CN104278101A - Specific ISSR-PCR primer group for identifying brasenia schreberi variety, molecular specific marker and method for identifying brasenia schreberi variety - Google Patents
Specific ISSR-PCR primer group for identifying brasenia schreberi variety, molecular specific marker and method for identifying brasenia schreberi variety Download PDFInfo
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Abstract
The invention discloses a specific ISSR-PCR primer group for identifying brasenia schreberi variety, a molecular specific marker and a method for identifying the brasenia schreberi variety. The specific ISSR-PCR primer group is composed of three specific primers. According to the method, the trouble that brasenia schreberi cannot be subjected to variety distinguishing only by the shape in introduction, breeding and production is effectively solved. The ISSR-PCR molecular marker is rapid, simple, convenient, reliable, high in stability, and low in DNA requirement amount, and the workload caused by a traditional variety distinguishing method can be relieved. The problems of adulterating, admixture of good and evil, mixing the false with the genuine in the production and popularization processes of the variety are greatly improved, the built ISSR-PCR product electrophoresis mode pattern can intuitively display the gene difference between the varieties, and the variety authenticity and consistency in production and scientific research are ensured.
Description
Technical field
The present invention relates to field of molecular marker, refer to a kind of method of specificity ISSR-PCR primer sets, molecular specific mark and the discriminating water shield kind thereof of identifying water shield kind particularly.
Background technology
Water shield (Brasenia schreberi) is perennial waterplant and important aquatic cash crop, is subordinate to Nymphaeceae or Cabombaceae or Cabombaceae.For a long time, particularly in recent decades, the Habitat destruction caused due to mankind's activity and excessively exploitation, group's area of water shield and population quantity sharply reduce, endangered, have been listed in country-level focused protection wild plant in imminent danger.
Water shield is regarded as gourmet food very early, also has very high pharmaceutical use.Containing abundant polysaccharide, protein, VITAMIN and trace element.It is mainly edible is partly the tender leaf roll of the children do not launched being rich in colloid.In China, water shield mainly originates in the areas such as area, the West Lake, Zhejiang, TAI HU AREA, Jiangsu and Lichuan County, Hubei.Its distribution shows as low altitude area, subtropical monsoon climate if area, the West Lake, Zhejiang, TAI HU AREA, Jiangsu and High aititude, extremely frigid zones, vertical zone sex climate are as Lichuan, Hubei Province (height above sea level 1200 ~ 1400m).Thus water shield is caused to have different variety and qualities.
At present to the discriminating of water shield kind mainly according to the morphological specificity of plant as the color etc. at pattern, face of blade and the back side, such as, but be sometimes difficult to difference come, the Huadu of West Lake water shield kind and Lichuan water shield kind is that the color at red, face of blade and the back side does not all have obvious difference.
Also there is no a kind of method of effective discriminating water shield kind at present.
Summary of the invention
Technical problem to be solved by this invention is just to provide a kind of method of specificity ISSR-PCR primer sets of specificity ISSR-PCR primer sets, molecular specific mark and the qualification water shield kind thereof of identifying water shield kind, molecular specific mark and discriminating water shield kind thereof.The difference that the present invention adopts ISSR molecular marking technique to build pure vegetable kind DNA fingerprinting, and form molecular identity card for the identification of the pure vegetable kind of difference, overcome the deficiency that morphology is differentiated.This authentication method has important directive significance to the scientific utilization of pure dish germ plasm resource and exploitation.
For solving the problems of the technologies described above, provided by the inventionly a kind ofly identify the specificity ISSR-PCR primer sets of water shield kind, described specificity ISSR-PCR primer sets is made up of three Auele Specific Primers, these three Auele Specific Primers are respectively:
1)P817：5’-CACACACACACACACAA-3’(5’-(CA)
8A-3’)；
2)P825：5’-ACACACACACACACACT-3’(5’-(AC)
8T-3’)；
3)P845：5’-CTCTCTCTCTCTCTCTRG-3’(5’-(CT)
8(A/T)G-3’)。
These three kinds of primers screen from 60 primers, therefrom chooses clear, repeated good 3 primers of amplified band for pcr amplification.
