INTEGRATING FORWARD AND REVERSE GENETIC TOOLS FOR FAST FORWARDING WHEAT IMPROVEMENT

Abstract

Bread wheat (Triticum aestivum) provides roughly 20% of the human daily caloric intake and is an important crop for global food security. Changing climatic conditions as well as biotic and abiotic stresses are threatening wheat production worldwide. Sustainable and continuous improvement of wheat using novel genes and alleles is critical to tackle wheat’s imminent challenges. Recent advances in wheat genomics have allowed researchers now to fast-track gene discovery pipelines by implementing strategies first developed in less complex model species. This thesis explores the use of forward and reverse genetic approaches to efficiently discover, map, and validate genes controlling important agronomic traits in bread wheat as well as describes a robust protocol to reduce the generation time in winter wheat. Speed breeding is an important tool that utilizes an increased photoperiod and growing temperature to increase vegetative growth and reduce the time from sowing to harvest. Chapter 1 of this thesis outlines a reproducible method to significantly reduce the generation time in winter wheat from over 120 days based on what has been previously reported to 93 days regardless of vernalization requirements or photoperiod sensitivity and provide a useful tool to increase the pace of the genetic gains in the winter wheat breeding programs. Tillering in wheat directly influences the major yield-related trait, spikes per unit area. Using the forward genetics approach, chapter 2 of thesis reports the identification of a novel tiller inhibition gene (tin6) to a small physical region of 2.1 Mb region on chromosome 2DS. This was the first example of using a genome coming from the pan-genome of wheat to perform MutMap. Using reverse genetics also has the potential to improve the end-use properties of wheat by knocking out genes which result in an increase of the nutritional value of the flour. Chapter 3 of this thesis, TILLING was used to identify knockouts in all three homeologous copies of the starch synthase gene SSIIa, which has been shown to increase the amount resistant starch in the endosperm of wheat which is known to have health benefits in humans. The grains coming from triple knockouts of SSIIa contained 118% higher resistant starch, and though they showed a decrease in thousand kernel weight, they did not have a shriveled phenotype which had been seen in other ssiia mutants. Chapter 4 of the thesis demonstrate reference genome enabled positional cloning of a tiller inhibition gene (tin3) in diploid wheat species Triticum monococcum. A MutMap population generated from a cross between tin3 and wildtype T. monococcum resulted in the identification of a single candidate gene, encoding a BLADE-ON-PETIOLE-Like protein, containing a splice-variant mutation. To show the power of using a diploid species for translational research in hexaploid wheat, the reverse genetics approach TILLING (Targeting Induced Local Lesions IN Genomes) was used to identify mutations in all three homeologous copies of tin3 in the Jagger mutant population. The full null mutant for the tin3 locus in wheat showed significantly reduced tillering in comparison to wildtype providing concrete evidence that genetic discoveries that are found in diploid wheat can be effectively translated to hexaploid wheat. There are some genes and QTLs have been identified that increase spike length, spikelets per spike, and grain size, very few studies have focused on increasing the number of grains per floret. Chapter 5 of the thesis was focused on positional cloning of the Mov-1 locus which is the underlying gene responsible for the multiovary (MOV) phenotype. The Mov-1 locus dominantly expresses as three ovaries per wheat floret, each of which develop into a grain. Using high resolution genetic mapping with the MOV-reference genome and gene expression data, we identified a single candidate gene that was localized to a small 144kb region on the Mov-1 physical region. To validate the role of the Mov-1 candidate gene in the MOV phenotype, ethyl methanesulfonate (EMS) and gamma radiation mutagenesis was performed to create deleterious point and deletions mutations, respectively. Using 5 independent TILLING and 5 deletion mutants this study demonstrate that Mov-1 candidate gene is required for the MOV phenotype in wheat. It is an exciting time to work in wheat research as the growing wheat genomic toolbox allows for researchers to efficiently identify and validate genes that have potential to improve wheat performance. The methods and findings in this body of work provide a breadth of knowledge that can be implemented in additional genetic studies in wheat in order to fast-track gene and trait discovery for the benefit of wheat geneticists and breeders alike.