CN111944922B

CN111944922B - EST-SSR primer set developed based on Paeonia lactiflora transcriptome sequence and its application

Info

Publication number: CN111944922B
Application number: CN202010906751.5A
Authority: CN
Inventors: 郭先锋; 左睿睿; 马燕
Original assignee: Shandong Agricultural University
Current assignee: Shandong Agricultural University
Priority date: 2020-09-02
Filing date: 2020-09-02
Publication date: 2022-03-22
Anticipated expiration: 2040-09-02
Also published as: CN111944922A

Abstract

The invention provides an EST-SSR primer set developed based on the peony transcriptome sequence and its application, belonging to the technical field of SSR molecular markers. The EST-SSR primer set provided by the present invention is composed of 13 pairs of primers, derived from the sequence of the Paeonia lactiflora transcriptome, and has the advantages of rich polymorphism, stable amplification, high versatility, good repeatability, etc., and enriches the number of Paeonia lactiflora EST-SSR primers. The invention also provides the application of the EST-SSR molecular marker primer set in the molecular identification of Paeonia lactiflora, which has the advantages of high efficiency, accuracy, practicality, and is not affected by seasons, and provides a new and reliable method for identification of Paeonia lactiflora germplasm resources.

Description

EST-SSR primer group developed based on peony transcriptome sequence and application thereof

Technical Field

The invention relates to the technical field of SSR molecular markers, in particular to an EST-SSR primer group developed based on a peony transcriptome sequence and application thereof.

Background

Paeonia lactiflora Pall belongs to Paeonia of Paeoniaceae, is a traditional famous flower with a very long cultivation history, is widely applied to gardens as landscaping and beautifying plants, and is an important cut flower in the international flower market. In the field of peony breeding, the breeding work is very active at home and abroad, and new varieties emerge endlessly. However, at present, the names of varieties in different peony producing areas and different production units are not completely the same. In the field of peony circulation, foreign matters with the same thing and the same name are abused, and the production, protection and further development and application of peony germplasm resources are seriously influenced.

In China, the work progress of identifying the peony variety is very slow all the time. The variety identification of the industry and institutions is only limited to the phenotype identification of the flowering phase, and the time is very short; some peony varieties have very similar morphological characteristics even in the flowering phase, and thus, the method also poses a serious challenge to the experiential appraisers. The traditional phenotype identification not only consumes a large amount of manpower, material resources and financial resources, but also has higher identification difficulty.

At present, molecular marker technologies such as RAPD, SRAP, AFLP and SSR have been applied to peony germplasm resource research, wherein SSR (simple repeat sequence repeat) is widely applied to researches on plant genetic evolution, breeding and the like due to the characteristics of abundant quantity, multiple alleles, high information providing amount, co-dominance and the like. However, the traditional SSR marker primer design process is slow, links such as construction of a genome library, identification and screening of repeated sequence cloning, sequencing, primer design and the like need to be carried out, and the SSR primer acquisition takes long time, is high in cost and is low in efficiency. An expressed sequence tag microsatellite (EST-SSR) is a novel molecular marker which is designed based on the simple sequence repetition of an expressed sequence tag, has the advantages of high polymorphism, co-dominant inheritance, good repeatability, low development cost and the like, is derived from a coding region of a genome, is closely linked with a functional gene, and is easier to obtain the information of gene expression. At present, EST-SSR is applied to various plants such as Chinese cabbage, cotton, oncidium, celery, peach, salvia miltiorrhiza, wax apple, waxberry and the like. It is also reported that EST-SSR markers and morphological characters are utilized to detect genetic diversity of 31 peony varieties, but EST-SSR markers derived from peony are utilized, and EST-SSR specific primers aiming at peony are few. In order to better protect and utilize peony germplasm resources, more specific primers need to be developed urgently.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide an EST-SSR primer group developed based on a peony transcriptome sequence and application thereof. The EST-SSR primer group can obviously amplify polymorphic bands and has the characteristics of stable amplification, good repeatability and the like; the EST-SSR primer group can realize molecular identification of peony germplasm, and has the advantages of rapidness, accuracy, practicability, no influence of flowering phase and the like.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect of the invention, an EST-SSR primer group developed based on a peony transcriptome sequence is provided, and comprises the following 13 pairs of primers:

