Use of clfci gene and promoter region tandem repeat sequence thereof in identifying watermelon flesh color intensity

Abstract

Provided is use of the watermelon flesh color intensity control gene C1FCI and a promoter region tandem repeat sequence thereof in identifying watermelon flesh color intensity. Aiming at the presence of the C1FCI gene promoter tandem repeat region in watermelon varieties, a PCR marker that is convenient to detect is designed, enabling rapid identification of possible flesh color phenotypes in the hybrid progeny of watermelons with light and dark flesh at the seedling stage. When applied in assisted breeding, the gene marker can improve watermelon fruit quality.

Description

Application of ClFCI gene and tandem repeat sequence in promoter region thereof in identifying flesh color intensity of watermelon TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to application of watermelon flesh color intensity control gene ClFCI gene and tandem repeat sequence in promoter region thereof in identifying flesh color intensity of watermelon. BACKGROUND

[0002] Watermelon (Citrullus lanatus) is one of the top ten fruits in the world, and is an important economic crop widely cultivated at home and abroad. China is the largest watermelon producing and consuming country. The fruit pulp color of watermelon is rich, and in different watermelon germplasm resources, the fruit pulp color can be generally divided into five types, i.e. red, pink, orange-red, yellow and white. The main reason for the formation of these fruit pulp colors is the difference in the content and types of various pigments.

[0003] In the process of breeding cultivated watermelon varieties, the pulp color has also undergone an improvement process from light to deep. However, the key gene controlling the pulp color intensity of cultivated watermelon is still unclear. Compared with watermelons with different pulp colors (red, orange, yellow and white), it is more common in watermelon resources to have watermelon varieties with the same pulp color but different depths. The content of various carotenoids in varieties with different pulp color depths is significantly different. For example, the content of lycopene in the fruit pulp of the red-pulp watermelon variety AU-Sweet Scarlet is higher than 50 mg / 1000 g fresh weight, while the content of lycopene in the pink-pulp watermelon variety Angeleno Black Seeded is 30 mg / 1000 g fresh weight. It can be seen that the nutritional value of watermelon fruits with different pulp colors is quite different, and it is also difficult to obtain offspring with deeper pulp color than parents in breeding work. Therefore, it is urgent to understand the internal molecular mechanism of controlling the difference in carotenoid accumulation ability of watermelon fruit pulp, and to identify the key watermelon pulp color intensity control gene (Flesh color intensity, FCI) to provide a solid foundation for further molecular improvement of watermelon. SUMMARY

[0004] The present application provides a watermelon ClFCI gene in controlling the pulp color intensity of watermelon.

[0005] The present application provides a watermelon ClFCI gene in controlling the pulp color intensity of watermelon.

[0006] The application further provides a method for deepening the color of watermelon pulp, which comprises the operation of overexpressing the ClFCI gene in a watermelon plant.

[0007] The application further provides a tandem repeat sequence related to the control of the color of watermelon pulp, which is located in the promoter region of the watermelon ClFCI gene, has a length of 1258 bp, and has a sequence composition as shown in sequence 1 in the sequence list.

[0008] The application further provides a method for identifying the color of watermelon pulp, which comprises the operation of detecting the number of tandem repeat sequences in the promoter region of the ClFCI gene of the watermelon to be tested; wherein the tandem repeat sequence has a length of 1258 bp, and has a sequence composition as shown in sequence 1 in the sequence list; when one of the tandem repeat sequences is detected, the pulp color of the watermelon to be tested is light; when two of the tandem repeat sequences are detected, the pulp color of the watermelon to be tested is intermediate or dark; and when three or more of the tandem repeat sequences are detected, the pulp color of the watermelon to be tested is dark.

[0009] The application discloses, for the first time, a key gene for controlling the color of watermelon pulp, and finds the molecular basis for the deepening of the pulp color of cultivated varieties through artificial selection. The application designs a convenient PCR marker for detection in view of the existence of the tandem repeat region of the ClFCI gene promoter in watermelon varieties, and can rapidly detect the possible pulp color phenotype of the hybrid offspring of deep and shallow pulp color at the seedling stage; the gene marker is applied to assisted breeding, and the fruit quality of watermelon can be improved. The application analyzes the molecular mechanism of the deepening of the pulp color of watermelon, and also provides a target gene for creating a high-quality watermelon variety with a higher content of carotene. BRIEF DESCRIPTION OF DRAWINGS

[0010] Fig. 1 is a sectional view of representative fruits of a watermelon ClFCI gene positioning parent and offspring population.

[0011] Fig. 2 is a fine positioning diagram of the watermelon ClFCI gene.