The invention provides a kind of molecular specific mark adopting specificity ISSR-PCR primer sets to obtain,
1) adopt specificity ISSR-PCR primer P817 to increase to water shield genome, obtain the DNA fragmentation of size 400bp, be molecular specific mark, or;
2) adopt specificity ISSR-PCR primer P825 to increase to water shield genome, obtain the DNA fragmentation of size 750bp, be molecular specific mark, or;
3) adopt specificity ISSR-PCR primer P845 to increase to water shield genome, obtain five DNA fragmentations that size is 2000bp, 700bp, 600bp, 500bp and 300bp; These five DNA fragmentations are all molecular specific mark.
Present invention also offers a kind of method adopting specificity ISSR-PCR primer sets to obtain molecular specific mark, comprise the following steps:
1) choose water shield young tender leaf, extract DNA, obtain water shield genome;
2) any one primer in above-mentioned specificity ISSR-PCR primer sets is chosen, with water shield genome for template,
Pcr amplification program reaction conditions:
Pcr amplification program is:
94 DEG C of denaturations 5 minutes, then carry out following circulation: 94 DEG C of sex change 30s, 54 DEG C of annealing 45s, and 72 DEG C extend 1.5min, amount to 35 circulations; After loop ends, 72 DEG C extend 7min, obtain PCR primer and be molecular specific mark.
Wherein, described PCR primer is:
1) adopt specificity ISSR-PCR primer P817 to increase to water shield genome, obtain the DNA fragmentation of size 400bp, be molecular specific mark, or;
2) adopt specificity ISSR-PCR primer P825 to increase to water shield genome, obtain the DNA fragmentation of size 750bp, be molecular specific mark, or;
3) adopt specificity ISSR-PCR primer P845 to increase to water shield genome, obtain five DNA fragmentations that size is 2000bp, 700bp, 600bp, 500bp and 300bp; These five DNA fragmentations are all molecular specific mark.
The invention provides a kind of method utilizing specificity ISSR-PCR primer sets to differentiate water shield kind, comprise the following steps:
1) with water shield genome for template, respectively with following three Auele Specific Primers,
P817：5’-CACACACACACACACAA-3’；
P825：5’-ACACACACACACACACT-3’；
P845:5 '-CTCTCTCTCTCTCTCTRG-3 '; Carry out PCR respectively, wherein,
Reaction system is:
| The dNTP of 0.25mM | 2.5μl |
| The primer of 1.5mM | 1.5μl |
| 2.5U Taq enzyme | 0.5μl |
| PCR?Buffer | 3.5μl |
| (10ng) water shield genome | 4μl |
| Ultrapure water | 13μl |
| Add up to | 25μl |
Pcr amplification program reaction conditions:
Pcr amplification program is:
94 DEG C of denaturations 5 minutes, then carry out following circulation: 94 DEG C of sex change 30s, 54 DEG C of annealing 45s, and 72 DEG C extend 1.5min, amount to 35 circulations; After loop ends, 72 DEG C extend 7min; Obtain PCR primer,
2) PCR primer will be obtained and be placed in 1.5% sepharose, and carry out electrophoretic separation, obtain electrophorogram;
3) P817, P825 and P845 tri-kinds of primer amplifications are obtained electrophorogram, be analyzed, obtain ISSR finger printing, thus identify different water shield kind.
Beneficial effect of the present invention is:
The present invention efficiently solve water shield introducing a fine variety, seed selection, depend merely on form in production and cannot carry out the so tired of kind differentiation.ISSR molecule marker is quick, easy, reliable, stability is high, DNA requirement is few, can alleviate the workload that traditional category district office brings.The present invention substantially improves kind in production extension process, with bad fill excellent, dragons and fishes jumbled together, the problem of mixing the spurious with the genuine, the ISSR-PCR product electrophoretic figure set up, can show gene difference between kind intuitively, ensures variety authentication, consistence in production scientific research.
