**3.1 PCR (Polymerase Chain Reaction)**

The technique of polymerase chain reaction (PCR) was developed in the late 80s (Mullis & Faloon, 1987; Saiki et al., 1988) and revolutionized the molecular genetic studies (Watson et al., 1997). The PCR allows the production of a large number of copies of specific DNA sequence without cloning. The impact of the PCR technique and methods derived from it Kary Mullis took to win the Nobel Prize in 1993. Many methods for cloning, sequencing and analysis of DNA polymorphism were accelerated or replaced by the use of the many derivations of the PCR technique (Ferreira & Grattapaglia, 1995).

The reaction has as its basic principle the natural replication of DNA molecules that occur in cells. After opening the double strand, obtained by heating, a primer specific base pairs is the region of interest, and then there is the amplification of a new single strand. The doublestranded DNA serves as template for the synthesis. The reaction is mediated by the action of Taq polymerase and results in amplification of a specific fragment.

According to Watson et al. (1997) PCR technique is relatively simple and versatile. The equipment required for initial reaction is the DNA that contains the sequence to be amplified, two oligonucleotides (primers) that direct the starting point of synthesis, DNA polymerase and the four deoxyribonucleotides (A, T, C, G). The mixture also contains a buffer solution, magnesium chloride and water.

The reaction is performed in an appropriate tube, heated (92-95°C) for 5 minutes, so that the double-stranded molecules are split to form single strands. These single strands serve as

and genes of interest. When the model is defined, the selection criterion becomes an RFLP marker and not the phenotype. This procedure is called marker-assisted selection and can be used for difficult to assess characteristics: resistance to nematodes, protein production. Some researchers have used RFLP technique (Apuy et al., 1988; Doyle, 1988; Doyle & Beachy, 1985) and reported at the time, the low level of polymorphism found in soybean plants. The correct choice of enzymes and determination of time of exposure of DNA to

Keim et al. (1990) developed a genetic map for soybean using RFLP marker associated with QTL (Quantitative Trait loci) and highlighted the quality of the technique. The same authors reported on the importance of maps for the improvement and understanding of plant

The RFLP technique has high consistency and repeatability of results. Several searches were performed for characterization of cultivars (Autrique et al., 1996; Gebhardt et al., 1989; O'Donoughue et al., 1994). Tozuka et al., (1990) conducted in 1097 collecting wild plants of soybean (*Glycine soja*) in Japan and applied the RFLP technique in mitochondrial DNA, in order to separate them into groups using two probes (*coxII* and *atp6*). The authors stressed

The resistance to cyst nematode (*Heterodera glycines* Ichinohe), due to the damage it causes in soybeans, is the subject of several studies. *Rhg4* gene was mapped on linkage group A2 by Weisemann et al. (1992) and Webb et al. (1995). Mahalingam & Skorupska (1995) obtained similar results working with RFLP and RAPD markers. Factors such as the large number of steps and use, in many cases, of radioactive probes, prevent the use of the technique on a

The technique of polymerase chain reaction (PCR) was developed in the late 80s (Mullis & Faloon, 1987; Saiki et al., 1988) and revolutionized the molecular genetic studies (Watson et al., 1997). The PCR allows the production of a large number of copies of specific DNA sequence without cloning. The impact of the PCR technique and methods derived from it Kary Mullis took to win the Nobel Prize in 1993. Many methods for cloning, sequencing and analysis of DNA polymorphism were accelerated or replaced by the use of the many

The reaction has as its basic principle the natural replication of DNA molecules that occur in cells. After opening the double strand, obtained by heating, a primer specific base pairs is the region of interest, and then there is the amplification of a new single strand. The doublestranded DNA serves as template for the synthesis. The reaction is mediated by the action of

According to Watson et al. (1997) PCR technique is relatively simple and versatile. The equipment required for initial reaction is the DNA that contains the sequence to be amplified, two oligonucleotides (primers) that direct the starting point of synthesis, DNA polymerase and the four deoxyribonucleotides (A, T, C, G). The mixture also contains a

The reaction is performed in an appropriate tube, heated (92-95°C) for 5 minutes, so that the double-stranded molecules are split to form single strands. These single strands serve as

enzymatic action, can determine the degree of polymorphism.

the quality of technical and plants classified in 18 groups.

