Method for early identification of pumpkin fruit type and application thereof

Abstract

The application belongs to the technical field of crop phenotype identification, and particularly relates to a method for early identification of pumpkin fruit type and application thereof. The application provides six pairs of specific primer pairs with high polymorphism, and the nucleotide sequences of the specific primer pairs are respectively shown in SEQ ID NO. 1-SEQ ID NO. 12. In the application, the DNA of the radicle, cotyledon, first new leaf or young leaf of pumpkin seed is subjected to PCR amplification through specific primer pairs, and then subjected to agarose electrophoresis separation, EB staining, and detection of polymorphism on an ultraviolet perspective instrument. According to the difference of the amplification product at the marker polymorphism site, long fruit type pumpkin and round fruit type pumpkin can be quickly identified at an early stage, so as to improve the detection efficiency of pumpkin fruit type. Moreover, the identification method provided by the application has accurate and reliable identification results and is not affected by environmental factors, and can effectively identify long fruit type and round fruit type pumpkin single plants before the development of fruits, so as to accelerate the screening process of the target single plant and significantly improve the breeding efficiency.

Description

TECHNICAL FIELD

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

[0002] Fruit shape is an important trait of melon crops, an important index for product classification, grading and evaluation, and an important direction of pumpkin breeding. Field observation of this trait is mainly carried out during the development period of the ovary or the fruiting period, and it needs to wait for 2-3 months from sowing to observation. At the same time, the pumpkin vine is vigorous, the planting area is large, and the cost of land and labor is high. The SSR (Simple Sequence Repeat) molecular marker technology is simple to operate and has good repeatability, and has been applied in breeding links such as crop purity identification and trait screening. However, there is no report on the early and rapid identification method of long fruit type and round fruit type of pumpkin. Therefore, it is necessary to develop specific primers with high polymorphism for early and rapid identification of pumpkin fruit type to improve the efficiency of fruit type identification of pumpkin in breeding, classification, evaluation and other processes. SUMMARY

[0003] The purpose of the present application is to provide six pairs of specific primer pairs for early identification of pumpkin fruit type. The DNA of the radicle, cotyledon, first new leaf or young leaf of the pumpkin seed is subjected to PCR amplification by the specific primer pairs to improve the detection efficiency of early pumpkin fruit type. The identification method provided by the present application has accurate and reliable results and is not affected by environmental factors. Long fruit type and round fruit type of pumpkin single plants can be effectively identified before fruit development, thereby accelerating the screening process of the target single plant and significantly improving the breeding efficiency.

[0004] In order to achieve the above purpose, the present application provides the following technical scheme:

[0005] Firstly, the present application provides a primer pair for identification of pumpkin fruit type. The primer is a primer pair for identification of pumpkin SSR molecular markers. The SSR molecular markers are SSR1-SSR6.

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

[0007] The primer pair for identifying SSR1 is composed of a forward primer with the nucleotide sequence of SEQ ID NO. 1 and a reverse primer with the nucleotide sequence of SEQ ID NO. 2.

[0008] The primer pair for identifying SSR2 is composed of a forward primer with the nucleotide sequence of SEQ ID NO. 3 and a reverse primer with the nucleotide sequence of SEQ ID NO. 4.

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

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

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

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

[0013] Preferably, the pumpkin fruit type is long fruit type and round fruit type.

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

[0015] Secondly, the present application provides a method for early identification of pumpkin fruit type, which is characterized by comprising the following steps:

[0016] (1) Sampling: sampling the radicle or cotyledon or leaf of pumpkin seeds and storing in cold storage;

[0017] (2) DNA extraction: extracting total genomic DNA of pumpkin by CTAB method;

[0018] (3) PCR amplification: using the DNA extracted in step (2) as a template, mixing the specific primer pairs for identifying the SSR1-SSR6 molecular markers independently, and using them for PCR amplification;

[0019] (4) PCR product detection: performing fluorescent staining on the PCR product amplified in step (3) and observing the results;

[0020] (5) Data collection and analysis.

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

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

[0023] The PCR amplification procedure is: pre-deformation 94℃ 1min, deformation 93℃ 1min, annealing 50℃ 1min, extension 72℃ 2min (40 cycles); extension 72℃ 1min.

