CN114381540A - Primer composition, kit and method for hemp composite identification polymorphic genetic marker - Google Patents

Primer composition, kit and method for hemp composite identification polymorphic genetic marker Download PDF

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CN114381540A
CN114381540A CN202111415390.5A CN202111415390A CN114381540A CN 114381540 A CN114381540 A CN 114381540A CN 202111415390 A CN202111415390 A CN 202111415390A CN 114381540 A CN114381540 A CN 114381540A
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张素华
李成涛
夏若成
于欢
陶瑞旸
陈安琪
刘希玲
阙庭志
林�源
赵珍敏
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Abstract

The invention provides a primer composition, a kit and a method capable of detecting 17 autosomal STR loci and 2 individual identification loci of a Cannabis sample in parallel, wherein the 17 autosomal STR loci comprise D02-CANN1, C11-CANN1, 4910, B01-CANN1, E07-CANN1, 9269, B05-CANN1, H06-CANN2, 5159, nH09, ANUCS501, CS1, ANUCS305, 3735, ANUCS 302, 1528 and 9043, and the 2 individual identification loci are DM029 and DM 016; the primer composition comprises a sequence of SEQ ID NO. 1-38. The primer composition, the kit and the method which contain the fluorescent marker and can detect 17 autosomal STR loci and 2 individual identification sites of the cannabis sample in parallel have the characteristics of high sensitivity, good specificity, strong stability, high accuracy and the like, and provide a brand new detection means for identification, individual identification, source deduction and STR database establishment in the forensic research of cannabis in China.

Description

Primer composition, kit and method for hemp composite identification polymorphic genetic marker
Technical Field
The invention relates to the technical field of molecular identification, in particular to a primer composition, a kit and a method for hemp composite identification polymorphic genetic markers.
Background
Cannabis is an annual plant of the genus Cannabis (Cannabiaceae) belonging to the family Cannabis, which is one of the oldest cultivated plants on earth, and is distributed mainly in Nepal, India, China, and other regions of Europe, Africa, and Asia. Hemp fiber is a good raw material for textile and paper making, cannabinoids contained in plants are widely applied to the field of medicine and health, and seeds are rich in various nutritional ingredients such as protein, amino acid, unsaturated fatty acid and trace elements which are easily absorbed by human bodies, and become important economic crops in many countries. However, because of the strong addictive and narcotic nature of Tetrahydrocannabinol (THC) contained in flowers and leaves, cannabis has been listed as one of the three drugs in parallel with heroin and cocaine by the banned convention in the united nations.
Cytological studies show that cannabis sativa is a diploid plant containing 20 chromosomes (18 of which are autosomes and 2 of which are sex chromosomes), the female chromosome type is XX, and the male chromosome type is XY. The utilization values of hemp male and female plants are different, the fiber quality of the male plant is obviously higher than that of the female plant, the male plant does not contain THC and Cannabidiol (CBD) or has less content, and the female plant has relatively higher content of THC and Cannabidiol (CBD), so the female plant has higher medicinal value and abuse potential.
In recent years, the number of people sucking or abusing cannabis in our country is continuously increasing due to the influence of the legalization of cannabis in some countries and regions in the world, and the number of cases for selling cannabis across countries is increasing. Therefore, the rapid and accurate judicial identification and toxic source tracking of cannabis has become an urgent social need. The conventional identification method of cannabis is mainly to analyze morphology and chemical components. However, in some cases, hemp is processed or mixed with tobacco leaves and the like, so that people cannot morphologically recognize such a material. For the analysis of chemical components of the hemp, the THC in the biological detection material is qualitatively and quantitatively analyzed by adopting a gas chromatography-mass spectrometry (GC-MS) method; however, the GC-MS method requires a large amount of fresh samples for the analysis of hemp chemical components because THC is easily oxidized in old samples and the content of THC is affected by the development stage, parts and planting environment of hemp plants. In addition, gender differences have a greater impact on cannabis THC content, however, old cannabis samples, processed cannabis samples, and cannabis prior to flowering stage do not allow for a morphologically or chemically accurate determination of cannabis plant gender. Finally, the individual identification and source inference of cannabis is also of great importance in practical cases, and it is precisely morphological and chemical analysis that is not possible. To better meet the needs of species identification, individual identification and source inference of cannabis in forensic identification, scientists have attempted to seek new approaches to address these problems.
With the rapid development of molecular biology technology, identification of cannabis on the DNA level is becoming a research hotspot and a new technological approach. At present, research on the hemp DNA genetic marker mainly focuses on RAPD, AFLP, SCAR, DNA barcode, STR and the like. Research data show that: the RAPD, AFLP and SCAR genetic markers can identify the species and sex of the hemp; the DNA bar code can accurately identify the hemp and the counterfeit products thereof, but the genetic markers can not carry out individualized identification and regional source inference on the hemp.
