Molecular markers tightly linked to a major QTL for wheat grain weight related to heat tolerance and their application

Abstract

The present invention discloses a molecular marker tightly linked to the main effect QTL of wheat grain weight related to heat tolerance and its application, which belongs to the technical field of wheat molecular genetic breeding. The molecular marker and the main effect QTkw / Hz‑4D of wheat grain weight are co-localized on the short arm of chromosome 4D of the wheat genome; the molecular marker AX‑111027348 is located at the 151st base of the nucleotide sequence shown in SEQ ID NO.1, and there is a C / T mutation, and it is developed into a KASP marker; the molecular marker provided by the present invention can be used for grain weight-assisted selection of Handan 6172 and Chinese Spring or their derivative materials, or for heat tolerance identification of ordinary hexaploid wheat materials. The molecular marker provided by the present invention has important applications in molecular marker-assisted breeding of wheat grain weight traits and heat-resistant wheat breeding.

Description

Technical Field

[0001] The present invention belongs to the field of wheat molecular breeding, and in particular relates to molecular markers and applications of QTLs related to wheat grain weight under thermal environments. Background Art

[0002] Wheat is a major global crop, providing food for one-third of the world's population. With global warming, wheat crops frequently experience extreme high temperatures during their later growth stages, severely threatening their yields. Improving wheat's heat tolerance and cultivating heat-tolerant varieties are of strategic importance for ensuring my country's food security and social stability.

[0003] The three key factors determining wheat yield are the number of ears per unit area, the number of grains per ear, and grain weight. Grain weight has a relatively independent effect on yield, and is primarily influenced by genetic factors. Among the three key factors, grain weight is the most stable and heritable of the three yield factors. Therefore, grain weight plays a crucial role in increasing wheat yield. Under heat stress conditions, reducing the decline in grain weight can minimize yield losses caused by heat damage.

[0004] In recent years, the development of high-throughput sequencing and gene chip detection technologies, and the subsequent major breakthroughs in wheat genomics, have laid a solid foundation for the detailed analysis of quantitative trait loci (QTLs) and the development of molecular markers. This has also opened up the possibility of wheat genetic improvement based on marker-assisted selection. Although many quantitative trait loci (QTLs) associated with thousand-grain weight have been reported, there are relatively few QTLs related to thousand-grain weight under heat stress conditions, and even fewer molecular markers for heat tolerance are available.

[0005] Marker-assisted selection directly targets target genotypes, unaffected by gene expression and environmental variations. This improves selection accuracy and accelerates breeding. Therefore, it is necessary to identify stable, major QTLs for 1000-grain weight under heat conditions and develop tightly linked molecular markers to improve the efficiency of heat-tolerant wheat breeding. Summary of the Invention

[0006] The purpose of the present invention is to provide a molecular marker for a major effect QTL of wheat grain weight related to heat tolerance and its application.

[0007] To achieve the above-mentioned purpose, the technical solution adopted by the present invention is: identifying the major effect gene locus QTkw / Hz-4D of wheat grain weight related to heat tolerance, which is located in the physical interval of 16.15Mb to 25.25Mb of wheat chromosome 4D.

[0008] The molecular marker AX-111027348 is tightly linked to the main effect QTkw / Hz-4D of grain weight related to wheat heat tolerance, characterized in that the molecular marker is located at the 151st base of the nucleotide sequence shown in SEQ ID NO.1, and has a C / T mutation.

[0009] Preferably, the KASP marker developed by the molecular marker comprises forward primers 1, 2 and a universal negative primer; the sequence of the forward primer 1 is shown as SEQ ID NO.2, the sequence of the forward primer 2 is shown as SEQ ID NO.3, and the sequence of the universal reverse primer is shown as SEQ ID NO.4.

[0010] Accordingly, the product prepared by using the KASP marker for screening wheat materials with high or low thousand-grain weight under a hot environment is the wheat material being Handan 6172, Chinese Spring or populations derived therefrom.

[0011] Correspondingly, in a method for screening wheat materials with high or low thousand-grain weight under a hot environment using the KASP marker, if the wheat material to be tested is identified as containing QTkw / Hz-4D and having a genotype of TT through KASP marker PCR detection, the wheat material to be tested is identified as wheat with high thousand-grain weight under a hot environment; if the wheat genotype to be tested is CC through KASP marker PCR detection, the sample is identified as wheat with low thousand-grain weight under a hot environment, and the wheat material is Handan 6172, Chinese Spring, or a population derived therefrom.

