CN111961740A - SSR primers and method for purity identification of Zaojia towel gourd hybrid seeds - Google Patents

SSR primers and method for purity identification of Zaojia towel gourd hybrid seeds Download PDF

Info

Publication number
CN111961740A
CN111961740A CN202010601289.8A CN202010601289A CN111961740A CN 111961740 A CN111961740 A CN 111961740A CN 202010601289 A CN202010601289 A CN 202010601289A CN 111961740 A CN111961740 A CN 111961740A
Authority
CN
China
Prior art keywords
purity
hybrid
luffa
zaojia
identifying
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN202010601289.8A
Other languages
Chinese (zh)
Other versions
CN111961740B (en
Inventor
韩小霞
胡新军
李勇奇
闵子扬
袁祖华
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Hunan vegetable research institute
Original Assignee
Hunan vegetable research institute
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Hunan vegetable research institute filed Critical Hunan vegetable research institute
Priority to CN202010601289.8A priority Critical patent/CN111961740B/en
Publication of CN111961740A publication Critical patent/CN111961740A/en
Application granted granted Critical
Publication of CN111961740B publication Critical patent/CN111961740B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • 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
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • 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
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/13—Plant traits
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Analytical Chemistry (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Physics & Mathematics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Genetics & Genomics (AREA)
  • Immunology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Botany (AREA)
  • Mycology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention belongs to the technical field of molecular detection, and particularly relates to an SSR primer and an SSR method for purity identification of early-quality towel gourd hybrid seeds. The method can be used for detecting the purity of the Zaojia towel gourd hybrid, can effectively distinguish the hybrid from a male parent and a female parent, and can quickly detect the purity of the hybrid. The method has the advantages of simplicity, rapidness, accuracy, low cost, simple operation and the like, can finish the detection of nearly 500 samples by one person in one day, can replace the traditional purity identification method of the towel gourd hybrid, is simple and convenient to operate, saves labor, does not need to occupy land, is accurate and reliable in result, greatly improves the identification efficiency, reduces the identification cost, and has higher commercial application value.