a primer pair PlE2, the sequence of which is shown as SEQ ID NO.1 and SEQ ID NO. 2;

a primer pair PlE6, the sequence of which is shown as SEQ ID NO.3 and SEQ ID NO. 4;

a primer pair PlE12, the sequence of which is shown as SEQ ID NO.5 and SEQ ID NO. 6;

a primer pair PlE20, the sequence of which is shown as SEQ ID NO.7 and SEQ ID NO. 8;

a primer pair PlE22, the sequence of which is shown as SEQ ID NO.9 and SEQ ID NO. 10;

a primer pair PlE23, the sequence of which is shown as SEQ ID NO.11 and SEQ ID NO. 12;

a primer pair PlE31, the sequence of which is shown as SEQ ID NO.13 and SEQ ID NO. 14;

a primer pair PlE33, the sequence of which is shown as SEQ ID NO.15 and SEQ ID NO. 16;

a primer pair PlE37, the sequence of which is shown as SEQ ID NO.17 and SEQ ID NO. 18;

a primer pair PlE39, the sequence of which is shown as SEQ ID NO.19 and SEQ ID NO. 20;

a primer pair PlE49, the sequence of which is shown as SEQ ID NO.21 and SEQ ID NO. 22;

a primer pair PlE53, the sequence of which is shown as SEQ ID NO.23 and SEQ ID NO. 24;

and the sequence of the primer pair PlE56 is shown as SEQ ID NO.25 and SEQ ID NO. 26.

In a second aspect of the present invention, there is provided a use of the above EST-SSR primer set in any one of (1) to (4) below:

(1) performing molecular identification on the peony variety;

(2) identifying the purity of the peony strain;

(3) analyzing genetic diversity of the peony variety;

(4) and (5) performing peony molecular breeding.

In a third aspect of the present invention, a method for identifying peony molecules by using the EST-SSR primer set is provided, which comprises the following steps:

(1) extracting genome DNA in a peony leaf sample;

(2) taking genome DNA in the peony leaf sample extracted in the step (1) as a template, performing PCR amplification by using at least one primer pair in an EST-SSR primer group, and modifying an upstream primer of the primer pair for PCR amplification by using a bioluminescence group;

(3) performing capillary fluorescence electrophoresis detection on the PCR amplification product, reading electrophoresis strip data, and counting detection sites; and (5) carrying out peony molecule identification according to the detection site.

Preferably, in step (2), the preferential utilization order of the primer pairs in the EST-SSR primer group is as follows: PlE20, PlE23, PlE49, PlE22, PlE31, PlE2, PlE6, PlE12, PlE37, PlE33, PlE53, PlE56, PlE 39.

Preferably, in step (2), the upstream primer of each primer pair is modified with 4 kinds of bioluminescent groups, FAM, HEX, TAMRA and ROX, respectively.

Preferably, in step (2), the PCR amplification system is 20 μ L, and comprises: mu.L of 30 ng/. mu.L template DNA, 10. mu.L of 2 XM 5 PAGE Taq Mix, 10. mu.M upstream and downstream primers, 0.5. mu.L each, and 8. mu.L ddH₂O。

Preferably, in step (2), the reaction procedure of PCR amplification is: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, renaturation at 51-59 ℃ for 30s, annealing at 72 ℃ for 30s, and 30 cycles; extending for 5min at 72 ℃; obtaining an amplification product.

