CN104561319A - Method for identifying purity of Jinghong No. 4 radish hybrid strain based on SSR (simple sequence repeat) label - Google Patents

Method for identifying purity of Jinghong No. 4 radish hybrid strain based on SSR (simple sequence repeat) label Download PDF

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CN104561319A
CN104561319A CN201510012415.5A CN201510012415A CN104561319A CN 104561319 A CN104561319 A CN 104561319A CN 201510012415 A CN201510012415 A CN 201510012415A CN 104561319 A CN104561319 A CN 104561319A
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seed
red
capital
purity
radish
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张丽
王庆彪
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Beijing Academy of Agriculture and Forestry Sciences
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

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Abstract

The invention discloses primer sequences and a method for detecting purity of a Jinghong No. 4 radish hybrid strain. The primer sequences are nucleotide sequences as shown in SEQ ID 1-2 in a sequence table; amplification band patterns of primers in the hybrid strain are complementary band patterns of parents; and according to the random sampling detection of 10 farmers in a seed production base, the detection of an SSR (simple sequence repeat) label coincides with the field result. The method disclosed by the invention has the advantages of quickness, simplicity, convenience, stability, reliability, low cost and the like, and the purity identification work of the Jinghong No. 4 radish hybrid strain can be completed within 3-4h, thereby having great application values.

