Genome-Edited Triple-Recessive Mutation AltersSeed Dormancy in Wheat

1Common wheat has three sets of sub-genomes, making mutations difficult to observe, especially for traits controlled by recessive genes. Here, we produced hexaploid wheat lines with loss of function of homeoalleles of Qsd1, which controls seed dormancy in barley, by Agrobacterium-mediated CRISPR/Cas9. Of the eight transformed wheat events produced, three independent events carrying multiple mutations in wheat Qsd1 homeoalleles were obtained. Notably, one line had mutations in every homeoallele. We crossed this plant with wild-type cultivar Fielder to generate a transgene-free triple-recessive mutant, as revealed by Mendelian segregation. The mutant showed a significantly longer seed dormancy period than wild-type, which may result in reduced pre-harvest sprouting of grains on spikes. PCR, southern blotting, and whole-genome shotgun sequencing revealed that this segregant lacked transgenes in its genomic sequence. This technique serves as a model for trait improvement in wheat, particularly for genetically recessive traits, based on locus information from diploid barley

タイリョウ EST オ リヨウシタ パンコムギ ニ オケル トランスクリプトーム カイセキ

京都大学0048新制・論文博士博士(農学)乙第11880号論農博第2610号新制||農||931(附属図書館)学位論文||H18||N4172(農学部図書室)UT51-2006-K278(主査)教授 谷坂 隆俊, 教授 内海 成, 教授 遠藤 隆学位規則第4条第2項該当Doctor of Agricultural ScienceKyoto UniversityDA

Expression Profile of Two Storage-Protein Gene Families in Hexaploid Wheat Revealed by Large-Scale Analysis of Expressed Sequence Tags

To discern expression patterns of individual storage-protein genes in hexaploid wheat (Triticum aestivum cv Chinese Spring), we analyzed comprehensive expressed sequence tags (ESTs) of common wheat using a bioinformatics technique. The gene families for α/β-gliadins and low molecular-weight glutenin subunit were selected from the EST database. The alignment of these genes enabled us to trace the single nucleotide polymorphism sites among both genes. The combinations of single nucleotide polymorphisms allowed us to assign haplotypes into their homoeologous chromosomes by allele-specific PCR. Phylogenetic analysis of these genes showed that both storage-protein gene families rapidly diverged after differentiation of the three genomes (A, B, and D). Expression patterns of these genes were estimated based on the frequencies of ESTs. The storage-protein genes were expressed only during seed development stages. The α/β-gliadin genes exhibited two distinct expression patterns during the course of seed maturation: early expression and late expression. Although the early expression genes among the α/β-gliadin and low molecular-weight glutenin subunit genes showed similar expression patterns, and both genes from the D genome were preferentially expressed rather than those from the A or B genome, substantial expression of two early expression genes from the A genome was observed. The phylogenetic relationships of the genes and their expression patterns were not correlated. These lines of evidence suggest that expression of the two storage-protein genes is independently regulated, and that the α/β-gliadin genes possess novel regulation systems in addition to the prolamin box

Multiple Wheat Genomes Reveal Novel Gli-2 Sublocus Location and Variation of Celiac Disease Epitopes in Duplicated α-Gliadin Genes

The seed protein α-gliadin is a major component of wheat flour and causes gluten-related diseases. However, due to the complexity of this multigene family with a genome structure composed of dozens of copies derived from tandem and genome duplications, little was known about the variation between accessions, and thus little effort has been made to explicitly target α-gliadin for bread wheat breeding. Here, we analyzed genomic variation in α-gliadins across 11 recently published chromosome-scale assemblies of hexaploid wheat, with validation using long-read data. We unexpectedly found that the Gli-B2 locus is not a single contiguous locus but is composed of two subloci, suggesting the possibility of recombination between the two during breeding. We confirmed that the number of immunogenic epitopes among 11 accessions varied. The D subgenome of a European spelt line also contained epitopes, in agreement with its hybridization history. Evolutionary analysis identified amino acid sites under diversifying selection, suggesting their functional importance. The analysis opens the way for improved grain quality and safety through wheat breeding

doi:10.1093/dnares/dsq009 Comparative Gene Expression Analysis of Susceptible and Resistant Near-Isogenic Lines in Common Wheat Infected by Puccinia triticina

Gene expression after leaf rust infection was compared in near-isogenic wheat lines differing in the Lr10 leaf rust resistance gene. RNA from susceptible and resistant plants was used for cDNA library construction. In total, 55 008 ESTs were sequenced from the two libraries, then combined and assembled into 14 268 unigenes for further analysis. Of these ESTs, 89 % encoded proteins similar to (E value of 10 25) characterized or annotated proteins from the NCBI non-redundant database representing diverse molecular functions, cellular localization and biological processes based on gene ontology classification. Further, the unigenes were classified into susceptible and resistant classes based on the EST members assembled from the respective libraries. Several genes from the resistant sample (14-3-3 protein, wali5 protein, actin-depolymerization factor and ADP-ribosylation factor) and the susceptible sample (brown plant hopper resistance protein, caffeic acid O-methyltransferase, pathogenesis-related protein and senescence-associated protein) were selected and their differential expression in the resistant and susceptible samples collected at different time points after leaf rust infection was confirmed by RT– PCR analysis. The molecular pathogenicity of leaf rust in wheat was studied and the EST data generated made a foundation for future studies