CN117821642A - Primer group and kit for identifying butterfly orchid variety and application of primer group and kit - Google Patents
Primer group and kit for identifying butterfly orchid variety and application of primer group and kit Download PDFInfo
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- 240000002292 Psychopsis papilio Species 0.000 title abstract 3
- 230000003321 amplification Effects 0.000 claims abstract description 27
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- 238000007403 mPCR Methods 0.000 claims abstract description 23
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- 241001505935 Phalaenopsis Species 0.000 claims description 9
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- 244000127818 Phalaenopsis amabilis Species 0.000 description 25
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Abstract
The present disclosure provides a primer set for identifying butterfly orchid variety, a kit and application thereof. The primer set includes: the 1 st primer pair to the 501 st primer pair, each primer pair comprises a forward primer and a reverse primer, and the forward primer of the 1 st primer pair, the reverse primer of the 1 st primer pair to the forward primer of the 501 st primer pair and the reverse primer of the 501 st primer pair are sequentially shown as SEQ ID NO:1 to SEQ ID NO: shown at 1002. The primer group can be used for obtaining DNA fingerprint data of samples to be detected through multiplex PCR amplification and high-throughput sequencing, and then obtaining variety identification conclusion by comparing the DNA fingerprint data among the samples to be detected, wherein the accuracy and the digitalization degree are high, hundreds of samples to be detected can be compared at one time through sequence analysis software, and the variety identification conclusion can be obtained rapidly, so that the accuracy and the efficiency of the variety identification of the butterfly orchid can be improved remarkably.
Description
Technical Field
The invention relates to the technical field of molecular identification, in particular to a primer group and a kit for identifying butterfly orchid varieties and application thereof.
Background
The butterfly orchid (Phalaenosis) has elegant shape and beautiful color, and has extremely high economic value. In recent years, the variety exchange is frequent in all parts of the world, the phenomena of homonymous foreign matters and homonymous foreign matters are serious, and the number of new hybrid varieties is multiplied along with the improvement of breeding technology, so that higher requirements are put on the management of the new varieties.
Currently, strain identification of butterfly orchid is based on a new plant strain (Distinctness Uniformity Stability, DUS) test, mainly based on morphological identification, and less using DNA (Deoxyribo Nucleic Acid ) molecular identification technology. A few researches report that analysis of variety genetic relationship is carried out by using an ISSR (Inter-simple sequence repeat, simple sequence repeat) molecular marker technology, however, the ISSR molecular marker technology is based on agarose gel electrophoresis after PCR (polymerase chain reaction ) amplification, and one site can only be detected by one PCR amplification, so that the flux is very low, the slipping genotype is easy to generate during the DNA polymerase reaction, and in the electrophoresis process, the slipping genotype is difficult to distinguish from the main genotype of a sample to be detected, thus easily leading to erroneous identification conclusion, and making the method unsuitable for variety identification with quite high accuracy requirements. Therefore, development of a detection technology capable of accurately identifying the strain of butterfly orchid is a technical problem to be solved.
BRIEF SUMMARY OF THE PRESENT DISCLOSURE
In order to solve the problems of the prior art, the embodiment of the disclosure provides a primer group and a kit for identifying the strain of the butterfly orchid and application thereof. The technical scheme is as follows:
in one aspect, an embodiment of the present invention provides a primer set for identifying a strain of phalaenopsis, the primer set comprising: the 1 st primer pair to the 501 st primer pair, each of which comprises a forward primer and a reverse primer, and the forward primer of the 1 st primer pair, the reverse primer of the 1 st primer pair to the forward primer of the 501 st primer pair and the reverse primer of the 501 st primer pair are sequentially as shown in SEQ ID NO:1 to SEQ ID NO: shown at 1002.
In another aspect, the embodiment of the invention provides a kit for identifying a strain of phalaenopsis, which comprises the primer set.
