KR20120063814A - Ssr primer isolated from lilum spp. and use thereof - Google Patents

Abstract

PURPOSE: An SSR primer isolated from Lilum spp. is provided to make DNA profile and to effectively evaluate gene source. CONSTITUTION: An SSR primer pair has two bases selected from sequence numbers 1 to 60. The SSR primer pair is derived from Lilum spp. A method for detecting SNP from Lilum spp. comprises: a step of isolating genome DNA from Lilum spp.; a step of performing PCR of the genome DNA using the SSR primer pair; and a step of classifying PCR products by size. A kit for detecting SNP from Lilum spp. contains the SSR primer pair, DNA polymerase, and buffer solution.

Description

SSR primer isolated from the genus Lilium plant and its use {SSR primer isolated from Lilum spp. and use kind}

The present invention relates to an SSR primer isolated from a plant of the genus Lilium, and more particularly, to an SSR primer pair having two base sequences selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 60, and PCR using the same. The present invention relates to a method for detecting DNA polymorphism of a genus Lilium plant and a method for identifying a variety of the plant.

Large quantities of genetic resources are collected annually in most major crops and cultivated crops used for breeding. However, on the other hand, the evaluation of genetic resources has not been sufficiently made. Rapid discrimination between species and subspecies varieties of collected genetic resources is very important for accurate management and protection of genetic resources. In addition to the identification of varieties, the identification and classification of trait traits and genetic relationships of genetic resources are very important for the conservation of genetic resources, the creation of new varieties, and the improvement of crops.

Recent rapid advances in molecular biology have led to the development of nucleic acid fingerprint analysis methods and various DNA markers that enable bio-diversity studies at the nucleic acid (DNA) level. This makes it possible to search useful traits, identify species of species, classify / identify varieties and analyze flexible relationships of populations in a simple and quick way with little influence on the external environment. Nucleic acid fingerprinting using polymerase chain reaction (PCR) developed so far includes randomly amplified polymorphic DNAs (RAPD), amplified fragment length polymorphic DNA (AFLP), and simple sequence repeat (SSR). The RAPD method has a disadvantage of poor reproducibility because the non-specific PCR product is amplified, and the AFLP method is spotlighted by high DNA polymorphism detection, but has a disadvantage of complicated appearance and analysis of a low reproducible band. In contrast, the SSR method is a method of constructing a PCR primer based on nucleotide sequence information of a microsatellite region, which is a DNA repeat sequence. Frequently used. In particular, the SSR method has the advantage that it is not affected by the external environment. Due to the superiority of this SSR method, studies are underway to develop SSR markers in several major crops and cultivated crops.

In rice, supersatellite markers have been developed that amplify SSR repeated in the rice genome by PCR technology to identify genotypes of rice varieties (Patent Application No. 2001-34003). Tang's team in the United States published a genetic map of sunflowers that developed 879 SSR markers (Tang et al., Theor. Appl. Genet., 105: 1124-1136, 2002). In addition, SSR markers have been developed in many plants such as barley, soybeans, yulmu, plums, tangerines, ginseng, ginger, amaranth, grass, crude, green beans, millet, corn, buckwheat, red beans, azalea, wolfberry, mushrooms, garlic, sesame and perilla It became. However, no SSR marker for lily has been developed.

Lilium (lily) is a cold-resistant bulb belonging to the botanical plants of Panerogamae, Angiospermae, Monocotyledonea, Liliales, Liliaceae and Lilium. , Diploid (2n) or triploid (3n), somatic chromosome number is 24 or 36. There are more than 130 species native to the northern hemisphere, 71 of which are known to be distributed in Asia, 22 to Europe and Eurasia, and 37 to the North American continent. There are as many as 4,000 species (Hebuk-gu, Han Yong-hee, Lee Soon-bong, Kim Sam-gon. 1994. Actuality and Theory of Nari Cultivation).

