CN108239675A - A kind of molecular marked compound TJcM02 and its application for being used to identify muskmelon unisexual flower - Google Patents

Abstract

The invention discloses a kind of for identifying molecular marked compound TJcM02 and its application of muskmelon unisexual flower, for current unisexuality female flower muskmelon selection and breeding the problem of, such as year limit for length, the shortcomings of efficiency is low and of high cost, the present invention provides a kind of TJcM02 molecular labelings and its primer special for identifying muskmelon unisexual flower, pass through PCR, the differentiation to unisexuality female flower and non-unisexuality female flower muskmelon material can be completed in Muskmelon Plants early stage, it is good with specificity, accuracy rate is high, it is not affected by environment the advantages of.

Description

A Molecular Marker TJcM02 for Identifying Parthenosexual Flowers of Melon and Its Application

technical field

The invention belongs to the field of biotechnology, and in particular relates to a molecular marker TJcM02 for identifying unisexual flowers of muskmelon and its application.

Background technique

Melon Cucumis melo L. belongs to Cucurbitaceae Cucurbitaceae, and cucumber belongs to Cucumis. According to the data of the Food and Agriculture Organization of the United Nations (FAO), my country's melon production and cultivation area both rank first in the world, which is of great significance to improving the economic benefits of farmers. The flower type of melon can be divided into two types according to the planting part of the flower organ, namely unisexual female flower and bisexual flower. The currently cultivated melons are male and hermaphroditic flowers. In the process of the first generation of hybrid seed production, manual detasseling is required for pollination, and the operation is relatively complicated. If the detasseling operation is not performed properly, the fruit setting rate and seed purity will be reduced. Therefore, breeding parthenogenetic melons is one of the important goals of current muskmelon genetics and breeding.

Adnane Boualem confirmed that the unisexual female flowers of melon are controlled by the dominant single gene A, and found that the male hermaphrodite flowers of melon are caused by the substitution of A57V due to the single nucleotide mutation of the A gene, resulting in the mutation of the ethylene biosynthesis enzyme encoded by CmACS-7. In addition, the expression patterns of floral types in Cucurbitaceae can be modified by hormones (such as ethylene) and environmental factors.

At present, the traditional hybridization method is generally used for the breeding of unisexual and female flowered melons, which has the disadvantages of long life, low efficiency and high cost. Molecular marker-assisted breeding can directly select traits from the DNA level, has the advantages of high efficiency, accuracy and economy, can greatly shorten the breeding cycle, and is a powerful supplement to traditional breeding techniques. InDel labeling has the advantages of high accuracy and good stability, and its detection is simple and convenient, with low requirements for equipment and technology, and specific primers can be designed based on the sequences on both sides of the insertion/deletion site for PCR amplification, directly in the electrophoresis It can be detected on the technical platform.

The development of suitable InDel molecular markers for the selection of unisexual female flowered melon varieties or lines can speed up the breeding process, improve the breeding efficiency, and reduce the breeding cost, which has important practical application value.

Contents of the invention

Aiming at the problems existing in the current breeding of unisexual melons, such as long years, low efficiency and high cost, the present invention provides a TJcM02 molecular marker for identifying unisexual flowers of melons and its special primers. Reaction, the distinction of parthenogenetic female flowers and non-parthenogenetic female flowered melon materials can be completed in the early stage of the melon plant, which has the advantages of good specificity, high accuracy and no environmental influence.

A special primer for identifying the molecular marker TJcM02 of unisexual flowers of melon, characterized in that the primer is a forward primer TTCAAATTCTTCACAACTTTACCGTATTGATCC from the 5' end to the 3' end of the 1-32 single-stranded DNA sequence; from the 5' end Reverse primer GGTGGTCATTTTCATAGAACTTTCCCATAC to the 1st-30th single-stranded DNA sequence at the 3' end.