Accompanying drawing explanation
Fig. 1 is the DNA collection of illustrative plates that primer P825 increases to Su Hang water shield kind;
Fig. 2 is the DNA collection of illustrative plates that primer P825 increases to Lichuan water shield kind;
Fig. 3 is the DNA collection of illustrative plates that primer P845 increases to Su Hang water shield kind;
Fig. 4 is the DNA collection of illustrative plates that primer P845 increases to Lichuan water shield kind;
Fig. 5 is the DNA collection of illustrative plates that primer P817 increases to Su Hang water shield kind;
Fig. 6 is the DNA collection of illustrative plates that primer P817 increases to Lichuan water shield kind;
In figure, M:2000bp marker;
Fig. 7 is Su Hang water shield kind and the Lichuan water shield variety source finger printing (molecular identity card) of ISSR mark structure;
In figure, dark squares representative bands of a spectrum exist, and figure the right P825, P845, P817 represent Primer respectively, the size of the DNA bands of a spectrum of the numeral amplification after "-" number.1-46 is sample number.
Fig. 8 is Hangzhou West Lake water shield kind and Lichuan good fortune Golconda water shield variety source finger printing (molecular identity card) of ISSR mark structure;
In figure, dark squares representative bands of a spectrum exist, and figure the right P825, P845, P817 represent Primer respectively, the size of the DNA bands of a spectrum of the numeral amplification after "-" number.1-23 representative samples Article Number.
Embodiment
In order to explain the present invention better, illustrate main contents of the present invention further below in conjunction with specific embodiment, but content of the present invention is not only confined to following examples.
Embodiment 1
1, sample
At the duration of flowering (May) of water shield, in Dongshan Town, Suzhou City of Jiangsu Province, good greatly paddy three ground in Hangzhou, Zhejiang province city Zhou Puren bridge and Hangzhou, Zhejiang province city gathers the young tender leaf of Su Hang water shield kind, at the young tender leaf of Lichuan Zhong Lu, Lichuan good fortune Golconda, Lichuan good fortune Golconda yu-kin bank and collection Lichuan, ground, the Baihe, Lichuan four water shield kind, some Young Plant tender leafs are got, every strain 5-10g in each place; Because water shield mainly seeks clone (asexual) breeding, and in the water that formed of its subterraneous stem, stem is shape of growing thickly, multi-branched, thus should sample to avoid adjacent to be sampled as same strain water shield every more than 5m.Every strain Young Plant tender leaf is placed in the sealing bag that silica gel is housed dry rapidly, to be used as to extract DNA's;
2, DNA extraction
CTAB method is adopted to extract the DNA of the water shield of the local different varieties of above-mentioned difference;
3, pcr amplification
1) respectively with above-mentioned differently square different varieties water shield genome for template, respectively with following three Auele Specific Primers,
P817：5’-CACACACACACACACAA-3’；
P825：5’-ACACACACACACACACT-3’；
P845:5 '-CTCTCTCTCTCTCTCTRG-3 '; Carry out PCR respectively, wherein,
Reaction system is:
| The dNTP of 0.25mM | 2.5μl |
| The primer of 1.5mM | 1.5μl |
| 2.5U Taq enzyme | 0.5μl |
| PCR?Buffer | 3.5μl |
| (10ng) water shield genome | 4μl |
| Ultrapure water | 13μl |
| Add up to | 25μl |
Pcr amplification program reaction conditions:
Pcr amplification program is:
94 DEG C of denaturations 5 minutes, then carry out following circulation: 94 DEG C of sex change 30s, 54 DEG C of annealing 45s, and 72 DEG C extend 1.5min, amount to 35 circulations; After loop ends, 72 DEG C extend 7min; Obtain PCR primer,
2) PCR primer will be obtained and be placed in 1.5% sepharose, carry out electrophoretic separation, obtain electrophorogram (as Fig. 1 ~ 6);
3) P817, P825 and P845 tri-kinds of primer amplifications are obtained electrophorogram, be analyzed, obtain ISSR finger printing, thus identify different water shield kind (as Fig. 7).
As shown in Fig. 1 ~ 7: in Dongshan Town, Suzhou City of Jiangsu Province, to gather Su Hang water shield kind be same kind to good greatly paddy three ground in Hangzhou, Zhejiang province city Zhou Puren bridge and Hangzhou, Zhejiang province city, be same kind in Lichuan Zhong Lu, Lichuan good fortune Golconda, Lichuan good fortune Golconda yu-kin bank and collection Lichuan, ground, the Baihe, Lichuan four water shield kind, and Su Hang water shield kind and Lichuan water shield kind are different varieties.
