Theoretical and Applied Genetics

, Volume 132, Issue 1, pp 257–272 | Cite as

Development and molecular cytogenetic identification of a new wheat-rye 4R chromosome disomic addition line with resistances to powdery mildew, stripe rust and sharp eyespot

  • Diaoguo AnEmail author
  • Pengtao Ma
  • Qi Zheng
  • Shulan Fu
  • Lihui Li
  • Fangpu Han
  • Guohao Han
  • Jing Wang
  • Yunfeng Xu
  • Yuli Jin
  • Qiaoling Luo
  • Xiaotian Zhang
Original Article


Key message

A wheat-rye 4R chromosome disomic addition line with resistances to powdery mildew, stripe rust, sharp eyespot and high kernel number per spike was developed and characterized by molecular cytogenetic method as novel resistant germplasm.


Rye (Secale cereale L.), a close relative of common wheat, is an important and valuable gene donor with multiple disease resistance for wheat improvement. However, resistance genes derived from rye have successively lost resistance to pathogens due to the coevolution of pathogen virulence and host resistance. Development and identification of new effective resistance gene sources from rye therefore are of special importance and urgency. In the present study, a wheat-rye line WR35 was produced through distant hybridization, embryo rescue culture, chromosome doubling and backcrossing. WR35 was then proven to be a new wheat-rye 4R disomic addition line using sequential GISH (genomic in situ hybridization), mc-FISH (multicolor fluorescence in situ hybridization) and ND-FISH (non-denaturing FISH) with multiple probes, mc-GISH (multicolor GISH), rye chromosome arm-specific marker analysis and SLAF-seq (specific-locus amplified fragment sequencing) analysis. At the adult stage, WR35 exhibited high levels of resistance to the powdery mildew (Blumeria graminis f. sp. tritici, Bgt) and stripe rust (Puccinia striiformis f. sp. tritici, Pst) pathogens prevalent in China, and a highly virulent isolate of Rhizoctonia cerealis, the cause of wheat sharp eyespot. At the seedling stage, it was highly resistant to 22 of 23 Bgt isolates and four Pst races. Based on its disease responses to different pathogen isolates, WR35 may possess resistance gene(s) for powdery mildew, stripe rust and sharp eyespot, which differed from the known resistance genes from rye. In addition, WR35 was cytologically stable and produced high kernel number per spike. Therefore, WR35 with multi-disease resistances and desirable agronomic traits should serve as a promising bridging parent for wheat chromosome engineering breeding.



Blumeria graminis f. sp. tritici


Chinese Spring wheat




Expressed sequence tag-simple sequence repeat


Fluorescence in situ hybridization


Genomic in situ hybridization


Infection type


Multicolor FISH


Non-denaturing FISH


Specific-locus amplified fragment sequencing


Polymerase chain reaction


Puccinia striiformis f. sp. tritici



The authors thank Dr. Yilin Zhou and Dr. Shichang Xu from the State Key Laboratory for Biology of Plant Disease and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China, and Dr. Shibin Cai from Institute of Food Crops, Jiangsu Academy of Agricultural Science, Nanjing, China, for conducting assessment of the reactions to powdery mildew, stripe rust and sharp eyespot. This research was supported by the National Key Research and Development Program of China (Nos. 2016YFD0102002 and 2016YFD0100102) and the National Natural Science Foundation of China (No. 31771793).

Compliance with ethical standards

Conflict of interest

The authors declare that our experiments comply with the current laws of China and we have no conflicts of interest.

Ethical approval

This article does not contain any studies that were performed with human participants or animals by any of the authors.

