Volume 10, Issue 5, Pages 670-684 (May 2017) A PP2C-1 Allele Underlying a Quantitative Trait Locus Enhances Soybean 100-Seed Weight  Xiang Lu, Qing Xiong, Tong Cheng, Qing-Tian Li, Xin-Lei Liu, Ying-Dong Bi, Wei Li, Wan-Ke Zhang, Biao Ma, Yong-Cai Lai, Wei-Guang Du, Wei-Qun Man, Shou-Yi Chen, Jin-Song Zhang  Molecular Plant  Volume 10, Issue 5, Pages 670-684 (May 2017) DOI: 10.1016/j.molp.2017.03.006 Copyright © 2017 The Author Terms and Conditions

Figure 1 Analysis of Seed Traits in RILs Derived from G. soja ZYD7 and G. max HN44. (A) Distribution of the 100-seed weight in the RILs. (B) Distribution of the seed oil contents in the RILs. (C) Seed fatty acid profile in RILs. (D) The line R245 with larger seeds is found from the RILs derived from ZYD7 and HN44. (E) Comparison of 100-seed weight. Error bars indicate SD (n = 4), and the asterisk indicates significant difference compared with the control (*P < 0.05). Molecular Plant 2017 10, 670-684DOI: (10.1016/j.molp.2017.03.006) Copyright © 2017 The Author Terms and Conditions

Figure 2 Genotyping Map and Genetic Map Constructed from Resequencing Data of the RILs. (A) The genotyping map was constructed from SNPs with high accuracy and quality called by using GATK. (B) Construction of the genetic map by using R/qtl based on the genotyping information. Molecular Plant 2017 10, 670-684DOI: (10.1016/j.molp.2017.03.006) Copyright © 2017 The Author Terms and Conditions

Figure 3 Quantitative Trait Loci Mapping. (A) QTLs related to semi-determinant, seed-coat color, leaflet shape, seed oil content, and 100-seed weight of soybean were mapped by using genetic map and information of each trait. For seed oil content and 100-seed weight, data from three years are presented. (B) Position and other information of each QTL. For the column of positive allele, ‘C’ indicates that the allele is derived from cultivar HN44 and ‘W’ indicates that the allele is derived from wild soybean ZYD7. Molecular Plant 2017 10, 670-684DOI: (10.1016/j.molp.2017.03.006) Copyright © 2017 The Author Terms and Conditions

Figure 4 Identification of Genes Related to 100-Seed Weight of Soybean. (A) Candidate genes related to 100-seed weight. By PCR amplification and sequencing, we found four genes (Glyma17g33631, Glyma17g33690, Glyma17g33790, and Glyma17g33800) with nucleotide variation at the coding regions between G. soja ZYD7 and G. max HN44, and variations at two of them (Glyma17g33690, Glyma17g33790) resulted in amino acid changes. These two genes (Glyma17g33690, Glyma17g33790) were identified as causal candidate genes. (B) Glyma17g33690 encodes a protein with PP2C domain and the protein was named PP2C. PP2C-1 was cloned from ZYD7 and PP2C-2 was cloned from HN44. PP2C-1 has a 27L and 37E, whereas PP2C-2 has four L residues and D at the two positions, respectively. (C) Glyma17g33790 encodes a protein with an EamA-like domain, and was named EAL. EAL-1 was cloned from ZYD7 and EAL-2 was cloned from HN44. EAL-1 has a 62N whereas EAL-2 has a 62S. (D) PP2C expression pattern in different soybean organs. (E) EAL expression pattern in different soybean organs. (F) Overexpression of PP2C-2 did not enhance seed weight of the transgenic plants. (G) Overexpression of PP2C-1 enhanced seed weight of the transgenic plants. (H) Overexpression of EAL-1 did not enhance seed weight of the transgenic plants. (I) The overexpression of EAL-2 did not enhance seed weight of the transgenic plants. For gene expression in (D) to (I), error bars indicate SD (n = 4). For seed weight in (F) to (I), error bars indicate SD (n = 3) and asterisks indicate significant difference compared with WT (*P < 0.05). Molecular Plant 2017 10, 670-684DOI: (10.1016/j.molp.2017.03.006) Copyright © 2017 The Author Terms and Conditions

Figure 5 Measurement of Seed Size, Cotyledon Size, and Integument Cell Size in PP2C-1 Transgenic Plant and Wild-Type Col-0. (A) Comparison of seed size between PP2C-1 transgenic plant and wild-type (WT) Col-0. (B) Comparison of cotyledon size from 5-day-old seedlings between PP2C-1 transgenic plant and WT Col-0. (C) Comparison of seed integument cells 15 days after pollination between PP2C-1 transgenic plant and WT Col-0. (D) Measurement of seed width in various transgenic plants. (E) Measurement of seed length in various plants. (F) Measurement of seedling cotyledon in various plants. (G) Measurement of seed integument cell size in various plants. (H) Expression of seed weight/size-related genes in transgenic plants. The siliques from WT Col-0 and PP2C-1-overexpressing plants were collected at 5 days after hand pollination.Error bars indicate SD (D–G, n = 6; H, n = 4), and asterisks indicate significant difference compared with the control (*P < 0.05). Molecular Plant 2017 10, 670-684DOI: (10.1016/j.molp.2017.03.006) Copyright © 2017 The Author Terms and Conditions

Figure 6 The Analysis of Interaction between PP2C-1 and GmBZRs. (A) PP2C-1 and GmBZR1 co-localized in nucleus in epidermal cells of tobacco. (B) PP2C-1 and GmBZR1 exhibited interaction in nucleus in epidermal cells of tobacco by BiFC analysis. (C) PP2C-1, but not PP2C-2, interacted with GmBZR1 and GmBZR2 as revealed by luciferase complementary imaging (LCI) analysis. Right panel: quantification of the luciferase activity in the left panel. I, II, III, and VI indicate different combinations of interactions. (D) Deletion of the N terminus from PP2C-1 disrupted the interaction between PP2C-1 and GmBZR1 or GmBZR2. dnPP2C-1 has a deletion of N terminus from PP2C-1. (E) PP2C-1 interacted with GmBZR3 and GmBZR4, whereas PP2C-2 was not revealed by LCI analysis. (F) Removal of the N terminus from PP2C-1 disrupted the interaction between PP2C-1 and GmBZR3 or GmBZR4. dnPP2C-1 has a deletion of N terminus from PP2C-1. (G) PP2C-1, but not PP2C-2, interacted with AtBZR1 and AtBES1 as revealed by LCI analysis. (H) Deletion of N terminus from PP2C-1 blocked the interaction between PP2C-1 with AtBZR1 or AtBES1. dnPP2C-1 has a deletion of N terminus from PP2C-1. (I) Anti-GFP immunoprecipitation (IP) of PP2C-1-GFP with GmBZR1-FLAG, GmBZR2-FLAG, GmBZR3-FLAG, and GmBZR4-FLAG, respectively. Wild-type (WT) is the sample from leaves of tobacco plants. PP2C-1-GFP was immunoprecipitated using GFP-trap agarose beads, and the immunoblots were probed with antibodies against GFP or FLAG. (J) Brassinolide treatment and PP2C-1 both promoted accumulation of dephosphorylated GmBZR1 whereas PP2C-2 did not have this function. Tobacco leaves from a 35S::GmBZR1-FLAG transgenic plant were treated with 250 nM brassinolide (BL) for 1 h. The immunoblots were probed with antibodies against GFP, FLAG, or actin. (K) Relative expression (RE) of GmBZR1 in the three lines of GmBZR1-overexpressing plants. Error bars indicate SD (n = 4). Asterisk indicates significant difference from WT (*P < 0.05). (L) The overexpression of GmBZR1 enhances seed weight of the transgenic plants. Error bars indicate SD (n = 4). Asterisk indicates significant difference from WT (*P < 0.05). (M) The seeds from WT and GmBZR1-overexpressing plant are shown. Molecular Plant 2017 10, 670-684DOI: (10.1016/j.molp.2017.03.006) Copyright © 2017 The Author Terms and Conditions

Figure 7 The Association and Linkage Analysis of PP2C Genotypes with Soybean 100-Seed Weight in Different Populations. (A) The phylogenetic tree of PP2C-1 and PP2C-2 genotypes from different cultivated soybeans and wild soybeans. Names in yellow indicate cultivated soybeans and names in black indicate wild soybeans. The blue lines on the left indicate that the connected accessions have PP2C-1 allele. The red lines on the right indicate that the connected accessions have PP2C-2 allele. (B) Association analysis of the 100-seed weight with PP2C haplotypes in cultivated soybeans and wild soybeans. There are 60 wild soybean accessions and 55 cultivated soybean accessions with PP2C-1 genotype, and there are 12 wild soybean accessions and 39 cultivated soybean accessions with PP2C-2 genotype. (C) Linkage analysis of the 100-seed weight with PP2C haplotypes in F2 populations (with F3 seeds) derived from a cross between cultivar HF47 and wild soybean ZYD203. Among F2 populations, there are 34 F2 individuals with PP2C-2 allele and 56 F2 individuals with PP2C-1 allele. (D) Linkage analysis of the 100-seed weight with PP2C haplotypes in RILs derived from a cross between cultivar HN44 and wild soybean ZYD7. Among RILs, there are 180 RILs with PP2C-2 allele and 18 RILs with PP2C-1 allele.Asterisks in (B) to (D) indicate significantly different distribution between two groups (*P < 0.05, Kruskal–Wallis test). Molecular Plant 2017 10, 670-684DOI: (10.1016/j.molp.2017.03.006) Copyright © 2017 The Author Terms and Conditions