Molecular marker related to nitrogen high-efficiency utilization trait and application thereof

Abstract

The application discloses a nitrogen high-efficiency utilization character related InDel molecular marker and application thereof, relates to the technical field of molecular biology.A kind of InDel molecular marker related to nitrogen high-efficiency utilization character of wheat, the InDel molecular marker is located in the 482963711bp site of 1A chromosome of wheat reference genome China Spring v1.0, and TC double-base insertion / deletion allelic variation exists in the site;The nucleotide sequence of the insertion allelic variation is as shown in SEQ ID NO:1, and the nucleotide sequence of the deletion allelic variation is as shown in SEQ ID NO:2.The application develops KASP molecular marker primer set based on the variation site, and through simple DNA extraction, PCR specific amplification and high-throughput genotyping operation, the precision identification and screening of nitrogen high-efficiency utilization character of wheat germplasm resources can be quickly completed.The molecular marker detection method is simple in operation and high in detection efficiency, and has a good application prospect in the field of wheat high-efficiency nitrogen breeding and germplasm resource screening.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, and in particular to a molecular marker associated with nitrogen-efficient utilization traits and its applications. Background Technology

[0002] Nitrogen is an essential macronutrient for plant growth and development. It participates in the formation of many important bioactive substances such as proteins, nucleic acids, phospholipids, chlorophyll and cytokinins. It plays a key role in physiological processes such as cell formation, plant growth, various metabolic processes, photosynthesis, energy metabolism and cell division and elongation (Noor, H. & Min, S. Effect of different sowing methods and Nitrogen rates on Yield and Quality of winterwheat in loess plateau of china. Appl. Ecol. Env. Res. 2020, 18(4), 5701–5726.).

[0003] Wheat (Triticum aestivum L.) is a major food crop in my country and the world, with an annual planting area exceeding 200 million hectares and an annual output of 749 million tons. With the continuous growth of the population, the global population is projected to reach 9.6 billion by 2050 (FAOSTAT, FAO, 2018). To meet the increasing demand for food consumption, total wheat production needs to increase by 60% from current levels to ensure global food security (https: / / www.un.org / ). High yield and quality of wheat are highly dependent on nitrogen fertilizer application, but only 30%–50% of the nitrogen fertilizer applied to the soil is absorbed and utilized by crops. A large amount of nitrogen fertilizer is easily lost from the soil through leaching, runoff, nitrification-denitrification, and ammonia volatilization. Low nitrogen fertilizer use efficiency not only increases agricultural production costs but also triggers a series of ecological and environmental problems such as soil acidification, water eutrophication, and exacerbation of the greenhouse effect (MJ Hawkesford, Genetic variation in traits for nitrogen use efficiency in wheat, J. Exp. Bot., 2017, 68: 2627–2632. MA Sutton, O. Oenema, JW Erisman, A. Leip, H. van Grinsven, W. Winiwarter, Too much of a good thing, Nature, 2011, 472: 159–161.). Compared with traits such as pests and diseases, grain size, and color, which are easily observed phenotypically, nitrogen efficiency is a complex quantitative trait that is easily affected by soil environment. Currently, the development of molecular markers for nitrogen efficiency traits in wheat is still relatively scarce. Therefore, exploring superior allelic variations in nitrogen efficiency, breeding new nitrogen-efficient crop varieties, improving crop nitrogen absorption and utilization efficiency, and reducing nitrogen fertilizer input and its environmental losses have important theoretical and practical value for promoting green and sustainable agricultural development. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a molecular marker related to nitrogen-efficient utilization traits and its application.

[0005] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows.

[0006] An InDel molecular marker associated with nitrogen-efficient utilization traits in wheat, wherein the InDel molecular marker is located at chromosome 482963711 bp on chromosome 1A of the wheat reference genome Chinese Spring v1.0, where a TC dibase insertion / deletion allelic variant exists; the nucleotide sequence of the insertion allelic variant is shown in SEQ ID NO: 1, and the nucleotide sequence of the deletion allelic variant is shown in SEQ ID NO: 2.

