CN108384884B - Corn SSR molecular markers and application thereof - Google Patents
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Abstract
The invention provides a group of corn SSR molecular markers and application thereof, wherein the number of the group of corn SSR molecular markers is 17, and the invention also provides a set of primer combination which is suitable for a capillary electrophoresis platform and is used for detecting the SSR molecular markers. The 17 SSR molecular markers can be used for: (1) constructing a corn variety SSR marker fingerprint database; (2) performing maternal traceability analysis on the corn sample; (3) identifying corn varieties and analyzing genetic diversity; (4) corn varieties of Demeia No.1 and Denghai 18 are distinguished. By applying the corn SSR molecular marker disclosed by the invention, the range of available marker loci of corn can be expanded on the genome level; the method provides a new tool for the researches of maize variety and germplasm resource identification, genetic relationship evaluation, cytoplasm genetic characteristics and the like, and has good application prospect.
Description
Technical Field
The invention belongs to the technical field of crop molecular biology, and particularly relates to a group of corn SSR molecular markers for a capillary electrophoresis detection technology and application thereof.
Background
The purity identification and variety differentiation of the corn are key links in a corn seed quality control system. The present corn purity identification and variety distinguishing method includes morphological identification, isoenzyme or seed storing protein electrophoresis technology, etc. Among them, the electrophoresis technique of protein stored in seeds has the advantages of simplicity, rapidness, accuracy, low cost, etc., and the relevant technical standards established according to the technique are being widely applied. However, due to the fact that polymorphism of seed storage protein among partial corn varieties is poor, or bands are represented as non-complementary types, and the like, characteristic bands are difficult to find by utilizing a seed storage protein electrophoresis technology, so that a rapid and reliable identification means is lacked in partial corn.
The emergence of DNA molecular marker technology provides a new means for the identification of corn varieties. Among them, SSR markers are widely regarded as having abundant amounts, high polymorphism, genetic co-dominant, stable amplification, easy exchange of primer sequences, and the like. One of the keys of the identification of varieties by Simple Sequence Repeats (SSR) marker technology is the amplified fragment detection technology. The existing research mostly utilizes more complex polyacrylamide gel electrophoresis combined with silver staining for detection. However, this technique is difficult to clearly distinguish the difference of several bases between amplified fragments due to its low resolution, and has relatively high requirements for instruments, equipment and personnel skills, which is not easy to popularize. While the SSR molecular marker technology based on the capillary electrophoresis detection technology can obtain quantitative DNA fragment analysis data. Compared with the conventional polyacrylamide gel electrophoresis detection method, the method has more accurate, sensitive and efficient result, and is more suitable for detection and analysis of large-batch varieties.
Disclosure of Invention
The invention aims to provide a group of corn SSR molecular markers suitable for a capillary electrophoresis detection technology.
In order to achieve the purpose of the invention, the corn genome of corresponding materials is sequenced and compared by collecting corn materials which are wide in source, rich in phenotype and genotype and strong in representativeness. The invention provides a group of corn SSR molecular markers suitable for capillary electrophoresis detection technology, wherein the molecular markers are one or more of the following 17 SSR molecular markers, and the 17 SSR molecular markers are respectively PHI015, PHI299852, UMC1245, UMC1632, UMC1134, NC030, PHI109275, PHI072, UMC2214, UMC1073, MMMC0481, UMC1066, PHI024, UMC1639, UMC1155, PHI053 and PHI 227562.
The 17 SSR molecular markers are obtained by amplifying the following primers respectively and sequentially: SEQ ID NO.1-2, SEQ ID NO.3-4, SEQ ID NO.5-6, SEQ ID NO.7-8, SEQ ID NO.9-10, SEQ ID NO.11-12, SEQ ID NO.13-14, SEQ ID NO.15-16, SEQ ID NO.17-18, SEQ ID NO.19-20, SEQ ID NO.21-22, SEQ ID NO.23-24, SEQ ID NO.25-26, SEQ ID NO.27-28, SEQ ID NO.29-30, SEQ ID NO.31-32, SEQ ID NO. 33-34.
Further, the invention provides a specific primer pair for amplifying the SSR molecular marker, which is any one of the following pairs: SEQ ID NO.1-2, SEQ ID NO.3-4, SEQ ID NO.5-6, SEQ ID NO.7-8, SEQ ID NO.9-10, SEQ ID NO.11-12, SEQ ID NO.13-14, SEQ ID NO.15-16, SEQ ID NO.17-18, SEQ ID NO.19-20, SEQ ID NO.21-22, SEQ ID NO.23-24, SEQ ID NO.25-26, SEQ ID NO.27-28, SEQ ID NO.29-30, SEQ ID NO.31-32, SEQ ID NO. 33-34.
