CN111549170A - Day lily microsatellite marker primer set and application thereof - Google Patents
Day lily microsatellite marker primer set and application thereof Download PDFInfo
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Abstract
The invention provides a day lily microsatellite marker primer set which comprises primers shown in SEQ ID NO. 1-40. The invention develops the daylily SSR core primer group for the first time, and solves the problem of daylily SSR marker deletion. The 20 pairs of SSR core primers developed by the invention have the advantages of high polymorphism, good repeatability, stable marking, clear band pattern, easy interpretation and the like, can be applied to the aspects of day lily variety identification, DNA fingerprint map construction, genetic diversity analysis and evaluation and the like, provides a new tool for the excavation of excellent genes of the day lily, molecular marker assisted breeding and variety identification, and has good application prospect.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and relates to a day lily microsatellite marker primer set and application thereof, in particular to an SSR core primer set developed by utilizing a day lily transcriptome sequence and application thereof in the aspects of day lily variety identification, fingerprint map construction and genetic diversity analysis and evaluation.
Background
Daylily is commonly called "golden lily" and belongs to the genus hemerocallis (hemerocallis L.) of the family Liliaceae in plant taxonomy. Day lily is an important economic crop. Its flower is steamed, dried in the sun, processed into dried vegetable, i.e. day lily or day lily, sold at home and abroad, is a very popular food, and has the efficacies of invigorating stomach, promoting urination, reducing swelling and the like; the root can be used for brewing wine; the leaves can be made into paper and woven straw mats; the dried scape can be used as paper coal and fuel, but the fresh flower is not suitable for eating too much, especially anther contains various alkaloids, which can cause poisoning phenomena such as diarrhea. With the high nutritive value of the day lily receiving wide attention of people, the day lily market is continuously heated, the variety resources of each local variety are various, the names are mixed, and the phenomenon of being good in the market is more. Therefore, the identification work of the daylily germplasm resources becomes very important.
The traditional identification method of general plants mainly comprises character identification, microscopic identification and chemical characteristic identification, and is greatly influenced by the growth environment, the growth period and the processing technology of the original plants. In recent years, molecular identification methods have been widely developed in various crops. The molecular marker can be used for understanding the genetic variation and germplasm characteristics of species, carrying out genotype identification and fingerprint analysis, estimating the genetic distance among groups, self-bred lines and breeding materials, positioning single gene and quantitative character sites, identifying candidate gene sequences and assisting breeding selection, but the application of the molecular marker on the daylily is quite lagged. The SSR is used as one of molecular markers, is widely applied to aspects such as pedigree analysis and evolution, genetic map construction, gene location cloning, variety identification and the like in the fields of botany and agronomy, and has the advantages of high repeatability, rich polymorphism, codominance, high reliability and the like.
At present, the day lily germplasm resource classification is mostly classified by morphology, but the classification method is limited by the growth period of plants (can be identified only in the flowering period).
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a daylily microsatellite marker primer group and application thereof, wherein SSR markers are developed based on a daylily transcriptome sequence, and a core primer group is screened, and the primer has the advantages of stable amplification, clear bands and rich polymorphism, and can be effectively used in the fields of daylily variety identification, DNA fingerprint map construction, genetic diversity analysis and evaluation and the like.
In recent years, the applicant collects day lily germplasm resources at home and abroad, establishes a day lily germplasm resource garden, develops SSR molecular markers by a transcriptome sequencing technology to classify the collected resources, and summarizes the collected resources to obtain a day lily germplasm resource identification core primer library and a set of corresponding identification systems. The technology can be used for identifying the germplasm resources of the day lily, analyzing and evaluating a DNA fingerprint spectrum and genetic diversity, provides scientific and technological support for the excavation of excellent genes of the day lily, molecular marker-assisted breeding, variety identification and brand authentication, and further has important significance for the standardization and standardization process in the application of the day lily industry.
In order to achieve the purpose, the invention provides the following technical scheme:
the day lily microsatellite marker primer group comprises primers shown as SEQ ID NO. 1-40.
The invention provides a method for detecting a day lily microsatellite marker, which comprises the following steps:
(1) extracting genome DNA of the daylily;
(2) synthesizing a primer group shown in SEQ ID NO.1-40, taking the genomic DNA extracted in the step (1) as a template, and performing PCR amplification by using the primer group to obtain an amplification product;
(3) and (4) separating and detecting polymorphism of the amplification product.
