**1. Introduction**

200 Soybean – Genetics and Novel Techniques for Yield Enhancement

Sinclair, T.R., Purcell, L.C., King, C.A. , Sneller, C.H., Chen, P. & Vadez, V. (2007). Drought

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common bean cultivars in relation to drought tolerance in environments with contrasting soil types. *Experimental Agriculture,* Vol. 25, No. 2, (April 1989), pp. 249-

Selection of soybean genotypes using morphological markers. *International Journal* 

and analysis of approximately 40,000 soybean cDNA clones from a full-lengthenriched cDNA library .*DNA Research,* Vol. 15, No. 6, (December 2008) pp. 333-346,

Beer, M., Schlüter, U., Kunert, K.J. & Foyer, C.H. (2008). Regulation of respiration and the oxygen diffusion barrier in soybean protect symbiotic nitrogen fixation from chilling-induced inhibition and shoots from premature senescence *Plant* 

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integrated high-density linkage map of soybean with RFLP, SSR, STS, and AFLP markers using a single F2 population. *DNA Research,* Vol. 14, No. 6, pp. 257–269, Soybean [*Glycine max* (*L.*) Merr.,] is considered a high quality source of oil and protein for food and feed. However, the several antinutritional factors ( lipoxygenase, trypsin inhibitor, lectin, and P34 allergen protein) present in raw mature soybean seeds. Soybean Kunitz trypsin inhibitor (KTI) protein has been proposed as one of the major antinutritional factor (Westfall and Hauge, 1948). KTI protein is a small, monomeric and non-glycosylated protein containing 181 amino acid residues. This 21.5 kDa non-glycosylated protein was first isolated and crystallized from soybean seeds by Kunitz (1945). KTI protein can cause the induction of pancreatic enzyme hypersecretion and a fast stimulation of pancreas growth, which is histologically described as pancreatic hypertrophy and hyperplasia (Liencer, 1995). Also, KTI may cause unfavorable physiological effects (Vasconcelos et al., 2001) and decrease weight gain in animals (Palacios et al., 2004). Proper heat processing is required to destroy KTI protein. However, excessive heat treatment may lower amino acid availability. The genetic removal of the KTI protein will improve the nutritional value of soybean. From the USDA germplasm collection, two soybean accessions (PI157440 and PI196168) lacking the KTI protein have been identified (Orf and Hymowitz, 1979). Based on the availability of soybean null lines lacking the KTI protein, it was suggested that KTI protein is not essential for soybean growth or development. Five electrophoretic forms of KTI have been discovered. The genetic control of four forms, *Ti a*, *Ti b*, *Ti c*, and *Ti d*, has been reported as a codominant multiple allelic series at a single locus (Singh et al., 1969; Hymowitz and Hadley, 1972; Orf and Hymowitz, 1979). Orf and Hymowitz (1979) found that the fifth form does not exhibit a soybean trypsin inhibitor-A2 band and is inherited as a recessive allele designated *ti*. Studies of amino acid and nucleotide sequences of polymorphic variants of KTI have revealed that there is a large sequence differences in nine amino acid residues between *Ti a* and *Ti b* (Song et al., 1993; Wang et al., 2004). Each *Ti c*, *Ti d* and *Ti e* differ by only one amino acid from *Ti a* type and *Ti f* differs by one amino acid from *Ti b* type (Wang et al., 2004). The *Ti* locus has been located on linkage group 9 in the classical linkage map of soybean (Hildebrand et al., 1980; Kiang, 1987), which is integrated in molecular linkage map A2 (chromosome number 8) of the USDA/Iowa State University soybean molecular linkage map (Cregan et al., 1999).

Identification and Confirmation of

**2.4 Genetic linkage analysis** 

**2.5 Detection of Satt228 marker** 

recessive gene.

**2.3 DNA extraction and DNA marker analysis** 

population 1 including *Ti* locus were used in population 2.

using the Kosambi (Kosambi, 1944) mapping function.

