A method and kit for detecting allelic variation of taagpl gene

Abstract

The invention discloses a primer pair, a kit and a detection method for detecting allelic variation of a TaAGPL gene. The primer pair is represented as the following 1): SEQ ID No.1 and SEQ ID No.3; and 2) SEQ ID No.2 and SEQ ID No.3. Through the primer pair for carrying out a PCR reaction, allelic variation of common wheat cytoplasm-type adenosine diphosphate glucose pyrophosphorylase gene can be identified easily and effectively. The method can be carried out just with common PCR instruments and DNA analyzers, is easy to operate, is simple and has high throughput.

Description

technical field

[0001] The invention belongs to the field of genetic engineering, and in particular relates to a method and a kit for detecting TaAGPL gene allelic variation. Background technique

[0002] Wheat is an allohexaploid, formed by hybridization of three chromosome groups during evolution, including three sets of chromosome groups A, B and D (AABBDD, 2n=6x=42). However, there are a large number of gene duplications between the A, B and D chromosome groups, and the repetitive sequences in wheat account for about 80% of the genome, especially for the isolation, identification and functional research of a gene controlling an important trait, which is easily affected by the homogeneity of different chromosomes. The influence of source genes has hindered the development of research on wheat genetics and breeding improvement. Wheat is one of the three most important food crops (wheat, rice and corn) in the world, with a total annual output of more than 600 million tons ...

Examples

Embodiment 1

[0046] Example 1. Detection of allelic variation of TaAGPL gene in wheat variety Zhongchunzhong and chromosome mapping

[0047] 1. Primer design

[0048] Firstly, the nucleotide sequences of TaAGPL (including cDNA and genomic DNA) of common wheat Xiaoyan 54 were compared by MegAlign software, and two characteristics were found in these gene sequences: (1) three homologous genes TaAGPL- 1A, TaAGPL-1B and TaAGPL-1D, the exon region is very conserved; (2) the intron region contains rich length polymorphisms and SNP differences ( figure 1 ). Depend on figure 1 It was found that there is a length difference between the genes TaAGPL-1A, TaAGPL-1B and TaAGPL-1D in the first intron (Intron I) (Table 1). Based on these two characteristics, a detection strategy for identifying TaAGPL genes was proposed: design intergenic conserved PCR primers according to the coding sequences of the conserved regions, amplify the DNA fragments of these TaAGPL genes by PCR, and then detect (using 3730...

Embodiment 2

[0070] Embodiment 2, detection of other wheat varieties TaAGPL gene allelic variation

[0071]Utilize the method of the present invention to have effectively analyzed the TaAGPL allelic variation type of backbone parent (more than 200 copies), modern variety and strain in wheat breeding, be used for guiding molecular breeding practice, pick 23 different allelic variation materials and carry out Display and PCR-based sequencing validation.

[0072] 1. Primer design

[0073] Two pairs of TaAGPL gene conservative primers designed in Example 1 were used (Table 3).

[0074] 2. PCR amplification

[0075] Genomic DNA was extracted using the conventional CTAB method.

[0076] Utilize the primers in Table 1 to carry out PCR with genomic DNA as a template, and the reaction system is as follows:

[0077]

[0078] Three, PCR amplification product type detection

[0079] Dilute the PCR product to 1 / 40 of the original concentration with ddH2O, take 5 μL of the diluted product into a...

technical field

[0001] The invention belongs to the field of genetic engineering, and in particular relates to a method and a kit for detecting TaAGPL gene allelic variation. Background technique

[0002] Wheat is an allohexaploid, formed by hybridization of three chromosome groups during evolution, including three sets of chromosome groups A, B and D (AABBDD, 2n=6x=42). However, there are a large number of gene duplications between the A, B and D chromosome groups, and the repetitive sequences in wheat account for about 80% of the genome, especially for the isolation, identification and functional research of a gene controlling an important trait, which is easily affected by the homogeneity of different chromosomes. The influence of source genes has hindered the development of research on wheat genetics and breeding improvement. Wheat is one of the three most important food crops (wheat, rice and corn) in the world, with a total annual output of more than 600 million tons ...

Examples

Embodiment 1

[0046] Example 1. Detection of allelic variation of TaAGPL gene in wheat variety Zhongchunzhong and chromosome mapping

[0047] 1. Primer design

[0048] Firstly, the nucleotide sequences of TaAGPL (including cDNA and genomic DNA) of common wheat Xiaoyan 54 were compared by MegAlign software, and two characteristics were found in these gene sequences: (1) three homologous genes TaAGPL- 1A, TaAGPL-1B and TaAGPL-1D, the exon region is very conserved; (2) the intron region contains rich length polymorphisms and SNP differences ( figure 1 ). Depend on figure 1 It was found that there is a length difference between the genes TaAGPL-1A, TaAGPL-1B and TaAGPL-1D in the first intron (Intron I) (Table 1). Based on these two characteristics, a detection strategy for identifying TaAGPL genes was proposed: design intergenic conserved PCR primers according to the coding sequences of the conserved regions, amplify the DNA fragments of these TaAGPL genes by PCR, and then detect (using 3730...

Embodiment 2

[0070] Embodiment 2, detection of other wheat varieties TaAGPL gene allelic variation

[0071]Utilize the method of the present invention to have effectively analyzed the TaAGPL allelic variation type of backbone parent (more than 200 copies), modern variety and strain in wheat breeding, be used for guiding molecular breeding practice, pick 23 different allelic variation materials and carry out Display and PCR-based sequencing validation.

[0072] 1. Primer design

[0073] Two pairs of TaAGPL gene conservative primers designed in Example 1 were used (Table 3).

[0074] 2. PCR amplification

[0075] Genomic DNA was extracted using the conventional CTAB method.

[0076] Utilize the primers in Table 1 to carry out PCR with genomic DNA as a template, and the reaction system is as follows:

[0077]

[0078] Three, PCR amplification product type detection

[0079] Dilute the PCR product to 1 / 40 of the original concentration with ddH2O, take 5 μL of the diluted product into a...

Images