CN116287364B - Primer group, kit and method for identifying cultivation type of Jiangxi local pomelo - Google Patents
Primer group, kit and method for identifying cultivation type of Jiangxi local pomelo Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 18
- 240000000560 Citrus x paradisi Species 0.000 title claims abstract 7
- 235000012907 honey Nutrition 0.000 claims abstract description 28
- 238000012408 PCR amplification Methods 0.000 claims description 4
- 238000000137 annealing Methods 0.000 claims description 3
- 238000004925 denaturation Methods 0.000 claims description 3
- 230000036425 denaturation Effects 0.000 claims description 3
- 238000003752 polymerase chain reaction Methods 0.000 claims description 3
- 238000012257 pre-denaturation Methods 0.000 claims description 3
- RZVAJINKPMORJF-UHFFFAOYSA-N Acetaminophen Chemical compound CC(=O)NC1=CC=C(O)C=C1 RZVAJINKPMORJF-UHFFFAOYSA-N 0.000 claims description 2
- 239000002773 nucleotide Substances 0.000 claims 4
- 125000003729 nucleotide group Chemical group 0.000 claims 4
- 238000001514 detection method Methods 0.000 abstract description 5
- 244000276331 Citrus maxima Species 0.000 description 38
- 238000010586 diagram Methods 0.000 description 4
- 239000000499 gel Substances 0.000 description 4
- 239000003550 marker Substances 0.000 description 4
- 238000012795 verification Methods 0.000 description 4
- 244000144730 Amygdalus persica Species 0.000 description 3
- 235000006040 Prunus persica var persica Nutrition 0.000 description 3
- 238000000246 agarose gel electrophoresis Methods 0.000 description 3
- 238000012217 deletion Methods 0.000 description 3
- 230000037430 deletion Effects 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 239000011543 agarose gel Substances 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 235000013399 edible fruits Nutrition 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 239000004576 sand Substances 0.000 description 2
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 1
- 241000207199 Citrus Species 0.000 description 1
- 235000001759 Citrus maxima Nutrition 0.000 description 1
- 239000012807 PCR reagent Substances 0.000 description 1
- 241000984945 Simona Species 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 235000020971 citrus fruits Nutrition 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000008774 maternal effect Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000003147 molecular marker Substances 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 230000008775 paternal effect Effects 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 238000013441 quality evaluation Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
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Abstract
The invention discloses a primer group, a kit and a method for identifying cultivation types of Jiangxi local pomelos, and relates to the technical field of molecular markers. The invention develops InDel marked primers with good polymorphism based on the whole genome resequencing result, wherein the primers can distinguish 2 main cultivated varieties in early stage by using any 1 pair, the detection rate is 92.31-100%, the detection accuracy is 100%, the invention provides basis for early and accurate identification of the variety of the Jinggang honey pomelo, and plays a positive role in producing clear variety names.
Description
The invention is as follows: a separate application of a primer group, a kit and an identification method for identifying the cultivation type of the Jinggang honey pomelo is provided, and the application date of the original application is as follows: 2022, 02, 22 days, application number: 202210163279.X, patent name: a primer group, a kit and an identification method for identifying the cultivation type of Jinggang honey pomelo.
Technical Field
The invention belongs to the technical field of molecular markers, and particularly relates to a primer group, a kit and a method for identifying variety of Jinggang honey pomelo seedlings.
Background
The Jinggang honey pomelo is a common name of a sweet pomelo variety produced and cultivated in Jian City of Jiangxi province by taking Jinggan mountain as a brand, is a geographic marking agricultural product in China, is one of six rich people industries in which Jian is supported and developed, and is one of three golden signs in Jiangxi province fruit industry. Up to now, the planting area of Jinggang honey pomelo reaches more than 40 mu, 3 main varieties are respectively golden sand pomelo, jin Lanyou and peach stream honey pomelo, and according to statistics, the planting area of the first two varieties reaches 71.27% of the total area of Jinggang honey pomelo by the year 2020, and is the dominant variety, and the planting area of peach stream honey pomelo accounts for 13.39%. The peach stream honey pomelo is a variety of Shatian pomelo in Jiangxi, has early maturity and remarkable fruit characteristics, and is easy to identify with other two Jinggang honey pomelo varieties. However, since Jin Shayou is a hybrid offspring of Jin Lanyou and Shatian pomelo, the two kinds of Jinggang honey pomelo are closely related and are difficult to distinguish only by morphological characteristics. In the rapid development process of Jinggang honey pomelo, jin Lanyou and Jin Shayou 2 main cultivated varieties are mixed, so that the production of seedlings is disordered, the names of the varieties are misused, and the health and sustainable development of the industry is seriously affected.
