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

[1]       CROSS-REFERENCE TO RELATED APPLICATIONS [2]           This application is proposed based on Chinese Patent Application No. 202410905164.2, filed on July 8, 2024, and claims the priority of the Chinese patent application, the entire contents of which are hereby incorporated herein by reference. TECHNICAL FIELD [3]            The present invention belongs to the technical field of crop genetics and breeding and agricultural biology, and specifically relates to a method for using Thinopyrum bessarabicum isochromosome i4JL in propagating a wheat recessive genic male sterile line and a maintainer line thereof. BACKGROUND [4]           Wheat (Triticum spp.) is one of the world's most important food crops, feeding approximately 40% of the global population and providing 20% of the calories and protein required by humans. The genetic improvement of wheat relies on abundant gene pool resources, including a primary gene pool (GP-1: material from the same species), a secondary gene pool (GP-2: material from closely related genera and species that share at least one of the wheat A, B, or D genomes), and a tertiary gene pool (GP-3: material from distantly related genera and species, i.e., other species that do not possess any of the wheat A, B, or D genomes). [5]           The genus Thinopyrum (Thinopyrum spp.) is a perennial genus in the Triticeae, comprising 11 species,including diploid Thinopyrum elongatum (2n = 2x = 14), Thinopyrum bessarabicum (2n = 2x = 14), polyploid Thinopyrum junceum (2n = 6x = 42 or 2n = 4x = 28), hexaploid Thinopyrum intermedium (2n = 6x = 42), and decaploid Thinopyrum elongatum (Th. ponticum Beauv., 2n = 10x = 70), and possesses characteristics of resistance to various biotic and abiotic stresses, serving as a tertiary gene pool for wheat variety improvement. Through distant hybridization and genomic-level introgression, breeders have created wheat-Thinopyrum introgression lines, translocation lines, substitution lines, addition lines, amphidiploid lines, and other valuable germplasm materials, and used them in wheat genetic improvement. [6]           In the prior art, Zhensheng Li et al. used the decaploid Thinopyrum elongatum (genome EeEeEbEbExExStStStSt) to develop a series of blue-grained wheats, including Triticum aestivum chromosome 4Ag (4D) alien substitution line ''Blue 58'', Triticum aestivum chromosome 4Ag monosomic or disomic alien addition lines, etc. (4Ag was formerly called 4E). Chinese scholars Kuanji Zhou et al. used the 4Ag recombinant chromosome to establish the 4E-ms1 hybrid wheat production system. This system takes the Triticum aestivumms1g homozygous male sterile mutant as the background germplasm, and introducing Thinopyrum elongatum chromosome 4Ag (also known as 4E; the long arm of 4E carries the blue-grained gene Ba, and the short arm of 4E carries the fertility restorer gene ThpMs1), thereby creating a Triticum aestivum (ms1g ms1g)-Thinopyrum elongatum chromosome 4E monosomic alien addition line, i.e., a light-blue-grained maintainer line. Chinese scholars Zhong'an Li et al. utilized the 4thS.4AgL recombinant chromosome, in which the short arm of chromosome 4 (4thS) of Triticum thaoudar (= Triticum boeoticum) carries a restorer gene for ms1b, and the chromosome long arm 4AgL of decaploid Thinopyrum elongatum carries the blue-grained gene Ba, to establish the 4thS.4AgL-ms1 hybrid wheat production system. Self-fertilization of the light-blue-grained maintainer line produces 66% white-grained genic male sterile lines, 30% medium-blue or light-blue-grained maintainer lines, and 4% dark-blue-grained seeds. Zhong'an Li et al. later further improved the blue-marked two-line hybrid wheat system, involving five novel alien chromosomes: T4AgL-4BL.4BS, T4AgL-4BL.4thS, T4AgL-4thS., T4thS-4AgL., and T4thS-4AgL-4BL. However, all the above-mentioned blue-marked two-line hybrid wheat systems produce a certain proportion of dark-blue-grained disomic alien addition lines; if medium / light-blue grains cannot be effectively separated from dark-blue grains, the purity of the male sterile lines and F1 hybrids will be affected. Therefore, it is highly necessary to develop a new generation of blue-marked two-line hybrid wheat production system. [7]          Thinopyrum bessarabicum (also known as Agropyron junceum; genome JJ, 2n = 2x = 14) is a perennial coastal wheatgrass with high salt tolerance. Artificial hybridization between Triticum aestivum L. and Thinopyrum bessarabicumcan be achieved, resulting in the formation of the Triticum aestivum-Thinopyrum bessarabicum amphidiploid (Tritipyrum; 2n = 8x = 56). Chinese Spring-Thinopyrum bessarabicum chromosome alien addition or substitution lines exhibit a high level of salt tolerance, and some germplasms are highly resistant to wheat stem rust; Roushan-Thinopyrum bessarabicum chromosome 6J alien substitution lines and T6JS.6DL translocation lines have relatively high grain iron and zinc contents; these derived germplasms will serve as important resources for wheat genetic improvement. However, to date, no report has been published on the use of the "J" genome chromosomes of Thinopyrum bessarabicum to develop a blue-marked hybrid wheat production technology. SUMMARY [8]            In view of the problems existing in the prior art, the objective of the present invention is to utilize Thinopyrum bessarabicum isochromosome i4JL to develop a blue-marked hybrid wheat technology system. In the Triticum aestivum-Thinopyrum bessarabicum isochromosome i4JL monosomic alien addition line developed in the present invention, the Thinopyrum bessarabicum isochromosome i4JL carries two copies of the blue-grained gene ThbBa, which results in relatively intense blue pigmentation, thereby facilitating optical sorting. This can greatly circumvent the drawbacks such as insufficient blue coloration or mottled blue pigmentation, facilitate obtaining high-purity male sterile line progeny, and enable more efficient propagation of male sterile lines. [9]            In order to achieve the above objective, the present invention adopts the following technical solution:

[10] In a first aspect of the present invention, provided is use of Thinopyrum bessarabicum isochromosome i4JL in at least one of the following (1) to (4):

[11] (1) restoring wheat stamen fertility;

[12] (2) maintaining a wheat recessive genic male sterile line;

[13] (3) propagating a wheat recessive genic male sterile line; and

[14] (4) creating a blue-marked two-line hybrid wheat production system.

[15] The blue-marked two-line hybrid wheat production system is a Triticum aestivum-Thinopyrum bessarabicum isochromosome i4JL monosomic alien addition line.

[16] In a second aspect of the present invention, provided is use of a Triticum aestivum-Thinopyrum bessarabicum isochromosome i4JL monosomic alien addition line in propagating a wheat genic male sterile line and a maintainer line.

[17] In the Triticum aestivum-Thinopyrum bessarabicum isochromosome i4JL monosomic alien addition line, genes related to stamen fertility in a Triticum aestivum background carry loss-of-function mutations.

[18] In the above-mentioned use, the genes related to stamen fertility include, but are not limited to, TaMSG47 gene.

[19] Preferably, all three pairs of theTaMSG47 gene in the Triticum aestivum background carry loss-of-function mutations.

[20] The Triticum aestivum-Thinopyrum bessarabicum isochromosome i4JL monosomic alien addition line is developed by introducing Thinopyrum bessarabicum isochromosome i4JL into a Triticum aestivum variety.

[21] In a third aspect of the present invention, provided is a method for propagating a wheat genic male sterile line and a maintainer line using Thinopyrum bessarabicum isochromosome i4JL, comprising the following steps:

[22] (1) constructing a Triticum aestivum-Thinopyrum bessarabicum isochromosome i4JL monosomic alien addition line, in which all three pairs of theTaMSG47 gene carry loss-of-function mutations;

[23] (2) selfing the Triticum aestivum-Thinopyrum bessarabicum isochromosome i4JL monosomic alien addition line, and performing optical sorting based on the color difference of the grains obtained from selfing.

[24] In step (1), a method for constructing the Triticum aestivum-Thinopyrum bessarabicum isochromosome i4JL monosomic alien addition line includes:

[25] using a wheat plant carrying a loss-of-function mutation in the TaMsg47 gene as a pollen donor to pollinate an artificially emasculated ''Triticum aestivum-Thinopyrum bessarabicum isochromosome i4JL monosomic alien addition line''; then, from a blue-grained F1 generation, identifying plants that are heterozygous for TaMsg47 in the A, B, and D genomes; and selfing the heterozygous plants, and then, from a blue-grained F2 generation, identifying plants that are homozygous for Tamsg47 in the A, B, and D genomes.

[26] In step (2), the optical sorting is performed according to the following criteria:

[27] (1) if a grain has a non-blue color, the grain develops into a Triticum aestivum euploid of the genic male sterile line, which does not carry Thinopyrum bessarabicum isochromosome i4JL, with 2n = 42; and

[28] (2) if a grain has a blue color, the grain develops into a Triticum aestivum-Thinopyrum bessarabicum isochromosome i4JL monosomic alien addition line of the maintainer line, which carries one Thinopyrum bessarabicum isochromosome i4JL, with 2n = 43.

