KR101518701B1 - Analysis method for polymorphism of Xanthoceras sorbifolium Bunge using RAPD primer - Google Patents

Abstract

The present invention relates to a method of analyzing polymorphism of mulberry tree using RAPD primer, and more particularly, to a method of measuring polymorphism of pollen of mulberry tree by PCR (polymerase chain reaction) of DNA (deoxyribonucleic acid) The present invention relates to a method for analyzing polymorphism of mulberry tree using RAPD primer.

Description

(Analysis method for polymorphism of Xanthoceras sorbifolium Bunge using RAPD primer)

The present invention uses RAPD primer for analysis of the polymorphism of mulberry tree, primer of SEQ ID NO: 1, and primer of SEQ ID NO: 5 using the primers of SEQ ID NO: 1 and SEQ ID NO: 5, respectively, The present invention relates to a method for analyzing the polymorphism of the mulberry tree using the polymorphism analysis kit and the RAPD primer of the mulberry tree. More specifically, the polymorphism of the pollen of the mulberry tree by PCR (deoxyribonucleic acid) Wherein the RAPD primer comprises at least one of RAPD primer and RAPD primer.

Xanthoceras sorbifolium Bunge belongs to the Sapindaceae family in the northern part of China and is called yellowhorn, shiny leaf yellowhorn, goldenhorn, or Chinese flowering chestnut in English. It has about 6 ~ 18 seeds in about 5 ~ 6cm thick perilla, which is composed of about 34% ~ 63% of oil. In China, it is used in various fields including edible oil and health food. (Zhou and Fu, 2010) have been used as raw materials. In addition, the growth characteristics of these species have cold tolerance that can withstand temperatures of minus 40 ° C. Therefore, they can be cultivated in extreme areas, and their value in use is also considered to be very high.

It has been reported that mulberry trees are widely distributed in the northeastern part of China from 28 ㅀ 34'N to 47 ㅀ 20'N and from 73 ㅀ 20'E to 120 ㅀ 25'E (Mou et al., 2008) Little is known about the characteristics, genetic structure and diversity of the population. However, only techniques that can be used in comparison with general necks have been reported by using RAPD (Random Amplified Polymorphic DNA) molecular markers to select specific markers in the selection neck (Guan et al., 2010). Therefore, if we study the characteristics of groups and individuals using molecular indices, we can help them to select good individuals and households.

Various genetic analysis methods such as RAPD, AFLP, I-SSR and SSR have been used for genetic characteristics and differentiation of cultivated population. Since RAPD was used by Williams et al. (1990), it was recognized as a marker to obtain high polymorphism with short nucleotide binding of 10 bp or less. Therefore, we analyzed specific indicator and group structure of garlic, mustard and ginseng, (2010), and the results of this study are summarized as follows.

Therefore, the present invention analyzes the polymorphism of the mulberry tree such as the genetic diversity and the group characteristics using the DNA obtained from the mulberry tree seed collected from the Inner Mongolia, Liaoning and Shandong farms in China as a RAPD indicator, And information on the polymorphism of mulberry trees such as the genetic diversity of the mulberry tree and the characteristics of the mulberry tree are used to establish basic data for the establishment of strategies for the collection and conservation of genetic resources. I hope it will be possible.

As a prior art related to the present invention, Korea published patent application No. 2011-0135835 analyzed 136 plums collected from domestic plums and apricot and plums using 12 primers having high reproducibility among 20 RAPD primer sequences, and found 7.1 polymorphisms By detecting bands, genetic distances between cultivars can be analyzed to efficiently evaluate the genetic resources of plums and to identify plum varieties.

However, the present invention and the preceding method differ from each other in the object of the invention and the detailed technical features thereof, and thus the inventions are different from each other.

The present invention provides a primer of SEQ ID NO: 1 and a primer of SEQ ID NO: 5, respectively, to provide a RAPD primer for analyzing the polymorphism of the mulberry tree.

The present invention is to provide a hornbilled polyphenolic acid kits (kit) comprising the RAPD primer using the primers of SEQ ID NO: 5 in the primers of SEQ ID NO: 1

The present invention provides a method for analyzing the polymorphism of mulberry tree using the RAPD primer of SEQ ID NO: 1 to SEQ ID NO: 5.