Utilize the special primers for molecular markers of the present invention, take the genomic DNA of the material to be detected as a template, obtain molecular markers by PCR amplification, and find through sequencing that in the muskmelon material of unisexual female flowers, the molecular marker size is a PCR product of 249bp, from 5 The single-stranded DNA sequence from `end to 3` is: TTCAAATTCTTCACAACTTTACCGTATTGATCCCTTAAAAATGAAGTAAAAACAATAATGAACGAACTAAGACAATCACCATTTGAAAGTTTAAGGACCAAATGAACCAAAGTTAAAAGTATAGAAACCAAAAATAAACATCGCTAAATAAACCAAATAAAACTTAGAATTACTTAATTGAAACAAAATAATATGAAATGGATCAAAGTATTTATTAGGACTGAATGGATCAAATCTTAGACGA.

Utilize special-purpose primer of the present invention to identify the molecular marker method of muskmelon, it is characterized in that the genomic DNA of the sample to be detected of muskmelon is extracted as template, utilize the special-purpose primer of molecular marker to carry out polymerase chain reaction (PCR) amplification, then by electrophoresis to amplify The product is tested.

1) Using the CTAB method to extract genomic DNA from any tissue or organ of the melon sample to be tested.

2) Using the genomic DNA of the sample to be tested obtained in step 1 as a template, perform PCR amplification with a pair of molecular marker-specific primers to obtain an amplification product.

3) Using polypropylene gel electrophoresis technology, the PCR amplification product obtained in step 2 is subjected to electrophoresis separation.

4) The PCR amplification product was stained by silver staining.

5) By judging the size of the amplified product, the larger amplified product is a 249bp DNA fragment, and the sample to be tested containing the 249bp amplified product is parthenogyne melon material; the sample to be tested not containing the 249bp amplified product is a non- Parthenogyne melon material.

The template used in the above PCR amplification is the melon genomic DNA.

The primers used for the above-mentioned PCR amplification are as follows:

1) A single-stranded DNA molecule shown in sequence 2 in the sequence listing, or a single-stranded DNA molecule in which one or several nucleotides are deleted, added or mutated in sequence 2 and has the same function as sequence 2.

2) A single-stranded DNA molecule represented by sequence 3 in the sequence listing, or a single-stranded DNA molecule in which one or several nucleotides are deleted, added or mutated in sequence 3 and has the same function as sequence 3.

Compared with the prior art, the positive effect of a special primer and molecular marker method for identifying unisexual flowers of melon disclosed by the present invention lies in:

1. When the traditional method is used for unisexual female flower selection, field testing is required, which is time-consuming and laborious, and is easily affected by the environment; however, this method uses DNA detection and is not affected by the environment.

2. In terms of detection time, the detection of traditional methods needs to be carried out at the flowering stage, but this method can be detected at the seedling stage or seed stage, and the detection time is earlier.

3. There are few markers linked with ACS-7 in the prior art, and no co-segregation markers, but the resistance gene in the present invention can co-segregate with ACS-7.

4. The traditional method can not reach 100% for the screening of unisexual female flowers, but the accuracy of this method can reach 100%.

To sum up, the method of the present invention has good specificity, high accuracy, has the advantage of not being affected by the environment, and can well distinguish parthenogenetic muskmelon materials, and can also distinguish homozygous and heterozygous. Therefore, the present invention obtains a molecular marker that can efficiently and accurately distinguish whether the muskmelon is parthenogenetic flower material.

Description of drawings

Figure 1 shows the field phenotype identification of unisexual and hermaphrodite flowers in muskmelon: Oa831 is a unisexual melon material, and Oa808 is a hermaphrodite melon material.

Figure 2 is the identification result of the molecular marker of the present invention: wherein M represents DNA Marker, and the remaining swimming lanes are the electrophoresis results of PCR amplification products of different samples, which are respectively: No. 1 represents the identification result of the unisexual female flower Oa831 material, and No. 2 represents The identification results of the hermaphrodite flower Oa808, No. 3 indicates the identification results of the F1 generation materials crossed between Oa831 and Oa808, and the rest are the identification results of the F2 generation materials.

For those skilled in the art, other related drawings can be obtained according to the above drawings without any creative effort.

