CN109439783A - A kind of SSR primer and method for sweet 25 Purities of muskmelon cenospecies gold - Google Patents
A kind of SSR primer and method for sweet 25 Purities of muskmelon cenospecies gold Download PDFInfo
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Abstract
The invention discloses a kind of SSR primers for sweet 25 Purities of muskmelon cenospecies gold, and the SSR primer includes primer pair gSSR15606 and/or primer pair gSSR45335；The SSR primer can generate specific marker, high specificity in sweet 25 Parents of gold.The present invention also provides a kind of method for identifying sweet 25 purity of muskmelon cenospecies gold, this method utilizes above-mentioned SSR primer, can quickly, accurately and efficiently identify muskmelon melon hybrid seed purity；Primer and method through the invention only need to simply extract muskmelon blade genomic DNA, then carry out PCR amplification and electrophoresis, can effectively identify the purity of muskmelon hybrid seed.Hybrid seed purity identification work can be completed in method of the invention in 5h, has the advantages such as quick, accurate, inexpensive, easy to operate, the method that can replace conventional hybridization Purity Identification, commercial application value with higher.
Description
Technical field
The invention belongs to molecular marking technique fields, and in particular to one kind is for sweet 25 Purities of muskmelon cenospecies gold
SSR primer and method.
Background technique
Muskmelon is Curcurbitaceae (Cucurbitaceae)) Cucumis (Cucumis) alienation pollination crop is important in the world
Fruit with vegetable crop, one of global ten big fruit.Muskmelon has stronger hybrid vigour, and first cross kind has growing way
By force, the advantages that disease resistance is good, yield is high, quality is good, muskmelon produces most commodity seeds and uses cenospecies in recent years.
Since most of cenospecies is artificial seed, purity is difficult to control, if emasculation not in time, be not thorough, just easily generate false miscellaneous
Kind, it causes hybrid seed purity to reduce, corresponding seed quality criteria is not achieved, reduce crop yield and quality.
Muskmelon hybrid seed purity identification conventional method is field shape identification, but the method qualification cycle is long, works
Amount is big, needs to expend a large amount of manpower and material resources, by seasonal effect, and available morphological characters is limited, and vulnerable to environmental condition shadow
Pilot causes qualification result inaccuracy, in addition, most parts of the north are because climate reasons have little time to make Purity Field identification, it cannot
Meet current year's production needs.Therefore, a set of method for quickly, accurately, efficiently identifying muskmelon hybrid seed genetic purity is built up, it is right
It is very necessary for improving muskmelon seeds quality management.
Summary of the invention
The present invention exists by field shape identification method by season mostly for the identification of current muskmelon hybrid seed purity
The defects of limitation is big, qualification cycle is long, the first purpose of this invention are to provide 2 pairs for identifying New melon variety gold honey
The SSR primer of 25 hybrid seed purities, these two pair primer can generate specific marker in sweet 25 Parents of gold, special
It is anisotropic strong.The object of the invention is also to provide a kind of method for identifying sweet 25 purity of muskmelon hybrid seed gold, the party
Method utilizes above-mentioned SSR primer, can quickly, accurately and efficiently identify muskmelon melon hybrid seed purity.
