KASP molecular marker associated with tomato fruit weight and its application

Abstract

The present invention discloses KASP molecular markers related to tomato fruit weight and their applications, and belongs to the technical field of nucleic acid determination methods. The present invention uses a whole genome association analysis method to obtain a SNP site related to tomato fruit weight, which is the 336003rd position of the full-length sequence of chromosome 9 of the tomato genome, and there is a polymorphism of T>C. The relevant KASP molecular markers and KASP primers are further developed for typing of tomato fruit weight, and a highly consistent and clearly defined group typing effect is verified in a tomato population. In the breeding process, the allelic variation of fruit weight is judged according to its genotype, and an excellent group that meets the breeding target size can be quickly selected at the seedling stage, realizing early screening of the breeding population and accelerating the molecular breeding process of tomato varieties.

Description

Technical Field

[0001] The invention belongs to the technical field of nucleic acid-containing determination methods, and in particular relates to a KASP molecular marker associated with tomato fruit weight and an application thereof. Background Art

[0002] tomato( Solanum lycopersicum L.) is an annual or perennial herb belonging to the Solanaceae family. The main edible part of the tomato is the fruit, which is rich in soluble sugars, carotenoids, vitamin C, lycopene, organic acids and other substances, and has many functions such as anti-oxidation, slowing down aging, enhancing immunity, and preventing cancer.

[0003] Tomato fruit weight is a quantitative trait controlled by multiple genes. It is easily affected by genetics, hormones, external environment, artificial cultivation measures, etc. during the development process. It has rich phenotypic variation and is a key factor controlling the size and yield of tomatoes. After continuous domestication and improvement, the fruit weight of tomatoes has increased from 1-2 grams of currant tomatoes, to 10-40 grams of cherry tomatoes, and to several hundred grams of cultivated tomatoes. The maximum difference is more than a hundred times.

[0004] At present, many quantitative trait loci (QTLs) controlling tomato fruit weight have been reported through molecular genetics studies. FW2.2 , a homologous gene encoding a P450 enzyme KLUH FW3.2 , encoding cell size regulatory factors FW11.3 , code SlCLV3 FAS (fasciated), encoding SlWUS LC (locule number) and the gene encoding brassinolide hydroxylase GLOBE 6 of them were precisely located and cloned. Another example is the study by Yu Ning et al. that identified a fw6.3 It is a major effect QTL (Fine Mapping of fw6.3, a Major-Effect Quantitative Trait Locus ThatControls Fruit Weight in Tomato. Plants, 2023,12(11):2065.).

[0005] Genome Wide Association Study (GWAS) is a method of discovering genes or genomic regions associated with traits by comparing single nucleotide polymorphism (SNP) sites between case and control populations. Tomato is a model plant for studying fruit development. It is self-pollinating, has a variety of variation types, and has a relatively large linkage disequilibrium decay rate, which can provide a large amount of genetic variation for genome-wide association analysis of tomatoes. GWAS can be used to more accurately discover excellent genes related to important agronomic traits in tomato germplasm resources.

[0006] Kompetitive Allele Specific PCR (KASP) is a fluorescence-based homogeneous genotyping technology based on traditional PCR and fluorescence detection, with the advantages of flexibility, simplicity, and high throughput. After using GWAS to mine target trait-related sites, combined with KASP molecular marker technology to assist breeding for selection, it can significantly improve breeders' efficiency in breeding varieties, solve the problem of low throughput of traditional molecular marker detection and inability to achieve large-scale screening, reduce field workload, and promote the development of related industries.

[0007] Patent documents CN 117683938 B and CN 118406794 A disclose KASP molecular markers that are closely linked to tomato fruit width and length, respectively. There is no report on KASP molecular markers for tomato fruit weight in the prior art. Tomato fruit weight is a quantitative trait controlled by multiple genes. Discovering KASP molecular markers related to it is of great significance for analyzing the regulatory mechanism of tomato fruit development and molecular breeding research of tomatoes. Summary of the invention

[0008] The purpose of the present invention is to provide a molecular marker related to tomato fruit weight, which has good genotyping effect and is used for screening and identifying materials with different tomato fruit weights. The fruit weight can be identified at the seedling stage to assist in selection breeding.

