Project Details:

Title:
Characterization and enhancement of soybean genetic resources for soilborne disease resistance

Parent Project: Characterization and enhancement of soybean genetic resources for soilborne disease resistance
Checkoff Organization:North Central Soybean Research Program
Categories:Breeding & genetics, Soybean diseases
Organization Project Code:CON55573
Project Year:2016
Lead Principal Investigator:James Kurle (University of Minnesota)
Co-Principal Investigators:
Jianxin Ma (Purdue University)
Katy M Rainey (Purdue University)
James Orf (University of Minnesota)
Nevin Young (University of Minnesota)
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Keywords: Disease Resistance, Fusarium graminearum, Glycine max, Partial resistance, Phytophthora root and stem rot, Phytophthora sojae, Pythium irregulare, Pythium ultimum, Rps gene, Soybean

Contributing Organizations

Funding Institutions

Information and Results

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Project Summary

The research described in this proposal is the second year of a systematic approach to identification and introgression of both partial and race specific resistance into cultivars adapted to the North Central region particularly very early maturity group soybean cultivars. It is coordinated with and complements the research conducted by a USB-funded soybean disease project entitled “Genes and Markers for Resistance to Phytophthora sojae, Pythium spp., and Fusarium graminearum in Soybean”, a research team led by Dr., Anne Dorrance is working in Ohio, Missouri, Virginia, and Iowa. This collaboration expands the number of options available for managing these diseases throughout the North Central Region by combining unique sources of resistance and pathogen isolates and the strengths and expertise of the our two research groups from Minnesota and Purdue with those of Missouri, Virginia, and Iowa.
Soilborne diseases caused by various oomycete and fungal pathogens have been a major limitation to soybean production. Each year the U.S. soybean crop is attacked by several pathogens that cause estimated annual losses valued at about 3.6 billion dollars (Wrather and Koenning, 2006). In the North Control Regions the root pathogens, Phytophthora sojae, Pythium ultimum, P. irregulare, and Fusarium graminearum are major culprits. Phytophthora stem and root rot caused by P. sojae was consistently ranked as the second-leading soybean disease during the past decade. P. ultimum and P. irregulare are frequently associated with seed and seedling diseases of soybean and corn and commonly isolated early in the season when the soils are moist and cool. F. graminearum causes seed decay and damping-off of soybean but is of particular importance since it is also the cause of "scab" in wheat and stalk and ear rot of corn. The severity of these diseases is exacerbated by their persistence as soilborne inoculum so that diseases caused by these pathogens are not effectively controlled by crop rotation and only to a limited extent by seed treatments. Development of disease-resistant soybean cultivars remains the most practical solution for limiting yield losses to these pathogens.

Project Objectives

Objective 1: Evaluation of soybean germplasm for resistance or partial resistance to P. sojae, P. irregulare, P. ultimum, and F. graminearum
280 soybean lines ancestral to soybean cultivars developed in Minnesota will be evaluated for resistance or partial resistance to four pathogens in greenhouses and growth chambers, using standardized growing conditions and inoculation techniques to determine varietal reaction to disease pressure.
Objective 2: Identification of QTLs underlying resistance to P. sojae, F. graminearum, P. irregulare, and P. ultimum by association mapping
Genotypic data for the 280 ancestral lines will be analyzed for significant marker-trait association with phenotypic resistance to map markers for resistance or partial resistance.
Objective 3: Fine mapping, isolation, and functional verification of two uncharacterized Rps genes conferring resistance to P. sojae .
PI567139B, possesses excellent resistance to the predominant pathotypes of P. sojae, including those able to defeat the main sources of resistance in contemporary soybean lines. A study of the resistance in PI567139B identified two potentially new Rps genes, RpsUN1 and RpsUN2. Location of genes will be pinpointed by fine mapping.
Objective 4: Development of highly adapted soybean cultivars or experimental lines with major resistance QTLs and Rps genes by marker-assisted selection
Locally adapted cultivars will be crossed with lines with the two Rps genes and multiple resistance QTLs. Major resistance genes will be identified by marker-assisted selection. Cultivars will also be used in the breeding programs at Purdue and Minnesota for enhancement of resistance in soybean cultivars or breeding lines.