More preferably, in step (2), the annealing temperature of primer pair PlE2 is 58 ℃; the annealing temperature of the primer pair PlE6 was 53 ℃; the annealing temperature of the primer pair PlE12 was 52 ℃; the annealing temperature of the primer pair PlE20 was 51 ℃; the annealing temperature of the primer pair PlE22 was 55 ℃; the annealing temperature of the primer pair PlE23 was 56 ℃; the annealing temperature of the primer pair PlE31 was 52 ℃; the annealing temperature of the primer pair PlE33 was 55 ℃; the annealing temperature of the primer pair PlE37 was 55 ℃; the annealing temperature of primer pair PlE39 was 59 ℃; the annealing temperature of the primer pair PlE49 was 53 ℃; the annealing temperature of the primer pair PlE53 was 53 ℃; the annealing temperature of primer pair PlE56 was 58 ℃.

Preferably, in the step (4), when peony molecule identification is performed, each primer pair is assigned with a corresponding code at an amplification site in a peony sample, and the same variety is determined when the codes of the two samples are completely the same; otherwise, the variety is determined to be different.

The invention has the beneficial effects that:

the invention develops and designs an EST-SSR primer group based on a peony transcriptome sequence, which consists of 13 pairs of primers, has the advantages of rich polymorphism, stable amplification, high universality, good repeatability and the like, and enriches the number of peony EST-SSR primers. The EST-SSR primer group can be used for identifying the peony molecules, has the characteristics of rapidness, reliability, practicability and the like, and provides important technical support for introduction and production of peony germplasm resources and DUS test of new varieties.

Drawings

FIG. 1: the peony variety 'ubiquitously red' utilizes a code obtained after 13 primer pairs in an EST-SSR primer group are amplified.

FIG. 2: a dot-plot of the amplification of the peony variety 'fuji' on primer pair PlE 2.

FIG. 3: paeonia lactiflora pall sample amplification site map on primer pair PlE 2.

FIG. 4: a dot-plot of the amplification of the peony variety 'fuji' on primer pair PlE 6.

FIG. 5: paeonia lactiflora pall sample amplification site map on primer pair PlE 6.

FIG. 6: a dot-plot of the amplification of the peony variety 'fuji' on primer PlE 31.

FIG. 7: paeonia lactiflora pall sample is subjected to amplification site diagram on the primer PlE 31.

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

As introduced in the background art, the work progress of identifying the peony variety in China is very slow, and although the peony is a traditional flower in China, the peony has very high application value. However, Chinese herbs 'heavy peony and light peony' lead the research of peony in new product identification, protection and molecular level to lag behind peony. The traditional phenotype identification not only consumes a great deal of manpower, material resources and financial resources, but also is only limited to the phenotype identification of the flowering phase, so that the time is very short, and the identification difficulty is high. EST-SSR has the advantages of high polymorphism, co-dominant inheritance, good repeatability, low development cost and the like, can be used for molecular identification of plant varieties, but has less development of related peony EST-SSR primers. However, the variety of peony is much larger than that of peony, and more primers are necessarily required to be developed to meet the variety identification requirement.

Based on this, the object of the present invention is to develop EST-SSR specific primers for peony based on the transcriptome sequence of peony. The ESTs database provides a source of SSRs sequences, but not all ESTs can be used to design SSR primers, which requires finding appropriate simple repeated sequences from the ESTs. When designing SSR primers, in order to improve the detection efficiency, SSRs with more repetition times need to be selected. In addition, since the coding region is conserved, SSRs in the 3 'or 5' UTRs which have large variations among different materials should be used, but ESTs whose adjacent sequences are too short to satisfy the primer design criteria should be removed. In order to enable each pair of primers to be specifically combined with a target region in the primer design process, the GC content, annealing temperature, primer length, product length and the like need to be considered and optimized. After primer design, validation of primers for EST-SSR is also required.

