CN110760610B - Polymorphic primer of camphor chloroplast SNP molecular marker and application thereof - Google Patents
Polymorphic primer of camphor chloroplast SNP molecular marker and application thereof Download PDFInfo
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Abstract
The invention discloses a polymorphic primer of camphor chloroplast SNP molecular marker and application thereof, belonging to the technical field of forestry molecular biology. The invention screens 11 pairs of polymorphic primers of cinnamomum camphora chloroplast SNP molecular markers, and applies the primers to cinnamomum camphora genetic diversity analysis and cinnamomum camphora lineage analysis, and chloroplast haplotype analysis shows that the distribution difference of different haplotypes is obvious, most haplotypes only exist in a specific population, only a few haplotypes are shared haplotypes, and the fact that certain geographical differentiation exists among the haplotypes is shown. The genetic differentiation coefficient Fst was 0.212 and the gene flow Nm was 0.929 for the 18 cinnamomum camphora populations, indicating a moderate gene flow between the cinnamomum camphora populations. The SNP molecular marker disclosed by the invention is good in repeatability, can comprehensively reveal the true level of genetic diversity of the cinnamomum camphora, and provides a theoretical basis for the protection and utilization of wild cinnamomum camphora resources.
Description
Technical Field
The invention belongs to the technical field of forestry molecular biology, and particularly relates to a polymorphic primer of camphor chloroplast SNP molecular markers and application thereof.
Background
Cinnamomum camphora (L.) Presl) is a tall tree of Lauraceae family, has important ecological, cultural and economic values, and is an important wood and special economic tree species in China. In recent years, many reports have been made on researches on taxonomy, physiology, chemical components and utilization, fine variety breeding, forest culture and management technologies of cinnamomum camphora, but the researches on genetic diversity of germplasm resources are not sufficient. The cinnamomum camphora has an early cultivation history in China, the introduction and cultivation conditions among regions are frequent, and the cinnamomum camphora has great difference aiming at the sample origin of population genetic analysis. In addition, in terms of research means, co-dominant DNA markers such as RAPD, ISSR and the like used in the early stage have poor repeatability and low polymorphism, and the real level of genetic diversity of the cinnamomum camphora cannot be completely revealed.
SNPs are called third generation molecular markers and are polymorphisms in DNA sequences caused by mutations of single nucleotides in the genome, which exist in the form of transversions, transitions, insertions and deletions of single bases. Compared with other molecular markers, SNPs have several following points: 1) the density is high, the distribution is wide, the SNP distribution is wide, and the SNP is distributed all over the whole genome; 2) the genetic stability of the SNP is higher than that of a repeated sequence polymorphic marker such as SSR; 3) the polymorphism, although SNP has only two allelic types and has less information amount in individuals compared with the SSR markers commonly used at present, the difference is made up by the high density and the stability of SNP; 4) representatively, some SNPs located inside genes may directly affect protein structure or expression level, and thus they may represent some of the contributing factors in the genetic mechanism of disease; 5) the method is easy to realize the automation of analysis, does not need to detect fragment length polymorphism, and is easy to realize automation. Due to the above characteristics, SNP is often used for studies on genetic diversity, construction of genetic linkage maps, germplasm resource identification, association analysis, and the like.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a polymorphic primer of camphor chloroplast SNP molecular marker, which is used for solving the problems of poor repeatability and low polymorphism of codominant DNA markers such as RAPD, ISSR and the like used in the prior art; the invention also aims to provide application of the polymorphic primer of the cinnamomum camphora chloroplast SNP molecular marker in the genetic diversity analysis and the archaea camphora lineage analysis of the cinnamomum camphora, so as to solve the problem that the prior art is difficult to comprehensively disclose the true level of the cinnamomum camphora genetic diversity.
