Use of thinopyrum bessarabicum isochromosome i4JL in maintaining and propagating recessive genic male sterile line in wheat

Abstract

Provided is a use of Thinopyrum bessarabicum isochromosome i4JL in maintaining and propagating a recessive genic male sterile line in wheat, relating to the technical field of crop genetics and breeding and agricultural biology. It has been verified that Thinopyrum bessarabicum isochromosome i4JL has a function of restoring stamen fertility in wheat, thereby establishing a foundation for the utilization thereof in wheat bio-breeding and molecular design breeding. The created common wheat-Thinopyrum bessarabicum isochromosome i4JL monosomic alien addition line exhibits male fertility and self-fruitfulness. Both a wheat nuclear male sterile line and a maintainer line can be simultaneously propagated by means of self-fertilization. The developed seeds show distinct color differences, enabling easy optical sorting, thereby facilitating obtaining a high-purity non-blue-grained male sterile line and a high-purity blue-grained maintainer line. Provided is a novel method for efficiently propagating a high-purity male sterile line and a maintainer line, laying a solid foundation for creating a new generation of blue-marked two-line hybrid wheat production system.

Description

Application of Isochromosome i4JL of Thinopyrum baicalensis in the Maintenance and Propagation of Recessive Genic Male Sterile Lines in Wheat

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application is based on the Chinese patent application with application number 202410905164.2 and application date of July 8, 2024, and claims the priority of the Chinese patent application. The entire content of the Chinese patent application is hereby introduced into this application as a reference. Technical Field

[0003] The present invention belongs to the field of crop genetic breeding and agricultural biotechnology, and specifically relates to a method for applying the isochromosome i4JL of Thinopyrum bessacharum to breeding a recessive nuclear male sterile line of wheat and its maintainer line. Background Art

[0004] Wheat (Triticum spp.) is one of the world's most important food crops, feeding approximately 40% of the world's population and providing 20% ​​of its energy and protein needs. Wheat genetic improvement relies on a rich gene pool, including primary gene pools (GP-1: conspecifics), secondary gene pools (GP-2: closely related species sharing at least one of the wheat A, B, and D genomes), and tertiary gene pools (GP-3: distantly related species sharing none of the wheat A, B, and D genomes).

[0005] Thinopyrum spp. is a perennial genus in the Triticeae family. It comprises 11 species, including diploid Thinopyrum ponticum (2n=2x=14), Thinopyrum bessa (2n=2x=14), polyploid Thinopyrum brittlebone (2n=6x=42 or 2n=4x=28), hexaploid Thinopyrum intermedia (2n=6x=42), and decaploid Thinopyrum ponticum (2n=10x=70). These species possess resistance to a variety of biotic and abiotic stresses and serve as a tertiary gene pool for wheat cultivar improvement. Through distant hybridization and genomic introgression, breeders have generated valuable wheat-Thinopyrum germplasm, including introgression lines, translocation lines, substitution lines, addition lines, and amphidiploid lines, for use in wheat genetic improvement.

[0006] In the prior art, Li Zhensheng et al. used decaploid Elytrigia longissima (genome E e E e E b E b E x E xStStStSt) has developed a series of blue-grained wheats, including the common wheat chromosome 4Ag (4D) substitution line "Blue 58," and common wheat chromosome 4Ag monosomic or disomic addition lines (4Ag was formerly known as 4E). Chinese scholars Zhou Kuanji and others used the 4Ag recombinant chromosome to establish the 4E–ms1 hybrid wheat production system. The 4E–ms1 technology system uses the common wheat ms1g homozygous male sterile mutant as the base germplasm. By adding the long-spiked Thinopsis chromosome 4Ag (or 4E: the long arm of 4E carries the blue grain gene Ba, and the short arm of 4E carries the fertility restorer gene ThpMs1), the common wheat (ms1g ms1g)–long-spiked Thinopsis chromosome 4E monosomic addition line, also known as the light blue grain maintainer line, was created. Chinese researchers Li Zhongan and others utilized the 4thS.4AgL recombinant chromosomes, which carry the restorer gene for ms1b on the short arm of chromosome 4 (4thS) of the einkorn wheat Triticum thaoudar (Triticum boeoticum) and the blue-grain gene Ba on the long arm of chromosome 4AgL of the decaploid Thinopsis elongata. They established the 4thS.4AgL–ms1 hybrid wheat production system. Selfing of the light-blue-grain maintainer line produced 66% white-grained genic male sterile lines, 30% medium (or light) blue-grain maintainer lines, and 4% dark-blue-grained seeds. Li Zhongan and others later improved the Blue Label two-line hybrid wheat system, involving five new exogenous chromosomes: T4AgL-4BL.4BS, T4AgL-4BL.4thS, T4AgL-4thS., T4thS-4AgL., and T4thS-4AgL-4BL. However, all of the aforementioned Blue Label two-line hybrid wheat systems produce a certain proportion of disomic addition lines with dark blue kernels. Failure to thoroughly separate medium / light blue kernels from dark blue kernels will compromise the purity of the male sterile lines and F1 hybrids. Therefore, the development of a new generation of Blue Label two-line hybrid wheat production systems is crucial.

[0007] Th. bessarabicum (also known as Agropyron junceum; genome JJ, 2n = 2x = 14) is a perennial coastal wheatgrass with high salt tolerance. Hybridization between common wheat (Triticum aestivum L.) and Thinopyrum bessarabicum (Thinopyrum bessarabicum) can be achieved artificially, resulting in common wheat-Thinopyrum amphidiploids (Tritipyrum; 2n = 8x = 56). Chinese Spring-Thinopyrum chromosomal addition or substitution lines exhibit high salt tolerance, and some germplasm is highly resistant to wheat stem rust. The Roushan-Thinopyrum chromosome 6J substitution line and T6JS.6DL translocation line exhibit high iron and zinc content in their grains. These derived germplasms will become important resources for wheat genetic improvement. However, to date, there have been no reports on the production of blue-label hybrid wheat using the "J" chromosomes of Thinopyrum bessarabicum. Summary of the Invention

[0008] To address the aforementioned issues in the existing technology, the present invention aims to develop a novel blue-label hybrid wheat technology system utilizing the isochromosome i4JL of Triticum aestivum. The resulting common wheat-Triticum aestivum isochromosome i4JL monosomic addition line carries two copies of the blue-grain gene ThbBa. This results in darker coloration, facilitating optical sorting and significantly avoiding issues such as "not blue when expected" or "blue with a hint of blue" in the expected blue. This facilitates the production of high-purity male sterile offspring and allows for more efficient breeding of male sterile plants.

