CN113736899A - Group of SSR molecular markers for identifying hemp varieties and application thereof - Google Patents
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Abstract
The invention provides a group of SSR molecular markers for identifying hemp varieties and application thereof. The invention also provides a set of primer combination which is suitable for a capillary electrophoresis platform and is used for detecting the SSR molecular markers, wherein the SSR molecular markers are 11 SSR molecular markers in total. The 11 SSR molecular markers can be used: identifying hemp varieties and analyzing genetic diversity analysis; the varieties of hemp Guglisia, hemp I, hemp 5, hemp 7, hemp 1 and YOUYA-31 are distinguished. By applying the hemp SSR molecular marker disclosed by the invention, the range of available marker sites of hemp can be expanded on the genome level; provides a new tool for the research of hemp variety and germplasm resource identification, genetic relationship evaluation, cytoplasm genetic characteristics and the like, and has good application prospect.
Description
Technical Field
The invention relates to the technical field of crop molecular breeding and identification, in particular to a group of SSR molecular markers for identifying hemp varieties and application thereof.
Background
Cannabis sativa L (Cannabis sativa L.) belonging to Urticales order Cannabis genus annual herbaceous plant of Cannabis genus of Cannabaceae family. The next three varieties are industrial, Indian and wild hemp, respectively. The industrial hemp is the most common type, and refers to the hemp variety with the tetrahydrocannabinol content of less than 0.3 percent, and has high economic utilization value. Because the hemp seed oil contains balanced omega-3 and omega-6 unsaturated fatty acid, the hemp seed oil is mostly applied to food, cosmetics and medicines; cannabidiol (CBD) of cannabis, which is a terpene phenolic compound, has a rich pharmacological action; the hemp fiber and the stalk core are excellent raw materials in the aspects of building materials, special military supplies, infant underwear, textiles and the like, and the hemp fiber has the advantages of radiation protection, antibiosis, good elasticity and the like, has wide market prospect and great economic benefit.
With the development of industrial hemp industry, a plurality of scientific research institutions breed a plurality of industrial hemp varieties according to the industrial development requirements. The germplasm resources of industrial hemp are richer, and the phenomena of foreign matters with the same name, synonyms with the same name and fish dragon mixing are easy to appear in the industrial hemp seed market. The industrial hemp seeds are easy to mix in the varieties and have low purity because the varieties of the industrial hemp are easy to accept foreign pollen in the cultivation process, and the quality of the industrial hemp seeds and the cultivation of new varieties are seriously influenced. Cannabis eucalyptus-31 is a high fiber content variety introduced in China from Ukrainian. The hybrid varieties of the Grissia, the Cannabis sativa I5, the Cannabis sativa I7 and the Cannabis sativa I1 are bred and improved in China. The hemp I has the advantages of lodging resistance, drought resistance and salt and alkali resistance besides high fiber content, and is a main cultivated variety of the hemp in Heilongjiang province. While the Grissian is also a fiber hemp, the advantage is not obvious from the hemp I. The two are very similar in seed appearance and plant appearance and are difficult to distinguish, so that the identification of the variety of the Grissia and the Cannabis I is an essential prerequisite for downstream markets or before breeding. The same is true for Longmariana No.1 and YOUYAO-31. Longmarianna No.1 has strong resistance to gray mold and stalk rot, and also has the advantages of lodging resistance, drought resistance and salt and alkali resistance. However, it is similar to Yushu-31 in appearance of seed morphology, and confusion during harvesting or seed preservation can affect seed purity. The medicinal cannabis longa cannabis No.5 and cannabis hemp No.7 belong to the dominant varieties with high CBD content and low THC content, have the same variety characteristics, and have the difference that the CBD content is different from the THC content. The difference in content directs the direction of the downstream application to be different. In order to avoid the confusion of the two varieties with similar characteristics, accurate variety identification and differentiation are important prerequisites. The characteristics of each variety and the origin of the variety identification are shown in Table 1.
Simple Sequence Repeat (SSR), also known as microsatellite DNA (microspatellite DNA), generally refers to a DNA sequence that repeats in tandem multiple times in units of 2-4 nucleotides. SSR is a novel DNA fingerprint technology developed in recent years and established on the basis of PCR, has the advantages of strong reliability, high repeatability, rich polymorphism and the like, is the most applied technology in variety identification at present, and is widely applied to variety approval of corn, rice, wheat, soybean and the like, construction of a germplasm resource core germplasm bank and genetic diversity analysis of germplasm resources. At present, there is a report on the construction of the hemp SSR fingerprint. The PCR-based SSR technology is utilized to identify hemp varieties, analyze genetic diversity and construct a DNA fingerprint database, and has important significance for hemp variety management, variety breeding and germplasm resource collection and protection.
TABLE 1 Properties and origin of variety identification of each variety
Disclosure of Invention
The invention aims to provide a group of SSR molecular markers for identifying hemp varieties and application thereof.
In order to realize the purpose of the invention, the invention obtains a group of SSR molecular markers for identifying the varieties of the hemp by collecting hemp materials with wide sources, rich phenotype and genotype types and strong representativeness, sequencing and comparing hemp genomes of corresponding materials.
