CN113604601B - InDel marker primer group and application thereof in identifying purity of broccoli 'DX-108' variety or seed - Google Patents
InDel marker primer group and application thereof in identifying purity of broccoli 'DX-108' variety or seed Download PDFInfo
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Abstract
The invention discloses an InDel marker primer group and application thereof in cauliflower variety DX-108' or seed purity identification. According to the invention, the differential InDel locus is screened in the whole genome range by utilizing the parental resequencing data of the broccoli variety DX-108', and a specific primer is designed to screen to obtain 12 pairs of InDel marker primers, wherein the nucleotide sequence of the primers is shown as SEQ ID NO.1-SEQ ID NO. 24. Based on PCR amplification reaction, the 12 pairs of InDel molecular marker primers can rapidly and accurately finish purity identification of the cauliflower variety and the hybrid seeds in a short time, the required detection sample amount is small, the period is short, the method is simple and rapid, the identification result is accurate, the seed production quality of the hybrid seeds DX-108 can be controlled, and the problems of long period, large labor investment, inaccurate identification and the like in the identification of the cauliflower variety are solved.
Description
Technical Field
The invention relates to a cauliflower molecular marker primer group and application thereof, in particular to a cauliflower InDel marker primer group and application thereof in identifying the purity of a cauliflower DX-108 variety or seed, belonging to the field of molecular identification of the purity of the cauliflower DX-108 variety or seed.
Background
Broccoli (Brassica oleracea var. Botrytis) is one of the most important vegetable varieties in the world, and is deeply favored by consumers in the middle and western world because of its rich nutrition and delicious flavor. China is the country of the world where cauliflower planting and yield are greatest. In 2018, the yield of chinese broccoli reached 1026 ten thousand tons, accounting for 40.67% of the total world yield (FAO, 2019). As the representative of high-quality healthy vegetables, the flower ball formed by the fleshy stem and inflorescence meristems of the high-quality healthy vegetables is rich in vitamin C and thioglucoside anticancer substances, and meets the requirements of current people on the healthy vegetables.
With the increase of the number of the broccoli varieties year by year, the centralized application of a few backbone parents, the genetic difference among varieties is smaller and smaller, effective identification is difficult, and the management and screening of the broccoli varieties with similar genetic backgrounds from the source are a great difficulty to be solved urgently. At present, the identification of the cauliflower variety is mainly carried out by traditional means such as morphological marking research, the time period is long, the obtained conclusion is often imperfect, and the influence of environment and human factors is difficult to remove. Therefore, it is necessary to establish a rapid genotyping-based identification method.
The molecular marker is a marker on the gene level, directly detects genetic variation on the DNA molecular level, is not influenced by various factors such as tissue and organ types, development stages, habitat conditions and the like, and has high polymorphism and stable inheritance. The InDel length polymorphism (InDel) marker is a length polymorphism variation generated by inserting or deleting a certain number of nucleotides at an allelic locus, belongs to a third generation molecular marker, is developed mainly based on whole genome sequencing, has the advantages of higher marker density than SSR (Simple Sequence Repeat) in a genome, has high marker specificity, good stability, simple and economical detection method and the like, has been successfully applied to the aspects of variety identification of rice, soybean, cotton and the like, and overcomes the uncertainty of identifying new varieties only according to morphological characteristics.
The cauliflower variety DX-108 is a high-yield and high-quality hybrid cauliflower variety cultivated by Tianjin agricultural academy of sciences, and belongs to late-maturing autumn varieties, and the maturing period of autumn varieties is about 85 days. The plant grows strongly, the leaf color is dark green, and the leaf is narrow; the plant type is compact in vertical uprush, long in middle inner leaf and good in ball protection performance; the flower ball is very compact, high round, white and smooth. The single ball weight is 1.63kg, the mu yield is 3912kg, and the yield is increased by 7.95% compared with the control LUCKY.
To date, there is no molecular marker and identification method for cauliflower variety DX-108, and development of molecular identification marker for cauliflower variety DX-108 is beneficial to standardizing cauliflower variety registration, enhancing market supervision, strengthening intellectual property protection, and the like.
