Conservation Genetics Resources

, Volume 8, Issue 2, pp 145–158 | Cite as

Genome-wide SNP discovery in the annual herb, Lasthenia fremontii (Asteraceae): genetic resources for the conservation and restoration of a California vernal pool endemic

  • Lorena Torres-Martínez
  • Nancy C. EmeryEmail author
Methods and Resources Article


California vernal pool (VP) ecosystems support a diverse community of endemic plants that are threatened by multiple anthropogenic pressures, generating a need for molecular tools to quantify the extent and distribution of genetic variation in native populations. Here, we used RADseq to discover single nucleotide polymorphisms (SNPs) for a widespread VP endemic plant species, Lasthenia fremontii. We discovered nuclear-based SNPs using a RAD-tag library of 12 individuals from different VP complexes using SbfI, a restriction enzyme that does not cleave the chloroplast genome in Lasthenia. A total of 316,106 catalog loci were obtained across all twelve individuals in the library. Of these, 713 loci were polymorphic, yielding 3918 candidate SNPs. Next, we genotyped an additional 285 additional plants to validate and characterize 71 of the candidate SNPs. Of these, 44 were polymorphic among VP complexes. A preliminary analysis of the distribution of genetic variation using these loci revealed significant isolation-by-distance across the species’ geographic range. Weaker, but in some cases significant, genetic differentiation was detected among subpopulations from different pools within a single VP complex. Thus, in this study, RADseq allowed the discovery of SNP markers that can characterize patterns of genetic variation at multiple spatial scales in L. fremontii, which can be used to inform the conservation and mitigation of VP populations.


Asteraceae California endemic Conservation genetics Ephemeral wetlands Lasthenia fremontii Population genetic structure RAD sequencing Vernal pools 



The authors gratefully acknowledge M. Hale and T. Hartwig for assistance standardizing the RAD-tag library, G. McKinney, M. Gribskov, and J. Thimmapuram for help with data analysis, and M. Levy for providing comments on an early draft of this manuscript. This work was supported by an NSF Doctoral Dissertation Improvement Grant (DEB # 1407011).