The SSR molecular markers for identifying the hemp varieties comprise one or more of the following 11 SSR molecular markers, wherein the 11 SSR molecular markers are respectively ANUCS304, CAN0031, 23 and 25, ANUCS201, B01CANN1, E07CANN1, ANUCS305 and 33, CAN0039 and CAN 0055.
The 11 SSR molecular markers are obtained by amplifying the following primers respectively and sequentially: 1-2 parts of SEQ ID NO.1, 3-4 parts of SEQ ID NO.5-6 parts of SEQ ID NO.7-8 parts of SEQ ID NO.9-10 parts of SEQ ID NO.11-12 parts of SEQ ID NO.13-14 parts of SEQ ID NO.15-16 parts of SEQ ID NO.17-18 parts of SEQ ID NO.19-20 parts of SEQ ID NO.21-22 parts of SEQ ID NO. 21.
The SSR molecular markers can be detected by adopting conventional technical means in the field, and meanwhile, the SSR molecular markers can be suitable for capillary electrophoresis detection technology. Quantitative DNA fragment analysis data can be obtained by the SSR molecular marker technology based on the capillary electrophoresis detection technology. Compared with the conventional polyacrylamide gel electrophoresis detection method, the method has more accurate, sensitive and efficient result, and is more suitable for detection and analysis of large-batch varieties.
When capillary electrophoresis detection is carried out, the SSR molecular markers can be divided into a plurality of different fluorescence marker groups, and the fluorescence markers in each group are the same.
As an embodiment of the present invention, the SSR molecular markers can be divided into four different fluorescence marker groups, where the fluorescence markers in each group are the same:
the first group consists of SSR molecular markers ANUCS304 and CAN 0031; the second group consists of SSR molecular markers 23, 25; the third group consists of SSR molecular markers ANUCS201 and B01CANN 1; the fourth group is composed of SSR molecular markers E07CANN1, ANUCS305, 33, CAN0039 and CAN 0055.
Further, the invention provides a specific primer pair for amplifying the SSR molecular marker.
Preferably, the specific primer pair comprises one or more of the following primer pairs: 1-2 parts of SEQ ID NO.1, 3-4 parts of SEQ ID NO.5-6 parts of SEQ ID NO.7-8 parts of SEQ ID NO.9-10 parts of SEQ ID NO.11-12 parts of SEQ ID NO.13-14 parts of SEQ ID NO.15-16 parts of SEQ ID NO.17-18 parts of SEQ ID NO.19-20 parts of SEQ ID NO.21-22 parts of SEQ ID NO. 21.
The invention also provides a kit which contains the specific primer pair for amplifying the SSR molecular marker.
The invention also provides a hemp genome chip which contains the hemp SSR molecular marker.
The invention provides the application of the SSR molecular marker, the specific primer, the kit and the hemp genome chip in constructing a hemp variety DNA fingerprint database.
The invention provides the application of the SSR molecular marker, the specific primer, the kit and the hemp genome chip in hemp germplasm resource genetic diversity analysis or seed quality detection.
The invention provides the application of the SSR molecular marker, the specific primer, the kit and the hemp genome chip in hemp variety identification, genetic relationship analysis and maternal traceability.
For the identification of hemp varieties, the invention provides, as an example, the use in distinguishing the varieties Griffonia and Cannabis I, Cannabis Longana 5 and Cannabis Hance 7, Cannabis Longana 1 and Euonymus alatus-31.
The invention provides a kit for distinguishing the divisions of Himalayan and Cannabis I, Cannabis 5 and Cannabis 7, Cannabis 1 and Euonymus alatus-31, which contains a specific primer combination aiming at the 11 hemp SSR molecular markers. Preferably, the nucleotide sequences of the specific primer combinations are respectively shown as SEQ ID NO. 1-22.
The invention provides the SSR molecular marker, the specific primer, the kit and the application of the hemp genome chip in hemp molecular marker-assisted breeding.
The invention also provides application of the hemp SSR molecular marker in preparation of a hemp genome chip.
The application specifically comprises the following steps:
1) extracting DNA of a hemp sample to be detected;
2) performing PCR amplification by using the DNA extracted in the step 1) as a template and utilizing the SSR molecular marker;
3) and detecting the PCR product by using a capillary electrophoresis system.
In step 2) of the above application, a reaction volume of 20. mu.L containing 10 to 40ng of sample DNA, 0.4. mu.M each of the forward primer and the reverse primer, and 10. mu.L of 2 Xpolymerase mixture was used for PCR amplification. The reaction procedure is as follows: pre-denaturation at 94 ℃ for 4 min; denaturation at 94 ℃ for 45s, annealing at 60 ℃ for 45s, and extension at 72 ℃ for 45s for 30 cycles; extension at 72 ℃ for 10 min.
In the step 3), the electrophoresis images of the obtained amplification products are compared by carrying out one-time capillary electrophoresis, and the marijuana variety is determined by analyzing the band conditions of the electrophoresis images.
