KR100277113B1 - New varieties of turf Anyang - Google Patents

Abstract

The present invention relates to a new breed of grass Anyang.

The present invention analyzes the variation of Anyang grass planted on the fairway of Anyang Benest Golf Club, selects representative species, classifies them by DNA fingerprinting method, identifies taxonomic locations, and contributes to Korean grass classification by providing new varieties of grass. In addition, the new varieties of grass turf of the present invention has excellent properties and texture, such as disease resistance, pressure resistance, dry resistance, etc. has an excellent effect of strengthening the competitiveness of the grass market.

Description

New varieties of turf Anyang

The present invention relates to a new breed of grass. More specifically, the present invention is to identify the taxonomic location of the grass planted on the fairway (BGC) of Anyang Benest Golf Club to register as a new breed of grass.

Zoysiagrass is a perennial herbaceous plant, belongs to the flower family, and reproduces with underground diameters, and shows many variations in form and characteristics depending on the region. Conventionally known Korean grass types include 5 to 6 species such as Z. japonica, Z. matrella, Z. tenuifolia, Z. sinica, and Z. macrostachya. Grass is known. Most of the grasses used in golf courses, stadiums, and parks in Korea are wild grasses, and some of them are used in the southern regions, but few varieties are systematically selected and bred. In other words, since most of the Korean grasses currently distributed in the market are randomly selected and multiplied in nature, the varieties of varieties are not clear, and various types of grass are mixed indiscriminately, resulting in very low uniformity. In addition, the turf imported from abroad is being distributed in recent years, reducing the market competitiveness of Korean turf. Therefore, the present invention has been made to solve the above problems, the object of the present invention is excellent in disease resistance, pressure resistance, dry resistance, etc., excellent texture and characteristics of varieties such as uniformity, differentiation and stability that are distinguished from other grass varieties This is an excellent lawn new varieties Anyang Stop. Another object of the present invention is to identify the new varieties of Anyang middle finger to register as a new breed.

The above object of the present invention is to analyze the variation of grass in Korea, planted on the fairway of Anyang BGC to select the representative Anyang grass species to classify the Korean grass species including the selected Anyang grass representative species by DNA fingerprinting to classify the location of Anyang grass This was accomplished by selecting an Anyang grass DNA label and demonstrating its distinction from other varieties.

Hereinafter, the configuration and operation of the present invention will be described in detail.

FIG. 1 is a photograph showing RAPD band profiles for each system amplified 36 Korean grasses selected from the fairway of Anyang Benest Golf Club with primers A03, A08, and A10, respectively.

Figure 2 is a RAPD analysis of Zoysia species with operon primers A03, A10, A11.

3 is a dendrogram prepared by cluster analysis of 19 strains of Zoysia species.

FIG. 4 is a label selection diagram of specific RAPD for the new turfgrass species of the present invention obtained using operon primers A03, A19, C10, D03, and D15.

Figure 5 is a photograph showing restriction enzyme digestion of recombinant plasmid DNAs including RAPD markers A03-700, A19-600, C10-600, D03-800, D15-750.

Figure 6 shows the nucleotide sequence of RAPD marker OPC10-600.

FIG. 7 is a photograph showing a STS (Sequence tagged site) marker for identifying a new breed of grass Anyang in the present invention.

8 is a photograph showing the morphological characteristics of the new grass species (A) and general species (B) of the present invention.

The present invention is the step of collecting Korean grass in Anyang BGC fairway; Morphological analysis of the collected Korean turfgrass, RAPD mutation analysis step of analysis; Identifying the taxonomic location of Anyang grass by the variance analysis; And selecting specific DNA labels of the Anyang grass and examining growth characteristics and asexual propagation means. In the following examples, materials for the investigation of Anyang's middle ground and DNA label development include S-94, a common species such as S-94, a common species, and American meyer, Zenith, Korean wild grass, Anyang paper was used, and reagents used for DNA extraction were manufactured by Sigma, USA. Tag DNA polymerase and dNTP were used by Boehringer Manheim. In addition, primers A, C, and D of operon were used as primers for RAPD analysis.

Hereinafter, the present invention will be described in detail with reference to the following examples, but the scope of the present invention is not limited to these examples.

Example 1

[Gathering of Anyang Grass]

Anyang BGC fairway grass was collected at Anyang Benest Golf Club using a 11.5cm diameter hole cutter. The number of samples taken is 1 in 4, 13 and 17 holes (short holes), 1, 2, 3, 5, 6, 7, 8, 11, 12, 14, 15, 18 holes (9) Three samples were taken from 10 and 16 holes, and the samples were grown in plastic pots of 16.5 cm diameter. If there are two or more samples per hole, to distinguish them, number them from the side closest to the tee and select 1-1, 1-2,... , 9-1, 9-2, 9-3, and the like. For the purpose of individual growth investigation, cut cloths containing one node per line were cut and planted in a plastic pot with a diameter of 10 cm. The planting soil was mixed with K-soil (product of cyan industry) and sand mixed with 12% of peat moss at a ratio of 1: 1.

