**2.1 RFLP (Restriction Fragment Length Polymorphism)**

Changes in DNA caused by changes in the nucleotides can be identified if they occur on a site of a restriction nuclease. If the DNA of plants, which differ in one or more of these nucleotides is digested by these enzymes, fragments of different sizes are generated and can be identified and subsequently cloned (Botstein et al., 1980). These fragments are called RFLP (Restriction Fragment Length Polymorphism).

The polymorphism revealed by restriction fragment analysis allows studies of biological phenomena and processes: characterization of germplasm, genetic map construction and assessment of genetic diversity.

In many studies were identified RFLP markers, which contributed to a broad coverage of the genome. Several genetic maps have been generated, allowing multiple applications within the plant breeding (Nodari et al., 1993). The first research related to RFLP markers were assigned to Helentjaris et al. (1986).

The principle of the technique is the extraction of DNA from a plant and its subsequent fragmentation through different restriction enzymes. Plants have, in most cases, more than one billion base pairs in its genome, and enzymatic digestion produces thousands of fragments that vary in length according to the distribution of restriction sites. The collective analysis is impossible, but the technique allows them to be separate and distinct.

The quality of DNA to be used is essential for the results obtained by RFLP technique. The genetic material is fragmented using restriction enzymes (Ferreira & Grattapaglia, 1995).

The DNA fragments obtained were separated by electrophoresis and subsequently transferred to a membrane using a technique called Southern blotting (Southern, 1975). The identification of the fragments is possible, using radioactive probes, which can be a

sample of plant tissue (usually leaves). No damage occurs and the plant can be conducted

Several breeding programs have used molecular biology techniques, aiming at the markerassisted selection (Alzate-Marin et al., 2003; Benchimol et al., 2003; Maluf et al., 2008). Features such as disease resistance, pest resistance, genetic purity, gene pyramiding, are some areas with possibilities of action research. Phenotypic characteristics difficult to

The marker-assisted selection is a process of indirect selection in which the character in question has a high heritability, since not influenced by environmental factors. There is increased efficiency of plant breeding, reducing the number of progenies and the number of generations for the stabilization of the genotypes. The selection can be performed in early

This is a useful tool in the genetic improvement of plants introduced differential pricing depending on the type of bullet used. The procedures require specialized laboratories with sophisticated equipment and qualified personnel. There is need to integrate multidisciplinary involving researchers with backgrounds in classical plant breeding, chemistry, biochemistry, plant physiology, statistics, computer science, bioinformatics and

The chapter aims to describe the key molecular markers, showing the evolution over recent decades and its practical applications in soybean. It also aims to relate key findings and

Changes in DNA caused by changes in the nucleotides can be identified if they occur on a site of a restriction nuclease. If the DNA of plants, which differ in one or more of these nucleotides is digested by these enzymes, fragments of different sizes are generated and can be identified and subsequently cloned (Botstein et al., 1980). These fragments are called

The polymorphism revealed by restriction fragment analysis allows studies of biological phenomena and processes: characterization of germplasm, genetic map construction and

In many studies were identified RFLP markers, which contributed to a broad coverage of the genome. Several genetic maps have been generated, allowing multiple applications within the plant breeding (Nodari et al., 1993). The first research related to RFLP markers

The principle of the technique is the extraction of DNA from a plant and its subsequent fragmentation through different restriction enzymes. Plants have, in most cases, more than one billion base pairs in its genome, and enzymatic digestion produces thousands of fragments that vary in length according to the distribution of restriction sites. The collective

The quality of DNA to be used is essential for the results obtained by RFLP technique. The genetic material is fragmented using restriction enzymes (Ferreira & Grattapaglia, 1995). The DNA fragments obtained were separated by electrophoresis and subsequently transferred to a membrane using a technique called Southern blotting (Southern, 1975). The identification of the fragments is possible, using radioactive probes, which can be a

analysis is impossible, but the technique allows them to be separate and distinct.

future possibilities of this tool that are joining forces for the development of soybean.

measure can also be evaluated using molecular markers.

