Research Project: Combining Phenotype, Genetics, and Genomics to Develop Common Beans with Resistance to Highly Variable Pathogens

Location: Soybean Genomics & Improvement Laboratory

2022 Annual Report

Objectives
Objective 1: Evaluate diverse common bean accessions, especially in the Andean gene pool, to discover genes and markers linked to these genes that confer resistance to the hyper-variable pathogens that cause rust, anthracnose, angular leaf spot, and other diseases of common bean. [NP301, C1, PS1A and PS1B] Objective 2: Use phenotypic approaches and molecular markers to develop common beans combining Andean and Mesoamerican gene pools to confer broad resistance to highly variable pathogens of common bean. [NP301, C1, PS1A and PS1B] Objective 3: Improve knowledge of virulence, genetic, and genomic diversity of the hyper-variable pathogens that cause common bean diseases. [NP301, C1, PS1A and PS1B; C3, PS3A]

Approach
The major objective of this project is to concurrently broaden the genetic base of common bean to decrease the vulnerability of this crop to the highly variable pathogens that cause the rust, anthracnose, and angular leaf spot diseases. This project is based on genetic solutions that use conventional (phenotype and genetics) and new (genomics) technologies to develop common bean cultivars with broad and durable resistance to these three pathogens. In objective 1, to discover new disease resistance genes, Andean and Mesoamerican common bean accessions will be inoculated under greenhouse conditions with numerous races of the three pathogens. Races known for their virulence will be used in these inoculations. Bean accessions with resistance to most races of three pathogens will be crossed with susceptible cultivars to characterize the new disease resistance genes. To develop DNA markers tagging the newly discovered resistance genes, DNA from the parents used in crosses and from segregating populations will genotyped with the BARCBEAD6K BeadChip. To validate the usefulness of the newly developed molecular markers, phenotypic and molecular approaches will be used. In objective 2, molecular methodologies will be used to accelerate the development of cultivars from various common bean market classes that combine sets of Andean and Mesoamerican genes and broad resistance. Multiple crosses will be performed and multiple races of these pathogens will be used to confirm the spectrum of resistance of the cultivars. In objective 3, to broaden the existing knowledge of the virulence, genetic, and genomic diversity of three mentioned pathogens, DNA from Mesoamerican and Andean strains with known virulence profiles will be used for sequencing and to obtain draft genomes of these pathogens. The sequences will be used to identify DNA markers that may tag specific strains of these pathogens. These markers can be used in genetic diversity studies, and can also be used to improve our understanding of the mechanisms that drive virulence changes in these pathogens.

Progress Report
Goal 1A, multiple disease resistance genes of Andean and Middle American origin that protect common bean from highly virulent pathogens have been identified. These genes are the foundation of a strategy to develop genetic solutions to control the pathogens that cause the anthracnose, rust, and angular leaf spot diseases, that recurrently produce new virulent strains. The following anthracnose resistance genes have been discovered on beans of Andean origin (name of the source of resistance gene in parenthesis): Co-1 4 (AND 277), Co-13 ( Jalo Listras Pretas), Co-PA (Paloma), and Co-BF (Beija Flo). We have also identified the Andean landrace G19833 as resistant to 19 different races of the anthracnose pathogen: 13 Mesoamerican and 6 Andean. G19833 was susceptible to one Andean race. The broad anthracnose resistance in G19833 is rare in Andean and in Middle American beans. The next step regarding the anthracnose resistance in G19833 would be to map and name the new anthracnose resistance gene. Because this landrace was used to sequence the first reference genome of common bean, there is a large quantity of sequence information associated with G19833 that would facilitate the mapping of the gene. We also found the Co-34 anthracnose resistance gene in the Middle American common bean named Ouro Negro. We have used fine mapping to find a more accurate position of the rust resistance genes Ur-3 (Aurora) and Ur-11 (PI181996), both of middle American origin. The rust resistance in Ur-11 is unique; it confers resistance to all but one of all known virulence races of the bean rust pathogen. We have also identified and mapped the Phg-1 (AND 277) angular leaf spot resistance gene of Andean origin and identified and mapped Phg-3 (Ouro Negro), an angular leaf spot resistance gene of Middle American origin. Oruro Negro also contains the Ur-14 rust resistance gene. We also discovered that the Andean landrace G19833 was broadly resistant to 17 races of the rust pathogen. None of the known sources of Andean and Middle American genes were resistant to these 17 Races. These anthracnose, rust, and angular leaf spot disease resistance genes will contribute to broaden the genetic base and protect common bean from the hyper virulent pathogens causing the anthracnose, rust, and angular leaf spot diseases. In Goal 1B, we developed highly accurate molecular markers that detect the presence of disease resistance genes in common bean. These markers were developed using plant pathology, genetics, genomics, and other technologies. Bulked segregant analysis performed using a chip containing thousands of single nucleotide polymorphism (SNP) markers to find the approximate location of multiple resistance genes on the genome of common bean. Using SNPs positively associated with the resistance genes, we developed Kompetitive allele specific PCR (KASP) markers to conduct the fine mapping and haplotype analysis to narrow the genomic region containing the resistance genes, which enabled the identification of KASP markers tightly linked to disease resistance genes. The accuracy of the developed molecular markers was validated with the purpose of selecting the KASP markers that most accurately identified the disease resistance genes. We developed KASP markers identifying the Ur-3, Ur-4, Ur-5, and Ur-11 rust resistance genes, which are the most important rust resistance genes used by breeders to develop common bean varieties with resistance to rust that are planted by farmers in North Dakota, Nebraska, and Puerto Rico. We also have developed KASP markers identifying the rust resistance genes in Andean landraces G19833 and PI 260418, that are broadly resistant to races of the bean rust pathogen. These two genes have not yet been named. We also develop a SSR (single sequence repeat) DNA marker that identifies the Ur-14 rust resistance gene present in Middle American bean Ouro Negro. In addition, we developed KASP markers tagging the Co-AC, Co-PA, and Co-BF anthracnose resistance genes present in the Andean beans Amendoim Cavalo, Paloma, and Bieja Flor landraces, respectively. Also, a KASP marker was developed that detects the presence of the Co-34 anthracnose resistance gene present in the Ouro Negro and an STS molecular marker that identified the Co-14 anthracnose resistance gene present in AND 277. Two molecular markers identifying angular leaf spot resistance genes were developed: a KASP marker linked to the Phg-3 gene present in Ouro Negro, and an STS marker linked to the Phg-1 gene present in AND 277. All the molecular markers identified will facilitate the introgression of multiple anthracnose, rust, and angular leaf spot resistance genes into new common bean varieties with resistance to pathogens. Hypothesis 2, in our project we have multiple dry and snap bean germplasm lines combining multiple genes conditioning resistance to the rust, bean common mosaic (BCMV) and bean common mosaic necrosis (BCMNV) viruses. These germplasm lines, which include pinto great northern, navy, and other market classes, have been used by breeders in different dry bean producing states to develop in collaboration with our project commercial common bean cultivars combining high yield, other desirable agronomic attributes, and resistance to pathogens. The following bean lines/cultivars have been registered in 2021 and 2022 in collaboration with common bean breeders in state universities. The black bean ND Twilight developed by Dr. Osorno of North Dakota state university. ND Twilight combines a new, still unnamed, rust resistance gene that confers resistance to all races of the rust pathogen under field conditions in North Dakota and resistance to the soybean cist nematode. The great northern White Pearl, developed by Dr. Carlos Urrea from the University of Nebraska, with the Ur-3 rust resistance gene and the I gene conferring resistance to all non-necrotic strains of the bean common mosaic virus. Dr. Urrea also developed the Wildcat pinto bean with the Ur-11 rust resistance gene and other genes conferring resistance to common bacterial blight and the I gene and the scar marker that together confer resistance to all known strains of the BCMV and BCMNV viral pathogens. The tepary bean TARS-Tep23, developed by Dr. Timothy Porch from the USDA-ARS Tropical Agriculture Research Station in Mayaguez, Puerto Rico, confers resistance to the rust pathogen. This resistance, found on a tepary bean, appears to be broader than the resistance spectrum of all known rust resistance genes in common bean. Tep 23 also exhibits wide adaptation to tropical and temperate regions and tolerance under heat and drought stress conditions. These common beans have been registered.