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

[0025] Finally, the application provides the application of the above-mentioned primer pair or the above-mentioned kit or the identification method in predicting the phenotype of pumpkin.

[0026] The application provides the application of the above-mentioned primer pair or the above-mentioned kit or the identification method in identifying or assisting in identifying the fruit type purity of pumpkin.

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

[0028] 1) The application provides six pairs of specific primer pairs for identifying SSR1-SSR6 molecular markers, the primer pairs have high polymorphism, and the PCR amplification identification of long fruit type pumpkin and round fruit type pumpkin shows better polymorphic amplification, which significantly improves the detection efficiency of long fruit type and round fruit type single plants.

[0029] 2) Compared with conventional morphological identification, the identification method provided by the application can more accurately identify the fruit type of pumpkin at any period of seed, seedling and plant, without waiting for the development of ovary or carrying out identification work after fruiting, and the results are reliable and not affected by the environment, saving manpower, material resources and land information, and improving the identification efficiency. BRIEF DESCRIPTION OF DRAWINGS

[0030] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed in the embodiments. Obviously, the drawings in the following description are only some embodiments of the application, and for those skilled in the art, other drawings can also be obtained without creative labor.

[0031] Figure 1Schematic diagram of amplification of SSR1 specific primer in 15 "long fruit type pumpkin" single plants and 15 "round fruit type pumpkin" single plants;

[0032] Figure 2 Schematic diagram of amplification of SSR2 specific primer in 15 "long fruit type pumpkin" single plants and 15 "round fruit type pumpkin" single plants;

[0033] Figure 3 Schematic diagram of amplification of SSR3 specific primer in 15 "long fruit type pumpkin" single plants and 15 "round fruit type pumpkin" single plants;

[0034] Figure 4 Schematic diagram of amplification of SSR4 specific primer in 15 "long fruit type pumpkin" single plants and 15 "round fruit type pumpkin" single plants;

[0035] Figure 5 Schematic diagram of amplification of SSR5 specific primer in 15 "long fruit type pumpkin" single plants and 15 "round fruit type pumpkin" single plants;

[0036] Figure 6 Schematic diagram of amplification of SSR6 specific primer in 15 "long fruit type pumpkin" single plants and 15 "round fruit type pumpkin" single plants;

[0037] Figures 1-6 In the figure, M: 50bp ladder; left 1-15: "long fruit type pumpkin" single plant number; right 16-30: "round fruit type pumpkin" single plant number.

[0038] Figure 7 Schematic diagram of amplification of SSR1 specific primer in 18 pumpkin single plants;

[0039] Figure 8 Schematic diagram of amplification of SSR2 specific primer in 18 pumpkin single plants;

[0040] Figure 9 Schematic diagram of amplification of SSR3 specific primer in 18 pumpkin single plants;

[0041] Figure 10 Schematic diagram of amplification of SSR4 specific primer in 18 pumpkin single plants;

[0042] Figure 11 Schematic diagram of amplification of SSR5 specific primer in 18 pumpkin single plants;

[0043] Figure 12 Schematic diagram of amplification of SSR6 specific primer in 18 pumpkin single plants;

[0044] Figures 7-12In the figure, the black vertical arrow indicates the single plant with the same zucchini type as the long fruit type pumpkin detected by the marker; the gray vertical arrow indicates the single plant with the same round fruit type pumpkin detected by the marker. DETAILED DESCRIPTION

[0045] The technical solutions provided by the present application are described in detail below in combination with the drawings and examples, but they should not be understood as limiting the scope of protection of the present application.

[0046] The production process, experimental method or detection method involved in the embodiments of the present application are all conventional methods in the prior art without special instructions, and the name and / or abbreviation thereof all belong to the conventional name in the art and are very clear and explicit in the related application field. The skilled person in the art can understand the conventional process steps and apply the corresponding equipment according to the name, and implement it according to the 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 application do not have special restrictions on the source, and are conventional products that can be purchased through normal commercial channels, or can be prepared according to the conventional method well known to those skilled in the art.

