CN116042897B - Molecular marker VEQ2 and application thereof - Google Patents
Molecular marker VEQ2 and application thereof Download PDFInfo
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/04—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection
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Abstract
The invention relates to the technical field of plant biology, in particular to a molecular marker VEQ2 and application thereof. The invention provides a locus VEQ2 for regulating and controlling the content of alpha-tocopherol in rice, a KASP (Kompetitive Alle-specific PCR) molecular marker for detecting the genotype of the locus and application thereof, which prove that the KASP marker is utilized to efficiently and accurately carry out genotyping detection on the locus VEQ2, predict the content of alpha-tocopherol, improve the breeding efficiency and shorten the breeding time. The method has the advantages of simple and convenient operation, rapid parting, accurate result, low cost and the like, can improve the selection efficiency of the target character, and meets the requirement of large-scale molecular marker assisted selection breeding.
Description
Technical Field
The invention relates to the technical field of plant biology, in particular to a molecular marker VEQ2 and application thereof.
Background
Rice (Oryza sativa L) is one of the most important food crops in the world, and is also the crop with the largest planting area and highest unit yield in the food crops in China. With the continuous development of the economic society in China, the market demand for nutritional, healthy and delicious functional rice is gradually increased. The nutritional quality of rice includes protein, fat, vitamins and trace mineral elements. Vitamin E has strong oxidation resistance and is a nutrient substance which is necessary for human body and has great benefit for human health. The most active tocopherol is alpha-tocopherol, which can help the human body to maintain normal operation of muscles, vascular systems and central nerves, provided that the physiological functions can be ensured to be normally operated only by absorbing 7-9mg of the tocopherol per day, and the alpha-tocopherol can also greatly help the physiological functions of the digestive system and the reproductive system. Vitamin E also plays a role in cell transduction and transcriptional regulation in animal and plant cells. People can ingest vitamin E through health products at present, can avoid the rise of cardiovascular diseases and cancer risks, and can effectively improve the immunity of human bodies. Developing a breeding technology for improving the vitamin E content of rice, cultivating rice varieties with high vitamin E content, meeting the requirements of the market on high-nutrition-quality rice, and being a new direction of rice nutrition quality breeding.
Currently, there are few studies and reports on improvement of alpha-tocopherol content in rice. There are many methods for increasing vitamin E content, including methods such as transgenesis, QTL for locating related vitamin E content, and molecular marker assistance. Some studies have been conducted to achieve the objective of increasing vitamin E content by combining a gene polymerization method with a molecular breeding or conventional crossbreeding method in order to provide genetic background for cloning genes related to vitamin E synthesis, and locating QTL related to vitamin E content. Studies have shown that alpha-tocopherol activity is highest in tocopherols and that transferring the gene encoding gamma-TMT into the target variety will theoretically increase the content of alpha-tocopherol. The gene encoding HPPD in Arabidopsis is transferred into rice, the content of tocotrienol is slightly increased, the total content of tocopherols is almost unchanged, the content of alpha-tocopherol is obviously increased, and the content of gamma-tocopherol is obviously reduced. The genes encoding HPT and gamma-TMT in Arabidopsis thaliana are independently transferred into lettuce, the total amount and the value of tocopherol are respectively increased, if the two genes are simultaneously transferred into lettuce, the total amount of tocopherol and the alpha/gamma value are simultaneously increased, and especially the total amount can be increased to 8 times of wild type and is higher than that of a single plant independently transferred with HPT.
Molecular markers are genetic markers based on nucleotide sequence variations within genetic material between individuals, and are a direct reflection of genetic polymorphisms at the DNA level. Single nucleotide polymorphisms (Single Nucleotide Polymorphism, SNP) are the third generation DNA molecular markers proposed by American scholars Lander E in 1996. SNP refers to the difference of only individual nucleotides or only small insertions, mutations or deletions between different alleles at the same site. SNPs are widely distributed in biological genomes, are abundant and genetically stable, and are the main genetic sources of phenotypic variation of different individuals in species.
Competitive allele-specific PCR (Kompetitive Allele Specific PCR, KASP) is a SNP genotyping technique that, since the advent, rapidly preempts the market with its ultra-high flexibility, accuracy and cost-performance, has been referred to in the industry as "genotyping investigator fingertip-jumping bead chain". KASP is the typing of SNPs based on specific matching of primer terminal bases. KASP contains three parts: DNA to be tested with target SNP; a KASP primer Mix (KASP Assay Mix) containing two allele-specific competitor forward primers for FAM or HEX adapter sequences, and a common reverse primer; KASP Master mix containing Universal FRET (fluorescence resonance energy transfer) cassette, ROX TM Passive reference dye, taq polymerase, free nucleotide and MgCl 2 . During the PCR reaction, the two allele-specific primers bind to and extend, respectively, target SNPs of different genotypes, thereby impregnating FAM or HEX adapter sequences onto the newly synthesized DNA strand. In the subsequent PCR reaction, FAM or HEX oligonucleotide primers with fluorescent groups of different colors can be amplified by using newly generated DNA with FAM or HEX linker sequences as templates to release fluorescent signals. The genotype can be judged by reading the fluorescent signal by an instrument. The KASP technique achieves bi-allele discrimination by competitive binding of two allele-specific forward primers. If the genotype of a given SNP is homozygous, only one of two possible fluorescent signals will be generated. If the genotypes are heterozygous, a mixed fluorescent signal is generated. KASP has the advantages of high polymorphism, abundant labels, low cost, simple operation and the like, and has become one of the mainstream methods of SNP detection analysis internationally.
The molecular marker assisted selection refers to the selection of a target trait genotype by means of molecular markers by analyzing the genotypes of the molecular markers closely linked to the target gene. The molecular marker assisted selection technology is utilized to improve the alpha-tocopherol content of rice, and has the advantages of no transgenic step, simple and convenient operation, rapid typing, flexible use and low cost, and can become an indispensable assisted breeding tool for wide breeders.
However, the molecular markers developed by improving the nutrition quality of the rice lines with good characters and stable background at present have the advantages of rapid typing, accurate results, low cost and the like, have low breeding efficiency and long breeding time, can not well improve the selection efficiency of the target characters and can not meet the requirement of large-scale molecular marker assisted selection breeding.
