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Son, Kim, Shin, Kim, Kim, Hyun, Shim, Lee, Park, and Kang: ISSR-derived Molecular Markers for Korean Wheat Cultivar Identification

Abstract

Inter simple sequence repeat (ISSR) is a technique that leads to the development of novel specific molecular markers and relationship analysis between species. In this study, 54 ISSRs were used to develop cultivar-specific DNA sequence characterized amplified regions (SCARs) for Korean wheat cultivars (Triticum aestivum). Using nine of 54 UBC ISSR primers, 16 cultivar-specific fragments were detected among 32 wheat cultivars. Of the 16 primers, 10 successfully amplified targets in 32 cultivars and six novel cultivar-specific markers were finally selected. A total of 13 Korean wheat cultivars (Ol, Dahong, Namhae, Jinpum, Anbaek, Jopoom, Jokyung, Sinmichal 1, Jeokjoong, Dajung, Goso and Joah) were classified by newly developed six SCAR markers. Cultivar-specific markers can be used not only for cultivar fingerprinting but also for marker-assisted selection in wheat breeding program.

INTRODUCTION

Triticum aestivum, also known as common wheat, is an important food crop worldwide (Zhang et al. 2012), consumed by more than 40% of the world’s population and is produced in more than 6,000,000 tons annually (Gupta et al. 2008). High quality elite cultivars that can adapt to climate change play a crucial role in wheat production (Zhu et al. 2011). Common wheat is a hexaploid (2n = 6× = 42) consisted of three distinct genomes A, B, and D which has seven pairs of chromosomes each genome (Gao et al. 2004). Although A, B and D genome are different, they are genetically related to each other. Both A and D genome are diploid in origin, but the origin of B genome is not yet clearly confirmed (Gill et al. 1991). Wheat has very large genome size which has more 80% of the entire genome consisted with repeat sequences like simple sequence repeat (SSR) or retrotransposon elements (Brenchley et al. 2012). Genes of each genome has tripled because common wheat germ cytological division is similar to that of normal diploid. These features facilitated the large genome research. Molecular markers have been used as a starter, genetic evolution research, and diagnosis in many crops including wheat. Using DNA-based molecular markers allows accurate, easy and rapid cultivar identification compare with phenotypic analysis and field growth test in wheat breeding program (Zhu et al. 2011). In addition, DNA-based molecular markers have been used in multi-purpose tools in various fields like taxonomy, physiology, embryology, and genetic engineering.
Simple sequence repeats (SSRs) or microsatellites were introduced by Litt and Lutty (1989). SSRs are short repeated sequence motifs of 1 to 6 bp, present numerously in eukaryotic genomes including plant and animals (Tauz and Renz 1984). Although SSR markers have many advantages like easy to use and detect higher degree polymorphism, SSRs in coding regions are less abundant than in intergenic regions. Moreover, the process of developing SSR markers are difficult and complex such as in selecting and sequencing clones containing putative SSR motifs for designing and testing the flanking primers (Gao et al. 2004). Inter simple sequence repeat (ISSR) markers were reported by Zietkiewicz et al. (1994) which are developed by utilizing repeated microsatellites primers to amplify inter-SSR DNA sequences. Various microsatellites anchored at 5′ end or 3′ end are useful for amplifying genomic DNA which increase primer’s specificity in PCR reaction (Godwin et al. 1997). Unlike other PCR-based techniques, ISSR does not require any information about target sequence of SSR regions (Tsumura et al. 1996). Advantages of ISSR include relatively low cost, high polymorphisms, and good reproducibility. ISSRs are highly rigid marker for scoring ISSR analysis because the random amplified markers are spurious or homoplasious bands (Bussell et al. 2005; Park et al. 2009). Kojima et al. (1998) showed that ISSR markers with Random amplified polymorphic DNA (RAPD) were used to construct genetic linkage map in Einkorn wheat. ISSR analysis is useful for detecting genetic diversity (Li and Ge 2001; Sica et al. 2005), and analyzing species relationship as well as marker-assisted selection (MAS) for molecular breeding in plant using a single primer composed of SSR (Hussain et al. 2000; Joshi et al. 2000; Pasakinskene et al. 2000). ISSR markers were also used for identification of abiotic stress like drought (Deshmukh et al. 2012). In addition, SSR and ISSRs markers are useful for cultivar identification (Crespel et al. 2009; Lu et al. 2009), specifically ISSR markers have been used for wheat cultivar identification (Nagaoka and Ogihara 1997) including others such as radish (Liu et al. 2008), lotus (Chen et al. 2008), and rose (Crespel et al. 2009).
In Korea, many kinds of wheat cultivars suitable for different baked products such as breads, cakes and cookies have been developed. Molecular markers are necessary tool to identify these Korean cultivars yet these remains to be investigated. In this study, we developed sequence characterization amplified region (SCAR) or sequence tagged site (STS) markers for identification of 32 Korean wheat cultivars based on specific alleles using ISSR technology.

