CN108330164B - Characteristic sequence, primer and identification method of apocarya variety Moore - Google Patents
Characteristic sequence, primer and identification method of apocarya variety Moore Download PDFInfo
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- CN108330164B CN108330164B CN201710782321.5A CN201710782321A CN108330164B CN 108330164 B CN108330164 B CN 108330164B CN 201710782321 A CN201710782321 A CN 201710782321A CN 108330164 B CN108330164 B CN 108330164B
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- 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
Abstract
The invention relates to a pair of molecular specificity marker primers of a pecan variety Moore with high specificity and a method capable of quickly identifying the pecan variety Moore. The primer sequences are as follows: an upstream primer: 5'-CGTGGCAAAAGCACAAAATGT-3', respectively; a downstream primer: 5'-AGTTGGGGAAAGTGGCTTCA-3' are provided. The molecular specific marker primer can be used for quickly and early identifying the variety Moore of the apocarya, is simple, quick and accurate, and is a molecular means which cannot be replaced by apparent characteristic identification of the apocarya variety.
Description
(I) technical field
The invention relates to a characteristic sequence of a carya illinoensis variety Moore, a molecular specificity marker primer and a method for rapidly identifying the carya illinoensis variety Moore by using the molecular specificity marker primer.
(II) background of the invention
Carya illinoensis (Carya)
K.koch) is the most economically valuable species of the hickory genus of the juglandaceae family. The apocarya is a typical outcrossing plant, and the current production practice shows that the variety configuration of the apocarya is one of the main factors influencing the yield of the apocarya. The United states is one of the original producing areas of the carya illinoensis and the central producing area thereof, and about 1000 named cultivars have been bred for years. The introduction of the apocarya in China has a history of more than 100 years, the varieties which are commonly used in the current production are dozens, and the production practice of many years proves that the majority of subtropical regions in China are suitable for the growth of the apocarya.
However, the main problems faced by the current apocarya producing area of China are low yield and instability, and the reason for the phenomenon has multiple aspects, one of which is that the current introduced varieties lack clear genetic relationship analysis, and some hybrids are misnamed disorderly, so that effective parent selection and reasonable configuration are difficult to perform, and variety identification, popularization, communication and new variety cultivation are inconvenient, therefore, the development of stable and specific DNA fingerprint markers of the varieties on a molecular level is a scientific way for realizing accurate and rapid identification of the apocarya varieties.
The variety identification and genetic relationship analysis of apocarya based on SSR molecular markers have been reported at home and abroad, but the detection is more complicated and the result is unstable.
Disclosure of the invention
The invention aims to provide a characteristic sequence and a molecular specificity marker primer of a carya illinoensis variety Moore and a method for rapidly identifying the carya illinoensis variety Moore by utilizing the pair of primers.
The technical scheme adopted by the invention is as follows:
the characteristic sequence of the apocarya variety Moore comprises the following sequences: 5'-AATTCGTGGCAAAAGCACAAAATGTTGTAGTGCGTCCCAATTACTGATATAATGCAGTACTACTGCTGAAGAAAAGTCAGATAAGTGCGTCCTATAATGCAGTACTACTGCTGAAGAAAAGTCAGATACTGGAGCTTGATGAAGCCACTTTCCCCAACTGAAACAATGCAATTATGCAGCAGATTCTATCATTCAACAGTGATCG-3' (SEQ ID No.1)
The invention also relates to a molecular specific marker primer of the apocarya variety Moore, wherein the primer sequence is as follows:
an upstream primer: 5'-CGTGGCAAAAGCACAAAATGT-3', respectively;
a downstream primer: 5'-AGTTGGGGAAAGTGGCTTCA-3' are provided.
The primer pair is characterized in that a PCR technology is adopted, varieties with large shape difference are screened from 24 common varieties to carry out simplified gene sequencing and comparative analysis, after a specific DNA fragment (SEQ ID No.1) of a carya illinoensis variety Moore is obtained through a large amount of screening tests, the fragment is cloned and sequenced, more than 1000 pairs of primers are designed for the gene fragment with large sequence difference, screening and verification are carried out in 24 samples, and finally the obtained molecular specific marker primer is subjected to PCR amplification on the carya illinoensis variety through primary screening and repeated screening of repeated sampling for more than three times, so that a specific fragment (SEQ ID No.2) with the size of 155bp can be obtained by only Moore, and no specific fragment can be obtained by other carya illinoensis varieties. It should be noted that the molecular specific marker primers of the invention are limited to the identification of the variety of apocarya, i.e. the sample to be tested is limited to apocarya.
