Application of i4JL in the breeding of wheat near-isogenic lines

Abstract

This patent discloses a breeding method for selecting near-isogenic wheat lines using the isoarm chromosome i4JL of *Thinopyrum besarmentosum*. The i4JL chromosome has a unique fluorescent band, making it easily distinguishable from wheat chromosomes. In a wheat background, it is primarily transmitted through female gametes, with extremely low transmission rates in male gametes. Furthermore, it contains two doses of the blue grain gene. Therefore, the self-crossing progeny of the wheat-*Thinopyrum besarmentosum* i4JL monosomatic addition line MAi4JL always exhibits easily separable blue and non-blue grains. By crossing MAi4JL with any non-blue grain wheat and selecting blue grain seeds from the hybrid progeny for five consecutive generations, near-isogenic wheat lines with different backgrounds and phenotypes (blue and non-blue grains) can be obtained, providing a new method for selecting near-isogenic wheat lines. Since i4JL significantly reduces plant height in different wheat backgrounds and significantly affects gene expression in wheat stems and grains, it has significant application potential in wheat genetic breeding research and applications.

Description

Technical Field

[0001] This invention discloses a breeding method for selecting near-isogenic lines of wheat using the isoarm chromosome i4JL of *Triticum aestivum*, belonging to the fields of crop genetics and breeding and agricultural biotechnology. Background Technology

[0002] Distant hybridization is one of the important approaches in wheat breeding and plays a crucial role in wheat breeding. The first key step in transferring and utilizing exogenous genes through distant hybridization is to add different exogenous chromosomes to the wheat background through hybridization and backcrossing between wheat and its related species, and to breed wheat heterologous addition lines in the same background. By comparing these addition lines with the background wheat, the genetic effects of specific exogenous chromosomes can be analyzed, and exogenous genes can be discovered and utilized. Therefore, breeding wheat heterologous addition lines in the same background is one of the most important tasks in wheat distant hybridization breeding. At present, the breeding of wheat heterologous addition lines usually requires continuous backcrossing and self-crossing, combined with cytological identification, to obtain homozygous disomic addition lines (Zhuang Lifang, Qi Zengjun, Ying Jia, Chen Peidu, Liu Dajun. Breeding and identification of disomic heterologous addition lines of common wheat-Thinopyrum bessarabicum[J]. Acta Genetica Sinica, 2003, 30(10): 919-925.). Diso addition lines obtained by backcrossing are identical to recurrent parents in all aspects except for the introduced exogenous chromosome from a wheat-related species. They can be called near-isogenic lines. However, to analyze the effect of exogenous chromosomes in different wheat backgrounds, it is usually necessary to backcross with other recurrent parents 5-6 times and then selfcross to obtain near-isogenic lines with different backgrounds. But these backgrounds are limited to the recurrent parent background and require multiple identifications and backcrosses, which is complicated and labor-intensive. Therefore, how to breed near-isogenic wheat lines with different backgrounds involving specific exogenous chromosomes in a simple and efficient manner is of great research and utilization value for the discovery and utilization of key exogenous genes.

[0003] Nanjing Agricultural University has long been engaged in wheat distant hybridization and chromosome engineering breeding, creating a number of special wheat germplasm, which provides important parents and tools for wheat genetic breeding (Zhuang Lifang, Qi Zengjun. Progress in research and application of plant chromosome mutagenesis [J]. Journal of Nanjing Agricultural University, 2018, 41(1):15; Wang Qing, He Ziming, Wang Libin, Qi Zengjun. Progress in the application of chromosome engineering in hybrid wheat breeding [J]. Chinese Science Bulletin, 2022, 67(26):11.). Bessarabic is a perennial coastal wheatgrass native to the Black Sea and Mediterranean regions. Due to its salt tolerance and resistance to various wheat diseases, it is an important genetic resource for wheat improvement (Gorham J, McDonnell E, Buderewicz E, Wynn Jones RG. Salt tolerance in the Triticeae: growth and solute accumulation in leaves of Thinopyrum bessarabicum[J]. J Exp Bot, 1985, 36(7): 1021-1031; Xu SS, Jin Y, Klindworth DL, Wang RR-C, Cai XW. Evaluation and characterization of seedling resistances to stem rust Ug99 races in wheat-alien species derivatives[J]. Crop Sci, 2009, 49(6): 2167-2175.). Currently, hexaploid and octoploid *Triticum aestivum* varieties and a series of supplementary lines (King IP, Forster BP, Law CC, Cant KA) have been developed.

[0004] Orford SE, Gorham J, Reader S, Miller TE. Introgression of salt-tolerance genes from Thinopyrum bessarabicuminto wheat[J]. New Phytol, 1997, 137(1): 75-81; Hassani HS, King IP, Reader SM, Caligari PDS, Miller TE. Can Tritipyrum, a new salt-tolerant potential amphiploid, be a successful cereal like Triticale[J]. J AgrSci Tech, 2000, 2: 177-195; Zhuang Lifang, Qi Zengjun, Ying Jia, Chen Peidu, Liu Dajun. Common wheat-Thinopyrum bessarabicuminto wheat[J]. New Phytol, 1997, 137(1): 75-81; Zhuang Lifang, Qi Zengjun, Ying Jia, Chen Peidu, Liu Dajun. Common wheat-Thinopyrum bessarabicuminto wheat[J]. New Phytol, 1997, 137(1): 75-81; Zhuang Lifang, Qi Zengjun, Ying Jia, Chen Peidu, Liu Dajun. Common wheat-Thinopyrum bessarabicuminto wheat[J]. New Phytol, 1997, 137(1): 75-81; Breeding and identification of novelwheat-Thinopyrum bessarabicum disomic alternating lines[J]. Acta Genetica Sinica, 2003, 30(10): 919-925.), and created a variety of different translocation lines (Du Pei. Breeding and effect analysis of common wheat-Thinopyrum bessarabicum chromosome translocation lines[D]. Nanjing: Nanjing Agricultural University, 2010; Wang Yanzhi. Creation, identification and gene mapping analysis of small chromosome fragment translocations of novelwheat-Thinopyrum bessarabicum[D]. Nanjing: Nanjing Agricultural University, 2013; Patokar C, Sepsi A, Schwarzacher T, Kishii M, Heslop-Harrison JS. Molecular cytogenetic characterization of novelwheat-Thinopyrum bessarabicum recombinant lines carrying intercalary translocations[J]. Chromosoma, 2016, 125(1): 163-172).Studies have shown that chromosome 4J of Thinopyrum bessarabicum contains the blue-grained gene BaThb (Shen YF, Shen J, Dawa, Zhuang LF, Wang YZ, Pu J, Feng YG, Chu CG, Wang XE, Qi ZJ. Physical localization of a novel blue-grained gene derived from Thinopyrum bessarabicum[J]. Mol Breeding, 2013, 31(1):195-204; Pu J, Wang Q, Shen YF, Zhuang LF, Li CX, Tan MF, Bie TD, Chu CG, Qi ZJ. Physical mapping of chromosome 4J of Thinopyrum bessarabicum using gamma radiation-induced aberrations[J]. Theor Appl Genet, 2015, 128(7): 1319-1328.), states that in wheat, blue aleurone layer wheat, or blue grain wheat, is formed by controlling the synthesis of anthocyanins.Shen et al. initially named the blue-grained gene BaThb and located it at FL0.52 on the long arm of chromosome 4J (Shen YF, Shen J, Dawa, Zhuang LF, Wang YZ, Pu J, Feng YG, Chu CG, Wang XE, Qi ZJ. Physical localization of a novel blue-grained gene derived from Thinopyrum bessarabicum[J]. Mol Breeding, 2013, 31(1):195-204.); Pu et al. used radiation mutagenesis to select multiple variants involving chromosome 4J, and used 101 chromosome-specific molecular markers for physical mapping to divide 4JS into 7 segments and 4JL into 17 segments, and further located BaThb in the 4JL-11 segment (see reference: Pu J, Wang Q, Shen YF, Zhuang LF, Li CX, Tan MF, Bie TD, Chu CG, QiZJ. Physical mapping of chromosome 4J of Thinopyrum bessarabicum using gammaradiation-induced aberrations[J]. Theor Appl Genet, 2015, 128(7): 1319-1328.). Analysis showed that there was a high degree of collinearity between chromosome 4J of Thinopyrum bessarabicum and chromosome 4D of wheat (Pu Jing. Physical mapping of chromosome 4J of Thinopyrum bessarabicum and fine mapping of blue grain gene[D]. Nanjing: Nanjing Agricultural University, 2014.).In the wheat-Thymus bessarabic 4J heterochromatic system bred by Nanjing Agricultural University, the wheat-Thymus bessarabic isochromosomal i4JL (also known as i4JL.4JL) monochromatic addition line, abbreviated as MAi4JL, has an additional 4JL isochromosomal chromosome. After systematic molecular marker, fluorescence in situ hybridization (FISH) and genome in situ hybridization (GISH) identification (Shen YF, Shen J, Dawa, Zhuang LF, Wang YZ, Pu J, Feng YG, Chu CG, Wang XE, Qi ZJ. Physical localization of a novel blue-grained gene derived from Thinopyrum bessarabicum[J]. Mol Breeding,2013,31(1):195-204; Pu J, Wang Q, Shen YF, Zhuang LF, Li CX, Tan MF, Bie TD, Chu CG, Qi ZJ. Physical mapping of chromosome 4J of Thinopyrum bessarabicum using gamma radiation-induced aberrations[J].Theor Appl Genet,2015,128(7):1319-1328;Chen JY,TangYQ,Yao LS,Wu H,Tu XY,Zhuang LF,Qi ZJ.Cytological and molecular characterization of Thinopyrum bessarabicum chromosomes and structural rearrangements introgressed in wheat[J].Molecular Breeding, 2019, 39:146; Wang Qing. DNA sequence analysis of the long arm of chromosome 4J of Wheatgrass baicalensis and fine localization of its blue grain gene [D]. Nanjing: Nanjing Agricultural University, 2017.), this chromosome only involves the long arm of chromosome 4J of Wheatgrass baicalensis, and has a distinct banding pattern that is significantly different from other chromosomes of wheat. Using the patented technology of Nanjing Agricultural University (Qi Zengjun; Chen Jianyong; Tang Yuqing; Yao Lesha, an oligonucleotide probe kit for simultaneous detection of chromosomes of wheat and Wheatgrass baicalensis and its usage, ZL201910474381.X, 20190530), this chromosome can be easily identified. Figure 1 ).

