LABA1, a Domestication Gene Associated with Long, Barbed Awns in Wild Rice

Abstract

Common wild rice (Oryza rufipogon), the wild relative of Asian cultivated rice (Oryza sativa), flaunts long, barbed awns, which are necessary for efficient propagation and dissemination of seeds. By contrast, O. sativa cultivars have been selected to be awnless or to harbor short, barbless awns, which facilitate seed processing and storage. The transition from long, barbed awns to short, barbless awns was a crucial event in rice domestication. Here, we show that the presence of long, barbed awns in wild rice is controlled by a major gene on chromosome 4, LONG AND BARBED AWN1 (LABA1), which encodes a cytokinin-activating enzyme. A frame-shift deletion in LABA1 of cultivated rice reduces the cytokinin concentration in awn primordia, disrupting barb formation and awn elongation. Sequencing analysis demonstrated low nucleotide diversity and a selective sweep encompassing an ∼800-kb region around the derived laba1 allele in cultivated rice. Haplotype analysis revealed that the laba1 allele originated in the japonica subspecies and moved into the indica gene pool via introgression, suggesting that humans selected for this locus in early rice domestication. Identification of LABA1 provides new insights into rice domestication and also sheds light on the molecular mechanism underlying awn development.

Figure 1.

The Awns of Wild Rice (O. rufipogon) and Cultivated Rice (O. sativa). (A) Wild rice typically bears a long, barbed awn, which aids in seed dispersal and deters large predators, such as birds. A panicle of wild rice (Tk212) is illustrated in the lower right corner. Bar = 10 cm. (B) Cultivated rice has either no awns or short and barbless awns and suffers from significant bird predation. A panicle of cultivated rice (93-11, indica) is illustrated in the lower left corner. Bar = 10 cm. (C) The awn of wild rice (Tk212) is long and barbed. Scanning electron micrograph in the lower right corner shows the surface of the awn of wild rice in the boxed region. Bar = 200 μm. (D) The awn of cultivated rice (93-11) is short and barbless. Scanning electron micrograph in the lower left corner shows the surface of the awn of cultivated rice in the boxed region. Bar = 200 μm.

Figure 2.

Phenotype of IL_9YIL304 and 93-11. (A) Panicles of the introgression line IL_9YIL304 and 93-11. Bar = 10 cm. (B) Comparison of awn length between IL_9YIL304 and 93-11. The awns of IL_9YIL304 (3.5 to 3.9 cm) were significantly longer than the awns of 93-11 (1.2 to 1.6 cm). (C) Comparison of awn proportion between IL_9YIL304 and 93-11. Awn proportion of IL_9YIL304 was almost 100%, whereas only ∼30% grains in a panicle of 93-11 showed awns. (D) Grains of IL_9YIL304 and 93-11. Grain of IL_9YIL304 exhibited a longer awn. Bar = 1 cm. (E) Scanning electron micrographs showing the surface of the awn of IL_9YIL304 and 93-11 in boxed regions of (D). IL_9YIL304 had upwardly oriented barbs on the awn surface, whereas the awn of 93-11 was barbless. Bar = 200 μm. (F) Longitudinal section in the boxed regions of (D) showed cell size in the awn of IL_9YIL304 and 93-11. Awn barb was derived from awn epidermal cell in IL_9YIL304. Arrows point to the layers of cells adjacent to vascular bundle whose size was measured. Bars = 200 μm. In (B) and (C), sampled size was n = 20. The statistical significance was at P < 0.05 based on a two-tailed Student’s t test. Error bars represent the sd.

Figure 3.

Map-Based Cloning of LABA1. (A) The target gene(s) for awn barbs and awn length mapped between RM1223 and RM17282 on the long arm of chromosome 4 based on linkage analysis using 300 F2 individuals. (B) LABA1 was further delimited to a 34.6-kb region between the marker M3 and RM17242 based on analysis of recombinant plants identified from an F3 population of 6655 individuals. (C) The Nipponbare genomic BAC clone (OSJNBb0061C13) shows five genes in this region. (D) The Yuanjiang common wild rice (YJCWR) genomic BAC clone (YJ0610808) shows deletion of three segments containing transposon elements and retains two gene models in this region. (E) An 8524-bp genomic fragment containing the entire ORF (4109 bp) of LOC_Os04g43840 was isolated by digesting the YJCWR genomic BAC (YJ0610808) DNA using EcoRI and BamHI and cloned into the binary vector pCAMBIA1300. (F) to (H) Complementation phenotypes of Y179: transgenic line CTP-1 showed increased awn length (G) and awn barbs on awn surface (H) compared with that the transgenic control. (I) to (K) RNAi phenotypes of IL_9YIL304: RNAi transgenic line RNAi-2 exhibited substantially reduced awn length (J) and shorter and smaller awn barbs (K) compared with IL_9YIL304. (L) and (M) Comparison of awn length (L) and awn proportion (M) between Y179 and two independent transgenic lines CTP-1 and CTP-2. (O) and (P) Comparison of awn length (O) and awn proportion (P) between IL_9YIL304 and two independent transgenic lines RNAi-1 and RNAi-2. CTP, LABA1 complementation transgenic plants. RNAi, LABA1 RNA interference plants. In (L) to (O), sampled size was n = 10. The statistical significance was at P < 0.05 based on a two-tailed Student’s t test. Error bars represent the sd. Bars = 10 cm in (F) and (I), 1 cm in (G) and (J), and 200 μm in (H) and (K).

