CN110885895A - Molecular marking method of rice gelatinization temperature gene ALK and special primer thereof - Google Patents
Molecular marking method of rice gelatinization temperature gene ALK and special primer thereof Download PDFInfo
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Abstract
The invention relates to the technical field of molecular biology, and discloses a molecular marking method of a rice gelatinization temperature gene ALK, which comprises the following steps: s1, extracting the genome DNA of the rice plant; s2, PCR amplification: simultaneously adding the primers into a PCR reaction system for amplification; s3, amplifying the ALK gene in the rice plant; s4, rapidly detecting the PCR product, reading a fluorescence intensity signal value, and then automatically genotyping to obtain a genotype result; s5, analyzing according to the fluorescence signal value; its special primer is also disclosed. According to the invention, an amplification hindered system formed by 5 primers is utilized, a detection sample is amplified only by one-time PCR (polymerase chain reaction), electrophoresis detection is not needed, amplification data is directly obtained on an original plate by using a fluorescence scanner, and genotype data of a rice gelatinization temperature gene ALK is obtained through software analysis. The invention can detect the growth of rice at any period and realize real closed-tube operation.
Description
Technical Field
The invention relates to the technical field of molecular biology, in particular to a fluorescent molecular marker of a gene ALK for controlling the characteristics of rice gelatinization temperature, cooking quality and the like and a special primer thereof.
Background
Rice is an important food crop, more than 100 countries in the world grow rice, and 2/3 people take rice as staple food. With the development of economy and the improvement of the living standard of people, the requirements of markets and consumers on the rice quality are higher and higher. The rice quality mainly comprises processing quality, appearance quality, cooking taste quality and nutrition quality, and specifically comprises transparency, chalkiness, grain type, amylose content, gel consistency, gelatinization temperature, protein content, aroma and the like. Gelatinization temperature is an important index for evaluating rice cooking quality next to amylose content, and many documents report that the character is controlled by a main effective gene, so that the cloning of the rice gelatinization temperature gene is beneficial to the molecular improvement of rice quality. The rice gelatinization temperature gene ALK is successfully separated by researchers of China Rice institute by using a map-based cloning method and is positioned near the Wx gene of the 6 th chromosome short arm of rice, and sequence analysis shows that the gene encodes soluble starch synthase II. Further comparing the DNA sequence of the gene between different varieties and the analysis result of the alkali digestion method, the base substitution in the coding region of the ALK gene is presumed to possibly cause the change of the crystal layer structure of the amylopectin, thereby causing the change of the gelatinization temperature. GC329 variation on 4329bp of an ALK gene sequence is a key factor influencing the gelatinization temperature, and the gelatinization temperature of TT type materials is lower, while the gelatinization temperature of GC type materials is higher.
The gelatinization temperature of rice is an important cooking quality character of rice, is a complex quantitative character controlled by a plurality of micro-effect genes together, is very easily influenced by other factors such as environment and the like, is difficult to be accurately selected directly through phenotype in rice breeding, can be identified after the rice gelatinization temperature is equal to mature harvest, needs to be rough before identification, and is time-consuming and labor-consuming. Different allelic gene types of the target gene can be rapidly and accurately identified by designing the functional marker of the target gene to carry out molecular marker-assisted selection, and a single plant containing the target gene can be rapidly and accurately screened.
Therefore, the obtained ALK gene functional marker can quickly and accurately identify different genotypes of a target gene, and is an important precondition for developing ALK gene molecular marker assisted selection.
The current molecular marker technical method for detecting gene SNP variation similar to the invention mainly comprises 2 methods: 1 based on the restriction enzyme digestion and agarose electrophoresis after PCR amplification, designing primers according to two sides of a connecting region of SNP variation of rice genes, digesting an amplification product by using restriction endonuclease after PCR amplification, and observing the digested product under UV light after dyeing. 2 designing three primers, amplifying to obtain fragments with different lengths by primer mismatching, detecting by polyacrylamide gel, and judging the genotype at the position of an observation band after silver staining and color development. Related patents for developing functional molecular markers of rice gelatinization temperature gene ALK are not searched.
