CN111455082A - ISSR molecular marking method for analyzing genetic diversity of cassytha indica - Google Patents
ISSR molecular marking method for analyzing genetic diversity of cassytha indica Download PDFInfo
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Abstract
The invention provides an ISSR molecular marking method for genetic diversity analysis of cassytha indica, belonging to the technical field of molecular biology. The method establishes an ISSR molecular marker primer system of the cassiteria tinctoria, amplifies genome DNA by ISSR-PCR reaction, analyzes strips according to ISSR-PCR results, establishes a DNA fingerprint of the cassiteria tinctoria, and establishes a genetic resource database of the cassiteria tinctoria. The invention optimizes ISSR reaction conditions of the cassiterite genome DNA, screens 9 primers with high polymorphism, clear spectral band and good repeatability, can reflect the intra-species genetic relationship of cassiterite of different populations, utilizes 9 primers to carry out cluster analysis on cassiterite, establishes a new method for variety identification and genetic diversity analysis of cassiterite, and provides technical guidance and theoretical support for scientific researches such as cassiterite resource identification and genetic diversity analysis.
Description
Technical Field
The invention relates to the technical field of molecular biology, in particular to an ISSR molecular marking method for analyzing genetic diversity of cassytha leaves.
Background
The Siberian cinquefoil herb is root of Tetracera asiatica (L our.) Hoogland of Dilleniaceae, is cool in nature and bitter in taste, has the effects of astringing to arrest diarrhea, relieving swelling and pain, and is commonly used for treating diarrhea, dysentery, rectocele and other symptoms, and the medicinal materials are rich in various bioactive components such as tannin, flavone, terpenoids and the like, and have obvious activities of bacteriostasis, anti-inflammation, anti-tumor and the like [ China herbal medicine administration of China Commission of Chinese materia Medica, China herbal medicine [ M ]. volume 3, Shanghai: Shanghai scientific and technical Press, 1999: 510 and 511 ].
The germplasm resources of the staphylium stannum have extremely rich genetic diversity on the DNA molecular level. The full understanding of the genetic diversity and pedigree relationship of the cassytha indica germplasm resources is the basic requirement and premise of germplasm innovation and genetic breeding. At present, the research on the cassiabarktree leaves is mostly concentrated on morphology, chemistry and pharmacology; in the aspect of molecules, researchers have studied the DNA extraction of the Siberian sylvestris.
The ISSR-PCR molecular marker technology combines the advantages of SSR and RAPD, and has the advantages of ① amplified genome DNA, suitability for any species rich in SSR repetitive units and widely distributed SSR, capability of simultaneously providing multi-site information and revealing the information of variation among different microsatellite loci, ② high genetic polymorphism and good repeatability, primers adopted by ISSR have stronger specificity, the strength of combination with templates is improved, and the repeatability of experimental results is enhanced, ③ ISSR markers are dominant markers, accord with Mendel genetic rules, less cost is brought to marking of ISSR, the using amount of template DNA is less, ⑤ does not need to know SSR background information of any target sequence, the ISSR markers can be amplified by using the primers without knowing DNA sequences in primer design, and can also reveal polymorphisms more than RF L P, RAPD and SSR, ④ is based on the specificity of the ISSR markers, have good stability and repeatability, can better reflect the genetic structure and genetic diversity of the species, and is considered as an ideal genetic marker method for researching ISSR and a wide variety of ISSR molecules in the traditional Chinese medicine and agriculture.
