KR20160097081A - Lilium formosanum CAPS marker identification method developed utilizing gene MatK - Google Patents
Lilium formosanum CAPS marker identification method developed utilizing gene MatK Download PDFInfo
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Abstract
Description
The present invention relates to a method for developing a white lily identification CAPS marker utilizing the MatK gene region, and more particularly, to a method for identifying a white lily identification CAPS marker using MatK gene region, The present invention relates to a method for developing a white-lined identification CAPS marker utilizing a MatK gene region, which is characterized by the development of a CAPS marker capable of recognizing a CAPS marker.
About 100 species of Lilium are known to grow in the temperate zone of the Northern Hemisphere and are classified into nine groups by the Royal Society of Horticulture in the UK and most of them are in the Sinomartagon, Martagon and Leucolirion groups. Lily is an economically important horticultural crop, which is the third highest in Korea following roses and chrysanthemums. It is an income crop with annual production of 192 million ha, of which 41 million was produced, with sales amounting to more than 32.2 billion won.
Most of the Asiatic strains found in Korea are genetic resources that can be useful for the development of new varieties through interspecific or interspecific hybridization, because they have excellent traits such as various colors and disease resistance. However, recent industrialization and lack of indiscriminate collection and management of indigenous plants have led to the rapid loss of these useful plants, and there is a need for conservation of genetic resources and systematic botanical classification. In January 2002, Korea became a member of the 50th Regular Member State with the introduction of a new plant variety protection system for the New International Plant Protection Alliance for the recognition of intellectual property rights for new varieties of plants. As a result, the importance of native plants has been emphasized, and in the case of cultivating new varieties using this, royalties have become obligatory. Therefore, in the case of domestic self-introduction and introduction self-introduction, it is necessary to prove origin and resource strategy and efforts.
Most varieties are Asiatic, Oriental and Longiflorum hybrids. Among them, LA (Longiflorum × Asiatic Hybrid), LO (Longiflorum × Oriental), OT (Oriental × Trumpet Hybrid) and OA (Oriental × Asiatic Hybrid) As the lines appear, attempts to substitute the traits of disadvantages have become active while maintaining the superior traits of each group. Among them, the white lily belongs to the Longiflorum group and has a white color, which is hard to find in the domestic native species Asiatic lily. In order to protect the new variety of LA hybrids between these two groups, accurate systematic identification will be required.
Molecular markers can be usefully used to identify genetically similar lily genomes, such as in-species varieties or environmentalized species that are purified to specific local environments, using a sequence change of a DNA-specific target region of a plant. The rbcL (rubisco gene region involved in photosynthetic carbon fixation) or matK (matulase) gene region, which utilizes the DNA region of the cell organelles inherited through the maternal system, such as chloroplasts, has a relatively high change in the length of the base sequence and the type of base It has been used as a barcode to distinguish genetic resources and to analyze the genetic relationship between similar genetic resources. In other words, the genetically unrelated genetic resources show many base changes in the DNA regions of these nuclei or chloroplasts, suggesting that genetic diversity is relatively high.
Cleaved amplified polymorphic sequences (CAPS), also called restriction amplification polymorphism sequences, are breed identification methods that detect sequence differences by restriction enzyme treatment.
Detection is carried out in advance for the purpose of a gene which knows that there is a difference in nucleotide sequence among varieties. Only the gene of the genome of the species is selected by PCR method and amplified. In most markers, there is no difference between the length of the amplified gene and the breed. However, since the internal sequence of the gene is different, restriction enzymes recognizing the sequence difference are selected and processed. It is possible to distinguish the difference between the varieties carrying the restriction enzyme-digested DNA and the varieties having the DNA not digested, and the difference in length is easily detected by electrophoresis.
Korean Patent Registration No. 10-1271367 (SSR primer isolated from Liliaceae plants and its uses) comprises a pair of SSR primers having two nucleotide sequences selected from the group consisting of SEQ ID NOS: 1 to 60 and PCR The present invention relates to a method for detecting a DNA polymorphism of a genus L. plant and a method for discriminating the genotype of the genus L., but it has a problem that it takes a long time to obtain a result because of the variety of kinds of discrimination markers. It is difficult to distinguish a certain religion in a short period of time.
In order to solve the above problems, a method for developing a white lily identification CAPS marker utilizing the matK gene region according to the present invention is used to identify the nucleotide sequence of the matK gene region from the mother line, The purpose of this study is to select the available restriction enzymes for the development of CAPS markers that can distinguish the native species and to verify the discrimination between genetic resources.
In order to achieve the above object, a method for developing a white lily identification CAPS marker using the MatK gene region according to the present invention comprises extracting DNA of a white lily, amplifying the MatK gene region with the DNA of the extracted white lily, And a step of verifying the CAPS marker of white flies based on the obtained MatK gene base sequence.
