CN116790790B - Wild jujube SSR molecular marker development method and application thereof in wild jujube germplasm resource identification - Google Patents
Wild jujube SSR molecular marker development method and application thereof in wild jujube germplasm resource identification Download PDFInfo
- Publication number
- CN116790790B CN116790790B CN202310758096.7A CN202310758096A CN116790790B CN 116790790 B CN116790790 B CN 116790790B CN 202310758096 A CN202310758096 A CN 202310758096A CN 116790790 B CN116790790 B CN 116790790B
- Authority
- CN
- China
- Prior art keywords
- wild jujube
- ssr
- molecular marker
- primer
- seqidno
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 240000008866 Ziziphus nummularia Species 0.000 title claims abstract description 73
- 239000003147 molecular marker Substances 0.000 title claims abstract description 22
- 238000000034 method Methods 0.000 title claims abstract description 12
- 238000011161 development Methods 0.000 title abstract description 10
- 238000012408 PCR amplification Methods 0.000 claims abstract description 9
- 238000013461 design Methods 0.000 claims abstract description 7
- 108020004414 DNA Proteins 0.000 claims description 20
- 238000000137 annealing Methods 0.000 claims description 5
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 claims description 5
- 238000011144 upstream manufacturing Methods 0.000 claims description 4
- 238000007400 DNA extraction Methods 0.000 claims description 3
- 108090000790 Enzymes Proteins 0.000 claims description 3
- 102000004190 Enzymes Human genes 0.000 claims description 3
- 230000001351 cycling effect Effects 0.000 claims description 3
- 238000004925 denaturation Methods 0.000 claims description 3
- 230000036425 denaturation Effects 0.000 claims description 3
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 2
- 238000012257 pre-denaturation Methods 0.000 claims description 2
- 238000010186 staining Methods 0.000 claims 1
- 230000002068 genetic effect Effects 0.000 abstract description 18
- 238000004458 analytical method Methods 0.000 abstract description 12
- 238000012163 sequencing technique Methods 0.000 abstract description 11
- 238000001514 detection method Methods 0.000 abstract description 8
- 238000009395 breeding Methods 0.000 abstract description 4
- 230000001488 breeding effect Effects 0.000 abstract description 4
- 239000000463 material Substances 0.000 abstract description 4
- 238000004519 manufacturing process Methods 0.000 abstract description 3
- 108091092878 Microsatellite Proteins 0.000 description 47
- 239000002773 nucleotide Substances 0.000 description 12
- 238000003752 polymerase chain reaction Methods 0.000 description 10
- PHTQWCKDNZKARW-UHFFFAOYSA-N isoamylol Chemical compound CC(C)CCO PHTQWCKDNZKARW-UHFFFAOYSA-N 0.000 description 8
- 108700028369 Alleles Proteins 0.000 description 7
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 230000003321 amplification Effects 0.000 description 4
- 238000003199 nucleic acid amplification method Methods 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 208000002109 Argyria Diseases 0.000 description 3
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 3
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 3
- 241001247821 Ziziphus Species 0.000 description 3
- 210000000582 semen Anatomy 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- 239000007984 Tris EDTA buffer Substances 0.000 description 2
- 238000000246 agarose gel electrophoresis Methods 0.000 description 2
- 230000000975 bioactive effect Effects 0.000 description 2
- 238000007621 cluster analysis Methods 0.000 description 2
- 230000000295 complement effect Effects 0.000 description 2
- 239000000539 dimer Substances 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- VGGSULWDCMWZPO-ODEMIOGVSA-N spinosin Chemical compound O([C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1C1=C(O)C=2C(=O)C=C(OC=2C=C1OC)C=1C=CC(O)=CC=1)[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O VGGSULWDCMWZPO-ODEMIOGVSA-N 0.000 description 2
- SMRPGWBDLOQHOS-UHFFFAOYSA-N 5-[4,5-dihydroxy-6-(hydroxymethyl)-3-(3,4,5-trihydroxy-6-methyloxan-2-yl)oxyoxan-2-yl]oxy-3,4-dihydroxy-6-[[9-hydroxy-4-(hydroxymethyl)-4,6a,6b,8a,11,11,14b-heptamethyl-14-oxo-2,3,4a,5,6,7,8,9,10,12,12a,14a-dodecahydro-1H-picen-3-yl]oxy]oxane-2-carboxylic acid Chemical compound OC1C(O)C(O)C(C)OC1OC1C(OC2C(OC(C(O)C2O)C(O)=O)OC2C(C3C(C4C(C5(CCC6(C)C(O)CC(C)(C)CC6C5=CC4=O)C)(C)CC3)(C)CC2)(C)CO)OC(CO)C(O)C1O SMRPGWBDLOQHOS-UHFFFAOYSA-N 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 235000021424 Jujube vinegar Nutrition 0.000 description 1
- 241000219100 Rhamnaceae Species 0.000 description 1
- 241000612118 Samolus valerandi Species 0.000 description 1
- 244000269722 Thea sinensis Species 0.000 description 1
- 229930013930 alkaloid Natural products 0.000 description 1
- 238000000861 blow drying Methods 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- VGGSULWDCMWZPO-UHFFFAOYSA-N flavoayamenin Natural products COC1=CC=2OC(C=3C=CC(O)=CC=3)=CC(=O)C=2C(O)=C1C1OC(CO)C(O)C(O)C1OC1OC(CO)C(O)C(O)C1O VGGSULWDCMWZPO-UHFFFAOYSA-N 0.000 description 1
- 235000013312 flour Nutrition 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000027939 micturition Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229920006316 polyvinylpyrrolidine Polymers 0.000 description 1
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 1
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 235000020418 red date juice Nutrition 0.000 description 1
- PPRSVUXPYPBULA-UHFFFAOYSA-N saponin A Natural products CC1(C)CCC2(CCC3(C)C(=CCC4C5(C)CCC(OC6OC(CO)C(O)C(O)C6=O)C(C)(C)C5CCC34C)C2C1)C(=O)O PPRSVUXPYPBULA-UHFFFAOYSA-N 0.000 description 1
- 229930000044 secondary metabolite Natural products 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000002936 tranquilizing effect Effects 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- 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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6858—Allele-specific amplification
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Immunology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Botany (AREA)
- Mycology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to the technical field of biology, and particularly relates to a wild jujube SSR molecular marker development method based on wild jujube transcriptome sequencing and application of the developed SSR molecular marker in wild jujube germplasm resource identification. The SSR molecular marker developed by the invention has the characteristics of good stability and high effectiveness. The invention takes the wild jujube as a material, 4 pairs of SSR molecular marker primer combinations are obtained from the wild jujube by transcriptome sequencing, SSR locus analysis and identification, SSR primer design, SSR-PCR reaction system establishment, SSR-PCR amplification and effectiveness detection, and wild jujube germplasm resources from different wild jujube main production areas nationwide can be effectively identified. The SSR molecular marker obtained by the invention can be used for germplasm resource identification, genetic diversity analysis and molecular auxiliary new variety breeding of wild jujube.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a wild jujube SSR molecular marker development method and application thereof in wild jujube germplasm resource identification.
Background
Wild jujube (ziziphus juuba Mill. Var. Spinosa (Bunge) Huex H.F.Chow) is a plant of the genus Ziziphus of the family Rhamnaceae, usually shrubs or small trees, has good temperature and drought resistance, and strong adaptability, and is widely distributed in vast northern areas of China. Wild jujube has a planting history of over 4000 years in China, and is a traditional Chinese medicine resource in China. Wild jujube is one of the first medicinal and edible medicinal plants issued by China in 2012, and the roots, the peels, the leaves, the pulp and the kernels of the wild jujube all contain a large amount of bioactive substances, so that the wild jujube has high development potential of medicines and health-care products. Wherein the wild jujube pulp can be processed into wild jujube juice, wild jujube vinegar, wild jujube flour, etc.; the wild jujube pit is a high-quality active carbon raw material and can be used as a craft; the wild jujube leaf can be used as tea raw material for development, and has health promotion effects of tranquilizing, promoting urination, and relieving inflammation; the semen Ziziphi Spinosae contains abundant bioactive secondary metabolites including semen Ziziphi Spinosae saponin A, B, spinosin, semen Ziziphi Spinosae oil, and alkaloids.
Along with the increase of the demand of the market for the wild jujube, the wild jujube main production area starts the introduction and cultivation work in different places, so that the wild jujube has unclear genetic background, mixed germplasm resources and similar morphology among multiple varieties, the seed selection of the wild jujube high-quality resources is affected, and the development of the wild jujube industry is challenged. SSR (Simple Sequence Repeats) is a molecular marker technology based on specific primer PCR developed in recent years. SSR markers have the following advantages over other molecular markers: (1) The number is abundant, the whole genome is covered, and the polymorphism is high; (2) The characteristic of multiple alleles provides high information; and (3) the operation is simple, the requirement on DNA is not high, and the repeatability is good. At present, no report related to the development of the wild jujube SSR molecular marker exists, so that the development of the SSR molecular marker with potential polymorphism based on the analysis of wild jujube transcriptome information by designing SSR sequence primers is particularly important for the genetic background analysis of wild jujube germplasm resources, the identification of variety authenticity and the molecular assistance of wild jujube breeding work.
Disclosure of Invention
The invention obtains a large amount of SSR information on the basis of wild jujube transcriptome sequencing, and performs verification and polymorphism analysis on the selected SSR primers through PCR amplification and polyacrylamide gel electrophoresis detection, so that the application of the effective SSR molecular marker primer combination in wild jujube germplasm resource identification is obtained, and a reliable technical means is provided for carrying out germplasm resource identification, genetic relationship and genetic diversity analysis, true-false product identification and molecular auxiliary wild jujube new variety breeding by utilizing SSR molecular markers.
The invention provides an application of an SSR molecular marker primer combination developed based on a wild jujube transcriptome in wild jujube germplasm resource identification, wherein the primers are SSR44, SSR48, SSR86 and SSR97 respectively, and upstream and downstream nucleotide sequences corresponding to the primers are as follows:
(1) SSR44 primer:
SEQ ID NO.1:SSR44-F:5`-CTCATCGGCGAGATCTTCCC-3`
SEQ ID NO.2:SSR44-R:5`-TACCGAAAAACTCGCCGGAA-3`
(2) SSR48 primer:
SEQ ID NO.3:SSR48-F:5`-AGGGAGAGATGGGAGCATGT-3`
SEQ ID NO.4:SSR48-R:5`-TGCACGACCCCACATTCTAC-3`
(3) SSR86 primer:
SEQ ID NO.5:SSR86-F:5`-GCATGGCGTGTTGGATTTGT-3`
SEQ ID NO.6:SSR86-R:5`-ATCCAACTTAGCTGCCCACC-3`
(4) SSR97 primer:
SEQ ID NO.7:SSR97-F:5`-GCCGATGATAAAACCGCCAC-3`
SEQ ID NO.8:SSR97-R:5`-TTGGTGAAGACGCTGAGGAC-3`
the invention provides application of the primer combination in wild jujube germplasm resource identification, genetic relationship and genetic diversity analysis. In order to achieve the above purpose, the present invention adopts the following technical scheme:
(1) Transcriptome sequencing is carried out on the wild jujube sample by transcriptome sequencing technology, sequencing data is filtered, a joint sequence and low-quality data in the sequencing data are removed, and sequence assembly is carried out to obtain high-quality wild jujube transcriptome data
(2) SSR detection from transcriptome data
(3) Primer design according to SSR information
(4) Wild jujube DNA extraction
(5) PCR system amplification primer screening
(6) Primer effectiveness identification by polyacrylamide gel electrophoresis and silver staining
The transcriptome sequencing sample in the step (1) is leaves of wild jujube which is a medicinal material of Taichen Cheng in Hebei province.
The transcriptome SSR detection in the step (2) is to identify genes in the transcriptome by adopting MISA software, and 6 types of SSRs are identified: single base (Mono-nucleotide) repeat SSR, double base (Di-nucleotide) repeat SSR, triple base (Tri-nucleotide) repeat SSR, tetra-nucleotide) repeat SSR, pentabase (Penta-nucleotide) repeat SSR, and hexabase (Hexa-nucleotide) repeat SSR.
The SSR primer design method in the step (3) comprises the following steps: primer3.0 (2.5.0 version) software is utilized to design primers at complementary sequences at two ends of SSR, the length of the primers is 20-25bp, the amplification length of the primers is 150-350 bp, the GC content is 40-60%, the annealing temperature is 50-55 ℃, the difference between the Tm values of the upstream and downstream primers is not more than 2 ℃, and Primer dimers, mismatch and hairpin structures are avoided.
The wild jujube DNA in the step (4) is extracted by adopting a CTAB method, the integrity of the DNA is detected by 1% agarose gel electrophoresis after the extraction, and the concentration and purity of the DNA are detected by a Nanodrop ND2000 spectrophotometer (Thermo Scientific, wilmington, DE). The A260/A280 detection value of all sample DNA is between 1.79 and 1.95. The concentration of all DNA was diluted to 100 ng/. Mu.L -1 The mixture is preserved in a refrigerator at the temperature of minus 20 ℃ for standby.
The PCR amplification system in the step (5) is 20 mu L: dNTP 0.8 mu L, tag enzyme 0.2 mu L, 10 Xbuffer 2 mu L, ddH 2 O15.6. Mu.L, forward and reverse primers each 0.2. Mu.L, DNA 1. Mu.L; the PCR reaction procedure was: 94 DEG CPre-denaturing for 3min; denaturation at 94℃for 30s, annealing at 53℃for 30s, extension at 72℃for 60s, and cycling for 35 times; extending at 72deg.C for 10min, and preserving at 4deg.C.
The primer validity in the step (6) is identified as follows: after PCR amplification, products are subjected to validity identification after 10% polyacrylamide gel electrophoresis and silver staining, then an original 0, 1 data matrix is constructed through Gene map 4.1 software, and genetic diversity indexes of SSR sites such as allele factors (na), effective allele factors (ne), shannon's information index (H0), nei's Gene diversity index (H) and the like are calculated and observed through POPGENE Version1.32 software. Cluster analysis was performed in UPGMA using NTSYS Version2.10 software.
The application of the wild jujube SSR molecular marker primer group in wild jujube germplasm resource identification, genetic relationship and genetic diversity analysis has the following effective effects:
1. the SSR molecular marker related to the identification of the wild jujube germplasm resources and the corresponding primer combination and application thereof are SSR molecular marker primers developed based on wild jujube transcriptome data, have multiple sites and comprehensive information, and have universality among wild jujube germplasm resources.
2. The materials used by the invention are derived from the main production areas (Shaanxi, shanxi, hebei, henan, liaoning and Gansu) of wild jujube in various places throughout the country, have wide coverage range and can comprehensively identify the genetic relationship of wild jujube germplasm resources.
3. Compared with the traditional molecular marker, the SSR molecular marker developed by the invention has accuracy, stability and effectiveness, provides a theoretical basis for germplasm resource identification, variety identification and molecular auxiliary breeding of wild jujube, and has good application prospect.
Drawings
FIG. 1 wild jujube germplasm resources SSR molecular marker development
FIG. 2 wild jujube germplasm resource Cluster map
Detailed Description
In order to make the content of the invention more understandable, the technical scheme of the invention is further described below with reference to specific embodiments.
1. Transcriptome sequencing
Transcriptome sequencing samples are leaves of wild jujube which is a river-north Chen-dao-di medicinal material, are collected in a wild jujube germplasm resource base in the inner hill city of river-north province in 2022 month, liquid nitrogen is quickly frozen after the samples are collected, and transcriptome sequencing is carried out in Beijing Baimaike biotechnology limited company to obtain 40Gb data quantity.
2. Transcriptome SSR detection
In the analysis, the MISA software is adopted to carry out SSR detection on unigene, and 6 types of SSRs can be identified: single base (Mono-nucleotide) repeat SSR, double base (Di-nucleotide) repeat SSR, triple base (Tri-nucleotide) repeat SSR, tetra-nucleotide) repeat SSR, pentabase (Penta-nucleotide) repeat SSR, and hexabase (Hexa-nucleotide) repeat SSR.
SSR primer design
The positions of SSR on transcriptome are different, but the sequences at two ends are mostly conserved single copy sequences, so that the primer is designed according to the complementary sequences at two ends of SSR, the single fragment is amplified by PCR reaction (PCR products with different lengths can be amplified by PCR method because of different numbers of tandem repeats of core sequence), and the obtained products are subjected to gel electrophoresis, thus displaying polymorphism of SSR locus. The Primer3.0 is adopted for SSR primer design, the primer length is 20-25bp, the primer amplification length is 150-350 bp, the GC content is 40-60%, the annealing temperature is 50-55 ℃, the difference between the Tm values of the upstream primer and the downstream primer is not more than 2 ℃, and the occurrence of primer dimer, mismatch and hairpin structure is avoided.
PCR reaction System
(1) Wild jujube DNA extraction
9 parts of wild jujube germplasm resource DNA (Table 1) are extracted by a modified CTAB method, and the steps are as follows:
1) Taking about 1g of fresh leaves of wild jujube, rapidly grinding the fresh leaves into powder under the freezing of liquid nitrogen, adding a small amount of PVP (polyvinylpyrrolidone K30) in the middle, transferring the ground powder into a 10mL centrifuge tube, adding 4mL CTAB preheated at 65 ℃, and preserving the heat for 30-60 min at 65 ℃ for a plurality of times during the light shaking.
2) An equal volume of chloroform was added: isoamyl alcohol (24:1) is gently mixed to be milky white, and centrifuged at 11,000rpm for 15min at 15-20 ℃.
3) The supernatant was transferred to a new 10mL centrifuge tube, added with 0.6 times of isopropanol and shaken well, and left at-20℃for 30min.
4) Centrifuging at 10,000rpm for 5min at 4deg.C, discarding supernatant, rinsing the precipitate with 70% ethanol for 2 times, and blow-drying on a clean bench.
5) The dried DNA was dissolved in 2mL of TE buffer, and 2.5. Mu.L of RNaseA was added thereto, followed by incubation at 37℃for 1 hour.
6) An equal volume of benzene was added: chloroform: isoamyl alcohol (25:24:1), chloroform: isoamyl alcohol (24:1) is extracted for 1 time respectively, and the isoamyl alcohol are mixed together by shaking, so that severe shaking is avoided, and DNA is prevented from being sheared mechanically.
7) Centrifuging at 11,000rpm at 4deg.C for 10min, transferring the supernatant to another clean centrifuge tube, adding 0.1 volume of NaAC and 2.5 times volume of frozen absolute ethanol, and precipitating at-20deg.C for 30min.
8) Centrifuge at 10,000rpm for 10min at 4 ℃. The supernatant was discarded, the DNA was transferred to a 1.5mL centrifuge tube, rinsed 2 times with 70% ethanol, and dried on a super clean bench.
9) The DNA was dissolved in an appropriate amount of TE buffer and stored in a refrigerator at 4℃or-20 ℃.
10 A small amount of the DNA solution was subjected to agarose electrophoresis, and DNA integrity was checked by 1% agarose gel electrophoresis, and DNA concentration and purity were checked by a NanoDrop ND2000 spectrophotometer (Thermo Scientific, wilmington, DE). The A260/A280 detection value of all sample DNA is between 1.79 and 1.95. The concentration of all DNA was diluted to 100 ng/. Mu.L -1 The mixture is preserved in a refrigerator at the temperature of minus 20 ℃ for standby.
TABLE 1 wild jujube germplasm resources sample Source
(2) PCR amplification system
Taking wild jujube DNA as a template, and a PCR amplification system is 20 mu L: dNTP 0.8 mu L, tag enzyme 0.2 mu L, 10 Xbuffer 2 mu L, ddH 2 O15.6. Mu.L, 0.2. Mu.L each of forward and reverse primers, and 1. Mu.L of DNA.
(3) PCR reaction procedure
Pre-denaturation at 94℃for 3min; denaturation at 94℃for 30s, annealing at 53℃for 30s, extension at 72℃for 60s, and cycling for 35 times; extending at 72deg.C for 10min, and preserving at 4deg.C.
5. Primer screening and polymorphism analysis
100 pairs of primers are randomly synthesized from SSR sites obtained from transcriptomes, PCR amplification is carried out on wild jujube germplasm resources, the amplified products are subjected to validity identification after 10% polyacrylamide gel electrophoresis and silver staining (figure 1), then an original 0-1 data matrix is constructed through Gene map 4.1 software, and genetic diversity indexes of SSR sites such as allele factors (na), effective allele factors (ne), shannon's information index (H0), nei's Gene diversity index (H) and the like are calculated and observed through POPGENEVERSION1.32 software. Cluster analysis was performed in terms of UPGMA using the NTSYS version2.10 software. 4 pairs of SSR molecular marker primer combinations with higher polymorphism are obtained through the steps. Carrying out PCR (polymerase chain reaction) amplification on 9 parts of wild jujube germplasm from different wild jujube producing areas nationwide by using 4 pairs of primers to obtain 9 alleles, wherein the effective allele factor variation range between SSR loci is 1.38-2, and the average value is 1.71; the variation range of the shannon coefficient is 0.45-0.83, and the average value is 0.62; nei's gene-like index was between 0.28 and 0.50, with an average value of 0.40 (Table 2).
TABLE 2 wild jujube primer information and polymorphism information
6. Analysis of genetic diversity of wild jujube germplasm resources
The genetic similarity coefficient reflects the genetic relationship among the wild jujube germplasm, and the 9 wild jujube germplasm similarity coefficients are 0.6364-1.0000 (table 3). Genetic similarity between Yanan and Gansu Qing Yang, yanan and Gansu Qing county, gansu Qing Yang and Gansu Qing county is 1.0000, which indicates that the germplasm has closer relationship. The similarity coefficient between Gansu Qing Yang and Shanxi Lin, gansu Qing Yang and Hebei Pingshan and Gansu Huan county is the lowest and 0.6364, which indicates that the genetic relationship is far.
TABLE 3 wild jujube germplasm resources genetic similarity analysis
As shown in fig. 2, the UPGMA clustering results performed on 9 parts of jujube germplasm showed that at a similarity coefficient of 0.87, 9 parts of germplasm could be clustered into 4 categories: the Yanan area of Shaanxi, the Qingyang area of Gansu and the Qingyang area of Gansu are class I; the Liaoning Chaoyang region and Liaoning Fuxin region are class II; the Shanxi Lin area, hebei Pingshan area and Hebei Chengtai area are class III; the Henan Luoyang area is IV type.
The foregoing describes embodiments of the present invention and all equivalent variations and modifications that come within the scope of the present invention.
Claims (4)
1. The SSR molecular marker primer combination for identifying the wild jujube germplasm resources is characterized by comprising 4 pairs of primers, including SSR44, SSR48, SSR86 and SSR97, wherein the upstream and downstream nucleotide sequences corresponding to the primers are as follows:
(1) SSR44 primer:
SEQIDNO.1:SSR44-F:5′-CTCATCGGCGAGATCTTCCC-3′
SEQIDNO.2:SSR44-R:5′-TACCGAAAAACTCGCCGGAA-3′
(2) SSR48 primer:
SEQIDNO.3:SSR48-F:5′-AGGGAGAGATGGGAGCATGT-3′
SEQIDNO.4:SSR48-R:5′-TGCACGACCCCACATTCTAC-3′
(3) SSR86 primer:
SEQIDNO.5:SSR86-F:5′-GCATGGCGTGTTGGATTTGT-3′
SEQIDNO.6:SSR86-R:5′-ATCCAACTTAGCTGCCCACC-3′
(4) SSR97 primer:
SEQIDNO.7:SSR97-F:5′-GCCGATGATAAAACCGCCAC-3′
SEQIDNO.8:SSR97-R:5′-TTGGTGAAGACGCTGAGGAC-3′。
2. the use of the SSR molecular marker primer combination of claim 1 in the identification of wild jujube germplasm resources.
3. The use according to claim 2, characterized in that: the method comprises the following steps: (1) SSR primer design; (2) wild jujube DNA extraction; (3) PCR amplification; (4) And (5) carrying out validity identification on the PCR product by polyacrylamide gel electrophoresis and staining.
4. A use according to claim 3, characterized in that: in step (3), the PCR amplification system was 20. Mu.L: dNTP 0.8 mu L, tag enzyme 0.2 mu L, 10 Xbuffer 2 mu L, ddH 2 O15.6. Mu.L, forward and reverse primers each 0.2. Mu.L, DNA 1. Mu.L; the PCR reaction procedure was: pre-denaturation at 94℃for 3min; denaturation at 94℃for 30s, annealing at 53℃for 30s, extension at 72℃for 60s, and cycling for 35 times; extending at 72deg.C for 10min, and preserving at 4deg.C.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310758096.7A CN116790790B (en) | 2023-06-26 | 2023-06-26 | Wild jujube SSR molecular marker development method and application thereof in wild jujube germplasm resource identification |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310758096.7A CN116790790B (en) | 2023-06-26 | 2023-06-26 | Wild jujube SSR molecular marker development method and application thereof in wild jujube germplasm resource identification |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN116790790A CN116790790A (en) | 2023-09-22 |
| CN116790790B true CN116790790B (en) | 2023-12-29 |
Family
ID=88037237
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310758096.7A Active CN116790790B (en) | 2023-06-26 | 2023-06-26 | Wild jujube SSR molecular marker development method and application thereof in wild jujube germplasm resource identification |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116790790B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR102335806B1 (en) * | 2020-11-04 | 2021-12-03 | 충북대학교 산학협력단 | Molecular marker based on chloroplast genome sequence for discriminating Zizyphus jujuba 'SanJo' cultivar and uses thereof |
| CN115786473A (en) * | 2022-09-13 | 2023-03-14 | 河北农业大学 | Screening method based on jujube genome polymorphism SSR locus and bar code generation |
| CN116240310A (en) * | 2023-04-04 | 2023-06-09 | 江西农业大学 | SSR molecular marker combination for evaluating genetic diversity of Choerospondias axillaris and application thereof |
-
2023
- 2023-06-26 CN CN202310758096.7A patent/CN116790790B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR102335806B1 (en) * | 2020-11-04 | 2021-12-03 | 충북대학교 산학협력단 | Molecular marker based on chloroplast genome sequence for discriminating Zizyphus jujuba 'SanJo' cultivar and uses thereof |
| CN115786473A (en) * | 2022-09-13 | 2023-03-14 | 河北农业大学 | Screening method based on jujube genome polymorphism SSR locus and bar code generation |
| CN116240310A (en) * | 2023-04-04 | 2023-06-09 | 江西农业大学 | SSR molecular marker combination for evaluating genetic diversity of Choerospondias axillaris and application thereof |
Non-Patent Citations (4)
| Title |
|---|
| 86份酸枣种质资源筛选和遗传多样性分析;李世鹏;陈叶;郭明欣;田珊;刘红霞;赵旭升;;安徽农业科学(11);全文 * |
| 基于SSR标记的255个枣品种亲缘关系和群体遗传结构分析;刘秀云;李慧;刘志国;赵锦;刘孟军;;中国农业科学(14);全文 * |
| 枣与酸枣的SSR遗传多样性研究;张鹏飞;尉东峰;刘亚令;宋美玲;张茹;雍鹏;;华北农学报(02);全文 * |
| 枣和酸枣杂交后代遗传多样性的SSR分析;闫芬芬;郑兴娟;罗智;王玖瑞;刘孟军;;西北林学院学报(03);第97-103+158页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN116790790A (en) | 2023-09-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Singh et al. | Evaluation of genetic diversity in turmeric (Curcuma longa L.) using RAPD and ISSR markers | |
| US11572594B2 (en) | Characteristic sequence, labeled primer and identification method of Carya illinoensis variety Davis | |
| Deepa et al. | A simple and efficient protocol for isolation of high quality functional RNA from different tissues of turmeric (Curcuma longa L.) | |
| CN111500763B (en) | SNP molecular marker related to palmitoleic acid content in oil tea seed oil and application thereof | |
| CN106636342B (en) | Acquisition method and application of EST-SSR labeled primer group developed based on ligusticum wallichii transcriptome sequence | |
| CN106978504B (en) | Preparation method and application of spinach SSR (simple sequence repeat) marker | |
| Kavas et al. | Discovery of simple sequence repeat (SSR) markers in hazelnut (Corylus avellana L.) by transcriptome sequencing and SSR-based characterization of hazelnut cultivars | |
| CN116790790B (en) | Wild jujube SSR molecular marker development method and application thereof in wild jujube germplasm resource identification | |
| CN110699480B (en) | Primer group for hybridization of EST-SSR (expressed sequence tag-simple sequence repeat) markers of cymbidium kanran and screening method | |
| CN116103429B (en) | Primer for rapidly identifying molecular markers of germplasm of rhizoma zingiberis, molecular markers and application | |
| CN101694696A (en) | Method for preparing molecular bar code system for describing and discriminating animals and plants and application thereof | |
| CN111534631A (en) | 2 SNP molecular markers related to oil content of oil-tea camellia kernel and application thereof | |
| CN110734996A (en) | group molecular marker linked with caffeine content of tea tree and application thereof | |
| CN111944917A (en) | Method for developing camellia plant SSR primers based on transcriptome sequencing | |
| Chen et al. | SSR marker development in Clerodendrum trichotomum using transcriptome sequencing | |
| CN108424975B (en) | Pueraria SSR marker primer pair developed based on transcriptome sequence and application thereof | |
| CN111518943B (en) | SNP molecular marker related to eicosenoic acid content in camellia seed oil and application thereof | |
| CN114766348A (en) | Method for identifying two parents of water lily hybrid based on ITS sequence and matK sequence | |
| CN116516042B (en) | SSR molecular marker related to fructus forsythiae germplasm resource identification, corresponding primer and application thereof | |
| Li et al. | Phylogenetic relationships in Actinidia as revealed by RAPDs and PCR-RFLPs of mtDNA | |
| CN113403417A (en) | SSR molecular marker AerM01 for sex identification of actinidia arguta and application thereof | |
| CN118389724A (en) | Mugwort SSR molecular marker development method and application thereof in mugwort interstitium identification | |
| Huang et al. | Comparative transcriptome analysis and simple sequence repeat marker development for two closely related Isodon species used as ‘Xihuangcao’herbs | |
| CN113151558B (en) | SSR molecular marker based on Ardisia crispa transcriptome as well as identification method and application thereof | |
| CN117210609B (en) | Primer pair, kit and identification method for identifying cymbidium sinense |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |