CN107586867B - Characteristic sequence, labeled primer and identification method of apocarya variety Pawnee - Google Patents
Characteristic sequence, labeled primer and identification method of apocarya variety Pawnee Download PDFInfo
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Abstract
The invention relates to a pair of characteristic sequences of a pecan variety Pawnee with high specificity, a molecular specificity marker primer and a method capable of quickly identifying the pecan variety Pawnee. The sequence of the molecular specific marker primer is as follows: an upstream primer: 5'-GCTCTTGTCACTTGGGCCTA-3', respectively; a downstream primer: 5'-TGAGGATGGAGGCTTTTAGGC-3' are provided. The molecular specific marker primer can be used for quickly and early identifying the pecan variety Pawnee, the method is simple, quick and accurate, and the molecular specific marker primer is a molecular means which cannot be replaced by apparent characteristic identification of the pecan variety.
Description
(I) technical field
The invention relates to a characteristic sequence of a carya illinoensis variety Pawnee, a molecular specificity marker primer and a method for rapidly identifying the carya illinoensis variety Pawnee 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 of a carya illinoensis variety Pawnee, a molecular specific marker primer and a method for rapidly identifying the carya illinoensis variety Pawnee by using the molecular specific marker primer.
The technical scheme adopted by the invention is as follows:
the characteristic sequence of the apocarya variety Pawnee is as follows: 5'-AATTCAATGGCTCTTGTCACTTGGGCCTATATTGTGGAATTTTGCTTCAGATGGAGATTTCTTATAAAGGTAAAGATTTATGTATTACTACAAAAGTATAATGGGGTTTTTGAAGAGCCTAAAAGCCTCCATCCTCATAGGACTCAAGACCATTAGATTGTGTTGAATGATGAGTCAGTTCCTATGATTTTAAGACCATATAGATACCCTTATTATCAAAAGACAGGGAT-3' (SEQ ID No.1)
The invention also relates to a molecular specific marker primer of the pecan variety Pawnee, wherein the primer sequence is as follows:
an upstream primer: 5'-TGTCAAGGACAAACGGGTGA-3', respectively;
a downstream primer: 5'-TGGAGAAGCGAAGTAGTCAACC-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 pecan variety Pawnee 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 after primary screening and repeated screening of repeated sampling for more than three times, finally obtained molecular specific marker primers are used for carrying out PCR amplification on the pecan variety, only Pawnee can obtain a specific fragment (SEQ ID No.2) with the size of 128bp, and other pecan varieties cannot obtain specific fragments. 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 128 bp: 5'-GCTCTTGTCACTTGGGCCTATATTGTGGAATTTTGCTTCAGATGGAGATTTCTTATAAAGGTAAAGATTTATGTATTACTACAAAAGTATAATGGGGTTTTTGAAGAGCCTAAAAGCCTCCATCCTCA-3' (SEQ ID No. 2).
Pawnee is a variety introduced by the United states department of agriculture scientific research (USDA-ARS) by cross breeding, released in 1984, selected from the progeny of a cross of 'Mohawk' × 'Starking Hardy Giant' in 1963. The tree is small and the growth potential is moderate. The male flowers are in a first-maturing type, but the probability of meeting the male flowers in the female flower period is high, and the male inflorescence is short. The nuts are early ripe, the average single fruit weight is about 10.0g, the nuts are oval, and the tops and the bottoms are round; the kernel is gold with black spots, the ridge in the back is wide, the main groove is wide, and the crack at the base part is obvious.
The invention also relates to a method for rapidly identifying the Carya illinoensis variety Pawnee by using the molecular specificity marker primer, which comprises the following steps: extracting genome DNA of 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, and if a DNA band with the size of 128bp appears in an electrophoresis result, determining that the carya illinoensis variety to be detected is a carya illinoensis variety Pawnee, otherwise, determining that the carya illinoensis variety is not the carya illinoensis variety Pawnee; the sequence of the molecular specific marker primer is as follows:
an upstream primer: 5'-TGTCAAGGACAAACGGGTGA-3', respectively;
a downstream primer: 5'-TGGAGAAGCGAAGTAGTCAACC-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 image analyzer, and if a 128bp DNA band appears in an electrophoresis result, determining that the variety of the carya illinoensis to be detected is Pawnee; otherwise, the result is no.
The invention has the following beneficial effects: the molecular specific marker primer can be used for quickly identifying the pecan variety Pawnee, the method is simple, quick and accurate, and is a molecular means which cannot be replaced by apparent characteristic identification of the pecan variety.
(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 19 is that a specific DNA strip with the molecular weight of 128bp is amplified by the Carya illinoensis variety Pawnee; the rest numbers are other apocarya varieties, and no specific DNA band with the size of 128bp 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 0.05g 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'-TGTCAAGGACAAACGGGTGA-3', respectively; and a downstream primer: 5'-TGGAGAAGCGAAGTAGTCAACC-3', synthesized by Shanghai Bioengineering technology, Inc.
