Molecular Ecology Resources (2008) 8,1371-1374 doi: 10.1111/j.1755-0998.2008.02294.x

PERMANENT GENETIC RESOURCES Isolation and characterization of microsatellite markers for Carolina hemlock ( caroliniana)

S. A. JOSSERAND,*K. M. POTTER,tC. S. ECHT*andC. D. NELSON* *US Deparhnent ofAgriculture Forest Service, Southern Research Station, Southern Institute of Forest Genetics, 23332 Mississippi 67, Saucier, MS 39574, USA, tNorth Carolina State University, 3041 Cornwallis Road, Research Triangle Park, NC 27709, USA

Abstract We describe the isolation and characterization of 31 polymorphic di- and trinucleotide micro- satellite marker loci for Carolina hemlock ( Englem.). In addition, primer pairs for 16 loci amplified scoreable alleles in six other Euga species. In eastern North America, both Carolina hemlock and eastern hemlock ( 1L.I Carr.) populations are declining due to infestation by , Adelges tsugae.The markers described here should enhance population genetic studies of hemlocks, providing valuable information for conserving and restoring these important forest species. Keywords: conservation genetics, hemlock, SSR markers Received 5 March 2008; revision accepted 15 May 2008

Carolina hemlock is endemic to the southern Appalachian M13forward(-29) sequence, CACGACGTTGTAAAACGAC, Mountains of eastern North America. It is a wind-pollinated allowing fluorescent dye (6-FAM, VIC, NED or PET, Applied species with a restricted range confined to isolated rocky Biosystems) inco'poration in PCR when using a dye-labelled slopes and ridges (Farjon 1990). In recent years, this rare and M13forward(-29) primer. All reverse primers were tailed vulnerable species, which has a G3 global conservation on their 5' end with a PIG tail, GTITCl'T (Brownstein et al. priority rank (www.mtureserve.org/explorer/),has begun 1996), forcing nonternplated dA additions to the amplified to experience mortality due to infestation by the hemlock fragments. woolly adelgid (HWA), Adelges tsugae Annand, an exotic Synthesized primer pairs were smened for polymorphism insect that has caused a rapid decline of eastern hemlock against 23 T. caroliniana samples collected from seven typical across much of its range (McClure et al. 2003). hemlock sites distributed across the mountainous area of Microsatellite markers were developed from fresh and adjacent . In addition, a tissue DNA collected from a single Tsuga caroliniana tree (tree subset of 63 markers that cleanly amplified in T. caroliniana #247, Linville Falls, North Carolina). Enrichment, cloning and wasevaluated for use in six additional Tsuga species: eastern sequencing were performed (Genetic Identification Services) hemlock (T.canadensis), n = 31; Chinese hemlock (T.chinensis), as previously described Uosserand et al. 2006), with the excep n = 5; mountain hemlock (T.mertensiana), n = 3; northern tion that the clone libraries were enriched for AC, GA and Japanese hemlock (T. diversifolia), n = 3; southern Japanese ATG simple sequence repeats (SSR). From each library, 53 hemlock (T. sieboklii), n = 2; and western hemlock (T. heto- recombinant colonies were selected at random and their phylla), n = 4. All DNA samples were isolated from fresh cloned fragments were sequenced. Relatively long SSRs that leaf samples using DNeasy 96 Kit (QIAGEN). PCR were centrally located in the sequence were identified for and allele separation were performed as previously 135of these inserts: 48,47 and 40 from the AC, GA and ATG described (Josserand et al. 2006), with the exception that libraries, respectively. Polymerase chain reaction (PCR) a hot-start monoclonal antibody (Genecraft, GmbH) was primer pairs for 99 of these inserts were selected using used in the PCR. An internal LIZ 600 size standard was Designerpc~version 1.03 (Research Genetics) and synthe- run with each sample and alleles were sized using the sized (Integrated DNA Technologies) for further evaluation. local southern algorithm and binned with GeneMapper All forward primers were tailed on their 5' end with the 3.7 software (Applied Biosystems). Analyses of genotype frequencies, genotypic disequilibrium and exact tests for P Correspondence: C.D. Nelson, Fax: 228-832-0130; values were conducted with GenePop 3.4 (http:// E-mail: [email protected] genepop.curtin.edu.au/).

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Of the 99 primer pairs tested with T. caroliniana samples, (average = 3.4), with observed and expected heterozygosities 34 amplified consistent allelic profiles and of these, 31 were ranging from 0.043 to 1.0 (average = 0.208) and from 0.043 polymorphic (Table 1).Among the polymorphic loci, the to 0.768 (average = 0.475), respectively. Tests of Hardy- number of alleles per locus ranged from two to seven Weinberg equilibrium found that 23 loci were not in equi-

Table 1 Results for 31 microsatellite loci isolated from Tsuga camliniana and tested on a sample of 23 T. caroliniana

Number of HWE Significant Locus Source GenBank Observed alleles: range (P LD with name library Accession repeat motif Primer sequences (5'4') in size (bp) Ho HE value) TGIlocus

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Table 1 Continued

Number of HWE Significant Locus Source GenBank Observed alleles: range (P LD with name library Accession repeat motif Primer sequences (5'3') insize (bp) H, HE value) TcSIlocus

TcSI-080 GA BV726524 (cr), F: ~ccrccr~~m- 4: 224-230 0.087m 0.717 O.OOOe 087

Repeat structure and primer sequences and observed number of alleles, allele size range (base pairs, bp), heterozygosity values (observed, Ho and expected, HE),and P values for exact tests of Hardy-Weinberg equilibrium (HWE), and TcSI locus number exhibiting significant (P c0.05) genotypic disequilibrium in paired tests (LD). Loci with H, values marked with = had an excess of homozygotes, as determined by MicroChecker analysis (van 0ostGhout et al. 2004). P values marked with * are significant following sequential Bonferroni correction for P < 0.05 (Holm 1979).