**3. Molecular markers based on DNA amplification** 

derivations of the PCR technique (Ferreira & Grattapaglia, 1995).

Taq polymerase and results in amplification of a specific fragment.

buffer solution, magnesium chloride and water.

**3.1 PCR (Polymerase Chain Reaction)** 

evolution.

large scale.

templates for the primers. Then the temperature is reduced (35 to 60°C) providing suitable conditions for pairing between primers and complementary sequences. In the next step the temperature is high (72°C) for up to 5 minutes. At the end of the temperature is again raised to 94°C for 20 seconds, for the separation of short strips of DNA that serve as templates for new cycles. For most protocols, the number of cycles varies between thirty and sixty.

The discovery of the bacterium *Thermus aquaticus* which lives in water temperature of 75°C and isolation of its polymerase (Saiki et al., 1988), enabled the automation of PCR by using thermocyclers (Watson et al., 1997 ), which are heating blocks that can be programmed to control the time and reaction temperature. They are equipment with the capacity to change the temperature quickly and repeat the cycle according to the protocol and the number of amplified fragments doubles every cycle. The Taq polymerase of bacteria supports high temperatures without being destroyed.

The enzyme requires a double-stranded fragment in the case, provided by specific primers to initiate amplification of the complementary strand of DNA. If two primers complementary to both strands of DNA are used, the amplification occurs in both directions and two new single strands are generated. Each will have as starting point the sequence of an initiator and extend beyond the other's position in initiating complementary strand. The extension process is always from the position 3' of the initiator. Millions of copies of a given follow-up of DNA, typically up to 4000pb can be synthesized using Taq polymerase.

According to Watson et al. (1997) in 1985 there were three research reports with PCR, some years later the PCR was being used in thousands of labs around the world. The technique caused a major revolution in the practice of molecular genetics.

### **3.2 RAPD (Random Amplified Polymorphic DNA)**

Molecular markers may characterize a plant from plant tissue samples. Welsh and McClelland (1990) and Williams et al. (1990) proposed a technique denominated RAPD (Random Amplified Polymorphic DNA) using primers of arbitrary sequence (10 nucleotides) and identifies polymorphic among different individuals. RAPD molecular markers can be used for studies related to genetic mapping, population genetics, molecular systematics, fingerprint genotypes and marker-assisted selection.

The technique allows the identification of numerous markers without prior notification, which may or may not be associated with traits of interest. If a particular marker is physically linked to a gene of agronomic interest, the selection of this results in indirect selection marker gene (Ferreira & Grattapaglia, 1995). The situation described is characterized as marker-assisted selection. If the distance between the marker and the gene is small, the efficiency of indirect selection will be higher.

The protocols used to show satisfactory results, but generally you should take steps to maintain the quality of DNA and reagents. The type of thermocycler can also be considered as a variable. The observation of the proposed action results in quality bands and the possibility of replication of results.

The principle of the technique is based on the PCR reaction: elevated temperature of the solution to the double-stranded DNA to open in their hydrogen bonds. Then the reduced temperature and allowing the pairing occurs between the primers and the DNA strand. Finally the temperature is again raised to the Taq DNA polymerase to amplify the fragments thereby generating bands that will be visualized in gel electrophoresis.

The temperature variations are possible using a thermal cycler that automatically performs the operations for each step: denaturation (92-95°C), pairing (35-60°C) and amplification

Molecular Markers: Assisted Selection in Soybeans 161

Once polymorphism observed between the groups, the reactions are being carried out individually with DNA from plants of groups, with the objective to confirm the results. A marker that shows Mendelian segregation can be considered a gene locus. Segregation of phenotypic characteristics assessed and polymorphic fragments should be individually

Then the analysis must be performed by testing the hypothesis of joint 9:3:3:1 segregation. If the hypothesis is rejected, the marker and the allele that determines the desired trait can be on the same chromosome. The question is which the distance between them, which can be