The STR is an oligonucleotide sequence formed by connecting 2-6 bp core sequences in series, has the advantages of high sensitivity, high identification capability, high species specificity, high accuracy of results, easiness in standardization and the like, is widely applied to individual identification, genetic identification and population survey in the field of judicial identification, and becomes the most widely applied genetic marker in the judicial identification. Based on this, some forensic scientists have attempted to apply STR genetic markers to the identification studies of cannabis and demonstrated the potential of STR genetic markers in the identification of cannabis, differentiation of cannabis varieties and inference of cannabis origin, etc. At present, 28 marijuana STR loci are discovered and reported at home and abroad. In 2003, Hsieh et al reported the first hemp STR locus CS1, observed the repetition times of the locus varied from 3 to 40 times in 108 hemp samples, and the heterozygosity was about 87.04%, which proves that the CS1 locus has high polymorphism. Later, more hemp polymorphic STR loci were developed, for example, Alghanim and others developed 11 hemp STR loci with polymorphisms, such as C11-CANN1, B01-CANN1 and D02-CANN1, and Valverde and others developed hemp STR loci with 6 four-base repeats, such as 5159, 4910 and 1528, which are also the first reported four-base repeat hemp STR loci. Based on the continuously developed hemp STR locus, the research of the hemp STR locus multiplex amplification system is also developed. In 2008, Howard and the like successfully constructed a multiplex amplification system with 10 STR loci, and the system was subjected to verification research on sensitivity, stability, species specificity and the like for the first time according to a DNA Analysis method Scientific Working Group (SWGDAM) verification guide, and the results show that the system can be used for establishing a hemp STR genetic database. Houston et al successfully constructs an STR multiplex amplification system of 13 loci by screening a hemp STR reported in earlier research and combining 6 four-base repeat STRs developed by Valverde et al, which is also the system researched and applied most at present. The research on hemp STR loci in China just started. In 2008, 3 loci ANUCS 301, ANUCS305 and CS1 with similar amplification conditions and higher heterozygosity are selected from Malaria and the like to investigate the individual and group genetic polymorphism of the cannabis sativa, and a theoretical basis is provided for deducing the variety and the producing area of the cannabis sativa of the drug protospecies by using STR genetic information.
In conclusion, the research on cannabis is mainly focused on identification of drug type cannabis and non-drug type cannabis in biochemical detection, and the research on cannabis DNA is relatively rare, and the research related to individual identification and source deduction is more rare. The research on the hemp STR multiplex amplification system is in the first stage abroad, and the research is hardly carried out at home.
Disclosure of Invention
The invention aims to fill the blank of the research of a hemp STR multiplex amplification system in China, provide a theoretical basis and a detection method for realizing hemp sex identification, individual identification and source deduction, and develop and establish a primer composition, a kit and a method capable of detecting 17 autosomal STR loci and 2 individual identification loci of a hemp sample in parallel.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the invention provides a primer composition for hemp complex identification of polymorphic genetic markers, which comprises amplification primers of 17 autosomal STR loci of hemp and amplification primers of 2 individual identification sites; wherein, the 17 autosomal STR loci comprise D02-CANN1, C11-CANN1, 4910, B01-CANN1, E07-CANN1, 9269, B05-CANN1, H06-CANN2, 5159, nH09, ANUCS501, CS1, ANUCS305, 3735, ANUCS 302, 1528 and 9043, and the 2 identification positioning points are DM029 and DM 016.
Further, the sequences of the amplification primers of the 17 autosomal STR loci and the amplification primers of the 2 individual identification loci of Cannabis sativa are as follows:
D02-CANN1：SEQ ID NO.1-2；C11-CANN1：SEQ ID NO.3-4；DM029：SEQ ID NO.5-6；DM016：SEQ ID NO.7-8；4910：SEQ ID NO.9-10；B01-CANN1：SEQ ID NO.11-12；E07-CANN1：SEQ ID NO.13-14；9269：SEQ ID NO.15-16；B05-CANN1：SEQ ID NO.17-18；H06-CANN2：SEQ ID NO.19-20；5159：SEQ ID NO.21-22；nH09：SEQ ID NO.23-24；ANUCS 501：SEQ ID NO.25-26；CS1：SEQ ID NO.27-28；ANUCS 305：SEQ ID NO.29-30；3735：SEQ ID NO.31-32；ANUCS 302：SEQ ID NO.33-34；1528：SEQ ID NO.35-36；9043：SEQ ID NO.37-38。
further, at least one primer in each pair of amplification primers is labeled with a fluorescent dye selected from one of FAM, HEX, TRMRA, and ROX.