Table 2 Marker transferability results for 16 Tsuga camliniam-derived markers tested with six Tslrga species of various sample sizes (n), observed number of alleles and allele size range in base pairs

T.canadensis T. chinensis T mertensiana T. diversifilia T. sieboldii T. ktemphylla Locus n =31 n=5 n=3 n=3 n = 2 n =4

-, no amplification or difficult to interpret amplification products. The following loci (GenBank Accession) were monomorphic in T. camliniana: TcSI-029 (BV726488), TcSI-052 (BV726500) and TcSI-07l (BV726517). librium (P < 0.05, Table 1).Tests for two-locus genotypic results of these three tests together are consistent with disequilibrium found 16 sigruficant locus pairs out of 435 inbreeding within geographically isolated stands (Farjon tests after sequential Bonferroni correction for P < 0.05 (Holm 1990) and do not support the presence of widesp~adlinkage 1979). Tests for null alleles, using the Dunn-Sidak correction disequilibrium or high null allele frequencies. for Monte Carlo simulations in the program Micro-Checker Microsatellite primers developed from some (van Oosterhoutet al. 2004), found 25 loci that have an excess species can be amplified in related taxa (Echt et al. 1999; of homozygotes (Table 1).Their mean estimated proportion Cha@ et al. 2004; Josserand et al. 2006). Of the 34 primer pairs of null alleles across all null estimators was 0.35 with a 95% that cleanly amplified in T. caroliniana, 16 (47%) amplified confidence interval of M.03, indicating little variation in the in the other six species (Table 2), with the greatest propor- proportion of excess homozygosity among the loci. The tion being transferable to T. chinensis (29%, 10 markers) and

Journal compilation O 2008 Blackwell Publishing Ltd No claim to original US government works 1374 PERMANENT GENETIC RESOURCES the least to T. canadensis (18%, 6 markers). Of the three References markers that were monomorphic for T. caroliniana, TcSI-029, Bentz SE, Riedel LGH, Pooler MR, Townsend AM (2002)Hybrid- TcSI-052 and TcSI-071, each successfully amplified poly- ization and self-compatibility in controlled pollinations of eastern morphic loci in at least one other Tsugs species. TcSI-029 North American and Asian hemlock (Tsuga) species. Journal of appeared polymorphic in five other species and TcSI-052 and Arboticulture, 28,200-205. TcSI-071 appeared polymorphic in one other species each Brownstein MJ,Carpten JD,Smith JR (1996) Modulation of non- (Table 2). templated nucleotide addition by Taq polymerase: primer modi- Clearly the microsatellite markers described in this fications that facilitate genotyping. BwTechniques, 20,1004-1010. paper will enhance population genetic studies of Carolina Chap6 D, Chaumeil P, Ramboer A et al. (2004)Cross-species trans- 'feiability and mapping of genomic and cDNA SSRs in pines. hemlock and other hemlock species, as well as assist inter- Theoretical and Applied Genetics, 109,12044214. and intraspecies breeding programmes (Bentz et al. 2002; Echt CS, Vendramin GG, Nelson CD, Marquardt P (1999)Mime Pooler et al. 2002) working to develop HWA resistant pop- satellite DNA as shared genetic markers among conifer species. ulations for species conservation and restoration. Canadian Journal of Forest Research, 29,345471. Farjon A (1990)Pinacme: Dmwngs and Descriptions ofthe Genera Abies, Cadrus, Pseudolnrir, Ketelemia, Nothotsuga, Ikga, Cathaya, Pd Acknowledgements stuga, Larkand Picea. Koeltz Scientific Books, Konigstein, Germany. Holm S (1979) A simple sequentially rejective multiple test pro- We thank the Southern Research Station, John Frampton and Bill cedure. Scandinavian Journal of Statistics, 6,670. Dvorak of North Carolina State University for their support of JosserandSA, Potter KM, JohnsonG, Bowen A, Frampton J, Nelsbn hemlock genetics research through Cooperative Agreement (SRS CD (2006)Isolation and characterizationof mi~~~atellitemarkers 05-CA-1133012&210) and Jim Vose (LIS Forest Service, Coweta in Fraser (Abiesfraseri).Molecular Ewlogy Notes, 6,6568. Hydrologic Laboratory). In addition, we thank Dennis Deemer McClm MS, Salom SM, Shields KS (2003)Hemlock WoollyAdelgid. (Southern Institute of Forest Genetics) for technical assistance and FHTET-200143. Forest Health Technology Enterprise Team, US the following for providing Tsuga spp. samples: Susan Bentz and Department of Agriculture, Forest Service, Morgantown, West Richard Olsen, National Arboretum, Washington, DC; Dan Moel- . ler, Hoyt Arboretum, Portland, OR; Barbara Crane, US Forest Pooler MR, Riedel LGH, Bentz SE, Townsend AM (2002)Molecular Service, Southern Region, Atlanta, GA; Valerie Hipkins, US Forest markers used to verify interspecific hybridizationbetween hem- Service, National Forest Genetics Laboratory, Placerville, CA; and lock (Tsuga)species.Journa1 of theAmmmmSociety ofHorticultura1 the staffs of Camcore and the JC Raulston Arboretum, North Science, 1227,623-627. Carolina State University, Raleigh, NC. van Oosterhout C, Hutchinson WF, Wills DPM, Shipley P (2004) h4iaxKhecker software for iden-g and cosrecting genotyping errors in microsatellite data. Molecular Ecology Notes, 4,535-538.

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