CN112442545A - Construction method of corylus plant variety SSR molecular marker fingerprint - Google Patents
Construction method of corylus plant variety SSR molecular marker fingerprint Download PDFInfo
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Abstract
The invention discloses a method for constructing a hazel plant variety SSR molecular marker fingerprint, which comprises the following steps: (1) extracting the corylus plant DNA of the ginseng variety; (2) performing multiplex PCR amplification on the variety to be detected by using 25 pairs of core SSR molecular marker primers respectively, (3) grouping the amplification products obtained in the step (2), and then performing capillary electrophoresis detection according to group mixing; (4) and carrying out data analysis on the detection result to construct a fingerprint. The core primer has strong polymorphism, is easy to read data, is compatible with various species of hazel plants, and has strong distinguishing function; by the primer mixed PCR technology, products of 2 groups of primers can be obtained by one-time PCR, and the PCR cost is saved by 1 time; by the PCR product mixed capillary electrophoresis technology, data information of 4-8 groups of primers can be obtained simultaneously by one-time electrophoresis, and the cost of capillary electrophoresis is saved by 3-7 times, so that the purposes of saving cost, economy and quickness in experimental operation are realized.
Description
Technical Field
The invention belongs to the field of molecular biology, and particularly relates to a method for constructing an SSR minute mark fingerprint of a hazel plant variety.
Background
The Corylus (Corylus) plant belongs to the family betulinaceae (Betulaceae) Corylus, and has 13 wild species widely accepted worldwide, and the widely cultivated Corylus species are Corylus avellana (c.avellana L.) and its filial generation with Corylus avellana (c.maxima Mill.), and the main species are: 'Tombul' (Turkish), 'TGDL' (Italy), 'Barcelona' (USA), 'Jefferson' (USA), etc.
The native corylus plants in China have 8 species and 2 varieties, and more than 90% of them are wild corylus heterophylla (C. heterophylla Fisch.). Mainly cultivated are hybrid hazelnuts of Pingyou and small amount of cultivated hazelnuts of horticulture. The hybrid hazel of Pingyu province (Corylushephyllla Fisch. times. Corylusavellana L.) is a filial generation obtained by domestic experts in the 80 th 20 th century by utilizing hybridization between Pingyu province hazel and overseas European hazel, 10 varieties and 30 excellent varieties are successively examined and screened in nearly 40 years so far, and the hybrid hazel has the characteristics of large fruit, high yield, high kernel rate, plump kernels, strong adaptability and the like, is widely introduced to all over the country, and the cultivation area of the country is nearly 120 mu of a mu at present.
With the rapid development of the hybrid hazel industry in Pingyou, some problems in scientific research and industry are to be solved: firstly, the existing Pingyou hybrid hazel variety (strain) is screened from a plurality of mixed progeny populations of female parents and male parents, the genetic relationship among the varieties (strains) is very complex, and some varieties (strains) are difficult to distinguish; secondly, cross breeding is a main means for hazelnut germplasm innovation, cross breeding work of each unit is continuously carried out all the time, and paternity test work of candidate groups needs to be carried out urgently; thirdly, at present, each unit successively begins to examine new varieties, and the identification work of the new varieties urgently needs an accurate and rapid technology; fourthly, the problems of variety mixing and synonym and synonymity of seedlings of Pingyou hybrid hazelnuts are particularly common, and the great economic loss is caused to the planters due to the mixed variety or poor adaptability or incompatibility with pollinated varieties.
Therefore, a simple, accurate and fast variety identification technology applied to hazel variety production and scientific research is urgently needed.
The apparent character identification technology is adopted by a plurality of basic level units and planters, and is distinguished by combining the nut appearance with tree body characters, such as nut size, character, color and appearance stripes; kernel shape, color, dehulling; tree vigor, branch opening angle and branch characteristics; color of branches, pores and fuzz; leaf size, shape, color; the length, shape and opening condition of the buds; shape, color, scale of leaf bud; the color of the female flowers and the number of pistils; the number, shape and color of the male inflorescence and the immature fruit. The apparent character identification method has long variety identification period and strong seasonality, and is influenced by cultivation factors, and the tree body development and the nut appearance in different areas have certain differences, so that the judgment of the phenotypic characters is influenced.
