CN108179222B - Nucleotide sequence of chorismate mutase related to high yield of rice and application thereof - Google Patents

Abstract

The invention discloses a chorismate mutase nucleotide sequence related to high yield of rice and its application. The method for screening rice with different yield traits provided by the present invention may include the following steps: (1) detecting the genotype of the rice to be tested based on a specific gene fragment; the specific gene fragment is located in the rice genome, is OSCM3, and has OSCM3_a and OSCM3_b two allelic forms, the OSCM3_a is shown in the sequence 1 of the sequence listing, and the OSCM3_b is shown in the sequence 2 of the sequence listing; (2) the following judgments are made: the two genotypes are compared under adjacent growth conditions, The average yield of rice populations homozygous for OSCM3_b was higher than that of rice populations homozygous for OSCM3_a. Experiments show that the method provided by the present invention can effectively screen rice with different yield traits per plant, and has simple operation and high accuracy, and has important application value in rice breeding.

Description

Nucleotide sequence of chorismate mutase related to high yield of rice and its application

technical field

The invention relates to the field of biotechnology, in particular to a chorismate mutase nucleotide sequence related to high yield of rice and its application.

Background technique

As the global population grows, the need to cultivate high-yielding crops increases. Rice (Oryza sativa L.) is one of the staple food sources on which most people on earth depend. Finding genomic fragments that affect high yield in rice and establishing a specific method to effectively evaluate the impact of this genomic fragment on yield will play an important role in promoting the development of molecular breeding. Targeted selection of parental materials in the breeding process is of great value for the ultimate cultivation of rice varieties with high-yielding traits. At present, the development of science and technology has made it possible to obtain the genomic components that affect high-yield traits in rice and to make deep use of the natural variation of rice genes.

In practice, breeding methods mostly rely on visible traits or/and molecular markers. The identification of yield-related genes is currently mostly limited to the analysis of mutants. Due to the complexity of factors affecting rice yield traits, the roles of multiple genes and environmental influences on them can make it difficult to effectively evaluate the impact of genomic segments on yield. Clearly, a limiting factor in molecular breeding in terms of yield is an efficient experimental design under field conditions in order to determine which genes or molecular fragments are suitable as selection conditions. Combining the performance of rice in the field and the quantitative study of its functional genes can play a guiding role in agricultural breeding.

SUMMARY OF THE INVENTION

The present invention provides a gene sequence which is proved to increase rice yield and its dedicated specific primer pair, and takes this as an example to establish an effective method for evaluating the influence of gene sequence on yield.

The screening method provided by the present invention is suitable for selecting high-yield rice (method A), comprising the steps of:

(1) Detecting the genotype of the rice to be tested based on a specific gene fragment; the specific gene fragment is located in the rice genome, and there are at least two allelic forms of OSCM3_a and OSCM3_b, and the OSCM3_a is shown in Sequence 1 of the Sequence Listing, and the OSCM3_b is shown in sequence 2 of the sequence listing;

(2) The following determination is made: under adjacent growth conditions, the average yield of the rice population homozygous for the OSCM3_b genotype is higher than the average yield of the rice population homozygous for the OSCM3_a genotype.

The adjacent growth conditions are adjacent and equal growth conditions within the same geographic area.

The screening method provided by the present invention is suitable for selecting high-yield rice (method B), and may include the following steps:

(1) Take the genomic DNA of the rice to be tested as a template, and use the following specific primer pairs to carry out PCR amplification; if there is only one PCR amplification product and as shown in sequence 1 in the sequence table, the genotype of the rice to be tested is OSCM3_a homozygous; if there is only one PCR amplification product and as shown in sequence 2 in the sequence table, the genotype of the rice to be tested is OSCM3_b homozygous;

(2) The following determination is made: under adjacent growth conditions, the average yield of the rice population homozygous for the OSCM3_b genotype is higher than the average yield of the rice population homozygous for the OSCM3_a genotype.

The present invention can also provide a screening method for high-yield rice, which is method A or method B;

The method A includes the following steps:

(1) detecting the genotype of the rice to be tested based on a specific gene fragment; the specific gene fragment is located in the rice genome, is OsCM3, and has two allelic forms of OsCM3_a and OsCM3_b, and the OsCM3_a is shown in Sequence 1 of the Sequence Listing, The OsCM3_b is shown in sequence 2 of the sequence listing;

(2) carry out the following judgment: under the same growth conditions, the average yield of the rice population whose genotype is OsCM3_b homozygous is higher than the average yield of the rice population whose genotype is OsCM3_a homozygous;

The method B includes the following steps:

(1) Take the genomic DNA of the rice to be tested as a template, and use the specific primer pair described in claim 4 to carry out PCR amplification; if there is only one PCR amplification product and as shown in sequence 1 in the sequence table, then the The genotype is OsCM3_a homozygous; if there is only one PCR amplification product and as shown in sequence 2 in the sequence table, the genotype of the rice to be tested is OsCM3_b homozygous;

(2) The following determination was made: under the same growth conditions, the average yield of the rice population with homozygous OsCM3_b genotype was higher than that of the rice population with homozygous OsCM3_a genotype.