[0009] To achieve the above object, the present invention adopts the following technical solution:

[0010] The present invention uses the whole genome association analysis method to obtain several single nucleotide polymorphism (SNP) sites related to tomato fruit weight, further designs KASP primers, and performs typing identification in tomato populations to obtain a molecular marker with good typing effect. The present invention uses the screened SNP site as a detection target for screening or identifying tomato fruit weight. The SNP site is the 336003rd position of the full-length sequence of chromosome 9 of the tomato genome, and there is a polymorphism of T>C; the tomato genome sequence number is GCF_000188115.5.

[0011] Specifically, the base at position 336003 of the full-length sequence of chromosome 9 of the tomato genome with sequence number GCF_000188115.5 includes two states, T and C. When the genotype is TT, the tomato fruit weight is larger; when the genotype is CC, the tomato fruit weight is smaller.

[0012] Furthermore, the application includes: using the genomic DNA of the tomato plant to be tested as a template, using a primer set that specifically detects the single nucleotide polymorphism site to perform a PCR reaction, judging the genotype according to the PCR product amplified by the corresponding paired primers, and then predicting the weight of the tomato fruit. This method can be used to screen germplasm at the tomato seedling stage or earlier.

[0013] The present invention develops a KASP molecular marker related to tomato fruit weight based on the above SNP site, takes the SNP site as the coordinate, intercepts the nucleotide fragments upstream and downstream of the genome, and designs a primer set based on the SNP site in the KASP molecular marker for specific detection of the SNP site in the tomato genome.

[0014] Furthermore, the KASP molecular marker uses the single nucleotide polymorphism site as a coordinate and selects 40-100 bp of nucleotide sequences upstream and downstream.

[0015] Furthermore, the nucleotide sequence of the KASP molecular marker is shown in SEQ ID NO.1.

[0016] The present invention can also develop a corresponding detection kit using the above SNP site as a detection target. Therefore, the present invention provides a detection kit for screening or identifying tomato fruit weight, and the kit includes a primer set for specifically amplifying the KASP molecular marker.

[0017] The primer set for amplifying the KASP molecular marker includes two specific forward primers and one universal reverse primer. The nucleotide sequence of the specific forward primer 1 is shown in SEQ ID NO.2, the nucleotide sequence of the specific forward primer 2 is shown in SEQ ID NO.3, and the nucleotide sequence of the universal reverse primer is shown in SEQ ID NO.4.

[0018] Furthermore, two different fluorescent label sequences are connected to the 5' ends of the specific forward primer 1 and the specific forward primer 2. In the present invention, different fluorescent labels are marked on the two specific primers, and identification of different genotypes can be achieved by identifying the fluorescent labels.

[0019] Furthermore, the 5' ends of the two specific forward primers were labeled with a FAM group and a HEX group, respectively.

[0020] The kit also includes a PCR reaction solution, which includes a PCR buffer, a TaqDNA polymerase and a dNTP mixture. The dosage relationship between the components in the PCR reaction solution is the ratio under conventional PCR conditions, which is common knowledge to those skilled in the art.

[0021] The present invention also provides a method for screening or identifying the fruit weights of different tomato varieties, comprising the following steps:

[0022] (1) Extracting genomic DNA from the tomato plants to be tested;

[0023] (2) using the genomic DNA of the tomato plant as a template and performing PCR amplification using a primer set for amplifying the KASP molecular marker;

[0024] (3) Detect the PCR products, perform genotyping on the tested tomatoes, and then determine the fruit weight.

[0025] Furthermore, in step (1), genomic DNA is extracted from leaves of seedlings of the tomato variety to be tested.

[0026] Furthermore, in step (2), the PCR reaction system is: 0.8 μL of DNA template, 0.75 μL of 2×KASP Master mix, and 0.05 μL of KASP Assay Mix. The KASP Assay Mix is ​​prepared by mixing 100 μM forward primer 1, forward primer 2, reverse primer, and ddH2O in a ratio of 12:12:30:46.

[0027] The PCR reaction program was as follows: step 1: pre-denaturation at 94°C for 15 min; step 2: denaturation at 94°C for 20 s, annealing and extension at 61-55°C for 60 s, 10 cycles, with the temperature decreasing by 0.6°C each cycle; step 3: denaturation at 94°C for 20 s, annealing and extension at 55°C for 60 s, for a total of 26 cycles.