The molecular marker technology has the advantages of accuracy, high efficiency and strong repeatability, is the most common method for researching plant genetic diversity, genetic relationship and variety identification, and domestic scholars successively adopt RAPD, ISSR and AFLP technologies to research the genetic relationship of hybrid hazel varieties (strains) of Pingyou, and report the genetic diversity of Pingyou hybrid hazel populations.
RAPD technique: liaodang and the like perform fingerprint analysis on 8 main cultivars of hybrid hazelnuts of Liaoning by using an RAPD labeling technology. 3 primers OPF05, OPG01 and OPH07 with good repeatability are screened from 60 10-base random primers, and 7 strips are selected as RAPD markers for variety identification, so that the fingerprint of the good variety of the hazelnut is constructed.
ISSR technology: chenxin and the like carry out genetic relationship research on 17 Pinghuo hybrid hazel main cultivars (lines) such as David, Liaoning hazel No. 3 and the like by adopting an ISSR molecular marker method. 7 ISSR primers are screened out from 60 ISSR primers for electrophoretic analysis, and the result shows that: a total of 58 bands were obtained for 7 ISSR primers, all samples were completely separated, and samples were divided into 3 clusters when the genetic similarity threshold was 0.687: the 14 varieties (lines) in the I group can be divided into 4 subclasses, 1 variety in the II group and 2 lines in the III group. The genetic similarity coefficient between samples is 0.448-0.879. The effective allelic factors, gene diversity, Shannon information indices of the populations were 1.6751, 0.3701, and 0.5308, respectively.
AFLP technology: carrying out AFLP analysis on 10 main cultivars such as David and the like serving as test materials by Maqinghua and the like, screening 15 pairs of Pst I/Mse I primers with strong polymorphism from 64 pairs of primers, and obtaining 1739 spectral bands in total, wherein the average polymorphic band ratio is 97.94%; the similarity coefficient of the main cultivar of the Pingyou hybrid hazel is 0.7556-0.8543, and the cultivar can be divided into 4 AFLP groups when the threshold value is 0.8398, wherein the Yuzhu (84-310), Liaohazel No. 4 (85-41) and Pingyou No. 69 (84-69) are 1 group independently, and the other cultivars are 1 group; the effective allelic gene factor, the gene diversity and the Shannon information index of the sample are 1.3921, 0.2482 and 0.3957 respectively, the genetic diversity is high, and the characteristic bands obtained by research can be used for quickly identifying the main cultivar of the hybrid hazel of Pingyou.
The molecular marking method has one or more of the following defects in the aspect of variety identification:
(1) RAPD, ISSR and AFLP markers are all dominant markers, the number of amplified fragments is large, false positive fragments are easy to exist, and the identification accuracy is reduced;
(2) the differences between samples are distinguished by the relative positions of the amplified bands and the cluster maps generated therefrom, the band sizes not being digitized;
(3) only relates to a limited number of varieties of hybrid hazelnut varieties (strains) of Pingyou, and does not have the universality of hazelnut plant varieties;
(4) the variety identification method has difference in different laboratories and different batches of experiments or depends on the control variety, and the method has no universality and convenience.
ZL201710385371.X discloses a method for identifying Pingyou hybrid hazel varieties by SSR markers, which adopts SSR molecular markers in combination with capillary electrophoresis technology to establish a Pingyou hybrid hazel variety identification method based on 4 pairs of EST-SSR markers, firstly releases SSR amplification data of 43 Pingyou hybrid hazel varieties (strains) generally introduced at home at present, provides comparable data results for identification of unknown varieties, and provides an effective method for identification of Pingyou hybrid hazel varieties. However, this method has the following disadvantages:
(1) the detection range only comprises a limited number of varieties of hybrid hazel varieties (strains) of Pingyou, and other hazel varieties are not involved;
(2) the source and the test range of the primer are Hazelnut hybrid hazelnuts, and the universality of the primer in all domestic and foreign corylus plant varieties is uncertain;
(3) the PIC value of the primer is low, and uncertainty exists in distinguishing similar varieties;
(4) the cost consumption of PCR and capillary electrophoresis technology in the test operation is large.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: how to provide an economical and rapid construction method of the hazel plant variety SSR molecular marker fingerprint.