[0009] To achieve the above object, the present invention adopts the following technical solutions:

[0010] In a first aspect, the present invention provides a use of the isochromosome i4JL of Thinopyrum baicalensis in at least one of the following (1)-(4):

[0011] (1) Restoring the function of wheat stamen fertility;

[0012] (2) Maintaining recessive genic male sterile lines in wheat;

[0013] (3) Breeding of recessive nuclear male sterile lines in wheat;

[0014] (4) Establish a blue label two-line hybrid wheat production system.

[0015] The blue label two-line hybrid wheat production system is a common wheat-Thinlygrass isochromosome i4JL monosomic alien addition line.

[0016] In a second aspect, the present invention provides the use of a common wheat-Thinlyleaf truncatula isochromosome i4JL monosomic alien addition line in breeding wheat nuclear male sterile lines and maintainer lines;

[0017] In the common wheat-Thinlygrass isochromosome i4JL monosomic alien addition line, an inactivating mutation occurred in a gene related to stamen fertility in the common wheat background.

[0018] In the above application, genes related to stamen fertility include but are not limited to the TaMSG47 gene.

[0019] Preferably, all three pairs of TaMSG47 genes in the common wheat background undergo inactivation mutations.

[0020] The common wheat-Thinlygrass isochromosome i4JL monomeric alien addition line is constructed by adding the common wheat variety to the isochromosome i4JL of Thinlygrass.

[0021] In a third aspect, the present invention provides a method for breeding wheat nuclear male sterile lines and maintainer lines using the isochromosome i4JL of Thinopyrum baicalensis, comprising the following steps:

[0022] (1) To construct a monosomic alien addition line on the common wheat–Thinlygrass isochromosome i4JL in which all three TaMSG47 genes had inactivating mutations;

[0023] (2) The common wheat-Thinlyleaf euryale isochromosome i4JL monosomic alien addition line was self-pollinated and optical sorting was performed based on the color difference of the self-pollinated grains.

[0024] In step (1), the method for constructing the common wheat-Thinlyleaf euryale isochromosome i4JL monomeric alien addition line is as follows:

[0025] Wheat plants with an inactivating mutation in the TaMsg47 gene were used as pollen donors to pollinate the artificially emasculated "common wheat-Thinnetgrass isochromosome i4JL monosomic alien addition line"; then, plants heterozygous for TaMsg47 in the A, B, and D genomes were identified from the blue-grain F1 generation; the TaMsg47 heterozygous plants were self-pollinated and then, plants homozygous for Tamsg47 in the A, B, and D genomes were identified from the blue-grain F2 generation.

[0026] In step (2), the criteria for optical sorting are:

[0027] (1) If the kernel color is not blue, it is a euploid common wheat of the nuclear male sterile line, does not carry the isochromosome i4JL of the Thinopyrum baessa, and 2n = 42;

[0028] (2) If the kernel color is blue, it is a maintainer wheat-Triticum aestivum isochromosome i4JL monosomic addition line, carrying one Triticum aestivum isochromosome i4JL, with 2n=43.

[0029] The non-blue color is white, amber or red, etc.

[0030] Beneficial effects of the present invention:

[0031] 1. The present invention experimentally verified that the evolutionary relationship between the isochromosome i4JL of Elysia baessa and the wheat 4A, 4B and 4D genomes is slightly distant. In addition, the special structure of the isochromosome can greatly avoid the recombination and exchange between the isochromosome i4JL of Elysia baessa and some homologous chromosomes of common wheat. It also verified that the isochromosome i4JL of Elysia baessa has the function of restoring the fertility of wheat stamens, and can restore the nuclear male sterility caused by three pairs of recessive homozygous genes (msg47msg47) on the 4A, 4B and 4D chromosomes of common wheat, creating conditions for using it to carry out wheat biological breeding and molecular design breeding.

[0032] 2. The "common wheat-Baisa grass isochromosome i4JL monosomic alien addition line" created by this invention is male-fertile and self-fruitful. Its isochromosome i4JL has a 35% chance of being transmitted through female gametes and only a 1.2% chance of being transmitted through male gametes, significantly reducing the formation of disomic alien addition lines with the common wheat-Baisa grass isochromosome i4JL. Furthermore, self-pollination of this common wheat-Baisa grass isochromosome i4JL monosomic alien addition line can simultaneously produce a wheat nuclear male sterile line and a maintainer line. The resulting kernels exhibit significant color differentiation, allowing different strains to be obtained through optical sorting. This significantly simplifies the breeding process for wheat sterile lines, facilitates the production of high-purity male sterile lines, and enables more efficient breeding of male sterile lines.

[0033] 3. The present invention provides a new method for breeding high-purity male sterile lines more efficiently, laying a solid foundation for creating a new generation of blue-label two-line hybrid wheat production system. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 shows a phylogenetic tree of Msg47 proteins from some Triticeae species. The text labels for each branch of the phylogenetic tree include: chromosome (e.g., "4J"), species name (e.g., "Thinopyrum bessarabicum"), ploidy (e.g., "(2x)"), and sequence source genotype (e.g., "PI 531711").

[0035] FIG2 shows two constructed plant binary expression vectors P1207 and P1209.

[0036] Figure 3 shows the seed color and karyotype analysis of the common wheat-Triticum aestivum isochromosome i4JL monosomic alien addition line (MAi4JL); A) shows the blue and non-blue grains produced by self-pollination of the MAi4JL line; B) shows the chromosome karyotype of the self-pollinated offspring of the MAi4JL line; "-A" and "-B" represent the common wheat euploid (2n=42) and the common wheat-Triticum aestivum isochromosome i4JL monosomic alien addition line (2n=43), respectively.

