**3.4 Microsatellites – SSR (Simple Sequence Repeat)**

Another type of molecular marker is the microsatellite or simple sequence repeat (SSR). Microsatellites or STR or SSRP (Simple Sequence Repeat Polymorphisms) or STMS (Sequence Tagged Microsatellite Sites) are some of the dominions assigned to these molecular markers. The use of these markers has been increasing, due to the fact that you use the PCR technique, co-dominant and present with a relatively high frequency within the plant genome (Akkaya et al., 1992).

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

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

MP 1 2 3 4 5 6 7 8

588 bp

531-bp

57 bp

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

in the evaluation of soybean plants resistant to stem canker.

**3.4 Microsatellites – SSR (Simple Sequence Repeat)** 

plant genome (Akkaya et al., 1992).

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)

Another type of molecular marker is the microsatellite or simple sequence repeat (SSR). Microsatellites or STR or SSRP (Simple Sequence Repeat Polymorphisms) or STMS (Sequence Tagged Microsatellite Sites) are some of the dominions assigned to these molecular markers. The use of these markers has been increasing, due to the fact that you use the PCR technique, co-dominant and present with a relatively high frequency within the

resistant.

564 bp

125 bp

*Hind III*.

use of SCAR marker.

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

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 amplified bands.

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

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

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 microsatellite markers in marker assisted selection in plants.

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 (Lanza et al., 2000).

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 (Cregan et al., 1999a).

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

Molecular Markers: Assisted Selection in Soybeans 167

of genetic purity of seed lots of soybean: Sat\_128, Satt070, Sat\_085, Satt079, Sat\_110, Satt184, Satt005, Satt141, Satt186, Satt146, Sat\_064, Sat\_094, Satt163, Satt181, Sat\_105, Satt162, Satt183,

The following is related in detail, the protocol used by Schuster et al. (2004): Total volume of 10 or 25 mL, containing 12.5 mM Tris-HCl (pH 8.3), 62.5 mM KCl, MgCl2, 2.5 mM, 125 mM of each deoxinucleotídios (dATP, dTTP, dGTP and dCTP), 0.2 µM of each primer (sense and antisense), a unit of Taq DNA polymerase and 30 ng of DNA. The total volume of the reaction was 25 mL when the separation was performed in 3% agarose gel and 10 mL when the separation was performed on 10% polyacrylamide gel. The amplifications were performed in Perkin Elmer 9600 thermocycler, programmed for an initial step of 7 min at 72°C, followed by 30 cycles of 1 min at 94°C, 1 min at 50°C and 2 min at 72°C. Finally, a step of 7 min at 72°C. The amplified fragments were separated by electrophoresis on 3% agarose gel containing ethidium bromide etídio (0,2 μg/mL) and 1X TBE buffer (90 mM Tris-borate, 2 mM EDTA), or 10% polyacrylamide gel in TBE buffer. In this case, the gels were stained after the run in TBE containing ethidium bromide (2μg/mL).The gels were photographed

The authors concluded that analysis of DNA extracted from seeds, considered atypical by the method of visual analysis with microsatellite markers is effective in determining the genetic purity of seed lots of soybean, and that the use of proteinase K and RNase in the process of extraction allows to obtain greater quantities and better quality of DNA for

Fuganti et al. (2004) conducted research towards the identification of microsatellite molecular markers for selection of soybean genotypes resistant to the nematode *Meloydogyne javanica*. The authors concluded at the time that the markers, SOYHSP 176 and Satt 114, the linkage group F of soybean, can be considered potential markers to be used in the process of

Hoshino et al. (2002) performed an extensive review of the microsatellite markers and reported that despite its widespread popularity there are certain limitations: 1. Identification of genotypes: usually performed by determining the length of the PCR product. The polymorphism is attributed to changes in the number of repeating units, however, it is possible that small insertions or deletions occur in adjacent regions that did not necessarily alter the number of repetitions, but change the length of the fragment generated. In such cases, determining the number of repetitions from a cloned fragment and its subsequent use for amplification in other species without sequencing, can lead to mistakes in the estimation of genetic distances; 2. null alleles: the occurrence of point mutations, insertions or deletions at the site of pairing of the initiator can prevent the amplification of a given microsatellite locus. If changes are not fixed in the population, only a portion of the alleles will be amplified; 3. mutation rate: Positive correlations between the length of the microsatellite sequence and mutation rate are not always true, and can cause errors in the estimation of genetic distances; 4. Frequency of different organisms: The application of this marker in a growing number of organisms has revealed that the abundance of repetitive sequences varies significantly between species. While some species contain a sufficient number of microsatellites for population studies, others have few sequences of this kind; 5. Artifacts PCR: Theoretically, the PCR technique allows the amplification of microsatellites from a single cell, but the amplification of microsatellites from small amounts of DNA are associated with high frequency of errors. Two common types of mistakes in this case are: the amplification of the alleles with incorrect length, and no amplification of one allele in

marker assisted selection of soybean genotypes resistant to root knot nematode

Satt167, Sat\_099, Satt156, Satt150, Satt175, and Satt136 Sat\_127.