The above-mentioned equivalent growth conditions may be equivalent growth conditions in the same geographical area or equivalent growth conditions in different geographical areas.

Growth conditions refer to the natural environment (soil, temperature, humidity and nutrients, etc.) and artificial management (fertilization, pesticide use, bird and pest control, etc.).

The present invention also provides a rice breeding method (method C), which may include the following steps:

(1) detect the genotype of the rice to be tested based on a specific gene fragment; the specific gene fragment is located in the rice genome, is OSCM3, and has two allelic forms of OSCM3_a and OSCM3_b, and the OSCM3_a is shown in sequence 1 of the sequence table, The OSCM3_b is shown in sequence 2 of the sequence listing;

(2) Rice whose genotype is OSCM3_b homozygous is the target rice for selection.

The present invention also provides a rice breeding method (method D), which may include the following steps:

(1) Take the genomic DNA of the rice to be tested as a template, and use the following specific primer pairs to carry out PCR amplification; if there is only one PCR amplification product and as shown in sequence 1 in the sequence table, the genotype of the rice to be tested is OSCM3_a homozygous; if there is only one PCR amplification product and as shown in sequence 2 in the sequence table, the genotype of the rice to be tested is OSCM3_b homozygous;

(2) Rice whose genotype is OSCM3_b homozygous is the target rice for selection.

The target rice is high-yield rice.

The present invention also protects a DNA fragment, which is OsCM3_b;

The OsCM3_b can be (d1) or (d2) as follows:

(d1) the DNA molecule shown in Sequence 2 of the Sequence Listing;

(d2) A DNA molecule having the same function as the sequence 2 through the substitution and/or deletion and/or addition of one or several nucleotides in sequence 2.

OsCM3_a gene and OsCM3_b gene are alleles.

In the above, "substitution and/or deletion and/or addition" occurs in a region other than the difference between the OsCM3_a gene shown in Sequence 1 of the Sequence Listing and Sequence 2 of the Sequence Listing.

The primer pair for amplifying the above-mentioned DNA fragment provided by the present invention consists of primer 1 and primer 2;

The primer 1 can be as follows (a1) or (a2):

(a1) the single-stranded DNA molecule shown in Sequence 3 of the Sequence Listing;

(a2) a single-stranded DNA molecule having the same function as sequence 3 by substitution and/or deletion and/or addition of one or several nucleotides in sequence 3;

The primer 2 can be (b1) or (b2) as follows:

(b1) the single-stranded DNA shown in Sequence 4 of the Sequence Listing;

(b2) A single-stranded DNA molecule with sequence 4 subjected to substitution and/or deletion and/or addition of one or several nucleotides and having the same function as sequence 4.

The application of the above-mentioned DNA fragment or the above-mentioned specific primer pair is also within the protection scope of the present invention, which is as follows (e1) or (e2) or (e3) or (e4):

(e1) screening high-yield rice;

(e2) screening rice with different yield traits;

(e3) identifying or assisting the identification of yield traits of rice;

(e4) Identifying or assisting in identifying rice with different yield traits.

In order to solve the above technical problems, the present invention also provides a kit including the specific primer pair. The kit may further include conventional reagents for extracting rice genomic DNA and/or conventional reagents for PCR amplification and/or conventional reagents for sequencing.

The use of the kit is as follows (e1) or (e2) or (e3) or (e4):

(e1) screening high-yield rice;

(e2) screening rice with different yield traits;

(e3) identifying or assisting the identification of yield traits of rice;

(e4) Identifying or assisting in identifying rice with different yield traits.

The preparation method of the kit also belongs to the protection scope of the present invention. The preparation method of the kit includes the step of packaging each primer in the kit separately.

The application of the DNA fragment, the specific primer pair, the kit or any one of the above methods in rice breeding also falls within the protection scope of the present invention. The breeding goal of the rice breeding is to obtain rice with high yield.

Any of the above-mentioned yields may be yield per plant. Any of the yields described above may be grain yields.

The present invention also provides a method for identifying a dominant allele, comprising the steps of: determining an allele having a dominant trait by comparing the differences in biological traits of different populations; in the different populations, each population is composed of the The composition of individuals who are homozygous for the allele.