SSR Marker Tightly Linked to the Ti Locus in Soybean [*Glycine max* (L.) Merr.] 203

F2 seeds tested for kunitz trypsin inhibitor protein were planted in the field on May, 2004. Young leaves were collected from the 94 individual F2 plants germinated among 98 F2 seeds and parent plants in population 1. In population 2, random 97 F2 seeds among 243 F2 seeds were planted in the greenhouse on April, 2005. Young leaves were collected from the 94 individual F2 plants. Genomic DNA was extracted from finely ground leaf tissue by means of a modified CTAB procedure (Saghai Maroof et al., 1984). For the analysis of random amplified polymorphic DNA (RAPD) markers, One-thousand 10-mer oligonucleotide primers were obtained from Operon Technologies (Alameda, U.S.A). For the analysis of simple sequence repeat (SSR) marker, total 35 SSR primers were selected from the A2 soybean molecular linkage map (Cregan et al., 1999) that contains *Ti* locus. Satt primers selected were synthesized by Bioneer, Inc. (Korea). For the analysis of amplified fragment length polymorphic (AFLP) markers, 342 primer sets were used. Amplification and electrophoresis for RAPD, SSR, and AFLP markers was performed as described by Kim et al., (2003). Based on the results of F1 seed genotype for kunitz trypsin inhibitor, the present and absent bulk populations from F2 plant population were made (Michelmore et al., 1991). The present and absent bulk population contained twenty F2 individuals each, which were selected on the basis of the kunitz trypsin inhibitor protein electrophoresis, respectively. RAPD, SSR, and AFLP markers were used in population 1. Only the markers linked in

Primers that distinguished the bulks and the parents were tested on the entire F2 population. Marker (RAPD, AFLP, and SSR) data obtained from 94 F2 progenies of population 1 and 2 were used to construct genetic linkage map including *Ti* locus using the computer program MAPMAKER v. 3.0 (Lander et al., 1987). Markers were assigned to group using the "Group" command, with a LOD score of 4.0 and maximum recombination distance of 50 cM. Once markers were assigned to a given linkage group, the most linkage marker order within the group was determined using the "Compare" command. Marker orders within each linkage group were ascertained by use of "Ripple" command. Map distance (cM) were computed

The banding patterns of kunitz trypsin inhibit protein (SKTI) that appeared in the parents and F2 seeds from the cross between cultivar Jinpumkong2 and C242 (population 1) are shown in Figure 1. Jinpumkong2 parent had band in 21.5 KDa position and the band was segregated in F2 seeds. The observed data for population 1 were 72 seeds with SKTI protein band and 26 seeds with no SKTI protein band (χ2=0.12, P=0.70-0.80). For population 2, the observed data were 185 seeds with SKTI protein band and 58 seeds with no SKTI protein band (χ2=0.17, P=0.70-0.80). These observations fit the expected 3 : 1 ratio for the presence or absence of the SKTI protein band. Earlier studies have shown that the null phenotype of SKTI is inherited as a recessive allele designated *ti* (Orf and Hymowitz, 1979). The segregation ratios of 3 : 1 observed in the F2 seed (population 1 and 2) and the Chi-square values strongly suggest that kunitz trypsin inhibitor protein band is controlled by a single

DNA markers have become fundamental tools for research involving soybean improvement programs. Microsatellites or simple sequence repeat (SSR) markers are highly polymorphic, abundant, and distributed throughout the genome (Cregan et al., 1999). With the development and public release of SSR primers, SSR markers have become available on molecular soybean linkage group (Cregan et al., 1999). Molecular markers tightly linked to desired genes are a valuable tool to detect genotypes of interest, saving time and resources. Marker assisted selection (MAS) using DNA markers instead of phenotypic assays reduces cost and increases the precision and efficiency of subsequent selection steps applied in breeding. To date, detection of the KTI protein free genotypes has been based on SDS-PAGE gel electrophoresis analysis of crude protein from mature seeds, however, with this method, test samples are restricted to proteins from mature soybean seeds. This is a time-consuming process, which is not possible in the early seedling stages of the corresponding population. SSR markers tightly linked to the *Ti* locus were identified and confirmed in soybean populations for marker assisted selection. If a marker linked to the *Ti* locus can be confirmed, then selection for KTI protein free genotypes might be performed at early seedling stages with relative ease.
