CN113046349B

CN113046349B - SNP molecular marker combination for detecting rice Wx gene and application thereof

Info

Publication number: CN113046349B
Application number: CN202011564137.1A
Authority: CN
Inventors: 彭佩; 李文博; 吴云天; 唐顺学; 肖金华; 田冰川
Original assignee: Huazhi Biotechnology Co Ltd
Current assignee: Huazhi Biotechnology Co Ltd
Priority date: 2020-12-25
Filing date: 2020-12-25
Publication date: 2021-11-16
Anticipated expiration: 2040-12-25
Also published as: CN113046349A

Abstract

The invention discloses an SNP molecular marker combination for detecting rice Wx gene, which comprises 4 molecular markers, namely Wx01, K _060585, K _060586 and K _ 060591; wherein the polymorphism of Wx01, the polymorphism of G/T, K _060585, the polymorphism of A/C, K _060586, the polymorphism of T/C, K _060591, is C/A. The molecular marker provided by the invention can be used for efficiently detecting the Wx gene, can be used for different haplotypes and assisted selective breeding of the Wx gene, can accelerate the breeding efficiency, reduce the breeding cost, shorten the breeding period, can also carry out purposeful polygene polymerization, improve the breeding efficiency and bring huge social and economic benefits. The marker is used for genotyping by applying the KASP technical principle, does not need enzyme digestion, electrophoresis, sequencing and the like, is simple and convenient to operate, and is beneficial to high-throughput rapid detection.

Description

SNP molecular marker combination for detecting rice Wx gene and application thereof

Technical Field

The invention belongs to the technical field of molecular biology and crop breeding, and particularly relates to a SNP molecular marker combination for detecting rice Wx genes and application thereof.

Background

Amylose Content is a key factor affecting rice quality, and rice Wx gene coded granular starch synthase (GBSS) is a main effective gene for controlling Amylose synthesis and directly affects Amylose Content (AC) in rice endosperm and pollen. The non-waxy gene (Wx) shows incomplete dominance to the waxy gene (Wx), and a relatively obvious dose effect exists. Differentiation of Wx genes to Wx in non-waxy varieties^aAnd Wx^bTwo alleles, of which wild rice is Wx^aIndica rice expressed as Wx^aMainly, the amylose content is higher; japonica rice is basically Wx^bThe amylose content is lower. In addition to the common 3-allelic forms, the Wx gene locus also has Wx^op、Wx^mp、Wxⁱⁿ、Wx^HH、Wx^IVAnd multiple allelic types, corresponding to different amylose content phenotypes.

In rice breeding, different Wx haplotypes of parents are often matched to improve rice quality, and detection of Wx haplotypes becomes a crucial link in the process. The traditional rice breeding method selects through identifying phenotypes, is long in time consumption, is easy to be limited by environmental conditions, is easy to cause errors in identification results, and is low in selection efficiency. The Wx gene molecular marker is used for assisting selection, so that the breeding efficiency can be accelerated, the breeding cost can be reduced, the breeding period can be shortened, purposeful polygene polymerization can be performed, the breeding efficiency is improved, and great social and economic benefits are brought. Most of molecular markers disclosed in the literature are SSR, Indel and CAPS markers, gel electrophoresis detection is required, reagents such as nucleic acid dyes and the like used in the detection process are harmful to the environment and human bodies, the automation degree is low, the detection flux is small, and in addition, the situation of non-specific amplification can occur sometimes, so that the detection result cannot be prepared and judged, and the detection efficiency is limited to a great extent.

Disclosure of Invention

It is a first object of the present invention to provide a molecular marker.

The second objective of the invention is to provide a group of specific primers.

A third object of the present invention is to provide a kit.

The fourth purpose of the invention is to provide the application of the molecular marker in auxiliary identification of rice Wx genotype and/or auxiliary breeding.

The fifth purpose of the invention is to provide the application of the primer in detecting the Wx genotype of the rice and/or assisting breeding.

It is a sixth object of the present invention to provide a method for detecting Wx genotype.