The specific primer amplification product designed aiming at the partial sequence of the characteristic sequence is 155 bp: 5'-CGTGGCAAAAGCACAAAATGTTGTAGTGCGTCCTATAATGCAGTACTACTGCTGAAGAAAAGTCAGATAAGTGCGTCCCAATTACTGATATAATGCAGTACTACTGCTGAAGAAAAGTCAGATACTGGAGCTTGATGAAGCCACTTTCCCCAACT-3' (SEQID No.2)
Moore is a species selected from the seedling trees in Florida, USA, and the tree body is small, and the growth potential is medium. The male flowers are of early maturity type, have short inflorescences and are good early pollination varieties. Small fruit type, average single fruit weight about 6.0g, nut oval shape, flat top and bottom, slightly flat cross section; the kernel is golden to light brown, the ridge in the back is narrow, and the main groove is tight.
The invention also relates to a method for rapidly identifying the apocarya variety Moore by using the molecular specific marker primer, which comprises the following steps: extracting genome DNA of the leaves of the carya illinoensis variety to be detected as a template, taking the molecular specificity marker primer as an amplification primer, performing PCR amplification, performing electrophoresis detection on an amplification product, wherein if an electrophoresis result shows a DNA strip with the size of 155bp, the carya illinoensis variety to be detected is Moore, otherwise, the carya illinoensis variety to be detected is not Moore; the sequence of the molecular specific marker primer is as follows:
an upstream primer: 5'-CGTGGCAAAAGCACAAAATGT-3', respectively;
a downstream primer: 5'-AGTTGGGGAAAGTGGCTTCA-3' are provided.
The method of the invention is characterized in that the selection of amplification primers, the DNA extraction, the determination of a PCR reaction system and reaction conditions, and the electrophoresis detection can be carried out according to the conventional method in the field.
Compared with the existing SSR marker identification method of the apocarya variety, the reliability of the method is improved greatly, and the method does not need electrophoresis analysis or sequencing with the resolution of 1-2bp after SSR marker amplification, and the method only needs common electrophoresis after common PCR to judge whether the band exists.
Preferably, the PCR amplification system of the present invention comprises:
the PCR amplification conditions were as follows: pre-denaturation at 94 ℃ for 300 s; denaturation at 95 ℃ for 30s, annealing at 56 ℃ for 60s, and extension at 72 ℃ for 50s for 30-40 cycles; finally, the temperature was leveled off at 72 ℃ for 300s, and the stopping temperature was 4 ℃.
The final concentration of the PCR Buffer is 1 x, which means that the concentration of each component in the PCR Buffer in the reaction system is the same as that of 1 x PCR Buffer, and 10 x PCR Buffer with the volume of 1/10 of the reaction system is usually selected. The 10 × PCR Buffer component is: 100mM Tris-HCl (pH 8.5), 500mM KCl, 25mM MgCl2And 1.0% Triton-X-100 in ddH2O。
Specifically, the method comprises the following steps:
(1) adding liquid nitrogen into the leaves of the carya illinoensis to be detected, grinding, and extracting the genomic DNA of the leaves of the carya illinoensis to be detected by an SDS-CTAB method;
(2) performing PCR amplification by taking the genomic DNA extracted in the step (1) as a template and the molecular specific marker primer as an amplification primer:
typically, a PCR amplification system consists of 25. mu.L each as follows:
alternatively, the composition of each 15. mu.L of the PCR reaction was as follows:
the PCR reaction conditions were as follows:
pre-denaturation at 94 ℃ for 300 s; denaturation at 95 ℃ for 30s, annealing at 56 ℃ for 60s, and extension at 72 ℃ for 50s for 30-40 cycles; finally, filling the mixture for 300s at 72 ℃, wherein the termination temperature is 4 ℃;
(3) taking 5 mu L of the amplified product in the step (2), uniformly mixing the amplified product with 1 mu L of 0.25% bromophenol blue buffer solution, spotting the mixed solution on 1.5% agarose gel, carrying out electrophoresis in 1 XTAE buffer solution at a voltage of 5V/cm, carrying out EB dyeing after the electrophoresis is finished, taking a picture on an automatic gel imaging system, and if a DNA band of 155bp appears in an electrophoresis result, determining that the variety of the carya illinoensis to be detected is Moore; otherwise, the result is no.
The invention has the following beneficial effects: the molecular specific marker primer can be used for quickly identifying the variety Moore of the carya illinoensis, is simple, quick and accurate, and is a molecular means which cannot be replaced by apparent characteristic identification of the variety of the carya illinoensis.
