Update:
Objective 1: Advanced generation breeding lines, selected from the results of previous year’s breeding efforts have been selected. These lines were selected based on their yield potential and response under IDC inducing conditions. These lines were planted in the 2016 growing season in the three target states: Minnesota, Nebraska, and North Dakota
Objective 2: Performance data was collected from multi-location, replicated IDC field trials performed on IDC inducing soils. SNP data was collected from a subset of lines using a 1.5K soybean SNP chip, and SNPs generated by genotype-by-sequencing. Multiple QTLwere identified by genome-wide association studies (GWAS) approaches. QTL were repeatedly located on chromosomes Gm03, Gm05, Gm07, Gm16, Gm17, Gm18, and Gm19. The following candidate genes were identified. Glyma.03G231200 (R^2=17%) encodes an enzymes that produces nicotianamine, a molecule that chelates iron and is involved in the transfer of iron via the phloem. Glyma.07G067700 (R^2=9%) encodes an enzyme that reduces iron to a state that can be taken up by the root system. Glyma.19G140000 (R^2=16%) encodes a protein that stores iron in vacuoles under iron deficiency conditions. Seven SNP markers located within the QTLs were developed to screen plants for IDC tolerance. These are available for testing from LGC Genomics (http://www.lgcgroup.com/services/genotyping/#.WVErfev1CJA)
Fiskeby III is a promising accession for the discovery of loci that confer unique tolerance to traits such as IDC. The Fiskeby III x Mandarin Ottawa RIL population was scored for IDC tolerance over several years and a single quantitative trait locus (QTL) on chromosome 5 was found to account for approximately 20% of the variation in the population. This indicated that there may be an important gene at this locus in Fiskeby III that contributes to enhanced IDC tolerance. This Gm05 QTL was cross-validated with association mapping results based on a large population of lines from the UMN soybean breeding program. This further suggests that the Fiskeby III gene has the potential to improve IDC tolerance by introducing the gene into elite varieties using standard breeding methods. With this goal in mind, the University of Minnesota research team identified a subset of near isogenic lines (NILs) for this trait. NILs are the genetic gold-standard for evaluating the effect of a single locus on a trait. Field evaluations in 2015 and 2016 in Danvers, MN validated the large IDC effect of the chromosome 5 QTL. Furthermore, plants heterozygous at the QTL were planted and genotyped in St. Paul, MN in 2015 and 2016 to recover individuals with novel recombinations that can narrow the QTL and map the gene responsible for this IDC tolerance. The new recombinants identified in 2015 were tested in Danvers in 2016; this effort reduced the QTL interval to 450 kb in size. The year (2017), the newest recombinants are currently under evaluation with the goal of reducing the QTL interval to 150 kb. Whole-genome resequencing on the parent lines, Fiskeby III x Mandarin Ottawa, identified a full set of DNA polymorphisms between the parents, and further analysis should reduce the QTL interval further that will enable the identification of gene responsible for IDC tolerance. With a marker very close to the gene, selection based on the marker will be highly accurate while if the responsible gene is discovered, molecular breeding will be error-proof.
Objective 3: Two near isogenic lines that are 98% genetically identical but differ in their iron response were studied. The major difference between the lines is ~120kb introgression that converted Clark, an iron efficient line, to an iron inefficient line (termed Isoclark). Because of their differential response to iron status, these are critical lines to better understand iron metabolism. Plants were grown hydroponically in either iron sufficient conditions, iron stress conditions for two days or iron stress conditions for 10 days. Plants were grown simultaneously to control for the age of the plants. RNA-seq was used to compare gene activity in Clark and Isoclark under the different treatments. Under this proposal, we are now characterizing the function of 19 genes that responded to iron stress in either Clark or Isoclark using Virus Induced Gene Silencing (VIGS). Using VIGS we can temporarily shut down the activity of specific genes and see how that change alters the ability of the plants to respond to iron stress. Ten genes were chosen because they responded the most to either short or long term iron stress, while another nine genes were selected because they responded to iron stress and showed sequence similarity to iron stress genes from other species. VIGS constructs have been constructed for all 19 genes and silenced plants have been preliminarily phenotyped in soil. These lines have undergone phenotyping under iron stress conditions and the data is currently being analyzed.