Molecular marker related to regulating the life of rose cutting and its application

Abstract

The application provides a molecular marker related to the bottle-planting life of Chinese rose and an application thereof, and belongs to the technical field of molecular markers. The application provides a method for screening a gene related to the bottle-planting life of Chinese rose based on whole genome association analysis RhNRT2.7 and exploring the function of the gene. The application also provides an application of the gene in the bottle-planting life of Chinese rose. Based on the current research on the function of the gene RhNRT2.7 , the gene is expected to be applied to cultivating new Chinese rose varieties with longer flowering periods. Based on the SNP variation of the gene, the application develops specific molecular markers, detection primers and a typing method based on KASP technology. These methods can quickly and accurately screen Chinese rose plants with long bottle-planting life potential at the seedling stage, provide efficient tools for molecular marker assisted breeding, and have important theoretical and application values.

Description

Technical Field

[0001] This invention relates to the field of molecular marker technology, specifically to a molecular marker for regulating the vase life of roses and its application. Background Technology

[0002] Genome-wide association analysis (GWAS), proposed in the 1990s, is a powerful tool for identifying associations between genotypes and phenotypes. Initially used primarily to study human diseases, GWAS has gradually become an important tool for revealing the basis of human genetic diseases. With technological advancements and increased sample sizes, GWAS has also been widely applied in plant and animal research. As a crucial method for identifying candidate genes, GWAS has yielded significant results in the study of important crops and Rosaceae plants. One study resequencing 336 peach varieties and performing association analysis with 26 agronomic traits identified numerous candidate variations. Based on the whole-genome resequencing of 815 walnut germplasms, a comprehensive walnut genome variation map was constructed, and genome association studies revealed associations between 27 genomic loci and 18 important agronomic traits. However, for roses, an important ornamental horticultural crop, GWAS research has been relatively limited until 2018, when Hibrand et al. published genomic data for diploid roses. Through GWAS, they identified candidate genes KSN, AP2, and TTG2 on chromosome 3 of roses, associated with continuous flowering, petal number, and thorn density.

[0003] Roses, as an important ornamental flower, have significant economic value, accounting for more than one-third of the global cut flower industry. The aging rate of cut roses directly affects post-harvest transportation and sales time; therefore, breeding varieties with longer vase life has important practical value.

[0004] Roses are fertilizer-loving plants, with nitrogen fertilizer playing a crucial role throughout their growth and development, especially in the post-harvest storage, transportation, and vase life of cut roses. Nitrogen supply is critical for their growth and development. Plants are highly sensitive to nitrogen; both excess and deficiency significantly impact normal plant growth. With adequate nitrogen supply, roses exhibit increased chlorophyll content, enhanced photosynthesis, stronger growth vigor, larger, bright green leaves, a longer functional period, more branching, and higher cut flower yield. However, insufficient nitrogen supply, due to its free movement within the plant, primarily affects older tissues. This leads to older leaves turning yellow first, poor new leaf development, reduced chlorophyll synthesis, decreased photosynthetic efficiency and light energy utilization, resulting in thinner flower branches, more blind shoots, and severely hindering plant growth and development.

[0005] Nitrate transporter 2 (NRT2) is a high-affinity nitrate transporter that plays a crucial role in nitrate uptake and transport in plants. NRT2 often exists as a gene family in plants. It has been well understood in Arabidopsis thaliana and rice. To date, researchers have identified seven NRT2 family members in Arabidopsis thaliana. AtNRT2.1 and AtNRT2.2 play key roles in high-affinity transport systems and plant adaptation to changes in nitrogen content. Under low-nitrogen conditions, AtNRT2.4 is significantly expressed primarily in roots and shoots, facilitating NO3 uptake by roots. - Bud phloem loaded with NO3 - Under nitrogen starvation conditions, the gene expression level of AtNRT2.4 initially increases and then decreases, while the gene expression level of AtNRT2.5 remains consistently high. Since they all mediate the uptake of high-affinity nitrate, AtNRT2.1, AtNRT2.2, AtNRT2.4, and AtNRT2 belong to the high-affinity nitrate transport system. Currently, there are few reports on the function of AtNRT2.3, but its expression in roots is independent of nitrate concentration. In Arabidopsis, different tissue expression characteristics exist at different vegetative growth stages, suggesting that AtNRT2 may possess a special function. Although the involvement of AtNRT2.6 in nitrate transport is not yet clear, studies suggest that it may need to work synergistically with other proteins to complete nitrate transport. Unlike other AtNRT2 members, AtNRT2.7's role is to increase nitrate accumulation in seeds. Besides AtNRT2.7, the other six members need to bind with AtNRT2.1 to form a complex to achieve their function as high-affinity transporters. Currently, four members of the NRT2 family have been identified in rice: OsNRT2.1, OsNRT2.2, OsNRT2.3, and OsNRT2.4. Overexpression of OsNRT2.1 can increase the length of the taproot and lateral roots, thereby promoting root growth. Simultaneously, the expression levels of auxin transporter genes (OsPINa / b / c and OsPIN2) also showed a similar upward trend, suggesting that OsNRT2.1 is involved in NO3-dependent NRT2 expression. -OsNRT2.3 plays a role in auxin transport. OsNRT2.3 contains two transcripts, NRT2.3a and NRT2.3b, with different biological functions. OsNRT2.3a transports nitrate from underground to aboveground parts, while overexpression of OsNRT2.3b significantly increases rice yield and nitrogen use efficiency, while also improving phosphorus absorption and accumulation. In nitrate uptake, OsNRT2.1, OsNRT2.2, and OsNRT2.3a work synergistically with OsNAR2.1 to achieve their functions, while OsNRT2.3b and OsNRT2.4 can operate relatively independently, completing nitrate uptake and transport without the participation of OsNAR2.1.

[0006] NRT2 has been well-studied in Arabidopsis and rice, and has also been identified and studied in various horticultural crops, such as rapeseed (Brassica campestris), spinach (Spinacia oleracea), and tomato (Solanumlycopersicum). In apple (Malus domestica) of the Rosaceae family, overexpression of the MdNRT2.1 gene significantly increases the number, length, surface area, and volume of roots in apple plants, suggesting a direct association between MdNRT2.1 and the formation and growth of adventitious roots. Currently, there are few reports on the expression regulation mechanism of the NRT2 gene in roses. In this study, the gene RhNRT2.7, which is associated with the vase life of roses, was obtained through GWAS screening, and its biological function was explored in depth. Based on the SNP variation of this gene, this invention developed specific molecular markers, detection primers, and a KASP-based genotyping method. These methods can rapidly and accurately screen rose plants with the potential for long vase life at the seedling stage, providing an efficient tool for molecular marker-assisted breeding, and have important theoretical and applied value. Summary of the Invention

[0007] The purpose of this invention is to screen for the rose nitrate transport-related gene RhNRT2.7 based on GWAS. This gene directly participates in the nitrate transport process and further affects the vase life of detached roses.

[0008] Therefore, in a first aspect, the present invention provides the RhNRT2.7 gene encoding a high-affinity nitrate transporter based on GWAS screening.

[0009] In a second aspect, the present invention provides that the biological function of the gene described in the first aspect of the present invention is to regulate the senescence and vase life of roses, and preliminarily explores its relationship with nitrate transport.

[0010] In this invention, the gene plays a role in regulating senescence and vase life in ornamental plants. More preferably, the Rosaceae plant is the rose.

[0011] The technical solution of this invention is implemented as follows:

[0012] This invention provides a primer set for identifying the genotype of SNP molecular markers related to the lifespan of rose flowers; wherein the SNP molecular markers for identifying the lifespan of rose flowers are located at position 82427489 on chromosome 82427489 of the 'Samantha' rose genome; the location of the molecular markers is determined based on the reference genome of the modern rose 'Samantha'.

[0013] As a further improvement of the present invention, the primer pair includes:

[0014] The specific nucleotide sequence of the first KASP forward primer (FAM) from the 5' end to the 3' end is shown in SEQ ID NO. 20;

[0015] The specific nucleotide sequence of the second KASP forward primer (VIC) from the 5' end to the 3' end is shown in SEQ ID NO. 21;

[0016] The specific nucleotide sequence of the KASP public reverse primer from the 5' end to the 3' end is shown in SEQ ID NO. 22.

[0017] As a further improvement of the present invention, the 5' ends of the forward primer and the reverse primer are respectively connected to fluorescent adapters that display different colors.

[0018] The present invention further protects a reagent kit containing the above-described primer set.

[0019] This invention further protects the use of the above-described primer set or the above-described kit in one of the following aspects:

[0020] (1) Assisted molecular design breeding;

[0021] (2) Select rose varieties with long vase life.

[0022] The present invention further provides a method for selecting rose varieties with long vase life, comprising the following steps:

[0023] (1) Extract genomic DNA from the tetraploid rose to be tested;

[0024] (2) Identify the PCR products obtained in step (1) by performing quantitative real-time PCR amplification using the above-mentioned kit to determine the genotype of the molecular marker, and then determine the vase life of the rose plant to be tested based on the genotype, wherein:

[0025] When the genotype of the molecular marker in the rose plant being tested is ProRhNRT2.7_T / T homozygous, the rose plant being tested has a shorter vase life.

[0026] When the genotype of the molecular marker in the rose plant under test is ProRhNRT2.7_T / C heterozygous, the rose plant under test has a medium vase life.

[0027] When the genotype of the molecular marker in the rose plant being tested is ProRhNRT2.7_C / C homozygous, the rose plant being tested has a longer vase life.

[0028] As a further improvement of the present invention, the bases at the Chr2B:82427489 site of the RhNRT2.7 promoter between homozygous sequences of the ProRhNRT2.7_T / T are all thymine.

[0029] As a further improvement of the present invention, in the ProRhNRT2.7_T / C heterozygous homologous chromosome, the Chr2B:82427489 position of the RhNRT2.7 promoter region of one chromatid is thymine, and the same position is cytosine on the other chromatid.

[0030] As a further improvement of the present invention, the bases at the Chr2B:82427489 site of the RhNRT2.7 promoter between homologous chromosomes of the ProRhNRT2.7_C / C homozygous sequence are all cytosine.

[0031] As a further improvement of the present invention, the identification method in step (2) is fluorescence identification: when the detected fluorescence signal is consistent with the fluorescence adapter signal at the 5' end of the first forward primer, the genotype of the molecular marker is determined to be ProRhNRT2.7_T / T homozygous; when the detected fluorescence signal is consistent with the fluorescence adapter signal at the 5' end of the second forward primer, the genotype of the molecular marker is determined to be ProRhNRT2.7_C / C homozygous; when the fluorescence adapter signals at the 5' ends of the two forward primers are detected simultaneously, the genotype of the molecular marker is determined to be ProRhNRT2.7_T / C heterozygous.

[0032] In some embodiments, the application is achieved by focusing on the vase life phenotype of 358 tetraploid rose materials, using GWAS to screen for candidate target genes, and further altering the expression of these genes in plants to verify their impact on the flower senescence process, thereby achieving the regulation. In some more specific embodiments, the regulation is achieved by influencing the function of nitrate nitrogen transport in plants.

[0033] Methods or techniques for altering gene expression in plants are known. Those skilled in the art, based on the disclosure of this application, can select methods to alter the expression of known genes in plants as needed. For example, virus-induced gene silencing technology can be used to reduce gene expression in plants. Therefore, in some embodiments, blocking or reducing the expression of said gene in plants is achieved through gene silencing technology.

[0034] Based on the researchers' findings, a high-affinity nitrate transporter protein, RhNRT2.7, associated with rose vase life was identified using GWAS. Silencing the expression of this gene or protein resulted in a shortened flower opening time and premature flower senescence. Cultivating roses under different nitrogen solutions revealed that nitrate nitrogen significantly prolonged the rose's shelf life compared to ammonium nitrogen. RhNRT2.7 may regulate rose vase life by affecting nitrate nitrogen transport. Therefore, in some embodiments, altering the expression of this gene or protein in the plant is achieved by reducing or blocking its expression, thereby regulating flower senescence and influencing vase life.

[0035] Premature senescence of rose petals severely impacts the ornamental value of roses. Understanding the senescence mechanism of rose petals, cultivating rose varieties with long vase life, and developing preservative solutions to extend the shelf life of cut roses have always been hot topics in postharvest rose research. This invention, through GWAS, identified the high-affinity nitrate transporter gene RhNRT2.7 as influencing rose vase life and its association with nitrate transport. This invention is based on the vase life phenotypes of 358 tetraploid roses, using core germplasm selected through analysis, ensuring representativeness. This invention provides a preliminary analysis of the molecular mechanism by which RhNRT2.7 participates in and regulates the flower opening process, offering a theoretical and technical basis for using genetic engineering to address the production problem of cut rose vase life. The discovery that RhNRT2.7 affects nitrate transport and thus rose vase life can provide a reference for inventing new preservative solutions. Moreover, extending the vase life of roses is an important breeding goal in many fields. Therefore, the discovery that RhNRT2.7 regulates the flower opening process and is related to the transport of nitrate nitrogen will also enable RhNRT2.7 to play a significant role in rose breeding.

[0036] This invention offers the following advantages: It develops specific molecular markers, detection primers, and a genotyping method based on KASP technology. These methods enable rapid and accurate screening of rose plants with long vase life potential during the seedling stage, providing an efficient tool for marker-assisted breeding and possessing significant theoretical and applied value. Attached Figure Description

[0037] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0038] Figure 1 Phylogenetic tree of 358 tetraploid roses;

[0039] Figure 2 A scatter plot of two-dimensional principal component analysis based on population variation sites;

[0040] Figure 3 Population structure analysis of 358 tetraploid roses;

[0041] Figure 4 Nucleotide diversity analysis for three different rose groups;

[0042] Figure 5 Linkage disequilibrium decay analysis for three different rose groups;

[0043] Figure 6 Manhattan plot for genome-wide association analysis of vase life traits in roses;

[0044] Figure 7 Selective removal analysis for ancient and modern European varieties;

[0045] Figure 8 To investigate the relationship between associated locus genotypes and the vase life trait of roses;

[0046] Figure 9 The relationship between associated locus genotypes and RhNRT2.7 gene expression levels;

[0047] Figure 10 A diagram illustrating the opening and aging stages of a rose flower;

[0048] Figure 11 The relative expression levels of RhNRT2.7 at different opening and senescence stages of rose;

[0049] Figure 12 Subcellular localization of RhNRT2.7 protein in tobacco leaves;

[0050] Figure 13 The effects of silencing RhNRT2.7 on the opening and senescence process of roses;

[0051] Figure 14 The expression levels of the silenced RhNRT2.7 gene and the control were compared.

[0052] Figure 15 The effect of silent RhNRT2.7 on the vase life of roses at different opening stages;

[0053] Figure 16 The relative expression level of the aging-related gene RhSAG12 in the silenced RhNRT2.7 sample;

[0054] Figure 17 The effects of different concentrations and types of nitrogen treatment on the vase life of roses;

[0055] Figure 18 Statistics on the vase life of roses treated with different concentrations and types of nitrogen;

[0056] Figure 19 KASP typing of the SNP site of the RhNRT2.7 promoter. Detailed Implementation

[0057] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0058] Example

[0059] The technical solutions of the present invention will be illustrated by examples below, but the scope of protection of the present invention is not limited to these examples.