The technical scheme adopted by the invention is that:
SSR primer provided by the present invention for sweet 25 Purity Identifications of muskmelon cenospecies gold, including 2 pairs of SSR primers
To sequence, the seed of muskmelon cenospecies gold honey 25 can be identified with any 1 pair or 2 pairs in this 2 pairs of primers application simultaneously respectively
Purity.The specific nucleotide sequence of above-mentioned primer is as follows:
Primer pair 1:
Upstream primer gSSR15606-F:5'- TCTGTTTGTACCACCAAAATC-3'(SEQ ID NO.1)
Downstream primer gSSR15606-R:5'- TCCTCCTCTTCTTTTACCAAT-3'(SEQ ID NO.2)
Primer pair 2:
Upstream primer gSSR45335-F:5'- TGGAATTAAATGTGGACAATG-3'(SEQ ID NO.3)
Downstream primer gSSR45335-R:5'- GTCCACCGTAAAAAGAGTTTC-3'(SEQ ID NO.4)
The present invention also provides a kind of methods for carrying out the identification of muskmelon hybrid seed purity using above-mentioned primer pair, specifically include following
Step:
(1) muskmelon Hybrid F1 and its corresponding parent's leaves genomic DNA are extracted；
(2) using the genomic DNA of extraction as template, PCR amplification is carried out using 1 ~ 2 pair of primer any in above-mentioned 2 pairs of primers；
(3) polyacrylamide gel electrophoresis for carrying out 8% to the pcr amplification product of step (2) detects；
(4) analyze step (3) electrophoresis detection result: the standard diagram of the real cenospecies of muskmelon is while having Parent
Otherwise the electrophorogram of specific band is pseudostationary, calculate hybrid seed purity using band statistical result；
If selecting primer pair gSSR15606 in step (2), can detect in step (4) 142bp maternal specific band, its
Nucleotides sequence list is as shown in SEQ ID NO.5 and the male parent specific band of 163bp, its nucleotides sequence list such as SEQ ID
NO.6 is then the real cenospecies of muskmelon, is otherwise pseudostationary；
If selecting primer pair gSSR45335 in step (2), can detect in step (4) 146bp maternal specific band, its
Nucleotides sequence list is as shown in SEQ ID NO.7 and the male parent specific band of 156bp, its nucleotides sequence list such as SEQ ID
Shown in NO.8, then it is the real cenospecies of muskmelon, is otherwise pseudostationary.
If selecting primer pair gSSR45335 and primer pair gSSR45335 in step (2) simultaneously, can be detected in step (4)
The maternal specific band of 142bp, its nucleotides sequence list are as shown in SEQ ID NO.5 and the male parent specificity item of 163bp out
Band, its nucleotides sequence list such as SEQ ID NO.6 can also detect maternal specific band, its core of 146bp in step (4)
Nucleotide sequence table is as shown in SEQ ID NO.7 and the male parent specific band of 156bp, nucleotides sequence list such as SEQ ID
Shown in NO.8, then it is the real cenospecies of muskmelon, is otherwise pseudostationary.
In above steps:
Muskmelon genomic DNA is extracted in step (1) of the present invention to extract using the CTAB method of improvement: 30mg tender leaf 1. being taken to be put into
The centrifuge tube of 2ml is added the steel ball of a diameter 4mm, covers tightly lid, is put into liquid nitrogen and freezes 90s, then utilizes tissue grinder
Instrument grind away；2. the CTAB extracting solution of 600 μ l 2% is added in the sample of milled, 55 DEG C of water-bath 20min, each 5min are jiggled
Once；3. 12000rpm is centrifuged 2min, 450 μ l supernatants are drawn in the centrifuge tube of clean 1.5ml, 300 μ l ratios are added
For the chloroform isoamyl alcohol mixed liquor of 24:1, mix well；4. 13000rpm is centrifuged 1min, 300 μ l supernatants are drawn in another new
1.5ml centrifuge tube, 550 μ l dehydrated alcohols and ammonium acetate is added, mixes well, is put into -20 DEG C of refrigerator 1h；5. 12000rpm from
The heart 10 minutes, supernatant is outwelled, is placed at room temperature for；6. 100 μ l ddH are added when alcohol-free taste in centrifuge tube2O dissolving DNA.
The reaction system used when PCR is expanded in step (2) of the present invention is preferred are as follows: the system of 15 μ l, including 7.5 μ l
The muskmelon DNA of the positive anti-primer of 2*Taq MasterMix, 10mM each 1 μ l, 1 μ l are template, the ultrapure water of 4.5 μ l sterilizing.
Amplification program is preferred when PCR is expanded in step (2) of the present invention are as follows: 94 DEG C of initial denaturation 3min；94 DEG C of denaturation 30s,
55 DEG C of annealing 30s, 72 DEG C of extension 30s are recycled 35 times；Finally extend 2min, 16 DEG C of termination PCR reactions.
In step (3) of the present invention when electrophoresis detection, is dyed using argentation, photograph to record characteristic bands under white light.
Beneficial effects of the present invention:
(1) any 1 ~ 2 pair of SSR primer in the present invention can generate maternal specific band and male parent specific band, and special
It is anisotropic strong, inheritance stability.
It (2), can be by muskmelon Hybrid with any 1 pair or 2 pairs in method of the invention and 2 pairs of primers of combination primer
The corresponding Parent seed of the hybrid seed of gold honey 25, other seeds mixed distinguish, and it is miscellaneous quickly to detect muskmelon
Hand over the purity of seed.
(3) primer and method through the invention only need to simply extract muskmelon blade genomic DNA, then carry out PCR amplification
And electrophoresis, it can effectively identify the purity of muskmelon hybrid seed.It is pure that hybrid seed can be completed in method of the invention in 5h
Identification work is spent, there are the advantages such as quick, accurate, inexpensive, easy to operate, conventional hybridization Purity Identification can be replaced
Method, commercial application value with higher.
Detailed description of the invention
Fig. 1 indicates the electrophoresis of gold sweet 25 parents and F1 cenospecies using SSR primer pair 1(gSSR15606) amplification
Figure；Wherein M indicates that Marker, P1 indicate maternal, and P2 indicates male parent, and F1 indicates cenospecies, and 1 indicates hybrid strain.
Fig. 2 indicates the electrophoretogram of gold sweet 25 parents and F1 cenospecies using SSR primer pair 2(gSSR45335) amplification
Spectrum, wherein M indicates that Marker, P1 indicate maternal, and P2 indicates male parent, and F1 indicates cenospecies, and 1 indicates hybrid strain.
Specific embodiment
With reference to embodiment, the present invention is described in further detail, but embodiments of the present invention are not
It is limited to this, cannot be limited the scope of protection of the present invention with following embodiments.
Experimental method used in following embodiments is conventional method unless otherwise specified；Used material,
Reagent etc., is commercially available unless otherwise specified.
(1) materials and methods
1. material
Gold honey 25, precocious thick-skinned melon, female flower, which is open into fruit maturation, needs 30-35d；The female flower open hour are more neat, pollination
And cultivation management saves labor；Plant growing way is vigorous, and blade is incised larger, and translucency is good, is conducive to that fruit annesl is mature and sugar
Accumulation；Fruit ellipse, pericarp is golden yellow to orange-yellow, and fruit surface smooth is without reticulate pattern；Pulp salmon pink, meat is crisp exquisiteness, mouthfeel
Crisp sweet tea, 18% or more solid content, there is stronger fragrance；Fruit caving is smaller, single fruit weight 2-2.5kg, and 10,000 jin of per mu yield or more.With gold
25 (F1) of honey and its parent are test test material.
2. research method
2.1 sowing
Cenospecies gold honey 25 and its parent are seeded in nursery in the hole tray in 72 holes, and wherein parent respectively sows 12, cenospecies 200
Grain, normal management.
The extraction of 2.2 genomic DNAs
Sweet 25 cenospecies of gold and its parent's tender leaf are taken, extracts genomic DNA, specific steps are as follows: 1. take with the CTAB method of improvement
30mg tender leaf is put into the centrifuge tube of 2ml, and the steel ball of a diameter 4mm is added, covers tightly lid, is put into liquid nitrogen and freezes 90s, then
Utilize tissue grinder instrument grind away；2. the CTAB extracting solution of 600 μ l 2% is added in the sample of milled, 55 DEG C of water-bath 20min, each
5min is jiggled once；3. 12000rpm is centrifuged 2min, 450 μ l supernatants are drawn in the centrifuge tube of clean 1.5ml, are added
Enter the chloroform isoamyl alcohol mixed liquor that 300 μ l ratios are 24:1, mixes well；4. 13000rpm is centrifuged 1min, draw on 300 μ l
Clear liquid is added 550 μ l dehydrated alcohols and ammonium acetate, mixes well, be put into -20 DEG C of refrigerator 1h in another new 1.5ml centrifuge tube；
5. 12000rpm is centrifuged 10 minutes, supernatant is outwelled, is placed at room temperature for；6. 100 μ l are added when alcohol-free taste in centrifuge tube
ddH2O dissolving DNA.
