Primer set for amplifying dwarf tomato InDel molecular marker

Abstract

The present application relates to the technical field of molecular marker development and molecular detection, discloses development of dwarf tomato InDel molecular marker primer, provides a group of dwarf tomato InDel molecular markers, the dwarf tomato InDel molecular marker includes 79 InDel markers shown in the following table, and the dwarf tomato InDel molecular marker is determined with reference to genome SL5.0.Using the dwarf tomato InDel molecular marker can provide technical support for carrying out target DNA sample rapid molecular detection in dwarf tomato;Since the set of markers is distributed on multiple chromosomes of the dwarf tomato genome, can be selected and used according to actual needs, the genomic DNA of the candidate material can be detected at any stage, has the advantages of flexible use, simple operation, high detection efficiency, less limiting factors, stable and accurate detection results, provides a simple, flexible and efficient way for genetic linkage map construction, genetic diversity analysis, resource identification, hybrid / purity identification and the like in the dwarf tomato.

Description

Technical Field

[0001] This invention relates to the field of molecular marker development and molecular detection technology, specifically to a primer set for amplifying the InDel molecular marker in dwarf tomatoes. Background Technology

[0002] Tomato (Solanum lycopersicum M.) belongs to the Solanaceae family and the Solanum genus. It originated in the western Pacific coast of South America and was domesticated in Mexico. As a global crop, tomatoes have advantages such as wide environmental adaptability and high yield, and are cultivated in various ways. Rich in lycopene, folic acid, potassium, vitamin C, flavonoids, vitamin E, and sugars, tomatoes have a wide range of uses in the consumer market. For over a century, tomatoes have developed into a global vegetable crop, ranking among the top 30 highest-yielding crops worldwide. Besides its significant economic value, tomatoes also play an important role in life science research. The first high-density genetic linkage map of tomatoes was constructed by Tanksley et al. using an F2 population from a cross between cultivated tomato VF36-Tm2a and Pannalia tomato LA0716. With the rapid development of modern genome sequencing technology, numerous molecular genetic linkage maps of tomatoes have been constructed, promoting the mapping and cloning of QTLs for important agronomic traits in tomatoes, thus facilitating genetic improvement of tomatoes. Tomatoes are not only an important economic crop, but also have advantages such as short growth cycle, strong self-reproduction and regeneration ability, easy control of pollination and fertilization, and easy genetic transformation. They have been widely used as a model plant in the fields of plant genetic engineering technology research.

[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 variations between individuals, which are a direct reflection of DNA-level genetic polymorphism. It is an effective tool for studying genetic diversity of germplasm resources, and is currently mainly used for classification of germplasm resources and screening of core germplasm. At present, molecular markers play a very important role in gene mapping and cloning, genetic resource analysis and utilization, variety authenticity and purity identification, and crop breeding. Insertion / deletion polymorphism (InDel, Insertion / Deletion) marker is a length polymorphism variation caused by the insertion / deletion of nucleotide fragments at the allelic site between different individuals. The distribution and density of InDel markers in the genome are only second to single nucleotide polymorphism markers (SNP, single nucleotide polymorphism). There are a large number of InDel markers in the genome of cultivated tomatoes that can be used as molecular markers. Previous studies have shown that 14%-22% of genes contain InDel, while genes containing SSR account for only 4.6% of the total number of genes. The amplification product of InDel marker has simple and clear banding, and the stability and product separation effect are obviously better than those of SSR marker; compared with the complex SNP typing system, InDel detection is simple and convenient, and has 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 been gradually applied to tomato genetic research. However, compared with other crops, the genetic background of dwarf tomatoes is narrow, and it is difficult to develop markers, which cannot meet the research needs of gene mapping, genetic diversity analysis, purity detection and consistency identification. At present, there is almost no report on the InDel variation characteristics of the whole genome of dwarf tomatoes, the development of molecular markers, and the application of InDel markers in hybrid seed purity detection and variety consistency identification;

[0004] Therefore, in order to meet the different research needs of the number and type of molecular markers, expand the application range of molecular markers in dwarf tomato research, and accelerate the process of molecular breeding of dwarf tomatoes, it is urgent to clarify the InDel variation characteristics of the whole genome of dwarf tomatoes. The primer set for amplifying the InDel molecular marker of dwarf tomato is used to solve the above problems. SUMMARY

[0005] TECHNICAL PROBLEMS SOLVED

[0006] In view of the deficiencies of the prior art, the primer set for amplifying the InDel molecular marker of dwarf tomato is provided, which has the advantages of being used for genetic linkage map construction, genetic diversity analysis, resource identification, hybrid seed / purity identification of dwarf tomatoes, and solves the above problems.