Description

The method of the red No. 4 radish Hybrid seed purity tests in a kind of capital based on SSR marker
Technical field
The present invention relates to primer sequence and the detection method of the red No. 4 radish Hybrid seed purity tests in a kind of capital, belong to biological technical field.
Background technology
Radish is with a long history in Chinese cultivated, widely distributed, and variety type is very abundant, in China's Vegetable produce supply, occupy critical role.But current radish seed operating unit gets more and more, and seed quality is uneven, the severe jamming sound development of radish kind industry.Happen occasionally because the reason such as biology hybrid and mechanical admixture causes purity of hybrid to reduce phenomenon in radish F1 generation cross breeding seed process, cause tremendous economic to lose to seed producers and manager.Traditional seed purity identification method is all carry out in field, observes feature and the consistence of main development phase kind, there is cycle long, the defect such as workload is large.In addition because Purity Field sense cycle is long, next year could sell to cause the cross-fertilize seed of current year's production often will wait until, not only can cause warehousing pressure, also can miss business opportunity.
In recent years along with molecular biological development, molecule marker based on DNA polymorphism becomes just gradually analyzes the multifarious powerful of biological heredity, because it has the advantage that not affected by environment, that test period is short, selective marker number is many, can carry out high-throughput test analysis, the extensive detection for Purity Identification and kind true or false.Especially micro-satellite (SSR) labeling technique has the advantages such as codominance, polymorphism is good, reproducible, experimental implementation is simple and is widely used.
" red No. 4 of capital " is one of serial radish varieties of red skin that Vegetable Research Centre, Beijing Academy of Agriculture and Forest Sciences is bred as, and has the advantages such as fleshy root color of the leather is pink, root shape is round just, disease resistance is strong, cooked quality is good, is suitable for North China and Northeast China region plantation in autumn.This kind cultivated area constantly expands, the also corresponding increase of seed demand and production of hybrid seeds area, and Rapid identification purity of hybrid will be conducive to the further genralrlization application of this kind.
Summary of the invention
The object of this invention is to provide a kind of primer sequence for detecting the red No. 4 radish purity of hybrid in capital.
Another object of the present invention is to provide a kind of method utilizing above-mentioned primer to carry out the red No. 4 radish Hybrid seed purity tests in capital.
For achieving the above object, the present invention adopts following technical scheme:
For detecting the primer sequence of the red No. 4 radish purity of hybrid in capital, comprise nucleotide sequence sequence as shown in SEQ ID No.1 ~ 2 in sequence table a pair.
Utilize above-mentioned primer to carry out the red No. 4 radish Hybrid seed purity tests in capital, method comprises the steps:
(1) the red No. 4 cross-fertilize seed genomic dnas in testing sample capital are extracted;
(2) primer shown in sequence table SEQ ID No.1 ~ 2 is adopted to carry out pcr amplification;
(3) be separated through 10% polyacrylamide gel electrophoresis by the amplified production in step (2), argentation dyes, and takes a picture and statistics;
(4) statistics of step (3) is utilized to calculate the purity of the red No. 4 radish cross-fertilize seed in capital.
Described step (1) adopts the red No. 4 radish cross-fertilize seed genomic dnas in alkaline lysis rapid extraction capital to be measured: get seed hypocotyl (germinateing 72 hours) and be placed in 2.0mL centrifuge tube; Add 0.4mol L -1naOH solution 100 μ l, steel ball 2; 48 hole mills grind 1min; After gently throwing away the heart, be put in temperature bath 1min in boiling water; The centrifugal 1min of 10000rmp; Get supernatant liquor 10 μ l and put into 96 hole PCR plate, add 200 μ l Tris damping fluid (PH=8.0), mix for subsequent use.
Described step (2) is pcr amplification: adopt primer as shown in sequence table SEQ ID No.1 ~ 2, and reaction system is: 1 μ l 10 × Buffer is (containing Mg 2+), 0.8 μ l 2.5mM dNTP, 1U Taq archaeal dna polymerase, 10 μMs of SSR upstream and downstream primers respectively add 0.5 μ l, template DNA 2 μ l, ddH 2o supplies 10 μ l; PCR response procedures is 94 DEG C of denaturation 5min; 35 amplification cycles, 94 DEG C of 30sec, 60 DEG C of 30sec, 72 DEG C of 1min; 72 DEG C extend 7min, 4 DEG C of preservations.
The detection of described step (3) amplified production: add 2 μ l sample-loading buffers in the amplified production of step (2), be separated through 10% native polyacrylamide gel electrophoresis, electrophoretic buffer is 1 × TBE, each loading wells adds 2 μ l samples, electrophoresis 60 minutes under 120V constant voltage.Argentation is utilized to dye after electrophoresis terminates.First gel is put into stationary liquid (450mL H 2o+50mL dehydrated alcohol+2.5mL Glacial acetic acid), jog 12min on shaking table, reclaims stationary liquid stand-by; Add silver-colored dye liquor (500ml H 2o+1g Silver Nitrate), jog 12min; Outwell silver-colored dye liquor, with 500ml distillation washing 30s, clean twice; After cleaning, outwell distilled water and add nitrite ion (500ml H 2o+7.5gNaOH+1500 μ l formaldehyde), jog is to DNA band shows; When band is clear, outwell developing solution, add stationary liquid, fix 2 minutes; Outwell stationary liquid, with distilled water rinsing 5 minutes; Take a picture and add up banding pattern.The relatively key band of red No. 4 cross-fertilize seed in capital and parents thereof, red No. 4 cross-fertilize seed in capital of the complementary banding pattern of performance codominance, what only have parent's banding pattern is non-cross-fertilize seed, and what occur other the third banding patterns is external hybrid;
