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Soybean cleistogamous flower molecular marker and application thereof

A molecular marker, soybean technology, applied in the direction of DNA/RNA fragment, microbial determination/inspection, recombinant DNA technology, etc., can solve the problem that soybean application has not made substantial progress, achieve excellent sensitivity and specificity, and prevent false positives Effect

Inactive Publication Date: 2020-09-15
INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Male sterility and seed sterility are not applicable to crops that reproduce by sexual reproduction; gene division and gene deletion have only been verified in tobacco; although apomixis is widely present in the plant world without normal fertilization A special mode of reproduction of seeds, but its application in soybeans has not yet made substantial progress

Method used

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  • Soybean cleistogamous flower molecular marker and application thereof
  • Soybean cleistogamous flower molecular marker and application thereof
  • Soybean cleistogamous flower molecular marker and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] A method for obtaining molecular markers of soybean atresia, including the following steps:

[0038] (1) To construct F with normal flower open soybean variety Ke1050 as the female parent and closed flower variety N639 as the male parent 2 Separated populations; field phenotypic identification results show that Ke1050 is a flower flag petal, which spreads out after pollination, and N639 is a flower flag petal, and the wing petals are closed for pollination. The qualitative statistics (open flower, closed flower) of the flower opening habits of the F2 population are carried out. Genetic analysisχ 2 =1.202 0.05 = 3.84, indicating that the atretic flower phenotype is controlled by a pair of recessive nuclear genes;

[0039] (2) Extract the parent and its F 2 Population leaf genomic DNA; use CTAB method to extract parents and F 2 The genomic DNA of the leaves of the segregated population of generations. Take an equal amount of mixing leaves in a 2mL centrifuge tube, put them int...

Embodiment 2

[0096] The process of identifying soybean atresia flower traits by molecular markers:

[0097] Extract the genomic DNA of the soybean to be tested, and use the genomic DNA as a template to carry out PCR amplification of SNP2 and SNP3 with primers (PCR reaction program: 95℃5min; 95℃20S, 58℃30S, 72℃30S, 34 cycles ;72℃5min.), take 1μL PCR product, Cutsmart Buffer 1μL, restriction enzyme (20,000U / mL) 0.2μL, add 7.8μL water, the total volume is 10μL, centrifuge and mix, SNP2 digestion at 65℃ for 2h, SNP3 was digested at 37°C for 2h. Then, 6% non-denaturing polyacrylamide gel electrophoresis and silver nitrate staining were performed sequentially. Then make the following judgments:

[0098] Using the genomic DNA of the soybean N639 and grams 1050 to be tested as a template, the specific primer SNP2 is used to perform PCR amplification. If a 342bp fragment is obtained, the flower opening habit of the soybean to be tested is or the candidate is a closed flower, If a 369bp fragment is ob...

Embodiment 3

[0105] Example 3 Primer Sensitivity Detection

[0106] Method: Use primer pair SNP2 and primer pair SNP3 to amplify parental DNA of different concentrations. The DNA concentrations of N639 and g 1050 are set to 100ng / μL, 80ng / μL, 60ng / μL, 40ng / μL, 20ng / μL, 10ng / μL, 5ng / μL, 2ng / μL, 1ng / μL, 0.5ng / μL, 0.2ng / μL, 0.1ng / μL.

[0107] PCR amplification system: DNA at the above concentration, primers (F+R) 1.5μL, Buffer 2μL, dNTP 1.5μL, EasyTaq enzyme 0.2μL, sterile water dilute to 20μL.

[0108] Reaction procedure: 95°C for 5min, 95°C for 20s, 58°C for 30s, 72°C for 30s, 72°C for 5min, 34 cycles.

[0109] The result is Figure 5 with Image 6 Shown.

[0110] It can be seen that the minimum amplification concentration of primer pairs SNP2 and SNP3 is 20ng / μL.

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Abstract

The invention discloses a soybean cleistogamous flower molecular marker. The molecular marker comprises a DNA molecule SNP2 obtained by using soybean genome DNA as a template and performing amplification by using a SNP2 primer pair, and a DNA molecule SNP3 obtained by using the soybean genome DNA as a template and performing amplification by using a SNP3 primer pair, wherein the SNP2 primer pair and the SNP3 primer pair are as shown in SEQ ID NO.1-SEQ ID NO.4. A method of map-based cloning is used to locate a cleistogamous flower trait decisive base sequence in cleistogamous flower soybean genome, and perform cloning, according to the cloning result, whether the trait of soybean is the cleistogamous flower is determined, and then the soybean is planted, so that the plant maintains a pure breed by avoiding the interference of foreign pollens, and the gene flow is controlled.

Description

Technical field [0001] The invention relates to the field of biotechnology, and more specifically to soybean atresia molecular markers and their applications. Background technique [0002] With the ever-increasing population, Jonathan and Tilman and others predict that current crop production must be doubled by 2050 in order to meet the food consumption demand brought about by world population growth. In order to meet this demand, crop production needs to grow at a rate of at least 2.4% per year. Cultivated soybean [Glycine max (L.) Merr.] belongs to Leguminosae (Leguminosae), Papilionate, Glycine, Soja sub-genus annual herb, is a plant protein in people's lives, The main source of oil is also an important feed crop, which accounts for about 56% of the global edible oil production. Ray et al. mentioned that the current annual increase rate of soybean production is about 1.3%, which is far from reaching the expected requirements. Therefore, finding an efficient breeding strategy...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12N15/11
CPCC12Q1/6895C12Q2600/156C12Q2600/13
Inventor 邱丽娟张勇洪慧龙孙如建
Owner INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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