Identification And Evaluation Of Novel Internal Control Gens And Fhb Resistance Genes By Microarray In Wheat

With the development of genomics and bioinformatics, more and more researchers focus on the analysis and applications of gene expression and regulation. Microarray and qRT-PCR are increasingly becoming of the good choice for high-throughput gene expression analysis. To get reliable results from both methods of analysis, it is vital to get accurate normalization of gene expression against one or more control genes. Thus, it is required that more ideal internal control genes are identified for normalization using various methods. Fusarium head blight (FHB) is a devastating fungal disease of common wheat (Triticum aestivum L.) and durum wheat (T. turgidum L. var. durum) worldwide. And FHB is one of the main factors that could reduce wheat yields and change milling, baking and pasta-making properties. The genetic control of FHB resistance has been analyzed through lots of QTL mapping studies. While FHB resistance genes were rarely identified and reported in wheat during recently years. Microarray provides abundant information of gene expression in whole genome, and the technology has been widely used in Wheat, In this study, novel internal control genes and FHB resistance genes were identified and evaluated in wheat using microarray.Novel internal control genes were selected and evaluated using microarray and Q-PCR.1. In this study, fifty candidate genes for internal control were obtained from the analysis of 333 Affymetrix GeneChip Wheat Genome arrays from nine independent experiments (salt toleration, drought toleration, aluminium toleration, nitrogene toleration, high temperature toleration, rust toleration, powdery mildew toleration and different development stages). The two-fold ratio method was used to select potential genes/fragments with minimal changes between samples.2. Expression levels of the 50 selected genes were then evaluated by quantitative real-time PCR with cDNA samples from different tissues, stages of development and environmental conditions. Seven novel internal control genes of 32 which were relatively stable were selected for revising the expression of target genes after comparing their respective expression profiles using NormFinder, geNorm, Pearson correlation coefficients and the 2-fold-change method.3. The expression stabilities of the novel internal control genes from this study were compared with 13 traditional ones for their expression stability. It was observed that 7 of the novel internal control genes were better than the traditional ones in expression stability under all the tested cDNA samples. Among the traditional internal control genes, the elongation factor 1-alpha exhibited strong expression stability, whereas the alpha-tubulin and GAPDH genes had very poor expression stability in the range of wheat samples tested.FHB resistance was evaluated in some wheat lines (Null,2DL,3BS,5A-QTL), and some QTL related resistance genes were selected and analyzed.using Microarray and Q-PCR.1. The disease resistance analysis showed that QTL lines (2DL,3BS,5A-QTL) had higher resistance to FHB than the Null-QTL ones (Null). Parental material (HC374) had the most obvious resistance, while other parental material (BW301) and.3251 (Null) presented worse resistance to FHB.2. One gene related with 2DL-QTL was identified in 2DL-QTL lines. The gene only expresses in lines which included 2DL-QTL. The gene is from Wuhan according to the expression pattern which is same as that in Wuhan. But the gene annotation is unknown till now.3. Four genes related with 3BS-QTL were identified in 3BS-QTL line. Those genes are lower expression or no expression in lines which included 3BS-QTL, and the genes annotation are also unknown up to now.4. Two genes related with 5A-QTL were identified in 5A-QTL line. One gene only expresses in lines which included 5A-QTL, and it is from Nyubay according to the expression pattern which is same as that in Nyubay. This gene encodes a lipid transfer protein (LTP) which plays an important role in resistance to pathogen and adaptation to environmental stresses. Another gene was only induced expression in lines which included 5A-QTL, and encodes an epoxide hydrolase which presents in all living organisms and plays important roles in proceeding metabolism, cellular protective mechanisms and signaling pathways.