Genes and molecular markers related to chlorophyll synthesis in eggplant fruit and their applications

Abstract

The present invention belongs to the technical field of molecular biology, and discloses a gene related to chlorophyll synthesis in eggplant fruit, a molecular marker and its application, and specifically discloses a gene related to chlorophyll synthesis in eggplant fruit, the gene is named SmAPRR2-Like, and the nucleotide sequence of the SmAPRR2-Like coding region is shown in SEQ ID NO: 1. The present invention firstly determines SmAPRR2-like as a candidate gene for determining chlorophyll synthesis in eggplant fruit based on the annotation and screening of candidate genes in the fine positioning interval of the eggplant green fruit color Gv1 gene, and through VIGS verification; then, through the amplification of DNA and cDNA sequences of the SmAPRR2-like gene of the father and mother, it is found that the gene has amplified polymorphism between the white fruit color mother and the green fruit color father, and on this basis, the internal molecular marker of the SmAPRR2-Like gene is developed.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology, and in particular relates to genes and molecular markers related to chlorophyll synthesis in eggplant fruits and applications thereof. Background Art

[0002] Eggplant (Solanum melongena L.) belongs to the genus Solanum (Solanum L.) of the Solanaceae family, and occupies an important position in the production of Solanaceae vegetables. Eggplant is a fruit product and is one of the world's top ten healthy vegetables recommended by WHO. The color of eggplant peel (hereinafter referred to as fruit color) is rich and varied, including purple-black, purple-red, green, white, pink, etc. Since different regions have different fruit color selection habits, fruit color is an important breeding target in the eggplant breeding process. Eggplant fruit color inheritance is a complex trait controlled by multiple genes. Among them, anthocyanins and chlorophyll are the main pigments that determine eggplant fruit color. The fruit skin of purple-red and purple-black eggplants is rich in anthocyanins, the skin of green fruit eggplants is rich in chlorophyll, and white fruit eggplants have no anthocyanin and chlorophyll synthesis visible to the naked eye. At the same time, in the genetic research on the relationship between eggplant fruit color and chlorophyll, Tatebe et al. (1939) found that the synthesis of chlorophyll in eggplant peel is controlled by the Gv gene (Tatebe T. On inheritance of color in Solanum melongena L. Japanese Journal of Genetics, 1939, 15: 261-271; Tatebe T. Further studies on inheritance of color in Solanum melongena Linn. Japanese Journal of Genetics, 1944, 20: 1~7).

[0003] In higher plants, the synthesis of chlorophyll starts from glutamyl-tRNA, and after 19 steps of reaction, chlorophyll a and chlorophyll b are finally generated. There are a total of 15 enzymes in the middle, which are encoded by 27 genes. Any problem with any gene in this process may cause changes in the activity of the enzyme, resulting in the accumulation of excessive intermediates, causing oxidative damage to the plant, and even cell death, thereby triggering the generation of different green-deficient mutants. In the eggplant breeding practice, the applicant found that a green fruit color inbred line 19143 (male parent) and a white flower and white fruit color inbred line 19141 (female parent) were hybridized, and the green fruit color and white fruit color single plant separation ratio of the F2 segregation population was 3:1.

[0004] Functional molecular markers (FMs) are dominant molecular markers developed based on polymorphic sequences in specific regions of functional genes that are closely related to the phenotype. Such markers can directly and quickly determine the presence or absence of alleles under different genetic backgrounds, greatly improving the efficiency of molecular marker-assisted selection. Summary of the invention

[0005] The first aspect of the present invention aims to provide a gene related to chlorophyll synthesis in eggplant fruit.

[0006] The second aspect of the present invention aims to provide a functional molecular marker related to chlorophyll synthesis in eggplant fruit.

[0007] The third aspect of the present invention aims to provide a primer set for amplifying the functional molecular marker of the second aspect.

[0008] The fourth aspect of the present invention aims to provide a kit.

[0009] The purpose of the fifth aspect of the present invention is to provide the use of the functional molecular marker of the second aspect of the present invention, the primer set of the third aspect and / or the kit of the fourth aspect.