[0012] Fig. 3 is a PCR detection diagram of the length of the ClFCI promoter in different watermelon materials detected at random.

[0013] Fig. 4 is a sectional view of fruits of four representative watermelon materials.

[0014] Fig. 5 is a schematic diagram of four types of ClFCI gene promoter structures existing in different watermelon materials.

[0015] Fig. 6 is an analysis diagram of the relative expression amount of the ClFCI gene in different organs of JLM.

[0016] Fig. 7 is an analysis diagram of the relative expression amount of the ClFCI gene in fruits of different pulp color materials.

[0017] Figure 8 is a chart of the flesh color phenotype of the offspring in the transgenic test of overexpression of ClFCI. DETAILED DESCRIPTION

[0018] In order to make the technical solutions, objectives and advantages of the present application clearer, the present application will be further described in detail below through specific examples. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present application, and are not used to limit the present application.

[0019] The first aspect of the present application provides application of the watermelon ClFCI gene in controlling the depth of the flesh color of watermelon.

[0020] The watermelon ClFCI gene can be referred to as a watermelon flesh color depth control gene, and its registered name in the Cucurbitaceae Genome Database is Cla97C06G121890 (full length 13908 bp), which can be obtained from http: / / cucurbitgenomics.org / v2 / feature / gene / Cla97C06G121890.

[0021] The second aspect of the present application provides a method for deepening the flesh color of watermelon, which comprises the operation of overexpressing the ClFCI gene in the watermelon plant.

[0022] According to the second aspect of the present application, the operation of overexpressing the ClFCI gene in the watermelon plant comprises the operation of constructing an overexpression vector of ClFCI by using a pYBA1302 vector, and then transforming the overexpression vector into the watermelon plant.

[0023] The third aspect of the present application provides a tandem repeat sequence related to the control of the depth of the flesh color of watermelon, which is located in the promoter region of the watermelon flesh color depth control gene (i.e. the ClFCI gene), has a length of 1258 bp, and its sequence composition is shown as sequence 1 in the sequence table.

[0024] The fourth aspect of the present application provides application of the tandem repeat sequence of the promoter region of the watermelon flesh color depth control gene ClFCI gene provided in the third aspect of the present application in identifying the depth of the flesh color of watermelon.

[0025] The fifth aspect of the present application provides a method for identifying the depth of the flesh color of watermelon, which comprises the operation of detecting the number of the tandem repeat sequence in the promoter region of the ClFCI gene of the watermelon to be tested; wherein the tandem repeat sequence has a length of 1258 bp, and its sequence composition is shown as sequence 1 in the sequence table.

[0026] According to the fifth aspect of the present application, when one of the tandem repeat sequences is detected, the flesh color of the watermelon to be tested is light; when two of the tandem repeat sequences are detected, the flesh color of the watermelon to be tested is intermediate or dark; and when three or more of the tandem repeat sequences are detected, the flesh color of the watermelon to be tested is dark.

[0027] The inventors of the present application found, through resequencing and PCR analysis, that the promoter region of the watermelon ClFCI gene is different in structure between deep and light flesh color parents; the difference is due to the fact that the sequence of 1258 bp before ATG of the ClFCI gene promoter of the light flesh color watermelons (JX-2, Cream S) is repeated three times in the deep flesh color varieties (Ming 58, JLM). After testing different flesh color watermelon materials, it was found that the sequence is repeated 1-4 times in different varieties, and the number of repetitions is related to the depth of the flesh color.

[0028] For example, watermelon variety 97103 (red flesh watermelon, flesh color is light), and watermelon variety Cream of Saskatchewan (Cream S, light yellow flesh watermelon, flesh color is light), the ClFCI gene promoter region has one of the tandem repeat sequences; specifically, the sequence of-1 bp to-3146 bp before ATG of the ClFCI gene promoter of watermelon variety 97103 has the nucleotide sequence consisting of sequence 2 in the sequence listing, and one of the tandem repeat sequences can be found.

[0029] Watermelon variety Xinhongbao (XHB, red flesh watermelon, flesh color is normal red, intermediate flesh color), the ClFCI gene promoter region has two of the tandem repeat sequences; specifically, the sequence of-1 bp to-4404 bp before ATG of the ClFCI gene promoter of Xinhongbao has the nucleotide sequence consisting of sequence 3 in the sequence listing, and two of the tandem repeat sequences can be found.

