A rare Waxy allele coordinately improves rice eating and cooking quality and grain transparency

Abstract

In rice (Oryza sativa), amylose content (AC) is the major factor that determines eating and cooking quality (ECQ). The diversity in AC is largely attributed to natural allelic variation at the Waxy (Wx) locus. Here we identified a rare Wx allele, Wx^mw , which combines a favorable AC, improved ECQ and grain transparency. Based on a phylogenetic analysis of Wx genomic sequences from 370 rice accessions, we speculated that Wx^mw may have derived from recombination between two important natural Wx alleles, Wxⁱⁿ and Wx^b . We validated the effects of Wx^mw on rice grain quality using both transgenic lines and near-isogenic lines (NILs). When introgressed into the japonica Nipponbare (NIP) background, Wx^mw resulted in a moderate AC that was intermediate between that of NILs carrying the Wx^b allele and NILs with the Wx^mp allele. Notably, mature grains of NILs fixed for Wx^mw had an improved transparent endosperm relative to soft rice. Further, we introduced Wx^mw into a high-yielding japonica cultivar via molecular marker-assisted selection: the introgressed lines exhibited clear improvements in ECQ and endosperm transparency. Our results suggest that Wx^mw is a promising allele to improve grain quality, especially ECQ and grain transparency of high-yielding japonica cultivars, in rice breeding programs.

Keywords: Oryza sativa L.; Waxy; allelic variation; amylose content (AC); eating and cooking quality (ECQ); grain appearance.

Figure 1

Map‐based cloning and functional characterization of Wx^mw (A–D) Comparison of grain appearance (A), amylose content (AC) (B), gel consistency (GC) (C) and rapid viscosity analysis (RVA) profiles (D), between Mowanggu (MWG) and other rice varieties carrying the various Wx alleles shown in panel (F). GLXN (Guanglingxiangnuo, glutenous rice with the null wx allele); NG46 (Nangeng46) and NIP (Nipponbare) are japonica, while IR64 and TQ (Teqing) are indica varieties. (E) Linkage analysis and mapping of Wx ^mw. (F) Genomic structure of Wx and alignment of several major polymorphic sequences among different Wx alleles. Representative rice cultivars for each Wx allele are listed in brackets. (G–I) Phenotypes of mature brown rice (G), AC (H) and GC (I) for transgenic rice lines and their wild type progenitor NIP(wx). NIP(wx)‐Wx ^b, NIP(wx)‐Wx ^mw, and NIP(wx)‐Wx ^mp represent transgenic lines with intact Wx ^b, Wx ^mw, or Wx ^mp transgenes, respectively, in the glutinous NIP(wx) background. All transgenic lines were homozygous and from the T₃ generation. Values labeled with different lowercase letters are significantly different by one‐way analysis of variance (ANOVA) multiple comparison (P < 0.05). Error bars indicate SD, n = 3.

Figure 2

Comparison of grain qualities and Wx expression among near‐isogenic lines (NILs) (A) Appearance of brown rice. (B) Amylose content (AC) of rice flours. (C) Taste value of cooked rice. (D) Rapid viscosity analysis (RVA) profiles of rice flours. (E–F) Relative expression levels of Wx messenger RNA (E) and granule‐bound starch synthase I (GBSSI) activity (F) in developing seeds at various days after flowering. (G–H) Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) assays of starch granule‐bound GBSSI (G) and total seed proteins (H) from immature seeds 15 d after flowering. NIP(Wx ^mw) and NIP(Wx ^mp) are the near‐isogenic lines introgressed with the Wx ^mw or Wx ^mp alleles, respectively, in the japonica Nipponbare (NIP(Wx ^b)) background. Values labeled with different lowercase letters are significantly different by one‐way analysis of variance (ANOVA) multiple comparison (P < 0.05). Error bars indicate SD, n = 3.

Figure 3

Comparison of endosperm appearance and morphology of grain transverse sections between near‐isogenic lines (NILs) carrying different Wx alleles (A) Milled rice after incubation at 40 °C for 2, 4, 6, 8, 12, and 24 h. The corresponding moisture contents are listed in Table S7. (B–E) Transverse sections of mature grains after 2 h of drying. (F–I) Transverse sections of grains after 24 h of drying. Red arrows indicate holes within starch granules. NIP(wx), NIP(Wx ^mw) and NIP(Wx ^mp) are NILs introgressed with the wx, Wx ^mw and Wx ^mp alleles, respectively, in the japonica Nipponbare (NIP(Wx ^b)) background.

Figure 4

Phylogenetic analysis and proposed evolutionary relationship among various Wx alleles in rice (A) Neighbor‐joining phylogenetic tree based on full‐length Wx genomic sequences from 370 rice accessions. The rice accessions and their Wx genotypes are given in Table S8. (B) Wx ^mw is derived from the recombination between Wx ⁱⁿ and Wx ^b alleles. Both alleles are evolved from the type I haplotype (Wx ^lv–I) of the ancestral Wx ^lv allele.

Figure 5

Comparison of plant morphology and grain qualities between 2661(Wx^b) and its near‐isogenic line 2661(Wx^mw) (A) Plant morphology during filling stage. (B) Endosperm appearance at different moisture contents. (C) Amylose content (AC). (D) Gel consistency (GC). (E) Taste value of cooked rice. (F) Rapid viscosity analysis (RVA) profiles of rice flours. Values with different lowercase letters are significantly different (P < 0.05, Student's t‐test). Error bars indicate SD, n = 3.