CN117683938B - KASP molecular marker closely linked with tomato fruit width and application thereof - Google Patents
KASP molecular marker closely linked with tomato fruit width and application thereof Download PDFInfo
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Abstract
The invention provides a KASP molecular marker closely linked with tomato fruit width and application thereof, belonging to the technical field of determination methods containing nucleic acid, wherein the KASP molecular marker is used for specifically detecting SNP locus at 3154269 th site of full-length sequence of chromosome 4 of tomato genome, and variation locus of SNP is C/T; the nucleotide sequence of the KASP molecular marker is shown as SEQ ID NO.1 in the sequence table. According to the invention, tomato materials with different genotypes can be obviously separated by using the marker, the difference of the fruit widths of the tomato materials with different genotypes is obvious at the level of P <0.0001, the CC genotype material is larger than the fruit width of the TT genotype material, the allelic variation of the sample fruit width is judged according to the genotype, high-throughput, rapid and accurate detection can be realized in the seedling stage, the breeding of different tomato fruit widths is assisted, and the breeding and improvement process of tomato varieties is accelerated.
Description
Technical Field
The invention belongs to the technical field of nucleic acid-containing determination methods, and relates to a KASP molecular marker closely linked with tomato fruit width and application thereof.
Background
Tomato (Solanum lycopersicum) is native to south america and is a worldwide vegetable. The tomatoes are light-loving and temperature-loving, have no strict cultivation conditions, strong environment adaptability, long growth period, long fruiting period, high yield, good flavor and large market demand, are rich in various nutritional ingredients such as soluble sugar, carotenoid, VC, lycopene, organic acid and the like, are widely cultivated in the north and south of China, and are one of main crops cultivated in various places of China.
Fruit width is one of the important economic traits of fruits, affecting crop yield, quality and commodity. However, fruit width is a relatively complex quantitative inherited agronomic trait that is susceptible to many aspects of inheritance, hormones, external environments, artificial cultivation measures, etc. during development. The traditional breeding is mainly used for selecting fruits according to phenotypes, and the accuracy of breeding results is greatly influenced by the environment and is time-consuming and labor-consuming. The whole genome association analysis (Genome Wide Association Study, GWAS) is based on quantitative trait linkage disequilibrium theory, by expression and genotype association analysis to obtain significant association markers affecting phenotypic traits. The GWAS is utilized to excavate relevant sites of target characters and then is combined with molecular markers to assist breeding, so that the breeding efficiency of breeders can be remarkably improved, the field workload is reduced, the breeding cost is reduced, the product quality and quality can be further improved, and the development of relevant industries is promoted.
In recent years, the third generation molecular marking technology, SNP markers are rapidly developed to cover the whole genome, and the advantages of high density and good stability are obtained. The competitive allelic variation specific PCR (Kompetitive ALLELE SPECIFIC PCR, KASP) can accurately judge the double alleles of SNPs and InDels on specific sites in a wide genome DNA sample, has the characteristics of high marking success rate, accurate typing, low development and detection cost, strong detection flux flexibility and the like, and is a mainstream marker for developing high-flux gene analysis at present.
No KASP molecular markers for tomato fruit width are reported in the prior art.
Disclosure of Invention
Aiming at the defects existing in the prior art, the invention provides a KASP molecular marker closely linked with the width of tomato fruits and application thereof, and the following aims are fulfilled: the genotyping effect is good, and can be used for detecting allele loci which are extremely obviously related to the width of tomato fruits.
In order to solve the technical problems, the invention adopts the following technical scheme:
The KASP molecular marker is used for specifically detecting SNP locus at 3154269 th site of full-length sequence of chromosome 4 of tomato genome, and variation locus of SNP is C/T.
The nucleotide sequence of the KASP molecular marker is shown as SEQ ID NO.1 in the sequence table.
The KASP molecular marker comprises 3 KASP primers, namely 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 as SEQ ID NO.2 in a sequence table; the nucleotide sequence of the specific forward primer 2 is shown as SEQ ID NO.3 in a sequence table; the nucleotide sequence of the universal reverse primer is shown as SEQ ID NO.4 in the sequence table.
The specific forward primer 1 and the specific forward primer 2 are connected with two different fluorescent tag sequences.
The 5' end of the specific forward primer 1 is connected with a fluorescence tag sequence of FAM group; and the 5' end of the specific forward primer 2 is connected with a fluorescent tag sequence of HEX group.
A detection kit or detection reagent prepared from KASP molecular marker.
