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

Vijaykumar Veerappan, Mehul Jani, Khem Kadel, Taylor Troiani, Ronny Gale, Tyler Mayes, Elena Shulaev, Jiangqi Wen, Kirankumar S. Mysore, Rajeev K. Azad, Rebecca Dickstein

In 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. Two 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. In 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.