title: Microspore culture reveals complex meiotic behaviour in a trigenomic Brassica hybrid.
abstract: BACKGROUND
  Development of synthetic allohexaploid Brassica (2n = AABBCC) would be beneficial for agriculture, as allelic contributions from three genomes could increase hybrid vigour and broaden adaptation. Microspore culture of a near-allohexaploid hybrid derived from the cross (B. napus × B. carinata) × B. juncea was undertaken in order to assess the frequency and distribution of homologous and homoeologous crossovers in this trigenomic hybrid. SNP and SSR molecular markers were used to detect inheritance of A, B and C genome alleles in microspore-derived (MD) progeny. SNP allele copy number was also assessed. The MD progeny were also compared to progeny derived by self-pollination and open-pollination for fertility (estimated by self-pollinated seed set and pollen viability) and DNA ploidy (measured by flow cytometry).
  
  
  RESULTS
  In the trigenomic hybrid, homologous chromosome pairs A(j)-A(n), B(j)-B(c) and C(n)-C(c) had similar meiotic crossover frequencies and segregation to that previously observed in established Brassica species, as demonstrated by marker haplotype analysis of the MD population. Homoeologous pairing between chromosomes A1-C1, A2-C2 and A7-C6 was detected at frequencies of 12-18 %, with other homoeologous chromosome regions associating from 8 % (A3-C3) to 0-1 % (A8-C8, A8-C9) of the time. Copy number analysis revealed eight instances of additional chromosomes and 20 instances of chromosomes present in one copy in somatically doubled MD progeny. Presence of chromosome A6 was positively correlated with self-pollinated seed set and pollen viability in the MD population. Many MD progeny were unable to produce self-pollinated seed (76 %) or viable pollen (53 %), although one MD plant produced 198 self-pollinated seeds. Average fertility was significantly lower in progeny obtained by microspore culture than progeny obtained by self-pollination or open-pollination, after excluding MD progeny which had not undergone chromosome doubling.
  
  
  CONCLUSIONS
  Based on SNP data analysis of the microspore-derived progeny, crossover frequency per chromosome in the allohexaploid hybrid was found to be similar to that in established Brassica species, suggesting that the higher chromosome number did not significantly disrupt cellular regulation of meiosis. SNP allele copy number analysis revealed the occurrence not only of homoeologous duplication/deletion events but also other cryptic duplications and deletions that may have been the result of mitotic instability. Microspore culture simplified the assessment of chromosome behaviour in the allohexaploid hybrid but yielded progeny with lower fertility and a greater range of ploidy levels compared to progeny obtained by self- or open-pollination.
authors.affiliation: School of Agriculture and Food Sciences, The University of Queensland, Brisbane, 4072, Australia. annaliese.mason@agrar.uni-giessen.de.|Centre for Integrative Legume Research, The University of Queensland, Brisbane, 4072, Australia. annaliese.mason@agrar.uni-giessen.de.|Department of Plant Breeding, IFZ Research Centre for Biosystems, Land Use and Nutrition, Justus Liebig University, Heinrich-Buff-Ring 26-32, 35392, Giessen, Germany. annaliese.mason@agrar.uni-giessen.de.
authors.forename: Annaliese S
authors.initials: AS
authors.lastname: Mason
authors.affiliation: School of Plant Biology, The University of Western Australia, 35 Stirling Highway, Crawley, 6009, Perth, Australia. junjunjun0729@gmail.com.
authors.forename: Junko
authors.initials: J
authors.lastname: Takahira
authors.affiliation: School of Plant Biology, The University of Western Australia, 35 Stirling Highway, Crawley, 6009, Perth, Australia. chaya_pau12@rediffmail.com.|Plant Breeding & Genetics Department, Punjab Agricultural University, Ferozepur Road, Ludhiana, Punjab, 141004, India. chaya_pau12@rediffmail.com.
authors.forename: Chhaya
authors.initials: C
authors.lastname: Atri
authors.affiliation: Department of Plant Breeding, IFZ Research Centre for Biosystems, Land Use and Nutrition, Justus Liebig University, Heinrich-Buff-Ring 26-32, 35392, Giessen, Germany. Birgit.Samans@agrar.uni-giessen.de.
authors.forename: Birgit
authors.initials: B
authors.lastname: Samans
authors.affiliation: School of Agriculture and Food Sciences, The University of Queensland, Brisbane, 4072, Australia. a.hayward@uq.edu.au.|Centre for Integrative Legume Research, The University of Queensland, Brisbane, 4072, Australia. a.hayward@uq.edu.au.
authors.forename: Alice
authors.initials: A
authors.lastname: Hayward
authors.affiliation: The UWA Institute of Agriculture, The University of Western Australia, 35 Stirling Highway, Crawley, 6009, Perth, Australia. wallace.cowling@uwa.edu.au.
authors.forename: Wallace A
authors.initials: WA
authors.lastname: Cowling
authors.affiliation: School of Agriculture and Food Sciences, The University of Queensland, Brisbane, 4072, Australia. jacqueline.batley@uwa.edu.au.|Centre for Integrative Legume Research, The University of Queensland, Brisbane, 4072, Australia. jacqueline.batley@uwa.edu.au.|School of Plant Biology, The University of Western Australia, 35 Stirling Highway, Crawley, 6009, Perth, Australia. jacqueline.batley@uwa.edu.au.
authors.forename: Jacqueline
authors.initials: J
authors.lastname: Batley
authors.affiliation: School of Plant Biology, The University of Western Australia, 35 Stirling Highway, Crawley, 6009, Perth, Australia. matthew.nelson@uwa.edu.au.|The UWA Institute of Agriculture, The University of Western Australia, 35 Stirling Highway, Crawley, 6009, Perth, Australia. matthew.nelson@uwa.edu.au.
authors.forename: Matthew N
authors.initials: MN
authors.lastname: Nelson
mesh_terms: D001937:Brassica / Q000235:genetics*; D003434:Crossing Over, Genetic; D006824:Hybridization, Genetic; D008540:Meiosis; D011003:Ploidies