The invention provides application of the corn SSR molecular marker in constructing a corn variety DNA fingerprint database.
The invention provides application of the corn SSR molecular marker in genetic diversity analysis of corn germplasm resources.
The invention provides application of the corn SSR molecular marker in corn identification.
The invention provides application of the corn SSR molecular marker in corn molecular marker-assisted breeding.
The invention provides application of the corn SSR molecular marker in preparation of a corn genome chip.
The invention provides a corn genome chip containing the corn SSR molecular marker.
The invention provides application of the corn SSR molecular marker in distinguishing corn Demeia No.1 and Denghai 18 varieties.
The application comprises the following steps:
1) extracting DNA of a corn sample to be detected;
2) performing PCR amplification by using the DNA extracted in the step 1) as a template and utilizing the SSR molecular marker;
3) and detecting the PCR product by using a capillary electrophoresis system.
In step 2) of the above application, 20. mu.L of reaction volume containing dNTP0.25mM, 0.4. mu.M of each of the forward and reverse primers, 1.0 unit of Taq DNA polymerase, and 1 XPCR buffer (not containing Mg) was used for PCR amplification2+)，MgCl21.5mM, sample DNA 10-40 ng. The reaction procedure is as follows: pre-denaturation at 94 ℃ for 4 min; denaturation at 94 ℃ for 45s, annealing at 60 ℃ for 45s, and extension at 72 ℃ for 45s for 30 cycles; extending for 10min at 72 ℃, and storing at 4 ℃.
Further, the present invention provides a kit for distinguishing corn varieties germania No.1 and denghai 18, which contains specific primer combinations for the 17 corn SSR molecular markers of the present invention. Preferably, the nucleotide sequences of the specific primer combinations are respectively shown as SEQ ID NO. 1-34.
The 17 pairs of SSR primers provided by the invention can realize the acquisition of genotyping data on a fluorescence capillary electrophoresis platform. The specific scheme is that the 5' end of one of each pair of primers is marked with a fluorescent group; preparing PCR reaction system and adding DNA, primer, dNTP and MgCl2Taq enzyme, Buffer; operating a reaction program; detecting the amplification product on a fluorescent capillary electrophoresis system; and collecting the original data by using capillary electrophoresis system matched software, and importing the original data into genotype software to analyze the original data to obtain the genotype data in a fragment length format.
Preferably, the 17 pairs of specific SSR primers of the invention are labeled by fluorescent dyes, and four fluorescent dyes of PET, NED, VIC and FAM are selected in total. Diluting the PCR product with ultrapure water by 30 times; respectively taking the 4 diluted solutions with the same volume, mixing to form a mixed solution, sucking 1 microliter of the mixed solution, adding 0.5 microliter of LZ500 molecular weight internal standard and 8.5 microliter of deionized formamide into a deep hole plate special for a DNA analyzer; then denaturalizing the mixture for 5min at 95 ℃ on a PCR instrument, taking out the mixture, immediately placing the mixture on ice, and cooling the mixture for more than 10 min; after being instantaneously centrifuged for 10s, the sample is placed on a DNA analyzer for capillary electrophoresis detection. The raw data collected was analyzed using GeneMapper software. The software system compares the position of the target peak with an internal standard LZ500 in the same lane, and directly gives the accurate size of the target DNA fragment.
In a preferred embodiment of the present embodiment, the SSR molecules of the FAM fluorescence labeling group are identified as PHI015, PHI299852, UMC1245, UMC 1632; SSR molecules of the VIC fluorescence labeling group are labeled as UMC1134, NC030, PHI109275, PHI072 and UMC 2214; SSR molecular markers of the NED fluorescence marker group are UMC1073, MMMC0481, UMC1066 and PHI 024; SSR molecules of the PET fluorescence labeling group are labeled as UMC1639, UMC1155, PHI053 and PHI 227562. Performing four times of capillary electrophoresis by using corn DNA No.1 as a template and four groups of primers marked by FAM, VIC, NED and PET fluorescence respectively to obtain four electrophoretogram results; then, DNA of German Asia No.1 is used as a template, and a total electrophoretogram result is obtained by performing capillary electrophoresis on all primer mixtures labeled by FAM, VIC, NED and PET fluorescence. Comparing the total electrophoretogram result with the electrophoresis results of four groups of fluorescence labeled primers of FAM, VIC, NED and PET respectively (figure 1), it can be seen that the target peaks appearing on the individual electrophoretograms of FAM, VIC, NED and PET can be distinguished on the total electrophoretogram, and the peaks of each color are not interfered with each other, namely, the primer combination (total 17 pairs of primers) provided by the invention can be used for one-time capillary electrophoresis, the target bands are not interfered with each other, and the result is easy to judge. The same conclusion can be reached by performing the same experiment using the maize denuding 18DNA as a template (FIG. 2).
