CN118460783A - A SNP site associated with rice α-tocopherol content and its application - Google Patents

Abstract

The invention provides an SNP locus related to rice alpha-tocopherol content and application thereof, wherein the SNP molecular marker is positioned at 22620372 th base of a chromosome 7 of rice, and the application comprises application in predicting rice alpha-tocopherol content and application in breeding of rice lines with high alpha-tocopherol content; the method has the advantages of simple and convenient operation, rapid parting, accurate result, low cost and the like, can improve the character selection efficiency, and meets the requirement of large-scale molecular marker auxiliary selection.

Description

A SNP site associated with rice α-tocopherol content and its application

Technical Field

The invention relates to the field of plant molecular biotechnology, and in particular to a SNP site associated with rice alpha-tocopherol content and an application thereof.

Background Art

Rice (Oryza sativa L) is one of the most important food crops in the world. It is also the crop with the largest planting area and the highest yield among my country's food crops. With the continuous development of the economy and society, the market demand for nutritious, healthy and delicious functional rice is gradually increasing. The nutritional quality of rice includes protein, fat, vitamins and trace mineral elements. Among them, vitamin E is an amphiphilic lipid vitamin. Its natural products are divided into eight types according to the saturation of the hydrophobic tail and the number and position of methyl groups on the aromatic ring, namely α, β, γ, δ-tocopherol and α, β, γ, δ tocotrienol. Among them, α-tocopherol has the highest biological activity, and the other seven types have only 30% to 50% of the activity of α-tocopherol due to different stereo configurations. As an important antioxidant, vitamin E is not only related to the reproductive system, but also closely related to the normal metabolism of the central nervous system, digestive system, cardiovascular system and muscle system, and is involved in the construction and maintenance of cell membranes, delaying cell aging (Traber and Sies, 1996). However, the human body cannot synthesize vitamin E on its own and must obtain it from plants.

Increasing the total vitamin E content of rice, especially the α-tocopherol content, will help increase the body's intake and absorption of vitamin E, but there are currently few research reports in this regard.

Sookwong et al. (2009) located the QTL related to tocotrienol content and proved that the tocotrienol content in the bran layer of rice can be increased by hybridization between varieties. In addition, there are some studies that use transgenic methods to increase the total vitamin E content in rice or change its isomer composition ratio (Zhang Guiyun et al. 2012a; Zhang Guiyun et al. 2012b; Zang et al. 2013), but there are few related reports. The main research results are as follows: The OSHGGT gene from Nipponbare was introduced into the japonica rice Wuyu No. 3 for overexpression. The results showed that the contents of total tocotrienols and γ-tocotrienol in the bran layer and endosperm of rice seeds after transgenic were 1.67 and 1.52 times of the original, respectively, and the content of total tocopherols did not decrease. The ratio of total tocotrienols to total tocopherols in the bran layer and endosperm increased to 0.82 and 1.8, respectively (Zhang Yun et al. 2012b). The rice OSHPT gene driven by the CaMV35S promoter was introduced into the rice Wuyu 3 using Agrobacterium-mediated transgenic rice. The results showed that the content of total tocopherol in the endosperm of transgenic rice was significantly increased, mainly due to the increase in α-tocopherol. The total tocopherol and total VE contents increased by up to 15 and 08 times, respectively (Zhang Guiyun et al. 2012c).

The study found that in the wild type and experimental blank control, α/γ-tocotrienol was only 0.7, far lower than α/γ-tocopherol. After constitutive overexpression of Arabidopsis γ-TMT (AtTMT) in Wuyujing 3, the results showed that the -isomer was converted to the α-isomer, the content of γ and β-tocotrienols was significantly reduced, and the content of -tocotrienol in transgenic seeds was significantly increased. After overexpression of ATTMT in the endosperm, the content of α-tocotrienol was also increased. The overall results showed that the α/γ-tocopherol in the seeds was increased after transgenic, and the -tocopherol content was not affected, which may be related to the low γ-tocopherol component in wild-type rice seeds. Overexpression of AtTMT had no effect on the total tocopherol or tocotrienol content (Zhang et al 2013). These studies indicate that overexpression of exogenous γ-TMT can change the synthesis of tocotrienols in rice, which is consistent with the results of related studies by other researchers such as Nan et al., providing good reference information for the improvement of vitamin E in rice.

