**3.3 SCAR (Sequence Characterized Amplified Regions)**

RAPD reactions can be performed easily, but problems related to standardization of equipment, reagents and protocols, often leads to difficulties for certain searches can be repeated. As referenced above, this may not be a big problem in a group of researchers careful (Alzate-Marin et al., 2005).

To overcome the inconsistent results with RAPD markers, polymorphic fragments obtained, have been cloned, sequenced and converted into SCAR markers (Sequence Characterized Amplified Regions) (Paran & Michelmore, 1992). According to the authors SCAR markers

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

MP 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 MP

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

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

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

RAPD reactions can be performed easily, but problems related to standardization of equipment, reagents and protocols, often leads to difficulties for certain searches can be repeated. As referenced above, this may not be a big problem in a group of researchers

To overcome the inconsistent results with RAPD markers, polymorphic fragments obtained, have been cloned, sequenced and converted into SCAR markers (Sequence Characterized Amplified Regions) (Paran & Michelmore, 1992). According to the authors SCAR markers

can be obtained in the presence or absence of the marker (Reiter et al., 1992).

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

determines resistance, respectively, 4.9 and 23.1 cM.

**3.3 SCAR (Sequence Characterized Amplified Regions)** 

careful (Alzate-Marin et al., 2005).

*Hind III*.

564 bp

performed in the early stages of development to occur without destroying the plants.

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., 2000).

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 characterize the new initiator.

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 obtained in Sambrook et al. (1989).

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 known that the bases are among the RAPD primers.

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

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

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 amplified fragments.

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. (1998) and Zhang & Stommel (2001).

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

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,

Molecular Markers: Assisted Selection in Soybeans 165

Are sequences consisting of repetitions of one to four nucleotides, which occur naturally in the genome, such as repeated (AT)n, (ATT)n. The genome of plants has, on average, ten times less than the microsatellite genome (Powell et al., 1996). The repeats are more common in plants (AT)n, (GA)n, (AC)n, (AAT)n and (AAC)n (Gupta & Varshney, 2000; Wang et al., 1994). The DNA from organelles has a low frequency of SSRs (1 per 317 Kb) (Wang et al., 1994). Microsatellites are present in coding regions and non-coding (Zane et al., 2002). The variation of n number of repeated elements generates a great amount of polymorphism. According Brondani et al. (1998), microsatellites have characteristics that result in benefits for the plant breeding: Nature co-dominant and multiallelic; highly polymorphic, allowing precise discrimination even of highly related; abundant and uniformly dispersed throughout the genome plants; can be analyzed by the PCR reaction. In plants one of the

Another important point is that the DNA sequences flanking the SSRs are conserved within the same species, allowing the selection of specific primers that amplify via PCR. The amplification using a pair of primers complementary to unique sequences that flank, resulting in an enormous fragment length polymorphism This size variation of PCR products is a consequence of the occurrence of different numbers of repeating units within structure of microsatellites (Ashkenazi et al., 2001; Cregan et al., 1999a; McCouch et al., 1997; Morgante & Olivieri, 1993). Thus, alleles may be determined for a given population. Homozygous individuals have the same number of repetitions in the chromosomes, while heterozygous individuals have different numbers of repeats in both chromosomes. Therefore, the locus is defined by the pair of primers and the various alleles by the size of

Some mistakes during DNA replication in different individuals of the same species, can provide a varying number of repeats within a microsatellite, which are different alleles. Currently, many species of plants already possess a set of microsatellite markers for use in

The practical use of microsatellite markers has occurred in human studies (Litt & Luty, 1989) and attracted the attention of plant breeders, since several studies have shown that microsatellites are widely distributed in the genome of the species (Brunel, 1994). According to Ferreira & Grattapaglia (1995), in eukaryotic genomes, these simple sequences are very

The initial protocols identified the microsatellite locus in clones of total genomic libraries using probes complementary to regions of interest, such as (AC) 10 and (AG) 20 (Rassmann et al., 1991). The amplification products are separated by electrophoresis, which in most cases, should be done on polyacrylamide gel because of the small size difference between fragments. The attainment of the primers is the most expensive step of the process of using

Each microsatellite locus can be analyzed individually or jointly with another, when the alleles of each locus have sizes sufficiently different to migrate into separate zones in the gel

Microsatellite markers are indicated to various kinds of analysis, because they are polymorphic in soybean, highly reproducible, co-dominant and by their low cost, considering that about 650 pairs of primers specific for soybeans are available on the market

Registration and the granting of rights to new varieties is usually done based on morphological and physiological characteristics, uniformity and stability, necessitating the

frequent, randomly distributed, besides being highly polymorphic genetic locus.

first findings was made by Nybom et al. (1992).

genetic studies (Akkaya et al., 1992; Cregan et al., 1999a).

microsatellite markers in marker assisted selection in plants.

amplified bands.

(Lanza et al., 2000).

(Cregan et al., 1999a).

which was resolved by digesting the fragments with the enzyme *HincII* (Figure 2). The technique was efficient because alleles from resistant parent were digested by *HincII* enzyme produced two fragments, one of 531 bp and another of 57 bp, while the susceptible parent stayed with the fragment of 588 bp. F1 plants heterozygous for the locus, showed a pattern of three bands. The same result was observed in F2 plants classified as heterozygous resistant.

The loss of polymorphism was recovered by enzymatic digestion and plants could be distinguished in homozygous recessive, homozygous dominant and heterozygous. The process becomes more expensive, but allows for the recovery of the polymorphism and the use of SCAR marker.

Fig. 2. SCAR monomorphic fragment of 588bp with and without enzymatic digestion (*HincII*): 1. resistant parent BR92-15454; 2. resistant parent BR92-15454 (digested); 3. susceptible parent IAC-11; 4.susceptible parent IAC-11(digested); 5. F1 plant, 6. F1 plant (digested); 7. heterozygotic resistant F2 plants; 8. heterozygotic resistant F2 plants (digested). Molecular pattern (MP), originating from the digestion of *λ* with the enzymes *EcoRI* and *Hind III*.

There are numerous studies to obtain SCAR markers linked to disease resistance of crops. In soybean there are the surveys conducted by Heer et al. (1998) who worked with resistance to soybean cyst nematode; Martins Filho et al. (2002), in studies on resistance to the fungus *Cercospora sojina*; Zheng et al. (2003) in relation to the mosaic virus and Carvalho et al. (2002) in the evaluation of soybean plants resistant to stem canker.
