CN109554501A - Amplimer, screening technique and the identification method of olive cultivar identification based on SNP site - Google Patents
Amplimer, screening technique and the identification method of olive cultivar identification based on SNP site Download PDFInfo
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Abstract
The present invention discloses amplimer, screening technique and the identification method of the olive cultivar identification based on SNP site, 8 pairs of olive amplimers.The present invention selects height reliability and the single copy segment of repeatability, menu copies segment and designs amplimer by being analyzed through digestion with restriction enzyme, cloning and sequencing, genome alignment to olive kind leaf DNA；Amplimer is designed with menu copy segment again, PCR amplification is carried out to olive kind leaf DNA, filters out amplification efficiency height, variation 8 pairs of single-copy nuclear gene markers abundant, 8 pairs of olive amplified productions are relatively pure, peak shape is clear, does not need fluorescent primer, detects cheap；Olive cultivar identification is carried out using 8 pairs of olive single-copy nuclear gene markers.
Description
Technical field
The present invention relates to field of biotechnology.The amplification of the specifically olive cultivar identification based on SNP site is drawn
Object, screening technique and identification method.
Background technique
Olive (Olea europaea L.) is the second-biggest-in-the-world traditional oil tree for being only second to oil palm, is Oleaceae
Olea aiphyllium mainly processes olive oil to fresh fruit, and olive oil is rich in monounsaturated fatty acids and antioxidant,
With important nutritive value, it is known as the good reputation of " liquid golden ".
Olive is distributed widely in Mediterranean Region, and olive cultivated area in the whole world is more than 8,800,000 hm2 at present.I
A large amount of external olive product have been collected and introduced in state since the 1960s introduces a fine variety olive from Albania for the first time
Kind resource, nearly 200 of existing registration kind.Frequent introduce a fine variety activity of the olive in country variant and ground section causes its kind mixed
Disorderly, synonym and homonym phenomenon are very serious, and cultivar identification work urgently carries out.Early period it is existing largely using RAPD,
AFLP, ISSR, SSR etc. mark the research identified olive kind, it is generally recognized that SSR marker is in olive cultivar identification
In more effectively.
But in practical applications SSR marker be not it is especially desirable, be mainly manifested in following aspect: (1) duplicate block is made
Increase at amplification error rate, expansion increases or lacked one to several repetitive units, causes product a variety of, and peak shape is chaotic, influences
Interpretation；(2) for, with the biggish Rare allele of major allele length difference, different researchers is subjective in group
Judgement is different, be easy to cause identification deviation；(3) SSR detection is expensive, and product detection mainly uses generation sequenator to examine
It surveys, needs one end mark fluorescent dyestuff (such as HEX) wherein, and such primer not storage endurance.
Summary of the invention
For this purpose, technical problem to be solved by the present invention lies in provide a kind of olive cultivar identification based on SNP site
Amplimer, screening technique and identification method.
In order to solve the above technical problems, the invention provides the following technical scheme: the olive cultivar identification based on SNP site
Amplimer, which is characterized in that including 8 pairs of olive amplimers；The forward primer and reverse primer of SWH1 is respectively such as SEQ
Shown in ID NO:1 and SEQ ID NO:2, the forward primer and reverse primer of SWH2 is respectively such as SEQ ID NO:3 and SEQ ID
Shown in NO:4, the forward primer and reverse primer of SWH3 is respectively as shown in SEQ ID NO:5 and SEQ ID NO:6, and SWH4 is just
To primer and reverse primer respectively as shown in SEQ ID NO:7 and SEQ ID NO:8, the forward primer and reverse primer point of SWH5
Not as shown in SEQ ID NO:9 and SEQ ID NO:10, the forward primer and reverse primer of SWH6 is respectively such as SEQ ID NO:11
With shown in SEQ ID NO:12, the forward primer and reverse primer of SWH7 is respectively such as SEQ ID NO:13 and SEQ ID NO:14 institute
Show, the forward primer and reverse primer of SWH8 is respectively as shown in SEQ ID NO:15 and SEQ ID NO:16.
The screening technique of the amplimer of olive cultivar identification based on SNP site extracts olive kind blade
DNA is analyzed through digestion with restriction enzyme, cloning and sequencing and genome alignment, selects single copy segment design primer, right
Olive kind leaf DNA carries out PCR amplification, filters out the list that amplification efficiency is high, progress olive cultivar identification is enriched in variation
It copies karyogene and marks amplimer.
