The origin of Wxla provides new insights into the improvement of grain quality in rice

Abstract

Appearance and taste are important factors in rice (Oryza sativa) grain quality. Here, we investigated the taste scores and related eating-quality traits of 533 diverse cultivars to assess the relationships between-and genetic basis of-rice taste and eating-quality. A genome-wide association study highlighted the Wx gene as the major factor underlying variation in taste and eating quality. Notably, a novel waxy (Wx) allele, Wx^la , which combined two mutations from Wx^b and Wxⁱⁿ , exhibited a unique phenotype. Reduced GBSSI activity conferred Wx^la rice with both a transparent appearance and good eating quality. Haplotype analysis revealed that Wx^la was derived from intragenic recombination. In fact, the recombination rate at the Wx locus was estimated to be 3.34 kb/cM, which was about 75-fold higher than the genome-wide mean, indicating that intragenic recombination is a major force driving diversity at the Wx locus. Based on our results, we propose a new network for Wx evolution, noting that new Wx alleles could easily be generated by crossing genotypes with different Wx alleles. This study thus provides insights into the evolution of the Wx locus and facilitates molecular breeding for quality in rice.

Keywords: Oryza sativa; allelic variation; appearance and eating quality; intragenic recombination; waxy.

Figure 1

Phenotypic analysis reveals trait relationships and subpopulation characteristics (A) Heatmap depicting Pearson's correlation coefficients between phenotype means for quality traits across all varieties within the study. Phenotypic distributions of quality traits are located within the diagonal. (B) Phenotypic distributions of quality traits divided by the aus (Aus), indica (Ind), and japonica (Jap) subpopulations. The number of varieties within each subpopulation was 50, 307, and 176, respectively. cP, centipoise.

Figure 2

Association study identification of multiple Wx alleles conferring different qualities (A) Manhattan plot for apparent amylose content (AAC) in the whole population. The dashed line represents the significance threshold (−log₁₀(P) = 7.18). (B) Local Manhattan plot (top) and LD heatmap (bottom) surrounding the peak on chromosome 6. The red dot indicates the position of nucleotide variation in the candidate gene. (C) Genotypes and phenotypes of different Wx alleles in 533 different rice varieties. (D) The appearance of brown rice and milled rice for representative cultivars carrying different Wx alleles. (E) Representative rapid visco‐analyzer (RVA) pasting properties for different Wx alleles. cP, centipoise.

Figure 3

The appearance and morphology of starch granules for different Wx alleles of white rice (A–D) Milled rice from genotypes ZN‐Wx ^b (A), ZN‐Wx ^la (B), ZN‐Wx ^mp (C) and ZN (D). (E–P) SEM images showing the morphology of the starch granules of ZN‐Wx ^b (E, I, M), ZN‐Wx ^la (F, J, N), ZN‐Wx ^mp (G, K, O) and ZN (H, L, P). Resolution, 40× (E–H), 500X (I–L) and 2,500X (M–P).

Figure 4

Different GBSSI enzyme activities lead to differences in qualities conferred by Wx^b, Wx^la and Wx^mp (A–E) The apparent amylose content (AAC) (A), gel consistency (GC) (B), gelatinization temperature (GT) (C), rapid visco‐analyzer (RVA) pasting properties (D) and taste score (E) of Wx ^b, Wx ^la and Wx ^mp in the ZN background. (F) The GBSSI activity of different Wx alleles during endosperm development. (G–I) Three‐dimensional homology modeling of GBSSI encoded by Wx ^b (G), Wx ^la (H) and Wx ^mp (I). ZN is a glutinous rice variety containing the wx allele. Error bars represent the SD of means (n = 5). Different letters above the bars indicate significant differences at P < 0.05, using Tukey's multiple‐comparison test. cP, centipoise.

Figure 5

Genetic diversity and evolutionary relationship among multiple Wx alleles in rice (A) Genotypes and distributions of haplotypes divided by 52 variants within the Wx gene. (B) Phylogenetic relationship of 200‐kb sequences flanking Wx generated from 500 cultivated rice accessions. (C) Extended haplotypes of Wx ⁱⁿ, Wx ^la, Wx ^b, and wx.

Figure 6

Proposed evolutionary relationships and estimation of the recombination rate at the Wx locus (A) The evolutionary relationships among Wx alleles and haplotypes in rice. (B) Estimates of population recombination rate (ρ) around the Wx locus in 533 cultivars. (C) Estimates of the intragenic recombination rate at the Wx locus. vg0601765887 and vg0601769158 are flanking markers used to screen for recombinants. The location of six functional variations are marked with red dashed lines.