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Aegilops umbellulata introgression carrying leaf rust and stripe rust resistance genes Lr76 and Yr70 located to 9.47-Mb region on 5DS telomeric end through a combination of chromosome sorting and sequencing


Key message

Lr76 and Yr70 have been fine mapped using the sequence of flow-sorted recombinant 5D chromosome from wheat-Ae. umbellulata introgression line. The alien introgression has been delineated to 9.47-Mb region on short arm of wheat chromosome 5D.


Leaf rust and stripe rust are among the most damaging diseases of wheat worldwide. Wheat cultivation based on limited number of rust resistance genes deployed over vast areas expedites the emergence of new pathotypes warranting a continuous deployment of new resistance genes. In this paper, fine mapping of Aegilops umbellulata-derived leaf rust and stripe rust resistance genes Lr76 and Yr70 is being reported. We flow sorted and paired-end sequenced 5U chromosome of Ae. umbellulata, recombinant chromosome 5D (5DIL) from wheat-Ae. umbellulata introgression line pau16057 and 5DRP of recurrent parent WL711. Chromosome 5U reads were mapped against the reference Chinese Spring chromosome 5D sequence, and alien-specific SNPs were identified. Chromosome 5DIL and 5DRP sequences were de novo assembled, and alien introgression-specific markers were designed by selecting 5U- and 5D-specific SNPs. Overall, 27 KASP markers were mapped in high-resolution population consisting of 1404 F5 RILs. The mapping population segregated for single gene each for leaf rust and stripe rust resistance. The physical order of the SNPs in pau16057 was defined by projecting the 27 SNPs against the IWGSC RefSeq v1.0 sequence. Based on this physical map, the size of Ae. umbellulata introgression was determined to be 9.47 Mb on the distal most end of the short arm of chromosome 5D. This non-recombining alien segment carries six NB-LRR encoding genes based on NLR annotation of assembled chromosome 5DIL sequence and IWGSC RefSeq v1.1 gene models. The presence of SNPs and other sequence variations in these genes between pau16057 and WL711 suggested that they are candidates for Lr76 and Yr70.

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  1. Adato O, Ninyo N, Gophna U, Snir S (2015) Detecting horizontal gene transfer between closely related taxa. PLoS Comput Biol. https://doi.org/10.1371/journal.pcbi.1004408

  2. Andrews S (2010) FastQC: a quality control tool for high throughput sequence data. http://www.bioinformatics.babraham.ac.uk/projects/fastqc

  3. Arzani A, Ashraf M (2017) Cultivated ancient wheats (Triticum spp.): a potential source of health-beneficial food products. Compr Rev Food Sci Food Saf 16:477–488

  4. Bansal M, Kaur S, Dhaliwal HS, Bains NS, Bariana HS, Chhuneja P, Bansal UK (2017) Mapping of Aegilops umbellulata-derived new leaf rust and stripe rust resistance loci in wheat. Plant Pathol 66:38–44

  5. Bulgarelli D, Biselli C, Collins NC, Consonni G, Stanca AM, Schulze-Lefert P, Vale G (2010) The CC-NB-LRR-Type Rdg2a resistance gene confers immunity to the seed-borne barley leaf stripe pathogen in the absence of hypersensitive cell death. PLoS ONE 5:e12599

  6. Bulos M, Echarte M, Sala C (2006) Occurrence of the rust resistance gene Lr37 from Aegilops ventricosa in Argentine cultivars of wheat. Electron J Biotechnol 9:580–586

  7. Ceoloni C, Kuzmanovic L, Ruggeri R, Rossini F, Forte P, Cuccurullo A, Bitti A (2017) Harnessing genetic diversity of wild gene pools to enhance wheat crop production and sustainability: Challenges and opportunities. Diversity 9:55. https://doi.org/10.3390/d9040055

  8. Cox TS (1998) Deepening the wheat gene pool. Journal of Crop Production 1:1–25

  9. Cox TS, Raupp WJ, Gill BS (1994) Leaf rust-resistance genes Lr41, Lr42, and Lr43 transferred from Triticum tauschii to common wheat. Crop Sci 34:339–343