The smaller the genetic distance between the marker and the allele of interest, the greater the efficiency of molecular markers, to the point that if the distance is close to zero, the marker is the gene itself. So there is a need for an excellent mechanism for phenotypic evaluation and qualified people to apply it, for all subsequent work depends on this step. Another point that needs to be analyzed is concerned for the fate of a marker allele is not in question but rather a region of chromosome near the gene of interest. The situation may lead to specific markers since the chromosomal region, classified as a molecular marker may not be present in different plants. The absence of amplification can be explained by mutations or rearrangements between the two sites, or on the site of hybridization of the initiator (Paran & Michelmore, 1993). Differences in only one base pair may be sufficient to inhibit

It is expected that with advances in functional genomics projects, some markers will lose its practical application, because knowing the sequence of the allele, the job is much more

Martins Filho et al. (2002) used the technique BSA, in a survey of three F2 populations of soybean exhibiting resistance to the fungus *Cercospora sojina*. The authors have built on resistant and susceptible groups, with six plants in each one of them and were able to identify RAPD markers in three populations. The DNA of plants assessed individually confirmed the identification observed in the groups, validating the methodology BSA. The distances between the markers found by the authors and the locus studied ranged between

Carvalho et al. (2002) identified two RAPD markers linked to gene for resistance to stem canker in soybeans, which were amplified by the same initiator: OPAB191320 present in all plants classified as homozygous resistant (coupling phase) and OPAB191150 present in susceptible plants (repulsion phase), whereas heterozygous plants classified as resistant showed the two fragments. The distance between the gene and the markers, was estimated

Gavioli et al. (2007) worked with F2 populations of soybean plants resistant and susceptible to the fungus that causes stem canker, which were evaluated in a greenhouse. The authors applied the methodology BSA (seven plants in each group) and identified a polymorphic RAPD band, with 588 bp, within the group of resistant plants. There were two replications of observed distances of 6.0 and 7.4 cM from the RAPD marker and the locus of interest. DNA from plants of each group was analyzed individually and the band found previously

Recently Costa et al. (2008) identified RAPD markers linked to genes conferring resistance to rust caused by *Phakopsora pachyrhizi*. The authors applied the methodology BSA (ten plants in each group) and related markers: OPBB16650, OPAK04800 OPR04850 and distant to 5.1 cM and 6.3 cM and 14.7 cM, respectively, the locus of resistance *Rpp4*. The authors concluded

assessed according to the 3:1 hypothesis.

accurate.

2.3 and 6.7 cM.

at 4.7 cm.

was confirmed (Figure 1).

determined by the number of recombinant individuals

amplification, especially at position 3 '(Williams et al., 1990).

(72°C). The cycle is repeated about 40 times and the final product of amplification can be visualized in agarose gel with ethidium bromide dye under ultraviolet light. RAPD markers behave as dominant, characterized by amplification of fragments or not.

The low temperature necessary for linking the primer to the template makes the process very dependent on the conditions of amplification. Therefore, modifications in the thermocycler used, in the DNA polymerase and other reagents of the reaction mixture can change the pattern of amplification. This may not representing a major problem in a group of researchers who carefully standardize the conditions of amplification, but may prevent data from being compared between different laboratories (Alzate-Marin et al., 2005).

The successful use of RAPD molecular marker is directly linked to quality of genomic DNA, which can be extracted using CTAB protocol. A soybean leaf stage provides enough DNA for several reactions. The leaf collected should be placed in contact with liquid nitrogen and then macerated. DNA can be extracted immediately or the leaves can be stored at – 80°C.

After extraction, the DNA must be quantified for standardization to occur in the reactions, which assures quality of the bands obtained. For RAPD reactions, quantities of DNA in the order of nanograms provide adequate conditions for the success of the process. DNA can be stored at low temperatures (-20°C to -80°C) depending on usage needs. Contaminant compounds such as proteins, polysaccharides and phenolic compounds can affect the quality of DNA and inhibit the action of Taq polymerase.

The observed differential to obtain RAPD markers, in relation to the PCR process is the use of arbitrary primers, and a large number of random amplification. Thus the bands obtained can not be directly related to a gene of interest. The primers are oligonucleotides of known sequence featuring 10bp. Currently a large number of primers is offered on the market, which allows an enormous amount of reactions with the same DNA.

