1a.Objectives (from AD-416):
To. 1)determine ploidy level and genome size of Carya species and selected geographic populations;. 2)validate the utility of selected Single Nucleotide Polymorphic DNA (SNP) markers for use across diverse Carya accessions; and. 3)design efficient and cost-effective standard assays for routine genetic profiling of NCGR-Carya accessions.
1b.Approach (from AD-416):
Diverse germplasm from the National Collection of Genetic Resources for Pecans and Hickories (NCGR-Carya) and from selected Pecan Breeding Program nurseries will be evaluated with flow cytometry to determine ploidy level and genome size. Using sequence information from a variety of sources, single nucleotide polymorphisms (SNPs) will be identified and will be qualified to ensure at least 20 clear base pairs on each side of the targeted variable base to allow unobstructed primer design. SNPs will be chosen to represent known genic regions that are well distributed across chromosomes in homologous species, as well as SNPs representing organelles. Selected sequence data will be used to generate PCR primers for validation of single nucleotide polymorphic DNA (SNPs) markers. Primers will be designed and SNPs validated using techniques of the KBiosciences competitive Allele Specific PCR assay (KASPar), performed in the AgriGenomics Lab at TAMU. SNPs will be validated for information content using a panel representing diverse species of Carya as well as geographically and phenotypically diverse pecan accessions. Validated SNPs will be configured for maximum utility within the diverse plate readers available in the AgriGenomics lab, recognizing that cost efficiency increases with increasing plate size. When the composition of the assay has been confirmed, DNA will be extracted (if necessary) from remaining NCGR accessions to be profiled and profiles determined.
This is a new project with the goal of obtaining information on the genetics of Carya (pecan/hickory) that is fundamental to future pecan improvement. Work in FY 2013 focused on the use of the well-established analytical technique known as flow cytometry to estimate genome size in diverse Carya accessions. Among the accessions to be examined by project work are specific pecan types whose chromosome number was possibly doubled (tetraploids) by use of experimental techniques. Confirmation of polyploidy (more than the normal genetic complement) raises the possibility of using such accessions in crosses with known tetraploid species of Carya. As work by this project continues, the generation of new information on ploidy level and genome size will likely prove useful in the characterization of diversity in native populations as well as in the development of improved rootstock materials for use by the pecan industry.