CN106498084A - A kind of SSR primers and authentication method for the identification of iron prop Fructus Benincasae hybrid seed purity - Google Patents
A kind of SSR primers and authentication method for the identification of iron prop Fructus Benincasae hybrid seed purity Download PDFInfo
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Abstract
The invention discloses a kind of SSR primer SSR327 for the identification of iron prop Fructus Benincasae hybrid seed purity, the primer SSR327 includes forward primer SSR327 F and downstream primer SSR327 R,, as shown in SEQ ID NO.1, the nucleotide sequence of the downstream primer SSR327 R is as shown in SEQ ID NO.2 for the nucleotide sequence of the forward primer SSR327 F.The authentication method of above-mentioned iron prop Fructus Benincasae hybrid seed purity is also disclosed, the primer can produce the maternal specific marker SSR327 of 184bp184Male parent specific marker SSR327 with 199bp199, reproducible, high specificity.The method utilizes above-mentioned SSR primers, can accurate, quick, convenient, effective identification iron prop Fructus Benincasae hybrid seed purity.
Description
Technical field
The invention belongs to hybrid seed purity identification technology field, and in particular to a kind of pure for iron prop Fructus Benincasae hybrid seed
The SSR primers SSR327 and authentication method of degree identification.
Technical background
Seed purity is to weigh an important indicator of seed quality.Quick and precisely identification of seed purity, for seed is given birth to
Product person, seller and user are respectively provided with significant application value.
Iron prop Fructus Benincasae is the first-filial generation Fructus Benincasae kind that Vegetables Inst., Guangdong Academy of Agricultural Sciences is bred as.As which has
The characteristics of high yield, disease-resistant, storage tolerance, welcome by grower deeply, usually supply falls short of demand for seed.The tradition of gourd seed Purity
Authentication method is identification of morphology method, by identifying the morphological characteristic of plant and fruit, tells because isolating in seed production process
The maternal selfing that the pollen that measure is not enough caused mixes individuality, maternal emasculation is not thoroughly caused is individual, and seed is harvested or added
The individuality of the mechanical admixture being mixed into during work, and then calculate hybrid seed purity.When the weak point of this method is identification
Between long, it usually needs can just obtain within 3 months or so qualification result after Fructus Benincasae plant result, and season is received due to melon growth
Limit with weather conditions, generally Purity cannot be carried out immediately after seed is harvested, cause the selling time of new production seed
Delay.Therefore it is badly in need of exploring a kind of Purity method that quick and precisely and not can be limited by seasonal climate.
The Fructus Benincasae variety Rapid identification that develops into of molecular marking technique provides brand-new means.Molecular marking technique
Including AFLP (amplified fragment length polymorphism) technology, RAPD (randomly amplified polymorphism) technology, SRAP, (correlated serieses expand
Increase state property) technology, RFLP (restriction fragment length polymorphism) technology etc., wherein SSR technology adopts PCR amplification method, only
Microcomponent need to be gathered and extract DNA, and less demanding to sample DNA quality, sample treatment technology is easy；As SSR marker is in
Codominant inheritance, and rich polymorphism, reproducible, as a result reliable and stable, it is highly suitable for hybrid seed purity detection.Point
Sub- labelling method is applied in cenospecies Purity Identification more and more extensively, is had and is gradually replaced becoming for traditional identification of morphology method
Gesture, is applied in the crop hybrid kind Purity such as Oryza sativa L., Fructus Cucumidis sativi, Fructus Cucurbitae moschatae, Fructus Benincasae chiehquae at present, but there is not yet should in Fructus Benincasae
With.
Content of the invention
First purpose of the present invention is to provide the SSR primers for iron prop gourd seed Purity, the primer energy
Produce maternal specific marker and male parent specific marker (i.e. codominant marker), reproducible, high specificity.
The present invention also aims to providing a kind of authentication method of iron prop gourd seed purity, the method is using above-mentioned
SSR primers, can accurate, quick, convenient, effective identification iron prop gourd seed purity.
First purpose of the present invention is achieved by the following technical solution：One kind is used for iron prop Fructus Benincasae hybrid seed
The SSR primers of Purity, the primer SSR327 include forward primer SSR327-F and downstream primer SSR327-R, described draw
The nucleotide sequence of thing SSR327 forward primer SSR327-F as shown in SEQ ID NO.1, draw by the downstream of the primer SSR327
The nucleotide sequence of thing SSR327-R is as shown in SEQ ID NO.2.
