CN114292940B - Combined barcode sequences and methods for identification of rehmannia and rehmannia plants - Google Patents

Abstract

The invention discloses a combined barcode sequence and a method for identifying rehmannia and rehmannia plants, wherein the combined barcode sequence is formed by splicing an R257 sequence and an ITS sequence, a nucleotide sequence of the R257 sequence is marked as SEQ ID No1, and a nucleotide sequence of the ITS sequence is marked as SEQ ID No2. The combined barcode sequence has good primer universality, high variability, capability of providing rich specific sites, low requirement on a template during PCR amplification reaction, easiness in operation and greater advantage in medicinal plant barcode identification.

Description

Combined barcode sequences and methods for identification of rehmannia and rehmannia plants

Technical Field

The invention belongs to the technical field of plant variety identification methods, and particularly relates to a combined barcode sequence and a method for identifying rehmannia and rehmannia plants.

Background

Rehmannia belongs to Orobanchaceae, is a perennial herb and comprises rehmannia, solanum leaf rehmannia, schizostachys, rehmannia of Tianmu, hubei rehmannia and Gao rehmannia, only 1 species of rehmannia is distributed in Korea and Japan, and the rest species are Chinese specific species. Rehmannia, which is a traditional Chinese medicine in China, has a long medicinal history and is recorded in Shen nong's herbal Jing in the east Han period, and the medicinal value of rehmannia, which is described in detail in Ben Cao gang mu, which is written by Li Shizhen by Ming dynasty medical scientist. Research shows that rehmannia contains iridoids, ionones, phenethyl alcohol glycosides, triterpenes, flavones, saccharides, rehmannia glycosides, dihydrocatalpol glycosides, aucubin, acteoside, 5-hydroxymethyl furfural, tyrosol, catalpol glycosides, various amino acids and the like. It has wide pharmacological effects, such as lowering blood sugar, resisting hypertension, osteoporosis, tumor, allergy, protecting cardiovascular system, relieving inflammation, stopping bleeding, and enhancing immunity.

Rehmannia belongs to the genus rehmannia and is one of the most studied traditional Chinese medicines in the field of academic now. However, the plants of the genus have wide distribution range and large change of appearance morphology, and the traditional classification mainly depends on morphological standards and needs special knowledge and subjective judgment. How to quickly and effectively distinguish the species of the genus still faces great difficulty in distinguishing true and false medicinal materials. Due to the medicinal value of rehmannia, the species of rehmannia such as Hubei rehmannia and schizophragma, etc., are often used as the traditional Chinese medicine rehmannia. How to distinguish the genuine herbs and give full play to the curative effect of rehmannia is also in need. At present, the identification of rehmannia is limited to morphological characteristics, and if the sample to be detected is a small part of defective leaves or rhizomes, a technician is required to give a conclusion by accumulation of substantial professional knowledge and long-term experience. The emerging molecular biology brings new methods for species identification, one is based on the polymerase chain reaction molecular marker technology, but the molecular markers made by this technology all identify rehmannia, but not rehmannia other species. The other is DNA barcode, which is a combined DNA barcode sequence based on the ITS of chloroplast gene and nuclear gene, but only the DNA barcode of psbA-trnH + ITS is disclosed to distinguish the species rehmanniae, and it is not said whether it can be used as a standard barcode sequence for identifying the respective species of the whole rehmannia genus. the DNA barcode of trnS-trnG + ITS can be used as a standard barcode sequence for identifying rehmannia species, but the chloroplast gene is uniparental gene, the DNA barcode of R257+ ITS is nuclear gene with parental genetic background, and R257 is more single-copy nuclear gene. The single copy nuclear gene can directly distinguish orthologous and paralogous, and the orthologous relationship between species is easy to establish, thereby providing convenience for revealing the genetic relationship problem of species and being widely applied to population genetics research.

Disclosure of Invention

The invention aims to provide a combined barcode sequence and a method for identifying rehmannia and rehmannia, wherein the combined barcode sequence R257+ ITS is used for accurately identifying the rehmannia and the rehmannia.

The technical scheme adopted by the invention is that the combined barcode sequence for identifying rehmannia and rehmannia plants is formed by splicing an R257 sequence and an ITS sequence, wherein the nucleotide sequence of the R257 sequence is marked as SEQ ID No1, and the nucleotide sequence of the ITS sequence is marked as SEQ ID No2.

The invention adopts another technical scheme that the method for identifying the rehmannia and the rehmannia plants is implemented according to the following steps:

step 1, extracting DNA of a sample to be detected; extracting the DNA of the sample by adopting a DNA extraction kit; or extracting sample DNA by adopting a CTAB method;

step 2: respectively carrying out PCR amplification reaction by using a primer of an R257 sequence and a primer of an ITS sequence to obtain an amplification product of the R257 sequence of the sample to be detected and an amplification product of the ITS sequence of the sample to be detected; the total volume of the reaction system for PCR amplification reaction was 20-30ng/uL of template DNA 20-30 uL, forward and reverse primers 0.3 uL (1. Mu. Mol/L), 10 uL of 2 XTaq PCR Mastermix (containing 0.3U Taq DNA polymerase, 1mmol/L dNTP,40mmol/L Tris-HCl (pH 8.3), 6mmol/L MgCl ₂ ) 8.4. Mu.L of double distilled water.

Step 3, performing gel electrophoresis on the amplification products respectively, and performing bidirectional sequencing on the sample with the bright single band to obtain a R257 sequence of the sample to be detected and an ITS zone sequence of the sample to be detected;

step 4, leading two sequences of bidirectional sequencing of the sample R257 to be detected into software to be spliced into a sequence, and obtaining the sequence of the sample R257 to be detected; introducing two sequences of ITS bidirectional sequencing of a sample to be detected into software to splice into one sequence to obtain the sequence of ITS of the sample to be detected;

step 5, aligning and cutting the R257 sequence of the sample to be detected and the R257 nucleotide sequence marked as SEQ ID No1, and aligning and cutting the ITS sequence of the sample to be detected and the ITS nucleotide sequence marked as SEQ ID No 2;

step 6, sequentially introducing the aligned R257 sequence and ITS zone sequence obtained in the step 5 into software to splice together to form a combined bar code sequence R257+ ITS;

and 7, importing the combined barcode sequence R257+ ITS into software to construct a phylogenetic tree, and if the sample to be detected and a certain species are gathered together, indicating that the sample to be detected is the species.

The invention is also characterized in that in the step 2, the PCR amplification condition of R257 is as follows: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 45 sec, annealing at 56 ℃ for 45 sec, extension at 72 ℃ for l min, 35 cycles, extension at 72 ℃ for 6 min, and heat preservation at 4 ℃; PCR amplification conditions for ITS: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 45 sec, annealing at 55 ℃ for 45 sec, extension at 72 ℃ for l min, 35 cycles, extension at 72 ℃ for 5min, and incubation at 4 ℃.

The combined barcode sequence has the advantages of good primer universality, high variability, capability of providing rich specific sites, low requirement on a template during PCR amplification reaction, easiness in operation and great advantage in medicinal plant barcode identification.

Drawings

FIG. 1 shows a phylogenetic tree of rehmannia plants constructed based on the distance method in the examples;

FIG. 2 is a phylogenetic tree constructed from standard DNA gene barcodes of the invention.

Detailed Description

The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

The ITS sequence is positioned in a gene transcription spacer region of ribosome DNA, the primer has good universality and high variability, and can provide rich specific sites. The R257 sequence is a single copy nuclear gene, and has the advantage that orthologs and paralogs can be directly distinguished. Chloroplast genes are uniparental inheritance, nuclear genes have the characteristics of parental inheritance and sequence conservation, the evolution speed is higher than that of plastid genomes, and DNA barcode sequence fragments which can be selected as identification species are more abundant. The R257 gene fragment has moderate length and provides more genetic information sites. Aiming at the two conditions, the combined DNA gene barcode sequence R257+ ITS is applied, the R257 sequence of the sample to be detected and the standard R257 sequence are aligned and aligned, the ITS sequence of the sample to be detected and the standard ITS sequence are aligned and aligned, the combined barcode R257+ ITS sequence of the sample to be detected is obtained after splicing, a phylogenetic tree is constructed, clustering analysis is carried out, and the type of the sample to be detected are clustered together, so that the identification accuracy is high, the operation is simple, the result is visual, and the identification degree is high.

As shown in fig. 2, a phylogenetic tree constructed with standard DNA gene barcodes, including RpBK schizophylla rehmannia (hubeibaokang); rpZS rehmannia glutinosa (Hubei bamboo mountain); rpHL schizophyllum rehmannia (salix after shanxi); rhYC Hubei rehmanniae radix (Yichang Hubei); rsCK radix rehmanniae solani leaf (chongqing city); rcLS rehmannia (zhejiang lishui); rcTX rehmannia glutinosa (crabapple stream, anhui); rcAH tianmu dihuang (am hui gao xi); rgTA rehmannia (santai' an, shandong); rgAK rehmannia (choke in the four kingdom); rgCZ rehmannia (shanxi changzhi); rgCC rehmannia (chongxin of gansu); tr cliff cabbage (foreign group), the species to be identified and that species are gathered together to form a single branch, and then the same species is proved. The number of samples to be tested is not limited, and only 10 samples to be tested are exemplified. This example is also a validation example, so the sample species name is known, but the true species identification is unknown, and the phylogenetic tree is constructed, and the cluster analysis is performed, and the sample to be tested and which species are together are the same.

The embodiment is as follows: obtaining R257 sequence and ITS sequence from rehmanniae sample

1. DNA extraction of a sample to be tested: (1) Samples of rehmannia were randomly collected from different origins, as shown in table 1;

TABLE 1 Experimental sample Collection information

(2) Provides two DNA extraction methods

1) The first DNA extraction method: and (2) performing DNA extraction on the sample to be detected in the table 1 by adopting a plant genome DNA kit method, wherein the method comprises the following steps:

1. 200mg of dried plant sample was placed in a 2ml centrifuge tube, 2-3 stainless steel balls were added, and ground on a tissue grinder at 28 Hz/s for 3 minutes.

2. 700 microliter 65 ℃ pre-heating buffer GP1 (added with mercaptoethanol before pre-heating) is rapidly added into the sample, the mixture is inverted and mixed evenly, the mixture is put into a 65 ℃ water bath kettle for warm bath for 20 minutes, and the centrifugal tube is inverted up and down to mix the sample 3 times every 7 to 8 minutes in the period for even mixing.

