CN114317812B - Rice salt tolerance related molecular marker and application thereof - Google Patents
Rice salt tolerance related molecular marker and application thereof Download PDFInfo
- Publication number
- CN114317812B CN114317812B CN202210201003.6A CN202210201003A CN114317812B CN 114317812 B CN114317812 B CN 114317812B CN 202210201003 A CN202210201003 A CN 202210201003A CN 114317812 B CN114317812 B CN 114317812B
- Authority
- CN
- China
- Prior art keywords
- rice
- primer
- molecular marker
- seq
- salt
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Abstract
The invention discloses a molecular marker related to salt tolerance of rice and application thereof. The molecular marker is obtained by whole genome association analysis and comprises a single nucleotide base mutation at 1796216bp, a single nucleotide base mutation at 1797072bp, two nucleotide base mutations at 1797131bp and 1797132bp and 9 nucleotide base deletion at 1797233bp on the No. 3 chromosome of a rice reference genome. By utilizing the molecular marker provided by the invention, the rice can be quickly and accurately detectedOsDSK2aThe salt-tolerant genotype of the gene is beneficial to the high-efficiency application of the gene in the breeding of the salt-tolerant molecules of rice.
Description
Technical Field
The invention belongs to the technical field of crop genetic breeding, and particularly relates to a molecular marker related to salt tolerance of rice.
Background
The saline-alkali soil available in China is about 5.5 hundred million acres, has huge development potential and is an important potential cultivated land resource in China. These saline and alkaline lands are mainly distributed in the inland areas of northeast, north China and northwest and the coastal areas of the Yangtze river. The method is an important precondition for developing and utilizing land resources by cultivating saline-alkali tolerant crop varieties and scientifically improving saline-alkali soil, and the method is the most economic and effective means for accelerating the cultivation process of the saline-alkali tolerant crop varieties by excavating and identifying important saline-alkali tolerant genes of the crops and developing molecular markers.
Rice is one of the important grain crops in China, is highly sensitive to salt stress, and the growth, development and yield of the rice are severely limited under the condition of salt stress. The present discovery shows that the gene for improving the salt tolerance of rice does not necessarily keep the stability of the yield,OsDSK2ais a key gene for balancing the growth and development and the salt tolerance of rice, and the low-level expression of the gene can ensure that the rice maintains lower metabolic level so as to improve the adaptation to salt stress.OsDSK2aThe content of active gibberellin in rice is influenced by regulating the stability of gibberellin metabolism regulating factor EUI, the influence on the growth and development of plants is small after the function of the gibberellin metabolism regulating factor EUI is lost, but the salt tolerance of rice can be improved, so that the rice can be subjected to salt toleranceThe yield of rice remains stable under salt stress.
Since natural variation sites of many genes often lead to enhancement or weakening of the functions of the genes, excellent allelic variation can be used for crop molecular breeding, and molecular marker technology can realize rapid high-energy screening of excellent traits, the gene is widely used in crop genetic improvement. The salt tolerance of crops is a very complicated quantitative character controlled by multiple sites, but the excavation of the salt-tolerant molecular markers of rice is far from enough at present.
Disclosure of Invention
The invention aims to provide a molecular marker related to rice salt tolerance and application thereof, and provides gene resources for rice salt tolerance genetic improvement. The invention also provides a salt-tolerant gene for riceOsDSK2aThe SNP molecular marker and the INDEL molecular marker respectively correspond to a primer group and a detection method, and a sample can be accurately identified by simultaneously combining a KASP method and an electrophoresis detection technology.
A molecular marker related to salt tolerance of rice, wherein the molecular marker comprises SNP _3_1796216_ T/C, SNP _3_1797072_ T/C, SNP _3_1797131_ GG/AT and INDEL _3_1797233_ GTATGAGGC/-;
the molecular marker SNP _3_1796216_ T/C is located at 1796216bp on the No. 3 chromosome of rice, and has single nucleotide base mutation T/C;
the molecular marker SNP _3_1797072_ T/C is located at 1797072bp on the No. 3 chromosome of rice, and has single nucleotide base mutation T/C;
the molecular marker SNP _3_1797131_ GG/AT is located AT 1797131bp on the No. 3 chromosome of rice, and continuous two nucleotide base mutations GG/AT exist;
the molecular marker INDEL _3_1797233_ GTATGAGGC/-is located at the position 1797233bp on the No. 3 chromosome of rice, and 9 nucleotide bases are deleted.
