CN109022612B - Rapid detection method for purity of broccoli hybrid and application - Google Patents
Rapid detection method for purity of broccoli hybrid and application Download PDFInfo
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Abstract
The invention belongs to the technical field of vegetable detection, and discloses a method for rapidly detecting the purity of broccoli hybrid seeds and application thereof, wherein broccoli genome DNA is extracted; numbering according to the published 4 pairs of primer sequences; determining a PCR amplification system, components and a reaction program; detecting the amplification primer; the purity of the hybrid "Tailv No. 5" was identified. The method for simultaneously identifying the purity of the broccoli 'Tailv No. 5' hybrid by using 4 pairs of SSR primers has high accuracy, the coincidence degree with the field identification result reaches 100%, and the result is reliable; the identification can be completed within 10 days, so that the complicated procedure of field planting identification is avoided, the labor force and the land resources are saved, and the identification cost is low; the genetic purity of the seeds is determined in the current year of seed production, so that grading and purchasing of the seeds are facilitated, and the identification efficiency is improved; the experimental operations such as DNA extraction, gel electrophoresis and the like are correspondingly simplified, and the method is more suitable for detecting large-scale batch samples.
Description
Technical Field
The invention belongs to the technical field of vegetable detection, and particularly relates to a method for rapidly detecting the purity of broccoli hybrid seeds and application thereof.
Background
Currently, the current state of the art commonly used in the industry is such that:
broccoli (Brassica oleracea var. italica), also known as broccoli, broccoli and the like, is a variety of Brassica species of the Brassica genus of the brassicaceae family. The edible part is fat and tender stems with bud groups, has rich nutrition and unique flavor, has anticancer and antioxidant effects, and is deeply loved by consumers. In recent years, the cultivation area of broccoli in China is increased year by year, the demand for dominant broccoli varieties is also increased continuously, and broccoli F is produced by utilizing heterosis1Hybrid species have become a trend. However, during the process of hybrid seed production, female parent selfing, mutual pollination in the operation process of people, cross pollination caused by birds, bees and the like can form false hybrids, and in addition, the broccoli hybrid seeds have small seeds and small difference among varieties, and are easy to cause mixing in the processes of harvesting, threshing, processing, storing, packaging and selling. At present, breeders mainly rely on the conventional DadaThe purity of the broccoli hybrid is identified by field planting, but the purity of the broccoli hybrid is long in period, labor-consuming, occupied in land and easily limited by seasons, the purity of the broccoli hybrid produced in the current year is difficult to determine, the variation caused by the influence of environmental conditions and the variation generated by the inheritance of the broccoli hybrid are difficult to distinguish, and the method is not suitable for the requirement of current production. The difference between plant varieties is the difference of genes at the bottom, and the purity of the hybrid detected by modern molecular biology means on the basis of heredity is more in line with market demands.
The molecular marker is used for DNA fingerprint identification to directly detect the difference of DNA levels among varieties, is not limited by environmental influence and seasons, has short test period and high accuracy, and is widely applied to purity identification of seeds. The SSR (simple Sequence sequences repeats) markers have the advantages of abundant quantity, uniform distribution, convenient operation, co-dominant inheritance, good stability and the like, and are ideal marker types for identifying the purity of hybrid seeds. At present, researchers have used SSR and EST-SSR markers to identify the purity of hybrids of various crops such as rice, watermelon, cucumber and the like, but the research on the genetic purity identification of broccoli hybrids is less, and the simultaneous identification of a plurality of pairs of primer markers is less.
In summary, the problems of the prior art are as follows:
(1) the purity of the broccoli hybrid seeds is identified by the existing field planting, the period is long, the labor is wasted, the land is occupied, and the method is easily limited by seasons;
(2) the purity of the hybrid seeds produced in the current year is difficult to determine by the existing detection method for the purity of the broccoli hybrid seeds.
(3) The purity identification of the broccoli is mostly carried out by only 1 pair of primers, the accuracy is low, and the broccoli is easy to be mixed with other varieties to cause loss.