[0028] The present invention uses a primer pair for specific detection of SNP No. 336003 for PCR amplification. Studies have shown that this site as a detection target has obtained a highly consistent and clearly defined population typing effect in a tomato population, and the typing efficiency is 95.7%.

[0029] Specifically, PCR amplification is performed using a primer set with a nucleotide sequence such as SEQ ID NO.2-4; when the identified genotype is CC, the tomato fruit weight is judged to be small; when the identified genotype is TT, the tomato fruit weight is judged to be large.

[0030] The present invention has the following beneficial effects:

[0031] (1) The present invention used the whole genome association analysis method to obtain the SNP site FW-336003 associated with tomato fruit weight, and further developed the related KASP molecular markers and KASP primers. It was verified in 231 tomato populations that highly consistent and clearly defined population typing results were obtained, with a typing efficiency of 95.7%.

[0032] (2) The present invention uses KASP molecular markers for typing tomato fruit weight, which can clearly separate tomato materials of different genotypes, and the difference in fruit weight of materials of different genotypes reaches a highly significant difference level. The fruit weight of TT genotype materials is heavier than that of CC genotype materials. In the breeding process, the allelic variation of fruit weight can be judged according to its genotype, and the excellent population that meets the breeding target size can be quickly selected at the seedling stage, realizing the early screening of the breeding population and accelerating the molecular breeding process of tomato varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is a Manhattan plot of the genome-wide association analysis results of tomato fruit weight traits in Example 1 of the present invention.

[0034] Figure 2 This is the KASP marker typing diagram of the related gene of the FW-336003 marker site in 20 materials.

[0035] Figure 3 This is the KASP marker typing diagram of the related gene of the FW-287848 marker site in 20 materials.

[0036] Figure 4 This is the KASP marker typing diagram of the related gene of the FW-301492 marker site in 20 materials.

[0037] Figure 5 This is the KASP marker typing diagram of the related gene of the FW-285173 marker site in 20 materials.

[0038] Figure 6 This is the KASP marker typing diagram of the related gene of the FW-293015 marker site in 20 materials.

[0039] Figure 7 This is the KASP marker typing diagram of the related gene of the FW-292352 marker site in 20 materials.

[0040] Figure 8 This is the KASP marker typing diagram of the related gene of the FW-339507 marker site in 20 materials.

[0041] Fig. 9 This is the KASP marker typing diagram of the related gene of the FW-297125 marker site in 20 materials.

[0042] Fig.10 This is the KASP marker typing diagram of the related gene of the FW-239835 marker site in 20 materials.

[0043] Fig.11 This is the KASP marker typing diagram of the related gene of the FW-296945 marker site in 20 materials.

[0044] Fig.12 This is the KASP marker typing diagram of the related gene of the FW-227918 marker site in 20 materials.

[0045] Fig.13 This is the KASP marker typing diagram of the related gene of the FW-222564 marker site in 20 materials.

[0046] Fig.14 This is the KASP marker typing diagram of the related gene of the FW-301410 marker site in 20 materials.

[0047] Fig.15 This is the genotyping diagram of different tomato materials using the KASP molecular marker FW-336003.

[0048] Fig.16 Genotypes identified by KASP molecular markers for FW-336003 and allelic variation in fruit weight. DETAILED DESCRIPTION

[0049] The present invention will be further described below in conjunction with specific embodiments. The following embodiments are only used to illustrate the present invention and are not intended to limit the scope of application of the present invention. Without departing from the spirit and essence of the present invention, modifications or replacements made to the inventive method, steps or conditions all belong to the scope of the present invention.

[0050] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0051] Example 1: Screening of SNP loci associated with tomato fruit weight

[0052] 1. Investigation of phenotypic data of tomato fruit weight

[0053] The natural population used in the present invention consists of 241 gooseberry tomatoes, cherry tomatoes and cultivated tomatoes. The material numbers are shown in Table 1. The materials come from the tomato planting resource library and the tomato and pepper group collection materials of the Vegetable Institute of Zhejiang Academy of Agricultural Sciences.

