Establishment of a soybean (Glycine max Merr. L) transposon-based mutagenesis repository

Abstract

Soybean is a major crop species providing valuable feedstock for food, feed and biofuel. In recent years, considerable progress has been made in developing genomic resources for soybean, including on-going efforts to sequence the genome. These efforts have identified a large number of soybean genes, most with unknown function. Therefore, a major research priority is determining the function of these genes, especially those involved in agronomic performance and seed traits. One means to study gene function is through mutagenesis and the study of the resulting phenotypes. Transposon-tagging has been used successfully in both model and crop plants to support studies of gene function. In this report, we describe efforts to generate a transposon-based mutant collection of soybean. The Ds transposon system was used to create activation-tagging, gene and enhancer trap elements. Currently, the repository houses approximately 900 soybean events, with flanking sequence data derived from 200 of these events. Analysis of the insertions revealed approximately 70% disrupted known genes, with the majority matching sequences derived from either Glycine max or Medicago truncatula sequences. Among the mutants generated, one resulted in male-sterility and was shown to disrupt the strictosidine synthase gene. This example clearly demonstrates that it is possible to disrupt soybean gene function by insertional mutagenesis and to derive useful mutants by this approach in spite of the tetraploid nature of the soybean genome.

Tnt1 Retrotransposon Mutagenesis: A Tool for Soybean Functional Genomics

“…The flanking sequences in this population can be readily identified using high-throughput sequencing methods to create a searchable database of insertion sites, comparable to those currently available for model species (WilliamsCarrier et al, 2010;Urba nski et al, 2012). Clearly, this approach has advantages over T-DNA or Ac/Ds mutagensis of soybean, in which a large number of independent transgenic lines would be needed (Scholte et al, 2002;Wessler, 2006;Mathieu et al, 2009). The Ac/ Ds system has the added limitation that most insertions occur within a short distance (within a few centimorgan) of the original insertion site (Jones et al, 1990;Ito et al, 1999;Parinov and Sundaresan, 2000).…”

“…However, clearly, phenotypes can be obtained by chemical or radiation mutagenesis (Cooper et al, 2008;Bolon et al, 2011). Moreover, we previously identified a male-sterile mutant of soybean using Ac/Ds mutagenesis (Mathieu et al, 2009). Even in the model plant Arabidopsis, the presence of multigene families can limit the ability to obtain phenotypes by mutating a single member of the family (Stacey et al, 2006).…”

“…Mutagenesis can modulate gene expression and create very useful loss-of-function mutants, whose phenotypes can validate and explore gene function. Insertional mutagenesis has been successfully used to study gene function in both model and crop plant species (Cowperthwaite et al, 2002;Alonso et al, 2003;An et al, 2003;Fladung et al, 2004;Tadege et al, 2008;Mathieu et al, 2009). A clear example is the use of T-DNA tagging to create large mutant populations of Arabidopsis (Arabidopsis thaliana; Alonso et al, 2003).…”

“…However, although the development of a T-DNA insertional mutant repository in soybean is technically possible, it would require a tremendous amount of labor, since each mutant line would require an independent transformation event. Thus, a transposon-tagging strategy, where many mutations could be derived from one primary transformation event, is an attractive approach for a plant such as soybean in which transformation requires a much longer time frame (roughly 1 year, from seed to seed; Parrott and Clemente, 2004;Mathieu et al, 2009).…”

“…For example, Mathieu et al (2009) utilized the well-characterized maize (Zea mays) transposon, Ac/Ds, to identify a soybean male-sterile line. Ac/Ds is a class II transposon, which transposes into new locations in plant genomes via a "cut-and-paste" mechanism (Wessler, 2006).…”