The concrete nucleotide sequence of the forward primer SSR327-F and downstream primer SSR327-R of primer SSR327 is respectively such as
Under：
SSR327-F：5’-AAACAACACCGTTTCTTCCG-3’(SEQ ID NO.1)；
SSR327-R：5’-TCAGCGACATCAGAAACACC-3’(SEQ ID NO.2)；
Above marker bands are clear, reproducible, reclaim DNA-PCR through specific fragment (polyacrylamide non denatured glue)
After amplification-agarose gel reclaims-TA cloning and sequencings, analysis result is learnt：Primer SSR327 can produce the maternal specificity of 184bp
Labelling SSR327184(SEQ ID NO.3) and the male parent specific marker SSR327 of 199bp199(SEQ ID NO.4).
Second object of the present invention is achieved through the following technical solutions：A kind of mirror of iron prop gourd seed purity
Determine method, containing following steps：
(1) Fructus Benincasae cotyledon is taken, Fructus Benincasae genomic DNA is extracted；
(2) with Fructus Benincasae genomic DNA as template, enter performing PCR amplification using above-mentioned primer SSR327, obtain PCR amplifications and produce
Thing；
(3) gel electrophoresiss are carried out to pcr amplification product, is developed the color, count electrophoresis result；
(4) electrophoresis result is analyzed, the individual plant only simultaneously with two parent's specific bands is just real
Cenospecies, lack any one band therein or the individual plant for non-parent's specific band is designated as pseudostationary, calculate seed purity,
Wherein primer SSR327 can produce the maternal specific marker SSR327 as shown in SEQ ID NO.3 of 184bp184And 199bp
The male parent specific marker SSR327 as shown in SEQ ID NO.4199.
In above-mentioned iron prop Fructus Benincasae hybrid seed purity authentication step：
When in step (2), PCR is expanded the 20 μ L reaction systems that adopt for：150ng·μL-11 μ L of genomic DNA, contain Mg2+
10×PCR buffer 2μL、2.5mmol·L-1dNTPs 1.5μL、0.25mmol·L-1SSR327-F 1μL、
0.25mmol·L-11 μ L of SSR327-R, 2U/ μ L Taq enzymes 1 μ L, ddH2O 12.5μL.
When in step (2), PCR is expanded, amplification program is：After 95 DEG C of denaturations 3min, 94 DEG C of degeneration 30s, 56 DEG C of annealing
30s, 72 DEG C of extension 1min, after 40 circulations, 72 DEG C extend 7min, 4 DEG C of preservations eventually.
Adopt during gel electrophoresiss in step (3) weight/mass percentage composition for 8% native polyacrylamide gel electrophoresises.
Compared with prior art, the invention has the advantages that：
(1) the SSR primer SSR327 in the present invention can produce maternal specific marker and male parent in the first generation of hybrid simultaneously
Specific marker, and high specificity；
(2) iron prop gourd seed can be made a distinction by the method for the present invention with its parent, and quick detection goes out hybrid seed
Purity；
(3) the inventive method has accurate, quick, low cost, short operation qualification cycle easy, can replace passing
The method of system hybrid seed purity identification, has broad application prospects.
Description of the drawings
Fig. 1 is the PCR primer polyacrylamide gel of primer SSR327 in iron prop gourd seed Purity in embodiment 2
Electrophoresis pattern, wherein M：I molecular weight standards of 100bp Ladder；P1：Iron prop Fructus Benincasae is maternal, P2：Iron prop Fructus Benincasae male parent；F1：Ferrum
Post Fructus Benincasae cenospecies；
Fig. 2 is by the 1-46 iron prop Fructus Benincasae cenospecies sample individual plant taking from Guangdong Suixi base in embodiment 3, circle
Represent the pseudostationary consistent with maternal banding pattern；
Fig. 3 is justified by the 47-57 iron prop Fructus Benincasae cenospecies sample individual plant taken from Guangdong Suixi base in embodiment 3
Circle represents the pseudostationary consistent with maternal banding pattern；
Fig. 4 is by the 1-46 iron prop Fructus Benincasae cenospecies sample individual plant taking from Guangdong Qingyuan base in embodiment 4, circle
Represent the pseudostationary consistent with maternal banding pattern；
Fig. 5 is by the 47-92 iron prop taking from Guangdong Qingyuan base in embodiment 4 ", Fructus Benincasae cenospecies sample individual plant,
Circle represents the pseudostationary consistent with maternal banding pattern；
Fig. 6 is justified by the 93-138 iron prop Fructus Benincasae cenospecies sample individual plant taken from Guangdong Qingyuan base in embodiment 4
Circle represents the pseudostationary consistent with maternal banding pattern；
Fig. 7 by the 139-159 iron prop Fructus Benincasae cenospecies sample individual plant taking from Guangdong Qingyuan base in embodiment 4,
Circle represents the pseudostationary consistent with maternal banding pattern.