[0030] Watermelon variety Ming 58 (deep red flesh watermelon, flesh color is dark), and watermelon variety JLM (bright yellow flesh watermelon, flesh color is dark), the ClFCI gene promoter region has three of the tandem repeat sequences; specifically, the sequence of-1 bp to-5662 bp before ATG of the ClFCI gene promoter of watermelon varieties Ming 58 and JLM has the nucleotide sequence consisting of sequence 4 in the sequence listing, and three of the tandem repeat sequences can be found.

[0031] The ClFCI gene promoter region of watermelon variety GS89 (black broken ribs, or GS89-HBJ, orange pulp, and deep pulp color) has four tandem repeat sequences. The sequence of the ClFCI gene promoter from -1 bp to -6920 bp of ATG has the nucleotide sequence shown in SEQ ID NO: 5 in the sequence listing, and four tandem repeat sequences can be found.

[0032] According to the fifth aspect of the present application, the method for identifying the pulp color of watermelon comprises using the genomic DNA of the watermelon to be tested as a template and using the following primers for PCR amplification:

[0033] upstream primer: 5'-CAAGGATAATTTTAAAATAATG-3' (SEQ ID NO: 6);

[0034] downstream primer: 5'-ATATACTAATATAATTTGTAGGG-3' (SEQ ID NO: 7);

[0035] If the amplified fragment contains one tandem repeat sequence shown in SEQ ID NO: 1, the pulp color of the watermelon to be tested is light; if the amplified fragment contains two tandem repeat sequences shown in SEQ ID NO: 1, the pulp color of the watermelon to be tested is intermediate or deep; and if the amplified fragment contains three or more tandem repeat sequences shown in SEQ ID NO: 1, the pulp color of the watermelon to be tested is deep.

[0036] For example, for watermelon varieties 97103 and Cream of Saskatchewan, a 1343 bp fragment can be amplified, and sequencing shows that the fragment contains one 1258 bp long tandem repeat sequence shown in SEQ ID NO: 1, and the sequence of the 1343 bp fragment is shown in SEQ ID NO: 2, 1-1343 in the sequence listing.

[0037] For watermelon variety Xinhongbao (XHB), a 2601 bp fragment can be amplified, and sequencing shows that the fragment contains two 1258 bp long tandem repeat sequences shown in SEQ ID NO: 1, and the sequence of the 2601 bp fragment is shown in SEQ ID NO: 3, 1-2601 in the sequence listing.

[0038] For watermelon variety Ming 58 and watermelon variety JLM, a 3859 bp fragment can be amplified, and sequencing shows that the fragment contains three 1258 bp long tandem repeat sequences shown in SEQ ID NO: 1, and the sequence of the 3859 bp fragment is shown in SEQ ID NO: 4, 1-3859 in the sequence listing.

[0039] For watermelon variety GS89, a 5117 bp fragment can be amplified, and sequencing shows that the fragment contains four 1258 bp long tandem repeat sequences shown in SEQ ID NO: 1, and the sequence of the 5117 bp fragment is shown in SEQ ID NO: 5, 1-5117 in the sequence listing.

[0040] According to the fifth aspect of the present application, the method for identifying the color depth of watermelon pulp comprises using the genomic DNA of the watermelon to be tested as a template and performing PCR amplification with the following primers:

[0041] The upstream sequence is 5'-CAAGGATAATTTTAAAATAATG-3' (sequence 6);

[0042] The downstream sequence is 5'-GTAAAGATGGGTTGGGTTGTTTAC-3' (sequence 8);

[0043] If the amplified fragment contains one tandem repeat sequence shown in sequence 1, the pulp color of the watermelon to be tested is light; if the amplified fragment contains two tandem repeat sequences shown in sequence 1, the pulp color of the watermelon to be tested is intermediate or dark; and if the amplified fragment contains three or more tandem repeat sequences shown in sequence 1, the pulp color of the watermelon to be tested is dark.

[0044] The primer combination of sequence 6 and sequence 8 can be used to amplify the promoter region of the ClFCI gene, and the size of the amplification product can be used to determine the number of tandem repeat sequences shown in sequence 1. For example, a 3146 bp fragment (the sequence composition is shown in sequence 2 in the sequence listing) contains one tandem repeat sequence shown in sequence 1, a 4404 bp fragment (the sequence composition is shown in sequence 3 in the sequence listing) contains two tandem repeat sequences shown in sequence 1, a 5662 bp fragment (the sequence composition is shown in sequence 4 in the sequence listing) contains three tandem repeat sequences shown in sequence 1, and a 6920 bp fragment (the sequence composition is shown in sequence 5 in the sequence listing) contains four tandem repeat sequences shown in sequence 1.

[0045] In the following examples, various reagents, materials, etc. are used, and if not specifically stated, they are products that can be obtained from commercial channels; and in the following examples, various tests and detection methods are used, and if not specifically stated, they are conventional tests and detection methods in the art, which can be obtained from textbooks, reference books or academic journals.