[0054] Table 1. 241 natural population materials of tomato

[0055]

[0056]

[0057]

[0058] The natural population was planted at the Yangdu Research and Innovation Base of Zhejiang Academy of Agricultural Sciences in 2021. Three holes were planted in the seedling tray for each tomato material, and 6 plants were retained in each hole. After germination, the strongest transplants were selected. Five plants were planted for each material. After the fruits were ripe, 7 complete and disease-free fruits in the commercial maturity stage were randomly selected. The weight of the fruit was accurately measured with an electronic balance, and the single fruit weight was taken as the final fruit weight phenotypic data.

[0059] 2. Genome-wide association analysis

[0060] 241 tomato materials were sent for resequencing, and the obtained SNP sites were filtered according to the following criteria:

[0061] (1) Consider the genotype of each individual at this site and only retain the genotypes with a GQ value greater than 20 and covered by more than 5 reads;

[0062] (2) Filter out multi-allelic SNPs markers and retain only biallelic sites;

[0063] (3) Filter out sites with a deletion rate higher than 20%;

[0064] (4) Filter out sites with minimum allele frequency (MAF) < 0.05.

[0065] Finally, 8,668,967 SNP loci were obtained for genome-wide association analysis.

[0066] The SNP loci obtained after screening were subjected to association analysis and significance test with the fruit weight phenotype, and finally the SNP loci associated with the target trait were obtained. The specific method was to use GEMMA software for genome-wide association analysis with P = 1 × 10 -8 The significant SNP loci were selected as the threshold level of significant correlation, and 4 segments significantly correlated with fruit weight were identified in the whole genome (such as Figure 1 ), among which the segment on chromosome 9 of the tomato genome has the greatest genetic contribution to fruit weight.

[0067] The above-mentioned tomato genome version is SL3.1, and the specific sequence number is GCF_000188115.5.

[0068] Thirteen SNP sites located in exons on chromosome 9 were initially selected, namely:

[0069] 1. FW-336003, located at position 336,003 of chromosome 9 of the tomato genome, with a polymorphic site of T / C;

[0070] 2. FW-287848, located at position 287,848 of chromosome 9 of the tomato genome, with a polymorphic site of G / T;

[0071] 3. FW-301492, located at position 301,492 of chromosome 9 of the tomato genome, with a polymorphic site of A / G;

[0072] 4. FW-285173, located at position 285,173 of chromosome 9 of the tomato genome, with a polymorphic site of A / G;

[0073] 5. FW-293015, located at position 293,015 of chromosome 9 of the tomato genome, with a polymorphic site of G / A;

[0074] 6. FW-292352, located at position 292,352 of chromosome 9 of the tomato genome, with a polymorphic site of C / T;

[0075] 7. FW-339507, located at position 339,507 of chromosome 9 of the tomato genome, with a polymorphic site of G / A;

[0076] 8. FW-297125, located at position 297,125 of chromosome 9 of the tomato genome, with a polymorphic site of C / G;

[0077] 9. FW-239835, located at position 239,835 of chromosome 9 of the tomato genome, with a polymorphic site of T / C;

[0078] 10. FW-296945, located at position 296,945 of chromosome 9 of the tomato genome, with a polymorphic site of T / A;

[0079] 11. FW-227918, located at position 227,918 of chromosome 9 of the tomato genome, with a polymorphic site of T / C;

[0080] 12. FW-222564, located at position 222,564 of chromosome 9 of the tomato genome, with a polymorphic site of T / C;

[0081] 13. FW-301410, located at position 301,410 of chromosome 9 of the tomato genome, with a polymorphic site of T / A.

[0082] Example 2: Development of KASP molecular markers associated with tomato fruit weight

[0083] 1. Referring to the sequence information of Phytozome, 100 bp sequences upstream and downstream of the relevant SNP sites were downloaded to design KASP primers. The primer set for each site included two specific forward primers F1 and F2, and a universal reverse primer R. The 5' ends of the two specific forward primers were connected to the fluorescent label sequence carrying the FAM group and the fluorescent label sequence carrying the HEX group, respectively. The specific sequences are shown in Table 2.

[0084] Table 2. Nucleotide sequences

[0085]

[0086]

[0087]

[0088] 2. Use KASP primers of the above 13 SNP sites to perform KASP genotyping on 20 tomato materials, and further select KASP molecular markers with good typing effects.

