A tetraploid R. longa 1e chromosome molecular marker and its application
A technology of Thinpyrum elongatum and chromosome, applied in the field of molecular markers of tetraploid Thinpyrum elongatum 1E chromosome
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Embodiment 1
[0064] Example 1 Based on GBS technology to obtain tetraploid Etyophony elongatum 1E chromosome-specific fragment
[0065]When the materials such as diploid E. elongatum, tetraploid E. elongatum, parent 8801 and common wheat-tetraploid E. elongatum 1-7E chromosome substitution line grow to the seedling stage, take the leaves and send them to Beijing Novogene Technology Co., Ltd. carried out GBS sequencing, and obtained the effective sequence of each sample through GBS sequencing.
[0066] The sequence of the obtained wheat-tetraploid E. elongatum 1E / 1D substitution line was first compared with the known sequence of Chinese spring, and the sequence with a similarity of 22.85% (23%) or more to wheat was eliminated, and the remaining The following sequence was then compared with the sequence of 8801 and tetraploid E. elongatum to obtain a sequence with a similarity greater than 22.85% (23%). The remaining sequences were then compared with diploid E. elongatum and other substituti...
Embodiment 2 4
[0067] Example 2 Development of Chromosome-Specific Molecular Markers of Tetraploid Echinopsis elongatum 1E
[0068] Primers were designed for the 597 specific sequences obtained above. The PCR primers were designed using the online software TTE3Plus, and all the primers were synthesized in Chengdu Qingke Weiye Biotechnology Co., Ltd. 235 pairs of primers were successfully designed.
[0069] The PCR reaction system is as follows: the total volume of the PCR reaction is 25uL, the DNA template (concentration needs to be diluted to about 100ng / uL) plus 1uL, the upper and lower primers each 1uL (10uM), 2×Taq Master Mix (P114) 12.5uL, ddH 2 O 9.5 uL.
[0070] The PCR program was: 94°C for 5h; 94°C for 30min, 50-60°C for 30min, 72°C for 2h, 35 cycles; 72°C for 10h; 12°C.
Embodiment 3
[0071] Embodiment 3 agarose gel electrophoresis detection
[0072] The PCR products were detected by 3% agarose gel electrophoresis, and the designed primers could amplify specific bands in tetraploid Thipotentia elongatum, parent 8801, and 1E / 1D substitution lines, and in CS, Shumai 482, Shumai 921, diploid E. elongatum, 2E / 2A substitution line, 3E / 3D substitution line, 4E / 4D substitution line, 5E / 5D substitution line, 6E / 6D substitution line, 7E / 7D substitution line No bands could be amplified in the substitution lines, and this primer was the specific primer for the 1E / 1D substitution line, that is, the specific molecular marker for the 1E chromosome of tetraploid E. elongatum. 235 pairs of primers were successfully designed, and 132 pairs of specific primers were obtained after PCR amplification, product recovery, and sequencing verification in the above materials, and the molecular marker development efficiency reached 56.17%.
[0073] attached figure 1 , attached fig...
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