Further, the amplification primers of D02-CANN1, C11-CANN1, DM029, DM016, 4910 and BO1-CANN1 are marked by FAM; the amplification primers of E07-CANN1, 9269, B05-CANN1, H06-CANN2, 5159 and nH09 are marked by HEX; the amplification primers of the ANUCS501, the CS1 and the ANUCS305 are marked by TRMRA; 3735. the amplification primers of ANUCS 302, 1528 and 9043 are marked by ROX.
In a second aspect, the present invention also provides a kit for multiplex identification of polymorphic genetic markers in cannabis comprising the above primer composition, further comprising 4 XPCR reaction premix VII [50mM Tris-HCl (pH 8.3), 50mM KCl,1.5mM MgCl2,0.2mM dNTPs,0.08mg/mL bovine serum albumin(BSA)]And deionized water.
In a third aspect, the invention also provides a method for carrying out compound identification on polymorphic genetic markers of cannabis by adopting the primer composition or the kit, which is particularly used for cannabis sex identification, individual identification and source deduction; the method comprises the following steps:
carrying out PCR composite amplification on a genome DNA sample of a sample to be detected by adopting an amplification primer with a sequence shown in SEQ ID NO. 1-38;
step two, mixing the PCR amplification product with a proper amount of molecular weight internal standard and formamide uniformly; denaturing the mixture and cooling;
and step three, adopting a genetic analyzer to carry out typing detection.
Further, the total volume of the amplification system for PCR multiplex amplification is 10. mu.L, and the amplification system comprises: 4 XPCR reaction premix VII 2.5 uL, primer mix (10 uL) 1 uL, deionized water 5.5 uL, 1 ng/. mu.L genomic DNA1 uL.
Further, the concentrations of the amplification primers in the amplification system are as follows: D02-CANN 1: 0.03 mu M; C11-CANN 1: 0.03 mu M; DM 029: 0.05 μ M; DM 016: 0.03 mu M; 4910: 0.04 μ M; B01-CANN 1: 0.06 μ M; E07-CANN 1: 0.05 μ M; 9269: 0.04 μ M; B05-CANN 1: 0.03 mu M; H06-CANN 2: 0.04 μ M; 5159: 0.04 μ M; nH 09: 0.04 μ M; ANUCS 501: 0.05 μ M; CS 1: 0.05 μ M; ANUCS 305: 0.05 μ M; 3735: 0.05 μ M; ANUCS 302: 0.04 μ M; 1528: 0.05 μ M; 9043: 0.05. mu.M.
Further, the procedure of the PCR multiplex amplification is as follows: pre-denaturation at 95 ℃ for 2 min; denaturation at 95 ℃ for 5 seconds, annealing at 56 ℃ for 1 minute, and extension at 60 ℃ for 30 seconds for 28 cycles; final extension at 60 ℃ for 5 min; keeping the temperature at 15 ℃.
Further, the PCR composite amplification adopts a reaction thermal cycler; the reaction thermal cycler is selected from one of ABI9700, ABI 9600, ABI2720, Bio-Rad iCycler, and Bio-Rad C1000.
Further, the molecular weight internal Standard is marked by T500Size Standard and LIZ fluorescence; the fluorescence was corrected using 5-Dye Matrix Standards.
Further, in the second step, the volume ratio of the PCR amplification product, the molecular weight internal standard and the formamide is 1:8.5: 0.5.
Further, the genetic analyzer is selected from one of 3100 series, 3130 series and 3500 series genetic analyzers.
Further, the first step also comprises the step of carrying out DNA extraction on flowers, stems, leaves and/or seeds of the sample to be detected to prepare a genome DNA sample.
Further, the denaturation condition in the second step is 95 ℃ for 3 minutes; the cooling conditions were ice cooling for 3 minutes.
By adopting the technical scheme, compared with the prior art, the invention has the following technical effects:
1. the invention simultaneously amplifies 17 autosomal STR loci and 2 individual identification points of the hemp, and is superior to a composite amplification system which contains at most 15 loci in earlier research; in addition, the invention firstly tries to add the sex identification site into the hemp STR composite amplification system.
2. The invention provides an effective method for further research of the hemp STR locus, establishment of a hemp STR database and detection and identification of hemp in a case.
3. The invention establishes a forensic medicine detection kit with high sensitivity, good specificity and strong stability, provides an effective detection tool for the sex identification, individual identification, source deduction and the like of the cannabis sativa, and has important significance for meeting the requirement of the cannabis judicial identification.
Drawings
FIG. 1 is a Ladder typing map of multiplex amplification for identification of hemp polymorphic genetic markers in one embodiment of the present invention;
FIG. 2 is a typing map of female sample DNA using multiplex amplification method for hemp multiplex identification comprising 17 autosomal STR loci and 2 identity discrimination sites according to one embodiment of the present invention;
FIG. 3 is a graph of the typing map of male sample DNA using the multiplex amplification method for the multiplex identification of cannabis containing 17 autosomal STR loci and 2 discriminatory loci according to one embodiment of the present invention.