[0007] More preferably, the insertion allelic variant corresponds to the HapⅠ haplotype, and the deletion allelic variant corresponds to the HapⅡ haplotype. The nitrogen-efficient use trait is manifested as follows: wheat carrying the HapⅠ haplotype has better nitrogen-efficient use trait than wheat carrying the HapⅡ haplotype.

[0008] The application of the above InDel molecular markers in identifying traits of high nitrogen utilization efficiency in wheat.

[0009] The above-mentioned InDel molecular markers were used in screening wheat varieties with excellent nitrogen-efficient utilization traits.

[0010] The primer set for amplifying the InDel molecular marker described above is characterized in that the primer set includes primer A, primer B and primer C; the nucleotide sequence of primer A is shown in SEQ ID NO: 3, the nucleotide sequence of primer B is shown in SEQ ID NO: 4, and the nucleotide sequence of primer C is shown in SEQ ID NO: 5.

[0011] Application of the above primer set in identifying traits of high nitrogen utilization efficiency in wheat.

[0012] The application of the above primer set in screening wheat varieties with excellent nitrogen-efficient utilization traits.

[0013] A kit for identifying traits of high nitrogen utilization efficiency in wheat, comprising the aforementioned primer set.

[0014] A kit for screening superior wheat varieties, comprising the aforementioned primer set.

[0015] A method for identifying nitrogen-efficient utilization traits in wheat includes the following steps: (1) extracting genomic DNA from the wheat to be tested; (2) using the genomic DNA as a template, performing PCR amplification using the primer set described above to obtain amplification products; (3) determining the salt tolerance of wheat based on the amplification products; detecting fluorescence signals on the amplification products, determining the wheat to be tested to be haplotype I when FAM fluorescence signal is detected, and determining the wheat to be tested to be haplotype II when HEX fluorescence signal is detected; (4) determining the nitrogen-efficient utilization traits of the wheat to be tested based on the haplotype determination results.

[0016] The beneficial effects of the above technical solution are as follows: This invention provides a molecular marker fragment related to nitrogen-efficient use traits in wheat, wherein the molecular marker fragment contains the coding region of the TaZF-1A gene; the nucleotide sequence of the coding region of the TaZF-1A gene is shown in SEQ ID NO: 1. This invention utilizes the molecular marker fragment to screen nitrogen-efficient wheat varieties, improving the screening efficiency of nitrogen-efficient wheat and benefiting wheat germplasm breeding. This invention utilizes the KASP molecular marker primer set, requiring only simple DNA extraction, PCR-specific amplification, and genotyping to identify samples, facilitating the detection and screening of nitrogen-efficient wheat varieties or lines, and greatly accelerating the breeding process of nitrogen-efficient wheat varieties. Attached Figure Description

[0017] Figure 1 This is an electrophoresis image of the TaZF-1A coding region amplification of the present invention.

[0018] Figure 2 This is a peak diagram of the sequencing results of this invention.

[0019] Figure 3 The nucleotide sequences are TaZF-1A-HapⅠ and TaZF-1A-HapⅡ.

[0020] Figure 4 The graph shows the differences in grain yield, grain nitrogen concentration, nitrogen accumulation, and NHI levels among different haplotype wheat varieties / lines in different years.

[0021] Figure 5 This is a graph showing the results of Kasp marker detection in different wheat varieties. Detailed Implementation

[0022] The following embodiments illustrate the present invention in detail. All raw materials and equipment used in the present invention are commercially available products and can be directly obtained through market purchase. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods.

[0023] It should be understood that, when used in this application specification and the appended claims, the term "comprising" indicates the presence of the described features, integrals, steps, operations, elements and / or components, but does not exclude the presence or addition of one or more other features, integrals, steps, operations, elements, components and / or a collection thereof.

[0024] It should also be understood that the term “and / or” as used in this application specification and the appended claims means any combination of one or more of the associated listed items and all possible combinations, and includes such combinations.

[0025] References to "one embodiment" or "some embodiments" as described in this application specification mean that one or more embodiments of this application include the features described in connection with that embodiment.

[0026] Defined features, structure, or characteristics. Therefore, the phrases "in one embodiment," "in some embodiments," "in other embodiments," "in still other embodiments," etc., appearing in different places in this specification do not necessarily refer to the same embodiment, but rather mean "one or more, but not all, embodiments," unless otherwise specifically emphasized. The terms "comprising," "including," "having," and variations thereof mean "including, but not limited to," unless otherwise specifically emphasized.

[0027] Furthermore, in the description of this application and the appended claims, the terms "first," "second," "third," etc., are used only to distinguish descriptions and should not be construed as indicating or implying relative importance.

[0028] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0029] Example 1: Mining of Superior Haplotypes

[0030] (1) Primer design

[0031] Based on the sequence differences of the three copies 1A, 1B and 1D of the TaZF-1A gene in the wheat genome database (http: / / plants.ensembl.org / Triticum_aestivum / Info / Index), primers TaZF-1A-CDS-F and TaZF-1A-CDS-R specifically for amplifying the coding region were designed. Primer synthesis and sequencing were performed by Beijing Qingke Biotechnology Co., Ltd.

[0032] TaZF-1A-CDS-F: 5′-GTGTGTGCTGGTTGTGCG-3′ (SEQ ID NO: 6);

[0033] TaZF-1A-CDS-R: 5′-TGGAACATGATCGAAAAGCACG-3′ (SEQ ID NO: 7);

[0034] (2) Extraction of total DNA from wheat

[0035] 358 wheat varieties (including modern cultivars and local varieties) were selected, and detailed information is shown in Table 1. Total DNA was extracted from wheat using the CTAB method: 0.1–0.2 g of wheat leaves were quick-frozen and ground in liquid nitrogen. The mixture was then removed from the centrifuge tube, cooled to room temperature, and 600 μL of CTAB extraction buffer preheated to 65 °C was added. The mixture was vigorously vortexed for 2 min and then incubated in a 65 °C water bath for 30 min. Next, 600 μL of a chloroform:isoamyl alcohol (24:1, v:v) mixture was added and vortexed. The mixture was centrifuged at 12000 rpm at room temperature for 10 min. The supernatant was transferred to a new tube, and an equal volume of pre-chilled isopropanol was added. The mixture was inverted and incubated at -20 °C for 30 min. Subsequently, the mixture was centrifuged at 12000 rpm at 4 °C for 10 min. The supernatant was discarded, and the precipitate was washed twice with 500 μL of 70% ethanol. After drying, 120 μL of sterile water was added to dissolve the DNA, yielding wheat DNA.

[0036] (3) Sequence alignment and haplotype analysis of the coding region of the TaZF-1A gene

[0037] The target fragment was amplified using the high-fidelity PCR enzyme 2*TransStart FastPfu Fly PCR SuperMix.

[0038] The coding region sequence of TaZF-1A was amplified using the TaZF-1A-CDS-F / R primer pair and 358 hexaploid wheat DNA samples as templates. The PCR system contained: 10 μL 2×PhantaMax Buffer, 0.6 μL PhantaMax Super-Fidelity DNA Polymerase, 0.6 μL dNTP Mix (10 μM), 0.8 μL each of forward and reverse primers (10 μM), 200 ng DNA template, and ddH2O to a total volume of 50 μL. The PCR reaction program for the coding region was as follows: Step 1, 98℃ pre-denaturation for 1 min; Step 2, 98℃ denaturation for 10 s, 59℃ annealing for 5 s, 72℃ extension for 1 min, 30 cycles; Step 3, 72℃ extension for 1 min. The electrophoresis results are shown below. Figure 1 As shown, the PCR amplification products were sequenced, and a partial sequencing result peak diagram is shown below. Figure 2 .

[0039] The sequencing results from step (3) were assembled and sequence aligned using DNAMAN software (http: / / www.lynnon.com). Sequence alignment analysis revealed one TC double-base deletion InDel variant (TC double-base insertion / deletion allelic variant located at chromosome 482963711bp on chromosome 1A of the wheat reference genome Chinese Spring v1.0) in the TaZF-1A coding region (1bp~724bp) of 358 wheat varieties. This deletion caused an open reading frame shift, altering the amino acid sequence and generating an early stop codon, leading to premature translation termination. Based on this variant site, the tested materials were classified into two haplotypes, named TaZF-1A-HapⅠ and TaZF-1A-HapⅡ, respectively. The nucleotide sequences of TaZF-1A-HapⅠ and TaZF-1A-HapⅡ are shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 2. Figure 3 As shown in Table 1, the haplotype results for different wheat varieties / lines are presented in Table 1.

[0040] Table 1. Performance of haplotypes, grain yield, and nitrogen-efficient use-related traits of different wheat varieties under normal nitrogen application conditions for two consecutive years.

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[0049] Table 2. Performance of haplotypes, grain yield, and nitrogen-efficient use-related traits of different wheat varieties under low-nitrogen conditions for two consecutive years.

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[0059] Example 2: Grain nitrogen content, grain yield, and nitrogen accumulation of different haplotype wheat varieties under different nitrogen levels.

[0060] Thirty-eight wheat varieties were planted for two consecutive years (October 2023 to June 2024 and October 2024 to June 2025) in the Dishang Experimental Field of the Institute of Grain and Oil Crops, Hebei Academy of Agricultural and Forestry Sciences. Two nitrogen application levels were set up: normal nitrogen application and low nitrogen application. The wheat was planted by broadcasting and a randomized complete block design was used. Each variety and each treatment was planted in 2-meter rows with 120 seeds per row. The normal nitrogen application group received 240 kg N / ha of nitrogen fertilizer, while the low nitrogen application group received 90 kg N / ha. Other wheat management practices were carried out according to conventional methods.

[0061] At maturity, plants of each variety and treatment were harvested in rows of 1 meter in length. The above-ground straw and ears were separated and dried at 80°C to constant weight. The grain yield and straw dry weight were weighed separately. After grinding, the nitrogen content of the straw and grain samples was determined using a fully automatic Kjeldahl nitrogen analyzer. The nitrogen accumulation per unit area and nitrogen harvest index (NHI) were calculated. The results are shown in Tables 1 and 2.

[0062] According to the results in Tables 1 and 2, in two consecutive years of field trials, regardless of whether the nitrogen application environment was normal or low, wheat varieties / lines carrying the TaZF-1A gene HapⅠ haplotype had significantly higher grain yield, grain nitrogen concentration, total nitrogen accumulation, and NHI than the HapⅡ haplotype in different years, and the differences between the two haplotypes were all highly significant. Figure 4 (See Table 3). The above results indicate that HapⅠ is a nitrogen-efficient and excellent haplotype.

[0063] Table 3. Statistical analysis results of the correlation between the two allele genotypes of TaZF-1A, a gene involved in nitrogen efficiency regulation in common wheat, and nitrogen efficiency use traits.

[0064]

[0065] Example 3: Development and Application of KASP Molecular Markers

[0066] Since the difference between the two haplotypes of TaZF-1A is a two-base InDel deletion variant, a competitive allele-specific PCR (KASP) molecular marker was developed based on this difference and named Kap_TaZF-1A. The KASP marker primer sequences are shown in Table 4.

[0067] Table 4 Primer sequences for the KASP marker Kasp_TaZF-1A used to detect nitrogen-efficient traits in wheat.

[0068]

[0069] The detection of two haplotypes of the TaZF-1A gene using the KASP marker mainly includes two steps: PCR-specific amplification and genotyping.

[0070] (1) PCR amplification system and procedure. DNA from 188 wheat varieties was used as template.

[0071] Preparation of KASP-labeled primer working solution: Take 12 μl each of the two upstream primers (primer A and B in Table 3, 100 μM) and 30 μl of the downstream primer (primer C in Table 3, 100 μM), add sterile ultrapure water to make up to 100 μl, mix well and use as KASP-labeled primer working solution, store at 4 ℃ for later use.

[0072] The PCR amplification system consisted of: 1.5 μl template DNA, 0.0417 μl primer working solution, 0.75 μl 2×KASP Master Mix (LGC, product number: KBS-1050-112), and sterile ultrapure water to a final volume of 3 μl.