[29] The non-blue color includes, but is not limited to, white, amber, and red.

[30] Beneficial effects of the present invention:

[31] 1. Through experiments, the present invention has demonstrated that the Thinopyrum bessarabicum isochromosome i4JL has a relatively distant evolutionary relationship with the wheat 4A, 4B, and 4Dgenomes, and this unique isochromosome structure can greatly suppress recombination between the Thinopyrum bessarabicum isochromosome i4JL and the homoeologous chromosomes of Triticum aestivum; the present invention has also verified that the Thinopyrum bessarabicum isochromosome i4JL possesses the function of restoring wheat stamen fertility and can restore the genic male sterility caused by three pairs of homozygous recessive genes (msg47 msg47) on the 4A, 4B, and 4D chromosomes of Triticum aestivum, thereby creating conditions for its application in wheat bio-breeding and molecular design breeding.

[32] 2. The Triticum aestivum-Thinopyrum bessarabicum isochromosome i4JL monosomic alien addition line developed in the present invention exhibits male fertility and self-fertility, and the Thinopyrum bessarabicum isochromosome i4JL has a 35% probability of being transmitted via female gametes, and only a 1.2% probability of being transmitted via male gametes, thereby greatly reducing the formation of the Triticum aestivum-Thinopyrum bessarabicum isochromosome i4JL disomic alien addition line. Furthermore, by selfing of the Triticum aestivum-Thinopyrum bessarabicum isochromosome i4JL monosomic alien addition line, both a wheat genic male sterile line and a maintainer line can be simultaneously propagated. The resulting grains exhibit distinct color differences, allowing different lines to be obtained by optical sorting, thereby greatly simplifying the propagation process of the wheat male sterile line, facilitating obtaining a high-purity male sterile line, and enabling more efficient propagation of the male sterile line.

[33] 3. The present invention provides a new method for more efficiently propagating a high-purity male sterile line, laying a solid foundation for the development of a new generation of blue-marked two-line hybrid wheat production system. BRIEF DESCRIPTION OF THE DRAWINGS

[34] FIG. 1 shows phylogenetic analysis of Msg47 proteins from some Triticeae species, where text labels for each branch of a 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'').

[35] FIG. 2 shows two constructed plant binary expression vectors P1207 and P1209.

[36] FIG. 3 shows seed color and karyotype analysis of the Triticum aestivum-Thinopyrum bessarabicum isochromosome i4JL monosomic alien addition line (MAi4JL), where A) shows blue and non-blue grains produced by selfing of the MAi4JL line; B) shows chromosome karyotypes of the selfed progenies of the MAi4JL line; ''-A'' and ''-B'' represent the Triticum aestivum euploid (2n = 42) and the Triticum aestivum-Thinopyrum bessarabicum isochromosome i4JL monosomic alien addition line (2n = 43), respectively.

[37] FIG. 4 shows fertility performance of selfed progenies of the Triticum aestivum (msg47 msg47)-Thinopyrum bessarabicum isochromosome i4JL monosomic alien addition line, where materials were developed using the vector P1207; a non-blue-grained group shows two F3 plants P1207-43-4-A22 and P1207-43-4-A23; a blue-grained group shows two F3 plants P1207-43-4-B35 and P1207-43-4-B36. Top-row images reflect the overall performance of plants, particularly the morphological characteristics of wheat spikes; middle-row images show the status of anther development, particularly the presence or absence of pollen grains; and bottom-row images show results of Alexander staining for fertile pollen grains. DESCRIPTION OF THE EMBODIMENTS

[38] It needs to be noted that the following detailed descriptions are all exemplary and are intended to provide further description of the present application. Unless specified otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present application pertains.

[39] As previously stated, the creation of wheat-Thinopyrum germplasm materials is a feasible approach for the genetic improvement of wheat, and under artificial conditions, Thinopyrum bessarabicum can be hybridized with Triticum aestivum. However, to date, there have been no reports on the use of Thinopyrum bessarabicum"J" genome chromosomes to develop a blue-marked hybrid wheat production technology.

[40] In the prior art, in the Triticum aestivum (mslg mslg)- Thinopyrum elongatum chromosome 4E monosomic alien addition line developed via the 4E-ms1 technology system, chromosome 4E carries only one copy of the blue-grained gene, resulting in light coloration. Moreover, grains with delayed development frequently exhibit phenomena such as insufficient blue coloration or mottled blue pigmentation. Furthermore, all existing blue-marked two-line hybrid wheat systems produce a certain proportion of dark-blue-grained disomic alien addition lines. If medium / light blue grains cannot be completely separated from dark blue grains, the male sterile line will become contaminated, which severely disrupts its production and ultimately reduces its purity.

[41] The team led by Dr. Zengjun Qi at Nanjing Agricultural University subjected the Chinese Spring-Thinopyrum bessarabicum chromosome 4J disomic alien addition line (DA4J) to 60Co irradiation and obtained various 4J chromosome variants, such as Thinopyrum bessarabicum isochromosome i4JL (isoarm chromosome 4JL4JL, also abbreviated as I4JL, i4JL.4JL, and I4JL.4JL), and used the 4J chromosome variants to map the blue aleurone layer gene ThbBa (formerly designated BaThb) to the 4JL-11 region. Moreover, it has been confirmed that wheat gene MSG47 is orthologous to rice gene dpw and maize gene ZmMs25. When the three pairs of TaMSG47 genes in the A, B, and D genomes of Triticum aestivum are knocked out (the genotype is simplified as msg47 msg47), the Triticum aestivum (msg47 msg47) exhibits pollen-free male sterility.

[42] In view of this, the present invention aims to utilize Thinopyrum bessarabicum isochromosome i4JL to innovate a blue-marked hybrid wheat technology system. Through experiments, the present invention has verified that the  Thinopyrum bessarabicum isochromosome i4JL has relatively distant phylogenetic relationships with the 4A, 4B, and 4D genomes of wheat, and has demonstrated that the Thinopyrum bessarabicum isochromosome i4JL possesses the function of restoring wheat stamen fertility and can restore the genic male sterility caused by three pairs of homozygous recessive genes (msg47 msg47) on the 4A, 4B, and 4D chromosomes of Triticum aestivum.

[43] In the Triticum aestivum (msg47 msg47)-Thinopyrum bessarabicum isochromosome i4JL monosomic alien addition line (or abbreviated as MAi4JL) in which all three pairs of TaMSG47 genes have undergone loss-of-function mutations, since the Thinopyrum bessarabicum isochromosome i4JL carries two symmetric 4JL arms and the Thinopyrum bessarabicum blue-grained gene ThbBa is located on 4JL, the Thinopyrum bessarabicum isochromosome i4JL carries two copies of the blue-grained gene. This leads to a distinct color difference in grains set by selfed progenies of the Triticum aestivum-Thinopyrum bessarabicum isochromosome i4JL monosomic alien addition line, so that the blue aleurone layer coloration exhibits a dosage effect, thereby greatly avoiding the drawbacks, such as insufficient blue coloration or mottled blue pigmentation, existing in the blue-marked two-line hybrid wheat systems in the prior art. By virtue of the characteristic of relatively deep blue coloration of the progeny grains, the optical sorting efficiency is enhanced, the propagation process of the wheat male sterile line is simplified, facilitating the acquisition of high-purity male sterile line progenies. The present invention thereby innovates wheat breeding technology and lays a solid foundation for the creation of a new generation of blue-marked two-line hybrid wheat production system.

[44] The specific embodiments of the present invention are described in further detail below in conjunction with the examples. The following detailed descriptions are all exemplary and are intended to provide further description of the present application, rather than to limit the scope of the present invention.

[45] The Thinopyrum bessarabicum isochromosome i4JL used in the examples was obtained by referring to the method for 60Co irradiation of a Chinese Spring-Thinopyrum bessarabicum chromosome 4J disomic alien addition line (DA4J) by the team led by Dr. Zengjun Qi at Nanjing Agricultural University (Physical mapping of chromosome 4J of Thinopyrum bessarabicum using gamma radiation-induced aberrations).

