Gene and whole-genome duplications are widespread in plant nuclear genomes, resulting in sequence heterogeneity. Identification of duplicated genes may be particularly challenging in highly redundant genomes, especially when there are no diploid parents as a reference. Here, we developed a pipeline to detect the different copies in the ribosomal RNA gene family in the hexaploid grass Spartina maritima from next-generation sequencing (Roche-454) reads. The heterogeneity of the different domains of the highly repeated 45S unit was explored by identifying single nucleotide polymorphisms (SNPs) and assembling reads based on shared polymorphisms. SNPs were validated using comparisons with Illumina sequence data sets and by cloning and Sanger (re)sequencing. Using this approach, 29 validated polymorphisms and 11 validated haplotypes were reported (out of 34 and 20, respectively, that were initially predicted by our program). The rDNA domains of S. maritima have similar lengths as those found in other Poaceae, apart from the 5'-ETS, which is approximately two-times longer in S. maritima. Sequence homogeneity was encountered in coding regions and both internal transcribed spacers (ITS), whereas high intragenomic variability was detected in the intergenic spacer (IGS) and the external transcribed spacer (ETS). Molecular cytogenetic analysis by fluorescent in situ hybridization (FISH) revealed the presence of one pair of 45S rDNA signals on the chromosomes of S. maritima instead of three expected pairs for a hexaploid genome, indicating loss of duplicated homeologous loci through the diploidization process. The procedure developed here may be used at any ploidy level and using different sequencing technologies.

Biology Centre ASCR Institute of Plant Molecular Biology Branišovská 31 České Budějovice CZ 37005 Czech Republic

UMR CNRS 6553 Ecobio OSUR University of Rennes 1 Bât 14A Campus Scientifique de Beaulieu 35 042 Rennes Cedex France

UMR CNRS 6553 Ecobio OSUR University of Rennes 1 Bât 14A Campus Scientifique de Beaulieu 35 042 Rennes Cedex France UMR Institut de Génétique Environnement et Protection des Plantes Institut National de la Recherche Agronomique BP35327 35653 Le Rheu Cedex France

UMR Institut de Génétique Environnement et Protection des Plantes Institut National de la Recherche Agronomique BP35327 35653 Le Rheu Cedex France

Zobrazit více v PubMed

Ainouche M. L., Chelaifa H., Ferreira de Carvalho J., Bellot S., Ainouche A. K., et al. , 2012.  Polyploid evolution in Spartina: Dealing with highly redundant hybrid genomes, pp. 225–243 in Polyploidy and Genome Evolution edited by E. Douglas, Springer, Berlin, Heidelberg.

Ainouche M. L., Fortune P. M., Salmon A., Parisod C., Grandbastien M.-A., et al. , 2008.  Hybridization, polyploidy and invasion: lessons from Spartina (Poaceae). Biol. Invasions 11: 1159–1173.

Akhunova A. R., Matniyazov R. T., Liang H., Akhunov E. D., 2010.  Homoeolog-specific transcriptional bias in allopolyploid wheat. BMC Genomics 11: 505. PubMed PMC

Altschul S. F., Madden T. L., Schäffer A. A., Zhang J., Zhang Z., et al. , 1997.  Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25: 3389–3402. PubMed PMC

Álvarez I., Wendel J. F., 2003.  Ribosomal ITS sequences and plant phylogenetic inference. Mol. Phylogenet. Evol. 29: 417–434. PubMed

Badaeva E. D., Friebe B., Gill B. S., 1996.  Genome differentiation in Aegilops. 2. Physical mapping of 5S and 18S–26S ribosomal RNA gene families in diploid species. Genome 39: 1150–1158. PubMed

Barker R. F., Harberd N. P., Jarvis M. G., Flavell R. B., 1988.  Structure and evolution of the intergenic region in a ribosomal DNA repeat unit of wheat. J. Mol. Biol. 201: 1–17. PubMed