MATERIALS AND METHODS

Plant materials and DNA extraction

Genomic DNAs were extracted from young leaves of 32 Korean wheat varieties using HiGeneTM Genomic DNA Prep Kit (SolGent, Korea). They were chosen as major cultivars in Korea and representative species in the classification of wheat variety (Table 1).

PCR amplification and cloning

Information of nucleotide sequences of the primers for inter simple sequence repeat (ISSR) PCR amplification is indicated in Table 2. PCR amplification was performed with 40 ng of template genomic DNA, 5 uM of primer, 2.5 mM of dNTP, and 1 unit of i-star maxII DNA polymerase (Intron, Korea) and Prime Taq DNA Polymerase (GENET Bio., Korea). The PCR annealing condition used consists 35 cycles of 94°C for 30 sec, 50°C for 30 sec and 72°C for 1 min. Amplified products were checked on 1.5% agarose gel electrophoresis where specific bands (300 – 1,000 bp) were eluted from the gel. The eluted DNA fragments were ligated into pGEM-T vector (Promega, USA), and cloned into DH5α competent Escherichia coli cells. Plasmid DNA were sent to Xenotech Inc., Korea for sequencing with the primer pairs M13 forward and SP6 using a PRISM3700 automatic DNA sequencer (ABI Inc.).

Sequence analysis, primer design and re-amplification

The sequence-analyzed DNA fragment length was determined from 300 to 1,000bp. These fragments were included in wheat species (Fig. 1). The sequence information which was used to design the SCAR primers using primer3 (version 0.4.0, http://frodo.wi.mit.edu/) was confirmed in GenBank (NCBI, http://www.ncbi.nlm.nih.gov). Designed primers were tested for classification of Korean wheat varieties. PCR reaction was performed with 40 ng of genomic DNA, 200 uM of each dNTP, 1× reaction buffer and 1 U i-star maxII DNA polymerase (Intron, Korea) in a total volume of 25 ul. PCR conditions include initial denaturation at 94°C for 3 min followed by 35 cycles of 94°C for 30 sec, 50°C for 30, 72°C for 1 min and the final extension of 72°C for 5 min. The amplified DNA fragments were confirmed on 1.2% agarose gel electrophoresis.

RESULTS AND DISCUSSION

ISSR amplifications

We have surveyed 54 ISSR primers for amplification using 32 Korean wheat cultivars, 14 of which showed consistent amplification and multiple polymorphism containing specific bands (Fig. 1). Other primers showing monomorphic banding pattern were not selected for further experiments in this study. ISSR primer UBC849 was profiled for identification of eight wheat cultivars in China wherein six different bands were amplified (Zhu et al. 2011), but when tested in Korean wheat cultivars there was no polymorphism detected UBC811 (Fig. 1). Although ISSR primer has been used for genetic linkage map in Einkorn wheat (Kojima et al. 1998), it was not used in this study because it failed to amplify the target allele. The 14 primers showing consistent and polymorphic amplification were consisted of all 3′-end anchoring (Fig. 1; Table 2). The number of amplified products varied from 1 to 8 with sizes that range from 200 to 1,000 bp depending on the primers and cultivars. The PCR reactions were carried out over three times in different thermocyclers (GeneAmp PCR System 9700, ABI, USA; Biometra, Germany) using different DNA polymerase (Prime Taq DNA Polymerase, GENET Bio, Korea; i-star maxII, Intron, Korea) to obtain reproducible results. Since many bands were detected, the specific bands were eluted from the agarose gel for the sequencing. Polymorphisms were observed not only among different species like Sumai3 (data not shown) but also among different 32 Korean wheat cultivars (Fig. 1).