[0005] The research of this invention shows that this chromosome is mainly transmitted through female gametes, with a transmission rate of only about 1 / 4, while the transmission rate through male gametes is extremely low. No homozygous stable disomic addition lines were observed. Therefore, the seeds of the self-pollinated offspring of MAi4JL show obvious segregation between blue and non-blue grains. Furthermore, because the blue grain gene is directly sensed by the endosperm, the copy number of the blue grain gene in the aleurone layer of the blue grain endosperm is 4, which is one more copy than the normal 4J chromosome disomic addition line. Seeds containing this chromosome are deep blue and stable, with minimal environmental influence. Figure 1 Taking advantage of the fact that i4JL is mainly transmitted through female gametes and has a stable deep blue grain color, MAi4JL can be crossed with any non-blue grain wheat variety (multiple backcrosses can also be combined for rapid improvement of agronomic traits) to obtain F1 hybrids with segregated grain color. Blue grains are selected and planted, and allowed to self-pollinate to obtain F2. The process of selecting blue grains and planting and self-pollinating continues until F6. The resulting grains are planted separately according to whether they are blue or non-blue, thus obtaining near-isogenic lines with significant differences in grain color between blue and non-blue grains. Since gene recombination in the offspring of two parents can produce a rich variety of variations, i4JL can be used to select a series of near-isogenic lines with different phenotypes in the same combination according to the breeding objectives. This method can be extended to multiple cross combinations. Compared to backcrossing, which can only select near-isogenic lines from specific backgrounds, this method utilizes the extremely low transmission rate of i4JL male gametes and the direct endosperm perception of the blue seed trait. It can distinguish between blue and non-blue seeds in the hybrid generation. Therefore, it can quickly, simply, and efficiently select near-isogenic wheat lines from different backgrounds through self-pollination combined with seed sorting. At the same time, it eliminates the need for continuous backcrossing and identification, saving manpower and resources. Among the selected near-isogenic lines, the blue-grained lines have a deeper blue grain color and significantly lower plant height than the non-blue-grained lines, providing important tools and materials for conventional wheat breeding and bio-breeding. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a breeding method for selecting near-isogenic lines of wheat using the isoarm chromosome i4JL of *Wheatgrass bisa*.

[0007] The technical solution adopted by this invention to solve its technical problem is:

[0008] In a first aspect, the present invention protects the application of the isochromosome i4JL of wheat or the wheat-wheat isochromosome i4JL monosomy supplementary line MAi4JL in the breeding of near-isogenic wheat lines.

[0009] Secondly, the present invention relates to the application of the isochromosome i4JL of wheat or the monosomy MAi4JL of wheat-wheat isochromosome i4JL in the preparation and breeding of near-isogenous wheat lines.

[0010] In a specific implementation plan, the wheat near-isogenic line is a dwarf blue-grained wheat near-isogenic line.

[0011] Thirdly, this invention protects the application of the isochromosome i4JL of wheat or the wheat-wheat isochromosome i4JL monosodium addition line MAi4JL in wheat crop breeding.

[0012] Fourthly, this invention protects the use of the isochromosome i4JL of wheat or the wheat-wheat isochromosome i4JL monosodium addition line MAi4JL in the preparation of products for wheat crop breeding.

[0013] In a specific implementation plan, the wheat crop breeding refers to the selection and breeding of short-stalked blue-grain wheat crops.

[0014] In a specific implementation plan, the cereal crop is wheat, rye, triticale, or wheatgrass.

[0015] In a more specific implementation, the wheat is common wheat, diploid wheat, or tetraploid wheat.

[0016] Fifthly, the present invention provides a method for breeding near-isogenic wheat lines using the wheat-wheatgrass isochromosome i4JL monosodium addition line MAi4JL. By hybridizing MAi4JL once and then selecting blue-grained seeds for continuous self-pollination, near-isogenic blue-grained wheat lines and near-isogenic non-blue-grained wheat lines can be bred.

[0017] Sixthly, the present invention also protects the application of the method described above in the breeding of near-isogenic wheat lines, dwarf blue-grained wheat varieties or strains.

[0018] In a specific implementation plan, the wheat near-isogenic line is a dwarf blue-grained wheat near-isogenic line.

[0019] Beneficial effects:

[0020] 1. This invention discloses a breeding method for selecting near-isogenic lines of dwarf blue-grained wheat using the special monomeric addition line MAi4JL of wheat-Baisayan wheatgrass. This method does not require backcrossing. After one hybridization, the target phenotype plants are selected for continuous self-pollination and blue-grained seeds are selected for planting to develop near-isogenic wheat systems with different phenotypic expressions. The blue-grained and non-blue-grained lines show clear separation of grain color and are easy to sort. At the same time, the plant height of the blue-grained line is significantly reduced. Therefore, this invention provides a new tool and method for discovering and utilizing key genes on 4JL and creating new wheat germplasm.

[0021] 2. The dwarf blue-grain near-isogenous lines bred by the breeding method disclosed in this invention contain a special isoarm chromosome i4JL of *Wheatgrass bisa*. Its genetic transmission is mainly through female gametes, and the transmission rate is significantly lower than the theoretical value, while the transmission rate of male gametes is extremely low. This provides an important genetic resource for studying the segregation of exogenous chromosomal variations and their genetic effects, and provides important inspiration for the creation, discovery and utilization of special exogenous chromosomal variations.

[0022] 3. The dwarf blue-grain near-isogenic lines bred by the breeding method disclosed in this invention can be combined with genetic engineering because i4JL has a special chromosome structure and is not closely related to wheat 4A, 4B and 4D, making recombination less likely. It has easily distinguishable grain color traits, which is conducive to the stable transmission of exogenous variant chromosomes and does not interfere with the genetic background composition of ordinary wheat. Therefore, the new wheat germplasm created will have significant application value.

[0023] 4. The dwarf blue-grain near-isogenic lines bred by the breeding method disclosed in this invention can be used as special-purpose wheat varieties. In particular, blue-grain seeds produce more anthocyanins due to the high copy number of the blue grain gene, and therefore can be used for efficient anthocyanin isolation and wheat nutrient breeding. Attached Figure Description

[0024] Figure 1 Comparison of grain color and karyotype between blue and red grain lines in the MAi4JL near-isogenic line (left: blue grain line; right: red grain line);

[0025] Figure 2 Differential gene expression and GO enrichment analysis of the second internode at the jointing stage of the MAi4JL near-isogenic line; among which... Figure 2 Figure A in the diagram is a volcano diagram, showing differentially expressed genes in the stems of the blue and red series; Figure 2 Figure B in the diagram is a gene expression heatmap; Figure 2 Figure C in the figure shows the GO enrichment of upregulated genes in wheat; Figure 2 Figure D in the diagram shows the GO enrichment of downregulated expression genes in wheat; Figure 2 Figure E in the figure shows GO enrichment of the 4JL-specific expressed gene;

