**3.2 Confirmation of Satt228 marker in four different soybean populations**

Cosegregation between Satt228 marker and Ti locus was confirmed in four different populations. Two cultivars (Jinpumkong2, Hannamkong) and two landraces (GS06, 20M183) have Kunitz trypsin inhibitor protein (*TiTi* genotype) in their mature seeds. The C242 parent is a clark-derived near isogenic line and does not have Kunitz trypsin inhibitor protein (*titi* genotype) in the mature seeds. Four different populations were developed. Four female parents (Jinpumkong2, Hannamkong, GS06, 20M183) and one male parent C242 were crossed in the greenhouse in June 2002. F1 seeds from the cross of Jinpumkong2 x C242, Hannamkong x C242, GS06 x C242, and 20M183 x C242 were obtained and planted in

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

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

3:PI196168 (*titi*), 4: William (*TiTi*), 5: C242 (*titi*), 6: W60 (*titi*), 7: PI157440 (*titi*). +: present

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

Cosegregation between Satt228 marker and Ti locus was confirmed in four different populations. Two cultivars (Jinpumkong2, Hannamkong) and two landraces (GS06, 20M183) have Kunitz trypsin inhibitor protein (*TiTi* genotype) in their mature seeds. The C242 parent is a clark-derived near isogenic line and does not have Kunitz trypsin inhibitor protein (*titi* genotype) in the mature seeds. Four different populations were developed. Four female parents (Jinpumkong2, Hannamkong, GS06, 20M183) and one male parent C242 were crossed in the greenhouse in June 2002. F1 seeds from the cross of Jinpumkong2 x C242, Hannamkong x C242, GS06 x C242, and 20M183 x C242 were obtained and planted in

protein (Sigma, product number: T6522 ). 1:Jinpumkong2(*TiTi*), 2:Clark (*TiTi*),

**3.2 Confirmation of Satt228 marker in four different soybean populations** 

of KTI protein and -: absent of KTI protein.

marker analysis.

DNA.

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 summarized in Figure 5.

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.

Identification and Confirmation of

absent of KTI protein.

SSR Marker Tightly Linked to the Ti Locus in Soybean [*Glycine max* (L.) Merr.] 209

Fig. 6. Pattern of genomic DNA amplification by Satt228 marker using leaf tissue of parent and individual F5 plants (A, C, E, G) and pattern of polyacrylamide protein gel using protein extracted from parents and 10 random seeds of individual F6 seed harvested (B, D, F, H). A and B, P1: 'Jinpumkong2' and P2: C242; C and D, P1: 'Hannamkong' and P2: C242; E and F, P1:GS06 and P2: C242; G and H, P1:20M183 and P2: C242. M; molecular marker, S; Kunitz trypsin inhibitor protein (Sigma, product number: T6522 ), +; present of KTI protein, -;

polyacrylamide gel banding patterns of protein extracted from the mixture of 10 random F6 seeds harvested from each F5 plant are shown in Figure 6 (G and H). The P1 parent (20M183) had the AA genotype while the P2 parent (C242) had the BB genotype (G of Figure 6). All 56 individual F5 plants were shown only to have the BB genotype pattern for the Satt228

Segregation patterns of genomic DNA amplification by the Satt228 marker using young leaf tissue of four parents and several individual F5 plants (A, C, E, G) and patterns of polyacrylamide protein gel using protein extracted from 10 random seeds of four parents and individual F6 harvested (B, D, F, H) are shown in Figure 6. The bands amplified by the Satt228 marker are clearly detecting the AA and BB genotypes. The seed protein band by the SDS-PAGE is a little different in color density according to each population, staining time of Coomassie blue and protein content. However, the detection of the presence or absence of the Kunitz trypsin inhibitor protein was very clear because the Kunitz trypsin inhibitor protein is controlled by single gene and is not influenced by environment.

