CN116083618A - EST-SSR primer developed based on Paeonia ostii waterlogged transcriptome sequence and application thereof - Google Patents
EST-SSR primer developed based on Paeonia ostii waterlogged transcriptome sequence and application thereof Download PDFInfo
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Abstract
The invention discloses an EST-SSR primer developed based on an Paeonia ostii waterlogging transcriptome sequence and application thereof, wherein the EST-SSR primer developed based on the Paeonia ostii waterlogging transcriptome sequence comprises 20 pairs of primers P1-P20, and the sequences of the P1-P20 are respectively shown as SEQ ID No.1 to SEQ ID No.40, and the primers have the advantages of clear amplified strips, high polymorphism, good repeatability, strong universality and the like.
Description
Technical Field
The invention relates to the technical field of SSR molecular marker development, in particular to an EST-SSR primer developed based on an Paeonia ostii waterlogging transcriptome sequence and application thereof.
Background
The oil peony is a novel woody oil crop and has the characteristics of drought resistance, barren resistance, severe cold resistance, shadow resistance and the like. The peony seed oil extracted from the seeds contains up to 92% of unsaturated fatty acid, wherein, the alpha-linolenic acid which is very beneficial to the health of human bodies is up to 43%. The peony for oil mainly comprises two strains of Paeonia ostii and Paeonia ostii, wherein Paeonia ostii is a main series suitable for planting in the south of the Yangtze river, the root system of Paeonia ostii belongs to the fleshy root, is sensitive to moisture, and is a type of plant which is intolerant to waterlogging. Because of more rainwater in the south of the river, especially when the Paeonia ostii barks in the pod stage, the Paeonia ostii barks just meet the season of plum rain, and the yield of Paeonia ostii barks is greatly affected. In this regard, scientific researchers try to successfully relieve disasters caused by rainwater to Paeonia ostii through modes such as rain-sheltering cultivation, root limiting cultivation, agricultural light complementary cultivation and the like. However, only partial alleviation is achieved, and certain difficulties exist in implementing the modes of cultivating Paeonia ostii in large-area mountain areas and interplanting Paeonia ostii under forests. Therefore, in order to fundamentally solve the problems, it is urgently required to select some varieties of oil peonies with waterlogging tolerance, so as to solve the problem of yield reduction caused by the fact that the oil peonies are intolerant in variety, facilitate popularization and application of the oil peonies in a faster and larger scale, and alleviate the shortage situation of oil in China.
Paeonia ostii belongs to woody plants, flowers and pods can only be formed three years after seeds are sown, and the adverse factors of long period, wide range, high cost and the like exist in field seed selection. Therefore, development of a molecular marker means capable of identifying a strain having a high waterlogging resistance at an early stage is urgently required. In addition, most Paeonia ostii varieties are self-pollination offspring, accurate parents are unknown, a plurality of uncertain factors are brought to breeding work, and the phenomena of confusion of genetic relationship, homonymous foreign matters or homonymous foreign matters and the like are often caused. Molecular marker assisted breeding is an important direction of research in modern genetic breeding science, and among a plurality of molecular markers, SSR markers have the advantages of high polymorphism, good repeatability, simplicity in operation and the like, and are widely applied to the aspects of variety identification, genetic diversity analysis, molecular marker assisted selection breeding and the like of plants such as corn, soybean, cabbage, cotton, chrysanthemum, chinese scholartree, magnolia, waxberry and the like. Regarding development of peony SSR markers, there are few reports, such as Han Pingdeng, in the multi-gene regulation of high accumulation of carbon-eighteen unsaturated fatty acids of peony seeds, on the basis of a high-throughput RNA-seq database of the peony, SSR markers for regulating and controlling the genes related to the oil synthesis, fatty acid accumulation, seed size, grain weight and other target characters are developed; luo Liuming et al develop SSR markers for regulating and controlling candidate genes related to flower color formation based on the transcriptome information of original peony petals in RNA-seq-based peony SSR marker development and universality analysis; hu Yonggong in the patent application of publication No. CN107881250A, a peony EST-SSR primer developed based on transcriptome sequencing and a development method thereof are provided, and EST-SSR primers for identifying wild peony molecules such as paeonia rockii, paeonia flavescens, paeonia suffruticosa and the like are developed based on petal transcriptome information of different development periods of the peony.