The significance of solving the technical problems is as follows:
(1) the seed purity can be rapidly identified, and reliable variety guarantee is obtained for farmers to plant Tailv No. 5;
(2) the purity of the seeds is simultaneously identified through a plurality of pairs of primers, the accuracy is high, the mixture of other varieties can be better distinguished, and the purity of parents is also ensured.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a method for rapidly detecting the purity of broccoli hybrid and application thereof,
the invention is realized in such a way that a method for rapidly detecting the purity of broccoli hybrids is provided, and the method for rapidly detecting the purity of the broccoli hybrids comprises the following steps:
the method comprises the following steps: quickly extracting the genomic DNA of the broccoli by adopting a CTAB simple extraction method;
step two: 3 pairs of SSR primers and 1 pair of EST-SSR primers with polymorphism are screened from the primers disclosed in the document and numbered;
step three: determining a PCR amplification system, components and a reaction program;
step four: detecting an amplification primer, adding 2 mu L of loading Buffer into an amplification product, uniformly mixing, taking the mixture, carrying out electrophoresis on the mixture on non-denatured polyacrylamide gel with the concentration of 8% on a double vertical plate, and taking a picture after dyeing and developing with a developing solution;
step five: the purity of the hybrid is identified by the band characteristics of the identified sample on the gel.
Further, in the first step, a CTAB simple extraction method is adopted to rapidly extract the genomic DNA of the broccoli seedling, and the specific steps comprise:
firstly, 0.2g of young and tender leaves are taken at the seedling stage, cut into small pieces and placed into 2mL centrifuge tubes, 1 stainless steel ball is added into each centrifuge tube, a centrifuge tube cover is covered, then the centrifuge tubes are placed into a foam box filled with liquid nitrogen to stand for 10min, the liquid nitrogen is poured out, and the foam box is rapidly and forcefully shaken for 5 min;
secondly, opening a centrifugal tube cover to pour out steel balls, adding 600 mu L of 2% CTAB lysate into the centrifugal tube, and carrying out water bath at 65 ℃ for 30 min;
③ adding chloroform and isoamyl alcohol (24:1) with the same volume, gently shaking for several times, and centrifuging at 12000rpm for 10 min;
fourthly, adding 2/3 volumes of precooled isopropanol into the supernatant, uniformly mixing, standing for 120min in a refrigerator at minus 20 ℃, and centrifuging for 10min at 12000 rpm;
fifth, abandoning the supernatant and precipitatingThe DNA thus obtained was washed twice with 70% ethanol, dried and then added with ddH2Storing at O-20 deg.C for use.
Further, in the second step, the numbers and sequences of the 4 pairs of primers are respectively as follows:
SSR 6: an upstream primer 5'-CGAACATCTTAGGCCGAATC-3'; a downstream primer 5'-GGTTAACCTGCGGGATATTG-3';
BoE 225: an upstream primer 5'-ATCTGATGTGGAGCCTGTTGTT-3'; a downstream primer 5'-CCTTACGATCTGGGAGTGAAAA-3';
CB 10028: an upstream primer 5'-CTGCACATTTGAAATTGGTC-3'; a downstream primer 5'-AAATCAACGCTTACCCACT-3';
CB 10526: an upstream primer 5'-TTCTTCTTTCCACCACCA-3'; the downstream primer 5'-ACTCGGCGGTTAGAGAAT-3'.
Further, in the third step, the total volume of the PCR amplification reaction is 10 μ L, and the system components are: genomic DNA 8ng, 10. mu.M upstream and downstream primers 0.2. mu.L, 10mM dNTP 0.2. mu.L, 5U. mu.L-1Taq DNA polymerase 0.15. mu.L, 10 XBuffer (containing Mg)2+) mu.L, and the sterile redistilled water is replenished to 7.45 mu.L.
Further, in the third step, the amplification procedure is as follows: pre-denaturation at 95 deg.C for 5min, denaturation at 95 deg.C for 0.5min, annealing at 55 deg.C for 0.5min, extension at 72 deg.C for 0.5min, 31 cycles, extension at 72 deg.C for 5min, and storage at 4 deg.C.
