CN111206113A - InDel molecular marker for assisting selection of early heading genes of rice and application of InDel molecular marker - Google Patents
InDel molecular marker for assisting selection of early heading genes of rice and application of InDel molecular marker Download PDFInfo
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Abstract
The invention discloses an InDel molecular marker for assisting in selecting early heading genes of rice and application thereof, belonging to the technical field of molecular markers. The invention preliminarily identifies the candidate region related to the heading stage of rice by using the whole genome re-sequencing BSA technology, and then utilizes the InDel marker developed by parent whole genome re-sequencing to hybridize and construct F of 9311/CL33 in the candidate region2500 early heading individuals in the segregating population are subjected to marker analysis, a PSM8-8 marker and a gene are co-segregated, and finally, the early heading stage gene qEH8-1 is finely positioned in a range of about 270kb between an InDel marker PSM8-6 and a PSM 8-9. The InDel molecular marker PSM8-8 designed according to the invention is used for amplifying the rice plant genome, and whether the detected plant has the early heading character or not is judged through a denatured polyacrylamide gel electrophoresis picture.
Description
Technical Field
The invention belongs to the technical field of molecular markers, and particularly relates to an InDel molecular marker for assisting in selection of rice early heading genes and application thereof.
Background
The heading period (growth period) of rice is one of important agronomic traits of rice, the heading period directly determines the regional adaptability and seasonal adaptability of rice varieties, plays an important role in high yield and stable yield of the rice varieties, and is an important target of rice breeding. The breeding of early-maturing and high-yield rice varieties has always been regarded by rice breeding workers. The heading period of the rice variety is mainly determined by the photosensitivity, the temperature sensitivity and the basic nutrition and health maintenance property of the rice variety; photosensitivity has a determining role in governing the maturity stage for strongly sensitive species. For varieties with the same light-temperature reaction in the same region, the heading period is mainly determined by the length of the basic vegetative growth period. The diversity and the strength of the combination of photosensitivity and basic vegetative growth are different, so that the heading stage shows various changes; on one hand, the method provides abundant resources for breeding of different ecotypes, and on the other hand, the genetic expression in the heading stage is abnormally complicated. Therefore, the genetic rule of the rice heading stage gene/QTL is deeply known, and the related heading stage gene/QTL is finely positioned and cloned, so that the method has important guiding and practical significance for breeding in the rice growth stage.
Heading stage is a complex genetic trait whose detection of QTL depends on the genetic structure of the segregating population used. The commonly used population is mainly F2/F3BC1, DH and RIL, the genetic background of the groups is complex, and the QTL positioning is influenced to a certain extent. The QTL of the heading stage detected by different parent combinations is different, and QTL loci influencing the heading stage detected by segregation populations of the same combination in different environments are not completely the same, so that the influence of different chromosome segments on the heading stage is difficult to accurately estimate. To make moreIn recent years, secondary mapping populations such as near Isogenic Lines (IL), chromosome fragment substitution lines (CSSLs) and single fragment substitution lines (SSSLs) for reducing genetic background interference have been established one after another and are widely applied to fine positioning of the rice heading QTL. Studies have shown that because there is only one homozygous chromosomal segment from the donor parent within the genome of each single-segmentally-substituted line, while the rest of the genome is identical to the recipient parent, the differences between all single-segmentally-substituted lines and their recipient parent, and all heritable variations between single-segmentally-substituted lines, are linked only to the substituted segment.
Indica rice variety 9311 (Yang rice No. 6) is an excellent hybrid rice backbone parent, 2 super rice combinations of Eryou Baojiu and Y Eryou No.1 which are popularized in a large area are cultivated by using the indica rice variety as a male parent, but in Guangxi and the whole double cropping rice growing region of south China, the whole growing period is too long, so that the indica rice variety is not suitable for late-production and planting, the popularization and application of the indica rice variety are seriously influenced, and the yield increase of grains is restricted.
