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