Satt228 marker analysis was conducted on the genomic DNA of the parents and the 273 individual F5 plants lacking the Kunitz trypsin inhibitor protein derived from the cross of 'Jinpumkong2' (*TiTi*) and C242 (*titi*). After harvesting at maturity, SDS-PAGE electrophoresis using crude protein extracted from ten random F6 seeds of each F5 plants and parents was performed to detect Kunitz trypsin inhibitor protein of size 21.5 kDa. DNA banding pattern of the Satt228 marker and polyacrylamide gel banding patterns of the protein is shown in Figure 6 (A and B). The P1 parent (Jinpumkong2) had the AA genotype (allele 1) and the P2 parent (C242) had BB genotype (allele 2) for Satt228 marker. All 273 individual F5 plants were shown only to have the BB genotype (A of Figure 6). This indicated all 273 F5 progenies had the *titi* genotype and contained no Kunitz trypsin inhibitor protein. Also, the P1 parent had Kunitz trypsin inhibitor protein of 21.5 kDa size and the P2 parent did not have the KTI protein (B of Figure 6). All 273 individual F5 plants did not have the Kunitz trypsin inhibitor protein of 21.5 kDa size (B of Figure 6). Amplification patterns obtained from the Satt228 marker using genomic DNA of 17 individual F5 plants derived from the cross of Hannamkong (*TiTi*) and C242 (*titi*) and polyacrylamide gel banding patterns of protein extracted from the mixture of 10 random F6 seeds harvested from each F5 plant are shown in Figure 6 (C and D). The P1 parent (Hannamkong) had a AA genotype while the P2 parent (C242) had the BB genotype (C of Figure 6). All 17 individual F5 plants derived from cross of Hannamkong and C242 showed only the BB genotype pattern for Satt228 marker analysis (C of Figure 6). This indicated all 17 F5 progenies had the *titi* genotype and no Kunitz trypsin inhibitor protein. For the protein analysis, the P1 parent had the 21.5 kDa Kunitz trypsin inhibitor protein while the P2 parent did not have the KTI protein in polyacrylamide protein (D of Figure 6). All 17 individual F5 plants did not have the 21.5 kDa Kunitz trypsin inhibitor protein within their F6 seed samples (D of Figure 6). Amplification patterns by the Satt228 marker using genomic DNA of 45 individual F5 plants derived from the cross of GS06 (*TiTi*) and C242 (*titi*) and polyacrylamide gel banding patterns of protein extracted from the mixture of 10 random F6 seeds harvested from each F5 plant are shown in Figure 6 (E and F). The P1 parent (GS06) had the AA genotype and the P2 parent (C242) had the BB genotype (E of Figure 6). All 45 individual F5 plants derived from the cross of GS06 and C242 displayed only the BB genotype pattern for marker Satt228 (E of Figure 6). Also, the P1 parent had the 21.5 kDa Kunitz trypsin inhibitor protein and the P2 parent did not have the KTI protein in polyacrylamide protein gel from mature seeds (F of Figure 6). All 45 individual F6 seeds harvested from same individual F5 plants did not have Kunitz trypsin inhibitor protein of 21.5 kDa size (F of Figure 6). This indicated all 45 F5 progenies had the *titi* genotype and contained no Kunitz trypsin inhibitor protein. Using marker Satt228, amplification patterns from 56 individual F5 plants derived from the cross of 20M183 (*TiTi*) and C242 (*titi*) and

Segregation patterns of genomic DNA amplification by the Satt228 marker using young leaf tissue of four parents and several individual F5 plants (A, C, E, G) and patterns of polyacrylamide protein gel using protein extracted from 10 random seeds of four parents and individual F6 harvested (B, D, F, H) are shown in Figure 6. The bands amplified by the Satt228 marker are clearly detecting the AA and BB genotypes. The seed protein band by the SDS-PAGE is a little different in color density according to each population, staining time of Coomassie blue and protein content. However, the detection of the presence or absence of the Kunitz trypsin inhibitor protein was very clear because the Kunitz trypsin inhibitor