Embodiment 2
1, sample
At the duration of flowering (May) of water shield, gather the young tender leaf of Hangzhou West Lake water shield kind at Hangzhou West Lake, gather the young tender leaf of Lichuan water shield kind at, Lichuan good fortune Golconda, get some Young Plant tender leafs in each place, every strain 5-10g; Because water shield mainly seeks clone (asexual) breeding, and in the water that formed of its subterraneous stem, stem is shape of growing thickly, multi-branched, thus should sample to avoid adjacent to be sampled as same strain water shield every more than 5m.Every strain Young Plant tender leaf is placed in the sealing bag that silica gel is housed dry rapidly, to be used as to extract DNA's;
2, DNA extraction
CTAB method is adopted to extract the DNA of the water shield of the local different varieties of above-mentioned difference;
3, pcr amplification
1) respectively with above-mentioned differently square different varieties water shield genome for template, respectively with following three Auele Specific Primers,
P817：5’-CACACACACACACACAA-3’；
P825：5’-ACACACACACACACACT-3’；
P845:5 '-CTCTCTCTCTCTCTCTRG-3 '; Carry out PCR respectively, wherein,
Reaction system is:
| The dNTP of 0.25mM | 2.5μl |
| The primer of 1.5mM | 1.5μl |
| 2.5U Taq enzyme | 0.5μl |
| PCR?Buffer | 3.5μl |
| (10ng) water shield genome | 4μl |
| Ultrapure water | 13μl |
| Add up to | 25μl |
Pcr amplification program reaction conditions:
Pcr amplification program is:
94 DEG C of denaturations 5 minutes, then carry out following circulation: 94 DEG C of sex change 30s, 54 DEG C of annealing 45s, and 72 DEG C extend 1.5min, amount to 35 circulations; After loop ends, 72 DEG C extend 7min; Obtain PCR primer,
2) PCR primer will be obtained and be placed in 1.5% sepharose, and carry out electrophoretic separation, obtain electrophorogram;
3) P817, P825 and P845 tri-kinds of primer amplifications are obtained electrophorogram, be analyzed, obtain ISSR finger printing, thus identify different water shield kind (as Fig. 8).
As shown in Figure 8: Hangzhou West Lake water shield kind and Lichuan good fortune Golconda water shield kind are different varieties.
Other unspecified part is prior art.Although above-described embodiment is to invention has been detailed description; but it is only the present invention's part embodiment; instead of whole embodiment, people can also obtain other embodiments according to the present embodiment under without creative prerequisite, and these embodiments all belong to scope.
Claims (5)
1. the specificity ISSR-PCR primer sets identifying water shield kind, it is characterized in that: described specificity ISSR-PCR primer sets is made up of three Auele Specific Primers, and these three Auele Specific Primers are respectively:
1)P817：5’-CACACACACACACACAA-3’；
2)P825：5’-ACACACACACACACACT-3’；
3)P845：5’-CTCTCTCTCTCTCTCTRG-3’。
2. adopt the molecular specific mark that described in claim 1, specificity ISSR-PCR primer sets obtains, it is characterized in that:
1) adopt specificity ISSR-PCR primer P817 to increase to water shield genome, obtain the DNA fragmentation of size 400bp, be molecular specific mark, or;
2) adopt specificity ISSR-PCR primer P825 to increase to water shield genome, obtain the DNA fragmentation of size 750bp, be molecular specific mark, or;
3) adopt specificity ISSR-PCR primer P845 to increase to water shield genome, obtain five DNA fragmentations that size is 2000bp, 700bp, 600bp, 500bp and 300bp; These five DNA fragmentations are all molecular specific mark.
3. adopt specificity ISSR-PCR primer sets to obtain a method for molecular specific mark described in claim 1, it is characterized in that: comprise the following steps:
1) choose water shield young tender leaf, extract DNA, obtain water shield genome;
2) choose any one primer in above-mentioned specificity ISSR-PCR primer sets, with water shield genome for template, reaction system is:
Pcr amplification program reaction conditions:
Pcr amplification program is:
94 DEG C of denaturations 5 minutes, then carry out following circulation: 94 DEG C of sex change 30s, 54 DEG C of annealing 45s, and 72 DEG C extend 1.5min, amount to 35 circulations; After loop ends, 72 DEG C extend 7min, obtain PCR primer and be molecular specific mark.
