Red clover (Trifolium pratense) is an important forage plant worldwide. This study was directed to broadening current knowledge of red clover's coding regions and enhancing its utilization in practice by specific reanalysis of previously published assembly. A total of 42,996 genes were characterized using Illumina paired-end sequencing after manual revision of Blast2GO annotation. Genes were classified into metabolic and biosynthetic pathways in response to biological processes, with 7,517 genes being assigned to specific pathways. Moreover, 17,727 enzymatic nodes in all pathways were described. We identified 6,749 potential microsatellite loci in red clover coding sequences, and we characterized 4,005 potential simple sequence repeat (SSR) markers as generating polymerase chain reaction products preferentially within 100-350 bp. Marker density of 1 SSR marker per 12.39 kbp was achieved. Aligning reads against predicted coding sequences resulted in the identification of 343,027 single nucleotide polymorphism (SNP) markers, providing marker density of one SNP marker per 144.6 bp. Altogether, 95 SSRs in coding sequences were analyzed for 50 red clover varieties and a collection of 22 highly polymorphic SSRs with pooled polymorphism information content >0.9 was generated, thus obtaining primer pairs for application to diversity studies in T. pratense. A set of 8,623 genome-wide distributed SNPs was developed and used for polymorphism evaluation in individual plants. The polymorphic information content ranged from 0 to 0.375. Temperature switch PCR was successfully used in single-marker SNP genotyping for targeted coding sequences and for heterozygosity or homozygosity confirmation in validated five loci. Predicted large sets of SSRs and SNPs throughout the genome are key to rapidly implementing genome-based breeding approaches, for identifying genes underlying key traits, and for genome-wide association studies. Detailed knowledge of genetic relationships among breeding material can also be useful for breeders in planning crosses or for plant variety protection. Single-marker assays are useful for diagnostic applications.

Agricultural Research Ltd Troubsko Czechia

Department of Experimental Biology Faculty of Science Masaryk University Brno Czechia

Department of Psychiatry University Hospital Brno and Masaryk University Brno Czechia

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Adams N. R. (1995). Detection of the effects of phytoestrogens on sheep and cattle. J. Anim. Sci. 73, 1509–1515. 10.2527/1995.7351509x PubMed DOI

Ashrafi H., Hill T., Stoffel K., Kozik A., Yao J., Chin-Wo S. R., et al. . (2012). De novo assembly of the pepper transcriptome (Capsicum annuum): a benchmark for in silico discovery of SNPs, SSRs and candidate genes. BMC Genomics 13:571. 10.1186/1471-2164-13-571 PubMed DOI PMC

Blanca J., Cañizares J., Roig C., Ziarsolo P., Nuez F., Picó B. (2011). Transcriptome characterization and high throughput SSRs and SNPs discovery in Cucurbita pepo (Cucurbitaceae). BMC Genomics 12:104. 10.1186/1471-2164-12-104 PubMed DOI PMC

Botstein D., White R. L., Skolnick M., Davis R. W. (1980). Construction of a genetic linkage map in man using restriction fragment length polymorphisms. Am. J. Hum. Genet. 32, 314–331. PubMed PMC

Cidade F. W., Vigna B. B., de Souza F. H., Valls J. F., Dall'Agnol M., Zucchi M. I., et al. . (2013). Genetic variation in polyploid forage grass: assessing the molecular genetic variability in the Paspalum genus. BMC Genet. 14:50. 10.1186/1471-2156-14-50 PubMed DOI PMC

da Maia L. C., Palmieri D. A., De Souza V. Q., Kopp M. M., de Carvalho F. I. F., Costa de Oliveira A. (2008). SSR Locator: tool for simple sequence repeat discovery integrated with primer design and PCR simulation. Int. J. Plant Genomics 2008:412696. 10.1155/2008/412696 PubMed DOI PMC

De Vega J. J., Ayling S., Hegarty M., Kudrna D., Goicoechea J. L., Ergon Å., et al. . (2015). Red clover (Trifolium pratense L.) draft genome provides a platform for trait improvement. Sci. Rep. 5:17394. 10.1038/srep17394 PubMed DOI PMC

Dellaporta S. L., Wood J., Hicks J. B. (1983). A plant DNA minipreparation: version II. Plant Mol. Biol. Report. 1, 19–21. 10.1007/BF02712670 DOI

