Differential Metabolite Profiles during Fruit Development in High-Yielding Oil Palm Mesocarp

To better understand lipid biosynthesis in oil palm mesocarp, in particular the differences in gene regulation leading to and including de novo fatty acid biosynthesis, a multi-platform metabolomics technology was used to profile mesocarp metabolites during six critical stages of fruit development in comparatively high- and low-yielding oil palm populations. Significantly higher amino acid levels preceding lipid biosynthesis and nucleosides during lipid biosynthesis were observed in a higher yielding commercial palm population. Levels of metabolites involved in glycolysis revealed interesting divergence of flux towards glycerol-3-phosphate, while carbon utilization differences in the TCA cycle were proven by an increase in malic acid/citric acid ratio. Apart from insights into the regulation of enhanced lipid production in oil palm, these results provide potentially useful metabolite yield markers and genes of interest for use in breeding programmes.     

Selection and evaluation of reference genes for quantitative gene expression analysis in broomcorn millet (<Emphasis Type="Italic">Panicum miliaceum</Emphasis> L.)

The appropriate reference genes are crucial for normalization of the target gene expression in qRT-PCR analysis. Broomcorn millet (Panicum miliaceum L.) is one of the most important crops in drought areas worldwide, while the systematical investigation and evaluation of reference genes has not been investigated in this species up to now. Here, 9 commonly used reference genes were selected to detect their expressional stability in different tissues and under different stresses in broomcorn millet. ΔC_t, BestKeeper, NormFinder and GeNorm approaches were used to evaluate the potentiality of these candidate genes as the reference gene in broomcorn millet. Taken together, results found that 18S and GAPDH were the suitable reference genes for gene expression normalization in different tissues and under stress treatment in broomcorn millet. This was the first study to investigate the reference genes for qRT-PCR analysis in broomcorn millet, which will facilitate the gene expression studies and also accelerate revealing the molecular mechanism of well-adapted extreme climatic conditions.     

Selection of reference genes for quantitative real-time PCR in six oil-tea camellia based on RNA-seq

qRT-PCR is becoming a routine tool in molecular biology to study gene expression. It is necessary to find stable reference genes when performing qRT-PCR. The expression of genes cloned in oil-tea camellia currently cannot be accurately analyzed due to a lack of suitable reference genes. We collected different tissues (including roots, stems, leaves, flowers and seeds) from six oil-tea camellia species to determine stable reference genes. Five novel and ten traditional reference gene sequences were selected from the RNA-seq database of Camellia oleifera Abel seeds and specific PCR Primers were designed for each. Cycle threshold (C _t) data were obtained from each reaction for all samples. Three different software tools, geNorm, NormFinder and Best-Keeper were applied to calculate the expression stability of the candidate reference genes according to the C _t values. The results were similar between the three software packages, and indicated that the traditional genes TUBα-3, ACT7α and the novel gene CESA were relatively stable in all species and tissues. However, no genes were sufficiently stable across all species and tissues, thus the optimal number of reference genes required for accurate normalization varied from 2 to 6. Finally, the relative expression of squalene synthase (SQS) and squalene epoxidase (SQE) genes related to important ingredients squalene and tea saponin in oil-tea camellia seeds were compared by using stable to less stable reference genes. The comparison results validated the selection of reference genes in the current study. In summary, for the different tissues of six oil-tea camellia species different optimal numbers of suitable reference genes were found.     

Genomic structure,QTL mapping,and molecular markers of lipase genes responsible for palm oil acidity in the oil palm (<Emphasis Type="Italic">Elaeis guineensis</Emphasis> Jacq.)

