Method for early identification of pumpkin fruit type and application thereof

Abstract

The invention belongs to the technical field of crop phenotype identification, and particularly relates to a method for early identification of pumpkin fruit types and application thereof. The invention provides six specific primer pairs with high polymorphism, and the nucleotide sequences of the specific primer pairs are respectively shown as SEQ ID NO. 1 to SEQ ID NO. 12. The method comprises the following steps: carrying out PCR amplification on DNA (deoxyribonucleic acid) of radicles, cotyledons, first new leaves or plant young leaves of pumpkin seeds through specific primers, carrying out agarose electrophoretic separation and EB dyeing, detecting polymorphism on an ultraviolet scenograph, and rapidly identifying long-fruit pumpkins and round-fruit pumpkins in an early stage according to the difference of amplified products on marked polymorphic sites. According to the identification method provided by the invention, the identification result is accurate and reliable and is not influenced by environmental factors, and long fruit type and round fruit type pumpkin single plants can be effectively identified before fruit development, so that the screening process of target single plants is accelerated, and the breeding efficiency is remarkably improved.

Description

Technical Field

[0001] The invention belongs to the technical field of crop phenotype identification, and particularly relates to a method for early identification of pumpkin fruit types and application thereof. Background Art

[0002] Fruit shape is an important trait of melon crops, an important indicator for product classification, grading and evaluation, and an important direction for pumpkin breeding. Field observation of this trait is mainly carried out during the ovary development period or the fruiting period. It takes 2-3 months from sowing to observation. At the same time, pumpkin vines are vigorous, the planting area is large, and the land and labor costs are high. SSR (Simple Sequence Repeat) molecular marker technology is simple to operate and has good repeatability. It has been applied in breeding links such as crop purity identification and trait screening, but there are no reports on early rapid identification methods for long and round fruit types of pumpkin. To this end, it is necessary to develop specific primers with high polymorphism for early rapid identification of pumpkin fruit types to improve the efficiency of pumpkin fruit type identification in breeding, classification, and evaluation processes. Summary of the invention

[0003] The purpose of the present invention is to provide six pairs of specific primers for early identification of pumpkin fruit types, and to improve the efficiency of early pumpkin fruit type detection by using specific primers to perform PCR amplification on pumpkin seed radicle, cotyledon, first new leaf or young leaf DNA. Through the identification method provided by the present invention, the identification result is accurate and reliable and is not affected by environmental factors, and can effectively identify long-fruit type and round-fruit type pumpkin plants before fruit development, thereby accelerating the screening process of target plants and significantly improving breeding efficiency.

[0004] In order to achieve the above object, the present invention provides the following technical solutions:

[0005] First, the present invention provides a primer pair for pumpkin fruit type identification, wherein the primers are a primer pair for identifying pumpkin SSR molecular markers, and the SSR molecular markers are SSR1-SSR6;

[0006] The primer pair sequence information for identifying SSR1-SSR6 are as follows:

[0007] The primer pair for identifying SSR1 consists of a forward primer having a nucleotide sequence as shown in SEQ ID NO.1 and a reverse primer having a nucleotide sequence as shown in SEQ ID NO.2;

[0008] The primer pair for identifying SSR2 consists of a forward primer having a nucleotide sequence as shown in SEQ ID NO.3 and a reverse primer having a nucleotide sequence as shown in SEQ ID NO.4;

[0009] The primer pair for identifying SSR3 consists of a forward primer having a nucleotide sequence as shown in SEQ ID NO.5 and a reverse primer having a nucleotide sequence as shown in SEQ ID NO.6;

[0010] The primer pair for identifying SSR4 consists of a forward primer having a nucleotide sequence as shown in SEQ ID NO.7 and a reverse primer having a nucleotide sequence as shown in SEQ ID NO.8;

[0011] The primer pair for identifying SSR5 consists of a forward primer having a nucleotide sequence as shown in SEQ ID NO.9 and a reverse primer having a nucleotide sequence as shown in SEQ ID NO.10;

[0012] The primer pair for identifying SSR6 consists of a forward primer having a nucleotide sequence as shown in SEQ ID NO.11 and a reverse primer having a nucleotide sequence as shown in SEQ ID NO.12.