Disclosure of Invention
In view of this, the present invention provides a molecular marker VEQ2 and its use.
The invention provides a molecular marker VEQ2 and application thereof, which can lead the genetic background to be stable, improve the content of alpha-tocopherol of rice with good comprehensive properties, and develop a KASP (Kompetitive Allele-Specific PCR) molecular marker in order to improve the nutrition quality of rice with good comprehensive properties, and further prove that the KASP marker is more efficient, the Q2 locus gene for controlling the content of the alpha-tocopherol in the rice polished rice can be accurately detected for genotyping detection, the breeding efficiency is improved, and the breeding time is shortened. The method has the advantages of simple and convenient operation, rapid parting, accurate result, low cost and the like, can improve the selection efficiency of the target character, and meets the requirement of large-scale molecular marker assisted selection breeding.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides the application of the molecular marker in the following aspects:
(I) Predicting or identifying the alpha-tocopherol content of a graminaceous plant; and/or
(II) improvement of the nutritional quality of Gramineae plants.
The molecular marker is positioned at the 28809100 base of the chromosome 2 of the gramineous plant;
the improvement comprises increasing the alpha-tocopherol content in the progeny gramineous plants by molecular marker-assisted selective breeding.
In some embodiments of the invention, the gramineous plant comprises rice; the rice includes polished rice or brown rice.
The invention also provides a primer set, which comprises:
(I) The forward primer Q2-F1 has a nucleotide sequence shown as SEQ ID NO. 1; and
(II) forward primer Q2-F2 has a nucleotide sequence shown as SEQ ID NO. 2; and
(III) the reverse primer Q2-R has a nucleotide sequence shown as SEQ ID NO. 3; or (b)
(IV) a nucleotide sequence which encodes the same protein as the nucleotide sequence set forth in any one of (I) to (III) but differs from the nucleotide sequence set forth in any one of (I) to (III) due to the degeneracy of the genetic code; or (b)
(v) a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotide sequences with the nucleotide sequence shown in any one of (I) to (iv), and functionally identical or similar to the nucleotide sequence shown in any one of (I) to (iv); or (b)
A nucleotide sequence having at least 90% sequence homology with the nucleotide sequence of any one of (I) to (V).
On the basis of the above study, the invention also provides application of the primer group in the following aspects:
(I) Predicting or identifying the alpha-tocopherol content of a graminaceous plant; and/or
(II) improvement of the nutritional quality of gramineous plants; and/or
(III) preparing a kit for predicting or identifying the alpha-tocopherol content of a graminaceous plant;
the improvement comprises increasing the alpha-tocopherol content in the progeny gramineous plants by molecular marker-assisted selective breeding.
In some embodiments of the invention, the gramineous plant comprises rice; the rice includes polished rice or brown rice.
The invention also provides a kit comprising the primer group.
The invention also provides application of the kit in the following aspects:
(I) Predicting or identifying the alpha-tocopherol content of a graminaceous plant; and/or
(II) improvement of the nutritional quality of gramineous plants;
the improvement comprises increasing the alpha-tocopherol content in the progeny gramineous plants by molecular marker-assisted selective breeding.
In some embodiments of the invention, the gramineous plant comprises rice; the rice includes polished rice or brown rice.
The invention also provides an identification method of the tocopherol content, which comprises the following steps: extracting genomic DNA of the gramineous plant, identifying the genotype of the gramineous plant by using the primer group and/or the kit, and detecting the tocopherol content.
In some embodiments of the invention, the tocopherol comprises alpha-tocopherol.
In some embodiments of the invention, the gramineous plant comprises rice; the rice includes polished rice or brown rice.
In some embodiments of the invention, the alpha-tocopherol content criteria based on the genotype are as follows:
(I) If the 28809100 base of chromosome 2 of the gramineous plant is CC, the gramineous plant has higher alpha-tocopherol content than if the base is TT, namely the average alpha-tocopherol content of the CC plant is obviously higher than that of the TT plant;
(II) if the base 28809100 of chromosome 2 of the gramineous plant is CT, the gramineous plant has a higher or similar level of alpha-tocopherol content than if the base is TT, i.e., the CT plant has no significant difference or higher mean alpha-tocopherol content than the TT plant.
In some embodiments of the invention, the gramineous plant comprises rice; the rice includes polished rice or brown rice.
The invention also provides a gramineous plant breeding method, which comprises the following steps: extracting genomic DNA of gramineous plants, carrying out PCR amplification by using the primer group and/or the kit, and judging according to fluorescent signals of PCR products to obtain corresponding varieties.
In some embodiments of the invention, the gramineous plant comprises rice; the rice includes polished rice or brown rice.
In some embodiments of the invention, the criteria for determining the genotype for evaluating the α -tocopherol content based on the fluorescence signal are as follows:
(I) If the 28809100 base of chromosome 2 of the gramineous plant is CC, the gramineous plant has higher alpha-tocopherol content than if the base is TT, namely the average alpha-tocopherol content of the CC plant is obviously higher than that of the TT plant;
(II) if the base 28809100 of chromosome 2 of the gramineous plant is CT, the gramineous plant has a higher or similar level of alpha-tocopherol content than if the base is TT, i.e., the CT plant has no significant difference or higher mean alpha-tocopherol content than the TT plant.
In some embodiments of the invention, the gramineous plant comprises rice; the rice includes polished rice or brown rice.
In some embodiments of the invention, the reaction procedure for PCR amplification comprises:
pre-denaturation at 94℃for 15min, denaturation at 94℃for 20s, annealing at 61-55℃for 60s,10 cycles (0.6℃decrease per cycle); and/or
(II) pre-denaturation at 94℃for 15min, denaturation at 94℃for 20s, annealing at 55℃for 60s,26 cycles; and/or
(III) pre-denaturation at 94 ℃ for 15min, denaturation at 94 ℃ for 20s, annealing at 57 ℃ for 60s, and 32-38 cycles;
preferably, said (III) is pre-denatured at 94℃for 15min, denatured at 94℃for 20s, annealed at 57℃for 60s,35 cycles.