Further, the CAPS marker verification is characterized by amplifying the DNA of a white louse with a matK primer, and reacting the DNA of the amplified white louse with an NruI restriction enzyme.
Also, in the method for developing a white lily identification CAPS marker utilizing the MatK gene region, the primer for amplifying the MatK gene region comprises a Lily_matK_F primer prepared from the nucleotide sequence of ATGGAAGAATTACAAGGG in the 5 'to 3' direction and a 5 'to 3' And the Lily_matK_R primer prepared in the nucleotide sequence of CTAATTATTCACCAGGTC in the direction of SEQ.
The present invention relates to a method of developing a CAPS marker capable of discriminating among various kinds of white flies using the matK gene region, and is capable of discriminating native flies from crosses crossed with white flies and white flies .
Also, it can be useful to determine whether or not genetic material is transferred from white flies to future generations when it is used as a cross breed in order to develop new varieties using white flies in the future.
FIG. 1A is a graph showing the PCR result of the MatK gene region of the white flies and the native flies. FIG.
Fig. 1B is a state diagram showing a base substitution site of MatK gene of white flies and native flies. Fig.
Fig. 2 is an analysis chart of similarity between white lily and wild lily.
FIG. 3A is a state diagram showing the NruI restriction enzyme site in the white lily and wild-type MatK genes. FIG.
FIG. 3B is a state diagram showing the gene cleaved by amplifying the MatK gene of the white flies and the wild flies and using the Nru I restriction enzyme as a CAPS marker.
The present invention relates to a method for developing a white-lined identification CAPS marker utilizing the MatK gene region.
The method for developing a white lily identification CAPS marker utilizing the MatK gene region comprises the steps of extracting DNA of a white lily, amplifying the MatK gene region with the DNA of the extracted white lily to obtain a base sequence, And verifying the CAPS marker of the white lily on the basis of the CAPS marker.
In addition, the CAPS marker verification is preferably performed by amplifying the DNA of the white lily with the matK primer, and reacting the amplified DNA of the white lily with the NruI restriction enzyme.
Also, in the method for developing a white lily identification CAPS marker utilizing the MatK gene region, the primer for amplifying the MatK gene region comprises a Lily_matK_F primer prepared from the nucleotide sequence of ATGGAAGAATTACAAGGG in the 5 'to 3' direction and a 5 'to 3' It is preferable to use the Lily_matK_R primer prepared in the nucleotide sequence of CTAATTATTCACCAGGTC in the direction of SEQ.
FIG. 1A is a DNA marker showing the PCR result of the MatK gene region of the white flies and the native flies, wherein M is a DNA marker, 1 is a winged lark, 2 is a sea urchin, 3 is a sea tangle, 4 is a sea louse, 5 is a sea lion, , 7 is tsunari, 8 is hairy body language, 9 is sky lily, 10 is sky louse and 11 is white lily.
FIG. 1B is a state diagram showing a base substitution site of MatK gene of white flies and wild flies. FIG.
Fig. 2 is an analysis chart of similarity of white lily and wild lily.
FIG. 3A is a state diagram showing NruI restriction enzyme sites in the white lily and naturally occurring MatK genes. FIG.
FIG. 3B is a state diagram showing the gene cleaved by amplifying the MatK gene of the white louse and the native Lari and using the Nru I restriction enzyme as a CAPS marker, wherein M is a DNA marker, 1 is a white lily, 2 is a winged lily, 4 represents the tsunami, 5 is the sea louse, 6 is the sea lion, 7 is the middle lady, 8 is the sea lion, 9 is the hairy middle louse, 10 is the sky lily and 11 is the sky lance.
The white lily used in the present invention belongs to the Longiflorum group, which is originated from Taiwan and is the origin of the white lily which has a large plant size in the classification of the Royal Horticultural Society of the United Kingdom, and has high affinity with the native species in Korea.
Hereinafter, the present invention will be described in more detail with reference to Examples and Experimental Examples. It will be apparent to those skilled in the art, however, that the following examples are illustrative of the present invention only and do not limit the scope of the present invention. That is, a simple modification or change of the present invention can be easily performed by those skilled in the art, and all such modifications and alterations can be considered to be included in the scope of the present invention.
Example 1: Preparation of white lily and wild lily
The white lily which is collected and managed by the lily-based Korean biotechnology research institute used in the present invention and the wing lily of the Chungnam National University, purchased from Pyongchang, Gangwon Province, Ten kinds of native flies, which consisted of flies, and flies, were used as comparative subjects.
Example 2 Leaf Sampling and DNA Extraction of White Lily and Native Lily
For leaf sampling and DNA extraction of white lily and native lily, the young leaves of the wild lily, which was being maintained in the greenhouse of Chungnam National University, were cut out, quenched by liquid nitrogen, and then subjected to genomic DNA preparation using the HiGene genomic DNA prep kit (BIOFACT, Daejeon) Respectively.