Disclosure of Invention
It is an object of the present invention to provide an InDel marker primer set for identifying the authenticity or seed purity of the broccoli variety `DX-108` variety;
the second object of the invention is to provide a PCR detection kit for identifying the authenticity of the broccoli variety 'DX-108' or the purity of the seeds of the broccoli hybrid 'DX-108';
the invention further aims to apply the InDel marker primer pair group to the aspects of identifying the authenticity or seed purity of the broccoli variety DX-108.
The above object of the present invention is achieved by the following technical solutions:
in one aspect, the invention provides an InDel marker-based primer pair group for identifying broccoli variety DX-108', wherein the primer pair group consists of 12 pairs of primers, namely the following primer pair 1-primer pair 12, and the nucleotide sequences of the 12 pairs of primers are respectively as follows:
primer pair 1: consists of a forward primer shown in SEQ ID NO.1 and a reverse primer shown in SEQ ID NO. 2;
primer pair 2: consists of a forward primer shown in SEQ ID NO.3 and a reverse primer shown in SEQ ID NO. 4;
primer pair 3: consists of a forward primer shown in SEQ ID NO.5 and a reverse primer shown in SEQ ID NO. 6;
primer pair 4: consists of a forward primer shown in SEQ ID NO.7 and a reverse primer shown in SEQ ID NO. 8;
primer pair 5: consists of a forward primer shown in SEQ ID NO.9 and a reverse primer shown in SEQ ID NO. 10;
primer pair 6: consists of a forward primer shown as SEQ ID NO.11 and a reverse primer shown as SEQ ID NO. 12;
primer pair 7: consists of a forward primer shown in SEQ ID NO.13 and a reverse primer shown in SEQ ID NO. 14;
primer pair 8: consists of a forward primer shown as SEQ ID NO.15 and a reverse primer shown as SEQ ID NO. 16;
primer pair 9: consists of a forward primer shown as SEQ ID NO.17 and a reverse primer shown as SEQ ID NO. 18;
primer pair 10: consists of a forward primer shown in SEQ ID NO.19 and a reverse primer shown in SEQ ID NO. 20;
primer pair 11: consists of a forward primer shown in SEQ ID NO.21 and a reverse primer shown in SEQ ID NO. 22;
primer pair 12: consists of a forward primer shown as SEQ ID NO.23 and a reverse primer shown as SEQ ID NO. 24.
Another aspect of the present invention is to provide a PCR detection kit for identifying the authenticity of a broccoli variety 'DX-108' or for identifying the purity of a broccoli hybrid 'DX-108' seed, the PCR detection kit comprising: dNTPs, taq enzyme and MgCl 2 A PCR primer pair consisting of a forward primer and a reverse primer, an amplification buffer and sterilized water; wherein the PCR primer pair is any one of the InDel-labeled primer pair sets (primer pair 1-primer pair 12 described above).
Yet another aspect of the invention is to provide a method of identifying the authenticity or seed purity of a broccoli variety 'DX-108': the polymorphism of the InDel locus among different individuals can be displayed by PCR amplification and agarose gel electrophoresis detection by using the primer pair group based on the InDel marker, so that the identification purpose is achieved.
Specifically, the invention provides an application of the InDel marker primer pair group in identifying the authenticity of a broccoli hybrid 'DX-108' variety, which comprises the following steps:
(1) Extracting genome DNA of a broccoli plant sample to be detected;
(2) Taking the extracted sample genome DNA as a template, respectively taking 12 pairs of primer pairs as forward and reverse primers, establishing a PCR amplification system, and respectively carrying out PCR amplification;
(3) If the banding pattern of each of the 12 amplification products simultaneously has the banding pattern of the parent and the parent of the broccoli hybrid 'DX-108', the broccoli variety to be detected is the broccoli hybrid 'DX-108'; if the banding of any one of the 12 amplification products does not simultaneously have the banding of the parent and parent of the broccoli hybrid 'DX-108', the broccoli variety to be tested is not the broccoli hybrid 'DX-108'.
Wherein, the PCR amplification system in the step (2) is preferably: 10 Xbuffer 1.0. Mu.L, 25mM MgCL 2 1.0. Mu.L, 2mM dNTPs 1.0. Mu.L, 10. Mu.M forward and reverse primers each 1.0. Mu.L, 0.5 units Taq DNA polymerase 0.2. Mu.L, template 50ng, add ddH 2 O to 10. Mu.L;
the PCR amplification procedure was: 3min at 95 ℃; cycling for 35 times at 95 ℃ for 15s,58 ℃ for 15s and 72 ℃ for 20 s; and at 72℃for 5min.