(4) statistics of step (3) is utilized to calculate for trying the red No. 4 radish purity of hybrid in capital: red No. 4 purity of hybrid (%) in capital=(always detecting seed grain number-non-cross-fertilize seed number-external cross-fertilize seed number)/total detection seed grain number × 100% is surveyed in censorship.
Beneficial effect of the present invention:
The present invention's screening obtains the primer that can be used in the red No. 4 radish Hybrid seed purity tests in capital for a pair, and provides the method for red No. 4 Hybrid seed purity tests in capital.This detection method is quick, easy, stable, reliable, cost is low, can complete the red No. 4 Purity Identification work in capital, be conducive to the quality control of the red No. 4 seed efficiently and accuratelies in capital within 3-4h.
Accompanying drawing explanation
Amplification figure in Fig. 1, SSR2202 red No. 4 cross-fertilize seed in Beijing and parents thereof.
Amplification figure (1 ~ 36 individual plant) in Fig. 2, SSR2202 red No. 4 cross-fertilize seed in Beijing.
Embodiment
Below in conjunction with accompanying drawing and preferred forms, the present invention will be further described, to make the public have overall to summary of the invention and understand fully, and not limiting the scope of the present invention.Embodiment is as follows:
One, materials and methods
1.1 material
Primer screening material therefor comprises red No. 4 cross-fertilize seed in capital, female parent, each 8 individual plants of male parent.
The red No. 4 Hybrid seed purity test material therefors in capital are, the red No. 4 radish cross-fertilize seed in capital that seed breeding base 10 peasant households produce, and each household gets 100 seeds at random.
1.2 research method
1.2.1 the extraction of genomic dna
Seed germination, after 72 hours, takes off plumular axis and is placed in 2.0mL centrifuge tube; Add 0.4mol L -1naOH solution 100 μ l, steel ball 2; 48 hole mills grind 1min; After gently throwing away the heart, be put in temperature bath 1min in boiling water; The centrifugal 1min of 10000rmp; Get supernatant liquor 10 μ l and put into 96 hole PCR plate, add 200 μ l Tris damping fluid (PH=8.0), mix for subsequent use.
1.2.2 pcr amplification and detection
Screening acquisition can be used in the special primer of red No. 4 Hybrid seed purity tests in capital for a pair.
Table 1 can be used for the primer sequence that red No. 4 purity of hybrid in capital detect
SEQ ID No. Primer Sequence 5 '-3 '
1 SSR2202F GGGAGCCGATCATCTAACAA
2 SSR2202R AGAAGGAGCAGCGAATTTGA
PCR reaction system is: 1 μ l 10 × Buffer is (containing Mg 2+), 0.8 μ l 2.5mM dNTP, 1U Taq archaeal dna polymerase, 10 μMs of SSR upstream and downstream primers respectively add 0.5 μ l, template DNA 2 μ l, ddH 2o supplies 10 μ l; PCR response procedures is 94 DEG C of denaturation 5min; 35 amplification cycles, 94 DEG C of 30sec, 60 DEG C of 30sec, 72 DEG C of 1min; 72 DEG C extend 7min, 4 DEG C of preservations.
The detection of amplified production: add 2 μ l sample-loading buffers in amplified production, be separated through 10% native polyacrylamide gel electrophoresis, electrophoretic buffer is 1 × TBE, and each loading wells adds 2 μ l samples, electrophoresis 60 minutes under 120V constant voltage.Argentation is utilized to dye after electrophoresis terminates.First gel is put into stationary liquid (450mL H 2o+50mL dehydrated alcohol+2.5mL Glacial acetic acid), jog 12min on shaking table, reclaims stationary liquid stand-by; Add silver-colored dye liquor (500ml H 2o+1g Silver Nitrate), jog 12min; Outwell silver-colored dye liquor, with 500ml distillation washing 30s, clean twice; After cleaning, outwell distilled water and add nitrite ion (500ml H 2o+7.5gNaOH+1500 μ l formaldehyde), jog is to DNA band shows; When band is clear, outwell developing solution, add stationary liquid, fix 2 minutes; Outwell stationary liquid, with distilled water rinsing 5 minutes; Take a picture and add up banding pattern.
1.2.3 data statistics
Select the primer showing the complementary banding pattern of codominance in red No. 4 cross-fertilize seed in Beijing and parents thereof for Hybrid seed purity test.For trying the red No. 4 radish purity of hybrid in capital: red No. 4 purity of hybrid (%) in capital=(total detection seed grain number-non-cross-fertilize seed number-external cross-fertilize seed number)/total detection seed grain number × 100% is surveyed in censorship.
Two, results and analysis
2.1 primer screening
Red No. 4 cross-fertilize seed in 300 pairs of primer pair capital of synthesis, female parent, each 8 individual plants of male parent are utilized to screen, wherein between primer SSR2202 red No. 4 cross-fertilize seed in Beijing, female parent, male parent different individual plant, performance is consistent, and red No. 4 cross-fertilize seed in capital and parents thereof show the complementary banding pattern of codominance, as shown in Figure 1.
The qualification of the red No. 4 cross-fertilize seed seed purities in 2.2 capital
Primer SSR2202 is utilized to carry out PCR detection to the red No. 4 radish cross-fertilize seed in each 100 capital produced from seed breeding base 10 peasant households.The results are shown in Table 2, red No. 4 purity of hybrid in capital of 8 peasant household's productions are the purity of hybrid that 100%, 2 peasant households produce is 99%, as shown in Fig. 2, the seed number with male parent banding pattern has 1 (band shown in arrow) to be non-cross-fertilize seed, and all the other seeds are red No. 4 cross-fertilize seed in capital.
The red No. 4 radish purity of hybrid detection case in capital that each peasant household of table 2 produces