Supplementary material

122_2018_3214_MOESM1_ESM.pdf (150 kb)
Supplementary material 1 (PDF 149 kb)
122_2018_3214_MOESM2_ESM.pdf (20 kb)
Supplementary material 2 (PDF 19 kb)


  1. Alkhimova AG, Heslop-Harrison JS, Shchapova AI, Vershinin AV (1999) Rye chromosome variability in wheat-rye addition and substitution lines. Chromosome Res 7:205–212CrossRefGoogle Scholar
  2. An DG, Zhong GC, Li JM, Wang ZG, Wang YM, Ji J (2003) Chromosome doubling methods of immature embryo plants of wheat distant hybridization. Acta Agron Sin 29:955–957Google Scholar
  3. An DG, Li LH, Li JM, Li HJ, Zhu YG (2006) Introgression of resistance to powdery mildew conferred by chromosome 2R by crossing wheat nullisomic 2D with rye. J Integr Plant Biol 48:838–847CrossRefGoogle Scholar
  4. An DG, Zheng Q, Zhou YL, Ma PT, Lv ZL, Li LH, Li B, Luo QL, Xu HX, Xu YF (2013) Molecular cytogenetic characterization of a new wheat-rye 4R chromosome translocation line resistant to powdery mildew. Chromosome Res 21:419–432CrossRefGoogle Scholar
  5. An DG, Zheng Q, Luo QL, Ma PT, Zhang HX, Li LH, Han FP, Xu HX, Xu YF, Zhang XT (2015) Molecular cytogenetic identification of a new wheat-rye 6R chromosome disomic addition line with powdery mildew resistance. PLoS ONE 10:e0134534CrossRefGoogle Scholar
  6. Bento M, Gustafson P, Viegas W, Silva M (2010) Genome merger: from sequence rearrangements in triticale to their elimination in wheat-rye addition lines. Theor Appl Genet 121:489–497CrossRefGoogle Scholar
  7. Borner A, Korzun V, Voylokov AV, Worland AJ, Weber WE (2000) Genetic mapping of quantitative trait loci in rye (Secale cereale L.). Euphytica 116:203–209CrossRefGoogle Scholar
  8. Chebotar S, Röder MS, Korzun V, Saal B, Weber WE, Börner A (2003) Molecular studies on genetic integrity of open-pollinating species rye (Secale cereale L.) after long-term genebank maintenance. Theor Appl Genet 107:1469–1476CrossRefGoogle Scholar
  9. Chen J, Li GH, Du ZY, Quan W, Zhang HY, Che MZ, Wang Z, Zhang ZJ (2013) Mapping of QTL conferring resistance to sharp eyespot (Rhizoctonia cerealis) in bread wheat at the adult plant growth stage. Theor Appl Genet 126:2865–2878CrossRefGoogle Scholar
  10. Cuadrado A, Schwarzacher T (1998) The chromosomal organization of simple sequence repeats in wheat and rye genomes. Chromosoma 107:587–594CrossRefGoogle Scholar
  11. Cuadrado A, Schwarzacher T, Jouve N (2000) Identification of different chromatin classes in wheat using in situ hybridization with simple sequence repeat oligonucleotides. Theor Appl Genet 101:711–717CrossRefGoogle Scholar
  12. Curtis CA, Lukaszewski AJ (1993) Localization of genes in rye that restore male fertility to hexaploid wheat with timopheevi cytoplasm. Plant Breeding 111:106–112CrossRefGoogle Scholar
  13. Dean R, Van Kan JAL, Pretorius ZA, Hammond-Kosack KE, Di Pietro A, Spanu PD, Rudd JJ, Dickman M, Kahmann R, Ellis J, Foster GD (2012) The top 10 fungal pathogens in molecular plant pathology. Mol Plant Pathol 13:414–430CrossRefGoogle Scholar
  14. Devos KM, Atkinson MD, Chinoy CN, Francis HA, Harcourt RL, Koebner RM, Liu CJ, Masojć P, Xie DX, Gale MD (1993) Chromosomal rearrangements in the rye genome relative to that of wheat. Theor Appl Genet 85:673–680CrossRefGoogle Scholar
