Development and application of dwarf tomato InDel molecular marker primer

Abstract

The invention relates to the technical field of molecular marker development and molecular detection, discloses dwarf tomato InDel molecular marker primer development, and provides a group of dwarf tomato InDel molecular markers, the dwarf tomato InDel molecular markers comprise 79 InDel markers shown in the following table, and the dwarf tomato InDel molecular markers are determined by reference to a genome SL5.0. The dwarf tomato InDel molecular marker can provide technical guarantee for rapid molecular detection of a target DNA sample in dwarf tomatoes; the set of markers are distributed on multiple chromosomes of the dwarf tomato genome, can be selected and used according to actual needs, can detect the genome DNA of a candidate material at any stage, and has the advantages of flexibility in use, simplicity in operation, high detection efficiency, few limiting factors and stable and accurate detection result; a simple, flexible and efficient way is provided for genetic linkage map construction, genetic diversity analysis, resource identification, hybrid / purity identification and the like in dwarf tomatoes.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular marker development and molecular detection, in particular to the development and application of dwarf tomato InDel molecular marker primers. Background Art

[0002] Tomato (Solanum lycopersicum) belongs to the Solanaceae family and the genus Solanum. It is native to the Pacific coast of western South America and was domesticated in Mexico. As a global crop, tomato boasts a wide range of environmental adaptability and high yield, resulting in a variety of cultivation methods. Rich in lycopene, folate, potassium, vitamin C, flavonoids, vitamin E, and carbohydrates, tomatoes enjoy a wide range of consumer uses. Over the past century, tomatoes have become a global vegetable crop, ranking among the 30 highest-yielded crops worldwide. In addition to its significant economic value, tomatoes also play a crucial role in life science research. The first high-density genetic linkage map of tomato was constructed by Tanksley et al. using an F2 population derived from a cross between the cultivated tomato cultivar VF36-Tm2a and the Pennellia tomato cultivar LA0716. With the rapid development of modern genome sequencing technologies, a large number of molecular genetic linkage maps have been constructed for tomato, facilitating the mapping and cloning of QTLs for important agronomic traits, thus facilitating genetic improvement. Tomato is not only an important economic crop, but also has the advantages of a short growth cycle, strong self-reproduction and regeneration ability, easy control of pollination and fertilization, and ease of genetic transformation. It has been widely used as a model plant in the research of plant genetic engineering technology.

[0003] In recent years, with the rapid development of molecular biology, molecular marker technology has been widely used in the classification of dwarf tomato germplasm. Molecular markers are genetic markers based on nucleotide sequence variation between individuals and directly reflect genetic polymorphism at the DNA level. They are effective tools for studying the genetic diversity of germplasm resources and are currently primarily used for classification and core collection screening. Molecular markers play a vital role in gene mapping and cloning, genetic resource analysis and utilization, variety authenticity and purity verification, and the breeding of new crop varieties. Insertion / deletion polymorphism (InDel) markers are length polymorphic variations caused by the insertion / deletion of nucleotide segments in the DNA sequence at the allelic locus between individuals. InDel markers are second only to single nucleotide polymorphisms (SNPs) in terms of their distribution and density within the genome. Numerous InDels exist in the cultivated tomato genome, which can be used as molecular markers. Previous studies have shown that 14% to 22% of genes contain InDels, while genes containing SSRs only account for 4.6% of the total number of genes. The amplification product of InDel markers has a simple and clear banding pattern, and its stability and product separation effect are significantly better than those of SSR markers. Compared with SNP markers with complex typing systems, InDel detection is simple and convenient, with lower requirements for instruments and technology, and can be performed on an electrophoresis technology platform. In tomatoes, with the completion of tomato genome sequencing, InDel markers have gradually been used in tomato genetic research. However, compared with other crops, dwarf tomatoes have a narrow genetic background and difficult to develop markers, which cannot meet research needs such as gene positioning, genetic diversity analysis, purity detection and consistency identification. At present, there are almost no reports on the InDel variation characteristics and molecular marker development of the whole genome of dwarf tomatoes, as well as the application of InDel markers in hybrid seed purity detection and variety consistency identification.

[0004] Therefore, in order to meet the needs of different studies on the number and types of molecular markers, expand the application scope of molecular markers in dwarf tomato research, and accelerate the process of dwarf tomato molecular breeding, it is urgent to clarify the InDel variation characteristics of the whole genome of dwarf tomatoes; the present invention provides the development of dwarf tomato InDel molecular marker primers and their application to solve the above-mentioned problems. Summary of the Invention

[0005] Technical problems solved

[0006] In response to the shortcomings of the existing technology, the present invention provides the development and application of dwarf tomato InDel molecular marker primers, which have the advantages of being used in the construction of dwarf tomato genetic linkage maps, genetic diversity analysis, resource identification, hybrid / purity identification, etc., and solve the above-mentioned problems.