The classification of Lilium was first distinguished by Linne in 1754, when Lilium was Only seven species, including candidum , L. bulbiferum, L. pomponium , L. chalcedonicum , L. martagon , L. canadence , L. camtschatcence , were found. Origin is included in Lilium. Wilson uses Leucolirion (orientated or downward or upward), Archelirion (Prunus), or Pseudolirion (4) subclassification (Wilson EH. 1925. The lilies of Eastern Asia: A Monograph. Dulau & Company, LTD. London). The comber classified the species in consideration of the species' shape, geographical distribution, bulb shape, leaf attachment, seed germination and development process (Comber HF. 1949. A New classification of the genus) Lilium RHS Lily Yearbook 15: 86-105) Although this classification is somewhat questionable in some species, it is widely used because of its similarity and mating affinity. After the hybridization between the cultivars, a number of horticultural varieties were raised, and horticultural classification was required. It is used universally. This taxonomy is Asian, Maltagon or Turk's Cap group, Candidum group, American group, Longiflorum group, Trumpet group, depending on hybridization affinity. ) And Oriental group. Among them, Asian, Trumpet, Oriental, etc. are widely used for cut flowers, differentiation and flower beds regardless of East and West. Since then, two additional horticultural varieties have been classified into eight families, with Oriental hybrids and other remaining species as limits (McRae, E, A. 1998. Lilies. Timber Press, Portland).

Thus, about 70 species of Nari grow in the temperate zone of the northern hemisphere, and about 11 species are distributed in Korea, and Korea is a native country of Nari. However, there are some studies on the native lilies of Korea, but some researches on the cultivation and reproduction are very poor. A Study on the Distribution, Ecology, and Morphological Characteristics of Korean Native Lilium). DNA markers not only reflect the genetic characteristics of varieties inherently, but are also universal characteristics of organisms, and can be developed and applied to all crops. can do. Moreover, when DNA markers are used for cultivar identification purposes, they can be defined by quantifying the distinction between varieties and varieties. (Park Sung-hwan, Takara Korea. Life Science & Biotechnology 14-15p). Lilies are a very important flower crop all over the world and are the third most produced in Korea. Korean native lilies have been used as an important model for the cultivation of lily horticulture varieties and are still used in many countries.

However, the development of SSR (Simple Sequence Repeats) markers for the Lilium has been insufficient. The development of unique markers of these native native lilies can be said to be very important because they can be used as a basis for assigning unique characteristics to Korean lilies. Therefore, research has been continuously conducted to study Lilium, which was studied only in horticultural aspects, in molecular genetics, and to develop SSR markers to analyze genetic similarity and diversity among Lilium species.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

Accordingly, the present inventors have completed the present invention by developing SSR primer pairs that show a large number of polymorphic variation in various country strains while researching to develop an SSR marker capable of efficiently evaluating the genetic resources of Lilium.

It is therefore an object of the present invention to provide an SSR marker and its use capable of efficiently evaluating the genetic resources of Lilium.

In order to achieve the above object, the present invention provides an SSR primer pair having two base sequences selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 60.

In order to achieve another object of the present invention, the present invention comprises the steps of (a) extracting genomic DNA from the genus Lilium plants to be analyzed; (b) using the extracted genomic DNA as a template and performing PCR using the SSR primer pair of claim 1; And (c) provides a method for detecting DNA polymorphism of the genus Lilium plant comprising the step of separating the PCR product by size.

In order to achieve another object of the present invention, the present invention comprises the steps of (a) extracting genomic DNA from Lilium and Lilium varieties; (b) using each extracted genomic DNA as a template and performing PCR using the SSR primer pair of claim 1; (c) separating each PCR product by size; And (d) provides a method for identifying the breed of lilies comprising the step of comparing the results of the separation according to the size of the lilies and the control lilies.

In order to achieve another object of the present invention, the present invention provides a kit for detecting DNA polymorphism of the genus Lilium plant comprising the SSR primer pair, DNA polymerase and PCR reaction buffer of the present invention.

In order to achieve another object of the present invention, the present invention provides a kit for identifying a breed of Lilium comprising the SSR primer pair of the present invention.

Hereinafter, the present invention will be described in detail.

The present invention is characterized in that it provides an SSR marker capable of specifically analyzing the genetic diversity of Lilium spp. Plants. In the present invention, a SSR marker was developed from a plant of the genus Lilium by a novel method combining AFLP and one way PCR. SSR markers isolated from the genus Lilium are the first to be provided by the present invention.

