CN112725521B - Dendrobium chrysotoxum SSR molecular marker primer composition and application thereof - Google Patents
Dendrobium chrysotoxum SSR molecular marker primer composition and application thereof Download PDFInfo
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Abstract
The invention provides a dendrobium chrysotoxum SSR molecular marker primer composition and application thereof, belonging to the technical field of molecular biology. The invention develops and screens 25 pairs of SSR molecular marker primers with good amplification effect and high polymorphism from dendrobium chrysotoxum transcriptome sequences, and by adopting the SSR molecular marker primers, the genetic diversity and genetic relationship of dendrobium chrysotoxum groups can be analyzed, and germplasm identification and genetic relationship analysis can be performed on dendrobium chrysotoxum clones or varieties, so that the invention has the advantages of high efficiency, accuracy, practicability, no influence by seasons and the like, and lays a foundation for the follow-up dendrobium chrysotoxum germplasm resource management, breeding, genetic protection, genetic diversity analysis among groups and in groups and the like.
Description
Technical Field
The invention relates to the technical field of molecular biology, in particular to a dendrobium chrysotoxum SSR molecular marker primer composition and application thereof.
Background
Dendrobium chrysotoxum (Dendrobium chrysotoxum) is a perennial epiphytic herb of the genus Dendrobium of the family Orchidaceae, is one of the widely cultivated dendrobium species integrating appreciation, eating and medication, and is recorded in the 'Chinese pharmacopoeia' 2020 edition. The stem and flower of the Chinese medicinal composition are dried and soaked in water for drinking, and the Chinese medicinal composition has the effects of beautifying and improving eyesight, soothing nerves and enriching blood, resisting oxidation, enhancing immunity and the like. Meanwhile, the dendrobium chrysotoxum stem meat is spindle-shaped, the leaves are evergreen in four seasons, the flower is golden yellow, the flower quantity is large, the fragrance is slightly brought, the flowering period is as long as 1 month, the dendrobium chrysotoxum stem flower has high ornamental value, and the dendrobium chrysotoxum stem meat can be used for various cultivation and application in large and small pot culture, vertical suspension, epiphytic stone or branches and the like. The dendrobium chrysotoxum is native to the southwest region of China, is grown at the elevation of 500 + 1600m, is on the trunk or rocks under sparse forest in evergreen broad-leaved forest with sufficient sunlight, and is abroad distributed in northeast of India, Burmese, Thailand, Laos and Vietnam.
The SSR molecular marker has the characteristics of simplicity and convenience in use, rapidness, high stability, high allele diversity, capability of performing co-dominant allele analysis and the like, is a main molecular marker technology at present, and is widely applied to the fields of various researches such as plant genetic diversity analysis, population structure analysis, variety identification, germplasm resource genetic relationship analysis and the like. For example, Shenying and the like adopt an ISSR-PCR method to identify 9 plants in the dendrobii genus, including dendrobium candidum, dendrobium chrysanthum, dendrobium imbricatum, dendrobium chrysotoxum, dendrobium nobile, dendrobium candidum, dendrobium roseum and dendrobium officinale (Shenying, slow process, Wanxiaofeng, and the like). the application of ISSR-PCR in the identification of dendrobium species [ J ] Chinese herbal medicine, 2005,36(3):423 + 427 ]. Patent CN104313162A discloses a molecular specific marker primer for identifying dendrobium chrysotoxum and dendrobium candidum and a method thereof.
In recent years, a large number of SSR primers are developed on dendrobium plants such as dendrobium officinale and dendrobium nobile, however, the developed SSR primers are not reported in dendrobium chrysotoxum of the same genus, and the genetic variation analysis still adopts RAPD and other analysis markers, for example, Tikendra and the like (2019) utilize 12 pairs of RAPD primers and 11 pairs of ISSR primers to evaluate the genetic stability of dendrobium chrysotoxum clone seedlings in the tissue culture process, and find that the clone seedlings have higher genetic singleness (96.3%) and lower polymorphism (3.6%) with mother plants. Dendrobium chrysotoxum is used as an ornamental and medicinal plant, currently, approved and widely cultivated Dendrobium chrysotoxum varieties are not seen in production, wild resources or tissue culture aseptic seeding obtained seedlings or cloned seedlings are mainly used in production, and identification, conservation, development and utilization of Dendrobium chrysotoxum germplasm resources are increasingly emphasized in various circles.