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  1. Baird NA, Etter PD, Atwood TS et al (2008) Rapid SNP discovery and genetic mapping using sequenced RAD markers. PLoS ONE 3:1–7. doi: 10.1371/journal.pone.0003376 CrossRefGoogle Scholar
  2. Barbour MG, Solomeshch AI, Holland RF et al (2005) Vernal pool vegetation of California: communities of long-inundated deep habitats. Phytocoenologia 35:177–200. doi: 10.1127/0340-269X/2005/0035-0177 CrossRefGoogle Scholar
  3. Barbour MG, Solomeshch AI, Buck-diaz JJ (2007) Classification, ecological characterization, and presence of listed plant taxa of vernal pool associations in California. United States and Wildlife service agreement study, Report No 814205G238. University of California, Davis, CA, pp 1–146Google Scholar
  4. Barchi L, Lanteri S, Portis E et al (2011) Identification of SNP and SSR markers in eggplant using RAD tag sequencing. BMC Genom 12:304. doi: 10.1186/1471-2164-12-304 CrossRefGoogle Scholar
  5. Baxter SW, Davey JW, Johnston JS et al (2011) Linkage mapping and comparative genomics using next-generation rad sequencing of a non-model organism. PLoS ONE 6:e19315. doi: 10.1371/journal.pone.0019315 CrossRefPubMedPubMedCentralGoogle Scholar
  6. Bolger AM, Lohse M, Usadel B (2014) Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30:2114–2120. doi: 10.1093/bioinformatics/btu170 CrossRefPubMedPubMedCentralGoogle Scholar
  7. Catchen JM, Amores A, Hohenlohe P et al (2011) Stacks: building and genotyping loci de novo from short-read sequences. G3: Genes Genomes Genetics 1:171–182. doi: 10.1534/g3.111.000240
  8. Catchen J, Bassham S, Wilson T et al (2013a) The population structure and recent colonization history of Oregon threespine stickleback determined using restriction-site associated DNA-sequencing. Mol Ecol 22:2864–2883. doi: 10.1111/mec.12330 CrossRefPubMedPubMedCentralGoogle Scholar
  9. Catchen J, Hohenlohe PA, Bassham S et al (2013b) Stacks: an analysis tool set for population genomics. Mol Ecol 22:3124–3140. doi: 10.1111/mec.12354 CrossRefPubMedPubMedCentralGoogle Scholar
  10. Chan R, Baldwin BG, Ornduff R (2001) Goldfields revisited: a molecular phylogenetic perspective on the evolution of Lasthenia (Compositae : Heliantheae sensu lato). Int J Plant Sci 162:1347–1360. doi: 10.1086/323277 CrossRefGoogle Scholar
  11. Corander J, Majander KK, Cheng L, Merilä J (2013) High degree of cryptic population differentiation in the Baltic Sea herring Clupea harengus. Mol Ecol 22:2931–2940. doi: 10.1111/mec.12174 CrossRefPubMedGoogle Scholar
  12. Crawford DJ, Ornduff R (1989) Enzyme Electropheresis and Evolutionary relationships among three species of Lasthenia (Asteraceae: Heliantheae). Am J Bot 76:289–296CrossRefGoogle Scholar
  13. Davey JL, Blaxter MW (2011) RADseq: next-generation population genetics. Brief Funct Genomics 9:416–423. doi: 10.1093/bfgp/elq031 CrossRefPubMedCentralGoogle Scholar
  14. Deokar A, Ramsay L, Sharpe AG et al (2014) Genome wide SNP identification in chickpea for use in development of a high density genetic map and improvement of chickpea reference genome assembly. BMC Genom 15:708. doi: 10.1186/1471-2164-15-708 CrossRefGoogle Scholar
  15. Eaton D, Ree R (2013) Inferring phylogeny and introgression using RADseq data: an example from flowering plants (Pedicularis: Orobanchaceae). Syst Biol 0:1–18. doi: 10.5061/dryad.bn281
  16. Elam DR (1998) Population genetics of vernal pool plants: theory, data and conservation implications. In: Witham CW, Bauder ET, Belk D, Ferren WR Jr, Ornduff R (eds) Ecology, conservation, and management of vernal pool ecosystems—proceedings from a 1996 conference. California Native Plant Society, SacramentoGoogle Scholar
