CN117568515B - SNP locus related to rice linoleic acid content and application thereof - Google Patents
SNP locus related to rice linoleic acid content and application thereof Download PDFInfo
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- 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
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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- C12Q2600/00—Oligonucleotides characterized by their use
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Abstract
The invention provides an SNP locus related to rice linoleic acid content and application thereof, comprising the steps of predicting, identifying or assisting in identifying the rice linoleic acid content, wherein the SNP molecular marker is positioned at 1731808 th base of a chromosome 6 of rice, and when the genotype of the SNP molecular marker of a sample to be detected is G, the probability of the sample to be detected for high linoleic acid content is obviously higher than that when the genotype is C. 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
Technical Field
The invention relates to the technical field of plant molecular biology, in particular to an SNP locus related to rice linoleic acid content and application thereof.
Background
The rice is rich in protein, fat, vitamins, trace minerals and other nutrients. Among them, linoleic acid (Linoleic acid) is an essential fatty acid which cannot be synthesized by human body by itself and can only be taken up by diet. Linoleic acid has the effects of reducing blood fat, improving vascular elasticity and promoting microcirculation, can prevent or reduce the incidence rate of cardiovascular diseases, and has remarkable prevention and treatment effects on diseases such as hypertension, hyperlipidemia, angina pectoris, coronary heart disease, atherosclerosis, senile obesity and the like, and is called vascular scavenger. Developing a breeding technology for improving the linoleic acid content in rice, cultivating rice varieties with high linoleic acid content, meeting the demand of the market on high-nutrition-quality rice, and being a new direction for improving the nutrition quality of rice.
The gene editing technology is a technical scheme mainly used for improving the linoleic acid content of rice at present. OsFAD3.1 and OsFAD3.2 are located on chromosome 11 and chromosome 12, respectively, and each encode a key enzyme in the rice fatty acid synthesis pathway, omega-3 fatty acid desaturase. Both cdnas were up to 97.32% homologous. Chen Xiao Legion uses gene editing technology to knock out OsFAD3.1 and OsFAD3.2 simultaneously, and blocks the way of converting linoleic acid into linolenic acid, so that the relative content of linoleic acid in seeds is improved (Li Zhiyuan, syngnathus, fang Zhiyuan, etc. cabbage SNP marker development and DNA fingerprint construction of main variety [ J ]. Chinese agricultural science, 2018,51 (14): 2771-27). ABE and other key enzyme genes for controlling oleic acid to be converted into linoleic acid by knocking out OsFAD2-1 by using gene editing technology, and the oleic acid content is found to be increased by 2 times compared with the wild type (ABE K,ARAKI E,SUZUKI Y,TOKI S,SAIKA H.Production of high oleic/low linoleic rice by genome editing.Plant Physiology and Biochemistry,2018,131:58-62).
Molecular marking is a genetic marking technology based on nucleotide sequence variation in genetic material among individuals, and directly reflects the genetic diversity of DNA level. Among them, single nucleotide polymorphism (Single Nucleotide Polymorphism, SNP) is a third generation DNA molecular marker, which was first proposed by American scholars Lander E in 1996. SNPs refer to differences in only individual nucleotides between different alleles at the same locus. SNPs are widely distributed, numerous and genetically stable in the biological genome, and are one of the major genetic sources of inter-individual phenotypic variation in species.
Competitive allele-specific PCR (Kompetitive ALLELE SPECIFIC PCR, KASP) is a technical approach for SNP genotyping. Since the advent of KASP, it has rapidly taken up the market with its high degree of flexibility, accuracy and economy, called the "indispensable tool for genotyping researchers". The KASP technology is based on the principle of specific matching of primer terminal bases, and can perform SNP typing and InDels (Insertions and Deletions) detection. The KASP technique includes three main components: target SNP contained in a DNA sample to be measured; a KASP primer Mix (KASP ASSAY Mix) comprising two specific competing forward primers with different tail sequences, and a common reverse primer; a KASP master mix comprising a universal FRET (fluorescence resonance energy transfer) probe, ROX TM passive reference dye, taq polymerase, free nucleotides and MgCl2. In the PCR reaction, the specific primer binds to the target SNP and extends, so that the newly synthesized strand is linked with a tail sequence for complementary replication. The FAM or HEX labeled oligonucleotides bind to complementary tail sequences on the newly synthesized strand and release fluorescent signals by multiple rounds of PCR amplification. As PCR amplification proceeds, the fluorescent signal gradually increases, eventually reading the data at the end of the reaction. By reading the terminal fluorescence, two potential genotypes at one site per sample can be determined. If the genotype of a given SNP is homozygous, only one fluorescent signal will be generated; if the genotypes are heterozygous, a mixed fluorescent signal is generated.
Molecular marker assisted selection refers to genotype assisted phenotypic selection using molecular markers closely linked to the target gene, thereby improving breeding efficiency. In the aspect of increasing the linoleic acid content of rice, the molecular marker assisted selection technology has a plurality of advantages, such as no need of transgenic operation, simple and convenient operation, rapid typing, flexible use and low cost. Therefore, developing a molecular marker technology for improving the content of the auxiliary linoleic acid provides a new auxiliary breeding tool for a large number of breeders.
Disclosure of Invention
In view of the above, the invention aims to provide an SNP locus related to the linoleic acid content of rice and application thereof, which can complete genotyping of the LAQ3 locus for controlling the linoleic acid content in rice and predict whether the linoleic acid content in rice is improved.
The technical scheme of the invention is realized as follows:
One of the purposes of the invention is to provide an application of detecting SNP molecular markers related to rice linoleic acid content in rice breeding, comprising prediction, identification or auxiliary identification of rice linoleic acid content, wherein the SNP molecular markers are positioned at 1731808 th base of chromosome 6 of rice, and when the genotype of a sample to be detected is G, the probability of the sample to be detected for high linoleic acid content is obviously higher than that when the genotype is C.
