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Molecular marker and application of intron 2-based identification of barley semi-dwarf multi-tiller gene hvhtd

A molecular marker, semi-dwarf technology, applied in the field of agricultural biology, can solve the problems of increasing fertilizer input, increasing production cost, environmental pollution, etc., and achieving the effect of improving selection efficiency and speeding up progress

Active Publication Date: 2022-03-29
ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Semi-dwarf crops increase the lodging resistance of crops, but at the same time increase the input of fertilizers, leading to problems such as increased production costs and environmental pollution

Method used

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  • Molecular marker and application of intron 2-based identification of barley semi-dwarf multi-tiller gene hvhtd
  • Molecular marker and application of intron 2-based identification of barley semi-dwarf multi-tiller gene hvhtd
  • Molecular marker and application of intron 2-based identification of barley semi-dwarf multi-tiller gene hvhtd

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1. Design of molecular marker SNP62

[0044] 1), test materials: mutant htd and its wild-type Hua30 (ie, Hua30, Hua30);

[0045] Mutant htd was obtained by EMS mutagenesis: using 0.03mol / L EMS to treat malting barley variety Hua 30, from 1710 individual plants M 2 According to the phenotype observation in the generation, a semi-dwarf and multi-tiller mutant was obtained. After multi-generation observation, it was found that the semi-dwarf and multi-tiller mutant was inherited stably, and we named it htd. Compared with its wild-type flower 30, its external manifestations are: the leaves become thinner and narrower, the leaf color becomes darker, the plant height becomes shorter, and the number of tillers increases.

[0046] A gene Hvhtd controlling semi-dwarf and multi-tiller traits was cloned from the mutant htd, and the nucleotide sequence of the gene is shown in SEQ ID NO:1. The protein encoded by the semi-dwarf multi-tiller gene Hvhtd has the amino acid seq...

Embodiment 2

[0054] Example 2. Using molecular markers to identify sequence differences between mutant htd and its wild-type flower 30:

[0055] 1), DNA extraction: Take 100 mg of barley leaves, and use the CTAB method to extract DNA. The specific steps are as follows:

[0056] ① Grind the sheared leaves in liquid nitrogen and put them into 1.5ml centrifuge tubes, add 600 μL of CTAB to each tube and place in a warm water bath at 65°C for 50-60 minutes (take out every 10 minutes and shake gently);

[0057] ②Add 600 microliters of chloroform / isoamyl alcohol (24:1) and mix well for 15 minutes. Slowly, weigh in pairs, and after balancing, centrifuge at 9600 rpm for 10 minutes at 4°C;

[0058] ③Pipe the supernatant to another 1.5ml new centrifuge tube, add 2 times the volume of absolute ethanol and mix slightly (precipitate in -20°C refrigerator for 30-60min);

[0059] ③ Pick out the precipitate with a pipette tip, put it in a 1.5ml centrifuge tube, wash it twice with 70% ethanol and air dry; ...

Embodiment 3

[0069] Example 3. Using the molecular marker SNP62 to detect 32 barley germplasm resources (including mutant htd and flower 30, 2 repetitions) according to the method described in Example 2, the barley germplasm resources include wild barley, local varieties and from around the world The barley varieties bred from various places and the F 1 (See Table 1);

[0070] The result is as Figure 4 shown.

[0071] The test results show that the F of htd / Zhepi No. 8 in these materials 1 The hybrid is heterozygous, and the mutant htd is a mutant genotype; other materials are wild-type genotypes, and their phenotypes are also different from the semi-dwarf multi-tiller phenotype of the mutant htd, indicating that they are not Containing the Hvthd mutant gene also proves that this molecular marker can be used in the detection of barley germplasm resources.

[0072] Table 1. Information on 32 barley germplasm resources detected by molecular marker SNP62

[0073]

[0074]

[0075]...

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Abstract

The invention discloses a gene Hvhtd controlling semi-dwarf and multi-tiller traits. The nucleotide sequence of the gene is as described in SEQ ID NO:1. The present invention also provides the protein encoded by the above-mentioned semi-dwarf multi-tiller gene Hvhtd, the amino acid sequence of which is as described in SEQ ID NO:3. The present invention also provides a molecular marker SNP62 for identifying the barley semi-dwarf multi-tiller gene Hvhtd based on intron 2; the molecular marker SNP62 obtained in the present invention can identify whether barley contains the Hvhtd semi-dwarf multi-tiller gene. The invention utilizes the marker to carry out auxiliary selection of semi-dwarf multi-tiller breeding of Hvhtd gene, which can ensure 100% accuracy rate and speed up the breeding progress of barley semi-dwarf multi-tiller breeding.

Description

technical field [0001] The invention relates to a gene marking method for identifying barley semi-dwarf multi-tiller gene Hvhtd and corresponding primers. The method belongs to the field of agricultural biotechnology and can be used for rapid screening of barley semi-dwarf multi-tiller germplasm and molecules containing Hvhtd mutant genes Marker Assisted Breeding. Background technique [0002] Many factors such as population growth, climate change and natural environment damage have led to global food shortages, and how to increase crop yields has become a common concern of breeders. In the 1960s, the application of semi-dwarf genes significantly increased crop yields, which was called the "Green Revolution" (Peng et al., 1999; Monna et al., 2002), and the "Green Revolution Gene" involved Gibberella pathways of metabolism. Semi-dwarf crops increase the lodging resistance of crops, but at the same time, they also increase the amount of fertilizer input, resulting in increas...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/29C12N15/11C07K14/415C12Q1/6895
CPCC12Q1/6895C07K14/415C12Q2600/13C12Q2600/156
Inventor 华为汪军妹朱靖环尚毅巫小建杨建明
Owner ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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