The female parent of the cauliflower hybrid 'DX-108' is PN-644, and the male parent of the cauliflower hybrid 'DX-108' is PN-643.
Specifically, the invention provides an identification method for the purity of seeds of broccoli hybrid 'DX-108' based on the InDel marker primer pair group, which comprises the following steps:
(1) Extracting genome DNA of a broccoli seed sample to be detected;
(2) Respectively taking the extracted genomic DNA of the seed sample as a template, respectively taking 12 pairs of primer pairs as forward and reverse primers, establishing a PCR amplification system, and respectively carrying out PCR amplification;
(3) If the banding patterns of each of the 12 amplification products simultaneously have the banding patterns of the parent and the parent of the broccoli hybrid 'DX-108', the broccoli seed to be detected is the broccoli hybrid 'DX-108'; if the band of any one of the 12 amplification products does not simultaneously have the band of the parent and the parent of the broccoli hybrid 'DX-108', the broccoli seed to be detected is not the broccoli hybrid 'DX-108';
(4) And calculating the ratio of the number of the real 'DX-108' cauliflower hybrid seeds to the total number of the detected seeds to obtain the seed purity of the 'DX-108' cauliflower hybrid seeds.
Wherein, the PCR amplification system in the step (2) is preferably: 10 Xbuffer 1.0. Mu.L, 25mM MgCL 2 1.0. Mu.L, 2mM dNTPs 1.0. Mu.L, 10. Mu.M forward and reverse primers each 1.0. Mu.L, 0.5 units Taq DNA polymerase 0.2. Mu.L, template 50ng, add ddH 2 O to 10. Mu.L;
the PCR amplification procedure was: 3min at 95 ℃; cycling for 35 times at 95 ℃ for 15s,58 ℃ for 15s and 72 ℃ for 20 s; and at 72℃for 5min.
The female parent of the cauliflower hybrid 'DX-108' is PN-644, and the male parent of the cauliflower hybrid 'DX-108' is PN-643.
The invention firstly extracts genome DNA of female parent fine inbred line PN-644', male parent fine inbred line PN-643', PN-601',PN-602 ',PN-603 ',PN-604 ',PN-737 ',GT-42 ',GS-57 ' and so on of cauliflower variety ' DX-108', carries out Illumina double end sequencing on genome DNA of each sterile line and inbred line, and after removing joint sequence and data quality control, the obtained original data is compared with cauliflower reference genome by BWA software, and the comparison result is removed repeatedly by SAMTOOLS. InDel variation in the samples was analyzed using a GATK HaplotypeCaller module.
Based on the Illumina high-throughput sequencing, the invention further adopts differential InDel locus screening and specific primer design: the unique InDel locus in the parent genome of the broccoli variety 'DX-108' is screened by using a Perl language script, the InDel is set to be 20-500bp, and the sequencing depth is more than 4. Based on the reference genome information, the selected site is positioned on the genome, and a nucleotide sequence 200bp upstream and downstream of the InDel marker site is extracted. Primers were designed in batches using Primer3.0 and self-programming Perl language scripts. Carrying out PCR amplification on the broccoli 'DX-108' variety and the parent and the mother thereof by using the designed and synthesized primer, and screening to obtain 12 pairs of InDel marked primers; the screened 12 pairs of InDel marker primers can be used for rapidly and accurately completing the purity identification of the broccoli variety 'DX-108' or hybrid seeds in a short time, so that the problems of long identification period, large labor investment and the like of the existing broccoli variety 'DX-108' are solved, and the 'DX-108' can be rapidly, efficiently, stably and accurately detected based on InDel molecular markers.
Compared with the prior art, the invention has the following advantages:
1. the invention utilizes the parental resequencing data of the broccoli variety DX-108' to screen the difference InDel locus in the whole genome range and designs the specific primer, thereby having high accuracy and stable and reliable result.
2. Compared with polyacrylamide gel electrophoresis, the method has the advantages of simple operation, low cost, high speed, wide application range and the like.
