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Cucumber female character related SNP (single nucleotide polymorphism) marker and InDel marker and application thereof
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A female and marker technology, applied in the field of molecular biology, can solve problems such as conflicting research results, achieve accurate and reliable results, save land and cost, and accelerate the breeding process.
Active Publication Date: 2016-01-20
VEGETABLE RES INST OF SHANDONG ACADEMY OF AGRI SCI
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Problems solved by technology
Previous studies believed that through the analysis of the copy number of ACS1 in the cucumber genome, we can judge whether it is female or not, but there are also research results that are contrary to this
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Embodiment 1
[0072] In the summer of 2014 and 2015, 8 pure female lines (A10h, A86h-1, M16, A72, ZQ3, SJ11, MC8-3, YN) and 3 non-female lines (AZ-1, BB, X8- 2) and 3 hybrids (Cuilong, BM28, DL102), the sexually stable pure female materials and non-female materials and the young leaves of the hybrids were used for DNA extraction, followed by PCR amplification and molecular marker detection analysis. Specific steps are as follows:
[0073] DNA extraction: use the fast plantgenomic DNA extraction system (DP321) produced by Tiangen Biochemical Technology (Beijing) Co., Ltd.
[0075] Take 100 mg of young cucumber leaves, add liquid nitrogen and grind them thoroughly. Add 400 μl of buffer FP1 and 6 μl of RNaseA (10 mg / ml), vortex for 1 min, and place at room temperature for 10 min.
[0076] 2. Add 130 μl buffer FP2, mix thoroughly, and vortex for 1 min.
[0077] 3. Centrifuge at 12,000 rpm (13,400×g) for 5 minutes, and transfer the supernatant to a new cent...
Embodiment 2
[0101] In order to further verify the effectiveness of the above method, four hybrid varieties Zhongnong26, Qingyan2, Jinchun2 and Jinyou35 were detected by the above method.
[0102] The method for identification of cucumber female line varieties, the steps are as follows:
[0103] (1) extracting the DNA of the cucumber genome to be tested;
[0104] (2) take the genomic DNA of the cucumber to be tested as a template, and use the detection primers of SNP markers and InDel markers to carry out PCR amplification reaction;
[0105]The reaction system of the PCR amplification reaction is 25 lμl, as follows:
[0106] 10ng / μl template DNA 1μl, 10pmol / μl upstream primer 1μl, 10pmol / μl downstream primer 1μl, 10mmol / ldNTPmix 0.5μl, Taq DNA polymerase 1U, 10×PCR reaction buffer 2.5μl, the balance is ultrapure water.
[0107] The amplification program of the PCR amplification reaction is:
[0108] Gradient descent PCR was carried out first, denatured at 95°C for 30s, annealing tempera...
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Abstract
The invention relates to cucumber female character related SNP (single nucleotide polymorphism) maker and InDel maker and an application thereof. The SNP maker and the InDel maker are positioned at 9 SNP sites and 4 InDel sites in a range of 800-1300bp of an ACS1 upstream sequence of a cucumber; sequences in an area where all the SNP and Indel sites are positioned can be amplified by using a pair of primers, and whether the cucumber is female or not can be determined according to result of sequence analysis. The method provided by the invention can be used for cucumber female line marker assisted selective breeding, and can greatly improve the efficiency of female line selection and shorten the breeding time.
Description
technical field [0001] The invention relates to SNP markers and InDel markers related to cucumber female traits and applications thereof, belonging to the technical field of molecular biology. Background technique [0002] Cucumber is one of the vegetable crops with the largest cultivated area in my country. Cucumber hybrids bred from female lines have the characteristics of early maturity and high yield. In addition, using the female line to prepare a generation of hybrids can greatly reduce the cost of seed production and improve the purity of seed production. However, due to the lack of corresponding female line germplasm resources in our country, using conventional breeding methods to backcross foreign female sources into female lines of the domestic cucumber type and South China type cucumber, the breeding cycle is long, labor-intensive and time-consuming. Moreover, the expression of cucumber sex types is also affected by environmental conditions, and there is still a...
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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/68
Inventor 李利斌曹齐卫王永强孙小镭杨桂兰张志强
Owner VEGETABLE RES INST OF SHANDONG ACADEMY OF AGRI SCI
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