In order to achieve the purpose of the invention, the invention adopts the technical scheme that:
the polymorphic primers of the cinnamomum camphora chloroplast SNP molecular marker comprise 11 pairs of polymorphic primers, and the nucleotide sequences of the primers are as follows:
the CpSNP1 upstream primer is shown as SEQ ID NO. 1;
the CpSNP1 downstream primer is shown as SEQ ID NO. 2;
the CpSNP2 upstream primer is shown as SEQ ID NO. 3;
the downstream primer of CpSNP2 is shown as SEQ ID NO. 4;
the CpSNP9 upstream primer is shown as SEQ ID NO. 5;
the downstream primer of CpSNP9 is shown as SEQ ID NO. 6;
the CpSNP13 upstream primer is shown as SEQ ID NO. 7;
the downstream primer of CpSNP13 is shown as SEQ ID NO. 8;
the CpSNP14 upstream primer is shown as SEQ ID NO. 9;
the CpSNP14 downstream primer is shown as SEQ ID NO. 10;
the CpSNP15 upstream primer is shown as SEQ ID NO. 11;
the downstream primer of CpSNP15 is shown as SEQ ID NO. 12;
the CpSNP16 upstream primer is shown as SEQ ID NO. 13;
the CpSNP16 downstream primer is shown as SEQ ID NO. 14;
the CpSNP18 upstream primer is shown as SEQ ID NO. 15;
the downstream primer of CpSNP18 is shown as SEQ ID NO. 16;
the CpSNP19 upstream primer is shown as SEQ ID NO. 17;
the CpSNP19 downstream primer is shown as SEQ ID NO. 18;
the CpSNP20 upstream primer is shown as SEQ ID NO. 19;
the CpSNP20 downstream primer is shown as SEQ ID NO. 20;
the CpSNP21 upstream primer is shown as SEQ ID NO. 21;
the downstream primer of CpSNP21 is shown as SEQ ID NO. 22.
The PCR-HRM amplification system of the polymorphic primers is as follows: the upstream and downstream primers are 10. mu.M and 1. mu.L respectively; 2 XFroget-Me-NotEva Green qPCR Master Mix 5. mu.L, H2O2. mu.L, DNA template 30ng, 1. mu.L, and the total volume of the system was 10. mu.L.
The PCR-HRM amplification program of the polymorphic primers is as follows: 2min at 95 ℃, 5s at 95 ℃, 30s at 60 ℃ and 30-35 cycles; 95 ℃ for 10s, 60 ℃ for 1min, 95 ℃ for 15s and 60 ℃ for 15 s.
The application of polymorphic primers of cinnamomum camphora chloroplast SNP molecular markers in the analysis of genetic diversity of cinnamomum camphora.
The application of polymorphic primers of cinnamomum camphora chloroplast SNP molecular markers in the analysis of the cinnamomum camphora lineage.
The application comprises the following steps:
1) extracting genome DNA of different camphor varieties;
2) carrying out PCR amplification and HRM analysis on the genomic DNA extracted in the step 1) by using primers shown in SEQ ID NO.1-SEQ ID NO.22 of claim 1;
3) and (3) analyzing the polymorphism of SNP sites of chloroplasts, analyzing the lineage of the cinnamomum camphora, and analyzing the genetic diversity of the cinnamomum camphora. Has the advantages that: compared with the prior art, the invention screens 11 pairs of polymorphic primers of the cinnamomum camphora chloroplast genome SNP molecular marker, and applies the primers to the genetic diversity analysis and the cinnamomum camphora lineage analysis of the cinnamomum camphora, and based on the chloroplast haplotype analysis, the distribution difference of different haplotypes is obvious, most haplotypes only exist in a specific group, and only a few haplotypes are shared haplotypes, which indicates that certain geographical differentiation exists among the haplotypes. The genetic differentiation coefficient Fst was 0.212 and the gene flow Nm was 0.929 for the 18 cinnamomum camphora populations, indicating a moderate gene flow between the cinnamomum camphora populations. The SNP molecular marker disclosed by the invention is good in repeatability, can comprehensively reveal the true level of genetic diversity of the cinnamomum camphora, and provides a theoretical basis for the protection and utilization of wild cinnamomum camphora resources.
Drawings
Figure 1 is a graph of a partial haploid network.
Detailed Description
The present invention is further illustrated by the following specific examples, which are not intended to be limiting.
Example 1
1) Test material
The tested materials are from 18 groups of natural distribution areas of cinnamomum camphora, including Sichuan Luzhou, Jiangxi Jingan, Jiangxi Ningdu, Sichuan Yibin, Guizhou Daizhen, Guangxi Guilin, Jiangxi Enjiang, Jiangxi Longgang, Anhui Anqing, Jiangxi Legan, Jiangxi Ruhong, Jiangxi Pingxiang, Guangdong Guangzhou, Jiangxi Nanchang, Guizhou Guiyang, Guizhou lichee, Fujian Wuyi mountain and Jiangsu Nanjing. Selecting an ancient camphor tree with the diameter at breast height of more than 60cm, collecting 5-10 fresh leaves for each individual, carrying out dry ice transportation, and quickly freezing in a refrigerator at-80 ℃ by using liquid nitrogen for storage. The specific collection group positions, longitude and latitude, sample numbers and the like are shown in table 1.
