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Mulberry EST-SSR (simple sequence repeat) molecular markers, and core primer group and application thereof

A technology of molecular markers and core primers, applied in biochemical equipment and methods, recombinant DNA technology, microbial measurement/inspection, etc., can solve problems such as limiting mulberry molecular genetics research, and achieve clear amplification results and good repeatability , Amplify the effect of stability

Active Publication Date: 2017-09-08
SERICULTURE & AGRI FOOD RES INST GUANGDONG ACAD OF AGRI SCI
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AI Technical Summary

Problems solved by technology

EST-SSR markers can be developed from expressed sequence tag libraries or transcriptome sequencing data, but currently the number of EST-SSR molecular markers in mulberry trees is not enough to meet the needs of germplasm genetic diversity, germplasm identification, genetic map construction and molecular Researches such as marker-assisted breeding have limited the molecular genetics research of mulberry trees, and more EST-SSR markers with high polymorphism need to be developed, which has important utilization value for genetic research and breeding practice

Method used

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  • Mulberry EST-SSR (simple sequence repeat) molecular markers, and core primer group and application thereof
  • Mulberry EST-SSR (simple sequence repeat) molecular markers, and core primer group and application thereof
  • Mulberry EST-SSR (simple sequence repeat) molecular markers, and core primer group and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0052] Example 1: Development of EST-SSR core primer set based on mulberry transcriptome sequence

[0053] 1. Design and synthesis of EST-SSR primers in the mulberry transcriptome sequence

[0054] The MISA software was used to search all the SSR sites in the mulberry unigenes database of the mulberry transcriptome. The length of the SSR motif repeating unit retrieved was 2 to 6 nucleotides, which were set to 2, 3, 4, 5, The minimum number of search repetitions for 6 nucleotides is 6, 5, 5, 4, and 4 times, and the length of the flanking sequence of the SSR site is ≥ 150 bp. According to the searched SSR site and flanking sequence, use Primer3v2.3.4 (http: / / primer3.sourcego-rge.net) software to design specific primers, the design criteria are: the primer length is 18-25bp, the annealing temperature is 57-63°C, the GC content is 40-70%, and the length of the PCR product is 100- 300bp, select SSR sites that can be compared with the NCBI non-redundant protein database to synthes...

Embodiment 2

[0078] Embodiment 2: the application of mulberry EST-SSR core primer set

[0079] 1. DNA extraction

[0080] Take the improved CTAB method to extract the DNA of the genomes of the above-mentioned 21 mulberry varieties (including 11 Guangdong mulberry varieties, 5 white mulberry germplasms, 4 Mengmulberry germplasms and 1 long-fruit mulberry germplasms); the specific steps are the same as in the examples 1 step 2.

[0081] 2. PCR amplification

[0082] Using the DNA extracted in step 1 as a template, use the 6 pairs of EST-SSR core primers developed in Example 1 (see Table 1), divide them into 3 groups according to the combination in Table 1, and perform multiplex PCR.

[0083] The three sets of EST-SSR molecular marker core primer set PCR reaction system are as follows:

[0084] Reaction system I: 50ng / μL DNA template 0.5μL, 10×PCR buffer (without Mg 2+ ) 2.5μL, 25mM MgCl 2 2.0 μL, 10 mM dNTPs 0.5 μL, 5U / μL Tap DNA polymerase 0.2 μL, 10 μM MulSSR5053 upstream primer 0.1 ...

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Abstract

The invention discloses mulberry EST-SSR (simple sequence repeat) molecular markers, and a core primer group and application thereof. A large amount of SSR molecular markers are developed by performing transcriptome sequencing and sequence information process on mulberries, 6 groups of polymorphic markers are screened from the SSR molecular markers, and a technological system according with SSR marker fluorescence detection is established. Through amplification and detection on the DNA of 21 mulberry varieties, the 6 groups of polymorphic markers have rich primer amplification polymorphism and high repeatability, the genetic diversity analysis result is identical to morphological morus plant classification, and the 6 groups of polymorphic markers are new markers existing stably and can be applied to mulberry germplasm resource genetic diversity analysis, variety identification, genetic map construction and molecular marker assistant breeding.

Description

[0001] Technical field: [0002] The invention belongs to the technical field of molecular marker technology development and application, and in particular relates to mulberry EST-SSR molecular marker and its core primer set and application. [0003] Background technique: [0004] Mulberry belongs to the genus Morus L. of the family Moraceae. It is a perennial woody plant for the purpose of harvesting mulberry leaves and raising silkworms. It is one of the important economic crops in my country. my country is one of the important origin centers of mulberry trees. According to statistics, the mulberry germplasm collected and preserved in my country belongs to 15 mulberry species and 3 varieties, with more than 3,000 germplasm resources. It is the country with the largest amount of mulberry germplasm preservation in the world. . After long-term natural and artificial selection, mulberry germplasm resources have rich genetic diversity. In the process of collection, preservation an...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6895C12Q2600/156C12Q2600/16
Inventor 王振江唐翠明戴凡炜罗国庆肖更生邝哲师黄静李智毅
Owner SERICULTURE & AGRI FOOD RES INST GUANGDONG ACAD OF AGRI SCI
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