The screening technique of the amplimer of the above-mentioned olive cultivar identification based on SNP site, includes the following steps:
(1) 20 olive kinds are chosen, blade is acquired, extract olive growing leaves piece DNA；
(2) DNA enzymatic is cut into 500- using restriction enzyme by the leaf DNA for selecting one of kind at random
The segment of 2000bp or so；
(3) endonuclease bamhi is packed at random using cloning vector, convert into Escherichia coli；
(4) multiple monoclonal bacterial plaque sequencings are chosen in coated plate culture at random；
(5) sequence measured is subjected to BLAST in announced olive genome, if certain segment is in olive base
Because, there are more than one copy, abandoning the segment in group, otherwise, which is single-copy nuclear gene segment；
(6) by the single-copy nuclear gene segment that screening obtains in step (5), amplimer is designed, expands target fragment control
System is between 500-1500bp；
(7) it is carried out using leaf DNA of the design amplimer in step (6) to 20 olive kinds in step (1)
PCR amplification, amplified production carry out agarose gel electrophoresis detection, and product can be expanded by choosing clear single, all kinds of band
Sequence, it is sequenced using ABI 3730XL sequenator；
(8) amplified production variation is abundant in selecting step (7), can distinguish the primer of at least two kinds as olive
Cultivar identification label, filters out totally 8 pairs of olive amplimers, and the forward primer and reverse primer for being respectively as follows: SWH1 are respectively such as
Shown in SEQ ID NO:1 and SEQ ID NO:2, the forward primer and reverse primer of SWH2 is respectively such as SEQ ID NO:3 and SEQ
Shown in ID NO:4, the forward primer and reverse primer of SWH3 respectively as shown in SEQ ID NO:5 and SEQ ID NO:6, SWH4's
Forward primer and reverse primer are respectively as shown in SEQ ID NO:7 and SEQ ID NO:8, the forward primer and reverse primer of SWH5
Respectively as shown in SEQ ID NO:9 and SEQ ID NO:10, the forward primer and reverse primer of SWH6 is respectively such as SEQ ID NO:
Shown in 11 and SEQ ID NO:12, the forward primer and reverse primer of SWH7 is respectively such as SEQ ID NO:13 and SEQ ID NO:14
Shown, the forward primer and reverse primer of SWH8 is respectively as shown in SEQ ID NO:15 and SEQ ID NO:16.
Olive cultivar identification method based on SNP site, includes the following steps:
(2-1) acquisition to detection olive kind blade material, extract olive growing leaves piece DNA；
(2-2) uses 8 pairs of olive amplimers described in claim 1, respectively the leaf DNA of olive to be detected
It is expanded, is sequenced；
(2-3) is removed the low region in sequence first and last end and sequencing quality using ContigExpress software, and is modified and be in
Existing bimodal degeneracy base；
Sequence is exported at fasta format, and by 8 sequences and is merged by (2-4), imports BioEdit software, and existing
Based in claim 18 pairs of olive amplimers construct olive kind information bank in other kind sequences to row；
(2-5) use ClustalX software building Neighbor-Joining tree, when olive to be detected with and it is existing
Some olive kind X is gathered in the olive kind information bank constructed based on 8 pairs of olive amplimers in claim 1
When together, illustrate that the genetic affinity of olive to be detected and this olive kind X is close；
(2-6) olive product close to olive to be detected and step (2-5) genetic affinity in BioEdit software again
The variant sites difference of kind X is checked, to be detected if both olive to be detected and olive kind X do not have difference
Olive is just olive kind X, if there are difference, oil olives to be detected for both olive to be detected and olive kind X
Olive kind in the existing olive kind information bank constructed based on 8 pairs of olive amplimers in claim 1, then should not
Kind to be detected is olive new varieties.