Disclosure of Invention
The invention aims to: aiming at the existing problems, the ISSR molecular marking method for the genetic diversity analysis of the cassytha leaves is provided. The invention firstly adopts a DNA molecular marking method to identify the strong-drug Siberian lygodium stems in the same and different producing areas, screens out the germplasm resource advantage producing areas of the strong-drug Siberian lygodium stems, analyzes the genetic relationship of each producing area of the strong-drug Siberian lygodium stems, and provides a molecular crude pharmacology identification method for the strong-drug Siberian lygodium stems.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
an ISSR molecular marker primer system for genetic diversity analysis of Siberian cinquefoil, wherein the primer sequence comprises:
UBC number 807: the nucleotide sequence is shown as SED ID No. 1;
UBC No. 810: the nucleotide sequence is shown as SED ID No. 2;
UBC number 811: the nucleotide sequence is shown as SED ID No. 3;
UBC No. 836: the nucleotide sequence is shown as SED ID No. 4;
UBC No. 840: the nucleotide sequence is shown as SED ID No. 5;
UBC No. 842: the nucleotide sequence is shown as SED ID No. 6;
UBC No. 880: the nucleotide sequence is shown as SED ID No. 7;
UBC 881: the nucleotide sequence is shown as SED ID No. 8;
UBC No. 888: the nucleotide sequence is shown as SED ID No. 9;
preferably, the method for analyzing the genetic diversity of the cassiabarktree leaves by using the primer system comprises the following specific steps:
1) extracting the genomic DNA of the casserole vine, and amplifying the genomic DNA of the casserole vine by using ISSR-PCR reaction;
2) carrying out electrophoresis and color development on a PCR product obtained by the PCR amplification in the step 1), and observing, photographing and storing in an ultraviolet gel imager after the color development is finished;
3) analyzing the band information obtained in the step 2), extracting data of a gel image by using GENESCAN3.1 software, selecting a proper internal standard and setting software analysis parameters, selecting the color of a corresponding fluorescent marker and selecting SIZISTANDARD, analyzing by using Binthere software to obtain a result, storing the result into an X L S format, converting a numerical value in an X L S table into 1 if the numerical value is not 0, generating an original matrix consisting of 'l' and '0', performing cluster analysis by using an UPGMA method of a SHAN program in NTSYSpc-2.11F software, and generating a cluster image;
preferably, the ISSR-PCR amplification procedure in step 1) is as follows: pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 30s, annealing at 45s, wherein the annealing temperature is determined by Tm of different primers, extension at 72 ℃ for 2min, 45 cycles, extension at 72 ℃ for 10min, and storage at 4 ℃;
preferably, the annealing temperature is determined according to the selected primer, and is as follows:
SED ID No.1 50.9℃
SED ID No.2 51.2℃
SED ID No.3 50.1℃
SED ID No.4 50.3℃
SED ID No.5 51.5℃
SED ID No.6 50.6℃
SED ID No.7 53.2℃
SED ID No.8 52.6℃
SED ID No.9 52.3℃
preferably, the electrophoresis in step 2) is performed under the following conditions: performing 0.8% agarose gel electrophoresis at 75V for 50 min;
preferably, the primer system is applied to auxiliary identification of the cassiteria tinctoria.
In summary, due to the adoption of the technical scheme, the invention has the beneficial effects that:
1) the invention utilizes 9 primers to perform cluster analysis on the cassiterite, and establishes a new method for variety identification and genetic diversity analysis of the cassiterite.
2) The invention optimizes ISSR reaction conditions of the cassiterite genomic DNA, and obtains 9 primers which have high polymorphism, clear spectral band and good repeatability and can reflect the intra-species genetic relationship of cassiterite with different populations by screening.
3) The ISSR banding pattern of the cassiterite leaf rattan obtained by amplification of the invention shows high polymorphism and has good repeatability, the genetic diversity among cassiterite leaf rattan groups can be well disclosed by using the technology, the genetic relationship among cassiterite leaf rattan germplasm resources can be distinguished, and the ISSR banding pattern of the cassiterite leaf rattan has important significance for protecting and utilizing the cassiterite leaf rattan resources. Meanwhile, the ISSR molecular marker technology is used for identifying the interspecific and intraspecies grades of the Dilleniaceae genus, which is effective and practical.
4) The invention adopts a DNA molecular marking method to identify 90 parts of the strong-drug Siberian cinquefoil herb in the same variety and different producing areas for the first time, screens out the germplasm resource advantage producing areas of the strong-drug Siberian cinquefoil herb, analyzes the genetic relationship of each producing area of the strong-drug Siberian cinquefoil herb, and provides a molecular biological and pharmaceutical identification method for the strong-drug Siberian cinquefoil herb.