Detailed ways

The present invention is described below through specific embodiments. Unless otherwise specified, the technical means used in the present invention are methods known to those skilled in the art. In addition, the embodiments should be considered as illustrative rather than limiting the scope of the invention, the spirit and scope of which is defined only by the claims. For those skilled in the art, on the premise of not departing from the spirit and scope of the present invention, various changes or modifications to the material components and dosage in these embodiments also belong to the protection scope of the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified; the instruments and reagents used, etc., can be obtained from commercial sources unless otherwise specified.

The melon materials Oa831, Oa808 and F1 generation materials in the following examples are available to the public from the Plant Resources and Breeding Laboratory of Tianjin University. These materials are only used for repeating the relevant experiments of the present invention and cannot be used for other purposes.

Example 1, the acquisition of molecular markers

The melon gene CmACS-7 is an important regulatory factor for regulating the flower type of melon. Using the resequencing information of the melon genome, an InDel molecular marker TJcM02 was obtained in the region near the position of the gene, and its sequence is shown in sequence 1 in the sequence listing. In order to utilize the InDel site, suitable specific primers were designed, including upstream primer F and downstream primer R, and the primer sequences are as follows:

Upstream primer F: TTCAAATTCTTCACAACTTTACCGTATTGATCC

Downstream primer R: GGTGGTCATTTTCATAGAACTTTCCCATAC

Example 2, the acquisition of melon materials Oa831, Oa808 and F2 generation segregated populations and the identification of material flower types

The melon material Oa831 is the female parent, and Oa808 is the male parent. The F1 generation was obtained by crossing, and the F2 generation melon material was obtained by selfing the F1 generation. Cultivate normally in the field, and observe the flower type of the materials at the flowering stage. According to whether the open flowers contain stamens and pistils, it is determined that Oa831 is a unisexual flower material, Oa808 is a bisexual flower material, and F1 is a unisexual flower material. 44 plants Among the F2 melon plants, 39 plants showed unisexual female floral material, and 5 plants showed hermaphrodite flower material.

Example 3, the application of molecular markers in identifying whether the muskmelon is unisexual female flowered melon

For the melon material in Example 2, use the CTAB method to extract the genomic DNA of the leaves. The specific steps are: take 0.2 g of young melon leaves and put them into a 1.5 mL centrifuge tube, add 50 μL of 2% CTAB extraction buffer, grind, and then add Make up to 400 μL, bathe in 65°C water for 30 minutes; add 400 μL of chloroform:isoamyl alcohol (24:1), and shake gently for 5 minutes. Centrifuge at 12,000 rpm for 5 minutes; take 200 μL of supernatant, add 200 μL of pre-cooled isopropanol to mix, and place at -20°C for 20 min; centrifuge at 12,000 rpm for 10 min; discard supernatant, add 150 μL of pre-cooled ethanol, mix gently and wash, 10,000 Centrifuge at rpm for 5 min; discard the supernatant, air dry or blow dry; add 100 μL of distilled water to dissolve the DNA, and place at room temperature for 1 h; dilute the DNA to 50 ng/μL with distilled water, and use it as a PCR template or store it at -20 °C for later use.

Using the above-mentioned muskmelon genomic DNA as a template, and the upstream primer F and downstream R in Example 1 as primers, PCR amplification was carried out, and a 10 μL reaction system was used, including:

The procedure used for PCR amplification was: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 57°C for 30 s, extension at 72°C for 30 s, and 35 cycles; final extension at 72°C for 5 min.

The PCR amplification product was electrophoresed at 140v constant voltage for 90min in 8% polyacrylamide gel, and the gel was stained to observe the result. The specific primer and other components of PCR can also be made into a kit for identifying whether the muskmelon is parthenotheca muskmelon.

