CN118326069A - InDel marker primer capable of identifying tomato variety and application thereof - Google Patents

Abstract

The invention discloses an InDel marker primer capable of identifying tomato varieties and application thereof, and belongs to the technical field of biology. The InDel marker primer, the kit and the identification method provided by the invention can be used for rapidly and accurately identifying specific tomato varieties, provide a new method and a new tool for subsequent tomato breeding and breeding, and have wide application prospects.

Description

InDel marker primer capable of identifying tomato variety and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to an InDel marker primer capable of identifying tomato varieties and application thereof.

Background

Heterosis is prevalent in the biological world, meaning the phenomenon that a hybrid is superior to two parents in one or more traits. Heterosis utilization is a typical representation of genetic applications and has achieved great success in rice, maize, and wheat crops. Along with the deep breeding work, the application of heterosis is not limited to grain crops, and the heterosis also applied to the fields of vegetables and the like. Cherry tomato (Lycopersicon esculentum var. Cerasiform A. Gray) is a variety of cultivars of tomato, and has unique fruit flavor and rich nutrients. The market in recent years provides series of new requirements for cherry tomato varieties, such as more bright color, more various shapes, more outstanding flavor quality, stronger comprehensive resistance and the like, and also provides new challenges for cherry tomato breeding.

In the process of hybrid seed production, the problems of parent selfing or exogenous pollen pollution and the like exist, so that the purity of seeds is reduced, and the yield and quality of crops are further affected. Therefore, purity identification is a necessary process for guaranteeing the quality of tomato hybrid varieties, and is also a solid guarantee for realizing high quality and high yield of crops. At present, tomato purity identification in China is mainly identified by a field phenotype, and the identification method is easily interfered by environmental factors, so that the problems that the field phenotype is not easy to grasp, the time consumption is too long and the like exist.

Disclosure of Invention

The invention aims to solve the problems of improving the accuracy and shortening the identification time of tomato variety identification.

In order to solve the problems, the first aspect of the invention provides an InDel marker primer capable of identifying tomato varieties, wherein the InDel marker primer comprises an upstream primer shown in SEQ ID No.1 and a downstream primer shown in SEQ ID No.2, the sequence of an InDel marker site corresponding to the InDel marker primer is shown in SEQ ID No.5,

SEQ ID No.1：TTCACTGTTTATTTTTCCTG；

SEQ ID No.2：CTCCATCCTTTGTATCTCAT；

SEQ ID No.5：

ATCCTAGATAAACATTAAGCAAGATTTAATACTTCTTCAACAG。

The second aspect of the invention provides an application of the InDel marker primer provided in the first aspect, namely, the InDel marker primer is applied to any one or more of the following fields:

a: identification or auxiliary identification of tomato varieties;

b: preparing a product for identifying or assisting in identifying tomato varieties;

c: selecting or assisting in selecting tomato varieties;

d: preparing a product for breeding or assisting in breeding tomato varieties;

e: breeding tomatoes;

f: preparing tomato breeding products.

In a third aspect the invention provides a kit for identifying a variety of tomato, said kit comprising the InDel marker primer provided in the first aspect.

Preferably, the kit further comprises reagents for a PCR reaction and/or reagents for agarose gel electrophoresis.

According to a fourth aspect of the present invention there is provided a method of identifying a tomato variety using the InDel marker primer provided in the first aspect or the kit provided in the third aspect.

Preferably, the identification method is used to identify any one or more of tomato varieties "Mu sweet 2", "XT2114" and "XT 2115".

Preferably, the authentication fee includes the steps of:

S1: extracting DNA of a sample tomato;

S2: performing PCR amplification by using the DNA of the sample tomato as a template and using the InDel marked primer provided in the first aspect or the kit provided in the third aspect;

s3: detecting the amplified product by agarose gel electrophoresis;

s4: and identifying the tomato variety according to the imaging result of agarose gel electrophoresis.

Preferably, in the step S4, if the imaging result is double-band, the molecular weight indicated by the upper band is 297bp, and the molecular weight indicated by the lower band is 254bp, the variety of the sample tomato is "Mu-sweet No. 2"; if the imaging result is single band type and the molecular weight shown by the band is 297bp, the variety of the sample tomato is 'XT 2114'; if the imaging result is single band type and the molecular weight shown by the band is 254bp, the variety of the sample tomato is 'XT 2115'.