In still another aspect, an embodiment of the present invention provides an application of the primer set described above in identifying a strain of phalaenopsis, where the application includes:
multiplex PCR amplification is carried out on DNA of a sample to be detected by using the primer set of claim 1, so as to obtain a multiplex PCR amplification product;
purifying the multiplex PCR amplification product;
constructing a high-throughput sequencing library by using the purified multiplex PCR amplification product to obtain a high-throughput library of the sample to be detected;
purifying the high throughput library of the test sample;
sequencing the high-throughput library of the sample to be tested to obtain sequencing data;
analyzing the sequencing data to obtain DNA fingerprint data;
comparing the DNA fingerprint data with a control sample to obtain a genetic similarity coefficient;
and obtaining a variety identification conclusion between the sample to be detected and the control variety according to the genetic similarity coefficient.
Specifically, the obtaining the variety identification conclusion between the sample to be tested and the control variety according to the genetic similarity coefficient includes: and when the genetic similarity coefficient is greater than or equal to 99%, judging that the sample to be tested and the control sample are of very similar varieties or identical varieties.
The technical scheme provided by the embodiment of the disclosure has the beneficial effects that: the embodiment of the invention provides a primer group and a kit for identifying butterfly orchid varieties and application thereof, wherein the primer group can be used for obtaining DNA fingerprint data of samples to be detected through multiplex PCR amplification and high-throughput sequencing, and then obtaining variety identification conclusion through comparing the DNA fingerprint data among the samples to be detected. The DNA fingerprint data are base sequences of a plurality of MNP marks obtained after sequencing, the resolution reaches a single base level, the accuracy and the digital degree are high, hundreds of samples to be tested can be compared at one time through sequence analysis software, and a variety identification conclusion can be quickly obtained, so that the accuracy and the efficiency of the variety identification of the phalaenopsis can be remarkably improved. The primer group provided by the embodiment of the invention is used for identifying the butterfly orchid variety, has good application value in the aspects of butterfly orchid variety identification, DNA fingerprint database construction and the like, provides technical support for intellectual property protection of butterfly orchid varieties in China, and can promote industrial healthy development.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present disclosure, the drawings required for the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present disclosure, and other drawings may be obtained according to these drawings without inventive effort for a person of ordinary skill in the art.
Fig. 1 is a diagram of a MNP marker detection site number distribution of a sample to be tested according to an embodiment of the present disclosure, wherein an abscissa is the sample to be tested and an ordinate is the MNP site number;
fig. 2 is a graph of the differential proportion distribution of MNP marker loci provided by embodiments of the present disclosure.
Detailed Description
For the purposes of clarity, technical solutions and advantages of the present disclosure, the following further details the embodiments of the present disclosure with reference to the accompanying drawings.
The embodiment of the invention provides a primer group for identifying butterfly orchid varieties, which comprises the following components: the 1 st primer pair to the 501 st primer pair, each primer pair comprises a forward primer and a reverse primer, and the forward primer of the 1 st primer pair, the reverse primer of the 1 st primer pair to the forward primer of the 501 st primer pair and the reverse primer of the 501 st primer pair are sequentially shown as SEQ ID NO:1 to SEQ ID NO:1002, specifically as shown in table 1.
Table 1 shows the primer pair sequences of 501 pairs
The primers in the primer group are not interfered, so that each primer pair can normally react in the same amplification reaction, and the target gene sequence is specifically amplified.
In another aspect, the embodiment of the invention provides a kit for identifying the strain of butterfly orchid, which adopts the primer set.
In still another aspect, an embodiment of the present invention provides a method for identifying a strain of phalaenopsis using the above primer set, the method comprising:
performing multiplex PCR amplification on the DNA of the sample to be detected by using the primer group to obtain a multiplex PCR amplification product;
purifying the multiplex PCR amplification products;
constructing a high-throughput sequencing library by using the purified multiplex PCR amplification product to obtain a high-throughput library of a sample to be detected;
purifying a high throughput library of the sample to be tested;
sequencing a high-throughput library of a sample to be tested to obtain sequencing data;
analyzing the sequencing data to obtain DNA fingerprint data;
comparing the DNA fingerprint data with a control sample to obtain a genetic similarity coefficient;
and obtaining a variety identification conclusion between the sample to be detected and the control variety according to the genetic similarity coefficient.
Specifically, obtaining a variety identification conclusion between a sample to be tested and a control variety according to the genetic similarity coefficient comprises the following steps: when the genetic similarity coefficient is greater than or equal to 99%, judging that the sample to be detected and the control sample are of very similar varieties or the same varieties.