Example 2

[Variation Analysis of Anyang Grass]

Experimental Example 1

[Morphological morphology investigation]

Length, head length, number of seeds per head, number of heads per square meter, length and width of seeds, length of grass, length of leaves, leaf length, leaf length of 36 system Anyang BGC fairway grass collected in Example 1, Leaf width, length of laparoscopic diameter, number of laparoscopic diameter, and number of internodes were examined, and five times were examined. As a result, as shown in Table 1, the lengths of the water exits were 4-1, 8-1, and 17-1 longer than those of other strains, and the length of seeds and the number of seeds per ear were 4-1, 8-1, The length of 17-1 was shorter than other strains, but the number was larger. For leaf width, 4-1 and 8-1 strains were 4.8 ~ 5.2cm, significantly wider than 3.1 ~ 4.0cm of other strains. In numbers, the 4-1 and 8-1 strains were shorter and fewer than the others, and no creep appeared in 17-1. Overall, 4-1, 8-1, 10-2, 16-3, and 17-1 showed distinct differences from other strains, and there was no significant difference between the remaining strains. Therefore, all strains except 4-1, 8-1 and 17-1 were uniform enough to be recognized as one cultivar.

Experimental Example 2

[RAPD Mutation Analysis]

36 materials collected from 18 holes of Anyang Benest Golf Course of Example 1 were analyzed by RAPD method to analyze the degree of variation between Anyang grass. Samples showing more than 3% of the variation according to the degree of variance were considered non-Anyang grass and were not used as DNA marker selection materials. In addition, nine primers such as operon primers A01, A02, A03, A07, A08, A09, A10, C01, and C03 were used for RAPD analysis. 2 g of the grass leaves growing on the test cloth were pulverized with liquid nitrogen, and 150 μl of the grass leaf was transferred to a 1.5 mL microtube. Here, DNA extraction buffer (0.35M sorbitol, 0.1M Tris-base, 5mM EDTA; pH 7.5), nuclear digestion buffer (0.2M tris-base, 0.05M EDTA, 2M NaCl, 2% CTAB), 5% sarkosy1, respectively The extract solution mixed at a ratio of 5: 5: 2 was heated to 60 ° C., 750 μl was added thereto, mixed well, and heated at 60 ° C. for 30 minutes. After 30 minutes, 500 µl of chloroform was added and mixed well for 15 minutes. After centrifugation at 10,000 rpm for 10 minutes, the supernatant was taken and precipitated with the same amount of isopropyl alcohol. The precipitated DNA was recovered by centrifugation at 10,000 rpm for 10 minutes, washed twice with 70% ethanol, and then ethanol was evaporated and the DNA pellet was dissolved in DNA buffer. Final concentration of DNA was measured using a Fluorometer. PCR was performed using a Perkin Elmer 9600. The PCR reaction was denatured at 94 ° C. for 3 minutes, followed by 45 times at 94 ° C. for 1 minute and 72 ° C. for 2 minutes. The 25 μl reaction solution contained 10ng of templete, 100μM of dNTP, and 0.2μM of primer, and 10 × reaction buffer was used containing 1.5mM of Mg ²⁺ . Electrophoresis was performed after the PCR reaction. At this time, electrophoresis at 100V on a 1.4% agarose gel containing TBE buffer for 4 hours, stained with EtBr, observed with a UV illuminator and photographed to obtain data. Flexibility analysis of the data was performed using unweighted pair-group centriod clustering by NTSYS. As shown in FIG. 1, 4-1, 8-1, and 17-1 were clearly distinguished from other strains in the RAPD band pattern, and the remaining strains did not show any significant difference. However, because there was a slight difference in some bands among the other strains, the difference between the strains was used to examine 18 polymorphic bands that appeared in 36 strains per primer, with 1 and 0 depending on the presence or absence of bands as shown in Table 2. Scoring and writing binomial data matrix code showed flexibility. As a result, the 4-1, 8-1, and 17-1 strains were classified as completely different from the rest of the strains, and within the remaining strains, 9-1, 15-1, 16-1, 16-3, 18 The -2 line was about 12% different from the other lines. Thus, lines 1-1, 2-1, 3-1, 6-2, 8-2, 9-3, 10-1, 14-1, 12-2, 13-1, 10- Except for 3, 11-1, 11-2 and 4-1, 8-1, 9-1, 10-2, 15-1, 16-1, 16-3, 17-1, and 18-2, which had large variations The remaining strains were selected and named Anyangji.

Example 3

[Classification of Anyang Stop Using RAPD Fingerprinting]

Representative Anyang stop system 1-1, 1-2, 2-1, 4-1, 8-1, 17-1 separated in Example 1 by the RAPD method of Example 2 the above embodiment It confirmed through the same RAPD method as 2. For reference, five kinds of DNA of Korean grass, including Z. japonica, Z. matrella, Z. tennuifolia, Z. sinica, and Z. machrostachya, were distributed by Professor Choi, Jun-Soo from the Department of Ornamental Horticulture, Dankook University. I received a lot of consideration. In addition, in order to investigate the distinction between Anyang Stop and other stops, Dongpai Stop, Sunkyung Stop, Seoul Jongmyo S-94 and Seoul Jongmyo General Grass were included in the analysis. At this time, the primers used for RAPD analysis were five such as operon primers A03, A09, A10, A11, A18, and the result of RAPD profile was as shown in FIG. In addition, a total of 50 markers were selected from the five random primers (A03, A09, A10, A11, and A18) as shown in Table 3 below. Indicated. Experimental results showed that the grass was divided into five species or groups, Z. sinia, Z. machrostachya and Z. tennuifolia, each of which was independently represented by one species, and Z. japonica and Z. matrella were included in one group. As shown in FIG. 4, Anyang Stop was formed as an independent group different from those of Korean grass.

Example 4

Selection of Anyang Stop Specific DNA Marks

Experimental Example 1

[Development of Anyang Stop Specific Label by RAPD]

Example 2 for S-94, General Jong, Meyer, Zenith, Yaji, a representative grass of Korea, and Anyang Jungji of the present invention, for a total of 60 primers (Operon primer A, C, D series) RAPD analysis was carried out in the same manner as in to select a marker that appeared only in Anyang stop. As a result, when Operon primer A03, A19, C10, D03, D15 were used, five markers that were specific to Anyang middle ground were selected as shown in Fig. 4, and these were respectively A03-700, A19-600, and C10-600. , D03-800 and D15-750 (FIG. 5).

Experimental Example 2

[Determination of DNA Sequences of Anyang Stop Specific Labels and Selection of STS (Sequence Tagged Site) Labels]

Anyangji specific DNA fragments obtained as a result of the RAPD of Experimental Example 1 were recovered from the agarose gel by freeze-thaw. The isolated DNA fragment was mixed with pGEM ^T easy vector (Promega Co., Ltd.) at a ratio of 3: 1 and ligated at 4 ° C. for 24 hours. J.109, a strain of E. coli host strain, was treated with TSS to produce competent cells. The ligation solution was mixed and transformed by heat shock at 42 ° C. for 90 seconds. The transformed E. coli were incubated for 12 hours in a medium containing 50 mg / mL of ampicillin X-gal and IPTG, and recombinants were selected. Plasmid DNA was isolated by alkaline lysis. The separated plasmid was digested with EcoRI and electrophoresed. As a result, as shown in FIG. 5, four or more recombinant clones, each of which was cloned into the desired size DNA fragment, were obtained. The sequencing of four clones obtained for each label was performed by Sanger's chain termination method. As a result, the DNA base sequence shown in FIG. 6 was obtained. In addition, the STS label was synthesized in the forward and reverse direction by adding 12 bases to the random primer sequencing used for RAPD analysis after sequencing, and the annealing temperature was determined by PCR by calculating the Tm synthesized primer PCR was performed for each label. As a result, as shown in Table 4, among the five selected markers, except for C10-600, the markers failed to switch to STS because the differences between varieties that appeared in RAPD were lost when switching to STS. Therefore, C10-600 was added to 25μl reaction solution, template 30ng, dNTP 200μM, primers 25pmol, 100mM Tris-HCl (pH 8.3), 1.25mM MgCl ₂ , 50mM KCl, 94 ℃ 45 seconds, 68 ℃ 45 seconds, 72 The reaction was carried out 35 times at 1 ° C. and 30 minutes, followed by 10 minutes at 72 ° C. to convert to STS. As a result, the Anyang stop specific label converted to the STS label as shown in FIG.

Example 5

[Silent breeding means of Anyang Stop]

Ansexual breeding method of the Anyang stop of the present invention was carried out as follows.

Experimental Example 1

[Proliferation method by sprigging]

Sprigs of a part of the grass plant including the ground crawling or underground diameter of the new varieties of Anyang Middle Area of the present invention are cut to include a node and planted at a depth of 20 to 40 cm at a depth of 20 to 40 cm. Provide plenty of moisture after planting. As a result, pedicles and roots emerged from each node to form a lawn.