(4) And (3) PCR amplification:
composition of PCR reaction solution (15. mu.L):
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.
A clear, bright and stable specific DNA band with the molecular weight of about 128bp is amplified from the thin-shell hickory varieties Pawnee with the numbers of 19 respectively, and the rest of the thin-shell hickory varieties do not generate a special DNA band with the size of 128bp and other non-target bands.
Sequence listing
<110> scientific institute of forestry in Zhejiang province
<120> characteristic sequence, labeled primer and identification method of apocarya variety Pawnee
<160>4
<170>SIPOSequenceListing 1.0
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<211>230
<212>DNA
<213>Carya illinoensis
<400>1
aattcaatgg ctcttgtcac ttgggcctat attgtggaat tttgcttcag atggagattt 60
cttataaagg taaagattta tgtattacta caaaagtata atggggtttt tgaagagcct 120
aaaagcctcc atcctcatag gactcaagac cattagattg tgttgaatga tgagtcagtt 180
cctatgattt taagaccata tagataccct tattatcaaa agacagggat 230
<210>2
<211>128
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<223> Artificial sequence
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gctcttgtca cttgggccta tattgtggaa ttttgcttca gatggagatt tcttataaag 60
gtaaagattt atgtattact acaaaagtat aatggggttt ttgaagagcc taaaagcctc 120
catcctca 128
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<211>20
<212>DNA
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<223> Artificial sequence
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<212>DNA
<213>Unknown
<220>
<223> Artificial sequence
<400>4
tgaggatgga ggcttttagg c 21
Claims (4)
1. The molecular specificity marker primer of the pecan variety Pawnee has the following primer sequences:
an upstream primer: 5'-GCTCTTGTCACTTGGGCCTA-3', respectively;
a downstream primer: 5'-TGAGGATGGAGGCTTTTAGGC-3' are provided.
2. A method for rapidly identifying the Carya illinoensis variety Pawnee by using the molecular specific marker primer of claim 1, which comprises the following steps: extracting genome DNA of leaves of the carya illinoensis variety to be detected as a template, taking the molecular specificity marker primer as an amplification primer, carrying out PCR amplification, carrying out electrophoresis detection on an amplification product, and if an electrophoresis result shows a unique 128bp specific DNA band, determining that the carya illinoensis variety to be detected is the carya illinoensis variety and is called Pawnee, otherwise, determining that the carya illinoensis variety is not the carya illinoensis variety; the sequence of the molecular specific marker primer is as follows:
an upstream primer: 5'-GCTCTTGTCACTTGGGCCTA-3', respectively;
a downstream primer: 5'-TGAGGATGGAGGCTTTTAGGC-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 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 128bp DNA band appears in an electrophoresis result, determining that the variety of the carya illinoensis to be detected is Pawnee, otherwise, determining that the variety is not Pawnee.
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| CN109652588B (en) * | 2019-02-28 | 2022-02-08 | 浙江省林业科学研究院 | Characteristic sequence, labeled primer and identification method of apocarya variety Elliott |
| CN109694865B (en) * | 2019-02-28 | 2022-02-11 | 浙江省林业科学研究院 | Characteristic sequence, labeled primer and identification method of apocarya variety Desirable |
| CN109652428B (en) * | 2019-02-28 | 2022-09-20 | 浙江省林业科学研究院 | Characteristic sequence, labeled primer and identification method of apocarya variety Sioux |
| CN109652415B (en) * | 2019-02-28 | 2021-12-17 | 浙江省林业科学研究院 | Characteristic sequence, labeled primer and identification method of pecan variety Osage |
| CN109652589B (en) * | 2019-02-28 | 2022-01-11 | 浙江省林业科学研究院 | Characteristic sequence, labeled primer and identification method of apocarya variety Gloria Grande |
| CN111719009B (en) * | 2019-03-22 | 2023-03-14 | 云南省林业科学院 | Primer group, kit and method for authenticating apomixis filial generation authenticity of paullo walnuts |
| CN114182034B (en) * | 2021-11-04 | 2023-08-04 | 中国林业科学研究院亚热带林业研究所 | SSR molecular marker of apocarya variety McMillian and application thereof |
| CN113981125B (en) * | 2021-11-04 | 2023-07-11 | 中国林业科学研究院亚热带林业研究所 | Molecular marker of apocarya variety Creek and application thereof |
| CN114182033B (en) * | 2021-11-04 | 2023-08-04 | 中国林业科学研究院亚热带林业研究所 | SSR molecular markers of apocarya Mahan, pawnee and Greenliver and application thereof |
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