[0037] Figure 4: Fertility of selfed progenies from a common wheat (msg47msg47)–Thinlygrass isochromosome i4JL monosomic alien addition line. This lineage was created using the P1207 vector. The non-blue grain group shows two F3 plants, P1207-43-4-A22 and P1207-43-4-A23; the blue grain group shows two F3 plants, P1207-43-4-B35 and P1207-43-4-B36. The top image depicts overall plant appearance, particularly ear morphology; the middle image shows anther development, particularly the presence of pollen grains; and the bottom image shows Alexander staining of fertile pollen grains. DETAILED DESCRIPTION

[0038] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present application belongs.

[0039] As previously mentioned, creating wheat-Thinoplasts germplasm is a feasible approach to wheat genetic improvement. Under artificial conditions, Thinoplasts bessagrass can be hybridized with common wheat. However, to date, there have been no reports of using the "J" chromosomes of Thinoplasts bessagrass to create blue-label hybrid wheat production technology.

[0040] In the existing technology, the common wheat (ms1g ms1g)-long-spiked wheat chromosome 4E monosomic alien addition line created by the 4E-ms1 technical system has only one blue grain gene on chromosome 4E, resulting in lighter coloration. In addition, the late-developing grains often show phenomena such as "not blue when they should be blue" or "blue with dots when they should be blue". In addition, the existing blue-label two-line hybrid wheat system will produce a certain proportion of dark blue grain disomic alien addition lines. If the medium / light blue grains and dark blue grains cannot be thoroughly sorted, the sterile lines will be mixed, which will greatly interfere with the production of the sterile lines and reduce the purity of the sterile lines.

[0041] Dr. Qi Zengjun's team from Nanjing Agricultural University conducted a study on the Chinese spring-Triticum aestivum chromosome 4J disomic alien addition line (DA4J). 60 Co irradiation has yielded various 4J chromosome variants, such as the isoarm chromosome 4JL·4JL (abbreviated as I4JL, i4JL.4JL, and I4JL.4JL) of Thinopyrum basilii. These 4J chromosome variants were used to localize the blue aleurone gene ThbBa (earlier named BaThb) to the 4JL-11 region. Furthermore, the wheat MSG47 has been confirmed to be an ortholog of the rice dpw gene and the maize ZmMs25 gene. When three pairs of TaMSG47 genes are knocked out in the A, B, and D genomes of common wheat (simplified genotype denoted as msg47 msg47), common wheat (msg47msg47) exhibits pollen-free male sterility.

[0042] Based on this, the present invention aims to utilize the isochromosome i4JL of Triticum aestivum to create an innovative blue-label hybrid wheat technology system. Experiments have verified that the evolutionary relationship between the isochromosome i4JL of Triticum aestivum and the wheat 4A, 4B, and 4D genomes is slightly distant. This also demonstrates that the isochromosome i4JL of Triticum aestivum has the function of restoring stamen fertility in wheat, potentially restoring the nuclear male sterility caused by three pairs of recessive homozygous genes (msg47msg47) on chromosomes 4A, 4B, and 4D of common wheat.

[0043] The present invention created a common wheat (msg47msg47)-Baisa grass isochromosome i4JL monomeric alien addition line (or abbreviated as MAi4JL) in which all three pairs of TaMSG47 genes have undergone inactivation mutations. Since the Baisa grass isochromosome i4JL carries two symmetrical 4JL arms and the Baisa grass blue grain gene ThbBa is located in 4JL, the Baisa grass isochromosome i4JL carries two copies of the blue grain gene, which will cause the common wheat-Baisa grass isochromosome i The grains produced by the self-pollinated offspring of the 4JL monosomic alien addition line have obvious color differences, which makes the blue endosperm coloring show a dosage effect, greatly avoiding the drawbacks of "not blue when it should be blue" or "blue spots when it should be blue" in the blue-label two-line hybrid wheat system in the existing technology. The darker blue grains in the offspring grains are used to improve the efficiency of optical sorting and simplify the breeding process of wheat sterile lines, which is conducive to obtaining high-purity male sterile line offspring, innovate wheat breeding technology, and lay a solid foundation for creating a new generation of blue-label two-line hybrid wheat production system.

[0044] The following detailed description is for illustrative purposes only and is intended to provide further explanation of the present invention, rather than to limit the scope of the present invention.

[0045] The isomeric chromosome i4JL of the Thinopyrum baicalensis used in the embodiment was obtained by the team of Dr. Qi Zengjun of Nanjing Agricultural University from the Chinese Spring-Thinopyrum baicalensis chromosome 4J disomic addition line (DA4J). 60 The results were obtained by Co irradiation (Physical mapping of chromosome 4J of Thinopyrum bessarabicum using gamma radiation-induced aberrations).

[0046] Example 1: Analysis of the i4JL Carrying Element, Double-Dose Advantage, and Evolutionary Relationship with Wheat Species on the Isochromosome of Thinopyrum baicalensis

[0047] 1. Analysis of the ThbBa gene element regulating anthocyanin synthesis on chromosome i4JL of Elytrigia baessa

[0048] The blue aleurone gene ThpBa of common wheat "Blue 58" comes from the decaploid Thinopyrum ponticum = Agropyron elongatum = Elytrigia elongata (2n = 10x = 70, genome E e E b E xThe long arm of chromosome 4Ag of the wheat variety StSt is located 0.71-0.80° from the centromere. 4Ag is a recombinant chromosome whose centromere and pericentromere regions originate from chromosomes of group E, but whose outer arms originate from chromosomes of group St. It is often referred to as chromosome 4E. ThpMYB1, ThpMYB2, ThpR1, and ThpR2, derived from the decaploid Thinopsis elongata, are the four key genes that determine the blue aleurone layer of wheat. ThpMYB1 and ThpMYB2 encode MYB transcription factors, while ThpR1 and ThpR2 encode bHLH transcription factors. Co-expression of ThpMYB1 and ThpR1 leads to the accumulation of blue anthocyanins in wheat grains. Therefore, ThpBa theoretically represents a combination of genes encoding MYB and bHLH transcription factors. The genome sequence of the hexaploid Thinopyrum intermedium (Thinopyrum intermedium v3.1 DOE-JGI, http: / / phytozome.jgi.doe.gov / ) indicates that ThpMYB1 and ThpMYB2 both point to the same Thinopyrum intermedium gene, but no orthologs of ThpR1 and ThpR2 have been found. Comparison of orthologs with Thinopyrum bessagrass suggests that ThpMYB1 and ThpMYB2 belong to different genomes of the decaploid Thinopyrum elongatum. Similarly, ThpR1 and ThpR2 also belong to different genomes, but the 3' end of the ThpR2 gene is incomplete and likely nonfunctional.

