CN109554501B - Amplification primer, screening method and identification method for identifying olive variety based on SNP locus - Google Patents

Abstract

The invention discloses an amplification primer, a screening method and an identification method for identifying olive varieties based on SNP sites, and 8 pairs of olive amplification primers. The method comprises the steps of carrying out restriction enzyme digestion, clone sequencing and genome comparison analysis on leaf DNA of the olive variety, selecting single copy fragments with high reliability and repeatability, and designing amplification primers by the single copy fragments; then designing an amplification primer by a single copy fragment, carrying out PCR amplification on the leaf DNA of the olive variety, screening out 8 pairs of single copy nuclear gene markers with high amplification efficiency and rich variation, wherein 8 pairs of olive amplification products are relatively pure, have clear peak shapes, do not need a fluorescent primer and have low detection cost; the olive variety identification can be carried out by using 8-pair olive single-copy nuclear gene markers.

Description

Amplification primer, screening method and identification method for identifying olive variety based on SNP locus

Technical Field

The invention relates to the field of biotechnology. In particular to an amplification primer, a screening method and an identification method for identifying the olive variety based on SNP loci.

Background

Olive (Olea europaea L.) is the second largest woody oil plant in the world after oil palm, is an evergreen tree of the genus Olea of the family oleaceae, is mainly used for processing olive oil from fresh fruits, is rich in monounsaturated fatty acids and antioxidant substances, has important nutritional value, and is in the reputation of "liquid gold".

Olives are widely distributed in mediterranean areas, and the olive growing area around the world is currently over 880 ten thousand hm 2. Since the introduction of olea europaea in Albania for the first time in the 60 th of 20 th century, China collects and introduces a large amount of foreign olive variety resources, and the number of the existing registered varieties is nearly 200. Frequent introduction activities of the olea europaea in different countries and regions cause chaotic varieties, the phenomena of homologies, synonyms and homonymy foreign matters are very serious, and the variety identification work is urgently needed. In the early stage, a large number of researches for identifying the olive variety by using markers such as RAPD, AFLP, ISSR, SSR and the like exist, and the SSR marker is generally considered to be more effective in identifying the olive variety.

However, in practical applications, SSR markers are not particularly desirable, and are mainly characterized by the following: (1) the amplification error rate is increased due to the repeated region, one or more repeated units are added or reduced, so that various products are generated, the peak shape is disordered, and the interpretation is influenced; (2) for rare alleles with large length difference with main alleles in a population, different researchers have different subjective judgments and are easy to cause identification deviation; (3) SSR detection is expensive, the detection of products is mainly detected by using a first-generation sequencer, a fluorescent dye (such as HEX) needs to be marked at one end of the SSR detection, and the primer is not storable.

Disclosure of Invention

Therefore, the technical problem to be solved by the invention is to provide an amplification primer, a screening method and an identification method for identifying the olive variety based on the SNP locus.

In order to solve the technical problems, the invention provides the following technical scheme: the amplification primer for identifying the olive variety based on the SNP locus is characterized by comprising 8 pairs of olive amplification primers; the forward primer and the reverse primer of SWH1 are shown as SEQ ID NO: 1 and SEQ ID NO: 2, the forward primer and the reverse primer of SWH2 are shown as SEQ ID NO: 3 and SEQ ID NO: 4, the forward primer and the reverse primer of SWH3 are shown as SEQ ID NO: 5 and SEQ ID NO: 6, the forward primer and the reverse primer of SWH4 are shown as SEQ ID NO: 7 and SEQ ID NO: 8, the forward primer and the reverse primer of SWH5 are shown as SEQ ID NO: 9 and SEQ ID NO: 10, the forward primer and the reverse primer of SWH6 are shown as SEQ ID NO: 11 and SEQ ID NO: 12, the forward primer and the reverse primer of SWH7 are shown as SEQ ID NO: 13 and SEQ ID NO: 14, the forward primer and the reverse primer of SWH8 are shown as SEQ ID NO: 15 and SEQ ID NO: shown at 16.

The method for screening the amplification primer for identifying the olive variety based on the SNP locus comprises the steps of extracting the leaf DNA of the olive variety, carrying out restriction enzyme digestion, clone sequencing and genome comparison analysis, selecting a single-copy fragment design primer, carrying out PCR amplification on the leaf DNA of the olive variety, and screening out the single-copy nuclear gene marker amplification primer which has high amplification efficiency and abundant variation and is used for identifying the olive variety.