The technical scheme of the invention is as follows: a method for constructing a hazel plant variety SSR molecular marker fingerprint comprises the following steps:
(1) extracting the corylus plant DNA of the ginseng variety;
(2) performing multiplex PCR amplification on varieties to be detected by using 25 pairs of core SSR molecular marker primers respectively, wherein each pair of primers is marked with a fluorescent dye, and the nucleotide sequences of the core SSR molecular marker primers are as follows:
(3) grouping the amplification products in the step (2), and then mixing the amplification products according to groups to perform capillary electrophoresis detection;
(4) carrying out data analysis on the detection result to construct a fingerprint;
the mixed PCR amplification is carried out on the same sample with similar amplified fragment size, consistent annealing temperature, similar amplification efficiency (agarose gel strip signal intensity) and different fluorescence labels, and the operation can save half of the PCR experiment cost and workload. The recommended primer combinations for multiplex PCR amplification are as follows:
capillary electrophoresis detection groups were as follows:
| group 1 | GK6.63 | BR423 | B613 | B664 | B654 |
| Group 2 | B029b | B733 | B777 | KG845 | BR483 |
| Group 3 | BR215 | B619 | B716 | B791 | GB332 |
| Group 4 | GB410 | B504 | B734 | B606 | KG817 |
| Group 5 | B720 | GB818 | KG811 | B657 | GB875 |
。
Performing fluorescent labeling on the forward primer, wherein the fluorescent labeling mainly comprises FAM (fluoroscein), HEX (Hexachlorofluoroscein), ROX (fluoroxyX rhodamine), TAMRA (carboxyetyl rhodamine) and the like, and from the viewpoint of cost control and subsequent practical operation, the FAM and HEX labeling is recommended, and the preferable fluorescent labeling of each pair of primers is as follows:
| primer name | Fluorescent markers | Primer name | Fluorescent markers |
| KG817 | Fam | GB818 | Hex |
| B791 | Hex | GK6.63 | Fam |
| GB332 | Fam | B777 | Fam |
| B619 | Hex | B720 | Fam |
| B613 | Fam | KG845 | Hex |
| BR215 | Fam | B733 | Hex |
| B734 | Fam | B654 | Fam |
| B029b | Fam | BR423 | Hex |
| B664 | Hex | B606 | Hex |
| KG811 | Fam | B716 | Fam |
| BR483 | Fam | GB410 | Fam |
| GB875 | Fam | B504 | Hex |
| B657 | Hex |
。
The parameters of the PCR amplification were as follows:
and (3) PCR reaction system: the total volume is 20.0 μ L, ddH2O 14.8.8 μ L, dNTP 0.4 μ L, Buffer 2.0 μ L, F-primer 0.3 μ L, R-primer 0.3 μ L, DNA 2.0 μ L, Taq 0.2 μ L; PCR reaction procedure: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 57-63 ℃ for 35s, and extension at 72 ℃ for 40s for 35 cycles, and extension at 72 ℃ for 3 min.
In the invention, the selected varieties are as follows:
starting from the core primer, the invention introduces the primer commonly used in foreign corylus germplasm resource research, and performs secondary screening in the domestic existing variety (strain), thereby ensuring the universality of the experimental primer in the corylus variety; in the process of primer screening, taking corylus avellana variety 'Barcelona' as a reference variety, and taking the site information of the corresponding primer in the database as reference to read and correct the amplification data, thereby ensuring the reliability of the read data and obtaining the site information of more varieties through data retrieval; the screening of the primers takes strong polymorphism, simple data reading and strong repeatability as standards, the optimal selection is preferred, the PIC value of the selected primer is higher, the average value is 0.7234, and meanwhile, the SSR loci are distributed in 11 different linkage groups, so that the primers are ensured to have strong distinguishing function.
