PCR/ LDR (polymerase chain reaction/ ligase detection reaction) molecular marker and method for authenticating Oryza sativa L. nitrate transport protein gene NRT1. 1B genotype

A NRT1.1B, nitrate transport technology, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve problems such as unfavorable high-throughput analysis, cumbersome detection process, etc., to achieve large applications Value, Ease of Use

Inactive Publication Date: 2020-07-03
SHANGHAI ACAD OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] The technical problem of the present invention: the present invention aims at the relatively cumbersome detection process of the traditional molecular marker technology based on PCR and electrophoresis technology, which is not conducive to the high-throughput analysis of many samples. A SNP site in the 980th position of the coding re

Method used

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  • PCR/ LDR (polymerase chain reaction/ ligase detection reaction) molecular marker and method for authenticating Oryza sativa L. nitrate transport protein gene NRT1. 1B genotype
  • PCR/ LDR (polymerase chain reaction/ ligase detection reaction) molecular marker and method for authenticating Oryza sativa L. nitrate transport protein gene NRT1. 1B genotype
  • PCR/ LDR (polymerase chain reaction/ ligase detection reaction) molecular marker and method for authenticating Oryza sativa L. nitrate transport protein gene NRT1. 1B genotype

Examples

Experimental program
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Effect test

Embodiment 1

[0043] 1. Rice material

[0044] Shenfan 14 is the restorer line of the three-line hybrid japonica rice Huayou 14, and 9311 is the super hybrid rice parent indica variety. my country completed the draft genome of 9311 in 2002, and found that Shenfan 14 and 9311NRT1.1B genes are in the coding region by sequencing The 980th bases are C and T respectively, indicating that Shenfan 14 and 9311 carry japonica and indica NRT1.1B ( figure 1 ), the F1 used was derived from the cross between Shenfan 14 and 9311.

[0045] 2. Rice genomic DNA extraction:

[0046] Shenfan 14, 9311 and their hybrid F1 generation rice plants each took about 50 mg of leaves, cut them into pieces with scissors, put them into a 2ml centrifuge tube, and added 600 μl of 1.5×CTAB solution (1.5% CTAB, 75mM Tris-HCl, 15mM EDTA, 1.05 M NaCl, PH8.0) and a steel ball with a diameter of 5 mm, vibrated and ground at a frequency of 70 Hz for 90 s on a fast grinding machine; the ground sample was incubated in a water bath...

Embodiment 2

[0064] The rice samples used in Example 2 were 48 strains from the F2 population of Shenfan 14 and 9311 hybrids, and other detection steps and methods were the same as in Example 1. The test results are shown in Table 1.

[0065]

[0066]

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Abstract

The invention relates to a PCR/ LDR (polymerase chain reaction/ ligase detection reaction) molecular marker and a method for authenticating an Oryza sativa L. nitrate transport protein gene NRT1. 1B genotype, and belongs to the technical field of Oryza sativa L. breeding. The molecular marker disclosed by the invention comprises one pair of PCR primers and three LDR probes, wherein the forward primer sequence of the PCR primer pair is disclosed in SEQ ID NO. 1, and the reverse primer sequence of the PCR primer pair is disclosed in SEQ ID NO. 2; and the LDR probes include a fluorescence labeling probe Probe-FAM of which the sequence is disclosed in SEQ ID NO. 3, an indica type allele specific probe Probe-ind of which the sequence is disclosed in SEQ ID NO. 4 and a japonica-type allele specific probe Probe-jap of which the sequence is disclosed in SEQ ID NO. 5. According to the method, an Oryza sativa L. variety or the nitrate transport protein gene NRT1. 1B genotype in each strain in abreeding population can be accurately and quickly authenticated, and in addition, the high-throughput detection of multiple samples can be realized.

Description

technical field [0001] The invention relates to the technical field of rice breeding, in particular to a PCR / LDR molecular marker and method for identifying rice nitrate transporter gene NRT1.1B genotype. Background technique [0002] Asian cultivated rice includes two subspecies of indica and japonica, which have been greatly differentiated in terms of morphology, development and physiology, and ecological environment adaptation. The study found that indica rice has higher nitrogen use efficiency than japonica rice, and its yield is relatively higher. [0003] In 2015, Chinese scientists found that the difference in nitrogen utilization efficiency between indica and japonica rice was caused by the differentiation of the nitrate transporter gene NRT1.1B. Sequence analysis showed that the NRT1.1B gene of indica rice and japonica rice had a base variation at position 980 in the coding region, which was T in indica rice and C in japonica rice. Studies have shown that introduc...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12Q1/6869C12N15/11
CPCC12Q1/6895C12Q1/6869C12Q2600/13C12Q2600/156C12Q2531/113C12Q2563/107C12Q2561/125
Inventor 储黄伟曹黎明程灿涂荣剑牛付安周继华孙滨
Owner SHANGHAI ACAD OF AGRI SCI
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