Tree Genetics & Genomes

, Volume 5, Issue 1, pp 247–255 | Cite as

Identification of COS markers in the Pinaceae

  • Cherdsak Liewlaksaneeyanawin
  • Jun Zhuang
  • Michelle Tang
  • Nima Farzaneh
  • Gillian Lueng
  • Claire Cullis
  • Susan Findlay
  • Carol E. Ritland
  • Jörg Bohlmann
  • Kermit RitlandEmail author
Original Paper


Conserved ortholog set (COS) markers are evolutionary conserved, single-copy genes, identified from large databases of express sequence tags (ESTs). They are of particular use for constructing syntenic genetic maps among species. In this study, we identified a set of 1,813 putative single-copy COS markers between spruce and loblolly pine, then designed primers for 931 of these markers and tested these primers with DNA from spruce, pine, and Douglas fir. Of these 931 primers, 56% (524) amplified a product in both spruce and pine, and 71% (373) of these were single-banded; 224 amplicons were single-banded in all three species. Even though these COS markers were selected from large EST databases, a substantial proportion (20–30%) of amplicons displayed multiple bands or smears, suggesting significant paralogy. Sequencing of three single-banded amplicons showed high nucleotide similarities among 29 conifer species, suggesting orthology of single-banded amplicons. Screening for COS marker polymorphism in two pedigrees of white spruce and two pedigrees of loblolly pine revealed an average informativeness of 36% for spruce and 24% for pine (e.g., at least one parent was heterozygous for a single-nucleotide polymorphism within the entire amplified product). This corresponds to an average nucleotide heterozygosity of 0.05% and 0.03%, respectively, which is considerably lower than that found in other studies of spruce and pine. Thus, the advantages of COS markers for constructing syntenic maps are offset by their lower polymorphism.


COS markers Conifers 



This work was funded by the Genome BC/Genome Canada “Conifer Forest Health Genomics” project to J.B. and K.R. We sincerely thank Barry Jaquish from the British Columbia Ministry of Forests for providing the spruce crosses, Craig Echt from USDA Forest Service for contributing the DNA of loblolly pine crosses, and Tristan Gillan from the University of British Columbia for sampling plant materials. We acknowledge the support of the Vancouver Genome Sciences Center for sequencing.

Supplementary material

11295_2008_189_MOESM1_ESM.xls (669 kb)
Table S1 Total of 1,815 putative single-copy COS between spruce and loblolly pine (XLS 672 KB)
11295_2008_189_MOESM2_ESM.xls (468 kb)
Table S2 Test of COS primers in loblolly pine (Lp), white spruce (Ws), and Douglas fir (Df). A = approximately size to the nearest 50 bp of the product size including forward (19 bp) and reverse (20) primer tails. 0 = no product, 1 = single product, 2 = multiple products with clear band, 3 = multiple products with smear band (XLS 468 KB)
11295_2008_189_Fig1_ESM.gif (254 kb)
Supplementary Fig. 1

Sequence alignment of COS marker at locus C20992 amplified from 29 conifer species. Loblolly pine and spruce EST sequences are indicated by EST. The remaining sequences are genomic sequences. The dots indicate conserved nucleotides and dashed lines indicate indels (relative to P. menziesii) (GIF 256 KB)

11295_2008_189_Fig1_ESM.tif (2.2 mb)
High-resolution image (TIF 2.16 MB)


