CN111286554A - SSR primer for identifying purity of hybrid seeds of Niubao white gourd and application of SSR primer - Google Patents
SSR primer for identifying purity of hybrid seeds of Niubao white gourd and application of SSR primer Download PDFInfo
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Abstract
The invention discloses an SSR primer for identifying purity of hybrid seeds of Niubao white gourd, which comprises an upstream primer SSR11c160-F and a downstream primer SSR11c160-R of a primer SSR11c160, wherein the nucleotide sequence of the upstream primer SSR11c160-F is shown as SEQ ID No.1, and the nucleotide sequence of the downstream primer SSR11c160-R is shown as SEQ ID No. 2. The primer can generate 193bp female parent specific markers and 225bp male parent specific markers, and has good repeatability and strong specificity. The method can accurately, quickly and effectively identify the purity of the hybrid seeds of the Niubao white gourd by using the SSR primers. Also discloses application of the primer in purity identification of the hybrid seeds of the Niubao white gourd and the hybrid seeds of the Niubao white gourd.
Description
Technical Field
The invention belongs to the technical field of purity identification of wax gourd hybrid seeds, and particularly relates to an SSR primer for purity identification of Niubao wax gourd hybrid seeds and application thereof.
Technical Field
Seed purity is an important index for measuring seed quality, and influences the yield and product quality of crops. The yield reduction due to low purity is all likely to offset the yield increase potential of a new variety, and low seed purity can cause significant losses for agricultural production. The seed purity identification mainly comprises methods such as conventional identification, biochemical identification, molecular marker identification and the like.
The conventional identification comprises field planting identification, kernel and seedling form identification and physicochemical identification, and the methods have the defects of long time consumption, easy influence of environmental factors, more land use, less marker characters for identification and the like.
The biochemical identification comprises methods such as isoenzyme electrophoresis, protein electrophoresis, high performance liquid chromatography and the like, and the technology is complicated.
The molecular marker identification method has the advantages of no environmental limitation, rich types, large quantity, high polymorphism, codominance and the like.
Common marker types include AFLP (amplified fragment length polymorphism) markers, RAPD (random primer amplification polymorphism) markers, SRAP (related sequence amplification polymorphism) markers, RFLP (restriction fragment length polymorphism) markers, and the like.
The SSR marker adopts a PCR amplification method, only trace tissues need to be collected to extract DNA, the requirement on the quality of the DNA of a sample is not high, and the sample processing technology is simple and convenient; the SSR marker is codominant heredity, has rich polymorphism, good repeatability and stable and reliable result, and is very suitable for purity detection of hybrid seeds.
The molecular marker method is more and more widely applied to purity identification of hybrid seeds, has a tendency of gradually replacing the traditional form identification method, and is applied to purity identification of hybrid seeds of crops such as rice, cucumber, pumpkin, joint melon and the like at present.
The Mubao white gourd is a first-filial generation small-fruit white gourd variety bred by the vegetable research institute of agricultural academy of sciences of Guangdong province. The Mobao white gourd has the characteristics of beautiful shape, compact meat quality, dark green skin color and the like, and is popular with farmers.
At present, a method for identifying the purity of the variety, which can be fast and accurately carried out and is not limited by seasonal climate, needs to be explored.
Disclosure of Invention
The invention aims to provide an SSR primer for identifying the purity of the seeds of the white gourd, which can generate a female parent specific marker and a male parent specific marker (i.e. a codominant marker), and has good repeatability and strong specificity.
The invention also aims to provide a method for identifying the purity of the black precious white gourd seeds, and the method can accurately, quickly, conveniently and effectively identify the purity of the black precious white gourd seeds by using the SSR primers.
The third purpose of the invention is to provide the application of the SSR primers in purity identification of the hybrid seeds of the Niubao white gourd and the hybrid seeds of the Niubao white gourd.
The first object of the present invention can be achieved by the following technical solutions: an SSR primer for identifying purity of a white gourd seed, which comprises an upstream primer SSR11c160-F and a downstream primer SSR11c160-R of a primer SSR11c160, wherein the nucleotide sequence of the upstream primer SSR11c160-F is shown as SEQ ID NO.1, and the nucleotide sequence of the downstream primer SSR11c160-R is shown as SEQ ID NO. 2.