under ultraviolet light.

analysis.

distinction in at least one of them. However, most morphological traits are quantitative traits, with its expression being altered by environmental factors. Moreover, the registration number is increasing rapidly, making it impossible for authorities to compare the new varieties efficiently with existing ones. The marker has been proven useful for identifying varieties and tested for various crops such as soybeans.

The microsatellite markers have been used in soybean for mapping specific genes that determine agronomic traits, and also to identify QTLs (Quantitative Trait Loci) of economic importance, involved in grain yield and genetic resistance to pests and diseases, which are characteristics of complex inheritance (Yuan et al., 2002).

The development of genetic maps is considered one of the applications of greatest impact in the technology of molecular markers for genetic analysis of species and, potentially, in plant breeding. In this context, the genetic maps enable: Full coverage and analysis of genomes; decomposition of complex genetic traits into their Mendelian components; location of genomic regions that control traits of importance; quantification of the effect in these regions studied feature, directing all this information to use in breeding programs.

Some quantitative trait loci (QTL) for resistance to some important diseases of soybean, were mapped in the chromosomal region adjacent to the locus of resistance to leaf rust *Rpp5.* Near the marker Satt009, were mapped QTL for resistance to the pathogen *Sclerotinia sclerotiorum*, the fungus that causes white mold in soybean (Arahana et al., 2001), and an adjacent chromosomal region, is mapped a QTL for resistance to the cyst nematode, which causes the most serious threat to soybean production (Concibido et al., 1997).

Mekesem et al. (2000) constructed a map of high saturation of three genomic regions in soybean, which contains the *Rhg4* and *rhg1* alleles that confer resistance to SCN, and the region containing the *Rfs* allele, which confers resistance to *Fusarim solani* f. sp. *glycines*.

In linkage group N, 3.2 cM from Satt009 marker was mapped a locus of resistance to *Phytophthora sojae*, the pathogen that causes root rot in soybean and marker linked to Satt080, and a QTL for resistance to *Fusarium solani* f. sp. *glycines* (Njiti et al., 2002).

Other microsatellite markers linked to various diseases of soybean have been reported in the literature. Mudge et al. (1997) concluded that microsatellite flanking Satt038 and Satt130 allele *rhg1*. Cregan et al. (1999b) detected the SSR Sat-168 and Satt 309 delimiting *rgh1*. The marker Satt215 was found to be linked to the gene *Rbs1*, which confers resistance to black pod-of-staff, with selection efficiency of 88% (Bachman et al., 2001). A new gene, RCSPeking, which confers resistance to stain-frog-eye, was mapped to 1.1 cM of marker Satt244, on linkage group G (Yang et al., 2001). Funganti et al. (2004) identified the marker Satt114, linked to the nematode resistance locus of the root knot nematode (*Meloydogyne javanica*), in linkage group F.

Garcia et al. (2008) mapped the locus *Rpp5* in three different populations of soybean (PI 200456, PI 471904 and PI 200526), and obtained: six markers linked to this locus in population PI 200456 (Satt530, Sat\_208, Sat\_166, Sat\_275, and Sat\_280 Sat\_266 ) and five in PI471904 (Satt530, Sat\_208, Sat\_166, Sat\_275, Sat\_280) and three in PI 200526 (Sat\_166, and Sat\_275 Sat\_280).

Morceli et al. (2008) identified two new microsatellite markers potentially associated with resistance of soybean to soybean rust caused by *Phakopsora pachyrhizi*. The authors evaluated the markers on individual plants, and found the link to *Rpp5* gene and are present on linkage group N of soybean. The efficiency of selection was determined for all markers linked to gene *Rpp5*, and the combination of the markers Sat\_275 + Sat\_280 was 100%.

Schuster et al. (2004) used markers for determining genetic purity of seed lots of soybean. The authors presented a table listing the primers used more frequently in the determination

distinction in at least one of them. However, most morphological traits are quantitative traits, with its expression being altered by environmental factors. Moreover, the registration number is increasing rapidly, making it impossible for authorities to compare the new varieties efficiently with existing ones. The marker has been proven useful for identifying

The microsatellite markers have been used in soybean for mapping specific genes that determine agronomic traits, and also to identify QTLs (Quantitative Trait Loci) of economic importance, involved in grain yield and genetic resistance to pests and diseases, which are

The development of genetic maps is considered one of the applications of greatest impact in the technology of molecular markers for genetic analysis of species and, potentially, in plant breeding. In this context, the genetic maps enable: Full coverage and analysis of genomes; decomposition of complex genetic traits into their Mendelian components; location of genomic regions that control traits of importance; quantification of the effect in these regions

Some quantitative trait loci (QTL) for resistance to some important diseases of soybean, were mapped in the chromosomal region adjacent to the locus of resistance to leaf rust *Rpp5.* Near the marker Satt009, were mapped QTL for resistance to the pathogen *Sclerotinia sclerotiorum*, the fungus that causes white mold in soybean (Arahana et al., 2001), and an adjacent chromosomal region, is mapped a QTL for resistance to the cyst nematode, which

Mekesem et al. (2000) constructed a map of high saturation of three genomic regions in soybean, which contains the *Rhg4* and *rhg1* alleles that confer resistance to SCN, and the region containing the *Rfs* allele, which confers resistance to *Fusarim solani* f. sp. *glycines*. In linkage group N, 3.2 cM from Satt009 marker was mapped a locus of resistance to *Phytophthora sojae*, the pathogen that causes root rot in soybean and marker linked to

Other microsatellite markers linked to various diseases of soybean have been reported in the literature. Mudge et al. (1997) concluded that microsatellite flanking Satt038 and Satt130 allele *rhg1*. Cregan et al. (1999b) detected the SSR Sat-168 and Satt 309 delimiting *rgh1*. The marker Satt215 was found to be linked to the gene *Rbs1*, which confers resistance to black pod-of-staff, with selection efficiency of 88% (Bachman et al., 2001). A new gene, RCSPeking, which confers resistance to stain-frog-eye, was mapped to 1.1 cM of marker Satt244, on linkage group G (Yang et al., 2001). Funganti et al. (2004) identified the marker Satt114, linked to the nematode resistance locus of the root knot nematode (*Meloydogyne javanica*), in

Garcia et al. (2008) mapped the locus *Rpp5* in three different populations of soybean (PI 200456, PI 471904 and PI 200526), and obtained: six markers linked to this locus in population PI 200456 (Satt530, Sat\_208, Sat\_166, Sat\_275, and Sat\_280 Sat\_266 ) and five in PI471904 (Satt530, Sat\_208, Sat\_166, Sat\_275, Sat\_280) and three in PI 200526 (Sat\_166, and

Morceli et al. (2008) identified two new microsatellite markers potentially associated with resistance of soybean to soybean rust caused by *Phakopsora pachyrhizi*. The authors evaluated the markers on individual plants, and found the link to *Rpp5* gene and are present on linkage group N of soybean. The efficiency of selection was determined for all markers linked to gene *Rpp5*, and the combination of the markers Sat\_275 + Sat\_280 was 100%. Schuster et al. (2004) used markers for determining genetic purity of seed lots of soybean. The authors presented a table listing the primers used more frequently in the determination

studied feature, directing all this information to use in breeding programs.

causes the most serious threat to soybean production (Concibido et al., 1997).

Satt080, and a QTL for resistance to *Fusarium solani* f. sp. *glycines* (Njiti et al., 2002).

varieties and tested for various crops such as soybeans.

characteristics of complex inheritance (Yuan et al., 2002).

linkage group F.

Sat\_275 Sat\_280).

of genetic purity of seed lots of soybean: Sat\_128, Satt070, Sat\_085, Satt079, Sat\_110, Satt184, Satt005, Satt141, Satt186, Satt146, Sat\_064, Sat\_094, Satt163, Satt181, Sat\_105, Satt162, Satt183, Satt167, Sat\_099, Satt156, Satt150, Satt175, and Satt136 Sat\_127.

The following is related in detail, the protocol used by Schuster et al. (2004): Total volume of 10 or 25 mL, containing 12.5 mM Tris-HCl (pH 8.3), 62.5 mM KCl, MgCl2, 2.5 mM, 125 mM of each deoxinucleotídios (dATP, dTTP, dGTP and dCTP), 0.2 µM of each primer (sense and antisense), a unit of Taq DNA polymerase and 30 ng of DNA. The total volume of the reaction was 25 mL when the separation was performed in 3% agarose gel and 10 mL when the separation was performed on 10% polyacrylamide gel. The amplifications were performed in Perkin Elmer 9600 thermocycler, programmed for an initial step of 7 min at 72°C, followed by 30 cycles of 1 min at 94°C, 1 min at 50°C and 2 min at 72°C. Finally, a step of 7 min at 72°C. The amplified fragments were separated by electrophoresis on 3% agarose gel containing ethidium bromide etídio (0,2 μg/mL) and 1X TBE buffer (90 mM Tris-borate, 2 mM EDTA), or 10% polyacrylamide gel in TBE buffer. In this case, the gels were stained after the run in TBE containing ethidium bromide (2μg/mL).The gels were photographed under ultraviolet light.

The authors concluded that analysis of DNA extracted from seeds, considered atypical by the method of visual analysis with microsatellite markers is effective in determining the genetic purity of seed lots of soybean, and that the use of proteinase K and RNase in the process of extraction allows to obtain greater quantities and better quality of DNA for analysis.

Fuganti et al. (2004) conducted research towards the identification of microsatellite molecular markers for selection of soybean genotypes resistant to the nematode *Meloydogyne javanica*. The authors concluded at the time that the markers, SOYHSP 176 and Satt 114, the linkage group F of soybean, can be considered potential markers to be used in the process of marker assisted selection of soybean genotypes resistant to root knot nematode

Hoshino et al. (2002) performed an extensive review of the microsatellite markers and reported that despite its widespread popularity there are certain limitations: 1. Identification of genotypes: usually performed by determining the length of the PCR product. The polymorphism is attributed to changes in the number of repeating units, however, it is possible that small insertions or deletions occur in adjacent regions that did not necessarily alter the number of repetitions, but change the length of the fragment generated. In such cases, determining the number of repetitions from a cloned fragment and its subsequent use for amplification in other species without sequencing, can lead to mistakes in the estimation of genetic distances; 2. null alleles: the occurrence of point mutations, insertions or deletions at the site of pairing of the initiator can prevent the amplification of a given microsatellite locus. If changes are not fixed in the population, only a portion of the alleles will be amplified; 3. mutation rate: Positive correlations between the length of the microsatellite sequence and mutation rate are not always true, and can cause errors in the estimation of genetic distances; 4. Frequency of different organisms: The application of this marker in a growing number of organisms has revealed that the abundance of repetitive sequences varies significantly between species. While some species contain a sufficient number of microsatellites for population studies, others have few sequences of this kind; 5. Artifacts PCR: Theoretically, the PCR technique allows the amplification of microsatellites from a single cell, but the amplification of microsatellites from small amounts of DNA are associated with high frequency of errors. Two common types of mistakes in this case are: the amplification of the alleles with incorrect length, and no amplification of one allele in

Molecular Markers: Assisted Selection in Soybeans 169

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

**/Primer** 

**Base sequence** 

Adapter 5'-C T C G T A G A C T G C G T

Adapter 5'-G A C G A T G A G T C C T G

Primer 5'-G A T G A G T C C T G A G T

Adapter 5'-C T C G T A G A C T G C G T A C A T G C A-3'

Primer 5'-G A C T G C G T A C A T G C

Adapter 5'-C T C G T A G A C T G C G T

Primer 5'-G A C T G C G T A C C A G C

C A G G C C-3'

Primer 5'-G A C T G C G T A C A G G C

Adapter 5'-G A C G A T G A G T C C T G

Primer 5'-C G A T G A G T C C T G A C C G A **E**-3'

3'-C A T C T G A C G C A T G G

3'-T A C T C A G G A C T C A T-

3'-C A T C T G A C G C A T G T-

3'-C T G A C G C A T G G T C G

3'-C A T C T G A C G C A T G T-

3'-T A C T C A G G A C T G G

A C C-3'

T T A A-5' Primer 5'-G A C T G C G T A C C A A T

T C **E**-3'

A G-3'

A A **E**-3'

A G **E**-3'

A C C-3'

T T **E**-3'

C C **E**-3'

A C-3'

C-5'

Table 1. Restriction sites, sequences of adapters and primers used for six enzymes in AFLP

A-5'

3'...C↑C C G G G...5' Adapter 5'-T C G T A G A C T G C G T A

5'

5'

5'

primers used for six enzymes in AFLP analysis (Table 1).

3'...C T T A A↑G...5'

3'...A A T↑T...5'

3'...G↑A C G T C...5'

3'...T T C G A↑A...5'

3'...AGC↑T...5'

analysis (E: arbitrary nucleotide used in the pre-amplification).

*EcoRI* 5'...G↓A A T T C...3'

*MseI* 5'...T↓T A A...3'

*PstI* 5'...C T G C A↓G...3'

*HindIII* 5'...A↓A G C T T...3'

*ApaI* 5'...G G G C C↓C...3'

*TaqI* 5'...T↓C G A...3'

**Restriction site Adapter** 

**Restriction enzyme** 

heterozygotes. The authors asserted that this type of marker is a valuable tool for analysis of genetic variability in germplasm of plant species and therefore its maintenance.

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

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 use.
