INTRODUCTION
Wheat (Triticum aestivum L.) is one of three important crops with rice and maize which are food and forage in the world (Musick et al., 1994; Howell et al., 1995). The total annual output of wheat is about 600 million tons in the world and 40% of the world’s population use wheat as food (Gupta et al., 2008). Korean wheat production was as low as about 44,000 tons from 13,000 ha field in 2014 (MAFRA, 2014). However, self-sufficiency rate of Korean wheat is as low as 2.2% because Korean wheat has low productivity (3.35 ton/ha) and higher price (about 38,000 KRW/kg) than imported wheat. Until now, actually most wheat (4 million tons/year) is imported from United Stated of America (USA), Canada and Australia.
35 wheat cultivars have been developed by Rural Developmental Agriculture (RDA) and wheat breeding program has focused on high grain yield and early maturation over the last 40 years in Korea (Park et al., 2011).
Recently breeding program of wheat is focusing on the high quality of wheat for good end-use products. Wheat cultivars were developed that were suitable for the each different end-use product like bread, noodles and others (Park et al., 2011). According to this reason, the specific markers are required for identifying wheat cultivars. Phenotypic identification by plant and seed shapes or color is not easy and it has a limit to distinguish between some cultivars.
DNA-based molecular markers are easy to treat and correct. DNA markers are important to distinguish between cultivars and it will be able to be adapted to marker assisted selection (MAS) as selectable factor in crop breeding process. DNA markers are useful tool and important for protection of genetic resource, proprietary germplasm and quantification of specific cultivars. Various DNA marker techniques like random amplification polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), simple sequence repeat (SSR), nucleotide polymorphism (SNP) and functional genes related particular traits have been used for developing marker or MAS in breeding process (Sadat et al., 2013).
Eleven DNA markers containing nine sequence specific amplification region (SCAR) and two SSR markers have been developed for Korean wheat identification through the previous study. Nine SCAR markers were derived from inter simple sequence repeat (ISSR) and AFLP analysis, and two SSRs were derived from transposable elements, respectively (Son et al., 2013; Son et al., 2014). Using the genotype analysis of 11 markers, 27 of 32 Korean wheat cultivars containing mainly cultivated varieties like Keumkang and Jokyoung were identified cor- rectly (Son et al., 2014). Because five cultivars were not identified from others, we searched SSR and SNP markers and evaluated them to develop and construct Korean wheat specific DNA marker set in this study.
SSR or microsatellite was one of the molecular bio-techniques and it was used generally for genotype analysis of cultivars (Chen et al., 1998). Some SSRs like (CT)10, (GAA)5 or (AAT)6 have been used in DNA fingerprinting for the detection of genetic variation and cultivar identification (Devos et al., 1995). Especially SSR marker has the advantages that can be useful in the initial separation of genetic generation as the co-dominant marker (Chen et al., 1998; Temnykh et al., 2001). Because of the possibility of the SSR as a molecular marker, Roder et al. (1995) investigated the SSR of allohexaploid wheat. SSR was used for wheat species relationship analysis, and was used in many traits and gene mapping and marker development (Roder et al., 1995). In recent years, a lot of SSRs related to some genes have been used in many studies, such as that used in the comparative genomic of other plant like rye and Gramineae (Varshney et al., 2005).
For this reason, we searched some SSRs and genes related to wheat agricultural trait that was reported by previous studies and we evaluated them in this study. We tried to find alternatively amplified band between 32 Korean wheat cultivars. As a result of this analysis, we constructed the complete DNA marker sets for Korean wheat cultivar identification.
MATERIALS AND METHODS
Plant materials and DNA extraction
Genomic DNAs were extracted from young leaves of 32 Korean wheat cultivars using HiGeneTM Genomic DNA Prep Kit (SolGent, Korea). They were chosen as major cultivars in Korea and representative species in the classification of wheat cultivars (Table 1).
PCR analysis
Information of primer sets for SSRs and SNPs analysis is indicated in Table 2. PCR was performed with 20ng diluted pre-amplified products, 5uM primer sets, 0.2mM dNTPs and 0.5U DNA Taq polymerase (Intronbiotechnology, Korea) in a total volume of 25ul. PCR condition was carried out for 35 cycles at 94°C for 30 s, 55-60°C for 30 s, 72°C for 60 s and the final extension of 72°C for 5 min. The amplified DNA fragments were loaded on 1.5% agarose gel and QIAxcel auto electrophoresis system (QIAgen, USA).