However, in the prior art: 1. the technical method for detecting the SNP variation of the gene based on the enzyme digestion after the PCR amplification and the agarose electrophoresis comprises the steps of carrying out the enzyme digestion and transferring to the agarose electrophoresis detection after the PCR amplification is finished, and then observing and counting by using a gel imaging system. 2. Genotypes were distinguished by primer mismatch amplification of different fragment sizes. Since the amplified fragments have small differences, the band differences must be detected by running the gel on a high resolution polyacrylamide gel. The preparation process of polyacrylamide gel is complex, and the used reagents acrylamide and methylene bisacrylamide are nerve agents and have great harm to human bodies. After gel electrophoresis, silver nitrate and formaldehyde are needed for dyeing, and the reagents are toxic reagents and carcinogens and have great harm to the environment and human bodies.
Disclosure of Invention
The invention aims to provide a molecular marking method of a rice gelatinization temperature gene ALK and a special primer thereof, which can realize the rapid detection of allelic variation types of the ALK gene in rice varieties by a simple and reliable method, provide ALK gene functional molecular marking primers for screening two genotypes of high gelatinization temperature (GC) and low gelatinization temperature (TT) of rice, and solve the problem that the development of a functional marker of the rice gelatinization temperature gene ALK is convenient for the application of the functional marker in rice breeding.
In order to achieve the purpose, the invention provides the following technical scheme:
a special molecular marker primer for a rice gelatinization temperature gene ALK comprises two general primers:
# 1: GAAGGTGACCAAGTTCATGCT, respectively; # 2: GAAGGTCGGAGTCAACGGATT, respectively; also comprises 3 specific primers of ALK gene:
forward primer ALK-Ftt:
GAAGGTGACCAAGTTCATGCTTACAAGGAGAGCTGGAGGGGTT；
forward primer ALK-Fgc:
GAAGGTCGGAGTCAACGGATTTACAAGGAGAGCTGGAGGGGGC；
the reverse primer ALK-R: CTGAGGTCCTGCGACATGC are provided.
A molecular marking method of rice gelatinization temperature gene ALK comprises the following steps:
s1, extracting the genome DNA of the rice plant;
s2, PCR amplification: adding the 3 primers ALK-Ftt, ALK-Fgc, ALK-R and two general primers # 1 and #2 into a PCR reaction system at the same time for amplification;
s3, amplifying the ALK gene in the rice plant;
s4, rapidly detecting the PCR product in an enzyme labeling instrument containing three fluorescence detection channels of FAM, HEX and ROX, reading a fluorescence intensity signal value, and then automatically genotyping the fluorescence signal value file by combining the labeling information through an SNP decoder tool to obtain a genotype result;
s5, analyzing according to the fluorescence signal value, and if HEX fluorescence signals are obtained by scanning, determining the HEX fluorescence signals to be TT genotypes with low gelatinization temperature; if FAM fluorescence signals are obtained by scanning, the FAM fluorescence signals are GC allelic type with high gelatinization temperature; the result of the scan is a red signal, which indicates that the sample is in a heterozygous state.
As a further scheme of the invention: in S2, the PCR reaction system was 10 μ L: 5 μ L of 2 XPAMS mastermix, 0.15 μ L of 10mM ALK-Ftt marker primer, 0.15 μ L of 10mM ALK-Fgc marker primer, 0.4 μ L of 10mM ALK-R universal reverse primer, 1 μ L of template DNA, 3.3 μ L of ddH2O。
As a still further scheme of the invention: in S3, the PCR reaction procedure is: 94 ℃ for 3 min; then 10 cycles at 94 ℃, 20sec, 65 ℃ (-0.8 ℃ per cycle), 1 min; then 30 cycles of 94 ℃, 20sec, 57 ℃, 1 min.
Compared with the prior art, the invention has the beneficial effects that:
an amplification hindered system formed by 5 primers is utilized, a detection sample is subjected to PCR amplification only once without electrophoresis detection, amplification data is directly obtained on an original plate by using a fluorescence scanner, and genotype data of the rice ALK is obtained through software analysis. The invention can detect rice at any growth period, and has the advantages of simple and rapid operation, low cost, accurate result and realization of real closed-tube operation. Therefore, while solving the disadvantages of long time consumption, complicated process, low efficiency and toxic substance use of the currently used method, the functional marker of the rice gelatinization temperature gene ALK is successfully developed, which is convenient for the application of the ALK gene in rice breeding.
