Application of elytrigia besa equal-arm chromosome i4JL in breeding of wheat near-isogenic line

Abstract

The invention discloses a breeding method for breeding a wheat near-isogenic line by utilizing an elytrigia besa equal-arm chromosome i4JL, the i4JL has specific fluorescent stripes, is easy to distinguish from wheat chromosomes, is mainly transferred through female gametes in a wheat background, is extremely low in male gamete transfer rate, contains two doses of blue-grain genes, and can be used for breeding wheat near-isogenic lines. Therefore, the selfing progeny of the wheat-elytrigia besa i4JL monomer addition line MAi4JL always shows separation of blue grains and non-blue grains which are easy to sort, the MAi4JL is hybridized with any non-blue grain wheat, and after blue grain seeds are selected from the hybrid progeny and continuously planted for five generations, wheat blue grain and non-blue grain near-isogenic lines with different backgrounds and phenotypes are obtained. A new method is provided for the breeding of the near-isogenic line of wheat; due to the fact that i4JL obviously reduces the plant height in different wheat backgrounds and obviously affects gene expression in wheat stalks and grains, the i4JL has important application potential in wheat genetic breeding research and application.

Description

Technical Field

[0001] The invention discloses a breeding method for selecting wheat near-isogenic lines by utilizing the isochromosome i4JL of Thinopyrum bessacharum, and belongs to the fields of crop genetic breeding and agricultural biotechnology. Background Art

[0002] Distant hybridization is one of the important approaches to wheat breeding and plays a significant 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 select wheat addition lines with the same background. These addition lines can be compared with the background wheat to analyze the genetic effects of specific exogenous chromosomes and to discover and utilize exogenous genes. Therefore, the selection of wheat addition lines with the same background is one of the most important tasks in wheat distant hybridization breeding. Currently, the selection of wheat addition lines usually requires continuous backcrossing and selfing, 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 addition lines of common wheat and Thinopyrum bessarabicum [J]. Acta Genetica Sinica, 2003, 30(10): 919-925). Compared with the recurrent parent, the disomic addition line obtained by the backcross method has the same background as the recurrent parent except for a certain exogenous chromosome of the introduced wheat related species. It can be called a near-isogenic line. However, if the effect of the exogenous chromosome in different wheat backgrounds is to be analyzed, it is usually necessary to backcross with other recurrent parents 5-6 times and then self-pollinate to obtain near-isogenic lines with different backgrounds. However, these backgrounds are limited to the recurrent parent background and require multiple identifications and backcrossings. The procedure is complicated and the workload is large. Therefore, how to simply and efficiently breed wheat near-isogenic lines involving specific exogenous chromosomes in different backgrounds has important research and utilization value for discovering and utilizing 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 germplasms, providing important parents and tool materials for wheat genetic breeding (Zhuang Lifang, Qi Zengjun. Research and application progress of plant chromosome mutagenesis [J]. Journal of Nanjing Agricultural University, 2018, 41(1):15; Wang Qing, He Ziming, Wang Libin, Qi Zengjun. Application progress of chromosome engineering in hybrid wheat breeding [J]. Science Bulletin, 2022, 67(26):11.). Thinopyrum bessarabicum 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, Budrewicz 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.). At present, hexaploid and octoploid wheat and a series of additional lines (King IP, Forster BP, Law CC, Cant KA,

