A molecular marker closely linked to the light semi-sensitivity trait of eggplant and application thereof

Abstract

The application discloses a molecular marker closely linked with a solanum melongena light semi-sensitivity character, a primer sequence of the molecular marker and application of the molecular marker. The molecular marker is a SmMYB113-Indel molecular marker, which can be used for detecting polymorphism or genotypes of SmMYB113 in a solanum melongena genome, thereby identifying or assisting in identifying whether anthocyanin synthesis in solanum melongena pericarp depends on light. The SmMYB113-Indel molecular marker exists at a position of -200 bp in a SmMYB113 promoter region, and contains 10-base insertion and 5 SNP variations. The molecular marker provided by the application has high stability, can be used for screening of the light semi-sensitivity type at a solanum melongena seedling stage in a simple and rapid manner, and a backcross breeding method can be used for rapidly cultivating a solanum melongena variety improvement line of the light semi-sensitivity type.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to an Indel molecular marker closely linked to the photosensitive phenotype of eggplant and its application. Background Technology

[0002] Anthocyanins are naturally occurring water-soluble pigments. Due to their antioxidant activity, they not only serve as a protective agent for plants against biotic and abiotic stresses, but also offer medicinal and health benefits in humans, particularly in combating diabetes, cancer, inflammation, bacteria, obesity, and preventing cardiovascular diseases. The higher the anthocyanin content of a plant, the greater its cultivation and economic value.

[0003] Anthocyanins are one of the main coloring substances in eggplant fruit, and their synthesis is light-induced (Araguirang and Richter 2022). Low-light environments such as greenhouse cultivation, open-field overwintering cultivation, and dense planting often lead to poor coloring of eggplant peels, seriously affecting fruit quality and posing a pressing problem in eggplant production. Based on the light dependence of eggplant peel coloring, purple eggplants can be classified into "photosensitive," "photosensitive-semi-photosensitive," and "non-photosensitive" types. In "photosensitive" eggplants, anthocyanin synthesis is highly susceptible to light conditions, resulting in poor or no coloring under low light. In "photosensitive-semi-photosensitive" eggplants, anthocyanin synthesis is relatively easily affected by light conditions, leading to uneven coloring and "shaded" areas under insufficient light. In "non-photosensitive" eggplants, anthocyanin synthesis is not significantly affected by light conditions, and the fruit can still color normally under low light (He et al. 2022). Although numerous studies have shown... SmMYB113 These genes are key to the regulation of anthocyanin synthesis in eggplant fruit (Yang et al. 2022a, You et al. 2022, Li et al. 2023), but current research results still cannot explain why "photosensitive" and "non-photosensitive" eggplants can still synthesize anthocyanins under dark conditions. To date, the role of anthocyanins in eggplant synthesis remains a key focus. SmMYB113 The screening of superior allelic variants and their molecular markers is almost non-existent, which greatly hinders research. SmMYB113 Application in improving eggplant fruit color under low light conditions. Summary of the Invention

[0004] To address the problems existing in the prior art, the purpose of this invention is to provide a molecular marker closely linked to the photosensitive phenotype of eggplant and its application for detection. SmMYB113Superior allelic variations in genes enable rapid and efficient screening and identification of photosensitive hemi-sensitive traits in eggplant seedlings, accelerating the eggplant breeding process. The molecular markers developed in this invention, closely linked to photosensitive hemi-sensitive traits in eggplant, are of great significance for improving the appearance quality of eggplant fruits and revealing the molecular mechanisms of anthocyanin accumulation and metabolism in eggplant under low-light conditions.

[0005] In a first aspect, the present invention provides a SmMYB113-Indel molecular marker closely linked to the "semi-photosensitive" trait of eggplant, wherein the SmMYB113-Indel molecular marker is located at -200 bp in the SmMYB113 promoter region of eggplant, and its nucleic acid sequence is shown in SEQ ID NO:5;

[0006] The criteria for determining the "semi-photosensitive" trait are that a small amount of anthocyanins can be synthesized in the pericarp under dark or low light conditions and in the dark part of the lower part of the calyx, and the pericarp color is significantly different from that under normal light conditions.

[0007] The SmMYB113-Indel molecular marker contains 10 base insertions and 5 SNP variations.

[0008] In a second aspect, the present invention provides the use of the above-described SmMYB113-Indel molecular marker in at least one of the following (1) or (2):

[0009] (1) Application of identifying the photosensitivity of anthocyanin synthesis in eggplant pericarp;

[0010] (2) Application in screening “photosensitive” eggplant.

[0011] In a third aspect, the present invention provides primer pairs for amplifying the above-mentioned SmMYB113-Indel molecular marker, the nucleic acid sequences of said primer pairs being shown in SEQ ID NO.6-SEQ ID NO.7.

