CN113736899B

CN113736899B - SSR molecular markers for identifying cannabis varieties and application thereof

Info

Publication number: CN113736899B
Application number: CN202110996568.3A
Authority: CN
Inventors: 关海涛; 张晓磊; 陈中华; 张瑞英; 尹阳; 兰青阔
Original assignee: SAFETY AND QUALITY INSTITUTE OF AGRICULTURAL PRODUCTS HEILONGJIANG ACADEMY OF AGRICULTURAL SCIENCES
Current assignee: SAFETY AND QUALITY INSTITUTE OF AGRICULTURAL PRODUCTS HEILONGJIANG ACADEMY OF AGRICULTURAL SCIENCES
Priority date: 2021-08-27
Filing date: 2021-08-27
Publication date: 2023-09-29
Anticipated expiration: 2041-08-27
Also published as: CN113736899A

Abstract

The application provides a group of SSR molecular markers for identifying cannabis varieties and application thereof. The application also provides a set of primer combination suitable for detecting the SSR molecular markers of the capillary electrophoresis platform based on 11 SSR molecular markers. Using these 11 SSR molecular markers: identifying cannabis varieties and analyzing genetic diversity; distinguishing cannabis from glissade and hemp one number varieties of Cannabis sativa No.5 and Cannabis sativa No.7, cannabis sativa No.1 and You Sha-31. Through the application of the cannabis sativa SSR molecular marker, the range of available marker loci of cannabis sativa can be expanded on the genome level; provides a new tool for researches such as cannabis sativa variety and germplasm resource identification, genetic relationship evaluation, cytoplasmatic genetic characteristics and the like, and has good application prospect.

Description

SSR molecular markers for identifying cannabis varieties and application thereof

Technical Field

The application relates to the technical field of crop molecular breeding and identification, in particular to a group of SSR molecular markers for identifying cannabis varieties and application thereof.

Background

Cannabis sativa L.A Cannabis sativa L.of Cannabis of Urtica order. There are three varieties below that are industrial hemp, indian hemp and wild hemp, respectively. Industrial cannabis is the most common type, namely cannabis varieties with tetrahydrocannabinol content below 0.3%, which has high economic utility. The hemp seed oil is mainly applied to foods, cosmetics and medicines due to the balanced omega-3 and omega-6 unsaturated fatty acids; cannabidiol (CBD) in cannabis, which is a terpene phenolic compound, has a rich pharmacological effect; the hemp fiber and stalk core are excellent raw materials in the aspects of building materials, special supply munitions, infant underclothes, textiles and the like, and the hemp fiber has the advantages of radiation protection, antibiosis, good elasticity and the like, and has wide market prospect and huge economic benefit.

With the development of industrial hemp industry, many industrial hemp varieties are cultivated by a plurality of scientific research institutions aiming at the industrial development requirements. The method enriches the germplasm resources of the industrial hemp and also makes the market of the industrial hemp seeds easy to have the phenomena of homonymous foreign matters, homonymous foreign matters and fish-dragon mixing. The industrial hemp is easy to accept external pollen in the cultivation process, so that the variety is easy to mix and has low purity, and the quality of industrial hemp seeds and the cultivation of new varieties are seriously affected. Hemp You Sha-31 is a high fiber content variety introduced from ukraine in china. The Glimenia, cannabis sativa No. one, cannabis sativa No.5, cannabis sativa No.7 and Cannabis sativa No.1 are hybrid varieties which are improved by Chinese breeding. Besides high fiber content, the hemp No. I has the advantages of lodging resistance, drought resistance and salt and alkali resistance, and is a main cultivated variety of hemp in Heilongjiang province. While glissade, while also a fiber cannabis, has no obvious advantage over cannabis one. Both are very similar in seed appearance and plant appearance and are difficult to distinguish, so that identification of varieties of glissade and cannabis one is a necessary precondition for downstream markets or breeding. The same is true for dragon hemp No.1 and You Sha-31. The cannabis sativa No.1 has strong gray mold and stalk rot resistance, and has the advantages of lodging resistance, drought resistance and salt and alkali resistance. However, it is very similar in morphological appearance to You Sha-31, such as confusion during the harvest stage or seed preservation stage, and affects seed purity. The medicinal cannabis sativa L.No. 5 and cannabis sativa L.No. 7 belong to dominant varieties with higher CBD content and low THC content, have the same variety characteristics and are different in CBD content and THC content. The different content indicates a different direction of downstream applications. In order to avoid confusion of two varieties with similar characteristics, accurate variety identification and distinction are important preconditions. The characteristics and variety identification reasons of each variety are shown in Table 1.

