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Genotype identification primer and method of rice amylose content control gene Wx and identification method

A technology of amylose content and identification method, applied in the field of genotype identification primers and identification of rice amylose content control gene Wx, can solve the problem of high cost and inability to distinguish homozygous individual plants from heterozygous individual plants , limited application and other issues, to achieve the effect of low cost

Active Publication Date: 2013-09-04
INST OF FOOD CROPS HUBEI ACAD OF AGRI SCI
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  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

According to the Wx In1 site sequence, Cai Xiuling et al. (Journal of Plant Physiology and Molecular Biology, 2002, 28(2):137-144) developed the PCR-AccⅠ molecular marker, which is a co-dominant marker, but the disadvantage is that the PCR fragment needs enzyme digestion, cumbersome procedures and high cost; Mao Xingxue et al. (China Rice Science, 2005, 19(3): 285-287) developed a one-step PCR detection marker, which can be completed by simple PCR electrophoresis, but it is not obvious Sex markers, the SNP type of the target site can only be judged by the presence or absence of electrophoresis bands, and it is impossible to distinguish homozygous individual plants from heterozygous individual plants, thus limiting its application in breeding

Method used

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  • Genotype identification primer and method of rice amylose content control gene Wx and identification method
  • Genotype identification primer and method of rice amylose content control gene Wx and identification method
  • Genotype identification primer and method of rice amylose content control gene Wx and identification method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1: a kind of genotype identification method of rice amylose content control gene Wx Its steps are as follows:

[0035] 1) Biomaterials

[0036] G-type materials are: Teqing, Jin 23B, Ejin B, Zhong 9B, Yangdao 4, Yuetai B, R287, HR141, HR820, Jinke 1B, R8006;

[0037] T-shaped materials are: 9311, Mianhui 725, Fuhui 838, HD9802S, Ezao 18, Minghui 63, Huanghuazhan, Yue4A, Y58S, Guangzhan 63S, Zaoyou 143, Yuetai A.

[0038] 2) DNA extraction from rice

[0039] The CTAB method was used to extract DNA from the above materials.

[0040] 3) PCR

[0041] The PCR reaction system is 20 μL, containing 2.0 μL 10×Buffer, 1.0 μL dNTPs (10 mmol / L), 0.5 μL each of 4 10 μmol / L primers (see Table 1 for primer sequences), 0.2 μL Taq enzyme (5U / μL), 2.0 μL template DNA, 12.8 μL ddHO 2 O; PCR reaction program: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 1 min, annealing at 58°C for 1 min, extension at 72°C for 1 min, a total of 35 cycles; extension at 72°C f...

Embodiment 2

[0044] Example 2: Wx In1 SNP site in F 2 Identification of single gene segregators in populations.

[0045] 1) Biomaterials

[0046] F 2 group, choose F 2 Group of 100 individual plants.

[0047] 2) CTAB method to extract DNA from the above materials.

[0048] 3) PCR

[0049] Refer to Example 1 for the process.

[0050] 4) Result analysis

[0051] Using the present invention to mark pair F 2 See image 3 , where P1 is the G-type parent Zhenshan 97, P2 is the T-type parent Minghui 63, and lanes 1-21 are 21 F strains constructed from the two parents. 2 , the homozygous G-type individual plant has the same 207bp band as the G-type parent Zhenshan 97, the homozygous T-type individual plant has the same 235bp band as the T-type parent Minghui 63, and the GT heterozygous individual plant has 207bp at the same time With the 235bp band, the three genotypes also have a 387bp common band, and the genotype of the individual plant is marked below the corresponding lane, so the m...

Embodiment 3

[0052] Example 3: Correlation analysis between the base type of In1SNP site and amylose content in different varieties

[0053] The results of Wx In1 base identification of 150 conventional rice varieties using this marker showed that there were 97 T-type varieties and 53 G-type varieties; The average amylose content of G-type rice is 22.5% ( Figure 4 ), the t-test showed that there was a very significant difference between the two (P<0.01), and the one-way analysis of variance showed that the Wx In1SNP site explained 77.8% of the phenotypic variation of the amylose content, so Wx is the main factor controlling the amylose content Effective gene, In1G / T replacement caused the difference in amylose content of two different genotype materials, using the markers developed in this study can determine the Wx genotype of different rice varieties to predict the level of amylose content of the variety, so that through the Wx Genetic selection aids in the selection of varieties with ...

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Abstract

The invention discloses a genotype identification primer and method of a rice amylose content control gene Wx. The level of AC (amylose content) depends on a basic group type of a first intron+1(Inl) site of the Wx gene. According to the genotype identification primer and method, a set of functional markers for identifying different basic group types at a WxInl site is developed. The method comprises the following steps of: carrying out PCR (Polymerase Chain Reaction) amplification by means of the markers and genomic DNA (Deoxyribonucleic Acid) of a rice variety to be detected; respectively amplifying 207bp or 235bp special stripes by using the Inl site as an isozygotic G or T basic group genotype material, wherein GT heterozygotic material has the two strips at the same time. The three strips share one 387bp stripe which can be used as reference of the existing Wx gene. Therefore, the Wx genotype identification method is simple and rapid and low in cost, and can be widely applied to molecular marker-assisted selection to cultivate varieties with different ACs applied to different industrial applications or assistant selection of moderate AC varieties applied to quality breeding of rice.

Description

technical field [0001] The invention belongs to the field of molecular genetics, and specifically relates to a genotype identification primer for rice amylose content control gene Wx, and also relates to a genotype identification method for rice amylose content gene Wx, which is simple, accurate, It has the advantages of low cost and non-destructive detection in the early growth stage of rice, and is suitable for mass screening of Wx genotypes in breeding so as to breed varieties with different amylose content. technical background [0002] Rice is a staple food for more than half of the world's population, and improving rice quality has always been a major goal of breeders. The main component of rice is starch (accounting for about 90% of polished rice), including two types of amylose and amylopectin. Amylose content (AC) is an important physical and chemical index of rice quality. The level of AC is related to the viscosity of rice, Softness, luster and eating quality are...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 蔡海亚徐得泽吴建平游艾青周雷程建平赵锋
Owner INST OF FOOD CROPS HUBEI ACAD OF AGRI SCI
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