RESULTS AND DISCUSSION
SSR fingerprinting of 32 Korean wheat cultivars
We used 14 SSRs to assess whether the markers were for a high temperature (Son et al., 2015) and we used them for development of cultivar specific DNA markers in this study. We used fourteen SSR primer pairs for polymorphic analysis to develop cultivar specific markers in Korean wheat. Each primer set produced between 1 and 4 detectable amplified bands that range in size from 50 to 400bp, depending on the primers and Korean wheat cultivars. Through SSR analysis, we selected three SSR primer sets, XbarC137, Xcfd43 and Gwm291 that were suitable for the cultivar identification marker (Table 2). These markers have been reported to be related to some QTL (quantitative trait loci) of wheat which were Grain-filling duration and HIS kernel weight (Mason et al., 2010). Three SSRs have been used for MAS to screen the heat tolerance wheat resources. We used 14 SSRs to assess whether the markers were for a high temperature (Son et al., 2015) and we used them for development of cultivar specific DNA markers in this study.
Two DNA bands of 295bp and 260bp were amplified by XbarC137 primer pairs. The 295bp band was detected in six cultivars, Geuru, Anbaek, Yeonbaek, Jeokjoong, Baekjoong and Sugang, and 260bp band was detected in the others (Fig. 1). Two DNA bands of 210bp and 180bp were amplified by Xcfd43. The 210bp band was amplified in only one of 32 cultivars, Jopoom (Fig 1.). Using Scfd43 primer, we can isolate Jopoom from other cultivars. Therefore, we expected that this marker is able to identify single cultivar, Jopoom. Five different DNA bands were amplified by Gwm291. Four cultivars, Tapdong, Alchan, Sinmichal 1 and Sugang, have a band of 169bp. 152bp and 124bp bands were detected in Gobun and Jonong, respectively. Two bands 182bp and 200bp have detected in other cultivars except for the six cultivars mentioned above.
These three SSR markers were suitable as DNA marker sets to distinguish Korean wheat cultivar, and we named them KWSM012, KWSM013 and KWSM014, respectively.
SNP polymorphism analysis of 32 Korean wheat cultivars
The partial DNA sequence of Hsp16.9 has been reported by Garg et al. (2012) in previous study. We used allelicspecific primer designing from the position of base transition (from A to G) in Hsp16.9 gene sequences (Garg et al., 2012) in wheat. They reported that amino acid sequence was changed from aspartic acid (Asp) to asparagine (Asn) residue by nucleotide transition. Allelic specific primer was designed to amplify 197 bp of DNA fragment from HSP16.9 (Garg et al., 2012). We used the A allelic specific primers and performed PCR reaction. Two cultivars, Uri and Jokyoung, resulted in presence of band of 197 bp but other 30 cultivars have no any amplified band (Fig. 1). Heat shock protein gene was known to plays a role to regulate defensive system of crops for heat or drought stress. It was reported that the heat susceptible wheat has allele A (Garg et al., 2012). This SNP marker was evaluated by using 23 Turkey wheat resources and 7 Korean wheat cultivars (Son et al., 2015).
Application of DNA marker to identification of Korean wheat cultivars
Eleven Korean wheat cultivar specific marker sets have been developed from ISSR and SSR analysis (Son et al. 2013; Son et al., 2014), previously. To use 11 marker sets, we can identify the 27 Koran wheat cultivars from 32 cultivars (Son et al., 2014). This technique using small number of marker combination was easy and simple to isolate some cultivar from various cultivars (Son et al., 2014). 27 of 32 cultivars (84%) were isolated by 11 primer sets. But now, we developed four DNA markers additionally in this study which contain three SSR and one SNP markers. We are able to distinguish 32 Korean wheat cultivars com- pletely using 15 DNA markers (Table 1; Fig. 2).
We easily produced one or more target DNA fragment by single PCR reaction. Especially, cultivar specific DNA marker construction was very useful technique for identification of cultivars as well as for protection of genetic resources and crop breeding program. Therefore, we coded using 15 Korean wheat cultivar specific marker polymorphisms to use them more easily (Fig. 2; Fig. 3). The presence or absence of amplified bands which were bands at the same position in the agarose gel was recorded as binary code of 1 or 0, and they were applied for the code.
적 요
이전 연구로부터 11개의 한국밀 품종판별 마커가 개발되어 32개 품종 중 27개를 판별할 수 있었다. 본 연구를 통해 개발 된 3개의 SSR과 1개의 SNP 마커(KWSM012-015)를 기존의 마커 세트에 추가하여 총 15개의 마커 세트를 이용하여 32개 품종 모두를 판별할 수 있게 되었다. 새로 개발된 마커의 정 보는 다음과 같다.
