Genome‐wide LORE1 retrotransposon mutagenesis and high‐throughput insertion detection in Lotus japonicus

Abstract

SUMMARYUse of insertion mutants facilitates functional analysis of genes, but it has been difficult to identify a suitable mutagen and to establish large populations for reverse genetics in most plant species. The main challenge is developing efficient high-throughput procedures for both mutagenesis and identification of insertion sites. To date, only floral-dip T-DNA transformation of Arabidopsis has produced independent germinal insertions, thereby allowing generation of mutant populations from seeds of single plants. In addition, advances in insertion detection have been hampered by a lack of protocols, including software for automated data analysis, that take full advantage of high-throughput next-generation sequencing. We have addressed these challenges by developing the FSTpoolit protocol and software package, and here we demonstrate its efficacy by detecting 8935 LORE1 insertions in 3744 Lotus japonicus plants. The identified insertions show that the endogenous LORE1 retrotransposon is well suited for insertion mutagenesis due to homogenous gene targeting and exonic insertion preference. As LORE1 transposition occurs in the germline, harvesting seeds from a single founder line and cultivating progeny generates a complete mutant population. This ease of LORE1 mutagenesis, combined with the efficient FSTpoolit protocol, which exploits 2D pooling, Illumina sequencing and automated data analysis, allows highly cost-efficient development of a comprehensive reverse genetic resource.

Tnt1 Retrotransposon Mutagenesis: A Tool for Soybean Functional Genomics

“…In this strategy, a higher number of insertions per line allows for a lower number of individual plants to be maintained in order to create a population suitable for mutant screening. 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).…”

CYTOKININ OXIDASE/DEHYDROGENASE3 Maintains Cytokinin Homeostasis during Root and Nodule Development in Lotus japonicus

“…Homozygous LORE1 inserts were genotyped with allele specific primers in combination with the P2 internal LORE1 primer as described (Urba nski et al, 2012). Primer sequences were obtained from the LORE1 resource page (carb.au.dk/lotus-base) or designed in the same region if amplification was unsuccessful (Supplemental Table S2).…”

“…To identify mutants in Ckx3, we searched the publicly available LORE1 insertional mutant resources (Fukai et al, 2012;Urba nski et al, 2012;Hirakawa et al, 2014; accessed via carb.au.dk/lotus-base/). We identified lines with insertions in exon 1 (5497) and exon 3 (17827) of Ckx3 and named these ckx3-1 and ckx3-2, respectively ( Fig.…”

“…The availability of the LORE1 insertion population makes L. japonicus an ideal system for reverse genetics (Fukai et al, 2012;Urba nski et al, 2012), and here, we make use of this resource to address the role of cytokinin breakdown during nodule development. We identify two insertion alleles in LjCkx3 and show that this gene is critical for maintaining cytokinin homeostasis required for efficient symbiotic infection, organogenesis, and nitrate-dependent regulation of nodulation.…”

Translational Genomics in Agriculture: Some Examples in Grain Legumes

“…However, systematic mutant populations have been developed in several species in order to address this issue. TILLING populations are available for L. japonicus, pea, M. truncatula and chickpea (Dalmais et al, 2008;Le-Signor et al, 2009;Perry et al, 2003;Varshney et al, unpublished), Fast Neutron generated deletion mutant populations for M. truncatula, pea and soybean (Bolon et al, 2011;Hofer et al, 2009;Rogers et al, 2009;Smykal et al, 2012) and insertion mutant populations generated using exogenous (Medicago, d 'Erfurth et al, 2003) and endogeneous retrotransposons (Lotus, Urbański et al, 2012) have been generated and have contributed to gene discovery in these species (see Domoney et al, 2013;Domonkos et al, 2013;Urbański et al, 2013).…”