CN111961740B - SSR primer and method for identifying purity of early-optimal luffa hybrid seeds - Google Patents

SSR primer and method for identifying purity of early-optimal luffa hybrid seeds Download PDF

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CN111961740B
CN111961740B CN202010601289.8A CN202010601289A CN111961740B CN 111961740 B CN111961740 B CN 111961740B CN 202010601289 A CN202010601289 A CN 202010601289A CN 111961740 B CN111961740 B CN 111961740B
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韩小霞
胡新军
李勇奇
闵子扬
袁祖华
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Hunan vegetable research institute
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Abstract

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

Description

SSR primer and method for identifying purity of early-optimal luffa hybrid seeds
Technical Field
The invention belongs to the technical field of molecular detection, and particularly relates to an SSR primer and a method for identifying purity of early-maturing luffa hybrid seeds.
Background
Loofah is a cultivar of the genus luffa in the Cucurbitaceae (cuurbitaceae), an annual climbing herb. There are two kinds of cultivated Luffa in our country in botanicals, namely, ordinary Luffa cylindrica (l.) m.j.roem.) and ribbed Luffa acutangula (l.) roxb. The early-maturing Luffa belongs to the meat Luffa of the common Luffa cylindrica (l.) m.j.roem.). In the process of producing the hybrid seeds of the luffa, a female parent emasculation mode is mainly adopted. Namely, the male parent and the female parent are respectively sowed according to the growth period, and all male flowers and buds of the female parent are manually removed before the female flowers and the male parent bloom. Pollen of the male parent is collected in the flowering period and pollinated to the column heads of female flowers of the female parent, so that the hybrid is obtained. Because the male flowers of the luffa are infinite cotton, the manual emasculation is easy and incomplete. If the female parent is not completely emasculated, pollen from the female parent may fall or be transmitted by insects to its own stigma, thereby producing false hybrids, which are a major factor in the production resulting in reduced seed purity. According to the relevant regulations of national standards of the people's republic of China (GB 8079-87), the purity of the luffa hybrid must reach 90% to be marketable.
The traditional towel gourd hybrid purity detection mainly adopts morphological identification, and is mainly based on the morphological identification of seeds and ovaries of female parent and hybrid, but the method has the defects of long period, large occupied area, large environmental influence and the like, and the practicability is strong, but the identification time is about 60 days. At present, molecular marker identification of hybrid purity has the advantages of good stability, large information quantity and the like, and is widely applied to crop seed purity quality detection, as in the prior art, the SSR markers of towel gourd are reported to be used for identifying the purity of the hybrid of the ribbed towel gourd 'YaLv No. 6' and the 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 the hybrid is still identified by the conventional loofah SSR molecular markers: (1) The genome extraction adopts a CTAB method or an SOD method, and the operation steps are complicated and time-consuming; (2) The cost of the PCR reaction system and the program which are not optimized is wasted; (3) And the silver dyeing step is complicated in dyeing and color development link operation, and the result is unstable.
Disclosure of Invention
Aiming at the technical problems, the invention aims to provide an SSR primer and a method for identifying the purity of the hybrid seeds of 'early-quality luffa', which can rapidly and accurately distinguish the hybrid seeds from male parent and female parent and detect the purity of the hybrid seeds.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
firstly, the invention provides an SSR primer for identifying the purity of early-maturing luffa 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 an identification method of the purity of the early-quality luffa hybrid seeds, which comprises the following steps:
(1) Extracting genome DNA of early-optimal luffa parents 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 to obtain an amplification product;
(3) Detecting the amplification product in the step (2) by adopting gel electrophoresis, developing color, and counting electrophoresis results;
(4) Analyzing the electrophoresis result in the step (3), wherein the single plant with the parent specific bands is a real hybrid, and the single plant lacking any band is a false hybrid.
Further, the extraction method of the genomic DNA in the step (1) specifically comprises the following steps: the sample to be extracted was placed in a PCR plate, 50. Mu.l of 0.25mol/L NaOH solution was added, the plate was boiled in boiling water for 3min, the PCR plate was removed with forceps, and 150. Mu.l of Tris. Sub.HCl with pH=8.0 was added for neutralization reaction.
The samples to be extracted include, but are not limited to radicle, hypocotyl, etc., and are suitable for hybrid seeds, germinated plant leaves and all parts.
