Molecular marker closely linked with heat-resistance-related wheat grain weight major QTL (Quantitative Trait Loci) and application of molecular marker

Abstract

The invention discloses a molecular marker closely linked with heat resistance-related wheat grain weight major QTL (quantitative trait loci) and application of the molecular marker, and belongs to the technical field of wheat molecular genetic breeding. The molecular marker and the wheat grain weight major QTkw / Hz-4D are co-localized on a 4D chromosome short arm of a wheat genome; the molecular marker AX-111027348 is located at the 151st basic group of a nucleotide sequence as shown in SEQ ID NO.1, C / T mutation exists, and a KASP marker is developed from the molecular marker AX-111027348; the molecular marker provided by the invention can be used for carrying out grain weight assisted selection on Han 6172 and Chinese spring or derivative materials thereof, or carrying out heat resistance identification on a common 6-ploid wheat material. The molecular marker provided by the invention has important application in wheat grain weight character molecular marker assisted breeding 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 environment. Background Art

[0002] Wheat is an important food crop in the world, providing food for one-third of the world's population. With global warming, wheat often encounters extreme high temperatures in the late growth period, which seriously threatens wheat production. Improving wheat's heat resistance and cultivating heat-resistant wheat varieties are of great strategic significance to ensuring my country's food security and social stability.

[0003] The three elements of wheat yield include: number of ears per unit area, number of grains per ear and grain weight. Among them, grain weight has a relatively independent effect on yield improvement, and grain weight is mainly affected by genetic factors. Among the three elements of yield, it can be stably inherited. Therefore, grain weight plays an important role in increasing wheat yield. Under heat stress conditions, reducing the decline in grain weight can minimize the yield loss caused by heat damage.

[0004] In recent years, the development of high-throughput sequencing technology, gene chip detection technology, and the subsequent major breakthroughs in wheat genomics have provided a solid foundation for the fine analysis of quantitative trait loci and the development of molecular markers, and also made it possible to carry out wheat genetic improvement based on molecular marker-assisted selection. Although many quantitative trait loci (QTL) related to thousand-grain weight have been reported, there are fewer QTLs related to thousand-grain weight under heat stress, and even fewer heat-resistant molecular markers are available.

[0005] Molecular marker-assisted selection directly selects the target genotype, which is not affected by gene expression and environmental changes, and can improve the accuracy of selection, thereby accelerating breeding. Therefore, it is necessary to identify stable and major 1000-grain weight QTLs under heat conditions, develop molecular markers closely linked to them, and improve the efficiency of heat-resistant wheat breeding. Summary of the invention

[0006] The purpose of the present invention is to provide a molecular marker of 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, wherein the gene locus is located in the physical interval of 16.15Mb to 25.25Mb of wheat chromosome 4D.

[0008] The molecular marker AX-111027348, which is tightly linked to the main effect QTkw / Hz-4D of grain weight related to wheat heat tolerance, is characterized in that the molecular marker is located at the 151st base of the nucleotide sequence shown in SEQ ID NO.1, and there is 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, the wheat materials are Handan 6172, Chinese Spring or populations derived therefrom.

[0011] Correspondingly, in the method of 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 through KASP marker PCR detection and the genotype is TT, 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 the heat resistance of wheat using the KASP marker is characterized in that: if the wheat material to be tested is identified as containing QTkw / Hz-4D through KASP marker PCR detection and the genotype is TT, then 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 resistance; if the wheat material to be tested is identified as containing QTkw / Hz-4D through KASP marker detection and the genotype is CC, then 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 resistance, 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 of more than 38° C. for at least 3 days during the wheat filling period, or an artificially created wheat growth environment with a maximum daily temperature of more than 38° C.

[0015] Beneficial effects of the present invention: Using the high-generation recombinant self-pollinated population of Handan 6172 and Chinese Spring, a wheat grain weight QTkw / Hz-4D related to heat tolerance was finally located through phenotypic collection and linkage analysis, and a SNP marker AX-111027348 closely linked to it was also located. The KASP marker was further developed based on the locus information, which has the advantages of stable amplification, accurate typing, and easy and fast detection. It can be used to assist in the selection of the thousand-grain weight of Handan 6172, Chinese Spring or its derivative materials under hot environments or to identify the heat tolerance of the materials. The KASP marker was significantly correlated with the rate of decrease in thousand-grain weight caused by heat environment in a natural population consisting 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 breeding heat-resistant wheat varieties and provide a molecular-assisted selection method. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the co-localization map of wheat grain weight QTkw / Hz-4D and molecular markers on chromosome 4D related to heat tolerance in Example 1.

[0017] Figure 2 Thousand-grain weight of different genotypes of RILs population in the field (A) and under heat environment (B).

[0018] Figure 3 The genotyping results of KASP markers for RILs population (A) and natural population wheat materials (B). DETAILED DESCRIPTION

[0019] 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, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

[0020] Example 1 Discovery of QTkw / Hz-4D related to wheat grain weight under thermal environment and development of KASP markers.

[0021] Identification of 1000-grain weight phenotype under heat stress

[0022] The wheat variety Han 6172 was used as the female parent and Chinese Spring as the male parent to obtain F1, which was self-pollinated to produce F2. The F7 generation recombinant inbred line (RILs) population containing 269 lines was obtained by single seed transmission. The thousand-grain weight trait of 269 lines in the RILs population was identified in 8 environments in 2020-2021, 2021-2022 and 2022-2023.

[0023] The field experiment was conducted in the Dahe Experimental Park of Hebei Academy of Agriculture and Forestry Sciences (Shijiazhuang, 38°05′ N, 114°44′ E). The experimental materials were arranged in sequence and sown manually, with a row spacing of 30 cm and a plant spacing of 10 cm. Each plant line was planted in one row with a total of 21 plants. Routine field management was performed, and there were no serious pests and lodging during the growth period. The experiment set up field conditions and two thermal environment conditions. The thermal environment conditions were covered with a shed to increase temperature during the flowering period of wheat (May 10-May 30), and the shed temperature was recorded at the same time. All wheat materials were harvested and dried and threshed after maturity. The thousand-grain weight of wheat was measured using a grain grain analyzer, and repeated three times.

[0024] 2. Construction of genetic linkage map and QTL positioning

[0025] 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.

[0026] Using the complete interval mapping method of IciMapping 4.1 software combined with the thousand-grain weight data and genetic linkage map, a stable major 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 ).

[0027] Under field conditions, QTkw / Hz-4D was identified in 2021 and 2022, with LOD values ​​of 5.0775 and 6.5649, respectively, and the contribution rates to phenotypic variation were 6.2465% and 0.8805%, respectively; under hot environments, QTkw / Hz-4D was identified five times from 2021 to 2023, with the minimum LOD value of 5.4482 and the maximum value of 22.1732, and the minimum contribution rate to the phenotype was 7.1627% and the maximum was 28.6358%. The contribution rate of this QTL under hot environment conditions is significantly higher (see Table 1).

[0028] Table 1 Identification of the main effect QTkw / Hz- 4D of wheat grain weight

[0029] 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.

[0030] According to the genotyping of this locus, the differences in thousand-grain weight of different genotypes in the recombinant inbred line population were statistically analyzed. Figure 2The results showed that under field planting conditions within three years, the average thousand-grain weight of the TT genotype strains was significantly higher than that of the CC genotype strains only in 2022, and there was no significant difference in the other two years; under five thermal environment conditions within three years, the average thousand-grain weight of the TT genotype strains was significantly higher than that of the CC genotype strains (p < 0.05).

[0031] From the perspective of the decrease rate of thousand-grain weight under heat environment in different genotype strains in the recombinant inbred line population in Table 2, 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 decrease rate of thousand-grain weight 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 decrease rate of thousand-grain weight between the TT genotype strains and the CC genotype strains reached an extremely significant level (p < 0.01).

[0032] Table 2 Variance analysis of the decrease rate of thousand-grain weight of different genotypes in the recombinant inbred line population constructed by Handan 6172 / Chinese Spring under hot environment

[0033] This indicates that the effect of this site 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.

[0034] 3. Development of KASP Marker

[0035] The flanking sequence of the 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 that is closely linked to QTkw / Hz-4D, and the polymorphism is T / C.

[0036] KASP primers were designed using Polymarker (http: / / www.polymarker.info / ) for the two alleles of the target SNP marker AX-111027348. The primers included two upstream primers and one universal downstream primer.

[0037] The upstream primer 1 is a DNA molecule with a fluorescent label FAM added to the 5' end, and the 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 the sequence is shown in SEQ ID NO.3; the universal downstream primer is a single-stranded DNA molecule of SEQ ID NO.4.

[0038] The developed KASP marker was used to perform PCR detection on 24 lines in the recombinant inbred line population, using PRAMS professional SNP detection reagent (Wuhan Jingpeptide Biotechnology Co., Ltd.). The amplification system is shown in Table 3, and the PCR amplification program is shown in Table 4.

[0039] Table 3 PCR amplification system

[0040] Table 4 PCR amplification program

[0041] The PCR reaction was carried out 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.

[0042] The results are as follows Figure 3 As shown in A, the upper left of the figure shows a line with the genotype of SNP marker AX-111027348 being CC, and the lower right shows a line with the genotype of SNP marker AX-111027348 being TT, indicating that the marker can distinguish different genotypes of SNP marker AX-111027348. After testing, the typing results of the KASP marker were consistent with the genotype results obtained by the wheat 55K SNP chip, proving that the developed KASP marker can achieve successful typing of the SNP marker AX-111027348.

[0043] Example 2 Verification in natural wheat populations

[0044] 272 wheat varieties (lines) were collected and planted in the Dahe Experimental Park of Hebei Academy of Agriculture and Forestry Sciences (Shijiazhuang, 38°05′ N, 114°44′ E) in 2023-2024. The seeds were sown manually with a row spacing of 30 cm and a plant spacing of 10 cm. Each plant line was planted in one row with a total of 21 plants. Routine field management was carried out, and the experiment was set up with field planting and one hot environment planting. The hot environment planting was covered with a shed to increase temperature during the flowering period of wheat (May 10-May 30), and the shed temperature was recorded at the same time. All wheat materials were harvested and dried after maturity, and the thousand-grain weight of wheat was measured using a grain grain analyzer, which was repeated three times.

[0045] DNA of 272 species was extracted, and SNP typing was performed on the 272 DNA samples using the steps in Example 1 ( Figure 3 B).

[0046] The results of KASP marker typing and thousand-grain weight under heat environment are shown in Table 5: 50 varieties (lines) (18.38%) have genotype TT, which is the same as China Spring; 219 varieties (lines) (80.51%) have genotype CC, which is the same as Han 6172, and 3 varieties (lines) (1.10%) have heterozygous genotype CT. According to the typing results combined with the analysis of its thousand-grain weight phenotypic data, it was found that the thousand-grain weight of wheat varieties (lines) with genotype TT had no significant difference with that of varieties (lines) with genotype CC under field conditions and heat environment, but the thousand-grain weight decline rate of wheat varieties (lines) with genotype TT under heat environment was significantly lower than that of varieties (lines) with genotype CC. Although the genetic background of varieties (lines) constituting natural populations is very different, and the thousand-grain weight variation between varieties is large, which masks the difference in thousand-grain weight between wheat varieties (lines) with genotype TT and varieties (lines) with genotype CC under heat environment, the contribution of TT genotype is still reflected in the decline rate of thousand-grain weight under heat environment. This indicates that the KASP marker developed in the present invention can be used to assist in the identification of heat tolerance in wheat.

[0047] Table 5 Variance analysis of thousand-grain weight and thousand-grain weight decline rate under heat environment in natural populations of wheat varieties (lines) with different genotypes

[0048] 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 implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. A molecular marker AX-111027348 closely linked to the main effect QTkw / Hz-4D of grain weight related to wheat heat tolerance, characterized in that: The molecular marker and the major effect QTkw / Hz-4D are co-localized in the physical interval of 16.15Mb to 25.25Mb of the short arm of chromosome 4D of the wheat genome; the molecular marker is located at the 151st base of the nucleotide sequence shown in SEQ ID NO.1, and there is a C / T mutation.

2. A KASP marker developed for the molecular marker according to claim 1, characterized in that: The KASP marker 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.

3. A product for screening high or low thousand-grain weight wheat materials under a hot environment prepared using the KASP marker of claim 2. The wheat material is Handan 6172, Chinese Spring or a population derived therefrom.

4. A method for screening wheat varieties with high or low thousand-grain weight under a hot environment using the KASP marker according to claim 2, characterized in that: If the wheat material to be tested contains QTkw / Hz-4D and the genotype is TT, the wheat material to be tested is identified as wheat with high thousand-grain weight under hot environment, and if the genotype is CC, the wheat material to be tested is identified as wheat with low thousand-grain weight under hot environment. The wheat material is Handan 6172, Chinese Spring or a population derived therefrom.

5. A method for identifying heat tolerance of wheat using the KASP marker according to claim 2, characterized in that: If the wheat material to be tested is identified as containing QTkw / Hz-4D through KASP marker PCR detection and the genotype is TT, then the wheat material to be tested is identified as wheat with a low rate of decrease in thousand-grain weight under heat, and the wheat material has strong heat resistance; if the wheat material to be tested is identified as containing QTkw / Hz-4D through KASP marker detection and the genotype is CC, then the wheat material to be tested is wheat with a high rate of decrease in thousand-grain weight under heat, and the wheat material has weak heat resistance, and the wheat material is all hexaploid common wheat materials.

6. Use of the QTL according to claim 1 or the KASP marker according to claim 2 in heat-tolerant wheat breeding.

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