1School of Agriculture, Charles Sturt University, Wagga Wagga, NSW 2650, 2NSW Agriculture, Agricultural Institute, Wagga Wagga, NSW 2650, Australia
The genes conferring disease resistance in plants exhibit significant homology in their structure. The degenerated primers specific to the conserved region of the resistance genes i.e, N in tobacco, RPS 2 in Arabidopsis, Pto in tomato, and L10 and Cre 3 in wheat, and Xa21 in rice, were used to amplify the homologous sequences from 18 genotypes of canola (Brassica napus L.). A number of resistance gene analogs (RGAs) were amplified in the different genotypes of canola. The denaturing gels (4%) revealed the highest level of polymorphism, followed by non-denaturing polyacrylamide gels. The silver staining based detection of RGAs was found to be efficient and safer to use as compared to the radioactive nucleotide based assays. The resistance gene analogs also segregated as single loci in the DH population (BLN 692 x YN90-1016) developed to ‘tag’ the genes for blackleg resistance. Besides, a few loci also showed distorted segregations. Association of these RGAs with genes conferring blackleg resistance is being investigated.
KEYWORDS: Disease resistance, Genotypes, Molecular diversity, Silver staining, Segregation
The sequence comparison of cloned genes for disease resistance has shown structural homology among themselves irrespective of fungal, bacterial or viral resistance. Different approaches have been used to study structural homology of resistance genes. RFLP analysis has been reported to be inefficient to detect cross hybridization. However, the conserved sequences have been used to design degenerated primers to amplify the similar sequences in other plants (Kanazin et al 1996, Leister et al 1996, Yu et al 1996). Using this approach, a number of RGAs have been mapped very close to the genes for resistance to nematodes (Seah et al 1998), late blight of potato (Leister et al 1996), several diseases of soyabean (Kanazin et al 1996), potyviruses of soyabean, Phytophthora root rot, powdery mildew (Yu et al 1996) and wheat leaf rusts (Feuillet et al 1997). In Brassica napus, degenerated primers based on the sequence of the P-loop and an internal hydrophobic region were used to amplify RGAs from Quantum, a cultivar resistant to blackleg disease in canada. The deduced amino acid sequence of the translated polypeptides has shown 15% homology to the NBS region of RPM1 and IC2-2 and 30 % to NBS region of RPS2 (Deng et al 1998). The cloning of ‘single’ amplified fragment of RGAs from the agarose gels often resulted in heterogeneity of the clones (Deng et al 1998, Ohmori et al 1998, Seah et al 1998). These clones could be better resolved on high-resolution gel electrophoresis, which increases the efficiency of detecting putative resistance genes of cereals (Chen et al 1998). Here we report the comparative analysis of resistance gene analogs in canola (Brassica napus L.) using agarose, polyacrylamide and denaturing gel electrophoresis and used that information to study molecular diversity for disease resistance genes in canola and inheritance of RGAs.
MATERIALS AND METHODS
Plant Materials: Eighteen cultivars of canola Oscar, Capitol, Dunkeld, Charlton, Columbus, Quantum, Shiralee, Maluka, Norin 20, Rainbow, Barossa, BLN 692, YN90-1016, Narendra, Pactol, Hyola 42, Karoo and Siren and a DH population (61 plants) of BLN 692 (resistant to the blackleg) x YN90-1016 (susceptible to the blackleg) were used in this investigation.
DNA Extraction: DNA extraction was carried-out as described earlier (Raman et al 1999).
PCR Amplification: PCR amplifications were performed in a 25 μl volume. Each PCR reaction contained 4 μM of each primer, 1 unit of Taq polymerase (Promega), 200 μM of each dNTP (Promega), 3-5 mM of MgCl2, 50 nanogram of template DNA, 1 x reaction buffer A (Promega). Amplifications were carried-out in heated lid OMN E thermocycler. The oligos (S1/AS1, S2/AS2. S2/AS3, XLRR-F/R, RLK-F/R, Pto Kinase 1/-2 and Kinase 2a/rpEGF) were used to amplify homologous sequences as described (Feuillet et al 1996, Leister et al 1996, Yu et al 1996, Ohmori et al 1998, Seah et al 1998).