Further, the PCR reaction system in the step (2) is 10 μl, wherein: 10 ng/. Mu.l of genomic 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 DNA polymerase 0.1. Mu.l, and sterilized ddH were added 2 O 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 extension at 72℃for 3min.
Further, the gel electrophoresis in the step (3) is 8% non-denaturing polyacrylamide gel electrophoresis.
Further, the color development in the step (3) specifically includes: after electrophoresis, separating two glass plates, placing gel in a special basin, adding distilled water to wash for 2-3 times, placing into a dyeing liquid, taking out the gel plate after dyeing, and rinsing with distilled water for 2-3 times; placing the rubber plate into developing solution, slightly shaking, quickly rinsing with distilled water for 2-5 times after the strips are clear, placing the rubber plate on a film observing lamp, and photographing and recording.
Finally, the invention also provides application of the SSR primers SF32 and SR32 in purity identification and molecular auxiliary breeding of early-maturing luffa hybrid.
Compared with the prior art, the invention has the following beneficial effects:
the method can be used for detecting the purity of the early-quality luffa hybrid, can effectively distinguish the hybrid from a male parent and a female parent, and can rapidly detect the purity of the hybrid. The method has the advantages of simplicity, rapidness, accuracy, low cost, simplicity in operation and the like, one person can finish detection of nearly 500 samples in one day, the method can replace the traditional purity identification method of the luffa hybrid seeds, is simple and convenient to operate, saves labor, does not occupy land, has accurate and reliable results, greatly improves identification efficiency, reduces identification cost and has higher commercial application value.
Drawings
FIG. 1 is a diagram showing the screening of the primers for identifying the purity of the early-maturing luffa hybrid.
FIG. 2 is a diagram of a polyacrylamide electrophoresis gel for identifying the purity of the early-maturing luffa hybrid.
FIG. 3 is a sample of hybrid purity identification.
FIG. 4 is a sample of hybrid purity identification.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions in the embodiments of the present invention will be clearly and completely described in the following in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The materials used in the examples below are all commercially available from conventional sources.
Example 1 screening of primers for purity identification of early-maturing Luffa hybrid
According to the SSR primer of the white gourd genome, and taking genomic DNA of the female parent 'S1' and the male parent 'S2' of the early-quality luffa as templates for primer screening, obtaining a mark S32 with co-dominant stripes, wherein corresponding primer pairs are SF32 and SR32 respectively
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 marking tape has clear shape and good repeatability. The PCR amplification of the primer pair S32 can generate a female parent specific marker with the length of about 100bp and a male parent specific marker with the length of about 80bp, as shown in figure 1, lanes 1-3 are early-best luffa female parent, lanes 4-6 are early-best luffa male parent, and lane 7 is a hybrid.
Example 2 early-good luffa hybrid seed purity identification method
In this example, the embryonal axis of germinated seeds was used as a sample for genomic DNA extraction
(1) Early-optimal luffa and rapid extraction of genomic DNA of male parent and female parent seed radicle
About 200 seeds of the early-maturing luffa hybrid to be tested are taken per part, 4-8 seeds of the father and mother are respectively taken, the seeds are soaked for 5 hours, and then the seeds are placed in a germination box filled with filter paper, and the temperature is kept constant at 37 ℃ and the moisture is preserved. About 6-7d of germination, taking the hypocotyl or root tip part of single germinated seed about 0.5 cm-1 cm, placing in 96-well PCR plate, and extracting its genome DNA by rapid alkaline cooking method. The extracted genomic DNA sample was placed in a 96-well PCR plate, and 50. Mu.l of a 0.25mol/L NaOH solution was added with a pipette, and boiled in boiling water for 3min, and the sample was gently held and gently placed during the operation to prevent boiling water from splashing into the sample. The 96-well PCR plate was removed with forceps, and 150. Mu.l of 0.1mol/L Tris-HCl (pH=8.0) was added for neutralization. The DNA extracted by the method is easy to degrade, and can be stored for 4 days at the maximum in a refrigerator at the temperature of 4 ℃.
(2) PCR amplification
Using the genomic DNA extracted in step (1) as a template, rapid amplification was performed using a simplified PCR amplification system using the primer pair SF32 and SR32 described in example 1. The PCR amplification reaction adopts a 10 mul system, and the specific ratio of the reagents is as follows: about 0.5. Mu.l of 10 ng/. Mu.l of DNA template, 1. Mu.l of 10 XPCR Buffer, 0.1. Mu.l of 10mmol/ldNTP, 0.1. Mu.l of 20pmol/l SSR upstream and downstream primers, 0.1. Mu.l of 2.5U/. Mu.l rTaq, and sterilized ddH2O were added to a total volume of 10. Mu.l. The PCR reaction procedure was: 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 extension at 72℃for 3min. After the PCR was completed, 2. Mu.l of 6 Xsample buffer was added to 10. Mu.l of the reaction system, and the mixture was homogenized and electrophoresis was performed.
