Are conjoined microspore-derived embryos of canola genetically identical?

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1 Are conjoined microspore-derived embryos of canola genetically identical? Anouska Cousin 1 and Matthew Nelson 1,2 1 Canola Breeders Western Australia Pty Ltd, Locked Bag 888, COMO, WA 6952, Australia 2 School of Plant Biology, Faculty of Natural and Agricultural Sciences, The University of Western Australia, 35 Stirling Highway, Crawley WA 6009, Australia ABSTRACT Microspore culture is used routinely in many Brassica breeding programmes to produce completely homozygous, doubled haploid lines. The CBWA canola (Brassica napus) breeding programme produces approximately 2500 fertile, doubled haploid lines from about 50 pairs of F 1 plants each year. We regularly observe the occurrence of conjoined embryos (twins and triplets) at the torpedo-shaped embryo stage, with embryos joined either at the base of the hypocotyl or around the middle section of the hypocotyl. We used 10 microsatellite markers to genetically characterise 13 sets of twins and 2 sets of triplets derived from three pairs of F 1 plants. We found that all twins/triplets sets were identical at all marker loci tested, a result that statistically supports that these twins/triplets are indeed genetically identical. We concluded that each set of twins/triplets were identical clones, possibly analogous to polyembryony previously reported in twn mutant lines of Arabidopsis thaliana in which additional embryos develop from suspensor cells of zygotic embryos. We discuss the practical outcomes of our findings. Key words: Doubled haploidy breeding Brassica napus microsatellite markers INTRODUCTION Microspore culture is a well established method in canola (Brassica napus) for rapid production completely homozygous, doubled haploid lines (DHLs). In the microspore culture programme of Canola Breeders Western Australia Pty Ltd, we produce approximately 2500 fertile DHLs per annum, which are derived from pairs of F 1 plants produced from 50 crosses between elite parents. During the course of DHL production we frequently observe conjoined microspore-derived embryos (MDEs) at the torpedo-shaped embryo stage. The conjoined MDEs generally are present as twins but also occasionally as triplets. Most other examples of twinning or polyembryony in the scientific literature refer to zygotic embryos, rather than MDEs. For example, the Arabidopsis thaliana mutant allele twn1 causes supernumerary embryos via suspensor transformation (Vernon et al. 2001). Similarly, the twn2 mutant of A. thaliana results in the basal cells of zygotic embryos to proliferate abnormally to produce multiple embryos rather than suspensor cells (Zhang and Somerville, 1997). No equivalent work has been reported for microspore derived embryos of A. thaliana. One report that may have some relevance to canola was reported by Prabhudesai and Bhaskaran (1993) who developed a system for repeated cycles of somatic embryogenesis from microspore-derived embryos of B. juncea (Indian mustard). In our study, we used a genetic approach to test three possible pathways that might lead to twinned MDEs: Firstly, each twin could be derived from two independent microspores produced by two distinct meiotic events which later fuse together during the course of the microspore culture process. Such embryos will be genetically distinct and have random allelic assortment compared to each other and would therefore would both be of value to a breeding programme. Secondly, each pair of twins may be derived from different gametes from the same tetrad (i.e. the same meiotic event); 50% of any given twinned embryos will be genetically identical (if they share sister chromatids that divided at Meiosis II) while 50% will be genetically contrasting (if they have non-sister chromatids that divided at Meiosis I). These MDEs would be of great basic research value as they would allow partial tetrad analysis (see review by Copenhaver et al. 2000). Thirdly, each pair of twins could be clonally-derived from the same microspore through post-meiotic, mitotic cell division. These twins would be genetically identical and therefore only one member of the twin would be required for breeding purposes.

