A molecular marker for chromosome structural variation related to tomato fruit color and its application

Abstract

This invention belongs to the field of plant genetic engineering and molecular biology, specifically relating to a molecular marker for chromosomal structural variation related to tomato fruit color and its application. The molecular marker for this chromosomal structural variation is an 8.4kb inversion on chromosome 3 of the white-fruited tomato mutant, with the inversion located at bases 4230030 to 4238459 of chromosome 3, and a reference genome version of SL4.0. Based on this molecular marker, specific primers are provided for identifying whether tomato hybrids have white fruit and whether they are heterozygous, as well as a method for identifying whether tomato hybrids have white fruit. Using the molecular marker, specific primers, and method provided by this invention, tomato materials with white fruit can be quickly selected, shortening the breeding cycle and improving breeding efficiency.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering and molecular biology technology, specifically relating to a molecular marker for chromosomal structural variation related to tomato fruit color and its application. Background Technology

[0002] tomato( Solanum lycopersicum Tomato (Solanum nigrum), belonging to the Solanaceae family and the Tomato genus, is a widely cultivated and important vegetable crop. It is highly popular due to its rich nutrition, delicious flavor, and vibrant color. Fruit color, as an important sensory quality, directly affects the marketability of the fruit.

[0003] The color of a tomato fruit is determined by both the flesh and the skin. During the ripening process of red tomatoes, chlorophyll gradually degrades, and chromoplasts are synthesized, transforming into plastids primarily composed of lycopene, resulting in red flesh. Tomato skin color is either yellow or transparent. Yellow skin is dominant over transparent, colorless skin, regulated by the transcription factor SlMYB12. When the gene is normally expressed, a large amount of flavonoids accumulates in the tomato skin, resulting in a yellow color, and ultimately, a red fruit. After a mutation in SlMYB12, the skin becomes colorless and transparent due to the lack of flavonoid accumulation, resulting in a pink fruit. White tomatoes have transparent skin, and the fruit is milky white or white, relatively soft, and has a better flavor and quality, making them suitable for breeding new white tomato varieties. Breeding white tomato varieties requires investigating fruit color during flowering and fruit setting, as well as during fruit ripening, which is labor-intensive and time-consuming.

[0004] With the development of molecular biology, marker-assisted selection breeding has become an important breeding technique. This technique allows for screening of hybrid offspring at the seedling stage, greatly reducing workload, improving breeding efficiency, and shortening the breeding cycle. Therefore, developing molecular markers for white tomato varieties can provide technical support for the breeding of new white tomato varieties. Summary of the Invention

[0005] To address the shortcomings of the existing technology, the purpose of this invention is to provide a molecular marker for chromosomal structural variations related to tomato fruit color and its application.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides the application of a detection reagent for a molecular marker of chromosomal structural variation in the identification of tomato fruit color, wherein the molecular marker of chromosomal structural variation is an 8.4 kb inversion occurring on chromosome 3 of the white tomato mutant, the physical location of the inversion being at bases 4230030 to 4238459 of chromosome 3, and the reference genome version is SL4.0.

[0007] Furthermore, the detection reagent includes primers that amplify molecular markers of chromosomal structural variations at inversion sites.

[0008] In a second aspect, the present invention provides specific primers for detecting molecular markers of chromosomal structural variations as described in the first aspect, the specific primers comprising two pairs of forward and reverse primers, wherein one pair of forward and reverse primers amplifies a sequence that spans an inversion site on the genome of a white-fruited tomato mutant, and the other pair of forward and reverse primers amplifies a sequence that is a site on the normal tomato genome within an inversion fragment region.

[0009] Furthermore, the forward and reverse primers for the amplified sequences spanning inversion sites on the mutant genome are shown in SEQ ID NO:3~4, and the forward and reverse primers for the amplified sequences spanning inversion fragment regions on sites on the normal tomato genome are shown in SEQ ID NO:5~6.

[0010] A third aspect of the invention provides a kit comprising the specific primers described in the second aspect.

[0011] A fourth aspect of the present invention provides the application of the specific primers described in the second aspect or the kits described in the third aspect in marker-assisted breeding of tomatoes.

[0012] A fifth aspect of the present invention provides the application of the specific primers described in the second aspect or the kit described in the third aspect in identifying whether the hybrid offspring of tomatoes are white fruits and whether they are heterozygous.

[0013] A sixth aspect of the present invention provides a method for identifying whether a tomato hybrid offspring has white fruit, comprising the following steps: Genomic DNA was extracted from the tomatoes to be tested; Using the genomic DNA of the tomato to be tested as a template, PCR amplification was performed using the specific primers described in the second aspect; the PCR amplification products were then detected.

[0014] Furthermore, the PCR amplification products can be detected by agarose gel electrophoresis or first-generation sequencing.

[0015] Furthermore, the detection method is agarose gel electrophoresis. If only the 649 bp characteristic band can be detected, it is a homozygous ginkgo tomato; if only the 458 bp characteristic band can be detected, it is a non-ginkgo tomato; if both the 649 bp and 458 bp characteristic bands can be detected, it is a heterozygous ginkgo tomato.

[0016] Furthermore, the PCR amplification products were separated by 1.5% agarose gel electrophoresis at 140 V for 30 minutes, and the band patterns were finally counted.

[0017] Furthermore, the detection method is first-generation sequencing. If the amplified sequence is as shown in SEQ ID NO:1, it is a homozygous ginkgo tomato; if the amplified sequence is as shown in SEQ ID NO:2, it is a non-ginkgo tomato; if the amplified sequence is as shown in both SEQ ID NO:1 and SEQ ID NO:2, it is a heterozygous ginkgo tomato.

[0018] Furthermore, the total volume of the PCR amplification reaction was 10 μL, including DNA template (50 ng•μL). -1 2 μL, primers for forward and reverse directions (10 μmol•L) -1 0.25 μL each of the following: 2×M5 HiPer plus Taq HiFi PCR Mix (Polymerex Corporation), 5 μL of each; and 2 μL of double-distilled water.

[0019] Furthermore, the PCR amplification procedure was as follows: pre-denaturation at 94℃ for 3 minutes; denaturation at 94℃ for 30 seconds, annealing at 55℃ for 30 seconds, extension at 72℃ for 30 seconds, for 32 cycles; incubation at 72℃ for 5 minutes, and storage at 16℃.

[0020] A seventh aspect of the present invention provides the application of the method described in the sixth aspect in marker-assisted breeding of tomatoes.

[0021] Compared with the prior art, the technical solution of the present invention has the following beneficial effects: This invention constructed F1 and F2 populations using the high-generation inbred line X23 (P1) of white-fruited tomato and the high-generation inbred line X22 (P2) of pink-fruited tomato as parents. By investigating the fruit color of the F1 and F2 populations, the genetic patterns of the white-fruited tomato trait were analyzed. Subsequently, the gene controlling white fruit in tomatoes was finely mapped using BSA-Seq combined with map-based cloning, and the chromosomal structural variations within the mapped region were sequenced. Markers were developed based on these variation characteristics, improving their accuracy. Furthermore, the markers were validated using a segregating F2 population of 264 plants, demonstrating a 100% accuracy rate. This invention not only lays the foundation for cloning and elucidating the mechanism of white-fruited tomato genes but also provides a theoretical basis for using molecular markers to assist in the breeding of new white-fruited tomato varieties.

[0022] This invention allows for the selection of tomato materials with white fruits based on genotype during the seedling stage, without having to wait until flowering and fruiting to observe traits. This reduces unnecessary planting area and the investment of manpower and resources, accelerates the breeding process, and improves breeding efficiency, thus having significant application value and social benefits. Attached Figure Description

[0023] Figure 1 Phenotypic diagrams of the flowers and fruits of the parental materials, Ginkgo biloba tomato X23 and Pink tomato X22, in Example 1 of this invention.

[0024] Figure 2 The images show the fruit phenotypes of white tomato X23 and pink tomato X22 at different stages in Example 1 of this invention, from left to right: green ripening stage, color-changing stage, and ripening stage.

[0025] Figure 3 The distribution of the two progeny pools and the ΔSNP-index across the entire genome is shown in the diagram.

[0026] Figure 4 This is a schematic diagram of the gene mapping of ginkgo tomato in Example 1 of the present invention.

[0027] Figure 5 This is a schematic diagram of the structural variation of chromosome 3 in the white tomato mutant.

[0028] Figure 6 The results of partial agarose gel electrophoresis of random individual plants from the tomato parental materials X23 (P1), X22 (P2), F1, and F2 populations used in Example 2 of this invention are shown. Detailed Implementation

[0029] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. 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 invention pertains.

[0030] Terminology Explanation: BSA-Seq: BSA-Seq (Bulked Segregant Analysis by Sequencing) is an analytical method that combines Bulked Segregant Analysis (BSA) and Next-Generation Sequencing (NGS) technologies. BSA-Seq analyzes DNA pools (i.e., two constructed gene pools) of individuals exhibiting extreme traits (such as high and low phenotypes) within a genetic population. High-throughput sequencing is then used to perform deep sequencing on these pools, thereby identifying trait-related genetic variations.