Disclosure of Invention
Therefore, the invention aims to provide the primer group, the kit and the method for identifying the cultivation type of the Jinggang honey pomelo, which can effectively distinguish 2 main cultivated varieties, provide a basis for early and accurate identification of the Jinggang honey pomelo varieties and play a positive role in producing clear variety names.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a primer for identifying the cultivation type of Jinggang honey pomelo, wherein the primer group comprises any 1 pair or a combination of a plurality of pairs of PA1, PA2, PA3, PA4, PA5, PA6, PA7 and PA 8; the sequences of the primers are shown in Table 1:
TABLE 1 sequences of primer sets
The invention develops the 8 InDel marked primers with good polymorphism based on the whole genome resequencing result, and the 8 pairs of primers can distinguish 2 main cultivated varieties by using any 1 pair, thereby providing basis for early and accurate identification of the Jinggang honey pomelo varieties and playing a positive role in producing clear variety names. The Jinggang honey pomelo cultivars of the present invention preferably include Jin Lanyou pomelo and Jin Shayou, wherein Jin Shayou is a filial generation of Jin Lanyou (female parent) and Shatian pomelo (male parent), and Jinsha pomelo is a offspring.
The method for acquiring the primer group is not particularly limited, preferably young leaves are placed in liquid nitrogen for grinding, then total genome DNA is extracted, quality evaluation is carried out on the obtained original data through FastQC, adapter sequences are removed through Fastp software, data quality control is carried out, index is established by taking a Citrus maxima (Burm.) Merr.cv.Wanbai (website: http:// Citrus. Hzau. Edu. Cn/original/download/index. Php) as a reference genome, and the sequenced fragments are compared with the reference genome sequence through BWA software, inDel detection and filtration are carried out through GATK. InDel marker screening was performed using stringent parameters (Simona et al, 2017): (1) the QUAL of the mutation site is greater than 400; (2) the GQ value of the mutation site is 99; (3) a depth of coverage greater than 20; (4) InDel insertions or deletions are between 5 and 8bp in length. The flanking sequence information of the variant position is screened out according to the annotation of the reference genome, and the amplification primer of the InDel marker is designed by using Primer3.0 (http:// bioinfo. Ut. Ee/primer3 /). Primer specific detection was then performed using e-PCR, and finally 8 pairs of specific InDel-labeled amplification primers were selected.
The invention also provides a kit for identifying the cultivation type of the Jinggang honey pomelo, which comprises the primer group.
The kit of the invention preferably further comprises PCR reagents, such as Mix solution and/or ddH 2 O。
The invention also provides a method for identifying the cultivation type of the Jinggang honey pomelo, which comprises the following steps: taking genomic DNA of a Jinggang honey pomelo cultivar as template DNA, carrying out PCR amplification by using the primers, and carrying out homozygosis when the amplified product has only one band, wherein the variety is male parent Shatian pomelo or female parent Jin Lanyou; two or more than two bands are heterozygous, and the variety is offspring.
Specifically, in the invention, the genotypes of the female parent Jin Lanyou, male parent Shatian pomelo and the offspring Jin Shayou are taken as references, and the variety type is judged according to the sample genotypes. The record consistent with the genotype of the female parent is 1, and the variety is Jin Lanyou; the record consistent with the male parent genotype is 2, and the variety is Shatian pomelo; the record consistent with the offspring genotype is 3, and the variety is golden sand pomelo; the record of other genotypes is 4, which is other varieties; genotype deletions were recorded as 0.
The primer group or the kit can be applied to early identification and can be used for utilizing young leaves of young trees, so that the genome DNA is preferably extracted from the young leaves. The method for extracting the genomic DNA according to the present invention is not particularly limited, and preferably a CTAB method or a kit method is used.
The system for PCR amplification according to the present invention preferably comprises, in 15. Mu.l: mu.l Mix, 0.3. Mu.l 10mmol/L forward primer, 0.3. Mu.l 10mmol/L reverse primer, 1.5. Mu.l template DNA and the balance ddH 2 O. The PCR amplification procedure of the present invention preferably comprises a pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s, annealing at 55℃for 30s, extension at 72℃for 30s, and reaction for 35 cycles; and then the extension is carried out for 5min at 72 ℃.
When the primer group or the kit is used for the identification, at least two rounds of PCR identification are preferably carried out, then agarose gel electrophoresis is used for retaining the primer with clear gel pattern and completely consistent size with the prediction, and the primer is amplified to obtain two bands, so that the locus is heterozygous, and the size difference which can be distinguished by the gel pattern exists, so that the locus can be determined to be the marker with good effect. The agarose gel electrophoresis of the invention preferably adopts 1.5-2% of agarose gel by mass percent, the voltage is 120V, and the electrophoresis is 15-20min.
The beneficial effects are that: according to the invention, 8 InDel marked primers with good polymorphism are developed based on the whole genome resequencing result, and 2 main cultivated varieties can be distinguished in early stage by using any 1 pair of 8 pairs of primers, so that a basis is provided for early and accurate identification of the Jinggang honey pomelo varieties, and positive effects are exerted on clarifying variety names in production.
Drawings
FIG. 1 is a diagram of the specificity verification of PA1 and PA 2; in each figure, lanes 1,2 are female parent Jin Lanyou, lane 3 are offspring Jin Shayou, lanes 4,5 are male parent Shatian pomelo, the remainder are jingang honey pomelo producing region cultivars, and the following is the same;
FIG. 2 is a diagram of the specificity verification of PA3 and PA 4;
FIG. 3 is a diagram of the specificity verification of PA5 and PA 6;
FIG. 4 is a diagram of the specificity verification of PA7 and PA 8.
Detailed Description
The primer set, the kit and the identification method for identifying the cultivation type of the Jinggang honey pomelo provided by the invention are described in detail below by combining with the examples, but are not to be construed as limiting the protection scope of the invention.
Example 1
The primers shown in Table 1 were synthesized by Beijing Engine biotechnology Co.
Materials: extracting the gene group DNA of the young leaf of the honey pomelo.
The PCR reaction system was 15. Mu.l: mu.l Mix, 0.3. Mu.l of 10mmol/L forward primer, 0.3. Mu.l of 10mmol/L reverse primer, 1.5. Mu.l of 100 ng/. Mu.l template DNA and 6. Mu.l ddH2O.
The PCR amplification procedure was: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s, annealing at Tm55℃for 30s, elongation at 72℃for 30s, and reaction for 35 cycles; finally, the extension is carried out at 72 ℃ for 5min.
Agarose gel electrophoresis adopts 1.5-2% of agarose gel with the mass percent, the voltage is 120V, and the electrophoresis is 15-20min. Through at least two rounds of PCR identification, the primer with clear gel pattern and completely consistent size with the prediction is reserved, and the result is shown in figures 1-4, the primer is amplified to obtain two bands, the locus is heterozygous, the size difference of the gel pattern can be distinguished, the marker with good effect can be determined, the detection rate is 92.31-100%, and the detection accuracy is 100% (Table 2).
Table 2 8 statistics of InDel molecular marker identification results
Note that: the records consistent with the maternal genotype were 1, with the paternal genotype were 2, with the offspring genotype were 3, with the other genotypes were 4, and the deletion was 0.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (4)
1. A method for identifying the cultivated type of jingang honey pomelo, which is characterized by comprising the following steps: taking genomic DNA of a Jinggang honey pomelo cultivar as template DNA, carrying out PCR amplification by using a primer, and carrying out homozygosity when the amplified product has only one band, wherein the variety is male parent Shatian pomelo or female parent Jin Lanyou; heterozygous when two or more than two bands exist, and the variety is offspring Jin Shayou;
the primer group comprises any 1 pair or combination of two pairs of PA4 and PA 5;
the PA4 comprises PA4-F with a nucleotide sequence shown as SEQ ID NO.7 and PA4-R with a nucleotide sequence shown as SEQ ID NO. 8;
the PA5 comprises PA5-F with a nucleotide sequence shown as SEQ ID NO.9 and PA5-R with a nucleotide sequence shown as SEQ ID NO. 10.
2. The method of claim 1, wherein the genomic DNA is extracted from young leaves of honey pomelo tree.
3. The method of claim 1, wherein the PCR amplification system is 15 μl, comprising: mu.l Mix, 0.3. Mu.l 10mmol/L forward primer, 0.3. Mu.l 10mmol/L reverse primer, 1.5. Mu.l template DNA and the balance ddH 2 O。
4. A method according to claim 1 or 3, wherein the PCR amplification procedure comprises a pre-denaturation at 94 ℃ for 5min; denaturation at 94℃for 30s, annealing at 55℃for 30s, extension at 72℃for 30s, and reaction for 35 cycles; and then the extension is carried out for 5min at 72 ℃.
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| CN202210163279.XA CN114438249B (en) | 2022-02-22 | 2022-02-22 | Primer group, kit and identification method for identifying cultivation types of valium odoratum honey pomelos |
| CN202211017099.7A CN116287364B (en) | 2022-02-22 | 2022-02-22 | Primer group, kit and method for identifying cultivation type of Jiangxi local pomelo |
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| KR101099624B1 (en) * | 2009-12-28 | 2011-12-28 | 대한민국 | Specific primer for discrimination of aborted anthers in Citrus tree and uses thereof |
| CN102492774A (en) * | 2011-12-09 | 2012-06-13 | 南京农业大学 | Primers and method for quickly distinguishing orange varieties |
| CN103725785B (en) * | 2014-01-09 | 2014-12-31 | 中国林业科学研究院热带林业研究所 | Construction method and application of teak clone fingerprint spectrums |
| KR101603156B1 (en) * | 2014-02-26 | 2016-03-17 | 대한민국 | A method for identifying citrus varieties using microsatellites markers |
| CN105603096B (en) * | 2016-02-25 | 2020-01-14 | 华中农业大学 | Codominant InDel molecular marker for identifying single embryo and multiple embryos of citrus and application thereof |
| KR101877532B1 (en) * | 2017-06-01 | 2018-07-12 | 대한민국(농촌진흥청장) | Primers for detecting SNP markers specific to Haryejosaeng and uses thereof |
| CN108660246B (en) * | 2018-05-21 | 2021-01-22 | 华中农业大学 | InDel molecular markers of Ma (Citrus paradisi) pomelos and application of InDel molecular markers in early-stage differentiation of rough-peel Ma pomelos of citrus varieties |
| KR101994831B1 (en) * | 2018-10-02 | 2019-07-01 | 주식회사 바이오메딕 | Multiplex primer sets for determining genotype of locus resistant to Citrus tristeza virus and uses thereof |
| CN110042172B (en) * | 2019-06-03 | 2020-12-04 | 广东省农业科学院果树研究所 | Rapid identification primer and method for citrus hybrids based on SNP markers |
| CN110468127B (en) * | 2019-09-11 | 2022-06-21 | 华中农业大学 | CAPS molecular marker suitable for identifying red-pulp honey pomelos, white-pulp honey pomelos and yellow-pulp honey pomelos and application thereof |
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| JP7398121B2 (en) * | 2020-05-29 | 2023-12-14 | 国立研究開発法人農業・食品産業技術総合研究機構 | Citrus variety identification methods and identification kits |
| JP7316668B2 (en) * | 2020-07-30 | 2023-07-28 | 国立研究開発法人農業・食品産業技術総合研究機構 | How to identify citrus varieties |
| CN113355448B (en) * | 2021-07-09 | 2022-04-08 | 华中农业大学 | InDel molecular marker for identifying phyllanthus emblica and application thereof |
| CN113528698B (en) * | 2021-07-14 | 2022-01-25 | 中国农业科学院油料作物研究所 | InDel molecular marker for identifying and/or distinguishing cabbage vegetables and application thereof |
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| CN109266776A (en) * | 2018-10-24 | 2019-01-25 | 华中农业大学 | Utilize the kit and method of InDel Marker Identification citrus shaddock hybrid generation |
| CN111286556A (en) * | 2020-04-03 | 2020-06-16 | 江西省农业科学院园艺研究所 | Method for identifying variety of golden orchid pomelo based on whole genome InDel marker |
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