Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Use of an expressed sequence tag-based method for single nucleotide polymorphism identification and discrimination of Citrus species and cultivars

  • 435 Accesses

  • 3 Citations

Abstract

Sweet orange [Citrus sinensis (L.) Osbeck] represents the most important Citrus species, followed by clementine (C. clementina Hort. ex Tan.). Citrus species and genotypes are difficult to recognize as they have a moderate level of diversity due to nucellar selection, vegetative propagation and origin by single spontaneous mutation. Despite the large number of available sequences and the existence of a draft assembly of sweet orange and clementine, there are currently no single nucleotide polymorphism (SNP) databases for Citrus species. For this purpose, the QualitySNP software was used to discover SNPs in 19 Citrus species starting from 540,000 expressed sequence tags (ESTs) assembled in 52,000 contigs. The vast majority of ESTs, contigs and SNPs were found in C. clementina and C. sinensis: 4,400 out of 16,000 contigs (27 %) of C. clementina and 4,100 out of 17,000 contigs (24 %) of C. sinensis contained putative SNPs. A total of 3,634 sequences were associated with enzymes belonging to 121 metabolic KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways, among which the secondary metabolite pathway was the most represented. A total of 163 SNPs from 52 contigs and genes of specific functional categories were validated and 81 polymorphic sites were found. Thirty-seven selected SNPs, validated by Sanger sequencing, confirmed that polymorphisms were mainly between species, while poor within-species variability was discovered. This work provides a collection of 15,879 putative SNP markers that could be exploited by the Citrus community. Furthermore, the validated SNPs associated with specific genes could be used for functional genetic studies in germplasm diversity analysis, mapping and breeding.

This is a preview of subscription content, log in to check access.

Fig. 1

References

  1. Anithakumari AM, Tang J, van Eck HJ, Visser RGF, Leunissen JAM, Vosman B, van der Linden CG (2010) A pipeline for high throughput detection and mapping of SNPs from EST databases. Mol Breed 26:65–75

  2. Barkley NA, Roose ML, Krueger RR, Federici CT (2006) Assessing genetic diversity and population structure in a citrus germplasm collection utilizing simple sequence repeat markers (SSRs). Theor Appl Genet 112:1519–1531

  3. Bernardi J, Licciardello C, Russo MP, Chiusano ML, Carletti G, Reforgiato Recupero G, Marocco A (2010) Use of a custom array to study differentially expressed genes during blood orange (Citrus sinensis L. Osbeck) ripening. J Plant Physiol 167:301–310

  4. Biswas MK, Baig MNR, Cheng Y, Deng XX (2010) Retro-transposon based genetic similarity within the genus Citrus and its relatives. Genet Res Crop Evol 57:963–972

  5. Bretó MP, Ruiz C, Pina JA, Asíns MJ (2001) The diversification of Citrus clementina Hort. ex Tan., a vegetatively propagated crop species. Mol Phylogen Evol 21:285–293

  6. Brookes AJ (1999) The essence of SNPs. Gene 234:177–186

  7. Chaisan T, Van K, Kim MY, Kim KD, Choi B-S, Lee S-H (2010) In silico single nucleotide polymorphism discovery and application to marker-assisted selection in soybean. Mol Breed 29:221–233

  8. Ching A, Caldwell KS, Jung M, Dolan M, Smith OS, Tingey S, Morgante M, Rafalski AJ (2002) SNP frequency, haplotype structure and linkage disequilibrium in elite maize inbred lines. BMC Genet 3:19

  9. Close TJ, Bhat PR, Lonardi S, Wu Y, Rostoks N, Ramsay L, Druka A, Stein N, Svensson JT, Wanamaker S, Bozdag S, Roose ML, Moscou MJ, Chao S, Varshney RK, Szűcs P, Sato K, Hayes PM, Matthews DE, Kleinhofs A, Muehlbauer GJ, DeYoung J, Marshall DF, Madishetty K, Fenton RD, Condamine P, Graner A, Waugh R (2009) Development and implementation of high-throughput SNP genotyping in barley. BMC Genomics 10:582