[0048] The pumpkin seeds used in the present application are commercially available round fruit type silver chestnut type pumpkin varieties and long fruit type sweet dragon type pumpkin varieties.

[0049] Example 1

[0050] Method for identifying the fruit type of long fruit type pumpkin and round fruit type pumpkin

[0051] (1) Sampling

[0052] Respectively, 15 long fruit type pumpkin seeds and 15 round fruit type pumpkin seeds were germinated, and when the plant cotyledon was flat and the first new leaf was fully expanded, 1 g of new leaf was collected and stored at-80℃.

[0053] (2) DNA extraction

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

[0055] (3) PCR amplification

[0056] Six pairs of specific primers in Table 1 were independently mixed for PCR amplification;

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

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

[0059] Table 1: Information of six pairs of specific molecules

[0060]

[0061] Note: The primer of SSR6 does not detect the site at 300 bp of long fruit type pumpkin.

[0062] (4) PCR product detection

[0063] A 2.5% high-resolution standard gel agarose (DNA fragment separation range 40-1000 bp) is used. 20 μl EB (ethidium bromide, ethidium bromide) is added for staining. 2 μl Loading Dye is added to the PCR product obtained after amplification, the voltage is 200 v, and electrophoresis is performed for 2-3 hours. After electrophoresis, the bands are observed by using a gel imaging system, and the size of each SSR marker fragment is recorded.

[0064] (5) Data collection and analysis

[0065] Polymorphism judgment: a single plant with long fruit type polymorphic site is judged as long fruit type; a single plant with round fruit type polymorphic site is judged as round fruit type.

[0066] Example 2

[0067] Detection results and analysis

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

[0069] In the horizontal solid arrow at 150 bp in Figure 1 , the position and fragment size of the long fruit type pumpkin difference site.

[0070] In the horizontal dotted arrow at 160 bp in Figure 1 , the position and fragment size of the round fruit type pumpkin difference site.

[0071] In the horizontal dotted arrow at 160 bp in Figure 2Horizontal solid arrow at 130bp: the location of the different site of long fruit type pumpkin and the size of the fragment.

[0072] In Figure 2 Horizontal dotted arrow at 150bp: the location of the different site of round fruit type pumpkin and the size of the fragment.

[0073] In Figure 3 Horizontal solid arrow at 110bp: the location of the different site of long fruit type pumpkin and the size of the fragment.

[0074] In Figure 3 Horizontal dotted arrow at 120bp: the location of the different site of round fruit type pumpkin and the size of the fragment.

[0075] In Figure 4 Horizontal solid arrow at 90bp: the location of the different site of long fruit type pumpkin and the size of the fragment.

[0076] In Figure 4 Horizontal dotted arrow at 70bp: the location of the different site of round fruit type pumpkin and the size of the fragment.

[0077] In Figure 5 Horizontal solid arrow at 140bp: the location of the different site of long fruit type pumpkin and the size of the fragment.

[0078] In Figure 5 Horizontal dotted arrow at 150bp: the location of the different site of round fruit type pumpkin and the size of the fragment.

[0079] In Figure 6 Horizontal solid arrow at 300bp: the long fruit type pumpkin is deleted at the different site.

[0080] In Figure 6 Horizontal dotted arrow at 300bp: the location of the different site of round fruit type pumpkin and the size of the fragment.

[0081] Therefore, the six pairs of specific primers of the SSR in the application are consistent in the difference of the polymorphic sites detected by PCR amplification.

[0082] Example 3: verification of polymorphism

[0083] DNA samples are extracted from 18 pumpkin cotyledons, and the extracted DNA samples are subjected to PCR amplification by using the specific primers of SSR1, SSR2, SSR3, SSR4, SSR5 and SSR6, and the PCR amplification products are subjected to electrophoresis separation, and the size and quantity of the amplified bands are observed and recorded.

[0084] Result analysis: according to the electrophoresis result, judging the fruit type according to the polymorphic site of the specific SSR1, SSR2, SSR3, SSR4, SSR5 and SSR6 primers, the amplification result is shown in Table 1. Figures 7-12 As long as one marker indicates that a single plant is long fruit type, it is judged as long fruit type.

[0085] Field observation comparison: comparing the fruit type screening result of SSR1, SSR2, SSR3, SSR4, SSR5 and SSR6 with the field observation phenotype result, verifying the consistency, and the comparison result is shown in Table 2.

[0086] Table 2 is the fruit type phenotype data of 18 single plants, and the screening result of long fruit type pumpkin and round fruit type pumpkin of the specific SSR1, SSR2, SSR3, SSR4, SSR5 and SSR6 primers in 18 single plants.

[0087] Table 2 is the fruit type phenotype data of 18 single plants, and the screening result of long fruit type pumpkin and round fruit type pumpkin of the specific SSR1, SSR2, SSR3, SSR4, SSR5 and SSR6 primers in 18 single plants.

[0088]

[0089]

[0090] From the result of Table 2, it can be seen that the combined use of marker detection result is consistent with the field observation result.

[0091] From the above, it can be obtained that the six pairs of specific SSR molecular marker primers in the embodiments 1-3 of the present application are used for PCR amplification, then separated by agarose electrophoresis, EB staining, and detecting the polymorphism on the ultraviolet perspective instrument, according to the difference of the amplification product at the marker polymorphic site, long fruit type pumpkin and round fruit type pumpkin can be identified early and quickly, time and labor are saved, the identification result is accurate, and the practical application value is great.

[0092] Although the above embodiments have made a detailed description of the present application, it is only a part of the embodiments of the present application, but not all the embodiments, and other embodiments can be obtained according to the present embodiments without creativity, and these embodiments all belong to the protection scope of the present application.

Claims

1. A primer pair combination for identifying pumpkin fruit shape, characterized in that, The primer pair combination consists of primer pairs for identifying SSR molecular markers of pumpkin, namely SSR1-SSR6; The primer pair sequences for identifying SSR1-SSR6 are shown below: The primer pair for identifying SSR1 consists of a forward primer with the nucleotide sequence shown in SEQ ID NO.1 and a reverse primer with the nucleotide sequence shown in SEQ ID NO.2; The primer pair for identifying SSR2 consists of a forward primer with the nucleotide sequence shown in SEQ ID NO.3 and a reverse primer with the nucleotide sequence shown in SEQ ID NO.4; The primer pair for identifying SSR3 consists of a forward primer with the nucleotide sequence shown in SEQ ID NO.5 and a reverse primer with the nucleotide sequence shown in SEQ ID NO.6; The primer pair for identifying SSR4 consists of a forward primer with the nucleotide sequence shown in SEQ ID NO.7 and a reverse primer with the nucleotide sequence shown in SEQ ID NO.8; The primer pair for identifying SSR5 consists of a forward primer with the nucleotide sequence shown in SEQ ID NO.9 and a reverse primer with the nucleotide sequence shown in SEQ ID NO.10; The primer pair for identifying SSR6 consists of a forward primer with the nucleotide sequence shown in SEQ ID NO.11 and a reverse primer with the nucleotide sequence shown in SEQ ID NO.

12.

2. The SSR primer pair combination for pumpkin fruit shape identification according to claim 1, characterized in that, The pumpkin fruit shapes are long and round.

3. A kit for early identification of pumpkin fruit shape, characterized in that, It contains the primers for identifying SSR1-SSR6 molecular markers as described in claim 1, and the reagents for detection.

4. A method for early identification of pumpkin fruit shape, characterized in that, The identification method includes the following steps: (1) Sampling: Take samples of the radicle, cotyledon or leaf of the pumpkin seed and store them in cold storage; (2) DNA extraction: Total genomic DNA was extracted from pumpkin using the CTAB method; (3) PCR amplification: Using the DNA extracted in step (2) as a template, the specific primer pairs for identifying SSR1~SSR6 molecular markers described in claim 1 are mixed separately for PCR amplification. (4) PCR product detection: The PCR products amplified in step (3) are subjected to fluorescent staining, and the results are observed; (5) Data collection and analysis; The pumpkin fruit shapes are long and round.

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

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

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

8. The application of the primer pair combination of claim 1 or 2, the kit of claim 3, or the identification method of any one of claims 4-7 in predicting pumpkin fruit shape, characterized in that, The pumpkin fruit shapes are long and round.

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