3. The samples after the water bath were taken out, and after cooling to room temperature, 700. Mu.l of chloroform was added thereto, and the centrifuge tube was inverted upside down in order to sufficiently mix the mixture, and after mixing was uniform, the mixture was centrifuged at 12000 rpm for 5min at room temperature.

4. The upper aqueous phase obtained after centrifugation was carefully poured into a new 2ml centrifuge tube, 700. Mu.l of buffer GP2 was added and mixed by inversion.

5. Pouring the fully and uniformly mixed solution into an adsorption column CB3 by times, wherein each time is 600-700 microliter, centrifuging at the normal temperature at 12000 r/m for 30s, discarding waste liquid, and putting the adsorption column CB3 into a collecting pipe.

6. Adding 500 microliters of buffer GD (absolute ethyl alcohol is added before the experiment) into the adsorption column CB3, centrifuging at the normal temperature at 12000 r/min for 30s, discarding the waste liquid, and putting the adsorption column CB3 into a collecting pipe.

7. Adding 600 microliters of rinsing liquid PW (absolute ethyl alcohol is added before the experiment) into an adsorption column CB3, centrifuging at the normal temperature at 12000 r/min for 30s, discarding waste liquid, placing the adsorption column CB3 into a collecting pipe, and repeating the step;

8. and placing the placed collecting pipe into a centrifuge, centrifuging at 12000 rpm for 2min at normal temperature, discarding the waste liquid, discarding the collecting pipe, and placing the adsorption column CB3 at room temperature for 2-3h to completely dry the residual rinsing liquid in the adsorption material.

9. Transferring the adsorption column CB3 into a clean 1.5ml centrifuge tube, adding 200 microliters of elution buffer TE, standing at room temperature for 5min, centrifuging at the speed of 12000 rpm at room temperature for 2min, and collecting the solution into a centrifuge tube of 1.5 microliters. Storing at-20 deg.C for use.

2) Carrying out DNA extraction on a sample to be detected in the table 1 by adopting a CTAB method, which specifically comprises the following steps:

1. 200mg of the dried sample were placed in a 2ml centrifuge tube, vitamin C, PVP and 2 small steel balls were added and ground on a tissue grinder at a frequency of 28 Hz per second for 3min.

2. Adding 1000 microliters of the pretreatment extract (STE) into a centrifuge tube, reversing for 2min, mixing uniformly, standing for 10min, placing in a centrifuge, centrifuging at room temperature at 8000 rpm for 10min, pouring out the supernatant, and repeating the steps once;

3. and (3) pouring 800 microliters of CTAB preheated at 65 ℃ into the mixed solution, reversing to mix the mixed solution uniformly, and carrying out warm bath in a 65 ℃ water bath kettle for 45min, wherein the centrifugal tube is turned upside down to mix the sample 3 times every 15min in order to mix the mixed solution uniformly. To the mixture was poured 600ul chloroform-isoamyl alcohol (24), to mix well and slowly reverse for 10min, centrifuged at 8000 rpm for 10min at room temperature, the supernatant was transferred to a 2.0ml centrifuge tube using a micropipette, and this step was repeated once;

4. the supernatant was transferred to a 1.5ml centrifuge tube using a micropipette, 800. Mu.l of absolute ethanol stored at 4 ℃ was added to the tube, and 2 to 3 drops of NaCl solution were added. Placing the centrifuge tube in a refrigerator at-20 deg.C for 60min, centrifuging at 4 deg.C at 2000 rpm for 10min, and discarding the waste liquid to obtain precipitate.

5. 500 microliters of 70% ethanol was added to the centrifuge tube twice repeatedly, and the precipitate was washed thoroughly. The centrifuge tube is placed at room temperature for 4-6h, and air-dried for precipitation.

6. Adding 200 microliters of TE solution into a centrifugal tube, standing at room temperature for 5min, centrifuging at 12000 rpm for 2min, fully mixing uniformly, and storing at-20 ℃ to obtain the DNA of the sample to be detected.

2. PCR amplification

(1) And respectively carrying out amplification reaction on the R257 sequence and the ITS sequence of the extracted DNA of the sample to be detected. The forward primer F and the reverse primer R of the R257 sequences are specifically as follows: f, CCCAGCAAGGATACAAAT, GGGTTATGCCAGATACAGTTA, and performing R257 sequence amplification reaction on a sample to be detected by using a R257 sequence primer F and a primer R to obtain a sample to be detected R257 amplification product. The forward primer F and the reverse primer R of the ITS zone sequence specifically comprise: f: TCCTCCGCTTATTGATATGC R, GGAAGTAAAA GTCGTAACAAGG uses an ITS sequence primer to carry out ITS sequence amplification reaction on the DNA of a sample to be detected, and an ITS amplification product of the sample to be detected is obtained.

(2) And (3) PCR reaction system: the total volume of the PCR amplification reaction system was 20. Mu.L, 1. Mu.L of 20-30ng/uL template DNA, forward and reverse primers were 0.3. Mu.L (1. Mu. Mol/L), 10. Mu.L of 2 XTaq PCR Mastermix, 8.4. Mu.L of double distilled water (ddH) ₂ O)。

(3) PCR amplification reaction process; after PCR amplification reaction, respectively carrying out gel electrophoresis on the obtained R257 amplification product and ITS amplification product of the sample to be detected, and sequencing the amplification product of the sample with a single bright band. The primers sequenced were identical to the PCR primers described above. To ensure the reliability of the DNA barcode sequence, forward and reverse sequencing is required.

And (3) introducing the forward sequencing result of the sample R257 into BioEdit software, looking at the peak shape of a peak chart interface, removing the initial or final set of peak mixed peaks, and re-sequencing if the initial or final set of peak mixed peaks are not good. And (3) leading the reverse sequencing result of the sample R257 into BioEdit software, observing the peak shape of a peak image interface, and splicing the forward and reverse sequences of the sample R257 to be detected if the forward and reverse sequences are spliced together to obtain a sample R257 sequence. And (3) introducing the ITS forward sequencing result of the sample into BioEdit software, looking at the peak shape of a peak diagram interface, removing the initial or final set of peak mixed peaks, and re-sequencing if the initial or final set of peak mixed peaks are not good. And (3) leading the reverse sequencing result of the ITS of the sample into BioEdit software, observing the peak shape of a peak diagram interface, and splicing forward and reverse sequences of the ITS of the sample to be detected together if the forward and reverse sequences are well spliced to obtain the ITS sequence of the sample. Respectively introducing a R257 sequence of a sample to be detected and a R257 nucleotide sequence marked as SEQ ID No1 into BioEdit software, and aligning the head and the tail of the R257 sequence of the sample to be detected with the R257 nucleotide sequence marked as SEQ ID No 1; respectively introducing an ITS sequence of a sample to be detected and an ITS nucleotide sequence marked as SEQ ID No2 into BioEdit software, aligning and cutting the head and the tail of the ITS sequence of the sample to be detected and the ITS nucleotide sequence marked as SEQ ID No2, respectively introducing the aligned and cut R257 sequence and the ITS sequence into Taxon DNA software for sequence splicing, and obtaining an R257+ ITS sequence. The R257+ ITS sequence is introduced into MEGA software, and a phylogenetic tree is constructed by using an NJ method. Because the software is an English interface, the species name of the obtained phylogenetic tree is changed into a Chinese name. As shown in fig. 1, sample 2, sample 3 and schizophragma are pooled together, indicating that sample 1, sample 2, sample 3 are schizophra; sample 4, sample 5, sample 6 and rehmannia of Tianmu are taken together, indicating that sample 4, sample 5, sample 6 are rehmannia of Tianmu; sample 7, sample 8, sample 9, sample 10 and rehmanniae radix are taken together, indicating that sample 7, sample 8, sample 9, sample 10 are rehmanniae radix.

<110> northwest university

<120> combinatorial barcode sequences and methods for identification of rehmannia and rehmannia

<130> do not

<160> 1

<170> PatentIn version 3.3

<210> 1

<211> 216

<212> SEQ ID No1

<213> Artificial sequence (Artificial sequence)

<400> 1

RpBK1. tagttactcg acgcttttga ctatgtatgt tgagaatcaa aagtttctag aggctctgtc tttgtttaaa gaaatgaggg agattaaatg ttcgcttgat

RpBK3. tagttactcg acgcttttga ctatgtatgt tgagaatcaa aagtttctag aggctctgtc tttgtttaaa gaaatgaggg agattaaatg ttcgcttgat

RpZS2. tagttactcg acgcttttga ctatgtatgt tgagaatcaa aagtttctag aggctctgtc tttgtttaaa gaaatgaggg agattaaatg ttcgcttgat

RpHL1. tagttactcg acgcttttga ctatgtatgt tgagaatcaa aagtttctag aggctctgtc tttgtttaaa gaaatgaggg agattaaatg ttcgcttgat

RpHL3. tagttactcg acgcttttga ctatgtatgt tgagaatcaa aagtttctag aggctctgtc tttgtttaaa gaaatgaggg agattaaatg ttcgcttgat

RhYC1. tagttactcg acgcttttga ctatgtatgt tgagaatcaa aagtttctag aggctctgtc tttgtttaaa gaaatgaggg agattaaatg ttcgcttgat

RhYC2. tagttactcg acgcttttga ctatgtatgt tgagaatcaa aagtttctag aggctctgtc tttgtttaaa gaaatgaggg agattaaatg ttcgcttgat

RhYC3. tagttactcg acgcttttga ctatgtatgt tgagaatcaa aagtttctag aggctctgtc tttgtttaaa gaaatgaggg agattaaatg ttcgcttgat

RhYC4. tagttactcg acgcttttga ctatgtatgt tgagaatcaa aagtttctag aggctctgtc tttgtttaaa gaaatgaggg agattaaatg ttcgcttgat

RhYC5. tagttactcg acgcttttga ctatgtatgt tgagaatcaa aagtttctag aggctctgtc tttgtttaaa gaaatgaggg agattaaatg ttcgcttgat

RsCK1. tagttactcg acgcttttga ctatgtatgt tgagaatcaa aagtttctag aggctctgtc tttgtttaaa gaaatgaagg agattaaatg tttgcttgat