The SSR marker provided by the present invention refers to a pair of PCR primers, and includes a forward primer and a reverse primer. SSR primer pair provided in the present invention has two base sequences selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 60. Preferably, L1 (primary pair represented by SEQ ID NOs: 1 and 2), L4 (primary pair represented by SEQ ID NOs: 3 and 4), L5 (primary pair represented by SEQ ID NOs: 5 and 6), L9 (SEQ ID NO: 7 And primer pairs represented by SEQ ID NO: 8), L18 (primer pairs represented by SEQ ID NOs: 9 and 10), L20 (primer pairs represented by SEQ ID NOs: 11 and 12), and L27 (primer pairs represented by SEQ ID NOs: 13 and 14) , L39 (primary pairs represented by SEQ ID NOs: 15 and 16), L54 (primary pairs represented by SEQ ID NOs: 17 and 18), L59 (primary pairs represented by SEQ ID NOs: 19 and 20), L60 (SEQ ID NOs: 21 and 22) Primer pairs represented by SEQ ID NO.), L61 (primer pairs represented by SEQ ID NOs: 23 and 24), L66 (primer pairs represented by SEQ ID NOs: 25 and 26), L67 (primer pairs represented by SEQ ID NOs: 27 and 28), eL16 (Primary pairs represented by SEQ ID NOs: 29 and 30), eL17 (primary pairs represented by SEQ ID NOs: 31 and 32), eL28 (SEQ ID NO: Primer pairs represented by SEQ ID NOs: 33 and 34), eL34 (primer pairs represented by SEQ ID NOs: 35 and 36), eL40 (primer pairs represented by SEQ ID NOs: 37 and 38), and eL42 (primers represented by SEQ ID NOs: 39 and 40) Pairs), eL44 (primary pairs represented by SEQ ID NOs 41 and 42), eL47 (primary pairs represented by SEQ ID NOs 43 and 44), eL57 (primary pairs represented by SEQ ID NOs 45 and 46), eL58 (SEQ ID NO 47) Primer pairs represented by and 48), eL64 (primer pairs represented by SEQ ID NOs: 49 and 50), eL65 (primer pairs represented by SEQ ID NOs: 51 and 52), eL70 (primer pairs represented by SEQ ID NOs: 53 and 54) , eL75 (primer pairs represented by SEQ ID NOs: 55 and 56), eL77 (primer pairs represented by SEQ ID NOs: 57 and 58), and eL81 (primer pairs represented by SEQ ID NOs: 59 and 60).

Primer set of the present invention is preferably at least one, at least two, at least three, at least four, at least five, at least six, at least seven, eight selected from the group consisting of the 30 SSR primer set More than 9, more than 9, more than 10, more than 11, more than 12, more than 13, more than 14, more than 15, more than 16, more than 17, more than 18, more than 19, more than 20 , At least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 SSR primer sets, most preferably Can use 30 SSR primer sets simultaneously. By using 30 sets of SSR primers simultaneously, it is possible to more efficiently distinguish the cultivars from other cultivars.

In the present invention, "primer" refers to a single stranded oligonucleotide sequence that is complementary to the nucleic acid strand to be copied and may serve as a starting point for the synthesis of the primer extension product. The length and sequence of the primer should allow to start the synthesis of the extension product. The specific length and sequence of the primer will depend on the primer utilization conditions such as temperature and ionic strength as well as the complexity of the DNA or RNA target required.

The primer pairs provided in the present invention and the information on the repeat motifs present in the supersatellites amplified by them, the size of the supersatellites to be amplified and the chromosomal alleles in which the supersatellites are present are shown in Table 1 below. .

SSR primer pairs provided in the present invention can be usefully used for detecting DNA polymorphism and analyzing genetic diversity in the genus Lilium plants. The SSR primer according to the present invention can be used to efficiently evaluate and preserve the genetic resources of the genus Lilium plant. Accordingly, the present invention provides a method for detecting DNA polymorphism of a Lilium plant comprising performing PCR using the SSR primer pair.