[0046] In the present application, the "deep and light" of the watermelon pulp color is detected and defined in the following way:

[0047] The CR-410 color difference meter produced by Konica minolta is used to detect the flesh color of watermelon, and the measurement results are composed of five data indexes: "L" represents the brightness of the object: 0-100 indicates from black to white; "a" represents the red-green color of the object: positive value indicates red, negative value indicates green; "b" represents the yellow-blue color of the object: positive value indicates yellow, negative value indicates blue; "C" represents color saturation; and "h" represents the hue angle. The present application is mainly to distinguish different shades of the same flesh color (most of the flesh colors of watermelon can be classified into red and yellow colors), so the "C" value measured is used to measure the shade of the flesh color of watermelon; the reading "C" represents the color saturation, and the judgment index of the shade of the flesh color of watermelon is: "C">30 is defined as "deep color" of the flesh color of watermelon, "C" value is between 25-30 (25≤"C"≤30) is defined as "intermediate color" of the flesh color of watermelon, and "C"<25 is defined as "light color" of the flesh color of watermelon.

[0048] In the following examples, the test materials used (a representative fruit section view is shown in FIG. 1) are as follows:

[0049] Deep and light red flesh separation population: the male parent is JX-2 (light red flesh watermelon, "C" value is 21, and the flesh color is light), and the female parent is Ming 58 (deep red flesh watermelon, "C" value is 33, and the flesh color is deep); and the F1 and F2 generations obtained by crossing the two;

[0050] Deep and light yellow flesh separation population: the male parent is Cream of Saskatchewan (abbreviated as Cream S, light yellow flesh watermelon, "C" value is 16, and the flesh color is light), and the female parent is JLM (bright yellow flesh watermelon, "C" value is 34, and the flesh color is deep); and the F1 and F2 generations obtained by crossing the two.

[0051] All the test materials in the present application are germplasm resource materials preserved by the Watermelon Germplasm Resource Bank of the Vegetable Research Institute of the Beijing Academy of Agriculture and Forestry Sciences, and anyone can freely obtain the relevant materials from there for the purpose of realizing the purpose of the present application. The contact address is: Vegetable Research Institute of the Beijing Academy of Agriculture and Forestry Sciences, West Suburb Banjing, Haidian District, Beijing, China, with a postal code of 100097, a contact person of Zhang Jie, and a contact telephone number of 01051503039.

[0052] Extraction of genomic DNA of the above test materials: refer to the method of Murray et al. (1980) (Murray M, Thompson W F. Rapid isolation of high molecular weight plant DNA [J]. Nucl Acid Res, 1980, 8: 668-673.).

[0053] The total RNA of the above-mentioned test materials was extracted by using the EASY spin Plus Plant RNA Kit plant RNA rapid extraction kit provided by Beijing Aidley Biotechnology Co., Ltd., and according to the instructions of the kit, the total RNA of the organs such as roots, stems, leaves, flowers and fruits of the above-mentioned test materials was extracted. The concentration of the extracted total RNA of the test materials was determined by ultraviolet spectrophotometer (Shimadzu UV-1201, Japan) with OD260 value, and the extraction quality of the total RNA of the test materials was detected by 1.2% agarose gel electrophoresis. The first strand of cDNA was synthesized by using the Reverse Transcriptase M-MLV (RNase H-) reverse transcription kit purchased from TAKARA company.

[0054] Example 1

[0055] This example is used to illustrate the genomic positioning, gene cloning of watermelon flesh color depth control gene ClFCI, and the discovery of tandem repeat sequence in the promoter region and the relationship between watermelon flesh color depth.

[0056] I. ClFCI gene positioning:

[0057] According to the resequencing results of 4 watermelon parents (JX-2, Ming58, Cream S, JLM), two pairs of SNP / InDel sites between the two parents were obtained, and specific high-throughput KASP primers were designed for genomic positioning of ClFCI gene.

[0058] Group analysis method (BSA) was used to amplify the parents and DNA pool, and polymorphic markers linked to the traits were screened to obtain the initial positioning interval, so as to position the ClFCI gene site to the 3.2Mb region (22400000 to 24600000bp) on the 6th chromosome of watermelon 97103v2.5 genome.

[0059] Further, using the resequencing data of watermelon genome, polymorphic markers in the above-mentioned interval were further designed, and KASP marker detection was carried out with the above-mentioned test material genomic DNA as template to construct genetic map. By 14 pairs of KASP markers (high-throughput KASP primer group as shown in Table 1) in the above-mentioned interval, the FCI site was positioned between marker 6-24.17 (located at 24.17Mb on Chr6) and marker 6-24.3 (located at 24.3Mb on Chr6). The interval contains 15 genes, including Cla97C06G121890. The fine mapping of watermelon flesh color depth control gene (i.e. ClFCI gene) is shown in Figure 2.

[0060] Table 1 KASP primer composition

[0061] II. Cloning of candidate genes, discovery of tandem repeats in the promoter region and relationship with flesh color depth of watermelon

[0062] According to the results of genome annotation, the sequence of 15 candidate genes in the initial positioning interval of the watermelon flesh color depth control gene site was analyzed using resequencing data, and the following primers were designed:

[0063] The specific primers (FCI primers) for amplifying the CDS sequence of the ClFCI gene are as follows:

[0064] The upstream sequence is: 5'-ATGGCTCCCAAAGCTGGAAAAAC-3';

[0065] The downstream sequence is: 5'-TCAACTTGAAACCTCAACAATC-3';

[0066] The specific primers (FCI-P) for amplifying the promoter region sequence of the ClFCI gene are as follows:

[0067] The upstream sequence is: 5'-CAAGGATAATTTTAAAATAATG-3' (sequence 6);

[0068] The downstream sequence is: 5'-GTAAAGATGGGTTGGGTTGTTTAC-3' (sequence 8);

[0069] The specific primers (FCI-SV) for amplifying the SV existing in the promoter region of the ClFCI gene are as follows:

[0070] The upstream sequence is: 5'-CAAGGATAATTTTAAAATAATG-3' (sequence 6);

[0071] The downstream sequence is: 5'-ATATACTAATATAATTTGTAGGG-3 (sequence 7).

[0072] Using 4 watermelon parent (JX-2, Ming 58, Cream S, JLM) genomic DNA as template, PCR amplification was performed with the above FCI primers, FCI-P primers, and FCI-SV primers, respectively.

[0073] The reaction system (20 μL) of PCR amplification reaction was as follows: 2 μL of 10× TransStart Taq Buffer containing 15 mM MgCl2; 0.8 μL of dNTPs with a concentration of 2.5 mM; 0.9 U of TransStart Taq DNA Polymerase; 0.5 μL of 10 mM upstream primer, 0.5 μL of 10 mM downstream primer; 20 ng of template DNA; and ddH2O, supplemented to 20 μL.

[0074] The PCR amplification reaction program was as follows: stage 1, pre-denaturation at 94 ℃ for 5 min; stage 2, 94 ℃ for 30 s, 55 ℃ for 30 s, 72 ℃ for 2 min 30 s, for a total of 38 cycles; stage 3, extension at 72 ℃ for 10 min; and stage 5, storage at 4 ℃.

[0075] The results showed that the length of the fragment obtained by amplification with the above FCI primer was 5547 bp, and sequencing found that it was exactly the CDS of the Cla97C06G121890 gene, which was consistent with the sequence in the genomic database (http: / / cucurbitgenomics.org / v2 / feature / gene / Cla97C06G121890) of Cucurbitaceae.

[0076] The promoter sequence with a length of 3146 bp was obtained by amplification with the above FCI-P primer in JX-2 and Cream S, which were parent materials with light-colored pulp, and the sequencing results showed that the sequence composition was as shown in sequence 2; the promoter sequence with a length of 5662 bp was obtained by amplification in Ming 58 and JLM, which were parent materials with dark-colored pulp, and the sequencing results showed that the sequence composition was as shown in sequence 4. By comparing sequence 2 and sequence 4, it can be found that sequence 2 has one tandem repeat sequence shown in sequence 1, and sequence 4 has three tandem repeat sequences shown in sequence 1.

[0077] The 1343 bp fragment amplified from the JX-2 and Cream S parents with light pulp color and the 3859 bp fragment amplified from the Ming 58 and JLM parents with dark pulp color were sequenced. It was found that there was a large structural variation (SV) between the two fragments. The part of the promoter in the dark pulp parent material had a repeated insertion sequence. Specifically, the 1343 bp fragment amplified from the light pulp parent material contained one tandem repeat sequence shown in SEQ ID NO: 1, and the 3859 bp fragment amplified from the dark pulp parent material contained three tandem repeat sequences shown in SEQ ID NO: 1. The difference of 2516 bp between the two fragments was due to the difference of two 1258 bp tandem repeat sequences shown in SEQ ID NO: 1. The amplification result was the same as the bioinformatics analysis result.