The screening method of the amplification primer for identifying the olive variety based on the SNP locus comprises the following steps:

(1) selecting 20 olive varieties, collecting leaves, and extracting DNA of the olive leaves;

(2) randomly selecting one variety of leaf DNA, and using restriction endonuclease to cut the DNA into fragments of about 500-2000 bp;

(3) randomly packaging enzyme digestion fragments by using a cloning vector, and transforming the enzyme digestion fragments into escherichia coli;

(4) plating and culturing, and randomly selecting a plurality of monoclonal bacterial plaques for sequencing;

(5) BLAST the determined sequence in a published olive genome, discarding a fragment if it has more than one copy in the olive genome, otherwise it is a single copy nuclear gene fragment;

(6) designing an amplification primer for the single-copy nuclear gene segment obtained by screening in the step (5), and controlling the amplification target segment to be between 500 and 1500 bp;

(7) carrying out PCR amplification on the leaf DNA of the 20 olive varieties in the step (1) by using the amplification primers designed in the step (6), carrying out agarose gel electrophoresis detection on amplification products, selecting sequences with clear and single bands and capable of amplifying the products of all varieties, and sequencing the sequences by using an ABI 3730XL sequencer;

(8) selecting the primers which are abundant in amplification product variation and capable of distinguishing at least two varieties in the step (7) as olive variety identification markers, and screening out 8 pairs of olive amplification primers, wherein the primers are respectively as follows: the forward primer and the reverse primer of SWH1 are shown as SEQ ID NO: 1 and SEQ ID NO: 2, the forward primer and the reverse primer of SWH2 are shown as SEQ ID NO: 3 and SEQ ID NO: 4, the forward primer and the reverse primer of SWH3 are shown as SEQ ID NO: 5 and SEQ ID NO: 6, the forward primer and the reverse primer of SWH4 are shown as SEQ ID NO: 7 and SEQ ID NO: 8, the forward primer and the reverse primer of SWH5 are shown as SEQ ID NO: 9 and SEQ ID NO: 10, the forward primer and the reverse primer of SWH6 are shown as SEQ ID NO: 11 and SEQ ID NO: 12, the forward primer and the reverse primer of SWH7 are shown as SEQ ID NO: 13 and SEQ ID NO: 14, the forward primer and the reverse primer of SWH8 are shown as SEQ ID NO: 15 and SEQ ID NO: shown at 16.

The olive variety identification method based on the SNP locus comprises the following steps:

(2-1) collecting leaf materials of olive varieties to be detected, and extracting DNA of olive leaves;

(2-2) amplifying and sequencing the DNA of the leaves of the olea europaea to be detected respectively by using the 8-pair olea europaea amplification primers of claim 1;

(2-3) removing the sequence head and tail ends and the regions with low sequencing quality by using Contigexpress software, and modifying degenerate bases showing double peaks;

(2-4) outputting the sequences into a fasta format, combining the 8 sequences, introducing the sequences into BioEdit software, and aligning the sequences with other variety sequences in an existing olive variety information base constructed on the basis of the 8 pairs of olive amplification primers in the claim 1;

(2-5) constructing a Neighbor-Joining tree by using ClustalX software, and when the to-be-detected olive is gathered with a certain olive variety X in the existing olive variety information base constructed on the basis of the 8 pairs of olive amplification primers in the claim 1, indicating that the genetic relationship between the to-be-detected olive and the olive variety X is very close;

(2-6) checking variation site difference between the to-be-detected olive and the olive variety X with a close genetic relationship in the step (2-5) in a BioEdit software, wherein if the to-be-detected olive and the olive variety X have no difference, the to-be-detected olive is the olive variety X, and if the to-be-detected olive and the olive variety X have a difference, the to-be-detected olive variety is not in an existing olive variety information base constructed on the basis of the 8 pairs of olive amplification primers in the claim 1, and the to-be-detected variety is a new olive variety.

In the above method for identifying the variety of the olive based on the SNP locus, in the step (2-2), PCR amplification all uses a 30uL reaction system: 20ng DNA, 3uL of 10 XBuffer buffer, 2.4uL of 2.4mM dNTPs, 2.4uL of 10uM primer, 1.2uL of each of forward primer and reverse primer, and 0.15uL of Taq DNA polymerase; PCR amplification was performed using an ABI 96U Thermo cycler PCR instrument; the reaction procedure for PCR amplification was:

pre-denaturation: 94 ℃ for 4 min;

denaturation: 94 ℃, 30s, annealing: 60 ℃, 30s, and extension: at 72 ℃ for 2min, for 10 cycles;

denaturation: 94 ℃, 30s, annealing: 55 ℃, 30s, and extension: at 72 ℃ for 2min, for a total of 26 cycles;

extension: 72 deg.C, 10 min.

