A molecular marker for identifying QTL tin7 for tiller number and its application

Abstract

The present invention discloses a molecular marker for identifying a tiller number QTL tin7 and its application, and relates to the technical field of molecular markers. The nucleotide sequence of the molecular marker is shown in SEQ ID NO.1, and there is a SNP site at the 36bp base of the nucleotide sequence, which is a T / C mutation. The present invention discloses for the first time a molecular marker CHS10_2B791664276 for accurately detecting the tiller number locus tin7 of wheat N2496 based on a fluorescent quantitative PCR platform, and the molecular marker detection is accurate and efficient, and the amplification is convenient and stable. The molecular marker CHS10_2B791664276 disclosed by the present invention is significantly correlated with the tiller number locus tin7, has high accuracy in molecular marker-assisted selection, can improve the selection and identification efficiency of wheat tiller number varieties adapted to different environments, and has a high success rate.

Description

Technical Field

[0001] The invention relates to the technical field of molecular markers, in particular to a molecular marker for identifying a tiller number QTL tin7 and an application thereof. Background Art

[0002] Wheat is the third largest crop in the world and one of the most traded commodities in the world market. It is also one of the main grain crops in my country, with a large planting area and a long planting history in my country. my country is one of the earliest countries in the world to plant wheat. China is the second largest wheat planting area and the largest wheat yield in the world. The annual planting area of ​​wheat in my country is more than 26.6667 million hectares, accounting for about 27% of the grain crop area; the total output is more than 100 million tons, accounting for about 22% of the grain crop output. Therefore, it is necessary to accelerate the wheat breeding process, select high-yield, stable and high-quality wheat varieties, increase grain production, ensure the basic needs of national life, and promote the improvement of national economic strength.

[0003] Wheat tiller refers to the branches of Gramineae plants growing below or near the ground. Tillering is closely related to wheat population structure, changes in physiological activities, material production and distribution, and the number of tillers directly affects wheat yield. Wheat tillering is a quantitative trait, controlled by multiple genes, and greatly affected by environmental effects. Since 1988, Richards et al. discovered a pair of recessive genes that inhibit the number of wheat tillers in "Israel uniculm" and named it tin1, research on genes controlling the number of wheat tillers has begun at home and abroad. At present, the loci related to wheat tillering that have been located include 1A, 1B, 1D, 2A, 2D, 3A, 3B, 4A, 4D, 5A, 5D, 6A, 6D, 7A, etc. Araki et al. located a tillering gene on chromosome 4A in 1999. Katoet et al. located a gene controlling tillering on chromosome 5A in 2000. Li et al. located a new gene locus controlling tiller number on chromosome 6AS in 2002. Huang et al. located eight tillering QTLs on 1B, 2A, 2D, 3B, 4D, 5D, 6D and 7A in 2003, which can explain more than 9% of phenotypic variation. An et al. located tillering QTLs on 1B, 3A, 3B and 6A in 2005, which can explain more than 6.7-12.9% of phenotypic variation. Kuraparthy et al. located a tillering gene on chromosome 3A in 2007 and named it tin3. Naruoka et al. located a tillering gene on chromosome 6B in 2011. Zhang et al. located a tillering-inhibiting gene on chromosome 1AS in 2012 and named it ftin. Wang et al. located three tillering QTLs on 2B, 2D and 5A in 2015, which can explain 19.1, 14.6 and 9.6% of the phenotypic variation, respectively. Therefore, it is necessary to locate the wheat tillering gene, which is of great significance for effectively regulating the number of wheat tillers and shaping new wheat lines with reasonable plant types. The present invention intends to develop a molecular marker for identifying the wheat tiller number QTL tin7 to improve the accuracy of wheat tiller number trait prediction. Summary of the invention

[0004] The purpose of the present invention is to provide a molecular marker for identifying tiller number QTL tin7 and its application to solve the problems existing in the above-mentioned prior art. The molecular marker is significantly correlated with the tiller number locus tin7, has high accuracy in molecular marker-assisted selection, can improve the selection and identification efficiency of wheat tiller number varieties adapted to different environments, and has a high success rate.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The invention provides a molecular marker CHS10_2B791664276 which is closely linked to a wheat tillering number locus tin7. The nucleotide sequence of the molecular marker CHS10_2B791664276 is shown in SEQ ID NO.1. There is a SNP site at the 36 bp base of the nucleotide sequence, which is a T / C mutation.

