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A primer set developed based on the transcriptome sequence of Macrobrachium rosenbergii and its application

A technology of transcriptome sequence and Macrobrachium rosenbergii, applied in the field of SSR primer sets, can solve the problems of limiting the analysis of genetic diversity of Macrobrachium rosenbergii, and achieve the effect of enriching marker sources

Active Publication Date: 2021-08-06
ZHEJIANG INST OF FRESH WATER FISHERIES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are relatively few reports on the development of SSR primers and related genetic studies of Macrobrachium rosenbergii, which greatly limits the genetic diversity analysis of Macrobrachium rosenbergii and other related research.

Method used

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  • A primer set developed based on the transcriptome sequence of Macrobrachium rosenbergii and its application
  • A primer set developed based on the transcriptome sequence of Macrobrachium rosenbergii and its application
  • A primer set developed based on the transcriptome sequence of Macrobrachium rosenbergii and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Transcriptome sequencing

[0034] Randomly selected ovarian tissue from 3 healthy individuals of Macrobrachium rosenbergii, using ReagentInvitrogen extracts RNA. Take equal amounts of RNA and mix them, construct a genomic library and sequence, and entrust Shanghai Lingen Biotechnology Co., Ltd. to complete the sequencing using the Illumina HiSeq 2500 sequencing platform.

[0035] Table 1 Statistics of transcriptome data of Macrobrachium rosenbergii

[0036]

Embodiment 2

[0037] Screening and identification of embodiment 2 SSR sequences

[0038] Use MISA v1.0 (http: / / pgrc.ipk-gatersleben.de / misa) to perform SSR detection on Unigene, and the corresponding minimum number of repeats for each Unit size is the number of repeats when a single nucleotide is used as the repeating unit At least 12 times can be detected; when the repeat unit is dinucleotide, the minimum repeat number is 6; when the repeat unit is trinucleotide, the repeat number is at least 5; when the repeat unit is tetranucleotide, the repeat number is at least 5; When a unit is used, the repeat number is at least 5; when a pentanucleotide is used as a repeat unit, the repeat number is at least 4; when a hexanucleotide is used as a repeat unit, the repeat number is at least 4.

[0039] Finally, statistical analysis was performed on the occurrence frequency of SSR, the type of repeating primitive, the number of repeats and its polymorphism, and the results are shown in Table 2.

[0040...

Embodiment 3

[0045] Design and synthesis of embodiment 3SSR primers

[0046] According to the sequences and sites detected in Example 2, use primer3 (https: / / sourceforge.net / projects / primer3 / ) to design SSR primers in batches. Primer design criteria are: there is no SSR in the primer sequence; the sequence is in the DNA conservative sequence region; the length is between 15-30bp; there is no complementary sequence in the upstream and downstream primers; there is no complementary sequence in the primer itself; the primer annealing temperature (Tm) is 45 Between -60°C; the difference between the Tm values ​​of the upstream and downstream primers is ≤5°C; the GC content is between 40% and 60%; the product size is 100-400bp. Primers for 2-6 nucleotide repeat motifs were synthesized by Shanghai Sangon Bioengineering Co., Ltd. and used for SSR primer screening.

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Abstract

The invention discloses a primer set developed based on the transcriptome sequence of Macrobrachium rosenbergii and its application, belonging to the technical field of molecular markers. The invention provides an SSR molecular marker primer set for the analysis of Macrobrachium rosenbergii genetic diversity, including 8 pairs of primers; the invention also provides a method for obtaining the SSR molecular marker primer set; the invention also provides the SSR molecule The application of the marker primer set in the genetic diversity analysis of Macrobrachium rosenbergii, the primer set can effectively carry out genetic diversity analysis and population clustering analysis on Macrobrachium rosenbergii germplasm resources. The 8 pairs of primers in the present invention have the advantages of rich polymorphism, stable amplification, good repeatability, convenient statistics, etc., and enrich the method for genetic diversity analysis of Macrobrachium rosenbergii germplasm resources.

Description

technical field [0001] The invention relates to the field of SSR primer sets, in particular to a primer set developed based on the transcriptome sequence of Macrobrachium rosenbergii and its application. Background technique [0002] Macrobrachium rosenbergii (Macrobrachium rosenbergii), also known as bighead shrimp and Malaysian prawn, belongs to the phylum Arthropoda, class Mollusca, order Decapod, family Longbrachidae, genus Macrobrachium, native to Southeast Asia, and is an important species in the world at present. It is one of the freshwater farmed shrimps and the largest freshwater shrimp in the world. It is widely distributed in aquatic ecosystems, distributed in both freshwater areas (such as rivers and lakes) and brackish water areas (such as estuaries), and is sensitive to changes in physical and chemical indicators in the water environment. It has the characteristics of fast growth, wide feeding habits, short growth cycle and strong disease resistance. Since the...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6888C12Q1/6858C12N15/11G16B20/20
CPCC12Q1/6858C12Q1/6888C12Q2600/156G16B20/20C12Q2531/113C12Q2565/125C12Q2563/107
Inventor 李喜莲陈雪峰顾志敏高强张宇飞慎佩晶徐洋蒋文枰徐宾朋程海华彭菲黄振远
Owner ZHEJIANG INST OF FRESH WATER FISHERIES
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