The invention provides a locus VEQ2 for regulating and controlling the content of alpha-tocopherol in rice, a KASP (Kompetitive Allele-Specific PCR) molecular marker for detecting the genotype of the locus and application thereof, and the locus VEQ2 is efficiently and accurately subjected to genotyping detection by using the KASP marker, so that the alpha-tocopherol content is predicted, the breeding efficiency is improved, and the breeding time is shortened. The method has the advantages of simple and convenient operation, rapid parting, accurate result, low cost and the like, can improve the selection efficiency of the target character, and meets the requirement of large-scale molecular marker assisted selection breeding.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIG. 1 shows the results of Sanger sequencing of the VEQ2 site in the PXB genome in example 2; blue highlights, VEQ2 sites;
FIG. 2 shows the results of the Sanger sequencing of the VEQ2 locus in the AKITAKOMACHI genome of example 2; blue highlights, VEQ2 sites;
FIG. 3 shows the results of the Sanger method of sequencing the VEQ2 site in the Domix genome in example 2; blue highlights, VEQ2 sites;
FIG. 4 shows the distribution of the relative content of alpha-tocopherol in 288 parts of the variety of the VEQ2 locus homozygous C genotype (CC) and 244 parts of the brown rice with the variety of the homozygous T genotype (TT) in example 3; the 5 horizontal lines from top to bottom of each bin represent the maximum, 75%, median, 25% and minimum values, respectively, the points represent outliers, "×" represents mean, "×" represents very significant;
FIG. 5 shows the distribution of the relative content of alpha-tocopherol in polished rice of 288 varieties of the VEQ2 locus homozygous C genotype (CC) and 244 varieties of the homozygous T genotype (TT) in example 4; the 5 horizontal lines from top to bottom of each bin represent the maximum, 75%, median, 25% and minimum values, respectively, the points represent outliers, "×" represents mean, "×" represents very significant;
FIG. 6 shows the use of the KASP markers to identify genotypes at the VEQ2 locus in 12 rice varieties in example 6; wherein blue dotsIs homozygous T genotype, red dot is homozygous C genotype, green dot is heterozygous genotype, black square dot is ddH 2 O；
FIG. 7 shows the detection of F of rice varieties Baikezaohe and AKITAKOMACHI using the molecular markers of the present invention in example 7 2 Genotype of VEQ2 locus in the generation-isolated plants; wherein the blue dot is homozygous T genotype, the red dot is homozygous C genotype, the green dot is heterozygous genotype, and the black square dot is ddH 2 O；
FIG. 8 shows the F of the VEQ2 genotype of example 8 for rice varieties Baikezaohe and AKITAKOMACHI 2 The influence of alpha-tocopherol content in brown rice of the generation isolated plants; CC. TT and CT represent the homozygous C genotype, homozygous T genotype and heterozygous genotype of the VEQ2 locus, respectively; the 5 horizontal lines from top to bottom of each bin represent the maximum, 75%, median, 25% and minimum values, respectively, "×" represents the mean value, "×" represents the most significant;
FIG. 9 shows the F of the VEQ2 genotype of example 9 for rice varieties Baikezaohe and AKITAKOMACHI 2 Influence of alpha-tocopherol content in polished rice of the generation isolated plants; CC. TT and CT represent the homozygous C genotype, homozygous T genotype and heterozygous genotype of the VEQ2 locus, respectively; the 5 horizontal lines from top to bottom of each bin represent maximum, 75%, median, 25% and minimum values, respectively, the points represent outliers, "×" represents mean, and "×" represents significance.
Detailed Description
The invention discloses a molecular marker VEQ2 and application thereof, and a person skilled in the art can refer to the content of the molecular marker VEQ2 and properly improve the technological parameters. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that variations and modifications can be made in the methods and applications described herein, and in the practice and application of the techniques of this invention, without departing from the spirit or scope of the invention.
The invention aims to overcome the defects and shortcomings of the prior art and provides a site VEQ2 related to the content of rice brown rice and polished rice alpha-tocopherol, and a detection method and application thereof.
The invention locates a new SNP molecular marker VEQ2 for regulating and controlling the alpha-tocopherol content of rice by a whole genome association analysis method. The VEQ2 site is located at base 28809100 of chromosome 2 of rice (Nipponbare genome version MSU v 6.1). When the genotype of the VEQ2 locus is C, the brown rice and polished rice of the rice have higher alpha-tocopherol content. When the genotype of the VEQ2 locus is T, the brown rice and polished rice of the rice have lower alpha-tocopherol content.
A specific primer combination MP0445Q2 (vg 0228814969) for detecting a KASP molecular marker of a SNP locus for regulating and controlling the alpha-tocopherol content of rice polished rice, wherein the SNP locus is positioned at 28809100 th base of chromosome 2 of rice, and the KASP marker is obtained by PCR amplification of a primer with a nucleotide sequence shown as MP0445Q2 ID NO. 1-3.
The second object of the present invention is to provide a method for identifying the genotype of the alpha-tocopherol content site VEQ2 in the regulation of rice brown rice and polished rice by using the above molecular markers and the application thereof.
The third object of the present invention is to provide a method for identifying the alpha-tocopherol content in brown rice and polished rice of rice using the above molecular markers, or simultaneously identifying the genotype of the VEQ2 site and the alpha-tocopherol content in brown rice and polished rice, and applications thereof.
The molecular marker for regulating and controlling the alpha-tocopherol content of rice and the VEQ2 locus gene is obtained by amplifying a primer with a nucleotide sequence shown as SEQ ID NO. 1-3.
The invention provides application of the molecular marker in identifying the genotype of the VEQ2 locus for regulating and controlling the content of alpha-tocopherol in rice polished rice.
The invention provides application of the molecular marker in rice breeding.
The invention provides a specific primer combination for detecting the genotype of a VEQ2 locus for regulating and controlling the content of alpha-tocopherol in rice brown rice and polished rice, which contains primers with nucleotide sequences shown in SEQ ID NO. 1-3.
Wherein, the forward primer Q2-F1 shown in SEQ ID NO. 1:
GAAGGTGACCAAGTTCATGCTCCATAAGCTTACGGCACAAT and the forward primer Q2-F2 shown in SEQ ID NO. 2:
GAAGGTGACCAAGTTCATGCTCCATAAGCTTACGGCACAAC, and the reverse primer Q2-R as shown in SEQ ID NO. 3: TAGGTCCTACCATACCATAATCTAT.