[0007] The present invention also provides a KASP primer combination for identifying the wheat tiller number trait, comprising two forward primers and a universal reverse primer; the nucleotide sequences of the two forward primers are shown as SEQ ID NO.2 and SEQ ID NO.3 respectively, and the nucleotide sequence of the universal reverse primer is shown as SEQ ID NO.4.

[0008] The present invention also provides the use of the above-mentioned KASP primer combination in preparing a product for identifying the tiller number trait.

[0009] Furthermore, the product is a kit.

[0010] The invention also provides a product for identifying the trait of wheat tiller number, comprising the above-mentioned KASP primer combination.

[0011] Furthermore, the product is a kit.

[0012] The present invention also provides the use of the molecular marker CHS10_2B791664276, KASP primer combination or product in identifying the wheat tiller number trait.

[0013] The present invention also provides a method for identifying the trait of wheat tiller number, comprising the following steps:

[0014] Extracting the DNA of the wheat to be tested;

[0015] The DNA is used as a template and the KASP primer combination is used to perform fluorescent quantitative PCR amplification to obtain the genotype of the wheat to be tested. The tiller number trait of the wheat to be tested is determined according to the genotype: the tiller number of CC genotype wheat is lower than that of TT genotype wheat.

[0016] Furthermore, the reaction system of the fluorescent quantitative PCR amplification is: 2×KASP Master mix 5 μL, KASPAssay Mix 0.14 μL, template DNA 50 ng, and DNase / RNase-free deionized water supplemented to 10 μL.

[0017] Furthermore, the reaction procedure of the fluorescent quantitative PCR amplification is: activation at 95°C for 15 min; denaturation at 95°C for 20 s, annealing and extension at 65°C for 60 s, 10 cycles, with the annealing and extension temperature decreasing by 1°C each time; denaturation at 94°C for 20 s, annealing and extension at 57°C for 60 s, 30 cycles; 37°C for 60 s, collecting fluorescence signals.

[0018] The present invention discloses the following technical effects:

[0019] The present invention discloses for the first time a locus tin7 from wheat N2496 that is significantly correlated with the tiller number, and is located on wheat chromosome 2B. This locus has a high utilization value in wheat yield (regulating the tiller number trait) breeding. The present invention discloses for the first time a molecular marker CHS10_2B791664276 for accurately detecting the tiller number locus tin7 of wheat N2496 based on a fluorescent quantitative PCR platform, and the molecular marker detection is accurate and efficient, and the amplification is convenient and stable. The molecular marker CHS10_2B791664276 disclosed by the present invention is significantly correlated with the tiller number locus tin7, has high accuracy for molecular marker-assisted selection, can improve the selection and identification efficiency of wheat tiller number varieties that adapt to different environments, and has a high success rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0021] Figure 1 The present invention is a linkage genetic map of the position of the tiller number locus tin7 on chromosome 2B and the molecular marker CHS10_2B791664276;

[0022] Figure 2 The results of genotyping leaf DNA of N2496, CN16 and CM107 at the three-leaf stage using fluorescent quantitative PCR primers in Example 2 of the present invention;

[0023] Figure 3 This is the result of genotyping the offspring of the F7 generation RIL population of N2496×CM107 in Example 3 of the present invention using fluorescent quantitative PCR primers. DETAILED DESCRIPTION

[0024] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0025] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0026] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0027] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0028] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0029] Example 1 Obtaining the tillering number locus tin7 and the molecular marker CHS10_2B791664276

[0030] In the present invention, the tillering number locus tin7 and the molecular marker CHS10_2B791664276 are obtained by the following method:

[0031] (1) The wheat N2496 with few tillers was used as the female parent and the wheat CN16 as the male parent to obtain the hybrid F1. The F1 generation of individual plants were self-pollinated to obtain an F2 population containing 177 lines, which constituted the genetic population.

