1Wagga Wagga Agricultural Institute, PMB, Wagga Wagga, NSW 2650, Australia
2Research Institute for Bioresources, Okayama University, Kurashiki, 710-0046, Japan
Introduction
Molecular maps serve many purposes including marker-assisted selection, gene tagging and map based cloning of agronomically desirable traits. Different approaches based upon RAPDs (William et al 1990), DAFs (Caetano-Anolles et al 1991), microsatellites (Becker and Heun 1995, Becker et al 1995), RFLPs (Cho et al 1998) and AFLPs (Vos et al 1995) have been used to generate linkage maps in a number of crops including barley. Being a self-pollinated crop, barley shows less variation with RFLPs (Heun et al 1991) and hence there is a need to evaluate more markers to discover the level of allelic variation. Furthermore, the RFLP markers generally represent single and low copy sequences, due to the nature of southern analysis. The multiloci based PCR marker assays (microsatellites, RAFs, RGAs and AFLPs) are more efficient for DNA mapping and fingerprinting studies as compared to RFLPs. Inter-simple sequence repeat analysis (ISSR) involves the use of microsatellite sequences directly in PCR amplification (Gupta et al 1994). These repeats are widely dispersed in the genome, and have a higher average heterozygosity index than any single locus approaches (Roder et al 1995, Powell et al 1996, Milbourne et al 1997). Microsatellites are inherited in a codominant Mendelian manner (Morgante et al 1993 and Thomas et al 1993). Resistance gene analogs (RGAs) are also members of multigene families and reveal polymorphism among different genotypes upon PCR amplification (Chen et al 1998). RGA markers were also found to segregate in Mendelian ratios (Raman et al 1999). The amplified fragment length polymorphism (AFLP) approach has the ability to expedite the construction of saturated linkage maps as it detects high levels of DNA polymorphism in single lanes. AFLP maps are currently being made in a number of crops (Becker et al 1995, Pot et al 1996). In this investigation, a preliminary attempt was made to evaluate different techniques such as ISSR, RAF, RGA, AFLP and sequence tagged microsatellite analyses for their ability to reveal DNA polymorphism in a doubled haploid (DH) population from the cross of Harrington x Brindabella. This information will become the basis for map construction and tagging genes especially for aluminium tolerance, and resistances to stripe rust and leaf rust in barley.
Materials and Methods
DNA Extraction: DNA of Harrington, Brindabella and their DH population (100 plants) made by the bulbosum method (Chen and Hayes, 1989) was extracted by using a standared CTAB method. The DNA was further used for different experiments.
ISSR Analysis: SSR primers were procured from the University of British Columbia (UBC 801-900) and 69 anchored primers were screened to find polymorphism between Harrington and Brindabella. Only primers revealing polymorphism were used further for single plant analyses. PCR amplifications were performed in a 25-μl volume. The PCR conditions were as described by Gupta et al (1995). Electrophoresis at 90V for 2 hr, using a 1.5% agarose gel, was used to separate the PCR products.
RGA and RAF Fingerprinting: Different degenerated primers specific to Pto kin of tomato, RPS2 of Arabidopsis, L10 gene of wheat, and Xa21 of rice (Chen et al 1998) were used for DNA fingerprinting. PCR amplifications were performed by following conditions described by Raman et al 1999. The PCR products were separated on 5 per cent polyacrylamide gels containing 7.5M urea. The gels were stained with silver as described by the supplier (Promega). Autoradiography was performed by using automatic processor compatible films (Promega). RAF analysis was performed as described by Waldron et al (1998).
AFLP Analysis: This was performed as described by Vos et al (1995) by using the Large Genome System AFLP I (Gibco BRL, USA). The 3 μl of denatured PCR products were loaded on 5 per cent polyacrylamide gels (0.4 mm) containing 7.5 M urea. Electrophoresis was carried-out at 120 W at 50 0C for 2 hrs. Thereafter, the gels were transferred to Whatman 3mm filter papers, dried on a gel dryer (Bio Rad) and used further for autoradiography.
Microsatellite Analysis: Thirty primers described by Liu et al 1996 were synthesized by Research Genetics and used to study polymorphism among microsatellites in Harrington, Brindabella and their DH population with PCR amplifications conditions as used by Liu et al (1996). The PCR products were separated on either agarose or polyacrylamide gels.
Results
Among different 69 primers screened, only 4 ISSR loci were found to be polymorphic between Harrington and Brindabella. All the loci segregated into 1:1 ratios in DH population (Table 1), except primer (CT)8RC which showed significantly distorted segregation ratios.
Table 1. Segregation of Inter-Simple Sequence Repeats Among a Subset of the Double Haploid Population from Harrington x Brindabella
* Significant Segregation Ratios
In contrast to ISSR and microsatellites, a number of bands were amplified (~90) when degenerated primers of RGAs and random primers were used. All the loci showed the normal expected segregation ratios. Among the different techniques, AFLP revealed maximum polymorphism. The level of polymorphism for different primer combinations ranged from 2 to 14.3 per cent (Table 2) among DHs. Using the AFLP technique, a maximum of 13 loci (with a single primer combination) were found to be segregating. Most of the AFLP loci segregated into 1:1 ratio. However, one locus detected by E-AGG/M-CTT showed distorted segregation and had the tendency to segregate towards one parent.
Table 2. Segregating AFLP Loci in a Doubled Haploid Population from Harrington x Brindabella.
*χ2 Significant at p=0.005
Among different 20 microsatellites screened, only 4 microsatellite loci were found to be segregating with a mean of 2.3 alleles per microsatellite (Table 3).
Table 3. Segregation of Microsatellites Among Subset of the DH Population from Harrington x Brindabella.
The allelic polymorphism was higher in AFLP (a mean of 5/primer) as compared to ISSR (1 allele/primer) {Table 1}. It was observed that polyacrylamide gels resolve the microsatellites better than agarose gels. Similar observations have been made previously (Becker and Heun 1995).
Conclusions
The results indicated that multilocus based markers such as AFLPs, RGAs and RAFs detect high levels of polymorphism in barley and show Mendelian segregation, and hence are suitable for map construction. The level of polymorphism was highly dependent upon the nature of the primers and techniques used. So far, the alleles present at more than 150 loci have been determined for the DH population. Further attempts are being made to saturate the linkage map. Polymorphic microsatellites and STS primers are being used to ‘anchor’ the linkage group on specific chromosomes. This linkage map of Harrington x Brindabella will be used for QTL mapping of genes conferring resistance to leaf and stripe rust and tolerance to aluminium.
Acknowledgments
The authors thank Acid Soil Action and the Grains Research and Development Corporation, Australia, for financial support.
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