[0049] The long arm of chromosome 4 (4JL) of Thinoplasmaceae (accession PI 531711) carries the dominant blue aleurone gene, ThbBa (earlier named BaThb), located in the 4JL-11 region. Therefore, the isochromosome i4JL of Thinoplasmaceae carries two copies of the blue-grain gene, and its grain blue color also exhibits a dosage effect. In the paper "DNA Sequence Analysis of the Long Arm of Chromosome 4J of Thinoplasmaceae and Fine Mapping of the Blue-Grain Gene," Wang Qing used a Chinese Spring-Thinoplasmaceae chromosome 4JL telodisomic addition line as material, obtained and sequenced the 4JL telomere, assembled the 4JL telosome genome sequence, precisely mapped the key ThbBa region, and cloned its key gene. Based on the location of ThbBa, the collinearity of wheat 4A, 4B, and 4D genes, and the sequences of the ThpMYB1 and ThpR1 genes, the corresponding orthologous genes ThbMYB1 and ThbR1 were identified by searching the 4JL telosome genome sequence database of Thinopyrum bessacharum. Overexpression of ThbMYB1 and ThbR1 resulted in anthocyanin accumulation in wheat coleoptiles, revealing the nature of the ThbBa gene and confirming its location in 4JL. Therefore, Thinopyrum bessacharum carries two copies of the blue-grain gene ThbBa on its isochromosome i4JL.

[0050] The full-length cDNA sequences of ThbMYB1 and ThbR1 of Thinopyrum bessapa are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

[0051] 2. Analysis of Double-dose Advantage of Isochromosome i4JL in Thinopyrum baicalensis

[0052] Because the i4JL chromosome in Triticum aestivum carries two symmetrical arms, the genes located in 4JL are naturally duplicated. The blue grain gene ThbBa is located in 4JL, meaning that i4JL carries two copies of the blue grain gene, resulting in a dosage effect in blue aleurone coloration. Therefore, i4JL could be transferred to other common wheat varieties through backcrossing, leveraging the darker blue grain coloration to improve optical sorting efficiency and innovate wheat breeding techniques.

[0053] 3. Analysis of the evolutionary relationship between chromosome i4JL of Elytrigia baessa and wheat species

[0054] In their study "Barley Male Sterility Gene HvMSG47 and Its Application," Ni Fei et al. found that the TaMsg47 gene in common wheat controls male development, and knocking out this gene can cause pollen-free male sterility in wheat. Based on recent research findings from various researchers, they hypothesized that the ThbMsg47 gene in Thinopyrum bessapa is located on the long arm of chromosome 4 (4JL) of the fourth homeogroup.

[0055] Using barley and wheat Msg47 gene sequences, a BLAST search of the 4JL telosome genomic sequence database for Thinopyrum thbsapa revealed a 6,085-bp spliced ​​sequence (PI 531711|20406194|6085|cvg_22.0_tip_0) containing the Thinopyrum ThbMsg47 gene. Bioinformatics analysis revealed that the genomic coding region for Thinopyrum thbMsg47 is 2,630 bp long (9 exons), encoding a 1,815-bp cDNA. The cDNA sequence is shown in SEQ ID NO: 3.

[0056] The ThbMsg47 gene of Thinopyrum baicalensis encodes a 604aa protein, and the amino acid sequence of the encoded protein is shown in SEQ ID NO:4.

[0057] The isochromosome i4JL of Thinopyrum baicalensis carries two copies of the ThbMsg47 gene, which may have the function of complementing the TaMSG47 gene of common wheat and may play a role in restoring stamen fertility in the isochromosome i4JL of Thinopyrum baicalensis.

[0058] Further database searches and phylogenetic tree analysis using the full-length Msg47 proteins of wheat, barley, Elygrove longissimus, Elygrove bessapa, and Elygrove intermedia (Figure 1) showed that barley was more distantly related to the other species. Among the three genomes of Elygrove intermedia, the genetic relationship between the 4V genome and wheat 4A and the diploid Elygrove longissimus 4E genome was relatively closer, the 4J genome and wheat 4B and 4D genomes were relatively closer, and the 4S genome and Elygrove bessapa 4J genome were relatively closer. Comparing the diploid Elytrigia elongata 4E genome with the Elytrigia intermedia 4J genome, the evolutionary relationship between the Elytrigia bessagras 4J genome and the wheat 4A, 4B, and 4D genomes is slightly distant (Figure 1). In addition, the special structure of the isochromosome i4JL of Elytrigia bessagras greatly avoids the recombination and exchange between the Elytrigia bessagras i4JL and the homologous chromosomes of common wheat, which better ensures the stability and reproducibility of the common wheat-Elytrigia bessagras isochromosome i4JL monomeric alien addition line.

[0059] Example 2: Creation of a Triticum aestivum (msg47msg47)-Triticum truncatum isochromosome i4JL monosomic addition line and functional analysis of Triticum truncatum isochromosome i4JL in restoring fertility

[0060] Common wheat is hexaploid, and some functional genes have three pairs of alleles, a total of six copies. The dominant TaMsg47 gene controls male fertility. Wheat A, B and D genomes each have a pair of TaMSG47 alleles. The three pairs of homozygous dominant allele genotypes can be expressed as TaMsg-A47 TaMsg-A47, TaMsg-B47 TaMsg-B47, TaMsg-D47TaMsg-D47, abbreviated as Msg47Msg47. This genotype determines wheat male fertility; the three pairs of heterozygous allele genotypes can be expressed as TaMsg-A47 Tamsg-A47, TaMsg-B47 Tamsg-B47, TaMsg-D47 Tamsg-D47, abbreviated as Msg47 msg47. This genotype also determines wheat male fertility; the three pairs of homozygous recessive allele genotypes can be expressed as Tamsg-A47 Tamsg-A47, Tamsg-B47 Tamsg-B47, Tamsg-D47 Tamsg-D47, abbreviated as msg47msg47, this genotype determines the pollenless male sterility of wheat.

[0061] If the fertile wheat (Msg47Msg47) is used as the background, the common wheat (Msg47Msg47)-Thinjagrass isochromosome i4JL monomeric alien addition line is self-pollinated, the seeds of different colors produced will theoretically all be normal and fertile. If the sterile wheat (msg47msg47) background is used, the stamen fertility of the common wheat (msg47msg47)-Baisa Thinoploid isochromosome i4JL monosomic alien addition line (MAi4JL) remains to be determined. If the Baisa Thinoploid isochromosome i4JL has the function of restoring fertility, this MAi4JL line will be male fertile and self-fertile, but the fertility of the different colored seeds produced by self-fertilization will be different: 1) Non-blue kernels (white, amber, or red, etc.) are common wheat euploids (msg47msg47; 2n=42), and the established plants are theoretically male sterile; 2) Blue kernels are common wheat (msg47msg47)-Baisa Thinoploid isochromosome i4JL monosomic alien addition line (MAi4JL; 2n=43), and the established plants are theoretically male fertile and self-fertile.

[0062] In the early stage, the isochromosome i4JL monosomic alien addition line of Yangmai No. 6 (Msg47Msg47)-Bai Sassiae and the isochromosome i4JL monosomic alien addition line of Yangmai No. 158 (Msg47Msg47)-Bai Sassiae were created through continuous backcrossing. Starting from the existing Yangmai 158 (Msg47Msg47)-Bai Sassiae isochromosome i4JL monomeric alien addition line, two sets of CRISPR / Cas9 gRNA target sites were designed (Table 1) to target and edit the TaMsg47 gene in the Yangmai 158 (Msg47Msg47)-Bai Sassiae isochromosome i4JL monomeric alien addition line. All six dominant TaMsg47 genes in the common wheat background were knocked out to recessive Tamsg47 genes, while the ThbMsg47 genotype on the Bai Sassiae isochromosome i4JL was retained.

[0063] Table 1: TaMSG47-specific gRNA target sites

[0064] Note: The above sequences are based on the MSG47 gene from wheat 4A, 4B, and 4D and thinopyrum 4J. Two gene-editing vectors, P1207 and P1209, were used, each equipped with dual target sites (with NGG PAM sequences). Bases highlighted in gray may affect the specific recognition of the gRNA target site or disrupt the NGG PAM site.

[0065] Two plant binary expression vectors, P1207 and P1209 (Figure 2), were constructed. Within their T-DNA regions, they contain two sets of gRNA expression cassettes, targeting gRNA target sites 1 and 2, respectively; they also include two constitutive expression cassettes, driving the Cas9 gene and the Bar gene, respectively. P1207 can cleave all TaMsg47 genes, but has a few base differences with the ThbMsg47 target site, which may affect gRNA recognition of the ThbMsg47 target site. P1209 can cleave all TaMsg47 genes in common wheat (only TaMsg-B47 target site 2 has a single base difference), but has a key base difference with the PAM NGG site of the ThbMsg47 gene, which may prevent it from binding to the ThbMsg47 target site. The plant binary expression vectors P1207 and P1209 were introduced into Agrobacterium EHA105, respectively, and then the Yangmai No. 158 (Msg47Msg47)-Bai Sassiae isochromosome i4JL monomeric alien addition line was used as the recipient for Agrobacterium genetic transformation and gene editing of wheat immature embryos. The Yangmai No. 6 (Msg47Msg47)-Bai Sassiae isochromosome i4JL monomeric alien addition line was occasionally used here.

[0066] Wheat Agrobacterium genetic transformation was carried out according to the method described by Ishida et al. in Wheat (Triticum aestivum L.) transformation using immature embryos (2015).

[0067] However, practice has shown that the Yangmai 158 (Msg47Msg47)-Baisaihuai isochromosome i4JL monomeric alien addition line is not suitable for genetic transformation. The Baisaihuai isochromosome i4JL may hinder the formation and redifferentiation of wheat callus, and it is difficult to obtain positive transgenic plants carrying the Baisaihuai isochromosome i4JL, let alone knocking out the three pairs of TaMsg47 genes in the Yangmai 158 (Msg47Msg47)-Baisaihuai isochromosome i4JL monomeric alien addition line.

[0068] To circumvent this technical barrier, the T-DNA regions of P1207 and P1209 were introduced into the common wheat variety "Fielder," which is easily transformable. Transgenic plants with the TaMsg47 gene edited were generated. Studies have shown that the fertility of transgenic Fielder plants is closely related to the TaMSG47 genotype. Complete male sterility occurs when all three TaMsg47 genes in the A, B, and D genomes are knocked out (the simplified genotype msg47msg47). Complete male fertility also occurs when only one TaMsg47 allele in each of the A, B, and D genomes is knocked out (the simplified genotype Msg47msg47). Transgenic Fielder plants with the TaMsg47 gene edited were self-pollinated or backcrossed, and their progeny were identified as heterozygous for all three genomes (Msg47msg47). Transgenic Fielder (Msg47msg47) was then used as a pollen donor to pollinate the artificially emasculated Yangmai 158–Bai Sa Yan Cao isochromosome i4JL monosomic alien addition line. This also occasionally involved the Yangmai 6 (Msg47Msg47)–Bai Sa Yan Cao isochromosome i4JL monosomic alien addition line. Plants heterozygous for TaMsg47 in the A, B, and D genomes (Msg47msg47+i4JL′) were then identified from the blue-grain F1 generation. The blue-grain F1 (Msg47msg47+i4JL′) was self-fertilized, and plants homozygous for Tamsg47 in the A, B, and D genomes (msg47msg47+i4JL′) were then identified from the blue-grain F2 generation. If the isochromosome i4JL of Thinopyrum baessa has the function of restoring fertility, the blue-seeded F2 (msg47msg47+i4JL′) will self-pollinate to produce fruit, thereby harvesting the F3 generation of blue-grained and non-blue-grained (white, amber or red, etc.) seeds, among which the blue-grained F3 seeds are the common wheat (msg47msg47)-isochromosome i4JL monomeric alien addition line (2n=43), and the non-blue-grained F3 seeds are the common wheat (msg47msg47) euploid (2n=42).