In addition, aiming at the problem of higher cost consumption of PCR and capillary electrophoresis technology in test operation, the invention provides two operation methods for saving the test cost: 1. for the same sample with similar amplified fragment size, consistent annealing temperature, similar amplification efficiency (signal intensity of agarose gel strip) and different fluorescence labels, mixed PCR amplification is carried out, and the operation can save half of PCR experiment cost and workload (generally 2 groups of primers with different fluorescence labels are mixed); 2. based on the existing SSR locus data information, the amplification fragment range of the same group of primers in different varieties is calibrated, and for the primers in different fragment ranges, the PCR product is properly diluted and mixed product capillary electrophoresis can be carried out (generally, 2-4 groups are mixed, the fragment ranges are not overlapped or covered, and the distance is 20bp or more). By combining the two operations, data information of 4-8 groups of primers can be obtained simultaneously by one-time capillary electrophoresis, so that the aims of saving cost, economy and quickness are fulfilled.
Compared with the prior art, the invention has the following beneficial effects:
the invention establishes an economic and rapid hazel plant variety identification technology based on multiple SSR molecular markers. In the technology, the core primer has strong polymorphism and simple and easy data reading, is compatible with various species of hazel plants, and has strong distinguishing function; in addition, by the primer mixed PCR technology, products of 2 groups of primers can be obtained by one-time PCR, and the PCR cost is saved by 1 time; by the PCR product mixed capillary electrophoresis technology, data information of 4-8 groups of primers can be obtained simultaneously by one-time electrophoresis, and the cost of capillary electrophoresis is saved by 3-7 times, so that the purposes of saving cost, economy and quickness in experimental operation are realized.
Drawings
FIG. 1 is a partial fluorescent primer peak pattern.
Detailed Description
The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were all commercially available unless otherwise specified.
(1) Selection of reference varieties: experiments were carried out with the domestic examined and named corylus species as reference species (table 1).
TABLE 1 list of reference varieties
(2) Collecting a hazel tree sample: preferably healthy, tender leaves, and is taken back to the laboratory in an ice box after being sampled, and stored at-80 ℃ for later use; the dehydrated and dried leaves can also be used, after sampling, the leaves and the package are placed in allochroic silica gel for storage at room temperature, and the silica gel is replaced periodically to keep the sample dry.
(3) Extracting and detecting genome DNA: extracting sample DNA by modified CTAB method, detecting DNA integrity by 0.8% agarose gel electrophoresis, detecting DNA purity and concentration by ultraviolet spectrophotometer, and detecting by ddH2O the DNA was diluted to 10 ng/. mu.L in bulk for further use.
(4) PCR reaction
PCR reaction (20.0. mu.L): 14.8 μ L of ddH2O 14.8, 0.4 μ L of dNTP, 2.0 μ L of Buffer, 0.3 μ L (20.0 μ M) of F-primer, 0.3 μ L (20.0 μ M) of R-primer, 2.0 μ L of DNA, and 0.2 μ L of Taq. PCR reaction procedure: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, renaturation at 57-63 ℃ (according to the annealing temperature in Table 2) for 35s, extension at 72 ℃ for 40s, for 35 cycles, and extension at 72 ℃ for 3 min.
The forward primer is fluorescently labeled with FAM (carboxyfluoroscecin), HEX (Hexachlorofluoroscecin), ROX (carboxyX rhodomine), TAMRA (carboxymethylrhodamine), etc., and the use of FAM and HEX labels is recommended from the viewpoint of cost control and subsequent practical operation (Table 2).
TABLE 2 Experimental synthetic fluorescent primer List (25 pairs)
Note: f represents a forward primer, and R represents a reverse primer; the choice of the fluorescent label can be self-adjusting depending on the different PCR amplification and capillary electrophoresis mixing protocols, and the types of fluorescent labels shown in the table are suitable for the PCR mixing protocol recommended by the present invention.
For the same sample with similar amplified fragment size, consistent annealing temperature, similar amplification efficiency (agarose gel strip signal intensity) and different fluorescence labels, mixed PCR amplification is carried out (Table 3 shows the technical scheme of primer mixed PCR recommended by the invention), and the operation can save half of the PCR experiment cost and workload.
TABLE 3 combination scheme of the Mixed PCR primers
(5) Capillary electrophoresis
The primers used in the invention are widely used in foreign corylus plant genetic relationship and population genetics research, and the early research of the invention uses Chinese corylus plant resources to further expand the amplification range, so that more comprehensive amplification range and allelic variation information of each primer are obtained (Table 4).
