A method for identifying the purity of thin-skinned melon hybrid seeds

Abstract

The invention discloses a method for identifying the purity of hybrid seeds of thin-skinned melons, the identification method comprising amplifying DNA extracted from thin-skinned melons with primer SSR1, primer SSR2 or primer SSR3; primer SSR1, upstream primer nucleotide sequence as shown in SEQ ID NO.1, downstream primer nucleotide sequence as shown in SEQ ID NO.2; primer SSR2, upstream primer nucleotide sequence as shown in SEQ ID NO.3, downstream primer nucleotide sequence as shown in SEQ ID NO.4; primer SSR3, upstream primer nucleotide sequence as shown in SEQ ID NO.5, downstream primer nucleotide sequence as shown in SEQ ID NO.6. The identification method disclosed in the patent can specifically identify the purity of hybrid seeds of Emerald Green Treasure, Fresh and Crunchy No. 1, Fresh and Sweet Crunchy Melon and White Beauty No. 1, and has high accuracy and fast speed, and is suitable for popularization and application.

Description

Technical Field

[0001] The invention relates to the technical field of seed purity identification, and in particular to a method for identifying the purity of thin-skinned melon hybrid seeds. Background Art

[0002] Melon is an annual herb. China's melon production accounts for a large proportion of the world's total. The planting area of ​​thin-skinned melons is also increasing. Therefore, the demand for thin-skinned melon seeds is increasing. Most melon seeds are hybrids, and it is urgent to ensure the purity of the seeds.

[0003] Seed purity is usually identified by field morphological identification, but it takes a long time, is difficult, requires a lot of land, and is not very reliable. With the rapid development of molecular biology technology, purity identification can be carried out using molecular marker technologies such as AFLP, RAPD, and SSR. Among them, SSR (Simple Sequence Repeat) molecular markers have the characteristics of high polymorphism, abundant quantity, good repeatability, and co-dominant inheritance, and have been widely used in the purity identification of various crop hybrids.

[0004] At present, the thin-skinned melon hybrid varieties, Emerald Green Treasure, Fresh and Crunchy No. 1, Fresh and Sweet Crunchy Melon and White Beauty No. 1, independently bred by the Vegetable Research Institute of Ningbo Academy of Agricultural Sciences, are highly favored by melon farmers for their high central sugar content, steady growth, excellent quality, high fruit setting rate and good disease resistance. It is particularly necessary to explore an identification method that can specifically identify the purity of the above four types of melon hybrid seeds. Summary of the invention

[0005] The purpose of the present invention is to provide a method for identifying the purity of thin-skinned melon hybrid seeds, so as to solve the problem that the purity of hybrid seeds of Emerald Green Treasure, Fresh Sweet Crunchy Melon, White Beauty No. 1 and Fresh Crunchy No. 1 melons cannot be identified quickly and accurately.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] A method for identifying the purity of thin-skinned melon hybrid seeds, the method comprising amplifying DNA extracted from thin-skinned melon using primer SSR1, primer SSR2 or primer SSR3;

[0008] The primers and their nucleotide sequence information are as follows:

[0009] Primer SSR1, the upstream primer nucleotide sequence is shown in SEQ ID NO.1, and the downstream primer nucleotide sequence is shown in SEQ ID NO.2;

[0010] Primer SSR2, the upstream primer nucleotide sequence is shown in SEQ ID NO.3, and the downstream primer nucleotide sequence is shown in SEQ ID NO.4;

[0011] Primer SSR3, the upstream primer nucleotide sequence is shown as SEQ ID NO.5, and the downstream primer nucleotide sequence is shown as SEQ ID NO.6.

[0012] Furthermore, the primer SSR1 is used to identify the purity of the hybrid seeds of Emerald Green Treasure; the primer SSR2 is used to identify the purity of the hybrid seeds of Fresh and Crunchy No. 1; and the primer SSR3 is used to identify the purity of the hybrid seeds of Fresh and Sweet Crunchy Melon and White Beauty No. 1.

[0013] Furthermore, the identification method specifically comprises the following steps:

[0014] (1) Extracting and testing the quality of melon genomic DNA from individual melon seedlings;

[0015] (2) using the genomic DNA extracted in step (1) as a template and performing PCR amplification using the primers described in claim 1;

[0016] (3) detecting the amplified product by agarose gel electrophoresis;

[0017] (4) Analyze the electrophoresis results and then calculate the purity of the seeds based on the electrophoresis results.

[0018] Furthermore, the specific method of melon genome extraction and quality detection in step (1) is as follows:

[0019] Select the hybrid varieties of Emerald Green Treasure, Fresh and Sweet Crunchy Melon, White Beauty No. 1 and Fresh and Sweet Crunchy Melon and their parental seeds for sowing. When the cotyledons grow out, grind them into pulp, extract genomic DNA by CTAB method, and then detect the DNA quality by Nanodrop2000 ultra-micro spectrophotometer.

[0020] Furthermore, the specific method of PCR amplification in step (2) is as follows:

[0021] The reaction system used for PCR amplification included: 1 μL of melon genomic DNA, 12.5 μL of 2×Easy Taq PCR Super Mix, 1 μL of each forward and reverse primer, and sterilized ultrapure water added to 25 μL;

[0022] The PCR amplification program was pre-denaturation at 94°C for 3 min; then 30 cycles were performed, each cycle including denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s; after extension for 2 min, the sample was stored at 4°C.

[0023] Furthermore, the electrophoresis analysis method and purity calculation method in step (4) are as follows:

[0024] When the female parent shows one characteristic band and the male parent shows another characteristic band different from that of the female parent, the true hybrid is a combination of the characteristic bands of both the female and male parents, otherwise it is a false hybrid.

[0025] Variety purity = number of true hybrid individuals in the tested melon hybrid seed population / number of individuals in the tested melon hybrid seed population × 100%

[0026] The beneficial effects of the present invention are:

[0027] 1. The present invention provides a method for identifying the purity of hybrid seeds of thin-skinned melons. The method uses three sets of primers SSR1, primer SSR2 and primer SSR3, which can quickly detect true hybrids from the first generation of hybrids of Emerald Green Treasure, Fresh and Crunchy No. 1, Fresh and Sweet Crunchy Melon and White Beauty No. 1, respectively, and has strong specificity.

[0028] 2. The method for identifying the purity of thin-skinned melon hybrid seeds provided by the present invention can distinguish thin-skinned melon hybrids from their parents and quickly detect the purity of hybrid seeds. It has the advantages of accuracy, speed, low cost, short identification cycle, simple operation, etc. and has broad application prospects.

[0029] 3. The present invention provides a method for identifying the purity of thin-skinned melon hybrid seeds. During the identification process, it is sufficient to extract the DNA of the cotyledons, thereby avoiding the step of preparing a complex extract during radicle extraction. At the same time, it is not necessary to use it, which reduces the operating steps of the experimenters and saves workload. On the other hand, it is not necessary to cultivate the seeds to the stage where new leaves grow, thus saving time overall. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is the agarose gel electrophoresis pattern of the PCR product amplified by the primer SSR1 in the purity identification of the Emerald Green Treasure melon seeds in Example 1 of the present invention;

[0031] Where M: DL 2000bp Marker; P1: Emerald Green Treasure female parent; P2: Emerald Green Treasure male parent; 4-52: Emerald Green Treasure hybrid number;

[0032] Figure 2 The agarose gel electrophoresis pattern of the PCR product amplified by the primer SSR2 in the purity identification of the fresh and crisp No. 1 melon seeds in Example 1;

[0033] Where M: DL 2000bp Marker; P1: Xiancui No. 1 female parent; P2: Xiancui No. 1 male parent; 4-52: Xiancui No. 1 hybrid number, the samples marked in the box are pseudo-hybrids with the same banding pattern as the female parent;

[0034] Figure 3This is the agarose gel electrophoresis pattern of the PCR product amplified by the primer SSR3 in the purity identification of the fresh sweet and crisp melon melon seeds in Example 1;

[0035] Where M: DL 2000bp Marker; P1: female parent of fresh and sweet crisp melon; P2: male parent of fresh and sweet crisp melon; 4-52: hybrid number of fresh and sweet crisp melon;

[0036] Figure 4 This is the agarose gel electrophoresis pattern of the PCR product amplified by the primer SSR3 in the purity identification of Baimei No. 1 melon seeds in Example 1;

[0037] Wherein, M: DL 2000bp Marker molecular weight standard; P1: Baimei No. 1 female parent; P2: Baimei No. 1 male parent; 4-52: Baimei No. 1 hybrid number. DETAILED DESCRIPTION

[0038] Example 1

[0039] This embodiment provides an SSR primer and method for identifying the purity of hybrid seeds of thin-skinned melon varieties Emerald Green Treasure, Fresh Sweet Crunchy Melon, White Beauty No. 1 and Fresh Crunchy No. 1 Melon:

[0040] (1) Extraction and detection of melon genomic DNA:

[0041] Select 100 seeds each of Emerald Green Treasure, Fresh and Sweet Crunchy Melon, White Beauty No. 1 and Fresh and Crunchy No. 1 melon hybrids with full and uniform seeds and 1 seed each of their corresponding male and female parents for sowing.

[0042] When cotyledons grow, 0.1 g of cotyledons corresponding to each melon variety are placed in a 2 mL centrifuge tube and ground into pulp. 1000 μL of CTAB extract is added and the mixture is incubated in a water bath at 65 °C for 15 min. During the mixing period, the mixture is inverted 2 to 3 times. Then, 1000 μL of chloroform is added to each centrifuge tube and the mixture is shaken vigorously. The tube is then centrifuged at 12000 r / min for 10 min and the centrifugation is repeated twice.

[0043] Take the supernatant after the above centrifugation into a new centrifuge tube, add 2 times the volume of anhydrous ethanol for precipitation, centrifuge at 12000r / min for 10min, discard the supernatant, wash the precipitate with 75% ethanol solution, centrifuge at 12000r / min for 5min, discard the supernatant, dry the precipitate at the bottom, and fully dissolve the precipitate with 50μL deionized water, and then use Nanodrop2000 ultramicro spectrophotometer to detect DNA quality.

[0044] (2) PCR amplification and detection

[0045] The reaction system used for PCR amplification includes: 1 μL of genomic DNA of Emerald Green, Fresh Sweet Crunchy Melon, White Beauty No. 1 and Fresh Crunchy No. 1 melons extracted in step (1), 12.5 μL of 2×EasyTaq PCR SuperMix (purchased from Quanshijin Biotechnology Co., Ltd.), 1 μL of each forward and reverse primer, and sterilized ultrapure water is added to 25 μL. Independent PCR amplification reaction systems are prepared for each of the above melon varieties for amplification.

[0046] The above PCR reaction system is amplified in a PCR instrument to obtain an amplified product.

[0047] The amplification program was pre-denaturation at 94°C for 3 min; then 30 cycles, each cycle including denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s; and finally extension for 2 min before storage at 4°C.

[0048] The amplified products were separated by 2% agarose gel electrophoresis and the band patterns were detected after EB staining.

[0049] The primers used above and the corresponding nucleotide sequences are as follows:

[0050] Development of SSR primers for identification of Emerald Green Treasure, Fresh and Sweet Crunchy Melon, White Beauty No. 1 and Fresh and Crunchy No. 1 melon genomes: First, based on the published melon genomes, 200 pairs of PCR primers spanning SSR loci were designed and synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0051] Finally, after screening, the primer SSR1 that can be used to identify the purity of the emerald green hybrid seeds was determined, wherein the upstream primer nucleotide sequence is shown in SEQ ID NO.1, and the downstream primer nucleotide sequence is shown in SEQ ID NO.2;

[0052] Primer SSR2 can be used to identify the purity of Xiancui No. 1 hybrid seeds, wherein the upstream primer nucleotide sequence is shown in SEQ ID NO.3, and the downstream primer nucleotide sequence is shown in SEQ ID NO.4;

[0053] The primer SSR3 can be used to identify the purity of hybrid seeds of fresh sweet and crisp melon and Baimei No. 1, wherein the upstream primer nucleotide sequence is shown in SEQ ID NO.5, and the downstream primer nucleotide sequence is shown in SEQ ID NO.6.

[0054] SEQ ID NO.1: 5'-ATATTCACCCAACCCCTAAG-3';

[0055] SEQ ID NO.2: 5'-GGATAGCAATTATCCCACC-3';

[0056] SEQ ID NO.3: 5'-CACGACGTGTAAAACGACCCCTATTTAAATATGATACCCTCA-3';

[0057] SEQ ID NO.4: 5'-GCCCATGACAACATATTAGCAA-3';

[0058] SEQ ID NO.5: 5'-AAAGGATGACCAAATGAAG-3';

[0059] SEQ ID NO.6: 5'-GACTTGTATCCAGGTTTGC-3';

[0060] (4) Electrophoresis band analysis

[0061] According to the analysis of the electrophoresis results, when the female parent shows one characteristic band and the male parent shows another characteristic band different from that of the female parent, the true hybrid is a combination of the characteristic bands of both the female and male parents, and other characteristics are false hybrids.

[0062] Variety purity = the number of true hybrid individuals in the tested melon hybrid seed population / the number of individuals in the tested melon hybrid seed population × 100%.

[0063] (5) Results analysis

[0064] Figure 1 To amplify the genomic DNA of Emerald Green and its parents using primer SSR1, Figure 1 It can be clearly seen that a specific band of 262bp was amplified in the Emerald Emerald female parent, and a specific band of 394bp was amplified in the male parent. The electrophoresis pattern of the Emerald Emerald standard hybrid seeds is a band 262 / 394bp with both parent-specific markers. Among the electrophoresis patterns of the 100 hybrid seeds tested, 100 were consistent with the electrophoresis band type of the Emerald Emerald standard hybrid seeds, and 0 lanes had electrophoresis band types inconsistent with the Emerald Emerald hybrid seed standard band type, indicating that 0 plants tested were hybrid plants, and the calculation showed that the purity of the Emerald Emerald hybrid seeds was 100% (the specific results are summarized in Table 1).

[0065] Figure 2 In order to amplify the genomic DNA of Xiancui No. 1 and its parents using primer SSR2, Figure 2It can be clearly seen that a specific band of 153bp was amplified in the female parent of Fresh and Crunchy No. 1, and a specific band of 145bp was amplified in the male parent. The electrophoresis pattern of the standard hybrid seeds of Fresh and Crunchy No. 1 was a band of 153 / 145bp with both parent-specific markers. Among the electrophoresis patterns of the 100 hybrid seeds tested, 99 were consistent with the electrophoresis band type of the standard hybrid seeds of Fresh and Crunchy No. 1, and the electrophoresis band type of one lane (the band marked with a box in the electrophoresis pattern) was inconsistent with the standard band type of the hybrid seeds of Fresh and Crunchy No. 1, indicating that this plant was a hybrid plant. The purity of the hybrid seeds of Fresh and Crunchy No. 1 was calculated to be 99% (the specific results are summarized in Table 1).

[0066] Figure 3 In order to amplify the genomic DNA of fresh sweet crisp melon and its parents using primer SSR3, Figure 3 It can be clearly seen that a specific band of 276bp was amplified in the female parent of the fresh sweet and crisp melon, and a specific band of 250bp was amplified in the male parent. The electrophoresis pattern of the standard hybrid seeds of the fresh sweet and crisp melon is a band 276 / 250bp with both parent-specific markers. Among the electrophoresis patterns of the 100 hybrid seeds tested, 100 were consistent with the electrophoresis band type of the standard hybrid seeds of the fresh sweet and crisp melon, and the electrophoresis band type of 0 lanes was inconsistent with the standard band type of the fresh sweet and crisp melon hybrid seeds, indicating that these 0 plants were hybrid plants. It was calculated that the purity of the fresh sweet and crisp melon hybrid seeds was 100% (the specific results are summarized in Table 1).

[0067] Figure 4 In order to amplify the genomic DNA of Baimei 1 and its parents using primer SSR3, Figure 4 It can be clearly seen that a specific band of 276bp was amplified in the female parent of Baimei No. 1, and a specific band of 248bp was amplified in the male parent. The electrophoresis pattern of the standard hybrid seeds of Baimei No. 1 was a band 276 / 248bp with both parent-specific markers. Among the electrophoresis patterns of the 100 hybrid seeds tested, 100 had the same electrophoresis band type as the standard hybrid seeds of Baimei No. 1, and 0 lanes had an electrophoresis band type that was inconsistent with the standard band type of the hybrid seeds of Baimei No. 1, indicating that these 0 plants were hybrid plants. It was calculated that the purity of the hybrid seeds of Baimei No. 1 was 100% (the specific results are summarized in Table 1).

[0068] Table 1

[0069]

[0070] from Figure 1 , Figure 2 , Figure 3 and Figure 4 It can be seen that the identification method of the present invention can effectively distinguish the thin-skinned melon varieties Emerald Green Treasure, Fresh and Crunchy No. 1, Fresh and Sweet Crunchy Melon and White Beauty No. 1 hybrid and their parents, and can also perform purity identification on the above-mentioned thin-skinned melon hybrid varieties.

[0071] The above description is a detailed description of the preferred feasible embodiments of the present invention, but the embodiments are not intended to limit the scope of the patent application of the present invention. All equivalent changes or modified changes completed under the technical spirit suggested by the present invention should fall within the patent scope covered by the present invention.

Claims

1. A method for identifying the purity of thin-skinned melon hybrid seeds, characterized in that: The identification method comprises amplifying DNA extracted from thin-skinned melon using primer SSR1, primer SSR2 or primer SSR3; The primers and their nucleotide sequence information are as follows: Primer SSR1, the upstream primer nucleotide sequence is shown in SEQ ID NO.1, and the downstream primer nucleotide sequence is shown in SEQ ID NO.2; Primer SSR2, the upstream primer nucleotide sequence is shown in SEQ ID NO.3, and the downstream primer nucleotide sequence is shown in SEQ ID NO.4; Primer SSR3, the upstream primer nucleotide sequence is shown in SEQ ID NO.5, and the downstream primer nucleotide sequence is shown in SEQ ID NO.6; The primer SSR1 is used to identify the purity of the hybrid seeds of Emerald Green Treasure; the primer SSR2 is used to identify the purity of the hybrid seeds of Fresh and Crunchy No. 1; and the primer SSR3 is used to identify the purity of the hybrid seeds of Fresh and Sweet Crunchy Melon and White Beauty No.

1.

2. The identification method according to claim 1, characterized in that: The specific steps include: (1) Extracting and testing the quality of melon genomic DNA from individual melon seedlings; (2) using the genomic DNA extracted in step (1) as a template and performing PCR amplification using the primers described in claim 1; (3) detecting the amplified product by agarose gel electrophoresis; (4) Analyze the electrophoresis results and then calculate the purity of the seeds based on the electrophoresis results.

3. The identification method according to claim 2, characterized in that: The specific method for melon genome extraction and quality detection in step (1) is as follows: Select the hybrid varieties of Emerald Green Treasure, Fresh and Sweet Crunchy Melon, White Beauty No. 1 and Fresh and Sweet Crunchy Melon and their parental seeds for sowing. When the cotyledons grow out, grind them into pulp, extract genomic DNA by CTAB method, and then detect the DNA quality by Nanodrop2000 ultra-micro spectrophotometer.

4. The identification method according to claim 2, characterized in that: The specific method of PCR amplification in step (2) is as follows: The reaction system used for PCR amplification included: 1 μL of melon genomic DNA, 12.5 μL of 2×Easy Taq PCR Super Mix, 1 μL of each forward and reverse primer, and sterilized ultrapure water added to 25 μL; The PCR amplification program was pre-denaturation at 94°C for 3 min; then 30 cycles, each cycle including denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s; after extension for 2 min, the sample was stored at 4°C.

5. The identification method according to claim 2, characterized in that: The electrophoresis analysis method and purity calculation method in step (4) are as follows: When the female parent shows one characteristic band and the male parent shows another characteristic band different from the female parent, the true hybrid shows a combination of the characteristic bands of both the female and male parents, and the other characteristics are false hybrids; Variety purity = the number of true hybrid individuals in the tested melon hybrid seed population / the number of individuals in the tested melon hybrid seed population × 100%.

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