[0078] Example 2

[0079] This example is used to illustrate the application of the tandem repeat sequence in the promoter region of the ClFCI gene in identifying the light and dark pulp color of watermelon.

[0080] Eighteen watermelon materials (Ming 58, AU, ZHT, L600, XHB, TS409, Sanbai, k no frost, PI296341, improved TWF, hard TWF, GS12, GS10, GS89, GS41, QM4K, Congo, 97103) were selected, and the genomic DNA was extracted, and the following primers were used for PCR amplification (the PCR system and reaction program were the same as in Example 1):

[0081] The upstream primer was 5'-CAAGGATAATTTTAAAATAATG-3' (SEQ ID NO: 6);

[0082] The downstream primer was 5'-ATATACTAATATAATTTGTAGGG-3' (SEQ ID NO: 7).

[0083] The PCR amplification products were electrophoresed, and the results showed that the promoter region in different watermelon materials appeared four types of PCR products with lengths of 1343 bp, 2601 bp, 3859 bp, and 5117 bp, respectively. Sequencing found that it was due to different numbers of the tandem repeats shown in sequence 1 of 1258 bp (the electrophoresis results are shown in Figure 3, in which the first lane and the 18th lane are DNA markers (unit: bp), and the 17th lane is a blank control without any DNA template; the pulp color is shown in Table 2). At the same time, the CR-410 color difference meter produced by Konica minolta Company was used to detect the pulp color of the watermelon, and the "C" value of each test watermelon material was detected (see Table 2).

[0084] Table 2 Length of amplified fragments and pulp color of 18 different watermelon materials

[0085] The electrophoresis fragments of the 18 watermelon materials were recovered and sequenced, and the results showed that the sequence compositions of the four types of PCR products with lengths of 1343 bp, 2601 bp, 3859 bp, and 5117 bp were 1-1343 shown in sequence 2, 1-2601 shown in sequence 3, 1-3859 shown in sequence 4, and 1-5117 shown in sequence 5 in the sequence list, respectively; that is, the 1343 bp fragment contains one sequence shown in sequence 1, the 2601 bp fragment contains two sequences shown in sequence 1, the 3859 bp fragment contains three sequences shown in sequence 1, and the 5117 bp fragment contains four sequences shown in sequence 1.

[0086] As can be seen from the above, when the ClFCI gene promoter region of the 18 watermelon materials contains one sequence shown in sequence 1, the pulp color is light; when it contains two sequences shown in sequence 1, the pulp color is intermediate or dark; and when it contains three to four sequences shown in sequence 1, the pulp color is dark.

[0087] Then, four representative materials were further selected: L600, XHB, Ming 58, and GS89 (the pulp color is shown in Figure 4), and the following primers were used for PCR amplification (the PCR system and reaction procedure are the same as in Example 1):

[0088] The upstream sequence is: 5'-CAAGGATAATTTTAAAATAATG-3' (sequence 6);

[0089] The downstream sequence is: 5'-GTAAAGATGGGTTGGGTTGTTTAC-3' (sequence 8).

[0090] The PCR amplification products were electrophoresed, and the amplification products were sequenced, and it was found that the sequence of the amplification fragments of L600, XHB, Ming58, and GS89 was sequentially as shown in 2, 3, 4, and 5 in the sequence table.

[0091] Meanwhile, according to the method of Petry FC and Mercadante AZ. New method for carotenoid extraction and analysis by HPLC-DAD-MS / MS in freeze-dried Citrus and Mango pulps. Journal of the Brazilian Chemical Society. 2018, 1: 205-215, the carotenoid content (unit: mg / 100g FW, representing the number of target substances mg per 100g fresh weight) in the pulp of different deep and shallow pulp color watermelon materials was detected by Ultra Performance Liquid Chromatography (UPLC) (ExionLC TM AD, https: / / sciex.com.cn / ) and Tandem Mass Spectrometry (MS / MS), and the results are shown in Table 3.

[0092] Table 3 Carotenoid content and colorimeter value in watermelon materials

[0093] As can be seen from FIG. 4 and Table 3, the number of sequences represented by sequence 1 contained in the promoter region of the ClFCI gene of the watermelon material has a significant relationship with the depth and shallowness of the pulp color: when only one sequence represented by sequence 1 is contained, the pulp color is light, when two sequences represented by sequence 1 are contained, the pulp color is intermediate or dark; when 3-4 sequences represented by sequence 1 are contained, the pulp color is dark; and the number of sequences represented by sequence 1 has a positive correlation with the content of carotenoids in the watermelon material that causes the depth and shallowness of the watermelon pulp color.

