Rape oil content character major gene resistance bit and application
A technology of main effect genes and gene loci, which is applied in the field of molecular biology and genetic breeding, can solve the problems of small effect value and difficult application of rapeseed breeding, and achieve the effect of high difficulty, convenient and fast detection, and clear position
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Embodiment 1
[0031] Example 1. Construction of high and low oil content rape strain combination F2 segregation population and determination of oil content
[0032] The population used in this example is the offspring of hybrids of high and low oil content parents (zy036 with oil content of 51% and Y817 with 35% respectively)—the F2 generation. The oil content of the parents, F1 and F2 seeds was determined by a near-infrared analyzer. The distribution of oil content data in the F2 generation segregation population shows that the distribution of oil content is continuous, and the variation distribution is normal, and the variation range is very wide, which proves that oil content is a quantitative trait ( figure 1 ).
Embodiment 2
[0033] Example 2. Extraction of total DNA from leaves of parent, F1 and F2 segregation populations
[0034] The total DNA of leaves was extracted by the CTAB method, and the specific steps were as follows:
[0035] A. An appropriate amount of leaf samples are taken from an ultra-low temperature refrigerator (-70°C), put them into a frozen mortar immediately, add liquid nitrogen and grind them into powder; quickly put them into a 50ml centrifuge tube, and add them to a water bath at 60°C for pretreatment. The heated extract (0.2M Tris-Cl, 0.25NaCl, 25mM EDTA, 0.5% (mass ratio) SDS, pH 7.5) was mixed evenly, and placed in a water bath at 60°C for 40 minutes;
[0036] B. Take out the centrifuge tube, add an equal volume of chloroform: isoamyl alcohol (24:1, V / V), slowly invert the centrifuge tube 30-50 times up and down to fully mix, and centrifuge at 1300g for 10 minutes;
[0037] C. Take the supernatant into another centrifuge tube, add an equal volume of chloroform: isoamyl a...
Embodiment 3
[0039] Example 3. Development of primers and screening of polymorphisms
[0040] The screening of primers was carried out according to the reported SSR marker primers and the self-developed molecular markers of functional genes related to oil content. Screening results showed that there were 180 pairs of primers that were different between the parents. The polymorphism screening procedure is as follows:
[0041] (1) The DNA templates of 5 strains were randomly selected from each of the parents and mixed in equal amounts to form the DNA samples of the two parents for primer screening.
[0042] (2) PCR system and procedures:
[0043] 10×buffer(MgCl2 free) 1ul
[0044] dNTPs mixture (10mM) 0.2ul
[0045] Left primer (50ng / ul) 1ul
[0046] Right primer (50ng / ul) 1ul
[0047] Taq polymerase (5U / ul) 0.1u
[0048] MgCl2 (25mM) 0.8ul
[0049] Rapeseed total DNA 2ul
[0050] ddH2o 5.9ul
[0051]
[0052] total 10ul
[0053] PCR reaction p...
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