  15. Endo TR (2007) The gametocidal chromosome as a tool for chromosome manipulation in wheat. Chromosome Res 15:67CrossRefGoogle Scholar
  16. Evanega SD, Singh RP, Coffman R, Pumphrey MO (2014) The Borlaug global rust initiative: Reducing the genetic vulnerability of wheat to rust. In: Tuberosa R, Graner A, Frison E (eds) Genomics of plant genetic resources. Springer, Dordrecht, pp 317–331CrossRefGoogle Scholar
  17. Falke KC, Sušić Z, Wilde P, Wortmann H, Möhring J, Piepho HP, Geiger HH, Miedaner T (2009) Testcross performance of rye introgression lines developed by marker-assisted backcrossing using an Iranian accession as donor. Theor Appl Genet 118:1225–1238CrossRefGoogle Scholar
  18. Friebe B (1996) Characterization of wheat-alien translocations conferring resistance to diseases and pests: current status. Euphytica 91:59–87CrossRefGoogle Scholar
  19. Friebe B, Hatchett JH, Gill BS, Mukai Y, Sebesta EE (1991) Transfer of Hessian fly resistance from rye to wheat via radiation-induced terminal and intercalary chromosomal translocations. Theor Appl Genet 83:33–40CrossRefGoogle Scholar
  20. Friebe B, Kynast RG, Gill BS (2000) Gametocidal factor-induced structural rearrangements in rye chromosomes added to common wheat. Chromosome Res 8:501–511CrossRefGoogle Scholar
  21. Fu SL, Lv ZL, Qi B, Guo X, Li J, Liu B, Han FP (2012) Molecular cytogenetic characterization of wheat-Thinopyrum elongatum addition, substitution and translocation lines with a novel source of resistance to wheat fusarium head blight. J Genet Genom 39:103–110CrossRefGoogle Scholar
  22. Fu SL, Yang MY, Fei YY, Tan FQ, Ren ZL, Yan BJ, Zhang HY, Tang ZX (2013) Alterations and abnormal mitosis of wheat chromosomes induced by wheat-rye monosomic addition lines. PLoS ONE 8:e70483CrossRefGoogle Scholar
  23. Fu SL, Ren ZL, Chen XM, Yan BJ, Tan FQ, Fu TH, Tang ZX (2014) New wheat-rye 5DS-4RS·4RL and 4RS-5DS·5DL translocation lines with powdery mildew resistance. J Plant Res 127:743–753CrossRefGoogle Scholar
  24. Fu SL, Chen L, Wang YY, Li M, Yang ZJ, Qiu L, Yan BJ, Ren ZL, Tang ZX (2015) Oligonucleotide probes for ND-FISH analysis to identify rye and wheat chromosomes. Sci Rep 5:10552CrossRefGoogle Scholar
  25. He ZH, Xia XC, Chen XM, Zhuang QS (2011) Progress of wheat breeding in China and the future perspective. Acta Agron Sin 37:202–215CrossRefGoogle Scholar
  26. He ZH, Xia XC, Chen XM, Zhang Y, Zhang Y, Yan J, Cao SH, Rasheed A (2015) Application of molecular markers in plant quality and disease resistance breeding. In: The seventh national symposium on wheat genetics and breeding, Zhengzhou, ChinaGoogle Scholar
  27. Heun M, Friebe B (1990) Introgression of powdery mildew resistance from rye into wheat. Phytopathology 80:242–245CrossRefGoogle Scholar
  28. Jiang J, Friebe B, Gill BS (1993) Recent advances in alien gene transfer in wheat. Euphytica 73:199–212CrossRefGoogle Scholar
  29. Jiang YJ, Zhu FF, Cai SB, Wu JZ, Zhang QF (2016) Quantitative trait loci for resistance to Sharp eyespot (Rhizoctonia cerealis) in recombinant inbred wheat lines from the cross Niavt 14 × Xuzhou 25. Czech J Genet Plant Breed 52:139–144CrossRefGoogle Scholar
