SNP molecular marker mk6398 and application thereof
A technology of molecular markers and progeny, applied in the direction of recombinant DNA technology, DNA/RNA fragments, microbial determination/inspection, etc.
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Embodiment 1
[0039] Example 1 Mapping Population and DNA Extraction
[0040] 1. Mapping groups
[0041] Wild-type jute (C. olitorius L.) germplasm J009 and jute (C. olitorius L.) variety Guangfengchangguo (GFG) with lower salt tolerance than J009 were used as parent plants. 150 individuals of the F2 generation were used to construct a high-resolution genetic map, and combined with the salt-tolerance traits of the F2:3 offspring for salt-tolerance QTLs mapping. Wild plant germplasm resources usually carry a large number of resistance genes, which are natural gene pools for stress-resistant breeding, so they are very suitable for the screening of jute salt-tolerant genes.
[0042] 2. DNA extraction
[0043] Genomic DNA was extracted from young leaf tissues of 150 F2 individuals and 2 parents to prepare a genotyping-by-sequencing (GBS) library. Wherein, genomic DNA was extracted using DNeasy PlantMini Kit (Tiangen, Beijing, China); DNA degradation and contamination were monitored by 1% aga...
Embodiment 2
[0044] The evaluation of embodiment 2 germination stage salt tolerance
[0045] Salt tolerance experiments were carried out in two different salt concentration environments (140mM and 160mM). The specific experiments are as follows:
[0046] The surface of the seeds of the mapping population (150 F2:3 families) was sterilized, and the seeds of each family were planted in two Petri dishes with the same sterilized paper in the amount of 30 seeds / dish and placed in the illumination culture Cultivate in the box (cultivation conditions: relative humidity 75%, 12h-12h day / night photoperiod, corresponding temperature is 28±0.5°C / 25±0.5°C), wherein, one culture dish is used for salt stress (salt stress group), and the other One Petri dish was used as a control (control group).
[0047] The design of the salt stress group and the control group is as follows: on the first day of cultivation, 3ml of sodium chloride solution was added to the culture dish of the salt stress group, and 3ml...
Embodiment 3
[0050] Example 3 library construction and sequencing
[0051] The GBS library of the DNA described in Example 1 was constructed according to the standard GBS scheme, specifically comprising:
[0052] 1. DNA was digested with restriction endonucleases MseI and HaeIII, and then barcoded adapters (barcodedadapters) were ligated to have individualized tags. The resulting restriction ligated samples with different barcode sequences were purified using AgencourtAMPure XP (Beckman);
[0053]2. Use Phusion Master Mix (NEB) universal primer (universal primer) and index primer (index primer) to perform PCR amplification on the purified sample to obtain complete i5 and i7 sequences;
[0054] 3. Use Agencourt AMPure XP (Beckman) to purify the PCR products and combine them, then electrophoresis on 2% agarose gel; use the gel extraction kit (Qiagen) to separate 375-400bp fragments (including index and aptamer);
[0055] 4. Use Agencourt AMPure XP (Beckman) to purify these fragment products...
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