Description

SSR primers and method for purity identification of Zaojia towel gourd hybrid seeds
Technical Field
The invention belongs to the technical field of molecular detection, and particularly relates to an SSR primer and an SSR method for purity identification of early-quality towel gourd hybrid seeds.
Background
Luffa cylindrica is a cultivar of Luffa genus of Cucurbitaceae (Cucurbitaceae), an annual climbing herbaceous plant. There are two botanicals of Luffa cultivated in China, namely common Luffa (Luffa cylindrica (L.) m.j.roem.) and Luffa acutangula (L.) Roxb.). The Zaojia towel gourd belongs to the shredded towel gourd in common towel gourd (Luffa cylindrica (L.) M.J.Roem.). In the production process of hybrid loofah seeds, a female parent castration mode is mainly adopted. The male parent and the female parent are respectively sown according to the growth period, and all male flower buds of the female parent and the female parent are manually picked off before the female parent and the female parent bloom. Pollen of the male parent is collected in the flowering period and pollinated to the stigmas of female flowers of the female parent, so that hybrid seeds are obtained. Because the male flowers of the towel gourd are unlimited catkins, artificial emasculation is easy and incomplete. If the emasculation of the female parent is incomplete, the pollen of the female parent may drift down or spread by the insect to its own stigma, thereby producing a pseudo hybrid, which is a major factor in the production resulting in a decrease in seed purity. According to the relevant regulations of the national standard of the people's republic of China (GB8079-87), the purity of the towel gourd hybrid seeds can be sold only when the purity reaches 90%.
The traditional loofah hybrid purity detection mainly adopts morphological identification which is mainly based on the morphological identification of the female parent and the seeds and ovaries of the hybrid, the method has strong practicability, but the identification time needs about 60 days, and the defects of long period, large occupied area, large environmental influence and the like exist. At present, the molecular marker is adopted to identify the purity of hybrid seeds, has the advantages of good stability, large information amount and the like, and is widely applied to the purity quality detection of crop seeds, for example, in the prior art, the SSR marker of the towel gourd is used for identifying the purity of hybrid seeds of luffa acutangula linnaeus 'Yalv No. 6' and common towel gourd 'Sisheng No. 2', but the primers and the method for identifying the purity of the early-quality towel gourd are not reported yet. Meanwhile, the purity of hybrid species identified by the current towel gourd SSR molecular markers still exists: (1) the genome extraction adopts a CTAB method or an SOD method, and the operation steps are complicated and time-consuming; (2) cost is wasted by a PCR reaction system and a program which are not optimized; (3) silver dyeing step and color development link are complex to operate, results are unstable, and the like.
Disclosure of Invention
Aiming at the technical problems, the invention aims to provide the SSR primer and the method for identifying the purity of the hybrid seeds of the 'Zaojia luffa', which can quickly and accurately distinguish the hybrid seeds from male parents and female parents and detect the purity of the hybrid seeds.
In order to achieve the purpose, the invention adopts the following technical scheme:
firstly, the invention provides an SSR primer for identifying purity of early-excellent towel gourd hybrid seeds, which comprises an upstream primer SF32 and a downstream primer SR32, wherein the nucleotide sequence of the upstream primer SF32 is shown as SEQ ID NO. 1, and the nucleotide sequence of the downstream primer SR32 is shown as SEQ ID NO. 2.
Secondly, the invention provides a method for identifying the purity of the Zaojia towel gourd hybrid seeds, which comprises the following steps:
(1) extracting genome DNA of the Zaojia luffa parent and filial generation;
(2) performing PCR amplification by using the genomic DNA extracted in the step (1) as a template and using the primers SF32 and SR32 of claim 1 to obtain an amplification product;
(3) detecting the amplification product in the step (2) by gel electrophoresis, developing, and counting the electrophoresis result;
(4) analyzing the electrophoresis result in the step (3), wherein the single plant with the parent specificity band is a true hybrid, and the single plant without any one band is a false hybrid.
Further, the method for extracting the genomic DNA in the step (1) specifically comprises the following steps: the sample to be extracted was put on a PCR plate, 50. mu.l of a 0.25mol/L NaOH solution was added thereto, boiled in boiling water for 3min, the PCR plate was taken out with tweezers, and 150. mu.l of Tris ∙ HCl (0.1 mol/L pH 8.0) was added thereto to neutralize the reaction.
The sample to be extracted includes but is not limited to radicle, hypocotyl, etc. and is suitable for hybrid seed, germinating plant leaf and other parts.
Further, the PCR reaction system in the step (2) is 10 μ l, wherein: 10 ng/. mu.l genome DNA 0.5. mu.l, 10 XPCR Buffer 1. mu.l, 10mmol/l dNTP 0.1. mu.l, 20pmol/l SSR upstream and downstream primers 0.1. mu.l, 2.5U/. mu.l rTaq DNADNPolymerase A0.1. mu.l, sterile ddH2O to a total volume of 10. mu.l.