Experimental Example 2

[Proliferation Method by Stolonizing]

The ground crawling or underground view of the new varieties of Anyang in the present invention is cut to include a node and scattered in the area where the grass forge is formed. To prevent drying after midwife, irrigation is done after surface covering. As a result, lawns were formed using more grass than sprigging.

Experimental Example 3

[Proliferation Method by Sodding]

The new varieties of Anyang stop in 60-180cm in length, 40-45cm in width and planted at a certain interval on a wide and flat square grass turf (sod). As a result, the space between the turf was filled by the ground crawler and the underground, forming a lawn.

Experimental Example 4

[Proliferation Method by Plugging]

The new varieties of Anyang Stop is planted at regular intervals with a plug (Plug), which is a cylindrical or block-shaped small piece of grass. As a result, the space between the plugs was filled by the ground crawler and the underground, forming a lawn.

Experimental Example 5

[Proliferation method using multishoot]

After disinfecting and disintegrating tissues with high cleavage, such as ground creep, underground section, and apex of new varieties of Anyang, BA (6-benzyladenin) 0.1 ~ 1.0mg / L and NAA (1-naphthaleneacetic acid) 0.05 ~ 0.1mg MS medium containing / L (KNO ₃ 1900mg / L, NH ₄ NO ₃ 1650mg / L, MgSO ₄ · 7H ₂ O 370mg / L, KH ₂ PO ₄ 170mg / L, CaCl ₂ · 2H ₂ O 440mg / L, _{H 2 BO 3 6.2mg / L,} MnSO 4 · 4H 2 O 22.3mg / L, ZnSO 4 · 7H 2 O 8.6mg / L, NaMoO 4 · 2H 2 O 0.25mg / L, CuSO 4 · 5H 2 O 0.025mg / L, CoCl ₂ · 6H ₂ O 0.025mg / L, KI 0.83mg / L, FeSO ₄ · 7H ₂ O 27.8mg / L, disodium EDTA 37.3Mg / L, Glysine 2mg / L, sucrose 30 × 103mg / L, Thiamine hydrochloride 0.5mg / L, Pyridoxine hydrochloride 0.5mg / L, Nicotinic acid 0.5mg / L, myo-inositol 100mg / L, pH 5.7) is induced to multishoot. The isolated multishoot is separated into individual individuals and transferred to a 1/2 concentration of MS medium without hormones for rooting. Rooted plants are purified and grown in greenhouses.

Experimental Example 6

[Method of Regeneration of Plant from Callus]

2,4-D (2,4-dichlorophenoxyacetic acid) 1 after harvesting and disinfecting tissues such as ground creep or underground part of the new varieties Anyang middle ground, immature inflorescence, immature embryo, fertilization, and the tip of the root. The callus is incubated on the MS medium of Example 5 containing ~ 5mg / L and 0.5-1.0mg / L kinetin. The separated callus is transferred to a 1/2 concentration of MS medium containing no hormone to regenerate the plant. Regenerated plants are purified and grown in greenhouses.

Example 6

[Formal Characteristics of Anyang Stop]

The morphological characteristics of varieties such as leaf length, leaf width, section length and cleavage rate of common grass (Z. japonica) and new varieties of turfgrass of the present invention were compared. As a result, as can be seen in Table 5, the leaf length and leaf width, internode of Anyang stopped short texture of the grass better than deuljandi as well as the division is fast with high capital increased density robustness to ² / cm 2 or more divisions may cushion . In addition, as shown in Fig. 8, Anyang Middle Land was distinguished from ordinary grass by the ratio of seed width and length of 4-5.

As can be seen from the above embodiment, the present invention has an effect of contributing to the classification of grass in Korea by identifying the taxonomic location of the system through DNA fingerprint analysis. In addition, the new varieties of grass Anyangjung of the present invention has an excellent uniformity, differentiation, stability and texture compared to the conventional Korean grass and general grass, and has a strong breeding power through various asexual reproduction methods, so it has the effect of strengthening the market competitiveness of grass. It is a very useful invention in the grass breeding industry.

Claims (2)

Leaf color is bright yellow green, leaf length is 6 ~ l2cm, leaf width is 3.5 ~ 4cm, intercution length is 4 ~ 6mm, density is 2 / cm ² or more. Anyangji grass is a new kind of grass, characterized by the fact that it grows or spreads by planting or cultivating grass vegetation or grass forge including 4 ~ 5 length and width ratio. The new turfgrass species according to claim 1, wherein the new turfgrass varieties Anyang Stop has a gene sequence of the following (I).