Molecular identification method of gossypium trilobum cytoplasm male sterile restoring gene
A technology for male sterility and gene restoration, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc. Breeding and other issues to achieve the effect of reducing workload, reducing breeding input costs, and reducing risks
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Embodiment 1
[0030] The InDel molecular marker of the present invention is a deletion sequence 5'-TATTTGCACCCTAAGCAATAGTTTTAGAACAATTAA-3' (SEQ ID NO.1) with a deletion of 36 bp. The deletion site of the InDel marker is in upland cotton chromosome ChrD05: 54213468-54213503 (refer to the upland cotton sequencing results of Nanjing Agricultural University, the download URL is http: / / mascotton.njau.edu.cn / info / 1054 / 1118.htm ).
[0031] In the upstream and downstream of the marker, according to the InDel site, sequence and PCR primer design principles, design primers, successfully convert it into InDel marker primers, and amplify the InDel molecular marker primer pair as shown in Table 1:
[0032] Table 1: InDel Molecular Labeling Primers
[0033]
[0034] In the genome of the homozygous restorer line of the three-lobed cotton cytoplasmic male sterile restorer line, the 239bp band was specifically amplified and deleted, and 275bp and A 239bp band, a 275bp band was specifically amplifie...
Embodiment 2
[0037] 1. The CTAB method was used to extract the seed DNA of the first-generation backcross population (BCF1) constructed by three lines of cotton male sterile (CMS-D8).
[0038] 2. Using the InDel molecular marker primers, the extracted DNA is amplified by PCR. The PCR reaction system is shown in Table 2.
[0039] Table 2 20μl PCR reaction system
[0040]
[0041] The PCR program was: pre-denaturation at 94°C for 2 min; denaturation at 94°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 15 s, and 35 cycles; then final extension at 72°C for 2 min, and storage at 4°C.
[0042] Perform agarose gel (2.5%) electrophoresis on the PCR product, electrophoresis detection in 1×TAE electrophoresis buffer, voltage 80-90V, current 100mA, 40min-60min, take pictures with a gel imaging system and save, and electrophoresis results Perform band analysis.
[0043] 3. Test results
[0044] Judgment criteria: In the statistics, if only 239bp band appears, it contains homozygo...
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