**3.1 Screening of** *titi* **genotype using Satt228 marker**

Only two genotypes (PI 157440 and PI 196168) and two near isogenic lines (C242, W60) have been known as soybean genotypes with lacking Kunitz trypsin inhibitor protein (*titi* genotype). C242 is a near isogenic line derived from cultivar 'Clark' and W60 is a near isogenic line derived from cultivar 'William'.

Satt228 marker very tightly linked to *Ti* locus at distance of 0 cM was used to screen germplasms with *titi* genotype (Kunitz trypsin inhibitor protein absent) for marker confimation and testing the possibility of marker-assisted selection (MAS). Amplification patterns obtained from Satt228 marker using genomic DNA of four soybean strains (PI157440, PI196168, W60, and C242) with *titi* genotype (Kunitz trypsin inhibitor protein absent) and three cultivars ('Jinpumkong2', 'Clark', and 'William') with *TiTi* genotype (Kunitz trypsin inhibitor protein present) are shown in Figure 4-1A. Also, polyacrylamide gel banding patterns of protein extracted from random 10 seeds of these seven germplasms used are shown in Figure 4-1B. *TiTi* genotypes ('Jinpumkong2', 'Clark', and 'William') had allele1, however, *titi* genotypes (PI196168, C242, W60 and PI157440) had allele2 in the result of PCR by Satt228 marker (Figure 4-1A). *TiTi* genotypes ('Jinpumkong2', 'Clark', and 'William') had 21.5 kDa band that indicates Kunitz trypsin inhibitor protein, however *titi* genotypes (PI196168, C242, W60 and PI157440) did not have the band in of protein gel electrophoresis from the mature seed (Figure 4-1B). From the comparison of gel electrophoresis for Kunitz trypsin inhibitor protein (Figure 4-1B) and banding pattern amplified by Satt228 marker from the genomic DNA (Figure 4-1A), there was a strong agreement between protein band (21.5 kDa) for Kunitz trypsin inhibitor protein and banding pattern by Satt228 marker. All *TiTi* genotypes ('Jinpumkong2', 'Clark', and 'William') which shown 21.5 kDa protein band in protein electrophoresis of mature seed had

Identification and Confirmation of

summarized in Figure 5.

SSR Marker Tightly Linked to the Ti Locus in Soybean [*Glycine max* (L.) Merr.] 207

the greenhouse. F2 seeds per each cross were harvested from several F1 plants in November 2002. All F2 seeds per each cross were planted in the field in May 2003. F2 plants per each cross were harvested individually. Random F3 seeds from individual F2 plant per each cross were tested by SDS-PAGE protein analysis to detect Kunitz trypsin inhibitor protein. Individual F2 plants (F3 seeds) with free Kunitz trypsin inhibitor protein (*titi* genotype) per each cross were planted in the greenhouse and harvested individually in June 2004. Random F4 seeds from individual F3 plant harvested per each cross were planted in the field in June 2004. At maturity, F4 plants (F5 seeds) were harvested individually per each cross in November 2004. Random F5 seeds from individual F4 plant harvested per each cross were planted in the field in May 2005. Five parents and individual F5 plants per each cross were used to confirm the SSR marker tightly linked to *Ti* locus. Agronomical traits except for the Kunitz trypsin inhibitor protein were not considered in each generation. The pedigree for the development of the four populations lacking the Kunitz trypsin inhibitor protein is

Fig. 5. The pedigree of the four population development to confirm cosegregation between marker Satt228 and the *Ti* locus. F2 plants lacking the KTI protein from each population were selected and advanced to the next generation. All F5 plants have the *titi* genotype

(lacking Kunitz trypsin inhibitor protein). G.H is greenhouse.

the allele1 amplified by Satt228 marker from the genomic DNA. However, all *titi* genotypes (PI196168, C242, W60 and PI157440) which shown no 21.5 kDa protein band in electrophoresis of mature seed had allele2 amplified by Satt228 marker from the genomic DNA.

Fig. 4. Pattern of genomic DNA amplification by Satt228 marker using leaf tissue of germplasms (1A) and pattern of polyacrylamide protein gel electrpophoresis extracted from 10 random seeds harvestes (1B). M; molecular marker, S; Kunitz trypsin inhibitor protein (Sigma, product number: T6522 ). 1:Jinpumkong2(*TiTi*), 2:Clark (*TiTi*), 3:PI196168 (*titi*), 4: William (*TiTi*), 5: C242 (*titi*), 6: W60 (*titi*), 7: PI157440 (*titi*). +: present of KTI protein and -: absent of KTI protein.

Moraes et al. (2006) reported specific DNA marker designed to detect the absence of SKTI protein. For markers to be most useful in breeding programs, they should reveal polymorphism in different genetic backgrounds, which is referred to as marker validation (Sharp et al., 2001). Specific DNA marker designed to detect the absence of SKTI protein reported by Moraes et al. (2006) was not valid between germplasms of *TiTi* (SKTI protein present) and *titi* (SKTI protein absent) genotype used in this study. No polymorphism was observed among germplasms used. However, Cosegregation between allele of Satt228 marker and presence or absence of SKTI protein in several soybean germplasms of *TiTi* and *titi* genotyes was observed (Figure 4-1A and 1B). This results indicate that selection of germplasms or lines with lacking Kunitz trypsin inhibitor protein is possible by Satt228 marker analysis.