Therefore, finding and developing SSR primers suitable for dendrobium chrysotoxum genetic research is a prerequisite basis for further carrying out researches on dendrobium chrysotoxum germplasm resource management, heredity, protection and the like.
Disclosure of Invention
Aiming at the defects, the invention provides a dendrobium chrysotoxum SSR molecular marker primer composition and application thereof. The invention screens 25 pairs of SSR molecular marker primers with good amplification effect and high polymorphism in dendrobium chrysotoxum, can analyze the genetic diversity and genetic relationship of dendrobium chrysotoxum groups, can also perform germplasm identification and genetic relationship analysis on dendrobium chrysotoxum clones or varieties, has the advantages of high efficiency, accuracy, practicability, no influence by seasons and the like, and lays a foundation for the follow-up dendrobium chrysotoxum germplasm resource management, breeding, genetic protection, genetic diversity analysis among groups and in groups and the like.
In order to achieve the above object, the technical solution of the present invention is as follows:
in one aspect, the invention provides a dendrobium chrysotoxum SSR molecular marker primer composition.
Specifically, the primer composition is shown in the following table 1:
TABLE 1 primer compositions
In another aspect, the invention provides a product for germplasm identification, genetic diversity or genetic relationship analysis of dendrobium chrysotoxum, wherein the product comprises the SSR molecular marker primer composition.
Specifically, the product is an independent reagent or a kit.
In another aspect, the invention provides an application of the SSR molecular marker primer composition in preparation of products for germplasm identification, genetic diversity or genetic relationship analysis of dendrobium chrysotoxum.
In another aspect, the invention provides an application of the SSR molecular marker primer composition or the SSR molecular marker primer product in germplasm identification, genetic diversity or genetic relationship analysis of dendrobium chrysotoxum.
In another aspect, the present invention provides a method for germplasm identification, genetic diversity or genetic relationship analysis of dendrobium chrysotoxum, wherein the method comprises the following steps:
(1) extracting the genome DNA of the dendrobium chrysotoxum sample to be detected;
(2) performing PCR amplification by taking the genomic DNA extracted in the step (1) as a template, and modifying an upstream primer of a primer pair for PCR amplification by adopting a bioluminescent group;
(3) performing capillary fluorescence electrophoresis detection on the PCR amplification product obtained in the step (2), and collecting data;
(4) and (4) carrying out statistical analysis on the genetic diversity or clustering and polymorphic information content of the dendrobium chrysotoxum population according to the data obtained in the step (3).
Specifically, the PCR amplification system in step (2) is: the total volume of the reaction system is 10. mu.L, and the reaction system comprises 1.0. mu.L of DNA template (50 ng/. mu.L), 1.0. mu.L of 10 XBuffer I Buffer solution, 0.1. mu. L TAKARA HS Taq enzyme (5U/. mu.L), 0.6. mu.L of primer (5. mu.M), 0.8. mu.L of 2.5mM dNTP, and deionized water to make up to 10. mu.L.
More specifically, the primer of the PCR amplification system is shown as SEQ ID NO. 1-SEQ ID NO. 50.
Further specifically, the PCR amplification reaction procedure: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 72 ℃ for 30s, and extension at 72 ℃ for 40 cycles; extension at 60 ℃ for 10 min.
In another aspect, the invention also provides application of the SSR molecular marker amplification primer composition or product in dendrobium chrysotoxum molecular marker assisted breeding.
Compared with the prior art, the invention has the advantages that:
1. the invention provides a dendrobium chrysotoxum SSR molecular marker and a primer composition, which can perform germplasm identification, genetic diversity or genetic relationship analysis on the dendrobium chrysotoxum and lay a foundation for germplasm resource management, breeding, genetic protection, genetic diversity analysis among dendrobium chrysotoxum groups and in the groups and the like.
