CN107299151B - DNA molecular marker for identifying rice fatty acid quality and application thereof - Google Patents
DNA molecular marker for identifying rice fatty acid quality and application thereof Download PDFInfo
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Abstract
The invention discloses a DNA molecular marker for identifying rice fatty acid quality, and develops a pair of primers (SEQ ID NO:1-2) for rice linolenic acid quality genotyping based on a PCR technology, wherein the rice linolenic acid quality gene is OsFAD 3. The gene marker and the primer thereof provided by the invention can be used for quickly detecting the allelic variation in the gene OsFAD3 which influences the linolenic acid content of rice in rice materials, and have the advantages of simple operation method and low cost. The DNA molecular marker of the invention can distinguish the rice genotypes with different linolenic acid contents, and the detection result is accurate and reliable; the method can effectively utilize the gene OsFAD3 functional marker to carry out molecular marker assisted breeding and carry out genetic improvement on the quality of the linolenic acid of the rice.
Description
Technical Field
The invention relates to the technical field of molecular genetic breeding, in particular to a DNA molecular marker for identifying the fatty acid quality of rice and application thereof.
Background
α -linolenic acid is one of essential fatty acids, has important effects in maintaining lipoprotein balance, reducing blood lipid, regulating cholesterol metabolism, lowering blood pressure, resisting thrombosis, and preventing canceration, and is a health product (Shimian Rubia 2014; Kim et al 2014).
α -linolenic acid can be converted into eicosapentaenoic acid (EPA) in human body, and then converted into docosahexaenoic acid (DHA) through β -oxidation, and EPA and DHA are involved in reducing platelet aggregation, inhibiting vasoconstriction and thrombosis, multiple dietary supplement α -linolenic acid test results related to heart protection show that α -linolenic acid has better prevention effect on cardiovascular diseases as a nutrient element (Gladine et al, 2013; Nozue et al, 2013; De Goede et al, 2013).
Human body can not convert linoleic acid into α -linolenic acid due to lack of omega-3 Fatty Acid Dehydrogenase (FAD), so it must supplement α -linolenic acid by diet (Yadav et al, 1993). Studies show that a class of omega-3 fatty acid dehydrogenases (FAD3) participate in the conversion of plant linoleic acid and linolenic acid, and influence the synthesis of linolenic acid.A significant decrease in the content of linolenic acid in seeds of Arabidopsis thaliana FAD3 mutant (James and Dooner, 1990; Lemieux et al, 1990), whereas overexpression of FAD3 or tissue-specific expression of FAD3 protein can significantly increase the content of linolenic acid (Damude et al, 2006; Eckert et al, 2006; Puttick et al, 2009).
Rice is an important food crop, more than half of the world population uses rice as staple food, the rice contains a plurality of fatty acids beneficial to human bodies, but α -linolenic acid content is low, so that the improvement of α -linolenic acid content in the rice has great significance for promoting the physical health of people using the rice as staple food, research shows that the rice OsFAD3 gene has activity of omega-3 fatty acid dehydrogenase, OsFAD3 is expressed in a tobacco root system, the α -linolenic acid content in the root system (Kodama et al, 1997) can be obviously improved, rice has rich germplasm resources, the content variation of α -linolenic acid in different rice varieties is wide and is greatly influenced by environment, currently, because the content determination needs special instruments such as liquid chromatography and other detection technologies, the cost is high, the operation is not easy, therefore, the gene marker is developed according to the specific sequence of the rice OsFAD3 gene, the relationship between different gene types of the OsFAD and the rice 493 23- α gene is identified, and the marker is developed for improving the rice molecular marker for assisting the rice molecular marker in breeding and selecting the rice marker for improving the linolenic acid content.
Disclosure of Invention
The invention aims to develop a functional molecular marker capable of identifying the quality of linoleic acid/linolenic acid of rice, apply the functional molecular marker to screening and identification of rice breeding materials, increase the accuracy of detecting α -linolenic acid of rice in a breeding improvement process and improve the selection efficiency.
In order to achieve the purpose of the invention, the DNA molecular marker for identifying the fatty acid quality of rice is positioned in the 5' UTR region of the rice OsFAD3 gene (MSU accession number is Loc _ Os11g01340), and the nucleotide sequence of the DNA molecular marker is as follows: 5 '-AGGAAGGA-3'.
