DNA fragment related to kernel yield of camellia oleifera seeds, SNP molecular marker and application of SNP molecular marker
A seed kernel rate and molecular marker technology, applied in the field of DNA fragments and SNP molecular markers, can solve the problems of slow breeding of new varieties, restricting the development of Camellia oleifera industry, and insufficiency of breeding speed of improved varieties.
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Embodiment 1
[0059] Example 1 Construction and character determination of the isolated population of Camellia oleifera seeds
[0060] In this example, Changlin No. 53 and Changlin No. 81 were used as the female parent and male parent, respectively, and the controlled pollination technology was used to create a hybrid F1 generation population with widely separated economic traits. The F1 population was kept in Dongfanghong Forest Farm in Wucheng District, Zhejiang Province using a randomized block design with 3 replicates. After 180 offspring individuals were fully ripened (5% fruit cracked), the seeds were collected and the seed kernel rate was determined. The operation steps are as follows:
[0061] (1) Take 10-15 seeds from each sample, bake at 105°C to constant mass, and record the seed mass (W 1 ).
[0062] (2) Peel off the hard seed coat from Camellia oleifera seeds, and record the total mass of the seed kernels (W 2 ).
[0063] (3) Calculate the seed kernel rate of each sample: ...
Embodiment 2
[0066] Example 2 Construction of Camellia oleifera linkage map
[0067] 1. Genomic DNA extraction
[0068] The spring leaves of 180 individuals of Changlin No. 53 × Changlin No. 81 family and their parents were collected in March, and total genomic DNA was extracted by KAC method (TaKaRa kit Code No. 9768). Specific steps are as follows:
[0069] (1) First, add 500 μl of Buffer HS II to a 1.5 ml centrifuge tube; accurately weigh 100 mg of Camellia oleifera young leaves for grinding with liquid nitrogen; quickly add the ground powder to the centrifuge tube and mix, then add 10 μl of RNase A ( 10 mg / ml), fully shaken and mixed, and incubated in a water bath at 56°C for 10 minutes.
[0070] (2) Add 62.5 μl of Buffer KAC and mix well. Place on ice for 5 minutes and centrifuge at 12,000 rpm for 5 minutes. Take the supernatant, add the same volume of Buffer GB as the supernatant, and mix well.
[0071] (3) Place the Spin Column in the Collection Tube, move the solution to the S...
Embodiment 3
[0093] Embodiment 3 Camellia oleifera seed kernel rate gene locus and linked SNP locus mining
[0094] The QTL Icimapping software was used to analyze the data, and the complete interval mapping method (ICIM) was used to locate the QTL for seed emergence rate. The scan step size was set to 1 cM; the probability of stepwise regression marker entry (PIN) was 0.002 (POUT=2*PIN=0.002); the LOD value was 2.5. LOD significance thresholds were determined by running 1000 permutation tests. The seed kernel rate gene locus rkf.04-1 of Camellia oleifera was located on chromosome 4 of Camellia oleifera, with a contribution rate of 15.1%, and its closely linked SNP molecular marker was chr04-77743434 (Table 1, figure 1 ).
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