[0069] The editing efficiency of the P1207 vector was slightly lower, and gene sequencing was used to determine the mutation status of the three pairs of TaMsg47 alleles in the candidate plants. First, Fielder gene-edited plants with the target genotype (Msg47msg47) were identified and used to pollinate the artificially emasculated Yangmai 158 (Msg47Msg47)–Thinlygrass isochromosome i4JL monosomic alien addition line. Following the above strategy, blue-grained F1 plants (Msg47msg47+i4JL′) and blue-grained F2 plants (msg47msg47+i4JL′) with the target genotype were obtained (Table 2): P1207-43 and P1207-43-4, respectively (Table 3). Experiments have shown that blue-grained F2 plants (msg47msg47+i4JL′) self-fertilize, demonstrating that the isochromosome i4JL in Thinopyrum basilii has the function of restoring fertility, making the common wheat MAi4JL line, which carries the male sterility genetic background (msg47msg47), male-fertile. F3 seeds produced by self-pollination of P1207-43-4 were harvested and sorted by color into non-blue grain groups (also known as white, amber, or red) and blue grain groups, P1207-43-4-A and P1207-43-4-B, respectively (Table 3). Of the 114 F3 grains harvested, 41 were blue, accounting for approximately 36%.

[0070] The P1209 vector has a high editing efficiency, resulting in mutations in individual alleles within the editing generation (i.e., the T0 generation), functionally equivalent to homozygous mutations. Similarly, gene sequencing was used to determine the mutational status of the three pairs of TaMsg47 alleles in candidate plants. For this purpose, three pairs of Fielder gene-edited plants, each harboring at least one Tamsg4 inactivating mutation but still exhibiting male fertility, were selected. These plants were used to pollinate the artificially emasculated Yangmai 158 (Msg47Mg47)–Thinnia baicalensis isochromosome i4JL monosomic alien addition line, yielding F1 blue-grained seeds. Three pairs of male-fertile F1 plants, each harboring at least one Tamsg4 inactivating mutation, were bagged and self-pollinated to obtain F2 blue-grained seeds. In the F2 generation, nine F2 plants were identified through genome sequencing: P1209-16-11, P1209-16-12, P1209-16-61, P1209-16-63, P1209-16-73, P1209-16-76, P1209-16-85, P1209-48-25, and P1209-48-30. Given the high editing efficiency of the P1209 vector and the diversity of editing events in the plants, only the genotypes and sequences of these nine selected F2 plants are presented here (Tables 3, 4-a, and 4-b). The experiment showed that blue-grained F2 plants (msg47msg47+i4JL′) self-fertilized, further demonstrating that the isochromosome i4JL in Thinopyrum bessaquah has the ability to restore fertility, making the common wheat MAi4JL line carrying the male sterility genetic background (msg47msg47) male fertile. F3 seeds produced by self-pollinating nine F2 plants were harvested and sorted into non-blue grain groups (also known as white, amber, or red grain groups) and blue grain groups. Of the 2,274 F3 grains harvested, 832 were blue, accounting for approximately 37%.

[0071] Table 2: New common wheat-Thinlygrass isochromosome i4JL monosomic alien addition lines based on P1207 and their genotypes Note: a Target site 2 did not undergo mutation and is therefore not shown; b Homozygous genotype, both alleles are identical; c i4JL unmutated genotype; d For heterozygous genotypes, only the mutant alleles are shown, not the non-mutated alleles. For the wild-type (WT) group, the gray background indicates polymorphic bases between different genomes. For the non-wild-type (F1, F2, and F3) group, the gray background indicates polymorphic bases within the same genome that differ from the wild-type sequence.

[0072] Table 3: New common wheat-Thinlygrass isochromosome i4JL monosomic alien addition lines and their fertility Note: a Still heterozygous genotype; b Only the alleles that are still in the heterozygous state are shown, and the other two pairs of alleles are equivalent to recessive homozygous; c Although all three pairs of alleles are recessive homozygous, the msg-B47 gene has a 9 or 12 bp deletion. Whether this deletion can cause functional inactivation remains to be verified. d The low self-fertilization rate may be due to the presence of the Cas9 and gRNA expression cassettes, and the low specificity of the P1207 dual target, which may affect the function of the ThbMsg47 gene on the i4JL chromosome; e Self-pollination may be caused by pollination of other fertile plants due to the lack of bagging and isolation; f The self-pollination fruit set rate was not counted, and was determined based on visual inspection of the actual flowering and fruiting conditions; g The average self-pollination fruit set rate is obtained based on approximately 10 blue-grain group plants or 30 non-blue-grain group plants; the genotype, transgene and chromosome status of the F3 line are also comprehensively evaluated based on approximately 10 blue-grain group plants or 30 non-blue-grain group plants.

[0073] Table 4a: New common wheat-Thinlygrass isochromosome i4JL monosomic alien addition lines based on P1209 and their genotypes Note: This table lists the major genotypes detected in the target individuals. In the wild-type (WT) group, polymorphic bases between different genomes are annotated with a gray background; in the non-wild-type (F2) group, polymorphic bases within the same genome but different from the wild-type sequence are annotated with a gray background. In the non-wild-type (F2) group, the wild-type gene sequence is underlined. Different types of single-base insertions within the same gene in the same individual are indicated by degenerate bases (H = A / C / T; W = A / T). *Rare mutations, found in only 1-5% of sequences.

[0074] Table 4b: New common wheat-Thinlygrass isochromosome i4JL monosomic alien addition lines based on P1209 and their genotypes Note: This table lists the major genotypes detected in the target individuals; Unknown indicates that the target sequence was not obtained. In the wild-type (WT) group, polymorphic bases between different genomes are annotated with a gray background; in the non-wild-type (F2) group, polymorphic bases within the same genome but different from the wild-type sequence are annotated with a gray background. In the non-wild-type (F2) group, wild-type gene sequences or Unknown are indicated with an underlined font. Different types of single-base insertions occurring in the same gene in the same individual are indicated with a degenerate base (H = A / C / T; W = A / T). *Rare mutations, found in only 1-5% of sequences.

