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Indel molecular marker of asparagus bean commonly used in different legume crops and development method and application thereof

A molecular marker, bean technology, applied in biochemical equipment and methods, microbial determination/inspection, recombinant DNA technology, etc., to save costs, improve accuracy and selection efficiency

Active Publication Date: 2019-07-12
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Marker-assisted breeding requires high-density genetic linkage maps, but studies using cowpea transcriptome data to develop InDel markers have not been reported

Method used

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  • Indel molecular marker of asparagus bean commonly used in different legume crops and development method and application thereof
  • Indel molecular marker of asparagus bean commonly used in different legume crops and development method and application thereof
  • Indel molecular marker of asparagus bean commonly used in different legume crops and development method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1 Extraction of Plant Material Leaf Genomic DNA

[0053] The seeds of bean materials were sterilized with 1% hypochlorous acid solution for 10 minutes, and then rinsed with distilled water for 5 times. Sow Su Zi 41, Su Cow 1419, 10 cowpea resources and 34 different bean crops in plastic cups filled with vermiculite (the lower diameter is 7 cm, the upper diameter is 9 cm, and the height is 15 cm), and then covered with 2 cm of vermiculite ;

[0054] DNA was extracted from Suzi 41 and Su cowpea 1419, 10 cowpea resources and 34 different bean plant leaves.

[0055] The total DNA of leaves was extracted by the CTAB method, and the specific steps were as follows:

[0056] (1) Take 0.1 gram of fresh leaf sample, add 650 μL of extract solution (1.4M NaCl, 100mM Tris, pH 8.0, 20mM EDTA, pH 8.0, 2% CTAB) to grind, then put it into a 1.5mL centrifuge tube and place it in a constant temperature water bath at 65°C 60 minutes, during which mixed 2-3 times;

[0057] (2) A...

Embodiment 2I

[0060] Example 2 Development of InDel molecular marker primers and screening of polymorphisms

[0061] (1) Use the lllumina Hiseq2000 platform to sequence the transcriptomes of cowpea varieties Suzi 41 and Su cowpea 1419, and perform BLAST comparison based on the transcriptome assembly sequences of Suzi 41 (salt-tolerant) and Su cowpea 1419 (salt-sensitive), and screen for Insertion / deletion (InDel) transcripts.

[0062] (2) Select sequences with relatively large differences in the sequence length of the insertion / deletion (InDel), and compare parameters: a. Alignment sequence ≥ 300bp, b. Insertion / deletion length ≥ 3bp, c. A pair of sequences of Suzi 41 and Suyu 1419 1. Alignment, d. The similarity of the two aligned sequences is greater than 90%. Then use Primer5.0 software to design InDel labeled primers, and the length of the amplified product is about 200bp.

[0063] (3) 845 pairs of InDel primers were synthesized by a biological company for polymorphism screening. The...

Embodiment 3

[0087] The feasibility of the developed InDel markers was analyzed using 10 cowpea resources (including long cowpea and rice cowpea). 107 pairs of marker primers were randomly selected from the InDel markers designed in Example 2, and PCR amplification and electrophoresis analysis were performed on 10 cowpea resources respectively, wherein the DNA extraction was the same as in Example 1, and the methods such as PCR amplification and electrophoresis were the same as in Example 2. The material names of 10 cowpea resources are shown in Table 2 (not limited to the materials in Table 2). After electrophoresis, the amplification rate was counted.

[0088] Table 2

[0089]

[0090]The results are shown in Table 3. There were 93 markers (87%) that could amplify bands in 10 cowpea materials, and 14 markers (13%) could only amplify bands in some cowpea materials. Among these markers, 98 markers (91.6%) had polymorphisms in two or more cowpea materials, and 9 markers (8.4%) had no p...

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Abstract

The invention discloses an Indel molecular marker of an asparagus bean commonly used in different legume crops and a development method and application thereof. The InDel molecular marker is preparedby using a legume DNA as a template and utilizing the following one or several sets of primer pairs as shown in SEQ ID NO.1-24 for PCR amplification. The method develops the InDel marker by performingtranscriptome sequencing on Suzi 41 and Susie 1419, performing BLAST alignment on transcriptome assembly sequences and screening for transcripts with insertion / deletion. After analyzing the feasibility of the developed InDel label, the universality of the different legume materials are analyzed by using the effective amplification marker obtained by screening. The InDel molecular marker has versatility on different legume materials, and can be used for genetic map construction, QTL localization, genetic diversity analysis and molecular marker assisted selection of the legume materials in thefuture.

Description

technical field [0001] The invention relates to the development and application of InDel molecular marker primers, in particular to a long cowpea InDel molecular marker universal in different bean crops and its development method and application. Background technique [0002] In the study of molecular markers of cowpea, DNA molecular markers based on PCR (Polymerase chain reaction, polymerase chain reaction) are mostly used. Using dominant molecular markers, such as RAPD, ISSR, etc., a batch of reproducible, stable and reliable molecular marker primers have been screened (Chen Chanyou et al., 2008; Chen C Y, et al., 2010). In recent years, the application of co-dominant molecular markers such as SSR and SNP has been favored. In 2009, 1 375 SNP sites based on EST sequences were first reported (Muchero W, et al., 2009). Li et al. (2001) took the lead in developing 27 SSR markers for cowpea research. Gupta et al. designed and screened 102 SSR markers from the cowpea unigenes...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12N15/11
CPCC12Q1/6895C12Q2600/156
Inventor 许文静张红梅陈景斌袁星星陈华涛刘晓庆
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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