CN113151559B - Method for identifying purity of muskmelon hybrid seeds - Google Patents

Abstract

The invention discloses a method for identifying the purity of muskmelon hybrid seeds, which solves the technical problems that the traditional seed purity identification period is long, the influence of human factors is large, and the market rapid and accurate identification needs are difficult to meet. The invention comprehensively considers multiple factors such as SSR sites, screens and obtains the EST-SSR primer SSR8, amplifies sample DNA to be tested by using the EST-SSR primer SSR8, obtains a band spectrum by gel electrophoresis, compares the band spectrum with bands of male parent and female parent spectra, and simultaneously has the specific bands of the male parent and the female parent which are true hybrid varieties, and is a false hybrid variety lacking any characteristic band in the two parents or being a non-parent specific band. The method can effectively distinguish parents and hybrid seeds of the Xuetong No. 8, and can rapidly and accurately identify the seed purity.

Description

Method for identifying purity of muskmelon hybrid seeds

Technical Field

The invention relates to the technical field of seed identification, in particular to a method for identifying the purity of muskmelon hybrid seeds.

Background

Seed is the primary material foundation for agricultural production, seed purity is the core index for measuring seed quality, and is the key factor for guaranteeing stable yield and increasing yield of good varieties.

The melon fruits are rich in nutrition and fragrant and sweet in taste, and are one of the very favorite fruits of the consumers. In recent years, along with the wide popularization and application of melon hybrid seeds, a large number of new melon varieties are introduced into the market, so that the authenticity of the varieties is difficult to guarantee, the problems caused by the purity of seeds occur at times, and the healthy development of melon industry is seriously influenced; therefore, the purity of the hybrid seeds is more and more paid attention to seed enterprises and melon farmers.

The Xuetong No. 8 is a hybrid thick-skin melon variety bred by a gardening institute of the agricultural academy of sciences in Henan province, the full growth period is about 104 days, the fruit development period is 28-35 days, the fruit is high and round, the peel is white, the fruit is medium and medium, the average single melon weight is 1.60-1.85 and kg, the pulp is 3.4-3.8 and cm, the pulp is light orange, the content of soluble solid in the center is more than 16.5%, the pulp is thin and crisp, the quality is excellent, the fruit is not fallen to the pedicel after being ripe, the commodity is good, the storage and transportation are durable, and the cultivation in a protected area is suitable; is popular once pushed out. The hybrid melon seed production needs artificial supplementary pollination, and adopts artificial emasculation, female flower and male flower bagging. In the process of seed production, the female parent is not thoroughly emasculated, the isolation measures are improper or the mechanical mixing is possible to cause the impurity of hybrid seeds.

The current traditional seed purity identification method is based on field morphology observation of plants after sowing, has long identification period, is greatly influenced by human factors, is unfavorable for timely selling of seeds, and is difficult to meet the identification requirement of the market. Therefore, the development of an accurate and efficient hybrid seed purity identification method has become a common concern for scientific research institutions and seed enterprises.

Disclosure of Invention

The invention aims to provide a method for identifying the purity of muskmelon hybrid seeds suitable for snow-red 8, which aims to solve the technical problems that the traditional method for identifying the purity of the seeds is long in period, greatly influenced by human factors and difficult to meet the requirements of fast and accurately identifying the purity of the hybrid seeds in the market.

In order to solve the technical problems, the invention adopts the following technical scheme:

designing a method for identifying the purity of muskmelon hybrid seeds, which comprises the following steps:

(1) Accelerating germination of seeds: soaking melon seeds in water at normal temperature for 4-6 hours, taking out, rubbing seed coat mucus, draining, wrapping with wet gauze, dark culturing at 28 ℃, sowing in a cave dish after sprouting, and growing into two leaves and one heart seedling for later use;

(2) DNA extraction: taking young leaves of the single seedling obtained in the previous step, and extracting DNA by using an SDS method;

(3) And (3) PCR amplification: the DNA of the sample to be tested was amplified using the following EST-SSR primers:

SSR forward primer: 5'-ATTGGTGAAACCTGAGATGG-3';

SSR reverse primer: 5'-TAAGGATGATTTCGGGTGTC-3';

(4) Gel electrophoresis detection: performing polyacrylamide gel electrophoresis detection on the PCR amplification product obtained in the above step to obtain a corresponding electrophoresis pattern;

(5) Amplification band type comparison: analyzing the obtained electrophoresis patterns, comparing the pattern strips of each hybrid to be tested with the male parent and the female parent, wherein the hybrid is true hybrid with the specific strips of the male parent and the female parent, and the hybrid is false hybrid without any characteristic strip in the two parents or with the specific strip of the non-parent;

(6) Purity calculation: the number of the hybrid seeds with the specific band type of the variety accounts for the percentage of the number of the seeds of the detection sample variety, and the calculation formula is as follows:

variety purity = number of hybrid seeds/number of test sample seeds x 100%.