Further, when the genotype of the SNP molecular marker of the sample to be detected is homozygous G, the sample to be detected is high in linoleic acid content, when the genotype of the SNP molecular marker of the sample to be detected is heterozygous GC, the sample to be detected is medium in linoleic acid content, and when the genotype of the SNP molecular marker of the sample to be detected is homozygous C, the sample to be detected is low in linoleic acid content.
Further, the SNP molecular marker is applied to rice variety breeding, and comprises the step of improving linoleic acid content in progeny rice in the rice variety breeding by molecular marker-assisted selection.
Further, the rice includes polished rice or brown rice.
The invention also provides a primer group for detecting SNP molecular markers related to the linoleic acid content of rice, wherein the SNP molecular markers are positioned at the 1731808 th base of chromosome 6 of rice, when the genotype of the SNP molecular markers of a sample to be detected is G, the probability of the sample to be detected with high linoleic acid content is higher than that of the genotype of the sample to be detected with high linoleic acid content is C, and the primer group comprises a primer 1, a primer 2 and/or a primer 3;
The primer 1 has:
(1) A nucleotide sequence as shown in SEQ ID No. 1; or (b)
(2) A nucleotide sequence obtained by substituting, deleting or adding one or more bases to the nucleotide sequence shown in (1), and having the same or similar function as (1); or (b)
(3) A nucleotide sequence having at least 70% homology with the nucleotide sequence as set forth in (1) or (2);
the primer 2 has:
(4) A nucleotide sequence as shown in SEQ ID No. 2; or (b)
(5) A nucleotide sequence obtained by substituting, deleting or adding one or more bases to the nucleotide sequence shown in (4), and having the same or similar function as (4); or (b)
(6) A nucleotide sequence having at least 70% homology with the nucleotide sequence as set forth in (4) or (5);
The primer 3 has:
(7) A nucleotide sequence as shown in SEQ ID No. 3; or (b)
(8) A nucleotide sequence obtained by substituting, deleting or adding one or more bases to the nucleotide sequence shown in (7), and having the same or similar function as (7); or (b)
(9) A nucleotide sequence having at least 70% homology with the nucleotide sequence as set forth in (7) or (8);
The plurality is 2 to 12.
Further, the primer group is applied to rice breeding with high linoleic acid content.
Further, the primer group is applied to detection of the linoleic acid content of rice.
The invention also aims to provide a kit for breeding rice varieties and/or detecting the linoleic acid content of rice, which comprises the primer group and a detection reagent; the detection reagent comprises a KASP kit; the rice includes polished rice or brown rice.
In some embodiments of the invention, the kit comprises 2X KASP MASTER mix and KASP ASSSY mix;
The KASP ASSSY mix is prepared by mixing 36. Mu.M forward primer and 90. Mu.M reverse primer in equal volume, wherein the forward primer comprises the primer 1 and/or the primer 2, and the reverse primer comprises the primer 3.
The fourth object of the present invention is to provide a method for predicting, identifying or assisting in identifying linoleic acid content in rice, wherein the method is to detect the 1731808 th base of chromosome 6 of rice, when the genotype of the molecular marker of the sample to be detected is G, the probability of the sample to be detected being high linoleic acid content is significantly higher than that when the genotype is C.
When the genotype of the SNP molecular marker of the sample to be detected is homozygous G, the sample to be detected is high in linoleic acid content, when the genotype of the SNP molecular marker of the sample to be detected is heterozygous GC, the sample to be detected is medium in linoleic acid content, and when the genotype of the SNP molecular marker of the sample to be detected is homozygous C, the sample to be detected is low in linoleic acid content.
Further, the method comprises the following steps: and (3) taking genomic DNA of the sample to be detected as a template, carrying out PCR amplification by adopting the primer group and/or the kit, and judging the linoleic acid content of the sample to be detected according to a reaction result.
In some embodiments of the invention, SNP typing detectors (LGC; fluostar Omega; SNP LINE LITE Omega F) are used for data reading, and if the corresponding fluorescence signal is low, the clusters are scattered and can be recycled. The procedure set up was as follows: denaturation at 94℃for 20s and annealing at 57℃for 60s,6 cycles.
Compared with the prior art, the invention has the beneficial effects that:
The invention designs and develops a three-primer molecular marker with short amplified fragment and strong specificity based on the mutation that the 1731808 th base C of the LAQ3 locus on the chromosome 6 of rice is replaced by G. By using the marker, PCR amplification is carried out by using detection primers with different fluorescence, the detection primers corresponding to specific sequences are exponentially increased along with PCR reaction, after signals are generated, the corresponding signals are detected, the genotypes of homozygous G, homozygous C and heterozygous GC can be clearly distinguished, and the genotyping of LAQ3 locus for regulating and controlling the linoleic acid content in rice can be completed, and whether the linoleic acid content in the rice is improved is predicted. 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, shorten the breeding period and meet the requirement of large-scale molecular marker auxiliary selection.
