EST-SSR marker characterization based on RNA-sequencing of Lolium multiflorum and cross transferability to related species


Simple sequence repeat (SSR) markers are considered very useful molecular markers for genetic and molecular breeding, specifically for non-model species. To develop a set of whole genome-wide distributed SSR marker for annual ryegrass (Lolium multiflorum L.), we obtained the first transcriptome dataset of annual ryegrass subjected to drought stress and identified a total of 11,254 EST-SSR. Fifty EST-SSR markers were selected to test the amplification and polymorphism rates in 23 accessions of Lolium and Festuca diploid species. The results showed that all of the 50 functionally relevant markers were polymorphic, with allele number and polymorphism content averaging 7.4 and 0.839, respectively, and fully transferable across 23 accessions of Lolium and Festuca species. Sequencing of PCR products showed that the nucleotide sequences of Lolium and Festuca species had a high level of conservation. Different types of tri-repeats as abundant SSR motifs were observed in repeat sequence regions. A number of EST-SSRs were mapped to genome sequences of Lolium perenne and chromosomes of Brachypodium distachyon. Among them, 973 and 1375 Lolium SSR markers were located on the five chromosomes of B. distachyon and L. perenne scaffolds, respectively. In addition, we confirmed that EST-SSRs as well as SRAP markers could clearly identify varieties of Lolium and Festuca species, and the two sets of molecular markers had a significant correlation (r = 0.74). This set of EST-SSR markers are valuable genetic tools for genetic diversity, evolutionary, and association mapping studies in Lolium, Festuca, and many other grass species.

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  1. Alisoltani A, Ebrahimi S, Azarian S, Hematyar M, Shiran B, Jahanbazi H, Fallahi H, Mousavi-Fard S, Rafiei F (2015) Parallel consideration of SSRs and differentially expressed genes under abiotic stress for targeted development of functional markers in almond and related Prunus species. Sci Hortic 198:462–472

    Article  CAS  Google Scholar 

  2. Altermann E, Klaenhammer TR (2005) PathwayVoyager: pathway mapping using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. BMC Genomics 6(1):60.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  3. Asp T, Frei UK, Didion T, Nielsen KK, Lübberstedt T (2007) Frequency, type, and distribution of EST-SSRs from three genotypes of Lolium perenne, and their conservation across orthologous sequences of Festuca arundinacea, Brachypodium distachyon, and Oryza sativa. BMC Plant Biol 7(1):36.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  4. Ayres NM, Mcclung AM, Larkin PD, Hfj B, Jones CA, Park WD (1997) Microsatellites and a single-nucleotide polymorphism differentiate apparent amylose classes in an extended pedigree of US rice germ plasm. Theor Appl Genet 94(6):773–781.

    Article  CAS  Google Scholar 

  5. Bajgain P, Rouse MN, Anderson JA (2015) Comparing genotyping-by-sequencing and single nucleotide polymorphism chip genotyping for quantitative trait loci mapping in wheat. Crop Sci 56(1):232–248

    Article  CAS  Google Scholar 

  6. Bhandawat A, Singh G, Raina AS, Kaur J, Sharma RK (2016) Development of genic SSR marker resource from RNA-Seq data in Dendrocalamus latiflorus. J Plant Biochem Biotechnol 25(2):179–190.

    Article  CAS  Google Scholar 

  7. Byrne S, Armstead I, Swain SC, Panitz F, Bendixen C, Hedegaard J, Nagy I, Farrell JD, Studer B, Caccamo M (2014) A draft sequence assembly of the perennial ryegrass (Lolium perenne) genome. In: International Plant and Animal Genome Conference Xxii

  8. Cheng Y, Ma X, Zhou K, Humphreys MW, Zhang XQ (2016) Phylogenetic analysis of Festuca–Lolium complex using SRAP markers. Genet Resour Crop Evol 63(1):7–18.

    Article  Google Scholar 

  9. Diekmann K, Hodkinson TR, Wolfe KH, Bekerom RVD, Dix PJ, Barth S (2009) Complete chloroplast genome sequence of a major allogamous forage species, perennial ryegrass (Lolium perenne L.) DNA Res 16(3):165–176.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  10. Fu N, Wang Q, Shen HL (2013) De novo assembly, gene annotation and marker development using Illumina paired-end transcriptome sequences in celery (Apium graveolens L.) PLoS One 8(2):e57686.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  11. Garg R, Patel RK, Tyagi AK, Jain M (2011) De novo assembly of chickpea transcriptome using short reads for gene discovery and marker identification. DNA Res 18(1):53–63.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  12. Gupta PK, Rustgi S, Sharma S, Singh R, Kumar N, Balyan HS (2003) Transferable EST-SSR markers for the study of polymorphism and genetic diversity in bread wheat. Mol Gen Genomics 270(4):315–323.

    Article  CAS  Google Scholar 

  13. Hand ML, Spangenberg GC, Forster JW, Cogan NOI (2013) Plastome sequence determination and comparative analysis for members of the Lolium-Festuca grass species complex. G3: Genes Genomes Genet 3(4):607–616.

    Article  CAS  Google Scholar 

  14. Kantety RV, La RM, Matthews DE, Sorrells ME (2002) Data mining for simple sequence repeats in expressed sequence tags from barley, maize, rice, sorghum and wheat. Plant Mol Biol 48(5):501–510.

    Article  PubMed  CAS  Google Scholar 

  15. Klein PE, Klein RR, Vrebalov J, Mullet JE (2003) Sequence-based alignment of sorghum chromosome 3 and rice chromosome 1 reveals extensive conservation of gene order and one major chromosomal rearrangement. Plant J Cell Mol Biol 34(5):605–621.