The primer combination is obtained by designing and screening the VEQ2 locus. The combined positions of Q2-F1 and Q2-F2 on the genome are the same, the bases at the 3' end are T and C respectively, and the primers can be specifically matched with the SNP of the VEQ2 locus respectively, so that the selective amplification of different genotypes of the VEQ2 locus is realized. To increase selection efficiency, a T to A mismatch was introduced at the 5 th base from the 3' end of Q2-F1 and Q2-F2, respectively. In addition, the 5' -end of Q2-F1 has FAM linker sequence
A1: GAAGGTGACCAAGTTCATGCT, 5' end of Q2-F2 carries HEX linker sequence A2: GAAGGTCGGAGTCAACGGATT. After several PCR cycles, FAM and HEX linkers permeate into the newly generated PCR fragments, allowing FAM or HEX oligonucleotide primers with blue and red fluorescent groups, respectively, to amplify using the newly generated PCR fragments as templates and to carry fluorescent signals into the PCR products. Finally, the Q2-F1 primer can amplify PCR products with blue fluorescence, and the Q2-F2 primer can amplify PCR products with red fluorescence. Q2-R is a public reverse primer, and can be respectively paired with Q2-F1 and Q2-F2 to amplify a PCR product of 91 bp.
The invention provides application of the specific primer combination in identifying genotypes of VEQ2 loci for regulating and controlling alpha-tocopherol content in rice polished rice.
The invention provides a method for identifying whether genotype variation of a VEQ2 locus for regulating and controlling alpha-tocopherol content exists in specific rice germplasm resources by using the specific primer combination.
The invention provides application of the specific primer combination in rice germplasm resource improvement.
Further, the invention provides a method for detecting the genotype of the VEQ2 locus for regulating and controlling the content of alpha-tocopherol in the brown rice and polished rice of rice. Firstly extracting genome DNA from a sample to be detected, then carrying out PCR amplification by using forward primers shown by Q2-F1 and Q2-F2 and reverse primers shown by Q2-R, and finally judging the genotype by analyzing fluorescent signals of PCR products.
If the fluorescent signal released by the amplified product is red, the genotype of the sample to be detected is homozygote C, and the sample to be detected has higher alpha-tocopherol content; if the released fluorescent signal is blue, the genotype of the sample to be detected is homozygote T, and the content of alpha-tocopherol is lower; if the released fluorescent signal is green, the sample to be detected is heterozygous genotype CT, and the alpha-tocopherol content is medium.
Further, the PCR reaction system used KASP-TFV 4.02X1sterMix kit. The volume of the reaction system was 5. Mu.L, and it contained 2 XSPMstermix 2.5. Mu.L, 36. Mu. Mol/L of Q2-F1, 36. Mu. Mol/L of Q2-F2 and 90. Mu. Mol/L of Q2-R primer each 0.023. Mu.L, 50 ng/. Mu.L of template DNA 1. Mu.L, ddH 2 O1.43. Mu.L was made up to 5. Mu.L.
The PCR reaction procedure was: pre-denaturation at 94℃for 15min; denaturation at 94℃for 20s, annealing at 61-55℃for 60s,10 cycles (0.6℃decrease per cycle); denaturation at 94℃for 20s and annealing at 55℃for 60s,26 cycles. If the parting effect is not obvious, the parting effect can be increased by 6 to 12 cycles: denaturation at 94℃for 20s and annealing at 57℃for 60s. Optimally, 9 cycles can be added: denaturation at 94℃for 20s and annealing at 57℃for 60s. PCR reactions and fluorescence detection were performed on an ABI Quantum studio7 Flexreal-Time PCR system (Applied Biosystem, foster City, calif., USA).
Kits containing the specific primer combinations of the invention are also within the scope of the invention.
The invention provides application of the kit containing the primers shown in Q2-F1, Q2-F2 and Q2-R in breeding of a Q2 locus for regulating and controlling VE content in rice.
The invention provides application of the kit containing the primers shown in Q2-F1, Q2-F2 and Q2-R in rice germplasm resource improvement.
The invention provides a molecular marker VEQ2 related to the content of alpha-tocopherol of an E-vitamin substance in rice and application thereof, belonging to the technical field of plant biology. SNP site Q2, which is located by the early laboratory (Hainan university Metabolic biology laboratory) through mGWAS (metagenome-based genome-wide association studies), is designed to regulate the alpha-tocopherol content of rice. On the basis, molecular markers are developed for the Q2 locus, a high-value parent AKITAKOMACHI, domsiah and a low-value parent PXB of the Q2 locus of vitamin E are screened, then backcross transformation is carried out on the PXB by taking AKITAKOMACHI and Domsiah as donors through molecular marker selection, and finally offspring plants with homozygous Q2 locus high-value alleles and high genetic background recovery rate are selected as improved varieties to be popularized and applied.
The invention provides a molecular marker for regulating and controlling rice polished rice alpha-tocopherol Q2 genotype identification and application thereof, which can ensure that the genetic background is stable, the content of alpha-tocopherol of rice with good comprehensive properties is improved, in order to improve the nutrition quality of rice lines with good and stable properties, the content of alpha-tocopherol of the rice lines is improved, KASP (Kompetitive Allele-Specific PCR) molecular markers are developed, specific primers are designed aiming at allele SNP loci, and under the technical support of LGC (Laboratory of the Government Chemist) companies, the fact that the Q2 locus genes for regulating and controlling the content of alpha-tocopherol in rice polished rice are detected by using the KASP markers more efficiently and accurately for genotyping detection is confirmed, the breeding efficiency is improved, and the breeding time is shortened.
The molecular marker of the invention comprises two forward primers SEQ ID NO.1 and SEQ ID NO.2 and one reverse primer SEQ ID NO.3. The SNP locus is positioned at 28809100 base of rice chromosome 2. This technique is based on specific matching of primer terminal bases to genotype SNPs and detect InDels (insertions and deletions). The method has the advantages of simple and convenient operation, rapid parting, accurate result, low cost and the like, can improve the selection efficiency of the target character, and meets the requirement of large-scale molecular marker assisted selection breeding.
