1 Page 1 of PERMANENT GENETIC RESOURCES Isolation and characterization of nine microsatellite loci in the Pale Pitcher Plant Sarracenia alata (Sarraceniaceae) MARGARET M. KOOPMAN*, ELIZABETH GALLAGHER, and BRYAN C. CARSTENS Department of Biological Sciences, Louisiana State University, 202 Life Sciences Building, Baton Rouge, LA *Correspondence: Margaret M. Koopman PH: ; FAX: ; Keywords: Microsatellites, Pitcher Plants, Sarracenia alata, Sarraceniaceae Running Title: Sarracenia microsatellites
2 Page 2 of Abstract We isolated and evaluated nine microsatellite loci, for the first time in the family Sarraceniaceae, from Sarracenia alata. Loci exhibit between one and thirteen alleles and a mean observed heterozygosity of 0.52 in one well-sampled population. Additionally, the primers reported here cross-amplify in four closely related species of Sarracenia. These markers will be used to explore patterns of population divergence in this ecologically well-known, rapidly evolving genus.
3 Page 3 of The genus Sarracenia comprises eleven species primarily restricted to the Gulf Coast States of North America, with one widespread species distributed through the mid- Atlantic region of the United States. This carnivorous genus has been a model system for addressing ecological questions for more than a century, however, to our knowledge no microsatellite markers have been developed in this family and no genetic studies focusing on Sarracenia alata Wood have been previously published. In this paper we report the development of nine polymorphic microsatellite markers in S. alata. These markers will be used to examine patterns of population divergence in the species throughout Louisiana. Microsatellite markers were developed using the protocol of Glenn and Schable (2005). High molecular weight DNA was extracted from dried leaf tissue of one individual of Sarracenia alata using the protocol of Sanchez-Hernandez and Gaytan- Oyarzun (2006), which utilizes both traditional CTAB methods and the DNeasy plant extraction kit (Qiagen, Valencia, CA). Genomic DNA (gdna) was digested with RsaI or Bsu (Table 1) and XmnI. SuperSNX24 linkers (Glenn & Schable 2005) were ligated to fragments of genomic DNA. To ensure that this ligation was successful a PCR was performed following Glenn and Schable (2005); this PCR product was then separately hybridized to three mixes of biotinylated oligo probes (listed in Glenn & Schable 2005: Mix2 (dinucleotide)/mix3(trinucleotide)/mix4(tetranucleotide)). This gdnabiotynylated complex was added to magnetic beads coated with streptavidin (Dynabeads M-280 Invitrogen, Carlsbad, CA) with a 2h incubation at 33 C in a rotating oven. The bead mixture was washed twice with 2xSSC, 0.1%SDS and four times
4 Page 4 of with 1xSSC, 0.1%SDS, with the latter two washes conducted at 50 C. A magnetic particle concentrator captured the beads (bound with biotin and gdna) after each wash. Enriched fragments were denatured at 95 C for 10min to separate them from the beads and precipitated with an ethanol wash. A recovery PCR was performed following Glenn and Schable (2005). These fragments were cloned using a Qiagen PCR cloning kit (Valencia, CA) following manufacturers protocols. Bacterial colonies that contained vectors with gdna were used as template for PCR. These products were cleaned using ExoAP (Glenn & Schable, 2005) and sequenced using BigDye Terminator v3.1 (Applied Biosystems, Foster City, CA). Sequencing reactions were cleaned with an ethanol precipitation and run on an ABI PRISM 3100 automated sequencer (Applied Biosystems, Foster City, CA). Sequences were edited in Sequencher v4.6 (Gene Codes, Ann Arbor, MI) and replicate sequences were removed. To search automatically for microsatellite repeats and develop primers, we exported double stranded products to MSATCOMMANDER (Faircloth, 2008), which automatically adds a M13: 5 -GGAAACAGCTATGACCAT-3 and a CAG: 5 -CAGTCGGGCGTCATCA-3 tag to the 5 end of the suggested forward and reverse primers. These universal sequence tags facilitate fluorescent labeling in later PCR reactions (Schuelke, 2000). The quality of each primer and its tag is then assessed. The forward and reverse tagged primer pair with the lowest penalty score was chosen for initial primer construction. Primers flanking thirty-four candidate microsatellites were developed. We report nine primer pairs that cleanly amplified products of expected size from 59 individuals of Sarracenia alata collected from five populations throughout the state of