[0089] Table 3. 20 tomato materials

[0090]

[0091] The specific operation of KASP genotyping is as follows:

[0092] Tomato genomic DNA was extracted by magnetic bead method, and the tomato genomic DNA to be tested was used as template. The genomic DNA samples of the above 20 tomato materials were amplified and fluorescently detected using 13 pairs of KASP primer sets using IntelliQube genotyping platform.

[0093] PCR reaction system: DNA 0.8 μL, 2x KASP Master mix 0.75 μL, KASP Assay Mix 0.05 μL. KASP Assay Mix was prepared by mixing 100 μM of specific forward primer F1, specific forward primer F2, universal reverse primer R and ddH2O in a volume ratio of 12:12:30:46.

[0094] PCR reaction program: pre-denaturation at 94°C for 15 min, denaturation at 94°C for 20 sec, annealing at 61-55°C (temperature decreased by 0.6°C each cycle) for 60 sec, for a total of 10 cycles; denaturation at 94°C for 20 sec, annealing at 55°C for 60 sec, for a total of 26 cycles.

[0095] The IntelliQube fully automatic PCR instrument was used to read and analyze the fluorescence data. The horizontal and vertical axes represent the magnitude of FAM and HEX fluorescence signals, respectively. If the fluorescence signal of the PCR amplification result is red, which is consistent with the color of the fluorescent connector of the forward primer F1, it means that the sample carries the FAM fluorescence signal. If the fluorescence signal of the PCR amplification result is blue, which is consistent with the color of the fluorescent connector of the forward primer F2, it means that the sample carries the HEX fluorescence signal. The black hollow dots represent the amplification signal of the negative control (NTC), and the gray hollow dots represent DNA samples with no detected amplification signal.

[0096] like Figure 2-Figure 14 As shown, the fluorescence signal of the PCR amplification product showed that markers FW-301492 and FW-297125 could not be typed; the typing efficiency of marker FW-301410 was 50%; the typing efficiency of markers FW-239835 and FW-227918 was 75%; the typing efficiency of markers FW-287848, FW-285173, FW-293015, FW-292352, FW-339507, and FW-222564 was 90%; the typing efficiency of markers FW-296945 and FW-336003 was 95% and 100%. Taking high typing efficiency and concentrated fluorescent signal points as typing standards, FW-336003 had the highest typing efficiency, concentrated fluorescent signals, and the two signals were connected to the origin at right angles, so the FW-336003 KASP molecular marker was finally selected.

[0097] Example 3: Application of KASP molecular markers for tomato fruit weight selection

[0098] Firstly, a natural population material containing 231 tomato samples was constructed using tomato varieties such as currant tomatoes, cherry tomatoes, and cultivated tomatoes (see Table 4). In 2024, population phenotypic identification was carried out in Hangzhou, Zhejiang. Five plants were planted in each material, and seven commercially mature tomatoes were randomly selected from each material. The fruits were accurately measured with an electronic balance, and the weight of a single fruit was taken as the identification result, finally obtaining the phenotypic data of fruit weight.

[0099] Table 4. 231 natural population materials of tomato

[0100]

[0101]

[0102]

[0103] The specific application method of using KASP molecular markers for tomato fruit weight selection is as follows:

[0104] The tomato genomic DNA was used as a template to extract the tomato genomic DNA by magnetic bead method, and the FW-336003KASP molecular marker primer set was used for PCR amplification.

[0105] PCR reaction system: contains 0.8 μL template DNA, 0.75 μL 2×KASP Master mix (from LGC), and 0.05 μL KASP Assay Mix. The preparation method of KASP Assay Mix is ​​to mix 100 μM specific forward primer F1, specific forward primer F2, universal reverse primer R and ddH2O in a volume ratio of 12:12:30:46;

[0106] PCR reaction program: pre-denaturation at 94°C for 15 min, denaturation at 94°C for 20 sec, annealing at 61-55°C (temperature decreased by 0.6°C each cycle) for 60 sec, for a total of 10 cycles; denaturation at 94°C for 20 sec, annealing at 55°C for 60 sec, for a total of 26 cycles.