  10. Conesa A, Götz S, Garcia-Gomez JM, Terol J, Talon M, Robles M (2005) Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics 21:3674–3676

  11. Davies F, Albrigo LG (1994) Taxonomy, cultivars and breeding. In: Hammerschlag FA, Litz RE (eds) Citrus. CAB International, Wallingford, pp 12–52

  12. Forment J et al (2005) Development of a citrus genome-wide EST collection and cDNA microarray as resources for genomic studies. Plant Mol Biol 57(3):375–391

  13. Fu J, Peng ZJ, Cai XD, Guo WW (2011) Regeneration and molecular characterization of interspecific somatic hybrids between Satsuma mandarin and two seedy sweet oranges for scion improvement. Plant Breed 130:287–290

  14. Fujii H, Shimada T, Nonaka K, Kita M, Kuniga T, Endo T, Ikoma Y, Omura M (2012) High-throughput genotyping in citrus accessions using a SNP genotyping array. Tree Genet Genomes. doi: 10.1007/s11295-012-0542-3

  15. Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl Acids Symp Ser 41:95–98

  16. Hamdy Amar M, Biswas MK, Zhang Z, Guo WW (2011) Exploitation of SSR, SRAP and CAPS-SNP markers for genetic diversity of Citrus germplasm collection. Sci Hort 128:220–227

  17. Hamilton JP, Hansey CN, Whitty BR, Stoffel K, Massa AN, Van Deynze A, De Jong WS, Douches DS, Buell CR (2011) Single nucleotide polymorphism discovery in elite North American potato germplasm. BMC Genomics 12:302

  18. Hodgson RW (1967) Horticultural varieties of Citrus. In: Reuther W, Webber HJ, Batchelor LD (eds) The Citrus industry, 1st edn., Chapt. 4, vol 1. University of California, California, p 483

  19. Jannati M, Fotouhi R, Abad AP, Salehi Z (2009) Genetic diversity analysis of Iranian citrus varieties using micro satellite (SSR) based markers. J Hort For 1:120–125

  20. Jiang D, Ye Q-L, Wang F-S, Cao L (2010) The mining of Citrus EST-SNP and its application in cultivar discrimination. Agric Sci China 9:179–190

  21. Licciardello C, Russo MP, Vale’ G, Recupero RG (2008) Identification of differentially expressed genes in the flesh of blood and common oranges. Tree Genet Genomes 4:315–331

  22. Liu Q, Zhu A, Chai L, Zhou W, Yu K, Ding J, Xu J, Deng X (2009) Transcriptome analysis of a spontaneous mutant in sweet orange [Citrus sinensis (L.) Osbeck] during fruit development. J Exp Bot 60(3):801–813

  23. Martinez-Godoy AM, Mauri N, Juarez J, Marques CM, Santiago J, Forment J, Gadea J (2008) A genome-wide 20 K citrus microarray for gene expression analysis. BMC Genomics 9:art. no. 318

  24. McNally KL, Childs KL, Bohnert R, Davidson RM, Zhao K, Ulat VJ, Zeller G, Clark RM, Hoen DR, Bureau TE, Stokowski R, Ballinger DG, Frazer KA, Cox DR, Padhukasahasram B, Bustamante CD, Weigel D, Mackill DJ, Bruskiewich RM, Rätsch G, Buell CR, Leung H, Leach JE (2009) Genome wide SNP variation reveals relationships among landraces and modern varieties of rice. Proc Natl Acad Sci USA 106:12273–12278

  25. Micheletti D, Troggio M, Salamini F, Viola R, Velasco R, Salvi S (2011) On the evolutionary history of the domesticated apple. Nat Genet 43:1044–1045

