Combining Fine Mapping, Whole-Genome Re-Sequencing, and RNA-Seq Unravels Candidate Genes for a Soybean Mutant with Short Petioles and Weakened Pulvini

Abstract

A short petiole is an important agronomic trait for the development of plant ideotypes with high yields. However, the genetic basis underlying this trait remains unclear. Here, we identified and characterized a novel soybean mutant with short petioles and weakened pulvini, designated as short petioles and weakened pulvini (spwp). Compared with the wild type (WT), the spwp mutant displayed shortened petioles, owing to the longitudinally decreased cell length, and exhibited a smaller pulvinus structure due to a reduction in motor cell proliferation and expansion. Genetic analysis showed that the phenotype of the spwp mutant was controlled by two recessive nuclear genes, named as spwp1 and spwp2. Using a map-based cloning strategy, the spwp1 locus was mapped in a 183 kb genomic region on chromosome 14 between markers S1413 and S1418, containing 15 annotated genes, whereas the spwp2 locus was mapped in a 195 kb genomic region on chromosome 11 between markers S1373 and S1385, containing 18 annotated genes. Based on the whole-genome re-sequencing and RNA-seq data, we identified two homologous genes, Glyma.11g230300 and Glyma.11g230600, as the most promising candidate genes for the spwp2 locus. In addition, the RNA-seq analysis revealed that the expression levels of genes involved in the cytokinin and auxin signaling transduction networks were altered in the spwp mutant compared with the WT. Our findings provide new gene resources for insights into the genetic mechanisms of petiole development and pulvinus establishment, as well as soybean ideotype breeding.

Keywords: RNA-seq; candidate genes; fine mapping; short petioles and weakened pulvini mutant; soybean; whole-genome re-sequencing.

Figure 1

Phenotype comparison between wild-type (WT) and spwp mutant-type (MT) plants. (A): Phenotype appearance of whole plants of the WT (left) and MT (right). The scale bar is 10 cm. (B): Comparison of petiole length from the first to third leaf position between the WT and MT. L1, L2, and L3 represent the first to third leaf petioles from the top to the bottom, respectively. (C,D): Longitudinal sections on the middle part of the petioles at the third node, counted from the top to the bottom of the WT (C) and MT (D) plants. The scale bars are 50 μm. (E): Comparison of the single-cell length of the leaf petioles between the WT and MT. The result represents the average value of five consecutive cells in a single longitudinal cell file. Significant differences were determined using the Student’s t-test: ** p < 0.01. All data are given as mean ± SD.

Figure 2

Cross-sectional microstructure of pulvini in wild-type (WT) and spwp mutant-type (MT) plants. A and B: Phenotypic characterization of pulvini in WT (A) and MT (B) plants, respectively. The scale bars are 1 cm. (C,D): Transection of pulvini in the WT (C) and MT (D), respectively. The scale bars are 500 μm. (E–L): Partial magnifying views of the transection of pulvini in the WT (E,G,I,K) and MT (F,H,J,L). The scale bars are 100 μm. (E,F) indicate the motor cells of the WT and MT on the abaxial side (AB), respectively. (I,J) indicate the motor cells of the WT and MT on the adaxial side (AD), respectively. (G,H) indicate the vascular cylinder of the WT and MT on the abaxial side, respectively. (K,L) indicate the vascular cylinder of the WT and MT on the adaxial side, respectively.

Figure 3

Fine-mapping of the spwp1 locus. (A): the spwp1 locus was initially mapped by single-sequence repeat (SSR) markers Satt063 and S1465 on chromosome (Chr.) 14 using 42 mutant plants derived from the F₂ population of KF1 × spwp. (B): the spwp1 locus was further mapped to a region between SSR markers S1382 and S1465 using theF₂ population of NN1138-2 × spwp. (C): the position of the spwp1 locus was finally narrowed down to a genomic region of 183 kb between SSR markers S1413 and S1418 using the F_2:3 population of KF1 × spwp. (D): Fifteen predicted open reading frames (ORFs) were located in this region according to the Williams 82 reference genome (Glycine max Wm82.a4.v1). The numerals below the markers indicate the number of identified recombinants (Rec.).

Figure 4

Fine-mapping of the spwp2 locus. (A): the spwp2 locus was initially mapped by single-sequence repeat (SSR) markers S1335 and Sat_331 on chromosome (Chr.) 11 using the F₂ population of KF1 × spwp. (B): the position of the spwp2 locus was finally delimited to a 195 kb region between SSR markers S1373 and S1385. The numerals below the markers indicate the number of identified recombinants (Rec.). (C): eighteen predicted open reading frames (ORFs) existed in the 195 kb fine-mapping interval according to the Williams 82 reference genome (Glycine max Wm82.a4.v1).

Figure 5

Distribution of delta SNP index values in 20 chromosomes of soybean. The x-axis indicates the physical position of chromosomes and the y-axis indicates delta SNP index values. Delta index = index (MT) − index (WT).

Figure 6

Differentially expressed genes (DEGs) in the spwp mutant compared with the wild-type (WT). (A): Volcano plot of DEGs determined by RNA-seq using the criteria p-value ≤ 0.05 and |log2 (Fold Change)| ≥ 1. Red dots indicate up-regulated genes and green dots indicate down-regulated genes. (B): enriched GO terms in the “molecular function” (MF) and “biological processes” (BP) categories of DEGs.

Figure 7

Analysis of the expression levels of all candidate genes using RNA-seq data, and six of these genes validated by RT-qPCR analysis. (A,B): Expression profiles of 18 and 15 genes predicted to be located in the spwp2 locus (A) and the spwp1 locus (B), respectively. The FPKM (fragments per kilobase of transcript per million mapped reads) value is the average of three biological replicates. (C): Relative expression levels of six genes detected by RT-qPCR in petioles and pulvini. Error bars indicate the SD (n = 3). Asterisks indicate a significant difference compared with the corresponding controls (**, p < 0.01; Student’s t-test).