The extraction of the daylily genome DNA adopts a CTAB method or a plant genome DNA extraction kit, and the extraction methods of which the quality of the obtained DNA meets the PCR amplification requirement are all suitable for the application.
Preferably, 15 μ l of the PCR amplification system is: 2 XTaq Master Mix 7.5ul, DNase-FreeWater 4.5 ul, Forward Primer 1ul, Reverse Primer 1ul, template DNA 1 ul.
Preferably, the PCR amplification procedure is: hot starting at 95 ℃ for 3min, denaturation at 95 ℃ for 45 s, annealing at 48-57 ℃ for 30 s, extension at 72 ℃ for 1min, 30 cycles, and final extension at 72 ℃ for 5 min.
The invention provides application of the day lily microsatellite marker primer group in day lily variety identification.
Preferably, 20 pairs of core primers are used for polyacrylamide gel electrophoresis detection, and according to the detection result, the number of interspecific differences = 1 is different from the number of interspecific differences "< 2 >, similar to the number of interspecific differences = 0, and the same species.
The invention provides application of the day lily microsatellite marker primer group in the construction of a day lily DNA fingerprint.
The invention provides application of the day lily microsatellite marker primer group in the aspects of analysis and evaluation of day lily genetic diversity.
The invention has the beneficial effects that: the invention develops the daylily SSR core primer group for the first time, and solves the problem of daylily SSR marker deletion. The 20 pairs of SSR core primers developed by the invention have the advantages of high polymorphism, good repeatability, stable marking, clear band pattern, easy interpretation and the like, can be applied to the aspects of day lily variety identification, DNA fingerprint map construction, genetic diversity analysis and evaluation and the like, provides a new tool for the excavation of excellent genes of the day lily, molecular marker assisted breeding and variety identification, and has good application prospect.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:
FIG. 1 is a silver-stained PAGE picture of 43 parts of daylily germplasm DNA samples amplified by SSR primer HHC 32;
FIG. 2 is a silver-stained PAGE picture of 43 parts of daylily germplasm DNA samples amplified by SSR primer HHC 35;
FIG. 3 is a silver-stained PAGE picture of 43 parts of daylily germplasm DNA samples amplified by SSR primer HHC 36;
FIG. 4 is a silver-stained PAGE picture of 43 parts of daylily germplasm DNA samples amplified by SSR primer HHC 40;
FIG. 5 is a silver-stained PAGE picture of 43 parts of daylily germplasm DNA samples amplified by SSR primer HHC 42;
FIG. 6 is a silver-stained PAGE picture of 43 parts of daylily germplasm DNA samples amplified by SSR primer HHC 43;
FIG. 7 is a silver-stained PAGE picture of 43 parts of daylily germplasm DNA samples amplified by SSR primer HHC 45;
FIG. 8 is a silver-stained PAGE picture of 43 parts of daylily germplasm DNA samples amplified by SSR primer HHC 52;
FIG. 9 is a silver-stained PAGE picture of 43 parts of daylily germplasm DNA samples amplified by SSR primer HHC 53;
FIG. 10 is a silver-stained PAGE picture of 43 parts of daylily germplasm DNA samples amplified by SSR primer HHC 65;
FIG. 11 is a silver-stained PAGE picture of 43 parts of daylily germplasm DNA samples amplified by SSR primer HHC 72;
FIG. 12 is a silver-stained PAGE picture of 43 parts of daylily germplasm DNA samples amplified by SSR primer HHC 78;
FIG. 13 is a silver-stained PAGE picture of 43 parts of daylily germplasm DNA samples amplified by SSR primer HHC 80;
FIG. 14 is a silver-stained PAGE picture of 43 parts of daylily germplasm DNA samples amplified by SSR primer HHC 90;
FIG. 15 is a silver-stained PAGE picture of 43 parts of daylily germplasm DNA samples amplified by SSR primer HHC 93;
FIG. 16 is a silver-stained PAGE picture of 43 parts of daylily germplasm DNA samples amplified by SSR primer HHC 95;
FIG. 17 is a silver-stained PAGE picture of 43 parts of daylily germplasm DNA samples amplified by SSR primer HHC 99;
FIG. 18 is a silver-stained PAGE picture of 43 parts of daylily germplasm DNA samples amplified by SSR primer HHC 101;
FIG. 19 is a silver-stained PAGE picture of 43 parts of daylily germplasm DNA samples amplified by SSR primer HHC 106;
FIG. 20 is a silver-stained PAGE picture of 43 parts of daylily germplasm DNA samples amplified by SSR primer HHC 109;
FIG. 21 is a diagram of 43 parts of daylily germplasm clusters obtained from the SSR primer amplification result.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from conventional biochemicals, unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.