Specifically, the method of the present invention

(a) extracting genomic DNA from the genus Lilium plant to be analyzed;

(b) performing PCR using the extracted genomic DNA as a template and the SSR primer pair provided by the present invention; And

(c) separating the PCR products by size.

The extraction of genomic DNA in step (a) is performed by phenol / chloroform extraction or SDS extraction (Tai et al., Plant Mol. Biol. Reporter, 8: 297-303, 1990) commonly used in the art. Trimethyl Ammonium Bromide; Murray et al., Nuc. Res., 4321-4325, 1980) or commercially available DNA extraction kits.

In addition, the PCR in the step (b) may be performed using a PCR reaction mixture containing a variety of components known in the art required for the PCR reaction. The PCR reaction mixture includes an appropriate amount of DNA polymerase, dNTP, PCR buffer solution and water (dH ₂ O) in addition to genomic DNA extracted from the genus Lilium plant to be analyzed and the SSR primer pair provided in the present invention. The PCR buffer solution includes Tris-HCl, MgCl ₂ , KCl and the like. At this time, the concentration of MgCl ₂ greatly affects the specificity and quantity of amplification. Preferably in the range of 1.5-2.5 mM. Generally, if an excess of the Mg ^{2 +} increase the non-specific PCR amplification products, and reduce the yield of a PCR product if the Mg ^{2 +} insufficient. The PCR buffer solution may further contain an appropriate amount of Triton X-100. PCR also denatured the template DNA at 94-95 ° C., followed by denaturation; Annealing; And a cycle of extension, followed by general PCR reaction conditions which are finally elongated at 72 ° C. The denaturation and amplification may be performed at 94-95 ° C. and 72 ° C., respectively, and the temperature at the time of binding may vary depending on the type of primer. Preferably it is 52-57 degreeC, More preferably, it is 55 degreeC. The time and number of cycles in each step can be determined according to the conditions generally made in the art. Optimal reaction conditions when performing PCR using the SSR primer pair according to the present invention are as follows: After denature the template DNA for 2 minutes at 95 ℃, 30 seconds at 95 ℃; 30 seconds at 55 ° C .; And 35 cycles of one minute at 72 ° C., followed by reaction at 72 ° C. for 5 minutes.

PCR amplification results can be identified by size separation by agarose gel (agarose gel), polyacrylamide gel electrophoresis or fluorescence analysis (ABI prism 3100 genetic analyzer-electropherogram) (step (c) ). At this time, in order to use the fluorescence analyzer PCR is performed in the step (b) using a primer pair attached to a fluorescent die known in the art. Preferably it can be confirmed by polyacrylamide gel electrophoresis, more preferably by modified polyacrylamide gel electrophoresis. Electrophoresis results can be analyzed by silver staining after electrophoresis. General PCR performance and resulting analysis methods are well known in the art.

In addition, the present invention provides a method for identifying a variety of lilies, including performing PCR using the SSR primer pair.

Specifically, how to identify varieties of Lilium

(a) extracting genomic DNA from Lilium and Lilium varieties;

(b) performing PCR using each of the extracted genomic DNA as a template and the SSR primer pair provided by the present invention;

(c) separating each PCR product by size; And

(d) comparing the separation results for each size of step (c) with each other.

Genomic DNA extraction, PCR execution and isolation of PCR products by size in steps (a) to (c) are as described above, and in step (d) comparisons of Li and control Li varieties are the same combination of SSR primers. This is done by comparing the size of each PCR product of the cultivars of the pair and the control cultivars of the pair with each other. By comparing the size comparison results for each pair of SSR primers, if the results are the same as those of the control and the control species, the sample can be identified as the same breed as the control group. The identification method of the variety can be useful when the identification of the breed, such as judging whether there is a violation of the labeling of origin through judging whether the overlapping when introducing a new genetic resource and confirm the origin.

Genomic DNA extraction, PCR, and size separation of PCR products for a control group of varieties may be performed at the same time as the sample of the group, but may be performed prior to the sample of the group and prepared as a reference for identification. Using such a reference table can be useful because it can be compared with the reference table by performing genomic DNA extraction, PCR and separation of the size of the PCR product for the Lili to be a sample.

