Functional diversity markers

The availability of sequence data of expressed DNA has enabled the development of markers that are physically associated with coding regions of the genome. Expressed Sequence Tags (ESTs) are the result of sequencing complementary (cDNA) clones and the information generated is generally stored in databases. These sequences can then be used for designing primers to readily generate polymorphic markers. The raw sequence information will also aid in screening for the occurrence of microsatellite sequences (EST-SSR) or single nucleotide polymorphisms (EST-SNP), after which markers can be developed that are targeted to transcribed regions of the genome. Complementary DNA can also be used as template for subsequent direct marker generation, for example through AFLP technology (cDNA-AFLP). cDNA-AFLP is commonly used in the identification of genetic polymorphisms between contrasting phenotypes under controlled conditions in order to facilitate the construction of linkage maps, or to identify candidate genes. In diversity studies, the application of cDNA-AFLP should be limited to the identification and comparison of specific gene-related patterns, as in general cDNA differences may be caused by differences in the developmental stage of plants and the environmental conditions rather than by existing DNA polymorphisms. A simple PCR-based marker technique that targets coding sequences in the genome is Sequence-Related Amplified Polymorphism (SRAP). SRAP uses forward primers consisting of an unspecific filler sequence of ten bases, the sequence CCGG and three selective nucleotides. Reverse primers also contain a filler sequence, but are followed by the sequence AATT and three selective nucleotides. The CCGG sequence is used to target GC-rich regions, such as exons in open reading frames, while the AATT sequence on the reverse primers is aimed at AT-rich regions, such as promotors and introns. The generally conserved nature of exon sequences, combined with the generally variable nature of introns, promotors and spacers, enables SRAP analysis to generate polymorphic bands. The use of selective nucleotides on the PCR primers results in the amplification of subsets of open reading frames that display multilocus band profiles following appropriate labelling of a primer, polyacrylamide gel electrophoresis and autoradiography. A related technique that uses EST sequence information is Target Region Amplification Polymorphism (TRAP). For TRAP analysis, a fixed primer designed from a targeted EST sequence is combined with an arbitrary primer having an AT- or CG-rich core sequence. For different plant species TRAP revealed multiple scorable fragments, and the technique may be well suited for determining the genotypes of germplasm and tagging genes for traits of interest. In case gene sequence data are available, markers can be developed for particular (groups of) genes. This is the case, for example, in a recently developed strategy for analyzing groups of resistance genes - Resistance Gene Homologue Polymorphism (RGHP). With this methodology, groups of resistance genes are targeted by PCR using primers aimed at conserved domains of resistance genes, such as the Leucine Rich Repeat (LRR) or the Nucleotide Binding Site (NBS), both involved in resistance mechanisms. In NBS-directed profiling (NBS-DP), one primer is targeted to a conserved sequence of the NBS, while the other primer is based on the presence of a nearby endonuclease restriction site. The highly conserved nature of these targets allows the NBS-DP primers to be used beyond the species level. Because resistance genes have often originated from gene duplications, variation may also be traced in analogs of the gene. Polyacrylamide gel electrophoresis and autoradiography are part of the NBS-DP technique, resulting in AFLP-like banding profiles that are scored as dominant markers. The appealing feature of NBS-DP is that it may also detect variation in resistance genes that were thus far unknown.

Suggested reading

Linden, C.G. van der, M.J.M. Smulders and B. Vosman, 2005. Motif-directed profiling: a glance at molecular evolution. In: Bakker, F.T., L.W. Chatrou, B. Gravendeel and P.B. Pelser (eds), Plant Species-Level Systematics: New Perspectives on Pattern and Process. Regnum Vegetabile 143. ARG Gantner Verlag, Ruggell, Liechtenstein; Koeltz, Königstein, Germany, pp. 291-303.

Linden, C.G. van der, D.C.A.E. Wouters, V. Mihalka, E.Z. Kochieva, M.J.M. Smulders and B. Vosman, 2004. Efficient targeting of plant disease resistance loci using NBS profiling. Theoretical and Applied Genetics, 109: 384-393.
Hu, J. and B.A. Vick, 2003. Target region amplification polymorphism: a novel marker technique for plant genotyping. Plant Molecular Biology Reporter 21: 289-294.

Thiel, T., W. Michalek, R.K. Varshney and A. Graner A. 2003. Exploiting EST databases for the development and characterization of gene-derived SSR-markers in barley (Hordeum vulgare L.). Theoretical and Applied Genetics, 106: 411-422.

Brugmans, B., A. Fernandez del Carmen, C.W.B. Bachem, H. van Os, H.J. van Eck and R.G.F. Visser, 2002. A novel method for the construction of genome wide transcriptome maps. Plant Journal, 31: 211-222.

Rafalski, J.A., 2002. Novel genetic mapping tools in plants: SNPs and LD-based approaches. Plant Science, 162: 329-333.

Li, G. and C.F. Quiros, 2001. Sequence-related amplified polymorphism (SRAP), a new marker system based on a simple PCR reaction: its application to mapping and gene tagging in Brassica. Theoretical and Applied Genetics, 103: 455-461.