The above-mentioned olive cultivar identification method based on SNP site, in step (2-2), PCR amplification uses 30uL anti-
Answer system: the primer 2 .4uL of the dNTPs 2.4uL, 10uM of 20ng DNA, 10 × buffer buffer 3uL, 2.4mM, forward direction are drawn
Object and reverse primer each 1.2uL, Taq DNA polymerase 0.15uL；PCR amplification uses ABI 96U Thermo cycler PCR
Instrument；The response procedures of PCR amplification are as follows:
Initial denaturation: 94 DEG C, 4min；
Denaturation: 94 DEG C, 30s, annealing: 60 DEG C, 30s, and extend: 72 DEG C, 2min amount to 10 circulations；
Denaturation: 94 DEG C, 30s, annealing: 55 DEG C, 30s, and extend: 72 DEG C, 2min amount to 26 circulations；
Extend: 72 DEG C, 10min.
Technical solution of the present invention achieves following beneficial technical effect:
The present invention is by olive kind leaf DNA, through digestion with restriction enzyme, cloning and sequencing, genome alignment
Analysis, selects height reliability and the single copy segment of repeatability, and menu copies segment and designs amplimer；It is copied again with menu
Segment designs amplimer, carries out PCR amplification to olive kind leaf DNA, filters out amplification efficiency height, variation abundant 8
To single-copy nuclear gene marker, 8 pairs of olive amplified productions are relatively pure, and peak shape is clear, do not need fluorescent primer, detect valence
Lattice are cheap；Olive cultivar identification is carried out to olive single-copy nuclear gene marker using 8 pairs.
The present invention by single-copy nuclear gene segment be applied to cultivar identification, have various advantages: (1) height reliability and
Repeatability.Single-copy nuclear gene segment, though being also based on round pcr, repetitive structure can give when design primer
It excludes, amplified production is relatively pure；(2) peak shape is clear, and there is no the differences of different researcher's subjectivity interpretations；(3) it does not need glimmering
Light primer send sequencing company after amplification, detect cheap.The present invention can be used as standardized for olive cultivar identification
Single-copy nuclear gene segment detection method.
Specific embodiment
One, the screening technique of the amplimer of the olive cultivar identification based on SNP site
(1) 20 olive kinds are chosen, blade is acquired, extract olive growing leaves piece DNA；
| Number | Variety name |
| 1 | ' Crow Lycra ' |
| 2 | ' soft A Si ' |
| 3 | ‘Argudell’ |
| 4 | ' Lu Kesi ' |
| 5 | ‘Callosina’ |
| 6 | ‘Mirtolia’ |
| 7 | ' Otto card ' |
| 8 | ‘Royal de Calatayud’ |
| 9 | ' Lay star ' |
| 10 | ‘Kerkiras’ |
| 11 | ' Ge Daer ' |
| 12 | ' Fo Ao ' |
| 13 | ‘Dolce Agogia’ |
| 14 | ' griggles Seville ' |
| 15 | ‘Chemlal de Kabilye’ |
| 16 | ' A Daya ' |
| 17 | ' beans fruit ' |
| 18 | ' Ke Nika ' |
| 19 | ' skin Guar ' |
| 20 | ' OK a karaoke club illiteracy ' |
(2) DNA enzymatic is cut into 500- using restriction enzyme by the leaf DNA for selecting one of kind at random
The segment of 2000bp or so；
(3) endonuclease bamhi is packed at random using cloning vector, convert into Escherichia coli；
(4) multiple monoclonal bacterial plaque sequencings are chosen in coated plate culture at random；
(5) sequence measured is subjected to BLAST in announced olive genome, if certain segment is in olive base
Because, there are more than one copy, abandoning the segment in group, otherwise, which is single-copy nuclear gene segment；
(6) by the single-copy nuclear gene segment that screening obtains in step (5), amplimer is designed, expands target fragment control
System is between 500-1500bp；
(7) it is carried out using leaf DNA of the design amplimer in step (6) to 20 olive kinds in step (1)
PCR amplification, amplified production carry out agarose gel electrophoresis detection, and product can be expanded by choosing clear single, all kinds of band
Sequence, it is sequenced using ABI 3730XL sequenator；
(8) amplified production variation is abundant in selecting step (7), can distinguish the primer of at least two kinds as olive
Cultivar identification label, filter out totally 8 pairs of olive amplimers and be respectively specifically shown in Table 1: the forward primer of SWH1 and
Reverse primer is respectively as shown in SEQ ID NO:1 and SEQ ID NO:2, and the forward primer and reverse primer of SWH2 is respectively such as SEQ
Shown in ID NO:3 and SEQ ID NO:4, the forward primer and reverse primer of SWH3 is respectively such as SEQ ID NO:5 and SEQ ID
Shown in NO:6, the forward primer and reverse primer of SWH4 is respectively as shown in SEQ ID NO:7 and SEQ ID NO:8, and SWH5 is just
To primer and reverse primer respectively as shown in SEQ ID NO:9 and SEQ ID NO:10, the forward primer and reverse primer of SWH6
Respectively as shown in SEQ ID NO:11 and SEQ ID NO:12, the forward primer and reverse primer of SWH7 is respectively such as SEQ ID NO:
Shown in 13 and SEQ ID NO:14, the forward primer and reverse primer of SWH8 is respectively such as SEQ ID NO:15 and SEQ ID NO:16
It is shown.