Electrophoresis and Autoradiography: To compare the precise resolution of RGAs, the PCR products were electrophoresed using 1.0 per cent agarose gels, 5 per cent polyacrylamide gels, and denaturing 4 per cent polyacrymaide gels containing 7.5 M urea. Electrophoresis of denaturing gels and autoradiography was performed as described (Raman et al 1999).
Silver Staining of Denaturing Polyacrylamide Gels: The silver staining and subsequently autoradiography was performed according to the instructions described by the manufacturer (Promega) using APC films.
All the 7 degenerated primer combinations amplified PCR products from the genomic DNA of different genotypes of canola. Using S1/AS1 primers, only a few bands (one clear band of ~ 500 bp and a few unclear bands) were detected in the agarose gels. Similar results were found in tomato using these primers (Ohmori et al 1998). However using denaturing gels, about 4 clear RGAs were detected. The same number of cloned RGAs have been reported upon cloning of the ‘single’ bands recovered from agarose gels (Ohmori et al 1998). The PCR products amplified using S1/AS1 primers could not be differentiated from each other, irrespective of the genotypes. However, the PCR products digested with Hpa II revealed heterogeneity among RGAs. No such variability was found when PCR products were digested with Rsa 1, Hae II, Taq 1, Hha 1 and Hinf 1. Denaturing gels revealed the highest level of polymorphism with all the primers used. Using XLRR-F/R primers, only 6 RGAs ranging from 350 bp to 1.0 Kb were detected in agarose gel as compared to 15 in non-denaturing polyacrylamide and 45 in denaturing polyacrylamide gels. These results indicated that PCR products were heterogeneous, amplified from multiple gene families that could not be resolved very well by conventional electrophoresis.
Among different primers, the maximum polymorphism was found with S2/AS3 primers specific to RPS2 and N gene in Arabidopsis. Among 18 genotypes resistant and susceptible to the blackleg caused by Leptosphaeria maculans, RGAs ranged from 45 to 95 using different primers. The level of polymorphism remained the same irrespective of method of the detection such as autoradiography using 32p or silver staining. However, the bands were sharp in silver stained gels. Using three primers, a total of 331 polymorphic bands were amplified. The maximum polymorphic bands (127) were observed by using S2/AS3 primers followed by 89 bands with RLK-F/R (Table 1).
Table 1: Segregation Patterns of different RGAs in doubled haploid population of BLN 692 and YN90-1016
Primer Band Scored bands Segregation of bands
Combination ---------- ----------------------------------------------------------------------
(No.) BLN 692 YN90-1016 DHs (BLN 692/YN90-1016)
S2/ AS3 a. 127 - + 19/26
b. + - 25/20
c. + - 12/33*
d. - + 23/22
e. + - 30/15*
f. + - 23/22
g. + - 26/19
h. - + 31/14*
RLK-F/R a. 89 + - 22/21
b. + - 17/28
c. + - 16/29
d. - + 21/24
S2/AS2 a. 95 + - 24/16
b. - + 26/19
c. + - 23/22
d. - + 36/9*
e. + - 13/32*
+: Present, -: Absent, *: Distortions
Among 331 bands, only 17 polymorphic bands were found to be segregated in the DH population of BLN 692 X YN90-1016 (Fig 1).
1 2 3 4 5 6 P1 P2
Fig 1.: Segregation of Polymorphic bands among doubled haploid population of BLN 692 and YN90-1016. P1: BLN 692, P2: YN90-1016, 1-6: DHs of BLN 692/YN90-1016, Primers: S2/AS3
About 70 per cent of them segregated as single loci in the DH population (Table 1). A few loci showed distorted segregation which may be due to the heterozygous nature of the parents, a small size of the population screened and preferential segregation towards one of the parents. Results of the present investigation have shown that canola has motifs of leucine rich repeats, nucleotide binding sites and protein kinases similar to RPS2, N gene, CCN resistance locus of wheat, and Xa21 gene of rice. The mapping of these RGAs and their association with other known resistance genes of canola is underway.
The authors are thankful to the Grains Research and Development Corporation, Australia for providing financial support in the project “Use of Molecular Markers To Enhance The Efficiency Of Oilseed Brassica”.
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