CN112609018B

CN112609018B - SNP molecular marker of rice grain type related gene GLW2 and application thereof

Info

Publication number: CN112609018B
Application number: CN202011443769.2A
Authority: CN
Inventors: 彭佩; 李文博; 郑秀婷; 周霆; 唐顺学; 肖金华; 田冰川
Original assignee: Huazhi Biotechnology Co ltd
Current assignee: Huazhi Biotechnology Co ltd
Priority date: 2020-12-11
Filing date: 2020-12-11
Publication date: 2022-09-13
Anticipated expiration: 2040-12-11
Also published as: CN112609018A

Abstract

The invention discloses an SNP molecular marker of a rice grain type related gene GLW2 and application thereof. The SNP molecular marker is K _020501, and the polymorphism of K _020501 is G or T. By utilizing the molecular marker, the rice grain type related gene GLW2 can be rapidly and accurately detected. The invention does not need complicated procedures such as enzyme digestion, electrophoresis, sequencing and the like in the detection process, reduces the pollution of aerosol and the use of toxic substances such as EB and the like, and is favorable for the efficient and environment-friendly application of the GLW2 gene in the commercial molecular breeding of rice.

Description

SNP molecular marker of rice grain type related gene GLW2 and application thereof

Technical Field

The invention relates to the field of rice breeding, in particular to an SNP (single nucleotide polymorphism) molecular marker of a rice grain type related gene GLW2 and application thereof.

Background

Rice is an important grain crop in China, and the rice has high yield and is safe to grain. In recent years, with the rapid increase of global population, the year-by-year decrease of cultivated land area, the trend of land circulation and non-grain transformation is increased, and the shortage of food is still a significant problem which puzzles many countries. Therefore, the development of rice genetic breeding research is always the central importance of the nation, and the maintenance of stable and high yield of grains becomes the primary goal of crop variety breeding and needs to be solved urgently.

The rice yield constituent elements mainly comprise effective spike number, spike grain number, grain weight, setting rate and the like, wherein the grain weight is mainly influenced by grain type and grouting. The yield character of the rice is a complex character determined by multiple genes, and is mainly regulated and controlled by 3 factors of the effective spike number of a single plant, the effective grain number of each spike and the thousand grain weight, the thousand grain weight heritability is highest, the grain size is mainly controlled by the grouting degree and the grain type, the grain type character is formed by the grain length, the grain width and the grain thickness, the grain weight can be selected in the rice seedling stage by selecting the rice grain type related genes through molecular markers, and the breeding efficiency can be greatly improved.

Disclosure of Invention

The present invention is directed to solving at least one of the problems of the prior art. Therefore, the invention provides an SNP molecular marker of a rice grain type related gene GLW 2.

The invention also provides a primer group for detecting the SNP molecular marker K _ 020501.

The invention also provides a detection method of the SNP molecular marker.

The invention also provides application of the SNP molecular marker.

The SNP molecular marker according to an embodiment of the first aspect of the invention, wherein the polymorphism of K _020501 is G or T, is K _ 020501.

According to some embodiments of the invention, the polymorphic site of the SNP molecular marker K _020501 is located at chr2.28865722(MSU7.0 position).

The Primer group for detecting the SNP molecular marker K _020501 according to the second embodiment of the invention comprises specific primers and universal primers, wherein the specific Primer sequences comprise Primer Seq Allele X and Primer Seq Allele Y, and the nucleotide sequence of the Primer Seq Allele X is shown in SEQ ID NO. 1; the nucleotide sequence of the Primer Seq Allle Y is shown in SEQ ID NO. 2; the nucleotide sequence of the universal primer is shown as SEQ ID NO. 3.

According to some embodiments of the present invention, preferably, the Primer Seq Allele X is connected to FAM or HEX fluorescent sequence at 5 'end, and Primer Seq Allele Y is connected to FAM and HEX fluorescent sequence at 5' end, respectively.

According to some embodiments of the invention, the primer set is used in rice breeding.

The method for detecting the SNP molecular marker according to the third aspect of the embodiment of the invention comprises the following steps:

s1, extracting genome DNA from rice leaves;

s2, detecting the K _020501 molecular marker by using the genomic DNA extracted in the step S1 as a template and using a primer of the SNP molecular marker K _ 020501;

s3, if the base of the SNP site of K _020501 is G, judging that the rice sample to be tested contains homozygous GLW2 gene; if the base of the detection site is T, judging that the tested rice sample contains homozygous GLW2 gene; if G, T is detected at the detection site, the rice to be detected is judged to contain heterozygous GLW2 gene.

According to some embodiments of the present invention, preferably, in step S1, the simplified CTAB method (cetyl trimethyl ammonium bromide method) is used to extract genomic DNA from rice leaves.

According to some embodiments of the present invention, preferably, in step S2, the SNP sites are detected using KASP (competitive allele specific PCR) technique.

The application of the SNP molecular marker according to the fourth embodiment of the invention is the application of the SNP molecular marker in rice breeding.

According to some embodiments of the invention, the application is detection using a K _020501 molecular marker, and the rice line carrying the GLW2 gene is selected for subsequent breeding.

According to some embodiments of the invention, the application is to provide a kit for detecting GLW2 gene SNP molecular markers, and the kit comprises primers with nucleotide sequences shown as SEQ ID NO. 1-3.

According to some embodiments of the invention, the kit is used for rice breeding.

According to some embodiments of the present invention, the application provides a gene chip, wherein the gene chip comprises primers with nucleotide sequences shown as SEQ ID NO. 1-3.

The SNP molecular marker of the rice grain type related gene GLW2 according to the embodiment of the invention has at least the following beneficial effects: by carrying out SNP molecular marking on the rice grain type related gene GLW2 and carrying out GLW2 gene detection on rice materials by applying KASP technical reaction, the rice grain type related gene GLW2 can be quickly and accurately detected in different germplasm resources of indica rice, japonica rice and the like. The invention carries out genotyping on the developed SNP marker by using the KASP technology, basically automates DNA extraction, PCR system construction, fluorescence signal detection and the like in the KASP technical process, can realize high-throughput detection of 96, 384 and 1536 pore plates, and is suitable for large-scale and high-throughput GLW2 gene identification and screening. The invention does not need complicated procedures such as enzyme digestion, electrophoresis, sequencing and the like in the detection process, reduces the pollution of PCR product aerosol and the use of toxic substances such as EB and the like, can simultaneously carry out foreground selection and background selection in the early stage of molecular marker-assisted breeding, improves the background recovery rate, reduces the scale of breeding groups, accelerates the breeding process, and is beneficial to the efficient and environment-friendly application of the GLW2 gene in the commercial molecular breeding of rice.

Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.

Drawings

FIG. 1 is a flow chart of the development of molecular markers according to an embodiment of the present invention;

FIG. 2 is a typing chart of the molecular marker K _020501 according to the embodiment of the present invention.

Detailed Description

In order to explain technical contents, achieved objects, and effects of the present invention in detail, the following description is made with reference to the accompanying drawings in combination with the embodiments. The test methods used in the examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are commercially available reagents and materials unless otherwise specified.

The embodiment of the invention is as follows: a design process of the molecular marker comprises the steps of determining a physical position through a cloned target gene GLW2, extracting SNP loci and flanking sequences, designing and synthesizing a primer sequence of the marker, and then carrying out screening test on the marker, wherein the design process is as follows:

1 primer design

The GLW2 (NCBI: LOC4330436) gene is determined to be within 15.3kb of the long arm end of chromosome 2, and the re-sequencing data of the donor material and the non-donor material of GLW2 are utilized to excavate SNP sites at two sides of the gene interval and the vicinity thereof. A miRNA396 binding site exists in the GLW2 gene interval, and in a gene donor material, the site is mutated and cannot be sheared by the miRNA396, so that the gene donor material is large in particle size. The selected SNP site was flanked by sequences extracted and primer-designed using the on-line primer design website BatchPrimer3(http:// probes. pw. usda. gov/BatchPrimer3 /). The primer design of K _020501 is shown in Table 1, each group is labeled with three primers, and the 5' ends of two specific primers are respectively connected with FAM and HEX fluorescent sequences. The primers were synthesized by Invitrogen corporation.

Table 1: primer design for K _020501

2 sample detection

DNA extraction: extracting genome DNA from rice leaf and adopting simplified CTAB method.

KASP reaction test: the KASP reaction assay was performed on the LGC SNPline genotyping platform. 20ng of DNA sample was added to the microplate, dried and added to the KASP reaction mixture, and the reaction system is shown in Table 2. PCR amplification is completed in a water bath thermal cycler, and the Touchdown PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 15 min; the first step of amplification reaction, denaturation at 94 ℃ for 20s, annealing at 61-55 ℃ and extension for 60s for 10 cycles, wherein the annealing and extension temperature in each cycle is reduced by 0.6 ℃; the second amplification reaction, denaturation at 94 ℃ for 20s, annealing at 57 ℃ and extension for 60s, 32 cycles. After the reaction is finished, a scanner Pherastar is used for reading fluorescence data of the KASP reaction product, and the result of fluorescence scanning can be automatically converted into a graph.

Table 2: KASP detection reaction system

	Final concentration	Volume (ul)
			100UM Primer C	0.42μM	0.0125
100UM Primer X	0.17μM	0.0050
			100UM Primer Y	0.17μM	0.0050
2x KASP Master Mix	1x	1.4792
			Ultrapure water		1.4983
Total volume		3

3 marking typing data

The results of the examination of KASP preliminary screening reaction of the rice variety containing the GLW2 gene and 23 rice varieties not containing it by the above-mentioned examination method using the marker K _020501 are shown in Table 3.

Table 3: k _020501 primary screening data

Material numbering	Name of Material	K_020501
			1	307R	T:T
2	Minghui 63	G:G
			3	Huanghuazhan (Huanghuazhan)	G:G
4	Small indica	G:G
			5	Shuhui 527	G:G
6	Zhenshan 97B	G:G
			7	Nanjing 11	G:G
8	9311	G:G
			9	IR24	G:G
10	IR64	G:G
			11	Perennial rice	G:G
12	Wild rice	G:G
			13	SLG1	G:G
14	Extra green tea	G:G
			15	Lemont	G:G
16	Kasalath	G:G
			17	Middle flower 11	G:G
18	Nipponbare	G:G
			19	Wuyujing 3	G:G
20	TP309	G:G
			21	294 of Liaojing tea	G:G
22	Pine and round-grained nonglutinous rice 6	G:G
			23	Asominori	G:G

As can be seen from Table 3, except that the large-particle material 307R has a base T as a test site detection result, 10 parts of rice materials detect a base G at the test site, and the accuracy of the invention in detecting the GLW2 gene is proved.

4 Natural population verification

The SNP marker K _020501 was subjected to natural population validation using 180 parts of the material. 180 parts of materials comprise known homozygous GLW2 gene-containing varieties and F1 materials constructed by common hybrid rice and core rice breeding and core breeding materials. The results of typing in the natural population are shown in fig. 2, only 1 part of the material was detected as a homozygous GLW2 genotype with large grain trait and as a known GLW2 donor, and the rest of the material was a homozygous GLW2 genotype with no large grains except for ambiguous typing and no amplification. Therefore, the SNP marker detection site is a high-specificity site, the GLW2 gene is not widely applied to the detection of common rice breeding, the GLW2 gene is introduced subsequently to directionally improve the rice gene, and K _020501 can be used for the efficient detection of the rice GLW2 gene.

The LGC SNpline genotyping platform used in the invention and the consumable materials of the reagents matched with the platform are purchased from LGC company in the United kingdom.

The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all equivalent changes made by using the contents of the present specification and the drawings, or applied directly or indirectly to the related technical fields, are included in the scope of the present invention.

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Claims

1. The application of the SNP molecular marker of the rice grain type related gene GLW2 in detecting the GLW2 gene is characterized in that: the SNP molecular marker is K _020501, wherein the polymorphism of K _020501 is G or T and is positioned at the chr2.28865722 of the rice in the MSU7.0 genome version.

2. A primer set for detecting the SNP molecular marker K _020501 as set forth in claim 1, characterized in that: the Primer group comprises specific primers and universal primers, wherein the specific primers comprise Primer Seq Allle X and Primer Seq Allle Y, and the nucleotide sequence of the Primer Seq Allle X is shown as SEQ ID No. 1; the nucleotide sequence of the Primer Seq Allle Y is shown in SEQ ID NO. 2; the nucleotide sequence of the universal primer is shown as SEQ ID NO. 3.

3. Use of the primer set according to claim 2 for rice breeding.

4. A method for detecting the SNP molecular marker according to claim 1, wherein: the detection method comprises the following steps:

s1, extracting genome DNA from rice leaves;

s2, using the genomic DNA extracted in the step S1 as a template, and using the primer group of claim 2 to detect the K _020501 molecular marker;

s3, if the base of the SNP site of K _020501 is G, judging that the rice sample to be tested contains homozygousGLW2A gene; if the base of the detection site is T, determining that the rice sample to be tested is homozygousGLW2A gene; if G, T are detected at the same time, determining that the rice to be detected contains heterozygosisGLW2A gene.

5. The detection method according to claim 4, characterized in that: in step S2, the SNP sites are detected by the KASP technique.

6. The SNP molecular markers according to claim 1 in rice breedingThe application is characterized in that: the application is to use K-020501 molecular marker for detection and selection of carrierGLW2And (4) carrying out subsequent breeding on the genic rice strain.

7. A kit, characterized in that: the kit comprises a primer with a nucleotide sequence shown as SEQ ID NO. 1-3.

8. A gene chip, which is characterized in that: the gene chip comprises a primer with a nucleotide sequence shown as SEQ ID NO. 1-3.