5 Page 5 of Louisiana (Table 1) as well as a single individual of four additional species in the genus (Sarracenia leucophylla, S. flava, S. minor and S. psittacina). Linkage disequilibrium and deviations from HWE was assessed in a single, well-sampled population (Lake Ramsey; 26 individuals). PCR conditions for primer pairs were initially optimized on one accession of S. alata. Loci were amplified under the following PCR conditions in 25µl volume reactions (BioRad PCR machine): 1X PCR buffer, 1.5mM MgCl 2, 0.2mM each dntp, 5mM BSA, 1 Unit Taq DNA polymerase, 0.16µM appropriately fluorescently FAM labeled primer (either M13 or CAG, see Table 1 for label and direction), 0.16µM unlabeled PCR primer, 0.04µM labeled PCR primer (with M13 or CAG tail) and 1-10ng genomic DNA. PCR conditions were as follows: 94 C for 4 min; 35 cycles of 94 C for 30 sec, T a C (Table 1) for 30 sec, 72 C for 45 sec; followed by 8 cycles of 94 C for 30 sec, 53 C for 30 sec, 72 C for 45 sec; with a final extension at 72 C for 10 min. PCR products and 500ROX- labeled size standard (GeneScan Warrington, UK) were suspended in formamide before running on an ABI PRISM 3100 automated sequencer (Applied Biosystems, Foster City, CA). Fragment analysis was conducted with GENEMAPPER version 4.0 (ABI, Foster City, CA). Data were analyzed in GENEPOP v (Rousset, 2008). The nine loci reported here were polymorphic across populations of S. alata (Table 1). Though all individuals of S. alata are homozygous at locus 36, each population sampled is fixed for one allele, suggesting population structure across the state. Within the Lake Ramsey population the number of alleles range from one to thirteen, observed heterozygosity ranges from 0 to 1.0 and F IS values range from to 1.0 (Table 1). The nine loci are in Hardy-
6 Page 6 of Weinberg equilibrium (Table 1) and no linkage disequilibrium was detected in all possible pairwise comparisons (Bonferroni-corrected P< 0.005) for this population. Preliminary analyses demonstrate the ability of these novel microsatellite markers to detect genetic diversity in S. alata. The microsatellite loci also amplified cleanly in four additional species of Sarracenia. Amplification of these loci in S. alata and other Sarracenia species will prove useful in population genetic studies throughout the genus and possibly in other closely related genera Acknowledgments Funding was provided by the Louisiana State University Faculty Research Program to BCC. We thank S. Hird for collecting S. alata material, S. Furches for providing DNA of the non-focal Sarracenia species, and the Whitehead lab for sharing equipment used in the protocol described above. 102
7 Page 7 of References Faircloth BC (2008) MSATCOMMANDER: detection of microsatellite repeat arrays and automated, locus-specific primer design. Molecular Ecology Resources 8, Glenn TC, Schable NA (2005) Isolating microsatellite DNA loci. Methods in Enzymology 395, Rousset F (2008) GENEPOP'007: a complete re-implementation of the GENEPOP software for Windows and Linux. Molecular Ecology Resources 8, Sanchez-Hernandez C, Gaytan-Oyarzun JC (2006) Two mini-preparation protocols to DNA extraction from plants with high polysaccharide and secondary metabolites. African Journal of Biotechnology 5, Schuelke M (2000) An economic method for the fluorescent labeling of PCR fragments. Nature Biotechnology 18,
8 Page 8 of Table 1 Characterization of nine microsatellite loci in 59 Sarracenia alata individuals for five populations collected throughout Louisiana (species-wide) as well as 26 individuals for a single population (Lake Ramsey; St. Tammany Parish, LA). Annealing temperature (T a ), universal fluorescent-labeled primer used (CAG or M13) and the direction (F or R) of the PCR primer that had an identical tag, the enzyme used (digest) in initial gdna digestion, size range of alleles. The following indices were calculated in GENEPOP: N A, number of alleles; H O, observed and H E, expected heterozygosity under HWE; F IS, inbreeding coefficient. Significance values for deviation from HWE are indicated for the Lake Ramsey population Cover photo caption The Pale Pitcher Plant, Sarracenia alata, is one of eleven species in the carnivorous genus restricted to eastern North America. In the current issue Koopman et al. present microsatellite loci for S. alata, these markers also amplify in four additional congeners. Photo: M. Koopman.
