Transferability of Microsatellite Markers across Eleven Species of Magnolia L.

in HortScience

The genus Magnolia (Magnoliaceae) comprises more than 130 species distributed predominantly in temperate and tropical regions in Southeast Asia and is valued worldwide for its ornamental traits as well as for timber and medicinal products, and in trade. Despite their favored status, many species of Magnolia are faced with threats from logging, agricultural land use, development, and collection, and are at risk of extinction. Conservation of these species through habitat preservation and in ex situ collections is needed to prevent extinction. To provide a tool for conservation of Magnolia species, microsatellite markers developed previously for Magnolia ashei were tested in 10 other species of Magnolia to determine their transferability across species. Of the 64 primer pairs tested, 21 amplified alleles in the expected size range in all samples; 11 primer pairs amplified clean products in most, but not all, species; 18 primer pairs consistently amplified a polymerase chain reaction (PCR) product in most species, but had either low peak height or other amplification issues; and 14 primers showed excessive stutter, nonspecific amplification, or no amplification. Cluster analysis using the 129 alleles amplified by these 21 simple sequence repeat (SSR) primer pairs generated groups that corresponded to the known taxonomic relationships in this genus.

Abstract

The genus Magnolia (Magnoliaceae) comprises more than 130 species distributed predominantly in temperate and tropical regions in Southeast Asia and is valued worldwide for its ornamental traits as well as for timber and medicinal products, and in trade. Despite their favored status, many species of Magnolia are faced with threats from logging, agricultural land use, development, and collection, and are at risk of extinction. Conservation of these species through habitat preservation and in ex situ collections is needed to prevent extinction. To provide a tool for conservation of Magnolia species, microsatellite markers developed previously for Magnolia ashei were tested in 10 other species of Magnolia to determine their transferability across species. Of the 64 primer pairs tested, 21 amplified alleles in the expected size range in all samples; 11 primer pairs amplified clean products in most, but not all, species; 18 primer pairs consistently amplified a polymerase chain reaction (PCR) product in most species, but had either low peak height or other amplification issues; and 14 primers showed excessive stutter, nonspecific amplification, or no amplification. Cluster analysis using the 129 alleles amplified by these 21 simple sequence repeat (SSR) primer pairs generated groups that corresponded to the known taxonomic relationships in this genus.

Magnolia L. (Magnoliaceae) is a popular genus comprising more than 130 species distributed predominantly in temperate and tropical regions in Southeast Asia (Azuma et al., 1999; Figlar and Nooteboom, 2004; Kim et al., 2001). They are valued worldwide for their ornamental traits as well as for timber and medicinal products, and in trade, and are well recognized botanically as one of the earliest flowering plants (Raven et al., 1986). Despite their favored status, many species of Magnolia are faced with threats from logging, agricultural land use, development, and collection, and are at risk of extinction. According to a recent study by Botanic Gardens Conservation International (BGCI), 48% of the species studied are threatened in the wild (critically endangered, endangered, or vulnerable), including 75% of the species from neotropical regions (Rivers et al., 2016). Conservation of these species in situ through habitat preservation and in ex situ collections is needed to prevent extinction.

Microsatellite markers, or SSRs, are an efficient method to assess the genetic diversity and population structure of plant populations [Powell et al. (1996); reviewed in Varshney et al. (2005); Wang et al. (2009)], and have proved useful for guiding conservation efforts in several Magnolia species, including M. ashei Weath. (von Kohn et al., 2018), M. obovata Thunb. (Isagi et al., 1999), M. stellata (Seibold & Zucc.) Maxim. (Ueno et al., 2005), M. sieboldii K. Koch (Kikuchi and Isagi, 2002), M. tripetala (L.) L. (Gilkison, 2013), M. sharpii Miranda, and M. schiedeana Schltdl. (Newton et al., 2008), among others. The availability of additional SSR markers that are useful in multiple Magnolia species could facilitate the characterization of threatened species for ex situ conservation. The objective of this study was to determine the transferability of genomic SSR primers developed for M. ashei (von Kohn et al., 2018) to 10 additional Magnolia species to provide a tool for population and conservation studies.

Materials and Methods

Plant material.