2 In this study, we used microsatellite markers to determine the nature of 13 sets of twinned MDEs and 2 sets of triplet MDEs derived from three different genetic crosses of canola. MATERIALS AND METHODS Plant material: Controlled crosses were performed between three pairs of elite canola lines. Two F 1 seedlings from each cross combination were grown in controlled environment growth rooms with 16 h photoperiod with a light intensity of 200 µmolm -2 s -1 at 15/5ºC (day/night). When the plants reached anthesis, flowers were trimmed back and the plants moved to a 10/5 ºC temperature regime. Approximately 80 buds (2-4 mm in length from the base to the tip of the sepal) were collected on ice from pairs of F 1 plants; buds were used immediately or stored at 4 ºC for 24 h. Leaf samples of the F 1 plants were harvested for subsequent DNA extraction. Microspore isolation: Microspores isolation and plant regeneration methods were similar to Nelson et al (2006) with some modifications. After microspore isolation microspores were resuspended in fresh NLN- 13 medium to give a density of 4 x 10 5 ml -1. This twinned embryo investigation was part of a larger microspore culture experiment aimed at determining the effect of colchicine concentration and heat-treatment on efficiency of doubled haploid production, the microspore solution was divided between six foil-wrapped Petri dishes (10 ml volumes) and subjected to six different treatments: three colchicine concentrations (25 µm, 50 µm or 100 µm) and two temperatures (25 ºC or 32.5 ºC) to induce microspores. After 24 h, another 10 ml NLN-13 solution was added to each plate to dilute the colchicine (down to 12.5 µm, 25 µm and 50 µm). They were then resealed, wrapped in foil and placed at 25 C for two weeks; on a shaker for 1 week and the foil was then removed and the dishes were shaken for a further week until the embryos became green. The resulting embryos were then cold-treated at 4 C for a minimum of 1 week to encourage regeneration. Embryos were then placed onto regeneration media; twin or triplet embryos were tracked from this point onwards. After 2-3 weeks on regeneration media the shooting embryos were trimmed and placed in culture vessels with no hormones to promote root growth. After 4 weeks the plantlets (including twins and triplets) that had roots were taken out of culture and transferred to pasteurised potting mix and kept under humidity covers for 1 week in a controlled environment room at a constant 15 ºC with a 12 h photoperiod and a light intensity of 120 µmol.m -2.s -1. After one week with no covers the plants were transferred to a controlled environment growth rooms with a 12 h photoperiod and light intensity of around 200 µmolm -2 s -1 at 15/5 ºC (day/night). Leaf samples were taken for subsequent DNA extraction. After another 2 weeks the plants are then transferred to a glasshouse where plants were grown to maturity with self-pollination of fertile plants enforced by bagging. Molecular marker analysis: DNA was extracted from F 1 and microspore-derived lines (MDLs) using Qiagen MagAttract Genomic DNA Extraction Kit and quantified relative to a 100bp DNA ladder (Promega) on a 1% agarose TBE gel, visualised using ethidium bromide and UV transillumination. Ten microsatellite markers (Brassica Microsatellite Consortium, Agriculture and Agri-Food Canada, Saskatoon Research Centre, Saskatoon, Canada) that we previously found to detect polymorphisms between parental lines (data not presented) were used to genotype the pairs of F 1 individuals and MDLs along with six parental DNA controls previously extracted from bulked leaf tissue of each variety. The ten microsatellite markers detected 15 loci distributed across 11 chromosomes. PCR amplification was conducted using AmpliTaq Gold Mastermix (Applied Biosystems, AB) in 15uL reactions with 40ng DNA and 5pmol of forward and reverse primers. Each forward primer was fluorescently labelled (6FAM, PET, NED or VIC), allowing the detection of fluorescently-labelled amplicons on an AB3730xl capillary DNA sequencer (AB). Marker allele sizes were estimated relative to an internal Genescan LIZ600 size standard (AB) using GeneMapper 3.7 software (AB). Statistical analysis: Microsatellite marker loci that were heterozygous in the F 1 microspore donors were scored for each twin/triplet set. The probability mass function (f) based on the expected binomial

