**1. Introduction**

154 Soybean – Genetics and Novel Techniques for Yield Enhancement

Świątkiewicz S., Świątkiewicz M. (2009). Second generation of genetically modified plants in

Teshima, R., Akiyama, H., Okunuki, H., Sakushima, J.-I., Goda, Y., Onodera, H,, Sawada, J.-

Theodorou, M.K., Williams, B.A., Dhanoa, M.S., McAllan, A.B., France, J. (1994). A simple

Tudisco, R., Calabro', S., Terzi, V., Cutrignelli, M.I., Bovera, F., Piccolo, V., Zicarelli, F.,

Tudisco, R., Lombardi, P., Bovera, F., d'Angelo, D., Cutrignelli, M.I., Ma stellone, V., Terzi,

Tudisco, R., Ma stellone, V., Cutrignelli, M.I., Lombardi, P., Bovera, F., Mirabella, N.,

Van den Eede, G., Aarts, H., Buhk, H.J., Corthier, G., Flint, H.J., Hammes, W., Jacobsen, B.,

Vecchio, L., Cisterna, B., Malatesta, M., Martin, T.E., Biggiogera, M. (2994). Ultrastructural

Wiedemann, S., Lutz, B., Kurtz, H. F., Schwarz, J., Albrecht, C. (2006). In situ studies on the

Warwick, S.I., Legere, A., Simard, M.J., James, T. (2008). Do escaped transgenes persist in

Zelaya, I.A., Owen, M.D.K., VanGessel, M.J. (2007). Transfer of glyphosate resistance: Evidence of hybridisation in Conyza (Asteraceae). *Amer. J. Botany*, 94, pp. 660-673.

modified (GM) plants. *Food Chemistry and Toxicology*, 42, pp. 1127-1156. Van Hall, G. (2000). Lactate as a fuel for mitochondrial respiration. *Acta Physiologica* 

*Journal of Agricultural and Food Chemistry*, 48, pp. 2305-2312.

animal nutrition. *Medycyna Wet*, 65, pp. 460-465.

*Animal Nutrition*, 57, pp. 235–252.

*Biotechnologies,* Naples, Italy, Giugno 2004.

in their offsprings. *Animal*, 4, pp. 1662-1671.

rumen. *Journal of Animal Science,* 84, pp. 135-144.

population. *Molecular Ecology*, 17, pp. 1387-1395.

*Scandinava*, 168, pp. 643-656.

*Histochemistry*, 48, pp. 448–454.

enzymatic analysis. *Animal Science*, 82, pp. 193-199.

193.

glyphosate-tolerant corn is equivalent to that of conventional corn (Zea mays L.).

I. and Toyoda, M. (2000). Effect of GM and non-GM soybeans on the immune system of BN rats and B10a mice. *Journal of Food Hygiene Society Japan*, 41,pp. 188–

gas production method using a pressure transducer to determine the fermentation kinetics of ruminant feeds. *Animal Feed Science and Technology*, 48, pp. 185-197. Tony, M.A., Butschke, A., Broll, A., Grohman, L., Zagon, J., Halle, I., Danicke, S., Schauzu,

M., Hafez, H.M., Flachowsky, G. (2003). Safety assessment of Bt 176 maize on broiler nutrition: degradation of maize-DNA and its metabolic fate. *Archive of* 

Piccolo, G., Infascelli, F. (2004). In vitro fermentation kinetics of some genetically modified feeds. *Proceeding of 2nd International Meeting on Veterinary morpho-functional* 

V., Avallone, L., Infascelli, F. (2006). Genetically modified soybean in rabbit feeding: detection of DNA fragments and evaluation of metabolic effects by

Piccolo, G., Calabrò, S., Avallone, L., Infascelli, F. (2010). Fate of transgenic DNA and evaluation of metabolic effects in goats fed genetically modified soybean and

Midtvedt, T., van der Vossen, J., von Wright, A., Wackernagel, W., Wilcks A. (2004). The relevance of gene transfer to the safety of food and feed derived from genetically

analysis of testes from mice fed on genetically modied soybean. *European Journal of* 

time-dependent degradation of recombinant corn DNA and protein in the bovine

nature? The case of an herbicide resistance transgene in a weedy Brassica napa

Modern agriculture seeks increasing gains in productivity, due to great demand for food and the reduction of new agricultural frontiers. A major concern relates to fungal diseases and pest damage, and productivity growth necessarily implies reducing losses caused by these organisms.

Genetic improvement provides plants with different degrees of resistance, which can be used by farmers, making the most economical and efficient management. The process of obtaining resistant cultivars is usually done by the transfer of resistance alleles from exotic sources, which need further evaluation. This strategy has been used successfully in breeding programs for many years.

The evaluation process in plants is an improvement methodology with high cost, complex and subject to environmental variations. Another problem encountered concerns the manipulation of plant pathogens in a place where they occur. As an alternative to overcome the problems mentioned above are used molecular markers. With the development of research in molecular biology, there was the possibility of having one more tool in breeding programs, using DNA as the basic material.

The markers can be classified according to the methodology used to identify them: hybridization - RFLP (Restriction Fragment Length Polymorphism) or amplification of DNA - RAPD (Random Amplified Polymorphic DNA); SCAR (Sequence Characterized Amplified Regions); microsatellites (or SSR - Simple Sequence Repeats) and AFLP (Amplified Fragment Length Polymorphism). The markers are based on natural variation in DNA sequence and have Mendelian segregation.

The use of molecular markers was initiated in the last century, when Bateson & Punnett (1905) indicated the possibility of linkage between genes controlling characteristics of petal color and shape of pollen grain.The strategy of using molecular markers requires basic knowledge about the genetic nature of the trait studied, classifying it as a qualitative or quantitative (Ferreira & Grattapaglia, 1995), whose difference is based on the magnitude of the effect of replacing one allele by another in a given locus.

Molecular markers can be a useful tool to monitor the transfer of alleles of interest. In the early stages of intermediate and improving the process is efficient, but final confirmation is essential in field conditions (Alzate-Marin et al., 2005).

This need for phenotypic analysis requires quality in the polls so that the marker may reflect the field conditions. The test is performed with molecular markers using only a small

Molecular Markers: Assisted Selection in Soybeans 157

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

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,

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

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

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

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

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

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

period the membranes are washed in SSC solution, dried and exposed to X-ray.

depending mainly on the base composition of DNA analyzed.

subjected to electrophoresis (Ferreira & Grattapaglia, 1995).

promote the visualization of the fragments under ultraviolet light.

three hours at 90°C and 6. Use immediately or store the membrane at 4°C.

between alleles.

within the genome.

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

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 measure can also be evaluated using molecular markers.

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

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 others.

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