The organism may be a sexually reproduced organism, specifically a plant, and more specifically, rice.

The trait may be a measurable trait, specifically a yield trait, more specifically a grain yield trait.

Experiments show that the method provided by the invention can be used to screen rice with different yield traits per plant, the operation is simple, the accuracy rate is high, and the method has important application value in rice breeding.

Detailed ways

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

The present invention will be further described in detail below with reference to the specific embodiments, and the given examples are only for illustrating the present invention, rather than for limiting the scope of the present invention.

Example 1. Design and synthesis of primers

Chromic acid is an important intermediate substance in rice. In the rice genome, several genes have been annotated as encoding chorismate mutase (EC5.4.99.5). For example, the gene encoding chorismate mutase 3 (CT829390.1; OSCM3) (hereinafter referred to as OSCM3 gene). Located on the first chromosome of rice. Through a large number of preliminary experiments and sequence comparison, the inventors of the present invention found that there are two allelic fragments in the OSCM3 gene (one allelic fragment is shown in Sequence 1 of the sequence table, named as the allelic fragment OSCM3_a; the other An allelic fragment is shown in SEQ ID NO: 2 of the sequence listing, named as the allelic fragment OSCM3_b), which is related to the yield per plant of rice.

Design specific primer pairs based on the above two allele fragments, consisting of primer 1 and primer 2.

Primer 1 (sequence 3 in the sequence listing): 5'-CTGTTGCCACCTCTCCAGT-3';

Primer 2 (sequence 4 in the sequence listing): 5'-TCTCCTCAGCAAGTAGGCTACT-3'.

Example 2. The establishment of a typing method based on the allelic fragment in rice

The method of establishment is as follows:

1. Using the genomic DNA (about 10-100 ng) of the rice to be tested as a template, a specific primer pair consisting of primer 1 and primer 2 is used for PCR amplification to obtain a PCR amplification product.

The reaction program of PCR amplification: 95°C for 5 minutes; 95°C for 30 seconds, 60°C for 1 minute, 72°C for 1 minute, 35 cycles; 72°C for 8 minutes.

2. After completing step 1, the PCR amplification product is sequenced, and the following determination is made according to the sequencing result: if there is only one PCR amplification product, and as shown in sequence 1 in the sequence table, the genotype of the rice to be tested is OSCM3_a Homozygous type; if there is only one PCR amplification product, and as shown in sequence 2 in the sequence table, the genotype of the rice to be tested is OSCM3_b homozygous type; if there are two PCR amplification products, one is as shown in the sequence table. The genotype of the rice to be tested is OSCM3_a/OSCM3_b heterozygous.

Since all rice varieties are cultivated or farmed, the proportion of individuals with homozygous genotype is high.

Example 3. Association analysis between the genotypes based on the allele fragment and the yield per plant in rice

1. According to the typing method established in Example 2, detect the genotype of each trial rice variety (see Table 1, Table 2 and Table 3, the fourth column for the results).

Table 1

2. Statistics on the yield per plant of different rice varieties

In 2017, a number of rice varieties were planted in the Xiangshan area of Beijing (see Table 1 for details, the name of the rice variety in the second column, and the origin of the rice variety in the third column). After the rice is mature, the rice plants are harvested per plant, repeated at least three times, weighed, and averaged to obtain the yield per plant of the variety (see Table 1, column 5 for the results).

The results in Table 1 show that the genotype of 17 of the 37 rice cultivars is OSCM3_a homozygous, and the average yield per plant of these 17 cultivars is 26.23±2.17 g; the genotype of 20 of the 37 rice cultivars is For OSCM3_b homozygous type, the average yield per plant of these 20 varieties was 31.75±2.18 g; the mean significance test of the two homozygous types was P=0.03; the paired t-test significance P=0.019 (N=12).

3. Establish a method for screening rice with different yield traits per plant

The method for screening rice with different yield traits per plant is as follows:

(1) Detecting the genotype of the rice to be tested based on a specific gene fragment; the specific gene fragment is located in the rice genome and is OSCM3, and there are two allelic forms of OSCM3_a (AP003239.3) and OSCM3_b (CP018157.1). OSCM3_a is shown in sequence 1 of the sequence listing, and OSCM3_b is shown in sequence 2 of the sequence listing;

(2) The following determination is made: under adjacent growth conditions, the average yield per plant of the rice population homozygous for OSCM3_b is higher than the average yield per plant for the rice population homozygous for OSCM3_a genotype.

sequence listing

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Claims (5)

