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EST-SSR marker primer developed based on platycladus orientalis transcriptome sequence and application of EST-SSR marker primer

A technology of sequence and orientalis, applied in the field of plant molecular markers, can solve the problem of less molecular markers of orientalis microsatellites

Active Publication Date: 2022-04-12
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are few microsatellite molecular markers in Thuja arborvitae, and there are even fewer markers for efficient polymorphic EST-SSR primers developed for Thuja arborvitae

Method used

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  • EST-SSR marker primer developed based on platycladus orientalis transcriptome sequence and application of EST-SSR marker primer
  • EST-SSR marker primer developed based on platycladus orientalis transcriptome sequence and application of EST-SSR marker primer
  • EST-SSR marker primer developed based on platycladus orientalis transcriptome sequence and application of EST-SSR marker primer

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Experimental program
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Effect test

Embodiment 1

[0082] Embodiment 1, development and screening method of orientalis EST-SSR primer set

[0083] 1. Primer development and design method

[0084] According to the sequencing data of the transcriptome of Arborvitae, MISA (Microsatellite) software was used to search for SSR sites, and the search criteria were set as: single nucleotide, dinucleotide, trinucleotide, tetranucleotide, pentanucleotide and Hexanucleotides, the minimum number of repetitions of each nucleotide is 10, 6, 5, 5, 5, 5, respectively. Then, the Primer 3.0 primer batch design program was used to design primers for the Unigene sequence containing the SSR site, and the length of the SSR site sequence was 17-28bp. The main parameters for primer design are as follows: the annealing temperature (Tm) is between 50-65°C; the size of the PCR product is 100-500bp, and the GC content is 40-60%.

[0085] 2. Polymorphic primer detection method

[0086] 1.1 Test materials

[0087] The test materials were the leaves of 4 a...

Embodiment 2

[0135] Embodiment 2, a kind of method utilizing the polymorphic EST-SSR marker primer provided in embodiment 1 to analyze the genetic diversity of arborvitae germplasm resources

[0136] Utilizing the EST-SSR labeled primers of PoE9, PoE64, PoE74, PoE84, PoE85, PoE89, PoE97, PoE105, PoE133, PoE139, PoE166 screened out in Example 1, 94 parts of arborvitae species were amplified by capillary electrophoresis with fluorescent primers The EST-SSR molecular markers of the germplasm materials, 94 arborvitae germplasm materials include arborvitae varieties (clones) and their cultivars weeping arborvitae, Sa Jinbai, the specific numbers and names are shown in Table 1.

[0137] The 94 germplasm materials of Thuja arborvitae in Table 1 were all sourced and preserved in the Thuja arborvitae germplasm resource nursery of the Institute of Forestry and Fruit Trees, Beijing Academy of Agriculture and Forestry Sciences. Capillary electrophoresis was used to detect the size of fluorescent PCR p...

Embodiment 3

[0159] Embodiment 3, a kind of method utilizing the polymorphic EST-SSR marker primer provided in embodiment 1 to identify the genetic relationship of arborvitae germplasm resources

[0160] Utilize the EST-SSR labeled primers of PoE9, PoE64, PoE74, PoE84, PoE85, PoE89, PoE97, PoE105, PoE133, PoE139, PoE166 that are screened out in embodiment 1, adopt fluorescent primer capillary electrophoresis to amplify the known species of arborvitae EST-SSR molecular markers of 'Arborvitae', 'Arborvitae' and their vegetative offspring, and three clones of Arborvitae to be tested. The above materials are all sourced from the oriental cypress germplasm resource nursery of the Institute of Forestry and Fruit Trees, Beijing Academy of Agriculture and Forestry Sciences. Capillary electrophoresis was used to detect the size of fluorescent PCR products, Popgene32 was used to statistically analyze genetic diversity, and Ntsys2.10 was used to construct a phylogenetic clustering map. Specific step...

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Abstract

The invention discloses an EST-SSR (expressed sequence tag-simple sequence repeat) marker primer developed based on a platycladus orientalis transcriptome sequence and application of the EST According to the EST-SSR marker primer, an SSR sequence is obtained on the basis of platycladus orientalis transcriptome sequencing data, a primer is designed, and the stably-amplified, efficient and polymorphic microsatellite marker primer is obtained through a triple screening method of agarose gel electrophoresis, polyacrylamide gel electrophoresis and capillary electrophoresis screening. The effectiveness of the developed EST-SSR marker primer in the aspects of genetic diversity analysis and genetic relationship identification is verified by utilizing different varieties and clone of platycladus orientalis, and the EST-SSR marker primer can be applied to genetic diversity analysis, genetic relationship and variety identification, molecular marker-assisted selective breeding, genetic relationship identification and genetic relationship identification of platycladus orientalis plants. And the research fields of new plant variety protection and the like.

Description

technical field [0001] The invention belongs to the technical field of plant molecular markers, and in particular relates to EST-SSR marker primers developed based on the transcriptome sequence of orientalis arborvitae and applications thereof. Background technique [0002] Platycladus orientalis (L.) Franco is an evergreen tree belonging to the genus Arborvitae of the Cupressaceae family and belongs to a single genus of coniferous plants. Arborvitae has the widest natural distribution range in my country and is an important ecological stress-resistant tree species with high ecological, economic and social value. Arborvitae is resistant to drought and barrenness, has strong stress resistance, and has a wide range of adaptability. It is an important tree species for afforestation in my country; The lifespan of arborvitae is the main ancient tree resource and cultural tree species in my country; the leaves of arborvitae have pharmacological effects such as antibacterial, anti-...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/6858C12N15/11
CPCY02A40/81
Inventor 刘国彬曹均廖婷郭丽琴王烨姚砚武
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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