[0004] Orford SE, Gorham J, Reader S, Miller TE. Introduction of salt-tolerance genes from Thinopyrum bessarabicum into 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. bessarabicum) disomic alien addition 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 positioning analysis of small chromosome translocation fragments in Thinopyrum bessarabicum [D]. Nanjing: Nanjing Agricultural University, 2013; Patokar C, Sepsi A, Schwarzacher T, Kishii M, Heslop-Harrison JS. Molecular cytogenetic characterization of novel wheat-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, ShenYF, 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.), in wheat, blue aleurone wheat, i.e. blue grain wheat, is formed by controlling the synthesis of anthocyanins.Shen Yuefeng et al. initially named the blue-grained gene BaThb and located it on the long arm of chromosome 4J at FL0.52 from the centromere (Shen YF, Shen J, Dawa, Zhuang LF, Wang YZ, Pu J, Feng YG, ChuCG, Wang XE, Qi ZJ. Physical localization of a novel blue-grained gene derivedfrom Thinopyrum bessarabicum[J]. Mol Breeding, 2013, 31(1):195-204.); Pu Jing et al. bred a variety of variants involving chromosome 4J using radiation mutagenesis, and used 101 chromosome-specific molecular markers for physical mapping, dividing 4JS into 7 segments and 4JL into 17 segments, and further located BaThb in segment 4JL-11 (see references: 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.). The analysis showed that chromosome 4J of Thinopyrum bessarabicum and chromosome 4D of wheat had a high degree of collinearity (Pu Jing. Physical mapping of chromosome 4J of Thinopyrum bessarabicum and fine positioning of blue grain genes[D]. Nanjing: Nanjing Agricultural University, 2014.).In the wheat-Thinopyrum bessarabicum 4J heterochromatic system bred at Nanjing Agricultural University, the wheat-Thinopyrum bessarabicum isochromosome i4JL (also known as i4JL.4JL) monomeric addition line, abbreviated as MAi4JL, has an additional isochromosome 4JL. This is characterized by systematic molecular labeling, fluorescence in situ hybridization (FISH), and genomic in situ hybridization (GISH) (Shen YF, Shen J, Dawa, Zhuang LF, Wang YZ, Pu J, Feng YG, Chu CG, Wang XE, Qi ZJ. Physical mapping 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 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 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 a novel blue-grained gene derived from Thinopyrum bessarabicum[J]. Mol Breeding, 2013, 31(1):195-204; 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 the 4J chromosome of Elytrium bessapensum and fine positioning of its blue grain gene [D]. Nanjing: Nanjing Agricultural University, 2017.). This chromosome only involves the long arm of the 4J chromosome of Elytrium bessapensum and has specific banding patterns that are significantly different from other wheat chromosomes. This chromosome can be easily identified using the patented technology of Nanjing Agricultural University (Qi Zengjun; Chen Jianyong; Tang Yuqing; Yao Lesha, An oligonucleotide probe kit for simultaneous detection of wheat and Elytrium bessapensum chromosomes and its use, ZL201910474381.X, 20190530). Figure 1 ).

[0005] The present invention's research shows that this chromosome is mainly transmitted through female gametes, and the transmission rate is only about 1 / 4, while the male gamete transmission rate is extremely low, and no homozygous stable disomic addition line is produced. Therefore, the seeds of the MAi4JL self-pollinated offspring show obvious separation of blue and non-blue grains. Moreover, because the blue grain gene is endosperm-sensitive, the number of blue grain gene copies in the endosperm aleurone layer of the blue grain is 4, which is one more copy than the normal 4J chromosome disomic addition line. The seeds containing this chromosome are dark blue and stable, and are less affected by the environment ( Figure 1 Taking advantage of the fact that i4JL is primarily transmitted through female gametes and has a stable dark blue grain color, MAi4JL can be hybridized with any non-blue-grain wheat variety (multiple backcrosses can also be used to rapidly improve agronomic traits) to obtain a hybrid F1 with segregating grain color. Blue grains are then selected for planting and self-pollinated to obtain an F2. Blue grains are then selected for planting and self-pollinated until the F6 generation. The resulting grains are then planted separately as blue and non-blue grains to obtain near-isogenic lines with distinct grain color differences between blue and non-blue grains. Because genetic recombination in the offspring of hybrids between two parents can produce a rich variety of variants, i4JL can be used to select a series of near-isogenic lines with different phenotypes within the same combination based on breeding objectives. This method can be extended to multiple cross combinations. Compared with backcrossing, which can only select near-isogenic lines with specific backgrounds, this method utilizes the characteristics of i4JL, which has an extremely low male gamete transmission rate and the blue seed trait that is directly perceptible to the endosperm, to distinguish blue grains from non-blue grain seeds in the current hybrid generation. Therefore, wheat near-isogenic lines with different backgrounds can be bred quickly, simply and efficiently through self-pollination combined with grain sorting. At the same time, the process of continuous backcrossing and identification is omitted, saving manpower and material resources. Among the near-isogenic lines selected, the blue grain line has a darker blue grain color and a significantly lower plant height than the non-blue grain line, providing important tool materials for conventional wheat breeding and biological breeding. Summary of the Invention

[0006] In view of the shortcomings of the prior art, the present invention aims to provide a breeding method for selecting near-isogenic lines of wheat using the isochromosome i4JL of Thinopyrum bessacharum.

[0007] The technical solution adopted by the present invention to solve the technical problem is:

[0008] In a first aspect, the present invention protects the use of the isochromosome i4JL of Thinopyrum truncatum or the wheat-Thinopyrum truncatum isochromosome i4JL monomeric addition line MAi4JL in breeding wheat near-isogenic lines.

[0009] In a second aspect, the present invention provides an application of the Thinopyrum isochromosome i4JL or the wheat-Thinopyrum isochromosome i4JL monosomic addition line MAi4JL in the preparation of a product for breeding wheat near-isogenic lines.

[0010] In a specific embodiment, the wheat near-isogenic line is a dwarf blue-grain wheat near-isogenic line.