[0107] The IntelliQube fully automatic PCR instrument was used to read and analyze the fluorescence data. The horizontal and vertical axes represent the magnitude of FAM and HEX fluorescence signals, respectively. If the fluorescence signal of the PCR amplification result is red, which is consistent with the color of the fluorescent connector of the forward primer F1, it means that the sample carries a FAM fluorescence signal. If the fluorescence signal of the PCR amplification result is blue, which is consistent with the color of the fluorescent connector of the forward primer F2, it means that the sample carries a HEX fluorescence signal. The black hollow dots represent the amplification signals of the negative control (NTC), and the gray hollow dots represent samples with no detected fluorescence signals.

[0108] The typing efficiency of FW-336003KASP molecular marker in 231 tomato populations was 95.7% (e.g. Fig.15 As shown in the figure, 221 materials were distinguished and clustered into two genotypes, and the FAM and HEX amplification signals were at right angles to the line connecting the origins, indicating a good typing effect.

[0109] The data of tomato fruit genotype detected by KASP molecular markers and fruit weight phenotype statistically analyzed in the field are shown in Table 5.

[0110] Table 5. Genotypic and phenotypic data of 231 materials

[0111]

[0112]

[0113]

[0114] Note: / in the table means no fluorescence signal was detected.

[0115] Combining the genotype and fruit weight data, it was found that the average single fruit weight of tomato materials carrying the CC (HapI) genotype was 56.72 g, while the average single fruit weight of tomato materials carrying the TT (HapII) genotype was 164.2 g. The difference in tomato fruit weight between the two genotypes reached an extremely significant difference level (p<0.0001) (e.g. Fig.16 As shown in the figure), the allele T / C at the FW-336003 locus is extremely significantly correlated with tomato fruit weight, indicating that this marker can be used for molecular marker-assisted selection breeding of tomato fruit weight.

Claims

1. Application of single nucleotide polymorphism sites as detection targets in screening or identifying tomato fruit weight, characterized in that: The single nucleotide polymorphism site is at position 336003 of the full-length sequence of chromosome 9 of the tomato genome, and there is a polymorphism of T>C; the tomato genome sequence number is GCF_000188115.5; when the genotyping is TT, the tomato fruit weight is larger; when the genotyping is CC, the tomato fruit weight is smaller.

2. A method for screening or identifying the fruit weight of different tomato varieties, characterized in that: The following steps are involved: (1) extracting genomic DNA from the tomato plants to be tested; (2) Using the genomic DNA of tomato plants as a template, PCR amplification was performed using a primer set for amplifying a KASP molecular marker related to tomato fruit weight, wherein the primer set for amplifying the KASP molecular marker was used to specifically detect the single nucleotide polymorphism site at position 336003 of the full-length sequence of chromosome 9 in the tomato genome, wherein the single nucleotide polymorphism site had a polymorphism of T>C; the sequence number of the tomato genome was GCF_000188115.5; (3) The PCR product is tested, and the genotype of the tomato to be tested is performed to determine the fruit weight. When the genotype is CC, the tomato fruit weight is judged to be small; when the genotype is TT, the tomato fruit weight is judged to be large.

3. The method for screening or identifying the fruit weight of different tomato varieties according to claim 2, characterized in that: The KASP molecular marker is a nucleotide sequence of 40-100 bp upstream and downstream, with the single nucleotide polymorphism site as the coordinate.

4. The method for screening or identifying the fruit weight of different tomato varieties according to claim 3, characterized in that: The nucleotide sequence of the KASP molecular marker is shown in SEQ ID NO.

1.

5. The method for screening or identifying the fruit weight of different tomato varieties according to claim 4, characterized in that: The primer set for amplifying the KASP molecular marker includes a specific forward primer 1, a specific forward primer 2 and a universal reverse primer, the nucleotide sequence of the specific forward primer 1 is shown in SEQ ID NO.2, the nucleotide sequence of the specific forward primer 2 is shown in SEQ ID NO.3, and the nucleotide sequence of the universal reverse primer is shown in SEQ ID NO.

4.

6. The method for screening or identifying the fruit weight of different tomato varieties according to claim 5, characterized in that: The 5' ends of the specific forward primer 1 and the specific forward primer 2 are respectively connected to two different fluorescent label sequences.

7. The method for screening or identifying the fruit weight of different tomato varieties according to claim 6, characterized in that: The 5' ends of the two specific forward primers were labeled with a FAM group and a HEX group, respectively.

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