118 cinnamomum camphora group sampling information
| Origin of origin | Sample size | Group of people | Longitude (G) | Latitude |
| Sichuan Luzhou | 11 | SCLZ | 105.26 | 28.58 |
| Sichuan Yibin | 5 | SCYB | 104.25 | 28.25 |
| Jing xi an | 7 | JXJA | 115.15 | 28.54 |
| All of Jiangxi Ningdu | 10 | JAND | 115.51 | 26.26 |
| Jiangxienjiang | 6 | JXEJ | 115.43 | 27.31 |
| Jiangxi dragon hillock | 5 | JXLG | 115.60 | 26.75 |
| Flood of river and west | 21 | JXRH | 116.43 | 28.88 |
| Jiangxi Pingxiang | 10 | JXPX | 114.06 | 27.64 |
| Jiangxi Nanchang | 31 | JXNC | 115.82 | 28.75 |
| Jiangxi le' an medicine | 11 | JXLA | 115.73 | 27.28 |
| Guizhou dai zhen | 5 | GZDZ | 107.43 | 28.45 |
| Guizhou Guiyang | 15 | GZGY | 106.72 | 26.63 |
| Guizhou litchi wave | 5 | GZLB | 107.89 | 25.42 |
| Guangxi Guilin | 5 | GXGL | 110.30 | 25.17 |
| Anhui Anqing | 14 | AHAQ | 117.04 | 30.51 |
| Guangdong province of Guangdong province | 5 | GDGZ | 113.36 | 23.19 |
| Fujian Wuyi mountain | 10 | FJWYS | 117.96 | 27.67 |
| Jiangsu Nanjing | 15 | JSNJ | 118.84 | 32.05 |
2) Extraction and detection of total DNA of cinnamomum camphora
The extraction of the total DNA of the cinnamomum camphora is carried out by adopting an improved CTAB method, and the specific operation steps are as follows:
opening the water bath kettle, and preheating at 65 ℃;
taking 10mL of a centrifuge tube, adding 4mL of CTAB lysate and 80 mu L of beta-mercaptoethanol, and putting the centrifuge tube into a water bath for preheating;
taking about 2g of cinnamomum camphora leaves, putting the leaves into a mortar prepared in advance, introducing liquid nitrogen for grinding until the sample is in a uniform powder state, adding the powder into the centrifuge tube in the previous step, putting the centrifuge tube into a water bath kettle, carrying out water bath at 65 ℃ for 30min, and reversing and uniformly mixing the materials every 5 min;
taking out the centrifuge tube, placing on ice for cooling for three minutes, adding a chloroform isoamyl alcohol mixed solution with the same volume (chloroform: isoamyl alcohol is 24: 1), fully and uniformly mixing, placing in a centrifuge, and centrifuging at 12000r for 10min at 4 ℃;
preparing a new 10mL centrifuge tube, sucking the supernatant into the new centrifuge tube by using a pipette, adding the chloroform isoamylol mixed solution with the same volume again, uniformly mixing, placing in a centrifuge, and centrifuging for 10min at 4 ℃ at 12000 r;
respectively sucking 500 μ L of supernatant, subpackaging in 3 centrifuge tubes of 1.5mL, respectively adding 1mL of ethanol and 50 μ L of sodium acetate solution, reversing, mixing, placing in a refrigerator at-20 deg.C, standing for 2h, and waiting for floccule to precipitate; transferring the rest supernatant to a 2mL centrifuge tube, and placing in a refrigerator at-20 ℃ for later use;
preparing new 1.5mL centrifuge tubes, respectively adding 1mL 75% ethanol solution, picking floccules precipitated from 3 centrifuge tubes into the new centrifuge tubes, vortexing for 1min, and centrifuging for 5min at 10000 r;
carefully pouring off the ethanol solution, adding a new 1mL of 75% ethanol solution, and repeating the previous step;
pouring off the ethanol solution again, and drying by using a vacuum drying instrument;
adding 200. mu.L of 1 XTE solution, dissolving for 3h, taking out and centrifuging, and obtaining supernatant which is the extracted DNA.
Taking a small amount of extracted total DNA of the cinnamomum camphora, and detecting the quality and concentration of a DNA sample by using NanoDrop 2000c (ThermoFisher scientific); meanwhile, the quality of the DNA is detected by using 1% agarose gel electrophoresis, and whether the degradation of the DNA is caused or not is detected by observing whether the bands are dispersed or not.