[0013] Preferably, the pumpkin fruit shape is elongated and round.

[0014] Secondly, the present invention also provides a kit for early identification of pumpkin fruit types, which contains the specific primer pairs for identifying the SSR1-SSR6 molecular markers described above and reagents for detection.

[0015] Secondly, the present invention provides a method for early identification of pumpkin fruit type, characterized in that the identification method comprises the following steps:

[0016] (1) Sampling: Sample the radicle, cotyledon or leaf of pumpkin seeds and store them in a refrigerator;

[0017] (2) DNA extraction: The total genomic DNA of pumpkin was extracted using the CTAB method;

[0018] (3) PCR amplification: using the DNA extracted in step (2) as a template, the specific primer pairs for identifying the SSR1 to SSR6 molecular markers described in claim 1 are independently mixed and used for PCR amplification;

[0019] (4) PCR product detection: The PCR product amplified in step (3) is subjected to fluorescent staining to observe the result;

[0020] (5) Data collection and analysis.

[0021] Preferably, the radicle or cotyledon of the pumpkin seed in step (1) refers to the part that grows during the germination process of the seed; the leaf is the first new leaf or the DNA of the young leaf of the plant.

[0022] Preferably, the PCR system in step (3) comprises: the total volume of the reaction system is 15 μl, including 1.5 μl dNTP (2.5 mM), 1.5 ul 10× buffer, 1.8 ul MgCl2 (25 mM), 2 ul primer (5 mM), 1.0 ul of each pair of SSR-specific primers, 5 ul DNA (10 ng / ul), 0.05 ul Taq (1 U), and 3.145 ul ultrapure water;

[0023] The PCR amplification program is: pre-deformation at 94°C for 1 min, deformation at 93°C for 1 min, annealing at 50°C for 1 min, extension at 72°C for 2 min (40 cycles); extension at 72°C for 1 min.

[0024] Preferably, the fluorescent staining in step (4) comprises: using 2.5% high-resolution standard gel agarose and adding 20 ul of ethidium bromide for staining.

[0025] Finally, the present invention provides the use of the primer pair or the kit or identification method described above in predicting pumpkin phenotype.

[0026] The present invention provides the application of the primer pair or the kit or identification method described above in identifying or assisting in identifying the purity of pumpkin fruit types.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] 1) The present invention provides six pairs of specific primer pairs for identifying SSR1~SSR6 molecular markers. The primer pairs have high polymorphism and show good polymorphic amplification in PCR amplification identification of long-fruit pumpkin and round-fruit pumpkin, which significantly improves the detection efficiency of long-fruit and round-fruit individual plants.

[0029] 2) Compared with conventional morphological identification, the identification method provided by the present invention can more accurately identify pumpkin fruit type at any stage of seeds, seedlings and plants, without waiting for ovary development or fruiting to carry out identification work. The results are reliable and not affected by the environment, saving manpower, material resources and land materials, and improving identification efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0031] Figure 1The schematic diagram is the amplification of SSR1 specific primers in 15 "long-fruit pumpkin" plants and 15 "round-fruit pumpkin" plants;

[0032] Figure 2 The schematic diagram is the amplification of SSR2 specific primers in 15 "long-fruit pumpkin" plants and 15 "round-fruit pumpkin" plants;

[0033] Figure 3 The schematic diagram is the amplification of SSR3 specific primers in 15 "long-fruit pumpkin" plants and 15 "round-fruit pumpkin" plants;

[0034] Figure 4 The schematic diagram is the amplification of SSR4 specific primers in 15 "long-fruit pumpkin" plants and 15 "round-fruit pumpkin" plants;

[0035] Figure 5 The schematic diagram is the amplification of SSR5 specific primers in 15 "long-fruit pumpkin" plants and 15 "round-fruit pumpkin" plants;

[0036] Figure 6 The schematic diagram is the amplification of SSR6 specific primers in 15 "long-fruit pumpkin" plants and 15 "round-fruit pumpkin" plants;

[0037] Figure 1-6 Middle, M: 50bp ladder; left 1-15: "long-fruit pumpkin" individual plant numbering; right 16-30: "round-fruit pumpkin" individual plant numbering.

[0038] Figure 7 The schematic diagram is the amplification of SSR1 specific primers in 18 pumpkin plants;

[0039] Figure 8 The schematic diagram is the amplification of SSR2 specific primers in 18 pumpkin plants;

[0040] Fig. 9 The schematic diagram is the amplification of SSR3 specific primers in 18 pumpkin plants;

[0041] Fig.10 The schematic diagram is the amplification of SSR4 specific primers in 18 pumpkin plants;

[0042] Fig.11 The schematic diagram is the amplification of SSR5 specific primers in 18 pumpkin plants;

[0043] Fig.12 The schematic diagram of the specific primers for SSR6 amplification in 18 pumpkin plants;

[0044] Figure 7-12In the middle, the black vertical arrows are the single plants with the same stripe pattern as the long-fruit pumpkin; the gray vertical arrows are the single plants with the same stripe pattern as the round-fruit pumpkin. DETAILED DESCRIPTION

[0045] The technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0046] The production processes, experimental methods or detection methods involved in the embodiments of the present invention, unless otherwise specified, are all conventional methods in the prior art, and their names and / or abbreviations are all conventional names in the field, and are very clear and unambiguous in the relevant application fields. Technical personnel in the field can understand the conventional process steps based on the names and apply the corresponding equipment, and implement them according to conventional conditions or the conditions recommended by the manufacturer.

[0047] The various instruments, equipment, raw materials or reagents used in the embodiments of the present invention are not particularly limited in terms of their sources, and are all conventional products that can be purchased through regular commercial channels, or can be prepared according to conventional methods well known to those skilled in the art.

[0048] The pumpkin seeds used in the present invention are commercially available round-fruit type silver chestnut type pumpkin varieties and long-fruit type Ganlong type pumpkin varieties.

[0049] Example 1

[0050] Identification method of long-fruit pumpkin and round-fruit pumpkin

[0051] (1) Sampling

[0052] Fifteen long-fruit pumpkin seeds and fifteen round-fruit pumpkin seeds were germinated respectively. When the cotyledons of the plants were flat and the first new leaf was fully unfolded, 1 g of new leaves were collected and stored at -80°C.

[0053] (2) DNA extraction

[0054] Total genomic DNA was extracted from new pumpkin leaves according to the CTAB method (Murray HG, Thompson WF. Rapid isolation of higher weight DNA. Nucleic Acids Res, 1980, 8: 4321).

[0055] (3) PCR amplification

[0056] The six pairs of specific primers in Table 1 were mixed separately and used for PCR amplification;

[0057] The mixed sample system of each pair of SSR specific primers is 15μl, including 1.5μl dNTP (2.5mM), 1.5ul 10× buffer, 1.8ul MgCl2 (25mM), 2ul primer (5mM; 1.0ul of each sequence of each pair of SSR specific primers is added respectively), 5ul DNA (10ng / ul), 0.05ul Taq (1U), and 3.145ul ultrapure water.

[0058] The PCR amplification program was as follows: pre-denaturation at 94°C for 1 min; denaturation at 93°C for 1 min, annealing at 50°C for 1 min, extension at 72°C for 2 min (40 cycles); and extension at 72°C for 1 min.

[0059] Table 1 Six pairs of specific molecular information

[0060]

[0061] Note: The primers for SSR6 did not detect the site at 300bp in long-fruit pumpkin.

[0062] (4) PCR product detection

[0063] Use 2.5% high-resolution standard gel agarose (DNA fragment separation range 40-1000bp). Add 20μl EB (ethidium bromide) for staining. Add 2μl Loading Dye to the PCR product obtained after amplification, voltage 200v, electrophoresis for 2-3 hours. After the electrophoresis, use a gel imaging system to observe the bands and record the size of each SSR marker fragment.

[0064] (5) Data collection and analysis

[0065] Polymorphism judgment: Individual plants with long-fruit polymorphic sites are judged as long-fruit type; individual plants with round-fruit polymorphic sites are judged as round-fruit type.

[0066] Example 2

[0067] Test results and analysis

[0068] According to the observation of gel imaging system, Figure 1 It can be seen that:

[0069] exist Figure 1 The horizontal solid arrow at 150 bp in the middle: the location of the differential site and the fragment size of the long-fruited pumpkin.

[0070] exist Figure 1 The horizontal dotted arrow at 160 bp in the middle shows the location of the differential site and fragment size in round-fruit pumpkin.

[0071] exist Figure 2The horizontal solid arrow at 130 bp in the middle: the location of the differential site and the fragment size of the long-fruit pumpkin.

[0072] exist Figure 2 The horizontal dotted arrow at 150 bp in the middle shows the location of the differential site and fragment size of round-fruit pumpkin.

[0073] exist Figure 3 The horizontal solid arrow at 110 bp in the middle: the location of the differential site and the fragment size of the long-fruit pumpkin.

[0074] exist Figure 3 The horizontal dotted arrow at 120 bp in the middle shows the location of the differential site and fragment size in round-fruit pumpkin.

[0075] exist Figure 4 The horizontal solid arrow at 90 bp in the middle: the location of the differential site and the fragment size of the long-fruited pumpkin.

[0076] exist Figure 4 The horizontal dotted arrow at 70 bp in the middle shows the location of the differential site and fragment size of round-fruit pumpkin.

[0077] exist Figure 5 The horizontal solid arrow at 140 bp in the middle shows the location of the differential site and fragment size of the long-fruit pumpkin.

[0078] exist Figure 5 The horizontal dotted arrow at 150 bp in the middle shows the location of the differential site and fragment size of round-fruit pumpkin.

[0079] exist Figure 6 The horizontal solid arrow at 300 bp in the middle: the long-fruited pumpkin is missing at this differential site.

[0080] exist Figure 6 The horizontal dotted arrow at 300 bp in the middle shows the location of the differential site and fragment size in round-fruit pumpkin.

[0081] It can be seen from this that the differences in polymorphic sites detected by PCR amplification using the six pairs of SSR specific primers described in the present invention are consistent.

[0082] Example 3 Polymorphism Verification

[0083] DNA samples were extracted from 18 pumpkin cotyledons, and PCR amplification was performed on the extracted DNA samples using specific SSR1, SSR2, SSR3, SSR4, SSR5 and SSR6 primers. The PCR amplification products were separated by electrophoresis, and the size and number of the amplified bands were observed and recorded.

[0084] Result analysis: According to the electrophoresis results, the fruit type was determined according to the polymorphic sites of the specific SSR1, SSR2, SSR3, SSR4, SSR5 and SSR6 primers. The amplification results were as follows: Figure 7-12 As shown in the figure, if there is one mark indicating that the plant is of long-fruit type, it is judged as long-fruit type.

[0085] Field observation comparison: The fruit type screening results of SSR1, SSR2, SSR3, SSR4, SSR5 and SSR6 were compared with the field observation phenotypic results to verify their consistency. The comparison results are shown in Table 2.

[0086] Table 2 shows the fruit phenotypic data of 18 individual plants, as well as the results of long-fruit pumpkin and round-fruit pumpkin screened out from the 18 individual plants using specific primers of SSR1, SSR2, SSR3, SSR4, SSR5 and SSR6.

[0087] Table 2 Comparison of 18 individual fruit phenotype data and screening results of six pairs of primers

[0088]

[0089]

[0090] From the results in Table 2, we can see that the results of comprehensive use of marker detection are consistent with the field observation results.

[0091] In summary, Examples 1-3 of the present invention use the six pairs of primers of the specific SSR molecular markers described in the present invention to perform PCR amplification, and then separate through agarose electrophoresis and EB staining, and detect polymorphism on a UV radiometer. According to the differences in the amplification products at the marker polymorphic sites, long-fruit pumpkins and round-fruit pumpkins can be quickly identified at an early stage, which saves time and labor, and the identification results are accurate, and the practical application value is great.