Example 1
1. Test material
35 parts of representative daylily varieties in different regions and 8 parts of daylily germplasm with unknown variety names or sources are selected, the total number is 43 parts, and the names and the collection places are shown in table 1.
TABLE 143 parts daylily variety information
2. Sample DNA extraction
Collecting fresh and tender day lily leaves, quickly drying the samples by silica gel particles and then taking back, taking about 50mg of each sample, grinding by a ball mill, extracting sample DNA by using a plant extraction genome kit of Tiangen, detecting the sample quality by using 1% agarose gel electrophoresis after extraction is finished, measuring the concentration and the purity of the sample by using NanoDrop, diluting the DNA to 20-50 ng/mu l, and storing the sample at 4 ℃ for later use.
3. SSR primer development, design and core primer screening
And performing transcriptome sequencing on the daylily by adopting a second-generation sequencing technology, and performing SSR locus search on all sequenced transcripts by using MISA software. The search criteria were: the minimum length of the SSR repeat is 18bp, the minimum times of the repeat of mononucleotide, dinucleotide, trinucleotide, tetranucleotide, pentanucleotide and hexanucleotide are 12, 6, 5, 4 and 4, and SSR sites with incompletely repeated intervals are not listed as search objects. And (2) designing batch primers by using Primer 3.0 software in combination with the characteristic of conservation at two ends of SSR sites, designing 11363 pairs in total, and selecting SSR primers in Unigene related to amino acid metabolism and terpenoid polyketide metabolism according to gene annotation classification to synthesize 89 pairs in total. In addition, 25 pairs of SSR primers commonly used by hemerocallis are searched from the literature, and the total number of the SSR primers is 114 for screening and identification.
The primer design principle is that the EST sequence length is more than 200 bp, the start and end positions of the SSR sequence are respectively not less than 20 bp from the 5 'end to the 3' end, the primer length is 18-24 bp, the annealing temperature Tm value is 40-60 ℃, the Tm value difference of the upstream primer and the downstream primer is not more than 5 ℃, the GC content is 40-60%, the GC content difference of the upstream primer and the downstream primer is not too large, and the expected length of the product is 100-500 bp. The optimal annealing temperature of each primer is tested, the melting temperature (Tm value) of each primer is taken as reference, the upper temperature and the lower temperature float by 5 ℃, 8 temperature gradients are automatically generated by a gradient PCR instrument, and the optimal temperature is selected when the amplified band is more, the amplified band is bright and the background is clear.
The method comprises the steps of preliminarily screening 114 pairs of primers through agarose electrophoresis, screening 70 pairs of primers with clear amplified bands, screening 33 pairs of primers with high polymorphism and stable amplification through 6% non-denaturing polyacrylamide gel electrophoresis, and further screening 20 pairs of primers with high polymorphism and clear bands among varieties and types as core primers for identifying the daylily germplasm (Table 2) by using 43 collected daylily germplasm materials.
TABLE 220 SSR core primer information
4. PCR amplification and product detection
Wherein the total reaction volume of the PCR amplification system is 15 mul, and comprises 2 XTaq Master Mix 7.5 mul, DNase-FreeWater 4.5 mul, Forward Primer 1 mul, Reverse Primer 1 mul and template DNA 1 mul. Reaction procedure: hot starting at 95 ℃ for 3min, denaturation at 95 ℃ for 45 s, annealing at 48-57 ℃ for 30 s, extension at 72 ℃ for 1min, 30 cycles, and final extension at 72 ℃ for 5 min. The PCR product was detected on 1% agarose gel, and the amplified product was separated by 6% native polyacrylamide gel electrophoresis, followed by polymorphism detection by silver staining.
