Three dominant awnless genes in common wheat: Fine mapping, interaction and contribution to diversity in awn shape and length

Abstract

The awn is a long needle-like structure formed at the tip of the lemma in the florets of some grass species. It plays a role in seed dispersal and protection against animals, and can contribute to the photosynthetic activity of spikes. Three main dominant inhibitors of awn development (Hd, B1 and B2) are known in hexaploid wheat, but the causal genes have not been cloned yet and a genetic association with awn length diversity has been found only for the B1 allele. To analyze the prevalence of these three awning inhibitors, we attempted to predict the genotypes of 189 hexaploid wheat varieties collected worldwide using markers tightly linked to these loci. Using recombinant inbred lines derived from two common wheat cultivars, Chinese Spring and Mironovskaya 808, both with short awns, and a high-density linkage map, we performed quantitative trait locus analysis to identify tightly linked markers. Because this linkage map was constructed with abundant array-based markers, we converted the linked markers to PCR-based markers and determined the genotypes of 189 hexaploids. A significant genotype-phenotype correlation was observed at the Hd and B1 regions. We also found that interaction among these three awning inhibitors is involved in development of a membranous outgrowth at the base of awn resembling the Hooded mutants of barley. For the hooded awn phenotype, presence of the Hd dominant allele was essential but not sufficient, so B2 and other factors appear to act epistatically to produce the ectopic tissue. On the other hand, the dominant B1 allele acted as a suppressor of the hooded phenotype. These three awning inhibitors largely contribute to the genetic variation in awn length and shape of common wheat.

Fig 1. Awn phenotypes observed in the RIL population and near-isogenic line (NIL) of S-615.

(A, B) Awns of the parental CS (A) and M808 (B) lines. (C-E) A hooded phenotype, defined as the presence of a membranous structure (C and D) or broadening of the base of awn (E) (indicated by red arrows), could be observed in some RILs but not in the parents. (F, G) Awn phenotype of a NIL of common wheat cv. S-615 with the Hd allele of CS (Hd-S615). The genotype of S-615 is hdhdb1b1b2b2, with long awns. (H) Frequency distribution of awn length at the middle (black bars) and top (grey bars) of the spike.

Fig 2. Two-way interaction plots for the three identified QTLs.

Two-locus genotypic effects for awn length at the top (A to C) and middle (D to F) and for the hooded phenotype (G to I) were plotted using the genotype data of the markers with the highest LOD score. Red lines represent a genotype homozygous for CS and blue lines represent a genotype homozygous for M808. Error bars are ± standard errors.

Fig 3. Comparison of awn length and hooded phenotype among different genotypes of RILs.

Comparison of awn length at (A) the top and (B) the middle of the spike for the eight combinations of Hd, B1, B2 and WT alleles in RILs. ANOVA with a post-hoc Tukey’s HSD test was performed and genotypes with the same letter did not present significant differences in awn length (P < 0.05). The phenotypic data of the parental lines (CS [Hd B2] and M808 [B1]) are indicated by red dots. (C) Scatter plot of awn length at the top (y-axis) with respect to that of the middle of the spike (x-axis). Three groups are observed: WT (blue dots), RILs with one of the dominant awning inhibitor alleles (green) and RILs with at least two dominant alleles (pink). Awn length of the parental lines are indicated by black dots. (D) Frequency distribution of RILs with hooded phenotype according to genotype.

Fig 4. Chromosomal synteny of Hd and orthologous regions among wheat, rice and barley.

Using the sequence of markers located around the Hd locus (left map), a Blast search was performed to find the corresponding genomic scaffolds (middle panel). Genes present on each scaffold were used to search for orthologs in rice and barley. Comparing our genetic map of wheat with the physical map of rice chromosome 3 (top of right panel), we located DL near the Hd locus. On the other hand, this region of chromosome 4A is known to have an inversion with respect to the corresponding region of chromosome 4H (bottom of right panel). Therefore, the ortholog of DL in barley might be around the Hd locus, but the exact location could not be determined.

Fig 5. Chromosomal synteny between wheat chromosome 6B and barley chromosome 6H.

The region around the B2 locus was compared with the corresponding region of the barley genome to predict the location of the RAE2 gene (MLOC_71047.1). The left map (genetic map) corresponds to chromosome 6B of wheat, the middle panel shows the genomic scaffolds of wheat containing the indicated markers and the right map (physical map) is barley chromosome 6H, where the orthologs of the genes on the wheat scaffolds are indicated.

Fig 6. Box and whisker plots for comparison of awn length at the top (A) and middle (B) of the spike in the six subspecies of T. aestivum.

Fig 7. Comparison of phenotype of RILs with known genotype and hexaploids with predicted genotypes.

The awn length of hexaploids with a putative Hd allele (A) and B1 (B) allele was compared with that of RILs with known genotypes. WT RILs are indicated by red dots, those with one dominant awning inhibitor by blue, RILs with at least two inhibitors by green and hexaploid wheat lines by black. Individuals with an awn length at the top and middle of the spike > 60 mm (red circles) were considered WT. The awn length of phenotypically WT hexaploids (C) and lines putatively containing inhibitor alleles (D) was also compared with the RILs. Hexaploids with the M808-allele at WABM232824 (near the B1 locus) are indicated by orange dots.

Fig 8. Model of interactions among Hd, B1 and B2 for development of the hooded phenotype.

The dominant Hd allele is necessary for development of the membranous lateral expansion at the base of awns. Presence of the dominant B2 allele and other factors contribute to the formation of this ectopic tissue. In contrast, presence of the dominant B1 allele suppresses its formation, leading to a complete lack of the hooded phenotype or to a broadening of the base of awn, depending probably on the genetic background.