In another aspect, the present invention provides a kit for detecting DNA polymorphism of the genus Lilium plants comprising SSR primer pair, DNA polymerase and PCR reaction buffer solution according to the present invention. The composition of the PCR reaction buffer is as described above.

In addition, the components necessary for performing electrophoresis capable of confirming amplification of PCR products may be further included in the kit of the present invention.

In addition, the present invention provides a kit for identifying a breed of Lilium comprising the SSR primer pair according to the present invention. The breed identification method is as described above. In addition to the DNA polymerase and the PCR reaction buffer of the above-described composition in order to be able to easily perform the PCR reaction may be further included, the components or known to perform electrophoresis to confirm whether the PCR product amplification or not Identification criteria for selected varieties may be further included in the kits of the present invention.

Lilium plants that can be applied to the present invention include Lilium amabile Palibin, Lilium callosum Sieb. Et Zucc., Lilium cernuum Komarov, Lilium lancifolium Thunb., Lilium leichtlinii var. maximowitzii, Lilium hansonii LeichtLilium Ex, Lilium tsingtauense Gilg, Lilium hybridaAsiatic 'Red ALERT', Lilium hybrida Asiatic 'Yellow' Asian hybrid hybrid 'Wieme' (Lilium hybrida Asiatic 'wermeer'), Lilium formosanum, Trumpet 'Lilium longiflorum' white American ', Lilium brownie, Lilium regale Wilson , Oriental hybrid 'Sorborne', Oriental hybrid hybrid 'Casablanca', and Oriental hybrid hybrid 'Reeveb'.

Therefore, the SSR primer according to the present invention can be very useful for preparing DNA profiles of the genus Lilium plants. In this way, by efficiently evaluating Nari genetic resources, it is possible to effectively conduct redundancy analysis and establish novelty with existing resources when introducing new genetic resources. In addition, the SSR primer according to the present invention can effectively detect the DNA polymorphism of the genus Lilium plant, it is possible to determine the varieties of Lilium.

Figure 1 shows the SSR profile in the genus Lilium plants using the SSR primer of the present invention.
Figure 2 shows the SSR profile in the genus Lilium plants using the SSR primer of the present invention.
Figure 3 shows the SSR profile in the genus Lilium plants using the SSR primer of the present invention.
Figure 4 shows the SSR profile in the genus Lilium plants using the SSR primer of the present invention.
5 shows a schematic of the genus Lilium plant based on SSR markers.

Below. The present invention will be described in detail by examples.

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

<Experimental Method>

1. Experimental material

The lilies used in the experiments used 17 individuals collected and grown at Kangwon National University (see Table 1).

Lilium sample used turn Classification Scientific name One Asiatic group Lilium amabile Palibin 2 Asiatic group Lilium callosum Sieb . Et Zucc . 3 Asiatic group Lilium cernuum Komarov 4 Asiatic group Lilium lancifolium Thunb . 5 Asiatic group Lilium leichtlinii var . maximowitzii 6 Martagon group Lilium hansonii Leichtl . Ex 7 Martagon group Lilium tsingtauense Gilg 8 Asiatic group Lilium hybridaAsiatic ‘ Red ALERT ’ 9 Asiatic group Lilium hybridaAsiatic   ‘ Yellow ’ 10 Asiatic group Lilium hybridaAsiatic   ‘ wermeer ’ 11 Longiflorum group Lilium formosanum 12 Longiflorum group Lilium longiflorum  ‘ white ’ 13 Asiatic group Lilium brownie 14 Trumpet group Lilium regale Wilson 15 Oriental group Lilium hybridaOriental ‘ sorbonne ’ 16 Oriental group Lilium hybridaOriental ‘ casablanca ’ 17 Oriental group Lilium hybridaOriental ‘ reneve ’

2. DNA  extraction( extraction )

Genomic DNA extraction of plants was carried out using a commercial kit (QiAGEN. DNeasy plant Maxi kit), which was then pre-grounded using young leaves. The extracted DNA was quantitatively analyzed by electrophoresis on 1% agarose gel.

3. EST - SSR  ( Simple Sequence Repeats ) Marker  Development and primer  design

Among the 3329 Lilium EST sequences, EST sequences with repeat motifs were selected from Longiflorum, Fomosanum, Reagale, and Hybrid . 166 EST-SSR primers were constructed using the ARGOS program with EST-sequences with selected repeat motifs.

