CN116904644A - Molecular marker for identifying M25 navel orange and application thereof - Google Patents
Molecular marker for identifying M25 navel orange and application thereof Download PDFInfo
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- 240000002319 Citrus sinensis Species 0.000 title claims abstract description 93
- 235000005976 Citrus sinensis Nutrition 0.000 title claims abstract description 93
- 239000003147 molecular marker Substances 0.000 title claims abstract description 42
- 210000000349 chromosome Anatomy 0.000 claims abstract description 14
- 239000002773 nucleotide Substances 0.000 claims abstract description 9
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 9
- 238000010586 diagram Methods 0.000 claims description 13
- 238000001962 electrophoresis Methods 0.000 claims description 12
- 238000000034 method Methods 0.000 claims description 12
- 239000000463 material Substances 0.000 claims description 10
- 238000009395 breeding Methods 0.000 claims description 5
- 230000003321 amplification Effects 0.000 claims description 3
- 230000001488 breeding effect Effects 0.000 claims description 3
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 3
- 238000012216 screening Methods 0.000 claims description 3
- 238000012408 PCR amplification Methods 0.000 claims description 2
- 210000001519 tissue Anatomy 0.000 claims description 2
- 101100240528 Caenorhabditis elegans nhr-23 gene Proteins 0.000 abstract description 11
- 108020004414 DNA Proteins 0.000 description 10
- 238000003780 insertion Methods 0.000 description 7
- 230000037431 insertion Effects 0.000 description 7
- 230000035772 mutation Effects 0.000 description 6
- 235000013399 edible fruits Nutrition 0.000 description 5
- 238000004925 denaturation Methods 0.000 description 4
- 230000036425 denaturation Effects 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 238000011161 development Methods 0.000 description 4
- 238000001502 gel electrophoresis Methods 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 3
- 108091081062 Repeated sequence (DNA) Proteins 0.000 description 3
- 229920000936 Agarose Polymers 0.000 description 2
- 241000207199 Citrus Species 0.000 description 2
- 238000000137 annealing Methods 0.000 description 2
- 235000020971 citrus fruits Nutrition 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 239000012154 double-distilled water Substances 0.000 description 2
- 238000003384 imaging method Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- 238000012271 agricultural production Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000007622 bioinformatic analysis Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 238000003976 plant breeding Methods 0.000 description 1
- 230000002028 premature Effects 0.000 description 1
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- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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Abstract
The invention discloses a molecular marker for identifying M25 navel orange and application thereof, wherein the nucleotide sequence of the molecular marker CsChr3-DG is shown as SEQ ID NO.1, and a primer pair for amplifying the molecular marker CsChr3-DG is positioned on a chromosome of the M25 navel orange Chr3, so that quick identification of seedlings of the M25 navel orange is facilitated.
Description
Technical Field
The invention relates to a molecular marker for identifying M25 navel orange and application thereof, belonging to the technical field of plant breeding.
Background
The sweet orange is an important pillar of economy in hilly and mountain areas in China, the variety structure and the ratio of the mature period of the product are seriously unbalanced, and the sweet orange is a factor for restricting the development of the navel orange industry. In order to break through the restriction and promote the industrial development, breeders create navel orange varieties, such as Gannan early navel orange variety, non-child sweet orange middle-breeding No. 7 'and middle-breeding No. 8', by utilizing multiple means. But the unbalance phenomenon of the variety structure and the maturation period proportion of the product still exists.
In bud mutation seed selection, the inventor breeds a new germplasm from 'New He' navel orange, and the germplasm is 10 days earlier in maturity period and larger in fruits compared with the bud mutation source 'New He' navel orange, and is named as M25 navel orange. Because citrus is long in childhood, it is difficult to identify fruit traits in childhood. Therefore, the development of the specific molecular marker for identifying the M25 navel orange can be used for rapidly identifying the M25 seedlings.
Disclosure of Invention
The invention aims to overcome the defects in the prior art, provides a molecular marker for identifying M25 navel oranges and application thereof, and is convenient for quick identification of M25 navel orange seedlings.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
in a first aspect, the invention provides a molecular marker for identifying M25 navel orange, the nucleotide sequence of the molecular marker CsChr3-DG is shown as SEQ ID NO.1, and a primer pair for amplifying the molecular marker CsChr3-DG is positioned on the chromosome of the M25 navel orange Chr 3.
Further, the primer pair for amplifying the molecular marker Cschr3-DG is Cschr3-DG-F and Cschr3-DG-R; the nucleotide sequence of the Cschr3-DG-F is shown as SEQ ID NO.2, and the nucleotide sequence of the Cschr3-DG-R is shown as SEQ ID NO. 3.
In a second aspect, the invention provides a kit comprising a primer pair for amplifying the molecular marker Cschr 3-DG.
In a third aspect, the invention provides a method for identifying M25 navel orange, comprising the step of detecting the primer pair of the molecular marker Cschr3-DG to amplify the genomic DNA of the navel orange.
With reference to the third aspect, further, a method for identifying M25 navel orange includes the steps of:
extracting genome DNA of navel orange varieties;
performing PCR amplification on the extracted genome DNA by using a primer pair of a molecular marker Cschr3-DG to obtain an amplification product;
electrophoresis is carried out on the molecular marker Cschr3-DG to obtain a control electrophoresis gel diagram;
electrophoresis is carried out on the amplified product, and an electrophoresis gel diagram is obtained;
and comparing the electrophoresis gel diagram with a control electrophoresis gel diagram, and screening to obtain the M25 navel orange.
The molecular marker, the method or the kit can be applied to detection of M25 navel orange tissue materials for breeding premature navel orange germplasm.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides a molecular marker Cschr3-DG capable of identifying a transposon insertion site on a chromosome of M25 navel orange Chr3, and can identify whether navel orange seedlings or other propagation materials are M25 germplasm or not, and has important value in detection of first generation and second generation of M25 navel orange.
Drawings
Fig. 1 is a schematic diagram showing fruit morphology comparison of an M25 navel orange and a 'new york' navel orange provided by an embodiment of the present invention;
FIG. 2 is a schematic diagram showing the difference between chromosome sequences of M25 navel orange and 'Newhall' navel orange Chr3 according to the embodiment of the present invention;
FIG. 3 is a gel electrophoresis diagram of molecular marker Cschr3-DG for detecting M25 navel orange and 'Newhol' navel orange;
FIG. 4 is a gel electrophoresis chart of molecular marker Cschr3-DG for detecting M25 navel orange and other types of mainly cultivated navel oranges in Gannan region.
Detailed Description
The invention is further described below with reference to the accompanying drawings. The following examples are only for more clearly illustrating the technical aspects of the present invention, and are not intended to limit the scope of the present invention.
Embodiment one:
the present example performs identification of the differential sites and development of molecular markers.
The M25 navel orange is obtained by screening the following steps:
in the process of continuously selecting the bud mutation of the navel orange for many years, a bud mutation is found in the 'New HEL' navel orange, and the early maturing navel orange variety M25 with larger fruits is obtained through breeding for many years. As shown in FIG. 1, the fruit morphology comparison of M25 navel orange and 'Neoher' navel orange provided by the embodiment of the invention is schematically shown.
(1) Identification of the differential sites.
Samples of M25 navel orange and 'Newhol' navel orange materials identified for use were taken from the national navel orange engineering research center nursery. And (3) extracting DNA of the M25 navel orange and the navel orange leaves by using a CTAB method, then establishing a library, and carrying out second generation sequencing with the depth of 30 multiplied.
Sequencing data were analyzed on a Linux server with the orange genome as reference (http:// citrus. Hzau. Edu. Cn). By alignment, an insertion/deletion site was identified at position 1501564-1501565 of the navel orange Chr3 chromosome. Wherein a 6927bp transposon sequence is inserted at the 1501564-1501565 th position on the chromosome of the Chur 3 of the M25 navel orange, and terminal repeated sequences formed by the Chur 3:1501556-1501564 (9 bp) sequence are formed at two sides of the inserted sequence.
(2) Differential site sequence clone analysis
The invention utilizes the resequencing technology to analyze and identify the sequence difference of 'Newhol' and its bud mutation variety M25, identifies the insertion of 6927bp transposon sequence on the 1501564-1501565 position of the Chr3 chromosome of M25, and forms terminal repeated sequences formed by the Chr3:1501556-1501564 (9 bp) sequence on two wings of the insertion sequence.
In order to further define the sequence variation characteristics, the invention designs primers according to sequences at two sides of a reference genome Chr3 chromosome 1501564-1501565, takes M25 navel orange and 'Newhol' navel orange DNA as templates, clones the sequences at the variation positions, and discovers that the 1501564-1501565 th position sequence of the 'Newhol' navel orange Chr3 chromosome is consistent with the reference genome, as shown in the upper graph in fig. 2.
A6927 bp sequence was inserted into the chromosome of the navel orange of M25 at the 1501564-1501565 th position, and terminal repeated sequences formed by the sequence of Chr3:1501556-1501564 were formed on both sides of the inserted sequence as shown in the lower graph of FIG. 2. Bioinformatic analysis indicated that the insert was a DNA transposon.
(3) Molecular marker design
According to the characteristics of the insertion sequence, the nucleotide sequence of the designed molecular marker CsChr3-DG is shown as SEQ ID NO.1, and a primer pair for amplifying the molecular marker CsChr3-DG is positioned on a chromosome of M25 navel orange Chr 3. As shown in the lower diagram of fig. 2. The designed molecular marker primer pair is Cschr3-DG-F and Cschr3-DG-R, and the sequences of the primer pair are respectively shown as SEQ ID NO.2 and SEQ ID NO. 3.
Embodiment two:
in the embodiment, molecular marker identification of M25 navel orange and 'Newhall' navel orange materials is carried out, and CTAB method is adopted to extract DNA of blades of the M25 navel orange and 'Newhall' navel orange materials, and the molecular marker identification of the M25 navel orange and 'Newhall' navel orange materials is used.
The PCR reaction system in this example was 10. Mu.l, which contained 0.5. Mu.l of the DNA template, 0.25. Mu.l of each of the upstream and downstream primers (1 mmol/L), 5. Mu.l of Mix, and 4. Mu.l of ddH2O.
PCR reaction procedure: denaturation at 98℃for 30s; then, 35 cycles of denaturation at 98℃for 10s, annealing at Tm (54.5 ℃) for 5s and elongation at 72℃for 3min were performed; then extending for 10min at 72 ℃; finally, the mixture is preserved at 10 ℃.
The PCR amplified products were electrophoresed using 1% agarose. Film was scanned and analyzed in a ChemiScope imaging system. FIG. 3 shows a gel electrophoresis chart of molecular marker Cschr3-DG for detecting M25 and 'Newhol' navel orange; wherein M is DL-15000marker,1-2 channels are Cschr3-DG to detect M25 navel orange banding pattern, and 3-4 channels are Cschr3-DG to detect 'Newhol' navel orange banding pattern.
As can be seen from fig. 3: the molecular marker Cschr3-DG can distinguish M25 navel orange from 'New HEL' navel orange of bud mutation source. In the identified gel, cschr3-DG is linearly labeled with two types of bands, the sizes of which are 7183bp and no bands, respectively. The band with 7183bp is M25 navel orange, and the band without band is 'Newhall' navel orange.
Embodiment III:
this example uses the molecular marker Cschr3-DG to identify M25 and other navel orange germplasm. And extracting the blade DNA of the M25 navel orange and 'Newhall' navel orange materials by adopting a CTAB method, and identifying molecular markers of the M25 and 'Newhall' navel orange materials.
The PCR reaction system was 10. Mu.l, which contained 0.5. Mu.l of the DNA template, 0.25. Mu.l of each of the upstream and downstream primers (1 mmol/L), 5. Mu.l of Mix, and 4. Mu.l of ddH2O.
PCR reaction procedure: denaturation at 98℃for 30s; then, 35 cycles of denaturation at 98℃for 10s, annealing at Tm (54.5 ℃) for 5s and elongation at 72℃for 3min were performed; then extending for 10min at 72 ℃; finally, the mixture is preserved at 10 ℃.
The PCR amplified products were electrophoresed on 1% agarose. Film was scanned and analyzed in a ChemiScope imaging system.
FIG. 4 is a gel electrophoresis diagram of molecular marker Cschr3-DG for detecting M25 and other main cultivated navel orange varieties in Gannan region; wherein, 1 channel is Cschr3-DG to detect M25 navel orange banding pattern, and 2-10 channels are Cschr3-DG to detect banding patterns of other main cultivated navel orange varieties in Gannan region.
As can be seen from fig. 4: the molecular marker Cschr3-DG can distinguish M25 navel orange from other navel orange germplasm. In the identified gel plots, cschr3-DG is linearly labeled with two bands, with the respective sizes of having a 7183bp band and no 7183bp band. M25 navel orange with 7183bp band, other navel orange germplasm are all without band.
According to the transposon insertion site on the chromosome of the M25 navel orange Chr3, the dominant molecular marker CsChr3-DG for detecting the M25 navel orange is developed, and the marker has very important value in the detection of the first generation and the second generation of the M25 navel orange.
And can identify the transposon insertion site on the chromosome of the M25 navel orange Chr3, thereby being convenient and fast to identify whether the navel orange seedlings or other propagation materials are M25 germplasm or not, and having great value for agricultural production detection or market supervision.
The foregoing is merely a preferred embodiment of the present invention, and it should be noted that modifications and variations could be made by those skilled in the art without departing from the technical principles of the present invention, and such modifications and variations should also be regarded as being within the scope of the invention.
The nucleotide sequence related to the invention is as follows:
SEQ ID NO.1
SEQ ID NO.2
5’-GCCATCTTTCACTCAGCAC-3’
SEQ ID NO.3
5’-CCGAACAAGGCAACAGC-3’。
Claims (6)
1. a molecular marker for identifying M25 navel orange is characterized in that the nucleotide sequence of the molecular marker CsChr3-DG is shown as SEQ ID NO.1, and a primer pair for amplifying the molecular marker CsChr3-DG is positioned on a chromosome of the M25 navel orange Chr 3.
2. The molecular marker of claim 1, wherein the primer pair for amplifying the molecular marker CsChr3-DG is CsChr3-DG-F and CsChr3-DG-R; the nucleotide sequence of the Cschr3-DG-F is shown as SEQ ID NO.2, and the nucleotide sequence of the Cschr3-DG-R is shown as SEQ ID NO. 3.
3. A kit comprising a primer pair for amplification of the molecular marker CsChr3-DG of claim 2.
4. A method for identifying M25 navel orange, comprising the step of amplifying genomic DNA of navel orange by detecting primer pair of molecular marker CsChr3-DG according to claim 1.
5. The method according to claim 4, comprising the steps of:
extracting genome DNA of navel orange varieties;
performing PCR amplification on the extracted genome DNA by using a primer pair of a molecular marker Cschr3-DG to obtain an amplification product;
electrophoresis is carried out on the molecular marker Cschr3-DG to obtain a control electrophoresis gel diagram;
electrophoresis is carried out on the amplified product, and an electrophoresis gel diagram is obtained;
and comparing the electrophoresis gel diagram with a control electrophoresis gel diagram, and screening to obtain the M25 navel orange.
6. Use of a molecular marker according to any one of claims 1-2, or a method according to any one of claims 4-5, or a kit according to claim 3 for detecting M25 navel orange tissue material for breeding pre-maturing navel orange germplasm.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108754014A (en) * | 2018-06-25 | 2018-11-06 | 华中农业大学 | Differentiate the molecular labeling of navel orange and its differentiates method, application and the kit of navel orange |
| CN109517924A (en) * | 2019-01-17 | 2019-03-26 | 四川省农业科学院园艺研究所 | A kind of citrus anthocyanin accumulates the molecular detecting method to form red meat character |
| CN110669863A (en) * | 2019-10-30 | 2020-01-10 | 华中农业大学 | Citrus mitochondria InDel molecular marker and application thereof |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108754014A (en) * | 2018-06-25 | 2018-11-06 | 华中农业大学 | Differentiate the molecular labeling of navel orange and its differentiates method, application and the kit of navel orange |
| CN109517924A (en) * | 2019-01-17 | 2019-03-26 | 四川省农业科学院园艺研究所 | A kind of citrus anthocyanin accumulates the molecular detecting method to form red meat character |
| CN110669863A (en) * | 2019-10-30 | 2020-01-10 | 华中农业大学 | Citrus mitochondria InDel molecular marker and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| HALA F. AHMED: "Phytohormones content and random amplified polymorphic DNA (RAPD) marker assessment of some Egyptian citrus cultivars", AFRICAN JOURNAL OF BIOTECHNOLOGY, vol. 11, no. 91, 13 November 2013 (2013-11-13), pages 15755 - 15762 * |
| 柯玲俊等: "利用转座子显示技术鉴别柑橘芽变品种", 园艺学报, vol. 44, no. 6, 25 June 2017 (2017-06-25), pages 1207 - 1216 * |
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