Loblolly pine SSR markers for shortleaf pine genetics

—Simple sequence repeats (SSR) are highly informative DNA-based markers widely used in population genetic and linkage mapping studies. We have been developing PCR primer pairs for amplifying SSR markers for loblolly pine (Pinus taeda L.) using loblolly pine DNA and EST sequence data as starting materials. Fifty primer pairs known to reliably amplify polymorphic markers in loblolly pine were screened for their use in shortleaf pine (P. echinata Mill.), longleaf pine (P. palustris Mill.), and slash pine (P. elliottii var. elliottii Engelm.). Thirty-four of these generated “high-quality” marker data for a small range-wide sample of shortleaf pines and 32 were polymorphic. Expected heterozygosities for the polymorphic markers averaged 0.71 and ranged from 0.40 to 0.88. A subset of the polymorphic markers should be very useful for determining identity or parentage of unknown trees while the whole set should provide excellent information on genetic diversity, gene fl ow, and population structure in shortleaf pine. 1Research Geneticist and Project Leader (CDN), Biological Science Technician (SJ), and Research Geneticist (CSE), Southern Institute of Forest Genetics, USDA Forest Service, 23332 Mississippi 67, Saucier, MS 39574; Resource Scientist (JK), Missouri Department of Conservation, Columbia, MO 65201. CDN is corresponding author: to contact call (228) 832-2747 or email at dananelson@fs.fed. us INTRODUCTION Microsatellite, or simple sequence repeat (SSR), markers have been used to characterize factors affecting pollen fl ow in shortleaf pine stands in southern Missouri (Dyer and Sork 2001), and to study local and range-wide population histories in loblolly pine (Al-Rabab’ah and Williams 2002, 2004). Although isozyme markers have proven useful in studying shortleaf pine populations (Edwards and Hamrick 1995, Huneycutt and Askew 1989, Raja and others 1997), it is desirable to develop DNA-based markers for use in detailed studies of local and/or range-wide population dynamics. Most SSR marker development work in pines to date has centered on loblolly pine (Pinus taeda L.), a close relative of shortleaf pine (P. echinata Mill.). Given these facts, we have begun developing SSR markers for use in various other southern pine species. In the current study we tested a selected set of 50 loblolly pine SSR markers on a sample of 6 shortleaf pine trees. In addition, our test included a loblolly pine control sample and two samples each of longleaf pine (P. palustris Mill.) and slash pine (P. elliottii var. elliottii Engelm.). MATERIALS AND METHODS Fifty SSR primer pairs that were developed from loblolly pine DNA and characterized as high-quality genetic markers based on their reliable and repeatable amplifi cation and detection in loblolly pine were tested in this study (Tables 1 and 2). Six shortleaf pine, two longleaf pine, two slash pine and two loblolly pine trees were used in testing the 50 primer pairs. The six shortleaf pine trees were all growing on the Harrison Experimental Forest in southeast Mississippi. Four trees were selected from four different provenances that were growing in a range-wide provenance test (Wells and Wakeley 1970). The sources included Southampton County, VA (source 455), Putnam County, GA (463), St. Helena Parish, LA (473), and Dent County, MO (485). The remaining two shortleaf pine trees were fi rst-generation parent trees growing in a clone bank. Both of these trees originated in the Ozark National Forest in Arkansas. Genomic DNA was isolated from fresh leaf samples from each tree using a DNeasytm 96 Plant Kit (Qiagen cat. no. 69181). The 50 primer pairs were screened with genomic DNA using the following PCR protocol for each 12 μl total volume reaction: 20 ng genomic DNA, 200 μM of each primer, 200 μM dNTPs, 1x Taq buffer (2.0 mM MgCl2, 10 Mm Tris-HCl, 50 mM KCl), and 0.5 U Taq DNA polymerase (Promega). The PCRs were completed using the following touchdown protocol on PTC-200 thermal cyclers (MJ Research): 2 min at 94 °C; followed by 20 cycles of 30 s at 94 °C, 30 s at X, and 30 s at 72 °C, where X = 65 °C in the fi rst cycle, decreasing by 0.5 °C every cycle thereafter; followed by 15 cycles of 30 s at 92 °C, 30 s at 55 °C, and 1 min at 72 °C; followed by a 15-min extension at 72 °C and an indefi nite hold at 4 °C. The resulting PCR products were separated on an ABI PRISM 3100 Genetic Analyser (Applied Biosystems) as recommended by the manufacturer. ABI PRISM LIZ500 was used as an internal size standard. Allele sizes (in base pairs [bp]) were determined using the local southern algorithm implemented by ABI Prism