CN115927727A - InDel molecular marker coseparated with fruit position and fruit size of muskmelon and application thereof - Google Patents

Abstract

The invention discloses an InDel molecular marker coseparated with fruit positions and fruit sizes of melons and application thereof, wherein an upstream primer sequence of a primer pair for amplifying the molecular marker is shown as SEQ ID No.1, and a downstream primer sequence is shown as SEQ ID No. 2. The molecular marker primer is not only co-separated from the fruit part and the fruit size character in a segregation population, but also has the amplification banding pattern consistent with the fruit part and the fruit size phenotype in different breeding materials, so that the molecular marker primer can be directly used for melon molecular marker assisted breeding and has a good application value in melon new variety cultivation.

Description

InDel molecular marker coseparated with fruit position and fruit size of muskmelon and application thereof

Technical Field

The invention belongs to the technical field of molecular breeding, and particularly relates to an InDel molecular marker coseparated with fruit positions and fruit sizes of melons and application thereof.

Background

The melon (Cucumismelo L.) is one of the main cucurbitaceae vegetable crops, has wide cultivation area and important economic value. China is a great country for producing melons, and the melons play an important role in fruit and vegetable production.

In recent years, molecular assisted selective breeding technology is widely developed and applied, and can rapidly screen required individuals on a molecular level, greatly shorten breeding time and greatly promote breeding work. Whether a marker closely linked with the character can be found or not and whether the marker can be used in different materials is a sign for the success of molecular marker-assisted selective breeding.

The fruit position and the fruit size of the melon are important agronomic characters of the melon, and directly determine the plant type and the yield of the melon. The fruiting part of the fruit is a very important agronomic character and an important component of the plant type, and directly determines the structure of the plant; the size of the fruit is an agronomic character of the fruit after the fruit is completely mature, is easily influenced by the environment and is a difficult point in character improvement. The conventional breeding needs to identify the fruit size after the fruit is mature, and is long in time consumption and high in cost. By constructing a genetic group, initially positioning BSA-seq, further encrypting a marker by using a large group, finely positioning and obtaining a molecular marker coseparated with the fruit size, the plant type of the melon can be directionally improved by a marker-assisted selection technology, the selection efficiency of the fruit size is obviously improved, the fruit size can be selected in the seedling stage, and the breeding process is accelerated.

Disclosure of Invention

One of the purposes of the invention is to provide an InDel molecular marker which is co-separated with the fruit position and the fruit size of the melon.

The invention also aims to provide the application of the molecular marker in melon molecular breeding.

Another object of the present invention is to provide a method for determining fruit bearing part and fruit size of melon.

In order to achieve the purpose, the invention provides the following technical scheme:

the nucleotide sequences of an upstream primer and a downstream primer of a primer pair for amplifying the InDel molecular marker are respectively as follows:

FpFs-F:5' -TGCCAAATGGAATACAACG (shown in SEQ ID NO. 1);

FpFs-R:5' -AATGGGACATGTCCTGGAATA (shown in SEQ ID NO. 2).

The invention also discloses application of the InDel molecular marker coseparated with the fruit position and the fruit size of the melon in molecular breeding of the melon. The method is mainly used for identifying or assisting in identifying the fruit position and the fruit size phenotype of the melon. By utilizing the InDel molecular marker primer, the fruit position and the fruit size of the melon can be accurately, quickly and effectively identified. Those skilled in the art will appreciate that the fruit size and position of melon can be determined, for example, by detecting the presence or absence of the molecular markers of the present invention. Specifically, the above-mentioned pair of molecular-labeled primers of the present invention can be used, and the detection can also be performed by a sequencing method.

The invention also discloses a method for judging the fruit position and the fruit size of the muskmelon, which comprises the following steps:

(4) Extracting the genomic DNA of the muskmelon to be detected;

(5) Performing PCR amplification on the template by using the genomic DNA extracted in the step (1) as the template and using the molecular marker primer pair of claim 1, and performing electrophoresis detection and/or sequencing on the PCR amplification product;

(6) And (3) judging according to the electrophoresis band and/or the sequencing result in the step (2), wherein the specific standard is as follows:

if the PCR amplification product only has a characteristic strip with the length of 195bp as shown in SEQ.ID.NO.3, the variety to be detected is pure and has the small fruit shape of the main vine melon; if the PCR amplification product only has a characteristic strip with the length of 202bp as shown in SEQ.ID.NO.4, the variety to be detected is homozygous lateral vine bearing melon big fruit type character; if the PCR amplification product has a characteristic strip with the length of 195bp as shown in SEQ ID No.3 and a characteristic strip with the length of 202bp as shown in SEQ ID No.4, the variety to be detected has the large fruit type character of the heterozygous lateral vine melon.