3. The method is based on PCR amplification reaction, utilizes InDel molecular markers on 12 sites, can rapidly and accurately finish purity identification of the 'DX-108' cauliflower variety and hybrid seeds in a short time, is simple and rapid, has short detection period, saves labor cost and has good application and popularization prospects.
4. According to the invention, besides identifying the broccoli variety 'DX-108' according to the amplified banding pattern of the primer group, different varieties including 'jin 56', 'jin 66', 'jin 69', 'jin 70', 'jin 74', 'jin 79', 'jin 80', 'DX-103', 'DX-142', 'Chun 30', 'CB-30', 'CY-17', 'CY-28', 'BE-2', 'B-11', 'BX-110', 'BX-120' or 'SX-60' can BE distinguished according to the different banding patterns of the primer group among the broccoli varieties.
Drawings
FIG. 1 is an electrophoretogram of PCR amplification products of 12 pairs of primer pairs of the broccoli variety `DX-108` female parent `PN-644` and `DX-108` male parent `PN-643` and `DX-108` species; lanes 1, 2, 3 are shown in the figure as 5 individual mixes of the parent of the broccoli variety `DX-108`, the parent of the broccoli variety `DX-108` and `DX-108`.
FIG. 2 is an electrophoretogram of PCR amplification products of the cauliflower variety 'DX-108' and 12 pairs of primers of 'body fluid 56', 'body fluid 66', 'body fluid 69', 'body fluid 70', 'body fluid 74', 'body fluid 79', 'body fluid 80', 'DX-103', 'DX-142', 'spring Se30', 'CB-30', 'CY-17', 'CY-28', 'BE-2', 'B-11', 'BX-110', 'BX-120', or 'SX-60'; in the figure, M is Marker, lanes 01-12 are 12 pairs of primers such as DX108-I3, DX108-I13, DX108-I15, DX108-I19, DX108-I24, DX108-I25, DX108-I30, DX108-I31, DX108-I33, DX108-I34, DX108-I47, DX108-I55, etc.
FIG. 3 is an electropherogram of the detection of seed purity of broccoli variety 'DX-108' using primer DX 108-I19; in the figure, M is Marker, lane 01 is male parent of broccoli variety 'DX-108', lane 02 is female parent of broccoli variety 'DX-108', and lanes 03-96 are 94 single plants of the sample to be detected.
FIG. 4 is an electrophoretogram of the detection of seed purity of broccoli variety 'DX-108' using primer DX 108-I24; in the figure, M is Marker, lane 01 is male parent of broccoli variety 'DX-108', lane 02 is female parent of broccoli variety 'DX-108', and lanes 03-96 are 94 single plants of the sample to be detected.
FIG. 5 is an electrophoretogram of the detection of seed purity of broccoli variety 'DX-108' using primer DX 108-I30; in the figure, M is Marker, lane 01 is male parent of broccoli variety 'DX-108', lane 02 is female parent of broccoli variety 'DX-108', and lanes 03-96 are 94 single plants of the sample to be detected.
Detailed Description
The invention will be further described with reference to specific embodiments, and advantages and features of the invention will become apparent from the description. These examples are merely exemplary and do not limit the scope of the invention in any way. It will be understood by those skilled in the art that various changes and substitutions can be made in the details and form of the invention without departing from the spirit and scope of the invention, but these modifications and substitutions are intended to be within the scope of the invention.
Test example 1 genome-wide screening for differential InDel sites and design of screening specific primers
1 test method
1.1 Illumina high throughput sequencing
Extracting genome DNA of female parent fine inbred line ' PN-644', male parent fine inbred line ' PN-643', PN-601', ' PN-602', ' PN-603', ' PN-604', ' PN-737', ' GT-42', ' GS-57' and the like of the broccoli variety ' DX-108', performing Illumina double-end sequencing on the genome DNA of each sterile line and inbred line, removing joint sequences and data quality control of the obtained original data, comparing the effective sequencing data with a broccoli reference genome through BWA software, and removing repetition of the comparison result through SAMTOOLS. Detecting and analyzing InDel variation in a sample by using a GATK HaplotypeCaller module;
1.2 differential InDel site selection and specific primer design
The unique InDel locus in the parent genome of the broccoli variety 'DX-108' is screened by using a Perl language script, the InDel is set to be 20-500bp, and the sequencing depth is more than 4. Based on the reference genome information, the selected site is positioned on the genome, and a nucleotide sequence 200bp upstream and downstream of the InDel marker site is extracted. Primers were designed in batches using Primer3.0 and self-programming Perl language scripts. And (3) carrying out PCR amplification on the broccoli 'DX-108' variety and the parent and parent thereof by using the designed and synthesized primer.
2 test results
FIG. 1 is an electrophoretogram of PCR amplification products of 12 pairs of primer pairs of the broccoli variety `DX-108` female parent `PN-644` and `DX-108` male parent `PN-643` and `DX-108` species; the 12 pairs of InDel primers can be simultaneously amplified to the characteristic bands of the parental genes in the 'DX-108', and can be used for identifying the broccoli variety 'DX-108' and seed purity thereof (figure 1).
The 12 pairs of InDel marker primer pairs used to identify the purity of the `DX-108` broccoli variety or `DX-108` broccoli hybrid seed in test example 1 and test example 2 below are shown in Table 1.
TABLE 1 12 primer sets for cauliflower 'DX-108' variety identification
Test example 2'DX-108' quick identification of broccoli variety
1 test method
1.1 extraction of leaf genomic DNA
Extracting 5 single plant mixed sample DNAs of broccoli hybrid ' DX-108' and female parent PN-644' and male parent PN-643', body fluid 56', ' body fluid 66', ' body fluid 69', ' body fluid 70', ' body fluid 74', ' body fluid 79', ' body fluid 80', ' body fluid 81', ' DX-103', ' DX-142', ' spring snow 30', ' CB-30', ' CY-17', ' CY-28', ' BE-2', ' B-11', ' BX-110', ' BX-120' and ' SX-60' respectively.
1.2 PCR amplification
PCR amplification was performed using the genomic DNAs of the respective broccoli varieties described in 1.1 as templates, and DX108-I3, DX108-I13, DX108-I15, DX108-I19, DX108-I24, DX108-I25, DX108-I30, DX108-I31, DX108-I33, DX108-I34, DX108-I47 and DX108-I55 as primers, respectively;
the PCR amplification system is as follows: 10 Xbuffer 1.0. Mu.L, 25mM MgCL 2 1.0. Mu.L, 2mM dNTPs 1.0. Mu.L, 10. Mu.M forward and reverse primers each 1.0. Mu.L, 0.5 units Taq DNA polymerase 0.2. Mu.L, template 50ng, add ddH 2 O to 10. Mu.L;
the PCR amplification procedure was: the reaction was repeated 35 times at 95℃for 3min, (95℃for 15s,58℃for 15s, and 72℃for 20 s) and at 72℃for 5min.
1.3 PCR amplification product detection assay
mu.L of 10 XLoding Buffer was added to the amplified product of 1.2, and after electrophoresis on a 2% agarose gel at a constant voltage of 120V/cM for 90min, gelred was stained and photographed by a gel imaging system. According to the electrophoretic band type identification variety, if the band type of 12 InDel loci simultaneously has the band type of the parent and the parent of the broccoli hybrid 'DX-108', determining that the broccoli variety to be detected is the broccoli hybrid 'DX-108'.
2 identification result
The method is used for carrying out variety identification on broccoli hybrid broccoli varieties 'DX-108' and 'jin 56', 'jin 66', 'jin 69', 'jin 70', 'jin 74', 'jin 79', 'jin 80', 'DX-103', 'DX-142', 'spring snow 30', 'CB-30', 'CY-17', 'CY-28', 'BE-2', 'B-11', 'BX-110', 'BX-120' or 'SX-60', and 12 pairs of primers are all 3 types of bands, and for visual representation, the same primers are generally classified in the amplification map according to the molecular weight of the minimum amplification product, and the second molecular weight is classified when the molecular weights of the minimum products are the same, and so on. The type of low molecular weight band is defined as 1, the type of high molecular weight band is defined as 2, and the hybrid co-band type is defined as 3. The corresponding pattern types are converted into unique codes like the identity card numbers, and the codes are used for distinguishing varieties. The agarose gel electrophoresis patterns amplified by the 12 pairs of primers of the broccoli hybrid broccoli variety 'DX-108', 'jin 56', 'jin 66', 'jin 69', 'jin 70', 'jin 74', 'jin 79', 'jin 80', 'DX-103', 'DX-142', 'spring snow 30', 'CB-30', 'CY-17', 'CY-28', 'BE-2', 'B-11', 'BX-110', 'BX-120' or 'SX-60' are shown in FIG. 2. In FIG. 2, M is Marker, lanes 01-12 are 12 pairs of primers, such as DX108-I3, DX108-I13, DX108-I15, DX108-I19, DX108-I24, DX108-I25, DX108-I30, DX108-I31, DX108-I33, DX108-I34, DX108-I47, DX108-I55, etc., respectively; the encoding table is shown in table 2.