Claims (2)

1. for a primer sequence for the red No. 4 radish Hybrid seed purity tests in capital, it is characterized in that, described primer nucleotide sequences is primer as shown in sequence table SEQ ID No.1 ~ 2.
2. utilize primer described in claim 1, detect the method for the red No. 4 radish purity of hybrid in capital, its feature comprises the following steps:
(1) the red No. 4 radish cross-fertilize seed genomic dnas in rapid extraction capital: get seed hypocotyl (seed germination 72 hours) and be placed in 2.0mL centrifuge tube; Add 0.4mol L -1naOH solution 100 μ l, steel ball 2; 48 hole mills grind 1min; After gently throwing away the heart, be put in temperature bath 1min in boiling water; The centrifugal 1min of 10000rmp; Get supernatant liquor 10 μ l and put into 96 hole PCR plate, add 200 μ l Tris damping fluid (PH=8.0), mix for subsequent use.
(2) primer as shown in sequence table SEQ ID No.1 ~ 2 is adopted to carry out pcr amplification: reaction system is: 1 μ l 10 × Buffer is (containing Mg 2+), 0.8 μ l 2.5mM dNTP, 1U Taq archaeal dna polymerase, 10 μMs of SSR upstream and downstream primers respectively add 0.5 μ l, template DNA 2 μ l, ddH 2o complements to 10 μ l; PCR response procedures is 94 DEG C of denaturation 5min; 35 amplification cycles, 94 DEG C of 30sec, 60 DEG C of 30sec, 72 DEG C of 1min; 72 DEG C extend 7min, 4 DEG C of preservations;
(3) amplified production of step (2) is separated through 10% polyacrylamide gel electrophoresis, argentation dyes, take a picture and statistics, the relatively key band of red No. 4 cross-fertilize seed in capital and parents thereof, what show the complementary banding pattern of codominance is red No. 4 cross-fertilize seed in capital, what only have one of parent banding pattern is non-cross-fertilize seed, and what occur other the third banding patterns is external hybrid;
(4) statistics of step (3) is utilized to calculate for trying the red No. 4 radish purity of hybrid in capital: red No. 4 purity of hybrid (%) in capital=(always detecting seed grain number-non-cross-fertilize seed number-external hybrid number)/total detection seed grain number × 100% is surveyed in censorship.
CN201510012415.5A 2015-01-04 2015-01-04 Method for identifying purity of Jinghong No. 4 radish hybrid strain based on SSR (simple sequence repeat) label Pending CN104561319A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106636403A (en) * 2016-12-22 2017-05-10 黄冈师范学院 EST-SSR (expressed sequence tag-simple sequence repeats) molecular markers for heat-resistant radish, primers of molecular markers and application thereof
CN108531637A (en) * 2018-03-13 2018-09-14 新疆农业科学院哈密瓜研究中心 A method of based on SSR molecular marker to Xinjiang thick-skinned melon " Calusena lansium 9818 " purity of hybrid high throughput identification

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CN103642906A (en) * 2013-11-22 2014-03-19 中国热带农业科学院热带作物品种资源研究所 Pepper hybrid seed purity testing EST-SSR (expressed sequence tag-simple sequence repeat) molecular marker and application thereof
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CN103642906A (en) * 2013-11-22 2014-03-19 中国热带农业科学院热带作物品种资源研究所 Pepper hybrid seed purity testing EST-SSR (expressed sequence tag-simple sequence repeat) molecular marker and application thereof
CN103882010A (en) * 2014-03-25 2014-06-25 北京市农林科学院 Method for rapidly extracting DNA (Desoxvribose Nucleic Acid) of plant genome with high flux

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106636403A (en) * 2016-12-22 2017-05-10 黄冈师范学院 EST-SSR (expressed sequence tag-simple sequence repeats) molecular markers for heat-resistant radish, primers of molecular markers and application thereof
CN108531637A (en) * 2018-03-13 2018-09-14 新疆农业科学院哈密瓜研究中心 A method of based on SSR molecular marker to Xinjiang thick-skinned melon " Calusena lansium 9818 " purity of hybrid high throughput identification
CN108531637B (en) * 2018-03-13 2022-08-30 新疆农业科学院哈密瓜研究中心 Method for high-throughput identification of Xinjiang muskmelon yellow skin 9818 hybrid purity based on SSR molecular marker

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Application publication date: 20150429