RsCK2. tagttactcg acgcttttga ctatgtatgt tgagaatcaa aagtttctag aggctctgtc tttgtttaaa gaaatgaagg agattaaatg tttgcttgat

RsCK3. tagttactcg acgcttttga ctatgtatgt tgagaatcaa aagtttctag aggctctgtc tttgtttaaa gaaatgaagg agattaaatg tttgcttgat

RsCK4. tagttactcg acgcttttga ctatgtatgt tgagaatcaa aagtttctag aggctctgtc tttgtttaaa gaaatgaagg agattaaatg tttgcttgat

RsCK5. tagttactcg acgcttttga ctatgtatgt tgagaatcaa aagtttctag aggctctgtc tttgtttaaa gaaatgaagg agattaaatg tttgcttgat

RcLS1. tagttactcg acgcttttga ctatgtatgt tgagaatcaa aagtttctag aggctctgtc tttgtttaaa gaaatgaggg agattaaatg ttcgcttgat

RcTX1. tagttactcg acgcttttga ctatgtatgt tgagaatcaa aagtttctag aggctctgtc tttgtttaaa gaaatgaggg agattaaatg ttcgcttgat

RcTX3. tagttactcg acgcttttga ctatgtatgt tgagaatcaa aagtttctag aggctctgtc tttgtttaaa gaaatgaggg agattaaatg ttcgcttgat

RcAH1. tagttactcg acgcttttga ctatgtatgt tgagaatcaa aagtttctag aggctttgtc tttgtttaaa gaaatgaggg agattaaatg ttcgcttgat

RcAH2. tagttactcg acgcttttga ctatgtatgt tgagaatcaa aagtttctag aggctttgtc tttgtttaaa gaaatgaggg agattaaatg ttcgcttgat

RgTA4. tagttactcg acgcttttga ctatgtatgt tgagaatcaa aagtttctag aggctctgtc tttgtttaaa gaaatgaagg agattaaatg tttgcttgat

RgAK1. tagttactcg acgcttttga ctatgtatgt tgagaatcaa aagtttctag aggctctgtc tttgtttaaa gaaatgaagg agattaaatg tttgcttgat

RgCZ1. tagttactcg acgcttttga ctatgtatgt tgagaatcaa aagtttctag aggctctgtc tttgtttaaa gaaatgaagg agattaaatg tttgcttgat

RgCC1. tagttactcg acgcttttga ctatgtatgt tgagaatcaa aagtttctag aggctctgtc tttgtttaaa gaaatgaagg agattaaatg tttgcttgat

RgCC5. tagttactcg acgcttttga ctatgtatgt tgagaatcaa aagtttctag aggctctgtc tttgtttaaa gaaatgaagg agattaaatg tttgcttgat

Tr. tagttactcg acgcttttga ctatgtatgt tgagaatcaa aagtttttag aggctctgtc tttgtttaaa gaaatgaggg agattcaatg ttcgattgat

RpBK1. ctcactacgt gtaatataat gatagatgtg tatgggcaac ttgatatggc gaaggaagcc gataagctgt tttggggtat gagaaaaatt gggattgagc

RpBK3. ctcactacgt gtaatataat gatagatgtg tatgggcaac ttgatatggc gaaggaagcc gataagctgt tttggggtat gagaaaaatt gggattgagc

RpZS2. ctcactacgt gtaatataat gatagatgtg tatgggcaac ttgatatggc gaaggaagcc gataagctgt tttggggtat gagaaaaatt gggattgagc

RpHL1. ctcactacgt gtaatataat gatagatgtg tatgggcaac ttgatatggc gaaggaagcc gataagctgt tttggggtat gagaaaaatt gggattgagc

RpHL3. ctcactacgt gtaatataat gatagatgtg tatgggcaac ttgatatggc gaaggaagcc gataagctgt tttggggtat gagaaaaatt gggattgagc

RhYC1. ctcactacgt gtaatataat gatagatgtg tatgggcaac ttgatatggc gaaggaagcc gataagctkt tttggggtat gagaaaaatt gggattgagc

RhYC2. ctcactacgt gtaatataat gatagatgtg tatgggcaac ttgatatggc gaaggaagcc gataagctkt tttggggtat gagaaaaatt gggattgagc

RhYC3. ctcactacgt gtaatataat gatagatgtg tatgggcaac ttgatatggc gaaggaagcc gataagctgt tttggggtat gagaaaaatt gggattgagc

RhYC4. ctcactacgt gtaatataat gatagatgtg tatgggcaac ttgatatggc gaaggaagcc gataagctkt tttggggtat gagaaaaatt gggattgagc

RhYC5. ctcactacgt gtaatataat gatagatgtg tatgggcaac ttgatatggc gaaggaagcc gataagctkt tttggggtat gagaaaaatt gggattgagc

RsCK1. ctcactacgt gtaatataat gatagatgtg tatgggcaac ttgatatggc gaaggaagcc gataagctgt tttggggtat gagaaaaatt gggattgagc

RsCK2. ctcactacgt gtaatataat gatagatgtg tatgggcaac ttgatatggc gaaggaagcc gataagctgt tttggggtat gagaaaaatt gggattgagc

RsCK3. ctcactacgt gtaatataat gatagatgtg tatgggcaac ttgatatggc gaaggaagcc gataagctgt tttggggtat gagaaaaatt gggattgagc

RsCK4. ctcactacgt gtaatataat gatagatgtg tatgggcaac ttgatatggc gaaggaagcc gataagctgt tttggggtat gagaaaaatt gggattgagc

RsCK5. ctcactacgt gtaatataat gatagatgtg tatgggcaac ttgatatggc gaaggaagcc gataagctgt tttggggtat gagaaaaatt gggattgagc

RcLS1. ctcactacgt gtaatataat gatagatgtg tatgggcaac ttgatatggc gaaggaagcc gataagctgt tttggggtat gagaaaaatt gggattgagc

RcTX1. ctcactacgt gtaatataat gatagatgtg tatgggcaac ttgatatggc gaaggaagcc gataagctgt tttggggtat gagaaaaatt gggattgagc

RcTX3. ctcactacgt gtaatataat gatagatgtg tatgggcaac ttgatatggc gaaggaagcc gataagctgt tttggggtat gagaaaaatt gggattgagc

RcAH1. ctcactacgt gtaatataat gatagatgtg tacgggcaac ttgatatggc gaaggaagcc gataagctgt tttggggtat gagaaaaatt gggattgagc

RcAH2. ctcactacgt gtaatataat gatagatgtg tacgggcaac ttgatatggc gaaggaagcc gataagctgt tttggggtat gagaaaaatt gggattgagc

RgTA4. ctcactacgt gtaatataat gatagatgtg tatgggcaac ttgatatggc gaaggaagcc gataagctgt tttggggtat gagaaaaatt gggattgagc

RgAK1. ctcactacgt gtaatataat gatagatgtg tatgggcaac ttgatatggc gaaggaagcc gataagctgt tttggggtat gagaaaaatt gggattgagc

RgCZ1. ctcactacgt gtaatataat gatagatgtg tatgggcaac ttgatatggc gaaggaagcc gataagctgt tttggggtat gagaaaaatt gggattgagc

RgCC1. ctcactacgt gtaatataat gatagatgtg tatgggcaac ttgatatggc gaaggaagcc gataagctgt tttggggtat gagaaaaatt gggattgagc

RgCC5. ctcactacgt gtaatataat gatagatgtg tatgggcaac ttgatatggc gaaggaagcc gataagctgt tttggggtat gagaaaaatt gggattgagc

Tr. ctcacgacgt gtaatataat gatagatgtg tatggacagc ttgatatggc gaaggaagct gataagctgt tttggggtat gagaaaaatt ggaattgagc

RpBK1. cgaatgttgt tagttacaat actcttttga gggtttatgg tgatgctgag ctttttgggg aggcaataca tctctttagg ttgatgcaga gaaaggatat

RpBK3. cgaatgttgt tagttacaat actcttttga gggtttatgg tgatgctgag ctttttgggg aggcaataca tctctttagg ttgatgcaga gaaaggatat

RpZS2. cgaatgttgt tagttacaat actcttttga gggtttatgg tgatgctgag ctttttgggg aggcaataca tctctttagg ttgatgcaga gaaaggatat

RpHL1. cgaatgttgt tagttacaat actcttttga gggtttatgg tgatgctgag ctttttgggg aggcaataca tctctttagg ttgatgcaga gaaaggatat

RpHL3. cgaatgttgt tagttacaat actcttttga gggtttatgg tgatgctgag ctttttgggg aggcaataca tctctttagg ttgatgcaga gaaaggatat

RhYC1. cgaatgttgt tagttacaat actcttttga gggtttatgg tgatgctgag ctttttgggg aggcaataca tctctttagg ttgatgcaga gaaaggatat

RhYC2. cgaatgttgt tagttacaat actcttttga gggtttatgg tgatgctgag ctttttgggg aggcaataca tctctttagg ttgatgcaga gaaaggatat

RhYC3. cgaatgttgt tagttacaat actcttttga gggtttatgg tgatgctgag ctttttgggg aggcaataca tctctttagg ttgatgcaga gaaaggatat

RhYC4. cgaatgttgt tagttacaat actcttttga gggtttatgg tgatgctgag ctttttgggg aggcaataca tctctttagg ttgatgcaga gaaaggatat

RhYC5. cgaatgttgt tagttacaat actcttttga gggtttatgg tgatgctgag ctttttgggg aggcaataca tctctttagg ttgatgcaga gaaaggatat

RsCK1. cgaatgttgt tagttacaat actcttttga gggtttatgg tgatgctgag ctttttgggg aggcaataca tctctttagg ttgatgcaga gaaaggatat

RsCK2. cgaatgttgt tagttacaat actcttttga gggtttatgg tgatgctgag ctttttgggg aggcaataca tctctttagg ttgatgcaga gaaaggatat

RsCK3. cgaatgttgt tagttacaat actcttttga gggtttatgg tgatgctgag ctttttgggg aggcaataca tctctttagg ttgatgcaga gaaaggatat

RsCK4. cgaatgttgt tagttacaat actcttttga gggtttatgg tgatgctgag ctttttgggg aggcaataca tctctttagg ttgatgcaga gaaaggatat

