Dear Dr Mohamed,

Both AFLP and SSR PCR DNA markers.

Amplified fragment length polymorphisms (AFLPs)

AFLP is a PCR based DNA fingerprint technique (Vos et al., 1995; Zabeau and Vos,

1993). It involves restriction of genomic DNA followed by ligation of adaptors to restricted fragments and pre-selective and selective PCR amplification of a subset of these fragments. The amplified fragments are resolved on a sequencing gel and visualized either by autoradiography or fluorescent sequencing equipment (Meksem et al., 1995; Zhang et al., 1999; Schwarz et al., 1999; Huang et al., 2000b; Huang and Sun 1999; Hartl and Seefelder, 1998), depending on the method of labelling or silver staining (Cho et al., 1996). In contrast to RAPD, AFLP is highly reproducible and also transferable between different populations (Jones et al., 1997; Yin et al., 1999; Waugh et al., 1997; Li et al., 1998; Rouppe van der Voort et al., 1997). One major limitation of AFLPs is the dominant nature and the difficulty in identifying allelic variants at a specific locus although co-dominant AFLP markers have been found, however, in frequencies of 4-15 % among all polymorphic AFLP markers (Waugh et al., 1997; Lu et al., 1998; Boivin et al., 1999).

Simple sequence repeats (SSRs) or microsatellite

DNA sequences with di-, tri-, tetra- or penta-nucleotide tandem repeats are described

either as microsatellites (Litt and Luty, 1989), as simple sequence repeats (SSRs; Hearne et al., 1992) or as short tandem repeats (STRs; Edwards et al., 1991). The first report of microsatellites in plants was made by Condit and Hubbell (1991). These markers appear to be hyper-variable, in addition to which their co-dominance and reproducibility make them ideal for genome mapping, as well as for population genetic studies (Dayanandan et al., 1998). Inter-SSRs are a variant of the RAPD technique, although the higher annealing temperatures probably mean that they are more rigorous than RAPDs. They are present in the vast majority of eukaryotic genomes. The total number of different dinucleotide blocks has been estimated for several species (Ma et al., 1996; Wu and Tanksley, 1993; Morgante and Olivieri, 1993). The number of sites ranged from 103 to 105 depending on the species and repeat motif. Polymorphism produced by a variable number of tandem repeats has been demonstrated in a large number of species. This feature has made microsatellites a very attractive molecular marker for species with a narrow genetic base such as wheat and barley. This methodology is based on the use of primers complementary to SSRs. Multilocus profiles have been generated using different kinds of oligonucleotide containing simple sequence repeats as single primer (Gupta et al., 1994; Nagaoka and Ogihara, 1997) or in combination with arbitrary sequence oligonucleotides (Wu et al., 1994). These studies have shown the reproducibility of the patterns generated the Mendelian inheritance of the polymorphic amplified bands and their usefulness in the investigation of the genetic relationships. Mapping of this kind of markers in barley (Becker and Heun, 1995) and rapeseed (Kresovich et al., 1995; Szewe-MeFadden et

al., 1996; Uzunova and Ecke, 1999; Plieske and Struss, 2001) have been conducted. In wheat, Devos et al. (1995) searched sequence database and converted two microsatellite sequences into PCR based markers. Röder et al. (1995), Ma et al. (1996) and Plaschke et al. (1995) investigated the potential of microsatellite sequences as genetic markers in hexaploid wheat.

AFLP relies on restriction sites and arbitrary nucleotide extensions, in such a way that you may get amplification and polymorphism for any combination in almost any organism, especially eukaryotic. On the other hand, in SSR, for microsatellite amplification you need to know the flanking sequences, so you can design the right primers.

The two step amplification in AFLPs is rather technical; the first one with a single nucleotide extension on each side, and the second one with two more nucleotides, so you reduce the number of fragments.

Hi Mohamed

Just to elaborate:

The reason why you don't have to know the sequense of your material working with AFLP is because you link adaptors onto the fragments after restriction. These adaptors serves as primer bindings sites.                                                                                                 In SSR you have to know the flanking sequense for primer design.

Dear Dr Mohamed,

Both AFLP and SSR PCR DNA markers.