Examples
In this embodiment, 26 butterfly orchid varieties are randomly selected from butterfly orchid samples collected in Jiang Handa as the samples to be measured, and the 26 butterfly orchid varieties are: orburg, earma, allazod, blon square, blumeton, gauss, cali, deban, pheprara, freund Long Tela, labas, lanbronzon, meng Feisi, merida, montre, naborg, norbuh, pittsburgh, deer Dane, st.Rosa, stuttgart, duling, tuber, raney and Woltaila.
Extracting DNA of a sample to be detected: the leaf DNA of the 26 butterfly orchid varieties is extracted by using a plant genome DNA extraction kit (product number: DP 320) produced by Tiangen biochemical technology (Beijing) limited company, the operation steps are detailed in the specification of the kit, the DNA of 26 samples to be detected is obtained, then 1 mu L of the DNA of the samples to be detected is respectively taken, the DNA concentration of the samples to be detected is measured by using a Qubit fluorescent quantitative instrument, and in the embodiment, the DNA concentration of the samples to be detected is measured to be 20 ng/. Mu.L-50 ng/. Mu.L.
The primer group provided by the embodiment of the invention is utilized to carry out multiplex PCR amplification on the DNA of the sample to be detected, and a multiplex PCR amplification product is obtained.
Specifically, the amplification reaction of each sample to be tested includes: 4 mu L of the primer set provided by the embodiment of the invention, 4 mu L of DNA of a sample to be detected, 10 mu L of Genoplexs 3 xT Master Mix (manufacturer: boruidi biotechnology Co., ltd.) and 12 mu L of water are mixed by shaking to obtain a mixture, and the mixture is used as a multiplex PCR amplification system for multiplex PCR amplification. Multiplex PCR amplification procedure: 3min at 95 ℃; (95 ℃ C. 20sec,60 ℃ C. 4 min). Times.17 cycles; and (3) obtaining multiple PCR amplification products at 72 ℃ for 4 min.
The multiplex PCR amplification products were purified.
Specifically, the amplified product after the end of the reaction was then purified using DNA purification magnetic beads (manufacturer: nanjinouzan Biotechnology Co., ltd.) and the procedure was detailed in the specification of the product.
And constructing a high-throughput sequencing library by using the purified multiplex PCR amplification product to obtain a high-throughput library of the sample to be detected.
Specifically, 10. Mu.L of Genoplexs 3 XT Master Mix, 2. Mu.L of illuminea sequencing adapter primer (produced by Boruidi Biotechnology Co., ltd.) at a concentration of 5. Mu.M, and 16. Mu.L of water were added to the purified multiplex PCR amplification product, and the PCR amplification reaction was performed as follows: 3min at 95 ℃; (95 ℃ 15s,58 ℃ 15s,70 ℃ 30 s). Times.8 cycles; the reaction was terminated at 16℃by final extension at 72℃for 5 min. After the reaction is finished, a high-throughput sequencing library of the sample to be tested is obtained.
Purifying the high throughput library of the test sample. Specifically, the high-flux sequencing library is purified by using DNA purification magnetic beads, so that a purified high-flux sequencing library is obtained, and the specific purification method refers to the specification of a DNA purification magnetic bead product.
Sequencing the high-flux library of the sample to be tested to obtain sequencing data. Specifically, an illumine NextSeq550 sequencer is adopted to sequence the high-throughput sequencing library, sequencing data of a sample to be tested are obtained, and detailed sequencing steps are referred to the using instruction of the sequencer.
And analyzing the sequencing data to obtain DNA fingerprint data. Specifically, sequence analysis is performed by using data alignment software Bowtie2 (version number 2.1.0) to obtain MNP-labeled DNA sequences of each sample to be tested.
Detection rate of MNP marker
The primer group provided by the embodiment of the invention carries out multiplex PCR amplification and construction of a sequencing library, the 26 butterfly orchid DNA samples are subjected to multiplex amplification, second-generation high-throughput sequencing and data analysis, the 501 markers can be detected in 26 samples to be detected, and specific detection information is shown in Table 2.
