Characterization of Microsatellite Loci in Festuca Gautieri

American Journal of Botany: e360–e362. 2011.

AJB Primer Notes & Protocols in the Plant Sciences

C HARACTERIZATION OF MICROSATELLITE LOCI IN F ESTUCA GAUTIERI (POACEAE) AND TRANSFERABILITY TO F. ESKIA AND F. × PICOEUROPEANA 1

Jos é Gabriel Segarra-Moragues2,4 and Pilar Catal á n3

2 Centro de Investigaciones sobre Desertiﬁ caci ó n (CIDE, CSIC-UV-GV), Carretera de Moncada-N á quera Km 4.5, E-46113 Moncada, Valencia, Spain; and 3 Departamento de Agricultura y Econom í a Agraria, Escuela Polit é cnica Superior de Huesca, Universidad de Zaragoza, C/ Carretera de Cuarte s/n, E-22071 Huesca, Spain

• Premise of the study: Enriched genomic libraries were used to isolate and characterize microsatellite loci in Festuca gautieri , an important plant component of subalpine calcareous grasslands of the eastern Iberian Peninsula, the Pyrenees, and the Can- tabrian Mountains. Microsatellites were required to investigate landscape genetics across its distribution range and at a nar- rower geographical scale within the Ordesa y Monte Perdido , Aig ü estortes, and Picos de Europa Spanish national parks. • Methods and Results: Ten polymorphic microsatellite loci were characterized. They ampliﬁ ed a total of 116 alleles in a sample of 30 individuals of F. gautieri , showing high levels of genetic diversity (expected heterozygosity = 0.821). Cross-species transferability to two other close congeners, F. eskia and F × picoeuropeana, increased the total number of alleles to 137. These taxa showed lower numbers of alleles but similar levels of genetic diversity to F. gautieri . • Conclusions: These microsatellite primers will be useful in population and landscape genetics and in establishing conservation strategies for these characteristic elements of subalpine pastures.

Key words: conservation genetics; Festuca gautieri ; landscape genetics; Loliinae; Pooideae; SSR.

Festuca L. (Poaceae), with more than 500 species, is the largest F. eskia Ramond ex DC. , an edaphic vicariant species occupying genus of the Loliinae. Species of Festuca are widespread in the similar habitats but on siliceous soils. In geographical areas where holarctic region but also inhabit cool and temperate areas in the both substrates come into contact, populations of F. gautieri and southern hemisphere. They grow in a large variety of different F. eskia naturally hybridize, producing the morphologically inter- habitats, from wetlands to xeric ecosystems, and are especially mediate taxon F. × picoeuropeana M. I. Gut. & Homet. well adapted to extreme conditions in mountains and in arctic and Such habitats have been subject to diverse human-induced subantarctic areas. Based on its wide taxonomic diversity and geo- degradations because of overexploitation of subalpine pastures graphical and ecological amplitude, different authors have specu- for cattle and sheep and the construction of touristic resources lated on the putative ancient origin of Festuca and postulated an such as ski resorts and have been identifi ed as potentially sensi- evolutionary trend from more basal broad-leaved lineages to more tive areas for loss of biodiversity under climatic change. recently evolved fi ne-leaved lineages, with some species with in- We have conducted a microsatellite characterization in F. termediate features forming an intermediate grade between these gautieri to investigate the genetic landscape and ecology of sub- two major clades ( Inda et al., 2008 and references therein). Festuca alpine grassland communities of F. gautieri, F. eskia, and their gautieri (Hack.) K. Richt. corresponds to a basal group of fi ne- hybrid F. ×picoeuropeana in the Spanish Pyrenean-Cantabrian leaved Festuca taxa (Festuca sect. Eskia Willk.) that are most di- national parks of Ordesa y Monte Perdido, Aig ü estortes i Estany versifi ed in Mediterranean subalpine mountains and a major de Sant Maurici, and Picos de Europa. In these areas, these taxa constitutive element of subalpine pastures. This calcareous species act as a shelter to numerous herbaceous plants and animals and, is diploid with 2n = 14 chromosomes and is capable of both sexual thus, play a key role in the subalpine plant community structure reproduction and vegetative spread through rhizomatous tillering. and in the maintenance of its global biodiversity ( Gonzalo-Turpin This species is morphologically and phylogenetically close to and Hazard, 2009; Gonzalo-Turpin et al., 2010). Our study is aimed at addressing conservation strategies for their biodiversity 1 Manuscript received 2 June 2011; revision accepted 15 July 2011. and at promoting ecological restoration of altered areas with The authors thank Antonio D í az-P é rez for his help with fi eld sampling, local genotypes. We will perform genotypic analysis of individu- and G. Sadowski and F. Fernando for information on the enrichment als based on this set of nuclear simple sequence repeat (SSR) process of the Festuca gautieri SSR library. Financial support was provided markers to describe the landscape genetics of each species. This by the Spanish projects UZ2008-TEC-14 from the University of Zaragoza, will enable us (1) to detect hybrids and introgression zones, (2) PI097/08 from the Arag ó n Government, GA-LC-012/2008 from the Arag ó n to interpret historical and ecological processes affecting the sub- Government– La Caixa, and 059/2009 from the Ministry of Environment (National Parks division). J.G.S.-M. was supported by a Spanish Ministry alpine landscapes, (3) to propose relevant genetic and ecological of Science and Innovation “ Ram ó n y Cajal ” postdoctoral contract. units for the conservation of each species, (4) to select local gen- 4 Author for correspondence: [email protected] otypes for conservation and ecological restoration, and (5) to build predictive models of distribution of these species regarding doi:10.3732/ajb.1100267 different scenarios of global climate change.

