CN103773760A - Universal SSR (simple sequence repeat) molecular marker CSSR1 for chrysanthemum and related plants and application thereof - Google Patents

Abstract

The present invention provides a universal SSR molecular marker CSSR1 for plants of Chrysanthemum and related genera, and the primer sequences for PCR amplification of the marker CSSR1 are shown in Seq ID No.1 and 2. The invention also provides the application of the SSR marker in the identification of intergeneric hybrids of Chrysanthemum and Chrysanthemum. In addition, the present invention also provides a method for amplifying the SSR molecular marker, and establishes a general SSR molecular marker technology system among plant materials of the genus Chrysanthemum, Chrysanthemum and Chrysanthemum, which can be used for It can be used in the identification of hybrids within and between genera of Chrysanthemum, Chrysanthemum and Taihang Chrysanthemum, analysis of plant genetic diversity, construction of fingerprints and molecular marker-assisted breeding, etc., with broad application prospects.

Description

General SSR Molecular Marker CSSR1 and Its Application in Chrysanthemum and Related Genera

technical field

The invention relates to the field of molecular biology, in particular to a universal SSR molecular marker CSSR1 for plants of Chrysanthemum and related genera and its application.

Background technique

Chrysanthemum (Chrysanthemum) belongs to the dicotyledonous class Asteraceae. Chrysanthemum plants native to my country mainly include: wild chrysanthemum (C. Chrysanthemum (C.lavandulifolium), small red chrysanthemum (C.chanetii), purple wild chrysanthemum (C.zawadskii), chrysanthemum brain (C.mankingense), etc. Among them, chrysanthemum (Chrysanthemum morrofolium) is the species with the highest ornamental value. Chrysanthemum is one of the top ten famous flowers in China and one of the four major cut flowers in the world. It has a cultivation history of more than 3,000 years in China and has formed a variety group with rich flower types and various colors. . In addition to ornamental value, Chrysanthemum plants also have high medicinal value and food value. Chrysanthemum contains stachydrine, locust glycoside, luteolin, cosmoside, adenosine, choline, glucoside and other ingredients, especially rich in volatile oil, and the oil is mainly composed of juniperone, borneol and borneol hexanoic acid esters etc. In terms of efficacy, chrysanthemum has the effects of calming the liver and improving eyesight, dispelling wind and clearing heat, relieving cough and relieving pain, and is effective in treating headaches, dizziness, red eyes, swelling and pain, wind-heat cold, cough and other diseases.

The genus Ajania belongs to the dicotyledonous class Asteraceae, and is mainly distributed in the vast areas except the southeastern half of my country. There are also a few species in Mongolia, the Soviet Union, northern Korea and northern Afghanistan. A.acificum, a wild species of the genus Chrysanthemum, is native to Taiwan and Japan, and has been widely used in the gardens of the southeast coast of my country, Japan, the Netherlands and other countries as ground cover and potted plants. The bright leather of its leaves has high ornamental value. Chrysanthemum breeders also use chrysanthemum as a parent to carry out cross breeding with plants of the genus Chrysanthemum to improve the ornamental properties of chrysanthemum branches and leaves.

Opisthopappus, Asteraceae, including O. taihangensis and O. longilobus, mainly distributed in Shanxi, Hebei, Henan and other places in my country. The plants grow prostrate, mostly in Steep slopes and crevices of rocks on cliffs are resistant to barrenness, cold and drought, and have become important breeding materials for horticultural workers.

Chrysanthemum and Taihang Chrysanthemum, as close relatives of Chrysanthemum, have become new germplasm resources and are used by chrysanthemum breeders for distant hybridization with chrysanthemums to improve the resistance and ornamental properties of existing chrysanthemum varieties.

Simple sequence repeat (SSR), also known as microsatellite DNA (microsatellite), is a sequence of dozens or even hundreds of nucleotides repeated in series with 2 to 5 nucleotides. sequence. These sequences widely exist in different loci of eukaryotic genomes, and the number of repeating units at each locus may not be exactly the same, thus forming polymorphisms. Due to its advantages of rich polymorphism, high repeatability, and co-dominant inheritance, it is widely used in the fields of plant population and evolutionary genetic research, genetic diversity analysis of germplasm resources, and fingerprint construction.

However, the development of SSR markers in chrysanthemum is currently in its infancy, and there is no report on the development of SSR markers in Ajania and Opisthopappus due to the lack of gene sequence information; therefore, currently Ajania and Opisthopappus No SSR molecular markers have been developed for Opisthopappus, and there is no SSR marker that can be used for general use between Chrysanthemum and related genera; For the development and utilization of wild resources of Chrysanthemum, Chrysanthemum and Taihang Chrysanthemum, it is of great significance to carry out intergeneric distant hybrid breeding and molecular level research.