Drawings
FIG. 1A DNA template of Selenia
FIG. 2 DNA map of primer 807 on Siberian vine No. 1-30 sample
FIG. 3 DNA map of primer 807 on Siberian vine No. 31-60 sample
FIG. 4 DNA map of primer 807 on Siberian vine No. 61-90 sample
FIG. 5 DNA map of primer 880 on Sinomenium acutum No. 1-30 sample
FIG. 6 DNA map of primer 880 on Siberian vine 31-60 sample
FIG. 7 DNA map of primer 880 on Sinomenium acutum No. 61-90 sample
[ legend: marker is ROX (red) fluorescence labeled Marker, and its name is: rox1000, fragment sizes from bottom to top on the gel plot are in order: 50. 75, 100, 125, 150, 200, 250, 300, 350, 400, 450, 475, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000(bp), for a total of 23 bands. Primers are labeled with FAM (blue) fluorescence
FIG. 8 IsR clustering tree of Siberian cinquefoil stems
FIG. 9 two-dimensional graph of PCoA of Sedum cassythe with different habitats
FIG. 10 three-dimensional view of PCoA of Sedum cassiabarkeri of different habitats
Detailed Description
In order that the invention may be more clearly expressed, the invention will now be further described by way of specific examples. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
(1) Experimental Material
90 parts of Siberian cinquefoil stems used in the test were collected from Guangxi, Guangdong, hong Kong, etc., and are shown in Table 1.
TABLE 1 test Material Numbers and sample points
(2) ISSR primer design
The 100 primers used in this experiment were universal primers published by the university of Columbia (UBC). The primers were synthesized by Beijing Ding Guoshang biotechnology, Inc. After the primers are synthesized, the primers are centrifuged and diluted and shaken to dissolve according to requirements, the primers are diluted to 10 mu M, and the primers are separated into devices for later use in a refrigerator at the temperature of-20 ℃.
(3) Extraction of DNA
Since the medicinal plant components are quite complex and usually contain a large amount of secondary metabolites such as protein, pigment, polysaccharide, polyphenol and the like and cell contents, particularly polysaccharide and polyphenol substances which seriously affect the extraction of DNA, the common method for extracting plant DNA is selected for extraction by a CTAB method in the research.
Extraction by CTAB method, which is carried out as follows:
1) weighing 50mg of the material, and grinding the material by liquid nitrogen for three to four times;
2) the powder was placed into a 2m L centrifuge tube containing 800 μ L2 × CTAB extraction buffer;
3) adding 60 mu L mercaptoethanol, mixing uniformly, and preheating at 60 ℃ for 30 min;
4) uniformly mixing the mixture for two to three times, taking out a sample, and standing the sample at room temperature;
5) after the sample cooled to room temperature, 800. mu. L chloroform isoamyl alcohol (24:1) was added;
6) oscillating and mixing evenly, and centrifuging for 15min at 12000 rpm;
7) taking the supernatant to a new 2m L centrifuge tube, adding equal volume of chloroform isoamyl alcohol (24:1), shaking and mixing uniformly, and centrifuging at 12000rpm for 15 min;
8) taking the supernatant to a new 2m L centrifuge tube, adding 1.5 times of 1 × CTAB precipitation solution, standing for 20-30min, and centrifuging at 12000rpm for 15 min;
9) the precipitate was air-dried and dissolved in 200. mu. L TE-buffer;
10) adding 400 mu L95% ethanol, 20 mu L3M NaAC, and precipitating at-20 ℃ for 1 h;
11) centrifuging at 4 ℃ and 12000rpm for 15 min;
12) washing with 500 μ L75% ethanol, and centrifuging at 12000rpm for 10 min;
13) adding 30-50 mu L TE-buffer;
14) and detecting by 0.8% agarose gel electrophoresis.
(4) ISSR-PCR amplification
The ISSR-PCR reaction system is shown in Table 2, and the ISSR-PCR amplification procedure is as follows: pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 30s, annealing at 45s (determined by different primer Tm values), extension at 72 ℃ for 2min, 45 cycles, extension at 72 ℃ for 10min, and storage at 4 ℃.