Satt228 marker analysis was conducted on the genomic DNA of the parents and the 273 individual F5 plants lacking the Kunitz trypsin inhibitor protein derived from the cross of 'Jinpumkong2' (*TiTi*) and C242 (*titi*). After harvesting at maturity, SDS-PAGE electrophoresis using crude protein extracted from ten random F6 seeds of each F5 plants and parents was performed to detect Kunitz trypsin inhibitor protein of size 21.5 kDa. DNA banding pattern of the Satt228 marker and polyacrylamide gel banding patterns of the protein is shown in Figure 6 (A and B). The P1 parent (Jinpumkong2) had the AA genotype (allele 1) and the P2 parent (C242) had BB genotype (allele 2) for Satt228 marker. All 273 individual F5 plants were shown only to have the BB genotype (A of Figure 6). This indicated all 273 F5 progenies had the *titi* genotype and contained no Kunitz trypsin inhibitor protein. Also, the P1 parent had Kunitz trypsin inhibitor protein of 21.5 kDa size and the P2 parent did not have the KTI protein (B of Figure 6). All 273 individual F5 plants did not have the Kunitz trypsin inhibitor protein of 21.5 kDa size (B of Figure 6). Amplification patterns obtained from the Satt228 marker using genomic DNA of 17 individual F5 plants derived from the cross of Hannamkong (*TiTi*) and C242 (*titi*) and polyacrylamide gel banding patterns of protein extracted from the mixture of 10 random F6 seeds harvested from each F5 plant are shown in Figure 6 (C and D). The P1 parent (Hannamkong) had a AA genotype while the P2 parent (C242) had the BB genotype (C of Figure 6). All 17 individual F5 plants derived from cross of Hannamkong and C242 showed only the BB genotype pattern for Satt228 marker analysis (C of Figure 6). This indicated all 17 F5 progenies had the *titi* genotype and no Kunitz trypsin inhibitor protein. For the protein analysis, the P1 parent had the 21.5 kDa Kunitz trypsin inhibitor protein while the P2 parent did not have the KTI protein in polyacrylamide protein (D of Figure 6). All 17 individual F5 plants did not have the 21.5 kDa Kunitz trypsin inhibitor protein within their F6 seed samples (D of Figure 6). Amplification patterns by the Satt228 marker using genomic DNA of 45 individual F5 plants derived from the cross of GS06 (*TiTi*) and C242 (*titi*) and polyacrylamide gel banding patterns of protein extracted from the mixture of 10 random F6 seeds harvested from each F5 plant are shown in Figure 6 (E and F). The P1 parent (GS06) had the AA genotype and the P2 parent (C242) had the BB genotype (E of Figure 6). All 45 individual F5 plants derived from the cross of GS06 and C242 displayed only the BB genotype pattern for marker Satt228 (E of Figure 6). Also, the P1 parent had the 21.5 kDa Kunitz trypsin inhibitor protein and the P2 parent did not have the KTI protein in polyacrylamide protein gel from mature seeds (F of Figure 6). All 45 individual F6 seeds harvested from same individual F5 plants did not have Kunitz trypsin inhibitor protein of 21.5 kDa size (F of Figure 6). This indicated all 45 F5 progenies had the *titi* genotype and contained no Kunitz trypsin inhibitor protein. Using marker Satt228, amplification patterns from 56 individual F5 plants derived from the cross of 20M183 (*TiTi*) and C242 (*titi*) and

protein is controlled by single gene and is not influenced by environment.

Fig. 6. Pattern of genomic DNA amplification by Satt228 marker using leaf tissue of parent and individual F5 plants (A, C, E, G) and pattern of polyacrylamide protein gel using protein extracted from parents and 10 random seeds of individual F6 seed harvested (B, D, F, H). A and B, P1: 'Jinpumkong2' and P2: C242; C and D, P1: 'Hannamkong' and P2: C242; E and F, P1:GS06 and P2: C242; G and H, P1:20M183 and P2: C242. M; molecular marker, S; Kunitz trypsin inhibitor protein (Sigma, product number: T6522 ), +; present of KTI protein, -; absent of KTI protein.

polyacrylamide gel banding patterns of protein extracted from the mixture of 10 random F6 seeds harvested from each F5 plant are shown in Figure 6 (G and H). The P1 parent (20M183) had the AA genotype while the P2 parent (C242) had the BB genotype (G of Figure 6). All 56 individual F5 plants were shown only to have the BB genotype pattern for the Satt228

Identification and Confirmation of

**5. Acknowledgment** 

83-85.

