Submitted to: Plant Molecular Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/26/2006
Publication Date: 10/1/2006
Citation: Singh, J., Zhang, S., Chen, C., Cooper, L., Bregitzer, P.P., Sturbaum, A.K., Hayes, P.M., Lemaux, P.G. 2006. High-frequency DS remobilization over multiple generations in barley facilitates gene tagging in large genome cereals. Plant Molecular Biology.62:937-950 Interpretive Summary: Mobile genetic elements, called transposons, are known to cause mutations when they move into a gene. One type of element, Ds, has been moved from corn to barley, where it functions similarly. Movement of the Ds element into barley genes will cause mutations that can be detected visually or by chemical or molecular measurements that detect changes in physiological functions. Because Ds has been studied for 80 years, its genetic sequence is well known. This knowledge can enable researchers to identify and locate the places in the barley genome to which Ds moves, and also to discover detailed information about the gene into which it has moved. This process of creating mutation-based changes in barley genetic functions, and going on to discovering precise information about the gene involved in the change, is called "gene tagging". Tagging genes ultimately gives researchers a greater understanding of what genes exist, where they are, and how they function. Because this system depends on relatively localized movements of Ds, and because the barley genome is very large, it is important that a number of specialized genetic stocks--each with single Ds element in a different location--be created, and that the location of the Ds element be mapped. Previous research described the process of creating and mapping the first 20 specialized Ds barley genetic stocks. This report represents a continuation of this work in which Ds lines have been carefully studied to determine whether this system is operable in barley, and to determine molecular features that can be used to determine which Ds lines are useful versus those that may not operation correctly. The experiments conducted for these reports showed that the majority of lines (> 80%) were useful--meaning that the Ds elements could move around--and that particular molecular features could be identified that were predictive of whether any particular line would be useful. Thus, these materials have been shown to be useful for their intended purpose, and are now ready to be used in studies which will attempt to identify genes of importance to barley and to other closely-related crops such as corn, rice, and wheat.
Technical Abstract: Different strategies are used to exploit the maize Ac/Ds transposon system for functional genomics studies in heterologous species. In one approach, large numbers of independent Ds insertion lines (TNPs) are generated and screened phenotypically. Alternatively, smaller numbers of TNPs are produced; Ds locations mapped; and those near genes of interest are reactivated for localized gene targeting. To use the latter strategy effectively, it is imperative to understand key characteristics of the system. We generated more than 100 single-copy primary, secondary, tertiary and quaternary Ds TNPs in barley. Frequencies of reactivation of primary, secondary and tertiary TNPs with intact terminal inverted repeats (TIRs) ranged from 11.8% to 17.1%. In 16% of TNPs, TIRs were damaged and no transposition could be detected among progeny of crosses using parental lines with imperfect TIRs. In half of the lines, the nature of flanking sequences and status of the 11 bp TIRs and 8-bp direct repeats were determined. BLAST searches using a gene prediction program indicate that 86% of TNP flanking sequences match either known or putative genes, confirming preferential insertion of Ds into genic regions in barley, critical for functional genomics efforts in species with large genomes like barley and wheat. The observed reactivation frequencies of primary, secondary, tertiary and quaternary TNPs, coupled with the tendency of Ds for localized transposition, validates the approach of saturation mutagenesis using Ds to tag and characterize genes linked to Ds insertions.