1. A method for screening high-yield rice, which is method A or method B; Described method A comprises the steps: (1) Detect the genotype of the rice to be tested based on a specific gene fragment; the specific gene fragment is located in the rice genome, which is OsCM3, and there are two allelic forms of OsCM3_a and OsCM3_b, and the OsCM3_a is shown in Sequence 1 of the sequence table, The OsCM3_b is shown in sequence 2 of the sequence listing; (2) Make the following judgment: under adjacent growth conditions, the average yield of the rice population with the genotype homozygous for OsCM3_b is higher than the average yield of the rice population with the genotype homozygous for OsCM3_a; The method B includes the following steps: (1) Take the genomic DNA of the rice to be tested as a template, and use specific primer pairs to carry out PCR amplification; if there is only one PCR amplification product as shown in sequence 1 in the sequence table, the genotype of the rice to be tested is OsCM3_a pure If there is only one PCR amplification product and as shown in sequence 2 in the sequence table, the genotype of the rice to be tested is OsCM3_b homozygous; The specific primer pair consists of primer 1 and primer 2; The primer 1 is the single-stranded DNA molecule shown in the sequence 3 of the sequence listing; The primer 2 is the single-stranded DNA shown in the sequence 4 of the sequence listing; (2) The following judgment is made: under the adjacent growth conditions, the average yield of the rice population homozygous for OsCM3_b genotype is higher than that of the rice population homozygous for OsCM3_a genotype. 2. A screening method for high-yield rice, which is method A or method B; The method A includes the following steps: (1) Detect the genotype of the rice to be tested based on a specific gene fragment; the specific gene fragment is located in the rice genome, which is OsCM3, and there are two allelic forms of OsCM3_a and OsCM3_b, and the OsCM3_a is shown in Sequence 1 of the sequence table, The OsCM3_b is shown in sequence 2 of the sequence listing; (2) Make the following judgment: under the same growth conditions, the average yield of the rice population with homozygous genotype OsCM3_b is higher than the average yield of the rice population with homozygous genotype OsCM3_a; The method B includes the following steps: (1) Take the genomic DNA of the rice to be tested as a template, and use specific primer pairs to carry out PCR amplification; if there is only one PCR amplification product as shown in sequence 1 in the sequence table, the genotype of the rice to be tested is OsCM3_a pure If there is only one PCR amplification product and as shown in sequence 2 in the sequence table, the genotype of the rice to be tested is OsCM3_b homozygous; The specific primer pair consists of primer 1 and primer 2; The primer 1 is the single-stranded DNA molecule shown in the sequence 3 of the sequence listing; The primer 2 is the single-stranded DNA shown in the sequence 4 of the sequence listing; (2) The following determinations were made: under the same growth conditions, the average yield of the rice population with homozygous OsCM3_b genotype was higher than that of the rice population with homozygous OsCM3_a genotype. 3. A rice breeding method, which is method C or method D; Described method C comprises the steps: (1) Detect the genotype of the rice to be tested based on a specific gene fragment; the specific gene fragment is located in the rice genome, which is OsCM3, and there are two allelic forms of OsCM3_a and OsCM3_b, and the OsCM3_a is shown in Sequence 1 of the sequence table, The OsCM3_b is shown in sequence 2 of the sequence listing; (2) Rice whose genotype is OsCM3_b homozygous is the target rice for selection; The method D comprises the following steps: (1) Take the genomic DNA of the rice to be tested as a template, and use specific primer pairs to carry out PCR amplification; if there is only one PCR amplification product as shown in sequence 1 in the sequence table, the genotype of the rice to be tested is OsCM3_a pure If there is only one PCR amplification product and as shown in sequence 2 in the sequence table, the genotype of the rice to be tested is OsCM3_b homozygous; The specific primer pair consists of primer 1 and primer 2; The primer 1 is the single-stranded DNA molecule shown in the sequence 3 of the sequence listing; The primer 2 is the single-stranded DNA shown in the sequence 4 of the sequence listing; (2) Rice whose genotype is homozygous for OsCM3_b is the target rice for selection. 4. The application of DNA fragments or specific primer pairs is as follows (e1) or (e2) or (e3) or (e4): The DNA fragment is OsCM3_b; Described OsCM3_b is the DNA molecule shown in sequence 2 of the sequence listing; The specific primer pair consists of primer 1 and primer 2; The primer 1 is the single-stranded DNA molecule shown in the sequence 3 of the sequence listing; The primer 2 is the single-stranded DNA shown in the sequence 4 of the sequence listing; (e1) Screening high-yield rice; (e2) Screening rice with different yield traits; (e3) Identification or auxiliary identification of yield traits of rice; (e4) Identification or assisted identification of rice with different yield traits. 5. Use of the method described in any one of claims 1-3 or the DNA fragment described in claim 4 or the specific primer pair described in claim 4 in high-yield rice breeding.