  30. Koeszegi B, Farshadfar E, Vagujfalvi A, Sutka J (1996) Drought tolerance studies on wheat/rye disomic chromosome addition lines. Acta Agron Hung 44:121–126Google Scholar
  31. Korzun V, Malyshev S, Voylokov A, Börner A (1997) RFLP-based mapping of three mutant loci in rye (Secale cereale L.) and their relation to homoeologous loci within the Gramineae. Theor Appl Genet 95:468–473CrossRefGoogle Scholar
  32. Lee TG, Hong MJ, Johnson JW, Bland DE, Kim DY, Seo YW (2009) Development and functional assessment of EST-derived 2RL-specific markers for 2BS.2RL translocations. Theor Appl Genet 119:663–673CrossRefGoogle Scholar
  33. Lemańczyk G, Kwaśna H (2013) Effects of sharp eyespot (Rhizoctonia cerealis) on yield and grain quality of winter wheat. Eur J Plant Pathol 135:187–200CrossRefGoogle Scholar
  34. Li ZS, Li B, Tong YP (2008) The contribution of distant hybridization with decaploid Agropyron elongatum to wheat improvement in China. J Genet Genom 35:451–456CrossRefGoogle Scholar
  35. Lind V (1982) Analysis of the resistance of wheat-rye addition lines to powdery mildew of wheat (Erysiphe graminis f. sp. tritici). Tagungsbericht Akademie Der Landwirtschaftswissenschaften Der DdrGoogle Scholar
  36. Liu Y, Zhang QF, Fu BS, Cai SB, Jiang YJ, Zhang ZL, Deng YY, Wu JZ, Dai TB (2015) Genetic diversity of wheat germplasm resistant to sharp eyespot and genotyping of resistance loci using SSR markers. Acta Agron Sin 41:1671CrossRefGoogle Scholar
  37. Liu LQ, Luo QL, Teng W, Li B, Li HW, Li YW, Li ZS, Zheng Q (2018) Development of Thinopyrum ponticum specific molecular markers and FISH probes based on SLAF-seq technology. Planta 247(5):1099–1108CrossRefGoogle Scholar
  38. Lukaszewski AJ, Porter DR, Baker CA, Rybka K, Lapinski B (2001) Attempts to transfer Russian wheat aphid resistance from a rye chromosome in Russian triticales to wheat. Crop Sci 41:1743–1749CrossRefGoogle Scholar
  39. Luo C, Shu B, Yao QS, Wu HX, Xu WT, Wang SB (2016) Construction of a high-density genetic map based on large-scale marker development in mango using specific-locus amplified fragment sequencing (SLAF-seq). Front Plant Sci 7:1310PubMedPubMedCentralGoogle Scholar
  40. Ma PT, Xu HX, Xu YF, Li LH, Qie YM, Luo QL, Zhang XT, Li XQ, Zhou YL, An DG (2015) Molecular mapping of a new powdery mildew resistance gene Pm2b in Chinese breeding line KM2939. Theor Appl Genet 128:613–622CrossRefGoogle Scholar
  41. Masoudi-Nejad A, Nasuda S, McIntosh RA, Endo TR (2002) Transfer of rye chromosome segments to wheat by a gametocidal system. Chromosome Res 10:349–357CrossRefGoogle Scholar
  42. McDonald BA, Linde C (2002) The population genetics of plant pathogens and breeding strategies for durable resistance. Euphytica 124:163–180CrossRefGoogle Scholar
  43. Miftahudin, Scoles GJ, Gustafson JP (2002) AFLP markers tightly linked to the aluminum-tolerance gene Alt3 in rye (Secale cereale L.). Theor Appl Genet 104:626–631CrossRefGoogle Scholar
  44. Mukai Y, Nakahara Y, Yamamoto M (1993) Simultaneous discrimination of the three genomes in hexaploid wheat by multicolor fluorescence in situ hybridization using total genomic and highly repeated DNA probes. Genome 36:489–494CrossRefGoogle Scholar
