Unleashing floret fertility in wheat through the mutation of a homeobox gene Shun Sakumaa,b,c,1, Guy Goland, Zifeng Guob, Taiichi Ogawaa,e, Akemi Tagiria, Kazuhiko Sugimotoa,f, Nadine Bernhardtg, Jonathan Brassacg, Martin Mascherh,i, Goetz Henselj, Shizen Ohnishik, Hironobu Jinnok, Yoko Yamashital, Idan Ayalond, Zvi Pelegd, Thorsten Schnurbuschb,m,1, and Takao Komatsudaa,f,1 aAgrogenomics Research Center, National Institute of Agrobiological Sciences, 305-8602 Tsukuba, Japan; bIndependent HEISENBERG Research Group Plant Architecture, Leibniz Institute of Plant Genetics and Crop Plant Research, 06466 Gatersleben, Germany; cFaculty of Agriculture, Tottori University, 680-8553 Tottori, Japan; dThe Robert H. Smith Institute of Plant Sciences and Genetics in Agriculture, The Hebrew University of Jerusalem, 7610001 Rehovot, Israel; eInstitute of Agrobiological Sciences, National Agriculture and Food Research Organization, 305-8518 Tsukuba, Japan; fInstitute of Crop Science, National Agriculture and Food Research Organization, 305-8518 Tsukuba, Japan; gResearch Group Experimental Taxonomy, Leibniz Institute of Plant Genetics and Crop Plant Research, 06466 Gatersleben, Germany; hIndependent Research Group Domestication Genomics, Leibniz Institute of Plant Genetics and Crop Plant Research, 06466 Gatersleben, Germany; iGerman Centre for Integrative Biodiversity Research Halle-Jena-Leipzig, 04103 Leipzig, Germany; jResearch Group Plant Reproductive Biology, Leibniz Institute of Plant Genetics and Crop Plant Research, 06466 Gatersleben, Germany; kKitami Agricultural Experiment Station, Hokkaido Research Organization, 099-1496 Kunneppu, Japan; lCentral Agricultural Experiment Station, Hokkaido Research Organization, 069-1395 Naganuma, Japan; and mInstitute of Agricultural and Nutritional Sciences, Faculty of Natural Sciences III, Martin Luther University Halle-Wittenberg, 06120 Halle, Germany Edited by Sarah Hake, University of California, Berkeley, CA, and approved January 24, 2019 (received for review September 7, 2018) Floret fertility is a key determinant of the number of grains per the three subgenomes B, A, and D (8, 9). The first event led to inflorescence in cereals. During the evolution of wheat (Triticum the formation of the allotetraploid wild emmer (Triticum turgidum sp.), floret fertility has increased, such that current bread wheat ssp. dicoccoides, genome formula BBAA) 0.36 to 0.5 Mya. Its A (Triticum aestivum) cultivars set three to five grains per spikelet. subgenome was inherited from the diploid species Triticum urartu However, little is known regarding the genetic basis of floret fer- and its B subgenome from Aegilops speltoides or an extinct tility. The locus Grain Number Increase 1 (GNI1) is shown here to closely related species. Domesticated emmer (Triticum tur- be an important contributor to floret fertility. GNI1 evolved in the gidum ssp. dicoccum) was selected by early farmers from Triticeae through gene duplication. The gene, which encodes a stands of wild emmer and is the progenitor of modern durum homeodomain leucine zipper class I (HD-Zip I) transcription factor, wheat, currently the most widely cultivated tetraploid wheats. was expressed most abundantly in the most apical floret primordia The second polyploidization event, which occurred ∼7,000 y and in parts of the rachilla, suggesting that it acts to inhibit rachilla ago, involved domesticated tetraploid wheat and the D sub- growth and development. The level of GNI1 expression has de- genome donor Aegilops tauschii. As the ploidy level increased, creased over the course of wheat evolution under domestication, the spikes evolved to produce a larger number of florets per leading to the production of spikes bearing more fertile florets and spikelet: Diploid wheats (T. urartu and Triticum monococcum) setting more grains per spikelet. Genetic analysis has revealed that GNI-A1 the reduced-function allele contributes to the increased Significance number of fertile florets per spikelet. The RNAi-based knockdown of GNI1 led to an increase in the number of both fertile florets and grains in hexaploid wheat. Mutants carrying an impaired GNI-A1 Grain number is a key determinant of cereal grain yield, but its allele out-yielded WT allele carriers under field conditions. The underlying genetic basis in wheat remains undefined. This data show that gene duplication generated evolutionary novelty study demonstrates a direct association between increased affecting floret fertility while mutations favoring increased grain floret fertility, higher grain number per spike, and higher plot GNI1 production have been under selection during wheat evolution yields of field-grown wheat. The gene, encoding an HD- under domestication. Zip I transcription factor, was identified as responsible for in- creased floret fertility. The WT allele acts specifically during rachilla development, with its product serving to lower grain floret fertility | grain number | duplication | HD-Zip I transcription factor | wheat yield potential; in contrast, the reduced-function