Further, a fifth aspect of the present invention provides a method for verifying the accuracy of an InDel marker primer, the InDel marker primer being shown in SEQ ID No.1 and SEQ ID No.2, the verification method comprising the steps of:

step1: planting target crops, and carrying out field phenotype identification;

step 2: performing PCR (polymerase chain reaction) amplification on target crops by using InDel marked primers and performing agarose gel electrophoresis on the PCR amplification result;

Step 3: and comparing the field phenotype identification result with the agarose gel electrophoresis result, and calculating the accuracy.

The invention has the beneficial effects that: tomato variety "sweet 2" is F ₁ produced by crossing female parent "XT2114" and male parent "XT 2115". The verification of the hybrid seeds is crucial in hybridization breeding, the InDel marker, the primer pair, the kit and the identification method provided by the invention are adopted to carry out PCR amplification on tomato DNA, and agarose gel electrophoresis is carried out on PCR products, so that related tomato varieties can be identified with high accuracy in extremely short time according to electrophoresis imaging bands, and a new method and a new tool are provided for subsequent tomato breeding and breeding, thus having wide application prospects.

Drawings

FIG. 1 is a photograph showing the result of gel electrophoresis imaging after PCR amplification by adding designed InDel primers to DNA samples of the hybrid variety "Mutaman No. 2" and parental individual thereof as templates in example 1 of the present invention;

FIG. 2 is a photograph showing the result of agarose gel electrophoresis imaging of the primer specificity verification experimental part in example 2 of the embodiment of the present invention;

FIG. 3 is a photograph of the field phenotype of "Mu sweet No. 2" and its parent and its parents in an embodiment of the present invention.

Detailed Description

In order that the above-recited objects, features and advantages of the present invention will become more readily apparent, a more particular description of embodiments of the invention will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings. It should be noted that the following examples are only for illustrating the implementation method and typical parameters of the present invention, and are not intended to limit the scope of the parameters described in the present invention, so that reasonable variations are introduced and still fall within the scope of the claims of the present invention.

It should be noted that endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and that such range or value should be understood to include values approaching such range or value. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein.

Unless defined otherwise, all terms, symbols and other scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In some cases, terms with commonly understood meanings are defined herein for either clarity or for ease of reference, such definitions herein should not be construed to represent a significant departure from the conventional understanding in the art. The technical methods described or cited herein are generally well known to those skilled in the art and are employed by conventional methods.

Description of the terminology:

InDel markers: insertion-deletion (InDel) refers to the insertion or deletion of nucleotide fragments of different sizes in the sequence at the same genomic locus between closely related species or different individuals of the same species, i.e., one or more bases in one sequence at a position that is homologous to another sequence. The InDel polymorphism molecular marker is a marker which designs a specific primer based on sequences at two sides of an insertion or deletion site and carries out PCR amplification, and has the advantages of clear and simple amplified product banding pattern, strong stability, obvious separation effect and the like. With the development of the second generation sequencing technology, experimenters can quickly mine variation information from the whole genome range, and the development and application of InDel markers are facilitated. InDel loci are widely distributed in genomic DNA of animals and plants, and compared with SNP, the InDel loci have better genetic stability. Compared with SSR markers, the InDel molecular markers are developed, the workload of corresponding development is greatly reduced, and the amplified products are clear and simple in band type and have better stability and separation effect. Compared with the traditional field cell phenotype identification method, the InDel molecular marker detection hybrid purity can avoid the problems of large test field, environmental mutation, artificial subjective judgment error, long period and the like, and can rapidly and accurately identify the purity of a large amount of hybrid seeds at the seedling or seed level.

"Mu sweet No. 2": tomato variety "sweet 2" is F ₁ produced by crossing female parent "XT2114" and male parent "XT 2115".

In order to solve the technical problems in the background art, the specific embodiment of the invention provides an InDel marker primer capable of identifying tomato varieties, which comprises an upstream primer shown in SEQ ID No.1 and a downstream primer shown in SEQ ID No.2, wherein the sequence of an InDel marker locus corresponding to the InDel marker primer is shown in SEQ ID No.5,

SEQ ID No.1：TTCACTGTTTATTTTTCCTG；

SEQ ID No.2：CTCCATCCTTTGTATCTCAT；

SEQ ID No.5：ATCCTAGATAAACATTAAGCAAGATTTAATACTTCT TCAACAG。

More specifically, the design procedure of the InDel marker primer of the present embodiment is as follows: and carrying out whole genome resequencing on the sample plant, and obtaining CLEAN DATA after data quality control. The tomato whole genome data were aligned with BWA software and parental mutation sites were aligned and screened. According to the resequencing detection result, selecting a male parent deletion, female parent non-variation or female parent deletion, and designing primers at conserved regions at two sides of a difference site by PRIMER PREMIER 5.0.0 software. In order to ensure the specificity of amplification, the primer design parameters especially consider that the GC content is between 40 and 50 percent, the annealing temperature is between 50 and 60 ℃, the length of the primer fragment is between 17 and 25bp, and the length of the primer PCR amplified product is between 200 and 400bp. And screening out specific primers through PCR amplification and gel electrophoresis imaging.

In some preferred embodiments, the InDel-labeled primers described above may be used in any one or more of the following fields:

a: identification or auxiliary identification of tomato varieties;

b: preparing a product for identifying or assisting in identifying tomato varieties;

c: selecting or assisting in selecting tomato varieties;

d: preparing a product for breeding or assisting in breeding tomato varieties;

e: breeding tomatoes;

f: preparing tomato breeding products.

The specific embodiment of the invention also provides a tomato variety identification method, the identification cost adopts the InDel marker primer or the kit containing the InDel marker primer to identify the tomato variety, and the identification method can accurately and rapidly identify any one or more of tomato varieties 'Mutaman No. 2', 'XT 2114' and 'XT 2115'.

More specifically, the steps of the above identification method are as follows:

S1: extracting DNA of a sample tomato;

S2: PCR amplification using the InDel marker primer of claim 1 or the kit of claim 3 or 4 using the DNA of the sample tomato as a template;

s3: detecting the amplified product by agarose gel electrophoresis;

S4: and identifying the tomato variety according to the agarose gel electrophoresis imaging result.

More specifically, in step S1 of the above embodiment, the organ from which the DNA is extracted may be selected from one or more of roots, stems, leaves, flowers, fruits, seeds.

More specifically, the DNA extraction method may be any method conforming to national standards or commercial kit, and of course, preferably a CTAB method, such as a CTAB method, is used to extract DNA, and the specific steps are as follows: plant tissue was placed in a 2ml centrifuge tube equipped with steel balls (diameter: 2 mm) and ground with a multiple sample tissue grinder (frequency: 60Hz, time: 90 s). Adding 700 mu L of CTAB solution of NaHSO ₃ (NaHSO ₃ concentration: 10.4 g.L ^-1) into the grinded centrifuge tube, fully shaking, putting into a 65 ℃ water bath kettle, carrying out water bath for 30min, and shaking once every 10 min. After completion of the water bath, 700. Mu.L of a mixture of isoamyl alcohol and chloroform (isoamyl alcohol: chloroform=1:24) was added to the centrifuge tube and centrifuged (rotation speed: 8000g, time: 10 min), and 400. Mu.L of the supernatant was extracted and placed in a 1.5ml centrifuge tube. The supernatant was added with 300. Mu.L of precooled isopropanol and centrifuged (rotation speed: 12000g, time: 10 min), the supernatant was poured out to leave a bottom, rinsed with 75% ethanol, and then air-dried, and dissolved with ddH ₂ O to obtain an aqueous DNA solution.

More specifically, in step S2 of the above embodiment, the PCR reaction system for PCR amplification comprises 5.5. Mu.L of ddH ₂ O, 10. Mu.L of 2×taq mix, 1.5. Mu.L of the upstream primer (concentration: 10 mM), 1.5. Mu.L of the downstream primer (concentration: 10 mM), 1.5. Mu.L of the single-strain DNA of the hybrid "Mu sweet No. 2" or its parent. The PCR reaction procedure in PCR amplification includes: pre-denaturation at 94℃for 5min, denaturation at 94℃for 20s, annealing for 20s (annealing temperature was set according to the design primer), extension at 72℃for 40s, cycling for 35 times, extension at 72℃for 10min, and preservation at 4 ℃.