[0007] Technical Solution

[0008] To achieve the above object, the present invention provides the following technical solution: development of dwarf tomato InDel molecular marker primers, wherein the dwarf tomato InDel molecular markers include the following 79 InDel molecular markers: primers for amplification of InDel-1 are shown in SEQ ID NOs. 1 to 2, primers for amplification of InDel-2 are shown in SEQ ID NOs. 3 to 4, primers for amplification of InDel-3 are shown in SEQ ID NOs. 5 to 6, primers for amplification of InDel-4 are shown in SEQ ID NOs. 7 to 8, primers for amplification of InDel-5 are shown in SEQ ID NOs. 9 to 10, primers for amplification of InDel-6 are shown in SEQ ID NOs. 11 to 12, primers for amplification of InDel-7 are shown in SEQ ID NOs. 13 to 14, primers for amplification of InDel-8 are shown in SEQ ID NOs. 15 to 16, primers for amplification of InDel-9 are shown in SEQ ID NOs. 17 to 18, primers for amplification of InDel-10 are shown in SEQ ID NOs. The primers for amplifying InDel-11 are shown in SEQ ID NOs. 21-22, the primers for amplifying InDel-12 are shown in SEQ ID NOs. 23-24, the primers for amplifying InDel-13 are shown in SEQ ID NOs. 25-26, the primers for amplifying InDel-14 are shown in SEQ ID NOs. 27-28, the primers for amplifying InDel-15 are shown in SEQ ID NOs. 29-30, the primers for amplifying InDel-16 are shown in SEQ ID NOs. 31-32, the primers for amplifying InDel-17 are shown in SEQ ID NOs. 33-34, the primers for amplifying InDel-18 are shown in SEQ ID NOs. 35-36, the primers for amplifying InDel-19 are shown in SEQ ID NOs. 37-38, and the primers for amplifying InDel-20 are shown in SEQ ID NOs. The primers for amplifying InDel-21 are shown in SEQ ID NOs. 41-42, the primers for amplifying InDel-22 are shown in SEQ ID NOs. 43-44, the primers for amplifying InDel-23 are shown in SEQ ID NOs. 45-46, the primers for amplifying InDel-24 are shown in SEQ ID NOs. 47-48, the primers for amplifying InDel-25 are shown in SEQ ID NOs. 49-50, the primers for amplifying InDel-26 are shown in SEQ ID NOs. 51-52, the primers for amplifying InDel-27 are shown in SEQ ID NOs. 53-54, the primers for amplifying InDel-28 are shown in SEQ ID NOs. 55-56, and the primers for amplifying InDel-29 are shown in SEQ ID NO.57-58, the primers for amplification of InDel-30 are shown in SEQ ID NOs. 59-60, the primers for amplification of InDel-31 are shown in SEQ ID NOs. 61-62, the primers for amplification of InDel-32 are shown in SEQ ID NOs. 63-64, the primers for amplification of InDel-33 are shown in SEQ ID NOs. 65-66, the primers for amplification of InDel-34 are shown in SEQ ID NOs. 67-68, the primers for amplification of InDel-35 are shown in SEQ ID NOs. 69-70, the primers for amplification of InDel-36 are shown in SEQ ID NOs. 71-72, the primers for amplification of InDel-37 are shown in SEQ ID NOs. 73-74, the primers for amplification of InDel-38 are shown in SEQ ID NOs. 75-76, and the primers for amplification of InDel-39 are shown in SEQ ID NOs. The primers for amplifying InDel-40 are shown in SEQ ID NOs. 79-80, the primers for amplifying InDel-41 are shown in SEQ ID NOs. 81-82, the primers for amplifying InDel-42 are shown in SEQ ID NOs. 83-84, the primers for amplifying InDel-43 are shown in SEQ ID NOs. 85-86, the primers for amplifying InDel-44 are shown in SEQ ID NOs. 87-88, the primers for amplifying InDel-45 are shown in SEQ ID NOs. 89-90, the primers for amplifying InDel-46 are shown in SEQ ID NOs. 91-92, the primers for amplifying InDel-47 are shown in SEQ ID NOs. 93-94, the primers for amplifying InDel-48 are shown in SEQ ID NOs. 95-96, and the primers for amplifying InDel-49 are shown in SEQ ID NOs. The primers for amplifying InDel-50 are shown in SEQ ID NOs. 99-100, the primers for amplifying InDel-51 are shown in SEQ ID NOs. 101-102, the primers for amplifying InDel-52 are shown in SEQ ID NOs. 103-104, the primers for amplifying InDel-53 are shown in SEQ ID NOs. 105-106, the primers for amplifying InDel-54 are shown in SEQ ID NOs. 107-108, the primers for amplifying InDel-55 are shown in SEQ ID NOs. 109-110, the primers for amplifying InDel-56 are shown in SEQ ID NOs. 111-112, the primers for amplifying InDel-57 are shown in SEQ ID NOs. 113-114, and the primers for amplifying InDel-58 are shown in SEQ ID NO.115-116, the primers for amplification of InDel-59 are shown in SEQ ID NOs. 117-118, the primers for amplification of InDel-60 are shown in SEQ ID NOs. 119-120, the primers for amplification of InDel-61 are shown in SEQ ID NOs. 121-122, the primers for amplification of InDel-62 are shown in SEQ ID NOs. 123-124, the primers for amplification of InDel-63 are shown in SEQ ID NOs. 125-126, the primers for amplification of InDel-64 are shown in SEQ ID NOs. 127-128, the primers for amplification of InDel-65 are shown in SEQ ID NOs. 129-130, the primers for amplification of InDel-66 are shown in SEQ ID NOs. 131-132, and the primers for amplification of InDel-67 are shown in SEQ ID NOs. The primers for amplifying InDel-68 are shown in SEQ ID NOs. 135-136, the primers for amplifying InDel-69 are shown in SEQ ID NOs. 137-138, the primers for amplifying InDel-70 are shown in SEQ ID NOs. 139-140, the primers for amplifying InDel-71 are shown in SEQ ID NOs. 141-142, the primers for amplifying InDel-72 are shown in SEQ ID NOs. 143-144, the primers for amplifying InDel-73 are shown in SEQ ID NOs. 145-146, the primers for amplifying InDel-74 are shown in SEQ ID NOs. 147-148, the primers for amplifying InDel-75 are shown in SEQ ID NOs. 149-150, and the primers for amplifying InDel-76 are shown in SEQ ID NOs. The primers for amplifying InDel-77 are shown in SEQ ID NOs. 153-154, the primers for amplifying InDel-78 are shown in SEQ ID NOs. 155-156, and the primers for amplifying InDel-79 are shown in SEQ ID NOs. 157-158.

[0009] Preferably, the dwarf tomato InDel molecular marker is determined with reference to the genome SL5.0.

[0010] The present invention also provides the use of the amplification primers described in the above technical solution in the positioning of genes related to important agronomic traits of dwarf tomatoes.

[0011] The present invention also provides the use of the amplification primers described in the above technical solution in constructing a genetic linkage map of dwarf tomatoes.

[0012] The present invention also provides the use of the amplification primers described in the above technical solution in detecting dwarf tomato resources.

[0013] The present invention also provides the use of the amplification primers described in the above technical solution in the genetic breeding of dwarf tomatoes.

[0014] The present invention also provides the use of the amplification primers described in the above technical solution in the genetic diversity analysis of dwarf tomatoes.

