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The est-ssr marker primer developed based on the hybrid orchid transcriptome and its application

A hybrid blue and primer set technology, applied in the biological field, can solve the problems of EST-SSR application research that has not been reported, and achieve the effects of easy identification, rich polymorphism, and stable amplification

Active Publication Date: 2021-07-27
CROP RES INST OF FUJIAN ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

With the continuous enrichment of EST database and the development of high-throughput sequencing technology, EST-SSR has been developed and applied in a large number of plants, but there are no reports on the application of EST-SSR in hybrid orchids.

Method used

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  • The est-ssr marker primer developed based on the hybrid orchid transcriptome and its application
  • The est-ssr marker primer developed based on the hybrid orchid transcriptome and its application
  • The est-ssr marker primer developed based on the hybrid orchid transcriptome and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 The target hybrid orchid sample is the rooted tissue culture seedling, annual plant or biennial plant leaf of 'Zi Yan' in the flower garden of the Crop Research Institute of the Fujian Academy of Agricultural Sciences. The Illumina high-throughput deep sequencing of the transcriptome contains a total of 88,734 Unigenes as the analysis background data.

Embodiment 2

[0044] Example 2 Identification of target hybrid orchid transcriptome SSR sites and SSR primer design

[0045] For the 88,734 Unigenes of the target hybrid orchid leaves, use the site mining tool MISA software to search for SSR sites. The search criteria are: the number of repeats of single, two, three, four, five, and hexanucleotides is at least 10, 6, 5, 5, 5, 5 times. 19728 SSR loci were found from 88734 Unigene sequences in the hybrid orchid transcriptome, distributed in 14469 Unigenes, and the occurrence frequency was 16.31%. Among them, there were 10350 Unigene sequences containing only one SSR site, and 4119 Unigene sequences containing two or more SSR sites, with an average of one SSR site per 4.79kb. The types of SSR sites detected in the hybrid orchid transcriptome include one, two, three, four, five, and six nucleotide repeat unit types and mixed SSRs, among which, the sites of one, two, and three nucleotide repeat units There are many, respectively 12111, 4382, a...

Embodiment 3

[0070] Example 3 Polymorphism Analysis of Hybrid Blue EST-SSR Marker

[0071] The 9 pairs of hybrid blue EST-SSR primers screened in Example 2 were subjected to polymorphism analysis, and the results are shown in Table 2.

[0072] Table 2 Hybrid blue EST-SSR marker polymorphism analysis table

[0073]

[0074]

[0075] It can be seen from Table 2 that a total of 145 bands were amplified by 9 pairs of primers, and the number of polymorphic bands was between 8 and 26. On average, 16.1 polymorphic bands were amplified by each pair of primers. 16.1 polymorphic bands were amplified, and the polymorphic ratio of each primer was 100.00%. The polymorphism information content analysis of each primer showed that the polymorphism information content ranged from 0.769 to 0.938, indicating that EST-SSR markers had relatively abundant genetic polymorphisms in hybrid orchid germplasm.

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Abstract

The invention discloses a hybrid orchid EST-SSR primer and its application in genetic diversity analysis, genetic map construction and marker-assisted breeding of hybrid orchid plant germplasm resources. The invention discloses 9 sets of primer pairs and a screening method for hybrid orchid EST-SSR primers, which specifically include: obtaining the original gene database of the target hybrid orchid leaf, performing SSR site search on the original gene database, and performing PCR on the target sequence by Primer 5.0 Design primers and generate candidate primers; perform PCR amplification on candidate primers, select PCR primers for primary screening, and finally screen hybrid blue EST-SSR primers. The 9 pairs of EST-SSR marker primers provided by the present invention come from the transcriptome sequence of hybrid orchid leaves. The primers are rich in polymorphism, stable in amplification, and easy to identify amplified bands. Transcriptome development blanks for SSR primers.

Description

technical field [0001] The invention relates to the field of biotechnology, and more specifically relates to EST-SSR marker primers developed based on hybrid orchid transcriptomes and their applications. Background technique [0002] Hybrid orchid (Cymbidium hybrid) is a special name for a class of orchids bred by crossbreeding Cymbidium and Cymbidium. Excellent characteristics, high ornamental, economic and cultural value, deeply loved by consumers in recent years, has become a new type of orchid with great development potential in the orchid market, with broad market prospects. At present, the research on hybrid orchids mainly focuses on tissue culture technology, cultivation technology, flowering regulation, etc. There are few studies on hybrid orchids' genetic diversity, genetic map construction, and gene mapping. The molecular markers used in the above research in hybrid orchids are only some general primers such as RAPD, SRAP, etc., which greatly limits the progress o...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12Q1/6858C12N15/11
CPCC12Q1/6858C12Q1/6895C12Q2600/156C12Q2531/113
Inventor 钟淮钦林榕燕林兵罗远华樊荣辉吴建设叶秀仙方能炎
Owner CROP RES INST OF FUJIAN ACAD OF AGRI SCI