Molecular characterization of the major wheat domestication gene Q

Abstract

The Q gene is largely responsible for the widespread cultivation of wheat because it confers the free-threshing character. It also pleiotropically influences many other domestication-related traits such as glume shape and tenacity, rachis fragility, spike length, plant height, and spike emergence time. We isolated the Q gene and verified its identity by analysis of knockout mutants and transformation. The Q gene has a high degree of similarity to members of the AP2 family of transcription factors. The Q allele is more abundantly transcribed than q, and the two alleles differ for a single amino acid. An isoleucine at position 329 in the Q protein leads to an abundance of homodimer formation in yeast cells, whereas a valine in the q protein appears to limit homodimer formation. Ectopic expression analysis allowed us to observe both silencing and overexpression effects of Q. Rachis fragility, glume shape, and glume tenacity mimicked the q phenotype in transgenic plants exhibiting post-transcriptional silencing of the transgene and the endogenous Q gene. Variation in spike compactness and plant height were associated with the level of transgene transcription due to the dosage effects of Q. The q allele is the more primitive, and the mutation that gave rise to Q occurred only once leading to the world's cultivated wheats.

Figure 1.

Analysis of EMS mutants. (A) Illustrated structure of the Q gene. Exons are depicted in red. Arrows indicate locations of point mutations in the EMS mutants. (B) Spike morphology of CS (Q); fndel-143 (null for Q); EMS-induced mutants mq36, mq125, and mq194; and CS-DIC 5A (q).

Figure 2.

Southern analysis of various wheat taxa hybridized with a fragment of WAP2 (Q). Left to right: N5AT5D, N5BT5D, N5DT5B, fndel-143 (Q-null), T. aestivum spp. aestivum cv. CS (Q), CS-DIC 5A (q), T. aestivum ssp. macha (q), European T. aestivum ssp. spelta (q; TA2603), Iranian T. aestivum ssp. spelta (Q), European T. aestivum ssp. spelta (q; DS 5A Europe), T. turgidum ssp. carthlicum (Q), T. turgidum ssp. durum cv. LDN (Q), and T. turgidum ssp. polonicum (Q).

Figure 3.

Locations of six conserved nucleotide differences between Q- and q-genotypes within the genomic sequence of the gene. Arrows represent single nucleotide polymorphisms between Q and q. Green, intron; red, exon; blue, 3′ UTR. The polymorphism indicated in red represents the amino acid difference between Q and q alleles at position 329 of the predicted protein. The green bar represents a variable microsatellite within intron 9.

Figure 4.

Relative transcription levels of Q in CS and q in CS-DIC 5A during spike development. Q was consistently transcribed at higher levels than q, but transcription of Q and q followed the same trend as the spike matured. Transcription levels in fndel-143 (null for Q) are shown to indicate the specificity of the Taqman system.

Figure 5.

Transcription levels of Q from CS, q from CS-DIC 5A, and the Q null allele from fndel-143 in root tips, young leaves of 1-month-old plants, and flag leaves. The transcription levels in immature spikes (fraction length, 0.1) are shown for comparison.

Figure 6.

The homodimers of Q protein in yeast two-hybrid analysis. (Left) The yeast cells after mating were grown in SD/-L-W medium to confirm the cells containing both bait and prey. (Right) The protein-protein interactions were examined by the growth under the selection of SD/-L-W-H-A and in the presence of X-Gal. The cells could grow only when the combination of bait∷prey resulted in the protein interaction and therefore the activation of reporter genes (HIS, ADE2, and lacZ).

Figure 7.

Analysis of T₁ transgenic plants. (A) T₁ transgenic speltoid spike (T32). (B) Untransformed Bobwhite square spike. (C) T₁ transgenic subcompactoid spike (T30). (D) T₁ transgenic compactoid spike (T39). (E) Whole plant view of T₁ transgenic individuals. Left to right: T32, Bobwhite, T30, and T39. (F) Differences in spikelet disarticulation pattern between untransformed Bobwhite (QQ) (left) and a T₁ speltoid transgenic (T32, Q silenced) (right). In Bobwhite, Q confers a tough rachis and abscission occurs at the base of the spikelet causing the seed to be free-threshing. When Q is silenced (and in qq genotypes) abscission occurs at the junction of the rachis and rachilla causing the spikelet to shatter from the rachis and the seed to be nonfree-threshing.

Figure 8.

Relative transcription levels of the fast-neutron induced deletion mutant fndel-143 (null for Q), CS (QQ), T32 (speltoid transgenic), untransformed Bobwhite, T30 (subcompactoid transgenic), and T39 (compactoid transgenic). Transcription levels in CS (Q) and fndel-143 (null for Q) are shown for comparison. Bars represent standard error.

Figure 9.

Phylogenetic tree of 12 Triticum genotypes (Table 1) based on full-length genomic DNA sequences (start to stop codon) of the Q/q gene calculated by the neighbor-joining method and rooted by the q homeoallele from T. turgidum ssp. durum chromosome 5B as an outgroup. Open circles indicate nodes supported by bootstrap values >70%.