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Primers for identifying types of rice GS5 gene and GLW7 gene and application of primers for identifying types of rice GS5 gene and GLW7 gene

A genotype and gene technology, applied in the field of primers to identify rice GS5 gene and GLW7 gene type, can solve the problems of high toxicity of gel components, cumbersome gel making process, and large fragment differences, so as to speed up the efficiency, process, and amplification Good effect, effect of reducing difficulty

Pending Publication Date: 2020-03-20
YANGZHOU UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The marker can be distinguished by PCR amplification. If the number of repeated sequences of the two varieties is very different, the fragments corresponding to the PCR amplification products will also have a large difference. Generally, the difference is greater than 10 bases and the amplified fragment does not exceed Corresponding to 10 times the number of different bases can be directly distinguished by agarose gel electrophoresis. When the number of SSR base differences is less than 10 bases, although PCR can be amplified, it is difficult to perform on agarose gel Differentiation must be done by means of acrylamide gel, and the latter has the disadvantages of cumbersome gel preparation process, long electrophoresis time and high toxicity of gel components

Method used

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  • Primers for identifying types of rice GS5 gene and GLW7 gene and application of primers for identifying types of rice GS5 gene and GLW7 gene
  • Primers for identifying types of rice GS5 gene and GLW7 gene and application of primers for identifying types of rice GS5 gene and GLW7 gene
  • Primers for identifying types of rice GS5 gene and GLW7 gene and application of primers for identifying types of rice GS5 gene and GLW7 gene

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Experimental program
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Effect test

Embodiment 1

[0035] Grain shape is an important trait that determines the yield and quality of rice. Larger grain shape can increase grain weight, thereby increasing yield, while longer grain shape can improve grain filling and filling degree, reduce chalkiness, and improve rice appearance quality. GS5 and GLW7 are two key grain shape genes that have been cloned in rice, corresponding to gene numbers Os05g0158500 and Os07g0505200, respectively. The core variation is mainly reflected in the difference of the simple repeat sequence (SSR) in the promoter region or 5UTR region, such as figure 1 As shown, the functional variation of GS5 is the AAAC sequence repeat difference at 324 to 327 bp upstream of ATG, variant 1 is CAAA, corresponding to wide-grained and heavy-grained phenotypes, and variant 2 is AAACAAAC, corresponding to normal phenotypes. The functional variation of GLW7 is the TCCACT sequence repeat difference at 133 to 150 bp upstream of ATG. Variant 1 is TCCACTTCCACT, corresponding t...

Embodiment 2

[0043] In order to verify the PCR amplification and enzyme digestion identification effects of the three pairs of markers, we selected eight varieties for analysis, namely YYP1, Nipponbare (NIP), Zhenshan 97 (ZS97), Daohuaxiang (DHX), Kasalath (Kasa) , WY3, Bao Da Li (BDL) and Katy.

[0044] Accelerate the germination and germination of the above-mentioned varieties, take samples of the leaves two weeks later, put them into a 2ml centrifuge tube with steel beads, and extract DNA by TPS small-scale extraction method. The specific steps are:

[0045] 1. Shake and crush the leaves with a ball mill, add 500ul TPS buffer, and place at 65°C for 45 minutes;

[0046] 2. Centrifuge at 12000 rpm for 10 minutes, suck 300ul supernatant into a new centrifuge tube, add an equal volume of isopropanol, and place at room temperature for 45 minutes;

[0047] 3. Centrifuge at 12,000 rpm for 10 minutes to obtain a DNA precipitate, discard the supernatant, and add 500ul of 75% ethanol;

[0048] ...

Embodiment 3

[0052] Some varieties may have variations in the primer binding sites, which may affect the amplification effect. We further clarified the identification effect of the two gene recognition primers on a large number of varieties. We collected 280 varieties distributed all over the world, planted them in the field, and extracted DNA from the leaves at the seedling stage. The extraction method is the same as above. Subsequently, the GS5 and GLW7 enzyme digestion identification markers were used for PCR amplification and enzyme digestion identification respectively, and finally the variation types of different varieties were obtained. The results of GS5 marker identification (Table 1) showed that bands were amplified in all 280 varieties, of which 52 varieties contained "AAAC" deletion wide-grain and heavy-grain variation, 224 varieties were wild type, and 3 varieties were heterozygous type, proving that the marker can be used for genotype identification of hybrid rice and F2 segr...

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Abstract

The invention discloses primers for identifying types of a GS5 gene and a GLW7 gene and application of the primers for identifying the types of the GS5 gene and the GLW7 gene, and belongs to the technical field of rice variety identification. According to the primers for identifying the types of the GS5 gene and the GLW7 gene and application of the primers for identifying the types of the GS5 geneand the GLW7 gene, by turning simply-repeated sequences of the rice GS5 gene and the rice GLW7 gene into digestion molecule markers, the types of the GS5 gene and the GLW7 gene are identified, and nucleotide sequences of the primers for identifying the types of the GS5 gene and the GLW7 gene are shown in SEQ ID NO.: 1-6 as shown in the description. By means of the molecule markers of the primersfor identifying the types of the GS5 gene and the GLW7 gene, the favorable variation types of the GS5 gene and the GLW7 gene can be accurately identified, the breeding screening efficiency can be improved, and a breeding screening process can be sped up; and the designed digestion molecule markers have good amplification effects, and selected enzymes are common enzymes which are low in price so that the identification cost can be greatly reduced, and the primers for identifying the types of the GS5 gene and the GLW7 gene are suitable for genotype analysis of a large number of varieties.

Description

technical field [0001] The invention belongs to the technical field of rice variety identification, and in particular relates to a primer for identifying rice GS5 gene and GLW7 gene type and application thereof. Background technique [0002] Rice is an important food crop. With the development of my country's economy and the improvement of people's living standards, higher requirements are put forward for rice varieties. However, the current traditional variety improvement takes a long time, and requires planting a large number of populations and screening target traits with the aid of naked eye observation. With the improvement of rice genome information and continuous breakthroughs in functional gene research, the use of molecular marker-assisted selection for directional improvement of varieties has become a effective means. SSR marker is a relatively common molecular marker, which is characterized by a simple repetition of a nucleic acid base sequence. The marker can b...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12N15/11
CPCC12Q1/6895C12Q2600/13C12Q2600/156
Inventor 张林谢东刘巧泉卞中邹怡婷范晓磊李钱峰张昌泉
Owner YANGZHOU UNIV
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