[0031] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments of the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, and / or combinations thereof.

[0032] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0033] Test materials: The white-fruited tomato used in this example (tomato high-generation inbred line X23, hereinafter referred to as X23) is a white-fruited mutant of tomato. It has been verified that this mutant is not controlled by the known Lutescent2(L2) gene. Currently, this white-fruited tomato gene has not been cloned, and its molecular mechanism remains unresolved. The normal tomato used (tomato high-generation inbred line X22, hereinafter referred to as X22) is a pink-fruited tomato. High-generation inbred lines X23 and X22 are from tomato resources preserved by the Shandong Academy of Agricultural Sciences. Verification materials used for the technical effects of this invention can be obtained from the Shandong Academy of Agricultural Sciences.

[0034] like Figure 1 and Figure 2 As shown, the white-fruited tomato mutant plants grow normally, with creamy-white petals. The fruit matures normally, with white flesh and a transparent skin, resulting in a creamy-white or white appearance. The pink-fruited tomato has yellow petals, and the immature fruit is light green, turning pink when ripe.

[0035] Example 1: Obtaining the linkage marker with the tomato ginkgo gene Using tomato inbred line X23 as the female parent (P1, white flowers and white fruit) and tomato inbred line X22 as the male parent (P2, yellow flowers and pink fruit), a segregating population was constructed. The F1 generation was obtained through crossbreeding of the parents, and the F1 generation was then self-pollinated to obtain the F2 population. All F1 fruits were white. Among the 264 F2 individual plants, 201 exhibited white fruit and 63 exhibited pink fruit, conforming to Mendelian segregation ratio of 3:1, indicating that the white fruit trait is controlled by a dominant single gene.

[0036] like Figure 3 and Figure 4 As shown, the gene controlling the white fruit trait was located within the 4Mb range of chromosome 3 of tomato using BSA-seq. Subsequently, this range was encrypted and labeled, and recombinant plants were screened. Based on the phenotypic traits of the recombinant plants, the gene was finally located within the 21.6 kb range.

[0037] Parental resequencing alignment revealed a chromosomal structural variation within this region. To clarify the type of this structural variation, long-fragment sequencing of the *Ginkgo biloba* mutant (X23) genome was performed using third-generation sequencing technology. The results showed that the chromosomal structural variation was an 8.4 kb inversion, located at bases 4230030 to 4238459 (SL4.0 version) of chromosome 3. A schematic diagram of the chromosomal structural variation is shown below. Figure 5 As shown.

[0038] Primers F1 and R1 were designed to cross the inversion sites across the mutant genome, and primers F2 and R2 were designed to cross the inversion sites on the normal tomato genome. The primer sequences are shown in Table 1.

[0039] Table 1 Primer Sequences

[0040] Young leaves from parental tomato plants (X23, X22) and F1 plants were collected, and genomic DNA was extracted from the parents using a modified CTAB (hexadecyltrimethylammonium bromide) method. The total volume of the PCR amplification reaction was 10 μL, containing DNA template (50 ng / μL). -1 2 μL, primers for forward and reverse directions (10 μmol·L⁻¹) -1 0.25 μL each of the following: 2×M5 HiPer plus Taq HiFi PCR Mix (Polymerex Corporation), 5 μL of each; and 2 μL of double-distilled water.

[0041] The PCR amplification program was as follows: 94℃ pre-denaturation for 3 minutes; 94℃ denaturation for 30 seconds, 55℃ annealing for 30 seconds, 72℃ extension for 30 seconds, 32 cycles; 72℃ incubation for 5 minutes, storage at 16℃.

[0042] PCR amplification products were separated by 1.5% agarose gel electrophoresis with 1×TBE buffer. Electrophoresis was performed at a constant power of 140 V for 30 min, and the band patterns were counted.

[0043] The genomic DNA of the selected tomato materials was amplified by PCR using the primer combinations shown in Table 1. The amplification products were detected by gel electrophoresis. Primers F1 and R1 amplified a specific fragment in the white-fruited mutant tomato. The specific fragment was recovered and sequenced, and the nucleotide sequence of the fragment is shown in SEQ ID NO:1, with a fragment size of 649 bp. Primers F2 and R2 amplified a specific fragment in non-white-fruited tomato. The specific fragment was recovered and sequenced, and the nucleotide sequence of the fragment is shown in SEQ ID NO:2, with a fragment size of 458 bp. When both primer pairs were used simultaneously, the above two specific fragments were amplified in F1. The two specific fragments were recovered and sequenced, and the nucleotide sequences are shown in SEQ ID NO:1 and SEQ ID NO:2, with fragment sizes of 649 bp and 458 bp, respectively.