Shoot tips from 22 samples representing 11 Magnolia species (Table 1) were collected or sent from cooperators and refrigerated before DNA extraction. Genomic DNA was extracted from 0.040 to 0.100 g vegetative buds using a PowerPlant Pro DNA Isolation Kit (MoBio Laboratories, Inc., Carlsbad, CA) according to the manufacturer’s instructions with the following modifications: the addition of a small amount of garnet matrix (BIO 101, Inc. Vista, CA) to aid in homogenization, inclusion of the optional phenolic separation solution, and an additional wash step before elution. DNA was quantified using a NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific, Wilmington, DE).

Table 1.

List of 11 Magnolia species used in this study, including section, ploidy, number of samples per species tested, and source of samples.

Table 1.

SSR primer evaluation and PCR.

Genomic SSR primers were developed and selected from Magnolia ashei as described in von Kohn et al. (2018). Briefly, SSRs were identified from Illumina sequence data (deposited in GenBank, accession no. PCNC00000000) using the MIcroSAtellite identification tool (Beier et al., 2017). Primers for 64 SSR loci with differing repeat units (Table 2) were identified using Primer 3 Plus (Untergasser et al., 2012). PCR primers were manufactured by Integrated DNA Technologies (Coralville, IA). The forward primers had an additional M13 (–21) universal sequence (TGTAAAACGACGGCCAGT) attached to the 5′ end to allow indirect fluorescent labeling of PCR products using a universal 6-carboxy-fluorescine (FAM)-labeled M13 primer (Schuelke, 2000). PCR was carried out in a Bio-Rad T100 Thermal Cycler (Bio-Rad Laboratories, Inc., Hercules, CA). The 15-µL PCR mixture contained 30 ng template genomic DNA, 0.25 µM of each reverse and universal FAM-labeled M13 (−21) primer, and 0.0625 µM of the forward primer with 1× Bioline MangoMix and 3.77 mm Bioline MgCl2 (Bioline Inc., Taunton, MA). PCR profiles consisted of initial denaturation at 94 °C for 5 min; followed by 30 cycles of 94 °C for 30 s, optimized annealing temperature of each primer pair (Table 2) for 30 s, and 72 °C for 60 s; followed by eight cycles of 94 °C for 30 s, 53 °C for 45 s, and 72 °C for 45 s; and a final extension at 72 °C for 10 min. Products were analyzed on an ABI 3730xl DNA Analyzer (Applied Biosystems, Foster City, CA) using 2 µL PCR product, 10 µL formamide (Applied Biosystems), and 0.2 µL GeneScan 500 LIZ Size Standard (Applied Biosystems). Allele sizes and number of alleles per locus were determined with GeneMarker version 2.6.3 (SoftGenetics, State College, PA). Reactions that resulted in unexpected, ambiguous, low, or no amplification were repeated.

Table 2.

Characteristics of the 50 polymorphic simple sequence repeat loci derived from M. ashei including sequence, repeat unit, annealing temperature, allelic statistics, and their transferability to 10 other Magnolia species. The 18 primers pairs from loci below the dashed horizontal line consistently amplified a polymerase chain reaction product in the indicated species, but had either low peak height, ambiguous alleles, unexpected number of repeats, or did not amplify a product in at least one sample in a species that otherwise had that locus.

Table 2.

Data analysis.

Allele frequency analysis for 21 loci that showed consistent and predicted amplification products in all samples tested was performed using Cervus 3.0.7 (Kalinowski et al., 2007), based on allelic data from the 17 diploid samples (Table 1). Data from these 21 loci for all species were converted to a binary matrix (presence/absence of allele) and used to generate a similarity matrix based on the Jaccard coefficient using NYSYSpc version 2.02 (Rohlf, 1998). Accessions were then clustered using the unweighted pair group method with arithmetic mean (UPGMA) algorithm in NTSYSpc. Cophenetic matrices were constructed and compared with the similarity matrices using the MXCOMP program to test the goodness of fit of a cluster (Rohlf, 1998).

