Gene relevant to wheat thousand seed weight, functional marker and application thereof
A thousand-grain weight and genetic technology, applied in the field of wheat molecular biotechnology and breeding applications, can solve problems affecting wheat thousand-grain weight
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Embodiment 1
[0049] Cloning of 1000-grain weight-related gene TaSnRK2.10 gene in wheat, the material used is Lumai 21.
[0050] 1) Electronic cloning of wheat TaSnRK2.10 cDNA sequence;
[0051] (1) Using the rice SAPK10 (Accession: AB125311) gene sequence submitted by NCBI as a probe, search the wheat EST database, splice the obtained sequences with DNAMAN software, and predict the open reading frame to obtain the complete wheat SnRK2.10cDNA sequence .
[0052] (2) According to the obtained full-length cDNA sequence, two pairs of specific primers SnRK2.10-1 and SnRK2.10-2 were designed and screened by Primer5 software, which were used to clone wheat SnRK2.10 cDNA and gDNA gene respectively.
[0053] TaSnRK2.10-1 forward primer: 5′-GCTTGCTCGGTTGCTTTGC-3′ (SEQ ID NO: 7)
[0054] Reverse primer: 5'-CATCCAAAAGGCCAAACCGT-3' (SEQ ID NO: 8)
[0055] TaSnRK2.10-2 forward primer: 5′-GTCAAGTACATCGAGCGAGGG-3′ (SEQ ID NO: 9)
[0056] Reverse primer: 5′-CGTCGTTCAGGAACTGGTTGA-3′ (SEQ ID NO: 10)
[...
Embodiment 2
[0082] Functional marker development and association analysis
[0083] 1) Use TaSnRK2.10-2 (forward primer sequence is SEQ ID NO: 9, reverse primer sequence is SEQ ID NO: 10) primer sequence to PCR amplify DNA from 10 materials in Huanghuai wheat region and analyze the genome sequence It was found that the TaSnRK-4A (SEQ ID NO: 4) sequence had SNP and InDel sequence differences among varieties.
[0084] 2) There are 3 SNP sites and one InDel site in the TaSnRK2.10-4A genome sequence, such as image 3 As shown in a: the 1123rd nucleotide from the 5' end is C or T, the 1321st nucleotide from the 5' end is A or T, the 1757th nucleotide from the 5' end is G or C, from Nucleotides 1832-1834 at the 5' end are deletions or insertions of TGA trinucleotides. Three SNP sites and one InDel site are closely linked: when the 1123rd nucleotide from the 5' end is C, the 1321st and 1757th nucleotides from the 5' end must be A and G, and at the same time The 1832-1834th nucleotide is the de...
Embodiment 3
[0100] Method for Detecting 1000-grain Weight of Wheat Varieties or Strains Using TaSnTK2.10-4A-caps Markers
[0101] 1) Extract the DNA of the wheat variety to be tested, and use TaSnTK2.10-4A-caps labeled primers (forward primer sequence: SEQ ID NO: 11, reverse primer sequence: SEQ ID NO: 12) to perform PCR on the wheat variety DNA Amplification, the PCR amplification system is 20 μl, including H 2 O 14.3 μl, 2×PCR buffer 2.0 μl, Taq enzyme 0.20 μl, left reverse primer 0.5 μl, cDNA 1.0 μl, dNTP 1.5 μl; amplification conditions are 94°C pre-denaturation for 4 minutes; 94°C denaturation for 40 seconds, 58°C Anneal for 45s, extend at 72°C for 1min, 35 cycles; extend at 72°C for 10min; store at 10°C.
[0102] 2) Digest the above-mentioned amplified products with SalI endonuclease respectively, and detect whether the SNP site at position 1757 of the 5' end of the wheat TaSnRK-4A genome sequence to be tested is C or G, and determine whether the genotype of the wheat to be tested ...
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