  45. Nguyen V, Fleury D, Timmins A, Laga H, Hayden M, Mather D, Okada T (2015) Addition of rye chromosome 4R to wheat increases anther length and pollen grain number. Theor Appl Genet 128:953–964CrossRefGoogle Scholar
  46. Pedersen C, Langridge P (1997) Identification of the entire chromosome complement of bread wheat by two-colour FISH. Genome 40:589–593CrossRefGoogle Scholar
  47. Qiu L, Tang ZX, Li M, Fu SL (2016) Development of new PCR-based markers specific for chromosome arms of rye (Secale cereale L.). Genome 59:159–165CrossRefGoogle Scholar
  48. Rabinovich SV (1998) Importance of wheat-rye translocations for breeding modern cultivars of Triticum aestivum L. (Reprinted from Wheat: prospects for global improvement). Euphytica 100:323–340CrossRefGoogle Scholar
  49. Rahmatov M, Rouse MN, Nirmala J, Danilova T, Friebe B, Steffenson BJ, Johansson E (2016) A new 2DS center dot 2RL Robertsonian translocation transfers stem rust resistance gene Sr59 into wheat. Theor Appl Genet 129:1383–1392CrossRefGoogle Scholar
  50. Schlegel RHJ (2016) Current list of wheats with rye and alien introgression. V05–16, pp 1–18. http://www.rye-gene-mapde/rye-introgression
  51. Schneider A, Rakszegi M, Molnár-Láng M, Szakács É (2016) Production and cytomolecular identification of new wheat-perennial rye (Secale cereanum) disomic addition lines with yellow rust resistance (6R) and increased arabinoxylan and protein content (1R, 4R, 6R). Theor Appl Genet 129:1045–1059CrossRefGoogle Scholar
  52. Sheng BQ, Duan XY (1991) Improvement of scale 0–9 method for scoring adult plant resistance to powdery mildew of wheat. Beijing Agric Sci 1:38–39Google Scholar
  53. Shi JR, Wang YZ, Chen HG, Shen YW (2000) Screening techniques and evaluation of wheat resistance to sharp eyespot caused by Rhizoctonia cerealis. Acta Phytophylacica Sin 27(2):107–112Google Scholar
  54. Si QM, Zhang XX, Duan XY, Sheng BQ, Zhou YL (1992) On gene analysis and classification of powdery mildew (Erysiphe graminis f. sp. tritici) resistant wheat varieties. Acta Phytopathol Sin 22:349–355Google Scholar
  55. Sidhu MC, Satija CK, Sharma I (2001) Screening of wheat-rye addition lines for Karnal bunt resistance. Crop Improv 28:214–217Google Scholar
  56. Sun XW, Liu DY, Zhang XF, Li WB, Liu H, Hong WG, Jiang CB, Guan N, Ma CX, Zeng HP (2013) SLAF-seq: an efficient method of large-scale de novo snp discovery and genotyping using high throughput sequencing. PLoS ONE 8:e58700CrossRefGoogle Scholar
  57. Szakács E, Molnár-Láng M (2010) Identification of new winter wheat—winter barley addition lines (6HS and 7H) using fluorescence in situ hybridization and the stability of the whole ‘Martonvásári 9 kr1’ − ‘Igri’ addition set. Genome 53:35–44CrossRefGoogle Scholar
  58. Tang ZX, Yang ZJ, Fu SL (2014) Oligonucleotides replacing the roles of repetitive sequences pAs1, pSc119.2, pTa-535, pTa71, CCS1, and pAWRC.1 for FISH analysis. J Appl Genet 55:313–318CrossRefGoogle Scholar
  59. Targońska M, Bolibok-Brągoszewska H, Rakoczy-Trojanowska M (2016) Assessment of genetic diversity in Secale cereale based on SSR markers. Plant Mol Biol Rep 34:37–51CrossRefGoogle Scholar
  60. Volin RB, Sharp EL (1973) Physiologic specialization and pathogen aggressiveness in stripe rust. Phytopathology 63:699–703CrossRefGoogle Scholar
