A kasp marker primer for detecting wx-d1 gene in waxy wheat k107wx1 and its application

A technology for labeling primers and waxy wheat, which is applied in the field of molecular genetics and breeding to achieve the effects of improving detection efficiency, shortening the transfer process and improving breeding efficiency

Active Publication Date: 2019-06-14
JIANGSU LIXIAHE REGION AGRI RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Waxy Wheat K107Wx1 Wx-A1b and Wx-B1b Genes have been reported and utilized as co-dominant markers, while Wx-D1 Gene co-dominant markers have not been reported yet

Method used

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  • A kasp marker primer for detecting wx-d1 gene in waxy wheat k107wx1 and its application
  • A kasp marker primer for detecting wx-d1 gene in waxy wheat k107wx1 and its application
  • A kasp marker primer for detecting wx-d1 gene in waxy wheat k107wx1 and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Embodiment 1, waxy wheat K107Wx1 Wx-D1 Gene high-throughput KASP marker design and its dedicated primer sequence development.

[0039] Based on the white fire wheat developed by Shariflou et al. (2001) Wx-D1bThe co-dominant markers of the genes amplified wheat K107Wx1, Kanto107, Ningmai 14 and Caiwx ( Wx-D1b ), the K107wx1 Wx-D1d There was no difference between the gene and common wheat Ning 14 amplified bands, both of which were 840bp, while the amplified band of waxy wheat Caiwx was 260bp, which was significantly different from the other three wheat varieties, such as figure 1 shown. The results indicated that the co-dominant marker could not be used for K107Wx1 Wx-D1 Gene molecular marker assisted breeding, and K107Wx1 Wx-D1 The type of gene deletion is different from that of white fire wheat Wx-D1b , further research is needed to develop SNP co-dominant molecular markers suitable for K107Wx1.

[0040] According to Hu Yaping (2008) used Wx-D1 Gene-specif...

Embodiment 2

[0049] Example 2, KASP molecular marker detection in waxy wheat K107Wx1 Wx-D1 Genetic method established.

[0050] 1. Genomic DNA extraction:

[0051] The leaf tissue of the wheat variety to be tested was taken, and the whole genome DNA was extracted by CTAB method.

[0052] 2. Using the DNA extracted in step 1 as a template, use the DNA developed in Example 1 for the detection of glutinous wheat K107Wx1 Wx- D1 The specific primers for the KASP marker of the gene were amplified by PCR to obtain the amplified product.

[0053] Preparation of KASP labeled primer working solution:

[0054] Take 12 μl (100 μM) of upstream primers and 30 μl (100 μM) of downstream primers, supplement them with sterile ultrapure water to 100 μl, and use them as KASP-labeled primer working solutions.

[0055] PCR amplification reaction system: Contains 2 μl of DNA template (20-30ng / μl), 0.08 μl of primer working solution, 2.5 μl of KASP 2×Master Mix (LGC Company, KBS-1016-002), supplemented with...

Embodiment 3

[0063] Example 3. Amplification verification between K107Wx1, Kanto107 and common wheat Ningmai 14 using KASP molecular markers and their use in breeding:

[0064] 1. Test materials:

[0065] The test materials include: waxy wheat K107Wx1 (AA genotype) as the donor parent, non-waxy wheat Ningmai 14 (GG genotype) as the reincarnated parent. Wx-D1 Transformation with deletion of protein subunits. K107Wx1 was crossed with Ningmai 14 to obtain the corresponding F1 generation, F1 was backcrossed with the recurrent parent Ningmai 14 for 8 generations to obtain BC8F1 population, and self-crossed to obtain BC8F2 population. choose Wx-D1 Gene normal Kanto107 (GG genotype) was used as the control for molecular marker-assisted screening.

[0066] 2. KASP molecular marker detection:

[0067] Using the above-mentioned high-throughput molecular marker system to analyze the genotypes of the parents and BC8F1 population, and using the known genotype Kanto107 as a control, the test results...

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Abstract

The invention discloses a KASP labeled primer for detecting a Wx-D1 gene in waxy wheat K107Wx1 and application of the KASP labeled primer, and belongs to the technical field of molecular genetic breeding; and three groups of KASP primers provided by the invention, when used for detecting the Wx-D1 genotype in the waxy wheat K107Wx1, are low in human error and high in analysis throughput, and are suitable for detecting a great amount of samples. A codominant molecular marker provided by the invention, when applied to transfer breeding of the Wx-D1 gene in the waxy wheat K107Wx1, has an important practical significance for accelerating genetic improvement by virtue of the Wx-D1 gene in the waxy wheat K107Wx1 and f improving a breeding efficiency.

Description

technical field [0001] The invention belongs to the technical field of molecular genetic breeding, in particular to waxy wheat Wx-D1 Development and application of high-throughput molecular markers for genes. Background technique [0002] Starch is the main component of wheat endosperm and plays a key role in food and non-food industries. Starch contains 20% to 30% amylose and 70% to 80% amylopectin, of which granule-bound starch synthase (GBSS I) is responsible for the synthesis of amylose, also known as Wx protein. It consists of the 7AS, 4AL and 7DS Wx-A1, Wx-B1, Wx-D1 Three genes code for synthesis. Deletion or inactivation of any Wx protein subunit will lead to a decrease in amylose content, and different contents of amylose directly affect the final use quality of different wheat products (Liu Yingchun, 2005). [0003] All three Wx protein subunits are naturally missing. For example, the Japanese wheat cultivar Kanto 107 (Kanto107) lacks the Wx-Al and Wx-Bl protei...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12N15/11
CPCC12Q1/6895C12Q2600/156
Inventor 程顺和裔新胡文静蒋正宁高德荣张勇别同德
Owner JIANGSU LIXIAHE REGION AGRI RES INST
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