The primer pair for detecting the molecular marker related to the salt tolerance of the rice comprises a primer pair for detecting the molecular marker SNP _3_1796216_ T/C, and comprises specific upstream primers X1F, Y1F and downstream universal primer C1R, wherein the primer sequences are respectively shown in a sequence table SEQ ID NO: 1-3;
the primer pair for detecting the molecular marker SNP _3_1797072_ T/C comprises a specific upstream primer X2F, a specific Y2F and a specific downstream universal primer C2R, wherein the primer sequences are respectively shown in a sequence table SEQ ID NO: 4-6;
the primer pair for detecting the molecular marker SNP _3_1797131_ GG/AT comprises specific upstream primers X3F, Y3F and downstream universal primer C3R, wherein the primer sequences are respectively shown in sequence tables SEQ ID NO: 7-9;
the primer pair for detecting the molecular marker INDEL _3_1797233_ GTATGAGGC/-comprises a specific upstream primer 4F and a specific downstream primer 4R, and the primer sequences are respectively shown in a sequence table SEQ ID NO: 10-11.
Rice geneOsDSK2aThe detection method of the salt-tolerant genotype takes the rice genome DNA as a template and utilizes the primer to carry out PCR reaction detection; if the first three groups of primers detect the nucleotide sequences of the primers SEQ ID NO: 1. SEQ ID NO: 4. SEQ ID NO: 7, the size of the PCR product detected by the fourth group of primers is 125bp, and the rice sample is judged to be homozygous and not salt-tolerantOsDSK2aThe genotype; if the first three groups of primers detect the nucleotide sequences of the primers SEQ ID NO: 2. SEQ ID NO: 5. SEQ ID NO: 8, the size of the PCR product detected by the fourth group of primers is 116bp, and the salt tolerance of the rice sample is determined to be homozygousOsDSK2aThe genotype; if the primer nucleotide sequences of SEQ ID NO: 1. the amino acid sequence of SEQ ID NO: 2. SEQ ID NO: 4. SEQ ID NO: 5. SEQ ID NO: 7. SEQ ID NO: 8 corresponding basic group and PCR products of 125bp and 116bp, and determining that the rice sample is heterozygous salt-resistantOsDSK2aThe genotype of the plant.
A kit for detecting the molecular marker comprises the primer.
Preferably, the SNP molecular marker specific upstream primer is respectively connected with FAM and/or HEX fluorescent joint sequences.
A gene chip comprises a nucleotide sequence shown as a sequence table SEQ ID NO: 1-11.
The rice salt tolerance related molecular marker is applied to rice breeding.
A rice breeding method, according to the detection method, the rice gene is detectedOsDSK2aDetecting the genotype of the plant, selecting the plant carrying salt toleranceOsDSK2aAnd carrying out subsequent breeding on the genotype rice sample.
The invention has the beneficial effects that: by using the inventionOsDSK2aThe molecular marker can quickly and accurately detect the salt-tolerant genes of the rice in germplasm resources of indica rice, japonica rice and the like and recombinant inbred line materials after hybridization of different parentsOsDSK2aThe genotype of (a). The SNP molecular marker and the INDEL molecular marker are utilized, and the invention can carry out large-scale identification on breeding groups through KASP technology or electrophoresis detection, accelerate the molecular breeding process and is beneficial to salt-tolerant genesOsDSK2aApplication in breeding.
Drawings
FIG. 1 shows rice genesOsDSK2aThe expression difference of different genotypes in different rice varieties.
FIG. 2 shows the application of the molecular marker in the identification of salt-tolerant materials of rice.
Detailed Description
In order that the invention may be more fully understood, reference will now be made to the following description. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
The test methods used in the examples are all conventional methods, and the used materials and yield data under salt stress are all stored or created by application units.
Example 1
Rice geneOsDSK2aThe molecular marker finds rice genes by analyzing yield data of rice core germplasm resources under salt stress and application thereofOsDSK2aSeveral polymorphic sites with salt tolerance, including 3 SNPs and 1 INDEL. The method comprises the following specific steps:
1. the molecular marker was primer-designed using the rice reference genome (os-Nipponbare-reference-IRGSp-1.0) sequence.