  17. Emerson KJ, Merz CR, Catchen JM et al (2010) Resolving postglacial phylogeography using high-throughput sequencing. Proc Natl Acad Sci USA 107:16196–16200. doi: 10.1073/pnas.1006538107 CrossRefPubMedPubMedCentralGoogle Scholar
  18. Emery NC (2009) Ecological limits and fitness consequences of cross-gradient pollen movement in Lasthenia fremontii. Am Nat 174:221–235. doi: 10.1086/600089 CrossRefPubMedGoogle Scholar
  19. Emery NC, Rice KJ, Stanton ML (2011a) Fitness variation and local distribution limits in an annual plant population. Evolution (NY) 65:1011–1020. doi: 10.1111/j.1558-5646.2010.01183.x CrossRefGoogle Scholar
  20. Emery NC, Torres-Martínez L, Forrestel E, Baldwin BG, Ackerly DD (2011b) The ecology, evolution and diversification of the vernal pool niche in Lasthenia (Madieae, Asteraceae). In: Alexander DG, Schlising RA (eds) Research and recovery in vernal pool landscapes. Studies from the Herbarium. Number 16. California State University, Chico, pp 39–57 Google Scholar
  21. Excoffier L, Lischer HEL (2010) Arlequin suite ver 3.5: a new series of programs to perform population genetics analyses under Linux and Windows. Mol Ecol Resour 10:564–567. doi: 10.1111/j.1755-0998.2010.02847.x CrossRefPubMedGoogle Scholar
  22. Faircloth BC, Glenn TC (2012) Not all sequence tags are created equal: designing and validating sequence identification tags robust to indels. PLoS ONE. doi: 10.1371/journal.pone.0042543 PubMedPubMedCentralGoogle Scholar
  23. Gaither MR, Bernal MA, Coleman RR et al (2015) Genomic signatures of geographic isolation and natural selection in coral reef fishes. Mol Ecol 24:1543–1557. doi: 10.1111/mec.13129 CrossRefPubMedGoogle Scholar
  24. Griggs FT, Jain SK (1983) Conservation of vernal pool plants in California: population biology of a rare and unique grass genus Orcuttia. Biol Conserv 27:171–193CrossRefGoogle Scholar
  25. Halbur MM, Sloop CM, Zanis MJ, Emery NC (2014) The population biology of mitigation: impacts of habitat creation on an endangered plant species. Conserv Genet 15:679–695. doi: 10.1007/s10592-014-0569-0 CrossRefGoogle Scholar
  26. Harter AV, Gardner KA, Falush D et al (2004) Origin of extant domesticated sunflowers in eastern North America. Nature 430:201–205. doi: 10.1038/nature02710 CrossRefPubMedGoogle Scholar
  27. Hecht BC, Matala AP, Hess JE, Narum SR (2015) Environmental adaptation in Chinook salmon (Oncorhynchus tshawytscha) throughout their North American range. Mol Ecol 24:5573–5595. doi: 10.1111/mec.13409 CrossRefPubMedGoogle Scholar
  28. Hohenlohe PA, Amish SJ, Catchen JM et al (2011) Next-generation RAD sequencing identifies thousands of SNPs for assessing hybridization between rainbow and westslope cutthroat trout. Mol Ecol Resour 11:117–122. doi: 10.1111/j.1755-0998.2010.02967.x CrossRefPubMedGoogle Scholar
  29. Holland RF, Jain SK (1981) Insular biogeography of vernal pools in the Central Valley of California. Amer Nat 117:24–37CrossRefGoogle Scholar
  30. Jombart T (2008) Adegenet: a R package for the multivariate analysis of genetic markers. Bioinformatics 24:1403–1405. doi: 10.1093/bioinformatics/btn129 CrossRefPubMedGoogle Scholar
  31. Jombart T, Ahmed I (2011) Adegenet 1. 3–1: new tools for the analysis of genome-wide SNP data. Bioinformatics 27:3070–3071. doi: 10.1093/bioinformatics/btr521 CrossRefPubMedPubMedCentralGoogle Scholar
  32. Karam M-J, Lefèvre F, Dagher-Kharrat MB et al (2015) Genomic exploration and molecular marker development in a large and complex conifer genome using RADseq and mRNAseq. Mol Ecol Resour 15:601–612. doi: 10.1111/1755-0998.12329 CrossRefPubMedGoogle Scholar
