CN116769953A - Molecular marker for identifying Gannan early navel orange and application thereof - Google Patents
Molecular marker for identifying Gannan early navel orange and application thereof Download PDFInfo
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- 240000002319 Citrus sinensis Species 0.000 title claims abstract description 99
- 235000005976 Citrus sinensis Nutrition 0.000 title claims abstract description 99
- 239000003147 molecular marker Substances 0.000 title claims abstract description 49
- 210000000349 chromosome Anatomy 0.000 claims abstract description 18
- 238000000034 method Methods 0.000 claims description 13
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- 230000003321 amplification Effects 0.000 claims description 5
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 5
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- 238000009395 breeding Methods 0.000 claims description 3
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- 239000002773 nucleotide Substances 0.000 claims description 2
- 125000003729 nucleotide group Chemical group 0.000 claims description 2
- 108020004414 DNA Proteins 0.000 description 16
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- 230000035772 mutation Effects 0.000 description 5
- 241000207199 Citrus Species 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 230000002759 chromosomal effect Effects 0.000 description 4
- 235000020971 citrus fruits Nutrition 0.000 description 4
- 238000004925 denaturation Methods 0.000 description 4
- 230000036425 denaturation Effects 0.000 description 4
- 238000003780 insertion Methods 0.000 description 4
- 230000037431 insertion Effects 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical class [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
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- 238000012163 sequencing technique Methods 0.000 description 3
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- 229920000936 Agarose Polymers 0.000 description 2
- 238000000137 annealing Methods 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 239000012154 double-distilled water Substances 0.000 description 2
- 235000013399 edible fruits Nutrition 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
- 230000002028 premature Effects 0.000 description 2
- 230000005070 ripening Effects 0.000 description 2
- 108091081062 Repeated sequence (DNA) Proteins 0.000 description 1
- 208000025865 Ulcer Diseases 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000003766 bioinformatics method Methods 0.000 description 1
- 230000034303 cell budding Effects 0.000 description 1
- 238000004040 coloring Methods 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
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- 239000000796 flavoring agent Substances 0.000 description 1
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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
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- C12Q2600/156—Polymorphic or mutational markers
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Abstract
The invention discloses a molecular marker for identifying Gannan early navel orange and application thereof, wherein a DNA transposon is inserted between 29470212 th and 29470213 th positions of a chromosome of the navel orange Chur 9 to form a molecular marker CsChur 9-GNZ, and primer pairs for amplifying the molecular marker CsChur 9-GNZ are CsChur 9-GNZ-F and CsChur 9-GNZ-R, so that the molecular marker CsChur 9-GNZ-R is convenient for rapid identification of Gannan early navel orange seedlings.
Description
Technical Field
The invention relates to a molecular marker for identifying Gannan early navel orange and application thereof, belonging to the technical field of plant breeding.
Background
At present, the main cultivated variety in the market is medium-ripe citrus, the ripe period structure is unreasonable, for example, the ripe period of the navel orange variety cultivated in the main in Jiangxi province is mainly concentrated in 11 months to 12 months, a large number of fresh navel oranges are concentrated in the market in a short period, and huge pressure is brought to storage, transportation, processing and the like.
In order to solve the phenomenon, breeders cultivate a plurality of high-quality citrus varieties, wherein the Gannan early navel orange is a super early maturing navel orange variety which is bud-changed and bred from a main cultivated navel orange variety 'Neoher'. The early ripening of the navel orange fruits of Gannan early ' is obvious, including the color turning and coloring of the fruit peel, the accumulation of soluble solids and soluble sugar is accelerated, the solid acid ratio is earlier than the ripening related characters of the jump period, and the time for reaching the same appearance color and luster and the internal quality level is more than 35 days earlier than that of the main cultivated navel orange variety ' Neohol '. The Gannan early navel orange has the advantages of moderate tree vigor, good early bearing and yield, early mature period, orange pulp, tender slag, sweet and slightly sour flavor, easy peeling and ulcer resistance. The citrus has a long childhood period, and a certain difficulty exists in identifying varieties through morphology in the childhood period.
Disclosure of Invention
The invention aims to overcome the defects in the prior art, and provides a molecular marker for identifying Gannan early navel oranges and application thereof, which are convenient for rapid identification of Gannan early 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 a Gannan early navel orange, wherein a DNA transposon is inserted between 29470212 th and 29470213 th positions of a chromosome of the navel orange Chr9 to form a molecular marker CsChr9-GNZ, and a primer pair for amplifying the molecular marker CsChr9-GNZ is CsChr9-GNZ-F and CsChr9-GNZ-R;
the sequence of Cschr9-GNZ-F is shown as SEQ ID NO.2, and the sequence of Cschr9-GNZ-R is shown as SEQ ID NO. 3.
Furthermore, the nucleotide sequence of the molecular marker Cschr9-GNZ is shown as SEQ ID NO. 1.
Further, the primer pair of the molecular marker Cschr9-GNZ is characterized in that one primer is positioned on the chromosome of the navel orange Chr9, and the other primer is positioned on the inserted DNA transposon.
In a second aspect, the invention provides a kit comprising the primer pair for amplifying the molecular marker Cschr9-GNZ.
In a third aspect, the present invention provides a method for identifying Gannan early navel orange, comprising the step of detecting the primer pair of the molecular marker Cschr9-GNZ to amplify the genomic DNA of navel orange.
With reference to the third aspect, further, the method includes the steps of:
extracting genome DNA from navel orange varieties;
respectively carrying out PCR amplification on the extracted genome DNA by using a primer pair of a molecular marker Cschr9-GNZ to obtain an amplification product;
respectively carrying out electrophoresis on the amplified products to obtain an electrophoresis gel diagram;
and comparing the electrophoresis gel diagram with the electrophoresis gel diagram of the molecular marker Cschr9-GNZ, and screening out the target navel orange.
In a fourth aspect, the invention provides an application of the molecular marker, the method or the kit in breeding of the germplasm of the early-maturing navel orange.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides a transposon insertion site on a chromosome of Gannan early navel orange Chr9, and a dominant molecular marker Cschr9-GNZ detected by Gannan early navel orange is developed based on the site, which is used for identifying whether navel orange seedlings or other propagation materials are Gannan early varieties or not;
provides a primer pair of a molecular marker Cschr9-GNZ for detecting Gannan early navel orange, which is convenient for rapid identification of Gannan early navel orange seedlings.
Drawings
FIG. 1 is a schematic diagram showing the difference between the chromosomal sequence of Gannan early navel orange Chur 9 and the chromosomal sequence of Gannan early navel orange Chur 9;
FIG. 2 shows the detection of the molecular markers Cschr9-GNZ of the 'Gannanza' navel orange and 'Newhall' navel orange;
FIG. 3 shows the detection of the molecular marker Cschr9-GNZ of other navel orange varieties in Gannan early and Gannan regions.
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.
The invention provides a difference site between a Chr9 chromosome on Gannan early navel orange and a navel orange from which bud mutation is derived, and provides a molecular marker Cschr9-GNZ for detecting Gannan early navel orange and a primer pair thereof.
The invention adopts the technical scheme that: the difference site of the Gannanza navel orange and the navel orange of which the bud mutation source is 'New HEL' is positioned on the chromosome of the Chur 9, and the reference genome sequence (the chromosome sequence of the Chur 9 of the sweet orange) is found in NCBI database (GenBank: CM 030748.1). A6927 bp transposon sequence was inserted between the 29470212 th and 29470213 th chromosomes of the 'Gannanza' navel orange, and terminal repeats formed by the Chr9:29470205-29470212 (9 bp) sequences were formed on both sides of the inserted sequence. The molecular marker Cschr9-GNZ was developed in close linkage with this site. Cschr9-GNZ is located on chromosome Chr9 and has the sequence shown in SEQ ID NO. 1.
Primer pair corresponding to the Gannan early navel orange identification molecular marker CsChr9-GNZ, wherein the upstream primer sequence of CsChr9-GNZ is CsChr9-GNZ-F (SEQ ID NO. 2) respectively, and the downstream primer sequence of CsChr9-GNZ (SEQ ID NO. 3) respectively
The method for detecting the 'Gannanza' navel orange by using the molecular marker or the primer pair comprises the steps of amplifying genome DNA of the navel orange by using the primer pair of the molecular marker Cschr9-GNZ, and obtaining an amplified fragment corresponding to variation in the 'Gannanza' after 1% agarose gel electrophoresis of an amplified product, wherein the amplified fragment is indicated as 'Gannanza' germplasm. If the amplified fragment corresponding to the variation in the Gannan early navel orange is not obtained, the specific variation site contained in the Gannan early is not existed, and the nursery stock is predicted not to be the early-matured Gannan early navel orange.
Specifically, the molecular marker Cschr9-GNZ for detecting the Gannan early navel orange disclosed by the invention comprises the following screening steps:
(1) In the process of continuously bud mutation seed selection of navel orange for many years, a premature bud mutation is found in 'New HEL' navel orange, and the variety 'Gannanzhao' of the premature navel orange is obtained through breeding for many years, and the variety is identified by agricultural rural parts, so that the variety protection is obtained.
(2) Analysis by re-sequencing techniques identified the sequence differences of 'newhol' and its budding variety 'Gannan early', identified the insertion of a 6927bp transposon sequence at position 29470212 on the 'Gannan early' Chr9 chromosome, and formed terminal repeats of the Chr9:29470205-29470212 (9 bp) sequence flanking the insertion.
(3) Based on the sequence difference found above, a molecular marker Cschr9-GNZ for detecting Gannanza navel orange is developed, one amplification primer of the marker is positioned on a Chr9 chromosome, and the other primer is positioned on an inserted transposon sequence, so that the specificity of the sequence is ensured.
The invention also provides application of the molecular marker Cschr9-GNZ of the 'Gannan early' navel orange or a primer pair, a screening step or a kit thereof in detecting the 'Gannan early' navel orange.
Embodiment one:
in this example, the identification of the differential sites and the development of the molecular markers were performed as follows.
(1) Identification of the differential sites
Samples of the 'Gannanza' and 'Neoher' navel orange materials used for identification were taken from the national navel orange engineering research center nursery, and three biological replicates were selected for different sample materials. Modified CTAB method is used to extract the DNA of the blades of the 'Gannan early' and 'Newhol' navel orange, and then the DNA is subjected to library establishment and second generation sequencing with the depth of 30 multiplied.
The invention takes the sweet orange genome as a reference (http:// citrus. Hzau. Edu. Cn), and analyzes sequencing data on a Linux server. By alignment, an insertion/deletion site was identified at the 29470212 position of the Chr9 chromosome.
(2) Differential site sequence clone analysis
In order to further define the sequence variation characteristics, primers are designed according to sequences at two sides of a chromosome 29470212 of a reference genome, the sequence of a 'Gannan early' navel orange and a 'Neohol' navel orange DNA are used as templates, the sequence at the variation position is cloned, and as a result, the sequence at the 29470212 position of the chromosome of the 'Neohol' navel orange is found to be consistent with the reference genome, a 6927bp sequence is inserted at the 29470212 position of the chromosome, terminal repeated sequences formed by the sequences of the Chr9:29470205-29470212 (9 bp) are formed at two sides of the inserted sequence, and the bioinformatics analysis shows that the inserted sequence is a DNA transposon. As shown in FIG. 1, the difference between the chromosomal sequence of the 'Gannanza' navel orange Chur 9 and the chromosomal sequence of the 'Newhall' navel orange Chur 9 is schematically shown.
(3) Molecular marker design
According to the characteristics of the inserted sequence, the invention designs a molecular marker primer pair, one amplification primer is positioned on the chromosome of the navel orange Chr9, and the other primer is positioned on the inserted transposon sequence. The sequences of the Cschr9-GNZ primer pair named Cschr9-GNZ-F and Cschr9-GNZ-R are shown as SEQ ID NO.2, and the sequences of the Cschr9-GNZ-R are shown as SEQ ID NO. 3.
(4) Molecular marker identification of Gannan early navel orange and Neohol navel orange material
The CTAB method is used for extracting the blade DNA of the Gannan early navel orange and the Neohol navel orange materials, and is used for molecular marker identification of the Gannan early navel orange and the Neohol navel orange materials.
The extracted leaf DNA was amplified, and a PCR reaction system (10. Mu.l) containing 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 was used.
PCR reaction procedure: denaturation at 95℃for 5min; followed by 35 cycles of denaturation at 95℃for 30s, annealing at Tm (56 ℃) for 30s and elongation at 72℃for 2min; then extending for 5min at 72 ℃; finally, the mixture is preserved at 10 ℃.
The PCR amplification product of the present invention was electrophoresed on 1% agarose. Film was scanned and analyzed in a ChemiScope imaging system.
(5) Results and analysis
The molecular marker Cschr9-GNZ can distinguish the 'Gannanza' navel orange from which the bud mutation source 'Newhall' navel orange. In the identified gel plots, cschr9-GNZ is a linear marker with two bands of 3310bp in size and no band. The navel orange with the 3310bp band is the 'Gannanza' navel orange, and the navel orange without the band is the 'Newhall' navel orange.