[0011] In a third aspect, the present invention protects the use of the isochromosome i4JL of Elytrigonum serratum or the wheat-Elytrigonum serratum isochromosome i4JL monosomic addition line MAi4JL in wheat crop breeding.

[0012] In a fourth aspect, the present invention protects the use of the isochromosome i4JL of Thinopyrum truncatum or the wheat-Thinopyrum isochromosome i4JL monosomic addition line MAi4JL in preparing products for wheat crop breeding.

[0013] In a specific embodiment, the wheat crop breeding is the breeding of dwarf blue-grain wheat crops.

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

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

[0016] In a fifth aspect, the present invention provides a method for breeding wheat near-isogenic lines using the wheat-Thinochloa baicalensis isochromosome i4JL monomeric addition line MAi4JL, wherein MAi4JL is hybridized once and blue-grain seeds are selected for continuous self-pollination, thereby breeding blue-grain wheat near-isogenic lines and non-blue-grain wheat near-isogenic lines.

[0017] In a sixth aspect, the present invention also protects the application of the method described above in breeding wheat near-isogenic lines, dwarf blue-grain wheat varieties or lines.

[0018] In a specific embodiment, the wheat near-isogenic line is a dwarf blue-grain wheat near-isogenic line.

[0019] Beneficial effects:

[0020] 1. The present invention discloses a breeding method for selecting a dwarf blue-grain wheat near-isogenic line using the wheat-Thinlygrain special monomeric addition line MAi4JL. This method does not require backcrossing. Instead, through a single hybridization, by selecting plants with the target phenotype, continuously self-pollinating, and planting blue-grain seeds, wheat near-isogenic lines with different traits can be bred. Furthermore, the grain color separation between the blue-grain line and the non-blue-grain line is clear, making sorting easy. Simultaneously, the plant height of the blue-grain line is significantly reduced. Therefore, this method provides a new tool and method for discovering and utilizing key genes in 4JL and creating new wheat germplasm.

[0021] 2. The dwarf blue-grain near-isogenic line bred by the breeding method disclosed in the present invention contains a special isochromosome i4JL of the thinleaf grass, and 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 skewed segregation of exogenous chromosome variations and their genetic effects, and provides important inspiration for the creation, discovery and utilization of special exogenous chromosome variations.

[0022] 3. The dwarf blue-grain near-isogenic lines bred using the breeding method disclosed in the present invention can be combined with genetic engineering. Because i4JL has a unique chromosome structure and is distantly related to wheat 4A, 4B, and 4D, it is less susceptible to recombination and has easily distinguishable grain color traits, which is conducive to the stable transmission of exogenous mutant 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 the present invention can be used as special wheat varieties. In particular, blue-grain seeds have more copies of the blue-grain gene and produce more anthocyanins, so they can be used for efficient anthocyanin separation and wheat nutritional breeding. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0025] Figure 2 Differentially expressed genes and GO enrichment analysis of the second internode of the MAi4JL near-isogenic line at the jointing stage; Figure 2 Figure A is a volcano plot showing differentially expressed genes in the stems of the blue and red lines; Figure 2 Panel B in the figure is a gene expression heat map; Figure 2 Figure C shows the GO enrichment of up-regulated genes in wheat; Figure 2 Figure D in the figure shows the GO enrichment of down-regulated genes in wheat; Figure 2 Figure E in the figure shows the GO enrichment of genes specifically expressed in 4JL;

[0026] Figure 3 Differentially expressed genes and GO enrichment analysis of the aleurone layer of blue-grain and non-blue-grain seeds produced by MAi4JL near-isogenic blue-grain lines 21 days after flowering; Figure 3 Figure A is a volcano plot of differentially expressed genes with blue and red particles; Figure 3 Panel B in the figure is the expression heat map of differentially expressed genes; Figure 3 Figure C shows the GO enrichment of up-regulated genes in wheat; Figure 3 Figure D in the figure shows the GO enrichment of down-regulated genes in wheat; Figure 3 Figure E in the figure shows the GO enrichment of genes specifically expressed in 4JL;

[0027] Figure 4 The isogenic chromosome i4JL of Thinopyrum baessa was used to assist in the breeding of wheat near-isogenic lines, among which, Figure 4 Figures A and B are the blue-grain and white-grain lines of MAi4JL / Yangmai 158F6, respectively. Figures C and D are the blue-grain and white-grain lines of MAi4JL / Yangmai 6F6. Figure 4 Figures E and F in the figure are the blue-grain and white-grain lines of MAi4JL / Yannong 19F6. In the figure, the blue-grain lines all contain one i4JL, and the non-blue-grain lines corresponding to the other wheat chromosomes are basically the same. Continuing to select blue-grain individual plants for self-pollination can isolate more strictly near-isogenic lines. DETAILED DESCRIPTION

[0028] The present invention is further described in detail below with reference to the examples. Reagents or instruments used without manufacturer's indication are considered to be conventional products that can be purchased on the market.