Project Deliverables

Screened 280 ancestral lines for resistance or partial resistance to P. ultimum pv ultimum, P. irregulare (Duplicate runs of rice inoculum assay) and F. graminearum (Duplicate runs - rice inoculum and rolled towel assays). Association mapping of phenotypic data with genotypic detected no significant marker trait associations for partial resistance to F. graminearum. Population size and/or marker density combined with environmental error in measurement of traits resulted in insufficient power to detect significant marker-trait associations
Measures of resistance included reduced root necrosis score and maintenance of relative root weight and relative root length when inoculated plants are compared to uninoculated. Association mapping detected significant marker associated with reduced root necrosis, relative root weight and root length at a single position and multiple markers for maintenance of relative root dry weight.

As alternative approach to association mapping, six crosses were made between diverse sources, resistant and susceptible lines, for use in biparental mapping. Growout of seed from crosses (F1) will be conducted in South American location to make seed available for 2016 growing season.

Pinpointed location of RpsUN1 to a 6.5-cM region between two SSR markers, Satt159 and 03_0250, on chromosome 3 and RpsUN2 to a 3.0-cM region between 16_1275 and Sat_144 on chromosome 16. Haplotype analysis comparing this region between PI 567139B and other genotyped soybean accessions has identified RpsUN1 and RpsUN2 as unique and are present only in PI567139B.
Introgressed RpsUN1 and RpsUN2 into elite breeding lines.

Progress of Work

Update:
Objective 1:

Multiple methods of inoculation were tested to validate the methodology to be used to conduct screening for resistance to seed, seedling, and root rot caused by F. graminearum and P. ultimum var.ultimum. Three replicates of 280 lines were also tested for F. graminearum response in growth pouches. Inoculated plants were assigned a root necrosis score, and the ratio of uninoculated to inoculated root length and root dry weight calculated. Correlation analysis was performed to compare results in rice inoculated seedling screens and growth pouch assays. Poor correlations were observed between the seedling and pouch assays for the measured traits. Growth pouch measurements lack sensitivity effects of F. graminearum inoculation. The rolled towel assay and seedling assay were selected as the most reliable screening methods Phenotyping is underway for three replicates of seedling P. irregulare assay.
We completed six crosses of lines with divergent resistance characteristics, resistant and susceptible, for biparental mapping of resistance and partial resistance to P. sojae. Seed was increased this winter in the Chilean nursery.

To correct possible genotyping errors 40 lines are being regenotyped with the 6k SNP assay. Once compiled, association mapping will be rerun with all phenotypic data which will improve the power of the tests performed for all traits.


Objective 2:

Association mapping on results of phenotyping for resistance to F. graminearum indicates location of markers for partial resistance to F. graminearum, root necrosis (1 marker identified on chromosome 8) and root rot resistance (10 markers identified which may be good targets for genomic selection) in 280 ancestral lines. Association mapping analysis has begun for 3 replicates of greenhouse P. ultimum var ultimum assays. Inoculation severely reduced emergence of seedlings. 35% of the 280 lines assayed had no seedlings emerge in any of the three replications. Only three soybean varieties had greater than an average of 50% emergence. Lack of emergence lessened our ability to assay relative root tolerance, but relative emergence measurements are being mapped in the population and the phenotyping will be repeated at a lower inoculum rate. Two putative loci were identified on chromosomes 2 and 20.

Objective 3:

Pinpointed location of RpsUN1 and RpsUN2. RpsUN1 was mapped to a 6.5-cM region between two SSR markers, Satt159 and 03_0250, on chromosome 3, suggesting the RpsUN1 was likely to be a novel allele at the Rps1 locus, and RpsUN2 was mapped to a 3.0-cM region between 16_1275 and Sat_144 on chromosome 16, which was also likely to be a novel Rps gene.

