Breeding method for cultivating high sprout emergence resistant wheat by using wheat-rye translocation line T5DL·5DS-2RS

Abstract

The application discloses a breeding method for cultivating high sprout emergence resistant wheat by using a wheat-rye translocation line T5DL·5DS-2RS. The method uses oligonucleotide probe set #6 and a molecular marker combination (Xlfz7772 and Xlfz7979) to accurately identify and track the wheat-rye translocation line T5DL·5DS-2RS, determines the translocation breakpoint and fragment size of the translocation line, and finds that the translocation line is highly resistant to sprout emergence, and the resistance is derived from the aggregation of the 2RS of rye and the huixuanhong resistance gene. Transcriptome analysis reveals that the introduction of T5DL·5DS-2RS into cultivated wheat enhances the sprout emergence resistance by regulating the related genes of hormone signal and starch metabolism. The method can improve the sprout emergence resistance and grain hardness of wheat, especially white wheat, realizes the synchronous improvement of resistance and grain quality, and is suitable for safe production and genetic improvement of wheat in rainy sprout emergence areas.

Description

Technical Field

[0001] This invention belongs to the field of crop genetics and breeding and molecular biology, specifically relating to a breeding method for creating wheat varieties highly resistant to pre-sprouting using a specific wheat-rye small-fragment translocation line T5DL·5DS-2RS. This invention accurately identifies the chromosomal composition of the T5DL·5DS-2RS translocation line and its hybrid offspring populations using oligonucleotide probe kit #6 and molecular marker combinations (Xlfz7772 and Xlfz7979). Simultaneously, pre-sprouting experiments confirm that T5DL·5DS-2RS and its hybrid offspring carrying this translocation chromosome enhance pre-sprouting resistance. This demonstrates that introducing the translocation chromosome can improve both pre-sprouting resistance and grain firmness in wheat, making it suitable for breeding high-hardness, pre-sprouting-resistant wheat varieties, especially white-skinned wheat. Furthermore, RNA-seq analysis preliminarily reveals the molecular basis for the enhanced pre-sprouting resistance of the T5DL·5DS-2RS translocation line. Background Technology

[0002] Pre-harvest sprouting (PHS) refers to the phenomenon where grains sprout on the ear of a crop during the late stages of maturity and before harvest when the crop is exposed to continuous rain or high humidity. PHS in wheat not only leads to a decrease in thousand-grain weight and bulk density, but also causes excessive activation of hydrolytic enzymes such as α-amylase, which damages the starch structure, resulting in poor dough viscoelasticity and deterioration in baking quality. In severe cases, it can render the grains unusable for processing and consumption (Simsek S, Ohm JB, Lu H, Rugg M, Berzonsky W, Alamri MS, Mergoum M (2014) Effect of pre-harvest sprouting on physicochemical properties of starch in wheat. Foods, 3:194–207. https: / / doi.org / 10.3390 / foods3020194). Global warming has led to extreme weather events, with frequent PHS (Proteinized Wildlife Species) events, especially affecting white wheat, which has become a significant factor impacting wheat production (Chang C, Zhang H, Lu J, Si H, Ma C (2023) Genetic improvement of wheat with pre-harvest sprouting resistance in China. Genes 14:837. https: / / doi.org / 10.3390 / genes14040837). For example, from late May to early June 2023, heavy rainfall in the Huanghuai region led to a 6.9% decrease in the total summer grain output in Henan Province compared to 2022 (Guo K, Zou C, Yu W, Hu L, Fang W, Tan Y, Zhang L, Cheng L (2024) Comprehensive assessment of the intensity of continuous rain during the 2023 mature period of winterwheat in Henan province. Meteorological and Environmental Sciences, 1:97–104. https: / / doi.org / 10.16765 / j.cnki.1673-7148.2024.01.012). Wheat PHS causes more than $1 billion in global economic losses annually.Therefore, breeding wheat varieties resistant to pre-harvest sprouting, especially white-skinned wheat, is crucial for the safe production of wheat (Chang C, Zhang H, Lu J, Si H, Ma C (2023) Genetic improvement of wheat with pre-harvest sprouting resistance in China. Genes 14:837. https: / / doi.org / 10.3390 / genes14040837).