According to the invention, a peony EST-SSR primer group is finally obtained through optimization design, and comprises 13 pairs of primers, which are as follows:

PlE2 F：ATGGAGCAACTAACAGAGGG；(SEQ ID NO.1)

R：GGAGAAAGACACGATGATGAG。(SEQ ID NO.2)

PlE6 F：AGCCAGTGTCAGGGTAGTC；(SEQ ID NO.3)

R：GTTGAAGAAGAAACAGAGGG。(SEQ ID NO.4)

PlE12 F：GTCAACGGGCTCCTTACCA；(SEQ ID NO.5)

R：TGCTTCCACCGCTTCTCA。(SEQ ID NO.6)

PlE20 F：TCAAGGCACGGTACATCT；(SEQ ID NO.7)

R：ATTGCAGCATAGTAAAGTCG。(SEQ ID NO.8)

PlE22 F：TTAGCAAGGTAAGGAGAACA；(SEQ ID NO.9)

R：CATCAAAGAGGTGGGGTA。(SEQ ID NO.10)

PlE23 F：CGGTAATCTGTAACACTTCATC；(SEQ ID NO.11)

R：GCTTGTAAGAGGCGTAACTAA。(SEQ ID NO.12)

PlE31 F：TCTTGCATCTCCCTAACCT；(SEQ ID NO.13)

R：ATTGCCAAACCTGCTGTC。(SEQ ID NO.14)

PlE33 F：TTTTATCCTCACTGTCCCCTCC；(SEQ ID NO.15)

R：TCCGCCTCAATTTCCGTC。(SEQ ID NO.16)

PlE37 F：GACGAGTAAAAGGACAAACG；(SEQ ID NO.17)

R：CAGAAACTGCCGCAAGAG。(SEQ ID NO.18)

PlE39 F：AATCCGCAGTTACAAGCC；(SEQ ID NO.19)

R：CGACGAGCACATACACCA。(SEQ ID NO.20)

PlE49 F：CAAAATAAGTAGGGAGTGAGTG；(SEQ ID NO.21)

R：GAATGGAGGGAAGAAGATAA。(SEQ ID NO.22)

PlE53 F：ATTGAAACCTTTGGACGAAC；(SEQ ID NO.23)

R：CACCACTAATGTCAGACGATG。(SEQ ID NO.24)

PlE56 F：CTGTCGTTGTTATTGGAGTTAG；(SEQ ID NO.25)

R：GGAGACCTACACCCTTGCT。(SEQ ID NO.26)

the peony EST-SSR primer group obtained by final optimization has the advantages of rich polymorphism, stable amplification, high identification efficiency and high universality, and particularly has remarkable advantages in the aspects of identification efficiency and universality. In terms of identification efficiency, the peony EST-SSR primers of the present invention have a lower 0 allele frequency in the inter-species amplification, and the 13 pairs of primers have an average 0 allele frequency of 0.059 (it is reported that the 0 allele frequency of the primers is generally between 0.2 and 0.4); wherein the allele frequency of 11 pairs of 0 is less than 0.03(7 pairs equal to 0), and the allele frequency of 0 of only two pairs of primers is between 0.2 and 0.4; therefore, the peony EST-SSR primer group can efficiently identify different peony varieties. In the aspect of universality, in the peony EST-SSR primers, 9 pairs of primers such as PLE2, PLE12, PLE22, PLE23, PLE31, PLE33, PLE37, PLE39 and PLE53 can also be used for identifying peony varieties, so that the peony EST-SSR primers have the advantage of high universality.

Based on the developed peony EST-SSR primer group, another embodiment of the invention provides a method for identifying peony molecules based on the EST-SSR primer group, which comprises the following steps:

1) extracting genome DNA in a peony leaf sample;

2) the upstream primers of each pair of primers are respectively modified by 4 biological fluorescent groups of FAM (blue), HEX (green), TAMRA (black) and ROX (red); the method specifically comprises the following steps: FAM fluorescent group is modified at 5' end of primer in upstream of PlE2 and PlE6 primer pairs; HEX is modified at the 5' end of the upstream primer of the primer pairs PlE22, PlE31, PlE33, PlE37 and PlE 39; ROX is modified at the 5' end of the upstream primer of the primer pairs PlE20, PlE49, PlE53 and PlE 56; TAMRA is modified at the 5' end of the upstream primer of the primer pair PlE12 and PlE 23.

