Rare allele of OsPPKL1 associated with grain length causes extra-large grain and a significant yield increase in rice

Abstract

Grain size and shape are important components determining rice grain yield, and they are controlled by quantitative trait loci (QTLs). Here, we report the cloning and functional characterization of a major grain length QTL, qGL3, which encodes a putative protein phosphatase with Kelch-like repeat domain (OsPPKL1). We found a rare allele qgl3 that leads to a long grain phenotype by an aspartate-to-glutamate transition in a conserved AVLDT motif of the second Kelch domain in OsPPKL1. The rice genome has other two OsPPKL1 homologs, OsPPKL2 and OsPPKL3. Transgenic studies showed that OsPPKL1 and OsPPKL3 function as negative regulators of grain length, whereas OsPPKL2 as a positive regulator. The Kelch domains are essential for the OsPPKL1 biological function. Field trials showed that the application of the qgl3 allele could significantly increase grain yield in both inbred and hybrid rice varieties, due to its favorable effect on grain length, filling, and weight.

Fig. 1.

Map-based cloning of qGL3. (A) Parental grains used for QTL analysis and fine-mapping. (Scale bar: 10 mm.) (B) QTLs affecting grain length and width identified in the N411 × N643 F₂ population. Red and blue circles show grain length and width, respectively. Circle sizes reflect their effects on phenotypic variations. The major loci, including three known loci (shown in bracket) and the qGL3 locus, are indicated by arrows. (C) A fine-linkage map generated by analyzing 2,968 N411 × 93-11 BC₂F₃ segregating plants. The recombinant numbers are given between markers. (D) High-resolution linkage analysis of phenotypes and marker genotypes. White bars represent chromosomal segments for 93-11 homozygote, black with spots for N411 homozygote, and grille for heterozygote, respectively. Progeny testing was used to confirm the genotypes at the qGL3 locus. S, segregation; D, desegregation. (E) Predicted ORFs based on the Nipponbare genome sequence. The horizontal arrows represent predicted five ORFs (ORF1, LOC_Os03g44460; ORF2, LOC_Os03g44470; ORF3, LOC_Os03g44484; ORF4, LOC_Os03g44500; and ORF5, LOC_Os03g44510). (F) The gene structure of ORF4. Empty boxes refer to 5′ and 3′ UTRs, gray boxes to exons, and the lines between boxes to introns. The SNPs in the OsPPKL1^N411 are shown by dashed lines; SNP1, c.+1092C→A; SNP2, c.+1495C→T; SNP3, c.+2643A→G; SNP4, c.+2838T→C. (G) The Kelch domains and the PP2A domain predicted in the protein encoded by ORF4. Solid lines show the positions of two amino acid transitions.

Fig. 2.

Functional analysis of OsPPKLs. (A) Grains of the transgenic plants (T₁) and ZH11. (Scale bar: 10 mm.) (B) Comparison of the grain length between ZH11 and the transgenic plants in A. P values from a t test of the transgenic plants against ZH11 were indicated. (C) The grains of OsPPKL knockout mutants in the Dongjin background. (D) Comparisons of the grain length of the mutant plants. P values from a t test of the mutant against Dongjin were indicated. (E) Schematic representation of constructs for overexpressing different domains.

Fig. 3.

Comparison of different traits between 93-11 and NIL-qgl3. (A) Grains of 93-11 and NIL-qgl3. (Scale bar: 10 mm.) (B) Panicles of 93-11 and NIL-qgl3. (Scale bar: 2 cm.) (C) Plants of 93-11 and NIL-qgl3. (Scale bar: 10 cm.) (D) Scanning electron and light microscope photos of glume outer surfaces of 93-11 and NIL-qgl3 spikelets at three stages. Stage 1, spikelet = 2 mm; stage 2, spikelet = 5 mm; stage 3, maturity. (Scale bars: yellow, 50 μm; blue, 1.0 mm.) (E) Cell density of glume outer surfaces of 93-11 and NIL-qgl3 spikelets at three stages. (F) Grain filling rate of 93-11 and NIL-qgl3 after fertilization, indicated by dry weight of 1,000 grains.

Fig. 4.

Evaluation of the qgl3 effects on hybrid rice. (A) Comparison of grains and brown rice of three pairs of hybrids. (Scale bar: 10 mm.) (B) Actual grain yields of different hybrids in plots. (C) Chalk rates of white rice of different hybrids. P values from a t test were indicated.