More specifically, in step S3 of the above embodiment, the PCR amplification product is subjected to agarose gel electrophoresis and imaged in a gel imaging system (model: BIO-RAD). When the primers are screened, a large-scale A3-1 gel electrophoresis system is adopted, 350V voltage and 500mA current are set, and 4% agarose gel electrophoresis is carried out for 50min. The nucleic acid staining is performed by gel staining, i.e. adding nucleic acid dye E×Red in a proportion of 5 μ L E ×Red 10000×stock solution per 50ml agarose solution.

More specifically, in step S4 of the above embodiment, if the imaging result is double-band and the molecular weight indicated by the upper band is 297bp and the molecular weight indicated by the lower band is 254bp, the variety of the sample tomato is "Mu-sweet No. 2"; if the imaging result is single band type and the molecular weight shown by the band is 297bp, the variety of the sample tomato is 'XT 2114'; if the imaging result is single band type and the molecular weight shown by the band is 254bp, the variety of the sample tomato is 'XT 2115'.

In order to further ensure the accuracy of screening primer assay purity, the specific embodiment of the invention also carries out field phenotype identification and laboratory molecular detection contrast test, wherein:

The field phenotype identification comprises the following steps: on the plots of the same experimental plot, the hybrid "Mu sweet No. 2" was planted for identification, and the female parent "XT2114" was planted as a control. And observing whether the phenotype of the plant to be identified is consistent with that of a normal 'Mu-sweet No. 2' plant at each growth stage, and comparing the phenotype with that of the female parent planted, so as to identify hybrid female parent inbred seeds in the hybrid seeds.

Laboratory molecular assays include: taking seedling leaves of hybrid 'Mu-sweet No. 2' plants, extracting single plant DNA by a CTAB method, adding screening primers for PCR amplification, and placing amplified products in a gel imaging system for imaging after agarose gel electrophoresis. And counting the banding according to the gel imaging result, recording hybrid non-Mu-sweet No. 2 plants in the plants to be identified, and calculating the purity. When the purity of the primer is detected, a large-scale A3-1 gel electrophoresis system is adopted to expand the detection quantity, 350V voltage and 500mA current are set, and the detection is carried out for 50min by 4% agarose gel electrophoresis.

The field phenotype identification is compared with the laboratory molecular detection result one by one, plants which cannot be identified due to growth stopping during the field phenotype identification period can be eliminated during the comparison, exogenous pollen pollution abnormal strains with field phenotypes different from 'sweet No. 2' and female parent 'XT 2114' can be eliminated, the field phenotype identification result is not generated, and the uncertainty of the exogenous pollen pollution abnormal strains is strong. In actual hybrid production, workers can effectively and manually control surrounding exogenous pollen sources.

The method comprises the steps of counting plants of hybrid seeds 'Mu-sweet No. 2' corresponding to the double bands in molecular detection and female parent 'XT 2114' corresponding to the upper bands in the bands, and if the molecular detection is completely consistent with the field phenotype identification result, successfully developing InDel molecular markers capable of accurately identifying the purity of the hybrid seeds 'Mu-sweet No. 2', wherein the subsequent purity identification of 'Mu-sweet No. 2' can be directly carried out by molecular detection.

The following description of specific embodiments of the invention will be further made with reference to specific examples and figures of the specification, in which, unless otherwise indicated, the use of commercially available kits and reagents, instruments are referred to in accordance with the protocols and parameters set forth by the manufacturer.

Example 1

Hybrid "Mu-sweet No. 2" and screening of primers specific for bands of PCR amplification products of parental primers

Taking seedling leaves of female parent XT2114 and male parent XT2115 of hybrid variety Mutaman No. 2 as samples, sending to Tianjin North He Zhuangyuan technology Co., ltd, carrying out whole genome resequencing, and obtaining CLEAN DATA after data quality control. The tomato whole genome data were aligned with BWA software and parental mutation sites were aligned and screened.

Selecting a male parent deletion, a female parent non-variation or a female parent deletion, designing primers at conserved regions at two sides of a difference site by PRIMER PREMIER 5.0.0 software, wherein the male parent non-variation InDel site is selected. In order to ensure the specificity of amplification, the design parameters of the primers specifically consider that the GC content is 40% -50%, the annealing temperature is 50-60 ℃, the length of the primer fragment is 17-25 bp, the length of the PCR amplified product of the primers is 200-400 bp, and the sequences of the primers are shown in the table 1.

TABLE 1 InDel primer sequences designed for hybridization "Mu-sweet No. 2" assay

The DNA sample of the hybrid variety ' Mu-sweet No. 2 ' and the parent and parent strain thereof are taken as templates, designed InDel primers are added, and primers with specificity of the Mu-sweet No. 2 ' and the parent and parent strain thereof are screened out through PCR amplification and gel electrophoresis imaging. The specific imaging results are shown in fig. 1, and each pair of primers in fig. 1 amplifies the same 12 DNA samples, namely 4 female parent 'XT 2114', 4 male parent 'XT 2115' and 4 hybrid variety 'Mu sweet No. 2' DNA samples in sequence from left to right. As can be seen from FIG. 1, the PCR amplification result of the primer No. 2 shows that the parental and hybrid bands thereof exhibit specificity.

The sequence of the amplified primer of the female parent 'XT 2114' with the number 2 is shown as SEQ ID No.3,

SEQ ID No.3：TTCACTGTTTATTTTTCCTGTCACTGTTTATTTTTCC TGTCTCGGCCACGTGTACTCATCAGAAAACCTGACTTAAAACAGAAAACCAGATCCTAGATAAACATTAAGCAAGATTTAATACTTCTTCAACAGAATTTGCACCAATCTATTGCTATTCTAGCTTCTGCACATCGTAAGAAAAGGACACTGAGGAAGGTTTACAAGAATATAAATAGCAACCTCATCTCTAAATAATATACAAGCTAAATAAATGCAAACTAAAGAGCATATGCTTTCAATGAGATACAAAGGATGGAG;

The sequence of the male parent 'XT 2115' amplified by the primer with the number of 2 is shown as SEQ ID No.4,

SEQ ID No.4：TTCACTGTTTATTTTTCCTGTCACTGTTTATTTTTCC TGTCTCGGCCACGTGTACTCATCAGAAAACCTGACTTAAAACAGAAAACCAGAATTTGCACCAATCTATTGCTATTCTAGCTTCTGCACATCGTAAGAAAAGGACACTGAGGAAGGTTTACAAGAATATAAATAGCAACCTCATCTCTAAATAATATACAAGCTAAATAAATGCAAACTAAAGAGCATATGCTTTCAATGAGATACAAAGGATGGAG;

The InDel marking site of the InDel marking primer with the number of 2 is shown as SEQ ID No.5,

SEQ ID No.5：ATCCTAGATAAACATTAAGCAAGATTTAATACTTCT TCAACAG。

Example 2

Verifying accuracy of purity detection of specific primer pair "Mu sweet No. 2

A batch of hybrid "Mu-sweet No. 2" seeds (quantity: 468) were randomly extracted and sown into 96-well trays with homozygous female parent "XT2114" seeds, one well after another. When the plant grows to two leaves and is planted in the plastic greenhouse at one heart, the plant needs to be fully prepared and planted on the same ridge in order to reduce the influence caused by uneven soil fertility and other factors.

The method comprises the steps of setting a number plate for field 'Mu-sweet No. 2' plants with identification groups, and cutting new leaves of 173 plants to extract DNA. The 173 DNA samples were subjected to amplification by using the screened primer 2-F/R, and the PCR amplified products were subjected to agarose gel electrophoresis and then placed in a BIO-RAD gel imaging system to obtain images. 173 DNA bands were counted, 173 of which were double band and 0 of which were used to obtain 100% of the purity of the "sweet 2" seeds of the hybrid (formula: purity (%) of tomato hybrid "sweet 2" =number of plants (seeds) amplified into two bands/number of detected populations (seeds) ×100%). The imaging results of part of the glue pattern are detailed in fig. 2, and the specific statistical results are detailed in table 2.

