CN112251535A - KASP marker for rapidly identifying fertility of onion nuclei in large groups and application thereof - Google Patents
KASP marker for rapidly identifying fertility of onion nuclei in large groups and application thereof Download PDFInfo
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Abstract
The invention provides a KASP marker for rapidly identifying fertility of cell nuclei of a large number of groups of onions and application thereof, belonging to the technical field of molecular biology. Wherein, the nucleotide sequence of the primer for amplifying the KASP marker is shown as the sequence 1-3 in the sequence table. The KASP marker is utilized to assist in breeding the onion male sterile line and the matched maintainer line, so that the method has the characteristics of high throughput, low cost and high accuracy, is accurate, reliable, convenient and quick in result, greatly improves the selection efficiency, saves time and cost, and plays an important role in detecting a large number of onion cell nucleus genotypes, thereby having good practical application value.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to a KASP marker for rapidly identifying fertility of cell nuclei of a large number of groups of onions and application thereof.
Background
The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
Onion (Allium cepa L.) is a biennial vegetable of alliaceae, belonging to the genus Allium, and is one of the important vegetables widely planted in the world, the planting area is second only to that of tomatoes and potatoes, and the total yield is third in all vegetable crops (FAO, 2018). The onion is an important export foreign exchange special vegetable in China, and the area and the yield of the onion are in the top of the world. Onion is not only fresh but also used in processed products in large quantities, and is rich in various sulfides and sugars, which are the main reasons for its unique flavor. From the perspective of medical care, onion has the effects of resisting thrombus, promoting blood circulation, resisting cancer and the like, and the nutritional and medical values of onion are gradually recognized all over the world.
The onion originates from the middle Asia region, the cultivation history is short in China, and the variety resources are relatively deficient. The breeding of hybrid species is far behind the breeding of countries such as Japan, America, Netherlands, etc. At present, high-end varieties generally need to be imported, and the price of seeds is 2-3 times or even more than that of domestic varieties of the same type. With the adjustment of agricultural industrial structure, the demand for good varieties of onions is increasing day by day, the production mainly takes variety introduction and conventional variety breeding as main parts, a first filial generation variety with the proprietary intellectual property right of China is lacked, a large amount of foreign exchange imported seeds from foreign countries is needed, and the production cost is high.
Onion is the vegetable crop which is selected and bred by the hybrid seed by using the cytoplasmic male sterile line at the earliest, the hybrid seed production mainly utilizes a CMS-S male sterile system of cytoplasmic-nuclear interaction, the sterility is jointly controlled by a sterile cytoplasmic factor (S) and a pair of nuclear recessive monogenes (msms), the genotype of the sterile line is S (msms), and the genotype of the maintainer line is N (msms). Therefore, the key link of hybrid breeding is the breeding of the sterile line and the maintainer line thereof. As the onion is a two-year cross-pollinated vegetable, the self-pollination decays seriously, the identification of the male sterile genotype by the traditional test cross method needs 4 to 8 years, and the method has the problems of time consumption, labor consumption and low efficiency. The conventional breeding and molecular marker assisted breeding are combined. The molecular marker assisted selection technology of the cytoplasmic male sterile gene can greatly improve the selection efficiency of the onion male sterile line and the onion maintainer line.
Because onion genomic DNA is large (17.9pg), 15,290Mbp is present in every 1 chromosome, which is 6 times that of maize genome, 16 times that of tomato, and 107 times that of Arabidopsis. The nuclear gene markers of the CMS-S system have been studied slowly, but have made substantial progress, compared to most other vegetable crops. Several molecular markers that are currently more effective are used for molecular markers of the Ms locus of the nuclear gene. The three markers AOB272, OPT and PsaO can be used for breeding maintainer lines in a segregation population linked with the markers, but cannot be applied to a population with unknown background, only can reduce the workload of testing and crossing of the segregation population, and cannot shorten the breeding process. The coseparation SCAR marker which is developed by the research team and is closely linked with the onion male sterility gene Ms and the fertility restorer gene Ms can identify the genotype of an onion single plant at the Ms locus, but can be determined only by two PCR amplifications. Recently, Huo et al (2015) developed a co-dominant PCR molecular marker, AcSKP1, and the genotype of the Ms site was identified by one-time PCR. The development and application of the markers effectively avoid the blindness of maintainer line screening and improve the selection efficiency. However, the inventors found that the markers are based on PCR and then subjected to electrophoresis to judge the fertility of the cell nucleus, and although the markers are more rapid than field identification, at most 96 samples can be detected each time, and for the detection of a large number of samples, the detection efficiency is low, the amplification result is not stable, and the method has certain limitations.