However, up to the present, the development of SSR (simple sequence repeat) markers for the waterlogged red-rooted salvia root of Paeonia ostii has not been reported yet. Therefore, by utilizing sequence information obtained by the water logging pest transcriptome sequencing of Paeonia ostii, the development of EST-SSR primers can provide important theoretical basis for screening of important character genes related to water logging pest, molecular marker assisted selective breeding, germplasm identification and the like.
Disclosure of Invention
In order to solve the technical problems, the invention aims to provide an EST-SSR primer developed based on an Paeonia ostii waterlogging transcriptome sequence and application thereof, wherein the EST-SSR primer developed by the invention can amplify clear, stable and high-polymorphism strips, can better identify Paeonia ostii waterlogging character related genes, screen out Paeonia ostii waterlogging-tolerant varieties, and can be used for molecular identification, genetic diversity analysis, genetic relationship analysis and molecular marker-assisted breeding of Paeonia ostii varieties.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
EST-SSR primers developed based on Paeonia ostii waterlogging transcriptome sequences comprise the following 20 pairs of primers:
the primer pair P1 has the sequences shown as SEQ ID NO.1 and SEQ ID NO. 2;
primer pair P2, the sequences of which are shown as SEQ ID NO.3 and SEQ ID NO. 4;
the primer pair P3 has the sequences shown in SEQ ID NO.5 and SEQ ID NO. 6;
the primer pair P4 has the sequences shown as SEQ ID NO.7 and SEQ ID NO. 8;
primer pair P5, the sequences of which are shown as SEQ ID NO.9 and SEQ ID NO. 10;
primer pair P6, the sequences of which are shown as SEQ ID NO.11 and SEQ ID NO. 12;
the primer pair P7 has the sequences shown as SEQ ID NO.13 and SEQ ID NO. 14;
primer pair P8, the sequences of which are shown as SEQ ID NO.15 and SEQ ID NO. 16;
the primer pair P9 has the sequences shown as SEQ ID NO.17 and SEQ ID NO. 18;
the primer pair P10 has the sequences shown as SEQ ID NO.19 and SEQ ID NO. 20;
the primer pair P11 has the sequences shown as SEQ ID NO.21 and SEQ ID NO. 22;
the primer pair P12 has the sequences shown in SEQ ID NO.23 and SEQ ID NO. 24;
the primer pair P13 has the sequences shown in SEQ ID NO.25 and SEQ ID NO. 26;
the primer pair P14 has the sequences shown in SEQ ID NO.27 and SEQ ID NO. 28;
the primer pair P15 has the sequences shown as SEQ ID NO.29 and SEQ ID NO. 30;
the primer pair P16 has the sequences shown in SEQ ID NO.31 and SEQ ID NO. 32;
the primer pair P17 has the sequences shown in SEQ ID NO.33 and SEQ ID NO. 34;
the primer pair P18 has the sequences shown as SEQ D No.35 and SEQ ID No. 36;
the primer pair P19 has the sequences shown as SEQ ID NO.37 and SEQ ID NO. 38;
the primer pair P20 has the sequences shown as SEQ ID NO.39 and SEQ ID NO. 40;
the application of the EST-SSR primer developed based on the Paeonia ostii waterlogging transcriptome sequence in any one of the following (1) - (4):
(1) Molecular identification of a tree peony variety in Jiangnan province;
(2) Analyzing the genetic diversity of the tree peony varieties in the south of the Yangtze river;
(3) Genetic relationship analysis of the tree peony varieties in the south of the Yangtze river;
(4) Molecular marker assisted breeding of tree peony in the south of the Yangtze river;
the tree peony varieties in the south of the Yangtze river are any one of the following 9 tree peony varieties: red lotus, poinsettia, yulouchun, dahua, paeonia ostii, red, xishi powder, purple hydrangea and Xiangxi powder.
The method for identifying the molecular of the tree peony variety in south China by utilizing the EST-SSR primer developed based on the water logging transcriptome sequence of the tree peony comprises the following steps:
(1) Extracting DNA of leaves of tree peony in south China by adopting an improved magnetic bead method, and extracting the DNA by adopting 0.4-0.6 g of sample for each sample;
(2) Taking the DNA of the leaves of the tree peony in the south China extracted in the step (1) as a template, and respectively carrying out PCR amplification by using P1-P20 pairs of primers in EST-SSR primers to obtain PCR amplification products;
(3) Detecting the PCR amplified product in the step (2) through 1.5% agarose electrophoresis, and if the product does not exist, performing secondary PCR amplification and secondary agarose detection, wherein the sample loading amount is 3 mu L; if the product exists, uniformly mixing 3 mu L of PCR amplification product with 5 mu LLoading Buffer, centrifuging, denaturing at 94 ℃ for 5min, freezing in an ice box for 1 min, performing vertical electrophoresis analysis on 6% denatured polyacrylamide gel, and observing after silver staining;
(4) And (3) reading the strip data by adopting software quantitone according to the silver staining result, counting the detection sites, and carrying out molecular identification of the tree peony varieties in the south of China according to the detection sites.
Preferably, in the step (2), the reaction system for PCR amplification is 20. Mu.L, comprising: ddH 2 O14.8. Mu.L, dNTP 0.4. Mu.L, buffer 2. Mu.L, 20. Mu.M upstream and downstream primers 0.3. Mu.L, DNA template 2. Mu.L, taq 0.2. Mu.L.
Preferably, in the step (2), the reaction procedure of the PCR amplification is: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s, renaturation at 54℃for 35s, extension at 72℃for 40s for 35 cycles; finally, the extension is carried out at 72 ℃ for 3min.
Preferably, in the step (4), when the molecular identification of the tree peony variety in the south of the Yangtze river is carried out, corresponding codes are assigned to amplification sites of each pair of primers in the tree peony sample, DNA bands are marked as 1, DNA-free bands are marked as 0, and the obtained mixture is converted into a 0,1 data matrix, and if the female parent of the sample to be detected is known, the male parent can be checked and judged according to whether the female parent has part or all fragments of the male parent.
Preferably, the tree peony species in the south of the river is any one of the following 9 tree peony species: red lotus, poinsettia, yulouchun, dahua, paeonia ostii, red, xishi powder, purple hydrangea and Xiangxi powder.
The invention has the following beneficial effects due to the adoption of the technical scheme:
the invention develops a set of EST-SSR primers based on the Paeonia ostii waterlogging transcriptome sequence, comprises 20 pairs of primers P1-P20, has the advantages of clear amplified strip, high polymorphism, good repeatability, strong universality and the like, fills up the blank of SSR markers of candidate genes related to Paeonia ostii waterlogging traits, enriches the quantity of the EST-SSR primers of Paeonia ostii, can utilize the EST-SSR primer set developed by the invention to carry out molecular identification, genetic diversity analysis, genetic relationship analysis, molecular marker assisted selection breeding and the like of the varieties of Paeonia ostii, and particularly provides a favorable technical means for early screening of waterlogging-tolerant varieties.
Drawings
FIG. 1 shows agarose electrophoresis results of amplified products of 9 varieties of Jiangnan peony by using primer pairs P1-P20 (9 varieties of Jiangnan peony are sequentially spotted in the sequence from left to right: hibiscus sabdariffa, yulouchun, darich, poinsettia, purple hydrangea, xiangxi powder, paeonia ostii, xishi powder and Paeonia ostii);
FIG. 2 is a PAGE electrophoresis of 9 varieties of Jiangnan peony on EST-SSR primer pairs P1-P20 (9 varieties of Jiangnan peony sample application sequences in the figure are, in order from left to right, red lotus, yulouchun, darich, poinsettia, purple hydrangea, xiangxi powder, paeonia ostii, xishi powder and Paeonia ostii);
FIG. 3 is a graph of cluster analysis of 9 varieties of S.jiangensis.
Detailed Description
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The experimental methods in the following examples are conventional methods unless otherwise specified. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Example 1 development of EST-SSR primers based on Paeonia ostii Lemonnieri Lepidium waterlogging transcriptome sequences
(1) The Paeonia ostii waterlogging pest transcriptome database is derived from high-throughput sequencing results of the inventor on Paeonia ostii waterlogging pest stress;
(2) Searching SSR sites of the database in the step (1) from http:// pgrc.ipk-gatersleben.de/MISA/download software MISA, and screening according to the following standard: SSR length is more than 12bp, amplified products are more than 200bp, the repetition number of dinucleotides is more than or equal to 6, the repetition number of trinucleotides is more than or equal to 5, and the repetition number of tetranucleotide, pentanucleotide and hexanucleotide is more than or equal to 4;
(3) And (3) designing primers on two sides of the SSR sequence screened in the step (2) by using Primer3 software, and finally determining 20 pairs of EST-SSR primers shown in the table 1:
table 1:20 pairs of EST-SSR primer information
According to the invention, 20 pairs of EST-SSR primers designed based on the Paeonia ostii waterlogging pest transcriptome sequence are utilized, the amplified bands are clear, the polymorphism is high, the repeatability is good, the universality is strong, the Paeonia ostii waterlogging pest trait related genes can be well identified, paeonia ostii waterlogging pest-resistant varieties are screened out, and the method has certain advantages in early identification of waterlogging pest-resistant germplasm.
Example 2 EST-SSR primer set P1-P20 for genetic diversity analysis of Oriental peony variety
(1) Extracting leaf genome DNA of 9 varieties of south-river peony, namely Hibiscus sabdariffa, paeonia ostii, sparassis crispa, porphyra tenera, yulouchun, xishi powder, paeonia ostii and Dachang;
(2) Taking the peony leaf DNA extracted in the step (1) as a template, and respectively carrying out PCR amplification by using 20 primer pairs of P1-P20 in the EST-SSR primer group; the reaction system for PCR amplification was 20. Mu.L, including: ddH 2 O14.8. Mu.L, dNTP 0.4. Mu.L, buffer 2. Mu.L, 20. Mu.M upstream and downstream primers 0.3. Mu.L, DNA template 2. Mu.L, taq 0.2. Mu.L; the reaction procedure for PCR amplification was: the reaction procedure for PCR amplification was: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s, renaturation at 54℃for 35s, extension at 72℃for 40s for 35 cycles; finally, the extension is carried out at 72 ℃ for 3min. Generally, the annealing temperature is fluctuating above and below 54 ℃.
(3) Detecting PCR amplified products by 1.5% agarose electrophoresis, wherein the sample loading amount is 3 mu L, the agarose electrophoresis results of 9 kinds of Jiangnan peony varieties amplified products by using the primer pair P1-P20 are shown in figure 1, and if no products exist, performing secondary PCR amplification and secondary agarose detection; if there is a product, 3. Mu.L of the PCR amplification product is mixed with 5. Mu.L of Loading Buffer. After centrifugation, denaturation is carried out for 5min at 94 ℃, vertical electrophoresis analysis is carried out on 6% denatured polyacrylamide gel after ice box freezing for 1 min, and after silver staining, the PAGE electrophoresis diagram of 9 varieties of the tree peony in the south of the Yangtze river on P1-P20 on the EST-SSR primer pair P1-P20 is shown in figure 2.
(4) According to silver staining results, the band data are read by adopting software quantitone, detection sites are counted, DNA bands are marked as 1 at the electrophoresis positions of PCR amplification products at certain same mobility positions of gel, no DNA bands are marked as 0, and then the DNA bands are converted into a 0,1 data matrix comparison result table shown in table 2. Genetic diversity analysis was performed using popkene 32 software according to the statistical results, and genetic differentiation sizes between the various populations were measured using the genetic identity I (genetic identity) and genetic distance D (genetic distance) of Nei. Based on the genetic similarity coefficient, the UPGMA method in NTSYSpcVersion2.10e software is used for carrying out cluster analysis on each sample, and a cluster analysis chart of 9 varieties of the tree peony in the south China is constructed, wherein the cluster analysis chart is shown in figure 3.
Table 2: 0,1 data matrix comparison result table of 9 kinds of Jiangnan peony varieties amplified results
Analysis of results: the result shows that the EST-SSR primer group developed based on the Paeonia ostii waterlogging transcriptome sequence has the advantages of clear amplified band, high polymorphism, good repeatability and the like. 20 pairs of primers have polymorphism in the genome DNA of the 9 varieties of the tree peony in the south of the Yangtze river, wherein the polymorphism ratio of 12 pairs of primers reaches 100%, and the polymorphism of 4 pairs of primers is more than 80%. The observed allele (Na) varied from 1.3333 to 2.0000 with an average value of 1.8307; effective allele (Ne) ranges from 1.0604 to 1.6926 with an average of 1.3884; the variation range of the Nei's gene diversity is 0.1481-0.3972, and the average value is 0.2561; the Shannon information index varies from 0.1096 to 0.5818 with an average value of 0.3720. Therefore, the 20 pairs of EST-SSR primers developed by the invention are effective primers and have universality, can be used for genetic diversity analysis, genetic relationship identification and the like of the tree peony varieties in the south of the Yangtze river, and particularly has strong advantages in early screening of waterlogging-tolerant varieties.
Example 3 identification of whether the father of the peony sample to be tested is the south-river peony variety "Hibiscus sabdariffa" using EST-SSR primer set developed based on Paeonia ostii waterlogging transcriptome sequence "
(1) Extracting leaf genome DNA of a peony sample to be detected and a south-river peony variety Hibiscus sabdariffa;
(2) Taking the peony leaf genome DNA extracted in the step (1) as a template, and respectively carrying out PCR amplification by using 20 primer pairs of P1-P20 in the EST-SSR primer group; the reaction system for PCR amplification was 20. Mu.L, including: ddH 2 O14.8. Mu.L, dNTP 0.4. Mu.L, buffer 2. Mu.L, 20. Mu.M upstream and downstream primers 0.3. Mu.L each, DNA template 2. Mu.L, taq 0.2. Mu.L; PCR amplification reaction processThe sequence is as follows: the reaction procedure for PCR amplification was: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s, renaturation at 54℃for 35s, extension at 72℃for 40s for 35 cycles; finally, the extension is carried out at 72 ℃ for 3min.
(3) Detecting the PCR amplification product by 1.5% agarose electrophoresis, and if the product does not exist, carrying out secondary PCR amplification and secondary agarose detection, wherein the sample loading amount is 3 mu L; if there is a product, 3. Mu.L of the PCR amplification product is mixed with 5. Mu.L of Loading Buffer. After centrifugation, denaturation was carried out at 94℃for 5min, ice box freezing for 1 min, vertical electrophoresis analysis was carried out on a 6% denatured polyacrylamide gel, and after silver staining, observation was carried out.
(4) According to silver staining results, software Quantityone is adopted to read strip data, detection sites are counted, corresponding codes are assigned to amplification sites of each primer pair in a peony sample to be detected and Hibiscus sabdariffa, DNA strips are marked as 1, DNA strips are marked as 0, and then the data are converted into a 0,1 data matrix comparison result table shown in table 3. According to the fact that the fragments of the offspring are derived from the male parent and the female parent, that is to say, the fragments of the offspring show part or all of the fragments of the male parent and the female parent at the same time, under the condition that the female parent of the sample to be detected is known, the male parent can be checked and judged according to whether the fragments of the offspring have part or all of the fragments of the male parent.
Table 3: comparison result table of 0,1 data matrix of amplification results of Hibiscus sabdariffa and peony sample to be detected
Analysis of results: according to the results in Table 2, the similarity of the amplified bands of the peony sample to be detected and the red lotus is high, and the father of the peony sample to be detected can be judged to be red lotus. Therefore, the EST-SSR primer group developed based on the Paeonia ostii waterlogging transcriptome sequence can be used for parent identification and detection of the Paeonia ostii variety.
The above embodiments are all preferred embodiments of the present invention, but are not limited thereto, and any other modifications, improvements, adaptations, etc. without departing from the spirit and scope of the present invention shall be included in the scope of the present application.
Sequence listing
SEQ ID NO.1:CTTTCGAGAACCGCATTTTC
SEQ ID NO.2:ATCGAAACACGACCCACTTC
SEQ ID NO.3:GAGCCGCGGTGATATATGTT
SEQ ID NO.4:CCGCGAAATTTCTTCGATAA
SEQ ID NO.5:TCCACGAACCAAACACTGAA
SEQ ID NO.6:TCTTTGTGATCTGGTGCTGC
SEQ ID NO.7:GAGAGAAGCTCCGTTGTTGG
SEQ ID NO.8:GAATTACAACACCCCATGCC
SEQ ID NO.9:AGGCCGAGCTCCACATGTAT
SEQ ID NO.10:TGCGGAGGATGAGTTCTTCT
SEQ ID NO.11:TACCTGTCTCGAACTCCCCA
SEQ ID NO.12:CTATGAGGGACCAGAGAGCG
SEQ ID NO.13:CCAGAGCCACATGCCTTATT
SEQ ID NO.14:ATGTCATCTGAGGTTTCGGC
SEQ ID NO.15:ACCTCCTTCTTTAGCGCTCC
SEQ ID NO.16:CCGCCAATTTCAGAGAGAGA
SEQ ID NO.17:TCCCACATTCTTGTGCAGAG
SEQ ID NO.18:GTTTGGGGAAATTGGGAACT
SEQ ID NO.19:CCGGGACTATTTTCTAGCCC
SEQ ID NO.20:CAAAACTCCATCAGCAGCAG
SEQ ID NO.21:TCTTGCCATTTCCTTGAAGC
SEQ ID NO.22:AAGGTGGCACGAGCATTAAC
SEQ ID NO.23:ATGGCCGTAGCATAGTGTCC
SEQ ID NO.24:CACAAGAGGCCAACACGTAA
SEQ ID NO.25:GCAGAGTCGATCCTGGCTAC
SEQ ID NO.26:GGGTACGTCTGCAACCTTGT
SEQ ID NO.27:AAAACGTGTGGCCTCAAAAC
SEQ ID NO.28:GCGACGAATCAGGAGAGAAC
SEQ ID NO.29:GAGAGAGATAGAGGGCGCAA
SEQ ID NO.30:TCACCTCCGATTCGTAAACC
SEQ ID NO.31:AGCCTTAATCGGTCGAACCT
SEQ ID NO.32:TTGTGCCAGACACCAAGAAG
SEQ ID NO.33:TCCATACGCATTCTTTTCCC
SEQ ID NO.34:GGAGCCCAAGCGTAACAATA
SEQ ID NO.35:AAGGCCGGTAAGAAGCTACC
SEQ ID NO.36:TTCCCTAGAGGTGATGGTGG
SEQ ID NO.37:TCGAGTACCTTGAGGAGCGT
SEQ ID NO.38:CAGATGGGCTTCTGCTTGTT
SEQ ID NO.39:GAACAAAAGCCTTGCCTCTG
SEQ ID NO.40:TACGATAGGCCACAACCACA
Claims (7)
1. EST-SSR primers developed based on Paeonia ostii waterlogging transcriptome sequences are characterized by comprising 20 pairs of primers:
the primer pair P1 has the sequences shown as SEQ ID NO.1 and SEQ ID NO. 2;
primer pair P2, the sequences of which are shown as SEQ ID NO.3 and SEQ ID NO. 4;
the primer pair P3 has the sequences shown in SEQ ID NO.5 and SEQ ID NO. 6;
the primer pair P4 has the sequences shown as SEQ ID NO.7 and SEQ ID NO. 8;
primer pair P5, the sequences of which are shown as SEQ ID NO.9 and SEQ ID NO. 10;
primer pair P6, the sequences of which are shown as SEQ ID NO.11 and SEQ ID NO. 12;
the primer pair P7 has the sequences shown as SEQ ID NO.13 and SEQ ID NO. 14;
primer pair P8, the sequences of which are shown as SEQ ID NO.15 and SEQ ID NO. 16;
the primer pair P9 has the sequences shown as SEQ ID NO.17 and SEQ ID NO. 18;
the primer pair P10 has the sequences shown as SEQ ID NO.19 and SEQ ID NO. 20;
the primer pair P11 has the sequences shown as SEQ ID NO.21 and SEQ ID NO. 22;
the primer pair P12 has the sequences shown as SEQ ID NO.23 and SEQ ID NO. 24;
the primer pair P13 has the sequences shown as SEQ ID NO.25 and SEQ ID NO. 26;
the primer pair P14 has the sequences shown as SEQ ID NO.27 and SEQ ID NO. 28;
the primer pair P15 has the sequences shown as SEQ ID NO.29 and SEQ ID NO. 30;
the primer pair P16 has the sequences shown as SEQ ID NO.31 and SEQ ID NO. 32;
the primer pair P17 has the sequences shown as SEQ ID NO.33 and SEQ ID NO. 34;
the primer pair P18 has the sequences shown as SEQ ID NO.35 and SEQ ID NO. 36;
the primer pair P19 has the sequences shown as SEQ ID NO.37 and SEQ ID NO. 38;
the primer pair P20 has the sequences shown as SEQ ID NO.39 and SEQ ID NO. 40.