Results

Of the 64 primer pairs tested, 21 amplified alleles in the expected size range in all samples of all 11 species tested (Table 2). Eleven primer pairs amplified clean products in most, but not all, species. An additional 18 primer pairs (below the dashed horizontal line in Table 2) consistently amplified a PCR product in the indicated species, but had either low peak height, ambiguous alleles, unexpected size of the repeated unit, or did not amplify a product in at least one sample in a species that otherwise had that locus. Finally, the remaining 14 primers (not listed) showed excessive stutter, nonspecific amplification, or no amplification.

A total of 129 alleles were scored from the 21 primer pairs that resulted in “complete” data. Allelic data from the 17 diploid samples were used to calculate allelic frequencies and polymorphic information content (PIC) for these 21 loci. PIC values ranged from 0.16 to 0.83 (Table 2). The 129 alleles were also used to generate a UPGMA dendrogram based on the Jaccard similarity coefficient (Fig. 1). Although the purpose of this study was not to confirm interspecific diversity or taxonomic classification in Magnolia, the phenogram provides strong evidence that the primers developed in M. ashei are representative of the allelic diversity in the genus. The cophenetic correlation coefficient for the dendrogram was 0.982, indicating that the dendrogram is a very good fit to the data set (Rohlf, 1998). Equally significant, the major clusters in the dendrogram followed the taxonomic relationships reported previously (Figlar and Nooteboom, 2004; Kim et al., 2001; Kim and Suh, 2013). Specifically, all samples grouped together by species, and all species clustered together by section (Table 1).

Fig. 1.
Fig. 1.

Unweighted pair group method with arithmetic mean dendrogram of 22 Magnolia samples based on Jaccard similarity data from 129 alleles from 21 simple sequence repeats loci. Numbers in boxes indicate the percentage of M. ashei primers that amplified a product in each species (of 50 primers listed in Table 2). Names following braces indicate the taxonomic section to which the species belongs. Cophenetic correlation coefficient (r) = 0.982.

Citation: HortScience horts 54, 2; 10.21273/HORTSCI13605-18

Discussion

Many studies have reported the utility and limitations of cross-species amplification of SSR primers in plants [e.g., reviewed by Barbara et al. (2007), Varshney et al. (2005), Wang et al. (2009)]. As would be expected and has been confirmed in other studies, the success of SSR primer transferability across species is correlated with how closely related the species are (Bruegmann and Fladung, 2013; Buzatti et al., 2016). We observed similar results in our study (Fig. 1), in which only one primer did not amplify in M. macrophylla Michx. and two primers did not amplify in M. dealbata Zucc. (Table 2), both of which are in the same section (sec. Macrophylla Figler & Noot.) as M. ashei (Table 1). Conversely, an average of 5.75 primers did not amplify in species outside the section.

The real test of how useful SSR markers will be in related species or genera is not whether they amplify, but how polymorphic the markers are (Barbara et al., 2007). Because our study used only one to three samples of each species, it is not possible to determine whether the SSR loci that showed only one allele in a species are truly monomorphic in that species. Some studies indicate that genomic SSRs may have more nonspecific amplification or stuttering than genic SSRs (Varshney et al., 2005), although they may be more polymorphic in closely related accessions [such as within a species (Song et al., 2012)]. The SSRs described here were developed to study population structure within one species (M. ashei) and were derived from genomic DNA, so they may prove to be less polymorphic, less transferable, or less clearly amplified in more distantly related taxa. Comparative studies indicate that successful cross-species amplification and polymorphism are more likely within genera such as Magnolia, which are perennial (vs. annual) and are outcrossing (vs. selfing) (Barbara et al., 2007).

