Camellia oleifera self-incompatibility-related genes, snp molecular markers and their applications
A molecular marker and affinity technology, applied in the fields of application, genetic engineering, plant gene improvement, etc., can solve problems affecting the agronomic traits of camellia oleifera
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Embodiment 1
[0038] Embodiment 1, the total DNA extraction of Camellia oleifera seed kernel, root or leaf and fruit setting rate calculation
[0039] DNA secure new plant genomic DNA extraction kit (TIANGEN kit Code No. DP320, spin column type) combined with laboratory modified CTAB method was used to extract total DNA from single plant. The specific operation steps are as follows:
[0040] (1) Take 600μL of 2×CTAB solution into a 1.5mL centrifuge tube, mix well, and keep it in a 65°C air heater for later use;
[0041] (2) Take out the oil tea seed kernels, roots or leaves from the liquid nitrogen rapidly, and grind them into fine powder rapidly in a pre-cooled mortar filled with liquid nitrogen;
[0042] (3) Transfer the ground powder to the prepared centrifuge tube (1), mix the powder with the solution on a vortexer, and place it in a water bath at 65°C for 10 minutes, during which it is evenly stirred every 2 minutes;
[0043] (4) Add 600 μL of chloroform:isoamyl alcohol (24:1), vorte...
Embodiment 2
[0049] Example 2, third-generation sequencing + second-generation sequencing samples and annotation analysis
[0050] 1. Sequencing sample preparation and collection:
[0051] Select the nationally approved Camellia oleifera seed oil 'Huaxin' and 'Huajin' (respectively: National S-SC-CO-009-2009 and National S-SC-CO-010-2009) self-pollination combination (Self -pollination: SP), cross-pollination (Cross-pollination: CP) and non-pollination (Non-pollination: NP) ('Huajin' and 'Huaxin' emasculated bagging). Cross-pollination was done by artificial Pollination is completed. Pick the flower buds of 'Huaxin' and 'Huajin' at the white stage, gently stroke the anthers with tweezers, spread the anthers in a sulfuric acid paper bag, fully crack the anthers at 25°C, and release the mature pollen (Pollen: Pn); store the pollen in a 1.5mL centrifuge tube at -80°C; before pollination, take the pollen out of the refrigerator to balance for 1h at room temperature. When pollinating, select t...
Embodiment 3
[0059] Example 3: CoMAPK gene acquisition and SNP site analysis
[0060] 1. Analysis of CoMAPK9 gene characteristics associated with self-incompatibility
[0061] Transcript annotation analysis and trend analysis and Pearson correlation analysis of differential transcripts and differential proteins were performed. CoMAPK9 was found to be a unique sequence of Camellia oleifera, which strongly responded to self-pollination and out-pollination. The functional characteristics of CoMAPK9 gene during the self-incompatibility process of Camellia oleifera were studied by sequence analysis, expression pattern analysis, prokaryotic expression, and subcellular localization.
[0062] Analysis of CoMAPK9 in different parts of flowers ( figure 2 A), the expression pattern of 2-84h in the styles of different treatments ( figure 2 B).
[0063] The accuracy of CoMAPK9 gene sequence was verified by inducing CoMAPK9 protein expression by prokaryotic expression experiments ( image 3 A), th...
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