Molecular characterisation of a population derived from microspores of Brassica napus B. carinata hybrids

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1 Molecular characterisation of a population derived from microspores of Brassica napus B. carinata hybrids Annaliese Mason 1, Matthew Nelson 1,2, Guijun Yan 1 and Wallace Cowling 1,2 1 School of Plant Biology, Faculty of Natural and Agricultural Sciences, The University of Western Australia, 35 Stirling Highway, Crawley WA 6009, Australia. 2 Canola Breeders Western Australia Pty Ltd, Locked Bag 888, COMO, WA 6952, Australia masona02@student.uwa.edu.au ABSTRACT Characterisation of meiosis and subsequent chromosome inheritance in a population of 84 F 1 microspore-derived progeny from interspecific hybrids of Brassica napus (AAC n C n ) B. carinata (BBC c C c ) was carried out through simple sequence repeat (SSR) molecular marker analysis. The majority (82%) of progeny in the population appeared to have a chromosome complement approximating that of the F 1 parent (ABC n C c ). Every individual in the data set was found to be missing at least two of the 106 genomic loci tested, with an average of 16% of loci missing in total. Almost half of the ABC n C c individuals produced at least one seed when selfed with or without application of colchicine. The most likely explanation for these results is the production of unreduced male gametes by the F 1 and subsequent selection for unreduced gametes during the microspore culture process. Key words: Interspecific hybridisation canola chromosome complements microsatellite markers unreduced gametes INTRODUCTION Interspecific hybridisation offers a means of introducing new genetic variation into the inbred Brassica napus (canola) breeding population in Australia. B. napus as a species is limited in genetic variation. This is due to its relatively recent origin as an agricultural species, thought to have formed through hybridisation events between diploid progenitor species B. rapa and B. oleracea (Osborn, 2004). Breeding and selection for agricultural traits has reduced the pool of genetic variation even further. Crossing B. napus with related species creates interspecific hybrids that act as a source for new, potentially useful alleles for breeding programs. Microspore culture and doubled haploidy have the potential to stabilise new interspecific Brassica hybrids within a single generation. Immature pollen cells (microspores) from F 1 interspecific hybrids may be submitted to culture conditions known to induce microspore embryogenesis. Chromosome doubling in the microspores during embryogenesis and culture may occur spontaneously or be induced through application of chemical agents. Doubling the chromosome number may improve the fertility and genomic stability of such progeny, as chromosome pairs, or bivalents, are far more likely to undergo standard inheritance than univalents (unpaired chromosomes), which may persist for a number of generations or be lost immediately during meiosis in the interspecific hybrid. B. carinata (Ethiopian mustard) is an amphidiploid crop species closely related to B. napus. The two species share a common C genome, which should facilitate interspecific hybridisation. In hybrid crosses involving the B. carinata (BBC c C c ) and B. napus (AAC n C n ), F 1 individuals will have an expected chromosome complement of ABC n C c. The meiotic process in these individuals is expected to result in microspores segregating for univalent A and B genome chromosomes. C genome chromosomes are expected to pair as homologues, resulting in recombination and segregation of C genome alleles (Nelson et al., 2007). Molecular markers of known genomic location offer a means of identifying which chromosomes are inherited in microspore-derived plants, and microsatellites, or short sequence repeats (SSRs), are particularly suited for this due to potential co-dominance, locus specificity and high levels of allelic polymorphism. The latter is necessary to differentiate between alleles from the A, B and C genome chromosomes given the known genomic homoeology between the Brassica genomes (Parkin et al., 2003); this is particularly important in differentiating between the shared C genome chromosomes. The aim of this research was

2 to characterise chromosome inheritance in a microspore-derived population resulting from B. napus B. carinata F 1 hybrids through molecular marker analysis. MATERIALS AND METHODS Plant material: The experimental cross B. napus B. carinata and subsequent F 1 microspore embryogenesis was carried out at The University of Western Australia (see Castello et al., these Proceedings). Hybrid F 1 plants were obtained through reciprocal hand crossing of the parent species B. napus Trilogy, Tristate, Trigold, Surpass DH and Surpass 501TT with Brassica carinata accessions , and Microspores were isolated from mature F 1 hybrids and subjected to isolated microspore culture for the purpose of obtaining embryos and subsequently microspore-derived lines (MDLs) as described by Nelson et al. (2006). Seed set under selfing and open pollination conditions was recorded. A subset of MDLs was clonally propagated from stem cuttings treated with 0.5% colchicine. Microsatellite marker analysis: DNA was purified from the 84 MDLs along with F 1 hybrids and parental controls using a Qiagen MagAttract genomic DNA extraction kit according to the manufacturer s instructions. Microsatellite markers were obtained under material transfer agreement from the Agriculture and AgriFood Canada (AAFC) Brassica Microsatellite Consortium (A and C genome markers) and the AAFC Brassica juncea Microsatellite Consortium (B genome markers). Marker locations in B. napus (A and C genome markers) and in B. juncea (B genome markers) were provided (Andrew Sharpe and Derek Lydiate, pers. comm., 2005). Microsatellite markers were amplified using 25 µl reactions with the final concentrations: 2 ng/µl DNA template, 1x PCR buffer (Promega), 0.04 U/µL Taq Polymerase, 2 mg/µl MgCl 2, 200 µm of each dntp and 0.2 µm of each primer. PCR was performed using an Eppendorf Mastercycler thermal cycler: initial denaturing of 5 minutes at 94 C, followed by 35 cycles of 30 s at 94 C, 30 s at 50 C and 60 s at 72 C, with final extension at 72 C for 7 minutes (B genome markers) or 15 minutes (A and C genome markers). The 5 nucleotide of the forward primers of the A and C genome markers were fluorescently labelled using 6FAM, PET, NED or VIC (Applied Biosystems, AB). Amplification products were assayed on an AB3730xl capillary DNA sequencer (AB) and fragment analysis conducted using Genemapper or Peak Scanner Software v1.0 software (AB). B genome marker amplification products were electrophoresed in TBE buffer using 2% agarose gels, stained with Ethidium bromide, viewed under UV transillumination and scored for presence or absence of amplification products. RESULTS Seventy-two microsatellite primer pairs detected a total of 133 alleles at 106 loci on all 38 chromosomes (10, 8, 9 and 9 chromosomes from the A, B, C n and C c genomes, respectively). A total of 24 to 31 A genome loci and 14 to 34 B genome loci were tested for each individual. A total of 19 to 22 C genome alleles of B. napus origin and 16 to 19 C genome alleles of B. carinata origin were tested for each individual. The marker loci genotypes were used to infer the chromosome complement of the 84 MDLs. Of the 84 individuals in the population, one group of 71 (82%) appeared to have chromosome complements similar, but not identical, to that of the F 1 parent (ABC n C c ). The second group of 13 individuals scored presence for C chromosome alleles of either B. napus (C n ) or B. carinata (C c ) origin, not both, at most loci. On average, total C genome allele presence was 86% for the first group and 55% for the second group (Fig 1.).

3 Number of individuals % 50 55% 55 60% 60 65% 65 70% 70 75% 75 80% 80 85% 85 90% 90 95% % Percentage of total C genome alleles present from microsatellite (SSR) marker results Fig. 1. Total C genome allele presence in 84 individuals of a population derived from F 1 (ABC n C c ) (Brassica napus (AAC n C n ) x B. carinata (BBC c C c )) microspores. Individuals showing evidence of parental C genome allele segregation (C n or C c ) for most loci are marked in dark grey, whereas individuals with two C genome alleles (C n and C c ) for most loci are marked in lighter grey. All individuals in the data set showed absence of at least two alleles tested. Approximately 50% of individuals in the dataset had at least one B genome chromosome where one locus was present and one locus was absent, and 60% of individuals had at least one A genome chromosome where one locus was present and one locus was absent. Twelve individuals in the population showed presence of all A genome loci tested (but were missing B loci or C- genome alleles), and 19 individuals showed presence of all B genome loci tested (but were missing A loci or C-genome alleles). Five individuals in the population showed presence of all A and B genome loci tested, but were missing C-genome alleles. Average proportions of total genomic loci present in the population are given in Table 1. Table 1. Genomic loci presence in a population derived from microspores of F 1 (Brassica napus x B. carinata) hybrids Average proportion of loci present from molecular marker results Genome Category 1* individuals (n=71) Category 2** individuals (n=13) All individuals (n=84) A B C (B. napus) C (B. carinata) All (total) *Category 1 individuals showed presence of both Brassica napus and B. carinata C genome alleles at most loci. **Category 2 individuals showed presence of only one allele of either B. napus or B. carinata origin at most loci. Category 1 progeny (Table 1) were thought to result from unreduced gametes. Category 1 progeny were found to have both B. napus and B. carinata C alleles at most loci tested, from which presence of both the maternal and paternal sets of C genome chromosomes can be inferred. Category 2 progeny (Table 1) were thought to result from reduced gametes, and showed primarily (>80% of loci tested) normal meiotic segregation, with either a B. napus C allele or a B. carinata C allele present at each locus, not both. This indicates presence of only one set of randomly assorted C chromosomes. Supplementary ploidy level data from flow cytometry (not shown) for three of the Category 2 progeny revealed that these progeny were haploid (DNA content agreed with putative reduced genome content) further indicating that reduction had occurred during meiosis in the F 1 hybrid to produce these individuals.

4 Supplementary ploidy level data from flow cytometry (not shown) for 33 of the Category 1 progeny indicated that these progeny had diploid or greater ploidy levels (DNA content agreed with putative unreduced genome content of ABCC or greater) supporting the hypothesis that these progeny had resulted from unreduced gametes produced by the F 1 hybrid. Twenty-seven individuals in the set of 84 produced at least one seed when selfed with or without application of colchicine. All of these individuals were derived from putative unreduced gametes (Category 1 in Table 1), with genome complements of approximately ABC n C c, and a minimum of 79% of total genomic loci present. None of the putative reduced gametes (Category 2 in Table 1) were fertile. DISCUSSION AND CONCLUSION Under normal meiotic conditions in the hybrid, the production of F 1 microspore-derived individuals with both C n and C c alleles for most loci is extremely unlikely. Gametes produced through normal pairing and segregation between C genome chromosomes during meiosis in the F 1 hybrid are expected to have either a C n or a C c allele at each locus but only 13/84 appeared to be the products of normal meiotic segregation in the C-genome. The presence of both alleles at a single locus indicates failure of homologous C genome chromosome separation during the meiotic process. The C genomes of B. napus and B. carinata are closely homologous and bivalent pairing has previously been observed between them (U, 1935). Therefore, asynapsis is unlikely to be the cause of the meiotic dysfunction. First division restitution (FDR) however is a mechanism known to occur in the Brassiceae (Heyn, 1977) that explains this result. FDR involves normal meiotic pairing of homologous chromosomes during Meiosis I, followed by either failure of the metaphase plate to separate homologous chromosome pairs or failure of the cytokinetic division. Meiosis II will then proceed normally, with sister chromatids separating in a mitosis-like division. Gametes produced by this method are referred to as unreduced or 2n. Very low frequencies of unreduced gametes would normally be produced by parental plants (Heyn, 1977). In this experiment, more than 80% of the progeny appeared to result from unreduced gametes - this may be due to selection pressure for viability and near-complete chromosome sets during the microspore culture process. Retention of most chromosomes could hypothetically increase survival and embryogenesis of microspores in culture. The observation of higher fertility in microspore-derived individuals from putative unreduced gametes compared to reduced gametes is consistent with this hypothesis. The most likely explanations for absence of loci in individuals in this population (Table 1) are abnormal meiotic events. A and B genome chromosomes, being present only in haploid form in the F 1 hybrid, have no homologous chromosomes with which to pair during Meiosis I. However, A, B and C Brassica genomes are highly homoeologous (Snowdon, 2007) and pairing between genomes has previously been observed under interspecific hybridisation conditions (Li et al., 2005). Loss of chromosome fragments is a possible outcome of homoeologous pairing, as recombination events between non-homologous chromosomes may result in creation of acentric chromosomes which are subsequently lost during mitotic division. Intra- and inter-genomic pairing resulting in non-reciprocal translocation events may also be responsible for the presence of both C genome alleles at some loci in individuals appearing to result from normal reduced gametes. Some mechanisms of FDR, such as irregular cytokinetic division, may also result in loss of whole chromosomes during meiosis. Further investigation of the meiotic process in Brassica interspecific hybrids will be required to determine the actual mechanisms underlying the inheritance of the observed chromosome complements in the experimental population. The most probable explanation for the observed results is production of low frequencies of unreduced gametes through FDR in the F 1 hybrid, and subsequent selection for these unreduced gametes during the microspore culture process. It is extremely interesting that several putative unreduced gametes were fertile and have remained fertile in subsequent generations.

5 ACKNOWLEDGEMENTS This work was supported by the Australian Research Council with industry partners NPZ Lembke (Germany) and COGGO Ltd (Australia). The bulk of the experimental work for this project was carried out by Marie-Claire Castello (growth, hybridisation and microspore culture) and by Clare O Lone (SSR molecular markers). This research was funded by Australian Research Council Linkage grant LP REFERENCES Heyn, F. J. (1977) Analysis of Unreduced Gametes in the Brassiceae by Crosses between Species and Ploidy Levels. Z. Pflanzenzüchtg, 78, Li, M. T., Li, Z. Y., Zhang, C. Y., Qian, W. & Meng, J. L. (2005) Reproduction and cytogenetic characterization of interspecific hybrids derived from crosses between Brassica carinata and B. rapa. Theoretical and Applied Genetics, 110, Nelson, M., Castello, M.-C., Thomson, L., Cousin, A., Yan, G. & Cowling, W. A. (2006) Microspore culture from interspecific hybrids of Brassica napus and B. carinata produces fertile progeny. IN Mercer, C. F. (Ed.) "Breeding for success: diversity in action", Proceedings of the 13th Australasian Plant Breeding Conference. Christchurch, New Zealand. Nelson, M., Mason, A., Castello, M., O'Lone, C., Yan, G., Chen, S. & Cowling, W. A. (2007) Molecular characterisation of microspore-derived progeny from the interspecific F1 of Brassica napus B. carinata. 12th International Rapeseed Congress. Wuhan, China. Osborn, T. C. (2004) The contribution of polyploidy to variation in Brassica species. Physiologia Plantarum, 121, Parkin, I. A. P., Sharpe, A. G. & Lydiate, D. J. (2003) Patterns of genome duplication within the Brassica napus genome. Genome, 46, Snowdon, R. (2007) Cytogenetics and genome analysis in Brassica crops. Chromosome research, 15, 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,

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