TABLE 2 ISSR-PCR reaction System
| Components | Volume of |
| Template DNA | 2μL |
| P1 | 0.5μL |
| dNTPs | 0.5μL |
| 10XPCR buffer | 2.5μL |
| Taq DNA polymease | 0.5μL |
| ddH2O | 19μL |
Centrifuging for several seconds, and performing PCR amplification according to the following parameters
(5) Screening of primers
Carrying out amplification screening on 100 ISSR primers by using a Stachys sieboldii sample, detecting a PCR product by using 0.8% agarose gel electrophoresis, and screening out primers with clear background, good amplification effect and high polymorphism for ISSR-PCR reaction; and simultaneously, screening the optimal annealing temperature of each primer according to the Tm value of each primer.
(6) Electrophoretic detection of ISSR amplification products
And (3) taking 8 mu L PCR products, carrying out electrophoresis by using 0.8% agarose gel (containing GelRed dye), carrying out voltage 75V and electrophoresis for 50min, using a D2000DNA Marker as a standard reference, and observing and photographing in an ultraviolet gel imager for storage after the electrophoresis is finished.
(7) Data statistics and analysis
1) GENESCAN3.1 software analysis: and (3) opening the glue picture by using GENESCAN3.1 software (the glue picture is a sample picture scanned by an automatic sequencer 377), extracting data of the glue picture, installing MATRIX of the glue picture, selecting a proper internal standard, namely SIZESTANDARD, setting proper analysis parameters of the software, and analyzing to obtain a result.
2) And (3) Binthere software analysis, namely extracting the result of each fragment size of the sample (obtained by analyzing a gel map by the GENESCAN3.1 software) through the Binthere software, opening the Binthere software, setting the range of the fragment size, importing data, selecting the color of a corresponding fluorescent marker, selecting a proper internal STANDARD, namely SIZI STANDARD, clicking for analysis, exporting the result and storing the result in an X L S format.
3) The EXCE L converts that the value in the table is not 0 into 1 (no conversion of the value 0), thereby generating an original matrix composed of "l" and "0".
4) NTSYSpc-2.11F software analysis: data analysis was performed using NTSYSpc-2.11F software. And solving the DICE similar coefficient matrix from the original matrix by using a SimQual program, and obtaining the similar coefficient matrix. And (4) performing cluster analysis by using a UPGMA method in the SHAN program, and generating a cluster map by using a Tree plot module.
Results and analysis
(1) Detection of DNA
The quality of DNA is an important factor affecting the result of PCR amplification. The experiment extracts partial genomic DNA of the cassiteria tinctoria by a CTAB method, and electrophoresis detection shows that the extracted cassiteria tinctoria DNA has bright and clear bands and unobvious trailing phenomenon, the molecular weight of the bands is more than 2000bp, which shows that the extracted cassiteria tinctoria genomic DNA can be used for ISSR analysis and SRAP analysis, and is shown in figure 1.
(2) ISSR primer screening
In the experiment, 9 primers with good repeatability, clear bands and high polymorphism are screened from 100 primers, and can be used for analyzing genetic diversity of the cassiteria tinctoria, and the table 3 shows.
TABLE 3 primer sequences used for ISSR analysis
| Primer and method for producing the same | Sequence of | Annealing temperature (. degree.C.) |
| UBC807 | AGA GAG AGA GAG AGA GT | 50.9 |
| UBC810 | GAG AGA GAG AGA GAG AT | 51.2 |
| UBC811 | GAG AGA GAG AGA GAG AC | 50.1 |
| UBC836 | AGAGAGAGAGAG AGA GYA | 50.3 |
| UBC840 | GAGAGAGAGAGA GAG AYT | 51.5 |
| UBC842 | GAGAGAGAGAGA GAG AYG | 50.6 |
| UBC880 | GGA GAG GAG AGG AGA | 53.2 |
| UBC881 | GGG TGG GGT GGG GTG | 52.6 |
| UBC888 | BDB CAC ACA CAC ACA CA | 52.3 |
Note that: y ═ C, T, B ═ C, G, T)
(3) ISSR primer amplification results
And (3) carrying out ISSR-PCR amplification on 90 collected Siberian cinquefoil stems in different producing areas according to corresponding numbers by using the screened 9 primers (all amplification are repeatedly tested to ensure the reliability of results, M is 100bp DNAsader), and co-amplifying 1666 sites, wherein the sizes of the fragments are distributed between 50 and 1000 bp. Wherein 1664 polymorphic sites are present, the percentage of the polymorphic sites is 99.86%, and 185 polymorphic sites can be amplified by each primer on average, and the results are shown in Table 4. The amplification site of the primer UBC880 is 256 at the most, and the amplification site of the primer UBC840 is 143 at the least. As a result of partial amplification, the DNA patterns of the primers 807 and 880 are as follows (see FIGS. 2 to 7). From the polymorphic data, the genetic diversity among the tested cassiteria tenuifolia samples is higher, which shows that the population has stronger environment adaptation capability and wider genetic basis, and can provide experimental basis for optimization and breeding.