Baumel A., Ainouche M. L., Levasseur J. E., 2001.  Molecular investigations in populations of Spartina anglica CE Hubbard (Poaceae) invading coastal Brittany (France). Mol. Ecol. 10: 1689–1701. PubMed

Baumel A., Ainouche M. L., Bayer R. J., Ainouche A. K., Misset M. T., 2002.  molecular phylogeny of hybridizing species from the genus Spartina Schreb. (Poaceae). Mol. Phylogenet. Evol. 22: 303–314. PubMed

Bena G., Jubier M.-F., Olivieri I., Lejeune B., 1998.  Ribosomal external and internal transcribed spacers: combined use in the phylogenetic analysis of Medicago (Leguminosae). J. Mol. Evol. 46: 299–306. PubMed

Chalhoub B., Denoeud F., Liu S., Parkin I. A. P., Tang H., et al. , 2014.  Early allopolyploid evolution in the post-Neolithic Brassica napus oilseed genome. Science 345: 950–953. PubMed

Chang K.-D., Fang S.-A., Chang F.-C., Chung M.-C., 2010.  Chromosomal conservation and sequence diversity of ribosomal RNA genes of two distant Oryza species. Genomics 96: 181–190. PubMed

Combes M.-C., Cenci A., Baraille H., Bertrand B., Lashermes P., 2011.  Homeologous gene expression in response to growing temperature in a recent allopolyploid (Coffea arabica L.). J. Hered. 103: 36–46. PubMed

Duchemin W., Dupont P.-Y., Campbell M. A., Ganley A. R., Cox M. P., 2014.  HyLiTE: accurate and flexible analysis of gene expression in hybrid and allopolyploid species. BMC Bioinformatics 16: 8. PubMed PMC

El-Metwally S., Ouda O. M., Helmy M., 2014.  Next generation sequencing technologies and challenges in sequence assembly, Springer, New York, NY.

El-Twab M. H. A., 2007.  Physical mapping of the 45S rDNA on the chromosomes of Triticum turgidum and T. aestivum using fluorescence in situ hybridization for chromosome ancestors. Arab J. Biotechnol. 10: 69–80.

Fan H., Yakura K., Miyanishi M., Sugita M., Sugiura M., 1995.  In vitro transcription of plant RNA polymerase I-dependent rRNA genes is species-specific. Plant J. 8: 295–298. PubMed

Flagel L., Udall J., Nettleton D., Wendel J., 2008.  Duplicate gene expression in allopolyploid Gossypium reveals two temporally distinct phases of expression evolution. BMC Biol. 6: 16. PubMed PMC

Fortune P. M., Schierenbeck K. A., Ainouche A. K., Jacquemin J., Wendel J. F., et al. , 2007.  Evolutionary dynamics of Waxy and the origin of hexaploid Spartina species (Poaceae). Mol. Phylogenet. Evol. 43: 1040–1055. PubMed

Fortune P. M., Schierenbeck K., Ayres D., Bortolus A., Catrice O., et al. , 2008.  The enigmatic invasive Spartina densiflora : A history of hybridizations in a polyploidy context. Mol. Ecol. 17: 4304–4316. PubMed

Foucaud, 1897 Un Spartina inédit. Ann. Soc. Sci. Nat. Char. Inf. 32: 220–222.

Frankowski J., Bastrop R., 2010.  Identification of Anguilla anguilla (L.) and Anguilla rostrata (Le Sueur) and their hybrids based on a diagnostic single nucleotide polymorphism in nuclear 18S rDNA. Mol. Ecol. Resour. 10: 173–176. PubMed

Gerlach W. L., Bedbrook J. R., 1979.  Cloning and characterization of ribosomal RNA genes from wheat and barley. Nucleic Acids Res. 7: 1869–1885. PubMed PMC

Groves, H., and J. Groves, 1880 Spartina x townsendii Nobis. Rep. Bot. Soc. Exch. club Br. Id. 1–37.

Hancock J. M., Dover G. A., 1988.  Molecular coevolution among cryptically simple expansion segments of eukaryotic 26S/28S rRNAs. Mol. Biol. Evol. 5: 377–391. PubMed

Ilut D. C., Coate J. E., Luciano A. K., Owens T. G., May G. D., et al. , 2012.  A comparative transcriptomic study of an allotetraploid and its diploid progenitors illustrates the unique advantages and challenges of RNA-seq in plant species. Am. J. Bot. 99: 383–396. PubMed

Jiao Y., Wickett N. J., Ayyampalayam S., Chanderbali A. S., Landherr L., et al. , 2011.  Ancestral polyploidy in seed plants and angiosperms. Nature 473: 97–100. PubMed

Katoh K., Toh H., 2010.  Parallelization of the MAFFT multiple sequence alignment program. Bioinformatics 26: 1899–1900. PubMed PMC

Koh J., Soltis P. S., Soltis D. E., 2010.  Homeolog loss and expression changes in natural populations of the recently and repeatedly formed allotetraploid Tragopogon mirus (Asteraceae). BMC Genomics 11: 97. PubMed PMC

Kovarik A., 2005.  Rapid concerted evolution of nuclear ribosomal DNA in two Tragopogon allopolyploids of recent and recurrent origin. Genetics 169: 931–944. PubMed PMC

Kovarik A., Dadejova M., Lim Y. K., Chase M. W., Clarkson J. J., et al. , 2008.  Evolution of rDNA in Nicotiana allopolyploids: a potential link between rDNA homogenization and epigenetics. Ann. Bot-London 101: 815–823. PubMed PMC

Kuzoff R. K., Sweere A. J., Soltis D. E., Soltis P. S., Zimmer E. A., 1998.  The phylogenetic potential of entire 26S rDNA sequences in plants. Mol. Biol. Evol. 15: 251–263. PubMed

Langmead B., Salzberg S. L., 2012.  Fast gapped-read alignment with Bowtie 2. Nat. Methods 9: 357–359. PubMed PMC

Leitch I. J., Bennett M. D., 2004.  Genome downsizing in polyploid plants. Biol. J. Linn. Soc. Lond. 82: 651–663.

Levy A. A., Feldman M., 2002.  The impact of polyploidy on grass genome evolution. Plant Physiol. 130: 1587–1593. PubMed PMC

Li H., Handsaker B., Wysoker A., Fennell T., Ruan J., et al. , 2009.  The sequence alignment/map format and SAMtools. Bioinformatics 25: 2078–2079. PubMed PMC

Liu B., Davis T. M., 2011.  Conservation and loss of ribosomal RNA gene sites in diploid and polyploid Fragaria (Rosaceae). BMC Plant Biol. 11: 157. PubMed PMC

Mable B. K., 2004.  “Why polyploidy is rarer in animals than in plants”: myths and mechanisms. Biol. J. Linn. Soc. Lond. 82: 453–466.

Mahé F., Pascual H., Coriton O., Huteau V., Navarro Perris A., et al. , 2010.  New data and phylogenetic placement of the enigmatic Old World lupin: Lupinus mariae-josephi H. Pascual. Genet. Resour. Crop Ev. 58: 101–114.

Maluszynska J., Heslop-Harrison J. S., 1993.  Physical mapping of rDNA loci in Brassica species. Genome 36: 774–781. PubMed

Marchant C., 1968.  Evolution in Spartina (Gramineae). II. Chromosomes, basic relationships and the problem of Spartina x townsendii agg. Biol. J. Linn. Soc. Lond. 60: 381–409.