[0007] Technical Solution

[0008] To achieve the above object, the present application provides the following technical scheme: dwarf tomato InDel molecular marker primer development, the dwarf tomato InDel molecular marker includes the following 79 InDel molecular markers: the amplification primer of InDel-1 is as shown in SEQ ID NO.1-2, the amplification primer of InDel-2 is as shown in SEQ ID NO.3-4, the amplification primer of InDel-3 is as shown in SEQ ID NO.5-6, the amplification primer of InDel-4 is as shown in SEQ ID NO.7-8, the amplification primer of InDel-5 is as shown in SEQ ID NO.9-10, the amplification primer of InDel-6 is as shown in SEQ ID NO.11-12, the amplification primer of InDel-7 is as shown in SEQ ID NO.13-14, the amplification primer of InDel-8 is as shown in SEQ ID NO.15-16, the amplification primer of InDel-9 is as shown in SEQ ID NO.17-18, the amplification primer of InDel-10 is as shown in SEQ ID NO.19-20, the amplification primer of InDel-11 is as shown in SEQ ID NO.21-22, the amplification primer of InDel-12 is as shown in SEQ ID NO.23-24, the amplification primer of InDel-13 is as shown in SEQ ID NO.25-26, the amplification primer of InDel-14 is as shown in SEQ ID NO.27-28, the amplification primer of InDel-15 is as shown in SEQ ID NO.29-30, the amplification primer of InDel-16 is as shown in SEQ ID NO.31-32, the amplification primer of InDel-17 is as shown in SEQ ID NO.33-34, the amplification primer of InDel-18 is as shown in SEQ ID NO.35-36, the amplification primer of InDel-19 is as shown in SEQ ID NO.37-38, the amplification primer of InDel-20 is as shown in SEQ ID NO.39-40, the amplification primer of InDel-21 is as shown in SEQ ID NO.41-42, the amplification primer of InDel-22 is as shown in SEQ ID NO.43-44, the amplification primer of InDel-23 is as shown in SEQ ID NO.45-46, the amplification primer of InDel-24 is as shown in SEQ ID NO.47-48, the amplification primer of InDel-25 is as shown in SEQ ID NO.49-50, the amplification primer of InDel-26 is as shown in SEQ ID NO.51-52, the amplification primer of InDel-27 is as shown in SEQ ID NO.53-54, the amplification primer of InDel-28 is as shown in SEQ ID NO.55-56, the amplification primer of InDel-29 is as shown in SEQ ID NO.57-58, the primers for amplifying InDel-30 are shown as SEQ ID NO. 59-60, the primers for amplifying InDel-31 are shown as SEQ ID NO. 61-62, the primers for amplifying InDel-32 are shown as SEQ ID NO. 63-64, the primers for amplifying InDel-33 are shown as SEQ ID NO. 65-66, the primers for amplifying InDel-34 are shown as SEQ ID NO. 67-68, the primers for amplifying InDel-35 are shown as SEQ ID NO. 69-70, the primers for amplifying InDel-36 are shown as SEQ ID NO. 71-72, the primers for amplifying InDel-37 are shown as SEQ ID NO. 73-74, the primers for amplifying InDel-38 are shown as SEQ ID NO. 75-76, the primers for amplifying InDel-39 are shown as SEQ ID NO. 77-78, the primers for amplifying InDel-40 are shown as SEQ ID NO. 79-80, the primers for amplifying InDel-41 are shown as SEQ ID NO. 81-82, the primers for amplifying InDel-42 are shown as SEQ ID NO. 83-84, the primers for amplifying InDel-43 are shown as SEQ ID NO. 85-86, the primers for amplifying InDel-44 are shown as SEQ ID NO. 87-88, the primers for amplifying InDel-45 are shown as SEQ ID NO. 89-90, the primers for amplifying InDel-46 are shown as SEQ ID NO. 91-92, the primers for amplifying InDel-47 are shown as SEQ ID NO. 93-94, the primers for amplifying InDel-48 are shown as SEQ ID NO. 95-96, the primers for amplifying InDel-49 are shown as SEQ ID NO. 97-98, the primers for amplifying InDel-50 are shown as SEQ ID NO. 99-100, the primers for amplifying InDel-51 are shown as SEQ ID NO. 101-102, the primers for amplifying InDel-52 are shown as SEQ ID NO. 103-104, the primers for amplifying InDel-53 are shown as SEQ ID NO. 105-106, the primers for amplifying InDel-54 are shown as SEQ ID NO. 107-108, the primers for amplifying InDel-55 are shown as SEQ ID NO. 109-110, the primers for amplifying InDel-56 are shown as SEQ ID NO. 111-112, the primers for amplifying InDel-57 are shown as SEQ ID NO. 113-114, the primers for amplifying InDel-58 are shown as SEQ ID NO.As shown in SEQ ID NOs 115-116, the primers for amplifying InDel-59 are shown in SEQ ID NOs 117-118; the primers for amplifying InDel-60 are shown in SEQ ID NOs 119-120; the primers for amplifying InDel-61 are shown in SEQ ID NOs 121-122; the primers for amplifying InDel-62 are shown in SEQ ID NOs 123-124; the primers for amplifying InDel-63 are shown in SEQ ID NOs 125-126; the primers for amplifying InDel-64 are shown in SEQ ID NOs 127-128; the primers for amplifying InDel-65 are shown in SEQ ID NOs 129-130; the primers for amplifying InDel-66 are shown in SEQ ID NOs 131-132; and the primers for amplifying InDel-67 are shown in SEQ ID NOs 115-116. As shown in SEQ ID NOs 133-134, 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 135-136. Primers for InDel-77 amplification are shown in SEQ ID NOs 152-152, primers for InDel-78 amplification are shown in SEQ ID NOs 153-154, primers for InDel-78 amplification are shown in SEQ ID NOs 155-156, and primers for InDel-79 amplification are shown in SEQ ID NOs 157-158.

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

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

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

[0012] This invention also provides the application of the amplification primers described in the above technical solution in the detection of dwarf tomato resources.

[0013] The application also provides the application of the primer for amplification in the genetic breeding of the dwarf tomato.

[0014] The application also provides the application of the primer for amplification in the genetic diversity analysis of the dwarf tomato.

[0015] The application also provides the application of the primer for amplification in the hybridization / purity identification of the dwarf tomato.

