066 Development of Simple Sequence Repeat DNA Markers for Use in Pecan Genetic Studies

Abstract

A microsatellite library has been developed from `Halbert', a native pecan selection from Coleman County, Texas, using methods developed at the Texas A&M Univ. Crop Biotechnology Center. A total of 6144 DNA fragment clones were archived in 384 well plates for screening. Four-hundred-thirty-nine clones were positive after Southern hybridization using di- and tri-nucleotide repeats as probes. One-hundred-twenty-five positive clones were sequenced on an ABI 377 automated DNA sequencer. Of these, 24 repeats had enough sequences at the two ends to design primers. Primers were designed using Primer Express software, and were synthesized by Genosys, USA. The simple sequence repeats (SSRs) chosen for primer analysis include di- (CA and GA) and tri-nucleotide repeats (CTT, GAA and GAT). The SSRs were amplified under high stringency conditions with temperatures based on length and GC content. Reproducibility was verified using `Halbert' DNA isolated from different inventories. Of the 24 primer pairs tested, 20 successfully amplified microsatellites from `Halbert'. DNA was isolated from 48 pecan and hickory accessions selected to strategically represent the genetic diversity of the NCGR Carya collections (a core collection). The accessions included parent-progeny combinations, individuals from geographically distant native populations, species, and interspecific hybrids. The 20 SSR primers that produced good amplification products in `Halbert' were used to evaluate the collection, with 11 revealing multiple sizes of the repeat. The number of bands amplified with different primer combinations ranged from 4 to 32 in the 48 genotypes tested. We used RFLPscan software to aid in gel scoring (sizing amplified fragments, and comparing amplification profiles), and NTSYSpc software to evaluate genetic similarities. Evaluation of the data confirms the utility of the primers in delimiting known relationships.