[0075] Example 3: Seed color and karyotype analysis of common wheat and its corresponding MAi4JL

[0076] 1. Experimental Methods

[0077] The results were based on the study of the Chinese spring-Triticum baicalensis chromosome 4J disomic addition line (DA4J) by Dr. Qi Zengjun's team from Nanjing Agricultural University. 60 Physical mapping of chromosome 4J of Thinopyrum bessarabicum using gamma radiation-induced aberrations (Co irradiation) was used to obtain the isochromosome i4JL of Thinopyrum bessarabicum. The resulting isochromosome i4JL was used to create the genotypes "Yang 6–B," "P1207-43-4-36-B," and "P1209-16-12-B." Seed color phenotypes of common wheat (Yang 6–A), "P1207-43-4-36-A," and "P1209-16-12-A" and their corresponding MAi4JL (Yang 6–B), "P1207-43-4-36-B," and "P1209-16-12-B) were observed, and karyotypes were further determined by FISH analysis of mitotic metaphase chromosomes in root tip cells using oligonucleotide probe sets.

[0078] in:

[0079] “Yang 6–A” is the wild-type Yangmai 6 (genotype Msg47Msg47), and “Yang 6–B” is the Yangmai 6 (Msg47Msg47)-Thinlygrass isochromosome i4JL monosomic alien addition line.

[0080] “P1207-43-4-36-A” is the common wheat “P1207-43-4-36-A” (genotype msg47 msg47), and “P1207-43-4-36-B” is the common wheat (msg47 msg47)-Triticum aestivum isochromosome i4JL monosomic alien addition line.

[0081] “P1209-16-12-A” is the common wheat “P1209-16-12-A” (genotype msg47 msg47), and “P1209-16-12-B” is the common wheat (msg47 msg47)-Thinoxylum bessacharum isochromosome i4JL monosomic alien addition line.

[0082] 2. Experimental Results

[0083] The results of the MAi4JL seed color phenotype observation are shown in Figure 3-A, and the results of the MAi4JL karyotype detection are shown in Figure 3-B.

[0084] The results showed that non-blue-grain plants ("Yang 6-A," "P1207-43-4-36-A," and "P1209-16-12-A") had a chromosome number of 2n = 42 and did not contain any extraneous chromosomes. Blue-grain plants ("Yang 6-B," "P1207-43-4-36-B," and "P1209-16-12-B") had a chromosome number of 2n = 43, with an additional isochromosome i4JL from the Elytrinum bessacharum. Furthermore, the chromosome composition of common wheat in non-blue-grain and blue-grain plants was similar. The additional isochromosome i4JL from the Elytrinum bessacharum in blue-grain wheat only involved the long arm of 4J, and its banding characteristics were significantly different from those of other common wheat chromosomes, laying a solid foundation for subsequent tracking and identification of the MAi4JL-specific germplasm.

[0085] Example 4: Detection of male-female gamete transmission rules using the common wheat-Thinlyleaf truncatula isochromosome i4JL monosomic alien addition line

[0086] Several non-blue-grained common wheat varieties ("Shannong 28", "Luyan 128", "Jimai 44", "Bainong 4199", etc.) were used as pollen donors to pollinate the artificially emasculated Yangmai No. 6 (Msg47Msg47)-Bai Sayancao isochromosome i4JL monosomic alien addition line. The artificially emasculated common wheat "Fielder" was pollinated using the Yangmai No. 6 (Msg47Msg47)-Bai Sayancao isochromosome i4JL monosomic alien addition line as a pollen donor. The proportion of blue grains in the set grains was counted to test the transmission pattern of the Bai Sayancao isochromosome i4JL through male and female gametes.

[0087] The test results showed that when non-blue-grain common wheat varieties were used as pollen donors to pollinate the artificially emasculated Yangmai No. 6-Bai Sa Yan Mao Cao isochromosome i4JL monomeric alien addition line, the BC3F1 generation blue grains ranged from 10% to 64% (mostly concentrated in 22-33%), and the BC4F1 generation blue grains ranged from 1.7% to 46% (mostly concentrated in 21-35%). For the sake of conservatism, the higher value of the general range was taken, and it was speculated that the isochromosome i4JL of Bai Sa Yan Mao Cao had a 35% chance of being transmitted through female gametes.

[0088] Using the Yangmai 6-Bai Sa Thin Wheatgrass isochromosome i4JL monosomic alien addition line as a pollen donor to pollinate the artificially emasculated common wheat "Fielder", only 45 of the 3,716 F1 hybrid kernels obtained were blue, indicating that the isochromosome i4JL of Bai Sa Thin Wheatgrass has approximately a 1.2% chance of being transmitted through male gametes.

[0089] Theoretically, the grains produced by self-pollination of the common wheat-Triticum aestivum isochromosome i4JL monosomic alien addition line should contain approximately 64.2% non-blue grains (white, amber, or red, etc., which are euploid common wheat and do not carry Triticum aestivum isochromosome i4JL; 2n=42), approximately 35.4% blue grains (which are the common wheat-Triticum aestivum isochromosome i4JL monosomic alien addition line; 2n=43), and approximately 0.4% darker blue grains (i.e., the expected common wheat-Triticum aestivum isochromosome i4JL disomic alien addition line; 2n=44). However, in the past two years of continuous self-pollination experiments, no stably inherited common wheat-Triticum aestivum isochromosome i4JL disomic alien addition line has been obtained.

[0090] If the wheat in the common wheat-Triticum aestivum isochromosome i4JL monosomic alien addition line is fertile, then theoretically all seeds of different colors produced by self-pollination of this monosomic alien addition line will be fertile. If the wheat in the common wheat-Triticum aestivum isochromosome i4JL monosomic alien addition line is male sterile, then theoretically all non-blue seeds produced by self-pollination of this monosomic alien addition line will be sterile.

[0091] The results of the comprehensive analysis of the double-dose advantage of the isochromosome i4JL of Elysia baicalensis in Example 1 and the detection of the male and female gamete transmission rules of the i4JL chromosome in the common wheat-Elysia baicalensis isochromosome i4JL monomeric alien addition line show that the isochromosome i4JL chromosome of Elysia baicalensis can be used to construct a blue-label maintainer line and simplify the maintenance and reproduction of sterile lines, providing a new technical means for the production of two-line hybrid wheat.

[0092] Example 5: Analysis of the application prospects of the common wheat (msg47msg47)-Thinlygrass isochromosome i4JL monosomic alien addition line

[0093] Focusing on the new common wheat (msg47msg47)-Triticum baicalensis isochromosome i4JL monomeric alien addition line, its fertility was further studied to determine its application prospects.