In the examples, DNA of Germany Asia No.1 and DNA of Denghai 18 were used as templates, and a combination of FAM, VIC, NED and PET fluorescence labeled primers was used to perform one-time capillary electrophoresis to obtain capillary electrophoresis patterns of Germany Asia No.1 and DNA of Denghai 18, respectively, and the capillary electrophoresis patterns were compared. As can be seen from FIG. 3, German No.1 shows FAM blue peaks at 134bp and 141bp, and Denmark 18 shows FAM blue peaks at 131bp and 145 bp; the German Asia No.1 has VIC green peaks at 83bp, 99bp and 126bp, and the Denmark No. 18 has VIC green peaks at 86bp, 104bp and 120 bp; the German Asia No.1 shows NED yellow peaks at 136bp and 166bp, and the Denmark 18 shows NED yellow peaks at 142bp and 161 bp; german No.1 shows PET red peaks at 107bp and 170bp, while Denghai 18 shows PET red peaks at 101bp and 175 bp. The peaks of each fluorescent marker of German Asia No.1 and Shanghai 18 are not overlapped and are clearly distinguished. Therefore, the fluorescence labeling primer combination provided by the invention can distinguish the corn Germania No.1 and the dengue-18 in one-time capillary electrophoresis.
The amplification products of different fluorescent markers can be subjected to electrophoresis in the same lane, signals are clear, the difference of the sizes of the amplified fragments is obvious, the sizes of the fragments can be accurately calculated, the electrophoresis peak types of each DNA sample are different, and the judgment is easy. The method has the advantages of high sensitivity, good resolution, accurate and reliable result, high efficiency, high speed and the like. The primer combination provided by the invention can be used for conveniently and rapidly distinguishing the corn Delia No.1 and the Shanghai 18 varieties, and has the advantages of cost saving, efficiency improvement, convenience in operation and accurate result. The primer set provided by the invention can be used for corn fingerprint map construction, variety identification, genetic diversity analysis and the like, and has a very wide application prospect.
Drawings
FIGS. 1A-1D are SSR fluorescence labeled capillary electrophoresis images of corn Demeia No.1, respectively, wherein FIG. 1A is a comparison of the combined electrophoresis result of the primer combination with FAM, VIC, NED and PET fluorescence labeling of Demeia No.1 with the electrophoresis result of the primer combination with FAM labeling only. FIG. 1B is a comparison of the results of the combined FAM, VIC, NED and PET fluorescence labeled primers of German Asia No.1 with those of primers labeled only by VIC. FIG. 1C shows the comparison of the combined electrophoresis results of FAM, VIC, NED and PET fluorescence labeled primers of German Asia No.1 with the electrophoresis results of NED labeled primers only. FIG. 1D shows the comparison of the combined electrophoresis results of FAM, VIC, NED and PET fluorescence labeled primers of German Asia No.1 with the electrophoresis results of PET labeled primers only. The comparison results of the figures respectively show that the target peaks appearing on the electrophoresis images of the single-color fluorescence labeling primers can be distinguished on the electrophoresis images of the four-color fluorescence labeling primer combination (the target peaks appearing are indicated by arrows), and the target peaks do not interfere with each other, so that the DNA fingerprint information of the varieties can be clearly read. Namely, the primer combination provided by the invention can be used for one-time capillary electrophoresis, target bands are not interfered with each other, results are easy to judge, and time, experimental reagents and consumables are saved.
