JP2020080761A - Method for identifying regional variation of plant species - Google Patents

Abstract

To quickly identify regional variation by DNA variation unique to a target plant species that naturally grows in a survey area.SOLUTION: Provided is a method for identifying a regional variation of a plant species, which comprises the following steps A to D: a step A of collecting a sample from a target plant species that naturally grows in a survey area, analyzing the whole base sequence of the chloroplast DNA of the target plant species, and using it as a reference sequence; a step B of collecting a sample from the target plant species that naturally grows in one or more other areas and grouping by region to create a DNA mixture containing the chloroplast DNA; a step C of amplifying a DNA fragment by a PCR method in which a primer set is applied to the DNA mixture created in the step B; and a step D of simultaneously analyzing the base sequences of the DNA fragments amplified in the step C using a next-generation sequencer, and identifying the regional variation in the reference sequence determined in the step A.SELECTED DRAWING: None

Description

The present invention relates to a method for rapidly identifying regional variations in plant species.

In recent years, as measures for biodiversity conservation, not only measures against alien species but also efforts to conserve local ecosystems and genes have become active in greening. For example, the Ministry of the Environment stated in the “Guideline for slope greening in natural parks” formulated in 2015 that one of the purposes of greening is “to contribute to the maintenance, restoration and conservation of natural ecosystems” and the purpose of greening. The basic principle for fulfilling the above is stated as "respecting the locality and uniqueness of nature".

Therefore, as an environmental conservation measure at large civil engineering sites such as tunnels and dams, in addition to using native species distributed around the site, seedlings with genetic characteristics unique to the area to which the site belongs, that is, local seedlings, are used. The greening used is required. However, it is difficult to identify the regional seedlings that have the same genetic characteristics as the plant strains distributed around the target area from the seedlings in circulation from the morphology, etc. It is necessary to determine regional characteristics.

For example, in Patent Document 1, a geographical distribution pattern for each genotype for the same plant species collected from a plurality of regions is generated in advance for each same plant species as a database, and the genotype of a plant to be planted is determined. , A method for discriminating the regional characteristics of plants to be planted, which discriminates whether or not it matches the geographical distribution pattern, has been proposed. The method proposed in Patent Document 1 needs to collect plant species from a plurality of areas in advance and analyze their genotypes to generate a geographical distribution pattern. Therefore, it is necessary to collect various plant species from Japan in advance, analyze their genotypes, and create a database, which requires enormous preparation.

Further, in the regional distinction of seeds and seedlings, until now, a base sequence analysis by a capillary sequencer was performed, but the capillary sequencer has a high analysis accuracy, but is characterized by accurately decoding the base sequence of one kind of DNA fragment. Therefore, it was a problem that it took time and labor for analysis. For example, if there are 10 types of candidate fragments and it is attempted to discriminate the regional characteristics of 15 strains, it is necessary to decode the nucleotide sequences of 150 types of DNA fragments one by one by a capillary sequencer. In particular, when trying to discriminate the locality of a plant species having poor information on gene sequences, it is necessary to find a fragment capable of discriminating the locality from a plurality of DNA fragments amplified by a PCR reaction using an arbitrary primer set. Yes, it was very troublesome.

JP, 2016-73223, A

It is an object of the present invention to quickly identify a regional variation based on a DNA variation unique to a target plant species that grows naturally in a survey area.

Means for solving the problems of the present invention are as follows.
1. A method for identifying a regional variation of a plant species, which comprises the following steps A to D.
Step A: a step of collecting a sample from a target plant species that grows naturally in the survey area, analyzing the entire base sequence of the chloroplast DNA of the target plant species, and using it as a reference sequence.
Step B: a step of collecting a sample from a target plant species that naturally grows in another region of 1 or more, and grouping each region into a DNA mixture containing chloroplast DNA.
Step C: A step of amplifying a DNA fragment by a PCR method in which a primer set is applied to the DNA mixture prepared in Step B.
Step D: a step of simultaneously analyzing the base sequences of the DNA fragments amplified in step C using a next-generation sequencer to identify the regional mutations in the reference sequence determined in step A.
2. 1. Collecting 10 or more samples in step A The method for identifying regional variation described in.
3. The other region in the process B is 10 or more regions. Or 2. The method for identifying regional variation described in.
4. In step B, 100 or more samples are collected in total. ~3. The method for identifying regional variation according to any one of 1.
5. 1. The primer set in step C is a primer mixture containing a plurality of primer sets. ~4. The method for identifying regional variation according to any one of 1.
6.1. ~5. A method for planting a plant, comprising planting a seedling having a regional variation identified by the method for identifying a regional variation according to any one of 1.