(IV) description of the drawings
FIG. 1 shows the results of PCR amplification of 24 Carya illinoensis varieties; m is Takara DL2000 marker; only the number 1 is that a specific DNA strip with the molecular weight of 155bp is amplified by the Moore of the carya illinoensis variety; the rest numbers are other apocarya varieties, and no specific DNA band with the size of 155bp is generated.
(V) detailed description of the preferred embodiments
The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:
example 1:
(1) extracting the genomic DNA of the apocarya variety:
taking 0.01g of young leaves of the apocarya variety to be detected, adding liquid nitrogen to thoroughly grind, extracting the genome DNA by adopting an SDS-CTAB method, and extracting for multiple times to obtain the crude extract of the genome DNA of the apocarya variety. The crude DNA extract was purified with a Magabio nucleic acid purification kit (Bori Bio, Hangzhou, China) and checked for integrity, purity and concentration by 1.5% agarose gel electrophoresis and DNA/RNA UV spectrophotometer (GeneQuant Pro, GE Healthcare). DNA samples with OD260/OD280>1.8 were used for subsequent PCR amplification. The DNA extract was stored at-20 ℃ in a refrigerator for further use.
(2) Designing specific PCR amplification primers, wherein the sequences of the primer pairs are as follows:
an upstream primer: 5'-CGTGGCAAAAGCACAAAATGT-3', respectively; and a downstream primer: 5'-AGTTGGGGAAAGTGGCTTCA-3', synthesized by Shanghai Bioengineering technology, Inc.
(4) And (3) PCR amplification:
composition of PCR reaction solution (15. mu.L):
2X TsingKE master mix (Ongko Bio, Beijing) 7.5. mu.L
10 μ M of each of the upstream and downstream primers 0.6 μ L
20 ng/. mu.L template DNA 2. mu.L
dd H2O 4.3μL；
The amplification reaction was carried out on a TC-XP type amplification apparatus. Amplification conditions: pre-denaturation at 94 ℃ for 300 s; denaturation at 95 ℃ for 30s, annealing at 56 ℃ for 60s, and extension at 72 ℃ for 50s for 30 cycles; finally, the temperature was leveled off at 72 ℃ for 300s, and the stopping temperature was 4 ℃.
(4) And (3) electrophoresis detection: and (3) taking 3 mu L of the PCR amplification product in the step (3), uniformly mixing with 1 mu L of 0.25% bromophenol blue buffer solution, spotting on 1.5% agarose Gel, carrying out electrophoresis in 1 XTAE buffer solution at a voltage of 5V/cm, staining in an aqueous solution containing 0.5 mu g/mL EB for 30 minutes after the electrophoresis is finished, and then taking pictures on a Bio-rad Gel imaging system Gel Doc.
According to the method, 24 pecan varieties (the numbers 1-24 represent the pecan varieties and are sequentially 1, Moore, 2, Nacono, 3, Van Deman, 4, Mohawk, 5, Davis, 6, Caddo, 7, Choctaw, 8, Osage, 9, Mahan, 10, Hirschi, 11, Peruque, 12, Gloria Grande, 13, Sioux, 14, dependendable, 15, Kiowa, 16, Hunan No.1, 17, Stuart, 18, Desirable, 19, Pawnee, 20, Elliott, 21, Pyzner, 22, Schley, 23, ShaoXing, 24 and Sumner are subjected to electrophoresis detection by PCR amplification maps, and the result is shown in figure 1.
Wherein, a clear, bright and stable specific DNA band with the molecular weight of about 155bp is amplified from the apocarya Moore with the number of 1 respectively, while the rest apocarya varieties have no generation of special DNA bands with the size of 155bp and no generation of other non-target bands.
Sequence listing
<110> scientific institute of forestry in Zhejiang province
<120> characteristic sequence, primer and identification method of apocarya variety Moore
<160>4
<170>SIPOSequenceListing 1.0
<210>1
<211>205
<212>DNA
<213>Carya illinoensis
<400>1
aattcgtggc aaaagcacaa aatgttgtag tgcgtcccaa ttactgatat aatgcagtac 60
tactgctgaa gaaaagtcag ataagtgcgt cctataatgc agtactactg ctgaagaaaa 120
gtcagatact ggagcttgat gaagccactt tccccaactg aaacaatgca attatgcagc 180
agattctatc attcaacagt gatcg 205
<210>2
<211>155
<212>DNA
<213>Unknown
<220>
<223> Artificial sequence
<400>2
cgtggcaaaa gcacaaaatg ttgtagtgcg tcctataatg cagtactact gctgaagaaa 60
agtcagataa gtgcgtccca attactgata taatgcagta ctactgctga agaaaagtca 120
gatactggag cttgatgaag ccactttccc caact 155
<210>3
<211>21
<212>DNA
<213>Unknown
<220>
<223> Artificial sequence
<400>3
cgtggcaaaa gcacaaaatg t 21
<210>4
<211>20
<212>DNA
<213>Unknown
<220>
<223> Artificial sequence
<400>4
agttggggaa agtggcttca 20
Claims (4)
1. The molecular specificity labeling primer of the apocarya variety Moore comprises the following primer sequences:
an upstream primer: 5'-CGTGGCAAAAGCACAAAATGT-3', respectively;
a downstream primer: 5'-AGTTGGGGAAAGTGGCTTCA-3' are provided.