[0012] Correspondingly, the method for identifying wheat heat tolerance using the KASP marker is characterized in that: if the wheat material to be tested is identified as containing QTkw / Hz-4D and the genotype is TT through KASP marker PCR detection, the wheat material to be tested is identified as wheat with a low rate of decrease in thousand-grain weight affected by heat, and the wheat material has strong heat tolerance; if the wheat material to be tested is identified as containing QTkw / Hz-4D and the genotype is CC through KASP marker detection, the wheat material to be tested is identified as wheat with a high rate of decrease in thousand-grain weight affected by heat, and the wheat material has weak heat tolerance, and the wheat material is all hexaploid common wheat materials.

[0013] Correspondingly, the gene locus QTkw / Hz-4D or the molecular marker is used in wheat breeding.

[0014] The thermal environment described in the present invention is a natural environment with a maximum daily temperature greater than 38°C for at least three days during the wheat filling period, or an artificially created wheat growth environment with a maximum daily temperature greater than 38°C.

[0015] Beneficial effects of the present invention:

[0016] Phenotypic collection and linkage analysis using high-generation recombinant inbred populations of Handan 6172 and Chinese Spring ultimately pinpointed a single nucleotide polymorphism (SNP) marker, QTkw / Hz-4D, associated with heat tolerance. The closely linked SNP marker, AX-111027348, was then closely linked to the marker. Furthermore, the KASP marker was developed based on this locus information. This marker offers the advantages of stable amplification, accurate typing, and rapid and easy detection. This marker can be used to assist in the selection of 1,000-grain weight for Handan 6172, Chinese Spring, or their derivatives in heat-resistant environments, or to identify heat-tolerant materials.

[0017] The KASP marker was significantly correlated with the rate of decrease in thousand-grain weight caused by heat environment in a natural population of 272 wheat varieties (lines), indicating that the KASP marker developed in the present invention is suitable for the identification of heat resistance of common hexaploid wheat materials, and can lay a theoretical foundation for the breeding of heat-resistant wheat varieties and provide a molecular-assisted selection method. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the co-localization map of wheat grain weight QTkw / Hz-4D and molecular markers related to heat tolerance on chromosome 4D in Example 1.

[0019] Figure 2 Thousand-grain weight of different genotypes of RILs population in the field (A) and under heat environment (B).

[0020] Figure 3 The genotyping results of KASP markers for RILs population (A) and natural population wheat materials (B). DETAILED DESCRIPTION

[0021] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials and reagents used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

[0022] Example 1 Discovery of QTkw / Hz-4D related to wheat grain weight under thermal environment and development of KASP markers.

[0023] 1. Identification of 1000-grain weight phenotypes under heat stress conditions

[0024] A population of F7 recombinant inbred lines (RILs) containing 269 lines was generated by crossing the wheat variety Han 6172 as the female parent and Chinese Spring as the male parent. The 1,000-grain weight trait of these 269 lines was evaluated across eight environments in the 2020-2021, 2021-2022, and 2022-2023 seasons.

[0025] A field experiment was conducted at the Dahe Experimental Park of the Hebei Academy of Agriculture and Forestry Sciences (Shijiazhuang, 38°05′ N, 114°44′ E). Wheat materials were arranged sequentially and seeded manually, with row spacing of 30 cm and plant spacing of 10 cm. Each line was planted in one row, totaling 21 plants. Routine field management was followed, and no serious pests, diseases, or lodging occurred during the growing season. The experiment was conducted under both field conditions and two thermal regimes. The thermal regime involved a covered greenhouse heat treatment during the flowering period (May 10–30), with greenhouse temperatures recorded. All wheat materials were harvested, air-dried, and threshed upon maturity. Thousand-grain weight was measured using a grain analyzer, with three replicates.

[0026] 2. Construction of genetic linkage maps and QTL mapping

[0027] The genotypes of the two parents and 269 lines were obtained using the wheat 55K SNP chip, and the genetic linkage map of the RIL population was constructed using the QTL analysis software IciMapping4.1.

[0028] Using the complete interval mapping method of the software IciMapping 4.1 combined with the thousand-grain weight data and the genetic linkage map, a stable major effect QTL related to grain weight under thermal environment was detected on chromosome 4D, named QTkw / Hz-4D, and a SNP marker AX-111027348 ( Figure 1 ).

[0029] Under field conditions, QTkw / Hz-4D was identified in 2021 and 2022, with LOD values ​​of 5.0775 and 6.5649, respectively, contributing 6.2465% and 0.8805% to the phenotypic variation, respectively. Under thermal conditions, QTkw / Hz-4D was identified five times from 2021 to 2023, with LOD values ​​ranging from a minimum of 5.4482 to a maximum of 22.1732, and contributions to the phenotypic variation ranging from a minimum of 7.1627% to a maximum of 28.6358%. The contribution of this QTL was significantly higher under thermal conditions (Table 1).

[0030] Table 1 Identification of the main effect QTkw / Hz- 4D of wheat grain weight

[0031]

[0032] A SNP marker AX-111027348 tightly linked to QTkw / Hz-4D was identified. This SNP is located at 19207375bp on the short arm of chromosome 4D of the Chinese Spring reference genome IWGSC RefSeq v1.0, and its polymorphism is T / C.

[0033] According to the genotyping of this locus, the differences in thousand-grain weight among different genotypes in the recombinant inbred line population were statistically analyzed. Figure 2 The results showed that under the field planting conditions in three years, the average thousand-grain weight of the TT genotype lines was significantly higher than that of the CC genotype lines only in 2022, and there was no significant difference in the other two years; under the five thermal environment conditions in three years, the average thousand-grain weight of the TT genotype lines was significantly higher than that of the CC genotype lines (p < 0.05).

[0034] Judging from the thousand-grain weight decline rate of different genotype strains under heat environment in Table 2 in the recombinant inbred line population, there was no significant difference between the TT genotype strains and the CC genotype strains in 2021; under the two heat environments in 2022, the thousand-grain weight decline rate between the TT genotype strains and the CC genotype strains reached extremely significant (p < 0.01) and significant levels (p < 0.05); under the two heat environments in 2023, the average value of the thousand-grain weight decline rate between the TT genotype strains and the CC genotype strains reached an extremely significant level (p < 0.01).

[0035] Table 2 Variance analysis of the 1000-grain weight reduction rate of different genotypes in the recombinant inbred line population constructed from Handan 6172 / China Spring under heat conditions

[0036]

[0037] This indicates that the effect of this locus under heat stress is reproducible and significant. Therefore, the SNP marker AX-111027348 was developed as a KASP marker to facilitate its application in other materials.

[0038] 3. Development of KASP Marker

[0039] The flanking sequence of the above-mentioned SNP is shown in SEQ ID NO. 1. The left end of the sequence is 5'. The 151st position of the sequence corresponds to a SNP tightly linked to QTkw / Hz-4D, and the polymorphism is T / C.

[0040] Polymarker (http: / / www.polymarker.info / ) was used to design KASP primers targeting the two alleles of the target SNP marker AX-111027348. The primers included two upstream primers and one universal downstream primer.

[0041] The upstream primer 1 is a DNA molecule with a fluorescent label FAM added to the 5' end, and its sequence is shown in SEQ ID NO.2; the upstream primer 2 is a DNA molecule with a fluorescent label HEX added to the 5' end, and its sequence is shown in SEQ ID NO.3; the universal downstream primer is a single-stranded DNA molecule of SEQ ID NO.4.

[0042] The developed KASP marker was used to perform PCR detection on 24 lines in the recombinant inbred line population. PRAMS professional SNP detection reagent (Wuhan Jingpeptide Biotechnology Co., Ltd.) was used. The amplification system is shown in Table 3, and the PCR amplification procedure is shown in Table 4.

[0043] Table 3 PCR amplification system

[0044]

[0045] Table 4 PCR amplification program

[0046]

[0047] PCR reactions were performed on an AB PCR instrument (manufactured by AB Company), and the fluorescence value of the PCR product was detected using an ABI 7500 real-time fluorescence quantitative PCR instrument for genotyping.

[0048] The results are as follows Figure 3 As shown in Figure A, the upper left image shows a line with the CC genotype for the SNP marker AX-111027348, and the lower right image shows a line with the TT genotype for the SNP marker AX-111027348. This indicates that the marker can distinguish different genotypes of the SNP marker AX-111027348. Testing showed that the KASP marker typing results were consistent with the genotype results obtained from the wheat 55K SNP array, demonstrating that the developed KASP marker can successfully genotype the SNP marker AX-111027348.

[0049] Example 2 Verification in natural wheat populations

[0050] A total of 272 wheat varieties (lines) were collected and planted in the Dahe Experimental Park of the Hebei Academy of Agriculture and Forestry Sciences (Shijiazhuang, 38°05′ N, 114°44′ E) during the 2023-2024 season. Seedling was performed manually, with row spacing of 30 cm and plant spacing of 10 cm. Each line was planted in one row, totaling 21 plants. Conventional field management was followed, with both field planting and a hot-zone planting. The hot-zone planting was subjected to a greenhouse-covered heat treatment during the flowering period (May 10–30), with greenhouse temperatures recorded. All wheat materials were harvested, air-dried, and threshed upon maturity. Thousand-grain weight was measured using a grain analyzer with three replicates.

[0051] DNA of 272 varieties was extracted and SNP typing was performed on the 272 DNA samples using the steps in Example 1 ( Figure 3 B).

[0052] Table 5 shows the results of KASP marker typing and thousand-grain weight under heat conditions. Fifty varieties (lines) (18.38%) had the genotype TT, identical to China Spring; 219 varieties (lines) (80.51%) had the genotype CC, identical to Han 6172; and three varieties (lines) (1.10%) had the heterozygous genotype CT. Based on the typing results and analysis of their thousand-grain weight phenotypic data, it was found that the thousand-grain weight of wheat varieties (lines) with the genotype TT did not differ significantly from that of varieties (lines) with the genotype CC under field conditions and heat conditions. However, the rate of decrease in thousand-grain weight of wheat varieties (lines) with the genotype TT under heat conditions was significantly lower than that of varieties (lines) with the genotype CC. Although the genetic background of the varieties (lines) constituting the natural population is highly diverse and the variation in thousand-grain weight between varieties (lines) masked the difference in thousand-grain weight between wheat varieties (lines) with the genotype TT and those with the genotype CC under heat conditions, the contribution of the genotype TT was still reflected in the rate of decrease in thousand-grain weight under heat conditions. This indicates that the KASP marker developed in the present invention can be used to assist in the identification of heat tolerance in wheat.

[0053] Table 5 Variance analysis of 1000-grain weight and 1000-grain weight decline rate under heat environment in natural populations of wheat varieties (lines) with different genotypes

[0054]

[0055] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be practiced over a wide range of parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although specific embodiments have been given herein, it should be understood that further modifications may be made to the present invention. In summary, this application is intended to encompass any variations, uses, or improvements to the present invention, including those made by conventional techniques known in the art that depart from the scope of the present invention. Applications of the essential features may be made within the scope of the following claims.

Claims

1. A reagent for detecting the SNP molecular marker AX-111027348 closely linked to the main effect QTkw / Hz-4D of wheat grain weight related to heat tolerance in detecting wheat grain weight traits under heat conditions, characterized in that: The SNP molecular marker AX-111027348 and QTkw / Hz-4D are co-localized in the physical interval of 16.15Mb to 25.25Mb on the short arm of chromosome 4D of the wheat genome; the nucleotide sequence of the SNP molecular marker AX-111027348 is shown in SEQ ID NO.1, and there is a C / T mutation at 151bp; the wheat materials are Handan 6172 and Chinese Spring.

2. A KASP primer set for amplifying the SNP molecular marker AX-111027348, which is tightly linked to the main effect QTkw / Hz-4D of wheat grain weight and is associated with wheat heat tolerance, for preparing a product for screening wheat materials with high or low thousand-grain weight under heat conditions, characterized in that: The KASP primer set comprises a forward primer 1, a forward primer 2 and a universal reverse primer; the sequence of the forward primer 1 is shown in SEQ ID NO.2, the sequence of the forward primer 2 is shown in SEQ ID NO.3, and the sequence of the universal reverse primer is shown in SEQ ID NO.4; the SNP molecular marker AX-111027348 and the main effect QTkw / Hz-4D of grain weight related to wheat heat tolerance are co-localized in the physical interval of 16.15Mb to 25.25Mb on the short arm of chromosome 4D of the wheat genome; the nucleotide sequence of the SNP molecular marker AX-111027348 is shown in SEQ ID NO.1, and a C / T mutation exists at 151bp of the SNP molecular marker; the wheat materials are Handan 6172 and Chinese Spring.

3. A method for screening wheat varieties with high or low thousand-grain weight under a hot environment, characterized in that: Using the genomic DNA of wheat to be tested as a template and the KASP primer set according to claim 2 as primers, PCR amplification is performed to obtain an amplified product; when the amplified product contains QTkw / Hz-4D and the genotype is TT, the wheat to be tested is wheat with high 1000-grain weight under a hot environment; if the amplified product contains QTkw / Hz-4D and the genotype is CC, the wheat to be tested is wheat with low 1000-grain weight under a hot environment; QTkw / Hz-4D is a major effect of grain weight related to wheat heat tolerance and is located in the physical interval of 16.15Mb to 25.25Mb on the short arm of chromosome 4D of the wheat genome; the nucleotide sequence of the SNP molecular marker AX-111027348 is shown in SEQ ID NO.1, and a C / T mutation exists at 151bp thereof; the wheat materials are Handan 6172 and Chinese Spring.

4. Use of a reagent for detecting the SNP molecular marker AX-111027348, which is tightly linked to the main effect QTkw / Hz-4D of wheat grain weight and is associated with heat tolerance, or a kit comprising the KASP primer set according to claim 2, in wheat grain weight breeding under assisted heat conditions; the nucleotide sequence of the SNP molecular marker AX-111027348 is shown in SEQ ID NO. 1, and a C / T mutation exists at position 151 bp; the wheat materials are Handan 6172 and Chinese Spring.

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