[0094] As can be seen from the above, the primers of sequence 6 and sequence 7 can detect the SV sequence of the promoter region of the ClFCI gene of the watermelon material with deep and light pulp color, and thereby identify whether the pulp color of the watermelon material is deep or light; the ClFCI gene promoter region fragment of the watermelon material can also be obtained by amplification of sequence 6 and sequence 8, and the number of tandem repeat sequences in the obtained ClFCI gene promoter region fragment can be used to identify whether the pulp color of the watermelon material is deep or light.

[0095] Example 3

[0096] This example is used to illustrate the spatial and temporal expression analysis of ClFCI gene, which confirms that the SV difference in the promoter region (i.e. the number of tandem repeats of sequence 1) leads to high expression of the target gene in deep-pulp varieties.

[0097] The expression of ClFCI gene in the root, stem, leaf, flower, and fruit organs of watermelon variety JLM was detected by fluorescence quantitative PCR, and the mechanism of ClFCI was preliminarily analyzed. At the same time, the cDNA of two parents Cream S, JLM and representative F2 fruits of the offspring were analyzed to analyze the expression of ClFCI gene in fruits of different pulp colors. Specifically as follows:

[0098] The cDNA of the root, stem, leaf, flower, and fruit of the test materials was used as the template for PCR amplification with the following fluorescence quantitative PCR primers.

[0099] Fluorescence quantitative PCR primers:

[0100] FCI_qPCR_F: 5'-CAGTAGGTGCATCATCTCCAG-3';

[0101] FCI_qPCR_R: 5'-CAATCTCAGCTTCATTGTCGC-3';

[0102] ACTIN_F: 5'-CCTACAACTCAATTATGAAGTGTG-3';

[0103] ACTIN_R: 5'-GAAATCCACATCTGCTGGAAGGTG-3'.

[0104] Among them, primer FCI_qPCR is a specific primer for spatial and temporal expression analysis of ClFCI, and primer ACTIN is an internal reference primer for fluorescence quantitative PCR.

[0105] The fluorescence quantitative PCR amplification reaction system (20 μL) is: 20 ng of cDNA template, 2x qPCR Master Mix 10 μL, 10 μM upstream primer 0.4 μL, 10 μM downstream primer 0.4 μL, Nuclear-Free Water to 20 μL.

[0106] The fluorescence quantitative PCR amplification reaction program is: stage 1: 95°C pre-denaturation: 5 min; stage 2: 95°C for 20 s, 58°C for 20 s, 72°C for 30 s, a total of 40 cycles; stage 3: 94°C release fluorescence for 8 min.

[0107] The results are shown in Figure 6, which shows that ClFCI gene is expressed in the root, stem, leaf, flower, and fruit of JLM. ​

[0108] The expression of the ClFCI gene in the fruits of 40 F2 generation watermelon materials used to construct deep-fleshed and light-fleshed pools for sequencing was further examined in the parental materials Cream S and JLM and their F2 populations. The results are shown in Figure 7 (in Figure 7, Y1-Y20 are representative F2 plants with deep yellow flesh, and W1-W20 are representative F2 plants with light yellow flesh). The results showed that in different flesh color parents and segregating populations, the expression level of the ClFCI gene in the fruits of deep-fleshed materials was significantly higher than that in light-fleshed varieties, with an expression level 2-3 times higher.

[0109] These results indicate that tandem repeats in the promoter region of the ClFCI gene can lead to high expression of the ClFCI gene, thereby causing a deepening of the watermelon flesh color.

[0110] Example 4

[0111] This example illustrates that overexpression of the ClFCI gene can deepen the color of watermelon flesh.

[0112] An overexpression vector for ClFCI was constructed using the pYBA1302 vector: the full-length CDS of ClFCI was amplified from the cDNA of watermelon material 97103 and then inserted into the EcoRI / XhoI site of pYBA1302. Following the watermelon genetic transformation method published in our laboratory (Zhang Jie; Guo Shaogui; Ren Yi; Zhang Haiying; Gong Guoyi; Zhou Ming; Wang Guizhang; Zong Mei; He Hongju; Liu Fan; Xu Yong*; High-level expression of a novel chromoplast phosphate transporter ClPHT4; 2is required for flesh color development in watermelon, New Phytologist, 2017, 213(3):1208-1221.), Agrobacterium carrying the target plasmid (FCI-OE) was transformed into watermelon material L600. The successful transformation of watermelon by the overexpression vector was confirmed using specific primers (upstream primer for the 35S promoter and downstream primer for the ClFCI gene) and Bar test strips. After obtaining the homozygous T2 line, the fruit pulp color phenotype was observed.

