SNP (single nucleotide polymorphism) molecular marker relevant to camellia oleifera seed oil content and application thereof
A molecular marker, oil content technology, applied in recombinant DNA technology, microbial determination/inspection, DNA/RNA fragments, etc., can solve the problems of difficult to screen markers, time-consuming, occupying a large area of forest land, etc., and achieve convenient detection methods. The effect of fast, improving selection efficiency and saving production costs
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Embodiment 1
[0043] Example 1 Construction and Character Measurement of Camellia oleifera Seed Oil Content Segregation Population
[0044] In this example, common Camellia oleifera resources are used to collect natural populations of 500 germplasm resources in the nursery, and their origins cover most of the main Camellia oleifera producing areas in my country, including Zhejiang Province, Hunan Province, Jiangxi Province, Guangxi District, Fujian Province, and Guangdong Province. Province etc. After the fruit of 500 individuals is fully mature (5% of the fruit is cracked), the seeds are collected, and the oil content of the seeds is determined by Soxhlet extraction. The operation steps are as follows:
[0045] (1) Prepare a medium-speed filter paper bag, put it in an aluminum box, bake at 105°C to constant mass, and record the quality of the aluminum box and filter paper bag (W 1 ).
[0046] (2) Peel off the hard seed coat of an appropriate amount of Camellia oleifera seeds, bake at 105...
Embodiment 2
[0050] Embodiment 2 Cofad2-1A gene fragment amplification
[0051] 1. Total DNA extraction from leaves:
[0052] Use the TaKaRa MiniBEST Plant Genomic DNA Extraction Kit to extract the total DNA of leaves by using the cracking system of plant materials rich in polysaccharides, polyphenols and oils. The specific steps are as follows:
[0053] (1) First add 500 μl of Buffer HSⅡ into a 1.5ml centrifuge tube. Take 0.1g of fresh leaves and add liquid nitrogen to fully grind, quickly add the ground leaf powder into a centrifuge tube and mix well, then add 10μl of RNaseA (10mg / ml), shake and mix well, and incubate in a water bath at 56°C for 10 minutes ;
[0054] (2) Add 62.5 μl of Buffer KAC and mix well. Place on ice for 5 minutes and centrifuge at 12000 rpm for 5 minutes. Take the supernatant, add the same volume of Buffer GB as the supernatant, and mix well.
[0055] (3) Place the Spin Column on the collection tube, move the solution to the Spin Column (the solution is too m...
Embodiment 3
[0079] Example 3 Screening of SNP sites related to Camellia oleifera seed oil content
[0080] Population structure analysis and linkage disequilibrium analysis, the steps are as follows:
[0081] (1) Import the SNPs data of all samples into the Structure2.3.4 software, set K=2~9, run 5 times for each K value, burnin 5000 times, and repeat 50000 times. When both LnP(D) and α values remain stable, and α Figure 4 ), K=4 in the present invention, and K (4) subgroup effect values of each sample are determined (Table 1).
[0082] Table 1 The four subgroup effect values of some individuals in the natural population
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[0087] (2) Import the SNPs site data, K subgroup effect value data, phenotype data (see Example 1) and Kinship matrix data of all samples into the TASSEL5.0 software, and use the MLM method to analyze the relationship between SNPs and oil content traits Linkage disequilibrium, screening molecular markers significan...
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