A kind of indel molecular marker closely linked with salt-tolerance-related traits of cowpea and its primers and applications
A cowpea and salt-tolerant technology, applied in the fields of molecular biology and genetic breeding, can solve problems such as poor repeatability, small effect value, and difficult application of cowpea breeding
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Embodiment 1
[0039] Embodiment 1. Construction and character of cowpea salt-tolerant segregation population;
[0040] The populations used in this example are the F2 populations of the offspring of salt-tolerant and salt-sensitive parents (Suzi 41 with salt tolerance level 1 and Su cowpea 1419 with level 5, respectively). STR and SPAD data distribution results show that: these two traits are continuous normal distribution, and the range of variation is very wide, which proves that STR and SPAD belong to quantitative traits ( figure 1 ).
Embodiment 2
[0041] Example 2. The extraction of total DNA from the leaves of parent Suzi 41, Su cowpea 1419, F1 and F2 segregated populations;
[0042] The total DNA of leaves was extracted by the CTAB method, and the specific steps were as follows:
[0043] A. Take 0.1 gram of fresh leaves and grind them, add 650 microliters of extract to grind them, then put them into a 1.5 milliliter centrifuge tube and place them in a constant temperature water bath at 65°C for 60 minutes, during which they mix 2-3 times;
[0044] B. Add an equal volume of phenol: chloroform: isoamyl alcohol (25:24:1, volume ratio), gently invert it to make it fully mixed; centrifuge at 12000rpm for 10 minutes to make it layered, and gently suck the supernatant into Another 1.5 ml centrifuge tube, add an equal volume of chloroform: isoamyl alcohol (24:1, volume ratio) and re-extract once;
[0045] C. Add 1 ml of -20°C pre-cooled absolute ethanol, freeze at 4°C for no more than 30 minutes to allow DNA to precipitate, ...
Embodiment 3
[0047] Example 3. Development of InDel molecular marker primers and screening of polymorphisms:
[0048] According to the assembly sequence of the transcriptomes of Suzi 41 and Sujiang 1419, BLAST alignment was performed to screen the transcripts with insertion / deletion (InDel);
[0049] Sequences with relatively large differences in sequence length of insertion / deletion (InDel) were selected, and then Primer5.0 software was used to design InDel marker primers.
[0050] 845 pairs of InDel primers were synthesized by a biological company. The screening results showed that 93% of the amplified products of the primers had length polymorphism differences between the parents, and 31% of the amplified products of the primers had co-dominance in F1. The polymorphism screening procedure is as follows:
[0051] (1) Randomly select 4 strains of DNA from each parent and mix them in equal amounts as templates for screening primers.
[0052] (2) PCR reaction system:
[0053]
[0054...
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