SNP discovery from next-generation transcriptome sequencing data and their validation using KASP assay in wheat (Triticum aestivum L.)
Single nucleotide polymorphisms (SNPs) are becoming the most amenable form of DNA-based molecular markers for genetic analysis. In hexaploid bread wheat (Triticum aestivum L.), it is difficult to discern true polymorphic SNPs due to homoeologous and paralogous genes. Two serial analysis of gene expression (SAGE) libraries were developed utilizing leaves from resistant plants carrying leaf rust resistance gene Lr28; one library was derived from leaves that were mock inoculated and the other was derived from leaves inoculated with the urediniospores of the leaf rust pathogen Puccinia triticina. Next-generation sequencing reads, after quality trimming and removal of fungal sequences, were mapped to wheat reference sequences at Ensembl Plants. CLC Genomics Workbench and Freebayes softwares were employed for SNP calling. A total of 611 SNPs were predicted to be common by both softwares, of which 207 varietal SNPs were identified by ConservedPrimer software. A subset of 100 SNPs was used for validation across 47 wheat genotypes using Kompetitive Allele Specific PCR (KASP) assay; 83 SNPs could be successfully validated. These SNPs were positioned on wheat subgenomes and chromosome arms. When functionally annotated, many sequences harboring SNPs showed homology to resistance and resistance-like genes listed in Plant Resistance Gene database (PRGdb) as well as pathogenesis-related (PR) and stress-responsive genes. The results of the present study involving discovery of SNPs associated with resistance to leaf rust, a major threat to wheat production worldwide, will be valuable for molecular breeding for rust resistance.
KeywordsSingle nucleotide polymorphisms Wheat Transcriptome KASP assay Leaf rust
The authors are thankful to Biotechnology Information System Network Distributed Information Sub Centre (BT/BI/04/065/04) for providing facilities for bioinformatics analyses. This work was supported by Department of Biotechnology, Government of India (Grant No. BT/PR6037/AGR/02/308/05 and BT/PR3925/BID/7/384/2011) and Centre of Excellence, Technical Education Quality Improvement Program-II (Grant No. NPIU/TEQIP II/FIN/31/158).
KM, MK, and PKG designed the experiment and wrote the manuscript; SC, DS, and SK performed SNP genotyping; JP and RP supported the bioinformatics analysis; HSB and KVP revised the manuscript. All the authors read and approved the final manuscript.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflicts of interest.
- Allen AM, Barker GL, Berry ST, Coghill JA, Gwilliam R, Kirby S, Robinson P, Brenchley RC, D’Amore R, McKenzie N, Waite D, Hall A, Bevan M, Hall N, Edwards KJ (2011) Transcript-specific, single nucleotide polymorphism discovery and linkage analysis in hexaploid bread wheat (Triticum aestivum L.) Plant Biotechnol J 9:1086–1099CrossRefPubMedGoogle Scholar
- Benjamini Y, Hochberg Y (1995) Controlling the false discovery rate: a practical and powerful approach to multiple testing. J R Stat Soc Series B Stat Methodol 57:289–300Google Scholar
- Boutet G, Carvalho SA, Falque M, Peterlongo P, Lhuillier E, Bouchez O, Lavaud C, Piley-Nayel M, Riviere N, Baranger A (2016) SNP discovery and genetic mapping using genotyping by sequencing of whole genome genomic DNA from a pea RIL population. BMC Genomics 17:121CrossRefPubMedPubMedCentralGoogle Scholar
- Braun HJ, Atlin G, Payne T (2010) Multi-location testing as a tool to identify plant response to global climate change. In: climate change and crop production. Ed: CRP Reynolds, CABI London pp: 115–138Google Scholar
- Cavanagh CR, Chao S, Wang S, Huang BE, Stephen S, Kiani S, Forrest K, Saintenac C, Brown-Guedira GL, Akhunova A, See D, Bai G, Pumphrey M, Tomar L, Wong D, Kong S, Reynolds M, Da Silva ML, Bockelman H, Talbert L, Anderson JA, Dreisigacker S, Baenziger S, Carter A, Korzun V, Morrell PL, Dubcovsky J, Morell MK, Sorrells ME, Hayden MJ, Akhunov E (2013) Genome-wide comparative diversity uncovers multiple targets of selection for improvement in hexaploid wheat landraces and cultivars. Proc Natl Acad Sci U S A 110:8057–8062CrossRefPubMedPubMedCentralGoogle Scholar
- Chapman JA, Mascher M, Bulu A, Barry K, Georganas E, Session A, Strnadova V, Jenkins J, Sehgal S, Oliker L, Schmutz J, Yelick KA, Scholz U, Waugh R, Poland JA, Muehlbauer GJ, Stein N, Rokhsar DS (2015) A whole-genome shotgun approach for assembling and anchoring the hexaploid bread wheat genome. Genome Biol 16:26CrossRefPubMedPubMedCentralGoogle Scholar
