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Application of a bayberry CAPS marker in detection of allo-related MrMYB1 Alleles in Chinese Bayberry

An allele and marker technology, applied in the field of CAPS markers, can solve the problems of time-consuming, laborious and low efficiency

Pending Publication Date: 2019-01-11
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, the commonly used method to identify the MrMYB1 allele of Myrica rubra is cloning and sequencing, but this method is time-consuming, laborious and inefficient.

Method used

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  • Application of a bayberry CAPS marker in detection of allo-related MrMYB1 Alleles in Chinese Bayberry
  • Application of a bayberry CAPS marker in detection of allo-related MrMYB1 Alleles in Chinese Bayberry
  • Application of a bayberry CAPS marker in detection of allo-related MrMYB1 Alleles in Chinese Bayberry

Examples

Experimental program
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Effect test

Embodiment 1

[0028] Example 1: CAPS mark development ideas

[0029] The research proved that MrMYB1 gene sequence contains 2 exons, 119bp and 625bp respectively, and 1 intron, 246bp. The allele MrMYB1d is missing a C at the 30th position after the start codon (ATG) (attached figure 1 ), resulting in a frameshift mutation that cannot synthesize normal MrMYB1 protein. It was found that this deletion mutation caused the loss of a HhaI recognition site (5'-GCGC-3') of the sequence, thus enabling the development of CAPS markers.

[0030] In order to make the PCR product of allele MrMYB1 produce well-separated bands after HhaI digestion and be applicable to both genomic DNA and cDNA, the selected upstream and downstream primers should have different distances from the restriction site, and the distance between the upstream and downstream primers should be different. No additional HhaI cleavage site should be present. Considering the above two requirements, the upstream primer was finally des...

Embodiment 2

[0031] Embodiment 2: CTAB method extracts Waxberry genome DNA

[0032] (1) Prepare CTAB genomic DNA extract, the formula is: 2% CTAB (hexadecyl triethyl ammonium bromide, Hexadecyl trimethyl ammonium bromide), 0.1M Tris, 20mM EDTA, 1.4M NaCl, pH 8.0.

[0033] (2) Collect the young leaves of red bayberry.

[0034] (3) Put it into liquid nitrogen and grind it into powder, weigh about 0.7g and transfer it into a 10ml centrifuge tube filled with 4ml CTAB solution and 80μL β-mercaptoethanol (preheated at 65°C), bathe in 65°C water for 1h; add 4ml Chloroform / isoamyl alcohol (24:1, v / v), mix well, centrifuge at 12000rpm for 10min, 15°C, take supernatant and add 4ml chloroform / isoamyl alcohol (24:1, v / v) again, mix well , centrifuged at 12000rpm for 10min. Aspirate the supernatant, add 2ml 5M NaCl, 4ml isopropanol (pre-cooled at -20°C), mix well, and place at -20°C for 2h. Centrifuge at 12000rpm for 15min, discard the supernatant, rinse the precipitate twice with 75% ethanol, and d...

Embodiment 3

[0035] Embodiment 3: RNA extraction and cDNA synthesis of red bayberry fruit

[0036](1) Prepare CTAB RNA extract, formula is: 2% CTAB, 2% PVP-K30 (vinylpyrrolidone iodine, polyvinylpyrrolidone), 100mL 1M Tris, 50mL 0.5M EDTA, 2M NaCl, 0.5% spermidine, pH 8.0.

[0037] (2) Gather ripe red bayberry fruit.

[0038] (3) Extract RNA. Put the bayberry fruit into liquid nitrogen and grind it into powder, weigh about 1.0g and transfer it into a 10mL centrifuge tube filled with 4mL CTAB solution and 80μL β-mercaptoethanol (preheated at 65°C), bathe in 65°C water for 5min; add 4ml chloroform / Isoamyl alcohol (24:1, v / v), mix well, centrifuge at 10000rpm for 10min, take the supernatant and add 4ml of chloroform / isoamylalcohol (24:1, v / v), mix well, and centrifuge at 12000rpm for 10min. Aspirate the supernatant, add 2M LiCl, mix well, and place at 4°C overnight. Centrifuge at 10,000 rpm for 20 min at 4°C, discard the supernatant, dissolve the precipitate with SSTE at 65°C, add 400 μL...

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Abstract

The invention provides application of a bayberry CAPS marker in detection of allo-related MrMYB1 Alleles in Chinese Bayberry, wherein the CAPS marker is a set of primer pairs and a restriction enzyme,the primer pair sequence is SEQ ID NO: 1-2, that restriction endonuclease is HhaI. The invention can easily, accurately and efficiently identify the alleles MrMYB1 and MrMYB1d of the transcription factor MrMYB1 gene of Myrica rubra regulating anthocyanin biosynthesis and different genotypes of Myrica rubra. The molecular marker can be used to predict the coloring ability of Myrica rubra fruits and molecular assisted selection.

Description

technical field [0001] The invention belongs to the fields of plant molecular biotechnology and genetic engineering, and relates to a CAPS marker for detecting MrMYB1 alleles related to the synthesis of myrica rubra anthocyanins. Background technique [0002] Red bayberry (Morella rubra) is a characteristic fruit in southern my country. Its fruit is bright in color, unique in flavor and rich in bioactive substances, and is deeply loved by consumers. Anthocyanins are the characteristic pigments of red bayberry fruit. Different varieties have different colors due to the different anthocyanin contents. For example, the fruit of 'Water Chestnut', which accumulates a large amount of anthocyanins, is purple-black, and the fruit of 'Dongkui', which accumulates a large amount of anthocyanins, is purple-black. The fruit of 'Pink' that accumulates a small amount of anthocyanins is pink, and the fruit of 'Crystal' that does not accumulate anthocyanins is white. [0003] Studies have s...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12N15/11
CPCC12Q1/6895C12Q2600/13C12Q2600/156
Inventor 刘晓芬徐昌杰王文丽朱长青陈昆松
Owner ZHEJIANG UNIV
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