Molecular marker closely linked with major QTL (quantitative trait loci) qSL2V of ear length of hairy wheat and application of molecular marker

Abstract

The invention discloses a molecular marker closely linked with a major QTL (Quantitative Trait Loci) qSL2V of the ear length of tufted wheat and application of the molecular marker. According to the invention, a new spike length major QTL is positioned from an artificially synthesized durum wheat-haynaldia villosa didiploid STH65-4 and is named as qSL2V, and a molecular marker I-M5 and a molecular marker I-M3 for identifying the locus are provided. The molecular marker can be used for molecular marker-assisted selective breeding of the ear length QTL locus qSL2V, a molecular tool for identification or auxiliary identification is provided for transferring and utilizing the ear length gene of hairy wheat, and the wheat germplasm resource creation and high-yield breeding process is accelerated.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and wheat molecular breeding, and relates to a molecular marker tightly linked to a major effect QTL (quantitative trait locus) site qSL2V of spike length on a 2VL chromosome of Haynaldiavillosa and an application thereof. Background Art

[0002] Wheat (Triticum aestivum L.) is the second largest grain crop in my country and plays an important role in ensuring my country's food security. With the total amount of cultivated land in my country restricted, the total amount of wheat consumption increasing, and adverse environments frequently occurring, it is particularly important to increase the yield per unit area and the total wheat yield. Wheat yield improvement mainly focuses on the improvement of traits such as the number of spikes, the number of grains per spike, and the thousand-grain weight. The spike traits related to yield include spike type, spike length, number of spikelets, number of grains per spike, and other traits. Modern breeding has made the genetic background between wheat germplasm more and more similar, which has brought certain restrictions to the improvement of wheat spike traits. Distant species are valuable gene banks for wheat genetic improvement, and have genetic diversity different from common wheat in terms of spike morphology and grain traits. Therefore, using the genetic diversity of distant species to improve wheat yield traits is an important way.

[0003] As an important trait of wheat spike, spike length directly or indirectly affects the number of grains per spike and grain weight, two factors related to wheat yield. In addition, the wheat spike length trait is correlated with the spikelet density. Low spikelet density can reduce the spread of fusarium head blight and effectively prevent wheat fusarium head blight. Therefore, it is of great significance to increase the number of wheat spikelets and obtain new high-yield wheat varieties by discovering spike length-related genes / QTL loci between wheat species or genera, developing tightly linked molecular markers, and utilizing them through molecular breeding technology. Summary of the invention

[0004] The purpose of the present invention is to identify a major QTL locus qSL2V for spike length from Triticum vulgare, and to provide a molecular marker closely linked to the QTL locus.

[0005] To achieve the above object, the present invention adopts the following scheme:

[0006] Molecular markers closely linked to the major QTL locus qSL2V for ear length in Elymus villosa, the molecular markers are I-M5 and I-M3.

[0007] The sequence of the molecular marker I-M5 is shown in SEQ ID NO:5, and the sequence of the molecular marker I-M3 is shown in SEQ ID NO:6.

[0008] The primer sequence of the molecular marker I-M5 consists of an upstream primer shown in SEQ ID NO:1 and a downstream primer shown in SEQ ID NO:2.

[0009] The primer sequence of the molecular marker I-M3 consists of an upstream primer shown in SEQ ID NO:3 and a downstream primer shown in SEQ ID NO:4.

[0010] The genetic distance between the molecular marker I-M5 and the main effect QTL site qSL2V of ear length is 1.27 cM, and the genetic distance between the molecular marker I-M3 and the main effect QTL site qSL2V of ear length is 6.10 cM.

[0011] The molecular markers are applied to durum wheat-Triticum villosa amphidiploid plants or their derivative lines, and the genomic DNA of the plants is amplified using the PCR method. When a 166bp band of the I-M5 molecular marker appears, or when a 189bp band of the I-M3 molecular marker appears, it indicates that the plant contains the main QTL site qSL2V for ear length from Triticum villosa.

[0012] PCR reaction system 10μL:

[0013] Contains 1 μL DNA template (concentration 80-150 ng / μL), 5 μL 2×Taq Mix, 0.2 μL each of left and right primers, and 3.6 μL ddH2O.

[0014] PCR reaction program: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 20 s, for a total of 35 cycles; extension at 72°C for 10 min; storage at 4°C for future use.

[0015] Detection of PCR amplification products: Take 2 μL of PCR amplification products and perform electrophoresis in 8% polyacrylamide gel at a constant voltage of 200 V for 50-60 min, stain with silver nitrate and perform labeling and typing.