Dice L. R. (1945). Measures of the amount of ecologic association between species. Ecology 26, 297 10.2307/1932409 DOI

Durand J., Bodénès C., Chancerel E., Frigerio J.-M., Vendramin G., Sebastiani F., et al. . (2010). A fast and cost-effective approach to develop and map EST-SSR markers: oak as a case study. BMC Genomics 11:570. 10.1186/1471-2164-11-570 PubMed DOI PMC

Forster J. W., Jones E. S., Kölliker R., Drayton M. C., Dupal M. P., Guthridge K. M., et al. (2001). Application of DNA profiling to an outbreeding forage species, in Plant Genotyping: The DNA Fingerprinting of Plants, ed Henry R. J.(Wallingford: CABI; ), 299–320. Available online at: http://www.cabi.org/cabebooks/ebook/20083015002 (Accessed February 9, 2016).

Ghamkhar K., Isobe S., Nichols P. G. H., Faithfull T., Ryan M. H., Snowball R., et al. (2012). The first genetic maps for subterranean clover (Trifolium subterraneum L.) and comparative genomics with T. pratense L. and Medicago truncatula Gaertn. to identify new molecular markers for breeding. Mol. Breed. 30, 213–226. 10.1007/s11032-011-9612-8 DOI

Graham P. H., Vance C. P. (2003). Legumes: importance and constraints to greater use. Plant Physiol. 131, 872–877. 10.1104/pp.017004 PubMed DOI PMC

Herrmann D., Boller B., Studer B., Widmer F., Kölliker R. (2008). Improving persistence in red clover: insights from QTL analysis and comparative phenotypic evaluation. Crop Sci. 48:269 10.2135/cropsci2007.03.0143 DOI

Isobe S., Kölliker R., Hisano H., Sasamoto S., Wada T., Klimenko I., et al. . (2009). Construction of a consensus linkage map for red clover (Trifolium pratense L.). BMC Plant Biol. 9:57. 10.1186/1471-2229-9-57 PubMed DOI PMC

Isobe S. N., Hisano H., Sato S., Hirakawa H., Okumura K., Shirasawa K., et al. . (2012). Comparative genetic mapping and discovery of linkage disequilibrium across linkage groups in white clover (Trifolium repens L.). G3 2, 607–617. 10.1534/g3.112.002600 PubMed DOI PMC

Ištvánek J., Jaroš M., Křenek A., Řepková J. (2014). Genome assembly and annotation for red clover (Trifolium pratense; Fabaceae). Am. J. Bot. 101, 327–337. 10.3732/ajb.1300340 PubMed DOI

Jaccard P. (1901). Étude comparative de la distribution florale dans une portion des Alpes et du Jura. Bull. Soc. Vaudoise Sci. Nat. 37, 547–579. 10.5169/seals-266450 DOI

Jakešová H., Řepková J., Nedělník J., Hampel D., Dluhošová J., Soldánová M., et al. (2015). Selecting plants with increased total polyphenol oxidases in the genus Trifolium. Czech J. Genet. Plant Breed. 51, 155–161. 10.17221/107/2015-CJGPB DOI

Jones B. A., Hatfield R. D., Muck R. E. (1995). Screening legume forages for soluble phenols, polyphenol oxidase and extract browning. J. Sci. Food Agric. 67, 109–112. 10.1002/jsfa.2740670117 DOI

Kataoka R., Hara M., Kato S., Isobe S., Sato S., Tabata S., et al. . (2012). Integration of linkage and chromosome maps of red clover (Trifolium pratense L.). Cytogenet. Genome Res. 137, 60–69. 10.1159/000339509 PubMed DOI

Klimenko I., Razgulayeva N., Gau M., Okumura K., Nakaya A., Tabata S., et al. . (2010). Mapping candidate QTLs related to plant persistency in red clover. Theor. Appl. Genet. 120, 1253–1263. 10.1007/s00122-009-1253-5 PubMed DOI PMC

Kongkiatngam P., Waterway M. J., Coulman B. E., Fortin M. G. (1996). Genetic variation among cultivars of red clover (Trifolium pratense L.) detected by RAPD markers amplified from bulk genomic DNA. Euphytica 89, 355–361.