4. adopt specificity ISSR-PCR primer sets to obtain the method for molecular specific mark according to claim 3, it is characterized in that: described PCR primer is:
1) adopt specificity ISSR-PCR primer P817 to increase to water shield genome, obtain the DNA fragmentation of size 400bp, be molecular specific mark, or;
2) adopt specificity ISSR-PCR primer P825 to increase to water shield genome, obtain the DNA fragmentation of size 750bp, be molecular specific mark, or;
3) adopt specificity ISSR-PCR primer P845 to increase to water shield genome, obtain five DNA fragmentations that size is 2000bp, 700bp, 600bp, 500bp and 300bp; These five DNA fragmentations are all molecular specific mark.
5. the method utilizing specificity ISSR-PCR primer sets described in claim 1 to differentiate water shield kind: it is characterized in that: comprise the following steps:
1) with water shield genome for template, respectively with following three Auele Specific Primers,
P817：5’-CACACACACACACACAA-3’；
P825：5’-ACACACACACACACACT-3’；
P845:5 '-CTCTCTCTCTCTCTCTWG-3 '; Carry out PCR respectively, wherein, reaction system is:
Pcr amplification program reaction conditions:
Pcr amplification program is:
94 DEG C of denaturations 5 minutes, then carry out following circulation: 94 DEG C of sex change 30s, 54 DEG C of annealing 45s, and 72 DEG C extend 1.5min, amount to 35 circulations; After loop ends, 72 DEG C extend 7min; Obtain PCR primer,
2) PCR primer will be obtained and be placed in 1.5% sepharose, and carry out electrophoretic separation, obtain electrophorogram;
3) P817, P825 and P845 tri-kinds of primer amplifications are obtained electrophorogram, be analyzed, obtain ISSR finger printing, thus identify different water shield kind.
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| CN201410561492.1A CN104278101A (en) | 2014-10-21 | 2014-10-21 | Specific ISSR-PCR primer group for identifying brasenia schreberi variety, molecular specific marker and method for identifying brasenia schreberi variety |
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| CN201410561492.1A CN104278101A (en) | 2014-10-21 | 2014-10-21 | Specific ISSR-PCR primer group for identifying brasenia schreberi variety, molecular specific marker and method for identifying brasenia schreberi variety |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104805190A (en) * | 2015-03-31 | 2015-07-29 | 江汉大学 | Method for testing distinctness, uniformity and stability of hybrid corn new variety |
| CN105063223A (en) * | 2015-09-02 | 2015-11-18 | 中国热带农业科学院香料饮料研究所 | Method for identifying coffee germplasm resource by using ISSR (inter-simple sequence repeat) fingerprint chromatography |
| CN110453005A (en) * | 2019-08-28 | 2019-11-15 | 广西壮族自治区农业科学院微生物研究所 | A kind of ISSR-SCAR label and its application method identifying low temperature stimulation type elegant precious mushroom |
-
2014
- 2014-10-21 CN CN201410561492.1A patent/CN104278101A/en active Pending
Non-Patent Citations (3)
| Title |
|---|
| YUAN-HUO DONG: "Genetic structure and gene flow in the endangered aquatic economic crop Brasenia schreberi J. F. Gmel. (Nymphaeaceae) in China", 《PROC. OF SPIE》 * |
| 张光富: "江浙莼菜遗传多样性和遗传结构的ISSR分析", 《湖泊科学》 * |
| 张青林: "ISSR及其在果树上的应用", 《果树学报》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104805190A (en) * | 2015-03-31 | 2015-07-29 | 江汉大学 | Method for testing distinctness, uniformity and stability of hybrid corn new variety |
| CN105063223A (en) * | 2015-09-02 | 2015-11-18 | 中国热带农业科学院香料饮料研究所 | Method for identifying coffee germplasm resource by using ISSR (inter-simple sequence repeat) fingerprint chromatography |
| CN105063223B (en) * | 2015-09-02 | 2018-03-23 | 中国热带农业科学院香料饮料研究所 | A kind of method using ISSR fingerprint identification coffee germ plasm resources |
| CN110453005A (en) * | 2019-08-28 | 2019-11-15 | 广西壮族自治区农业科学院微生物研究所 | A kind of ISSR-SCAR label and its application method identifying low temperature stimulation type elegant precious mushroom |
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Application publication date: 20150114 |