CN108130379A - Affiliation carries out mirror method for distinguishing between fortune paulownia and congener - Google Patents
Affiliation carries out mirror method for distinguishing between fortune paulownia and congener Download PDFInfo
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- CN108130379A CN108130379A CN201710865015.8A CN201710865015A CN108130379A CN 108130379 A CN108130379 A CN 108130379A CN 201710865015 A CN201710865015 A CN 201710865015A CN 108130379 A CN108130379 A CN 108130379A
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Abstract
The present invention relates to a kind of affiliations between fortune paulownia and congener to carry out mirror method for distinguishing, the method includes：（a）Extract the total DNA of fortune paulownia blade；（b）With step（a）The total DNA extracted carries out PCR amplification for template, wherein, the primer of PCR amplification is rpl16 primer pairs and petL psbE primer pairs；（c）Extract the total DNA of Paulownia sibling species blade；（d）With with step（b）Identical method, with step（c）The total DNA extracted carries out PCR amplification respectively for template；（e）To step（b）With（d）Amplified production carry out sequence alignment respectively, build UPGMA phylogenetic trees.
Description
Technical field
The invention belongs to species authentication technique fields, and in particular to utilize cpDNA（Chloroplast DNA）Molecular labeling is to white flower
Paulownia carry out mirror method for distinguishing with its sibling species affiliation.
Background technology
Paulownia are as the excellent forest type for originating in China, and germ plasm resource is also extremely abundant, morphological variation and geographical variation
Type largely exists, paulownia naturally can cross-pollination breeding offspring characteristic so that the hybridization between paulownia is extremely easy.With gas
, there is different ecotypes, inter-species has accumulated in kind in the alternating of time and prolonged backcrossing and interspecific hybridization repeatedly
A large amount of phenotype transitionality hybrid swarms, show as more complicated interspecies relation；Despite the presence of relationship it is complicated, but with regard to its kind
Between difference present in terms of biological character and morphology be not it is very big, this evolve for research Paulownia botanical system and
Considerably increase difficulty in terms of classification, therefore, just currently for, certain kinds of affiliation of Paulownia and taxonomically
Dispute present in position etc. is also bigger.
In plant, due to chloroplast DNA category matrocliny, mutation rate is little, and heredity is relatively stablized, and evolutionary rate is slow,
And molecular weight is small, simple in structure, chloroplast gene spacer region be non-coding sequence, influenced by Environmental variations it is minimum, can
It is higher to preserve the degree of its variation, preferable resolution capability can be provided in system position analysis is carried out, this is for illustrating
The relationship of inter-species in category, and the accuracy for system position analysis provides guarantee.It is currently reported to utilize cpDNA molecular labelings
The method for carrying out persimmon Germplasm Identification（Number of patent application：201610228088.1）, however, for increasingly complex fortune paulownia and
The affiliation of its congener there is no the report of reliable identification method.
Invention content
In order to solve the above technical problems, utilize cpDNA the present invention provides a kind of（Chloroplast DNA）Molecular labeling is to white flower
Affiliation between paulownia and congener carries out mirror method for distinguishing, this is the affiliation of fortune paulownia and congener
Identification and development provide reliable theoretical foundation, also provide theoretical direction and technical support for the Genetic conservation of the species.
Therefore, the present invention provides the sides that a kind of affiliation between fortune paulownia and congener is differentiated
Method, the method includes：
（a）Extract the total DNA of fortune paulownia blade；
（b）With step（a）The total DNA extracted carries out PCR amplification for template, wherein, the primer of PCR amplification is rpl16 primers
Pair and petL-psbE primer pairs；
（c）Extract the total DNA of Paulownia sibling species blade；
（d）With with step（b）Identical method, with step（c）The total DNA extracted carries out PCR amplification respectively for template；
（e）To step（b）With（d）Amplified production carry out sequence alignment respectively, build UPGMA phylogenetic trees.
The analysis result of the present invention can be as identification fortune paulownia and the foundation of sibling species affiliation.