[0094] In the P1207 F3 generation, stamen development and seed set were compared between plants with non-blue grains (also known as white, amber, or red grains; P1207-43-4-A, Table 3) and plants with blue grains (P1207-43-4-B, Table 3). During the flowering stage of wheat, mature, undehiscent anthers were soaked in Alexander stain (Solabo #G3050) for 12 hours. Anther and pollen grain morphology was observed using an Olympus microscope (BX53F2). Alexander stain is a multi-color pollen stain that stains viable pollen grains purple-red and underdeveloped or inactive pollen grains green. The results showed that plants with non-blue grains were male sterile, as indicated by hollow anther locules devoid of pollen grains (Figure 4). Plants with blue grains were male fertile or partially fertile, as indicated by anther locules filled with mature pollen grains (Figure 4). In the F3 generation, the self-fertility rate of plants in the non-blue-grain group was 0.1%, while that of plants in the blue-grain group was 30.9%, a highly significant difference. The non-blue-grain group was a euploid common wheat (msg47msg47), theoretically characterized by male sterility and self-fertility. However, 8 of the 8,086 florets tested produced fruit, likely due to false positives caused by pollen crosstalk due to the lack of bagging. The low self-fertility rate in the blue-grain group may be due to the continued presence of the Cas9 and gRNA transgene expression cassettes in the P1207-43-4-B plants, and the lack of specificity for the dual P1207 targets. Perhaps transient ThbMsg47 gene editing interfered with its normal function, thereby inhibiting the fertility restoration function of chromosome i4JL in the thinning wheat grass, leading to the low self-fertility rate in the blue-grain group.

[0095] Stamen development and seed set were compared between plants with non-blue grains (also known as white, amber, or red grains) and blue grains in the F3 generation of P1209. Alexander staining revealed that non-blue grains were male sterile, characterized by hollow anther locules devoid of pollen grains, while blue grains were male fertile or partially fertile, characterized by anther locules filled with mature pollen grains. In the F3 generation, the self-fertility rate of non-blue grains was 0% (with the exception of line P1209-16-61-A, which had a self-fertility rate of approximately 7%), while that of blue grains ranged from 62% to 93%. All eight independent F3 lines had plants with self-fertility rates exceeding 85% (with the exception of line P1209-16-76-B, which had the highest self-fertility rate of 78%), indicating a highly significant difference.

[0096] In the F3 generation, the only difference between the non-blue-grained and blue-grained plants was that the former were euploids of common wheat (msg47msg47) (2n = 42), while the latter were monosomic alien addition lines of common wheat (msg47msg47)–Elysium serratum with isochromosome i4JL (2n = 43; Figure 3, Tables 2 and 3). This indicates that the isochromosome i4JL of Elysium serratum has the ability to restore male fertility in common wheat (msg47msg47), making it suitable for creating a new wheat genic male sterile maintainer line that combines restoration and maintenance functions. Self-pollination of this new genic male sterile maintainer line produces non-blue-grained seeds that are wheat genic male sterile lines, while blue-grained seeds maintain the new wheat genic male sterile maintainer line. Self-pollination of this new wheat (msg47msg47)–Elysium serratum with isochromosome i4JL monosomic alien addition line can be used to propagate wheat genic male sterile lines and their maintainers.

[0097] The above description is merely a preferred embodiment of the present application and is not intended to limit the present application. Various modifications and variations are possible for those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present application shall be included within the scope of protection of the present application.

Claims

1. Use of the isochromosome i4JL of Thinopyrum baicalensis in at least one of the following (1)-(4): (1) Restoring the fertility of wheat stamens; (2) Maintaining recessive genic male sterile lines in wheat; (3) Breeding of recessive nuclear male sterile lines in wheat; (4) Establish a blue label two-line hybrid wheat production system.

2. The use according to claim 1, characterized in that The blue label two-line hybrid wheat production system is a common wheat-Thinlygrass isochromosome i4JL monosomic alien addition line.

3. [Article 26, 10.12.2024] Application of the common wheat–Thinlygrass isochromosome i4JL monosomic alien addition line in the breeding of wheat nuclear male sterile lines and maintainer lines.

4. The use according to claim 3, characterized in that In the common wheat-Thinlygrass isochromosome i4JL monosomic alien addition line, a gene related to stamen fertility in the common wheat background has undergone an inactivating mutation; Preferably, the genes related to stamen fertility are three pairs of TaMSG47 genes.

5. The use according to claim 3, characterized in that The common wheat-Thinlyleaf truncatum isochromosome i4JL monomeric alien addition line is constructed by adding the common wheat variety to the isochromosome i4JL of Thinlyleaf truncatum.

6. A method for breeding wheat nuclear male sterile lines and maintainer lines using the isochromosome i4JL of Thinopyrum baicalensis, characterized in that: The following steps are involved: (1) constructing a common wheat-Triticum aestivum isochromosome i4JL monomeric alien addition line, wherein a gene related to stamen fertility in the common wheat background has an inactivating mutation; (2) The common wheat-Thinlyleaf euryale isochromosome i4JL monosomic alien addition line was self-pollinated and optical sorting was performed based on the color difference of the self-pollinated grains.

7. The method according to claim 6, characterized in that The genes related to stamen fertility are three pairs of TaMSG47 genes.

8. The method according to claim 7, characterized in that In step (1), the method for constructing the common wheat-Thinlyleaf truncatula isochromosome i4JL monosomic alien addition line is as follows: Wheat plants with an inactivating mutation in the TaMsg47 gene were used as pollen donors to pollinate the artificially emasculated "common wheat-Thinnetgrass isochromosome i4JL monosomic alien addition line"; then, plants heterozygous for TaMsg47 in the A, B, and D genomes were identified from the blue-grain F1 generation; these TaMsg47 heterozygous plants were self-pollinated and then, plants homozygous for Tamsg47 in the A, B, and D genomes were identified from the blue-grain F2 generation.

9. The method according to claim 6, characterized in that In step (2), the criteria for optical sorting are: (1) If the kernel color is not blue, it is a euploid common wheat of the nuclear male sterile line, does not carry the isochromosome i4JL of the Thinopyrum baessa, and 2n = 42; (2) If the kernel color is blue, it is a maintainer wheat-Triticum aestivum isochromosome i4JL monosomic addition line, carrying one Triticum aestivum isochromosome i4JL, with 2n=43.

10. The method according to claim 6, characterized in that The non-blue color is white, amber or red, etc.

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