CN110628930A - dCAPS molecular marker for identifying panicle length character of rice and application thereof - Google Patents
dCAPS molecular marker for identifying panicle length character of rice and application thereof Download PDFInfo
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Abstract
The invention provides a dCAPS molecular marker for identifying the panicle length property of rice and application thereof, wherein the dCAPS molecular marker is a PL-dCAPS marker, a 316bp strip can be amplified in the genomic DNA of the rice by using a primer, and the rice amplification strip of a long panicle variety is only one strip after being cut by restriction enzyme Xba I; the rice amplification strips of the short spike variety are subjected to enzyme digestion by restriction enzyme Xba I to obtain two strips, and the specific primer sequence for amplifying the molecular marker is shown in SEQ.ID.NO. 1-2. The molecular marker for identifying the panicle length character of the rice can be applied to molecular marker-assisted breeding of high-yield characters of the rice, the rice with the long panicle character can be quickly screened out without field statistics, the influence of environmental factors on phenotype identification is avoided, the breeding period is greatly shortened, and the molecular marker has important theoretical and practical significance for breeding new varieties of the long panicle character of the rice.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and relates to a dCAPS molecular marker for identifying the trait of rice panicle length and application thereof.
Background
Rice (Oryza sativa L.) is an important food crop, and lives more than half of the population worldwide. China is a large country for rice production and consumption, so that rice plays an important role in food production and safety in China and even the world. The growth and development of rice and the physiological regulation and control process influence the final yield and quality, so the research of breeding rice by utilizing the molecular biology technology to enhance the quality and improve the yield becomes an effective way for improving the production level of rice.
The yield of the rice has close relation with various panicle characters of the rice, the panicle length is a panicle character selection index positively correlated with the yield of the rice, and the gene for controlling the panicle length is cloned, so that a foundation can be laid for solving the development mechanism of the panicle of the rice and cultivating high-yield rice varieties. Although a large number of rice panicle length QTLs were identified and a small number were cloned, there are still many unknown genes to be explored.
The Single Nucleotide Polymorphism (SNP) marker is rich in genetic polymorphism in a genome and high in internal frequency of the gene, so that the SNP marker is more favorable for developing a marker co-separated from a functional gene. An economical and simple method for detecting SNPs is to splice amplification polymorphism (CAPS) markers or derivative CAPS markers. The two methods combine PCR amplification and enzyme digestion reaction, utilize restriction enzyme to identify target SNP site sequence for enzyme digestion, and then carry out electrophoresis typing identification. CAPS markers can be developed for SNP sequences of naturally occurring cleavage sites, while dCAPS can be developed for SNPs of non-naturally occurring cleavage sites after artificially introducing mutant bases. CAPS has been developed only to a limited extent due to the low probability of naturally occurring recognition by restriction endonucleases. However, almost all SNP sites can be converted into a label that can be detected by electrophoresis by designing corresponding PCR primers after artificially introducing a mutant base. The method has the characteristics of co-dominance, site specificity, simplicity in operation, quickness in detection, low cost, independence of precision instruments and equipment and the like, and can be used for plant genotyping, positioning, genetic diversity analysis, variety identification and the like.
In the previous work, the inventors located a new ear length QTL PL9 based on the developed specific-loci amplified fragment sequencing (SLAF-Seq) marker. If a proper dCAPS molecular marker can be developed on the QTL, the breeding efficiency of the selection of the important yield character of the rice panicle length can be improved by screening the marker, the breeding cost is saved, and the economic benefit of the rice industry is improved.
Disclosure of Invention
The invention aims to provide a dCAPS molecular marker for identifying the trait of rice panicle length;
the invention also aims to provide a method for identifying the panicle length character of rice by using the molecular marker.
In order to realize the purpose of the invention, firstly, rice variety G1025 (short spike) and K1561 (long spike) are hybridized to construct a Recombinant Inbred Line (RILs) group, the genomic DNA of the RILs group is extracted, a high-density genetic linkage map is constructed by utilizing a specific site amplification fragment (SLAF) sequencing technology to obtain SNP sites which are obviously associated with the rice spike length character, the fragments linked with the SNP sites are cloned and sequenced and are converted into dCAPS molecular markers, the rice spike length character is identified in a natural group and the Recombinant Inbred Line (RILs) group by utilizing the dCAPS molecular markers which are successfully converted, and the molecular marker identification result is consistent with the phenotype identification result.
The dCAPS molecular marker for identifying the panicle length property of the rice is named as PL-dCAPS marker, a primer can be used in the genomic DNA of the rice to amplify a 316bp strip, and the rice amplified strip of the long panicle variety is only provided with one 316bp strip after being digested by restriction enzyme Xba I; after the rice amplified bands of the short spike variety are cut by restriction enzyme Xba I, two bands of 274bp and 42bp are obtained.
Further, the PL-dCAPS markers of the present invention were amplified by the following specific primer pairs:
an upstream primer F: 5'-CGACGACAAGACCCTTTCAGAAGGGATTTACATGGTCAATCCTC-3' (SEQ. ID. NO. 1);
a downstream primer R: 5'-GCCACACATGACCTCCAAAA-3' (SEQ. ID. NO. 2);
the invention provides a dCAPS molecular marker specific primer pair for identifying the panicle length character of rice, which comprises the following components in part by weight:
an upstream primer F: 5'-CGACGACAAGACCCTTTCAGAAGGGATTTACATGGTCAATCCTC-3' (SEQ. ID. NO. 1);
a downstream primer R: 5'-GCCACACATGACCTCCAAAA-3' (SEQ. ID. NO. 2);
the invention provides a kit containing the specific primer pair. Further, the kit of the present invention further contains a restriction enzyme Xba I.
The invention provides application of a molecular marker PL-dCAPS in identification of a rice panicle length trait.
The invention provides application of a molecular marker PL-dCAPS in rice molecular marker assisted breeding.
The invention provides an application of a specific primer pair shown in SEQ.ID.NO. 1-2 or a kit containing the specific primer pair in identification of the panicle length trait of rice.
The invention provides an application of a specific primer pair shown in SEQ.ID.NO. 1-2 or a kit containing the specific primer pair in rice molecular marker-assisted breeding.
The invention also provides a method for identifying the panicle length character of rice, which comprises the following steps:
(1) extracting the genome DNA of the rice to be detected;
(2) taking the genomic DNA of rice to be detected as a template, and carrying out PCR amplification reaction by using a specific primer pair shown in SEQ.ID.NO. 1-2;
(3) and carrying out enzyme digestion on the amplification product by using a restriction enzyme Xba I, carrying out parting on the enzyme digestion product by using agarose gel electrophoresis, wherein the enzyme digestion product only comprises a 316bp strip, the rice to be detected is a long-ear variety, and the enzyme digestion product comprises two strips of 274bp and 42bp, so the rice to be detected is a short-ear variety.
In the method, the 20-microliter system of the PCR amplification reaction in the step (2) is as follows: 1 mu L of genome DNA to be detected, 0.5 mu L of each of upstream and downstream primers, 2 XEs Taq DNA Polymerase Reaction mix10 mu L and 8 mu L of distilled water;
the PCR amplification reaction program is as follows: denaturation at 94 deg.C for 5 min; 30s at 94 ℃, 30s at 57 ℃ and 30s at 72 ℃ for 35 cycles; extending for 5min at 72 ℃;
wherein the 20 mu L enzyme digestion system in the step (3) is as follows: 10 mu L of PCR product, 1 mu L of Xba I and 9 mu L of distilled water, and the enzyme digestion reaction condition is 37 ℃ for 4 hours;
after the enzyme digestion, 10. mu.L of the enzyme digestion product is subjected to typing by 2% agarose gel electrophoresis.
The invention provides application of the method for identifying the panicle length character of rice in rice breeding.
The invention has the advantages of
The molecular marker for identifying the panicle length character of the rice can be applied to molecular marker-assisted breeding of high-yield characters of the rice, the rice with the long panicle character can be quickly screened out without field statistics, the influence of environmental factors on phenotype identification is avoided, the breeding period is greatly shortened, and the molecular marker has important theoretical and practical significance for breeding new varieties of the long panicle character of the rice.