2. Use of an EST-SSR primer developed based on an Paeonia ostii waterlogging transcriptome sequence as set forth in claim 1 in any one of the following (1) - (4):
(1) Molecular identification of a tree peony variety in Jiangnan province;
(2) Analyzing the genetic diversity of the tree peony varieties in the south of the Yangtze river;
(3) Genetic relationship analysis of the tree peony varieties in the south of the Yangtze river;
(4) Molecular marker assisted breeding of tree peony in the south of the Yangtze river;
the tree peony varieties in the south of the Yangtze river are any one of the following 9 tree peony varieties: red lotus, poinsettia, yulouchun, dahua, paeonia ostii, red, xishi powder, purple hydrangea and Xiangxi powder.
3. A method for molecular identification of a tree peony variety in south China by using the EST-SSR primer developed based on the water logging transcriptome sequence of tree peony according to claim 1, which is characterized by comprising the following steps:
(1) Extracting DNA of leaves of tree peony in south China by adopting an improved magnetic bead method, and extracting the DNA by adopting 0.4-0.6 g of sample for each sample;
(2) Taking the DNA of the leaves of the tree peony in the south China extracted in the step (1) as a template, and respectively carrying out PCR amplification by using P1-P20 pairs of primers in EST-SSR primers to obtain PCR amplification products;
(3) Detecting the PCR amplified product in the step (2) through 1.5% agarose electrophoresis, and if the product does not exist, performing secondary PCR amplification and secondary agarose detection, wherein the sample loading amount is 3 mu L; if the product exists, 3 mu L of PCR amplification product is uniformly mixed with 5 mu L of Loading Buffer, after centrifugation, denaturation is carried out for 5min at 94 ℃, vertical electrophoresis analysis is carried out on 6% denatured polyacrylamide gel after ice box freezing for 1 min, and observation is carried out after silver staining;
(4) And (3) reading the strip data by adopting software quantitone according to the silver staining result, counting the detection sites, and carrying out molecular identification of the tree peony varieties in the south of China according to the detection sites.
4. The method for molecular identification of a variety of Paeonia ostii according to claim 3, wherein in the step (2), the reaction system for PCR amplification is 20. Mu.L, comprising: ddH 2 O14.8. Mu.L, dNTP 0.4. Mu.L, buffer 2. Mu.L, 20. Mu.M upstream and downstream primers 0.3. Mu.L, DNA template 2. Mu.L, taq 0.2. Mu.L.
5. The method for molecular identification of a variety of Paeonia ostii according to claim 3, wherein in the step (2), the reaction procedure of PCR amplification is as follows: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s, renaturation at 54℃for 35s, extension at 72℃for 40s for 35 cycles; finally, the extension is carried out at 72 ℃ for 3min.
6. The method for molecular identification of a tree peony variety based on EST-SSR primers developed by a tree peony waterlogged transcriptome sequence according to claim 3, wherein in the step (4), when molecular identification of the tree peony variety is carried out, corresponding codes are assigned to amplification sites of each pair of primers in a tree peony sample, DNA bands are marked as 1, no DNA bands are marked as 0, the obtained mixture is converted into a 0,1 data matrix, and under the condition that the female parent of a sample to be detected is known, the male parent can be checked and judged according to whether the fragments of the offspring have part or all of the male parent.
7. The method for molecular identification of a tree peony variety in south China according to claim 3 based on EST-SSR primers developed by water logging transcriptome sequences of tree peony, wherein the tree peony variety in south China is any one of the following 9 tree peony varieties: red lotus, poinsettia, yulouchun, dahua, paeonia ostii, red, xishi powder, purple hydrangea and Xiangxi powder.
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