, Volume 128, Issue 4, pp 521–532 | Cite as

“Doubled-haploid” allohexaploid Brassica lines lose fertility and viability and accumulate genetic variation due to genomic instability

  • Margaret W. Mwathi
  • Sarah V. Schiessl
  • Jacqueline Batley
  • Annaliese S. MasonEmail author
Original Article


Microspore culture stimulates immature pollen grains to develop into plants via tissue culture and is used routinely in many crop species to produce “doubled haploids”: homozygous, true-breeding lines. However, microspore culture is also often used on material that does not have stable meiosis, such as interspecific hybrids. In this case, the resulting progeny may lose their “doubled haploid” homozygous status as a result of chromosome missegregation and homoeologous exchanges. However, little is known about the frequency of these effects. We assessed fertility, meiosis and genetic variability in self-pollinated progeny sets (the MDL2 population) resulting from first-generation plants (the MDL1 population) derived from microspores of a near-allohexaploid interspecific hybrid from the cross (Brassica napus × B. carinata) × B. juncea. Allelic inheritance and copy number variation were predicted using single nucleotide polymorphism marker data from the Illumina Infinium 60K Brassica array. Seed fertility and viability decreased substantially from the MDL1 to the MDL2 generation. In the MDL2 population, 87% of individuals differed genetically from their MDL1 parent. These genetic differences resulted from novel homoeologous exchanges between chromosomes, chromosome loss and gain, and segregation and instability of pre-existing karyotype abnormalities. Novel karyotype change was extremely common, with 2.2 new variants observed per MDL2 individual. Significant differences between progeny sets in the number of novel genetic variants were also observed. Meiotic instability clearly has the potential to dramatically change karyotypes (often without detectable effects on the presence or absence of alleles) in putatively homozygous, microspore-derived lines, resulting in loss of fertility and viability.


Microspore culture Brassica Meiosis Interspecific hybrids Allopolyploidy Copy number variation 



MM’s PhD study was supported by the Research and Higher Degree and the Tuition Scholarships from the University of Queensland, Australia, and by an Australia-India Strategic Research Fund: Biotechnology grant. Dr. Ning Cheng assisted with SNP array preparations. 60K Infinium SNP data imaging analysis was done at the Translational Research Institute (TRI) in Brisbane, Australia. ASM is supported by DFG Emmy Noether grant MA6473/1-1.

Author contribution

MM conducted the experiments and collected the data, and co-wrote the manuscript and analysed the data with AM. SVS assisted with data analysis and interpretation and visualization of the data. MM, AM and JB contributed to manuscript revisions and provided input into experimental design. AM and JB supervised MM.

Compliance with ethical standards

The authors declare that this paper complies with all relevant ethical standards.

Conflict of interest

The authors declare that they have no conflicts of interest.