The DNA was diluted to 30 ng/. mu.L and stored at-20 ℃.
3) Chloroplast SNP primer amplification and screening
Based on the existing five chemotypes of cinnamomum camphora chloroplast genomes, SNP loci are obtained by comparing mafft software, primer design is carried out on the obtained loci by utilizing Oligo software, each pair of primers only contains one SNP locus, the length of a product is between 100bp and 350bp, and the Tm values of all the primers are 60 ℃. In total, 23 pairs of SNP primers (synthesized by Nanjing King-Smith Biotechnology Ltd.) were designed and screened to obtain 11 pairs of SNP primers with high specificity and polymorphism (Table 2). PCR amplification and HRM (high resolution melting curve) typing were performed using a ViiA 7Real-Time PCR System with fast 96-Well Block Real-Time fluorescent quantitative PCR instrument.
TABLE 2 chloroplast SNP primer sequence information
Note: f: an upstream primer; r: downstream primer
The PCR-HRM amplification system of the polymorphic primers is as follows: DNA template 30ng, 1 μ L; the upstream and downstream primers are 10. mu.M and 1. mu.L respectively; 2 XFroget-Me-Not EvaGreen qPCR Master Mix 5. mu.L, H2O2. mu.L, the total volume of the system was 10. mu.L.
The PCR-HRM amplification program of the polymorphic primers is as follows: 2min at 95 ℃, 5s at 95 ℃, 30s at 60 ℃ and 30-35 cycles; 95 ℃ for 10s, 60 ℃ for 1min, 95 ℃ for 15s and 60 ℃ for 15 s.
4) Data analysis
MAF (minimum allele frequency), h (Nei diversity index) and I (shannon diversity index) were estimated using the software popgen32, and PIC (polymorphic information content) values were estimated using the software powermarker.
Software SHESIS is used for counting the number and the type of haplotypes of the 11 SNP loci. Haplotype polymorphism analysis, neutral test and mismatch analysis were performed using DNAsp and Fst and Nm were calculated, and genetic differentiation parameters Nst and Gst were calculated using PERMUT.
The haplotype data was constructed using a central-connectivity Network (media-joining) model using software Network version 5.0.1.1. In order to obtain a simpler and higher-quality Network diagram, Frequency >1 is selected to eliminate possible error information sites, and the high-variance sites are subjected to weight reduction processing and further analyzed by Reduced-media.
5) Chloroplast genome SNP site (cpSNP) polymorphism analysis
Using high resolution melting curve (HRM) technique, 11 SNP sites of 18 cinnamomum camphora populations were genotyped. Typing results were evaluated for Minimum Allele Frequency (MAF), Nei diversity index (h), shannon diversity index (I) and Polymorphic Information Content (PIC) using popgen32 and powermarker software (table 3). The highest values of MAF, h, I and PIC among the 11 SNP sites were SNP1(0.479, 0.499, 0.692, 0.375), the lowest ones were SNP18(0.027, 0.052, 0.124, 0.051), and the average values of MAF, h, I and PIC were 0.175, 0.168, 0.400 and 0.208, respectively, all showing low genetic diversity. The MAF for SNP9 and SNP18 was below 0.1, a low frequency allele.
TABLE 3 SNP site diversity detection
| SNP | MAF | h | I | PIC |
| SNP1 | 0.479 | 0.499 | 0.692 | 0.375 |
| SNP2 | 0.151 | 0.256 | 0.424 | 0.223 |
| SNP9 | 0.097 | 0.175 | 0.318 | 0.160 |
| SNP13 | 0.129 | 0.225 | 0.385 | 0.200 |
| SNP14 | 0.075 | 0.139 | 0.267 | 0.130 |
| SNP15 | 0.102 | 0.183 | 0.330 | 0.167 |
| SNP16 | 0.124 | 0.217 | 0.374 | 0.193 |
| SNP18 | 0.027 | 0.052 | 0.124 | 0.051 |
| SNP19 | 0.172 | 0.285 | 0.459 | 0.244 |
| SNP20 | 0.108 | 0.192 | 0.341 | 0.174 |
| SNP21 | 0.462 | 0.497 | 0.690 | 0.374 |
| Average | 0.175 | 0.168 | 0.400 | 0.208 |
Note: MAF: a minimum allele frequency; h: a Nei diversity index; i: a shannon diversity index; PIC: polymorphic information content.
