CN113981124A - Oriental cherry SSR molecular marker primer and application thereof in identification of 42 oriental cherry varieties - Google Patents
Oriental cherry SSR molecular marker primer and application thereof in identification of 42 oriental cherry varieties Download PDFInfo
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
Abstract
The invention discloses a oriental cherry SSR molecular marker primer and application thereof in identification of 42 oriental cherry varieties, wherein the invention independently develops oriental cherry SSR related molecular marker primers from tail leaf oriental cherry genome data and 23 pairs of primers screened in the literature, wherein the total number of common Prunus SSR markers is 39 pairs, the primers are amplified and subjected to polymorphism screening, the common 42 oriental cherry varieties are subjected to genetic diversity research, 16 pairs of primers are screened, 2 pairs of primers PTCR22 and SC7YT with the highest distinguishing rate are selected from the 16 pairs of primers and combined, the combination can identify 39 of the 42 oriental cherry varieties, the distinguishing rate can reach 92.9 percent, and then any primer in the rest 14 pairs of primers is combined randomly, so that all samples can be distinguished. The optimal variety identification primer combination PTCR22+ SC7YT + PS12A02 is screened out, and the SSR characteristic fingerprint of the oriental cherry variety is constructed, so that an important basis is provided for oriental cherry variety identification and molecular marker-assisted breeding.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a oriental cherry SSR molecular marker primer and application thereof in identification of 42 oriental cherry varieties.
Background
Cherry blossom is a general name of plants of Prunus (Prunus senula) of Rosaceae (Rosaceae) and Prunus (subgenus Cerasus). Cherry blossom is a world famous flower tree, and flowers are full of trees when blooming, and fall into colorful flowers when not blooming; the product has wide development and utilization prospect as fruit trees with higher economic value and elegant garden flowers and trees. In the last hundred years, more than 600 oriental cherry horticultural varieties are cultivated through natural variation screening and artificial hybridization. However, since the morphological similarity between many varieties is high, the variety sources are not described in detail, and the phenomena of synonyms and foreign homonyms occur frequently; most oriental cherry flowers are firstly blossomed and then are in different phases, are easy to hybridize, have rich and diverse morphological variations, long life history and relatively short flowering phase, and lack a rapid and accurate identification method (wanxian honor, 2014, Shi et al, 2013). In recent centuries, more than 600 sakura horticultural varieties (Yuan Dong Ming et al, 2018) were obtained by natural mutation screening and hybridization. For the horticultural varieties, taxonomists propose three-level and five-level classification standards and classification standards of flowering phases and flower colors according to morphological characteristics of trees, flowers, fruits, leaves, winter buds and the like (Shiyu sister-in-law, 2007, Teng Junxiong, 2009, Zhang Qiong and the like, 2012). As different scholars have different judgment standards for morphological characters, characters among varieties are crossed and overlapped, and strong plasticity exists in different environments, the morphological identification is difficult, so that the phenomena of variety name confusion, synonym, foreign body synonym and the like frequently occur.
The genetic diversity of plant varieties is mainly reflected by the polymorphism of genetic markers, and molecular markers are genetic markers based on the polymorphism of nucleic acids. It is not interfered by tissue type, development period, environmental conditions, etc., has the advantages of extremely large quantity, high polymorphism, etc., and is an ideal genetic marker. Molecular markers have been widely used in germplasm resource research, hybrid identification, genetic map construction, target gene localization, genetic diversity, genetic relationship research, and marker-assisted selective breeding. Wherein, microsatellite (simple sequence repeat, SSR) is a simple repeat sequence which is composed of 2-6 nucleotide tandem repeat segments and is uniformly distributed in a genome; has the advantages of high polymorphism, good stability, multiple alleles, codominance, abundant quantity, good genome coverage, simple operation and the like. Has been applied to the research of genetic relationship, genetic diversity analysis, variety fingerprint drawing, variety identification, DNA fingerprint construction and the like of plants such as Chinese plum (Prunus salicina), peach (Prunus persica), Lycium ruthenicum (Lycium ruthenicum), loquat (Eriobotrya japonica) and the like. The early stage of the research group utilizes 2 pairs of SSR molecular markers developed from genome data of cerasus serrulata to successfully identify 20 varieties of cerasus serrulata, only two primers are utilized to identify 20 varieties of cerasus serrulata, and great progress is achieved. Therefore, the invention further develops molecular marker primers on the basis of the previous research, and lays a foundation for identifying more cherry blossom varieties. Carry out the genetic diversity research to the common 42 oriental cherry variety materials of collection, continue to utilize the SSR mark that develops in the cerasus serrulata genome data of tail leaf and the polymorphic primer that the part was gathered and was screened out equally, appraise these 42 oriental cherry varieties, screen the optimal variety and appraise primer combination to establish oriental cherry variety SSR characteristic fingerprint, provide important basis for oriental cherry variety appraisal and molecular marker assisted breeding.