[0003] Wheat pre-harvest sprouting resistance is a complex quantitative trait controlled by multiple genes. Its regulatory mechanisms involve multiple dimensions such as seed dormancy maintenance, hormone signal balance, nutrient metabolism, and stress response (Tai L, Wang HJ, Xu XJ, Sun WH, Ju L, Liu WT, Li WQ, Sun J, Chen KM (2021) Pre-harvest sprouting incereals: genetic and biochemical mechanisms. Journal of Experimental Botany, 72(8), 2857–2876. https: / / doi.org / 10.1093 / jxb / erab024; Vetch JM, Stougaard RN, Martin JM, Giroux MJ (2019) Review: revealing the genetic mechanisms of pre-harvest sprouting in hexaploid wheat (Triticum aestivum L.). Plant Science, 281, 180–185.). https: / / doi.org / 10.1016 / j.plantsci.2019.01.004. First, seed dormancy, as the core physiological basis for resisting pre-harvest sprouting, can prevent germination by inhibiting premature embryo activation. The difference in dormancy intensity is mainly dominated by genetic factors, and is also dynamically regulated by environmental signals (such as temperature, light, and humidity) (Chang C, Zhang H, Lu J, Si H, Ma C (2023) Genetic improvement of wheat with pre-harvestsprouting resistance in China. Genes, 14(4), 837. https: / / doi.org / 10.3390 / genes14040837).The antagonistic balance between abscisic acid (ABA) and gibberellins (GAs) is a core pathway regulating the dormancy-germination transition: ABA inhibits the activity of seed germination-related enzymes (such as α-amylase) and maintains a low metabolic state by activating downstream signaling factors (such as the PYR / PYL receptor family), while GAs promote the synthesis of α-amylase, promote the degradation of nutrients, and provide energy for embryo growth (Toorop PE, Barroco RM, Engler G, Groot SPC, Hilhorst HWM (2005) Differentially expressed genes associated with dormancy or germination of Arabidopsis thaliana seeds. Planta, 221:637–647. https: / / doi.org / 10.1007 / s00425-004-1477-1). In addition, hormones such as ethylene (ETH), brassinolide (BR), auxin (IAA), and cytokinin (CTK) also affect the ability to resist pre-harvest sprouting by cross-regulating the levels and signal transduction of GA and ABA (Jhanji S, Goyal E, Chumber M, & Kaur G (2024) Exploring fine tuning between phytohormones and ROS signaling cascade in regulation of seeddormancy, germination and seedling development. Plant Physiology and Biochemistry, 207, 108352. https: / / doi.org / 10.1016 / j.plaphy.2024.108352).Starch, as the main energy storage substance in wheat grains, has its degradation efficiency directly determining the energy supply for germination initiation. Starch hydrolysis requires the synergistic action of multiple enzymes, including α-amylase, β-amylase, and α-glucosidase. Current research has demonstrated a negative correlation between α-amylase activity and seed dormancy. Upregulation of α-amylase inhibitor expression can significantly delay starch decomposition, cutting off the energy source for seed germination (Sun ZT, & Henson CA (1991). A quantitative assessment of the importance of barley seed alpha-amylase, beta-amylase, debranching enzyme, and alpha-glucosidase in starch degradation. Archives of Biochemistry and Biophysics, 284(2), 298–305. https: / / doi.org / 10.1016 / 0003-9861(91)90299-x; Mares, DJ, & Mrva, K. (2014). Wheat grain preharvest sprouting and latematurity). alpha-amylase. Planta, 240(6), 1167–1178. https: / / doi.org / 10.1007 / s00425-014-2172-5). Reactive oxygen species (ROS) play a signaling role in breaking seed dormancy. For example, Xu et al. found that the interaction between PHS9 and OsGAP can couple the transduction of ROS signal and ABA signal, regulating rice germination before harvest. Therefore, the removal of ROS increases the resistance to pre-harvest sprouting to a certain extent (Xu F, Tang J, Gao S, Cheng X, Du L, & Chu C (2019) Control of rice pre-harvest sprouting by glutaredoxin-mediated abscisic acid signaling. The Plant Journal, 100(5), 1036–1051. https: / / doi.org / 10.1111 / tpj.14501).In addition, since Tamyb10 promotes the synthesis of ABA in seeds and regulates the accumulation of flavonoids to give seeds color, red wheat generally has a stronger resistance to pre-harvest sprouting than white wheat, but this is not absolute (Lang J, Jiang H, Cheng M, Wang M, Gu J, Dong H, Li M, GuoX, Chen Q & Wang J (2024) Variation of TaMyb10 and their function on graincolor and pre-harvest sprouting resistance of wheat. The Plant Journal, 118(5), 1388–1399. https: / / doi.org / 10.1111 / tpj.16676).

[0004] Based on genomics and map-based cloning methods, nine clearly defined wheat genes related to pre-harvest sprouting resistance (TaMKK3-A, TaSdr, TaPHS1, TaDOG1, TaDFR, Tamyb10, TaQsd1, TaSRO1, and TaVp-1) were cloned, distributed across axes 2A, 2B, 3A, 3B, 3D, and 4A. These genes function in wheat pre-harvest sprouting resistance through different regulatory pathways (Vetch JM, Stougaard RN, Martin JM, & Giroux MJ (2019) Revealing the genetic mechanisms of pre-harvest sprouting in hexaploid wheat (Triticum aestivum L.). Plant Science, 281, 180–185. https: / / doi.org / 10.1016 / j.plantsci.2019.01.004; Chang C, Zhang H, Lu J, Si H, Ma C (2023) Genetic improvement of wheat with pre-harvest sprouting resistance in China. Genes, 14(4), 837. https: / / doi.org / 10.3390 / genes14040837).For example, TaMKK3-A encodes a mitogen-activated protein kinase (MAPK) kinase 3, which is involved in the phosphorylation of important proteins in the ABA signaling pathway in Arabidopsis thaliana (Danquah A, de Zélicourt A, Boudsocq M, Neubauer J, Frei Dit Frey N, Leonhardt N, Pateyron S, Gwinner F, Tamby JP, Ortiz-Masia D, Marcote MJ, Hirt H, & Colcombet J (2015) Identification and characterization of an ABA-activated MAP kinase cascade in Arabidopsis thaliana. The Plant Journal, 82(2), 232–244. https: / / doi.org / 10.1111 / tpj.12808; Torada A, Koike M, Ogawa T, Takenouchi Y, Tadamura K, Wu J, Matsumoto T, Kawaura K & Ogihara Y (2016)). ACausal Gene for Seed Dormancy on Wheat Chromosome 4A Encodes a MAP KinaseKinase. Current Biology, 26(6), 782–787. https: / / doi.org / 10.1016 / j.cub.2016.01.063; TaSRO1 interacts with TaVP-1, with TaVP-1 acting as a positive regulator and TaSRO-1 as a negative regulator, regulating seed dormancy and pre-harvest sprouting resistance in wheat by modulating the ABA signaling pathway (Liu S, Li L, Wang W, Xia G & Liu S (2024) TaSRO1 interacts with TaVP1 to modulate seed dormancy and pre-harvest sprouting resistance in wheat. Journal of Integrative Plant Biology, 66(1), 36–53. https: / / doi.org / 10.1111 / jipb.13600).Furthermore, through quantitative trait locus (QTL) mapping and genome-wide association analysis (GWAS), more loci associated with wheat resistance to sprouting have been located. For example, Tai et al. (Tai L, Wang, HJ, Xu XJ, Sun WH, Ju L, Liu WT, Li WQ, Sun J, & Chen KM (2021) Pre-harvest sprouting in cereals: genetic and biochemical mechanisms. Journal of Experimental Botany, 72(8),2857–2876. https: / / doi.org / 10.1093 / jxb / erab024; Tai L, Wu J, Jing Y, Liu H, Zeng Q, Xu X, Shi S, Wang H, Liu W, Sun J, Han DJ, Chen KM (2024) A genome-wide association study uncovers that TaPI4K-2A regulates pre-harvestsprouting in wheat. Plant Communications, 5(5), 100739.) (https: / / doi.org / 10.1016 / j.xplc.2023.100739) Through inductive analysis of 188 valid QTLs related to PHS, 66 MetaQTLs were obtained that were distributed across all 21 chromosomes of wheat, of which 44 MetaQTLs did not have a target gene. Subsequently, GWAS analysis of the PHS phenotype of 680 wheat materials revealed 102 PHS-related QTLs, of which 53 were newly discovered.In addition, in rye, Rakoczy-Trojanowska et al. identified 71 SNPs associated with resistance to pre-harvest sprouting in rye (Secale cereale L.) through GWAS analysis of 149 different rye inbred lines, including 5 SNPs associated with α-amylase activity (Rakoczy-Trojanowska M, Krajewski P, Bocianowski J, Schollenberger M, Wakuliński W, Milczarski P, Masojć P, Targońska-Karasek M, Banaszak Z, Banaszak K, Brukwiński W, Orczyk W, Kilian A (2017). Identification of single nucleotide polymorphisms associated with brown rust resistance, α-amylase activity and pre-harvest sprouting in rye (Secale cereale L.). Plant Molecular Biology Reporter, 35(3), 366–378. https: / / doi.org / 10.1007 / s11105-017-1030-6; Myśków et al. found one SNP (7R) related to α-amylase activity and three SNPs (1R, 4R, 7R) related to resistance to ear sprouting (Myśków B, Góralska M, Lenarczyk N, Czyczyło-Mysza I & Stojałowski S (2018) Putative candidate genes responsible for leaf rolling in rye (Secale cereale L.). BMC Genetics, 19(1), 57. https: / / doi.org / 10.1186 / s12863-018-0665-0).

