Genome-wide association studies reveal that members of bHLH subfamily 16 share a conserved function in regulating flag leaf angle in rice <i>(Oryza sativa)</i>

<div><p>As a major component of ideal plant architecture, leaf angle especially flag leaf angle (FLA) makes a large contribution to grain yield in rice. We utilized a worldwide germplasm collection to elucidate the genetic basis of FLA that would be helpful for molecular design breeding in rice. Genome-wide association studies (GWAS) identified a total of 40 and 32 QTLs for FLA in Wuhan and Hainan, respectively. Eight QTLs were commonly detected in both conditions. Of these, 2 and 3 QTLs were identified in the <i>indica</i> and <i>japonica</i> subpopulations, respectively. In addition, the candidates of 5 FLA QTLs were verified by haplotype-level association analysis. These results indicate diverse genetic bases for FLA between the <i>indica</i> and <i>japonica</i> subpopulations. Three candidates, <i>OsbHLH153</i>, <i>OsbHLH173</i> and <i>OsbHLH174</i>, quickly responded to BR and IAA involved in plant architecture except for <i>OsbHLH173</i>, whose expression level was too low to be detected; their overexpression in plants increased rice leaf angle. Together with previous studies, it was concluded that all 6 members in bHLH subfamily 16 had the conserved function in regulating FLA in rice. A comparison with our previous GWAS for tiller angle (TA) showed only one QTL had pleiotropic effects on FLA and TA, which explained low similarity of the genetic basis between FLA and TA. An ideal plant architecture is expected to be efficiently developed by combining favorable alleles for FLA from <i>indica</i> with favorable alleles for TA from <i>japonica</i> by inter-subspecies hybridization.</p></div

<i>OsbHLH174</i> overexpression transgenic plants showed an increase in the leaf angle.

<p>(A) The morphology of wild type (WT) and <i>OsbHLH174</i>: OX plants, at the seedling, tillering and heading stages. (B) quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) analysis of <i>OsbHLH174</i> transcripts in WT and <i>OsbHLH174</i>: OX at the seedling stage. (C) FLA and TSLA of the wild type and <i>OsbHLH174</i>:OX-1 and -2 at the heading stage (<i>P</i>< 0.001, n≥ 5).</p

Summary of variances resolved by two-way analysis of variance for flag leaf angle in two environments.

<p>Summary of variances resolved by two-way analysis of variance for flag leaf angle in two environments.</p

Haplotype analysis of <i>qFLA1d</i>/<i>OsBRI1</i>.

<p>(A) Major haplotypes (haplotypes each carried by more than 5accessions) of <i>OsBRI1</i> in the full population according to SNPs data from RiceVarMap version 1. The SNPs in red and bold are non-Synonymous SNPs. (B) Comparison of FLA between Hap2 and Hap3 in <i>indica</i> rice by an independent <i>t</i>-test. (C) Comparison of FLA among Hap4-Hap6 in <i>japonica</i> rice by a Duncan’s test (<i>P</i>< 0.01), respectively.</p

Significant association loci for rice flag leaf angle detected in both Hainan and Wuhan using the linear mixed model in the germplasm collection.

<p>Significant association loci for rice flag leaf angle detected in both Hainan and Wuhan using the linear mixed model in the germplasm collection.</p

A Novel Tiller Angle Gene, <i>TAC3</i>, together with <i>TAC1</i> and <i>D2</i> Largely Determine the Natural Variation of Tiller Angle in Rice Cultivars