Molecular markers are genetic markers based on nucleotide sequence variation within genetic material between individuals, and are a direct reflection of genetic polymorphism at the DNA level. Single Nucleotide Polymorphism (SNP) is the third generation of DNA molecular markers proposed by American scholar Lander E in 1996. SNP refers to the difference between different alleles at the same site with only individual nucleotides. SNP is widely distributed in the genome of organisms, with a large number and genetic stability, and is the main genetic source of phenotypic variation among different individuals in a species.

Competitive allele-specific PCR (KASP) is a SNP genotyping technology. Since its introduction, it has quickly seized the market with its ultra-high flexibility, accuracy and cost-effectiveness. It is called "the string of beads jumping at the fingertips of genotyping researchers" in the industry. KASP technology is based on the specific matching of primer terminal bases to type SNPs and detect InDels (Insertions and Deletions). KASP consists of three parts: DNA to be tested with target SNPs; KASP primer mixture (KASP Assay Mix), which contains two different allele-specific competitive forward primers with unique tail sequences and a shared reverse primer; KASP master mix, which contains a universal FRET (fluorescence resonance energy transfer) box, ROX™ passive reference dye, Taq polymerase, free nucleotides and MgCl2. During the PCR reaction, an allele-specific primer binds to the target SNP and extends, thereby connecting the tail sequence to the newly synthesized chain for complementary replication. After multiple rounds of PCR amplification, the FAM or HEX labeled oligonucleotides bind to the new complementary tail sequence, release the fluorophore from the quenching group, and generate a fluorescent signal. The sequence is continuously amplified, and the fluorescent signal is continuously enhanced, eventually reaching the end point of the reaction, and the data is read. Through terminal fluorescence reading judgment, each well uses dual-color fluorescence to detect two possible genotypes of a site in a sample. If the genotype of a given SNP is homozygous, only one fluorescent signal will be generated. If the genotype is heterozygous, a mixed fluorescent signal will be generated.

Molecular marker-assisted selection refers to the selection of target trait genotypes by analyzing the genotypes of molecular markers that are closely linked to the target gene. Using molecular marker-assisted selection technology to increase the α-tocopherol content of rice has the advantages of not going through the genetic modification step, being easy to operate, fast typing, flexible to use, and low cost. It will become an indispensable auxiliary breeding tool for breeders. Therefore, providing rice molecular markers has important practical significance.

Summary of the invention

In view of the shortcomings of the prior art, the present invention proposes a SNP site related to the α-tocopherol content in rice and its application. The α-tocopherol content in rice is predicted by genotyping the VEQ7L site that regulates the α-tocopherol content in rice.

The technical solution of the present invention is as follows:

One of the purposes of the present invention is to provide an application of a VEQ7L molecular marker or a reagent for detecting a VEQ7L molecular marker, wherein the application comprises the following (I) or (II):

(I) Application in predicting α-tocopherol content in rice;

(II) Application in the breeding of rice lines with high α-tocopherol content;

The VEQ7L molecular marker is located at the 22620372nd base of chromosome 7 of the rice genome version Os-Nipponbare-Reference-IRGSP-1.0.

Furthermore, the nucleotide sequence containing the VEQ7L molecular marker is shown as SEQ ID NO.6, wherein the VEQ7L molecular marker is located at position 10001 of the sequence shown in SEQ ID NO.6.

Furthermore, the rice includes polished rice or brown rice.

Furthermore, the reagent includes a primer set, and the nucleotide sequence of the primer set is shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3.

Furthermore, the method for predicting the α-tocopherol content of rice is: detecting the genotype of the sample to be tested at the VEQ7L locus. If the genotype of the sample to be tested is a homozygous GTAC genotype, the probability that the sample to be tested has a high α-tocopherol content is significantly higher than other genotypes.

Furthermore, the method for predicting the α-tocopherol content of rice is: detecting the genotype of the sample to be tested at the VEQ7L locus; if the genotype of the sample to be tested is a homozygous GTAC genotype, the sample to be tested has a high α-tocopherol content; if the genotype of the sample to be tested is a heterozygous H genotype, the sample to be tested has a medium α-tocopherol content; if the genotype of the SNP molecular marker of the sample to be tested is a homozygous GG genotype, the sample to be tested has a low α-tocopherol content.

The second object of the present invention is to provide a primer set for detecting a SNP molecular marker related to the α-tocopherol content in rice, wherein the molecular marker is located at the 22620372nd base of chromosome 7 of the rice genome version Os-Nipponbare-Reference-IRGSP-1.0; the nucleotide sequence of the primer set is shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3.