The extracted DNA is preferably subjected to electrophoresis on a DU-730 UV / Vis spectrophotometer (Beckman coulter, CA) and 1% agarose gel for quantitative and qualitative analysis of extracted DNA.
Example 3: Amplification and nucleotide sequence analysis of the white Lily and naturally occurring matK domains
After obtaining the nucleotide sequence of the DNA extracted from the white lily and the wild lily, the matK nucleotide sequence was obtained using the genetic information disclosed in the US NCBI.
A primer was prepared according to the obtained matK nucleotide sequence and subjected to PCR. The PCR conditions consisted of denaturation at 94 ° C for 5 minutes, denaturation at 94 ° C for 30 seconds, annealing at 53 ° C for 30 seconds, and stabilization of the PCR product at 72 ° C for 5 minutes.
After completion of the PCR, electrophoresis was performed on 1% agarose gel to confirm amplification of the PCR products. PCR products were inserted into T-Blunt vector (Biofact), and then the base sequence was amplified using M13 (-20) Respectively.
As a result of analyzing the nucleotide sequences of the white flies and the native flies matK according to Example 3, the matK genes in the white flies and the wild flies were PCR amplified with a length of 1,539 bp as shown in FIG. 1A, . As shown in FIG. 1B, the nucleotide sequences of 165, 272, 347, 739, 1050 and 1071 were substituted.
Example 4: Analysis of nucleotide sequence similarity using matK nucleotide sequences of the white flies and wild flies
The similarity analysis using the matK nucleotide sequence of the above-described white lily and native lily was carried out using the UPGMA method of Molecular Evolutionary Genetics Analysis (MEGA) software.
As a result of classification of genetic rootlets based on the nucleotide sequences of the matL gene regions of the white and native nares, the white and native naturally occurring genetic resources could be classified into six groups as shown in FIG. In the first group, the matK gene was 100% identical to the nucleotide sequence. The second group is the group with the land louse. Third, it was found that the nucleotide sequences of the two species belonging to the Martagon group are the same, and the nucleotide sequence of 1,037 differs from other species in common. In the fourth and fifth groups, the nucleotide sequence was changed from 1,125 to 1,172 and 1,251. Finally, in the sixth group, white lily was included, and it was confirmed that base change was observed in 6 regions of the whole region of the matK gene specifically with the 10 species of native lily.
Example 5: Conversion and validation of white lily identification CAPS markers
The position of all restriction enzymes in the nucleotide sequence of matK obtained in the present invention was determined by GENETYX-WIN Software Ver. 5.0 (Genetyx), and the matK gene region was amplified by performing PCR for verification with the CAPS marker utilizing the matK gene region capable of identifying the white lily. Then, TCGCGA Of the NruI restriction enzyme.
The PCR was performed using DNA amplifiers based on matK primer information and the PCR conditions were the same as those for confirming the nucleotide sequence.
In addition, 10 μl of the PCR product obtained by performing the PCR, 20 μl of a reaction solution containing NruI restriction enzyme (Enzynomics, Daejeon) and 1 × Ez Buffer III was reacted at 37 ° C. for 2 hours, and electrophoresis was performed on 1.2% agarose gel And the CAPS marker was verified.
The matK gene region was amplified using the primer used in Example 3, followed by treatment with Nru I restriction enzyme, as shown in FIGS. 3A and 3B. In the 10 species of native species, 1,196 bp and 343 bp It was confirmed that it was cleaved by Nru I restriction enzyme and divided into two pieces. In the case of white lily, base substitution of 343 base sequence from G to T was impossible and Nru I restriction enzyme was not able to function. Thus, the existing 1,539 bp single gene fragment Can be confirmed in FIG. 3B.
Claims (3)
The method of developing a white lily identification CAPS marker comprises the steps of extracting white lily DNA;
Amplifying the MatK gene region with the DNA of the extracted white flies to obtain a base sequence; And
And a step of verifying the CAPS marker of the white flies based on the obtained MatK gene nucleotide sequence.
The CAPS marker verification includes amplifying DNA of a white flock with matK primer; And
And a step of reacting the amplified white lily DNA with NruI restriction enzyme. The method for developing a white lily identification CAPS marker utilizing the MatK gene region.
The primer for amplifying the MatK gene region was a Lily_matK_F primer prepared from the nucleotide sequence of ATGGAAGAATTACAAGGG in the 5 'to 3'direction; And
Wherein the Lily_matK_R primer is prepared using a base sequence of CTAATTATTCACCAGGTC in the 5 'to 3' direction.
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| KR101271367B1 (en) | 2010-12-08 | 2013-06-07 | 강원대학교산학협력단 | SSR primer isolated from Lilum spp. and use thereof |
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| KR101271367B1 (en) | 2010-12-08 | 2013-06-07 | 강원대학교산학협력단 | SSR primer isolated from Lilum spp. and use thereof |
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