The specific nucleotide sequences of the upstream primer SSR11c160-F and the downstream primer SSR11c160-R of the primer SS327 are respectively as follows:
SSR11c160-F：5’-GCCCACACTCACCTTACACA-3’(SEQ ID NO.1)；
SSR11c160-R：5’-AGCCCCACCGAATTAATGCT-3’(SEQ ID NO.2)。
the marker band has clear shape and good repeatability, and the analysis result shows that after the DNA is recovered by a specific fragment (polyacrylamide non-denatured gel), the PCR amplification, the agarose gel recovery and the cloning sequencing are carried out: the primer SSR11c160 can generate 193bp female parent specific marker SSR11c160193(SEQ ID NO.3) and 225bp male parent specific marker SSR11c160225(SEQ IDNO.4)。
The second purpose of the invention can be realized by the following technical scheme: a method for identifying the purity of the seeds of the Niubao white gourd comprises the following steps:
(1) taking wax gourd cotyledons, and extracting wax gourd genome DNA;
(2) performing PCR amplification by using the wax gourd genome DNA as a template and adopting the SSR primer SSR11c160 to obtain an amplification product;
(3) detecting the amplification product by utilizing polyacrylamide gel electrophoresis;
(4) analyzing the electrophoresis result, the primer SSR11c160 can generate 193bp female parent specific marker SSR11c160193 and 225bp male parent specific marker SSR11c160225, a single plant with the specific bands of the female parent and the male parent (namely with the co-dominant marker) is a true hybrid, a single plant lacking any one band is a false hybrid, and then the purity of the seed is calculated according to the electrophoresis result.
In the method for identifying the purity of the black gourd seeds:
preferably, in the PCR amplification in step (2), the reaction system used comprises: the concentration is 200 ng. mu.L-1The wax gourd genome DNA is 1 mu L, contains Mg2+10 XPCR buffer 2. mu.L, 2.5mM dNTPs 1.5. mu.L, upstream primer SSR11c 160-F1. mu.L at a concentration of 10mM, downstream primer SSR11c 160-R1. mu.L at a concentration of 10mM, Taq enzyme 1. mu.L at a concentration of 2U/. mu.L, ddH2Make up to 20. mu.L of O.
Preferably, when performing PCR amplification in step (2), the amplification procedure is: pre-denaturation at 95 deg.C for 3min, denaturation at 94 deg.C for 30s, annealing at 55 deg.C for 30s, extension at 72 deg.C for 30s, final extension at 72 deg.C for 7min after 35 cycles, and storing at 4 deg.C.
Preferably, in the step (3), 8% by mass of non-denatured polyacrylamide gel electrophoresis is used for detecting the polyacrylamide gel electrophoresis.
The last object of the present invention can be achieved by the following technical solutions: the SSR primers are applied to purity identification of hybrid seeds of the Niubao white gourd and hybrid seeds of the Niubao white gourd.
Compared with the prior art, the invention has the following advantages:
(1) the SSR primer SSR11c160 in the invention can simultaneously generate a female parent specific marker and a male parent specific marker in a first hybrid generation, and has strong specificity;
(2) the method can distinguish the hybrid Niubao from the parent thereof, and quickly detect the purity of the hybrid seed;
(3) the method has the advantages of accuracy, rapidness, low cost, short identification period, simple operation and the like, can replace the traditional method for identifying the purity of the hybrid seeds, and has wide application prospect.
Drawings
FIG. 1 is a polyacrylamide gel electrophoresis pattern (M: 100bp Ladder I molecular weight standard; P) of the PCR product of primer SSR11c160 in identification of purity of white gourd seeds of Takara Shuzo in example 1-21: female parent of Niubao white gourd, P2: male parent of black gourd; f1: hybrid black-white gourd);
FIG. 2 is a sample individual plant from a hybrid of Benincasa hispida in example 3, and the box represents a pseudo hybrid consistent with the maternal banding pattern.
Detailed description of the preferred embodiment
Example 1
The screening process of the SSR primer SSR11c160 for purity identification of the hybrid seeds of the Niubao white gourd provided by the embodiment is as follows:
a plurality of pairs of primers designed according to sequencing results of wax gourd genome are arranged at parents (Mobao winter)Male and female melon) and F1And (2) screening to select a primer SSR11c160 of a codominant differential marker band, wherein the marker band has clear type and good repeatability, and the sequence is as follows:
SSR11c160-F：5’-GCCCACACTCACCTTACACA-3’(SEQ ID NO.1)；
SSR11c160-R：5’-AGCCCCACCGAATTAATGCT-3’(SEQ ID NO.2)。
by taking wax gourd genome DNA as a template and using the SSR primer SSR11c160 to perform PCR amplification, performing polyacrylamide gel electrophoresis on an amplification product, recovering DNA from a specific fragment (polyacrylamide non-denatured gel), performing PCR amplification, recovering agarose gel, and performing TA cloning sequencing, the analysis result shows that: primer SSR11c160 can generate 193 maternal specific marker SSR11c160193(SEQ ID NO.3) and 225bp male parent specific marker SSR11c160225(SEQ ID NO.4) as shown in FIG. 1.