    Article  CAS  Google Scholar 

  16. Li DJ, Deng Z, Qin B, Liu X, Men ZH (2012) De novo assembly and characterization of bark transcriptome using Illumina sequencing and development of EST-SSR markers in rubber tree (Hevea brasiliensis Muell. Arg.) BMC Genomics 13(1):192.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  17. Mccouch SR, Temnykh S, Lukashova A, Coburn J, Declerck G, Cartinhour S, Harrington S, Thomson M, Septiningsih E, Semon M (2001) Microsatellite markers in rice: abundance, diversity, and applications. Rice Genet IV:117–135

    Google Scholar 

  18. Pan L, Zhang X, Wang J, Ma X, Zhou M, Huang LK, Nie G, Wang P, Yang Z, Li J (2016) Transcriptional profiles of drought-related genes in modulating metabolic processes and antioxidant defenses in Lolium multiflorum. Front Plant Sci 7(170).

  19. Saha MC, Mian MA, Eujayl I, Zwonitzer JC, Wang L, May GD (2004) Tall fescue EST-SSR markers with transferability across several grass species. Theor Appl Genet 109(4):783–791.

    Article  PubMed  Google Scholar 

  20. Salem M, Vallejo RL, Leeds TD, Palti Y, Liu S, Sabbagh A, Yao J (2012) RNA-Seq identifies SNP markers for growth traits in rainbow trout. PLoS One 7(5):e36264.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  21. Thiel T, Michalek W, Varshney RK, Graner A (2003) Exploiting EST databases for the development and characterization of gene-derived SSR-markers in barley (Hordeum vulgare L.) Theor Appl Genet 106(3):411–422.

    Article  PubMed  CAS  Google Scholar 

  22. Wang FZ, Wang QB, Kwon SY, Kwak SS, WA S (2005) Enhanced drought tolerance of transgenic rice plants expressing a pea manganese superoxide dismutase. J Plant Physiol 162(4):465–472.

    Article  PubMed  CAS  Google Scholar 

  23. Wang S, Wang X, He Q, Liu X, Xu W, Li L, Gao J, Wang F (2012) Transcriptome analysis of the roots at early and late seedling stages using Illumina paired-end sequencing and development of EST-SSR markers in radish. Plant Cell Rep 31(8):1437–1447.

    Article  PubMed  CAS  Google Scholar 

  24. Wang Z, Yu G, Shi B, Wang X, Qiang H, Gao H (2014) Development and characterization of simple sequence repeat (SSR) markers based on RNA-sequencing of Medicago sativa and in silico mapping onto the M. truncatula genome. PLoS One 9(3):e92029.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  25. Wit F (1958) Tetraploid Italian ryegrass (Lolium multiflorum Lam.) Euphytica 7(1):47–58

    CAS  Google Scholar 

  26. Wong MM, Cannon CH, Wickneswari R (2012) Development of high-throughput SNP-based genotyping in Acacia auriculiformis x A. mangium hybrids using short-read transcriptome data. BMC Genomics 13(1):726.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  27. Xia EH, Yao QY, Zhang HB, Jiang JJ, Zhang LP, Gao LZ (2016) CandiSSR: an efficient pipeline used for identifying candidate polymorphic SSRs based on multiple assembled sequences. Front Plant Sci 6(796):1171

    PubMed  PubMed Central  Google Scholar 

  28. Yang SS, Tu ZJ, Cheung F, Xu WW, Lamb JFS, Jung HJG, Vance CP, Gronwald JW (2011) Using RNA-Seq for gene identification, polymorphism detection and transcript profiling in two alfalfa genotypes with divergent cell wall composition in stems. BMC Genomics 12(1):19

    Article  Google Scholar 

  29. Yu Y, Yuan D, Liang S, Li X, Wang X, Lin Z, Zhang X (2011) Genome structure of cotton revealed by a genome-wide SSR genetic map constructed from a BC1 population between gossypium hirsutum and G. barbadense. BMC Genomics 12(1):15.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  30. Zhang H, Wei L, Miao H, Zhang T, Wang C (2012a) Development and validation of genic-SSR markers in sesame by RNA-seq. BMC Genomics 13(1):316.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  31. Zhang J, Shan L, Duan J, Jin W, Chen S, Cheng Z, Qiang Z, Liang X, Li Y (2012b) De novo assembly and characterisation of the transcriptome during seed development, and generation of genic-SSR markers in peanut ( Arachis hypogaea L.) BMC Genomics 13(1):90.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

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Authors and contributors

LP collected data and wrote the manuscript. XZ and XM designed the project. JW participated in technical editing of the manuscript. TH, ZY, and LT analyzed the data. All authors have read the manuscript.


This research work was funded by the earmarked fund for Modern Agro-industry Technology Research System (No. CARS-35-05), the National Basic Research Program (973 program) in China (No. 2014CB138705), and the National Natural Science Foundation of China (NSFC 31372363).

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Correspondence to Xinquan Zhang.

Electronic supplementary material

Fig S1

The distribution of repeated nucleotide types of SSR motifs in L. multiflorum (A-B) and the number of unigenes that are distributed on an annotated species (C). (PNG 423 kb)

Fig S2

Principal coordinate analysis of EST-SSR markers variation among 23 accessions of Lolium and Festuca species. (PNG 360 kb)

High resolution image (TIFF 1683 kb)

High resolution image (TIFF 1426 kb)

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Pan, L., Huang, T., Yang, Z. et al. EST-SSR marker characterization based on RNA-sequencing of Lolium multiflorum and cross transferability to related species. Mol Breeding 38, 80 (2018).

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  • Lolium and Festuca diploid species
  • Transcriptome
  • EST-SSR markers
  • Transferability and polymorphism analysis
  • Genetic relationship