It is utilized respectively the quality and concentration of agarose gel electrophoresis and the proposed DNA of UV spectrophotometer measuring.
2.3 PCR amplification and detection
Using the muskmelon genomic DNA of extraction as template, any 1 ~ 2 pair of primer of gSSR15606 and gSSR45335 primer pair is utilized
To progress PCR amplification.PCR reaction system are as follows: each 1 μ l of positive anti-primer of 7.5 μ l 2*Taq MasterMix, 10mM, 1 μ l's
Muskmelon DNA is template, and the ultrapure water of sterilizing is added to complement to 15 μ l.
Response procedures are as follows: 94 DEG C of initial denaturation 3min；94 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 30s, circulation 35
It is secondary；Finally extend 2min, 16 DEG C of termination PCR reactions, 4 DEG C of refrigerators are stored to be detected.
The detection of amplified production: pcr amplification product is electrophoresis on 8% polyacrylamide gel, 160V pressure stabilizing electrophoresis in concentration
1.5 hours, argentation dyeing was carried out after electrophoresis, characteristic bands are then photographed to record under white light and are analyzed.
(2) result and analysis
187 single plants are shown using the electrophoresis result of label gSSR15606 amplification while detecting the spy of 142bp and 163bp
Band (see figure 1) is levied, parent's sequencing result also indicates that maternal specific sequence (the SEQ ID for producing that size is 142bp
) and the male parent specific sequence of 163bp (SEQ ID NO.6) NO.5；And with this 187 lists after label gSSR45335 amplification
Strain also detects the characteristic bands (see figure 2) of 146bp and 156bp simultaneously, and parent's sequencing result also indicates that generating size is 146bp
Maternal specific sequence (SEQ ID NO.7) and 156bp male parent specific sequence (SEQ ID NO.8), show this 187
Single plant is true hybrid, purity are as follows: 187/188*100% ≈ 99.5%, the result fit like a glove with field shape investigation result, accurately
Rate is 100%.
Sequence table
<110>Jiangsu Lv Gang modern agricultural development Co., Ltd
<120>a kind of SSR primer and method for sweet 25 Purities of muskmelon hybrid seed gold
<160> 8
<170> Patentin version 3.3
<210> 1
<211> 21
<212> DNA
<213>artificial sequence
<400> 1
TCTGTTTGTA CCACCAAAAT C 21
<210> 2
<211> 21
<212> DNA
<213>artificial sequence
<400> 2
TCCTCCTCTT CTTTTACCAA T 21
<210> 3
<211> 21
<212> DNA
<213>artificial sequence
<400> 3
TGGAATTAAA TGTGGACAAT G 21
<210> 4
<211> 21
<212> DNA
<213>artificial sequence
<400> 4
GTCCACCGTA AAAAGAGTT TC 21
<210> 5
<211> 142
<212> DNA
<213>muskmelon (Cucumis melo L.)
<400>5
tctgtttgta ccaccaaaat cctcagcagt tccaaaaaga agaagaagaa gaagaagaat 60
tcaaacaaaa atgtcgtctg ttggaacttt tcaactacta ctacctccat ttccttcttc 120
ctcctcctct tcttttacca at 142
<210> 6
<211> 163
<212> DNA
<213>muskmelon (Cucumis melo L.)