In addition, the kit comprising the primer pair can be used for identifying the fruit bearing part and the fruit size character of the melon, and in the specific application, the reagent containing the molecular marker primer pair can be selected to prepare the kit.

In addition, the use of the reagent for detecting the presence or absence of the InDel molecular marker for gene mapping controlling the fruit position and fruit size of melon, which can be performed according to the conventional method, can be used for mapping the gene controlling the fruit position and fruit size of melon using the molecular marker of the present invention.

The invention also protects a carrier containing the molecular marker. The recombinant vector may be an expression vector or a cloning vector into which the molecular marker of the present invention is inserted. After obtaining the above recombinant vector, one skilled in the art can transform the recombinant vector into a suitable cell according to different needs to obtain a recombinant cell containing the recombinant vector. Accordingly, the invention also protects a recombinant cell containing the recombinant vector.

The invention has the advantages that:

according to the invention, a main vine bearing melon small fruit type melon 470B and a side vine bearing melon large fruit type melon HNA24 are used as parents to prepare a hybrid combination for researching fruit positions and fruit sizes of melons. Constructing a gene pool through extreme single plants in the F2 population, carrying out BSA-seq, and preliminarily positioning the result part and the fruit size gene on the No. 5 chromosome of the melon. An F2 population containing 513 individuals was further constructed for marker encryption, and the resulting site and fruit size genes were mapped to the region of 101.232kb (995, 848-1,097, 080) on chromosome 5. An InDel molecular marker primer is obtained in the interval, and the molecular marker primer is not only co-separated from the fruiting part and the fruit size character in a segregation population, but also has the same amplification banding pattern as the fruiting part and the fruit size phenotype in different breeding materials, so that the molecular marker primer can be directly used for melon molecular marker-assisted breeding and has a good application value in melon new variety breeding. In addition, the molecular marker can be used for accurately and rapidly identifying the fruit position and the fruit size character of the melon in the bud stage or seedling stage, and has the advantages of convenience in detection and stable amplification product.

By utilizing the molecular marker primer, screening of muskmelon fruiting parts and fruit size genes can be carried out in the seedling stage, a breeding population can be reduced, the breeding process can be accelerated, the breeding efficiency can be improved, and the molecular marker primer has an important application value.

In the selective breeding process of melons, a large number of segregation populations are generated, and by adopting the molecular marker, required plants can be identified in the seedling stage, so that the occupied area in the breeding process is reduced, the manpower and material resources required by identification after the plants grow up are reduced, the breeding efficiency is greatly improved, and the selection period is shortened.

Drawings

FIG. 1 is a polyacrylamide gel electrophoresis pattern of PCR products marked by melon fruit position and fruit size gene InDel;

FIG. 2 is a verification of fruit fruiting part and fruit size co-segregation marker in natural populations.

Detailed Description

The principles and features of this invention are described below in conjunction with the following drawings, which are set forth by way of illustration only and are not intended to limit the scope of the invention.

Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. The test methods in the following examples are conventional methods unless otherwise specified. Unless otherwise indicated, all reagents and materials used are commercially available.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention. The preferred embodiments and materials described herein are intended to be exemplary only.

The biological material adopted by the invention is as follows:

melon material 470B, which exhibits a major vine bearing melon fruitlet trait;

melon material HNA24, which exhibits lateral vine bearing melon fruity traits;

two materials were provided by the zhengzhou fruit tree institute (it is to be explained that using this material as a research basis is only a reason for the convenience of obtaining experimental materials, and it is not understood that the implementation of the related art solution of the present application must rely on the experimental materials);

the above biological materials are stored in the laboratories of the applicant units and can be distributed to the public for verification tests within twenty years from the filing date or can be obtained by the public by purchase.

Example 1 development of InDel molecular markers cosegregating with melon fruit parts and fruit size

(ii) construction of genetically isolated populations

Preparing a hybrid combination by taking main vine bearing melon small fruit type melon 470B and lateral vine bearing melon large fruit type melon HNA24 as parents; f ₂ In the population, 549 lateral vines have large fruits, 171 main vines have small fruits, and the separation ratio (x) of Mendelian genetic interest rate of 3: 1 is met by a chi-square test (shown in Table 1) ² Is more than 0.60 x ² _0.05 = 3.84). The result shows that the main vine bearing small fruit character is controlled by a pair of recessive genes. By F ₂ Extreme single plants in the population construct a gene pool, BSA-seq is carried out, and the genes of the fruit position and the fruit size are preliminarily positioned on the No. 5 chromosome of the melon. Further construction of F containing 513 individuals ₂ The group is marked and encrypted, and the genes for controlling fruit fruiting part and fruit size are positioned in the 101.232kb (995, 848bp-1,097, 080bp) interval of chromosome 5.