[0015] The present invention also provides the use of the amplification primers described in the above technical solution in the identification of dwarf tomato hybrids and purity.

[0016] Compared with the prior art, the present invention provides the development and application of dwarf tomato InDel molecular marker primers, which have the following beneficial effects:

[0017] The development and application of the dwarf tomato InDel molecular marker primers can provide technical support for the rapid molecular detection of target DNA samples in dwarf tomatoes using dwarf tomato InDel molecular markers. Since this set of markers is distributed on multiple chromosomes in the dwarf tomato genome, they can be selected and used according to actual needs, and the genomic DNA of candidate materials can be detected at any stage. It has the advantages of flexible use, simple operation, high detection efficiency, few limiting factors, and stable and accurate detection results. It provides a simple, flexible and efficient approach for the construction of genetic linkage maps, genetic diversity analysis, resource identification, hybrid / purity identification, etc. in dwarf tomatoes. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Schematic diagram of some InDel primer screening results provided by the present invention;

[0019] Figure 2 This is a graph showing the agarose gel electrophoresis results of PCR products of 79 InDel markers in dwarf tomatoes of the present invention;

[0020] Figure 3 The physical map of dwarf tomato constructed by the 79 InDel markers provided by the present invention;

[0021] Figure 4 The present invention provides an electrophoresis diagram for genetic diversity analysis of dwarf tomatoes using InDel molecular markers. DETAILED DESCRIPTION

[0022] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0023] See also Figures 1 - 4 ;

[0024] The present invention provides a set of dwarf tomato InDel molecular markers, which include 79 InDel markers shown in the following table: In the present invention, the dwarf tomato InDel molecular markers are determined with reference to the genome SL5.0.

[0025] Compared to previous SSR markers, the dwarf tomato InDel molecular markers developed in this paper are distributed across multiple chromosomes in the dwarf tomato genome. They exhibit unique amplification, offer excellent typing efficiency, and are highly representative, enabling high-throughput testing with a short detection cycle. For variety authentication or purity testing, the InDel markers are simple and rapid to use, with stable and reliable results. Flexible marker selection is possible based on the number of samples and loci tested. Therefore, the InDel markers developed in this paper can be standardized and scaled for testing of varying sample sizes.

[0026] The present invention also provides primers for amplifying the InDel molecular marker of the above technical solution, wherein the primers for amplifying InDel-1 are shown as SEQ ID NOs. 1 to 2, the primers for amplifying InDel-2 are shown as SEQ ID NOs. 3 to 4, the primers for amplifying InDel-3 are shown as SEQ ID NOs. 5 to 6, the primers for amplifying InDel-4 are shown as SEQ ID NOs. 7 to 8, the primers for amplifying InDel-5 are shown as SEQ ID NOs. 9 to 10, the primers for amplifying InDel-6 are shown as SEQ ID NOs. 11 to 12, the primers for amplifying InDel-7 are shown as SEQ ID NOs. 13 to 14, and the primers for amplifying InDel-8 are shown as SEQ ID NOs. The primers for amplification of InDel-9 are shown in SEQ ID NOs. 17 to 18, the primers for amplification of InDel-10 are shown in SEQ ID NOs. 19 to 20, the primers for amplification of InDel-11 are shown in SEQ ID NOs. 21 to 22, the primers for amplification of InDel-12 are shown in SEQ ID NOs. 23 to 24, the primers for amplification of InDel-13 are shown in SEQ ID NOs. 25 to 26, the primers for amplification of InDel-14 are shown in SEQ ID NOs. 27 to 28, the primers for amplification of InDel-15 are shown in SEQ ID NOs. 29 to 30, the primers for amplification of InDel-16 are shown in SEQ ID NOs. .31-32, the primers for amplification of InDel-17 are shown in SEQ ID NOs.33-34, the primers for amplification of InDel-18 are shown in SEQ ID NOs.35-36, the primers for amplification of InDel-19 are shown in SEQ ID NOs.37-38, the primers for amplification of InDel-20 are shown in SEQ ID NOs.39-40, the primers for amplification of InDel-21 are shown in SEQ ID NOs.41-42, the primers for amplification of InDel-22 are shown in SEQ ID NOs.43-44, the primers for amplification of InDel-23 are shown in SEQ ID NOs.45-46, the primers for amplification of InDel-24 are shown in SEQ ID NO. .47-48, the primers for amplification of InDel-25 are shown in SEQ ID NOs.49-50, the primers for amplification of InDel-26 are shown in SEQ ID NOs.51-52, the primers for amplification of InDel-27 are shown in SEQ ID NOs.53-54, the primers for amplification of InDel-28 are shown in SEQ ID NOs.55-56, the primers for amplification of InDel-29 are shown in SEQ ID NOs.57-58, the primers for amplification of InDel-30 are shown in SEQ ID NOs.59-60, the primers for amplification of InDel-31 are shown in SEQ ID NOs.61-62, and the primers for amplification of InDel-32 are shown in SEQ ID NO.The primers for amplifying InDel-33 are shown in SEQ ID NOs. 65 to 66, the primers for amplifying InDel-34 are shown in SEQ ID NOs. 67 to 68, the primers for amplifying InDel-35 are shown in SEQ ID NOs. 69 to 70, the primers for amplifying InDel-36 are shown in SEQ ID NOs. 71 to 72, the primers for amplifying InDel-37 are shown in SEQ ID NOs. 73 to 74, the primers for amplifying InDel-38 are shown in SEQ ID NOs. 75 to 76, the primers for amplifying InDel-39 are shown in SEQ ID NOs. 77 to 78, and the primers for amplifying InDel-40 are shown in SEQ ID NOs. The primers for amplifying InDel-41 are shown in SEQ ID NOs. 81 to 82, the primers for amplifying InDel-42 are shown in SEQ ID NOs. 83 to 84, the primers for amplifying InDel-43 are shown in SEQ ID NOs. 85 to 86, the primers for amplifying InDel-44 are shown in SEQ ID NOs. 87 to 88, the primers for amplifying InDel-45 are shown in SEQ ID NOs. 89 to 90, the primers for amplifying InDel-46 are shown in SEQ ID NOs. 91 to 92, the primers for amplifying InDel-47 are shown in SEQ ID NOs. 93 to 94, and the primers for amplifying InDel-48 are shown in SEQ ID NOs. The primers for amplifying InDel-49 are shown in SEQ ID NOs. 97 to 98, the primers for amplifying InDel-50 are shown in SEQ ID NOs. 99 to 100, the primers for amplifying InDel-51 are shown in SEQ ID NOs. 101 to 102, the primers for amplifying InDel-52 are shown in SEQ ID NOs. 103 to 104, the primers for amplifying InDel-53 are shown in SEQ ID NOs. 105 to 106, the primers for amplifying InDel-54 are shown in SEQ ID NOs. 107 to 108, the primers for amplifying InDel-55 are shown in SEQ ID NOs. 109 to 110, and the primers for amplifying InDel-56 are shown in SEQ ID NOs. 111 to 112. The primers for amplifying InDel-56 are shown in SEQ ID NOs. 111 to 112, the primers for amplifying InDel-57 are shown in SEQ ID NOs. 113 to 114, the primers for amplifying InDel-58 are shown in SEQ ID NOs. 115 to 116, the primers for amplifying InDel-59 are shown in SEQ ID NOs. 117 to 118, the primers for amplifying InDel-60 are shown in SEQ ID NOs. 119 to 120, the primers for amplifying InDel-61 are shown in SEQ ID NOs. 121 to 122, the primers for amplifying InDel-62 are shown in SEQ ID NOs. 123 to 124, and the primers for amplifying InDel-63 are shown in SEQ ID NO.125-126, the primers for amplification of InDel-64 are shown in SEQ ID NOs. 127-128, the primers for amplification of InDel-65 are shown in SEQ ID NOs. 129-130, the primers for amplification of InDel-66 are shown in SEQ ID NOs. 131-132, the primers for amplification of InDel-67 are shown in SEQ ID NOs. 133-134, the primers for amplification of InDel-68 are shown in SEQ ID NOs. 135-136, the primers for amplification of InDel-69 are shown in SEQ ID NOs. 137-138, the primers for amplification of InDel-70 are shown in SEQ ID NOs. 139-140, and the primers for amplification of InDel-71 are shown in SEQ ID NOs. 141-142. 42, primers for amplification of InDel-72 are shown in SEQ ID NOs. 143-144, primers for amplification of InDel-73 are shown in SEQ ID NOs. 145-146, primers for amplification of InDel-74 are shown in SEQ ID NOs. 147-148, primers for amplification of InDel-75 are shown in SEQ ID NOs. 149-150, primers for amplification of InDel-76 are shown in SEQ ID NOs. 152-153, primers for amplification of InDel-77 are shown in SEQ ID NOs. 153-154, primers for amplification of InDel-78 are shown in SEQ ID NOs. 155-156, and primers for amplification of InDel-79 are shown in SEQ ID NOs. 157-158.