[46] Example 1: Analysis of Elements Carried by Thinopyrum bessarabicum Isochromosome i4JL, Double- Dose Advantage, and Evolutionary Relationships with Triticeae Species

[47] 1. Analysis of ThbBa Gene Element Regulating Anthocyanin Synthesis Carried by Thinopyrum bessarabicum Isochromosome i4JL

[48] The blue aleurone layer gene ThpBa of Triticum aestivum ''Blue 58'' is derived from the long arm of chromosome 4Ag of decaploid Thinopyrum elongatum (Thinopyrum ponticum = Agropyron elongatum = Elytrigia elongata, 2n = 10x = 70, genome EeEbExStSt), located in the 0.71-0.80 region relative to the centromere. Chromosome4Ag is a recombinant chromosome; its centromeric and pericentromeric regions are derived from E genome chromosomes, whereas the distal ends of both arms thereof are derived from St genome chromosomes; it is often abbreviated as chromosome 4E. ThpMYB1, ThpMYB2, ThpR1, and ThpR2 derived from decaploid Thinopyrum elongatum are four key genes that determine the blue aleurone layer in wheat, wherein ThpMYB1 and ThpMYB2 encode MYB transcription factors, and ThpR1 and ThpR2 encode bHLH transcription factors. Co-expression of ThpMYB1 and ThpR1 induces blue anthocyanin accumulation in wheat grains. Therefore, ThpBa theoretically represents a combination of genes encoding MYB and bHLH transcription factors. According to the genome sequence of hexaploid Thinopyrum intermedium (Thinopyrum intermedium v3.1 DOE-JGI, http: / / phytozome.jgi.doe.gov / ), both ThpMYB1 and ThpMYB2 map to the same Thinopyrum intermedium gene, whereas no orthologous genes of ThpR1 and ThpR2 were identified. Comparison with the orthologous genes of Thinopyrum bessarabicum suggests that ThpMYB1 and ThpMYB2are derived from different genomes of decaploid Thinopyrum elongatum; similarly, ThpR1 and ThpR2 also belong to different genomes. However, the 3' end of the ThpR2 gene is incomplete and is likely non-functional.

[49] The long arm of chromosome 4 (4JL) of Thinopyrum bessarabicum (germplasm PI 531711) carries the dominant blue aleurone layer gene ThbBa (formerly designated BaThb), which is located in the 4JL-11 region. The Thinopyrum bessarabicum isochromosome i4JL therefore carries two copies of the blue-grained gene ThbBa, and the blue coloration of its grains also exhibits a dosage effect. Qing Wang, in ''Genomic Sequence Analysis of The Long Arm of Chromosome 4J and Fine Mapping of The Blue-Grained Gene from Thinopyrum Bessarabicum,'' used the Chinese Spring-Thinopyrum bessarabicum chromosome 4JL ditelosomic alien addition line as material to perform flow cytometric sorting to obtain the 4JL telosome, subjected it to sequencing, assembled the genome sequence of the 4JL telosome, precisely mapped the critical region of ThbBa, and cloned its key gene. Integrating the mapping information of ThbBa, the collinearity of wheat 4A, 4B, and 4D genes, and the sequences of the ThpMYB1 and ThpR1 genes, a search against the Thinopyrum bessarabicum 4JL telosome genome sequence database identified the corresponding orthologous genes ThbMYB1 and ThbR1. Co-overexpression of ThbMYB1 and ThbR1 can induce anthocyanin accumulation in wheat coleoptiles, thereby revealing the nature of the ThbBa gene and confirming that the ThbBa gene is located on 4JL. Therefore, the Thinopyrum bessarabicum isochromosome i4JL carries two copies of the blue-grained gene ThbBa.

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

[51] 2. Analysis of Double-Dosage Advantage of Thinopyrum bessarabicum Isochromosome i4JL

[52] Since the Thinopyrum bessarabicum isochromosome i4JL carries two symmetric 4JL arms, the genes located on 4JL are naturally duplicated. The Thinopyrum bessarabicum blue-grained gene ThbBa is located on 4JL. That is, the Thinopyrum bessarabicum isochromosome i4JL carries two copies of the blue-grained gene, and the coloration of the blue aleurone layer consequently exhibits a dosage effect. Therefore, the Thinopyrum bessarabicum isochromosome i4JL can be transferred into other Triticum aestivum varieties through backcrossing, and by means of the characteristic of relatively deep blue-grained coloration, the optical sorting efficiency is enhanced, and wheat breeding technology is innovated.

[53] 3. Analysis of Evolutionary Relationships between Thinopyrum bessarabicum Isochromosome i4JL and Triticeae Species

[54] In the study by Fei Ni et al. titled"Barley Male Sterility Gene HvMSG47 and Use Thereof", it was found that the Triticum aestivum TaMsg47 gene controls male development, and knockout of this gene leads to pollen-free male sterility in wheat. Based on recent research findings, it is inferred that the Thinopyrum bessarabicumThbMsg47 gene is located on the long arm of the homoeologous group 4 chromosome (4JL).

[55] Using the barley and wheat Msg47 gene sequences, a BLAST search was performed against the Thinopyrum bessarabicum 4JL telosome genome sequence database. One assembled sequence of 6,085 bp assembled sequence (PI 531711|20406194|6085|cvg_22.0_tip_0) was identified to contain the Thinopyrum bessarabicum ThbMsg47 gene. Bioinformatics analysis revealed that the Thinopyrum bessarabicum ThbMsg47 coding region has a full genomic length of 2,630 bp (totaling 9 exons), the full length of the cDNA is 1,815 bp, and the cDNA sequence thereof is shown in SEQ ID NO: 3.

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

[57] The Thinopyrum bessarabicum isochromosome i4JL carries two copies of the ThbMsg47 gene, which may function to complement the Triticum aestivum TaMSG47 gene and may play a role in restoring stamen fertility by the Thinopyrum bessarabicum isochromosome i4JL.

[58] Further database searches were carried out, and phylogenetic analysis was performed using the full-length Msg47 proteins from wheat, barley, Thinopyrum elongatum, Thinopyrum bessarabicum, and Thinopyrum intermedium (FIG. 1). The results revealed that barley has a relatively distant genetic relationship with the other species; among the three genomes of Thinopyrum intermedium, the 4V genome has a relatively closer genetic relationship with the 4A genome of wheat and the 4E genome of diploid Thinopyrum elongatum, the 4J genome is more closely related to the 4B and 4D genomes of wheat, and the 4S genome is more closely related to the 4J genome of Thinopyrum bessarabicum. Compared with the 4E genome of diploid Thinopyrum elongatum and the 4J genome of Thinopyrum intermedium, the 4J genome of Thinopyrum bessarabicum exhibits a slightly distant evolutionary relationship with the 4A, 4B, and 4D genomes of wheat (FIG. 1). Combined with the unique structure of the Thinopyrum bessarabicum isochromosome i4JL, greatly avoids recombination and exchange between the Thinopyrum bessarabicum i4JL and the homoeologous chromosomes of Triticum aestivum, thereby better ensuring the stability and reproducibility of the Triticum aestivum-Thinopyrum bessarabicum isochromosome i4JL monosomic alien addition line.

[59] Example 2: Development of "Triticum aestivum (msg47 msg47)-Thinopyrum bessarabicum Isochromosome i4JL Monosomic Alien Addition Line" and Functional Analysis of Fertility Restoration of Thinopyrum bessarabicum Isochromosome i4JL

[60] Triticum aestivum is a hexaploid, and some functional genes possess three pairs of alleles, i.e., six copies in total. The dominant TaMsg47 gene controls male fertility. Each of the A, B, and D genomes of wheat includes one pair of TaMSG47 alleles. The genotype with three pairs of homozygous dominant alleles can be represented as TaMsg-A47 TaMsg-A47, TaMsg-B47 TaMsg-B47, and TaMsg-D47 TaMsg-D47, abbreviated as Msg47 Msg47, and this genotype confers male fertility in wheat. The genotype with three pairs of heterozygous alleles can be represented as TaMsg-A47 Tamsg-A47, TaMsg-B47 Tamsg-B47, and TaMsg-D47 Tamsg-D47, abbreviated as Msg47 msg47, and this genotype also confers male fertility in wheat. The genotype with three pairs of homozygous recessive alleles can be represented as Tamsg-A47 Tamsg-A47, Tamsg-B47 Tamsg-B47, and Tamsg-D47 Tamsg-D47, abbreviated as msg47 msg47, and this genotype confers pollen-free male sterility in wheat.

[61] When a fertile wheat (Msg47 Msg47) background is used, self-fertilization of the Triticum aestivum (Msg47 Msg47)-Thinopyrum bessarabicum isochromosome i4JL monosomic alien addition line results in seed setting, and seeds of different colors produced are all theoretically normally fertile. when a sterile wheat (msg47 msg47) background is used, the stamen fertility of the Triticum aestivum (msg47 msg47)-Thinopyrum bessarabicum isochromosome i4JL monosomic alien addition line (MAi4JL) remains to be determined. when the Thinopyrum bessarabicum isochromosome i4JL possesses the function of restoring fertility, this MAi4JL line exhibits male fertility and self-fruitfulness; however, the seeds of different colors produced from self-fertilization differ in fertility: 1) non-blue grains (white, amber, red, etc.) are Triticum aestivum euploids (msg47 msg47; 2n = 42), and the plants established are theoretically male sterile; 2) blue grains are Triticum aestivum (msg47 msg47)-Thinopyrum bessarabicum isochromosome i4JL monosomic alien addition lines (MAi4JL; 2n = 43), and the plants established are theoretically male fertile and self-fertile.