[0073] The PCR reaction program was as follows: pre-denaturation at 94 °C for 15 min; followed by 10 cycles, each cycle consisting of denaturation at 94 °C for 20 s and annealing for 20 s (the first annealing temperature was 61 °C, and the temperature was decreased by 0.6 °C each cycle); then 26 cycles, each cycle consisting of denaturation at 94 °C for 20 s and annealing at 55 °C for 1 min; finally, extension at 72 °C for 3 min, and storage at 4 °C.

[0074] (2) After the PCR reaction was completed, the fluorescence signal was converted into analyzable values ​​using a fluorescence signal reader (Omega, BMG LABTECH, Germany) and a fluorescence detection system (Araya), and the fluorescence data of the reaction products were read. The fluorescence scanning results were graphically displayed using GraphPad Prism: the type carrying TC bases (HapⅠ haplotype) showed FAM fluorescence, distributed near the x-axis; the type carrying TG bases (HapⅡ haplotype) showed HEX fluorescence, distributed near the y-axis; the negative control (CK) showed no detectable signal and was located near the origin (see...). Figure 5 The genotyping results for different wheat varieties were consistent with the sequencing results, as detailed in Table 5. Therefore, the Kasp_TaZF-1A molecular marker can accurately distinguish between HapⅠ and HapⅡ and can be applied to the molecular design breeding of nitrogen-efficient wheat varieties.

[0075] Table 5. KASP marker genotyping of different wheat varieties

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[0079] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these examples without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

[0080] In the above embodiments, the descriptions of each embodiment have different focuses. For parts that are not described in detail or recorded in a certain embodiment, please refer to the relevant descriptions of other embodiments.

[0081] The above-described embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention, and should all be included within the protection scope of the present invention.

Claims

1. An InDel molecular marker associated with nitrogen-efficient utilization traits in wheat, characterized in that, The InDel molecular marker is located at chromosome 482963711 bp on the wheat reference genome Chinese Spring v1.0, where a TC double-base insertion / deletion allelic variant exists. The nucleotide sequence of the insertion allelic variant is shown in SEQ ID NO: 1, and the nucleotide sequence of the deletion allelic variant is shown in SEQ ID NO:

2.

2. The InDel molecular marker according to claim 1, characterized in that, The insertion allelic variant corresponds to the HapⅠ haplotype, and the deletion allelic variant corresponds to the HapⅡ haplotype. The nitrogen-efficient use trait is as follows: wheat carrying the HapⅠ haplotype has a better nitrogen-efficient use trait than wheat carrying the HapⅡ haplotype.

3. The application of the InDel molecular marker as described in claim 1 or 2 in identifying nitrogen-efficient utilization traits in wheat.

4. The application of the InDel molecular marker as described in claim 1 or 2 in screening wheat varieties with excellent nitrogen-efficient utilization traits.

5. The primer set for amplifying the InDel molecular marker as described in claim 1 or 2, characterized in that, The primer set includes primer A, primer B and primer C; the nucleotide sequence of primer A is shown in SEQ ID NO: 3, the nucleotide sequence of primer B is shown in SEQ ID NO: 4 and the nucleotide sequence of primer C is shown in SEQ ID NO:

5.

6. The application of the primer set according to claim 5 in identifying the nitrogen-efficient utilization trait in wheat.

7. The application of the primer set described in claim 5 in screening wheat varieties with excellent nitrogen-efficient utilization traits.

8. A kit for identifying traits of high nitrogen utilization efficiency in wheat, characterized in that, Includes the primer set as described in claim 5.

9. A kit for screening superior wheat varieties, characterized in that, Includes the primer set as described in claim 5.

10. A method for identifying nitrogen-efficient utilization traits in wheat, characterized in that, The method includes the following steps: (1) extracting the genomic DNA of the wheat to be tested; (2) using the genomic DNA as a template, performing PCR amplification using the primer set described in claim 5 to obtain the amplification product; (3) determining the salt tolerance of the wheat based on the amplification product; detecting the fluorescence signal of the amplification product, determining that the wheat to be tested is a Hap I haplotype when a FAM fluorescence signal is detected, and determining that the wheat to be tested is a Hap II haplotype when a HEX fluorescence signal is detected; (4) determining the nitrogen-efficient utilization trait of the wheat to be tested based on the haplotype determination result.

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