Compared with the prior art, the invention has the following beneficial effects:
1) The invention discovers a new locus VEQ2 for regulating and controlling the content of alpha-tocopherol in rice brown rice and polished rice. The average content of alpha-tocopherol in the pure and C-shaped plants of VEQ2 locus is 82.2 percent (P=0.0061) and 112.8 percent (P=0.027) higher than that in the pure and T-shaped brown rice and polished rice, respectively, and the average content of alpha-tocopherol in the hybrid plants is 33.8 percent higher than that in the pure and T-shaped brown rice and polished rice, respectively
(p=0.083) and 78.8% (p=0.018).
2) A KASP marker was designed and developed based on the VEQ2 locus. The marker can be used for conveniently, flexibly and accurately genotyping the VEQ2 locus, predicting the content of alpha-tocopherol in the brown rice and polished rice, improving the character selection efficiency and meeting the requirement of large-scale molecular marker assisted selection.
The molecular marker VEQ2 and the raw materials and reagents used in the application of the molecular marker VEQ2 can be purchased from the market.
The invention is further illustrated by the following examples:
example 1 localization of the VEQ2 site controlling alpha-tocopherol content Using Whole genome correlation analysis
The relative content of brown rice alpha-tocopherol was determined using a high performance liquid chromatography-mass spectrometry combined with a widely targeted metabolite determination technique (table 1) using 533 parts of rice germplasm resources collected from around the world as a study object, using a resequencing technique to determine the nuclear genome sequence of each variety. Performing metabolite-based whole genome correlation analysis based on the above genome and alpha-tocopherol relative content data, and locating a SNP site VEQ2 (P=1.79×10 -13 ). The VEQ2 site is located at base 28809100 of chromosome 2 of rice (Nipponbare genome version MSUv 6.1). Resequencing data indicated (table 1) that both genotypes were detected at the VEQ2 locus in 533 cultivars. Wherein 288 parts of homozygous T genotype varieties and 244 parts of homozygous C genotype varieties are used.
TABLE 1 genotype of Rice and relative content of alpha-tocopherol
Example 2VEQ2 site genotype Sanger sequencing validation
From the 533 varieties, a variety PXB having a homozygous T genotype and a variety AKITAKOMACHI, domish having a homozygous C genotype, respectively, were selected based on the resequencing data. Primers VEQ2CXF were designed based on the 1kb sequence upstream and downstream of the VEQ2 site:
GGCATAAGGTTCAATTCTTTCTCAT (as shown in SEQ ID No. 4), and VEQ2CXR: TATCTCCTCCCATATTAAGGGCAAC (SEQ ID No. 5). The leaf genomic DNA of AKITAKOMACHI, domish and PXB was used as templates, and primers VEQ2CXF and VEQ2CXR were used to amplify PCR, and the amplified products were subjected to Sanger sequencing. Sequencing results show that the genotype of the VEQ2 locus of the variety PXB is homozygous T (see figure 1), and the genotype of the VEQ2 locus of the variety AKITAKOMACHI (see figure 2) and Domish (see figure 3) is homozygous C.
Example 3 Effect of the VEQ2 site on the alpha-tocopherol content in brown rice
The 533 varieties are divided into two groups of homozygous T genotypes and homozygous C genotypes according to the genotypes of the VEQ2 loci. The brown rice of homozygous T genotype variety has a relative alpha-tocopherol content of 4535 at the lowest value and 295520 at the highest value and 69351 at the average value. Brown rice of homozygous genotype C variety had a relative alpha-tocopherol content of 24927 at the lowest value, 413397 at the highest value and 147636 at the average value (fig. 4, table 1). T-tests showed that the average relative content of alpha-tocopherol in homozygous T and C genotype varieties was very different (p=7.36×10 -49 ). The above results indicate that the VEQ2 site is significantly correlated with the alpha-tocopherol content of rice and brown rice. Wherein, the VEQ2 genotype is a group with homozygous T, the average content of alpha-tocopherol in the brown rice is lower, and the VEQ2 locus genotype is a group with homozygous C, and the average content of alpha-tocopherol in the brown rice is higher.
Example 4 influence of the VEQ2 site on the alpha-tocopherol content in polished rice
The 533 varieties are divided into two groups of homozygous T genotypes and homozygous C genotypes according to the genotypes of the VEQ2 loci. The polished rice of homozygous T genotype variety has a minimum value of 4786, a maximum value of 328971 and an average value of 47161 for the relative alpha-tocopherol content. The polished rice of the homozygous genotype C variety had a relative alpha-tocopherol content of 2836 at a minimum and 1034214 at a maximum and 104321 (FIG. 5, table 1). T-test showed that the average relative content of alpha-tocopherol in homozygous T and C genotype varieties was very different (p=1.78×10 -10 ). The above results indicate that the VEQ2 locus is also significantly associated with the alpha-tocopherol content of rice polished rice. Wherein, the VEQ2 genotype is a group of homozygous T, the average content of alpha-tocopherol in the polished rice is lower, and the VEQ2 locus genotype is a group of homozygous C, and the average content of alpha-tocopherol in the polished rice is higher.
EXAMPLE 5 development of the VEQ2 site KASP marker
1. Primer design
A three primer combination capable of discriminating the genotype of the vitamin EVEQ2 site was designed based on the instructions of the KASP kit (KASP-TF V4.0 XMaster Mix) from LGC (Laboratory of the Government Chemist) company and the sequence of 1kb genomic DNA upstream and downstream of the VEQ2 site:
Q2-F1: GAAGGTGACCAAGTTCATGCTCCATAAGCTTACGGCACAAT (shown as SEQ ID No. 1)
Q2-F2: GAAGGTGACCAAGTTCATGCTCCATAAGCTTACGGCACAAC (shown as SEQ ID No. 2)
Q2-R TAGGTCCTACCATACCATAATCTAT (shown as SEQ ID No. 3).
2. Analysis of expected amplification results
The genomic DNA of rice varieties is amplified by the primer combination, and the amplified product is detected by a SNP genotyping detector (ABI quantsurio 7 Flexreal-Time PCR system (Applied Biosystem, foster City, calif., USA). If the amplified product releases a blue fluorescent signal, the amplified product indicates that the sample to be detected has a homozygous T genotype, namely TT, if the released fluorescent signal is red, the amplified product indicates that the sample to be detected has a homozygous C genotype, namely CC, and if the released fluorescent signal is green, the amplified product indicates that the sample to be detected has a heterozygous genotype CT.
