18-BTT: High-throughput fluorescent crossover reporters to dissect control of tomato meiotic recombination

A major challenge facing society is to improve crop yield and quality, in order to meet the needs of the global population. One means to address this challenge is to breed and further improve crop germ plasm. For example, disease and stress resistance traits from wild populations can be beneficial to introduce into elite domesticated varieties. In this case beneficial traits must then be introgressed over many generations to achieve a desirable, improved strain.

The process of crop improvement relies on the trait re-assortment in offspring that occurs naturally during sexual reproduction. Specifically, diversity is generated during a specialised cell division called meiosis, which results in production of gametes (sex cells). One mechanism that creates genetic diversity during meiosis is called crossover recombination. During meiosis, homologous chromosomes physically pair and exchange reciprocal regions, which generates new combinations of genetic variation. However, crossover recombination patterns along chromosomes are highly skewed in many of the most important crop species, including wheat, maize, barley and tomato. As a consequence, some regions of the genome are inaccessible for breeding, despite containing important genes with effects on agronomically important traits.

The specific crop this proposal will investigate is Solanum lycopersicum (tomato). Tomato is a major global crop with an annual yield of over 130 million tons. Domesticated tomato belongs to a tribe of 13 related Solanaceous species, which represent an important source of wild diversity for improving yield and disease resistance traits. Following wide crosses, breeders must complete multiple cycles of introgression to obtain useful lines, and this process relies on meiosis and recombination. However, low numbers of recombination events and skewed genomic distributions limits the efficiency of this process. Tools that boost or unlock recombination will accelerate improvement of tomato germ plasm. The tomato FTL system that we will develop will enable academic and industrial researchers to rapidly quantify recombination frequency. This will facilitate the development of technology to modulate meiotic recombination to enhance breeding efforts and increase food security. All of the tools we develop with be shared on an open-access basis.

During meiosis homologous chromosomes undergo programmed DNA double-strand breaks (DSBs), which may be repaired as reciprocal crossovers (COs). In addition to generating genetic diversity, COs are required for proper chromosome segregation. Despite their importance, most species only undergo 1-2 COs per chromosome per meiosis. CO distributions are also non-random, with the majority of events occurring in narrow 'hotspots', with intervening cold regions. The limited number of COs slows breeding, and favourable traits located in recombination-cold regions can suffer linkage drag with nearby unfavourable traits. Therefore, there is an urgent need to understand and manipulate meiotic recombination in ways that accelerate breeding and crop improvement. In the proposed work we will build on our track record of developing fluorescent CO reporters and apply this to the context of tomato, a key crop species. Tomato has specific benefits for the study of meiotic recombination including, (i) a well assembled and annotated genome, (ii) excellent meiotic immunocytology with pronounced euchromatin/heterochromatin differentiation, and (iii) existing maps of the CO landscape generated by genotyping-by-sequencing. In the proposed work we will further strengthen this suite of characteristics by developing high-throughput fluorescent FTL CO reporters. The FTL system will provide academic and industrial researchers with powerful tools to quantify CO frequencies in different regions of the tomato genome rapidly and at low cost. This will allow investigation of genetic mechanisms that govern CO distributions in a complex plant genome, as well as optimization of growth conditions and genetic backgrounds that maximize useful recombination during breeding.

In addition to expanding our understanding of a fundamental biological mechanism, the anticipated results of this project have industrial and commercial impact. Solanum lycopersicum (tomato) is a major global crop with an annual yield of over 130 million tons. Domesticated tomato belongs to a tribe of 13 related Solanaceous species, which represent an important source of wild diversity for improving yield and disease resistance traits. Following wide crosses, breeders must complete multiple cycles of introgression to obtain useful lines, and this process relies on meiosis and recombination. However, low CO numbers and skewed genomic distributions limits the efficiency of this process. Tools that boost CO numbers, or unlock recombination within heterochromatin, will accelerate improvement of tomato germ plasm. Our proposed tomato FTL system will enable academic and industrial researchers to rapidly quantify CO frequency. This will facilitate the development of technology to modulate meiotic recombination to enhance breeding efforts and increase food security. All of the tools we develop with be shared on an open-access basis. The PIs are also actively engaged in educating students, including thosefrom under-served communities, and the public, about plant reproductive biology and its impact on agriculture and food security.

Outreach and Inclusion: The Copenhaver lab has a long track record of mentoring undergraduates, graduates and postdoctoral associates, including those from under-represented groups (URMs). Dr. Copenhaver serves as faculty mentor in the UNC Post-Baccalaureate Research Education Program (PREP), which provides additional research experience for URMs. Dr. Copenhaver also participates in the UNC Initiative for Maximizing Student Diversity (IMSD). He is actively engaged in public outreach relating to plant biotechnology, including service on public panels discussing CRISPR (Parr Center for Ethics) and agricultural genetic engineering (North Carolina Animal Agriculture Coalition). The proposed work would provide Dr. Copenhaver with a basis for expanded engagement in the crop genetics community. Dr Henderson participates in outreach activities at the University of Cambridge, which includes presenting research to URM students interested in applying to study. He has also participated in public science events, including the University Open Day and Festival of Plants. (iii) Broad

Dissemination of Results: Dr Copenhaver is Editor-in-Chief of PLoS Genetics and thus plays a leading role in promoting open access to scientific knowledge. We intend to publish our results in leading scientific journals and deposit publications in open-access collections. We are increasingly using preprints to release our work coincident with journal submission. The team members will also present our findings at relevant scientific meetings.