A splice acceptor site mutation in TaGW2-A1 increases thousand grain weight in tetraploid and hexaploid wheat through wider and longer grains

Abstract

Across 13 experiments the gw2 - A1 mutant allele shifts grain size distribution consistently across all grains significantly increasing grain weight (6.6 %), width (2.8 %) and length (2.1 %) in tetraploid and hexaploid wheat. There is an urgent need to identify, understand and incorporate alleles that benefit yield in polyploid wheat. The rice OsGW2 gene functions as a negative regulator of grain weight and width and is homologous to the wheat TaGW2 gene. Previously it was shown that transcript levels of the A-genome homoeologue, TaGW2-A1, are negatively associated with grain width in hexaploid wheat. In this study we screened the tetraploid Kronos TILLING population to identify mutants in TaGW2-A1. We identified a G to A transition in the splice acceptor site of exon 5 which leads to mis-splicing in TaGW2-A1. We backcrossed the mutant allele into tetraploid and hexaploid wheat and generated a series of backcross derived isogenic lines which were evaluated in glasshouse and field conditions. Across 13 experiments the GW2-A1 mutant allele significantly increased thousand grain weight (6.6 %), grain width (2.8 %) and grain length (2.1 %) in tetraploid and hexaploid wheat compared to the wild type allele. In hexaploid wheat, this led to an increase in spike yield since no differences were detected for spikelet or grain number between isogenic lines. The increase in grain width and length was consistent across grains of different sizes, suggesting that the effect of the mutation is stable across the ear and within spikelets. Differences in carpel size and weight between alleles were identified as early as 5 days before anthesis, suggesting that TaGW2-A1 acts on maternal tissue before anthesis to restrict seed size. A single nucleotide polymorphism marker was developed to aid the deployment of the mutant allele into breeding programmes.

Fig. 1

G2373A results in mis-splicing of TaGW2-A1. a Diagram of TaGW2-A1 target region including exons 2–6 (black and grey numbered boxes) and introns (thin line). The position of the G>A transition at position 2373 (G2373A) of IWGSC_CSS_6AS_scaff_4408273 is indicated. b Sequence alignment of gDNA from wild type (top) and mutant line T4-2235 (middle and bottom) which includes the G2373A transition. The G2373A mutation in line T4-2235 is in red font and yellow highlight. Exon sequences are in uppercase letters (exon 4 in black; exon 5 in grey), whereas intron sequences adjacent to the splice sites (red font) are in lowercase. The 78-bp of intron four are represented by the number 78 between parentheses. Note that the mutation leads to a change in the AG splice acceptor site in T4-2235 which removes either four bp (GAAG) or nine bp (GAAGAACAG) from exon 5. c Nucleotide sequence of cDNA spanning exons 4 and 5 in wild type (top sequence) and the two variants of mutant T4-2235 (middle and bottom sequences), with their corresponding amino acid translations. The −4 bp mutant allele is missing four nucleotides which disrupts the reading frame (red amino acid residues) leading to a premature termination codon (red asterisk) in the T4-2235 sequence. The −9 bp mutant allele is missing nine nucleotides which leads to the loss of three amino acids (EEQ) from the mutant protein

Fig. 2

Crossing scheme used to introduce the G2373A allele (gw2-A) into tetraploid wheat Kronos a and hexaploid wheat Paragon b both carrying a wild type GW2-A allele. Small crossed circles indicate self-pollination, whereas straight arrows indicate back-cross to either Kronos (a) or Paragon (b). For the Kronos stream (a), F₂-, BC₂-, and BC₄-derived lines were evaluated under glasshouse (GH) and field environments, whereas for the Paragon stream (b) only BC₂- and BC₄-derived lines were evaluated. The results of each generation are indicated by the outline colour (red dash: Table 1; blue: Table 2; Fig. 4a; green Tables 3, 4; Fig. 4b)

Fig. 3

Representative grains from field grown BC₂F₄ near isogenic lines carrying the wild type GW2-A1 or G2373A gw2-A1 mutant allele in tetraploid wheat Kronos and hexaploid wheat Paragon. Grains are aligned to show differences in width (20 grains) and length (10 grains). Scale bar = 1 cm

Fig. 4

Box and whisker plots of grain morphometric parameters of BC-derived lines in tetraploid wheat Kronos a and hexaploid wheat Paragon b, with the wild type GW2-A (grey) or G2373A gw2-A mutant allele (blue and green in tetraploid and hexaploid, respectively). Each row represents a separate experiment and the columns represents grain area (left), grain width (middle) and grain length (right). The left boundary of the box indicates the 25th percentile, the black line within the box marks the median (50th percentile), and the right boundary of the box indicates the 75th percentile. The error bars (whiskers) on either side of the box indicate the 10th and 90th percentiles. The red line within the box marks the mean

Fig. 5

Carpel/grain developmental time course of G2373A gw2-A1 NILs. Carpel/grain width (a), length (b), and dry weight (c) in Paragon BC₂ NILs carrying either the Paragon wild type GW2-A (dark grey) or the G2373A gw2-A1 mutant allele (green). Samples were taken at heading [−5 days post anthesis (dpa)], anthesis (0 dpa) and 3, 9, 16, and 23 dpa. Inset in (c) magnifies the first three time points to help visualise differences. Asterisks indicate significance of pairwise comparisons at each time point: *P < 0.05; **P < 0.01; ***P < 0.001