4. Simple Sequence Repeats (SSR) Analysis

PCR amplification was performed by adding 3 μl of quantitative DNA, 5 μM primer pair, 10X buffer, 2.5 mM dNTPs, 5 U / μl i-StarMAX II Taq DNA polymerase (iNtRON, Korea) to 25 μl volume. 95 ° C., total denaturation for 2 minutes; 35 cycles of 95 ° C. 30 seconds, 55 ° C. 30 seconds, and 72 ° C. 1 minute as 1 cycle; And 72 ° C. for 5 minutes. To identify the amplified PCR product, 5 μl was mixed with 5 μl (1: 1) of electrophoresis loading buffer (98% formamide, 0.02% BPB, 0.02% Xylene, and 5 mM NaOH) to 6% polyacrylic acid. The result was confirmed by silver-staining after electrophoresis (PAGE) for 120 minutes on an amide gel (polyacrylamide gel).

<Experimental Results>

1.EST-SSR Analysis

A total of 166 SSR primers were designed from those with SSR motifs among the known EST sequences in Lilium. Among the 166 EST-SSR primer sets, 81 EST-SSR primer sets were selected and identified by PCR amplification to see if they are useful as markers that can distinguish 17 species of lichens (see FIGS. 1 and 2). In addition, 85 EST-SSR primer sets were analyzed for polymorphism through electrophoresis after PCR amplification using only 12 species except for oriental (see FIGS. 3 and 4).

As a result, it was confirmed that at least one species was amplified in 131 primers among 166 EST-SSR primer sets. The result is described in more detail as follows.

1) Analysis of all 17 species including Oriental and Asiatic resulted in amplification of 26 EST primer sets. Among them, 14 primers were amplified in all 17 species, and 12 were selected by amplifying at least 10 or more species among 17 species. Finally, primers usable as markers were finally selected from 14 primer sets (L_primer).

2) 47 primer sets were amplified in 12 species except 3 Oriental species and 2 Asiatic species to show polymorphism. Of these, 30 primers were amplified in all 12 species, and 17 primers were confirmed to be amplified in at least 10 species but not all. Finally, 16 primers were finally selected (eL_primer) usable as markers.

From these results, 30 SSR Markers that could be used to distinguish between species were selected (see Table 2).