Figure 4.

Expression Pattern of LABA1 and Function of LABA1. (A) The expression pattern of LABA1 in IL_9YIL304 and 93-11. (B) Enzyme activity of LABA1 as a cytokinin nucleoside 5′-monophosphate phosphoribohydrolase was measured with iPRMP as substrate. Standards (Std) of iPRMP and iP and reaction products of iPRMP without LABA1 and with LABA1 were separated by HPLC. (C) Comparison of cytokinin concentration in 1- to ∼2-cm young panicles of transgenic plants and control. (D) Expression of rice RR1 to RR11 in 1- to ∼2-cm young panicles of Y179 and CTP-1. The expression of RR2, RR3, RR4, RR6, RR7, and RR11 was significantly increased in CTP-1. (E) RNA in situ hybridization of RR6 in the awn of 93-11 and IL_9YIL304 at the Sp7 stage. The transcripts of RR6 were present in the epidermal cells of the awn primordia in IL_9YIL304, but not in 93-11. Bar = 100 μm.

Figure 5.

The Effect of LABA1 on Awn Development. (A1) to (A4) Scanning electron microscopy images showed spikelet development of 93-11 from the Sp7 to Sp8l stage. (B1) to (B4) Scanning electron microscopy images showed spikelet development of IL_9YIL304 from the Sp7 to Sp8l stage. (C1) to (C4) RNA in situ hybridization of laba1 during spikelet development of 93-11 from the Sp7 to Sp8l stage. (D1) to (D4) RNA in situ hybridization of LABA1 during spikelet development of IL_9YIL304 from the Sp7 to Sp8l stages. (E1) to (E4) Expression of Histone H4 in developing spikelets of 93-11. (F1) to (F4) Expression of Histone H4 in developing spikelets of IL_9YIL304. (A1), (B1), (C1), (D1), (E1), and (F1) Sp6, formation of stamen primordia stage. (A2), (B2), (C2), (D2), (E2), and (F2) Sp7, formation of carpel primordia stage. (A3), (B3), (C3), (D3), (E3), and (F3) Sp8e (Sp8 early), differentiation of ovule and pollen stage. (A4), (B4), (C4), (D4), (E4), and (F4) Sp8l (Sp8 late), differentiation of ovule and pollen stage. Arrows point to awn primordia. Bars = 100 μm.

Figure 6.

Nucleotide Diversity across LABA1 Genomic Region on Chromosome 4. (A) The location of sampled loci in genomic region of ∼1.4 Mb around LABA1 on chromosome 4. (B) Nucleotide diversity π of cultivated rice and wild rice at the sampled loci. (C) The relative ratio of π in cultivated rice to wild rice shows a selective sweep of ∼800 kb surrounding laba1 in cultivated rice.

Figure 7.

EH Analysis of Awned and Awnless O. sativa Accessions across a 1.4-Mb Region Flanking LABA1. Physical positions on chromosome 4 are indicated across the top of the figure in base pairs. Blue cells match the allele found in the reference genome, Nipponbare (temperate japonica), and yellow cells indicate a nonreference allele. All polymorphisms are SNP data, which were extracted from the high-density rice array data set. Haplotypes are clustered based on whether they were found more in the japonica varietal group (japonica haplotypes) or the indica varietal group (indica haplotypes). Haplotypes specific to the aus subpopulation are grouped separately (aus haplotypes). The allele found at the functional nucleotide polymorphism is indicated in white and red: the wild type C in white cells and derived laba1 in red cells. The numbers of accessions that harbored each haplotype are indicated in the right-hand table (tej = temperate japonica, trj = tropical japonica, aro = aromatic, ind = indica, aus = aus, admix = admixture, determined by HDRA genome-wide information). Awnless accessions are grouped with their predicted barb trait groups; red asterisks indicate the presence of at least one awnless accession with a specific haplotype.