It is embodied as example
Embodiment 1
The screening process of the SSR primer SSR327 of the iron prop Fructus Benincasae hybrid seed purity identification that the present embodiment is provided is as follows：
Some in parent's (iron prop Fructus Benincasae male parent and female parent) and F1 according to the primer of Fructus Benincasae transcript profile sequencing result design
Screened between (iron prop Fructus Benincasae cenospecies), selected the primer SSR327 of codominance difference slug band, the marker bands are clear,
Reproducible, sequence is as follows：
SSR327-F：5’-AAACAACACCGTTTCTTCCG-3’(SEQ ID NO.1)；
SSR327-R：5’-TCAGCGACATCAGAAACACC-3’(SEQ ID NO.2).
With Fructus Benincasae genomic DNA as template, enter performing PCR amplification using above-mentioned SSR primers SSR327 and amplified production is entered
Row polyacrylamide gel electrophoresis specific fragment (polyacrylamide non denatured glue) reclaims DNA PCR amplification agaroses
After glue reclaim TA cloning and sequencing, analysis result is learnt：Primer SSR327 can produce the maternal specific marker of 184bp
SSR327184(its nucleotide sequence is as shown in SEQ ID NO.3) and the male parent specific marker SSR327 of 199bp199(its nucleoside
Acid sequence such as SEQ ID NO.4).
Maternal specific marker SSR327184, as shown in SEQ ID NO.3：
Male parent specific marker SSR327199, as shown in SEQ ID NO.4：
Embodiment 2
" iron prop " Fructus Benincasae hybrid seed purity authentication method that the present embodiment is provided, comprises the following steps：
(1) extraction of Fructus Benincasae DNA
Experiment material is iron prop Fructus Benincasae cenospecies and its male parent, maternal cotyledon, extracts cotyledon DNA steps as follows：
1. take a piece of cotyledon to be put in 2mL centrifuge tubes, add liquid nitrogen grinding pestle grinding, add when liquid nitrogen is evaporated soon rapidly
Enter 800 μ L 2%CTAB extracting solution, after mixing, be placed in 65 DEG C of water-bath 45min (shaking up once every 10min)；
2. the chloroform of 800 μ L, after standing to room temperature, is added：Isoamyl alcohol (24:1), soft mixing, is centrifuged after standing 2min,
12000rmp, 15min, take supernatant (about 510 μ L) and are transferred to new 1.5mL centrifuge tubes；
3. the NaAc (3mol/l) of 1/3 volume of supernatant is added, (- 20 DEG C of the dehydrated alcohol of 1.5 times of volumes of supernatant is added
Pre-cooling), -20 DEG C of 30min～1h are placed in after soft mixing；
4. 12000rmp, is centrifuged 10min, abandons supernatant；
5. in centrifuge tube, add 75% ethanol (pre-cooling) washing DNA to precipitate 2 times, then washed 1 time with dehydrated alcohol, be placed on
Dry up on superclean bench；
6. 50 μ L TE (or ddH are added2O) dissolve, standby as Semen Benincasae leaf genomic DNA.
(2) with Semen Benincasae leaf genomic DNA as template, performing PCR expansion is entered using the primer SSR327 designed in embodiment 1
Increase.
PCR system (20 μ L)
PCR amplification program be：After 95 DEG C of denaturations 3min, 94 DEG C of degeneration 30s, 56 DEG C of annealing 30s, 72 DEG C of extensions
1min, after 40 circulations, 72 DEG C extend 7min eventually, are placed in 4 DEG C of preservations.
(3) polyacrylamide gel electrophoresis (PAGE) detection is carried out to pcr amplification product
1. PCR primer is added 4 μ L 6 × Loading buffer, gets rid of lower rear vortex and mix；
2. 2 μ L amplified productions, 8% (mass percent) native polyacrylamide gel electrophoresises, 180V voltage stabilizings are taken
60min；
3. PAGE glue is pulled down, first with distillation washing one time, then uses 0.1%AgNO3Solution dyes 10min；
4. with distillation washing 2 times, then with 2%NaOH, 0.04%Na2CO3, 0.4% formaldehyde colour developing 10min, after colour developing with from
To wash twice, then photographic analysis on lamp box.
(4) amplification
Primer SSR327 amplifies two band of 184bp and 199bp in iron prop Fructus Benincasae cenospecies, amplifies in male parent B96
The band of 199bp, maternal B96 amplify the band (see Fig. 1) of 184bp.
