1School of Agriculture, Charles Sturt University, Wagga Wagga, NSW 2650, 2NSW Agriculture, Agricultural Institute, Wagga Wagga, NSW 2650, Australia
Molecular maps are very important to 'tag' the genes for agronomic importance and to perform map based cloning of target genes. The level and nature of DNA polymorphism are basically required to construct the linkage maps. The polymorphism among inter simple sequence repeats and amplified fragment lengths of DNA (AFLP) was analysed among a DH population of BLN 692 (resistant to the blackleg disease) X YN90-1016 (susceptible to the blackleg). Thirty combinations of EcoR1/Mse1 primers and twenty polymorphic inter-simple sequence repeats (di-, tri-, tetra-nucleotide and complex) were used to study polymorphism among DH plants. So far, more than 120 segregating loci were identified. Most of the ISSR and AFLP loci segregated into Mendelian ratios. However, some of them had a tendency to segregate towards one parent. Attempts are being made to find more polymorphic loci, which will be used to generate a linkage map of 'Australian canola.'
KEYWORDS: Polymorphism, Molecular mapping, Microsatellite, Inter simple sequence repeats, AFLP analysis, and PCR
During the last decade, a number of molecular maps have been made in number of crop plants. Linkage maps are very important to 'tag' the genes for agronomic importance, and to perform marker assisted selection and map based cloning of target genes. Linkage maps of canola have been constructed using isozymes, RAPDs, RFLPs (Ferreira et al 1994, Foisset et al 1996) and to a limited extent using microsatellites (Kresovich et al 1995). No such maps have been made in Australia especially for QTL analyses of complex traits like yield, oil content and quality, where a large G X E component is involved. Different approaches have been used to generate linkage maps including RAPD analysis (William et al 1990), DAF (Caetano-Anolles et al 1991) microsatellite analysis, RFLP and AFLP analysis (Cho et al 1998). PCR based assays are more efficient as compared to RFLP analysis. Moreover, the RFLP markers generally represent single and low copy sequences. SSRs are widespread in the genome and the average heterozygosity index of these markers has been reported to be higher than any other single locus approach (Roder et al 1995. Powell et al 1996, Milbourne et al 1997). AFLP approach has the ability to expedite the construction of a saturated linkage map as it detects a high level of polymorphisms in DNA in a single lane. The segregating loci mostly map on the independent locations (Chi, 1998). AFLP maps are currently being made in a number of crops (Becker et al 1995, Pot et al 1996). Herein we report the polymorphism using simple sequence repeats and AFLPs in a doubled haploid population aiming to generate a linkage map of Australian canola.
MATERIALS AND METHODS
Plant Materials: The parents; BLN 692 (Resistant to the blackleg) and YN90-1061 (Susceptible to the blackleg) and their DH population (61 plants) were used to study DNA polymorphism.
DNA Extraction: About 250 to 300 mg of leaves were collected from 2 wk-old glasshouse raised seedlings and were used for DNA extraction in 2 ml eppendorf tubes. The frozen tubes containing tissues were ground into powder with mini pestle and mortar (Eppendorf) and the DNA extraction was carried-out as described by Guidet et al (1991). The homogenates were extracted twice with phenol : chloroform : iso-amyl alcohol (25: 24: 1). The supernatants were precipitated with sodium acetate and iso-propanol. The DNA pellet was washed with 70 per cent ethanol and air-dried. The DNA was dissolved into 30 μl of TE buffer.
PCR Amplification of ISSRs: PCR amplifications were performed in a 20 μl volume. Each PCR reaction contained 0.2 μM each primer, 1 unit of Taq polymerase (Promega), 200 μM of each dNTP (Promega), 1.5 mM of MgCl2, 50 nanogram of template DNA, and 1 x reaction buffer A (Promega). Amplifications were carried-out in heated lid OMN E thermocycler programmed at 940C for 5 min for initial denaturation, followed by 940C for 1 min, 450C for 2 min and 72 0C for 2 min for 45 cycles followed by a final step of extension at 720C for 5 min. The annealing temperature of individual primer was calculated as 5 0C less than the melting temperature of each oligo. SSR primers were synthesized from University of British Columbia (UBC 801-900), Canada and 50 primers were screened to find-out polymorphism among parents. Only primers revealing polymorphism were used for single plant analysis.
Electrophoresis: The PCR products were electrophoresed using 1.5 per cent agarose gels. The agarose gels were run at 90 V for 2 hr and then stained with ethidium bromide (0.5 μg/ml) and photographed under UV transilluminator.
AFLP ANALYSIS: AFLP analysis was performed as described by Vos et al (1995) using Large Genome System AFLP I (Gibco BRL). About 250 nanogram of DNA was digested with EcoR1 and Mse1 enzymes and digested fragments were ligated with adaptors. The ligated DNA fragments were amplified in 20 μl volume by using 32P labelled different E- and unlabelled M- primers. The amplified products were equally mixed with formamide loading buffer (98% formamide, 10 mM EDTA pH 8.0, 0.5% bromophenol blue and 0.5% xylene cyanol and were denatured at 900C for 3 min. Electrophoresis was carried out at 120 W at 50 0C for 2 hrs using 5 per cent polyacrylamide gels (0.4 mm). containing 7.5 M urea. The denatured PCR products (3 μl) were loaded on the gels. After electrophoresis, the gels were transferred on Whatman 3mm filter papers and, after drying on gel dryer (Bio Rad), were used further for autoradiography.
All the 50 SSRs primers and 64 combinations of E-/M- primers were screened for polymorphism against the two parents (BLN 692 and YN90-1016). Most of the SSR primers amplified the homologous sequences from genomic DNA of canola. A number of polymorphic di-nucleotide repeats were found in canola and these segregated in the DH population (Table1).Tri-,tetra-, and complex nucleotide repeats such as (AGC)6, (GATA)4, were also present. Besides the nature of repeat, 'anchor' on 3' end also influenced the level of polymorphism (Table 1). Using different simple sequence repeats, only a few loci (1-3) segregated in the DH population . However, using different AFLP primers more loci (2-11) were found to be segregating (Table 1). Like SSR primers, the level of polymorphism in AFLPs was also influenced by the nature of primers. Most of the ISSRs and AFLP loci segregated in Mendelian fashion. In a doubled haploid population, a 1:1 Mendelian segregation ratio was expected
Table 1: Segregation of ISSR and AFLP Loci in Doubled Haploid Population of BLN 692 x YN90-1016
*SSR Segregating *AFLP Segregating
Primer loci (No.) Primer loci (No.)
(AG)8T 3 E-AGG/M-CTA 5
(GA) 8T 2 E-AGC/M-CAT 4
(GA) 8AC 3 E-AGC/M-CTC 6
(GA) 8AA 1 E-ACT/M-CAC 9
(CT) 8T 1 E-ACT/M-CTA 8
(CT) 8A 1 E-ACC/M-CAC 2
(CA) 8A 2 E-AAC/M-CAT 9
(GT) 8T 1 E-AAG/M-CAG 11
(TC) 8A 1 E-ACA/M-CAC 5
(AC) 8T 2 E-ACT/M-CAA 6
(TG) 8A 1
(AG) 8YC 2
(GA) 8YG 2
Others 5 25
where there was not any selection. However, some of loci showed distorted segregation ratios. These loci have shown preferential segregation toward one of the parents. The polymorphic loci will be used to develop a linkage map of canola, which will be further used to identify QTLs involved in resistance against blackleg disease in canola.
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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