FIG. 4 is a profile of identity studies for multiplex identification of multiplex amplification methods comprising 17 autosomal STR loci and 2 discriminatory loci according to one embodiment of the present invention;
FIG. 5 is a graph showing the results of a sensitivity study of a multiplex amplification method for multiplex identification of cannabis plants comprising 17 autosomal STR loci and 2 discriminatory loci according to one embodiment of the present invention;
FIG. 6 is a typing map for species-specific studies of multiplex amplification methods for complex identification of cannabis sativa containing 17 autosomal STR loci and 2 discriminatory loci in accordance with one embodiment of the present invention.
Detailed Description
The invention provides a primer composition, a kit and a method for hemp composite identification polymorphic genetic markers, and is particularly used for hemp gender identification, individual identification and source deduction. The primer composition comprises amplification primers of 17 hemp autosomal STR loci and amplification primers of 2 individual identification sites; wherein, the 17 autosomal STR loci comprise D02-CANN1, C11-CANN1, 4910, B01-CANN1, E07-CANN1, 9269, B05-CANN1, H06-CANN2, 5159, nH09, ANUCS501, CS1, ANUCS305, 3735, ANUCS 302, 1528 and 9043, and the 2 identification positioning points are DM029 and DM 016.
In a preferred embodiment of the present invention, the sequences of the amplification primers for the 17 autosomal STR loci and the amplification primers for the 2 individualized loci are as follows:
D02-CANN1：SEQ ID NO.1-2；C11-CANN1：SEQ ID NO.3-4；DM029：SEQ ID NO.5-6；DM016：SEQ ID NO.7-8；4910：SEQ ID NO.9-10；B01-CANN1：SEQ ID NO.11-12；E07-CANN1：SEQ ID NO.13-14；9269：SEQ ID NO.15-16；B05-CANN1：SEQ ID NO.17-18；H06-CANN2：SEQ ID NO.19-20；5159：SEQ ID NO.21-22；nH09：SEQ ID NO.23-24；ANUCS 501：SEQ ID NO.25-26；CS1：SEQ ID NO.27-28；ANUCS 305：SEQ ID NO.29-30；3735：SEQ ID NO.31-32；ANUCS 302：SEQ ID NO.33-34；1528：SEQ ID NO.35-36；9043：SEQ ID NO.37-38。
in a preferred embodiment of the present invention, at least one primer of each pair of amplification primers is labeled with a fluorescent dye selected from one of FAM, HEX, TRMRA, and ROX.
In a preferred embodiment of the invention, the amplification primers of D02-CANN1, C11-CANN1, DM029, DM016, 4910, BO1-CANN1 are labeled with FAM; the amplification primers of E07-CANN1, 9269, B05-CANN1, H06-CANN2, 5159 and nH09 are marked by HEX; the amplification primers of the ANUCS501, the CS1 and the ANUCS305 are marked by TRMRA; 3735. the amplification primers of ANUCS 302, 1528 and 9043 are marked by ROX.
In addition, the kit comprises the primer composition for hemp composite identification of polymorphic genetic markers, 4 XPCR reaction premixed liquid VII and deionized water.
The present invention will be described in detail and specifically with reference to the following examples and drawings so as to provide a better understanding of the invention, but the following examples do not limit the scope of the invention.
In the examples, the conventional methods were used unless otherwise specified, and reagents used were those conventionally commercially available or formulated according to the conventional methods without specifically specified.
Example 1
The embodiment provides a method for the compound identification of polymorphic genetic markers of cannabis, which comprises the following steps:
1. screening of cannabis STR loci suitable for forensic applications;
according to literature reports and gene databases, the currently developed marijuana STR loci are searched, but not all the STR loci are suitable for the research of a marijuana multiplex amplification system. Polymorphic STR loci are screened to knock out dinucleotide repeats in the loci. And finally, selecting 17 optimal autosomal STR loci and 2 individual identification loci for kit development, wherein the loci are D02-CANN1, C11-CANN1, DM029, DM016, 4910, B01-CANN1, E07-CANN1, 9269, B05-CANN1, H06-CANN2, 5159, nH09, ANUCS501, CS1, ANUCS305, 3735, ANUCS 302, 1528 and 9043. Wherein, the sex determination site DM029 is related to the X chromosome, all samples show a single peak, the sex determination site DM016 is related to the Y chromosome, all male samples show a single peak, and female samples do not show a peak.