[0026] Figure 3 Differential gene expression and GO enrichment analysis of the aleurone layer of blue and non-blue seeds from the near-isogenic blue-grained line MAi4JL, 21 days after flowering; among which... Figure 3 Figure A in the diagram is a volcano diagram of differentially expressed genes in blue and red granules; Figure 3 Figure B in the figure is a heatmap of differentially expressed genes; Figure 3 Figure C in the figure shows the GO enrichment of upregulated genes in wheat; Figure 3 Figure D in the diagram shows the GO enrichment of downregulated expression genes in wheat; Figure 3 Figure E in the figure shows GO enrichment of the 4JL-specific expressed gene;

[0027] Figure 4 Near-isogenic wheat lines were bred using the isoarm chromosome i4JL of *Thinopyrum cymosum*, among which... Figure 4 Figures A and B in the diagram represent the blue and white grain lines of MAi4JL / Yangmai 158F6, respectively; Figures C and D represent the blue and white grain lines of MAi4JL / Yangmai 6 F6. Figure 4 Figures E and F in the figure represent the blue-grained and white-grained lines of MAi4JL / Yannong 19F6. In addition to each blue-grained line containing one i4JL chromosome, the non-blue-grained lines corresponding to other wheat chromosomes are basically the same. Further self-pollination of blue-grained single plants can isolate more strictly isogenetic lines. Detailed Implementation

[0028] The present invention will be further described in detail below with reference to the embodiments. Unless otherwise specified, all reagents or instruments used are considered to be conventional products that can be purchased on the market.

[0029] First, Nanjing Agricultural University created a monosomy 4JL addition line of wheat-*Leymus chinensis* isochromosomal i4JL, named MAi4JL, through distant hybridization and systematic molecular marker, FISH, and GISH identification. This addition line has normal fertility. After eight generations of continuous self-pollination, each time blue seeds were selected for planting, the seeds of the self-pollinated offspring still showed seed color segregation, with blue and red seeds respectively. The blue and red seeds showed uniform traits within the lines, and except for a significant difference in plant height, other traits were similar. Referring to the reference karyotypes of *Leymus chinensis* and *Leymus chinensis*, an oligonucleotide probe kit was used. (Qi Zengjun; Chen Jianyong; Tang Yuqing; Yao Lesha, An oligonucleotide probe kit for simultaneous detection of chromosomes of wheat and *Leymus chinensis* and its usage, ZL201910474381.X, 20190530; Chen JY, Tang YQ, Yao LS, Wu H, Tu XY, Zhuang LF, Qi ZJ. Cytological and molecular characterization of Thinopyrum bessarabicumchromosomes and structural rearrangements introgressed in FISH analysis of wheat[J].MolecularBreeding,2019,39:146. showed that the blue-grained line had a chromosome number of 2n=6x=43, with the addition of an isomeric chromosome i4JL from *Wheatgrass spp.*, making it a monosomic addition line. Its exogenous chromosome exhibits unique fluorescent signal bands, unlike any wheat chromosome, and the two arms of the signal bands are identical, indicating it is a long-arm 4JL isomeric chromosome derived from the *Wheatgrass spp.* chromosome 4J. Since i4JL carries two copies of the blue-grain gene BaThb, seeds carrying this chromosome are deep blue with a uniform and stable color, easily distinguishable from red grains. This suggests that i4JL can be stably transmitted across generations, but it is difficult to produce homozygous stable disomydial addition lines. The non-blue-grained line plants all had a chromosome composition of 2n=6x=42, containing no exogenous chromosomes. Their grains were red, and except for the exogenous chromosome, the chromosomes of the blue-grained and non-blue-grained wheat lines were identical. Figure 1 The i4JL line is a strictly isogenetic line for blue and red grains, and is an important genetic material for studying the transmission of exogenous chromosomes, phenotypic effects and gene expression characteristics. It also provides an important tool for breeding isogenetic lines of blue and non-blue grain wheat with different backgrounds using i4JL.

[0030] Secondly, analysis of self-cross and backcross progeny clarified the genetic transmission characteristics of i4JL. Years of investigation showed that the seeds of the blue-grained line plants exhibited a segregation of blue and red grains, with blue seeds accounting for 27.05% (2013), 23.88% (2014), 26.78% (2023 field survey), and 29.14% (2023 greenhouse survey) of all seeds (Table 1). The blue seeds were darker and more uniform in color, showing a significant difference from non-blue seeds. Figure 1 Orthogonal experiments using MAi4JL as the female parent and Yangmai 158, Yangmai 6, and Yannong 19 showed that the transmission rate of i4JL through female gametes was 29.93% (Yangmai 158), 29.17% (Yangmai 6), and 31.56% (Yannong 19), respectively. The frequency of female gametes containing i4JL was significantly lower than that of normal female gametes. However, no blue seeds were found in the offspring of hybrids between MAi4JL and the three wheat varieties, indicating that the male gamete transmission rate of this chromosome is extremely low (Table 2). To further clarify the male gamete transmission rate of this chromosome, in 2023, a large number of hybrids were conducted using MAi4JL as the male parent and a K-type sterile line as the female parent, resulting in 839 hybrid seeds. It was determined that only 2 of these seeds contained i4JL, therefore its transmission rate through male gametes was only 0.24%.

