CN111304360B - Rice good taste gene REQ2 codominant molecular marker and amplification primer and application thereof - Google Patents

Abstract

The invention belongs to the field of molecular biology and genetic breeding, and particularly relates to a rice good taste gene REQ2 codominant molecular marker, and an amplification primer and application thereof. The codominant molecular marker comprises RM13990 and RM14045, a specific amplification primer is designed aiming at the molecular marker, the excellent taste gene REQ2 of the rice is detected through PCR amplification, and the rice variety or material with obviously improved taste can be obtained through the auxiliary screening of the REQ2 gene molecular marker.

Description

Rice good taste gene REQ2 codominant molecular marker and amplification primer and application thereof

Technical Field

The invention belongs to the field of molecular biology and genetic breeding, and particularly relates to a rice fine taste gene REQ2 codominant molecular marker, and an amplification primer and application thereof.

Background

In recent years, with the development of socioeconomic and the improvement of living standard, the requirements of people on rice quality are increased correspondingly, and especially the requirements on taste quality are higher and higher. Therefore, good taste becomes an important target for rice breeding, and the quality of the taste is a key for determining whether rice is sold successfully and can occupy the market for a long time. The Heilongjiang province is in the dominant production area of japonica rice in China, and is the core production area of high-quality rice in the northeast, and brands such as Wuchang rice, xiangshui rice and the like are known in China. How to rapidly and efficiently breed varieties with good taste becomes the most important topic in the national rice breeding field including Heilongjiang.

Molecular genetic studies have shown that the taste of rice is a quantitative trait controlled by multiple genes. Compared with the appearance quality traits of rice grain shape, chalkiness and the like, the taste trait of rice is very important, and the conventional improvement difficulty is very high. At present, only a few genes or loci have been located which control rice taste. The application of the loci in rice molecular breeding is rarely reported.

The existing genes for improving the taste of rice are few, and most of the existing special benefits for improving the taste are the use of alleles of amylose regulatory sites of rice, such as a breeding method of good-taste rice in a patent CN 101268757A; a polymerization breeding method of a rice variety with good taste and rice blast resistance disclosed in patent CN105613255A and a polymerization breeding method of a rice variety with good taste and rice blast resistance disclosed in patent CN 110169383A are used for improving taste by using allele of Wx. It is different from the method of directly using taste gene to improve.

In addition, the taste improvement of the rice at the present stage is still based on the conventional selection at the early stage, and the identification and screening at the later stage are the main technical means. On one hand, the selection efficiency is low, and the purpose is not strong; on the other hand, the selection period is long and time and labor are consumed. Therefore, the development of the taste gene and the functional codominant molecular marker thereof has important significance for breeding rice with excellent taste traits.

Disclosure of Invention

In view of the above technical problems, the present invention provides a co-dominant molecular marker of rice superior taste gene REQ2, and an amplification primer and application thereof.

The invention has the following conception:

the invention firstly locates a gene REQ2 from 'Longdao 18' for controlling good taste on the 2 nd chromosome, identifies PCR practical economic markers RM13990 and RM14045 closely linked with the good taste gene, and can effectively carry out molecular-assisted selective breeding research on the good taste of rice.

The invention aims at providing a rice superior taste gene REQ2 codominant molecular marker, which is RM13990 and/or RM14045, wherein the nucleotide sequence of RM13990 is shown as SEQ ID NO.1, and the nucleotide sequence of RM14045 is shown as SEQ ID NO. 2.

The second purpose of the invention is to provide a primer for amplifying the co-dominant molecular marker, wherein the primer for amplifying RM13990 is RM13990-F and RM13990-R, the nucleotide sequence is shown as SEQ ID NO.3-4, the primer for amplifying RM14045 is RM14045-F and RM14045-R, and the nucleotide sequence is shown as SEQ ID NO. 5-6.

The third object of the present invention is to provide a detection reagent or a kit containing the primer.