Drawings
FIG. 1 is a schematic diagram of rice gelatinization temperature gene ALK allele amplification.
FIG. 2 is a diagram of the results of the genotyping of 93 rice parent varieties by the molecular marking method of rice gelatinization temperature gene ALK.
FIG. 3 is a schematic representation of the TT genotype at low gelatinization temperature in the present invention.
FIG. 4 is a schematic representation of GC alleles at high gelatinization temperature in the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
A functional molecular marker PM-ALK4329 of a rice gelatinization temperature gene ALK is composed of three primers:
1. forward primer ALK-Ftt:
GAAGGTGACCAAGTTCATGCTTACAAGGAGAGCTGGAGGGGTT(3#)；
2. forward primer ALK-Fgc:
GAAGGTCGGAGTCAACGGATTTACAAGGAGAGCTGGAGGGGGC(4#)；
3. the reverse primer ALK-R: CTGAGGTCCTGCGACATGC (5 #).
The forward primer ALK-Ftt end corresponds to TT variant gene type with low gelatinization temperature, and the forward primer ALK-Fgc end corresponds to GC variant gene type with high gelatinization temperature.
Two universal primers are also included, which are identical to the underlined parts of the two forward primers, respectively, and the tails of the two universal primers are labeled with different fluorescent labels:
#1：GAAGGTGACCAAGTTCATGCT-FAM，
#2：GAAGGTCGGAGTCAACGGATT-HEX。
the sequence of the #1 marker is identical to the underlined part of primer ALK-Ftt, and the sequence of the #2 marker is identical to the underlined part of primer ALK-Fgc. The primer design for PCR amplification is shown in FIG. 1 (note: #1, Allle 1HEX fluorescence universal primer; #2, Allle 2FAM fluorescence universal primer; #3, SNP Allle 1 specific amplification primer (marker primer ALK-Ftt); #4, SNPallele 2 specific amplification primer (marker primer ALK-Fgc); #5, rice DNA template shown by the universal reverse primer (marker primer ALK-R) is amplified, HEX fluorescence signal obtained by fluorescence scanning is low gelatinization temperature material, FAM fluorescence signal obtained by fluorescence scanning is high gelatinization temperature material.
By adopting the technical scheme, 3 marked specific primers designed according to the ALK gene sequence and two general fluorescent primers are simultaneously added into a PCR reaction system for amplification. The ALK allele sequence can be matched with a forward primer ALK-Ftt according to SNP difference to obtain a HEX fluorescence signal value through amplification; and matching the forward primer ALK-Fgc to amplify to obtain a FAM fluorescence signal value. If the rice sample is in a heterozygous state at the locus, the two forward primers are simultaneously amplified, the variety is marked as red in the typing result chart, and the analysis result is shown in the chart 2 (note: the variety containing GC genotype is detected by FAM fluorescence signals (right-side point), the rice variety containing TT genotype is detected by HEX fluorescence signals (left-side point), and gray dots are negative control CK).
The functional marker of the rice gelatinization temperature gene ALK is subjected to molecular marker detection by adopting the following steps:
s1, extracting the genome DNA of the rice plant;
s2, PCR amplification: the 3 primers ALK-Ftt, ALK-Fgc and ALK-R and the two universal primers # 1 and #2 are added into a PCR reaction system at the same time, and the PCR reaction system is 10 muL: mu.L of 2 XPAMS master mix (the reagent is purchased from Wuhan city scenery peptide Biotechnology Co., Ltd., mainly comprises 2 universal fluorescent primers, buffer, DNTP and Taq enzyme, internal standard ROX and the like), 0.15. mu.L of 10mM ALK-Ftt labeled primer, 0.15. mu.L of 10mM ALK-Fgc labeled primer, 0.4. mu.L of 10mM ALK-R universal reverse primer, 1. mu.L of template DNA, and 3.3. mu.L of ddH 2O.
S3, amplifying the ALK gene in the rice plant; the PCR reaction program is as follows: 94 ℃ for 3 min; then 10 cycles at 94 ℃, 20sec, 65 ℃ (-0.8 ℃ per cycle), 1 min; then 30 cycles of 94 ℃, 20sec, 57 ℃ and 1 min;
s4, rapidly detecting the PCR product in an enzyme labeling instrument containing three fluorescence detection channels of FAM, HEX and ROX, reading a fluorescence intensity signal value, and then automatically genotyping by passing a fluorescence signal value file through an SNP decoder (http:// www.snpway.com/snpdecoder01/) tool in combination with the labeling information to obtain a genotype result;
s5, analyzing according to the fluorescence signal value, and if HEX fluorescence signals are obtained by scanning, determining the HEX fluorescence signals to be TT genotypes with low gelatinization temperature; if FAM fluorescence signals are obtained by scanning, the FAM fluorescence signals are GC allelic type with high gelatinization temperature; the result of the scan is a red signal, which indicates that the sample is in a heterozygous state.
Example (c): detection of ALK alleles of 93 rice parent varieties by using molecular marker PM-ALK4329
The PCR reaction system is 10 ul: mu.L of 2 XPAMS master mix (containing 2 universal fluorescent primers), 0.15. mu.L of 10mM ALK-Ftt labeled primer, 0.15. mu.L of 10mM ALK-Fgc labeled primer, 0.4. mu.L of 10mM ALK-R universal reverse primer, 1. mu.L of template DNA, 3.3. mu.L of ddH 2O. PCR reaction procedure: 94 ℃ for 3 min; then 10 cycles at 94 ℃, 20sec, 65 ℃ (-0.8 ℃ per cycle), 1 min; then 30 cycles of 94 ℃, 20sec, 57 ℃, 1 min. The amplified product is rapidly detected in a microplate reader containing three fluorescence detection channels of FAM, HEX and ROX, the fluorescence intensity signal value is read, and then the fluorescence signal value file is automatically genotyped by an SNP decoder (http:// www.snpway.com/snpdecoder01/) tool in combination with the labeling information, so as to obtain the genotype result, as shown in FIG. 3 and FIG. 4.
The specific implementation steps are as follows:
(1) extraction of genomic DNA from Rice
93 parts of rice leaves planted in Nanning are respectively taken, and genomic DNA of rice is obtained by a CTAB extraction method (a hexadecyl trimethyl ammonium bromide method);
(2) PCR amplification
The PCR reaction system is 10 ul: mu.L of 2 XPAMS master mix, 0.15. mu.L of 10mM ALK-Ftt labeled primer, 0.15. mu.L of 10mM ALK-Fgc labeled primer, 0.4. mu.L of 10mM ALK-R universal reverse primer, 1. mu.L of template DNA, 3.3. mu.L of LddH 2O. PCR reaction procedure: 94 ℃ for 3 min; then 10 cycles at 94 ℃, 20sec, 65 ℃ (-0.8 ℃ per cycle), 1 min; then 30 cycles of 94 ℃, 20sec, 57 ℃ and 1 min;
(3) rapidly detecting the PCR product in an enzyme labeling instrument comprising three fluorescence detection channels of FAM, HEX and ROX, reading a fluorescence intensity signal value, and then automatically carrying out genotyping on a fluorescence signal value file through an SNP decoder (http:// www.snpway.com/snpdecoder01/) tool in combination with marking information to obtain a genotype result;
(4) analyzing according to the fluorescence signal value, and if HEX fluorescence signals are obtained by scanning, determining the HEX fluorescence signals to be TT genotypes with low gelatinization temperature; if FAM fluorescence signals are obtained by scanning, the FAM fluorescence signals are GC allelic type with high gelatinization temperature; the result of the scan is a red signal, which indicates that the sample is in a heterozygous state.
(5) Results and analysis
The gene typing detection of the molecular markers of 93 rice parent varieties is shown in FIG. 2, FIG. 3 and FIG. 4. As can be seen from the figure: the green dots that detect the HEX fluorescence signal are the parent material of the TT genotype at low gelatinization temperature, while the blue dots that detect the FAM fluorescence signal are the parent material of the GC allele at high gelatinization temperature. Parent material without heterozygote
The specificity of the functional marker of the rice gelatinization temperature gene ALK will be described below with reference to examples.