6) Genetic diversity analysis of cinnamomum camphora based on cpNP haplotypes
SNP haplotype statistics were performed on all Cinnamomum camphora using software SHESIS, and a total of 52 haplotypes were detected in 186 individuals (Table 4). The SNP haplotypes have larger distribution difference among the cinnamomum camphora groups, wherein the H4 has the widest distribution range and is distributed in 12 groups; secondly H13, with distribution among 11 populations; h18, H17, H3, H16, H42, H9, H14, H15, H39, H6, H8 and H28 have distributions in 9, 8, 6, 5, 4, 3, 2 populations, respectively, and the remaining haplotypes are distributed in only one population.
The distribution of SNP haplotypes in the population is also very different (table 5), 18 populations all contain more than one haplotype, the guru population with the most distributed haplotype types is jiangxi nanchang (JXNC) and has 17 haplotypes distributed, and the jiangxi rehong (JXRH) population has 13 haplotypes, which may be caused by the larger number of samples in the two populations compared to the other populations. The number of haplotypes contained in Jiangxi Longgang (JXLG) and Guizhou litchi (GZLB) populations is the least, 2.
The SNP haplotype polymorphism index (Hd) of the population was estimated using DNAsp (Table 5), wherein the haplotype polymorphism was the highest in the SCYB population (Hd of 1) and the haplotype polymorphism was the lowest in the GZLB population (Hd of 0.4). The overall SNP haplotype polymorphism for the 18 populations was 0.817, relatively high.
Based on the 52 SNP haplotypes, DNAsp software is used for estimating the genetic differentiation coefficient of the cinnamomum camphora population, and the result shows that Fst is 0.212, the gene flow Nm is 0.929, and that the populations have a moderate level of gene flow. Meanwhile, software PERMUT is used for calculating Nst and Gst, and the result shows that Gst (0.105) is less than Nst (0.239), but the P value is more than 0.05, and the result is not obvious, so that whether the distribution of the cinnamomum camphora population has an obvious pedigree geographic pattern or not can not be determined by SNP haplotype information.
TABLE 4 haplotype distribution
TABLE 5 haplotype diversity statistics
| POP ID | Number of individuals N | Haplotype number Nh | Haplotype polymorphism index Hd |
| SCLZ | 11 | 7 | 0.891 |
| SCYB | 5 | 5 | 1.00 |
| JXJA | 7 | 5 | 0.905 |
| JXND | 10 | 7 | 0.867 |
| JXEJ | 6 | 3 | 0.600 |
| JXLG | 5 | 2 | 0.600 |
| JXRH | 21 | 13 | 0.952 |
| JXPX | 10 | 8 | 0.956 |
| JXNC | 31 | 17 | 0.929 |
| JXLA | 11 | 5 | 0.618 |
| GZDZ | 5 | 4 | 0.900 |
| GZGY | 10 | 8 | 0.956 |
| GZLB | 5 | 2 | 0.400 |
| GXGL | 5 | 4 | 0.900 |
| AHAQ | 14 | 6 | 0.747 |
| GZDZ | 5 | 3 | 0.800 |
| FJWYS | 10 | 5 | 0.800 |
| JSNJ | 15 | 9 | 0.886 |
7) cpSNP haplotype-based gurnus camphora lineage analysis
The result of the haplotype net graph is shown in FIG. 1, which has a mesh structure, and haplotype H3 is located at the central position of the whole network, and may be the ancient haplotype of the core. The deleted haplotype is used as a boundary and can be divided into an upper branch system and a lower branch system, wherein Clade I consists of H8, H6 and H27, and the other haplotypes consist of Clade II. The haplotype contained in Clade I is mainly distributed in Sichuan Luzhou (SCLZ), Sichuan Yibin (SCYB) and Guizhou Guiyang (GZGY), and the haplotype is also distributed in the Jiangxi group in small part. The haplotype of Clade II is widely distributed throughout the country. The population haplotypes in the western region are the most abundant, and all haplotypes except H6 and H8 are the distribution centers of haplotypes.
It is to be noted that the above-mentioned list is only a few specific embodiments of the present invention. It is obvious that the invention is not limited to the above embodiments, but that many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.