Disclosure of Invention
Aiming at the prior art, long-term technical and practical exploration shows that the invention provides the KASP marker for rapidly identifying the fertility of the onion cell nucleus of a large number of populations and the application thereof, the KASP marker can be used for assisting in breeding the onion male sterile line and the matched maintainer line, has the characteristics of high throughput, low cost and high accuracy, and plays an important role in the detection of the large number of populations of the onion cell nucleus genotype, so the KASP marker has good practical application value.
The invention is realized by the following technical scheme:
in a first aspect of the invention, there is provided a KASP marker for rapidly identifying fertility of a large population of onion nuclei, wherein the nucleotide sequence of primers for amplifying the KASP marker is as follows:
SEQ ID NO.1(KASP-MS)：
5'-GCAACATCTTCTTCTACTTGTTGTATAGA-3'；
SEQ ID NO.2(KASP-ms)：
5'-GCAACATCTTCTTCTACTTGTTGTATAGT-3'；
SEQ ID NO.3(KASP-C)：
5'-TCATTTATATGGRTAAAAACTGCTGATGGC-3'。
preferably, the 5' ends of the sequences 1 and 2 are also designed with fluorescent labels;
more preferably, the 5 'end of the sequence 1 is designed with a first fluorescent label, the 5' end of the sequence 2 is designed with a second fluorescent label, and the first fluorescent label and the second fluorescent label are not the same; in one embodiment of the present invention, the first fluorescent tag may be FAM and the second fluorescent tag may be HEX.
Thus, the nucleotide sequences of the primers for amplifying the KASP tag are as follows:
SEQ ID NO. 4: the FAM fluorescent tag sequence is underlined
5'-GAAGGTGACCAAGTTCATGCGCAACATCTTCTTCTACTTGTTGTATAGA-3'；
SEQ ID No. 5: HEX fluorescent tag sequence is underlined
5'-GAAGGTCGGAGTCAACGGAGCAACATCTTCTTCTACTTGTTGTATAGT-3'；
SEQ ID NO.3：
5'-TCATTTATATGGRTAAAAACTGCTGATGGC-3'。
In a second aspect of the present invention, there is provided the above KASP-labeled amplification primer, comprising:
SEQ ID NO.1(KASP-MS)：
5'-GCAACATCTTCTTCTACTTGTTGTATAGA-3'；
SEQ ID NO.2(KASP-ms)：
5'-GCAACATCTTCTTCTACTTGTTGTATAGT-3'；
SEQ ID NO.3(KASP-C)：
5'-TCATTTATATGGRTAAAAACTGCTGATGGC-3'。
preferably, the 5' ends of the sequences 1 and 2 are also designed with fluorescent labels;
more preferably, the 5 'end of the sequence 1 is designed with a first fluorescent label, the 5' end of the sequence 2 is designed with a second fluorescent label, and the first fluorescent label and the second fluorescent label are not the same; in one embodiment of the present invention, the first fluorescent tag may be FAM and the second fluorescent tag may be HEX.
Thus, the nucleotide sequences of the primers for amplifying the KASP tag are as follows:
SEQ ID NO. 4: the FAM fluorescent tag sequence is underlined
5'-GAAGGTGACCAAGTTCATGCGCAACATCTTCTTCTACTTGTTGTATAGA-3'；
SEQ ID No. 5: HEX fluorescent tag sequence is underlined
5'-GAAGGTCGGAGTCAACGGAGCAACATCTTCTTCTACTTGTTGTATAGT-3'；
SEQ IDNO.3：
5'-TCATTTATATGGRTAAAAACTGCTGATGGC-3'。
In a third aspect of the invention, there is provided the use of the above KASP marker and/or KASP marker amplification primer for rapidly identifying fertility in the nucleus of a large population of onions.
In a fourth aspect of the invention, there is provided a method for rapidly identifying fertility in a plurality of onion cell nuclei of a population, the method comprising:
carrying out PCR amplification on the genomic DNA of the onion material to be detected based on the KASP labeled amplification primer; and detecting and judging the genotype of the polymorphic site in the PCR amplification product.