[0012] In a fourth aspect, the present invention provides the use of the above primer pair in any of the following (1)-(3):

[0013] (1) Identify the light sensitivity of anthocyanin synthesis in eggplant pericarp;

[0014] (2) Screening for "photosensitive" eggplants;

[0015] (3) Prepare a kit for detecting “photosensitive” eggplant.

[0016] In a fifth aspect, the present invention provides a kit for detecting photosensitive eggplant, the kit containing the aforementioned primer pair.

[0017] The application of the above-mentioned reagent kit in screening "photosensitive" eggplants also falls within the scope of protection of this invention.

[0018] In a sixth aspect, the present invention provides a molecular marker-assisted breeding method for photosensitive eggplant, wherein the method uses the above-mentioned kit to perform molecular markers on the target eggplant.

[0019] The molecular marker-assisted selection method includes the following steps:

[0020] (1) Use the SmMYB113-Indel molecular marker primers in the kit to perform PCR amplification on the genomic DNA of the test material to obtain PCR amplification products;

[0021] (2) PAGE gel electrophoresis was used to detect the polymorphism of PCR amplification products, and the photosensitive type of eggplant in the test material was determined based on the electrophoresis results.

[0022] The criteria for determining the electrophoresis results are as follows: when the detection result is a single 66bp fragment, the plant to be tested is a homozygous "photosensitive" or "non-photosensitive" eggplant; when the detection result is two fragments, 66bp and 76bp, the plant to be tested is a heterozygous "photosensitive-semi-photosensitive" eggplant; and when the detection result is a single 76bp fragment, the plant to be tested is a homozygous "photosensitive-semi-photosensitive" eggplant.

[0023] The beneficial effects of this invention are:

[0024] 1. The Indel region provided by this invention is located at -200 bp in the eggplant SmMYB113 promoter region, and its polymorphism is...

[0025] Whether it contains a 10-base insertion and a 5-SNP variation, if present, indicates a "photosensitive semi-sensitive" eggplant. Using the Indel region described in this invention, "photosensitive semi-sensitive" eggplants can be distinguished from "photosensitive" and "non-photosensitive" eggplants.

[0026] 2. This invention uses SmMYB113-Indel molecular marker primers to detect allelic variations of the SmMYB113 gene related to anthocyanin synthesis in eggplant. The identification of samples can be completed simply by DNA extraction, PCR-specific amplification, PAGE gel electrophoresis or agarose gel electrophoresis, which facilitates the detection and screening of photosensitive eggplant varieties or lines and can greatly accelerate the breeding process of high-quality eggplant varieties. Attached Figure Description

[0027] Figure 1 This study investigated the color, anthocyanin content, and expression levels of anthocyanin synthesis-related genes in the shaded (below the calyx) and illuminated (middle of the pericarp) parts of eggplant fruits with different photosensitivity. PS: "Photosensitive" eggplant; SPS: "Photosemi-photosensitive" eggplant; NPS: "Non-photosensitive" eggplant.

[0028] in, Figure 1 A is a schematic diagram showing the color comparison between the shaded (below the calyx) and the shaded (middle of the peel) parts of eggplant fruits with five different photosensitivity. Figure 1 B shows the color intensity of anthocyanins extracted from the shaded (below the calyx) and the exposed (middle of the pericarp) parts of eggplant fruits with five different photosensitivity levels. Figure 1 C represents the anthocyanin content in the shaded (below the calyx) and shaded (middle of the pericarp) parts of eggplant fruits with five different photosensitivity levels. Figure 1 D represents the fold difference in expression levels of anthocyanin synthesis-related genes between the light-exposed (middle of the pericarp) and dark-exposed (below the calyx) parts of eggplant fruits with five different photosensitivity levels.

[0029] Figure 2 Sequence alignment at -200 bp in the promoter region of eggplant gene SmMYB113. PS: "Photosensitive" eggplant; SPS: "Photosensitive-semi-photosensitive" eggplant; NPS: "Non-photosensitive" eggplant; WE108: White eggplant; GE133: Green eggplant; SmMYB113-V4.1: Eggplant genome sequence ("Photosensitive" eggplant).

[0030] Figure 3 Electrophoretic bands of the SmMYB113-Indel molecular marker in the PCR products of populations P1, P2, F1, and F2, where P1 represents the photosensitive eggplant line '76', P2 represents the white eggplant line '54', F1 is the first generation of hybridization between P1 and P2, F2 is the self-crossed offspring of F1, and the F2 photosensitive and F2 photosensitive populations represent 3 white eggplants, 4 green eggplants, 7 photosensitive plants, and 44 photosensitive plants randomly selected from the F2 population, respectively.