9 Page 9 of 28 Species-wide (N=59) Lake Ramsey (N=26) Locus Primer Sequence(5'-3') T a Repeat Unit Label, direction Digest Size (bp) N A H O H E F IS N A H O H E F IS P Genbank Accession # 5 F GAACAAGAGCACTACATTTGC 60 (GA)^11 CAG, F Rsa GQ R TCGAGCTTCCTCCTTGTGG 7 F GAAGCTGGTTGGATCGTC 60 (CT)^16 (ATCT)^4 CAG, F Rsa GQ R ACGTTAGCAGAACCAGAACC 18 F CACGCTCTTTTGGGCAATTC 60 (GTTTT)^6 CAG, R Bsu GQ R GTGCCTTCAATCTGGGTTCG 19 F CTGTGAATATCGCCGACGC 54 (CT)^17 M13, R Rsa GQ R ATAGTCGCCGTTCGGTC 21 F TTTTGGATTGGACCCAGCG 60 (GT)^22 CAG, F Rsa GQ R TCAAAGGGTAGGGCACCTG 27 F GTGAGTTTTGAGGAATTTCGTTTTG 60 (GT)^6 CAG, F Rsa GQ R GTCTGGTCTCAACCCGTTATG 36 F CTAGCACCTCCGGAACTCTC 60 (GTTT)^5 M13, F Bsu GQ R GATGTCCATGACGTGTGCG 44 F GGGCCTAGCTATGTTGGG 54 (TG)^8 CAG, F Rsa GQ R CCGAAGGCCAAATGGAGAC 47 F ATCACCCACCAGAAACGGG 60 (GAAAA)^2 CAG, R Bsu GQ R GCGTGGTAGGCAGGTAAATG
10 Page 10 of 28 Pop: 9L Locus: MSAT Expected number of homozygotes : Observed number of homozygotes : 9 Expected number of heterozygotes: Observed number of heterozygotes: Tot msat21 Expected number of homozygotes : Observed number of homozygotes : 1
11 Page 11 of 28 Allele frequencies and : Tot
12 Page 12 of 28 Observed number of heterozygotes: 16 Allele frequencies and : Tot Expected number of homozygotes : Observed number of homozygotes : 25 Expected number of heterozygotes: Observed number of heterozygotes: 0
13 Page 13 of Tot Expected number of homozygotes : Observed number of homozygotes : 24 Expected number of heterozygotes: Observed number of heterozygotes: 0 Allele frequencies and :
14 Page 14 of 28 Expected number of heterozygotes: Observed number of heterozygotes: 16 Allele frequencies and : Tot Expected number of homozygotes : Observed number of homozygotes : 9 Expected number of heterozygotes: Observed number of heterozygotes: 10
15 Page 15 of Tot Expected number of homozygotes : Observed number of homozygotes : 9 Expected number of heterozygotes: Observed number of heterozygotes: 14 Allele frequencies and :
16 Page 16 of Tot Expected number of homozygotes : Observed number of homozygotes : 8 Expected number of heterozygotes: Observed number of heterozygotes: 9 Allele frequencies and :
17 Page 17 of 28 e/o het. Def. tests Pop : 9L estimates locus P-val S.E. W&C R&H Steps MSAT19 MSAT switches switches
19 Page 19 of Expected number of homozygotes : Observed number of homozygotes : Expected number of heterozygotes: Observed number of heterozygotes: Allele frequencies and : Hardy Weinberg test when ******** ================= Results by population
20 Page 20 of esti locus P-val S.E. W& Tot MSAT Expected number of homozygotes : MSAT Observed number of homozygotes : 5 MSAT Expected number of heterozygotes: MSAT Observed number of heterozygotes: 48 MSAT MSAT MSAT MSAT Allele frequencies and : MSAT
21 Page 21 of Tot Expected number of homozygotes : Observed number of homozygotes : 22 Expected number of heterozygotes: Observed number of heterozygotes: 32 Allele frequencies and :
22 Page 22 of Tot Expected number of homozygotes : Observed number of homozygotes : 49 Expected number of heterozygotes: Observed number of heterozygotes: 0 Allele frequencies and :
23 Page 23 of 28 Observed number of homozygotes : 48 Expected number of heterozygotes: Observed number of heterozygotes: 0 Allele frequencies and : Tot Expected number of homozygotes : Observed number of homozygotes : 19 Expected number of heterozygotes: Observed number of heterozygotes: 32
24 Page 24 of Tot Expected number of homozygotes : Observed number of homozygotes : 21 Expected number of heterozygotes: Observed number of heterozygotes: 25 Allele frequencies and :
25 Page 25 of Tot Expected number of homozygotes : Observed number of homozygotes : 24 Expected number of heterozygotes: Observed number of heterozygotes: 22 Allele frequencies and :
26 Page 26 of Expected number of homozygotes : Observed number of homozygotes : 21 Expected number of heterozygotes: Observed number of heterozygotes: 17 Allele frequencies and : Tot
27 Page 27 of rdy Weinberg test when H1= heterozygote deficit ************************ ======================================== Results by population
28 Page 28 of estimates us P-val S.E. W&C R&H Steps AT19 AT21 AT18 AT27 AT36 AT44 AT7 AT switches switches switches switches switches switches switches switches switches
Supplemental Text/Tables PCR Amplification and Sequencing PCR was carried out in a reaction volume of 20 µl using the ABI AmpliTaq GOLD kit (ABI, Foster City, CA). Each PCR reaction contained 20 ng genomic
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