[0027] In the present invention, when the product is used, the PCR reaction system is preferably as shown in Table 4. The PCR program is preferably: pre-denaturation: 94°C, 4 min; cyclic amplification: 94°C, 30 s; 55°C, 30 s; 72°C, 30 s; 35 cycles; cyclic extension: 72°C, 7 min, stored at 4°C;

[0028] The present invention also provides the use of the amplification primers of the above technical solution in constructing a genetic linkage map of dwarf tomatoes.

[0029] InDel molecular markers are an important genetic marker and have been widely used in the construction of crop linkage maps. The present invention uses QTLmapping software to draw the physical map of these 79 InDel molecular markers on the chromosomes of dwarf tomato ( Figure 2 ), laying the foundation for the construction of the genetic linkage map of dwarf tomato.

[0030] The present invention also provides the application of the amplification primers of the above technical solution in detecting dwarf tomato resources.

[0031] As a high-throughput DNA fingerprinting marker, InDel molecular marker technology can better reveal polymorphisms in the genome, and therefore has outstanding advantages in germplasm resource identification and has been widely used in crops such as corn, rice, and Chinese cabbage. This study utilized developed polymorphic InDel primers, randomly selected two pairs of polymorphic primers (InDel-7 and InDel-11), and combined them with a rapid method for extracting genomic DNA from dwarf tomato seedlings to identify 24 dwarf tomato varieties. PCR amplification results showed that different varieties exhibited variety-specificity when tested with different primers ( Figure 3 The results showed that InDel technology is a rapid, accurate, and efficient method for large-scale DNA fingerprint analysis. The InDel molecular markers developed in this invention can play an important role in the identification of dwarf tomato germplasm resources.

[0032] The present invention also provides the application of the amplification primers of the above technical solution in the genetic breeding of dwarf tomatoes.

[0033] The present invention also provides the application of the amplification primers of the above technical solution in the genetic diversity analysis of dwarf tomatoes.

[0034] The present invention also provides the application of the amplification primers of the above technical solution in the identification of dwarf tomato hybrids and purity.

[0035] To further illustrate the present invention, a set of dwarf tomato InDel molecular markers, primers and applications provided by the present invention will be described in detail below with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0036] If specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in the field or in the product instructions were used. All reagents and instruments used are those whose manufacturers are indicated and are commercially available through regular channels.

[0037] Example 1:

[0038] Extraction of genomic DNA from dwarf tomato

[0039] At the three-leaf stage of dwarf tomato, 1 g of young leaves were collected and placed in a 2 mL Eppendorf centrifuge tube. Two steel balls and 250 μL of a modified CTAB lysis buffer were added. The sample was crushed with a plant tissue crusher at 27 r / s for 90 s. The crushed sample was centrifuged at 12000 r / min in a greenhouse for 1 min. 500 μL of CTAB lysis buffer was added. After thorough mixing, the sample was placed in a 65 ° C water bath for 30 min. After cooling to room temperature, an equal volume of chloroform was added and gently shaken for 5 min. The emulsion was stirred at 1200 r / min. Centrifuge at 0 rpm for 8–10 min at room temperature. Transfer 400 μL of the supernatant to a new 1.5 mL centrifuge tube, add 600 μL of anhydrous ethanol, and incubate on ice for 1 h. After standing, centrifuge the mixture at 12,000 rpm for 5 min, discard the supernatant, and wash the precipitate two to three times with 75% ethanol. After each wash, centrifuge at 12,000 rpm for 3 min, discard the supernatant, and dry the DNA at room temperature. Add 100 μL of ddH₂O, dissolve thoroughly, and store at 4°C until ready for use. DNA purity and quantification were analyzed using a UV / Vis spectrophotometer.