Disclosure of Invention
One of the purposes of the invention is to provide a oriental cherry SSR molecular marker primer.
The invention also aims to provide the function of the oriental cherry SSR molecular marker primer in oriental cherry variety identification.
The invention also aims to provide a kit for identifying oriental cherry.
In order to achieve the purpose, the invention provides the following technical scheme:
oriental cherry SSR molecular marker primers are at least 3 pairs of primer combinations, specifically, PTCR22, SC7YT, ZJF01, ZJF02, ZJF03, BPPCT008, BPPCT040, CPSCT021, EMPaS11A, M9a, MA016b, PS12A02, MA007a, PceGA25, UDP97-403, UDP96-005, PTCR22 and SC7YT in 14 pairs, wherein the nucleotide sequences of the forward primer and the reverse primer are specifically as follows:
(1) ZJF 01: the sequence of the forward primer is shown as SEQ ID NO.1, and the sequence of the reverse primer is shown as SEQ ID NO. 2;
(2) ZJF 02: the sequence of the forward primer is shown as SEQ ID NO.3, and the sequence of the reverse primer is shown as SEQ ID NO. 4;
(3) ZJF 03: the sequence of the forward primer is shown as SEQ ID NO.5, and the sequence of the reverse primer is shown as SEQ ID NO. 6;
(4) BPPCT 008: the sequence of the forward primer is shown as SEQ ID NO.7, and the sequence of the reverse primer is shown as SEQ ID NO. 8;
(5) BPPCT 040: the sequence of the forward primer is shown as SEQ ID NO.9, and the sequence of the reverse primer is shown as SEQ ID NO. 10;
(6) CPSCT 021: the sequence of the forward primer is shown as SEQ ID NO.11, and the sequence of the reverse primer is shown as SEQ ID NO. 12;
(7) EMPaS 11A: the sequence of the forward primer is shown as SEQ ID NO.13, and the sequence of the reverse primer is shown as SEQ ID NO. 14;
(8) m9 a: the sequence of the forward primer is shown as SEQ ID NO.15, and the sequence of the reverse primer is shown as SEQ ID NO. 16;
(9) MA016 b: the sequence of the forward primer is shown as SEQ ID NO.17, and the sequence of the reverse primer is shown as SEQ ID NO. 18;
(10) PS12a 02: the sequence of the forward primer is shown as SEQ ID NO.19, and the sequence of the reverse primer is shown as SEQ ID NO. 20;
(11) MA007 a: the sequence of the forward primer is shown as SEQ ID NO.21, and the sequence of the reverse primer is shown as SEQ ID NO. 22;
(12) PceGA 25: the sequence of the forward primer is shown as SEQ ID NO.23, and the sequence of the reverse primer is shown as SEQ ID NO. 24;
(13) UDP 97-403: the sequence of the forward primer is shown as SEQ ID NO.25, and the sequence of the reverse primer is shown as SEQ ID NO. 26;
(14) UDP 96-005: the sequence of the forward primer is shown as SEQ ID NO.27, and the sequence of the reverse primer is shown as SEQ ID NO. 28;
(15) PTCR 22: the sequence of the forward primer is shown as SEQ ID NO.29, and the sequence of the reverse primer is shown as SEQ ID NO. 30;
(16) SC7 YT: the sequence of the forward primer is shown as SEQ ID NO.31, and the sequence of the reverse primer is shown as SEQ ID NO. 32.