Example 6 identification of genotypes at the VEQ2 locus in different Rice varieties Using the KASP marker
1. Experimental materials
Rice germplasm resources 88B-2, lucaihao, sankecui, jiabala, huke3hao, taizhang65hao, liusha1hao, benbanggu, hanmadao, mowanggui, gang46B, M401. All experimental materials were stored and supplied by the university of hainan tropical crop academy of metabolism biology laboratory.
2. Extraction of genomic DNA from rice
The method for extracting rice genome DNA by adopting CTAB method comprises the following specific steps: rice leaves 3cm long were harvested and extracted in 800. Mu.L of extraction buffer [1.5% (w/V) CTAB,1.05mol/LNaCl,75mmol/LTris-HCl (pH 8.0), 15mmol/LEDTA (pH 8.0)]Is collected in a 1.5mL centrifuge tube. Water bath at 65 ℃ for 30min, and mixing evenly in a reverse way. 800. Mu.L of chloroform to isoamyl alcohol (volume ratio 24:1) was added and mixed by inversion for 15min.12000r/min, and centrifuging at room temperature (20-30 ℃) for 10min. The supernatant was pipetted into a new 1.5mL centrifuge tube, 2 volumes of 95% ethanol were added, mixed well and precipitated at-20℃for 30min. Centrifuge at 12000r/min for 15min. The 95% ethanol was decanted and the precipitate was washed with 75% ethanol. Pouring out 75% ethanol, drying, adding 100 μl sterilized ddH 2 O dissolves DNA.
3. PCR amplification and detection
The variety PXB of example 2 was used as homozygous T genotype control, the variety AKITAKOMACHI was used as homozygous C genotype control, the genomic DNA of PXB and AKITAKOMACHI were mixed in equal amounts to obtain heterozygous genotype control, and ddH was used 2 O is a negative control. The DNA of the 12 rice varieties/lines described in this example was PCR amplified using the specific primer combinations (Q2-F1, Q2-F2, Q2-R) obtained by the screening in example 5. Amplification was performed using the KASP-TFV 4.02XMasterMix kit from LGCGenomics, 5. Mu.L of PCR reaction system comprising 2 XKASPMasterMix 2.5. Mu.L, 36. Mu. Mol/L of Q2-F1, 36. Mu. Mol/L of Q2-F2 and 90. Mu. Mol/L of Q2-R primers each 0.023. Mu.L, 50 ng/. Mu.L of template DNA 1. Mu.L, ddH 2 O1.43. Mu.L was made up to 5. Mu.L.
The PCR reaction procedure was: pre-denaturation at 94℃for 15min; denaturation at 94℃for 20s, annealing at 61-55℃for 60s,10 cycles (0.6℃decrease per cycle); denaturation at 94℃for 20s, annealing at 55℃for 60s,26 cycles; denaturation at 94℃for 20s and annealing at 57℃for 60s,9 cycles.
After the PCR reaction was completed, the fluorescent signal was read on an ABIQuantum studio7Flexreal-TimePCRsystem (Applied Biosystem, fosterCity, CA, USA).
4. Results and analysis
As shown in FIG. 6, the PCR products of 11 lines, 88B-2, lucaihao, sankecui, jiabala, huke3hao, taizhong65hao, liusha1hao, benbanggu, hanmadao, mowanggui, gang46B, etc., emit blue fluorescent signals, and the signal points thereof are clustered together with the homozygous T-type strain PXB, which also emits blue fluorescent signals, in a coordinate system, indicating that the genotype of the VEQ2 site thereof is TT. M401 and homozygous C line AKITAKOMACHI emitting red fluorescent signal are clustered together, indicating that the genotype of its VEQ2 locus is CC. The 2 heterozygous genotype controls gave a green fluorescent signal, but none of the varieties and the heterozygous controls clustered together, indicating that none of the VEQ2 loci were heterozygous varieties.
EXAMPLE 7 identification of F with KASP marker 2 Genotyping of VEQ2 sites in isolated populations
1. Experimental materials
Crossing the recipient parent, e.g. Baikezaohe, with the donor parent AKITAKOMACHI to obtain F 1 ，F 1 Selfing to obtain F 2 Isolating the population. The recipient parent is stored and provided by Metabolic biology laboratory at the university of Henan Tropical crop university, F 1 And F 2 Provided by the invention.
2. Extraction of genomic DNA from rice
Reference example 6.
3. PCR amplification and detection
The parental variety Baikezaohe is used as a homozygous T genotype control, the parental variety AKITAKOMACHI is used as a homozygous C genotype control, the genomic DNA of Baikezaohe and AKITAKOMACHI are mixed in equal amounts to be used as a heterozygous genotype control, and ddH is used 2 O is a negative control. By implementation ofEXAMPLE 5 KASP primer combinations (Q2-F1, Q2-F2, Q2-R) screened for F as described in this example 2 And respectively extracting leaf genome DNA of individual plants of the population for PCR amplification. Amplification was performed using the KASP-TFV 4.02X1sterMix kit from LGCGenomics. The PCR reaction system, the reaction procedure and the method for detecting the products were the same as in example 6.
4. Results and analysis
The genotype test results are shown in FIG. 7. There are 27F 2 The PCR product of the single plant emits blue fluorescent signals, and the signal points of the PCR product are gathered together with the parent variety Baikezaohe which emits blue fluorescent signals in a coordinate system, so that the genotype of the VEQ2 locus of the PCR product is TT. There are 26F 2 The PCR product of the single plant emits red fluorescent signals, and the signal points of the PCR product are gathered together with a parent variety AKITAKOMACHI which emits red fluorescent signals in a coordinate system, so that the genotype of the VEQ2 locus of the PCR product is CC. There are 63F 2 The PCR product of the single plant emits green fluorescent signals, and the signal points of the PCR product are gathered together with the heterozygous control which emits the green fluorescent signals in a coordinate system, so that the genotype of the VEQ2 locus of the PCR product is CT.
Example 8VEQ2 site pair F 2 Influence of alpha-tocopherol content in brown rice plants
1. Experimental materials
Example 7 describes F 2 More than 10g of 52 individual plants of seeds can be harvested in the population.