<400>6
tctgtttgta ccaccaaaat cctcagcagt tccaaaaaga agaagaagaa gaagaagaag 60
aagaagaaga agaagaagaa ttcaaacaaa aatgtcgtct gttggaactt ttcaactact 120
actacctcca tttccttctt cctcctcctc ttcttttacc aat 163
<210> 7
<211> 146
<212> DNA
<213>muskmelon (Cucumis melo L.)
<400>7
tggaattaaa tgtggacaat ggtatataaa ttaaaagaat aataataaaa caaaaacaaa 60
gggggaaaaa aaataataat aataagaaga aaaaaagggg agtctctgac caagcaggat 120
gtggggtcca ccgtaaaaag agtttc 146
<210> 8
<211> 156
<212> DNA
<213>muskmelon (Cucumis melo L.)
<400>8
tggaattaaa tgtggacaat ggtatataaa ttaaaagaat aataataaaa caaaaacaaa 60
gggggaaaaa aaataataat aataagaaga ataataagaa aaaaaagggg agtctctgac 120
caagcaggat gtggggtcca ccgtaaaaag agtttc 156
<110>Jiangsu Lv Gang modern agricultural development Co., Ltd
<120>a kind of SSR primer and method for sweet 25 Purities of muskmelon cenospecies gold
<140>201810785348.4
<160> 8
<170> Patentin version 3.3
<210> 1
<211> 21
<212> DNA
<213>artificial sequence
<400> 1
TCTGTTTGTA CCACCAAAAT C 21
<210> 2
<211> 21
<212> DNA
<213>artificial sequence
<400> 2
TCCTCCTCTT CTTTTACCAA T 21
<210> 3
<211> 21
<212> DNA
<213>artificial sequence
<400> 3
TGGAATTAAA TGTGGACAAT G 21
<210> 4
<211> 21
<212> DNA
<213>artificial sequence
<400> 4
GTCCACCGTA AAAAGAGTTT C 21
<210> 5
<211> 142
<212> DNA
<213>muskmelon (Cucumis melo L.)
<400>5
tctgtttgta ccaccaaaat cctcagcagt tccaaaaaga agaagaagaa gaagaagaat 60
tcaaacaaaa atgtcgtctg ttggaacttt tcaactacta ctacctccat ttccttcttc 120
ctcctcctct tcttttacca at 142
<210> 6
<211> 163
<212> DNA
<213>muskmelon (Cucumis melo L.)
<400>6
tctgtttgta ccaccaaaat cctcagcagt tccaaaaaga agaagaagaa gaagaagaag 60
aagaagaaga agaagaagaa ttcaaacaaa aatgtcgtct gttggaactt ttcaactact 120
actacctcca tttccttctt cctcctcctc ttcttttacc aat 163
<210> 7
<211> 146
<212> DNA
<213>muskmelon (Cucumis melo L.)
<400>7
tggaattaaa tgtggacaat ggtatataaa ttaaaagaat aataataaaa caaaaacaaa 60
gggggaaaaa aaataataat aataagaaga aaaaaagggg agtctctgac caagcaggat 120
gtggggtcca ccgtaaaaag agtttc 146
<210> 8
<211> 156
<212> DNA
<213>muskmelon (Cucumis melo L.)
<400>8
tggaattaaa tgtggacaat ggtatataaa ttaaaagaat aataataaaa caaaaacaaa 60
gggggaaaaa aaataataat aataagaaga ataataagaa aaaaaagggg agtctctgac 120
caagcaggat gtggggtcca ccgtaaaaag agtttc 156
Claims (3)
1. a kind of SSR primer for sweet 25 Purities of muskmelon cenospecies gold, it is characterised in that: the SSR primer includes
Primer pair gSSR15606 and/or primer pair gSSR45335；The core of the upstream primer gSSR15606-F of primer pair gSSR15606
Nucleotide sequence is as shown in SEQ ID NO.1, and the nucleotide sequence of the downstream primer gSSR15606-R of primer pair gSSR15606 is such as
Shown in SEQ ID NO.2;The nucleotide sequence of the upstream primer gSSR45335-F of primer pair gSSR45335 such as SEQ ID NO.3
Shown, the nucleotide sequence of the downstream primer gSSR45335-R of primer pair gSSR45335 is as shown in SEQ ID NO.4.