Further, the PCR reaction procedure in the step (2) is as follows: pre-denaturation at 94 ℃ for 4min, denaturation at 94 ℃ for 30s, annealing at 52.5 ℃ for 30s, extension at 72 ℃ for 30s, 25 cycles total, and final extension at 72 ℃ for 3 min.
Further, the gel electrophoresis in the step (3) is 8% native polyacrylamide gel electrophoresis.
Further, the color development in the step (3) is specifically as follows: after electrophoresis is finished, separating two glass plates, putting gel into a special basin, adding distilled water for cleaning for 2-3 times, putting the gel into dyeing liquid, taking out the gel plate after dyeing is finished, and rinsing the gel plate for 2-3 times by using distilled water; and (3) placing the rubber plate into a developing solution, slightly shaking, quickly rinsing for 2-5 times by using distilled water after strips are clear, placing the rubber plate on a film observation lamp, and taking a picture for recording.
Finally, the invention also provides application of the SSR primers SF32 and SR32 in purity identification of early and good towel gourd hybrid and molecular assisted breeding.
Compared with the prior art, the invention has the following beneficial effects:
the method can be used for detecting the purity of the Zaojia towel gourd hybrid, can effectively distinguish the hybrid from a male parent and a female parent, and can quickly detect the purity of the hybrid. The method has the advantages of simplicity, rapidness, accuracy, low cost, simple operation and the like, can finish the detection of nearly 500 samples by one person in one day, can replace the traditional purity identification method of the towel gourd hybrid, is simple and convenient to operate, saves labor, does not need to occupy land, is accurate and reliable in result, greatly improves the identification efficiency, reduces the identification cost, and has higher commercial application value.
Drawings
FIG. 1 is a screening diagram of the purity identification primer for Zaojia Luffa hybrid.
FIG. 2 is a polyacrylamide electrophoresis gel diagram of the PCR product for purity determination of the Zaojia loofah hybrid.
FIG. 3 shows a sample for identifying the purity of hybrid.
FIG. 4 shows a sample for identifying the purity of hybrids.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The materials used in the following examples are all commercially available from conventional sources.
Example 1 screening of primers for identifying purity of Zaojia Luffa hybrid
Performing primer screening according to SSR primers of wax gourd genome and using genome DNA of the female parent 'S1' and the male parent 'S2' of the Zaojia towel gourd as templates to obtain a marker S32 with co-dominant bands, wherein corresponding primer pairs are SF32 and SR32 respectively, wherein
The nucleotide sequence of the upstream primer SF32 is as follows: 5'-CATCACCACCACCACAACC-3' (SEQ ID NO:1)
The nucleotide sequence of the downstream primer SR32 is: 5'-GCCACCAAAATCAGCTCTATG-3' (SEQ ID NO: 2).
The mark belt has clear shape and good repeatability. PCR amplification of the primer pair S32 can generate female specific marker with a length of about 100bp and male specific marker with a length of about 80bp, as shown in FIG. 1, wherein lanes 1-3 are female parents of Zajia Luffa cylindrica, lanes 4-6 are male parents of Zajia Luffa cylindrica, and lane 7 is hybrid species.
Example 2 method for identifying purity of Zaojia Luffa hybrid seed
In this example, the embryonic axis of germinated seeds was used as the sample for extracting genomic DNA
(1) Quick extraction of early-good towel gourd and male parent and female parent seed radicle genome DNA
The method comprises collecting about 200 seeds of the Zaojia Luffa hybrid, soaking the seeds in 4-8 seeds of male parent and female parent for 5 hr, placing in a germination box filled with filter paper, and keeping constant temperature and moisture at 37 deg.C. Germinating for about 6-7 days, taking the hypocotyl or root tip part of single germinated seed about 0.5-1 cm, placing on 96-well PCR plate, and extracting its genome DNA by rapid alkaline cooking method. The extracted genomic DNA sample is put into a 96-well PCR plate, 50 mu L of 0.25mol/L NaOH solution is added by a pipette, boiled in boiling water for 3min, and gently held during operation to prevent the boiling water from splashing into the sample. The 96-well PCR plate was removed with tweezers, and 150. mu.l of Tris ∙ HCl (pH 8.0) was added thereto at a concentration of 0.1mol/L to neutralize the reaction. The DNA extracted by the method is easy to degrade, and can be stored for 4 days at the maximum in a refrigerator at 4 ℃.
(2) PCR amplification
The genomic DNA extracted in step (1) was used as a template, and the primer pair SF32 and SR32 described in example 1 was used to perform rapid amplification using a simplified PCR amplification system. A10-microliter system is adopted in PCR amplification reaction, and the specific ratio of each reagent is as follows: 10 ng/. mu.l DNA template about 0.5. mu.l, 10 XPCR Buffer 1. mu.l, 10mmol/l dNTP 0.1. mu.l, 20pmol/l SSR upstream and downstream primers 0.1. mu.l, 2.5U/. mu.l rTaq 0.1. mu.l, add sterile ddH2O to total volume 10. mu.l. The PCR reaction program is: pre-denaturation at 94 ℃ for 4min, denaturation at 94 ℃ for 30s, annealing at 52.5 ℃ for 30s, extension at 72 ℃ for 30s, 25 cycles total, and final extension at 72 ℃ for 3 min. After the PCR is finished, 2. mu.l of 6 Xsample-adding buffer solution is added into 10. mu.l of the reaction system, and the mixture is mixed evenly for electrophoresis.
(3) Gel electrophoresis
And carrying out electrophoresis on the PCR amplification product by adopting 8% polyacrylamide gel, and carrying out silver staining and color development by adopting a simplified silver staining step. The specific operation steps are as follows:
A. and (3) washing the glass plate under clean water by using a cleaning solution for treating the glass plate and the groove sealing, washing the glass plate by using distilled water for 2-3 times until the glass plate has no fine impurities and is completely dried under a light-transmitting condition, covering the glass plate with a groove, placing the matched glass plate in a plastic parting strip, and carefully aligning the glass plate to enable the glass plate to be tightly matched with the plastic parting strip. Weighing 0.24g of agar powder, adding 30ml of 1 XTBE buffer solution, heating on a microwave oven to completely dissolve, taking a proper amount of agar solution by using a 5ml pipette, adding the agar solution into a gap below a glass plate, enabling the agar solution to permeate into a groove of the glass plate for about 3mm, and standing at room temperature for 20-40 min until the agar is completely solidified. And placing the sealed glass plate in an electrophoresis tank, enabling the plastic strips on the periphery of the glass plate to be tightly matched with the clamping positions of the electrophoresis tank, screwing down the screws, and connecting the anode and the cathode of the electrophoresis tank with an electrophoresis apparatus.
B. Pouring gel, namely, slightly shaking up the prepared 8% PAGE (polyacrylamide) gel, slowly pouring the gel into a glass gel chamber, inserting a sample comb after the gel is filled in the whole gel chamber, and polymerizing for 90min to 1h at room temperature. After the gel was completely polymerized, 0.5 × TAE running buffer was added and the comb was gently pulled out.
C. Sample application and electrophoresis 5. mu.l of 6 Xsample application buffer solution is added into each PCR product, the mixture is mixed, 3 to 4. mu.l of sample is added into each sample application hole, and standard molecular weight DNA is set as a fragment size standard. And (4) carrying out constant temperature electrophoresis at 180V for about 90min, and stopping electrophoresis when the indicator (6 times xylene in the sample adding buffer solution) approaches the bottom of the gel plate.
D. After dyeing and color development electrophoresis, separating two glass plates, placing the gel in a special basin, adding 500ml of distilled water for cleaning for 2-3 times, placing the gel in a dyeing solution, dyeing for 5min, taking out the gel plate, and rinsing with distilled water for 2-3 times. And (3) placing the rubber plate into 400ml of developing solution, slightly shaking, quickly rinsing with distilled water for 2-5 times after strips are clear, placing the rubber plate on a film observation lamp, and taking a picture for recording.
(4) Statistical analysis
And (4) carrying out statistical analysis on the electrophoresis result, wherein the single plant with the parent specificity band is a true hybrid, and the single plant lacking any one band is a false hybrid. According to the formula: SSR purity (total number of assays-number of individuals of non-hybrid band type)/total number of assays × 100%, hybrid purity was calculated.
Example 3 verification of the primers for identifying purity of Zaojia Luffa hybrid
(1) By adopting the method of the embodiment 2 and utilizing the specific primers of the embodiment 1, the seedling stage samples of the early and good towel gourds planted in the fields of 100 base plants are detected, 1 hybrid plant is found, the purity is 99%, and the identification result is matched with the field identification result. FIG. 2 is a diagram of a purity identification gel of a part of early-good loofah hybrid seeds, in which lane 1 is 50bp marker, lane 2 is female parent, lane 3 is male parent, and the rest lanes are samples to be detected.
(2) The method of example 2 is adopted to sample the seeds of the Hunan bimodal seed production base, the purity identification is carried out, 190 plants germinate, and the DNA sample is extracted by a simplified method for detection, wherein the detection results are all true hybrid seeds, the seed purity is 100 percent, the detection results are consistent with the field investigation results, and the accuracy is 100 percent, as shown in figure 3.
(3) The method of example 2 is adopted to sample the seeds in Xinjiang seed production base, purity identification is carried out, 190 plants germinate, DNA samples are extracted by a simplified method for detection, and the detection result shows that 188 plants are hybrid seeds, 2 plants are female parent banding patterns, the seed purity is 98.95 percent, the method is consistent with the field investigation result, the accuracy is 100 percent, and is shown in figure 4.
The above examples show that the method of the present invention can rapidly and effectively identify the early-quality luffa hybrid seed and the male and female parent seeds.
Sequence listing
<110> vegetable research institute of Hunan province
<120> SSR primers and method for purity identification of early-excellent towel gourd hybrid seeds
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
catcaccacc accacaacc 19
<210> 2
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
gccaccaaaa tcagctctat g 21

Claims (8)

1. The SSR primer for identifying the purity of the early-excellent luffa hybrid seed is characterized by comprising an upstream primer SF32 and a downstream primer SR32, wherein the nucleotide sequence of the upstream primer SF32 is shown as SEQ ID NO. 1, and the nucleotide sequence of the downstream primer SR32 is shown as SEQ ID NO. 2.
2. The method for identifying the purity of the Zaojia towel gourd hybrid seeds is characterized by comprising the following steps of:
(1) extracting genome DNA of the Zaojia luffa parent and filial generation;
(2) performing PCR amplification by using the genomic DNA extracted in the step (1) as a template and using the primers SF32 and SR32 of claim 1 to obtain an amplification product;
(3) detecting the amplification product in the step (2) by gel electrophoresis, developing, and counting the electrophoresis result;
(4) analyzing the electrophoresis result in the step (3), wherein the single plant with the parental specificity strip is a hybrid, and the single plant lacking any one strip is a false hybrid.
3. The method for identifying the purity of the Zaojia luffa hybrid seed according to claim 2, wherein the method for extracting the genomic DNA in the step (1) is specifically: the sample to be extracted was put on a PCR plate, 50. mu.l of a 0.25mol/L NaOH solution was added thereto, boiled in boiling water for 3min, the PCR plate was taken out with tweezers, and 150. mu.l of Tris ∙ HCl (0.1 mol/L pH 8.0) was added thereto to neutralize the reaction.
4. The method for identifying the purity of the hybrid seeds of the zao jia luffa as claimed in claim 2, wherein the PCR reaction system in the step (2) is 10 μ l, wherein: 0.5. mu.l of 10 ng/. mu.l of genomic DNA, 1. mu.l of 10 XPCR Buffer, 0.1. mu.l of 10mmol/l dNTP, 0.1. mu.l of 20pmol/l SSR upstream and downstream primers, 0.1. mu.l of 2.5U/. mu.l rTaq DNADNA polymerase, and sterilized ddH2O to a total volume of 10. mu.l.
5. The method for identifying the purity of the hybrid seeds of the early-quality luffa as claimed in claim 2 or 4, wherein the PCR reaction procedure in step (2) is as follows: pre-denaturation at 94 ℃ for 4min, denaturation at 94 ℃ for 30s, annealing at 52.5 ℃ for 30s, extension at 72 ℃ for 30s, 25 cycles total, and final extension at 72 ℃ for 3 min.
6. The method for identifying the purity of the Zaojia luffa hybrid seed according to claim 2, wherein the gel electrophoresis in step (3) is 8% native polyacrylamide gel electrophoresis.
7. The method for identifying the purity of the Zaojia luffa hybrid seed according to claim 2, wherein the color development in the step (3) is specifically: after electrophoresis is finished, separating two glass plates, putting gel into a special basin, adding distilled water for cleaning for 2-3 times, putting the gel into dyeing liquid, taking out the gel plate after dyeing is finished, and rinsing the gel plate for 2-3 times by using distilled water; and (3) placing the rubber plate into a developing solution, slightly shaking, quickly rinsing for 2-5 times by using distilled water after strips are clear, placing the rubber plate on a film observation lamp, and taking a picture for recording.
8. The use of SSR primers SF32 and SR32 in the purity identification of early-quality Luffa hybrid and molecular assisted breeding according to claim 1.
CN202010601289.8A 2020-06-29 2020-06-29 SSR primer and method for identifying purity of early-optimal luffa hybrid seeds Active CN111961740B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202010601289.8A CN111961740B (en) 2020-06-29 2020-06-29 SSR primer and method for identifying purity of early-optimal luffa hybrid seeds

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202010601289.8A CN111961740B (en) 2020-06-29 2020-06-29 SSR primer and method for identifying purity of early-optimal luffa hybrid seeds

Publications (2)

Publication Number Publication Date
CN111961740A true CN111961740A (en) 2020-11-20
CN111961740B CN111961740B (en) 2024-03-26

Family

ID=73360902

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202010601289.8A Active CN111961740B (en) 2020-06-29 2020-06-29 SSR primer and method for identifying purity of early-optimal luffa hybrid seeds

Country Status (1)

Country Link
CN (1) CN111961740B (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114457184A (en) * 2022-02-21 2022-05-10 广东省农业科学院蔬菜研究所 SNP molecular marker and KASP primer for identifying color of luffa seed coat and application thereof
CN118441086A (en) * 2024-05-15 2024-08-06 湖南省蔬菜研究所 Molecular marker linked with browning resistance of luffa fruits and application thereof

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104004833A (en) * 2014-05-09 2014-08-27 广东省农业科学院蔬菜研究所 EST-SSR core primer group developed on basis of transcriptome sequence of towel gourd and application thereof
CN105359777A (en) * 2015-10-19 2016-03-02 西藏自治区农牧科学院蔬菜研究所 Cultivation method for year-circle production of loofahs in plateau area
CN105755117A (en) * 2016-03-03 2016-07-13 广东省农业科学院蔬菜研究所 Nucleotide primer composition for quickly identifying purity of 'Yalv No.6' hybrid seeds of luffa acutangula roxb and detection method
CN105821154A (en) * 2016-06-03 2016-08-03 福建省农业科学院作物研究所 SSR primers and method for purity identification of luffa hybrid seeds
CN107312868A (en) * 2017-08-30 2017-11-03 福建省农业科学院作物研究所 The SSR primer sets developed based on american pumpkin transcript profile sequence and its application
CN109439783A (en) * 2018-07-17 2019-03-08 江苏绿港现代农业发展有限公司 A kind of SSR primer and method for sweet 25 Purities of muskmelon cenospecies gold

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104004833A (en) * 2014-05-09 2014-08-27 广东省农业科学院蔬菜研究所 EST-SSR core primer group developed on basis of transcriptome sequence of towel gourd and application thereof
CN105359777A (en) * 2015-10-19 2016-03-02 西藏自治区农牧科学院蔬菜研究所 Cultivation method for year-circle production of loofahs in plateau area
CN105755117A (en) * 2016-03-03 2016-07-13 广东省农业科学院蔬菜研究所 Nucleotide primer composition for quickly identifying purity of 'Yalv No.6' hybrid seeds of luffa acutangula roxb and detection method
CN105821154A (en) * 2016-06-03 2016-08-03 福建省农业科学院作物研究所 SSR primers and method for purity identification of luffa hybrid seeds
CN107312868A (en) * 2017-08-30 2017-11-03 福建省农业科学院作物研究所 The SSR primer sets developed based on american pumpkin transcript profile sequence and its application
CN109439783A (en) * 2018-07-17 2019-03-08 江苏绿港现代农业发展有限公司 A kind of SSR primer and method for sweet 25 Purities of muskmelon cenospecies gold