  10. Eitas TK, Nimchuk ZL, Dangl JL (2008) Arabidopsis TAO1 is a TIR-NB-LRR protein that contributes to disease resistance induced by the Pseudomonas syringae effector AvrB. PNAS 105:6475–6480

  11. Feldman M, Levy AA (2012) Genome evolution due to allopolyploidization. Genetics 192:763–774

  12. Feuillet C, Langridge P, Waugh R (2008) Cereal breeding takes a walk on the wild side. Trends Genet 24:24–32

  13. Forrest K, Pujol V, Bulli P, Pumphrey M, Wellings C, Herrera-Foessel S, Huerta-Espino J, Singh R, Lagudah E, Hayden M, Spielmeyer W (2014) Development of a SNP marker assay for the Lr67 gene of wheat using a genotyping by sequencing approach. Mol Breed 34:2109–2118

  14. Giorgi D, Farina A, Grosso V, Gennaro A, Ceoloni C, Lucretti S (2013) FISHIS: fluorescence In situ hybridization in suspension and chromosome flow sorting made easy. PLoS ONE 8:e57994. https://doi.org/10.1371/journal.pone.0057994

  15. Hall TA (1999) Bioedit: a user-friendly biological sequence alignment editor and analysis program for Windows 97/98/NT. Nucl Acid Symp 41:95–98

  16. Hussain W, Baenziger PS, Belamkar V, Guttieri MJ, Venegas JP, Easterly A, Sallam A, Poland J (2017) Genotyping-by-Sequencing derived high-density linkage map and its application to QTL mapping of flag leaf traits in bread wheat. Sci Rep 7:16394. https://doi.org/10.1038/s41598-017-16006-z

  17. Hussien T, Bowden RL, Gill BS, Cox TS (1997) Chromosome location of leaf rust resistance gene Lr43 from Aegilops tauschii in common wheat. Crop Sci 37:1764–1766

  18. International Wheat Genome Sequencing Consortium (2014) A chromosome-based draft sequence of the hexaploid bread wheat (Triticum aestivum) genome. Science 345:1251788-1–125178811

  19. International Wheat Genome Sequencing Consortium (2018) Shifting the limits in wheat research and breeding using a fully annotated reference genome. Science. https://doi.org/10.1126/science.aar7191

  20. Joshi NA, Fass JN (2011) Sickle: a sliding-window, adaptive, quality-based trimming tool for FastQ files (Version 1.33) [Software]. https://github.com/najoshi/sickle

  21. Jupe F, Pritchard L, Etherington GJ, MacKenzie K, Cock PJA, Wright F, Sharma SK, Bolser D, Bryan GJ, Jones JDG, Hein I (2012) Identification and localisation of the NB-LRR gene family within the potato genome. BMC Genom 13:75

  22. Kerber ER, Dyck PL (1969) Inheritance in hexaploid wheat of leaf rust resistance and other characters derived from Aegilops squarrosa. Can J Genet Cytol 11:639–647

  23. Klymiuk V, Yaniv E, Huang L, Raats D, Fatiukha A, Chen S, Feng L, Frenkel Z, Krugman T, Lidzbarsky G, Chang W, Jääskeläinen MJ, Schudoma C, Paulin L, Laine P, Bariana H, Sela H, Saleem K, Sørensen CK, Hovmøller MS, Distelfeld A, Chalhoub B, Dubcovsky J, Korol AB, Schulman AH, Fahima T (2018) Cloning of the wheat Yr15 resistance gene sheds light on the plant tandem kinase-pseudokinase family. Nat Commun 9:3735

  24. Kubaláková M, Macas J, Doležel J (1997) Mapping of repeated DNA sequences in plant chromosomes by PRINS and C-PRINS. Theor Appl Genet 94:758–763

  25. Kumari N, Gina BG, Li H (2013) Development and validation of a breeder-friendly KASPar marker for wheat leaf rust resistance locus Lr21. Mol Breed 31:233–237

  26. Kuraparthy V, Chhuneja P, Dhaliwal HS, Kaur S, Bowden RL, Gill BS (2007a) Characterization and mapping of cryptic alien introgression from Aegilops geniculata with novel leaf rust and stripe rust resistance genes Lr57 and Yr40 in wheat. Theor Appl Genet 114:1379–1389

