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, inability to apply broad-spectrum herbicides in grain fields, large quantities, etc., and achieve the effect of high-throughput application
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Embodiment 1
[0043] Example 1: Construction of F2 Segregated Population of Millet
[0044] The male parent is Zhanggu No. 1: Kangnatujing type (plant height, about 150cm), the flag leaf is long and narrow, the bristles are red, the glume is red, fertile, and the leaf color is greener.
[0045] The female parent is A2 sterile line: non-resistant Natujing type (short plant type, plant height is about 100cm), flag leaves are short and wide, bristles are green, glumes are green, partly sterile, and leaves are yellowish.
[0046] F1 (Zhangzagu No. 3, some anti-Natujing type, plant height is about 130cm) is obtained by crossing the male parent and the female parent.
[0047] The F1 generation selfed produces the F2 generation population, and a total of 480 individual plants were obtained. 480 individuals of the F2 generation were analyzed according to the aforementioned method for determining the resistance type, and a total of 360 strains of the resistance type and part of the resistance type were fou...
Embodiment 2
[0048] Example 2: Extraction of genomic DNA from parents and individuals of F1 and F2 generations
[0049] The CTAB method was used to extract the genomic DNA of the parents, F1 generation, and 480 F2 generation individuals in Example 1. The specific methods are as follows:
[0050] (1) Weigh 1.0g of fresh leaves, cut them into pieces, put them in a mortar, grind them with liquid nitrogen and add 3ml 1.5×CTAB, grind them into a homogenate and transfer them into a 15ml centrifuge tube, then add 1ml 1.5× into the mortar Flush with CTAB and transfer to centrifuge tube. After mixing, in a 65°C water bath for 30 minutes, shaking slowly from time to time.
[0051] 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 the volume to 1L, and add mercaptoethanol with a final concentration of 0.2% (2ml) before use.
[0057] (2) After cooling to room temperature, add an equal vo...
Embodiment 3
[0062] Example 3: Preparation of molecular markers
[0063] Using the genomic DNA of the anti-naptogyne type or part of the anti-naptogyne type extracted in Example 2 from the paternal parent, F1 generation, or F2 generation as a template, molecularly labeled primers (SEQ ID NO: 2 and SEQ ID NO: 3 ) Perform PCR amplification.
[0064] The PCR reaction system is as follows:
[0065] Sterile water 20.2μl
[0066] 10*Buffer (including Mg 2+ ) 2.5μl
[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, 72 extension for 40 seconds, running for 35 cycles; finally, extension at 72°C for 3 minutes. PCR products can be stored at 4°C.
[0075] The amplified products are purified to obtain molecular markers. Sequenc...
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