RsCK5. cgaatgttgt tagttacaat actcttttga gggtttatgg tgatgctgag ctttttgggg aggcaataca tctctttagg ttgatgcaga gaaaggatat

RcLS1. cgaatgttgt tagttacaat actcttttga gggtttatgg tgatgctgag ctttttgggg aggcaataca tctctttagg ttgatgcaga gaaaggatat

RcTX1. cgaatgttgt tagttacaat actcttttga gggtttatgg tgatgctgag ctttttgggg aggcaataca tctctttagg ttgatgcaga gaaaggatat

RcTX3. cgaatgttgt tagttacaat actcttttga gggtttatgg tgatgctgag ctttttgggg aggcaataca tctctttagg ttgatgcaga gaaaggatat

RcAH1. cgaatgttgt tagttacaat actcttttga gggtttatgg tgatgctgag ctttttgggg aggcaataca tctctttagg ttgatgcaga gaaaggatat

RcAH2. cgaatgttgt tagttacaat actcttttga gggtttatgg tgatgctgag ctttttgggg aggcaataca tctctttagg ttgatgcaga gaaaggatat

RgTA4. cgaatgttgt tagttacaat actcttttga gggtttatgg tgatgctgag ctttttgggg aggcaataca tctctttagg ttgatgcaga gaaaggatat

RgAK1. cgaatgttgt tagttacaat actcttttga gggtttatgg tgatgctgag ctttttgggg aggcaataca tctctttagg ttgatgcaga gaaaggatat

RgCZ1. cgaatgttgt tagttacaat actcttttga gggtttatgg tgatgctgag ctttttgggg aggcaataca tctctttagg ttgatgcaga gaaaggatat

RgCC1. cgaatgttgt tagttacaat actcttttga gggtttatgg tgatgctgag ctttttgggg aggcaataca tctctttagg ttgatgcaga gaaaggatat

RgCC5. cgaatgttgt tagttacaat actcttttga gggtttatgg tgatgctgag ctttttgggg aggcaataca tctctttagg ttgatgcaga gaaaggatat

Tr. caaatgttgt tagttacaat actcttttga gggtttatgg tgatgcagag ctttttgggg aggcaataca tctctttaga ttaatgcaga gaaaggatat

RpBK1. tgagcagaat gttgtgacat ataacacgat gattatgatt tatgggaaga cgcttgagca tgaaaaagct aataatctta tccaggagat gcatagtaga

RpBK3. tgagcagaat gttgtgacat ataacacgat gattatgatt tatgggaaga cgcttgagca tgaaaaagct aataatctta tccaggagat gcatagtaga

RpZS2. tgagcagaat gttgtgacat ataacacgat gattatgatt tatgggaaga cgcttgagca tgaaaaagct aataatctta tccaggagat gcatagtaga

RpHL1. tgagcagaat gttgtgacat ataacacgat gattatgatt tatgggaaga cgcttgagca tgaaaaagct aataatctta tccaggagat gcatagtaga

RpHL3. tgagcagaat gttgtgacat ataacacgat gattatgatt tatgggaaga cgcttgagca tgaaaaagct aataatctta tccaggagat gcatagtaga

RhYC1. tgagcagaat gttgtgacat ataacacgat gattatgatt tatgggaaga cgctcgagca tgaaaaagct aataatctta tccaggagat gcatagtaga

RhYC2. tgagcagaat gttgtgacat ataacacgat gattatgatt tatgggaaga cgctcgagca tgaaaaagct aataatctta tccaggagat gcatagtaga

RhYC3. tgagcagaat gttgtgacat ataacacgat gattatgatt tatgggaaga cgctcgagca tgaaaaagct aataatctta tccaggagat gcatagtaga

RhYC4. tgagcagaat gttgtgacat ataacacgat gattatgatt tatgggaaga cgctcgagca tgaaaaagct aataatctta tccaggagat gcatagtaga

RhYC5. tgagcagaat gttgtgacat ataacacgat gattatgatt tatgggaaga cgctcgagca tgaaaaagct aataatctta tccaggagat gcatagtaga

RsCK1. tgagcagaat gttgtgacat ataacacgat gattatgatt tatgggaaga cgcttgagca tgaaaaagct aataatctta tccaggagat gcatagtaga

RsCK2. tgagcagaat gttgtgacat ataacacgat gattatgatt tatgggaaga cgcttgagca tgaaaaagct aataatctta tccaggagat gcatagtaga

RsCK3. tgagcagaat gttgtgacat ataacacgat gattatgatt tatgggaaga cgcttgagca tgaaaaagct aataatctta tccaggagat gcatagtaga

RsCK4. tgagcagaat gttgtgacat ataacacgat gattatgatt tatgggaaga cgcttgagca tgaaaaagct aataatctta tccaggagat gcatagtaga

RsCK5. tgagcagaat gttgtgacat ataacacgat gattatgatt tatgggaaga cgcttgagca tgaaaaagct aataatctta tccaggagat gcatagtaga

RcLS1. tgagcagaat gttgtgacat ataacacgat gattatgatt tatgggaaga cgctcgagca tgaaaaagct aataatctta tccaggagat gcatagtaga

RcTX1. tgagcagaat gttgtgacat ataacacgat gattatgatt tatgggaaga cgctcgagca tgaaaaagct aataatctta tccaggagat gcatagtaga

RcTX3. tgagcagaat gttgtgacat ataacacgat gattatgatt tatgggaaga cgctcgagca tgaaaaagct aataatctta tccaggagat gcatagtaga

RcAH1. tgagcagaat gttgtgacat ataacacgat gattatgatt tatgggaaga cgctcgagca tgaaaaagct aataatctta tccaggagat gcatagtaga

RcAH2. tgagcagaat gttgtgacat ataacacgat gattatgatt tatgggaaga cgctcgagca tgaaaaagct aataatctta tccaggagat gcatagtaga

RgTA4. tgagcagaat gttgtgacat ataacacgat gattatgatt tatgggaaga cgcttgagca tgaaaaagct aataatctta tccaggagat gcatagtaga

RgAK1. tgagcagaat gttgtgacat ataacacgat gattatgatt tatgggaaga cgcttgagca tgaaaaagct aataatctta tccaggagat gcatagtaga

RgCZ1. tgagcagaat gttgtgacat ataacacgat gattatgatt tatgggaaga cgcttgagca tgaaaaagct aataatctta tccaggagat gcatagtaga

RgCC1. tgagcagaat gttgtgacat ataacacgat gattatgatt tatgggaaga cgcttgagca tgaaaaagct aataatctta tccaggagat gcatagtaga

RgCC5. tgagcagaat gttgtgacat ataacacgat gattatgatt tatgggaaga cgcttgagca tgaaaaagct aataatctta tccaggagat gcatagtaga

Tr. tgagcagaac gttgtgacgt ataacacgat gattatgatt tatgggaaga cgcttgagca tgaaaaagct aataatctta tccaggagat gcatagtaga

RpBK1. ggcattgagc ccaatgcagt tacttattcc acaataatat cgatatgggg taaagtggga aaattagatc gggcggcgat gctttttcag aagcttcgaa

RpBK3. ggcattgagc ccaatgcagt tacttattcc acaataatat cgatatgggg taaagtggga aaattagatc gggcggcgat gctttttcag aagcttcgaa

RpZS2. ggcattgagc ccaatgcaat tacttattcc acaataatat cgatatgggg taaagtggga aaattagatc gggcggcgat gctttttcag aagcttcgaa

RpHL1. ggcattgagc ccaatgcaat tacttattcc acaataatat cgatatgggg taaagtggga aaattagatc gggcggcgat gctttttcag aagcttcgaa

RpHL3. ggcattgagc ccaatgcaat tacttattcc acaataatat cgatatgggg taaagtggga aaattagatc gggcggcgat gctttttcag aagcttcgaa

RhYC1. ggcattgagc ccaatgcaat tacttattcc acaataatat cgatatgggg taaagyggga aaattagatc gggcggcgat gctttttcag aagcttcgaa

RhYC2. ggcattgagc ccaatgcaat tacttattcc acaataatat cgatatgggg taaagtggga aaattagatc gggcggcgat gctttttcag aagcttcgaa

RhYC3. ggcattgagc ccaatgcaat tacttattcc acaataatat cgatatgggg taaagyggga aaattagatc gggcggcgat gctttttcag aagcttcgaa

RhYC4. ggcattgagc ccaatgcaat tacttattcc acaataatat cgatatgggg taaagtggga aaattagatc gggcggcgat gctttttcag aagcttcgaa

RhYC5. ggcattgagc ccaatgcaat tacttattcc acaataatat cgatatgggg taaagtggga aaattagatc gggcggcgat gctttttcag aagcttcgaa

RsCK1. ggcattgagc ccaatgcaat tacttattcc acaataatat cgatatgggg taaagtggga aaattagatc gggcggcgat gctttttcag aagcttcgaa

RsCK2. ggcattgagc ccaatgcaat tacttattcc acaataatat cgatatgggg taaagtggga aaattagatc gggcggcgat gctttttcag aagcttcgaa

RsCK3. ggcattgagc ccaatgcaat tacttattcc acaataatat cgatatgggg taaagtggga aaattagatc gggcggcgat gctttttcag aagcttcgaa

RsCK4. ggcattgagc ccaatgcaat tacttattcc acaataatat cgatatgggg taaagtggga aaattagatc gggcggcgat gctttttcag aagcttcgaa

RsCK5. ggcattgagc ccaatgcaat tacttattcc acaataatat cgatatgggg taaagtggga aaattagatc gggcggcgat gctttttcag aagcttcgaa

RcLS1. ggcattgagc ccaatgcaat tacttattcc acaataatat cgatatgggg taaagtggga aaattagatc gggcggcgat tctttttcag aagcttcgaa

RcTX1. ggcattgagc ccaatgcaat tacttattcc acaataatat cgatatgggg taaagtggga aaattagatc gggcggcgat tctttttcag aagcttcgaa

RcTX3. ggcattgagc ccaatgcaat tacttattcc acaataatat cgatatgggg taaagtggga aaattagatc gggcggcgat tctttttcag aagcttcgaa

RcAH1. ggcattgagc ccaatgcaat tacttattcc acaataatat cgatatgggg taaagtggga aaattagatc gggcggcgat gctttttcag aagcttcgaa

RcAH2. ggcattgagc ccaatgcaat tacttattcc acaataatat cgatatgggg taaagtggga aaattagatc gggcggcgat gctttttcag aagcttcgaa

RgTA4. ggcattgagc ccaatgcaat tacttattcc acaataatat cgatatgggg taaagtggga aaattagatc gggcggcgat gctttttcag aagcttcgaa

RgAK1. ggcattgagc ccaatgcaat tacttattcc acaataatat cgatatgggg taaagtggga aaattagatc gggcggcgat gctttttcag aagcttcgaa

RgCZ1. ggcattgagc ccaatgcaat tacttattcc acaataatat cgatatgggg taaagtggga aaattagatc gggcggcgat gctttttcag aagcttcgaa

RgCC1. ggcattgagc ccaatgcaat tacttattcc acaataatat cgatatgggg taaagtggga aaattagatc gggcggcgat gctttttcag aagcttcgaa

RgCC5. ggcattgagc ccaatgcaat tacttattcc acaataatat cgatatgggg taaagtggga aaattagatc gggcggcgat gctttttcag aagcttcgaa

Tr. ggcgttgagc ccaatgcaat tacttattcc acaataatat caatatgggg taaagtggga aaattagatc gggcggcaat gctttttcag aagcttcgaa

RpBK1. gctctggtat tgaaattgat caggttcttt atcagacaat gattgttgcg tacgagaggg ctggtttagt tgcg

RpBK3. gctctggtat tgaaattgat caggttcttt atcagacaat gattgttgcg tacgagaggg ctggtttagt tgcg

RpZS2. gctctggtat tgaaattgat caggttcttt atcagacaat gattgttgcg tacgagaggg ctggtttagt tgcg

RpHL1. gctctggtat tgaaattgat caggttcttt atcagacaat gattgttgcg tacgagaggg ctggtttagt tgcg

RpHL3. gctctggtat tgaaattgat caggttcttt atcagacaat gattgttgcg tacgagaggg ctggtttagt tgcg

RhYC1. gttctggtat tgaaattgat caggttcttt atcagacaat gattgtagcg tatgagaggg ctggtttagt tgcg

RhYC2. gttctggtat tgaaattgat caggttcttt atcagacaat gattgtagcg tatgagaggg ctggtttagt tgcg

RhYC3. gttctggtat tgaaattgat caggttcttt atcagacaat gattgtagcg tatgagaggg ctggtttagt tgcg

RhYC4. gttctggtat tgaaattgat caggttcttt atcagacaat gattgtagcg tatgagaggg ctggtttagt tgcg

RhYC5. gttctggtat tgaaattgat caggttcttt atcagacaat gattgtagcg tatgagaggg ctggtttagt tgcg

RsCK1. gctctggtat tgaaattgat caggttcttt atcagacaat gattgtagcg tacgagaggg ctggtttagt tgcg

RsCK2. gctctggtat tgaaattgat caggttcttt atcagacaat gattgtagcg tacgagaggg ctggtttagt tgcg

RsCK3. gctctggtat tgaaattgat caggttcttt atcagacaat gattgtagcg tacgagaggg ctggtttagt tgcg

RsCK4. gctctggtat tgaaattgat caggttcttt atcagacaat gattgtagcg tacgagaggg ctggtttagt tgcg

RsCK5. gctctggtat tgaaattgat caggttcttt atcagacaat gattgtagcg tacgagaggg ctggtttagt tgcg

RcLS1. gttctggtat tgaaattgat caggttcttt atcagacaat gattgtagcg tatgagaggg ctggtttagt tgcg

RcTX1. gttctggtat tgaaattgat caggttcttt atcagacaat gattgtagcg tatgagaggg ctggtttagt tgcg

RcTX3. gttctggtat tgaaattgat caggttcttt atcagacaat gattgtagcg tatgagaggg ctggtttagt tgcg

RcAH1. gttctggtat tgaaattgat caggttcttt atcagacaat gattgtagcg tatgagaggg ctggtttagt tgcg

RcAH2. gttctggtat tgaaattgat caggttcttt atcagacaat gattgtagcg tatgagaggg ctggtttagt tgcg

RgTA4. gctctggtat tgaaattgat caggttcttt atcagacaat gattgtagcg tacgagaggg ctggtttagt tgcg

RgAK1. gctctggtat tgaaattgat caggttcttt atcagacaat gattgtagcg tacgagaggg ctggtttagt tgcg

RgCZ1. gctctggtat tgaaattgat caggttcttt atcagacaat gattgtagcg tacgagaggg ctggtttagt tgcg

RgCC1. gctctggtat tgaaattgat caggttcttt atcagacaat gattgtagcg tacgagaggg ctggtttagt tgcg

RgCC5. gctctggtat tgaaattgat caggttcttt atcagacaat gattgtagcg tacgagaggg ctggtttagt tgcg

Tr. gctctggtgt tgaaattgat cagattcttt atcagacaat gattgtagca tatgagaggg ctggtttagt tgcg

<212> SEQ ID No2

<213> Artificial sequence (Artificial sequence)

<400> 1

RpBK1. tggggtcgcg gtcgtaagcg cgaatgactc gcaccattgg gtctgtgacg atgcccgagc agacgacgtc gtccggcacg acagcacgcg agttgagtgt

RpBK3. tggggtcgcg gtcgtaagcg cgaatgactc gcaccattgg gtctgtgacg atgcccgagc agacgacgtc gtccggcacg acagcacgcg agttgagtgt

RpZS2. tggggtcgcg gtcgtaagcg cgaatgactc gcaccattgg gtctgtgacg atgcccgagc agacgacgtc gtccggcacg acagcacgcg agttgagtgt

RpHL1. tggggtcgcg gtcgtaagcg cgaatgactc gcaccattgg gtctgtgacg atgcccgagc agacgacgtc gtccggcacg acagcacgcg agttgagtgt

RpHL3. tggggtcgcg gtcgtaagcg cgaatgactc gcaccattgg gtctgtgacg atgcccgagc agacgacgtc gtccggcacg acagcacgcg agttgagtgt

RhYC1. tggggtcgcg gtcgtaagcg cgaatgtctc gcacaattgg gtctgtgacg atgcccgagc agacgacgtc gtccggcacg acagcacgcg agttgagtgt

RhYC2. tggggtcgcg gtcgtaagcg cgaatgtctc gcacaattgg gtctgtgacg atgcccgagc agacgacgtc gtccggcacg acagcacgcg agttgagtgt

RhYC3. tggggtcgcg gtcgtaagcg cgaatgtctc gcacaattgg gtctgtgacg atgcccgagc agacgacgtc gtccggcacg acagcacgcg agttgagtgt

RhYC4. tggggtcgcg gtcgtaagcg cgaatgtctc gcacaattgg gtctgtgacg atgcccgagc agacgacgtc gtccggcacg acagcacgcg agttgagtgt

RhYC5. tggggtcgcg gtcgtaagcg cgaatgtctc gcacaattgg gtctgtgacg atgcccgagc agacgacgtc gtccggcacg acagcacgcg agttgagtgt

RsCK1. tggggtcgcg gtcgtaagcg cgaatgactc gcaccattgg gtctgtgacg atgcccgaga agacgacgtc gtccggcacg acagcacgcg agttgagtgt

RsCK2. tggggtcgcg gtcgtaagcg cgaatgactc gcaccattgg gtctgtgacg atgcccgaga agacgacgtc gtccggcacg acagcacgcg agttgagtgt

RsCK3. tggggtcgcg gtcgtaagcg cgaatgactc gcaccattgg gtctgtgacg atgcccgaga agacgacgtc gtccggcacg acagcacgcg agttgagtgt

RsCK4. tggggtcgcg gtcgtaagcg cgaatgactc gcaccattgg gtctgtgacg atgcccgaga agacgacgtc gtccggcacg acagcacgcg agttgagtgt

RsCK5. tggggtcgcg gtcgtaagcg cgaatgactc gcaccattgg gtctgtgacg atgcccgaga agacgacgtc gtccggcacg acagcacgcg agttgagtgt

RcLS1. tggggtcgcg gtcgtaagtg cgaatggctc gcacaattgg gtctgtgacg atgcccgagc agacgacgtc gtccggcacg acagcacgcg agttgagtgt

RcTX1. tggggtcgcg gtcgtaagcg cgaatggctc gcacaattgg gtctgtgacg atgcccgagc agacgacgtc gtccggcacg acagcacgcg agttgagtgt

RcTX3. tggggtcgcg gtcgtaagcg cgaatggctc gcacaattgg gtctgtgacg atgcccgagc agacgacgtc gtccggcacg acagcacgcg agttgagtgt

RcAH1. tggggtcgcg gtcgtaagcg cgaatggctc gcacaattgg gtctgtgacg atgcccgagc agacgacgtc gtccggcacg acagcacgcg agttgagtgt

RcAH2. tggggtcgcg gtcgtaagcg cgaatggctc gcacaattgg gtctgtgacg atgcccgagc agacgacgtc gtccggcacg acagcacgcg agttgagtgt

RgTA4. tggggtcgcg gtcgtaagcg cgaatgactc gcaccattgg gtctgtgacg atgcccgaga agacgacgtc gtccggcacg acagcacgcg agttgagtgt

RgAK1. tggggtcgcg gtcgtaagcg cgaatgactc gcaccattgg gtctgtgacg atgcccgaga agacgacgtc gtccggcacg acagcacgcg agttgagtgt

RgCZ1. tggggtcgcg gtcgtaagcg cgaatgactc gcaccattgg gtctgtgacg atgcccgaga agacgacgtc gtccggcacg acagcacgcg agttgagtgt

RgCC1. tggggtcgcg gtcgtaagcg cgaatgactc gcaccattgg gtctgtgacg atgcccgaga agacgacgtc gtccggcacg acagcacgcg agttgagtgt

RgCC5. tggggtcgcg gtcgtaagcg cgaatgactc gcaccattgg gtctgtgacg atgcccgaga agacgacgtc gtccggcacg acagcacgcg agttgagtgt

Tr. ggtaggaagc gcaattggct cacgccattg ggtctttaac gatggacgag cggacgacgt cgcccggcac gacagcacgc gagttgagtt tgcttcaacc

RpBK1. tcaaccacca ctggtcgtga cgtgcgccgc cgcgggatcg catttgggcc agccgcgcaa cgctgtgcac gggaggccat tatccgcccc cacccaacga

RpBK3. tcaaccacca ctggtcgtga cgtgcgccgc cgcgggatcg catttgggcc agccgcgcaa cgctgtgcac gggaggccat tatccgcccc cacccaacga

RpZS2. tcaaccacca ctggtcgtga cgtgcgccgc cgcgggatcg catttgggcc agccgcgcaa cgctgtgcac gggaggccat tatccgcccc cacccaacga

RpHL1. tcaaccacca ctggtcgtga cgtgcgccgc cgcgggatcg catttgggcc agccgcgcaa cgctgtgcac gggaggccat tatccgcccc cacccaacga

RpHL3. tcaaccacca ctggtcgtga cgtgcgccgc cgcgggatcg catttgggcc agccgcacaa cgctgtgcac gggaggccat tatccgcccc cacccaacga

RhYC1. tcaaccacca ctggtcgtga cgtgcgccgc cgcgggatcg catttgggcc agccgcgcaa caccgcgcac gggaggccat tatccgcccc cacccaacga

RhYC2. tcaaccacca ctggtcgtga cgtgcgccgc cgcgggatcg catttgggcc agccgcgcaa caccgcgcac gggaggccat tatccgcccc cacccaacga

RhYC3. tcaaccacca ctggtcgtga cgtgcgccgc cgcgggatcg catttgggcc agccgcgcaa caccgcgcac gggaggccat tatccgcccc cacccaacga

RhYC4. tcaaccacca ctggtcgtga cgtgcgccgc cgcgggatcg catttgggcc agccgcgcaa caccgcgcac gggaggccat tatccgcccc cacccaacga

RhYC5. tcaaccacca ctggtcgtga cgtgcgccgc cgcgggatcg catttgggcc agccgcgcaa caccgcgcac gggaggccat tatccgcccc cacccaacga

RsCK1. tcaaccacca ctggtcgtga cgtgcgccgc cgcgggatcg catttgggcc agccgcgcaa cactgcgcac gggaggccat tatccgcccc cacccaacga

RsCK2. tcaaccacca ctggtcgtga cgtgcgccgc cgcgggatcg catttgggcc agccgcgcaa cactgcgcac gggaggccat tatccgcccc cacccaacga

RsCK3. tcaaccacca ctggtcgtga cgtgcgccgc cgcgggatcg catttgggcc agccgcgcaa cactgcgcac gggaggccat tatccgcccc cacccaacga

RsCK4. tcaaccacca ctggtcgtga cgtgcgccgc cgcgggatcg catttgggcc agccgcgcaa cactgcgcac gggaggccat tatccgcccc cacccaacga

RsCK5. tcaaccacca ctggtcgtga cgtgcgccgc cgcgggatcg catttgggcc agccgcgcaa cactgcgcac gggaggccat tatccgcccc cacccaacga

RcLS1. tcaaccacca ctggtcgtga cgtgcgccgc cgcgggatcg catttgggcc agccgcgcaa caccgcgcac gggaggccat tatccgcccc ccacccaacg

RcTX1. tcaaccacca ctggtcgtga cgtgcgccgc cgcgggatcg catttgggcc agccgcgcaa caccgcgcac gggaggccat tatccgcccc cacccaacga

RcTX3. tcaaccacca ctggtcgtga cgtgcgccgc cgcgggatcg catttgggcc agccgcgcaa caccgcgcac gggaggccat tatccgcccc cacccaacga

RcAH1. tcaaccacca ctggtcgtga cgtgcgccgc cgcgggatcg catttgggcc agccgcgcaa caccgcgcac gggaggccat tatccgcccc cacccaacga

RcAH2. tcaaccacca ctggtcgtga cgtgcgccgc cgcgggatcg catttgggcc agccgcgcaa caccgcgcac gggaggccat tatccgcccc cacccaacga

RgTA4. tcaaccacca ctggtcgtga cgtgcgccgc cgcgggatcg catttgggcc agccgcgcaa cactgcgcac gggaggccat tatccgcccc cacccaacga

RgAK1. tcaaccacca ctggtcgtga cgtgcgccgc cgcgggatcg catttgggcc agccgcgcaa cactgcgcac gggaggccat tatccgcccc cacccaacga

RgCZ1. tcaaccacca ctggtcgtga cgtgcgccgc cgcgggatcg catttgggcc agccgcgcaa cactgcgcac gggaggccat tatccgcccc cacccaacga

RgCC1. tcaaccacca ctggtcgtga cgtgcgccgc cgcgggatcg catttgggcc agccgcgcaa cactgcgcac gggaggccat tatccgcccc cacccaacga

RgCC5. tcaaccacca ctggtcgtga cgtgcgccgc cgcgggatcg catttgggcc agccgcgcaa cactgcgcac gggaggccat tatccgcccc cacccaacga

Tr. accactggtc gtgacgcgcg tcgccgcagg atcgcatttg ggccggccat gcgtccctgc gcacgggggg ccattatccg cccccacacg acgatcgcct

RpBK1. tcgccgcata gggacgagag tgggggcgac gcgatgcgtg acgcccaggc agacgtgccc tcggcctaaa ggcttcgggc gcaacttgcg ttcaaagact

RpBK3. tcgccgcata gggacgagag tgggggcgac gcgatgcgtg acgcccaggc agacgtgccc tcggcctaaa ggcttcgggc gcaacttgcg ttcaaagact

RpZS2. tcgccgcata gggacgagag tgggggcgac gcgatgcgtg acgcccaggc agacgtgccc tcggcctaaa ggcttcgggc gcaacttgcg ttcaaagact

RpHL1. tcgccgcata gggacgagag tgggggcgac gcgatgcgtg acgcccaggc agacgtgccc tcggcctaaa ggcttcgggc gcaacttgcg ttcaaagact

RpHL3. tcgccgcata gggacgagag tgggggcgac gcgatgcgtg acgcccaggc agacgtgccc tcggcctaaa ggcttcgggc gcaacttgcg ttcaaagact

RhYC1. tcgccacata gggacgaggg tgggggcgac gcgatgcgtg acgcccaggc agacgtgccc tcggcctaaa ggcttcgggc gcaacttgcg ttcaaagact

RhYC2. tcgccacata gggacgaggg tgggggcgac gcgatgcgtg acgcccaggc agacgtgccc tcggcctaaa ggcttcgggc gcaacttgcg ttcaaagact

RhYC3. tcgccacata gggacgaggg tgggggcgac gcgatgcgtg acgcccaggc agacgtgccc tcggcctaaa ggcttcgggc gcaacttgcg ttcaaagact

RhYC4. tcgccacata gggacgaggg tgggggcgac gcgatgcgtg acgcccaggc agacgtgccc tcggcctaaa ggcttcgggc gcaacttgcg ttcaaagact

RhYC5. tcgccacata gggacgaggg tgggggcgac gcgatgcgtg acgcccaggc agacgtgccc tcggcctaaa ggcttcgggc gcaacttgcg ttcaaagact

RsCK1. tcgccacata gggacgaggg gagggggcga cgcgatgcgt gacgcccagg cagacgtgcc ctcggcctaa aggcttcggg cgcaacttgc gttcaaagac

RsCK2. tcgccacata gggacgaggg gagggggcga cgcgatgcgt gacgcccagg cagacgtgcc ctcggcctaa aggcttcggg cgcaacttgc gttcaaagac

RsCK3. tcgccacata gggacgaggg gagggggcga cgcgatgcgt gacgcccagg cagacgtgcc ctcggcctaa aggcttcggg cgcaacttgc gttcaaagac

RsCK4. tcgccacata gggacgaggg gggggggcga cgcgatgcgt gacgcccagg cagacgtgcc ctcggcctaa aggcttcggg cgcaacttgc gttcaaagac

RsCK5. tcgccacata gggacgaggg gagggggcga cgcgatgcgt gacgcccagg cagacgtgcc ctcggcctaa aggcttcggg cgcaacttgc gttcaaagac

RcLS1. atcgccacat agggacgagg gtgggggcga cgcgatgcgt gacgcccagg cagacgtgcc ctcggcctaa aggcttcggg cgcaacttgc gttcaaagac

RcTX1. tcgccacata gggacgaggg tgggggcgac gcgatgcgtg acgcccaggc agacgtgccc tcggcctaaa ggcttcgggc gcaacttgcg ttcaaagact

RcTX3. tcgccacata gggacgaggg tgggggcgac gcgatgcgtg acgcccaggc agacgtgccc tcggcctaaa ggcttcgggc gcaacttgcg ttcaaagact

RcAH1. tcgccacata gggacgaggg tgggggcgac gcgatgcgtg acgcccaggc agacgtgccc tcggcctaaa ggcttcgggc gcaacttgcg ttcaaagact

RcAH2. tcgccacata gggacgaggg tgggggcgac gcgatgcgtg acgcccaggc agacgtgccc tcggcctaaa ggcttcgggc gcaacttgcg ttcaaagact

RgTA4. tcgccacata gggacgaggg gagggggcga cgcgatgcgt gacgcccagg cagacgtgcc ctcggcctaa aggcttcggg cgcaacttgc gttcaaagac

RgAK1. tcgccacata gggacgaggg gagggggcga cgcgatgcgt gacgcccagg cagacgtgcc ctcggcctaa aggcttcggg cgcaacttgc gttcaaagac

RgCZ1. tcgccacata gggacgaggg gagggggcga cgcgatgcgt gacgcccagg cagacgtgcc ctcggcctaa aggcttcggg cgcaacttgc gttcaaagac

RgCC1. tcgccacata gggacgaggg gagggggcga cgcgatgcgt gacgcccagg cagacgtgcc ctcggcctaa aggcttcggg cgcaacttgc gttcaaagac

RgCC5. tcgccacata gggacgaggg gagggggcga cgcgatgcgt gacgcccagg cagacgtgcc ctcggcctaa aggcttcggg cgcaacttgc gttcaaagac

Tr. tggtagggcg agagtggggg cgacgctatg tgtgacgccc aggcagacgt gccctcggcc taaaggcttc gggcgcaact tgcgttcaaa gactcgatgg

RpBK1. cgatggttca cgggattctg caattcacac caagtatcgc atttcgctac gttcttcatc gatgcgagag ccgagatatc cgttgccgag agtcgttttg

RpBK3. cgatggttca cgggattctg caattcacac caagtatcgc atttcgctac gttcttcatc gatgcgagag ccgagatatc cgttgccgag agtcgttttg

RpZS2. cgatggttca cgggattctg caattcacac caagtatcgc atttcgctac gttcttcatc gatgcgagag ccgagatatc cgttgccgag agtcgttttg

RpHL1. cgatggttca cgggattctg caattcacac caagtatcgc atttcgctac gttcttcatc gatgcgagag ccgagatatc cgttgccgag agtcgttttg

RpHL3. cgatggttca cgggattctg caattcacac caagtatcgc atttcgctac gttcttcatc gatgcgagag ccgagatatc cgttgccgag agtcgttttg

RhYC1. cgatggttca cgggattctg caattcacac caagtatcgc atttcgctac gttcttcatc gatgcgagag ccgagatatc cgttgccgag agtcgttttg

RhYC2. cgatggttca cgggattctg caattcacac caagtatcgc atttcgctac gttcttcatc gatgcgagag ccgagatatc cgttgccgag agtcgttttg

RhYC3. cgatggttca cgggattctg caattcacac caagtatcgc atttcgctac gttcttcatc gatgcgagag ccgagatatc cgttgccgag agtcgttttg

RhYC4. cgatggttca cgggattctg caattcacac caagtatcgc atttcgctac gttcttcatc gatgcgagag ccgagatatc cgttgccgag agtcgttttg

RhYC5. cgatggttca cgggattctg caattcacac caagtatcgc atttcgctac gttcttcatc gatgcgagag ccgagatatc cgttgccgag agtcgttttg

RsCK1. tcgatggttc acgggattct gcaattcaca ccaagtatcg catttcgcta cgttcttcat cgatgcgaga gccgagatat ccattgccga gagtcgtttt

RsCK2. tcgatggttc acgggattct gcaattcaca ccaagtatcg catttcgcta cgttcttcat cgatgcgaga gccgagatat ccgttgccga gagtcgtttt

RsCK3. tcgatggttc acgggattct gcaattcaca ccaagtatcg catttcgcta cgttcttcat cgatgcgaga gccgagatat ccattgccga gagtcgtttt

RsCK4. tcgatggttc acgggattct gcaattcaca ccaagtatcg catttcgcta cgttcttcat cgatgcgaga gccgagatat ccattgccga gagtcgtttt

RsCK5. tcgatggttc acgggattct gcaattcaca ccaagtatcg catttcgcta cgttcttcat cgatgcgaga gccgagatat ccattgccga gagtcgtttt

RcLS1. tcgatggttc acgggattct gcaattcaca ccaagtatcg catttcgcta cgttcttcat cgatgcgaga gccgagatat ccgttgccga gagtcgtttt

RcTX1. cgatggttca cgggattctg caattcacac caagtatcgc atttcgctac gttcttcatc gatgcgagag ccgagatatc cgttgccgag agtcgttttg

RcTX3. cgatggttca cgggattctg caattcacac caagtatcgc atttcgctac gttcttcatc gatgcgagag ccgagatatc cgttgccgag agtcgttttg

RcAH1. cgatggttca cgggattctg caattcacac caagtatcgc atttcgctac gttcttcatc gatgcgagag ccgagatatc cgttgccgag agtcgttttg

RcAH2. cgatggttca cgggattctg caattcacac caagtatcgc atttcgctac gttcttcatc gatgcgagag ccgagatatc cgttgccgag agtcgttttg

RgTA4. tcgatggttc acgggattct gcaattcaca ccaagtatcg catttcgcta cgttcttcat cgatgcgaga gccgagatat ccgttgccga gagtcgtttt

RgAK1. tcgatggttc acgggattct gcaattcaca ccaagtatcg catttcgcta cgttcttcat cgatgcgaga gccgagatat ccgttgccga gagtcgtttt

RgCZ1. tcgatggttc acgggattct gcaattcaca ccaagtatcg catttcgcta cgttcttcat cgatgcgaga gccgagatat ccgttgccga gagtcgtttt

RgCC1. tcgatggttc acgggattct gcaattcaca ccaagtatcg catttcgcta cgttcttcat cgatgcgaga gccgagatat ccgttgccga gagtcgtttt

RgCC5. tcgatggttc acgggattct gcaattcaca ccaagtatcg catttcgcta cgttcttcat cgatgcgaga gccgagatat ccgttgccga gagtcgtttt

Tr. ttcacgggat tctgcaattc acaccaagta tcgcatttcg ctacgttctt catcgatgcg agagccgaga tatccttgcc gagagtcatt ttgacattcg

RpBK1. acattcgaga gacatccttt ccccccctcg cgcaccgcgg acggggcgat ggaaggcgga tgcttctttg agttttcctt ggcgcatgcc gcgccggggt

RpBK3. acattcgaga gacatccttt ccccccctcg cgcaccgcgg acggggcgat ggaaggcgga tgcttctttg agttttcctt ggcgcatgcc gcgccggggt

RpZS2. acattcgaga gacatccttt ccccccctcg cgcaccgcgg acggggcgat ggaaggcgga tgcttctttg agttttcctt ggcgcatgcc gcgccggggt

RpHL1. acattcgaga gacatccttt ccccccctcg cacaccgcgg acggggcgat ggaaggcgga tgcttctttg agttttcctt ggcgcatgcc gcgccggggt

RpHL3. acattcgaga gacatccttt ccccccctcg cacaccgcgg acggggcgat ggaaggcgga tgcttctttg agttttcctt ggcgcatgcc gcgccggggt

RhYC1. acattcgaga gacatccttc cccccctcgc gcaccgcgga cggggcgatg ggaggcggat gcttctttga gttttccttg gcgcatgccg cgccggggtt

RhYC2. acattcgaga gacatccttc cccccctcgc gcaccgcgga cggggcgatg ggaggcggat gcttctttga gttttccttg gcgcatgccg cgccggggtt

RhYC3. acattcgaga gacatccttc cccccctcgc gcaccgcgga cggggcgatg ggaggcggat gcttctttga gttttccttg gcgcatgccg cgccggggtt

RhYC4. acattcgaga gacatccttc cccccctcgc gcaccgcgga cggggcgatg ggaggcggat gcttctttga gttttccttg gcgcatgccg cgccggggtt

RhYC5. acattcgaga gacatccttc cccccctcgc gcaccgcgga cggggcgatg ggaggcggat gcttctttga gttttccttg gcgcatgccg cgccggggtt

RsCK1. gacattcgag agacatcctt tcccccctcg cgcaccgcgg acggggcgat ggaaggcgga tgcttctttt agttttcctt ggcgcatgcc gcgccggggt

RsCK2. gacattcgag agacatcctt tcccccctcg cgcaccgcgg acggggcgat ggaaggcgga tgcttctttt agttttcctt ggcgcatgcc gcgccggggt

RsCK3. gacattcgag agacatcctt tcccccctcg cgcaccgcgg acggggcgat ggaaggcgga tgcttctttt agttttcctt ggcgcatgcc gcgccggggt

RsCK4. gacattcgag agacatcctt tcccccctcg cgcaccgcgg acggggcgat ggaaggcgga tgcttctttt agttttcctt ggcgcatgcc gcgccggggt

RsCK5. gacattcgag agacatcctt tcccccctcg cgcaccgcgg acggggcgat ggaaggcgga tgcttctttt agttttcctt ggcgcatgcc gcgccggggt

RcLS1. gacattcgag agacatcctt ccccccctcg cgcaccgcgg acggggcgat ggaaggcgga tgcttctttg agttttcctt ggcgcatgcc gcgccggggt

RcTX1. acattcgaga gacatccttc cccccctcgc gcaccgcgga cggggcgatg ggaggcggat gcttctttga gttttccttg gcgcatgccg cgccggggtt

RcTX3. acattcgaga gacatccttc cccccctcgc gcaccgcgga cggggcgatg ggaggcggat gcttctttga gttttccttg gcgcatgccg cgccggggtt

RcAH1. acattcgaga gacatccttc cccccctcgc gcaccgcgga cggggcgatg ggaggcggat gcttctttga gttttccttg gcgcatgccg cgccggggtt

RcAH2. acattcgaga gacatccttc cccccctcgc gcaccgcgga cggggcgatg ggaggcggat gcttctttga gttttccttg gcgcatgccg cgccggggtt

RgTA4. gacattcgag agacatcctt tcccccctcg cgcaccgcgg acggggcgat ggaaggcgga tgcttctttg agttttcctt ggcgcatgcc gcgccggggt

RgAK1. gacattcgag agacatcctt tcccccctcg cgcaccgcgg acggggcgat ggaaggcgga tgcttctttg agttttcctt ggcgcatgcc gcgccggggt

RgCZ1. gacattcgag agacatcctt tcccccctcg cgcaccgcgg acggggcgat ggaaggcgga tgcttctttg agttttcctt ggcgcatgcc gcgccggggt

RgCC1. gacattcgag agacatcctt tcccccctcg cgcaccgcgg acggggcgat ggaaggcgga tgcttctttg agttttcctt ggcgcatgcc gcgccggggt

RgCC5. gacattcgag agacatcctt tcccccctcg cgcaccgcgg acggggcgat ggaaggcgga tgcttctttg agttttcctt ggcgcatgcc gcgccggggt

Tr. agagacaccc tttcccccct cgcgcgccac aaacggggcg acgggaggcg atgcttcttt gagttttcct tggcacatgc cgcgccgggg tttgttagcc

RpBK1. tcgttagccc gcacgacgag cactcacatg cacgccggca ggaagggaac gcggcgagag gttgcccccc caccacggtc ccgatgcggt taaacatgtt

RpBK3. tcgttagccc gcacgacgag cactcacatg cacgccggca ggaagggaac gcggcgagag gttgcccccc caccacggtc ccgatgcggt taaacatgtt

RpZS2. tcgttagccc gcacgacgag cactcacgtg cacgccggca ggaagggaac gcggcgagag gttgcccccc caccacggtc ccgatgcggt taaacatgtt

RpHL1. tcgttagccc gcacgacgag cactcacgtg cacgccggca ggaagggaac gcggcgagag gttgcccccc caccacggtc ccgatgcggt taaacatgtt

RpHL3. tcgttagccc gcacgacgag cactcacgtg cacgccggca ggaagggaac gcggcgagag gttgcccccc caccacggtc ccgatgcggt taaacatgtt

