Research Project: Enhancing Resistance to Biotic and Abiotic Stresses in Alfalfa

Location: Plant Germplasm Introduction and Testing Research

2021 Annual Report

Objectives
Objective 1: Identify DNA markers associated with resistance to soil borne diseases in alfalfa to clearly define the genetic basis of resistance to disease and accelerate breeding programs. (NP215 2A) Objective 2: Identify alfalfa DNA markers and germplasm associated with drought and salt tolerance to clearly define the genetic basis of resistance to these stressors and accelerate breeding programs. (NP215 2A).

Approach
Approach 1: Marker-assisted selection for disease resistance will increase selection accuracy and reduce selection cycles in alfalfa breeding programs. First, genome-wide association mapping will be used to identify loci associated with VW resistance. Then, genetic regions responsible for VW resistance will be sequenced and compared among different genotypes using haplotyping and comparative genomics approaches. Significant SNP markers linked to VW resistance loci will be validated in various breeding populations provided by collaborators. High throughput platforms such as Kompetitive Allele Specific PCR (KASP) (www.lgcgenomics.com) or Taqman (www.thermofisher.com) assays will be used to test the cosegregation of marker loci and disease resistance scores. Flanking sequences for the significant SNP markers will be used for designing specific primers for array-based genotyping platforms (KASP or Taqman). Multiplex primer combinations will be used for evaluating the resistance locus or candidate gene, and all markers will be scored in a given genotype. Single markers with two character states will be tested for significant phenotypic differences between genotype groups by the t test for each trait, and Mann–Whitney U test for chip quality. Marker combinations will be analyzed using analysis of variance (ANOVA) for each trait, and Kruskal–Wallis test for chip quality. Statistical analyses will use SAS software (SAS Institute Inc. 2011, SAS OnlineDoc 9.3, Cary, NC, USA). Approach 2: Breeding for abiotic stress tolerance is challenged by genotype x environment interactions (G x E). Genomic selection provides greater gain and increased selection accuracy than conventional breeding. To develop a genome-wide marker platform and statistical models for genomic selection of drought tolerant alfalfa. BC1 populations have been developed and will be screened for drought tolerance. Selected plants will be randomly intermated in the greenhouse in order to generate an elite base population. The population will used for associated mapping and genomic selection for alleles that affect drought tolerance, salt tolerance, forage quality and other economical traits. We will test statistic models by using the majority of the training population to create a prediction model, which is then used to predict a Genomic Estimated Breeding Value (GEBV) for each of the remaining individuals in the training population based only on their genotype data. Once validated, the model can then be applied to a breeding population to calculate GEBVs of each individual based only on a plant’s genotype information. Such GEBVs represent the overall predicted value of an individual as a potential parent for crossing.

Progress Report
In support of Objective 1, research continued on determining the function of genes associated with resistance to Verticillium wilt (VW) of alfalfa, which is a devastating disease that reduces forage yields by up to 50% in the Northern United States and Canada. The best method for controlling the disease is to develop and grow resistant varieties. To understand the genetic basis of resistance in alfalfa to Verticillium wilt, an ARS scientist in Prosser, Washington, used DNA markers to identify genes associated in disease resistance. Two genes possibly associated with VW resistance have been identified and confirmed using mutants of a relative of alfalfa, M. truncatula, which is widely used as a model plant for genetic studies. We found that one of the resistance genes identified may function better than the other in conferring disease resistance. Three DNA markers derived from the better resistance gene have been developed and transferred to commercial alfalfa breeders and pathologists for rapid and reliable detection of resistance to VW in alfalfa. The results were reported this year at the American Societies of Agronomy, Crop Science and Soil Science Annual Virtual Meeting and will be published in a peer-reviewed journal. In support of Sub-objective 2A, progress was made on developing molecular markers associated with drought resistance. In the Western United States, the great majority of alfalfa is produced with supplemental irrigation water, which represents a large part of the total costs for a producer. An ARS scientist in Prosser, Washington, in collaboration with scientists from alfalfa seed companies and universities, tested 200 alfalfa breeding lines in the field for drought resistance. We used genome-wide association studies (GWAS) and genomic selection to improve the breeding processes. The genotypes of the alfalfa breeding lines were determined using more than 10,000 DNA markers. Datasets were analyzed using new methods so each line could be selected based on genotypes associated with drought resistance, an approach generally referred to as “genomic selection.” This genomic selection approach allowed us to identify an ideal group of alfalfa materials (accessions) with high yield under drought conditions that can be used for developing improved varieties. This is the first report in which this new method of data analysis was shown to improve the accuracy of genomic selection and this approach can be broadly applied to a wide range of other crops and traits. In support of Sub-objective 2B, progress was made on developing alfalfa populations with improved salt tolerance. Many agricultural lands in the Western United States have high soil salt concentrations that are detrimental to alfalfa growth and production. Developing alfalfa varieties with salt tolerance is critical for sustainable production under increasing soil salinity. An ARS scientist in Prosser, Washington, in collaboration with another ARS scientist in Logan, Utah, evaluated traits related to salt tolerance in alfalfa. They identified differences in DNA sequences called “single nucleotide polymorphisms” (SNPs) between salt tolerant and salt susceptible alfalfa plants and identified 27 SNP markers associated with salt tolerance. SNP markers were analyzed using eight different genomic selection models to identify the best model for selecting for salt tolerance. The best model (support vector machine and random forest) was 80% accurate at predicting alfalfa yield based on SNP markers associated with salt tolerance. The SNP markers and identification of the best model to use for predicting salt tolerance based on SNP markers are useful for more efficient development of new alfalfa varieties with improved salt tolerance.