[0092] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A primer pair for pumpkin fruit type identification, characterized in that: The primers are a primer pair for identifying pumpkin SSR molecular markers, and the SSR molecular markers are SSR1-SSR6; The primer pair sequence information for identifying SSR1-SSR6 are as follows: The primer pair for identifying SSR1 consists of a forward primer having a nucleotide sequence as shown in SEQ ID NO.1 and a reverse primer having a nucleotide sequence as shown in SEQ ID NO.2; The primer pair for identifying SSR2 consists of a forward primer having a nucleotide sequence as shown in SEQ ID NO.3 and a reverse primer having a nucleotide sequence as shown in SEQ ID NO.4; The primer pair for identifying SSR3 consists of a forward primer having a nucleotide sequence as shown in SEQ ID NO.5 and a reverse primer having a nucleotide sequence as shown in SEQ ID NO.6; The primer pair for identifying SSR4 consists of a forward primer having a nucleotide sequence as shown in SEQ ID NO.7 and a reverse primer having a nucleotide sequence as shown in SEQ ID NO.8; The primer pair for identifying SSR5 consists of a forward primer having a nucleotide sequence as shown in SEQ ID NO.9 and a reverse primer having a nucleotide sequence as shown in SEQ ID NO.10; The primer pair for identifying SSR6 consists of a forward primer having a nucleotide sequence as shown in SEQ ID NO.11 and a reverse primer having a nucleotide sequence as shown in SEQ ID NO.

12.

2. The SSR primer pair for pumpkin fruit type identification according to claim 1, characterized in that: The pumpkin fruit types are long fruit type and round fruit type.

3. A kit for early identification of pumpkin fruit type, characterized in that: Contains the primers for identifying the SSR1-SSR6 molecular markers as described in claim 1 and a reagent for detection.

4. A method for early identification of pumpkin fruit type, characterized in that: The identification method comprises the following steps: (1) Sampling: Sample the radicle, cotyledon or leaf of pumpkin seeds and store them in a refrigerator; (2) DNA extraction: The total genomic DNA of pumpkin was extracted using the CTAB method; (3) PCR amplification: using the DNA extracted in step (2) as a template, the specific primer pairs for identifying the SSR1 to SSR6 molecular markers described in claim 1 are independently mixed and used for PCR amplification; (4) PCR product detection: The PCR product amplified in step (3) is subjected to fluorescent staining to observe the result; (5) Data collection and analysis.

5. The identification method according to claim 4, characterized in that: The radicle or cotyledon of the pumpkin seed in step (1) refers to the part that grows during the germination process of the seed; the leaf is the first new leaf or the DNA of the young leaf of the plant.

6. The identification method according to claim 4, characterized in that: The PCR system in step (3) includes: the total volume of the reaction system is 15 μl, including 1.5 μl dNTP (2.5 mM), 1.5 ul 10× buffer, 1.8 ul MgCl2 (25 mM), 2 ul primer (5 mM), 5 ul DNA (10 ng / ul), 0.05 ul Taq (1 U), and 3.145 ul ultrapure water; The PCR amplification program is: pre-deformation at 94°C for 1 min, deformation at 93°C for 1 min, annealing at 50°C for 1 min, extension at 72°C for 2 min (40 cycles); extension at 72°C for 1 min.

7. The identification method according to claim 4, characterized in that: The step (4) of fluorescent staining includes: using 2.5% high-resolution standard gel agarose and adding 20ul of ethidium bromide for staining.

8. Use of the primer pair according to claim 1 or 2, the kit according to claim 3, or the identification method according to any one of claims 4 to 7 in predicting pumpkin phenotype.

9. Use of the primer pair described in claim 1 or 2, the kit described in claim 3, or the identification method described in any one of claims 4 to 7 in identifying or assisting in identifying the purity of pumpkin fruit types.

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