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Molecular marker SIsv1223 closely linked with millet herbicide-resistant gene

A herbicide-resistant gene and molecular marker technology, applied in the field of molecular biology, can solve the problems of lack of herbicide resistance, obstacles to millet production, and inability to apply broad-spectrum herbicides in corn fields, and achieve high-throughput application effects

Active Publication Date: 2012-07-25
深圳华大基因农业控股有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the large number and variety of weeds in corn fields, and the lack of herbicide-resistant varieties in production, broad-spectrum herbicides cannot be used to kill weeds in corn fields, which has become a major problem hindering millet production.

Method used

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  • Molecular marker SIsv1223 closely linked with millet herbicide-resistant gene
  • Molecular marker SIsv1223 closely linked with millet herbicide-resistant gene
  • Molecular marker SIsv1223 closely linked with millet herbicide-resistant gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1: Construction of millet F2 generation segregation population

[0044] The male parent is Zhanggu No. 1: Anti-Nabujing type (high plant type, plant height is about 150cm), long and narrow flag leaves, red bristles, red glumes, fertile, greenish leaf color.

[0045] The female parent is the A2 sterile line: non-resistant Nabujing type (short plant type, plant height about 100cm), short and wide flag leaves, green bristles, green glumes, partly sterile, yellowish leaf color.

[0046] F1 (Zhang Zagu No. 3, partially anti-Nabu net type, plant height of about 130cm) was obtained by crossing the male parent and the female parent.

[0047] The F1 generation was self-crossed to generate the F2 generation population, and a total of 480 individual plants were obtained. 480 individuals of the F2 generation were analyzed according to the method for determining the type of anti-Natura, and a total of 360 strains were found to be anti-Natten and partly anti-Nart (includin...

Embodiment 2

[0048] Example 2: Extraction of parental and F1 generation, F2 generation individual genomic DNA

[0049] Genomic DNA of the parents, the F1 generation, and 480 F2 generation individuals in Example 1 were extracted by the CTAB method, and the specific methods were as follows:

[0050] (1) Weigh 1.0g of fresh leaves, cut them into pieces and put them in a mortar, grind them with liquid nitrogen, add 3ml of 1.5×CTAB, grind them into a homogenate and transfer them to a 15ml centrifuge tube, then add 1ml of 1.5× CTAB into the mortar Rinse with CTAB and transfer to a centrifuge tube. After mixing, place in a water bath at 65°C for 30 minutes, and shake slowly from time to time.

[0051] Among them, the formula of 1.5×CTAB is as follows (1L):

[0052] CTAB 15g

[0053] 1M Tris.Cl (pH 8.0) 75ml

[0054] 0.5M EDTA 30ml

[0055] NaCl 61.4g

[0056] Add deionized water to make up to 1 L, and add mercaptoethanol with a final concentration of 0.2% (2 ml) before use.

[0057] (2) ...

Embodiment 3

[0062] Example 3: Preparation of Molecular Markers

[0063] Taking the Genomic DNA of the anti-Narth net type or part of the anti-Natur net type in the male parent, F1 generation, or F2 generation extracted in Example 2 as a template, with molecular marker primers (SEQ ID NO: 2 and SEQ ID NO: 3 ) for PCR amplification.

[0064] The PCR reaction system is as follows:

[0065] Sterile water 20.2μl

[0066] 10*Buffer (with Mg 2+ ) 2.5 μl

[0067] dNTPs (25mM) 0.15μl

[0068] Taq enzyme (5U / μl) 0.15μl

[0069] Forward primer 0.5μl

[0070] Reverse primer 0.5μl

[0071] Template 1.0μl

[0072] Total volume 25μl

[0073] The PCR reaction procedure is as follows:

[0074] Pre-denaturation at 94°C for 5 minutes; denaturation at 94°C for 30 seconds, annealing at 60°C for 30 seconds, extension at 72°C for 40 seconds, and 35 cycles; final extension at 72°C for 3 minutes. PCR amplification products can be stored at 4°C.

[0075] The amplified product is purified to obtain mo...

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Abstract

The invention belongs to the field of molecular biology and relates to a molecular marker, in particular to a molecular marker closely linked with a millet herbicide-resistant gene. The nucleotide sequence of the molecular marker is shown as SEQID NO:1, or the molecular marker is a deoxyribonucleic acid (DNA) segment which contains the nucleotide sequence shown as SEQ ID NO:1 in a millet genome. The invention also relates to a primer of the molecular marker, the application of the molecular marker to millet herbicide-resistant gene positioning or millet genetic breeding, a millet herbicide-resistant gene positioning method and a millet breeding method. In the invention, a molecular marker SIsv1223 closely linked with the millet herbicide-resistant gene is found, so that a millet genome DNA sequence and the millet herbicide-resistant gene are linked with each other and the establishment of a millet molecular marker-assisted breeding system is further facilitated.

Description

technical field [0001] The invention belongs to the field of molecular biology and relates to a molecular marker, in particular to a molecular marker closely linked with a millet resistance gene. The present invention also relates to the primer of the molecular marker, the use of the molecular marker in the mapping of the herbicide resistance gene of millet or the genetic breeding of millet, a method of mapping the herbicide resistance gene of millet, and a method of breeding of millet. Background technique [0002] Millet (Setaria italica L.Beauv.) is an important food crop originating in my country. It is mainly planted in the north of my country and plays an important role in national food production and food security. Millet is a diploid self-pollinated crop, and its genome is small, about 470Mb. These characteristics make it very suitable as the object of genome research. [0003] The use of herbicides to kill weeds has become an important measure to effectively contro...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12N15/63C12N5/10C12N15/10C12Q1/68
Inventor 张耕耘全志武夏秋菊倪雪梅
Owner 深圳华大基因农业控股有限公司
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