TABLE 4 analysis of polymorphism of amplification products of each primer
| Primer and method for producing the same | Amplification of Total sites | Polymorphic site | Ratio of polymorphism/% |
| UBC807 | 159 | 157 | 98.74 |
| UBC810 | 160 | 160 | 100 |
| UBC811 | 177 | 177 | 100 |
| UBC836 | 212 | 212 | 100 |
| UBC840 | 143 | 143 | 100 |
| UBC842 | 187 | 187 | 100 |
| UBC880 | 256 | 256 | 100 |
| UBC881 | 207 | 207 | 100 |
| UBC888 | 165 | 165 | 100 |
| Total number of | 1666 | 1664 | 99.86 |
(4) Genetic diversity analysis
The POPGENE1.32 software is used for analyzing genetic diversity of samples of 90 different populations, and genetic distance (D) and genetic similarity (I) are important parameters for measuring the genetic differentiation degree and genetic difference between species, populations and systems. The larger the genetic distance, the smaller the genetic similarity, which indicates the farther the genetic relationship, and conversely, the closer the genetic relationship. And (3) carrying out data processing according to the ISSR molecular polymorphism DNA fragment analysis result, and calculating Nei genetic similarity and genetic distance among 90 cassiterite floras, wherein the data of No. 1-8 parts are shown in Table 5. The results show that: the genetic similarity of 90 test drugs ranges from 0.8512 to 0.9317, and the genetic variation is small; the genetic distance ranges from 0.4915 to 1.9289. According to the genetic distance, the method comprises the following steps: the genetic distance (D is 0.4915) and the genetic similarity (I is 0.9317) in the Siberian cassis are the Sihualanxiang in Guangxi, and the genetic relationship is very close; the maximum genetic distance (D ═ 1.9289) is 42 (Hualanxiang county in Guangxi defense city, harbor city, Cin) and 2 (Wuming district monument in Guangxi Nanning city), and the minimum genetic similarity (I ═ 0.8512) is 79 (Shatian of Hongkong Huang great fairy district, Hongkong)
Village) and No.7 (Guangnaning city, Yongxian county, lotus pond, town, arborvitae, etc.), which indicate that the relationship is far away; shows the abundant genetic diversity of different populations under different geographic conditions.
TABLE 5 ISSR-based Stachys sieboldii similarity coefficient and genetic distance (part No. 1-8)
| Numbering | C1 | C2 | C3 | C4 | C5 | C6 | C7 | C8 |
| C1 | **** | 0.8905 | 0.8833 | 0.8858 | 0.8805 | 0.8716 | 0.8589 | 0.8802 |
| C2 | 1.0560 | **** | 0.8945 | 0.8919 | 0.8940 | 0.8777 | 0.8720 | 0.8821 |
| C3 | 1.1015 | 1.0844 | **** | 0.8898 | 0.8905 | 0.8774 | 0.8676 | 0.8856 |
| C4 | 1.0557 | 1.1303 | 1.0805 | **** | 0.8940 | 0.8791 | 0.8735 | 0.8868 |
| C5 | 1.1344 | 1.0756 | 1.0550 | 0.9943 | **** | 0.8863 | 0.8774 | 0.8908 |
| C6 | 1.2777 | 1.3943 | 1.2791 | 1.2417 | 1.0904 | **** | 0.8746 | 0.8781 |
| C7 | 1.6342 | 1.6127 | 1.5737 | 1.4132 | 1.3011 | 1.3327 | **** | 0.8814 |
| C8 | 1.2446 | 1.4828 | 1.2577 | 1.2287 | 1.1313 | 1.3744 | 1.3466 | **** |
Note: the genetic similarity of the Nei's (at), the genetic distance of the Nei's (at).