The third object of the present invention is to provide a method for predicting the α-tocopherol content of rice, which predicts the α-tocopherol content of rice by detecting the genotype of the VEQ7L molecular marker of the sample to be tested; if the genotype is a homozygous GTAC genotype, the probability that the sample to be tested has a high α-tocopherol content is significantly higher than other genotypes.

Furthermore, the detection method includes: using the genomic DNA of the sample to be tested as a template, using the primer set to perform PCR amplification, determining the genotype of the VEQ7L molecular marker of the sample to be tested according to the PCR reaction result, and judging the α-tocopherol content of the sample to be tested according to the genotype.

A fourth object of the present invention is to provide a method for breeding rice strains with high α-tocopherol content, comprising the following steps:

(1) Detect the genotype of the VEQ7L molecular marker in rice, and use the recipient parent with the genotype of GTAC as the male parent and cross it with the rice with the genotype of GG to obtain F1;

(2) using primers with sequences such as SEQ ID No. 1 to 3 to detect the genotype of the F1 hybrid, selecting a true hybrid as the female parent, and backcrossing it with a recipient parent with a genotype of GTAC to obtain BC1F1;

(3) Planting BC1F1, using primers with sequences such as SEQ ID No. 1-3 to detect the genotype of the BC1F1 plants; selecting plants with a heterozygous genotype for the VEQ7L molecular marker;

(4) Identify the genetic background of the individual plants selected in step (3) and select plants with high genotype similarity to the recurrent parent;

(5) backcrossing the plant selected in step (4) with the recipient parent with the genotype of GTAC to obtain BC2F1;

(6) Planting BC2F1, repeating steps (3) and (4), selecting plants with heterozygous genotype at the VEQ7L locus and high genetic background recovery rate, and collecting them from the hybrid BC2F2;

(7) Planting BC2F2, repeating steps (3) and (4), selecting plants with the GTAC genotype at the VEQ7L locus and the highest genetic background homozygosity rate, and harvesting them from the hybrid BC2F3;

(8) Select individual plants with significantly increased α-tocopherol content in brown rice and polished rice from the BC2F3 self-bred varieties and breed them into rice lines with high α-tocopherol content.

Furthermore, the recipient parent with the genotype of GTAC is Akita Komachi.

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

The present invention designs and develops a three-primer combination with a short amplified fragment and strong specificity based on the mutation of the VEQ7L locus in which the base G at position 22620372 of chromosome 7 of rice is replaced by GTAC. The marker is used to perform PCR amplification by using detection primers with different fluorescence, and the detection primers corresponding to the specific sequence grow exponentially with the PCR reaction. After the signal is generated, the corresponding signal is detected, and the homozygous GTAC, homozygous GG and heterozygous H genotypes can be clearly distinguished, so that the genotyping of the VEQ7L locus regulating the α-tocopherol content in rice can be completed, and whether the α-tocopherol content in rice is increased can be predicted. The present invention has the advantages of simple operation, rapid typing, accurate results, low cost, etc., can improve the efficiency of trait selection, shorten the breeding period, and meet the needs of large-scale molecular marker-assisted selection.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: PXB sequencing results showed homozygous GG genotype;

Figure 2: AKITAKOMACHI sequencing results showed a homozygous GTAC genotype;

Figure 3: Box plot of α-tocopherol content in polished rice of homozygous GTAC and homozygous GG genotype varieties; the five horizontal lines from top to bottom of each box represent the maximum value, 75% value, median value, 25% value and minimum value respectively; points represent outliers; "×" represents the average value; the blue box is the homozygous GTAC genotype, and the yellow box is the homozygous GG genotype; the distribution and average content of α-tocopherol in polished rice of the homozygous GTAC population are significantly higher than those of the homozygous GG population;

Figure 4: Fluorescence signal reading results of different rice genotypes detected by primer combinations Q7L-F1, Q7L-F2 and Q7L-Ra; the red, blue, green and black dots represent homozygous GG, homozygous GTAC, heterozygous H genotypes, and ddH ₂ O, respectively;

Figure 5: Fluorescence signal reading results of different rice variety genotypes detected using primer combinations Q7L-F1, Q7L-F2 and Q7L-Ra.