The 11 pairs of SSR primers provided by the invention can realize the acquisition of genotyping data on a fluorescence capillary electrophoresis platform. The specific scheme is that the 5' end of one of each pair of primers is marked with a fluorescent group; preparing a PCR reaction system and adding a mixture of DNA, a primer and polymerase; operating a reaction program; detecting the amplification product on a fluorescent capillary electrophoresis system; and collecting the original data by using capillary electrophoresis system matched software, and importing the original data into genotype software to analyze the original data to obtain the genotype data in a fragment length format.
Preferably, the specific SSR primer pair 11 of the invention is labeled by fluorescent dyes, and four fluorescent dyes of PET, NED, VIC and FAM are selected in total. Diluting the PCR product with ultrapure water by 30 times; respectively taking the 4 diluted solutions with the same volume, mixing to form a mixed solution, sucking 1 microliter of the mixed solution, adding 0.5 microliter of LIZ500 molecular weight internal standard and 8.5 microliter of deionized formamide into a deep hole plate special for a DNA analyzer; then denaturalizing the mixture for 5min at 95 ℃ on a PCR instrument, taking out the mixture, immediately placing the mixture on ice, and cooling the mixture for more than 10 min; after being instantaneously centrifuged for 10s, the sample is placed on a DNA analyzer for capillary electrophoresis detection. The raw data collected was analyzed using GeneMapper software. The software system compares the position of the target peak with the internal standard LIZ500 in the same lane, and directly gives the accurate size of the target DNA fragment.
In a preferred embodiment of the embodiments of the present invention, the SSR molecule markers of the FAM fluorescence marker panel are ANUCS304, CAN 0031; the SSR molecular markers of the VIC fluorescent marker group are 23 and 25; the SSR molecule marker of the NED fluorescence marker group is ANUCS201, B01CANN 1; SSR molecules of the PET fluorescence labeling group are labeled E07CANN1, ANUCS305, 33, CAN0039 and CAN 0055. Taking the Grignard DNA as a template, and respectively carrying out four times of capillary electrophoresis on PCR products of four groups of primers which are fluorescently labeled by FAM, VIC, NED and PET to obtain four electrophoretogram results; and performing primary capillary electrophoresis on a PCR product mixture of four groups of primers which are fluorescently labeled by FAM, VIC, NED and PET by using DNA of Grignard as a template to obtain a total electrophoretogram result. Comparing the total electrophoretogram result with the electrophoresis results of four groups of fluorescence labeled primers of FAM, VIC, NED and PET respectively (fig. 1A-fig. 1D), it can be seen that the target peaks appearing on the FAM, VIC, NED and PET individual electrophoretograms can be distinguished on the total electrophoretogram, and the peaks of each color are not interfered with each other, that is, the primer combination (total 11 pairs of primers) provided by the invention can be used for one-time capillary electrophoresis, the target bands are not interfered with each other, and the result is easy to judge. The same experiments were performed using DNA of Cannabis sativa No. I (FIGS. 2A-2D), Cannabis sativa No.5 (FIGS. 4A-4D), Cannabis sativa No.7 (FIGS. 5A-5D), Cannabis sativa No.1 (FIGS. 7A-7D) and Euonymus alatus-31 (FIGS. 8A-8D) as templates, and the same conclusions were obtained.
In the embodiment, DNA of Grignard and Cannabis I is respectively taken as a template, FAM, VIC, NED and PET fluorescence labeling primer combination are used for carrying out one-time capillary electrophoresis, and capillary electrophoresis images of Grignard and Cannabis I are respectively obtained and compared. As can be seen from FIG. 3, FAM blue peaks appear in 189, 195, 296 and 301bp in Greek Asia, and FAM blue peaks appear in 160, 212, 294 and 298bp in Cannabis I; VIC green peaks appear in Gelistia at 171, 174, 177, 208, 213 and 216bp, and VIC green peaks appear in Cannabis I at 171, 175, 177 and 213 bp; NED yellow peaks appear in Gelistia at 188, 196, 330, 334 and 351bp, and NED yellow peaks appear in Cannabis I at 180, 328, 340 and 342 bp; PET red peaks appear in Gelistia at 129, 230 and 291bp, and PET red peaks appear in Cannabis I at 123, 126, 129, 132, 196, 230, 236, 242 and 291 bp. At least 2-3 different peaks appear in each fluorescent mark of the Grignard reagent and the Cannabis I, and the fluorescent marks are not overlapped and are clearly distinguished. Therefore, the fluorescence labeling primer combination provided by the invention can be used for distinguishing Cannabis glaseria and Cannabis sativa I in one-time capillary electrophoresis. Similarly, the fluorescence labeling primer combination provided by the patent of the invention can distinguish the hemp 5 and hemp 7 (figure 6), the hemp 1 and the eucalyptus-31 (figure 9) in one-time capillary electrophoresis.
The amplification products of different fluorescent markers can be subjected to electrophoresis in the same lane, signals are clear, the difference of the sizes of the amplified fragments is obvious, the sizes of the fragments can be accurately calculated, the electrophoresis peak types of each DNA sample are different, and the judgment is easy. The method has the advantages of high sensitivity, good resolution, accurate and reliable result, high efficiency, high speed and the like. The primer combination provided by the invention can be used for conveniently and rapidly distinguishing the varieties of Grissia, Cannabis sativa I, Cannabis sativa 5, Cannabis sativa 7, Cannabis sativa 1 and Euonymus alatus-31, and has the advantages of cost saving, efficiency improvement, convenience in operation and accurate result. The primer provided by the invention can be used for hemp fingerprint map construction, variety identification, genetic diversity analysis and the like, and has a very wide application prospect.