Table 1. Characteristics of 18 microsatellite loci developed in Festuca gautieri . For each locus, the primer pair sequences, repeat motif, size of the original fragment (bp), annealing temperature, and GenBank accession numbers are shown.

a ′ ′ b ° Locus Primer sequence (5 – 3 ) Repeat motifSizeT a ( C) GenBank Accession No.

FgauA02* F: 6FAM-CGTTTCAGTGTCGTTGATGTC (CA)13 176 56 JN040543 R: TTCTCTGCGTGGTCTGTATTG

FgauA04* F: VIC-AAGGAAGCACACTACCTACACG (CA)10 294 56 JN040544 R: ATCCCAATCTGAACCCAATC

FgauA101 F: NED-CACGAAGAACGAACAAGAAC (CA)13 280 51 JN040553 R: ATTCAAGGCAACCCTCTCTA

FgauA104 F: VIC-ACCACGGTCTCTCACATAGG (GT)13 226 56 JN040555 R: CCGATGTTCACGAATTGAC

FgauA111* F: VIC-TGACCTAAACTGTTCCCAAATG (GT)23 209 51 JN040545 R: CATGCAAGGTTGTATCTCACG

FgauA115 F: VIC-TCCGATTTGTGGTCAATCTC (GT)13 206 56 JN040557 R: CATGCAATCTTCACGAATTG

FgauA117 F: NED-TCAAACCTTTGTGAACAACAG (CA)9 298 56 JN040558 R: GGGATGGTGTGGTTAGTAAAT

FgauA119 F: NED-CGGTCTCTCCTTGGATGA (GT)21 149 56 JN040559 R: GGGAACACGAAGAACGAA

FgauA121* F: VIC-TGGAGAGGAACTTAGTTGAAAG (CA)13 119 56 JN040546 R: TGTACGACATGCTGATCTACA

FgauB07* F: PET-TCATCGCTGACAAACTCTTC (CT)16 275 56 JN040547 R: CTGACGGGTATTACTTCCAAC

FgauB101 F: VIC-GGAAGAGCCAAGATCCAATCT (GA)11 262 51 JN040560 R: CGTGCTGATTTAGATCCCATC

FgauB103* F: VIC-CCACCTGTCATAAGCCTTTC (GA)6 G(GA)11 138 51 JN040548 R: GCTGATGTCCTCTTCTCGTC

FgauB104 F: NED-AGTGTTCAGCGTTTGTCAGTG (GA)11 172 56 JN040561 R: CCCAGCAATCTTTATCGTGAG

FgauB109* F: PET-CATGGCTTGACACTCTATGAG (GA)13 217 51 JN040549 R: TTTCAGTAAAGGGAACATCTTG

FgauB119* F: 6FAM-GGGACACAAGCACTAAAGTTG (GA)15 146 51 JN040550 R: CCAAAAACAAAATAGGACGAAG

FgauB125* F: NED-AAAGCACCCAGAATATAATGAG (CT)15 211 56 JN040551 R: ACTTGCTGTTACCATGTCAAC

FgauB130* F: PET-GGAAAAGCCTAGAGAGAGGTG (GA)3 GG(GA)8 176 56 JN040552 R: CAAAGGGCACATCAGTTAAAG

FgauC105 F: NED-TACCTTTGTAGGGACGATACG (CAA)13 294 56 JN040565 R: GGTCATTTTGATGGGAGATAC a Polymorphic loci are denoted with an asterisk (*). b 6FAM, NED, PET, and VIC are ﬂ uorescent dyes from Applied Biosystems.

METHODS AND RESULTS (Biotools, Madrid, Spain), 2 mM MgCl2 , 0.4 mM of each dNTP, 5 pmol each of the labeled (forward) and unlabeled (reverse) primers, 1 U Taq polymerase (Bio- tools), and 20 ng of template DNA. The PCR program consisted of one step of 4 One hundred micrograms of total DNA was extracted from silica gel– dried ° ° young leaves of an individual pool using the DNeasy Plant Minikit (QIAGEN, min at 94 C; followed by 35 cycles each of 1 min at 94 C, 1 min at annealing temperature ( Table 1 ) , and 45 s at 72 ° C; and a fi nal extension step of 7 min at Barcelona, Spain) following the manufacturer’ s instructions. This DNA served as a ° template for enriched microsatellite library construction for CT, GT, CTT, and GTT 72 C. The products were run on an ABI 3730 automated sequencer (Applied motifs developed by Genetic Identifi cation Services (Chatsworth, California, USA ; Biosystems) using LIZ500 as the internal lane size standard and the amplifi ed http://www.genetic-id-services.com) following Jones et