From the results, it can be found that only one 249bp band was amplified in the unisexual female flower material Oa831, only one 228bp band was amplified in the hermaphroditic flower material Oa808, and 228 and 249bp bands were amplified in the F1 generation material at the same time . In the F2 population, only one 228bp band was amplified among the 5 plants identified as hermaphroditic flowers, which was the same as the hermaphrodite material Oa808; among the 39 plants identified as unisexual female flowers, 6 plants Only the 249bp band was amplified, which is the same as the parthenogyne flower material Oa831, which is a homozygous parthenogyne flower melon material. The 249bp and 228bp bands were amplified at the same time in 33 plants, which was the same as the F1 generation melon material, which was heterozygous parthenogenetic melon material. The molecular marker identification results were consistent with the phenotypic identification results of the melon flower type.

Based on the above results, it can be known that using the InDel marker TJcM02 shown in Sequence 1 in the sequence table, and the specific primers shown in Sequence 2 and Sequence 3, the genomic DNA of the muskmelon material to be identified was subjected to PCR amplification, separated by electrophoresis, and detected by silver staining. It was found that the identification result of the floral type of melon was consistent with the identification result of the field phenotype, indicating that the InDel marker TJcM02 provided by the present invention and its specific primers can be used for auxiliary screening and identification of the floral type of the melon, screening or auxiliary screening for unisexual female flowers Melon strains or varieties.

The present invention has been described as an example above, and it should be noted that, without departing from the core of the present invention, any simple deformation, modification or other equivalent replacements that can be made by those skilled in the art without creative labor all fall within the scope of this invention. protection scope of the invention.

sequence listing

<110> Tianjin University

<120> A Molecular Marker TJcM02 for Identifying Parthenosexual Flowers of Melon and Its Application

<160> 3

<170> PatentIn version 3.5

<210> 1

<211> 249

<212>DNA

<213> PCR amplification product

<400> 1

TTCAAATCTT CACAACTTTA CCGTATTGAT CCCTTAAAAA TGAAGTAAAA ACAATAATGA 60

ACGAACTAAG ACAATCACCA TTTGAAAGTT TAAGGACCAA ATGAACCAAA GTTAAAAGTA 120

TAGAAACAAA AATAAACATC GCTAAATAAA CCAAATAAAAA CTAGAATTAC TTAATTGAAA 180

CAAAATAATA TGAAATGGAT CAAATCTTTA GACTTTAGTG TATGGGAAAG TTCTATGAAA 240

ATGACCACC 249

<210> 2

<211> 32

<212>DNA

<213> Artificial sequence

<400> 2

TTCAAATTCTTCACAACTTTACCGTATTGATCC 32

<210> 3

<211> 30

<212>DNA

<213> Artificial sequence

<400> 3

GGTGGTCATTTTCATAGAACTTTCCCATAC 30

Claims (3)

1. it is a kind of identify muskmelon unisexual flower molecular labeling TJcM02 primer special it is characterized in that：The primer is from 5` ends To the forward primer TTCAAATCTTCACAACTTTACCGTATTGATCC of the single-stranded DNA sequence of 1-32 at 3` ends；From 5` Hold the reverse primer GGTGGTCATTTTCATAGAACTTTCCCATAC of the 1-30 single-stranded DNA sequences at 3` ends.

2. the molecule labelling method of the primer special identification muskmelon unisexual flower using claim 1, extracts muskmelon test sample to be checked The genomic DNA of product carries out PCR (PCR) using molecular labeling primer special and expands, then pass through as template Electrophoresis is detected amplified production.

3. method as claimed in claim 2, it is characterized in that step is as follows：

1) the muskmelon sample to be measured arbitrarily genomic DNA of tissue or organ is extracted using CTAB methods；

2) using the sample to be tested genomic DNA obtained by step 1 as template, using molecular labeling specific primer to carrying out PCR expansions Increase, obtain amplified production；

3) using polyacrylate hydrogel electrophoretic techniques, the pcr amplification product obtained by step 2 is separated by electrophoresis；

4) pcr amplification product is dyed using silver staining；

5) by judging the size of amplified production, the larger DNA fragmentation for 249bp of amplified production contains 249bp amplified productions Sample to be tested be unisexuality female flower muskmelon material；The sample to be tested for not containing 249bp amplified productions is non-unisexuality female flower muskmelon material Material.