A brassinosteroid-6-oxidase 1 mutein of compact plant type puccinellia and use thereof

Abstract

The application discloses a compact type of euphorbia humilis brassinosteroid-6-oxidase 1 mutant protein and application thereof, and belongs to the technical field of crop genetic breeding. The mutation mode of the brassinosteroid-6-oxidase 1 mutant protein is that tryptophan at the 437th position is mutated into a stop codon, so that the protein translation is prematurely terminated. The compact type of euphorbia humilis mutant is obtained through chemical mutagenesis, and the euphorbia humilis mutant obtained after mutation is characterized in that the leaf tip thorn is short, the hypocotyl is short, the plant height is reduced, the leaf width, the leaf area and the leaf density are all significantly increased, and the ear density is increased, so that the overall plant type structure is more compact, and the plant is more suitable for cultivation. Therefore, the mutant can be used as new germplasm material for plant type improvement, and has a good application prospect.

Description

Technical Field

[0001] This invention relates to the field of crop genetics and breeding technology, and more specifically, to a brassinolide-6-oxidase 1 mutant protein of a compact plant species, *Potentilla anserina*, and its application. Background Technology

[0002] sand bon ( Agriophyllum squarrosum (L.) Moq.) is an annual herbaceous plant belonging to the genus *Lysimachia* in the broad family Amaranthaceae. As a pioneer plant for shifting sand dunes, *Lysimachia* exhibits excellent resistance to adversity, drought, and poor soil conditions. The establishment of its population can lay the material environmental foundation for dune vegetation succession and ecological restoration. Before flowering, *Lysimachia* plants are highly palatable, making them an important forage in arid regions. *Lysimachia* seeds are nutritionally complete and balanced, have a history of consumption spanning over a thousand years, and are a famous local delicacy in the Hexi Corridor and Alashan Plateau of northern China, showing great potential in the green functional food market. Therefore, *Lysimachia* can be introduced, domesticated, and genetically improved as an environmentally friendly future crop to fully utilize sandy land resources and promote ecological restoration and sustainable development in arid areas.

[0003] Over a long period of adaptation to the wind erosion and sand burial environment of shifting sand dunes, *Arundinaria salsa* has evolved a loose plant structure. This structure includes the preferential development of four lateral branches at the base, with the apical dominance of the main stem not being obvious in the early stages. The lateral branches are well-developed and extend outwards, and the hypocotyl can plastically elongate to cope with sand burial. While this structure is important for seedling emergence and survival during the windy spring season when sand covers the plants, it also easily causes lodging, which is detrimental to field cultivation. In addition, the sharp spines formed at the leaf tips of *Arundinaria salsa*, especially the spines of the hard leaves in the mature stage, can serve as a defensive trait to prevent animal grazing, but they severely affect the artificial harvesting of seeds. Therefore, improving the traits of wild weeds and selecting strains with compact plant structures suitable for field cultivation are the main goals of the current introduction, domestication, and genetic improvement of *Arundinaria salsa*.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] The purpose of this invention is to provide a brassinolide-6-oxidase 1 mutant protein of compact plant type *Aquilaria sinensis* and its application. Using the mutant protein provided by this invention, *Aquilaria sinensis* plants with traits such as short hypocotyl, dwarfed plants, compact plant type, and lodging resistance can be bred, providing a new approach for the selection of suitable *Aquilaria sinensis* varieties.

[0006] This invention is implemented as follows: In a first aspect, the present invention provides a brassinolide-6-oxidase 1 mutant protein of a compact plant type of *Erigeron breviscapus*, the brassinolide-6-oxidase 1 mutant protein having the following mutation: W437(TGG)→Stop(TAG), the amino acid sequence of which is shown in SEQ ID NO.3.