Disclosure of Invention
In order to solve the technical problems, the invention provides an InDel molecular marker for assisting in selecting rice early heading genes, a primer for amplifying the molecular marker and application of the molecular marker. The heading stage identification is carried out on the chromosome fragment substitution line library which takes Guangxi common wild rice as a donor and indica rice 9311 as a receptor, and the heading stage of one chromosome fragment substitution line is found to be about 15 days earlier than that of the receptor 9311. It is contemplated that F can be used by hybridizing the chromosome fragment replacement line with the receptor 93112The segregation population finely positions the QTL of the early-maturing heading stage within 1cM, and carries out molecular marker-assisted selection on the QTL to culture an early-maturing restorer line variety which adapts to the ecological conditions of the double-cropping rice region in south China and a hybridization combination.
In order to achieve the purpose, the invention adopts the following technical scheme:
according to one aspect of the invention, the invention provides an oligonucleotide primer pair for amplifying a rice InDel molecular marker, wherein the primer pair is a nucleotide sequence shown as SEQ ID No.1 and a downstream primer SEQ ID No. 2.
The invention also claims other oligonucleotide primer pairs for amplifying the InDel molecular marker.
According to one aspect of the invention, the invention also relates to a method for identifying the early heading genotype of a rice plant, comprising the following steps:
(1) using rice sample DNA as a template, and amplifying InDel molecular markers by using SEQ ID No.1 and SEQ ID No.2 or other primer pairs;
(2) and identifying the genotype of the rice sample at the heading stage.
Further, the step (1) of extracting and identifying the rice sample DNA comprises the following steps: (1) extracting DNA of each rice sample according to a CTAB method; (2) analyzing the purity and integrity of DNA of each sample by agarose gel electrophoresis; (3) detecting the purity of the sample DNA, requiring OD260/280The ratio is between 1.8 and 2.0; (4) detecting the DNA concentration of the sample, requiring the concentration of the sample>20ng/μL。
Further, the amplification step in the step (1) is as follows: 20 mu L of reaction system comprises 0.1-0.2 mu M of upstream primer, 0.1-0.2 mu M of downstream primer, 200 mu M dNTP, 1 XPCR reaction buffer solution, 50-100 ng of DNA template and 1UTaq enzyme; the reaction procedure used was: pre-denaturation at 94 deg.C for 5min, circulation at 94 deg.C for 1min, 55 deg.C for 1min, and 72 deg.C for 1min for 35 times, and extension at 72 deg.C for 5 min;
the 1 XPCR reaction buffer comprises 50mM KCl, 10mM Tris-HCl pH8.3, 1.5mM MgCl20.01% gelatin.
Furthermore, the concentration of the upstream primer is 0.15 mu M, and the concentration of the downstream primer is 0.15 mu M.
Further, the PCR product is identified by polyacrylamide denaturing gel electrophoresis.
Further, the concentration of the polyacrylamide denatured gel is 6%.
Further, when the band is 178bp, the early heading gene is contained in the rice sample.
Compared with the prior art, the invention has the following beneficial effects:
the primer pair provided by the invention can be used for detecting the molecular marker of a rice sample, identifying the heading stage genotype of a rice plant, and accelerating the cultivation of a premature variety and a hybrid rice combination which are suitable for ecological conditions of double-cropping rice regions in south China.
Drawings
FIG. 1 is a diagram showing the identification of the chromosome fragment substitution line CL33 and the heading date of the receptor parent 9311 in example 1.
FIG. 2 shows 9311/CL 33F in example 12Heading date profile of segregating populations.
FIG. 3 is a chromosome map of SNP-index of 30 pools E1-30 of the very early heading individuals of example 1.
FIG. 4 is a chromosome map of SNP-index of 30 very late heading single-plant pool L1-30 in example 1.
FIG. 5 is a chromosome map of △ SNP-index of the very early heading single plant mixed pool E1-30 and the very late heading single plant mixed pool L1-30 in example 1.
FIG. 6 is the gel electrophoresis chart of the modified polyacrylamide in example 2.
Detailed Description
Example 1
1. Chromosome fragment substitution line heading period identification
By performing heading date identification on 113 chromosome fragment replacement lines with indica rice 9311 as genetic background, CL33 of one replacement line is found to heading 15-20 days earlier than the receptor parent 9311, as shown in FIG. 1.