Drawings
FIGS. 1A-1D are schematic diagrams of SSR fluorescence labeled capillary electrophoresis of Grignard, respectively, wherein FIG. 1A is a comparison of the electrophoresis result of the Grignard by FAM, VIC, NED and PET fluorescence labeled primer combination with the electrophoresis result of the FAM labeled primer only. FIG. 1B is a comparison of the results of combined FAM, VIC, NED and PET fluorescently labeled primers in Grignard with those labeled only with VIC. FIG. 1C shows a comparison of the results of FACM, VIC, NED and PET fluorescence labeled primer combinations in Grignard with the results of NED labeled primer only. FIG. 1D shows a comparison of the results of combined FAM, VIC, NED and PET fluorescently labeled primers in Grignard with those of primers labeled only with PET. The comparison results of the figures respectively show that the target peaks appearing on the electrophoresis images of the single-color fluorescence labeling primers can be distinguished on the electrophoresis images of the four-color fluorescence labeling primer combination (the target peaks appearing are indicated by arrows), and the target peaks do not interfere with each other, so that the DNA fingerprint information of the varieties can be clearly read. Namely, the primer combination provided by the invention can be used for one-time capillary electrophoresis, target bands are not interfered with each other, results are easy to judge, and time, experimental reagents and consumables are saved.
FIGS. 2A-2D show the result of SSR fluorescence labeling capillary electrophoresis detection of Cannabis sativa I. FIG. 2A is a comparison of the electrophoresis results of the FAM, VIC, NED and PET fluorescence labeled primer combination of Cannabis sativa I and the electrophoresis results of the FAM labeled primer only. FIG. 2B is a comparison of the electrophoresis results of the FAM, VIC, NED and PET fluorescence labeled primer combination of Cannabis sativa I and the electrophoresis results of the VIC labeled primer only. FIG. 2C is a comparison of the electrophoresis results of the FAM, VIC, NED and PET fluorescence labeled primer combination of Cannabis sativa I and the electrophoresis results of the NED labeled primer only. FIG. 2D is a comparison of the electrophoresis results of the FAM, VIC, NED and PET fluorescence labeled primer combination of Cannabis sativa I and the electrophoresis results of the PET labeled primer only. The comparison results of the figures respectively show that the target peaks appearing on the electrophoresis images of the single-color fluorescence labeling primers can be distinguished on the electrophoresis images of the four-color fluorescence labeling primer combination (the target peaks appearing are indicated by arrows), and the target peaks do not interfere with each other, so that the DNA fingerprint information of the varieties can be clearly read. Namely, the primer combination provided by the invention can be used for one-time capillary electrophoresis, target bands are not interfered with each other, results are easy to judge, and time, experimental reagents and consumables are saved. FIGS. 1A-1D and FIGS. 2A-2D both illustrate the same objective, and the experimental results of two varieties (Grignard and Cannabis I) can further illustrate the practicability and reliability of the primer combination provided by the present invention.
FIG. 3 is a capillary electrophoresis image of the 11 cannabis SSR molecular markers provided herein for the treatment of Grignard and cannabis I, and the results of comparing the two. The results show that the FAM target peak (blue) shown in Grignard (upper panel) and the FAM target peak (both marked by arrows) shown in Cannabis I (lower panel) are clearly distinguishable, and the molecular weights of the target peaks are different. Similarly, the two varieties have different peak molecular weights of VIC, NED and PET meshes and are clearly distinguished. The primer combination provided by the patent can be used for distinguishing varieties of Grissia and Cannabis I.
FIGS. 4A-4D are SSR fluorescence labeled capillary electrophoresis images of Longmarianna 5, respectively, wherein FIG. 4A is a comparison of the combined electrophoresis results of FAM, VIC, NED and PET fluorescence labeled primers of Longmarianna 5 with the electrophoresis results of FAM labeled primers only. FIG. 4B is a comparison of the results of FAM, VIC, NED and PET fluorescence labeled primer combinations on Cannabis sativa No.5 with the results of electrophoresis on the primers labeled only by VIC. FIG. 4C is a comparison of the results of FAM, VIC, NED and PET fluorescence labeled primer combinations on Cannabis sativa No.5 with the results of the primer combinations labeled only with NED. FIG. 4D is a comparison of the results of FAM, VIC, NED and PET fluorescence labeled primer combinations on Cannabis sativa No.5 with the results of electrophoresis on PET labeled primer only.
FIGS. 5A-5D are SSR fluorescence labeled capillary electrophoresis images of Hanma No.7, respectively, wherein FIG. 5A is a comparison of the combined electrophoresis results of FAM, VIC, NED and PET fluorescence labeled primers of Hanma No.7 with the electrophoresis results of FAM labeled primers only. FIG. 5B is a comparison of the electrophoresis results of FAM, VIC, NED and PET fluorescence labeled primer combination of Hanma No.7 with the electrophoresis results of VIC labeled primer only. FIG. 5C is a comparison of the electrophoresis results of FAM, VIC, NED and PET fluorescence labeled primer combination of Hanma No.7 with the electrophoresis results of NED labeled primer only. FIG. 5D is a comparison of the electrophoresis results of FAM, VIC, NED and PET fluorescence labeled primer combination of Hanma No.7 with the electrophoresis results of PET labeled primer only.
FIG. 6 is a capillary electrophoresis image of 11 SSR molecular markers of Cannabis sativa No.5 and Cannabis sativa No.7, compared with each other. The two varieties show different and distinct molecular weights of FAM, VIC, NED and PET meshes. The primer combination provided by the patent can be used for distinguishing varieties of the cannabis sativa No.5 and the cannabis sativa No. 7.
FIGS. 7A-7D are SSR fluorescence labeled capillary electrophoresis images of Longmarianna 1, respectively, wherein FIG. 7A is a comparison of the combined electrophoresis results of FAM, VIC, NED and PET fluorescence labeled primers of Longmarianna 1 with the electrophoresis results of FAM labeled primers only. FIG. 7B is a comparison of the results of FAM, VIC, NED and PET fluorescence labeled primer combinations on Cannabis sativa No.1 with the results of electrophoresis on the primers labeled only by VIC. FIG. 7C is a comparison of the results of FAM, VIC, NED and PET fluorescence labeled primer combinations on Cannabis sativa No.1 with the results of the electrophoresis with NED labeled primer only. FIG. 7D is a comparison of the results of FAM, VIC, NED and PET fluorescence labeled primer combinations on Cannabis sativa No.1 with the results of electrophoresis on PET labeled primer only.
FIGS. 8A-8D are fluorescence labeling capillary electrophoresis images of Youshan-31 SSR, respectively, wherein FIG. 8A is a comparison of the electrophoresis results of Youshan-31 by the FAM, VIC, NED and PET fluorescence labeling primer combinations with the electrophoresis results of the FAM labeling primer alone. FIG. 8B is a comparison of the combined electrophoresis results of FAM, VIC, NED and PET fluorescence labeled primers for Youyan-31 with the electrophoresis results of VIC labeled primers alone. FIG. 8C is a comparison of the combined electrophoresis results of YOUYA-31 with FAM, VIC, NED and PET fluorescence labeled primers and the electrophoresis results with NED labeled primers only. FIG. 8D is a comparison of the combined electrophoresis results of YOUYA-31 with FAM, VIC, NED and PET fluorescence labeled primers and the electrophoresis results with PET labeled primers only.
FIG. 9 is a capillary electrophoresis image of Cannabis sativa No.1 and Ewing-31 using the 11 hemp SSR molecular markers provided herein, and comparing the two. Similarly, the two varieties show different and distinct molecular weights of FAM, VIC, NED and PET meshes. The primer combination provided by the patent can be used for distinguishing varieties of the Longmarihuan No.1 and the Euonymus alatus-31.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the examples follow conventional experimental conditions. Those skilled in the art will appreciate that the details of the present invention not described in detail herein are well within the skill of those in the art.
Unless otherwise specified, the biochemical reagents used in the examples of the present invention are commercially available, and the hemp materials used are publicly known in the art, among which, the variety approval registration number of Youyan yarn-31 is 2006005, the variety approval registration number of Longhan No.1 is Black registration 2011003, the variety approval registration number of Longhan No.2 is Black registration 2013009, the variety approval registration number of Cannabis No. one is Black registration 2015005, the variety approval registration number of Genghua No.1 is Black registration 2016012, the variety approval registration number of Longhan No.3 is Black registration 3513, the variety approval registration number of Longhan No.1 is Black confirmation 2017002, the variety approval registration number of Han No.2 is Black confirmation 2017003, the variety approval registration number of Han No.1 is Black confirmation 2017004, the variety approval registration number of Longhan No.2 is Black confirmation 2017005, the variety approval registration number of Grey Seya is Black confirmation 2017006, the variety approval registration number of china hemp 5 is black approval 2018001, the variety approval registration number of china hemp 4 is black approval 2018002, the variety approval registration number of mu ma 1 is black approval 2018003, the variety approval registration number of Qingda ma 2 is black approval 2018004, the variety approval registration number of line hemp 1 is black approval 2018005, the variety approval registration number of Longhan 4 is black approval 2019001, the variety approval registration number of Qingda ma 3 is black approval 2019002, the variety approval registration number of Qingda hemp 4 is black approval 2019003, the variety approval registration number of china hemp 6 is black approval 2019004, the variety approval registration number of china hemp 7 is black approval 2019005, and the variety approval registration number of Longhan 5 is black approval 2019006.