Simple repeat sequences (Simple sequence repeat, SSR), also known as microsatellites DNA (Microsatellite DNA), generally refer to DNA sequences that are repeated in tandem multiple times in units of 2-4 nucleotides. The SSR is a novel DNA fingerprint technology developed in recent years and based on PCR, has the advantages of strong reliability, high repeatability, rich polymorphism and the like, is the most applied technology in the current variety identification, and has been widely applied to the construction of core germplasm libraries of corn, rice, wheat, soybean and other varieties and the genetic diversity analysis of germplasm resources. Currently, there are few reports on construction of cannabis sativa SSR fingerprint. And the PCR-based SSR technology is utilized to identify the cannabis sativa varieties, analyze genetic diversity and construct a DNA fingerprint database, and has important significance for cannabis sativa variety management, variety breeding and germplasm resource collection and protection.

TABLE 1 characterization of the varieties and their origin

Disclosure of Invention

The application aims to provide a group of SSR molecular markers for identifying cannabis varieties and application thereof.

In order to achieve the aim of the application, the application obtains a group of SSR molecular markers for identifying cannabis varieties by collecting cannabis materials with wide sources, abundant phenotype and genotype and strong representativeness, sequencing and comparing the cannabis genomes of the corresponding materials.

The SSR molecular markers for identifying the cannabis sativa varieties provided by the application comprise one or more of the following 11 SSR molecular markers, wherein the 11 SSR molecular markers are ANUCS304, CAN0031, 23, 25, ANUCS201, B01CANN1, E07CANN1, ANUCS305, 33, CAN0039 and CAN0055 respectively.

The 11 SSR molecular markers are respectively obtained by sequentially amplifying the following primers: SEQ ID No.1-2, SEQ ID No.3-4, SEQ ID No.5-6, SEQ ID No.7-8, SEQ ID No.9-10, SEQ ID No.11-12, SEQ ID No.13-14, SEQ ID No.15-16, SEQ ID No.17-18, SEQ ID No.19-20, SEQ ID No.21-22.

The SSR molecular marker can be detected by adopting a conventional technical means in the field, and can be applied to a capillary electrophoresis detection technology. Quantitative DNA fragment analysis data can be obtained by an SSR molecular marker technology based on a capillary electrophoresis detection technology. Compared with the conventional polyacrylamide gel electrophoresis detection method, the method has the advantages of more accurate, sensitive and efficient result, and is more suitable for detection and analysis of large-scale varieties.

When capillary electrophoresis detection is carried out, the SSR molecular markers can be divided into a plurality of different fluorescent marker groups, and the fluorescent markers in each group are the same.

As an embodiment of the application, the SSR molecules described above can be labeled into four distinct groups of fluorescent labels, the fluorescent labels within each group being identical:

the first group consists of SSR molecular markers ANUCS304 and CAN0031; the second group consists of SSR molecular markers 23, 25; the third group consists of SSR molecular markers ANUCS201 and B01CANN1; the fourth group consisted of SSR molecular markers E07CANN1, ANUCS305, 33, CAN0039, and CAN0055.

Further, the application 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: SEQ ID No.1-2, SEQ ID No.3-4, SEQ ID No.5-6, SEQ ID No.7-8, SEQ ID No.9-10, SEQ ID No.11-12, SEQ ID No.13-14, SEQ ID No.15-16, SEQ ID No.17-18, SEQ ID No.19-20, SEQ ID No.21-22.

The application also provides a kit which contains the specific primer pair for amplifying the SSR molecular marker.

The application also provides a cannabis genome chip which contains the cannabis SSR molecular marker.

The application provides application of the SSR molecular marker, the specific primer, the kit and the cannabis sativa genome chip in constructing a cannabis sativa variety DNA fingerprint database.

The application provides application of the SSR molecular marker, the specific primer, the kit and the cannabis genome chip in cannabis germplasm genetic diversity analysis or seed quality detection.

The application provides application of the SSR molecular marker, the specific primer, the kit and the cannabis genome chip in cannabis variety identification, genetic relationship analysis and maternal tracing.

For identification of cannabis varieties, as an example, the application provides for use in differentiating between glissade and cannabis varieties one, cannabis 5 and cannabis 7, cannabis 1 and You Sha-31.

The application provides a kit for distinguishing between glissade and cannabis one, cannabis 5 and cannabis 7, cannabis 1 and You Sha-31, which contains a specific primer combination for 11 cannabis SSR molecular markers of the application. Preferably, the nucleotide sequences of the specific primer combinations are shown in SEQ ID NOS.1-22, respectively.

The application provides application of the SSR molecular marker, the specific primer, the kit and the cannabis genome chip in cannabis molecular marker assisted breeding.

The application also provides application of the cannabis sativa SSR molecular marker in preparing cannabis sativa genome chips.

