CN111549170B - Daylily microsatellite marker primer group and application thereof - Google Patents
Daylily microsatellite marker primer group and application thereof Download PDFInfo
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Abstract
The invention provides a day lily microsatellite marked primer group, which comprises primers shown in SEQ ID NO. 1-40. The invention develops the day lily SSR core primer group for the first time, and solves the problem of day lily SSR mark deletion. The 20 pairs of SSR core primers developed by the invention have the advantages of high polymorphism, good repeatability, stable marking, clear bands, easy interpretation and the like, can be applied to aspects of day lily variety identification, DNA fingerprint construction, genetic diversity analysis and evaluation and the like, provides a new tool for the excavation of day lily excellent genes, 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, relates to a day lily microsatellite marker primer group and application thereof, and in particular relates to an application of utilizing day lily transcriptome sequences to develop an SSR core primer group in the aspects of day lily variety identification, fingerprint construction and genetic diversity analysis and evaluation.
Background
Daylily is commonly called "day lily", and belongs to plants of the genus hemerocallis (Hemerocaliss l.) of the family liliaceae in plant taxonomy. Daylily is an important commercial crop. The flower is steamed and dried to be processed into dried vegetable, namely the golden dish or the daylily, which is a popular food at home and abroad, and has the effects of invigorating stomach, promoting urination, reducing swelling and the like; the root can make wine; the leaves can be used for paper making and weaving straw mats; the dried flower can be used as paper coal and fuel, but the fresh flower is not suitable for eating more, especially for anther, and contains various alkaloids, which can cause poisoning phenomena such as diarrhea. Along with the high nutritive value of day lily, the day lily market is continuously heated, the variety of germplasm resources of various local varieties is various, the names are mixed, and the market is frequently filled with the phenomenon. Therefore, the identification work of the daylily germplasm resources becomes very important.
The conventional identification method of the general plant mainly comprises character identification, microscopic identification and chemical characteristic identification, and is greatly influenced by the growth environment, the growing period and the processing technology of the original plant. In recent years, molecular identification methods have been widely developed in various crops. The molecular marker can be used for knowing genetic variation and germplasm characteristics of species, carrying out genotype identification and fingerprint analysis, estimating genetic distances among populations, inbred lines and breeding materials, positioning single genes and quantitative trait loci, identifying candidate gene sequences and assisting breeding selection, however, the application of the molecular marker on daylily is quite lag. SSR is used as one of molecular markers, and is widely applied to the aspects of pedigree analysis and evolution, genetic map construction, gene location cloning, variety identification and the like in the fields of botanic and agronomy, and has the advantages of high repeatability, abundant polymorphism, co-dominance, high reliability and the like.
Currently, the classification of the germplasm resources of day lily is classified by morphology, but this classification method is limited to the growing period of plants (identifiable only in the flowering period).
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a day lily microsatellite marker primer set and application thereof, SSR markers are developed based on day lily transcriptome sequences, and a core primer set is screened, so that the primer has the advantages of stable amplification, clear strips and rich polymorphism, and can be effectively used in the fields of day lily variety identification, DNA fingerprint construction, genetic diversity analysis and evaluation and the like.
In recent years, the applicant collects the day lily germplasm resources at home and abroad and establishes a day lily germplasm resource nursery, develops SSR molecular markers to classify the collected resources through a transcriptome sequencing technology, and summarizes to obtain a day lily germplasm resource identification core primer library and a set of corresponding identification systems. The technology can be used for daylily germplasm resource identification, DNA fingerprint spectrum and genetic diversity analysis and evaluation, provides scientific and technological support for daylily excellent gene excavation, molecular marker assisted breeding, variety identification and brand authentication, and further has important significance for standardization and standardization processes in daylily industrial application.
In order to achieve the above object, the present invention provides the following technical solutions:
the day lily microsatellite marked primer group comprises primers shown in SEQ ID NO. 1-40.
The invention provides a detection method of day lily microsatellite markers, which comprises the following steps:
(1) Extracting daylily genome DNA;
(2) Synthesizing a primer group shown in SEQ ID NO.1-40, and carrying out PCR amplification by using the genomic DNA extracted in the step (1) as a template and using the primer group to obtain an amplification product;
(3) And separating the amplified products and detecting polymorphism.
The extraction method of the daylily genome DNA, which adopts a CTAB method or a plant genome DNA extraction kit, and the quality of the obtained DNA meets the PCR amplification requirement, is suitable for the patent.
Preferably, 15. Mu.l of the PCR amplification system is: 2 XTaq Master Mix 7.5ul, DNase-Free Water 4.5 ul,Forward Primer 1 ul,Reverse Primer 1 ul, template DNA 1 ul.
Preferably, the PCR amplification procedure is: the thermal start is carried out for 3min at 95 ℃, denaturation is carried out for 45 s at 95 ℃, annealing is carried out for 30 s at 48-57 ℃, extension is carried out for 1min at 72 ℃,30 cycles are carried out, and the final extension is carried out for 5 min at 72 ℃.
The invention provides application of the daylily microsatellite marked primer group in daylily variety identification.
Preferably, 20 pairs of core primers are adopted for polyacrylamide gel electrophoresis detection, and according to the detection result, the number of inter-species difference sites 2 is different varieties, the number of inter-species difference sites=1 is approximate variety, and the number of inter-species difference sites=0 is the same variety.
The invention provides application of the daylily microsatellite marked primer group in the construction of daylily DNA fingerprint.
The invention provides application of the daylily microsatellite marked primer group in analysis and evaluation of daylily genetic diversity.
The beneficial effects of the invention are as follows: the invention develops the day lily SSR core primer group for the first time, and solves the problem of day lily SSR mark deletion. The 20 pairs of SSR core primers developed by the invention have the advantages of high polymorphism, good repeatability, stable marking, clear bands, easy interpretation and the like, can be applied to aspects of day lily variety identification, DNA fingerprint construction, genetic diversity analysis and evaluation and the like, provides a new tool for the excavation of day lily excellent genes, molecular marker assisted breeding and variety identification, and has good application prospect.
Drawings
The accompanying drawings are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate the invention and together with the embodiments of the invention, serve to explain the invention. In the drawings:
FIG. 1 is a silver-stained PAGE of SSR primer HHC32 amplified 43 daylily germplasm DNA samples;
FIG. 2 is a silver-stained PAGE of SSR primer HHC35 amplified 43 daylily germplasm DNA samples;
FIG. 3 is a silver-stained PAGE of SSR primer HHC36 amplified 43 daylily germplasm DNA samples;
FIG. 4 is a silver-stained PAGE of SSR primer HHC40 amplified 43 daylily germplasm DNA samples;
FIG. 5 is a silver-stained PAGE of SSR primer HHC42 amplified 43 daylily germplasm DNA samples;
FIG. 6 is a silver-stained PAGE of SSR primer HHC43 amplified 43 daylily germplasm DNA samples;
FIG. 7 is a silver-stained PAGE of SSR primer HHC45 amplified 43 daylily germplasm DNA samples;
FIG. 8 is a silver-stained PAGE of SSR primer HHC52 amplified 43 daylily germplasm DNA samples;
FIG. 9 is a silver-stained PAGE of SSR primer HHC53 amplified 43 daylily germplasm DNA samples;
FIG. 10 is a silver-stained PAGE of SSR primer HHC65 amplified 43 daylily germplasm DNA samples;
FIG. 11 is a silver-stained PAGE of SSR primer HHC72 amplified 43 daylily germplasm DNA samples;
FIG. 12 is a silver-stained PAGE of SSR primer HHC78 amplified 43 daylily germplasm DNA samples;
FIG. 13 is a silver-stained PAGE of SSR primer HHC80 amplified 43 daylily germplasm DNA samples;
FIG. 14 is a silver stain PAGE of SSR primer HHC90 amplified 43 day lily germplasm DNA samples;
FIG. 15 is a silver-stained PAGE of SSR primer HHC93 for amplifying 43 daylily germplasm DNA samples;
FIG. 16 is a silver-stained PAGE of SSR primer HHC95 amplified 43 day lily germplasm DNA samples;
FIG. 17 is a silver-stained PAGE of SSR primer HHC99 amplified 43 daylily germplasm DNA samples;
FIG. 18 is a silver-stained PAGE of SSR primer HHC101 amplified 43 daylily germplasm DNA samples;
FIG. 19 is a silver stain PAGE of SSR primer HHC106 amplified 43 day lily germplasm DNA samples;
FIG. 20 is a silver-stained PAGE of SSR primer HHC109 amplified 43 daylily germplasm DNA samples;
FIG. 21 is a cluster map of 43 daylily germplasm obtained by the result of SSR primer amplification.
Detailed Description
The following examples facilitate a better understanding of the present invention, but are not intended to limit the same. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the examples described below, unless otherwise specified, were purchased from conventional Biochemical reagents. The quantitative tests in the following examples were all set up in triplicate and the results averaged.
Example 1
1. Test material
35 parts of representative day lily varieties in different areas and 8 parts of day lily varieties with unknown variety names or sources are selected, 43 parts are added, and the names and the collection places are shown in table 1.
