Molecular markers tightly linked to the major QTL for zinc content in wheat grains and their application

Abstract

The present invention discloses a molecular marker tightly linked to a major QTL for zinc content in wheat grains and its application, and belongs to the technical field of wheat molecular genetic breeding. The molecular marker and the major QTL for zinc content in wheat grains QGrZnc.sau‑4A are co-localized on the short arm of chromosome 4A of the wheat genome; the molecular marker is located at the 36th base of the nucleotide sequence shown in SEQ ID NO.1, and there is a C / T mutation. The major QTL for zinc content in wheat grains QGrZnc.sau‑4A of the present invention is located on chromosome 4A, and has the effect of significantly improving the zinc content in wheat grains. The molecular marker provided by the present invention is tightly linked to the QTL, and can be used for molecular marker-assisted detection of QTL and molecular breeding, thereby improving detection efficiency and breeding efficiency.

Description

Technical Field

[0001] The invention relates to the technical field of wheat molecular genetic breeding, and in particular to a molecular marker tightly linked to a major effect QTL of zinc content in wheat grains and an application thereof. Background Art

[0002] Wheat (Triticum sp.) is one of the most important food crops in the world. Faced with the increase in the global population, most breeders focus their research on wheat yield traits and ignore the improvement of nutritional elements. Related studies have shown that the reduction of zinc content in wheat grains has become a global problem. Current strategies to improve human nutrition mainly include dietary diversity, drug supplementation, industrial fortification and biofortification. Due to the limited diet of people in poor areas and insufficient industrial investment in fortification, biofortification has become the most promising, cost-effective and sustainable strategy to increase the zinc content of wheat grains to solve the problem of malnutrition.

[0003] Relevant studies have shown that the zinc content of existing wheat varieties in China is generally low. Therefore, it is necessary to cultivate wheat varieties with high zinc content. In the process of improving common wheat, due to the use of a few backbone parent resources, the genetic diversity among varieties has gradually decreased, and the improvement of zinc is limited by a narrow genetic basis. The adverse effects on future wheat improvement breeding are obvious. The large amount of untapped genetic resources in wheat relatives can effectively help us face the challenges of future wheat production. Accelerating the use of unique genetic resources in relatives is very important for increasing the zinc content of common wheat grains. Different genotypes of wheat have different abilities to absorb and utilize nutrients. Therefore, it is committed to the study of nutritional genetic positioning of zinc-efficient genotypes, improving and improving the efficiency of wheat absorption and utilization of soil zinc, and effective measures to cultivate zinc-efficient wheat varieties.

[0004] Single Nucleotide Polymorphism (SNP) marker refers to a mutation in a single nucleotide site A, T, G or C at a specific position in the nucleotide sequence, which changes the DNA sequence and causes polymorphism. SNPs are widely distributed in plant genomes and are present in large numbers in both the coding and non-coding regions of genes. For organisms with simpler genomes, SNPs are often used to scan the entire genome, construct high-density genetic maps, and perform QTL detection and analysis of important traits; for wheat with a more complex genome, SNPs are often used in combination with gene chips, such as 9K, 55K and 660K SNP chips, to detect genomic regions related to target traits and promote the breeding process.

[0005] Competitive allele-specific PCR (KASP) technology is a new type of SNP detection technology that has emerged with the development of modern science and technology. Its working principle is to design specific primers for SNP sites, and to perform labeling and detection through the specificity of SNP. It can be applied to a wide range of genomic DNA. Its Master Mix is ​​usually based on ordinary PCR and uses two special fluorescent groups and quenching groups as complementary probes. KASP technology has the characteristics of high throughput, accurate results, low cost, simple operation, and no need for electrophoresis detection, which well solves the shortcomings of other markers in application.