At the li EST - SSR marker of primer  order turn Phra
Emer order order
number Repeating motifs Range of size of supersatellite to be amplified (bp) Number of alleles One L1 AACCTACACTTCCCTCTTCTTT One (CAC) ₅ 384 5 TTATTAGCAGCAACATTCAACT 2 2 L4 GTCTCACAGCCCTCCTACAC 3 (CCA) ₅ 286 10 ACTTTTCTTCGAGAATCAAGTG 4 3 L5 AACTCCACAATAAGAGGGAAG 5 (GA) ₉ 292 8 TGTTGTACTTGGCTGTTACATT 6 4 L9 CAATCCTCTGTGTCAATAACTG 7 (CAG) ₆ 187 4 GTAACAACCGGATCTTTAACTC 8 5 L18 ACAGCCCTCCTACACAACTC 9 (CCA) ₅ 180 7 GTCATAAACGGGTAGGGTTT 10 6 L20 CCAACAATTTTGATTACATGG 11 (GGA) ₉ 213 6 ATTCAAGCAATATCTCATCCTC 12 7 L27 CCTACATGTGCATCTCAAATAC 13 (CAA) ₄ 236 5 TAACAGATCCAGCAAAGATATG 14 8 L39 CTGAAGCAAACCTAATTCCTAC 15 (CTT) ₄ 274 9 GATATGATAAAGGGCAAGACTC 16 9 L54 CGGTAGTCTTAAGCAAGAAGTT 17 (ATC) ₅ 303 5 ACTGATATGGAGTTGGATGAGT 18 10 L59 ACTGGGGAGAATATCAAGAAC 19 (CAC) ₉ 288 9 AAAAACCAACTACAACACATCA 20 11 L60 CCACAATAAACGATGATGTCT 21 (TCC) ₆ 400 9 TAAGCATCATATCAAGCATAGC 22 12 L61 GATTGCACTCTATCAGTCACAG 23 (GCC) ₅ 243 8 TAATCCCTTTATGAAGATGGTC 24 13 L66 CTATTTCCCCTCCTTTGACC 25 (CT) ₇ 160 6 AGATGGTGTCTGTTGAAGTTTT 26 14 L67 CAGAGATACAAAGCAAAAACAA 27 (CT) ₈ 154 9 AAGAGTGGAGGATCTGAAGAG 28 15 eL16 TTTCTCGGTTGGCCCCTATG 29 (CTC) ₄ 188 10 AGATGAGACATTGCCGGCTG 30 16 eL17 CTGATCTGGTAGACGAGCACGA 31 (AAT) ₄ 219 6 AGATGCTCACAAACACCGTCAA 32 17 eL28 TGCGCTCTGTAGTGTGTTCCAT 33 (TAA) ₆ 191 7 CAGACATGCCATGAAAACGAAG 34 18 eL34 CCCGTCAAGCAAGGATATCAAG 35 (TGG) ₄ 248 3 CCTTCTCTTCCTTCTTCGGCTC 36 19 eL40 TGACTTCCGCAGAGATAGAGGC 37 (GGC) ₅ 181 8 CTCATGTCAGTCCCATGCACTC 38 20 eL42 AAGCATGCTGAGCTGTTGTCAG 39 (GCA) ₄ 150 4 CTGCTTGAGTTGGTGTTGTTCG 40 21 eL44 TGGTGGTAGAGGGCAATCATCT 41 (GTG) ₄ 203 4 CTTGAGCAAAACAGACATCCCC 42 22 eL47 TCCCAATGAAGAACACCCTCTC 43 (CTT) ₄ 169 7 GACCTGGAAGAAGTCGGTGATG 44 23 eL57 GAACCGGTCTTCTTCCCTCAAC 45 (CCT) ₄ 162 7 GCCTCTCCACTGCAACCAGTAA 46 24 eL58 CAAAGGAGAAGCGATGAGTCGT 47 (CTC) ₄ 229 4 GGAACCATCGGTGAGAAGAGTG 48 25 eL64 AGTCAGATGCAGGAGAGGATGG 49 (GAG) ₄ 250 6 GTCCTCCGCTTCCACAAGTTC 50 26 eL65 CGAAATTAGGGTTAGGGTTCCG 51 (GCG) ₄ 205 6 GTCGGAGAAATTGCTCGAATTG 52 27 eL70 CAGGAGCTTAGGTGCTGCTGTT 53 (GCG) ₄ 184 7 TAGTGCTGCTCAGTTGTGTGGG 54 28 eL75 TACATCTGCTGGGTCCATCCTT 55 (TGC) ₄ 182 7 TGACAGCATTGTGAATGGAAGC 56 29 eL77 TTCCATTTCTAAACCCACACCG 57 (CGC) ₄ 152 3 TGATTTAGCTTTCAGCGCAGTG 58 30 eL81 CCCTTTGATGAAGCAGAAGTGC 59 (GCC) ₄ 157 4 TTGCACAGAAAATCACGATGCT 60

In addition, based on the EST-SSR primer amplification results of each species of the genus plants are shown in Table 3 that can be classified according to the set.

Classification marker set Lilium species EST-SSR primer set Lilium amabile Palibin L4, L5, L59, L60, L61, eL16 Lilium callosum Sieb. Et Zucc. L5, L20, eL44, eL47 Lilium cernuum Komarov L5, L18, L20, L59, L60, L67, eL28, eL75, eL77, eL81 Lilium lancifolium Thunb. eL40, eL65, eL75 Lilium leichtlinii var. maximowitzii L9 L20, L59, L67, eL75 Lilium hansonii Leichtl . Ex L5, L9, L20, L27, L59, eL16, eL40, eL44 Lilium tsingtauense Gilg L5, L20, L67, eL16, eL70, eL77 Lilium hybrida Asiatic ‘Red ALERT’ L20, L27, L39, L67 Lilium hybrida Asiatic ‘Yellow’ L20, L39, L61, L67 Lilium hybrida Asiatic  ‘ wermeer ’ L20, L39, L61, L67 Lilium formosanum L9, L67, eL57, eL58, eL65, eL81 Lilium longiflorum ‘white’ L67, L40, eL65 Lilium brownie L4, L18, L39, eL13, eL47, eL70, eL81 Lilium regale Wilson L9, L20, L39, eL16, eL47, eL70, eL75, eL81 Lilium hybrida Oriental ‘sorbonne’ L9 Lilium hybrida Oriental ‘casablanca’ L4, L18, L20, L60, L67 Lilium hybrida Oriental ‘reneve’ L59, L67