Supplementary material

412_2019_720_MOESM1_ESM.pptx (6.1 mb)
Supplementary Figure 1 Graphed Log R Ratios (from −1.5 to 1.5) output from Illumina Genome Studio (normalized) for the Illumina Infinium Brassica 60 K SNP array SNPs used to genotype and derive copy-number data for six allohexaploid lines (MDL07, MDL23, MDL28, MDL30, MDL60 and MDL64) derived from microspore culture of a single hybrid plant of the cross (Brassica napus × B. carinata) × B. juncea and their self-pollinated progeny. (PPTX 6283 kb)
412_2019_720_MOESM2_ESM.pdf (220 kb)
Supplementary Figure 2 Pollen viability in second-generation individuals derived from microspores of a Brassica napus × B. carinata× B. juncea hybrid. First generation parent pollen viability is indicated with a blue star for each line. Different lowercase letters indicate significant differences in pollen viability between progeny sets (Tukey’s HSD; p < 0.05). Supplementary Figure 3 Number of novel karyotype changes (whole or segmental chromosome duplication and deletion events) in second-generation individuals derived from microspores of a Brassica napus × B. carinata× B. juncea hybrid. Different lowercase letters indicate significant differences in the number of novel karyotype changes between progeny sets (Tukey’s HSD; p < 0.05). (PDF 220 kb)
412_2019_720_MOESM3_ESM.xlsx (23 mb)
ESM 1 Supplementary Table 1: Phased A- and C-genome allele calls from cleaned SNP data for microspore-derived allohexaploid lines (MDLs) from the cross (Brassica napus × B. carinata× B. juncea and their self-pollinated progeny (highlighted in blue). Supplementary Table 2: Log R Ratio output from Illumina Genome Studio (normalized) for the Illumina Infinium Brassica 60 K SNP array SNPs used to genotype and derive copy-number data for allohexaploid lines derived from self-pollination (SP) and microspore culture (MDL) of a single hybrid plant of the cross (Brassica napus × B. carinata× B. juncea. Self-pollinated progeny of each microspore derived line are highlighted in pale blue. Putative duplications (values >0.2 or > 0.5) are highlighted in blue, putative deletions of one chromosome copy (values between −0.2/−0.5 and − 2.0) are highlighted in orange, and putative deletions of both chromosome copies are highlighted in red (values < −2.0). Supplementary Table 3: Loss of both chromosome copies for a whole chromosome (del) or part of a chromosome (del – p), loss of one chromosome copy for a whole chromosome (miss) or part of a chromosome (miss – p), and duplication of a whole chromosome (dup) or part of a chromosome (dup – p) of A and C genome chromosomes in allohexaploid lines produced by microspore culture (MDL) and self-pollination (SP) of an allohexaploid hybrid from the cross combination (Brassica napus × B. carinata× B. juncea), as predicted from Log R Ratios output by Illumina Genome Studio from Illumina Infinium Brassica 60 K SNP array data. (XLSX 23561 kb)


  1. Abbott R, Albach D, Ansell S, Arntzen JW, Baird SJE, Bierne N, Boughman JW, Brelsford A, Buerkle CA, Buggs R, Butlin RK, Dieckmann U, Eroukhmanoff F, Grill A, Cahan SH, Hermansen JS, Hewitt G, Hudson AG, Jiggins C, Jones J, Keller B, Marczewski T, Mallet J, Martinez-Rodriguez P, Most M, Mullen S, Nichols R, Nolte AW, Parisod C, Pfennig K, Rice AM, Ritchie MG, Seifert B, Smadja CM, Stelkens R, Szymura JM, Vainola R, Wolf JBW, Zinner D (2013) Hybridization and speciation. J Evol Biol 26:229–246Google Scholar
  2. Bayer PE, Hurgobin B, Golicz AA, Chan CKK, Yuan YX, Lee H, Renton M, Meng JL, Li RY, Long Y, Zou J, Bancroft I, Chalhoub B, King GJ, Batley J, Edwards D (2017) Assembly and comparison of two closely related Brassica napus genomes. Plant Biotechnol J 15:1602–1610PubMedPubMedCentralGoogle Scholar
  3. Bretagnolle F, Thompson JD (1995) Tansley review no. 78. Gametes with the stomatic (sic) chromosome number: mechanisms of their formation and role in the evolution of autopolypoid plants. New Phytol 129:1–22Google Scholar
  4. Chen S, Nelson MN, Chèvre A-M, Jenczewski E, Li Z, Mason AS, Meng J, Plummer JA, Pradhan A, Siddique KHM, Snowdon RJ, Yan G, Zhou W, Cowling WA (2011) Trigenomic bridges for Brassica improvement. Crit Rev Plant Sci 30:524–547Google Scholar
  5. Chester M, Lipman MJ, Gallagher JP, Soltis PS, Soltis DE (2013) An assessment of karyotype restructuring in the neoallotetraploid Tragopogon miscellus (Asteraceae). Chromosom Res 21:75–85Google Scholar
  6. Clarke WE, Higgins EE, Plieske J, Wieseke R, Sidebottom C, Khedikar Y, Batley J, Edwards D, Meng J, Li R, Lawley CT, Pauquet J, Laga B, Cheung W, Iniguez-Luy F, Dyrszka E, Rae S, Stich B, Snowdon RJ, Sharpe AG, Ganal MW, Parkin IAP (2016) A high-density SNP genotyping array for Brassica napus and its ancestral diploid species based on optimised selection of single-locus markers in the allotetraploid genome. Theor Appl Genet 129:1887–1899PubMedPubMedCentralGoogle Scholar
  7. Comai L (2005) The advantages and disadvantages of being polyploid. Nat Rev Genet 6:836–846PubMedGoogle Scholar
  8. De Storme N, Geelen D (2013) Sexual polyploidization in plants--cytological mechanisms and molecular regulation. New Phytol 198:670–684PubMedPubMedCentralGoogle Scholar
  9. De Storme N, Mason AS (2014) Plant speciation through chromosome instability and ploidy change: cellular mechanisms, molecular factors and evolutionary relevance. Current Plant Biology 1:10–33Google Scholar
  10. Dolatabadian A, Patel DA, Edwards D, Batley J (2017) Copy number variation and disease resistance in plants. Theor Appl Genet 130:2479–2490PubMedGoogle Scholar
  11. Gaeta RT, Pires JC (2010) Homoelogous recombination in allopolyploids: the polyploid ratchet. New Phytol 186:18–28PubMedGoogle Scholar
  12. Geng XX, Chen S, Astarini IA, Yan GJ, Tian E, Meng J, Li ZY, Ge XH, Nelson MN, Mason AS, Pradhan A, Zhou WJ, Cowling WA (2013) Doubled haploids of novel trigenomic Brassica derived from various interspecific crosses. Plant Cell Tiss Org 113:501–511Google Scholar
  13. Gupta M, Atri C, Agarwal N, Banga SS (2016) Development and molecular-genetic characterization of a stable Brassica allohexaploid. Theor Appl Genet 129:2085–2100PubMedGoogle Scholar
  14. Howard HW (1942) The effect of polyploidy and hybridity on seed size in crosses between Brassica chinensis, B. carinata, amphidiploid B. chinensis-carinata and autotetraploid B. chinensis. J Genet 43:105–119Google Scholar
  15. Iwasa S (1964) Cytogenetic studies on the artificially raised trigenomic hexaploid hybrid forms in the genus Brassica. J Fac Agric Kyushu Univ 13:309–352Google Scholar
  16. Jahne A, Lorz H (1995) Cereal microspore culture. Plant Sci 109:1–12Google Scholar
  17. Lagercrantz U, Lydiate DJ (1996) Comparative genome mapping in Brassica. Genetics 144:1903–1910PubMedPubMedCentralGoogle Scholar
  18. Leitch AR, Leitch IJ (2008) Genomic plasticity and the diversity of polyploid plants. Science 320:481–483PubMedGoogle Scholar
  19. Li QF, Chen YG, Yue F, Qian W, Song HY (2018) Microspore culture reveals high fitness of B. napus-like gametes in an interspecific hybrid between Brassica napus and B. oleracea. PLoS One 13:e0193548PubMedPubMedCentralGoogle Scholar
  20. Mallet J (2007) Hybrid speciation. Nat Rev 446:279–283Google Scholar
  21. Mason AS, Batley J (2015) Creating new interspecific hybrid and polyploid crops. Trends Biotechnol 33:436–441PubMedGoogle Scholar
  22. Mason AS, Batley J, Bayer PE, Hayward A, Cowling WA, Nelson MN (2014a) High-resolution molecular karyotyping uncovers pairing between ancestrally related Brassica chromosomes. New Phytol 202:964–974PubMedGoogle Scholar
  23. Mason AS, Higgins EE, Snowdon RJ, Batley J, Stein A, Werner C, Parkin IAP (2017) A user guide to the Brassica 60K Illumina Infinium (TM) SNP genotyping array. Theor Appl Genet 130:621–633PubMedGoogle Scholar
  24. Mason AS, Huteau V, Eber F, Coriton O, Yan G, Nelson MN, Cowling WA, Chèvre A-M (2010) Genome structure affects the rate of autosyndesis and allosyndesis in AABC, BBAC and CCAB Brassica interspecific hybrids. Chromosom Res 18:655–666Google Scholar
  25. Mason AS, Nelson MN, Castello M-C, Yan G, Cowling WA (2011) Genotypic effects on the frequency of homoeologous and homologous recombination in Brassica napus × B. carinata hybrids. Theor Appl Genet 122:543–553PubMedGoogle Scholar
  26. Mason AS, Nelson MN, Takahira J, Cowling WA, Moreira Alves G, Chaudhuri A, Chen N, Ragu ME, Dalton-Morgan J, Coriton O, Huteau V, Eber F, Chèvre A-M, Batley J (2014b) The fate of chromosomes and alleles in an allohexaploid Brassica population. Genetics 197:273–283PubMedPubMedCentralGoogle Scholar
  27. Mason AS, Takahira J, Atri C, Samans B, Hayward A, Cowling WA, Batley J, Nelson MN (2015) Microspore culture reveals complex meiotic behaviour in a trigenomic Brassica hybrid. BMC Plant Biol 15:173PubMedPubMedCentralGoogle Scholar
  28. Mason AS, Yan GJ, Cowling WA, Nelson MN (2012) A new method for producing allohexaploid Brassica through unreduced gametes. Euphytica 186:277–287Google Scholar
  29. Mwathi MW, Gupta M, Atri C, Banga SS, Batley J, Mason AS (2017) Segregation for fertility and meiotic stability in novel Brassica allohexaploids. Theor Appl Genet 130:767–776PubMedGoogle Scholar
  30. Navabi ZK, Stead KE, Pires JC, Xiong Z, Sharpe AG, Parkin IAP, Rahman MH, Good AG (2011) Analysis of B-genome chromosome introgression in interspecific hybrids of Brassica napus x B. carinata. Genetics 187:659–673PubMedPubMedCentralGoogle Scholar
  31. Navabi ZK, Strelkov SE, Good AG, Thiagarajah MR, Rahman MH (2010) Brassica B-genome resistance to stem rot (Sclerotinia sclerotiorum) in a doubled haploid population of Brassica napus x Brassica carinata. Can J Plant Pathol 32:237–246Google Scholar
  32. Nelson MN, Mason AS, Castello MC, Thomson L, Yan GJ, Cowling WA (2009) Microspore culture preferentially selects unreduced (2n) gametes from an interspecific hybrid of Brassica napus L. x Brassica carinata Braun. Theor Appl Genet 119:497–505PubMedGoogle Scholar
  33. Nicolas SD, Le Mignon G, Eber F, Coriton O, Monod H, Clouet V, Huteau V, Lostanlen A, Delourme R, Chalhoub B, Ryder CD, Chèvre AM, Jenczewski E (2007) Homeologous recombination plays a major role in chromosome rearrangements that occur during meiosis of Brassica napus haploids. Genetics 175:487–503PubMedPubMedCentralGoogle Scholar
  34. Nicolas SD, Monod H, Eber F, Chèvre AM, Jenczewski E (2012) Non-random distribution of extensive chromosome rearrangements in Brassica napus depends on genome organization. Plant J 70:691–703PubMedGoogle Scholar
  35. Péle A, Rousseau-Gueutin M, Chèvre AM (2018) Speciation success of polyploid plants closely relates to the regulation of meiotic recombination. Front Plant Sci 9:907PubMedPubMedCentralGoogle Scholar
  36. R_Core_Team (2017) R: A language and environment for statistical computing., 2.10.1 edn. R Foundation for Statistical Computing, Vienna, AustriaGoogle Scholar
  37. Samans B, Chalhoub B, Snowdon RJ (2017) Surviving a genome collision: genomic signatures of allopolyploidization in the recent crop species Brassica napus. Plant Genome 10:3Google Scholar
  38. Schiessl S-V, Katche E, Lhien E, Singh Chawla H, Mason AS (2018) The role of genomic structural variation in the genetic improvement of polyploid crops. The Crop Journal 7:127–140Google Scholar
  39. Schubert I, Lysak MA (2011) Interpretation of karyotype evolution should consider chromosome structural constraints. Trends Genet 27:207–216PubMedGoogle Scholar
  40. Seymour DK, Filiault DL, Henry IM, Monson-Miller J, Ravi M, Pang AD, Comai L, Chan SWL, Maloof JN (2012) Rapid creation of Arabidopsis doubled haploid lines for quantitative trait locus mapping. Proc Natl Acad Sci U S A 109:4227–4232PubMedPubMedCentralGoogle Scholar
  41. Song KM, Lu P, Tang KL, Osborn TC (1995) Rapid genome change in synthetic polyploids of Brassica and its implications for polyploid evolution. Proc Natl Acad Sci U S A 92:7719–7723PubMedPubMedCentralGoogle Scholar
  42. Stein A, Coriton O, Rousseau-Gueutin M, Samans B, Schiessl SV, Obermeier C, Parkin IAP, Chevre AM, Snowdon RJ (2017) Mapping of homoeologous chromosome exchanges influencing quantitative trait variation in Brassica napus. Plant Biotechnol J 15:1478–1489PubMedPubMedCentralGoogle Scholar
  43. Szadkowski E, Eber F, Huteau V, Lodé M, Huneau C, Belcram H, Coriton O, Manzanares-Dauleux MJ, Delourme R, King GJ, Chalhoub B, Jenczewski E, Chèvre AM (2010) The first meiosis of resynthesized Brassica napus, a genome blender. New Phytol 186:102–112PubMedGoogle Scholar
  44. Takahira J, Cousin A, Nelson MN, Cowling WA (2011) Improvement in efficiency of microspore culture to produce doubled haploid canola (Brassica napus L.) by flow cytometry. Plant Cell Tiss Org 104:51–59Google Scholar
  45. Tian E, Jiang Y, Chen L, Zou J, Liu F, Meng J (2010) Synthesis of a Brassica trigenomic allohexaploid (B. carinata × B. rapa) de novo and its stability in subsequent generations. Theor Appl Genet 121:1431–1440PubMedGoogle Scholar
  46. U N (1935) Genome-analysis in Brassica with special reference to the experimental formation of B. napus and peculiar mode of fertilization. Japanese Journal of Botany 7:389–452Google Scholar
  47. Udall JA, Wendel JF (2006) Polyploidy and crop improvement. Crop Sci 46:S3–S14Google Scholar
  48. Xiong ZY, Gaeta RT, Pires JC (2011) Homoeologous shuffling and chromosome compensation maintain genome balance in resynthesized allopolyploid Brassica napus. Proc Natl Acad Sci U S A 108:7908–7913PubMedPubMedCentralGoogle Scholar
  49. Yang S, Chen S, Zhang KN, Li L, Yin YL, Gill RA, Yan GJ, Meng JL, Cowling WA, Zhou WJ (2018) A high-density genetic map of an allohexaploid Brassica doubled haploid population reveals quantitative trait loci for pollen viability and fertility. Front Plant Sci 9:1161PubMedPubMedCentralGoogle Scholar
  50. Zhou JN, Tan C, Cui C, Ge XH, Li ZY (2016) Distinct subgenome stabilities in synthesized Brassica allohexaploids. Theor Appl Genet 129:1257–1271PubMedGoogle Scholar
  51. Ziolkowski PA, Berchowitz LE, Lambing C, Yelina NE, Zhao X, Kelly KA, Choi K, Ziolkowska L, June V, Sanchez-Moran E, Franklin C, Copenhaver GP, Henderson IR (2015) Juxtaposition of heterozygous and homozygous regions causes reciprocal crossover remodelling via interference during Arabidopsis meiosis. eLife 4:1–29Google Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.School of Agriculture and Food SciencesUniversity of QueenslandBrisbaneAustralia
  2. 2.School of Biological SciencesThe University of Western AustraliaCrawleyAustralia
  3. 3.Department of Plant Breeding, Research Centre for Biosystems, Land Use and NutritionJustus Liebig UniversityGiessenGermany

Personalised recommendations