In a preferred embodiment, step（a）And step（c）Total DNA extraction method using modified CTAB method, specifically
Step is：
（1）Dry blade 50mg is weighed, 3% soluble PVP powder is added in, fine powder is ground into after pouring into liquid nitrogen；
（2）The fine powder is fitted into 2 mL Eppendorf pipes, 800 2 × CTAB of μ L with passing through 60 DEG C of preheatings in pipe
Extracting solution mixes；
（3）60 μ L mercaptoethanols are added in into Eppendorf pipes, gentle agitation preheats 30 min at 60 DEG C；
（4）It is primary that mixing is gently overturned per 10-15min, places sample at room temperature after 3-5 times；
（5）Then reach to temperature and 800 μ L chloroforms are added in the sample of room temperature：Isoamyl alcohol（24：1）Mixed liquor, after mixing
In 12 000 r min-115 min of lower centrifugation；
（6）Transfer supernatant is placed in a 2 new mL Eppendorf pipes, adds in isometric chloroform：Isoamyl alcohol（24：1）
Mixed liquor vibrates mixing, in 12 000 r min-115 min of lower centrifugation；
（7）Again in Aspirate supernatant to another 2mL Eppendorf pipe, isometric 1 × CTAB precipitated liquids mixing is added in
Uniformly, 20-30 min are placed at room temperature, and 15 min are centrifuged under 12 000 r min-1；
（8）Supernatant is discarded, precipitation is dried at room temperature, is dissolved in 200 μ L TE-buffer；
（9）Then 400 μ L, 95% ethyl alcohol is added in, 60 min are stood at -20 DEG C, generates precipitation；
（10）At 4 DEG C, in 12000 r min-115 min of lower centrifugation；
（11）Liquid is discarded supernatant, 75% ethyl alcohol for adding in 500 μ L precoolings is washed, and 12000 r min-1 centrifuge 10 min；
（12）Liquid is discarded supernatant, is spontaneously dried at room temperature, 30-50 μ 1 × TE-buffer of L are dissolved in, at -20 DEG C
Lower preservation.
In a preferred embodiment, in step（b）And step（d）In, select cpDNA primer pairsrpl16WithpetL- psbEFor fortune paulownia and the identification for belonging to Interspecific relationship.It is describedrpl16WithpetL-psbEThe sequence of primer pair such as table
Shown in 1：
Table 1
Preferably, in step（b）And step（d）In PCR amplification condition it is as follows：
10×PCR buffer 5 μL
dNTPs 4 μL
R primers（10 μM） 0.4 μL
F primers（10 μM） 0.4 μL
DNA profiling（10 ng/μL） 5.0 μL
Taq DNA Polymerase（5 U/μL） 0.4 μL
ddH2O 34.8 μL。
In further preferred embodiment, in step（b）And step（d）In, the program of PCR amplification is as follows：
rpl16Amplification program：94 DEG C of 5 min of pre-degeneration, 94 DEG C are denaturalized 45 seconds, and 55 DEG C are annealed 30 seconds, 35 cycles, 72 DEG C of extensions
2min, 72 DEG C extend 10 min, are preserved at 4 DEG C；
petL-psbEAmplification program：94 DEG C of 5 min of pre-degeneration, 94 DEG C of 1 min of denaturation, 52 DEG C of 1 min of annealing, 35 recycle, and 72
DEG C extension 5 min, 72 DEG C of 8 min of extension are preserved at 4 DEG C.
Preferably, the product after PCR amplification is detected with 1.5% agarose gel electrophoresis, can be obtained clear, single
One target stripe carries out sequencing after purification.
The present invention uses cpDNA（Chloroplast DNA）Molecular labeling builds UPGMA systematic growths to fortune paulownia and sibling species
Tree（As shown in Figure 1）, the identification and development of the affiliation for paulownia platymiscium, which provide, can refer to foundation.
Description of the drawings
Fig. 1 is based on cpDNA（Chloroplast DNA）The Paulownia germplasm UPGMA phylogenetic trees of molecular labeling.
Specific embodiment
The method of the present invention is described in detail below by way of specific embodiment.
Using in 3 populations of the fortune paulownia for being distributed in different geographical and 9 Paulownias kinds totally 77 parts in the present embodiment
Material is used for the identification of affiliation.Specific seeds and locality as shown in table 2：
Table 2
| Number | Kind（Mutation, modification） | Number of individuals | Acquired original |
| 1 | Fortune paulownia 1,P. fortunei | 6 | Yichang, Yichang, Hubei |
| 2 | Fortune paulownia 2,P. fortunei | 6 | Guizhou is all even, Duyun, Guizhou |
| 3 | Fortune paulownia 3,P. fortunei | 6 | Lishui of Zhejiang, Lishui, Zhejiang |
| 4 | Royal paulownia,P. tomentosa | 8 | Zhengzhou, henan, Zhengzhou, Henan |
| 5 | Bright leaf royal paulownia,P. tomentosa var.lucida | 6 | Dalian, Dalian, Liaoning |
| 6 | River paulownia,P. fargesii | 6 | Jianshi, hubei, Jianshi Country, Hubei |
| 7 | Paulownia elongata,P. elongata | 7 | Jiangxi Komsomolsk, Gongqingcheng, Jiangxi |
| 8 | Formosan paulownia (Cortex seu Radix Paulowniae kawakamii),P. kawakamii | 6 | Fujian Shaowu, Shaowu Country, Fujian |
| 9 | Paulownia australis,P. australis | 8 | Jiangxi Komsomolsk, Gongqingcheng, Jiangxi |
| 10 | Paulownia catalpifolia,P. catalpifolia | 6 | Taian Shandong, Tai ' an Shandong |
| 11 | Yichang paulownia,P. ichangensis | 6 | Yichang, Yichang, Hubei |
| 12 | Chengdu paulownia,P.albiphloea var.chengtuensis | 6 | Sichuan Chengdu, Chengdu, Sichuan |
Using following methods, the identification of progress more than paulownia Interspecific relationship：
（a）Extract the total DNA of 3 blade of fortune paulownia 1, fortune paulownia 2 and fortune paulownia；
（b）With step（a）The total DNA extracted carries out PCR amplification for template, wherein, the primer of PCR amplification is rpl16 primers
Pair and petL-psbE primer pairs；
（c）Extract the total DNA of other germplasm blades in addition to fortune paulownia；
（d）With with step（b）Identical method, with step（c）The total DNA extracted carries out PCR amplification respectively for template；
（e）To step（b）With（d）Amplified production carry out sequence alignment respectively, build UPGMA phylogenetic trees.
Specifically, step（a）And step（c）Total DNA extraction method using step in detail below：
（1）Dry blade 50mg is weighed respectively, adds in 3% soluble PVP powder, fine powder is ground into after pouring into liquid nitrogen；
（2）The fine powder is fitted into 2 mL Eppendorf pipes, 800 2 × CTAB of μ L with passing through 60 DEG C of preheatings in pipe
Extracting solution mixes；
（3）60 μ L mercaptoethanols are added in into Eppendorf pipes, gentle agitation preheats 30 min at 60 DEG C；
（4）It is primary that mixing is gently overturned per 10-15min, places sample at room temperature after 3-5 times；
（5）Then reach to temperature and 800 μ L chloroforms are added in the sample of room temperature：Isoamyl alcohol（24：1）Mixed liquor, after mixing
In 12 000 r min-115 min of lower centrifugation；
（6）Transfer supernatant is placed in a 2 new mL Eppendorf pipes, adds in isometric chloroform：Isoamyl alcohol（24：1）
Mixed liquor vibrates mixing, in 12 000 r min-115 min of lower centrifugation；
（7）Again in Aspirate supernatant to another 2mL Eppendorf pipe, isometric 1 × CTAB precipitated liquids mixing is added in
Uniformly, 20-30 min are placed at room temperature, and 15 min are centrifuged under 12 000 r min-1；
（8）Supernatant is discarded, precipitation is dried at room temperature, is dissolved in 200 μ L TE-buffer；
（9）Then 400 μ L, 95% ethyl alcohol is added in, 60 min are stood at -20 DEG C, generates precipitation；
（10）At 4 DEG C, in 12000 r min-115 min of lower centrifugation；
（11）Liquid is discarded supernatant, 75% ethyl alcohol for adding in 500 μ L precoolings is washed, and 12000 r min-1 centrifuge 10 min；
（12）Liquid is discarded supernatant, is spontaneously dried at room temperature, 30-50 μ 1 × TE-buffer of L are dissolved in, at -20 DEG C
Lower preservation.
In step（b）And step（d）In, it is specifically chosen cpDNA primer pairsrpl16WithpetL-psbEFor fortune paulownia
Identification with belonging to Interspecific relationship.It is describedrpl16WithpetL-psbEThe sequence of primer pair is as shown in table 1：
Table 1
In step（b）And step（d）In PCR amplification condition it is as follows：
10×PCR buffer 5 μL
dNTPs 4 μL
R primers（10 μM） 0.4 μL
F primers（10 μM） 0.4 μL
DNA profiling（10 ng/μL） 5.0 μL
Taq DNA Polymerase（5 U/μL） 0.4 μL
ddH2O 34.8 μL。
In step（b）And step（d）In, the program of PCR amplification is as follows：
rpl16Amplification program：94 DEG C of 5 min of pre-degeneration, 94 DEG C are denaturalized 45 seconds, and 55 DEG C are annealed 30 seconds, 35 cycles, 72 DEG C of extensions
2min, 72 DEG C extend 10 min, are preserved at 4 DEG C；
petL-psbEAmplification program：94 DEG C of 5 min of pre-degeneration, 94 DEG C of 1 min of denaturation, 52 DEG C of 1 min of annealing, 35 recycle, and 72
DEG C extension 5 min, 72 DEG C of 8 min of extension are preserved at 4 DEG C.
Product after PCR amplification is detected with 1.5% agarose gel electrophoresis, can obtain clear, single mesh
Band is marked, carries out sequencing after purification.
The present invention uses cpDNA（Chloroplast DNA）Molecular labeling builds UPGMA systematic growths to fortune paulownia and sibling species
Tree（As shown in Figure 1）, the identification and development of the affiliation for paulownia platymiscium, which provide, can refer to foundation.
With cpDNA（Chloroplast DNA）Molecular labeling builds UPGMA phylogenetic trees to fortune paulownia and sibling species（Such as
Shown in Fig. 1）, it is copolymerized as three classes, the first kind：White flower 2, white flower 3, white flower 1, paulownia australis, Yichang paulownia, Chengdu paulownia and Lankao
Paulownia；Second class is royal paulownia, bright leaf royal paulownia and paulownia catalpifolia；Third class：Formosan paulownia (Cortex seu Radix Paulowniae kawakamii)；4th class：River paulownia.
In terms of the 4th class of phylogenetic tree, three fortune paulownia plants gather for together, therefore, it is considered that fortune paulownia is one
Kind that is independent and stablizing.
The method of the present invention provides for the identification and development of the affiliation of paulownia platymiscium and can refer to foundation.
Sequence table
<110>North China University of Water Resources and Electric Power
<120>Affiliation between fortune paulownia and congener carries out mirror method for distinguishing
<150> 2017105403534
<151> 2017-07-05
<160> 4
<170> SIPOSequenceListing 1.0
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<213>Artificial sequence (Artificial Sequence)
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gctatgctta gtgtgtgact cgttg 25
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<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
cccttcatta ttcctctatg ttg 23
<210> 3
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<212> DNA
<213>Artificial sequence (Artificial Sequence)
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agtagaaaac cgaaataact agtta 25
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<213>Artificial sequence (Artificial Sequence)
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tatcgaatac tggtaataat atcagc 26
Claims (6)
1. a kind of affiliation between fortune paulownia and congener carries out mirror method for distinguishing, the method includes：
（a）Extract the total DNA of fortune paulownia blade；
（b）With step（a）The total DNA extracted carries out PCR amplification for template, wherein, the primer of PCR amplification is rpl16 primers
Pair and petL-psbE primer pairs；
（c）Extract the total DNA of Paulownia sibling species blade；
（d）With with step（b）Identical method, with step（c）The total DNA extracted carries out PCR amplification respectively for template；
（e）To step（b）With（d）Amplified production carry out sequence alignment respectively, build UPGMA phylogenetic trees.
2. according to the method described in claim 1, it is characterized in that, step（a）And step（c）Total DNA extraction method tool
Body step is：
（1）Dry blade 50mg is weighed, 3% soluble PVP powder is added in, fine powder is ground into after pouring into liquid nitrogen；
（2）The fine powder is fitted into 2 mL Eppendorf pipes, 800 2 × CTAB of μ L with passing through 60 DEG C of preheatings in pipe
Extracting solution mixes；
（3）60 μ L mercaptoethanols are added in into Eppendorf pipes, gentle agitation preheats 30 min at 60 DEG C；
（4）It is primary that mixing is gently overturned per 10-15min, places sample at room temperature after 3-5 times；
（5）Then reach to temperature and 800 μ L chloroform-isoamyl alcohol mixed liquors are added in the sample of room temperature, after mixing 12
000 r•min-115 min of lower centrifugation；
（6）Transfer supernatant is placed in a 2 new mL Eppendorf pipes, adds in isometric chloroform：Isoamyl alcohol（24：1）
Mixed liquor vibrates mixing, in 12 000 r min-115 min of lower centrifugation；
（7）Again in Aspirate supernatant to another 2mL Eppendorf pipe, isometric 1 × CTAB precipitated liquids mixing is added in
Uniformly, 20-30 min are placed at room temperature, and 15 min are centrifuged under 12 000 r min-1；
（8）Supernatant is discarded, precipitation is dried at room temperature, is dissolved in 200 μ L TE-buffer；
（9）Then 400 μ L, 95% ethyl alcohol is added in, 60 min are stood at -20 DEG C, generates precipitation；
（10）At 4 DEG C, in 12000 r min-115 min of lower centrifugation；
（11）Liquid is discarded supernatant, 500 μ L, 75% ethyl alcohol is added in and is washed, 12000 r min-1 centrifuge 10 min；
（12）Liquid is discarded supernatant, is spontaneously dried at room temperature, 30-50 μ 1 × TE-buffer of L are dissolved in, at -20 DEG C
Lower preservation.
3. according to the method described in claim 2, it is characterized in that, step（5）In middle chloroform-isoamyl alcohol mixed liquor, chloroform：It is different
The volume ratio of amylalcohol is 24:1.
4. according to the method described in claim 1, it is characterized in that, in step（b）And step（d）In, select cpDNA primer pairsrpl16WithpetL-psbEFor fortune paulownia and the identification for belonging to Interspecific relationship.
5. according to the method described in claim 1, it is characterized in that, in step（b）And step（d）In PCR amplification condition such as
Under：
10×PCR buffer 5 μL
dNTPs 4 μL
R primers（10 μM） 0.4 μL
F primers（10 μM） 0.4 μL
DNA profiling（10 ng/μL） 5.0 μL
Taq DNA Polymerase（5 U/μL） 0.4 μL
ddH2O 34.8 μL。
6. according to the method described in claim 1, it is characterized in that, in step（b）And step（d）In, the program of PCR amplification is such as
Under：
rpl16Amplification program：94 DEG C of 5 min of pre-degeneration, 94 DEG C are denaturalized 45 seconds, and 55 DEG C are annealed 30 seconds, 35 cycles, 72 DEG C of extensions
2min, 72 DEG C extend 10 min, are preserved at 4 DEG C；
petL-psbEAmplification program：94 DEG C of 5 min of pre-degeneration, 94 DEG C of 1 min of denaturation, 52 DEG C of 1 min of annealing, 35 recycle, and 72
DEG C extension 5 min, 72 DEG C of 8 min of extension are preserved at 4 DEG C.
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