The SSR primers reported in this article and those from other Magnolia studies contribute to an increasingly valuable set of tools to gather genetic and population data on Magnolia species. Of the 304 Magnoliaceae species studied in the 2016 BGCI report, 20% could not be assessed as a result of a lack of data. As development of SSR markers becomes more economical (Zalapa et al., 2012), it may be feasible to develop markers specifically for data-deficient species with pressing conservation needs. However, in many cases, especially when quick action is needed and resources are limited, the use of SSR markers developed for other species will be critical in obtaining sufficient data to guide effective conservation of threatened species. The Global Strategy for Plant Conservation, adopted as part of the Convention on Biological Diversity in 2002, includes as one of its targets the conservation of at least 75% of the world’s threatened plants in ex situ collections by 2020 (Hird and Kramer, 2013). Currently, only 43% of threatened Magnolia species are preserved in ex situ collections (Rivers et al., 2016). Many Magnolia species have recalcitrant seeds, so must be conserved via resource- and space-intensive plantings. Data derived from SSR markers can be used to determine population structure or genetic diversity, which enables maximizing the amount of genetic diversity held in ex situ collections while minimizing the number of individuals required. This information will allow curation of ex situ collections to capture and reflect the genetic diversity of the wild populations (Cires et al., 2013; IUCN/SSC, 2014; Rivers et al., 2016). The SSR primers described here provide a valuable tool for developing conservation strategies for this important genus.

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Contributor Notes

The mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendations or endorsement by the U.S. Department of Agriculture.

Current address: Irrigated Agriculture Research and Extension Center, Washington State University, 24106 N. Bunn Road, Prosser, WA 99350.

Corresponding author. E-mail: Margaret.Pooler@ars.usda.gov.

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    Unweighted pair group method with arithmetic mean dendrogram of 22 Magnolia samples based on Jaccard similarity data from 129 alleles from 21 simple sequence repeats loci. Numbers in boxes indicate the percentage of M. ashei primers that amplified a product in each species (of 50 primers listed in Table 2). Names following braces indicate the taxonomic section to which the species belongs. Cophenetic correlation coefficient (r) = 0.982.

  • AzumaH.ThienL.B.KawanoS.1999Molecular phylogeny of Magnolia (Magnoliaceae) inferred from cpDNA sequences and evolutionary divergence of the floral scentsJ. Plant Res.112291306

    • Search Google Scholar
    • Export Citation
  • BarbaraT.Palma-SilvaC.PaggiG.M.BeredF.FayM.F.LexerC.2007Cross-species transfer of nuclear microsatellite markers: Potential and limitationsMol. Ecol.1637593767

    • Search Google Scholar
    • Export Citation
  • BeierS.ThielT.MünchT.ScholzU.MascherM.2017MISA-web: A web server for microsatellite predictionBioinformatics3325832585

  • BruegmannT.FladungM.2013Potentials and limitations of the cross-species transfer of nuclear microsatellite makers in six species belonging to three sections of the genus Populus LTree Genet. Genomes914131421

    • Search Google Scholar
    • Export Citation
  • BuzattiR.S.O.ChicataF.S.L.LovatoM.B.2016Transferability of microsatellite markers across six Dalbergia (Fabaceae) species and their characterization for Dalbergia miscolobiumBiochem. Syst. Ecol.69161165

    • Search Google Scholar
    • Export Citation
  • CiresE.DeSmetY.CuestaG.GoetghebeurP.SharrockS.GibbsD.OldfieldS.KramerA.SamainM.2013Gap analyses to support ex situ conservation of genetic diversity in Magnolia, a flagship groupBiodivers. Conserv.22567590

    • Search Google Scholar
    • Export Citation
  • FiglarR.B.NooteboomH.P.2004Notes on Magnoliaceae IVBlumea4987100

  • GilkisonV.A.2013Comparisons of genetic diversity among disjunct populations of Magnolia tripetala. Honors project thesis Western Kentucky University. 17 Sept. 2018. <http://digitalcommons.wku.edu/stu_hon_theses/423>

  • HirdA.KramerA.T.2013Achieving target 8 of the global strategy for plant conservation: Lessons learned from the North American collections assessmentAnn. Mo. Bot. Gard.99161166

    • Search Google Scholar
    • Export Citation
  • IsagiY.KanazashiT.SuzukiW.TanakaH.AbeT.1999Polymorphic microsatellite DNA markers for Magnolia obovata Thunb. and their utility in related speciesMol. Ecol.8685702

    • Search Google Scholar
    • Export Citation
  • IUCN/SSC2014Guidelines on the use of ex situ management for species conservation. Version 2.0. IUCN Species Survival Commission Gland Switzerland. 18 Sept. 2018. <https://portals.iucn.org/library/sites/library/files/documents/2014-064.pdf>

  • KalinowskiS.T.TaperM.L.MarshallT.C.2007Revising how the computer program CERVUS accommodates genotyping error increases success in paternity assignmentMol. Ecol.1610991106

    • Search Google Scholar
    • Export Citation
  • KikuchiS.IsagiY.2002Microsatellite genetic variation in small and isolated populations of Magnolia sieboldii ssp. japonicaHeredity88313321

    • Search Google Scholar
    • Export Citation
  • KimS.ParkC.W.KimY.D.SuhY.2001Phylogenetic relationships in family Magnoliaceae inferred from NDHF sequencesAmer. J. Bot.88717728

  • KimS.SuhY.2013Phylogeny of Magnoliaceae based on ten chloroplast DNA regionsJ. Plant Biol.56290305

  • NewtonA.C.GowJ.RobertsonA.Williams-LineraG.Ramírez-MarcialN.González-EspinosaM.AllnuttT.R.EnnosR.2008Genetic variation in two rare endemic Mexican trees, Magnolia sharpii and Magnolia schiedeanaSilvae Genet.57348356

    • Search Google Scholar
    • Export Citation
  • ParrisJ.K.RanneyT.G.KnapH.T.BairdW.V.2010Ploidy levels, relative genome sizes, and base pair composition in MagnoliaJ. Amer. Soc. Hort. Sci.135533547

    • Search Google Scholar
    • Export Citation
  • PowellW.MachrayG.C.ProvanJ.1996Polymorphism revealed by simple sequence repeatsTrends Plant Sci.1215222

  • RavenP.H.EvertR.F.EichhornS.E.1986Biology of plants. 4th ed. Worth Publishers New York NY

  • RiversM.BeechE.MurphyL.OldfieldS.2016The red list of Magnoliaceae. Botanic Gardens Conservation International Surrey UK

  • RohlfF.J.1998NTSYS-pc 2.02: Numerical taxonomy and multivariate analysis system. Exeter Software Applied Biostatistics Inc. Setauket New York NY

  • SchuelkeM.2000An economic method for the fluorescent labeling of PCR fragmentsNat. Biotechnol.18233234

  • SongY.P.JiangX.B.ZhangM.WangZ.L.BoW.H.AnX.M.ZhangD.Q.ZhangZ.Y.2012Differences of EST-SSR and genomic-SSR markers in assessing genetic diversity in poplarFor. Stud. China1417

    • Search Google Scholar
    • Export Citation
  • UenoS.SetsukoS.KawaharaT.YoshimaruH.2005Genetic diversity and differentiation of the endangered Japanese endemic tree Magnolia stellata using nuclear and chloroplast microsatellite markersConserv. Genet.6563574

    • Search Google Scholar
    • Export Citation
  • UntergasserA.CutcutacheI.KoressaarT.YeJ.FairclothB.C.RemmM.RozenS.G.2012Primer3: New capabilities and interfacesNucl. Acids Res.40E115

    • Search Google Scholar
    • Export Citation
  • U.S Department of Agriculture Agricultural Research Service2018U.S. National Plant Germplasm System: GrinGlobal version 1.9.7.1. 18 Sept. 2018. <https://npgsweb.ars-grin.gov/gringlobal/taxon/taxonomysearch.aspx>

  • VarshneyR.K.GranerA.SorrellsM.E.2005Genic microsatellite markers in plants: Features and applicationsTrends Biotechnol.234855

  • von KohnC.ConradK.KramerM.PoolerM.2018Genetic diversity of Magnolia ashei characterized by SSR markersConserv. Genet.19923936

  • WangM.L.BarkleyN.A.JenkinsT.M.2009Microsatellite markers in plants and insects. Part I: Applications of biotechnologyGenes Genomes Genomics35467

    • Search Google Scholar
    • Export Citation
  • ZalapaJ.E.CuevasH.ZhuH.SteffanS.SenalikD.ZeldinE.McCownB.HarbutR.SimonP.2012Using next-generation sequencing approaches to isolate simple sequence repeat (SSR) loci in the plant sciencesAmer. J. Bot.99193208

    • Search Google Scholar
    • Export Citation
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