Sequence listing
110 university of Nanjing forestry, institute of biological resources of academy of sciences of Jiangxi province
Polymorphic primer of <120> cinnamomum camphora chloroplast SNP molecular marker and application thereof
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cccaatccat gaatctaa 18
Claims (3)
1. The application of the polymorphic primer of the cinnamomum camphora chloroplast SNP molecular marker in the cinnamomum camphora lineage analysis is characterized by comprising the following steps:
1) extracting genome DNA of different camphor varieties;
2) carrying out PCR-HRM amplification and HRM analysis on the genomic DNA extracted in the step 1) by using polymorphic primers of the camphor chloroplast SNP molecular markers;
3) analyzing polymorphism of SNP sites of chloroplasts, counting the number and types of haploids of the SNP sites, analyzing SNP haplotype distribution in a cinnamomum camphora population, calculating SNP haplotype polymorphism index of the cinnamomum camphora population, calculating genetic differentiation coefficient of the cinnamomum camphora population, then constructing a haplotype network map for haplotype data by adopting a central connection network model, and analyzing the cinnamomum camphora lineage;
the polymorphic primers of the cinnamomum camphora chloroplast SNP molecular marker comprise 11 pairs of polymorphic primers, wherein the nucleotide sequences of the primers are as follows:
the CpSNP1 upstream primer is shown as SEQ ID NO. 1;
the CpSNP1 downstream primer is shown as SEQ ID NO. 2;
the CpSNP2 upstream primer is shown as SEQ ID NO. 3;
the downstream primer of CpSNP2 is shown as SEQ ID NO. 4;
the CpSNP9 upstream primer is shown as SEQ ID NO. 5;
the downstream primer of CpSNP9 is shown as SEQ ID NO. 6;
the CpSNP13 upstream primer is shown as SEQ ID NO. 7;
the downstream primer of CpSNP13 is shown as SEQ ID NO. 8;
the CpSNP14 upstream primer is shown as SEQ ID NO. 9;
the CpSNP14 downstream primer is shown as SEQ ID NO. 10;
the CpSNP15 upstream primer is shown as SEQ ID NO. 11;
the downstream primer of CpSNP15 is shown as SEQ ID NO. 12;
the CpSNP16 upstream primer is shown as SEQ ID NO. 13;
the CpSNP16 downstream primer is shown as SEQ ID NO. 14;
the CpSNP18 upstream primer is shown as SEQ ID NO. 15;
the downstream primer of CpSNP18 is shown as SEQ ID NO. 16;
the CpSNP19 upstream primer is shown as SEQ ID NO. 17;
the CpSNP19 downstream primer is shown as SEQ ID NO. 18;
the CpSNP20 upstream primer is shown as SEQ ID NO. 19;
the CpSNP20 downstream primer is shown as SEQ ID NO. 20;
the CpSNP21 upstream primer is shown as SEQ ID NO. 21;
the downstream primer of CpSNP21 is shown as SEQ ID NO. 22.
2. The use of the polymorphic primers of the cinnamomum camphora chloroplast SNP molecular marker in the cinnamomum camphora lineage analysis according to claim 1, wherein in the step 2), the PCR-HRM amplification system is: upstream and downstream guide1 μ L of each 10 μ M; 2 XFroget-Me-Not EvaGreen qPCR Master Mix 5. mu.L, H2O2. mu.L, DNA template 30ng, 1. mu.L, the total volume of the system being 10. mu.L.
3. The use of the polymorphic primers of the cinnamomum camphora chloroplast SNP molecular marker in the cinnamomum camphora lineage analysis according to claim 1, wherein in the step 2), the PCR-HRM amplification procedure is as follows: 2min at 95 ℃, 5s at 95 ℃, 30s at 60 ℃ and 30-35 cycles; 95 ℃ for 10s, 60 ℃ for 1min, 95 ℃ for 15s and 60 ℃ for 15 s.
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Application publication date: 20200207 Assignee: Nanjing dehetang Biotechnology Co.,Ltd. Assignor: NANJING FORESTRY University Contract record no.: X2020320000335 Denomination of invention: Polymorphic primers for SNP molecular markers in chloroplast of Cinnamomum camphora and their application Granted publication date: 20201030 License type: Common License Record date: 20201211 Application publication date: 20200207 Assignee: Nanjing peptide crystal Biotechnology Co.,Ltd. Assignor: NANJING FORESTRY University Contract record no.: X2020320000333 Denomination of invention: Polymorphic primers for SNP molecular markers in chloroplast of Cinnamomum camphora and their application Granted publication date: 20201030 License type: Common License Record date: 20201211 Application publication date: 20200207 Assignee: Nanjing Jiangbei new district zhuangdi League biotechnology service center Assignor: NANJING FORESTRY University Contract record no.: X2020320000334 Denomination of invention: Polymorphic primers for SNP molecular markers in chloroplast of Cinnamomum camphora and their application Granted publication date: 20201030 License type: Common License Record date: 20201211 |