  26. Novelli VM, Takita MA, Machado MA (2004) Identification and analysis of single nucleotide polymorphisms (SNPs) in citrus. Euphytica 138:227–237

  27. Ollitrault P, Terol J, Garcia-Lor A, Berard A, Chauveau A, Froelicher Y, Belzile C, Morillon R, Navarro L, Brunel D, Talon M (2012) SNP mining in C. clementina BAC end sequences; transferability in the Citrus genus (Rutaceae), phylogenetic inferences and perspectives for genetic mapping. BMC Genomics 13:13

  28. Singhal D, Gupta P, Sharma P, Kashyap N, Anand S, Sharma H (2011) In-silico single nucleotide polymorphisms (SNP) mining of Sorghum bicolor genome. Afr J Biotechnol 10:580–583

  29. Specht CA, DiRusso CC, Novotny CP, Ullrich RC (1982) A method for extracting high-molecular-weight deoxyribonucleic acid from fungi. Anal Biochem 119:158–163

  30. Tang J, Vosman B, Voorrips RE, van der Linden CG, Leunissen JAM (2006) QualitySNP: a pipeline for detecting single nucleotide polymorphisms and insertions/deletions in EST data from diploid and polyploid species. BMC Bioinform 7:438

  31. Terol J et al (2007) Analysis of 13000 unique Citrus clusters associated with fruit quality, production and salinity tolerance. BMC Genomics 8:art. no. 31

  32. Terol J, Naranjo MA, Ollitrault P, Talon M (2008) Development of genomic resources for Citrus clementina: characterization of three deep-coverage BAC libraries and analysis of 46,000 BAC end sequences. BMC Genomics 9:423

  33. Trebbi D, Maccaferri M, de Heer P, Sørensen A, Giuliani S, Salvi S, Sanguineti MC, Massi A, Andries E, van der Vossen G, Tuberosa R (2011) High-throughput SNP discovery and genotyping in durum wheat (Triticum durum Desf.). Theor Appl Genet 123:555–569

  34. Van Inghelandt D, Melchinger AE, Lebreton C, Stich B (2010) Population structure and genetic diversity in a commercial maize breeding program assessed with SSR and SNP markers. Theor Appl Genet 120:1289–1299

  35. Vezzulli S, Micheletti D, Riaz S, Pindo M, Viola R, This P, Walker MA, Troggio M, Velasco R (2008) A SNP transferability survey within the genus Vitis. BMC Plant Biol 8:128

  36. Vignal A, Milan D, Cristobal MS, Eggen A (2002) A review on SNP and other types of molecular markers and their use in animal genetics. Genet Sel Evol 34:275–305

  37. Xu Q et al (2012) The draft genome of sweet orange (Citrus sinensis). Nat Genet. doi:10.1038/ng.2472

  38. Ye J-L, Zhu A-D, Tao N-G, Xu Q, Xu J, Deng X-X (2010) Comprehensive analysis of expressed sequence tags from the pulp of the red mutant ‘Cara Cara’ Navel orange (Citrus sinensis Osbeck). J Integr Plant Biol 52:856–867

Download references

Acknowledgments

This work was supported by the following Italian Projects of the MiPAAF: RAVAGRU (Ricerche avanzate in agrumicultura e loro applicazioni) D.M. 187/7303/2007 and ESPLORA (ESPLORAzione della biodiversità vegetale ed animale alla ricerca di alleli superiori da inserire nei programmi avanzati di miglioramento genetico a sostegno dell’agricoltura nazionale) D.M. 14658/7303/2010.

Author information

Correspondence to Jamila Bernardi.

Electronic supplementary material

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Bernardi, J., Mazza, R., Caruso, P. et al. Use of an expressed sequence tag-based method for single nucleotide polymorphism identification and discrimination of Citrus species and cultivars. Mol Breeding 31, 705–718 (2013). https://doi.org/10.1007/s11032-012-9828-2

Download citation

Keywords

  • SNP
  • Citrus
  • QualitySNP
  • EST