To associate a characteristic phenotypic marker for a particular molecularly was proposed by Michelmore et al. (1991) methodology BSA (Bulked Segregant Analysis).

Once determined the phenotypic trait of interest, plant a segregating F2 population are classified according to the established criteria, such as resistant or susceptible to a given disease.

The plant DNA can be extracted from a small leaf sample, taken at the beginning of plant development, individually stored refrigerated. All plants of the F2 population provide DNA for further RAPD reactions.

After the end of the phenotypic evaluations plants should be classified into distinct groups, contrasting in relation to the characteristic under study. Each group is composed of a small number of plants following the theory. For a dominant RAPD marker segreganting in an F2 population, the probability of a bulk of n individuals having a band and a second bulk of equal size not having a band will be 2(1-[1/4]n )(1/4)n when the locus is unlinked to the target gene. Therefore low individuals per book are required. For example, the probability of an unlinked locus being polymorphic between bulks of 10 such individuals is 2 x 10-6. Even when many loci are screened, the chances of detecting an unlinked locus are small. As smaller bulks are utilized, the frequency of false positives will increase. However, as the linkage of all polymorphisms is confirmed by analysis of a segregating populations, bulked segregant analysis with only small numbers of individuals in one or both bulks will provide great enrichment for marker linked to target loci (Michelmore et al., 1991).

Two solutions containing DNA should be prepared in equimolar concentrations, for the continuation of the technique. Example: A group with DNA from plants resistant to disease and another group with the DNA of plants susceptible to disease. Initially the RAPD reactions are performed with the DNA of each group. Thus polymorphism in a group may be associated with the characteristic that distinguishes them.

(72°C). The cycle is repeated about 40 times and the final product of amplification can be visualized in agarose gel with ethidium bromide dye under ultraviolet light. RAPD markers

The low temperature necessary for linking the primer to the template makes the process very dependent on the conditions of amplification. Therefore, modifications in the thermocycler used, in the DNA polymerase and other reagents of the reaction mixture can change the pattern of amplification. This may not representing a major problem in a group of researchers who carefully standardize the conditions of amplification, but may prevent

The successful use of RAPD molecular marker is directly linked to quality of genomic DNA, which can be extracted using CTAB protocol. A soybean leaf stage provides enough DNA for several reactions. The leaf collected should be placed in contact with liquid nitrogen and then macerated. DNA can be extracted immediately or the leaves can be stored at – 80°C. After extraction, the DNA must be quantified for standardization to occur in the reactions, which assures quality of the bands obtained. For RAPD reactions, quantities of DNA in the order of nanograms provide adequate conditions for the success of the process. DNA can be stored at low temperatures (-20°C to -80°C) depending on usage needs. Contaminant compounds such as proteins, polysaccharides and phenolic compounds can affect the

The observed differential to obtain RAPD markers, in relation to the PCR process is the use of arbitrary primers, and a large number of random amplification. Thus the bands obtained can not be directly related to a gene of interest. The primers are oligonucleotides of known sequence featuring 10bp. Currently a large number of primers is offered on the market,

To associate a characteristic phenotypic marker for a particular molecularly was proposed

Once determined the phenotypic trait of interest, plant a segregating F2 population are classified according to the established criteria, such as resistant or susceptible to a given

The plant DNA can be extracted from a small leaf sample, taken at the beginning of plant development, individually stored refrigerated. All plants of the F2 population provide DNA

After the end of the phenotypic evaluations plants should be classified into distinct groups, contrasting in relation to the characteristic under study. Each group is composed of a small number of plants following the theory. For a dominant RAPD marker segreganting in an F2 population, the probability of a bulk of n individuals having a band and a second bulk of equal size not having a band will be 2(1-[1/4]n )(1/4)n when the locus is unlinked to the target gene. Therefore low individuals per book are required. For example, the probability of an unlinked locus being polymorphic between bulks of 10 such individuals is 2 x 10-6. Even when many loci are screened, the chances of detecting an unlinked locus are small. As smaller bulks are utilized, the frequency of false positives will increase. However, as the linkage of all polymorphisms is confirmed by analysis of a segregating populations, bulked segregant analysis with only small numbers of individuals in one or both bulks will provide

