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Chromosoma

, 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

Abstract

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.

Keywords

Microspore culture Brassica Meiosis Interspecific hybrids Allopolyploidy Copy number variation 

Notes

Acknowledgements

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)

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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

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