Sequence analysis and designing the SCAR primers

We eluted 16 different DNA fragments from the agarose gel and performed the sequencing process. The number of eluted bands was one each in UBC808, UBC815, UBC816, UBC823 and UBC840, two in UBC807, and three each in UBC812, UBC813, and UBC819 (Fig. 1). BLAST analysis, querying the Triticum sequences deposited at NCBI, confirmed that the ISSR fragments were real DNA fragments from Wheat species, and further analysis of these primer pairs is therefore likely. However, all of the sequences are not coded with any functional genes because ISSR amplifies genomic sequences between SSR loci or non-coding regions (Son et al. 2012). Hence, we designed the SCAR primers using primer3 input (Ver. 0.4.0) program from 16 analyzed DNA sequences based on ISSR amplicons. The primer length was fixed 20 to 24 bp to increase primer specificity and efficiency.

Application of SCAR markers to 32 Korean wheat cultivars

The newly developed markers were tested for reliability in classifying Korean wheat varieties. A total of ten cultivar-specific primer pairs which did not amplify or have generated non-specific band were discarded. With this, we used the remaining six primer pairs as SCARs (Table 3) which successfully amplified target sequences in Korean wheat cultivars with sizes of the amplicons corresponded well to expected length in sequence analysis (Fig. 2). Six SCAR primer pairs are generated from UBC808, UBC812 and UBC816. We tested these primer pairs over three times using different DNA polymerase and PCR machines to validate the application of these SCARs for the identification of 32 Korean wheat cultivars. Although these markers have little disadvantage due to different annealing temperature in PCR condition, it did not affect the efficiency of the PCR assay. With these new marker combinations, a total of 13 out of 32 Korean wheat cultivars were identified as unique from each other (Fig. 2). These include Ol, Dahong, Chungkye, Tapdong, Namhae, Olgeuru, Gobun, Jopoom, Jokyoung, Younbaek, Sinmichal 1, Jeokjoong and Hanbaek.
Unlike the other 12 cultivars, Younbaek was identified distinctly from the others because of unamplified reactions from all primer sets. The 812-1 SCAR marker was amplified in 12 cultivars, Ol, Dahong, Namhae, Jinpum, Anbaek, Jopoom, Jokyung, Sinmichal 1, Jeokjoong, Dajung, Goso and Joah. Interestingly, no coded functions has been designated yet to this gene marker in Triticum. However, some of these cultivars have been reported to exhibit traits such as Jeokjoong which is known to have moderate resistance to Fusarium and is used for making noodle (Park et al. 2008), Baegjoong and Joah which are resistant to pre-harvest sprouting, and Goso which is very useful for making cookie as well as it has resistance to pre-harvest sprouting like Baegjoong, Joa and Sugang (Park et al. 2009). Similar to 812-1, the 812-4 is functionally non-coded gene marker which was specifically amplified in nine Korean wheat cultivars (Ol, Olgeuru, Gobun, Jopoom, Jokyung, Sinmichal 1, Dajung and Goso). Additionally, the number of Korean wheat cultivars with amplifications were six for 816-1, 11 for 816-2, 21 for 808R, and 25 for 808A primer. The complete detail of the cultivar and primer pair combination with amplifications is shown in Fig. 2.
Both 816-1 and 816-2 are allele-specific primers. A nucleotide of 3′-end is different, 816-1 is A (T allele-specific primer) and 816-2 is T (A allele-specific primer). With this, Chungkye contains T allele because it showed amplification with 816-1but not with 816-2 primer. On the otherhand, Ol, Dahong, Eunpa, Tapdong, Joeun and Jopoom have A allele while Namhae, Gobun, Seodun, Milseong, Jeokjoong, have both T and A alleles.
Since19 Korean wheat cultivars were not clearly identified and classified by the six primer sets, we planned to proceed with both amplified fragment length polymorphism (AFLP) and transposon display (TD) techniques to detect more polymorphism. With this technique, we expect to classify more cultivars using variable molecular markers in the future. Nonetheless in this study we were able to identify 13 out of 32 Korean wheat cultivars using the 6 primer pairs. The newly developed cultivar-specific SCAR markers can be used not only for cultivar fingerprinting but also for marker-assisted selection and inter-specific hybridization to combine important traits in the wheat breeding program (Burton 1992; Brilman 2001). This technique also saves time and money by performing only one PCR reaction.