TABLE 4 primer amplification efficiency and data read information
Based on the SSR locus data information, primers in different fragment ranges are optimally combined. The range of each primer combination fragment is arranged from small to large, the condition of mutual superposition or coverage can not occur, and the distance between the ranges of the fragments is 20bp or more. The capillary electrophoresis method comprises the following steps: after formamide and the molecular weight internal standard are mixed uniformly according to the volume ratio of 100:1, 9.0 mu L of the mixture is added into the upper sample plate, 1.0 mu L of PCR product (PCR product of 5 groups of primers mixed in the invention) diluted by 10 times is added, and a sequencer is used for carrying out capillary electrophoresis. The cost of capillary electrophoresis can be saved by 4 times.
TABLE 5 primer mix capillary electrophoresis grouping
Note: the 5 groups of primers in the square frame are the recommended mixed electrophoresis combination (data of the 5 groups of primers can be obtained by one-time electrophoresis), and can also be combined with the range of the primer fragments according to the needs.
(6) Data reading and statistics
And reading the original data derived from the capillary electrophoresis sequencer by using GeneMarker V2.2.0 fragment analysis software, and comparing and analyzing the position of the molecular weight internal standard of each lane with the position of the peak value of each sample to obtain the size of the fragment. In order to ensure the accuracy of the data, a manual reading mode or a manual reinspection mode is recommended for all experimental data. Data reading main peaks and the direction of numerical rounding were selected for unimodal and multimodal (fig. 1) in the manner recommended in table 4. The final fingerprint information is shown in table 6.
TABLE 6 fingerprint information of the selected varieties
Sequence listing
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ctacccgcct gcgagaac 18
<210> 11
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
tgaaatcttc acctcttaaa agatcc 26
<210> 12
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
ggaatctgag ctgccaagtc 20
<210> 13
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
aaggtcctgt ttgttggatc tc 22
<210> 14
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
tgtttctttg acaacctgca tt 22
<210> 15
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
caatttacac ctcagggaag ag 22
<210> 16
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
aagttcaccc aagaaatcca c 21
<210> 17
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
caaagccgtc gacaacag 18
<210> 18
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
tttgcatttg atgccgataa 20
<210> 19
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
gaacaactga agacagcaaa g 21
<210> 20
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
aaggcggcac tcgctcac 18
<210> 21
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
ttaccaccac ttttcaacac ca 22
<210> 22
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
ggtacatcaa agaagggagc ac 22
<210> 23
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
atgatgatga ggaggaggag aa 22
<210> 24
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
caaaatcagg catacagaac ca 22
<210> 25
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
gagagtgcgt cttcctctgg 20
<210> 26
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
agcctcacct ccaacgaac 19
<210> 27
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
gaagttgggt tggaagcagt t 21
<210> 28
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
cgtcctctgc acactctcat ac 22
<210> 29
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
gcaaacttcc agaaaaccaa 20
<210> 30
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 30
aatgttcgta ggacaactgc at 22
<210> 31
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 31
agggaagggt gtaggacgtt 20
<210> 32
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 32
tcgttttctc cacatcacca 20
<210> 33
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 33
ctctgtgtcg gctttctggt 20
<210> 34
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 34
ataaacctca cgccacacct 20
<210> 35
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 35
tatagatgcc atgggtgcaa acaaaa 26
<210> 36
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 36
actatcactt gacccacctt ccctcttt 28
<210> 37
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 37
caccctcttc accacctcat 20
<210> 38
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 38
catcccctgt tggagttttc 20
<210> 39
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 39