(5) ISSR-based clustering analysis of cassiabarktree leaves in different producing areas
Clustering analysis was performed on 90 different origins using NTSYS2.10 software and the clustering map is shown in fig. 8. The cassis vines can be distinguished between 0.87 and 0.93, the genetic similarity is 0.87 (I), and the cassis vines can be divided into two major classes, wherein the first class comprises 71 samples, samples with numbers 1 to 41 and 61 to 90 (from Guangdong, hong Kong, Guangxi Wuming, Lingshan, Qinzhou, Capacity and the like); the second category included 19 samples from 42-60 (thought county and city defense, Guangxi).
The genetic similarity is 0.876 (II), the first class can be divided into 5 subclasses, and the first subclass is a sample of No.1 (Guangxi Wuming district monument); the second subclass comprises 28 samples such as No. 2-6 and No. 8-30 (Guangxi Wuming district monument, Guangxi Dingxian county lotus pond town, Guangxi Qinzhou northern district Guitai town, Guangxi Lingshan county Shaping town, Guangxi Xuan county bottom town, Guangxi Qinxi City Acacia bamboo town, Guangdong Shenzhen Futian district pen rack mountain); the third subclass is sample No.7 (guangxi shou lian tang town); the fourth subclass is sample No. 31-41 (Guangdong Shenzhen fairy lake botanical garden, phoenix tree mountain, Meilin reservoir); the fifth subclass was sample No. 72-90 (southern hong Kong, Haemaria xanthata).
The genetic similarity is 0.883(III), the second subclass can be divided into 8 branches, the first branch is No. 2-5 (Guangxi Wuming area monument, Guangxi Xingning area stand-off); the second branch is No. 26-29 (Shaping town of Guangxi Lingshan county, Acacia lentiscus trifoliatus town of Guangxi Lingshan county, Acacia lentiscus trifoliata town of Guangxi Ling xi city); the third branch is No. 30 (Guangdong Shenzhen Futian zone penholder mountain); the fourth branch is 8-16 and 21 (bottom town of Guangxi Bing county, Guitai town of North Guangxi province); the fifth branch is 19, 20, 23 (Guitai town of northern district of Guangxi Qinzi, Shaping town of Guangxi Lingshan county); the sixth branch is 17-18 (the town of the county of Guangxi capacitive county, the Town of the Guitai town of the northern district of Guangxi Qinzi); the seventh branch is No. 22, No. 24 (Shaping town of Guangxi Lingshan county); the eighth branch is 25 (sanden town, san shan county, guangxi). The fifth sub-group is divided into 6 branches, the first branch is No. 61, 66, 68-71 (Guangxi city protection town of Nacizu); the second branch is 62-65 (Guangxi city protection town); the third branch is 67 (Guangxi city protection town); the fourth branch is 72-87 and 89-90 (southern hong Kong, Longqian mountain, yellow great fairy zone); the fifth branch is No. 79 (hong kong huang fairy site); the sixth branch is number 88 (Hongkong yellow fairy area). After being further subdivided, the medicine is found in No. 59-60 (Guangxi city protection town); 10-12, 15, 16, 21 (the bottom town of Guangxi Capacity county, the Guitai town of the northern district of Guangxi Qinzhou); number 72-87 (south hong Kong, yellow fairy area); no. 44-51 (Hualanxiang, Miss prefecture, Guangxi); number 52-56 (Guangxi city protection town); no. 34-39 (Guangdong Shenzhen Shenhu plantain and Guangdong Shenzhen Juhu region phoenix tree mountain).