Amplified fragment length polymorphisms (AFLPs)

AFLP is a PCR based DNA fingerprint technique (Vos et al., 1995; Zabeau and Vos,

1993). It involves restriction of genomic DNA followed by ligation of adaptors to restricted fragments and pre-selective and selective PCR amplification of a subset of these fragments. The amplified fragments are resolved on a sequencing gel and visualized either by autoradiography or fluorescent sequencing equipment (Meksem et al., 1995; Zhang et al., 1999; Schwarz et al., 1999; Huang et al., 2000b; Huang and Sun 1999; Hartl and Seefelder, 1998), depending on the method of labelling or silver staining (Cho et al., 1996). In contrast to RAPD, AFLP is highly reproducible and also transferable between different populations (Jones et al., 1997; Yin et al., 1999; Waugh et al., 1997; Li et al., 1998; Rouppe van der Voort et al., 1997). One major limitation of AFLPs is the dominant nature and the difficulty in identifying allelic variants at a specific locus although co-dominant AFLP markers have been found, however, in frequencies of 4-15 % among all polymorphic AFLP markers (Waugh et al., 1997; Lu et al., 1998; Boivin et al., 1999).

Simple sequence repeats (SSRs) or microsatellite

DNA sequences with di-, tri-, tetra- or penta-nucleotide tandem repeats are described

either as microsatellites (Litt and Luty, 1989), as simple sequence repeats (SSRs; Hearne et al., 1992) or as short tandem repeats (STRs; Edwards et al., 1991). The first report of microsatellites in plants was made by Condit and Hubbell (1991). These markers appear to be hyper-variable, in addition to which their co-dominance and reproducibility make them ideal for genome mapping, as well as for population genetic studies (Dayanandan et al., 1998). Inter-SSRs are a variant of the RAPD technique, although the higher annealing temperatures probably mean that they are more rigorous than RAPDs. They are present in the vast majority of eukaryotic genomes. The total number of different dinucleotide blocks has been estimated for several species (Ma et al., 1996; Wu and Tanksley, 1993; Morgante and Olivieri, 1993). The number of sites ranged from 103 to 105 depending on the species and repeat motif. Polymorphism produced by a variable number of tandem repeats has been demonstrated in a large number of species. This feature has made microsatellites a very attractive molecular marker for species with a narrow genetic base such as wheat and barley. This methodology is based on the use of primers complementary to SSRs. Multilocus profiles have been generated using different kinds of oligonucleotide containing simple sequence repeats as single primer (Gupta et al., 1994; Nagaoka and Ogihara, 1997) or in combination with arbitrary sequence oligonucleotides (Wu et al., 1994). These studies have shown the reproducibility of the patterns generated the Mendelian inheritance of the polymorphic amplified bands and their usefulness in the investigation of the genetic relationships. Mapping of this kind of markers in barley (Becker and Heun, 1995) and rapeseed (Kresovich et al., 1995; Szewe-MeFadden et

al., 1996; Uzunova and Ecke, 1999; Plieske and Struss, 2001) have been conducted. In wheat, Devos et al. (1995) searched sequence database and converted two microsatellite sequences into PCR based markers. Röder et al. (1995), Ma et al. (1996) and Plaschke et al. (1995) investigated the potential of microsatellite sequences as genetic markers in hexaploid wheat.

Many thanks Prof. Reyes-Valdes, Dr. Thestrup, and Prof. Khaled F M Salem for these detailed and valuable answers!

Dr.Thestrup and Dr Khaled have given nice elaboration to your question but I will come to the last part of your question i.e Why does SSR require only one step for PCR amplification and AFLP requires two?

Basically in AFLP we can run the product on denatured gel after first amplication also but it will look seamier type so to reduce it we use selective amplification For instance if we use 1 base extra so out of four fragments only 1 will get amplified (because of 4 NT) that is why AFLP needs selective amplification I,e 2 steps of PCR

AFLP  is dominant marker and SSR is co-dominant marker. AFLP is combination of RFLP and RAPD.

Dear Mohamed,

! You need to convert aflp marker to scar marker for mas studies, but ssr is a co dDear Mohammed,

AFLP is a genomic marker, you can screen whole genome with aflp (both introns and exons), on the other hand, ssr is a genic marker, and you can also screen ests. Besides,aflp is a dominant marker

As mentioned earlier, AFLP is dominant marker while SSR is codominant marker. IN ssr we need to have prior knowledge on sequence and we need to design the primers. There is no need for primer designing in AFLP.... just need to screen for best combinations. The choice of technique will depend on your study objectives.