[0016] Compared with the prior art, the dwarf tomato InDel molecular marker primer development and application provided by the application has the following beneficial effects:

[0017] The primer set for amplifying the dwarf tomato InDel molecular marker can provide technical support for the rapid molecular detection of target DNA samples in the dwarf tomato by using the dwarf tomato InDel molecular marker; since the markers are distributed on multiple chromosomes of the dwarf tomato genome, the markers can be selected and used according to actual needs, the genomic DNA of the candidate material can be detected at any stage, and the markers have the advantages of flexible use, simple operation, high detection efficiency, few limiting factors, stable and accurate detection results, and the like, and provide a simple, flexible and efficient way for the genetic linkage map construction, genetic diversity analysis, resource identification, hybridization / purity identification and the like of the dwarf tomato. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 Part of the InDel primer screening results provided by the application are shown in the figure;

[0019] Figure 2 The dwarf tomato physical map constructed by the 79 InDel markers provided by the application is shown in the figure;

[0020] Figure 3 The electropherogram of the genetic diversity analysis of the dwarf tomato by the InDel molecular marker provided by the application is shown in the figure.

[0021] Figure 4 The electropherogram of the genetic diversity analysis of the dwarf tomato by the InDel molecular marker provided by the application is shown in the figure. DETAILED DESCRIPTION

[0022] The technical solutions in the embodiments of the application will be clearly and completely described below with reference to the drawings in the embodiments of the application. Obviously, the described embodiments are only part of the embodiments of the application, rather than all the embodiments of the application. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative work fall within the protection scope of the application.

[0023] Please refer toFigures 1-4 ;

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

[0025] Compared with the previous markers such as SSR, the dwarf tomato InDel molecular markers developed in the application are distributed on multiple chromosomes of the dwarf tomato genome, have the characteristics of unique amplification, good typing effect, strong representation, can realize high-throughput detection, and have a short detection period; when performing variety right confirmation or purity detection, the operation is simple and rapid, the detection result is stable and reliable, and the markers can be flexibly selected according to the detection sample and the number of sites. Therefore, the InDel markers developed in the application can realize standardization and scaling for detection of different numbers of sample amounts.

[0026] The application also provides primers for amplifying the InDel molecular markers in the above technical solution, the primers for amplifying InDel-1 are shown as SEQ ID NO. 1-2, the primers for amplifying InDel-2 are shown as SEQ ID NO. 3-4, the primers for amplifying InDel-3 are shown as SEQ ID NO. 5-6, the primers for amplifying InDel-4 are shown as SEQ ID NO. 7-8, the primers for amplifying InDel-5 are shown as SEQ ID NO. 9-10, the primers for amplifying InDel-6 are shown as SEQ ID NO. 11-12, the primers for amplifying InDel-7 are shown as SEQ ID NO. 13-14, the primers for amplifying InDel-8 are shown as SEQ ID NO. 15-16, the primers for amplifying InDel-9 are shown as SEQ ID NO. 17-18, the primers for amplifying InDel-10 are shown as SEQ ID NO. 19-20, the primers for amplifying InDel-11 are shown as SEQ ID NO. 21-22, the primers for amplifying InDel-12 are shown as SEQ ID NO. 23-24, the primers for amplifying InDel-13 are shown as SEQ ID NO. 25-26, the primers for amplifying InDel-14 are shown as SEQ ID NO. 27-28, the primers for amplifying InDel-15 are shown as SEQ ID NO. 29-30, the primers for amplifying InDel-16 are shown as SEQ ID NO. 31-32, the primers for amplifying InDel-17 are shown as SEQ ID NO. 33-34, the primers for amplifying InDel-18 are shown as SEQ ID NO. 35-36, the primers for amplifying InDel-19 are shown as SEQ ID NO. 37-38, the primers for amplifying InDel-20 are shown as SEQ ID NO. 39-40, the primers for amplifying InDel-21 are shown as SEQ ID NO. 41-42, the primers for amplifying InDel-22 are shown as SEQ ID NO. 43-44, the primers for amplifying InDel-23 are shown as SEQ ID NO. 45-46, the primers for amplifying InDel-24 are shown as SEQ ID NO. 47-48, the primers for amplifying InDel-25 are shown as SEQ ID NO. 49-50, the primers for amplifying InDel-26 are shown as SEQ ID NO. 51-52, the primers for amplifying InDel-27 are shown as SEQ ID NO. 53-54, the primers for amplifying InDel-28 are shown as SEQ ID NO. 55-56, the primers for amplifying InDel-29 are shown as SEQ ID NO. 57-58, the primers for amplifying InDel-30 are shown as SEQ ID NO. 59-60, the primers for amplifying InDel-31 are shown as SEQ ID NO. 61-62, the primers for amplifying InDel-32 are shown as SEQ ID NO.63-64, the primers for amplifying InDel-33 are shown as SEQ ID NO. 65-66, the primers for amplifying InDel-34 are shown as SEQ ID NO. 67-68, the primers for amplifying InDel-35 are shown as SEQ ID NO. 69-70, the primers for amplifying InDel-36 are shown as SEQ ID NO. 71-72, the primers for amplifying InDel-37 are shown as SEQ ID NO. 73-74, the primers for amplifying InDel-38 are shown as SEQ ID NO. 75-76, the primers for amplifying InDel-39 are shown as SEQ ID NO. 77-78, the primers for amplifying InDel-40 are shown as SEQ ID NO. 79-80, the primers for amplifying InDel-41 are shown as SEQ ID NO. 81-82, the primers for amplifying InDel-42 are shown as SEQ ID NO. 83-84, the primers for amplifying InDel-43 are shown as SEQ ID NO. 85-86, the primers for amplifying InDel-44 are shown as SEQ ID NO. 87-88, the primers for amplifying InDel-45 are shown as SEQ ID NO. 89-90, the primers for amplifying InDel-46 are shown as SEQ ID NO. 91-92, the primers for amplifying InDel-47 are shown as SEQ ID NO. 93-94, the primers for amplifying InDel-48 are shown as SEQ ID NO. 95-96, the primers for amplifying InDel-49 are shown as SEQ ID NO. 97-98, the primers for amplifying InDel-50 are shown as SEQ ID NO. 99-100, the primers for amplifying InDel-51 are shown as SEQ ID NO. 101-102, the primers for amplifying InDel-52 are shown as SEQ ID NO. 103-104, the primers for amplifying InDel-53 are shown as SEQ ID NO. 105-106, the primers for amplifying InDel-54 are shown as SEQ ID NO. 107-108, the primers for amplifying InDel-55 are shown as SEQ ID NO. 109-110, the primers for amplifying InDel-56 are shown as SEQ ID NO. 111-112, the primers for amplifying InDel-57 are shown as SEQ ID NO. 113-114, the primers for amplifying InDel-58 are shown as SEQ ID NO. 115-116, the primers for amplifying InDel-59 are shown as SEQ ID NO. 117-118, the primers for amplifying InDel-60 are shown as SEQ ID NO. 119-120, the primers for amplifying InDel-61 are shown as SEQ ID NO. 121-122, the primers for amplifying InDel-62 are shown as SEQ ID NO. 123-124, the primers for amplifying InDel-63 are shown as SEQ ID NO.SEQ ID NO. 127-128, the amplification primer of InDel-65 is shown as SEQ ID NO. 129-130, the amplification primer of InDel-66 is shown as SEQ ID NO. 131-132, the amplification primer of InDel-67 is shown as SEQ ID NO. 133-134, the amplification primer of InDel-68 is shown as SEQ ID NO. 135-136, the amplification primer of InDel-69 is shown as SEQ ID NO. 137-138, the amplification primer of InDel-70 is shown as SEQ ID NO. 139-140, the amplification primer of InDel-71 is shown as SEQ ID NO. 141-142, the amplification primer of InDel-72 is shown as SEQ ID NO. 143-144, the amplification primer of InDel-73 is shown as SEQ ID NO. 145-146, the amplification primer of InDel-74 is shown as SEQ ID NO. 147-148, the amplification primer of InDel-75 is shown as SEQ ID NO. 149-150, the amplification primer of InDel-76 is shown as SEQ ID NO. 152-152, the amplification primer of InDel-77 is shown as SEQ ID NO. 153-154, the amplification primer of InDel-78 is shown as SEQ ID NO. 155-156, and the amplification primer of InDel-79 is shown as SEQ ID NO. 157-158.