Krzywinski M., Schein J., Birol I., Connors J., Gascoyne R., Horsman D., et al. . (2009). Circos: an information aesthetic for comparative genomics. Genome Res. 19, 1639–1645. 10.1101/gr.092759.109 PubMed DOI PMC

Kulikova O., Geurts R., Lamine M., Kim D.-J., Cook D. R., Leunissen J., et al. . (2004). Satellite repeats in the functional centromere and pericentromeric heterochromatin of Medicago truncatula. Chromosoma 113, 276–283. 10.1007/s00412-004-0315-3 PubMed DOI

Lee Y. G., Jeong N., Kim J. H., Lee K., Kim K. H., Pirani A., et al. . (2015). Development, validation and genetic analysis of a large soybean SNP genotyping array. Plant J. 81, 625–636. 10.1111/tpj.12755 PubMed DOI

Li H., Durbin R. (2010). Fast and accurate long-read alignment with Burrows-Wheeler transform. Bioinformatics 26, 589–595. 10.1093/bioinformatics/btp698 PubMed DOI PMC

Li H., Handsaker B., Wysoker A., Fennell T., Ruan J., Homer N., et al. . (2009). The sequence alignment/map format and SAMtools. Bioinformatics 25, 2078–2079. 10.1093/bioinformatics/btp352 PubMed DOI PMC

Lynch M., Bost D., Wilson S., Maruki T., Harrison S. (2014). Population-genetic inference from pooled-sequencing data. Genome Biol. Evol. 6, 1210–1218. 10.1093/gbe/evu085 PubMed DOI PMC

Metzgar D., Bytof J., Wills C. (2000). Selection against frameshift mutations limits microsatellite expansion in coding DNA. Genome Res. 10, 72–80. 10.1101/gr.10.1.72 PubMed DOI PMC

Mullen M. P., Creevey C. J., Berry D. P., McCabe M. S., Magee D. A., Howard D. J., et al. . (2012). Polymorphism discovery and allele frequency estimation using high-throughput DNA sequencing of target-enriched pooled DNA samples. BMC Genomics 13:16. 10.1186/1471-2164-13-16 PubMed DOI PMC

Novaes E., Drost D. R., Farmerie W. G., Pappas G. J., Jr., Grattapaglia D., Sederoff R. R., et al. . (2008). High-throughput gene and SNP discovery in Eucalyptus grandis, an uncharacterized genome. BMC Genomics 9:312. 10.1186/1471-2164-9-312 PubMed DOI PMC

Pandey M. K., Agarwal G., Kale S. M., Clevenger J., Nayak S. N., Sriswathi M., et al. . (2017). Development and evaluation of a high density genotyping 'Axiom_Arachis' array with 58 K SNPs for accelerating genetics and breeding in groundnut. Sci. Rep. 7:40577. 10.1038/srep40577 PubMed DOI PMC

Park C. Y., Weaver C. M. (2012). Vitamin D interactions with soy isoflavones on bone after menopause: a review. Nutrients 4, 1610–1621. 10.3390/nu4111610 PubMed DOI PMC

Qi L. L., Ma G. J., Long Y. M., Hulke B. S., Gong L., Markell S. G. (2015). Relocation of a rust resistance gene R2 and its marker-assisted gene pyramiding in confection sunflower (Helianthus annuus L.). Theor. Appl. Genet. 128, 477–488. 10.1007/s00122-014-2446-0 PubMed DOI

Raveendar S., Lee G.-A., Jeon Y.-A., Lee Y. J., Lee J.-R., Cho G.-T., et al. . (2015). Cross-amplification of Vicia sativa subsp. sativa microsatellites across 22 other Vicia species. Molecules 20, 1543–1550. 10.3390/molecules20011543 PubMed DOI PMC

Ritchie M. E., Phipson B., Wu D., Hu Y., Law C. W., Shi W., et al. . (2015). limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res. 43:e47. 10.1093/nar/gkv007 PubMed DOI PMC

Rogers S. O., Bendich A. J. (1989). Extraction of DNA from plant tissues, in Plant Molecular Biology Manual, eds Gelvin S. B., Schilperoort R. A., Verma D. P. S.(Dordrecht: Springer; ), 73–83. Available online at: http://www.springerlink.com/index/10.1007/978-94-009-0951-9_6 (Accessed February 9, 2016). DOI

RStudio Team (2015). RStudio: Integrated Development for R. Boston, MA: RStudio, Inc; Available online at: http://www.rstudio.com/ (Accessed February 6, 2017).