[0005] Breeders have developed excellent cultivars such as Yumai 18, Yumai 7698, and Bainong 3217 through various breeding strategies. However, the PHS resistance of many local varieties (such as Lanhuamai and Huixianhong) has not been explored and utilized. (Chang C, Zhang H, Lu J, Si H, Ma C (2023) Genetic improvement of wheat with pre-harvest sprouting resistance in China. Genes, 14(4), 837. https: / / doi.org / 10.3390 / genes14040837; Zhou Y, Bai S, Li H, Sun G, Zhang D, Ma F, Zhao X NieF, Li J, Chen L, Lv L, Zhu L, Fan, R, Ge Y, Shaheen A, Guo, G, Zhang Z, Ma J, Liang H, Qiu X, Song CP (2021) Introgressing the Aegilops tauschii genome into wheat as a basis for cereal improvement. Nature) Plants, 7(6), 774–786. https: / / doi.org / 10.1038 / s41477-021-00934-w. In addition, the richer PHS resistance genetic resources in wheat's close relatives can also be introduced into wheat, for example, by crossing jointed goatgrass, which is resistant to pre-sprouting, with tetraploid wheat and then doubling the number of crosses to obtain synthetic wheat with PHS resistance (Imtiaz M, Ogbonnaya FC, Oman J & van Ginkel M (2008) Characterization of quantitative trait loci controlling genetic variation for preharvest sprouting in synthetic backcross-derived wheatlines. Genetics, 178(3), 1725–1736. https: / / doi.org / 10.1534 / genetics.107.084939).

[0006] Nanjing Agricultural University has developed a wheat-rye translocation line, T5DL·5DS-2RS (Zhuang L, Liu P, Liu Z, Chen T, Wu N, Sun L, Qi Z (2015) Multiple structural aberrations and physical mapping of rye chromosome 2R introgressed into wheat[J]. Molecular Breeding, 35: 133-144. https: / / doi.org / 10.1007 / s11032-015-0333-2.). Patent CN112273221B has reported that the translocation line T5DL·5DS-2RS exhibits high grain hardness due to the deletion of the 5D fragment and can significantly improve grain hardness in different wheat backgrounds. It also preliminarily determined the translocation breakpoint and disclosed a combination of molecular markers that can identify T5DL·5DS-2RS. In this invention, the translocation breakpoint was further refined, and it was found that T5DL·5DS-2RS possesses high resistance to PHS. After PHS treatment, the germination rate of T5DL·5DS-2RS (3.64%) was significantly lower than that of the parental Huixian Red (51.38%) and the white-skinned wheat cultivars Yannong 19 (YN19, 94.18%) and Zhengmai 9023 (ZM9023, 95.56%). In the backcross progeny of T5DL·5DS-2RS and YN19 / ZM9023, homozygous translocation individuals also exhibited PHS resistance. The germination rate of homozygous translocation individuals was on average 39.5% lower than that of heterozygous translocation individuals and on average 60.5% lower than that of non-translocation individuals. This demonstrates that the translocation line T5DL·5DS-2RS can be used to improve PHS resistance in wheat with different backgrounds. Simultaneously, transcriptome sequencing was performed on the ZM9023 / T5DL·5DS-2RS BC3F3 population and its parents. Analysis showed that the translocation significantly altered gene expression patterns. Differentially expressed genes related to PHS resistance were identified in both wheat and rye chromosomes. These genes were enriched in pathways such as plant hormone signal transduction, starch and sucrose metabolism, etc. Among them, ABA and GA signal transduction-related genes, especially the rye gene ScWN2R01G148500 (PER1), may be key factors regulating PHS resistance. The wheat-rye translocation line T5DL·5DS-2RS integrates the PHS resistance genes of wheat HXH and rye 2RS, significantly improving resistance to pre-sprouting sprouting and also significantly increasing grain hardness, providing new germplasm for breeding high-hardness, pre-sprouting sprouting-resistant wheat, especially white-skinned wheat. Summary of the Invention

[0007] The purpose of this invention is to disclose a breeding method for cultivating wheat with high resistance to pre-sprouting using the wheat-rye small fragment translocation line T5DL·5DS-2RS.

[0008] The objective of this invention can be achieved through the following technical solutions:

[0009] In a first aspect, the present invention claims protection for a breeding method for wheat with high resistance to ear sprouting, the method comprising the following steps:

[0010] (1) Using the wheat-Jingzhou rye small fragment translocation line T5DL·5DS-2RS as the donor parent, hybridization and continuous backcrossing were carried out with the wheat cultivar to be improved to obtain generation populations;

[0011] (2) Genotyping of the generation population obtained in step (1) was performed using molecular marker combination and / or oligonucleotide probe kit #6, and homozygous single plants carrying two T5DL·5DS-2RS translocation chromosomes and heterozygous single plants carrying one T5DL·5DS-2RS translocation chromosome were screened.

[0012] (3) The homozygous single plants obtained in step (2) were subjected to indoor or outdoor germination resistance identification, and highly resistant individuals with low germination rate were selected.

[0013] (4) The highly resistant individuals selected in step (3) are evaluated for agronomic traits, and homozygous translocation lines with excellent comprehensive traits are selected to obtain new wheat varieties with high resistance to ear sprouting.