  33. Kardos M, Luikart G, Allendorf FW (2015) Measuring individual inbreeding in the age of genomics: marker-based measures are better than pedigrees. Heredity (Edinb) 115:63–72. doi: 10.1038/hdy.2015.17 CrossRefGoogle Scholar
  34. Keeler-Wolf T, Elam DR, Lewis K, Flint SA (1998) California vernal pool assessment: preliminary report. Department of Fish and Game, SacramentoGoogle Scholar
  35. Keenan K, Mcginnity P, Cross TF et al (2013) DiveRsity: an R package for the estimation and exploration of population genetics parameters and their associated errors. Methods Ecol Evol 4:782–788. doi: 10.1111/2041-210X.12067 CrossRefGoogle Scholar
  36. Lai Z, Kane NC, Kozik A et al (2012) Genomics of compositae weeds: EST libraries, microarrays, and evidence of introgression. Am J Bot 99:209–218. doi: 10.3732/ajb.1100313 CrossRefPubMedGoogle Scholar
  37. Loarie SR, Duffy PB, Hamilton H et al (2009) The velocity of climate change. Nature 462:1052–1055. doi: 10.1038/nature08649 CrossRefPubMedGoogle Scholar
  38. Mastretta-Yanes A, Arrigo N, Alvarez N et al (2014) Restriction site-associated DNA sequencing, genotyping error estimation and de novo assembly optimization for population genetic inference. Mol Ecol Resourc 1–14. doi: 10.1111/1755-0998.12291
  39. Miller M, Dunham J, Amores A et al (2007) Rapid and cost-effective polymorphism identification and genotyping using restriction site associated DNA (RAD) markers. Genome Res 17:240–248. doi: 10.1101/gr.5681207 CrossRefPubMedPubMedCentralGoogle Scholar
  40. Morin PA, Luikart G, Wayne RK (2004) SNPs in ecology, evolution and conservation. Trends Ecol Evol 19:208–216. doi: 10.1016/j.tree.2004.01.009 CrossRefGoogle Scholar
  41. Morin PA, Martien KK, Taylor BL (2009) Assessing statistical power of SNPs for population structure and conservation studies. Mol Ecol Resour 9:66–73. doi: 10.1111/j.1755-0998.2008.02392.x CrossRefPubMedGoogle Scholar
  42. Narum SR, Buerkle CA, Davey JW et al (2013) Genotyping-by-sequencing in ecological and conservation genomics. Mol Ecol 22:2841–2847. doi: 10.1111/mec.12350
  43. Ornduff R (1966) A biosystematic survey of the Goldfield Genus Lasthenia. University of California Publications in Botany, vol 40, p 92Google Scholar
  44. Peakall R, Smouse PE (2006) GENALEX 6: genetic analysis in Excel. Population genetic software for teaching and research. Mol Ecol Notes 6:288–295. doi: 10.1111/j.1471-8286.2005.01155.x CrossRefGoogle Scholar
  45. Peakall R, Smouse PE (2012) GenALEx 6.5: genetic analysis in Excel. Population genetic software for teaching and research-an update. Bioinformatics 28:2537–2539. doi: 10.1093/bioinformatics/bts460 CrossRefPubMedPubMedCentralGoogle Scholar
  46. Pegadaraju V, Nipper R, Hulke B et al (2013) De novo sequencing of sunflower genome for SNP discovery using RAD (Restriction site Associated DNA) approach. BMC Genom 14:556. doi: 10.1186/1471-2164-14-556 CrossRefGoogle Scholar
  47. Petrov DA, Hartl DL (1999) Patterns of nucleotide substitution in Drosophila and mammalian genomes. Proc Natl Acad Sci USA 96:1475–1479. doi: 10.1073/pnas.96.4.1475 CrossRefPubMedPubMedCentralGoogle Scholar
  48. Pujolar JM, Jacobsen MW, Frydenberg J et al (2013) A resource of genome-wide single-nucleotide polymorphisms generated by RAD tag sequencing in the critically endangered European eel. Mol Ecol Resour 13:706–714. doi: 10.1111/1755-0998.12117 CrossRefPubMedGoogle Scholar
  49. Rajakaruna N (2003) Edaphic differentiation in Lasthenia: a model for studies in evolutionary ecology. Madroño 50:34–40Google Scholar
  50. Ramp JM, Collinge SK, Ranker TA (2006) Restoration genetics of the vernal pool endemic Lasthenia conjugens (Asteraceae). Conserv Genet 7:631–649. doi: 10.1007/s10592-005-9052-2