[0040] Example 2:

[0041] Screening of InDel markers for dwarf tomato

[0042] Nine dwarf tomatoes collected by the Tomato Breeding Research Group of Hebei University of Science and Technology were subjected to 10× resequencing, and whole-genome genetic variation analysis was performed using SL5.0 as the reference genome. A total of 476,708 high-quality InDels were obtained at the chromosome level. InDel sites were selected on 12 chromosomes based on the physical location and function of the chromosome where the variant site was located. A self-written script in Perl language was used to combine the InDel variant sites obtained from the parental resequencing data, and the reference genome sequence of 100 bp upstream and downstream of the InDel variant site in the candidate interval was extracted. InDel primers were designed using Primer5.0 software. Based on the chromosome length and the number and type of variant sites on each chromosome, 144 variant sites that can be designed as InDel markers were screened. The applicability of the 144 InDel markers was screened using 9 different types of dwarf tomato resource materials, and 81 markers with stable amplification, clear typing, and strong representativeness were screened (Table 1). The results of some InDel primer screening are shown in Figure 1. Figure 1 As shown; A is the screening result of the non-polymorphic primer ZD01-1, B is the screening result of the polymorphic primer InDel-7, the numbers represent different dwarf tomato varieties, M represents Marker, and the numbers 1-24 represent the amplification results of dwarf tomato samples NO.1-NO.24 respectively. Figure 1 As shown in A, the bands amplified from samples No.1-No.24 are the same, indicating that there is no polymorphism. Figure 1In the B, A band was amplified, B band was amplified, and H band was amplified, indicating that the primers are polymorphic.