<div><p>Tiller angle is one of the most important components of the ideal plant architecture that can greatly enhance rice grain yield. Understanding the genetic basis of tiller angle and mining favorable alleles will be helpful for breeding new plant-type varieties. Here, we performed genome-wide association studies (GWAS) to identify genes controlling tiller angle using 529 diverse accessions of <i>Oryza sativa</i> including 295 <i>indica</i> and 156 <i>japonica</i> accessions in two environments. We identified 7 common quantitative trait loci (QTLs), including the previously reported major gene <i>Tiller Angle Control 1</i> (<i>TAC1</i>), in the two environments, 10 and 13 unique QTLs in Hainan and Wuhan, respectively. More QTLs were identified in <i>indica</i> than in <i>japonica</i>, and three major QTLs (<i>qTA3</i>, <i>qTA1b</i>/<i>DWARF2</i> (<i>D2</i>) and <i>qTA9c</i>/<i>TAC1</i>) were fixed in <i>japonica</i> but segregating in <i>indica</i>, which explained the wider variation observed in <i>indica</i> compared with that in <i>japonica</i>. No common QTLs were identified between the <i>indica</i> and <i>japonica</i> subpopulations. Mutant analysis for the candidate gene of <i>qTA3</i> on chromosome 3 indicated a novel gene, <i>Tiller Angle Control 3</i> (<i>TAC3</i>), encoding a conserved hypothetical protein controlling tiller angle. <i>TAC3</i> is preferentially expressed in the tiller base. The <i>ebisu dwarf</i> (<i>d2</i>) mutant exhibited a decreased tiller angle, in addition to its previously described abnormal phenotype. A nucleotide diversity analysis revealed that <i>TAC3</i>, <i>D2</i> and <i>TAC1</i> have been subjected to selection during <i>japonica</i> domestication. A haplotype analysis identified favorable alleles of <i>TAC3</i>, <i>D2</i> and <i>TAC1</i>, which may be used for breeding plants with an ideal architecture. In conclusion, there is a diverse genetic basis for tiller angle between the two subpopulations, and it is the novel gene <i>TAC3</i> together with <i>TAC1</i>, <i>D2</i>, and other newly identified genes in this study that controls tiller angle in rice cultivars.</p></div

Significant association loci for rice tiller angle detected in both Hainan and Wuhan using the LMM and LR.

<p>Significant association loci for rice tiller angle detected in both Hainan and Wuhan using the LMM and LR.</p

The mutant of <i>D2</i>, the candidate gene of <i>qTA1b</i>, showing decreased tiller angle.

<p>(a) A representation of pairwise <i>r²</i> values (a measure of LD) all polymorphic sites in <i>D2</i>, where the darkness of the color of each box corresponds to the <i>r²</i> value according to the legend. The line in red color represent lead SNP. (b) Schematic diagram of <i>D2</i> gene structure with the T-DNA insertion. The black squares represent exons, and the black lines represent introns. The red arrow indicates the position of lead SNP of <i>qTA1b</i>; the triangle indicates the position of T-DNA insertion; L and R, left and right genomic primers; N, vector primer. (c) genotyping of the <i>d2-3</i> mutant. W, the wild type; H, heterozygote; M, homozygote. (d) qRT-PCR expression analysis of <i>D2</i> in wild type ZH11, heterozygote (<i>d2-3</i> H)and homozygote (<i>d2-3</i> M) mutant using the tissues aboveground at the seedling stage; the number of plants in each genotype (n)≥3 plants, *** significant difference between mutants and wild type at p<0.001.(e)-(g) Phenotypes of ZH11 and <i>d2-3</i> mutant (right) at tillering stage and heading stage; the total plant number (n) = 96 plants, *** significant difference between mutants and wild type at p<0.001 (h)-(j) Phenotypes of T65, <i>d2-1</i> and <i>d2-2</i> mutant at tillering stage and heading stage; the number of plants in each genotype (n)≥15 plants, *** significant difference between mutants and wild type at p<0.001.</p

Comparison of genetic effects among <i>qTA3</i>/<i>TAC3</i> haplotypes on tiller angle in <i>indica</i> subpopulation.

<p>Comparison of genetic effects among <i>qTA3</i>/<i>TAC3</i> haplotypes on tiller angle in <i>indica</i> subpopulation.</p

Phenotypic distribution of rice tiller angle.

<p>Histogram showing distributions of tiller angle in the full population (a), in <i>indica</i> accessions (b) and in <i>japonica</i> accessions (c) in Hainan and Wuhan, respectively.</p