[0044] (SEQ ID NO:1).

[0045] CAAAGCGTAAAATAATAGTGGATGAAATGTAGAATTTCGTGAAAAACTATATTTTATATTCGACGATAAAATTACTTTAATGTATATATAGAGTATATATTAAATTCCTTTAACTTCTTCATATTTTAAATTTTGTTAATGAAAATTTTGACTATGCTATCATACGTGGATCGAAATATAGGCACAAGCACACTTCTCACATTATACACGTATATATGGTCATCGATTTGTT CTAATTTAATTTGGTATAAGCTGAAAAATTTACAACAATTTTTTCTCCCTTCTTAGAAAAGTTTTAGTAATGCATTTTATCAAAAATAAATTAGCATGTGATTGTGATAAATATTATTTCAACGTGTTTTTACGTTACTTGAACATGCTTTTTTCTCAATTTATATAAAAAAGAAAGACCGTTGAAAGAAAAAAAAAATTAGTTTTTTGGGGATTTCTAATTTGGTCAA (SEQ ID NO:2).

[0046] Therefore, this embodiment obtained a linkage marker that can be applied to the identification of white fruits in tomato hybrid offspring.

[0047] Example 2: Validation of linkage markers with tomato ginkgo genes The marker linked to the tomato ginkgo gene obtained in Example 1 was used to validate the parents, F1 and 264 F2 individual plants to determine the accuracy of the marker for marker-assisted selection.

[0048] The PCR amplification system and detection method are the same as in Example 1.

[0049] Compared with the fruit color survey results of F2 individual plants, the accuracy of this marker was found to be 100% in 264 F2 individual plants, and some agarose gel electrophoresis results were as follows. Figure 6 As shown.

[0050] In summary, this study obtained markers linked to the tomato white fruit gene, which not only lays the foundation for cloning the gene controlling white fruit in tomatoes, but also provides a theoretical basis for using molecular markers to assist in the breeding of new white-fruited tomato varieties.

[0051] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of them. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of a reagent for detecting molecular markers of chromosome structural variations in the identification of tomato fruit color, characterized in that, The molecular marker for the chromosomal structural variation is an 8.4 kb inversion on chromosome 3 of the white tomato mutant, with the physical location of the inversion at bases 4230030 to 4238459 on chromosome 3, and the reference genome version is SL4.

0.

2. Specific primers for detecting the molecular markers of chromosomal structural variations as described in claim 1, characterized in that, The specific primers include two pairs of forward and reverse primers. One pair of forward and reverse primers amplifies sequences that cross inverted sites on the genome of the white-fruited tomato mutant, while the other pair of forward and reverse primers amplifies sequences that are sites on the normal tomato genome within the inverted segment region. The forward and reverse primers that amplify sequences that cross inverted sites on the mutant genome are shown in SEQ ID NO:3~4, and the forward and reverse primers that amplify sequences that are sites on the normal tomato genome within the inverted segment region are shown in SEQ ID NO:5~6.

3. A reagent kit, characterized in that, Includes the specific primers described in claim 2.

4. The application of the specific primers of claim 2 or the kit of claim 3 in molecular marker-assisted breeding of tomatoes.

5. The application of the specific primers of claim 2 or the kit of claim 3 in identifying whether the hybrid offspring of tomatoes are white fruits and whether they are heterozygous.

6. A method for identifying whether a tomato hybrid offspring has white fruit, characterized in that, Includes the following steps: Genomic DNA was extracted from the tomatoes to be tested; Using the genomic DNA of the tomato to be tested as a template, a PCR amplification reaction was performed using the specific primers described in claim 2; Detect PCR amplification products.

7. The method as described in claim 6, characterized in that, The methods for detecting PCR amplification products are agarose gel electrophoresis or first-generation sequencing.

8. The method as described in claim 7, characterized in that, The detection method is agarose gel electrophoresis. If only the 649 bp characteristic band can be detected, it is a homozygous ginkgo tomato; if only the 458 bp characteristic band can be detected, it is a non-ginkgo tomato; if both the 649 bp and 458 bp characteristic bands can be detected, it is a heterozygous ginkgo tomato.

9. The method as described in claim 7, characterized in that, The detection method is first-generation sequencing. If the amplified sequence is as shown in SEQ ID NO:1, it is a homozygous ginkgo tomato; if the amplified sequence is as shown in SEQ ID NO:2, it is a non-ginkgo tomato; if the amplified sequence is as shown in both SEQ ID NO:1 and SEQ ID NO:2, it is a heterozygous ginkgo tomato.

10. The application of the method according to any one of claims 6 to 9 in molecular marker-assisted breeding of tomatoes.

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