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 using the SSR molecular marker;

3) PCR products were detected using a capillary electrophoresis system.

In step 2) of the above application, a reaction volume of 20. Mu.L was used for PCR amplification, containing 10-40ng of sample DNA, 0.4. Mu.M each of forward primer and reverse primer, and 10. Mu.L of 2 Xpolymerase mixture. The reaction procedure is: pre-denaturation at 94℃for 4min; denaturation at 94℃for 45s, annealing at 60℃for 45s, extension at 72℃for 45s for 30 cycles; extending at 72℃for 10min.

In step 3), the electrophoresis patterns of the obtained amplification products can be compared by performing one-time capillary electrophoresis, and the hemp variety can be determined by analyzing the band conditions of the electrophoresis patterns.

The 11 pairs of SSR primers provided by the application can realize the acquisition of genotyping data on a fluorescent 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 DNA, a primer and a polymerase mixture; running a reaction program; detecting the amplified product on a fluorescent capillary electrophoresis system; and collecting the original data by using software matched with a capillary electrophoresis system, and importing genotype software to analyze the original data to obtain genotype data in a fragment length format.

Preferably, the 11 pairs of specific SSR primers of the application are labeled with fluorescent dyes, and PET, NED, VIC, FAM fluorescent dyes are adopted in total. Diluting the fluorescence-labeled PCR product with ultrapure water for 30 times; respectively mixing the 4 diluted solutions with equal volumes to form a mixed solution, sucking 1 mu L of the mixed solution, adding 0.5 mu L of LIZ500 molecular weight internal standard and 8.5 mu L of deionized formamide into a special deep pore plate of a DNA analyzer; then denaturing the sample on a PCR instrument at 95 ℃ for 5min, taking out, immediately placing on ice, and cooling for more than 10min; after 10s of instantaneous centrifugation, the sample is placed on a DNA analyzer for capillary electrophoresis detection. The raw data collected were analyzed using GeneMapper software. The software system will directly give the exact size of the target DNA fragment by comparing the position of the target peak with the internal standard LIZ500 in the same lane.

In a preferred implementation of the examples of the present application, the SSR molecular markers of the FAM fluorescent marker set are anccs 304, CAN0031; SSR molecular markers of the VIC fluorescent marker group are 23 and 25; the SSR molecular marker of the NED fluorescent marker group is ANUCS201, B01CANN1; the SSR molecular markers of the PET fluorescent marker group are E07CANN1, ANUCS305, 33, CAN0039 and CAN0055. Performing four times of capillary electrophoresis by using the PCR products of four groups of primers respectively marked by FAM, VIC, NED, PET fluorescence by taking Glioclada DNA as a template to obtain four electrophoresis chart results; and performing capillary electrophoresis once by using DNA of Gliocladium as a template and using a PCR product mixture of the four groups of primers which are fluorescently marked by FAM, VIC, NED, PET to obtain a total electrophoresis pattern result. Comparing the result of the total electrophoresis pattern with the result of FAM, VIC, NED, PET four groups of fluorescence labeling primers (fig. 1A-1D), it can be seen that the objective peaks appearing on FAM, VIC, NED, PET single electrophoresis patterns can be distinguished on the total electrophoresis pattern, and the peaks of each color are not interfered with each other, which means that the primer combination (11 pairs of primers in total) provided by the application can be used for primary capillary electrophoresis, the objective bands are not interfered with each other, and the result is easy to judge. The same experiment was performed using cannabis one (fig. 2A-2D), cannabis 5 (fig. 4A-4D), cannabis 7 (fig. 5A-5D), cannabis 1 (fig. 7A-7D) and You Sha-31 (fig. 8A-8D) DNA as templates, and the same conclusion could be reached.

In the examples, DNA of Glioclada and Cannabis sativa is used as a template, and a FAM, VIC, NED, PET fluorescent labeled primer combination is used for performing disposable capillary electrophoresis, so that capillary electrophoresis patterns of Glioclada and Cannabis sativa are obtained respectively and compared. As can be seen from FIG. 3, glioclada exhibits FAM blue peaks at 189, 195, 296 and 301bp, and Cannabis number one exhibits FAM blue peaks at 160, 212, 294 and 298 bp; glioclada shows VIC green peaks at 171, 174, 177, 208, 213 and 216bp, cannabis No. 171, 175, 177 and 213 bp; NED yellow peaks appear in Gleasia at 188, 196, 330, 334 and 351bp, and NED yellow peaks appear in Cannabis No. 180, 328, 340 and 342 bp; the glissade showed PET red peaks at 129, 230 and 291bp, cannabis No. 123, 126, 129, 132, 196, 230, 236, 242 and 291 bp. At least 2-3 different peaks appear in each fluorescent label of Glioclada and Cannabis sativa, and the fluorescent labels are not overlapped and are clearly distinguished. Thus, the fluorescent-labeled primer combinations provided by the present application can distinguish Cannabis, glioclada, from Cannabis one in a single capillary electrophoresis. Similarly, the combination of fluorescent-labeled primers provided by the present application can distinguish between cannabis 5 and cannabis 7 (fig. 6), cannabis 1 and You Sha-31 (fig. 9) in a single capillary electrophoresis.