2. Designing an amplification primer component;
establishing a composite amplification detection kit containing five fluorescent labels (FAM, HEX, TAMRA, ROX and LIZ), wherein the LIZ fluorescent label (T500) is adopted as the molecular weight internal standard.
The genetic markers and their corresponding amplification primer sequences, marker fluorescence and final reaction concentrations described in this example are shown in Table 1.
TABLE 1 primer sequences, final reaction concentrations and labeled fluorescence used for multiplex amplification
Figure BDA0003375071970000081
Figure BDA0003375071970000091
Figure BDA0003375071970000101
3. Constructing and optimizing a multiplex PCR amplification system;
and adjusting and optimizing PCR reaction conditions including primer proportioning concentration, DNA template amount, annealing temperature, cycle number and the like on the constructed composite amplification parting system to obtain a balanced and stable PCR product parting result and realize the composite amplification of 19 genetic markers.
The multiplex PCR amplification system described in this example, comprising 17 autosomal STR loci from cannabis and 2 unique loci, is shown in table 2, wherein DNA is extracted from cannabis samples, such as flowers, stems, leaves, seeds.
TABLE 2 multiplex PCR amplification System comprising 17 autosomal STR loci from Cannabis sativa and 2 discriminatory loci
Figure BDA0003375071970000102
The reaction system can obtain good results on various reaction thermal cyclers (such as ABI9700, ABI 9600, ABI2720, Bio-RadiCycler, etc.) by adopting the following procedures: pre-denaturation at 95 ℃ for 2 min; denaturation at 95 ℃ for 5 seconds, annealing at 56 ℃ for 1 minute, and extension at 60 ℃ for 30 seconds for 28 cycles; final extension at 60 ℃ for 5 min; keeping the temperature at 15 ℃.
4. Establishing an analysis method and detecting a composite amplification product;
a 3100 series, 3130 series, or 3500 series genetic analyzer spectral calibration (Matrix) file was created. When sample is added by capillary electrophoresis, 1 mu L of PCR is amplifiedThe amplification product was mixed with 8.5. mu.L formamide, 0.5. mu.L molecular weight internal Standard (T500Size Standard); the mixture was denatured at 95 ℃ for 3 minutes, then placed on ice to cool for 3 minutes; and (3) carrying out typing detection on the 19 genetic markers by adopting the genetic analyzer with the model. Obtaining electrophoretic migration parameters of different alleles of each marker by capillary electrophoresis, and respectively obtaining the electrophoretic migration parameters based on the electrophoretic migration parameters according to GeneMapperID v3.2.1 and GeneMapperID v3.2.1
Figure BDA0003375071970000111
The format of the ID-X v1.5 software requires that corresponding Bin and Panel files be written to create an electrophoretic analysis method.
Wherein, the Ladder typing map using the above method is shown in FIG. 1, the typing map of the DNA of the female sample using the above method is shown in FIG. 2, and the typing map of the DNA of the male sample using the above method is shown in FIG. 3.
Example 2
This example illustrates the detection of cannabis sample DNA using the method provided in example 1.
Different tissues (flowers, stems, leaves and seeds) of one cannabis sample were collected for DNA extraction and quantification. The above DNA samples were subjected to multiplex amplification of 19 genetic markers using the kit constructed in example 1. All samples gave efficient amplification products on the genetic markers.
Example 3
In this embodiment, forensic verification (identity, sensitivity, species specificity, and forensic parameter calculation) is performed on the method provided in embodiment 1 according to the SWGDAM requirement, and then the superiority of the method is determined, specifically, the following operations are performed:
(1) identity studies: genomic DNA from the flower, leaf and stem of the same plants was quantified to 1 ng/. mu.L and detected separately using the method provided in example 1.
The results show (fig. 4) that the kit constructed in example 1 has good tissue identity: flowers, leaves and stems of the same plant have the same genotype.
(2) Sensitivity study: cannabis sativa genomic DNA was diluted in multiples of 2ng/μ L, 1ng/μ L, 0.5ng/μ L, 0.25ng/μ L, 0.125ng/μ L, 0.0625ng/μ L, 0.03125ng/μ L and 0.015625ng/μ L, and the detection was performed on cannabis sativa genomic DNA at the above concentrations using the method provided in example 1, and the detection was repeated 3 times for each template amount sample.
The results show (fig. 5) that the kit constructed in example 1 has high sensitivity: complete genotyping of genetic markers can be obtained at DNA template amounts as low as 0.125 ng.
(3) Species-specific studies: genomic DNAs from dogs, cats, mice, sheep, pigs, cattle, rabbits, chickens, ducks, monkeys, nightshade, corn poppy, mulberry leaves, poppy, sage, and humulus were quantified to 5 ng/. mu.L, and the above-mentioned genomic DNAs not derived from hemp were each detected by the method provided in example 1.