2. The dendrobium chrysotoxum SSR molecular marker and the primer composition have the advantages of good amplification effect and high polymorphism in dendrobium chrysotoxum, can accurately, efficiently and stably identify dendrobium chrysotoxum germplasm resources, and are simple and convenient to operate.
3. The dendrobium chrysotoxum SSR molecular marker disclosed by the invention can be used for assisting in selective breeding, so that the breeding process of dendrobium chrysotoxum is accelerated.
Drawings
FIG. 1 is an amplification locus diagram of SSR molecular marker primer DC 11 in 7 Dendrobium chrysotoxum plants.
Fig. 2 is a clustering analysis graph of 8 dendrobium chrysotoxum populations.
FIG. 3 is a graph of cluster analysis of clones of 12 Dendrobium chrysotoxum.
Detailed Description
The present invention will be further illustrated in detail with reference to the following specific examples, which are not intended to limit the present invention but are merely illustrative thereof. The experimental methods used in the following examples are not specifically described, and the materials, reagents and the like used in the following examples are generally commercially available under the usual conditions without specific descriptions.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.
Example 1 Dendrobium chrysotoxum SSR primer screening
1. The invention utilizes a dendrobium chrysotoxum transcriptome sequence, 100 pairs of SSR primers are designed and screened, the SSR primers are synthesized in a biological engineering (Shanghai) corporation and then serve as candidate primers of the dendrobium chrysotoxum, and the upstream primers of each pair of primers are respectively modified by 2 biological fluorescent groups of FAM (blue) and HEX (green).
2. Randomly selecting 7 dendrobium chrysotoxum plants (seedlings) as test materials, cutting tender leaves of the dendrobium chrysotoxum plants, extracting genome DNA of the dendrobium chrysotoxum plants by using a DP305 kit (Chinese Tiangen), and detecting the quality and the concentration of the DNA by respectively adopting 1.2% agarose gel electrophoresis and a NanoDrop spectrophotometer.
3. Taking sample DNA as a template, and carrying out PCR amplification by using a fluorescence modified primer and a downstream primer, wherein the PCR amplification reaction system is as follows: the total volume of the reaction system is 10. mu.L, and the reaction system comprises 1.0. mu.L of DNA template (50 ng/. mu.L), 1.0. mu.L of 10 XBuffer I Buffer solution, 0.1. mu. L TAKARA HS Taq enzyme (5U/. mu.L), 0.6. mu.L of primer (5. mu.M), 0.8. mu.L of 2.5mM dNTP, and deionized water to make up to 10. mu.L. The PCR amplification procedure was: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 72 ℃ for 30s, and extension at 72 ℃ for 40 cycles; and (3) extending for 10min at 60 ℃ to obtain an amplification product.
4. And (3) carrying out capillary fluorescence electrophoresis detection on the PCR amplification product, namely detecting the amplification product by using an ABI 3730xl DNA Analyzer gene Analyzer, reading the result by using GeneMarker 2.2.0 software, counting the sizes of characteristic bands and polymorphic bands amplified by each pair of primers, and screening SSR molecular marker primers with polymorphism.
5. The final screening yielded the following 25 pairs of SSR molecular marker primers (Table 2). Taking the primer DC 11 as an example, the capillary electrophoresis detection chart of the SSR primer pair amplified in 7 dendrobium chrysotoxum plants is shown in figure 1.
TABLE 2SSR primers
Example 2 application of SSR primer composition in dendrobium chrysotoxum population genetic diversity analysis
1. The test material was 8 populations of dendrobium chrysotoxum from different sources (table 3), each population sampled 8-14 plants, each plant individually numbered as CA1-CA12, CB1-CB12, CC1-CC10, CD1-CD14, CE1-CE8, CF1-CF8, CG1-CG12, CH1-CH8, and 84 single plants were sampled altogether.