From the above, most of the samples in the Guangxi producing area are gathered together, and the samples in Guangdong and hong Kong are gathered together; the genetic distance is relatively short because the genetic positions are adjacent, which indicates that the genetic diversity of most samples is related to the geographic positions. Meanwhile, the results of the molecular markers can identify samples from different producing areas. Except for city prevention, the relation between the Shansi and samples in other Guangxi areas is farthest, probably because the samples collected by the Shansi cannot be subjected to gene communication with other samples in a remote hundred thousand mountain areas; the Guangdong Shenzhen Yintai mountain and Guangxi Xiyeteng collected in the same category, probably because the two are collected from the roadside, the growth environment is similar, and the shape of the leaf is similar to the character of the Guangxi sample. A part of samples of Guangxi Fangchana town, Shenzhen and hong Kong samples are gathered in the same category, and have closer genetic relationship with hong Kong, probably because the geographic positions of the samples are similar and all have harbors, and in the growing process of day, month and month accumulation, the climate and the like are similar to those of Guangxi, and the genetic communication with the samples is more. The cassiterite clustering result shows that the genetic distance among most of the colonies is relatively obvious regionality, and the genetic distances among the colonies which are relatively close to each other are relatively small and are clustered into one group. However, some geographically distant communities are grouped into one category, which indicates that the clustering of the communities is not completely related to the geographic location.
(6) Principal coordinate analysis of Stachys sieboldii of different producing areas
Principal coordinate analysis (PCoA) can more intuitively reflect the spatial distance relationship of the population. Based on the genetic distance and the genetic similarity, the PCoA analysis of each colony of Siberian staphylos (Siberian sylvestris) was performed by using the software NTSYS-pc 2.1, and the results are shown in FIGS. 9 and 10. The main coordinate result is not completely consistent with the previous clustering result, as shown in fig. 9, all cassiterite florists are roughly divided into three categories, the first category is the uppermost 1-30 (south Guangxi ning city, Qinzhou city, Yulin, Cenxi city, Guangdong pen-frame mountain park) florists, and the category is more consistent with the clustering analysis result when the similarity of the front face is 0.872; the middle 31-40 (Guangdong Shenzhen city), No. 61-90 (Guangxi Fangcheng harbor city, hong Kong) and the like are gathered into one category; no. 41-60 (Merlin reservoir, Shangsi Guangxi, Guangxi City-protective harbor) and the like. In the latter two categories, the attribution of No. 41 (Guangdong Meilin reservoir) is different from that of the cluster analysis, and samples in Shangxiang and Fangcheng harbor in the cluster analysis are grouped into one category.
The first three principal coordinates in FIG. 10 and 3D are improved in interpretation, and the analysis results show that 72-90 (hong Kong) cluster is at the top left, and 61-71 (Guangxi defense against city, harbor, city, and shuttle town) cluster is at the top left; no. 1-30 (south-West Guanning, Qinzhou, Yulin, Cenxi, Guangdong pengshan park) and No. 42-60 (Shansi, Guangxi defense harbor); no. 31-41 (Guangdong Shenzhen city) is arranged below the reactor. The genetic distance and the relationship of each population of the cassiteria filiformis in the three-dimensional space are consistent with the result of the similarity of 0.874 in the previous cluster analysis, and the genetic relationship among the populations of the cassiteria filiformis is better explained.
The above description is intended to describe in detail the preferred embodiments of the present invention, but the embodiments are not intended to limit the scope of the claims of the present invention, and all equivalent changes and modifications made within the technical spirit of the present invention should fall within the scope of the claims of the present invention.
Although the invention has been described in detail hereinabove by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that many modifications and improvements can be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Sequence listing
<110> university of traditional Chinese medicine in Guangxi
<120> ISSR molecular marking method for cassis vine genetic diversity analysis
<130>2018
<160>9
<170>SIPOSequenceListing 1.0
<210>1
<211>17
<212>DNA
<213> Artificial series (Artificial sequence)
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<212>DNA
<213> Artificial series (Artificial sequence)
<400>2
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<212>DNA
<213> Artificial series (Artificial sequence)
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<212>DNA
<213> Artificial series (Artificial sequence)
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<212>DNA
<213> Artificial series (Artificial sequence)
<400>5
<210>6
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<212>DNA
<213> Artificial series (Artificial sequence)
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<210>7
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<212>DNA
<213> Artificial series (Artificial sequence)
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<213> Artificial series (Artificial sequence)
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Claims (6)
1. An ISSR molecular marker primer system for analyzing genetic diversity of Siberian cinquefoil, wherein the primer sequence comprises:
UBC number 807: the nucleotide sequence is shown as SED ID No. 1;
UBC No. 810: the nucleotide sequence is shown as SED ID No. 2;
UBC number 811: the nucleotide sequence is shown as SED ID No. 3;
UBC No. 836: the nucleotide sequence is shown as SED ID No. 4;
UBC No. 840: the nucleotide sequence is shown as SED ID No. 5;
UBC No. 842: the nucleotide sequence is shown as SED ID No. 6;
UBC No. 880: the nucleotide sequence is shown as SED ID No. 7;
UBC 881: the nucleotide sequence is shown as SED ID No. 8;
UBC No. 888: the nucleotide sequence is shown in SED ID No. 9.