Example (b):
purpose of the experiment: 93 rice parent materials are detected to be TT or GC types of ALK genes, and the accuracy of the scheme is verified by combining the phenotypic analysis of the detected materials.
The implementation scheme is as follows: according to literature reports, GC → TT variation on 4329bp influencing ALK gene sequence is a key factor influencing rice gelatinization temperature, and TT type material has lower gelatinization temperature and higher GC type gelatinization temperature.
The invention discloses a fluorescent molecular marker for distinguishing allele types of TT or GC two ALK genes, which is applied to 93 collected rice parent materials for detection, and the detection results are shown in a figure 2, a figure 3 and a figure 4. Simultaneously, the gelatinization temperature (alkali digestion value) of the corresponding parent material is measured, and the combined statistics is carried out with the detection result of the fluorescence marker, and the statistical result shows that the molecular marker is consistent with the phenotype result: the rice parent material fluorescence signal with the alkali extinction value higher than 6.0 (including 6.0) is HEX (TT), and the rice parent material fluorescence signal with the alkali extinction value lower than 6.0 (not including 6.0) is FAM (GC) (Table 1). The method can be applied to efficiently and accurately breeding the rice varieties with low gelatinization temperature, and greatly saves time and workload.
TABLE 193 parts of rice parent material for alkali digestion and PM-ALK4329 detection typing
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.
Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.
Claims (4)
1. A special molecular marker primer for a rice gelatinization temperature gene ALK comprises two general primers:
# 1: GAAGGTGACCAAGTTCATGCT, respectively; # 2: GAAGGTCGGAGTCAACGGATT, characterized in that,
also comprises 3 ALK gene specific primers:
forward primer ALK-Ftt:
GAAGGTGACCAAGTTCATGCTTACAAGGAGAGCTGGAGGGGTT；
forward primer ALK-Fgc:
GAAGGTCGGAGTCAACGGATTTACAAGGAGAGCTGGAGGGGGC；
the reverse primer ALK-R: CTGAGGTCCTGCGACATGC are provided.
2. A molecular marking method of rice gelatinization temperature gene ALK is characterized by comprising the following steps:
s1, extracting the genome DNA of the rice plant;
s2, PCR amplification: simultaneously adding the 3 primers ALK-Ftt, ALK-Fgc and ALK-R as well as the two universal primers #1 and #2 in the PCR reaction system for amplification;
s3, amplifying the ALK gene in the rice plant;
s4, rapidly detecting the PCR product in an enzyme labeling instrument containing three fluorescence detection channels of FAM, HEX and ROX, reading a fluorescence intensity signal value, and then automatically genotyping the fluorescence signal value file by combining the labeling information through an SNP decoder tool to obtain a genotype result;
s5, analyzing according to the fluorescence signal value, and if HEX fluorescence signals are obtained by scanning, determining the HEX fluorescence signals to be TT genotypes with low gelatinization temperature; if FAM fluorescence signals are obtained by scanning, the FAM fluorescence signals are GC allelic type with high gelatinization temperature; the result of the scan is a red signal, which indicates that the sample is in a heterozygous state.