The present invention relates to a method for collecting and preserving genetic resources of mulberry tree by measuring the information about the genetic polymorphism of mulberry tree such as the genetic diversity and the characteristics of the mulberry tree by carrying out the PCR of the DNA extracted from the mulberry tree seed Can be established.

The present invention provides a method for analyzing the polymorphism of mulberry tree using RAPD primer, comprising the step of polymerase chain reaction (PCR) of deoxyribonucleic acid (DNA) extracted from a seedling tree seed and measuring the polymorphism of the mulberry tree .

The present invention can analyze the genetic polymorphism of mulberry tree such as genetic diversity and group characteristics using DNA obtained from mulberry tree seed collected from Inner Mongolia Autonomous Region, Liaoning Province and Shandong Province cultivation farms as mulberry tree RAPD indicator. In addition, the present invention can be used to establish basic data for the establishment of strategies for the collection and conservation of genetic resources of mulberry trees by measuring information on the genetic polymorphisms of mulberry trees such as the genetic diversity and characteristics of mulberry trees have.

Fig. 1 is a Dendrogram of 36 Chinese cabbage seeds classified by the UPGMA method. [SD is a Chinese cabbage seed collected from Shandong Province, IM China, collected from Inner Mongolia Autonomous Region of China, LN collected from Liaoning Province, China Which means tree seeds.

The present invention shows a RAPD primer for analysis of the polymorphism of mulberry tree using the primers of SEQ ID NO: 1 and SEQ ID NO: 5, respectively.

The present invention shows a haploid polychromatic analysis kit (kit) comprising RAPD primers using the primers of SEQ ID NO: 5 and SEQ ID NO: 5, respectively.

The present invention shows a method for analyzing the polymorphism of mulberry tree using RAPD primer.

The present invention provides a method for analyzing the polymorphism of mulberry tree using a RAPD primer, which comprises PCR of DNA extracted from a mulberry tree seed and measuring the polymorphism of the mulberry tree.

DNA extracted from seedlings can be subjected to PCR using primers of SEQ ID NO: 1 and SEQ ID NO: 5, respectively, using primers.

CCGCCCAAAC ... SEQ ID NO: 1

ACCTCAGCTC ... SEQ ID NO: 2

CAGGCCCTTC ... SEQ ID NO: 3

GGAGGGTGTT ... SEQ ID NO: 4

TCGTCGAGGT ... SEQ ID NO: 5

In the above, the PCR for the DNA extracted from the seedlings of the mulberry tree was carried out by using the primers of SEQ ID NO: 1 and SEQ ID NO: 5, respectively, and using the primer and the PCR reaction solution, PCR can be carried out.

As an example of PCR for the DNA extracted from the seedlings of the seedlings, 1.5 mM MgCl ₂ , 200 uM dNTP, 0.025% BSA, 40 ng primer (the primers of SEQ ID NO: 1 and SEQ ID NO: 5 are included in each reaction solution) 1 unit Taq. Polymerase and 25 ng template DNA were used for the PCR of the DNA extracted from the seedlings. The PCR conditions were as follows: 45 sec at 94 ° C, 45 sec at 34 ° C, 1 min at 72 ° C The conditions were repeated 45 cycles and then incubated at 72 ° C for 5 minutes to display the number of amplified bands (1) and no (0) after electrophoresis on 1% agarose gel .

In the above, PCR of the DNA extracted from the seedlings of the mulberry tree can be used to measure the number of observed heterozygotes (Nm) in the mulberry tree.

In the above, PCR of the DNA extracted from the seedlings of the seedlings can be performed to measure the number of the alleles (Ne) in the seedlings of the seedlings.

In the above, PCR of the DNA extracted from the seedlings of the seedlings can be used to measure the polymorphism of the seedlings of the seedlings of the ratio of the polymorphic locus to the seedlings.

In the above, PCR of the DNA extracted from the seedlings of the mulberry tree can be used to measure the average structure and distance characteristics of mulberry trees.

In the above, PCR was carried out on the DNA extracted from the seedlings of the mulberry tree to determine the number (Nm) of observed heterozygotes for the mulberry tree, the number of effective alleles (Ne) per gene site, the ratio of polymorphic loci, One or more characteristics can be used to measure the mountain polymorphism of the mulberry tree.

The number of observed heterozygotes (Nm), the number of valid alleles per gene site (Ne), and the ratio of polymorphic loci among the polymorphic properties of the mulberry tree can be analyzed and / or measured using the POPGENE 1.32 program.

Among the polymorphic properties of the above mulberry tree, structural and distance analysis can be analyzed and / or measured using Nei's gene diversity.

Among these polymorphic properties, the distance estimation can be analyzed and / or measured by UPGMA using genetic distance and NTSYSpc 2.2 (Applied Biostatistics, USA).

In the above, the mulberry tree is subjected to the PCR of the seed DNA of the mulberry tree collected from the region using the Shandong province of China, the Inner Mongolia Autonomous Region of China and the Liaoning Province of China, and then the polymorphism of the mulberry tree is measured to identify the collected region of the mulberry tree and / It can be judged.

Genomic DNA extraction kit (TOYOBO, Japan) can be used for the extraction of DNA from seedlings collected from Shinto shrubs in China.

Genomic DNA extraction kit (TOYOBO, Japan) can be used for the extraction of DNA from seedlings collected from Inner Mongolia Autonomous Region of China.

Genomic DNA extraction kit (TOYOBO, Japan) can be used for the extraction of DNA from seedlings collected from Lycian tree in China.

In order to accomplish the object of the present invention, a method for analyzing the polymorphism of the mulberry tree using the RAPD primer according to the present invention is provided. .

Hereinafter, the present invention will be described in detail with reference to Examples and Test Examples. However, these are for the purpose of illustrating the present invention in more detail, and the scope of the present invention is not limited thereto.

<Experimental Example>

[Experimental Method]

(1) Characteristics of mulberry trees collected in Shandong, China

The genomic DNA extraction kit (TOYOBO, Japan) was used to obtain mulberry tree DNA from the mulberry tree seeds collected from Shandong, China.

With reference 20㎕ _{1.5mM MgCl 2, 200uM dNTP, 0.025} % BSA, 40ng primer ( SEQ ID NO: 1 in the primer of SEQ ID NO: 5 is included in the reaction solution, gagak each), 1 unit Taq. Polymerase, 25 ng template DNA from Edible seeds collected in Shandong, China, was added with hot start PCR for 45 sec at 94 ° C, 45 sec at 34 ° C and 1 min at 72 ° C. After repeating 45 cycles, PCR was carried out at 72 ° C for 5 minutes. The number of heterozygotes observed (Nm), the number of effective alleles per gene (Ne), and the number of heterozygotes (Ne) observed by displaying the amplitudes of the amplified bands after electrophoresis on 1% agarose gel, , Ratio of polymorphic loci, structure and distance analysis.

The characteristics such as the number of heterozygotes (Nm), the number of valid alleles (Ne), and the ratio of polymorphic loci in the polymorphism data of the mulberry tree were analyzed using the POPGENE 1.32 program. Nei gene diversity was used for distance estimation and UPGMA was performed using genetic distance and NTSYSpc 2.2 (Applied Biostatistics, USA).

(2) Characteristics of mulberry trees collected in the Inner Mongolia Autonomous Region of China

The genomic DNA extraction kit (TOYOBO, Japan) was used to obtain the mulberry tree DNA from the mulberry tree seed collected from the Inner Mongolia Autonomous Region of China.

With reference 20㎕ _{1.5mM MgCl 2, 200uM dNTP, 0.025} % BSA, 40ng primer ( SEQ ID NO: 1 in the primer of SEQ ID NO: 5 is included in the reaction solution, gagak each), 1 unit Taq. polymerase, 25 ng template DNA obtained from the seedlings collected from Inner Mongolia, China, was added to the reaction solution. The reaction was carried out by hot start PCR at 45 ° C. for 45 seconds at 34 ° C., 45 seconds at 34 ° C. and 1 minute at 72 ° C. After repeated cycles, PCR was carried out at 72 ° C for 5 minutes. The number of heterozygotes observed (Nm), the number of effective alleles per gene (Ne), and the number of heterozygotes (Ne) observed by displaying the amplitudes of the amplified bands after electrophoresis on 1% agarose gel, , Ratio of polymorphic loci, structure and distance analysis.