tcgcatgggt aattttctca c 21
<210> 40
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 40
tcatcatttg ggtgcttcaa 20
<210> 41
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 41
acaaaccaaa gggagtgtgg 20
<210> 42
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 42
caagctttcc atcatcgtca 20
<210> 43
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 43
tcttgtggtt tagcatactt ctcg 24
<210> 44
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 44
gaagaaagca agaagagagg aga 23
<210> 45
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 45
gaacattgtc gtatgcggac t 21
<210> 46
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 46
tctgtttgtt gcgcatgatt 20
<210> 47
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 47
cctctactat ctaggaagcc cca 23
<210> 48
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 48
actttggcct tttggacttt g 21
<210> 49
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 49
gccatctcca tttcccaac 19
<210> 50
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 50
cggaatggtt ttctgcttca g 21
Claims (4)
1. A method for constructing a hazel plant variety SSR molecular marker fingerprint is characterized by comprising the following steps:
(1) extracting the corylus plant DNA of the ginseng variety;
(2) performing multiplex PCR amplification on varieties to be detected by using 25 pairs of core SSR molecular marker primers respectively, wherein each pair of primers is marked with a fluorescent dye, and the nucleotide sequences of the core SSR molecular marker primers are as follows:
(3) grouping the amplification products in the step (2), and then mixing the amplification products according to groups to perform capillary electrophoresis detection;
(4) carrying out data analysis on the detection result to construct a fingerprint;
the primer combinations used for the multiplex PCR amplification are as follows:
capillary electrophoresis detection groups were as follows:
。
2. The method of claim 1, wherein each pair of primers is fluorescently labeled as follows:
。
3. The method of claim 1, wherein the PCR reaction system: the total volume is 20.0 μ L, ddH2O 14.8.8 μ L, dNTP 0.4 μ L, Buffer 2.0 μ L, F-primer 0.3 μ L, R-primer 0.3 μ L, DNA 2.0 μ L, Taq 0.2 μ L; PCR reaction procedure: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 57-63 ℃ for 35s, and extension at 72 ℃ for 40s for 35 cycles, and extension at 72 ℃ for 3 min.
4. The construction method according to claim 1, wherein the reference varieties are:
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113049661A (en) * | 2021-05-27 | 2021-06-29 | 广州市农业科学研究院 | Rice variety identification method and system |
| CN113096734A (en) * | 2021-05-11 | 2021-07-09 | 中国科学院水生生物研究所 | Method for screening molecular marker combination for diploid population paternity test |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107130034A (en) * | 2017-05-26 | 2017-09-05 | 中国林业科学研究院林业研究所 | A kind of method that utilization SSR marker identifies flat Europe hybrid hazel kind |
-
2020
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107130034A (en) * | 2017-05-26 | 2017-09-05 | 中国林业科学研究院林业研究所 | A kind of method that utilization SSR marker identifies flat Europe hybrid hazel kind |
Non-Patent Citations (8)
| Title |
|---|
| BARBARA FUSSI等: "Molecular differentiation of Turkish and Common hazels (Corylus colurna L. and Corylus avellana L.) using multiplexed nuclear microsatellite markers", 《ANN. FOR. RES.》 * |
| JOSE A. FREIXAS‑COUTIN等: "Development of a reliable Corylus sp. reference database through the implementation of a DNA fingerprinting test", 《PLANTA》 * |
| M. AKIN等: "A multiplexed microsatellite fingerprinting set for hazelnut", 《EUR. J. HORTIC. SCI.》 * |
| N.V. BASSIL等: "SSR Fingerprinting Panel Verifies Identities of Clones in Backup Hazelnut Collection of USDA Genebank", 《ACTA HORTICULTURAE》 * |
| 王艳梅等: "中国榛属植物DNA提取与SSR初步分析", 《河南师范大学学报(自然科学版)》 * |
| 程云清等: "平欧杂交榛转录组中SSR信息分析和引物筛选", 《园艺学报》 * |
| 饶龙兵等: "基于桤木属转录组测序的SSR分子标记的开发", 《林业科学研究》 * |
| 马庆华等: "基于EST-SSR标记的平欧杂种榛品种鉴定", 《植物遗传资源学报》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113096734A (en) * | 2021-05-11 | 2021-07-09 | 中国科学院水生生物研究所 | Method for screening molecular marker combination for diploid population paternity test |
| CN113096734B (en) * | 2021-05-11 | 2021-12-14 | 中国科学院水生生物研究所 | Method for screening molecular marker combination for diploid population paternity test |
| CN113049661A (en) * | 2021-05-27 | 2021-06-29 | 广州市农业科学研究院 | Rice variety identification method and system |
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| CN112442545B (en) | 2022-04-22 |
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