The method for identifying regional variation of the present invention is a target plant that grows naturally in a survey area by analyzing and comparing chloroplast DNA of a target plant species that grows naturally in a survey area such as a civil engineering site and other areas. It is not necessary to collect the target plant species from all over Japan and analyze the genotype of each to create a geographical database in order to identify the DNA mutations specific to the species, greatly reducing the work in the previous stage. be able to. Since the method for identifying regional variation of the present invention uses the chloroplast DNA sequence of a plant species that naturally grows in the survey area as a reference sequence, the chloroplast DNA sequence has not been analyzed so far and is registered in public databases and the like. Even in plant species that have not been investigated, it is possible to identify DNA mutations unique to the surveyed area.

The method for identifying regional variation of the present invention is characterized by simultaneously analyzing the nucleotide sequences of many DNA fragments by utilizing a next-generation sequencer, and between groups including a plurality of samples grouped for each collected region. It is possible to greatly analyze the difference in the genes of the above and significantly reduce the time required to identify the DNA mutation unique to the investigated region. Further, by using a primer mixture containing a plurality of primer sets during PCR, the time required to identify a DNA mutation can be further shortened.

By identifying a DNA mutation unique to the survey area by the method for identifying regional variation of the present invention, and selecting a population having this DNA mutation and planting in this survey area, so-called contamination of domestic and foreign species is prevented, The local ecosystem can be preserved.

-Regional variation identification method The regional variation identification method of the plant species of the present invention is characterized by comprising the following steps A to D.
Step A: a step of collecting a sample from a target plant species that grows naturally in the survey area, analyzing the entire base sequence of the chloroplast DNA of the target plant species, and using it as a reference sequence.
Step B: a step of collecting a sample from a target plant species that naturally grows in another region of 1 or more and grouping the regions into groups to prepare a DNA mixture containing chloroplast DNA.
Step C: A step of amplifying a DNA fragment by a PCR method in which a primer set is applied to the DNA mixture prepared in Step B.
Step D: a step of simultaneously analyzing the nucleotide sequences of the DNA fragments amplified in step C using a next-generation sequencer to identify the regional mutations in the reference sequence determined in step A.

Hereinafter, the regional mutation identification method of the present invention will be described along with its steps.
"Process A"
A sample is collected from a target plant species that grows naturally in the survey area, and the entire nucleotide sequence of the chloroplast DNA of the target plant species is analyzed and used as a reference sequence.
The survey area is not particularly limited, but it may be a basin or mountain area where a large-scale civil engineering construction site is located, a municipality unit such as a municipality, a prefecture, a regional division such as the Kanto region, the Kyushu region, or Western Japan and Eastern Japan. There are large categories. If the plant species are highly adaptable and live in environments that differ greatly, areas can be divided according to their habitat, such as dry land and wet land, or flat land and highlands.
From among the plant species that grow naturally in such a survey area, the target plant species is selected according to the need for planting, etc., a sample is collected, the entire base sequence of the chloroplast DNA is analyzed, and the reference sequence To do. The number of samples is not particularly limited, but the number of samples should be 10 or more in order to identify the DNA mutations unique to the target plant species that grows naturally in the survey area, not the DNA mutations unique to the individual who collected the sample. Is preferable, and 20 or more is more preferable. The analysis for determining the reference sequence can be performed by an arbitrary device such as a capillary sequencer or a next-generation sequencer.

"Process B"
A sample is collected from a target plant species that grows naturally in one or more other regions, and is grouped for each region to create a DNA mixture containing chloroplast DNA.
For the target plant species, samples are collected from the indigenous strains distributed in other areas. When collecting samples, samples should be taken from naturally occurring overgrown lines rather than artificially planted ones.
Other areas can be determined, for example, in the same manner as in the above step A. It is preferable to select, for example, a region far from the survey region, a region having a different altitude, a region having a significantly different environment, or the like, as the other region for collecting the sample. Specifically, it is preferable to select in consideration of the horizontal direction (north/south limit) including the distribution range of the target plant species, the vertical direction from the coastal area to the mountainous area, geology, rainfall, sunshine duration, etc. .. The other area is preferably 10 areas or more selected from various parts of Japan, more preferably 20 areas or more, and further preferably 30 areas or more. The total number of samples to be collected is preferably 100 or more, more preferably 200 or more.

