Identification of a major QTL controlling the content of B-type starch granules in Aegilops

Abstract

Starch within the endosperm of most species of the Triticeae has a unique bimodal granule morphology comprising large lenticular A-type granules and smaller near-spherical B-type granules. However, a few wild wheat species (Aegilops) are known to lack B-granules. Ae. peregrina and a synthetic tetraploid Aegilops with the same genome composition (SU) were found to differ in B-granule number. The synthetic tetraploid had normal A- and B-type starch granules whilst Ae. peregrina had only A-granules because the B-granules failed to initiate. A population segregating for B-granule number was generated by crossing these two accessions and was used to study the genetic basis of B-granule initiation. A combination of Bulked Segregant Analysis and QTL mapping identified a major QTL located on the short arm of chromosome 4S that accounted for 44.4% of the phenotypic variation. The lack of B-granules in polyploid Aegilops with diverse genomes suggests that the B-granule locus has been lost several times independently during the evolution of the Triticeae. It is proposed that the B-granule locus is susceptible to silencing during polyploidization and a model is presented to explain the observed data based on the assumption that the initiation of B-granules is controlled by a single major locus per haploid genome.

Fig. 1.

Starch granule morphology. Light micrographs of iodine-stained starch from mature endosperms of the synthetic tetraploids KU37 and KU41, the natural tetraploid Ae. peregrina and a typical F₁ seed from a cross between KU37 (female) and Ae. peregrina (pollen donor). The scale bar is 100 μm.

Fig. 2.

Starch granule number during grain development. Grains were harvested at various stages of development from 4 days after anthesis (DAA) through to maturity. (A) Images of developing grains (scale bar 5 mm) and iodine-stained starch (scale bar 50 μm) from KU37 (left) and Ae. peregrina (right) from 4 DAA to maturity. (B) Fresh weight, (C) number of starch granules, and (D) percentage of granules less that 10 μm diameter were also recorded. Values are the means ±SE of measurements on a minimum of three separate grains. (This figure is available in colour at JXB online.)

Fig. 3.

Analysis of granule-size distribution. The F₃ seeds were harvested from individual F₂ plants. Starch granules were extracted from the endosperm of 4–16 individual F₃ seeds per plant. The proportion of small-granules (10 μm or less in diameter) in the endosperm was examined by microscopy and quantified by image analysis. (A) Examples of micrographs of seeds with low, medium, and high numbers of small granules. The percentage of small granules is indicated. The scale bar is 100 μm. (B) The percentage of small granules in individual F₃ seeds from 84 separate F₂ plants. Open squares are values for individual seeds and closed diamonds are means for individual F₂ plants. (C) The frequency of F₂ plants per granule size category. Mean granules sizes for individual F₂ plants are plotted against granule-size bins of three units.

Fig. 4.

Genetic location of the B-granule QTL on the short arm of chromosome 4S. (A) Genotype data for the linked markers shown in Table 2 together with the granule-size phenotypes for 84 F₂ plants (average granule size per F₂ plant) shown in Fig. 3B were used to calculate the position of a QTL responsible for variation in B-granule number. (B) The F₂ plants were grouped according to the genotype of markers 4G and TR129 and the mean value of the associated phenotype was assessed. Group AP, both markers are homozygous for the Ae. peregrina genotype; KU, both markers are homozygous for the KU37 genotype; and AP/KU both marker are heterozygous. Means are all significantly different at P <0.01. Values are means ±SE of the percentage of small granules per seed.

Fig. 5.

A model of the evolution of the B-granule-less trait in Aegilops. The diploid progenitors (panel 1) have seven chromosomes each, but for simplicity, only the group 4 chromosomes are shown. The position of the B-locus is indicated by a black bar. This bar is omitted in panels 2 and 3 where the B-locus is presumed inactive or deleted. Immediately following formation of the tetraploids by polyploidization (panel 2, left-hand side), the B-loci are present as in the diploid progenitors. However, genome rearrangements following polyploidization lead to the loss of some of the B-loci (panel 2, right-hand side). The cross between Ae. peregrina and KU37 (panel 3) results in an F₁ that is heterozygous for the B-locus on chromosome 4S. The B-locus on chromosome 4U is inactive. Thus, although the Aegilops used in this study are tetraploid, only a single B-loci is responsible for the initiation of B-granules.