FIGS. 2A-2D show the results of SSR fluorescence-labeled capillary electrophoresis detection of Zea mays Shanghai 18. FIG. 2A is a comparison of the electrophoresis results of the fluorescence labeled primer combinations of FAM, VIC, NED and PET for Denghai 18 with the electrophoresis results of the fluorescence labeled primer combinations of FAM only. FIG. 2B is a comparison of the electrophoresis results of the fluorescence labeled primer combination of FAM, VIC, NED and PET for Denghai 18 with the electrophoresis results of the fluorescence labeled primer combination of VIC only. FIG. 2C is a comparison of the electrophoresis results of the fluorescence labeled primer combination FAM, VIC, NED and PET for Denghai 18 with the electrophoresis results of the fluorescence labeled primer combination NED only. FIG. 2D is a comparison of the electrophoresis results of the fluorescence labeled primer combination of FAM, VIC, NED and PET for Denghai 18 with the electrophoresis results of the fluorescence labeled primer only with PET. The comparison results of the figures respectively show that the target peaks appearing on the electrophoresis images of the single-color fluorescence labeling primers can be distinguished on the electrophoresis images of the four-color fluorescence labeling primer combination (the target peaks appearing are indicated by arrows), and the target peaks do not interfere with each other, so that the DNA fingerprint information of the varieties can be clearly read. Namely, the primer combination provided by the invention can be used for one-time capillary electrophoresis, target bands are not interfered with each other, results are easy to judge, and time, experimental reagents and consumables are saved. FIGS. 1A-1D and 2A-2D both illustrate the same objective, and the results of two experiments (Demeia No.1 and Denghai 18) further illustrate the utility and reliability of the primer combinations provided by this patent.
FIG. 3 is a capillary electrophoresis image of corn Demeia No.1 and Denghai 18 using 17 corn SSR molecular markers provided herein, and comparing the results. The results show that the peak of FAM (blue) shown in Germany Asia No.1 (upper panel) and the peak of FAM (both marked with arrows) shown in Denghai 18 (lower panel) are clearly distinguishable and the molecular weights of the peaks of interest are different. Similarly, the two varieties have different peak molecular weights of VIC, NED and PET meshes and are clearly distinguished. The primer combination provided by the patent can be used for carrying out variety differentiation on corn Germania No.1 and Denghai 18.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the examples follow conventional experimental conditions. Those skilled in the art will appreciate that the details of the present invention not described in detail herein are well within the skill of those in the art.
If not otherwise stated, the biochemical reagents used in the examples of the present invention are commercially available, and the corn materials used are all commonly used corn known in the art.
Example 1 determination of SSR molecular markers and primers for maize
Taking 36 corn varieties to carry out SSR primer screening. Primers with distinct band patterns and higher polymorphism, which can be amplified by selecting a site from 987 pairs of SSR primers (https:// FTP. MaizeGDB. org/MaizeGDB/FTP/SSRs /), are used as selectable primers by denaturing polyacrylamide gel electrophoresis. According to the molecular weight range, PIC value and the like of alleles in 36 varieties of selectable primers, namely, each combination is composed of as many primers as possible, and the conditions that all the allele ranges of the primers in the combination are not overlapped are adopted, four primer combinations are composed and the primers with FAM, VIC, NED and PET fluorophores are respectively synthesized. See table 1. Those skilled in the art will appreciate that other fluorophores including FAM, VIC, NED, PET may be selected by those skilled in the art to modify the four sets of primers in table 1, as long as the same fluorophore is labeled by the label in each set, and is not limited to which fluorophore is selected.
TABLE 1 corn SSR capillary electrophoresis primers and primer combinations
Example 2 corn variety differentiation Using SSR molecular markers provided by the invention
(1) Rapid extraction of DNA
And (3) carrying out DNA extraction on the corn seeds by adopting an alkaline cooking method. The method is rapid and simple to operate, has no toxic or harmful reagent, is suitable for DNA preparation in the field of plant molecular biology, and has important significance for greatly shortening the seed purity detection and transgene detection time, improving the detection efficiency and reducing the detection cost. The specific operation steps are as follows: taking a plurality of corn seeds, and placing the corn seeds in a 1.5mL centrifuge tube; adding 400 μ L NaOH (1M) into the centrifuge tube to ensure that the seeds are completely soaked, and carrying out boiling water bath for 5 min; adding 200 μ L Tris-HCl (1M, pH 8.0) into a centrifuge tube, and boiling in water bath for 1 min; 200. mu.L of TE buffer (pH 8.0) was added thereto and the mixture was fully dissolved for further use.
(2) Quality and quantity of DNA sample
Detecting OD values of 260nm and 280nm of DNA sample on ultraviolet spectrophotometer, and selecting OD260/280Samples with values of 1.8-1.9 were used for the assay.
(3) Core primer selection
The primers determined in example 1 to satisfy the capillary fluorescence detection technique were selected by analyzing the distribution of primers on the chromosome, the level of polymorphism, the PCR amplification stability, and the banding pattern of the amplification product, as shown in table 1.