Two solutions containing DNA should be prepared in equimolar concentrations, for the continuation of the technique. Example: A group with DNA from plants resistant to disease and another group with the DNA of plants susceptible to disease. Initially the RAPD reactions are performed with the DNA of each group. Thus polymorphism in a group may

data from being compared between different laboratories (Alzate-Marin et al., 2005).

behave as dominant, characterized by amplification of fragments or not.

quality of DNA and inhibit the action of Taq polymerase.

disease.

for further RAPD reactions.

which allows an enormous amount of reactions with the same DNA.

by Michelmore et al. (1991) methodology BSA (Bulked Segregant Analysis).

great enrichment for marker linked to target loci (Michelmore et al., 1991).

be associated with the characteristic that distinguishes them.

Once polymorphism observed between the groups, the reactions are being carried out individually with DNA from plants of groups, with the objective to confirm the results.

A marker that shows Mendelian segregation can be considered a gene locus. Segregation of phenotypic characteristics assessed and polymorphic fragments should be individually assessed according to the 3:1 hypothesis.

Then the analysis must be performed by testing the hypothesis of joint 9:3:3:1 segregation. If the hypothesis is rejected, the marker and the allele that determines the desired trait can be on the same chromosome. The question is which the distance between them, which can be determined by the number of recombinant individuals

The smaller the genetic distance between the marker and the allele of interest, the greater the efficiency of molecular markers, to the point that if the distance is close to zero, the marker is the gene itself. So there is a need for an excellent mechanism for phenotypic evaluation and qualified people to apply it, for all subsequent work depends on this step.

Another point that needs to be analyzed is concerned for the fate of a marker allele is not in question but rather a region of chromosome near the gene of interest. The situation may lead to specific markers since the chromosomal region, classified as a molecular marker may not be present in different plants. The absence of amplification can be explained by mutations or rearrangements between the two sites, or on the site of hybridization of the initiator (Paran & Michelmore, 1993). Differences in only one base pair may be sufficient to inhibit amplification, especially at position 3 '(Williams et al., 1990).

It is expected that with advances in functional genomics projects, some markers will lose its practical application, because knowing the sequence of the allele, the job is much more accurate.

Martins Filho et al. (2002) used the technique BSA, in a survey of three F2 populations of soybean exhibiting resistance to the fungus *Cercospora sojina*. The authors have built on resistant and susceptible groups, with six plants in each one of them and were able to identify RAPD markers in three populations. The DNA of plants assessed individually confirmed the identification observed in the groups, validating the methodology BSA. The distances between the markers found by the authors and the locus studied ranged between 2.3 and 6.7 cM.

Carvalho et al. (2002) identified two RAPD markers linked to gene for resistance to stem canker in soybeans, which were amplified by the same initiator: OPAB191320 present in all plants classified as homozygous resistant (coupling phase) and OPAB191150 present in susceptible plants (repulsion phase), whereas heterozygous plants classified as resistant showed the two fragments. The distance between the gene and the markers, was estimated at 4.7 cm.

Gavioli et al. (2007) worked with F2 populations of soybean plants resistant and susceptible to the fungus that causes stem canker, which were evaluated in a greenhouse. The authors applied the methodology BSA (seven plants in each group) and identified a polymorphic RAPD band, with 588 bp, within the group of resistant plants. There were two replications of observed distances of 6.0 and 7.4 cM from the RAPD marker and the locus of interest. DNA from plants of each group was analyzed individually and the band found previously was confirmed (Figure 1).

Recently Costa et al. (2008) identified RAPD markers linked to genes conferring resistance to rust caused by *Phakopsora pachyrhizi*. The authors applied the methodology BSA (ten plants in each group) and related markers: OPBB16650, OPAK04800 OPR04850 and distant to 5.1 cM and 6.3 cM and 14.7 cM, respectively, the locus of resistance *Rpp4*. The authors concluded

Molecular Markers: Assisted Selection in Soybeans 163

have advantages: specificity for detecting a single locus and less sensitivity to variations of the reactions. SCAR markers have co-dominant nature and were defined as fragments of genomic DNA, located in a defined locus, which are identified by PCR amplification using a pair of specific oligonucleotides as primers (Ferreira & Grattapaglia, 1998; Nietzsche et al.,