The seventh purpose of the invention is to provide the application of the molecular marker or the primer or the kit in improving the rice quality.

The eighth purpose of the present invention is to provide the application of the above molecular marker combination, primer combination or kit in cultivation of Wx haplotype rice.

The technical scheme adopted by the invention is as follows:

in a first aspect of the present invention, a SNP molecular marker is provided, which includes one or more of the following four molecular markers: wx01, K _060585, K _060586, and K _ 060591; wherein the polymorphism of Wx01, the polymorphism of G/T, K _060585, the polymorphism of A/C, K _060586, the polymorphism of T/C, K _060591, is C/A.

According to the SNP molecular marker of the first aspect, the SNP locus of Wx01 is located at the 1765760 th base on the No. 6 chromosome of rice; the SNP locus of the K _060585 is located at the 1768006 th base on the No. 6 chromosome of rice; the SNP locus of the K _060586 is located at 1768998 th base on No. 6 chromosome of rice; the SNP locus of the K _060591 is located at 1767032 th base on the No. 6 chromosome of rice.

In a second aspect of the invention, a set of primers is provided, said primers being used for amplifying the SNP molecular markers according to the first aspect of the invention.

According to a second aspect of the present invention,

the primer sequences used to amplify Wx01 were:

specific primers:

Primer X：5’-CAGGAAGAACATCTGCAAGG-3’(SEQ ID NO.1)；

Primer Y：5’-TCAGGAAGAACATCTGCAAGT-3’(SEQ ID NO.2)；

the general primer is as follows:

Primer C：5’-TCTGAATAAGAGGGGAAACAA-3’(SEQ ID NO.3)；

the primer sequence for amplifying K _060585 was:

specific primers:

Primer X：5’-GAGATCAATTRTAACTCACCAT-3’(SEQ ID NO.4)；

Primer Y：5’-AGATCAATTRTAACTCACCAG-3’(SEQ ID NO.5)；

the general primer is as follows:

Primer C：5’-ACCTCAACAACAACCCATAC-3’(SEQ ID NO.6)；

the primer sequences used for amplification of K _060586 were:

specific primers:

Primer X：5’-CTGGAGGAACAGAAGGGCT-3’(SEQ ID NO.7)；

Primer Y：5’-CTGGAGGAACAGAAGGGCC-3’(SEQ ID NO.8)；

the general primer is as follows:

Primer C：5’-TCCTGCATGAGCTCCGGGAT-3’(SEQ ID NO.9)；

the primer sequences used for amplifying K _060591 were:

specific primers:

Primer X：5’-TTCCAGGGCCTCAAGCCCC-3’(SEQ ID NO.10)；

Primer Y：5’-GTTCCAGGGCCTCAAGCCCA-3’(SEQ ID NO.11)；

the general primer is as follows:

Primer C：5’-TCGTCACGCTGAGCGACGTC-3’(SEQ ID NO.12)。

preferably, the specific primers are respectively connected with different fluorescent linker sequences.

Further, the fluorescent linker sequences are FAM and HEX fluorescent linker sequences.

Further, the FAM and HEX fluorescent linker sequences are respectively connected to the 5' ends of the two specific primers.

In a third aspect of the invention, a kit is provided, wherein the kit comprises the primer combination of the second aspect of the invention.

In a fourth aspect of the invention, the application of the molecular marker combination of the first aspect of the invention in assisting identification of rice Wx genotype and/or assisting breeding is provided.

In a fifth aspect of the invention, the application of the primer combination of the second aspect of the invention or the kit of the third aspect of the invention in the detection of the Wx genotype of rice and/or in assisted breeding is provided.

Specifically, the primer or the kit can be used for detecting the Wx genotype, so that the breeding efficiency can be accelerated, the breeding cost can be reduced, the breeding period can be shortened, purposeful polygene polymerization can be performed, the breeding efficiency can be improved, and the breeding can be assisted.

In a sixth aspect of the present invention, there is provided a method for detecting Wx genotype, comprising the steps of:

s1: performing KASP reaction detection on a rice sample genome by using the primer of the second aspect of the invention or the kit of the third aspect of the invention; wherein, the two specific primers are respectively connected with different fluorescent joint sequences;

s2: if only the fluorescent signal corresponding to the fluorescent joint sequence connected with the Primer Y is detected, judging the rice sample to be a Y-type allele type; if only detecting the fluorescent signal corresponding to the fluorescent joint sequence connected with the Primer X, judging the rice sample to be X-type allelic gene type; if two kinds of fluorescence are detected simultaneously, the rice sample is judged to be heterozygous genotype, the typing conditions of 4 markers are combined, and the type of the Wx gene in the tested rice sample is judged according to the combined haplotype.

Further, the rice to be detected is judged to be a heterozygous genotype, the genotype results of 4 markers are combined, the Wx type of the sample is judged according to the haplotype, if the TAAC haplotype is detected by Wx, K _060591, K _060585 and K _060586, the sample is Wx type, and if the GCAC haplotype is detected by Wx, K _060591, K _060585 and K _060586, the sample is Wx type^aType, if Wx, K _060591, K _060585 and K _060586 detect GCAT haplotype, the sample is Wx^HHType, if Wx, K _060591, K _060585 and K _060586 detect GCCC haplotype, the sample is WxⁱⁿType, if Wx, K _060591, K _060585 and K _060586 detect TCAC haplotype, the sample is Wx^bAnd (4) molding.

In a seventh aspect of the invention, there is provided the use of a molecular marker combination according to the first aspect of the invention or a primer combination according to the second aspect of the invention or a kit according to the third aspect of the invention for improving rice quality.

Specifically, Wx genotypes can be detected by molecular marker combination auxiliary detection or primer combination or kit, so that Wx haplotypes are obtained, and rice quality is improved by using different parent pairing of Wx haplotypes.

In an eighth aspect of the present invention, there is provided a use of the molecular marker combination of the first aspect of the present invention or the primer combination of the second aspect of the present invention or the kit of the third aspect of the present invention in breeding rice with different Wx haplotypes.

The invention has the beneficial effects that:

the invention provides an SNP molecular marker which can be used for an automatic instrument to efficiently detect Wx genes and can be used for different haplotypes and auxiliary selective breeding of the Wx genes. The selection is carried out by identifying the genotype, the required time is short, the identification result is accurate, the selection efficiency is low, the Wx haplotype is accurately detected, and the rice quality is further improved by conveniently matching parents. The Wx gene molecular marker is used for assisting selection, so that the breeding efficiency can be accelerated, the breeding cost can be reduced, the breeding period can be shortened, purposeful polygene polymerization can be performed, the breeding efficiency can be improved, and great social and economic benefits can be brought. The marker is used for genotyping by applying the KASP technical principle, does not need enzyme digestion, electrophoresis, sequencing and the like, is simple and convenient to operate, and is beneficial to high-throughput rapid detection.

Drawings

FIG. 1 is a flow chart of development of SNP molecular markers.

FIG. 2 is a typing diagram of molecular marker Wx 01.

FIG. 3 is a typing diagram of a K _060591 molecular marker.

FIG. 4 is a typing diagram of the molecular marker K _ 060586.

FIG. 5 is a typing chart of the molecular marker K-060585.

Detailed Description

Preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 development of SNP molecular markers of Wx Gene

Early studies show that Wx has various allelic forms, SNPs corresponding to different haplotypes are found according to published sequences of the articles, the position of Wx is determined in 1766194-1770656 interval of chromosome 6 of rice according to literature information, and the flanking sequences of variant SNP sites are extracted by referring to the position of Nipponbare genome (MSU 7.0). Aiming at the candidate SNP markers, the marker designed based on the KASP reaction principle and the single base difference of the materials can be used for carrying out Wx gene detection on the rice material in high flux, 4 SNP markers Wx, K _060591, K _060586 and K _060585 which can distinguish different material types and have good amplification effect are selected, and different haplotypes of the Wx gene are distinguished in a combined mode. The scheme for developing the SNP molecular marker is shown in figure 1.

And primer design was performed using the online primer design website BatchPrimer3(https:// probes. pw. usda. gov/batchPrimer3 /). Each group is marked with three primers, and the 5' ends of two specific primers are respectively connected with FAM and HEX fluorescent joint sequences. The primers were synthesized by Invitrogen corporation. Each label consists of three primers, the 5' ends of the two specific primers are respectively connected with a fluorescent joint sequence specific to a KASP reaction reagent of LGC company, and a universal primer. Specific primer information is shown in table 1.

If only the fluorescence signal corresponding to the primer PrimerX is detected from the PCR product of the sample, the detection site is the X-type allele type; if only the fluorescence signal corresponding to the primer PrimerY is detected, the detection site is Y-type allele type; if two fluorescence signals are detected simultaneously, detecting the locus, judging that the rice to be detected is a heterozygous genotype, combining genotype results of 4 markers, judging the Wx type of the sample according to the haplotype, if Wx, K _060591, K _060585 and K _060586 detect TAAC haplotype, the sample is Wx type, if Wx, K _060591, K _060585 and K _060586 detect GCAC haplotype, the sample is Wx type^aType, if Wx, K _060591, K _060585 and K _060586 detect GCAT haplotype, the sample is Wx^HHType, if Wx, K _060591, K _060585 and K _060586 detect GCCC haplotype, the sample is WxⁱⁿType, if Wx, K _060591, K _060585 and K _060586 detect TCAC haplotype, the sample is Wx^bAnd (4) molding.

TABLE 1 Wx tag information

Example 2 detection of KASP reaction of SNP molecular marker

1. The primer combination is designed aiming at the molecular marker combination, the KASP reaction can be used for detecting whether the rice variety contains the Wx gene in high flux, and the primer combination designed in the embodiment 1 is adopted.

2. Extracting genome DNA from rice leaves by a 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; performing a first-step amplification reaction, namely performing denaturation at 94 ℃ for 20 seconds, annealing at 65-57 ℃ and extending for 60 seconds for 10 cycles, wherein the annealing and extending temperature of each cycle is reduced by 0.8 ℃; the second amplification reaction, denaturation at 94 ℃ for 20 seconds, annealing at 57 ℃ and extension for 60 seconds, 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. The LGC SNpline genotyping platform used in this example and its associated reagent consumables were purchased from LGC, Inc., UK.

TABLE 2 reaction System for KASP detection

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

Example 3 Natural population validation of SNP molecular markers

KASP reaction verification was performed using 96 rice varieties labeled with Wx01 molecular marker in the graph shown in FIG. 2, K _060591 molecular marker in the graph shown in FIG. 3, K _060586 molecular marker in the graph shown in FIG. 4, and K _060585 molecular marker in the graph shown in FIG. 5, and the results are shown in Table 3, except for ambiguous materials, 84 rice varieties were classified into Wx, Wx^a、Wx^HH、WxⁱⁿAnd Wx^bFive haplotypes, the most of which are Wx^bThe type and japonica rice material are all of the type, and the results are consistent with the results of the previous researches, which also indicates that the molecular marker combination in the example 1 can accurately detect the Wx genotype.

TABLE 3 Mark typing data for Wx

The above embodiments are merely preferred examples to illustrate the present invention, and it should be apparent to those skilled in the art that any obvious variations and modifications can be made without departing from the spirit of the present invention.

SEQUENCE LISTING

<110> Huazhi Biotechnology Ltd

<120> SNP molecular marker combination for detecting rice Wx gene and application thereof

<130>

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<170> PatentIn version 3.5

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Claims

1. A set of primers, wherein the primers are used for amplifying SNP molecular markers, and the SNP molecular markers are composed of the following four molecular markers: wx01, K _060585, K _060586 and K _ 060591; wherein the polymorphism of Wx01, the polymorphism of G/T, K _060585, the polymorphism of A/C, K _060586, the polymorphism of T/C, K _060591, is C/A; the SNP locus of Wx01 is located at 1765760 th base on the No. 6 rice chromosome; the SNP locus of the K _060585 is located at the 1768006 th base on the No. 6 chromosome of rice; the SNP locus of the K _060586 is located at 1768998 th base on No. 6 chromosome of rice; the SNP locus of the K _060591 is located at 1767032 th base on the No. 6 chromosome of rice.

2. The primer according to claim 1,

the primer sequences used to amplify Wx01 were:

specific primers:

Primer X：5’-CAGGAAGAACATCTGCAAGG-3’（SEQ ID NO.1）；

Primer Y：5’-TCAGGAAGAACATCTGCAAGT-3’（SEQ ID NO.2）；

the general primer is as follows:

Primer C：5’-TCTGAATAAGAGGGGAAACAA-3’（SEQ ID NO.3）；

the primer sequence for amplifying K _060585 was:

specific primers:

Primer X：5’-GAGATCAATTRTAACTCACCAT-3’（SEQ ID NO.4）；

Primer Y：5’-AGATCAATTRTAACTCACCAG-3’（SEQ ID NO.5）；

the general primer is as follows:

Primer C：5’-ACCTCAACAACAACCCATAC-3’（SEQ ID NO.6）；

the primer sequences used for amplification of K _060586 were:

specific primers:

Primer X：5’-CTGGAGGAACAGAAGGGCT-3’（SEQ ID NO.7）；

Primer Y：5’-CTGGAGGAACAGAAGGGCC-3’（SEQ ID NO.8）；

the general primer is as follows:

Primer C：5’-TCCTGCATGAGCTCCGGGAT-3’（SEQ ID NO.9）；

the primer sequences used for amplifying K _060591 were:

specific primers:

Primer X：5’-TTCCAGGGCCTCAAGCCCC-3’（SEQ ID NO.10）；

Primer Y：5’-GTTCCAGGGCCTCAAGCCCA-3’（SEQ ID NO.11）；

the general primer is as follows:

Primer C：5’-TCGTCACGCTGAGCGACGTC-3’（SEQ ID NO.12）。

3. the primer of claim 2, wherein the specific primers are linked to different fluorescent linker sequences.

4. A kit comprising the primer according to any one of claims 1 to 3.

The application of the SNP molecular marker in the auxiliary identification of the rice Wx genotype and/or the auxiliary breeding is characterized in that the SNP molecular marker comprises the following four molecular markers: wx01, K _060585, K _060586 and K _ 060591; wherein the polymorphism of Wx01, the polymorphism of G/T, K _060585, the polymorphism of A/C, K _060586, the polymorphism of T/C, K _060591, is C/A; the SNP locus of Wx01 is located at 1765760 th base on the No. 6 rice chromosome; the SNP locus of the K _060585 is located at the 1768006 th base on the No. 6 chromosome of rice; the SNP locus of the K _060586 is located at 1768998 th base on No. 6 chromosome of rice; the SNP locus of the K _060591 is located at 1767032 th base on the No. 6 chromosome of rice.

6. Use of the primer according to any one of claims 1 to 3 or the kit according to claim 4 for detecting the Wx genotype of rice and/or assisting in breeding.

7. A method for detecting the genotype of Wx comprising the steps of:

s1: performing KASP reaction detection on a rice sample genome by using the primer according to any one of claims 1 to 3 or the kit according to claim 4; wherein, the two specific primers are respectively connected with different fluorescent joint sequences;

The application of the SNP molecular marker in improving the rice quality is characterized in that the SNP molecular marker consists of the following four molecular markers: wx01, K _060585, K _060586 and K _ 060591; wherein the polymorphism of Wx01, the polymorphism of G/T, K _060585, the polymorphism of A/C, K _060586, the polymorphism of T/C, K _060591, is C/A; the SNP locus of Wx01 is located at 1765760 th base on the No. 6 rice chromosome; the SNP locus of the K _060585 is located at the 1768006 th base on the No. 6 chromosome of rice; the SNP locus of the K _060586 is located at 1768998 th base on No. 6 chromosome of rice; the SNP locus of the K _060591 is located at 1767032 th base on the No. 6 chromosome of rice.

9. Use of the primer according to any one of claims 1 to 3 or the kit according to claim 4 for improving rice quality.