[0027] In the present application, the PCR reaction system is preferably shown in Table 4 when the product is applied. The PCR program is preferably as follows: pre-denaturation: 94℃, 4 min; cycle amplification: 94℃, 30 s; 55℃, 30 s; 72℃, 30 s; cycle 35 times; cycle extension: 72℃, 7 min; and storage at 4℃.

[0028] The present application also provides the application of the above technical solution amplification primer in constructing the genetic linkage map of dwarf tomato.

[0029] As an important genetic marker, the InDel molecular marker has been widely used in the construction of crop linkage map. The present application uses QTL mapping software to draw the physical map of the 79 InDel molecular markers on the chromosome of dwarf tomato ( Figure 2 ), which lays a foundation for the construction of the genetic linkage map of dwarf tomato.

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

[0031] As a high-throughput DNA fingerprint marker, InDel molecular marker technology can better reveal the polymorphism in the genome, thus having more prominent advantages in germplasm identification, and is widely used in crops such as corn, rice and Chinese cabbage. The present application uses the developed polymorphic InDel primers, randomly selects two pairs of polymorphic primers (InDel-7 and InDel-11), and combines the rapid extraction method of dwarf tomato seedling genomic DNA to identify 24 dwarf tomato varieties, and the PCR amplification result shows that different varieties present variety specificity under the detection of different primers (Table 1) Figure 3 ). The results show that InDel technology, as a rapid, accurate and efficient method, can be used for large-scale DNA fingerprint analysis. The InDel molecular marker developed in the present application can play an important role in the identification of dwarf tomato germplasm resources.

[0032] The present application also provides the application of the above-mentioned primer for amplification in the genetic breeding of dwarf tomato.

[0033] The present application also provides the application of the above-mentioned primer for amplification in the genetic diversity analysis of dwarf tomato.

[0034] The present application also provides the application of the above-mentioned primer for amplification in the hybridization / purity identification of dwarf tomato.

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

[0036] If the specific technology or condition is not specified in the examples, the technology or condition described in the literature in the art is used, or the product instruction is followed. The reagents or instruments used are the conventional products that can be commercially available from the regular channels, and the manufacturers are indicated.

[0037] Example 1:

[0038] Extraction of genomic DNA of dwarf tomato

[0039] In 2 mL Eppendorf centrifuge tube, 1 g of young leaves of dwarf tomato at three-leaf stage were taken, two steel balls and 250 μL of modified CTAB lysis solution were added, and the sample was printed with a plant tissue crusher at 27 r / s for 90 s. The crushed sample was centrifuged at 12000 r / min for 1 min in a greenhouse, 500 μL of CTAB lysis solution was added, and the mixture was thoroughly mixed and placed in a 65°C water bath for 30 min. Then the sample was taken out and cooled to room temperature, an equal volume of chloroform was added, and the mixture was gently shaken for 5 min. The emulsion was centrifuged at 12000 r / min for 8-10 min at room temperature, 400 μL of supernatant was transferred to a new 1.5 mL centrifuge tube, 600 μL of anhydrous ethanol was added, and the mixture was placed in an ice bath for 1 h. The mixture was centrifuged at 12000 r / min for 5 min, the supernatant was discarded, and the precipitate was washed with 75% ethanol for 2-3 times. Each time after washing, the mixture was centrifuged at 12000 r / min for 3 min, the supernatant was discarded, and the DNA was dried at room temperature. 100 μL of ddH2O was added, and the mixture was thoroughly dissolved and stored at 4°C for later use. The DNA purity and quantity were analyzed and detected by ultraviolet-visible spectrophotometer.