[0010] The sixth aspect of the present invention aims to provide a molecular marker breeding method for eggplant fruit color.

[0011] The seventh aspect of the present invention aims to provide a method for identifying or assisting in identifying the color of eggplant fruit.

[0012] The purpose of the eighth aspect of the present invention is to provide a method for detecting the eggplant ginkgo color gv1gv1 genotype.

[0013] In order to achieve the above object, the technical solution adopted by the present invention is:

[0014] In a first aspect, the present invention provides a gene related to chlorophyll synthesis in eggplant fruit, the gene is named SmAPRR2-Like, and the nucleotide sequence of the SmAPRR2-Like coding region is shown in SEQ ID NO:1.

[0015] Preferably, the CDS of SmAPRR2-Like is 1674 bp in length, encoding 557 amino acids, including a response regulation receiving domain (20 to 129 amino acids) and a Myb-like DNA binding domain (318 to 368 amino acids).

[0016] The second aspect of the present invention provides a functional molecular marker related to chlorophyll synthesis in eggplant fruit, wherein the functional molecular marker is characterized by a functional deficiency of the SmAPRR2-Like gene, i.e., the SmAPRR2-Like gene fragment of the first aspect cannot be amplified.

[0017] The third aspect of the present invention provides a primer set for amplifying the functional molecular marker of the second aspect of the present invention, the primer set comprising primer set 1 and / or primer set 2, the primer set 1 comprising an upstream primer as shown in sequence SEQ ID NO:8 and a downstream primer as shown in SEQ ID NO:9; the primer set 2 comprises an upstream primer as shown in SEQ ID NO:12 and a downstream primer as shown in SEQ ID NO:13.

[0018] A fourth aspect of the present invention provides a kit comprising the primer pair of the third aspect of the present invention.

[0019] Preferably, the kit also includes common reagents for PCR amplification.

[0020] The fifth aspect of the present invention provides the use of the functional molecular marker of the second aspect of the present invention, the primer set of the third aspect of the present invention and / or the kit of the fourth aspect of the present invention in any one of (1) to (4):

[0021] (1) Breeding or assisting in breeding eggplant varieties with different colors;

[0022] (2) preparing products for breeding or assisting in breeding eggplant varieties of different colors;

[0023] (3) Identify or assist in identifying the color of eggplant fruit;

[0024] (4) Prepare products for identifying or assisting in identifying the color of eggplant fruit.

[0025] Preferably, the fruit color includes green fruit color and white fruit color.

[0026] The sixth aspect of the present invention provides a molecular marker breeding method for eggplant fruit color, comprising the step of detecting the functional molecular marker of the second aspect of the present invention or performing molecular marker detection using the primer set of the third aspect of the present invention or the kit of the fourth aspect of the present invention.

[0027] A seventh aspect of the present invention provides a method for identifying or assisting in identifying eggplant fruit color, wherein the primer set of the third aspect of the present invention or the kit of the fourth aspect of the present invention is used to perform PCR amplification on the eggplant genome to be detected to obtain a PCR product, and the eggplant fruit color is identified based on the fragment size of the PCR product.

[0028] Preferably, the reaction procedure of the PCR amplification is 90-95°C for 2-5 min; 90-95°C for 20-40 ses, 50-55°C for 1-3 min, 70-74°C for 1-4 min, 35 cycles; 70-75°C for 10-15 min.

[0029] Preferably, the concentration of the upstream and downstream primers in the PCR amplification reaction system is 0.1-0.3 mM.

[0030] Preferably, if primer set 1 contains a 706bp amplification band, the fruit color of the eggplant plant is green; if primer set 2 has no amplification band at 706bp, the fruit color of the eggplant plant is white; if primer set 2 contains a 1303bp amplification band, the fruit color of the eggplant plant is green; if primer set 2 has no amplification band at 1303bp, the fruit color of the eggplant plant is white.

[0031] The eighth aspect of the present invention provides a method for detecting the gv1gv1 genotype of eggplant ginkgo color, using the primer set of the second aspect of the present invention to perform PCR amplification on the DNA of the eggplant plant to be tested, and perform electrophoresis detection. If the target band is not amplified, the genotype of the plant is gv1gv1.