Specific band is reclaimed, Song Sheng works company is sequenced.As a result show to be amplified in iron prop Fructus Benincasae cenospecies respectively 184bp,
199bp bands, are consistent with the sequence of maternal, male parent amplified production.
Embodiment 3
Field planting and sampling, sample are carried out to the iron prop Fructus Benincasae cenospecies that Guangdong Suixi is planted using the method for embodiment 2
Product amount to 57 plants, to its individual plant numbering, extract individual plant DNA and are detected (see Fig. 2, Fig. 3)；Carried out using Morphology observation method simultaneously
Purity.Testing result：54 plants of hybrid seed, maternal 3 plants, seed purity is 95%, consistent with Fields detection result.
Embodiment 4
The iron prop Fructus Benincasae cenospecies that Guangdong Qingyuan is planted using the method for embodiment 2 carry out field planting and sampling, sample
Product amount to 159 plants, to its individual plant numbering, extract individual plant DNA and are detected (see Fig. 4-Fig. 7)；Entered using Morphology observation method simultaneously
Row Purity.Testing result：139 plants of hybrid seed, maternal 20 plants, seed purity is 87.42%, with Fields detection result one
Cause.
Above example shows that iron prop gourd seed effectively can be distinguished by the method for the present invention with its parental seed,
Accurately simultaneously quick detection goes out seed purity.
Specific embodiment listed above remarks additionally to the present invention, it should be pointed out that above example is served only for
The invention will be further described, does not represent protection scope of the present invention, and other people are make non-of prompting according to the present invention
The modification and adjustment of matter, still falls within protection scope of the present invention.
<110>Vegetables Inst., Guangdong Academy of Agricultural Sciences
<120>A kind of SSR primers and authentication method for the identification of iron prop Fructus Benincasae hybrid seed purity
<210> 1
<211> 20
<212> DNA
<221>Forward primer SSR327-F
<400> 1
AAACAACACC GTTTCTTCCG 20
<210> 2
<211> 20
<212> DNA
<221>Downstream primer SSR327-R
<400> 2
TCAGCGACAT CAGAAACACC 20
<210> 3
<211> 184
<212> DNA
<221>Iron prop Fructus Benincasae female parent specific marker SSR327184
TCAGCGACAT CAGAAACACC TTTACTACAG TAGTGTCCCA TGGAGGAAGA GTTAAGGTGG 60
AAGTTGCAAG ATCAGAGAAT GGGGAAGAAG AAGAAGAAGA AAGAGGAGAA GGTAGGCGTT 120
GAAAATGAAG GTTGGAAAGT GGGAGGGAGA AAGAAGGCAG TAATCGGAAG AAACGGTGTT 180
GTTT 184
<210> 4
<211> 199
<212> DNA
<221>Iron prop Fructus Benincasae male parent specific marker SSR3271994
TCAGCGACAT CAGAAACACC TTTACTATAG TAGTGTCCCA TGGAGGAAGA GTTAAGGTGG 60
AAGTTGCAAG ATCAGAGAAT GGGGAAGAAG AAGAAGAAGA AGAAGAAGAA GAAGAAAGAG 120
GAGAAGGGAG GCGTTGAAAA TGGAGGTTGG AAAGTGGGAG GGAGAAAGAA GGCAGTAATC 180
GGAAGAAACG GTGTTGTTT 199
Claims (5)
1. a kind of SSR primers for the identification of iron prop Fructus Benincasae hybrid seed purity, is characterized in that：The primer SSR327 includes
Trip primer SSR327-F and downstream primer SSR327-R, the nucleotide sequence of the primer SSR327 forward primer SSR327-F is such as
Shown in SEQ ID NO.1, the nucleotide sequence such as SEQ ID NO.2 institutes of the downstream primer SSR327-R of the primer SSR327
Show.
2. a kind of authentication method of iron prop Fructus Benincasae hybrid seed purity, is characterized in that containing following steps：
(1) Fructus Benincasae cotyledon is taken, Fructus Benincasae genomic DNA is extracted；
(2) with Fructus Benincasae genomic DNA as template, usage right requires that the primer SSR327 in 1 enters performing PCR amplification, obtains PCR expansions
Volume increase thing；
(3) gel electrophoresiss are carried out to pcr amplification product, is developed the color, count electrophoresis result；
(4) electrophoresis result is analyzed, the individual plant only simultaneously with two parent's specific bands is just real hybridization
Kind, lack any one band therein or the individual plant for non-parent's specific band is designated as pseudostationary, calculate seed purity, wherein
Primer SSR327 can produce the maternal specific marker SSR327 as shown in SEQ ID NO.3 of 184bp184And 199bp as
Male parent specific marker SSR327 shown in SEQ ID NO.4199.