[0113] The upstream primer for the 35S promoter and the downstream primer for the ClFCI gene are as follows:

[0114] 35S-F: 5'-GAAGTTTCATTTCATTTGGAGAGG-3';

[0115] ClFCI-R: 5'-ACTTGAAACCTCAACAATCTC-3'.

[0116] By constructing the trans-ClFCI gene overexpression lines, observing the phenotype of the offspring pulp color, it is found that (as shown in Figure 8, wherein the first "control" is a blank control, and the following three are different transgenic lines) overexpression of the ClFCI gene in the shallow pulp color variety L600 can make the pulp color of the watermelon pulp deepen, and the detection finds that the carotenoid content in the pulp is significantly improved. Therefore, ClFCI transgenic overexpression can improve the deepening of the watermelon pulp color.

[0117] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. Application of watermelon ClFCI gene in controlling flesh color of watermelon.

2. A method for deepening flesh color of watermelon, comprising the operation of overexpressing ClFCI gene in watermelon plant.

3. The method of claim 2, wherein the operation of overexpressing ClFCI gene in watermelon plant comprises the operation of constructing overexpression vector of ClFCI by using pYBA1302 vector, and then transforming the overexpression vector into watermelon plant.

4. A tandem repeat sequence related to control of flesh color of watermelon, wherein the tandem repeat sequence is located in the promoter region of watermelon ClFCI gene, has a length of 1258 bp, and the sequence composition is shown as sequence 1 in the sequence table.

5. Application of the tandem repeat sequence of the promoter region of watermelon ClFCI gene in claim 4 in identifying flesh color of watermelon, wherein the tandem repeat sequence has a length of 1258 bp, and the sequence composition is shown as sequence 1 in the sequence table; when 1 tandem repeat sequence is detected, the flesh color of the watermelon to be tested is light; when 2 tandem repeat sequences are detected, the flesh color of the watermelon to be tested is intermediate or dark; and when 3 or more tandem repeat sequences are detected, the flesh color of the watermelon to be tested is dark.

7. The method of claim 6, wherein the method for identifying flesh color of watermelon comprises using the genomic DNA of the watermelon to be tested as a template, and performing PCR amplification by using the following primers: an upstream primer shown as sequence 6: 5'-CAAGGATAATTTTAAAATAATG-3'; and a downstream primer shown as sequence 7: 5'-ATATACTAATATAATTTGTAGGG-3'; if the amplified fragment contains 1 tandem repeat sequence shown as sequence 1, the flesh color of the watermelon to be tested is light; if the amplified fragment contains 2 tandem repeat sequences shown as sequence 1, the flesh color of the watermelon to be tested is intermediate or dark; and if the amplified fragment contains 3 or more tandem repeat sequences shown as sequence 1, the flesh color of the watermelon to be tested is dark.

6. A method for identifying the depth of flesh color of watermelon, the method comprising the operation of detecting the number of tandem repeats in the promoter region of the ClFCI gene of the watermelon to be tested; wherein, 8. The method of claim 6, wherein the method for identifying flesh color of watermelon comprises using the genomic DNA of the watermelon to be tested as a template, and performing PCR amplification by using the following primers: an upstream primer shown as sequence 6: 5'-CAAGGATAATTTTAAAATAATG-3'; and a downstream primer shown as sequence 8: 5'-GTAAAGATGGGTTGGGTTGTTTAC-3'; if the amplified fragment contains 1 tandem repeat sequence shown as sequence 1, the flesh color of the watermelon to be tested is light; if the amplified fragment contains 2 tandem repeat sequences shown as sequence 1, the flesh color of the watermelon to be tested is intermediate or dark; and if the amplified fragment contains 3 or more tandem repeat sequences shown as sequence 1, the flesh color of the watermelon to be tested is dark.

9. The method of claim 8, wherein the method for identifying flesh color of watermelon comprises using the genomic DNA of the watermelon to be tested as a template, and performing PCR amplification by using the following primers: ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ Upstream sequence shown in SEQ ID NO: 6: 5'-CAAGGATAATTTTAAAATAATG-3'; Downstream sequence shown in SEQ ID NO: 8: 5'-GTAAAGATGGGTTGGGTTGTTTAC-3'; If the amplified fragment is 3146 bp in size, it contains one tandem repeat sequence shown in SEQ ID NO: 2; If the amplified fragment is 4404 bp in size, it contains two tandem repeat sequences shown in SEQ ID NO: 1; If the amplified fragment is 5662 bp in size, it contains three tandem repeat sequences shown in SEQ ID NO: 1; If the amplified fragment is 6920 bp in size, it contains four tandem repeat sequences shown in SEQ ID NO: 1.

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