Method for authenticating whether to-be-detected variety is Ruiza816 and special primer group thereof
A primer set and Ruizha technology, applied in the field of identifying whether the tested variety is Ruiza 816, can solve the problems of poor seed quality, adulteration of female parent, long time, etc., and achieve fast speed, intellectual property protection, and clear map. Effect
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Embodiment 1
[0033] Example 1, Construction of the DNA Fingerprint of Ruiza 816
[0034]1. Screening of SSR primer pairs
[0035] After screening a large number of cotton SSR primers, it was found that 15 SSR primer pairs (see Table 1 for the nucleotide sequence) could be used to make the DNA fingerprint of Ruiza 816.
[0036] Nucleotide sequences of table 115 primer pairs
[0037] serial number
Forward primer (5'→3')
Reverse primer (5'→3')
1
CS62 primer pair
GATGGCTACCTCCCCTTTGTA (sequence 1)
CGTAAGGAAGCCTAGCAAAA (sequence 2)
2
NAU1103 primer pair
GGAGCCAGAAGTTGAGAAAA (sequence 3)
TTCGGCTTCTGCTTTTACTT (sequence 4)
3
NAU1186 primer pair
AATGGTCCTGCTCCAGATT (sequence 5)
AATCGTCGTCGTCGAATTAT (Sequence 6)
4
NAU1233 primer pair
TTCGGGAAAGTTAGAGGAGA (Sequence 7)
TCCTCAGAGCTCGGAATAGT (sequence 8)
5
NAU2026 primer pair
GAATCTCGAAAACCCCCATCT (sequence 9)
ATTTGGAAGCGAAGTACCAG (Seque...
Embodiment 2
[0085] Example 2, Application of the DNA Fingerprint of Ruiza 816
[0086] Using the genomic DNA of Ruiza 816, Zhongzhimian 2 or Lumianyan 28 as templates, 15 SSR primer pairs were used for PCR identification, followed by 8% polyacrylamide gel electrophoresis and silver staining.
[0087] The PCR system was 10.0 μL, containing 0.5U Taq enzyme, and the concentrations of the forward primer and the reverse primer were both 0.5 μM.
[0088] PCR reaction program: pre-denaturation at 94°C for 3 minutes; denaturation at 94°C for 30 s, annealing at 56°C for 45 s, extension at 72°C for 45 s, 30 cycles; denaturation at 94°C for 1 min, annealing at 56°C for 45 s, extension at 72°C for 2 min, and storage at 4°C.
[0089] The parameters of polyacrylamide gel electrophoresis are as follows: the sample volume per well is 1.8 μL; 200v voltage, 50min.
[0090] The results are shown in Table 4.
[0091] Table 4 The results of this embodiment (band type)
[0092]
[0093] Using the CS62 pr...
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