The amplified products of different fluorescent markers can be electrophoresed in the same lane, the signals are clear, the amplified fragments have obvious size difference, the sizes of the fragments can be accurately calculated, and the electrophoresis peak types of each DNA sample are different, so that 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 application can be used for conveniently and rapidly distinguishing the species of Glioclada from Cannabis sativa No. one, cannabis sativa No.5 and Cannabis sativa No.7 and Cannabis sativa No.1 and You Sha-31, thereby realizing the advantages of cost saving, efficiency improvement, convenient operation and accurate result. The set of primers provided by the application can be used for cannabis fingerprint construction, variety identification, genetic diversity analysis and the like, and has a very broad application prospect.

Drawings

FIGS. 1A-1D are graphs of SSR fluorescence-labeled capillary electrophoresis of Glioclada, respectively, wherein FIG. 1A is a comparison of the results of electrophoresis of the combination of Glioclada primer FAM, VIC, NED and PET fluorescence-labeled primer with the result of electrophoresis of FAM-labeled primer alone. FIG. 1B is a comparison of the results of electrophoresis of combinations of primers labeled with VIC alone in Glioclada via FAM, VIC, NED and PET. FIG. 1C is a comparison of the results of electrophoresis of the combination of primers in Glioclada via FAM, VIC, NED and PET fluorescence labeled with the primers only labeled with NED. FIG. 1D is a comparison of the electrophoresis results of the combination of the primers by way of FAM, VIC, NED and PET fluorescence labeling in Glioclada with the electrophoresis results of the primers by way of PET labeling only. The comparison results of the figures respectively show that the target peaks appearing on the electrophoresis patterns of the single-color fluorescent-labeled primers can be distinguished on the electrophoresis patterns of the four-color fluorescent-labeled primer combination (the target peaks appear as indicated by arrows), and each target peak is not interfered with each other, so that the DNA fingerprint information of the variety can be clearly read. The primer combination provided by the application can be used for primary capillary electrophoresis, target bands are not mutually interfered, the result is easy to judge, and the time, experimental reagent and consumable are saved.

FIGS. 2A-2D show the results of SSR fluorescence-labeled capillary electrophoresis detection of Cannabis sativa number one. Wherein, FIG. 2A is a comparison of the electrophoresis result of Cannabis sativa No. FAM, VIC, NED and PET fluorescent-labeled primer combination with the electrophoresis result of FAM-labeled primer alone. FIG. 2B is a comparison of the electrophoresis results of Cannabis sativa No. FAM, VIC, NED and PET fluorescent-labeled primer combinations with the electrophoresis results of VIC-labeled primers alone. FIG. 2C is a comparison of the electrophoresis results of Cannabis sativa No. FAM, VIC, NED and PET fluorescent-labeled primer combinations with NED-labeled primer alone. FIG. 2D is a comparison of the electrophoresis results of Cannabis sativa No. FAM, VIC, NED and PET fluorescent-labeled primer combinations with the electrophoresis results of PET-labeled primers alone. The comparison results of the figures respectively show that the target peaks appearing on the electrophoresis diagram of the single-color fluorescent-labeled primer can be distinguished on the electrophoresis diagram of the four-color fluorescent-labeled primer combination (the target peaks appear as indicated by arrows), and each target peak is not interfered with each other, so that the DNA fingerprint information of the variety can be clearly read. The primer combination provided by the application can be used for primary capillary electrophoresis, target bands are not mutually interfered, the result is easy to judge, and the time, experimental reagent and consumable are saved. The experimental results of two varieties (Glioclada and Cannabis-A) are presented in FIGS. 1A-1D and FIGS. 2A-2D for the same purpose to further illustrate the utility and reliability of the primer combinations provided by the present application.

FIG. 3 shows the results of capillary electrophoresis of Glioclada and Cannabis sativa I using the 11 SSR molecular markers provided by the present application, and comparing the two. The results demonstrate that FAM peaks (blue) shown in glissade (upper panel) are clearly distinguishable from FAM peaks (both marked with arrows) shown in cannabis number one (lower panel), and that the peak molecular weights of interest are different. Similarly, the two varieties showed different peak molecular weights of VIC, NED, PET mesh and are clearly distinguishable. The primer combination provided by the patent can be used for distinguishing varieties of glibenclamide and cannabis.

Fig. 4A-4D are respectively SSR fluorescence-labeled capillary electrophoresis charts of kenaf No.5, wherein fig. 4A is a comparison of the electrophoresis results of the combination of the kenaf No.5, FAM, VIC, NED and PET fluorescence-labeled primers with the electrophoresis results of the FAM-labeled primers alone. FIG. 4B is a comparison of the result of the electrophoresis of the combination of the primer and the PET fluorescence-labeled primer of the Cannabis sativa No.5 through FAM, VIC, NED and the result of the electrophoresis of the primer and the primer through VIC labeling only. FIG. 4C is a comparison of the result of electrophoresis of the combination of No.5 Dragon hemp with FAM, VIC, NED and PET fluorescent-labeled primer and the result of electrophoresis of NED-labeled primer alone. FIG. 4D is a comparison of the result of electrophoresis of the combination of No.5 Dragon hemp through FAM, VIC, NED and the PET fluorescent-labeled primer, and the result of electrophoresis of the PET-labeled primer alone.

FIGS. 5A-5D are schematic diagrams of SSR fluorescence-labeled capillary electrophoresis of China-hemp No.7, respectively, wherein FIG. 5A is a comparison of the electrophoresis results of the combination of the China-hemp No.7, FAM, VIC, NED and PET fluorescence-labeled primers with the FAM-labeled primers alone. FIG. 5B is a comparison of the electrophoresis results of China hemp No.7 through FAM, VIC, NED and PET fluorescent marker primer combination with the electrophoresis results of the VIC-only marker primer. FIG. 5C is a comparison of the electrophoresis results of China hemp No.7 through FAM, VIC, NED and PET fluorescent marker primer combination with the electrophoresis results of NED-only marker primers. FIG. 5D is a comparison of the electrophoresis results of China hemp No.7 through FAM, VIC, NED and PET fluorescent labeled primer combination with the electrophoresis results of the PET labeled primer alone.

FIG. 6 shows the results of capillary electrophoresis of Cannabis sativa No.5 and Cannabis sativa No.7 with 11 SSR molecular markers of Cannabis sativa provided by the application, and comparing the two. The two varieties showed different peak molecular weights of FAM, VIC, NED, PET mesh and were clearly distinguishable. The primer combination provided by the patent can be used for distinguishing varieties of Long Da hemp No.5 and China hemp No. 7.

Fig. 7A-7D are respectively SSR fluorescence labeled capillary electrophoresis charts of kenaf No.1, wherein fig. 7A is a comparison of the electrophoresis results of the combination of the kenaf No.1, FAM, VIC, NED and PET fluorescence labeled primers with the electrophoresis results of the FAM labeled primers alone. FIG. 7B is a comparison of the results of the electrophoresis of the combination of the primer sets of Cannabis sativa No.1 through FAM, VIC, NED and PET fluorescence labeled with the primer set of Cannabis sativa, and the results of the electrophoresis of the primer set of Cannabis sativa through VIC only. FIG. 7C is a comparison of the results of the electrophoresis of the combination of No.1 Dragon/hemp primer and the electrophoresis of the NED-only labeled primer by FAM, VIC, NED and PET fluorescent labeled primer. FIG. 7D is a comparison of the result of electrophoresis of the combination of the FAM, VIC, NED and PET fluorescent-labeled primers of Dragon hemp No.1 with the result of electrophoresis of the PET-labeled primers alone.

FIGS. 8A-8D are, respectively, a capillary electrophoresis chart of a fluorescent marker of Geranium-31 SSR, wherein FIG. 8A is a comparison of the electrophoresis results of Geranium-31 via FAM, VIC, NED and PET fluorescent marker primer combinations with the electrophoresis results of FAM-only marker primers. FIG. 8B is a comparison of the electrophoresis results of the combination of the primer set FAM, VIC, NED and PET fluorescent label with the primer set VIC-labeled alone. FIG. 8C is a comparison of the electrophoresis results of the combination of the Neem-31 primer by FAM, VIC, NED and PET fluorescent-labeled primers with the NED-labeled primer alone. FIG. 8D shows a comparison of the electrophoresis results of the combination of the primer set FAM, VIC, NED and the PET fluorescent label with the primer set labeled with PET alone for especially yarn-31.

FIG. 9 is a capillary electrophoresis chart of Cannabis sativa No.1 and Cannabis sativa No. You Sha-31 using the 11 SSR molecular markers provided by the application, and comparing the two. Similarly, the two varieties showed different peak molecular weights of FAM, VIC, NED, PET mesh and are clearly distinguishable. The primer combination provided by the patent can be used for variety discrimination of Long Da hemp No.1 and You Sha-31.