The technical scheme of the invention achieves the following beneficial technical effects:

the method comprises the steps of carrying out restriction enzyme digestion, clone sequencing and genome comparison analysis on leaf DNA of the olive variety, selecting single copy fragments with high reliability and repeatability, and designing amplification primers by the single copy fragments; then designing an amplification primer by a single copy fragment, carrying out PCR amplification on the leaf DNA of the olive variety, screening out 8 pairs of single copy nuclear gene markers with high amplification efficiency and rich variation, wherein 8 pairs of olive amplification products are relatively pure, have clear peak shapes, do not need a fluorescent primer and have low detection cost; the olive variety identification can be carried out by using 8 pairs of olive single-copy nuclear gene markers.

The invention applies the single copy nuclear gene fragment to variety identification, and has the advantages of multiple aspects: (1) high reliability and repeatability. The single copy nuclear gene fragment is based on the PCR technology, but the repetitive structure can be eliminated when designing the primer, and the amplification product is relatively pure; (2) the peak shape is clear, and the difference of subjective interpretation of different researchers does not exist; (3) fluorescent primers are not needed, the amplified product is sent to a sequencing company, and the detection cost is low. The invention can be used as a standardized single-copy nuclear gene fragment detection method for identifying olive varieties.

Detailed Description

Screening method of amplification primers for identifying olive varieties based on SNP sites

(1) Selecting 20 olive varieties, collecting leaves, and extracting DNA of the olive leaves;

numbering	Name of breed
		1	'Krollai'
2	'Soft Ash'
		3	‘Argudell’
4	'Lux'
		5	‘Callosina’
6	‘Mirtolia’
		7	'ao Toka'
8	‘Royal de Calatayud’
		9	'Laixing'
10	‘Kerkiras’
		11	'Goldal'
12	' Buddha ' of '
		13	‘Dolce Agogia’
14	'apple-sevelavia'
		15	‘Chemlal de Kabilye’
16	' adaya ' food '
		17	'Douguo'
18	'Kenican'
		19	'Guo'
20	'Carla Mongolia'

(2) Randomly selecting one variety of leaf DNA, and using restriction endonuclease to cut the DNA into fragments of about 500-2000 bp;

(3) randomly packaging enzyme digestion fragments by using a cloning vector, and transforming the enzyme digestion fragments into escherichia coli;

(4) plating and culturing, and randomly selecting a plurality of monoclonal bacterial plaques for sequencing;

(5) BLAST the determined sequence in a published olive genome, discarding a fragment if it has more than one copy in the olive genome, otherwise it is a single copy nuclear gene fragment;

(6) designing an amplification primer for the single-copy nuclear gene segment obtained by screening in the step (5), and controlling the amplification target segment to be between 500 and 1500 bp;

(7) carrying out PCR amplification on the leaf DNA of the 20 olive varieties in the step (1) by using the amplification primers designed in the step (6), carrying out agarose gel electrophoresis detection on amplification products, selecting sequences with clear and single bands and capable of amplifying the products of all varieties, and sequencing the sequences by using an ABI 3730XL sequencer;

(8) selecting the primers which are abundant in amplification product variation and capable of distinguishing at least two varieties in the step (7) as olive variety identification markers, and screening out 8 pairs of olive amplification primers, wherein the primers are shown in table 1: the forward primer and the reverse primer of SWH1 are shown as SEQ ID NO: 1 and SEQ ID NO: 2, the forward primer and the reverse primer of SWH2 are shown as SEQ ID NO: 3 and SEQ ID NO: 4, the forward primer and the reverse primer of SWH3 are shown as SEQ ID NO: 5 and SEQ ID NO: 6, the forward primer and the reverse primer of SWH4 are shown as SEQ ID NO: 7 and SEQ ID NO: 8, the forward primer and the reverse primer of SWH5 are shown as SEQ ID NO: 9 and SEQ ID NO: 10, the forward primer and the reverse primer of SWH6 are shown as SEQ ID NO: 11 and SEQ ID NO: 12, the forward primer and the reverse primer of SWH7 are shown as SEQ ID NO: 13 and SEQ ID NO: 14, the forward primer and the reverse primer of SWH8 are shown as SEQ ID NO: 15 and SEQ ID NO: shown at 16.

TABLE 1 primer information for Olive variety identification

Secondly, an olive variety identification method based on SNP loci,

example 1:

in 2016, a part of olive variety resource A is collected from a planting base of Wudu olive in Gansu by the forestry research institute of China forestry science, and the phenotypic character of the olive variety resource A is similar to that of the existing olive variety 'pea fruit', but the olive variety resource A is different in the characters such as fruit color and the like. To identify whether this variety resource a is 'legume', the following treatment analysis was performed:

1. respectively collecting leaf materials of an olive variety A and a known variety 'pea fruit' and extracting DNA;

2. amplifying and sequencing the two DNA fragments by using 8 pairs of olive amplification primers in the table 1 of the invention;

PCR amplification used a 30uL reaction: 20ng DNA, 3uL of 10 XBuffer (Promega, USA) buffer, 2.4uL of 2.4mM dNTPs, 2.4uL of 10uM primer, 1.2uL each of forward and reverse primers, and 0.15uL of Taq DNA polymerase (5U/uL, Takara, Shiga, Japan). PCR amplification was performed using an ABI 96U Thermo cycler PCR instrument.

The reaction procedure for PCR amplification was:

3. removing regions with low sequencing quality, such as the head end and the tail end of the sequence, by using ContigExpress software, and modifying degenerate bases presenting double peaks;

4. the sequences were exported to fasta format, and 8 sequences from two varieties were merged and opened using bioedit.

5. By comparing the variation site difference of the two varieties on each fragment, no difference is found in the result, so that the variety resource A is identified as 'pea fruit'.

Example 2

In 2016, the forestry research institute of the national forestry science research institute collects a part of olive variety resource B from the planting base of Mianyang olive in Sichuan, and the important variety classification characters such as fruits and the like are not seen, so that the variety is unclear. To identify this breed resource B, the following treatment analysis was performed:

1. collecting leaf material of olive variety B, and extracting DNA;

2. amplifying and sequencing the DNA of the leaves of the variety resource by using 8 pairs of olive amplification primers in the table 1 of the invention;

PCR amplification used a 30uL reaction: 20ng DNA, 3uL of 10 XBuffer (Promega, USA) buffer, 2.4uL of 2.4mM dNTPs, 2.4uL of 10uM primer, 1.2uL each of forward and reverse primers, and 0.15uL of Taq DNA polymerase (5U/uL, Takara, Shiga, Japan). PCR amplification was performed using an ABI 96U Thermo cycler PCR instrument.

The reaction procedure for PCR amplification was:

3. removing regions with low sequencing quality, such as the head end and the tail end of the sequence, by using ContigExpress software, and modifying degenerate bases presenting double peaks;

4. outputting the sequences into a fasta format, combining 8 sequences, introducing the sequences into BioEdit software, and aligning the sequences with other variety sequences in an existing olive variety information base constructed on the basis of 8 pairs of olive amplification primers in the table 1 of the invention;

5. a Neighbor-Joining tree (NJ tree) was constructed using ClustalX software. The result shows that the variety B is gathered with the Foao in the olive variety information base, the branch length is the same, and the genetic relationship between the B and the Foao is very close.

6. And then the variation site difference of the two varieties B and 'Foo' is checked in BioEdit software, and no difference is found, so that the variety resource B is identified as 'Foo'.

Example 3

In 2017, a subtropical forestry research institute of China forestry science institute collected one part of olive variety resource C from Lishui olive planting base in Zhejiang. The variety resource is introduced from Sichuan, the phenotypic character of the variety resource is similar to that of the existing olive variety 'laixing', but the phenotypic character of the variety resource is different from that of the leaf texture and the like. To identify if this breed resource C is 'laixing', the following treatment analysis was performed:

1. collecting leaf material of olive variety C, and extracting DNA;

2. amplifying and sequencing the DNA of the leaves of the variety resource by using 8 pairs of olive amplification primers in the table 1 of the invention;

PCR amplification used a 30uL reaction: 20ng DNA, 3uL of 10 XBuffer (Promega, USA) buffer, 2.4uL of 2.4mM dNTPs, 2.4uL of 10uM primer, 1.2uL each of forward and reverse primers, and 0.15uL of Taq DNA polymerase (5U/uL, Takara, Shiga, Japan). PCR amplification was performed using an ABI 96U Thermo cycler PCR instrument.