[0014] The molecular marker combination includes marker Xlfz7772 with primer sequences as shown in SEQ ID NO: 1 and SEQ ID NO: 2, and marker Xlfz7979 with primer sequences as shown in SEQ ID NO: 3 and SEQ ID NO: 4.

[0015] Further, in step (2), the oligonucleotide probe kit #6 includes probe pAs1-1 with the sequence shown in SEQ ID NO: 5, probe pAs1-3 with the sequence shown in SEQ ID NO: 6, probe pAs1-4 with the sequence shown in SEQ ID NO: 7, probe pAs1-6 with the sequence shown in SEQ ID NO: 8, probe AFA-3 with the sequence shown in SEQ ID NO: 9, probe AFA-4 with the sequence shown in SEQ ID NO: 10, probe oligo-6 with the sequence shown in SEQ ID NO: 11, probe pSc119.2-1 with the sequence shown in SEQ ID NO: 12, and probe (GAA) with the sequence shown in SEQ ID NO: 13. 10 ;

[0016] The 5' end of the sequences shown in SEQ ID NO: 5 to SEQ ID NO: 11 is modified with a TAMRA fluorescent group, and the 5' end of the sequences shown in SEQ ID NO: 12 and SEQ ID NO: 13 is modified with a FAM fluorescent group.

[0017] Furthermore, the wheat cultivar mentioned is Yannong19 (YN19) and / or Zhengmai9023 (ZM9023).

[0018] Furthermore, the new wheat variety with high resistance to ear sprouting has a high hardness trait of ≥70 grain hardness.

[0019] Secondly, the present invention claims protection for a molecular marker and probe primer composition for detecting high resistance to ear sprouting in wheat, the composition comprising a molecular marker combination and an oligonucleotide probe sleeve #6; the molecular marker combination and the oligonucleotide probe sleeve #6 are respectively as described above.

[0020] Thirdly, the present invention claims protection for a kit for detecting wheat with high resistance to ear sprouting, the kit comprising the above-mentioned molecular marker and probe primer composition.

[0021] Fourthly, the present invention seeks protection for the application of the aforementioned breeding method in the commercial breeding of wheat with high resistance to ear sprouting.

[0022] Fifthly, this invention seeks protection for the application of the wheat-rye translocation line T5DL·5DS-2RS in commercial breeding of wheat with high resistance to pre-sprouting sprouting.

[0023] Sixthly, the present invention seeks protection for the application of the aforementioned molecular marker and probe primer composition in molecular marker-assisted selection breeding of wheat with high resistance to ear sprouting.

[0024] Seventhly, the present invention seeks protection for the application of the aforementioned kit in molecular marker-assisted selection breeding of wheat with high resistance to ear sprouting.

[0025] In a specific embodiment of the present invention, the development process of a breeding method for highly resistant wheat to ear sprouting includes the following steps:

[0026] (1) The materials include Huixian Red (HXH) from Xinxiang City, Henan Province, Jingzhou Rye (JZHM) from Jingzhou City, Hubei Province, artificially synthesized triticale Jinghui No. 1 (JH1) and wheat Huixian Red-Jingzhou Rye dimer addition line DA2R, and well-known white wheat cultivars YN19 and ZM9023. Using the wheat-rye translocation line T5DL·5DS-2RS (Zhuang L, Liu P, Liu Z, Chen T, Wu N, Sun L, Qi Z (2015) Multiple structural aberrations and physical mapping of rye chromosome 2R introgressed into wheat[J]. Molecular Breeding, 35: 133-144.https: / / doi.org / 10.1007 / s11032-015-0333-2.) created by Nanjing Agricultural University as the donor parent, it was hybridized and backcrossed with YN19 and ZM9023, and continuously planted to obtain BC3F2 and BC3F3 population seeds. T5DL·5DS-2RS was then introduced into different wheat backgrounds.

[0027] (2) This invention uses rye genome probes and oligonucleotide probe kit #6 (as shown in Table 1) published in patent CN110358859B to identify T5DL·5DS-2RS and its hybrid offspring populations using chromosomal fluorescence in situ hybridization (FISH) and genomic fluorescence in situ hybridization (GISH). Figure 1 ). FISH / GISH method reference (Du P, Zhuang L, Wang Y, YuanL, Wang Q, Wang D, Dawadondup T, Shen J, Xu H, Zhao H, Chu C, Qi Z (2017) Development of oligonucleotides and multiplex probes for quick and accurate identification of wheat and Thinopyrum bessarabicum chromosomes. Genome, 60:93–103. https: / / doi.org / 10.1139 / gen-2016-0095).

[0028] Table 1. Probe sequences of oligonucleotide probe kit #6

[0029]

[0030] (3) Based on the T5DL·5DS-2RS chromosome breakpoint location in patent CN112273221B, the 3,752,285 ~ 4,310,326 bp interval on 5DS and the 211,015,368 bp-218,513,466 bp interval on 2RS were extracted, and primers were designed using NCBI Primer-BLAST (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). DNA was extracted using the CTAB method (Springer NM (2010) Isolation of plant DNA for PCR and genotyping using organic extraction and CTAB. Cold Spring Harb Protoc 2010:pdb.prot5515. https: / / doi.org / 10.1101 / pdb.prot5515). The marker combination (Xlfz7772 and Xlfz7979, primer sequences are shown in Table 2) reported in patent CN112273221B was used to identify T5DL·5DS-2RS and its hybrid progeny population.

[0031] Table 2 Primer sequences for the molecular marker combinations Xlfz7772 and Xlfz7979

[0032]

[0033] (4) Referring to the wheat germination resistance test method (NY / T1739-2009) issued by the Ministry of Agriculture and Rural Affairs of the People's Republic of China, the resistance to PHS of HXH, JZHM, DA2R, YN19, ZM9023, T5DL·5DS-2RS and their hybrid offspring populations was tested. Germination rate calculation method: Germination rate = (Number of germinated grains / Total number of grains) × 100%. Relative germination index (RGI) = Germination rate of tested varieties / Germination rate of susceptible control varieties. The resistance identification standard is based on ZM9023 as a susceptible control. According to RGI, the resistance to ear sprouting is divided into 5 levels: high resistance to ear sprouting (HR; RGI < 0.05), resistance to ear sprouting (R; 0.05 ≤ RGI ≤ 0.20), moderate resistance to ear sprouting (MR; 0.21 ≤ RGI ≤ 0.40), susceptible to ear sprouting (S; 0.41 ≤ RGI ≤ 0.60), and highly susceptible to ear sprouting (HS; RGI > 0.60).

[0034] (5) Grain samples after 24 h of PHS treatment were collected. Fourteen homozygous translocation strains and fourteen non-translocation strains were obtained from the ZM9023 / T5DL・5DS-2RS BC3F3 segregating population. Six biological replicates of ZM9023 and T5DL・5DS-2RS were used for transcriptome sequencing on the DNBSEQ-T7 platform of Beijing Novogene Biotechnology Co., Ltd. The sequencing data volume was 10G (Q30≥85%), and the average number of reads was 88,110,283. The CS-2R genome, formed by combining the genome of “Chinese Spring” (https: / / wheat-urgi.versailles.inra.fr / Seq-Repository / Assemblies) with the genome of rye “Weining” 2R chromosome (Li G, Wang L, Yang J, He H, Jin H, Li X, Ren T, Ren Z, Li F, Han X, Zhao X, Dong L, Li Y, Song Z, Yan Z, Zheng N, Shi C, Wang Z, Yang S, Xiong Z, Zhang M, Sun G, Zheng X, GouM, Ji C, Du J, Zheng H, Doležel J, Deng XW, Stein N, Yang Q, Zhang K, Wang D (2021), highlights rye genomic characteristics and agronomically important genes. Nature Genetics, 53(4): 574–584. https: / / doi.org / 10.1038 / s41588-021-00808-z), is a high-quality genome assembly that highlights rye genomic characteristics and agronomically important genes. As an integrated reference genome, the raw sequencing data were quality controlled and tested using FastQC (version 0.12.2). Low-quality sequences were filtered using Trimmomatic (version 0.42). High-quality clean reads were aligned with the CS-2R integrated reference genome using HISAT2 (version 2.1.0) to generate BAM files. The BAM files were sorted using Samtools (version 1.10). The sorted BAM files were assembled using StringTie (version 2.2.3), and gene expression levels were quantified. Read coverage analysis was performed using bedtools (v2.30.0). Subsequently, differentially expressed genes were analyzed using edgeR (version 4.0.2).For the two groups of significantly differentially expressed genes, the intersection was screened out, and then gene GO (Gene Ontology) enrichment analysis and annotation were performed using the online tool Triticeae-GeneTribe (Chen Y, Song W, Xie X, Wang Z, Guan P, Peng H, Jiao Y, Ni Z, Sun Q, Guo W (2020) A collinearity-incorporating homology inference strategy for connecting emerging assemblies in the Triticeae tribe as a pilot practice in the plant pangenomic era. Molecular Plant, 13(12), 1694–1708. https: / / doi.org / 10.1016 / j.molp.2020.09.019). KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analysis was performed using R (version 4.3.3).

[0035] (6) Agronomic traits of T5DL·5DS-2RS and its hybrid offspring (YN19 / T5DL·5DS-2RS and ZM9023 / T5DL·5DS-2RS) were evaluated and screened. The genetic effects of translocation homozygotes were comprehensively evaluated, and their application potential in wheat quality breeding was clarified.

[0036] The beneficial effects of this invention are:

[0037] (1) This invention discloses a breeding method for wheat with high resistance to ear sprouting. The anti-ear sprouting gene of T5DL·5DS-2RS is applied to breeding practice. The wheat bred by this method has significantly improved resistance to ear sprouting, broadened the current genetic basis of wheat resistance to ear sprouting, and provided a new method to solve the problem of poor resistance to ear sprouting in wheat, especially white wheat.

[0038] (2) By using the developed codominant molecular marker combination, individuals carrying the target translocation chromosome can be screened quickly and accurately during the seedling stage, thus reducing the population size for later phenotypic identification.

[0039] (3) The anti-sprouting effect is significant and stable. The germination rate of T5DL·5DS-2RS itself is only 3.64%, which is significantly lower than that of its red-skinned parent HXH (51.38%) and white-skinned wheat YN19 (94.18%) and ZM9023 (95.56%). In the hybrid offspring, the germination rate of homozygous translocation is 39.5% lower than that of heterozygous plants and 60.5% lower than that of non-translocation plants. Among them, the germination rate of the homozygous population of YN19 / T5DL·5DS-2RSBC3F2 is 22.41%, and the germination rate of the homozygous population of ZM9023 / T5DL·5DS-2RSBC3F3 is 16.04%, both of which significantly improved the PHS resistance level.

[0040] (4) Through transcriptome analysis, this invention clarified the potential molecular basis of T5DL·5DS-2RS for resistance to pre-sprouting. The introduction of T5DL·5DS-2RS changed the wheat gene expression pattern, with wheat germination-related genes tending to be downregulated and dormancy-promoting genes tending to be upregulated. Among them, the TraesCS5D02G004900 (KAO1) gene missing in 5DS and the ScWN2R01G148500 (PER1) gene infiltrated by 2RS may be key factors, providing information for the discovery of genes that resist pre-sprouting.

[0041] (5) It can be improved simultaneously with other excellent traits: T5DL·5DS-2RS itself has high hardness characteristics (see CN112273221B). New varieties (lines) bred using the method of this invention can obtain high resistance to ear sprouting while also having high grain hardness. Quality and resistance are improved simultaneously, and ear sprouting resistant hard wheat is cultivated.

[0042] (6) Broad application prospects: The method provided by this invention has strong universality and can be hybridized with any wheat variety that needs to improve its resistance to ear sprouting, especially white wheat. It is particularly suitable for promotion and application in rainy and ear sprouting areas such as the Huang-Huai winter wheat region and the middle and lower reaches of the Yangtze River winter wheat region in my country. It is of great significance for ensuring the safety and high-quality development of wheat production in my country. Attached Figure Description

[0043] Figure 1 FISH / GISH analysis of chromosomes in wheat-rye translocation line T5DL·5DS-2RS.

[0044] a: Blue represents chromosomes stained with DAPI, and green signals represent FAM (6-carboxyfluorescein) modified pSc119.2-1 and (GAA) chromosomes. 10The red signal represents the production of TAMR (6-carboxytetramethylrhodamine) modified oligonucleotides pAs1-1, pAs1-3, pAs1-4, pAs1-6, AFA-3, AFA-4, and Oligo-6. The arrow points to T5DL·5DS-2RS. b: T5DL·5DS-2RS FISH karyotype. c: Blue represents chromosomes stained with DAPI, and green represents FAM-modified pSc119.2-1 and (GAA). 10 The red lines represent the production of rye genome probes, with the red lines indicating the production of TAMR-modified oligonucleotides pAs1-1, pAs1-3, pAs1-4, pAs1-6, AFA-3, AFA-4, and Oligo-6. The arrows point to T5DL·5DS-2RS. d: FISH / GISH karyotype of T5DL·5DS-2RS.