  1. Achere V, Faivre-Rampant P, Jeandroz S, Besnard G, Markussen T, Aragones A, Fladung M, Ritter E, Favre JM (2004) A full saturated linkage map of Picea abies including AFLP, SSR, ESTP, 5S rDNA and morphological markers. Theor Appl Genet 108:1602–1613PubMedCrossRefGoogle Scholar
  2. Ahuja MR, Neale DB (2005) Evolution of genome size in conifers. Silvae Genet 54:126–137Google Scholar
  3. Bouille M, Bousquet J (2005) Trans-species shared polymorphisms at orthologous nuclear gene loci among distant species in the conifer Picea (Pinaceae): implications for the long-term maintenance of genetic diversity in trees. Am J Bot 92:63–73CrossRefGoogle Scholar
  4. Brown GR, Kadel EE, Bassoni DL, Kiehne KL, Temesgen B, van Buijtenen JP, Sewell MM, Marshall KA, Neale DB (2001) Anchored reference loci in loblolly pine (Pinus taeda L.) for integrating pine genomics. Genetics 159:799–809PubMedGoogle Scholar
  5. Brown GR, Gill GP, Kuntz RJ, Langley CH, Neale DB (2004) Nucleotide diversity and linkage disequilibrium in loblolly pine. Proc Natl Acad Sci U S A 101:15255–15260PubMedCrossRefGoogle Scholar
  6. Chagné D, Brown G, Lalanne C, Madur D, Pot D, Neale D, Plomion C (2003) Comparative genome and QTL mapping between maritime and loblolly pines. Mol Breed 12:185–195CrossRefGoogle Scholar
  7. Chapman MA, Chang J, Weisman D, Kesseli RV, Burke JM (2007) Universal markers for comparative mapping and phylogenetic analysis in the Asteraceae (Compositae). Theor Appl Genet 115:747–755PubMedCrossRefGoogle Scholar
  8. Devey ME, Sewell MM, Uren TL, Neale DB (1999) Comparative mapping in loblolly and radiata pine using RFLP and microsatellite markers. Theor Appl Genet 99:656–662CrossRefGoogle Scholar
  9. Doyle JJ, Doyle JL (1987) A rapid DNA isolation procedure for small quantities of fresh tissue. Phytochem Bull 19:11–15Google Scholar
  10. Emrich SJ, Li L, Wen T-J, Yandeau-Nelson MD, Fu Y, Guo L, Chou H-H, Aluru S, Ashlock DA, Schnable PS (2007) Nearly identical paralogs: implications for maize (Zea mays L.) genome evolution. Genetics 175:429–439PubMedCrossRefGoogle Scholar
  11. Farjon A (2001) World checklist and bibliography of conifers, 2nd edn. Kew Publishing, Kew, p 310Google Scholar
  12. Florin R (1963) The distribution of conifer and taxad genera in time and space. Acta Horti Bergiani 20:121–312Google Scholar
  13. Fredslund J, Madsen L, Hougaard B, Nielsen A, Bertioli D, Sandal N, Stougaard J, Schauser L (2006) A general pipeline for the development of anchor markers for comparative genomics in plants. BMC Genomics 7:207PubMedCrossRefGoogle Scholar
  14. Fulton TM, Van der Hoeven R, Eannetta NT, Tanksley SD (2002) Identification, analysis, and utilization of conserved ortholog set markers for comparative genomics in higher plants. Plant Cell 14:1457–1467PubMedCrossRefGoogle Scholar
  15. Gernandt DS, Lopez GG, Garcia SO, Liston A (2005) Phylogeny and classification of Pinus. Taxon 54:29–42Google Scholar
  16. Hughes CE, Eastwood RJ, Donovan Bailey C (2006) From famine to feast? Selecting nuclear DNA sequence loci for plant species-level phylogeny reconstruction. Philos Trans R Soc Lond Ser B Biol Sci 361:211–225CrossRefGoogle Scholar
  17. Kinlaw CS, Neale DB (1997) Complex gene families in pine genomes. Trends Plant Sci 2:356–359CrossRefGoogle Scholar
  18. Komulainen P, Brown GR, Mikkonen M, Karhu A, García-Gil MR, O’Malley D, Lee B, Neale DB, Savolainen O (2003) Comparing EST-based genetic maps between Pinus sylvestris and Pinus taeda. Theor Appl Genet 107:667–678PubMedCrossRefGoogle Scholar