Drawings
FIG. 1 is an electrophoretogram of PL-dCAPS labeled primer amplifications G1025 and K1561; m is Marker, 1 is rice variety G1025, 2 is rice variety K1561;
FIG. 2 is a validation of PL-dCAPS markers in the natural population; m is Marker, 1-12 are 12 long spike varieties, 13-24 are 12 short spike varieties;
FIG. 3 is a graph showing the validation of PL-dCAPS markers in RILs populations; m is Marker, 12 is rice variety G1025, 13 is rice variety K1561, and the others are different individuals in RILs;
Detailed Description
The following examples further illustrate the present invention but should not be construed as limiting the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 development of dCAPS molecular marker for identifying trait of panicle Length of Rice
1. SNP (Single nucleotide polymorphism) sites obviously associated with rice long-ear traits are excavated by an SLAF-Seq method
(1) 2 rice varieties G1025 (short spike) and K1561 (long spike) with different sources and obvious spike length traits are selected to be hybridized to construct a Recombinant Inbred Line (RILs) population;
(2) constructing a high-density genetic linkage map by using a Specific Locus Amplification Fragment (SLAF) sequencing technology and utilizing genotype data of RILs (Rils populations);
(3) analyzing Quantitative Trait Loci (QTL) of the spike length of RILs population on the basis of genetic linkage map, detecting a main effect QTL (named PL9) for controlling the spike length, and obtaining SNP loci obviously associated with the rice spike length trait;
2. SNP site-specific enzyme cutting site analysis and dCAPS primer design
(1) Selecting SNP loci closely associated with the rice panicle length traits from the SNP loci obtained in the step 1, and carrying out subsequent dCAPS Marker development on SNP loci Marker775977-30 with the minimum P value and the high contribution rate, wherein the sequence of the SLAF label Marker775977 is shown as SEQ.ID.NO. 3;
(2) searching restriction enzyme information caused by SNP site mutation selected in the step (1) by using online enzyme digestion recognition software dCAPS Finder 2.0(http:// helix.wustluedu.edu/dCAPS/dcaps.html), selecting a proper endonuclease Xba I, wherein the enzyme digestion recognition sequence of the endonuclease is TCTAGA, and artificially introducing a mismatched base T at the position of Marker 775977-28;
(3) carrying out genotype analysis on Marker775977-30 sites in RILs population, wherein the total number of genotypes is C, T and Y, wherein Y represents CT degenerate basic group, if the genotype of a certain material is Y, the material is in a heterozygous state at the sites; according to the recognition sequence of the restriction enzyme Xba I, if the SNP site is C, the restriction enzyme can not be cut; if T is, the enzyme can be cut; if Y, partial enzyme digestion can be carried out; the average phenotype values of the two genotypes of the Marker775977-30 sites are respectively calculated, and the spike length of the C genotype material is found to be significantly larger than that of the Y genotype (P < 0.01), so that the PCR product of the short-spike rice variety is considered to be more likely to be enzyme-cut;
(4) the converted dCAPS molecular Marker at the Marker775977-30 site is named as PL-dCAPS Marker, and a primer is designed: firstly, designing an upstream Primer by using online enzyme digestion recognition software dCAPS Finder 2.0(http:// helix.wustl.edu/dCAPS/dcaps.html) in a mode of introducing mismatched bases, and designing a downstream PCR Primer by using Primer 5.0 software; the design standard of the downstream primer is 18-25 bp in length, 40-60% of GC content and 55-65 ℃ of annealing temperature, the downstream primer is close to the GC content and the annealing temperature of the upstream primer as much as possible, no primer dimer and hairpin structure exist, and the downstream primer can be paired with the upstream primer to amplify a target gene product. Considering that the upstream primer only cuts 27 bases at the 5 ' end after the enzyme cutting site is cut, and the PCR product only has 301bp, in order to better distinguish the products which are cut and are not cut by agarose gel, a 15bp linker sequence is added at the 5 ' end of the upstream primer, so that the upstream primer only cuts 42 bases at the 5 ' end after the enzyme cutting site is cut. The sequence of the upstream primer designed according to the principle is as follows:
an upstream primer F: 5' -CGACGACAAGACCCTTTCAGAAGGGATTTACATGGTCAATCCTC-3’(SEQ.ID.NO.1)；
A downstream primer R: 5'-GCCACACATGACCTCCAAAA-3' (SEQ. ID. NO. 2);
wherein, the underlined part of the upstream primer F is an adaptor sequence;
the primer pair is used for carrying out PCR amplification on the genomic DNA of the rice variety G1025 (short panicle) and K1561 (long panicle), 316bp bands can be obtained by amplification (figure 1), and the designed primers are good in specificity.
The dCAPS molecular marker for identifying the panicle length property of the rice and the specific primer pair for the molecular marker are obtained through the design, 316bp bands can be amplified in the genomic DNA of the rice by using the primers, and only one 316bp band exists in the amplified bands of the rice of the long panicle variety after the amplified bands of the rice are cut by restriction enzyme Xba I; after the rice amplified bands of the short spike variety are cut by restriction enzyme Xba I, two bands of 274bp and 42bp are obtained.
Example 2 verification of dCAPS molecular markers for identifying panicle length trait of Rice
(1) Extracting genome DNA of 12 short-ear rice varieties and 12 long-ear rice varieties by a CTAB method, detecting the concentration and purity of the DNA by a nucleic acid instrument, and then using ddH2Diluting O to 300ng/L for later use;
(2) the specific primer pair designed in the example 1 is used for carrying out PCR amplification on 24 parts of materials, and the 20-microliter system of the PCR amplification reaction comprises: 1 μ L of genome DNA to be detected, 0.5 μ L of each of the upstream and downstream primers, 10 μ L of 2 × Es Taq DNA polymerasereaction mix (TIANGEN, Beijing), and 8 μ L of distilled water. The PCR amplification reaction program is as follows: denaturation at 94 deg.C for 5 min; 30s at 94 ℃, 30s at 57 ℃ and 30s at 72 ℃ for 35 cycles; extending for 5min at 72 ℃;
(3) after the PCR reaction is finished, 10 microliter of reaction product is detected by 2% agarose gel electrophoresis, and 24 samples all obtain a single band, which indicates that the specificity of the primer is good; sequencing the amplified product, wherein the band is 316bp long, the sequence is shown as SEQ.ID.NO.4, the sequencing of the PCR product in the step is not an essential step, but the accuracy of the design of the primer can be verified through sequencing the product, and the size of the enzyme digestion band can be deduced;
(4) and (3) digesting the PCR product obtained in the step (2) by using a restriction enzyme Xba I, wherein 20 mu L of digestion system is as follows: 10 μ L of the PCR product, 1 μ L of Xba I (Takara, Dalian) and 9 μ L of distilled water, and the digestion reaction was carried out at 37 ℃ for 4 hours; after the completion of the digestion, 10. mu.L of the digested product was fractionated by 2% agarose gel electrophoresis (FIG. 2), and the polymorphism analysis of the digestion amplification revealed: 10 parts of 12 long-ear rice variety materials cannot be subjected to enzyme digestion (83.3 percent), 11 parts of 12 short-ear rice varieties can be subjected to partial enzyme digestion (91.7 percent), the size of an enzyme digestion strip accords with the expectation of molecular marker design (because the other 42bp enzyme digestion strip is too small, the enzyme digestion strip can not be displayed after being deposited into an electrophoresis solution during electrophoresis), and the accuracy rate of the developed PL-dCAPS marker co-separated from the rice long-ear character is 87.5 percent;
(5) from the RILs group constructed by crossing the rice variety G1025 (short panicle) and K1561 (long panicle) in the example 1, 22 individuals were randomly selected to further verify the coseparation of the PL-dCAPS marker and the rice panicle length trait;
the DNA extraction, PCR amplification, enzyme digestion reaction, electrophoresis detection and the like are the same as the verification method of the 22 natural varieties;
enzyme-cleaved amplification polymorphism analysis found (FIG. 3): 10 of 11 long ear lines could not be digested (90.9%), while 11 of 11 short ear lines could be partially digested (100%), with an average accuracy of 95%.
The verification in natural population and RILs population shows that the coincidence rate of the identification result of the marker on the rice spike length and the field identification result is extremely high, and the marker has good application value.