[0045] Figure 2 Molecular marker analysis of T5DL·5DS-2RS involving 5DS and 2RS breakpoints.

[0046] ab: Amplification analysis of the breakpoint locations of chromosomes 2R and 5D in T5DL·5DS-2RS. M: DL 2000; 1: HXH; 2: JZHM; 3: JH1; 4: DA2R; 5: T5DL·5DS-2RS. c: Schematic diagram of the breakpoint locations of chromosomes 2R and 5D in T5DL·5DS-2RS.

[0047] Figure 3 GO enrichment analysis of T5DL·5DS-2RS deletion and introgression regions.

[0048] a: GO enrichment analysis of genes in the 5DS deletion region. b: GO enrichment analysis of genes in the 2RS translocation region.

[0049] Figure 4 Amplification of the molecular marker combination (Xlf7979 + Xlf7772) in the ZM9023 / T5DL·5DS-2RS BC3F3 population

[0050] Lanes: M: DL2000; 1-57: ZM9023 / T5DL·5DS-2RS BC3F3 group.

[0051] Figure 5 PHS performance of HXH, JZHM, DA2R, YN19, ZM9023, T5DL·5DS-2RS and their progeny populations after PHS treatment.

[0052] Among them, a: PHS performance of HXH, JZHM, DA2R, T5DL·5DS-2RS, YN19, and ZM9023 (ear image). b: PHS performance of HXH, JZHM, DA2R, T5DL·5DS-2RS, YN19, and ZM9023 (grain image). c: PHS performance of the YN19 / T5DL·5DS-2RS BC3F2 segregating population (ear image). d: PHS performance of the ZM9023 / T5DL·5DS-2RS BC3F3 segregating population (ear image).

[0053] Figure 6 Comparison of plant height and grain characteristics between individual plants selected from the ZM9023 / T5DL・5DS-2RS backcross population and their parents. 1: T5DL・5DS-2RS, 2: ZM9023, 3-11: Superior individual plants selected from the ZM9023 / T5DL・5DS-2RS backcross population.

[0054] Figure 7 Venn diagrams showing differentially expressed genes after budding treatment of ZM9023 and T5DL・5DS-2RS spikelets, and differentially expressed genes between homozygous translocation and non-translocation plant samples in the ZM9023 / T5DL・5DS-2RS BC3F3 population.

[0055] Figure 8 This is a graph showing the enrichment analysis of differentially expressed genes GO and KEGG after PHS treatment. Gene names are on the left and enrichment pathways are on the right.

[0056] Figure 9 This is a heatmap showing the expression levels of key differentially expressed genes. T5D represents T5DL・5DS-2RS, HOT represents homozygous translocation, and NT represents non-translocation. Detailed Implementation

[0057] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0058] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0059] Example 1

[0060] 1. Precise identification of translocation breakpoints in the wheat-Jingzhou rye translocation line T5DL·5DS-2RS

[0061] This invention uses oligonucleotide probe kit #6 (ONPM#6, as shown in Table 1) to perform FISH identification of T5DL·5DS-2RS and GISH identification using rye genome probes to confirm the chromosome identification results in patent CN112273221B. Figure 1 ).

[0062] Based on the chromosome breakpoint location determined in patent CN112273221B, sequences from the 3,752,285bp-4,310,326bp region on 5DS and the 211,015,368bp-218,513,466bp region on 2RS were selected. Primers were designed using NCBI Primer-BLAST. Sixteen rye-specific markers were developed and localized in the aforementioned 2R region. Finally, using two markers, Xlfz9440 and Xlfz9374 (as shown in Table 2), the breakpoint was precisely located within a region of approximately 57 kb between 217,979,408 bp and 218,036,385 bp. The length of the 2RS chromosome translocation fragment in this translocation line is approximately 218 Mb (Table 3; Figure 2). Annotation results for this region show that it contains a total of 1657 rye genes, with GO enrichment in 45 entries, including the regulation of translation and gene expression by diurnal rhythm, oxidative stress response, hydrogen peroxide decomposition and metabolism, pollen development, etc. (Figure 3).

[0063] Sixteen markers were developed within the aforementioned 5D deletion region. Two new markers, Xlfz8882 and Xlfz8884, further narrowed the breakpoint to approximately 12 kb within the range of 4,055,799 bp to 4,067,739 bp. The 5DS chromosome deletion fragment in the translocation line was approximately 4.05 Mb in length (Table 4; Figure 2). Annotation of this region revealed a total of 52 genes enriched in 21 entries, including C-8 steroid isomerase activity, steroid δ-isomerase activity, seed maturation, and fruit development (Figure 3).

[0064] Table 3 Information on primers for 2R-specific molecular markers in rye

[0065]

[0066] Table 4. Information on wheat 5D-specific primer molecular markers

[0067]

[0068] 2. Transfer of T5DL·5DS-2RS into white wheat

[0069] This invention involves crossing and backcrossing the T5DL·5DS-2RS translocation line with YN19 and ZM9023, respectively. The resulting YN19 / T5DL·5DS-2RS BC3F2 and ZM9023 / T5DL·5DS-2RS BC3F3 populations were used for subsequent PHS resistance experiments and for breeding translocation lines with superior overall traits. The marker combination (Xlfz7772+Xlfz7979) reported in patent number CN112273221B was used to identify the T5DL·5DS-2RS identity in the YN19 / T5DL·5DS-2RS BC3F2 and ZM9023 / T5DL·5DS-2RS BC3F3 populations. Figure 4 ).

[0070] 3. PHS resistance identification

[0071] During field planting in Henan Province in 2023, following continuous heavy rainfall, we observed that homozygous translocation plants in the T5DL·5DS-2RS translocation line and its hybrid progeny exhibited lower PHS resistance than other varieties. Therefore, PHS resistance was assessed in mature ears of HXH, JZHM, YN19, ZM9023, DA2R, T5DL·5DS-2RS, the YN19 / T5DL·5DS-2RS BC3F2 population, and the ZM9023 / T5DL·5DS-2RS BC3F3 population. The results showed that compared to HXH (51.38%), YN19 (94.18%), and ZM9023 (95.56%), the germination rate of DA2R was 0.00%, the germination rate of the T5DL·5DS-2RS translocation line was 3.64%, and the germination rate of Jingzhou rye was 10.7%. Figure 5 Table 5). The T5D·5DS-2RS translocation line showed HR (RGI = 0.04 < 0.05), while the parental HXH showed S and DA2R showed HR. Identification using the ZM9023 / T5DL·5DS-2RS BC3F3 and YN19 / T5DL·5DS-2RS BC3F2 populations showed ( Figure 5(Table 5) The average germination rates of homozygous translocation plants in both populations (22.41% and 16.04%) were significantly lower than those of heterozygous translocation plants (56.86% and 61.02%) and non-translocation plants (83.13% and 76.42%), indicating that the introduction of T5DL·5DS-2RS improved PHS resistance. Since HXH is a red-skinned wheat with certain PHS resistance, the large-sample tested population ZM9023 / T5DL·5DS-2RS BC3F3 was divided into red-grained and white-grained subgroups based on grain color. The comparison revealed that, overall, the germination rate of the red-skinned subgroup (40.11%) was lower than that of the white-skinned subgroup (65.85%). Further statistical analysis revealed that the average germination rates of the homozygous translocation (12.13%), heterozygous translocation (50.99%), and non-translocation plant populations (74.47%) were all lower than those of the corresponding homozygous translocation (23.30%), heterozygous translocation (75.55%), and non-translocation subpopulations (79.98%), respectively. Moreover, the PHS resistance of both the homozygous translocation and non-translocation populations was higher than that of the corresponding non-translocation populations. These results indicate that the PHS resistance of the translocation line T5DL·5DS-2RS originates from its two parents, namely the 2RS segment and the 5DS deletion segment in DA2R, as well as resistance genes such as HXH red skin. The aggregation of resistance genes from the two species significantly improved the PHS resistance of the translocation line T5DL·5DS-2RS, providing new germplasm for breeding wheat with high resistance to ear germination.

[0072] Table 5 Germination rates of spikelets after germination treatment in HXH, JZHM, YN19, ZM9023, DA2R, T5DL·5DS-2RS, YN19 / T5DL·5DS-2RS BC3F2 and ZM9023 / T5DL·5DS-2RS BC3F3 populations.

[0073]

[0074] Note: RGI was calculated using ZM9023 as a sensitive control.

[0075] 4. Analysis of the effects of T5DL·5DS-2RS on agronomic traits

[0076] T5DL·5DS-2RS is induced by ionizing radiation, and its effects on wheat agronomic traits need to be evaluated in different wheat backgrounds. The thousand-grain weight, plant height, tiller number, spike length, spikelet number, grains per spike, grain width, and grain length of HXH, YM158, YN19, ZM9023, T5DL·5DS-2RS, and their segregating populations were investigated (Table 6).

[0077] Analysis showed that in YM158 / T5DL·5DS-2RS F3, the number of tillers in homozygous translocation was significantly less than that in non-translocation plants. Similarly, in YM158 / T5DL·5DS-2RS F5, the number of grains per ear in homozygous translocation was significantly less than that in non-translocation plants. However, these traits did not show significant differences in other populations (Table 8). In most populations, the thousand-grain weight of homozygous translocation plants was significantly lower than that in heterozygous translocation and non-translocation populations. Furthermore, the grain width of homozygous translocation plants was significantly lower than that in heterozygous translocation and non-translocation populations, while the grain length of homozygous translocation plants was longer than that in heterozygous translocation and non-translocation populations (Table 8). This indicates that T5DL·5DS-2RS has a significant effect on increasing grain length and decreasing grain width and thousand-grain weight, but it has no consistent effect on protein content, tiller number, and other traits. Thousand-grain weight and grain width showed a high positive correlation in all populations (r=0.67-0.91, mean 0.83; p<0.0001), and the decrease in thousand-grain weight in homozygous translocation plants was mainly due to the decrease in grain width.

[0078] Because the T5DL·5DS-2RS translocation line has poor agronomic traits, especially a reduced thousand-grain weight, it is necessary to continuously select homozygous translocation lines with superior traits, particularly high thousand-grain weight, from the offspring population. From BC3F3 and BC2F4, this invention selected 41 YN19 / T5DL·5DS-2RS derivative lines and 59 ZM9023 / T5DL·5DS-2RS derivative lines with relatively good traits, among which the highest single-plant thousand-grain weight reached 51.4g (Table 7). This demonstrates that the undesirable traits of T5DL·5DS-2RS can be improved through backcrossing and selection. These superior lines are currently undergoing further selection and yield testing.

[0079] Table 6. Comparison of traits between HXH, YM158, YN19, ZM9023 and T5DL·5DS-2RS (2023, Nanjing)

[0080]

[0081] Table 7. Trait analysis of the preferred homozygous T5DL·5DS-2RS lines

[0082]

[0083]

[0084]

[0085]

[0086] Table 8. Population trait analysis of T5DL·5DS-2RS hybrid progeny

[0087]

[0088] 5. Differential gene expression analysis after PHS treatment

[0089] To investigate the molecular response of T5DL·5DS-2RS to PHS treatment, transcriptome sequencing was performed on wheat grains treated with PHS for 24 h, including 6 biological replicates each of the parental ZM9023 and T5DL·5DS-2RS, as well as 14 homozygous translocated and 14 non-translocated single plants from the ZM9023 / T5DL·5DS-2RS BC3F3 generation. Clean reads were aligned and sorted with the CS-2R reference genome, and quantitative analysis was performed to analyze differentially expressed genes (DEGs) between the two populations of homozygous translocated and non-translocated single plants and between the parents T5DL·5DS-2RS and ZM9023. A total of 2379 co-upregulated genes and 6763 co-downregulated genes were screened (|logFC|>=1, p < 0.05; Figure 7). GO and KEGG enrichment analyses showed that differentially expressed genes (DEGs) were significantly enriched in PHS-related biological processes and pathways, and the relevant genes were annotated (Figure 8). Among the 6,763 downregulated wheat genes, many germination-related genes were suppressed, including the TraesCS5D02G004900 gene (kauronic acid oxidase 1; KAO1) located in the 5DS deletion fragment and involved in gibberellin (GA) synthesis, GA biosynthesis and transformation-related genes (GA3ox, GA2OX1, and GA20OX), abscisic acid (ABA) signal receptors and inhibitors (WRKY24, FER, and PYL), genes that regulate and inhibit ethylene (ETH) biosynthesis (ISS1, ACO, and ACS), brassinolide (BR) biosynthesis and signal receptor-related genes (BRL2, BAK1, CYP, and BZR1), α-amylase involved in starch degradation to provide energy for spikelet budding, aquaporin (PIP2), and peroxidase (PER) (Figure 9). Among the 1258 upregulated genes from rye 2RS, ScWN2R01G148500 had the highest average expression level and was labeled as 1-cysteine ​​peroxidase (PER1), which can scavenge reactive oxygen species (ROS), inhibit ABA degradation, and block GA biosynthesis (Haslekås et al., 2003; Chen H et al., 2020). Other upregulated genes included ABA receptor (PYL5) and regulatory enzymes (RHA2B, CIPK2, and CIPK21), peroxidase (APX2), and its input regulatory protein (SP1) (Figure 9). The 1121 upregulated wheat genes included α-amylase inhibitors (α-AI), ABA signaling pathway-related genes (BRE1, ABI5, and RHA2B), seed maturation protein (SMP), and seipin protein (SEI) (Figure 9).Transcriptome analysis revealed that the molecular mechanism of budding resistance in the T5DL・5DS-2RS translocation line mainly involves the interaction of genes deleted in the 5DS fragment, genes introduced into the 2RS fragment, and genes in the wheat background. This significantly altered the gene expression pattern after PHS induction, namely, the upregulation of wheat dormancy-related genes and the downregulation of germination-related genes. Through the balanced regulation of hormone signals such as ABA and GA, PHS resistance was ultimately enhanced. This result further proves that the high PHS resistance of the T5DL・5DS-2RS translocation line originates from the interaction of genes from multiple sources.

Claims

1. A breeding method for wheat with high resistance to pre-sprouting sprouting, characterized in that, The method includes the following steps: (1) Using the wheat-Jingzhou rye small fragment translocation line T5DL·5DS-2RS as the donor parent, hybridization and continuous backcrossing were carried out with the wheat cultivar to be improved to obtain generation populations; (2) Genotyping of the generation population obtained in step (1) was performed using molecular marker combination and / or oligonucleotide probe kit #6, and homozygous single plants carrying two T5DL·5DS-2RS translocation chromosomes and heterozygous single plants carrying one T5DL·5DS-2RS translocation chromosome were screened. (3) The homozygous single plants obtained in step (2) were subjected to indoor or outdoor germination resistance identification, and highly resistant individuals with low germination rate were selected; (4) The highly resistant individuals selected in step (3) are evaluated for agronomic traits, and homozygous translocation lines with excellent comprehensive traits are selected to obtain new wheat varieties with high resistance to ear sprouting. The molecular marker combination includes marker Xlfz7772 with primer sequences as shown in SEQ ID NO: 1 and SEQ ID NO: 2, and marker Xlfz7979 with primer sequences as shown in SEQ ID NO: 3 and SEQ ID NO:

4.

2. The method according to claim 1, characterized in that, In step (2), the oligonucleotide probe kit #6 includes probe pAs1-1 with the sequence shown in SEQ ID NO: 5, probe pAs1-3 with the sequence shown in SEQ ID NO: 6, probe pAs1-4 with the sequence shown in SEQ ID NO: 7, probe pAs1-6 with the sequence shown in SEQ ID NO: 8, probe AFA-3 with the sequence shown in SEQ ID NO: 9, probe AFA-4 with the sequence shown in SEQ ID NO: 10, probe oligo-6 with the sequence shown in SEQ ID NO: 11, probe pSc119.2-1 with the sequence shown in SEQ ID NO: 12, and probe (GAA) with the sequence shown in SEQ ID NO:

13. 10 ; The 5' end of the sequences shown in SEQ ID NO: 5 to SEQ ID NO: 11 is modified with a TAMRA fluorescent group, and the 5' end of the sequences shown in SEQ ID NO: 12 and SEQ ID NO: 13 is modified with a FAM fluorescent group.

3. The method according to claim 1, characterized in that, The wheat cultivars mentioned are Yannong 19 and / or Zhengmai 9023.

4. The method according to claim 1, characterized in that, The new wheat variety with high resistance to ear sprouting has a high hardness trait of ≥70 grain hardness.

5. A molecular marker and probe primer composition for detecting high resistance to ear sprouting in wheat, characterized in that, The composition comprises a molecular marker assembly and an oligonucleotide probe kit #6; The molecular marker combination includes marker Xlfz7772 with primer sequences as shown in SEQ ID NO: 1 and SEQ ID NO: 2, and marker Xlfz7979 with primer sequences as shown in SEQ ID NO: 3 and SEQ ID NO: 4; The oligonucleotide probe kit #6 comprises probe pAs1-1 with the sequence shown in SEQ ID NO: 5, probe pAs1-3 with the sequence shown in SEQ ID NO: 6, probe pAs1-4 with the sequence shown in SEQ ID NO: 7, probe pAs1-6 with the sequence shown in SEQ ID NO: 8, probe AFA-3 with the sequence shown in SEQ ID NO: 9, probe AFA-4 with the sequence shown in SEQ ID NO: 10, probe oligo-6 with the sequence shown in SEQ ID NO: 11, probe pSc119.2-1 with the sequence shown in SEQ ID NO: 12, and probe (GAA) with the sequence shown in SEQ ID NO:

13. 10 ; The 5' end of the sequences shown in SEQ ID NO: 5 to SEQ ID NO: 11 is modified with a TAMRA fluorescent group, and the 5' end of the sequences shown in SEQ ID NO: 12 and SEQ ID NO: 13 is modified with a FAM fluorescent group.

6. A kit for detecting high resistance to ear sprouting in wheat, characterized in that, This kit contains the molecular marker and probe primer composition as described in claim 5.

7. The application of the breeding method according to any one of claims 1-4 in the commercial breeding of wheat with high resistance to ear sprouting.

8. Application of the wheat-rye translocation line T5DL·5DS-2RS in commercial breeding of wheat with high resistance to pre-ear sprouting.

9. The application of the molecular marker and probe primer composition according to claim 5 in molecular marker-assisted selection breeding of wheat with high resistance to ear sprouting.

10. The application of the kit according to claim 6 in molecular marker-assisted selection breeding of wheat with high resistance to ear sprouting.

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