Then, at least chloroplast DNA is extracted from the collected individual samples. The DNA to be extracted may include chloroplast DNA, and all DNA may be extracted. The origin of chloroplasts is cyanobacteria that have co-existed intracellularly during evolution, but during the process of intracellular symbiotic evolution, many of the genes that cyanobacteria have migrated to the host's nuclear genome. As the somatic DNA, only the DNAs involved in chloroplast essential functions such as chloroplast formation, maintenance, and core reactions of photosynthesis were left. Chloroplast DNA is as short as 120,000 to 200,000 base pairs as compared with nuclear DNA (100 million base pairs or more), has a slow evolution rate, and has small interspecific variation among land plants, and thus is suitably used for phylogenetic analysis. be able to.

The method of extracting DNA is not particularly limited, and a general method of applying a surfactant such as CTAB (hexadecyltrimethylammonium bromide) or a method of using a commercially available DNA extraction reagent kit can be applied. it can.
It is preferable that the extracted DNA is dissolved in a DNA storage buffer such as TE buffer, and then the concentration and purity of the extracted DNA is quantified for each sample using a spectrophotometer or the like to prepare a constant concentration. Then, the DNA extracted from each sample is grouped into regions and mixed to prepare a DNA mixture containing chloroplast DNA. At this time, if the extracted DNA solution is adjusted to have the same concentration for each sample, by mixing equal volumes of the DNA solutions, the DNA mixture in which the DNAs from each sample are mixed in the same amount is prepared. Can be created.

"C process"
A DNA fragment is amplified by the PCR method in which a primer set is applied to the DNA mixture created in step B.
The primer set used is not particularly limited, but it is preferable to use a primer set for amplifying a region where intraspecific mutation is likely to occur. When a region where intraspecific mutation is likely to occur is reported in the target plant species, a primer set for amplifying the region can be used. If the target plant species has not been reported for its chloroplast DNA, intraspecific variation, etc., the chloroplast DNA has high homology between land plants, so that the chloroplast DNA in other plants is Previous literature on chloroplast DNA (for example, "The Tortoise and the Hare II, Relative Utility of 21 Noncoding Chloroplast DNA Sequences for Phylogenetic Analysis" (Joey Shaw, et al. American Journal of Botany, pp.142-166, 2005. ), "Comparison of whole chloroplast genome sequences to choose noncoding regions for phylogenetic studies in angiosperms: the tortoise and the hare III. "(Joey Shaw, et al. American Journal of Botany, pp.275-288, 2007.)," Leaves created from Primer pairs suitable for PCR-SSCP analysis of chloroplast DNA in angiosperms" (Nishizawa, T. The Journal of phytogeography and taxonomy, pp63-66, 2000.), etc. Based on the reference sequence of chloroplast DNA, a primer set for amplifying a region where intraspecific mutation is reported can be designed and prepared.

At this time, it is also possible to use a primer mixture in which a plurality of primer sets are mixed. At this time, it is preferable to combine primer sets to be mixed according to the annealing temperature of the primers and the length of the DNA fragment to be amplified. If you mix primer sets with different annealing temperatures or primer sets with different lengths of amplified fragments, only specific fragments will increase, and you may miss DNA fragments useful for regional distinction. It will become more sexual. When preparing the primer mixture, it is preferable to adjust the concentration of each primer solution to be constant and mix them in a constant volume.

1st PCR which amplifies the DNA fragment which analyzes a base sequence by PCR reaction is performed using a primer set. At that time, depending on the amount of DNA generally applied to the PCR reaction, a PCR is performed by adding a primer set or a primer mixture to a DNA solution that is divided into groups for each region and mixed. Since the method for identifying regional variation of the present invention uses a next-generation sequencer, a mixture of many DNA fragments can be analyzed simultaneously. Therefore, for example, if 15 samples are divided into 3 groups and 3 primer mixtures prepared from 10 kinds of preselected primer sets are used, amplification is performed by PCR reaction using 3 primer mixtures for 3 groups. Nucleotide sequence analysis may be performed on the 9 types of DNA fragments. On the other hand, the conventional method using a capillary sequencer needs to analyze 150 kinds of DNA fragments.

The temperature condition for carrying out the PCR reaction is not particularly limited, but for example, denaturation reaction: 94° C. for 30 seconds→annealing reaction: temperature corresponding to the primer mixture (50-60° C.) for 1 minute→amplification reaction: 72° C. for 1 minute, etc. Can be mentioned. By repeating this cycle about 30 times, the target DNA fragment can be amplified. When performing the PCR reaction, it is preferable to carry out the PCR reaction in about 30 cycles in order to minimize the risk of inserting an erroneous base. As for the reagent used in the PCR reaction such as DNA polymerase, it is preferable to use a highly pure reagent such as PrimeStar HS Polymerase (manufactured by Takara Bio Inc.).