  27. Kuraparthy V, Sood S, Chhuneja P, Dhaliwal HS, Kaur S, Bowden RL, Gill BS (2007b) A cryptic wheat-Aegilops triuncialis translocation with leaf rust resistance gene Lr58. Crop Sci 47:1995–2003

  28. Lee HA, Yeom SI (2015) Plant NB-LRR proteins: tightly regulated sensors in a complex manner. Briefings Funct Genom 14:233–242

  29. Li H (2011) A statistical framework for SNP calling, mutation discovery, association mapping and population genetical parameter estimation from sequencing data. Bioinformatics 27:2987–2993. https://doi.org/10.1093/bioinformatics/btr509

  30. Li H (2013) Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM [arXiv preprint]. arXiv:1303.3997

  31. Li H, Durbin R (2009) Fast and accurate short read alignment with Burrows–Wheeler Transform. Bioinformatics 25:1754–1760

  32. Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G, Durbin R, 1000 Genome Project Data Processing Subgroup (2009) The sequence alignment/map (SAM) format and SAMtools. Bioinformatics 25:2078–2079. https://doi.org/10.1093/bioinformatics/btp352

  33. Lu G, Moriyama EN (2004) Vector NTI, a balanced all-in-one sequence analysis suite. Brief Bioinform 5:378–388. https://doi.org/10.1093/bib/5.4.378

  34. Marchal C, Zhang J, Zhang P, Fenwick P, Steuernagel B, Adamski N, Boyd L, Mcintosh R, Wulff B, Berry S, Lagudah E, Uauy C (2018) BED-domain containing immune receptors confer diverse resistance spectra to yellow rust. Nat Plants 4:662–668

  35. Mascher M, Muehlbauer GJ, Rokhsar DS, Chapman J, Schmutz J, Barry K, Muñoz-Amatriaín M, Close TJ, Wise RP, Schulman AH, Himmelbach VA, Mayer KFX, Scholz U, Poland JA, Stein N, Waugh R (2013) Anchoring and ordering NGS contig assemblies by population sequencing (POPSEQ). Plant J 76:718–727

  36. McIntosh RA (1983) Genetic and cytogenetic studies involving Lr18 resistance to Puccinia recondita. In: Sakamoto M (ed) Proceedings 6th International Wheat Symposium, pp 777–783, Kyoto, Japan

  37. McIntosh RA, Yamazaki Y, Dubcovsky J, Rogers W J, Morris C, Appels R, Xia XC (2013). Catalogue of gene symbols for Wheat: 2013 supplement. In: KOMUGI–Integrated Wheat Science http://www.shigen.nig.ac.jp/wheat/komugi/genes/macgene/2013/GeneCatalogueIntroduction.pdf

  38. Michelmore RW, Meyers BC (1998) Clusters of resistance genes in plants evolve by divergent selection and a birth-and-death process. Genome Res 8:1113–1130

  39. Molnár I, Kubaláková M, Šimková H, Cseh A, Molnár-Láng M, Doležel J (2011) Chromosome isolation by flow sorting in Aegilops umbellulata and Ae. comosa and their allotetraploid hybrids Ae. biuncialis and Ae. geniculata. Plos One 6:e27708

  40. Mondal S, Rutkoski JE, Velu G, Singh PK, Crespo-Herrera LA, Guzmán C, Bhavani S, Lan C, He X, Singh RP (2016) Harnessing diversity in wheat to enhance grain yield, climate resilience, disease and insect pest resistance and nutrition through conventional and modern breeding approaches. Frontiers in Plant Science 7:991

  41. Narang D, Kaur S, Steuernagel B, Ghosh S, Dhillon R, Bansal M, Uauy C, Wulff BBH, Chhuneja P (2019) Fine mapping of Aegilops peregrina co-segregating leaf and stripe rust resistance genes to distal most end of 5DS. Theoret Appl Genet. https://doi.org/10.1007/s00122-019-03293-5

  42. Peterson RF, Campbell AB, Hannah AE (1948) A diagnostic scale for estimating rust severity on leaves and stem of cereals. Can J Res Sect C Bot Sci 26:490–500