variant in- creased both floret and grain number. GNI1 evolved through gene duplication in the Triticeae, and its mutations have been he tribe Triticeae (subfamily Pooideae, family Poaceae) en- ∼ under parallel selection in both wheat and barley over the Tcompasses 30 genera and 360 species, including the eco- course of domestication. nomically important cereal crops bread wheat (Triticum aestivum), durum wheat (Triticum turgidum ssp. durum), barley (Hordeum Author contributions: S.S., G.G., Z.P., T.S., and T.K. designed research; S.S., G.G., Z.G., T.O., vulgare), and rye (Secale cereale) (1). Triticeae plants produce an A.T., K.S., N.B., J.B., G.H., S.O., H.J., Y.Y., I.A., and Z.P. performed research; S.S. and M.M. unbranched inflorescence, referred to as a spike. Whereas the ma- analyzed data; and S.S., T.S., and T.K. wrote the paper. jority of species, including wheat, develop a single spikelet on each The authors declare no conflict of interest. rachis node, some species produce two or more (2). The wheat spike This article is a PNAS Direct Submission. is made up of a number of spikelets, with a terminal spikelet at its This open access article is distributed under Creative Commons Attribution-NonCommercial- apex; each spikelet generates an indeterminate number of florets NoDerivatives License 4.0 (CC BY-NC-ND). attached to a secondary axis, the rachilla (3, 4). The number of grains Data deposition: Gene sequences generated in this study have been deposited in the DNA set per spikelet is determined by the fertility of each floret (5, 6). At Data Bank of Japan (DDBJ) (accession nos. AB711370–AB711394 and AB711888– AB711913) and in the NCBI GenBank database (accession nos. MH134165–MH134483). the white anther stage, a wheat spikelet normally produces up to 12 The RNA-seq data have been deposited in the European Nucleotide Archive (accession floret primordia (Fig. 1A); however, during development, more than no. PRJEB25119). 70% of the florets abort (6, 7). Despite its importance for grain 1To whom correspondence may be addressed. Email: [email protected], number determination and the potential for grain yield improvement, [email protected], or [email protected]. the genetic basis of floret fertility in wheat is largely unknown. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. Two polyploidization events have been responsible for the 1073/pnas.1815465116/-/DCSupplemental. appearance of bread wheat, an allohexaploid which harbors Published online February 21, 2019. 5182–5187 | PNAS | March 12, 2019 | vol. 116 | no. 11 www.pnas.org/cgi/doi/10.1073/pnas.1815465116 Downloaded by guest on September 26, 2021 LDN produced 2.4 (Fig. 2A). The increased grain number per A sm BCD f7 spikelet in LDN was largely driven by the higher number of f5 f6 f3 f4 f5 grains set in the basal and central parts of the spike (Fig. 2B). A f2 f4 f1 f3 f2 f1 f2 single major QTL, associated with a log10 odds (LOD) score of gl f1 f3 f4 gl 18.71 was mapped to chromosome 2AL; it accounted for 61% of f1 f2 gl gl gl gl gl gl the phenotypic variance (Fig. 2C). To further narrow the target genomic region, a backcross recombinant line population was E FGHf6 f5 f5 developed, which allowed the locus to be mendelized as the gene f4 f4 Grain Number Increase 1-A (GNI-A1) (Fig. 2D). Fine mapping f3 f3 f3 f3 f2 located GNI-A1 within a 5.4-Mbp region which harbors 26 pu- f1 f2 f2 f2 f1 f1 f1 tative genes, including one encoding an HD-Zip I transcription gl gl gl gl gl gl gl gl factor, the closest wheat homolog to the barley Six-rowed spike 1 gene (vrs1)(SI Appendix, Table S1) (21). A sequence comparison Fig. 1. Structure of the wheat spikelet. (A) A schematic model illustrating of the two parental GNI-A1 alleles revealed a polymorphism the spike at the white anther stage. (B–D) The diploid progenitors of bread responsible for a single amino acid substitution (N105Y: 105 wheat: (B) Triticum urartu,(C) Aegilops speltoides,(D) Ae. tauschii.(E–G) asparagine to tyrosine) within the highly conserved homeo- Tetraploid wheats: (E) wild emmer (T. turgidum ssp. dicoccoides), (F) do- domain (Fig. 2E). The recombinant plants carrying the LDN mesticated emmer (T. turgidum ssp. dicoccum), (G) durum (T. turgidum ssp. allele (generating the 105Y variant) displayed a significantly durum). (H) Hexaploid bread wheat (T. aestivum). Fertile florets are marked in yellow. f, floret; gl, glume; sm, spikelet meristem. higher grain number per spikelet than those carrying the DIC-2A allele (105N variant, ancestral) (Fig. 2F). Notably, the mutation in LDN was identical to that found in the barley six-rowed spike set one or two grains per spikelet, tetraploid wheats two or three, mutant Int-d.41 allele at vrs1 (21), suggesting that the function of and hexaploid wheats more than three (Fig. 1 B–H)(10). the resulting HD-Zip I protein was lost or attenuated in LDN. The genetic diversity of grass inflorescences determines its To verify the inhibitory role, GNI-A1 was silenced using RNA reproduction and therefore, the resulting number of branches, interference (RNAi).