Drought is the most important constraint that effects chickpea production globally. RNA-Seq has great potential to dissect the molecular mechanisms of tolerance to environmental stresses. Transcriptome profiles in roots and shoots of two contrasting Iranian kabuli chickpea genotypes (Bivanij and Hashem) were investigated under water-limited conditions at early flowering stage using RNA-Seq approach. A total of 4,572 differentially expressed genes (DEGs) were identified. Of these, 261 and 169 drought stress responsive genes were identified in the shoots and the roots, respectively, and 17 genes were common in the shoots and the roots. Gene Ontology (GO) analysis revealed several sub-categories related to the stress, including response to stress, defense response and response to stimulus in the tolerant genotype Bivanij as compared to the sensitive genotype Hashem under drought stress. In addition, several Transcription factors (TFs) were identified in major metabolic pathways such as, ABA, proline and flavonoid biosynthesis. Furthermore, a number of the DEGs were observed in “QTL-hotspot” regions which were reported earlier in chickpea. Drought tolerance dissection in the genotypes revealed that the genes and the pathways involved in shoots of Bivanij were the most important factor to make a difference between the genotypes for drought tolerance. The identified TFs in the experiment, particularly those which were up-regulated in shoots of Bivanij during drought stress, were potential candidates for enhancing tolerance to drought.
The aim of this study was to evaluate the effect of different durations of flaxseed oil consumption on broiler performance, fatty acid content of meat and expression of FADS2 gene in the liver of chicken. A total of 336 unsexed day old chicks were randomly assigned to 7 dietary treatments with 4 replicates of 12 chickens in each. Body weight, weight gain, feed intake and feed conversion ratio were not affected by dietary treatments. Longer consumption of flaxseed oil resulted in higher deposition of long chain n‐3 fatty acids in broiler breast and thigh (P < 0.05). Regression analysis showed that feeding broilers with diet enriched with flaxseed oil 17 and 7 days prior to slaughter for breast and thigh respectively, is enough for producing n‐3 labeled meat. Weekly replacement of oil source of diet (soybean oil) with flaxseed oil increased expression of FADS2 gene. To incorporate enough n‐3 fatty acids into chicken meat through addition of flaxseed oil to broiler diet and to maximize de novo long chain n‐3 fatty acid synthesis in broiler body, we recommend adding flaxseed oil to broiler diets 17 days prior to slaughter. Practical applications: Omega‐3 enrichment of broiler chickens depends on both nutrition and gene expression. Flaxseed oil is an omega‐3 oil source that can be supplemented to broiler diet to enrich chicken meat with the beneficial n‐3 fatty acids. However, adding such oil sources to broiler rations is restricted because of their negative effects on broiler performance and meat quality. Therefore, shorter period of such oil sources consumption is preferred. Flaxseed oil consumption can affect gene expression too. Thus, it is necessary to determine the optimum duration of flaxseed oil supplementation to broiler diets to produce omega‐3 labeled chicken using both nutrition and gene expression. Flaxseed oil consumption can affect fatty acid profile of chicken meat through manipulation of fatty acid deposition, de novo fatty acid synthesis, fatty acid oxidation and expression of genes involved in fatty acid metabolism. To incorporate enough n‐3 fatty acids into chicken, providing at least 3 g n‐3 fatty acid per kilogram of meat, by adding flaxseed oil to broiler diet, we recommend adding flaxseed oil to broiler diets 17 days prior to slaughter.
BackgroundThe use of stably expressed genes as normalizers has crucial role in accurate and reliable expression analysis estimated by quantitative real-time polymerase chain reaction (qPCR). Recent studies have shown that, the expression levels of common housekeeping genes are varying in different tissues and experimental conditions. The genomic DNA contamination in RNA samples is another important factor that also influence the interpretation of the data obtained from qPCR. It is estimated that the gDNA contamination in gene expression analysis lead to an overestimation of the RNA transcript level. The aim of this study was to validate the most stably expressed reference genes in two different tissues of Aeluropus littoralis—halophyte grass at salt stress and recovery condition. Also, a qPCR-based approach for monitoring contamination with gDNA was conducted.ResultsTen candidate reference genes participating in different biological processes were analyzed in four groups of samples including root and leaf tissues, salt stress and recovery condition. To determine the most stably expressed reference genes, three statistical methods (geNorm, NormFinder and BestKeeper) were applied. According to results obtained, ten candidate reference genes were ranked based on the stability of their expression. Here, our results show that a set of four housekeeping genes (HKGs) e.g. RPS3, EF1A, GTF and RPS12 could be used as general reference genes for the all selected conditions and tissues. Also, four set of reference genes were proposed for each tissue and condition including: RPS3, EF1A and UBQ for salt stress and root samples; RPS3, EF1A, UBQ as well as GAPDH for recovery condition; U2SURP and GTF for leaf samples. Additionally, for assessing DNA contamination in RNA samples, a set of unique primers were designed based on the conserved region of ribosomal DNA (rDNA). The universality, specificity and sensitivity of these primer pairs were also evaluated in Poaceae.ConclusionsOverall, the sets of reference genes proposed in this study are ideal normalizers for qPCR analysis in A.littoralis transcriptome. The novel reference gene e.g. RPS3 that applied this study had higher expression stability than commonly used housekeeping genes. The application of rDNA-based primers in qPCR analysis was addressed.Electronic supplementary materialThe online version of this article (doi:10.1186/s40709-016-0053-8) contains supplementary material, which is available to authorized users.
One method extensively used for the quantification of gene expression changes and transcript abundances is reverse-transcription quantitative real-time PCR (RT-qPCR). It provides accurate, sensitive, reliable, and reproducible results. Several factors can affect the sensitivity and specificity of RT-qPCR. Residual genomic DNA (gDNA) contaminating RNA samples is one of them. In gene expression analysis, non-specific amplification due to gDNA contamination will overestimate the abundance of transcript levels and can affect the RT-qPCR results. Generally, gDNA is detected by qRT-PCR using primer pairs annealing to intergenic regions or an intron of the gene of interest. Unfortunately, intron/exon annotations are not yet known for all genes from vertebrate, bacteria, protist, fungi, plant, and invertebrate metazoan species.Here we present a protocol for detection of gDNA contamination in RNA samples by using ribosomal DNA (rDNA)-based primers. The method is based on the unique features of rDNA: their multigene nature, highly conserved sequences, and high frequency in the genome. Also as a case study, a unique set of primers were designed based on the conserved region of ribosomal DNA (rDNA) in the Poaceae family. The universality of these primer pairs was tested by melt curve analysis and agarose gel electrophoresis. Although our method explains how rDNA-based primers can be applied for the gDNA contamination assay in the Poaceae family, it could be easily used to other prokaryote and eukaryote species
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