Example 2
The method for identifying the purity of the hybrid seeds of the Niubao white gourd comprises the following steps of:
(1) extraction of DNA of white gourd
The experimental material is a hybrid of the Mubao white gourd and cotyledons of male parent and female parent, and the step of extracting the cotyledons DNA is as follows:
① placing a cotyledon into a 2mL centrifuge tube, adding liquid nitrogen, grinding with a pestle, quickly adding 800 μ L of 2% CTAB extractive solution when liquid nitrogen is evaporated to dryness, mixing, and placing in 65 deg.C water bath for 45min (shaking every 10 min);
② standing to room temperature, adding 800 μ L chloroform isoamyl alcohol (24:1), mixing gently, standing for 2min, centrifuging, 12000rmp, 15min, collecting supernatant (510 μ L), and transferring to new 1.5mL centrifuge tube;
③ adding NaAc (3mol/l) in the volume of 1/3 of the supernatant, adding absolute ethyl alcohol (precooling at the temperature of minus 20 ℃) in the volume of 1.5 times of the supernatant, gently mixing uniformly, and placing at the temperature of minus 20 ℃ for 30min-1 h;
④ 12000rmp, centrifuging for 10min, and discarding the supernatant;
⑤ washing DNA precipitate with 75% ethanol (precooled) for 2 times, washing with anhydrous ethanol for 1 time, and drying on a clean bench;
⑥ mu.L of TE (or ddH) was added2O) dissolving, and using the dissolved DNA as wax gourd cotyledon genome DNA for later use.
(2) PCR amplification is carried out by using the white gourd cotyledon genomic DNA as a template and adopting the primer SSR11c160 designed in the example 1.
PCR System (20. mu.L)
The procedure for PCR amplification was: pre-denaturation at 95 deg.C for 3min, denaturation at 94 deg.C for 30s, annealing at 55 deg.C for 30s, extension at 72 deg.C for 30s, final extension at 72 deg.C for 7min after 35 cycles, and storing at 4 deg.C.
(3) Performing polyacrylamide gel electrophoresis (PAGE) detection on PCR amplification products
① adding the PCR product into 4 μ L6 × Loading buffer, mixing by vortex;
② collecting 2 μ L of amplification product, performing electrophoresis with 8% (by mass) non-denaturing polyacrylamide gel, and stabilizing the pressure at 180V for 60 min;
③ the PAGE gel was removed, washed once with distilled water and then with 0.1% AgNO3Dyeing for 10min by using the solution;
④ washing with distilled water for 2 times, and adding 2% NaOH and 0.04% Na2CO30.4% formaldehyde for 10min, washing twice with tap water after color development, and then taking pictures on a lamp box for analysis.
(4) Amplification results
The primer SSR11c160 amplifies 193bp and 225bp bands in the hybrid of the Niubao white gourd; a 225bp band is amplified in the male parent; the mother amplified a 193bp band (see FIG. 1).
The specific band was recovered and sent to the Producer for sequencing. The result shows that 193bp and 225bp bands are respectively amplified in the hybrid of the Niubao white gourd and are consistent with the sequences of the amplification products of the female parent and the male parent.
Wherein, the single plant with the specificity bands of the female parent and the male parent is a true hybrid, the single plant lacking any one band is a false hybrid, and then the purity of the seed can be calculated according to the further electrophoresis result.
Example 3
Carrying out field planting and sampling on the hybrid seeds of the Niubao white gourd by adopting the method of the embodiment 2, wherein the total number of samples is 80, numbering the individual plants, and extracting DNA of the individual plants for detection (see figure 2); and simultaneously, performing purity identification by using a morphological detection method. And (3) detection results: the purity of the hybrid seeds of 74 plants and 6 female parent plants (parent plants in red boxes) is 92.5 percent, which is consistent with the field detection result.
The above examples show that the method of the invention can effectively distinguish the Changbai white gourd seeds from the parent seeds thereof, and accurately and rapidly detect the seed purity.
The present invention is described in detail by the embodiments, which should be pointed out in the above description, but the embodiments are only used for further explanation of the invention, and do not represent the scope of the invention, and other non-essential modifications and adjustments made according to the teachings of the present invention still belong to the scope of the invention.
Sequence listing
<110> vegetable research institute of academy of agricultural sciences of Guangdong province
<120> SSR primer for identifying purity of hybrid seeds of Niubao white gourd and application thereof
<160>4
<170>SIPOSequenceListing 1.0
<210>1
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>1
gcccacactc accttacaca 20
<210>2
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>2
agccccaccg aattaatgct 20
<210>3
<211>193
<212>DNA
<213> SSR11c160193(Simple Sequence Repeats) as a female parent specific marker
<400>3
gcccacactc accttacaca ggccccattt cctaccaatt ctcgtcatat ttcgaatcac 60
aatccttttg tcacctgata gtttaattgt acaattcgtg aagagaaaga agaagaagaa 120
ggaagaagag cctattcctt gcatccatca gtactgtttg ttaatgttcg tccagcatta 180
attcggtggg gct 193
<210>4
<211>225
<212>DNA
<213> SSR11c160225(Simple Sequence reports)
<400>4
gcccacactc accttacaca ggccccattt cctaccaatt ctcgtcatat ttcgaatcac 60
aatccttttg tcacctgata gtttaattgt acaattcgtg aagagaaaga agaagaagaa 120
ggagaagaag aagaagaaga agaagaggac gaatccagta atcctattcc ttgcatccat 180
cagtactgtt tgttaatgtt cgtccagcat taattcggtg gggct 225
Claims (6)
1. An SSR primer for identifying purity of hybrid seeds of Niubao white gourd is characterized in that: the primers comprise an upstream primer SSR11c160-F and a downstream primer SSR11c160-R of a primer SSR11c160, wherein the nucleotide sequence of the upstream primer SSR11c160-F is shown as SEQ ID NO.1, and the nucleotide sequence of the downstream primer SSR11c160-R is shown as SEQ ID NO. 2.
2. The method for identifying the purity of the hybrid seeds of the Niubao white gourd is characterized by comprising the following steps of:
(1) taking wax gourd cotyledons, and extracting wax gourd genome DNA;
(2) performing PCR amplification by using a white gourd genome DNA as a template and adopting a primer SSR11c160 in claim 1 to obtain an amplification product;
(3) detecting the amplification product by utilizing polyacrylamide gel electrophoresis;
(4) analyzing the electrophoresis result, the primer SSR11c160 can generate 193bp female parent specific marker SSR11c160193And 225bp male parent specific marker SSR11c160225The single plant with the specificity bands of the female parent and the male parent is a true hybrid, the single plant lacking any one band is a false hybrid, and then the purity of the seed is calculated according to the electrophoresis result.
3. The method for identifying the purity of the hybrid seeds of the Niubao white gourd according to claim 2, wherein the method comprises the following steps: during PCR amplification in the step (2), the adopted reaction system comprises: the concentration is 200 ng. mu.L-1The wax gourd genome DNA is 1 mu L, contains Mg2+10 XPCR buffer 2 uL, 2.5mM dNTPs 1.5 uL, upstream primer SSR11c 160-F1 uL with the concentration of 10mM, downstream primer SSR11c 160-R1 uL with the concentration of 10mM, Taq enzyme 1 uL with the concentration of 2U/. mu.L, ddH2Make up to 20. mu.L of O.
4. The method for identifying the purity of the hybrid seeds of the Niubao white gourd according to claim 2, wherein the method comprises the following steps: when PCR amplification is carried out in the step (2), the amplification procedure is as follows: pre-denaturation at 95 deg.C for 3min, denaturation at 94 deg.C for 30s, annealing at 55 deg.C for 30s, extension at 72 deg.C for 30s, final extension at 72 deg.C for 7min after 35 cycles, and storing at 4 deg.C.
5. The method for identifying the purity of the hybrid Niubao seed according to claim 2, wherein: and (4) adopting non-denatured polyacrylamide gel electrophoresis with the mass percentage of 8% for polyacrylamide gel electrophoresis detection in the step (3).
6. The SSR primer of claim 1 is applied to purity identification of hybrid seeds of Niubao white gourd and hybrid seeds of Niubao white gourd.
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Cited By (1)
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| CN112048567A (en) * | 2020-09-29 | 2020-12-08 | 湖南省作物研究所 | Specific SSR marker primer for purity identification of rape Pol-CMS hybrid or parent seed thereof and application thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112048567A (en) * | 2020-09-29 | 2020-12-08 | 湖南省作物研究所 | Specific SSR marker primer for purity identification of rape Pol-CMS hybrid or parent seed thereof and application thereof |
| CN112048567B (en) * | 2020-09-29 | 2024-01-16 | 湖南省作物研究所 | Specific SSR marker primer for purity identification of rape Pol-CMS hybrid or parent seed thereof and application thereof |
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