Example 1 Cannabis SSR molecular markers and primer identification
According to 22 hemp varieties (Youyan-31, Longmaria No.1, Longmaria No.2, Cannabis I, Qingmaria No.1, Longmaria No.3, Longmaria No.1, China hemp No.2, China hemp No.1, Longmaria No.2, Greessian, China hemp No.5, China hemp No. 4, Japanese hemp No.1, Qingmaria No.2, Cannabis No.1, Longmaria No. 4, Qingdama No.3, Qingmaria No. 4, China hemp No. 6, China hemp No.7 and Longmaria No. 5), gene sequences are searched for SSR sites, and 300 pairs of SSR primers are designed; extracting DNA of Grignard, Cannabis I, Cannabis 5, Cannabis 7, Cannabis 1 and YOUYA-31, respectively performing PCR and polyacrylamide gel electrophoresis on the six varieties with the 300 pairs of primers, analyzing and comparing parameters such as annealing temperature, amplifiable property, band specificity and definition of each pair of primers, whether stable single band can be obtained in the six varieties, and preliminarily screening 210 pairs of primers; carrying out fluorescence labeling on the primers, carrying out PCR and capillary electrophoresis on the DNA of the six varieties, and screening 50 pairs of primers according to conditions such as peak intensity, genotype reading difficulty and the like of each primer; and preliminarily grouping the primers according to the molecular weight range, PIC value and the like of the alleles of each pair of primers, wherein each combination consists of as many primers as possible, the ranges of all the primers in the combination are not overlapped, and the primers do not interfere with each other, and most importantly, the genotype data of Grignard, Cannabis sativa No.5, Cannabis sativa No.7, Cannabis sativa No.1 and Euonymus alatus-31 can be accurately read, and the conditions that the corresponding variety combinations can be distinguished are adopted, so that the total 11 pairs of SSR primers in four groups are finally determined. These primers were combined and primers with FAM, VIC, NED, PET fluorophores were synthesized, respectively (see Table 2). Those skilled in the art will appreciate that other fluorophores including FAM, VIC, NED, PET may be selected by those skilled in the art to modify the four sets of primers in table 2, as long as the same fluorophore is labeled by the label in each set, and is not limited to which fluorophore is selected.
TABLE 2 hemp SSR capillary electrophoresis primers and primer combinations
Example 2 discrimination of hemp varieties by SSR molecular markers provided by the invention
(1) Rapid extraction of DNA
DNA extraction of hemp seeds was performed by CTAB method. The method has the advantages of quick and simple operation, high quality of extracted DNA, suitability for DNA preparation in the field of plant molecular biology, and great significance for greatly shortening the time of seed purity inspection and transgene detection, improving the detection efficiency and reducing the detection cost. The specific operation steps are as follows: grinding a plurality of hemp seeds into powder, and placing the powder into a 1.5mL centrifuge tube; adding 700 mu L CTAB extracting solution into a centrifugal tube, and incubating for 30min at 65 ℃; adding 500 μ L chloroform-isoamyl alcohol (24: 1) into a centrifuge tube, shaking violently, and centrifuging at 12000rpm for 10 min; taking the supernatant, adding isopropanol with 0.7 times volume, and centrifuging at 12000rpm for 5 min; removing supernatant, washing precipitate with 75% ethanol for 2 times; the precipitate was dried at room temperature, and 200. mu.L of TE buffer (pH 8.0) was added thereto, and the mixture was sufficiently dissolved for use.
(2) Quality and quantity of DNA sample
Detecting OD values of 260nm and 280nm of DNA sample on ultraviolet spectrophotometer, and selecting OD260/280Samples with values of 1.8-1.9 were used for the assay.
(3) Core primer selection
The primers determined in example 1 to satisfy the capillary fluorescence detection technique were selected by analyzing the distribution of primers on the chromosome, the level of polymorphism, the PCR amplification stability, and the banding pattern of the amplification product, as shown in table 2.
(4) PCR amplification and capillary electrophoresis detection
And (3) carrying out fluorescent dye marking on the screened specific SSR primer, and selecting four fluorescent dyes of PET, NED, VIC and FAM in total. PCR amplification used a 20. mu.L reaction volume containing 10-40ng of sample DNA, 0.4. mu.M each of forward and reverse primers, and 10. mu.L of 2 × polymerase mix. The reaction procedure is as follows: pre-denaturation at 94 ℃ for 4 min; denaturation at 94 ℃ for 45s, annealing at 60 ℃ for 45s, and extension at 72 ℃ for 45s for 30 cycles; extension at 72 ℃ for 10 min.
Diluting the PCR product with ultrapure water by 30 times; respectively taking the 4 diluted solutions with the same volume, mixing to form a mixed solution, sucking 1 microliter of the mixed solution, adding 0.5 microliter of LIZ500 molecular weight internal standard and 8.5 microliter of deionized formamide into the deep hole plate special for the DNA analyzer; then denaturalizing the mixture for 5min at 95 ℃ on a PCR instrument, taking out the mixture, immediately placing the mixture on ice, and cooling the mixture for more than 10 min; after being instantaneously centrifuged for 10s, the sample is placed on a DNA analyzer for capillary electrophoresis detection. The raw data collected was analyzed using GeneMapper software. The software system compares the position of the target peak with the internal standard LIZ500 in the same lane to directly give the accurate size of the target DNA fragment. The band sizes of the amplification products obtained by amplifying primer sets in Grignard and Cannabis I in Table 2 are shown in Table 3. The band sizes of the amplification products obtained by amplifying the primer sets of the primer. The band sizes of the amplification products obtained by amplifying the primer sets of the Cannabis sativa 1 and the Euonymus alatus-31 in the table 2 are shown in the table 5.