Margulies M., Egholm M., Altman W. E., Attiya S., Bader J. S., et al. , 2005.  Genome sequencing in microfabricated high-density picolitre reactors. Nature 437: 376–380. PubMed PMC

Matyášek R., Renny-Byfield S., Fulneček J., Macas J., Grandbastien M.-A., et al. , 2012.  Next generation sequencing analysis reveals a relationship between rDNA unit diversity and locus number in Nicotiana diploids. BMC Genomics 13: 722. PubMed PMC

Mentewab A. B., Jacobsen M. J., Flowers R. A., 2011.  Incomplete homogenization of 18S ribosomal DNA coding regions in Arabidopsis thaliana. BMC Res. Notes 4: 93. PubMed PMC

Metzker M. L., 2009.  Sequencing technologies—the next generation. Nat. Rev. Genet. 11: 31–46. PubMed

Milne I., Bayer M., Cardle L., Shaw P., Stephen G., et al. , 2009.  Tablet–next generation sequence assembly visualization. Bioinformatics 26: 401–402. PubMed PMC

Nei M., Rooney A. P., 2005.  Concerted and birth-and-death evolution of multigene families. Annu. Rev. Genet. 39: 121. PubMed PMC

Novák P., Neumann P., Macas J., 2010.  Graph-based clustering and characterization of repetitive sequences in next-generation sequencing data. BMC Bioinformatics 11: 378. PubMed PMC

Ohno S., 1970.  Evolution by Gene Duplication. Springer-Verlag, New York.

Page J. T., Gingle A. R., Udall J. A., 2013a PolyCat: A resource for genome categorization of sequencing reads from allopolyploid organisms. G3 (Bethesda) 3: 517–525. PubMed PMC

Page J. T., Huynh M. D., Liechty Z. S., Grupp K., Stelly D., et al. , 2013b Insights into the evolution of cotton diploids and polyploids from whole-genome re-sequencing. G3 (Bethesda) 3: 1809–1818. PubMed PMC

Page J. T., Liechty Z. S., Huynh M. D., Udall J. A., 2014.  BamBam: genome sequence analysis tools for biologists. BMC Res. Notes 7: 829. PubMed PMC

Peralta M., Combes M.-C., Cenci A., Lashermes P., Dereeper A., 2013.  SNiPloid: A utility to exploit high-throughput SNP data derived from RNA-Seq in allopolyploid species. Int. J. Plant Genomics 2013: 1–6. PubMed PMC

Peterson P. M., Romaschenko K., Arrieta Y. H., Saarela J. M., 2014.  A molecular phylogeny and new subgeneric classification of Sporobolus (Poaceae: Chloridoideae: Sporobolinae). Taxon 63: 1212–1243.

Poczai P., Hyvönen J., 2010.  Nuclear ribosomal spacer regions in plant phylogenetics: problems and prospects. Mol. Biol. Rep. 37: 1897–1912. PubMed

Prokopowich C. D., Gregory T. R., Crease T. J., 2003.  The correlation between rDNA copy number and genome size in eukaryotes. Genome 46: 48–50. PubMed

Ramsey J., Schemske D. W., 1998.  Pathways, mechanisms, and rates of polyploid formation in flowering plants. Annu. Rev. Ecol. Syst. 29: 467–501.

Rogers, S. O., and A. J. Bendich. 1987 Ribosomal RNA genes in plants: variability in copy number and in the intergenic spacer. Plant Mol. Biol. 9:509:520. PubMed

Rosato M., Castro M., Rossello J. A., 2008.  Relationships of the woody Medicago species (Section Dendrotelis) assessed by molecular cytogenetic analyses. Ann. Bot-London 102: 15–22. PubMed PMC

Rousseau-Gueutin M., Bellot S., Martin G. E., Boutte J., Chelaifa H., et al. , 2015.  The chloroplast genome of the hexaploid Spartina maritima (Poaceae, Chloridoideae): Comparative analyses and molecular dating. Mol. Phylogenet. Evol. 93: 5–16. PubMed

Rozen S., Skaletsky H., 2000.  Primer3 on the WWW for general users and for biologist programmers. Methods Mol. Biol. 132: 365–386. PubMed

Salmon A., Flagel L., Ying B., Udall J. A., Wendel J. F., 2010.  Homoeologous nonreciprocal recombination in polyploid cotton. New Phytol. 186: 123–134. PubMed

Schaal B. A., Learn G. H., 1988.  Ribosomal DNA variation within and among plant populations. Ann. Mo. Bot. Gard. 75: 1207–1216.