TABLE 3.8 Dendrobium chrysotoxum population test materials
| Dendrobium chrysotoxum population number | Sampling single plant number (plant) | Origin of |
| CA | ||
| 12 | Myanmar eighty | |
| CB | ||
| 12 | Burmese dried orange berry leaf | |
| CC | 10 | Yunnan Xishuangbanna Meng Hai county |
| CD | 14 | |
| CE | ||
| 8 | Guizhou | |
| CF | ||
| 8 | Yunnan Pu' er | |
| CG | ||
| 12 | De hong Long Chuan county in | |
| CH | ||
| 8 | Yunnan Cangyuan county | |
| Total | 84 | - |
2. Cutting tender leaves of each plant, extracting dendrobium chrysotoxum genome DNA by using a DP305 kit (Chinese Tiangen), and detecting the quality and the concentration of the DNA by respectively adopting 1.2% agarose gel electrophoresis and a NanoDrop spectrophotometer.
3. 25 pairs of SSR primers in Table 2 were synthesized in Biotechnology engineering (Shanghai) GmbH, and the upstream primers of each pair of primers were modified with 2 bioluminescence groups, FAM (blue) and HEX (green), respectively. And carrying out PCR amplification by using the primers and the annealing temperature.
4. And detecting the amplification product by using an ABI 3730xl DNA Analyzer gene Analyzer, reading the result by using GeneMarker 2.2.0 software, and counting the sizes of characteristic bands and polymorphic bands amplified by each pair of primers. Calculating the number of alleles (Na), effective allele factor (Ne), observation heterozygosity (Ho), expected heterozygosity (He) and breeding coefficient (F) in the group of each SSR locus by using software POPGENE 1.32IS) Shannon information index (I), thereby obtaining the related data of SSR locus. As can be seen from Table 4 below, the number of alleles (Na) at each locus varied from 2 to 26, with an average of 9.84. The desired heterozygosity (He) is 0.036-0.917, the observed heterozygosity (Ho) is 0.083-0.964, and the Shannon information index (I) is 0.101-2.931.
TABLE 4.25 genetic analysis results of SSR loci
5. Referring to step 4, the number of alleles (Na), effective allele count (Ne), observed heterozygosity (Ho), expected heterozygosity (He), and intra-population breeding coefficient (F) of each population were calculated using the software POPGENE 1.32IS) The average ratio (M ratio) of the number of alleles to the size range of alleles was calculated for each population using the software Arlequin 3.5.2.2. The results of the genetic diversity analysis of each population are shown in Table 5, at the species level, Na, Ne, Ho, He, FISAnd M ratio has an average value of 4.87, 3.24, 0.50, 0.600.14 and 0.24. At the same time, F of 8 groupsISThe values are all low (0.09-0.18), which indicates that dendrobium chrysotoxum populations show heterozygote deficiency and relatively more inbred reproduction in the populations. The M ratio values for the 8 populations were all less than 0.68, which also indicates that the effective population size of the 8 dendrobium chrysotoxum population is decreasing.
TABLE 5 genetic diversity of Dendrobium chrysotoxum populations
| Group numbering | Na | Ne | Ho | He | FIS | M ratio |
| CA | 5.36 | 3.47 | 0.51 | 0.58 | 0.12 | 0.27 |
| CB | 4.80 | 3.14 | 0.53 | 0.61 | 0.11 | 0.26 |
| CC | 4.84 | 3.21 | 0.48 | 0.58 | 0.13 | 0.23 |
| CD | 5.56 | 3.39 | 0.48 | 0.57 | 0.15 | 0.25 |
| CE | 4.48 | 3.16 | 0.48 | 0.59 | 0.12 | 0.22 |
| CF | 4.08 | 2.89 | 0.45 | 0.55 | 0.19 | 0.21 |
| CG | 5.00 | 3.35 | 0.54 | 0.60 | 0.09 | 0.23 |
| CH | 4.84 | 3.32 | 0.51 | 0.65 | 0.18 | 0.24 |
| Average | 4.87 | 3.24 | 0.50 | 0.60 | 0.14 | 0.24 |
6. Calculation of paired Reiter fixed indices (F) Using the software Arlequin 3.5.2.2ST) Cavalli-Sforza and Edwards genetic distance (D), and molecular variance analysis (AMOVAs). As can be seen from Table 6, the paired Reiter fixed index (F) among the 8 Dendrobium chrysotoxum populationsST) 0.010-0.162, and 0.071-0.288 of Cavalli-Sforza and Edwards genetic distance (D), which indicates that the relative relationship between dendrobium chrysotoxum populations is relatively close, and the genetic distance between CA and CE populations is minimum. Molecular analysis of variance at 25 SSR sites showed (Table 7) that the genetic variation was mainly due to individuals (82.6%), and the genetic variation among populations was only 4.79%. Clustering analysis (fig. 2) was performed based on genetic distance (D), and 8 dendrobium chrysotoxum populations could be grouped into 2 categories, wherein populations CB and CH were one category, and the remaining populations were one category.
TABLE 6 paired Reiter fixed index F for Dendrobium chrysotoxum populationST(upper diagonal) and genetic distance D (lower diagonal)
Note: significance (P < 0.01).
TABLE 7.25 molecular analysis of variance of SSR loci
| Source of variation | Degree of freedom | Sum of squared deviations | Variance component | Proportion of components |
| Between |
7 | 91.411 | 0.301 | 4.79 |
| Between individuals within a group | 76 | 514.851 | 0.792 | 12.60 |
| Between individuals | 83 | 436 | 5.190 | 82.60 |
| Sum of | 166 | 1042.262 | 6.284 | - |
Example 3 identification and genetic relationship analysis of clones of different Dendrobium chrysotoxum
1. Because the approved widely cultivated dendrobium chrysotoxum variety does not exist in the current market, the dendrobium chrysotoxum is produced mainly by sowing propagation (seedling), division or tissue culture for breeding seedlings. The application obtains 12 excellent individual plants of dendrobium chrysotoxum through screening, 12 clones of dendrobium chrysotoxum are constructed by means of plant division and tissue culture, the clones are respectively numbered as Line 1-Line 12, and the main morphological characteristics of the clones are shown in table 8. Taking the 12 clones as test materials, cutting young leaves of each clone, extracting dendrobium chrysotoxum genome DNA by using a DP305 kit (Chinese Tiangen), and detecting the quality and the concentration of the DNA by respectively adopting 1.2% agarose gel electrophoresis and a NanoDrop spectrophotometer.
2. PCR amplification was performed using the 25 pairs of SSR primers selected in example 1 and their annealing temperatures.
3. And detecting the amplification product by using an ABI 3730xl DNA Analyzer gene Analyzer, reading the result by using GeneMarker 2.2.0 software, and counting the sizes of characteristic bands and polymorphic bands amplified by each pair of primers.
4. Clustering analysis was performed using NTSYS pc Version 2.10e software, and the results are shown in fig. 3. As can be seen from FIG. 3, 25 pairs of SSR primers can completely identify and distinguish the 12 clones of Dendrobium chrysotoxum to be tested. At a relative distance of 0.763, 12 tested dendrobium chrysotoxum clones can be divided into 4 groups, clone lines 1, 4, 5, 7, 6, 8, 9 are grouped into one group, clone lines 2, 11, 12 are grouped into one group, clone Line 3 is one group, clone Line 10 is one group, which also indicates that the relationship between clone lines 1 and 4 is the closest, and the relationship between clone lines 3 and 10 and the other 10 clones is further.
TABLE 8.12 statistics of major morphological characteristics of Dendrobium chrysotoxum clones
Experimental example 1 accuracy and repeatability test
According to the SSR molecular marker primer composition, 50 samples of 8 dendrobium chrysotoxum populations in example 2 are selected for genetic diversity analysis, the genetic diversity analysis is compared with the genetic diversity result in example 2, the consistency and the accuracy of the genetic diversity analysis are detected, and the detection results are shown in the following table 9.