[0029] First, Nanjing Agricultural University created a monosomic addition line on the isochromosome i4JL of wheat and Thinopyrum baessa through distant hybridization and systematic molecular marker, FISH, and GISH identification. This line, named MAi4JL, has normal fertility. After eight generations of continuous self-pollination, with blue seeds selected for planting each time, the selfed progeny still showed color segregation, with blue and red seeds, respectively. The blue and red seed lines exhibited consistent traits within the lineage, and except for a significant difference in plant height, all other traits were similar. Using reference to the Chinese Spring and Thinopyrum baessa reference karyotypes, oligonucleotide probe set #7 (Qi Zengjun; Chen Jianyong; Tang Yuqing; Yao Lesha, An oligonucleotide probe kit for simultaneous detection of wheat and Thinopyrum chromosomes and its use, 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 wheat[J].Molecular Breeding,2019,39:146.) FISH identification showed that the blue-grain line had a chromosome number of 2n=6x=43, with an additional isomeric chromosome i4JL from Triticum aestivum, which was a monosomic addition line. Its exogenous chromosome had a specific fluorescent signal band, which was different from any other wheat chromosome, and the signal bands on both arms were exactly the same. It was the long-arm 4JL isomeric chromosome derived from the 4J chromosome of Triticum aestivum. Since i4JL carries two copies of the blue-grain gene BaThb, the seeds carrying this chromosome are dark blue and the color is uniform and stable, which can be easily distinguished from red grains. This shows that i4JL can be stably transmitted between generations, but it is difficult to produce a homozygous and stable disomic addition line. The chromosome composition of non-blue-grain line plants is 2n=6x=42, without any exogenous chromosomes, and the grains are red. Except for the exogenous chromosomes, the blue-grain and non-blue-grain lines have exactly the same chromosomes ( Figure 1 ), which are strictly blue-grain and red-grain near-isogenic lines, are important genetic materials for studying the genetic transmission, phenotypic effects and gene expression characteristics of exogenous chromosomes. They also provide an important tool for breeding blue-grain and non-blue-grain near-isogenic lines of wheat with different backgrounds using i4JL.

[0030] Secondly, the genetic transmission characteristics of i4JL were clarified by analyzing the progeny of self-crosses and backcrosses. Years of investigation showed that the seeds of the offspring of blue-grain plants were separated into blue and red seeds. Among them, the proportion of blue seeds in all seeds was 27.05% (2013), 23.88% (2014), 26.78% (2023 field) and 29.14% (2023 greenhouse) (Table 1). The blue seeds were dark and uniform in color, which was significantly different from non-blue seeds ( Figure 1 Orthogonal experiments using MAi4JL as the female parent with Yangmai 158, Yangmai 6, and Yannong 19 showed that the transmission rates of i4JL through female gametes were 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. No blue-grained seeds were found in the hybrid offspring with MAi4JL as the male parent 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, a large number of hybrids were carried out in 2023 using MAi4JL as the male parent and the K-type sterile line as the female parent to obtain 839 hybrid seeds. After identification, only 2 seeds containing i4JL were found, indicating that its transmission rate through male gametes was only 0.24%.

[0031] Third, to clarify the phenotypic effects of i4JL, traits of MAi4JL near-isogenic lines were investigated in the field and in a greenhouse. Four replicates were used in the field. Analysis of variance revealed significant and extremely significant differences between the blue- and red-grain lines in plant height, number of spikelets per ear, number of grains per ear, internodes 1–4, and 1000-grain weight. The blue-grain line had extremely significant reductions in plant height, 1000-grain weight, and significantly or extremely significant reductions in internode length 1–4, with no significant effects on other traits. Compared with the red-grain line, the blue-grain line had a 13.45% (11.28 cm) decrease in plant height, primarily due to a decrease in internode length 1–4, and a 12.92% (4.31 g) decrease in 1000-grain weight. However, the number of spikelets per ear increased by 2.75%, and the number of grains per ear increased by 16.01% (Table 3).

[0032] Under the conditions of the net house, 10 replicates were set up, and the variance analysis showed that except for the internode from the bottom 1, which did not reach significance, there were significant and extremely significant differences in plant height, number of spikelets per ear, number of grains per ear, internodes from the bottom 2 to the bottom 4, and 1000-grain weight between the blue-grain and red-grain lines. The specific decrease is shown in Table 4.