The content of the linolenic acid in the rice produced by the rice plant fructification containing the DNA molecular marker is obviously lower than that of the rice produced by the rice plant lacking the DNA molecular marker; the ratio of the content of linoleic acid to the content of linolenic acid in the rice produced by the fructification of the rice plant containing the DNA molecular marker is obviously higher than that of the rice produced by the rice plant lacking the DNA molecular marker.
The invention also provides a PCR primer pair for amplifying the nucleic acid fragment containing the DNA molecular marker, which comprises:
OSFA3F:5′-GGCGGAAAGGGAGAAGAG-3′(SEQ ID NO:1)
OSFA3R:5′-TAGGGACATAGCAAAGCAAAGG-3′(SEQ ID NO:2)
the invention also provides a kit containing the primer pair and used for detecting the DNA molecular marker.
The invention also provides application of the primer pair or the kit in rice linoleic acid/linolenic acid quality genotyping. The method comprises the following steps:
1) extracting DNA of a rice sample to be detected;
2) performing PCR amplification by using the primers OSFA3F and OSFA3R and using the DNA as a template;
3) analyzing the PCR amplification product.
The PCR reaction system is as follows: DNA template 100ng, 10 XPCR buffer 2ul, 2ul of 2mM dNTP mix, 0.3ul of each of 10uM primers OSFA3F and OSFA3R, 0.1ul of rTaq DNA polymerase, ddH2And O is supplemented to 20 ul.
The PCR amplification program is set as follows: pre-denaturation at 94 ℃ for 4 min; denaturation at 94 ℃ for 30 seconds, annealing at 55 ℃ for 30 seconds, extension at 72 ℃ for 30 seconds, and circulation for 32 times; the reaction was terminated by extension at 72 ℃ for 7 minutes.
Performing electrophoresis detection on the PCR amplification product in 4% polyacrylamide gel, wherein if a characteristic strip with the size of 182bp appears in the electrophoresis result, the linolenic acid content of the rice to be detected is higher, and the ratio of the linoleic acid/linolenic acid content is lower; if a characteristic strip with the size of 190bp appears in the electrophoresis result, the linolenic acid content of the rice to be detected is lower, and the ratio of the linoleic acid/linolenic acid content is higher; and if the electrophoresis result shows the two characteristic bands, the rice to be detected is in a heterozygous type.
The invention also provides application of the DNA molecular marker in improving the quality of linoleic acid/linolenic acid of rice varieties.
The invention also provides application of the DNA molecular marker in identifying or breeding high-quality linoleic acid/linolenic acid rice varieties.
The invention further provides application of the DNA molecular marker in rice molecular marker assisted breeding.
Compared with the prior art, the invention has the following advantages:
the invention discovers a DNA molecular marker for identifying the quality of fatty acid of rice for the first time, and develops a pair of primers for genotyping the quality of linolenic acid of rice based on a PCR technology, wherein the linolenic acid quality gene of rice is OsFAD 3. The gene marker and the primer thereof provided by the invention can be used for quickly detecting the allelic variation in the gene OsFAD3 which influences the linolenic acid content of rice in rice materials, and have the advantages of simple operation method and low cost. The DNA molecular marker of the invention can distinguish the rice genotypes with different linolenic acid contents, and the detection result is accurate and reliable; the method can effectively utilize the gene OsFAD3 functional marker to carry out molecular marker assisted breeding and carry out genetic improvement on the quality of the linolenic acid of the rice.
Drawings
FIG. 1 is a gel diagram of the polymorphism of the marker OSFA3 in the rice parent in example 2 of the present invention, namely, the detection of the allelic form of OsFAD3 gene of different rice varieties by using the gene marker OSFA 3; wherein, from left to right, the rice is Zhenshan 97, Minghui 63, Nipponbare 9311, ACC9 (African cultivated rice), ACC10 (common wild rice), Zhonghua 11, KASALATH, CYPRESS, red rice, strain 233 and precocious black.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise indicated, the examples follow conventional experimental conditions, such as the Molecular Cloning handbook, Sambrook et al (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual,2001), or the conditions as recommended by the manufacturer's instructions.