Specifically, the reaction procedure of the PCR amplification comprises the following steps: pre-denaturation at 94 ℃ for 15 min; denaturation at 94 ℃ for 20s, annealing at 61-55 ℃ for 1min (0.6 ℃ per cycle), 10 cycles; denaturation at 94 ℃ for 20s, annealing at 55 ℃ for 1min, and 26-29 cycles.
More specifically, the method comprises:
a) extracting total DNA of the onion material to be detected;
b) transferring the sample from the 96-well plate to a 384-well plate, and finally transferring the sample to a 1536-well plate to ensure that the final concentration of the sample DNA is about 10 ng/. mu.l;
c) drying the 1536 pore plate filled with the DNA sample;
d) constructing a PCR system on the dried DNA sample; preferably, only 1. mu.l of reaction system is required for each reaction, including 10ng of DNA, 0.5. mu.l of KASP-labeled amplification primer, 2 × Master Mix standard ROX, 0.014. mu.l of KASP-by-Design assay Mix, ddH2Supplementing O to 1 μ l;
e) sealing the membrane of the pore plate added with the reaction system, and quickly centrifuging at a low speed;
f) carrying out water bath PCR after centrifugation, wherein the reaction program comprises pre-denaturation at 94 ℃ for 15 min; denaturation at 94 ℃ for 20s, annealing at 61-55 ℃ for 1min (0.6 ℃ per cycle), 10 cycles; denaturation at 94 deg.C for 20s, annealing at 55 deg.C for 1min, and 26-29 cycles;
g) and drying the reacted pore plate, cooling, reading the plate on a microplate reader Pheastar, analyzing the scanning data by adopting SNPviewer2 software, and determining the genotype of the onion cell nucleus according to the analysis result, namely detecting the cell nucleus fertility of the onion sample.
More specifically, if the fluorescence signal of the amplification product of the individual to be detected is marked by a red dot, the cell nucleus genotype of the onion sample is invisible homozygous msms; if the fluorescence signal of the onion amplification product to be detected is marked by a blue dot, the cell nucleus genotype of the onion sample is dominant homozygous MsMs; and if the fluorescence signal of the onion amplification product to be detected is marked by a green dot, determining that the genotype of the onion sample is heterozygous Msms.
In a fifth aspect of the invention, the KASP marker amplification primer and/or the identification method are applied to breeding of onion male sterile lines and matched maintainer lines.
In a sixth aspect of the invention, there is provided a detection kit comprising the above KASP-labeled amplification primer.
Specifically, the kit further comprises a fluorescent probe and a reagent required by the construction of a PCR system.
The beneficial technical effects obtained by one or more technical schemes are as follows:
(1) fast, efficient, high throughput: the KASP marker primer is used for detecting the genotype of the onion cell nucleus, can be used for high-throughput PCR of 96-well plates, 384-well plates and 1536-well plates, can quickly judge the cell nucleus type in high throughput through data display after reading the plates, greatly improves the detection efficiency, reduces the time and labor cost, is very beneficial to high-throughput genotyping selection of onion field breeding materials, and is particularly suitable for operation of a large number of samples.
(2) The marking is stable and convenient: the onion material with known cell nucleus genotype is verified, and the identification result is completely consistent with the phenotype. Compared with the existing SCAR marker, the screening method of the KASP marker primer in the technical scheme is simple and easy to implement, the genotype of the onion cell nucleus can be determined only by reading a plate by a microplate reader without any electrophoresis after the PCR reaction is finished, the amplification efficiency is high, the result is accurate, reliable, convenient and quick, the cost is low, and the batch, automation and standardization of experimental operation can be realized, so that the method has good value of practical application.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.
FIG. 1 is a chart of typing clusters of KASP molecular markers in example 1 of the present invention.
FIG. 2 is the verification of the detection result of KASP marker by using mature SCAR molecular marker in example 2 of the present invention. Wherein: 1-19, 41-59, 81-95 are hybrids 152, 172, 153, respectively, from the OP population; 21-39, 61-79 are hybrid seeds 121, 132 from different inbred lines, respectively; 20. 40 is a positive control 303, and the cell nucleus genotype is MsMs; 60. 80 is positive control 203, the genotype of the cell nucleus is msms; 96 is no template control NTC; m is DNA molecular weight standard DL 2000.