Drawings
FIG. 1 shows that in-Mummy valley sequencing results are genotype CC, with LAQ3 sites shaded;
FIG. 2 shows Yuyannuo sequencing results genotype GG, shaded LAQ3 sites;
FIG. 3 shows a bin pattern of linoleic acid content in polished rice of homozygous C (CC) and homozygous G (GG) genotype varieties; wherein 5 horizontal lines from top to bottom of each box type respectively represent a maximum value, a 75% value, a median value, a 25% value and a minimum value; points represent outliers; "×" represents the average value; "x" means very significant; blue box is homozygous C genotype, red orange box is homozygous G genotype; the distribution and average content of linoleic acid in polished rice of homozygous G group are obviously higher than those of homozygous C group;
FIG. 4 shows the fluorescent signal reading results of the detection of genotypes of different rice varieties using the primer combinations Q3-F1, Q3-F2 and Q3-R; wherein red, blue, green and black dots represent homozygous G, homozygous C, heterozygous GC genotypes, and ddH 2 O, respectively;
FIG. 5 shows the fluorescent signal readings of the detection of genotypes of different rice varieties using the primer combinations Q3-F1, Q3-F2 and Q3-Ra; genotypes of different samples are shown as "x", but cannot be automatically distinguished by the detection instrument into red, blue, green, representing homozygous G, homozygous C, heterozygous GC genotypes, respectively. Black dots represent ddH 2 O, respectively;
FIG. 6 shows the fluorescent signal reading results of the detection of genotypes of different rice varieties using the primer combinations Q3-F1, Q3-F2 and Q3-Rb; genotypes of different samples are shown as "x", but cannot be automatically distinguished by the detection instrument into red, blue, green, representing homozygous G, homozygous C, heterozygous GC genotypes, respectively. Black dots represent ddH 2 O, respectively;
FIGS. 7, 8 and 9 show the use of primer combinations Q3-F1, Q3-F2 and Q3-R to identify genotypes at LAQ3 locus in different rice germplasm resources; wherein red, blue, green and black dots represent homozygous G, homozygous C, heterozygous GC genotypes, and ddH 2 O, respectively;
FIG. 10 shows the fluorescent signal scan results of detection of 150 BC 2F2 hybrid samples, homozygous G control Yuyannuo, homozygous C control in the Moamn valleys, mixed DNA in the heterozygous control Yuyannuo and Moamn valleys, and negative control ddH 2 O with primer combinations Q3-F1, Q3-F2 and Q3-R; wherein, 70 hybrid samples show green fluorescence, which is heterozygous genotype; 22 hybrid samples showed red fluorescence, which is homozygous genotype C; 58 hybrid samples show blue fluorescence, which is homozygous G genotype; in the Moscht valley, yuyannuo, heterozygous controls showed blue, red and green fluorescence, respectively; ddH 2 O does not show fluorescence, is a black dot;
FIG. 11 is a box plot showing the linoleic acid content distribution of LAQ 3-labeled brown rice of different genotypes; wherein 5 horizontal lines from top to bottom of each box type respectively represent a maximum value, a 75% value, a median value, a 25% value and a minimum value; "×" represents the average value; "x" means very significant; blue boxes are homozygous G (GG) genotypes, orange boxes are homozygous C (CC) genotypes, and ash boxes are heterozygous GC genotypes; the distribution and average content of linoleic acid in the population with the LAQ3 marker genotype homozygous C is significantly lower than that in the population with the linoleic acid marker genotype homozygous G or heterozygous GC.
FIG. 12 shows a bin pattern of linoleic acid content profiles of LAQ3 labeled polished rice of different genotypes; wherein 5 horizontal lines from top to bottom of each box type respectively represent a maximum value, a 75% value, a median value, a 25% value and a minimum value; "×" represents the average value; "x" means very significant; blue boxes are homozygous G (GG) genotypes, orange boxes are homozygous C (CC) genotypes, and ash boxes are heterozygous GC genotypes; the distribution and average content of linoleic acid in the population with the LAQ3 marker genotype homozygous C is significantly lower than that in the population with the linoleic acid marker genotype homozygous G or heterozygous GC.
Detailed Description
In order to better understand the technical content of the present invention, the following provides specific examples to further illustrate the present invention.
The experimental methods used in the embodiment of the invention are conventional methods unless otherwise specified.
Materials, reagents, and the like used in the examples of the present invention are commercially available unless otherwise specified.
The invention provides a molecular marker LAQ3 related to rice linoleic acid content and application thereof, belonging to the technical field of plant molecular biology. SNP locus LAQ3 which is located by a laboratory (university of Hainan Metabolic biology laboratory) through mGWAS (metabolome-based genome-wide association studies) and used for regulating and controlling the linoleic acid content of rice. On the basis, molecular markers are developed for LAQ3 loci, LAQ3 locus high-value parent PXB and low-value parent AKITAKOMACHI of linoleic acid are screened, then backcross transformation is carried out on AKITAKOMACHI by taking PXB as a donor through molecular marker selection, and finally offspring plants with homozygous LAQ3 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 brown rice and polished rice LAQ3 genotype identification and application thereof, which can ensure that the genetic background is stable, the content of linoleic acid in rice with good comprehensive properties is improved, in order to improve the nutrition quality of rice with good comprehensive properties and stable backgrounds, the content of the linoleic acid is improved, KASP (Kompetitive Allele-SPECIFIC PCR) molecular markers are developed, specific primers are designed for allele SNP loci, and under the technical support of LGC (Laboratory of the Government Chemist) company, the fact that the Q3 locus genes for regulating and controlling the linoleic acid content in rice polished rice can be detected and controlled more efficiently and accurately by using the KASP markers for genotyping detection is verified, 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 the 1731808 th base of the chromosome 6 of the rice. The technique is based on specific matching of primer terminal bases to genotype SNPs. 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.
The invention aims to overcome the defects and shortcomings in the prior art and provides a molecular marker LAQ3 related to the content of rice brown rice and polished rice linoleic acid, and a detection method and application thereof.
According to the invention, a novel SNP molecular marker LAQ3 for regulating and controlling the linoleic acid content of rice is positioned by a whole genome association analysis method. The LAQ3 site is located at base 1731808 of chromosome 6 of rice (genomic version Os-Nipponbare-Reference-IRGSP-1.0). When the LAQ3 locus genotype is G, the brown rice and polished rice of the rice have higher linoleic acid content. When the genotype of LAQ3 locus is C, the brown rice and polished rice of the rice have lower linoleic acid content.
The second object of the invention is to provide a method for identifying the LAQ3 genotype of the site for regulating and controlling the linoleic acid content of rice and polished rice by utilizing the molecular marker and application thereof.
The third object of the present invention is to provide a method for identifying linoleic acid content in brown rice and polished rice of rice using the above molecular markers, or simultaneously identifying the gene of the LAQ3 locus and linoleic acid content in brown rice and polished rice, and applications thereof.