3) Sample DNA is taken as a template, and PCR amplification is carried out by using a fluorescence modified primer and a downstream primer, wherein the amplification system is 20 mu L, and the amplification system comprises 1 mu L of template DNA with the concentration of 30 ng/mu L, 10 mu L of 2 xM 5 PAGE Taq Mix, 0.5 mu L of each upstream primer with the concentration of 10 mu M and 8 mu L of ddH₂O, the reaction procedure of amplification is as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, renaturation at 51-59 ℃ for 30s, annealing at 72 ℃ for 30s, and 30 cycles; extending for 5min at 72 ℃; obtaining an amplification product; compared with a three-primer method, the method provided by the invention has the advantages that two primers are used for PCR amplification, the background is clear, and the detection efficiency is higher.

4) Performing capillary fluorescence electrophoresis detection on the PCR amplification product, reading electrophoresis strip data, and counting detection sites;

5) and (5) carrying out peony molecule identification according to the detection site.

When identifying peony molecules, sometimes it is not necessary to use all 13 primer pairs, at least one primer pair may be used in the following order of primer pairs, and the order of preferential use of primer pairs is in order: PlE20, PlE23, PlE49, PlE22, PlE31, PlE2, PlE6, PlE12, PlE37, PlE33, PlE53, PlE56, PlE 39.

And sequentially carrying out PCR amplification according to the sequence of the primer pairs, determining detection sites after amplification, then comparing whether the detection sites are the same, and if so, continuing to carry out PCR amplification by using the next primer pair. If the detection sites are completely the same, judging the same variety; otherwise, the variety is determined to be different.

For comparison between two samples, comparison of the amplification sites directly by the primers can be performed. When a large number of samples are compared, the comparison of amplification sites is particularly complicated, the numbers can be sequentially carried out according to the molecular weights corresponding to different varieties of amplification fragments of each pair of primers, and the comparison of the coded molecular identity cards is simpler and quicker.

In order to make the technical solutions of the present application more clearly understood by those skilled in the art, the technical solutions of the present application will be described in detail below with reference to specific embodiments.

The test materials used in the examples of the present invention, which were not specifically described, were all those conventional in the art and commercially available. The experimental conditions and methods are not specified in the examples of the present invention, and are generally performed under conventional conditions or conditions recommended by the manufacturer.

Example 1: development of peony EST-SSR primer group

1. Design and Synthesis of primers

(1) The peony transcriptome database is derived from the high-throughput sequencing result of peony buds (NCBI No. SRP059306) of the inventor subject group;

(2) searching and analyzing SSR sites of the database in the step (1) by using Trinity 2.4.0 software, screening an EST sequence with the SSR length of 18bp or more, and specifically screening a standard: the minimum number of times of repeat of mononucleotide is 20; dinucleotide is repeated for 10 times at the minimum; the minimum number of times of repetition of the three, four, five and six nucleotides is 6;

(3) designing upstream and downstream primers for the EST sequences containing the SSR sites screened by using Primer Premier 5.0 software, and detecting the base complementarity of the primers by using the 'self-complementary' function of DNAMAN 7.0.2 software to avoid the influence of mismatching, Primer dimer and hairpin structure on PCR reaction; finally, 60 pairs of candidate primers are determined;

2. preliminary screening of primers and optimization of annealing temperature

(1) Selecting 12 peony resources with large morphological difference, extracting peony genome DNA by using a DP305 kit (Chinese Tiangen), and detecting the quality and concentration of the DNA by respectively adopting agarose gel electrophoresis with the concentration of 1% and a spectrophotometer;

