**2.3 DNA extraction and DNA marker analysis**

202 Soybean – Genetics and Novel Techniques for Yield Enhancement

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

Soybean genotype C242 (clark derived near isogenic line) has the *ti* allele and lacks a soybean kunitz trypsin inhibitor. C242 was a generous gift from J. Specht, professor of Agronomy, University of Nebraska-Lincoln, USA. Cultivar Jinpumkong2 and Clark has kunitz trypsin inhibitor protein band (*TiTi*). Two mapping populations were developed. Population 1 was derived from a cross between cultivar Jinpumkong2 and C242. Population 2 was made from a mating between cultivar Clark and C242. The F1 plants from two

98 F2 seed from F1 plants for population 1 and 243 F2 seed from F1 plants for population 2 was analysed electrophoretically to determine the presence (SKTI-`+') or absence (SKTI- 'null') of kunitz trypsin inhibitor. A piece of cotyledon from each F2 seed was removed and the remaining embryo germinated to given a F2 mapping population. The separated cotyledon tissue was incubated for 30 min (room temperature) in 1 ml Tris-HCl, pH 8.0, containing 1.56 % v/v β-mercaptoethanol. After centrifugation, 50 l of the supernatant were added to an equivalent amount of 5X sample buffer [10% w/v sodium dodecyl sulphate (SDS), 50% v/v glycerol, 1.96% v/v β-mercaptoethanol, 1 M Tris-HCl, pH 6.8]. The samples were boiling at 97°C for 5 min and then centrifuged. Two microlitre of the supernatant were used for electrophoresis on 12% acrylamide SDS polyacrylamide gel electrophoresis (SDS-PAGE) medium gels in Owl Separation Systems. Inc(Model: P9DS, Portsmouth, NH USA). Electrophoresis was practiced at 120 V for 7 hr. Gels were stained overnight in an aqueous solution of 0.25 g coomassie brilliant blue R250, 10% acetic acid, 45% methanol and destaining solution (5% acetic acid, 14 methanol) for several hours. A Wide-Range SDS-PAGE molecular mass standard (Sigma MarkerTM, Product Code : M4038) containing the 21.5kDa soybean trypsin inhibitor protein, was used to aid recognition of

populations were grown in the greenhouse to produce F2 seeds.

**2.2 Determination of kunitz trypsin inhibitor genotype** 

samples lacking the kunitz trypsin inhibitor.

seedling stages with relative ease.

**2.1 Plant genotypes** 

**2. Identification of SSR marker** 

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