CN116042905A - SV (space velocity) marker for identifying citrus varieties and application thereof - Google Patents
SV (space velocity) marker for identifying citrus varieties and application thereof Download PDFInfo
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- 241000207199 Citrus Species 0.000 title claims abstract description 53
- 235000020971 citrus fruits Nutrition 0.000 title claims abstract description 53
- 239000003550 marker Substances 0.000 title claims abstract description 10
- 238000000034 method Methods 0.000 claims abstract description 16
- 210000000349 chromosome Anatomy 0.000 claims abstract description 5
- 238000012408 PCR amplification Methods 0.000 claims description 13
- 235000013399 edible fruits Nutrition 0.000 claims description 11
- 244000276331 Citrus maxima Species 0.000 claims description 6
- 235000009508 confectionery Nutrition 0.000 claims description 6
- 244000144730 Amygdalus persica Species 0.000 claims description 5
- 235000005976 Citrus sinensis Nutrition 0.000 claims description 5
- 240000002319 Citrus sinensis Species 0.000 claims description 5
- 235000006040 Prunus persica var persica Nutrition 0.000 claims description 5
- 241000270708 Testudinidae Species 0.000 claims description 5
- 210000000481 breast Anatomy 0.000 claims description 5
- 238000001962 electrophoresis Methods 0.000 claims description 5
- 239000010389 wendan Substances 0.000 claims description 5
- 241000253115 Carpesium Species 0.000 claims description 4
- 235000001759 Citrus maxima Nutrition 0.000 claims description 4
- 235000019605 sweet taste sensations Nutrition 0.000 claims description 4
- 238000001514 detection method Methods 0.000 abstract description 9
- 239000003147 molecular marker Substances 0.000 abstract description 7
- 238000009395 breeding Methods 0.000 abstract description 4
- 230000001488 breeding effect Effects 0.000 abstract description 4
- 239000012634 fragment Substances 0.000 abstract description 3
- 239000011543 agarose gel Substances 0.000 abstract description 2
- 238000009396 hybridization Methods 0.000 abstract description 2
- 239000000047 product Substances 0.000 description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical class [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- PHTQWCKDNZKARW-UHFFFAOYSA-N isoamylol Chemical compound CC(C)CCO PHTQWCKDNZKARW-UHFFFAOYSA-N 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 235000005979 Citrus limon Nutrition 0.000 description 1
- 244000131522 Citrus pyriformis Species 0.000 description 1
- HEFNNWSXXWATRW-UHFFFAOYSA-N Ibuprofen Chemical compound CC(C)CC1=CC=C(C(C)C(O)=O)C=C1 HEFNNWSXXWATRW-UHFFFAOYSA-N 0.000 description 1
- 241001093501 Rutaceae Species 0.000 description 1
- 208000005652 acute fatty liver of pregnancy Diseases 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000009402 cross-breeding Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 239000003517 fume Substances 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000004570 mortar (masonry) Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000007894 restriction fragment length polymorphism technique Methods 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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Abstract
The invention relates to a structural variation marker combination for identifying citrus varieties, which comprises 9 structural variation sites respectively positioned on chromosome 1-9, and reference sequences are respectively shown in SEQ ID NO. 1-9, and a method for identifying citrus varieties by using the structural variation marker combination. The molecular marker combination based on large-fragment structural variation developed by the invention is used for assigning values to corresponding detection results by utilizing a specific assignment rule, is used for constructing citrus molecular identity cards, and can effectively distinguish different citrus varieties and be used for assisting hybridization breeding. The method can complete detection by adopting the common agarose gel, and has the advantages of simple operation, low cost and high detection efficiency.
Description
Technical Field
The invention relates to the field of citrus breeding, and more particularly relates to an SV marker for identifying citrus varieties and application thereof.
Background
Citrus belongs to the family of rutaceae, is a generic term for citrus and related plants, and is mainly composed of citrus (Citrus ret i cu l ata B l anco), orange (C itrus s i nens i sOsbeck), pomelo (Citrus grand i s Osbeck), lemon (Citrus l imon Osbeck) and the like, and evergreen fruit trees cultivated in tropical and subtropical regions.
China is a large citrus production country, and the citrus planting area and the total yield are in the first place in the world. The scale of the citrus industry in China has rapidly developed in recent decades, and the total yield of citrus in China reaches 4584.5 ten thousand tons by 2019.
In production, citrus varieties are various, and 1753 citrus variety resources are saved in China at present. The multi-embryo nature of citrus severely hampers the cross-breeding effort, so that new citrus hybrid varieties mainly select a few single-embryo varieties as the female parent, such as 'kriman Ding Ju', 'qing' and 'Wang Gan', resulting in many new citrus varieties that differ in plant morphology and are indistinguishable. Therefore, the molecular markers which can identify different citrus varieties are developed, and the molecular identity card of the citrus varieties is constructed, so that the molecular marker has important practical application value for identifying the purity of citrus seedlings and assisting in breeding by the molecular markers.
The traditional RFLP, RAPD, SSR and AFLP molecular markers have the advantages of less number, high detection difficulty and high SNP molecular marker development cost and detection cost. Thus, there is a need for a new method and marker for identifying and marking citrus varieties.
Disclosure of Invention
In order to solve the problems, the invention provides a structural variation marker combination for identifying citrus varieties, which is characterized by comprising 9 structural variation sites respectively positioned on chromosome 1-9, and reference sequences are respectively shown in SEQ ID NO. 1-9.
The invention also provides a method for identifying citrus varieties, which comprises the step of detecting PCR amplification products corresponding to the 9 structural variation marker combinations.
In a specific embodiment, the primer pairs for amplifying the 9 structural variation sites are primer pairs 1-9, respectively, and the sequences are shown in SEQ ID NOs 10 and 11, 12 and 13, 14 and 15, 16 and 17, 18 and 19, 20 and 21, 22 and 23, 24 and 25, 26 and 27, respectively.
In a specific embodiment, the method comprises the steps of:
s1: extracting genomic DNA from the citrus variety;
s2: respectively carrying out PCR amplification on the genome DNA by using the primer pairs to obtain 9 PCR amplification products;
s3: respectively carrying out electrophoresis observation on the 9 parts of PCR amplification products;
s4: and respectively assigning values to 9 parts of PCR amplification products according to the electrophoresis band type, sequentially arranging the PCR amplification products, and combining the variety codes to obtain the finished product variety code, wherein the variety code can be used as an identification mark of the citrus variety.
In a specific embodiment, in S4, the assignment is performed by:
assigning 0 when the amplified product contains only the A band, assigning 1 when the amplified product contains only the B band, assigning 2 when the amplified product contains both the A band and the B band, and assigning X when the amplified product contains neither the A band nor the B band;
wherein, the size of the A band corresponding to the primer pair 1 is 383bp, and the size of the B band is 443bp;
the size of the A band corresponding to the primer pair 2 is 379bp, and the size of the B band is 459bp;
the size of the A band corresponding to the primer pair 3 is 366bp, and the size of the B band is 445bp;
the size of the A band corresponding to the primer pair 4 is 321bp, and the size of the B band is 398bp;
the size of the A band corresponding to the primer pair 5 is 346bp, and the size of the B band is 414bp;
the size of the A band corresponding to the primer pair 6 is 332bp, and the size of the B band is 395bp;
the size of the A band corresponding to the primer pair 7 is 400bp, and the size of the B band is 533bp;
the size of the A band corresponding to the primer pair 8 is 358bp, and the size of the B band is 402bp;
the size of the A band corresponding to the primer pair 9 is 383bp, and the size of the B band is 443bp.
In a specific embodiment, the citrus variety is selected from the group consisting of: sweet taste, sweet taste-detoxication, peak-clearing, large fruit orange, modified orange, yang Xiang, breast orange, xingchun ponkan, fuben navel orange, P420527032, lude red summer orange, mapo orange, carpesium orange, modified orange-detoxication, jin Shuiju ×peach leaf orange, seedless local early, large fruit seedless ponkan, QJ07-5, wen Dan shaddock, tortoise well, ming day, qing see X Jin Shuigan, jin Shuiju ×sugar orange, loving 30 numbers.
The invention also provides application of the structural variation marker combination in identifying citrus hybrid offspring.
In a specific embodiment, the citrus hybrid progeny to be identified are hybrid progeny of one or more of the following varieties: sweet mouth, sweet mouth-detoxication, peak clearing, large fruit orange, modified orange, yang Xiang, breast orange, xingchun ponkan, fuben navel orange, P420527032, lude red summer orange, mapo orange, carpesium orange, modified orange-detoxication, jin Shuiju x peach leaf orange, seedless local early, large fruit seedless ponkan, QJ07-5, wen Dan shaddock, tortoise well, ming day, qing see X Jin Shuigan, jin Shuiju x sugar orange, loving 30 #
The molecular marker combination based on large fragment Structural Variation (SV) developed by the invention is used for assigning values to corresponding detection results by utilizing a specific assignment rule, is used for constructing citrus molecular identity cards, and can effectively distinguish different citrus varieties and be used for assisting hybridization breeding. The method can complete detection by adopting the common agarose gel, and has the advantages of simple operation, low cost and high detection efficiency.
Drawings
FIGS. 1-9 are electrophoretograms of amplified products obtained by PCR of 24 citrus varieties using corresponding SV primer pairs on 9 chromosomes.
Detailed Description
The principles and features of the present invention are described below with reference to the drawings, the examples are illustrated for the purpose of illustrating the invention and are not to be construed as limiting the scope of the invention.
1. SV molecular marker acquisition
The team performs comparison and evaluation on genome sequences of multiple citrus varieties, respectively finds a large fragment-based Structural Variation (SV) on 9 chromosomes of citrus, and the reference sequences are shown as SEQ ID NO. 1-9, and the 9 SV combinations can be used for identifying citrus varieties as shown in Table 1
TABLE 1 primer sequences for SSR molecular markers CHR2-2 and CHR8-4
Assignment rule for SV molecular marker amplified bands
We assigned the amplified corresponding band pattern based on the characteristics of each SV, as shown in table 2.
TABLE 2 assignment of SV primer amplification bands for each pair
CV combination for identification of citrus varieties
1) Selection of samples
We selected 24 common citrus varieties for generating corresponding SV "identification card codes" including the following varieties: sweet taste, sweet taste-detoxication, peak-clearing, large fruit orange, modified orange, yang Xiang, breast orange, xingchun ponkan, fuben navel orange, P420527032, lude red summer orange, mapo orange, carpeo orange, modified orange-detoxication, jin Shuiju ×peach leaf orange, seedless local early, large fruit seedless ponkan, QJ07-5, wen Dan pomelo, tortoise well, ming day, qing see X Jin Shuigan, jin Shuiju ×sugar orange, aijiu 30 (corresponding to lanes to 2-25 in FIGS. 1-9, respectively).
2) Extraction of Total DNA
The method for extracting the total DNA of the citrus sample by using the modified CTAB method comprises the following specific operation steps: after sufficiently grinding the citrus leaf sample in a mortar using liquid nitrogen, 0.1g of the sample was taken in a 1.5mL centrifuge tube;600. Mu.L of CTAB extract (containing 1.5% CTAB and 1.5% beta-mercaptoethanol, preheated to 65℃for use in a water bath) was added to the milled sample; placing the centrifuge tube into a 65 ℃ water bath kettle for water bath for 60 min, taking out the centrifuge tube every 30 min, and slightly mixing the centrifuge tube up and down for several times; samples were removed and 700 μl of chloroform was added in a fume hood: isoamyl alcohol (24:1) solution, gently mix 10 min, centrifuge 10000g for 15 min, aspirate supernatant into another new centrifuge tube and repeat the procedure once more; adding 60 mu LNaC (5M) solution and 1mL frozen absolute ethanol, gently inverting for several times, mixing, and freezing for 30 min at-20 ℃ in a refrigerator to precipitate DNA;10000g of the mixture is centrifuged for 5 min, the supernatant is discarded, 1mL of prepared 70% ethanol is added for soaking 30 min, and DNA is washed; 10000g of the solution is centrifuged for 2 min, the solution is discarded and then put into a centrifuge for idle centrifugation for 2 min, the precipitate is properly air-dried, and then 50 mu L of ddH is added 2 O dissolves DNA, and the DNA is stored in a refrigerator at-20 ℃ for standby after concentration detection by the NanoDrop.
3) PCR reaction system and reaction program
PCR amplification was performed using the citrus leaf DNA as template and SV molecular marker primers, respectively. The 20. Mu.L reaction system was as follows: 2 XVazyme Mi.times.10. Mu.L, each of the forward primer and the reverse primer was 0.5. Mu.L, and the template DNA (100-200 ng) was 0.5. Mu.L, the remainder was supplemented with water.
The reaction procedure was as follows: 94 ℃ 2 min; 94 ℃ for 30s,55-60 ℃ for 30s,72 ℃ for 30s,30 cycles; 2mi n at 72 ℃. After the reaction, the amplified product was stored at 4℃for use.
4) Identification results of each variety
The SV identity cards of the respective varieties obtained according to the amplification bands (FIGS. 1 to 9) for the respective varieties and the assignment rules of Table 2 are shown in Table 3. 24 common citrus varieties can be identified and distinguished by the SV identity card code of the invention.
TABLE 3 SV identification card coding statistics for each variety
The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the invention.
Claims (8)
1. The structural variation marker combination for identifying citrus varieties is characterized by comprising 9 structural variation sites respectively positioned on chromosome 1-9, and reference sequences are respectively shown in SEQ ID NO. 1-9.
2. A method for identifying citrus varieties, comprising the step of detecting PCR amplification products corresponding to the combination of 9 structural variation markers of claim 1.
3. The method of claim 2, wherein the primer pairs for amplifying the 9 structural variation sites are primer pairs 1-9, respectively, having sequences as shown in SEQ ID NOS 10 and 11, 12 and 13, 14 and 15, 16 and 17, 18 and 19, 20 and 21, 22 and 23, 24 and 25, 26 and 27, respectively.
4. A method according to claim 3, comprising the steps of:
s1: extracting genomic DNA from the citrus variety;
s2: respectively carrying out PCR amplification on the genome DNA by using the primer pairs to obtain 9 PCR amplification products;
s3: respectively carrying out electrophoresis observation on the 9 parts of PCR amplification products;
s4: and respectively assigning values to 9 parts of PCR amplification products according to the electrophoresis band type, sequentially arranging the PCR amplification products, and combining the variety codes to obtain the finished product variety code, wherein the variety code can be used as an identification mark of the citrus variety.
5. The method of claim 4, wherein in S4, the assigning is performed by:
assigning 0 when the amplified product contains only the A band, assigning 1 when the amplified product contains only the B band, assigning 2 when the amplified product contains both the A band and the B band, and assigning X when the amplified product contains neither the A band nor the B band;
wherein, the size of the A band corresponding to the primer pair 1 is 383bp, and the size of the B band is 443bp;
the size of the A band corresponding to the primer pair 2 is 379bp, and the size of the B band is 459bp;
the size of the A band corresponding to the primer pair 3 is 366bp, and the size of the B band is 445bp;
the size of the A band corresponding to the primer pair 4 is 321bp, and the size of the B band is 398bp;
the size of the A band corresponding to the primer pair 5 is 346bp, and the size of the B band is 414bp;
the size of the A band corresponding to the primer pair 6 is 332bp, and the size of the B band is 395bp;
the size of the A band corresponding to the primer pair 7 is 400bp, and the size of the B band is 533bp;
the size of the A band corresponding to the primer pair 8 is 358bp, and the size of the B band is 402bp;
the size of the A band corresponding to the primer pair 9 is 383bp, and the size of the B band is 443bp.
6. A method according to any one of claims 2-5, wherein said citrus varieties are selected from the group consisting of: sweet taste, sweet taste-detoxication, peak-clearing, large fruit orange, modified orange, yang Xiang, breast orange, xingchun ponkan, fuben navel orange, P420527032, lude red summer orange, mapo orange, carpesium orange, modified orange-detoxication, jin Shuiju ×peach leaf orange, seedless local early, large fruit seedless ponkan, QJ07-5, wen Dan shaddock, tortoise well, ming day, qing see X Jin Shuigan, jin Shuiju ×sugar orange, loving 30 numbers.
7. Use of the combination of structural variation markers of claim 1 for identifying citrus hybrid offspring.
8. The method of claim 7, wherein the citrus hybrid offspring to be identified is a hybrid offspring of one or more of the following varieties: sweet taste, sweet taste-detoxication, peak-clearing, large fruit orange, modified orange, yang Xiang, breast orange, xingchun ponkan, fuben navel orange, P420527032, lude red summer orange, mapo orange, carpesium orange, modified orange-detoxication, jin Shuiju ×peach leaf orange, seedless local early, large fruit seedless ponkan, QJ07-5, wen Dan shaddock, tortoise well, ming day, qing see X Jin Shuigan, jin Shuiju ×sugar orange, loving 30 numbers.
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Citations (5)
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|---|---|---|---|---|
| CN102492774A (en) * | 2011-12-09 | 2012-06-13 | 南京农业大学 | Primers and method for quickly distinguishing orange varieties |
| CN110273020A (en) * | 2019-06-27 | 2019-09-24 | 华中农业大学 | For distinguishing the SNP marker and application of citrus summer orange and common sweet orange |
| CN113186332A (en) * | 2021-05-09 | 2021-07-30 | 湖北省农业科学院经济作物研究所 | SV molecular marker for constructing radish molecular identity card and application thereof |
| CN113462810A (en) * | 2021-08-16 | 2021-10-01 | 湖北省农业科学院果树茶叶研究所 | SSR marker for identifying citrus varieties and application thereof |
| JP2021185904A (en) * | 2020-05-29 | 2021-12-13 | 国立研究開発法人農業・食品産業技術総合研究機構 | Identification method and identification kit for breed of citrus |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102492774A (en) * | 2011-12-09 | 2012-06-13 | 南京农业大学 | Primers and method for quickly distinguishing orange varieties |
| CN110273020A (en) * | 2019-06-27 | 2019-09-24 | 华中农业大学 | For distinguishing the SNP marker and application of citrus summer orange and common sweet orange |
| JP2021185904A (en) * | 2020-05-29 | 2021-12-13 | 国立研究開発法人農業・食品産業技術総合研究機構 | Identification method and identification kit for breed of citrus |
| CN113186332A (en) * | 2021-05-09 | 2021-07-30 | 湖北省农业科学院经济作物研究所 | SV molecular marker for constructing radish molecular identity card and application thereof |
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Non-Patent Citations (1)
| Title |
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| 常爱玲: "‘无子瓯柑’InDel和SV标记开发", 中国硕士学位论文电子期刊农业科技, 15 April 2018 (2018-04-15), pages 1 - 58 * |
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