(4) Capillary electrophoresis detection
And (3) carrying out fluorescent dye marking on the screened specific SSR primer, and selecting four fluorescent dyes of PET, NED, VIC and FAM in total. Diluting the PCR product with ultrapure water by 30 times; respectively taking the 4 diluted solutions with the same volume, mixing to form a mixed solution, sucking 1 microliter of the mixed solution, adding 0.5 microliter of LZ500 molecular weight internal standard and 8.5 microliter of deionized formamide into a deep hole plate special for a DNA analyzer; then denaturalizing the mixture for 5min at 95 ℃ on a PCR instrument, taking out the mixture, immediately placing the mixture on ice, and cooling the mixture for more than 10 min; after being instantaneously centrifuged for 10s, the sample is placed on a DNA analyzer for capillary electrophoresis detection. The raw data collected was analyzed using GeneMapper software. The software system compares the position of the target peak with an internal standard LZ500 in the same lane, and directly gives the accurate size of the target DNA fragment. The band sizes of the amplified products obtained by amplifying maize German Asia No.1 and Denghai 18 with each primer set in Table 1 are shown in Table 2.
TABLE 2 bands and sizes of the difference peaks of corn Demeia No.1 and Denghai 18
(5) Determination of primer combination
Performing four times of capillary electrophoresis by using corn DNA No.1 as a template and four groups of primers marked by FAM, VIC, NED and PET fluorescence respectively to obtain four electrophoretogram results; then, DNA No.1 Demeia is used as a template, and a total electrophoretogram result is obtained by performing capillary electrophoresis on all primer mixtures labeled by FAM, VIC, NED and PET fluorescence. Comparing the total electrophoretogram result with the electrophoresis results of four groups of fluorescence labeled primers of FAM, VIC, NED and PET respectively (fig. 1A-fig. 1D), it can be seen that the target peaks appearing on the FAM, VIC, NED and PET individual electrophoretograms can be distinguished on the total electrophoretogram, and the peaks of each color are not interfered with each other, namely, the primer mixture provided by the invention can be used for one-time capillary electrophoresis, the target bands are not interfered with each other, and the result is easy to judge. The same conclusions can be drawn by performing the same experiment using maize denuding 18DNA as a template (FIGS. 2A-2D).
(6) Distinguishing between Demeia No.1 and Denghai 18 varieties
In the examples, DNA of Germany Asia No.1 and DNA of Denghai 18 were used as templates, and a combination of FAM, VIC, NED and PET fluorescence-labeled primers (see Table 1) was used to perform one-time capillary electrophoresis, to obtain capillary electrophoretograms of Germany Asia No.1 and DNA of Denghai 18, respectively, and to compare them. As can be seen from FIG. 3, German No.1 shows FAM blue peaks at 134bp and 141bp, and Denmark 18 shows FAM blue peaks at 131bp and 145 bp; the German Asia No.1 has VIC green peaks at 83bp, 99bp and 126bp, and the Denmark No. 18 has VIC green peaks at 86bp, 104bp and 120 bp; the German Asia No.1 shows NED yellow peaks at 136bp and 166bp, and the Denmark 18 shows NED yellow peaks at 142bp and 161 bp; german No.1 shows PET red peaks at 107bp and 170bp, while Denghai 18 shows PET red peaks at 101bp and 175 bp. The peaks of each fluorescent marker of German Asia No.1 and Shanghai 18 are not overlapped and are clearly distinguished. Therefore, the fluorescence labeling primer combination (table 1) provided by the invention patent can be used for distinguishing the corn Germania No.1 and the Shanghai No. 18 in one capillary electrophoresis.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
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Claims (5)
1. The SSR molecular marker combination is suitable for capillary electrophoresis detection technology, can be used for constructing a corn DNA fingerprint database, analyzing genetic diversity of corn germplasm resources and/or identifying corn varieties and is characterized by consisting of the following 17 SSR molecular markers, namely PHI015, PHI299852, UMC1245, UMC1632, UMC1134, NC030, PHI109275, PHI072, UMC2214, UMC1073, MMMC0481, UMC1066, PHI024, UMC1639, UMC1155, PHI053 and PHI 227562; the molecular marker combination can obtain respective capillary electrophoresis images of corn varieties through one-time capillary electrophoresis and compare the capillary electrophoresis images;
in the molecular marker combination, the 17 SSR molecular markers are divided into four different fluorescence marker groups, the fluorescence markers in each group are the same,
the first group consists of the following SSR molecular markers: PHI015, PHI299852, UMC1245, UMC 1632;
the second group consists of the following SSR molecular markers: UMC1134, NC030, PHI109275, PHI072, UMC 2214;
the third group consists of the following SSR molecular markers: UMC1073, MMMC0481, UMC1066, PHI 024;
the fourth group consisted of the following SSR molecular markers: UMC1639, UMC1155, PHI053, PHI 227562;
the 17 SSR molecular markers are obtained by amplifying the following primers respectively and sequentially: SEQ ID NO.1-2, SEQ ID NO.3-4, SEQ ID NO.5-6, SEQ ID NO.7-8, SEQ ID NO.9-10, SEQ ID NO.11-12, SEQ ID NO.13-14, SEQ ID NO.15-16, SEQ ID NO.17-18, SEQ ID NO.19-20, SEQ ID NO.21-22, SEQ ID NO.23-24, SEQ ID NO.25-26, SEQ ID NO.27-28, SEQ ID NO.29-30, SEQ ID NO.31-32, SEQ ID NO. 33-34.
