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Paphiopedilum Henryanum EST-SSR (expressed sequence tag-simple sequence repeat) marker primers and their development method and application

A Paphiopedilum and labeling technology, which is applied in biochemical equipment and methods, microbiological determination/inspection, DNA/RNA fragments, etc., can solve problems that have not been reported, and achieve good repeatability, low production cost, and polymorphism. sexual enrichment effect

Active Publication Date: 2017-11-21
INST OF VEGETABLE & FLOWERS CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, so far, the research on EST-SSR markers of Paphiopedilum has not been reported

Method used

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  • Paphiopedilum Henryanum EST-SSR (expressed sequence tag-simple sequence repeat) marker primers and their development method and application
  • Paphiopedilum Henryanum EST-SSR (expressed sequence tag-simple sequence repeat) marker primers and their development method and application
  • Paphiopedilum Henryanum EST-SSR (expressed sequence tag-simple sequence repeat) marker primers and their development method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1. Development of P. henrypipes EST-SSR primers

[0051] 1. EST-SSR primer design

[0052] Experimental materials: P. henrylis organs were obtained from the greenhouse of the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences; various reagents or medium components used in all the examples of the present invention were obtained commercially.

[0053] 1) Construction of transcriptome library: Extract total RNA from Papudendron henrylium organs, isolate mRNA, synthesize and purify cDNA by reverse transcription, end repair, add adenine nucleosides to connect sequencing adapters, and recover the size of 200-700bp by agarose gel electrophoresis The recovered fragments were amplified by PCR to construct a transcriptome library.

[0054] 2) Acquisition of transcriptome data: Sequencing the transcriptome library obtained in step 1) to obtain transcriptome sequencing data, and splicing the sequencing data to obtain sequence data of unigenes. ...

Embodiment 2

[0075] Example 2. Analysis of Genetic Diversity Using P. henrypipes EST-SSR Primers

[0076] Experimental materials: The experimental samples of P. henryi were obtained from 8 plants of Taiyang Chong population (TYC) and 25 plants of Dalong population (DN) in Malipo County, Wenshan Prefecture, Yunnan Province.

[0077] Analysis of the amplified polymorphism information of these 34 pairs of EST-SSR primers (see Table 3 below) shows that the observed and expected heterozygosity ranges are 0.0303-1.000 and 0.0597-0.6793, respectively, and the distribution range of the Shannon information index is 0.14- 1.25, with an average value of 0.61, indicating that the genetic differentiation of these 33 P. henryii materials is relatively rich.

[0078] Table 3 Analysis of polymorphism information amplified by 34 pairs of EST-SSR primers

[0079]

[0080]

[0081] In addition, these 34 pairs of primers were also used to perform cluster analysis on 33 different P. henley species from ...

Embodiment 3

[0082] Example 3. Generic analysis in Paphiopedilum using EST-SSR primers

[0083] The generality analysis of other six species of P. genus from the genus Paphiopedilum was carried out using microsatellites of P. henley. The results (see Table 4 below) showed that 34 pairs of EST-SSR primers were used in P. villosum, The successful expansion rate of P. venustum, P. wardii, P. dianthum, P. concolor and P. micranthum are 0.85, 0.82, 0.74, 0.68, 0.56, and 0.56, respectively, indicating that these 34 pairs of EST-SSR primers have high versatility in the genus Paphiopedilum, especially in the subgenus Paphiopedilum, which shows that The closer the genetic distance to P. henlensis, the higher the amplification efficiency of SSR primers. It was proved that 34 pairs of EST-SSR primers developed by using the transcriptome sequencing of P. henrifolia can be used for the study of genetic diversity and genetic structure of closely related species.

[0084] Table 4 Amplification of 34 ...

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Abstract

The invention discloses an EST-SSR (expressed sequence tag-simple sequence repeat) marker primer group developed based on Paphiopedilum henryanum transcriptome sequences; the group includes 34 pairs of primers having nucleotide sequences shown as in SEQ ID NO. 1 to SEQ ID NO. 68. The invention also discloses an agent comprising the Paphiopedilum henryanum EST-SSR marker primers. The invention further discloses a method of developing the Paphiopedilum henryanum EST-SSR marker primers. The Paphiopedilum henryanum EST-SSR marker primers provided herein have the advantages such as rich polymorphism, good repeatability, good amplification stability and good static convenience, and are applicable to genetic diversity analysis and molecular mark assisted breeding of germplasm resources of Paphiopedilum henryanum and other species in Paphiopedilum, especially subgenus Paphiopedilum, as well as to molecular research relating to plants in Paphiopedilum.

Description

technical field [0001] The invention relates to an EST-SSR marker primer developed based on the transcriptome sequence of Paphiopedilum henryanum, a development method and an application thereof, and belongs to the field of biotechnology. Background technique [0002] Henry Paphiopedilum belongs to Orchidaceae (Orchidaceae) Paphiopedilum plant. It is native to southwestern Guangxi, southeastern Yunnan and northern Vietnam. It grows in hidden crevices in limestone areas at an altitude of 900-1300m or in evergreen broad-leaved forests and Rocky or well-drained places in shrubs. Now all Paphiopedilum plants are listed in Appendix I of the Convention on International Trade in Endangered Species of Wild Fauna and Flora and are banned from trade. As an endangered species, the study of its genetic diversity has become very important, and the conservation of Paphiopedilum is even more urgent. [0003] Simple sequence repeat (SSR) markers have the characteristics of high polymorphi...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11C12N15/10
CPCC12N15/1003C12Q1/6806C12Q1/6895C12Q2600/156
Inventor 贾瑞冬徐玉凤葛红周妍慧程浩杨树华赵鑫
Owner INST OF VEGETABLE & FLOWERS CHINESE ACAD OF AGRI SCI
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