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

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

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