[0032] Preferably, the reaction procedure of the PCR amplification is 90-95°C for 2-5 min; 90-95°C for 20-40 ses, 50-55°C for 1-3 min, 70-74°C for 1-4 min, 35 cycles; 70-75°C for 10-15 min.

[0033] Preferably, the concentration of the upstream and downstream primers in the PCR amplification reaction system is 0.1-0.3 mM.

[0034] The beneficial effects of the present invention are:

[0035] The present invention firstly determines SmAPRR2-like as a candidate gene for determining chlorophyll synthesis in eggplant fruit based on annotation and screening of candidate genes in the fine positioning interval of the eggplant green fruit color Gv1 gene and verification through VIGS; then, through DNA and cDNA sequence amplification of the paternal and maternal SmAPRR2-like genes, it is found that the gene has an amplified polymorphism between the white fruit color female parent and the green fruit color male parent; on this basis, an internal molecular marker (functional molecular marker) of the SmAPRR2-Like gene is developed.

[0036] The present invention designs a specific primer set based on the internal molecular markers of the SmAPRR2-like gene. The primer set can accurately distinguish the genotypes of individual plants in a segregating population under a specific genetic background (such as the F2 population used for positioning and its pedigree segregating offspring), thereby making a judgment on the fruit color phenotype. At the same time, it can distinguish between green fruit color and white fruit color resources in natural populations, and explore white fruit color eggplant resources with gv1gv1 gene mutations.

[0037] The intragenic molecular markers and molecular marker amplification primers of the present invention can be simply, quickly and accurately applied to eggplant fruit color breeding practice, thereby accelerating the improvement process of eggplant fruit color traits.

[0038] The screening results of candidate genes for the fine positioning interval of the eggplant green fruit color Gv1 gene in the present invention lay a foundation for the cloning of the eggplant green fruit color Gv1 candidate gene SmAPRR2-Like and the study of its role in the synthesis of anthocyanins in eggplant fruits; the molecular markers within the SmAPRR2-Like gene and the molecular marker amplification primers can be simply, quickly and accurately applied to the practice of eggplant fruit color breeding, accelerating the improvement process of eggplant fruit color traits. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 From left to right are the female parent 19141, the male parent 19143 and 20183F1; among them, the fruit color of the female parent 19141 is white, and no anthocyanin synthesis was observed in the hypocotyl, leaf veins, flowers and sepals during the entire growth period; the fruit color of the male parent 19143 is green, and no anthocyanin synthesis was observed in the hypocotyl, leaf veins, flowers and sepals during the entire growth period; 20183F1 is the hybrid F1 generation of the female parent 19141 and the male parent 19143, and no anthocyanin synthesis was observed in the hypocotyl, leaf veins, flowers and fruit peel.

[0040] Figure 2 The phenotype of eggplant fruit after VIGS silencing of the SmAPRR2-Like gene related to fruit chlorophyll synthesis; A is the untreated control, B is the negative control (pTRV2), and C is the silencing effect of the pTRV2-SmAPRR2-Like vector.

[0041] Figure 3 The amplification results of primer combination P3 and primer combination P5 of SmAPRR2-Like gene on paternal 19143, maternal 19141, and 20 20183F2 individual plants with different fruit colors; wherein A is the amplification result of primer combination P3, B is the amplification result of primer combination P5, and C is the fruits corresponding to paternal 19143, maternal 19141, and 20 20183F2 individual plants.

[0042] Figure 4The results of amplification of the primer combination P3 of the SmAPRR2-Like gene in various materials of the green fruit color and white fruit color natural populations; among them, A is the material of the green fruit color natural population, and B is the material of the white fruit color natural population. DETAILED DESCRIPTION

[0043] The present invention is further described in detail below through specific examples.