[62] Previously, the Yangmai 6 (Msg47 Msg47)-Thinopyrum bessarabicum isochromosome i4JL monosomic alien addition line and the Yangmai 158 (Msg47 Msg47)-Thinopyrum bessarabicum isochromosome i4JL monosomic alien addition line were developed through successive backcrossing. Starting from the existing Yangmai 158 (Msg47 Msg47)-Thinopyrum bessarabicum isochromosome i4JL monosomic alien addition line, two sets of CRISPR / Cas9 gRNA target sites (Table 1) were designed to perform targeted editing on the TaMsg47 gene in the Yangmai 158 (Msg47 Msg47)-Thinopyrum bessarabicum isochromosome i4JL monosomic alien addition line. All six dominant TaMsg47 genes in the Triticum aestivum background were knocked out to produce recessive Tamsg47genes, while the ThbMsg47 genotype carried by the Thinopyrum bessarabicum isochromosome i4JL was retained.

[63] Table 1: TaMSG47-Specific gRNA Target Sites Target site 1 (PAM|) Target site 2 (|PAM) P1207 CCtITGCCGGTGCACGGCAAGAGC GGAGCTGTTCAAGCGCCTGCIaGG 4A CCtITGCCGGTGCACGGCAAGAGC GGAGCTGTTCAAGCGCCTGCIaGG 4B CCtITGCCGGTGCACGGCAAGAGC GGAGCTGTTCAAGCGCCTGCIaGG 4D CCtITGCCGGTGCACGGCAAGAGC GGAGCTGTTCAAGCGCCTGCIaGG 4J cctitgccggtgcacggcaagGgT GGAGCTGTTCAAGCGCTTGCIaGG P1209 CCcigagctggccgatgagatcgc GACGATGGCGAAGCACGGGCIgGG 4A CCcigagctggccgatgagatcgc GACGATGGCGAAGCACGGGCIgGG 4B CCcigagctggccgatgagatcgc GACGATGGCGAAGCACGGcCIaGG 4D CCcigagctggccgatgagatcgc GACGATGGCGAAGCACGGGCIgGG 4J TCc|GAGCTGGCCGATGAGATCGC GACGATGGCGAAGCACGGGCIgGA

[64] Note: The above sequences are based on the MSG47 genes from Triticum aestivum chromosomes 4A, 4B, and 4D and Thinopyrum bessarabicum chromosome 4J. Two gene editing vectors P1207 and P1209 are involved, each carrying dual target sites (with NGG PAM sequences). Bases highlighted with gray shading may affect the specific recognition of the gRNA target sites or disrupt the NGG PAM sites.

[65] Two plant binary expression vectors P1207 and P1209 were constructed (FIG. 2). Within their T-DNA regions, there are two sets of gRNA expression cassettes targeting gRNA target site 1 and target site 2, respectively, and two sets of constitutive expression cassettes respectively driving the Cas9 gene and the Bar gene. P1207 can cleave all TaMsg47 genes, but there are a few base differences at the ThbMsg47 target site, which may affect the recognition of the ThbMsg47 target site by gRNA. P1209 can cleave all TaMsg47 genes of Triticum aestivum (with only a single base difference at target site 2 of TaMsg-B47), but it has a key base difference at the PAM NGG site of the ThbMsg47 gene and may not bind to the ThbMsg47 target site. The plant binary expression vectors P1207 and P1209 were separately introduced into Agrobacterium tumefaciens strain EHA105. Subsequently, Agrobacterium-mediated genetic transformation and gene editing of wheat immature embryos were carried out, using the Yangmai 158 (Msg47 Msg47)-Thinopyrum bessarabicum isochromosome i4JL monosomic alien addition line as a recipient. Occasionally, the Yangmai 6 (Msg47 Msg47)-Thinopyrum bessarabicum isochromosome i4JL monosomic alien addition line was also used.

[66] Referring to the method described by Ishida et al. in "Wheat (Triticum aestivum L.) transformation using immature embryos" (2015), Agrobacterium-mediated genetic transformation of wheat was carried out.

[67] However, practical results demonstrated that the Yangmai 158 (Msg47 Msg47)-Thinopyrum bessarabicum isochromosome i4JL monosomic alien addition line is not suitable for genetic transformation. The Thinopyrum bessarabicum isochromosome i4JL may hinder wheat callus formation and redifferentiation, making it difficult to obtain positive transgenic plants carrying the Thinopyrum bessarabicum isochromosome i4JL, let alone achieve knockout of the three pairs of TaMsg47 genes in the Yangmai 158 (Msg47 Msg47)-Thinopyrum bessarabicum isochromosome i4JL monosomic alien addition line.

[68] To circumvent this technical barrier, the T-DNA regions of P1207 and P1209 were separately introduced into the Triticum aestivum variety ''Fielder'', which is amenable to genetic transformation, and transgenic plants with edited TaMsg47 genes were obtained. Studies have shown that the fertility of transgenic Fielder plants is closely correlated with the TaMSG47 genotype. If all three pairs of TaMsg47 genes in the A, B, and D genomes are knocked out (simplified genotype msg47 msg47), the plants exhibit complete male sterility. If one TaMsg47 allele is knocked out in each of the A, B, and D genomes (simplified genotype Msg47 msg47), the plants also exhibit complete male fertility. For the transgenic Fielder plants with edited TaMsg47 genes, after selfing or backcrossing, a genotype (Msg47 msg47) heterozygous for the A, B, and D genomes was identified from their progenies. Then, the transgenic Fielder (Msg47 msg47) was used as a pollen donor to pollinate an artificially emasculated Yangmai 158-Thinopyrum bessarabicum isochromosome i4JL monosomic alien addition line; occasionally, the Yangmai 6 (Msg47 Msg47)-Thinopyrum bessarabicum isochromosome i4JL monosomic alien addition line was also used. Subsequently, from the blue-grained F1 generation, plants heterozygous for TaMsg47 in the A, B, and D genomes (Msg47 msg47 + i4JL') were identified. The blue-grained Fi (Msg47 msg47 + i4JL') self-fertilized and set seeds, and then, from the blue-grained F2 generation, plants homozygous for Tamsg47 in the A, B, and D genomes (msg47 msg47 + i4JL') were identified. When the Thinopyrum bessarabicum isochromosome i4JL possesses the function of restoring fertility, the blue-grained F2 (msg47 msg47 + i4JL') self-fertilized and sets seeds, thereby harvesting F3-generation blue-grained and non-blue-grained (white, amber, red, etc.) seeds, wherein the blue-grained F3 seeds were the Triticum aestivum (msg47 msg47)-Thinopyrum bessarabicum isochromosome i4JL monosomic alien addition line (2n = 43), and the non-blue-grained F3 seeds were Triticum aestivum (msg47 msg47) euploids (2n = 42).

[69] The editing efficiency of the Pi207 vector was slightly low, and the mutation status of the three pairs of TaMsg47 alleles in the candidate plants was determined by gene sequencing. First, Fielder gene-edited plants with the target genotype (Msg47 msg47) were identified and used to pollinate the artificially emasculated Yangmai i58 (Msg47 Msg47)-Thinopyrum bessarabicum isochromosome i4JL monosomic alien addition line. Following the strategy described above, blue-grained Fi plants (Msg47 msg47 + i4JL') and blue-grained F2 plants (msg47 msg47 + i4JL') with the target genotype were obtained (Table 2), designated as Pi207-43 and Pi207-43-4, respectively (Table 3). Experimental results demonstrated that the blue-grained F2 plants (msg47 msg47 + i4JL') self-fertilized and set seeds, indicating that the Thinopyrum bessarabicum isochromosome i4JL possessed the function of restoring fertility, thus rendering the Triticum aestivum MAi4JL line carrying a male sterility genetic background (msg47 msg47) male fertile. F3 seeds produced from the self-fertilization of Pi207-43-4 were harvested and sorted by color into a non-blue-grained group (also referred to as white-grained group, amber-grained group, or red-grained group) and a blue-grained group, designated as Pi207-43-4-A and Pi207-43-4-B, respectively (Table 3). Among the ii4 F3 grains harvested, 4i were blue grains, accounting for approximately 36%.