Further, in the fourth step, 2. mu.L of loading Buffer is added into the amplification product and mixed evenly, then 2.5. mu.L of the mixture is taken out to be electrophoresed on 8 percent double vertical plate non-denaturing polyacrylamide gel, the electrophoresis is carried out for 70min under the constant voltage of 220V, and the gel is processed by AgNO3And taking a picture after dyeing and developing by the developing liquid.
Another objective of the present invention is to provide an application of the rapid detection method for the purity of broccoli hybrid in the detection of the purity of other vegetable hybrids, wherein the application method for the purity of vegetable hybrids comprises:
quickly extracting vegetable genome DNA by adopting a CTAB simple extraction method;
3 pairs of SSR primers and 1 pair of EST-SSR primers with polymorphism are screened from the primers disclosed in the document and numbered;
determining a PCR amplification system, components and a reaction program;
detecting an amplification primer, adding 2 mu L of loading Buffer into an amplification product, uniformly mixing, taking the mixture, carrying out electrophoresis on the mixture on non-denatured polyacrylamide gel with the concentration of 8% on a double vertical plate, and taking a picture after dyeing and developing with a developing solution;
the purity of the hybrid is identified by the band characteristics of the identified sample on the gel.
In summary, the advantages and positive effects of the invention are:
(1) the 3 pairs of SSR primers SSR6, CB10526, CB10028 and 1 pair of EST-SSR primers BoE225 used by the invention have high accuracy for simultaneously identifying the purity of the broccoli 'Tailv No. 5' hybrid, the coincidence degree with the field identification result reaches 100%, and the result is reliable;
(2) the method can definitely distinguish and distinguish different varieties of broccoli, the identification can be completed within 10 days, the complex procedure of field planting identification is avoided, labor force and land resources are saved, and the identification cost is reduced. Therefore, the genetic purity of the seeds can be determined in the same year of seed production, the grading and purchasing of the seeds are facilitated, and the identification efficiency is improved;
(3) the characters of the broccoli are easily influenced by the environment, and the field identification is sometimes difficult, but the method can be carried out indoors without field or environment influence;
(4) compared with the existing method for identifying the purity of 1 pair of primers, the method for identifying the purity of the broccoli seeds by 4 pairs of primers has the advantages that the identification result is more accurate and reliable;
(5) the experimental identification uses 205 samples for purity detection, and the identification population is large. In addition, field verification in the prior art is rarely performed, and the test passes one-to-one field verification, so that the corresponding primer marks are more accurate and reliable.
The invention simplifies the experiment operations such as DNA extraction, gel electrophoresis and the like correspondingly, and is more suitable for detecting large-scale batch samples.
Experiments prove that 24 mixed single plants are contained in 205 samples to be detected by using a conventional field identification method, and the seed purity is 88.3%; by using the invention, 24 mixed single plants are totally contained in 205 samples to be detected, the seed purity is 88.3 percent, and the coincidence degree of the two results reaches 100 percent. The time from sowing to identification is 5-6 months by adopting field identification, but the method only needs 10 days from sowing to identification; the field planting identification cost including labor force, pesticide, fertilizer, land and the like is more than five times of the required cost of the method. Therefore, the method has obvious advantages in the aspects of feasibility, reliability and timeliness of hybrid identification, and is suitable for popularization and application in seed production, seed reproduction and distribution enterprises of Tailv No. 5'.
Drawings
FIG. 1 is a flow chart of a method for rapidly detecting the purity of broccoli hybrids according to an embodiment of the present invention.
FIG. 2 is a PCR (polymerase chain reaction) electropherogram of SSR primer SSR6 for detecting characteristics of broccoli seed detection characteristics of Tailv No. 5 provided by the embodiment of the invention.
In the figure: 2 is Marker, 4 is female parent, 5 is male parent, 12 and 13 are other broccoli materials artificially mixed, and the rest are hybrid seeds of Tailv No. 5.
FIG. 3 is a PCR electropherogram of EST-SSR primer BoE225 for detecting characteristics of broccoli seed "Tailv No. 5" provided by the embodiment of the invention.
In the figure: 2 is Marker, 4 is female parent, 5 is male parent, 20 is other broccoli material mixed artificially, and the rest is Tailv No. 5 hybrid.
FIG. 4 is a PCR electropherogram of SSR primer CB10028, a seed detection characteristic of "Tailv No. 5" of broccoli provided by the embodiment of the present invention.
In the figure: 1 is Marker, 4 is female parent, 5 is male parent, 12 is other broccoli material mixed artificially, and the rest is Tailv No. 5 hybrid.
FIG. 5 is a PCR electrophoresis map of SSR primer CB10526, which is a seed detection characteristic of "Tailv No. 5" of broccoli provided by the embodiment of the invention.
In the figure: 3 is Marker, 1 is female parent, 2 is male parent, 11 is other broccoli material mixed artificially, the rest is Tailv No. 5 hybrid.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The invention provides a method and a special primer for quickly, accurately and efficiently performing molecular identification on genetic purity of a broccoli variety 'Tailv No. 5' seed, thereby greatly improving purity identification efficiency of the broccoli commercial variety.
The application principle of the present invention is described in detail with reference to the accompanying drawings;
as shown in fig. 1, the method for rapidly detecting the purity of broccoli hybrid provided by the embodiment of the invention comprises the following steps:
s101: quickly extracting the genomic DNA of the broccoli by adopting a CTAB simple extraction method;
s102: 3 pairs of SSR primers and 1 pair of EST-SSR primers with polymorphism are screened from the primers disclosed in the document and numbered;
s103: determining a PCR amplification system, components and a reaction program;
s104: detecting the amplification primers, adding 2 mu L of loading Buffer into the amplification products, mixing uniformly, taking the mixture, carrying out electrophoresis on the mixture on double-vertical-plate non-denatured polyacrylamide gel, and taking pictures after dyeing and developing with developing solution;
s105: the purity of the hybrid "Tailv No. 5" was identified by the band characteristics of the identified sample on the gel.
In step S101, the method for rapidly extracting genomic DNA of broccoli seedling provided by the embodiment of the present invention using CTAB simple extraction method includes the specific steps of:
firstly, 0.2g of young and tender leaves are taken at the seedling stage, cut into small pieces and put into 2mL centrifuge tubes, 1 stainless steel ball (the diameter is about 0.5cm) is added into each centrifuge tube, a centrifuge tube cover is covered, then the centrifuge tube cover is put into a foam box filled with liquid nitrogen to stand for 10min, the liquid nitrogen is poured out, and the foam box is quickly and forcibly shaken for 5 min;
secondly, opening a centrifugal tube cover to pour out steel balls, adding 600 mu L of 2% CTAB lysate into the centrifugal tube, and carrying out water bath at 65 ℃ for 30 min;
③ adding chloroform and isoamyl alcohol (24:1) with the same volume, gently shaking for several times, and centrifuging at 12000rpm for 10 min;
fourthly, adding 2/3 volumes of precooled isopropanol into the supernatant, uniformly mixing, standing for 120min in a refrigerator at minus 20 ℃, and centrifuging for 10min at 12000 rpm;
fifthly, abandoning the supernatant, washing the precipitated DNA twice by 70 percent ethanol, adding ddH after drying2Storing at O-20 deg.C for use.
In step S102, the numbers and sequences of the 4 pairs of primers provided in the embodiments of the present invention are as follows:
number SSR 6: upstream primer 5'-CGAACATCTTAGGCCGAATC-3', SEQ ID NO: 1; downstream primer 5'-GGTTAACCTGCGGGATATTG-3', SEQ ID NO: 2;
number BoE 225: upstream primer 5'-ATCTGATGTGGAGCCTGTTGTT-3', SEQ ID NO: 3;
downstream primer 5'-CCTTACGATCTGGGAGTGAAAA-3', SEQ ID NO: 4;
number CB 10028: upstream primer 5'-CTGCACATTTGAAATTGGTC-3', SEQ ID NO: 5; downstream primer 5'-AAATCAACGCTTACCCACT-3', SEQ ID NO: 6;
number CB 10526: upstream primer 5'-TTCTTCTTTCCACCACCA-3', SEQ ID NO: 7; downstream primer 5'-ACTCGGCGGTTAGAGAAT-3', SEQ ID NO: 8.
in step S103, the total PCR amplification reaction system provided by the embodiment of the present invention is 10 μ L, and the system components are: genomic DNA 8ng, 10. mu.M upstream and downstream primers 0.2. mu.L, 10mM dNTP 0.2. mu.L, 5U. mu.L-1Taq DNA polymerase 0.15. mu.L, 10 XBuffer (containing Mg)2+) mu.L, and the sterile redistilled water is replenished to 7.45 mu.L.
In step S103, the amplification procedure provided in the embodiment of the present invention is: pre-denaturation at 95 deg.C for 5min, denaturation at 95 deg.C for 0.5min, annealing at 55 deg.C for 0.5min, extension at 72 deg.C for 0.5min, 31 cycles, extension at 72 deg.C for 5min, and storage at 4 deg.C.
In step S104, 2. mu.L of loading Buffer (containing 1% GelRad nucleic acid dye) is added to the amplification product provided by the embodiment of the present invention and mixed, and then 2.5. mu.L of the mixture is taken in the concentration of 8%Performing electrophoresis on double vertical plate non-denaturing polyacrylamide gel at constant voltage of 220V for 70min, and subjecting the gel to AgNO3And taking a picture after dyeing and developing by the developing liquid.
The application of the rapid detection method for the purity of the broccoli hybrid provided by the embodiment of the invention in the detection of the purity of other vegetable hybrids comprises the following steps:
quickly extracting vegetable genome DNA by adopting a CTAB simple extraction method;
designing 4 pairs of primer sequences according to the genomic DNA of the vegetables, and numbering the primer sequences;
determining a PCR amplification system, components and a reaction program;
detecting the amplification primers, adding 2 mu L of loading Buffer into the amplification products, mixing uniformly, taking the mixture, carrying out electrophoresis on the mixture on double-vertical-plate non-denatured polyacrylamide gel, and taking pictures after dyeing and developing with developing solution;
the purity of the hybrid is identified by the band characteristics of the identified sample on the gel.
The application principle of the present invention will be further described in detail with reference to the following specific embodiments;
example 1;
the 'Tailv No. 5' sample seeds artificially mixed with the false hybrid are used as materials to prove the feasibility of the method and the special primer thereof for identifying the purity of the 'Tailv No. 5' hybrid seeds of broccoli.
(1) Extraction of seedling genomic DNA from a sample of ` Tuo Green No. 5 ` artificially mixed with a pseudohybrid. The method comprises the following steps of firstly, preparing a male parent and a female parent of broccoli, wherein the male parent and the female parent of the broccoli are respectively one part, the hybrid seed of the broccoli is No. 1', the other random hybrid seeds are 1-2, the individual plant is numbered, and the genomic DNA of the broccoli seedling is quickly extracted by adopting a CTAB simple extraction method, and the method comprises the following specific steps:
firstly, 0.2g of young and tender leaves are taken at the seedling stage, cut into small pieces and put into 2mL centrifuge tubes, 1 stainless steel ball (the diameter is about 0.5cm) is added into each centrifuge tube, a centrifuge tube cover is covered, then the centrifuge tube cover is put into a foam box filled with liquid nitrogen to stand for 10min, the liquid nitrogen is poured out, and the foam box is quickly and forcibly shaken for 5 min;
secondly, opening a centrifugal tube cover to pour out steel balls, adding 600 mu L of 2% CTAB lysate into the centrifugal tube, and carrying out water bath at 65 ℃ for 30 min;
③ adding chloroform and isoamyl alcohol (24:1) with the same volume, gently shaking for several times, and centrifuging at 12000rpm for 10 min;
fourthly, adding 2/3 volumes of precooled isopropanol into the supernatant, uniformly mixing, standing for 120min in a refrigerator at minus 20 ℃, and centrifuging for 10min at 12000 rpm;
fifthly, abandoning the supernatant, washing the precipitated DNA twice by 70 percent ethanol, adding ddH after drying2Storing at O-20 deg.C for use.
(2) The numbers and sequences of the 4 pairs of primers are respectively as follows:
number SSR 6: an upstream primer 5'-CGAACATCTTAGGCCGAATC-3'; the downstream primer 5'-GGTTAACCTGCGGGATATTG-3'.
Number BoE 225: an upstream primer 5'-ATCTGATGTGGAGCCTGTTGTT-3'; the downstream primer 5'-CCTTACGATCTGGGAGTGAAAA-3'.
Number CB 10028: an upstream primer 5'-CTGCACATTTGAAATTGGTC-3'; the downstream primer 5'-AAATCAACGCTTACCCACT-3'.
Number CB 10526: an upstream primer 5'-TTCTTCTTTCCACCACCA-3'; the downstream primer 5'-ACTCGGCGGTTAGAGAAT-3'.
(3) PCR amplification system, components and reaction procedure. The total PCR amplification reaction system is 10 mu L, and the system components are as follows: genomic DNA 8ng, 10. mu.M upstream and downstream primers 0.2. mu.L, 10mM dNTP 0.2. mu.L, 5U. mu.L-1Taq DNA polymerase 0.15. mu.L, 10 XBuffer (containing Mg)2+) mu.L, and the sterile redistilled water is replenished to 7.45 mu.L.
The amplification procedure was: pre-denaturation at 95 deg.C for 5min, denaturation at 95 deg.C for 0.5min, annealing at 55 deg.C for 0.5min, extension at 72 deg.C for 0.5min, 31 cycles, extension at 72 deg.C for 5min, and storage at 4 deg.C.
(4) And (5) detecting an amplification product. Adding 2 μ L loading Buffer (containing 1% GelRad nucleic acid dye) into amplification product, mixing, performing electrophoresis on 2.5 μ L mixture on 8% double vertical plate non-denaturing polyacrylamide gel at constant voltage of 220V for 70min, and passing the gel through AgNO3And taking a picture after dyeing and developing by the developing liquid.
(5) Analyzing the electrophoresis result, and identifying the purity of the hybrid 'Tailv No. 5' through the strip characteristics of the identified sample on the gel:
the size of a female parent specific marker band generated by the SSR primer SSR6 is about 180bp, and the size of a generated male parent specific marker band is about 100 bp; the size of a female parent specific marker band generated by the EST-SSR primer BoE225 is about 210bp, and the size of a generated male parent specific marker band is about 230 bp; the size of a female parent specific marker band generated by the SSR primer CB10028 is about 230bp, and the size of a generated male parent specific marker band is about 210 bp; the size of the female parent specific marker band generated by the SSR primer CB10526 is about 290bp, and the size of the generated male parent specific marker band is about 305 bp.
Only the single plant which is simultaneously provided with the respective father and mother specific markers of 4 pairs of primers is the true 'Tailv No. 5' hybrid single plant, any one band which is lacked is marked as a false hybrid, and the genetic purity of the seed to be detected is calculated according to the electrophoresis result.
Example 2;
the method is characterized in that Tailv No. 5 hybrid seeds produced in 2017 years in a large field by a broccoli breeding base of Taizhou academy of agricultural sciences are used as samples to be tested, the purity of the broccoli 'Tailv No. 5' hybrid seeds is identified by a conventional field planting identification method and the method, the identification results of the two methods are compared, and the feasibility, reliability and timeliness of the method are further investigated.
In example 2, 205 parts of the sample to be tested are counted, and the single plant is numbered and subjected to conventional field planting identification and identification by the method.
(1) And (5) performing conventional field planting identification. And (2) performing plug seedling on 205 parts of the material in 2017 in 8-10 th month, transplanting in 9-20 th month, and performing morphological observation on 205 parts of the material in 2017 in 12-2018 in 2 nd month to determine the number of the mixed plants and the genetic purity of the sample to be detected.
(2) The method of the invention is used for identification. Except the following operations, the other operation steps and analysis and identification are the same as those in the embodiment 1, and are not described again.
(3) Results
As shown in fig. 2, the PCR electropherogram of SSR primer SSR6 for detecting characteristics of broccoli seed "tai lv 5" provided in the embodiments of the present invention.
Wherein 2 is Marker, 4 is female parent, 5 is male parent, 12 and 13 are other broccoli materials artificially mixed, and the rest are hybrid seeds of Tailv No. 5.
As shown in FIG. 3, the PCR electropherogram of EST-SSR primer BoE225 is a detection characteristic feature of broccoli seed "Tailv No. 5" provided by the embodiment of the invention.
Wherein 2 is Marker, 4 is female parent, 5 is male parent, 20 is other broccoli material mixed artificially, and the rest is Tailv No. 5 hybrid.
As shown in fig. 4, the PCR electropherogram of SSR primer CB10028 for detecting seeds of broccoli "tai lv 5" provided in the embodiment of the present invention.
Wherein 1 is Marker, 4 is female parent, 5 is male parent, 12 is other broccoli material mixed artificially, and the rest is Tailv No. 5 hybrid.
As shown in fig. 5, the PCR electropherogram of SSR primer CB10526 is a characteristic of detecting seeds of broccoli "tai lv 5" provided in the embodiments of the present invention.
Wherein 3 is Marker, 1 is female parent, 2 is male parent, 11 is other broccoli material mixed artificially, and the rest is Tailv No. 5 hybrid.
As shown in fig. 2-5; by using a conventional field identification method, 24 mixed single plants are contained in 205 samples to be detected, and the seed purity is 88.3%; by using the invention, 24 mixed single plants are totally contained in 205 samples to be detected, the seed purity is 88.3 percent, and the coincidence degree of the two results reaches 100 percent. The time from sowing to identification is 5-6 months by adopting field identification, but the method only needs 10 days from sowing to identification; the field planting identification cost including labor force, pesticide, fertilizer, land and the like is more than five times of the required cost of the method. Therefore, the method has obvious advantages in the aspects of feasibility, reliability and timeliness of hybrid identification, and is suitable for popularization and application in seed production, seed reproduction and distribution enterprises of Tailv No. 5'.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Sequence listing
<110> Taizhou city institute of agricultural science
<120> quick detection method for purity of broccoli hybrid and application thereof
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<141> 2018-09-19
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<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
ccttacgatc tgggagtgaa aa 22
<210> 5
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
ctgcacattt gaaattggtc 20
<210> 6
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
<210> 7
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
ttcttctttc caccacca 18
<210> 8
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
Claims (4)
1. A method for rapidly detecting the purity of broccoli hybrids is characterized by comprising the following steps:
the method comprises the following steps: quickly extracting the genomic DNA of the broccoli by adopting a CTAB simple extraction method;
step two: screening 3 pairs of SSR primers and 1 pair of EST-SSR primers with polymorphism according to the published primers, and numbering;
step three: determining a PCR amplification system, components and a reaction program;
step four: detecting an amplification primer, adding 2 muL of loading Buffer into an amplification product, uniformly mixing, carrying out electrophoresis on the mixture on non-denatured polyacrylamide gel with the concentration of 8% on a double vertical plate, and taking a picture after dyeing and developing with a developing solution;
step five: identifying the purity of the hybrid through the strip characteristics of the identified sample on the gel;
in the first step, the genomic DNA of the broccoli seedling is rapidly extracted by adopting a CTAB simple extraction method, and the method specifically comprises the following steps:
firstly, 0.2g of young and tender leaves are taken at the seedling stage, cut into small pieces and placed into 2mL centrifuge tubes, 1 stainless steel ball is added into each centrifuge tube, a centrifuge tube cover is covered, then the centrifuge tubes are placed into a foam box filled with liquid nitrogen to stand for 10min, the liquid nitrogen is poured out, and the foam box is rapidly and forcefully shaken for 5 min;
secondly, opening a centrifugal tube cover to pour out steel balls, adding 600 mu L of 2% CTAB lysate into the centrifugal tube, and carrying out water bath at 65 ℃ for 30 min;
③ adding chloroform and isoamyl alcohol (24:1) with the same volume, gently shaking for several times, and centrifuging at 12000rpm for 10 min;
fourthly, adding 2/3 volumes of precooled isopropanol into the supernatant, uniformly mixing, standing for 120min in a refrigerator at minus 20 ℃, and centrifuging for 10min at 12000 rpm;
fifthly, abandoning the supernatant, washing the precipitated DNA twice by 70 percent ethanol, adding ddH after drying2Storing at O-20 deg.C;
in the second step, the number and the sequence of 4 pairs of primers are respectively as follows:
SSR 6: an upstream primer 5'-CGAACATCTTAGGCCGAATC-3'; a downstream primer 5'-GGTTAACCTGCGGGATATTG-3';
BoE 225: an upstream primer 5'-ATCTGATGTGGAGCCTGTTGTT-3'; a downstream primer 5'-CCTTACGATCTGGGAGTGAAAA-3';
CB 10028: an upstream primer 5'-CTGCACATTTGAAATTGGTC-3'; a downstream primer 5'-AAATCAACGCTTACCCACT-3';
CB 10526: an upstream primer 5'-TTCTTCTTTCCACCACCA-3'; the downstream primer 5'-ACTCGGCGGTTAGAGAAT-3'.
2. Rapid purity of broccoli hybrid according to claim 1The detection method is characterized in that in the third step, the total system of PCR amplification reaction is 10 mu L, and the system components are as follows: genome DNA 8ng, 10 μ M upstream and downstream primers are respectively 0.2 μ L, 10mM dNTP 0.2 μ L, 5U μ L-1Taq DNA polymerase 0.15 μ L, 10 XBuffer (containing Mg)2+)1 mu L, and supplementing sterile redistilled water to 7.45 mu L.
3. The method for rapidly detecting the purity of the broccoli hybrid of claim 1, wherein in the third step, the amplification procedure comprises: pre-denaturation at 95 deg.C for 5min, denaturation at 95 deg.C for 0.5min, annealing at 55 deg.C for 0.5min, extension at 72 deg.C for 0.5min, 31 cycles, extension at 72 deg.C for 5min, and storage at 4 deg.C.
4. The method for rapidly detecting the purity of broccoli hybrids according to claim 1, wherein in the fourth step, 2 μ L of loading Buffer is added into the amplification product and mixed uniformly, then the 2.5 μ L mixture is subjected to electrophoresis on 8% double-vertical-plate non-denaturing polyacrylamide gel at a constant voltage of 220V for 70min, and the gel is subjected to AgNO treatment3And taking a picture after dyeing and developing by the developing liquid.
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| CN110592256B (en) * | 2019-09-27 | 2022-12-13 | 台州市农业科学研究院 | Brassica oleracea 'Tailv No. 6' hybrid seed purity EST-SSR molecular marker system, identification method and application thereof |
| CN112391488B (en) * | 2020-10-21 | 2022-11-08 | 浙江省农业科学院 | SNP marker for identifying broccoli variety Zhe Qing 80 |
| CN114196778A (en) * | 2021-12-25 | 2022-03-18 | 云南省农业科学院粮食作物研究所 | Method for making broccoli variety DNA molecular identity card |
| CN114561489B (en) * | 2022-04-15 | 2024-06-04 | 浙江美之奥种业股份有限公司 | A primer pair for identifying the self-incompatible line type II S unit type broccoli and its application and identification method |
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