Wxlv, the Ancestral Allele of Rice Waxy Gene Changquan Zhang, Jihui Zhu, Shengjie Chen, Xiaolei Fan, Qianfeng Li, Yan Lu, Min Wang, Hengxiu Yu, Chuandeng Yi, Shuzhu Tang, Minghong Gu, Qiaoquan Liu  Molecular Plant  Volume 12, Issue 8, Pages 1157-1166 (August 2019) DOI: 10.1016/j.molp.2019.05.011 Copyright © 2019 The Authors Terms and Conditions

Figure 1 Identification of Wxlv in Oryza sativa. (A–E) Comparison of the eating and cooking qualities (ECQs) of Q11 with Nip and GC2 carrying the Wxb or Wxa allele: appearance of cooked rice (A), taste value (B), AAC (C), GC (D), and RVA profile (E) of milled rice. GLXN in (E) is a glutinous cultivar with the null wx allele. (F–H) Map-based cloning of Wxlv. Location of the LV locus for low viscosity in a 10.17-kb genomic region on chromosome 6 determined using 40 F2 plants from Nip*Q11 (F) and 3000 BC6F2 individuals with low starch viscosity (G). The numbers below the bar in (F) and (G) indicate the numbers of recombinants. (H) The genotypes and phenotypes of the recombinants and the allelic variation in the mapped Wx gene between Q11 with Nip and GC2. (I–N) Transgenic verification of Wxlv. The NILs were Nip(wx), Nip(Wxlv), and Nip(Wxa) in the Nip background introgressed with the wx, Wxa, and Wxlv alleles, respectively. (I) and (J) show the appearances of brown rice from the NILs and the corresponding derived transgenic lines Nip(wx)-Wxa, Nip(wx)-Wxlv, and Nip(Wxa)-Wxlv, respectively. (K–N) The RVA profiles, AAC, and GC of milled rice from the plants indicated above. Error bars are mean ± SD (n = 3). Significant differences were determined by Student's t-test (*P < 0.05, **P < 0.01). Molecular Plant 2019 12, 1157-1166DOI: (10.1016/j.molp.2019.05.011) Copyright © 2019 The Authors Terms and Conditions

Figure 2 Expression and Activity of Wxlv-Encoded GBSSI in the Nip Background. (A and B) Profiles of Wx mature mRNAs (A) and GBSSI activity (B) during endosperm development for Nip and its NILs Nip(Wxa) and Nip(Wxlv). (C) SDS–PAGE assay of starch granule-bound GBSSI (top) and total seed proteins (bottom) in mature seeds. (D–H) ECQs of milled rice in the Nip(wx) background carrying the transgene Wxb (Nip(wx)-Wxb) or its site-specific mutant Wxb(P415S) with the C–T substitution at Ex10-115 (Nip(wx)-Wxb(P415S)): (D) appearance of brown rice; (E) AAC; (F) GC; (G) GT; and (H) RVA profile. (I and J) Three-dimensional homology modeling of GBSSI and its phosphorylation at the 415th residue encoded by Wxlv (I) and Wxa (J). Error bars are mean ± SD (n = 3). Significant differences were determined by one-way ANOVA (*P < 0.05, **P < 0.01). Molecular Plant 2019 12, 1157-1166DOI: (10.1016/j.molp.2019.05.011) Copyright © 2019 The Authors Terms and Conditions

Figure 3 Morphology and Fine Structure of Starch Granules before and after Cooking. (A–O) All samples were from the mature grains of NILs carrying different Wx alleles in the Nip background: Nip(Wxb) (A, D, G, J, and M), Nip(Wxlv) (B, E, H, K, and N), and Nip(Wxa) (C, F, I, L, and O). (A–C) SEM images showing the morphologies of isolated starch granules. (D–F) SEM images showing the morphologies of paste starch granules after hot gelatinization and subsequent cooling during the RVA process. (G–I) Images of whole cooked rice kernels after cooling. (J–L) SEM images showing transverse sections of cooked rice after cooling. (M–O) SEM images showing the morphologies of cooked rice, with red arrows indicating some ungelatinized starch components. (P–R) Molecular weight distributions of debranched total starches (P), isolated native amylose (Q), and debranched amylose (R) determined by GPC. MW represents the apparent molecular weight relative to the standards. Molecular Plant 2019 12, 1157-1166DOI: (10.1016/j.molp.2019.05.011) Copyright © 2019 The Authors Terms and Conditions

Figure 4 Genetic Diversity and Evolutionary Relationship among Multiple Wx Alleles in Rice. (A) Genotypes and phenotypes of different Wx alleles. The genomic structure of the Wx gene is shown on the top, and the nucleotide sequences and their encoded residues (in parentheses) of six functional polymorphic sites are listed for each allele. The right side presents the AAC and GC values of mature grains from the NILs carrying corresponding Wx alleles. (B) Distributions of polymorphic nucleotides at five functional sites of the Wx gene in tested wild rice samples. The locations of the five polymorphic sites are the same as in (A), and the numbers at the bottom indicate the numbers of the wild rice samples used in the analysis of each polymorphic site (details can be found in Supplemental Data 1 and 2; n = 121 use accessions W001 to W121 and n = 391 use accessions W001 to W121 and 270 accessions from published data). (C) Proposed evolutionary relationship among various Wx alleles in rice. Wxlv-I, Wxlv-II, Wxlv-III, and Wxlv-IV indicate the four haplotypes of Wxlv alleles in O. sativa. (D) Worldwide distributions of different Wx alleles in O. sativa. Different colors represent the various Wx alleles, and each pie chart indicates the proportions of different Wx alleles in a certain area. The size of each pie chart represents the amount of samples, with a larger pie chart indicating more samples. The pie charts at the bottom summarize the distributions of multiple Wx alleles throughout the world or on different continents; the numbers in parentheses indicate the numbers of tested cultivars carrying a certain Wx allele. Molecular Plant 2019 12, 1157-1166DOI: (10.1016/j.molp.2019.05.011) Copyright © 2019 The Authors Terms and Conditions