Accomplishments
1. Development of DNA markers for Verticillium wilt resistance in alfalfa. Verticillium wilt is an alfalfa disease that reduces forage yields by up to 50 percent. Current breeding strategies primarily rely on field or greenhouse screening to identify disease resistant plants, which is a time-consuming process requiring specific conditions to produce reliable results. An ARS scientist in Prosser, Washington, developed three DNA markers that accurately identify alfalfa plants that are resistant to Verticillium wilt. The DNA markers are being used by a major commercial producer of alfalfa varieties to accelerate breeding efforts to develop new varieties with enhanced resistance to Verticillium wilt.

2. Development of DNA markers for drought tolerance in alfalfa. In the Western United States, the great majority of alfalfa is produced with supplemental irrigation water, which represents a large part of the total costs for a producer. An ARS scientist in Prosser, Washington, in collaboration with scientists from alfalfa seed companies and universities, generated more than 10,000 DNA markers and analyzed variation in the field for drought tolerance across 200 alfalfa breeding lines. The scientist used the markers and tested different methods of data analysis and was able to predict drought tolerance with 90% accuracy based on the presence of DNA markers. The DNA markers and suggested methods for data analysis can be used by alfalfa breeding programs to accelerate the development of new varieties with enhanced drought tolerance.

Review Publications
Zhang, F., Kang, J., Long, R., Yu, L., Sun, Y., Wang, Z., Zhao, Z., Zhang, T., Yang, Q. 2020. Construction of high-density genetic linkage map and mapping quantitative trait loci (QTL) for flowering time in autotetraploid alfalfa (Medicago sativa L.) using genotyping by sequencing. The Plant Genome. 13(3). Article e20045. https://doi.org/10.1002/tpg2.20045.
Lin, S., Medina, C., Norberg, S., Combs, D., Wang, G., Shewmaker, G., Fransen, S., Llewellyn, D., Yu, L. 2021. Genome-wide association studies identifying multiple loci associated with alfalfa forage quality. Journal of Genetics and Genomics. 12. Article 648192. https://doi.org/10.3389/fpls.2021.648192.
He, F., Long, R., Zhang, T., Zhang, F., Wang, Z., Yang, X., Jiang, X., Yang, C., Zhi, X., Li, M., Yu, L., Kang, J., Yang, Q. 2020. Quantitative trait locus mapping of yield and plant height in autotetraploid alfalfa (Medicago sativa L.). The Crop Journal. 8(5):812-818. https://doi.org/10.1016/j.cj.2020.05.003.
Tang, Z., Parajuli, A., Chen, C., Hu, Y., Revolinski, S., Medina, C., Lin, S., Zhang, Z., Yu, L. 2021. Validation of UAV-based alfalfa biomass predictability using photogrammetry with fully automatic plot segmentation. Scientific Reports. 11(1). Article 3336. https://doi.org/10.1038/s41598-021-82797-x.
Chandel, A., Khot, L., Yu, L. 2021. Alfalfa (Medicago sativa L.) crop vigor and yield characterization using high resolution aerial multispectral and thermal infrared imaging technique. Computers and Electronics in Agriculture. 182. Article 105999. https://doi.org/10.1016/j.compag.2021.105999.
Yu, L., Medina, C., Peel, M. 2021. Genetic and genomic assessments for improving drought resilience in alfalfa. In: Yu LX., Kole C., editors. The Alfalfa Genome. Compendium of Plant Genomes. Springer. Cham, Switzerland. p.235-253. https://doi.org/10.1007/978-3-030-74466-3_14.
Samac, D.A., Yu, L., Missaoui, A.M. 2021. Identification and characterization of disease resistance genes in alfalfa and Medicago truncatula for breeding improved cultivars. In: Yu,X. and Kole, C., editors. The Alfalfa Genome, Compendium of Plant Genomics. Springer, Cham:Switzerland. p. 211-233. https://doi.org/10.1007/978-3-030-74466-3_13.
Medina, C., Yu, L. 2021. Developing SNPs and strategies for genomic analysis in alfalfa. In: Yu LX., Kole C., editors. The Alfalfa Genome. Compendium of Plant Genomes. Springer. Cham, Switzerland. pp.159-175. https://doi.org/10.1007/978-3-030-74466-3_10.
Parajuli, A., Yu, L., Peel, M., See, D.R., Wager, S., Norberg, S., Zhang, Z. 2021. Self-incompatibility, inbreeding depression, and potential to develop inbred lines in alfalfa. In: Yu LX., Kole C., editors. The Alfalfa Genome. Compendium of Plant Genomes. Springer. Cham, Switzerland. p.255-269. https://doi.org/10.1007/978-3-030-74466-3_15.