Example 1 development of Gene markers for linolenic acid quality in Rice
1. According to the literature
Kodama H, Akagi H, Kusumi K, Fujimura T, Iba K. (1997), Structure, chromosol location and expression of a rice gene encoding the micro ω -3fatty acid desaturase plant Molecular Biology,33: 493-. The gene sequences of OsFAD3 of the sequenced germplasm were aligned between websites ricevarmap (http:// ricevarmap. ncpgr. cn) and RIGW (http:// rice. hzau. edu. cn/cgi-bin/gb2/gbrowse _ syn), and an 8bp (AGGAAGGA) insertion/deletion variation was found in the 5' UTR region of the OsFAD3 gene.
2. Downloading a gene sequence of OsFAD3 from an MSU website (http:// rice. plant biology. MSU. edu /), intercepting a sequence of 400bp upstream and downstream with the 8bp insertion deletion variation as a center, designing a primer by using software primer 5 to amplify a fragment comprising the 8bp insertion deletion site, wherein the designed gene marker is named as OSFA3, and the corresponding primer sequences are OSFA3F: 5'-GGCGGAAAGGGAGAAGAG-3' and OSFA3R: 5'-TAGGGACATAGCAAAGCAAAGG-3' respectively. The primers were synthesized by Shanghai Biopsis, Inc.
Example 2 detection of Gene marker OSFA3 and polymorphisms in Rice parents
1. Extracting DNA of rice samples to be detected including conventional rice varieties, common wild rice and African cultivated rice
2. The sample DNA was PCR-amplified using the designed gene marker OSFA3 to detect the polymorphism of the gene marker OSFA 3.
And (3) PCR reaction system: the total volume of 20ul included: DNA template 100ng, 2ul 10 XPCR buffer, 2ul 2mM dNTP mixture, 10uM primers OSFA3F and OSFA3R each 0.3ul, 0.1ul rTaq DNA polymerase. The PCR reaction was performed on a PCR thermal cycler. The PCR amplification program is set as follows: the reaction was terminated by pre-denaturation at 94 ℃ for 4 min, followed by denaturation at 94 ℃ for 30sec, annealing at 55 ℃ for 30sec, extension at 72 ℃ for 30sec, and cycling for 32 cycles, and final extension at 72 ℃ for 7 min.
The PCR product was electrophoresed in a 4% polyacrylamide gel for 40 minutes (electrophoresis parameters: voltage: 2000V, current: 200mA, power: 90W), developed with silver nitrate and sodium hydroxide solution and fixed. The strip pattern was read by taking a picture in front of the light box. The results listed in FIG. 1 were obtained (FIG. 1). As can be seen from FIG. 1, the gene OsFAD3 has polymorphism among different rice varieties, the gene marker OSFA3 has successful amplification, clear banding pattern, obvious polymorphism and moderate amplified fragment size (200bp), which indicates that the developed marker can successfully detect the genetic variation of different varieties.
A characteristic strip with the size of 182bp (SEQ ID NO:3) appears in the electrophoresis result, the linolenic acid content of the rice correspondingly to be detected is higher, and the ratio of the linoleic acid/linolenic acid content is lower; a characteristic strip with the size of 190bp (SEQ ID NO:4) appears in the electrophoresis result, the corresponding rice to be detected has lower linolenic acid content, and the ratio of the linoleic acid to the linolenic acid content is higher; and if the electrophoresis result shows the two characteristic bands, the rice to be detected is in a heterozygous type.
Example 3 application of Gene marker OSFA3 to linolenic acid quality selection in a Breeding population
In order to evaluate the effect of molecular marker-assisted selection of the quality of linolenic acid in rice by using the marker OSFA3, a breeding segregation population constructed by hybridization of Nippon japonica rice (NIP) and Zhenshan indica rice 97(ZS97) is analyzed as the marker OSFA3, and a genotype with high content of linoleic acid/linolenic acid in rice is selected. The application comprises the following steps:
1. DNA was extracted from 107 individuals.
2. The genotype of the OsFAD3 gene is detected by using a marker OSFA3, the gene type is consistent with the parent ZS97 and is marked as 11 (the size of an amplification product is 182bp), the gene type is consistent with the NIP gene and is marked as 22 (the size of the amplification product is 190bp), and the gene type containing both ZS97 and NIP gene is marked as heterozygous 12. The genotype of the marker OSFA3 among 107 individuals appeared as 11, 22, 12 genotype segmentations.
