Molecular Breeding

, 39:146 | Cite as

Cytological and molecular characterization of Thinopyrum bessarabicum chromosomes and structural rearrangements introgressed in wheat

  • Jianyong Chen
  • Yuqing Tang
  • Lesha Yao
  • Hao Wu
  • Xinyu Tu
  • Lifang Zhuang
  • Zengjun QiEmail author


Thinopyrum bessarabicum is an important genetic resource for wheat improvement by chromosome engineering. However, the present low-resolution karyotype limits identification of its chromosomes. Oligonucleotide probes to identify tandem repeats provide an efficient way to produce high-resolution karyotypes in many species. In this study, putative tandem repeats were identified using unassembled sequence reads of Th. bessarabicum, and 306 repeat clusters were identified. Among them, 17 had conserved motifs that varied in size from 71 to 856 bp and occupied 0.01% to 1.30% of the genome. Thirty-nine oligonucleotides from 17 clusters were developed, and 21 from 8 clusters produced clear and stable signals in Th. bessarabicum chromosomes. Five tandem repeat clusters were distributed only at the telomeric or subtelomeric regions, and the BSCL242 probe produced signals only on chromosome 7JL. The other three were mainly in intercalary and centromeric regions with a few weak signals in telomeric regions. A new multiplex oligonucleotide probe (ONPM#7) containing 13 oligonucloetides distinguished all wheat and Th. bessarabicum chromosomes after one round of FISH. The high-resolution karyotype of Th. bessarabicum in corresponding with the seven homoeologous group chromosomes of wheat has been developed. Three spontaneous translocations and one isochromosome among Th. bessarabicum chromosomes introgressed into wheat thus have been characterized in combined with molecular marker analysis. The ONPM#7 probe and molecular markers provide powerful tools for engineering transfer of chromosomal segments from Th. bessarabicum to wheat.


Fluorescence in situ hybridization Structural chromosome variation Chromosome engineering 



We thank R. McIntosh, University of Sydney, Australia, and Masahiro Kishii, CIMMYT, Mexico, for the English editing of the manuscript.

Author contributions

Project design: ZQ and JC; experimental work: JC, YT, LY, HW, and XT; data analysis: JC, YT, LY, and ZQ; manuscript preparation: JC, YT, LY, LZ, and ZQ. All authors reviewed and approved the manuscript.

Funding information

This project was funded by the Fundamental Research Funds for the Central Universities (Y0201700147). Bioinformatics analyses were supported by the Bioinformatics Center of Nanjing Agricultural University, China.

Compliance with ethical standards

Conflicts of interest

The authors declare that they have no conflicts of interest.

Supplementary material

11032_2019_1054_Fig6_ESM.png (6.9 mb)
Fig. S1

Physical mapping of tandem repeats BSCL5 (a-d), BSCL75 (e-h), BSCL156 (i-l), and BSCL158 (m-p) in Th. bessarabicum. Blue, chromosomes counterstained with DAPI; green, probes modified with FAM; red, probes modified with TAMRA. (PNG 7115 kb)

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High resolution image (TIF 57876 kb)
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Fig. S2 Idiograms of the genomic organization of six tandem repeats in Th. bessarabicum chromosomes. (PNG 11 kb)
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Fig. S3

Chromosomes of CS-Th. bessarabicum amphiploid after ONPM#7 FISH (a, c) and sequential GISH (b, d). (PNG 6650 kb)

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High resolution image (TIF 26657 kb)
11032_2019_1054_Fig8_ESM.png (1.3 mb)
Fig. S4

Chromosome painting with ONPM#7 and sequential GISH in CS-Th. bessarabicum disomic substitution line DS1J(1B). Arrows indicate chromosome 1J. Images order: chromosomes counterstained with DAPI (blue), chromosome painting with FAM modfied probes (green), chromosome painting with TAMRA modified probes (red), merged figures of blue, green and red, and sequential GISH (green) with Fluorescein-12-dUTP labeled genomic DNA of Th. bessarabicum as probe. (PNG 1356 kb)

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High resolution image (TIF 19305 kb)
11032_2019_1054_Fig9_ESM.png (1.5 mb)
Fig. S5

Chromosome painting in CS-Th. bessarabicum monosomic addition line MA2J with ONPM#7. Arrow indicates chromosome 2J. Images order is the same as Fig. S4. (PNG 1540 kb)

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High resolution image (TIF 20920 kb)
11032_2019_1054_Fig10_ESM.png (1.1 mb)
Fig. S6

Chromosome painting with ONPM#7 and sequential GISH in CS-Th. bessarabicum monosomic addition line MA3J. Arrow indicates chromosome 3J. Images order is the same as Fig. S4. (PNG 1145 kb)

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High resolution image (TIF 15707 kb)
11032_2019_1054_Fig11_ESM.png (1.3 mb)
Fig. S7

Chromosome painting with ONPM#7 and sequential GISH in CS-Th. bessarabicum disomic substitution line DS5J(5A). Arrows indicate chromosomes 5J. Images order is the same as Fig. S4. (PNG 1321 kb)

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High resolution image (TIF 19000 kb)
11032_2019_1054_Fig12_ESM.png (1.4 mb)
Fig. S8

Chromosome painting with ONPM#7 and sequential GISH in CS-Th. bessarabicum disomic substitution line DS6J(6A). Arrows indicate chromosomes 6J. Images order is the same as Fig. S4. (PNG 1470 kb)

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High resolution image (TIF 20003 kb)
11032_2019_1054_Fig13_ESM.png (130 kb)
Fig. S9

Chromosome painting with ONPM#7 and sequential GISH in CS-Th. bessarabicum line M5B/MS5J(5A)/MS7J(7D). Arrow indicates chromosome 7J. Images order is the same as Fig. S4. (PNG 130 kb)

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High resolution image (TIF 15380 kb)
11032_2019_1054_Fig14_ESM.png (3.7 mb)
Fig. S10

Chromosome painting in CS-Th. bessarabicum disomic addition line DA6JS·2JL with ONPM#7. Arrows indicate chromosomes 6JS·2JL. Images order is: chromosome painting (multi-color), and sequential GISH (green). (PNG 3834 kb)

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High resolution image (TIF 10073 kb)
11032_2019_1054_Fig15_ESM.png (3 mb)
Fig. S11

Chromosome painting in CS-Th. bessarabicum disomic addition line DA3JS·4JL with ONPM#7. Arrows indicate chromosomes 3JS·4JL. Images order is the same as Fig. S10. (PNG 3083 kb)

11032_2019_1054_MOESM11_ESM.tif (9.3 mb)
High resolution image (TIF 9483 kb)
11032_2019_1054_Fig16_ESM.png (3.6 mb)
Fig. S12

Chromosome painting in CS-Th. bessarabicum disomic addition line DA4JL·3JS with ONPM#7. Arrows indicate chromosomes 4JL·3JS. Images order is the same as Fig. S10. (PNG 3672 kb)

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High resolution image (TIF 9702 kb)
11032_2019_1054_MOESM13_ESM.doc (39 kb)
ESM 1 (DOC 39 kb)
11032_2019_1054_MOESM14_ESM.xls (39 kb)
ESM 2 (XLS 39 kb)


  1. Albert PS, Gao Z, Danilova TV, Birchler JA (2010) Diversity of chromosomal karyotypes in maize and its relatives. Cytogenet Genome Res 129:6–16PubMedCrossRefGoogle Scholar
  2. Benson G (1999) Tandem Repeats Finder: a program to analyze DNA sequences. Nuc Acids Res 27:573–580CrossRefGoogle Scholar
  3. Cuadrado Á, Jouve N (2010) Chromosomal detection of simple sequence repeats (SSRs) using nondenaturing FISH (ND-FISH). Chromosoma 119:495–503PubMedCrossRefGoogle Scholar
  4. Cuadrado Á, Vitellozzi F, Jouve N, Ceoloni C (1997) Fluorescence in situ hybridization with multiple repeated DNA probes applied to the analysis of wheat–rye chromosome pairing. Theor Appl Genet 94:347–355CrossRefGoogle Scholar
  5. Dawadundup (2009) Development and molecular characterization of wheat-Thinopyrum bessarabicum alien chromosome lines involving 4J and 5J. Master’s thesis, Nanjing Agricultural University, Nanjing. [In Chinese with English abstract.]Google Scholar
  6. Doležel J, Číhalíková J, Lucretti S (1992) A high-yield procedure for isolation of metaphase chromosomes from root tips of Vicia faba L. Planta 188:93–98PubMedCrossRefGoogle Scholar
  7. Du P, Zhuang LF, Wang YZ, Yuan L, Wang Q, Wang DR, Dawadondup, Tan LJ, Shen J, Xu HB, Zhao H, Chu CG, Qi ZJ (2017) Development of oligonucleotides and multiplex probes for quick and accurate identification of wheat and Thinopyrum bessarabicum chromosomes. Genome 60:93–103PubMedCrossRefGoogle Scholar
  8. Du P, Li L, Liu H, Fu L, Qin L, Zhang Z, Dai X, Huang B, Dong W, Tang F, Zhuang L, Han Y, Qi Z, Zhang X (2018) High-resolution chromosome painting with repetitive and single-copy oligonucleotides in Arachis species identifies structural rearrangements and genome differentiation. BMC Plant Biol 18:240PubMedPubMedCentralCrossRefGoogle Scholar
  9. Gaál E, Valárik M, Molnár I, Farkas A, Linc G (2018) Identification of COS markers specific for Thinopyrum elongatum chromosomes preliminary revealed high level of macrosyntenic relationship between the wheat and Th. elongatum genomes. PLoS One 13:e0208840PubMedPubMedCentralCrossRefGoogle Scholar
  10. Gill B, Friebe B, Endo T (1991) Standard karyotype and nomenclature system for description of chromosome bands and structural aberrations in wheat (Triticum aestivum). Genome 34:830–839CrossRefGoogle Scholar
  11. Grewal S, Yang C, Edwards SH, Scholefield D, Ashling S, Burridge AJ, Burridge AJ, King IP, King J (2018) Characterisation of Thinopyrum bessarabicum chromosomes through genome-wide introgressions into wheat. Theor Appl Genet 131:389–406PubMedCrossRefGoogle Scholar
  12. Han Y, Zhang T, Thammapichai P, Weng Y, Jiang J (2015) Chromosome-specific painting in Cucumis species using bulked oligonucleotides. Genetics 200:771–779PubMedPubMedCentralCrossRefGoogle Scholar
  13. Hassani HS, King IP, Reader SM, Caligari PDS, Miller TE (2010) Can Tritipyrum, a new salt tolerant potential amphiploid, be a successful cereal like Triticale? J Agr Sci Tech 2:177–195Google Scholar
  14. Heniko S, Ahmad K, Malik HS (2001) The centromere paradox: stable inheritance with rapidly evolving DNA. Science 293:1098–1102CrossRefGoogle Scholar
  15. Hsiao C, Wang RRC, Dewey DR (1986) Karyotype analysis and genome relationships of 22 diploid species in the tribe Triticeae. Can J Genet Cytol 28:109–120CrossRefGoogle Scholar
  16. Huang X, Zhu M, Zhuang L, Zhang S, Wang J, Chen X, Wang D, Chen J, Bao Y, Guo J, Zhang J, Feng Y, Chu C, Du P, Qi Z, Wang H, Chen P (2018) Structural chromosome rearrangements and polymorphisms identified in Chinese wheat cultivars by high-resolution multiplex oligonucleotide FISH. Theor Appl Genet 131:1967–1986PubMedCrossRefPubMedCentralGoogle Scholar
  17. Jiang J, Gill B (2006) Current status and the future of fluorescence in situ hybridization (FISH) in plant genome research. Genome 49:1057–1068PubMedCrossRefPubMedCentralGoogle Scholar
  18. Jiang J, Friebe B, Gill BS (1994) Recent advances in alien gene transfer in wheat. Euphytica 73:199–212CrossRefGoogle Scholar
  19. Jurka J, Kapitonov VV, Kohany O, Jurka MV (2007) Repetitive sequences in complex genomes: structure and evolution. Annu Rev Genomics Hum Genet 8:241–259PubMedCrossRefPubMedCentralGoogle Scholar
  20. Kato A (1999) Air drying method using nitrous oxide for chromosome counting in maize. Biotech Histochem 74:160–166PubMedCrossRefGoogle Scholar
  21. Kato A, Lamb J, Birchler J (2004) Chromosome painting using repetitive DNA sequences as probes for somatic chromosome identification in maize. Proc Natl Acad Sci USA 101:13554–13559PubMedCrossRefGoogle Scholar
  22. King IP, Orford SE, Cant KA, Reader SM, Miller TE (1996) An assessment of the salt tolerance of wheat/Thinopyrum bessarabicum 5Eb addition and substitution lines. Plant Breed 115:77–78CrossRefGoogle Scholar
  23. King IP, Forster BP, Law CC, Cant KA, Orford SE, Gorham J, Reader S, Miller TE (1997a) Introgression of salt-tolerance genes from Thinopyrum bessarabicum into wheat. New Phytol 137:75–81CrossRefGoogle Scholar
  24. King IP, Law CN, Cant KA, Orford SE, Reader SM, Miller TE (1997b) Tritipyrum, a potential new salt-tolerant cereal. Plant Breeding 116:127–132CrossRefGoogle Scholar
  25. Komuro S, Endo R, Shikata K, Kato A (2013) Genomic and chromosomal distribution patterns of various repeated DNA sequences in wheat revealed by a fluorescence in situ hybridization procedure. Genome 56:131–137PubMedCrossRefGoogle Scholar
  26. Lapitan N (1992) Organization and evolution of higher plant genomes. Genome 35:171–181CrossRefGoogle Scholar
  27. Li C (2014) Development and application of molecular markers specific for Thinopyrum bessarabicum Löve based on RNA-seq. Master’s thesis, Nanjing Agricultural University, Nanjing. [In Chinese with English abstract.]Google Scholar
  28. Li D, Li T, Wu Y, Zhang X, Zhu W, Wang Y, Zeng J, Xu L, Fan X, Sha L, Zhang H, Zhou Y, Kang H (2018) FISH-based markers enable identification of chromosomes derived from tetraploid Thinopyrum elongatum in hybrid lines. Front Plant Sci 9:526PubMedPubMedCentralCrossRefGoogle Scholar
  29. Liu Z (2015) Development and physical mapping of EST-SSR markers identified from transcriptome sequences of Thinopyrum bessarabicum. Master’s thesis, Nanjing Agricultural University, Nanjing. [In Chinese with English abstract.]Google Scholar
  30. Mehrotra S, Goyal V (2014) Repetitive sequences in plant nuclear DNA: types, distribution, evolution and function. Genom Proteom Bioinf 12:164–171CrossRefGoogle Scholar
  31. Mirzaghaderi G, Hassani H, Karimzadeh G (2010) C-banded karyotype of Thinopyrum bessarabicum and identification of its chromosomes in wheat background. Genet Resour Crop Evol 57:319–324CrossRefGoogle Scholar
  32. 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 36:489–494PubMedCrossRefGoogle Scholar
  33. Novák P, Neumann P, Pech J, Steinhaisl J, Macas J (2013) RepeatExplorer: a Galaxy-based web server for genome-wide characterization of eukaryotic repetitive elements from next-generation sequence reads. Bioinformatics 29:792–793PubMedCrossRefGoogle Scholar
  34. Novák P, Ávila Robledillo L, Koblížková A, Vrbová I, Neumann P, Macas J (2017) TAREAN: a computational tool for identification and characterization of satellite DNA from unassembled short reads. Nuc Acids Res 45(12):e111CrossRefGoogle Scholar
  35. Patokar C, Sepsi A, Schwarzacher T, Kishii M, Heslop-Harrison J (2016) Molecular cytogenetic characterization of novel wheat-Thinopyrum bessarabicum recombinant lines carrying intercalary translocations. Chromosoma 125:163–172PubMedCrossRefGoogle Scholar
  36. Pedersen C, Langridge P (1997) Identification of the entire chromosome complement of bread wheat by two-colour FISH. Genome 40:589–593PubMedCrossRefPubMedCentralGoogle Scholar
  37. Pu J, Wang Q, Shen Y, Zhuang L, Li C, Tan M, Bie T, Chu C, Qi Z (2015) Physical mapping of chromosome 4J of Thinopyrum bessarabicum using gamma radiation-induced aberrations. Theor Appl Genet 128:1319–1328PubMedCrossRefPubMedCentralGoogle Scholar
  38. Qi Z, Du P, Qian B, Zhuang L, Chen H, Chen T, Shen J, Guo J, Feng Y, Pei Z (2010) Characterization of a wheat-Thinopyrum bessarabicum (T2JS-2BS·2BL) translocation line. Theor Appl Genet 121:589–597PubMedCrossRefPubMedCentralGoogle Scholar
  39. Qian B (2007) Chromosome structure of Thinopyrum bessarabicum Löve introduced into common wheat revealed by molecular markers. Master’s thesis, Nanjing Agricultural University, Nanjing. [In Chinese with English abstract.]Google Scholar
  40. Ribeiro-Carvalho C, Guedes-Pinto H, Heslop-Harrison JS, Schwarzacher T (2001) Introgression of rye chromatin on chromosome 2D in the Portuguese wheat landrace ‘Barbela’. Genome 44:1122–1128PubMedCrossRefGoogle Scholar
  41. Rychlik W (2007) OLIGO 7 primer analysis software. Methods Mol Biol 402:35PubMedCrossRefGoogle Scholar
  42. Schneider A, Linc G, Molnár-Láng M, Graner A (2003) Fluorescence in situ hybridization polymorphism using two repetitive DNA clones in different cultivars of wheat. Plant Breed 122:396–400CrossRefGoogle Scholar
  43. Shen J (2011) Development and molecular cytogenetic characterization of wheat-Thinopyrum bessarabicum alien chromosome lines. Master’s thesis, Nanjing Agricultural University, Nanjing. [In Chinese with English abstract.]Google Scholar
  44. Shen Y, Shen J, Dawadondup, Zhuang L, Wang Y, Pu J, Feng Y, Chu C, Wang X, Qi Z (2013) Physical localization of a novel blue-grained gene derived from Thinopyrum bessarabicum. Mol Breeding 31:195–204CrossRefGoogle Scholar
  45. Tan (2011) Development and identification of structural variations involving chromosome 1J and 5J of Th. bessarabicum Löve. Master thesis, Nanjing Agricultural University, Nanjing, China. [In Chinese with English abstract.]Google Scholar
  46. Tang Z, Yang Z, Fu S (2014) Oligonucleotides replacing the roles of repetitive sequences pAs1, pSc119.2, pTa-535, pTa71, CCS1, and pAWRC.1 for FISH analysis. J Appl Genet 55:313–318PubMedCrossRefGoogle Scholar
  47. Wang RRC (1985) Genome analysis of Thinopyrum bessarabicum and Th. elongatum. Can J Genet Cytol 27:722–728CrossRefGoogle Scholar
  48. Wang Y (2013) Development and characterization of small segment translocations of Thinopyrum bessarabicum and cytological mapping of interest genes. Master’s thesis, Nanjing Agricultural University, Nanjing. [In Chinese with English abstract.]Google Scholar
  49. Wang H, Dai K, Xiao J, Yuan C, Zhao R, Doležel J, Wang X (2017) Development of intron targeting (IT) markers specific for chromosome arm 4VS of Haynaldia villosa by chromosome sorting and next-generation sequencing. BMC Genomics 18:167PubMedPubMedCentralCrossRefGoogle Scholar
  50. William M, Mujeeb-Kazi A (1993) Thinopyrum bessarabicum: biochemical and cytological markers for the detection of genetic introgression in its hybrid derivatives with Triticum aestivum L. Theor Appl Genet 86:365–370PubMedCrossRefGoogle Scholar
  51. William M, Mujeeb-Kazi A (1995) Biochemical and molecular diagnostics of Thinopyrum bessarabicum chromosomes in Triticum aestivum germplasm. Theor Appl Genet 90:952–956PubMedCrossRefGoogle Scholar
  52. Yang J, Liu Z, Chen J, Wang Y, Zhuang L, Qi Z (2018) Accurate characterization of Wheat-Thinopyrum bessarabicum alien chromosome translocations using oligonucleotide multiplex painting combined with genomic in situ hybridization and molecular marker analysis. J Triticeae Crops 38:253–261. [In Chinese with English abstract.]Google Scholar
  53. Zhang P, Li W, Fellers J, Friebe B, Gill BS (2004) BAC-FISH in wheat identifies chromosome landmarks consisting of different types of transposable elements. Chromosoma 112:288–299PubMedCrossRefGoogle Scholar
  54. Zhang X, Wei X, Xiao J, Yuan C, Wu Y, Cao A, Xing L, Chen P, Zhang S, Wang X, Wang H (2017) Whole genome development of intron targeting (IT) markers specific for Dasypyrum villosum chromosomes based on next-generation sequencing technology. Mol Breed 37:115CrossRefGoogle Scholar
  55. Zhang S, Zhu M, Shang Y, Wang J, Dawadundup, Zhuang L, Zhang J, Chu C, Qi Z (2019) Physical organization of repetitive sequences and chromosome diversity of barley revealed by fluorescence in situ hybridization (FISH). Genome. PubMedCrossRefGoogle Scholar
  56. Zhu M, Du P, Zhuang L, Chu C, Zhao QZ (2017) A simple and efficient non-denaturing FISH method for maize chromosome differentiation using single-strand oligonucleotide probes. Genome 60:657–664PubMedCrossRefGoogle Scholar
  57. Zhuang L, Qi Z, Ying J, Chen P, Liu D (2003) Development and identification of a set of Triticum aestivum-Thinopyrum bessarabicum disomic alien addition lines. Acta Genetica Sin 30:919–925. [In Chinese with English abstract.]Google Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  • Jianyong Chen
    • 1
  • Yuqing Tang
    • 1
  • Lesha Yao
    • 1
  • Hao Wu
    • 1
  • Xinyu Tu
    • 1
  • Lifang Zhuang
    • 1
  • Zengjun Qi
    • 1
    Email author
  1. 1.State Key Laboratory of Crop Genetics and Germplasm EnhancementNanjing Agricultural UniversityNanjingChina

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