Seo J. H., Bae H. G., Park D. H., Kim B. S., Lee J. W., et al. , 2013.  Sequence polymorphisms in ribosomal RNA genes and variations in chromosomal loci of Oenothera odorata and O. laciniata. Genes Genomics 35: 117–124.

Snowdon R. J., Köhler W., Köhler A., 1997.  Chromosomal localization and characterization of rDNA loci in the Brassica A and C genomes. Genome 40: 582–587. PubMed

Soltis D. E., Albert V. A., Leebens-Mack J., Bell C. D., Paterson A. H., et al. , 2009.  Polyploidy and angiosperm diversification. Am. J. Bot. 96: 336–348. PubMed

Soreng R. J., Peterson P. M., Romaschenko K., Davidse G., Zuloaga F. O., et al. , 2015.  A worldwide phylogenetic classification of the Poaceae (Gramineae): Phylogenetic classification of the grasses. J. Syst. Evol. 53: 117–137.

Strong D. R., Ayres D. R., 2013.  Ecological and evolutionary misadventures of Spartina. Annu. Rev. Ecol. Evol. Syst. 44: 389–410.

Tennessen J. A., Govindarajulu R., Ashman T.-L., Liston A., 2014.  Evolutionary origins and dynamics of octoploid strawberry subgenomes revealed by dense targeted capture linkage maps. Genome Biol. Evol. 6: 3295–3313. PubMed PMC

Toloczyki C., Feix G., 1986.  Occurrence of 9 homologous repeat units in the external spacer region of a nuclear maize rRNA gene unit. Nucleic Acids Res. 14: 4969–4986. PubMed PMC

Udall J. A., Swanson J. M., Haller K., Rapp R. A., Sparks M. E., et al. , 2006.  A global assembly of cotton ESTs. Genome Res. 16: 441–450. PubMed PMC

Van de Peer Y., Maere S., Meyer A., 2009.  The evolutionary significance of ancient genome duplications. Nat. Rev. Genet. 10: 725–732. PubMed

Volkov R., Kostishin S., Ehrendorfer F., Schweizer D., 1996.  Molecular organization and evolution of the external transcribed rDNA spacer region in two diploid relatives of Nicotiana tabacum (Solanaceae). Plant Syst. Evol. 201: 117–129.

Volkov R. A., Borisjuk N. V., Panchuk I. I., Schweizer D., Hemleben V., 1999.  Elimination and rearrangement of parental rDNA in the allotetraploid Nicotiana tabacum. Mol. Biol. Evol. 16: 311–320. PubMed

Waterhouse A. M., Procter J. B., Martin D. M. A., Clamp M., Barton G. J., 2009.  Jalview Version 2–a multiple sequence alignment editor and analysis workbench. Bioinformatics 25: 1189–1191. PubMed PMC

Wendel J. F., 2000.  Genome evolution in polyploids. Plant Mol. Niol. 42: 225–249. PubMed

Wendel J. F., Schnabel A., Seelanan T., 1995.  Bidirectional interlocus concerted evolution following allopolyploid speciation in cotton (Gossypium). Proc. Natl. Acad. Sci. USA 92: 280–284. PubMed PMC

Zentgraf U., Velasco R., Hemleben V., 1998.  Different Transcriptional Activities in the Nucleus, pp. 131–168 in Progress in Botany, edited by H.-D. Behnke K., Esser J. W., Kadereit U., Lüttge, Runge M. Springer, Berlin.

The Utility of Graph Clustering of 5S Ribosomal DNA Homoeologs in Plant Allopolyploids, Homoploid Hybrids, and Cryptic Introgressants

Remarkable variation of ribosomal DNA organization and copy number in gnetophytes, a distinct lineage of gymnosperms