Haploid plants containing chromosomes from only one parent can greatly accelerate plant breeding. A novel method for producing haploid Arabidopsis thaliana has been developed based on the observation that, when wildtype plants are crossed to mutant plants in which a centromere protein (CENH3) is replaced with an altered centromere variant, chromosomes from the mutant are eliminated, yielding haploid progeny at a high frequency. Unlike most existing methods for producing haploids, this method creates haploids through seed and thus does not require tissue culture, a major source of genotype dependence. In addition, CENH3 is universally required for centromere function in eukaryotes, providing a practical basis for transferring the method into any plant that can be transformed.
This EAGER project will leverage this preliminary work to adapt the technology to produce haploids in tomato. The work will use A. thaliana to determine how genome elimination can be engineered without a cenh3 null mutation, information critical for the potential use of the method in crop plants. RNA interference will be used first to down-regulate CENH3 in tomato. Finally, altered CENH3 transgenes will be introduced into tomato plants where endogenous CENH3 has been silenced to test for their ability to induce haploids when crossed to wild type.
Broader Impacts
Tomato has tremendous economic importance, and is rapidly becoming a model for basic plant biology both within and outside the Solanaceae. Despite these advantages, it completely lacks a haploid production method. Development of such a protocol would synergize with existing genetic tools (including large banks of wild germplasm) and emerging genomic resources. The combination of these strengths will facilitate studies that use natural variation to understand agriculturally important traits and will provide for improved tomato breeding, which could have profound effects on food security in the many countries including developing countries in Africa and Asia where tomato and its close relatives eggplant and potato are important crops.
The proposed research will provide research training opportunities for postdoctoral fellows and disadvantaged and underrepresented undergraduate students that integrate genetic and molecular methods with statistical and mathematical rigor. These young scientists will also receive training in plant breeding as well as in quantitative genomic methods.
A major challenge in plant breeding is to rapidly create true breeding lines that combine favorable traits from two different parents. Like humans, plants contain two versions of each gene, one from their mother and one from their father. True breeding or "homozygous" lines are those in which the two versions of each gene are identical over most or all of the genome. Such lines can be made by laborious rounds of inbreeding. However, if a plant with only one version of each gene can be created, then converted back into a plant with two identical versions of each gene, a true breeding variety is made in just two steps. This project developed novel technology for creating plants with only one version of each gene (termed "haploid" plants). We used the laboratory plant Arabidopsis thaliana, a weedy relative of mustard and cabbage, to refine previous methods for creating haploid plants. Chromosomes contain DNA, the genetic material, and also proteins. We engineered a protein that is essential for genetic inheritance, so that chromosomes from one parent are selectively discarded after fertilization. This created offspring with only one version of each gene. In the work funded by this grant, we found many alterations to inheritance proteins that created haploid plants in a genetic cross. We also studied how the process of chromosome loss proceeded after fertilization, to better understand how haploid plants are generated. Lastly, we began to export our haploid production method into tomato, a highly valuable crop for basic research and for agriculture that completely lacks a haploid production method. While studying the process of chromosome loss that gives rise to haploid plants, we discovered a very straightforward way to make minichromosomes. These are variants of normal chromosomes in which large regions of the genetic material have been deleted. Minichromosomes are appealing to plant geneticists, because they help us to understand the minimal machinery required for accurate inheritance of the genetic material. In addition, they could have profound applications in biotechnology, as they would be able to easily transfer genes between different plant varieties.
