CN107312848A - The method that hazel Germplasm Identification is carried out using cpDNA sequences - Google Patents
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Abstract
The invention discloses a kind of method that hazel Germplasm Identification is carried out using cpDNA sequences, PCR amplifications are carried out using 3 pairs of chloroplaset cpDNA primer pair standards germplasm, PCR amplifications are carried out using 3 pairs of primer pairs target germplasm to be identified, the extension increasing sequence of the extension increasing sequence and standard germplasm of obtained target germplasm is compared, whether hazel Germplasm Identification is unanimously carried out according to comparison result, the cpDNA sequence mark distinguishing abilities utilized are strong, it is reproducible, it can effectively differentiate and easily obscure hazel germ plasm resource or other platymisciums, inter-species classification to China's Coryluss resource, Genetic relationship and phylogenetic analysis provide important technical support.
Description
Technical field
CpDNA sequences are utilized to carry out hazel germplasm mirror the present invention relates to a kind of method of hazel Germplasm Identification, more particularly to one kind
Fixed method.
Background technology
Hazel be corylaceae (Corylaceae) Corylus (Corylus) plant, both included in these hazel kinds arbor type or
Cover shrub type (Zhang Yu and wait, 2005).World's Coryluss there are about 20 kinds, be widely distributed in the Northern Hemisphere Asia,
Europe, the Temperate Region in China in North America.China's Coryluss aboundresources, China's natural distributed has 8 kinds and 2 mutation, point
Also very extensively, economic use value is higher for cloth scope.
Since 1950s, the classification of China's Coryluss is still that complete foundation morphological feature is divided, different
Systematist due to based on morphological size it is different with the standard of foundation, thus classification results are not quite similar.Particularly with China
There are many differences in Coryluss kind and the division of mutation, systematists.Hu Xianxiao (1948) is what is write《Chinese tree figure
Will》In propose that Genus Corylus in China should belong to corylaceae (Corylaceae) Corylus (Corylus) first, including 6 kinds 2
Mutation.Chen Rong (1975) exists《Chinese tree taxology》In think that Genus Corylus in China should be divided into 3 kinds and 3 mutation.Yu
Moral is dredged (1979) and existed《Fruit tree taxology》It is middle that Genus Corylus in China is divided into 4 kinds, 2 mutation.《Chinese Plants will》
China's Coryluss have been divided 7 kinds, 2 mutation by (1979 editions), and refer to a kind that leaves a question open first.Zheng Wanjun (1985) exists
《Chinese tree will》In introduce Genus Corylus in China comprising 7 kinds and 3 mutation.Liang Weijian (1988,2005) thinks that river hazel should
It is an independent kind, and has assert the river hazel mutation short handle river hazel with typical short handle feature.As can be seen here, Plant Taxonomy
There are many disputes in classification of the family to China's Coryluss, Coryluss resource category is recorded and differed, classification position is indefinite,
This collection to China's Coryluss resource, preserve and using affecting, and then influence Coryluss crossbreeding
The development of work.
Chloroplast gene (cpDNA) is quite guarded, and has independent evolution line, is independent of other any data
Molecular evolutionary trees can be built, (such as inter-species) plant classification discriminating problem can be solved in relatively low taxonomic category.Which overcome biography
The weakness for research method of uniting, improves the level of plant germplasm resource discriminating.
Prior art one：
Morphology mark is that, using the phenotypic character of plant as genetic marker, current Coryluss classification relies primarily on form
Research, the difference dependent on phenotype.In terms of hereditary angle, phenotypic character is divided into quantitative character and the class of qualitative character two.But
It is that phenotypic character is influenceed by many factors such as environment, weather, nutrition conditions, the information often provided is not comprehensive enough, and according to shape
State progress discriminating classification workload is big, cycle length, cost are high.For example：Both peaceful hazels of river hazel are in tree-like, blade, inflorescence and fruit
It is closely similar in real form, it is not easily distinguishable, especially often obscures in both cross-distribution areas, profit is investigated and produced to scientific research
With bringing very big puzzlement.And for example, the Ostryopsis davidiana of Ostryopsis davidiana category and the hair hazel of Corylus are also very close in phenotype, are difficult area
Point.