Detailed Description
It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise. It is to be understood that the scope of the invention is not to be limited to the specific embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention.
The present invention is further illustrated by reference to specific examples, which are intended to be illustrative only and not limiting. If the experimental conditions not specified in the examples are specified, the conditions are generally as usual or as recommended by the reagents company; reagents, consumables and the like used in the following examples are commercially available unless otherwise specified.
As mentioned above, the existing molecular marker for onion cytoplasmic male sterility genes has low detection efficiency, and amplification results are not stable, so that the method has certain limitations.
In view of the above, the invention provides a KASP marker and a corresponding amplification primer for rapidly and accurately identifying a large number of populations of onion cell nucleus types, which can be used for assisted breeding of onion male sterile lines and matched maintainer lines, have the characteristics of high throughput, low cost and high accuracy, and play an important role in the detection of a large number of populations of onion cell nucleus genotypes.
Specifically, the KASP marker for identifying the onion cell nucleus type comprises KASP-MS, KASP-MS and KASP-C, and the nucleotide sequence of a primer for amplifying the marker is shown as a sequence 3-5 in a sequence table:
SEQ ID NO. 4: the FAM fluorescent tag sequence is underlined
5'-GAAGGTGACCAAGTTCATGCGCAACATCTTCTTCTACTTGTTGTATAGA-3'；
SEQ ID No. 5: HEX fluorescent tag sequence is underlined
5'-GAAGGTCGGAGTCAACGGAGCAACATCTTCTTCTACTTGTTGTATAGT-3'；
SEQ ID NO.3：
5'-TCATTTATATGGRTAAAAACTGCTGATGGC-3'。
It should be noted that other fluorescent tag sequences commonly used in the art can also be used as the fluorescent tag sequence.
KASP is a competitive allele specific PCR marker (KASP) that allows for accurate biallelic determination of SNPs and InDels at specific sites in a wide range of genomic DNA samples (even DNA samples of complex genomes). The marker has the characteristics of high throughput, low cost and high accuracy, and plays an important role in molecular marker-assisted breeding application.
In the invention, a specific set of KASP primers is designed according to SNP mutation site information of onion skp1-Ms (GenBank No. KM065384.1) and skp1-Ms (GenBank No. KM065385.1) gene sequences in a GenBank database, and consists of two upstream primers (KASP-MS and KASP-Ms) and a downstream primer (KASP-C), wherein the 5 'ends of the two upstream primers are connected with fluorescent tag sequences, the 3' end comprises allelic variant base T/A which is used for distinguishing alleles, the primers KASP-MS and KASP-C are amplified to read fertile cell nucleus type MsMs, and the primers KASP-Ms and KASP-C are amplified to read sterile cell nucleus type MsMs, and if the primers are heterozygous, the KASP-MS and KASP-Ms can be amplified with the KASP-C. After the PCR reaction is finished, reading the plate on a microplate reader Pheastar, analyzing the scanning data by adopting SNPviewer2 software, determining the genotype of the cell nucleus of the onion according to the analysis result, and if the fluorescence signal data of the amplification product of the individual to be detected is analyzed by SNPviewer2 and then is marked as a red dot, then the genotype of the cell nucleus of the onion sample is invisible and homozygous msms; if the fluorescence signal of the onion amplification product to be detected is marked by a blue dot, the cell nucleus genotype of the onion sample is dominant homozygous MsMs; and if the fluorescence signal of the onion amplification product to be detected is marked by a green dot, determining that the genotype of the onion sample is heterozygous Msms. And if the fluorescence signal data of the amplification product of the sample to be detected is pink after being analyzed by SNPviewer2, the result is invalid.
The invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
Example 1 establishment of KASP marker and detection method
(I) obtaining of KASP-tagged primer sequences
According to the SNP mutation site information of onion skp1-Ms (GenBank No. KM065384.1) and skp1-Ms (GenBank No. KM065385.1) gene sequences in a GenBank database, a special set of KASP marker primers is designed by using Primer Premier 5.0 and Primer 3.0(http:// biolnfo. ut. ee/Primer3-0.4.0/Primer3/input. htm), and consists of two upstream primers (KASP-MS and KASP-Ms) and a downstream Primer (KASP-C), wherein the nucleotide sequences of the primers are shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 in a sequence table.