[0016] A primer pair used to amplify a molecular marker tightly linked to the major QTL site qSL2V for length of pubescent wheat ears, wherein the molecular marker tightly linked to the major QTL site qSL2V for length of pubescent wheat ears is selected from I-M5 or I-M3, the specific primer pair sequence of I-M5 is shown in SEQ ID NO:1 and SEQ ID NO:2, and the specific primer pair sequence of I-M3 is shown in SEQ ID NO:3 and SEQ ID NO:4.

[0017] The primer pair is used in detecting the main effect QTL locus qSL2V of hairy wheat ear length.

[0018] As a preferred embodiment of the present invention, durum wheat-Triticum villosa amphidiploid plants or their derived wheat lines are taken as the objects, and the plant genomic DNA is amplified by PCR using the primer pair. When a 166bp band of the I-M5 molecular marker appears, or when a 189bp band of the I-M3 molecular marker appears, it indicates that the major QTL site qSL2V for the length of the villous wheat ear exists in the plant.

[0019] Beneficial Effects

[0020] The present invention locates a major QTL site qSL2V for spike length from a synthetic durum wheat-villous wheat amphidiploid germplasm resource, and obtains two closely linked molecular markers I-M5 and I-M3. These two molecular markers are co-dominant InDel markers, which can accurately identify the presence or absence of the major QTL site qSL2V for spike length in the durum wheat-villous wheat amphidiploid material and its derived heterochromatic system, and predict whether it has long spikes, thereby effectively detecting the major QTL site qSL2V for spike length in wheat molecular marker-assisted breeding. At the same time, the present invention also lays a good foundation for cloning the major QTL site qSL2V for spike length and studying its function. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The ear length phenotype of STH65-4 and STH79-3 in the mature plant stage in Example 1 of the present invention.

[0022] Figure 2 In Example 1 of the present invention, STH65-4 / STH79-3 F 2 Histogram of ear length of 203 individual plants in the population.

[0023] Figure 3 BSE-seq analysis results in Example 1 of the present invention.

[0024] Figure 4 Genetic linkage map of the major QTL locus qSL2V for ear length in Example 1 of the present invention.

[0025] Figure 5 Analysis of the genetic effect of qSL2V on ear length in Example 2 of the present invention and analysis of the association between its tightly linked marker I-M5 and ear length; Note: A / H genotype represents that a 166bp band can be amplified, and B genotype represents that a 166bp band cannot be amplified.

[0026] Figure 6 In Example 2 of the present invention, markers I-M5 and I-M3 were used to detect the F of STH65-4 / STH79-3. 2 Group results. DETAILED DESCRIPTION

[0027] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0028] Example 1 Location of the major QTL locus qSL2V for hairy wheat ear length and development of its tightly linked Indel markers I-M5 and I-M3

[0029] 1. Experimental Materials

[0030] Plant materials: Two parents, durum wheat-haynaldia villosa amphidiploids STH65-4 and STH79-3 (reference: Liu YQ, Liu JH, Huang ZP, et al. (2024) Phenotypic characterization and gene mapping of hybrid necrosis in Triticum durum-Haynaldia villosa amphiploids. Theor Appl Genet 137, 185.), of which STH65-4 is the long-spike parent and STH79-3 is the short-spike parent. The F of STH65-4 / STH79-3 2 Population creation: The long-eared amphidiploid STH65-4 was used as the female parent and the short-eared amphidiploid STH79-3 was used as the male parent. The F 1 , F 1 A single plant self-pollination yielded 203 F 2 The F 2 The population was used to construct a genetic map of the qSL2V target interval for ear length.

[0031] 2. Identification of ear length traits

[0032] The ear length is measured at the mature stage, and the length from the base to the top of the ear (excluding the awn length) is the ear length. 2 The spike length data of 203 plants in the group were obtained and statistically analyzed, and a frequency distribution diagram was drawn. The results are as follows Figure 1 and Figure 2 shown.

[0033] 3.BSE-seq(Bulked segregant exome capture sequencing)

[0034] Using Bulked Segregant Analysis (BSA), we analyzed the 2Ten extremely long-ear plants and ten extremely short-ear plants were selected from the segregating population. An equal amount of DNA was taken from each long-ear plant and mixed to construct a long-ear mixed pool, and an equal amount of DNA was taken from each short-ear plant and mixed to construct a short-ear mixed pool (Reference: Dong CH, Zhang LC, Chen ZX, et al. (2020) Combining a New Exome Capture Panel With an Effective varBScore Algorithm Accelerates BSA-Based Gene Cloning in Wheat. Front Plant Sci 11: 1249.). The constructed long-spike mixed pool, short-spike mixed pool and DNA of the two parents were sent to Shijiazhuang Boridi Biotechnology Co., Ltd. for exon capture sequencing (reference: Liu YQ, Liu JH, Huang ZP, et al. (2024) Phenotypic characterization and gene mapping of hybrid necrosis in Triticum durum-Haynaldia villosa amphiploids. Theor Appl Genet 137, 185.).