The reaction procedure for PCR amplification was:

3. removing regions with low sequencing quality, such as the head end and the tail end of the sequence, by using ContigExpress software, and modifying degenerate bases presenting double peaks;

4. outputting the sequences into a fasta format, combining 8 sequences, introducing the sequences into BioEdit software, and aligning the sequences with other variety sequences in an existing olive variety information base constructed on the basis of 8 pairs of olive amplification primers in the table 1 of the invention;

5. a Neighbor-Joining tree (NJ tree) was constructed using ClustalX software. The result shows that the 'Luxingx' of the variety C and the olive variety information base are gathered in the same branch, but the branch length is different, which indicates that the C is not the 'Luxingx'.

6. And then the variation site difference of the two varieties C and Leixing is checked in BioEdit software, and 2 SNP site differences exist between the two varieties, so that the variety resource C is a new variety.

It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications are possible which remain within the scope of the appended claims.

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Claims (3)

1. The amplification primer for identifying the olive variety based on the SNP locus is characterized by comprising 8 pairs of olive amplification primers; the forward primer and the reverse primer of SWH1 are shown as SEQ ID NO: 1 and SEQ ID NO: as shown in figure 2, the first and second,

the forward primer and the reverse primer of SWH2 are shown as SEQ ID NO: 3 and SEQ ID NO: as shown in (4) in the figure,

the forward primer and the reverse primer of SWH3 are shown as SEQ ID NO: 5 and SEQ ID NO: as shown in figure 6, the flow of the gas,

the forward primer and the reverse primer of SWH4 are shown as SEQ ID NO: 7 and SEQ ID NO: as shown in figure 8, the flow of air,

the forward primer and the reverse primer of SWH5 are shown as SEQ ID NO: 9 and SEQ ID NO: as shown in figure 10 of the drawings,

the forward primer and the reverse primer of SWH6 are shown as SEQ ID NO: 11 and SEQ ID NO: as shown in figure 12 of the drawings,

the forward primer and the reverse primer of SWH7 are shown as SEQ ID NO: 13 and SEQ ID NO: as shown in figure 14, the first and second,

the forward primer and the reverse primer of SWH8 are shown as SEQ ID NO: 15 and SEQ ID NO: shown at 16.

2. The olive variety identification method based on the SNP locus is characterized by comprising the following steps:

(2-1) collecting leaf materials of olive varieties to be detected, and extracting DNA of olive leaves;

(2-2) amplifying and sequencing the DNA of the leaves of the olea europaea to be detected respectively by using the 8-pair olea europaea amplification primers of claim 1;

(2-3) removing the sequence head and tail ends and the regions with low sequencing quality by using Contigexpress software, and modifying degenerate bases showing double peaks;

(2-4) outputting the sequences into a fasta format, combining the 8 sequences, introducing the sequences into BioEdit software, and aligning the sequences with other variety sequences in an existing olive variety information base constructed on the basis of the 8 pairs of olive amplification primers in the claim 1;

(2-5) constructing a Neighbor-Joining tree by using ClustalX software, and when the to-be-detected olive is gathered with a certain olive variety X in the existing olive variety information base constructed on the basis of the 8 pairs of olive amplification primers in the claim 1, indicating that the genetic relationship between the to-be-detected olive and the olive variety X is very close;

(2-6) checking variation site difference between the to-be-detected olive and the olive variety X with a close genetic relationship in the step (2-5) in a BioEdit software, wherein if the to-be-detected olive and the olive variety X have no difference, the to-be-detected olive is the olive variety X, and if the to-be-detected olive and the olive variety X have a difference, the to-be-detected olive variety is not in an existing olive variety information base constructed on the basis of the 8 pairs of olive amplification primers in the claim 1, and the to-be-detected variety is a new olive variety.

3. The method for identifying an olive variety based on a SNP site according to claim 2, wherein in the step (2-2), PCR amplification is performed by using a 30uL reaction system: 20ng DNA, 3uL of 10 XBuffer buffer, 2.4uL of 2.4mM dNTPs, 2.4uL of 10uM primer, 1.2uL of each of forward primer and reverse primer, and 0.15uL of Taq DNA polymerase; PCR amplification was performed using an ABI 96U Thermo cycler PCR instrument;

the reaction procedure for PCR amplification was:

pre-denaturation: 94 ℃ for 4 min;

denaturation: 94 ℃, 30s, annealing: 60 ℃, 30s, and extension: at 72 ℃ for 2min, for 10 cycles;

denaturation: 94 ℃, 30s, annealing: 55 ℃, 30s, and extension: at 72 ℃ for 2min, for a total of 26 cycles;

extension: 72 deg.C, 10 min.