[70] The editing efficiency of the Pi209 vector was very high, and in the editing generation (i.e., T0 generation), it might have caused independent mutations in each allele, which were functionally equivalent to homozygous mutations; likewise, the mutation status of the three pairs of TaMsg47 alleles in the candidate plants was determined by gene sequencing. Therefore, Fielder gene-edited plants, in which each of the three pairs of TaMsg47 alleles included at least one Tamsg4 loss-of-function mutation but still exhibited male fertility, were selected and used to pollinate the artificially emasculated Yangmai i58 (Msg47 Msg47)-Thinopyrum bessarabicum isochromosome i4JL monosomic alien addition line, and Fi blue-grained seeds were harvested. From these seeds, blue-grained Fi plants, in which each of the three pairs of TaMsg47 alleles included at least one Tamsg4 loss-of-function mutation and exhibited male fertility, were identified, bagged, and self-fertilized to obtain F2 blue-grained seeds. In the F2 plant generation, 9 individual F2 plants were identified by gene sequencing, namely Pi209-i6-ii, Pi209-i6-i2, Pi209-i6-6i, Pi209-i6-63, Pi209-i6-73, Pi209-i6-76, Pi209-i6-85, Pi209-48-25, and Pi209-48-30. In view of the high editing efficiency of the Pi209 vector and the diversity of editing events in plants, only the genotypes and sequences of the 9 selected individual F2 plants are presented herein (Table 3, Table 4a, and Table 4b). Experimental results demonstrated that the blue-grained F2 plants (msg47 msg47 + i4JL') self-fertilized and set seeds, once again indicating that the Thinopyrum bessarabicum isochromosome i4JL possessed the function of restoring fertility, thus rendering the Triticum aestivum MAi4JL line carrying a male sterility genetic background (msg47 msg47) male fertile. F3 seeds produced by selfing of each of the above 9 individual F2 plants were harvested and then sorted separately into a non-blue-grained group (also referred to as white-grained group, amber-grained group, or red-grained group) and a blue-grained group. Among the 2,274 F3 grains harvested, 832 were blue grains, accounting for approximately 37%.

[71] Table 2: P1207-Based Novel Triticum aestivum-Thinopyrum bessarabicum Isochromosome i4JL Monosomic Alien Addition Lines and Genotypes Thereof Germplasm material Genome MSG47 gene target site region sequence PAM Target site 1 a Chinese    Spring 4A cggccgccttccTtgccggtgcacggcaagagctccgggcccg  b (WT) 4B cggccgccttcct|tgccggtgcacggcaagagc|tccgggcccg  b 4D cggccgccttcct|tgccggtgcacggcaagagc|tccgggcccg  b PI 531711(WT)         4J CGGCCGCCTTCCTTGCCGGTGCACGGCAAGGGTTCCGGGCCCG  c P1207-43(F1)         4A CGGCCGCCTTCCTTGC----GCACGGCAAGAGCTCCGGGCCCG  d 4B CGGCCGCCTTCCTTG-CGGTGCACGGCAAGAGCTCCGGGCCCG  d 4D CGGCCGCCTTCCTTG-CGGTGCACGGCAAGAGCTCCGGGCCCG  d 4J cGGccGccTTCCTTGccGGTGcAcGGcAAGGGTTccGGGcccG  c P1207-43-4(F2)       4A CGGCCGCCTTCCTTGC----GCACGGCAAGAGCTCCGGGCCCG  b 4B CGGCCGCCTTCCTTG-CGGTGCACGGCAAGAGCTCCGGGCCCG  b 4D CGGCCGCCTTCCTTG-CGGTGCACGGCAAGAGCTCCGGGCCCG  b 4J CGGCCGCCTTCCTTGCCGGTGCACGGCAAGGGTTCCGGGCCCG c P1207-43-4-B (F3) 4A CGGCCGCCTTCCTTGC----GCACGGCAAGAGCTCCGGGCCCG b 4B CGGCCGCCTTCCTTG-CGGTGCACGGCAAGAGCTCCGGGCCCG b 4D CGGCCGCCTTCCTTG-CGGTGCACGGCAAGAGCTCCGGGCCCG b 4J CGGCCGCCTTCCTTGCCGGTGCACGGCAAGGGTTCCGGGCCCG c P1207-43-4-A (F3) 4A CGGCCGCCTTCCTTGC----GCACGGCAAGAGCTCCGGGCCCG b 4B CGGCCGCCTTCCTTG-CGGTGCACGGCAAGAGCTCCGGGCCCG b 4D CGGCCGCCTTCCTTG-CGGTGCACGGCAAGAGCTCCGGGCCCG b 4J                   i4JL chromosome absent Note: a : no mutations were detected at Target Site 2, so relevant data are omitted; b : homozygous genotype, and the two alleles are identical; c : unmutated genotype of i4JL; d : heterozygous genotype, only the mutated allele is shown, and the unmutated allele is not shown. In the wild-type (WT) group, polymorphic bases among different genomes are highlighted with gray background; in the non-wild-type (F1, F2, and F3) groups, only polymorphic bases within the same genome that differ from the wild-type sequence are highlighted with gray background.

[72] Table 3: Novel Triticum aestivum-Thinopyrum bessarabicum Isochromosome i4JL Monosomic Alien Addition Lines and Their Fertility Plant Plant TaMSG47 Cas9 i4JL Seed Plant Self-seed setting No. gener genotype transgen chromoso color stamen rate ation e me P1207-43 F1 Msg47msg47 a Present Present Blue-grai Fertile Not ned counted P1207-43- F2 msg47 msg47 Present Present Blue-grai Partiall 46% d ned y fertile P1207-43- 4-A F3 msg47 msg47 Present Absent White-gr ained Sterile 0.1% e P1207-43- 4-B F3 msg47 msg47 Present Present Blue-grai ned Partiall y fertile 30.9% d P1209-16 11 F2 msg47 msg47 Present Present Blue-grai ned Fertile >85% f P1209-16 12 F2 msg47 msg47 Present Present Blue-grai ned Fertile >85% f P1209-16 61 F2 Msg-A47msg-A 47 ab Absent Present Blue-grai ned Fertile >85% f P1209-16 63 F2 msg47 msg47 c Absent Present Blue-grai ned Fertile >85% f P1209-16 73 F2 msg47 msg47 Absent Present Blue-grai ned Fertile >85% f P1209-16 76 F2 Msg-B47msg-B 47 ab Absent Present Blue-grai ned Fertile >85% f P1209-16 85 F2 Msg-B47msg-B 47 ab Absent Present Blue-grai ned Fertile >85% f P1209-48 25 F2 msg47 msg47 Present Present Blue-grai ned Fertile >85% f P1209-48 30 F2 msg47 msg47 Present Present Blue-grai ned Fertile >85% f P1209-16- 11-A F3 msg47 msg47 Present Absent White-gr ained Sterile 0% g P1209-16- F3 msg47 msg47 Present Present Blue-grai Fertile 89% g 11-B                                                ned P1209-16- 12-A F3 msg47 msg47 Present Absent White-gr ained Sterile 0% g P1209-16- 12-B F3 msg47 msg47 Present Present Blue-grai ned Fertile 62% g P1209-16- 61-A F3 Msg-A47msg-A 47 ab Absent Absent White-gr ained Sterile 7% g P1209-16- 61-B F3 Msg-A47msg-A 47 ab Absent Present Blue-grai ned Fertile 68% g P1209-16- 63-A F3 msg47 msg47 c Absent Absent White-gr ained Sterile 0% g P1209-16- 63-B F3 msg47 msg47 c Absent Present Blue-grai ned Fertile 93% g P1209-16- 73-A F3 msg47 msg47 Absent Absent White-gr ained Sterile 0% g P1209-16- 73-B F3 msg47 msg47 Absent Present Blue-grai ned Fertile 81% g P1209-16- 76-A F3 Msg-B47msg-B 47 ab Absent Absent White-gr ained Sterile 0% g P1209-16- 76-B F3 Msg-B47msg-B 47 ab Absent Present Blue-grai ned Fertile 67% g P1209-16- 85-A F3 Msg-B47msg-B 47 ab Absent Absent White-gr ained Sterile 0% g P1209-16- 85-B F3 Msg-B47msg-B 47 ab Absent Present Blue-grai ned Fertile 73% g P1209-48- F3 msg47 msg47 Present Absent White-gr Sterile 0% g 25-A                                                 ained P1209-48- 25-B F3 msg47 msg47 Present Present Blue-grai ned Fertile 91% g P1209-48- 30-A F3 msg47 msg47 Present Absent White-gr ained Sterile 0% g P1209-48- F3 msg47 msg47 Present Present Blue-grai Fertile 76% g 30-B                                                ned Note: a : genotype that remains heterozygous; b : only the heterozygous allele is shown, and the other two pairs of alleles are equivalent to homozygous recessive; c : although all three pairs of alleles are equivalent to homozygous recessive, the msg-B47 gene has a 9- or 12-bp deletion, and whether this deletion causes loss of function remains to be verified; d : self-seed setting rate is relatively low, possibly because the Cas9 and gRNA expression cassettes are still present, and the specificity of the dual target sites of P1207 is not strong, which may affect the function of the ThbMsg47 gene on the i4JL chromosome; e : self-seed setting rate may be due to cross-pollination from other fertile plants due to the absence of bagging isolation; f : self-seed setting rate was not statistically calculated but was based on visual determination of the actual flowering and seed setting status; g : the average self-seed setting rate was obtained from approximately 10 plants in the blue-grained group or 30 plants in the non-blue-grained group; the genotype, transgene status, and chromosome status of this F3 line were also based on a comprehensive evaluation of approximately 10 plants in the blue-grained group or 30 plants in the non-blue-grained group.

[73] Table 4a: P1209-Based Novel Triticum aestivum-Thinopyrum bessarabicum Isochromosome i4JL Monosomic Alien Addition Lines and Genotypes Thereof Germplasm                 MSG47 gene target site region sequence Genome___________-_____-z 2_________ material                                  PAM I Target site 1| Chinese Spring (wt) 4a cggcattgccccc|ga G CTGGCCGATGAGATCGCGGAGCGGGTG 4B cggcattgccccc|ga G CTGGCCGATGAGATCGCGGAGCGGGTG 4D cggcattgccccc|ga G CTGGCCGATGAGATCGCCGAGCGGGTG PI 531711 (WT) 4J CGGCATTGCCTCCGA G CTGGCCGATGAGATCGCGGAGCGGGTG P1209-16-11 (F2) 4A CGGCATTGCCCCCGA G CTGGCCGATGAGATCGCGGAGCGGGTG 4B CGGCATTGCCCCCGA GTCTGGCCGATGAGATCGCGGAGCGGGTG 4D CGGCATTGCCCCCGA G —TGGCCGATGAGATCGCCGAGCGGGTG 4J CGGCATTGCCTCCGA G CTGGCCGATGAGATCGCGGAGCGGGTG P1209-16-12 (F2) 4A CGGCATTGCCCCCGA G CTGGCCGATGAGATCGCGGAGCGGGTG 4B CGGCATTGCCCCCGAGG CTGGCCGATGAGATCGCGGAGCGGGTG CGGCATTGCCCCCGA G CTGGCCGATGAGATCGCCGAGCGGGTG 4D CGGCATTGCCCCCGA - —GGCCGATGAGATCGCCGAGCGGGTG CGGCATTGCCCCCGAGG CTGGCCGATGAGATCGCCGAGCGGGTG* 4J CGGCATTGCCTCCGA G CTGGCCGATGAGATCGCGGAGCGGGTG P1209-16-61 (F2) 4A CGGCATTGCCCCCGA G CTGGCCGATGAGATCGCGGAGCGGGTG 4B CGGCATTGCCCCCGA GACTGGCCGATGAGATCGCGGAGCGGGTG CGGCATTGCCCCCGA G CTGGCCGATGAGATCGCCGAGCGGGTG 4D CGGCATTGCCCCCGA G —TGGCCGATGAGATCGCCGAGCGGGTG 4J CGGCATTGCCTCCGA G CTGGCCGATGAGATCGCGGAGCGGGTG CGGCATTGCCCCCGA G CTGGCCGATGAGATCGCGGAGCGGGTG P1209-16-63 (F2) 4A CGGCATTGCCCCCGA GACTGGCCGATGAGATCGCGGAGCGGGTG CGGCATTGCCC---- — ----CCGATGAGATCGCGGAGCGGGTG 4B CGGCATT-------- — ---GCCGATGAGATCGCGGAGCGGGTG CGGCATTGCCCCCGA G CTGGCCGATGAGATCGCCGAGCGGGTG 4D CGGCATTGCCCCCGA GTCTGGCCGATGAGATCGCCGAGCGGGTG 4J CGGCATTGCCTCCGA G CTGGCCGATGAGATCGCGGAGCGGGTG P1209-16-73 (F2) 4A CGGCATTGCCCCCGA G CTGGCCGATGAGATCGCGGAGCGGGTG 4B CGGCATTGCCCCCGA GTCTGGCCGATGAGATCGCGGAGCGGGTG CGGCATTGCCCCCGA G CTGGCCGATGAGATCGCCGAGCGGGTG 4D CGGCATTGCCCCCGA G —TGGCCGATGAGATCGCCGAGCGGGTG 4J CGGCATTGCCTCCGA G CTGGCCGATGAGATCGCGGAGCGGGTG CGGCATTGCCCCCGA G CTGGCCGATGAGATCGCGGAGCGGGTG P1209-16-76 (F2) 4A CGGCATTGCCCCCGA - ---------gagatcgcggagcgggtg CGGCATTGCCCCCGA G CTGGCCGATGAGATCGCGGAGCGGGTG 4B CGGCATTGCCCCCGA GTCTGGCCGATGAGATCGCGGAGCGGGTG CGGCATTGCCCCCGA G -TGGCCGATGAGATCGCCGAGCGGGTG 4D CGGCATTGCCCCCGA GACTGGCCGATGAGATCGCCGAGCGGGTG 4J CGGCATTGCCTCCGA_G_CTGGCCGATGAGATCGCGGAGCGGGTG P1209-16-85 (F2) 4A CGGCATTGCCCCCGA G CTGGCCGATGAGATCGCGGAGCGGGTG CGGCATTGCCCCCGA G CTGGCCGATGAGATCGCGGAGCGGGTG 4B CGGCATTGCCCCCGA GTCTGGCCGATGAGATCGCGGAGCGGGTG 4D CGGCATTGCCCCCGA G -TGGCCGATGAGATCGCCGAGCGGGTG 4J CGGCATTGCCTCCGA G CTGGCCGATGAGATCGCGGAGCGGGTG CGGCATTGCCCCCGA GACTGGCCGATGAGATCGCGGAGCGGGTG P1209-48-25 (F2) 4A CGGCATTGCCCCCGA ----------GAGATCGCGGAGCGGGTG 4B CGGCATTGCCCCCGAGG CTGGCCGATGAGATCGCGGAGCGGGTG 4D CGGCATTGCCCCCGA GACTGGCCGATGAGATCGCCGAGCGGGTG 4J CGGCATTGCCTCCGA G CTGGCCGATGAGATCGCGGAGCGGGTG P1209-48-30(F2) 4A CGGCATTGCCCCCGA GACTGGCCGATGAGATCGCGGAGCGGGTG 4B CGGCATTGCCCCCGA GACTGGCCGATGAGATCGCGGAGCGGGTG 4D CGGCATTGCCCCCGA GACTGGCCGATGAGATCGCCGAGCGGGTG 4J CGGCATTGCCTCCGA G CTGGCCGATGAGATCGCGGAGCGGGTG Note: This table lists the major genotypes detected in the target individuals. In the wild-type (WT) group, polymorphic bases among different genomes are highlighted with gray background; in the non-wild-type (F2) group, only polymorphic bases within the same genome that differ from the wild-type sequence are highlighted with gray background. In the non-wild-type (F2) group, the wild-type gene sequence is represented in underlined font. Different types of single-base insertions occurring in the same gene of the same individual are represented by degenerate bases (H = A / C / T; W = A / T). * : rare mutation, present in only 1-5% of the sequences.

[74] Table 4b: P1209-Based Novel Tr Isochromosome i4JL Monosomic Alien Addition Germplasm   Genome___MSG47 material iticum aestiv Lines and Ge gene target Target site 2 um-Thinopyrum bessarabicum notypes Thereof site region sequence PAM Chinese Spring (wt) 4a       cgacgctggcgacgatggcgaagcacg ggcGggcggcggaggg 4B       CGACGCTGGCGACGATGGCGAAGCACG GCCAGGCGGCGGAGGG 4D CGACGCTGGCGACGATGGCGAAGCACG GGCGGGCGGCGGAGGG PI 531711 (WT) 4J CGACGCTGGCGACGATGGCGAAGCACG GGCGGACGGCGGAGGG P1209-16-11 (F2) 4A CGACGCTGGCGACGATGGCGAAGCACGT GGCGGGCGGCGGAGGG 4B CGACGCTGGCGACGATGGCGAAGCACG GCCAGGCGGCGGAGGG 4D CGACGCTGGCGACGATGGCGAAGCACGTTGGCGGGCGGCGGAGGG 4J Unknown P1209-16-12 (F2) 4A CGACGCTGGCGACGATGGCGAAGCAC- GGCGGGCGGCGGAGGG 4B Unknown 4D CGACGCTGGCGACGATGGCGAAGCACGW GGCGGGCGGCGGAGGG 4J Unknown CGACGCTGGCGACGATGGCGAAGCACG GGCGGGCGGCGGAGGG P1209-16-61 (F2) 4A CGACGCTGGCGACGATGGCGAAGCACGT GGCGGGCGGCGGAGGG 4B Unknown CGACGCTGGCGACGATGGCGAAGCACGA GGCGGGCGGCGGAGGG 4D CGACGCTGGCGACGATGGCGAAGCACGTTGGCGGGCGGCGGAGGG 4J Unknown P1209-16-63 (F2) 4A CGACGCTGGCGACGATGGCGAAGCAC- GGCGGGCGGCGGAGGG 4B Unknown CGACGCTGGCGACGATGGCGAAGCAC- GGCGGGCGGCGGAGGG 4D CGACGCT-------------------- GGCGGGCGGCGGAGGG 4J Unknown P1209-16-73 (F2) 4A CGACGCTGGCGACGATGGCGAAGCACGT GGCGGGCGGCGGAGGG 4B CGACGCTGGCGACGATGGCGAAGCACG GCCAGGCGGCGGAGGG CGACGCTGGCGACGATGGCGAAGCAC- GGCGGGCGGCGGAGGG 4D CGACGCTGGCGACGATGGCGAAGCACGTTGGCGGGCGGCGGAGGG 4J Unknown (F2) CGACGCTGGCGACGATGGCGAAGCA— GGCGGGCGGCGGAGGG P1209-16-76 4A CGACGCTGGCGACGATGGCGAAGCACGT GGCGGGCGGCGGAGGG 4B CGACGCTGGCGACGATGGCGAAGCACG GCCAGGCGGCGGAGGG CGACGCTGGCGACGATGGCGAAGCACGA GGCGGGCGGCGGAGGG 4D CGACGCTGGCGACGATGGCGAAGCACGTTGGCGGGCGGCGGAGGG 4J Unknown P1209-16-85 (F2) 4A CGACGCTGGCGACGATGGCGAAGCACGA GGCGGGCGGCGGAGGG 4B CGACGCTGGCGACGATGGCGAAGCACG GCCAGGCGGCGGAGGG 4D CGACGCTGGCGACGATGGCGAAGCACGTTGGCGGGCGGCGGAGGG 4J Unknown P1209-48-25 (F2) 4A CGACGCTGGCGACGATGGCGAAGCACGA GGCGGGCGGCGGAGGG CGACGCTGGCGACGATGGCGAAGCACG GCCAGGCGGCGGAGGG 4B CGACGCTGGCGACGATGGCGAAGCAC- GCCAGGCGGCGGAGGG* CGACGCTGGCGACGATGGCGAAGCACGH GCCAGGCGGCGGAGGG* 4D CGACGCTGGCGACGATGGCGAAGCACGTTGGCGGGCGGCGGAGGG 4J Unknown P1209-48-30(F2) 4A CGACGCTGGCGACGATGGCGAAGCACGA GGCGGGCGGCGGAGGG CGACGCTGGCGACGATGGCGAAGCACG GCCAGGCGGCGGAGGG 4B CGACGCTGGCGACGATGGCGAAGCAC- GCCAGGCGGCGGAGGG* CGACGCTGGCGACGATGGCGAAGCACGT GCCAGGCGGCGGAGGG* 4D CGACGCTGGCGACGATGGCGAAGCACGTTGGCGGGCGGCGGAGGG 4J Unknown Note: This table lists the major genotypes detected in the target individuals. Unknown represents that the target site sequence at that position was not obtained. In the wild-type (WT) group, polymorphic bases among different genomes are highlighted with gray background; in the non-wild-type (F2) group, only polymorphic bases within the same genome that differ from the wild-type sequence are highlighted with gray background. In the non-wild-type (F2) group, the wild-type gene sequence and “Unknown” are represented in underlined font. Different types of single-base insertions occurring in the same gene of the same individual are represented by degenerate bases (H = A / C / T; W = A / T). * : rare mutation, present in only 1-5% of the sequences.

[75] Example 3: Seed Color and Karyotype Analysis of Triticum aestivum and Its Corresponding MAi4JL

[76] 1. Experimental Method

[77] The Thinopyrum bessarabicum isochromosome i4JL was obtained by referring to the method for 60Co irradiation of a Chinese Spring-Thinopyrum bessarabicum chromosome 4J disomic alien addition line (DA4J) by the team led by Dr. Zengjun Qi at Nanjing Agricultural University (Physical mapping of chromosome 4J of Thinopyrum bessarabicum using gamma radiation-induced aberrations). The obtained Thinopyrum bessarabicum isochromosome i4JL was used to develop the lines "Yang 6-B", "P1207-43-4-36-B", and "P1209-16-12-B". Furthermore, seed color phenotype observation was carried out on Triticum aestivum: "Yang 6-A", "P1207-43-4-36-A", and "P1209-16-12-A", as well as their corresponding MAi4JL: "Yang 6-B", "P1207-43-4-36-B", and "P1209-16-12-B". In addition, fluorescence in situ hybridization (FISH) analysis was performed on root tip cell mitotic metaphase chromosomes with an oligonucleotide probe set for further karyotype analysis.

[78] Wherein:

[79] "Yang 6-A" is wild-type Yangmai 6 (genotype Msg47 Msg47), and "Yang 6-B" is the Yangmai 6 (Msg47 Msg47)-Thinopyrum bessarabicum isochromosome i4JL monosomic alien addition line.

[80] "P1207-43-4-36-A" is Triticum aestivum "P1207-43-4-36-A" (genotype msg47 msg47), and "P1207-43-4-36-B" is the Triticum aestivum (msg47 msg47)-Thinopyrum bessarabicum isochromosome i4JL monosomic alien addition line.

[81] "P1209-16-12-A" is Triticum aestivum "P1209-16-12-A" (genotype msg47 msg47), and "P1209-16-12-B" is the Triticum aestivum (msg47 msg47)-Thinopyrum bessarabicum isochromosome i4JL monosomic alien addition line.

[82] 2. Experimental Results

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

[84] The  results showed that the  non-blue-grained 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 alien chromosomes, while the blue-grained plants ("Yang 6-B", "P1207-43-4-36-B", and "P1209-16-12-B") had a chromosome number of 2n = 43 and contained one added Thinopyrum bessarabicum isochromosome i4JL. Furthermore, the chromosome constitution of Triticum aestivum was similar between the non-blue-grained plants and the blue-grained plants. The added Thinopyrum bessarabicum isochromosome i4JL in the blue-grained wheat involved only the long arm of 4J, and its banding pattern was distinctly different from those of the other Triticum aestivum chromosomes, thereby establishing a solid foundation for the subsequent tracking and identification of the MAi4JL-specific germplasm.

[85] Example 4: Detection of Female and Male Gamete Transmission Patterns Using a Triticum aestivum-Thinopyrum bessarabicum Isochromosome i4JL Monosomic Alien Addition Line

[86] Multiple non-blue-grained Triticum aestivum varieties (such as "Shannong 28", "Luyan 128", "Jimai 44", and "Bainong 4199") were used as pollen donors to pollinate an artificially emasculated Yangmai 6  (Msg47 Msg47)-Thinopyrum bessarabicum isochromosome i4JL monosomic alien addition line. The Yangmai 6  (Msg47 Msg47)-Thinopyrum bessarabicum isochromosome i4JL monosomic alien addition line was used as a pollen donor to pollinate artificially emasculated Triticum aestivum ''Fielder''. The proportion of blue grains among the set seeds was counted to detect the transmission patterns of the Thinopyrum bessarabicum isochromosome i4JL via female and male gametes.

[87] The test results showed that when non-blue-grained Triticum aestivum varieties were used as pollen donors to pollinate the artificially emasculated Yangmai 6-Thinopyrum bessarabicum isochromosome i4JL monosomic alien addition line, the proportion of blue grains in the BC3F1 generation ranged from 10% to 64% (mostly concentrated between 22% and 33%), and the proportion of blue grains in the BC4F1 generation ranged from 1.7% to 46% (mostly concentrated between 21% and 35%). Conservatively, by adopting the upper value of the commonly observed range, it is inferred that the Thinopyrum bessarabicum isochromosome i4JL has a 35% transmission rate through female gametes.

[88] When the Yangmai 6-Thinopyrum bessarabicum isochromosome i4JL monosomic alien addition line was used as a pollen donor to pollinate the artificially emasculated Triticum aestivum ''Fielder'', among the 3,716 F1 hybrid grains obtained, only 45 F1 hybrid grains exhibited blue, indicating that the Thinopyrum bessarabicum isochromosome i4JL has approximately a 1.2% transmission rate through male gametes.

[89] Theoretically, among the grains produced by selfing of the Triticum aestivum-Thinopyrum bessarabicum isochromosome i4JL monosomic alien addition line, approximately 64.2% are non-blue grains (white, amber, red, etc., which are Triticum aestivum euploids that do not carry the Thinopyrum bessarabicum isochromosome i4JL; 2n = 42), approximately 35.4% are blue grains (Triticum aestivum-Thinopyrum bessarabicum isochromosome i4JL monosomic alien addition line; 2n = 43), and approximately 0.4% are dark-blue grains (i.e., the expected Triticum aestivum-Thinopyrum bessarabicum isochromosome i4JL disomic alien addition line; 2n = 44). However, in the past two years of continuous selfing experiments, a stably inherited Triticum aestivum-Thinopyrum bessarabicum isochromosome i4JL disomic alien addition line has not yet been obtained.