al. (2002) . Genomic DNA fragment lengths assigned to allelic sizes with GENEMARKER version 1.85 was partially restricted with a cocktail of seven blunt-end cutting enzymes (Rsa I, software (SoftGenetics, State College, Pennsylvania, USA). After an initial HaeIII, BsrB1, PvuII, StuI, ScaI, and Eco RV). Fragments in the size range of 300 screening of individuals, 10 out of 24 loci (JN040543, JN040544, JN040545, to 750 bp were adapted and subjected to magnetic bead capture using biotinylated JN040546, JN040547, JN040548, JN040549, JN040550, JN040551, and capture molecules (CPG, Lincoln Park, New Jersey, USA). Libraries were prepared JN040552) were polymorphic, whereas eight loci (JN040553, JN040555, JN040557, JN040558, JN040559, JN040560, JN040561, and JN040565) were in parallel using Biotin-(CA)15 , Biotin-(GA)15 , Biotin-(AAC) 12 , and Biotin-(AAG) 12 . Captured molecules were amplifi ed and restricted with Hind III to remove the adapt- monomorphic ( Table 1 ) and six loci (JN040554, JN040556, JN040562, ers. The resulting fragments were ligated into the Hind III site of pUC19 plasmid JN040563, JN040564, and JN040566, not shown) showed multibanding patterns and cloned by electroporation into E. coli DH5 α strain (ElectroMaxJ, Invitrogen, suggesting nonspecifi c priming, and were subsequently excluded from analysis. Barcelona, Spain) and screened on X-gal/IPTG/ampicillin-Luria– Bertani agar Genotypic data were obtained for 30 individuals per one population each of plates. Sequences were obtained on an ABI 3730, using ABI PRISM BigDye Ter- F. gautieri , F. eskia, and their hybrid F. × picoeuropeana in a sympatric locality minator cycle sequencing methodology (Applied Biosystems, Madrid, Spain). (Spain: Torla, Pyrenees, Ordesa y Monte Perdido National Park, Punta Acuta, One hundred and forty-four clones were sequenced, of which 121 were unique 42 ° 38 ′ 14.84 ″ N, 0 ° 3 ′ 45.25 ″W, 1850– 1900 m, 11/06/1997, P. Catal á n , JACA sequences containing microsatellites. After discarding clones with too short R288082, JACA R288084, and JACA R288085, respectively) for 10 microsat- fl anking sequences, primers were designed for 95 clones using DesignerPCR, ellite loci (Tables 1 and 2) . Number of alleles (N a ) and observed (H o) and unbi- version 1.03 (Research Genetics, Huntsville, Alabama, USA). From the 40 primer ased expected (H e ) heterozygosities (Nei, 1978) were calculated with GENETIX pairs assayed, 24 produced clear amplicons of the expected size in 2% agarose version 4.05 (Belkhir et al., 1996 – 2004). Inbreeding coeffi cients (F IS ) and de- gels and were subsequently selected for analysis on automated sequencers. For- viations from Hardy – Weinberg equilibrium and linkage disequilibrium be- ward primers were labeled with fl uorescent dyes for automated electrophoresis. tween pairs of microsatellite loci, using 1000 permutations, were calculated PCR amplifi cations were performed in a 20 μ L mix containing 1× Taq buffer using GENEPOP version 4.0 software ( Rousset, 2008 ). e362 American Journal of Botany )