TABLE 9 accuracy test results
From table 9, it can be seen that the SSR molecular marker primer composition of the present invention can accurately identify individual plants in dendrobium chrysotoxum populations with an accuracy and a repeatability of 100%.
The SSR molecular marker disclosed by the invention can be used for assisting in selective breeding, so that the breeding process of dendrobium chrysotoxum is accelerated.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
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<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 43
ctctacctca ccgtgttccg 20
<210> 44
<211> 20
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 44
ctcttgccat tgctctgctt 20
<210> 45
<211> 20
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 45
atgacgccat gtaccactcc 20
<210> 46
<211> 20
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 46
acaacgattc gcaccagttc 20
<210> 47
<211> 21
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 47
ctttctacca ccaggttgtc a 21
<210> 48
<211> 20
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 48
ccataaagtc ggtccatagc 20
<210> 49
<211> 20
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 49
tttgttttcc actatttctg 20
<210> 50
<211> 20
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 50
tctaacatta caagctctgc 20
Claims (7)
1. A dendrobium chrysotoxum SSR molecular marker primer composition is characterized in that:
the primer composition comprises:
2. a dendrobium chrysotoxum germplasm identification, genetic diversity or genetic relationship analysis product is characterized in that: the product comprises the primer composition of claim 1.
3. The product of claim 2, wherein: the product is an independent reagent or a kit.
4. The application of the primer composition of claim 1 in preparation of dendrobium chrysotoxum germplasm identification, genetic diversity or genetic relationship analysis products.
5. Use of the primer composition of claim 1 or the product of claim 2 in germplasm identification, genetic diversity or genetic relationship analysis of dendrobium chrysotoxum.
6. A dendrobium chrysotoxum germplasm identification and genetic diversity or genetic relationship analysis method is characterized by comprising the following steps: the method comprises the following steps:
(1) extracting the genome DNA of the dendrobium chrysotoxum sample to be detected;
(2) performing PCR amplification by taking the genomic DNA extracted in the step (1) as a template, and modifying an upstream primer of a primer pair for PCR amplification by adopting a bioluminescent group;
(3) performing capillary fluorescence electrophoresis detection on the PCR amplification product obtained in the step (2), and collecting data;
(4) analyzing the genetic diversity or clustering and polymorphic information content of the dendrobium chrysotoxum population according to the data obtained in the step (3);
the PCR amplification system in the step (2) is as follows: the total volume of the reaction system is 10 muL, and the reaction system comprises 1.0 muL of DNA template, 1.0 muL of 10 XBuffer I Buffer solution, 0.1 muL of L TAKARA HS Taq enzyme, 0.6 muL of primer, 0.8 muL of 2.5mM dNTP, and deionized water to make up to 10 muL;
the primer of the PCR amplification system in the step (2) is the primer composition shown in SEQ ID NO. 1-SEQ ID NO. 50 in the claim 1;
the PCR amplification reaction program in the step (2): pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 72 ℃ for 30s, and extension at 72 ℃ for 40 cycles; extension at 60 ℃ for 10 min.
7. The primer composition of claim 1 or the product of claim 2, for use in dendrobium chrysotoxum molecular marker-assisted breeding.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106119409A (en) * | 2016-09-23 | 2016-11-16 | 皖西学院 | A kind of Chloroplast Simple primer method to identifying Herba Dendrobii |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106119409A (en) * | 2016-09-23 | 2016-11-16 | 皖西学院 | A kind of Chloroplast Simple primer method to identifying Herba Dendrobii |
Non-Patent Citations (2)
| Title |
|---|
| 《Identification of the Geographic Origin of Dendrobium thyrsiflorum on Chinese Herbal Medicine Market Using Trinucleotide Microsatellite Markers》;Yuan Ying Hui等;《BIOLOGICAL & PHARMACEUTICAL BULLETIN》;20111231;第34卷(第12期);第1794-1800页 * |
| 《基于SSR的8个平欧杂交榛品种指纹图谱构建》;王泽亮等;《四川林业科技》;20190430;第40卷(第2期);第5-8页 * |
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