The characteristics such as the number of heterozygotes (Nm), the number of valid alleles (Ne), and the ratio of polymorphic loci in the polymorphism data of the mulberry tree were analyzed using the POPGENE 1.32 program. Nei gene diversity was used for distance estimation and UPGMA was performed using genetic distance and NTSYSpc 2.2 (Applied Biostatistics, USA).

(3) Characteristics of mulberry trees collected in Liaoning, China

The genomic DNA extraction kit (TOYOBO, Japan) was used to obtain mulberry tree DNA from the mulberry seed collected from Liaoning, China.

With reference 20㎕ _{1.5mM MgCl 2, 200uM dNTP, 0.025} % BSA, 40ng primer ( SEQ ID NO: 1 in the primer of SEQ ID NO: 5 is included in the reaction solution, gagak each), 1 unit Taq. Polymerase and 25 ng template DNA obtained from the seeds of the seedlings collected from Liaoning Province, China were added to the reaction mixture. The reaction was performed by hot start PCR for 45 seconds at 94 ° C, 45 seconds at 34 ° C, and 1 minute at 72 ° C ) And PCR was carried out at 72 ° C for 5 minutes. The number of heterozygotes observed (Nm), the number of effective alleles per gene (Ne), and the number of heterozygotes (Ne) observed by displaying the amplitudes of the amplified bands after electrophoresis on 1% agarose gel, , Ratio of polymorphic loci, structure and distance analysis.

The characteristics such as the number of heterozygotes (Nm), the number of valid alleles (Ne), and the ratio of polymorphic loci in the polymorphism data of the mulberry tree were analyzed using the POPGENE 1.32 program. Nei gene diversity was used for distance estimation and UPGMA was performed using genetic distance and NTSYSpc 2.2 (Applied Biostatistics, USA).

[Experiment result]

(1) The ratio (P) of polymorphic loci in 19 loci collected to shrubs collected from Shandong, Inner Mongolia and Liaoning province was close to 58% in Shandong Province (SD) (Hamrick and Sherman-Broyles, 1992), but it was somewhat lower in seed collected from the Inner Mongolia Autonomous Region (IM) and Liaoning Province (LN) (Table 1) ). As mentioned above, the RAPD method is simple because of its simple analysis method and many amplification products are available. However, considering all two alleles, the homogeneity of the allele frequency distribution observed in one allele is high The genetic structure of the native pine tree population has also been reported. Therefore, it should be considered in the analysis of polymorphic locus using RAPD markers (Lee, Seokwoo et al., 2007).

(2) The number of observed heterozygotes (Observed number of alleles, Nm) and the number of effective alleles of gene locus collected from Shandong Province, Inner Mongolia Autonomous Region and Liaoning Province, as shown in Table 1 below (Effective number of alleles, Ne ) Were found to be 1.42 and 1.27 in the three regions, respectively. Seed collected in Shandong province showed the highest value. The genetic diversity of domesticated mustard populations in Korea is comparable to the average number of alleles observed in 17 domestic populations (1.22) and the average number of effective alleles (1.17) (Oh et al. 2010). However, it seems that the cultivar will be different from the natural group because it is possible that the cultivated species has been crossed with the individuals that have been artificially transferred from other regions, or on the contrary, they have been isolated for a long time. The flowering of the flower is very active, but the productivity of the seed is low (Ding and Ao, 2008), and seed harvesting starts from 3 to 4 years after the planting Since the harvested seeds are used or sold for other purposes due to the continuous sale, the observed number of alleles is likely to be low when compared with the natural population in which seed production, germination and growth are active. These results indicate that the genetic diversity of cultivars (P = 14.8%) is lower than that of wild species in the genetic analysis of Lauhanguo ( Siraitia grosvenorii [swingle]) used for edible and medicinal use in China and wild species (Tang et al ., 2007).

(3) The mean genetic diversity (Hs) was 0.16 and the genetic diversity (Ht) of the whole group was 0.27 in Nei 's genetic diversity analysis of Seedlings collected from Shandong Province, Inner Mongolia Autonomous Region and Liaoning Province. The genetic diversity of Shandong province was 0.21, which is more than twice as high as Liaoning (see Table 2). The coefficient of gene differentiation (Gst), which indicates the degree of differentiation between groups, was 0.42. The high degree of genetic differentiation suggests that gene migration between groups is low (Moon & Hee, 2008). In the case of the Shandong cultivated group, it is assumed that genetic exchange is less likely to occur unless the plant is reorganized because it is geographically isolated from the other two groups.

(4) Cluster analysis (UPGMA) was conducted to investigate the genetic distances between the groups of mulberry trees collected in Shandong, Inner Mongolia and Liaoning province. As a result, Inner Mongolia and Liaoning, Seeds collected in Shandong showed genetic distance (see Table 3, see Fig. 1). In the case of Inner Mongolia group and Liaoning province, it is considered that the genetic distance did not show a large difference because of the possibility that the movement of the cultivar was carried out because it is geographically adjacent. However, unlike herbaceous plants, mulberry trees are harvested for 10 to 20 years or more when they are planted. Therefore, if not geographically close to each other, And it is not possible to exclude the possibility that the seeds of the seeds were the same area when planting. As mentioned earlier, seedlings are not uniform in seedlings. Therefore, if the genetic diversity of the group is lowered, it may lead to the loss of excellent resources such as seed loss and the loss of genetic resources. Therefore, Is required. In addition, it will be necessary to secure an excellent genetic resource through in-situ or off-site conservation through investigation of genetic groups and genetic characterization.

It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the present invention as defined by the appended claims. It will be understood that the invention may be variously modified and changed.

The present invention analyzes polymorphism of mulberry tree such as genetic diversity and group characteristics using DNA obtained from mulberry tree seed collected from Inner Mongolia Autonomous Region, Liaoning Province and Shandong Provincial Farm in China as a mulberry tree RAPD indicator, To analyze the polymorphism and to measure the information about the polymorphism of the mulberry tree such as the genetic diversity of the mulberry tree and the characteristics of the mulberry tree to establish the basic data for establishing strategies for the collection and conservation of the genetic resources of mulberry tree And it is industrially applicable.

<110> KNU-Industry Cooperation Foundation <120> Analysis method for polymorphism of Xanthoceras sorbifolium Bunge <160> 5 <170> Kopatentin 1.71 <210> 1 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> artificial RAPD primer <400> 1 ccgcccaaac 10 <210> 2 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> artificial RAPD primer <400> 2 acctcagctc 10 <210> 3 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> artificial RAPD primer <400> 3 caggcccttc 10 <210> 4 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> artificial RAPD primer <400> 4 ggagggtgtt 10 <210> 5 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> artificial RAPD primer <400> 5 tcgtcgaggt 10

Claims (5)

A RAPD primer composition for analyzing the polymorphism of a mulberry tree, comprising at least one primer selected from SEQ ID NOS: 1 to 5. 11. A cultivar polymorphism analysis kit of a mulberry tree comprising the RAPD primer composition for analyzing the locus polymorphism of the mulberry tree of claim 1. Wherein the step of PCR is carried out by PCR using at least one primer selected from the group consisting of SEQ ID NOS: 1 to 5 of DNA extracted from a seedling of a mulberry tree, and a step of measuring the polymorphism of the mulberry tree. Analysis method. The method of claim 3,
Wherein the polymorphism of the mulberry tree is determined by measuring at least one selected from the number of observed heterozygotes (Nm), the number of effective alleles (Ne) per genetic locus, the ratio of polymorphic loci, structure and distance analysis. Analysis of Mountain Polymorphism in. The method of claim 3,
A method for analyzing the polymorphism of mulberry tree using RAPD primer characterized in that the DNA extracted from the mulberry tree seed is the seed DNA of mulberry tree collected in the Shandong province, the Inner Mongolia Autonomous Region of China and the Liaoning Province of China.

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