Specific primer pair for identification of spermatophyte species and applications of specific primer pair

A technology for specific primer pairs and species identification, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., and can solve the problems of errors, unreliable identification results, and unsatisfactory primer versatility

Inactive Publication Date: 2013-03-20
INST OF BOTANY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, a large number of evaluation studies in recent years have found that matK and rbcL have very limited discriminative power or primer generality is not ideal
Although trnH-psbA evolves rapidly, there are structures such as inversions and SSR sites in some groups, which cause problems such as difficult sequencing and unreliable identification results
ITS is an ideal candidate DNA barcode, especially suitable for the distinction of lower taxonomic levels, but there are the following problems: (1) Some taxa have incomplete concerted evolution, and paralogous copies may cause errors; ( 2) Existing primers cannot eliminate fungal contamination; (3) The generality of primers is not good, and PCR amplification and sequencing are difficult

Method used

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  • Specific primer pair for identification of spermatophyte species and applications of specific primer pair
  • Specific primer pair for identification of spermatophyte species and applications of specific primer pair
  • Specific primer pair for identification of spermatophyte species and applications of specific primer pair

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1. Discovery of specific primer pairs for identifying angiosperm species

[0022] After studying the entire plant chloroplast genome, the inventor found that there are regions in the chloroplast genome that evolve faster than known marker genes, which are suitable for phylogenetic analysis and DNA barcoding. Among them, the fastest evolution is the ycf1 gene (also known as the Ycf1 gene).

[0023] The ycf1 gene is one of the few genes in the chloroplast genome with unclear functions. The ycf1 gene is located in the SSC region and the IR region, and the part located in the IR region is relatively short, generally less than 1KB, with less sequence variation. The copies located in IRb are often pseudogenized or lost in some taxa. The ycf1 gene evolves quickly, the base substitution rate is high, and the degree of variation is higher than that of the matK gene, which can be used for phylogenetic analysis. Because the ycf1 gene is longer (for example, 5706bp in the tob...

Embodiment 2

[0029] Example 2. Application of the specific primer pair designed in Example 1 to identify apricot plants

[0030] The Apricot plants used in this embodiment are as follows: Prunus armeniaca L.; Prunus holosericea (Batalin) Kostina; Prunus sibirica L.; Prunus mume Sieb.; Prunus zhengheensis J.Y. Zheng et M.N. Lu. See Table 1 for specific material information.

[0031] Table 1 Specific material information of apricot plants used in this embodiment

[0032]

[0033] 1. Take plant leaves and extract genomic DNA.

[0034] 2. Using the genomic DNA extracted in step 1 as a template, a primer pair consisting of ycf1bF and ycf1bR is used for PCR amplification to obtain PCR amplification products.

[0035] PCR reaction system (25ul): 2.5μL dNTP (2.5mmol / L), 2.5μL 10×PCR buffer (containing Mg 2+ ), the upstream primer and the downstream primer each consist of 1.25μL (5μmol / L), 0.25U polymerase, 2μL genomic DNA (20-30ng) and water.

[0036] PCR reaction program: denaturation at 94°C for 4min; den...

Embodiment 3

[0041] Example 3. Application of the specific primers designed in Example 1 to identify Chimonanthus plants

[0042] The Chimonanthus plants used in this example are as follows: Calycan thus chinensis; Calycan thusfloridus; Calycan thus occiden talis; Chimonan thus campanula tus; Chimonan thus gramma tus; Chimonan thusnitens; Chimonan thus praecox; Chimonan thus saljiangensis; Chimonan thus salicifolius; Idiospermus a traliense. See Table 2 for specific material information.

[0043] Table 2 Specific material information of the Chimonanthaceae plants used in this example

[0044]

[0045] The detection method is the same as in Example 2.

[0046] See the sequencing results Figure 11 to Figure 19 ,will Figure 11 to Figure 19 Arranged in order from left to right.

[0047] The phylogenetic tree constructed by using the primer pair composed of ycf1bF and ycf1bR to carry out PCR amplification of the specific fragments Figure 20 . The results show that the ycf1b fragment amplified from ...

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Abstract

The invention discloses a specific primer pair for identification of spermatophyte species and applications of the specific primer pair. The invention provides a pair of specific primers consisting of a single stranded DNA (Deoxyribose Nucleic Acid) A and a single stranded DNA B. The single stranded DNA A is 15-40bp and has a DNA fragment same as the DNA fragment shown as a sequence I in a sequence table. The single stranded DNA B is 15-40bp and has a DNA fragment same as the DNA fragment shown as a sequence II of the sequence table. In the invention, with the ycflb gene of a plant to be tested as a template, the DNA fragment obtained by PCR (Polymerase Chain Reaction) amplification with the specific primer pair is also protected. The DNA fragment can be used for assisting identification of the spermatophyte species. The specific primer pair can be used for developing general kits, effectively identifying land plant species, and promoting the development of plant DNA bar codes, contributing to social progress.

Description

Technical field [0001] The invention relates to a specific primer pair for identifying seed plant species and its application. Background technique [0002] Many social occasions need to know the species names of materials, such as medicinal materials, samples to be inspected, seeds, wood, etc., because only knowing the species names can people understand the materials. However, in many occasions, general morphological and anatomical features cannot accurately identify species, and higher resolution and more accurate genetic information must be used. Using DNA barcode technology to identify species is becoming a routine technology, and the US Food and Drug Administration has listed DNA barcode technology as one of the applied technologies. To make DNA barcode technology truly serve the society, it is imperative to reduce technical difficulty and cost, and improve identification capabilities. Finding the right DNA barcode is the sacred mission of scientists. For plants, althoug...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/68
Inventor 周世良董文攀徐超
Owner INST OF BOTANY CHINESE ACAD OF SCI
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