**4. Conclusion**

Soybean Kunitz trypsin inhibitor (KTI) protein is a small, monomeric and non -glycosylated protein containing 181 amino acid residues and is responsible for the inferior nutritional quality of unheated or incompletely heated soybean meal. Ti gene controls the presence or absence of KTI protein. SSR marker tightly linked to the Ti locus was identified and was confirmed in two ways. Two mapping populations were developed. Population 1 was derived from a cross between cultivar Jinpumkong2 (*TiTi*) and C242 (*titi*). Population 2 was made from a mating between cultivar Clark (*TiTi*) and C242. Each F2 seed from F1 plants was analysed electrophoretically to determine the presence of the KTI protein band. Twelve DNA markers (4 RAPD, 4 AFLP, and 3 SSR) and *Ti* locus were found to be genetically linked in population 1 consisted with 94 F2 individual plants. Three SSR markers (Satt409, Satt228, and Satt429) were linked with *Ti* locus within 10 cM. Satt228 marker was tightly linked with *Ti* locus. Satt228 marker was tightly linked within 0 - 3.7 cM of the *Ti* locus. Using several germplasms with *TiTi* or *titi* genotypes, Satt228 marker was confirmed. *TiTi* genotypes ('Jinpumkong2', 'Clark', and 'William') had allele1 and *titi* genotypes (PI196168, C242, W60 and PI157440) had allele2 in Satt228 marker analysis. 'Jinpumkong2', 'Clark', and 'William' (*TiTi* genotype) had Kunitz trypsin inhibitor protein of 21.5 kDa size and PI196168, C242, W60, and PI157440 (*titi* genotype) did not have the band in protein gel electrophoresis from the mature seed. Cosegregation between KTI protein (21.5 kDa size) and allele of Satt228 marker was observed in seven germplasms with different genetic background. This result indicates that Satt228 marker may effectively utilized to select the plant with *titi*  genotype. Also, Satt228 marker tightly linked to the *Ti* locus was confirmed in four different F5 populations. Four female parents (*Glycine max* L. cv. Jinpumkong2, Hannamkong, GS06, 20M183) of *TiTi* (KTI protein present) genotype and one male parent C242 of *titi* (KTI protein absent) genotype were used. Four different populations of F2 plants free of KTI protein were advanced to the F5 generation. Satt228 marker analysis showed the four female parents had the allele 1 (AA genotype) while the C242 male parent has the allele 2 (BB genotype). In seed, four parents had KTI protein and C242 had not KTI protein of 21.5 kDa. A total of 391 F5 plants derived from the four crosses (273 plants from Jinpunkong2 x C242, 17 plants from Hannamkong x C242, 45 plants from GS06 x C242, and 56 plants from 20M183 x C242) all have the allele 2 (BB genotype) for Satt228 marker. The 391 individual F6 seeds harvested from same individual F5 plants are also absent of the KTI protein. Complete cosegregation between the Satt228 marker allele and the *Ti* locus was observed in these four different populations. The objective of this research was to identify and to confirm a SSR marker tightly linked to the *Ti* locus for MAS breeding in different genetic populations and germplasms. So far, KTI free new soybean cultivars (Gaechuck#1, Gaechuck#2 and Jinnong#1) have been developed using Satt228 marker.