Table 2 shows 26 kinds of information on detecting butterfly orchid
| Sequence number | Sample numbering | Variety name | Number of detected positions | Detection rate of |
| 1 | HDL220727001 | Orburg | 477 | 95.40％ |
| 2 | HDL220727002 | High heat | 475 | 95.00％ |
| 3 | HDL220727003 | Arlazomib | 470 | 94.00％ |
| 4 | HDL220727004 | Bloom square pill | 479 | 95.80％ |
| 5 | HDL220727005 | Blumton (Brighton) | 473 | 94.60％ |
| 6 | HDL220727006 | Gauss (Gauss) | 489 | 97.80％ |
| 7 | HDL220727007 | Cali (Cari) | 472 | 94.40％ |
| 8 | HDL220727008 | Deban | 477 | 95.40％ |
| 9 | HDL220727009 | Phenanthramide | 455 | 91.00％ |
| 10 | HDL220727010 | Fu Long Tela | 465 | 93.00％ |
| 11 | HDL220727011 | Labas | 455 | 91.00％ |
| 12 | HDL220727014 | Langbaone | 458 | 91.60％ |
| 13 | HDL220727015 | Memphis | 469 | 93.80％ |
| 14 | HDL220727016 | Merida | 491 | 98.20％ |
| 15 | HDL220727018 | Monte mine | 484 | 96.80％ |
| 16 | HDL220727019 | Montreeli | 456 | 91.20％ |
| 17 | HDL220727020 | Naobo (a Chinese character of 'Naobo' type of music) | 464 | 92.80％ |
| 18 | HDL220727021 | North Buddha | 455 | 91.00％ |
| 19 | HDL220727022 | Pittsburgh | 469 | 93.80％ |
| 20 | HDL220727023 | Deer pill | 482 | 96.40％ |
| 21 | HDL220727024 | Holy rosea | 481 | 96.20％ |
| 22 | HDL220727025 | Stuttgart | 475 | 95.00％ |
| 23 | HDL220727026 | Turin | 457 | 91.40％ |
| 24 | HDL220727028 | Fig. l bank | 461 | 92.20％ |
| 25 | HDL220727029 | Ranies (R) S | 472 | 94.40％ |
| 26 | HDL220727030 | Volterra | 473 | 94.60％ |
As shown in Table 2, the number of detected marks of the Merida variety is 491 at most, 470.5 MNP marks can be detected for each variety on average, the average detection rate is 94.11%, and the distribution of the number of detected positions of the MNP marks of the sample to be detected is shown in FIG. 1.
Accuracy analysis
In order to examine the accuracy of the primer pairs, reproducibility experiments (including two independent experiments performed under different personnel, different batch reagents and different instruments) were performed on the butterfly orchid varieties (fli Long Tela, deer dan, monte-mine, langobon, marcande, meng Feisi, lapis, tanis, phenanthrara, nobto, pittsburgh, napera and stuttgart) among 13 samples to be tested, and the 2 experimental data of each sample to be tested were compared and analyzed, and the accuracy of the typing was calculated according to the formula of accuracy = 1- (1-accuracy)/2. The accuracy refers to the proportion of MNP marking sites which are consistent in the typing results of the two experiments to all MNP marking sites. The statistical results are shown in Table 3.
Table 3 shows the accuracy of 501 butterfly orchid MNP marking sites
As shown in Table 3, a total of 6034 MNP markers were compared, the number of unrepeated sites was 14, and the typing accuracy was 99.88%. The high marking accuracy indicates that the identification result is not influenced by different personnel, different batches of reagents and different instruments, which provides technical support for sharing DNA fingerprint data.
Discrimination of MNP-tagged varieties
And comparing all detected MNP marker genotypes of the 26 samples to be detected in pairs, and counting the number of the MNP markers of the 26 samples to be detected in pairs according to the principle that at least 1 SNP is judged to be different on the allelic type of the same MNP marker locus in different varieties, wherein 325 comparison results are obtained, the average difference of each sample to be detected is 375 marker loci, the average difference proportion is 83%, and the difference proportion distribution is shown in figure 2. As can be seen from FIG. 2, the selected MNP markers have high polymorphism, and any strain of butterfly orchid can be distinguished significantly.