[90] When the Triticum aestivum in the Triticum aestivum-Thinopyrum bessarabicum isochromosome i4JL monosomic alien addition line is normally fertile itself, then the seeds of different colors produced by selfing of this monosomic alien addition line are theoretically all normally fertile. When the Triticum aestivum in the Triticum aestivum-Thinopyrum bessarabicum isochromosome i4JL monosomic alien addition line is male sterile itself, then the non-blue-grained seeds produced by selfing of this monosomic alien addition line are theoretically all sterile.

[91] Based on the analysis of the double-dosage advantage of Thinopyrum bessarabicum isochromosome i4JL in Example 1 and the detection results of female and male gamete transmission patterns of the i4JL chromosome in the Triticum aestivum-Thinopyrum bessarabicum isochromosome i4JL monosomic alien addition line, it is shown that the Thinopyrum bessarabicum isochromosome i4JL can be used to construct a blue-marked maintainer line and simplify the maintenance and propagation of the male sterile line, thereby providing an entirely new technical means for two-line hybrid wheat production.

[92] Example 5: Analysis of Application Prospects of Triticum aestivum (msg47 msg47)-Thinopyrum bessarabicum Isochromosome i4JL Monosomic Alien Addition Line

[93] Focusing on the novel Triticum aestivum (msg47 msg47)-Thinopyrum bessarabicum isochromosome i4JL monosomic alien addition line, further research was conducted on its fertility to determine its application prospects.

[94] Corresponding to the P1207 F3 generation, the stamen development and seed setting statuses of plants were compared between the non-blue-grained group (also referred to as white-grained group, amber-grained group, or red-grained group, etc.; P1207-43-4-A, Table 3) and the blue-grained group (P1207-43-4-B, Table 3). At the wheat flowering stage, mature non-dehiscent anthers were collected and soaked in Alexander's stain (Solarbio #G3050) for 12 hours; the morphology of the anthers and pollen grains was observed using an Olympus microscope (BX53F2). Alexander's stain is a polychromatic stain for plant pollen that stains viable pollen grains purple-red or stains poorly developed or non-viable pollen grains green. The results showed that plants in the non-blue-grained group were male sterile, exhibiting empty anther locules and no pollen grains (FIG. 4); plants in the blue-grained group were male fertile or partially fertile, exhibiting anther locules filled with mature pollen grains (FIG. 4). In the F3 generation, the self-seed setting rate of plants in the non-blue-grained group was 0.1%, and the self-seed setting rate of plants in the blue-grained group was 30.9%; the difference was highly significant. Plants in the non-blue-grained group are Triticum aestivum (msg47 msg47) euploids, which are theoretically male sterile and do not set seed upon selfing. However, among the 8,086 florets detected, 8 florets set seed, which is most likely attributable to false positives caused by cross-pollination due to the absence of bagging. The low self-seed setting rate of plants in the blue-grained group may be due to the fact that the P1207-43-4-B plants still carry the Cas9 and gRNA transgene expression cassettes. Furthermore, the specificity of the dual target sites of P1207 is not strong. Transient editing of the ThbMsg47 gene may have interfered with its normal function, thereby suppressing the fertility restoration function of the Thinopyrum bessarabicum isochromosome i4JL, which in turn resulted in a relatively low self-seed setting rate of plants in the blue-grained group.

[95] Corresponding to the P1209 F3 generation, the stamen development and seed setting statuses of plants between the non-blue-grained group (also referred to as white-grained group, amber-grained group, or red-grained group, etc.) and the blue-grained group were compared. Staining by Alexander's stain showed that plants in the non-blue-grained group were male sterile, exhibiting empty anther locules and no pollen grains; plants in the blue-grained group were male fertile or partially fertile, exhibiting anther locules filled with mature pollen grains. In the F3 generation, the self-seed setting rate of plants in the non-blue-grained group was 0% (with the exception of the P1209-16-61-A line, which had a self-seed setting rate of approximately 7%), and the self-seed setting rate of plants in the blue-grained group ranged from 62% to 93%. Among these, each of 8 independent F3 lines contained plants with a self-seed setting rate of > 85% (with the exception of the P1209-16-76-B line, in which the maximum self-seed setting rate of a single plant was 78%). The difference was highly significant.

[96] In the F3 generation, the only difference between plants in the non-blue-grained group and the blue-grained group is that the former are Triticum aestivum (msg47 msg47) euploids (2n = 42), while the latter are Triticum aestivum (msg47 msg47)-Thinopyrum bessarabicum isochromosome i4JL monosomic alien addition lines (2n = 43; FIG. 3, Tables 2 and 3). Therefore, the Thinopyrum bessarabicum isochromosome i4JL possesses the function of restoring male fertility in Triticum aestivum (msg47 msg47), and can be used to develop a novel wheat genic male sterile maintainer line that combines both restoration and maintenance functions. The non-blue-grained seeds produced from the self-fertilization of the novel genic male sterile maintainer line develop into wheat genic male sterile lines, and the blue-grained seeds enable the self-maintenance of the novel wheat genic male sterile maintainer line. The novel Triticum aestivum (msg47 msg47)-Thinopyrum bessarabicum isochromosome i4JL monosomic alien addition line, upon selfing, can be used to propagate the wheat genic male sterile line and its maintainer line.

[97] The foregoing descriptions are merely preferred embodiments of the present application and are not intended to limit the present application. For those skilled in the art, various changes and modifications may be made to the present application. Any modifications, equivalent substitutions, improvements, and the like made within the spirit and principle of the present application shall fall within the scope of protection of the present application.

Claims

1. Use of Thinopyrum bessarabicum isochromosome i4JL in at least one of the following (1) to (4):(1) restoring a wheat stamen fertility function;(2) maintaining a wheat recessive genic male sterile line;(3) propagating a wheat recessive genic male sterile line; and(4) creating a blue-marked two-line hybrid wheat production system.

2. The use according to claim 1, wherein the blue-marked two-line hybrid wheat production system is a Triticum aestivum-Thinopyrum bessarabicum isochromosome i4JL monosomic alien addition line.

3. Use of a Triticum aestivum-Thinopyrum bessarabicum isochromosome i4JL monosomic alien addition line in propagating a wheat genic male sterile line and a maintainer line.

4. The use according to claim 3, wherein in the Triticum aestivum-Thinopyrum bessarabicum isochromosome i4JL monosomic alien addition line, genes related to stamen fertility in a Triticum aestivum background carry loss-of-function mutations; andpreferably, the genes related to stamen fertility are three pairs of TaMSG47 genes.

5. The use according to claim 3, wherein the Triticum aestivum-Thinopyrum bessarabicum isochromosome i4JL monosomic alien addition line is developed by introducing Thinopyrum bessarabicum isochromosome i4JL into a Triticum aestivum variety.

6. A method for propagating a wheat genic male sterile line and a maintainer line using Thinopyrum bessarabicum isochromosome i4JL, comprising the following steps:(1) constructing a Triticum aestivum-Thinopyrum bessarabicum isochromosome i4JL monosomic alien addition line, wherein in the Triticum aestivum-Thinopyrum bessarabicum isochromosome i4JL monosomic alien addition line, genes related to stamen fertility in a Triticum aestivum background carry loss-of-function mutations; and(2) selfing the Triticum aestivum-Thinopyrum bessarabicum isochromosome i4JL monosomic alien addition line, and performing optical sorting based on the color difference of grains obtained from selfing.

7. The method according to claim 6, wherein the genes related to stamen fertility arethree pairs of TaMSG47 genes.

8. The method according to claim 7, wherein in step (1), a method for constructing the Triticum aestivum-Thinopyrum bessarabicum isochromosome i4JL monosomic alien addition line comprises:using a wheat plant carrying a loss-of-function mutation in the TaMsg47 gene as a pollen donor to pollinate an artificially emasculated ''Triticum aestivum-Thinopyrum bessarabicum isochromosome i4JL monosomic alien addition line''; then, from a blue-grained F1 generation, identifying plants heterozygous for TaMsg47 in A, B, and D genomes; and selfing the plants heterozygous for TaMsg47 to set seeds, and then, from a blue-grained F2 generation, identifying plants homozygous for Tamsg47 in the A, B, and D genomes.

9. The method according to claim 6, wherein in step (2), criteria for the optical sorting are as follows:(1) if a grain has a non-blue color, the grain develops into a Triticum aestivum euploid of a genic male sterile line, which does not carry Thinopyrum bessarabicum isochromosome i4JL, with 2n = 42; and(2) if a grain has a blue color, the grain develops into a Triticum aestivum-Thinopyrum bessarabicum isochromosome i4JL monosomic alien addition line of a maintainer line, which carries one Thinopyrum bessarabicum isochromosome i4JL, with 2n = 43.

10. The method according to claim 6, wherein the non-blue color is white, amber, or red, etc.

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