[0031] Third, to clarify the phenotypic effect of i4JL, the traits of near-isogenic lines of MAi4JL were investigated in both field and greenhouse experiments. Four replicates were designed for the field experiment. Analysis of variance showed significant and highly significant differences between the blue-grained and red-grained lines in plant height, number of spikelets per panicle, number of grains per panicle, length of internodes from the top 1 to the bottom 4, and thousand-grain weight. The blue-grained line showed a highly significant reduction in plant height, a significant reduction in thousand-grain weight, and a significant or highly significant reduction in the length of internodes from the top 1 to the bottom 4, while having no significant effect on other traits. Compared to the red-grained line, the blue-grained line showed a 13.45% decrease in plant height (11.28 cm), mainly due to a decrease in the length of internodes from the top 1 to the bottom 4, and a 12.92% decrease in thousand-grain weight (4.31 g), but a 2.75% increase in the number of spikelets per panicle and a 16.01% increase in the number of grains per panicle (Table 3).

[0032] Under net-house conditions, 10 replicates were set up. Analysis of variance showed that, except for the first internode from the bottom which did not reach a significant value, there were significant and highly significant differences between the blue-grained and red-grained lines in plant height, number of spikelets per panicle, number of grains per panicle, second to fourth internodes from the bottom, and thousand-grain weight. The specific decreases are shown in Table 4.

[0033] Phenotypic identification in 14 replicates across two pilot trials showed that i4JL significantly or extremely significantly reduced plant height, length of the second to fourth internode from the top, thousand-grain weight, and increased the number of spikelets and grains per spike.

[0034] Fourth, to understand the impact of exogenous chromosome addition on gene expression, RNA-seq analysis of internodes in blue-grain wheat plants at the jointing stage revealed that the introduction of blue-grain monochromatograms significantly affected wheat gene expression. 668 differentially expressed genes were detected between the blue-grain and non-blue-grain lines, with 297 downregulated and 371 upregulated genes in the blue-grain line. GO enrichment analysis showed that the upregulated genes in wheat were mainly enriched in biological processes (BP) such as reactive oxygen species response and cellular stimulation by salicylic acid, while the downregulated genes were significantly enriched in BP such as gibberellin response and lignin biosynthesis. Furthermore, 2530 4JL-specific transcripts were identified in the blue-grain line. GO enrichment analysis indicated that these 4JL-specific transcripts were mainly enriched in BP such as heat shock response, messenger RNA transport, translational elongation, and peroxisome fission. Figure 2 ).

[0035] RNA-seq analysis of the aleurone layers of blue and non-blue seeds from blue-grained plants 21 days after flowering revealed that 1202 genes were upregulated and 408 genes were downregulated in the aleurone layer of blue-grained plants compared to the red-grained aleurone layer. These genes were mainly enriched in light-response-related BP entries and auxin activation signal-related BP entries, respectively. 2314 genes were specifically expressed in *Wheatgrass bisaenopsis* 4JL, among which the transcripts with the highest expression levels were all identified as bHLH-MYC transcription factors, and their sequences were consistent with the key factor ThbbHLH of the blue grain gene BaThb identified by Wang Qing (Wang Qing. DNA sequence analysis of the long arm of chromosome 4J of *Wheatgrass bisaenopsis* and its fine mapping of blue grain genes [D]. Nanjing: Nanjing Agricultural University, 2017.). The remaining transcripts were mainly enriched in entries related to phosphatidylserine biosynthesis, regulation of salt stress response, and light intensity response. Figure 3 ).

[0036] Finally, based on the transmission characteristics of i4JL, a method for breeding near-isogenic lines of blue-grained and non-blue-grained wheat with different genetic backgrounds using MAi4JL was established. MAi4JL was crossed with Yangmai 6, Yangmai 158, and Yannong 19 to obtain F1 hybrid seeds with segregated grain color. Blue-grained seeds were selected and planted separately. F6 hybrid seeds were obtained through continuous self-pollination. From each of the three combinations, one family was selected, and chromosome identification was performed on the blue-grained and non-blue-grained seeds. The blue-grained line contained one i4JL chromosome, while the non-blue-grained line did not contain any exogenous chromosome. Except for the presence or absence of an exogenous chromosome, the chromosome composition of the non-blue-grained line was identical to that of the blue-grained line. Figure 4Therefore, through continuous self-pollination, near-isogenic lines of blue-grained wheat were obtained in different backgrounds. Grain color survey showed that the occurrence frequency of blue-grained seeds in different wheat backgrounds was 30.23% (Yangmai 158), 22.11% (Yangmai 6), and 22.39% (Yannong 19) (Table 4). Ten biological replicates were planted for phenotypic surveys of blue-grained and non-blue-grained seeds respectively. The results showed that plant height showed a highly significant decreasing trend between blue-grained and non-blue-grained lines, while the decreasing or increasing trends of other traits were inconsistent in different backgrounds (Table 5).

[0037] The following examples illustrate preferred embodiments of the present invention, but the present invention is not limited thereto.

[0038] Example 1: Genetic transmission analysis of isoarm chromosome i4JL of *Leymus chinensis*.

[0039] Oligonucleotide probe kit #7 can produce abundant signals on the chromosomes of wheat and *Thinopyrum bessarabicum*, and can simultaneously distinguish all chromosomes of the two species (Qi Zengjun; Chen Jianyong; Tang Yuqing; Yao Lesha, An oligonucleotide probe kit for simultaneous detection of chromosomes of wheat and *Thinopyrum bessarabicum* and its usage, ZL201910474381.X, 20190530; Chen JY, Tang YQ, Yao LS, Wu H, Tu XY, Zhuang LF, Qi ZJ. Cytological and molecular characterization of *Thinopyrum bessarabicum* chromosomes and structural rearrangements introduced in wheat[J]. Molecular Breeding, 2019, 39:146.). Using this probe kit, the special blue-grained monosomy wheat line MAi4JL (Shen YF, Shen J, Dawa, Zhuang LF, Wang YZ, Pu J, Feng YG, Chu) bred by Nanjing Agricultural University was analyzed. CG, Wang XE, QiZJ. Physical localization of a novel blue-grained gene derived from Thinopyrum bessarabicum[J]. Mol Breeding, 2013, 31(1): 195-204) Chromosome FISH analysis of metaphase chromosomes in root tip cells of blue and red seeds after eight consecutive generations of self-pollination showed that the chromosome number of the red seed line was 2n = 6x = 42, containing no exogenous chromosomes, while the chromosome number of the blue seed line was 2n = 6x = 43, with the addition of an isochromosomal chromosome i4JL. Further karyotype analysis showed that the chromosomes of the red and blue seed lines were completely identical. Figure 1 The red-grained line is a strictly near-isogenic line. The seeds of the offspring of the red-grained line are all red, while the seeds of the offspring of the blue-grained line show a separation of blue and red grain colors.