[0032] (2) The tillering number of the F2 population plants was identified in the field during the wheat maturity period.

[0033] (3) The DNA of each strain of the genetic population was extracted by the CTAB method, and genotyping was performed using the 55K chip technology with the DNA of the parents N2496 and CN16 as templates to obtain the genotype data of the F2 population. The banding type of the parent N2496 was recorded as A (i.e., the single base difference site was C, and the genotype was CC), and the banding type of the parent CN16 was recorded as B (i.e., the single base difference site was T, and the genotype was TT). The banding type of the F2 population strains derived from the banding type of N2496 was recorded as A, and the banding type derived from CN16 was recorded as B.

[0034] (4) The obtained F2 population genotype data were used to construct a wheat molecular linkage map using JoinMap4.0 mapping software to find the optimal number of markers and marker sequence and determine the linkage group to be used subsequently. The tiller number locus tin7 was located in the section of CHS10_2B791664276 to CHS10_2B796343172 on chromosome 2B using the interval mapping method of Map QTL6.0 software and combined with the tiller number phenotypic data of the F2 population. The CHS10_2B791664276 marker was closely linked to the tiller number locus tin7.

[0035] (5) Convert the SNP sites in the target segment into fluorescent quantitative PCR primers for subsequent screening. Five pairs of fluorescent quantitative PCR primers were designed using the PolyMarker website (Table 1). Fluorescent quantitative PCR primer design standards: amplification primer length 18-30 bp, amplification product length 53-87 bp, annealing temperature 57-62°C, GC content between 40% and 60%.

[0036] The synthetic primer sequences are:

[0037] Forward primer 1: F probe + amplification primer sequence;

[0038] Forward primer 2: H probe + amplification primer sequence;

[0039] Reverse primer: amplification primer sequence;

[0040] Among them, F probe: 5'-GAAGGTGACCAAGTTCATGCT-3' (SEQ ID NO.17, 5' end is bound to FAM fluorescent group);

[0041] H probe: 5'-GAAGGTCGGAGTCAACGGATT-3' (SEQ ID NO. 18, with a HEX fluorescent group bound to the 5' end).

[0042] Table 15 KASP primer sequences and amplified fragment lengths

[0043]

[0044]

[0045] Note: Primers with "-1" in their names are forward primers 1; primers with "-2" in their names are forward primers 2; primers with "-3" in their names are reverse primers.

[0046] (6) Competitive allele-specific PCR (KASP) analysis

[0047] a) Screening of polymorphic molecular markers between parents: The five pairs of primers designed above (as shown in Table 1) were selected, and DNA of parents N2496 and CN16 were used as templates for PCR amplification, and a pair of molecular marker primers with good effects were obtained, named CHS10_2B791664276-1 / 2 / 3 (nucleotide sequences are shown in SEQ ID NO.2-4, respectively). The amplified product is the molecular marker CHS10_2B791664276 with polymorphism, and the nucleotide sequence is shown in SEQ ID NO.1.

[0048] SEQ ID NO.1:

[0049] 5'-GCCCCAGATCCGTCATTATGGCTTGGTTGGGGTCTYTGGCTTTAGTGATAGGGTTTGGTGCAAAGTTTGTT-3'; where Y is C or T.