The fourth object of the present invention is to provide any one of the following applications of the co-dominant molecular marker:

1) The application in the genotype identification of the rice good taste gene REQ 2;

2) The application in identifying the excellent taste character of rice;

3) The application in identifying and screening the rice germplasm resources with good taste;

4) The application in molecular marker assisted breeding of rice fine taste variety.

The fifth purpose of the invention is to provide any one of the following applications of the primer:

1) The application in the genotype identification of the rice good taste gene REQ 2;

2) The application in identifying the excellent taste character of rice;

3) The application in identifying and screening the rice germplasm resources with good taste;

4) The application in molecular marker assisted breeding of rice fine taste variety.

Further, the genotype identification of the rice fine taste gene REQ2 comprises the following steps:

performing PCR amplification on rice by using RM13990-F and RM13990-R, amplifying a 405bp nucleotide sequence shown in SEQ ID NO.1 from a material carrying REQ2, and amplifying a 397bp nucleotide sequence from a material without REQ 2;

performing PCR amplification on rice by using RM14045-F and RM14045-R, amplifying a 121bp nucleotide sequence shown in SEQ ID NO.2 from a material carrying REQ2, and amplifying a 131bp nucleotide sequence from a material without REQ 2;

utilizes RM13990-F, RM13990-R, RM14045-F and RM14045-R to carry out PCR amplification on rice, amplifies a nucleotide sequence of 405bp shown in SEQ ID NO.1 and a nucleotide sequence of 121bp shown in SEQ ID NO.2 from a material carrying REQ2, and amplifies a nucleotide sequence of 397bp and a nucleotide sequence of 131bp from a material without REQ 2.

Further, the PCR amplification system was 15. Mu.L containing 50mM KCl,10mM Tris-HCl (pH 8.8), 0.1% Triton-X,1.5mM MgCl ₂ ，200μM each of dNTPs, 0.2. Mu.M of reach primer and 0.5U Taq DNA polymerase.

Further, the PCR amplification procedure was pre-denaturation at 94 ℃ for 5min; denaturation at 94 ℃ for 1min, annealing at 55-60 ℃ for 1min, extension at 72 ℃ for 2min and 30 cycles; finally, extension is carried out for 8min at 72 ℃.

The sixth object of the present invention is to provide any one of the following applications of the detection reagent or the kit:

1) The application in the genotype identification of the rice fine taste gene REQ 2;

2) The application in identifying the excellent taste character of rice;

3) The application in identifying and screening the rice germplasm resources with good taste;

4) The application in molecular marker assisted breeding of rice varieties with good taste.

Compared with the prior art, the invention has the following beneficial effects:

1. the present invention identifies a novel taste gene (REQ 2) on chromosome 2 and a co-dominant molecular marker for genotyping the same. Different from the currently reported influence on taste, REQ2 is a new gene locus for controlling the taste of rice, which is derived from a high-quality japonica rice variety 'Longdao 18' in Heilongjiang province in China, and provides an excellent gene for molecular improvement of the taste of rice.

2. By the auxiliary screening of the REQ2 gene marker, rice varieties or materials with obviously improved taste can be obtained.

3. The molecular marker can be used for selecting the excellent taste genotype of the hybrid progeny of the Longdao 18 and the hybrid progeny breeding population of the derivative material of the Longdao 18, can effectively identify the excellent taste individuals with the gene, is convenient for timely hybridization transformation, improves the breeding efficiency and accelerates the breeding process.

Drawings

FIG. 1 is a schematic diagram showing the location of REQ2 on the second chromosome of rice in an example of the present invention.

Fig. 2 is a schematic diagram of a detection result of an RM13990 molecular marker in the embodiment of the present invention, where M: molecular marking standard; 1: 18 parts of dragon rice; 2: dongnong 415;3-10: f6 generation strain with good taste; 11-17: f6 generation strain with bad taste.

FIG. 3 is a schematic diagram of the detection result of the RM14045 molecular marker in the embodiment of the present invention, wherein M: molecular marker standard; 1: 18 parts of dragon rice; 2: dongnong 415;3-10: f6 generation strain with good taste; 11-17: f6 generation strain with bad taste.