The degradation of triglycerides in oil palm fruit due to an endogenous lipase in the pulp is the main reason for acidification of palm oil, which causes major economic losses and is currently mainly associated with the FLL1 gene. We designed this study to identify all the major genes controlling differences in acidity and lipase activity in the oil palm fruit mesocarp and determine a molecular markers kit to allow marker-assisted selection of commercial varieties with low acidity. Not only one gene (FLL1) but three closely linked genes including FLL1 were found and characterized in LM2T_EgCIR184O12c, a bacterial artificial chromosome sequence of 231 kb. Intra-gene PCR-based markers were designed for these genes. A QTL gene co-localization analysis for oil acidity (percentage of fatty acids released) was performed on two mapping populations. It evidenced a single major QTL at our lipase gene loci, explaining 84 to 92% of phenotypic variation, and validating the main genetic control of palm oil acidification by FLL1 and/or by the two new lipase genes. The three lipase genes had high homology to demonstrated triacylglycerol lipases. While FLL1 shows the highest expression levels, the two other genes may also contribute to oil acidity. Our molecular markers of lipase genes and the associated major QTL is an important step towards marker-assisted selection of commercial varieties with low acidity.     

Selection and Evaluation of Reference Genes for Expression Analysis Using qRT-PCR in the Beet Armyworm Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae)

Quantitative real-time PCR (qRT-PCR) is a reliable and reproducible technique for measuring and evaluating changes in gene expression. The most common method for analyzing qRT-PCR data is to normalize mRNA levels of target genes to internal reference genes. Evaluating and selecting stable reference genes on a case-by-case basis is critical. The present study aimed to facilitate gene expression studies by identifying the most suitable reference genes for normalization of mRNA expression in qRT-PCR analysis of the beet armyworm Spodoptera exigua (Lepidoptera: Noctuidae). For this purpose, three software tools (geNorm, NormFinder and BestKeeper) were used to investigate 10 candidate reference genes in nine developmental stages and five different tissues (epidermis, head, midgut, fat body and hemolymph) in three larval physiological stages (molting, feeding and wandering stages) of, S. exigua. With the exception of 18S ribosomal RNA (18S), all other candidate genes evaluated, β-actin1(ACT1), β-actin2 (ACT2), elongation factor1(EF1), elongation factor 2 (EF2), Glyceralde hyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein L10 (L10), ribosomal protein L17A (L17A), superoxide dismutase (SOD), α-tubulin (TUB),proved to be acceptable reference genes. However, their suitability partly differed between physiological stages and different tissues. L10, EF2 and L17A ranked highest in all tissue sample sets. SOD, ACT2, GAPDH, EF1 and ACT1 were stably expressed in all developmental stage sample sets; ACT2, ACT1 and L10 for larvae sample sets; GAPDH, ACT1 and ACT2 for pupae and adults; SOD and L17A for males; and EF2 and SOD for females. The expression stability of genes varied in different conditions. The findings provided here demonstrated, with a few exceptions, the suitability of most of the 10 reference genes tested in tissues and life developmental stages. Overall, this study emphasizes the importance of validating reference genes for qRT-PCR analysis in S. exigua.     

Evaluation of appropriate reference genes for gene expression studies in pepper by quantitative real-time PCR

Quantitative real-time polymerase chain reaction (qRT-PCR) has been extensively used in several plant species as an accurate technique for gene expression analysis. However, the expression level of a target gene may be misconstrued due to unstable expression of the reference genes under different experimental conditions. Therefore, it is necessary to systematically evaluate these reference genes before experiments are conducted. Recently, more and more studies have focused on gene expression in pepper (Capsicum annuum L.). In this study, ten putative reference genes were chosen to identify expression stability by using geNorm and NormFinder statistical algorithms in ten different pepper sample pools, including those from different plant tissues (root, stem, leaf and flower) and from plants treated with hormones (salicylic acid and gibberellic acid) and abiotic stresses (cold, heat, salt and drought). EF1?? and UEP exhibited the most stable expression across all of the tested pepper samples. For abiotic stress or different hormone treatment, the ranking of candidate reference genes was not completely consistent, except for EF1?? which showed a relatively stable expression level. For different tissues, the expression of Actin1 was stable and it was considered an appropriate reference gene. It is concluded that EF1??, UEP and Actin1 are suitable reference genes for reliable qRT-PCR data normalization for the tissues and experimental conditions used in this experiment.