The application of the oriental cherry SSR molecular marker primer in oriental cherry variety identification is that 16 pairs of developed SSR primers and a total of 39 pairs of primers of 23 pairs of common Prunus SSR markers screened in the literature are amplified and subjected to polymorphism screening. Finally, 16 pairs of primers are screened out, so that 42 varieties can be distinguished and identified, and the selected oriental cherry varieties are shown in table 1.
The 2 pairs of primers PTCR22 and SC7YT with the highest discrimination rate are selected from 16 pairs of primers and combined, 39 of 42 cherry blossom varieties can be identified by the combination, and the discrimination rate can reach 92.9%. In which, the tail leaf cherry could not be amplified to obtain a band, and P17 red Pink cherry could not be distinguished from P41 mountain cherry. Any one of the remaining 14 pairs of primers can be arbitrarily combined to distinguish all samples. The optimal combination is PTCR22+ SC7YT + PS12A02, taking into account the amplification of cerasus serrulata and the polymorphism of each primer.
Table 142 oriental cherry variety information
A kit for identifying sakura varieties comprises PTCR22, SC7YT and sakura SSR molecular marker primers combined by any 1-14 pairs of ZJF01, ZJF02, ZJF03, BPPCT008, BPPCT040, CPSCT021, EMPaS11A, M9a, MA016b, PS12A02, MA007a, PceGA25, UDP97-403, UDP96-005, PTCR22 and SC7YT in 14 pairs.
The invention has the beneficial effects that:
according to the invention, molecular marker primers related to cherry blossom SSR are independently developed from cerasus genome data, 23 pairs of commonly-used SSR markers screened in the literature are subjected to amplification and polymorphism screening, genetic diversity research is carried out on common 42 cerasus varieties, 16 pairs of primers are screened, 2 pairs of primers PTCR22 and SC7YT with the highest distinguishing rate are selected from the 16 pairs of primers to be combined, the combination can identify 39 of the 42 cerasus varieties, the distinguishing rate can reach 92.9%, and then any primer in the rest 14 pairs of primers is combined randomly, so that all samples can be distinguished. The optimal variety identification primer combination PTCR22+ SC7YT + PS12A02 is screened out, and the SSR characteristic fingerprint of the oriental cherry variety is constructed, so that an important basis is provided for oriental cherry variety identification and molecular marker-assisted breeding. Enriches the molecular marker identification types of the oriental cherry varieties and improves the identification efficiency.
Drawings
FIG. 1 is a 42 cherry blossom variety clustering dendrogram constructed based on 16 pairs of SSR markers.
Detailed Description
The principles and features of this invention are described below in conjunction with the following drawings, which are set forth by way of illustration only and are not intended to limit the scope of the invention.
Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. The test methods in the following examples are conventional methods unless otherwise specified. Unless otherwise indicated, all reagents and materials used are commercially available.
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. In addition, any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention. The preferred embodiments and materials described herein are intended to be exemplary only.
1 materials and methods
1.1 test site and test Material
The test field is arranged in a cherry breeding garden of scientific research institute of forestry in Zhejiang province, 30 degrees of north latitude 13 '12 degrees and 120 degrees of east longitude 01' 11 degrees. The method belongs to subtropical monsoon climate areas, is clear in four seasons and moderate in annual temperature, and has the average annual temperature of 15.9-17.0 ℃, the extreme highest temperature of 39.8-42.9 ℃ and the extreme lowest temperature of-7.1-15.0 ℃; the illumination is sufficient, and the annual average sunshine hours are 1710-2100 h; air is wet, and the annual average relative humidity is 76% -81%; the rainfall is sufficient, and the average annual rainfall is 980-2000 mm; and the frost period is 199-328 days.
In 12 months of 2015, cherry varieties were collected from cherry breeding bases of 7 provinces of Fujian province, Guizhou province, Shandong province, Hubei province, Shanghai city, Yunnan province and Zhejiang province and planted in cherry breeding gardens of scientific research institute of forestry of Zhejiang province (see Table 1). Collecting DNA samples of the varieties, respectively placing fresh and disease-free young leaves in self-sealing bags filled with silica gel for rapid drying, and then storing in a freezer at-20 ℃ for later use.
1.2 DNA extraction method
208 parts of genomic DNA of a test material was extracted using a rapid plant genomic DNA extraction kit (model DP3112) manufactured by Bioteke, and a sample was pretreated with a buffer solution before extraction to remove a large amount of impurities such as polysaccharides and pigments. DNA quality was checked by 1% agarose gel electrophoresis, and DNA concentration was determined by Nanodrop ultramicro spectrophotometer and diluted to 50 ng. mu.L-1And storing in a refrigerator at-20 deg.C for use.
1.3 SSR primer development
Based on the sequencing result of the whole genome of cerasus serrulata, Primer 5.0 software was used to develop SSR primers. 16 pairs of developed SSR primers and 23 pairs of common SSR markers of Prunus screened in the literature are amplified and subjected to polymorphism screening, wherein the total number of the primers is 39. Finally, 16 pairs of primers are screened to identify 42 varieties (SSR primer information is shown in Table 2). The primers were synthesized by Biotechnology engineering (Shanghai) Inc.
TABLE 2 SSR primer information
1.4 PCR amplification
The PCR amplification system was 25. mu.L:50ng·μL-1DNA template 2. mu.L, 2 XTSINGKE Master MIX (blue) 12.5. mu.L (produced by Biotech Co., Ltd., Pryotokyo Opticeae), forward primer 0.5. mu.L, reverse primer 0.5. mu.L, ddH2O9.5. mu.L. The PCR reaction was carried out on an Applied Biosystem Veriti Thermal Cycler PCR (Gene Company Limited, hong Kong, China) instrument, with the following reaction program: 5min at 95 ℃; 35 cycles of 45s at 95 ℃, 45s at 55-58 ℃ and 45s at 72 ℃; 7min at 72 ℃; finally, the mixture is stored at 4 ℃. The PCR amplification products were screened for clear products by electrophoresis on a 2% agarose gel.
1.5 capillary electrophoresis
The clearly stabilized PCR product was pipetted 1. mu.L through Qsep100TMAnd (3) carrying out capillary electrophoresis detection by using a full-automatic nucleic acid protein analyzer. The cassette used for capillary electrophoresis was S1 high resolution card holder, the molecular Size standard reference was 1K Size marker, both produced by Guangzhou Jiyuan Biotech, Inc.
1.6 data analysis
Comparing and analyzing various kinds of peak values output by the full-automatic nucleic acid protein analyzer to obtain the size of fragments, and analyzing data by using GeneMarker 2.2.0 software. Sequence length data were converted to genotype using Covert software. Calculating the number of alleles (Na) of SSR primer, and observing heterozygosity (H)o) Nei's genetic diversity (H), Shannon's information index (I) and Polymorphism Information Content (PIC), PopGene 32 software was used for statistical analysis. The MG software is used for converting the numerical value of the fragment size into a0 and 1 data matrix, Ntsys 2.1 software is used for calculating the genetic similarity Coefficient (Coefficient) among samples, and the UPGMA method is used for clustering.
2 results
2.1 molecular marker polymorphism analysis
Detecting the extracted DNA by using a NanoDrop ultramicro spectrophotometer and 1.0% agarose gel electrophoresis, wherein A260/A280 of leaf extracts of 24 oriental cherry varieties are all between 1.8 and 2.0, A260/A230 is more than 2.0, and the concentration range of the DNA is 230 ng.mu.L-1-550ng·μL-1The contents of protein, pigment, phenolic substances and the like in the extracted DNA sample are low, the purity of the DNA sample is high, and the subsequent detection requirements can be met. Randomly selecting 6 sakura varieties of Yuethai cherry, eight-red-branch Sakura, Sakura kukura, Yinhe cherry, Dayu cherry and Dahan cherry, and carrying out polymorphism screening on the developed 16 pairs of SSR primers and 39 pairs of SSR primers screened in the literature to obtain 16 pairs of polymorphism primers (Table 2) with clear, stable and good repeatability amplified bands.
The 16 pairs of SSR primers are used for respectively carrying out amplification and capillary electrophoresis on the genome DNA of 42 oriental cherry varieties to obtain 8-36 polymorphic alleles detected by different primers, 17.5 polymorphic alleles can be detected by each pair of primers on average, and the effective alleles are 9.21 on average. The primer SC7YT has the most polymorphic allele factors and effective allele factors amplified for all varieties, and the primer can better reflect the difference of different varieties. The observed degree of hybridization and the observed degree of heterozygosity of 16 pairs of primers are 0.02 to 0.98, the desired degree of hybridization is 0.03 to 0.37, and the desired degree of heterozygosity is 0.63 to 0.97. Shannon's information index is 1.23-3.38, average is 2.30; the Nei's genetic diversity index was 0.63-0.96, with ZJF03 highest both for the Shannon diversity index and the Nei's genetic diversity (Table 3).
TABLE 316 genetic information for SSR markers in 42 oriental cherry varieties
2.2 discrimination of Oriental cherry varieties by molecular markers
According to the analysis of the genotypes of all varieties of oriental cherry, the varieties which can be distinguished by 16 pairs of SSR primers are different from 4 to 30, and the average number is 13.12. The number of varieties distinguished by M9a and MA016b is minimum, the distinguishing rate is 9.52%, the primer M9a can distinguish 4 varieties of cerasus campanulata excellent plant, cerasus serrulata, cerasus heaven and cerasus serrulata, and the primer MA016b can distinguish 4 varieties of cerasus serrulata, cerasus sinense, cerasus pini-virens and cerasus serrulata. The primer SC7YT has the highest distinguishing rate which reaches 71.43 percent and can distinguish 30 varieties; secondly, the primers PTCR22 and ZJF03 show that the distinguishing rate is 69.05 percent and 61.90 percent respectively, and 29 varieties and 26 varieties can be distinguished respectively.
TABLE 4 differentiation of Oriental cherry varieties by molecular markers
2.3 SSR-marker-based oriental cherry variety genetic relationship analysis
The genetic similarity coefficient range of 42 oriental cherry varieties is 0.80-0.94, wherein the similarity of Chimaphila arenarium and Chimaphila caudal pedunculata is the lowest, which indicates that the two varieties have the farthest genetic relationship. The similarity of the cerasus serrulata and the cerasus serrulata, the cerasus serrulata and the cerasus serrulata is the highest; the green cherry is a variety bred from the bean cherry, and the American cherry and the Jiye from the same parents prove that the 16 pairs of primers selected by people can correctly reflect the genetic relationship among the varieties to a certain extent. When the genetic similarity coefficient is 0.852, the 42 oriental cherry varieties are gathered together; when the genetic similarity coefficient was 0.94, the 42 cherry blossom varieties were grouped into 42 groups, which were completely distinguishable (FIG. 1).
2.4 screening of optimal primer combinations
The 2 pairs of primers PTCR22 and SC7YT with the highest discrimination rate are selected from 16 pairs of primers and combined, 39 of 42 cherry blossom varieties can be identified by the combination, and the discrimination rate can reach 92.9%. In which, the tail leaf cherry could not be amplified to obtain a band, and P17 red Pink cherry could not be distinguished from P41 mountain cherry. Any one of the remaining 14 pairs of primers can be arbitrarily combined to distinguish all samples. The optimal combination of the 42 cherry varieties is identified as PTCR22+ SC7YT + PS12A02 by comprehensively considering the amplification condition of the cerasus serrulata and the polymorphism of each primer.
2.5 identification of SSR characteristic fingerprint and variety of oriental cherry variety
42 different genotypes were detected out of 42 sakura varieties based on the primer combination PTCR22+ SC7YT + PS12A 02. Based on the 3 pairs of primers and the corresponding genotypes of each variety, the combination of (primer _ genotype/primer _ genotype) is used as the SSR characteristic fingerprints of 42 oriental cherry varieties, and specifically, referring to Table 5, molecular basis is provided for identification of each oriental cherry variety.
SSR characteristic fingerprints (primer + genotype) of table 542 oriental cherry varieties
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
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<213> Prunus dielsiana
<400> 12
tccatatctc ctcctgcttg a 21
<210> 13
<211> 20
<212> DNA
<213> Prunus dielsiana
<400> 13
accactttga ggaacttggg 20
<210> 14
<211> 20
<212> DNA
<213> Prunus dielsiana
<400> 14
ctgcctggaa gagcaataac 20
<210> 15
<211> 20
<212> DNA
<213> Prunus dielsiana
<400> 15
gccgaaaccc taggtgagcg 20
<210> 16
<211> 20
<212> DNA
<213> Prunus dielsiana
<400> 16
caggatgctt gcggtgcttg 20
<210> 17
<211> 20
<212> DNA
<213> Prunus dielsiana
<400> 17
tggctggtgg agacggagga 20
<210> 18
<211> 20
<212> DNA
<213> Prunus dielsiana
<400> 18
atgataccca gcctcccggg 20
<210> 19
<211> 18
<212> DNA
<213> Prunus dielsiana
<400> 19
gccaccaatg gttcttcc 18
<210> 20
<211> 18
<212> DNA
<213> Prunus dielsiana
<400> 20
agcaccagat gcacctga 18
<210> 21
<211> 20
<212> DNA
<213> Prunus dielsiana
<400> 21
gtgcatcgtt aggaactgcc 20
<210> 22
<211> 20
<212> DNA
<213> Prunus dielsiana
<400> 22
gcccctgaga tacaactgca 20
<210> 23
<211> 24
<212> DNA
<213> Prunus dielsiana
<400> 23
gcaattcgag ctgtatttca gatg 24
<210> 24
<211> 24
<212> DNA
<213> Prunus dielsiana
<400> 24
cagttggcgg ctatcatgtc ttac 24
<210> 25
<211> 20
<212> DNA
<213> Prunus dielsiana
<400> 25
ctggcttaca actcgcaagc 20
<210> 26
<211> 20
<212> DNA
<213> Prunus dielsiana
<400> 26
cgtcgaccaa ctgagactca 20
<210> 27
<211> 20
<212> DNA
<213> Prunus dielsiana
<400> 27
gtaacgctcg ctaccacaaa 20
<210> 28
<211> 19
<212> DNA
<213> Prunus dielsiana
<400> 28
cctgcatatc accacccag 19
<210> 29
<211> 21
<212> DNA
<213> Prunus dielsiana
<400> 29
tgtgctaatg ccaaaaatac c 21
<210> 30
<211> 20
<212> DNA
<213> Prunus dielsiana
<400> 30
acatgcattt caacccactc 20
<210> 31
<211> 19
<212> DNA
<213> Prunus dielsiana
<400> 31
tgcatgagaa acttgtggc 19
<210> 32
<211> 19
<212> DNA
<213> Prunus dielsiana
<400> 32
tgcatgagaa acttgtggc 19
Claims (6)
1. Oriental cherry SSR molecular marker primers are at least 3 pairs of primer combinations, specifically, PTCR22, SC7YT, ZJF01, ZJF02, ZJF03, BPPCT008, BPPCT040, CPSCT021, EMPaS11A, M9a, MA016b, PS12A02, MA007a, PceGA25, UDP97-403, UDP96-005, PTCR22 and SC7YT in 14 pairs, wherein the nucleotide sequences of the forward primer and the reverse primer are specifically as follows:
(1) ZJF 01: the sequence of the forward primer is shown as SEQ ID NO.1, and the sequence of the reverse primer is shown as SEQ ID NO. 2;
(2) ZJF 02: the sequence of the forward primer is shown as SEQ ID NO.3, and the sequence of the reverse primer is shown as SEQ ID NO. 4;
(3) ZJF 03: the sequence of the forward primer is shown as SEQ ID NO.5, and the sequence of the reverse primer is shown as SEQ ID NO. 6;
(4) BPPCT 008: the sequence of the forward primer is shown as SEQ ID NO.7, and the sequence of the reverse primer is shown as SEQ ID NO. 8;
(5) BPPCT 040: the sequence of the forward primer is shown as SEQ ID NO.9, and the sequence of the reverse primer is shown as SEQ ID NO. 10;
(6) CPSCT 021: the sequence of the forward primer is shown as SEQ ID NO.11, and the sequence of the reverse primer is shown as SEQ ID NO. 12;
(7) EMPaS 11A: the sequence of the forward primer is shown as SEQ ID NO.13, and the sequence of the reverse primer is shown as SEQ ID NO. 14;
(8) m9 a: the sequence of the forward primer is shown as SEQ ID NO.15, and the sequence of the reverse primer is shown as SEQ ID NO. 16;
(9) MA016 b: the sequence of the forward primer is shown as SEQ ID NO.17, and the sequence of the reverse primer is shown as SEQ ID NO. 18;
(10) PS12a 02: the sequence of the forward primer is shown as SEQ ID NO.19, and the sequence of the reverse primer is shown as SEQ ID NO. 20;
(11) MA007 a: the sequence of the forward primer is shown as SEQ ID NO.21, and the sequence of the reverse primer is shown as SEQ ID NO. 22;
(12) PceGA 25: the sequence of the forward primer is shown as SEQ ID NO.23, and the sequence of the reverse primer is shown as SEQ ID NO. 24;
(13) UDP 97-403: the sequence of the forward primer is shown as SEQ ID NO.25, and the sequence of the reverse primer is shown as SEQ ID NO. 26;
(14) UDP 96-005: the sequence of the forward primer is shown as SEQ ID NO.27, and the sequence of the reverse primer is shown as SEQ ID NO. 28;
(15) PTCR 22: the sequence of the forward primer is shown as SEQ ID NO.29, and the sequence of the reverse primer is shown as SEQ ID NO. 30;
(16) SC7 YT: the sequence of the forward primer is shown as SEQ ID NO.31, and the sequence of the reverse primer is shown as SEQ ID NO. 32.