107 seeds of the single plants are harvested, and the contents of linoleic acid and linolenic acid in mature embryos are measured by using a gas chromatography-mass spectrometer. The anova results showed (table 1) that there was no difference in linoleic acid content between the 3 genotypes, while there was a significant difference in linolenic acid content and in the ratio of linoleic acid to linolenic acid. Multiple comparisons showed no significant difference in linolenic acid content and the ratio of linoleic acid to linolenic acid content between genotypes 11 and 12, while the difference in linolenic acid content and the ratio of linoleic acid to linolenic acid content between genotypes 22 and 12 was significant. Therefore, the gene marker OSFA3 can be used for well distinguishing genotypes with different linolenic acid qualities in breeding populations, and can be used for effectively selecting single plants with high linolenic acid content and low ratio of linoleic acid to linolenic acid content.
TABLE 1 analysis of OsFAD3 genotype and linolenic acid content of isolated population
Note: the capital letters A, B in table 1 indicate the significant level of difference of 0.01 for the Duncan test.
Example 4 application of marking OSFA3 in identification of linolenic acid content of natural rice germplasm material
The gene marker OSFA3 is used for identifying the linolenic acid content of natural rice germplasm, the ratio of linoleic acid to linolenic acid content and the like.
1. 52 parts of germplasm materials are selected, the genotype of OsFAD3 in the 52 parts of rice germplasm materials is analyzed by using a gene marker OSFA3, and simultaneously, the contents of linoleic acid and linolenic acid in mature embryos are determined by using a gas chromatography-mass spectrometer.
2. Marker OSFA3 divided 52 germplasm materials into two groups, one group consistent with the genotype of ZS97, designated 11, and the other group consistent with the genotype of NIP, designated 22. The analysis results of the contents of linolenic acid and the like of the two groups of germplasm materials show that the content difference of linoleic acid between the two genotypes is not obvious, and the content difference of the linolenic acid and the ratio of the contents of the linoleic acid and the linolenic acid is obvious, which shows that the gene marker OSFA3 can also effectively identify the content of the linolenic acid in natural germplasm groups of rice.
TABLE 2 analysis of OsFAD3 genotype and linolenic acid content in natural germplasm population
The fatty acid content determination method used in the present invention is described in the references (Garg et al, 2016) as follows:
(1) selecting 100 mature seeds, removing the glumes of the seeds by a brown rice machine (model: JLGL-45) to obtain brown rice, dividing the brown rice into seed embryos and endosperm by a scalpel, and respectively grinding the embryo tissues and the endosperm tissues into powder by liquid nitrogen;
(2) weighing 50mg of the powder, placing the powder in a 10ml glass tube with a cover, adding 100ul of fatty acid internal standard (0.04g of oleic acid internal standard C17: 0 dissolved in 100ml of chloroform) and 3ml of fatty acid extract 1(100ml of methanol: 10ml of saturated hydrochloric acid: 10ml of chloroform), covering the bottle, and placing the bottle in a 90 ℃ water bath kettle for water bath for 60 min;
(3) cooling to room temperature, adding 1ml of double distilled water and 2ml of fatty acid extract 2(400 ml of hexane: 100ml of chloroform), and shaking for 30 sec;
(4) placing the mixed solution in a centrifuge (JS-5.3), setting the revolution number to be 2000r/min, and setting the centrifugation time to be 10 min;
(5) the supernatant organic reagent is sucked, filtered by a 0.2um filter membrane and placed in a 1.5ml sample bottle. Storing at-20 deg.C, and standing for use;
(6) placing 1ml of sample in a Gas chromatography-mass spectrometer (Gas chromatography-Mass spectrometer-computer, GC-MS) (chromatographic column: SH Stabil wax DA) in sequence, setting the split ratio to be 1:10, setting the initial temperature of a column box to be 170 ℃, keeping for 1min, then heating to 230 ℃ at the speed of 3 ℃/min, and keeping for 3 min;
(7) and (3) utilizing GC-MS re-analysis software (a GC-MS analysis system) to carry out library screening retrieval on the appeared peaks, selecting a required target peak to carry out manual integration, and deriving an analysis result.
The invention has the following advantages:
1. the method provided by the invention is simple and rapid to operate, has low cost, and can be implemented in general biological laboratories.
2. The gene marker provided by the invention can accurately distinguish different allelic forms of the OsFAD3 gene in different rice materials, the content difference of linolenic acid between different genotypes is determined, and the detection result is accurate and reliable.
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.
Reference to the literature
Shimiarubia, α -linolenic acid improves the action and mechanism research of the hypertension insulin resisting rat vascular endothelial cell injury [ doctor academic thesis ]. fourth university of military medical science, 2014
Kim K,Nam Y,Kim H,Hayes A,Lee B.(2014).α-Linolenic acid:nutraceutical, pharmacological and toxicological evaluation.FoodChem.Toxicol.70:163-178.
Gladine C,Combe N,Vaysse C,et al.(2013).Optimized rapeseed oilenriched with healthy micronutrients:a relevant nutritional approach toprevent cardiovascular diseases.Results of the Optim'Oils randomizedintervention trial.J.Nutr. Biochem.24:544-549.
Nozue T,Yamamoto S,Tohyama S,et al.(2013).Effects of statins on serumn-3 to n-6polyunsaturated fatty acid ratios in patients with coronary arterydisease. J.Cardiovasc.Pharmacol.Ther.18:320-326.
De Goede J,Verschuren W,Boer J,Kromhout D,Geleijnse J.(2013).N-6andn-3fatty acid cholesteryl esters in relation to incident stroke in a Dutchadult population:a nested case-control study.Nutr.Metab.Cardiovasc.Dis.23:737-743.
Yadav N,Wierzbicki A,Aegerter M,et al.(1993).Cloning of higher plantx-3fatty acid desaturases.Plant Physiol.103:467-476.
James DW,Dooner HK.1990.Isolation of EMS-induced mutants inArabidopsis altered in seed fatty acid composition.Theor.Appl.Genet.80:241–245.
Lemieux B,Miquel M,Somerville C,Browse J.1990.Mutants of Arabidopsiswith alterations in seed lipid fatty acid composition.Theor.Appl.Genet.80:234–240.
Damude H,Zhang H,Farrall L,Ripp K,Tomb J,Hollerbach D,Yadav N.(2006).Identification of bifunctional D12/x-3fatty acid desaturases for improving theratio of x-3to x-6fatty acid in microbes and plants.Proc.Natl.Acad.Sci.USA.103:9446–9451.
Eckert H,LaVallee B,Schweiger B,Kinney A,Cahoon E,Clemente T.(2006).Co-expression of the borage D6desaturase and the Arabidopsis D15desaturaseresults in high accumulation of stearidonic acid in the seeds of transgenicsoybean.Planta 224:1050–1057.
Puttick D,Dauk M,Lozinsky S,Smith M.2009.Overexpression of aFAD3desaturase increases synthesis of a polymethyleneinterrupted dienoicfatty acid in seeds of Arabidopsis thaliana L.Lipids 44:753–757.
Kodama H,Akagi H,Kusumi K,Fujimura T,Iba K.(1997).Structure,chromosomal location and expression of a rice gene encoding the microsomeω-3fatty acid desaturase.Plant Mol.Biol.33:493-502
Sambrook J&Russell DW,Molecular Cloning:a Laboratory Manual,2001
Garg S,Rizhsky L,Jin H,Yu X,Jing F,Yandeau-Nelson M,Nikolau B.(2016).Microbial production of bi-functional molecules by diversification of thefatty acid pathway. Metab.Eng.35:9-20。
Sequence listing
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Claims (6)
1. The rice linoleic acid/linolenic acid quality genotyping method is characterized by comprising the following steps of:
1) extracting DNA of a rice sample to be detected;
2) performing PCR amplification by using the primers OSFA3F and OSFA3R and using the DNA as a template;
3) analyzing the PCR amplification product;
the sequences of primers OSFA3F and OSFA3R are as follows:
OSFA3F:5′-GGCGGAAAGGGAGAAGAG-3′
OSFA3R:5′-TAGGGACATAGCAAAGCAAAGG-3′。
2. the method of claim 1, wherein the PCR reaction system in step 2) is: DNA template 100ng, 10 XPCR buffer 2ul, 2ul of 2mM dNTP mix, 0.3ul of each of 10uM primers OSFA3F and OSFA3R, 0.1ul of rTaq DNA polymerase, ddH2Supplementing O to 20 ul;
the PCR amplification program is set as follows: pre-denaturation at 94 ℃ for 4 min; denaturation at 94 ℃ for 30 seconds, annealing at 55 ℃ for 30 seconds, extension at 72 ℃ for 30 seconds, and circulation for 32 times; the reaction was terminated by extension at 72 ℃ for 7 minutes.
