Establishing method of cymbidium microsatellite labels, core fingerprint label database and kit

A technology of microsatellite marker and establishment method, which is applied in the field of fingerprint marker library of main species of Orchid, can solve the problems of large overall information of the genome, unknown, large manpower and material resources, etc., and achieves good versatility, good versatility and conservation. high effect

Inactive Publication Date: 2015-01-21
ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the genome of Orchid plants is relatively large and the overall information is unknown. It is unrealistic to analyze and identify the SSR sites of the genome for the tested germplasm one by one.

Method used

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  • Establishing method of cymbidium microsatellite labels, core fingerprint label database and kit
  • Establishing method of cymbidium microsatellite labels, core fingerprint label database and kit
  • Establishing method of cymbidium microsatellite labels, core fingerprint label database and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 The preparation and screening method of SSR markers in the genus Orchid

[0033] The present embodiment is the preparation and screening method of SSR marker of Orchid, which mainly includes the following steps:

[0034] 1. Acquisition of Jianlan gene expression sequence:

[0035] Select the buds of Jianlan to extract total RNA, use the standard Solexa sequencing method to obtain transcriptome Reads, remove low-quality, carrier and non-coding sequences (such as rRNA, tRNA and miRNA), use Trinity splicing software to complete the sequence assembly, and finally obtain 101423 Jianlan The non-redundant Unigene sequence of .

[0036] 2. Retrieval of SSR sites in Jianlan transcriptome sequence:

[0037] Using MSATCOMMANDER to analyze the occurrence frequency of SSR. The minimum number of repeating motifs was set to 10 times for single nucleotides, 6 times for dinucleotides, 4 times for trinucleotides, tetranucleotides, pentanucleotides, and hexanucleotides, and a...

Embodiment 2

[0048] Embodiment 2: the universality test of blue genus mark

[0049] 1. Experimental materials:

[0050] A total of 48 materials from five species of Orchid (Chunlan, Cymbidium, Jianlan, Molan and Hanlan) were selected to test the generality of Jianlan SSR markers in Orchid plants. For specific material information, see Table 1.

[0051] 48 pieces of test material information for carrying out primer screening and genetic analysis in the present invention in table 1

[0052]

[0053] 2. Genome extraction and SSR primer amplification and screening

[0054] Select about 200mg of young leaves for each material, put them into a 2.0ml centrifuge tube, and put steel balls with a diameter of 0.5cm into them. After being quickly frozen in liquid nitrogen, shake them on a homogenizer for 35s to grind the tissues into powder. CTAB method for extraction. Prepare 8% polyacrylamide gel in the DYCZ-30 type electrophoresis tank (Beijing Liuyi Instrument Factory) for electrophoresis se...

Embodiment 3

[0061] Embodiment 3: the establishment of Orchid genus core marker library

[0062] 1. SSR primer amplification results statistics and polymorphism analysis

[0063] According to the electrophoresis results of step (2) in Example 2, the different genotypes are marked as 1, 2, 3, etc., and then input into Powermarker to count the polymorphisms of these markers. Among the 61 pairs of primers, 49 pairs had polymorphisms (see Table 2 for 49 markers), and a total of 216 genotypes were detected in 48 test materials, with an average of 3.7 genotypes per pair of primers. It shows that Jianlan SSR has high applicability in Orchid, and can be used to analyze the genetic diversity, phylogenetic relationship and genetic mapping of Orchid plants.

[0064] Table 2 49 orchid polymorphic marker sequences and related information in the present invention

[0065]

[0066]

[0067]

[0068] 2. Analysis of SSR primers' interspecies discrimination in Orchid:

[0069] For the above 49 p...

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Abstract

The invention discloses an establishing method of cymbidium microsatellite labels, a core fingerprint label database and a kit. The method comprises the following steps: exploiting SSR primers based on Cymbidium ensifolium transcription group information, and carrying out versatility and polymorphism analysis on labels to obtain 49 cymbidium polymorphism labels. The labels have high polymorphism and good repeatability, and are very suitable for the genetic study of cymbidium plants. Additionally, the core fingerprint label database is constructed according to the distinguishing ability of the labels in cymbidium species. The SSR fingerprint label database obtained in the invention has the characteristics of stable and reliable labels, small quantity and strong cymbidium species distinguishing ability, can maximally reflect the interspecific difference (not intraspecies difference) of the cymbidium species, is convenient for realizing statistics, and can be used for identifying cymbidium main species.

Description

technical field [0001] The invention belongs to the field of molecular genetics, and relates to the development of a small-number and high-resolution fingerprint marker library of main species of the genus Orchid by using Jianlan's internal SSR preparation and biostatistical methods. technical background [0002] Chinese orchids are commonly known as national orchids, which belong to the genus Cymbidium of the family Orchidaceae (Oehidaeeae), and mainly include Chunlan, Cymbidium, Hanlan, Jianlan and Molan. Because of its high ornamental value and economic value, current Chinese orchid lovers often cross between species within a genus and even between genus in order to create more germplasm resources (so-called technological grasses), but at the same time it also causes In addition to blurring the boundaries of orchid species, it is difficult to distinguish these species when they are not blooming, which has caused some troubles for collectors of orchids, and an industry and...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12N15/10C12Q1/68
Inventor 李小白金亮向林秦德辉金凤孙崇波
Owner ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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