Detailed Description

The following examples are illustrative of the application and are not intended to limit the scope of the application. Unless otherwise indicated, the examples were all under conventional experimental conditions. What is not described in detail in the present specification belongs to the prior art known to those skilled in the art.

If not specified, the biochemical reagents used in the examples of the present application are all commercially available, and the hemp materials used are all common hemp materials known in the art, wherein, the variety approval registration number of You Sha-31 is black registration 2006005, the variety approval registration number of kenaf 1 is black registration 2011003, the variety approval registration number of kenaf 2 is black registration 2013009, the variety approval registration number of kenaf one is black registration 20151005, the breed approval registration number of Qing hemp 1 is black registration 2016012, the breed approval registration number of Longhemp 3 is black registration 2016013, the breed approval registration number of Longhemp 1 is black registration 2017002, the breed approval registration number of China hemp 2 is black registration 2017003, the breed approval registration number of China hemp 1 is black registration 2017004, the breed approval registration number of Longhemp 2 is black registration 2017005, the black identification number 2017006, the black identification number 2018001, the black identification number 2018002, the black identification number 2018003, the black identification number 2018004, the black identification number 2018005, the black identification number 2019001, the black identification number 2019002, the black identification number 2019003, the black identification number 2019004, the black identification number 2019005, the black identification number 2019006, and the black identification number 2019006.

Example 1 determination of SSR molecular markers and primers for cannabis sativa

Searching SSR sites according to the gene sequences of 22 cannabis varieties (You Sha-31, kenaf 1, kenaf 2, kenaf one, kenaf 1, kenaf 3, kenaf 1, kenaf 2, glioclada, kenaf 5, kenaf 4, cannabis 1, kenaf 2, kenaf 1, kenaf 4, kenaf 3, kenaf 4, kenaf 6, kenaf 7, and kenaf 5), and designing 300 pairs of SSR primers; extracting DNA of Glimenia, cannabis sativa No.5, cannabis sativa No.7, cannabis sativa No.1 and You Sha-31, respectively carrying out PCR and polyacrylamide gel electrophoresis on the six varieties by using the 300 pairs of primers, analyzing and comparing parameters such as annealing temperature, amplification possibility, band specificity, definition and whether stable single bands can be obtained in the six varieties of each pair of primers, and primarily screening out 210 pairs of primers; carrying out fluorescent marking on the primers, then carrying out PCR and capillary electrophoresis on DNA of the six varieties, and screening out 50 pairs of primers according to the conditions of peak intensity, genotype reading difficulty and the like of each primer; the primers are initially grouped according to the molecular weight range, PIC value and the like of alleles of each pair of primers, and four groups of 11 pairs of SSR primers are finally determined according to the conditions that each group consists of as many primers as possible, all the primer allele ranges in the group are not overlapped and the primer ranges are not interfered with each other, and most importantly, the genotype data of Glioclada, cannabis sativa No.5, cannabis sativa No.7, cannabis sativa No.1 and You Sha-31 can be accurately read and the conditions that the corresponding variety combinations can be distinguished. This primer was combined and primers with FAM, VIC, NED, PET fluorophores were synthesized separately (see table 2). Those skilled in the art will appreciate that other fluorophores, including FAM, VIC, NED, PET, may be selected in the art to modify the four sets of primers in Table 2, provided that the same fluorophores are labeled for each set of primers, and are not limited to which fluorophores are selected.

TABLE 2 hemp SSR capillary electrophoresis primers and primer combinations

Example 2 differentiation of Cannabis sativa varieties Using the SSR molecular markers provided by the application

(1) DNA rapid extraction

The DNA extraction of hemp seeds is carried out by adopting a CTAB method. The method has the advantages of rapid 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 seed purity inspection and transgene detection time, improving detection efficiency and reducing detection cost. The specific operation steps are as follows: grinding several hemp seeds into powder, and placing into a 1.5mL centrifuge tube; 700 mu L of CTAB extract is added into a centrifuge tube, and incubated for 30min at 65 ℃; adding 500 mu L of chloroform-isoamyl alcohol (24:1) into a centrifuge tube, shaking vigorously, and centrifuging at 12000rpm for 10min; taking the supernatant, adding 0.7 times of isopropanol, and centrifuging at 12000rpm for 5min; removing the supernatant, and washing the 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 samples

Detecting OD values of DNA samples at 260nm and 280nm on an ultraviolet spectrophotometer, selecting OD _260/280 Samples with values of 1.8-1.9 were used for detection.

(3) Core primer selection

The primers determined in example 1, which can satisfy the capillary fluorescent detection technique, were selected by analyzing the distribution of the primers on the chromosome, the polymorphism level, the stability of PCR amplification and the band type of the amplified product, and are specifically shown in Table 2.