2. a kind of method for carrying out sweet 25 Purities of muskmelon cenospecies gold using SSR primer described in claim 1, special
Sign is, specifically includes the following steps:
(1) muskmelon Hybrid F1 and its corresponding parent's leaves genomic DNA are extracted；
(2) using the genomic DNA of extraction as template, PCR amplification is carried out using SSR primer described in claim 1；
(3) polyacrylamide gel electrophoresis for carrying out 8% to the pcr amplification product of step (2) detects；
(4) analyze step (3) electrophoresis detection result: the standard diagram of the real cenospecies of muskmelon is while having parent
Otherwise the electrophorogram of this specific band is pseudostationary, calculate hybrid seed purity using band statistical result；
If selecting primer pair gSSR15606 in step (2), can detect in step (4) 142bp maternal specific band, its
Nucleotides sequence list is as shown in SEQ ID NO.5 and the male parent specific band of 163bp, its nucleotides sequence list such as SEQ ID
NO.6 is then the real cenospecies of muskmelon, is otherwise pseudostationary；
If selecting primer pair gSSR45335 in step (2), can detect in step (4) 146bp maternal specific band, its
Nucleotides sequence list is as shown in SEQ ID NO.7 and the male parent specific band of 156bp, its nucleotides sequence list such as SEQ ID
Shown in NO.8, then it is the real cenospecies of muskmelon, is otherwise pseudostationary；
If selecting primer pair gSSR45335 and primer pair gSSR45335 simultaneously in step (2), step can detect in (4)
The maternal specific band of 142bp, its nucleotides sequence list are as shown in SEQ ID NO.5 and the male parent specificity item of 163bp
Band, its nucleotides sequence list such as SEQ ID NO.6 can also detect maternal specific band, its core of 146bp in step (4)
Nucleotide sequence table is as shown in SEQ ID NO.7 and the male parent specific band of 156bp, nucleotides sequence list such as SEQ ID
Shown in NO.8, then it is the real cenospecies of muskmelon, is otherwise pseudostationary.
3. according to method described in right 2, it is characterised in that:
The reaction system used when PCR is expanded in the step (2) are as follows: the system of 15 μ l, including 7.5 μ l 2*Taq
The muskmelon DNA of the positive anti-primer of MasterMix, 10mM each 1 μ l, 1 μ l are template, the ultrapure water of 4.5 μ l sterilizing；
Amplification program when PCR is expanded in the step (2) are as follows: 94 DEG C of initial denaturation 3min；94 DEG C of denaturation 30s, 55 DEG C of annealing
30s, 72 DEG C of extension 30s are recycled 35 times；Finally extend 2min, 16 DEG C of termination PCR reactions；
In the step (3) when electrophoresis detection, is dyed using argentation, photograph to record characteristic bands under white light.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111961740A (en) * | 2020-06-29 | 2020-11-20 | 湖南省蔬菜研究所 | SSR primers and method for purity identification of Zaojia towel gourd hybrid seeds |
| CN112961931A (en) * | 2021-03-02 | 2021-06-15 | 宁波市农业科学研究院 | Rapid identification method for purity of Yongtian No.5 melon seeds |
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Cited By (3)
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| CN111961740A (en) * | 2020-06-29 | 2020-11-20 | 湖南省蔬菜研究所 | SSR primers and method for purity identification of Zaojia towel gourd hybrid seeds |
| CN111961740B (en) * | 2020-06-29 | 2024-03-26 | 湖南省蔬菜研究所 | SSR primer and method for identifying purity of early-optimal luffa hybrid seeds |
| CN112961931A (en) * | 2021-03-02 | 2021-06-15 | 宁波市农业科学研究院 | Rapid identification method for purity of Yongtian No.5 melon seeds |
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