1 Saskatoon Research Centre, Agriculture and Agri-Food Canada, 107 Science Place, Saskatoon, Saskatchewan S7N OX2, Canada
2Eastern Cereal and Oilseed Research Centre, Agriculture and Agri-Food Canada, Central Experimental Farm, Ottawa, Ontario K1A OC6, Canada
The transfer of genes for C3C4 intermediate metabolism from Moricandia into Brassica is expected to lead to improved drought tolerance through higher water use efficiency. The first step to the introgression of C3C4 metabolism into Brassica was the successful hybridization of Moricandia nitens with Brassica oleracea and B. napus. A genetic map of M. nitens, based on RPLP loci, is being developed and used for comparative mapping of Brassica and Moricandia. This comparative mapping will allow the marker assisted interspecies transfer of the genes controlling C3C4 intermediate metabolism.
KEYWORDS C3C4 intermediate plants, genetic linkage map, intergeneric hybridization, Moricandia nitens, Brassica rapa
C3C4 photosynthesis, which is found in several species within the genus Moricandia, can improve water-use-efficiency as it increases the assimilation of carbon in conditions where water availability is limiting the rate of photosynthesis (McVetty et al. 1989). The transfer of C3C4 physiology into Brassica crops to improve drought tolerance is an attractive proposition. Intergeneric Moricandia/Brassica hybrids exhibit photosynthesis that is intermediate between C3 and C3C4 (Apel et al. 1984), and the possibility of assaying C3C4 physiology directly in the progeny of these hybrids could assist in the transfer of this character to related species.
Molecular taxonomy has shown that B. rapa and B. oleracea are more closely related to Moricandia species than to several Brassica species, including B. nigra (Warwick and Black 1994), and evidence has already been presented for recombination between M. arvensis chromosomes and Brassica A and B genome chromosomes in intergeneric amphihaploid hybrids (Takahata and Takeda 1990). For the presented project we chose Moricandia nitens (Viv.) Durieu &Barr. which is one of the C3C4 intermediate species within the genus Moricandia with a CO2 compensation point (Γ) of 8μl CO2 ⋅ l-1 which is comparable to C4 species (Hylton et al. 1988).
For the intergeneric hybridization a crossing strategy was followed which should maximise the amount of intergeneric recombination between M. nitens and B. rapa chromosomes and increase the survival rate of gametes carrying recombinant M. nitens/B. rapa chromosomes. In order to identify these recombinant chromosomes and to map genes influencing the efficiency of C3C4 photorespiration, a genetic linkage map of the M. nitens genome has been developed. This map will allow comparative mapping between M. nitens and B. rapa which can be used to analyse substitution lines of 1) M. nitens with segments of B. rapa that disrupt C3C4 photosynthesis and 2) B. rapa with segments of M. nitens that promote C3C4 photosynthesis in Brassica.
Two accessions of M. nitens (Viviana) Durieu &Barr. (2n=28)(‘M. nit. 1’, ‘M. nit. 3’) and M. arvensis (L.) DC. (2n=28) (‘M. arv. 1’, ‘M. arv. 3’) kindly provided by Dr. Stephen Rawsthorne, John Innes Centre Norwich, UK, were used for both intergeneric crossing and RFLP mapping. Two B. oleracea lines (‘A12’, ‘GD’), one B. rapa line (‘MBR2’) and one resynthesized B. napus line (‘MBI2’) were used for the intergeneric crosses. All accessions were grown in a greenhouse with 16 hours photoperiod.
B. oleracea was pollinated with M. nitens and the intergeneric hybrids were rescued by ovary culture followed by in ovule-embryo culture (Beschorner, M., K. Ford and D.J. Lydiate, unpublished). MMCC amphidiploids were produced by colchicine treatment. These hybrids were crossed with a newly resynthesized B. napus produced by crossing B. oleracea with a canola quality B. rapa followed by chromosome doubling. The resulting MACC hybrid was rescued by the same in vitro techniques as the MC hybrid and will be backcrossed with both B. rapa and M. nitens (Fig. 1).
All three Brassica accessions could be hybridized successfully with M. nitens. Most of the hybrids were obtained when Brassica was used as the female parent. Hybrid plantlets were colchicined in vitro and the genotype of the hybrids was confirmed by morpho-logical and cytological studies. Meiosis studies of the chromosome doubled B. oleracea/M. nitens hybrids showed regular pairing with 23 bivalents. This result shows that the hybrids contained a complete set of the parental chromosomes, which is consistent with no intergeneric pairing between the C and M genomes. However, M and C chromosomes may be able to pair as revealed by the presence of up to three bivalents in meiosis studies of the CM amphihaploid hybrids. The MACC hybrid will now be backcrossed with both B. rapa and M. nitens to develop substitution lines (Fig. 1).
Fig. 1. Strategy for intergeneric transfer of C3C4 metabolism from M. nitens into B. rapa
Moricandia arvensis and M. nitens are closely related, and chloroplast DNA restriction site data provides evidence for the monophyly of these two taxa (Warwick and Black 1994). As intercrosses lead to full seed set and preliminary RFLP studies had shown an extremely high level of polymorphism between M. arvensis and M. nitens, a M. arvensis x M. nitens cross was used to develop a genetic linkage map of Moricandia. A single F1 plant was backcrossed with M. arvensis using the F1 as both pollen donor and pollen recipient. RFLP analysis was carried out on 120 B1 individuals, 60 from each of the backcross directions. The use of this reciprocal backcross strategy will enable the future study of female and male meiosis events. Southern hybridization filters, containing DNA of the parental lines, the F1 and 120 B1 individuals, were probed with 135 highly informative Brassica clones (Sharpe et al. 1995) which detected a high level of polymorphism, giving an average of 2.0 loci per probe.
The restriction patterns allowed the identification of 260 polymorphic loci. The genetic linkage analysis of these data has resulted in a M. arvensis/M. nitens genome map with 15 linkage groups, a total length of 1002 cM and a mean marker distance of 3.96 cM (Beschorner, M., S.I. Warwick and D.J. Lydiate, unpublished data). As both M. arvensis and M. nitens have 2N=28 chromosomes, two of the 15 linkage groups are expected to join for a final number of 14 linkage groups. The genetic linkage map will be used for comparative mapping between Moricandia and Brassica and for marker-assisted selection of B. rapa/M. nitens substitution lines.
This work was supported by the UK Biotechnology and Biological Sciences Research Council and by the Saskatchewan Agri-Food Innovation Fund.
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