(4) PCR amplification and capillary electrophoresis detection

And (3) marking the screened specific SSR primer with fluorescent dyes, and selecting PET, NED, VIC, FAM fluorescent dyes in total. The PCR was performed using a reaction volume of 20. Mu.L, 10-40ng of sample DNA, 0.4. Mu.M each of forward primer and reverse primer, and 10. Mu.L of 2 Xpolymerase mixture. The reaction procedure is: pre-denaturation at 94℃for 4min; denaturation at 94℃for 45s, annealing at 60℃for 45s, extension at 72℃for 45s for 30 cycles; extending at 72℃for 10min.

Diluting the fluorescence-labeled PCR product with ultrapure water for 30 times; respectively mixing the 4 diluted solutions with equal volumes to form a mixed solution, sucking 1 mu L of the mixed solution, adding 0.5 mu L of LIZ500 molecular weight internal standard and 8.5 mu L of deionized formamide into a special deep pore plate of a DNA analyzer; then denaturing the sample on a PCR instrument at 95 ℃ for 5min, taking out, immediately placing on ice, and cooling for more than 10min; after 10s of instantaneous centrifugation, the sample is placed on a DNA analyzer for capillary electrophoresis detection. The raw data collected were analyzed using GeneMapper software. The software system directly gives the exact size of the target DNA fragment by comparing the position of the target peak with the internal standard LIZ500 in the same lane. The target band sizes of the amplified products obtained by amplifying Glioclada and Cannabis sativa No. I by the respective sets of primers in Table 2 are shown in Table 3. The target band sizes of the amplified products obtained by amplifying the cannabis sativa No.5 and the cannabis sativa No.7 by the primers in Table 2 are shown in Table 4. The target band sizes of the amplified products obtained by amplifying the cannabis sativa No.1 and You Sha-31 by the respective sets of primers in Table 2 are shown in Table 5.

TABLE 3 comparison of target band sizes for Glioclada and Cannabis A

Table 4 comparison of the band sizes of the mesh of Cannabis sativa No.5 and Cannabis sativa No.7

Table 5 comparison of the mesh band sizes of Dragon hemp No.1 and You Sha-31

(5) Determination of primer combinations

Four times of capillary electrophoresis are respectively carried out by using four groups of primers of FAM, VIC, NED, PET fluorescent label by taking Glioclada DNA as a template, so as to obtain four electrophoresis chart results; and performing capillary electrophoresis by using the Gliocladium DNA as a template and using a FAM, VIC, NED, PET fluorescent-labeled all primer mixture to obtain a total electrophoresis pattern result. Comparing the result of the total electrophoresis pattern with the result of FAM, VIC, NED, PET four groups of fluorescence labeling primers (fig. 1A-1D), it can be seen that the target peaks appearing on FAM, VIC, NED, PET single electrophoresis patterns can be distinguished on the total electrophoresis pattern, and the peaks of each color are not interfered with each other, which means that the primer mixture provided by the application can be used for primary capillary electrophoresis, the target bands are not interfered with each other, and the result is easy to judge. The same experiment was performed using the DNA of cannabis one (fig. 2A-2D), cannabis 5 (fig. 4A-4D), cannabis 7 (fig. 5A-5D), cannabis 1 (fig. 7A-7D) and You Sha-31 (fig. 8A-8D) as templates, respectively, to obtain the same conclusion.

(6) Differentiation of hemp varieties

The capillary electrophoresis was performed once with the DNA of Glioclada and Cannabis sativa as templates, respectively, using a FAM, VIC, NED, PET fluorescent labeled primer set (see Table 2), and capillary electrophoresis patterns of Glioclada and Cannabis sativa were obtained, respectively, and compared. As can be seen from FIG. 3, glioclada exhibits FAM blue peaks at 189, 195, 296 and 301bp, and Cannabis number one exhibits FAM blue peaks at 160, 212, 294 and 298 bp; glioclada shows VIC green peaks at 171, 174, 177, 208, 213 and 216bp, cannabis No. 171, 175, 177 and 213 bp; NED yellow peaks appear in Gleasia at 188, 196, 330, 334 and 351bp, and NED yellow peaks appear in Cannabis No. 180, 328, 340 and 342 bp; the glissade showed PET red peaks at 129, 230 and 291bp, cannabis No. 123, 126, 129, 132, 196, 230, 236, 242 and 291 bp. At least 2-3 different peaks appear in each fluorescent label of Glioclada and Cannabis sativa, and the two peaks are not overlapped and are clearly distinguished. Thus, the fluorescent labeled primer combinations provided by the present application (Table 2) can distinguish between Glioclada and Cannabis in a single capillary electrophoresis.