Prior art two：
With the fast development of molecular labeling, as RFLP marks (Restriction fragment length
Polymorphism), DNArandom amplified polymorphic DNA RAPD (Random amplification polymorphism DNA), simple
Repetitive sequence SSR (Simple sequence repeats), AFLP AFLP (amplified
Fragment length polymorphism), sequence tagged site STS (sequence-tagged site) etc., for wood
In the taxonomic history of this plant.But these molecule labelling methods have certain defect, such as RFLP marking operations are cumbersome, and technology will
Ask high, price is also higher；The result of RAPD labeled analysis is not very accurate；SNP marker informative site is limited, costly
Deng.
Above-mentioned mark is come out based on the technological development without whole genome sequence information mostly, there is certain application value,
But randomness is strong, less stable.
The content of the invention
It is an object of the invention to provide a kind of method that hazel Germplasm Identification is carried out using cpDNA sequences.
The purpose of the present invention is achieved through the following technical solutions：
The method that hazel Germplasm Identification is carried out using cpDNA sequences of the present invention, utilizes following 3 pairs of chloroplasets cpDNA primers
PCR amplifications are carried out to standard germplasm, PCR amplifications are carried out using 3 pairs of primer pairs target germplasm to be identified, by obtained target species
The extension increasing sequence of the extension increasing sequence of matter and standard germplasm is compared, and whether hazel Germplasm Identification is unanimously carried out according to comparison result；
3 pairs of chloroplasets cpDNA primers include：
First pair：Forward primer：5'-GGCGATCAGTTGGACCTTTG-3'；
Reverse primer：5'-TTGGTAGCGCGTTTGTTTTG-3'；
Second pair：Forward primer：5'-GCTACATCCGCCCCTATACT-3'；
Reverse primer：5'-CCCTCTAGACTTAGCTGCTGT-3'；
3rd pair：Forward primer：5'-GCCAGTAATACCTGTGCTATTT-3'；
Reverse primer：5'-TATTCTTCACGTCCAGGATTAC-3'.
As seen from the above technical solution provided by the invention, utilization cpDNA sequences provided in an embodiment of the present invention are entered
The method of row hazel Germplasm Identification, the cpDNA sequence mark distinguishing abilities utilized are strong, reproducible, can effectively differentiate easily mixed
Hazel germ plasm resource of confusing or other platymisciums, inter-species classification, Genetic relationship and genetic evolution to China's Coryluss resource
Analysis provides important technical support, selects excellent and crossbreeding to have while carrying out the investigation of Coryluss wild resource to China
Great meaning.
Brief description of the drawings
Fig. 1 is the cpDNA sequence difference figures of different hazel kinds in the embodiment of the present invention.
Fig. 2 is the dendrogram of different hazel kind germplasm in the embodiment of the present invention.
Embodiment
With reference to the accompanying drawing in the embodiment of the present invention, the technical scheme in the embodiment of the present invention is carried out clear, complete
Ground is described, it is clear that described embodiment is only a part of embodiment of the invention, rather than whole embodiments.Based on this
The embodiment of invention, the every other implementation that those of ordinary skill in the art are obtained under the premise of creative work is not made
Example, belongs to protection scope of the present invention.
The method that hazel Germplasm Identification is carried out using cpDNA sequences of the present invention, its preferably embodiment is：
PCR amplifications are carried out using following 3 pairs of chloroplasets cpDNA primer pair standards germplasm, it is to be identified using 3 pairs of primer pairs
Target germplasm carries out PCR amplifications, the extension increasing sequence of the extension increasing sequence and standard germplasm of obtained target germplasm is compared, root
Whether hazel Germplasm Identification is unanimously carried out according to comparison result；
3 pairs of chloroplasets cpDNA primers include：
First pair：Forward primer：5'-GGCGATCAGTTGGACCTTTG-3'；
Reverse primer：5'-TTGGTAGCGCGTTTGTTTTG-3'；
Second pair：Forward primer：5'-GCTACATCCGCCCCTATACT-3'；
Reverse primer：5'-CCCTCTAGACTTAGCTGCTGT-3'；
3rd pair：Forward primer：5'-GCCAGTAATACCTGTGCTATTT-3'；
Reverse primer：5'-TATTCTTCACGTCCAGGATTAC-3'.
It is described to be entered using 3 pairs of chloroplaset cpDNA primers before performing PCR amplification step, in addition it is also necessary to extraction standard germplasm and target
The complete genome DNA of germplasm.
The complete genome DNA of the extraction standard germplasm and target germplasm comprises the following steps：
Step 1, preheating 2%CTAB extract solutions and 2% β-thin base ethanol in 65 DEG C of water-baths；
Step 2, take 0.2 gram of blade, add liquid nitrogen and be fully ground, be then transferred quickly in 2mL centrifuge tubes, add preheating
CTAB extract solutions 1ml, 65 DEG C of water-bath 60min, every 10min gently overturn mix；
After step 3, water-bath terminate, take out centrifuge tube and be cooled to room temperature；5min is centrifuged under the conditions of 13000rpm, supernatant is taken
800 μ L are transferred to new centrifuge tube；
Step 4, addition 800 μ L chloroforms/isoamyl alcohol (V:V=24: 1), fully mix, 13000rpm centrifugation 5min take
Clear liquid is transferred to the new centrifuge tubes of 2mL；
Step 5, repeat step 4, add isometric chloroform/isoamyl alcohol (V:V=24: 1), fully mix, 13000rpm from
Heart 5min, takes supernatant to be transferred to the new centrifuge tubes of 2mL；
The 3M NaAc of step 6, the isopropanol for adding the volume of supernatant 2/3 and 1/10 volume, gently overturn and mix, stand
15min is to producing flocculent deposit；
Supernatant is abandoned after centrifuging 6min under the conditions of step 7,12000rpm, bottom white DNA pellet is carefully taken out, is transferred to
In centrifuge tube new 1.5mL, washed twice, each 13000rpm centrifuges 6min, abandons ethanol, surpassed with 1000 μ L 70% ethanol
Net workbench air-dries the alcohol of residual；
Step 8, extraction product take 2~3 μ L to be detected with 1.5% agarose gel electrophoresis with 100 μ L TE buffer solutions,
Remaining is positioned over -20 DEG C and saved backup.
The reaction system of the PCR amplifications is as follows：
The program of PCR amplification is：
(1) 94 DEG C of pre-degeneration 4min；
(2) 94 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 2min, 10 circulations；
(3) 94 DEG C of 30s, 50 DEG C of 30s, 72 DEG C of 2min, 26 circulations；
(4) 72 DEG C of extension 10min；
(5) 4 DEG C of preservations.
The method that hazel Germplasm Identification is carried out using cpDNA sequences of the present invention, the cpDNA sequence mark distinguishing abilities utilized
By force, it is reproducible, it can effectively differentiate and easily obscure hazel germ plasm resource or other platymisciums, to the kind of China's Coryluss resource
Between classification, Genetic relationship and phylogenetic analysis provide important technical support, while carrying out Coryluss to China
Wild resource investigation selects excellent and crossbreeding to be significant.The present invention can utilize the amplification of the hazel cpDNA sequences
Primer carries out Amplification Analysis.
The present invention is according to universal primer, and design primer amplification obtains Coryluss cpDNA sequences, and amplification obtains leading to after sequence
Comparison analysis is crossed, it is not of the same race for material with China's Coryluss that primer is designed as cpDNA sequences, then using the sequence for determining to obtain
Enter performing PCR amplification, purifying and sequencing, screening obtains 3 to (IRF ID NO:A/B、IRF ID NO:C/D and IRF ID NO:E/
F it) can be used for the special primer for differentiating different hazel kind plants, the special primer, which can effectively differentiate, obscures unknown material for which kind of
Hazel kind is accredited as non-Coryluss.The characteristics of this method has simple to operate, reproducible, efficient, to China's Coryluss
Inter-species classification, Genetic relationship and the phylogenetic analysis of resource provide important technical support, while carrying out to China
The investigation of Coryluss wild resource selects excellent and crossbreeding to be significant.
In concrete application, the cpDNA sequences obtained using the present invention, which can be designed, is different from IRF ID NO of the present invention:A/
B、IRF ID NO:C/D or IRF ID NO:E/F primer or degenerate primer, or correlation cpDNA primers are utilized, enter
The amplification not of the same race of row Coryluss carries out discriminatory analysis to obtain cpDNA sequences.
Specific embodiment：
First embodiment, the acquisition of Coryluss material：
14 parts of Coryluss materials are gathered from China's different geographical according to factual survey, 14 parts of standard germplasm materials information are such as
Shown in table 1.1 part of Coryluss materials A to be identified is gathered from Shaanxi Taibai County.Qinling Mountains in Shaanxi mountain range is located at the friendship of the peaceful hazel of river hazel
Distributed areas, root are pitched it is documented that flat hazel and river hazel are all distributed in this region, foundation traditional form can not be accurate
Which kind of Corylus differentiates material to be identified is.In addition, also 1 part material picks up from Chifeng, its form and hair hazel and tiger
Hazel is similar, is material B to be identified.
The specifying information of 1 14 Coryluss materials of table
Second embodiment, the extraction of Coryluss material genomic DNA：
Complete genome DNA is extracted from above-mentioned 16 parts of material samples using the CTAB methods after optimization, specific method is as follows：
Step 201, preheating 2%CTAB extract solutions and 2% β-thin base ethanol in 65 DEG C of water-baths；
Step 202, take 0.2 gram of blade, add liquid nitrogen and be fully ground, be then transferred quickly in 2mL centrifuge tubes, add pre-
CTAB extract solutions 1ml, the 65 DEG C of water-bath 60min of heat, gently overturn every 10min and mix；
After step 203, water-bath terminate, take out centrifuge tube and be cooled to room temperature；5min is centrifuged under the conditions of 13000rpm, supernatant is taken
The μ L of liquid 800 are transferred to new centrifuge tube；
Step 204, addition 800 μ L chloroforms/isoamyl alcohol (V:V=24: 1), fully mix, 13000rpm centrifugation 5min take
Supernatant is transferred to the new centrifuge tubes of 2mL；
Step 205, repeat step 204, add isometric chloroform/isoamyl alcohol (V:V=24: 1), fully mix,
13000rpm centrifuges 5min, takes supernatant to be transferred to the new centrifuge tubes of 2mL；
The 3M NaAc of step 206, the isopropanol for adding the volume of supernatant 2/3 and 1/10 volume, gently overturn and mix, quiet
15min is put to producing flocculent deposit；
Supernatant is abandoned after centrifuging 6min under the conditions of step 207,12000rpm, bottom white DNA pellet is carefully taken out, is turned
Enter in the new centrifuge tubes of 1.5mL, washed twice with 1000 μ L 70% ethanol, each 13000rpm centrifuges 6min, abandons ethanol,
Superclean bench air-dries the alcohol of residual；
Step 208, extraction product take 2~3 μ L to be examined with 1.5% agarose gel electrophoresis with 100 μ L TE buffer solutions
Survey, remaining is positioned over -20 DEG C and saved backup.
3rd embodiment, the screening of primer：
1st, target gene fragment is obtained
Using the flat hazel material DNA of representative standard germplasm as template, with reference to petG-trnP (Mu et al, 2006),
TrnH-psbA (Hamilton et al, 1999), psbK-psbI (Zhang et al, 2014) etc. cpDNA sequences Designs are drawn
Thing, enters performing PCR amplification, and product purification respectively obtains 467bp, 524bp, 422bp genetic fragment after sequencing.
2nd, primer is screened
Primer is separately designed according to above-mentioned 3 genetic fragments, using 14 parts of Coryluss standard germplasm DNA as template, according to
Following systems (as shown in table 2) and program (as shown in table 3) enter performing PCR amplification：
Table 2, PCR reaction systems
Table 3, PCR amplification programs
The PCR primer that the above method is obtained detects that Ago-Gel contains Gelred by 1.5% agarose gel electrophoresis
Dyestuff.Utilize gel imaging system observed and recorded electrophoretic band.Utilize glue reclaim kit (Tiangeng biochemical technology Co., Ltd)
Recovery purifying PCR primer, then carries out two-way sequencing on ABI 3730xl sequenators.
Expand and be sequenced by above-mentioned PCR, finally filter out 3 pairs of specificity that can be used for differentiating the different hazel kind resources of China
Primer.Primer information is as follows：
IRF ID NO:A：Forward primer：5'-GGCGATCAGTTGGACCTTTG-3'；
IRF ID NO:B：Reverse primer：5'-TTGGTAGCGCGTTTGTTTTG-3'；
IRF ID NO:C：Forward primer：5'-GCTACATCCGCCCCTATACT-3'；
IRF ID NO:D：Reverse primer：5'-CCCTCTAGACTTAGCTGCTGT-3'；
IRF ID NO:E：Forward primer：5'-GCCAGTAATACCTGTGCTATTT-3'；
IRF ID NO:F：Reverse primer：5'-TATTCTTCACGTCCAGGATTAC-3'.
Fourth embodiment, the structure of phylogenetic tree：
Treated using method described in 3rd embodiment and obtained 3 pairs of primer pairs, the 14 parts of Coryluss materials of screening and 2 parts
Detection vegetable material (A and B) enters performing PCR amplification, product purification and sequencing.
Contig- is used to the sequencing sequence of 14 parts of Coryluss materials and 2 parts of vegetable materials (A and B) to be detected
After Express (version 9.1) check and correction, Multiple Sequence Alignment is carried out using Clustal X (version 2.11).Sequence alignment
Afterwards, sequence association is got up, built using the maximum likelihood method (Maximum Likelihood) of MEGA (version 5.0) software
Vertical most likelihood tree.
Fig. 1 results show：The sequence difference of different hazel kinds substantially, material not of the same race can be clearly differentiated by sequence difference
Material.And fibert material indifference in sequence mutually of the same race of different geographical, the hair hazel material sequence indifference of such as 3 different geographicals
It is different, the flat hazel material sequence indifference of 2 different geographicals.Materials A to be detected indifference consistent with river hazel sequence.This method is high
Effect, accurate, stability are high.
Fig. 2 results show：Together with materials A to be detected is gathered with the river hazel and short handle river hazel of Coryluss, material B to be detected
It is distant with all Coryluss, including the similar hair hazel of form.
Identified through the above method, materials A to be detected is consistent with river hazel (numbering 9) sequence, and with river hazel cluster together with, mirror
Determine the river hazel that it is Coryluss；Material B to be detected and hair hazel and other Coryluss germplasm are apart from each other, and it is brave hazel to differentiate it
Son category material.
The foregoing is only a preferred embodiment of the present invention, but protection scope of the present invention be not limited thereto,
Any one skilled in the art is in the technical scope of present disclosure, the change or replacement that can be readily occurred in,
It should all be included within the scope of the present invention.Therefore, protection scope of the present invention should be with the protection model of claims
Enclose and be defined.
Claims (5)
1. a kind of method that hazel Germplasm Identification is carried out using cpDNA sequences, it is characterised in that utilize following 3 couples of chloroplaset cpDNA
Primer pair standard germplasm carries out PCR amplifications, PCR amplifications is carried out using 3 pairs of primer pairs target germplasm to be identified, by obtained mesh
The extension increasing sequence for marking the extension increasing sequence and standard germplasm of germplasm is compared, and whether hazel germplasm mirror is unanimously carried out according to comparison result
It is fixed；
3 pairs of chloroplasets cpDNA primers include：
First pair：Forward primer：5'-GGCGATCAGTTGGACCTTTG-3'；
Reverse primer：5'-TTGGTAGCGCGTTTGTTTTG-3'；
Second pair：Forward primer：5'-GCTACATCCGCCCCTATACT-3'；
Reverse primer：5'-CCCTCTAGACTTAGCTGCTGT-3'；
3rd pair：Forward primer：5'-GCCAGTAATACCTGTGCTATTT-3'；
Reverse primer：5'-TATTCTTCACGTCCAGGATTAC-3'.
2. the method according to claim 1 that hazel Germplasm Identification is carried out using cpDNA sequences, it is characterised in that the profit
Entered with 3 pairs of chloroplaset cpDNA primers before performing PCR amplification step, in addition it is also necessary to the full-length genome of extraction standard germplasm and target germplasm
DNA。
3. the method according to claim 2 that hazel Germplasm Identification is carried out using cpDNA sequences, it is characterised in that described to carry
Standard germplasm and the complete genome DNA of target germplasm is taken to comprise the following steps：
Step 1, preheating 2%CTAB extract solutions and 2% β-thin base ethanol in 65 DEG C of water-baths；
Step 2, take 0.2 gram of blade, add liquid nitrogen and be fully ground, be then transferred quickly in 2mL centrifuge tubes, add preheating
CTAB extract solutions 1ml, 65 DEG C of water-bath 60min, gently overturn every 10min and mix；
After step 3, water-bath terminate, take out centrifuge tube and be cooled to room temperature；5min is centrifuged under the conditions of 13000rpm, the μ of supernatant 800 is taken
L is transferred to new centrifuge tube；
Step 4, addition 800 μ L chloroforms/isoamyl alcohol (V:V=24: 1), fully mix, 13000rpm centrifugation 5min take supernatant
It is transferred to the new centrifuge tubes of 2mL；
Step 5, repeat step 4, add isometric chloroform/isoamyl alcohol (V:V=24: 1), fully mix, 13000rpm centrifugations
5min, takes supernatant to be transferred to the new centrifuge tubes of 2mL；
The 3M NaAc of step 6, the isopropanol for adding the volume of supernatant 2/3 and 1/10 volume, gently overturn and mix, stand 15min
To generation flocculent deposit；
Supernatant is abandoned after centrifuging 6min under the conditions of step 7,12000rpm, bottom white DNA pellet is carefully taken out, is transferred to
In centrifuge tube new 1.5mL, washed twice, each 13000rpm centrifuges 6min, abandons ethanol, surpassed with 1000 μ L 70% ethanol
Net workbench air-dries the alcohol of residual；
Step 8, extraction product take 2~3 μ L to be detected with 1.5% agarose gel electrophoresis with 100 μ L TE buffer solutions, remaining
- 20 DEG C are positioned over to save backup.
4. the method according to claim 1 that hazel Germplasm Identification is carried out using cpDNA sequences, it is characterised in that the PCR
The reaction system of amplification is as follows：
5. the method according to claim 4 that hazel Germplasm Identification is carried out using cpDNA sequences, it is characterised in that the PCR
The program of amplification is：
(1) 94 DEG C of pre-degeneration 4min；
(2) 94 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 2min, 10 circulations；
(3) 94 DEG C of 30s, 50 DEG C of 30s, 72 DEG C of 2min, 26 circulations；
(4) 72 DEG C of extension 10min；
(5) 4 DEG C of preservations.
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2017
- 2017-07-13 CN CN201710571443.XA patent/CN107312848B/en not_active Expired - Fee Related
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|---|---|---|---|---|
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