**2. Molecular markers based on DNA hybridization 2.1 RFLP (Restriction Fragment Length Polymorphism)** 

RFLP (Restriction Fragment Length Polymorphism).

assessment of genetic diversity.

were assigned to Helentjaris et al. (1986).

generations (Barbosa Neto, 1998; Federizzi, 1998).

normally.

others.

fragment of the plant itself, complementary to the fragment of interest. At the end of the process the membranes are exposed to an X-ray film, showing the hybridization due to emission of radiation by the probe. The polymorphism observed among the plants may be related to genetic differences. The marker behaves as co-dominant, where at each locus studied is possible to identify individuals homozygous or heterozygous. The amount of information produced is large and allows the analysis of gene action and interactions between alleles.

The restriction nucleases are enzymes capable of breaking the DNA strand cutting it systematically in specific locations. The first enzyme found in the bacterium *Haemophilus influenzae*, showed ability to cut the genetic material of *Escherichia coli* and was named *HindII*. Enzymes have been isolated from bacterial strains differ by cleavage at specific restriction sites. The enzymes recognize sequences of four to eight bases. Enzymes that recognize restriction sites composed of four base pairs cleave DNA on average every 256 nucleotides (44 = 256). Those who recognize sites with 6 and 8 bp cleave DNA on average every 4096 and 65536 bp, respectively. However, this average can vary significantly, depending mainly on the base composition of DNA analyzed.

Preliminary evaluations of the cleavage sites, the relationship between the amount of enzyme and DNA, and exposure time are factors that determine the success of this step of RFLP technique. After digestion the samples receiving the loading buffer (0.25% bromophenol blue, 0.25% xylene cianol and 25% ficoll type - 400 - in water) and are subjected to electrophoresis (Ferreira & Grattapaglia, 1995).

The concentration of agarose gel used varies between 0.5% and 2% depending on the size of the fragments generated by digestion. For larger fragments, we use a lower concentration and when the fragments generated have low molecular weight, is used gels with higher concentration. The dye ethidium bromide (0.5 ug/ml) is added to the gel heated in order to promote the visualization of the fragments under ultraviolet light.

At the end of electrophoresis, the fragments should be transferred to a nylon membrane or nitrocellulose filter, according to the methodology Southern blotting (Southern, 1975): 1. Place the gel in alkaline solution (0.5 N NaOH, 1.5 M NaCl) for thirty minutes to break the hydrogen bonds of the double helix and allow hybridization with probes; 2. Transfer the gel to a neutral buffer solution (0.5 M Tris-Cl, 1.5 M NaCl, pH 7.5) for thirty minutes; 3. Transfer the fragments to the membrane by capillary action, which could last more than six hours; 4. Rinse the membrane in 10X SSC solutions and set in oven for thirty seconds ultraviolet and 5. Place the membrane to dry at room temperature and then subjected to treatment in vacuum furnace for three hours at 90°C and 6. Use immediately or store the membrane at 4°C.

The last step of the RFLP technique is the process of hybridization between the fragments generated by enzymatic digestion and the probes and subsequent exposure to X-ray. According to Ferreira & Grattapaglia (1995), the probes can be clones obtained by reverse transcription of mRNA, fragments of genomic DNA, fragments generated by amplification of known sequences or RAPD bands. The selection of clones is critical to the success of the technique. Depending on the type of probe used RFLP markers can show good results within the genome.

The membrane and the probes are placed in a common solution to the homologous sequences may hybridizing. This step is conducted for about twelve hours at 60°C. After this period the membranes are washed in SSC solution, dried and exposed to X-ray.

Through RFLP markers can be generated linkage maps used for mapping other traits of agronomic importance. Through them it is possible to detect associations between markers

Molecular Markers: Assisted Selection in Soybeans 159

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

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

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

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

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

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

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

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

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

follow-up of DNA, typically up to 4000pb can be synthesized using Taq polymerase.

caused a major revolution in the practice of molecular genetics.

systematics, fingerprint genotypes and marker-assisted selection.

thereby generating bands that will be visualized in gel electrophoresis.

is small, the efficiency of indirect selection will be higher.

possibility of replication of results.

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

new cycles. For most protocols, the number of cycles varies between thirty and sixty.

temperatures without being destroyed.

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 enzymatic action, can determine the degree of polymorphism.

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 evolution.

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 quality of technical and plants classified in 18 groups.

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 large scale.
