A splice acceptor site mutation in Emphasis Type=

Authors: James Simmonds Peter Scott Jemima Brinton Teresa C Mestre Max Bush Alicia del Blanco Jorge Dubcovsky Cristobal Uauy

Publish Date: 2016/02/16

Volume: 129, Issue: 6, Pages: 1099-1112

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Abstract

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 Agenome homoeologue TaGW2A1 are negatively associated with grain width in hexaploid wheat In this study we screened the tetraploid Kronos TILLING population to identify mutants in TaGW2A1 We identified a G to A transition in the splice acceptor site of exon 5 which leads to missplicing in TaGW2A1 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 GW2A1 mutant allele significantly increased thousand grain weight 66  grain width 28  and grain length 21  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 TaGW2A1 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 programmesWheat provides approximately 20  of the calories and 25  of protein consumed by humankind FAOSTAT 2015 and is an important source of micronutrients There are two major types of polyploid wheat hexaploid wheat 2n = 6x = 42 ~17 Mb AABBDD genomes primarily used for bread and biscuit products and tetraploid durum wheat 2n = 4x = 28 ~12 Mb AABB genomes used for pasta The three genomes A B and D referred to as homoeologues have a complementary set of collinear genes that share between 96 and 98  sequence identity across coding regions IWGSC 2014 Krasileva et al 2013 Since most wheat genes are present as two or three close homoeologous copies IWGSC 2014 mutations in individual genes can be masked by functional complementation by homoeologous genes present in the other genomes Borrill et al 2015 Lawrence and Pikaard 2003 This gene redundancy has limited the use of forward genetic screens in wheat and limited the detection of phenotypic effects of natural variantsDespite a growing human population and rising demand for wheat low rates of genetic gains for wheat yields Ray et al 2013 pose a real threat to global food security Therefore there is an urgent need to identify understand and incorporate alleles that increase wheat yield potential across multiple environments This task is complicated by the polygenic regulation of grain yield and the strong effect of the environment To facilitate its study overall grain yield can be broken down into its individual components This study focuses on thousand grain weight TGW and its physical parameters grain length width area which are inherited in a relatively stable manner and show higher heritability values than overall yield Kuchel et al 2007Significant advances have been made in our understanding of the genes controlling seed size in the model species rice reviewed in Xing and Zhang 2010 and Arabidopsis reviewed in Li and Li 2015 The characterised genes have been shown to function in Gprotein signalling Huang et al 2009 phytohormone biosynthesis/signalling Jiang et al 2013 or in the ubiquitin–proteasome pathway Li et al 2008 Song et al 2007 Xia et al 2013 Specifically in rice genes with large effects on grain size have been identified revealing an independent genetic control of grain length and width in this species Fan et al 2006 Weng et al 2008 This differs from our limited understanding in polyploid wheat where quantitative trait loci QTL for grain size and shape have been identified Breseghello and Sorrells 2007 Maphosa et al 2014 Rustgi et al 2013 Simmonds et al 2014 Sun et al 2009 Williams and Sorrells 2014 Zhang et al 2010 but no gene affecting TGW has yet been clonedThe rice Grain Width and Weight 2 OsGW2 gene encodes a previously unknown RINGtype E3 ubiquitin ligase and functions as a negative regulator of grain width and weight in rice Song et al 2007 The wheat genome has three GW2 homoeologues with the A genome copy TaGW2A1 being most closely examined to date Two studies have identified a significant association between a TaGW2A1 promoter single nucleotide polymorphism SNP and TGW and grain width in Chinese germplasm Su et al 2011 Zhang et al 2013 Despite discrepancies in the haplotype analysis both studies show a negative relationship between TaGW2A1 transcript levels and grain width A separate study identified a single base insertion in the last exon of TaGW2A1 which results in a truncated protein This allele was associated with increased TGW and grain width in hexaploid wheat compared to the full length wild type allele in a F23 population Yang et al 2012 These results support a conserved role of GW2 between wheat and rice as a negative regulator of grain size and weight However contradictory results have been reported from RNA interference RNAi studies of all three TaGW2 homoeologues in wheat whereas Bednarek et al 2012 reported a reduction in grain weight and size by downregulation of TaGW2 homoeologues Hong et al 2014 found an increase in equivalent transgenic lines

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