[0035] 4. Discovery and linkage map of the spike length QTL locus qSL2V

[0036] (1) Total DNA extraction

[0037] The modified CTAB method was used (reference: Carey SJ, Becklund LE, Fabre PP, et al. (2023) Optimizing the lysis step in CTAB DNA extractions of silica-dried andherbarium leaf tissues. Appl Plant Sci 11(3): e11522.).

[0038] (2) Primer design

[0039] According to the SNP and InDel polymorphic sites that coexist between parents and mixed pools obtained by BSE-seq analysis, with reference to the genome sequence of Dasypyrum villosum (reference: Zhang X, Wang HY, Sun HJ, et al. (2023) A chromosome-scale genome assembly of Dasypyrum villosum provides insights into its application as a broad-spectrum disease resistance resource for wheat improvement. Mol Plant 16: 432-451.), 10 co-dominant molecular markers with polymorphism between parents were screened in the 521Mb-587Mb segment of chromosome 2V for genetic map construction. All marker primer pairs were synthesized by General Bio (Anhui) Co., Ltd.

[0040] (3) PCR reaction and identification of amplified products

[0041] The reaction system for PCR amplification: contains 1 μL DNA template (concentration 80-150 ng / μL), 5 μL 2× Taq Mix, 0.2 μL left and right primers, 3.6 μL ddH2O;

[0042] PCR program: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 20 s, for a total of 35 cycles; extension at 72°C for 10 min; storage at 4°C for future use.

[0043] Take 2 μL of PCR amplification product and perform electrophoresis in 8% polyacrylamide gel at a constant voltage of 200 V for 50-60 min, stain with silver nitrate and perform labeling and typing.

[0044] The 10 polymorphic markers were used to identify 203 F 2 PCR amplification and genotyping were performed on individual strains in the population.

[0045] (4) Discovery of the ear length QTL locus qSL2V and construction of linkage map

[0046] 203 strain F 2The genotyping results of individual plants and the results of individual plant ear length were imported into QTL IciMapping4.2 software (reference: Dwiningsih Y, Kumar A, Thomas J, et al. (2021) Identification of genomic regions controlling chalkiness and grain characteristics in a recombinant inbred line rice population based on high-throughput SNP markers. Genes 12(11): 1690.), and the ear length QTL loci in STH65-4 were detected by composite interval mapping.

[0047] like Figure 3 As shown, the major QTL locus qSL2V for ear length was preliminarily located on chromosome 2VL.

[0048] like Figure 4 As shown, the main QTL locus qSL2V for ear length was located between molecular markers I-M5 and I-M3, with genetic distances of 1.27 cM and 6.10 cM, respectively. The LOD value was 3.82, which could explain 20.08% of the phenotypic variation in ear length. qSL2V was located between 584 Mb and 591 Mb on the long arm of chromosome 2V of E. villosa.

[0049] Example 2 Verification of the effect of the major QTL locus qSL2V for hairy wheat ear length

[0050] In order to verify the selection effect of molecular markers I-M5 and I-M3 in hybrid offspring and the genetic effect of qSL2V in offspring, we combined the F 2 The marker typing results of the population were used to verify the effect of qSL2V. The specific steps are as follows:

[0051] 1. Genotype identification

[0052] The reaction system for PCR amplification: contains 1 μL DNA template (concentration 80-150 ng / μ), 5 μL 2× Taq Mix, 0.2 μL left and right primers, 3.6 μL ddH2O;

[0053] PCR program: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 20 s, for a total of 35 cycles; extension at 72°C for 10 min; storage at 4°C for future use.

[0054] Detection of PCR amplification products: Take 2 μL of PCR amplification products and perform electrophoresis in 8% polyacrylamide gel at a constant voltage of 200 V for 50-60 min, stain with silver nitrate and perform labeling and typing.

[0055] If the molecular marker I-M5 can amplify a 166bp band, or the molecular marker I-M3 can amplify a 189bp band, it means that the main effect QTL site qSL2V of ear length exists in the tested germplasm; if it cannot be amplified, it means that the main effect QTL site qSL2V of ear length does not exist in the tested germplasm.

[0056] 2. Association Analysis

[0057] Based on the molecular markers I-M5 and I-M3, 2 The results of genotyping of individual plants in the population were 2 All the plants in the population were divided into two groups. All the plants in group A carried the qSL2V locus (genotypes A and H), and all the plants in group B did not carry the qSL2V locus (genotype B). The spike length data of all the plants in group A and the spike length data of all the plants in group B were used to analyze the correlation between the qSL2V locus and spike length (reference: Yu Q, Feng B, Xu Z, et al. (2022) Genetic dissection of three major quantitative trait loci for spike compactness and length in bread wheat (Triticum aestivum L.). Frontiers in Plant Science, 13: 882655.). The specific data are shown in Table 1. The results are as follows Figure 5 and Figure 6 As shown in the figure, the qSL2V locus is highly correlated with ear length, and the ear length of the group with the qSL2V locus is significantly higher than that of the group without the qSL2V locus. The above results show that qSL2V has a genetic effect of increasing ear length, and both I-M5 and I-M3 can be used as molecular markers to screen the major effect QTL locus qSL2V for ear length, and can also be used for molecular marker-assisted selection breeding for the major effect QTL locus qSL2V for ear length.

[0058] Table 1 203 strains F of STH65-4 / STH79-3 2 The ear length phenotype data of the population and the genotyping data of marker I-M5

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[0068] The above embodiments are merely exemplary implementations used to illustrate the principles of the invention, but the invention is not limited thereto. Those skilled in the art may make various improvements and changes without departing from the essence of the invention, and these improvements and changes also fall within the protection scope of the invention.

Claims

1. A molecular marker closely linked to the major QTL locus qSL2V for hairy wheat ear length, characterized in that: The molecular markers are I-M5 and I-M3, and the sequence information of the molecular markers are SEQ ID NO:5 and SEQ ID NO:6, respectively. The genetic distances between the molecular markers and the main QTL locus qSL2V of hairy wheat ear length are 1.27 cM and 6.10 cM, respectively.

2. The molecular marker according to claim 1, characterized in that The specific primer pair sequences of I-M5 are shown in SEQ ID NO: 1 and SEQ ID NO:

2.

3. The molecular marker according to claim 1, characterized in that The specific primer pair sequences of I-M3 are shown in SEQ ID NO: 3 and SEQ ID NO:

4.

4. Use of the molecular marker according to any one of claims 1 to 3 in molecular marker-assisted selection breeding for the major QTL locus qSL2V for hairy wheat ear length.

5. The use according to claim 4, characterized in that Using durum wheat-Hylotrichum villosa amphidiploid plants or their derived wheat lines as the objects, PCR was used to amplify the plant genomic DNA. When a 166bp band of the I-M5 molecular marker or a 189bp band of the I-M3 molecular marker appeared, it indicated that the major QTL locus qSL2V for H. villosa ear length existed in the plant.

6. The use according to claim 4, characterized in that The specific primer pair sequences of the I-M5 molecular marker are shown in SEQ ID NO: 1 and SEQ ID NO: 2; the specific primer pair sequences of the I-M3 molecular marker are shown in SEQ ID NO: 3 and SEQ ID NO:

4.

7. A primer pair for amplifying a molecular marker tightly linked to the major QTL locus qSL2V for hairy wheat ear length, characterized in that: The molecular marker tightly linked to the main QTL site qSL2V of hairy wheat ear length is selected from I-M5 or I-M3, the specific primer pair sequence of I-M5 is shown in SEQ ID NO:1 and SEQ ID NO:2, and the specific primer pair sequence of I-M3 is shown in SEQ ID NO:3 and SEQ ID NO:

4.

8. Use of the primer pair according to claim 7 in detecting the major QTL locus qSL2V for hairy wheat ear length.

9. The use according to claim 8, characterized in that: Taking durum wheat-Hylotrichum villosa amphidiploid plants or their derived wheat lines as the objects, the primer pair described in claim 7 is used to PCR amplify the plant genomic DNA. When a 166bp band of the I-M5 molecular marker appears, or when a 189bp band of the I-M3 molecular marker appears, it indicates that the main effect QTL site qSL2V of Hylotrichum villosa ear length exists in the plant.

10. The use according to claim 8, characterized in that: PCR reaction system 10μL: containing 1μL DNA template (concentration 80-150ng / μL), 5μL 2×Taq Mix, 0.2μL left and right primers, 3.6μL ddH2O; PCR reaction program: 95℃ pre-denaturation for 3min; 95℃ denaturation for 30s, 55℃ renaturation for 30s, 72℃ extension for 20s, a total of 35 cycles; 72℃ extension for 10min; 4℃ storage for future use; Detection of PCR amplification products: take 2μL PCR amplification products and perform electrophoresis in 8% polyacrylamide gel, electrophoresis at a constant voltage of 200V for 50-60min, silver nitrate staining and marked typing.

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