[0006] Previously, some scholars reported on the QTL positioning of wheat grain zinc content. However, there are currently not many molecular markers that are tightly linked to the grain zinc content trait and can be used for actual molecular breeding. Therefore, it is of great guiding significance to further identify more practical QTLs or genes for grain zinc content, use molecular biology techniques to select wheat strains with suitable grain zinc content, and use molecular biology techniques for wheat breeding. Summary of the invention

[0007] The purpose of the present invention is to provide a molecular marker tightly linked to a major QTL for zinc content in wheat grains and its application, so as to solve the problems existing in the above-mentioned prior art. The major QTL for zinc content in wheat grains, QGrZnc.sau-4A, of the present invention is located on chromosome 4A, and has the effect of significantly increasing the zinc content in wheat grains. The molecular marker provided by the present invention is tightly linked to the QTL, and can be used for molecular marker-assisted detection of QTL and molecular breeding, thereby improving detection efficiency and breeding efficiency.

[0008] To achieve the above object, the present invention provides the following solutions:

[0009] The present invention provides a molecular marker tightly linked to a major QTL QGrZnc.sau-4A for zinc content in wheat grains, wherein the molecular marker and the major QTL QGrZnc.sau-4A for zinc content in wheat grains are co-localized on the short arm of chromosome 4A of the wheat genome;

[0010] The molecular marker is located at the 36th base of the nucleotide sequence shown in SEQ ID NO.1, and there is a C / T mutation.

[0011] Preferably, the major QTL QGrZnc.sau-4A for wheat grain zinc content is located at 60.96–78.86 Mbp of chromosome 4A of Chinese spring, or 54.43–60.02 Mbp of chromosome 4A of wild two-grain wheat.

[0012] The present invention also provides an application of the above molecular marker in any of the following items:

[0013] A1. Detection of the major QTL QGrZnc.sau-4A for zinc content in wheat grains;

[0014] A2. Early identification or selection of wheat varieties with high grain zinc content;

[0015] A3. Wheat molecular genetics breeding;

[0016] A4. Improvement of wheat germplasm resources;

[0017] A5. Conduct genetic analysis and fine mapping of genes related to zinc content in wheat grains;

[0018] The major QTL QGrZnc.sau-4A for zinc content in wheat grains is located at 60.96–78.86 Mbp of chromosome 4A of Chinese spring wheat or 54.43–60.02 Mbp of chromosome 4A of wild two-grain wheat.

[0019] The present invention also provides a KASP primer set targeting the above molecular markers, wherein the KASP primer set comprises primers as shown in SEQ ID NO. 2-4.

[0020] The present invention also provides an application of the KASP primer set in any of the following items:

[0021] B1. Detection of the major QTL QGrZnc.sau-4A for zinc content in wheat grains;

[0022] B2. Early identification or selection of wheat varieties with high grain zinc content;

[0023] B3. Wheat molecular genetics breeding;

[0024] B4. Improvement of wheat germplasm resources;

[0025] B5. Conduct genetic analysis and fine positioning of genes related to zinc content in wheat grains;

[0026] B6. Prepare reagents, kits or chips for detecting the major QTL QGrZnc.sau-4A for zinc content in wheat grains;

[0027] The major QTL QGrZnc.sau-4A for zinc content in wheat grains is located at 60.96–78.86 Mbp of chromosome 4A of Chinese spring wheat or 54.43–60.02 Mbp of chromosome 4A of wild two-grain wheat.

[0028] The present invention also provides a kit for detecting the major QTL QGrZnc.sau-4A of zinc content in wheat grains, wherein the kit comprises the above-mentioned KASP primer set; the major QTL QGrZnc.sau-4A of zinc content in wheat grains is located at 60.96-78.86Mbp of chromosome 4A of Chinese spring, or 54.43-60.02Mbp of chromosome 4A of wild two-grain.

[0029] The present invention also provides a method for detecting the main effect QTL QGrZnc.sau-4A of zinc content in wheat grains, comprising the following steps:

[0030] Using the genomic DNA of the plant to be tested as a template, the KASP primer set is used to perform fluorescence quantitative PCR amplification, and the determination is made based on the fluorescence reading results;

[0031] The plants in which the fluorescent group marked by SEQ ID NO.2 was read were identified as plants containing the major QTL QGrZnc.sau-4A for the zinc content of wheat grains; the plants in which the fluorescent group marked by SEQ ID NO.3 was read were identified as plants not containing the major QTL QGrZnc.sau-4A for the zinc content of wheat grains;

[0032] The major QTL QGrZnc.sau-4A for zinc content in wheat grains is located at 60.96–78.86 Mbp of chromosome 4A of Chinese spring wheat or 54.43–60.02 Mbp of chromosome 4A of wild two-grain wheat.

[0033] Preferably, the reaction system of the fluorescent quantitative PCR is: 5 μL Master Mix, 5 ng template DNA, 1.4 μL mixed primers, ddH 2 O was added to a total volume of 10 μL;

[0034] The mixed primers consist of 10 ng / µL of the primer shown in SEQ ID NO.2, 10 ng / µL of the primer shown in SEQ ID NO.3, 10 ng / µL of the primer shown in SEQ ID NO.4 and ddH 2 O is mixed in a volume ratio of 6:6:15:23.

[0035] Preferably, the fluorescent quantitative PCR reaction procedure is: pre-denaturation at 94°C for 15 min; denaturation at 94°C for 20 s, annealing / extension at 61°C for 60 s, for a total of 10 cycles; denaturation at 94°C for 20 s, annealing / extension at 55°C for 60 s, for a total of 26 cycles.

[0036] The present invention discloses the following technical effects:

[0037] (1) The present invention discloses for the first time the major QTL QGrZnc.sau-4A of zinc content in grains from wild emmer wheat 'LM001', which is located on the short arm of chromosome 4A of tetraploid wheat and can significantly increase the zinc content in wheat grains. This QTL has a high utilization value in wheat breeding for regulating zinc content in grains.

[0038] (2) The present invention discloses a molecular marker KASP-GrZnc-sau9087 for accurately detecting QTLQGrZnc.sau-4A of the zinc content in the grain of wheat 'LM001' based on a fluorescent quantitative PCR platform. The marker is a co-dominant marker with accurate and efficient detection and convenient and stable amplification.

[0039] (3) The molecular marker KASP-GrZnc-sau9087 disclosed in the present invention is significantly correlated with the grain zinc content QTL QGrZnc.sau-4A, showing the characteristics of a tightly linked marker, and can be used to detect the major effect QTL for grain zinc content on wheat chromosome 4A, and to quickly screen plants with this locus with high accuracy, which can significantly improve the efficiency of selecting and identifying wheat varieties with higher grain zinc content that are adapted to different environments, and has a high success rate.

[0040] (4) The molecular marker KASP-GrZnc-sau9087 provided by the present invention is closely linked to the grain zinc content QTL QGrZnc.sau-4A on wheat 4A, and can be used to locate the wheat grain zinc content trait, thereby eliminating plants with low grain zinc content during the breeding process, improving breeding efficiency, and providing a basis for the study of wheat grain zinc content genes. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] 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.

[0042] Figure 1 The co-localization map of the major QTL QGrZnc.sau-4A and molecular marker KASP-GrZnc-sau9087 for zinc content in wheat grains on chromosome 4A in Example 1;

[0043] Figure 2 The fluorescence reading results of the Chinese local wheat population detected by the molecular marker KASP-GrZnc-sau9087 in Example 2; wherein, the FAM (circle) fluorescence is the strain with the same genotype as 'LM001', the HEX (square) fluorescence is the strain with the same genotype as 'Ailanmai'; the black diamond fluorescence is the blank control;

[0044] Figure 3 The phenotypic detection results of the grain zinc content of the Chinese local wheat population in Example 2; wherein Allele 1 is a population strain consistent with the 'Ailanmai' genotype, and Allele 2 is a population strain consistent with the 'LM001' genotype. DETAILED DESCRIPTION

[0045] 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.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0050] The wheat lines 'Ailanmai', 'LM001' and 'Chinese local wheat' used in the following examples were all provided by the Wheat Department of Sichuan Agricultural University.

[0051] Example 1

[0052] Obtaining the major QTL QGrZnc.sau-4A for zinc content in wheat grains and its molecular marker KASP-GrZnc-sau9087:

[0053] (1) The wheat line 'Ailanmai' was used as the female parent and the wheat line 'LM001' as the male parent to obtain the hybrid F1. The F1 generation was self-pollinated to obtain the F2. The single ear method was used in the F2 generation until the F9 generation. A total of 121 recombinant inbred lines were obtained, which constituted the genetic mapping population.

[0054] (2) Phenotypic identification of zinc content in grains of recombinant inbred line populations: A field experiment was conducted, with a total of 5 ecological sites (Wenjiang in 2020, Ya'an in 2020, Wenjiang in 2021, Wenjiang in 2022, and Chongzhou in 2022). One row of each material was planted at each environmental site (row length 1.5 m, row spacing 30 cm, plant spacing 10 cm). At maturity, three plants were randomly selected from each row, and the harvested grains were placed at 105°C for 30 min and then dried at 80°C to constant weight. After weighing the dry weight, grind and sieve, weigh 0.20 g of the sample and add 5 mL of mixed acid [HClO 4 / HNO 3 (v / v=4 / 1)] digestion for 12 hours, heating at 220℃ for 4-5 hours, and stopping when the solution is colorless and transparent and there is 1.00mL. After the liquid cools down, use 1% HNO 3 The volume was fixed and filtered. The zinc content was determined using an inductively coupled plasma mass spectrometer (ICP-MS; Nexlon 2000, PerkinElmer, USA).

[0055] (3) 55K SNP chip analysis

[0056] a) DNA extraction: DNA of the parental 'Ailanmai', 'LM001' and recombinant inbred line population plants was extracted using the CTAB method.

[0057] b) The extracted DNA was tested for quality using an ultra-micro spectrophotometer, and qualified samples were sent to the company for genotype analysis. In this embodiment, the genotype analysis of the parent-mapping population was completed by the wheat 55K SNP chip developed in collaboration between Beijing Capital Biotech Co., Ltd. (http: / / www.capitalbiotech.com) and Jia Jizeng's research group.

[0058] c) Construction of genetic linkage map: Based on the 55K SNP chip data, a genetic linkage map was constructed using JoinMap4.0. Combined with the maximum root length phenotype data of the population, the Inclusive Composite Interval Mapping-ADD (ICIM-ADD) method in QTL IciMapping version 4.2 was used to detect QTLs with a threshold of LOD ≥ 2.5. The BLUP (best linear unbiased prediction) value of grain zinc content at five ecological points was used to locate the major QTL QGrZnc.sau-4A for wheat grain zinc content, and the position of QGrZnc.sau-4A and the genetic distance between molecular markers were calculated. QGrZnc.sau-4A was finally located at 60.96–78.86 Mbp on chromosome 4A of Chinese spring or 54.43–60.02 Mbp on chromosome 4A of wild two-grain.

[0059] d) Comparison of grain zinc content loci and acquisition of molecular markers: Previous reports have shown that there are few QTLs or genes related to grain zinc content. Crespo-Herrera et al. reported that QGZn.cimmyt-4A-P2 (123.5-124.5 Mbp) controlling grain zinc content is located on chromosome 4A (Crespo-Herrera L et al. 2017, Frontiers in Plant Science, 8:1800), Velu et al. identified the QTL QGzncpk.cimmyt-4AS (24.0-26.3 Mbp) controlling zinc content on chromosome 4A (Velu G et al. 2017, Plant and Soil, 411:81-99), and Shariatipour detected MQTL / 3 (704.3-733.5 Mbp) controlling zinc content on the long arm of chromosome 4A (Shariatipour N et al. 2021, Euphytica, 217:86), Ren et al. detected QGZn.haust-4AL (720.1-731.0 Mbp) controlling the zinc content of grains on chromosome 4A (Ren P et al. 2022, Molecular Breeding, 42:49). This indicates that QGrZnc.sau-4A detected in this example is a new stable QTL.

[0060] In order to further densify the map and obtain molecular markers that are closely linked to the major QTL QGrZnc.sau-4A for zinc content in wheat grains, the 660K SNP chip data positioning results were used to physically locate the flanking markers and screen the genes located within the interval. The genes were further sequenced and polymorphic sites were mined to develop efficient KASP molecular markers. Finally, after multiple cloning sequencing, primer design and amplification, a total of 5 pairs of KASP primers were designed (see Table 1). By performing fluorescent quantitative PCR on the plants of the recombinant inbred line population, it was finally found that the marker KASP-GrZnc-sau9087 (polymorphic C / T) was closely linked to the major QTL QGrZnc.sau-4A for zinc content in grains. The co-localization map of this molecular marker and QGrZnc.sau-4A is shown in the figure. Figure 1 As shown, there is a C / T mutation at the 36th base ("R" represents C or T) in the sequence shown in SEQ ID NO.1.

[0061] SEQ ID NO. 1: CAATAACAGTATCATCAATAGAAAAAACATGGCATRACACATCTACCTGGCTGAGAAGACACTAACAACAC.

[0062] Table 1 Primer sequences of molecular marker KASP

[0063]

[0064] Example 2

[0065] Application of molecular marker KASP-GrZnc-sau9087 in identifying the major QTL QGrZnc.sau-4A controlling the zinc content in grains:

[0066] (1) 100 accessions were randomly selected from 717 Chinese local wheat accessions for verification.

[0067] (2) The 100 strains obtained were subjected to KASP-GrZnc-sau9087 marker detection. The specific method was as follows: DNA of the 100 strains was extracted; the DNA was used as a template and a specific primer pair (SEQ ID NO. 2-4) of the molecular marker KASP-GrZnc-sau9087 was used as a primer for fluorescence quantitative PCR.

[0068] The reaction system of the above fluorescence quantitative PCR is: 5μL Master Mix, 5ng template DNA, 1.4μL mixed primers (primers SEQ ID NO.2-4 are all at a concentration of 10ng / μL, 120μL, 120μL and 300μL are used respectively, and 460μL ddH 2O for mixing), ddH 2 Add ddHO to a total of 10 μL. 2 O replaced the blank of DNA template; the reaction program of fluorescence quantitative PCR was: pre-denaturation at 94℃ for 15min; denaturation at 94℃ for 20s, annealing / extension at 61℃ for 60s, for a total of 10 cycles; denaturation at 94℃ for 20s, annealing / extension at 55℃ for 60s, for a total of 26 cycles; fluorescence reading was performed after completion.

[0069] Valid results were detected in 100 strains, and the fluorescence readings were as follows Figure 2 As shown in the figure, the genotype of the plant with FAM (circle) fluorescence is consistent with 'LM001', and the genotype of the plant with HEX (square) fluorescence is consistent with 'Ailanmai'. The grain zinc content phenotypes of these lines were further investigated and an independent sample t-test was performed. The results are shown in Figure 3 As shown in the figure, it can be seen that the zinc content in the grains of the line with the genotype 'LM001' (Allele 2) is significantly higher than that of the line with the genotype 'Ailanmai' (Allele 1). This indicates that QGrZnc.sau-4A does have the effect of significantly increasing the zinc content in wheat grains.