[0060] Materials and Methods

[0061] I. Plant materials and treatment

[0062] 1. 358 tetraploid rose germplasm resources were all planted in greenhouses at the China Agricultural University Science and Technology Park.

[0063] 2. The propagation process for subculture seedlings of the 'Samantha' rose (Rosa hybrida 'Samantha') is as follows: Select a 2-3 cm long stem segment of the 'Samantha' rose with at least one leaf bud and insert it into a propagation subculture medium. When the seedling grows to 10-12 cm, transfer it to a rooting medium. When the root system of the seedling grows to 3-5 cm, remove the seedling, gently rinse the roots to remove the medium, and maintain humidity to prevent drying. Subsequently, plant the seedlings with mature root systems in a nutrient pot containing a mixture of potting soil and vermiculite (2:1), and cover them with a transparent heat-insulating cover for 15-20 days for acclimatization. After removing the cover, maintain 8 hours of darkness and 16 hours of light, keep the air humidity at around 60%, and control the temperature at 21-23℃.

[0064] 3. The propagation method for Nicotiana benthamiana is as follows: Sow the seeds evenly on the surface of soil containing sufficient moisture and a mixture of nutrient soil and vermiculite (1:1). After the seeds germinate within 7-10 days, transplant them. Place one seedling in each individual seedling pot. When the tobacco has grown to 4-6 leaves, it is ready for injection. Maintain the temperature of the cultivation environment at 23-26℃ and the humidity at 60-70%.

[0065] II. Phenotypic Data Analysis

[0066] Statistical analysis was conducted on the phenotypic characteristics of varieties from different sources. Microsoft Excel 2007 was used to organize and analyze the data, calculating the mean, standard deviation, coefficient of variation, kurtosis, and skewness for each trait. This patent primarily focuses on descriptive statistics and diversity analysis of the vase senescence phenotype of 358 tetraploid rose germplasm resources. The vase life of each variety was recorded by taking the average of the vase lifespans of five flowers, which was then used as the vase life phenotype for that variety. The time when the flowers showed obvious wilting was determined as the end of vase life.

[0067] III. Constructing a System Evolutionary Tree

[0068] To classify 358 tetraploid rose germplasm resources according to their phylogenetic relationships, the data were first processed using sequence alignment units in PHYLIP software, then resampled using the Bootstrap method, and finally a phylogenetic tree was constructed using FastTree software for more intuitive observation.

[0069] IV. Principal Component Analysis

[0070] Principal component analysis (PCA) in PLINK software was used to explore and identify potential genetic structural differences among tetraploid rose germplasm resources. First, based on the high-quality SNPs selected, the top 10 principal components were analyzed, and the explanatory power of each principal component was calculated. Finally, the results were visualized using ggplot2 software.

[0071] V. Group Structure Analysis

[0072] The population structure of tetraploid rose germplasm resources was analyzed using ADMIXTURE software to elucidate the genetic structure of the population. The analysis was based on a Bayesian model, using maximum likelihood estimation to determine the proportion of ancestral components in individuals, thereby revealing the genetic relationships between populations. First, a population size K was set; in this study, it was set from 2 to 10 for ADMIXTURE analysis. Then, the optimal K value was selected by comparing the cross-validation errors at different K values.

[0073] VI. Nucleotide Diversity Analysis

[0074] This patent utilizes VCFtools software to analyze nucleotide diversity, which reflects the degree of genotypic variation within a population. First, a single window is set to 100 kb, and then the average nucleotide diversity value is calculated for each 10 kb non-overlapping window.

[0075] VII. Chaining Imbalance Decay Analysis

[0076] Typically, when the correlation coefficient of a population variation site decreases to half of its maximum value, the genetic decay distance corresponding to that point is considered to be the genetic disequilibrium decay distance of the population. This patent mainly conducts linkage disequilibrium decay analysis on ancient European varieties, transitional varieties, and modern varieties in tetraploid rose germplasm resources, and uses PopLDdecay software to draw linkage disequilibrium decay maps.

[0077] 8. Genome-wide association analysis (GWAS)

[0078] Based on the identified high-quality SNP loci, a total of 27,206,170 high-quality SNP loci were selected by filtering according to the criteria of minor allele frequency (MAF) less than 0.05 and genotype deletion rate (GENO) greater than 10%. These loci will be used for subsequent GWAS.

[0079] Genome-wide association analysis (GWA) was performed using a mixture of linear models in GEMMA software. First, PLINK software was used to convert VCF files containing variant sites generated from population variation detection into BED files, aiming to convert files containing variant site information for subsequent analysis. Then, the previously obtained rose vase life phenotypic data were organized into FAM format files, followed by phylogenetic analysis. Finally, a mixture of linear models was used to analyze the association between variant sites and the target trait. The formula was: Y = Xβ + Zu + e (phenotypic value), where X is the fixed-effects design matrix, Z is the random-effects design matrix, β is the estimated coefficient of the fixed effects, u is the coefficient of the random effects, and e is the error term.

