InDel molecular marker for identifying elongation capacity of mesocotyl of rice, application and method

Abstract

The invention discloses an InDel molecular marker for identifying the elongation capacity of mesocotyl in rice, application and a method. The nucleotide sequence of the InDel marker is as shown in SEQ ID No. 4; the molecular marker is located at the 981 site to the 2040 site of a nucleotide sequence of SEQ ID No. 1 and is insertion and deletion of 1060 bp; the primer pair for detecting the InDel marker comprises a forward primer with a nucleotide sequence as shown in SEQ ID No. 2 and a reverse primer with a nucleotide sequence as shown in SEQ ID No. 3. The primer designed by the invention can effectively amplify specific gene segments of rice, and the specific gene segments are distinguished through agarose gel electrophoresis, so that mesocotyl length genotypes of different rice germplasms can be effectively distinguished, and identification of target genes in rice germplasm resources and breeding progenies can be conveniently and quickly realized; the labor cost is greatly reduced, the time is saved, and a foundation is laid for rice breeding work.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of rice molecular breeding, and particularly relates to an InDel molecular marker for identifying the hypocotyl elongation ability of rice, application and method. BACKGROUND

[0002] As a global important food crop, the yield of rice is of great significance to food security. With the development of agricultural planting mode, direct seeding of rice is widely adopted due to its advantages of labor saving and effort saving.

[0003] However, direct seeding of rice needs to be deeply sown to avoid bird damage and lodging, which puts higher requirements on the seed emergence ability. Studies have shown that the hypocotyl elongation ability of rice is a key factor to determine the penetration depth of seedling into the soil, and the hypocotyl length is significantly positively correlated with the emergence rate.

[0004] Therefore, it is of great application value to mine the genetic loci related to hypocotyl elongation and develop efficient molecular markers for breeding rice varieties suitable for direct seeding.

[0005] At present, specific InDel markers for the hypocotyl elongation ability of rice are still relatively lacking, which limits the efficiency of molecular marker assisted breeding.

[0006] Therefore, it is of great significance to develop new InDel markers closely linked to the hypocotyl elongation ability and establish a rapid and accurate genotyping system to accelerate the breeding process of direct seeding rice varieties and reduce the breeding cost. SUMMARY

[0007] Therefore, the present application provides an InDel molecular marker for identifying the hypocotyl elongation ability of rice, application and method.

[0008] To solve the above technical problems, the present application adopts the following technical solutions: An InDel molecular marker for identifying the hypocotyl elongation ability of rice, wherein the nucleotide sequence of the InDel marker is shown in SEQ ID No. 4.

[0009] Preferably, the molecular marker is located at positions 981-2040 of the nucleotide sequence shown in SEQ ID No. 1, and is an insertion and deletion of 1060 bp.

[0010] A primer pair for detecting the InDel marker, wherein the primer pair comprises a forward primer with a nucleotide sequence shown in SEQ ID No. 2, 5'-gaagaaatgaccaaaataaaagttgtatgtcttaatgagttc-3', and a reverse primer with a nucleotide sequence shown in SEQ ID No. 3, 5'-gcacggactgctttga-3'.

[0011] A product for detecting InDel marker, which is a kit.

[0012] Application of the product in rice breeding.

[0013] Preferably, any one of the following is included: increasing the length of the embryonic axis of rice, increasing the rate of rice seedling emergence, and improving rice varieties.

[0014] A method for detecting the elongation ability of the embryonic axis of rice, the InDel molecular marker is used for detecting the elongation ability of the embryonic axis of rice, and the method comprises the following steps: S1: extracting genomic DNA of a rice sample to be detected; S2: using the extracted genomic DNA as a template, and performing PCR amplification by using the primer pair; S3: performing electrophoresis detection on the PCR amplification product, when the amplification product is 665bp, the electrophoretic strain is long embryonic axis, and when the amplification product is 1725bp, the electrophoretic strain is short embryonic axis.

[0015] Preferably, in the step S3, The PCR amplification system is: 2x Taq Master Mix 5 μL, forward primer 0.5 μL, reverse primer 0.5 μL, template DNA 1 μL, and double-distilled water 3 μL; The PCR amplification program is: 94℃ pre-denaturation for 5min, 94℃ denaturation for 30s, 55℃ annealing for 30s, 72℃ extension for 2min, a total of 35 cycles, finally 72℃ complete extension for 7min, and storage at 4℃; The electrophoresis program is: 1.2% agarose gel electrophoresis, constant voltage 250v, 500A, electrophoresis time 15min.

[0016] Preferably, the primer pair is used to detect the rice sample, when the amplification product is 665bp, the electrophoretic strain is long embryonic axis, and when the amplification product is 1725bp, the electrophoretic strain is short embryonic axis.

[0017] The present application has the following technical effects relative to the prior art: (1) The primer provided by the present application is used for rice variety identification, and the results show that the long embryonic axis rice variety and the short embryonic axis rice variety have specific differences, and the band of the long embryonic axis rice variety is smaller than that of the short embryonic axis rice variety. (2) The primer designed in the application can effectively amplify the specific gene fragment of rice, and the specific gene fragment is distinguished by agarose gel electrophoresis; the different rice germplasm genotypes of the length of the mesocotyl can be effectively distinguished, so that the identification of the target gene in the rice germplasm resources and the breeding offspring can be conveniently and quickly realized, the labor cost is greatly reduced, the time is saved, and the foundation is laid for the rice breeding work; (3) The InDel molecular marker provided by the application only needs to be amplified by PCR and combined with agarose gel electrophoresis in actual application, and has the advantages of low cost, high throughput and high specificity, and is suitable for rice breeding and production practice. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 Figure 1 is a diagram of the position of the 1060bp insertion and deletion relative to the gene of the application; Figure 2 Figure 2 is a diagram of the amplification of the long and short mesocotyl genotypes of Kasalath and Peiaung by the primer set of the application; Figure 3 Figure 3 is a statistical diagram of the mesocotyl phenotype of the long mesocotyl material Kasalath and the short mesocotyl material Peiaung; Figure 4 Figure 4 is a diagram of the amplification band type of the above-mentioned primer set in 24 rice varieties, and the PCR amplification fragment size is 1725bp (upper band type) and 665bp (lower band type), respectively; Figure 5 Figure 5 is a statistical diagram of the mesocotyl length of the above-mentioned 24 rice varieties. DETAILED DESCRIPTION

[0019] The technical solutions in the embodiments of the application will be clearly and completely described below with reference to the drawings in the embodiments of the application. Obviously, the described embodiments are only part of the embodiments of the application, rather than all the embodiments. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the application.

[0020] The application discloses an InDel molecular marker for identifying the elongation ability of the mesocotyl of rice.

[0021] The molecular marker is located at 981-2040 of the nucleotide sequence shown in SEQ ID No. 1, and is an insertion and deletion of 1060bp.

[0022] The application further discloses a primer pair for detecting the InDel marker, wherein the primer pair comprises a forward primer 5'-gaagaaatgaccaaaataaaagttgtatgtcttaatgagttc-3' with a nucleotide sequence as shown in SEQ ID No. 2 and a reverse primer 5'-gcacggactgctttga-3' with a nucleotide sequence as shown in SEQ ID No. 3.

[0023] The application further discloses a product for detecting the InDel marker, wherein the product is a kit.

[0024] The application further discloses application of the product in rice breeding.

[0025] The application comprises any one of the following: improving the length of the embryonic axis of rice, improving the rate of rice seedling emergence, and improving rice varieties.

[0026] The application further discloses a method for detecting the elongation capacity of the embryonic axis of rice, wherein the InDel molecular marker is used to detect the elongation capacity of the embryonic axis of rice. S1: extracting genomic DNA of a rice sample to be detected; S2: using the extracted genomic DNA as a template and using the primer pair to perform PCR amplification; S3: performing electrophoresis detection on the PCR amplification product, wherein when the amplification product is 665bp, the electrophoretic strain is a long embryonic axis, and when the amplification product is 1725bp, the electrophoretic strain is a short embryonic axis; The PCR amplification system is as follows: 2x Taq Master Mix 5ul, forward primer 0.5ul, reverse primer 0.5ul, template DNA 1ul and double-distilled water 3ul. The PCR amplification program is as follows: 94 DEG C pre-denaturation for 5min, 94 DEG C denaturation for 30s, 55 DEG C annealing for 30s, 72 DEG C extension for 2min, a total of 35 cycles, finally 72 DEG C complete extension for 7min, and preservation at 4 DEG C; The electrophoresis program is as follows: 1.2% agarose gel electrophoresis, constant voltage 250V, 500A, and electrophoresis time 15min.

[0027] The primer pair is used to detect the rice sample, wherein when the amplification product is 665bp, the electrophoretic strain is a long embryonic axis, and when the amplification product is 1725bp, the electrophoretic strain is a short embryonic axis.

[0028] Example 1: Based on public resequencing data analysis of 3000 rice germplasms, it is found that the rice gene embryonic axis elongation gene located in SEQ ID No. 1 OsDET1-LThe 1060 bp insertion-deletion in the promoter region of 981-2040 is the key mutation site.

[0029] As shown in Figure 1 , a forward primer shown as SEQ ID No. 2 was designed according to the position of the inserted sequence: 5'-gaagaaatgaccaaaataaaag ttgtatgtcttaatgagttc-3'; a reverse primer shown as SEQ ID No. 3: 5'- gcacggactgctttga-3'.

[0030] The genomic DNA of Kasalath with long mesocotyl and Nipponbare with short mesocotyl was amplified by the designed primers. In the specific implementation, the rice was cultivated to two-leaf-one-heart stage, and the leaf was collected to extract the genomic DNA by the improved CTAB method; The PCR amplification system was as follows: 2x Taq Master Mix 5 μL, forward primer 0.5 μL, reverse primer 0.5 μL, template DNA 1 μL, and double-distilled water 3 μL.

[0031] The PCR amplification program was as follows: 94℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 2 min, a total of 35 cycles, and finally 72℃ complete extension for 7 min, and storage at 4℃. The amplification product was subjected to 1.2% agarose gel electrophoresis, and the electrophoresis program was as follows: 1.2% agarose gel electrophoresis, constant voltage 250 v, 500 A, electrophoresis time 15 min.

[0032] As shown in Figure 2 , the product of Kasalath with long mesocotyl was a smaller band, and the product of Nipponbare with short mesocotyl was a larger band.

[0033] The amplification product was subjected to Sanger sequencing, and the difference InDel molecular marker was determined as SEQ ID No. 4; that is, the specific band of the mesocotyl elongation ability could be distinguished by electrophoresis.

[0034] The rice seeds of Kasalath and Nipponbare were harvested at 30-40 days after flowering and air-dried under natural conditions; the seeds were treated at 45℃ for 3 days to remove dormancy; the sterile seeds were pretreated under humid conditions for 2 days to ensure uniform germination, and then sowed on the surface of 0.6% agar medium, and cultured in a dark underground culture room at 30℃ and 75% humidity for 7 days, and the length of the mesocotyl of the seedlings was identified, as shown in Figure 3 , the mesocotyl of Kasalath was long, corresponding to Figure 2 a short band; the mesocotyl of Nipponbare was short, corresponding to Figure 2 a long band.

[0035] Example 2: Take 24 rice materials, as shown in Table 1, for verification.

[0036] Table 1 - 24 rice variety name and InDel insertion missing information table

[0037] The above rice was respectively cultured to two-leaf-one-heart stage, and the leaf was collected to extract the genomic DNA by improved CTAB method. The genomic DNA of the rice to be tested was used as a template, and the primer pair, amplification system and program designed in Example 1 were used for PCR amplification to obtain the amplification product.

[0038] The PCR product obtained by amplification was detected by gel imaging system after agarose gel electrophoresis, and the electrophoresis result was shown in Figure 4 The electrophoresis band result was consistent with the genotype.

[0039] The above 24 materials were phenotypically identified, as shown in Figure 5 It can be seen that the genotype amplified by the forward primer shown in SEQ ID No. 2 and the reverse primer shown in SEQ ID No. 3 is also consistent with the mesocotyl phenotype.

[0040] SEQ ID No. 1: SEQ ID No. 2: 5'-gaagaaatgaccaaaataaaagttgtatgtcttaatgag ttc-3' SEQ ID No. 3: 5'-gcacggactgctttga-3' SEQ ID No. 4: The above merely illustrates the preferred embodiments of the present application, but is not intended to limit the technical scope of the present application, and any slight modification, equivalent change and modification made to the above embodiments according to the technical essence of the present application shall still fall within the technical scope of the present application.

Claims

1. An InDel molecular marker for identifying the ability of embryonic axis elongation in rice, characterized in that, The nucleotide sequence of the InDel marker is shown as SEQ ID No.

4. 2.The InDel molecular marker for identifying the embryonic axis elongation ability of rice according to claim 1, characterized in that, The molecular marker is located at 981-2040 of the nucleotide sequence shown in SEQ ID No. 1, which is an insertion and deletion of 1060 bp.

3. A primer pair for detecting the InDel marker of claims 1-2, characterized in that, The primer pair comprises a forward primer with a nucleotide sequence shown as SEQ ID No. 2, 5'-gaagaaatgaccaaaataaaagttgtatgtcttaatgagttc-3' and a reverse primer with a nucleotide sequence shown as SEQ ID No. 3, 5'-gcacggactgctttga-3'.

4. A product for detecting the InDel marker of claims 1-2, characterized in that, The product is a kit.

5. The product of claim 4 in rice breeding.

6. Use according to claim 5, characterized in that, Comprise: Any one of increasing the length of the embryonic axis of rice, increasing the rate of rice breaking soil emergence, and improving rice varieties.

7. A method for detecting the ability of hypocotyl elongation in rice, characterized by, The InDel molecular marker is used for detecting the elongation ability of the embryonic axis of rice, comprising the following steps: S1: extracting genomic DNA of the rice sample to be detected; S2: using the extracted genomic DNA as a template, and using the primer pair described above to perform PCR amplification; S3: performing electrophoresis detection on the PCR amplification product, when the amplification product is 665 bp, the electrophoresis strain is long embryonic axis, and when the amplification product is 1725 bp, the strain is short embryonic axis.

8. The method for detecting the capability of plumule elongation in rice according to claim 7, wherein, In the step S3, The PCR amplification system is: 2x Taq Master Mix 5 μL, forward primer 0.5 μL, reverse primer 0.5 μL, template DNA 1 μL, and double distilled water 3 μL; The PCR amplification program is: 94℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 2 min, a total of 35 cycles, finally 72℃ complete extension for 7 min, and storage at 4℃; The electrophoresis program is: 1.2% agarose gel electrophoresis, constant voltage 250V, 500A, electrophoresis time 15 min.

9. The method for detecting the capability of plumule elongation in rice according to claim 7, wherein, Using the primer pair of claim 3 to detect the rice sample, when the amplification product is 665 bp, the electrophoresis strain is long embryonic axis, and when the amplification product is 1725 bp, the strain is short embryonic axis.

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