2. A method for analyzing genetic diversity of Siberian cinquefoil by using the primer system of claim 1, which comprises the following steps:
1) extracting the genomic DNA of the casserole vine, and amplifying the genomic DNA of the casserole vine by using ISSR-PCR reaction;
2) carrying out electrophoresis and color development on a PCR product obtained by the PCR amplification in the step 1), and observing, photographing and storing in an ultraviolet gel imager after the color development is finished;
3) analyzing the band information obtained in the step 2), extracting data of a gel image by using GENESCAN3.1 software, selecting a proper internal STANDARD and setting software analysis parameters, selecting the color of a corresponding fluorescent marker and selecting SIZI STANDARD, analyzing the obtained result by using Binthere software, storing the result in an X L S format, converting a numerical value in an X L S table into 1 if the numerical value is not 0, generating an original matrix consisting of 'l' and '0', and performing cluster analysis by using a UPGMA method of a SHAN program in NTSYSpc-2.11F software to generate a cluster map.
3. The method for ISSR molecular marking of Sedum stannum genetic diversity analysis according to claim 2, wherein the ISSR-PCR amplification procedure in step 1) is as follows: pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 30s, annealing at 45s, wherein the annealing temperature is determined by Tm of different primers, extension at 72 ℃ for 2min, 45 cycles, extension at 72 ℃ for 10min, and storage at 4 ℃.
4. The ISSR molecular marker method for genetic diversity analysis of Sedum stannum according to claim 2, wherein the annealing temperature is determined according to the selected primers as follows:
SED ID No.150.9℃
SED ID No.251.2℃
SED ID No.350.1℃
SED ID No.450.3℃
SED ID No.551.5℃
SED ID No.650.6℃
SED ID No.753.2℃
SED ID No.852.6℃
SED ID No.952.3℃。
5. the ISSR molecular labeling method for genetic diversity analysis of cassis vines according to claim 2, wherein the electrophoresis in step 2) is performed under the conditions: electrophoresis was performed on 0.8% agarose gel at 75V for 50 min.
6. The use of the primer system of claim 1 for assisting in the identification of cassytha leaves.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101942504A (en) * | 2010-03-18 | 2011-01-12 | 河南科技大学 | DNA molecular labeling method |
| CN107164476A (en) * | 2017-05-26 | 2017-09-15 | 红河学院 | A kind of method that utilization ISSR reaction systems analyze tsaoko genetic diversity |
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2019
- 2019-09-05 CN CN201910836151.3A patent/CN111455082A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101942504A (en) * | 2010-03-18 | 2011-01-12 | 河南科技大学 | DNA molecular labeling method |
| CN107164476A (en) * | 2017-05-26 | 2017-09-15 | 红河学院 | A kind of method that utilization ISSR reaction systems analyze tsaoko genetic diversity |
Non-Patent Citations (4)
| Title |
|---|
| E. HRISTOVA 等: "Application of ISSR Methods in Studying Broomrape"S (Orobanchaceae) Biodiversity in Bulgaria", 《BIOTECHNOLOGY & BIOTECHNOLOGICAL EQUIPMENT》 * |
| 李娟玲 等: "鹧鸪茶种质资源ISSR分子标记中的引物筛选", 《安徽农业科学》 * |
| 杨和川 等: "金针菇菌株农艺性状评价及遗传多样性分析", 《南方农业学报》 * |
| 胡生福 等: "车前种质资源遗传多样性ISSR分析", 《安徽农业科学》 * |
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Application publication date: 20200728 |