Sequence listing
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ttcagaaggg atttacatgg tcaatccccy agaacgatcc aattcaagaa acccattaca 60
accagtaaaa gtgatgtcaa tagatgggtt aaaaagggct ttgaggtatg aaatactatc 120
ccagctgcca tctttgcata ttgttctgtt cacatttcat gttctttcct gacaaaagtc 180
aacttcaata gcgaatgaaa cagtgttgaa atatggaaat gattgtatta cagttgtcct 240
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ttcctgacaa aagtcaactt caatagcgaa tgaaacagtg ttgaaatatg gaaatgattg 240
tattacagtt gtcctgctag ataaagtaag cacatacttc atttgtagtc aaatcttttt 300
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Claims (10)
1. The dCAPS molecular marker for identifying the panicle length character of the rice is characterized by being a PL-dCAPS marker, a 316bp strip can be amplified in the genomic DNA of the rice by using a primer pair, and only one 316bp strip is obtained after the rice amplification strip of the long panicle variety is subjected to enzyme digestion by restriction enzyme Xba I; the rice amplified band of the short spike variety is cut by restriction enzyme Xba I to obtain two bands of 274bp and 42 bp;
the sequence of the upstream primer of the primer pair is shown as SEQ.ID.NO.1, and the sequence of the downstream primer is shown as SEQ.ID.NO. 2.
2. The specific primer pair for detecting the molecular marker of claim 1, wherein the sequence of the upstream primer in the specific primer pair is shown as SEQ.ID.NO.1, and the sequence of the downstream primer is shown as SEQ.ID.NO. 2.
3. A kit comprising the specific primer set according to claim 2.
4. The kit according to claim 3, wherein the kit further comprises a restriction enzyme Xba I.
5. The use of the molecular marker of claim 1 for identifying panicle length trait in rice.
6. The use of the molecular marker of rice according to claim 1 for molecular marker-assisted breeding.
7. Use of the specific primer pair according to claim 2 or the kit according to claim 3 or the kit according to claim 4 for identifying a trait of rice panicle length.
8. A method for identifying the trait of the panicle length of rice is characterized by comprising the following steps:
(1) extracting the genome DNA of the rice to be detected;
(2) taking the genomic DNA of rice to be detected as a template, and carrying out PCR amplification reaction by using a specific primer pair shown in SEQ.ID.NO. 1-2;
(3) and carrying out enzyme digestion on the PCR amplification product by using a restriction enzyme Xba I, carrying out parting on the enzyme digestion product by using agarose gel electrophoresis, wherein the enzyme digestion product only comprises one main band of 316bp, so that the rice to be detected is a long-ear variety, and the enzyme digestion product comprises two main bands of 274bp and 42bp, so that the rice to be detected is a short-ear variety.
9. The method of claim 8, wherein:
the 20-mu-L PCR amplification reaction system in the step (2) comprises: 1 mu L of genome DNA to be detected, 0.5 mu L of each of upstream and downstream primers, 10 mu L of 2 XEs Taq DNA Polymerase Reaction mix and 8 mu L of distilled water;
the PCR amplification reaction program is as follows: denaturation at 94 deg.C for 5 min; 30s at 94 ℃, 30s at 57 ℃ and 30s at 72 ℃ for 35 cycles; extending for 5min at 72 ℃;
the 20 mu L enzyme digestion system in the step (3) is as follows: 10 mu L of PCR product, 1 mu L of Xba I and 9 mu L of distilled water, and the enzyme digestion reaction condition is 37 ℃ for 4 hours;
after the completion of the digestion, 10. mu.L of the digested product was fractionated by 2% agarose gel electrophoresis.
10. Use of the method of claim 8 in rice breeding.
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| CN113549616A (en) * | 2021-08-06 | 2021-10-26 | 福建省农业科学院作物研究所 | CAPS molecular marker for identifying oncidium hybridum variety, screening method and application |
| CN113549616B (en) * | 2021-08-06 | 2023-03-24 | 福建省农业科学院作物研究所 | CAPS molecular marker for identifying oncidium hybridum variety, screening method and application |
| CN116103433A (en) * | 2023-02-03 | 2023-05-12 | 广西壮族自治区农业科学院 | CAPS molecular marker for identifying rice spike length character and application thereof |
| CN116103433B (en) * | 2023-02-03 | 2023-09-29 | 广西壮族自治区农业科学院 | CAPS molecular marker for identifying rice spike length character and application thereof |
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