[0050] b) KASP analysis of F2 population: PCR primers of molecular marker CHS10_2B791664276 with polymorphism obtained in the above steps were used to amplify DNA of parent N2496, CN16 and F2 population plants, and genotype identification was performed to obtain molecular marker data. The type of parent N2496 was recorded as A (i.e. CC genotype), the amplified fragment size was 53 bp, and the single base difference site was C. The type of parent CN16 was recorded as B (i.e. TT genotype), the amplified fragment length was 53 bp, and the single base difference site was T. The type of F2 population strains derived from N2496 was recorded as A, and those derived from CN16 were recorded as B.

[0051] c) Using the interval mapping method of MapQTL6.0 software and combining the tiller number phenotypic data of the F2 population, it was found that the molecular marker CHS10_2B791664276 was closely linked to the tiller number locus tin7. The results are as follows: Figure 1 As shown. Figure 1It can be seen that the tiller number locus tin7 is located in the 2.43 cM segment between markers CHS10_2B791664276 and CHS10_2B796343172, and the molecular marker CHS10_2B791664276 is closely linked to the tiller number locus tin7.

[0052] The location of tiller number locus tin7 on chromosome 2B and its linkage genetic map with molecular marker CHS10_2B791664276 are shown in Figure 1 .

[0053] Example 2 Application of molecular marker CHS10_2B791664276 tightly linked to tiller number locus tin7

[0054] 1. DNA extraction

[0055] The experimental materials were N2496, CN16 and CM107, among which CN16 and CM107 were multi-tillering varieties, and N2496 was a oligo-tillering variety. The leaf DNA of wheat samples at the three-leaf stage was extracted using the CTAB method.

[0056] 2. Primers and fluorescence quantitative PCR detection method for detecting tiller number locus tin7

[0057] (1) Fluorescence quantitative PCR primers

[0058] The primers CHS10_2B791664276-1 / 2 / 3 of Example 1 were used to detect the tillering number locus tin7, wherein the 5' ends of CHS10_2B791664276-1 and CHS10_2B791664276-2 were connected with a FAM fluorescent group and a HEX fluorescent group, respectively.

[0059] (2) Fluorescence quantitative PCR amplification reaction system: 2×KASP Master mix 5 μL, KASPAssay Mix 0.14 μL, template DNA 50 ng, DNase / RNase-free deionized water to 10 μL; wherein KASPAssay Mix contains primers CHS10_2B791664276-1, CHS10_2B791664276-2 and CHS10_2B791664276-3 in a volume ratio of 2:2:5. That is, in KASPAssay Mix, primers CHS10_2B791664276-1, CHS10_2B791664276-2 and CHS10_2B791664276-3 at a concentration of 100 μM are mixed in a volume ratio of 2:2:5.

[0060] (3) Fluorescence quantitative PCR program: activation at 95°C for 15 min; denaturation at 95°C for 20 s, annealing and extension at 65°C for 60 s, 10 cycles, with the annealing and extension temperature decreasing by 1°C each time; denaturation at 94°C for 20 s, annealing and extension at 57°C for 60 s, 30 cycles; 37°C for 60 s, collecting fluorescence signals.

[0061] (4) The specific method for analyzing PCR products is as follows: Wheat samples containing the tiller number locus tin7 all showed the same genotype as wheat N2496, recorded as type A (i.e., CC genotype), while wheat samples without the tiller number locus tin7 all showed fluorescent signals that were significantly different from wheat N2496, such as CN16 and CM107, recorded as type B (i.e., TT genotype). The results of genotyping N2496, CN16, and CM107 using KASP primers are shown in Figure 2 .

[0062] Example 3

[0063] Suitability of primer sequence CHS10_2B791664276-1 / 2 / 3 in population detection:

[0064] (1) The hybridization of N2496 and CM107 was carried out to obtain F1, and the F1 was self-crossed continuously to the F7 generation to obtain the validation population. The leaf DNA of each strain in the population was extracted at the three-leaf stage.

[0065] (2) Using the DNA obtained in step (1) as a template, fluorescent quantitative PCR amplification was performed using the primers CHS10_2B791664276-1 / 2 / 3 of Example 1. The fluorescent quantitative PCR amplification reaction system and procedure were the same as those of Example 2.

