CN111705157B - PCR marker and primer for screening I-type S haplotypes of cabbage self-incompatible line - Google Patents
PCR marker and primer for screening I-type S haplotypes of cabbage self-incompatible line Download PDFInfo
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Abstract
The invention relates to a cabbage breeding technology, in particular to a PCR marker and a primer for screening I-type S haplotypes of a cabbage self-incompatible line. The position of the PCR marker corresponds TO chromosome 6 of the brassica oleracea reference genome TO1000, 32,097,032bp-32,097,506bp. The nucleotide sequence of the primer is shown as SEQ ID NO. 1 and SEQ ID NO. 2. The invention also provides a method for screening the I-type S haplotypes of the cabbage self-incompatible line, and the method is simple, convenient and easy to implement, high in accuracy and good in stability, and can shorten the breeding period of the cabbage and improve the breeding efficiency.
Description
Technical Field
The invention relates to a cabbage breeding technology, in particular to a PCR marker and a primer for I-type S haplotype screening of a cabbage self-incompatible line.
Background
Cabbage (Brassica oleracea var. Capitata l.) belongs to Brassica of brassicaceae, and plays an important role in annual supply of vegetables in China. Cabbage belongs to the classical sporophyte type of self-incompatible plant, whose self-incompatibility is controlled by a highly polymorphic S locus with multiple alleles, where genes encoding male and female S determinants are present, and an S locus containing multiple S alleles, also known as S haplotypes (Nasrallah and Nasrallah, 1993). 3 classes of genes linked to this site have been identified: an S-site glycoprotein (S-loci glycoprotein) gene (SLG), an S-Site Receptor Kinase (SRK), an S-site cysteine-rich protein (SCR) gene and an S-site protein 11 (S-loci protein 11) gene (SP 11). Based on the similarity of the S-site sequences, brassica self-incompatibility can be divided into two broad categories: class i and class ii, wherein the class i S haplotype exhibits strong self-incompatibility; class II S haplotypes exhibit poor self-incompatibility (Nasrallah and Nasrallah, 1993).
The cabbage heterosis is obvious, the proportion of hybrid seeds produced in recent years accounts for more than 96% (Wangqingbuso et al, 2012), and the seed production by using the self-incompatible line is an important way for utilizing the heterosis. Generally, a large number of self-incompatible lines with excellent properties (disease resistance, cold resistance and the like) are firstly cultivated, and then a first hybrid generation suitable for market demands is cultivated by using different self-incompatible lines as parents. If the parents of the first hybrid generation are self-incompatible lines with the same S haplotype, bee pollination cannot be performed to produce seeds of the first hybrid generation, so that the parents of the first hybrid generation need to have different S haplotypes for bee pollination. Therefore, it is very necessary to perform a class I S haplotype selection of self-incompatible lines in cabbage cross-breeding work.
The predecessors have designed specific primers PK1/PK4 (Nishio et al, 1997) for screening the type I S haplotypes of the self-incompatible line and specific primers KD4/KD7 (Park et al, 2002) for screening the type II S haplotypes according to the SRK gene sequence, and successfully used for screening the S haplotypes of Brassica crops such as Chinese cabbage and the like. However, when the primer PK1/PK4 is used for amplification of cabbage materials, the problem that an amplification band is unstable is found, the primer cannot be used for marker-assisted screening of the I-type S haplotypes of the cabbage self-incompatibility line, and sequence comparison shows that the primer has low matching degree or cannot be matched in most of the I-type S haplotypes of the cabbage, so that a new primer needs to be developed for screening of the I-type S haplotypes of the cabbage self-incompatibility line.
Disclosure of Invention
According to the requirements of the field, the PCR marker for screening the I type S haplotypes of the cabbage self-incompatible line is developed, and the I type S haplotypes of the cabbage self-incompatible line can be accurately identified. The length of the nucleotide sequence of the PCR marker is 472-475bp, and the PCR marker can be obtained by amplifying the genomic DNA of the I-type S haplotype cabbage plant by adopting a specific primer.
In one aspect, the invention provides a PCR marker for screening I-type S haplotypes of a cabbage self-incompatible line, which is characterized in that: the position of the PCR marker in the cabbage genome corresponds TO chromosome 6 of the cabbage reference genome TO1000, 32,097,032bp-32,097,506bp.
In another aspect, the invention provides a primer for screening I-type S haplotypes of a cabbage self-incompatible line, wherein the nucleotide sequence of the primer is as follows:
the upstream primer PKC6F:5 'CAATTTCACAGAGAATAGTGA-3',
the downstream primer PKC6R:5 'ACCATTCCGGATATCCGCATTT-3'.
The invention also provides a kit for screening the I-type S haplotypes of the cabbage self-incompatible line, which is characterized by comprising the following steps of: including liquid or powder form of the primer.
Preferably, the kit further comprises universal reagents for performing a PCR reaction.
The invention also provides a method for screening I-type S haplotypes of the cabbage self-incompatible line, which comprises the following steps:
(1) Extracting genome DNA of a cabbage material to be detected;
(2) Carrying out PCR reaction by using the primer by using the genome DNA as a template to obtain a PCR product;
(3) And detecting the PCR product by using agarose gel electrophoresis, wherein if a characteristic band of 472bp-475bp appears in the PCR product, the cabbage material to be detected contains the type I S haplotype.
In a preferred embodiment of the method of the present invention, the PCR reaction system is: mu.l of 10. Mu. Mol/L upstream primer, 0.5. Mu.l of 10. Mu. Mol/L downstream primer,
PCR Supermix 5. Mu.L, template DNA 2. Mu.L, ddH 2 O 2μL。
In a preferred embodiment of the method of the invention, the procedure of the PCR reaction is: pre-denaturation at 94 deg.C for 5min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 45s, and 33 cycles; extension at 72 ℃ for 5min.
The application of the primer or the kit in the auxiliary screening of the I-type S haplotypes of the cabbage self-incompatible line also belongs to the protection scope of the invention.
In order to develop a PCR marker capable of accurately identifying the type I S haplotypes of the cabbage self-incompatibility line, 24 type I S haplotypes and 3 type II S haplotypes in the published cabbage (table 1) are downloaded on an NCBI website (https: w.ncbi.nlm.nih.gov /), and the difference sites which can distinguish the type II S haplotypes and are highly conserved in the type I S haplotypes are searched for between the type I S haplotype and the type II S haplotype sequences through sequence comparison. And then designing primers for DNA sequences of the differential sites by using software Primer 5.0, verifying each index of the primers, including the length of the primers, annealing temperature, G + C content, whether Primer dimer and hairpin structure exist, and screening out the primers meeting the conditions. The screened primers are identified by using the cabbage varieties 'late Feng', 'Zhonggan 11' and 'Zhonggan 23' with known S haplotypes, and finally, a PCR marker which can accurately identify whether the cabbage plant to be detected contains the self-incompatible line type I S haplotypes and a primer PKC6F/PKC6R for detecting the PCR marker are obtained. The length of the nucleotide sequence of the PCR marker is 472-475bp, taking the SRK gene of an S12 haplotype as an example, the nucleotide sequence of the PCR marker is shown as SEQ ID NO. 3; the nucleotide sequences of the primers PKC6F/PKC6R are shown in SEQ ID NO. 1 and SEQ ID NO. 2.
The detection accuracy of the primers PKC6F/PKC6R is verified by using the existing cabbage self-bred line materials of Z164, Z157, 10-357, 10-361, 10-364 and 10-503. The PCR results showed that the primers PKC6F/PKC6R amplified 475 bp-specific bands from the plants "Z164", "10-357", "10-361", "10-364" and "10-503" with the type I S haplotype (FIGS. 2-4), and no amplified band in the plant "Z157" without the type I S haplotype. Therefore, the primer PKC6F/PKC6R provided by the invention has strong specificity and good stability, does not produce non-specific amplification at 55 ℃, and can be used for very accurately detecting whether the cabbage plant to be detected contains the I-type S haplotype.
In general, plants containing the self-incompatible class I S haplotype were determined by manual flowering and observation of seed set, which took 4-6 months after seedling. The primer PKC6F/PKC6R provided by the invention is used for carrying out auxiliary screening on the cabbage type I S haplotypes through a PCR method, and a target strain can be rapidly screened at a seedling stage. Therefore, the PCR marker and the primer of the invention are used for auxiliary selection of cabbage breeding, so that the breeding period can be greatly shortened, and the breeding efficiency can be improved.
The kit provided by the invention comprises a primer PKC6F/PKC6R. The kit may further comprise reagents for extracting genomic DNA of the brassica oleracea material, general-purpose reagents for performing a PCR reaction (e.g. DNA polymerase, dntps, reaction buffers, etc.), associated consumables (e.g. centrifuge tubes), and kit instructions describing the methods for class i S haplotype assisted screening of the brassica oleracea self-incompatible line (genomic DNA extraction method, PCR system and procedure).