2. Alpha-tocopherol relative content detection
Each F 2 The single plant is harvested for selfing, 10g of rice is taken, shelled by a rice huller and ground into powder. 0.1g of the powder was weighed, 1ml of an extract (70% methanol) was added to extract the metabolite, and the relative content of alpha-tocopherol was detected using a widely targeted metabonomics detection method in a high performance liquid chromatography-triple quadrupole-linear ion trap mass spectrometry detection system (HPLC-6500 QTRAP, ABSCIEX). The detection conditions include: chromatographic column, shim-packVP-ODSC18 (pore 5.0 μm, length 2X 150 mm)); mobile phase, phase A is ddH 2 O+0.04% acetic acid, phase B methanol+0.04% acetic acid; elution gradient, 0min:5% of B mobile phase, 95% of B mobile phase in 11.0-15.0 min, and 5% of B mobile phase in 15.5-20 min; flow of The speed is 0.25mL/min; column temperature is 40 ℃; the sample loading was 2. Mu.L.
3. Results and analysis
According to the genotype of the VEQ2 site in example 7, 52F are described in this example 2 Individuals are divided into TT, CC and CT genotypes. As shown in table 2 and fig. 8, in brown rice of TT genotype plants, the relative content of α -tocopherol was at a minimum of 43486, at a maximum of 653754, and at an average of 247963. The brown rice of CC genotype plant has a minimum value of 207801, a maximum value of 783413 and an average value of 435485. The brown rice of CT genotype plant has relative alpha-tocopherol content of 20213, 621463 and 331643. t-tests show that the relative content of alpha-tocopherol in brown rice of CC genotype plants is significantly higher than that of TT genotype plants (P=6.09×10 -3 ) The relative content of alpha-tocopherol in brown rice of CT genotype plants was not significant although higher than that of TT genotype plants (p=8, 34×10 -2 ). The result shows that the VEQ2 locus has the effect of regulating the alpha-tocopherol content of the rice brown rice. Wherein, when the genotype of the VEQ2 locus is TT, the average content of alpha-tocopherol in the brown rice is lower, and when the genotype of the VEQ2 locus is CC, the average content of alpha-tocopherol in the brown rice is higher.
TABLE 2F 2 Genotype of population and relative content of alpha-tocopherol
Example 9VEQ2 site pair F 2 Influence of alpha-tocopherol content in polished rice plants
1. Experimental materials
Example 7 describes F 2 More than 20g of 46 individual plants of seeds can be harvested in the population.
2. Alpha-tocopherol relative content detection
Each F 2 The single plant is harvested from the inbred seeds, 10g of paddy is taken out of the seed, and is milled into polished rice by a rice polisher, and then is ground into powder. The relative alpha-tocopherol content was measured as in example 8.
3. Results and analysis
According to the genotype of the VEQ2 site in example 7, the 46F are described in this example 2 Individuals are classified into CC, TT and CT genotypes. As shown in table 2 and fig. 9, in polished rice of CC genotype plants, the relative content of α -tocopherol was at a minimum value of 17300, at a maximum value of 137000, and at an average value of 71982. Among polished rice of TT genotype plants, the minimum value of the relative content of alpha-tocopherol is 17600, the maximum value is 91600, and the average value is 42444. The polished rice of CT genotype plants had a relative alpha-tocopherol content of 21200 at a minimum and 256000 at a maximum and 75504 at an average. t-tests showed that the average alpha-tocopherol relative content in polished rice of both CC genotype (p=0.0028) and CT genotype (p=0.018) plants was significantly higher than that of TT genotype plants. The result shows that the VEQ2 locus has the effect of regulating the content of alpha-tocopherol in polished rice. Wherein, when the genotype of the VEQ2 locus is TT, the average content of alpha-tocopherol in polished rice is lower, and when the genotype of the VEQ2 locus is CC and CT, the average content of alpha-tocopherol in polished rice is higher.
Example 10 increasing alpha-tocopherol content of Rice Using VEQ2 markers
Hybridization, backcrossing and selfing are performed with donor parents, such as AKITAKOMACHI, domish, and acceptor parents with normal fertility, such as PXB, and in the process, a molecular marker is used for selecting a VEQ2 locus and a genetic background, so that a strain with CC genotype at the VEQ2 locus and increased alpha-tocopherol content in brown rice and polished rice under the PXB background is finally obtained. The parent materials are all stored and provided by the Metabolic biology laboratory at the university of Tropical crops, hainan university. The specific implementation steps are as follows:
1. hybridization of a parent receptor, e.g. PXB, with AKITAKOMACHI, domish to obtain F 1 。
2. Using primer combinationsDetection of F by Q2-F1, Q2-F2 and Q2-R 1 The genotype of the hybrid seeds is selected, the plant with PCR product producing green fluorescent signal is used as mother parent and acceptor parent, such as PXB, and back cross to obtain BC 1 F 1 。
3. Planting BC 1 F 1 Detection of BC Using primer combinations Q2-F1, Q2-F2 and Q2-R 1 F 1 Plant genotype. VEQ2 locus heterozygous genotype plants were selected.
4. Using a set of genotypes (e.g., 100, or 200, etc.) with polymorphisms between the donor parent and acceptor parent genomes and uniformly distributed molecular markers (which may be, but are not limited to, SSR, SNP, INDEL, EST, RFLP, AFLP, RAPD, SCAR, etc.) on the genomes, the individual plants selected in step 3 are subjected to genetic background identification, and plants with high similarity (e.g., greater than 88% similarity, or 2% medium selection, etc.) to the recurrent parent genotypes are selected.