The results show (fig. 6) that the kit constructed in example 1 has species specificity: no product peaks were detected from DNA from species other than sage and humulus, and although some product peaks were observed in the sage and humulus samples, most were not in allelic positions and were not the same as the normal marijuana peak patterns, and did not affect the interpretation of the results.
(4) And (3) calculating medical parameters: 126 parts of cannabis genomic DNA were quantified to 1 ng/. mu.L and detected by the method provided in example 1. And counting and calculating heterozygosity, polymorphism information content, individual identification probability, non-father exclusion probability, accumulated individual identification probability, accumulated non-father exclusion probability and sex information of each sample of the 17 autosomal STR loci.
The results show (table 3) that the kit constructed in example 1 has better system efficacy: the heterozygosity range of the 17 autosomal STR loci in 126 cannabis sativa samples is 0.2381-0.7937, the content range of polymorphism information is 0.2754-0.9419, the individual identification probability range is 0.4624-0.9855, the non-father exclusion probability range is 0.0410-0.5873, and the cumulative individual identification probability is 1-3.0 x 10-15Cumulative non-parent exclusion probability of 1-7.4 × 10-3(ii) a The marijuana sex determination sites DM029 and DM016 can meet the requirement of sex determination in a detection sample, two peaks DM029 and DM016 are detected in all male samples, and only a single peak DM029 exists in a female sample.
TABLE 3 forensic parameters of the Cannabis 17 autosomal STR loci
Figure BDA0003375071970000121
Figure BDA0003375071970000131
The embodiment shows that the primer composition, the kit and the method which contain the fluorescent marker and can detect 17 autosomal STR loci and 2 individual identification sites of the cannabis sample in parallel provide a brand-new detection means for identification, individual identification, source deduction and STR database establishment in the forensic research of cannabis in China.
The embodiments of the present invention have been described in detail, but the embodiments are merely examples, and the present invention is not limited to the embodiments described above. Any equivalent modifications and substitutions to those skilled in the art are also within the scope of the present invention. Accordingly, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered by the present invention.
Sequence listing
<110> institute of science and technology for judicial identification
<120> primer composition, kit and method for hemp complex identification of polymorphic genetic markers
<150> 2021102127562
<151> 2021-02-25
<160> 38
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA
<213> primers for amplifying D02-CANN1 (Artificial Sequence)
<400> 1
ggttgggatg ttgttgttgt g 21
<210> 2
<211> 21
<212> DNA
<213> primers for amplifying D02-CANN1 (Artificial Sequence)
<400> 2
agaaatccaa ggtcctgatg g 21
<210> 3
<211> 22
<212> DNA
<213> primers for amplification of C11-CANN1 (Artificial Sequence)
<400> 3
gtggtggtga tgatgataat gg 22
<210> 4
<211> 19
<212> DNA
<213> primers for amplification of C11-CANN1 (Artificial Sequence)
<400> 4
tgaattggtt acgatggcg 19
<210> 5
<211> 20
<212> DNA
<213> primers for the amplification of DM029 (Artificial Sequence)
<400> 5
gatgacagac ttcctgattg 20
<210> 6
<211> 20
<212> DNA
<213> primers for the amplification of DM029 (Artificial Sequence)
<400> 6
gtctaagagt gggaatgcta 20
<210> 7
<211> 18
<212> DNA
<213> primers for amplification of DM016 (Artificial Sequence)
<400> 7
gcccaagttg ctgctgag 18
<210> 8
<211> 18
<212> DNA
<213> primers for amplification of DM016 (Artificial Sequence)
<400> 8
cccaccgttt agggagca 18
<210> 9
<211> 19
<212> DNA
<213> primer for amplification 4910 (Artificial Sequence)
<400> 9
agattcccaa gatgagcaa 19
<210> 10
<211> 20
<212> DNA
<213> primer for amplification 4910 (Artificial Sequence)
