CN117385087B - SNP molecular marker Me-5676929 linked with microspore embryogenic gene, application thereof and special primer - Google Patents
SNP molecular marker Me-5676929 linked with microspore embryogenic gene, application thereof and special primer Download PDFInfo
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Abstract
The invention discloses a SNP molecular marker Me-5676929 linked with microspore embryogenesis genes, application thereof and a special primer. The SNP molecular marker is positioned at 166621892bp of chromosome 2, and the base is A or G; the location of the molecular markers is determined based on the capsicum reference genome CM 334. The substance for detecting the molecular marker can be used for assisting in breeding pepper varieties with high microspore embryogenesis frequency or identifying the microspore embryogenesis frequency of pepper varieties to be detected. The method provides a practical molecular marker for establishing a high-efficiency and stable chilli haploid cultivation technical system.
Description
Technical Field
The invention relates to the technical field of pepper cultivation, in particular to an SNP molecular marker Me-5676929 linked with microspore embryogenesis genes, and application and a special primer thereof.
Background
The capsicum is an important vegetable and economic crop widely cultivated in the world and is also a main economic prop crop in many areas of China. Haploid breeding technology plays an increasingly important role in plant variety improvement as one of the strong supports of modern breeding technology. By haploid culture, rich isolation, variation and recombination can be demonstrated in the current generation and permanently stabilized in homozygous form by chromosome doubling. The fully homozygous and stable inbred line can be obtained by applying the haploid breeding technology for one generation, the crop breeding period can be obviously shortened by 5-6 generations, the breeding process can be greatly accelerated, and the breeding efficiency can be obviously improved. And the recessive gene doubling haploid can be normally expressed without being covered by dominant genes, so that the selection efficiency and the selection accuracy of genotypes can be remarkably improved, and the method is favorable for polygene recombination and recessive gene selection. The mature haploid breeding technology has important application and research value in genetic transformation, mutant induction, genetic analysis, species evolution and other aspects. The pepper hybrid vigor is obvious, and the cross breeding is a main breeding mode. The acquisition of a stable pepper inbred line requires at least 7-8 successive generations of selfing and makes it difficult to select for the recessive trait. The combination of the pepper haploid breeding technology and the conventional breeding technology can obviously improve the breeding efficiency, and has important application value in pepper breeding. Microspore culture is an important way to obtain a pepper haploid. However, the too low frequency of embryogenesis of part of genotype microspores becomes a limiting bottleneck for the application of pepper haploid breeding technology. If a specific method can be adopted to identify whether plants belong to genotype varieties with high microspore embryogenesis frequency, namely, the varieties are primarily screened from the angle of microspore embryogenesis frequency, and then the varieties with high microspore embryogenesis frequency are utilized to carry out pepper haploid culture, the working efficiency can be greatly improved, the breeding speed is accelerated, and the stable pepper haploid breeding technology system is facilitated to be obtained, so that it is important to find a method or tool for rapidly identifying whether plants belong to varieties with high microspore embryogenesis frequency.
Disclosure of Invention
Aiming at the defects of the prior art, one of the purposes of the invention is to provide a SNP molecular marker Me-5676929 linked with microspore embryogenesis genes, and to provide a practical molecular marker for establishing a high-efficiency and stable chilli haploid cultivation technical system.
The second object of the invention is to provide a KASP specific primer group for detecting genotype of SNP molecular marker Me-5676929.
The invention also aims to provide a kit for detecting the genotype of the SNP molecular marker Me-5676929.
The fourth object of the invention is to provide the application of SNP molecular markers Me-5676929, KASP specific primer set or kit.
The invention aims at providing a method for identifying the frequency of microspore embryogenesis of a pepper variety.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
In a first aspect, the invention provides a SNP molecular marker Me-5676929 linked to microspore embryogenic gene, wherein the molecular marker is positioned at 166621892bp of chromosome 2, and the base is A or G; the location of the molecular marker is determined based on the capsicum reference genome CM 334;
Genotype corresponding to the molecular marker: a is genotype with low microspore embryogenesis frequency; g is the genotype of high microspore embryogenesis frequency; g is the genotype with high microspore embryogenesis frequency.
Specifically, SNP molecular marker Me-5676929 is located on chromosome 2 of pepper reference genome CM334, physical position is 166621892, the allele base of the position in genome is G, the allele base of the position in genome is A.