2. A corn genomic chip comprising only the combination of corn SSR molecular markers of claim 1.
3. Use of the corn SSR molecular marker combination of claim 1 for distinguishing corn germania No.1 from denghai 18 varieties.
4. Use according to claim 3, characterized in that it comprises the following steps:
1) extracting DNA of a corn sample to be detected;
2) performing PCR amplification by using the DNA extracted in the step 1) as a template according to the corn SSR molecular marker combination;
3) and detecting the PCR product by using a capillary electrophoresis system.
5. A kit for identifying corn Demeia No.1 and Denghai 18 varieties, which is characterized by only containing a specific primer combination aiming at the corn SSR molecular marker combination in claim 1.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101545004A (en) * | 2008-03-28 | 2009-09-30 | 上海市农业科学院 | Waxy maize inbred line Shen-W22 specific molecular marker and application thereof in offspring variety identification |
| CN105132420A (en) * | 2015-09-23 | 2015-12-09 | 北京市农林科学院 | Complete set of primers for identifying purity of maize varieties and application |
| CN106326962A (en) * | 2016-08-15 | 2017-01-11 | 安徽农业大学 | Corn two-dimensional code molecule identity card and construction method therefor |
| CN107513567A (en) * | 2017-09-15 | 2017-12-26 | 中国农业科学院作物科学研究所 | The construction method of chick-pea SSR finger-prints and application |
-
2018
- 2018-05-21 CN CN201810488914.5A patent/CN108384884B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101545004A (en) * | 2008-03-28 | 2009-09-30 | 上海市农业科学院 | Waxy maize inbred line Shen-W22 specific molecular marker and application thereof in offspring variety identification |
| CN105132420A (en) * | 2015-09-23 | 2015-12-09 | 北京市农林科学院 | Complete set of primers for identifying purity of maize varieties and application |
| CN106326962A (en) * | 2016-08-15 | 2017-01-11 | 安徽农业大学 | Corn two-dimensional code molecule identity card and construction method therefor |
| CN107513567A (en) * | 2017-09-15 | 2017-12-26 | 中国农业科学院作物科学研究所 | The construction method of chick-pea SSR finger-prints and application |
Non-Patent Citations (5)
| Title |
|---|
| 中国玉米新品种DNA指纹库建立系列研究*Ⅱ.适于玉米自交系和杂交种指纹图谱绘制的SSR核心引物的确定;赵久然等;《玉米科学》;20030228;第11卷(第2期);摘要，第3页第1段,第1.2节,第3节 * |
| 基于荧光SSR标记的玉米自交系遗传结构解析;张全芳等;《植物遗传资源学报》;20170131;第18卷(第1期);摘要，表3，第1.2节、1.3节 * |
| 张全芳等.基于荧光SSR标记的玉米自交系遗传结构解析.《植物遗传资源学报》.2017,第18卷(第1期),摘要，第1.2、1.3节，表3. * |
| 玉米通用SSR核心引物筛选及高通量多重PCR复合扩增体系建立;王凤格;《科学通报》;20061231;第51卷(第23期);第2738-2746页 * |
| 赵久然等.中国玉米新品种DNA指纹库建立系列研究*Ⅱ.适于玉米自交系和杂交种指纹图谱绘制的SSR核心引物的确定.《玉米科学》.2003,第11卷(第2期),第3-5、8页. * |
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