“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

Segregation for fertility and meiotic stability in novel Brassica allohexaploids

Key message: AllohexaploidBrassicapopulations reveal ongoing segregation for fertility, while genotype influences fertility and meiotic stability. Abstract: Creation of a new Brassica allohexaploid species is of interest for the development of a crop type with increased heterosis and adaptability. At present, no naturally occurring, meiotically stable Brassica allohexaploid exists, with little data available on chromosome behaviour and meiotic control in allohexaploid germplasm. In this study, 100 plants from the cross B. carinata × B. rapa (A2 allohexaploid population) and 69 plants from the cross (B. napus × B. carinata) × B. juncea (H2 allohexaploid population) were assessed for fertility and meiotic behaviour. Estimated pollen viability, self-pollinated seed set, number of seeds on the main shoot, number of pods on the main shoot, seeds per ten pods and plant height were measured for both the A2 and H2 populations and for a set of reference control cultivars. The H2 population had high segregation for pollen viability and meiotic stability, while the A2 population was characterised by low pollen fertility and a high level of chromosome loss. Both populations were taller, but had lower average fertility trait values than the control cultivar samples. The study also characterises fertility and meiotic chromosome behaviour in genotypes and progeny sets in heterozygous allotetraploid Brassica derived lines, and indicates that genotypes of the parents and H1 hybrids are affecting chromosome pairing and fertility phenotypes in the H2 population. The identification and characterisation of factors influencing stability in novel allohexaploid Brassica populations will assist in the development of this as a new crop species for food and agricultural benefit

Fertile allohexaploid Brassica hybrids obtained from crosses between B. oleracea and B. juncea via ovule rescue and colchicine treatment of cuttings

An allohexaploid Brassica crop (2n = AABBCC) does not exist naturally, but is of interest for its potential to combine useful traits found in the six cultivated Brassica species which share combinations of the A, B and C genomes with additional allelic heterosis. In this study, we aimed to produce 2n = AABBCC hybrids by crosses between B. juncea and a number of Brassica C genome species. We used ovule rescue to overcome hybridization barriers and different colchicine treatment methods to induce chromosome doubling of ABC hybrids to AABBCC allohexaploids, thus restoring fertility. Only the cross B. oleracea x B. juncea was successful, with six triploid hybrids produced from one genotype combination. Colchicine-containing regeneration media was unsuccessful in doubling chromosome number in these hybrids, but treatment of cuttings with 0.05 to 0.25% colchicine successfully produced 200 S-1 allohexaploid seeds. The S-1 plants produced 7-84% viable pollen and set 0-390 seeds per plant, with 23-27 bivalents and 0-3 univalents during metaphase I of meiosis. Our results highlight the difficulties in working with the wild C genome species, but showed tha t our methods have utility for producing euploid, chromosome-doubled progeny in this cross combination. Further, Brassica oleracea x B. juncea allohexaploid hybrids may contain useful genetic factors for improved meiotic stability and fertility in allohexaploid germplasm pools.Key message Ovule rescue followed by 0.05-0.25% colchicine treatment of cuttings successfully produces fertile, partially stable allohexaploid Brassica from the cross B. juncea x B. oleracea