Characterization and Rapid Gene-Mapping of Leaf Lesion Mimic Phenotype of spl-1 Mutant in Soybean (Glycine max (L.) Merr.)

Abstract

In plants, lesion mimic mutants (LMMs) reveal spontaneous disease-like lesions in the absence of pathogen that constitutes powerful genetic material to unravel genes underlying programmed cell death (PCD), particularly the hypersensitive response (HR). However, only a few LMMs are reported in soybean, and no related gene has been cloned until now. In the present study, we isolated a new LMM named spotted leaf-1 (<i>spl-1</i>) from NN1138-2 cultivar through ethyl methanesulfonate (EMS) treatment. The present study revealed that lesion formation might result from PCD and excessive reactive oxygen species (ROS) accumulation. The chlorophyll content was significantly reduced but antioxidant activities, viz., superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT), as well as the malondialdehyde (MDA) contents, were detected higher in <i>spl-1</i> than in the wild-type. According to segregation analysis of mutant phenotype in two genetic populations, viz., W82×<i>spl-1</i> and PI378692×<i>spl-1</i>, the spotted leaf phenotype of <i>spl-1</i> is controlled by a single recessive gene named <i>lm1</i>. The <i>lm1</i> locus governing mutant phenotype of <i>spl-1</i> was first identified in 3.15 Mb genomic region on chromosome 04 through MutMap analysis, which was further verified and fine mapped by simple sequence repeat (SSR) marker-based genetic mapping. Genetic linkage analysis narrowed the genomic region (<i>lm1</i> locus) for mutant phenotype to a physical distance of ~76.23 kb. By searching against the Phytozome database, eight annotated candidate genes were found within the <i>lm1</i> region. qRT-PCR expression analysis revealed that, among these eight genes, only <i>Glyma.04g242300</i> showed highly significant expression levels in wild-type relative to the <i>spl-1</i> mutant. However, sequencing data of the CDS region showed no nucleotide difference between <i>spl-1</i> and its wild type within the coding regions of these genes but might be in the non-coding regions such as 5' or 3' UTR. Hence, the data of the present study are in favor of <i>Glyma.04g242300</i> being the possible candidate genes regulating the mutant phenotype of <i>spl-1</i>. However, further validation is needed to prove this function of the gene as well as its role in PCD, which in turn would be helpful to understand the mechanism and pathways involved in HR disease resistance of soybean.