Method for detecting TaAGPS gene allelic variation relevant to thousand kernel weight

An allelic variation and 1000-grain weight technology, applied in the field of in situ variation, can solve the problems of reduced endosperm starch accumulation, no instantaneous starch synthesis, and reduced AGPase enzyme activity, achieving the effect of easy operation.

Inactive Publication Date: 2016-08-31
INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI
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Problems solved by technology

The study by Yano et al. showed that the mutation of rice AGPS2 gene led to the decrease of AGPase activity and the accumulation of endosperm starch. Ventriglia et al. analyzed the T-DNA insertion mutant aps1 of AGPase small subunit (APS1) in Arabidopsis and found that AGPase activity was completely lost, and the leaf There is almost no amount of transient starch synthesis in
Therefore, it is particularly important to study the small cytoplasmic AGPase subunit (TaAGPS) in wheat, but so far, there are almost no reports on the screening of good allelic variants of the TaAGPS gene and its molecular markers in wheat, which greatly hinders the development of wheat. Application of AGPase, a key gene of starch synthesis, in improvement of wheat yield traits

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  • Method for detecting TaAGPS gene allelic variation relevant to thousand kernel weight
  • Method for detecting TaAGPS gene allelic variation relevant to thousand kernel weight
  • Method for detecting TaAGPS gene allelic variation relevant to thousand kernel weight

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Embodiment 1

[0033] Embodiment 1, different 1000-grain weight materials TaAGPS gene sequence difference analysis

[0034] 1. Primer design

[0035] By using MegAlign software to compare the genomic DNA sequences and cDNA sequences on different chromosomes of common wheat Chinese spring TaAGPS, it was found that these gene sequences have the following characteristics: (1) Three homologous genes TaAGPS-7A, TaAGPS- 7B and TaAGPS-7D have some SNP polymorphisms in the exon region; (2) The exon and intron-dense regions of the three homologous genes TaAGPS-7A, TaAGPS-7B and TaAGPS-7D are distributed in the back of the gene half ( figure 1 ). Therefore, we proposed a scheme to analyze the difference of TaAGPS gene sequence in different 1000-grain weight materials: select the second half region covering the exon and intron-intensive regions of TaAGPS gene to design intergenic conserved PCR primers, and obtain them by gene cloning. Contains the sequence information of different TaAGPS genes, thro...

Embodiment 2

[0050] Embodiment 2, detection of other wheat varieties TaAGPS gene allelic variation

[0051] By comparing the sequence differences of the two alleles, it was found that, compared with the high thousand-grain weight allele, the low thousand-grain weight allele TaAGPS-7A-T contains A DdeI endonuclease site (CTCAG), but the high thousand-grain weight allele TaAGPS-7A-G (CGCAG) has no such endonuclease site. Therefore, according to the SNP at position 5092, a CAPS marker using DdeI endonuclease as a tool was developed to distinguish the two allelic variants of TaAGPS-7A. For the convenience of operation, the conservative primer pair (SEQ ID No: 1 and SEQ ID No: 2) was used to amplify a 636bp fragment containing three homologous genes. After digestion with DdeI, the TaAGPS-7A-T type produced 234bp and 402bp Two short fragments, while no short fragments were produced for the TaAGPS-7A-G type. After electrophoresis in 2% agarose gel, two allelic variations of TaAGPS-7A-G and TaAG...

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Abstract

The invention discloses a method for detecting wheat cytoplasmic adp glucose pyrophosphorylase small subunit (TaAGPS) gene excellent allelic variation relevant to thousand kernel weight by using CAPS (cleaved amplified polymorphic sequence) marks. According to the method, the corresponding basic groups of two allelic variations (TaAGPS-7A-T and TaAGPS-7A-G) in the 5092 site of the TaAGPS-7A exon region III are respectively T and G; the corresponding amino acid is Ser and Ala; firstly, a primer is used for performing PCR (polymerase chain reaction) amplification on SEQ ID No: 1 and SEQ ID No: 2; after the enzyme digestion by using DdeI incision enzyme, electrophoresis detection is performed; the authentication of different allelic variations of the TaAGPS genes in wheat germplasm resources and later generation breeding can be conveniently and efficiently realized. The operation can be completed through conventional PCR and agarose gel electrophoresis methods; the operation is convenient; stability and feasibility are realized.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a method for using CAPS (Cleaved Amplified Polymorphic Sequence) markers to detect the superior allelic variation of TaAGPS gene related to thousand-grain weight. Background technique [0002] The genome of wheat is an allohexaploid, consisting of three highly homologous genomes, A, B, and D. The genome is large and complex, which provides a challenge for the development of wheat genetics and breeding research. Wheat is one of the most important food crops in the world and is widely grown around the globe. Studies such as Rajaram show that: with the increase of the world population, it is expected that the global demand for wheat will further increase by 40% in 2020, so cultivating high-yield wheat varieties has become one of the main breeding goals of current breeders. The yield of wheat is composed of three elements: the number of spikelets, the number of grains ...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 刘冬成张爱民马晓玲阳文龙孙家柱余慷
Owner INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI
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