TABLE 1F ₂ Population phenotypic isolation analysis

According to the melon fruit position and the fruit size gene positioning interval, 18 pairs of InDel molecular marker primers are developed and amplified in the lateral vine big fruit parent HNA24 and the main vine small fruit parent 470B, and polymorphic markers are screened (figure 1).

The result shows that the 15 th pair of InDel molecular marker primers constructed according to the No. 5 chromosome are coseparated with the fruit size phenotype, have clear banding patterns and good repeatability, are named as FpFs, and have the following sequences:

FpFs-F:5' -TGCCAAATGGAATACAACG (shown in SEQ ID NO. 1);

FpFs-R:5' -AATGGGACATGTCCTGGAATA (shown in SEQ ID NO. 2).

Therefore, an InDel molecular marker primer is obtained within the interval of about 101.232kb (995,848bp-1,097,080bp) of the No. 5 chromosome, and the molecular marker primer is not only cosegregated with a fruiting part and a fruit size character in a segregation population, but also has an amplification banding pattern which is consistent with the fruiting part and the fruit size phenotype in different breeding materials. By utilizing the molecular marker primer, screening of muskmelon fruiting parts and fruit size genes can be carried out in the seedling stage, a breeding population can be reduced, the breeding process can be accelerated, the breeding efficiency can be improved, and the molecular marker primer has an important application value.

Example 2 amplification validation of melon InDel molecular markers

The polymorphisms obtained in example 1 were labeled at 178F ₂ Amplification verification in a group individual plant comprises the following steps:

1. extraction of muskmelon DNA to be detected in seedling stage by improved CTAB method

The experimental materials are lateral vine bearing melon big fruit type melon HNA24 and main vine bearing melon small fruit type melon 470B and F ₂ Fresh leaves of a group individual plant, and the steps of extracting genome DNA are as follows:

(1) When three leaves and one heart of the plant are used, the young leaves are taken out of a 2mL centrifuge tube and are brought back by a liquid nitrogen barrel. Before grinding, the leaves are frozen and crisp by using liquid nitrogen, and then are ground into powder by using a grinder;

(2) Adding 800 μ L of 2% CTAB preheated in 65 deg.C water bath for 1 hr into a centrifuge tube, shaking to make the folium melo powder fully contact with the liquid, placing in 65 deg.C water bath for 1 hr, and mixing every 10min;

(3) Cooling to room temperature after water bath, adding 800 μ L chloroform/isoamyl alcohol (volume ratio 24: 1) solution, shaking thoroughly, centrifuging at 4 deg.C at 12000r/min for 10min;

(4) Sucking out 600 μ L of supernatant, placing in 1.5mL centrifuge tube, adding 600 μ L of pre-cooled isopropanol at-20 deg.C, shaking thoroughly, and precipitating at-20 deg.C for 1h;

(6) Taking out the centrifugal tube, centrifuging at 4 ℃ for 10min at a rotating speed of 12000r/min, sucking out supernatant after centrifugation, and keeping precipitate;

(7) Washing with 75% ethanol for 2-3 times, drying at room temperature, adding distilled water to 80 μ L to obtain a DNA sample of the melon to be tested, and storing the sample in a refrigerator at-80 deg.C permanently.

2. PCR amplification was performed using the molecular InDel labeled primers selected in example 1 using Cucumis melo genomic DNA as a template.

The 10. Mu.L reaction system adopted during PCR amplification comprises: the concentration is 200 ng. Mu.L ^-1 1. Mu.L of genomic DNA of (1), 5. Mu.L of 2 XTaq PCR StarMixwith Loading Day (For PAGE), 0.5. Mu.L of forward primer, 0.5. Mu.L of reverse primer, ddH ₂ O3. Mu.L, wherein Taq PCR StarMixwith Loading Day (For PAGE) comprises Taq DNA Polymerase, mg ²⁺ And dNTPs.

The amplification procedure used in PCR amplification includes: after pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 30s, after 30 cycles, final extension at 72 ℃ for 10min, and preservation at 4 ℃.

3. Performing polyacrylamide gel electrophoresis (PAGE) detection on the PCR amplification product

Polyacrylamide gels were prepared with the reagents and amounts shown in Table 2.

TABLE 2 reagents and amounts used for Polyacrylamide gel preparation

Carefully and uniformly mixing the solution (avoiding bubbles), slowly pouring the mixture into a glass plate at a constant speed, slowly inserting a comb at a constant speed, and waiting for the solution to solidify. Pouring a proper amount of 0.5xTBE into the electrophoresis tank, then removing the glue clip and putting the glue clip into the electrophoresis tank, ensuring that no air bubbles exist in the glue plate, pouring a proper amount of 0.5xTBE into the upper part of the electrophoresis tank, and slowly pulling out the comb at a constant speed. Then, the PCR products after the amplification are sequentially added into corresponding comb holes of a polyacrylamide gel plate, 1.0uL of each PCR product is added, and 1 uL of 2000DNA Marker is added before the first sample hole of which the sample application is started on the gel. And connecting the line with an electrophoresis apparatus and an electrophoresis tank, and performing electrophoresis.

Cutting off power supply after electrophoresis, discharging the buffer solution from the electrophoresis tank, taking off the gel plate, carefully taking out the gel from the glass plate, putting the gel plate into a plastic tank filled with distilled water, and pouring out the distilled water. 1000ml of an aqueous solution containing 1.2g of AgNO3 was poured and the mixture was stained on a shaking table for 2min. Then pouring out the dyeing liquid, adding a small amount of distilled water into a plastic tank for quick rinsing, and then pouring out the distilled water. And pouring a developing solution for developing, wherein the developing solution comprises 15g of NaOH and 15mL of formaldehyde solution, fixing the volume to 1000mL, and dyeing until a strip appears. Rinsed with distilled water and then analyzed by photography on a light box.

4. Amplification results

And specifically, during verification:

when the molecular marker InDel is used for detecting the fruitlet parents of the main vine melon, a 195bp characteristic strip can be obtained through PCR amplification, and the specific base sequence of the 195bp characteristic strip is as follows:

the molecular marker InDel is used for detecting the big fruit type parents of the lateral vine melons, a 202bp characteristic strip can be obtained through PCR amplification, and the specific base sequence of the 202bp characteristic strip is as follows:

sequence alignment shows that a TATCGTT is inserted into a 111bp position, in other words, a product obtained after amplification of the large-fruit type variety of the lateral vine melon is a 202bp fragment through PCR; and the small fruit type parent of the main vine has a deletion of TATCGTT, so that the length of the product after amplification is 195bp. (FIG. 2)

The above examples show that the method of the present invention can effectively distinguish the big-fruit type material of the lateral vine melon from the small-fruit type material of the main vine melon, and accurately and rapidly detect the result part and the fruit size phenotype of the material.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims (6)

1. The InDel molecular marker co-separated with the fruit part and the fruit size of the melon is characterized in that the sequence of an upstream primer of a primer pair for amplifying the molecular marker is shown as SEQ ID NO.1, and the sequence of a downstream primer is shown as SEQ ID NO. 2.

2. Use of the InDel molecular marker co-segregating with melon fruiting part and fruit size as claimed in claim 1 in melon molecular breeding.

3. The use of claim 2, wherein the InDel molecular marker is used to identify or assist in identifying melon fruit part and fruit size phenotype.

4. A method for judging fruit position and fruit size character of melon, which is characterized in that the judging method comprises the following steps:

(1) Extracting the genomic DNA of the muskmelon to be detected;

(2) Taking the genomic DNA extracted in the step (1) as a template, carrying out PCR amplification on the template by using the molecular marker primer pair of claim 1, and carrying out electrophoresis detection and/or sequencing on the PCR amplification product;

(3) And (3) judging according to the electrophoresis band and/or the sequencing result in the step (2), wherein the specific standard is as follows:

if the PCR amplification product only has a characteristic strip with the length of 195bp as shown in SEQ.ID.NO.3, the variety to be detected is pure and has the small fruit shape of the main vine melon; if the PCR amplification product only has a characteristic strip with the length of 202bp as shown in SEQ.ID.NO.4, the variety to be detected is homozygous lateral vine melon large fruit type character; if the PCR amplification product has a characteristic strip with the length of 195bp as shown in SEQ.ID.NO.3 and a characteristic strip with the length of 202bp as shown in SEQ.ID.NO.4, the variety to be detected is the heterozygous lateral vine fruitlet type trait.

5. A kit for identifying melon fruiting part and fruit size character, which comprises the primer pair of claim 1.

6. The application of the reagent for detecting whether the InDel molecular marker exists in the gene positioning for controlling the fruit position and the fruit size of the melon is characterized in that the sequence of an upstream primer of a primer pair for amplifying the marker is shown as SEQ ID NO.1, and the sequence of a downstream primer is shown as SEQ ID NO. 2.

Images