The method for auxiliary screening of the I-type S haplotypes of the cabbage self-incompatible line provided by the invention only needs to extract the genomic DNA of the cabbage material to be detected, adopts the primer PKC6F/PKC6R designed by the invention to carry out PCR reaction, and detects whether the PCR product contains a characteristic strip of about 475bp through agarose gel electrophoresis, so that whether the I-type S haplotypes of the self-incompatible line are contained in the cabbage material to be detected can be accurately judged. Compared with the traditional screening method, the method has the advantages of simple and easy operation, strong specificity, good stability and the like, and can greatly shorten the breeding period of the cabbage, thereby improving the breeding efficiency.
Drawings
FIG. 1 shows the distribution of the primers PKC6F/PKC6R in the SRK gene (S-site receptor kinase gene).
FIG. 2 amplification of known S haplotype varieties with primers PKC6F/PKC 6R; in the figure, M is DNAladder of 2000bp, and the sizes of the bands from top to bottom are 2000bp,1000bp,750bp,500bp and 250bp in sequence; lanes numbered 1-3 show the amplification of primers PKC6F/PKC6R in cabbage varieties "late abundance", "Zhonggan 11" and "Zhonggan 23" in sequence; the black arrows indicate the amplification band of the primers PKC6F/PKC6R, and the size is about 475bp.
FIG. 3 shows the amplification of primers PKC6F/PKC6R on different cabbage inbred line materials; in the figure, M is DNA Ladder of 2000bp, and the sizes of the bands are 2000bp,1000bp,750bp,500bp and 250bp in sequence from top to bottom; the lanes numbered 1 to 6 were sequentially amplified with "Zhonggan 23" (control), "Zhonggan 11" (control), "10-357", "10-361", "10-364" and "10-503"; the black arrows indicate the amplification band of the primers PKC6F/PKC6R, and the size is about 475bp.
FIG. 4 primer PKC6F/PKC6R on cabbage BC 1 Progeny [ (Z157 XZ 164)]Screening results of (1); in the figure, M is DNA Ladder of 2000bp, and the sizes of the bands from top to bottom are 2000bp,1000bp,750bp,500bp and 250bp in sequence; lanes numbered 1 are "Z157", lanes numbered 2 are "Z164", lanes numbered 3-19 are BC where the primers PKC6F/PKC6R amplify positive 1 Generation [ (Z157 XZ 164). Times.Z 164)]Carrying out single plant cultivation; black arrows indicate the amplified bands of primers PKC6F/PKC6R, with a size of 475bp.
Detailed Description
The present invention is described in detail below with reference to examples, it being understood that the following examples are only illustrative and illustrative of the present invention and do not limit the scope of the present invention in any way.
Biological material
"late feng", "zhonggan 11" and "zhonggan 23" are all known varieties of cabbage (Brassica oleracea var. Capitata l.) and are commercially available. The S haplotype of "late abundance" is S68S2 (class I + class II), "Zhonggan 11" is S2S15 (class II), "Zhonggan 23" is S28S7 (class I), see non-patent documents "Miao Wen, tian Yi, zhuang mu, etc.. Distribution of first generation hybrid S haplotype of cabbage. Chinese vegetables, 2013,1 (10): 23-28".
Cabbage self-bred line materials "10-357", "10-361", "10-364", "10-503" are known cabbage materials, see non-patent document "the identification of cabbage self-incompatible S haplotypes, major paper of Chinese academy of agricultural sciences 2012" in the field.
The cabbage inbred line materials "Z164" and "Z157" are known cabbage materials, and refer to non-patent documents "Leizhiyuan, high-throughput SNP marker for cabbage fingerprint construction and heterosis group division, master thesis of Chinese academy of agricultural sciences 2018".
The above-mentioned biological materials are available from the group of subjects in cabbage institute of vegetable and flower, institute of agricultural science, and the applicant states that they can be distributed to the public for verification experiments within twenty years from the date of application.
The main reagents are as follows:
DNA Ladder was purchased from rema biotechnology limited, lanzhou under catalog No.: AG11904.
Unless otherwise specified, the reagents used in the following examples are conventional in the art, and are either commercially available or formulated according to methods conventional in the art, and may be of laboratory pure grade. Unless otherwise specified, the experimental methods and experimental conditions used in the following examples are all conventional in the art, and reference may be made to relevant experimental manuals, well-known literature, or manufacturer's instructions. 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.
Example 1 development of PCR markers for selection of class I S haplotypes of Brassica oleracea self-incompatible lines
We downloaded the 24 class I S haplotypes and 3 class II S haplotypes (shown in Table 1) from published cabbage on NCBI website (https:// www.ncbi.nlm.nih.gov. /), and searched for the difference sites between the class I S haplotype and the class II S haplotype sequences by sequence alignment, which should distinguish the class II S haplotypes (3) and have strong conservation in the class I S haplotypes (24). The software Primer 5.0 is utilized to design primers through DNA sequences of different sites, various indexes of the primers are verified, including the length of the primers, annealing temperature, (G + C) content, whether Primer dimers and hairpin structures exist or not, and Primer sequences meeting conditions are screened out from the indexes and handed to Shanghai Jieri Biotech Co., ltd for Primer synthesis.
TABLE 1 published class I and class II S haplotypes in Brassica oleracea
* : geneBank accession numbers are 2, indicating that these two sequences belong to two sequences at different positions of the SRK gene (S site receptor kinase gene).
New primers designed by screening cabbage varieties 'late Feng', 'Zhonggan 11' and 'Zhonggan 23' with known S haplotypes stored in the laboratory are utilized. "late abundance" is known as S haplotype of S68S2 (type i + type ii), "zhonggan 11" is known as S haplotype of S2S15 (type ii), "zhonggan 23" is known as S28S7 (type i), see "seedling Wen, tianyi, zhuang, etc.. Distribution of the first generation hybrid S haplotype of cabbage. 23-28".
The screening method comprises the following steps:
leaf genomic DNA of Brassica oleracea varieties "late abundant", "Zhonggan 11", zhonggan 23 "were extracted by the CTAB (cetyl triethylammonium Bromide, hexadecyltrimethyl ammonium Bromide) method, respectively, see Murray MG, thompson WF (1980) Rapid isolation of high molecular weight plant DNA. Nucleic acids Res 8, 4321-4325. The PCR reaction was carried out using the genomic DNA as a template and the designed novel primers according to the following system and procedure.
PCR reaction (10. Mu.L): 0.5. Mu.L of upstream primer (10. Mu. Mol/L), 0.5. Mu.L of downstream primer (10. Mu. Mol/L),
PCR SuperMix 5. Mu.L, template DNA (20 ng/. Mu.L) 2. Mu.L, ddH 2 O 2μL。
PCR reaction procedure: pre-denaturation at 94 ℃ for 5min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 45s, and 33 cycles; extension at 72 ℃ for 5min.
The PCR product was detected by electrophoresis using 1.2% agarose gel, constant pressure of 150V for 30min, and photographed using an ultraviolet light gel imager.
Primers were selected that amplified the band of interest in plants with the class I S haplotype but not the amplification product in plants without the class I S haplotype. Finally, PCR primers PKC6F/PKC6R for screening the I-type S haplotypes of the cabbage self-incompatibility line are screened, and the nucleotide sequences of the PCR primers are as follows:
PKC6F：5’-CAATTTCACAGAGAATAGTGA-3’(SEQ ID NO:1)；
PKC6R：5’-ACCATTCCGGATATCCGCATTT-3’(SEQ ID NO:2)。
the size of a characteristic band amplified by the primers PKC6F/PKC6R is about 475bp, and the nucleotide sequence of the characteristic band is shown as SEQ ID NO. 3 by taking the SRK gene of an S12 haplotype as an example. The position of the primer amplified fragment is shown in FIG. 1, and is located at the 5' end of the SRK gene, and the marker is located on chromosome 6 32.10Mb (C06: 32,097,032bp-32,097, 506bp) of the cabbage reference genome TO1000 (http:// plants. Ensembl. Org/index. Html).
As shown in FIG. 2, lanes numbered 1-3 show the amplification of primers PKC6F/PKC6R in the cabbage varieties "late abundance", "Zhonggan 11" and "Zhonggan 23" in sequence. The results show that the primers PKC6F/PKC6R present amplification products with fragment sizes of about 475bp in cabbage plants with the type I S haplotype ("late abundant", "Zhonggan 23"), but not in cabbage plants without the type I S haplotype ("Zhonggan 11").
Example 2 accuracy verification of primers PKC6F/PKC6R
Leaf genomic DNAs of Brassica oleracea inbred line materials "10-357", "10-361", "10-364", "10-503" were extracted by the CTAB (cetyl triethylammonium Bromide) method, respectively. The self-bred material number is introduced in Tianli, the identification of self-incompatible S haplotypes of cabbage, master thesis of Chinese academy of agricultural sciences in 2012. For genomic DNA extraction methods see Murray MG, thompson WF (1980) Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res 8, 4321-4325.
The Kawawa Dageno corporation was entrusted with the synthesis of primers PKC6F/PKC6R (SEQ ID NO:1 and SEQ ID NO: 2) for the identification of primer amplification in cabbage. And (3) carrying out PCR according to the following reaction system and reaction program by using the genomic DNA extracted in the step (1) as a template.
PCR reaction (10. Mu.L): the upstream primer PKC6F (10. Mu. Mol/L) is 0.5. Mu.L, the downstream primer PKC6R (10. Mu. Mol/L) is 0.5. Mu.L,
PCR SuperMix 5. Mu.L, template DNA (20 ng/. Mu.L) 2. Mu.L, ddH 2 O 2μL。
PCR reaction procedure: pre-denaturation at 94 ℃ for 5min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 45s, and 33 cycles; extension at 72 ℃ for 5min.
The amplified products were electrophoretically detected using 1.2% agarose gel, held at constant pressure of 150V for 30min, and photographed using an ultraviolet light gel imager.
As shown in FIG. 3, when primers PKC6F/PKC6R were used for amplification of inbred cabbage material, 475bp characteristic bands were detected from inbred cabbage plants "10-357", "10-361", "10-364" and "10-503" with class I S haplotypes.
Example 3 marker-assisted screening with primers PKC6F/PKC6R
Cabbage material: from the cabbage inbred line "Z164 "(class I S haplotype plants, recurrent parent) and" Z157 "(class II S haplotype plants) 1 And (4) a group. The construction method comprises the following steps: taking a cabbage inbred line Z157 as a female parent and Z164 as a male parent for hybridization, and backcrossing the hybridized cabbage inbred line Z164 with the female parent and Z157 to obtain BC 1 Obtaining 310 cabbage BC in total 1 And (5) plant growing. Cabbage inbred lines "Z164" and "Z157" are known cabbage materials and are described in the non-patent document Leizhiyuan, high-throughput SNP marker for cabbage fingerprint construction and heterosis group division, 2018 Master thesis of Chinese academy of agricultural sciences.
The primer PKC6F/PKC6R designed by the invention and the existing primer KD4/KD7 are utilized to pair the BC 1 The population was subjected to marker assisted screening. Primers KD4/KD7 are specific primers for screening class II S haplotypes, see non-patent documents "Park J I, lee S, watanabe M, takahata Y, nou I S.2002.Identification of S-allolines using polymerase chain reaction-cleared amplified polymorphic sequence of the S-specimen promoter in cloning lines of Brassica oleracea. Plant Breeding,121, having the following nucleotide sequences:
primer KD4 (5 '-3'): GAGGGCGAGATCTTAATT (SEQ ID NO: 4);
primer KD7 (5 '-3'): AAGACKATCATTACCGAGC (SEQ ID NO: 5).
Extracting the cabbage BC with CTAB (Hexadecyl triethyl ammonium Bromide, hexadecacyl trimethyl ammonium Bromide) 1 Leaf genomic DNA of plants, genomic DNA extraction methods are described in Murray MG, thompson WF (1980) Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res 8.
With cabbage BC 1 The leaf genome DNA of the plant is taken as a template, primers PKC6F/PKC6R and KD4/KD7 are respectively adopted, and PCR reaction is carried out according to the following system and program:
the PCR system (10. Mu.L) was: upstream primer (10. Mu. Mol/L) 0.5. Mu.L, downstream primer (10. Mu. Mol/L) 0.5. Mu.L,
PCR SuperMix 5. Mu.L, template DNA (20 ng/. Mu.L)L)2μL，ddH 2 O 2μL。
The procedure for PCR using primers PKC6F/PKC6R was: pre-denaturation at 94 ℃ for 5min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 45s, and 33 cycles; extension at 72 ℃ for 5min.
The procedure for PCR using primers KD4/KD7 was: pre-denaturation at 94 ℃ for 5min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 60s, and 33 cycles; extension at 72 ℃ for 5min.
After the PCR is finished, the amplification product is subjected to electrophoresis detection by using 1.2% agarose gel, constant voltage of 140V is kept for 30min, and the ultraviolet gel imager is used for photographing.
The results show that at 310 cabbage BC 1 128 plants are hybrid S haplotype plants of I type and II type, and target bands can be amplified by adopting primers PKC6F/PKC6R and KD4/KD7, wherein the size of the target band amplified by the primers PKC6F/PKC6R is 475bp, and the size of the target band amplified by the primers KD4/KD7 is about 1100bp; the other 182 plants are I-type S haplotype plants, only the primers PKC6F/PKC6R can amplify a target band, and the primers KD4/KD7 have no amplification product, which indicates that the primers PKC6F/PKC6R can effectively identify the cabbage plants with I-type S haplotype. FIG. 4 shows primer PKC6F/PKC6R on a portion of cabbage BC 1 And (5) amplification result of the plant.
Example 4 kit for auxiliary screening of class I S haplotypes of Brassica oleracea self-incompatible lines
1. Kit assembly
Liquid or powder primers PKC6F/PKC6R (SEQ ID NO:1 and SEQ ID NO: 2) and kit instructions are loaded into the kit, and a label is attached to the outer shell of the kit.
2. Kit use instruction
Brassica oleracea plants with a class I S haplotype were selected according to the following procedure:
(1) And extracting the genome DNA of the cabbage material to be detected.
(2) Performing PCR reaction by using the genomic DNA as a template and adopting a primer PKC6F/PKC6R to obtain a PCR product;
the PCR system is as follows: upstream primer (10. Mu. Mol/L) 0.5. Mu.L, downstream primer (10. Mu. Mol/L) 0.5μL，
PCR Supermix 5. Mu.L, template DNA (20 ng/. Mu.L) 2. Mu.L, ddH 2 O 2μL；
The PCR procedure was: pre-denaturation at 94 ℃ for 5min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 45s, and 33 cycles; extension at 72 ℃ for 5min.
(3) And (3) detecting the PCR product by using 1.2% agarose gel electrophoresis, wherein if a characteristic band of 472bp-475bp appears in the PCR product, the cabbage material to be detected contains the type I S haplotype.
Sequence listing
<110> vegetable and flower institute of Chinese academy of agricultural sciences
<120> PCR marker and primer for screening I-type S haplotypes of cabbage self-incompatible line
<130> P200406-SCH
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
caatttcaca gagaatagtg a 21
<210> 2
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
accattccgg atatccgcat tt 22
<210> 3
<211> 475
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
caatttcaca gagaatagtg aggaggtcgc ttatacattc cgaatgacca acaacagttt 60
ctactcgaga ttgacactaa gctccacagg gtattttgag cgactgacgt gggctccgtc 120
atcagtgata tggaacgtct tctggtcttc tccagcaaac ccccagtgcg atatgtacag 180
gatgtgtggg ccttactctt actgtgacgt gaacacatca ccatcgtgta actgtataca 240
agggttcgat cccaggaatt tgcagcagtg ggctctgaga atctcattaa gggggtgtaa 300
aaggaggacg ctgctgagct gcaatggaga tggttttacc aggatgaaga atatgaagtt 360
gccagaaact acgatggcta ttgtcgaccg cagtattggt ttgaaagaat gtgagaagag 420
gtgccttagc gattgtaatt gtaccgcgtt tgcaaatgcg gatatccgga atggt 475
<210> 4
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
gagggcgaga agatcttaat t 21
<210> 5
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
aagackatca tattaccgag c 21
Claims (3)
1. The method for screening I-type S haplotypes of the cabbage self-incompatible line comprises the following steps:
(1) Extracting genome DNA of a cabbage material to be detected;
(2) Carrying out PCR reaction by using the genome DNA as a template and adopting a primer to obtain a PCR product; the nucleotide sequence of the primer is as follows:
the upstream primer PKC6F:5 'CAATTTCACAGAGAATAGTGA-3',
the downstream primer PKC6R: 5-;
(3) And detecting the PCR product by using agarose gel electrophoresis, wherein if a characteristic band of 472-475bp appears in the PCR product, the cabbage material to be detected contains the type I S haplotype.
2. The method of claim 1, wherein: the PCR reaction system comprises: 10 μmol/L forward primer 0.5 μ L,10 μmol/L reverse primer 0.5 μ L,2 XPCR Supermix 5 μ L, template DNA 2 μ L, ddH 2 O 2μL。
3. The method according to claim 1 or 2, characterized in that: the procedure for the PCR reaction was: pre-denaturation at 94 ℃ for 5min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 45s, and 33 cycles; extension at 72 ℃ for 5min.
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| CN109554497A (en) * | 2018-12-20 | 2019-04-02 | 中国农业科学院蔬菜花卉研究所 | PCR primer, kit and its application for the screening of wild cabbage Recessive male sterile gene |
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