2. A method for rapidly identifying the apocarya variety Moore by using the molecular specific marker primer as claimed in claim 1, the method comprises the following steps: extracting genome DNA of leaves of a to-be-detected carya illinoensis variety as a template, taking the molecular specific marker primer as an amplification primer, performing PCR amplification, performing electrophoresis detection on an amplification product, and if an electrophoresis result shows a unique specific DNA strip of 155bp, determining that the carya illinoensis variety to be detected is a carya illinoensis variety Moore, otherwise, determining that the carya illinoensis variety to be detected is not a carya illinoensis variety Moore; the sequence of the molecular specific marker primer is as follows:
an upstream primer: 5'-CGTGGCAAAAGCACAAAATGT-3', respectively;
a downstream primer: 5'-AGTTGGGGAAAGTGGCTTCA-3' are provided.
3. The method of claim 2, wherein the PCR amplification conditions are as follows: pre-denaturation at 94 ℃ for 300 s; denaturation at 95 ℃ for 30s, annealing at 56 ℃ for 60s, and extension at 72 ℃ for 50s for 30-40 cycles; finally, the temperature was leveled off at 72 ℃ for 300s, and the stopping temperature was 4 ℃.
4. The method of claim 2, characterized in that the method is as follows:
(1) adding liquid nitrogen into the leaves of the carya illinoensis to be detected, grinding, and extracting the genomic DNA of the leaves of the carya illinoensis to be detected by an SDS-CTAB method;
(2) performing PCR amplification by taking the genomic DNA extracted in the step (1) as a template and the molecular specific marker primer as an amplification primer:
the composition of each 25. mu.L of the PCR amplification system was as follows:
10×PCR Buffer 2.5μL
10 mmol/L dNTPs 2.5μL
25 mmol/L MgCl22.5μL
5U/. mu.L Taq enzyme 0.2. mu.L
10 μ M of each of the upstream and downstream primers 1 μ L
20 ng/. mu.L template DNA 3. mu.L
ddH2O is complemented to 25 mu L;
the PCR amplification conditions were as follows:
pre-denaturation at 94 ℃ for 300 s; denaturation at 95 ℃ for 30s, annealing at 56 ℃ for 60s, and extension at 72 ℃ for 50s for 30-40 cycles; finally, filling the mixture for 300s at 72 ℃, wherein the termination temperature is 4 ℃;
(3) and (3) taking 3 mu L of the amplification product obtained in the step (2), uniformly mixing with 1 mu L of 0.25% bromophenol blue buffer solution, spotting the mixture on 1.5% agarose gel, performing electrophoresis in 1 XTAE buffer solution at a voltage of 5V/cm, performing EB (electron beam) dyeing after the electrophoresis is finished, taking a picture on an automatic gel imaging system, and if a unique 155bp DNA band appears in an electrophoresis result, determining that the variety of the carya illinoensis to be detected is Moore, otherwise, determining that the variety is not Moore.
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| CN104946640A (en) * | 2015-07-10 | 2015-09-30 | 浙江省林业科学研究院 | Specificity labeling primer for tea-oil tree improved variety Chang Lin 18 and detection method |
| CN105154529A (en) * | 2015-07-23 | 2015-12-16 | 浙江省林业科学研究院 | Molecular specific marker primer and identification method for fine varieties of camellia oleifera |
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| CN104946640A (en) * | 2015-07-10 | 2015-09-30 | 浙江省林业科学研究院 | Specificity labeling primer for tea-oil tree improved variety Chang Lin 18 and detection method |
| CN105154529A (en) * | 2015-07-23 | 2015-12-16 | 浙江省林业科学研究院 | Molecular specific marker primer and identification method for fine varieties of camellia oleifera |
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| Developing microsatellite DNA markers in pecan;Grauke L J等;《Amer Soc Hort Sci》;20031231;第128卷(第3期);374-380 * |
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