3. A method as claimed in claim 2The molecular marking method of the rice gelatinization temperature gene ALK is characterized in that in S2, the PCR reaction system is 10 muL: 5. mu.L of 2 XPAMS master mix, 0.15. mu.L of 10mM ALK-Ftt labeled primer, 0.15. mu.L of 10mM ALK-Fgc labeled primer, 0.4. mu.L of 10mM ALK-R universal reverse primer, 1. mu.L of template DNA, 3.3. mu.L of ddH2O。
4. The molecular marking method of rice gelatinization temperature gene ALK as claimed in claim 2, wherein in S3, the PCR reaction program is: 94 ℃ for 3 min; then 10 cycles of 94 ℃, 20sec, 65 ℃ and-0.8 ℃ are carried out for 1min per cycle; then 30 cycles of 94 ℃, 20sec, 57 ℃, 1 min.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111621591A (en) * | 2020-07-10 | 2020-09-04 | 四川农业大学 | Molecular marker for detecting rice alkali elimination value and detection method |
| CN114480720A (en) * | 2022-03-16 | 2022-05-13 | 四川省农业科学院作物研究所 | Molecular marker of rice gelatinization temperature gene ALK, primer and application thereof |
| CN114717348A (en) * | 2021-01-05 | 2022-07-08 | 中国科学院分子植物科学卓越创新中心 | Gene marker for distinguishing rice starch quality |
| CN114480720B (en) * | 2022-03-16 | 2024-06-04 | 四川省农业科学院作物研究所 | Molecular marker of rice gelatinization temperature gene ALK, primer and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1570110A (en) * | 2003-07-21 | 2005-01-26 | 中国科学院遗传与发育生物学研究所 | Rice grain gelatinization temperature main control gene ALK and its uses |
| CN104789558A (en) * | 2015-03-23 | 2015-07-22 | 华南农业大学 | Molecular marker for rice gelatinization temperature control gene and application thereof |
| JP2017038588A (en) * | 2015-08-18 | 2017-02-23 | 公立大学法人秋田県立大学 | Variant of oryza sativa l., rice flour, resistant starch, food, and production method of variant of oryza sativa l. |
| CN109735653A (en) * | 2019-03-15 | 2019-05-10 | 广西壮族自治区农业科学院 | The fluorescent molecule label of rice waxy gene Wx a kind of and its application |
-
2019
- 2019-11-01 CN CN201911061242.0A patent/CN110885895A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1570110A (en) * | 2003-07-21 | 2005-01-26 | 中国科学院遗传与发育生物学研究所 | Rice grain gelatinization temperature main control gene ALK and its uses |
| CN104789558A (en) * | 2015-03-23 | 2015-07-22 | 华南农业大学 | Molecular marker for rice gelatinization temperature control gene and application thereof |
| JP2017038588A (en) * | 2015-08-18 | 2017-02-23 | 公立大学法人秋田県立大学 | Variant of oryza sativa l., rice flour, resistant starch, food, and production method of variant of oryza sativa l. |
| CN109735653A (en) * | 2019-03-15 | 2019-05-10 | 广西壮族自治区农业科学院 | The fluorescent molecule label of rice waxy gene Wx a kind of and its application |
Non-Patent Citations (4)
| Title |
|---|
| J. S. BAO等: ""Nucleotide diversity in starch synthase IIa and validation of single nucleotide polymorphisms in relation to starch gelatinization temperature and other physicochemical properties in rice (Oryza sativa L.)"", 《THEOR APPL GENET》 * |
| JUAN HUANG等: ""Genetic editing of GS3 via CRISPR/Cas9 Accelerates the Breeding of Three-line Hybrid Rice With Superior Yield and Grain Quality"", 《RESEARCH SQUARE》 * |
| YING ZHOU等: ""Nucleotide Diversity and Molecular Evolution of the ALK Gene in Cultivated Rice and its Wild Relatives"", 《PLANT MOL BIOL REP》 * |
| 肖鹏等: ""稻米糊化温度的遗传与分子机理研究进展"", 《中国农业科技导报》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111621591A (en) * | 2020-07-10 | 2020-09-04 | 四川农业大学 | Molecular marker for detecting rice alkali elimination value and detection method |
| CN114717348A (en) * | 2021-01-05 | 2022-07-08 | 中国科学院分子植物科学卓越创新中心 | Gene marker for distinguishing rice starch quality |
| CN114717348B (en) * | 2021-01-05 | 2023-09-12 | 中国科学院分子植物科学卓越创新中心 | Gene marker for distinguishing rice starch quality |
| CN114480720A (en) * | 2022-03-16 | 2022-05-13 | 四川省农业科学院作物研究所 | Molecular marker of rice gelatinization temperature gene ALK, primer and application thereof |
| CN114480720B (en) * | 2022-03-16 | 2024-06-04 | 四川省农业科学院作物研究所 | Molecular marker of rice gelatinization temperature gene ALK, primer and application thereof |
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Application publication date: 20200317 |