Location: Forage and Range Research
Project Number: 2080-21500-002-020-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Jun 1, 2023
End Date: May 31, 2025
Objective:
Determine approaches for high-density genotyping of intermediate wheatgrass and other perennial plants using high-quality genome reference sequences and low-coverage resequencing of individual plants. Use high-density genotypes to conduct genetic analyses such as genome-wide association analysis and develop genomic estimated breeding values.
Approach:
Genomic DNA samples will be extracted from individual plants from USDA breeding populations in 96-well plate format. Sequencing adapters and unique combinations of DNA sequence barcodes will be added to the ends of randomly segmented pieces of DNA from each plant using a one-step enzymatic (transposase) procedure. The resulting DNA samples, from many hundreds of plants, are pooled (multiplexed) into libraries and size selected to a range of 600 to 800 DNA base-pairs that are amenable to high-throughput parallel sequencing of 400 million to over one billion individual DNA molecules. The resulting DNA sequences are separated (demultiplexed) out based on unique barcodes that were incorporated into each plant DNA sample and then aligned to similar sequences of the corresponding genome reference sequence. Minor mismatches between the DNA sample and genome reference sequences, known as single-nucleotide polymorphisms (SNPs), are called for each plant are tablulated across thousands of plants. A nearly complete compliment of the genome reference sequence is then imputed from each plant based on patterns similarity across thousands of incomplete genome alignments from many different plants. The resulting data is used to interrogate the genome for DNA variants associated with functionally important trait variation, such as differences in plant height for seed size, among different plants and develop overall genome-wide estimates of breeding value for each plant.