Table 1 is used for the primer information of olive cultivar identification
Two, the olive cultivar identification method based on SNP site,
Embodiment 1:
2016, Forestry Institue, Chinese Inst.of Forestry Sciences collected portion from Gansu Wudu olive planting base
Olive variety source A, phenotypic character and existing olive kind ' beans fruit ' are more similar, but still have in the characters such as fruit color
Difference.In order to identify whether variety source A is ' beans fruit ', following processing analysis has been carried out:
1. acquiring the blade material of olive kind A and known kind ' beans fruit ' respectively, DNA is extracted；
2. being expanded respectively to two parts of DNA using 8 pairs of olive amplimers in table 1 of the present invention, it is sequenced；
PCR amplification uses 30uL reaction system: 20ng DNA, 10 × buffer (Promega, USA) buffer 3uL,
The primer 2 .4uL of the dNTPs 2.4uL, 10uM of 2.4mM, forward primer and reverse primer each 1.2uL, Taq DNA polymerase (5U/
UL, Takara, Shiga, Japan) 0.15uL.PCR amplification uses ABI 96U Thermo cycler PCR instrument.
The response procedures of PCR amplification are as follows:
3. removing the low region of sequencing qualities such as sequence first and last end using ContigExpress software, and modify presentation
Bimodal degeneracy base；
8 sequences of two kinds are merged 4. exporting sequence at fasta format, and respectively, are beaten using bioedit
It opens.
5. by comparing variant sites difference of two kind in each segment, as a result without discovery difference, therefore
Identify that variety source A is ' beans fruit '.
Embodiment 2
2016, Forestry Institue, Chinese Inst.of Forestry Sciences collected portion from Mianyang, Sichuan olive planting base
Olive variety source B due to having no the important specieses taxonomic character such as fruit, therefore does not know what kind it is.In order to identify this
Variety source B has carried out following processing analysis:
1. acquiring the blade material of olive kind B, DNA is extracted；
2. being carried out respectively to the leaf DNA of this part of variety source using 8 pairs of olive amplimers in table 1 of the present invention
Amplification, sequencing；
PCR amplification uses 30uL reaction system: 20ng DNA, 10 × buffer (Promega, USA) buffer 3uL,
The primer 2 .4uL of the dNTPs 2.4uL, 10uM of 2.4mM, forward primer and reverse primer each 1.2uL, Taq DNA polymerase (5U/
UL, Takara, Shiga, Japan) 0.15uL.PCR amplification uses ABI 96U Thermo cycler PCR instrument.
The response procedures of PCR amplification are as follows:
3. removing the low region of sequencing qualities such as sequence first and last end using ContigExpress software, and modify presentation
Bimodal degeneracy base；
It merges 4. exporting sequence at fasta format, and by 8 sequences, imports BioEdit software and existing
Other kind sequences are to row in the olive kind information bank constructed based on 8 pairs of olive amplimers in table 1 of the present invention；
5. using ClustalX software building Neighbor-Joining tree (NJ tree).The result shows that kind B and oily olive
' Fo Ao ' in olive kind information bank gets together, and long branch is identical, illustrates that B and ' Fo Ao ' genetic affinity are close.
6. checking that discovery does not have to the variant sites difference of B He ' Fo Ao ' the two kinds in BioEdit software again
There is difference, therefore identifies that variety source B is ' Fo Ao '.
Embodiment 3
2017, Inst. of Subtropical Forestry, Chinese Academy of Forestry Sciences acquired from Lishui of Zhejiang olive planting base
To a olive variety source C.The variety source is introduced from Sichuan, phenotypic character and existing olive kind ' Lay star ' compared with
To be similar, but still have any different in the characters such as blade quality.In order to identify whether variety source C is ' Lay star ', carried out with
Lower processing analysis:
1. acquiring the blade material of olive kind C, DNA is extracted；
2. being carried out respectively to the leaf DNA of this part of variety source using 8 pairs of olive amplimers in table 1 of the present invention
Amplification, sequencing；
PCR amplification uses 30uL reaction system: 20ng DNA, 10 × buffer (Promega, USA) buffer 3uL,
The primer 2 .4uL of the dNTPs 2.4uL, 10uM of 2.4mM, forward primer and reverse primer each 1.2uL, Taq DNA polymerase (5U/
UL, Takara, Shiga, Japan) 0.15uL.PCR amplification uses ABI 96U Thermo cycler PCR instrument.
The response procedures of PCR amplification are as follows:
3. removing the low region of sequencing qualities such as sequence first and last end using ContigExpress software, and modify presentation
Bimodal degeneracy base；
It merges 4. exporting sequence at fasta format, and by 8 sequences, imports BioEdit software and existing
Other kind sequences are to row in the olive kind information bank constructed based on 8 pairs of olive amplimers in table 1 of the present invention；
5. using ClustalX software building Neighbor-Joining tree (NJ tree).The result shows that kind C and oily olive
' Lay star ' gathers in same branch in olive kind information bank, but long branch is different, illustrates that C is not ' Lay star '.
6. again in BioEdit software to C and ' the variant sites difference of Lay star ' the two kinds checks, discovery two
There are 2 SNP site differences by person, therefore variety source C is a new varieties.
Obviously, the above embodiments are merely examples for clarifying the description, and does not limit the embodiments.It is right
For those of ordinary skill in the art, can also make on the basis of the above description it is other it is various forms of variation or
It changes.There is no necessity and possibility to exhaust all the enbodiments.And it is extended from this it is obvious variation or
It changes among still in present patent application scope of protection of the claims.
Sequence table
<110>Inst. of Subtropical Forestry, Chinese Academy of Forestry Sciences
Forestry Institue, Chinese Inst.of Forestry Sciences
<120>amplimer, screening technique and the identification method of the olive cultivar identification based on SNP site
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Claims (5)
1. the amplimer of the olive cultivar identification based on SNP site, which is characterized in that including 8 pairs of olive amplimers；
The forward primer and reverse primer of SWH1 respectively as shown in SEQ ID NO:1 and SEQ ID NO:2,
The forward primer and reverse primer of SWH2 respectively as shown in SEQ ID NO:3 and SEQ ID NO:4,
The forward primer and reverse primer of SWH3 respectively as shown in SEQ ID NO:5 and SEQ ID NO:6,
The forward primer and reverse primer of SWH4 respectively as shown in SEQ ID NO:7 and SEQ ID NO:8,
The forward primer and reverse primer of SWH5 respectively as shown in SEQ ID NO:9 and SEQ ID NO:10,
The forward primer and reverse primer of SWH6 respectively as shown in SEQ ID NO:11 and SEQ ID NO:12,
The forward primer and reverse primer of SWH7 respectively as shown in SEQ ID NO:13 and SEQ ID NO:14,
The forward primer and reverse primer of SWH8 is respectively as shown in SEQ ID NO:15 and SEQ ID NO:16.
2. the screening technique of the amplimer of the olive cultivar identification based on SNP site, which is characterized in that extract olive product
Kind leaf DNA, is analyzed through digestion with restriction enzyme, cloning and sequencing and genome alignment, is selected single copy segment design and is drawn
Object carries out PCR amplification to olive kind leaf DNA, filters out amplification efficiency height, the abundant progress olive cultivar identification of variation
Single-copy nuclear gene mark amplimer.
3. the screening technique of the amplimer of the olive cultivar identification according to claim 2 based on SNP site, special
Sign is, includes the following steps:
(1) 20 olive kinds are chosen, blade is acquired, extract olive growing leaves piece DNA；
(2) DNA enzymatic is cut into the left side 500-2000bp using restriction enzyme by the leaf DNA for selecting one of kind at random
Right segment；
(3) endonuclease bamhi is packed at random using cloning vector, convert into Escherichia coli；
(4) multiple monoclonal bacterial plaque sequencings are chosen in coated plate culture at random；
(5) sequence measured is subjected to BLAST in announced olive genome, if certain segment is in olive genome
In there are more than one copy, abandon the segment, otherwise, the segment be single-copy nuclear gene segment；
(6) by the single-copy nuclear gene segment that screening obtains in step (5), amplimer is designed, amplification target fragment control exists
Between 500-1500bp；
(7) PCR expansion is carried out using leaf DNA of the design amplimer in step (6) to 20 olive kinds in step (1)
Increase, amplified production carries out agarose gel electrophoresis detection, and the sequence of product can be expanded by choosing clear single, all kinds of band
Column, are sequenced it using ABI 3730XL sequenator；
(8) amplified production variation is abundant in selecting step (7), can distinguish the primer of at least two kinds as olive kind
Identification marking filters out totally 8 pairs of olive amplimers, is respectively as follows:
The forward primer and reverse primer of SWH1 respectively as shown in SEQ ID NO:1 and SEQ ID NO:2,
The forward primer and reverse primer of SWH2 respectively as shown in SEQ ID NO:3 and SEQ ID NO:4,
The forward primer and reverse primer of SWH3 respectively as shown in SEQ ID NO:5 and SEQ ID NO:6,
The forward primer and reverse primer of SWH4 respectively as shown in SEQ ID NO:7 and SEQ ID NO:8,
The forward primer and reverse primer of SWH5 respectively as shown in SEQ ID NO:9 and SEQ ID NO:10,
The forward primer and reverse primer of SWH6 respectively as shown in SEQ ID NO:11 and SEQ ID NO:12,
The forward primer and reverse primer of SWH7 respectively as shown in SEQ ID NO:13 and SEQ ID NO:14,
The forward primer and reverse primer of SWH8 is respectively as shown in SEQ ID NO:15 and SEQ ID NO:16.
4. the olive cultivar identification method based on SNP site, which comprises the steps of:
(2-1) acquisition to detection olive kind blade material, extract olive growing leaves piece DNA；
(2-2) uses 8 pairs of olive amplimers described in claim 1, and the leaf DNA of olive to be detected carries out respectively
Amplification, sequencing；
(2-3) is removed the low region in sequence first and last end and sequencing quality using ContigExpress software, and it is double to modify presentation
The degeneracy base at peak；
Sequence is exported at fasta format, and by 8 sequences and is merged by (2-4), imports BioEdit software and existing base
Other kind sequences in olive kind information bank that 8 pairs of olive amplimers construct in claim 1 are to row；
(2-5) uses ClustalX software building Neighbor-Joining tree, when olive to be detected with and existing be based on
Some olive kind X flocks together in the olive kind information bank that 8 pairs of olive amplimers construct in claim 1
When, illustrate that the genetic affinity of olive to be detected and this olive kind X is close；
(2-6) olive kind X close to olive to be detected and step (2-5) genetic affinity in BioEdit software again
Variant sites difference checked, if both olive to be detected and olive kind X do not have difference, it is to be detected oil olive
Olive is just olive kind X, if there are difference, olive product to be detected for both olive to be detected and olive kind X
Kind is not in the existing olive kind information bank constructed based on 8 pairs of olive amplimers in claim 1, then this is to be checked
Survey kind is olive new varieties.
5. the olive cultivar identification method according to claim 4 based on SNP site, which is characterized in that in step (2-
2) in, PCR amplification uses 30uL reaction system: the dNTPs of 20ng DNA, 10 × buffer buffer 3uL, 2.4mM
The primer 2 .4uL of 2.4uL, 10uM, forward primer and reverse primer each 1.2uL, Taq DNA polymerase 0.15uL；PCR amplification makes
With ABI 96U Thermo cycler PCR instrument；The response procedures of PCR amplification are as follows:
Initial denaturation: 94 DEG C, 4min；
Denaturation: 94 DEG C, 30s, annealing: 60 DEG C, 30s, and extend: 72 DEG C, 2min amount to 10 circulations；
Denaturation: 94 DEG C, 30s, annealing: 55 DEG C, 30s, and extend: 72 DEG C, 2min amount to 26 circulations；
Extend: 72 DEG C, 10min.
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