In the step (2), 80-100mg of young leaves of the single seedling are taken, and the young leaves are placed in a centrifuge tube after vein removal; adding 1mLDNA extracting solution into each tube, putting into a plant tissue crusher, crushing for 2-4 min, and putting into a shaking table for slowly shaking for 15-25 min after crushing; centrifuging for 8-12 min at 11000-13000 r/min, transferring 600-700 mu L of supernatant to a centrifuge tube, adding isopropanol to 1.5mL scale, shaking slightly for 3-5min, centrifuging for 25-35 s at 11000-13000 r/min, discarding supernatant, adding 70% ethanol, centrifuging for 4-6 min at 11000-13000 r/min, retaining precipitate, adding 60 mu M LRNA enzyme water into the centrifuge tube to dissolve DNA and mixing uniformly when the residual solution is evaporated completely.

In said step (3), the one involved

PCR reaction system: 2X Taq Plus Master Mix 6 mu m L, SSR forward primer 0.4 mu m L, SSR reverse primer 0.4 mu m L, sample DNA 1.0 mu m L, and sterilized double distilled water 7.2 mu m L;

PCR reaction procedure: pre-denaturation at 94℃for 5min; denaturation at 94℃for 50s, annealing at 55℃for 50s, extension at 72℃for 50s, and cycling for 35 times; extending at 72 ℃ for 7min; preserving at 4 ℃.

In the step (4), it includes:

(1) and (3) glue preparation: taking 30mL of polyacrylamide gel mother solution in a beaker, adding 20mL of 10 XTBE buffer solution, 50mL of double distilled water, 75 [ mu ] L of TEMED and 750 [ mu ] L of 10% ammonium persulfate; pouring glue after the glue is mixed evenly, inserting a comb into a glue pouring opening after the glass plate is filled with the glue, and enabling the gel time to be not less than 1h;

(2) spotting: adding a 3 [ mu ] L6X loading buffer solution into a 15 [ mu ] L system and uniformly mixing; pouring the 1 XTBE buffer solution into an electrophoresis tank, mounting a glue plate, and taking down a comb; and adding 2 mu L of the PCR amplification product into each sample adding hole.

(3) Electrophoresis: the power is 300W, the voltage is 200V, and the current is 1500mA for electrophoresis;

(4) washing the glue: after electrophoresis, the rubber plate is taken down, the rubber is cut along the edge of the glass plate by a graver, the rubber is put into a fixed liquid, the rubber is slowly shaken on a shaking table for 8-12 min, the rubber is dyed in a dyeing liquid for 14-16 min, the rubber is quickly washed for 2-4 times by double distilled water, the rubber is shaken in a developing liquid for 4-6 min until the rubber strips are clear, and the rubber is put on a film observation lamp for observation after being washed by double distilled water.

Compared with the prior art, the invention has the main beneficial technical effects that:

1. the invention identifies the purity of the melon Xuetong No. 8 hybrid seeds based on EST-SSR molecular markers, has the advantages of co-dominance, simple operation, high polymorphism and the like, and can well distinguish the specific bands of male parent, female parent and hybrid seeds.

2. The invention designs the EST-SSR primer based on abundant melon EST sequences (129240 ten thousand melon EST sequences only published by the International cucurbitaceae genome project) and comprehensively considering factors such as SSR sites, GC content, secondary structure, hairpin structure and the like. Through multiple rounds of primer amplification and screening, 1 pair of EST-SSR primers SSR8 suitable for the snow-red No. 8 is obtained, the parents and the hybrid seeds can be effectively distinguished, and the seed purity of the muskmelon hybrid seeds can be rapidly and accurately detected.

3. The DNA extraction method (SDS) of the invention is simple and convenient to operate, and omits the steps of extracting and removing impurities such as protein, polysaccharide, phenols and the like from phenol, chloroform and isoamyl alcohol, thereby greatly saving time and cost.

4. The method removes the time required by germination and seeding, only takes 1-2 days from DNA extraction to obtaining an electropherogram result, is not influenced by environmental conditions, growth and development periods and the like, is simple and easy to implement, is economical and efficient, and has great application value in seed enterprise sales.

Drawings

Fig. 1 is a purity identification electropherogram of a hybrid seed primer SSR8 of Xuetong 8; wherein M:100bp Ladder; p1: father (Xuetong No. 8 father); p2: female parent (Xuetong No. 8 female parent); 1-20: hybrid (Xuetong No. 8); 21: false hybrids consistent with male parent bands.

Detailed Description

The following examples are given to illustrate the invention in detail, but are not intended to limit the scope of the invention in any way.

The instruments and devices referred to in the following examples are conventional instruments and devices unless otherwise specified; the related reagents and raw materials are all conventional products sold in the market unless specified; the detection, test and preparation methods are all conventional methods unless otherwise specified.

Embodiment one: application of muskmelon hybrid seed purity identification method

EST-SSR primer acquisition

Based on the abundant melon EST sequences published by GenBank database and the genome project of the cucurbitaceae, factors such as SSR sites, GC content, primer space structures and the like (for example, the starting position and the ending position of the SSR sequences are respectively at least 20bp away from the 5 'end and the 3' end, the primer length is 18-25 bp, the Tm value of the primer annealing temperature is 50-65 ℃, the Tm value of the upstream primer and the downstream primer is not more than 5 ℃, the GC content is 40-60%, the PCR amplification product is 100-350 bp, the secondary structure and the hairpin structure of the primers are avoided as much as possible, and the like), and a series of EST-SSR primers are designed independently; through primer amplification screening, the stable EST-SSR primer SSR8 (shown as SEQ ID NO. 1-2) suitable for the snow-red No. 8 is obtained, and can be used for identifying the purity of the hybrid seeds:

forward primer: 5'-ATTGGTGAAACCTGAGATGG-3';

reverse primer: 5'-TAAGGATGATTTCGGGTGTC-3'.

The primer was synthesized by the biosciences of gold sri.

Identification and verification of Xuetong No. 8 hybrid variety

1. DNA extraction

And (3) taking Xuetong No. 8 hybrid seeds, sprouting and sowing until two leaves and one heart are grown, taking 60 strains of the tested variety, and carrying out DNA extraction on 2 strains of each of the male parent and the female parent by using an SDS method. Taking 80-100mg of young leaves of a single seedling, removing veins, putting the young leaves into a 2mL centrifuge tube, and putting into a steel ball. 1ml of the extract of LDNA (10% SDS,200mmol/L NaCl,50mmol/L EDTA-Na) was added to each tube ₂ 200mmol/L Tris-HCl), placing into a plant crusher for crushing for 3min, placing on a shaking table for slowly shaking for 20min after crushing, and then taking out the steel balls. Centrifuging at 12000r/min for 10min, sucking 600-700 [ mu ] L of supernatant, transferring to a 1.5mL centrifuge tube, adding isopropanol to 1.5mL scale, and slightly shaking for 3-5min. Centrifuging at 12000r/min for 30s, discarding supernatant, adding a little 70% ethanol, centrifuging at 12000r/min for 5min, and retaining precipitate. And after the residual solution is completely evaporated, adding 60 mu LRNA enzyme water into the centrifuge tube to dissolve DNA, and uniformly mixing.

2. PCR amplification

PCR reaction system: 2X Taq Plus Master Mix 6 mu L, SSR forward primer 0.4 mu L, SSR reverse primer 0.4 mu L, sample DNA 1.0 mu L and sterilized double distilled water 7.2 mu L. The whole reaction system is 15 mu L.

PCR reaction procedure: pre-denaturation at 94℃for 5min; denaturation at 94℃for 50s, annealing at 55℃for 50s, extension at 72℃for 50s, and cycling for 35 times; extending at 72 ℃ for 7min; preserving at 4 ℃. Plus Master Mix was purchased from BioTeke corporation.