As described above, the SSR primer according to the present invention can be very useful for preparing a DNA profile of a plant of the genus Lilium. In this way, by efficiently evaluating Nari genetic resources, it is possible to effectively conduct redundancy analysis and establish novelty with existing resources when introducing new genetic resources. In addition, the SSR primer according to the present invention can effectively detect the DNA polymorphism of the genus Lilium plant, it is possible to determine the varieties of Lilium.

Claims (7)

SSR primer pair having two nucleotide sequences selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 60.
The method of claim 1, wherein the primer pair represented by SEQ ID NO: 1 and 2; Primer pairs represented by SEQ ID NOs: 3 and 4; Primer pairs represented by SEQ ID NOs: 5 and 6; Primer pairs represented by SEQ ID NOs: 7 and 8; Primer pairs represented by SEQ ID NOs: 9 and 10; Primer pairs represented by SEQ ID NOs: 11 and 12; Primer pairs represented by SEQ ID NOs: 13 and 14; Primer pairs represented by SEQ ID NOs: 15 and 16; Primer pairs represented by SEQ ID NOs: 17 and 18; Primer pairs represented by SEQ ID NOs: 19 and 20; Primer pairs represented by SEQ ID NOs: 21 and 22; Primer pairs represented by SEQ ID NOs: 23 and 24; Primer pairs represented by SEQ ID NOs: 25 and 26; Primer pairs represented by SEQ ID NOs: 27 and 28; Primer pairs represented by SEQ ID NOs: 29 and 30; Primer pairs represented by SEQ ID NOs: 31 and 32; Primer pairs represented by SEQ ID NOs: 33 and 34; Primer pairs represented by SEQ ID NOs: 35 and 36; Primer pairs represented by SEQ ID NOs: 37 and 38; Primer pairs represented by SEQ ID NOs: 39 and 40; Primer pairs represented by SEQ ID NOs: 41 and 42; Primer pairs represented by SEQ ID NOs: 43 and 44; Primer pairs represented by SEQ ID NOs: 45 and 46; Primer pairs represented by SEQ ID NOs: 47 and 48; Primer pairs represented by SEQ ID NOs: 49 and 50; Primer pairs represented by SEQ ID NOs: 51 and 52; Primer pairs represented by SEQ ID NOs: 53 and 54; Primer pairs represented by SEQ ID NOs: 55 and 56; Primer pairs represented by SEQ ID NOs: 57 and 58; And an SSR primer pair selected from the group consisting of primer pairs represented by SEQ ID NOs: 59 and 60.
The SSR primer pair according to claim 1 or 2, wherein the SSR primer pair is derived from a plant of Lilium spp.
(a) extracting genomic DNA from the genus Lilium plant to be analyzed;
(b) using the extracted genomic DNA as a template and performing PCR using the SSR primer pair of claim 1; And
(C) method for detecting DNA polymorphism of the genus Lilium plant comprising the step of separating the PCR product by size.
(a) extracting genomic DNA from Lilium and Lilium varieties;
(b) using each extracted genomic DNA as a template and performing PCR using the SSR primer pair of claim 1;
(c) separating each PCR product by size; And
(d) a method of identifying varieties of lilies comprising the step of comparing the results of the separation according to the size of the lilies and the control lilies.
A kit for detecting DNA polymorphism of a genus Lilium plant comprising the SSR primer pair of claim 1 or 2, a DNA polymerase and a PCR reaction buffer.
A kit for identifying a variety of lilies comprising the SSR primer pair of claim 1.

Images