Accomplishments
1. Genetic resistance to control highly virulent pathogens of common bean. The development of common bean varieties with effective genetic resistance to pathogens that recurrently produce new virulent strains is difficult. These pathogens include those that cause bean rust, anthracnose, and angular leaf spot diseases. ARS scientists in Beltsville, Maryland, have recently identified new Andean and Middle American resistance genes. Using genomics and other advanced technologies, they have developed molecular markers, mostly KASP markers, that accurately detect the presence of resistance genes in common bean. KASP markers have been developed for Ur-11 (rust resistance genes of Middle American origin), Ur-G19833, and Ur-PI260418 (rust resistance genes of Andean origin), and Co-BF (anthracnose resistance genes of Andean Origin). These markers have been made available to breeders to facilitate combining multiple genes into new breeding lines and cultivars for durable pathogen resistance. Public common been breeders, particularly those in North Dakota, Nebraska, Michigan, Colorado, and Puerto Rico with active bean breeding programs, as well private breeders developing elite commercial cultivars, are direct beneficiaries of this research.

Review Publications
Urrea, C.A., Pastor Corrales, M.A., Valentini, G., Xavier, L.S., Sanchez-Betancourt, E. 2022. Registration of ‘White Pearl’ great northern common bean cultivar with upright plant architecture and high yield. Journal of Plant Registrations. 16(1):6-12. https://doi.org/10.1002/plr2.20167.
Xavier, L.F., Poletine, J.P., Goncalves-Vidigal, M.C., Valentini, G., Vidigal Filho, P.S., Pastor Corrales, M.A. 2021. Characterization of diversity in Colletotrichum lindemuthianum in Parana, Brazil, suggest breeding strategies for anthracnose resistance in common bean. European Journal of Plant Pathology. https://doi.org/10.1007/s10658-021-02295-8.
Porch, T.G., Barrera, S., Berny Mier Y Teran, J.C., Diaz-Ramirez, J., Pastor Corrales, M.A., Gepts, P., Urrea, C.A., Rosas, J.C. 2022. Release of tepary bean TARS-Tep 23 germplasm with broad abiotic stress tolerance and rust and common bacterial blight resistance. Journal of Plant Registrations. 16:109-119. https://doi.org/10.1002/plr2.20180.
Urrea, C.A., Pastor Corrales, M.A., Valentini, G., Xavier, L., Sanchez-Betancourt, E. 2021. Registration of the slow darkening pinto common bean cultivar ‘Wildcat’. Journal of Plant Registrations. 16:220-228. https://doi.org/10.1002/plr2.20198.
Kumari, H., Weebadde, C., Bandaranayake3, P., Pastor Corrales, M.A., Rajapakshe, R. 2022. Snap bean breeding for rust resistance: validation of molecular markers for the ur-11 gene introgression. Journal of Semi-Arid Tropical Agricultural Research (Journal of SAT Research). 33(1):57-66. http://doi.org/10.4038/tar.v33i1.8535.