Rapid identification of causative insertions underlying Medicago truncatula Tnt1 mutants defective in symbiotic nitrogen fixation from a forward genetic screen by whole genome sequencing

Abstract

BackgroundIn the model legume Medicago truncatula, the near saturation genome-wide Tnt1 insertion mutant population in ecotype R108 is a valuable tool in functional genomics studies. Forward genetic screens have identified many Tnt1 mutants defective in nodule development and symbiotic nitrogen fixation (SNF). However, progress toward identifying the causative mutations of these symbiotic mutants has been slow because of the high copy number of Tnt1 insertions in some mutant plants and inefficient recovery of flanking sequence tags (FSTs) by thermal asymmetric interlaced PCR (TAIL-PCR) and other techniques.ResultsTwo Tnt1 symbiotic mutants, NF11217 and NF10547, with defects in nodulation and SNF were isolated during a forward genetic screen. Both TAIL-PCR and whole genome sequencing (WGS) approaches were used in attempts to find the relevant mutant genes in NF11217 and NF10547. Illumina paired-end WGS generated ~16 Gb of sequence data from a 500 bp insert library for each mutant, yielding ~40X genome coverage. Bioinformatics analysis of the sequence data identified 97 and 65 high confidence independent Tnt1 insertion loci in NF11217 and NF10547, respectively. In comparison to TAIL-PCR, WGS recovered more Tnt1 insertions. From the WGS data, we found Tnt1 insertions in the exons of the previously described PHOSPHOLIPASE C (PLC)-like and NODULE INCEPTION (NIN) genes in NF11217 and NF10547 mutants, respectively. Co-segregation analyses confirmed that the symbiotic phenotypes of NF11217 and NF10547 are tightly linked to the Tnt1 insertions in PLC-like and NIN genes, respectively.ConclusionsIn this work, we demonstrate that WGS is an efficient approach for identification of causative genes underlying SNF defective phenotypes in M. truncatula Tnt1 insertion mutants obtained via forward genetic screens.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2452-5) contains supplementary material, which is available to authorized users.

Validating Genome-Wide Association Candidates Controlling Quantitative Variation in Nodulation

“…S9). On average, there are 25 insertions in each Tnt1 line and as many as 97 independent Tnt1 loci have been reported (Veerappan et al, 2016). These background mutations can make phenotyping data potentially unreliable, complicating their use as the sole validation tool in reverse genetics phenotyping experiments.…”

Tnt1 retrotransposon as an efficient tool for development of an insertional mutant collection of Lotus japonicus

“…In the last decade, Tnt1 has been successfully used as a valuable tool for building M. truncatula mutant collections and applied in forward and reverse genetic studies (Tadege et al 2008;Zhou et al 2011;Bourcy et al 2013;Veerappan et al 2016). Phenotypic screening of the Tnt1 mutant lines is an efficient way to explore functions of new genes or new regulatory pathways.…”

Insertional mutagenesis of Brachypodium distachyon using the Tnt1 retrotransposable element

“…From our previous analyses of FSTs in M. truncatula Tnt1 insertion lines we know that TAIL-PCR can recover <50% of total FSTs in any given line (Sun et al, 2019). Encouraged by the TAIL-PCR results for B. distachyon, we sought to identify most of the insertions in all the R1 lines by taking advantage of two methodologies, sequence capture (Sun et al, 2019) and WGS (Veerappan et al, 2016). For WGS, DNA samples were extracted from the whole leaves of B. distachyon R0 mother plants (MP2 and MP15) and R1 (nc15-006, nc15-007 and nc15-008) plants using the Qiagen DNA extraction kit according to the manufacturer's instructions.…”