[0033] Phenotypic identification of 14 replicates in two pilot sites showed that i4JL had the effect of significantly or extremely significantly reducing plant height, length of the 2nd to 4th internodes, and 1000-grain weight, and increasing the number of spikelets per panicle and the number of grains per panicle.

[0034] Fourth, to understand the effect of exogenous chromosome addition on gene expression, RNA-seq analysis was performed on the internodes of blue-grain plants at the jointing stage. It was found that the introduction of blue-grain monomer chromosomes had an important effect on wheat gene expression. Among them, 668 differentially expressed genes were detected in the blue-grain line and the non-blue-grain line, of which 297 were down-regulated genes and 371 were up-regulated genes in the blue-grain line. GO enrichment analysis found that the up-regulated genes in wheat were mainly enriched in biological process (BP) items such as reactive oxygen species response and cell stimulation to salicylic acid, while the down-regulated genes in wheat were significantly enriched in BP items such as gibberellin response and lignin biosynthesis. At the same time, 2530 4JL-specifically expressed transcripts were identified in the blue-grain line. GO enrichment analysis showed that these 4JL-specifically expressed transcripts were mainly enriched in BP items such as heat shock response, messenger RNA transport, translation elongation and peroxisome fission ( Figure 2 ).

[0035] RNA-seq analysis of the aleurone layer of blue-grain and non-blue-grain seeds produced by blue-grain plants 21 days after flowering revealed that the aleurone layer of blue-grain plants had 1202 upregulated genes and 408 downregulated genes compared with the aleurone layer of red-grain seeds, which were mainly enriched in light response-related BP items and auxin activation signal-related BP items, respectively. There were 2314 genes specifically expressed in the 4JL of Thinopyrum baessa, among which the transcripts with the highest expression levels were all annotated 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 Thinopyrum baessa and fine positioning of blue-grain genes [D]. Nanjing: Nanjing Agricultural University, 2017.). The remaining transcripts were mainly enriched in phosphatidylserine biosynthesis process, regulation of salt stress response and response to light intensity ( Figure 3 ).

[0036] Finally, based on the transmission characteristics of i4JL, a method for breeding blue-grain and non-blue-grain wheat near-isogenic lines with different genetic backgrounds using MAi4JL was established. Hybrid F1 seeds with segregated grain color were obtained by hybridizing MAi4JL with Yangmai 6, Yangmai 158, and Yannong 19. Blue-grain seeds were selected and planted separately, and hybrid F6 seeds were obtained through continuous self-pollination. Chromosome identification of blue-grain and non-blue-grain seeds from one family of each of the three combinations revealed that the blue-grain line contained one i4JL chromosome, while the non-blue line did not contain any exogenous chromosomes. Their chromosome composition was identical except for the presence or absence of exogenous chromosomes. Figure 4), blue-grained near-isogenic lines with different backgrounds were obtained through continuous self-pollination. Grain color surveys showed that the frequencies of blue-grained seeds in different wheat backgrounds were 30.23% (Yangmai 158), 22.11% (Yangmai 6), and 22.39% (Yannong 19), respectively (Table 4). Ten biological replicates of blue-grained and non-blue-grained seeds were planted for phenotypic investigation. 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 Isochromosome i4JL in Thinopyrum baicalensis

[0039] Oligonucleotide probe set #7 can generate abundant signals on the chromosomes of wheat and Thinopyrum bessarabicum and can distinguish all chromosomes of the two species simultaneously (Qi Zengjun; Chen Jianyong; Tang Yuqing; Yao Lesha, An oligonucleotide probe kit for simultaneous detection of wheat and Thinopyrum bessarabicum chromosomes and its use, 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.). This probe set was used to characterize the blue-grained monosomic addition line MAi4JL of wheat bred by Nanjing Agricultural University (Shen YF, Shen J, Dawa, Zhuang LF, Wang YZ, Pu J, Feng YG, Chu CG, Wang XE, QiZJ. Physical localization of a novel blue-grained gene derived from Thinopyrum bessarabicum[J]. Mol Breeding, 2013, 31(1):195-204) FISH analysis of the root tip cells of blue-grained and red-grained seeds from eight generations of continuous self-pollination showed that the chromosome number of the red-grained line was 2n=6x=42 and did not contain any exogenous chromosomes, while the chromosome number of the blue-grained line was 2n=6x=43 and had an additional isochromosome i4JL of Thinopyrum bessarabicum. Further karyotype analysis showed that the wheat chromosomes of the red-grained and blue-grained lines were exactly the same ( Figure 1 ), which is a strict near-isogenic line. The seeds of the self-pollinated offspring of the red-grain line are all red, while the seeds of the self-pollinated offspring of the blue-grain line show blue and red color separation.

[0040] To clarify the genetic transmission characteristics of the variant chromosome i4JL, the color segregation of seeds of self-pollinated progenies of MAi4JL planted in the field and in a net house at the Nanjing Agricultural University Base, National Agricultural Science and Technology Park, Baima Town, Lishui District, Nanjing (hereinafter referred to as Nanjing Baima) in 2023 was investigated. A total of 4 replicates were investigated in the field, with 10 plants investigated in each replicate. The results showed that among the 40 plants surveyed, totaling 27,722 seeds, all showed segregation of blue and red grains, and no homozygous blue-grained plant was found, indicating that i4JL controlling the blue-grain trait is difficult to produce a homozygous disomical addition line DAi4JL through self-pollination. Among the individual plants, the proportion of blue seeds in the total number of seeds was an average of 26.78%, with a range of 14.78% to 33.88% (Table 1). The average proportion of blue seeds in the four replicates was 25.28%, 26.28%, 27.28% and 28.28%, respectively, and the rest were red seeds. Under the net house conditions, three replicates of 5 plants in each were investigated. Among the 15 individual plants with a total of 7638 seeds, the average proportion of blue seeds was 29.14%, with a range of 24.25% to 34.06%. The average proportion of blue seeds in the three replicates was 29.03%, 29.02% and 29.36%, respectively. The above results show that the proportion of blue-grain seeds in the self-pollinated offspring of MAi4JL is much lower than that of red-grain seeds, that is, the proportion of n+1 gametes containing i4JL formed by MAi4JL is far less than that of normal n gametes, resulting in the transmission rate of i4JL not conforming to Mendel's law of segregation. Since no homozygous blue-grained individual plants have been found in many years of planting practice, it is speculated that the gametes containing i4JL may have the characteristics of parthenogametic transmission.

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

[0042]

[0043]

[0044] To reveal the gamete transmission rate of i4JL, we designed a reciprocal cross experiment. In the progeny of hybrids using 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). In contrast, no blue grains were found in the progeny of hybrids using MAi4JL as the paternal parent and Yangmai 158, Yangmai 6, and Yannong 19, indicating that the transmission rate of i4JL through male gametes was extremely low. However, due to the limited amount of hybrid seeds obtained in the early stage (Table 2), to further clarify the male gamete transmission rate of this chromosome, 839 seeds were obtained through backcrossing using MAi4JL as the paternal parent and a wheat K-type sterile line as the female parent. Grain color and chromosome identification revealed that only two seeds contained i4JL, resulting in a male gamete transmission rate of only 0.24%. This result shows that due to the duplication and dosage effect of the gene involved, the transmission rate of male gametes containing i4JL is extremely low, and the transmission rate of female gametes containing i4JL is also far lower than that of normal gametes.

[0045] Table 2 Transmission rates of isochromosome i4JL of Thinopyrum baicalensis in different wheat backgrounds

[0046]

[0047] Example 2: Analysis of the phenotypic effects of chromosome i4JL in Thinopyrum baicalensis

[0048] To clarify the phenotypic effects of i4JL, the traits of the MAi4JL near-isogenic line were investigated in the field and in a greenhouse. Four replicates, each with three rows, were designed in the field, and approximately 15 plants were investigated in each replicate. The traits, including plant height, number of effective tillers, panicle length, number of effective spikelets, number of ineffective spikelets, and number of grains per spike, were investigated. Analysis of variance showed that there were significant and extremely significant differences between the blue-grain and red-grain lines in plant height, number of spikelets per spikelet, number of grains per spikelet, internodes from the top 1 to the top 4, and 1000-grain weight. The blue-grain line had the effect of extremely significantly reducing plant height, significantly reducing 1000-grain weight, and significantly or extremely significantly reducing the length of the internodes from the top 1 to the top 4, but had no significant effect on other traits. Compared with the red-grain line, the plant height of the blue-grain line decreased by 13.45% (11.28 cm), mainly due to the reduction in the length of the internodes from the top 1 to the top 4. The 1000-grain weight decreased by 12.92% (4.31 g), but the number of spikelets per ear increased by 2.75%, and the number of grains per ear increased by 16.01% (Table 3).

[0049] Under the conditions of the net house, 10 replicates were set up, with one row in each replicate and about 10 plants surveyed in each replicate. The variance analysis showed that except for the decrease in the internode from the bottom, which was not significant, there were significant and extremely significant differences in plant height, number of spikelets per ear, number of grains per ear, internodes from the bottom 2 to the bottom 4 and 1000-grain weight between the blue grain series and the red grain series. The specific decrease is 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, the lengths of the second to fourth internodes from the top, and increasing the number of spikelets per spike and the number of grains per spike.