Table 2 results of laboratory molecular detection and field phenotypic identification alignment of "Mutaman No. 2

In summary, the specific embodiment of the invention is based on genome resequencing sequence of the obtained hybrid variety 'Mu-sweet No. 2' father and mother, and the father deletion, the mother non-variation or the mother deletion, the father non-variation InDel locus are screened out through analysis and comparison, and the primer is designed. And (3) screening the designed primers by taking the DNA sample of the hybrid variety 'Mu-sweet No. 2' and the parent and parent single strain thereof as templates, and screening out primers with specificity of the parent and the hybrid PCR amplified products. Screening primer detection field hybrid 'Mu-sweet No. 2' with identification group, and combining field phenotype identification to confirm the accuracy of primer detection. When the molecular detection result in the laboratory is completely consistent with the field phenotype identification result, namely, the hybrid seeds of Mutaman No. 2 and the hybrid seeds of the female parent inbred seeds mixed therein can be accurately identified by molecular detection, the InDel molecular marker primer can be directly adopted for purity identification in the follow-up process. Although the present disclosure is described above, the scope of protection of the present disclosure is not limited thereto. Various changes and modifications may be made by one skilled in the art without departing from the spirit and scope of the disclosure, and these changes and modifications will fall within the scope of the invention.

Claims (9)

1. The InDel marker primer capable of identifying tomato varieties is characterized by comprising an upstream primer shown in SEQ ID No.1 and a downstream primer shown in SEQ ID No.2, wherein the sequence of an InDel marker locus corresponding to the InDel marker primer is shown in SEQ ID No.5,

SEQ ID No.1：TTCACTGTTTATTTTTCCTG；

SEQ ID No.2：CTCCATCCTTTGTATCTCAT；

SEQ ID No.5：ATCCTAGATAAACATTAAGCAAGATTTAATACTTCT TCAACAG。

2. Use of the InDel-labeled primer of claim 1, wherein the InDel-labeled primer is applied to any one or more of the following fields:

a: identification or auxiliary identification of tomato varieties;

b: preparing a product for identifying or assisting in identifying tomato varieties;

c: selecting or assisting in selecting tomato varieties;

d: preparing a product for breeding or assisting in breeding tomato varieties;

e: breeding tomatoes;

f: preparing tomato breeding products.

3. A kit for identifying tomato varieties, comprising the InDel marker primer of claim 1.

4. A kit according to claim 3, wherein the kit further comprises reagents for a PCR reaction and/or reagents for agarose gel electrophoresis.

5. A method for identifying tomato varieties, characterized in that the identification method adopts the InDel marking primer of claim 1 or the kit of claim 3 or 4 to identify tomato varieties.

6. The identification method of claim 5, wherein the identification method is used to identify any one or more of tomato variety "sweet 2", "XT2114" and "XT 2115".

7. The authentication method according to claim 5, wherein the authentication method comprises the steps of:

S1: extracting DNA of a sample tomato;

S2: PCR amplification using the InDel marker primer of claim 1 or the kit of claim 3 or 4 using the DNA of the sample tomato as a template;

s3: detecting the amplified product by agarose gel electrophoresis;

S4: and identifying the tomato variety according to the agarose gel electrophoresis imaging result.

8. The method according to claim 7, wherein in the step S4, if the imaging result is double-band and the molecular weight indicated by the upper band is 297bp and the molecular weight indicated by the lower band is 254bp, the variety of the sample tomato is "Mu-sweet 2"; if the imaging result is single band type and the molecular weight shown by the band is 297bp, the variety of the sample tomato is 'XT 2114'; if the imaging result is single band type and the molecular weight shown by the band is 254bp, the variety of the sample tomato is 'XT 2115'.

9. A method for verifying the accuracy of an InDel marker primer, wherein the InDel marker primer is shown as SEQ ID No.1 and SEQ ID No.2, the verification method comprising the steps of:

step1: planting target crops, and carrying out field phenotype identification;

step 2: performing PCR (polymerase chain reaction) amplification on target crops by using InDel marked primers and performing agarose gel electrophoresis on the PCR amplification result;

Step 3: and comparing the field phenotype identification result with the agarose gel electrophoresis result, and calculating the accuracy.