The invention provides a molecular marker for regulating and controlling LAQ3 locus gene of rice linoleic acid content, which 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 LAQ3 locus genotype for regulating and controlling linoleic acid content in rice polished rice.
The invention provides application of the molecular marker in rice breeding.
The invention provides a specific primer combination for detecting LAQ3 locus genotype for regulating linoleic acid content in rice polished rice, which contains primers with nucleotide sequences shown in SEQ ID No. 1-3.
The primer combination is obtained by designing and screening the LAQ3 locus. The 3' -terminal bases of Q3-F1 and Q3-F2 are G and C, respectively, and SNP is typed by specific matching of primer terminal bases. In addition, the 5' end of Q3-F1 is augmented with FAM linker sequence A1 (SEQ ID No. 4): GAAGGTGACCAAGTTCATGCT, Q3 5' to F2 was added HEX linker sequence A2 (SEQ ID No. 5): GAAGGTCGGAGTCAACGGATT, through multiple rounds of PCR amplification, FAM or HEX labeled oligonucleotides bind to the new complementary tail sequence, releasing the fluorescent material from the quenching group, generating a fluorescent signal. Wherein the FAM linker sequence releases blue fluorescence and the HEX linker sequence releases red fluorescence. Q3-R is a common reverse primer, and can be respectively paired with Q3-F1 and Q3-F2 to amplify a PCR product of 109 bp.
The invention provides application of the specific primer combination in identifying genotypes of LAQ3 loci for regulating and controlling linoleic acid content in rice polished rice.
The invention provides a method for identifying whether genotype variation of LAQ3 locus for regulating linoleic acid 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.
Furthermore, the invention provides a method for detecting the genotype of the Q3 locus for regulating and controlling the linoleic acid content in the brown rice and polished rice and the phenotype change caused by the locus gene mutation, firstly extracting genome DNA from a sample to be detected, then carrying out PCR (polymerase chain reaction) by using forward primers shown by Q3-F1 and Q3-F2 and reverse primers shown by Q3-R, and analyzing data corresponding to fluorescent signals released in the PCR amplification process.
If the fluorescent signal released by the amplified product is blue, the gene of the sample to be detected is homozygously G, and the sample to be detected has higher linoleic acid content; if the released fluorescent signal is red, the genotype of the sample to be detected is homozygote C, and the sample to be detected has lower linoleic acid content; if the green fluorescent signal is released, the sample to be detected is heterozygous genotype GC, and the linoleic acid content is medium.
After the completion of the PCR reaction, data were read using a SNP typing detector (LGC; fluostar Omega; SNP LINE LITE Omega F). If the fluorescence signal corresponding to the data is low, the clusters are scattered, and the data can be recycled. The procedure set up was as follows: denaturation at 94℃for 20s and annealing at 57℃for 60s,6 cycles. The fluorescent signal clusters read after 6 cycles of this site increase are all more concentrated and the fluorescent signal is stronger.
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 primer shown in Q3-R in breeding of a linoleic acid content Q3 locus in regulating and controlling rice polished rice.
The invention provides application of the kit containing the primer shown in Q3-R in rice germplasm resource improvement.
Example 1-localization of LAQ3 sites associated with linoleic acid content Using Whole genome correlation analysis
533 Parts of rice germplasm resources collected from all parts of the world are taken as research objects, the re-sequencing technology is used for determining the genome sequence of each variety of cell nuclei, and the high performance liquid chromatography-mass spectrometry is used for determining the relative content of the polished rice linoleic acid by combining with the broad-range targeted metabolite determination technology.
A metabolite-based whole genome association analysis was performed based on the above genome and linoleic acid relative content data, targeting one SNP locus LAQ3 (p=0.151×10 -14) that was significantly correlated with rice linoleic acid relative content. The LAQ3 site is located at base 1731808 of chromosome 6 of rice (genomic version Os-Nipponbare-Reference-IRGSP-1.0). Resequencing data indicated that, of the 533 cultivars, both genotypes were detected at the LAQ3 locus. Wherein 146 parts of homozygous G genotype varieties and 380 parts of homozygous C genotype varieties are adopted.
Example 2-LAQ3 site genotype Sanger sequencing validation
From the 533 parts of varieties, a variety Yuyannuo and a Mosha valley having homozygous G and homozygous C genotypes, respectively, were selected.
The 1KB sequence upstream and downstream of the linoleic acid Q3 SNP site is as follows (underlined and bolded is the SNP site):
GCTGCTAAAGATTTGCTGAACTTGCAACCCGAAGGAGTACATTGTCTGAAGAAACATTGCTGATCAGAGCAATCACCAAAGAAGATCAAACAGAATTTGGCCAGAGGTGACATTGAAAATTTGAAATACACTCCAGGTGCAGTATCATGGTCACTTCATGAAACTATTGTGCAGTAAATTTTGTGCAAAAATGCTACTGAGGTGCAGTAAAATGACCAGTGCATAGAACATCTGTTGTCTAAAAGTGAAACATAAGAAAAACAAATACTCCTCTAGTTAAGAGTGTTGTGCCTGTAGTGCAAACTGTGCAACAATTGAACCTGCTTAAAAAAAAACTTTTTGCATCTACACTCTAAAGTATGAATCCTGATTAATGTTGTATCCCAAATTCCCAGTGTTACAAACACAAGGAGTTCGTTTAGGCATTACAAGAACTCTGAATACTCTGCCGTGTGCACCCACAGAAGCCAAAAAACCTGGTTTTACTCATGCATCAACAACTGGGAAGTATCCCAATCTACCGGAAAAACCTGTTGTTTCCTTCTCTCTAAAGGCATCAAAGCACCAAAATGACCATGGTATGCATCATTCTCTTCGCCTCCTCAAGCTTTGGTTTCTTCTTGCTTCAGTTCACTAATCCTACACAGGTCTGAAAAAAAGCTGTCACATCCAGGGGATCTTGTTAAACTGTTCATAACTGTTAAGCATGCTGAATCCTAACCTGAATCCAACGTGCATTTGCCTATCGAATCTGAATCCTGATCGGTACTGTACTCCAAATCCCCAATGGGTTCAAGCCTGAACTGTACTAAGGCTGAGTTTGAGAGAGGGAGATAGAGAAGATTGGGGAGATACGCAAAATGAGGTAAGCTATTAGCGCATGATTAATTGAGTATTAACTATTTTAAATTTTAAAAATGAACTAATATGATTTTTTAAAGCAACTTTTCTATAGAAATTTTTTGCAAAAAACGCACCGTTTAGTAGTTTGGGAAGCGTGGACGTGGAAAACGAGTCACAATCTCCCCAATCTCCTTTGAAACAAAGCAGCCAGAACTCTCAAATTCTCGATGCGATTTACCAATACAATCTGGAGGAATCCTATGATGCATTTTGAAATATGAATTCAAATTAGCGCTGTGATCTACGCTTCGCTTCTACTGGGGGTGGTGAGTGATGGTGCTTGTCGACGAGCTGCTTCACCCGGAGCTCCAGCTCCCGGATCCTCGTCGCCCACTCGCGCCTTATCTCGATCGGCCTCGCCGCCCCCGCCGCCGCCAGCTCGCCGCCCCGCCGCTCGCCGGCGTCCTGCCGCCGCGCCGCTGGCCGCGCCCCCGCTCTCTTCCTCCTCTTGGCCTCCTTCGCCGCCGCCGCTCTCTCCAGCGCCCGCAGCACGTCCGACCCCCGCAGCTGCCCCGCGGGCTCCTGCTCCGGCGCCGGCGAGGAGGGCCGCCGCCCCGAGGCAGCCGCATCCTCCGGCTCCGGGTCTGGCTCGTCCCGCGCGTCACCGAAGAAGTTGGCGTCGATGCGGTCGTCGGCGGCGCCGTCCTCGTCGTCGCCGCCGCCGTCTCCCCACGAGCGGTTGCGGCGGGAGCCGCCGCGGCGTGGCCTCGCCGAGACGGCGACCAGGTATGGCTTGCGGCCGCCGGAGATCGTGGCGTCCGGCCGGGGCCGGACGGGGAGGAAGAAACGTGCGGTGGAGAACATCAGCGATATCTCTATCTCGTGCTGGTAATTTGGAAATGGGAATGAATGAATATCTCGGGGTTAAGGCCACAGGAGACTATAAGCATGTGTTGGATGGGATAAAACCGTTCCTGATTTTAGAGATAGAGTGATCCCACTTATTTGTTTAGTTTATAGGATAGAATGATCCTAGTTTTTTGTTTGGTAGAAGGAATAGGGGTAAGAATGAGATGAACGACGTTTGGAGACGTTAGCGCACCACATCCACAGATCCACTCCTAGCTTGGAGGAGCGGCGAACGGCCGGCGGT GAAAG(SEQ ID No.8)
primers were designed based on the 1kb sequence upstream and downstream of the LAQ3 site:
LAQ3CXF(SEQ ID No.6)：TTAGCGCATGATTAATTGAGTATTAAC
LAQ3CXR(SEQ ID No.7)：TAGGATTCCTCCAGATTGTATTGGTA
PCR amplification was performed using genomic DNA from the variety Murray Valley and Yuyannuo as templates, respectively, using primer pairs LAQ3CXF and LAQ3CXR, and Sanger sequencing was performed on the amplified products. Sequencing results show that the genotype of the LAQ3 locus in the Mummy valley of the variety is homozygote C (see figure 1), and the genotype of the LAQ3 locus of the variety Yuyannuo is homozygote G (see figure 2).
Example 3 Effect of LAQ3 site on linoleic acid content
The 533 cultivars were divided into two groups, homozygous G genotype and homozygous C genotype, according to the genotype of LAQ3 locus. The minimum value of linoleic acid relative content in polished rice of homozygous G genotype variety is 1356036, the maximum value is 2655059, and the average value is 2084524. The polished rice of homozygous genotype C variety had a minimum of 1329203 linoleic acid relative content, a maximum of 2502838, and an average of 1907869 (see FIG. 3). t-test showed that the average relative content of linoleic acid in homozygous C and G genotype varieties was very different (p=0.151×10 -14). The results show that the LAQ3 locus is obviously related to the content of the rice linoleic acid in the rice. Wherein, the LAQ3 genotype is a group of homozygous C, the average content of linoleic acid in the polished rice is lower, and the LAQ3 locus genotype is a group of homozygous G, the average content of linoleic acid in the polished rice is higher.
EXAMPLE 4 LAQ3 site KASP marker development
1. Primer design
Three sets of three primer combinations, i.e., primer combinations Q3-F1, Q3-F2 and Q3-R, Q3-F1, Q3-F2 and Q3-Ra, and Q3-F1, Q3-F2 and Q3-Rb were designed based on the instructions of LGC (Laboratory of the Government Chemist) company KASP kit (KASP-TF V4.0X Master Mix) and the sequence of 1kb genomic DNA upstream and downstream of the LAQ3 site. The primer sequences were as follows:
Q3-F1(SEQ ID No.1)：
GAAGGTGACCAAGTTCATGCTGTTTAGTAGTTTGGGAAGCGTGG
Q3-F2(SEQ ID No.2)：
GAAGGTCGGAGTCAACGGATTGTTTAGTAGTTTGGGAAGCGTGC
Q3-R(SEQ ID No.3)：
GTATTGGTAAATCGCATCGAGAATT
the 5' end of Q3-F1 is added with FAM linker sequence A1 (SEQ ID No. 4):
GAAGGTGACCAAGTTCATGCT
addition of HEX linker sequence A2 (SEQ ID No. 5) to the 5' end of Q3-F2:
GAAGGTCGGAGTCAACGGATT
Q3-Ra(SEQ ID No.9)：
ATTTCAAAATGCATCATAGGATTCC
Q3-Rb(SEQ ID No.10)：
TAGATCACAGCGCTAATTTGAATTC
2. Expected amplification results for different primer combinations
The three primer combinations amplify rice variety genomic DNA according to the primer sequences, and the amplified products are detected by a SNP genotyping detector (LGC; fluostar Omega; SNP LINE LITE Omega F). If the fluorescent signal released by the amplified product is red, the sample to be detected has homozygous G genotype, namely GG; if the blue fluorescent signal is released, the sample to be detected is indicated to be homozygous C genotype, namely CC; if the green fluorescent signal is released, the sample to be detected is indicated to be heterozygous genotype GC.
3. Actual amplification Effect of different primer combinations
FIG. 4 is the result of detection of KASP fluorescence signals of genomic DNA of 13 rice samples using Q3-F1, Q3-F2 and Q3-R. Wherein 1 sample is in the Mozuku valley, and KASP fluorescence is blue, which indicates that the genotype is homozygote C.1 sample was Yuyannuo, its KASP fluorescence was red, indicating its genotype was homozygous G. A sample is prepared by mixing the interior of the Murray valley and the genome DNA of Yuyannuo in equal concentration and equal volume, and KASP fluorescence is green, which shows that the genotype is GC. The black dot is the KASP result for negative control ddH 2 O.
FIGS. 5 and 6 are fluorescent signal readings from 10 rice varieties (excluding control varieties) detected with primer sets Q3-F1, Q3-F2 and Q3-Ra, and primer sets Q3-F1, Q3-F2 and Q3-Rb. Also included in FIGS. 5 and 6 are fluorescent signal reads of homozygous G control variety Yuyannuo, homozygous C control variety in and mixed DNA with Yuyannuo in the heterozygous control in the Mozuku valley, and two ddH 2 O negative controls. As shown in fig. 5 and 6, the fluorescence signals of the two ddhs 2 O are black, indicating that no fluorescence is generated. However, the data points of the Moscht valley, yuyannuo and the heterozygous control, as well as the detected sample, are mixed together to generate a fluorescence signal disorder, and the genotype of the LAQ3 locus cannot be accurately distinguished.
In summary, only the Q3-F1, Q3-F2 and Q3-R primer sets are effective in discriminating the genotype of LAQ3 locus.
Example 5-optimization of PCR reaction procedure for KASP marker and genotyping of LAQ3 locus in different rice varieties
1. Experimental materials
Wild fragrant and excellent sea-silk, chang Liangyou and excellent abdomen-silk, yue Liangyou 2024, jin Longyou and soft silk, mei you 5431, fu you 36, tai Feng you 1002, mei Liangyou and autumn-silk, long-looking Liangyou 889, jin Longyou and 1826, wild fragrant and excellent 9, jiu Xin Xiang, shou Xiang 1, han Liangyou 219, wild fragrant and excellent 2998, mei Xiang 2, qing Xiang you and soft silk, xin Liangyou B, R and 1761, fu Gui Jia Xiang Mi, mo Ling Gu and Yuyannuo.
2. Extraction of genomic DNA from rice
The method for extracting rice genome DNA by adopting CTAB method comprises the following specific steps: 3cm long rice leaves were taken, ground in 800. Mu.L of extraction buffer [1.5% (w/V) CTAB,1.05mol/L NaCl,75mmol/L Tris-HCl (pH 8.0), 15mmol/L EDTA (pH 8.0) ] and 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/isoamyl alcohol (volume ratio 24:1) was added and mixed by inversion for 15min. Centrifuge at 12000r/min for 10min at room temperature. 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. After 75% ethanol was poured off, 100. Mu.L of sterilized ddH 2 O was added to dissolve DNA after drying.
3. PCR amplification and detection
The variety Yuyannuo in example 2 was used as a homozygous G genotype control, the variety in the amnesia valley was used as a homozygous C genotype control, and Yuyannuo and genomic DNA in the amnesia valley were mixed in equal amounts to be used as a heterozygous genotype control. The DNA of the 20 rice varieties/lines described in this example was PCR amplified using the specific primer combinations (Q3-F1, Q3-F2, Q3-R) obtained by the screening in example 4. Amplification was performed using the KASP-TF V4.0X Master Mix kit from LGC Genomics, 10. Mu.L of a PCR reaction system comprising 2X KASP MASTER Mix 4.86. Mu.L of a 36. Mu.M Q3-F1, Q3-F2 primer, 90. Mu.M of a Q3-R primer, and mixing the three primers at a volume ratio of 1:1:1 to obtain KASP ASSSY Mix of 0.14. Mu.L KASP ASSSY Mix and 10 ng/. Mu.L of template DNA of 5. Mu.L.
The PCR reaction procedure was: pre-denaturation at 94℃for 15min; denaturation at 94℃for 20s, annealing at 61℃for 60s,10 cycles; denaturation at 94℃for 20s and annealing at 55℃for 60s,26 cycles.
If the fluorescence signal corresponding to the data is low, the clusters are scattered, and the data can be recycled. The procedure set up was as follows: denaturation at 94℃for 20s and annealing at 57℃for 60s,3-12 cycles.
After the completion of the PCR reaction, the fluorescent signal was read on a SNP typing detector (LGC; fluostar Omega; SNP LINE LITE Omega F).
4. Results and analysis
Fig. 7, 8 and 9 show the results of adding 6 and 9 cycles to the non-cycle. As shown in fig. 7 and 8, the data points representing 20 materials were distributed randomly without cycling, and genotyping could not be clearly performed. After more than 6 cycles, the data points aggregate rapidly and fluorescence representing different genotypes is generated (fig. 9).
As shown in FIG. 9 (see Table 2 for specific results), PCR products of 17 varieties such as wild fragrant excellent sea silk and the like emit blue fluorescent signals, and signal points thereof are gathered together with Yuyannuo varieties which emit blue fluorescent signals in a coordinate system, which shows that the genotype of the LAQ3 locus is GG. The PCR products of 1 variety in Moshaggy and the like emit red fluorescent signals, which indicates that the genotype of the LAQ3 locus is CC. The heterozygote control emits green fluorescent signals, and the signal points of 5 varieties of Chang Liuyou Fuxiang and the like are gathered together with the heterozygote control, so that the genotype of the LAQ3 locus is GC.