[0066] (3) The specific method for analyzing PCR products is as follows: Wheat samples containing the tiller number locus tin7 all showed the same genotype as wheat N2496, recorded as type A (i.e., CC genotype), while wheat samples without the tiller number locus tin7 all showed fluorescent signals that were significantly different from wheat N2496, recorded as type B (i.e., TT genotype). The results are shown in Figure 3 125 strains were tested, of which 57 strains could amplify the same type of fragment as N2496, which are plants containing the tiller number locus tin7, and it is predicted that the strains will have few tillers at maturity. 68 strains could amplify the same B-type fragment as CN16 and CM107, which are plants without the tiller number locus tin7, and it is predicted that these plants will have more tillers at maturity.

[0067] (4) At the wheat maturity stage, the tiller number of 125 F7 plants was identified, and the results are shown in Table 2. The results showed that the molecular marker CHS10_2B791664276 of the tiller number locus tin7 can predict the tiller number of the genetic population. Wheat varieties containing the tiller number locus tin7 all showed the same fluorescence signal as the fluorescent probe connected to the primer shown in SEQ ID NO.2, while wheat varieties not containing the tiller number locus tin7 all showed a fluorescence signal significantly different from the fluorescent probe connected to the primer shown in SEQ ID NO.2. The average tiller number of plants of the same type as N2496 was 3.41, which was significantly lower than the average tiller number of plants of the same type as CN16 and CM107, which was 5.99. The actual results were consistent with the expected results, indicating that the tiller number locus tin7 of the present invention does have the effect of significantly improving the tiller number, and the molecular marker CHS10_2B791664276 can be used to track and identify the tiller number locus tin7.

[0068] Table 2 Statistics of genotypes and tiller numbers of 125 F7 plants

[0069]

[0070]

[0071]

[0072]

[0073] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A KASP primer combination for identifying the tiller number trait of wheat, characterized in that: It comprises two forward primers and a universal reverse primer; the nucleotide sequences of the two forward primers are shown as SEQ ID NO.2 and SEQ ID NO.3 respectively, and the nucleotide sequence of the universal reverse primer is shown as SEQ ID NO.

4.

2. Use of the KASP primer combination as claimed in claim 1 in preparing a product for identifying the trait of tiller number of wheat.

3. The use according to claim 2, characterized in that: The product is a test kit.

4. A product for identifying the trait of wheat tiller number, characterized in that: Comprising the KASP primer combination according to claim 1.

5. The product according to claim 4, characterized in that The product is a test kit.

6. Use of the KASP primer combination according to claim 1 or the product according to claim 4 or 5 in identifying the trait of tiller number in wheat.

7. A method for identifying the trait of tiller number in wheat, characterized in that: The following steps are involved: Extracting the DNA of the wheat to be tested; The DNA is used as a template and the KASP primer combination of claim 1 is used to perform fluorescent quantitative PCR amplification to obtain the genotype of the wheat to be tested, and the tiller number trait of the wheat to be tested is determined according to the genotype: the tiller number of CC genotype wheat is lower than that of TT genotype wheat.

8. The method according to claim 7, characterized in that The reaction system of the fluorescent quantitative PCR amplification is: 2×KASP Master mix 5 μL, KASP Assay Mix 0.14 μL, template DNA 50 ng, and DNase / RNase-free deionized water to 10 μL.

9. The method according to claim 7, characterized in that: The reaction procedure of the fluorescent quantitative PCR amplification is as follows: activation at 95°C for 15 min; denaturation at 95°C for 20 s, annealing and extension at 65°C for 60 s, 10 cycles, with the annealing and extension temperature decreasing by 1°C each time; denaturation at 94°C for 20 s, annealing and extension at 57°C for 60 s, 30 cycles; 37°C for 60 s, collecting fluorescence signals.

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