172-175.

101:668-669.

125: 744-750.

88:9828-9832.

**6. References** 

Jinnong#1) have been developed using Satt228 marker.

Agronomy, University of Nebraska-Lincoln, U.S.A.

of soybeans. Crop Sci. 12:197-198.

markers. KoreanJ. Crop Sci. 48(4):297-302.

SSR Marker Tightly Linked to the Ti Locus in Soybean [*Glycine max* (L.) Merr.] 211

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

Soybean genotype C242 and W60 was a generous gift from James E. Specht, professor of

Cregan, P.B., T. Jarvik, A.L. Bush, R.C. Shoemaker, K.G. Lark, A.L. Kahler, N. Kaya, T.T.

Hidebrand, D.F., J.H. Orf & T. Hymowitz. 1980. Inheritance of an acid phosphatase and its

Hymowitz, T. & H.H. Hadley, 1972. Inheritance of a trypsin inhibitor variant in seed protein

Kim, M.S., Y.J. Cho, D.J. Park, S.J. Han, J.H. Oh, J.G. Hwang, M.S. Ko & J.I. Chung, 2003.

Kim, MS, Hur MR, Jeong WH, Park MS, Lee KJ, Shim SI, Kim MC, Jung WS, Lee JH, and

Kiang, Y.T., 1987. Mapping three protein loci on a soybean chromosome. Crop Sci. 27: 44-46. Kosambi, D.D., 1944. The estimation of map distance from recombinationAnn. Eugen. 12:

Kunitz, M., 1945. Crystallization of a soybean trypsin inhibitor from soybean. Science

Lander, E., P. Green, J. Abrahamson, A. Barlow, M.J. Daly, S.E. Lincoln & L. Newburg, 1987.

Liencer, I.E. (1995) Possible adverse effects of soybean anticarcinogens. Journal of Nutrition

Michelmore, R.W., J. Paran & R.V. Kesseli, 1991. Identification of markers linked to disease

Moraes, R., Soares, T., Colombo, L., Salla, M., Barros, J., Piovesan, N., Barros, E. and Moreira,

kunitz trypsin inhibitor and lectin in soybean seeds. Euphytica149:221-226.

different soybean populations. Genes &Genomics 30(4):329-336.

of experiment and natural populations. Genomics 1:174-181.

linkage map of the soybean genome. Crop Sci. 39:1464-1490.

VanToai, D.G. Lohnes, Jongil Chung & J.E. Specht, 1999. An integrated genetic

linkage with the Kunitz trypsin inhibitor in seed protein of soybeans. Crop Sci. 20:

Construction of genetic linkage map for korean soybean genotypes using molecular

Chung JI. 2008. Confirmation of Satt228 marker tightly linked to the Ti locus in four

MAPMAKER: An interactive computer package forprimary genetic linkage maps

resistance genes by bulked segregant analysis: A rapid method to detect markers in specific genomic regions by using segregation populations. Proc. Natl. Acad. Sci.

M. (2006)Assisted selection by specific DNA markers for genetic eliminationof the

marker (G of Figure 6). This indicated all 56 F5 progenies had the *titi* genotype and contained no Kunitz trypsin inhibitor protein. For the protein analysis the P1 parent had the Kunitz trypsin inhibitor protein of 21.5 kDa, while the P2 parent did not have the KTI protein (H of Figure 2). All 56 individual F5 plants did not have the 21.5 kDa Kunitz trypsin inhibitor protein based upon their 10 random F6 seed samples (H of Figure 6).

Segregation patterns of genomic DNA amplification by the Satt228 marker using young leaf tissue of four parents and several individual F5 plants (A, C, E, G) and patterns of polyacrylamide protein gel using protein extracted from 10 random seeds of four parents and individual F6 harvested (B, D, F, H) are shown in Figure 6. 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 (Kim et al., 2008).