2. Sample detection
Extraction of rice leaf gene by CTAB methodThe group DNA is detected by adopting a KASP technology aiming AT a molecular marker SNP _3_1796216_ T/C, SNP _3_1797072_ T/C, SNP _3_1797131_ GG/AT, the KASP reaction is carried out on an LGC SNpline genotyping platform, and the reaction system is shown in Table 1. The PCR amplification conditions were: 15 minutes at 94 ℃; the annealing temperature is reduced by 0.6 ℃ for 10 cycles in each cycle at 94 ℃ for 20 seconds and 58 ℃ for 60 seconds; 26 cycles of 94 ℃ for 20 seconds and 55 ℃ for 60 seconds. After the reaction is finished, reading fluorescence data, and if the sample amplification product only detects the fluorescence signal corresponding to the primer X, the detection site is homozygousOsDSK2aSalt-tolerant genotype; if only the fluorescence signal corresponding to the primer Y is detected, the detection site is homozygousOsDSK2aA salt-tolerant genotype; if two fluorescent signals are detected simultaneously, the detection site is heterozygousOsDSK2aThe genotype of the plant. The molecular marker INDEL _3_1797233_ GTATGAGGC/-was detected by ordinary PCR, and the reaction system is shown in Table 2. The PCR amplification conditions were: 5 minutes at 95 ℃; 35 cycles of 95 ℃ for 10 seconds, 52 ℃ for 10 seconds and 68 ℃ for 10 seconds; the temperature is 68 ℃ for 5 minutes and the temperature is kept at 4 ℃. Electrophoresis detection, if the size of the PCR product is 125bp, the detection site is homozygous and salt-freeOsDSK2aThe genotype; if the size of the PCR product is 116bp, the salt tolerance of the homozygous detection site is realizedOsDSK2aThe genotype; if PCR products of 125bp and 116bp are detected simultaneously, the detection site is heterozygousOsDSK2aThe genotype of the plant.
TABLE 1 KASP reaction System
TABLE 2 PCR reaction System
3. Natural population verification
The molecular markers SNP _3_1796216_ T/C, SNP _3_1797072_ T/C, SNP _3_1797131_ GG/AT and the molecular markers INDEL _3_1797233_ GTATGAGGC/-were verified by using 10 parts of rice germplasm resource materials, and the detection results of the molecular markers are shown in Table 3, and 5 parts of materials are foundIs detected asOsDSK2aSalt tolerant genotype, and 5 additional materials areOsDSK2aSalt-tolerant genotype. Because of the fact thatOsDSK2aThe rice salt tolerance is reduced when the expression level is higher, andOsDSK2athe salt tolerance of rice is enhanced when the expression quantity is lower, so that the detection of 10 parts of materials is carried out by fluorescent quantitative PCROsDSK2aThe results are shown in FIG. 1 and containOsDSK2aIn materials not tolerant to salt genotypesOsDSK2aThe expression is higher than that of the total content ofOsDSK2aMaterials of salt-tolerant genotype. Therefore, the molecular marker can be used for riceOsDSK2aAnd (3) high-efficiency detection of salt-tolerant genotypes.
TABLE 3.10 germplasm resources materialsOsDSK2aMolecular marker typing data
4. Application of molecular marker in screening of salt-tolerant materials
The germ plasm resource quantity of natural colony is enlarged, and the molecular marker of the invention is utilized to carry out salt-tolerant gene of riceOsDSK2aAfter genotyping, further analysis using yield statistics under salt stress (0.4%) is performed, and the results are shown in FIG. 2, which containsOsDSK2aThe yield of the material without the salt genotype is lower than that of the material containing the salt genotypeOsDSK2aMaterials of salt-tolerant genotype. Therefore, the molecular marker can be used for identification of the rice salt-resistant material and subsequent breeding application thereof.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent should be subject to the appended claims.
