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Molecular marker SIsv1363 closely linked with Setaria italica L. Beauv. leaf color gene

A technology of molecular markers and leaf color, applied in the field of molecular biology, can solve problems such as no literature reports

Inactive Publication Date: 2012-09-26
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, there are basically no literature reports on the study of the millet leaf color gene and its closely linked molecular markers

Method used

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  • Molecular marker SIsv1363 closely linked with Setaria italica L. Beauv. leaf color gene
  • Molecular marker SIsv1363 closely linked with Setaria italica L. Beauv. leaf color gene
  • Molecular marker SIsv1363 closely linked with Setaria italica L. Beauv. leaf color gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1: Construction of millet F2 generation segregation population

[0039] Male parent: Kangnabujing, high plant type, long and narrow flag leaves, red bristles, red glumes, fertile, greenish leaves, yellow-white pollen, late heading stage. The male parent is Zhang Gu No. 1 seed.

[0040] Female parent: Not resistant to Nabujing, short plant type, short and wide flag leaves, green setae, green glumes, partly sterile, yellowish leaf color, brown pollen, early heading stage. The female parent is the seed of millet A2 male sterile line.

[0041] F2 population construction: the male parent and the female parent were crossed to obtain the F1 generation (the leaf color of F1 is greenish), and F1 was self-crossed to obtain F2. Among them, F1 is the No. 3 seed of Zhang Zagu. A total of 480 individual plants of the F2 generation were obtained. The above-mentioned Zhanggu No. 1 seeds, millet A2 CMS seeds and Zhangzagu No. 3 seeds can be found in the Chinese patent applica...

Embodiment 2

[0042] Example 2: Extraction of parental and F1 generation, F2 generation individual genomic DNA

[0043] 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:

[0044] (1) Weigh 1.0g of fresh leaves, cut them into pieces and put them in a mortar, grind them with liquid nitrogen, add 3mL 1.5×CTAB, grind them into a homogenate and transfer them to a 15mL centrifuge tube, then add 1mL 1.5×CTAB into the mortar Rinse 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 during this period.

[0045] Among them, the formula of 1.5×CTAB is as follows (1L):

[0046]

[0047] Add deionized water to make up to 1 L, and add mercaptoethanol with a final concentration of 0.2% (2 ml) before use.

[0048] (2) After cooling to room temperature, an equal volume of chloroform / isoamyl alcohol (24:1) ...

Embodiment 3

[0053] Example 3: Preparation of Molecular Markers

[0054] Using the genomic DNA of the male parent, the F1 generation, or the F2 generation extracted in Example 2 as a template, PCR amplification was performed with a pair of molecular marker amplification primers (Seq ID No.2 and Seq ID No.3).

[0055] The PCR reaction system is as follows:

[0056]

[0057]

[0058] The PCR reaction procedure is as follows:

[0059] 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.

[0060] Molecular markers are obtained through the above amplification process, and the amplification product is preferably purified after amplification. Sequenced after purification, the result is shown in Seq ID No.1.

[0061] Those skilled in the art can understand that the molecular marker can also be ob...

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Abstract

The invention belongs to the field of molecular biology, relates to a molecular marker and especially relates to a molecular marker SIsv1363 closely linked with a Setaria italica L. Beauv. leaf color gene. The molecular marker SIsv1363 has a sequence shown in the Seq ID NO.1. The invention also relates to primers for amplification of the molecular marker SIsv1363, a use of the molecular marker SIsv1363 and the primers in Setaria italica L. Beauv. leaf color gene mapping or Setaria italica L. Beauv. genetic breeding, and a Setaria italica L. Beauv. breeding method. The molecular marker SIsv1363 links a genomic DNA sequence and the Setaria italica L. Beauv. leaf color gene, and is conducive to establishment of a Setaria italica L. Beauv. molecular marker auxiliary breeding system. A genetic close linkage distance between the molecular marker SIsv1363 and the Setaria italica L. Beauv. leaf color gene is 0.15cM. In Setaria italica L. Beauv. breeding practices and resource and variety identification, the simple, fast and high throughput utilization of the molecular marker SIsv1363 and the primers for amplification of the molecular marker SIsv1363 can be realized.

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 the millet leaf color gene. The invention also relates to primers for amplifying the molecular markers, and the use of the molecular markers and primers in the location of millet leaf color genes or millet genetics and breeding. Background technique [0002] my country is the country of origin of millet (Setaria italica L.Beauv.) and the concentrated planting country of millet in the world. Millet occupies an important position in my country's national economy and social production, and is of great significance to the construction of dry farming ecological agriculture. Therefore, it is particularly important to accelerate the breeding process of millet. Since millet is only a regionally important crop, the current research methods and methods related to millet are relatively backward. How to apply advanced...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12N15/63C12N5/10C12Q1/68
Inventor 张耕耘全志武夏秋菊张厚宝
Owner 深圳华大基因农业控股有限公司
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