  43. Pirseyedi SM, Somo M, Poudel RS, Cai X, McCallum B, Saville B, Fetch T, Chao S, Marais F (2015) Characterization of recombinants of the Aegilops peregrina-derived Lr59 translocation of common wheat. Theor Appl Genet 128:2403–2414

  44. Poland JA, Brown PJ, Sorrells ME, Jannink JE (2012) Development of high-density genetic maps for barley and wheat using a novel two-enzyme genotyping-by- sequencing approach. PLoS ONE 7:e32253

  45. Qiagen (2016) White paper on de novo assembly in CLC Assembly Cell4.0 [White paper]. retrieved from: https://www.qiagenbioinformatics.com/files/whitepapers/whitepaper-denovo-assembly.pdf

  46. Ramirez-Gonzalez RH, Uauy C, Caccamo M (2015a) PolyMarker: a fast polyploid primer design pipeline. Bioinformatics 31:2038–2039

  47. Ramirez-Gonzalez RH, Segovia V, Bird N, Fenwick P, Holdgate S, Berry S, Jack P, Caccamo M, Uauy C (2015b) RNA-Seq bulked segregant analysis enables the identification of high-resolution genetic markers for breeding in hexaploid wheat. Plant Biotechnol J 13:613–624

  48. Raupp WJ, Singh S, Brown-Guedira GL, Gill BS (2001) Cytogenetic and molecular mapping of the leaf rust resistance gene Lr39 in wheat. Theor Appl Genet 102:347–352

  49. Riley R, Chapman V (1958) Genetic control of the cytologically diploid behaviour of hexaploid wheat. Nature 182:713–715

  50. Rowland GG, Kerber ER (1974) Telocentric mapping hexaploid wheat of genes for leaf rust resistance and other characters derived from Aegilops squarrosa. Can J Genet Cytol 16:137–144

  51. Šafář J, Šimková H, Kubaláková M, Číhalíková J, Suchánková P, Bartoš J, Doležel J (2010) Development of chromosome-specific BAC resources for genomics of bread wheat. Cytogenet Genome Res 129:211–223

  52. Sánchez-Martín J, Steuernagel B, Ghosh S, Herren G, Hurni S, Adamski N, Vrána J, Kubaláková M, Krattinger SG, Wicker T, Doležel J, Keller B, Wulff BBH (2016) Rapid gene isolation in barley and wheat by mutant chromosome sequencing. Genome Biol 17:221–227

  53. Sears ER (1956) The transfer of leaf rust resistance from Aegilops umbellulata to wheat. Brookhaven Symp in Biol. No. 9, Genetics in Plant Breeding, pp 1–22

  54. Sears ER, Okamoto M (1958) Intergenomic chromosome relationship in hexaploid wheat. In: Paper presented at the proceedings of the 10th international. Congress of Genetics, Montreal, pp 258–259

  55. Šimková H, Svensson JT, Condamine P, Hřibová E, Suchánková P, Bhat PR, Bartoš J, Šafář J, Close TJ, Doležel J (2008) Coupling amplified DNA from flow-sorted chromosomes to high-density SNP mapping in barley. BMC Genom 9:294

  56. Singh H, Dhaliwal HS (2000) Intraspecific genetic diversity for resistance to wheat rusts in wild Triticum and Aegilops species. Wheat Inf Serv 90:21–30

  57. Steuernagel B, Jupe F, Witek K, Jones JDG, Wulff BBH (2015) NLR-parser: rapid annotation of plant NLR complements. Bioinformatics 31:1665–1667

  58. Thind AK, Wicker T, Šimková H, Fossati D, Moullet O, Brabant C, Vrána J, Doležel J, Krattinger SG (2017) Rapid cloning of genes in hexaploid wheat using cultivar-specific long-range chromosome assembly. Nat Biotechnol 35:793–796

  59. Tiwari VK, Wang S, Sehgal S, Vrána J, Friebe B, Kubaláková M, Chhuneja P, Doležel J, Akhunov E, Kalia B, Sabir J, Gill BS (2014) SNP Discovery for mapping alien introgressions in wheat. BMC Genom 15:273–284