3 distribution (see was used to test the probability of obtaining the observed results assuming random assortment of alleles within twin/triplet sets: where n = number of polymorphic loci, k = number of loci with identical alleles at polymorphic loci, and p = 0.5 for twins and p = 0.25 for triplets (that is, the probability of finding the same allele at each polymorphic locus). The same test was used to calculate the probability that the observed number of identical twins could have been obtained by chance if each set of twins was derived from the same tetrad family. In this case, p = 0.5 because there is an equal probability that any two members of a tetrad family will be identical (divided at Meiosis II) or non-identical (divided at Meiosis I). RESULTS AND DISCUSSION We isolated 13 sets of twins and 2 sets of triplets from various cross/treatment combinations (Table 1). Colchicine concentration and temperature regime in the first 24 h after microspore isolation did not appear to affect the frequency of twins/triplets in microspore cultures (data not presented). We observed two types of twinned MDEs: those joined at the base of the hypocotyl and those joined along the hypocotyl (Fig. 1). Each twin type occurred in approximately equal frequency, while both sets of triplet embryos were joined at the base of the hypocotyl (Table 1). Fig. 1 Dissecting microscope image of two types of twinned embryos observed in this study. AH twins are conjoined along the hypocotyl and BH twins are conjoined at the base of the hypocotyl. Table 1: sets of twins and triplets from three cross combinations, the types of twins/triplets (BH = attached at base of hypocotyls; AH = attached along the hypocotyls), number of polymorphic marker loci and the probability mass function (f) of these results occurring by chance. Microspore isolation twins triplets polymorphic marker loci Probability mass function (f) of observed genotypes 1 Cross-1 4 BH, 3 AH x 10-3 / - Cross-2 4 AH 1 BH x 10-4 / 6.0 x 10-8 Cross-3 2 BH 1 BH x 10-3 / 1.5 x Probabilities (where applicable) shown for twins / triplets, respectively. Using 10 microsatellite markers, we detected 9, 12 and 8 heterozygous loci in the three respective sets of F 1 hybrids derive from three crosses (Table 1). All MDLs were

4 homozygous at all marker loci that were heterozygous in the F 1 individuals, confirming that all embryos tested were derived from haploid gametic tissue and not from diploid somatic tissue. Allelic segregation appeared normal between twin/triplet sets but there was no variation within twin/triplet sets. We used the probability mass function (f) to test whether or not the observed results could have been obtained by random binomial assortment of marker alleles within each twin/triplet set and the results are shown in Table 1. We concluded that it was highly probable that each twin/ triplet set was indeed genetically identical and that these twins/triplets could not have been obtained by the fusing of random microspores during the microspore isolation and culture process. Genetically identical twins could have been produced via two pathways: either they were members of the same tetrad family that had divided at Meiosis II (i.e. shared sister chromatids) or they were clones derived from the same microspore. In the former pathway, there is an equal probability of pairs of tetrad family members being identical or non-identical (divided at Meiosis II or Meiosis I, respectively). The probability mass function was again used, this time to calculate the probability that all 13 sets of twins were products of Meiosis II and the result was: f = 1.2 x This low probability indicated that it was very unlikely that each of the 13 sets of twins could be derived from 13 tetrad families. Furthermore, the two sets of triplets were completely identical; if each set was derived from tetrad families, they would not all be identical. Therefore, it is highly likely that each twin/triplet is derived from a single microspore, possibly from embryogenic suspensor cells as has been observed in zygotic mutants of A. thaliana (Zhang and Somerville, 1997; Vernon et al. 2001). Cytological examination of early stages of microspore embryo development would be required to confirm this hypothesis. A practical outcome of our discovery that twinned/triplet MDEs are genetically identical is that we can recommend to commercial canola breeding programmes that there is no genetic value in regenerating plants from more than one member of a set of twinned or triplet embryos because they will not provide any new combination of alleles. ACKNOWLEDGEMENTS The authors would like to thank Priya Krishnamurthy for technical assistance and Wallace Cowling for his support in conducting this study. REFERENCES Copenhaver GP, Keith KC, Preuss D (2000) Tetrad analysis in higher plants. A budding technology. Plant Physiology 124, Nelson M, Castello M-C, Thomson L, Cousin A, Yan G, Cowling WA (2006) Microspore culture from interspecific hybrids of Brassica napus and B. carinata produces fertile progeny, In 'Breeding for Success: Diversity in Action. Proceedings of the 13th Australasian Plant Breeding Conference, Christchurch, New Zealand (Ed. CF Mercer). Prabhudesai V, Bhaskaran S (1993) A continuous culture system of direct somatic embryogenesis in microspore-derived embryos of Brassica juncea. Plant Cell Reports 12, Vernon DM, Hannon MJ, Le M, Forsthoefel NR (2001) An expanded role for the TWN1 gene in embryogenesis: defects in cotyledon pattern and morphology in the twn1 mutant of Arabidopsis (Brassicaceae). Am. J. Bot. 88, Zhang JZ, Somerville CR (1997) Suspensor-derived polyembryony caused by altered expression of valyl-trna synthetase in the twn2 mutant of Arabidopsis. PNAS 94,

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