Contents of the invention

The purpose of the present invention is to provide a universal SSR molecular marker CSSR1 for plants of Chrysanthemum and related genera and its application.

In order to achieve the purpose of the present invention, a general SSR molecular marker CSSR1 of Chrysanthemum and related plants of the present invention, the primer pair for PCR amplification molecular marker CSSR1 is:

Upstream primers: 5'-TTCAAGGGCTGGATGCCTTATT-3' and

Downstream primer: 5'-GTGTTTGCGATGTTCCATTCTC-3'.

The related genera involved in the present invention include but not limited to Chrysanthemum, Ajania, Opisthopappus and the like.

The invention also provides the application of the molecular marker in the identification of hybrids within or between genera of Chrysanthemum and related genera, analysis of genetic diversity of plant materials, construction of fingerprints, molecular marker assisted breeding, and the like.

The present invention further provides a method for identifying species of Chrysanthemum and related genera, comprising the following steps:

1) Extract the genomic DNA of the plant to be tested;

2) Use the genomic DNA of the plant to be tested as a template, and perform PCR amplification using the above-mentioned primer pair for PCR amplification of the molecular marker CSSR1;

3) Detection of PCR amplification products.

The PCR reaction system in step 2) is: each primer 0.32 μM/μL, Taq DNA polymerase 0.2 U/μL, dNTP 0.4 μM/μL, Mg ²⁺ 0.65 μM/μL, template DNA 2-4 ng/μL, ddH ₂ O preparation.

The PCR amplification program in step 2) is: 94°C for 5min; 94°C for 0.5min, 50-62°C for 0.5min, 72°C for 1min, a total of 30 cycles; 72°C for 5min. Preferably, the PCR amplification program is: 94°C for 5 min; 94°C for 0.5 min, 58°C for 0.5 min, 72°C for 1 min, a total of 30 cycles; 72°C for 5 min.

The present invention provides for the first time a general SSR molecular marker—CSSR1 among the plant materials of Chrysanthemum, Chrysanthemum and Chrysanthemum. The invention also provides the application of the SSR marker in the identification of intergeneric hybrids of Chrysanthemum and Chrysanthemum. In addition, the present invention also provides primer sequences and amplification methods for amplifying the SSR site, and establishes a general SSR molecular marker technology system among plant materials of Chrysanthemum, Chrysanthemum, and Chrysanthemum. It can be used for identification of hybrids within and between genera of Chrysanthemum, Chrysanthemum, and Chrysanthemum, analysis of plant genetic diversity, construction of fingerprints, and molecular marker-assisted breeding, etc., with broad application prospects.

Description of drawings

Fig. 1 is the STR typing diagram of the genomic DNA of Chrysanthemum, Chrysanthemum and Taihang Chrysanthemum plant material amplified by the SSR primer of CSSR1 site of the present invention; The 28 kinds of plant materials of the genus Chrysanthemum and Chrysanthemum, the abscissa represents the size of the amplification product, and the ordinate represents the amplification intensity.

Figure 2 shows the wild species of Chrysanthemum chrysanthemum (female parent), the ground cover chrysanthemum variety "Pudi Danfen" (male parent), and their hybrid offspring AP1, AP2, and AP3.

Detailed ways

The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the examples are all in accordance with conventional experimental conditions, such as Sambrook et al. Molecular cloning experiment manual (Sambrook J&Russell DW, Molecular cloning: a laboratory manual, 2001), or in accordance with the conditions suggested by the manufacturer's instructions.

Example 1 Acquisition of General SSR Molecular Marker CSSR1 of Chrysanthemum and Related Genus Plants

(1) Download the 7180 ESTs of all Chrysanthemum plants published on the website of the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/Genbank/) as of April 2012 Sequences were spliced using CAP3 software to obtain spliced gene sequences. Then use SSRIT software (http://www.gramene.org/db/markers/ssrtool) to search for SSR sites, and then use Primer Premier5.0 software to design upstream and downstream primers for SSR sites.

Wherein the primers for amplifying the molecular marker CSSR1 are the following primer pairs:

CSSR1 upstream primer F: 5'-TTCAAGGGCTGGATGCCTTATT-3'

CSSR1 downstream primer R: 5'-GTGTTTGCGATGTTCCATTCTC-3'

(2) DNA samples of common plant materials of Chrysanthemum, Ajania and Opisthopappus were extracted (see Table 1).