TABLE 3 comparison of the size of the bands of interest for Gelseia and Cannabis I
TABLE 4 comparison of the target band sizes for Longmarijuana No.5 and Hanma No.7
TABLE 5 comparison of the band sizes of Longmariana No.1 and YOUYAKU-31
(5) Determination of primer combination
Taking the Grignard DNA as a template, and respectively carrying out four times of capillary electrophoresis by using four groups of primers of FAM, VIC, NED and PET fluorescence labels to obtain four electrophoretogram results; and performing capillary electrophoresis by using the primer mixture of FAM, VIC, NED and PET fluorescence labels by using the Grignard DNA as a template to obtain a total electrophoretogram result. Comparing the total electrophoretogram result with the electrophoresis results of four groups of fluorescence labeled primers of FAM, VIC, NED and PET respectively (fig. 1A-fig. 1D), it can be seen that the target peaks appearing on the FAM, VIC, NED and PET individual electrophoretograms can be distinguished on the total electrophoretogram, and the peaks of each color are not interfered with each other, namely, the primer mixture provided by the invention can be used for one-time capillary electrophoresis, the target bands are not interfered with each other, and the result is easy to judge. The same conclusion can be obtained by performing the same experiment using DNA of Cannabis sativa I (FIG. 2A-FIG. 2D), Cannabis sativa 5 (FIG. 4A-FIG. 4D), Cannabis sativa 7 (FIG. 5A-FIG. 5D), Cannabis sativa 1 (FIG. 7A-FIG. 7D) and Euonymus alatus-31 (FIG. 8A-FIG. 8D) as templates.
(6) Differentiation of cannabis varieties
Taking DNA of Grignard and Cannabis I as templates, respectively, performing one-time capillary electrophoresis with FAM, VIC, NED and PET fluorescence labeling primer combination (see Table 2), respectively obtaining capillary electrophoresis images of Grignard and Cannabis I, and comparing. As can be seen from FIG. 3, FAM blue peaks appear in 189, 195, 296 and 301bp in Greek Asia, and FAM blue peaks appear in 160, 212, 294 and 298bp in Cannabis I; VIC green peaks appear in Gelistia at 171, 174, 177, 208, 213 and 216bp, and VIC green peaks appear in Cannabis I at 171, 175, 177 and 213 bp; NED yellow peaks appear in Gelistia at 188, 196, 330, 334 and 351bp, and NED yellow peaks appear in Cannabis I at 180, 328, 340 and 342 bp; PET red peaks appear in Gelistia at 129, 230 and 291bp, and PET red peaks appear in Cannabis I at 123, 126, 129, 132, 196, 230, 236, 242 and 291 bp. At least 2-3 different peaks appear in each fluorescent mark of the Grignard reagent and the Cannabis I, are not overlapped and are clearly distinguished. Therefore, the fluorescence labeling primer combination (table 2) provided by the invention patent can be used for distinguishing the Siya and the Cannabis I in one-time capillary electrophoresis.
Respectively taking DNA of the Longmariana No.5 and the Hanma No.7 as templates, and carrying out one-time capillary electrophoresis by using FAM, VIC, NED and PET fluorescence labeling primer combinations (shown in table 2) to respectively obtain capillary electrophoresis images of the Longmariana No.5 and the Hanma No.7 and comparing the capillary electrophoresis images. As can be seen from FIG. 6, FAM blue peaks appear at 189 and 296bp for Longmarihuan No.5, and FAM blue peaks appear at 220, 300 and 306bp for Hanma No. 7; the Longmarihuan No.5 has VIC green peaks at 172, 174, 177, 213 and 215bp, and the Hanma No.7 has VIC green peaks at 174, 213 and 219 bp; the Longmariana No.5 has NED yellow peaks at 182, 206, 334 and 346bp, and the Hanma No.7 has NED yellow peaks at 172, 188, 328 and 342 bp; the red peaks of PET appear in 129, 190, 230 and 291bp of Longmarihuan No.5, and the red peaks of PET appear in 123, 175, 196, 230, 245 and 291bp of Hanma No. 7. At least 2-3 difference peaks appear on each fluorescent mark of the Longmarijuana No.5 and the Hanma No.7, which are not overlapped and are clearly distinguished. Therefore, the fluorescence labeling primer combination (table 2) provided by the invention patent can be used for distinguishing the hemp 5 and the hemp 7 in one-time capillary electrophoresis.