  51. Ramp Neale JM, Ranker TA, Collinge SK (2008) Conservation of rare species with island-like distributions: a case study of Lasthenia conjugens (Asteraceae) using population genetic structure and the distribution of rare markers. Plant Species Biol 23:97–110. doi: 10.1111/j.1442-1984.2008.00211.x CrossRefGoogle Scholar
  52. Reitzel AM, Herrera S, Layden MJ et al (2013) Going where traditional markers have not gone before: utility of and promise for RAD sequencing in marine invertebrate phylogeography and population genomics. Mol Ecol 22:2953–2970. doi: 10.1111/mec.12228 CrossRefPubMedPubMedCentralGoogle Scholar
  53. Scaglione D, Acquadro A, Portis E et al (2012) RAD tag sequencing as a source of SNP markers in Cynara cardunculus L. BMC Genom 13:3. doi: 10.1186/1471-2164-13-3 CrossRefGoogle Scholar
  54. Semagn K, Babu R, Hearne S, Olsen M (2014) Single nucleotide polymorphism genotyping using Kompetitive Allele Specific PCR (KASP): overview of the technology and its application in crop improvement. Mol Breed 33:1–14. doi: 10.1007/s11032-013-9917-x CrossRefGoogle Scholar
  55. Slatkin M (1995) A measure of population subdivision based on microsatellite allele frequencies. Genetics 139:457–462PubMedPubMedCentralGoogle Scholar
  56. Slatkin M, Takahata N (1985) The average frequency of private alleles in a partially isolated population. Theor Popul Biol 28:314–331. doi: 10.1016/0040-5809(85)90032-2 CrossRefGoogle Scholar
  57. Sloop CM, Ayres DR (2010) Conservation genetics of two endangered vernal pool plants of the Santa Rosa plain, Sonoma County, CA. In: Proceedings of the CNPS conservation conference, California Plant SocietyGoogle Scholar
  58. Sloop CM, Eberl R, Ayres DR (2012) Genetic diversity and structure in the annual vernal pool endemic Limnanthes vinculans Ornduff (Limnanthaceae): implications of breeding system and restoration practices. Conserv Genet 13:1365–1379. doi: 10.1007/s10592-012-0380-8 CrossRefGoogle Scholar
  59. Staton SE, Bakken BH, Blackman BK, Chapman MA, Kane NC, Tang S, Ungerer MC, Knapp SJ, Rieseberg LH, Burke JM (2012) The sunflower (Helianthus annuus L.) genome reflects a recent history of biased accumulation of transposable elements. Plant J 72:142–153Google Scholar
  60. Stone DR (1990) California’s endemic vernal pool plants: some factors influencing their rarity and endangerment In: Ikeda DH, Schlising RA (eds) Vernal pool plants—their habitat and biology. California State University, Studies from the Herbarium No. 8, California State University, Chico, pp 89–107Google Scholar
  61. Walker JF, Zanis MJ, Emery NC (2014) Comparative analysis of complete chloroplast genome sequence and inversion variation in Lasthenia burkei (Madieae, Asteraceae). Am J Bot 101:722–729. doi: 10.3732/ajb.1400049 CrossRefPubMedGoogle Scholar
  62. Weir BS, Cockerham CC (1984) Estimating F-Statistics for the analysis of population structure. Evolution 38:1358–1370CrossRefGoogle Scholar
  63. Wu J, Li L-T, Li M et al (2014) High-density genetic linkage map construction and identification of fruit-related QTLs in pear using SNP and SSR markers. J Exp Bot 65:eru311. doi: 10.1093/jxb/eru311
  64. Xu P, Xu S, Wu X et al (2014) Population genomic analyses from low-coverage RAD-Seq data: a case study on the non-model cucurbit bottle gourd. Plant J 77:430–442. doi: 10.1111/tpj.12370 CrossRefPubMedGoogle Scholar
  65. Zedler PH (2003) Vernal pools and the concept of “isolated wetlands”. Wetlands 23:597–607. doi: 10.1672/0277 CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2016

Authors and Affiliations

  1. 1.Department of Biological SciencesPurdue UniversityWest LafayetteUSA
  2. 2.Department of Ecology and Evolutionary BiologyUniversity of Colorado BoulderBoulderUSA

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