CONCLUSION

Inter simple sequence repeat (ISSR) is a useful technique to analyze the relationship between species and to develop specific molecular markers without the need of target sequence information. In this study, we used ISSR primers to develop cultivar-specific DNA SCARs for Korean wheat cultivars. Six novel cultivar-specific markers (designated 812-1, 812-4, 816-1, 816-2, 808R, and 808A) were developed to identify 13 cultivars using 54 ISSR primers. A total of 13 cultivars were identified as unique based on specific alleles amplified which include Ol, Dahong, Namhae, Jinpum, Anbaek, Jopoom, Jokyung, Sinmichal 1, Jeokjoong, Dajung, Goso and Joah. Of which, Jokyung and Goso are few of the major cultivated wheat cultivars together with Keumkang in Korea. Cultivar-specific markers can be used not only for cultivar fingerprinting but also for marker-assisted selection in wheat breeding program. Furthermore, because ISSRs are based on universal repeat motifs in the genome, ISSRs can be used in molecular bio-techniques for development of novel marker and species relationship analysis in plants.

ACKNOWLEDGMENT

This study was supported by 2013 Postdoctoral Fellowship Program of National Institute of Crop Science, Rural Development Administration, Republic of Korea.

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Fig. 1
ISSR profiles of 32 Korean wheat cultivars (1–32, Table 1). The amplified products were represented by the sizes ranging from 200 to 1,000 bp. The yellow Arrows indicate the eluted DNA fragment. A; UBC807, B; UBC808, C; UBC812, D; UBC813, E; UBC816, F; UBC815, G; UBC819, H; UBC823, I; UBC836, J; 840, K; 844, L; 846, M; UBC849, N; UBC880.
pbb-01-262f1.gif
Fig. 2
The profiles of PCR amplified DNA fragments using developed SCAR markers in 32 Korean wheat cultivars. The + indicates amplified bands and − is absence. Red + indicated the distinguished 12 cultivars, 1; Ol, 3; Dahong, 4; Chungkye, 6; Tapdong, 7; Namhae, 9; Olguru, 11; Gobun, 19; Jopoom, 22; Jokyoung, 23; Younbaek, 24; Sinmichal 1, 26; Jeokjoong, 28; Hanbaek, by six novel markers combination. A; 812-1, B; 812-4, C; 816-1, D; 816-2, E; 808R and F; 808A.
pbb-01-262f2.gif
Table 1
The 32 Korean wheat cultivars used in this study and information of PCR amplification patterns.
No. Variety SCAR marker name

812-1 812-4 816-1 816-2 808R 808A
1 Ol + + +
2 Geuru + +
3 Dahong + +
4 Chungkye + + +
5 Eunpa + + +
6 Tapdong +
7 Namhae + + +
8 Uri + +
9 Olgeuru + + +
10 Alchan + +
11 Gobun + + + + +
12 Keumkang +
13 Seodun + + + +
14 Saeol + +
15 Jinpoom + + + +
16 Milsung + + + +
17 Joeun + + +
18 Anbaek + + +
19 Jopoom + + + + +
20 Sinmichal + +
21 Jonong + +
22 Jokyung + + +
23 Younbaek
24 Sinmichal 1 + +
25 Baekjung +
26 Jeokjoong + +
27 Sugang + +
28 Hanbaek + +
29 Suan + +
30 Dajoon + + + +
31 Goso + + +
32 Joa + + + +