3. Gel electrophoresis detection and gel washing

(1) And (3) glue preparation: repeatedly scrubbing the glass plate with a scrubber, flushing with double distilled water, assembling after thoroughly drying, aligning and clamping the two sides with a clamp, and horizontally placing the glass plate with the groove on a laboratory bench. 30mL of polyacrylamide gel mother liquor (290 g of acrylamide is weighed in a 1000mL beaker, 10g of N, N-bis is weighed and embedded in the acrylamide, double distilled water is firstly added to about 800mL, the mixture is placed in a 65 ℃ water bath for heating until the mixture is dissolved, the volume is fixed to 1000 mL), 10 XTBE buffer (108 g of Tris, 7.44g of EDTA-Na) is added in the beaker ₂ And respectively adding a proper amount of distilled water into 55g of boric acid in a beaker for dissolution, pouring the three solutions into a volumetric flask, and fixing the volume to 1000 mL) 20mL, 50mL of double distilled water, 75 mu L of TEMED and 750 mu L of 10% Ammonium Persulfate (APS). And (3) pouring glue after rapid and uniform mixing, lightly pouring the glue along a glue pouring opening at a uniform speed to prevent bubbles, and inserting a comb into the glue pouring opening after the glue is filled with the glass plate, wherein the gel time is not less than 1h.

(2) Spotting: and adding 3 mu L of 6 Xloading buffer solution into the 15 mu L system and uniformly mixing. Pouring the 1 XTBE buffer solution into an electrophoresis tank, mounting a glue plate, and taking down a comb; 2 mu L of PCR amplification products are added to each sample adding hole.

(3) Electrophoresis: the electrophoresis was performed at 300W, 200V and 1500mA current, and the electrophoresis was performed until the upper indicator tape reached the middle lower part of the gel plate for about 2h20min.

(4) Washing the glue: after electrophoresis, the gel plate was removed, the gel was cut along the edge of the glass plate with a nicking tool, the gel was put into a fixing solution (450 mL double distilled water, 50mL absolute ethyl alcohol and 2.5mL glacial acetic acid were added, mixed well), slowly shaken on a shaker for 10min, stained in a staining solution (1 g silver nitrate was dissolved in 500mL double distilled water) for 15min (note to block light), quickly washed 3 times with double distilled water, and shaken in a developing solution (7.5 g NaOH was dissolved in 500mL double distilled water, after dissolution was completed, 1.5mL formaldehyde was added until the bands were clear, washed with double distilled water, and then placed on a film observation lamp for observation.

4. Amplification band analysis: analyzing the electropherogram, comparing the male parent, the female parent and the map strips of the hybrid, wherein the hybrid is a true hybrid with the specific strips of the male parent and the female parent, and the hybrid is a false hybrid without any characteristic strip of the two parents or with the specific strip of the non-parent. The invention obtains 1 pair of primer SSR8 through screening, which can identify the purity of the muskmelon hybrid, the hybrid band is the complementary band of male parent and female parent (see figure 1), and the electrophoresis pattern is easy to observe and identify.

5. Purity calculation: the DNA of the test sample is amplified by EST-SSR primer, and the percentage of the number of the hybrid seeds with the specific band of the variety to the number of the seeds of the test sample represents the purity. The banding pattern of one of the 60 test samples was identical to that of the male parent, and was a false hybrid, and the seed purity of the test sample was calculated to be=59/60×100% =98.3%. The embodiment shows that the primer SSR8 designed and screened by the invention can effectively distinguish parent and hybrid seeds and can rapidly and accurately detect seed purity. The DNA extraction method is simple and convenient, omits the steps of extracting and removing impurities such as protein, polysaccharide, phenols and the like from phenol, chloroform and isoamyl alcohol, and saves time and cost. The method removes the time required by germination and seeding, only needs 1-2 days from DNA extraction to obtaining an electropherogram result, is not influenced by environmental conditions, growth and development periods and the like, is simple and easy to implement, is economical and efficient, and has great application value in seed enterprise sales.

While the invention has been described in detail with reference to the drawings and embodiments, those skilled in the art will understand that various specific parameters in the embodiments may be changed or equivalents may be substituted for related methods, steps and materials without departing from the spirit of the invention, so as to form a plurality of specific embodiments, which are common variations of the invention and will not be described in detail herein.

SEQUENCE LISTING

<110> institute of gardening at the academy of agricultural sciences in Henan province

<120> method for identifying purity of hybrid seed of muskmelon

<130> /

<160> 2

<170> PatentIn version 3.2

<210> 1

<211> 20

<212> DNA

<213> Artificial design

<400> 1

attggtgaaa cctgagatgg 20

<210> 2

<211> 20

<212> DNA

<213> Artificial design

<400> 2

taaggatgat ttcgggtgtc 20

Claims (4)

1. The method for identifying the purity of the muskmelon hybrid seeds suitable for Xuetong No. 8 is characterized by comprising the following steps:

