LYS3 encodes a prolamin-box-binding transcription factor that controls embryo growth in barley and wheat.

Mutations at the LYS3 locus in barley have multiple effects on grain development, including an increase in embryo size and a decrease in endosperm starch content. The gene underlying LYS3 was identified by genetic mapping and mutations in this gene were identified in all four barley lys3 alleles. LYS3 encodes a transcription factor called Prolamin Binding Factor (PBF). Its role in controlling embryo size was confirmed using wheat TILLING mutants. To understand how PBF controls embryo development, we studied its spatial and temporal patterns of expression in developing grains. The PBF gene is expressed in both the endosperm and the embryos, but the timing of expression in these organs differs. PBF expression in wild-type embryos precedes the onset of embryo enlargement in lys3 mutants, suggesting that PBF suppresses embryo growth. We predicted the down-stream target genes of PBF in wheat and found them to be involved in a wide range of biological processes, including organ development and starch metabolism. Our work suggests that PBF may influence embryo size and endosperm starch synthesis via separate gene control networks.

Identifying and Isolating Meiotic Mutants in a Polyploid Brassica Crop.

This chapter provides a detailed description of TILLING and CRISPR-Cas9 approaches for the purpose of studying genes/factors involved in meiotic recombination in the polyploid species B. napus. The TILLING approach involves the screening and identification of EMS-mutagenized M2 B. napus plants. The strategy for high-throughput plant pooling, the set up for microfluidic PCR and sequencing is provided and the parameters for the analysis of sequence results and the detection of mutants are explained. The CRISPR-Cas system relies on the optimal design of guide RNAs and their efficient expression. The procedure for the generation and detection of knockout mutants is described with the aims to simultaneously target homologous genes.

High-resolution melting-based TILLING of γ ray-induced mutations in rice.

Targeting Induced Local Lesions IN Genomes (TILLING) is a reverse genetics strategy for the high-throughput screening of induced mutations. γ radiation, which often induces both insertion/deletion (Indel) and point mutations, has been widely used in mutation induction and crop breeding. The present study aimed to develop a simple, high-throughput TILLING system for screening γ ray-induced mutations using high-resolution melting (HRM) analysis. Pooled rice (Oryza sativa) samples mixed at a 1:7 ratio of Indel mutant to wild-type DNA could be distinguished from the wild-type controls by HRM analysis. Thus, an HRM-TILLING system that analyzes pooled samples of four M2 plants is recommended for screening γ ray-induced mutants in rice. For demonstration, a γ ray-mutagenized M2 rice population (n=4560) was screened for mutations in two genes, OsLCT1 and SPDT, using this HRM-TILLING system. Mutations including one single nucleotide substitution (G→A) and one single nucleotide insertion (A) were identified in OsLCT1, and one trinucleotide (TTC) deletion was identified in SPDT. These mutants can be used in rice breeding and genetic studies, and the findings are of importance for the application of γ ray mutagenesis to the breeding of rice and other seed crops.

mlo-based powdery mildew resistance in hexaploid bread wheat generated by a non-transgenic TILLING approach.

Wheat is one of the most widely grown cereal crops in the world and is an important food grain source for humans. However, wheat yields can be reduced by many abiotic and biotic stress factors, including powdery mildew disease caused by Blumeria graminis f.sp. tritici (Bgt). Generating resistant varieties is thus a major effort in plant breeding. Here, we took advantage of the non-transgenic Targeting Induced Lesions IN Genomes (TILLING) technology to select partial loss-of-function alleles of TaMlo, the orthologue of the barley Mlo (Mildew resistance locus o) gene. Natural and induced loss-of-function alleles (mlo) of barley Mlo are known to confer durable broad-spectrum powdery mildew resistance, typically at the expense of pleiotropic phenotypes such as premature leaf senescence. We identified 16 missense mutations in the three wheat TaMlo homoeologues, TaMlo-A1, TaMlo-B1 and TaMlo-D1 that each lead to single amino acid exchanges. Using transient gene expression assays in barley single cells, we functionally analysed the different missense mutants and identified the most promising candidates affecting powdery mildew susceptibility. By stacking of selected mutant alleles we generated four independent lines with non-conservative mutations in each of the three TaMlo homoeologues. Homozygous triple mutant lines and surprisingly also some of the homozygous double mutant lines showed enhanced, yet incomplete, Bgt resistance without the occurrence of discernible pleiotropic phenotypes. These lines thus represent an important step towards the production of commercial non-transgenic, powdery mildew-resistant bread wheat varieties.

Highly efficient de novo mutant identification in a Sorghum bicolor TILLING population using the ComSeq approach.

Screening large populations for carriers of known or de novo rare single nucleotide polymorphisms (SNPs) is required both in Targeting induced local lesions in genomes (TILLING) experiments in plants and in screening of human populations. We previously suggested an approach that combines the mathematical field of compressed sensing with next-generation sequencing to allow such large-scale screening. Based on pooled measurements, this method identifies multiple carriers of heterozygous or homozygous rare alleles while using only a small fraction of resources. Its rigorous mathematical foundations allow scalable and robust detection, and provide error correction and resilience to experimental noise. Here we present a large-scale experimental demonstration of our computational approach, in which we targeted a TILLING population of 1024 Sorghum bicolor lines to detect carriers of de novo SNPs whose frequency was less than 0.1%, using only 48 pools. Subsequent validation confirmed that all detected lines were indeed carriers of the predicted mutations. This novel approach provides a highly cost-effective and robust tool for biologists and breeders to allow identification of novel alleles and subsequent functional analysis.

Genomics as the key to unlocking the polyploid potential of wheat.

Polyploidy has played a central role in plant genome evolution and in the formation of new species such as tetraploid pasta wheat and hexaploid bread wheat. Until recently, the high sequence conservation between homoeologous genes, together with the large genome size of polyploid wheat, had hindered genomic analyses in this important crop species. In the past 5 yr, however, the advent of next-generation sequencing has radically changed the wheat genomics landscape. Here, we review a series of advances in genomic resources and tools for functional genomics that are shifting the paradigm of what is possible in wheat molecular genetics and breeding. We discuss how understanding the relationship between homoeologues can inform approaches to modulate the response of quantitative traits in polyploid wheat; we also argue that functional redundancy has 'locked up' a wide range of phenotypic variation in wheat. We explore how genomics provides key tools to inform targeted manipulation of multiple homoeologues, thereby allowing researchers and plant breeders to unlock the full polyploid potential of wheat.