Development and Characterization of Genic Microsatellites for the Ornamental Plant Green and Gold (Chrysogonum virginianum)

in HortScience

The genus Chrysogonum is native to the eastern United States. Three entities have been recognized—either as three varieties of Chrysogonum virginianum or as two species, one of them with two varieties. The current study suggests that a fourth entity should be recognized. Several forms of the complex are in commercial trade as ornamentals. As very limited molecular information on Chrysogonum is available, we developed a set of genic simple sequence repeat markers (eSSRs) from de novo transcriptome sequencing. We tested a set of 17 eSSRs on a collection of C. virginianum genomic DNA samples from the three botanical varieties, and a new putative type observed in Tennessee, dubbed “Ocoee-type” for its geographic origin. The polymerase chain reaction and capillary electrophoresis analyses with downstream population genetics tools verified the usefulness of the eSSRs. By applying this approach, we showed recognizable variation within Chrysogonum, although it did not correspond exactly to previous infraspecific classifications. Finally, as demonstrated for the commercial cultivar Pierre included in the study, the eSSRs can be used for enhancing the future breeding or hybridization efforts of this ornamental plant.

Contributor Notes

This study was funded fully by an Agricultural Research Service–U.S. Department of Agriculture grant (58-6062-6) to the Trigiano laboratory for ornamental plants research. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Use of trade names is for identification purposes only and does not imply their endorsement by the authors or the study funding entities. The authors are grateful to the plant material collectors and herbaria for making them available to this study. The RNA sequencing raw read files are available as National Center for Biotechnology Information Sequence Read Archive accession SRR8181940.

Corresponding author. E-mail: mnowicki@utk.edu

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    Genic simple sequence repeats (eSSRs) discovered in the transcriptome of Chrysogonum virginianum ‘Chry_2’. Assembled RNA sequencing reads were screened for presence of di-, tri-, and tetra-motif repeat SSRs. Frequency (counts) of top 20 eSSR nonredundant motifs found in the assembled transcriptome are depicted in white; SSRs with primers constructed as per our algorithm are shown in gray. Insert: Frequency (counts) of di-, tri-, and tetra-motif repeat eSSRs. Note: Frequencies of nonredundant tetra-repeat eSSRs were not inspected before devising the primers for them. bp = base pair.

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    Linkage disequilibrium of the tested genic simple sequence repeats (eSSRs) of Chrysogonum virginianum. (A) Pairwise Index of Association (d) was tested to infer the eSSR distribution over C. virginianum genome by the feature of the probability of their cosegregation. The d values are color-coded, according to the legend depicted on the right side. (B) Collection-wise d was inferred over 1000 permutations of the entire dataset (n = 32). The overall d (black dashed line) and the corresponding P value are denoted.

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    Discriminant Analysis of Principal Components of the Chrysogonum virginianum genic simple sequence repeat (eSSR) dataset. All samples of C. virginianum used in this study are presented, with varieties labeled. The analysis was cross-checked by optimizing the number of principal components used from 1 to 32 (1000 permutations). C. virginianum ‘Pierre’ was added as a proof of concept for the subspecies placement by eSSR. The specimen ‘Chry_2’ that was used for the RNA sequencing is marked. (Insert) C. virginianum variety-wise unrooted tree of genetic distances. Samples used in the study were grouped by variety to infer the genetic distances [FST] among them (here, ranging from 0.02 to 0.11). The analysis was permuted 1000 times, and the bootstrap support [%] of each split is presented. PCA = principal components analysis.

Article References

  • Abu-AsabM.S.PetersonP.M.ShetlerS.G.OrliS.S.2001Earlier plant flowering in spring as a response to global warming in the Washington, DC, areaBiodivers. Conserv.10597612

    • Search Google Scholar
    • Export Citation
  • AmosW.HoffmanJ.FrodshamA.ZhangL.BestS.HillA.2007Automated binning of microsatellite alleles: Problems and solutionsMol. Ecol. Resour.71014

    • Search Google Scholar
    • Export Citation
  • AndrewsS.2014FastQC: A quality control tool for high throughput sequence data. Version 0.11.2. Babraham Institute Cambridge UK. 1 Nov. 2018. <http://www.bioinformatics.babraham.ac.uk/projects/fastqc>

  • ArcherF.I.AdamsP.E.SchneidersB.B.2017stratag: An R package for manipulating, summarizing and analysing population genetic dataMol. Ecol. Resour.17511

    • Search Google Scholar
    • Export Citation
  • AriasR.S.TechenN.RinehartT.A.OlsenR.T.KirkbrideJ.H.SchefflerB.E.2011Development of simple sequence repeat markers for Chionanthus retusus (Oleaceae) and effective discrimination of closely related taxaHortScience462329

    • Search Google Scholar
    • Export Citation
  • CBOL Plant Working Group2009A DNA barcode for land plantsProc. Natl. Acad. Sci. USA1061279412797

  • ChenX.SunM.LiangJ.XueH.ZhangQ.2013Genetic diversity of species of Chrysanthemum and related genera and groundcover cultivars assessed by amplified fragment length polymorphic markersHortScience48539546

    • Search Google Scholar
    • Export Citation
  • ClausenJ.KeckD.D.HieseyW.M.1939The concept of species based on experimentAmer. J. Bot.26103106

  • ClevingerJ.A.PaneroJ.L.2000Phylogenetic analysis of Silphium and subtribe Engelmanniinae (Asteraceae: Heliantheae) based on ITS and ETS sequence dataAmer. J. Bot.87565572

    • Search Google Scholar
    • Export Citation
  • DuminilJ.Di MicheleM.2009Plant species delimitation: A comparison of morphological and molecular markersPlant Biosyst.143528542

  • DupuisJ.R.RoeA.D.SperlingF.A.2012Multi-locus species delimitation in closely related animals and fungi: One marker is not enoughMol. Ecol.2144224436

    • Search Google Scholar
    • Export Citation
  • EllisJ.BurkeJ.2007EST-SSRs as a resource for population genetic analysesHeredity99125–132

  • Esfandani BozchaloyiS.SheidaiM.KeshavarziM.NoormohammadiZ.2017Species delimitation in Geranium sect. Batrachioidea: Morphological and molecularActa Bot. Hung.59319334

    • Search Google Scholar
    • Export Citation
  • EujaylI.SorrellsM.BaumM.WoltersP.PowellW.2001Assessment of genotypic variation among cultivated durum wheat based on EST-SSRs and genomic SSRsEuphytica1193943

    • Search Google Scholar
    • Export Citation
  • GaddyL.1986Twelve new ant-dispersed species from the southern AppalachiansBull. Torrey Bot. Club113247251

  • GoudetJ.2005Hierfstat, a package for R to compute and test hierarchical F-statisticsMol. Ecol. Resour.5184186

  • GoudetJ.RaymondM.de MeeüsT.RoussetF.1996Testing differentiation in diploid populationsGenetics14419331940

  • GrayA.1882Chrysogonum virginianum, var. dentatumBot. Gaz.73132

  • HawksK.T.2008Chrysogonum plant named ‘Quinn’s Gold’. U.S. Patent Application 11/906656 filed 7 Oct. 2008

  • HoffmanM.H.A.HopM.E.C.M.GeersF.A.M.2005List of names of perennials section Trees. International Standard 2005-2010. Applied Plant Research Lisse The Netherlands

  • JiangH.LeiR.DingS.-W.ZhuS.2014Skewer: A fast and accurate adapter trimmer for next-generation sequencing paired-end readsBMC Bioinformatics15182

    • Search Google Scholar
    • Export Citation
  • JombartT.DevillardS.BallouxF.2010Discriminant analysis of principal components: A new method for the analysis of genetically structured populationsBMC Genet.1194

    • Search Google Scholar
    • Export Citation
  • JombartT.KamvarZ.N.CollinsC.LustrikR.BeuginM.-P.KnausB.J.JombartM.T.2018Package ‘adegenet’. Github repository: <https://github.com/thibautjombart/adegenet>

  • KamvarZ.N.BrooksJ.C.GrünwaldN.J.2015Novel R tools for analysis of genome-wide population genetic data with emphasis on clonalityFront. Genet.6208

    • Search Google Scholar
    • Export Citation
  • KamvarZ.N.TabimaJ.F.GrünwaldN.J.2014Poppr: An R package for genetic analysis of populations with clonal, partially clonal, and/or sexual reproductionPeerJ2e281

    • Search Google Scholar
    • Export Citation
  • LinnaeusC.1754Genera plantarum exhibentes p. 391. 5th ed. Salvius Stockholm

  • LowryD.B.2012Ecotypes and the controversy over stages in the formation of new speciesBiol. J. Linn. Soc. Lond.106241257

  • MeudtH.M.LockhartP.J.BryantD.2009Species delimitation and phylogeny of a New Zealand plant species radiationBMC Evol. Biol.9111

  • MorgulisA.GertzE.M.SchäfferA.A.AgarwalaR.2006A fast and symmetric DUST implementation to mask low-complexity DNA sequencesJ. Comput. Biol.1310281040

    • Search Google Scholar
    • Export Citation
  • NesomG.1978Myrmecochory in Chrysogonum virginianum L. (Compositae)Bot. Soc. Amer. Abstr. Misc. Ser. Publ15621

  • NesomG.2001Taxonomic review of Chrysogonum (Asteraceae: Heliantheae)SIDA Contrib. Bot.19811820

  • PaneroJ.L.JansenR.K.1997Chloroplast DNA restriction site study of Verbesina (Asteraceae: Heliantheae)Amer. J. Bot.84382392

  • PaneroJ.L.JansenR.K.ClevingerJ.A.1999Phylogenetic relationships of subtribe Ecliptinae (Asteraceae: Heliantheae) based on chloroplast DNA restriction site dataAmer. J. Bot.86413427

    • Search Google Scholar
    • Export Citation
  • PeakallR.SmouseP.E.2012GenAlEx 6.5: Genetic analysis in Excel. Population genetic software for teaching and research—an updateBioinformatics2825372539

    • Search Google Scholar
    • Export Citation
  • PengY.GaoX.LiR.CaoG.2014Transcriptome sequencing and de novo analysis of Youngia japonica using the Illumina platformPLoS One9e90636

    • Search Google Scholar
    • Export Citation
  • ReedD.H.FrankhamR.2001How closely correlated are molecular and quantitative measures of genetic variation? A meta-analysisEvolution5510951103

    • Search Google Scholar
    • Export Citation
  • SegattoA.L.A.Reck-KortmannM.TurchettoC.FreitasL.B.2017Multiple markers, niche modelling, and bioregions analyses to evaluate the genetic diversity of a plant species complexBMC Evol. Biol.17234

    • Search Google Scholar
    • Export Citation
  • SimpsonJ.T.WongK.JackmanS.D.ScheinJ.E.JonesS.J.BirolI.2009ABySS: A parallel assembler for short read sequence dataGenome Res.1911171123

    • Search Google Scholar
    • Export Citation
  • SolbrigO.T.KyhosD.W.PowellM.RavenP.H.1972Chromosome numbers in Compositae VIII: HeliantheaeAmer. J. Bot.59869878

  • StatonM.E.FicklinS.2018Finding SSRs – findssrs_altered.pl Github repository: <https://github.com/statonlab/Finding-SSRs>

  • StuessyT.F.1973A systematic review of the subtribe Melampodiinae (Compositae Heliantheae). Contr. Gray Herb. 203:65–80

  • StuessyT.F.1977Revision of Chrysogonum (Compositae, Heliantheae)Rhodora79190202

  • The R Core Team2014R: A language and environment for statistical computing. R Foundation for Statistical Computing Vienna Austria

  • TrigianoR.BoggessS.BernardE.2018aFirst report of powdery mildew caused by Golovinomyces spadiceus on Green and Gold (Chrysogonum virginianum) in the United StatesPlant Dis.102252

    • Search Google Scholar
    • Export Citation
  • TrigianoR.BoggessS.OwnleyB.2018bFirst report of an aerial blight of Chrysogonum virginianum (Green and Gold) caused by Sclerotinia sclerotiorum in the United StatesPlant Dis.102450

    • Search Google Scholar
    • Export Citation
  • UntergasserA.CutcutacheI.KoressaarT.YeJ.FairclothB.C.RemmM.RozenS.G.2012Primer3—new capabilities and interfacesNucleic Acids Res.40e115

    • Search Google Scholar
    • Export Citation
  • VarshneyR.K.GranerA.SorrellsM.E.2005Genic microsatellite markers in plants: Features and applicationsTrends Biotechnol.234855

  • VarshneyR.K.ThielT.SteinN.LangridgeP.GranerA.2002In silico analysis on frequency and distribution of microsatellites in ESTs of some cereal speciesCell. Mol. Biol. Lett.7537546

    • Search Google Scholar
    • Export Citation
  • WeakleyA.S.2015Flora of the southern and mid-Atlantic states. University of North Carolina Herbarium North Carolina Botanical Garden University of North Carolina Chapel Hill

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