Molecular marker of wheat ta brd2-7a gene and application thereof

Abstract

The application discloses a wheat TaBRD2-7A gene molecular marker and application thereof, and belongs to the technical field of crop selection and cultivation. The molecular marker is a KASP marker, which is amplified by a primer group shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, the 5' ends of the two front primers are marked with FAM and HEX respectively, the PCR amplification product is detected by using a multifunctional enzyme marker, and genotyping is performed by using KlusterCaller software. The application has the advantages that: by applying the KASP molecular marker developed by the application, wheat excellent haplotypes can be identified in wheat line selection and breeding, not only the cost is saved, but also the selection efficiency is greatly improved, the breeding process is accelerated, and new possibilities are provided for efficient screening of excellent alleles of the TaBRD2-7A gene and cultivation of high-yield wheat varieties.

Description

TECHNICAL FIELD

[0001] The present application relates to molecular markers and their applications, in particular to a molecular marker of a wheat TaBRD2-7A gene and its application in assisted breeding, and belongs to the technical field of crop selection and cultivation. BACKGROUND

[0002] Wheat (Triticum aestivum L.) is the most widely distributed and largest grain crop in the world, and it is of great significance to improve wheat yield. In the early stage, the homologous gene TaBRD2-7A of rice OsBRD2 was obtained by homologous cloning method, and its biological function was analyzed by mutant identification and genetic transformation technology. The research results show that the wheat TaBRD2-7A gene has a positive regulation effect on plant height, thousand kernel weight and kernel length. However, the effect of natural variation of wheat TaBRD2-7A gene on wheat yield and related molecular markers have not been reported. Therefore, it is of great significance to mine the haplotype of TaBRD2-7A gene and develop useful molecular markers for wheat molecular genetic improvement and yield improvement. SUMMARY

[0003] The purpose of the present application is to provide a molecular marker of TaBRD2-7A gene related to wheat plant height, thousand kernel weight and kernel length and its application in identifying wheat haplotype, aiming to provide effective gene resources and molecular markers for crop genetic improvement and molecular marker assisted selection.

[0004] In order to achieve the above-mentioned goal, the technical scheme adopted by the present application is as follows:

[0005] A molecular marker of a wheat TaBRD2-7A gene, the TaBRD2-7A gene comprising two haplotypes of TaBRD2-7A-HapI and TaBRD2-7A-HapII, wherein the allelic variant bases of G, C, T, G, C, T, G, A, A, A, A, C, G, G, G, C, G, G, A, C and C at positions 738268917, 738268911, 738268853, 738268818, 738268815, 738268796, 738265454, 738265450, 738265128, 738265120, 738265115, 738264128, 738264092, 738263741, 738263738, 738263681, 738263663, 738263660, 738262835, 738262832 and 738262827 in the Chinese Spring wheat reference genome sequence RefSeq v2.1 are respectively G, C, T, G, C, T, G, A, A, A, A, C, G, G, G, C, G, G, A, C and C, and the allelic variant bases of G, A, A, A, A, C, G, G, G, C, G, G, A, C and C at positions 738262829, 738262826, 738262823, 738262820, 738262817, 738262814, 738262811, 738262808, 738262805, 738262802, 738262799, 738262796, 738262793, 738262790, 738262787, 738262784, 738262781, 738262778, 738262775, 738262772, 738262769, 738262766, 738262763, 738262760, 738262757, 738262754, 738262751, 738262748, 738262745, 738262742, 738262739, 738262736, 738262733, 738262730, 738262727, 738262724, 738262721, 738262718, 738262715, 738262712, 738262709, 738262706, 738262703, 738262700, 738262697, 738262694, 738262691, 738262688, 738262685, 738262682, 738262679, 738262676, 738262673, 738262670, 738262667, 738262664,The allelic variant bases of the molecular markers of the 738268917th, 738268911th, 738268853th, 738268818th, 738268815th, 738268796th, 738265454th, 738265450th, 738265128th, 738265120th, 738265115th, 738264128th, 738264092th, 738263741th, 738263738th, 738263681th, 738263663th, 738263660th, 738262835th, 738262832th and 738262827th in SEQ ID NO: 1 and SEQ ID NO: 2 are A, G, C, A, G, C, C, G, G, G, G, G, A, A, A, G, A, C, G, G and A respectively, and the molecular marker is a KASP marker, which is amplified by two forward primers shown in SEQ ID NO: 1 and SEQ ID NO: 2 and one reverse primer shown in SEQ ID NO: 3, wherein the 5' end of the forward primer shown in SEQ ID NO: 1 is labeled with FAM, the 5' end of the forward primer shown in SEQ ID NO: 2 is labeled with HEX, the PCR amplification product is detected by using a multifunctional enzyme marker, and the KlusterCaller software is used for genotyping, when the result of the KlusterCaller is blue, the haplotype of the wheat TaBRD2-7A gene is TaBRD2-7A-HapI, and when the result of the KlusterCaller is red, the haplotype of the wheat TaBRD2-7A gene is TaBRD2-7A-HapII.

