Molecular marker indel15-1 of soybean early-flowering-season major QTL (quantitative trait locus) and application thereof
A molecular marker, first flowering technology, applied in the determination/inspection of microorganisms, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of long breeding cycle and low selection efficiency
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Embodiment 1
[0035] Example 1: Acquisition of the main QTL for the initial flowering stage of soybean
[0036] (1) 'NJZNRIL' group building
[0037] The hybrid F 1 , construct the population through the "single-seed transmission" method, and obtain 236 F 2:8 Generation of a recombinant inbred line population ('NJZNRIL').
[0038] The steps of the "single-seed transmission" method are as follows: a seed harvested from the female plant in the parental hybrid generation grows into a F 1 Generation single plant, its self-crossing (i.e. self-pollination) yields fruit and harvests 1 F 2 Generation seeds, which grow into a F containing segregating traits 2 Generation line, each individual plant of it self-fertilizes and harvests F 3 For generation seeds, the isolated plants of the same traits are harvested and threshed into bags, and each seed is planted separately in the next year. 3 F 4 Generation seeds, ... until the characters of different plants in each family are completely stable an...
Embodiment 2
[0047] Example 2: Acquisition of the main QTL closely linked marker indel15-1 for soybean flowering stage
[0048] (1) Molecular marker development
[0049] Using the published soybean physical map information and the sequence information of the sequencing materials of the National Soybean Improvement Center, molecular markers were designed in the Gm15:48600kb-49850kb region of chromosome 15.
[0050] (2) Secondary population phenotype determination
[0051] (3) Secondary population molecular marker identification
[0052] Genomic DNA from leaves of NJZNRIL-derived secondary population soybean material was extracted by CTAB method, and the amplification experiment was carried out with indel15-1 primer pair. PCR reaction system (10ul), which contains 3ul DNA template (15ng), upstream and downstream primers (0.2mmol / L) each 1.5ul, 1.2ulMgCl 2 (2.5mmol / L), 0.24uldNTP (10mmol / L, N=A, C, G, T), 0.12ulTaq enzyme (5U / ul) and 1.4ulddH 2 O. PCR reaction program: denaturation at 95...
Embodiment 3
[0057] Embodiment 3: the application of indel15-1 primer pair in the selection of soybean initial flowering stage
[0058] (1) Genome amplification detection of both parents
[0059] Two groups of materials with longer and shorter flowering stages were randomly selected from the NJZNRIL population to verify the QTL for soybean flowering stage.
[0060] (2) Population amplification detection and marker analysis
[0061] The genomic DNA of leaves of each material was extracted by CTAB, and the amplification experiment was carried out with the indel15-1 primer pair. PCR reaction system (10ul), which contains 3ul DNA template (15ng), upstream and downstream primers (0.2mmol / L) each 1.5ul, 1.2ulMgCl 2 (2.5mmol / L), 0.24uldNTP (10mmol / L, N=A, C, G, T), 0.12ulTaq enzyme (5U / ul) and 1.4ulddH 2 O. PCR reaction program: denaturation at 95°C for 5 min; followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 40 s, extension at 72°C for 50 s; extension at 72°C for ...
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