Molecular marker sisv0503 closely linked to millet fertility gene
A technology of molecular markers and fertility genes, applied in the field of molecular biology, can solve problems such as backwardness of millet
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Embodiment 1
[0039] Embodiment 1: the construction of millet F2 generation segregating population
[0040] Male parent: Resistant to Nachajing, tall plant type, long and narrow flag leaves, red bristles, red glumes, fertile, greenish leaf color, yellow-white pollen, late heading stage. The male parent is Zhang Gu No. 1 seed.
[0041] Female parent: not resistant to Nachajing, short plant type, short and wide flag leaves, green bristles, green glume, partially sterile, yellowish leaf color, brown pollen, early heading stage. The female parent is millet A2 sterile line seeds. In the present invention, the above-mentioned partial sterility of the female parent means that about 5% of the grains in each ear are fertile, and the rest are sterile.
[0042] F2 population construction: the male parent and the female parent are crossed to obtain F1 generation (the phenotype of F1 is the same as that of the male parent), and F1 is self-crossed to obtain F2. Among them, F1 is the No. 3 seed of Zhan...
Embodiment 2
[0044] Example 2: Extraction of parental and individual genomic DNA of F1 and F2 generations
[0045] The genomic DNAs of the parental parent, F1 generation, and 480 F2 generation individuals in Example 1 were extracted with the CTAB method, and the specific methods were as follows:
[0046] (1) Weigh 1.0g of fresh leaves, cut them into pieces and put them in a mortar, grind with liquid nitrogen, add 3mL of 1.5×CTAB, grind into a homogenate and transfer it to a 15mL centrifuge tube, then add 1mL of 1.5 ×CTAB rinsed and transferred to a centrifuge tube. After mixing, place in a water bath at 65°C for 30 min, and shake slowly from time to time.
[0047] The formula of 1.5×CTAB is as follows (1L):
[0048]
[0049] Add deionized water to make up to 1 L, and add mercaptoethanol with a final concentration of 0.2% (2 mL) before use.
[0050] (2) After cooling to room temperature, add an equal volume of chloroform / isoamyl alcohol (24:1) and mix gently until the lower layer liqu...
Embodiment 3
[0055] Example 3: Preparation of molecular markers
[0056] Using the genomic DNA of the male parent, F1 generation, or F2 generation extracted in Example 2 as a template, PCR amplification was performed with a pair of molecular marker amplification primers (SeqIDNo.2 and SeqIDNo.3).
[0057] The PCR reaction system is as follows:
[0058]
[0059] The PCR reaction program is as follows:
[0060] 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, running for 35 cycles; final extension at 72°C for 3 minutes. PCR amplification products can be stored at 4°C.
[0061] The molecular marker is obtained through the above amplification process, and preferably, the amplification product is purified after amplification. Sequencing was performed after purification, and the results were shown in SeqIDNo.1.
[0062] For those skilled in the art, it can be understood that the molecular marke...
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