[0040] Example 2

[0041] InDel marker screening of dwarf tomato

[0042] 9 kinds of dwarf tomatoes collected by the tomato breeding team of Hebei Science and Technology Normal College were subjected to 10x resequencing. Based on the reference genome SL5.0, whole genome genetic variation analysis was performed, and 476708 high-quality InDels were obtained on the chromosome level. In combination with the physical location and function of the variation sites, InDel sites were selected on 12 chromosomes. Using Perl language self-compiled script combined with InDel variation sites obtained from parent resequencing data, the reference genome sequence of 100 bp upstream and downstream of the InDel variation site in the candidate interval was extracted, and InDel primers were designed using Primer5.0 software. According to the length of the chromosome and the number and type of variation sites on each chromosome, 144 variation sites that can be designed into InDel markers were screened out. 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 representation were screened out (Table 1). Part of the InDel primer screening results are shown in Figure 1 ; wherein A is the screening result of primer ZD01-1 without polymorphism, B is the screening result of primer InDel-7 with polymorphism, and the numbers represent the amplification results of different dwarf tomato varieties. According to A in Figure 1 , the bands amplified from NO.1-NO.24 samples are the same, indicating that there is no polymorphism in Gaiwei; Figure 1The A band shape, the B band shape and the H band shape were amplified in B, and the A band shape, the B band shape and the H band shape were amplified in B, indicating that the primer has polymorphism.

[0043] Table 1

[0044] InDel primer name Upstream primer SEQ ID NO. Downstream primer SEQ ID NO. InDel-1 TCACCTCCGATGTGGAAC Seq ID NO. 1 GTGTTGGGTGGTCTCCTT Seq ID NO. 2 InDel-2 TCGTTACACTAGCATACG Seq ID NO. 3 ATATGCCAATGAACTAGG Seq ID NO. 4 InDel-3 CCACACCATCATTTCGTG Seq ID NO. 5 AAAGAACTGTCTCACTCT Seq ID NO. 6 InDel-4 AGCGAACTATTGAGAGTC Seq ID NO. 7 GGTTAAATGGGATGGTCT Seq ID NO. 8 InDel-5 TGAGCATCCTAACTGCTT Seq ID NO. 9 TCCTACTGCTAACTGACA Seq ID NO. 10 InDel-6 TCTACTACGTTTGCTACA Seq ID NO. 11 AGTGAGGAAAAGTGTAGA Seq ID NO. 12 InDel-7 ACGCGCCTCAGCAAGTAG Seq ID NO. 13 ATAAGTGAACGAAGATCG Seq ID NO. 14 InDel-8 TATTGGCAGTACAGGTTCGA Seq ID NO. 15 AAGGGCGGAGGTTAAAGT Seq ID NO. 16 InDel-9 TAGATTCCGGACCAAGGA Seq ID NO. 17 ATCGTTTCCACAAGGGAA Seq ID NO. 18 InDel-10 AGAACGTGAGAGATAGAA Seq ID NO. 19 GCAAGGGTTCACACACAT Seq ID NO. 20 InDel-11 CGAGCTACAACAAGGCGAACAATT Seq ID NO. 21 GTATCGAACAGATACACCT Seq ID NO. 22 InDel-12 GGATGAATTTTGGTGTGAGG Seq ID NO. 23 GACCTACGTTTTTACACTCA Seq ID NO. 24 InDel-13 AATAAACGGGTCTCGCGT Seq ID NO. 25 GGAGTATGGACCAAGTCG Seq ID NO. 26 InDel-14 GACTAATTTCAGGGCAAC Seq ID NO. 27 CATGAGGCTTGTGGACTC Seq ID NO. 28 InDel-15 AGGCCGAACACTGATAGG Seq ID NO. 29 AAAGCTCGACACCAAACG Seq ID NO. 30 InDel-16 GGTCGTATAGGATGCTCT Seq ID NO. 31 CTTGCATAATTCACTAGGACC Seq ID NO. 32 InDel-17 TCACGCATACAGCAAGTA Seq ID NO. 33 TTTGGTTCCCATATGCGTG Seq ID NO. 34 InDel-18 AGAGATACAAGATCAACGC Seq ID NO. 35 GAACATACTCCACTAGCGTA Seq ID NO. 36 InDel-19 CATTATCTTGTTCGCAGCCA Seq ID NO. 37 GAATCGCCTAGGTTCAAAA Seq ID NO. 38 InDel-20 CACTAGTGTACCCCATGG Seq ID NO. 39 CAAATCGGCAGACTATTTC Seq ID NO. 40 InDel-21 CGTCATTGGGGATTCATT Seq ID NO. 41 TGAGAGGTAGGACATTGG Seq ID NO. 42 InDel-22 GATACCTATCCTATCCAAG Seq ID NO. 43 TGTGTGTGCGATTGGACG Seq ID NO. 44 InDel-23 TACATCTCCCTCCTAGAG Seq ID NO. 45 AGTATTTCCCCCGTACCA Seq ID NO. 46 InDel-24 GCTTAACCATGTACAACCCA Seq ID NO. 47 GTATTTAGTGTGCCAGCC Seq ID NO. 48 InDel-25 ACAAGACTGCTCATACCC Seq ID NO. 49 CAAGCTAACATGATCTCTG Seq ID NO. 50 InDel-26 TCATCGATCTTAGGTGGAG Seq ID NO. 51 TGCCATTAGTTTGCAGTAG Seq ID NO. 52 InDel-27 CAGAGAATTGAGCTAAGG Seq ID NO. 53 TGACAGTATTAGCTGCCA Seq ID NO. 54 InDel-28 GTCTTAGAGAAGTGTAAGTC Seq ID NO. 55 GATAGGTTATAGTGTAGGC Seq ID NO. 56 InDel-29 AGTATTAAAGGAGTCACGGG Seq ID NO. 57 CACGTCGATACTATGTCCA Seq ID NO. 58 InDel-30 TCAGGGACTAACATTCCCA Seq ID NO. 59 GAAGTTATTCGAGAGAGGTT Seq ID NO. 60 InDel-31 ATCACTGTCAGAGGGACC Seq ID NO. 61 GAGCATATCCCCTAGCATG Seq ID NO. 62 InDel-32 AGCATTGCAACATGACCAT Seq ID NO. 63 GAGTTATTATATTGGCAGGAG Seq ID NO. 64 InDel-33 TGAGAGCTATTGTAGTGTG Seq ID NO. 65 AAGGATCTGTGAGCTCTCC Seq ID NO. 66 InDel-34 TCCTCCCACACACTTCCCA Seq ID NO. 67 ACGACTCCCCAATGGACCA Seq ID NO. 68 InDel-35 ACCGAGACATGTCTGCTTC Seq ID NO. 69 TGCTCCTTCTAATGCAGT Seq ID NO. 70 InDel-36 GTTCATTGACCTCGAGAA Seq ID NO. 71 CGGTTACACCTCTTGCCTC Seq ID NO. 72 InDel-37 AACGCAATACAACTGCTC Seq ID NO. 73 ATGAAAGTGCACGGAGGGG Seq ID NO. 74 InDel-38 ACTAGTGTGGATCTTCAAG Seq ID NO. 75 AACACCTGCAAGACATTC Seq ID NO. 76 InDel-39 TGGATCCACGTTATCAGT Seq ID NO. 77 GTGGCGTTTTGGATTAATG Seq ID NO. 78 InDel-40 CTAATGCAAATACAGGTGG Seq ID NO. 79 GTGACGCTTAGATTATGTTC Seq ID NO. 80 InDel-41 CTCACTTCGATACCATCGA Seq ID NO. 81 CCATGCGTTTGGCCTTGTA Seq ID NO. 82 InDel-42 TTCGGAGTGCAGATTGGC Seq ID NO. 83 AGCAGGCCACTTAATTTC Seq ID NO. 84 InDel-43 GACGCAGAGAGTAGGTACG Seq ID NO. 85 CTAATGAGTTGTCCTAACG Seq ID NO. 86 InDel-44 GGAAGTTGCGGGTTGGTAA Seq ID NO. 87 GCAATGAGGGTCCGACCAA Seq ID NO. 88 InDel-45 GTTCCCCGAGTTGATTACA Seq ID NO. 89 TGTCAAGGGCCATATTGT Seq ID NO. 90 InDel-46 TTTGAACCTCGTGCACTTG Seq ID NO. 91 GTGGATACGATTCAGTAGA Seq ID NO. 92 InDel-47 CATAGCACGTAAAGTCTAG Seq ID NO. 93 ATGACACTTGTCAGGGTGG Seq ID NO. 94 InDel-48 CACTCCTCCCTCATCTAGC Seq ID NO. 95 TTTAATGGAGGTGCGCTGTTG Seq ID NO. 96 InDel-49 GCCCTTTAACTTCGACTCA Seq ID NO. 97 AATTGCCATGTAGGATGTC Seq ID NO. 98 InDel-50 GACGTGGTTTAAGGGGGAAT Seq ID NO. 99 CATCTTGAGGAGAGCCTCC Seq ID NO. 100 InDel-51 TGTGTGTCTAGTGCTGGG Seq ID NO. 101 CTCCAACTCGGGTTTTAAAG Seq ID NO. 102 InDel-52 GAGGAGTAGTTCTAGGTT Seq ID NO. 103 AACCAGATTACAACCAAC Seq ID NO. 104 InDel-53 TTTGACTCATTGCGAAGAG Seq ID NO. 105 CAGAGACACACTCACAAGT Seq ID NO. 106 InDel-54 GGATCATGCACAATAAGAAC Seq ID NO. 107 GGTTTGTCCTTCGAGTTGG Seq ID NO. 108 InDel-55 ACACAAGTATGGGGTTGTG Seq ID NO. 109 CCGAAACCACATTTAGGT Seq ID NO. 110 InDel-56 CAACATACGCACAACTATAG Seq ID NO. 111 ATCCACTCGAACGACACGAT Seq ID NO. 112 InDel-57 TTGGTCGTGTGAGCTCTGAC Seq ID NO. 113 CCAAAAAAATGCATGCTTAGGC Seq ID NO. 114 InDel-58 CACTAAAAGCAATGCTAAGACC Seq ID NO. 115 TTTCTTGAGGGTCATTGTG Seq ID NO. 116 InDel-59 ATGGAATTCCACGTCTTGC Seq ID NO. 117 CACATACTATAGATCAGAGTTCGG Seq ID NO. 118 InDel-60 GAACACAAGGATTGGTCA Seq ID NO. 119 TTGGTCGTTTAATGTGTGCA Seq ID No. 120 InDel-61 TTACTGGACACTGCAAAC Seq ID No. 121 CTCCTTCGTCTCCCAGTGGTT Seq ID No. 122 InDel-62 GAAACATTGAACTGGAGGGTAC Seq ID No. 123 ACGACCAGCCGGATCTCTTCCAG Seq ID No. 124 InDel-63 CCAAACATACTTCGAGTTGATGTG Seq ID No. 125 CTTCTTAGTACATGGACCAA Seq ID No. 126 InDel-64 ACCAACATGATCGGTGAGGA Seq ID No. 127 TGCATTTGCCTTACTAGTTGAG Seq ID No. 128 InDel-65 TAGTAAGGAGAGCTTACATG Seq ID No. 129 AACGCGGACAAACTATACTTG Seq ID No. 130 InDel-66 TATCTTGATCGAGCTCAACCC Seq ID No. 131 TGCTTTGATGCACGTGCA Seq ID No. 132 InDel-67 ACTTCAACCGTCCTCTCGAAC Seq ID No. 133 TACACCACTAAGCTTTATGC Seq ID No. 134 InDel-68 GAGTCACAGTACACCAATCTCTC Seq ID No. 135 TTCCTTCCTACTGGCCAAC Seq ID No. 136 InDel-69 CTCTCTTGTCAAGACCGTAGTCT Seq ID No. 137 CTCACTATGAAGTCGAAGT Seq ID No. 138 InDel-70 TTCAGCAAGAGTGGGACCA Seq ID No. 139 ATGTCACACCTAAAACCC Seq ID No. 140 InDel-71 GAATGTACAATGGATACCGT Seq ID No. 141 AGAGTTGCACGGTATTGA Seq ID No. 142 InDel-72 TCACGCCAAGTAGAACAATC Seq ID No. 143 TCTAGTAGGCTAATGGGTGT Seq ID No. 144 InDel-73 GTGCAACTCACATTGTGCC Seq ID No. 145 TGAATATCGGTCATCTTGAGGA Seq ID No. 146 InDel-74 AACCCTGCACCACAGATAGG Seq ID No. 147 CAAAGTCACCGACAGGGTGAA Seq ID No. 148 InDel-75 CCTTCTCGATCAAACGCAA Seq ID No. 149 ATGTCAGTCCTTCTTCTTG Seq ID No. 150 InDel-76 CTCTACAAAGTTACACTCC Seq ID No. 151 CCTCGCCTTTTTACCATCGCC Seq ID No. 152 InDel-77 TAGAAGCGTCCATTGTTG Seq ID No. 153 GGTCACTTAGAGATGAAAC Seq ID No. 154 InDel-78 CAGACTCTGGCACCCTAT Seq ID No. 155 GGTGCAGCACACAGAGAGGATG Seq ID No. 156 InDel-79 CTCACTTGACTGAGTTATG Seq ID No. 157 GGTTTTATGCTGCGTAATGA Seq ID No. 158

