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Molecular marker for wheat functional genes NAM-B1 and application of molecular marker

A technology of NAM-B1 and molecular markers, applied in the direction of recombinant DNA technology, DNA / RNA fragments, etc., can solve the problems of indistinguishability, limitation, cumbersome operation, etc.

Inactive Publication Date: 2017-08-04
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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Problems solved by technology

The molecular markers used in current reports are not designed based on the sequence characteristics of the functional NAM-B1 gene itself, but are designed based on other sequence sites linked to the functional NAM-B1 gene site. There are the following shortcomings in application: 1 , chromosomal recombination during mitosis may lead to the separation of molecular markers and linked functional NAM-B1 gene loci, that is, molecular markers cannot continue to anchor functional NAM-B1 genes, especially when the design site of molecular markers is far from the functional 2. These molecular markers are designed for a specific wheat donor material and recipient material, due to the genetic background of wheat germplasm resources However, the stability of the site characteristics of these molecular markers in other wheat materials cannot be guaranteed, that is, the site characteristics of molecular markers may vary among different materials, resulting in inapplicability in other materials, making the molecular marker only in certain wheat materials. Applicable between a specific donor material and recipient material; 3. As mentioned above, these molecular markers are not suitable for screening and identifying wheat materials containing functional NAM-B1 genes from a large number of wheat germplasm resources
4. Most of these molecular markers are of the CAPS (Cleavage Amplified Polymorphic Sequences) type. After PCR amplification, specific restriction enzyme digestion reactions are required, which is cumbersome, time-consuming, and costly to operate.
Since the molecular marker is designed in a conserved region, it is impossible to effectively distinguish the functional NAM-B1 gene with only individual base differences from the mutated NAM-B1 gene
In addition, since functional genes such as NAM-A1, NAM-D1, NAM-B2, and NAM-D2 already exist in wheat, there is no need to obtain these genes through molecular marker-assisted selection. Its application in discovering functional NAM-B1 gene and molecular marker-assisted breeding in wheat

Method used

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  • Molecular marker for wheat functional genes NAM-B1 and application of molecular marker
  • Molecular marker for wheat functional genes NAM-B1 and application of molecular marker

Examples

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Effect test

Embodiment 1

[0028] Example 1 Development of Wheat Functional NAM-B1 Gene Molecular Marker

[0029] Forward primers: NAM-A1 (KM820886), NAM-B1 (KM820887), NAM-D1 (KM820889), NAM-B2 (KM820888) and NAM-D2 (KM820890) cloned in wheat according to Wu Dan et al. (2015) The cDNA sequence of the gene was compared and analyzed by BLAST in the wheat genome database (http: / / www.wheatgenome.org / ) to obtain the gene sequence of each member. Since these genes have relatively high homology between the start codon ATG and the stop codon TGA, while the sequences on both sides have low homology, the NAM-B1 gene sequence in the upstream promoter region of ATG was selected to be compatible with other genes. Forward PCR amplification primers were designed for the differential sites of the 4 functional homologous gene sequences ( figure 1 a) to ensure specific amplification of the NAM-B1 gene sequence. The primer sequence is SpNAM-B1-F: 5'-CATAGAAGGAAGTGGCGGAATACT-3', located 424bp - 446bp upstream of the AT...

Embodiment 2

[0032] Example 2 Using molecular marker SpNAM-B1 to screen and identify wheat materials containing functional NAM-B1 gene.

[0033] Select 22 Chinese wheat varieties (Ningmai 3, Ningmai 9, Ningmai 10, Ningmai 11, Ningmai 13, Ningmai 16, Ningmai 17, Ningmai 19, Ningmai 20, Ningmai 21, Ningmai 22, Ningmai 23, Yangmai 9, Yangmai 16, Yangmai 20, Huamai 5, Huamai 6, Ningnuomai 1, Ningyan 1, Nongfeng 126, Zhenmai 5 , Zhenmai No. 6) and two wheat varieties (Jathti. Keratchna and Tankokeratchua) from Northern Europe (Finland) were used as research materials, and the genomic DNA of each wheat variety was extracted. All samples were amplified by PCR using microsatellite marker (microsatellite marker) WMS261 to detect the integrity of the template DNA ( figure 2 a) PCR amplification using universal primers of the NAM-B1 gene to detect whether it contains the NAM-B1 gene sequence ( figure 2 b); use molecular marker SpNAM-B1 to carry out PCR amplification to detect whether it contains ...

Embodiment 3

[0057] Example 3 Application of Molecular Marker SpNAM-B1 in Wheat Molecular Marker-Assisted Breeding.

[0058] In the wheat breeding process, molecular markers can be used to select the segregated progeny in each generation, and the individuals containing the functional NAM-B1 gene can be accurately obtained to enter the next breeding generation, which can greatly improve the breeding efficiency. The involved experimental technique and process are the same as in Example 2.

[0059] Although the present invention has been described in detail with general descriptions and specific embodiments above, some modifications or improvements can be made on the basis of the present invention. The present invention is characterized by using the difference sites between the NAM-B1 gene and the other four functional homologous genes (NAM-A1, NAM-D1, NAM-B2 and NAM-D2) sequences and the functional NAM - Design the forward primer and reverse primer at the SNP site between the B1 gene and th...

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Abstract

The invention relates to a molecular marker for wheat functional genes NAM-B1 and application of the molecular marker. Primer pair sequences for carrying out specific PCR (polymerase chain reaction) amplification on the molecular marker SpNAM-B1 for the wheat functional genes NAM-B1 are shown as SpNAM-B1-F and SpNAM-B1-R. The molecular marker and the application have the advantages that whether wheat contains the functional genes NAM-B1 or not can be identified by the aid of the molecular marker; the molecular marker can be used for carrying out high-protein molecular breeding on the wheat, the functional genes NAM-B1 for controlling grain protein contents can be transferred into receptor materials without the functional genes under assisted selection effects of the molecular markers, and accordingly the molecular marker and the application are beneficial to cultivating high-protein new wheat varieties with high iron and zinc contents.

Description

technical field [0001] The invention relates to a molecular marker of wheat functional NAM-B1 gene and its application in wheat variety identification and molecular marker assisted breeding. Background technique [0002] wheat( Triticum aestivum ) are important food crops, the staple food for about 30% of the world's population and nearly 20% of total global calorie consumption (IWGSC, 2014; Pfeifer et al., 2014). Wheat is an allohexaploid, and its genome consists of three sets of chromosomes (AABBDD), which is derived from tetraploid wild emmer wheat ( T. turgidum , genome type AABB) and the diploid G. A. tauschii , genome type DD) hybridization, domesticated wheat has accounted for more than 95% of the planting area of ​​different types of wheat crops worldwide (IWGSC, 2014; Marcusse et al., 2014). [0003] Wheat is an important source of protein and trace elements iron and zinc. The protein it provides accounts for 25% of the total consumption, and the iron and zinc ...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/68
Inventor 王化敦马鸿翔姚金保高春蕾
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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