Location: Plant Science Research
Project Number: 6070-22000-019-013-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Nov 1, 2022
End Date: Oct 30, 2027
The objective of this research is to develop genomic resources that can be applied for improvement of winter wheat germplasm adapted to the eastern United States. Research done in the parent project relies heavily on a reference quality genome sequence, however relevant winter wheat germplasm is not currently represented in genome assembly projects. Specific objectives include: (1) Obtain and assemble long read DNA sequences of eastern winter wheat cultivars at low coverage, (2) Use annotation software to identify the gene content of an eastern winter wheat cultivar that will can serve as a relevant reference assembly, (3) Utilize these new genomic resources to identify gene variants underlying traits of importance in soft winter wheat, (4) Incorporate the new genomic resources into the development of improved high-throughput genotyping approaches used for selection in breeding populations.
Approach obj 1: Plants of selected wheat varieties will be grown in a controlled environment chamber. Tissue for DNA isolation and library preparation will be harvested multiple times from a single plant of each cultivar that are maintained as a pure seed source for downstream analyses. Library sequencing will be done by collaborators using long read sequencing technology to obtain a genome coverage in the range of 4X to 8X. Sequence assembling will be done by the post-doc using high performance computing resources available through NCSU and the USDA. Approach obj 2: An existing preliminary assembly of wheat cultivar AGS2000 has been developed that requires gene annotation. This work will incorporate existing RNA sequencing data and annotation will be done using BRAKER2 or another suitable package. Alternatively, gene projections from ‘Chinese Spring’ RefSeq v2.1 will be done following published methods. Approach Obj 3: Contigs developed from the long read data produced in this project will be queried with sequences from regions of mapped genes/QTL to determine gene content in regions of interest and to identify variants in relevant wheat germplasm. These data will be used for identification of candidate genes and causal polymorphisms. Approach Obj 4: Sequences flanking variants between cultivars will be used to develop new markers for either targeted amplicon sequencing or Kompetitive allele specific polymerase chain reaction genotyping approaches that can be cost-effectively applied to large numbers of breeding samples.