Butterfly orchid variety identification
Comparing the DNA fingerprint data with a control sample to obtain a genetic similarity coefficient; and identifying varieties of the sample to be tested according to the genetic similarity coefficient. Obtaining the variety identification conclusion between the sample to be detected and the control variety according to the genetic similarity coefficient comprises the following steps: when the genetic similarity coefficient is greater than or equal to 99%, judging that the sample to be detected and the control sample are of very similar varieties or the same varieties. The control sample can be amplified and sequenced together with the sample to be tested to obtain DNA fingerprint data, or can be compared with the sample to be tested after being amplified and sequenced in advance, and can be determined according to actual conditions.
Specifically, another collected phalaenopsis variety, named DC1, is detected according to the experimental procedure, and then compared with the DNA fingerprint data of the 26 samples to be detected to obtain a genetic similarity coefficient, the genetic similarity coefficient is used as the basis for judging the conclusion of variety identification by referring to the national standard of the existing "plant variety identification MNP labeling method", and when the genetic similarity coefficient (GS) is greater than or equal to 99%, the samples to be detected and the control sample are judged to be of a very similar variety or the same variety. The results are shown in Table 4.
Table 4 shows the comparison result of DNA fingerprint data of the sample to be tested and 26 samples of the Toril DC1
As shown in table 4, the number of the difference sites of the curcandid variety in the curcandid DC1 and 26 samples to be tested is 0, and the gs value reaches 100%, and the same variety or the similar variety is determined; and the number of different sites among other butterfly orchid samples is remarkable (more than or equal to 331 sites), and the GS value is extremely low (less than or equal to 27.09%), and the butterfly orchid samples are judged to be different varieties. Therefore, the primer group provided by the embodiment of the invention can compare and analyze the collected DNA fingerprint data of the sample to be detected with the existing data, and is used for accurately identifying the butterfly orchid variety.
The foregoing description of the preferred embodiments of the present disclosure is provided for the purpose of illustration only, and is not intended to limit the disclosure to the particular embodiments disclosed, but on the contrary, the intention is to cover all modifications, equivalents, alternatives, and alternatives falling within the spirit and principles of the disclosure.
Claims (4)
1. A primer set for identifying a strain of phalaenopsis, the primer set comprising: the 1 st primer pair to the 501 st primer pair, each of which comprises a forward primer and a reverse primer, and the forward primer of the 1 st primer pair, the reverse primer of the 1 st primer pair to the forward primer of the 501 st primer pair and the reverse primer of the 501 st primer pair are sequentially as shown in SEQ ID NO:1 to SEQ ID NO: shown at 1002.
2. A kit for identifying a strain of phalaenopsis, comprising the primer set of claim 1.
3. Use of the primer set according to claim 1 for identifying a strain of phalaenopsis, comprising:
multiplex PCR amplification is carried out on DNA of a sample to be detected by using the primer set of claim 1, so as to obtain a multiplex PCR amplification product;
purifying the multiplex PCR amplification product;
constructing a high-throughput sequencing library by using the purified multiplex PCR amplification product to obtain a high-throughput library of the sample to be detected;
purifying the high throughput library of the test sample;
sequencing the high-throughput library of the sample to be tested to obtain sequencing data;
analyzing the sequencing data to obtain DNA fingerprint data;
comparing the DNA fingerprint data with a control sample to obtain a genetic similarity coefficient;
and obtaining a variety identification conclusion between the sample to be detected and the control variety according to the genetic similarity coefficient.
4. The use according to claim 3, wherein obtaining a variety identification conclusion between the test sample and the control variety based on the genetic similarity coefficient comprises: and when the genetic similarity coefficient is greater than or equal to 99%, judging that the sample to be tested and the control sample are of very similar varieties or identical varieties.
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| AR082253A1 (en) * | 2010-07-19 | 2012-11-21 | Basf Plant Science Co Gmbh | PLANTS THAT HAVE BETTER FEATURES RELATED TO PERFORMANCE AND A METHOD FOR PRODUCING |
| EP2562267A2 (en) * | 2011-08-24 | 2013-02-27 | National Cheng Kung University | Method and kit for identifying Phalaenopsis varieties |
| KR101736670B1 (en) * | 2016-06-07 | 2017-05-17 | 경북대학교 산학협력단 | Primer sets for identification of Phalaenopsis and composition of marker comprising the same |
| CN112695124A (en) * | 2021-01-29 | 2021-04-23 | 广东省农业科学院环境园艺研究所 | Phalaenopsis SSR molecular marker primer composition and application thereof |
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