Rice flooding-tolerant gene Sub1 co-dominant molecular marker and application

A technology of molecular markers and co-dominance, which is applied in the direction of DNA/RNA fragments, microbial detection/inspection, recombinant DNA technology, etc., can solve problems such as limiting the efficiency of flood-tolerant rice variety breeding, and achieve convenient, quick, and shortened detection procedures. The effect of breeding cycle and low cost

Active Publication Date: 2019-03-15
YUAN LONGPING HIGH TECH AGRI CO LTD +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are few reports on the use of MAS to breed flood-tolerant rice varieties, and most of them are screened using Sub1-linked, dominant or enzyme-cut markers, which limits th

Method used

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  • Rice flooding-tolerant gene Sub1 co-dominant molecular marker and application
  • Rice flooding-tolerant gene Sub1 co-dominant molecular marker and application
  • Rice flooding-tolerant gene Sub1 co-dominant molecular marker and application

Examples

Experimental program
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Effect test

Embodiment 1

[0036] Example 1 Development of Sub1 Gene Co-dominant Molecular Marker

[0037] The rice Sub1 gene is located on chromosome 9. It is a gene cluster composed of Sub1A, Sub1B and Sub1C. Currently, it is known that Sub1A, Sub1B and Sub1C have 2 (or missing), 9 and 7 alleles respectively: SubA1 ~2, Sub1B1~9, Sub1C1~7, rice flood tolerance traits are mainly controlled by Sub1A-1, and Sub1A-1 only appears in the combination with Sub1C-1, therefore, by analyzing the Sub1A and Sub1C in different rice materials The sequence was amplified and sequenced, and DNAMAN and other software and NCBI database and other sequence analysis tools were used for local and online sequence comparison analysis, and a SNP site was obtained in the Sub1A gene interval, in which the flood tolerance allele Sub1A-1 was G, Allele Sub1A-2 of intolerance to flooding is C (eg figure 1 A); A specific nucleotide sequence was found in the Sub1C gene interval, wherein the Sub1C-1 allele was 5'-GCCGTCG-3', and the res...

Embodiment 2

[0038] The detection primer (ARMS-PCR primer) design of embodiment 2Sub1 gene co-dominant molecular marker

[0039] For the two Sub1 gene-specific molecular primers developed in Example 1, ARMS-PCR detection primers were designed, and the principles of primer design are as follows:

[0040] First, design primers for specific amplification of the Sub1A-1 allele for the specific SNP site in the Sub1A gene, use the C base complementary to the G base in the SNP as the 3' end of the primer to design the reverse primer Sub1A-R, and combine 3 The second base at the ' end is changed to G, and then the forward primer Sub1A-F paired with it is designed upstream, and the primers Sub1A-R and Sub1A-F (SEQ ID NO:2-3) can only be specifically amplified The Sub1A-1 allele type produced a 586bp band, while the Sub1A-2 allele type and the Sub1A gene deletion type amplified no band.

[0041] Secondly, design specific amplification intolerance allelic primers for the specific nucleotide sequence...

Embodiment 3

[0045] Example 3 Establishment of detection method for rice sub1 specific molecular marker for submergence tolerance gene

[0046] According to the detection primers of two molecular markers of Sub1 designed in Example 2, the reaction program and reaction system of PCR were designed, and through continuous optimization, the following reaction program and system were determined:

[0047] PCR reaction system (10 μL): 10×PCR reaction buffer 1 μL, 10 mM dNTP 0.8 μL, 4 primers (10 μM) 0.15 μL, 0.1 μL Taq DNA polymerase (2.5U / ul); 2 μL DNA template, double-distilled Water to make up the balance.

[0048] The PCR reaction conditions were: pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 30 seconds, annealing at 58°C for 30 seconds, extension at 72°C for 45 seconds, a total of 35 cycles; extension at 72°C for 8 minutes.

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Abstract

The invention provides a rice flooding-tolerant gene Sub1 co-dominant molecular marker and application. The molecular marker comprises an SNP marker and an InDel marker which are separately located onrice No. 9 chromosome genes Sub1A and Sub1C, wherein the polymorphic site of the SNP marker on the gene Sub1A is G/C; and the polymorphic site of the InDel marker on the gene Sub1C is 5'-GCCGTCG-3'/5'-CA-3'. For the molecular marker, specific amplification primers are designed, and Sub1 genotype detection is carried out through PCR amplification. The provided molecular marker and an amplificationprimer thereof for Sub1 can be used for identifying the genotype of rice Sub1, a flooding-tolerant rice resource is bred, the rice flooding-tolerant gene Sub1 co-dominant molecular marker has the advantages of high identification accuracy, simplicity in operation, low cost and the like, the breeding period of the flooding-tolerant rice can be shortened, and the breeding cost is reduced.

Description

technical field [0001] The invention belongs to the fields of molecular biology and plant molecular breeding, and in particular relates to a co-dominant molecular marker and application of rice sub1 sub1 tolerance gene. Background technique [0002] Rice is one of the most important food crops in the world, and more than half of the world's population uses rice as a staple food. During the growth of rice, various adversities may be encountered, affecting its yield and quality, including cold, flooding, drought, pests and diseases and other adversities that will have a serious impact on rice growth. Therefore, research on improving stress resistance is the key to high and stable rice yields. important ways and means. China's rice planting is almost all over the country, with a wide distribution, but it has the characteristics of more and more concentrated in the south and less and scattered in the north. However, due to the impact of global warming, the rainfall in the sout...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/6895
CPCC12Q1/6895C12Q2600/13C12Q2600/156
Inventor 杨远柱邓钊丁文豪王凯石媛媛秦鹏符辰建严天泽
Owner YUAN LONGPING HIGH TECH AGRI CO LTD
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