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

[0052]

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

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

[0055]

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

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

[0058] To clarify the effects of the addition of the mutant chromosome i4JL on wheat gene expression, transcriptome sequencing was performed on the MAi4JL near-isogenic line. Samples were collected from the aleurone layer 21 days after anthesis. Three biological replicates of 10 blue and red kernels were collected from individual ears of the blue-grain line, each for RNA extraction and transcriptome sequencing (NovaSeq 6000 platform, Illumina, USA). The second sample was collected from internodes at the jointing stage of the stem. Three biological replicates of 10 internodes were collected from the second internode of the jointing stage of the blue-grain line and the red-grain line, each grown in the field, for RNA extraction and transcriptome sequencing (DNBSEQ-T7 platform, BGI, China).The clean data provided by the company were aligned using the Chinese Spring (IWGSC v1.1) reference genome with HISAT2 v2.2.1 using default parameters. FeatureCounts v1.5.0-p3 was then used with Triticum_aestivum.IWGSC.56.gtf as a reference for quantification 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.). The 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 differentially expressed genes using the Triticeae-GeneTribe (http: / / wheat.cau.edu.cn / TGT) online website (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 Plant Pangenomic Era[J]. Molecular Plant, 2020, 13(12): 1694-1708). Using this method, 668 differentially expressed genes were detected in the blue and red lines of the second internode of the jointing stage, of which 297 were down-regulated genes and 371 were up-regulated genes in the blue line.The three most upregulated genes were TraesCS4A02G359800, encoding lipoxygenases; TraesCS5B02G553900, encoding an F-box domain-containing protein; and TraesCS4A02G141500, encoding a SAR DNA-binding protein. The most downregulated genes were TraesCS7A02G162700, encoding a pathogen-related protein; TraesCS1B02G048100, encoding a phenylalanine ammonia-lyase (PAL); and TraesCS5D02G216400, encoding a protein kinase. GO enrichment analysis revealed that the upregulated genes in wheat were primarily enriched in biological process (BP) terms such as reactive oxygen species response and cellular stimulation with salicylic acid, while the downregulated genes were significantly enriched in BP terms such as gibberellin response and lignin biosynthesis. Figure 2 ).

[0059] Furthermore, 1,610 differentially expressed genes were detected in the blue and red lines of the grain aleurone layer 21 days after anthesis. Of these, 1,202 genes were upregulated and 408 genes were downregulated in the blue line relative to the red line. The three most highly upregulated genes were TraesCS4A02G039100, encoding the Pumilio protein; TraesCS4D02G224600, encoding the bHLH transcription factor; and TraesCS5B02G162800, encoding the ribulose-1,5-bisphosphate carboxylase enzyme. These genes are associated with seed maturation, anthocyanin synthesis, and photosynthesis, respectively. The gene with the highest down-regulated expression, TraesCS6B02G014300, encodes an unknown protein, followed by TraesCS5B02G007100, which encodes cytochrome P450, and TraesCS6D02G370100, which encodes cytochrome C. GO enrichment analysis showed that the up-regulated genes were mainly enriched in light response-related BP entries, while the down-regulated genes were mainly enriched in auxin activation signal-related BP entries ( Figure 3 ).

[0060] In order to mine the transcripts specifically expressed in 4JL during the two 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 further used to remove all reads aligned to the reference gene, and then Trinity v2.13.2 (Haas et al., 2013) was used to splice the filtered reads under the default parameters. The BUSCO ( 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) assessed the assembled transcripts. Quantification and differential expression analysis were performed using RSEM (Li et al., 2011b) and DEGseq2 (screening criteria: FDR < 0.05 and no expression in white grain samples). The differentially expressed transcripts were annotated using the eggNOG-mapper (http: / / eggnog-mapper.embl.de / ) online website (Huerta-Cepas et al., 2017). The OrgDb database (https: / / bioconductor.org / packages / release / bioc / html / AnnotationHub.html) was constructed based on the annotation result files using the R package AnnotationHub. GO enrichment analysis was performed based on this database using the R package ClusterProfiler (Yu G, Wang LG, Han Y, et al. clusterProfiler: An R package for comparing biological themes among gene clusters [J]. OMICS: A Journal of Integrative Biology, 2012, 16(5): 284-287.). Using this method, 2530 4JL-specifically expressed transcripts were identified in the blue-grained lines of internode samples at the jointing stage. GO enrichment analysis showed that these 4JL-specific expressed transcripts were mainly enriched in BP terms such as heat shock response, messenger RNA transport, translation elongation, and peroxisome fission ( Figure 2At the same time, 2314 4JL-specific expression genes were screened in the aleurone layer of the grains 21 days after flowering. Among them, the transcripts with the highest expression levels were all annotated as bHLH-MYC transcription factors, and their sequences were consistent with the blue grain gene BaThb key factor ThbbHLH identified by previous researchers. The remaining transcripts were mainly enriched in items such as phosphatidylserine biosynthesis process, regulation of salt stress response, and response to light intensity ( Figure 3 ).