[0031] Figure 4 Electrophoretic bands of SmMYB113-Indel molecular marker in PCR products of natural eggplant populations.

[0032] Table 1 shows the statistical results of phenotypic and molecular marker detection in 91 natural eggplant populations. Detailed Implementation

[0033] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0034] The standard for "photosensitive" eggplants is that the pericarp cannot synthesize anthocyanins in dark or low light conditions and in the dark part of the calyx, but can synthesize anthocyanins under normal light conditions.

[0035] The standard for "photosensitive semi-sensitive" eggplant: In dark or low light conditions and in the part of the calyx that is not exposed to light, a small amount of anthocyanins can be synthesized in the peel, and the peel color is significantly different from that under normal light conditions;

[0036] The standard for "non-photosensitive" eggplants is that in dark or low light conditions and in the part of the calyx that is not exposed to light, the peel can synthesize more anthocyanins, and the color of the peel is not significantly different from that under normal light conditions.

[0037] The standard for white or green eggplants is that no anthocyanins can be synthesized in the peel, regardless of whether it is in darkness, low light, or normal light conditions, and the peel is white (white eggplant) or green (green eggplant).

[0038] To enable those skilled in the art to better understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments.

[0039] Example 1: Expression level analysis of SmMYB113 in different photosensitive eggplant types

[0040] The pericarp (the part not exposed to light) and the middle pericarp (the part exposed to light) of five commonly available eggplant cultivars—one photosensitive type (PS1), two photosensitive semi-photosensitive types (SPS1 and SPS2), and two non-photosensitive types (NPS1 and NPS2)—were used as materials. Figure 1 A), the anthocyanin content was determined respectively. Figure 1 (BC) It was found that the anthocyanin content in the peel of the sun-exposed parts of five eggplant varieties was significantly higher than that in the shade-free parts. Compared with two non-photosensitive eggplant varieties, the changes in anthocyanin content in the peel of photosensitive and photosemi-photosensitive eggplant varieties were more pronounced after exposure to light. Subsequent transcriptome sequencing analysis revealed known anthocyanin synthesis structural genes ( SmCHS , SmCHI , SmF3H , SmF3'5'H , SmDFR , SmANS , Sm3GT , Sm5GT ) and key regulatory genes for anthocyanin synthesis ( SmMYB113 , SmTT8 , SmWRKY44 The expression level of ) in the peel of the light-exposed parts was significantly higher than that in the dark-exposed parts, and SmMYB113 The trends in expression and anthocyanin content were significantly correlated among varieties. Figure 1 D). Based on the conclusion drawn from previous studies that "SmMYB113 is a key factor regulating anthocyanin synthesis in eggplant," it is speculated that... SmMYB113 The expression level of anthocyanins directly determines their content, therefore further analysis is needed. SmMYB113 Does the promoter sequence differ among different varieties?

[0041] Example 2: Amplification of the SmMYB113 gene promoter sequence

[0042] Genomic DNA was extracted from the five eggplant species using the conventional CTAB method. The promoter sequence of the SmMYB113 gene was amplified (2000 bp). The amplification primers used were the upstream primer proSmMYB113-F and the downstream primer proSmMYB113-R, and their nucleotide sequences are shown in SEQ ID No. 1-SEQ ID No. 2, respectively.

[0043] Upstream primer proSmMYB113-F: 5'-TTTTCATTGCAAAATCATAAAAATCTAAA-3'; SEQ ID No. 1.

[0044] Downstream primer proSmMYB113-R: 5'-TTTTATTTTATTTATAATATATAGTTTATTTCAAGTCAT-3'; SEQ ID No. 2.

[0045] After amplification, agarose gel electrophoresis was performed, and the amplified fragments were recovered, ligated into the pEASY-blunt Simple vector (purchased from Beijing TransGen Biotech Co., Ltd.), and sequenced. By comparing with the eggplant genome database (https: / / solgenomics.net / organism / Solanum_melongena / genome), two allelic variants of the SmMYB113 gene in eggplant were identified, with sequences shown as SEQ ID No. 3 or SEQ ID No. 4, respectively.

