CN113684304A - Primer, kit and PCR method for identifying isodon longituba - Google Patents
Primer, kit and PCR method for identifying isodon longituba Download PDFInfo
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Abstract
The invention discloses a primer, a kit and a PCR method for identifying isodon longituba. The invention obtains a pair of universal primers UBC-812 capable of identifying Isodon longituba by screening ISSR marked universal primers, and on the basis of sequencing results of the universal primers and amplification products thereof, the primers capable of identifying Isodon longituba quickly and accurately are obtained by design and screening. By utilizing the primer, the genome of the Isodon longituba can be specifically amplified, and the primer has strong specificity and good stability, so that the quick identification of the Isodon longituba is realized, and the occurrence of false positive caused by diversity among groups is avoided. Meanwhile, the identification method is simple and convenient to operate, is suitable for rapid identification of the Isodon longituba in the market and production, and ensures the accuracy of medicine injection.
Description
Technical Field
The invention belongs to the field of biotechnology. More particularly, relates to a primer, a kit and a PCR method for identifying Isodon longituba.
Background
Isodon longituba (Rabdosia stracheyi) is a plant of the genus Isodon of the family Labiatae (Labiatae), and its dry aerial parts are widely used as a medicinal herb of Rabdosia serra in the south of the Ridge, because it is relatively similar to the plant morphological characteristics of the isodon lophanthide, namely Rabdosia lophanthides (Rabdosia lophathoides) and Isodon longituba (Rabdosia lophathoides var. graciliflora). However, the rabdosia longissima as a basic plant of the rabdosia lophanthide medicinal material is not recorded in the Chinese pharmacopoeia and the Chinese medicinal material standard in Guangdong province, so that the rabdosia lophanthide medicinal material needs to be distinguished from the rabdosia lophanthide to ensure the accuracy of the medicinal basic plant.
In view of the morphological characteristics that the Isodon longituba is difficult to distinguish from other plants of the same genus, the molecular identification method for the Isodon longituba is of great significance. Chinese patent CN 105349651A discloses a method and primers for identifying the variety of traditional Chinese medicine rabdosia lophanthide by EST-SSR marker, further constructs the EST sequence of rabdosia lophanthide by constructing a rabdosia lophanthide transcriptome information base, and further screens out 24 pairs of EST-SSR core primers with higher polymorphism among species and varieties and single amplification band type, and the core primers can be used for effectively and rapidly identifying the medicinal material base variety and mixed counterfeit of common rabdosia lophanthide. However, no molecular identification method for Isodon longituba is available at present.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a primer, a kit and a PCR method for identifying isodon longituba.
The first purpose of the invention is to provide a primer for identifying isodon longituba.
The second purpose of the invention is to provide a kit for identifying rabdosia longissima.
The third purpose of the invention is to provide a PCR method for identifying isodon longituba.
The above purpose of the invention is realized by the following technical scheme:
the invention obtains a pair of universal primers UBC-812 capable of identifying Isodon longituba by screening ISSR marked universal primers, and on the basis of sequencing results of the universal primers and amplification products thereof, the primers capable of identifying Isodon longituba quickly and accurately are obtained by design and screening.
The invention firstly provides a primer for identifying Isodon longituba, which comprises an upstream primer and a downstream primer, wherein the sequence of the upstream primer is shown as SEQ ID NO.1, and the sequence of the downstream primer is shown as SEQ ID NO. 2.
The invention also provides application of the primer in identifying isodon longituba.
The invention also provides application of the primer in preparation of a product for identifying isodon longituba.
The invention also provides application of the ISSR universal primer UBC-812 in identifying or preparing products for identifying Isodon longituba.
The invention also provides application of the product in identifying isodon longituba.
The invention also provides a kit for identifying Isodon longituba, which contains primers shown in SEQ ID NO.1 and SEQ ID NO. 2.
Specifically, the kit also contains reagents required by PCR amplification reaction.
The invention also provides application of the kit in identification of Isodon longituba.
The invention also provides a PCR method for identifying isodon longituba, which is characterized by comprising the following steps:
s1, extracting genome DNA of a sample to be detected;
s2, using the genome DNA obtained in the step S1 as a template, carrying out PCR amplification by using the primer of claim 1, and carrying out electrophoresis detection on an amplification product;
and S3, if the electrophoresis result shows that only one single band with the size of 1643bp exists, judging that the detected sample is the isodon longituba.
Preferably, the PCR amplification system is 25. mu.L, where mix is 12.5. mu.L, DNA template is 1. mu.L, forward and reverse primers are 1.5. mu.L, and the remainder is made up with water, see example 2.
Preferably, the final concentration of the forward primer and the final concentration of the reverse primer both amplified by PCR in step S2 are 0.6. mu. mol. L-1See example 2.
The use of primers at the above concentrations is advantageous for efficient PCR amplification.
Preferably, the amplification reaction procedure is pre-denaturation at 94 ℃ for 7min, denaturation at 94 ℃ for 2min, annealing at 59.8 ℃ for 45s, extension at 72 ℃ for 2min, and extension at 72 ℃ for 10min after 30 cycles, see example 2.
Preferably, the PCR amplification product obtained in step S2 is also sequenced, and if the sequencing result is the same as the sequence shown in SEQ ID NO.3, the sample is the Isodon longituba, see example 2.
The invention also provides application of the PCR method in identifying Isodon longituba.
The invention has the following beneficial effects:
the invention obtains a pair of universal primers UBC-812 capable of identifying Isodon longituba by screening ISSR marked universal primers, and on the basis of sequencing results of the universal primers and amplification products thereof, the primers capable of identifying Isodon longituba quickly and accurately are obtained by design and screening. By utilizing the primer, the genome of the Isodon longituba can be specifically amplified, and the primer has strong specificity and good stability, so that the quick identification of the Isodon longituba is realized, and the occurrence of false positive caused by diversity among groups is avoided. Meanwhile, the identification method is simple and convenient to operate, is suitable for rapid identification of the Isodon longituba in the market and production, and ensures the accuracy of medicine injection.
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FIG. 1 shows the results of PCR amplification of 12 known samples using primers 4 of Table 2; wherein the samples tested in lanes No. 1-4 are Isodon longituba; samples tested in lanes 5-8 are all rabdosia striifolia; the samples measured in lanes 9-12 are all the floral-scented tea vegetables.
FIG. 2 shows the results of PCR amplification of 12 known samples using primers in Table 2 except primer 4; wherein the primers used by A-L are UBC807, UBC810, UBC811, UBC815, UBC821, UBC823, UBC836, UBC840, UBC842, UBC846, UBC847 and UBC864 in sequence; samples measured in lanes 1-4 in the figure are all rabdosia longituba; samples tested in lanes 5-8 are all rabdosia striifolia; samples measured in lanes 9-12 are all the floral-scented tea vegetables; the used marker is the same, and the sizes of the bands are 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp from top to bottom in sequence.
FIG. 3 shows the result of the annealing temperature optimization of ISSR-SCAR primers.
FIG. 4 shows the results of PCR amplification of 12 known samples using ISSR-SCAR primers; wherein the samples tested in lanes No. 1-4 are Isodon longituba; samples tested in lanes 5-8 are all rabdosia striifolia; the samples measured in lanes 9-12 are all the floral-scented tea vegetables.
Detailed Description
The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
Example 1 screening of ISSR molecular marker Universal primer
The invention firstly screens ISSR molecular marker universal primers by extracting the genome of a known sample. The known samples are plants selected from wild and cultivated rabdosia species populations in different environments, and the collection information of the rabdosia medicinal material germplasm resources is shown in table 1. The samples in table 1 were identified as rabdosia filiformis, rabdosia fibraurea and rabdosia longifolia by the national professor of liu army, university of traditional Chinese medicine, and were all planted in the resource garden of the germplasm of zhen shanxi yellow grass, at the time of university of traditional Chinese medicine, guangzhou, and the sample specimens were stored in the traditional Chinese medicine resource research laboratory of the university of traditional Chinese medicine, guangzhou.
TABLE 1 Collection of Isodon herbs germplasm resources
Samples with numbers P30 and P31 were selected from Table 1 as the extraction targets of the longleaf rabdosia species genome, samples with numbers P9 and P10 were selected as the extraction targets of the stringy rabdosia species genome, and samples with numbers P13 and P24 were selected as the extraction targets of the stringy rabdosia species genome. The genomes of 2 plants were extracted for each number and split into Ep tubes.
The method for extracting plant genomic DNA was performed according to the instructions of DNA extraction kit (cat # DP320) from Tiangen Biochemical technology Ltd.
13 pairs of primers are screened from ISSR molecular marker universal primers, shown in table 2, and then the primers in table 2 are used for respectively carrying out PCR amplification on the genomic DNA of isodon longituba, isodon lophanthoides and isodon longituba, so that the total number of the genomic DNA of 12 samples is 12.
Table 213 pairs ISSR Universal primer sequences
The invention takes the extracted genome DNA of the known sample as a template, and uses 2 XTSINGKE Master Mix (TSE004, Beijing Ongke New Biotech Co., Ltd.) to carry out PCR amplification. The PCR amplification system was 25. mu.L, where mix 12.5. mu.L, DNA template 1. mu.L, forward and reverse primers were 1.5. mu.L, and the balance was made up with water. The PCR amplification reaction program comprises pre-denaturation at 94 ℃ for 7min, denaturation at 94 ℃ for 2min, annealing at 52.7 ℃ for 45s, extension at 72 ℃ for 2min, 30 cycles, and final extension at 72 ℃ for 10 min.
The PCR amplification electrophoresis result of the primer 4UBC-812 is shown in FIG. 1, wherein the sample detected in lanes 1-4 in the figure is Isodon longituba, wherein 1 and 2 are samples P30, and 3 and 4 are samples P31; the samples tested in lanes 5-8 are Rabdosia trichocarpa, wherein 5 and 6 are samples P9, and 7 and 8 are samples P10; the samples tested in lanes 9-12 are Ficus awkeotsang, where 9 and 10 are samples P24, and 11 and 12 are samples P13. As can be seen from FIG. 1, in the range of 1000-2000 bp, the primer 4 has amplification specificity to Isodon longituba, but has no amplification specificity to Isodon longituba and Isodon glaucoides.
The results of PCR amplification electrophoresis of primer 1UBC-807, primer 2UBC810, primer 3UBC-811, primer 5UBC815, primer 6UBC821, primer 7UBC823, primer 8UBC836, primer 9UBC840, primer 10UBC842, primer 11UBC846, primer 12UBC847, and primer 13UBC864 are shown in FIG. 2. The amplification results correspond to FIG. 2, Panel A, Panel B, Panel C, Panel D, Panel E, Panel F, Panel G, Panel H, Panel I, Panel J, Panel K and Panel L in the order of the primers, and the lane numbers and sample varieties corresponding to those in FIG. 2 are the same as those in FIG. 1. As can be seen from FIGS. 1 and 2, the primers 4 had amplification specificity for Isodon longituba, but had no amplification specificity for Isodon longituba and Isodon japonicus, and the remaining primers were not able to specifically amplify Isodon longituba alone. Therefore, primer 4 and its amplification product were subsequently selected for further study. Example 2 design of primers and detection of specificity
Because the ISSR molecular marker universal primer obtained by screening is easy to generate false positive and the detection specificity cannot be maintained, the method recycles and sequences the amplification product of the primer 4, and designs and screens the primer again on the basis.
And (3) cutting and recovering the specific amplified bands within the range of 1000-2000 bp in the graph 1. The gel cutting recovery kit is a mass agarose gel DNA recovery kit (DP210), and the specific operation steps are carried out according to the specification of the gel cutting recovery kit.
The recovered target fragment was ligated to the cloning Vector according to the instructions of pClone007 Vector Kit (TSV-007), as follows:
1. connection of
Placing a sterilized 0.2mL centrifuge tube on a plate, sucking 1 uL pClone007 Vector to the bottom of the centrifuge tube, replacing a gun head to suck 1 uL 10 XTopo Mix, adding the mixed solution into the Vector, lightly blowing and beating the mixture by using the gun head for 2-3 times, adding 8 uL recovered DNA fragment, and connecting the mixture at room temperature for 5 min.
2. Transformation of
mu.L of the ligation was added to 100. mu.L of DH 5. alpha. competent cells and placed on ice for 30 min. After the water bath heat shock at 42 ℃ for 90s, the mixture is quickly transferred into an ice bath and is subjected to the ice bath for 5 min. 400 μ L of LB liquid medium was added to the centrifuge tube on a clean bench, covered tightly and shaken well, and resuscitated at 150rpm for 1h at 37 ℃. After recovery, the mixture is centrifuged at low speed for 10s, 300. mu.L of supernatant is discarded, and the remaining 200. mu.L of supernatant is completely smeared on an Amp-resistant LB plate on an ultra-clean bench and cultured in an incubator at 37 ℃ for 16-18 h.
Finally, on the clean bench, from Amp+The plate was randomly picked up 10 single colonies with a sterilized pipette tip and inoculated to LB (Amp)+) After shaking the bacteria for 16h in the liquid medium, 1. mu.L of the bacterial solution was taken and PCR-amplified with the universal vector primers M13F/M13R. And detecting whether the amplification product contains a target band or not by 1.0% agarose gel electrophoresis, and if a single target band appears, taking 20 mu L of corresponding bacterial liquid and sending the corresponding bacterial liquid to Beijing Optimalaceae New industry biotechnology limited for sequencing.
The sequencing result is shown in SEQ ID NO.3, and the length is 1643 bp.
ISSR-SCAR marker primers were designed using Primer5.0 based on the above sequencing results. When in design, 7-11 bases are extended inwards on the basis of the original primer 4, 1 pair of primers are designed and screened according to the primer design principle, the primer sequences are shown in Table 3, wherein the thickened part is the newly designed primer sequence.
TABLE 3ISSR-SCAR primer sequences and annealing temperatures
In order to verify whether the designed ISSR-SCAR marker primer still maintains the specificity of the band, the invention respectively amplifies 12 known samples in example 1 by using the primers in the table 3, optimizes the annealing temperature of a reaction system and the primers, designs a gradient annealing temperature according to the Tm value of a primer synthesis report list, and takes the highest temperature of the band of interest as the annealing temperature.
The PCR reaction system used for detection was 25. mu.L, where mix was 12.5. mu.L, DNA template was 1. mu.L, forward and reverse primers were 1.5. mu.L, and the balance was made up with water.
Wherein the concentration of the forward and reverse primers is 0.6. mu. mol. L-1Introduction of this concentrationThe product is favorable for efficient PCR amplification.
The reaction program comprises pre-denaturation at 94 ℃ for 7min, denaturation at 94 ℃ for 2min, annealing at 56-66 ℃ for 45s, extension at 72 ℃ for 2min, and extension at 72 ℃ for 10min after 30 cycles.
The optimization results of the annealing temperatures are shown in fig. 3, wherein the annealing temperatures corresponding to lanes 1-8 are 66.0, 65.3, 64.0, 62.1, 59.8, 57.9, 56.7 and 56.0 ℃. As can be seen from FIG. 3, a single band with the same size appears in all lanes 5-8, and the optimal annealing temperature is selected to be 59.8 ℃ in the invention in consideration of increasing the annealing temperature and reducing the mismatch.
The optimized PCR reaction program comprises pre-denaturation at 94 ℃ for 7min, denaturation at 94 ℃ for 2min, annealing at 59.8 ℃ for 45s, extension at 72 ℃ for 2min, and extension at 72 ℃ for 10min after 30 cycles.
In order to verify whether the designed ISSR-SCAR marker primer still maintains the specificity of the strip, the primers shown in SEQ ID NO. 1-2 are used for carrying out PCR amplification on 12 known samples in example 1 respectively. The results are shown in FIG. 4, in which the samples measured in lanes 1-4 are Isodon longituba, the samples measured in lanes 5-8 are Isodon lophanthoides, and the samples measured in lanes 9-12 are Isodon lophanthoides. The result shows that the designed ISSR-SCAR primer is successfully converted into a specific marker and can be used for identifying the Isodon longituba.
Besides electrophoresis detection, the amplified product can be sequenced, and if the sequencing result is the same as the sequence shown in SEQ ID NO.3, the detected sample is the Isodon longituba.
When the ISSR-SCAR marker primer provided by the invention is used for identifying varieties of samples to be detected, only the judgment on whether the strips of the samples subjected to PCR amplification are single or not and whether the sizes of the strips accord with each other is needed, and the identification method is simple and rapid and has wide production and application values.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Sequence listing
<110> Guangzhou college of traditional Chinese medicine (Guangzhou institute of traditional Chinese medicine)
<120> primer, kit and PCR method for identifying Isodon longituba
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<170> SIPOSequenceListing 1.0
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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gagagagaga gagagaatac tgt 23
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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gagagagaga gagagaataa aagaatc 27
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<213> Isodon longifolia (Rabdosia stracheyi)
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gagagagaga gagagaatac tgtgatggtt agatctgcct cataatattc atgaatatat 60
atattcgtgc agtagtagat catatcagat ttttctgcat tggttctagg attcttttaa 120
cttgtaataa ttcactatta agttgttgcg acttagtcga agtgtgaata aactgtgatc 180
gtatcgagtt tatgattttt aagccctcta gctgtaaagt ttctttcttt ctttcaataa 240
tctgcaagat ttagtgttga ggggatttga tgtttgtaac aaaaaaatat ctacctcaca 300
ataatgtata tgcaagtttt gttgatgtca aactagatca aagactgatg ttgaattttc 360
tcagcaaata tatattcatc aaaacataaa tattcttgtt ttttcaagaa ctgattattt 420
acattttgtt tgtcatcaag cagcgtgcaa atgtgccaaa atttggcaac tgggaaaacg 480
atgacaatgt gccttacaca gtttatttcg agaaggcaag aaaacacaaa ggtgggaaaa 540
tgataaaccc caatgatcct caagaaaatc cggacatgtt ccccgatgcc gatcctccac 600
ctccagcagc tgcccctccg gcaaagcctc gtactcgacc agacgagcct aacggaaggg 660
gagcagccgg ggtgaagcca gaacatggag tgagcaaaga tgatggtgat tttcagcact 720
tgggcaactc tggtgaaaat ccgggccaaa gggctgctag tgagccgagc tatggtgctc 780
gggccccaag ggtagggcga cctgcccgtc ctagtggagg atccgagcag agcttcgagc 840
gttcgcctct tcatcctcat tatcaggcga aagtagcggg acggggcagt ggatcccctg 900
cttgggaagg aaagagccat gatagtagcc atggcacccc tgggaggtcc aagctaaggc 960
cgaccaatcg aggcgacgag agtgtaagtc ttttactgaa caattcaaaa taccttgact 1020
atttgaatag ctgcaacgac tgattatgtt gcaatggcag cctgataaag gtgcagctgt 1080
gccaagattc ggcgaatgga acgaaaatga cccccagtcg gccgagaact tcacgcacat 1140
tttcaacaaa gtgcgtgaag agaggaatgc cggagcagga aatgtttccg ggactcccag 1200
gcatccagct tacggcacac ctgggaggca gacagaacag cctaaggtac gtcgtgctca 1260
tgctgtgtat gtttagtctg agtggctttg actcgggctt atgttgtttt cttgaccttc 1320
gagtgcagaa atgcttctgc ggattgtggt aggtggaaga caaggaagga gaaggacacg 1380
aaaatggaga cgaagaacta gagtgatggc aacagatggt gcattgtttg attttgaact 1440
agtgttgttg cttgtggaaa taaacacaga gggtcttatc tgctttctgc atcatcttct 1500
ccgtcgtcgt ctccgtcttc cctgtagttt ctgggattac acctacttag tttgctcttc 1560
tactgtccag ttttccactt gtagtcatgt atggtataca acgattgctt atcctagatt 1620
cttttattct ctctctctct ctc 1643
Claims (10)
1. A primer for identifying Isodon longituba is characterized by comprising an upstream primer and a downstream primer, wherein the sequence of the upstream primer is shown as SEQ ID NO.1, and the sequence of the downstream primer is shown as SEQ ID NO. 2.
2. Use of the primer of claim 1 for identifying or preparing a product for identifying Isodon longituba.
Application of ISSR universal primer UBC-812 in identifying or preparing products for identifying Isodon longituba.
4. A kit for identifying Isodon longituba, comprising the primer of claim 1.
5. Use of the kit of claim 4 for identifying Isodon longituba.
6. A PCR method for identifying Isodon longituba is characterized by comprising the following steps:
s1, extracting genome DNA of a sample to be detected;
s2, using the genome DNA obtained in the step S1 as a template, carrying out PCR amplification by using the primer of claim 1, and carrying out electrophoresis detection on an amplification product;
and S3, if the electrophoresis result shows that only one single band with the size of 1643bp exists, judging that the detected sample is the isodon longituba.
7. The method of claim 6, wherein the PCR amplification reaction in step S2 is performed in a volume of 25. mu.L, where mix is 12.5. mu.L, DNA template is 1. mu.L, forward and reverse primers are 1.5. mu.L, and the balance is made up with water.
8. The method of claim 6, wherein the PCR amplification procedure in step S2 comprises pre-denaturation at 94 ℃ for 7min, denaturation at 94 ℃ for 2min and annealing at 59.8 ℃ for 45S, extension at 72 ℃ for 2min, and extension at 72 ℃ for 10min after 30 cycles.
9. The method of claim 6, wherein the PCR amplification product obtained in step S2 is further sequenced, and if the sequencing result is the same as the sequence shown in SEQ ID NO.3, the sample is Isodon longituba.
10. Use of the method of any one of claims 6 to 9 for identifying Isodon longituba.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101328500A (en) * | 2008-06-27 | 2008-12-24 | 山西医科大学 | Chinese hamster microsatellite genetic marker and screening method thereof |
| CN104894124A (en) * | 2015-06-19 | 2015-09-09 | 苏州市种子管理站 | ISSR-SCAR (inter simple sequence repeat-sequence characterized amplified region) marker capable of identifying Wujiang brassica chinensis and identification method of marker |
| CN105349651A (en) * | 2015-11-18 | 2016-02-24 | 广东省中药研究所 | Method utilizing EST-SSR marker for identification of traditional Chinese medicine serrate rabdosia herb varieties and primers |
| CN110760609A (en) * | 2019-11-29 | 2020-02-07 | 广州中医药大学(广州中医药研究院) | ISSR-SCAR primer, kit, identification method and application for identifying Isodon lophanthoides |
| CN111304219A (en) * | 2020-03-26 | 2020-06-19 | 华中农业大学 | GL1 gene separated from rice WZ1 and application thereof in increasing rice grain length |
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2021
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101328500A (en) * | 2008-06-27 | 2008-12-24 | 山西医科大学 | Chinese hamster microsatellite genetic marker and screening method thereof |
| CN104894124A (en) * | 2015-06-19 | 2015-09-09 | 苏州市种子管理站 | ISSR-SCAR (inter simple sequence repeat-sequence characterized amplified region) marker capable of identifying Wujiang brassica chinensis and identification method of marker |
| CN105349651A (en) * | 2015-11-18 | 2016-02-24 | 广东省中药研究所 | Method utilizing EST-SSR marker for identification of traditional Chinese medicine serrate rabdosia herb varieties and primers |
| CN110760609A (en) * | 2019-11-29 | 2020-02-07 | 广州中医药大学(广州中医药研究院) | ISSR-SCAR primer, kit, identification method and application for identifying Isodon lophanthoides |
| CN111304219A (en) * | 2020-03-26 | 2020-06-19 | 华中农业大学 | GL1 gene separated from rice WZ1 and application thereof in increasing rice grain length |
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