Gene mapping and candidate gene analysis of a sorghum sheathed panicle-I mutant

Abstract

Panicle exsertion is essential for crop yield and quality, and understanding its molecular mechanisms is crucial for optimizing plant architecture. In this study, the sheathed panicle-I (shp-I) mutant was identified from the ethyl methane sulfonate mutant population of the sorghum [Sorghum bicolor (L.) Moench] variety Hongyingzi (HYZ). While phenotypically similar to the wild type during the seedling stage, shp-I exhibits a significantly shorter peduncle internode at the heading stage. Cytomorphological analysis revealed reduced parenchyma cell size within the mutant's peduncle internode. Phytohormonal profiling showed lower levels of indole-3-acetic acid and higher concentrations of brassinosteroid in the mutant compared to the wild type at the peduncle internode. Genetic analysis confirmed that the mutant phenotype was caused by a recessive single-gene mutation. Through bulked segregant analysis sequencing (BSA-seq) genetic mapping, the causative locus for the mutant phenotype was localized to a 59.65-59.92 Mb interval on chromosome 10, which contains 28 putative genes. Additionally, the gene SbiHYZ.10G230700, which encodes a BTB/POZ and MATH (BPM) domain protein, was identified as a candidate gene. Further analysis revealed that the non-synonymous mutations in the candidate gene were located within the MATH domain, affecting the 3D structure of the protein. In summary, this study provides a new genetic material and candidate genes for future research into the molecular regulation of sorghum peduncle length.

FIGURE 1

Phenotypic analysis of wild type and sheathed panicle‐I (shp‐I) mutant. (A) Phenotypes of wild type and mutant at heading and maturity stages. The white arrows indicate the panicle exsertion status in wild type and mutant. (B) Panicle exsertion status of the wild type (left) and the mutant (right) at different development stages. DAH, day after heading. (C) Internodes of wild type (left) and the mutant (right) at maturity stage. IN1 to IN9 denote the first internode to the ninth internode from the apex to the base of plant morphology. (D) The quantification of internode lengths between the wild type and the mutant (**p < 0.001, two‐sided unpaired Student's t‐test). Data presents mean ± SD (n = 10). Scale bars in A, B, and C are 20 cm.

FIGURE 2

Morphological analysis and the quantification of parenchyma cells dimensions and quantities within the longitudinal section of peduncle. (A) Visual field of randomly selected parenchyma cells within the peduncle of wild type. (B) Visual field of randomly selected parenchyma cells within the peduncle of mutant. Scale bars in A and B are 50 µm. (C) The length of parenchyma cells in wild type and mutant. (D) The width of parenchyma cells in wild type and mutant. (E) The number of pith parenchyma cells in the same visual field. ***Statistically significant difference at the p < 0.001 level.

FIGURE 3

(A) Isolated tissues utilized for the quantification of phytohormone levels. FL, flag leaf; PE, peduncle; P, panicle. (B) The concentrations of gibberellins (GA₃) in different tissues of wild type and mutant. Scale bar in A is 20 cm. (C) The concentrations of brassinosteroid (BR) in different tissues of wild type and mutant. (D) The concentrations of indole‐3‐acetic acid (IAA) in different tissues of wild type and mutant. ***Statistically significant difference at p < 0.001 level.

FIGURE 4

The BSA‐seq analysis of F₂ population derived from mutant (♀) and wild type (♂). (A) Manhattan Plot generated based on single‐nucleotide polymorphism (SNP)‐index algorithm. The X‐axis represents the chromosomes of the reference genome, and the Y‐axis denotes the ΔSNP index value between the mutant and wild‐type pools. The blue and green lines indicate the 99% and 95% confidence intervals, respectively. The black dots denote the ΔSNP‐index for each SNP, and the red line is the fitted curve of the mean values of ΔSNP‐index derived from the sliding window analysis. The red arrow indicates the peak above the threshold on the Chromosome 10. (B) Manhattan Plot generated based on Euclidean distance (ED) algorithm. The X‐axis represents the chromosomes of the reference genome, the Y‐axis denotes the ED^4 value. The grey line represents the 99% threshold; the black line is the fitted curve of the ED^4 values. The red arrow indicates the candidates significantly above the threshold. BSA‐seq, bulked segregant analysis sequencing.

FIGURE 5

The expression levels of five candidate BTB/POZ and MATH (BPM) domain‐containing genes in flag leaf, peduncle, and panicle at two development stages in wild type and mutant. (A) The heatmap of the expression values (FPKM [fragments per kilobase of transcript per million mapped reads] values in RNA sequencing analysis) of five candidate genes. FL, flag leaf; PE, peduncle; P, panicle. The S1 indicates booting stage, and S2 indicates 4 days after heading. (B) The expression level of SbiHYZ.10G230700 at booting stage. (C) The expression level of SbiHYZ.10G230700 at 4 days after heading. *, **, and ***Statistically significant differences at p < 0.05, p < 0.01, and p < 0.001 levels, respectively. ns denotes no statistically significant difference.

FIGURE 6

Protein sequence analysis of SbiHYZ.10G230700 in wild type and mutant forms. (A) Amino acid sequence alignment of the SbiHYZ.10G230700 proteins in wild type (Sbi.10G230700‐WT) and mutant (Sbi.10G230700‐mut) variants. Amino acid residues highlighted indicate the non‐synonymous mutant sites. The identical amino acid residues were present in a purple background. MATE and BTB/POZ represent the conserved MATE and BTB/POZ domains, respectively. (B) The 3D structure of the SbiHYZ.10G230700 protein monomer in wild type and mutant forms. The wild‐type structure is depicted in green, while the mutant form is shown in red. (C) Close‐up view of the surface topology at the residues corresponding to non‐synonymous mutation sites from an identical perspective. (D) Close‐up view of the surface topology at the non‐synonymous mutation sites in the mutant SbiHYZ.10G230700 protein from an identical perspective. Green asterisks (*) mark residues corresponding to non‐synonymous mutation sites in the wild‐type protein, and red asterisks (*) denote the non‐synonymous mutant residues in the mutant protein. The orientation axes for the protein 3D structures are displayed in the right‐hand corners.