Five genes were predicted in the RpsUN1 mapping region as annotated from reference genome (Glyma.Wm82.a2 assembly). Four genes were predicted in the RpsUN2 mapping region as annotated from reference genome (Glyma.Wm82.a2 assembly). All of these genes encode NB-LRR type of genes.
Glyma.03g034600 in the RpsUN1 region showed increased expression after inoculation with P. sojae race 17 while three genes in the RpsUN2 region, Glyma.16g215200, Glyma.16g215000 and Glyma.16g214900, showed increased expression after inoculation with P. sojae race 25. RpsUN1 and RpsUN2 are present only in PI567139B.

Objective 4:
Introgressed RpsUN1 and RpsUN2 into prebreeding lines and elite breeding lines.

Update:
Objective 1:
To correct possible genotyping errors 40 lines were regenotyped with the 6k SNP assay.

A Pythium ultimum var. ultimum seed rot assay was developed and tested twice in two reps on a 13-line subset of the Ancestral Lines in order to screen lines for resistance or susceptibility to P. ultimum. The assay uses both P. ultimum inoculated plates and non-inoculated plates (as controls). Seven days after plating, the seeds are screened for root length, root weight, total weight (cotyledon and root), and visual rated for root rot severity using a 1 to 12 visual scale. The short time frame between plating and scoring could allow many lines to be quickly screened. The results of the two seed rot assay trials are being compared for repeatability. The results of the seed rot assay are being compared with results in the seedling rot assay conducted in the previous reporting period.

Objective 2:
The preliminary trial results of the seed rot screening assay for resistance to Pythium ultimum var. ultimum were analyzed for distribution of results obtained from different rating variables and for correlation with results obtained from earlier test of seedling rot assay.

Objective 3:
Progress report - Purdue
1. Refined location of RpsUN1 and RpsUN2 previously mapped to a 6.5-cM region between two SSR markers, Satt159 and 03_0250, on chromosome 3. RpsUN1 was mapped to a 6.5-cM region between two SSR markers, Satt159 and 03_0250, on chromosome 3Pinpointed location of RpsUN1 and RpsUN2. RpsUN1 was mapped to a 151 kb region between Markers BARCSOYSSR_03_0233 and BARCSOYSSR_03_0246 on chromosome 03 that harbors five genes including three NBS-LRR like gene models according the Williams 82 reference genome. RpsUN2 was mapped to a 36 kb region between two markers CAPS3 and CAPS4 on chromosome 16 that harbors four gene2, all of which are NBS-LRR like gene models according the Williams 82 reference genome. Based on RNA-sequencing data, it is suggested that Glyma.03g034400 and Glyma.16g214900 may be the candidate genes for RpsUN1 and RpsUN2. A manuscript has been accepted for publication in Theoretical and Applied Genetics.
2. Developed hairy root transformation system and obtained a special Phytophthora sojae strain with a molecular (Green Florescence Protein) tag that can be used to visualize the process of Phytophthora sojae infection. This system is being used for validation of the two candidate genes, in addition to the whole plant transformation system for functional validation.
Objective 4:

Progress - Purdue
At Purdue continued the backcrossing process assisted with marker assisted selection towards the introgression of RpsUN1 and RpsUN2 into four Purdue elite cultivars. The backcrossing progeny seeds have just been harvested and will be planted in greenhouse shortly.

Progress - Minnesota
At Minnesota Crosses were conducted during the summer of 2016 between northern adapted germplasm (MG00 to MGI) and lines containing the RpsUN1 and RpsUN2 resistance genes identified at Purdue University. Seven northern adapted lines were crossed with a line containing RpsUN1, and ten northern adapted lines were crossed with a line containing RpsUN2. By placing these resistance genes into multiple parents from MG00 to MGI, these resistance genes can be readily utilized in the breeding of elite cultivars. The progeny seeds have just been harvested.
Progeny seed from six crosses of lines with divergent resistance characteristics, resistant and susceptible, to be used for biparental mapping of resistance and partial resistance to P. sojae were increased and harvested.

Final Project Results

Update:

View uploaded report Word file

Minnesota
During this project year tests of multiple methods of inoculation with F. graminearum were conducted to validate the methodology, effectiveness, and consistency, to be used to conduct screening for resistance to seed, seedling, and root rot caused by F. graminearum. Based on these tests, the rolled towel assay and rice inoculum method were selected as the most reliable screening methods for assessing root rot and root development.

Utilizing these methods, rolled towel assay and rice inoculum methods, we completed phenotyping and association mapping of first and second runs of screening in 280 ancestral lines for resistance or partial resistance to P. sojae and F. graminearum.

Association mapping analysis was conducted on results obtained in P. sojae and F. graminearum phenotyping indicating the location of markers for partial resistance to F. graminearum. In 280 ancestral lines two categories of marker were identified that indicated resistance to F. graminearum, one marker identified on chromosome 8 indicating resistance to root necrosis and 10 markers associated with reduced susceptibility for reduction in root mass. These markers may also be good targets for genomic selection for root rot resistance. Association mapping of phenotypic data for P.sojae partial resistance did not detect significant marker train associations for resistance to P. sojae although phenotyping indicated that 280 lines differed widely in response to P.sojae inoculation. To examine this phenomenon more closely we have elected to examine the results of biparental crosses between cultivars exhibiting a wide divergence of responses to P.sojae inoculation. Completed six crosses of lines with divergent resistance characteristics, resistant and susceptible, for use in biparental mapping of resistance and partial resistance to P. sojae.

First run consisting of one rep of evaluation of 280 lines for resistance or partial resistance to P. ultimum is underway.


Purdue:
Generate a higher resolution genetic and physical map and finer mapping of these two genes, RpsUN1 and RpsUN2. RpsUN1 gene has been narrowed from an ~1,300-kb region to an ~150.8-kb region. RpsUN2 gene was narrowed from an ~ 64-kb region down to ~35.6-kb region.

Identify possible candidate genes in the interval region by quantitative real-time PCR gene expression and further analysis of candidate genes in the RpsUN1 and RpsUN2 regions. Expression data demonstrate that only a single gene Glyma.03g034600 in the RpsUN1 region showed enhanced expression pattern after inoculation with P. sojae race 17. By contrast, three genes Glyma.16g215200, Glyma.16g215000 and Glyma.16g214900, in the RpsUN2 region showed enhanced pattern in response to P. sojae race 25. These are candidates for further validation.

Crossed and backcrossed RpsUN1 and RpsUN2 donor lines with four Purdue elite lines, and got the three backcrossing introgression lines with RpsUN1 and RpsUN2 genes, respectively.

Benefit to Soybean Farmers

The goal of this research is to capitalize on molecular methods for identifying novel resistance to seed and seedling rot pathogens. The potential impact of this research would add to the options available to soybean farmers for managing these diseases. The potential benefit for American soybean farmers would be an increase in yield of as much as 10% based on estimates of yield lost to P. sojae and the complex of seedling diseases that include Fusarium and Pythium species.
The research is necessitated by changing patterns in the occurrence of diseases common in the corn-soybean cropping system. Identification of additional highly effective Rps genes is a necessity because of the appearance of new and complex P. sojae pathotypes capable of overcoming currently available resistance genes. Resistance to F. graminearum is necessitated by the increased prevalence of this fungus as a pathogen of soybeans grown in rotation with corn. Resistance to Pythium species is increasingly important as the prevalence of Pythium species that infect both corn and soybean become more common with planting of sequences consisting of only these two crops. Because resistance would be available as a varietal trait, the benefit would be available at the lowest cost. In addition, effective control of seed and seedling rots would protect the value of seed, currently the largest single expense in soybean production. In combination with seed treatment fungicides and management practices, varietal resistance would enhance the ability of these tactics to limit losses caused by seed, seedling, and root rots.

Performance Metrics

The success of this project would be indicated by 1) identification of markers for partial resistance to P. sojae, F. graminearum, P. ultimum, and P. irregulare; 2) incorporation of traits associated with these markers into breeding lines, 3) successful adoption and use of the resistance genes by both public and private breeders for control of the root rots caused by P. sojae, F. graminearum, P. ultimum, and P. irregulare and 4) adoption of RpsUN1 and RpsUN2 as effective genes for protection against P. sojae in adapted soybean lines grown throughout the North Central Region.

Project Years