Table 2: identification of the genotype of LAQ3 locus in different rice varieties Using the KASP marker
Example 6 identification of the genotype of LAQ3 site in BC 2F2 isolate with KASP markers
1. Experimental materials
Crossing an acceptor parent, such as AKITAKOMACHI, with a donor parent PXB to obtain F1, backcrossing the F1 to obtain BC 1F1,BC1F1, backcrossing to obtain BC 2F1,BC2F1, and selfing to obtain BC 2F2.
2. Extraction of genomic DNA from rice
The extraction method of rice genomic DNA was the same as in example 5.
3. PCR amplification and detection
The parent variety PXB is used as a homozygous G genotype control, the parent variety AKITAKOMACHI is used as a homozygous C genotype control, and the genomic DNA of the PXB and AKITAKOMACHI are mixed in equal amounts to form a heterozygous genotype control. The individual strains of the BC 2F2 population described in this example were individually extracted with the KASP primer combination (Q3-F1, Q3-F2, Q3-R) obtained by screening in example 4 and subjected to PCR amplification. Amplification was performed using the KASP-TF V4.0X Master Mix kit from LGC Genomics. The PCR reaction system was 10. Mu.L, including 2X KASP MASTER mix 4.86. Mu.L, 36. Mu.M of Q3-F1, Q3-F2 primers, 90. Mu.M of Q3-R primer, the three primers were mixed at a volume ratio of 1:1:1 to KASP ASSSY mix, 0.14. Mu.L of KASP ASSSY mix,10 ng/. Mu.L of template DNA was 5. Mu.L.
The PCR reaction procedure and product detection were as in example 5.
4. Results and analysis
The genotype test results are shown in FIG. 10 (see Table 3 for specific results). The PCR products of 58 individual plants emit blue fluorescent signals, and the signal points thereof are gathered together with the parent strain PXB which emits blue fluorescent signals in a coordinate system, which shows that the genotype of the LAQ3 locus is GG. The PCR products of 22 individual plants emit red fluorescent signals, and the signal points thereof are gathered together with the parent variety AKITAKOMACHI which also emits red fluorescent signals in a coordinate system, which shows that the genotype of the LAQ3 locus is CC. The PCR products of 70 individual plants emit green fluorescent signals, and the signal points are gathered together with the heterozygous control which emits green fluorescent signals in a coordinate system, so that the genotype of the LAQ3 locus is GC.
Table 3: genotype of LAQ3 locus in BC 2F2 isolate
Example 7-Effect of LAQ3 site on linoleic acid content in Brown rice of BC 2F2 plants
1. Experimental materials
The BC 2F2 population described in example 6.
2. Detection of relative content of linoleic acid
Each BC 2F2 individual plant was harvested from the inbred, 10g of rice was dehulled and ground to a powder. 0.1g of the powder was weighed, 1mL of an extract (70% methanol) was added to extract metabolites, and linoleic acid relative content 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-6500QTRAP,AB SCIEX).
3. Results and analysis
The 77 BC 2F2 individuals described in this example were divided into GG, CC and GC genotypes according to the genotype of LAQ3 locus in example 6. FIG. 11 shows the effect of LAQ3 genotype on linoleic acid content in brown rice (see Table 4 for specific data), with the exception of outliers, the relative linoleic acid content in brown rice of GG genotype plants was at a minimum of 22635009, at a maximum of 28119416, and at an average of 26143290. In brown rice of CC genotype plants, the minimum value of linoleic acid relative content is 18118516, the maximum value is 22728126, and the average value is 20871699 except for abnormal value. In brown rice of GC genotype plants, the minimum value of linoleic acid relative content was 19216750, the maximum value was 25025171, and the average value was 22674225, except for abnormal value. t tests show that the average value of the linoleic acid relative content in brown rice of GG genotype plants is extremely higher than that of CC genotype plants (P=1.48×10 -15), and the average value of the linoleic acid relative content in brown rice of GC genotype plants is extremely higher than that of CC genotype plants (P=5.85×10 -5). The result shows that the LAQ3 locus has the function of regulating and controlling the linoleic acid content of the rice brown rice. Wherein, the average content of linoleic acid in brown rice is lower when the genotype of the LAQ3 locus is CC, and the average content of linoleic acid in brown rice is higher when the genotype of the LAQ3 locus is GG.
Table 4: relative content of linoleic acid in brown rice with different LAQ3 loci
Example 8-influence of LAQ3 locus on linoleic acid content in polished rice of BC 2F2 plants
1. Experimental materials
The BC 2F2 population described in example 6.
2. Detection of relative content of linoleic acid
Each BC 2F2 individual plant was harvested from the inbred, 10g of rice was dehulled and ground to a powder. 0.1g of the powder was weighed, 1mL of an extract (70% methanol) was added to extract metabolites, and linoleic acid relative content 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-6500QTRAP,AB SCIEX).
3. Results and analysis
The 77 BC 2F2 individuals described in this example were divided into GG, CC and GC genotypes according to the genotype of LAQ3 locus in example 6. FIG. 12 shows the effect of LAQ3 genotype on linoleic acid content in polished rice (specific data are shown in Table 5), with the exception of outliers, the relative linoleic acid content in polished rice of GG genotype plants was found to be at a minimum of 24243999, a maximum of 29981951, and an average of 28378556. In polished rice of CC genotype plants, the minimum value of linoleic acid relative content is 22877469, the maximum value is 26634267, and the average value is 24118861 except for abnormal value. The polished rice of GC genotype plants, except for abnormal values, had a linoleic acid relative content of 21798441, a highest value of 29040804 and an average value of 25153614.t tests show that the average value of the linoleic acid relative content in polished rice of GG genotype plants is extremely higher than that of CC genotype plants (P=2.4X10 -15), and the average value of the linoleic acid relative content in polished rice of GC genotype plants is extremely higher than that of CC genotype plants (P=3.44X10 -13). The results show that the LAQ3 locus has the function of regulating and controlling the linoleic acid content of the polished rice. Wherein, when the genotype of the LAQ3 locus is CC, the average content of linoleic acid in polished rice is lower, and when the genotypes of the LAQ3 locus are GG and GC, the average content of linoleic acid in polished rice is higher.
