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Marker Types






Mapping can be conducted with a variety of marker types (Table 12.2). Two general classes can be identifi ed: markers that gen- erally are expressed in a co-dominant fash- ion so that the heterozygote is distinguishable from both homozygotes, and dominant markers in which the het- erozygote is identical to one homozygote. Isozymes, RFLPs (restriction fragment length polymorphisms), CAPS (cleaved amplified polymorphisms) and SSRs (simple sequence repeats) are usually co- dominant markers, while many polymerase chain reaction (PCR)-based markers such as RAPDs (random amplified polymorphic DNAs), ISSRs (inter-simple sequence repeats) and AFLPs (amplifi ed fragment


 

 

 
 

Table 12.2. Characteristics of some common types of DNA markers.

AFLP Amplifi ed fragment length polymorphism – PCR based, gel analysis, multilocus, sequence anonymous

CAPS Cleaved amplifi ed polymorphic sequence – PCR followed by restriction enzyme digestion, gel analysis, single locus per gel, sequence known, can target genes

ISSR Inter-simple sequence repeat – PCR based, gel analysis, multilocus, sequence anonymous

RAPD Random amplifi ed polymorphic DNA – PCR based, gel analysis, single to a few loci per gel, sequence anonymous

RFLP Restriction fragment length polymorphism – restriction enzyme and gel based, single to a few loci per gel, can target genes

SNP Single nucleotide polymorphism – PCR based, gel, plate or chip analysis; single locus, but multiplexing possible, sequence known, can target genes

STS Sequence-tagged site – PCR based, gel analysis, single locus, sequence known SSR (STMS, STR) Simple sequence repeat – PCR based, gel analysis, single locus, sequence

 
 

known, can target some genes

The method of detection of the DNA sequence (e.g. PCR) is listed, the method for distinguishing alleles (e.g. gel analysis), whether the method generally detects one or several to many marker loci in a single analysis, whether the DNA sequences detected are known or anonymous, and whether the method can target specifi c genes.


 

 

Fig. 12.1. Relationship between DNA sequence differences between two alleles and banding patterns observed for fi ve types of gel-based marker systems.

 


 

length polymorphisms) are dominant. For a cross in which most markers are expected to have 1: 1 segregation, dominance does not affect mapping. However, for an F2-type population, or one in which a variety of seg- regation types occurs (as is usual in citrus), co-dominant markers are more informative, meaning that, for a given population size, the accuracy of a linkage estimate from co- dominant markers is greater than that for dominant markers. Figure 12.1 illustrates the relationship between DNA sequence variation and banding patterns for several gel-based marker systems. Note how domi- nant markers arise when sequence variation in the PCR primer-binding site prevents amplification of one allele. Figure 12.2 shows examples of each marker system in citrus.

A second classifi cation of markers is based on the number of loci detected per analysis. Maps can be developed much more rapidly and inexpensively using high-


 

throughput markers where several to many markers can be detected in a single analysis. This is often called multiplexing. Marker systems in this class include AFLPs, ISSRs and RAPDs, all PCR-based techniques in which anonymous DNA sequences are analysed. In contrast, RFLPs, CAPS and SSR markers are based on detection of known DNA sequences through hybridization or PCR amplifi cation. These markers are typi- cally analysed ‘one at a time’.

An advantage of the sequence-based markers such as RFLPs and CAPS is that the primers or probes can be designed to target genes, while most anonymous DNA sequences are not likely to be genes. Mapping genes allows comparison of maps with those of other plant species, a process known as synteny analysis. Such markers are also relatively useful for quantitative trait locus (QTL) analysis because markers may themselves be candidate genes to con- trol the trait (see below).


 

 

Fig. 12.2. Examples of marker variation for fi ve types of DNA marker systems. The RFLP and RAPD mark- ers were separated on agarose gels. The SSR and AFLP patterns were analysed on an automated DNA analyser. The ISSRs were analysed on a silver-stained polyacrylamide gel and show patterns in seedlings from different source trees, not a single segregating population.

 

 


At present, an efficient mapping approach is to use a moderate number (100) of co-dominant markers such as SSRs as anchors for the map, and a larger number

(200) of AFLP or other high-throughput markers to increase marker density. SSRs are particularly informative because they have many alleles and are heterozygous in many individuals. These characteristics allow them to be used to combine and com- pare maps developed in different popula- tions. CAPS or single nucleotide polymorphism (SNP) markers could also be used for this purpose if they are suffi ciently polymorphic.

On the horizon, but not yet developed in citrus, are very high-throughput map- ping methods that involve parallel analysis of many markers (e.g. SNPs) per individual, or many individuals per marker. These approaches are being developed in humans, and in model organisms such as Arabidopsis and rice. These methods could revolutionize citrus mapping by facilitating analysis of much larger populations and/or development of much denser maps.

 

 


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