Thirty-three out of 135 pairwise comparisons between loci and populations H ns ns ns ns ns ns

showed signiﬁ cant linkage disequilibrium (P < 0.05 ); however, these were not

F consistent across loci and species. The 10 polymorphic microsatellite loci detected a total of 116 different SSR alleles in the 30 individuals of F. gautieri that were analyzed. The number of alleles ranged from a minimum of ﬁ ve alleles for locus FgauB130 to a maximum of 17 alleles for locus FgauB07, and the mean number of alleles per ) and expected ( ) and expected 0.169 +0.141***

o locus was 11.6 (Table 2). Observed heterozygosities ranged from 0.200 (locus

e ± H H FgauB130) to 0.800 (locus FgauB125) and expected heterozygosities ranged from 0.672 (locus FgauB130) to 0.918 (locus FgauB07). Eight out of 10 loci

showed signifi cant heterozygote defi ciency, with an overall inbreeding coeffi - cient value of FIS = 0.379 ( Table 2 ).

F. eskia F. All 10 microsatellite were successfully transferred to F. eskia and F. × pi- ), observed ( ), observed 0.221 0.701

± coeuropeana ( Table 2 ). The number of alleles scored compared to F. gautieri N H decreased from the intermediate value of 97 alleles in F. × picoeuropeana to 77 in F. eskia, whereas heterozygosity and FIS values were in the range of those reported for F. gautieri (Table 2). Considering all three taxa, a total of 137 dif-

a ferent alleles were scored from the 10 SSR loci. Of these, 58 were shared among

N the three taxa. Only three alleles were shared between F. gautieri and F. eskia , whereas F. × picoeuropeana shared more alleles with both F. gautieri (26 al-

F. eskia r leles) and (eight alleles). Twenty-nine, eight, and only ﬁ ve alleles were from the Spanish Ordesa y Monte Perdido National from the Spanish Ordesa y Monte Perdido National

A exclusive from F. gautieri , F. eskia , and F. × picoeuropeana , respectively. These ), number of alleles ( r F. × picoeuropeana A results are consistent with the proposed hybrid origin of . Genetic diversity indices as measured by observed and expected heterozygosi- F. eskia F. 187 – 223 6 114 – 142 8 0.300 197 – 225 9 0.733 0.305 0.933 0.745 +0.015 0.819 +0.015 − 0.143 ns ns ns ties and inbreeding coefﬁ cients were similar across the three taxa ( Table 2 ).

IS F , and

CONCLUSIONS 0.106 +0.332*** 7.70 0.603

± The results obtained in this exploratory analysis of the H

picoeuropeana genetic diversity in F. gautieri with 10 novel nuclear polymor- × phic microsatellite loci speciﬁ cally designed for this species F. ,

. For each locus, allele range ( . For support their use for conducting population genetics and landscape genetics studies. The successful cross-transferability of picoeuropeana 0.278 0.771

o × ± all 10 markers to the other two close taxa of this complex, F. H F. Festuca eskia and F. × picoeuropeana, further expand their usefulness to F. gautieri F. address similar initiatives in them. The genetic information

compiled for the whole complex of these subalpine Festuca

N taxa will implement more efﬁ cient conservation programs on these key components of subalpine ecosystems.