(2) performing PCR amplification on the candidate primers by using the determined 60 pairs, and preliminarily screening out primers which are stable in amplification, contain clear target bands and have high polymorphism by 8% polyacrylamide gel electrophoresis;

(3) carrying out annealing temperature screening on the primers screened in the step (2) and determining the optimal annealing temperature of each pair of primers; the following 13 primer pairs and their appropriate annealing temperatures were obtained by screening (table 1):

table 1: 13 pairs of EST-SSR primer information

Example 2: detection site coding of EST-SSR primer group for peony molecule identification and priority utilization sequencing of each primer pair

(1) Extracting genome DNA of 135 peony varieties;

(2) performing PCR amplification by using the primers and the annealing temperature in the table 1, and detecting by using an ABI 3730xl DNA Analyzer gene Analyzer after amplification;

(3) counting the sizes of characteristic spectral bands and polymorphic spectral bands amplified by each pair of primers according to the detection result of a gene analyzer;

(4) according to the molecular weight corresponding to the amplified fragments of different varieties of each pair of primers, the molecular weight is sequentially numbered as 0-9, if the molecular weight exceeds 9, the molecular weight is assigned as A-Z, and 0 represents zero allele without band of the lane due to loss of gene fragments (Table 2);

it should be noted that, the encoding is based on the existing sequence of the sizes of the amplified fragments, and since '1, 2, 3' in the encoding sequence does not represent a clear size relationship, the occurrence of a new length of the amplified fragments in the actual identification process can add a new encoding to the existing encoding.

(5) The allelic factor and Polymorphism Information Content (PIC) were analyzed using the Microlatelite Toolkit,

wherein Pij represents the jth allele of site iThe frequency of occurrence of the variation, reflecting the polymorphism level of each EST-SSR locus;

13 pairs of primers were sequenced from high to low PIC values to obtain the primer pair sequence of the present invention (table 3).

When the EST-SSR primer group is used for identifying the peony molecules, the sample can be coded according to the sequence of the primer pairs, and the specific coding rule is 'universal red' (FIG. 1).

Table 2: amplification sites and site codes of 13 pairs of primers in 135 peony varieties

Table 3: amplification polymorphism of 13 pairs of primers in 135 peony samples

Example 3: identification of whether certain plant A to be detected is a peony variety ' Fuji ' by utilizing EST-SSR primer group '

The specific implementation method comprises the following steps:

(1) respectively extracting DNA of a known peony variety 'Fuji' and a plant to be detected;

(2) utilizing 13 primer pairs in the table 1, wherein the upstream of each primer pair is modified by 4 biological fluorescent groups of FAM (blue), HEX (green), TAMRA (black) and ROX (red);

(3) after fluorescent modification, PCR amplification is carried out;

(4) detecting the amplified product by capillary fluorescence electrophoresis to obtain an amplified electrophoresis strip chart, and respectively counting the detection sites;

(5) and (3) coding the detected locus according to the sequence of each primer pair in the primer group, and judging the variety according to the coding consistency. In this example, the breed code of the known breed 'fuji' is: 00C29E8153AA 5; the variety code of the plant to be detected is as follows: 00C2C77153AA5, wherein the 3-bit codes of the two are different, namely the two codes are different at the amplification sites corresponding to 3 pairs of primers (shown in figures 2-7), so that the plant to be detected is not the peony 'Fuji'.

The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.