5. Analysis of results
And (3) adopting an artificial tape reading method, recording repeatable and easily-resolved bands on the electrophoretogram as '1', recording no band at the same position as '0', and establishing an original data matrix. And (3) calculating the genetic similarity coefficient among the varieties by using NTSYS software, carrying out cluster analysis by using a UPGMA method, and drawing a dendrogram. The results show that the genetic distance between 43 parts of daylily germplasm material is calculated according to the NTSYS software genetic similarityThe range of variation from 0 to 0.8138 and the range of variation of genetic similarity from 0.4432-1, as can be seen from the cluster map (FIG. 21), among which there are 5 groups (
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) The similarity between each group of materials is 1, and the materials cannot be distinguished among groups, and the similarity is as follows:
the groups are ditch county flower, Shanxi Daibian flower, unknown 2 and unknown 4;
comprises flos Lonicerae, radix astragali Complanati, and flos Curculiginis;
the composition comprises QIZHEN flower and unknown daylily;
the group comprises Hippocampus japonicas, Muyang collected germplasm and golden bud No. 1;
the composition comprises Chongqing Carthami flos and Longyou Carthami flos. In addition to the above 14 germplasm resources, the remaining 29 germplasm pools were distinguishable, and the above 5 material sets were still effectively distinguishable. According to the detection results, 2 indicates different varieties, = 1 indicates similar varieties, and = 0 indicates the same varieties.
Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
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Claims (8)
1. The day lily microsatellite marker primer group is characterized in that: comprises primers shown in SEQ ID NO. 1-40.
2. A method for detecting a day lily microsatellite marker is characterized by comprising the following steps: the method comprises the following steps:
(1) extracting genome DNA of the daylily;
(2) synthesizing a primer group shown in SEQ ID NO.1-40, taking the genomic DNA extracted in the step (1) as a template, and performing PCR amplification by using the primer group to obtain an amplification product;
(3) and (4) separating and detecting polymorphism of the amplification product.
3. The detection method according to claim 2, characterized in that: 15 μ l of the PCR amplification system was: 2 XTaqMaster Mix 7.5ul, DNase-Free Water 4.5 ul, Forward Primer 1ul, Reverse Primer 1ul, template DNA 1 ul.
4. The detection method according to claim 2, characterized in that: the PCR amplification procedure was: hot starting at 95 ℃ for 3min, denaturation at 95 ℃ for 45 s, annealing at 48-57 ℃ for 30 s, extension at 72 ℃ for 1min, 30 cycles, and final extension at 72 ℃ for 5 min.
5. The use of a day lily microsatellite marker primer set according to claim 1 in the identification of day lily varieties.
6. Use according to claim 5, characterized in that: and (3) performing polyacrylamide gel electrophoresis detection by using 20 pairs of core primers, wherein according to a detection result, the number of the interspecific difference ectopic spots of the variety 2 is different varieties, the number of the interspecific difference ectopic spots = 1 is an approximate variety, and the number of the interspecific difference ectopic spots = 0 is the same variety.
7. The application of the daylily microsatellite marker primer set in the aspect of construction of the daylily DNA fingerprint spectrum of claim 1.
8. The use of the microsatellite marker primer set of daylily of claim 1 for the analysis and evaluation of the genetic diversity of daylily.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116083629A (en) * | 2022-12-30 | 2023-05-09 | 江苏省农业科学院宿迁农科所 | SSR molecular marker primer combination for identifying hemerocallis species and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106011251A (en) * | 2016-06-07 | 2016-10-12 | 山西农业大学 | Primer group, kit and method for identifying population genetic structure of Hemerocallis L. plants |
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2020
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106011251A (en) * | 2016-06-07 | 2016-10-12 | 山西农业大学 | Primer group, kit and method for identifying population genetic structure of Hemerocallis L. plants |
Non-Patent Citations (2)
| Title |
|---|
| D.M. CAO等: "Genetic Diversity of Wild Daylily in Taihang Mountain Areas Based on ISSR Markers" * |
| 刘新星等: "食用黄花菜 SSR 标记开发及指纹图谱库构建" * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116083629A (en) * | 2022-12-30 | 2023-05-09 | 江苏省农业科学院宿迁农科所 | SSR molecular marker primer combination for identifying hemerocallis species and application thereof |
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| CN111549170B (en) | 2023-07-25 |
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