[0043] Table 1

[0044] InDel Primer Name Forward Primer Sequence Number Reverse Primer Sequence Number InDel-1 TCACCTCCGATGTGGAAC SeqIDNO.1 GTGTTGGGTGGTCTCCTT SeqIDNO.2 InDel-2 TCGTTACACTAGCATACG SeqIDNO.3 ATATGCCAATGAACTAGG SeqIDNO.4 InDel-3 CCACACCATCATTTCGTG SeqIDNO.5 AAAGAACTGTCTCACTCT SeqIDNO.6 InDel-4 AGCGAACTATTGAGAGTC SeqIDNO.7 GGTTAAATGGGATGGTCT SeqIDNO.8 InDel-5 TGAGCATCCTAACTGCTT SeqIDNO.9 TCCTACTGCTAACTGACA SeqIDNO.10 InDel-6 TCTACTACGTTTGCTACA SeqIDNO.11 AGTGAGGAAAAGTGTAGA SeqIDNO.12 InDel-7 ACGCGCCTCAGCAAGTAG SeqIDNO.13 ATAAGTGAACGAAGATCG SeqIDNO.14 InDel-8 TATTGGCAGTACAGGTTCGA SeqIDNO.15 AAGGGCGGAGGTTAAAGT SeqIDNO.16 InDel-9 TAGATTCCGGACCAAGGA SeqIDNO.17 ATCGTTTCCACAAGGGAA SeqIDNO.18 InDel-10 AGAACGTGAGAGATAGAA SeqIDNO.19 GCAAGGGTTCACACACAT SeqIDNO.20 InDel-11 CGAGCTACAACAAGGCGAACAATT SeqIDNO.21 GTATCGAACAGATACACCT SeqIDNO.22 InDel-12 GGATGAATTTTGGTGTGAGG SeqIDNO.23 GACCTACGTTTTTACACTCA SeqIDNO.24 InDel-13 AATAAACGGGTCTCGCGT SeqIDNO.25 GGAGTATGGACCAAGTCG SeqIDNO.26 InDel-14 GACTAATTTCAGGGCAAC SeqIDNO.27 CATGAGGCTTGTGGACTC SeqIDNO.28 InDel-15 AGGCCGAACACTGATAGG SeqIDNO.29 AAAGCTCGACACCAAACG SeqIDNO.30 InDel-16 GGTCGTATAGGATGCTCT SeqIDNO.31 CTTGCATAATTCACTAGGACC SeqIDNO.32 InDel-17 TCACGCATACAGCAAGTA SeqIDNO.33 TTTGGTTCCCATATGCGTG SeqIDNO.34 InDel-18 AGAGATACAAGATCAACGC SeqIDNO.35 GAACATACTCCACTAGCGTA SeqIDNO.36 InDel-19 CATTATCTTGTTCGCAGCCA SeqIDNO.37 GAATCGCCTAGGTTCAAAA SeqIDNO.38 InDel-20 CACTAGTGTACCCCATGG SeqIDNO.39 CAAATCGGCAGACTATTTC SeqIDNO.40 InDel-21 CGTCATTGGGGATTCATT SeqIDNO.41 TGAGAGGTAGGACATTGG SeqIDNO.42 InDel-22 GATACCTATCCTATCCAAG SeqIDNO.43 TGTGTGTGCGATTGGACG SeqIDNO.44 InDel-23 TACATCTCCCTCCTAGAG SeqIDNO.45 AGTATTTCCCCCGTACCA SeqIDNO.46 InDel-24 GCTTAACCATGTACAACCCA SeqIDNO.47 GTATTTAGTGTGCCAGCC SeqIDNO.48 InDel-25 ACAAGACTGCTCATACCC SeqIDNO.49 CAAGCTAACATGATCTCTG SeqIDNO.50 InDel-26 TCATCGATCTTAGGTGGAG SeqIDNO.51 TGCCATTAGTTTGCAGTAG SeqIDNO.52 InDel-27 CAGAGAATTGAGCTAAGG SeqIDNO.53 TGACAGTATTAGCTGCCA SeqIDNO.54 InDel-28 GTCTTAGAGAAGTGTAAGTC SeqIDNO.55 GATAGGTTATAGTGTAGGC SeqIDNO.56 InDel-29 AGTATTAAAGGAGTCACGGG SeqIDNO.57 CACGTCGATACTATGTCCA SeqIDNO.58 InDel-30 TCAGGGACTAACATTCCCA SeqIDNO.59 GAAGTTATTCGAGAGAGGTT SeqIDNO.60 InDel-31 ATCACTGTCAGAGGGACC SeqIDNO.61 GAGCATATCCCCTAGCATG SeqIDNO.62 InDel-32 AGCATTGCAACATGACCAT SeqIDNO.63 GAGTTATTATATTGGCAGGAG SeqIDNO.64 InDel-33 TGAGAGCTATTGTAGTGTG SeqIDNO.65 AAGGATCTGTGAGCTCTCC SeqIDNO.66 InDel-34 TCCTCCCACACACTTCCCA SeqIDNO.67 ACGACTCCCCAATGGACCA SeqIDNO.68 InDel-35 ACCGAGACATGTCTGCTTC SeqIDNO.69 TGCTCCTTCTAATGCAGT SeqIDNO.70 InDel-36 GTTCATTGACCTCGAGAA SeqIDNO.71 CGGTTACACCTCTTGCCTC SeqIDNO.72 InDel-37 AACGCAATACAACTGCTC SeqIDNO.73 ATGAAAGTGCACGGAGGGG SeqIDNO.74 InDel-38 ACTAGTGTGGATCTTCAAG SeqIDNO.75 AACACCTGCAAGACATTC SeqIDNO.76 InDel-39 TGGATCCACGTTATCAGT SeqIDNO.77 GTGGCGTTTTGGATTAATG SeqIDNO.78 InDel-40 CTAATGCAAATACAGGTGG SeqIDNO.79 GTGACGCTTAGATTATGTTC SeqIDNO.80 InDel-41 CTCACTTCGATACCATCGA SeqIDNO.81 CCATGCGTTTGGCCTTGTA SeqIDNO.82 InDel-42 TTCGGAGTGCAGATTGGC SeqIDNO.83 AGCAGGCCACTTAATTTC SeqIDNO.84 InDel-43 GACGCAGAGAGTAGGTACG SeqIDNO.85 CTAATGAGTTGTCCTAACG SeqIDNO.86 InDel-44 GGAAGTTGCGGGTTGGTAA SeqIDNO.87 GCAATGAGGGTCCGACCAA SeqIDNO.88 InDel-45 GTTCCCCGAGTTGATTACA SeqIDNO.89 TGTCAAGGGCCATATTGT SeqIDNO.90 InDel-46 TTTGAACCTCGTGCACTTG SeqIDNO.91 GTGGATACGATTCAGTAGA SeqIDNO.92 InDel-47 CATAGCACGTAAAGTCTAG SeqIDNO.93 ATGACACTTGTCAGGGTGG SeqIDNO.94 InDel-48 CACTCCTCCCTCATCTAGC SeqIDNO.95 TTTAATGGAGGTGCGCTGTTG SeqIDNO.96 InDel-49 GCCCTTTAACTTCGACTCA SeqIDNO.97 AATTGCCATGTAGGATGTC SeqIDNO.98 InDel-50 GACGTGGTTTAAGGGGGAAT SeqIDNO.99 CATCTTGAGGAGAGCCTCC SeqIDNO.100 InDel-51 TGTGTGTCTAGTGCTGGG SeqIDNO.101 CTCCAACTCGGGTTTTAAAG SeqIDNO.102 InDel-52 GAGGAGTAGTTCTAGGTT SeqIDNO.103 AACCAGATTACAACCAAC SeqIDNO.104 InDel-53 TTTGACTCATTGCGAAGAG SeqIDNO.105 CAGAGACACACTCACAAGT SeqIDNO.106 InDel-54 GGATCATGCACAATAAGAAC SeqIDNO.107 GGTTTGTCCTTCGAGTTGG SeqIDNO.108 InDel-55 ACACAAGTATGGGGTTGTG SeqIDNO.109 CCGAAACCACATTTAGGT SeqIDNO.110 InDel-56 CAACATACGCACAACTATAG SeqIDNO.111 ATCCACTCGAACGACACGAT SeqIDNO.112 InDel-57 TTGGTCGTGTGAGCTCTGAC SeqIDNO.113 CCAAAAAAATGCATGCTTAGGC SeqIDNO.114 InDel-58 CACTAAAAGCAATGCTAAGACC SeqIDNO.115 TTTCTTGAGGGTCATTGTG SeqIDNO.116 InDel-59 ATGGAATTCCACGTCTTGC SeqIDNO.117 CACATACTATAGATCAGAGTTCGG SeqIDNO.118 InDel-60 GAACACAAGGATTGGTCA SeqIDNO.119 TTGGTCGTTTAATGTGTGCA SeqIDNO.120 InDel-61 TTACTGGACACTGCAAAC SeqIDNO.121 CTCCTTCGTCTCCCAGTGGTT SeqIDNO.122 InDel-62 GAAACATTGAACTGGAGGGTAC SeqIDNO.123 ACGACCAGCCGGATCTCTTCCAG SeqIDNO.124 InDel-63 CCAAACATACTTCGAGTTGATGTG SeqIDNO.125 CTTCTTAGTACATGGACCAA SeqIDNO.126 InDel-64 ACCAACATGATCGGTGAGGA SeqIDNO.127 TGCATTTGCCTTACTAGTTGAG SeqIDNO.128 InDel-65 TAGTAAGGAGAGCTTACATG SeqIDNO.129 AACGCGGACAAACTATACTTG SeqIDNO.130 InDel-66 TATCTTGATCGAGCTCAACCC SeqIDNO.131 TGCTTTGATGCACGTGCA SeqIDNO.132 InDel-67 ACTTCAACCGTCCTCTCGAAC SeqIDNO.133 TACACCACTAAGCTTTATGC SeqIDNO.134 InDel-68 GAGTCACAGTACACCAATCTCTC SeqIDNO.135 TTCCTTCCTACTGGCCAAC SeqIDNO.136 InDel-69 CTCTCTTGTCAAGACCGTAGTCT SeqIDNO.137 CTCACTATGAAGTCGAAGT SeqIDNO.138 InDel-70 TTCAGCAAGAGTGGGACCA SeqIDNO.139 ATGTCACACCTAAAACCC SeqIDNO.140 InDel-71 GAATGTACAATGGATACCGT SeqIDNO.141 AGAGTTGCACGGTATTGA SeqIDNO.142 InDel-72 TCACGCCAAGTAGAACAATC SeqIDNO.143 TCTAGTAGGCTAATGGGTGT SeqIDNO.144 InDel-73 GTGCAACTCACATTGTGCC SeqIDNO.145 TGAATATCGGTCATCTTGAGGA SeqIDNO.146 InDel-74 AACCCTGCACCACAGATAGG SeqIDNO.147 CAAAGTCACCGACAGGGTGAA SeqIDNO.148 InDel-75 CCTTCTCGATCAAACGCAA SeqIDNO.149 ATGTCAGTCCTTCTTCTTG SeqIDNO.150 InDel-76 CTCTACAAAGTTACACTCC SeqIDNO.151 CCTCGCCTTTTTACCATCGCC SeqIDNO.152 InDel-77 TAGAAGCGTCCATTGTTG SeqIDNO.153 GGTCACTTAGAGATGAAAC SeqIDNO.154 InDel-78 CAGACTCTGGCACCCTAT SeqIDNO.155 GGTGCAGCACACAGAGAGGATG SeqIDNO.156 InDel-79 CTCACTTGACTGAGTTATG SeqIDNO.157 GGTTTTATGCTGCGTAATGA SeqIDNO.158

