ABSTRACT
Background: Q-PCR is the method of choice for PCR- based transcriptomics and validating microarray-based and RNA-seq results. Proper application of this technology requires proper normalization to correct as much as possible errors propagating during RNA extraction and cDNA synthesis. Objectives: The investigation was performed to find stable reference genes in sunflower under shifting in ambient temperature. Materials and Methods: Sequences of five well-known reference genes of Arabidopsis (Actin, Ubiquitin, Elongation factor-1, GAPDH, and SAND) and one well-known reference gene inhuman, Importin, were subjected to BLASTX against sunflower databases and the relevant genes were subjected to primer designing for q-PCR. Two sunflower inbred lines were cultivated at two dates so that anthesis occurred at nearly 30 °C and 40 °C (heat stress). The experiment was repeated for two years. Q-PCR was run on samples taken for two planting date separately at the beginning of anthesis for each genotype from leaf, taproots, receptacle base, immature and mature disc flowers and on pooled samples comprising of the tissues for each genotype, planting dates and also all tissues for both genotypes and both planting dates. Basic statistical properties of each candidate gene across all the samples were calculated. Furthermore, gene expression stability analysis was done for six candidate reference genes on Cq mean of two years using three independent algorithms, geNorm, Bestkeeper, and Refinder. Results: Designed primers for Actin2, SAND, GAPDH, Ubiquitin, EF-1a, and Importin yielded a single peak in melting curve analysis indicating specificity of the PCR reaction. Basic statistical analysis showed that Actin2 and EF-1a had the highest and lowest expression levels across all the samples, respectively. Actin2 appeared to be the most stable reference gene across all the samples based on the three used algorithms. Pairwise variation analysis revealed that for samples taken under ambient temperature of 30 °C, Actin2, EF-1a, SAND and for those taken under ambient temperature of 40 °C, Actin2, EF-1a, Importin and SAND have to be used for normalization in q-PCR studies. Moreover, it is suggested that normalization to be based on Actin2, SAND and EF-1a for vegetative tissues and Actin2, EF-1a, SAND and Importin for reproductive tissues. Conclusions: In the present research, proper reference genes for normalization of gene expression studies under heat stress conditions were introduced. Moreover, the presence of genotype-by- planting date interaction effects and tissue specific gene expression pattern on the behavior of the most three stable reference genes was indicated.
ABSTRACT
BACKGROUND: In plant breeding program to produce hybrid varieties, pair of male sterile and restorer fertility lines are required. Differentiation of lines possessing restorer fertility allele from the lines lacking it remove the need for the progeny test, and thus reducing the time and the cost in the hybrid production procedure. Canola breeding program in Iran has concentrated toward production of domestic hybrid varieties, however, it suffers from lack of molecular information in restore fertility status of lines, and therefore it needs time and tedious activities. OBJECTIVES: To design gene-based markers for distinguishing R-, A- lines and hybrids in sunflower breeding programs. MATERIAL AND METHODS: Aligning sequences of locus responsible for male sterility and that of male fertility resulted in finding differences in the loci, which used to define two set of suitable primer pairs. Genomic DNA from 25 R-lines (23 inbred lines and two commercial lines), 9 A-lines (7 inbred lines and two commercial lines), one B-line and two commercial hybrids were extracted and used in PCR as template. RESULTS: Using one-primer pairs, a band of nearly 1500 bp was amplified in restorer lines but not in A-, B- lines. Another primer pair used to distinguish hybrids (heterozygout) from restorer lines. Results of the report is predicted to be used in canola breeding for hybrid production. CONCLUSIONS: Although the molecular bases for the male sterility and fertility restoration in rapeseed is not published, taking advantages of gene-based markers, make rapeseed breeding program more efficient regarding time and costs.
ABSTRACT
BACKGROUND: Downy mildew caused by Plasmopara halstedii is a devastating disease in sunflower worldwide. Several dominant resistance genes designated as Pl have been identified and linked molecular markers have been demonstrated. However, no information on theresistance genes is available forIranian lines. OBJECTIVES: The presence of three map-based molecular markers previously proved to be linked to different resistance genes were evaluated in sunflower inbred lines. MATERIALS AND METHODS: Using PCR-based and CAPS molecular markers, 26 sunflower inbred lines with different responses to P. halstedii race 100 were used to detect the presence of three resistance loci including Pl1 , Pl6 and Pl13 within the lines. RESULTS: Molecular marker linked to Pl13 was present in some of the sunflower lines but was not correlated with the phenotypic reaction of the lines to race 100. Despite the use of three markers linked to Pl6 , PCR failed to amplify any corresponding product. This data may suggest that none of the genotypes possessed Pl6 locus. Pl1 -linked cleaved amplified polymorphic sequences (CAPS) were present in several resistance lines and effectively differentiated susceptible and resistant sunflower lines. CONCLUSIONS: Applicability of molecular markers in breeding programs revisited in disease management.