3. The method according to claim 1 or 2, characterized in that, in the step 3), the PCR amplification product is detected by electrophoresis in 4% polyacrylamide gel, if a characteristic band with the size of 182bp appears in the electrophoresis result, the rice to be detected has high linolenic acid content, and the ratio of linoleic acid/linolenic acid content is lower; if a characteristic band with the size of 190bp appears in the electrophoresis result, the linolenic acid content of the rice to be detected is low, and the ratio of the linoleic acid/linolenic acid content is higher; and if the electrophoresis result shows the two characteristic bands, the rice to be detected is in a heterozygous type.
4. The application of the DNA molecular marker for identifying the fatty acid quality of rice in improving the quality of linoleic acid/linolenic acid of rice varieties; the DNA molecular marker is located in the 5' UTR region of the rice OsFAD3 gene, and the nucleotide sequence is as follows: 5 '-AGGAAGGA-3';
the content of the linolenic acid in the rice produced by the rice plant fructification containing the DNA molecular marker is obviously lower than that of the rice produced by the rice plant lacking the DNA molecular marker; the ratio of the content of linoleic acid to the content of linolenic acid in the rice produced by the fructification of the rice plant containing the DNA molecular marker is obviously higher than that of the rice produced by the rice plant lacking the DNA molecular marker.
5. The application of the DNA molecular marker for identifying the fatty acid quality of rice in identifying or breeding linoleic acid/linolenic acid high-quality rice varieties;
the DNA molecular marker is located in the 5' UTR region of the rice OsFAD3 gene, and the nucleotide sequence is as follows: 5 '-AGGAAGGA-3';
the content of the linolenic acid in the rice produced by the rice plant fructification containing the DNA molecular marker is obviously lower than that of the rice produced by the rice plant lacking the DNA molecular marker; the ratio of the content of linoleic acid to the content of linolenic acid in the rice produced by the fructification of the rice plant containing the DNA molecular marker is obviously higher than that of the rice produced by the rice plant lacking the DNA molecular marker.
6. The application of the DNA molecular marker for identifying the fatty acid quality of rice in the molecular marker-assisted breeding of linoleic acid/linolenic acid quality rice;
the DNA molecular marker is located in the 5' UTR region of the rice OsFAD3 gene, and the nucleotide sequence is as follows: 5 '-AGGAAGGA-3';
the content of the linolenic acid in the rice produced by the rice plant fructification containing the DNA molecular marker is obviously lower than that of the rice produced by the rice plant lacking the DNA molecular marker; the ratio of the content of linoleic acid to the content of linolenic acid in the rice produced by the fructification of the rice plant containing the DNA molecular marker is obviously higher than that of the rice produced by the rice plant lacking the DNA molecular marker.
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| CN1193357A (en) * | 1995-06-07 | 1998-09-16 | 林克斯治疗公司 | Oligonucleotide tags for sorting and identification |
| CN1230226A (en) * | 1996-06-06 | 1999-09-29 | 林克斯治疗公司 | Sequencing by ligation of encoded adaptors |
| CN101643784A (en) * | 2009-09-15 | 2010-02-10 | 江苏省农业科学院 | Molecule marking method for identifying paddy low glutelin gene Lgcl |
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2017
- 2017-08-28 CN CN201710751127.0A patent/CN107299151B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1193357A (en) * | 1995-06-07 | 1998-09-16 | 林克斯治疗公司 | Oligonucleotide tags for sorting and identification |
| CN1230226A (en) * | 1996-06-06 | 1999-09-29 | 林克斯治疗公司 | Sequencing by ligation of encoded adaptors |
| CN101643784A (en) * | 2009-09-15 | 2010-02-10 | 江苏省农业科学院 | Molecule marking method for identifying paddy low glutelin gene Lgcl |
Non-Patent Citations (2)
| Title |
|---|
| Identification and evaluation of v-3 fatty acid desaturase genes for hyperfortifying a-linolenic acid in transgenic rice seed;Hua Liang Liu et,al;《Journal of Experimental Botany》;20121231;第63卷(第8期);摘要、3280页 Table 1、第3258页 讨论 * |
| 水稻功能基因组育种数据库(RFGB):3K水稻SNP与InDel子数据库;郑天清等;《科学通报》;20151231;第60卷(第4期);第367-371页 * |
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