Respectively taking the DNA of the Longmarihuan No.1 and the Euonymus alatus-31 as templates, and performing one-time capillary electrophoresis by using FAM, VIC, NED and PET fluorescence labeling primer combinations (shown in Table 2) to respectively obtain capillary electrophoresis images of the Longmarihuan No.1 and the Euonymus alatus-31 and comparing the capillary electrophoresis images. As can be seen from FIG. 9, LONGmariana No.1 shows FAM blue peaks at 160 and 294bp, YOUYA-31 shows FAM blue peaks at 189, 195, 296 and 300 bp; the Longmarihuan No.1 shows VIC green peaks at 172, 174, 177 and 214bp, and the YOUYA-31 shows VIC green peaks at 172, 175, 177, 214 and 216 bp; longmariana No.1 shows NED yellow peaks at 172, 177, 334 and 340bp, while Yuyousha-31 shows NED yellow peaks at 188, 196 and 334 bp; longmarian No.1 shows PET red peaks at 123, 129, 190, 242 and 291bp, while Yu Yao-31 shows PET red peaks at 129, 198, 230, 291 and 296 bp. At least 2-3 different peaks appear on each fluorescent mark of the Longmarihuan No.1 and the Youyan-31, which are not overlapped and clearly distinguished. Therefore, the fluorescence labeling primer combination (table 2) provided by the patent of the invention can be used for distinguishing the hemp seed No.1 and the Euonymus alatus-31 in one capillary electrophoresis.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
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Claims (10)
1. A group of SSR molecular markers for identifying hemp varieties is characterized in that the SSR molecular markers comprise one or more of the following 11 SSR molecular markers, namely ANUCS304, CAN0031, 23 and 25, ANUCS201, B01CANN1, E07CANN1, ANUCS305 and 33, CAN0039 and CAN 0055.
2. The cannabis SSR molecular marker according to claim 1, wherein the 11 SSR molecular markers are obtained by amplification sequentially with the following primers: 1-2 parts of SEQ ID NO.1, 3-4 parts of SEQ ID NO.5-6 parts of SEQ ID NO.7-8 parts of SEQ ID NO.9-10 parts of SEQ ID NO.11-12 parts of SEQ ID NO.13-14 parts of SEQ ID NO.15-16 parts of SEQ ID NO.17-18 parts of SEQ ID NO.19-20 parts of SEQ ID NO.21-22 parts of SEQ ID NO. 21.
3. Specific primers for amplifying the SSR molecular marker of claim 1 or 2;
preferably, the specific primers comprise one or more of the following primer pairs: 1-2 parts of SEQ ID NO.1, 3-4 parts of SEQ ID NO.5-6 parts of SEQ ID NO.7-8 parts of SEQ ID NO.9-10 parts of SEQ ID NO.11-12 parts of SEQ ID NO.13-14 parts of SEQ ID NO.15-16 parts of SEQ ID NO.17-18 parts of SEQ ID NO.19-20 parts of SEQ ID NO.21-22 parts of SEQ ID NO. 21.
4. A kit comprising the specific primer according to claim 3.
5. A Cannabis genome chip comprising the SSR molecular marker of claim 1 or 2.
6. Use of a SSR molecular marker according to claim 1 or 2 or a specific primer according to claim 3 or a kit according to claim 4 or a cannabis genome chip according to claim 5 for the construction of a cannabis variety DNA fingerprint database.
7. Use of a SSR molecular marker according to claim 1 or 2 or a specific primer according to claim 3 or a kit according to claim 4 or a cannabis genome chip according to claim 5 in analysis of genetic diversity of cannabis germplasm resources or in seed quality detection.
8. Use of a SSR molecular marker according to claim 1 or 2 or a specific primer according to claim 3 or a kit according to claim 4 or a cannabis genomic chip according to claim 5 for variety identification, genetic relationship analysis, maternal traceability of cannabis.
9. Use of a SSR molecular marker according to claim 1 or 2 or a specific primer according to claim 3 or a kit according to claim 4 or a cannabis genomic chip according to claim 5 in cannabis molecular marker assisted breeding.
10. Use according to any one of claims 6 to 9, characterized in that it comprises the following steps:
1) extracting DNA of a hemp sample to be detected;
2) performing PCR amplification by using the DNA extracted in the step 1) as a template according to the hemp SSR molecular marker;
3) and detecting the PCR product by using a capillary electrophoresis system.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004008841A2 (en) * | 2002-07-19 | 2004-01-29 | Arizona Board Of Regents, Acting For And On Behalf Of Arizona State University | Dna fingerprinting for cannabis sativa (marijuana) using short tandem repeat (str) markers |
| CN104450888A (en) * | 2014-11-10 | 2015-03-25 | 公安部物证鉴定中心 | Marihuana DNA fluorescent multiplex amplification system |
| CA2989194A1 (en) * | 2015-06-12 | 2016-12-15 | Anandia Laboratories Inc. | Methods and compositions for cannabis characterization |
| CN113025740A (en) * | 2021-02-25 | 2021-06-25 | 司法鉴定科学研究院 | Primer composition, kit and method for industrial hemp composite identification polymorphic genetic marker |
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2021
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004008841A2 (en) * | 2002-07-19 | 2004-01-29 | Arizona Board Of Regents, Acting For And On Behalf Of Arizona State University | Dna fingerprinting for cannabis sativa (marijuana) using short tandem repeat (str) markers |
| CN104450888A (en) * | 2014-11-10 | 2015-03-25 | 公安部物证鉴定中心 | Marihuana DNA fluorescent multiplex amplification system |
| CA2989194A1 (en) * | 2015-06-12 | 2016-12-15 | Anandia Laboratories Inc. | Methods and compositions for cannabis characterization |
| CN113025740A (en) * | 2021-02-25 | 2021-06-25 | 司法鉴定科学研究院 | Primer composition, kit and method for industrial hemp composite identification polymorphic genetic marker |
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