  60. Toor PI, Kaur S, Bansal M, Yadav B, Chhuneja P (2016) Genetic mapping of an adult plant stripe rust resistance gene transferred from Aegilops caudata into bread wheat. J Genet 95:933–938

  61. Uauy C, Brevis JC, Chen X, Khan I, Jackson L, Chicaiza O, Distelfeld A, Fahima T, Dubcovsky J (2005) High-temperature adult-plant (HTAP) stripe rust resistance gene Yr36 from Triticum turgidum ssp. dicoccoides is closely linked to the grain protein content locus Gpc-B1. Theor Appl Genet 112:97–105

  62. Valkoun J, Hammer K, Kučerová D, Bartoš P (1985) Disease resistance in the genus Aegilops L.—stem rust, leaf rust, stripe rust and powdery mildew. Kulturpflanze 33:133–153

  63. Vrána J, Kubaláková M, Šimková H, Číhalíková J, Lysák MA, Doležel J (2000) Flow-sorting of mitotic chromosomes in common wheat (Triticum aestivum L.). Genetics 156:2033–2041

  64. Wang S, Wong D, Forrest K, Allen A, Chao S, Huang BE et al (2014) Characterization of polyploid wheat genomic diversity using a high-density 90000 single nucleotide polymorphism array. Plant Biotechnol J 12:787–796

  65. Winfield M, Allen AM, Burridge AJ, Barker GLA, Benbow HR, Wilkinson PA et al (2016) High density SNP genotyping array for hexaploid wheat and its secondary and tertiary gene pool. Plant Biotechnol J 14:1195–1206. https://doi.org/10.1111/pbi.12485

  66. Wulff BBH, Moscou MJ (2014) Strategies for transferring resistance into wheat: from wide crosses to GM cassettes. Front Plant Sci 5:692

  67. Picard Tools. Broad Institute. http://broadinstitute.github.io/picard/

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We thank Marie Kubaláková, Romana Šperková and Jitka Weiserová for technical assistance with chromosome flow sorting and chromosome DNA amplification. This work was carried out under the Sustainable Crop Production Research for International Development (SCPRID) grant. The financial support provided by the Department of Biotechnology, Ministry of Science and Technology, Government of India (Grant No. BT/IN/UK/08/PC/2012), and the UK Biotechnology and Biological Sciences Research Council (Grants No. BB/JO12017/1 and BB/P016855/1) is gratefully acknowledged. MB, PC and CU gratefully acknowledge the support provided by Monsanto Beachell Borlaug International Scholars Programme (MBBISP). IM, KH, JV, JD and MV were supported by the ERDF project ‘Plants as a tool for sustainable global development’ (No. CZ.02.1.01/0.0/0.0/16_019/0000827). IM also gratefully acknowledges the support from a Marie Curie Fellowship Grant ‘AEGILWHEAT’ (H2020-MSCA-IF-2016-746253) under the H2020 framework programme of the European Union and from the Hungarian National Research, Development and Innovation Office (K116277). MB also gratefully acknowledges Dr Kuldeep Singh, Director, NBPGR, New Delhi, for his guidance in writing the project proposal for MBBISP.

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MB carried out the phenotyping of the germplasm, analysed both genotype and phenotype data and wrote the draft of the manuscript; NA helped in the assembly of flow-sorted chromosome sequence and SNP detection; PIK helped in phenotyping, DNA extraction and harvesting of high-resolution mapping population; SK maintained the germplasm and populations in the field and facilitated rust screening; JV and JD sorted the chromosomes, IM sequenced the 5U chromosome; MV and KH sequenced chromosomes 5D of WL711 and pau16057; CU helped in analysis of 5U chromosome data, marker generation and supervised the study; PC conceived the idea, designed and supervised the study, prepared the draft of the manuscript and submitted it. All the authors have read the manuscript and approved it.

Correspondence to Parveen Chhuneja.

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Bansal, M., Adamski, N.M., Toor, P.I. et al. Aegilops umbellulata introgression carrying leaf rust and stripe rust resistance genes Lr76 and Yr70 located to 9.47-Mb region on 5DS telomeric end through a combination of chromosome sorting and sequencing. Theor Appl Genet 133, 903–915 (2020). https://doi.org/10.1007/s00122-019-03514-x

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