De novo transcriptome assembly of the eight major organs of Sacha Inchi (Plukenetia volubilis) and the identification of genes involved in α-linolenic acid metabolism

Abstract Background Sacha Inchi (Plukenetia volubilis L.), which belongs to the Euphorbiaceae, has been considered a new potential oil crop because of its high content of polyunsaturated fatty acids in its seed oil. The seed oil especially contains high amounts of α-linolenic acid (ALA), which is useful for the prevention of various diseases. However, little is known about the genetic information and genome sequence of Sacha Inchi, which has largely hindered functional genomics and molecular breeding studies. Results In this study, a de novo transcriptome assembly based on transcripts sequenced in eight major organs, including roots, stems, shoot apexes, mature leaves, male flowers, female flowers, fruits, and seeds of Sacha Inchi was performed, resulting in a set of 124,750 non-redundant putative transcripts having an average length of 851 bp and an N50 value of 1909 bp. Organ-specific unigenes analysis revealed that the most organ-specific transcripts are found in female flowers (2244 unigenes), whereas a relatively small amount of unigenes are detected to be expressed specifically in other organs with the least in stems (24 unigenes). A total of 42,987 simple sequence repeats (SSRs) were detected, which will contribute to the marker assisted selection breeding of Sacha Inchi. We analyzed expression of genes related to the α-linolenic acid metabolism based on the de novo assembly and annotation transcriptome in Sacha Inchi. It appears that Sacha Inchi accumulates high level of ALA in seeds by strong expression of biosynthesis-related genes and weak expression of degradation-related genes. In particular, the up-regulation of FAD3 and FAD7 is consistent with high level of ALA in seeds of Sacha Inchi compared with in other organs. Meanwhile, several transcription factors (ABI3, LEC1 and FUS3) may regulate key genes involved in oil accumulation in seeds of Sacha Inchi. Conclusions The transcriptome of major organs of Sacha Inchi has been sequenced and de novo assembled, which will expand the genetic information for functional genomic studies of Sacha Inchi. In addition, the identification of candidate genes involved in ALA metabolism will provide useful resources for the genetic improvement of Sacha Inchi and the metabolic engineering of ALA biosynthesis in other plants

Global Quantitative Mapping of Enhancers in Rice by STARR-seq

Enhancers activate transcription in a distance-, orientation-, and position-independent manner, which makes them difficult to be identified. Self-transcribing active regulatory region sequencing (STARR-seq) measures the enhancer activity of millions of DNA fragments in parallel. Here we used STARR-seq to generate a quantitative global map of rice enhancers. Most enhancers were mapped within genes, especially at the 5′ untranslated regions (5′UTR) and in coding sequences. Enhancers were also frequently mapped proximal to silent and lowly-expressed genes in transposable element (TE)-rich regions. Analysis of the epigenetic features of enhancers at their endogenous loci revealed that most enhancers do not co-localize with DNase I hypersensitive sites (DHSs) and lack the enhancer mark of histone modification H3K4me1. Clustering analysis of enhancers according to their epigenetic marks revealed that about 40% of identified enhancers carried one or more epigenetic marks. Repressive H3K27me3 was frequently enriched with positive marks, H3K4me3 and/or H3K27ac, which together label enhancers. Intergenic enhancers were also predicted based on the location of DHS regions relative to genes, which overlap poorly with STARR-seq enhancers. In summary, we quantitatively identified enhancers by functional analysis in the genome of rice, an important model plant. This work provides a valuable resource for further mechanistic studies in different biological contexts. Keywords: Plant, Enhancer, Functional analysis, Epigenetic modification, Gene expressio

Physicochemical Composition and Energy Property Changes of Wheat Straw Cultivars with Advancing Growth Days at Maturity