  19. Krutovsky KV, Troggio M, Brown GR, Jermstad KD, Neale DB (2004) Comparative mapping in the Pinaceae. Genetics 168:447–461PubMedCrossRefGoogle Scholar
  20. Krutovsky KV, Elsik CG, Matvienko M, Kozik A, Neale DB (2006) Conserved ortholog sets in forest trees. Tree Genet Genom 3:61–70CrossRefGoogle Scholar
  21. Lin H, Zhu W, Silva JC, Gu X, Buell CR (2006) Intron gain and loss in segmentally duplicated genes in rice. Genome Biol 7:R41PubMedCrossRefGoogle Scholar
  22. Pelgas B, Beauseigle S, Achere V, Jeandroz S, Bousquet J, Isabel N (2006) Comparative genome mapping among Picea glauca, P. mariana x P. rubens and P. abies, and correspondence with other Pinaceae. Theor Appl Genet 113:1371–1393PubMedCrossRefGoogle Scholar
  23. Perry DJ, Bousquet J (1998) Sequence-tagged-site (STS) markers of arbitrary genes: the utility of black spruce-derived STS primers in other conifers. Theor Appl Genet 97:735–743CrossRefGoogle Scholar
  24. Ralph SG, Yueh H, Friedmann M, Aeschliman D, Zeznik JA, Nelson CC, Butterfield YS, Kirkpatrick R, Liu J, Jones SJ, Marra MA, Douglas CJ, Ritland K, Bohlmann J (2006) Conifer defence against insects: microarray gene expression profiling of Sitka spruce (Picea sitchensis) induced by mechanical wounding or feeding by spruce budworms (Choristoneura occidentalis) or white pine weevils (Pissodes strobi) reveals large-scale changes of the host transcriptome. Plant Cell Environ 29:1545–1570PubMedCrossRefGoogle Scholar
  25. Roy SW, Gilbert W (2005) Rates of intron loss and gain: implications for early eukaryotic evolution. Proc Natl Acad Sci U S A 102:5773–5778PubMedCrossRefGoogle Scholar
  26. Roy SW, Penny D (2007) Patterns of intron loss and gain in plants: intron loss-dominated evolution and genome-wide comparison of O. sativa and A. thaliana. Mol Biol Evol 24:171–181PubMedCrossRefGoogle Scholar
  27. Rozas J, Sanchez-DelBarrio JC, Messeguer X, Rozas R (2003) DnaSP, DNA polymorphism analyses by the coalescent and other methods. Bioinformatics 19:2496–2497PubMedCrossRefGoogle Scholar
  28. Rozen S, Skaletsky H (2000) Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol 132:365–386PubMedGoogle Scholar
  29. Rungis D, Hamberger B, Berube Y, Wilkin J, Bohlmann J, Ritland K (2005) Efficient genetic mapping of single nucleotide polymorphisms based upon DNA mismatch digestion. Mol Breed 16:261–270CrossRefGoogle Scholar
  30. Syring J, Willyard A, Cronn R, Liston A (2005) Evolutionary relationships among Pinus (Pinaceae) subsections inferred from multiple low-copy nuclear loci. Am J Bot 92:2086–2100CrossRefGoogle Scholar
  31. Wu F, Mueller LA, Crouzillat D, Petiard V, Tanksley SD (2006) Combining bioinformatics and phylogenetics to identify large sets of single-copy orthologous genes (COSII) for comparative, evolutionary and systematic studies: a test case in the euasterid plant clade. Genetics 174:1407–1420PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  • Cherdsak Liewlaksaneeyanawin
    • 1
  • Jun Zhuang
    • 2
  • Michelle Tang
    • 1
  • Nima Farzaneh
    • 1
  • Gillian Lueng
    • 1
  • Claire Cullis
    • 1
  • Susan Findlay
    • 1
  • Carol E. Ritland
    • 1
  • Jörg Bohlmann
    • 3
  • Kermit Ritland
    • 1
    Email author
  1. 1.Department of Forest SciencesUniversity of British ColumbiaVancouverCanada
  2. 2.The Institute for Genomic ResearchRockvilleUSA
  3. 3.Michael Smith LaboratoriesUniversity of British ColumbiaVancouverCanada

Personalised recommendations