<400> 10
acaaactggt atcaagagcc 20
<210> 11
<211> 22
<212> DNA
<213> primers for amplification of B01-CANN1 (Artificial Sequence)
<400> 11
atgacatacc agacagaaac tc 22
<210> 12
<211> 21
<212> DNA
<213> primers for amplification of B01-CANN1 (Artificial Sequence)
<400> 12
catccatagc attatcccac t 21
<210> 13
<211> 19
<212> DNA
<213> primers for amplification of E07-CANN1 (Artificial Sequence)
<400> 13
caaatgccac accaccttc 19
<210> 14
<211> 21
<212> DNA
<213> primers for amplification of E07-CANN1 (Artificial Sequence)
<400> 14
gtggtagcca ggtataggta g 21
<210> 15
<211> 20
<212> DNA
<213> primers for amplifying 9269 (Artificial Sequence)
<400> 15
cccaaactac tgtttgtgcc 20
<210> 16
<211> 22
<212> DNA
<213> primers for amplifying 9269 (Artificial Sequence)
<400> 16
acttgcacgt gatgttagat cc 22
<210> 17
<211> 19
<212> DNA
<213> primers for amplification of B05-CANN1 (Artificial Sequence)
<400> 17
ttgatggtgg tgaaacggc 19
<210> 18
<211> 21
<212> DNA
<213> primers for amplification of B05-CANN1 (Artificial Sequence)
<400> 18
ccccaatctc aatctcaacc c 21
<210> 19
<211> 19
<212> DNA
<213> primers for amplification of H06-CANN2 (Artificial Sequence)
<400> 19
tggtttcagt ggtcctctc 19
<210> 20
<211> 19
<212> DNA
<213> primers for amplification of H06-CANN2 (Artificial Sequence)
<400> 20
acgtgagtga tgacacgag 19
<210> 21
<211> 20
<212> DNA
<213> primer for amplification 5159 (Artificial Sequence)
<400> 21
ccagagcttg tggatctcct 20
<210> 22
<211> 20
<212> DNA
<213> primer for amplification 5159 (Artificial Sequence)
<400> 22
agtacgaaag ggcactgagg 20
<210> 23
<211> 21
<212> DNA
<213> primers for amplifying nH09 (Artificial Sequence)
<400> 23
ccaacatttt ctcagaaccc a 21
<210> 24
<211> 21
<212> DNA
<213> primers for amplifying nH09 (Artificial Sequence)
<400> 24
tcttgactgt agtaatccag c 21
<210> 25
<211> 22
<212> DNA
<213> primers for amplifying ANUCS501 (Artificial Sequence)
<400> 25
agcaataatg gagtgagtga ac 22
<210> 26
<211> 23
<212> DNA
<213> primers for amplifying ANUCS501 (Artificial Sequence)
<400> 26
agagatcaag aaattgagat tcc 23
<210> 27
<211> 20
<212> DNA
<213> primers for amplifying CS1 (Artificial Sequence)
<400> 27
aagcaactcc aattccagcc 20
<210> 28
<211> 23
<212> DNA
<213> primers for amplifying CS1 (Artificial Sequence)
<400> 28
taatgatgag acgagtgaga acg 23
<210> 29
<211> 16
<212> DNA
<213> primer for amplifying ANUCS305 (Artificial Sequence)
<400> 29
agcccgaccg tgaaga 16
<210> 30
<211> 17
<212> DNA
<213> primer for amplifying ANUCS305 (Artificial Sequence)
<400> 30
tgaagccgat gccctat 17
<210> 31
<211> 22
<212> DNA
<213> primer for amplification of 3735 (Artificial Sequence)
<400> 31
tgattctgtg tttgtgtgca at 22
<210> 32
<211> 20
<212> DNA
<213> primer for amplification of 3735 (Artificial Sequence)
<400> 32
catcgcaccc acaggttagt 20
<210> 33
<211> 21
<212> DNA
<213> primers for amplifying ANUCS 302 (Artificial Sequence)
<400> 33
aacataaaca ccaacaactg c 21
<210> 34
<211> 20
<212> DNA
<213> primers for amplifying ANUCS 302 (Artificial Sequence)
<400> 34
atggttgatg ttttgatggt 20
<210> 35
<211> 21
<212> DNA
<213> primer for amplification 1528 (Artificial Sequence)
<400> 35
ggactttgtc tagtgccttt g 21
<210> 36
<211> 20
<212> DNA
<213> primer for amplification 1528 (Artificial Sequence)
<400> 36
gagtacttgg ctgatgatgg 20
<210> 37
<211> 21
<212> DNA
<213> primer for amplification 9043 (Artificial Sequence)
<400> 37
aggtctgcgt tgtgcattat t 21
<210> 38
<211> 19
<212> DNA
<213> primer for amplification 9043 (Artificial Sequence)
<400> 38
agggctggtt tcagtttcg 19

Claims (14)

1. A primer combination for hemp composite identification polymorphism genetic marker is characterized in that the primer combination comprises amplification primers of 17 hemp autosomal STR loci and amplification primers of 2 individual identification sites; wherein the 17 autosomal STR loci comprise D02-CANN1, C11-CANN1, 4910, B01-CANN1, E07-CANN1, 9269, B05-CANN1, H06-CANN2, 5159, nH09, ANUCS501, CS1, ANUCS305, 3735, ANUCS 302, 1528 and 9043, and the 2 individual identification anchor points are DM029 and DM 016.