2. Genetic analysis of CL33 heading stage genes
By the pair 9311/CL 33F2The results of the heading date survey of 524 isolates are shown in FIG. 2, the variation range is 80-109 days, the early and late heading segregation ratio is 127:394, the segregation ratio is 1:3, and genetic analysis shows that: the early panicle of CL33 is controlled by 1 pair of recessive genes, with the late panicle being dominant over the early panicle.
3. Based on CL33, 9311 and its progeny mixed pool whole gene re-sequencing and BSA analysis
For rapid gene mapping and candidate gene Analysis, the study used mixed pools E1-30 and L1-30 constructed from individual populations (30 strains each) of CL33(ZP1) and 9311 and an extreme early spike (80-85 days) and an extreme late spike (105-.
(1) Deep analysis of genome re-sequencing
The reference indica rice genome size is 427,004,890bp (reference genome download address: ftp:// ftp. ensible genomes. org/pub/release-28/plantations/fasta/oryza _ indica), the alignment ratio of all samples is 93.83-98.74%, the average coverage depth of the reference genome (excluding N region) is 8.9-25.84X, the 1X coverage (coverage of at least one base) is more than 92.42%, and the data are shown in Table 1. The comparison result is normal, and can be used for subsequent variation detection and related analysis.
TABLE 1 statistics of sequencing depth and coverage
Remark ① Sample name.
② Mappedreads the number of reads aligned to the reference (including both single-ended and double-ended alignments).
③ Totalreads total number of reads for valid sequencing data.
④ Mappingrate, alignment ratio, number of reads aligned to the reference genome divided by the number of reads for valid sequencing data.
⑤ Average depth of sequencing, total number of bases aligned to the reference genome divided by genome size.
⑥ Coverage atleast 1X percentage of sites in the reference genome that are covered by at least 1 base.
⑦ Coverage atleast4X percentage of sites in the genome covered by at least4 bases of the reference genome.
(2) SNP frequency difference analysis of extremely early heading single plant mixing pool E1-30 and extremely late heading single plant mixing pool
Based on the result of genotyping, screening homozygous SNP sites in parents, selecting 16,491 polymorphic markers in total, selecting a parent CL33(ZP1) as a reference parent, analyzing and calculating SNP-index (i.e. frequency of SNP) of 16,491 marker sites between parents of two offspring, wherein, SNP-index is 0, SNP-index is 1 which is completely different from the SNP-index, in order to visually reflect the distribution of SNP-index of offspring on chromosome, mapping the distribution of SNP-index on chromosome, default selecting 1Mb as a window and 1kb as a step size, calculating the average value of SNP-index in each window to reflect the SNP-index distribution of offspring, fig. 3 is the distribution of SNP-index on chromosome of 30 very early single-plant mixed pools E1-30, fig. 4 is the distribution of SNP-index on chromosome of 30 very late single-plant mixed pools L1-30, calculating 5 (SNP-index distribution) as the result of SNP-index on chromosome, namely, selecting a difference of SNP-index of two offspring as a threshold value, and selecting a blue character (SNP-index) as a result of a 95% displacement test (SNP-index) as a map, wherein, the result is shown in the test of extreme.
(3) Early heading gene localization and candidate gene analysis
Selecting a window larger than a threshold value at a confidence level of 95% as a candidate interval, selecting SNP sites with significant differences of SNP-indexes of two offspring in a whole genome range in order to not ignore the influence of micro-effect QTL, namely selecting sites with SNP-indexes close to 1 (0.79 is selected in the item) of offspring 2 (extremely late heading stage) and SNP-index close to 0 (0.05 is selected in the item) of offspring 1 (extremely early heading stage), extracting annotation results of ANNOVAR for 121 candidate polymorphic marker sites, preferentially selecting genes causing stop loss or stop gain or nonsynonymous mutation or alternative splicing as candidate genes, and identifying 8 candidate genes (table 2) which are respectively Os08g0173600, Os08g0162000, Os08g0175600, Os08g 0101400, Os08g 0108 g 500, Os 017108 g 6708 and Os7108 g 017108 and Os7208 in reference genome sequence range corresponding to 820kb of 8 chromosomes.