Sato S., Isobe S., Asamizu E., Ohmido N., Kataoka R., Nakamura Y., et al. . (2005). Comprehensive structural analysis of the genome of red clover (Trifolium pratense L.). DNA Res. 12, 301–364. 10.1093/dnares/dsi018 PubMed DOI

Sato S., Nakamura Y., Kaneko T., Asamizu E., Kato T., Nakao M., et al. . (2008). Genome structure of the legume, Lotus japonicus. DNA Res. 15, 227–239. 10.1093/dnares/dsn008 PubMed DOI PMC

Schena M., Shalon D., Heller R., Chai A., Brown P. O., Davis R. W. (1996). Parallel human genome analysis: micromicroarray-based expression monitoring of 1,000 genes. Proc. Natl. Acad. Sci. U.S.A. 93, 10614–10619. PubMed PMC

Schmutz J., Cannon S. B., Schlueter J., Ma J., Mitros T., Nelson W., et al. . (2010). Genome sequence of the palaeopolyploid soybean. Nature 463, 178–183. 10.1038/nature08670 PubMed DOI

Shrivastava D., Verma P., Bhatia S. (2014). Expanding the repertoire of microsatellite markers for polymorphism studies in Indian accessions of mung bean (Vigna radiata L. Wilczek). Mol. Biol. Rep. 41, 5669–5680. 10.1007/s11033-014-3436-7 PubMed DOI

Sindhu A., Ramsay L., Sanderson L. A., Stonehouse R., Li R., Condie J., et al. . (2014). Gene-based SNP discovery and genetic mapping in pea. Theor. Appl. Genet. 127, 2225–2241. 10.1007/s00122-014-2375-y PubMed DOI PMC

Song Q., Hyten D. L., Jia G., Quigley C. V., Fickus E. W., Nelson R. L., et al. . (2013). Development and evaluation of SoySNP50K, a high-density genotyping array for soybean. PLoS ONE 8:e54985. 10.1371/journal.pone.0054985 PubMed DOI PMC

Sørenson T. (1948). A Method of Establishing Groups of Equal Amplitude in Plant Sociology Based on Similarity of Species Content and Its Application to Analyses of the Vegetation on Danish Commons. I kommission hos E. Munksgaard. Available online at: https://books.google.co.in/books?id=rpS8GAAACAAJ

Sprent J. I. (2009). Legume Nodulation. Oxford, UK: Wiley-Blackwell; (Accessed March 3, 2016).

Stasolla C., Katahira R., Thorpe T. A., Ashihara H. (2003). Purine and pyrimidine nucleotide metabolism in higher plants. J. Plant Physiol. 160, 1271–1295. 10.1078/0176-1617-01169 PubMed DOI

Tabone T., Mather D. E., Hayden M. J. (2009). Temperature switch PCR (TSP): robust assay design for reliable amplification and genotyping of SNPs. BMC Genomics 10:580. 10.1186/1471-2164-10-580 PubMed DOI PMC

Tayeh N., Aluome C., Falque M., Jacquin F., Klein A., Chauveau A., et al. . (2015). Development of two major resources for pea genomics: the GenoPea 13.2K SNP array and a high-density, high-resolution consensus genetic map. Plant J. 84, 1257–1273. 10.1111/tpj.13070 PubMed DOI

Teuscher E., Lindequist U. (2010). Biogene Gifte: Biologie, Chemie, Pharmakologie, Toxikologie, 3 neu bearb. Stuttgart: Wiss. Verl.-Ges.