Physicochemical properties of wheat straw play crucial roles in bioenergy and chemical conversion processes. Four wheat straw cultivars at different maturity levels were collected in Beijing, China, and the growth days (GDs) ranged from 236 to 263 days. The physicochemical compositions and energy properties were analyzed. Cellulose (Cel), hemicelluloses (Hem), monosaccharidic composition of hemicelluloses (xylan, arabinan, hemicellulosic glucan, galactan, and mannan), and dry matter (DM) increased from 236 to 263 days, which can be explained by the increasing lignification of the cell walls in wheat straw with maturity. Water-soluble carbohydrates (WSC), crude protein (CP), nitrogen (N), phosphorus (P), and copper (Cu) decreased initially and then leveled off, with leaves decreasing. Lignin (Lig), ash, sulfur (S), potassium (K), and sodium (Na) increased first and then decreased slightly to 263 days. Carbon (C), hydrogen (H), oxygen (O), volatile matter (VM), fixed carbon (FC), and higher heating value (HHV) had litter variation with advancing maturity. A number of significant correlations were found among different physicochemical compositions. Regression equations for Cel and DM based on GDs showed excellent performance for prediction, while models for WSC and CP showed good prediction

Selection of Reliable Reference Genes for Gene Expression Studies of a Promising Oilseed Crop, Plukenetia volubilis, by Real-Time Quantitative PCR

Real-time quantitative PCR (RT-qPCR) is a reliable and widely used method for gene expression analysis. The accuracy of the determination of a target gene expression level by RT-qPCR demands the use of appropriate reference genes to normalize the mRNA levels among different samples. However, suitable reference genes for RT-qPCR have not been identified in Sacha inchi (Plukenetia volubilis), a promising oilseed crop known for its polyunsaturated fatty acid (PUFA)-rich seeds. In this study, using RT-qPCR, twelve candidate reference genes were examined in seedlings and adult plants, during flower and seed development and for the entire growth cycle of Sacha inchi. Four statistical algorithms (delta cycle threshold (ΔCt), BestKeeper, geNorm, and NormFinder) were used to assess the expression stabilities of the candidate genes. The results showed that ubiquitin-conjugating enzyme (UCE), actin (ACT) and phospholipase A22 (PLA) were the most stable genes in Sacha inchi seedlings. For roots, stems, leaves, flowers, and seeds from adult plants, 30S ribosomal protein S13 (RPS13), cyclophilin (CYC) and elongation factor-1alpha (EF1α) were recommended as reference genes for RT-qPCR. During the development of reproductive organs, PLA, ACT and UCE were the optimal reference genes for flower development, whereas UCE, RPS13 and RNA polymerase II subunit (RPII) were optimal for seed development. Considering the entire growth cycle of Sacha inchi, UCE, ACT and EF1α were sufficient for the purpose of normalization. Our results provide useful guidelines for the selection of reliable reference genes for the normalization of RT-qPCR data for seedlings and adult plants, for reproductive organs, and for the entire growth cycle of Sacha inchi

Additional file 17: of De novo transcriptome assembly of the eight major organs of Sacha Inchi (Plukenetia volubilis) and the identification of genes involved in α-linolenic acid metabolism

Figure S9. Pearson correlation analysis of the gene expression ratios obtained from RNA-Seq and qPCR data. The qPCR log10 values (expression ratios; y-axis) were plotted against the RNA-Seq log10 values (x-axis). The Pearson correlation coefficient (r) is given in the plot, and the circle indicates the extremely significant difference at p < 0.01. (TIF 337 kb

Additional file 7: of De novo transcriptome assembly of the eight major organs of Sacha Inchi (Plukenetia volubilis) and the identification of genes involved in Îą-linolenic acid metabolism

Pathways identified in the Sacha Inchi transcriptome. Three hundred and fifty-three KEGG pathways identified in Sacha Inchi and the corresponding unigene numbers of each pathway are shown. (XLS 60 kb

Additional file 10: of De novo transcriptome assembly of the eight major organs of Sacha Inchi (Plukenetia volubilis) and the identification of genes involved in Îą-linolenic acid metabolism

The list of GO terms that were significantly enriched in male flowers, female flowers, roots, seeds and stems. Gene ontology (GO) terms were assigned to organ-specific unigenes based on the top hits against the NR database. (XLS 33 kb

Additional file 15: of De novo transcriptome assembly of the eight major organs of Sacha Inchi (Plukenetia volubilis) and the identification of genes involved in Îą-linolenic acid metabolism

Overview of the SSRs detected in the assembled unigenes of Sacha Inchi. (XLS 4872 kb