RhYC1. cgttagcccg cacgacgagc actcacgtgc acgccagcag gaagggaacg cggcgagagg ttgccccccc accacggtcc cgatgcggtt aaacatgttc

RhYC2. cgttagcccg cacgacgagc actcacgtgc acgccagcag gaagggaacg cggcgagagg ttgccccccc accacggtcc cgatgcggtt aaacatgttc

RhYC3. cgttagcccg cacgacgagc actcacgtgc acgccagcag gaagggaacg cggcgagagg ttgccccccc accacggtcc cgatgcggtt aaacatgttc

RhYC4. cgttagcccg cacgacgagc actcacgtgc acgccagcag gaagggaacg cggcgagagg ttgccccccc accacggtcc cgatgcggtt aaacatgttc

RhYC5. cgttagcccg cacgacgagc actcacgtgc acgccagcag gaagggaacg cggcgagagg ttgccccccc accacggtcc cgatgcggtt aaacatgttc

RsCK1. tcgttagccc gcacgacgag cactcacgtg cacgccagca ggaagggaac gcggcgagag gttgcccccc acaccacggt cccgatgtgg taaaacatgt

RsCK2. tcgttagccc gcacgacgag cactcacgtg cacgccagca ggaagggaac gcggcgagag gttgccccca caccacggtc ccgatgtggt aaaacatgtt

RsCK3. tcgttagccc gcacgacgag cactcacgtg cacgccagca ggaagggaac gcggcgagag gttgcccccc acaccacggt cccgatgtgg taaaacatgt

RsCK4. tcgttagccc gcacgacgag cactcacgtg cacgccagca ggaagggaac gcggcgagag gttgcccccc acaccacggt cccgatgtgg taaaacatgt

RsCK5. tcgttagccc gcacgacgag cactcacgtg cacgccagca ggaagggaac gcggcgagag gttgccccca acaccacggt cccgatgtgg taaaacatgt

RcLS1. tcgttagccc gcacgacgag cactcacgtg cacgccagca ggaagggaac gcggcgagag gttgcccccc caccacggtc ccgatgcggt taacatgttc

RcTX1. cgttagcccg cacgacgagc actcacgtgc acgccagcag gaagggaacg cggcgagagg ttgccccccc accacggtcc cgatgcggtt aaacatgttc

RcTX3. cgttagcccg cacgacgagc actcacgtgc acgccagcag gaagggaacg cggcgagagg ttgccccccc accacggtcc cgatgcggtt aaacatgttc

RcAH1. cgttagcccg cacgacgagc acttacgtgc acgccagcag gaagggaacg cggcgagagg ttgccccccc accacggtcc cgatgcggtt aaacatgttc

RcAH2. cgttagcccg cacgacgagc acttacgtgc acgccagcag gaagggaacg cggcgagagg ttgccccccc accacggtcc cgatgcggtt aaacatgttc

RgTA4. tcgttagccc gcacgacgag cactcacgtg cacgccagca ggaagggaac gcggcgagag gttgccccca caccacggtc ccgatgtggt taaacatgtt

RgAK1. tcgttagccc gcacgacgag cactcacgtg cacgccagca ggaagggaac gcggcgagag gttgccccca caccacggtc ccgatgtggt taaacatgtt

RgCZ1. tcgttagccc gcacgacgag cactcacgtg cacgccagca ggaagggaac gcggcgagag gttgccccca caccacggtc ccgatgtggt taaacatgtt

RgCC1. tcgttagccc gcacgacgag cactcacgtg cacgccagca ggaagggaac gcggcgagag gttgccccca caccacggtc ccgatgtggt taaacatgtt

RgCC5. tcgttagccc gcacgacgag cactcacgtg cacgccagca ggaagggaac gcggcgagag gttgccccca caccacggtc ccgatgtggt taaacatgtt

Tr. cacacggcac acgctcgcgc gcgcgccggc aggaagggaa aacgggaaga ggttgccccc cgcaccgtag cgatgagatc aaacatgttc gcggtctgc.

RpBK1. cgcggtctgc tttgcaggtt tcgacaatga tccttccgca ggttcaccta cggaaacctt gttacgactt ctccttcctc t.

RpBK3. cgcggtctgc tttgcaggtt tcgacaatga tccttccgca ggttcaccta cggaaacctt gttacgactt ctccttcctc t.

RpZS2. cgcggtctgc tttgcaggtt tcgacaatga tccttccgca ggttcaccta cggaaacctt gttacgactt ctccttcctc t.

RpHL1. cgcggtctgc tttgcaggtt tcgacaatga tccttccgca ggttcaccta cggaaacctt gttacgactt ctccttcctc t.

RpHL3. cgcggtctgc tttgcaggtt tcgacaatga tccttccgca ggttcaccta cggaaacctt gttacgactt ctccttcctc t.

RhYC1. gcggtctgct ttgcaggttt cgacaatgat ccttccgcag gttcacctac ggaaaccttg ttacgacttc tccttcctct ..

RhYC2. gcggtctgct ttgcaggttt cgacaatgat ccttccgcag gttcacctac ggaaaccttg ttacgacttc tccttcctct ..

RhYC3. gcggtctgct ttgcaggttt cgacaatgat ccttccgcag gttcacctac ggaaaccttg ttacgacttc tccttcctct ..

RhYC4. gcggtctgct ttgcaggttt cgacaatgat ccttccgcag gttcacctac ggaaaccttg ttacgacttc tccttcctct ..

RhYC5. gcggtctgct ttgcaggttt cgacaatgat ccttccgcag gttcacctac ggaaaccttg ttacgacttc tccttcctct ..

RsCK1. tcgcggtctg ctttgcaggt ttcgacaatg atccttccgc aggttcacct acggaaacct tgttacgact tctccttcct ct

RsCK2. cgcggtctgc tttgcaggtt tcgacaatga tccttccgca ggttcaccta cggaaacctt gttacgactt ctccttcctc t.

RsCK3. tcgcggtctg ctttgcaggt ttcgacaatg atccttccgc aggttcacct acggaaacct tgttacgact tctccttcct ct

RsCK4. tcgcggtctg ctttgcaggt ttcgacaatg atccttccgc aggttcacct acggaaacct tgttacgact tctccttcct ct

RsCK5. tcgcggtctg ctttgcaggt ttcgacaatg atccttccgc aggttcacct acggaaacct tgttacgact tctccttcct ct

RcLS1. gcggtctgct ttgcaggttt cgacaatgat ccttccgcag gttcacctac ggaaaccttg ttacgacttc tccttcctct ..

RcTX1. gcggtctgct ttgcaggttt cgacaatgat ccttccgcag gttcacctac ggaaaccttg ttacgacttc tccttcctct ..

RcTX3. gcggtctgct ttgcaggttt cgacaatgat ccttccgcag gttcacctac ggaaaccttg ttacgacttc tccttcctct ..

RcAH1. gcggtctgct ttgcaggttt cgacaatgat ccttccgcag gttcacctac ggaaaccttg ttacgacttc tccttcctct ..

RcAH2. gcggtctgct ttgcaggttt cgacaatgat ccttccgcag gttcacctac ggaaaccttg ttacgacttc tccttcctct ..

RgTA4. cgcggtctgc tttgcaggtt tcgacaatga tccttccgca ggttcaccta cggaaacctt gttacgactt ctccttcctc t.

RgAK1. cgcggtctgc tttgcaggtt tcgacaatga tccttccgca ggttcaccta cggaaacctt gttacgactt ctccttcctc t.

RgCZ1. cgcggtctgc tttgcaggtt tcgacaatga tccttccgca ggttcaccta cggaaacctt gttacgactt ctccttcctc t.

RgCC1. cgcggtctgc tttgcaggtt tcgacaatga tccttccgca ggttcaccta cggaaacctt gttacgactt ctccttcctc t.

RgCC5. cgcggtctgc tttgcaggtt tcgacaatga tccttccgca ggttcaccta cggaaacctt gttacgactt ctccttcctc t.

Tr. .......... .......... .......... .......... .......... .......... .......... .......... ..

Claims (2)

1. The combined barcode for identifying the rehmannia and the rehmannia is characterized by being formed by splicing an R257 sequence and an ITS sequence, wherein the nucleotide sequence of the R257 sequence is represented as SEQ ID No1, and the nucleotide sequence of the ITS sequence is represented as SEQ ID No2.

2. The method for identifying the rehmannia and the rehmannia plants is characterized by comprising the following steps of:

step 1, extracting DNA of a sample to be detected;

and 2, step: respectively carrying out PCR amplification reaction by using a primer of the R257 sequence and a primer of the ITS sequence to obtain an amplification product of the R257 sequence of the sample to be detected and an amplification product of the ITS sequence of the sample to be detected;

PCR amplification conditions for R257: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 45 sec, annealing at 56 ℃ for 45 sec, extension at 72 ℃ for l min, 35 cycles, extension at 72 ℃ for 6 min, and heat preservation at 4 ℃;

PCR amplification conditions for ITS: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 45 sec, annealing at 55 ℃ for 45 sec, extension at 72 ℃ for l min, 35 cycles, extension at 72 ℃ for 5min, and heat preservation at 4 ℃;

step 3, performing gel electrophoresis on the amplification products respectively, and performing bidirectional sequencing on the sample with the bright single band to obtain a to-be-detected sample R257 sequence and a to-be-detected sample ITS zone sequence;

step 4, introducing two sequences of bidirectional sequencing of the sample R257 to be detected into software to splice into one sequence, and obtaining a sequence of the sample R257 to be detected; leading two sequences of ITS bidirectional sequencing of a sample to be detected into software to be spliced into a sequence to obtain the sequence of ITS of the sample to be detected;

step 5, aligning and cutting the R257 sequence of the sample to be detected and the R257 nucleotide sequence marked as SEQ ID No1, and aligning and cutting the ITS sequence of the sample to be detected and the ITS nucleotide sequence marked as SEQ ID No 2;

step 6, sequentially importing the aligned R257 sequence and ITS zone sequence obtained in the step 5 into software to splice together to form a combined bar code sequence R257+ ITS;

and 7, importing the combined barcode sequence R257+ ITS into software to construct a phylogenetic tree, and if the sample to be detected and a certain species are gathered together, indicating that the sample to be detected is the species.

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