to ten populations, in which East Showa and West Harerghe revealed higher genetic diversity, and can be considered as the hotspots for in-situ conservation of V. galamensis. In addition, the values of SSR markers such as heterozygosity, Shannon‘s index, polymorphic information content and population clusters are an important baseline information for future V. galamensis cultivation, breeding and genetic resource conservation endeavors in Ethiopia.