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PCR detection primer for coix lacryma-jobi Waxy gene mutation, and application thereof

A technology for detecting primers and Coix coix, which is applied in the field of PCR detection primers for Coix coix Waxy gene mutations, can solve the problems of high heterogeneity of Coix coix varieties, unusable, difficult purification and rejuvenation, and variety selection, achieving good stability and accuracy The effect of high sex and accelerated breeding process

Pending Publication Date: 2020-05-26
贵州黔西南喀斯特区域发展研究院
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, the molecular detection technology of Waxy also has specificity in different crops, and the Waxy genotyping technology of wheat and other crops cannot be used for the Waxy genotyping of Coix.
For a long time, Coix has been basically in a wild or semi-wild state, and genetic improvement has rarely been carried out. Coupled with the characteristics of alienated pollination crops, the heterogeneity of Coix varieties is high, and it is difficult to purify, rejuvenate and breed.

Method used

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  • PCR detection primer for coix lacryma-jobi Waxy gene mutation, and application thereof
  • PCR detection primer for coix lacryma-jobi Waxy gene mutation, and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Coix selection:

[0032] (1) Select the mature and full grains of 'Xingren Baike' coix variety, and sow them by direct seeding, with a row spacing of 50 cm and a plant spacing of 30 cm, with a seeding rate of 7 grains per litter to ensure the emergence rate.

[0033] (2) Randomly select 30 Coix seedlings, numbered 1-30, take a small amount of Coix leaves to extract Coix genome DNA, use the extracted Coix genome DNA as a template, and use PCR primers to perform specific PCR amplification reaction;

[0034] The total volume of the PCR reaction system is 25 μL, containing 12.5 μL of Taq PCR MasterMix, 1 μL of 10 μM forward primer, 1 μL of 10 μM reverse primer, ddH 2 O 9.5 μL, DNA template 30-50 μg.

[0035] PCR reaction program: pre-denaturation at 94°C for 3 min, denaturation at 94°C for 50 s, annealing at 65°C for 50 s, extension at 72°C for 5 min, 32 cycles, final extension at 72°C for 10 min, storage at 12°C.

[0036] The PCR products were electrophoresed on 0.8% aga...

Embodiment 2

[0041] Coix selection:

[0042] (1) Select mature and plump seeds of the 'Guangxi Baiyi' variety, and sow them by direct seeding, with a row spacing of 50 cm and a plant spacing of 30 cm, with a seeding rate of 7 grains per litter to ensure the emergence rate, and seedlings should be reserved at the 3-leaf stage.

[0043] (2) Randomly select 10 Coix seedlings, numbered 31-40, take a small amount of Coix leaves to extract Coix genome DNA, use the extracted Coix genome DNA as a template, and use PCR primers to carry out specific PCR amplification reaction;

[0044] The total volume of the PCR reaction system is 25 μL, containing 12.5 μL of Taq PCR MasterMix, 1 μL of 10 μM forward primer, 1 μL of 10 μM reverse primer, ddH 2 O 9.5 μL, DNA template 30-50 μg.

[0045] PCR reaction program: pre-denaturation at 94°C for 3min, denaturation at 94°C for 50s, annealing at 67°C for 50s, extension at 72°C for 5min, 32 cycles, final extension at 72°C for 10min, storage at 12°C.

[0046] Th...

Embodiment 3

[0051] Coix selection:

[0052] (1) Select the mature and plump seeds of the 'An Guo Yi Yi' variety, sow by direct seeding method, row spacing 80cm, plant spacing 50cm, sowing rate 10 grains / litter to ensure emergence rate, 6-leaf stage fixed seedlings and reserved seedlings.

[0053] (2) randomly select 8 Coix seedlings, numbered 40-48, take a small amount of Coix leaves to extract Coix genome DNA, use the extracted Coix genome DNA as a template, and use PCR primers to perform specific PCR amplification reaction;

[0054] The total volume of the PCR reaction system is 25 μL, containing 12.5 μL of Taq PCR Master Mix, 1 μL of 10 μM forward primer, 1 μL of 10 μM reverse primer, ddH 2 O 9.5 μL, DNA template 30-50 μg.

[0055] PCR reaction program: pre-denaturation at 94°C for 3 min, denaturation at 94°C for 60 s, annealing at 69.7°C for 50 s, extension at 72°C for 5 min, 32 cycles, final extension at 72°C for 10 min, storage at 12°C. The PCR products were electrophoresed on 0.8...

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Abstract

The invention belongs to the field of plant breeding, and relates to a PCR detection primer for coix lacryma-jobi Waxy gene mutation, and an application thereof. The detection primer comprises a PCR forward primer and a reverse primer, wherein the PCR forward primer is 5'-GGACGTCAGCGAGTGGACC-3', and the binding region is a ninth exon conserved region; the PCR reverse primer is 5'-ACGAGTCACACAGGGTGGACG-3', and the binding region of the PCR reverse primer is an eleventh exon conserved region. The invention further discloses the application of the PCR detection primer in coix lacryma-jobi breeding. The PCR detection primer is used for japonica and waxy typing of a single coix lacryma-jobi plant. The mutation site of the Waxy gene of the coix lacryma-jobi can be specifically identified, and genotyping can be carried out. The typing marker of the Waxy gene belongs to a co-dominant marker, so that whether japonica rice and waxy rice of the single coix lacryma-jobi plant are heterozygous or not can be effectively distinguished, and the PCR detection primer can be used for breeding japonica rice and waxy rice varieties, thereby accelerating the breeding process. Japonica rice and glutinousrice identification and screening are carried out on the coix lacryma-jobi single plant at a molecular level, the influence of a plant growth environment is avoided, the stability is good, and the accuracy is high.

Description

technical field [0001] The invention belongs to the field of plant breeding, and relates to a PCR detection primer for mutation of Coix Waxy gene and application thereof. Background technique [0002] Coix is ​​an annual or perennial herb of the genus Coix (Coix L.) of the Poaceae family. It is a typical representative of "medicine (medicine) and food" crops with the same origin. It has anti-tumor, lowering blood sugar, inhibiting tumor angiogenesis, improving immunity, and beautifying the skin And other pharmacological effects, can treat edema, chronic gastrointestinal ulcers, lung impotence, warts, arthritis, xenon and uterine tumors and other diseases. [0003] Coix is ​​the same as other cereal crops, its grains are also divided into japonica and waxy, which is determined by the content and ratio of amylose and amylopectin in the endosperm. The synthesis of starch in the endosperm is a complex metabolic process, and granule-boundstarch synthase (GBSS) is the key enzyme ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12Q1/6858C12N15/11
CPCC12Q1/6895C12Q1/6858C12Q2600/13C12Q2600/156C12Q2531/113C12Q2565/125
Inventor 李祥栋潘虹魏心元陆秀娟
Owner 贵州黔西南喀斯特区域发展研究院
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