Development and molecular characterization of rapeseed (brassica napus l.) Resistant against beet cyst nematodes
Institute of Crop Science and Plant Breeding I, Justus-Liebig-University,
Ludwigstr. 23, D-35390 Giessen, Germany
An introgression breeding program, initiated by the lack of oilseed rape cultivars (Brassica napus, genome AACC) resistant against Heterodera schachtii (beet cyst nematode), was started a couple of years ago. Oilradish genotypes (Raphanus sativus, RR) possessing a high level of nematode resistance served as gene donors. Some highly resistant F1 hybrids (ACR) derived from intergeneric crosses via embryo rescue were detected. After colchicine treatment the progeny was used in a backcross program with spring rapeseed as pollen donor. Selection of resistant individuals was performed by nematode infection tests in vivo. Following the testing of 51 BC2 (2nd backcross generation) plants, derived from a single highly resistant BC1-individual, a BC3-progeny was generated and tested for the level of nematode resistance. In contrast to earlier generations some of the plants were fertile, phenotypically very similar to oilseed rape due to the reduced number of added R-chromosomes, and still resistant to nematodes. Further backcrossing is in progress and the first BC4-seeds derived from different resistant BC3-individuals are going to be tested in order to screen for resistant translocation lines. Molecular characterization of the material is performed by PCR fingerprinting (AFLP) and genomic in situ hybridization (GISH) assays, showing a loss of R-genome at each backcross step to 1-3 R-Chromosomes in the BC3-generation. In order to avoid laborious testing procedures in vivo molecular marker(s) for detecting individuals containing a gene or genes for nematode resistance would be very useful. A segregating oilradish population is being produced for bulked segregant analysis. Identified markers are to be applied in marker-assisted selection to accelerate the development of backcross progenies without running nematode resistance tests at every step.
KEYWORDS: nematode resistance, Heterodera schachtii, oilradish, oilseed rape
Nematodes are responsible for massive damage to plant crops every year. In addition to a number of migrating species (Pratylenchus spp.) some sedentary nematodes (Globodera spp., Ditylenchus spp., Heterodera spp.) are of major importance under temperate climates. The beet cyst nematode (BCN) Heterodera schachtii belongs to this group. The broad spectrum of host plants including members of the Chenopodiaceae and Brassicaceae families and the long persistence of the cysts lead to a sustaining problem in agriculture. In an accommodating environment the cysts, containing up to 500 eggs and L2 larvae, can persist over 20 years and remain infectious in the soil. Most plants can tolerate an attack and propagation of BCN. In sugar beet (Beta vulgaris) crop rotations BCN can cause major problems. The beets develop numerous small roots after infection with BCN leading to substantially loss of sugar yield. Various attempts have been made to control BCN. However, due to major environmental problems caused by broad spectrum nematicides, these have been prohibited. A wide crop rotation including non-host plants is one possibility to lower infection potential, as well as controlling weedy hosts. Resistant catch crops inducing the sliding of nematodes, but preventing the propagation of BCN as cysts, are another very useful method to control this pest.
Development of plant material
Due to the sources of nematode resistance against BCN used in highly resistant oilradish varieties (Baukloh 1976) cv. 'Fortissimo' was chosen as a donor of resistance gene(s) against H. schachtii. Wide hybridizations between the above mentioned resistant oilradish (RR; 2n = 18) and spring type oilseed rape (AACC; 2n = 38) cv. 'Drakkar', carried out as conventional crosses followed by embryo rescue technique (ovule culture, according to Thierfelder 1995), led to a number of hybrid plants (ACR; n = 26) possessing haploid chromosome sets of oilradish and rapeseed. These were tested for their level of nematode resistance according to the nematode resistance test of Toxopeus and Lubberts (1979). The selected individuals were colchicine treated to obtain fertile amphidiploids (AACCRR; 2n = 56) and back-crossed with oilseed rape applying ovule culture. The generated BC1-generation suffered from a lack of fertility similar to the BC0-plants (Pan 1997; cf. Tokumasu et al. 1988). A variation from bright yellow to white flowers and small anthers without any pollen were all in common for these individuals. After the selection of the highly resistant BC1-individual 2062/16 with yellow flowers and a good vitality, the BC2-generation consisting of 51 individuals was produced, again via ovule culture. Similarly the progeny of the highly resistant BC2-31 (BC3) was generated; this progeny was the main object of the investigations described in this paper.
Nematode resistance test
For the resistance test five cuttings per individual were taken and cultured in 96cm2 PVC boxes filled with sterilized sand according to Toxopeus & Lubberts (1978). Plants were fertilized once a week with a special solution containing usual micro and macro nutrients. After three weeks the well established plants were inoculated with 1000 L2 larvae each and allowed to grow under greenhouse conditions for seven weeks. After that the plants were washed from sand and cysts. The resulting mixture of sand and cysts was separated by floating in a 1.7M MgSO4 solution. Cysts swam on the surface of the solution, allowing them to be collected and quantified.
The number of cysts is an indication of the level of nematode resistance of a plant. Looking at the cysts it becomes obvious that their size differs greatly (0.8 to 2mm). According to this the number of eggs also varies. Due to this fact the total number of eggs and larvae inside the cysts is a better measure for the level of resistance of a plant as it expresses the real potential of pest propagation in a host plant. The corresponding suitable criterion is the Pf/Pi value, which is the quotient of final population density 'Pf ' (counted eggs and larvae resulting from crushed cyst suspension) to the initial population density 'Pi' (1,000 inoculated larvae in our case). If the quotient is >1 propagation of the nematodes has occured, if it is <1 the population density is lowered, if it is <0.2 the plants are considered resistant (BSA 1998).
The negative side effects of the Raphanus genome in the described hybrid plants on fertility, seed quality (i.e. glucosinolate content) and yield performance caused by genes with unknown and non-controllable functions is a characteristic problem in the course of a backcross program based on wide hybridization. As the number of alien chromosomes is not easily predictable with conventional molecular methods like AFLPs (Vos et al. 1995) a cytological method was applied to count and distinguish between the chromosomes of different origin (Snowdon et al. 1997, 1998). The use of total genomic DNA as a fluorescent probe (genomic in situ hybridization, GISH) is especially useful for diagnostic studies of the amount and integration of foreign chromatin in interspecific and intergeneric plant hybrids (Heslop-Harrison and Schwarzacher 1996). Although the size of Brassica chromosomes is extremely small, making a distinction based on morphological characteristics very hard, the number of added R-chromosomes in the BC-material was scored visually (Snowdon et al. 1997).
Nematode resistance level of hybrid plants
In the course of a previous trial in 1997/98 51 BC2-individuals and some control genotypes, such as susceptible and resistant oilradish and yellow mustard varieties, were assessed. There was a wide variation from an average number of 270 cysts/cutting to individual plants without cysts. The oilseed rape varieties were highly susceptible, but the resistant mother plant 2062/16 (BC1) was scored highly resistant as well as the four BC2-individuals BC2-3, -15, -20 and -31 (Voss et al. in press). Due to a high level of resistance and acceptable fertility the individual progeny BC2-31 was used to develop via embryo rescue the next backcross generation (BC3), which was expected to be more fertile and subsequently more similar to oilseed rape. The nematode resistance test of the BC3-generation derived from BC2-31 led to five highly resistant BC3-individuals (Fig. 1). Similar to the previous test a wide variation of susceptibility occured. The average number of cysts per cutting varied from 0 to 261. The resistant BC2-individuals repeatedly showed a very high level of resistance which was also expressed in the related BC3-progeny. The Pf/Pi values display all levels of susceptibility/resistance within the tested material (Fig. 2).
Figure 1: Results of nematode resistance test 1998, mean values of counted cysts per cutting, dotted lines indicate standard deviation
Four of the resistant BC3-individuals were back-crossed and for the first time no embryo rescue was necessary to receive seed material (BC4). The appearance of the seeds varies greatly according to the parent. Whereas the seeds of individual BC3-32 appear tight and numerous (Fig. 3) this is not the same for all other individuals. Especially BC3-34 has many aborted seeds and small pods.
BC4 seed samples obtained from backcrosses of different BC3-individuals resistant against Heterodera schachtii
Figure 2: Number of added R-chromosomes and results of nematode resistance test 1998; mean Pf/Pi values
Presence of Raphanus chromosomes in hybrid plants and progeny
With the help of the described GISH method (Snowdon et al. 1997) a number of the developed BC-individuals were characterized cytologically. The amount of added R-chromosomes in the BC1-mother was 9, as expected due to the allohexaploid hexaploid parent. In the BC2-generation number of R-chromosomes varied from 3 to 5, whereas in the BC3 only 1 to 3 R-chromosomes were detected (Fig. 2). Nevertheless, some of these plant were highly resistant. In one case (BC3-10) we were able to detect a chromosome translocation (Fig. 2; marked by a star) and 2 complete R-chromosomes.
The results of nematode resistance tests show that it is possible to select individuals with complete or very strong nematode resistance in higher generations of a backcross breeding program (Fig. 1). The accompanying results of the GISH analysis indicate that resistance against BCN in oilradish is inherited by genes on 1-2 chromosomes according to the highly resistant individuals BC3-32, -33, -34 and –22 (Fig. 2).
The phenotypic plant characteristics indicate that due to the reduces amount of R-genome the hybrid plants appear already very rapeseed-like, show a good pod development with fully matured BC4-seeds in the cases of BC3-21 and BC3-32 (Fig. 3). As expected the different R-chromosomes do not have the same impact on the fertility of the BC-plants. As the BC3-individuals 32, 33 and 34 have both the same number of added R-chromosomes and the same degree of nematode resistance they still exhibit a completely different level of fertility.
In order to to continue with the backcross breeding program to achieve highly resistant translocation lines one has to decide whether it is necessary to apply artificial enhancement through irradiation or simply to expect natural homoeologous chromosome recombinations. With the result of the GISH analysis of the plant BC3-10 where a translocation accompanied by two R-chromosomes was detected it becomes obvious that natural recombination events between R- and A- or C-genome, respectively, do occur. As a result the strategy for the next generations is the consequent back-crossing with high performance rapeseed pollen donors of winter and spring type. A strict selection for resistance combined with cytological analyses applied to a large number of BCn-seeds provides a good perspective for finding highly resistant rapeseed translocation lines.
Figure 3: Comparison of BC3-pods after crossing (BC3-34, left) and open pollinated BC2-inflorescence, without insertion of pods (right)
Following this course, a number of 160 BC4-seeds is going to be tested for its level of nematode resistance and content of R-chromatin. We expect to find a resistant translocation line within the next generations because the number of harvested seeds is in a range of 4,000 so far and can be increased optionally.
The authors would like to thank Prof. J. Müller, BBA, Münster/Germany for providing nematode material and helpful hints and the Gemeinschaft zur Förderung der privaten deutschen Pflanzenzüchtung e.V. (GFP), Bonn/Germany, for financial support (FKZ: ÖE 103/96 HS).
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