In the present invention, a microspore embryogenesis frequency of less than 2 is counted as a low microspore embryogenesis frequency, and a microspore embryogenesis frequency of greater than or equal to 2 is counted as a high microspore embryogenesis frequency.
In a second aspect, the invention provides a KASP specific primer set for detecting or determining the genotype corresponding to the SNP molecular marker Me-5676929 linked to microspore embryogenic genes.
In a preferred embodiment of the KASP specific primer set of the second aspect provided by the present invention, the primer set comprises:
The nucleotide sequence of the forward primer_X is shown in a sequence table SEQ ID NO.1, and specifically comprises the following steps: 5'-CATCACACTGATGCCTGAAGTC-3';
the nucleotide sequence of the forward primer_Y is shown in a sequence table SEQ ID NO.2, and specifically comprises the following steps: 5'-GCATCACACTGATGCCTGAAGTT-3';
The nucleotide sequence of the reverse primer primer_C is shown in a sequence table SEQ ID NO.3, and specifically comprises the following steps: 5'-CAATAAATGTATTAATCTAGAACACTAGAGGAG-3'.
Preferably, the 5' ends of the forward primer primer_X and the forward primer primer_Y are respectively connected with different fluorescent linker sequences.
Specifically, the fluorescent linker sequence may be one of FAM, HEX, FITC, RED, TET, JOE, R to 110.
In a third aspect the invention provides a kit comprising a set of KASP-specific primers as described above and PCR reaction reagents. Specifically, the PCR reaction reagent may include at least one of PCR buffer, DNA polymerase, dNTPs.
In a fourth aspect, the present invention provides the use of the above-described KASP-specific primer set and kit for detecting the genotype of the SNP molecular marker Me-5676929 linked to microspore embryogenic gene, said use being as follows (1) or (2):
(1) Auxiliary breeding of pepper varieties with high microspore embryogenesis frequency;
(2) And (5) identifying the frequency of microspore embryogenesis of the pepper variety to be detected.
The substance for detecting the genotype of the SNP molecular marker Me-5676929 linked with the microspore embryogenesis gene can be used for assisting in selecting pepper varieties with high microspore embryogenesis frequency in pepper haploid cultivation, and the varieties are used for haploid cultivation, so that a stable and efficient haploid cultivation system can be established.
The fifth aspect of the invention provides a method for identifying the frequency of microspore embryogenesis of a pepper variety to be detected, comprising the following steps:
extracting genome DNA of a pepper variety to be detected;
Taking genome DNA of a pepper variety to be detected as a template, and carrying out PCR amplification by utilizing the KASP specific primer group;
Performing fluorescence detection and analysis on the amplified product to obtain the genotype of the pepper variety to be detected, thereby determining the microspore embryogenesis frequency of the pepper variety to be detected;
When the genotype is A, the microspore embryogenesis frequency of the pepper variety to be detected is low; when the genotype is G, the microspore embryogenesis frequency of the pepper variety to be detected is high; when the genotype is A to G, the microspore embryogenesis frequency of the pepper variety to be detected is high.
In the method of the fifth aspect, as a preferred embodiment, the PCR is Touch-down PCR;
Further, the PCR procedure was pre-denatured at 94℃for 15 minutes; then denatured at 94℃for 20s; annealing at 61-55 ℃ for 60s, 10 cycles altogether, wherein 61 ℃ is the annealing temperature of the first cycle, and then the annealing temperature of each cycle is reduced by 0.6 ℃; then denatured at 94℃for 20s; renaturation/extension at 55℃for 60s, 26 cycles total.
The SNP molecular marker Me-5676929 linked with microspore embryogenesis genes and the application thereof provided by the invention have the following beneficial effects:
The invention uses the F10 generation RIL group which is constructed by the capsicum type material PM702 and the sweet pepper type material FS871 and contains 146 strains as a test material, and utilizes the simplified genome sequencing technology (SLAF-seq technology) to construct the capsicum high-density complete linkage genetic map. Based on the constructed high-density complete linkage genetic map of the capsicum, the ICIMAPPING software is utilized to carry out QTL positioning analysis of the microspore embryogenesis regulatory gene of the capsicum. The main effect QTL for controlling the embryogenesis of the microspores of the peppers is positioned on chromosome 2, and a tightly linked molecular marker Me-5676929 is obtained and is positioned on chromosome 2 by 166621892bp. Provides a practical molecular marker for establishing a high-efficiency and stable chilli haploid cultivation technical system.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a high-density complete linkage genetic map of capsicum provided in example 1 of the present invention, wherein black horizontal lines represent SLAF markers; the x-axis and the y-axis represent linkage group number and genetic distance (in centimorgan), respectively;
FIG. 2 is a QTL distribution diagram of the embryogenesis frequency of microspores of capsicum provided in example 1 of the present invention, wherein QTL ANALYSIS of TRAIT CHILLI is the QTL analysis of the target trait of capsicum; the x-axis represents linkage group number and the y-axis is LOD value;
FIG. 3 is a graph showing the genotyping result or KASP labeling result of 100 parts of capsicum material according to example 2 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions in the embodiments of the present invention will be clearly and completely described in the following in conjunction with the embodiments of the present invention. It will be apparent to those skilled in the art that the examples are merely to aid in understanding the invention and are not to be construed as a specific limitation thereof. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention. The process parameters for the specific conditions not noted in the examples below are generally as usual.