[0045] Example 3:

[0046] PCR amplification with InDel primers

[0047] The genomic DNA extracted from dwarf tomatoes was amplified by PCR.

[0048] The amplification reaction system is as follows:

[0049] Component volume (μL)

[0050] Buffer, DNA polymerase, dNTPs, and Mg 2+ A mixture of 5

[0051] Forward primer (10 μM) 0.5

[0052] Forward primer (10 μM) 0.5

[0053] Genomic template (30ng / μL)1

[0054] Water 3

[0055] Total volume 10

[0056] The amplification procedure is as follows:

[0057] Pre-denaturation: 94°C, 4 min;

[0058] Cyclic amplification: 94°C, 30 s; 55°C, 30 s; 72°C, 30 s; 35 cycles;

[0059] Extension cycle: 72°C, 7 min, stored at 10°C.

[0060] Example 4:

[0061] Preparation of agarose gel: Weigh 10.5g of agarose, add 350mL of 0.5×TBE, heat for 5min, cool slightly, add 10μL of 0.5μg / mL EB solution and mix well. Install the electrophoresis tank, pour the gel, install the sample comb, wait for the gel to cool and solidify, and pour 0.5×TBE into the electrophoresis tank. Take 5μL of PCR amplification product into the sample well, and separate it by 130V constant voltage electrophoresis for 1h~1.5h until the amplified DNA band is fully developed. After electrophoresis, the band characteristics are observed by imaging using a gel imaging system (Bio-Rad, Hercules, CA, USA). The results are as follows: Figure 2 shown.

[0062] The polymorphism of InDel primers in dwarf tomato varieties was screened by agarose gel electrophoresis of PCR amplification products of 144 pairs of InDel primers. Finally, 79 pairs of primers with good amplification effect and polymorphic InDel markers were screened ( Figure 1 These InDel markers can be applied to research such as identification of dwarf tomato varieties, construction of genetic maps, mapping of functional genes / QTLs, molecular-assisted breeding, and genetic diversity analysis.

[0063] The beneficial effects of the present invention are as follows: the use of dwarf tomato InDel molecular markers can provide technical support for the rapid molecular detection of target DNA samples in dwarf tomatoes; since this set of markers is distributed on multiple chromosomes of the dwarf tomato genome, they can be selected and used according to actual needs, and the genomic DNA of candidate materials can be detected at any stage. The present invention has the advantages of flexible use, simple operation, high detection efficiency, few limiting factors, and stable and accurate detection results, providing a simple, flexible and efficient approach for the construction of genetic linkage maps, genetic diversity analysis, resource identification, hybrid / purity identification, etc. in dwarf tomatoes.