Table 5: relative content of linoleic acid in polished rice with different LAQ3 loci
Example 9-increasing linoleic acid content in Rice Using LAQ3 marker
Hybridization, backcrossing and selfing are carried out by using a donor parent PXB and a normal fertility receptor, such as AKITAKOMACHI, and in the process, a LAQ3 locus and a genetic background are selected by using a molecular marker, so that a strain with GG genotype at the LAQ3 locus and increased linoleic acid content in polished rice under the AKITAKOMACHI background is finally obtained. The specific implementation steps are as follows:
1. F 1 is obtained by crossing a parent of a receptor, such as AKITAKOMACHI, with PXB.
2. Detecting the genotype of the F 1 generation hybrid by using a primer with the sequence shown as SEQ ID No. 1-3, selecting a true hybrid, namely, a plant with a PCR product generating a green fluorescent signal as a female parent and a receptor parent, such as AKITAKOMACHI, and backcrossing to obtain BC 1F1.
3. Planting BC 1F1, and detecting the genotype of the BC 1F1 plant by using a primer with the sequence shown as SEQ ID No. 1-3. LAQ3 locus heterozygous genotype plants were selected.
4. And (3) carrying out genetic background identification on the single plant selected in the step (3) by using a group of genotypes (for example, 100 or 200, etc.) with polymorphism between PXB and a receptor parent, such as AKITAKOMACHI, genome and uniformly distributed molecular markers (which can be types of markers such as SSR, SNP, INDEL, EST, RFLP, AFLP, RAPD, SCAR, etc.), and selecting plants with high similarity (such as similarity of more than 88 percent or medium selection rate of 2 percent, etc.) with the recurrent parent genotypes.
5. The BC 2F1 is obtained by backcrossing the plant selected in step 4 with a recipient parent, such as AKITAKOMACHI.
6. Planting BC 2F1, repeating the step 3 and the step 4, selecting out plants with heterozygous LAQ3 genotype and high genetic background recovery rate (such as more than 98 percent or 2 percent of selection rate and the like), and collecting the selfing seed BC 2F2.
7. And (3) planting BC 2F2, repeating the step (3) and the step (4), selecting a plant with the LAQ3 genotype of GG and highest homozygous rate of genetic background, and collecting the selfing seed BC 2F3.
8. And (3) determining the linoleic acid content of the brown rice and the polished rice in the BC 2F2 inbred seeds and the AKITAKOMACHI by using a wide targeting method, and selecting a single plant with obviously improved linoleic acid content of the brown rice and the polished rice in the BC 2F2 inbred seeds to reproduce into a rice line with high linoleic acid content.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
Claims (8)
1. The application of detecting SNP molecular markers related to rice linoleic acid content in rice breeding is characterized by comprising the steps of predicting, identifying or assisting in identifying the rice linoleic acid content, wherein the SNP molecular markers are positioned at 1731808 th base of a rice chromosome 6, and when the genotype of the SNP molecular markers of a sample to be detected is G, the probability of the sample to be detected for high linoleic acid content is obviously higher than that when the genotype is C;
The genome version number of the rice is Os-Nipponbare-Reference-IRGSP-1.0.
2. The use of the SNP molecular marker related to rice linoleic acid content for rice breeding according to claim 1, wherein the sample to be tested is high in linoleic acid content when the genotype of the SNP molecular marker in the sample to be tested is homozygous G, the sample to be tested is medium in linoleic acid content when the genotype of the SNP molecular marker in the sample to be tested is heterozygous GC, and the sample to be tested is low in linoleic acid content when the genotype of the SNP molecular marker in the sample to be tested is homozygous C.
3. The use of the SNP molecular marker for detecting the linoleic acid content of rice according to claim 1, wherein the SNP molecular marker comprises the use in rice variety breeding, comprising increasing the linoleic acid content in progeny rice in the variety breeding by molecular marker-assisted selection.
4. The use of the SNP molecular marker for detecting the content of linoleic acid in rice according to any one of claims 1 to 3, wherein the rice comprises polished rice or brown rice.
5. The application of detecting SNP molecular markers related to the linoleic acid content of rice in rice breeding according to any one of claims 1 to 3, wherein the primer set for detecting the SNP molecular markers related to the linoleic acid content of rice comprises a primer 1, a primer 2 and a primer 3; the primer 1 has a nucleotide sequence shown as SEQ ID No. 1;
the nucleotide sequence of the primer 2 is shown as SEQ ID No. 2;
The nucleotide sequence of the primer 3 is shown as SEQ ID No. 3.
6. The use of the SNP molecular marker for detecting the content of linoleic acid in rice according to claim 5, wherein the primer set is used in rice breeding with high linoleic acid content.
7. The use of SNP molecular markers for detecting the content of linoleic acid in rice according to claim 5, wherein the primer set is used for detecting the content of linoleic acid in rice.
8. A method for predicting, identifying or assisting in identifying the linoleic acid content of rice, comprising the steps of: the genome DNA of a sample to be detected is used as a template, the primer set as claimed in claim 5 is used for PCR amplification, the linoleic acid content of the sample to be detected is judged according to the reaction result, the 1731808 th base of a chromosome 6 of rice is detected, when the genotype of a molecular marker of the sample to be detected is G, the probability of the high linoleic acid content of the sample to be detected is obviously higher than that of the genotype of the sample to be detected is C, and the genome version number of the rice is Os-Nipponbare-Reference-IRGSP-1.0.
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