The endpoints of the ranges and any values disclosed in the present invention are not limited to the precise range or value, and the range or value should be understood to include values close to the range or value. For numerical ranges, one or more new numerical ranges may be obtained in combination with each other between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point values, and are to be considered as specifically disclosed in the present invention.
Example 1 acquisition of molecular markers Me-5676929
1.1 Test materials:
The invention constructs an F 10 generation recombinant inbred line comprising 146 strains as a test material by using a sweet pepper type material FS871 (bred by a hot pepper breeding subject group of the national institute of sciences and forestry, beijing) and a hot pepper type material PM702 (introduced by a American germplasm resource library) as parents. The green house is planted in winter 2015 in Beijing and the vegetable institute of the national academy of sciences of agriculture and forestry, and the green house is planted in 2016 and 2017 in Beijing and the vegetable institute of the national academy of sciences of agriculture and forestry. 3 repeats of each strain are planted in FS871, PM702 and 146 strains in each season, 10 strains are planted in each repetition, and the strains are designed according to random granules.
1.2 Constructing a high-density genetic map of the capsicum:
1.2.1 extraction of genomic DNA and construction of SLAF library
The total genomic DNA of all the test materials is extracted by using a plant genomic DNA extraction kit (TIANGEN, beijin, china), and the quality of the extracted genomic DNA is detected, wherein the undegraded and high-quality qualified DNA has D260/280 of 1.8-2.0 and D260/230 of 1.8-2.2. And detecting qualified genome DNA of each sample by utilizing haeIII enzyme digestion. Performing 3' end addition A treatment (3 '. Fwdarw.5 ' end) on the obtained enzyme slice segment by utilizing Klenow Fragment and dATP at 37 ℃; the Dual-index sequencing adaptors were ligated using T4 DNA ligase (PAGE-purified, life Technologies, USA); then PCR amplification is carried out (forward primer F1:5'-ATGATACGGCGACCACCGA-3' (shown as SEQ ID NO.6 of the sequence table), reverse primer R1:5'-CAAGCAGAGAGAGGCATACG-3' (shown as SEQ ID NO.7 of the sequence table)); PCR products were purified using Agencourt AMPure XP (beads Beckman Coulter, high Wycombe, UK) and mixed; the mixed sample is subjected to agarose gel electrophoresis and gel cutting, and a target fragment with the length of 314-394bp is selected and defined as SLAF label. PE125bp sequencing was performed using Illumina Hiseq TM 2500 (Illumina, inc.; san Diego, calif., USA) after the library quality was checked.
1.2.2SLAF-seq data analysis and polymorphic SLAF tags
And (3) identifying the raw data obtained by sequencing by using the Dual-index, and filtering the raw sequencing data to obtain reads of each sample. Sequencing mass values and GC content of sequencing reads of the filtered adaptors were assessed. Sequencing reads were aligned onto the capsicum reference genome CM334 by BWA software, and reads aligned double-ended to the same location were considered to originate from the same SLAF tag. Polymorphism analysis was performed using GATK software based on differences between allele and gene sequences, yielding 3 types of SLAF tags altogether: polymorphism, non-polymorphism and repetition. Polymorphic SLAF tags with genotype encoded aa×bb were selected for construction of genetic maps. To ensure the quality of the genetic map, polymorphic SLAF tags were filtered according to the following rules:
A filters out data below 10X of the parent and parent sequencing depth. The offspring is typed according to parent offspring genes, and the accuracy of offspring typing can be guaranteed only by the high-depth parent sequencing depth;
The number of B SNPs is greater than 8.SLAF label sequencing length is 200bp, if excessive SNP appears, the SNP is regarded as sequencing high-frequency variation region;
c integrity filtering. Screening markers whose genotypes cover at least 55% or more of all offspring individuals;
D bias separation mark filtering. Polymorphic markers with severe bias (chi-square test P < 0.001) were filtered out. The filtered high-quality polymorphic SLAF tag is used as SLAF tag for constructing genetic map.