- Charpe A, Koul S, Gupta SK, Singh A, Pallavi JK, Prabhu KV (2012) Marker assisted gene pyramiding of leaf rust resistance genes Lr9, Lr24 and Lr 28 in a bread wheat cultivar HD2329. J Wheat Res 4:20–28Google Scholar
- Cingolani P, Platts A, Wang LL, Coon M, Nguyen T, Wang L, Land SJ, Lu X, Ruden DM (2012) A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w1118; iso-2; iso-3. Fly 6:80–92CrossRefPubMedPubMedCentralGoogle Scholar
- Edwards D, Wilcox S, Barrero RA, Fleury D, Cavanagh CR, Forrest KL, Hayden MJ, Moolhuijzen P, Keeble-Gagnère G, Bellgard MI, Lorenc MT, Shang CA, Baumann U, Taylor JM, Morell MK, Langridge P, Appels R, Fitzgerald A (2012) Bread matters: a national initiative to profile the genetic diversity of Australian wheat. Plant Biotechnol J 10:703–708CrossRefPubMedGoogle Scholar
- Garrison E, Marth G (2012) Haplotype-based variant detection from short-read sequencing. arXiv preprint arXiv. 1207: 3907Google Scholar
- Jordan KW, Wang S, Lun Y, Gardiner LJ, MacLachlan R, Hucl P, Wiebe K, Wong D, Forrest KL, IWGSC, Sharpe AG, CHD S, Hall N, Toomajian C, Close T, Dubcovsky J, Akhunova A, Talbert L, Bansal UK, Bariana HS, Hayden MJ, Pozniak C, Jeddeloh JA, Hall A, Akhunova E (2015) A haplotype map of allohexaploid wheat reveals distinct patterns of selection on homoeologous genomes. Genome Biol 16:48CrossRefPubMedPubMedCentralGoogle Scholar
- Kal AJ, van Zonneveld AJ, Benes V, den Berg MV, Koerkamp MG, Albermann K, Strack N, Ruijter JM, Richter A, Dujon B, Ansorge W, Tabak HF (1999) Dynamics of gene expression revealed by comparison of serial analysis of gene expression transcript profiles from yeast grown on two different carbon sources. Mol Biol Cell 10:1859–1872CrossRefPubMedPubMedCentralGoogle Scholar
- Kassa MT, You FM, Hiebert CW, Pozniak CJ, Fobert PR, Sharpe AG, Menzies JG, Humphreys DG, Rezac HN, Fellers JP, McCallum BD, McCartney CA (2017) Highly predictive SNP markers for efficient selection of the wheat leaf rust resistance gene Lr16. BMC Plant Biol 17:45CrossRefPubMedPubMedCentralGoogle Scholar
- Lai K, Lorenc MT, Lee HC, Berkman PJ, Bayer PE, Visendi P, Ruperao P, Fitzgerald TL, Zander M, Chan CK, Manoli S, Stiller J, Batley J, Edwards D (2015) Identification and characterization of more than 4 million intervarietal SNPs across the group 7 chromosomes of bread wheat. Plant Biotechnol J 13:97–104CrossRefPubMedGoogle Scholar
- Li G, Xu X, Bai G, Carver BF, Hunger R, Bonman JM, Kolmer J, Dong H (2016) Genome-wide association mapping reveals novel QTL for seedling leaf rust resistance in a worldwide collection of winter wheat. Plant Genome 9:3Google Scholar
- Maccaferri M, Zhang J, Bulli P, Abate Z, Chao S, Cantu D, Bossolini E, Chen X, Pumphrey M, Dubcovsky J (2015) A genome-wide association study of resistance to stripe rust (Puccinia striiformis f. sp. tritici) in a worldwide collection of hexaploid spring wheat (Triticum aestivum L.) G3 5:449–465CrossRefPubMedPubMedCentralGoogle Scholar
- 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, Ratsch 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 U S A 106:12273–12278CrossRefPubMedPubMedCentralGoogle Scholar
- Wang S, Wong D, Forrest K, Allen A, Chao S, Huang BE, Maccaferri M, Salvi S, Milner SG, Cattivelli L, Mastrangelo AM, Whan AM, Stephe S, Barker G, Wieseke R, Plieske J, International Wheat Genome Sequencing Consortium, Lillemo M, Mather D, Appels R, Dolferus R, Brown-Guedira G, Korol A, Akhunova AR, Feuillet C, Salse J, MorganteM PC, Luo M-C, Dvorak J, Morell M, Dubcovsky J, Ganal M, Tuberosa R, Lawley C, Mikoulitch I, Cavanagh C, Edwards KJ, Hayden M, Akhunov E (2014) Characterization of polyploid wheat genomic diversity using a high-density 90,000 single nucleotide polymorphism array. Plant Biotech J 12:787–796CrossRefGoogle Scholar
- Wicker T, Mayer KFX, Gundlach H, Martis M, Steuernage B, Scholz U, Simková H, Kubaláková M, Choulet F, Taudien S, Platzer M, Feuillet C, Fahima T, Budak H, Dolezel J, Keller B, Stein N (2011) Frequent gene movement and pseudogene evolution is common to the large and complex genomes of wheat, barley, and their relatives. Plant Cell 23:1706–1719CrossRefPubMedPubMedCentralGoogle Scholar
- You FM, Huo N, Gu YQ, Lazo GR, Dvorak J, Anderson OD (2009) ConservedPrimers 2.0: a high-throughput pipeline for comparative genome referenced intron-flanking PCR primer design and its application in wheat SNP discovery. BMC Bioinformatics. 13: 331Google Scholar
- You FM, Huo N, Deal KR, Gu YQ, Luo M, McGuire PE, Dvorak J, Anderson OD (2011) Annotation-based genome-wide SNP discovery in the large and complex Aegilops tauschii genome using next-generation sequencing without a reference genome sequence. BMC Genomics 12:59CrossRefPubMedPubMedCentralGoogle Scholar