Sequence listing
<110> institute of biotechnology of Chinese academy of agricultural sciences
<120> molecular marker related to salt tolerance of rice and application thereof
<160> 11
<170> SIPOSequenceListing 1.0
<210> 1
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
tgcctcttcc ccgtccccgc cgct 24
<210> 2
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
tgcctcttcc ccgtccccgc cgcc 24
<210> 3
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
ccgcacgcgt cgaaggatcg ggaag 25
<210> 4
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
taatcatgaa acaaacaaat gatat 25
<210> 5
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
taatcatgaa acaaacaaat gatac 25
<210> 6
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
cctcacacca tttgcacaat atttt 25
<210> 7
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
tagatcttat aaaaatggta gatgg 25
<210> 8
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
tagatcttat aaaaatggta gatat 25
<210> 9
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
catcatctac aatttattta tttct 25
<210> 10
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
atcacccaat agcaataatt a 21
<210> 11
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
ttgtgcaaat ggtgtgaggg t 21
Claims (6)
1. The application of the molecular marker related to the salt tolerance of rice in rice breeding is characterized in that the molecular marker is a combination of SNP _3_1796216_ T/C, SNP _3_1797072_ T/C, SNP _3_1797131_ GG/AT and INDEL _3_1797233_ GTATGAGGC/-s;
the molecular marker SNP _3_1796216_ T/C is located at 1796216bp on the No. 3 chromosome of rice, and has single nucleotide base mutation T/C;
the molecular marker SNP _3_1797072_ T/C is located at 1797072bp on the No. 3 chromosome of rice, and has single nucleotide base mutation T/C;
the molecular marker SNP _3_1797131_ GG/AT is located AT 1797131bp on the No. 3 chromosome of rice, and continuous two nucleotide base mutations GG/AT exist;
the molecular marker INDEL _3_1797233_ GTATGAGGC/-is located at the position 1797233bp on the No. 3 chromosome of rice, and 9 nucleotide bases are deleted.
2. The primer pair for detecting the molecular marker related to the salt tolerance of the rice as claimed in claim 1, wherein the primer pair for detecting the molecular marker SNP _3_1796216_ T/C is a specific upstream primer X1F, a specific Y1F and a specific downstream universal primer C1R, and the primer sequences are respectively shown in the sequence table SEQ ID NO: 1-3;
the primer pair for detecting the molecular marker SNP _3_1797072_ T/C is a specific upstream primer X2F, a specific downstream primer Y2F and a specific downstream universal primer C2R, and the primer sequences are respectively shown in a sequence table SEQ ID NO: 4-6;
the primer pair for detecting the molecular marker SNP _3_1797131_ GG/AT is a specific upstream primer X3F, a specific downstream primer Y3F and a specific downstream universal primer C3R, and the primer sequences are respectively shown in a sequence table SEQ ID NO: 7-9;
the primer pair for detecting the molecular marker INDEL _3_1797233_ GTATGAGGC/-is a specific upstream primer 4F and a specific downstream primer 4R, and the primer sequences are respectively shown in a sequence table SEQ ID NO: 10-11.
3. A method for detecting the salt-tolerant genotype of a rice gene OsDSK2a is characterized in that the PCR reaction detection is carried out by taking the rice genome DNA as a template and using the primer of claim 2; if the first three groups of primers detect the nucleotide sequences of the primers SEQ ID NO: 1. SEQ ID NO: 4. SEQ ID NO: 7, the size of the PCR product detected by the fourth group of primers is 125bp, and the rice sample is judged to be homozygous salt-free OsDSK2a genotype; if the first three groups of primers detect the nucleotide sequences of the primers SEQ ID NO: 2. SEQ ID NO: 5. SEQ ID NO: 8, detecting that the size of the PCR product is 116bp by using the fourth group of primers, and judging that the rice sample is homozygous salt-tolerant OsDSK2a genotype; if the primer nucleotide sequences of SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 4. SEQ ID NO: 5. SEQ ID NO: 7. SEQ ID NO: and (3) judging that the rice sample is a heterozygous salt-tolerant OsDSK2a genotype by using the base corresponding to 8 and PCR products of 125bp and 116 bp.