[0040] To clarify the genetic transmission characteristics of the variant chromosome i4JL, the seed color segregation of MAi4JL self-pollinated progeny at the Nanjing Agricultural University Base (hereinafter referred to as Nanjing Baima) in the National Agricultural Science and Technology Park, Baima Town, Lishui District, Nanjing, was investigated in 2023. Four replicates were investigated in the field, with 10 plants in each replicate. The results showed that among the 27,722 seeds from the 40 individual plants investigated, blue and red seeds were segregated in all cases. No homozygous blue-seed plants were found, indicating that i4JL, which controls the blue seed trait, is unlikely to produce a homozygous disomic addition line DAi4JL through self-pollination. In the individual plants, the proportion of blue seeds averaged 26.78% of the total number of seeds, ranging from 14.78% to 33.88% (Table 1). The average proportions of blue seeds in the four replicates were 25.28%, 26.28%, 27.28%, and 28.28%, respectively, with the remainder being red seeds. Under the conditions of a net house, a total of 5 plants in each of the three replicates were investigated. Among the 7638 seeds in the 15 individual plants investigated, the average proportion of blue seeds was 29.14%, ranging from 24.25% to 34.06%. The average proportions of blue seeds in the three replicates were 29.03%, 29.02%, and 29.36%, respectively. The above results indicate that the proportion of blue seeds in the self-pollinated offspring of MAi4JL is much lower than that of red seeds. In other words, the proportion of n+1 gametes containing i4JL formed by MAi4JL is much lower than that of normal n gametes. This results in the i4JL transmission rate not conforming to Mendel's law of segregation. Since no homozygous blue-seed plants have been found in many years of planting practice, it is speculated that gametes containing i4JL may have the characteristic of parthenogenetic gamete transmission.

[0041] Table 1. Transmission of i4JL in near-isogenic lines under field conditions (Nanjing Baima, 2023)

[0042]

[0043]

[0044] To reveal the gamete transmission rate of i4JL, we designed a reciprocal cross experiment. In the offspring of crosses between MAi4JL as the female parent and Yangmai 158, Yangmai 6, and Yannong 19, we found that the transmission rates of i4JL through female gametes were 29.93%, 29.17%, and 31.56%, respectively (Table 2). However, in the offspring of crosses between MAi4JL as the male parent and Yangmai 158, Yangmai 6, and Yannong 19, no blue seeds were found, indicating that the transmission rate of i4JL through male gametes was extremely low. However, due to the small amount of hybrid seeds obtained in the previous stage (Table 2), in order to further clarify the transmission rate of this chromosome through male gametes, we used MAi4JL as the male parent and wheat K-type sterile lines as the female parent, and obtained 839 seeds through backcrossing. After identification by grain color and chromosome, only 2 plants were found to contain i4JL. Therefore, the transmission rate of i4JL through male gametes was only 0.24%. This result indicates that i4JL involves gene duplication and dosage effects, resulting in extremely low transmission rates in male gametes containing i4JL, and also in female gametes containing i4JL, which are far lower than normal gametes.

[0045] Table 2. Transmission rate of isoarm chromosome i4JL of *Bai Sa Yan* wheat in different wheat backgrounds.

[0046]

[0047] Example 2: Phenotypic Effect Analysis of the Isochromosome i4JL of *Leymus chinensis*

[0048] To clarify the phenotypic effect of i4JL, the traits of near-isogenic lines of MAi4JL were investigated in both field and greenhouse. In the field, four replicates were designed, with three rows per replicate and about 15 plants per replicate. The traits included plant height, number of effective tillers, panicle length, number of effective spikelets, number of ineffective spikelets, and number of grains per panicle. Analysis of variance showed that there were significant and highly significant differences between the blue-grained and red-grained lines in plant height, number of spikelets per panicle, number of grains per panicle, internodes from the top 1 to the top 4, and thousand-grain weight. The blue-grained line had a highly significant effect on reducing plant height, significantly reduced thousand-grain weight, and significantly or highly significantly reduced the length of internodes from the top 1 to the top 4, but had no significant effect on other traits. Compared to the red-grained series, the blue-grained series had a plant height decrease of 13.45% (11.28 cm), mainly due to a reduction in the length of the first to fourth internodes from the top. The thousand-grain weight decreased by 12.92% (4.31 g), but the number of spikelets per panicle increased by 2.75%, and the number of grains per panicle increased by 16.01% (Table 3).

[0049] Under net-house conditions, 10 replicates were set up, with each replicate consisting of approximately 10 plants. Analysis of variance showed that, except for the first internode from the bottom which did not show a significant decrease, there were significant and highly significant differences between the blue-grained and red-grained lines in plant height, number of spikelets per panicle, number of grains per panicle, second to fourth internodes from the bottom, and thousand-grain weight. The specific decreases are shown in Table 4.

[0050] Phenotypic identification of 14 replicates in two pilot trials showed that i4JL had a significant or highly significant effect on reducing plant height, lengths of the second to fourth internodes from the top, and increasing the number of spikelets per spike and grains per spike.

[0051] Table 3 Phenotypic traits of red-grained and blue-grained lines in the field of near-isogenic chromosome lines (Nanjing Baima, 2023)

[0052]

[0053] Numbers in the table are mean ± standard deviation. ** indicates p < 0.01, highly significant difference between blue and red lines; * indicates 0.01 < p < 0.05, significant difference between blue and red lines.

[0054] Table 4 Phenotypic traits of red-grained and blue-grained lines in the net chamber of near-isogenic chromosome lines (Nanjing Baima, 2023)

[0055]

[0056] Numbers in the table are mean ± standard deviation. ** indicates p < 0.01, highly significant difference between blue and red lines; * indicates 0.01 < p < 0.05, significant difference between blue and red lines.

[0057] Example 3. Effects of the isochromosome i4JL of Thinopyrum bessarabicum on wheat gene expression

[0058] To clarify the impact of adding the variant chromosome i4JL on wheat gene expression, transcriptome sequencing analysis was performed on near-isogenic lines of MAi4JL. Samples were collected at two time points. The first time point was the aleurone layer of the grains 21 days after flowering. Blue and red grains from single ears of the blue-grained line were collected, with three biological replicates of 10 grains each, for RNA extraction and transcriptome sequencing (NovaSeq 6000 platform, Illumina, USA). The second time point was the internodes at the jointing stage. The second internodes of the blue-grained and red-grained lines with similar growth in the field were collected, with three biological replicates of 10 internodes each, for RNA extraction and transcriptome sequencing (DNBSEQ-T7 platform, BGI, China).Using the Chinese spring genome (IWGSC v1.1) as the reference genome, HISAT2 v2.2.1 was used to align the clean data delivered by the company with default parameters. Subsequently, featureCounts v1.5.0-p3 was used with Triticum_aestivum.IWGSC.56.gtf as the reference, and quantification was performed based on the alignment results (Liao Y, Smyth GK, Shi W. FeatureCounts: an efficient general purpose program for assigning sequence reads to genomic features[J]. Bioinformatics, 2014, 30(7):923-930.). Differentially expressed genes were analyzed using the R package DESeq2 v1.20.0 (screening criteria: |log2(FoldChange)|>2, FDR<0.05) (Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2[J]. Genome Biology, 2014, 15(12): 550.), and finally, GO enrichment analysis (FDR < 0.05) was performed on the Triticeae-GeneTribe online website (http: / / wheat.cau.edu.cn / TGT) to detect differentially expressed genes (Chen Y, Song W, Xie X, et al. A Collinearity-Incorporating Homology Inference Strategy for Connecting Emerging Assemblies in the Triticeae Tribe as a Pilot Practice in the PlantPangenomic Era[J]. Molecular Plant, 2020, 13(12): 1694-1708). Using this method, 668 differentially expressed genes were detected in the blue and red lines at the second internode of the jointing stage, of which 297 genes were downregulated and 371 genes were upregulated in the blue line.The three most upregulated genes were TraesCS4A02G359800 (encoding lipoxygenases), TraesCS5B02G553900 (encoding a protein containing an F-box domain), and TraesCS4A02G141500 (encoding a SARDNA-binding protein). The most downregulated genes were TraesCS7A02G162700 (encoding a pathogen-related protein), TraesCS1B02G048100 (encoding phenylalanine ammonia-lyase, PAL), and TraesCS5D02G216400 (encoding a protein kinase). GO enrichment analysis revealed that the upregulated genes in wheat were mainly enriched in biological processes (BP) such as reactive oxygen species response and cellular response to salicylic acid stimulation, while the downregulated genes in wheat were significantly enriched in BP items such as gibberellin response and lignin biosynthesis. Figure 2 ).

[0059] Furthermore, 1610 differentially expressed genes were detected in the aleurone layer of blue and red seeds 21 days after flowering. Among them, 1202 genes were upregulated and 408 genes were downregulated in the blue seed line compared to the red seed line. The three genes with the highest upregulated expression were TraesCS4A02G039100 encoding Pumilio protein, TraesCS4D02G224600 encoding bHLH transcription factor, and TraesCS5B02G162800 encoding ribulose-1,5-bisphosphate carboxylase. These three genes are respectively related to seed maturation, anthocyanin synthesis, and photosynthesis. The gene with the highest downregulated expression, TraesCS6B02G014300, encodes an unknown protein, followed by TraesCS5B02G007100, which encodes cytochrome P450, and TraesCS6D02G370100, which encodes cytochrome C. GO enrichment analysis showed that upregulated genes were mainly enriched in light-responsive BP entries, while downregulated genes were mainly enriched in auxin activation signaling-related BP entries. Figure 3 ).

[0060] To extract transcripts specifically expressed by 4JL in the two time periods, SAMtools v1.5 (Petr D, James KB, Jennifer L, et al. Twelve years of SAMtools and BCFtools[J]. GigaScience, 2021, 10(2): giab008.) was used to remove all reads aligned to the reference gene. Then, Trinity v2.13.2 (Haas et al., 2013) was used to splice the filtered reads with default parameters, and BUSCO was used to analyze the results. FA, Waterhouse RM, Ioannidis P, et al. BUSCO: assessing genome assembly and annotation completeness with single-copy orthologs[J]. Bioinformatics, 2015, 31(19):3210-3212) The assembled transcripts were evaluated. Quantitative and differential expression analyses were performed using RSEM (Li et al., 2011b) and DEGseq2 (screening criteria: FDR < 0.05 and no expression in white grain samples). Differentially expressed transcripts were annotated using the online website eggNOG-mapper (http: / / eggnog-mapper.embl.de / ) (Huerta-Cepas et al., 2017). An OrgDb database was constructed based on the annotation results files using the R package AnnotationHub (https: / / bioconductor.org / packages / release / bioc / html / AnnotationHub.html). GO enrichment analysis was then performed on this sub-database using the R package ClusterProfiler (Yu G,Wang LG,Han Y,etal.clusterProfiler:An R package for comparing biological themes among geneclusters[J].OMICS:A Journal of Integrative Biology,2012,16(5):284-287.). Using this method, 2530 4JL-specific transcripts were identified in the blue grain line from internode samples at the jointing stage. GO enrichment analysis showed that these 4JL-specific transcripts were mainly enriched for BP items such as heat shock response, messenger RNA transport, translational elongation, and peroxisome fission. Figure 2Meanwhile, 2314 4JL-specific expression genes were screened from the aleurone layer of the grains 21 days after flowering. Among them, the transcripts with the highest expression levels were all identified as bHLH-MYC transcription factors, and their sequences were consistent with the key factor ThbbHLH of the blue grain gene BaThb identified by previous researchers. The remaining transcripts were mainly enriched with items related to phosphatidylserine biosynthesis, regulation of salt stress response, and response to light intensity. Figure 3 ).