5. Backcrossing the plant selected in step 4 with a recipient parent, e.g., PXB, to obtain BC 2 F 1 。
6. Planting BC 2 F 1 Repeating the steps 3 and 4, selecting the plant with the VEQ2 genotype heterozygous and high genetic background recovery rate (such as more than 98 percent or 2 percent selection rate) and the like, and collecting the selfing seed BC 2 F 2 。
7. Planting BC 2 F 2 Repeating the step 3 and the step 4, selecting a plant with the VEQ2 genotype of CC and highest homozygous rate of genetic background, and collecting the inbred seed BC 2 F 3 。
8. Determination of BC using a broad targeting method 2 F 2 Selection of BC for the content of alpha-tocopherol in inbred and donor parents, such as brown rice and polished rice in AKITAKOMACHI, domish 2 F 2 The single plant with obviously improved content of alpha-tocopherol in brown rice and polished rice in the selfing seeds is propagated into rice strain with high relative content of alpha-tocopherol.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (9)
1. Use of an isolated molecular marker in:
(I) Predicting or identifying the alpha-tocopherol content of rice; and/or
(II) improvement of the nutritional quality of gramineous plants;
The molecular marker is CT polymorphism of 28809100 th base of rice chromosome 2 of Nipponbare genome version MSU v 6.1;
the improvement is that the alpha-tocopherol content in the filial generation rice is improved through molecular marker assisted selective breeding;
the rice comprises polished rice or brown rice.
2. Use of a primer set for detecting a molecular marker in:
(I) Predicting or identifying the alpha-tocopherol content of rice; and/or
(II) improving the nutrition quality of rice; and/or
(III) preparing a kit for predicting or identifying the alpha-tocopherol content of rice;
the improvement is that the alpha-tocopherol content in the filial generation rice is improved through molecular marker assisted selective breeding;
the rice comprises polished rice or brown rice;
the molecular marker is CT polymorphism of 28809100 th base of rice chromosome 2 of Nipponbare genome version MSU v 6.1;
the primer set includes:
(I) The forward primer Q2-F1 has a nucleotide sequence shown as SEQ ID NO. 1;
(II) forward primer Q2-F2 has a nucleotide sequence shown as SEQ ID NO. 2;
and
(III) reverse primer Q2-R has the nucleotide sequence shown as SEQ ID NO. 3.
3. Use of a kit in:
(I) Predicting or identifying the alpha-tocopherol content of rice; and/or
(II) improving the nutrition quality of rice;
the improvement is that the alpha-tocopherol content in the filial generation rice is improved through molecular marker assisted selective breeding;
the rice comprises polished rice or brown rice;
the kit comprises a primer group for detecting a molecular marker;
the molecular marker is CT polymorphism of 28809100 th base of rice chromosome 2 of Nipponbare genome version MSU v 6.1;
the primer set includes:
(I) The forward primer Q2-F1 has a nucleotide sequence shown as SEQ ID NO. 1;
(II) forward primer Q2-F2 has a nucleotide sequence shown as SEQ ID NO. 2;
and
(III) reverse primer Q2-R has the nucleotide sequence shown as SEQ ID NO. 3.
4. The method for identifying the tocopherol content is characterized by comprising the following steps: extracting genome DNA of rice, identifying the genotype of the rice by using a primer group and/or a kit containing the primer group, and evaluating the tocopherol content;
the rice comprises polished rice or brown rice;
the primer group is used for detecting a molecular marker;
The molecular marker is CT polymorphism of 28809100 th base of rice chromosome 2 of Nipponbare genome version MSU v6.1;
the primer set includes:
(I) The forward primer Q2-F1 has a nucleotide sequence shown as SEQ ID NO. 1; or (b)
(II) forward primer Q2-F2 has a nucleotide sequence shown as SEQ ID NO. 2;
and
(III) reverse primer Q2-R has the nucleotide sequence shown as SEQ ID NO. 3.
5. The method of identification of claim 4, wherein the criteria for evaluating alpha-tocopherol content based on the genotype is as follows:
the rice genome is Nipponbare genome version MSU v6.1;
(I) If the 28809100 base of the chromosome 2 of the rice is CC, the rice has higher alpha-tocopherol content than the TT base, namely the average alpha-tocopherol content of CC plants is obviously higher than that of TT plants;
(II) if the base 28809100 of chromosome 2 of the rice is CT, the rice has a higher or similar level of alpha-tocopherol content as compared to when the base is TT, i.e., the CT plant contains no significant difference or higher average alpha-tocopherol content as compared to the TT plant;
The rice comprises polished rice or brown rice.
6. The rice breeding method is characterized by comprising the following steps: extracting rice genome DNA, and performing PCR amplification by using a primer group and/or a kit containing the primer group, and judging according to fluorescent signals of PCR products to obtain corresponding varieties;
the breeding is to evaluate the content of alpha-tocopherol;
the rice comprises polished rice or brown rice;
the primer group is used for detecting a molecular marker;
the molecular marker is CT polymorphism of 28809100 th base of rice chromosome 2 of Nipponbare genome version MSU v6.1;
the primer set includes:
(I) The forward primer Q2-F1 has a nucleotide sequence shown as SEQ ID NO. 1; or (b)
(II) forward primer Q2-F2 has a nucleotide sequence shown as SEQ ID NO. 2;
and
(III) reverse primer Q2-R has the nucleotide sequence shown as SEQ ID NO. 3.
7. The method of claim 6, wherein the genotyping based on the fluorescent signal is evaluated for alpha-tocopherol content as follows:
the rice genome is Nipponbare genome version MSU v6.1;
(I) If the 28809100 base of the chromosome 2 of the rice is CC, the rice has higher alpha-tocopherol content than the TT base, namely the average alpha-tocopherol content of CC plants is obviously higher than that of TT plants;
(II) if the base 28809100 of chromosome 2 of the rice is CT, the rice has a higher or similar level of alpha-tocopherol content as compared to when the base is TT, i.e., the CT plant contains no significant difference or higher average alpha-tocopherol content as compared to the TT plant;
the rice comprises polished rice or brown rice.
8. The method of claim 7, wherein the reaction procedure for PCR amplification comprises:
pre-denaturation at 94 ℃ for 15 min, denaturation at 94 ℃ for 20 s, annealing at 61-55 ℃ for 60 s,10 cycles, each cycle being reduced by 0.6 ℃; or (b)
(II) pre-denaturation at 94 ℃ for 15 min, denaturation at 94 ℃ for 20 s, annealing at 55 ℃ for 60 s,26 cycles; or (b)
(III) pre-denaturation at 94 ℃ for 15 min, denaturation at 94 ℃ for 20 s, annealing at 57 ℃ for 60 s, and 32-38 cycles.
9. The method of claim 8, wherein (iii) is pre-denatured at 94 ℃ for 15 min, denatured at 94 ℃ for 20 s, annealed at 57 ℃ for 60 s,35 cycles.
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