1.2.3 Construction of high Density genetic map
And calculating MLOD values between every two of the obtained polymorphic SLAF markers through filtering, arranging the marks according to MLOD values of the marks from small to large, wherein the highest MLOD values of the marks are in the same linkage group, filtering out the marks with MLOD values lower than 8 of other SLAF marks, and classifying all the marks into 12 groups. According to the positioning of the tag on the chromosome, the tag on the chromosome of chr1-chr12 is divided into 12 linkage groups (linkage group numbers and chromosome numbers are in one-to-one correspondence), and a final grouping result is obtained. And (3) mapping SLAF marks by MSTmap software, correcting genotype errors or deleting the SLAF marks by using the SMOOTH algorithm, and constructing a high-quality high-density genetic map by using HighMap software after at least four cycles of mapping, correction and mapping. The genetic distance between adjacent markers is calculated by a Kosambi mapping function.
Specifically, the high-density complete linkage genetic map of the capsicum of the invention is constructed by the method, wherein SLAF-seq sequencing is carried out on FS871, PM702 and RILs, the average sequencing depth of PM702 is 57.04X, the average sequencing depth of FS871 is 72.39X, and the average sequencing depth of RILs is 15.65X. A high-density complete linkage genetic map of capsicum containing 9328 high quality SLAF markers was constructed with a total map distance of 2009.69cM, an average map distance of 0.22cM using SLAF-seq (see FIG. 1).
1.2.4 Positioning of the embryogenesis regulatory Gene QTL for microspores of Capsici fructus
A high-density genetic map constructed based on SLAF-seq technology is adopted to carry out multi-environment QTL positioning by ICIMAPPING V3.3.3 software. And (3) respectively inputting three character values of different environments, and comprehensively analyzing and positioning QTL for the microspore embryogenesis frequency genes of the peppers. LOD values were estimated using the complete interval mapping (ICIM), and the threshold for LOD values was determined by permutations times over 95% confidence intervals.
Specifically, QTL positioning analysis was performed using ICIMAPPING V3.3.3 software, ICIMAPPING V3.3.3 software being trait positioning software specific to multiple years of repeated trials. And ICIMAPPING QTL, applying a multi-environment test QTL positioning method in positioning, and outputting an accurate result by integrating data of a plurality of repeated environments by software. The 1000 PT tests were applied to check the 0.95 confidence interval and determine that the LOD value threshold was 3.4217. In total, 7 QTLs were detected in this project and distributed on the 2, 6,8, 11, 12 chromosomes (see fig. 2, table 1). The major QTL interval with the highest contribution rate is located in Me2.2 of chromosome 2, and the interval size is 0.42Mb.
TABLE 1 results of positioning of microspore embryogenesis frequency QTL of Capsicum
| Name | Chr | Start(bp) | End(bp) | LOD | PVE | ADD |
| Me2.1 | 2 | 130092883 | 167615129 | 3.7374 | 5.0202 | 1.8945 |
| Me2.2 | 2 | 167038469 | 166621892 | 6.6564 | 18.299 | 2.5586 |
| Me6.1 | 6 | 209127509 | 209335290 | 4.0079 | 5.562 | 1.9714 |
| Me8.1 | 8 | 129982981 | 132037948 | 3.5837 | 4.8846 | 1.8611 |
| Me8.2 | 8 | 132543379 | 132924979 | 3.506 | 4.7588 | 1.8362 |
| Me11.1 | 11 | 174890572 | 204021481 | 3.5437 | 4.811 | 1.8439 |
| Me12.1 | 12 | 7239786 | 8480026 | 3.8342 | 5.2516 | 1.9193 |
Note that: chr: linkage group numbering; LOD: an associated maximum LOD value for the trait; PVE: phenotype contribution rate; ADD: additive effect value.