[0061] Example 4: Breeding near-isogenic wheat lines using the isoarm chromosome i4JL of *Thinopyrum bisacodylon*.

[0062] Before flowering, MAi4JL was artificially emasculated. Two to three days later, pollen was collected from Yangmai 6, Yangmai 158, and Yannong 19 for pollination, yielding F1 hybrid seeds with segregated grain color. Blue-grained seeds were selected for cytological identification, confirming the presence of one i4JL chromosome. These blue-grained seeds were then planted, and a portion were allowed to self-pollinate. Each year, only blue-grained seeds were selected for further planting. Cytological identification was performed on seeds obtained after six generations of continuous self-pollination using an oligonucleotide probe (#7). The results showed that non-blue-grained lines did not contain the exogenous i4JL chromosome, while all blue-grained lines contained one i4JL chromosome. Figure 4 The blue-grained and non-blue-grained lines were identical in all chromosomes except for the presence or absence of i4JL, indicating that near-isogenic lines were obtained through continuous self-pollination. One F6 family was selected from the offspring of each of the three combinations. Ten replicates of blue-grained and non-blue-grained seeds were planted in one row per replicate, and phenotypic identification was conducted in the Nanjing Baima net greenhouse. First, three replicates were randomly selected from each combination, and five plants were randomly selected from each replicate to investigate grain color segregation. The results are shown in Table 5. All 45 investigated individual plants showed segregation between blue and non-blue grains. The proportion of blue grains in the near-isogenic lines of the three combinations was 30.23% (Yangmai 158), 22.11% (Yangmai 6), and 22.39% (Yannong 19), respectively, similar to the proportion of blue grains in the original near-isogenic line MAi4JL. This further illustrates the female gamete transmission characteristics of i4JL, making it difficult to produce homozygous DAi4JL. Secondly, using 10 replicates, 8-15 plants were investigated per replicate. The phenotypic results of the plants showed that, among the three pairs of near-isogenic lines bred from the three backgrounds, the plant height of the blue-grained lines was significantly lower than that of the non-blue-grained lines (Table 6), with reductions of 14.56% (17.95 cm) (Yangmai 158 background), 16.37% (17.63 cm) (Yangmai 6 background), and 24.55% (27.30 cm) (Yannong 19 background), respectively. Other traits showed inconsistent performance among the near-isogenic lines in different backgrounds (Table 6).

[0063] Table 5. Transmission statistics of i4JL in different wheat backgrounds

[0064]

[0065]

[0066] Table 6 Comparison of traits in near-isogenous lines with different wheat backgrounds

[0067]

[0068] The figures in the table are mean ± standard deviation. ** indicates p < 0.01, meaning the difference between the blue and red groups is highly significant; * indicates p < 0.01.

Claims

1. The application of the wheat-wheat isochromosome i4JL or wheat-wheat isochromosome i4JL monosomy supplementary line MAi4JL in the breeding of near-isogenic wheat lines, among which, The wheat near-isogenic line is a dwarf blue-grained wheat near-isogenic line with reduced thousand-grain weight.

2. The application of the wheat-wheat isochromosome i4JL or wheat-wheat isochromosome i4JL monosomy supplemental line MAi4JL in the preparation and breeding of near-isogenic wheat lines, wherein, The wheat near-isogenic line is a dwarf blue-grained wheat near-isogenic line with reduced thousand-grain weight.

3. Application of the isochromosome i4JL of *Triticum aestivum* or the wheat-*Triticum aestivum* isochromosome i4JL monosomy supplemental line MAi4JL in cereal crop breeding, among which, The wheat crop breeding program involves selecting and breeding short-stalked blue-grain wheat crops with reduced thousand-grain weight.

4. The application of the isochromosome i4JL of *Triticum aestivum* or the monosomy addition line MAi4JL of wheat-*Triticum aestivum* isochromosome i4JL in the preparation of products for cereal crop breeding, among which, The wheat crop breeding program involves selecting and breeding short-stalked blue-grain wheat crops with reduced thousand-grain weight.

5. The application according to claim 3 or 4, characterized in that, The cereal crops mentioned are wheat, rye, triticale, or malt.

6. The application according to claim 5, characterized in that, The wheat in question is common wheat, diploid wheat, or tetraploid wheat.

7. A method for breeding near-ischemic wheat lines using the wheat-wheatgrass isochromosome i4JL monosomy supplemental line MAi4JL, characterized in that, By hybridizing MAi4JL once and then continuously self-pollinating blue seeds, a near-isogenic line of dwarf blue-grained wheat with reduced thousand-grain weight and a near-isogenic line of non-blue-grained wheat can be bred.

8. The application of the method of claim 7 in the breeding of near-isogenic wheat lines and dwarf blue-grain wheat varieties or lines with reduced thousand-grain weight; in, The wheat near-isogenic line is a dwarf blue-grained wheat near-isogenic line with reduced thousand-grain weight.

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