3. the authentication method of iron prop Fructus Benincasae hybrid seed purity according to claim 2, is characterized in that：PCR in step (2)
The 20 μ L reaction systems adopted during amplification for：150ng·μL-11 μ L of genomic DNA, contain Mg2+10×PCR buffer 2μL、
2.5mmol·L-1dNTPs 1.5μL、0.25mmol·L-11 μ L of forward primer SSR327-F, 0.25mmol L-1Downstream primer
1 μ L of SSR327-R, 2U/ μ L Taq enzymes 1 μ L, ddH2O 12.5μL.
4. the authentication method of iron prop Fructus Benincasae hybrid seed purity according to claim 2, is characterized in that：PCR in step (2)
During amplification, amplification program is：After 95 DEG C of denaturations 3min, 94 DEG C of degeneration 30s, 56 DEG C of annealing 30s, 72 DEG C of extension 1min, 40 are followed
After ring, 72 DEG C extend 7min, 4 DEG C of preservations eventually.
5. the authentication method of iron prop Fructus Benincasae hybrid seed purity according to claim 2, is characterized in that：Coagulate in step (3)
Adopt during gel electrophoresis weight/mass percentage composition for 8% native polyacrylamide gel electrophoresises.
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| CN109234433A (en) * | 2018-10-19 | 2019-01-18 | 广东省农业科学院蔬菜研究所 | A kind of SNP marker and its application of wax gourd seed type gene |
| CN109486991A (en) * | 2018-12-06 | 2019-03-19 | 南京农业大学 | Identify molecular labeling primer composition and its application of pears and apple intergeneric conjugal transfer |
| CN110029184A (en) * | 2019-03-05 | 2019-07-19 | 广西大学 | Identify SSR molecular marker primer and its application of wax gourd hybrid seed purity |
| CN111286554A (en) * | 2020-03-18 | 2020-06-16 | 广东省农业科学院蔬菜研究所 | SSR primer for identifying purity of hybrid seeds of Niubao white gourd and application of SSR primer |
| CN117385086A (en) * | 2023-11-27 | 2024-01-12 | 广东省农业科学院蔬菜研究所 | SSR (simple sequence repeat) marker primer for melon germplasm resources and construction method of fingerprint |
| CN117887893A (en) * | 2024-03-12 | 2024-04-16 | 广东省农业科学院蔬菜研究所 | KASP molecular marker closely linked with size of wax gourd seeds and application thereof |
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| CN109234433A (en) * | 2018-10-19 | 2019-01-18 | 广东省农业科学院蔬菜研究所 | A kind of SNP marker and its application of wax gourd seed type gene |
| CN109234433B (en) * | 2018-10-19 | 2019-06-21 | 广东省农业科学院蔬菜研究所 | A kind of SNP marker and its application of wax gourd seed type gene |
| CN109486991A (en) * | 2018-12-06 | 2019-03-19 | 南京农业大学 | Identify molecular labeling primer composition and its application of pears and apple intergeneric conjugal transfer |
| CN109486991B (en) * | 2018-12-06 | 2021-11-30 | 南京农业大学 | Molecular marker primer composition for identifying intergeneric hybrid of pear and apple and application thereof |
| CN110029184A (en) * | 2019-03-05 | 2019-07-19 | 广西大学 | Identify SSR molecular marker primer and its application of wax gourd hybrid seed purity |
| CN110029184B (en) * | 2019-03-05 | 2020-11-03 | 广西大学 | SSR molecular marker primer for identifying purity of hybrid seeds of white gourd and application thereof |
| CN111286554A (en) * | 2020-03-18 | 2020-06-16 | 广东省农业科学院蔬菜研究所 | SSR primer for identifying purity of hybrid seeds of Niubao white gourd and application of SSR primer |
| CN117385086A (en) * | 2023-11-27 | 2024-01-12 | 广东省农业科学院蔬菜研究所 | SSR (simple sequence repeat) marker primer for melon germplasm resources and construction method of fingerprint |
| CN117385086B (en) * | 2023-11-27 | 2024-04-16 | 广东省农业科学院蔬菜研究所 | SSR (simple sequence repeat) marker primer for melon germplasm resources and construction method of fingerprint |
| CN117887893A (en) * | 2024-03-12 | 2024-04-16 | 广东省农业科学院蔬菜研究所 | KASP molecular marker closely linked with size of wax gourd seeds and application thereof |
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