Developing microsatellite DNA markers in pecan

A microsatellite-enriched library was developed from 'Halbert', a native pecan [Carya illinoinensis (Wangenh.) K. Koch] selection from Coleman County, Texas. A genomic library enriched for simple sequence repeats (SSR) containing 6144 clones was archived in 384 well plates for screening. In total, 439 clones were identified after Southern hybridization using di- and tri-nucleotide repeats as probes. In total, 125 positive clones were sequenced and primers were designed for 24 repeats. The SSR markers chosen for analysis include di- (CT and GA) and tri-nucleotide repeats (CTT, GAA and GAT). Of the 24 primer pairs tested, 19 successfully amplified microsatellites from 'Halbert'. DNA was isolated from 48 pecan and hickory accessions selected to strategically represent the genetic diversity of the National Clonal Germplasm Repository (NCGR) Carya collections. The 19 SSR primers that produced good amplification products in 'Halbert' were used to evaluate the collection, with 11 revealing polymorphism. The number of fragments amplified with different primer combinations ranged from 4 to 32 in the 48 genotypes tested. Evaluation of the data confirms the utility of the microsatellites in delimiting known relationships.