[0007] This invention utilizes ethyl methanesulfonate (EMS) mutagenesis to construct a mutant library of *Ligustrum lucidum*, from which mutants exhibiting dwarfing, short hypocotyls, and compact plant structure were screened (and named as follows). dc1 To investigate the cause of the above-mentioned mutants, this invention first clarifies that the mutant phenotype is caused by a single-gene recessive mutation. Based on this, a genetic population was constructed, and through sequencing localization and map-based cloning, the mutation site was precisely located between the molecular markers Indel_7212684 and Indel_7614904 on chromosome 9 of *Sapium sebiferum*, with a genetic distance of 1.2 cM. It was found that the molecular marker dCAPS_7404132 was tightly linked to and co-segregated with the mutant phenotype. Based on the single nucleotide polymorphism (SNP) effect, the SNP of the molecular marker dCAPS_7404132 was found to be a non-synonymous mutation, and the gene EVM0006946 containing this molecular marker was preliminarily identified as... dc1 The target gene of the mutant was identified. Secondly, a new F2 mapping population was constructed to further confirm the target gene. dc1 The phenotypic variation in the mutant was caused by a single-gene recessive mutation. Whole-genome resequencing was used to screen SNPs and Indels based on Mendelian segregation. Both variations showed significant clustering at positions 0-20 Mbp on chromosome 9, validating the localization interval obtained by the first method. Molecular markers were redesigned based on the SNP and Indel variation information within the interval. Further map-based cloning was conducted using recombinant plants obtained through the first method, ultimately narrowing the localization interval to between molecular markers Indel_7212684 and Indel_7526517. The molecular marker dCAPS_7404132, located within this interval, remained tightly linked and co-segregated with the mutant phenotype in this mapping population. Comparison with the *Prunus armeniaca* reference genome revealed that the localization interval contained 10 coding genes. Only the SNPs corresponding to dCAPS_7404132 were non-synonymous mutations, ruling out the possibility of mutations in other gene coding regions. This confirmed that the EVM0006946 gene from which dCAPS_7404132 originates is... dc1 The target gene of the mutant. Third, transcriptome sequencing analysis of the 10 coding genes within the localization region revealed that only EVM0006946 showed significant expression differences between wild-type and mutant plants; other genes showed no statistically significant differences. Therefore, the EVM0006946 gene is the target gene. dc1The conclusion that the mutant targets a specific gene provides molecular evidence at the gene expression level. Fourth, through years of indoor and field cultivation experiments and phenotypic observations, it was confirmed that the mutant and the wild type have stable and heritable differences in plant structure and plant height. Based on the above evidence, the mutant gene was finally identified as originating from the EVM0006946 gene on chromosome 9, encoding brassinolide-6-oxidase 1 (AsBR6ox1). The specific mutation mode is: a GA transition occurs at the 3254th base of the 8th exon in the coding region of the EVM0006946 gene, causing the codon encoded at that position to change from TGG to TGA, and the corresponding amino acid mutation is tryptophan W437 becoming a stop codon. The amino acid sequence of this mutant is shown in SEQ ID NO.3.

[0008] This invention provides a technical approach to effectively shorten the breeding cycle of *Scutellaria barbata* by constructing a mutant library through chemical mutagenesis, followed by targeted phenotypic variation screening, genetic analysis, gene cloning, and field evaluation. This method integrates genetic analysis, whole-genome resequencing localization, map-based cloning, and transcriptome sequencing. Based on the phenotypic segregation ratio of multiple independent segregating populations, it determines that the mutation is controlled by a single-gene recessive mutation. By cross-validating two independent populations, candidate regions are accurately located. Co-segregating molecular markers are obtained using map-based cloning. Unique candidate genes are screened based on the base variation effect, and cross-validation is performed using expression differences and stable phenotypes, forming a highly efficient, low-false-positive strategy suitable for identifying mutant genes in plants.

[0009] The above mutant was obtained by mutating the amino acid sequence shown in SEQ ID NO.1. The mutation mode of this mutant is: tryptophan at position 437 is mutated to a stop codon. It should be understood that different wild-type *Prunus armeniaca* species... AsBR6ox1 The specific gene sequences may differ in wild-type *Pyrrosia lingua* from other sources. AsBR6ox1 In the gene, this mutation is located at the corresponding homologous position. Therefore, all genes originating from chromosome 9 of *Sargassum fusiforme*... AsBR6ox1 Gene mutations are not limited to the amino acid at position 437 in the 8th exon. Any mutation in the 8th exon that causes premature termination of translation, especially a mutation of tryptophan around position 437 in the 8th exon that causes premature termination of translation, should fall within the scope of protection of this invention. The sequences described above are merely examples for illustrative purposes and are not intended to limit specific sequences.

[0010] Secondly, the present invention provides a nucleic acid molecule encoding the aforementioned brassinolide-6-oxidase 1 mutant protein. In some embodiments, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.4.

[0011] As used in this invention, the term "nucleic acid molecule" is a collective term for deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), a biological macromolecular compound composed of many nucleotide monomers, and one of the most fundamental substances of life. A nucleotide sequence refers to the arrangement of bases in DNA or RNA. Nucleic acid molecules contain cDNA, and in some cases, nucleic acid molecules can be modified for use in the vectors of this invention, such as for codon optimization. In some cases, for the purpose of cloning into a vector, the sequence can be designed to contain terminal restriction sites. Nucleic acid molecules can be obtained from a variety of sources, such as by polymerase chain reaction (PCR) amplification of encoding nucleic acids from one or more given cells or isolated from said one or more given cells.

[0012] Thirdly, the present invention provides biological materials containing the above-mentioned brassinolide-6-oxidase 1 mutant protein or containing a gene encoding the above-mentioned brassinolide-6-oxidase 1 mutant protein, which are vectors, host bacteria, transformed cells, plant lines or seeds.

[0013] As used in this invention, the term "vector" refers to a self-replicating DNA molecule used in recombinant DNA technology to transfer a DNA fragment (target gene) to a recipient cell. Besides the commonly used *E. coli* plasmid vector, there are many other artificially constructed plasmid vectors suitable for microorganisms, yeast, plants, etc. Vectors include, but are not limited to: single-stranded, double-stranded, or partially double-stranded nucleic acid molecules; nucleic acid molecules containing one or more free ends, or without free ends (e.g., circular); nucleic acid molecules containing DNA, RNA, or both; and other types of polynucleotides known in the art. The most commonly used vector type is the "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA fragments can be inserted, for example, through standard molecular cloning techniques. Recombinant expression vectors can contain a form suitable for expressing nucleic acids in host cells, meaning that recombinant expression vectors include one or more regulatory elements that can be selected based on the host cell used for expression and which can be operatively linked to the nucleic acid sequence to be expressed.

[0014] As used in this invention, the term "host bacterium" refers to a bacterial strain used in biological experiments or industrial production to receive, replicate, and express exogenous genes (such as plasmids or recombinant DNA). The host bacterium can be a strain useful in the production of a related protein. To produce the related protein, the nucleic acid encoding the protein can be isolated and inserted into one or more vectors for further cloning and / or expression in the host bacterium. Methods for introducing vectors into the host bacterium are well known, and this invention does not specifically limit these methods.

[0015] The term "transformed cell" as used in this invention generally refers to a cell that has received exogenous nucleic acids (such as plasmids or recombinant genes) and undergone genetic alterations. This cell can be any cell transformed or transfected with exogenous nucleic acids, including prokaryotic cells (such as *E. coli*) and eukaryotic cells (mammalian cells, yeast, plant cells, especially plant protoplasts, suspension cells, callus cells, etc.). Plant cells are particularly preferred, such as cells derived from bryophytes like *Bryophytum comosum*, monocotyledons like rice, wheat, corn, and *Brachys breviscapus*, dicotyledons like *Arabidopsis thaliana*, poplar, soybean, and grape. Cells can originate from multiple tissue types and include primary cells and cell lines. Nucleic acids can exist in the host cell in single-copy or two- or more-copy forms, and in one embodiment, are expressed in the transformed cell.

[0016] Fourthly, the present invention provides the application of the above-mentioned brassinolide-6-oxidase 1 mutant protein, the gene encoding the above-mentioned brassinolide-6-oxidase 1 mutant protein, or any of the above-mentioned biological materials, wherein the brassinolide-6-oxidase 1 mutant gene is found in *Spatholobus suberectus*. AsBR6ox1 The 3254th base in the 8th exon of the gene's coding region is mutated from G to A, resulting in *Sargassum fusiforme*. AsBR6ox1 The reference sequence of the gene is shown in SEQ ID NO.5; (1) Application in shortening the leaf tips of *Spathiphyllum*; (2) Application in shortening the hypocotyl of Podocarpus santalinus; (3) Application in reducing the height of *Erigeron canadensis* plants; (4) Application in increasing the width of leaves of *Sempervivum tectorum*; (5) Application in increasing leaf area; (6) Application in increasing leaf density; (7) Application in increasing ear density; (8) Application in cultivating compact plant types of *Sargassum fusiforme*.

[0017] The agronomic traits of the mutant were verified through cultivation trials over several consecutive years, and it was found that... dc1 The mutant's plant height and other traits were significantly lower than those of the wild type; while its leaf width, leaf area, leaf density, and ear density were significantly higher than those of the wild type.

[0018] In particular, the shortening of the hypocotyl in *Polygonum multiflorum* is generally positively correlated with a decrease in plant height; that is, the shorter the hypocotyl, the shorter the plant tends to be. Shortening the hypocotyl and reducing plant height can improve the plant structure of *Polygonum multiflorum* and alleviate its tendency to lodging.

[0019] The mutant of this invention has increased leaf width, leaf area and leaf density, which can enhance photosynthetic capacity, increase crop yield potential, improve transpiration heat dissipation and water transport and optimize resource utilization efficiency.

[0020] The mutant of this invention has increased ear density, which can enhance resource utilization efficiency, improve population structure, and promote domestication and standardized production.

[0021] The mutant of this invention, through the improvement of the above traits, can obtain a phenotype of dwarfing and compact plant structure, weaken herbaceous traits and enhance agronomic traits. Therefore, it can be used as a germplasm resource for plant type improvement.

[0022] In some embodiments of the present invention, the above-mentioned application includes: mutagenesis and screening of plant cells, tissues, individuals or populations to encode brassinolide-6-oxidase 1 mutant protein.

[0023] In one alternative implementation, the plants are mutated using a non-lethal dose physicochemical mutagenesis method to obtain plant material.

[0024] The aforementioned non-lethal dose refers to the dose being controlled within a range of 20% above or below the median lethal dose.

[0025] Physicochemical mutagenesis methods include one or more combinations of the following physical mutagenesis and chemical mutagenesis methods: physical mutagenesis includes ultraviolet mutagenesis, X-ray mutagenesis, gamma-ray mutagenesis, beta-ray mutagenesis, alpha-ray mutagenesis, high-energy particle mutagenesis, cosmic ray mutagenesis, and microgravity mutagenesis; chemical mutagenesis includes alkylating agent mutagenesis, azide mutagenesis, base analog mutagenesis, lithium chloride mutagenesis, antibiotic mutagenesis, and intercalation dye mutagenesis; alkylating agent mutagenesis includes ethyl methanesulfonate mutagenesis, diethyl sulfate mutagenesis, and ethyleneimine mutagenesis.

[0026] In some embodiments of the present invention, the above application includes: modifying the endogenous source of *Polygonum hydropiper*. AsBR6ox1 The gene was modified to encode a mutant protein of brassinolide-6-oxidase 1.

[0027] Endogenous modification of the target plant AsBR6ox1Genes refer to those readily conceived by those skilled in the art, who modify target plants using conventional transgenic techniques, gene editing techniques (such as zinc-finger nucleases, transcription activator-like effector nucleases, or CRISPR / Cas9), mutation breeding techniques (such as biological [e.g., T-DNA, transposon Ds insertion, etc.], chemical, and physical mutagenesis, etc.), to imbue them with genes encoding the brassinolide-6-oxidase 1 mutant protein or similar non-synonymous mutations, thereby obtaining new plant varieties with a compact plant structure and the ability to grow and develop normally. Therefore, regardless of the technique used, as long as it utilizes the brassinolide-6-oxidase 1 mutant protein provided by this invention to impart the trait of a compact plant structure, it falls within the scope of protection of this invention.

[0028] Fifthly, the present invention provides a method for producing compact *Erigeron brevicorum*, comprising introducing into the genome of *Erigeron brevicorum* a gene encoding the aforementioned brassinolide-6-oxidase 1 mutant protein.

[0029] In some implementations, the introduced method is selected from genetic transformation methods, genome editing methods, or gene mutation methods (including biological, chemical, and physical mutagenesis methods).

[0030] The above-mentioned genetic transformation methods include, but are not limited to: producing individuals with mutated genes through self-pollination of parent plants carrying mutated genes or hybridization with other plant individuals.

[0031] In other embodiments, the methods introduced above can be Agrobacterium-mediated gene transformation, gene gun transformation, pollen tube pathway transformation, or rapid treatment of Agrobacterium rhizogenes co-culture and direct delivery. Any method that can obtain compact plantlets of *Agrobacterium tumefaciens* falls within the protection scope of this invention.

[0032] The present invention has the following beneficial effects: This invention obtained a compact plant type mutant of *Ligustrum lucidum* through chemical mutagenesis. The mutant exhibited reduced plant height, significantly increased leaf width, leaf area, and leaf density, and increased panicle density, resulting in a more compact overall plant structure, making it more suitable for cultivation. Therefore, this mutant selected in this invention can be utilized as a new germplasm material for plant type improvement and has good application prospects. Attached Figure Description

[0033] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0034] Figure 1 for dc1 Comparison of phenotypic characteristics of mutant and wild-type WT potted plants dc1 Genetic map; Figure 2 for dc1 Comparison of field and seedling phenotypes of mutants with wild-type WT; A represents mutants planted at Gaolan Station in 2022. dc1 Plants and wild-type F1 plants, seedling age 68 days; B represents the F2 segregating population numbered 1-1-11 grown in the laboratory. dc1 Mutant and wild-type WT seedlings, 13 days old; C represents seedlings 18 days old. dc1 Mutant and wild-type WT seedlings; D represents seedlings measured at 13 days old. dc1 Hypocotyl length and width of mutant (n=14) and wild-type WT (n=55) seedlings; Figure 3 To utilize the sequencing results of the first F2 segregating population, the distribution of SNPs (10,699) and Indels (2341) obtained by screening according to the law of segregation is shown on the 9 chromosomes of *Saccharomyces cerevisiae*; X-axis unit × 1e+07 bp; Figure 4 To utilize the sequencing and localization results of the second F2 segregating population, the distribution of SNPs (4219) and Indels (869) obtained by screening according to the law of segregation on the chromosome is shown; X-axis unit × 1e+07 bp; Figure 5 for dc1 Results of mutant gene cloning; A represents the results of map-based cloning technology. Figure 1 Four recombinants obtained from B screening (DC-14-1, 1071-1_d4, 1071-2_D4, 1072-2_D4) and the wild-type control 1071-4_D4 were used to design molecular markers based on SNPs and Indels variant information obtained from the second F2 segregating population sequencing. This narrowed the localization interval, and the target site was located between the molecular markers Indel_7212684 and Indel_7526517. The rectangle colors represent different genotypes, with MT representing the homozygous mutant genotype. dc1 / dc1 HT represents the heterozygous genotype WT / dc1WT represents the homozygous wild-type WT / WT; B shows the region encoding gene information. According to the genome annotation information, there are 10 coding genes in this region, only the EVM0006946 gene has a nonsynonymous mutation; C is the structure of the EVM0006946 gene. The full-length genomic DNA is 3491 bp. The reference sequence is shown in SEQ ID NO.5. A GA base substitution occurs in the 8th exon, forming an early stop codon. The encoded amino acid changes from tryptophan W437 to the stop codon. Figure 6 for dc1 Comparison of expression levels (FPKM) of 10 coding genes within the mutant localization region between mutant and wild types showed that only the expression level of the target gene EVM0006946 showed a significant difference (T-test). <0.01, ns>0.05); Figure 7 For the comparative analysis of agronomic traits under pot conditions in 2020, A is plant height (cm), B is main stem diameter (mm), C is seed size (mm), D is yield (g / plant), E is aboveground biomass (g / plant), F is harvest index (%), G is leaf length (cm), H is leaf width (cm), I is spike length (cm), and J is internode length (cm). Figure 8 The agronomic traits evaluated under field cultivation conditions at the Gaolan Experimental Station in 2021 are as follows: A is plant height (cm), B is main stem diameter (mm), C is number of lateral branches, D is number of leaves, and E is leaf area (cm²). 2 F is the leaf density (cm³) 2 G represents the number of ears, and H represents the ear density (cm²). 2 I represents the number of florets per spike, J represents the number of seeds per spike, K represents spikelet fertility (%), L represents seed size (mm), M represents the weight of 1000 seeds (g), N represents the yield (g / plant), O represents the aboveground biomass (g / plant), and P represents the harvest index (%). Figure 9 The agronomic traits of the plants under field cultivation conditions at the Gaolan Experimental Station in 2022 are evaluated as follows: A is plant height (cm), B is main stem diameter (mm), C is aboveground biomass (g / plant), D is leaf length (cm), E is leaf width (cm), F is spike length (cm), and G is internode length (cm). Figure 10The agronomic traits of the plants under field cultivation conditions at the Gaolan Experimental Station in 2023 are evaluated as follows: A is plant height (cm), B is main stem diameter (mm), C is seed size (mm), D is yield (g / plant), E is aboveground biomass (g / plant), F is harvest index (%), G is leaf length (cm), H is leaf width (cm), I is spike length (cm), and J is internode length (cm). Figure 11 For comparative analysis of endogenous hormone levels. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0036] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0037] Example 1 This example demonstrates the acquisition of a compact plant type mutant. Wild seeds of *Sargassum fusiforme* were collected from the Shapotou Desert Experimental Station in the Tengger Desert. The SPT strain was obtained through individual selection and purification. In 2014, experiments were conducted at the Shapotou Desert Experimental Station, selecting approximately 20,000 seeds (M0) from individual SPT plants. These seeds were treated in the dark for 8 hours with a 0.5% concentration of ethyl methanesulfonate (EMS) mutagen. The mutated M1 seeds were then used to cultivate seedlings, which were then transplanted to sandy land. After the plants matured, seeds were harvested from individual plants, forming a mutant library containing 1,500 independent M2 families.

[0038] In 2015, 573 M2 seeds were randomly selected and planted at the Shapotou Station. Plant phenotypes were observed, and a mutant with short spines, dwarfism, short hypocotyl, compact plant type, and larger leaves was found. After two generations of planting, the mutant phenotype was stably inherited, and it was named [name missing]. dc1 .

[0039] Sowing at the Shapotou Desert Experimental Station dc1 During the process, three plants with wild-type phenotypes were found in the mutants, which were produced by natural hybridization. After the seeds of each plant were harvested, they were sown again, and it was found that their offspring showed phenotypic segregation. Therefore, these three plants were identified as F1 plants, and the seeds of one of them were selected to construct the first F2 mapping population.

[0040] Example 2 This example demonstrates the whole-genome resequencing and transcriptome sequencing of the mutant strain screened in Example 1. The specific details are as follows: (1) dc1 Phenotypic variation and genetic analysis of mutants Genetic analysis based on the first F2 segregating population: dc1 The mutant plant height was significantly lower than the wild type. After four generations of cultivation, the phenotype was shown to be stably inherited (statistical segregation ratio 116:44, χ²=0.53, p=0.47). This confirmed that the dwarfing and compact plant type phenotype of this mutant was controlled by a single-gene recessive mutant. The overall phenotype exhibited short plant height, short hypocotyl, compact plant structure, and shorter leaf tip spines. Figure 2 As shown.

[0041] Genetic analysis based on the second F2 segregating population: Choose Gaolan Station dc1 Seeds from F1 plants (numbered 1-3-6, 1-1-11, and 1-1-13) harvested from the mutant plots (F2 generation) were sown, and phenotypic segregation was observed. Chi-square test revealed that the phenotypic segregation ratios of the three F2 generation populations conformed to Mendel's law of 3:1 segregation (as shown in Table 1). This result further confirms the compact plant type mutant. dc1 It is caused by a single gene recessive mutation.

[0042] Table 1 dc1 Statistical analysis of phenotypic segregation ratio in mutant F2 population

[0043] (2) dc1 Sequencing localization and map-based cloning This experiment employed two sequencing localization methods, combined with map-based cloning, variant site effect analysis, and gene expression level analysis to determine... dc1 The target gene.

[0044] Sequencing localization and map-based cloning 1: Select 3 from the first F2 segregation group dc1Illumina sequencing was performed on the mutant and four wild-type WT samples, yielding a total of 73.13 Gbp of clean reads with an average Q30 value of 92.97%, an average alignment rate of 98.59% with the reference genome, an average coverage depth of 10×, and a genome coverage of 91.31%. 3,251,982 SNPs and 650,660 Indels were identified. On chromosomes, the target mutation site and its neighboring genes are often tightly linked during cell division. In other words, variations near the target mutation tend to segregate within the mutant population, exhibiting clustering on the chromosome. Therefore, when using multiple mutants and wild-type individuals for sequencing localization, the identification of localization intervals should prioritize locations with significant SNP or Indel clustering. Based on Mendel's law of segregation, all mutant sites were screened for homozygous variants of the same type, and whose genotypes differed from both the wild-type and reference genomes at the same location. This resulted in 10,699 SNPs and 2,341 Indels. Based on the clustered distribution of variant sites on chromosomes, dc1 Preliminary localization is within the 1-40 Mbp region on chromosome 9 (e.g. Figure 3 (As shown).

[0045] Molecular markers were designed based on SNP and Indel information, and 42 markers from the F2 generation mapping population were used. dc1 The mutant and 14 molecular markers located the mutation site between the Indel_7085316 and Indel_7895116 molecular markers on chromosome 9, with a genetic distance of 1.9 cM (e.g., ...). Figure 1 (As shown). The mapping population was further expanded, and using 156 plants from the F2 segregating population and 6 molecular markers, the mutation site was located between Indel_7212684 and Indel_7614904, with a genetic distance of 1.2 cM. The dCAPS_7404132 molecular marker was located within this mapping interval and... dc1 The mutant phenotype exhibits tight linkage and co-segregation (e.g. Figure 1 (As shown). Meanwhile, based on the genotyping results of molecular markers Indel_7085316 and Indel_7895116, four recombinants were obtained from the F2 segregating population.

[0046] Based on the single nucleotide variant (SNP) effect, the SNP corresponding to the molecular marker dCAPS_7404132 was found to be a non-synonymous mutation, and the gene containing this molecular marker, EVM0006946, was preliminarily identified as the gene containing this molecular marker. dc1 The target gene of the mutant.

[0047] Sequencing localization and map-based cloning 2: To verify the accuracy and reliability of sequencing localization and the localization region of map-based clone 1, five mutant and five wild-type plants were selected from the second F2 mapping population constructed from seeds of plant 1-1-11 for whole-genome resequencing, with increased sequencing depth. Considering the mutant... dc1 There are differences between the genetic background and the reference genome of *Prunus salicina*. This experiment simultaneously selected two F1 parental plants (1-1-11 and 1-1-13) and three independent... dc1 Resequencing of the mutant plants yielded 181.42 Gb of clean reads with an average Q30 value of 94.27%, an average alignment rate of 98.42% with the reference genome, an average coverage depth of 12×, and a genome coverage of 91.32%. Analysis of the raw data yielded 4,880,261 SNPs and 1,009,030 Indels. A similar Mendelian law of segregation was used to filter variant sites, while ensuring that the obtained variant sites were heterozygous in both F1 parent plants. This significantly reduced interference from genetic background differences and the number of effective variant sites, ultimately yielding 4,219 SNPs and 869 Indels. Consistent with the 2020 sequencing results, variant sites were significantly clustered on chromosome 9 (0-20 Mbp interval, e.g., ...). Figure 4 As shown in the figure, this illustrates the feasibility of the sequencing localization method.

[0048] New molecular markers were designed based on SNP and Indel variant site information. Using phenotypic and genotyping data from four recombinant organisms, the mapping interval was narrowed down to between Indel_7212684 and Indel_7526517, which contains 10 coding genes (e.g., ...). Figure 5 As shown), the dCAPS_7404132 molecular marker is still associated with... dc1 Mutant phenotypes co-segregate.

[0049] Based on resequencing data, genes with nonsynonymous mutations or frameshift mutation sites were screened. The results showed that only the EVM0006946 gene had a nonsynonymous mutation in this localization region. This mutation site is also the SNP corresponding to the molecular marker dCAPS_7404132. According to genome annotation information, this gene encodes brassinolide-6-oxidase 1 (AsBR6ox1). The full-length DNA of this gene is 3491 bp, and the reference sequence is shown in SEQ ID NO.5, containing 8 exons. The CDS sequence length is 1395 bp, and the reference sequence is shown in SEQ ID NO.2, encoding a total of 465 amino acids. The base substitution in dCAPS_7404132 caused a GA transition at the 3254th base in the 8th exon, causing the codon at this position to change from TGG to TGA, i.e., from tryptophan W437 to a premature terminator (e.g., ...). Figure 5 (As shown).

[0050] Gene expression variation analysis: While performing sequencing localization and map-based cloning 1, young main stem apical tissues and mature leaves of mutant and wild-type individuals were selected for transcriptome sequencing analysis of gene expression variations. Analysis of the expression levels of 10 genes within the localization region revealed that only the EVM0006946 gene showed variation. dc1 There are significant differences between the mutant and the wild type; this gene... dc1 Significantly upregulated in mutants (e.g.) Figure 6 As shown), based on the above results, EVM0006946 ( AsBR6ox1 ) determined as dc1 The target gene.

[0051] Example 3 This embodiment measures the agronomic traits of the mutant strain, as detailed below: 1. Potted Planting Transplant the F2 seedlings, sown on January 9, 2020, in disposable paper cups, into large flowerpots (20 cm in diameter and 30 cm in height). Place the pots on an outdoor lawn for cultivation. Water moderately every 2-3 days during the early growth stage. Once side branches have developed, water once a week. After the plants mature, select... dc1 Six mutant plants and six mature plants with wild-type phenotypes were used for comparative agronomic trait determination.

[0052] like Figure 7 As shown, comparing the F2 generation population grown in flowerpots in 2020 dc1 Phenotypic findings of mutant and wild-type plants dc1 The mutant's plant height was significantly reduced compared to the wild type, averaging 37.15 cm, which was only half that of the wild type. Figure 7 A); The stem diameter at the base of the main stem is significantly larger than that of the wild type (average 4.43 mm vs. 3.00 mm). Figure 7 B); Seed size was significantly larger than that of the wild type (average 1.93 mm vs. 1.78 mm). Figure 7 C); the harvest index was significantly higher than that of the wild type (average 14.60% vs. 9.10%). Figure 7 F). Furthermore... dc1 The lengths of the 7th-8th leaves from bottom to top and the 1st leaf near the top of the mutant main stem ( Figure 7 G), the length of the 6th-7th panicle from top to bottom on the main stem ( Figure 7 The lengths of the 7th and 5th and 12th internodes from bottom to top on the main stem were significantly shorter than those of the wild type (I). Figure 7 J), the leaf width on the main stem is significantly larger than that of the wild type ( Figure 7 H). Yield per plant and aboveground biomass in dc1 There was no significant difference between the mutant and the wild type. Figure 7 D、 Figure 7 E).

[0053] 2. Field planting In order to evaluate dc1 The field adaptability of the mutant was assessed through a field experiment conducted for three consecutive years from 2021 to 2023 on loess soil at Gaolan Station. Seeds were sourced from healthy individual plants harvested the previous year. Comparisons were made... dc1 Agronomic traits of mutants and wild types.

[0054] May 1, 2021, selection dc1 Seeds of mutant and wild-type plants were germinated indoors and then transplanted into PVC plastic tubes for seedling cultivation. Once the true leaves were fully expanded, the seedling pots were transplanted to the loess soil at the Gaolan Station. In November, after the plants were fully mature, they were selected... dc1 Seven mutant and seven wild-type plants were used for agronomic trait evaluation.

[0055] In mid-April 2022, the mutant and wild-type seeds were directly sown in PVC plastic pipes, and the subsequent transplanting and field management processes were the same as in 2021. Five mutant and five wild-type plants were randomly selected for agronomic trait testing.

[0056] On May 27-28, 2023, wild-type and single-plant-harvested varieties from 2022 were sown in the field at Gaolan Station. dc1 The mutant seeds were directly sown in disposable paper cups (7 cm in diameter and 10 cm in height) with the bottom removed, and water was supplemented by drip irrigation. Around mid-November 2023, 10 mutant plants and 7 wild-type plants were randomly selected for agronomic trait evaluation and determination.

[0057] Field cultivation experiments have shown that, dc1 The overall plant size of both the mutant and wild-type plants was significantly larger than that of the potted plants in 2020. Consistent with 2020, dc1 The mutants were significantly shorter than the wild type, with even greater differences in plant height in the field, ranging from only 33.65% to 41.08% of the wild type. Figure 8 A, Figure 9 A, Figure 10 A); dc1 The mutant had significantly shorter leaf length, spike length, and internode length than the wild type. Figure 9 D、 Figure 9 F and Figure 9 G, Figure 10 G, Figure 10 I and Figure 10J), the leaf width is significantly larger than that of the wild type ( Figure 9 E, Figure 10 H). Unlike pot experiments, field cultivation... dc1 The stem diameter at the base of the main stem is significantly thinner than that of the wild type, averaging 7.51-7.89 mm. vs. 9.93-12.75 mm ( Figure 8 B, Figure 9 B, Figure 10 B). There were no significant differences in seed size and harvest index. Figure 8 L and Figure 8 P, Figure 10 C and Figure 10 F). dc1 The yield per plant and aboveground biomass were significantly lower than those of the wild type, with an average yield of 10.07-15.99 g / plant. vs. 47.02-73.65 g / plant ( Figure 8 N, Figure 10 (D) The average aboveground biomass is 50.97-92.06 g / plant. vs. 248.79-480.71 g / plant ( Figure 8 O, Figure 9 C, Figure 10 E). Furthermore, comparisons of other agronomic traits measured in 2021 revealed that... dc1 The number of lateral branches, leaves, spikes, florets per spike, and seeds per spike showed highly significant differences compared to the wild type, averaging only 30.88%, 5.01%, 5.53%, 35.52%, and 32.64% of the wild type, respectively. Figure 8 C Figure 8 D、 Figure 8 G, Figure 8 I, Figure 8 J). dc1 The mutant's leaf area, leaf density per unit area, ear density, and thousand-grain weight were significantly greater than those of the wild type, being 1.37, 2.38, 2.54, and 1.22 times that of the wild type, respectively. Figure 8 E, Figure 8 F, Figure 8 H, Figure 8 M), there was no significant difference in the fertility of spikelets on the plant ( Figure 8 K).

[0058] Overall phenotypic results indicate that, regardless of whether it is pot cultivation or field cultivation, dc1 The mutants consistently showed significantly shorter plant heights than the wild type, suggesting that the dwarfing and compact plant type phenotypes of this mutant are reproducible and heritable stable traits. dc1The mutant plant exhibits weak overall growth, resulting in a significant reduction in yield. However, its phenotypes, such as compact plant structure, dwarfing, shortened hypocotyl, and shortened leaf spines, are ideal traits in crop genetics and breeding. Therefore, the mutant strain screened in this invention can be utilized as a new germplasm material for plant type improvement.

[0059] 3. Hormone level variation Analysis using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MC / MS) revealed that the precursor compound 6-deoxystigmasterol (6DCS) of brassinolide (BL) was present in the mutant. dc1 Significant accumulation in the body, while BL is present in mutants. dc1 The content in [the sample] was lower than that in wild-type plants, showing statistical marginal significance, such as [the percentage of the sample in the wild-type plant]. Figure 11 As shown above, the results demonstrate the accuracy and reliability of the gene cloning results in this study from a physiological perspective.

[0060] In the above verification process, the steps of genetic analysis, whole-genome resequencing localization, map-based cloning, and transcriptome sequencing mutually verify the accuracy of the screening. Specifically, genetic analysis preliminarily determines the mutation type based on the segregation ratio; whole-genome resequencing and map-based cloning locate candidate regions at the genomic level; and transcriptome sequencing verifies whether candidate genes are truly related to the phenotype at the expression level. The localization results from multiple independent populations corroborate each other, and the cross-validation of expression differences and stable phenotypes forms a multi-layered chain of evidence, thus mutually proving that the screened mutated genes are real and reliable.

[0061] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A mutant protein of brassinolide-6-oxidase 1 from a compact plant type of *Erigeron breviscapus*, characterized in that, The brassinolide-6-oxidase 1 mutant protein has the following mutation: W437(TGG)→Stop(TAG), and its amino acid sequence is shown in SEQ ID NO.

3.

2. A nucleic acid molecule, characterized in that, It encodes the brassinolide-6-oxidase 1 mutant protein as described in claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that, The nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO.

4.

4. A biological material containing the brassinolide-6-oxidase 1 mutant protein of claim 1 or containing a gene encoding the brassinolide-6-oxidase 1 mutant protein of claim 1, characterized in that, The biological materials include carriers, host bacteria, transformed cells, plant lines, and seeds.

5. The use of the brassinolide-6-oxidase 1 mutant protein as described in claim 1, the gene encoding the brassinolide-6-oxidase 1 mutant protein as described in claim 1, or the biomaterial as described in claim 4, in any of the following, characterized in that, The brassinolide-6-oxidase 1 mutant gene is located in *Polygonum cuspidatum*. AsBR6ox1 In the coding region of the gene, the 3254th base of the 8th exon is mutated from G to A, as described in the text. AsBR6ox1 The reference sequence of the gene is shown in SEQ ID NO.5; (1) Application in shortening the leaf tips of *Spathiphyllum*; (2) Application in shortening the hypocotyl of Podocarpus santalinus; (3) Application in reducing the height of *Erigeron canadensis* plants; (4) Application in increasing the width of leaves of *Sempervivum tectorum*; (5) Application in increasing leaf area; (6) Application in increasing leaf density; (7) Application in increasing ear density; (8) Application in cultivating compact plant types of *Sargassum fusiforme*.

6. The application according to claim 5, characterized in that, Mutagenesis and screening were performed on cells, tissues, individuals or populations of *Polygonum cuspidatum* to encode the brassinolide-6-oxidase 1 mutant protein.

7. The application according to claim 5, characterized in that, It includes: the endogenous source of modifying Podocarpus santalinus. AsBR6ox1 The gene was modified to encode the brassinolide-6-oxidase 1 mutant protein.

8. A method for producing a compact plant type of *Erigeron canadensis*, characterized in that, This includes introducing a gene encoding the brassinolide-6-oxidase 1 mutant protein as described in claim 1 into the genome of *Polygonum cuspidatum*.

9. The method according to claim 8, characterized in that, The methods introduced are selected from genetic transformation methods, genome editing methods, gene mutation or hybridization methods.

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