Identification & integration of sudden death syndrome resistance genomic regions into Ontario-adapted food grade soybeans

Objectives:

• To develop Ontario-adapted food-grade soybean commercial cultivars and germplasm in which sudden death syndrome (SDS) resistant genes will be stacked with soybean cyst nematode (SCN) resistant genes.
• To identify quantitative trait loci (QTL) associated with the root and foliar resistance to SDS in Canadian food-grade soybean germplasm using linkage mapping, genome-wide association study (GWAS), and bulked segregant analyses.

• To develop reliable molecular marker(s) for SDS resistance that are desirable for marker-assisted selection (MAS).

Impacts:

• The development of Ontario-adapted commercial soybean cultivars that are resistant to SDS and SCN will provide Ontario’s soybean growers with new tools to fight against the most yield-limiting pests of soybean in the region and increase their soybean production.
• The enhancement of Canadian food-grade soybean germplasm through stacking SDS resistance genes with SCN resistance genes will provide Ontario-based soybean breeding programs with adapted SDS- and SCN-resistant sources, which can be used in cultivar development programs as these two diseases continue to spread across the province.
• The development of defined marker-based selection tools for discriminating genotypes that are resistant to SDS, at early generations, will help the breeding programs at the University of Guelph, and other interested public and private soybean breeding programs across Ontario and Canada, to accelerate the development of SDS-resistant cultivars without the need for the laborious and time-consuming process of greenhouse bioassays.
• Through this project, two graduate students will be trained and gain hands-on experience in plant breeding and genetics.

Project Overview:

Soybean sudden death syndrome (SDS) has been established in southwestern Ontario as the 2^nd most devastating pest, after soybean cyst nematode (SCN), and continues to spread across the province. The most effective countermeasure is using SDS-resistant cultivars to combat the disease and minimize its impact on yield; however, currently, there are no specific and sustained breeding efforts within Ontario food-grade breeding programs to develop disease-resistant cultivars based on innovative breeding approaches for continuous cultivar development for Ontario’s farmers. This lack of breeding activity can be due partly to limited information about the inheritance and mode of action of the resistance to SDS in Canadian soybean germplasm. Although there is no study on the genetic control of the resistance to SDS in Canadian soybean germplasm, several studies on exotic germplasm have shown that resistance is controlled by multiple genes. Mapping and isolating the genomic regions or quantitative trait loci (QTL) underlying the resistance is necessary for a better understanding of the molecular mechanisms governing the resistance, and for establishing efficient marker-assisted selection (MAS)-based breeding programs that will facilitate the development of resistant cultivars.

To identify molecular markers associated with soybean SDS, a panel of 250 distinct soybean lines was utilized, comprising 125 lines from Dr. Rajcan’s lab at the University of Guelph and 125 lines from Dr. Eskandari’s lab at the Ridgetown campus. The panel was evaluated for SDS resistance by calculating the disease index (DI) in an SDS-prone field in Rodney during 2022, in collaboration with Mr. Tenuta. In the summer of 2023, the second year of field evaluations focused on foliar symptoms was conducted in two SDS-prone fields: Rodney (in collaboration with Mr. Tenuta) and Chatham (in collaboration with Dr. Wally). Additionally, an SDS bioassay for root rot was carried out at the Harrow Research and Development Centre. Through genome-wide association studies (GWAS) analyses conducted by MSc student Kelsey Boucher, three markers on chromosomes 9, 13, and 17 were identified as being associated with root rot resistance, while two markers on chromosomes 13 and 15 were linked to foliar resistance. These markers hold potential for use in breeding programs to facilitate the development of new cultivars with enhanced SDS resistance.

Results:

Objective 1: To develop Ontario-adapted elite commercial cultivars and germplasm that are resistant to sudden death syndrome (SDS).