Figure 6: Fluorescence signal scanning results of F2 hybrid samples, homozygous GTAC control AKITAKOMACHI, homozygous GG control PXB, mixed DNA of heterozygous control PXB and AKITAKOMACHI, and negative control ddH ₂ O detected by primer combination Q7L-F1, Q7L-F2 and Q7L-Ra;

Figure 7: Box plot of the distribution of α-tocopherol content in brown rice of different genotypes marked with VEQ7L; wherein, the five horizontal lines from top to bottom of each box represent the maximum value, 75% value, median value, 25% value and minimum value, respectively; "×" represents the average value; the blue box is the homozygous GTAC genotype, the yellow box is the homozygous GG genotype, and the orange box is the heterozygous H genotype; the distribution and average content of α-tocopherol in the group with the VEQ7L marker genotype of homozygous GTAC were significantly higher than those in the group with the α-tocopherol marker genotype of homozygous GG or heterozygous H.

Figure 8: Box plot of the distribution of α-tocopherol content in polished rice of different genotypes marked with VEQ7L; wherein the five horizontal lines from top to bottom of each box represent the maximum value, 75% value, median value, 25% value and minimum value, respectively; "×" represents the average value; the blue box is the homozygous GTAC genotype, the orange box is the homozygous GG genotype, and the gray box is the heterozygous H genotype; the distribution and average content of α-tocopherol in the group with the VEQ7L marker genotype of homozygous GG were significantly lower than those in the group with the α-tocopherol marker genotype of homozygous GTAC or heterozygous H.

DETAILED DESCRIPTION

In order to enable those skilled in the art to better understand the technical content of the present invention, the present invention is further described below in conjunction with specific embodiments and drawings.

Example 1 - Localization of VEQ7L loci associated with α-tocopherol content using genome-wide association analysis

A total of 533 rice germplasm resources collected from all over the world were used as the research objects. The nuclear genome sequences of various varieties were determined using resequencing technology, and the relative content of α-tocopherol in polished rice was determined using high-performance liquid chromatography-mass spectrometry combined with broad targeted metabolite determination technology.

Based on the above genome and α-tocopherol relative content data, a metabolite-based genome-wide association analysis was conducted, and a SNP site VEQ7L (P=0.151× ^10-14 ) that was significantly correlated with the relative content of rice α-tocopherol was located. The VEQ7L site is located at base 22620372 of chromosome 7 of rice (genome version Os-Nipponbare-Reference-IRGSP-1.0). Resequencing data showed that two genotypes were detected at the VEQ7L site in the 533 varieties. Among them, there were 349 varieties with homozygous GG genotypes and 184 varieties with homozygous GTAC genotypes. (See Table 1)

Table 1: Genotypes of VEQ7L locus and relative content of α-tocopherol in 533 rice materials

Example 2-Verification of VEQ7L locus genotype by Sanger sequencing

From the above 533 varieties, varieties PXB (Pingxiang White) and AKITAKOMACHI (Akita Komachi) having homozygous GTAC and homozygous GG genotypes, respectively, were selected.

PXB (Pingxiang White) is a strain bred by the applicant through hybridization and backcrossing of disease-resistant local varieties collected from Pingxiang with Huang Huazhan.

The 1KB sequence upstream and downstream of the α-tocopherol VEQ7L SNP site is as follows (the underlined and bolded parts are SNP sites):

GTACTAC ATGACCGAGACGATAGGAAAGATTAGTCGTTCTTCGTTCCGCTATATCGACTAACTTTTTTTTTTTTGGTTTTAACACTAAATCGTTGCAGTACGGCGTGAACCTGGTGCGCGGCGTGGTGAAGGAGGTGAAGCCGAGGGATCGAGCTGAGCGACGGGAGCCGCGTGCCGTACGGCGTCCTGGTGTGGTCGACGGGCGTGGGGCCGTCGGAGTTCGTGAGATCGCTGCCGCTCCCCAAGT CCCCC GGCGGGAGGATCGGCGTCGACGAGTGGCTCCGCGTGCCGTCGGTGGAGGACGTGTTCGCGCTGGGCGACTGCGCCGGGTTCCTGGAAGGGACGGGGCGGGCCGTGCTGCCGGCCTGGCGCAGGTGGCGGAGCGGGAGGGGAGGTACCTGGCGCGGGTGATGTCGAGGATCGCGGCGCAGGATGGCGGCAGGGCGGGGCGCGCGGTGGGGAGCGCGGAGCTCGGGGAGCCGTTCGTGTGTACAAGCA ( SEQ ID No.6)

Primers were designed based on the 1 kb sequence upstream and downstream of the above VEQ7L site:

VEQ7LCXF (SEQ ID No.4):GCTCACGTCAAGGACTACGTCA

VEQ7LCXR (SEQ ID No. 5): TTGCTCGATCAGTTTCTTGATCGT

Using the genomic DNA of varieties PXB and AKITAKOMACHI as templates, primers VEQ7LCXF and VEQ7LCXR were used for PCR amplification, and the amplified products were sequenced by Sanger sequencing. The sequencing results showed that the genotype of the VEQ7L locus of variety PXB was homozygous GG (see Figure 1), and the genotype of the VEQ7L locus of variety AKITAKOMACHI was homozygous GTAC (see Figure 2).

Example 3 - Effect of VEQ7L site on α-tocopherol content

According to the genotype of the VEQ7L locus, the 533 varieties were divided into two groups: homozygous GTAC genotype and homozygous GG genotype. In the polished rice of the homozygous GTAC genotype variety, excluding the abnormal values, the minimum value of the relative content of α-tocopherol was 44228.2064, the maximum value was 168726.7959, and the average value was 99622. In the polished rice of the homozygous GG genotype variety, excluding the abnormal values, the minimum value of the relative content of α-tocopherol was 51271.74836, the maximum value was 123529.3883, and the average value was 84836 (see Figure 3). The t test showed that the average relative content of α-tocopherol in the homozygous GTAC and GG genotype varieties was significantly different (P=0.01925208). The above results indicate that the VEQ7L locus is significantly associated with the α-tocopherol content of rice polished rice. Among them, the average content of α-tocopherol in polished rice was lower in the group with homozygous GG genotype of VEQ7L locus, while the average content of α-tocopherol in polished rice was higher in the group with homozygous GTAC genotype of VEQ7L locus.

Example 4 - Development of KASP markers at the VEQ7L site

1. Primer design

According to the instructions of the KASP kit (KASP-TFV4.0 2X Master Mix) of LGC (Laboratory of the Government Chemist) and the sequence of 1 kb genomic DNA upstream and downstream of the VEQ7L site, three sets of three primer combinations for identifying the genotype of the α-tocopherol VEQ7L site were designed, namely the primer combinations VEQ7L-F1, VEQ7L-F2 and VEQ7L-Ra. The primer sequences are as follows:

Q7L-F1 (SEQ ID No. 1):

GAAGGTGACCAAGTTCATGCTGGACCACCTCTCCAAGGTACTAC

Q7L-F2 (SEQ ID No. 2):

GAAGGTCGGAGTCAACGGATTGGACCACCTCTCCAAGGTACATG

Q7L-Ra (SEQ ID No. 3):

GAACGAAGAACGACTAATCTTTCC

2. Expected amplification results of different primer combinations

According to the primer sequences, the three sets of primers were combined to amplify the genomic DNA of rice varieties, and the amplified products were detected by SNP genotyping detector (LGC; Fluostar Omega; SNP Line Lite Omega F) for fluorescence signals. If the fluorescence signal released by the amplified product is red, it indicates that the sample to be tested has a homozygous G genotype, that is, GG; if it is a blue fluorescence signal, it indicates that the sample to be tested is a homozygous GTAC genotype, that is, GTAC; if it is a green fluorescence signal, it indicates that the sample to be tested is a heterozygous genotype H.

3. Actual amplification effects of different primer combinations

Figure 4 shows the results of detecting KASP fluorescence signals of genomic DNA of 12 rice samples using VEQ7L-F1, VEQ7L-F2 and VEQ7L-Ra. Three samples were AKITAKOMACHI, and their KASP fluorescence was blue, indicating that their genotype was homozygous GTAC. Eight samples were PXB, and their KASP fluorescence was red, indicating that their genotype was homozygous GG. One sample was made by mixing genomic DNA of AKITAKOMACHI and PXB at equal concentrations and volumes, and its KASP fluorescence was green, indicating that its genotype was H. The black dots are the KASP results of the negative control ddH ₂ O.

Figure 5 shows the fluorescence signal reading results of 10 rice varieties (excluding the control variety) using the primer set VEQ7L-F1, VEQ7L-F2 and VEQ7L-Ra. Figure 5 also includes the fluorescence signal reading results of the homozygous GG control variety PXB, the homozygous GTAC control variety AKITAKOMACHI, the mixed DNA of the heterozygous control PXB and AKITAKOMACHI, and the negative control two ddH ₂ O. As shown in Figure 5, the fluorescence signal of ddH ₂ O is black, indicating that no fluorescence is generated.

In summary, the VEQ7L-F1, VEQ7L-F2, and VEQ7L-Ra primer sets can effectively distinguish the genotypes of the VEQ7L locus.

Example 5 - Identification of the genotype of the VEQ7L locus in the F2 segregating population using KASP markers

1. Experimental Materials

The recipient parent, such as AKITAKOMACHI, is crossed with the donor parent PXB to obtain F1, and F1 is self-pollinated to obtain F2.

2. PCR amplification and detection

The parent variety PXB was used as the homozygous GG genotype control, the parent variety AKITAKOMACHI was used as the homozygous GTAC genotype control, and the genomic DNA of PXB and AKITAKOMACHI was mixed in equal amounts as the heterozygous genotype control. The leaf genomic DNA of the individual plants of the F2 population described in this example was extracted and PCR amplified using the KASP primer combination (VEQ7L-F1, VEQ7L-F2, VEQ7L-Ra) screened in Example 4. The amplification was performed using the KASP-TF V4.0 2X Master Mix kit from LGC Genomics. The PCR reaction system was 10 μL, including 4.86 μL of 2×KASP Master mix, 36 μM of VEQ7L-F1 and VEQ7L-F2 primers, and 90 μM of VEQ7L-Ra primer. The three primers were mixed at a volume ratio of 1:1:1 to form KASP Asssy mix. 0.14 μL of KASP Asssy mix and 5 μL of 10 ng/μL template DNA were taken.

3. Results and analysis

The results of genotype detection are shown in Figure 6 (see Table 2 for specific results). The PCR products of 26 individual plants emitted blue fluorescent signals, and their signal points were clustered together with the parent variety AKITAKOMACHI that also emitted blue fluorescent signals in the coordinate system, indicating that the genotype of its VEQ7L locus was GTAC. The PCR products of 16 individual plants emitted red fluorescent signals, and their signal points were clustered together with the parent variety PXB that also emitted red fluorescent signals in the coordinate system, indicating that the genotype of its VEQ7L locus was homozygous GG. The PCR products of 44 individual plants emitted green fluorescent signals, and their signal points were clustered together with the heterozygous control that also emitted green fluorescent signals in the coordinate system, indicating that the genotype of its VEQ7L locus was H.

Table 2: Genotypes of the VEQ7L locus in the F2 segregating population

Example 6 - Effect of VEQ7L locus on α-tocopherol content in brown rice of F2 plants

1. Experimental Materials

The F2 population described in Example 5.

2. Detection of relative content of α-tocopherol

Each F2 plant was harvested from the cross, and 10 g of rice was husked and ground into powder. 0.1 g of the powder was weighed and 1 mL of extraction solution (70% methanol) was added to extract the metabolites. The relative content of α-tocopherol was detected using a high performance liquid chromatography-triple quadrupole-linear ion trap mass spectrometry detection system (HPLC-6500 QTRAP, AB SCIEX) using a broad targeted metabolomics detection method.

3. Results and analysis

According to the genotype of the VEQ7L locus in Example 5, the 86 F2 plants described in this example were divided into GTAC, GG and H genotype groups. Figure 7 shows the effect of the VEQ7L genotype on the α-tocopherol content in brown rice (see Table 3 for specific data). In the brown rice of the GTAC genotype plants, except for the abnormal values, the minimum value of the relative content of α-tocopherol was 64186.9, the maximum value was 195234.23, and the average value was 130688. In the brown rice of the GG genotype plants, except for the abnormal values, the minimum value of the relative content of α-tocopherol was 11741, the maximum value was 147913, and the average value was 103287. In the brown rice of the H genotype plants, except for the abnormal values, the minimum value of the relative content of α-tocopherol was 411245, the maximum value was 220387, and the average value was 33551. The t test showed that the relative content of α-tocopherol in brown rice of plants with homozygous GTAC genotype was significantly higher than that of plants with homozygous GG genotype (P=130688), and the average relative content of α-tocopherol in brown rice of plants with H genotype was significantly higher than that of plants with GG genotype (P=5.85×10-5). The above results indicate that the VEQ7L locus has the function of regulating the content of α-tocopherol in brown rice. Among them, the average content of α-tocopherol in brown rice was lower when the genotype of the VEQ7L locus was homozygous GG genotype, while the average content of α-tocopherol in brown rice was higher when the genotype of the VEQ7L locus was GTAC.

