A triallelic system of S5 is a major regulator of the reproductive barrier and compatibility of indica-japonica hybrids in rice

Abstract

Hybrid sterility is a major form of postzygotic reproductive isolation. Although reproductive isolation has been a key issue in evolutionary biology for many decades in a wide range of organisms, only very recently a few genes for reproductive isolation were identified. The Asian cultivated rice (Oryza sativa L.) is divided into two subspecies, indica and japonica. Hybrids between indica and japonica varieties are usually highly sterile. A special group of rice germplasm, referred to as wide-compatibility varieties, is able to produce highly fertile hybrids when crossed to both indica and japonica. In this study, we cloned S5, a major locus for indica-japonica hybrid sterility and wide compatibility, using a map-based cloning approach. We show that S5 encodes an aspartic protease conditioning embryo-sac fertility. The indica (S5-i) and japonica (S5-j) alleles differ by two nucleotides. The wide compatibility gene (S5-n) has a large deletion in the N terminus of the predicted S5 protein, causing subcellular mislocalization of the protein, and thus is presumably nonfunctional. This triallelic system has a profound implication in the evolution and artificial breeding of cultivated rice. Genetic differentiation between indica and japonica would have been enforced because of the reproductive barrier caused by S5-i and S5-j, and species coherence would have been maintained by gene flow enabled by the wide compatibility gene.

Fig. 1.

Fertility of ORF5-NJ11 transgenic-positive and -negative plants with Balilla as the recipient. (A) Spikelet fertility of positive (left, sterile) and negative (right, fertile) T₁ segregants. (B) Top, embryo sacs of negative (left, normal) and positive (right, abortive) plants; Bottom, pollen of negative (left) and positive (right) plants. Scale bars: 50 μm. (C) Fertility scores (error bar = 1 SEM) of transgene positive and negative plants in T₀ (Top) and a T₁ family (Bottom). There are 29 positive and 4 negative plants in T₀ and 15 positive and 14 negative segregants in the T₁ family (B10).

Fig. 2.

The sequence features of the S5 transcripts from 02428 (WCV), Balilla (japonica), and Nanjing 11 (indica).

Fig. 3.

In vitro assay of protease activity of S5. (A) Absolute activity measured as the spectrophotometer reading with that of water control set to zero (Top), and relative activity by using the highest activity (pH 3.0) as the reference (Bottom). The open bars are the readings of protease activity assays and the shaded bars are the readings of the pepstatin inhibition assays. Error bars = 1 SEM. (B) SDS-PAGE showing the protein bands of the autolytic products in a series of pH buffers (Top), and immunoblot confirmation of the S5 autolytic products by using Anti-CS5 and Anti-NS5 as the probes (Bottom). The lanes marked Ct are the controls. The molecular weight on the right side of the top section and the left side of the bottom section was estimated on the basis of size markers (as shown on the left side of the top section).

Fig. 4.

Expression of the S5 gene and subcellular localization of S5. (Top) In situ hybridization using an antisense probe showing expression in nucellar and archesporial cells (02428) (A), integuments and megasporocytes (Balilla) (B), and integuments and megaspores (Nanjing 11) (C), with the sense probe as the negative control (D). A is a transection, and B–D are longitudinal sections. Scale bars: 25 μm. (Bottom) Subcellular localization of the S5 protein in nucellar cells by immunogold and TEM techniques showing a large quantity of gold particles in the cell wall of Nanjing 11 (E) and Balilla (F) and few particles in the cell wall of 02428 (G). The one without the primary antibody is the negative control (H). Scale bars: 0.5 μm. Arrows indicate the gold particles. C, cytosol; CW, cell wall; M, mitochondrion; V, vacuole.