[0044] It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0045] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

[0046] The common molecular biology experimental techniques used in the following examples, such as DNA extraction, PCR amplification, etc., are usually performed according to conventional methods unless otherwise specified. For details, please refer to "Molecular Cloning Experiment Guide" (3rd edition) (Sambrook J, Russell DW, Janssen K, Argentine J. Translated by Huang Peitang et al., 2002, Beijing: Science Press), or implement according to the operating methods recommended by the manufacturer.

[0047] Example 1

[0048] The inventors used Super-BSA technology to locate the eggplant green fruit color Gv1 gene. Based on the initial location, the green fruit color gene Gv1 was further located between SNP80595470 and SNP80724027 through InDel and SNP molecular marker encryption ( Figure 3), with a size of 173.2kb. A total of 16 candidate genes were annotated within the 173.2kb Gv1 gene localization interval. Sequence comparison analysis found that the coding region sequences of the 16 genes had no differences that affected the gene-encoded proteins, except for Smechr0802033 (encoding a mitochondrial protein of unknown function) which had a missense mutation between the two parents. Using transcriptome sequencing information, it was found that the expression of 3 of the 16 candidate genes was different between the green fruit color male parent 19143 and the white fruit color female parent 19141 (differential gene screening criteria: |log2(FoldChange)|>0&padj<=0.05), of which 1 gene (Smechr0802018) was up-regulated in green fruit peel, and the other 2 genes (Smechr0802030, encoding power-related protein; Smechr0802033, encoding non-characteristic mitochondrial protein ymf11) were down-regulated. The candidate gene Smechr0802018 encodes a two-component response regulator protein, named SmAPRR2-Like ((Arabidopsis Pseudo Response Regulator 2, APRR2)-Like), which is most likely the candidate gene for the Gv1 locus of green fruit color in eggplant.

[0049] Example 2 Genes and Molecular Verification Related to Chlorophyll Synthesis in Eggplant Fruit

[0050] 1. Amplification of SmAPRR2-Like gene cDNA and DNA sequences

[0051] Based on the reference genome (https: / / db.cngb.org / search / project / CNP0000734) cDNA sequence (EGP19168), primers were designed in sequence for SmAPRR2-Like gene amplification and splicing. The amplification primers are shown in Table 1. The purified inbred lines 19141 of white-fruited eggplant and 19143 of green-fruited eggplant, which were bred by the Vegetable Research Institute of Guangdong Academy of Agricultural Sciences through multiple generations of strict bagging self-pollination, were selected. Figure 1) and cDNA of fruit peel in the commercial fruit period were used as templates, and the designed primers were used to amplify them. The results are shown in Table 1. All primer combinations of green fruit color 19143 have DNA and cDNA amplifications that meet the expected fragment size, while in white fruit color 19141, primer combinations P3 and P5 have no DNA amplification of the corresponding section of SmAPRR2-Like, and P3, P4, P5 and P6 have no cDNA amplification of the corresponding section. Using P2 upstream primer F and P4 downstream primer R for amplification, 2206bp of DNA and 1119bp of cDNA were amplified in 19143, while no corresponding DNA and cDNA amplification was performed in 19141. Therefore, it is speculated that there are two large fragments of SmAPRR2-Like gene in white fruit color 19141 in the amplification interval of P3 primer combination and the amplification interval of P5 primer combination, resulting in interruption of gene transcription and loss of gene function.

[0052] Table 1 SmAPRR2-Like gene amplification primers and amplification results

[0053]

[0054] Note: “+” represents amplification, and “-” represents no amplification.

[0055] 2. SmAPRR2-Like gene coding region and amino acid structure characteristics

[0056] The results of analyzing the mAPRR2-Like gene showed that the SmAPRR2-Like gene consisted of 12 exons and 11 introns. The nucleotide sequence of the SmAPRR2-Like gene coding region in Lvguose 19143 is shown in SEQ ID NO: 1, with a total length of 1674 bp, encoding 557 amino acids, including the response regulatory receiving domain (20 to 129 amino acids) at the N-terminus of the two-component response regulatory factor and the Myb-like DNA binding domain (318 to 368 amino acids) at the C-terminus.