TABLE 2 father and mother of broccoli variety 'DX-108', hybrid 'DX-108' and 18 parts of broccoli hybrid fingerprint code
According to the detection result, the band type of 12 InDel loci of the broccoli hybrid 'DX-108' simultaneously has the band type of the parent of the broccoli hybrid 'DX-108', the band type of 12 InDel loci of 'jin 56', 'jin 66', 'jin 69', 'jin 70', 'jin 74', 'jin 79', 'jin 80', 'DX-103', 'DX-142', 'spring Se30', 'CB-30', 'CY-17', 'CY-28', 'BE-2', 'B-11', 'BX-110', 'BX-120' or 'SX-60' simultaneously has the band type of the parent of the broccoli hybrid 'DX-108'; therefore, the primer group designed by the invention can accurately identify the cauliflower hybrid 'DX-108' and other cauliflower varieties.
Test example 3'DX-108' identification of purity of Cauliflower hybrid seed
1 test method
1.1 extraction of leaf genomic DNA
94 samples of hybrid seeds were randomly selected from a batch of `DX-108` cauliflower hybrid seeds.
Sowing seeds of a parent (PN-644 and PN-643) of the broccoli hybrid 'DX-108' and sample seeds in a 72-hole tray at the same time, and watering periodically until two true leaves grow; the 5 individual plants of the parent and the mother are respectively mixed and sampled, 94 hybrid sample seeds are respectively individual plant sampling leaves. Genomic DNA was extracted by the conventional CTAB method and stored at-20℃until use.
1.2 PCR amplification
PCR amplification as in test example 2.
1.3 PCR amplification product detection assay
The PCR amplification product detection and hybrid variety identification method were the same as in test example 2.
2 identification result
The partial results of agarose gel electrophoresis for identifying seed purity of broccoli hybrid 'DX-108' by the method are shown in FIG. 3, FIG. 4 and FIG. 5. In the figure, M is a Marker, lane 01 is a female parent of the broccoli hybrid 'DX-108', lane 02 is a male parent of the broccoli hybrid 'DX-108', and lanes 03-96 are 94 single plants of samples to be detected.
94 true hybrid seeds in 94 sample seeds, so that the purity of the batch of 'DX-108' hybrid seeds is 100%, meets the national standard and is consistent with the field investigation result.
SEQUENCE LISTING
<110> Tianjin agricultural academy of sciences
<120> InDel marker primer set and application thereof in identifying purity of broccoli 'DX-108' variety or seed
<130> TJ-2002-210718A
<160> 24
<170> PatentIn version 3.5
<210> 1
<211> 20
<212> DNA
<213> Artifical seqeence
<400> 1
ctcccgttca cacaagtcga 20
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<213> Artifical seqeence
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ccaggcggtt gataaggtgt 20
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<212> DNA
<213> Artifical seqeence
<400> 3
tctcttagac ctctcccggc 20
<210> 4
<211> 20
<212> DNA
<213> Artifical seqeence
<400> 4
gactgcttcc ttttctgcgc 20
<210> 5
<211> 20
<212> DNA
<213> Artifical seqeence
<400> 5
ttgacttcga tcaagcggca 20
<210> 6
<211> 20
<212> DNA
<213> Artifical seqeence
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<213> Artifical seqeence
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<211> 22
<212> DNA
<213> Artifical seqeence
<400> 8
cctctgatct tccattgcct ca 22
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<211> 22
<212> DNA
<213> Artifical seqeence
<400> 9
tcagtagcat tttgtcggaa ct 22
<210> 10
<211> 20
<212> DNA
<213> Artifical seqeence
<400> 10
cgtggtcctc atcaccttgg 20
<210> 11
<211> 22
<212> DNA
<213> Artifical seqeence
<400> 11
tgcgaatttt gtgaagttgg ca 22
<210> 12
<211> 20
<212> DNA
<213> Artifical seqeence
<400> 12
gctctggagg aaacagaccc 20
<210> 13
<211> 20
<212> DNA
<213> Artifical seqeence
<400> 13
agcagcgtct cggttatctg 20
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<212> DNA
<213> Artifical seqeence
<400> 14
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<213> Artifical seqeence
<400> 15
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<212> DNA
<213> Artifical seqeence
<400> 16
tgcgctctga catcttgagc 20
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<211> 21
<212> DNA
<213> Artifical seqeence
<400> 17
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<211> 20
<212> DNA
<213> Artifical seqeence
<400> 18
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<213> Artifical seqeence
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<213> Artifical seqeence
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cctgatgatc gtgggacctc 20
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<213> Artifical seqeence
<400> 21
tgtgctatgt gcttcacctc a 21
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<212> DNA
<213> Artifical seqeence
<400> 22
actgaatcgg cctacttgca 20
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<211> 20
<212> DNA
<213> Artifical seqeence
<400> 23
ggctctgtga tcaggcagtt 20
<210> 24
<211> 20
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<213> Artifical seqeence
<400> 24
cagcccaacc agacgtgaaa 20
Claims (8)
1. The InDel marker primer pair group of the broccoli variety 'DX-108' is characterized by comprising the following primer pair 1-primer pair 12:
primer pair 1: consists of a forward primer shown in SEQ ID NO.1 and a reverse primer shown in SEQ ID NO. 2;
primer pair 2: consists of a forward primer shown in SEQ ID NO.3 and a reverse primer shown in SEQ ID NO. 4;
primer pair 3: consists of a forward primer shown in SEQ ID NO.5 and a reverse primer shown in SEQ ID NO. 6;
primer pair 4: consists of a forward primer shown in SEQ ID NO.7 and a reverse primer shown in SEQ ID NO. 8;
primer pair 5: consists of a forward primer shown in SEQ ID NO.9 and a reverse primer shown in SEQ ID NO. 10;
primer pair 6: consists of a forward primer shown as SEQ ID NO.11 and a reverse primer shown as SEQ ID NO. 12;
primer pair 7: consists of a forward primer shown in SEQ ID NO.13 and a reverse primer shown in SEQ ID NO. 14;
primer pair 8: consists of a forward primer shown as SEQ ID NO.15 and a reverse primer shown as SEQ ID NO. 16;
primer pair 9: consists of a forward primer shown as SEQ ID NO.17 and a reverse primer shown as SEQ ID NO. 18;
primer pair 10: consists of a forward primer shown in SEQ ID NO.19 and a reverse primer shown in SEQ ID NO. 20;
primer pair 11: consists of a forward primer shown in SEQ ID NO.21 and a reverse primer shown in SEQ ID NO. 22;
primer pair 12: consists of a forward primer shown as SEQ ID NO.23 and a reverse primer shown as SEQ ID NO. 24.
2. For authentication ofA PCR detection kit for verifying the authenticity of broccoli variety 'DX-108' or for identifying the purity of broccoli hybrid 'DX-108' seed, the PCR detection kit comprising: dNTPs, taq enzyme and MgCl 2 A PCR primer pair consisting of a forward primer and a reverse primer, an amplification buffer solution and sterilized water; wherein the PCR primer pair is the primer pair 1-primer pair 12 in claim 1.
3. Use of the InDel marker primer set of claim 1 in the authenticity identification of broccoli hybrid 'DX-108' variety.
4. A use according to claim 3, comprising:
(1) Extracting genome DNA of a broccoli plant sample to be detected;
(2) Respectively establishing PCR amplification systems by taking the extracted sample genome DNA as a template and respectively taking the 12 pairs of primers as forward and reverse primers of claim 1 to respectively carry out PCR amplification;
(3) If the banding pattern of each of the 12 amplification products simultaneously has the banding pattern of the parent and the parent of the broccoli hybrid 'DX-108', the broccoli variety to be detected is the broccoli hybrid 'DX-108'; if the band of any one of the 12 amplification products does not simultaneously have the band of the parent and parent of the broccoli hybrid 'DX-108', the broccoli variety to be detected is not the broccoli hybrid 'DX-108'.
5. The use according to claim 4, wherein the PCR amplification system of step (2) is: 10 Xbuffer 1.0. Mu.L, 25mM MgCL 2 1.0. Mu.L, 2mM dNTPs 1.0. Mu.L, 10. Mu.M forward and reverse primers each 1.0. Mu.L, 0.5 units Taq DNA polymerase 0.2. Mu.L, template 50ng, add ddH 2 O to 10. Mu.L;
the PCR amplification procedure was: 3min at 95 ℃; cycling for 35 times at 95 ℃ for 15s,58 ℃ for 15s and 72 ℃ for 20 s; and at 72℃for 5min.
6. Use of the InDel marker primer set of claim 1 for identifying seed purity of broccoli hybrid 'DX-108'.
7. The use according to claim 6, comprising:
(1) Extracting genome DNA of a broccoli seed sample to be detected;
(2) Respectively establishing PCR amplification systems by taking the extracted genomic DNA of the seed sample as a template and respectively taking the 12 pairs of primers of claim 1 as forward and reverse primers to respectively carry out PCR amplification;
(3) If the banding pattern of each of the 12 amplification products simultaneously has the banding pattern of the parent and parent of the broccoli hybrid 'DX-108', the broccoli seed to be detected is the broccoli hybrid
'DX-108' seed; if the banding pattern of any one of the 12 amplification products is different from that of the parent and the parent of the broccoli hybrid 'DX-108', the broccoli seed to be detected is not the broccoli hybrid 'DX-108';
(4) And calculating the ratio of the number of the real 'DX-108' cauliflower hybrid seeds to the total number of the detected seeds to obtain the seed purity of the 'DX-108' cauliflower hybrid seeds.
8. The use according to claim 7, wherein the PCR amplification system of step (2) is: 10 Xbuffer 1.0. Mu.L, 25mM MgCL 2 1.0. Mu.L, 2mM dNTPs 1.0. Mu.L, 10. Mu.M forward and reverse primers each 1.0. Mu.L, 0.5 units Taq DNA polymerase 0.2. Mu.L, template 50ng, add ddH 2 O to 10. Mu.L;
the PCR amplification procedure was: 3min at 95 ℃; cycling for 35 times at 95 ℃ for 15s,58 ℃ for 15s and 72 ℃ for 20 s; and at 72℃for 5min.
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| CN102443654A (en) * | 2012-01-17 | 2012-05-09 | 天津市农业科学院中心实验室 | Cauliflower hybrid seed purity identification method based on EST-SSR (expressed sequence tag-simple sequence repeat) marker |
| CN102643913A (en) * | 2012-04-16 | 2012-08-22 | 温州市农业科学研究院 | Fast cauliflower genetic purity detection method based on polymerase chain reaction (PCR) technology |
| CN111793709A (en) * | 2020-07-01 | 2020-10-20 | 浙江省农业科学院 | Primer pair for identifying 88 days of broccoli variety of Zhenong pine pollen and method and application thereof |
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| WO2002077277A2 (en) * | 2001-03-26 | 2002-10-03 | Council Of Scientific And Industrial Research | Dna markers for assessing seed purity and a method of using dna sequences for assessing seed purity |
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| CN102443654A (en) * | 2012-01-17 | 2012-05-09 | 天津市农业科学院中心实验室 | Cauliflower hybrid seed purity identification method based on EST-SSR (expressed sequence tag-simple sequence repeat) marker |
| CN102643913A (en) * | 2012-04-16 | 2012-08-22 | 温州市农业科学研究院 | Fast cauliflower genetic purity detection method based on polymerase chain reaction (PCR) technology |
| CN111793709A (en) * | 2020-07-01 | 2020-10-20 | 浙江省农业科学院 | Primer pair for identifying 88 days of broccoli variety of Zhenong pine pollen and method and application thereof |
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