(1) Accelerating germination of seeds: soaking melon seeds in water at normal temperature for 4-6 hours, taking out, rubbing seed coat mucus, draining, wrapping with wet gauze, dark culturing at 25-29 ℃, sprouting, sowing in a cave dish, and growing into two leaves and one seedling for later use;

(2) DNA extraction: taking young leaves of the single seedling obtained in the previous step, and extracting DNA by using an SDS method;

(3) And (3) PCR amplification: the DNA of the sample to be tested was amplified using the following EST-SSR primers:

SSR forward primer: 5'-ATTGGTGAAACCTGAGATGG-3';

SSR reverse primer: 5'-TAAGGATGATTTCGGGTGTC-3';

(4) Gel electrophoresis detection: performing polyacrylamide gel electrophoresis detection on the PCR amplification product obtained in the above step to obtain a corresponding electrophoresis pattern;

(5) Amplification band type comparison: analyzing the obtained electrophoresis patterns, comparing the pattern strips of each hybrid to be tested with the male parent and the female parent, wherein the hybrid is true hybrid with the specific strips of the male parent and the female parent, and the hybrid is false hybrid without any characteristic strip in the two parents or with the specific strip of the non-parent;

(6) Purity calculation: the number of the hybrid seeds with the specific band type of the variety accounts for the percentage of the number of the seeds of the detection sample variety, and the calculation formula is as follows:

variety purity = number of hybrid seeds/number of test sample seeds x 100%.

2. The method for identifying the purity of hybrid seeds of muskmelon according to claim 1, wherein in the step (2), 80-100mg of young leaves of single seedlings are taken, and the young leaves are placed in a centrifuge tube after removing veins; adding 1mLDNA extracting solution into each tube, putting into a plant tissue crusher, crushing for 2-4 min, and putting into a shaking table for slowly shaking for 15-25 min after crushing; centrifuging for 8-12 min at 11000-13000 r/min, transferring 600-700 mu L of supernatant to a centrifuge tube, adding isopropanol to 1.5mL scale, shaking slightly for 3-5min, centrifuging for 25-35 s at 11000-13000 r/min, discarding supernatant, adding 70% ethanol, centrifuging for 4-6 min at 11000-13000 r/min, retaining precipitate, adding 60 mu M LRNA enzyme water into the centrifuge tube to dissolve DNA and mixing uniformly when the residual solution is evaporated completely.

3. The method for identifying the purity of hybrid seeds of muskmelon according to claim 1, wherein in said step (3), the reference is made to

PCR reaction system: 2X Taq Plus Master Mix 6 mu m L, SSR forward primer 0.4 mu m L, SSR reverse primer 0.4 mu m L, sample DNA 1.0 mu m L, and sterilized double distilled water 7.2 mu m L;

PCR reaction procedure: pre-denaturation at 94℃for 5min; denaturation at 94℃for 50s, annealing at 55℃for 50s, extension at 72℃for 50s, and cycling for 35 times; extending at 72 ℃ for 7min; preserving at 4 ℃.

4. The method for identifying the purity of hybrid seeds of muskmelon according to claim 1, wherein in the step (4), it comprises:

(1) and (3) glue preparation: taking 30mL of polyacrylamide gel mother solution in a beaker, adding 20mL of 10 XTBE buffer solution, 50mL of double distilled water, 75 [ mu ] L of TEMED and 750 [ mu ] L of 10% ammonium persulfate; pouring glue after the glue is mixed evenly, inserting a comb into a glue pouring opening after the glass plate is filled with the glue, and enabling the gel time to be not less than 1h;

(2) spotting: adding 3 [ mu ] L of 6 Xloading buffer solution into a 15 [ mu ] L system, uniformly mixing, pouring the 1 XTBE buffer solution into an electrophoresis tank, mounting a rubber plate, and taking down a comb; adding 2 mu L of PCR amplification products obtained in the previous step into each sample adding hole;

(3) electrophoresis: electrophoresis is carried out with power of 300W, voltage of 200V and current of 1500 mA;

(4) washing the glue: after electrophoresis, the rubber plate is taken down, the rubber is cut along the edge of the glass plate by a graver, the rubber is put into a fixed liquid, the rubber is slowly shaken on a shaking table for 8-12 min, the rubber is dyed in a dyeing liquid for 14-16 min, the rubber is quickly washed for 2-4 times by double distilled water, the rubber is shaken in a developing liquid for 4-6 min until the rubber strips are clear, and the rubber is put on a film observation lamp for observation after being washed by double distilled water.

Images