DNA amplification fingerprinting
molecular markers pathogen identification |
amplification methods
In essence, MAAP involves the use of a short, arbitrarily chosen oligonucleotide primer which, annealed to DNA, will direct DNA amplification of multiple genome regions (amplicons; Mullis, 1991). Temperature cycling and the use of a thermostable DNA polymerase are common components with the more specific and targeted PCR. In contrast to PCR, MAAP procedures use a single primer which is of arbitrary sequence. MAAP intentionally generates multiple products, which itself would be a rather undesirable result in a PCR reaction. MAAP is general, so that a primer used for one species can be used repeatedly for others, even if evolutionary distances between the template DNAs are large.
Amplification products are separated and recorded by a variety of detection methods; in all cases, a linear array of signals generates a profile, which is representative for the target DNA and specified by the DNA sequence of the primer. Variations in primer sites on the target DNA, length variations between primer sites, and possibly changes in the secondary structure of target DNA between or flanking the primer recognition sites, generate molecular polymorphisms. These amplification polymorphisms define molecular regions of the plant genome and thus can be used as (1) potential sequence tagged sites for positional cloning approaches, or (2) components of profile used in DNA profiling and diagnostics.
Three Techniques
MAAP procedures were developed independently, and apparently concurrently, in three laboratories. Welsh and McClelland (1990) developed AP-PCR, which uses PCR-length primers [18 to 32 nt] of arbitrary sequence to amplify target DNA under low stringency annealing conditions for two amplification cycles. This allows abundant mismatching and the generation of multiple amplification products (equivalent to a PCR reaction having gone wrong). Increased stringency of annealing at later amplification cycles generated reproducible products which were resolved on polyacrylamide gels and detected by autoradiography.
Williams et al (1990) invented the RAPD procedure, in which an arbitrary primer of either 9 or 10 nt produced amplification products after temperature cycling. RAPD products are routinely resolved on agarose gels and visualized by ethidium bromide. This provides a rapid method of scanning a genome. Alternative methods of detection, such as PAGE and silver-staining, coupled with careful optimization of amplification parameters (Collins and Symons, 1993) improved the utility of the approach. RAPD is widely used because of its simplicity and low-cost instrumentation.
Caetano-Anollés et al. (1991a,b) developed DNA Amplification Fingerprinting (DAF). Of all MAAP procedures, DAF utilizes the shortest primers, down to 5 nt in length. The optimal length was found to be 8 nt, a length which does not produce efficient amplification with RAPD. Informative amplification profiles were generated with 5 nt primers (5-mers), using soybean DNA as a template (Caetano-Anollés et al., 1993).
DAF products are routinely separated by thin polyacrylamide gels, backed onto plastic Gel-Bond film. This gel-plastic support, which provides support during the washing steps and helps preserve the original gel, is stained by an improved silverstaining method (Bassam et al., 1991; Caetano-Anollés and Gresshoff, 1994a), which detects DNA at about 1 pg mm-2. Resultant gels are air-dried and kept for permanent record and evaluation.
Pattern Detection
The PAGE/silver-staining technique provides a low-cost, high-throughput analytical method of DAF products. DAF products were also resolved by alternative methods. Agarose gels give clear resolution, but fewer products (Prabhu and Gresshoff, 1994). Fluorochrome labeled octamer primers were generated which then directed amplification of plant DNA (Caetano- Anollés et al., 1992a). The resultant amplification products were separated on an ABI Sequencer using Gene Scanner software. Single nucleotide resolution was obtained for lower sized amplification products. Tests using capillary electrophoresis have been promising (Dr. Patrick Williams, DNA Testing Laboratory, AFIP, Gaithersburg, MD; personal communication), providing separation of single samples in 30 minutes. In general, DAF generates scoreable polymorphisms in the molecular size range from 100 to 800 bp. Recently, we have used the pre-cast and automated PhastGel system (Pharmacia Inc.) to obtain profiles for pathogenic nematodes on soybean (Baum et al., 1994). Bands at higher molecular weight (up to 1500 bp) were scoreable; species and race-specific polymorphisms were detected. Denaturing gradient gel electrophoresis (DGGE) is another method which would help to distinguish polymorphic products of wheat (He et al., 1992).
Genetic Uses of DAF
The ability to detect molecular markers closely associated with genes of agricultural importance makes marker-based breeding an attractive proposition. The need for maintaining large plant populations through advanced breeding cycles can be reduced by detecting heterozygotes. MAAP markers converted through cloning, partial sequence analysis and specific PCR primer synthesis may provide SCARs (sequence characterized amplified regions), which are diagnostic for either a gene region in a plant or a pathogen. Figure 2 cartoons the utility of RFLPs and MAAP markers in generating diagnostic tools. For example, it may be possible to find markers specific for a soybean nematode race (see Baum et al., 1994), to convert it to a SCAR, then use a diagnostic, proactive test on agricultural soil to predict which nematode race is predominant in the field prior to planting.
Figure 2: RFLP and MAAP markers used as diagnostic tools for genome
analysis. Partial sequencing of clones, which are linked to your favorite
gene (yfg!) provides information for specific PCR primers, which in turn
generate a diagnostic product. A sequence characterized amplified region
(SCAR) was demonstrated for the supernodulation gene of soybean by
Kolchinsky et. al. (1994)
The ability to generate many amplification products means that DAF is very efficient in scanning the genome of an organism for variable sites. In a survey of 25 primers (all octamers), Prabhu and Gresshoff (1994), working with G. max and G. soja, detected an average of 1.5 AFLPs per primer. Interestingly, RAPD gels of soybean produce an average of 5 to 7 scoreable bands, while DAF in soybean produced an average of 20 to 25 bands. Accordingly, the ratio of scored polymorphism to scoreable band is nearly the same, that DAF is not picking up more AFLPs because of the shorter primer length, but because of the detection method.
DAF markers were shown to be repeatable polymorphisms in different DNA isolations, operators, time periods, and amplifications. They are heritable, as are about 75% of AFLPs between G. max and G. soja segregated as dominant Mendelian markers in F2 populations (Prabhu and Gresshoff, 1994; Caetano-Anollés et al., 1993). Interestingly, the other 25% segregated in a uniparental way, being either maternal or paternal. Maternal inheritance presumably stems from amplification of cytoplasmic replicons. As yet, paternal replication is unexplained, and may represent either highly repeated chromosomal replicons or possibly alterations from normal cytoplasmic inheritance patterns in soybean.
Recombinant Inbred Lines
Several DAF polymorphisms were mapped in recombinant inbred lines of soybean (Prabhu and Gresshoff, 1994). The use of inbred lines is very convenient for DAF, as the lines are predominantly homozygous. Since DAF markers are dominant, it is impossible to distinguish the dominant homozygote from the heterozygote. Accordingly, in normal F2 populations, larger sample numbers are required to obtain data equivalent to data obtained from the analysis of a codominant (e.g., RFLP) marker. In recombinant inbreds, however, DAF and RFLP markers share the same statistical advantages. Figure 3 provides a summary of some RIL mapping data (conducted in collaboration with Dr. Gordon Lark, Utah).
The large number of products allows a high-density genotyping and genotype differentiation (Gresshoff, 1992). This form of fingerprinting is similar to the Universal Product Code, in which bars and spaces define a product. Reliable exclusion is obtained when one or more bands differ between samples. Inclusion is more difficult, as many primers need to be tested, frequency of variation within the sampled species needs to be known, and careful statistical statements need to be generated. One cannot declare with 100% certainty that two things are the same; the statement must always be probabilistic. It is up to the user (society, courts, scientists) to concur on acceptable levels of confidence for such probabilities.
Fingerprint Applications
DAF allowed the easy distinction of variant turfgrass material in commercial plots (Callahan et al., 1993). For example, foundation stock from several geographic locations gave identical profiles for Bermudagrass Tifway 419, while samples analyzed from golf course owners repeatedly showed major variation. The application of DNA tests to the turfgrass industry is a major challenge in an area of repeated vegetative propagation, triploidy and genetic instability. Using DAF markers, Weaver (1994) developed a phylogenetic tree of centipedegrasses.
Sunflower material provided by a seed company was categorized into several groups. Some common bands permitted the suggestion of a possible pedigree. This type of analysis has utility for product verification and plant variety rights.
The determination of genetic identity is also essential for the determination of plant product quality, as many food manufacturers use processes directly optimized for a specific biological feedstock. This industry relies on biological material; it is essential that quality biological feedstock enters the manufacturing process. Often it is impossible to inspect the source plant as one looks at a harvested product. It is for these industrial and related horticultural applications that a new technology was needed. DNA analysis has provided an additional way by which closely related organisms are distinguished for industrial., manufacturing, and retailing purposes.
DAF markers are useful in defining closely linked regions in bulked segregant analysis (Michelmore et al., 1991). The availability of large primer sets and the generation of multiple amplification products result in the efficient screening of the genome.
Induced plant mutations have the advantage of being in near-isogenic background as the genetic difference between parent and mutant is minimal. Using 25 DAF primers, Caetano- Anollés et al. (1993) showed that the induced supernodulation mutant nts382 and its wild- type parent cv. "Bragg" did not show polymorphisms despite the pairwise comparison of nearly 500 amplification products. Only in the use of MAAP, in which the target DNA was predigested with two restriction nucleases (four base cutters) and then amplified with a single octamer, could polymorphisms be detected between mutant and wild-type parent. Only 19 primers were needed to reveal 42 AFLPs. Fourteen of these segregated at 100% with the supernodulation phenotype in G. soja (wild-type) and G. max (mutant ) derived F2 populations. Some AFLPs distinguished between the nts382 and nts1007 alleles. It is likely that these are valuable markers close to the nts locus and their cloning and further characterization will facilitate the isolation and ordering of yeast artificial chromosomes (YACs) in that region.
Mini-hairpin Primers
Funke and Kolchinsky (1994) demonstrated that stable YACs carrying soybean genomic DNA can be constructed, with an average size of about 200 kb (maximum 900 kb). About 7% represented chloroplastic DNA. The combination of clustered molecular markers, the ability to generate medium-sized YAC clones, end-clones and possible contigs, increase the chances of isolating soybean regions carrying developmentally significant genes. Caetano-Anollés and Gresshoff (1994b) used mini-hairpin primers in a DAF reaction to profile such soybean YACs. The mini-hairpin primers are interesting, because they contain on their 5 end a 7 nucleotide fold-back loop (4 nt in stem, 3 nt in the loop). The 3 end can be as short as 3 nt, allowing the generation of a small set of 64 primers, which are useful for the characterization especially of small genomes or genome components such as plasmids or YACs.
These findings show that single primer DNA amplification analysis of plant genomes adds a further genetic tool to construct high-density maps needed for positional cloning and marker-based breeding approaches.
DAF Parameters
Primer length: 5 nt minimum; 8 nt optimum Primer 3 end most important for specificity of reaction Primer concentration: 3æM for 8-mer primer and up to 30æM for 5-mer primer 2mM MgCl2 optimum for soybean genome and 6mM optimum for bacterial genome Taq polymerase produces good amplification results for large fragments Truncated Stoffel fragment should be used to amplify fragments in the 50-200 bp range
Excess template DNA (>25 ng/25 æl reaction) reduces intrinsic amplification products
For a more complete discussion of these parameters please gopher to: gopher.nalusda.gov. Select Information Centers from the menu. Next select Plant Genome Data and Information Center. If you would like a hard copy of the paper please contact the Plant Genome Data and Information Center at the address on page.
Abbreviations: PCR=polymerase chain reaction; DAF=DNA amplification fingerprinting; AP- PCR=arbitrary primer-PCR; MAAP=multiple arbitrary amplicon profiling; nt=nucleotide; bp=base pair; PAGE=polyacrylamide gel electrophoresis; RAPD=random amplified polymorphic DNA; RFLP=restriction fragment length polymorphism
References:
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