Table 3: Relative content of α-tocopherol in brown rice of different genotypes at the VEQ7L locus

Example 7 - Effect of VEQ7L locus on α-tocopherol content in polished rice of F2 plants

1. Experimental Materials

The F2 population described in Example 5.

2. Detection of relative content of α-tocopherol

Each F2 plant was harvested from the hybrid, and 10 g of rice was husked and ground into powder. 0.1 g of the powder was weighed and 1 mL of extraction solution (70% methanol) was added to extract the metabolites. The relative content of α-tocopherol was detected using a high performance liquid chromatography-triple quadrupole-linear ion trap mass spectrometry detection system (HPLC-6500 QTRAP, AB SCIEX) using a broad targeted metabolomics detection method.

3. Results and analysis

According to the genotype of the VEQ7L locus in Example 5, the 86 F2 plants described in this example were divided into GTAC, GG and H genotype groups. Figure 8 shows the effect of VEQ7L genotype on the α-tocopherol content in polished rice (specific data are shown in Table 5). In the polished rice of GTAC genotype plants, except for abnormal values, the minimum value of the relative content of α-tocopherol was 44228, the maximum value was 168727, and the average value was 99622. In the polished rice of GG genotype plants, except for abnormal values, the minimum value of the relative content of α-tocopherol was 11144, the maximum value was 123529, and the average value was 80230. In the polished rice of H genotype plants, except for abnormal values, the minimum value of the relative content of α-tocopherol was 43305, the maximum value was 143579, and the average value was 21088. The t test showed that the relative content of α-tocopherol in polished rice of plants with GTAC genotype was significantly higher than that of plants with GG genotype (P=0.01921), and the relative content of α-tocopherol in polished rice of plants with H genotype was significantly higher than that of plants with GG genotype (P=3.44×10-13). The above results indicate that the VEQ7L locus has the function of regulating the content of α-tocopherol in polished rice. Among them, the average content of α-tocopherol in polished rice was lower when the genotype of VEQ7L locus was GG, while the average content of α-tocopherol in polished rice was higher when the genotype of VEQ7L locus was GTAC and H.

Table 4: Relative content of α-tocopherol in polished rice of different genotypes at the VEQ7L locus

Example 8 - Using VEQ7L marker to increase the α-tocopherol content of rice

The donor parent PXB is hybridized, backcrossed and selfed with a recipient with normal fertility, such as AKITAKOMACHI, and molecular markers are used to select the VEQ7L locus and genetic background during the process, and finally a strain with a GTAC genotype at the VEQ7L locus in the AKITAKOMACHI background and an increased α-tocopherol content in polished rice is obtained. The specific implementation steps are as follows:

(1) Use the recipient parent (such as AKITAKOMACHI) as the male parent and hybridize it with PXB to obtain F1.

(2) Use primers with sequences such as SEQ ID No. 1 to 3 to detect the genotype of the F1 hybrid, select a true hybrid (i.e., a plant whose PCR product produces a green fluorescent signal) as the female parent, and backcross it with the recipient parent (such as AKITAKOMACHI) to obtain BC1F1.

(3) Plant BC1F1 and detect the genotype of the BC1F1 plant using primers with sequences such as SEQ ID No. 1 to 3. Select plants with heterozygous genotype at the VEQ7L locus.

(4) Using a set of molecular markers (such as but not limited to SSR, SNP, INDEL, EST, RFLP, AFLP, RAPD, SCAR, etc.) that are polymorphic and evenly distributed between the genomes of PXB and the recipient parent (such as AKITAKOMACHI), the genetic background of the individual plants selected in step (3) is identified, and plants with high genotype similarity to the recurrent parent (such as greater than 88% similarity, or 2% selection rate, etc.) are selected.

(5) Backcross the plant selected in step (4) with the recipient parent (such as AKITAKOMACHI) to obtain BC2F1.

(6) Plant BC2F1 and repeat steps (3) and (4) to select plants with heterozygous VEQ7L genotype and high genetic background recovery rate (e.g., greater than 98%, or 2% selection rate, etc.) and collect them as self-cross BC2F2.

(7) Plant BC2F2 and repeat steps (3) and (4) to select plants with a VEQ7L genotype of GTAC and the highest genetic background homozygosity rate, and collect them from the hybrid BC2F3.

(8) Determine the α-tocopherol content in brown rice and polished rice of BC2F3 inbred varieties and AKITAKOMACHI; and select individual plants with significantly increased α-tocopherol content in brown rice and polished rice of BC2F3 inbred varieties for breeding into rice lines with high α-tocopherol content by comparing them with the α-tocopherol content of AKITAKOMACHI.

The above descriptions are only some embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims (10)

1. Application of a VEQ7L molecular marker or a reagent for detecting a VEQ7L molecular marker, characterized in that the application comprises the following (I) or (II): (I) Application in predicting α-tocopherol content in rice; (II) Application in the breeding of rice lines with high α-tocopherol content; The VEQ7L molecular marker is located at the 22620372nd base of chromosome 7 of the rice genome version Os-Nipponbare-Reference-IRGSP-1.0. 2. The use according to claim 1, characterized in that the rice comprises polished rice or brown rice. 3. The use according to claim 1, characterized in that the reagent comprises a primer set, and the nucleotide sequence of the primer set is shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3. 4. The use according to claim 1, characterized in that the method for predicting the α-tocopherol content of rice is: detecting the genotype of the sample to be tested at the VEQ7L locus, if the genotype of the sample to be tested is a homozygous GTAC genotype, then the probability that the sample to be tested has a high α-tocopherol content is significantly higher than other genotypes. 5. The use according to claim 1, characterized in that the method for predicting the α-tocopherol content of rice is: detecting the genotype of the sample to be tested at the VEQ7L site, if the genotype of the sample to be tested is a homozygous GTAC genotype, the sample to be tested has a high α-tocopherol content, if the genotype of the sample to be tested is a heterozygous H genotype, the sample to be tested has a medium α-tocopherol content, and if the genotype of the sample to be tested is a homozygous GG genotype, the sample to be tested has a low α-tocopherol content. 6. A primer set for detecting a SNP molecular marker associated with the α-tocopherol content in rice, characterized in that the molecular marker is located at base 22620372 of chromosome 7 of the rice genome version Os-Nipponbare-Reference-IRGSP-1.0; the nucleotide sequence of the primer set is shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3. 7. A method for predicting the α-tocopherol content of rice, characterized in that the α-tocopherol content of rice is predicted by detecting the genotype of the VEQ7L molecular marker of the sample to be tested; if the genotype is a homozygous GTAC genotype, the probability that the sample to be tested has a high α-tocopherol content is significantly higher than that of other genotypes; the VEQ7L molecular marker is the VEQ7L molecular marker described in claim 1. 8. The method according to claim 7 is characterized in that the detection method comprises: using the genomic DNA of the sample to be tested as a template, using the primer set according to claim 6 to perform PCR amplification, determining the genotype of the VEQ7L molecular marker of the sample to be tested according to the PCR reaction result, and judging the α-tocopherol content of the sample to be tested according to the genotype. 9. A method for breeding rice lines with high α-tocopherol content, characterized by comprising the following steps: (1) Detecting the genotype of the VEQ7L molecular marker as claimed in claim 1 in rice, using a recipient parent with a genotype of GTAC as the male parent and hybridizing it with a rice with a genotype of GG to obtain F1; (2) using primers with sequences such as SEQ ID No. 1 to 3 to detect the genotype of the F1 hybrid, selecting a true hybrid as the female parent, and backcrossing it with a recipient parent with a genotype of GTAC to obtain BC1F1; (3) Planting BC1F1, using primers with sequences such as SEQ ID No. 1-3 to detect the genotype of the BC1F1 plants; selecting plants with a heterozygous genotype for the VEQ7L molecular marker; (4) Identify the genetic background of the individual plants selected in step (3) and select plants with high genotype similarity to the recurrent parent; (5) backcrossing the plant selected in step (4) with the recipient parent with the genotype of GTAC to obtain BC2F1; (6) Planting BC2F1, repeating steps (3) and (4), selecting plants with heterozygous genotype at the VEQ7L locus and high genetic background recovery rate, and collecting them from the hybrid BC2F2; (7) Planting BC2F2, repeating steps (3) and (4), selecting plants with the GTAC genotype at the VEQ7L locus and the highest genetic background homozygosity rate, and harvesting them from the hybrid BC2F3; (8) Select individual plants with significantly increased α-tocopherol content in brown rice and polished rice from the BC2F3 self-bred varieties and breed them into rice lines with high α-tocopherol content. 10 . The breeding method according to claim 9 , wherein the recipient parent with the genotype of GTAC is Akita Komachi.