**2.5 Detection of Satt228 marker**

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 recessive gene.

Identification and Confirmation of

23.0

19.8

25.6

4.5 **0.0** 5.1

cM

**MLG A2**

OPAR15

CGTATG

CATACG

Satt409 **Satt228** *Ti* Satt429

**Population 1 Population 2**

classical linkage group 9 (Hildebrand et al., 1980; Kiang 1987).

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

**3. Confirmation of Satt228 marker** 

isogenic line derived from cultivar 'William'.

SSR Marker Tightly Linked to the Ti Locus in Soybean [*Glycine max* (L.) Merr.] 205

**MLG A2**

Fig. 3. Molecular linkage map A2 (Cregan et al., 1999) of *Ti* locus defined using population 1 and population 2. Population 1 was derived from the cross of Jinpumkong2 (*TiTi*) and C242 (*titi*). Population 2 was derived from the cross of Clark (*TiTi*) and C242 (*titi*). Map was constructed using MAPMAKER/EXP (LOD 4.0 maximum distance 50 cM). Marker loci

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

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

3.7

18.2

cM

names are on the right and kosambi map distances are on the left. CLG09 is the

Satt409

cM

21.0

**CLG 09**

21.0 11.0 12.0

*Pgd2*

*Lap1*

*Ti Ap Fr3*

**Satt228** *Ti*

Fig. 1. Polyacrylamide gels of protein extracted from parents and F2 seeds. P1 (Jinpumkong2) and P2 (C242) are parents and arrow points to the Kunitz trypsin inhibitor band (21.5 KDa).

Of the 1,000 RAPD primers tested on two parents of population 1 (Jinpumkong2 and C242), approximately 12 % (124) primers produced polymorphic DNA fragment differences between the parents. Only 35 primers were identified as being polymorphic between bulked DNA samples with SKTI protein and bulked DNA samples with no SKTI protein. Of those 35 primers, only 16 also exhibited polymorphism between parents. Among 342 primer sets of AFLP analysis, only 10 primers were shown polymorphism between parents. Three SSR primers (Satt409, Satt228 and Satt429) among 35 primers selected were shown polymorphism between parents. Total 48 markes (35 RAPD, 10 AFLP, and 3 SSR) were used to obtain segregation data from 94 F2 individuals of population 1. Figure 2 represents some example of segregating DNA fragment for SSR markers (Satt228) in parents, bulked samples and F2 population.

Fig. 2. Patterns of segregating DNA fragment for SSR primer Satt228 in parents, bulked samples, and F2 population. P1 is Jinpumkong2 (*TiTi*) and P2 is C242 (*titi*). B1 is bulked of present kunitz trypsin inhibitor protein individuals and B2 is bulked of absent individuals.

A genetic map was constructed from the 48 segregating DNA markers and *Ti* locus. A total 11 DNA markers (4 RAPD, 4 AFLP, and 3 SSR) and *Ti* locus was found to be genetically linked in population 1. Three SSR markers, Satt409, Satt228, and Satt429 linked with *Ti* locus within 10 cM (Figure 3). Satt228 marker was very tightly linked with *Ti* locus at 0 cM. Three SSR markers linked with *Ti* locus in population 1 were applied in population 2. Only two SSR markers, Satt228 and Satt409 were linked with *Ti* locus. Satt228 marker was linked with *Ti* locus in 3.7 cM distance (Figure 3). The order and map distances of SSR markers and *Ti* locus differed between populations 1 and 2.

Fig. 3. Molecular linkage map A2 (Cregan et al., 1999) of *Ti* locus defined using population 1 and population 2. Population 1 was derived from the cross of Jinpumkong2 (*TiTi*) and C242 (*titi*). Population 2 was derived from the cross of Clark (*TiTi*) and C242 (*titi*). Map was constructed using MAPMAKER/EXP (LOD 4.0 maximum distance 50 cM). Marker loci names are on the right and kosambi map distances are on the left. CLG09 is the classical linkage group 9 (Hildebrand et al., 1980; Kiang 1987).