4. A kit for detecting the molecular marker of claim 1, wherein the primer of claim 2 is included in the kit.
5. A gene chip is characterized by comprising a nucleotide sequence shown in a sequence table SEQ ID NO: 1-11.
6. A rice breeding method is characterized in that the genotype of a rice gene OsDSK2a is detected according to the detection method of claim 3, and a rice sample carrying a salt-tolerant OsDSK2a genotype is selected for subsequent breeding.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210201003.6A CN114317812B (en) | 2022-03-03 | 2022-03-03 | Rice salt tolerance related molecular marker and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210201003.6A CN114317812B (en) | 2022-03-03 | 2022-03-03 | Rice salt tolerance related molecular marker and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114317812A CN114317812A (en) | 2022-04-12 |
| CN114317812B true CN114317812B (en) | 2022-05-20 |
Family
ID=81029916
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210201003.6A Active CN114317812B (en) | 2022-03-03 | 2022-03-03 | Rice salt tolerance related molecular marker and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114317812B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108486273A (en) * | 2018-04-08 | 2018-09-04 | 江苏强农农业技术服务有限公司 | On No. 5 chromosomes with the excavation and application of the SSR marker of Rice Salt QTL close linkages |
| CN109371162A (en) * | 2018-12-14 | 2019-02-22 | 中国农业科学院作物科学研究所 | SNP marker relevant to Salt Resistance of Rice and its application |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080028480A1 (en) * | 2004-06-14 | 2008-01-31 | Lindsay Megan P | Markers for Salinity Tolerance in Wheat Plants and the Use Thereof in Breeding Programs |
| CN104164450B (en) * | 2014-07-24 | 2017-01-18 | 中国农业科学院生物技术研究所 | Use of ubiquitin acceptor protein OsDSK2b in improvement of stress tolerance of plants |
| CN109371160A (en) * | 2018-12-14 | 2019-02-22 | 中国农业科学院作物科学研究所 | SNP marker relevant to Salt Resistance of Rice and Low DO tolerance and its application |
| CN110423840A (en) * | 2019-08-24 | 2019-11-08 | 武汉海稻国际生物科技有限公司 | A kind of Rice Salt QTL, localization method and its application |
| CN112458199B (en) * | 2020-12-24 | 2021-11-16 | 华智生物技术有限公司 | SNP molecular marker of rice salt-tolerant gene SKC1 and application thereof |
-
2022
- 2022-03-03 CN CN202210201003.6A patent/CN114317812B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108486273A (en) * | 2018-04-08 | 2018-09-04 | 江苏强农农业技术服务有限公司 | On No. 5 chromosomes with the excavation and application of the SSR marker of Rice Salt QTL close linkages |
| CN109371162A (en) * | 2018-12-14 | 2019-02-22 | 中国农业科学院作物科学研究所 | SNP marker relevant to Salt Resistance of Rice and its application |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114317812A (en) | 2022-04-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110241248B (en) | KASP marker related to wheat grain weight under salt stress condition and application thereof | |
| Singh et al. | Identification of sugarcane microsatellites associated to sugar content in sugarcane and transferability to other cereal genomes | |
| CN108165554B (en) | Corn leaf width control gene ZmNL4 and application thereof | |
| CN109593881A (en) | Detect rice low amylose content gene WxmqKASP molecular labeling and method | |
| Wang et al. | Development of intron targeting (IT) markers specific for chromosome arm 4VS of Haynaldia villosa by chromosome sorting and next-generation sequencing | |
| CN111979345A (en) | Development and application of KASP (Kaempferi protein) marker related to wheat biomass and yield under salt stress condition | |
| CN111893209B (en) | Indel site detection marker related to thousand grain weight of wheat and application thereof | |
| CN110747288A (en) | Rice large grain gene function marker and application | |
| CN111394508B (en) | Molecular marker linked with capsicum frutescens gene and application thereof | |
| CN115927703A (en) | Primer group for detecting specific molecular markers of rice grain type genes GS3 and GW5 and application thereof | |
| Pan et al. | Natural variation in OsMKK3 contributes to grain size and chalkiness in rice | |
| CN113736910A (en) | Linkage molecular marker of peanut single plant pod number main effect QTL site qPN7 and application thereof | |
| CN109251932A (en) | The bud green control gene PE-1 of eary maturity of rice and its application | |
| CN104711254B (en) | The low-phosphorous responsive genes ZmARF31 of corn INDEL molecular labelings and its application | |
| CN114317812B (en) | Rice salt tolerance related molecular marker and application thereof | |
| CN110846432A (en) | Codominant fluorescent molecular marker and detection method of brown planthopper resistant gene Bph3 | |
| CN111334597B (en) | SNP (Single nucleotide polymorphism) site and KASP (Kaempferi protein) marker for detecting powdery mildew resistance of watermelon and application thereof | |
| CN106399495B (en) | SNP marker closely linked with soybean short stalk character and application thereof | |
| CN112457385B (en) | Application of gene LJP1 for controlling rice growth period | |
| CN105925587B (en) | Early rice chloroplast development gene subjected to low-temperature response and detection method and application thereof | |
| CN109811077B (en) | KASP marker closely linked with wheat dwarf gene and application thereof | |
| CN106995846A (en) | The detection and application of a kind of Henan south aromatic rice Badh2 gene functions mark | |
| Hu et al. | Spike-stalk injection method causes extensive phenotypic and genotypic variations for rice germplasm | |
| CN116254364B (en) | SNP (Single nucleotide polymorphism) marker related to peanut fat content traits and application thereof | |
| CN116694811B (en) | Molecular marker closely linked with wheat seedling stage seed root number QTL and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |