CN112695124A - Phalaenopsis SSR molecular marker primer composition and application thereof - Google Patents
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Abstract
The invention provides a phalaenopsis SSR molecular marker primer composition, a fingerprint code and application thereof in phalaenopsis germplasm resource identification, belonging to the technical field of molecular biology. The invention provides 20 SSR molecular markers of phalaenopsis and amplification primer compositions thereof, analyzes genetic diversity of 38 different germplasm resources in phalaenopsis and constructs a molecular identity card by utilizing TP-M13-SSR technology, can accurately, efficiently and stably identify the phalaenopsis germplasm resources, and lays a foundation for phalaenopsis germplasm resource identification, genetic relationship analysis, character gene positioning, molecular marker assisted breeding, phalaenopsis related molecular research and the like.
Description
Technical Field
The invention relates to the technical field of molecular biology, in particular to a phalaenopsis SSR molecular marker primer composition, a fingerprint code and application thereof in identifying phalaenopsis germplasm resources.
Background
Phalaenopsis (Phalaenopsis), also known as butterfly orchid, is an evergreen herbaceous plant of Phalaenopsis (Phalaenopsis) in Orchidaceae, is one of the most famous ornamental species in world flowers, and has high economic value. The phalaenopsis is native to southeast Asia, Taiwan China and the like, and is transferred to mainland China after 90 years of 20 th century. The phalaenopsis tropical aerial orchid has a peculiar and beautiful flower type, is a dual-purpose flower for potted plant and cut flower, is a good name of orchids queen, and is popular with the public.
The butterfly orchid is provided with over seventy original seeds, and the seedlings are propagated mainly through two ways of producing seedlings by aseptic seeding of seeds and producing separate seedlings by tissue culture of pedicel axillary buds, so that large-scale production is realized. Many varieties of commercially cultivated phalaenopsis are obtained by long-term artificial hybridization breeding of original phalaenopsis for years, the varieties are various, and the provenance is complex, so that the molecular marker technology is widely applied to the aspects of phalaenopsis germplasm resource identification, genetic diversity analysis and the like. Patent CN108456720A discloses a method for identifying phalaenopsis varieties based on SRAP markers, which can distinguish 7 phalaenopsis varieties at one time. Patent CN107058487B discloses a method for evaluating genetic diversity of phalaenopsis by using two markers, namely Genomic-SSR and EST-SSR, which can make up for the limitation of detection only by using a single molecular marker. Hexietum et al used ISSR molecular marker technology to analyze genetic diversity and genetic relationship of 36 butterfly orchid varieties (Hexietum et al. ISSR analysis of genetic relationship of 36 butterfly orchid varieties [ J ]. Guangdong agricultural science. 2014,21: 140-.
Microsatellite markers (microsatellites), also known as Short Tandem Repeats (STRs) or simple repeats (SSRs), are repeats of several nucleotides (2-5) in repeat units, up to tens of nucleotides, widely distributed over different locations throughout the genome of eukaryotes, and cause polymorphisms at each locus due to differences in the number of repeats and incompleteness of the degree of repeats. A pair of specific primers is designed by utilizing conserved sequences at two ends of a certain microsatellite DNA, the microsatellite DNA sequence of the site is amplified, and the polymorphism of different genotype individuals on the SSR site can be displayed through electrophoretic detection. TP-M13-SSR (simple sequence repeat with tagged primer M13) technology combines SSR molecular marker technology and fluorescence sequencing technology, has the advantages of high repeatability, accurate result and the like, and solves a series of problems of low analysis flux, complicated detection process of amplification products, large workload of data recording and the like to a greater extent (Lihui et al, 2005).
Therefore, the method utilizes the genome sequence of the phalaenopsis to develop a new SSR molecular marker and a primer composition thereof, and utilizes the TP-M13-SSR technology to analyze genetic diversity and construct a molecular identity card, thereby playing an important role in germplasm resource identification, genetic relationship analysis, trait gene positioning, molecular marker-assisted breeding and phalaenopsis-related molecular research of the phalaenopsis.
Disclosure of Invention
Aiming at the defects, the invention provides a phalaenopsis SSR molecular marker primer composition, a fingerprint code and application thereof in phalaenopsis germplasm resource identification. The invention provides a Phalaenopsis SSR primer composition by using a simplified genome sequencing technology, analyzes genetic diversity and constructs a molecular identity card of 38 different germplasm resources in a Phalaenopsis by using a TP-M13-SSR technology, can accurately, efficiently and stably identify the Phalaenopsis germplasm resources, and lays a foundation for Phalaenopsis germplasm resource identification, genetic relationship analysis, trait gene positioning, molecular marker assisted breeding, Phalaenopsis related molecular research and the like.
In order to achieve the above object, the technical solution of the present invention is as follows:
in one aspect, the invention provides a primer composition for identifying a Phalaenopsis SSR molecular marker, wherein the SSR molecular marker comprises 6-N10137440, 9-N10001991, 31-N10013849, 37-N10016842, 52-11200965, 68-N2965390, 97-N032454, 98-N034003, 99-N034673, 101-N032870, 102-N033385, 106-N035168, P-018164443-13, P-018164443-20, P-018164443-30, P-018164443-31, P-018164443-34, P-018164443-43, P-018164443-52 and U6190.
Specifically, the primer composition comprises:
(1) amplifying primers of SSR molecular marker 6-N10137440:
SEQ ID NO:1：6-N10137440-F：5'-TGGCTTCAGTGAGACAATCG-3'
SEQ ID NO:2：6-N10137440-R：5'-CCTCGCAGACCATCAAACTA-3'；
(2) amplifying primers of SSR molecular marker 9-N10001991:
SEQ ID NO:3：9-N10001991-F：5'-TAGCAAAAGGGCGAGGAGTA-3'
SEQ ID NO:4：9-N10001991-R：5'-ATAAGTGGTTGGGCTTCACG-3'；
(3) primer for amplifying SSR molecular marker 31-N10013849:
SEQ ID NO:5：31-N10013849-F：5'-CCTTAGCTGCCGCTCTTCTA-3'
SEQ ID NO:6：31-N10013849-R：5'-CATCACAGCCCTTCACAGAA-3'；
(4) primer for amplifying SSR molecular marker 37-N10016842:
SEQ ID NO:7：37-N10016842-F：5'-GAGGAGGTGGTAGGGAGGAG-3'
SEQ ID NO:8：37-N10016842-R：5'-AGTCCTCTGATCGTTAGCCG-3'；
(5) primers for amplifying SSR molecular markers 52-11200965:
SEQ ID NO:9：52-11200965-F：5'-AACAAACACAAAAACAAACTGCAT-3'
SEQ ID NO:10：52-11200965-R：5'-CGGGATGAAGATGAGGAAGA-3'；
(6) primer for amplifying SSR molecular marker 68-N2965390:
SEQ ID NO:11：68-N2965390-F：5'-TCAATGATATCACATTCCTTGTACG-3'
SEQ ID NO:12：68-N2965390-R：5'-GCTAGGATTTTGTTAATTATTGTGC-3'；
(7) primers for amplifying SSR molecular marker 97-N032454:
SEQ ID NO:13：97-N032454-F：5'-ACCTGCAAATGAACTGAAGTGG-3'
SEQ ID NO:14：97-N032454-R：5'-GCCAACATCAAACAAACAGACC-3'；
(8) amplifying primers of SSR molecular markers 98-N034003:
SEQ ID NO:15：98-N034003-F：5'-GTGTCTACTTCTAACGCATCTTGT-3'
SEQ ID NO:16：98-N034003-R：5'-GGAGCTGAGTATATCAGACTTCG-3'；
(9) amplifying primers of SSR molecular marker 99-N034673:
SEQ ID NO:17：99-N034673-F：5'-GATATTATGTGATCAATGCGACG-3'
SEQ ID NO:18：99-N034673-R：5'-CCAAAATAAACCGCTTAATAAGC-3'；
(10) primers for amplifying SSR molecular marker 101-N032870:
SEQ ID NO:19：101-N032870-F：5'-TCCGCGCTATCTTTCTCTTAACT-3'
SEQ ID NO:20：101-N032870-R：5'-TTCCTGGTAATTAGAAAGGACGG-3'；
(11) primers for amplifying SSR molecular marker 102-N033385:
SEQ ID NO:21：102-N033385-F：5'-CTCCGCGCTATCTTTCTCTTAAC-3'
SEQ ID NO:22：102-N033385-R：5'-TCCTGGTAATTAGAAAGGACGGT-3'；
(12) primer for amplifying SSR molecular marker 106-N035168:
SEQ ID NO:23：106-N035168-F：5'-GCCTTCCAACCTTTTCTTCTTT-3'
SEQ ID NO:24：106-N035168-R：5'-CGTATGAGTCCCGATGAATCC-3'；
(13) primers for amplifying SSR molecular marker P-018164443-13:
SEQ ID NO:25：P-018164443-13-F：5'-ATTGCACGCTCTCATTCCTT-3'
SEQ ID NO:26：P-018164443-13-R：5'-TCTTCCCATTTCTCAATGCC-3'；
(14) primers for amplifying SSR molecular marker P-018164443-20:
SEQ ID NO:27：P-018164443-20-F：5'-CAGAAGCCGGAGAAGACAAG-3'
SEQ ID NO:28：P-018164443-20-R：5'-ATAAGCGAATCAGCAGCGTT-3'；
(15) primers for amplifying SSR molecular marker P-018164443-30:
SEQ ID NO:29：P-018164443-30-F：5'-CGTTCAGTCCATCCCAAGAT-3'
SEQ ID NO:30：P-018164443-30-R：5'-ACTGGCGATGTTTGGTGATT-3'；
(16) primers for amplifying SSR molecular marker P-018164443-31:
SEQ ID NO:31：P-018164443-31-F：5'-CATGTAGCCGGTCTCACGTA-3'
SEQ ID NO:32：P-018164443-31-R：5'-TGCCTCAGGAATCCTATTGA-3'；
(17) primers for amplifying SSR molecular marker P-018164443-34:
SEQ ID NO:33：P-018164443-34-F：5'-ACACAGACGTCTCCCAATCC-3'
SEQ ID NO:34：P-018164443-34-R：5'-GCTACGGTGAGGGAGTTCAG-3'；
(18) primers for amplifying SSR molecular marker P-018164443-43:
SEQ ID NO:35：P-018164443-43-F：5'-AAGCGGGAAACTTAAGCTGG-3'
SEQ ID NO:36：P-018164443-43-R：5'-TGCAAAAGGCCATAGAGCTT-3'；
(19) primers for amplifying SSR molecular marker P-018164443-52:
SEQ ID NO:37：P-018164443-52-F：5'-AAGAGGGAACGAGGAAGCA-3'
SEQ ID NO:38：P-018164443-52-R：5'-AATGGCTGTAGACCACCTGC-3'；
(20) primers for amplifying SSR molecular marker U6190:
SEQ ID NO:39：U6190-F：5'-CCATTAAAGCTCAGGAGGGA-3'
SEQ ID NO:40：U6190-R：5'-CGAAGAAATAGCCATCGAGC-3'。
on the other hand, the invention provides a phalaenopsis SSR molecular marker fingerprint code, wherein the fingerprint code comprises a fingerprint spectrum and a molecular identity card, and the SSR molecular marker sequence of the molecular identity card is as follows: 6-N10137440, 9-N10001991, 31-N10013849, 37-N10016842, 52-11200965, 68-N2965390, 97-N032454, 98-N034003, 99-N034673, 101-N032870, 102-N033385, 106-N035168, P-018164443-13, P-018164443-20, P-018164443-30, P-018164443-31, P-018164443-34, P-018164443-43, P-018164443-52 and U6190.
Specifically, the molecular identity card is shown in the following table 1.
TABLE 1
More specifically, the molecular identity card is constructed according to a diploid standard, and data encoding is performed on fingerprint data according to SSR detection results (amplified fragments of each locus are arranged according to molecular weight, the amplified fragments (alleles) are marked by Arabic numerals 1-9 from small to large, more than 9 alleles are marked by 120 alphabets A-Z in capital English), if the locus is not amplified in a certain variety, the locus is marked as 0, and each locus occupies two bits.
Specifically, the fingerprint is shown in the following table 2.
TABLE 2
In another aspect, the invention provides a kit comprising the SSR molecular marker amplification primer composition or the SSR molecular marker fingerprint code.
In another aspect, the invention provides an application of the SSR molecular marker amplification primer composition, the SSR molecular marker fingerprint code or the kit in phalaenopsis screening or germplasm resource identification.
In another aspect, the present invention provides the method for screening SSR molecular markers, wherein the method comprises the following steps: taking total DNA of phalaenopsis as a template, randomly breaking the DNA into fragments with the length of about 350bp by using an ultrasonic crusher, finishing library preparation through the steps of end repairing, tail adding A, sequencing joint adding, purification, PCR amplification and the like, carrying out primary quantification by using Qubit 2.0, diluting the library to 2 ng/mu L, then detecting the inserted fragments of the library by using Agilent 2100, and accurately quantifying the effective concentration of the library by using a Q-PCR method so as to ensure the quality of the library. Sequencing by using Illumina Hiseq 2500, firstly, performing quality detection on original off-line data by using FASTQC software, removing a linker and a low-quality base sequence, splicing double-end reads by using Flash software according to overlapped bases, screening a sequencing result by using MISA, and reserving a sequence with a microsatellite marker SSR.
In another aspect, the present invention provides a method for butterfly orchid screening or germplasm resource identification, wherein the method comprises the following steps:
(1) extracting total DNA of a plant to be detected;
(2) performing SSR molecular marker PCR amplification by taking the total DNA extracted in the step (1) as a template;
(3) detecting the PCR amplification product in the step (2) by capillary electrophoresis, and collecting data;
(4) and (4) performing genetic diversity index, clustering and polymorphic information content PIC calculation analysis according to the data obtained in the step (3).
Specifically, the PCR amplification system in step (2) is: the total volume of the reaction system is 10. mu.L, and the reaction system comprises 1.2. mu.L of DNA template (50 ng/. mu.L), 1.0. mu.L of 10 XBuffer I Buffer solution, 0.1. mu. L TAKARA HS Taq enzyme (5U/. mu.L), 0.6. mu.L of primer (5. mu.M), 0.8. mu.L of 2.5mM dNTP, 0.5. mu.L of TP-M13 (5. mu.M), and deionized water to make up to 10. mu.L.
Further specifically, the PCR amplification reaction procedure: 5min at 95 ℃; 30 cycles of 95 ℃ for 30s, 60 ℃ for 30s, and 72 ℃ for 30 s; 30s at 95 ℃, 30s at 53 ℃, 30s at 72 ℃ and 10 cycles; 30min at 60 ℃; storing at 4 ℃.
More specifically, the primer of the PCR amplification system comprises a sequence shown as SEQ ID NO. 1-SEQ ID NO. 40.
In another aspect, the invention also provides the application of the SSR molecular marker amplification primer composition, the SSR molecular marker fingerprint code or the kit in genetic relationship analysis, trait gene positioning and molecular marker assisted breeding of phalaenopsis.
Compared with the prior art, the invention has the advantages that:
1. the invention provides a Phalaenopsis SSR molecular marker and a primer composition, and provides a basis for screening and germplasm resource identification of Phalaenopsis by analyzing genetic diversity and constructing a molecular identity card for 38 different germplasm resources in Phalaenopsis by utilizing a TP-M13-SSR technology.
2. The Phalaenopsis SSR molecular marker and the primer composition can accurately, efficiently and stably identify Phalaenopsis germplasm resources, and are simple and convenient to operate.
3. The Phalaenopsis SSR molecular marker can be used for assisting in selective breeding, and early selection in the seedling stage is realized, so that the breeding process of Phalaenopsis is accelerated.
Drawings
FIG. 1 is a peak diagram of the result of SSR molecular marker primer amplification.
FIG. 2 is a genetic analysis and clustering chart.
Detailed Description
The present invention will be further illustrated in detail with reference to the following specific examples, which are not intended to limit the present invention but are merely illustrative thereof. The experimental methods used in the following examples are not specifically described, and the materials, reagents and the like used in the following examples are generally commercially available under the usual conditions without specific descriptions.
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.
Example 1 Phalaenopsis germplasm resources
The invention adopts 38 species of phalaenopsis germplasm resources which are collected and stored by an orchid resource garden of environmental horticulture research institute of Guangdong province academy of agricultural sciences, and the specific information is shown in the following table 3. All experimental materials are planted in the greenhouse of environmental gardening research institute of Guangdong province academy of agricultural sciences in the Guangdong province of agricultural modernization scientific and technological demonstration.
Table 338 germplasm resources basic information
Example 2 identification of moth orchid planting resources
1. Total DNA extraction
The extraction of the total DNA of the butterfly orchid plant is carried out according to the operation steps of a novel plant genome DNA extraction kit of Tiangen Biotechnology (Beijing) Co., Ltd, and the specific steps are as follows:
(1) adding liquid nitrogen into young root tips or young leaf tissues of phalaenopsis plants, fully grinding, and weighing about 100mg of fresh plant tissues.
(2) 400 μ L of buffer GPS and 10 μ L of RNase A (10mg/mL) were quickly added to the ground powder, vortexed quickly and mixed well, and then the tube was placed in a 65 ℃ water bath for 15min, and the tube was inverted during the water bath to mix the sample several times.
(3) Add 100. mu.L of buffer GPA, vortex for 1min, centrifuge at 12000rpm for 5min, transfer supernatant to filtration column CS (filtration column CS placed in collection tube), centrifuge at 12000rpm for 1min, transfer filtrate to new centrifuge tube.
(4) An equal volume of absolute ethanol was added and mixed well, at which time a flocculent precipitate may appear.
(5) Transferring the solution and flocculent precipitate obtained in the previous step to RNase-Free adsorption column CR2 (adsorption column CR2 is placed in a collection tube), centrifuging at 12000rpm for 1min, removing waste liquid, and placing RNase-Free adsorption column CR2 in the collection tube.
(6) Adding 550 μ L deproteinizing solution RD (checking whether anhydrous ethanol is added before use) into RNase-Free adsorption column CR2, centrifuging at 12000rpm for 1min, removing waste liquid, and placing RNase-Free adsorption column CR2 into collection tube.
(7) Adding 700 μ L of rinsing solution PW (to which anhydrous ethanol is added before use) into RNase-Free adsorption column CR2, centrifuging at 12000rpm for 1min, removing waste liquid, and placing RNase-Free adsorption column CR2 into a collection tube.
(8) And (5) repeating the step (7).
(9) And (3) putting the RNase-Free adsorption column CR2 back into the collection tube, centrifuging at 12000rpm for 2min, discarding the collection tube, transferring the RNase-Free adsorption column CR2 into a new centrifuge tube, and airing at room temperature for 5-10 min.
(10) Adding 50-100 μ L of elution buffer TB into RNase-Free adsorption column CR2, standing at room temperature for 3-5min, centrifuging at 12000rpm for 2min, and collecting the solution in a centrifuge tube.
(11) mu.L of DNA was used for 1.2% agarose gel electrophoresis detection, and 2. mu.L of DNA was used for NanoDrop spectrophotometry.
SSR molecular markers and primer compositions
2.1. Uniformly mixing 38 detected qualified Phalaenopsis germplasm resource DNA samples in equal quantity, randomly breaking a fragment with the length of about 350bp by using a Bioruptor ultrasonic crusher, and performing sequencing according to the sequence
After library preparation is completed by the steps of end repair, A tail addition, sequencing linker addition, purification, PCR amplification and the like in a Rapid DNA-Seq Kit (Bio Scientific, 5144-08) Kit step, primary quantification is carried out by using Qubit 2.0, the library is diluted to 2 ng/mu L, then the inserted fragment of the library is detected by using Agilent 2100, and the effective concentration of the library is accurately quantified by using a Q-PCR method so as to ensure the quality of the library. Sequencing by using Illumina Hiseq 2500, firstly performing quality detection on original off-line data by using FASTQC software, removing a linker and a low-quality base sequence, splicing double-end reads by using Flash software according to overlapped bases, screening a sequencing result by using MISA, and reserving a sequence with a microsatellite marker as an SSR molecular marker.
2.2. Primers were designed based on the SSR molecular markers and are shown in Table 4 below.
TABLE 4 SSR molecular marker primers
3. Sample amplification
PCR amplification of samples was performed using the primers in Table 4.
The total volume of the reaction system is 10. mu.L, and the reaction system comprises 1.2. mu.L of DNA template (50 ng/. mu.L), 1.0. mu.L of 10 XBuffer I Buffer solution, 0.1. mu. L TAKARA HS Taq enzyme (5U/. mu.L), 0.6. mu.L of primer (5. mu.M), 0.8. mu.L of 2.5mM dNTP, 0.5. mu.L of TP-M13 (5. mu.M), and deionized water to make up to 10. mu.L.
Reaction procedure: 5min at 95 ℃; 30 cycles of 95 ℃ for 30s, 60 ℃ for 30s, and 72 ℃ for 30 s; 30s at 95 ℃, 30s at 53 ℃, 30s at 72 ℃ and 10 cycles; 30min at 60 ℃; storing at 4 ℃.
4. Detection of
The amplification products were detected by 1.2% agarose gel electrophoresis. Adding 1.0 mu L of amplification product, 9 mu L of ROX-500 molecular weight internal standard and formamide mixed liquor (volume ratio is 0.5:8.5) into each hole of a 96-hole plate, performing denaturation at 95 ℃ for 3min, detecting by using an ABI 3730XL detector, injecting sample at 1kV voltage for 10s, and performing electrophoresis at 15kV for 30 min. The original Data file collected by the Data cloning software is imported into GeneMapper 3.2 software for analysis, the position of each peak is compared with the molecular weight internal standard in the lane, and the accurate size of the target DNA fragment is calculated. The capillary electrophoresis detection was performed independently for 3 replicates on each fluorescently labeled locus, and the average of the 3 replicates was taken and rounded up as the data for the experimental material on that locus.
5. Data analysis
And (4) performing genetic diversity index, clustering and polymorphic information content PIC calculation analysis on the sorted data by using NTSYS software.
EXAMPLE 3 primer amplification results
The results of the SSR molecular marker primer amplification are detailed in the following table 5. As SSR molecular marker primers and phalaenopsis are more in types, a peak diagram of an amplification result of 9-N10001991 is shown as an example, and a peak diagram of an amplification result of 9-N10001991 is shown as a diagram in figure 1. As can be seen from Table 5 and FIG. 1, the SSR molecular marker primers of the present invention have good amplification effect and high detectable rate, and can amplify stable DNA bands.
TABLE 5 primer amplification results
Example 4 genetic analysis and clustering
The genetic diversity index, clustering and polymorphic information content PIC calculation analysis results are shown in table 6 below and fig. 2.
TABLE 6
Example 5 molecular identification card construction
The method is constructed according to the diploid standard, and data encoding is carried out on fingerprint data according to SSR detection results (amplification fragments of each locus are arranged according to the molecular weight, the amplification fragments (alleles) are marked by Arabic numerals 1-9 from small to large, more than 9 alleles are marked by capital English letters A-Z), if the locus is not amplified in a certain variety, the locus is marked as 0, and each locus occupies two bits. Wherein, the SSR molecular marker sequence of the molecular identity card is as follows: 6-N10137440, 9-N10001991, 31-N10013849, 37-N10016842, 52-11200965, 68-N2965390, 97-N032454, 98-N034003, 99-N034673, 101-N032870, 102-N033385, 106-N035168, P-018164443-13, P-018164443-20, P-018164443-30, P-018164443-31, P-018164443-34, P-018164443-43, P-018164443-52 and U6190.
The molecular identity card information is shown in the following table 7, and the fingerprint spectrum is shown in the following table 8.
TABLE 7
TABLE 8
Experimental example 1 accuracy test
According to the SSR molecular marker primer composition and the germplasm resource identification method, 10 samples of 38 phalaenopsis plants are selected for detection, and the detection results are shown in the following table 9.
TABLE 9 accuracy test results
As can be seen from table 9, 38 phalaenopsis can be accurately identified by using the SSR molecular marker primer composition and the germplasm identification method of the present invention, with an accuracy of 100%.
The SSR molecular marker can be used for assisting in selective breeding and realizing early selection in the seedling stage, so that the breeding process of phalaenopsis is accelerated.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
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<110> environmental gardening institute of academy of agricultural sciences of Guangdong province
<120> Phalaenopsis SSR molecular marker primer composition and application thereof
<130> 20200114
<160> 40
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 1
tggcttcagt gagacaatcg 20
<210> 2
<211> 20
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 2
cctcgcagac catcaaacta 20
<210> 3
<211> 20
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 3
tagcaaaagg gcgaggagta 20
<210> 4
<211> 20
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 4
ataagtggtt gggcttcacg 20
<210> 5
<211> 20
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 5
ccttagctgc cgctcttcta 20
<210> 6
<211> 20
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 6
catcacagcc cttcacagaa 20
<210> 7
<211> 20
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 7
gaggaggtgg tagggaggag 20
<210> 8
<211> 20
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 8
agtcctctga tcgttagccg 20
<210> 9
<211> 24
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 9
aacaaacaca aaaacaaact gcat 24
<210> 10
<211> 20
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 10
cgggatgaag atgaggaaga 20
<210> 11
<211> 25
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 11
tcaatgatat cacattcctt gtacg 25
<210> 12
<211> 25
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 12
gctaggattt tgttaattat tgtgc 25
<210> 13
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 13
acctgcaaat gaactgaagt gg 22
<210> 14
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 14
gccaacatca aacaaacaga cc 22
<210> 15
<211> 24
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 15
gtgtctactt ctaacgcatc ttgt 24
<210> 16
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 16
ggagctgagt atatcagact tcg 23
<210> 17
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 17
gatattatgt gatcaatgcg acg 23
<210> 18
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 18
ccaaaataaa ccgcttaata agc 23
<210> 19
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 19
tccgcgctat ctttctctta act 23
<210> 20
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 20
ttcctggtaa ttagaaagga cgg 23
<210> 21
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 21
ctccgcgcta tctttctctt aac 23
<210> 22
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 22
tcctggtaat tagaaaggac ggt 23
<210> 23
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 23
gccttccaac cttttcttct tt 22
<210> 24
<211> 21
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 24
cgtatgagtc ccgatgaatc c 21
<210> 25
<211> 20
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 25
attgcacgct ctcattcctt 20
<210> 26
<211> 20
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 26
tcttcccatt tctcaatgcc 20
<210> 27
<211> 20
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 27
cagaagccgg agaagacaag 20
<210> 28
<211> 20
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 28
ataagcgaat cagcagcgtt 20
<210> 29
<211> 20
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 29
cgttcagtcc atcccaagat 20
<210> 30
<211> 20
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 30
actggcgatg tttggtgatt 20
<210> 31
<211> 20
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 31
catgtagccg gtctcacgta 20
<210> 32
<211> 20
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 32
tgcctcagga atcctattga 20
<210> 33
<211> 20
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 33
acacagacgt ctcccaatcc 20
<210> 34
<211> 20
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 34
gctacggtga gggagttcag 20
<210> 35
<211> 20
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 35
aagcgggaaa cttaagctgg 20
<210> 36
<211> 20
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 36
tgcaaaaggc catagagctt 20
<210> 37
<211> 19
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 37
aagagggaac gaggaagca 19
<210> 38
<211> 20
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 38
aatggctgta gaccacctgc 20
<210> 39
<211> 20
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 39
ccattaaagc tcaggaggga 20
<210> 40
<211> 20
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 40
cgaagaaata gccatcgagc 20
Claims (10)
1. A primer composition of a Phalaenopsis SSR molecular marker is characterized in that the SSR molecular marker comprises 6-N10137440, 9-N10001991, 31-N10013849, 37-N10016842, 52-11200965, 68-N2965390, 97-N032454, 98-N034003, 99-N034673, 101-N032870, 102-N033385, 106-N035168, P-018164443-13, P-018164443-20, P-018164443-30, P-018164443-31, P-018164443-34, P-018164443-43, P-018164443-52 and U6190;
the primer composition comprises:
(1) amplifying primers of SSR molecular marker 6-N10137440:
SEQ ID NO:1：6-N10137440-F：5'-TGGCTTCAGTGAGACAATCG-3'
SEQ ID NO:2：6-N10137440-R：5'-CCTCGCAGACCATCAAACTA-3'；
(2) amplifying primers of SSR molecular marker 9-N10001991:
SEQ ID NO:3：9-N10001991-F：5'-TAGCAAAAGGGCGAGGAGTA-3'
SEQ ID NO:4：9-N10001991-R：5'-ATAAGTGGTTGGGCTTCACG-3'；
(3) primer for amplifying SSR molecular marker 31-N10013849:
SEQ ID NO:5：31-N10013849-F：5'-CCTTAGCTGCCGCTCTTCTA-3'
SEQ ID NO:6：31-N10013849-R：5'-CATCACAGCCCTTCACAGAA-3'；
(4) primer for amplifying SSR molecular marker 37-N10016842:
SEQ ID NO:7：37-N10016842-F：5'-GAGGAGGTGGTAGGGAGGAG-3'
SEQ ID NO:8：37-N10016842-R：5'-AGTCCTCTGATCGTTAGCCG-3'；
(5) primers for amplifying SSR molecular markers 52-11200965:
SEQ ID NO:9：52-11200965-F：5'-AACAAACACAAAAACAAACTGCAT-3'
SEQ ID NO:10：52-11200965-R：5'-CGGGATGAAGATGAGGAAGA-3'；
(6) primer for amplifying SSR molecular marker 68-N2965390:
SEQ ID NO:11：68-N2965390-F：5'-TCAATGATATCACATTCCTTGTACG-3'
SEQ ID NO:12：68-N2965390-R：5'-GCTAGGATTTTGTTAATTATTGTGC-3'；
(7) primers for amplifying SSR molecular marker 97-N032454:
SEQ ID NO:13：97-N032454-F：5'-ACCTGCAAATGAACTGAAGTGG-3'
SEQ ID NO:14：97-N032454-R：5'-GCCAACATCAAACAAACAGACC-3'；
(8) amplifying primers of SSR molecular markers 98-N034003:
SEQ ID NO:15：98-N034003-F：5'-GTGTCTACTTCTAACGCATCTTGT-3'
SEQ ID NO:16：98-N034003-R：5'-GGAGCTGAGTATATCAGACTTCG-3'；
(9) amplifying primers of SSR molecular marker 99-N034673:
SEQ ID NO:17：99-N034673-F：5'-GATATTATGTGATCAATGCGACG-3'
SEQ ID NO:18：99-N034673-R：5'-CCAAAATAAACCGCTTAATAAGC-3'；
(10) primers for amplifying SSR molecular marker 101-N032870:
SEQ ID NO:19：101-N032870-F：5'-TCCGCGCTATCTTTCTCTTAACT-3'
SEQ ID NO:20：101-N032870-R：5'-TTCCTGGTAATTAGAAAGGACGG-3'；
(11) primers for amplifying SSR molecular marker 102-N033385:
SEQ ID NO:21：102-N033385-F：5'-CTCCGCGCTATCTTTCTCTTAAC-3'
SEQ ID NO:22：102-N033385-R：5'-TCCTGGTAATTAGAAAGGACGGT-3'；
(12) primer for amplifying SSR molecular marker 106-N035168:
SEQ ID NO:23：106-N035168-F：5'-GCCTTCCAACCTTTTCTTCTTT-3'
SEQ ID NO:24：106-N035168-R：5'-CGTATGAGTCCCGATGAATCC-3'；
(13) primers for amplifying SSR molecular marker P-018164443-13:
SEQ ID NO:25：P-018164443-13-F：5'-ATTGCACGCTCTCATTCCTT-3'
SEQ ID NO:26：P-018164443-13-R：5'-TCTTCCCATTTCTCAATGCC-3'；
(14) primers for amplifying SSR molecular marker P-018164443-20:
SEQ ID NO:27：P-018164443-20-F：5'-CAGAAGCCGGAGAAGACAAG-3'
SEQ ID NO:28：P-018164443-20-R：5'-ATAAGCGAATCAGCAGCGTT-3'；
(15) primers for amplifying SSR molecular marker P-018164443-30:
SEQ ID NO:29：P-018164443-30-F：5'-CGTTCAGTCCATCCCAAGAT-3'
SEQ ID NO:30：P-018164443-30-R：5'-ACTGGCGATGTTTGGTGATT-3'；
(16) primers for amplifying SSR molecular marker P-018164443-31:
SEQ ID NO:31：P-018164443-31-F：5'-CATGTAGCCGGTCTCACGTA-3'
SEQ ID NO:32：P-018164443-31-R：5'-TGCCTCAGGAATCCTATTGA-3'；
(17) primers for amplifying SSR molecular marker P-018164443-34:
SEQ ID NO:33：P-018164443-34-F：5'-ACACAGACGTCTCCCAATCC-3'
SEQ ID NO:34：P-018164443-34-R：5'-GCTACGGTGAGGGAGTTCAG-3'；
(18) primers for amplifying SSR molecular marker P-018164443-43:
SEQ ID NO:35：P-018164443-43-F：5'-AAGCGGGAAACTTAAGCTGG-3'
SEQ ID NO:36：P-018164443-43-R：5'-TGCAAAAGGCCATAGAGCTT-3'；
(19) primers for amplifying SSR molecular marker P-018164443-52:
SEQ ID NO:37：P-018164443-52-F：5'-AAGAGGGAACGAGGAAGCA-3'
SEQ ID NO:38：P-018164443-52-R：5'-AATGGCTGTAGACCACCTGC-3'；
(20) primers for amplifying SSR molecular marker U6190:
SEQ ID NO:39：U6190-F：5'-CCATTAAAGCTCAGGAGGGA-3'
SEQ ID NO:40：U6190-R：5'-CGAAGAAATAGCCATCGAGC-3'。
2. the Phalaenopsis SSR molecular marker fingerprint code is characterized by comprising a fingerprint spectrum and a molecular identity card, wherein the sequence of SSR molecular markers of the molecular identity card is as follows: 6-N10137440, 9-N10001991, 31-N10013849, 37-N10016842, 52-11200965, 68-N2965390, 97-N032454, 98-N034003, 99-N034673, 101-N032870, 102-N033385, 106-N035168, P-018164443-13, P-018164443-20, P-018164443-30, P-018164443-31, P-018164443-34, P-018164443-43, P-018164443-52 and U6190.
3. The fingerprint code of claim 2, wherein the molecular identification card is:
4. the fingerprint code according to claim 3, wherein the fingerprint map is:
5. a kit for phalaenopsis screening or germplasm resource identification, wherein the kit comprises the SSR molecular marker primer composition of claim 1 or the SSR molecular marker fingerprint code of claim 2.
6. Use of an SSR molecular marker primer composition according to claim 1, an SSR molecular marker fingerprint code according to claim 2, or a kit according to claim 5 in moth orchid screening or germplasm resource identification.
7. A method for screening SSR molecular markers according to claim 1, comprising the steps of: taking total DNA of phalaenopsis as a template, randomly breaking the DNA into fragments with the length of about 350bp by using an ultrasonic crusher, finishing library preparation through the steps of end repairing, tail adding A, sequencing joint adding, purification, PCR amplification and the like, carrying out primary quantification by using Qubit 2.0, diluting the library to 2 ng/mu L, then detecting the inserted fragments of the library by using Agilent 2100, and accurately quantifying the effective concentration of the library by using a Q-PCR method so as to ensure the quality of the library. Sequencing by using Illumina Hiseq 2500, firstly, performing quality detection on original off-line data by using FASTQC software, removing a linker and a low-quality base sequence, splicing double-end reads by using Flash software according to overlapped bases, screening a sequencing result by using MISA, and reserving a sequence with a microsatellite marker SSR.
8. A method for butterfly orchid screening or germplasm resource identification is characterized by comprising the following steps:
(1) extracting total DNA of a plant to be detected;
(2) performing SSR molecular marker PCR amplification by taking the total DNA extracted in the step (1) as a template;
(3) detecting the PCR amplification product in the step (2) by capillary electrophoresis, and collecting data;
(4) and (4) performing genetic diversity index, clustering and polymorphic information content PIC calculation analysis according to the data obtained in the step (3).
9. The method of claim 8,
the PCR amplification system in the step (2) is as follows: the total volume of the reaction system is 10 muL, and the reaction system comprises 1.2 muL of DNA template (50 ng/. mu.L), 1.0 muL of 10 XBuffer I Buffer solution, 0.1 mu L TAKARA HS Taq enzyme (5U/. mu.L), 0.6 muL of primer (5 muM), 0.8 muL of 2.5mM dNTP, 0.5 muL of TP-M13(5 muM), and deionized water to make up to 10 muL;
the PCR amplification reaction program comprises the following steps: 5min at 95 ℃; 30 cycles of 95 ℃ for 30s, 60 ℃ for 30s, and 72 ℃ for 30 s; 30s at 95 ℃, 30s at 53 ℃, 30s at 72 ℃ and 10 cycles; 30min at 60 ℃; storing at 4 deg.C;
the PCR amplification system primer comprises a sequence shown as SEQ ID NO. 1-SEQ ID NO. 40.
10. Use of an SSR molecular marker primer composition according to claim 1, an SSR molecular marker fingerprint code according to claim 2, or a kit according to claim 5 for genetic relationship analysis, trait gene mapping, and molecular marker-assisted breeding in phalaenopsis.
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