Further, when the base sequence is analyzed by a next-generation sequencer, 2nd PCR that gives an index sequence is performed in order to identify the group from which the amplified DNA fragment is derived. When performing the 2nd PCR, the primer set and temperature conditions to be used are performed according to the protocol published by the manufacturer of the next-generation sequencer.
In the obtained PCR product, it is confirmed by electrophoresis whether a DNA fragment of a desired length is amplified, and the DNA concentration is quantified by a spectrophotometer or the like.

"D process"
A next-generation sequencer is used to simultaneously analyze the nucleotide sequences of the DNA fragments amplified in the step C to identify the regional mutation contained in the reference sequence determined in the step A.
After adjusting the concentration of the PCR product obtained in the step C to the predetermined concentration described in the protocol published by the manufacturer of the next-generation sequencer and performing the pretreatment reaction, set the sample in the next-generation sequencer. Simultaneous analysis of base sequences is performed. The nucleotide sequence information of the DNA fragment decoded by the next-generation sequencer is organized and analyzed using the analysis software attached to the next-generation sequencer. For the analysis, for example, the mutation analysis software Miseq Reporter installed in the next-generation sequencer: MiSeq manufactured by Illumina can be used.

Then, the reference sequence determined in the step A is compared with the sequence of the DNA fragment amplified by each primer set in each analyzed group, and a DNA mutation specific to the reference sequence is spontaneously grown in the survey area in the target plant. Identified as a "regional variation" that is a species-specific variation.

-Plant planting method By the regional variation identification method of the present invention, it is possible to identify the regional variation unique to the target plant species that grows naturally in the surveyed area. Therefore, by transplanting a seedling having a genetic background peculiar to the region, which is represented by the regional variation identified by the regional variation identifying method of the present invention, into the survey area, the greening is performed while preserving the regional ecosystem. be able to.
For example, in the case of greening after large-scale civil engineering work, when purchasing seeds from a trader, it is often unclear which region the seeds originate from. Therefore, by selecting and planting only the seedlings having the regional variation unique to the area where the greening work is performed, it is possible to prevent the so-called domestic and foreign species from being mixed and to preserve the regional ecosystem.

"Process A"
The target plant was Dianthus superbus L. var. longicalycinus(Maxim.) FNWilliams. Kawaranadeshiko is a herbaceous plant that grows in sunny grasslands and rivers, and is a constituent species of the Todashiba-Susuki community, which is often seen in the Kanto suburbs. Kawaranadeshiko has been cultivated for ornamental use since the Edo period, and overseas relatives have been brought into Japan for a long time, so there are many varieties and relatives even today. It is also often used as a planting species for greening with consideration for biodiversity.

Regarding this Kawarana deco, the survey area was set as the coastal area of Miyagi prefecture, and 4 samples were collected. In determining the total nucleotide sequence of the chloroplast DNA of these 4 samples, first, a genomic library was prepared from the DNA containing the chloroplast DNA extracted from each sample. The DNA solution of each sample is cut into pieces of DNA having a length of about 300 bp, and each piece is treated with a reagent kit for attaching a tag for the next-generation sequencer analysis, for example, using the Illumina Nextera XT kit, and genomic live. I created a rally. An index sequence for identification was attached to the genomic library prepared from each sample by PCR reaction, and then the nucleotide sequences of all DNA fragments contained in the genomic library prepared from 4 samples were simultaneously analyzed by a next-generation sequencer: MiSeq. .. After sorting the obtained nucleotide sequence information for each sample according to the index sequence, the nucleotide sequence of the tobacco chloroplast DNA, for which the entire nucleotide sequence of the chloroplast DNA has already been decoded, is loaded on MiSeq as a reference sequence. Using the analysis software provided in the above, the DNA fragments derived from the chloroplast DNA of each sample were aligned and the entire nucleotide sequence was decoded.
As a result, the sequence of one sample was significantly different from the other three samples. Samples differing in this sequence are likely to be transplanted species or horticultural varieties from other areas, and it is presumed that they do not have a regional variation unique to the survey area. Was used as the reference sequence.