[0061] Example 4: Breeding wheat near-isogenic lines using the isochromosome i4JL of Thinopyrum baicalensis

[0062] MAi4JL was artificially emasculated before flowering. 2-3 days later, pollen from Yangmai No. 6, Yangmai 158, and Yannong 19 was collected and pollinated to obtain F1 hybrid seeds with segregated grain color. Cytological identification of blue-grained seeds confirmed that they contained one i4JL chromosome. These blue-grained seeds were then planted separately, and a portion of them were self-pollinated. Only blue-grained seeds were selected for continued planting each year. Cytological identification of seeds obtained after six consecutive selfing generations using oligonucleotide probe set #7 revealed that non-blue-grained lines did not contain the exogenous i4JL chromosome, while blue-grained lines all contained one i4JL chromosome. Figure 4 ), and the blue grain line and the non-blue grain line have exactly the same wheat chromosomes except for the presence of i4JL, and are near-isogenic lines obtained through continuous self-pollination. One F6 family was selected from the offspring of each of the three combinations, and 10 replicates of blue grain seeds and non-blue grain line seeds were planted, with 1 row per replicate, and the traits were identified in the Baima net room in Nanjing. First, three replicates were randomly selected from each combination, and 5 plants were randomly selected from each replicate to investigate the grain color segregation. The results are shown in Table 5. The grain color of the 45 individual plants investigated showed segregation between blue grains and non-blue grains, of which the proportion of blue grains in the near-isogenic lines of the three combinations was 30.23% (Yangmai 158), 22.11% (Yangmai No. 6), and 22.39% (Yannong 19), respectively, which is similar to the proportion of blue grains in the original near-isogenic line MAi4JL, further illustrating the female gamete transmission characteristics of i4JL, which makes it difficult to produce homozygous DAi4JL; secondly, 10 replicates were used to investigate 8-15 The phenotypic investigation results of the three pairs of near-isogenic lines bred from three backgrounds showed that the plant height of the blue-grain lines was significantly lower than that of the non-blue-grain lines (Table 6). The reduction rates were 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. The other traits showed inconsistent performance among the near-isogenic lines with different backgrounds (Table 6).

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

[0064]

[0065]

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

[0067]

[0068] The numbers in the table are mean ± standard deviation, ** indicates p < 0.01, and the difference between the blue and red series is extremely significant; * indicates 0.01

Claims

1. Application of the isochromosome i4JL of Thinopyrum baicalensis or the wheat-Thinopyrum baicalensis isochromosome i4JL monosomic addition line MAi4JL in breeding wheat near-isogenic lines.

2. Application of the isochromosome i4JL of Thinopyrum baicalensis or the wheat-Thinopyrum baicalensis isochromosome i4JL monomeric addition line MAi4JL in the preparation and breeding of wheat near-isogenic lines.

3. The use according to claim 1 or 2, characterized in that The wheat near-isogenic line is a dwarf blue-grain wheat near-isogenic line.

4. Application of the isochromosome i4JL of Elytrigonum truncatum or the wheat-Elytrigonum truncatum isochromosome i4JL monosomic addition line MAi4JL in wheat crop breeding.

5. Application of the isochromosome i4JL of Elytrigia baicalensis or the wheat-Elytrigia baicalensis isochromosome i4JL monosomic addition line MAi4JL in preparing products for wheat crop breeding.

6. The use according to claim 4 or 5, characterized in that The wheat crop breeding is to breed short-stalked blue-grain wheat crops.

7. The use according to claim 4 or 5, characterized in that The wheat crops are wheat, rye, triticale or triticale.

8. The use according to claim 7, characterized in that The wheat is common wheat, diploid wheat or tetraploid wheat.

9. A method for breeding wheat near-isogenic lines using the wheat-Thinlygrass isochromosome i4JL monosomic addition line MAi4JL, characterized in that: By hybridizing MAi4JL once and then selecting blue-grain seeds for continuous self-pollination, blue-grain wheat near-isogenic lines and non-blue-grain wheat near-isogenic lines can be bred.

10. Use of the method according to claim 9 in breeding wheat near-isogenic lines, dwarf blue-grain wheat varieties or lines; Preferably, the wheat near-isogenic line is a dwarf blue-grain wheat near-isogenic line.

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