Method for quickly identifying hybrid of miscanthus floridulus with silvergrass through using SSR molecule markers

A technology of molecular markers and five-jointed awns, which is applied in the direction of biochemical equipment and methods, and microbial measurement/inspection, can solve the problems of long identification time, rapid identification of unfavorable hybrids, and environmental impact of morphological traits. Wide distribution range, simple operation and good repeatability

Inactive Publication Date: 2013-05-01
HUNAN AGRICULTURAL UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Morphological characters are the basis of hybrid identification, especially the performance of some male-specific traits in hybrids, which are important morphological markers for identification of hybrids, but the identification takes a long time and takes up a lot of land resources, and morphological characters are affected by the environment. impact, etc., which is not conducive to the rapid identification of hybrids

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  • Method for quickly identifying hybrid of miscanthus floridulus with silvergrass through using SSR molecule markers
  • Method for quickly identifying hybrid of miscanthus floridulus with silvergrass through using SSR molecule markers
  • Method for quickly identifying hybrid of miscanthus floridulus with silvergrass through using SSR molecule markers

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: Primer Screening

[0034] 1) Extraction of genomic DNA

[0035] Select the Miscanth and Grass from various regions and the obtained artificial hybrid plants of Miscanth and Grass (the experimental materials are stored in the Miscanth Plant Resource Garden of Hunan Agricultural University), and when the plants grow to 4-5 leaves, select the young Leaves without diseased spots were rinsed with distilled water for 2 to 3 times, and then the genomic DNA was extracted by the conventional CTAB method, and the pH value of the TE solution for dissolving the DNA was 8.0;

[0036] Store at 4°C for later use. After the genomic DNA was completely dissolved, the DNA concentration was determined by 0.8% agarose gel electrophoresis.

[0037] 2) PCR-based SSR molecular marker analysis

[0038] The PCR reaction volume is 15 μL, including: 10×Buffer1.5 μL, MgCl 2 (25mmol·L -1 ) 1.5μL, dNTPs (10mmol L -1 ) 0.3 μL, Taq polymerase (5U·μL -1 )0.1μL, DNA template (20-25ng.μL...

Embodiment 2

[0059] Materials: Wujiemans (collected from Shaoyang, Hunan), Di (collected from Fengxian, Shaanxi) and their 8 true hybrids.

[0060] 1. Genomic DNA extraction: cut young leaves, use CTAB method to extract genomic DNA of each individual plant, dissolve in TE (pH8.0) solution, and store at 4°C for later use. DNA concentration was determined by 0.8% agarose gel electrophoresis.

[0061] 2. Analysis of SSR molecular markers based on PCR: PCR reaction volume is 15 μL, including: 10×Buffer1.5 μL, MgCl 2 (25mmol·L -1 ) 1.5μL, dNTPs (10mmol L -1 ) 0.3 μL, Taq polymerase (5U·μL -1 )0.1μL, DNA template (20-25ng.μL -1 ) 2 μL, the HAU-170 primer screened in Example 1 (2 μmol L -1 ) 1.5 μL, supplemented with ultrapure water. The PCR reaction program was as follows: pre-denaturation at 94°C for 5 min, 35 cycles (denaturation at 94°C for 30 s, annealing at 54°C for 30 s, extension at 72°C for 1 min), and finally extension at 72°C for 7 min and storage at 4°C. Reactions were performe...

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Abstract

The invention discloses a method for quickly identifying an interspecific distant hybrid of a herbaceous energy plant, namely miscanthus floridulus with silvergrass through using SSR (Simple Sequence Repeat) molecule markers. The method comprises the steps that tender leaves of the plant are selected, cleaned with distilled water, and then subjected to genome DNA (Deoxyribonucleic Acid) extraction by the conventional CTAB (Cetyl Trimethyl Ammonium Bromide) method; specific bands of the miscanthus floridulus and the silvergrass in various regions are analyzed respectively based on PCR (Polymerase Chain Reaction) and the SSR molecule markers; a pair of appropriate SSR markers is screened out; the markers generate one specific band (about 100bp) in the miscanthus floridulus, and generate the other specific band (about 123bp) in the silvergrass; and the markers show codominance in a filial generation of the miscanthus floridulus and the silvergrass. Therefore, the SSR markers can be used for molecule identification of the filial generation of the miscanthus floridulus and the silvergrass. With the method, the authenticity of the hybrid of the miscanthus floridulus with the silvergrass can be identified quickly and precisely. The method for identifying the hybrid of the miscanthus floridulus with the silvergrass is simple to operate, quick, accurate, good in repeatability, and free from an influence of an environmental factor.

Description

technical field [0001] The invention belongs to the technical field of identification of plant hybrids, in particular to a molecular identification method of Miscanthus plant hybrids. Background technique [0002] Miscanthus is a tall perennial herbaceous C4 plant in the Poaceae family. As the energy plant with the most development potential, it has attracted increasing attention. China is the origin and distribution center of Miscanthus, with rich wild resources and many ecological types. Miscanth plants have the characteristics of wide adaptability, strong stress resistance, high yield, high net energy output, good biomass quality, wide conversion and utilization channels, and good ecological value. They are suitable for planting and utilization on marginal land. Ecologically and economically efficient herbal energy plants. Grass (Miscanthus Sacchariflorus) in the genus Miscanthus is distributed in the northeast, northwest, north and east of China. It has strong stress r...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 易自力朱玉叶艾辛蒋建雄
Owner HUNAN AGRICULTURAL UNIV
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