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 (spl-1) 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 spl-1 than in the wild-type. According to segregation analysis of mutant phenotype in two genetic populations, viz., W82×spl-1 and PI378692×spl-1, the spotted leaf phenotype of spl-1 is controlled by a single recessive gene named lm1. The lm1 locus governing mutant phenotype of spl-1 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 (lm1 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 lm1 region. qRT-PCR expression analysis revealed that, among these eight genes, only Glyma.04g242300 showed highly significant expression levels in wild-type relative to the spl-1 mutant. However, sequencing data of the CDS region showed no nucleotide difference between spl-1 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 Glyma.04g242300 being the possible candidate genes regulating the mutant phenotype of spl-1. 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.

Keywords: MutMap mapping; candidate gene; physio-chemical performance; soybean; spotted leaf mutant.

Figure 1

Morphological characteristics of wild-type and spl-1 mutant soybean genotypes: (A,C) leaf phenotype characteristic of wild-type; and (B,D) leaf phenotype characteristic of spl-1 mutant plants. Scale bars: (A,B) 1.0 cm; and (C,D) 1.5 cm.

Figure 2

Comparison of leaf photosynthetic pigment contents in wild-type and spl-1 mutant plants at: seedling stage (A); and mature stage (B). Chla, chlorophyll a; Chlb, chlorophyll b; Caro, carotenoids; Total Chl, total chlorophyll; FW, Fresh weight. The error bars indicate the mean ± SE (n = 3). SPSS software was used for statistical analysis. * significantly different at p < 0.05; ** significantly different at p < 0.01.

Figure 3

Leaf anatomical structure of wild-type and spl-1 mutant soybean genotypes: (A,C) leaf anatomical structure of wild-type plant; and (B,D) leaf anatomical structure of spl-1 mutant plants. p, palisade parenchyma; S, spongy parenchyma; VB, vascular bundle. Arrows show linear arrangement of VB in wild-type (C) and non-linear/distorted arrangement of VB in spl-1 mutant (D).

Figure 4

Histochemical staining analysis for leaves of wild-type and spl-1 mutant soybean genotypes: (A) Trypan blue staining for cell death. The spots indicated the ROS accumulated area in the spl-1 mutant. (B) DAB staining for H₂O₂ accumulation. Scale bars: (A,B) 1 cm.

Figure 5

Graph showing different physiological characteristics/parameters determined for both the wild-type and spl-1 mutant plants: (A) activity of superoxide dismutase (SOD); (B) activity of peroxidase (POD); (C) activity of Catalase (CAT); and (D) content of the malondialdehyde (MDA). The upper second leaves (lower lesion mimic (LLM)) and upper third leaves (higher lesion mimic (HLM)) of plants were used the estimation of these parameters at six weeks after sowing in pots. The data represent the means ± SE of three replicates. * significantly different at p < 0.05; ** significantly different at p < 0.01.

Figure 6

Identification of candidate genomic region (lm1 locus) through MutMap analysis at a genomic interval of 45.80–48.95 Mb (Version Glyma v1.a1) on chromosome 04 of soybean: (A,B) the SNP-index of wild-type (A-Pool) and spl-1 mutant (B-Pool) pools, respectively, for chromosome 04; and (C) the ∆ (SNP-index) plot for chromosome 04. x-axis indicates the physical position of chromosome and y-axis indicates the average SNP-index in a 2-Mb interval with a 50-kb sliding window. The Δ (SNP-index) graph was plotted with statistical confidence intervals under the null hypothesis of no QTL (p < 0.05). The candidate region (lm1 locus) identified for spl-1 mutant phenotype is marked by two red dash border lines in ∆ (SNP-index) plot.

Figure 7

Mapping and fine mapping of lm1 locus. (A) Location of lm1 locus identified by MutMap-based BSA method on chromosome 04. (B) Dashes line indicated rough mapping of spl-1 locus from cross of W82×spl-1. Vertical lines indicate polymorphic markers. Names of markers are shown above the line and the recombinants between lm1 and each marker are shown below the line. (C) Fine mapping of lm1 with genotyping data from newly developed polymorphic markers in the cross of PI378692×spl-1. (D) Eight candidate genes in the fine-mapped region.

Figure 8

Relative gene expression of eight candidate genes in the leaves of wild-type and mutant (spl-1) plants at three developmental growth stages V1, V3 and R1 using qRT-PCR. Mean values of expression data of wild-type and spl-1 mutant plants were analyzed for statistical significance at p < 0.01 (**) level, as indicated by asterisks on top of bars.