2. The primer composition of claim 1, wherein the amplification primers for the 17 autosomal STR loci and the amplification primers for the 2 individualized loci are specifically as follows:
D02-CANN1：SEQ ID NO.1-2；C11-CANN1：SEQ ID NO.3-4；DM029：SEQ ID NO.5-6；DM016：SEQ ID NO.7-8；4910：SEQ ID NO.9-10；B01-CANN1：SEQ ID NO.11-12；E07-CANN1：SEQ ID NO.13-14；9269：SEQ ID NO.15-16；B05-CANN1：SEQ ID NO.17-18；H06-CANN2：SEQ ID NO.19-20；5159：SEQ ID NO.21-22；nH09：SEQ ID NO.23-24；ANUCS 501：SEQ ID NO.25-26；CS1：SEQ ID NO.27-28；ANUCS 305：SEQ ID NO.29-30；3735：SEQ ID NO.31-32；ANUCS 302：SEQ ID NO.33-34；1528：SEQ ID NO.35-36；9043：SEQ ID NO.37-38。
3. the primer composition of claim 1, wherein at least one primer of each amplification primer pair is labeled with a fluorescent dye selected from the group consisting of FAM, HEX, TRMRA, and ROX.
4. The primer composition of claim 3, wherein the amplification primers of D02-CANN1, C11-CANN1, DM029, DM016, 4910, BO1-CANN1 are labeled with FAM; the amplification primers of E07-CANN1, 9269, B05-CANN1, H06-CANN2, 5159 and nH09 are marked by HEX; the amplification primers of the ANUCS501, the CS1 and the ANUCS305 are marked by TRMRA; 3735. the amplification primers of ANUCS 302, 1528 and 9043 are marked by ROX.
5. A kit for multiplex identification of polymorphic genetic markers in cannabis sativa comprising a primer composition according to any one of claims 1-4, further comprising 4 XPCR premix VII, deionized water.
6. Method for the complex identification of polymorphic genetic markers in cannabis, using a primer composition according to any one of claims 1 to 4 or a kit according to claim 5, in particular for cannabis sexing, individual identification and source deduction; the method comprises the following steps:
carrying out PCR composite amplification on a genome DNA sample of a sample to be detected by adopting an amplification primer with a sequence shown in SEQ ID NO. 1-38;
step two, mixing the PCR amplification product with a proper amount of molecular weight internal standard and formamide uniformly; denaturing the mixture and cooling;
and step three, adopting a genetic analyzer to carry out typing detection.
7. The method according to claim 6, wherein the total volume of the PCR multiplex amplification system is 10 μ L, and comprises: 4 XPCR reaction premix VII 2.5 uL, 10 XPCR primer mixture 1 uL, deionized water 5.5 uL, 1 ng/. mu.L genomic DNA1 uL.
8. The method of claim 6, wherein the concentrations of the amplification primers in the amplification system are as follows: D02-CANN 1: 0.03 mu M; C11-CANN 1: 0.03 mu M; DM 029: 0.05 μ M; DM 016: 0.03 mu M; 4910: 0.04 μ M; B01-CANN 1: 0.06 μ M; E07-CANN 1: 0.05 μ M; 9269: 0.04 μ M; B05-CANN 1: 0.03 mu M; H06-CANN 2: 0.04 μ M; 5159: 0.04 μ M; nH 09: 0.04 μ M; ANUCS 501: 0.05 μ M; CS 1: 0.05 μ M; ANUCS 305: 0.05 μ M; 3735: 0.05 μ M; ANUCS 302: 0.04 μ M; 1528: 0.05 μ M; 9043: 0.05. mu.M.
9. The method of claim 6, wherein the PCR multiplex amplification procedure is: pre-denaturation at 95 ℃ for 2 min; denaturation at 95 ℃ for 5 seconds, annealing at 56 ℃ for 1 minute, and extension at 60 ℃ for 30 seconds for 28 cycles; final extension at 60 ℃ for 5 min; keeping the temperature at 15 ℃.
10. The method of claim 6, wherein the PCR multiplex amplification is performed using a reaction thermal cycler; the reaction thermal cycler is selected from one of ABI9700, ABI 9600, ABI2720, Bio-Rad iCycler and Bio-Rad C1000.
11. The method according to claim 6, wherein the internal molecular weight Standard is labeled with T500Size Standard and with LIZ fluorescence; the fluorescence was corrected using 5-Dye Matrix Standards.
12. The method of claim 6, wherein in step two, the volume ratio of the PCR amplification product, the molecular weight internal standard and the formamide is 1:8.5: 0.5.
13. The method of claim 6, wherein the genetic analyzer is selected from one of the 3100 series, 3130 series, and 3500 series genetic analyzers.
14. The method of claim 6, wherein step one further comprises performing DNA extraction on flowers, stems, leaves and/or seeds of the sample to be tested to prepare a genomic DNA sample.
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