SEQ ID NO.1(KASP-MS)：
5'-GCAACATCTTCTTCTACTTGTTGTATAGA-3'；
SEQ ID NO.2(KASP-ms)：
5'-GCAACATCTTCTTCTACTTGTTGTATAGT-3'；
SEQ ID NO.3(KASP-C)：
5'-TCATTTATATGGRTAAAAACTGCTGATGGC-3'。
The 5' ends of the two upstream primers are connected with a fluorescent label sequence, the 3' ends comprise allelic variant base T/A, wherein the 5' end of the primer KASP-MS is connected with a FAM fluorescent label sequence: 5'-GAAGGTGACCAAGTTCATGC-3' (SEQ ID NO.6), the 5' end of the primer KASP-ms is connected with a HEX fluorescent tag sequence: 5'-GAAGGTCGGAGTCAACGGA-3' (SEQ ID NO. 7). The primer sequence was synthesized by Shanghai shellfish crystal biotechnology, Inc.
Detection of the (II) KASP marker
Utilizing male parent inbred line 303 (genotype is S (MsMs))]Sterile line 203[ genotype is S (msms)]F obtained after hybridization1Generation, 96 samples were randomly selected as a sample population among the parental and filial generations.
a) The total DNA extraction method of onion material adopts a PlantZol genome extraction kit produced by Beijing all-purpose gold biotechnology limited, and the extraction method refers to the instruction. Agarose gel electrophoresis was used to determine the concentration and quality of the DNA.
b) The PCR reaction is carried out by using primers with nucleotide sequences shown as SEQ NO.4, SEQ NO.5 and SEQ NO.3 in the sequence table.
c) Transferring the sample from a 96-well plate to a 384-well plate by Replikator, and finally transferring to a 1536-well plate, ensuring that the final concentration of sample DNA is about 10 ng/. mu.l;
d) placing 1536 well plates filled with DNA in a 65 ℃ oven for drying for 30 min;
e) the dried DNA was subjected to PCR system construction, each reaction requiring only 1. mu.l of a reaction system comprising 10ng of DNA, 0.5. mu.l of KASP-labeled primer, 2 × Master Mix standard ROX (LCG Genomics, Teddington, Middlesex, UK, Beverly, MA, USA) and 0.014. mu.l of KASP-by-Design assay Mix (LGC Genomics, Beverly, MA, USA), ddH2Supplementing O to 1 μ l;
f) sealing the membrane of the pore plate added with the reaction system, and quickly centrifuging at a low speed;
g) carrying out water bath PCR after centrifugation, wherein the reaction program comprises pre-denaturation at 94 ℃ for 15 min; denaturation at 94 ℃ for 20s, annealing at 61-55 ℃ for 1min (0.6 ℃ per cycle), 10 cycles; denaturation at 94 deg.C for 20s, annealing at 55 deg.C for 1min, and 26-29 cycles; drying the reacted pore plate, cooling, reading the plate on a microplate reader Pheastar, analyzing the scanning data by adopting SNPviewer2 software, determining the genotype of the cell nucleus of the onion according to the analysis result, and if the fluorescence signal data of the amplification product of the individual to be detected is analyzed by SNPviewer2 and is marked by red dots, the genotype of the cell nucleus of the onion sample is invisible homozygous msms; if the fluorescence signal of the onion amplification product to be detected is marked by a blue dot, the cell nucleus genotype of the onion sample is dominant homozygous MsMs; if the fluorescence signal of the onion amplification product to be detected is marked by a green dot, the genotype of the onion sample is heterozygous Msms (FIG. 1).
Example 2 the results of the detection of KASP molecular markers were verified using SCAR molecular markers.
The onion hybrids tested were 5 groups: wherein 121 and 132 are derived from inbred lines of different genetic backgrounds [ genotype s (msms) ], 152, 153 and 172 are hybrids derived from OP populations.
Performing KASP marker verification on the 5 groups of hybrid materials from different sources, wherein the fluorescence signals of the msms onion individual amplification products with the cytoblast genotypes being invisible homozygous are marked by red dots; the fluorescent signal of the onion individual amplification product with the cell nucleus genotype of dominant homozygous MsMs is marked by a blue dot; the fluorescence signal of the onion individual amplification product with genotype heterozygous Msms is marked by green dots. The results of the genotypic survey are shown in Table 1.
TABLE 1 results of KASP molecular marker typing
The developed SCAR molecular marker capable of accurately identifying the onion cell nucleus genotype is used for identifying the cell nucleus genotype of the 5 groups of hybrids from different sources, and the specific method for detecting the onion cell nucleus genotype by the SCAR molecular marker refers to an authorized invention patent ZL201410256193.7, and specifically comprises the following steps:
PCR amplification primers:
FN1：5'-ATACACAGCTTCTAGCTGAATTTTTA-3'(SEQ ID NO.8)；
FN2：5'-TGTGTGTGTAATTTCTCTGTGCG-3'(SEQ ID NO.9)；
RN1：5'-ACAGAGTGAGAAATTTTATATATATAGGAAT-3'(SEQ ID NO.10)；
RN2：5'-CGGAAGATTAATATTTTGCGTATACAT-3'(SEQ ID NO.11)。
the above primers are used to amplify DNA from different hybrid populations. The PCR product was analyzed by 1% agarose gel electrophoresis to detect polymorphisms. The sample with the cell nucleus genotype of Msms only amplifies a segment of 886bp, the sample with the cell nucleus genotype of Msms amplifies two spectral bands of 886bp and 621bp, and the sample with the cell nucleus genotype of MsMs only amplifies a spectral band of 621bp (figure 2).
The analysis results of the hybrid population from different sources by using the SCAR molecular marker are shown in figure 2, and the judgment result of the invention is completely consistent with the KASP typing result.
It should be noted that the above-mentioned embodiments are only preferred embodiments of the present invention, and the present invention is not limited thereto, and although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications and equivalents can be made in the technical solutions described in the foregoing embodiments, or equivalents thereof. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention. Although the embodiments of the present invention have been described with reference to the accompanying drawings, it is not intended to limit the scope of the present invention, and it should be understood by those skilled in the art that various modifications and variations can be made without inventive efforts by those skilled in the art based on the technical solution of the present invention.
SEQUENCE LISTING
<110> institute of vegetables and flowers of academy of agricultural sciences of Shandong province
<120> KASP marker for rapidly identifying fertility of cell nucleus of a large number of groups of onions and application thereof
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<170> PatentIn version 3.3
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<211> 19
<212> DNA
<213> HEX fluorescent tag
<400> 7
<210> 8
<211> 26
<212> DNA
<213> Artificial sequence
<400> 8
atacacagct tctagctgaa ttttta 26
<210> 9
<211> 23
<212> DNA
<213> Artificial sequence
<400> 9
tgtgtgtgta atttctctgt gcg 23
<210> 10
<211> 31
<212> DNA
<213> Artificial sequence
<400> 10
acagagtgag aaattttata tatataggaa t 31
<210> 11
<211> 27
<212> DNA
<213> Artificial sequence
<400> 11
cggaagatta atattttgcg tatacat 27
Claims (10)
1. A KASP marker for rapidly identifying the fertility of a large number of onion cell nuclei is characterized in that a primer nucleotide sequence for amplifying the KASP marker is shown as a sequence 1-3 in a sequence table.
2. The KASP marker of claim 1, wherein the 5' ends of SEQ ID NO.1 and SEQ ID NO.2 are further designed with a fluorescent tag;
preferably, the 5 'end of the sequence 1 is designed with a first fluorescent label, the 5' end of the sequence 2 is designed with a second fluorescent label, and the first fluorescent label and the second fluorescent label are not the same;
further preferably, the first fluorescent label is FAM and the second fluorescent label is HEX.
3. The KASP-labeled amplification primer of claim 1, wherein the nucleotide sequence of said amplification primer is represented by sequences 1-3 in the sequence Listing;
preferably, the 5' ends of the sequences 1 and 2 are also designed with fluorescent labels;
more preferably, the 5 'end of the sequence 1 is designed with a first fluorescent label, the 5' end of the sequence 2 is designed with a second fluorescent label, and the first fluorescent label and the second fluorescent label are not the same;
further preferably, the first fluorescent label is FAM and the second fluorescent label is HEX.
4. Use of a KASP marker of claim 1 or 2 and/or an amplification primer of a KASP marker of claim 3 for rapidly identifying nuclear fertility in a large population of onion nuclei.
5. A method for rapidly identifying fertility in the nucleus of a plurality of onion nuclei in a population, said method comprising:
carrying out PCR amplification on the genomic DNA of the onion material to be detected based on the KASP-labeled amplification primer as defined in claim 3; and detecting and judging the genotype of the polymorphic site in the PCR amplification product.
6. The method of claim 5, wherein the reaction sequence for PCR amplification comprises: pre-denaturation at 94 ℃ for 15 min; denaturation at 94 deg.C for 20s, annealing at 61-55 deg.C for 1min, and reducing each cycle by 0.6 deg.C for 10 cycles; denaturation at 94 ℃ for 20s, annealing at 55 ℃ for 1min, and 26-29 cycles.
7. The method of claim 5 or 6, wherein the method comprises:
a) extracting total DNA of the onion material to be detected;
b) transferring the sample from a 96-well plate to a 384-well plate, and finally transferring the sample to a 1536-well plate to ensure that the final concentration of the sample DNA is 10 ng/. mu.l;
c) drying the 1536 pore plate filled with the DNA sample;
d) constructing a PCR system on the dried DNA sample;
e) sealing the membrane of the pore plate added with the reaction system, and quickly centrifuging at a low speed;
f) performing water bath PCR after centrifugation, wherein the PCR reaction program comprises pre-denaturation at 94 ℃ for 15 min; denaturation at 94 deg.C for 20s, annealing at 61-55 deg.C for 1min, and reducing each cycle by 0.6 deg.C for 10 cycles; denaturation at 94 deg.C for 20s, annealing at 55 deg.C for 1min, and 26-29 cycles;
g) and drying the reacted pore plate, cooling, reading the plate on a microplate reader Pheastar, analyzing the scanning data by adopting SNPviewer2 software, and determining the genotype of the onion cell nucleus according to the analysis result, namely detecting the cell nucleus fertility of the onion sample.
8. Use of the KASP marker of claim 1 or 2, the amplification primer of the KASP marker of claim 3, and/or the identification method of any one of claims 5 to 7 for the selective breeding of onion male sterile lines and matched maintainer lines.
9. A test kit comprising KASP-labeled amplification primers of claim 3.
10. The test kit of claim 9, further comprising a fluorescent probe and reagents required for PCR architecture.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113736864A (en) * | 2021-09-09 | 2021-12-03 | 辽宁省农业科学院 | Method for rapidly identifying purity of hybrid seeds of green Chinese onions |
| CN115852017A (en) * | 2022-08-31 | 2023-03-28 | 山东省农业科学院 | Method for identifying purity of onion male sterility three-line matched hybrid based on SNP molecular marker |
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| CN103184293A (en) * | 2013-04-11 | 2013-07-03 | 山东省农业科学院蔬菜研究所 | Codominant SCAR marker for identifying onion male sterility gene and application thereof |
| CN106834480A (en) * | 2017-02-24 | 2017-06-13 | 山东省农业科学院蔬菜花卉研究所 | A kind of KASP marks of a large amount of colony's onion cytoplasm fertilities of Rapid identification and its application |
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| CN103184293A (en) * | 2013-04-11 | 2013-07-03 | 山东省农业科学院蔬菜研究所 | Codominant SCAR marker for identifying onion male sterility gene and application thereof |
| CN106834480A (en) * | 2017-02-24 | 2017-06-13 | 山东省农业科学院蔬菜花卉研究所 | A kind of KASP marks of a large amount of colony's onion cytoplasm fertilities of Rapid identification and its application |
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| YU MENG HUO ET AL.: "AcSKP1, a multiplex PCR-based co-dominant marker in complete linkage disequilibrium with the male-fertility restoration (Ms) locus, and its application in open-pollinated populations of onion", 《EUPHYTICA》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113736864A (en) * | 2021-09-09 | 2021-12-03 | 辽宁省农业科学院 | Method for rapidly identifying purity of hybrid seeds of green Chinese onions |
| CN113736864B (en) * | 2021-09-09 | 2024-05-17 | 辽宁省农业科学院 | Method for rapidly identifying purity of green Chinese onion hybrid |
| CN115852017A (en) * | 2022-08-31 | 2023-03-28 | 山东省农业科学院 | Method for identifying purity of onion male sterility three-line matched hybrid based on SNP molecular marker |
| CN115852017B (en) * | 2022-08-31 | 2023-09-15 | 山东省农业科学院 | Method for identifying purity of onion male sterile three-line matched hybrid based on SNP molecular marker |
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| CN112251535B (en) | 2022-09-23 |
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