TABLE 2 Whole Gene resequencing BSA analysis identified heading-stage Gene-related candidate regions
4. Fine localization of genes and candidate gene validation
To verify the accuracy of candidate gene annotation results, the F of the parent whole genome re-sequencing developed InDel marker pair 9311/CL33 was used within the candidate gene interval2The marker analysis (Table 3) was carried out on 500 individuals of the segregating population, and it was found that the PSM8-8 marker was coseparated with the gene, and finally the early heading gene qEH8-1 was finely localized in the range of about 270kb between the InDel marker PSM8-6 and PSM8-9 (rice genome 1cM was about 250 kb). By utilizing the molecular marker PSM8-8 in the following table for detection, whether the rice plant has the early heading gene or not is judged, and the corresponding amplification upstream primers are the nucleotide sequences shown as SEQID No.1 and downstream primer SEQ ID No. 2.
SEQ ID No.1：5’-GTATTGTGGCATCAGGACTT-3’
SEQ ID No.2：5’-GCTCATTAGAGAAAACGAGG-3’
TABLE 3 InDel marker sequence information
Example 2
(1) Rice sample
Early-late ear-cutting parents CL33 and 9311, 9311/CL33 hybrid F231 plants (80-85 days) appeared as early panicles, 2 plants (105-106 days) appeared as late panicles and 1 plant with a panicle stage of 94 days in the genetically segregating population.
(2) DNA sample extraction and detection
According to the CTAB method (Murray)&Thompson 1980) extracted DNA from each rice sample. Analyzing the purity and integrity of DNA of each sample by agarose gel electrophoresis; nanodrop microspectrophotometer detection of sample DNA purity (OD)260/280The ratio is between 1.8 and 2.0); the Qubit accurately quantifies the sample DNA concentration (sample concentration)>20ng/μL)。
(3) PCR amplification
PCR amplification was performed according to the method of Panaud et al (1996) with minor modifications. 20 μ L of the reaction system included 0.15 μ M of primer, 200 μ M dNTP, 1 XPCR reaction buffer (50mM KCl, 10mM Tris-HCl pH8.3, 1.5mM MgCl)20.01% gelatin), 50-100 ng of DNA template and 1UTaq enzyme. The reaction is carried out in a 9700 type DNA amplification instrument or PTC-100TMThe method is carried out in a Programmmable Thermal Controller. The reaction procedure used was: pre-denaturation at 94 deg.C for 5min, circulation (94 deg.C for 1min, 55 deg.C for 1min, 72 deg.C for 1min) for 35 times, and final extension at 72 deg.C for 5 min.
(4) Electrophoresis
The PCR product is identified by polyacrylamide denaturing gel electrophoresis with the concentration of 6 percent, and the basic operation comprises the following three steps: (1) preparing glue: weighing 9.6g of urea, adding 45mL of distilled water and 8mL of 10 xTBE (108g of Tris-HCl, 55g of boric acid and 7.44g of EDTA added with distilled water for dissolving, and keeping the volume to 1L), stirring the urea by using a glass stick to fully dissolve the urea, then adding 12mL of 40% acrylamide solution (38g of acrylamide, 2g N and N' -methylene bisacrylamide are added with water to keep the volume to 100mL) by using a small cylinder, fully mixing the solution, adding 0.8mL of 10% ammonium persulfate and 35 muL of TEMED (refer to the thesis of the university of Gong-Fu, south China, 2016), quickly injecting the mixture into the middle of two glass plates with the sealed bottoms by agarose after mixing, immediately inserting a comb after filling, enabling the comb to be placed against a wall with a certain inclination with a table top, and standing the gel for solidification for 30-45 min. (2) Sample application: after the gel is fully coagulated, the comb is pulled out, and the sample application hole is kept intact as much as possible. Fixing the glass plate on a vertical electrophoresis tank (model: Dyy III-28D), adding a proper amount of 1 × TBE; repeatedly washing with TBE, and washing the sample application hole until no solidified substance exists; and taking out PCR amplification products, adding 4 mu L of sample loading buffer solution (0.25% bromophenol blue, 0.25% xylene cyanide and 50% glycerol) into each PCR product, uniformly mixing, and sucking 3-4 mu L of sample adding point sample holes by using a 10 mu L of pipette. (3) Electrophoresis: and (3) using a voltage-stabilizing and current-stabilizing electrophoresis apparatus, wherein the 250V constant voltage electrophoresis time is about 1-2 h.
(5) Silver staining and results recording
After completion of electrophoresis, the glass plate was taken out from the electrophoresis tank and carefully peeled offDischarging gel, and washing twice with distilled water; transfer to 0.1% AgNO3Dyeing in the solution, and slightly shaking on a shaking table for about 10 min; then transferring the gel into distilled water to rinse twice; transferring into color developing solution (6g sodium hydroxide, 0.076g sodium tetraborate, 1.6mL formaldehyde, adding water to a constant volume of 400mL) for developing for about 10min (refer to Gong Yongfu, 2016, master's academic paper of southern agriculture university), transferring into tap water for storage after coloring, and the result is shown in FIG. 6, wherein M represents DNA marker; 1 and 2 represent the late heading parent 9311 and the early heading chromosome fragment CL33, respectively; the remaining 34 samples were 9311/CL33 hybrid F2Genetically segregating a population of individual plants, wherein the occurrence of the same banding pattern (146bp) as that of the parent 9311 indicates that the plants are late-heading; the appearance of the same band type (178bp) as that of the chromosome fragment substitution line CL33 indicates that the plant shoots early; the emergence of heterozygous banding pattern at the same time indicates that the heading stage of the plant is located between the two.
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.
Claims (10)
1. The oligonucleotide primer pair for amplifying the rice InDel molecular marker is characterized in that the primer pair is a nucleotide sequence shown in SEQ ID No.1 and a downstream primer SEQ ID No. 2.
2. An InDel molecular marker co-isolated with a heading stage gene amplified by the primer pair of claim 1.
3. A pair of oligonucleotide primers for amplifying the molecular marker of claim 2.
4. The method for identifying the genotype of the rice plant at the heading stage is characterized by comprising the following steps of:
(1) amplifying an InDel molecular marker by using the rice sample DNA as a template and at least one primer pair selected from the group consisting of the primers in claim 1 or claim 3;
(2) and identifying the genotype of the rice sample at the heading stage.
5. The method for genotyping rice at the heading stage of a plant as claimed in claim 4, wherein the step (1) of extracting and identifying the DNA from the rice sample comprises: (1) extracting DNA of each rice sample according to a CTAB method; (2) analyzing the purity and integrity of DNA of each sample by agarose gel electrophoresis; (3) detecting the purity of the sample DNA, requiring OD260/280The ratio is between 1.8 and 2.0; (4) detecting the DNA concentration of the sample, requiring the concentration of the sample>20ng/μL。
6. The method for genotyping the heading date of a rice plant as claimed in claim 4, wherein the amplification in step (1) is: 20 mu L of reaction system comprises 0.1-0.2 mu M of upstream primer, 0.1-0.2 mu M of downstream primer, 200 mu M dNTP, 1 XPCR reaction buffer solution, 50-100 ng of DNA template and 1UTaq enzyme; the reaction procedure used was: pre-denaturation at 94 deg.C for 5min, circulation at 94 deg.C for 1min, 55 deg.C for 1min, and 72 deg.C for 1min for 35 times, and extension at 72 deg.C for 5 min;
the 1 XPCR reaction buffer comprises 50mM KCl, 10mM Tris-HCl pH8.3, 1.5mM MgCl20.01% gelatin.
7. The method for identifying the heading date genotype of rice plants as claimed in claim 6, wherein the concentration of the upstream primer is 0.15 μ M and the concentration of the downstream primer is 0.15 μ M.
8. The method for genotyping the heading date of rice plants as claimed in claim 6 or 7, wherein the PCR products are identified by denaturing polyacrylamide gel electrophoresis.
9. The method for genotyping a rice plant at heading stage according to claim 8, wherein the polyacrylamide denaturing gel is present at a concentration of 6%.
10. The method for genotyping the heading date of a rice plant of claim 9, wherein a strip of 178bp indicates that the rice sample contains the early heading gene.
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| CN114854893A (en) * | 2021-12-23 | 2022-08-05 | 山西农业大学农业基因资源研究中心 | SNPs (single nucleotide polymorphism) marker associated with millet heading stage character and identification method thereof |
| CN118116451A (en) * | 2024-03-13 | 2024-05-31 | 广东省农业科学院水稻研究所 | InDel background molecular marker design method based on resequencing and application |
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