[0046] SEQ ID No. 3:

[0047]

[0048] SEQ ID No.4:

[0049]

[0050] Example 3: Polymorphism analysis of the mMYB113 gene promoter sequence

[0051] DNA was extracted from five photosensitive (PS1-5), five photosemi-photosensitive (SPS1-5), five non-photosensitive (NPS1-5), one white eggplant, and one green eggplant using the conventional CTAB method. The promoter sequence of SmMYB113 (2000 bp upstream of the ATG) was cloned and sequenced. The amplification primers were proSmMYB113-F (upstream) and proSmMYB113-R (downstream). Sequence alignment was performed using DNAMAN, and the results are as follows: Figure 2 As shown: The promoter sequence of SmMYB113 differs only between photosensitive eggplant and other eggplant varieties. Specifically, in photosensitive eggplant, the SmMYB113 promoter sequence (200 bp upstream of ATG) contains a 10-base insertion and a 5-base mutation. Figure 2 The nucleic acid sequence of the SmMYB113-Indel molecular marker is shown in SEQ ID NO. 5:

[0052] The nucleic acid sequence of the SmMYB113-Indel molecular marker is: CTACCTTTCGTGGGCAT; SEQ ID NO.5.

[0053] Example 4: Development of molecular markers for the mMYB113 gene

[0054] Based on the InDel region in the SmMYB113 promoter sequence, SmMYB113-Indel molecular marker primer pairs were designed. The nucleic acid sequences of the primer pairs are shown in SEQ ID NO.6-SEQ ID NO.7:

[0055] SmMYB113-Indel-F: 5'-GATTCCGCTGTGTCGTACAAGG-3'; SEQ ID NO. 6.

[0056] SmMYB113-Indel-R: 5'-AACAGAAACATCCAAACTAGCCCA-3'; SEQ ID NO. 7.

[0057] Example 5: Application Validation of MYB113-Indel Molecular Marker

[0058] 1) Construction of the F2 population

[0059] In this study, the high-generation pure inbred line '54' (P1) of white eggplant and the "photosensitive-semi-sensitive" eggplant line '76' (P2), both cultivated and preserved in our laboratory, were selected as hybrid parents to construct a segregating population. The F1 generation exhibited the "photosensitive-semi-sensitive" phenotype. Self-pollination of the F1 generation produced the F2 segregating population, with eggplant skin colors of white, green, and purple, in a segregation ratio of 1:3:12, consistent with the study by Zhang et al. (2020). The purple skin was further divided into "photosensitive-semi-sensitive" and "photosensitive" phenotypes, in a segregation ratio of 2:1. Therefore, the "photosensitive-semi-sensitive" trait in eggplant is a dominant trait controlled by a single gene, while the "photosensitive" trait is a recessive trait.

[0060] 2) SmMYB113-Indel molecular marker scanning F2 segregation population

[0061] Using single-plant DNA from the P1, P2, F1, and F2 populations as templates, PCR amplification was performed using the developed SmMYB113-Indel molecular marker primer pair. Electrophoresis and silver staining results were statistically analyzed. Figure 3 As shown, the SmMYB113-Indel molecular marker is closely linked to the "photosensitive" eggplant in the F2 population with an accuracy of 100%.

[0062] 3) SmMYB113-Indel molecular marker scanning of eggplant natural populations

[0063] Genomic DNA was extracted from 91 natural eggplant populations using the conventional CTAB method. PCR amplification of the InDel region of the SmMYB113 promoter sequence was performed using the SmMYB113-Indel molecular marker primer pair. Electrophoresis and silver staining results were statistically analyzed. Figure 4 As shown, the SmMYB113-Indel molecular marker is closely linked to the "photosensitive semi-sensitive" eggplant type trait in natural population materials, with an accuracy rate of 81.23% (the field resource trait observation results and molecular marker detection results in 91 eggplant natural population materials in Table 1 are consistent).

[0064] Table 1: Comparison of field resource trait observation results and molecular marker detection results in 91 natural eggplant populations

[0065]

[0066]

[0067] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. The application of the SmMYB113-Indel molecular marker in the identification or screening of "photosensitive" eggplant, characterized in that, The term "photosensitive" refers to the photosensitive nature of anthocyanin synthesis in eggplant pericarp. The SmMYB113-Indel molecular marker is located at -200 bp in the SmMYB113 promoter region of eggplant, and its nucleic acid sequence is shown in SEQ ID NO:5; The SmMYB113-Indel molecular marker was amplified using primer pairs with nucleotide sequences as shown in SEQ ID NO.6-SEQ ID NO.

7.

2. A method for identifying or screening "photosensitive" eggplant, characterized in that, Includes the following steps: (1) Using primer pairs with nucleotide sequences as shown in SEQ ID NO.6-SEQ ID NO.7, genomic DNA of the material to be tested is amplified by PCR to obtain PCR amplification products; the primer pairs are used to amplify the SmMYB113-Indel molecular marker as described in claim 1. (2) PAGE gel electrophoresis was used to detect the polymorphism of PCR amplification products, and the photosensitivity type of eggplant in the test material was determined based on the electrophoresis results; The term "photosensitive" refers to the photosensitive nature of anthocyanin synthesis in eggplant peel.

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