[0045] Example Three

[0046] PCR amplification of InDel primers

[0047] The genomic DNA of the compact tomato extracted was subjected to PCR amplification.

[0048] The reaction system for amplification was as follows:

[0049] Component volume (μL)

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

[0051] Forward primer (10 μM) 0.5

[0052] Forward primer (10 μM) 0.5

[0053] Genomic template (30 ng / μL) 1

[0054] Water 3

[0055] Total volume 10

[0056] The amplification program was as follows:

[0057] Pre-denaturation: 94℃, 4 min;

[0058] Cycle amplification: 94℃, 30 s; 55℃, 30 s; 72℃, 30 s; cycle 35 times;

[0059] Cycle extension: 72℃, 7 min, storage at 10℃.

[0060] Example Four

[0061] Preparation of agarose gel: 10.5 g of agarose was weighed, 350 mL of 0.5×TBE was added, heated for 5 min, and after slight cooling, 10 μL of 0.5 μg / mL EB solution was added and mixed. The electrophoresis tank was installed, the gel was poured, the sample comb was installed, and after the gel cooled and coagulated, 0.5×TBE was poured into the electrophoresis tank. 5 μL of PCR amplification product was taken to the sample well, and electrophoresis separation was carried out at 130 V constant voltage for 1 h to 1.5 h until the amplified DNA band was fully developed. After electrophoresis, the band characteristics were observed by gel imaging system (Bio-Rad, Hercules, CA, USA), and the results are shown in Figure 2 .

[0062] The polymorphism of the InDel primer in the compact tomato variety is screened by agarose gel electrophoresis detection of 144 pairs of InDel primer PCR amplification products, and finally 79 pairs of primers with good amplification effect and showing polymorphism are screened (Table 1). Figure 1 These InDel markers can be applied to compact tomato variety identification, genetic map construction, functional gene / QTL positioning, molecular assisted breeding and genetic diversity analysis research.

[0063] The beneficial effects of the present application are: the compact tomato InDel molecular markers can provide technical support for the rapid molecular detection of target DNA samples in compact tomatoes; since the markers are distributed on multiple chromosomes of the compact tomato genome, they can be selected and used according to actual needs, and the genomic DNA of the candidate material can be detected at any stage, with the advantages of flexible use, simple operation, high detection efficiency, few limiting factors, stable and accurate detection results, providing a simple, flexible and efficient way for genetic linkage map construction, genetic diversity analysis, resource identification, hybridization / purity identification and the like in compact tomatoes.

[0064] Although the embodiments of the present application have been shown and described, it can be understood by those of ordinary skill in the art that various changes, modifications, replacements and variations can be made to the embodiments without departing from the principles and spirits of the present application, and the scope of the present application is defined by the appended claims and their equivalents.

Claims

1. A primer set for amplifying an InDel molecular marker of dwarf tomato, characterized in that, The dwarf tomato InDel molecular markers are composed of 79 InDel molecular markers of InDel-1 to InDel-79; the primer for amplification of InDel-1 is shown as SEQ ID NO. 1-2, the primer for amplification of InDel-2 is shown as SEQ ID NO. 3-4, the primer for amplification of InDel-3 is shown as SEQ ID NO. 5-6, the primer for amplification of InDel-4 is shown as SEQ ID NO. 7-8, the primer for amplification of InDel-5 is shown as SEQ ID NO. 9-10, the primer for amplification of InDel-6 is shown as SEQ ID NO. 11-12, the primer for amplification of InDel-7 is shown as SEQ ID NO. 13-14, the primer for amplification of InDel-8 is shown as SEQ ID NO. 15-16, the primer for amplification of InDel-9 is shown as SEQ ID NO. 17-18, the primer for amplification of InDel-10 is shown as SEQ ID NO. 19-20, the primer for amplification of InDel-11 is shown as SEQ ID NO. 21-22, the primer for amplification of InDel-12 is shown as SEQ ID NO. 23-24, the primer for amplification of InDel-13 is shown as SEQ ID NO. 25-26, the primer for amplification of InDel-14 is shown as SEQ ID NO. 27-28, the primer for amplification of InDel-15 is shown as SEQ ID NO. 29-30, the primer for amplification of InDel-16 is shown as SEQ ID NO. 31-32, the primer for amplification of InDel-17 is shown as SEQ ID NO. 33-34, the primer for amplification of InDel-18 is shown as SEQ ID NO. 35-36, the primer for amplification of InDel-19 is shown as SEQ ID NO. 37-38, the primer for amplification of InDel-20 is shown as SEQ ID NO. 39-40, the primer for amplification of InDel-21 is shown as SEQ ID NO. 41-42, the primer for amplification of InDel-22 is shown as SEQ ID NO. 43-44, the primer for amplification of InDel-23 is shown as SEQ ID NO. 45-46, the primer for amplification of InDel-24 is shown as SEQ ID NO. 47-48, the primer for amplification of InDel-25 is shown as SEQ ID NO. 49-50, the primer for amplification of InDel-26 is shown as SEQ ID NO. 51-52, the primer for amplification of InDel-27 is shown as SEQ ID NO. 53-54, the primer for amplification of InDel-28 is shown as SEQ ID NO. 55-56, the primer for amplification of InDel-29 is shown as SEQ ID NO.57-58, the primers for amplifying InDel-30 are shown as SEQ ID NO. 59-60, the primers for amplifying InDel-31 are shown as SEQ ID NO. 61-62, the primers for amplifying InDel-32 are shown as SEQ ID NO. 63-64, the primers for amplifying InDel-33 are shown as SEQ ID NO. 65-66, the primers for amplifying InDel-34 are shown as SEQ ID NO. 67-68, the primers for amplifying InDel-35 are shown as SEQ ID NO. 69-70, the primers for amplifying InDel-36 are shown as SEQ ID NO. 71-72, the primers for amplifying InDel-37 are shown as SEQ ID NO. 73-74, the primers for amplifying InDel-38 are shown as SEQ ID NO. 75-76, the primers for amplifying InDel-39 are shown as SEQ ID NO. 77-78, the primers for amplifying InDel-40 are shown as SEQ ID NO. 79-80, the primers for amplifying InDel-41 are shown as SEQ ID NO. 81-82, the primers for amplifying InDel-42 are shown as SEQ ID NO. 83-84, the primers for amplifying InDel-43 are shown as SEQ ID NO. 85-86, the primers for amplifying InDel-44 are shown as SEQ ID NO. 87-88, the primers for amplifying InDel-45 are shown as SEQ ID NO. 89-90, the primers for amplifying InDel-46 are shown as SEQ ID NO. 91-92, the primers for amplifying InDel-47 are shown as SEQ ID NO. 93-94, the primers for amplifying InDel-48 are shown as SEQ ID NO. 95-96, the primers for amplifying InDel-49 are shown as SEQ ID NO. 97-98, the primers for amplifying InDel-50 are shown as SEQ ID NO. 99-100, the primers for amplifying InDel-51 are shown as SEQ ID NO. 101-102, the primers for amplifying InDel-52 are shown as SEQ ID NO. 103-104, the primers for amplifying InDel-53 are shown as SEQ ID NO. 105-106, the primers for amplifying InDel-54 are shown as SEQ ID NO. 107-108, the primers for amplifying InDel-55 are shown as SEQ ID NO. 109-110, the primers for amplifying InDel-56 are shown as SEQ ID NO. 111-112, the primers for amplifying InDel-57 are shown as SEQ ID NO. 113-114, the primers for amplifying InDel-58 are shown as SEQ ID NO.As shown in SEQ ID NOs 115-116, the primers for amplifying InDel-59 are shown in SEQ ID NOs 117-118; the primers for amplifying InDel-60 are shown in SEQ ID NOs 119-120; the primers for amplifying InDel-61 are shown in SEQ ID NOs 121-122; the primers for amplifying InDel-62 are shown in SEQ ID NOs 123-124; the primers for amplifying InDel-63 are shown in SEQ ID NOs 125-126; the primers for amplifying InDel-64 are shown in SEQ ID NOs 127-128; the primers for amplifying InDel-65 are shown in SEQ ID NOs 129-130; the primers for amplifying InDel-66 are shown in SEQ ID NOs 131-132; and the primers for amplifying InDel-67 are shown in SEQ ID NOs 115-116. As shown in SEQ ID NOs 133-134, 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 135-136. Primers for amplification of InDel-77 are shown in SEQ ID NO. 152-152, primers for amplification of InDel-78 are shown in SEQ ID NO. 155-156, and primers for amplification of InDel-79 are shown in SEQ ID NO. 157-158. The dwarf tomato InDel molecular marker is determined with reference genome SL5.

0.

2. Use of primer set for amplifying dwarf tomato InDel molecular marker according to claim 1, characterized in that, The application is used in constructing a genetic linkage map of the dwarf tomato.

3. Use of primer set for amplifying dwarf tomato InDel molecular marker according to claim 1, characterized in that, The application is used in detecting the germplasm resources of the dwarf tomato.

4. Use of primer set for amplifying compact tomato InDel molecular marker according to claim 1, characterized in that, The application is used in analyzing the genetic diversity of the dwarf tomato. The application is used in constructing a genetic linkage map of the dwarf tomato. The application is used in detecting the germplasm resources of the dwarf tomato. The application is used in analyzing the genetic diversity of the dwarf tomato.

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