[0080] IX. Gene Cloning

[0081] Using rose cDNA as a template, specific primers were designed, and the target fragment was obtained by PCR amplification. The PCR product was purified after agarose gel electrophoresis confirmed the correct band size. The purified PCR product was directly ligated with a T vector to construct a recombinant plasmid. The ligation product was transformed into competent *E. coli* cells and plated on ampicillin-containing plates for culture. Single colonies were picked, and the inserted fragment was initially screened by colony PCR or plasmid PCR. Positive clones were further sequenced to confirm the integrity and accuracy of the target gene sequence (SEQ ID NO. 1-5).

[0082] 10. Virus-induced gene silencing (VIGS)

[0083] A VIGS silencing vector was constructed, and a specific RhNRT2.7 fragment of approximately 400 bp was cloned into the pTRV2 vector and transformed into Agrobacterium GV3101. Subsequently, the transformed Agrobacterium and Agrobacterium containing pTRV1 were mixed at a 1:1 ratio and used to infect rooted rose tissue culture seedlings. Plants infected with the empty pTRV2 vector served as a control. After one month of growth, RNA was extracted from petals to assess the silencing efficiency. The flower opening process of the RhNRT2.7 VIGS transformed lines was further observed (SEQ ID NO. 6-11).

[0084] XI. Quantitative Real-Time PCR Analysis

[0085] Total RNA was extracted from rose petals using the hot boron method. First-strand cDNA was synthesized using the HiScript II Q RT SuperMix for qPCR reverse transcription kit with 1 μg of total RNA as a template. Subsequently, qRT-PCR reactions were performed using the StepOne Real-Time PCR System and the KAPA SYBR FAST Universal qRT-PCR kit, with each reaction volume being 20 µL containing 1 µL of cDNA template (SEQ ID NO. 12-17).

[0086] XII. Subcellular localization

[0087] The coding region of the target gene was inserted into the enzyme-digested vector to construct the pRI101-target gene-GFP expression vector (SEQ ID NO. 18-19). pRI101-target gene-GFP, pRI101-GFP, the localization marker, and the P19 plasmid were transformed into competent Agrobacterium GV3101 cells. The transformed Agrobacterium was plated on solid LB agar plates containing kanamycin and rifampin and incubated upside down at 28°C for 3 days. Single colonies were picked and inoculated into liquid LB medium containing the corresponding antibiotics and cultured with shaking at 28°C for 8–12 h. After confirmation of correct bacterial culture, the culture was expanded to 10 mL of resistant LB medium and cultured with shaking at 28°C for 8–12 h. After the culture was completed, the cells were collected by centrifugation at 5,000 rpm for 8 min, resuspended, and OD adjusted. 600 Adjust the concentration to 1.5 (to 1.2 for bacterial solutions containing the silencing repressor P19). Mix the positioning marker and P19 bacterial solutions with equal volumes of pRI101-target gene-GFP or pRI101-GFP bacterial solutions, respectively. The experimental group consisted of a mixture of pRI101-target gene-GFP, positioning marker, and P19 bacterial solutions, while the control group consisted of a mixture of pRI101-GFP, positioning marker, and P19 bacterial solutions. Incubate in the dark for 3-4 hours. Select healthy leaves of Nicotiana benthamiana at the 4-6 leaf stage for injection. Use a syringe without a needle to inject the bacterial solution into the tender and healthy leaves, ensuring the entire leaf is fully saturated. Incubate in the dark for 1 day after injection, then transfer to light conditions for 2 days. After the culture period, observe the fluorescence signal using a laser confocal microscope, with GFP excitation light at 488 nm and mCherry excitation light at 552 nm.

[0088] XIII. Methods of treating roses with different nitrogen elements

[0089] Using 'Diana', 'Carola', and 'Brilliant' roses as materials, the flowers were first awakened by cutting the stems at a 45° angle, leaving 35 cm of stem. Thorns and leaves at the base of the stem were removed using a pruning tool, leaving 2-3 compound leaves at the top of the stem. All the flowers were then placed in a container filled with water (water level 30 cm above the stem) and soaked for 6 hours to ensure optimal physiological condition. Next, treatment solutions were prepared, and different concentrations (2.5 mM, 5 mM, 10 mM) of different solvents (NH4NO3, KNO3, NH4Cl) were tested, resulting in 10 treatment groups. Each treatment group contained 6 cut roses, and each treatment was repeated twice. Next, the roses were arranged in vases. Flower branches with fully developed buds and a grade of three bloom after thawing were selected. Each branch was individually placed in a transparent vase, and the corresponding treatment solution was added. The vases were sealed with cotton balls to prevent moisture evaporation. The vases were maintained at (20 ± 1)°C and 60% ± 5% relative humidity, and exposed to fluorescent light for 8 hours / 16 hours daily (dark / light). The treatment solution was changed every two days during the vase arrangement period. The vase lifespan of the roses was then observed and recorded. The opening time of the roses in each treatment group was observed and recorded daily, along with the duration from the start of vase arrangement to the withering of the flowers. The end of the vase lifespan was marked by the wilting of the flowers and their loss of ornamental value.

[0090] Results and Analysis

[0091] I. Phylogenetic analysis of 358 tetraploid rose germplasm resources

[0092] Based on resequencing data from 358 tetraploid rose germplasm resources, a total of 27,206,170 high-quality SNP loci were identified, and a phylogenetic tree was constructed accordingly. The results showed that these 358 germplasm resources could be clearly divided into three groups: ancient European varieties, transitional varieties, and modern varieties. Figure 1 In subsequent studies, a systematic analysis will be conducted on the above three groups to explore in depth their genetic differentiation characteristics and differences during the artificial domestication process.

[0093] II. Principal component analysis of different groups in tetraploid rose germplasm resources

[0094] Principal component analysis was conducted based on high-quality SNP loci obtained through identification. The results showed that the dispersion of the ancient European cultivar population was the greatest, followed by the transitional cultivar population, while the modern cultivar population exhibited significant clustering characteristics. Figure 2This phenomenon indicates that, under the continuous influence of human intervention and targeted breeding, the domestication direction of modern varieties tends to be consistent, and the genetic similarity among varieties is significantly increased compared to historical varieties, resulting in a decrease in nucleotide diversity within the population. These results further demonstrate that artificial selection plays a crucial role in shaping the genetic structure of roses.

[0095] III. Population Structure Analysis of Different Groups in Tetraploid Rose Germplasm Resources

[0096] Figure 3 In the graph, the horizontal axis represents different germplasm populations, and the vertical axis represents the Q value. Different colored areas reflect the genetic composition of each population. The results show that when K=2, the 358 tetraploid rose germplasm resources can be optimally divided into two major categories at the genomic level. Significant differentiation exists between the ancient European cultivar population and the modern cultivar population: green components account for approximately 70% in the ancient European cultivar population, while the modern cultivar population is almost entirely composed of yellow components, indicating significant differences in genotypic background between the two groups.

[0097] IV. Nucleotide diversity analysis of different groups in tetraploid rose germplasm resources

[0098] To investigate the genomic regions selected during the breeding and domestication process of modern roses, nucleotide diversity analysis was performed on ancient and modern European varieties. The results showed that the nucleotide diversity of the ancient European variety population was higher than that of the modern variety population. Figure 4 This indicates that as the domestication process progresses, the nucleotide diversity within rose populations gradually decreases, reflecting the significant impact of human intervention and selection on rose variety improvement.

[0099] V. Linkage disequilibrium decay analysis of different groups in tetraploid rose germplasm resources

[0100] The chain imbalance decay analysis results showed that the chain imbalance coefficient was highest in old European roses, followed by transitional varieties, and lowest in modern varieties. In terms of the chain imbalance decay rate—the physical distance from which the chain imbalance coefficient decays to half its maximum average—the old European varieties decayed the fastest among these 358 populations, followed by transitional varieties, while modern varieties decayed the slowest. Figure 5 The faster the linkage disequilibrium decays, the greater the diversity of a group of species, and the less pronounced the linkage between genes. This result is consistent with previous population structure analyses that have shown a decrease in genetic diversity among modern varieties.

[0101] VI. Screening for candidate genes affecting vase life of roses using GWAS and selective sweep analysis

[0102] Vase life traits were determined for 358 tetraploid rose germplasm resources, recording the number of days it took for flowers to develop from the tertiary flowering stage to the appearance of obvious senescence phenotypes. Based on this phenotypic data, a mixture linear model was constructed using GEMMA software to conduct GWAS. The GWAS results are shown in Figure 6. Only three associated loci significantly above the threshold were detected in the Manhattan plot. Among them, the Chr2B:82427489 locus (P = 1.72 × 10⁻⁻⁶) was the most significant. 9 The mutation was located in the promoter region of the RhNRT2.7 gene, where a T-to-C base mutation occurred. RhNRT2.7 encodes a high-affinity nitrate transporter, suggesting it may play an important role in regulating the vase life of roses. Selective clearance analysis further indicated that the RhNRT2.7 gene is located in a selected region compared to ancient European varieties, indicating that this gene has undergone significant selection during domestication and improvement. Figure 7 ).

[0103] There were significant differences in vase life among different genotypes of roses. The homozygous ProRhNRT2.7_T / T variety had a shorter vase life, the heterozygous ProRhNRT2.7_T / C variety had a medium vase life, and the homozygous ProRhNRT2.7_C / C variety had the longest vase life. Figure 8 Significant differences were also observed in the expression levels of RhNRT2.7 among different rose genotypes. The expression level of RhNRT2.7 was lowest in the ProRhNRT2.7_T / T homozygous type, followed by the ProRhNRT2.7_T / C heterozygous type, and the gene expression level was highest in the ProRhNRT2.7_C / C homozygous type. Figure 9 ).

[0104] VII. Analysis of RhNRT2.7 gene expression levels at different stages of rose bloom

[0105] To analyze the spatiotemporal expression specificity of the RhNRT2.7 gene, quantitative real-time PCR was used to detect the expression of 'Samantha' rose at different stages (S0–S6) during the flower opening process. Figure 10 The expression level of RhNRT2.7 in petals was investigated. The results showed that RhNRT2.7 expression was low in petals at stages S0 and S1, significantly increased at stage S2, peaked at stage S3, and then significantly decreased at stages S4 to S6. Figure 11 The significant decrease in the expression level of RhNRT2.7 during the flower senescence stage suggests that this gene may be involved in the regulation of rose petal senescence.

[0106] VIII. Subcellular localization analysis of RhNRT2.7 in tobacco

[0107] Subcellular localization analysis of transient expression in tobacco cells showed that RhNRT2.7 is localized to the plasma membrane. As shown in Figure 12, Agrobacterium carrying the pRI101-RhNRT2.7-GFP recombinant vector was co-injected into tobacco lower epidermal cells along with Agrobacterium carrying the plasma membrane localization marker vector PM-mCherry and the P19 repressor vector. After 3 days of co-expression, the green fluorescence signal of RhNRT2.7-GFP and the red fluorescence signal of the plasma membrane marker were highly correlated under a confocal microscope, indicating that the RhNRT2.7 protein is localized to the plasma membrane and is a typical membrane-localized protein.

[0108] IX. The effect of RhNRT2.7 silencing on rose flowering period

[0109] Nitrogen is a mobile element in plant tissues, and under nitrogen-deficient stress conditions, corresponding tissues will exhibit nitrogen deficiency symptoms. Considering that RhNRT2.7 was screened from the vase life phenotype of roses in GWAS, it was hypothesized that it might be involved in regulating the opening and senescence processes of roses. To further investigate the specific function of RhNRT2.7 in the opening and senescence processes of roses, the TRV2-RhNRT2.7 vector was constructed, and the RhNRT2.7 gene in roses was transiently silenced using VIGS technology with 'Samantha' tissue culture seedlings as material. The outermost petals were collected in stage S4 for subsequent RNA extraction to test the gene silencing efficiency and provide reliability. The results showed that the expression of RhNRT2.7 in the silenced plants was significantly reduced (…). Figure 13 This indicates that the silencing effect of the RhNRT2.7 gene is sufficient to support subsequent conclusions.

[0110] Fifteen silent lines and 15 control lines were selected, and their phenotypic characteristics were observed and the flowering process was recorded. The stages of rose flowers from S1 (sepals slightly open, flower showing color) to S6 (petals wilting) were observed and continuously photographed. Results showed that, compared with the TRV control, silencing RhNRT2.7 shortened the entire flowering period of roses, leading to premature flower senescence. Figure 14-15 The expression level of the senescence-related gene RhSAG12 in petals was determined by quantitative real-time PCR. The results showed that silencing RhNRT2.7 led to high expression of the senescence gene RhSAG12. Figure 16 ).

[0111] 10. Effects of different nitrogen solution treatments on cut roses

[0112] To further explore the relationship between nitrogen nutrition and rose flower senescence, nine different concentrations and nitrogen forms of 'Diana', 'Brilliant', and 'Carola' roses were used as experimental materials. The effects of these treatments on the vase life of cut roses were systematically evaluated, with flower wilting as the senescence criterion. As shown in Figure 17, compared with the water control, all nitrogen treatments prolonged the vase life of cut roses to varying degrees. Among the three varieties, treatments containing NH4NO3 or KNO3 significantly prolonged the time from the tertiary flowering stage to the appearance of senescence phenotypes. In contrast, NH4Cl treatments at certain concentrations only prolonged the vase life by about 1 day, and the overall effect was not significantly different from the water control. These results indicate that in cut roses, NO3⁻ is more beneficial than NH4⁺ in delaying flower senescence. Further analysis revealed that among different concentrations of NO3⁻ treatment solutions, 2.5 mM and 5 mM treatments had the most significant effects on delaying aging and extending the viewing period: 'Diana' and 'Brilliant' roses were extended by approximately 3 days compared to the control group (9 days), while 'Carola' was extended by approximately 2 days compared to the control group (Figure 18). These results provide important reference for optimizing the formulation of rose cut flower preservative solutions.

[0113] XI. Development of SNP Molecular Markers and Application of KASP Typing

[0114] Based on the SNP variation at Chr2B:82427489, KASP genotyping-specific primer pairs were designed. The two forward primers specifically recognize T and C base types, respectively, and their 5' ends are linked to different fluorescent tags. The first KASP forward primer is shown in SEQ ID NO. 20, the second KASP forward primer is shown in SEQ ID NO. 21, and the common KASP reverse primer is shown in SEQ ID NO. 22.

[0115] Using the primer combinations described above, PCR amplification was performed on the genomic DNA of unknown rose varieties, and KASP genotyping was conducted. The PCR amplification system for the KASP genotyping reagent is shown in Table 1, and the PCR amplification program is shown in Table 2. If the genotyping clusters were not clear enough after the initial PCR amplification, the amplification was continued for 3 more cycles according to the cycling conditions in step 3 of Table 2, and the fluorescence signal values ​​were read and analyzed again. If the clustering effect was still unsatisfactory, the number of PCR cycles could be further increased until tightly packed and well-separated genotyping clusters were obtained. To ensure the accuracy of the genotyping results, at least 15 samples were tested for each SNP locus, and the DNA samples should contain at least three different genotypes.

[0116] As shown in Figure 19, in the genotyping scatter plot, the ProRhNRT2.7_T / T homozygous samples mainly clustered on the FAM fluorescence axis, the ProRhNRT2.7_T / C heterozygous samples were distributed in the middle area of ​​the scatter plot, while the ProRhNRT2.7_C / C homozygous samples clustered on the VIC fluorescence axis. This KASP genotyping system can achieve rapid, accurate, and high-throughput genotyping, and is suitable for efficient screening of rose plants with long vase life at the seedling stage.

[0117] Table 1. KASP Genotyping Detection Reagent PCR Amplification System

[0118]

[0119] Table 2. KASP Genotyping Detection Reagent PCR Amplification Procedure

[0120]

[0121] Discussion and Conclusion

[0122] Premature senescence of rose petals severely impacts the ornamental value of roses. Therefore, elucidating the mechanism of rose flower senescence, cultivating rose varieties with long vase life, and developing novel rose preservative solutions have always been hot topics in postharvest rose research. Previous studies have demonstrated that rose flower senescence is regulated by plant hormones auxin and ethylene; however, the mechanisms by which nutrients (such as nitrogen) affect the rose senescence process remain unclear. This invention identifies a gene, RhNRT2.7, highly associated with rose vase life through genome-wide association analysis. Figure 7 This indicates that RhNRT2.7 may play an important role in regulating the senescence process of roses and affecting vase life. To further investigate whether RhNRT2.7 participates in regulating petal senescence, further verification revealed that compared to the TRV2 control plants, plants with silenced RhNRT2.7 exhibited a shortened flower opening cycle and premature flower senescence. Silencing RhNRT2.7 significantly shortened the opening time from S1 to S2 and the senescence time from S3 to S6, both by approximately 2 days.

[0123] The primary biological function of NRT2 family members is nitrate transport. To further investigate the relationship between nitrogen, RhNRT2.7, and rose flower senescence, different concentrations (2.5 mM, 5 mM, 10 mM) of different varieties of 'Diana', 'Brilliant', and 'Carola' roses were compared. - NH4 +The roses were treated with a nitrogen solution containing nitrate nitrogen. The results showed that nitrate nitrogen significantly extended the shelf life of roses compared to ammonium nitrogen, and that there may be an optimal concentration of rose preservative solution between 2.5 and 5 mM. It is speculated that RhNRT2.7 may play a key role in this process by influencing the transport of nitrate nitrogen.

[0124] In summary, the inventors have found that transient silencing of RhNRT2.7 shortens the rose flower development cycle and causes premature senescence; RhNRT2.7 may regulate flower opening time and petal senescence by affecting the transport of nitrate nitrogen.

[0125] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A primer set, characterized in that, The primer set was used to identify the genotype of SNP molecular markers related to the vase life of roses; wherein, the SNP molecular markers related to the vase life of roses are located at position 82427489 on chromosome 82427489 of the 'Samantha' rose genome, and their polymorphism is T / C; the location of the molecular markers was determined based on the reference genome of the modern rose 'Samantha'. The primer set includes: The specific nucleotide sequence of the first KASP forward primer from the 5' end to the 3' end is shown in SEQ ID NO. 20; The specific nucleotide sequence of the second KASP forward primer from the 5' end to the 3' end is shown in SEQ ID NO. 21; The specific nucleotide sequence of the KASP public reverse primer from the 5' end to the 3' end is shown in SEQ ID NO.

22.

2. A reagent kit, characterized in that, The kit contains the primer set as described in claim 1.

3. The application of the primer set of claim 1 or the kit of claim 2 in screening rose varieties with long vase life.

4. A method for selecting rose varieties with long vase life, characterized in that, Includes the following steps: (1) Extract genomic DNA from the tetraploid rose plants to be tested; (2) Identify the genomic DNA obtained in step (1) and perform real-time PCR amplification using the kit described in claim 2 to determine the genotype of the molecular marker described in claim 1. Then determine the vase life of the rose plant to be tested based on the genotype. When the genotype of the molecular marker in the rose plant to be tested is T / T homozygous, the vase life of the rose plant to be tested is shorter. When the genotype of the molecular marker in the rose plant to be tested is T / C heterozygous, the vase life of the rose plant to be tested is medium. When the genotype of the molecular marker in the rose plant being tested is C / C homozygous, the rose plant being tested has a longer vase life.

5. The method according to claim 4, characterized in that, The identification in step (2) is a fluorescence identification method: when the detected fluorescence signal is consistent with the fluorescence adapter signal at the 5' end of the first forward primer, the genotype of the molecular marker is determined to be T / T homozygous; when the detected fluorescence signal is consistent with the fluorescence adapter signal at the 5' end of the second forward primer, the genotype of the molecular marker is determined to be C / C homozygous; when the fluorescence adapter signals at the 5' ends of the two forward primers are detected simultaneously, the genotype of the molecular marker is determined to be T / C heterozygous.

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