In winter 2020, two breeding populations were generated through crosses between two SDS-resistant cultivars (AR17-178033 and M12-421024) with OAC Bruton in the growth room at University of Guelph. All three parental lines are SCN-resistant elite cultivars, resulting in SCN-resistant progeny. From each cross, 500 F2 seeds were sent to Costa Rica for a two-generation single seed descent (SSD) advance over winter. The F4 generation seeds were grown in the field at Ridgetown in spring 2021 to generate two breeding populations of 200 recombinant inbred lines (RIL). In summer 2022, F5 seeds of each single plant were grown in Ridgetown to evaluate them for important agronomic and seed composition traits and maturing date using randomized complete block designs with two replications, and to produce F6 seeds. F6 RILs from both populations were planted in replicated trials in two locations for yield, agronomic and seed composition trait evaluations in 2023. The F6 RILs from both populations were screened for SDS resistance using a greenhouse bioassay in fall 2023. The results demonstrated a broad spectrum of reactions to SDS, ranging from susceptibility to resistance, with some plants showing no symptoms of the disease. Based on the results of SDS bioassay and field trials in 2023, a total of six promising lines were selected and advanced to yield trials in 2024.

Objective 2: To identify QTL associated with resistance to SDS in Canadian food-grade soybean germplasm using linkage mapping, genome-wide association study and bulked segregant analyses (BSA).

GWAS analyses were conducted on a total of 250 diverse soybean genotypes, including elite and experimental lines for the University of Guelph breeding programs, plant introduction (PIs), ethylmethane- sulfonate (EMS) and fast neutron (FN) induced mutants, Chinese cultivars and parental lines. After establishing the GWAS diversity panel, the evaluation of diverse panel was implemented for their level of the resistance to SDS in SDS-prone fields in Rodney, ON (in collaboration with Tenuta’s lab) in summer 2022 and 2023. In addition, the lines were also evaluated for the resistance to SDS in an SDS-prone field in Chatham, ON (in collaboration with Wally’s lab). Lines were rated for foliar symptoms using the Southern Illinois University–Carbondale disease index (DX). DX was calculated as DX=(DI×DS)/9, where DI is disease incidence and DS is disease severity (rated on a 1–9 scale). Lines were then classified into SDS response groups: resistant (DX ≤ 10), intermediate (DX = 11–28), and susceptible (DX ≥ 29).

Alongside recording foliar symptoms in field for calculation of DI, greenhouse SDS bioassay of panel were completed in Harrow, ON in fall 2023 evaluating root rot symptoms. SDS evaluation or greenhouse SDS bioassay of two bio-parental populations were also implemented in Harrow, ON in early 2024. MSc student Kelsey Boucher conducted genome-wide association studies (GWAS) analyses, identifying three markers on chromosomes 9, 13, and 17 associated with root rot resistance, and two markers on chromosomes 13 and 15 linked to foliar resistance. These markers offer significant potential for incorporation into breeding programs aimed at developing new cultivars with improved resistance to SDS.

Objective 3: Designing Kompetitive Allele-Specific PCR (KASP) markers for the identified QTL (Obj.2) and developing reliable molecular marker(s) desirable for marker-assisted selection (MAS).

The identified marker-trait associations (MTAs) across six genomic regions will be utilized to develop KASP markers for the most influential and significant loci, enabling marker-assisted selection in future SDS-resistant cultivar development. These markers are expected to enhance the efficiency of our breeding programs and support other breeding initiatives interested in developing SDS-resistant soybeans for Ontario and Canada.

Recommendations:

Based on our evaluation of the panel of 250 University of Guelph-based soybean lines, we are convinced that available soybean cultivars vary in their susceptibility and resistance to SDS, which can be true among the Ontario Soybean Variety Trial (OSVT) entries as well. Therefore, if farmers have access to reliable data, they may want to consider resistance to SDS as one of the criteria when selecting their cultivars. The results of this study, including the most resistant and susceptible soybean lines tested for both foliar and root rot resistance, can be found in Kelsey Boucher’s MSc thesis through the following link: https://hdl.handle.net/10214/28694 (Identification of Marker Trait Associations (MTAs) for Resistance to Sudden Death Syndrome in a Panel of Canadian Elite Genotypes).

Acknowledgements:

This project was funded in part by SeCan and by the Ontario Agri-Food Innovation Alliance, a collaboration between the Government of Ontario and the University of Guelph.

Project Related Publications:

Boucher, K., I., Rajcan, M., and Eskandari. 2024. Identification of Marker Trait Associations (MTAs) for Resistance to Sudden Death Syndrome in a Panel of Canadian Elite Genotypes. https://hdl.handle.net/10214/28694