A = 30) each of N LITERATURE CITED 116 – 142 8 199 – 223 11 0.900 1.000 0.793 − 0.137 0.850 − 0.180

ns ns Belkhir , K. , P. Borsa , L. Chikhi , N. Raufaste , and F. Bonhomme . 1996 –

F 2004. GENETIX 4.05, logiciel sous Windows pour la g é n é tique des

cant. populations. Laboratoire Gé nome, Populations, Interactions, CNRS UMR 5171, Universit é de Montpellier II, Montpellier, France. Gonzalo-Turpin , H. , and L. Hazard. 2009 . Local adaptation occurs

0.082 +0.379*** 9.70 0.533 e along altitudinal gradient despite the existence of gene ﬂ ow in the ± H alpine plant species Festuca eskia. Journal of Ecology 97 : 742 – 751 .

Gonzalo-Turpin , H. , P. Barre , A. Gilbert , A. Grisard , C. P. West , and L. Hazard . 2010 . Co-occurring patterns of endophyte infection and genetic structure in the alpine grass, Festuca eskia: Implications for seed sourcing in ecological restoration. Conservation Genetics 1 1 :

F. gautieri F. 0.203 0.821

o < 0.001 ; ns = not signiﬁ 0.001 < ±

H 877 – 887 . P Inda , L. A. , J. G. Segarra-Moragues , J. M ü ller , P. M. Peterson ,

values are reported for one population ( values and P. Catal á n . 2008 . Dated historical biogeography of the temper- IS

F ate Loliinae (Poaceae, Pooideae) grasses in the northern and southern

a hemispheres. Molecular Phylogenetics and Evolution 46 : 932 – 957 . N < 0.01; ***

P Jones , K. C. , K. F. Levine , and J. D. Banks . 2002 . Characterization of 11 polymorphic tetranucleotide microsatellites for forensic applications Acuta.

r in California elk (Cervus elaphus canadensis ). Molecular Ecology A Notes 2 : 425 – 427 .

< 0.05, ** Nei , M. 1978 . Estimation of average heterozygosity and genetic distance P from a small number of individuals. Genetics 89 : 583 – 590 . : * 2. Results of initial primer screening for 10 polymorphic loci in three populations Rousset , F. 2008 . GENEPOP ’ 007: A complete re-implementation of Park, Punta Park, heterozygosities, and

Note the GENEPOP software for Windows and Linux. Molecular Ecology Table FgauA02FgauA04 140 – 194FgauA111 10 254 – 314 185 – 223FgauA121 13 14 101 – 119FgauB07FgauB103 9 277 – 327 0.367 110 – 142FgauB109 17 0.467 10 217 – 239FgauB119 0.500FgauB125 9 82 – 236 0.467 195 – 221FgauB130 17 0.829 0.600 12 172 – 182Mean 0.733 0.905 0.835 5 +0.562*** 0.567 0.844 140 – 194 +0.489*** 0.733 +0.405*** 0.800 0.918 286 – 324 8 0.754 185 – 223 13 +0.451*** 0.200 9 0.851 101 – 119 +0.350*** +0.027 0.890 11.60 277 – 311 8 0.715 0.267 0.513 16 0.633 +0.690*** 0.672 0.600 217 – 239 +0.178** − 0.122 7 0.233 102 – 186 0.667 +0.706*** 0.701 11 0.798 172 – 182 0.558 6 +0.624*** 0.200 +0.209* 0.804 184 – 194 0.500 0.917 − 0.076 5 288 – 324 +0.713*** 0.333 11 +0.276*** 0.677 101 – 119 275 – 331 0.872 6 11 0.567 +0.708*** 0.633 0.736 213 – 239 +0.431*** 132 – 190 8 0.233 0.700 0.699 +0.551*** 9 172 – 178 0.816 4 0.733 +0.192** 0.505 0.847 0.433 +0.227** 0.767 +0.542*** 0.731 +0.176 0.797 0.743 − 0.004 +0.461*** − 0.033 Locus Resources 8 : 103 – 106 .