[0070] 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. Application of a molecular marker tightly linked to the major QTL QGrZnc.sau-4A for zinc content in wheat grains in the following A1 or A2: A1. Detection of the major QTL QGrZnc.sau-4A for zinc content in wheat grains; A2. Early identification or selection of wheat varieties with high grain zinc content; The molecular marker and the major QTL QGrZnc.sau-4A of zinc content in wheat grains are co-localized on the short arm of chromosome 4A of the wheat genome; The molecular marker is located at the 36th base of the nucleotide sequence shown in SEQ ID NO.1, and there is a C / T mutation; the zinc content of wheat grains with the mutation base site being C is significantly higher than the zinc content of wheat grains with the mutation base site being T; The major QTL QGrZnc.sau-4A for wheat grain zinc content is located at 60.96-78.86 Mbp of chromosome 4A of Chinese spring wheat, or 54.43-60.02 Mbp of chromosome 4A of wild two-grain wheat.

2. Use of a KASP primer set targeting a molecular marker tightly linked to the major QTL QGrZnc.sau-4A for zinc content in wheat grains in any of the following: B1. Detection of the major QTL QGrZnc.sau-4A for zinc content in wheat grains; B2. Early identification or selection of wheat varieties with high grain zinc content; B3. Prepare reagents, kits or chips for detecting the major QTL QGrZnc.sau-4A for zinc content in wheat grains; The molecular marker and the major QTL QGrZnc.sau-4A of zinc content in wheat grains are co-localized on the short arm of chromosome 4A of the wheat genome; The molecular marker is located at the 36th base of the nucleotide sequence shown in SEQ ID NO.1, and there is a C / T mutation; the zinc content of wheat grains with the mutation base site being C is significantly higher than the zinc content of wheat grains with the mutation base site being T; The major QTL QGrZnc.sau-4A for zinc content in wheat grains is located at 60.96-78.86 Mbp of chromosome 4A of Chinese spring, or 54.43-60.02 Mbp of chromosome 4A of wild emmer; The KASP primer set includes primers shown as SEQ ID NO. 2-4.

3. A method for detecting the major effect QTL QGrZnc.sau-4A of zinc content in wheat grains, characterized in that: The steps include: Using the genomic DNA of the plant to be tested as a template, a KASP primer set targeting a molecular marker closely linked to the major QTL QGrZnc.sau-4A of zinc content in wheat grains is used for fluorescence quantitative PCR amplification, and determination is made based on the fluorescence reading result; the KASP primer set includes primers shown in SEQ ID NO.2-4; The plant in which the fluorescent group marked by SEQ ID NO.2 was read was identified as a plant containing the major QTL QGrZnc.sau-4A for zinc content in wheat grains, and the genotype was CC; the plant in which the fluorescent group marked by SEQ ID NO.3 was read was identified as a plant not containing the major QTL QGrZnc.sau-4A for zinc content in wheat grains, and the genotype was TT; the plant containing the major QTL QGrZnc.sau-4A for zinc content in wheat grains had a higher zinc content in grains than the plant not containing the major QTL QGrZnc.sau-4A for zinc content in wheat grains; The major QTL QGrZnc.sau-4A for wheat grain zinc content is located at 60.96-78.86 Mbp of chromosome 4A of Chinese spring wheat, or 54.43-60.02 Mbp of chromosome 4A of wild two-grain wheat.

4. The method according to claim 3, characterized in that: The reaction system of the fluorescent quantitative PCR is: 5 μL Master Mix, 5 ng template DNA, 1.4 μL mixed primers, and ddH2O added to a total volume of 10 μL; The mixed primer is obtained by mixing 10 ng / μL of the primer shown in SEQ ID NO.2, 10 ng / μL of the primer shown in SEQ ID NO.3, 10 ng / μL of the primer shown in SEQ ID NO.4 and ddH2O in a volume ratio of 6:6:15:

23.

5. The method according to claim 3, characterized in that: The fluorescence quantitative PCR reaction procedure is: pre-denaturation at 94°C for 15 min; denaturation at 94°C for 20 s, annealing / extension at 61°C for 60 s, for a total of 10 cycles; denaturation at 94°C for 20 s, annealing / extension at 55°C for 60 s, for a total of 26 cycles.

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