TABLE 1 day lily variety information 43 parts
2. Sample DNA extraction
Collecting fresh and tender day lily leaves, taking samples, carrying back after the samples are quickly dried by silica gel particles, taking about 50mg of each sample, extracting sample DNA by using a plant extraction genome kit of a day root after grinding by a ball mill, detecting the quality of the samples by using 1% agarose gel electrophoresis after the extraction is finished, measuring the concentration and the purity of the samples by using NanoDrop, diluting the DNA to 20-50 ng/mu l, and preserving the DNA at 4 ℃ for later use.
3. SSR primer development, design and core primer screening
Transcriptome sequencing is performed on day lily by adopting a second-generation sequencing technology, and SSR site searching is performed on all transcripts obtained by sequencing by using MISA software. The search criteria were: the minimum length of SSR repetition is 18bp, and the minimum times of single nucleotide, dinucleotide, trinucleotide, tetranucleotide, pentanucleotide and hexanucleotide repetition are 12, 6, 5, 4 and 4 times, and SSR sites with incomplete repetition interval are not listed as search objects. Based on the characteristic of conservation at two ends of the SSR locus, primer 3.0 software is utilized to carry out batch Primer design, primer 11363 pairs are designed altogether, and SSR primers in Unigene related to amino acid metabolism and terpene polyketide metabolism are selected for synthesis according to gene annotation classification, wherein the total number of the SSR primers is 89. In addition, 25 pairs of universal SSR primers of hemerocallis are obtained from the literature by searching, and 114 pairs of primers are screened and identified.
The primer design principle is that the length of EST sequence is more than 200 bp, the starting position and the ending position of SSR sequence are respectively at least 20 bp from the 5 'end and the 3' end, the primer length is 18-24 bp, the annealing temperature Tm value is 40-60 ℃, the difference between the Tm values of the upstream primer and the downstream primer is not more than 5 ℃, the GC content is 40-60%, the GC content of the upstream primer and the downstream primer is not too great, 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 a reference, the upper and lower temperature are respectively floated by 5 ℃, 8 temperature gradients are automatically generated by a gradient PCR instrument, and the optimal temperature of the amplified primers with more bands and clear background is selected.
The primers 114 are initially screened by agarose electrophoresis, 70 pairs of primers with clear amplified bands are screened, then 6% non-denaturing polyacrylamide gel electrophoresis is used for polymorphic primer screening, 33 pairs of primers with high polymorphism and stable amplification are screened, and 20 pairs of primers with high polymorphism and clear bands among varieties are screened as core primers for daylily germplasm identification by using 43 daylily germplasm materials collected (Table 2).
TABLE 2 SSR core primer information for 20 pairs
4. PCR amplification and product detection
The total reaction volume of the PCR amplification system was 15. Mu.l, including 2×Taq Master Mix 7.5. Mu.l, DNase-Free Water 4.5. Mu.l, forward Primer 1. Mu.l, reverse Primer 1. Mu.l, and template DNA 1. Mu.l. The reaction procedure: the thermal start is carried out for 3min at 95 ℃, denaturation is carried out for 45 s at 95 ℃, annealing is carried out for 30 s at 48-57 ℃, extension is carried out for 1min at 72 ℃,30 cycles are carried out, and the final extension is carried out for 5 min at 72 ℃. The PCR products were detected on a 1% agarose gel, and the amplified products were separated by 6% non-denaturing polyacrylamide gel electrophoresis, and polymorphism detection was performed by silver staining.
5. Analysis of results
The method of manually reading the bands is adopted, the repeatable and easily-resolved bands on the electrophoresis chart are marked as '1', the same position of the bands is not marked as '0', and an original data matrix is established. And calculating genetic similarity coefficients among varieties by using NTSYS software, performing cluster analysis by using a UPGMA method, and drawing a dendrogram. The result shows that the genetic distance between 43 daylily germplasm materials is 0-0.8138, the genetic similarity is 0.4432-1 according to NTSYS software genemic similiarity, and the clustering chart shows that 5 groups of daylily germplasm materials are shown (figure 21)-/>) The similarity between each group of materials is 1, and the groups cannot be distinguished, and the materials are respectively: />The group is canal county flowers, shanxi big flowers and unknown flowers2 and unknown 4; />The composition comprises rhizoma Dioscoreae flower, semen astragali Complanati needle and Zhejiang Xianju flower; />Group Qi Zhen flowers and unknown Huang Huaxing; />The group is fiveleaf gynostemma herb, sun-washing collecting germplasm and golden bud No. 1; />The group is Chongqing safflower and Longyuan safflower. The remaining 29 germplasm can be distinguished by removing the 14 germplasm resources, and the 5 groups of materials can be effectively distinguished. According to the detection result, the number of inter-variety difference sites 2 is different varieties, the number of inter-variety difference sites=1 is similar varieties, and the number of inter-variety difference sites=0 is the same varieties.