1.2.5 Development of SNP molecular markers in Capsici fructus linked to microspore embryogenesis genes
According to SLAF-seq sequencing data, the end 166621892 of the main locating interval is marked as Marker5676929, and the sequence of 70bp before and after the main locating interval is sequenced, so that the result shows that the parent PM702 with high microspore embryogenesis frequency is obtained, the allele base at the position is G, the parent FS871 with low microspore embryogenesis frequency is obtained, and the allele base at the position is A. Microspore embryogenesis frequency was less than 2 and less. FS871 microspore embryogenesis frequency was low at 0.07 and pm702 microspore embryogenesis frequency was high at 52.55. Based on the sequencing results, KASP primer Me-5676929 was developed for Marker5676929SNP site, the primer sequences are shown in Table 2.
TABLE 2 primer sequences for molecular Marker5676929
The underlined portion in Table 2 is the fluorescent linker sequence attached to the 5' end of the corresponding forward primer, FAM for the 5' linker of forward primer primer_X and HEX for the 5' linker of forward primer primer_Y.
Example 2 verification of reliability of SNP molecular marker Me-5676929 linked to microspore embryogenic Gene
100 Parts of material is randomly selected from the test material RILs constructed in the example 1 to serve as verification materials, and group verification is carried out on the molecular marker Me-5676929 closely linked with the gene for controlling the embryogenesis frequency of the capsicum microspores and the accuracy of the detection of a primer group developed for the molecular marker.
Extracting genome DNA of the verification material, and taking the genome DNA as a template of a PCR system;
The PCR reaction was performed on a Hydrocycler water bath PCR instrument and the fluorescence detection was performed on a BMG LABTECH GMbH platform.
The PCR amplification system is as follows: genomic DNA (20 ng/. Mu.L) 2.5ul,2xKASP Master mix (available from Gene Co.) 2.5. Mu.L; KBD Assay mix (i.e., a mixture of primers at a concentration of 10. Mu. Mol/. Mu.L for each primer) was 0.07. Mu.L by mixing forward primer X, forward primer Y and reverse primer in a molar ratio of 2:2:5;
The amplification conditions were in order: pre-denaturation at 94 ℃ for 15 min; denaturation at 94℃for 20s; annealing at 61-55 ℃ for 60s, 10 cycles altogether, wherein 61 ℃ is the annealing temperature of the first cycle, and then the annealing temperature of each cycle is reduced by 0.6 ℃; denaturation at 94℃for 20s; renaturation/extension at 55℃for 60s, 26 cycles total.
When the amplified product is subjected to fluorescence detection, if the sample PCR product only detects a fluorescence signal (blue fluorescence) corresponding to a forward primer X connected with a fluorescent linker sequence, the corresponding genotype is indicated as G, and the microspore embryogenesis frequency of the pepper strain is judged to be high; if the sample PCR product only detects a fluorescence signal (red fluorescence) corresponding to a forward primer Y connected with a fluorescence joint sequence, the corresponding genotype is A, and the microspore embryogenesis frequency of the pepper strain is judged to be low; if the fluorescent signal corresponding to the forward primer X and the fluorescent signal corresponding to the forward primer Y are detected simultaneously (namely green), the corresponding genotype is A to G, and the microspore embryogenesis frequency of the pepper strain is judged to be high.
The method for phenotypically obtaining microspore embryogenesis frequency is as follows:
The verification material with good growth vigor and no plant diseases and insect pests is selected as a donor plant, and buds in the single-core close period-double-core early period of microspore development period are strictly selected according to microscopic examination results. The buds were pretreated at 4℃for 1 day. Removing the calyx of bud, soaking in 75% alcohol for 30s, sterilizing with 10% sodium hypochlorite solution for 10min, and cleaning with sterile water for 3-4 times and 5min each time. The anthers were stripped intact with forceps on a bench top and inoculated onto N4-3 medium at a density of 12 anthers per 60mm diameter dish. The N4-3 culture medium is a solid-liquid double-layer culture medium (the final concentration of solid layer components and each component in the solid layer is NTH basic culture medium+sucrose 3% +active carbon 0.5% +agar powder 0.8 percent, the final concentration of liquid layer components and each component in the liquid layer is NTH basic culture medium+sucrose 3% +IAA 0.8mg/L+6-BA 0.5mg/L, suction filtration sterilization is carried out, wherein the solvent of the NTH basic culture medium is water, and the solute and the concentration are as follows, ammonia nitrate 825mg/L, potassium nitrate 950mg/L, calcium chloride dihydrate 166mg/L, magnesium sulfate heptahydrate 185mg/L, potassium phosphate 680mg/L, boric acid 6.20mg/L, manganese sulfate tetrahydrate 25mg/L, zinc sulfate heptahydrate 10mg/L, sodium molybdate dihydrate 0.25mg/L, copper sulfate pentahydrate 0.025mg/L, cobalt chloride hexahydrate 0.03mg/L, inositol 5mg/L, niacin 5mg/L, 5mg, folic acid 0.5mg, and folic acid 0.5 mg. The anther is firstly dark-cultured for 7 days at 35 ℃, and then transferred to a condition of 25 ℃ for continuous dark culture. Culturing for 6-9 weeks to obtain a large amount of microspore embryogenesis. The number of microspore embryogenesis per verification material can be counted at this time, and the microspore embryogenesis frequency can be calculated by the number of microspore embryogenesis/the number of inoculated anthers.
According to the typing result of the molecular marker Me-5676929, the 100 parts of material were classified into the same type (A: A) as the parent KASP with low microspore embryogenesis frequency, the same type (G: G) as the parent KASP with high microspore embryogenesis frequency, and two groups of KASP types were examined for microspore embryogenesis frequency according to t-test, resulting in a P value of 0.000034, and the two groups were very significantly different (Table 3, FIG. 3).
Comparing the genotyping result of each capsicum material with the actual calculated microspore embryogenesis frequency data, if the genotyping of the material is G, and if the actual calculated microspore embryogenesis frequency is higher than or equal to 2, determining that the genotyping identification result is accurate, otherwise, determining that the identification result is inaccurate; if the genotyping of the material is A to G, actually calculating that the microspore embryogenesis frequency is higher than 2 or equal to 2, determining that the genotyping identification result is accurate, otherwise, determining that the identification result is inaccurate; if the genotyping of the strain is A: A, actually calculating that the microspore embryogenesis frequency is lower than 2, determining that the genotyping identification result is accurate, otherwise, determining that the identification result is inaccurate. As can be obtained from the data in Table 3, the molecular marker Me-5676929 has a molecular detection accuracy of 92% and can be applied to detection of the microspore embryogenesis frequency of the capsicum variety.
Table 3 verification of the accuracy of the marks with 100 parts of material
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.
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| US6013859A (en) * | 1994-07-14 | 2000-01-11 | Pioneer Hi-Bred International, Inc. | Molecular methods of hybrid seed production |
| EP3328185B1 (en) * | 2015-07-28 | 2020-06-24 | Vilmorin & Cie | Method for producing haploid, dihaploid and doubled haploid plants by isolated microspore culture |
| BR112020007343A2 (en) * | 2017-10-13 | 2020-10-06 | Pioneer Hi-Bred International, Inc. | method for generating a haploid plant embryo, embryogenic microspore, embryoid or embryogenic tissue, plant cell comprising an expression cassette, plant cell population |
| CN111394509B (en) * | 2020-05-31 | 2023-06-06 | 湖南农业大学 | A Molecular Marker Linked to Pepper Reverse Thermosensitive Sterility Gene and Its Application |
| CN113881802B (en) * | 2021-11-08 | 2022-05-06 | 北京市农林科学院 | SNP Molecular Marker KQ8-3918 Linked to Gene Controlling Vc Content in Pepper Fruit and Its Application and Special Primers |
| CN113981126B (en) * | 2021-11-10 | 2022-09-02 | 北京市农林科学院 | SNP molecular marker KQ8-4472 linked with gene for controlling Vc content of pepper fruits, application and special primer |
| WO2023192132A1 (en) * | 2022-03-31 | 2023-10-05 | Seminis Vegetable Seeds, Inc. | Methods for improved microspore embryogenesis and production of doubled haploid microspore-derived embryos |
| CN116287387B (en) * | 2023-02-10 | 2025-09-23 | 中国农业科学院蔬菜花卉研究所 | A SNP site tightly linked to the PMMoV resistance gene L3 in pepper, KASP molecular marker and its application |
| CN116732235A (en) * | 2023-07-31 | 2023-09-12 | 华中农业大学 | InDel molecular marker for white pepper variety breeding and application thereof |
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| Mapping of quantitative trait loci for microspore embryogenesis-related traits in the oilseed rape doubled haploid population DH4069 x Express 617;Valdés, A 等;《Molecular Breeding》;20180530;第38卷(第5期);第1-15页 * |
| QTL mapping and candidate gene analysis of microspore embryogenesis in Capsicum;Bai, JY 等;《EUPHYTICA》;20230115;第219卷(第1期);第1-10页 * |
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