Table 1 Plant materials of the genera Chrysanthemum, Chrysanthemum and Chrysanthemum

(3) A common SSR-labeled PCR reaction system between Chrysanthemum, Ajania and Opisthopappus plant materials was obtained after a lot of experiments: upstream and downstream primer concentrations were 0.32 μM each /μL, the concentration of Taq DNA polymerase is 0.2U/μL, the concentration of dNTP is 0.4μM/μL, the concentration of Mg ²⁺ is 0.65μM/μL, and the concentration of template DNA is 2～4ng/μL.

The PCR amplification reaction conditions were: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 0.5 min, annealing at 58°C for 0.5 min, extension at 72°C for 1 min, 30 cycles; extension at 72°C for 5 min.

(4) Using the extracted plant material DNA samples of Chrysanthemum, Ajania, and Opisthopappus as templates, the designed upstream and downstream primers of the SSR site were used for PCR reaction, The polymorphic upstream and downstream primers were screened out, and the stable and polymorphic SSR sites CSSR1 and CSSR2 were screened out.

Example 2 Using the SSR molecular marker CSSR1 to carry out hybridization (AP1, AP2, AP3) between the ground cover chrysanthemum variety "Pudifendai" (C.Morifolium "Pudifendai") and A.Acificum identification

(1) Extract the genomic DNA samples of the ground cover chrysanthemum variety "Pudi Danfen", the chrysanthemum of the genus Chrysanthemum and the hybrid offspring AP1, AP2, and AP3.

(2) Using the PCR reaction system for amplifying the SSR molecular marker in Example 1, using the upstream and downstream primers of the CSSR1 site and using the DNA of the hybrid parents and hybrid offspring as templates, PCR reactions were performed.

(3) STR typing analysis was carried out on the parents and hybrid offspring (Figure 1). The hybrid offspring all showed the characteristic bands of the male and female parents, proving that AP1, AP2, and AP3 are ground cover chrysanthemum varieties "Pave ground light powder" and isochrysanthemum (Fig. 2).

Table 2 Characteristic bands of parents and hybrid offspring

Although the present invention has been described in detail with general descriptions and specific embodiments above, it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, the modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the protection scope of the present invention.

Claims (8)

1. Chrysanthemum and the general SSR molecule marker of relative genus plant CSSR1, is characterized in that, for the primer pair of pcr amplification molecule marker CSSR1 is:

Upstream primer: 5'-TTCAAGGGCTGGATGCCTTATT-3' and

Downstream primer: 5'-GTGTTTGCGATGTTCCATTCTC-3'.

2. molecule marker according to claim 1, is characterized in that, described relative genus plant includes but not limited to Chrysanthemum (Chrysanthemum), sub-Chrysanthemum (Ajania), Taihang Chrysanthemum (Opisthopappus).

3. the application of molecule marker in Chrysanthemum and relative genus plant genus or in the evaluation of bigenering, vegetable material analysis of genetic diversity, fingerprint map construction and molecular mark described in claim 1 or 2.

4. a method of identifying Chrysanthemum and relative genus plant variety, is characterized in that, comprises the following steps:

1) extract the genomic dna for the treatment of measuring plants;

2), take the genomic dna for the treatment of measuring plants as template, utilize the primer pair for pcr amplification molecule marker CSSR1 described in claim 1 to carry out pcr amplification;

3) detect pcr amplification product.

5. method according to claim 4, is characterized in that step 2) in PCR reaction system be: the each 0.32 μ M/ μ L of primer, Taq archaeal dna polymerase 0.2U/ μ L, dNTP0.4 μ M/ μ L, Mg ²⁺0.65 μ M/ μ L, template 2～4ng/ μ L, with ddH ₂o preparation.

6. method according to claim 4, is characterized in that step 2) in pcr amplification program be: 94 ℃ of 5min; 94 ℃ of 0.5min, 50～62 ℃ of 0.5min, 72 ℃ of 1min, totally 30 circulations; 72 ℃ of 5min.

7. method according to claim 6, is characterized in that step 2) in pcr amplification program be: 94 ℃ of 5min; 94 ℃ of 0.5min, 58 ℃ of 0.5min, 72 ℃ of 1min, totally 30 circulations; 72 ℃ of 5min.

8. method according to claim 4, is characterized in that, described relative genus plant includes but not limited to Chrysanthemum, sub-Chrysanthemum, Taihang Chrysanthemum.

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