Finally, it should be noted that: the foregoing description is only a preferred embodiment of the present invention, and the present invention is not limited thereto, but it is to be understood that modifications and equivalents of some of the technical features described in the foregoing embodiments may be made by those skilled in the art, although the present invention has been described in detail with reference to the foregoing embodiments. Any modification, equivalent replacement, improvement, etc. 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 (9)
1. Application of daylily microsatellite marker primer group in daylily variety identification; performing PCR (polymerase chain reaction) amplification on the daylily genome DNA by adopting 20 pairs of core primers to obtain an amplification product; then, performing polyacrylamide gel electrophoresis detection on the amplified product, wherein the number of inter-species difference sites is more than or equal to 2 and is different from the detection result, the number of inter-species difference sites=1 and is similar to the variety, and the number of inter-species difference sites=0 and is the same variety; the nucleotide sequence of the day lily microsatellite marker primer group is shown in SEQ ID NO. 1-40.
2. The use according to claim 1, characterized in that: 15 μl of the PCR amplification system was: 2 XTaq Master Mix 7.5ul, DNase-Free Water 4.5 ul,Forward Primer 1 ul,Reverse Primer 1 ul, template DNA 1 ul.
3. The use according to claim 1, characterized in that: the PCR amplification procedure was: the thermal start is carried out for 3min at 95 ℃, denaturation is carried out for 45 s at 95 ℃, annealing is carried out for 30 s at 48-57 ℃, extension is carried out for 1min at 72 ℃,30 cycles are carried out, and the final extension is carried out for 5 min at 72 ℃.
4. Application of daylily microsatellite marked primer group in construction of daylily DNA fingerprint; performing PCR (polymerase chain reaction) amplification on the daylily genome DNA by adopting 20 pairs of core primers to obtain an amplification product; then, performing polyacrylamide gel electrophoresis detection on the amplified product, wherein the number of inter-species difference sites is more than or equal to 2 and is different from the detection result, the number of inter-species difference sites=1 and is similar to the variety, and the number of inter-species difference sites=0 and is the same variety; the nucleotide sequence of the day lily microsatellite marker primer group is shown in SEQ ID NO. 1-40.
5. The use according to claim 4, characterized in that: 15 μl of the PCR amplification system was: 2 XTaq Master Mix 7.5ul, DNase-Free Water 4.5 ul,Forward Primer 1 ul,Reverse Primer 1 ul, template DNA 1 ul.
6. The use according to claim 4, characterized in that: the PCR amplification procedure was: the thermal start is carried out for 3min at 95 ℃, denaturation is carried out for 45 s at 95 ℃, annealing is carried out for 30 s at 48-57 ℃, extension is carried out for 1min at 72 ℃,30 cycles are carried out, and the final extension is carried out for 5 min at 72 ℃.
7. The application of the daylily microsatellite marked primer group in the aspect of analysis and evaluation of genetic diversity of daylily; performing PCR (polymerase chain reaction) amplification on the daylily genome DNA by adopting 20 pairs of core primers to obtain an amplification product; then, performing polyacrylamide gel electrophoresis detection on the amplified product, wherein the number of inter-species difference sites is more than or equal to 2 and is different from the detection result, the number of inter-species difference sites=1 and is similar to the variety, and the number of inter-species difference sites=0 and is the same variety; the nucleotide sequence of the day lily microsatellite marker primer group is shown in SEQ ID NO. 1-40.
8. The use according to claim 7, characterized in that: 15 μl of the PCR amplification system was: 2 XTaq Master Mix 7.5ul, DNase-Free Water 4.5 ul,Forward Primer 1 ul,Reverse Primer 1 ul, template DNA 1 ul.
9. The use according to claim 7, characterized in that: the PCR amplification procedure was: the thermal start is carried out for 3min at 95 ℃, denaturation is carried out for 45 s at 95 ℃, annealing is carried out for 30 s at 48-57 ℃, extension is carried out for 1min at 72 ℃,30 cycles are carried out, and the final extension is carried out for 5 min at 72 ℃.
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|---|---|---|---|
| CN202010518531.5A CN111549170B (en) | 2020-06-09 | 2020-06-09 | Daylily microsatellite marker primer group and application thereof |
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| 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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| 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.《International Conference on Germplasm of Ornamentals》.2013,第299-306页. * |
| 刘新星等.食用黄花菜 SSR 标记开发及指纹图谱库构建.《食品与发酵工业》.2021,第48卷(第12期),第188-195页. * |
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