(3) Gel electrophoresis
The PCR amplified product was electrophoresed on an 8% polyacrylamide gel and silver stained and developed using a simplified silver staining procedure. The specific operation steps are as follows:
A. the glass plate and the groove sealing treatment are washed cleanly under clean water by using a cleaning liquid, and then washed for 2-3 times by using distilled water until no tiny impurities exist on the glass plate under the light transmission condition, and after the glass plate is thoroughly dried, the groove glass plate is covered, the paired glass plates are placed in the plastic parting bead, and the glass plates are carefully aligned, so that the glass plates are closely matched with the plastic parting bead. Weighing 0.24g of agar powder, adding 30ml of 1 XTBE buffer solution, heating on a microwave oven to dissolve completely, taking a proper amount of the agar powder into a gap below a glass plate by using a 5ml liquid transfer device, penetrating the agar powder into a groove of the glass plate by about 3mm, and standing at room temperature for 20-40 min until the agar powder 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 closely matched with the clamping positions of the electrophoresis tank, tightening screws, and connecting the anode and the cathode of the electrophoresis tank with an electrophoresis apparatus.
B. Pouring the gel, namely pouring the prepared 8% PAGE (polyacrylamide) gel into a glass gel chamber slowly after shaking gently, inserting a sample comb after filling the whole gel chamber, and polymerizing for 90min to 1h at room temperature. After the gel was completely polymerized, 0.5 xTAE running buffer was added and the sample comb was gently pulled out.
C. And adding 5 μl of 6 Xsample buffer into each PCR product, mixing, adding 3-4 μl sample into each sample well, and setting standard molecular weight DNA as fragment size standard. And (3) stopping electrophoresis when the indicator (the xylene blue in the 6 Xsample adding buffer solution) approaches the bottom of the gel plate after the electrophoresis is performed at the constant temperature of 180V for about 90 min.
D. After the dyeing and the color development electrophoresis are finished, 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 liquid, dyeing for 5min, taking out the gel plate, and rinsing with distilled water for 2-3 times. Placing the rubber plate into 400ml of developing solution, gently shaking, quickly rinsing with distilled water for 2-5 times after the strips are clear, placing the rubber plate on a film observing lamp, and photographing and recording.
(4) Statistical analysis
And (3) carrying out statistical analysis on electrophoresis results, wherein a single plant with a parent specific band is a true hybrid, and a false hybrid is absent from any band. According to the formula: SSR purity= (total number of assays-number of individuals of non-hybrid band type)/total number of assays x 100%, hybrid purity is calculated.
Example 3 verification of purity identification primers for early-maturing Luffa hybrid
(1) By adopting the method of example 2 and using the specific primer described in example 1, the sample detection of 100 early-best luffa seedling stage in the field planting of the base is carried out, and the purity of 1 hybrid plant is found to be 99%, and the identification result is identical with the field identification result. FIG. 2 is a gel diagram for identifying the purity of partial early-excellent luffa hybrid seeds, wherein a lane 1 is a 50bp marker, a lane 2 is a female parent, a lane 3 is a male parent, and the rest are samples to be detected.
(2) The seeds of the Hunan double-peak seed production base were sampled by the method of example 2, the purity was identified, 190 plants were germinated, the DNA samples were extracted by a simplified method and detected, the detection results were all true hybrids, the seed purity was 100%, the accuracy was 100% consistent with the field investigation result, as shown in FIG. 3.
(3) Seeds of the Xinjiang seed production base are sampled by the method of the example 2, purity identification is carried out, 190 strains are germinated, a DNA sample is extracted by a simplified method for detection, the detection result shows that 188 strains are hybrid seeds, 2 strains are female parent bands, the seed purity is 98.95%, the field investigation result is consistent, and the accuracy is 100%, as shown in figure 4.
The above examples show that the method of the invention can rapidly and effectively identify the hybrid seeds of early-maturing luffa and the parent seeds of early-maturing luffa.
Sequence listing
<110> Hunan province vegetable institute
<120> SSR primer and method for purity identification of early-optimal luffa hybrid seeds
<160> 2
<170> SIPOSequenceListing 1.0
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catcaccacc accacaacc 19
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gccaccaaaa tcagctctat g 21