[0006] The aforementioned molecular marker of the wheat TaBRD2-7A gene is applied to breeding the TaBRD2-7A-HapI haplotype of wheat, and the TaBRD2-7A-HapI haplotype has lower plant height, larger thousand-grain weight and longer grain length compared with the TaBRD2-7A-HapII haplotype.

[0007] The present application has the advantages that:

[0008] (1) The present application develops a new KASP molecular marker, and the KASP molecular marker developed in the present application is derived from the genetic variation analysis of the TaBRD2-7A gene in a natural population of wheat, provides unique genetic information, and provides a new tool for wheat breeding;

[0009] (2) The KASP molecular marker developed by applying the application can identify excellent haplotypes of wheat in wheat line breeding, not only saving cost, but also greatly improving selection efficiency and accelerating breeding process, thereby providing new possibility for efficient screening of excellent alleles of TaBRD2-7A gene and cultivation of high-yield wheat varieties. BRIEF DESCRIPTION OF DRAWINGS

[0010] Figure 1 is a SNP variation site of the wheat TaBRD2-7A gene and a haplotype typing result diagram;

[0011] Figure 2 is a KASP marker genotype typing result diagram of part of the wheat TaBRD2-7A gene, wherein blue represents GG, red represents AA, and green represents heterozygous. DETAILED DESCRIPTION

[0012] The application will be specifically introduced below in combination with the drawings and examples.

[0013] I. Acquisition of wheat TaBRD2-7A gene polymorphic sites and haplotypes

[0014] 1. Acquisition of wheat TaBRD2-7A gene polymorphic sites

[0015] The wheat TaBRD2-7A gene segment (2 kb of promoter + full length of gene, SEQ ID NO: 4) was selected, sequence difference analysis in the interval was performed in the wheat genome variation union database (http: / / wheat.cau.edu.cn / WheatUnion / b_3 / ), 1726 samples in the database were selected, and the variation type was SNP.

[0016] The sequence difference analysis results show that there are 21 co-segregation SNP sites in the wheat TaBRD2-7A gene, which are respectively marked as SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, SNP7, SNP8, SNP9, SNP10, SNP11, SNP12, SNP13, SNP14, SNP15, SNP16, SNP17, SNP18, SNP19, SNP20, SNP21. The physical positions of the 21 SNP variation sites in the Chinese spring wheat reference genome sequence RefSeq v2.1 are respectively 738268917, 738268911, 738268853, 738268818, 738268815, 738268796, 738265454, 738265450, 738265128, 738265120, 738265115, 738264128, 738264092, 738263741, 738263738, 738263681, 738263663, 738263660, 73826835, 738262832, 738262827.

[0017] 2, Obtaining of wheat TaBRD2-7A gene haplotype

[0018] The sequence difference analysis in the interval is carried out by integrating the wheat genome variation union database network (http: / / wheat.cau.edu.cn / WheatUnion / b_3 / ), 1726 samples in the database are selected, and the haplotype is predicted. In combination with the existing sequencing data of the laboratory, one-generation sequencing is carried out on 20 wheat varieties with high genetic diversity to verify the sequence polymorphism and determine the haplotype.

[0019] The sequence analysis results show that there are 21 SNP variations in the wheat TaBRD2-7A gene, of which 6 SNP variations exist in the promoter region, 5 SNP variations exist in the first intron region, 7 SNP variations exist in the first exon region, and 3 SNP variations exist in the second exon region.

[0020] According to the comprehensive variation type, 2 haplotypes (Table 1-1, Table 1-2) are detected: TaBRD2-7A-HapI, TaBRD2-7A-HapII. Figure 1

[0021] Table 1-1 Sequence difference analysis results of 1726 samples (TaBRD2-7A-HapI)

[0022]

[0023] ​Table 1-2 1726 sample sequence difference analysis results (TaBRD2-7A-HapII)

[0024]

[0025] Two, identification of wheat TaBRD2-7A gene haplotype

[0026] Kompetitive allele-specific PCR (KASP) molecular marker assisted selection breeding can select target traits at the DNA level, not only the results are stable, reduce the cost of phenotype evaluation, but also can improve the efficiency of wheat breeding.

[0027] In order to further verify and use the SNP variation sites obtained, further develop KASP markers. From SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, SNP7, SNP8, SNP9, SNP10, SNP11, SNP12, SNP13, SNP14, SNP15, SNP16, SNP17, SNP18, SNP19, SNP20 and SNP21, SNP4 is selected, the primer sequence is designed by using WheatOmics (http: / / wheatomics.sdau.edu.cn / PrimerServer / ), and the primer sequence is evaluated by using WheatOmics, and finally verified in 232 materials.

[0028] For SNP4, the designed primer is composed of two forward primers (Primer Allele X, Primer Allele Y) and one reverse primer (Common), wherein the specific sequences of the two forward primers are SEQ ID NO: 1 and SEQ ID NO: 2 respectively, and the specific sequence of the reverse primer is SEQ ID NO: 3. In order to detect the PCR amplification product by using the enzyme label instrument, the FAM fluorescent group is labeled at the 5' end of the forward primer (Primer Allele X) shown in SEQ ID NO: 1, and the labeled primer is recorded as AlleleFAM. The HEX fluorescent group is labeled at the 5' end of the forward primer (Primer Allele Y) shown in SEQ ID NO: 2, and the labeled primer is recorded as AlleleHEX.

[0029] The DNA of 232 common hexaploid wheat varieties (seedlings) in Table 2-1, Table 2-2, Table 2-3 and Table 2-4 is subjected to PCR amplification reaction by using the above two labeled forward primers (AlleleFAM, AlleleHEX) and one reverse primer (Common), and the PCR reaction system and amplification program are as follows:

[0030] (1) The PCR reaction system specifically has: 2 μL of DNA template (30 ng / μL), 2.5 μL of KASP Master Mix (LGC Genomics, KBS-1016-002), and 0.08 μL of primer working solution, supplemented with sterile ultrapure water to 5 μL, wherein the preparation method of the primer working solution is as follows: 12 μL of AlleleFAM with a concentration of 100 μM, 12 μL of AlleleHEX with a concentration of 100 μM, and 30 μL of the latter primer (Common) with a concentration of 100 μM are mixed, and ddH2O is added to 100 μL;

[0031] (2) The amplification program specifically as follows: 95°C pre-denaturation for 15 min; 95°C denaturation for 20 s, 65-57°C annealing and extension for 60 s, 9 cycles, each cycle decreasing by 1°C; 95°C denaturation for 20 s, 57°C recombination for 60 s, 32 cycles; 10°C preservation.

[0032] The PCR product is sequenced, and the TaBRD2-7A gene is typed according to the sequencing result, specifically:

[0033] When the allelic variation base of SNP4 is G, the typing result of the wheat TaBRD2-7A gene is TaBRD2-7A-HapI;

[0034] When the allelic variation base of SNP4 is A, the typing result of the wheat TaBRD2-7A gene is TaBRD2-7A-HapII.

[0035] The typing results of the TaBRD2-7A gene of 232 wheat natural populations are shown in Tables 2-1, 2-2, 2-3, and 2-4.

[0036] Table 2-1 Typing results of the TaBRD2-7A gene of wheat from the 1st to the 60th

[0037]

[0038] Table 2-2 Typing results of the TaBRD2-7A gene of wheat from the 61st to the 120th

[0039]

[0040] Table 2-3 Typing results of the TaBRD2-7A gene of wheat from the 121st to the 180th

[0041]

[0042] Table 2-4 Typing results of the TaBRD2-7A gene of wheat from the 181st to the 232nd

[0043]

[0044] KASP marker genotyping was performed on 232 common hexaploid wheat varieties listed in Table 2-1, Table 2-2, Table 2-3 and Table 2-4. The operation steps are as follows:

[0045] (1) PCR amplification

[0046] Using the genomic DNA of the wheat to be tested as the template, the above two forward primers (AlleleFAM, AlleleHEX) and one reverse primer (Common) were used for PCR amplification reaction, and the PCR reaction system and amplification program were the same as before, which will not be repeated here.

[0047] The experiment also set up a blank control (NTC) without adding DNA template in the PCR reaction system, and 1 or more blank controls were set for each plate.

[0048] The PCR amplification products were scanned by a multifunctional enzyme marker (PHERAstar Plus, BMG LABTECH, Germany), the FAM excitation wavelength was 485 nm, the emission wavelength was 520 nm; the HEX excitation wavelength was 535 nm, the emission wavelength was 556 nm, and the system reference fluorescence ROX excitation wavelength was 575 nm, the emission wavelength was 610 nm.

[0049] The scanning data of the multifunctional enzyme marker was analyzed by KlusterCaller software (LGC Genomics, Beverly, USA), and the genotype was analyzed according to the fluorescence signal, specifically:

[0050] When the result of KlusterCaller is blue, the genotype of wheat TaBRD2-7A gene at SNP 4 site is GG, and the haplotype is TaBRD2-7A-HapI;

[0051] When the result of KlusterCaller is red, the genotype of wheat TaBRD2-7A gene at SNP 4 site is AA, and the haplotype is TaBRD2-7A-HapII;

[0052] When the result of KlusterCaller is green, the genotype of wheat TaBRD2-7A gene at SNP 4 site is hybrid.

[0053] The KASP marker genotyping results of TaBRD2-7A gene of some wheat varieties are shown in Table 2-5, Table 2-6, Table 2-7 and Table 2-8. Figure 2 .

[0054] KASP marker genotyping results show that the KASP marker genotyping results are completely consistent with the network data haplotype prediction results, and the KASP marker genotyping results are good.

[0055] This shows that the KASP marker development is successful, and can be further applied to breeding material detection.

[0056] Three, correlation analysis of wheat TaBRD2-7A gene haplotype and yield traits

[0057] Statistical 3 years 7 environments (E1: 2020 Yantai Lai Mountain, E2: 2020 in the school, E3: 2021 Hebei Shijiazhuang, E4: 2021 Yantai Lai Mountain, E5: 2021 Yantai Lai Mountain, E6: 2022 Yantai Muyu Village, E7: 2022 Yantai Lai Mountain) of wheat plant height, thousand kernel weight and grain length of natural population phenotype data, using R package lme4 to calculate the best linear unbiased estimate (BLUE) of each trait in multiple environments.

[0058] Statistical analysis of data using Excel software, Student's t-test for significance analysis, wheat TaBRD2-7A gene multi-environment agronomic traits (thousand kernel weight, plant height, grain length) correlation analysis results are shown in Table 3-1, Table 3-2 and Table 3-3.