"Process B"
Leaves of Japanese cotton crab, which grows naturally in three places in Shizuoka prefecture, were collected as a sample, which was taken as a Shizuoka group. In addition, leaves of planted seeds in the Technical Center of Taisei Corporation (Totsuka Ward, Yokohama City), leaves of commercially available horticultural varieties, and leaves of Ezokawaraneshiko, which is a closely related species of Kawaranadeshiko, were collected as samples and made into other groups. Since it was difficult to distinguish Ezokawa Ranadeshiko from the appearance, it was considered that it might have been mixed in the collected samples. Therefore, it was analyzed to confirm the presence or absence of contamination.

The sample was dried by encapsulating it in a plastic bag that could be sealed with silica gel, and then weighed 50 mg. This was frozen in a deep freezer at −80° C., crushed with zirconia beads, and then DNA was extracted using DNeasy Plant Mini Kit (manufactured by QIAGEN). The concentration of the DNA extract was adjusted to 10 ng/μL, and the Shizuoka group and the other groups were mixed in equal amounts to prepare a DNA mixture.

"C process"
Since there are no studies on intraspecific mutations in the chloroplast DNA of Aedes aegypti, a region that is considered to be more likely to detect an intraspecific mutation is selected from the existing literature on gene analysis of other plants. A kind of primer set was created with reference to the reference sequence of Kawanadeshiko collected from the surveyed area. The tested primer sets are shown in Table 1 below.

PCR reaction using PrimeStar HS Polymerase (manufactured by TAKARA BIO INC.) and the above 8 kinds of primer sets, using GeneAmp (system 9700) manufactured by Applied Biosystems, using the two kinds of DNA mixture prepared above as templates. Then, the DNA fragment was amplified. Amplification of the obtained PCR product was confirmed by the presence or absence of an electrophoretic band.

The amplified PCR product was purified using Agencourt AMPure XP (BECKMAN COULTER), the PCR products amplified with 8 primer sets were mixed in equal amounts for each group, and two samples from the Shizuoka group and other groups were mixed with each other. did.
Next, the obtained PCR products were mixed for each group, and 2nd PCR with an index sequence was performed.
After confirming the concentration of each group by Agilent 4200 Tape Station (manufactured by Agilent Technology), the concentration of each sample was diluted to 4 nM with 10 mM Tris buffer (pH 8.5). The diluted sample was mixed with 0.2N NaOH, the DNA was denatured into a single strand, and a hybridization buffer was added to further dilute it to obtain a final sample with a concentration of 4 pM.

"D process"
The final sample was added to Miseq Reagent Kit Nano V2 (manufactured by Illumina), nucleotide sequence analysis was performed using a next-generation sequencer (Miseq), and leaves were prepared from samples collected from the coastal area of Miyagi prefecture, which is the survey target. Based on the reference sequence of chloroplast DNA, alignment and mutation analysis were automatically performed. Furthermore, the number of mutations and the readability of sequence information were confirmed using an IGV (Integrative Genomics Viewer) capable of visualizing the sequence. The results are shown in Table 2.

As shown in Table 2, mutations could be confirmed in the sets A, D, E, G, and H of the eight primer sets. There were a total of 20 mutations, of which 19 were substitutions and 1 was insertion. Among them, it was confirmed that the region amplified by the primer sets A, D, G, and H had a geographical mutation unique to Kawaranadeshiko native to the coastal area of Miyagi prefecture, which is the survey area.

Claims (6)

A method for identifying a regional variation of a plant species, which comprises the following steps A to D.
Step A: a step of collecting a sample from a target plant species that grows naturally in the survey area, analyzing the entire base sequence of the chloroplast DNA of the target plant species, and using it as a reference sequence.
Step B: a step of collecting a sample from a target plant species that naturally grows in another region of 1 or more and grouping the regions into groups to prepare a DNA mixture containing chloroplast DNA.
Step C: A step of amplifying a DNA fragment by a PCR method in which a primer set is applied to the DNA mixture prepared in Step B.
Step D: a step of simultaneously analyzing the nucleotide sequences of the DNA fragments amplified in step C using a next-generation sequencer to identify the regional mutations in the reference sequence determined in step A. The regional mutation identification method according to claim 1, wherein 10 or more samples are collected in the step A. The regional variation identification method according to claim 1 or 2, wherein the other region in the step B is 10 or more regions. In the step B, a total of 100 or more samples are collected, and the method for identifying regional mutation according to claim 1. The regional mutation identification method according to any one of claims 1 to 4, wherein the primer set in the step C is a primer mixture containing a plurality of primer sets. A plant planting method, comprising planting a seedling having a regional variation identified by the regional variation identifying method according to any one of claims 1 to 5 in the survey area.