Torales S. L., Rivarola M., Pomponio M. F., Fernández P., Acuña C. V., Marchelli P., et al. . (2012). Transcriptome survey of Patagonian southern beech Nothofagus nervosa (= N. alpina): assembly, annotation and molecular marker discovery. BMC Genomics 13:291. 10.1186/1471-2164-13-291 PubMed DOI PMC

Torales S. L., Rivarola M., Pomponio M. F., Gonzalez S., Acuña C. V., Fernández P., et al. . (2013). De novo assembly and characterization of leaf transcriptome for the development of functional molecular markers of the extremophile multipurpose tree species Prosopis alba. BMC Genomics 14:705. 10.1186/1471-2164-14-705 PubMed DOI PMC

Ueno S., Le Provost G., Léger V., Klopp C., Noirot C., Frigerio J.-M., et al. . (2010). Bioinformatic analysis of ESTs collected by Sanger and pyrosequencing methods for a keystone forest tree species: oak. BMC Genomics 11:650. 10.1186/1471-2164-11-650 PubMed DOI PMC

Varshney R. K., Ribaut J.-M., Buckler E. S., Tuberosa R., Rafalski J. A., Langridge P. (2012). Can genomics boost productivity of orphan crops? Nat. Biotechnol. 30, 1172–1176. 10.1038/nbt.2440 PubMed DOI

Varshney R. K., Song C., Saxena R. K., Azam S., Yu S., Sharpe A. G., et al. . (2013). Draft genome sequence of chickpea (Cicer arietinum) provides a resource for trait improvement. Nat. Biotechnol. 31, 240–246. 10.1038/nbt.2491 PubMed DOI

Verma P., Chandra A., Roy A. K., Malaviya D. R., Kaushal P., Pandey D., et al. (2015). Development, characterization and cross-species transferability of genomic SSR markers in berseem (Trifolium alexandrinum L.), an important multi-cut annual forage legume. Mol. Breed. 35, 1–14. 10.1007/s11032-015-0223-7 DOI

Víquez-Zamora M., Vosman B., van de Geest H., Bovy A., Visser R. G., Finkers R., et al. . (2013). Tomato breeding in the genomics era: insights from a SNP array. BMC Genomics 14:354. 10.1186/1471-2164-14-354 PubMed DOI PMC

Vižintin L., Javornik B., Bohanec B. (2006). Genetic characterization of selected Trifolium species as revealed by nuclear DNA content and ITS rDNA region analysis. Plant Sci. 170, 859–866. 10.1016/j.plantsci.2005.12.007 DOI

Wink M. (2013). Evolution of secondary metabolites in legumes (Fabaceae). South Afr. J. Bot. 89, 164–175. 10.1016/j.sajb.2013.06.006 DOI

Yates S. A., Swain M. T., Hegarty M. J., Chernukin I., Lowe M., Allison G. G., et al. . (2014). De novo assembly of red clover transcriptome based on RNA-Seq data provides insight into drought response, gene discovery and marker identification. BMC Genomics 15:453. 10.1186/1471-2164-15-453 PubMed DOI PMC

Younas M., Xiao Y., Cai D., Yang W., Ye W., Wu J., et al. . (2012). Molecular characterization of oilseed rape accessions collected from multi continents for exploitation of potential heterotic group through SSR markers. Mol. Biol. Rep. 39, 5105–5113. 10.1007/s11033-011-1306-0 PubMed DOI

Young N. D., Debellé F., Oldroyd G. E., Geurts R., Cannon S. B., Udvardi M. K., et al. . (2011). The Medicago genome provides insight into the evolution of rhizobial symbioses. Nature 480, 520–524. 10.1038/nature10625 PubMed DOI PMC

Yu H., Xie W., Li J., Zhou F., Zhang Q. (2014). A whole-genome SNP array (RICE6K) for genomic breeding in rice. Plant Biotechnol. J. 12, 28–37. 10.1111/pbi.12113 PubMed DOI

Zalapa J. E., Cuevas H., Zhu H., Steffan S., Senalik D., Zeldin E., et al. . (2012). Using next-generation sequencing approaches to isolate simple sequence repeat (SSR) loci in the plant sciences. Am. J. Bot. 99, 193–208. 10.3732/ajb.1100394 PubMed DOI

Zhao P., Zhang G., Wu X., Li N., Shi D., Zhang D., et al. . (2013). Fine mapping of RppP25, a southern rust resistance gene in maize. J. Integr. Plant Biol. 55, 462–472. 10.1111/jipb.12027 PubMed DOI

Zrenner R., Stitt M., Sonnewald U., Boldt R. (2006). Pyrimidine and purine biosynthesis and degradation in plants. Annu. Rev. Plant Biol. 57, 805–836. 10.1146/annurev.arplant.57.032905.105421 PubMed DOI

Red Clover (Trifolium pratense) and Zigzag Clover (T. medium) - A Picture of Genomic Similarities and Differences