* The + indicates amplified bands and − is absence

Table 2
Sequence information of ISSR primers used in this study.
No. Primer Sequence
1 UBC801 ATATATATATATATATT
2 UBC802 ATATATATATATATATG
3 UBC803 ATATATATATATATATC
4 UBC804 TATATATATATATATAA
5 UBC805 TATATATATATATATAC
6 UBC806 TATATATATATATATAG
7 UBC807 AGAGAGAGAGAGAGAGT
8 UBC808 AGAGAGAGAGAGAGAGC
9 UBC809 AGAGAGAGAGAGAGAGG
10 UBC810 GAGAGAGAGAGAGAGAT
11 UBC811 GAGAGAGAGAGAGAGAC
12 UBC812 GAGAGAGAGAGAGAGAA
13 UBC813 CTCTCTCTCTCTCTCTT
14 UBC814 CTCTCTCTCTCTCTCTA
15 UBC815 CTCTCTCTCTCTCTCTG
16 UBC816 CACACACACACACACAT
17 UBC817 CACACACACACACACAA
18 UBC818 CACACACACACACACAG
19 UBC819 GTGTGTGTGTGTGTGTA
20 UBC820 GTGTGTGTGTGTGTGTC
21 UBC821 GTGTGTGTGTGTGTGTT
22 UBC822 TCTCTCTCTCTCTCTCA
23 UBC823 TCTCTCTCTCTCTCTCC
24 UBC824 TCTCTCTCTCTCTCTCG
25 UBC825 ACACACACACACACACT
26 UBC826 ACACACACACACACACC
27 UBC827 ACACACACACACACACG
28 UBC828 TGTGTGTGTGTGTGTGA
29 UBC829 TGTGTGTGTGTGTGTGG
30 UBC830 TGTGTGTGTGTGTGTGC
31 UBC831 ATATATATATATATATYA
32 UBC832 ATATATATATATATATYC
33 UBC831 ATATATATATATATATYA
34 UBC832 ATATATATATATATATYC
35 UBC833 ATATATATATATATATYG
36 UBC834 AGAGAGAGAGAGAGAGYT
37 UBC835 AGAGAGAGAGAGAGAGYC
38 UBC836 AGAGAGAGAGAGAGAGYA
39 UBC837 TATATATATATATATART
40 UBC838 TATATATATATATATARC
41 UBC839 TATATATATATATATARG
42 UBC840 GAGAGAGAGAGAGAGAYT
43 UBC841 GAGAGAGAGAGAGAGAYC
44 UBC842 GAGAGAGAGAGAGAGAYG
45 UBC843 CTCTCTCTCTCTCTCTRA
46 UBC844 CTCTCTCTCTCTCTCTRC
47 UBC845 CTCTCTCTCTCTCTCTRG
48 UBC846 CACACACACACACACART
49 UBC847 CACACACACACACACARC
50 UBC848 CACACACACACACACARG
51 UBC849 GTGTGTGTGTGTGTGTYA
52 UBC850 GTGTGTGTGTGTGTGTYC
53 UBC876 GATAGATAGACAGACA
54 UBC880 GGAGAGGAGAGGAGA

* Y and R are single letter abbreviation for mixed base positions. Y is presenting C or T, R is presenting A or G. The red letters indicate the selected UBC primers for eluting specific bands.

Table 3
The PCR condition information of six novel SCAR markers for the identification of 32 Korean wheat cultivars.
Marker name Forward sequences Reverse sequences Annealing temperature (°C) Product size (bp) Number of Polymorphic band
812-1 AAGAAGAAAAAGAAAGAGAGTTGAG GTTGTGTTTTTATTTGTTTTTGAC 55 359 1
812-4 GCCGAGATTAAATTTGTTACTACT AACATTTATGTTTGGTAAATTGAC 55 312 1
816-1 TGCTGAGGGCATATCTCAACA GTGTTGCACGACGCTGTACT 58 682 1
816-2 TGCTGAGGGCATATCTCAACG GTGTTGCACGACGCTGTACT 58 682 1
808R GGCTTACTACCTACACTTGACC GCAACAACAACCTTCTCCCAAT 58 392 1
808A GGCTTACTACCTACACTTGACC AACAACCTTCTCCCAATCCCCT 58 392 1
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