[0057] 3. Verification of SmAPRR2-Like gene as a gene related to fruit chlorophyll synthesis

[0058] The VIGS (Virus Induced Gene Silence) gene silencing system was further used to verify that the SmAPRR2-Like gene was a gene related to fruit chlorophyll synthesis. According to the CDS sequence of SmAPRR2-Like related to eggplant peel chlorophyll synthesis, specific primers were designed: SmRR-EF6: 5'-GCTAGCGAATTCATCGGGCTGTGATAAATACC-3' (SEQID NO: 16), SmRR-BR6: 5'-GCTAGCGGATCCGGGCAGTCATTACTTTGAGA-3' (SEQ ID NO: 17), and EcoR I and BamH I restriction sites were added to the 5' ends of the upstream and downstream primers, respectively, to construct the pTRV2-SmAPRR2-Like silencing expression vector (for the specific vector construction process and infection process, refer to the patent disclosure document CN201910120540.6). The hybrid combination 20183F1 (green fruit color) was prepared with the purified inbred line 19141 of white fruit color as the female parent and the purified inbred line 19143 of green fruit color as the male parent. Figure 1 ) was used as plant material (no anthocyanin synthesis was observed in the hypocotyl, veins, flowers, and sepals of the male, female, and F1 during the entire growth period). The constructed pTRV2-SmAPRR2-Like silencing expression vector was transformed with Agrobacterium and then infected with 20183F1 young fruits (the infection process refers to patent disclosure document CN201910120540.6), interfering with the expression of SmAPRR2-Like in the fruit, and observing the color change of the fruit skin one week after injection. At the same time, pTRV2 (equal volumes of TRV1 vector and TRV2 vector mixed) Agrobacterium bacterial solution was used as a negative control to infect eggplant fruits.

[0059] The results are as follows Figure 2 As shown, the fruit skin color of eggplant fruits infected with negative control pTRV2 Agrobacterium solution did not change significantly compared with the uninfected fruit (A in Figure 2) ( Figure 2 (B). After the VIGS vector pTRV2-SmAPRR2-Like, which was constructed with the gene SmAPRR2 related to chlorophyll synthesis in eggplant fruit, was transformed with Agrobacterium, the chlorophyll synthesis in the pericarp around the injection infection area was significantly inhibited, and the pericarp color changed from green to white-green ( Figure 2 C), indicating that silencing the SmAPRR2-Like gene in eggplant peel significantly inhibited chlorophyll synthesis in eggplant peel. The above results verify that the SmAPRR2-Like gene is closely related to chlorophyll synthesis in eggplant fruit.

[0060] Example 3 Development of molecular markers within the SmAPRR2-Like gene and their application in F2 segregating populations

[0061] The primer combination P3 and P5 of the SmAPRR2-Like gene (Table 1) was used to amplify individual plants with different fruit colors, including the male parent (purified inbred line 19143 with green fruit color), the female parent (purified inbred line 19141 with white fruit color eggplant) and 20183F2 (obtained by self-pollination of 20183F1, the segregation ratio of green fruit color and white fruit color plants in the F2 segregation generation was 3:1 according to the chi-square test).

[0062] The results showed that primer combination P3 amplified the 706bp target band in both the green fruit parent and the F2 green fruit plant, but not in the white fruit parent and the F2 white fruit plant ( Figure 3 A and C); Primer combination P5 amplified the 1303bp target band in both the green fruit parent and the F2 green fruit plant, but not in the white fruit parent and the F2 white fruit plant ( Figure 3 In B and C), the results of fruit color differentiation of 20 F2 plants are 100% consistent. This shows that the SmAPRR2-Like gene intragenic marker primer combinations P3 and P5 can efficiently and accurately differentiate between green fruit color and white fruit color plants, and the amplification results of the two are consistent.

[0063] Example 4 Application of SmAPRR2-Like intragenic molecular marker primer combination P3 in natural population identification

[0064] PCR detection was performed on 80 natural populations of green fruit color and white fruit color using primer combination P3 of SmAPRR2-Like gene. Specifically, the reaction system of PCR amplification included 10 μL 2×PCR mix, 1 μL DNA template (500 ng / μL), 0.5 μL 10 mM SmAPRR2-Like gene specific labeled upstream and downstream primers (primer combination P3), 8 μL ddH2O; the reaction program of PCR amplification was 94℃3min; 94℃30ses, 51℃1min, 72℃1min, 35 cycles; then 72℃10min.

[0065] from Figure 4 As shown in Table 2, all green fruit materials (such as green fruit parent 19143) had amplified target bands of 706 bp, 22 of 26 white fruit materials had no amplification, and 4 white fruit materials (19142, 21260, 21393 and E4701) had amplification. Further cloning and sequencing of 4 white fruit materials, including 19142, 21260, 21393 and E4701, proved that there were other InDels in the SmAPRR2-Like gene that caused frameshift mutations. It can be seen that the white fruit eggplant resources can be explored in natural populations through molecular markers in the SmAPRR2-Like gene.

[0066] Table 2 PCR detection results of the SmAPRR2-Like gene intragenic molecular marker primer combination P3 in natural populations

[0067]

[0068]

[0069] Note: "Yes" indicates that the primer combination P3 has a 706bp target band amplified, and "No" indicates that the primer combination P3 has no band amplified.

[0070] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. Use of a kit for identifying or assisting in identifying green and white fruit colors of eggplant in any one of the following (1) to (4); (1) Breeding or assisting in breeding eggplant varieties with different colors; (2) preparing products for breeding or assisting in breeding eggplant varieties of different colors; (3) Identify or assist in identifying the color of eggplant fruit; (4) preparing products for identifying or assisting in identifying the color of eggplant fruit; It is characterized in that The eggplants in (1) to (4) are F2 segregating populations with the white-fruited eggplant purified inbred line 19141 as the female parent and the green-fruited purified inbred line 19143 as the male parent; The kit includes a primer set, which is a combination of an upstream primer as shown in SEQ ID NO:8 and a downstream primer as shown in SEQ ID NO:9, or a combination of an upstream primer as shown in SEQ ID NO:12 and a downstream primer as shown in SEQ ID NO:13; The specific selection or identification criteria are as follows: If the primer set used is a combination of an upstream primer as shown in SEQ ID NO:8 and a downstream primer as shown in SEQ ID NO:9, and after amplification, an amplified band of 706 bp is contained, the fruit color of the eggplant plant is green; if no amplified band is contained at 706 bp, the fruit color of the eggplant plant is white; If the primer set used is a combination of an upstream primer as shown in SEQ ID NO: 12 and a downstream primer as shown in SEQ ID NO: 13, and a 1303 bp amplified band is contained after amplification, the fruit color of the eggplant plant is green; if there is no amplified band at 1303 bp, the fruit color of the eggplant plant is white.

2. A method for identifying or assisting in identifying the green fruit color and the white fruit color of eggplant, characterized in that: Using the kit in the application of claim 1 to perform PCR amplification on the eggplant genome to be detected to obtain a PCR product, and identifying the eggplant fruit color according to the fragment size of the PCR product; The reaction procedure of the PCR amplification is 90-95°C for 2-5 min; 90-95°C for 20-40 ses, 50-55°C for 1-3 min, 70-74°C for 1-4 min, 35 cycles; 70-75°C for 10-15 min; The results are determined as follows: If the primer set used is a combination of an upstream primer as shown in SEQ ID NO:8 and a downstream primer as shown in SEQ ID NO:9, and after amplification, an amplified band of 706 bp is contained, the fruit color of the eggplant plant is green; if no amplified band is contained at 706 bp, the fruit color of the eggplant plant is white; If the primer set used is a combination of an upstream primer as shown in SEQ ID NO: 12 and a downstream primer as shown in SEQ ID NO: 13, and after amplification, an amplified band of 1303 bp is contained, the fruit color of the eggplant plant is green; if no amplified band is found at 1303 bp, the fruit color of the eggplant plant is white; The eggplants to be tested are an F2 segregating population with the white-fruited eggplant purified inbred line 19141 as the female parent and the green-fruited eggplant purified inbred line 19143 as the male parent.

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