Abstract: Intergeneric hybridization is an important strategy to introgress alien genes into common wheat for its improvement. But presence of cross ability barrier mechanism regulated by Kr1 gene played a major destructive role for hybridization than other reported genes. In order to know the underlying molecular mechanism and to dissect out this barrier, a new annealing system, ACP (anneling control primer) system was used in chromosome 5B (containing Kr1 gene) specific Recombinant Inbred Line (RIL) population. Two differentially expressed fragments for Kr1 gene was identified, cloned and sequenced. Further the expression was confirmed by northern blotting analysis. Sequence analysis of the resulted clones revealed classes of putative genes, including stress responsive and signal transduction.

Abstract: Many methods are available for total RNA extraction from plants, except the floral organs like wheat pistils containing high levels of polysaccharides that bind/or co-precipitate with RNA. In this protocol, a simple and effective method for extracting total RNA from small and feathery wheat pistils has been developed. Lithium chloride (LiCl) and phenol:chloroform:isoamylalcohol (PCI) were employed and the samples were ground in microcentrifuge tube using plastic pestle. A jacket of liquid nitrogen and simplified procedures were applied to ensure thorough grinding of the pistils and to minimize the samples loss. These measures substantially increased the recovery of total RNA (approximately 50%) in the extraction process. Reliable differential display by cDNA-AFLP was successfully achieved with the total RNA after DNase treatment and reverse transcription. This method is also practicable for gene expression and gene regulation studies in floral parts of other plants.