[0059] Table 3-1 Correlation analysis results of wheat TaBRD2-7A gene in multiple environments

[0060]

[0061] Note: BLUE_ Thousand kernel weight represents the best linear unbiased estimate of thousand kernel weight in 7 environments; * represents P <0.05, ** represents P <0.01.

[0062] Table 3-2 Correlation analysis results of wheat TaBRD2-7A gene in multiple environments

[0063]

[0064] Note: BLUE_ Plant height represents the best linear unbiased estimate of plant height in 7 environments; * represents P <0.05, ** represents P <0.01.

[0065] Table 3-3 Correlation analysis results of wheat TaBRD2-7A gene in multiple environments

[0066]

[0067] Note: BLUE_grain length represents the best linear unbiased estimate of grain length in 7 environments; * represents P <0.05, ** represents P <0.01.

[0068] In summary, haplotype analysis results show that:

[0069] (1) Compared with TaBRD2-7A-HapII type, the average height of TaBRD2-7A-HapI is significantly reduced by 6.70%;

[0070] (2) Compared with TaBRD2-7A-HapII type, the average thousand-grain weight of TaBRD2-7A-HapI is significantly increased by 6.67%;

[0071] (3) Compared with TaBRD2-7A-HapII type, the grain length of TaBRD2-7A-HapI is significantly increased by 2.20%.

[0072] It can be seen that the two haplotypes of wheat TaBRD2-7A gene have significant regulatory effects on the plant height, thousand-grain weight and grain length of wheat. In combination with the performance of various traits, the wheat of TaBRD2-7A-HapI type has relatively excellent agronomic traits, and TaBRD2-7A-HapI type is an excellent haplotype of wheat, which has potential value in high-yield variety breeding.

[0073] In summary, the two haplotypes of wheat TaBRD2-7A gene identified by the application are significantly associated with the plant height, thousand-grain weight and grain length of wheat, and have important application value for improving the yield traits of wheat.

[0074] It should be noted that the above examples are merely examples for clearly illustrating the present application, and are not intended to limit the embodiments of the present application. Based on the above description, other different forms of changes or modifications can be made by those skilled in the art. Here, all the embodiments cannot be exhausted. Any obvious changes or modifications derived from the technical scheme of the present application are still within the protection scope of the present application.

Claims

1. Application of a molecular marker of a wheat TaBRD2-7A gene in identifying plant height, thousand kernel weight and kernel length of wheat, characterized in that, The TaBRD2-7A gene comprises two haplotypes of TaBRD2-7A-HapI and TaBRD2-7A-HapII, wherein the allelic variation base at position 738268818 in the Chinese Spring wheat reference genome sequence RefSeq v2.1 of TaBRD2-7A-HapI is G, the allelic variation base at position 738268818 in the Chinese Spring wheat reference genome sequence RefSeq v2.1 of TaBRD2-7A-HapII is A, the molecular marker is a KASP marker, which is amplified by two forward primers represented by SEQ ID NO: 1 and SEQ ID NO: 2 and one reverse primer represented by SEQ ID NO: 3, wherein the 5' end of the forward primer represented by SEQ ID NO: 1 is labeled with FAM, and the 5' end of the forward primer represented by SEQ ID NO: 2 is labeled with HEX, the PCR amplification product is detected by using a multifunctional enzyme marker, and genotyping is performed by using KlusterCaller software, when the result of KlusterCaller is blue, the allelic variation base at position 738268818 of the wheat TaBRD2-7A gene is G, and the haplotype is TaBRD2-7A-HapI, when the result of KlusterCaller is red, the allelic variation base at position 738268818 of the wheat TaBRD2-7A gene is A, and the haplotype is TaBRD2-7A-HapII; Compared with the TaBRD2-7A-HapII haplotype, the TaBRD2-7A-HapI haplotype has lower plant height, larger thousand-grain weight and longer grain length.

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