The RAPD polymorphic fragments have to be isolated, cloned and sequenced and then will be used for the synthesis of new primers. Usually the SCAR primers have at their 5 'end, the initiator used in RAPD reactions and its initial 3 'end, the additional bases that will

The SCAR markers can be synthesized starting from a molecular marker RAPD. The isolation of the RAPD fragment is accomplished through a direct cut in the agarose gel containing the desired band. The isolated fragment must be inserted into a vector, usually a plasmid, which will be used in the process of bacterial transformation. Transformed colonies is necessary to separate the fragment that contains, those not containing the desired DNA fragment. After this step the selected colonies are grown for growth and subsequent multiplication of the fragment. Detailed protocols on bacterial transformation can be

The fragments should be extracted and purified from plasmids. Restriction enzymes are able to cut the plasmid at sites flanking the fragment. The insert should be sequenced to be

For the synthesis of new initiators is considered some parameters such as GC percentage (minimum 50%) and pairing temperature (more than 56°C). Usually the new primers have between 16 and 24 base pairs. There are computer programs that help in developing new primers. Martins Filho et al. (2002) worked with two primers containing 18 nucleotides in

At the end of the process, the new SCAR primers should be tested with the plants of the F2 population and parents to prove the link between the marker and the locus of interest. The

Often there is loss of polymorphism of RAPD, when converted into SCAR markers. The problem results from the amplification of two alleles of that locus, which prevents the differentiation between plants. Paran & Michelmore (1992) reported that the polymorphism observed in the RAPD reaction, can be caused by differences in the nucleotide sequence of the site of annealing or rearrangement in the internal sequence of the amplification. A mis-pairing, mainly at the 3' primer, prevented the amplification of a fragment of the genotypes. In a SCAR primer, with the largest number of nucleotides, the end without pairing, is positioned in its middle region and may not interfere with the

The loss of polymorphism can be solved through the use of restriction enzymes, which promote the cut at specific sites in one allele of a given locus. The technique was successfully used by several authors, among them: Weng et al. (1998), Lahogue et al. (1998), Dax et al.

The choice of restriction enzyme based on the sequencing of the fragments SCAR. The sequences are evaluated within and should be sought restriction sites that can differentiate

The fragments are then PCR amplified and digested individually. The result of electrophoresis may reveal again the initial polymorphism. Gavioli et al. (2007) converted a RAPD polymorphic in a SCAR marker. The process resulted in the loss of polymorphism,

them. Monomorphic fragments in molecular weight may differ in base sequence.

2000).

characterize the new initiator.

obtained in Sambrook et al. (1989).

amplified fragments.

(1998) and Zhang & Stommel (2001).

known that the bases are among the RAPD primers.

each, and determined the temperature from 62°C as an ideal pairing.

confirmation allows the use of marker assisted selection in the process.

that the RAPD primers identified in the survey are indicated for assisted selection of soybean genotypes with the same source of resistance in this study. The selection can be performed in the early stages of development to occur without destroying the plants.

Fig. 1. RAPD polymorphic fragment of 588bp observed in: 1. resistant parent BR92-15454; 2. F1 plant; 4. resistant bulk and 6-9. F2 resistants progenies and absent in: 3. susceptible parent IAC-11; 5. susceptible bulk and 10-15. F2 susceptible progenies. 16. Negative control. Molecular pattern (MP), originating from the digestion of λ with the enzymes *EcoRI* and *Hind III*.

On soybeans, other authors have worked with RAPD markers and were able to identify some of them linked to genes of interest. Heer et al. (1998) identified markers for genes that determine resistance to cyst nematode. Chowdhury et al. (2002) worked with plants resistant to downy mildew and reported the existence of two markers linked to the gene that determines resistance, respectively, 4.9 and 23.1 cM.

Segregating progeny may be evaluated for the presence or absence of a particular marker. The heterozygote cannot be distinguished and this represents a loss of information in relation to RFLP markers. Populations consisting of backcross progeny, recombinant strains and di-haploids do not undergo this loss of information, since the full information available can be obtained in the presence or absence of the marker (Reiter et al., 1992).
