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KASP labeled primer for detecting Wx-D1 gene in waxy wheat K107Wx1 and application of KASP labeled primer

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

Active Publication Date: 2016-07-27
JIANGSU LIXIAHE REGION AGRI RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Co-dominant markers of Wx-A1b and Wx-B1b genes in waxy wheat K107Wx1 have been reported and utilized, but co-dominant markers of Wx-D1 genes have not been reported

Method used

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  • KASP labeled primer for detecting Wx-D1 gene in waxy wheat K107Wx1 and application of KASP labeled primer
  • KASP labeled primer for detecting Wx-D1 gene in waxy wheat K107Wx1 and application of KASP labeled primer
  • KASP labeled primer for detecting Wx-D1 gene in waxy wheat K107Wx1 and application of KASP labeled primer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1. Design of high-throughput KASP marker for Wx-D1 gene in waxy wheat K107Wx1 and development of its special primer sequence.

[0039] When the co-dominant marker based on the Wx-D1b gene developed by Shariflou et al. There was no difference in the amplified bands of Ning 14, 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 1shown. The results show that the co-dominant marker cannot be used for molecular marker-assisted breeding of the Wx-D1 gene of K107Wx1, and the type of Wx-D1 gene deletion in K107Wx1 is different from that of Wx-D1b in white fire wheat. Molecular markers.

[0040] According to the Wx-D1 gene-specific primers D1, D2, D3, and D4 (Table 1) used by Hu Yaping (2008), the full length of the Wx-D1 gene sequence in Kanto 107 and K107Wx1 was amplified, and the gel was recovered and sequenced. The two sequences were compared, and...

Embodiment 2

[0049] Example 2. Establishment of a method for detecting Wx-D1 gene in waxy wheat K107Wx1 by KASP molecular marker.

[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 special primers developed in Example 1 for detecting the KASP marker of the Wx-D1 gene in waxy wheat K107Wx1 to amplify 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, add 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 KASP2×MasterMix (LGC Company, KBS-1016-002), supplemented to 5 μl with sterile ultrapure water . Among...

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 recurrent parent, and the Wx-D1 protein subunit deletion was transgenic. 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. Kanto107 (GG genotype) with normal Wx-D1 gene was selected 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 of ...

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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 the development and application of high-throughput molecular markers for the Wx-D1 gene of waxy wheat. 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 (Granule-boundstarchsynthase, GBSSI) is responsible for the synthesis of amylose, also known as Wx protein. It is coded and synthesized by three genes Wx-A1, Wx-B1 and Wx-D1 located at 7AS, 4AL and 7DS. 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...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
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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