DNA of Cannabis sativa No.5 and DNA of Cannabis sativa No.7 are used as templates, and FAM, VIC, NED, PET fluorescence labeled primer combination (see Table 2) is used for performing disposable capillary electrophoresis to obtain capillary electrophoresis patterns of Cannabis sativa No.5 and DNA of Cannabis sativa No.7 respectively, and comparing. As can be seen from FIG. 6, the kenaf No.5 shows FAM blue peaks at 189 and 296bp, and the kenaf No.7 shows FAM blue peaks at 220, 300 and 306 bp; the green peaks of VIC appear in the No.5 of the dragon hemp at 172, 174, 177, 213 and 215bp, and the green peaks of VIC appear in the No.7 of the hemp at 174, 213 and 219 bp; the No.5 of the hemp shows NED yellow peaks at 182, 206, 334 and 346bp, and the No.7 of the hemp shows NED yellow peaks at 172, 188, 328 and 342 bp; the kenaf No.5 shows PET red peaks at 129, 190, 230 and 291bp, and the kenaf No.7 shows PET red peaks at 123, 175, 196, 230, 245 and 291 bp. At least 2-3 different peaks appear in each fluorescent label of the hemp No.5 and the hemp No.7, and the two peaks are not overlapped and are clear and distinguishable. Thus, the combination of fluorescent-labeled primers provided by the present application (Table 2) can distinguish between Cannabis sativa No.5 and Cannabis sativa No.7 in one capillary electrophoresis.

The DNA of Cannabis sativa No.1 and You Sha-31 were used as templates, and a FAM, VIC, NED, PET fluorescent labeled primer set (see Table 2) was used for one-time capillary electrophoresis to obtain capillary electrophoresis patterns of Cannabis sativa No.1 and You Sha-31, respectively, and comparison was made. As can be seen from FIG. 9, the Dragon hemp No.1 has FAM blue peaks at 160 and 294bp, and You Sha-31 has FAM blue peaks at 189, 195, 296 and 300 bp; the kenaf No.1 shows VIC green peaks at 172, 174, 177 and 214bp, and You Sha-31 shows VIC green peaks at 172, 175, 177, 214 and 216 bp; the No.1 of the cannabis sativa has NED yellow peaks at 172, 177, 334 and 340bp, and the You Sha-31 has NED yellow peaks at 188, 196 and 334 bp; the No.1 of the Cannabis sativa has PET red peaks at 123, 129, 190, 242 and 291bp, and You Sha-31 has PET red peaks at 129, 198, 230, 291 and 296 bp. At least 2-3 different peaks appear in each fluorescent label of the cannabis sativa No.1 and You Sha-31, and the fluorescent labels are not overlapped and are clearly distinguished. Thus, the combination of fluorescent-labeled primers provided by the present application (Table 2) can distinguish between Dragon hemp No.1 and You Sha-31 in one capillary electrophoresis.

While the application has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the application and are intended to be within the scope of the application as claimed.

Sequence listing

<110> institute of agricultural quality safety at academy of agricultural sciences of Heilongjiang province

<120> a set of SSR molecular markers for identifying cannabis varieties and uses thereof

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Claims

1. A group of specific primers for identifying SSR molecular markers of cannabis varieties, which is characterized in that the specific primers are as follows: SEQ ID No.1-2, SEQ ID No.3-4, SEQ ID No.5-6, SEQ ID No.7-8, SEQ ID No.9-10, SEQ ID No.11-12, SEQ ID No.13-14, SEQ ID No.15-16, SEQ ID No.17-18, SEQ ID No.19-20, SEQ ID No.21-22.

2. A kit for identifying a cannabis variety, comprising the specific primer of claim 1.

3. A cannabis genomic chip for identifying cannabis varieties, comprising the specific primer of claim 1.

4. Use of the specific primer of claim 1 or the kit of claim 2 or the cannabis genomic chip of claim 3 for constructing a cannabis variety DNA fingerprint database.

5. Use of the specific primer of claim 1 or the kit of claim 2 or the cannabis genomic chip of claim 3 in cannabis germplasm resources genetic diversity analysis or seed quality detection.

6. Use of the specific primer of claim 1 or the kit of claim 2 or the cannabis genomic chip of claim 3 in variety identification, genetic relationship analysis, maternal traceability of cannabis.

7. Use of the specific primer of claim 1 or the kit of claim 2 or the cannabis genomic chip of claim 3 in cannabis molecular marker assisted breeding.

8. Use according to any of claims 4-7, characterized by the following steps:

1) Extracting DNA of a hemp sample to be detected;

2) Performing PCR amplification by using the specific primer according to claim 1 by using the DNA extracted in the step 1) as a template;

3) PCR products were detected using a capillary electrophoresis system.