Detailed Description

The invention is described in detail below with reference to the figures and the specific embodiments, but the invention should not be construed as being limited thereto. The technical means used in the following examples are conventional means well known to those skilled in the art, and materials, reagents and the like used in the following examples can be commercially available unless otherwise specified.

Example 1

Discovery and molecular marker positioning of rice good taste gene REQ2

The Longdao 18 is a high-quality rice variety with a taste value of more than 90 points bred in Heilongjiang province, the Longdao 18 with good taste characteristics is taken as a male parent, a high-yield disease-resistant variety Dongnong 415 is taken as a female parent for hybridization, and a single-seed-transmission method is utilized to obtain an F6 generation recombinant inbred line population containing 190 lines. 2016-2017, the population is used for carrying out BSA mixed pool sequencing analysis on the taste quality characters of rice in two consecutive years, a stably inherited major QTL (figure 1) is detected among 32, 10Mb-33 and 10Mb on No.2 chromosome of the rice, the synergistic allele of the major QTL comes from a high-quality variety 'Longdao 18', the analysis shows that the major QTL controls the taste value and the taste of rice, and the introduction of the gene can improve the taste value of the rice by 5 minutes on average. In the F6 generation population, the taste value of the F6 generation strain carrying the gene is 85 +/-3 minutes, the taste value of the strain without the gene is 76 +/-3 minutes, and the strain has important application value and is named as Rice aging Quality 2 (REQ 2).

According to the positioning result of the previous stage, the target regions of the oryza sativa 18 and the Dongnong 415 are respectively subjected to alignment analysis, and 2 co-dominant molecular markers RM13990 and RM14045 which are closely linked with the REQ2 gene are screened out. Primers were designed for the molecular markers, the sequences are shown in table 1:

TABLE 1 REQ2 codominant molecular marker primer information

The two markers are applied to breeding progeny groups of the oryza sativa 18 and the Dongnon 415 for verification,

using RM13990, the progeny material carrying REQ2 (taste values between 85 and 90) was able to amplify a band of 405bp (shown in SEQ ID NO. 1), while the progeny without REQ2 (73 to 77) was able to amplify a band of 397 bp. When tested with RM14045, 121bp (SEQ ID NO. 2) bands of progeny material (taste values of 85-90) carrying REQ2, while the 131bp bands could be amplified by progeny (73-77) without REQ 2. Further validation of the F6 recombinant inbred line revealed that these two molecular markers co-segregate with REQ 2. In conclusion, the two molecular markers can be used for identifying whether the material to be detected contains the good taste allele of the oryza sativa 18 on the REQ2 locus.

Example 2

Application of molecular marker assisted breeding of rice fine taste variety

The method comprises the steps of taking the oryza sativa 18 containing the REQ2 gene or a derivative progeny of the oryza sativa 18 as a parent, hybridizing with a variety to be improved which does not contain the gene, then hybridizing or backcrossing, carrying out auxiliary selection by utilizing the molecular marker of the invention, and transferring the REQ2 into a breeding material to be improved.

The method comprises the following specific steps: according to the growth period of the oryza sativa 18 or progeny derived therefrom, the variety to be improved is planted in batches. In the heading and flowering period, a variety containing the REQ2 gene is taken as a male parent, and a variety needing improvement is taken as a female parent for hybridization. The obtained hybrid is self-crossed or backcrossed, and the molecular marker of the invention is used for tracking selection until the breeding material is stable.

Application examples

The method comprises the following steps of hybridizing 18 Longdao as a male parent and 415 Dongnong as a female parent, and performing auxiliary selection on high-yield and disease-resistant F2-F6 generation materials by using the molecular marker disclosed by the invention:

the DNA extraction adopts a conventional CTAB method,

PCR amplification system15 μ L of 50mM KCl,10mM Tris-HCl (pH 8.8), 0.1% Triton-X,1.5mM MgCl ₂ 200. Mu.M of each of the aforementioned nucleic acids of dNTPs, 0.2. Mu.M of each of the reach primers and 0.5U of Taq DNA polymerase. The reaction program is pre-denaturation at 94 ℃ for 5min; denaturation at 94 ℃ for 1min, annealing at 55-60 ℃ for 1min, extension at 72 ℃ for 2min and 30 cycles; finally, extension is carried out for 8min at 72 ℃.

The PCR amplification products were separated by 3.5% agarose gel electrophoresis.

The gel imaging system observes and records the results.

8 stable strains of material containing the REQ2 gene were selected in the F6 generation and these strains were able to amplify a band of 405bp instead of 397bp using the marker RM13990, as shown in FIG. 2; when the marker RM14045 is used, a 121bp band can be amplified instead of a 131bp band, which is shown in FIG. 3. The 8 portions of material were all measured to have a taste value between 86 and 90 points.

While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.

It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.

Sequence listing

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Claims (9)

1. The rice superior taste gene REQ2 codominant molecular marker is characterized in that the molecular markers are RM13990 and RM14045, the nucleotide sequence of the RM13990 is shown as SEQ ID NO.1, and the nucleotide sequence of the RM14045 is shown as SEQ ID NO. 2.

2. The primer for amplifying the codominant molecular marker of claim 1, wherein the primer for amplifying RM13990 is RM13990-F and RM13990-R, the nucleotide sequence is shown as SEQ ID NO.3-4, the primer for amplifying RM14045 is RM14045-F and RM14045-R, and the nucleotide sequence is shown as SEQ ID NO. 5-6.

3. A detection reagent or kit comprising the primer of claim 2.

4. The co-dominant molecular marker of claim 1, for any one of the following uses:

1) The application in the genotype identification of the rice good taste gene REQ 2;

2) The application in identifying the excellent taste character of rice;

3) The application in identifying and screening the rice germplasm resources with excellent taste;

4) The application in molecular marker assisted breeding of rice fine taste variety.

5. The primer of claim 2, wherein any one of the following applications:

1) The application in the genotype identification of the rice good taste gene REQ 2;

2) The application in identifying the excellent taste character of rice;

3) The application in identifying and screening the rice germplasm resources with good taste;

4) The application in molecular marker assisted breeding of rice varieties with good taste.

6. The use according to claim 5, wherein the genotyping of the rice elite taste gene REQ2 comprises the following steps:

performing PCR amplification on rice by using RM13990-F and RM13990-R, amplifying a 405bp nucleotide sequence shown in SEQ ID NO.1 from a material carrying REQ2, and amplifying a 397bp nucleotide sequence from a material without REQ 2;

performing PCR amplification on rice by using RM14045-F and RM14045-R, amplifying a 121bp nucleotide sequence shown in SEQ ID NO.2 from a material carrying REQ2, and amplifying a 131bp nucleotide sequence from a material without REQ 2;

utilizes RM13990-F, RM13990-R, RM14045-F and RM14045-R to carry out PCR amplification on rice, amplifies a nucleotide sequence of 405bp shown in SEQ ID NO.1 and a nucleotide sequence of 121bp shown in SEQ ID NO.2 from a material carrying REQ2, and amplifies a nucleotide sequence of 397bp and a nucleotide sequence of 131bp from a material without REQ 2.

7. The use of claim 6, wherein the PCR amplification system is 15 μ L, 50mM KCl,10mM Tris-HCl pH8.8, 0.1% Triton-X,1.5mM MgCl ₂ 200 μ Meach of dNTPs,0.2 μ M of reach primer and 0.5U of Taq DNA polymerase.

8. The use of claim 7, wherein the PCR amplification procedure is 94 ℃ pre-denaturation for 5min; denaturation at 94 ℃ for 1min, annealing at 55-60 ℃ for 1min, extension at 72 ℃ for 2min and 30 cycles; finally, extension is carried out for 8min at 72 ℃.

9. The use of the detection reagent or kit of claim 3 for any one of:

1) The application in the genotype identification of the rice good taste gene REQ 2;

2) The application in identifying the rice excellent taste character;

3) The application in identifying and screening the rice germplasm resources with excellent taste;

4) The application in molecular marker assisted breeding of rice fine taste variety.

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