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EST-SNP genotyping of citrus species using high-resolution melting curve analysis

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Abstract

Citrus taxonomy is very complex mainly due to specific aspects of its reproductive biology. A number of studies have been performed using various molecular markers in order to evaluate the level of genetic variability in Citrus. SNP markers have been used for genetic diversity assessment using a variety of different methods. Recently, the availability of EST database and whole genome sequences has made it possible to develop more markers such as SNPs. In the present study, the high-resolution melting curve analysis (HRM) was used to detect SNPs or INDELs in Citrus genus for the first time. We aimed to develop a panel of SNPs to differentiate Citrus genotypes which can also be applied to Citrus biodiversity studies. The results showed that 21 SNP containing markers produced distinct polymorphic melting curves among the Citrus spp. investigated through HRM analysis. It was proved that HRM is an efficient, cost-effective, and accurate method for discriminating citrus SNPs as well as a method to analyze more polymorphisms in a single PCR amplicon, representing a useful tool for genetic, biodiversity, and breeding studies. SNPs developed based on Citrus sinensis EST database showed a good transferability within the Citrus genus. Moreover, HRM analysis allowed the discrimination of citrus genotypes at specific level and the resulting genetic distance analysis clustered these genotypes into three main branches. The results suggested that the panel of SNP markers could be used in a variety of applications in citrus biodiversity assessment and breeding programs using HRM analysis.

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References

  • Amar MH, Biswas MK, Zhang ZW, Guo WW (2011) Exploitation of SSR, SRAP and CAPS–SNP markers for genetic diversity of Citrus germplasm collection. Sci Hortic 128:220–227

    Article  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • Batley J, Edwards D (2007) SNP applications in plants. In: Oraguzie N, Rikkerink E, Gardiner S, De Silva NH (eds) Association mapping in plants. Springer, New York, pp 95–102

    Chapter  Google Scholar 

  • Biswas MK, Chai LJ, Qiang X, Deng XX (2012) Generation, functional analysis and utility of Citrus grandis EST from a flower-derived cDNA library. Mol Biol Rep 39:7221–7235

    Article  PubMed  CAS  Google Scholar 

  • Cameron JW, Frost HB (1968) Genetics, breeding and nucellar embryony. In: Reuthen W, Webber HJ, Batchelor LD (eds) The citrus industry. University of California Press, Berkeley, pp 325–370

    Google Scholar 

  • Chagne D, Gasic K, Crowhurst RN, Han Y, Bassett HC, Bowatte DR, Lawrence TJ, Rikkerink EH, Gardiner SE, Korban SS (2008) Development of a set of SNP markers present in expressed genes of the apple. Genomics 92:353–358

    Article  PubMed  CAS  Google Scholar 

  • Chaisan T, Van K, Kim MY, Kim KD, Choi BS, Lee SH (2012) In silico single nucleotide polymorphism discovery and application to marker-assisted selection in soybean. Mol Breeding 29:221–233

    Article  Google Scholar 

  • Chen C, Zhou P, Choi YA, Huang S, Gmitter FG (2006) Mining and characterizing microsatellites from citrus ESTs. Theor Appl Genet 112:1248–1257

    Article  PubMed  CAS  Google Scholar 

  • Corbett Research (2006) High resolution melt assay design and analysis CorProtocolTM. Corbett Research, Sydney

    Google Scholar 

  • Crifò T, Petrone G, Lo Cicero L, Lo Piero AR (2012) Short cold storage enhances the anthocyanin contents and level of transcripts related to their biosynthesis in blood oranges. J Agr Food Chem 60:476–481

    Article  Google Scholar 

  • Deng ZN, Gentile A, Nicolosi E, Domina F, Vardi A, Tribulato E (1995) Identification of in vivo and in vitro lemon mutants by RAPD markers. J Hortic Sci 70:117–125

    CAS  Google Scholar 

  • Distefano G, Caruso M, La Malfa S, Gentile A, Wu S-B (2012) High resolution melting analysis is a more sensitive and effective alternative to gel-based platforms in analysis of SSR—an example in Citrus. Plos One 7(8):e44202. doi:10.1371/journal.pone.0044202

    Article  PubMed  CAS  Google Scholar 

  • Doyle JJ, Doyle JL (1987) A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem Bull 19:11–15

    Google Scholar 

  • Fang DQ, Roose ML (1997) Identification of closely related Citrus cultivars with inter-simple sequence repeat markers. Theor Appl Genet 95:408–417

    Article  CAS  Google Scholar 

  • Federici CT, Fang DQ, Scora RW, Roose ML (1998) Phylogenetic relationships within the genus Citrus (Rutaceae) and related genera as revealed by RFLP and RAPD analysis. Theor Appl Genet 94:812–822

    Article  Google Scholar 

  • Flot JF (2007) Champuru 1.0: a computer software for unraveling mixtures of two DNA sequences of unequal lengths. Mol Ecol Notes 7:974–977

    Article  CAS  Google Scholar 

  • Fujii H, Shimada T, Nonaka K, Kita M, Kuniga T, Endo T, Ikoma Y, Omura M (2012) High-throughput genotyping in citrus accessions using an SNP genotyping array. Tree Genet Genomes: 1–9

  • Ganal MW, Altmann T, Roder MS (2009) SNP identification in crop plants. Curr Opin Plant Biol 12:211–217

    Article  PubMed  CAS  Google Scholar 

  • Garcia-Lor A, Luro F, Navarro L, Ollitrault P (2012) Comparative use of InDel and SSR markers in deciphering the interspecific structure of cultivated citrus genetic diversity; a perspective for genetic association studies. Mol Genet Genomics 287:77–94

    Article  PubMed  CAS  Google Scholar 

  • Garritano S, Gemignani F, Voegele C, Nguyen-Dumont T, Le Calvez-Kelm F et al (2009) Determining the effectiveness of high resolution melting analysis for SNP genotyping and mutation scanning at the TP53 locus. BMC Genet 10:5

    Article  PubMed  Google Scholar 

  • Garvin MR, Saitoh K, Gharret AJ (2010) Application of single nucleotide polymorphisms to non-model species: a technical review. Mol Ecol Res 10:915–934

    Article  CAS  Google Scholar 

  • Gmitter FG, Chen C, Machado MA, de Souza AA, Ollitrault P, Froehlicher Y, Shimizu T (2012) Citrus genomics. Tree Genet Genomes 8:611–626

    Article  Google Scholar 

  • Goodstein DM, Shu S, Howson R, Neupane R, Hayes RD, Fazo J, Mitros T, Dirks W, Hellsten U, Putnam N, Rokhsar DS (2012) Phytozome: a comparative platform for green plant genomics. Nucleic Acids Res 40:78–86

    Article  Google Scholar 

  • Gupta PK, Roy JK, Prasad M (2001) Single nucleotide polymorphisms: a new paradigm for molecular marker technology and DNA polymorphism detection with emphasis on their use in plants. Curr Science 80:524–535

    CAS  Google Scholar 

  • Han Y, Khu DM, Monteros MJ (2012) High-resolution melting analysis for SNP genotyping and mapping in tetraploid alfalfa (Medicago sativa L.). Mol Breeding 29:489–501

    Article  Google Scholar 

  • Hayashi K, Hashimoto N, Daigen M, Ashikawa I (2004) Development of PCR-based SNP markers for rice blast resistance genes at the Piz locus. Theor Appl Genet 108:1212–1220

    Article  PubMed  CAS  Google Scholar 

  • Herrero R, Asins MJ, Carbonell EA, Navarro L (1996) Genetic diversity in the orange subfamily Aurantioideae. I. Intraspecies and intragenus genetic variability. Theor Appl Genet 92:599–609

    Article  CAS  Google Scholar 

  • Herrmann MG, Durtschi JD, Wittwer CT, Voelkerding KV (2007) Expanded instrument comparison of amplicon DNA melting analysis for mutation scanning and genotyping. Clin Chem 53:1544–1548

    Article  PubMed  CAS  Google Scholar 

  • Jeong HJ, Jo YD, Kang BC (2010) Identification of Capsicum species using SNP markers based on high resolution melting analysis. Genome 53:1029–1040

    Article  PubMed  CAS  Google Scholar 

  • Jiang D, Ye QL, Wang FS, Cao L (2010) The mining of citrus EST–SNP and its application in cultivar discrimination. Agricultural Sciences in China 9:179–190

    Article  CAS  Google Scholar 

  • Lehmensiek A, Sutherland M, McNamara R (2008) The use of high resolution melting (HRM) to map single nucleotide polymorphism markers linked to a covered smut resistance gene in barley. Theor Appl Genet 117:721–728

    Article  PubMed  CAS  Google Scholar 

  • Liew M, Pryor R, Palais R, Meadows C, Erali M, Lyon E, Wittwer C (2004) Genotyping of single-nucleotide polymorphisms by high-resolution melting of small amplicons. Clin Chem 50:1156–1164

    Article  PubMed  CAS  Google Scholar 

  • Liu K, Muse SV (2005) Powermarker: an integrated analysis environment for genetic marker analysis. Bioinformatics 21:2128–2129

    Article  PubMed  CAS  Google Scholar 

  • Luro FL, Laigret F, Bove JM, Ollitrault P (1995) DNA amplified fingerprinting, a useful tool for determination of genetic origin and diversity analysis in Citrus. Hort Sci 30:1063–1067

    CAS  Google Scholar 

  • Luro FL, Costantino G, Terol J, Argout X, Allario T, Wincker P, Talon M, Ollitrault P, Morillon R (2008) Transferability of the EST–SSRs developed on Nules clementine (Citrus clementina Hort ex Tan) to other Citrus species and their effectiveness for genetic mapping. BMC Genom 9:287–299

    Article  Google Scholar 

  • Mackay JF, Wright CD, Bonfiglioli RG (2008) A new approach to varietal identification in plants by microsatellite high resolution melting analysis: application to the verification of grapevine and olive cultivars. Plant Methods 4:8

    Article  PubMed  Google Scholar 

  • Markham NR, Zuker M (2005) DINAMelt web server for nucleic acid melting prediction. Nucleic Acids Res 33:W577–W581

    Article  PubMed  CAS  Google Scholar 

  • Moore GA (2001) Oranges and lemons: clues to the taxonomy of Citrus from molecular markers. Trends in Genet 17:536–540

    Article  CAS  Google Scholar 

  • Muleo R, Colao MC, Miano D, Cirilli M, Intrieri MC, Baldoni L, Rugini E (2009) Mutation scanning and genotyping by high-resolution DNA melting analysis in olive germplasm. Genome 52:252–260

    Article  PubMed  CAS  Google Scholar 

  • Nakano M, Shimizu T, Fujii H, Shimada T, Endo T, Nesumi H, Kuniga T, Omura M (2008) Marker enrichment and construction of haplotype-specific BAC contigs for the polyembryony genomic region in Citrus. Breeding Sci 58:375–383

    Article  CAS  Google Scholar 

  • Nicolosi E, Deng ZN, Gentile A, La Malfa S, Continella G, Tribulato E (2000) Citrus phylogeny and genetic origin of important species as investigated by molecular markers. Theor Appl Genet 100:1155–1166

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  • Novelli VM, Cristofani M, Souza AA, Machado MA (2006) Development and characterization of polymorphic microsatellite markers for the sweet orange (Citrus sinensis L. Osbeck). Genet Mol Biol 29:90–96

    Article  CAS  Google Scholar 

  • Ohnishi Y, Tanaka T, Ozaki K, Yamada R, Suzuki H, Nakamura Y (2001) A high-throughput SNP typing system for genome-wide association studies. J Hum Genet 46:471–477

    Article  PubMed  CAS  Google Scholar 

  • Oliver RE, Lazo GR, Lutz JD, Rubenfield MJ, Tinker NA, Anderson JM, Wisniewski Morehead NH, Adhikary D, Jellen EN, Maughan PJ, Brown Guedira GL, Chao S, Beattie AD, Carson ML, Rines HW, Obert DE, Bonman JM, Jackson EW (2011) Model SNP development for complex genomes based on hexaploid oat using high-throughput 454 sequencing technology. BMC Genom 12:77

    Article  CAS  Google Scholar 

  • Ollitrault F, Terol J, Pina JA, Navarro L, Talon M, Ollitrault P (2010) Development of SSR markers from Citrus clementina (Rutaceae) BAC end sequences and interspecific transferability in Citrus. Am J Bot 97:e124–e129

    Article  PubMed  CAS  Google Scholar 

  • 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 Genom 13:13

    Google Scholar 

  • Raghuvanshi SS (1962) Cytological studies in the genus Citrus. IV. Evolution in the genus Citrus. Cytologia 27:172–188

    Article  Google Scholar 

  • Rozen S, Skaletsky H (2000) Primer3 on the WWW for general users and for biologist programmers. In: Krawetz S, Misener S (eds) Bioinformatics methods and protocols: methods in molecular biology. Humana Press, Totowa, pp 365–386

    Google Scholar 

  • Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425

    PubMed  CAS  Google Scholar 

  • Seipp MT, Durtschi JD, Voelkerding KV, Wittwer CT (2009) Multiplex amplicon genotyping by high-resolution melting. J Biomol Tech 20:160–164

    PubMed  Google Scholar 

  • 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 Genom 9:423

    Article  Google Scholar 

  • van Tienderen PH, de Haan AA, van der Linden CG, Vosman B (2002) Biodiversity assessment using markers for ecologically important traits. Trends Ecol Evol 17:577–582

    Article  Google Scholar 

  • Wang HT, Kim MK, Kim YJ, Lee HN, Jin HZ, Chen J, Yang DC (2012) Molecular authentication of the Oriental medicines Pericarpium Citri Reticulatae and Citri Unshius Pericarpium using SNP markers. Gene 494:92–95

    Article  PubMed  CAS  Google Scholar 

  • White HE, Hall VJ, Cross NCP (2007) Methylation-sensitive high resolution melting-curve analysis of the SNRPN gene as a diagnostic screen for Prader–Willi and Angelman syndromes. Clin Chem 53:1960–1962

    Article  PubMed  CAS  Google Scholar 

  • Wu S-B, Wirthensohn MG, Hunt P, Gibson JP, Sedgley M (2008) High resolution melting analysis of almond SNPs derived from ESTs. Theor Appl Genet 118:1–14

    Article  PubMed  CAS  Google Scholar 

  • Wu S-B, Tavassolian I, Rabiei G, Hunt P, Wirthensohn M, Gibson J, Ford CM, Sedgley M (2009) Mapping SNP anchored genes using high resolution melting analysis in almond. Mol Genet Genomics 282:273–281

    Article  PubMed  CAS  Google Scholar 

  • Wu S-B, Franks T, Hunt P, Wirthensohn M, Gibson J, Sedgley M (2010) Discrimination of SNP genotypes associated with complex haplotypes by high resolution melting analysis in almond: implications for improved marker efficiencies. Mol Breed 25:351–357

    Article  CAS  Google Scholar 

  • Wu B, Yang R-T, Zhu S-P, Zhong Y, Jiang B, Zeng J-W, Zhong G (2012) Genotyping single nucleotide polymorphisms in mandarin cultivars using high resolution melting analysis. Acta Hort Sinica 39:777–782

    CAS  Google Scholar 

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Acknowledgments

This research was supported by Regione Siciliana, Project “Innovazioni bioagronomiche e fitopatologiche per il contenimento di CTV e per il rinnovamento dell’agrumicoltura siciliana”.

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Authors and Affiliations

  1. Dipartimento di Scienze delle Produzioni Agrarie e Alimentari, sez. Arboricoltura, University of Catania, Via Valdisavoia 5, Catania, 95123, Italy

    Gaetano Distefano, Stefano La Malfa & Alessandra Gentile

  2. School of Environmental and Rural Science, University of New England, Armidale, NSW, 2351, Australia

    Gaetano Distefano & Shu-Biao Wu

Authors
  1. Gaetano Distefano
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  2. Stefano La Malfa
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  3. Alessandra Gentile
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  4. Shu-Biao Wu
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Corresponding authors

Correspondence to Stefano La Malfa or Shu-Biao Wu.

Additional information

Communicated by W.-W. Guo

Electronic supplementary material

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Online Resource 1

Identification and reference number of the ESTs (HarvEST: Citrus 1.32 Assembly: #C54CS) forming the contigs used for SNPs identification (PDF 192 kb)

Online Resource 2

SNP haplotypes in 18 citrus genotypes as revealed by HRM analysis performed with 21 selected EST-SNPs markers (PDF 20 kb)

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Distefano, G., La Malfa, S., Gentile, A. et al. EST-SNP genotyping of citrus species using high-resolution melting curve analysis. Tree Genetics & Genomes 9, 1271–1281 (2013). https://doi.org/10.1007/s11295-013-0636-6

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