**3.5 AFLP (Amplified Fragment Length Polymorphisms)**

Since its development and publishing, this technique has been used to characterize genotypes, genetic mapping, especially in species with low DNA polymorphism (Zabeau, 1993). The use of this marker is based on the technique that combines DNA fragmentation with restriction enzymes type II, that cleave DNA at specific sites of rare cutting (recognize sites of 6-8 bases, ex: *ApaI*, *EcoRI*, *HindIII* and *PstI*) and frequent cutting (recognize sites of four bases, ex: *MseI* and *TaqI*) and amplification of these fragments by PCR (Vos et al., 1995).

The use of specific restriction enzymes, allows the knowledge of the cohesive ends generated, in which they are linked adapters that serve as binding sites for primers in a PCR reaction. It is essential that the DNA digestion is complete, because the partial digestion can reveal false polymorphisms. The purity of DNA used is a fundamental requirement for obtaining good results.

In the process of digestion, the two enzymes (unusual cutting / frequent cutting) can be used simultaneously (double digestion) or in two steps, if there is a reaction buffer common to both enzymes. Three classes of fragments are generated: frequent/frequent; unusual/frequent and unusual/unusual.

From the digestion of genomic DNA with *EcoRI* and *MseI* is expected that most of the fragments are cut *MseI*/*MseI* (frequent/frequent). Fragments cut *EcoRI*/*MseI* (unusual/frequent) occur in approximately equal frequency to twice the number of restriction enzyme sites of *EcoRI* and fragments cut *EcoRI*/*EcoRI* (unusual/unusual) occur in low volume (Ferreira & Grattapaglia, 1995).

The fragments generated by enzymatic digestion should be linked to specific adapters that have additional terminals to the ends resulting from cleavage. The process of connecting the adapters involves using ligases that enables DNA fragments to bind to the adapters.

With this procedure, both the sequence of adapters, as the result of the restriction site are known, allowing the construction of specific primers to these sequences for preamplification restriction fragment through PCR reactions. The primers consist of a sequence complementary to the adapter, followed by another site-specific restriction enzyme, and an extension of selective nucleotides at the 3' end (Lopes et al., 2002).

In the first stage of amplification, called pre-amplification, a selective nucleotide is used in the 3' end of primers. In the second phase of amplification, called selective amplification, three selective nucleotides are used in the terminal 3' primers.

The second amplification is done with a sample of the first. Therefore, only the fragments that have complementary nucleotides to the selective nucleotides will be amplified. Thus, the alleles of AFLP loci (presence or absence of a specific fragment or band) are from the loss

heterozygotes. The authors asserted that this type of marker is a valuable tool for analysis of

Microsatellite markers have many advantages compared to other types of markers (RFLP, RAPD, AFLP) are highly polymorphic and informative; the co-dominant inheritance, which allows discrimination between homozygous and heterozygous; are multi-allelic; occurring abundantly in genomes of eukaryotes; are based on PCR and thus need small amounts of DNA; are highly reproducible; require no radioactivity; are well dispersed in the genome in

The microsatellite markers have been used extensively to the major species of agronomic importance and have potential to occupy a prominent place among the markers of greatest

Since its development and publishing, this technique has been used to characterize genotypes, genetic mapping, especially in species with low DNA polymorphism (Zabeau, 1993). The use of this marker is based on the technique that combines DNA fragmentation with restriction enzymes type II, that cleave DNA at specific sites of rare cutting (recognize sites of 6-8 bases, ex: *ApaI*, *EcoRI*, *HindIII* and *PstI*) and frequent cutting (recognize sites of four bases, ex: *MseI* and *TaqI*) and amplification of these fragments by PCR (Vos et al., 1995). The use of specific restriction enzymes, allows the knowledge of the cohesive ends generated, in which they are linked adapters that serve as binding sites for primers in a PCR reaction. It is essential that the DNA digestion is complete, because the partial digestion can reveal false polymorphisms. The purity of DNA used is a fundamental requirement for

In the process of digestion, the two enzymes (unusual cutting / frequent cutting) can be used simultaneously (double digestion) or in two steps, if there is a reaction buffer common to both enzymes. Three classes of fragments are generated: frequent/frequent;

From the digestion of genomic DNA with *EcoRI* and *MseI* is expected that most of the fragments are cut *MseI*/*MseI* (frequent/frequent). Fragments cut *EcoRI*/*MseI* (unusual/frequent) occur in approximately equal frequency to twice the number of restriction enzyme sites of *EcoRI* and fragments cut *EcoRI*/*EcoRI* (unusual/unusual) occur in

The fragments generated by enzymatic digestion should be linked to specific adapters that have additional terminals to the ends resulting from cleavage. The process of connecting the

With this procedure, both the sequence of adapters, as the result of the restriction site are known, allowing the construction of specific primers to these sequences for preamplification restriction fragment through PCR reactions. The primers consist of a sequence complementary to the adapter, followed by another site-specific restriction enzyme, and an

In the first stage of amplification, called pre-amplification, a selective nucleotide is used in the 3' end of primers. In the second phase of amplification, called selective amplification,

The second amplification is done with a sample of the first. Therefore, only the fragments that have complementary nucleotides to the selective nucleotides will be amplified. Thus, the alleles of AFLP loci (presence or absence of a specific fragment or band) are from the loss

adapters involves using ligases that enables DNA fragments to bind to the adapters.

extension of selective nucleotides at the 3' end (Lopes et al., 2002).

three selective nucleotides are used in the terminal 3' primers.

genetic variability in germplasm of plant species and therefore its maintenance.

coding regions and non-coding; loci are often conserved between related species.

**3.5 AFLP (Amplified Fragment Length Polymorphisms)** 

use.

obtaining good results.

unusual/frequent and unusual/unusual.

low volume (Ferreira & Grattapaglia, 1995).

or gain of a restriction site or the complementary bases selective or not used at the terminals 3 'primers where one starts PCR with the region, which flanks the restriction site (Lopes et al., 2002). The authors presented a table with the restriction sites, sequences of adapters and primers used for six enzymes in AFLP analysis (Table 1).


Table 1. Restriction sites, sequences of adapters and primers used for six enzymes in AFLP analysis (E: arbitrary nucleotide used in the pre-amplification).

Molecular Markers: Assisted Selection in Soybeans 171

The markers presented are some similarities between themselves regarding the level of polymorphism, random distribution in the genome stability, gene expression among others. The comparison between them is presented in Table 2 adapted from Ferreira & Grattapaglia

**Characteristics RFLP RAPD Microsatellites AFLP** 

polymorphism Low - High Low - High Very High Very High

stability High High High High Number of locus High High High High Gene expression Co-dominant Dominant Co-dominant Dominant

per locus Multiallelic Two Multiallelic Two

genome Multiple Random Random Random

Technology Moderate Very High Very Low Moderate

genotypes High Very High Very High Very High

breeding Mean cost Low Cost Expensive Low Cost Specific mapping Moderate Very High Moderate Very High

germplasm High High High Very High Genetic mapping High High Very High High Genetics autogamy Moderate High Very High Very High Genetic allogamous Moderate High Very High Very High

analysis Very High Moderate High Moderate

The molecular markers reported so far in the chapter became important tools in the genetic improvement of plants. The following is a brief account made of the possible uses for these

Construction of genetic maps: The Mendelian segregation observed in molecular markers, allows them to be used to construct genetic maps of connection and enable the location of

Characterization of genetic variability: the polymorphism generated from DNA and observed in the form of bands, allows us to study and confirm the genetic variability among

Monitoring of genes: Genes may be screened in breeding programs that use the technique of backcrossing. Some applications of molecular markers linked to breeding programs can be facilitated through the use of this tool. In programs of backcross markers can maximize the efficiency of programs by increasing the probability of conversion of individuals and

Table 2. Comparative analysis for some characteristics of molecular markers.

**4. Relationship between markers and use practices** 

(1995).

Level of

Environmental

Number of alleles

Distribution in

Accessibility

Identification of

Application in

Evaluation of

Phylogenetic

genes within the chromosomes. QTLs mapping;

markers.

plants;

Fragments cut *EcoRI*/*MseI* are preferentially amplified, and this is due to lower efficiency of hybridization of primers *MseI* compared to that with primers *EcoRI*, and also the fact that the fragments *MseI*/*MseI* have terminal inverted sequence being amplified by a single initiator, enhancing the formation of a loop structure that competes with the annealing of primers (Vos et al., 1995).

For separation and identification of AFLP amplification products, the best method is to electrophoresis in denaturing polyacrylamide gel, which provides a high level of resolution, being effective for detecting single nucleotide differences. The number of fragments visualized in a polyacrylamide gel is variable and can reach values higher than one hundred. Theoretically the larger the genome size, the greater the amount of fragments. The readings of the gels can be manual or automated. The manual reading is performed after the revelation of the banding pattern by staining with silver nitrate or revelation in autoradiographs, in this case using primers labeled with radioisotopes. The second type is performed by DNA analyzers and requires fluorescent labeling.

The AFLP technique detects a greater number of fragments compared to other techniques that reveal molecular markers and provides comprehensive coverage of the genome. The use of restriction enzymes combined with appropriate conditions for hybridization of primers for the amplification reactions combines the robustness of the RFLP technique with the practicality of PCR.

 Just as the RAPD technique, the methodology requires no prior information of DNA sequences. AFLP markers present together, the exploratory capacity of RFLP polymorphisms (presence or absence of restriction sites) with the advantage of PCR. AFLP markers are dominant and heterozygous genotypes can not be directly discriminated against the homozygotes.

Malone et al. (2003), analyzing genetic contamination in soybean determined that, in addition to the cultivars presented a high degree of genetic similarity, analysis of the banding pattern obtained by AFLP revealed the presence of additional bands in all samples when compared with the strains pure (seed genetics). The variation in the fingerprinting of cultivars suggested to be related to genetic contamination occurred between the batches studied, and exogenous contamination.

Mertz et al. (2009) tried to verify the effectiveness of the technique of cDNA-AFLP in obtaining fragments of genes differentially expressed in soybean seed coats with contrasting permeability and have concluded that the technique could be a promising alternative for studies aimed at identifying genes related to seed quality. According to the authors, the cDNA-AFLP technique is effective in identifying genes expressed, because it allowed the taking of 47 differentially expressed cDNA fragments between the coats of soybean genotypes CD-202 and TP.

Colombari Filho et al. (2010) undertook a study to evaluate the heterosis for grain production in soybean and its relationship with genetic distances obtained with the AFLP molecular marker. The authors concluded that heterosis for grain production in soybean is correlated with genetic distances obtained with AFLP markers and it is possible to select from crosses from the molecular genetics distance between the parents.

The quality of DNA needed is an important factor for the success of the AFLP technique. During the extraction of a large number of samples of genomic DNA preparations can be varied in quantity and in quality. A DNA of high purity is required to ensure a complete digestion by restriction enzymes in all DNA samples. The quality of the enzymatic digestion can lead to errors of interpretations (Ferreira & Grattapaglia, 1995).

Fragments cut *EcoRI*/*MseI* are preferentially amplified, and this is due to lower efficiency of hybridization of primers *MseI* compared to that with primers *EcoRI*, and also the fact that the fragments *MseI*/*MseI* have terminal inverted sequence being amplified by a single initiator, enhancing the formation of a loop structure that competes with the annealing of

For separation and identification of AFLP amplification products, the best method is to electrophoresis in denaturing polyacrylamide gel, which provides a high level of resolution, being effective for detecting single nucleotide differences. The number of fragments visualized in a polyacrylamide gel is variable and can reach values higher than one hundred. Theoretically the larger the genome size, the greater the amount of fragments. The readings of the gels can be manual or automated. The manual reading is performed after the revelation of the banding pattern by staining with silver nitrate or revelation in autoradiographs, in this case using primers labeled with radioisotopes. The second type is

The AFLP technique detects a greater number of fragments compared to other techniques that reveal molecular markers and provides comprehensive coverage of the genome. The use of restriction enzymes combined with appropriate conditions for hybridization of primers for the amplification reactions combines the robustness of the RFLP technique with

 Just as the RAPD technique, the methodology requires no prior information of DNA sequences. AFLP markers present together, the exploratory capacity of RFLP polymorphisms (presence or absence of restriction sites) with the advantage of PCR. AFLP markers are dominant and heterozygous genotypes can not be directly discriminated

Malone et al. (2003), analyzing genetic contamination in soybean determined that, in addition to the cultivars presented a high degree of genetic similarity, analysis of the banding pattern obtained by AFLP revealed the presence of additional bands in all samples when compared with the strains pure (seed genetics). The variation in the fingerprinting of cultivars suggested to be related to genetic contamination occurred between the batches

Mertz et al. (2009) tried to verify the effectiveness of the technique of cDNA-AFLP in obtaining fragments of genes differentially expressed in soybean seed coats with contrasting permeability and have concluded that the technique could be a promising alternative for studies aimed at identifying genes related to seed quality. According to the authors, the cDNA-AFLP technique is effective in identifying genes expressed, because it allowed the taking of 47 differentially expressed cDNA fragments between the coats of soybean

Colombari Filho et al. (2010) undertook a study to evaluate the heterosis for grain production in soybean and its relationship with genetic distances obtained with the AFLP molecular marker. The authors concluded that heterosis for grain production in soybean is correlated with genetic distances obtained with AFLP markers and it is possible to select

The quality of DNA needed is an important factor for the success of the AFLP technique. During the extraction of a large number of samples of genomic DNA preparations can be varied in quantity and in quality. A DNA of high purity is required to ensure a complete digestion by restriction enzymes in all DNA samples. The quality of the enzymatic digestion

from crosses from the molecular genetics distance between the parents.

can lead to errors of interpretations (Ferreira & Grattapaglia, 1995).

performed by DNA analyzers and requires fluorescent labeling.

primers (Vos et al., 1995).

the practicality of PCR.

against the homozygotes.

genotypes CD-202 and TP.

studied, and exogenous contamination.
