CMS molecular tag based on hibiscus cannabinus mitochondrial gene non-coding region and primer pair and application of CMS molecular tag
A mitochondrial gene, non-coding region technology, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve problems such as single cytoplasmic type of kenaf and large-scale occurrence of diseased physiological races
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Embodiment 1
[0078] Extraction of Total DNA from Kenaf
[0079] The tips, mortar, and extraction buffer used in the extraction process of kenaf total genomic DNA were first autoclaved for 20 minutes, and then taken out for later use. Extract DNA according to the following steps:
[0080] (1) Preheat CTAB (0.1 mol / L Tris-HCl pH8.0, 0.025 mol / L EDTA-Na2pH8.0, 1.4 mol / LNaCl, 2% CTAB (w / v)) extract in a 65°C water bath;
[0081] (2) Grind 2 to 3 g of fresh or -80°C frozen leaves in liquid nitrogen;
[0082] (3) Quickly add CTAB extract, mix well, pour into a centrifuge tube, and place in a water bath at 65°C for 30 min. gently shaking in between;
[0083] (4) Add an equal volume of chloroform / isoamyl alcohol, mix well, and centrifuge at 12000 rpm for 10 min at room temperature;
[0084] (5) Take the supernatant, add 5M NaCl and absolute ethanol according to the ratio of extract: 5M NaCl: dehydrated ethanol = 1:1:1 to prepare a mixture, invert and mix well;
[0085] (5) Aspirate the superna...
Embodiment 2
[0099] Application of HMBQ102 Marker in Breeding Kenaf Cytoplasmic Male Sterile Line
[0100] Using this molecular marker to detect the existing known kenaf cytoplasmic male sterile line and maintainer line materials in our research group, the results are as follows figure 2 The size of the band amplified by the molecular tag in the known cytoplasmic male sterile lines of kenaf with different cytoplasmic types is 980bp (lane 1-10), and the band size amplified in the maintenance is 1082bp (lane 12- 19), indicating that this molecular signature can accurately identify the cytoplasmic male sterile line resources of different cytoplasmic types of kenaf.
Embodiment 3
[0102] Repeat experiment for stability of HMBQ102 labeling
[0103] According to the stable characteristic that the kenaf HMBQ102 molecular tag can amplify a band with a size of 980bp in the sterile cytoplasm and a band with a size of 1082bp in the maintainer line, the field planting resources of kenaf were screened and identified. The result is as image 3 Shown: In this example, a total of 16 germplasm resources with consistent cytoplasmic characteristics with UG93A and 10 germplasm resources with consistent cytoplasmic characteristics with UG93B were identified. The identification of the above materials provides a molecular basis for the selection and breeding of kenaf cytoplasmic androgenic lines. On the other hand, UG93BS1 and UG93BS2 ( image 3 a Lane 5, Lane 6) These two germplasm resources are two sterile individuals found in the maintainer line UG93B population. HMBQ102 molecular label detection shows that these two individuals have the same cytoplasmic type as the ...
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