Claims (7)

1. The method for identifying the purity of the early-maturing luffa hybrid seeds is characterized by comprising the following steps of:
(1) Extracting genome DNA of early-optimal luffa parents and filial generation;
(2) Performing PCR amplification by using the genomic DNA extracted in the step (1) as a template and using SSR primers to obtain an amplification product; the SSR primer 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;
(3) Detecting the amplification product in the step (2) by adopting gel electrophoresis, developing color, and counting electrophoresis results;
(4) Analyzing the electrophoresis result in the step (3), wherein the single plant with the parent specific bands is a hybrid, and the single plant lacking any band is a false hybrid.
2. The method for identifying purity of hybrid seeds of luffa as claimed in claim 1, wherein the method for extracting genomic DNA in step (1) comprises the following steps: placing a sample to be extracted into a PCR plate, adding 50 mu L of 0.25mol/L NaOH solution, boiling for 3min, taking out the PCR plate by forceps, adding 0.1mol/L Tris (hydrogen chloride) with pH=8.0, and carrying out neutralization reaction by 150 mu L of HCl.
3. The method for identifying purity of early-maturing luffa hybrid seeds according to claim 1, wherein the PCR reaction system in the step (2) is 10 μl, and 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 respectively 0.1 mu L, 2.5U/mu L rTaq DNA polymerase 0.1 mu L, and sterilizing ddH 2 O to a total volume of 10 mu L.
4. The method for identifying purity of hybrid seeds of luffa as claimed in claim 1 or 3, 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 for 25 cycles, and extension at 72℃for 3min.
5. The method for identifying purity of hybrid seeds of luffa as claimed in claim 1, wherein the gel electrophoresis in the step (3) is 8% non-denaturing polyacrylamide gel electrophoresis.
6. The method for identifying the purity of hybrid seeds of early-maturing luffa according to claim 1, wherein the color development in the step (3) is specifically: after electrophoresis, separating two glass plates, placing gel in a special basin, adding distilled water to wash for 2-3 times, placing into a dyeing liquid, taking out the gel plate after dyeing, and rinsing with distilled water for 2-3 times; placing the rubber plate into developing solution, slightly shaking, quickly rinsing with distilled water for 2-5 times after the strips are clear, placing the rubber plate on a film observing lamp, and photographing and recording.
The application of the SSR primer in early-optimal luffa hybrid purity identification and molecular assisted breeding is characterized in that the SSR primer comprises an upstream primer SF32 and a downstream primer SR32, 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.
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