As shown in figure 2, the molecular markers Cschr9-GNZ provided by the invention detect the 'Gannanza' navel orange and the 'Neoher' navel orange; wherein M is DL-5000marker,1-3 channels are Cschr9-GNZ for detecting the 'Gannanza' navel orange band, and 4-6 channels are Cschr9-GNZ for detecting the 'Newhol' navel orange band;
embodiment two:
in this example, molecular marker Cschr9-GNZ was used to identify Gannanza' and other navel orange germplasm.
(1) Molecular markers detect Gannanza' and other navel orange germplasm.
The CTAB method is used for extracting the blade DNA of the navel orange materials of Gannanza and Neohol and is used for molecular marker identification of the navel orange materials of Gannanza and Neohol.
The extracted DNA was subjected to PCR amplification by a specific PCR reaction system (10. Mu.l) containing 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 95℃for 5min; followed by 35 cycles of denaturation at 95℃for 30s, annealing at Tm (56 ℃) for 30s and elongation at 72℃for 2min; then extending for 5min 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.
(2) Results and analysis.
The molecular marker Cschr9-GNZ can distinguish the 'Gannanza' navel orange from other navel orange germplasm. In the identified gel plots, cschr9-GNZ is a linear marker with two bands of 3310bp in size and no band. The navel orange with the 217bp band is the 'Gannanza' navel orange, and other navel orange germplasm is not provided with the band.
As shown in FIG. 3, the molecular marker Cschr9-GNZ provided by the invention is used for detecting other navel orange varieties in the Gannan early and Gannan regions; wherein M is DL-5000marker,1-2 channels are Cschr9-GNZ to detect the banding pattern of the 'Gannanzao' navel orange, and 3-24 channels are Cschr9-GNZ to detect the banding patterns of other navel orange varieties.
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.
Claims (7)
1. A molecular marker for identifying Gannan early navel orange is characterized in that a DNA transposon is inserted between 29470212 th and 29470213 th chromosome of navel orange Chur 9 to form a molecular marker CsChur 9-GNZ, and a primer pair for amplifying the molecular marker CsChur 9-GNZ is CsChur 9-GNZ-F and CsChur 9-GNZ-R;
the sequence of Cschr9-GNZ-F is shown as SEQ ID NO.2, and the sequence of Cschr9-GNZ-R is shown as SEQ ID NO. 3.
2. The molecular marker according to claim 1, wherein the nucleotide sequence of the molecular marker Cschr9-GNZ is shown in SEQ ID NO. 1.
3. The molecular marker of claim 1, wherein the primer pair of molecular markers CsChr9-GNZ, one primer on the navel orange Chr9 chromosome and the other primer on the inserted DNA transposon.
4. A kit comprising a primer pair for use in the amplification of the molecular marker CsChr9-GNZ of claim 1.
5. A method for identifying a gan early navel orange, comprising the step of amplifying genomic DNA of the navel orange by detecting the primer pair of molecular marker CsChr9-GNZ according to claim 1.
6. The method according to claim 5, comprising the steps of:
extracting genome DNA from navel orange varieties;
respectively carrying out PCR amplification on the extracted genome DNA by using a primer pair of a molecular marker Cschr9-GNZ to obtain an amplification product;
respectively carrying out electrophoresis on the amplified products to obtain an electrophoresis gel diagram;
and comparing the electrophoresis gel diagram with the electrophoresis gel diagram of the molecular marker Cschr9-GNZ, and screening out the target navel orange.
7. Use of a molecular marker according to any one of claims 1-3, or a method according to any one of claims 5-6, or a kit according to claim 4, for breeding a pre-maturing navel orange germplasm.
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| 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 |
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| 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 |
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| Title |
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| 赖春旺 等: "脐橙早熟芽变及其早熟性状回复型材料的AFLP和MSAP分析", 果树学报, vol. 39, no. 08, 11 April 2022 (2022-04-11), pages 1346 - 1357 * |
| 陈健美;谢丽红;周娟;江小美;钟八莲;李淑惠;杨斌华;喻芳琴;: "‘赣南早’脐橙早熟性状回复型突变体的生理与转录组分析", 果树学报, no. 04, 11 April 2018 (2018-04-11), pages 401 - 409 * |
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