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
HAIBIN WU 等: "Genetic Linkage Map Construction and QTL Analysis of Two Interspecific Reproductive Isolation Traits in Sponge Gourd", 《FRONTIERSINPLANTSCIENCE》, vol. 7, pages 1 - 11 *
朱海生 等: "丝瓜转录组SSR位点分析及其分子标记开发", 《中国细胞生物学学报》, vol. 38, no. 9, pages 1118 *
王莎: "瓠瓜SSR和InDel分子标记的开发及应用", 《中国优秀硕士学位论文全文数据库 农业科技辑》, no. 03, pages 19 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114457184A (en) * 2022-02-21 2022-05-10 广东省农业科学院蔬菜研究所 SNP molecular marker and KASP primer for identifying color of luffa seed coat and application thereof
CN118441086A (en) * 2024-05-15 2024-08-06 湖南省蔬菜研究所 Molecular marker linked with browning resistance of luffa fruits and application thereof

Also Published As

Publication number Publication date
CN111961740B (en) 2024-03-26

Similar Documents

Publication Publication Date Title
CN105154550B (en) A kind of method using 1217 purity of EST-SSR molecular labeling Rapid identification variety of watermelon treasure sweet tea
CN111961740B (en) SSR primer and method for identifying purity of early-optimal luffa hybrid seeds
CN107058529B (en) Method for breeding durable resistance material of wheat stripe rust
CN109652587B (en) Method for identifying holly germplasm by using SSR molecular marker of transcriptome sequencing
Meiners et al. Analysis of the taxonomic subdivision within the genus Helleborus by nuclear DNA content and genome-wide DNA markers
CN107447012B (en) Identification of hybrid F between cultivated eggplant and wild eggplant by SSR molecular marker1Method of hybridization
CN106967797B (en) Specific sequence for detecting purity of melon seeds, detection method and application
CN103798132B (en) A kind of method of scale initiative wheat alien translocation line
CN114525364B (en) Molecular marker AhyWptc tightly linked with peanut white seed coat and application thereof
CN104928396A (en) Method for rapidly identifying hot pepper species and golden pepper purity degree by using EST-SSR molecular markers
CN107058518B (en) SSR molecular marker closely linked with sesame stem blight-resistant major gene locus and application thereof
CN107400703B (en) Molecular marker linked with major QTL qFSW-2 and qFSW-5 of corn seed storage tolerance and application thereof
CN107164539B (en) SSR molecular marker for identifying purity of pepper variety XingShu Lvyan hybrid and method thereof
CN113862392B (en) SSR molecular marker primer linked with Chinese cabbage yellow cotyledon gene Bryc and application thereof
CN112725521B (en) Dendrobium chrysotoxum SSR molecular marker primer composition and application thereof
CN108410967B (en) A kind of method of Rapid identification river camellia tradition famous-object
CN115109864A (en) SSR molecular marker E201 for identifying Chinese pumpkin &#39;Zhongchuanu No. 1&#39; hybrid, and primer, kit and method thereof
CN111349716B (en) SSR primer and method for identifying purity of Yun radish No. 2 hybrid seeds
CN109628633B (en) Method for rapidly identifying Carex assicata and Carex brachypodium by utilizing SSR molecular markers
CN109811085B (en) Primer for identifying purity of autumn-sown Chinese cabbage Zhenlun No. 6 seeds and application of primer
CN109825637B (en) Primer for identifying purity of Saijing 316 seeds of non-heading Chinese cabbage and application
CN106755387A (en) A kind of utilization molecular labeling Rapid identification cucumber stock is made a concerted effort the method for two purity
CN101974516A (en) Method for discriminating Chinese milk vetch variety by using SSR (Simple Sequence Repeat)fingerprint spectrum
CN105695587A (en) SSR molecular marker primer set for identifying purity of variety Shandong cotton research number 34 and application thereof
CN112662798B (en) InDel molecular marker for identifying seed purity of muskmelon floral bovine variety and application thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant