Wheat awn length qtl positioning and closely linked molecular marker and application thereof

Abstract

The application belongs to the field of genetic engineering and molecular breeding, and particularly relates to a wheat awn length QTL positioning and closely linked molecular marker and application. The application takes long awn variety Jimei 262 and short awn variety Yannong 24 as parents to construct a DH population, constructs a high-density genetic linkage map based on a wheat 55K SNP chip, and performs awn length QTL positioning. It is found that 9 QTLs related to awn length are detected, which are distributed on 5A, 4B, 3D, 4D, 4A, 7A, 2A, 1B and 7B chromosomes. The application first finds new QTL sites on 4B, 7A and 7B chromosomes. The QAl.saas-5A flanking marker is successfully converted into a KASP functional marker, and the verification result is consistent with the phenotype. The application enriches the wheat awn length genetic site resource, digs out new stable QTLs and develops practical molecular markers, and provides a basis for fine positioning, cloning and molecular breeding of awn length genes.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering and molecular breeding, specifically relating to a molecular marker for QTL mapping of wheat awn length and its close linkage, and its application. Background Technology

[0002] Wheat awns are an important morphological structure of the wheat spike, extending from the tip of the lemma of the florets. They are a significant product of wheat's long-term adaptation to the environment and artificial selection, playing a crucial role in plant growth and development, yield formation, and stress resistance. Removing awns from different parts of the wheat spike leads to a significant decrease in the thousand-grain weight and the number of grains per spike. Awns have a significant regulatory effect on wheat grain weight, and this effect varies significantly among different varieties. Generally, awned varieties can yield more than 10% more than awnless varieties, making awns a vital trait for increasing wheat yield. Wheat uses spikelets as its dispersal unit; the barbs on the surface of the awn protect the grains after they fall to the ground and promote soil penetration and propagation, which is important for natural wheat propagation and field colony establishment. Simultaneously, awns are crucial for enhancing wheat's stress resistance. Under drought conditions, leaf transpiration and photosynthetic rates decrease significantly, while the photosynthetic rate of awns remains relatively stable, resulting in awned wheat yielding 15%–30% more than awnless wheat.

[0003] The development of wheat awn growth is mainly regulated by five genes ( A , B1 , B2 , B3 and Hd It is determined by multiple minor-effect genes. Among them, A Genes promote the elongation of awns, while B1 , B2 and B3 This is an awn repressor gene, with a repression strength of [missing information]. B1 > B2 > B3 . Hd Genes control the hook-shaped curvature of the awns (Sourdille et al., 2002). Previous studies have confirmed that... Hd , B1 , B2 They are located in different chromosomal regions. B1 Located on chromosome 5AL, B2 Located on chromosome 6BL, Hd Located on 4AS staining, if B1 , B2 and Hd The presence of all three genes simultaneously will result in wheat that is completely awnless.

[0004] Traditional quantitative trait locus (QTL) mapping methods have identified multiple awn length-related genetic loci. However, the application of techniques such as gene SNP microarrays, genome-wide association studies (GWAS), and cluster segregation analysis (BSA) has significantly accelerated the development of genetic research on wheat crops. Currently, a large number of awn development-related QTLs have been mapped in wheat, and research on three key awn suppression genes is relatively in-depth, but only awn suppression genes have been identified. B1 It was successfully cloned. Therefore, further exploration of new loci for awn growth and analysis of their genetic effects and functions remain of great significance. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides a QTL mapping method for wheat awn length. This invention constructs a DH population using the long-awned material Jimai 262 and the short-awned material Yannong 24 as parents, and uses a 55K SNP chip to construct a high-density genetic linkage map for QTL mapping of wheat awn length.

[0006] This invention further provides a functional molecular marker closely linked to the QTL mapping of wheat awn length, providing a theoretical basis and technical support for the fine mapping, cloning, and molecular breeding of the wheat awn length gene.

[0007] Another objective of this invention is to provide the application of molecular markers based on the above-mentioned close linkage with QTL mapping in screening wheat varieties or lines with awn length traits.

[0008] The technical solution adopted by the present invention to achieve the above-mentioned objectives is as follows: (Claims) This invention provides a QTL mapping for wheat awn length, wherein the wheat awn length QTLs are located on chromosomes 5A, 4B, 3D, 4D, 4A, 7A, 2A, 1B, and 7B, respectively, and are named QAl.saas-5A, QAl.saas-4B, QAl.saas-3D, QAl.saas-4D, QAl.saas-4A, QAl.saas-7A, QAl.saas-2A, QAl.saas-1B, and QAl.saas-7B.

[0009] Furthermore, the marker intervals for the above-mentioned wheat awn length QTLs are as follows:

[0010] Preferably, the above-mentioned wheat awn length QTLs are located on chromosomes 4B, 7A, and 7B, respectively.

[0011] Another objective of this invention is to provide a molecular marker closely linked to the QTL mapping of wheat awn length, by converting the left-wing marker AX-110475033 of QAl.saas-5A into the KASP marker; the left-wing marker AX-108913575 of QAl.sass-4A into the KASP marker; the left-wing marker AX-110667361 of QAl.sass-4B into the KASP marker; and the left-wing marker AX-110526555 of QAl.sass-7A into the KASP marker.

[0012] Preferably, the KASP molecular marker is obtained by amplification using the following primer combination, the sequence of which is: QAl.saas-5A: Fa (as shown in SEQ ID NO.1): 5'-GAAGGTCGGAGTCAACGGATTGAGATTAAATGGAAACAATAGCGC-3', Fb (as shown in SEQ ID NO.2): GAAGGTGACCAAGTTCATGCTGAGATTAAATGGAAACAATAGCGT-3', R (as shown in SEQ ID NO.3): 5'-ACTCCCATCCATTTGTCA-3'; QAl.sass-4A: Fa (as shown in SEQ ID NO.4): 5'-GAAGGTCGGAGTCAACGGATTTGCTTCCTTGGCAGATTCTCTACGGCC-3', Fb (as shown in SEQ ID NO.5): 5'-GAAGGTGACCAAGTTCATGCTTTCCTTGGCAGATTCTCTACGGCG-3', R (as shown in SEQ ID NO.6): 5'-CAGAATCTGCAGGGGCCT-3' QAl.sass-4B: Fa (as shown in SEQ ID NO.7): 5'-GAAGGTCGGAGTCAACGGATTGACCGTGCATACAACAACTGCGGG-3', Fb (as shown in SEQ ID NO.8): 5'-GAAGGTGACCAAGTTCATGCTGACCGTGCATACAACAACTGCGGT-3', R (as shown in SEQ ID NO.9): 5'-TCCCGCTAAGCCACCCGAC-3'); QAl.sass-7A: Fa (as shown in SEQ ID NO.10): 5'-GAAGGTCGGAGTCAACGGATTTGCGATTGTGTGGGACAAGCTATGCAC-3', Fb (as shown in SEQ ID NO.11): 5'-GAAGGTGACCAAGTTCATGCTGATTGTGTGGGACAAGCTATGCAT-3', R (as shown in SEQ ID NO.12): 5'-AATTTGGGATCCCCAGGC-3'.

[0013] The present invention also provides a primer composition containing the primers indicated by the KASP molecular marker described above.

[0014] The present invention also provides the application of the above-mentioned molecular marker or primer composition in screening wheat varieties or lines with awn length related traits.

[0015] The beneficial effects of this invention are as follows: (1) In this invention, a DH population was constructed using the long-awned variety Jimai 262 and the short-awned variety Yannong 24 as parents. A high-density genetic linkage map was constructed based on the wheat 55K SNP chip to locate awn length QTLs. The results showed that a total of 9 awn length-related QTLs were detected, distributed on chromosomes 5A, 4B, 3D, 4D, 4A, 7A, 2A, 1B, and 7B. Among them, QAl.saas-5A was stably expressed in both environments, with a phenotypic contribution rate of 72.34% to 75.99%, and was a stable major-effect awn length QTL, with an additive effect from Jimai 262; QAl.saas-4B and QAl.saas-3D had phenotypic contribution rates greater than 3%, and were important minor-effect loci.

[0016] (2) This invention is the first to discover new QTL loci on chromosomes 4B, 7A, and 7B. The QAl.saas-5A flanking marker was successfully converted into a KASP functional marker, and the verification results were consistent with the phenotype. This invention enriches the genetic locus resources for wheat awn length, discovers new stable QTLs and develops practical molecular markers, providing a foundation for fine mapping, cloning, and molecular breeding of awn length genes. Attached Figure Description

[0017] Figure 1 Distribution of wheat awn length in the DH population at two different locations: A: Chengdu; B: Jinan. Figure 2KASP marker genotyping; A: KASP marker genotyping results for QAl.saas-5A; B: KASP marker genotyping results for QAl.sass-4A; C: KASP marker genotyping results for QAl.sass-4B; D: KASP marker genotyping results for QAl.sass-7A. Blue indicates materials phenotypically consistent with the parent Yannong 24, red indicates materials phenotypically consistent with the parent Jimai 262, green indicates heterozygous, and black indicates blank control. Detailed Implementation

[0018] The technical solution of the present invention will be further explained and described below through specific embodiments.

[0019] Example 1

[0020] 1.1 Experimental Materials and Cultivation The test materials were the long-awned parent Jimai 262, the short-awned parent Yannong 24, and 143 DH (Double Haploid) lines constructed from their hybridization, provided by Researcher Li Haosheng of the Crop Research Institute of Shandong Academy of Agricultural Sciences. In 2023 and 2024, these materials were planted at the experimental base of the Crop Research Institute of Shandong Academy of Agricultural Sciences (Jinan) and the Modern Agricultural Science and Technology Innovation Demonstration Park of Sichuan Academy of Agricultural Sciences (Chengdu), with two replicates for each environment.

[0021] During the late grain-filling stage of wheat, awn length was measured in both environments for all materials. The awn length was measured from spikes on 10 randomly selected individual plants from each DH line for subsequent genetic analysis.

[0022] 1.3 DNA extraction and construction of wheat genetic linkage map Genomic DNA was extracted from two wheat parents and 143 DH lines using the CTAB method. The DNA was then sent to Beijing Compson Biotechnology Co., Ltd. for 55K microarray analysis. SNP detection and analysis followed the guidelines of Ren et al. (2012). Marker genotyping and Bin map construction were performed using QTLIciMapping software, resulting in a high-density genetic linkage map covering all 21 wheat chromosomes.

[0023] 1.4 QTL Positioning Analysis of Wheat Awn Length Agronomic trait values ​​(awn length) were entered using the BIP function in the QTL Icimapping software. In the QTL mapping study of wheat awn length, the Complete Interval Mapping (ICIM) method was used for genetic analysis. QTL nomenclature followed the international genetic nomenclature rules (http: / / wheat.pw.usda.gov / ggpages / wgc / 98 / Intro.htm).

[0024] 2 Results and Analysis 2.1 Phenotypic characteristics of DH population awn length

[0025] In the Jinan environment, the awn length of Jimai 262 was 5.63 cm, and that of Yannong 24 was 1.25 cm. The awn length ranged from 0.67 to 7.66 cm across 143 DH lines, with an average awn length of 3.29 cm and a population coefficient of variation of 64.30%. In the Chengdu environment, the awn length of Jimai 262 was 5.65 cm, and that of Yannong 24 was 0.33 cm. The awn length ranged from 0.38 to 6.85 cm across 143 DH lines, with an average awn length of 2.84 cm and a population coefficient of variation of 76.18%.

[0026] In both Jinan and Chengdu environments, statistical results of awn length from 143 strains showed a significant segregation pattern beyond parentages, with a normal distribution. Statistical analysis revealed that the absolute values ​​of population skewness were all less than 3, and the absolute values ​​of kurtosis were less than 10. (Specific details are as follows...) Figure 1 As shown in the figure. This result indicates that wheat awn length conforms to the genetic characteristics of quantitative traits and can be applied to QTL mapping studies.

[0027] 2.2 Construction of high-density genetic linkage maps The wheat 55K chip contains 53,063 markers. After initial filtering, 16,515 SNP markers showed polymorphism. These valid markers were integrated into 401 Bin markers, and a genetic linkage map covering all 21 chromosomes of the wheat genome was successfully constructed based on this. The number of markers on each chromosome, genetic distance, and average interval are shown in Table 1.

[0028] Table 1. Genetic linkage map of longevity

[0029] 2.3 QTL Positioning Results of Mangchang Based on phenotypic data from two environments and a high-density genetic map, a total of nine QTLs associated with wheat awn length were detected, located on chromosomes 5A, 4B, 3D, 4D, 4A, 7A, 2A, 1B, and 7B. Under both different environmental conditions, one QTL located on chromosome 5A, namely QAl.saas-5A, was detected. This QTL explained 72.34%–75.99% of the phenotypic variation, with a LOD value of 66.91–69.60. Its additive effect site originated from Jimai 262 and is the major awn-promoting QTL. Three major-effect QTLs with a phenotypic contribution rate greater than 3% were identified: QAl.saas-5A, QAl.saas-4B, and QAl.saas-3D, with phenotypic contribution rates of 72.34%–75.99%, 2.76%–3.24%, and 1.36%–3.24%, respectively, and LOD values ​​of 66.91–69.60, 7.09–9.50, and 3.64–7.59, respectively. Their additive effects all originated from Jimai 262. One additive-effect QTL, QAl.saas-7B, originated from the parent Yannong 24, with a phenotypic contribution rate of 2.04% and an LOD value of 4.68. The marker intervals, LOD values, phenotypic contribution rates, and additive effects of each QTL are shown in Table 2. The QTL distribution for each chromosome is shown in Table 3. Figure 2 .

[0030] Table 2. Summary of stable QTL loci for awn length in the wheat DH population “Jimai 262 × Yannong 24”

[0031] 2.4 Development of KASP markers related to wheat awn length The left-wing marker AX-110475033 of the major-effect QTL QAl.saas-5A was converted into a KASP marker (Fa: 5'-GAAGGTCGGAGTCAACGGATTGAGATTAAATGGAAACAATAGCGC-3', Fb: 5' GAAGGTGACCAAGTTCATGCTGAGATTAAATGGAAACAATAGCGT-3', R: 5'-ACTCCCATCCATTTGTCA-3'). Validation was performed using existing DH lines. 68 lines carried the positive effect locus of QAl.saas-5A, and 75 lines carried the negative effect locus. Genotypic typing was highly consistent with the awn length phenotype, indicating that this marker can be stably used for molecular-assisted selection of the awn length trait. In addition, the left wing marker AX-108913575 of QAl.sass-4A was transformed (Fa: 5'-GAAGGTCGGAGTCAACGGATTTGCTTCCTTGGCAGATTCTCTACGGCC-3', Fb: 5'-GAAGGTGACCAAGTTCATGCTTTCCTTGGCAGATTCTCTACGGCG-3', R: 5'-CAGAATCTGCAGGGGCCT-3'), left wing tag AX-110667361 of QAl.sass-4B (Fa: 5'-GAAGGTCGGAGTCAACGGATTGACCGTGCATACAACAACTGCGGG-3', Fb: 5'-GAAGGTGACCAAGTTCATGCTGACCGTGCATACAACAACTGCGGT-3', R: The genotypic profiles of the markers 5'-TCCGCTAAGCCACCCGAC-3' and the left-wing marker AX-110526555 of QAl.sass-7A (Fa: 5'-GAAGGTCGGAGTCAACGGATTTGCGATTGTGTGGGACAAGCTATGCAC-3', Fb: 5'-GAAGGTGACCAAGTTCATGCTGATTGTGTGGGACAAGCTATGCAT-3', R: 5'-AATTTGGGATCCCCAGGC-3') were highly consistent with the awn length phenotype, indicating that these markers can be stably used for molecular-assisted selection of the awn length trait.

[0032] 3. Discussion Wheat awns play important biological functions in photosynthesis, yield formation, and drought and stress resistance, making them a key target trait in wheat breeding. This invention utilizes a DH population constructed from Jimai 262 and Yannong 24, and employs 55K microarray technology to construct a high-density genetic linkage map. Nine QTLs related to awn length were located under two different environments, involving nine chromosomes: 5A, 4B, 3D, 4D, 4A, 7A, 2A, 1B, and 7B. The QTLs on 4B and 7A are novel loci not previously reported, enriching the genetic locus resources for wheat awn length.

[0033] The additive effects of QAl.saas-4B and QAl.saas-7A both originate from Jimai 262, while the additive effect of QAl.saas-7B originates from Yannong 24. QAl.saas-5A on 5A staining is stably expressed in both environments, located between markers AX-110475033 and AX-110552728, with a genetic distance of 6 cM. This gene locus contributes 72.34%–75.99% to the phenotype, and its additive effect is provided by Jimai 262, making it a stable major-effect awn length QTL. This locus promotes awn elongation and may correspond to... B1 Allelic variation, regulatory variation or related B1 Tightly linked variants. This invention is the first to locate QA1.saas-4A on chromosome 4A, with a positive additive effect, promoting awn elongation, and... Hd The effects are different, suggesting that they may be: A Alleles of the gene. This invention detected QAl.saas-1B on chromosome 1B, with the enhancing allele originating from Jimai 262, contributing 1.72% to the phenotype. Furthermore, the QTLs detected on chromosomes 2A, 3D, and 4D all promoted awn elongation. This invention transformed the tightly linked marker of the major QTL QAl.saas-5A into a KASP functional marker, demonstrating high accuracy in population validation, and can be directly used for high-throughput molecularly assisted selection of wheat awn elongation.

[0034] In summary, this invention has identified a stable major QTL for wheat awn length and a novel locus, and developed practical functional markers, providing an important foundation for the fine localization, map-based cloning, and molecular breeding of high-yield and stress-resistant wheat awn length genes.

Claims

1. A QTL positioning method for wheat awn length, characterized in that, The wheat awn length QTLs are located on chromosomes 5A, 4B, 3D, 4D, 4A, 7A, 2A, 1B, and 7B, respectively, and are named QAl.saas-5A, QAl.saas-4B, QAl.saas-3D, QAl.saas-4D, QAl.saas-4A, QAl.saas-7A, QAl.saas-2A, QAl.saas-1B, and QAl.saas-7B.

2. The wheat awn length QTL positioning according to claim 1, characterized in that, The labeled intervals for the QTL of wheat awn length are as follows: 。 3. The wheat awn length QTL positioning according to claim 1 or 2, characterized in that, The QTLs for wheat awn length are located on chromosomes 4B, 7A, and 7B, respectively.

4. A molecular marker based on the wheat awn length QTL localization tightly linked according to any one of claims 1-3, characterized in that, Convert the left-wing designation AX-110475033 of QAl.saas-5A to KASP designation; convert the left-wing designation AX-108913575 of QAl.sass-4A to KASP designation; convert the left-wing designation AX-110667361 of QAl.sass-4B to KASP designation; convert the left-wing designation AX-110526555 of QAl.sass-7A to KASP designation.

5. The molecular marker according to claim 4, characterized in that, The KASP molecular marker was amplified using the following primer combination, and its sequence is as follows: QAl.saas-5A: Fa: 5'-GAAGGTCGGAGTCAACGGATTGAGATTAAATGGAAACAATAGCGC-3', Fb: GAAGGTGACCAAGTTCATGCTGAGATTAAATGGAAACAATAGCGT-3', R: 5'-ACTCCCATCCATTTGTCA-3'; QAl.sass-4A: Fa: 5'-GAAGGTCGGAGTCAACGGATTTGCTTCCTTGGCAGATTCTCTACGGCC-3', Fb: 5'-GAAGGTGACCAAGTTCATGCTTTCCTTGGCAGATTCTCTACGGCG-3', R: 5'-CAGAATCTGCAGGGGCCT-3' QAl.sass-4B: Fa: 5'-GAAGGTCGGAGTCAACGGATTGACCGTGCATACAACAACTGCGGG-3', Fb: 5'-GAAGGTGACCAAGTTCATGCTGACCGTGCATACAACAACTGCGGT-3', R: 5'-TCCGCTAAGCCACCCGAC-3'); QAl.sass-7A: Fa: 5'-GAAGGTCGGAGTCAACGGATTTGCGATTGTGTGGGACAAGCTATGCAC-3', Fb: 5'-GAAGGTGACCAAGTTCATGCTGATTGTGTGGGACAAGCTATGCAT-3', R: 5'-AATTTGGGATCCCCAGGC-3'.

6. A primer composition, characterized in that, It contains primers represented by the KASP molecular marker as described in claim 5.

7. The use of a molecular marker as described in claim 5 or a primer composition as described in claim 6 in screening wheat varieties or lines with awn length associated with the trait.

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