SEQUENCE LISTING

<110> Shandong university of agriculture

<120> EST-SSR primer group developed based on peony transcriptome sequence and application thereof

<130> 2020

<160> 26

<170> PatentIn version 3.5

<210> 1

<211> 20

<212> DNA

<213> Artificial sequence

<400> 1

atggagcaac taacagaggg 20

<210> 2

<211> 21

<212> DNA

<213> Artificial sequence

<400> 2

ggagaaagac acgatgatga g 21

<210> 3

<211> 19

<212> DNA

<213> Artificial sequence

<400> 3

agccagtgtc agggtagtc 19

<210> 4

<211> 20

<212> DNA

<213> Artificial sequence

<400> 4

gttgaagaag aaacagaggg 20

<210> 5

<211> 19

<212> DNA

<213> Artificial sequence

<400> 5

gtcaacgggc tccttacca 19

<210> 6

<211> 18

<212> DNA

<213> Artificial sequence

<400> 6

tgcttccacc gcttctca 18

<210> 7

<211> 18

<212> DNA

<213> Artificial sequence

<400> 7

tcaaggcacg gtacatct 18

<210> 8

<211> 20

<212> DNA

<213> Artificial sequence

<400> 8

attgcagcat agtaaagtcg 20

<210> 9

<211> 20

<212> DNA

<213> Artificial sequence

<400> 9

ttagcaaggt aaggagaaca 20

<210> 10

<211> 18

<212> DNA

<213> Artificial sequence

<400> 10

catcaaagag gtggggta 18

<210> 11

<211> 22

<212> DNA

<213> Artificial sequence

<400> 11

cggtaatctg taacacttca tc 22

<210> 12

<211> 21

<212> DNA

<213> Artificial sequence

<400> 12

gcttgtaaga ggcgtaacta a 21

<210> 13

<211> 19

<212> DNA

<213> Artificial sequence

<400> 13

tcttgcatct ccctaacct 19

<210> 14

<211> 18

<212> DNA

<213> Artificial sequence

<400> 14

attgccaaac ctgctgtc 18

<210> 15

<211> 22

<212> DNA

<213> Artificial sequence

<400> 15

ttttatcctc actgtcccct cc 22

<210> 16

<211> 18

<212> DNA

<213> Artificial sequence

<400> 16

tccgcctcaa tttccgtc 18

<210> 17

<211> 20

<212> DNA

<213> Artificial sequence

<400> 17

gacgagtaaa aggacaaacg 20

<210> 18

<211> 18

<212> DNA

<213> Artificial sequence

<400> 18

cagaaactgc cgcaagag 18

<210> 19

<211> 18

<212> DNA

<213> Artificial sequence

<400> 19

aatccgcagt tacaagcc 18

<210> 20

<211> 18

<212> DNA

<213> Artificial sequence

<400> 20

cgacgagcac atacacca 18

<210> 21

<211> 22

<212> DNA

<213> Artificial sequence

<400> 21

caaaataagt agggagtgag tg 22

<210> 22

<211> 20

<212> DNA

<213> Artificial sequence

<400> 22

gaatggaggg aagaagataa 20

<210> 23

<211> 20

<212> DNA

<213> Artificial sequence

<400> 23

attgaaacct ttggacgaac 20

<210> 24

<211> 21

<212> DNA

<213> Artificial sequence

<400> 24

caccactaat gtcagacgat g 21

<210> 25

<211> 22

<212> DNA

<213> Artificial sequence

<400> 25

ctgtcgttgt tattggagtt ag 22

<210> 26

<211> 19

<212> DNA

<213> Artificial sequence

<400> 26

ggagacctac acccttgct 19

Claims

1. an EST-SSR primer set based on Paeonia lactiflora's transcriptome sequence development, is characterized in that, described EST-SSR primer set comprises following 13 pairs of primers:

Primer pair PlE2, its sequence is as shown in SEQ ID NO.1 and SEQ ID NO.2;

Primer pair PlE6, its sequence is as shown in SEQ ID NO.3 and SEQ ID NO.4;

Primer pair PlE12, its sequence is as shown in SEQ ID NO.5 and SEQ ID NO.6;

Primer pair PlE20, its sequence is as shown in SEQ ID NO.7 and SEQ ID NO.8;

Primer pair PlE22, its sequence is as shown in SEQ ID NO.9 and SEQ ID NO.10;

Primer pair PlE23, its sequence is as shown in SEQ ID NO.11 and SEQ ID NO.12;

Primer pair PlE31, its sequence is as shown in SEQ ID NO.13 and SEQ ID NO.14;

Primer pair P1E33, its sequence is as shown in SEQ ID NO.15 and SEQ ID NO.16;

Primer pair PlE37, its sequence is as shown in SEQ ID NO.17 and SEQ ID NO.18;

Primer pair PlE39, its sequence is as shown in SEQ ID NO.19 and SEQ ID NO.20;

Primer pair PlE49, its sequence is as shown in SEQ ID NO.21 and SEQ ID NO.22;

Primer pair PlE53, its sequence is as shown in SEQ ID NO.23 and SEQ ID NO.24;

Primer pair PlE56, its sequence is shown as SEQ ID NO.25 and SEQ ID NO.26.

2. the application of the EST-SSR primer set described in claim 1 in any one of following (1)-(4):

(1) Molecular identification of peony varieties;

(2) Purity identification of peony products;

(3) Genetic diversity analysis of peony varieties;

(4) Molecular breeding of peony.

3. a method utilizing the EST-SSR primer set described in claim 1 to carry out the identification of peony molecule, is characterized in that, comprises the following steps:

(1) Extracting genomic DNA from Paeonia lactiflora leaf samples;

(2) Using the genomic DNA in the peony leaf sample extracted in step (1) as a template, using 13 pairs of primers in the EST-SSR primer set according to claim 1 to carry out PCR amplification respectively, the primer pairs used for PCR amplification The upstream primers are modified with biological fluorescent groups;

(3) Perform capillary fluorescence electrophoresis detection on the PCR amplification product, read the electrophoresis band data, and count the detection sites; carry out molecular identification of peony according to the detection sites;

In step (2), the utilization sequence of the 13 pairs of primers in the EST-SSR primer set is: PlE20, PlE23, PlE49, PlE22, PlE31, PlE2, PlE6, PlE12, PlE37, PlE33, PlE53, PlE56, PlE39;

In step (2), the PCR amplification system is 20 μL, including: 1 μL 30 ng/μL template DNA, 10 μL 2 × M5PAGE Taq Mix, 10 μM upstream and downstream primers, 0.5 μL each, and 8 μL ddH ₂ O;

In step (2), the reaction program of PCR amplification was as follows: 95°C pre-denaturation for 5 min; 95°C denaturation for 30 s, annealing at 51°C-59°C for 30 s, 72°C for 30 s, 30 cycles; 72 Extend at ℃ for 5 min; get the amplified product;

In step (2), the annealing temperature of the primer pair PlE2 is 58°C; the annealing temperature of the primer pair PlE6 is 53°C; the annealing temperature of the primer pair PlE12 is 52°C; the annealing temperature of the primer pair PlE20 is 51°C; The annealing temperature of primer pair PlE23 is 56 ℃; the annealing temperature of primer pair PlE31 is 52 ℃; the annealing temperature of primer pair PlE33 is 55 ℃; the annealing temperature of primer pair PlE37 is 55 ℃; the annealing temperature of primer pair PlE39 is 55 ℃; The annealing temperature was 59°C; the annealing temperature of the primer pair PlE49 was 53°C; the annealing temperature of the primer pair PlE53 was 53°C; the annealing temperature of the primer pair PlE56 was 58°C.

4 . The method according to claim 3 , wherein in step (2), the upstream primers of each pair of primers are respectively modified with any kind of biological fluorescent groups in FAM, HEX, TAMRA and ROX4. 5 .

5. The method according to claim 3, wherein in step (3), when performing molecular identification of peony, each primer pair is assigned a corresponding code to the amplification site in the peony sample, and the two samples When the codes are exactly the same, it is judged as the same variety; otherwise, it is judged as a different variety.