  61. Wang CM, Zheng Q, Li LH, Niu YC, Wang HB, Li B, Zhang XT, Xu YF, An DG (2009) Molecular cytogenetic characterization of a new T2BL·1RS wheat-rye chromosome translocation line resistant to stripe rust and powdery mildew. Plant Dis 93:124–129CrossRefGoogle Scholar
  62. Wang D, Zhuang LF, Sun L, Feng YG, Pei ZY, Qi ZJ (2010) Allocation of a powdery mildew resistance locus to the chromosome arm 6RL of Secale cereale L. cv. ‘Jingzhouheimai’. Euphytica 176:157–166CrossRefGoogle Scholar
  63. Xu HX, Yin DD, Li LH, Wang QX, Li XQ, Yang XM, Liu WH, An DG (2012) Development and application of EST-based markers specific for chromosome arms of rye (Secale cereale L.). Cytogenet Genome Res 136:220–228CrossRefGoogle Scholar
  64. Xu HX, Zhang J, Zhang P, Qie YM, Niu YC, Li HJ, Ma PT, Xu YF, An DG (2014) Development and validation of molecular markers closely linked to the wheat stripe rust resistance gene YrC591 for marker-assisted selection. Euphytica 198:317–323CrossRefGoogle Scholar
  65. Zhang J, Zhang JP, Liu WH, Han HM, Lu YQ, Yang XM, Li XQ, Li LH (2015) Introgression of Agropyron cristatum 6P chromosome segment into common wheat for enhanced thousand-grain weight and spike length. Theor Appl Genet 128:1827–1837CrossRefGoogle Scholar
  66. Zhao RH, Wang HY, Xiao J, Bie TD, Cheng SH, Jia Q, Yuan CX, Zhang RQ, Cao AZ, Chen PD, Wang XE (2013) Induction of 4VS chromosome recombinants using the CS ph1b mutant and mapping of the wheat yellow mosaic virus resistance gene from Haynaldia villosa. Theor Appl Genet 126:2921–2930CrossRefGoogle Scholar
  67. Zheng Q, Lv ZL, Niu ZX, Li B, Li HW, Xu SS, Han FP (2014) Molecular cytogenetic characterization and stem rust resistance of five wheat-T. thinopyrum ponticum partial amphiploids. J Genet Genom 41:591–599CrossRefGoogle Scholar
  68. Zhong GC, Mu SM, Zhang ZB (2002) Wheat distant hybridization. Chinese Science Press, BeijingGoogle Scholar
  69. Zhou YL, Duan XY, Gang C, Sheng BQ, Ying Z (2002) Analyses of resistance genes of 40 wheat cultivars or lines to wheat powdery mildew. Acta Phytopathol Sin 32:301–305Google Scholar
  70. Zhuang QS (2003) Chinese wheat improvement and pedigree analysis. Chinese Agriculture Press, BeijingGoogle Scholar
  71. Zhuang LF, Sun L, Li AX, Chen TT, Qi ZJ (2011) Identification and development of diagnostic markers for a powdery mildew resistance gene on chromosome 2R of Chinese rye cultivar Jingzhouheimai. Mol Breed 27:455–465CrossRefGoogle Scholar
  72. Zhuang LF, Liu P, Liu ZQ, Chen TT, Wu N, Sun L, Qi ZJ (2015) Multiple structural aberrations and physical mapping of rye chromosome 2R introgressed into wheat. Mol Breed 35:133CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Center for Agricultural Resources Research, Institute of Genetics and Developmental BiologyChinese Academy of SciencesShijiazhuangChina
  2. 2.The State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental BiologyChinese Academy of SciencesBeijingChina
  3. 3.The National Key Facility for Crop Gene Resources and Genetic Improvement, Institute of Crop ScienceChinese Academy of Agricultural SciencesBeijingChina
  4. 4.Province Key Laboratory of Plant Breeding and GeneticsSichuan Agriculture UniversityChengduChina

Personalised recommendations