[0064] Although the embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. Development of InDel molecular marker primers for dwarf tomato, characterized by: The dwarf tomato InDel molecular markers include the following 79 InDel molecular markers: primers for amplification of InDel-1 are shown in SEQ ID NOs. 1 to 2, primers for amplification of InDel-2 are shown in SEQ ID NOs. 3 to 4, primers for amplification of InDel-3 are shown in SEQ ID NOs. 5 to 6, primers for amplification of InDel-4 are shown in SEQ ID NOs. 7 to 8, primers for amplification of InDel-5 are shown in SEQ ID NOs. 9 to 10, primers for amplification of InDel-6 are shown in SEQ ID NOs. 11 to 12, primers for amplification of InDel-7 are shown in SEQ ID NOs. 13 to 14, primers for amplification of InDel-8 are shown in SEQ ID NOs. 15 to 16, primers for amplification of InDel-9 are shown in SEQ ID NOs. 17 to 18, primers for amplification of InDel-10 are shown in SEQ ID NOs. 19 to 20, primers for amplification of InDel-11 are shown in SEQ ID NOs. The primers for amplifying InDel-1 are shown in SEQ ID NOs. 21-22, the primers for amplifying InDel-12 are shown in SEQ ID NOs. 23-24, the primers for amplifying InDel-13 are shown in SEQ ID NOs. 25-26, the primers for amplifying InDel-14 are shown in SEQ ID NOs. 27-28, the primers for amplifying InDel-15 are shown in SEQ ID NOs. 29-30, the primers for amplifying InDel-16 are shown in SEQ ID NOs. 31-32, the primers for amplifying InDel-17 are shown in SEQ ID NOs. 33-34, the primers for amplifying InDel-18 are shown in SEQ ID NOs. 35-36, the primers for amplifying InDel-19 are shown in SEQ ID NOs. 37-38, the primers for amplifying InDel-20 are shown in SEQ ID NOs. 39-40, and the primers for amplifying InDel-21 are shown in SEQ ID NOs. The primers for amplifying InDel-22 are shown in SEQ ID NOs. 43-44, the primers for amplifying InDel-23 are shown in SEQ ID NOs. 45-46, the primers for amplifying InDel-24 are shown in SEQ ID NOs. 47-48, the primers for amplifying InDel-25 are shown in SEQ ID NOs. 49-50, the primers for amplifying InDel-26 are shown in SEQ ID NOs. 51-52, the primers for amplifying InDel-27 are shown in SEQ ID NOs. 53-54, the primers for amplifying InDel-28 are shown in SEQ ID NOs. 55-56, the primers for amplifying InDel-29 are shown in SEQ ID NOs. 57-58, and the primers for amplifying InDel-30 are shown in SEQ ID NO.59 to 60, the primers for amplification of InDel-31 are shown in SEQ ID NOs. 61 to 62, the primers for amplification of InDel-32 are shown in SEQ ID NOs. 63 to 64, the primers for amplification of InDel-33 are shown in SEQ ID NOs. 65 to 66, the primers for amplification of InDel-34 are shown in SEQ ID NOs. 67 to 68, the primers for amplification of InDel-35 are shown in SEQ ID NOs. 69 to 70, the primers for amplification of InDel-36 are shown in SEQ ID NOs. 71 to 72, the primers for amplification of InDel-37 are shown in SEQ ID NOs. 73 to 74, the primers for amplification of InDel-38 are shown in SEQ ID NOs. 75 to 76, the primers for amplification of InDel-39 are shown in SEQ ID NOs. 77 to 78, and the primers for amplification of InDel-40 are shown in SEQ ID NOs. The primers for amplifying InDel-41 are shown in SEQ ID NOs. 81-82, the primers for amplifying InDel-42 are shown in SEQ ID NOs. 83-84, the primers for amplifying InDel-43 are shown in SEQ ID NOs. 85-86, the primers for amplifying InDel-44 are shown in SEQ ID NOs. 87-88, the primers for amplifying InDel-45 are shown in SEQ ID NOs. 89-90, the primers for amplifying InDel-46 are shown in SEQ ID NOs. 91-92, the primers for amplifying InDel-47 are shown in SEQ ID NOs. 93-94, the primers for amplifying InDel-48 are shown in SEQ ID NOs. 95-96, the primers for amplifying InDel-49 are shown in SEQ ID NOs. 97-98, and the primers for amplifying InDel-50 are shown in SEQ ID NOs. The primers for amplifying InDel-51 are shown in SEQ ID NOs. 99 to 100, the primers for amplifying InDel-51 are shown in SEQ ID NOs. 101 to 102, the primers for amplifying InDel-52 are shown in SEQ ID NOs. 103 to 104, the primers for amplifying InDel-53 are shown in SEQ ID NOs. 105 to 106, the primers for amplifying InDel-54 are shown in SEQ ID NOs. 107 to 108, the primers for amplifying InDel-55 are shown in SEQ ID NOs. 109 to 110, the primers for amplifying InDel-56 are shown in SEQ ID NOs. 111 to 112, the primers for amplifying InDel-57 are shown in SEQ ID NOs. 113 to 114, the primers for amplifying InDel-58 are shown in SEQ ID NOs. 115 to 116, and the primers for amplifying InDel-59 are shown in SEQ ID NO.117-118, the primers for amplification of InDel-60 are shown in SEQ ID NOs. 119-120, the primers for amplification of InDel-61 are shown in SEQ ID NOs. 121-122, the primers for amplification of InDel-62 are shown in SEQ ID NOs. 123-124, the primers for amplification of InDel-63 are shown in SEQ ID NOs. 125-126, the primers for amplification of InDel-64 are shown in SEQ ID NOs. 127-128, the primers for amplification of InDel-65 are shown in SEQ ID NOs. 129-130, the primers for amplification of InDel-66 are shown in SEQ ID NOs. 131-132, the primers for amplification of InDel-67 are shown in SEQ ID NOs. 133-134, and the primers for amplification of InDel-68 are shown in SEQ ID NOs. The primers for amplifying InDel-69 are shown in SEQ ID NOs. 137-138, the primers for amplifying InDel-70 are shown in SEQ ID NOs. 139-140, the primers for amplifying InDel-71 are shown in SEQ ID NOs. 141-142, the primers for amplifying InDel-72 are shown in SEQ ID NOs. 143-144, the primers for amplifying InDel-73 are shown in SEQ ID NOs. 145-146, the primers for amplifying InDel-74 are shown in SEQ ID NOs. 147-148, the primers for amplifying InDel-75 are shown in SEQ ID NOs. 149-150, the primers for amplifying InDel-76 are shown in SEQ ID NOs. 152-153, and the primers for amplifying InDel-77 are shown in SEQ ID NOs. The primers for amplifying InDel-78 are shown in SEQ ID NOs. 153-154, the primers for amplifying InDel-79 are shown in SEQ ID NOs. 157-158.

2. The development of InDel molecular marker primers for dwarf tomato according to claim 1, characterized in that: The dwarf tomato InDel molecular marker is determined with reference to the genome SL5.

0.

3. The development and application of the dwarf tomato InDel molecular marker primer according to claim 1, characterized in that: This includes the application of amplification primers in the positioning of genes related to important agronomic traits of dwarf tomatoes.

4. The application of the dwarf tomato InDel molecular marker primer development according to claim 1, characterized in that: This includes the application of amplification primers in constructing a genetic linkage map of dwarf tomatoes.

5. The application of the dwarf tomato InDel molecular marker primer development according to claim 1, characterized in that: Including the application of amplification primers in detecting dwarf tomato resources.

6. The application of the dwarf tomato InDel molecular marker primer development according to claim 1, characterized in that: Including the application of amplification primers in dwarf tomato genetic breeding.

7. The application of the InDel molecular marker primers developed for dwarf tomato according to claim 1, characterized in that: Including the application of amplification primers in the analysis of genetic diversity of dwarf tomatoes.

8. The application of the dwarf tomato InDel molecular marker primer development according to claim 1, characterized in that: Includes the application of amplification primers in dwarf tomato hybrid / purity identification.

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