2. The oriental cherry SSR molecular marker primer according to claim 1, wherein the molecular marker primer is a combination of three pairs of PTCR22, SC7YT and PS12A02, and the nucleotide sequences of the forward primer and the reverse primer are as follows:
(1) PTCR 22: the sequence of the forward primer is shown as SEQ ID NO.29, and the sequence of the reverse primer is shown as SEQ ID NO. 30;
(2) SC7 YT: the sequence of the forward primer is shown as SEQ ID NO.31, and the sequence of the reverse primer is shown as SEQ ID NO. 32;
(3) PS12a 02: the sequence of the forward primer is shown as SEQ ID NO.19, and the sequence of the reverse primer is shown as SEQ ID NO. 20.
3. The oriental cherry SSR molecular marker primer is characterized in that the molecular marker primer is ZJF03, and the nucleotide sequences of a forward primer and a reverse primer are as follows: the sequence of the forward primer is shown as SEQ ID NO.5, and the sequence of the reverse primer is shown as SEQ ID NO. 6.
4. The use of oriental cherry SSR molecular marker primers according to claim 1 or 2 in the identification of 42 oriental cherry varieties, wherein the varieties are 42 as follows:
5. the use of oriental cherry SSR molecular marker primers according to claim 3 for identifying 26 oriental cherry varieties, wherein the varieties are 26 as follows:
6. a kit for identifying oriental cherry varieties, which comprises oriental cherry SSR molecular marker primers of any one of claims 1-3.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106434646A (en) * | 2016-11-30 | 2017-02-22 | 宁波城市职业技术学院 | Four pairs of EST-SSR (Expressed Sequence Tag-Simple Sequence Repeat) primers as well as preparation method thereof and application thereof to construction of fingerprint map of cerasus plants |
| CN106957914A (en) * | 2017-04-08 | 2017-07-18 | 中国农业科学院郑州果树研究所 | The authentication method of cherry variety |
| CN111349717A (en) * | 2020-05-06 | 2020-06-30 | 北京市林业果树科学研究院 | SSR (simple sequence repeat) markers of sweet cherry stock resources and fingerprint spectrum database thereof |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106434646A (en) * | 2016-11-30 | 2017-02-22 | 宁波城市职业技术学院 | Four pairs of EST-SSR (Expressed Sequence Tag-Simple Sequence Repeat) primers as well as preparation method thereof and application thereof to construction of fingerprint map of cerasus plants |
| CN106957914A (en) * | 2017-04-08 | 2017-07-18 | 中国农业科学院郑州果树研究所 | The authentication method of cherry variety |
| CN111349717A (en) * | 2020-05-06 | 2020-06-30 | 北京市林业果树科学研究院 | SSR (simple sequence repeat) markers of sweet cherry stock resources and fingerprint spectrum database thereof |
Non-Patent Citations (2)
| Title |
|---|
| Z.TURKOGLU等: "Simple sequence repeat-based assessment of genetic relationships among Prunus rootstocks", GENETICS AND MOLECULAR RESEARCH, vol. 9, no. 4, pages 4 * |
| 张文昕: "樱属植物茎、叶结构及SSR遗传多样性分析", 中国优秀硕士学位论文全文数据库 农业科技辑, pages 4 * |
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