Performing Department
(N/A)
Non Technical Summary
Using genomics technology to identify molecular markers that can be used to select for traits of interest is still not as efficient as it can be. Sequencing technology has outpaced the methods used to connect genotypes to phenotypes effectively across a wide range of species. One major time constraint in breeding programs is the bifurcation of the applied breeding program with the genetic discovery program. Using breeding relevant structured populations in an innovative way has the potential to increase the resolution of genetic mapping and decrease the time it takes to develop important genomic tools. The ability to react quickly to emerging threats in agriculture with native genetic variation being identified, selected for, and deployed is an important goal for translation genomics research.We will use advanced sequencing technology to construct a pangenome for a newly developed peanut multi parent advanced generation intercross population (MAGIC). We will assess the advantages of constructing a MAGIC population in an inbred polyploid crop that has minimal genetic diversity. We will map two very important traits in peanut; stem rot resistance and drought tolerance. We will use a modified strategy to attain higher genetic resolution; i.e. increase our ability to detect variation that is tightly linked to our trait of interest. This strategy will rely on the genetic power of the pangenome to shift the resource allocation from population advancement and replication to one year of intensive phenotyping and genetic mapping. We anticipate that the shift in resources will allow the identification of beneficial genomic tools quickly, within breeding relevant populations, that will effectively combine breeding with genetic discovery. Agronomically relevant germplasm will be able to be selected for and tested within the genetic experiment, vastly decreasing the time from discovery to crop improvement.The goal of this project is to demonstrate this approach is valuable as a new tool to more quickly develop genomic resources and improve crops more effectively to feed a growing population in our evolving environment.
Animal Health Component
0%
Research Effort Categories
Basic
25%
Applied
25%
Developmental
50%
Goals / Objectives
The major goals of the project are to demonstrate the efficiacy of developing pangenomic resources for multi parent structured populations that are directly relevant for breeding programs and to demonstrate that pangenomics in breeding populations helps acheive functional marker discovery and deployment in advanced germplasm lines. The specific objectives of the goals are as follows:1) Develop a pangenome graph for an 18-way multi parent advanced generation intercross population (MAGIC) developed in peanut using PacBio HIFI assembled genomes.2) Obtain genome wide variation from low coverage whole genome sequencing data on all the F1 hybrids that were used to construct the population across the 4 rounds of crossing.3) Analyze the variants using the pangenome graph and obtain high resolution map of cross overs during the construction of the population to eveluate the impact of rounds of intercrossing4) Obtain genome wide variation on the final MAGIC hybrid families that are a result of the 4 rounds of intercrossing to provide genotype information for functional marker discovery using the population5) Demonstrate howutilizing pangenomics increases the resolution to identify functional variation by mapping two important traits in peanut; stem rot resistance and tolerance to late season drought stress.
Project Methods
The main objectives of the project aim to develop and utilize a pangenome to map two important traits in peanut using low coverage whole genome sequencing (wgs). We have sequenced and assembled genome sequences from the 18 parent genotypes. Those assemblies will be used to construct a pangenome graph using PanPipes. All of the plants that were used during crossing will be sequenced and genome wide variantion will be assessed using the pangenome graph. Chromosomal crossing over sites will be quantified after each round of crossing from the initial round to the fourth round of crosses. The final set of hybrid families will be genotyped in the same way using low coverage wgs. The unique aspect of this is the use of low coverage sequencing to genotype a polyploid crop species. We have specially developed informatics to analyze these data effectively but it is not commonplace.There are two different traits of interest we will map using the MAGIC population; stem rot resistance and drought tolerance. Because the final set of families need to be advanced to fixation, we will map using remnant seed of the 8-way hybrids (3 rounds of crossing).For stem rot resistance, we will germinate 500 F2 seeds from a set of 8-way hybrid plants. We will take cuttings of each plant to recover seed. We will take tissue from each plant and hold for DNA extraction. Rows of a tolerant stem rot variety will be sowed per agronomic standards in a field used exclusively for stem rot evaluation. The 500 plants will be randomly transplanted in the rows, 5 to a row, and marked with flags. When the row canopy is mature, we will inoculate the flagged plants with an agar disc of sclerotium at the base of the plant and water the plants every day for 5 days to produce high humidity. The plants will be scored after sufficient disease pressure has occurred on a 1 to 5 scale for stem rot resistance. The 100 most resistance plants and the 100 most susceptible plants will be sequenced and a QTL-seq analysis will be conducted by bulking in silico to map potential resistance-linked loci using the pangenome graph. There are three unique departures from usual methods in this analysis; 1) we will use un-replicated single plants and will instead "replicate alleles" instead of discrete genotypes 2) we will sequence individuals separately and analyze the bulks in silico instead of bulking DNA and sequencing together and 3) we will use a pangenome to analyze bulk sequencing data.For drought tolerance, we will use a similar strategy in that we will germinate plants in the greenhouse, take cuttings to retain seed from each line, and transplant into the field. We will transplant 1,000 plants in 100 ft rows spaced 2 plants per foot. At about 100 days post planting, we will restrict water for 40 days by using drought shelters over the plots. The plots will be rated as a 1 to 5 visual score over the course of the drought stress. We will test two methods to map drought linked haplotypes; 1) We will use bulk analysis as described above to test the difference between drought tolerant plants and drought susceptible plants and 2) we will test drought tolerant plants versus a randomly sampled set that represents a null model.The outputs will be evaluated by the ability to successfully map field-based traits and identify strongly associated loci with high value diseae resistance and stress tolerance quickly. The results of the mapping efforts will be used to select disease resistant lines and drought tolerant lines that will be tested in replicated trials in the final year of the project. The key milestones are that we have successfully identified molecular targets for improvement and we have tested the efficacy of using genomic data to select for those targets in a genome assisted breeding program.