Application of KASP molecular marker in identification of tomato fruit width
The application comprises the following steps:
(1) Extracting genome DNA of a tomato plant to be detected;
(2) Taking genomic DNA of tomato plants as a template, and performing PCR amplification and KASP reaction detection by using KASP primers corresponding to KASP molecular markers;
(3) Reading a fluorescent signal detected by KASP reaction, and judging that the sample carries genotype CC when the genomic DNA of the tomato plant sample shows a fluorescent group signal carried by the specific forward primer 1, wherein the fruit width is larger; if the genomic DNA of the tomato plant sample shows the signal of the fluorescent group carried by the specific forward primer 2, the sample can be judged to carry genotype TT, and the fruit width is smaller.
Compared with the prior art, the invention has the following beneficial effects:
(1) The invention provides a SNP locus SlFW-3154269 closely linked with tomato fruit width genes, further develops related KASP molecular markers and KASP primers, obtains a highly consistent and clearly-defined group parting effect in 109 tomato groups, has parting efficiency of 100%, and provides a rapid and reliable detection means for genetic improvement of tomato fruit width characters.
(2) The KASP molecular marker is used for typing tomato fruit widths, obviously separates tomato materials with different genotypes, the difference of the fruit widths of the tomato materials with different genotypes is obvious at the level of P <0.0001, the CC genotype material is larger than the fruit width of the TT genotype material, the allelic variation of the sample fruit width is judged according to the genotype of the CC genotype material, high-throughput, rapid and accurate detection can be realized in a seedling stage, the breeding of different tomato fruit widths is assisted, and the breeding and improvement process of tomato varieties is accelerated.
Drawings
FIG. 1 is a population analysis result;
Wherein FIG. 1A is a diagram of linkage disequilibrium analysis, and FIG. 1B is a two-dimensional diagram of principal component analysis;
FIG. 2 is a Manhattan plot and Q-Q plot (Quantile-quantile plot) of the results of whole genome correlation analysis of tomato fruit width traits in example 1 of the present invention;
Wherein fig. 2A is a manhattan diagram; FIG. 2B is a Q-Q diagram;
FIG. 3 is a graph of the relative gene KASP signature at the SlFW4_3154269 signature site in 22 material parts;
FIG. 4 is a graph of the relative gene KASP signature at the SlFW4_2986588 signature site in 22 material parts;
FIG. 5 is a graph of the relative gene KASP signature at the SlFW4_2997658 signature site in 22 material parts;
FIG. 6 is a graph of the relative gene KASP signature at the SlFW4_3116566 signature site in 22 material parts;
FIG. 7 is a graph of the relative gene KASP signature at the SlFW4_3192067 signature site in 22 material parts;
FIG. 8 is a genotyping chart of KASP molecular markers of SlFW4_3154269 for different tomato germplasm;
FIG. 9 is a graph showing allelic variation of the identified genotypes of the KASP molecular markers of SlFW4_3154269 versus fruit width.
Detailed Description
Materials, reagents, etc. used in the examples described below are commercially available unless otherwise specified;
the version of tomato genome adopted by the invention is SL3.1, and the specific serial number is GCF_000188115.5.
EXAMPLE 1 screening of SNP loci related to tomato fruit width
1) Material
The invention adopts 241 parts of tomato core germplasm population materials composed of wild tomatoes, small tomatoes and large tomatoes, and the material numbers are shown in tables 1-3. A part of materials are collected and created by tomato capsicum groups in vegetable stations of Zhejiang national academy of sciences, and a part of materials are derived from tomato germplasm resource libraries.
TABLE 1 st to 81 st tomato core germplasm population Material
Table 2 82-162 parts tomato core germplasm population Material
Table 3 163-241 parts tomato core germplasm population Material
。
2) Fruit phenotype identification
The identification experiment of the fruit width phenotype is carried out in Hangzhou in 2021, a random block design is adopted, 3 holes are planted in each tomato material, 6 plants are reserved in each hole, 6 complete and disease-free fruits in the commodity maturity stage are randomly selected in each tomato material according to tomato germplasm resource description Specification, the width of the fruits is accurately measured by a vernier caliper, the average value is taken as an identification result, and finally the phenotype data of the fruit width is obtained.
3) High quality SNP locus screening
Re-sequencing 241 tomato core germplasm groups by using a re-sequencing technology, wherein the GQ value on the genotype is more than 20 and more than 5 reads are covered; a bi-allele; the loss rate is less than or equal to 20 percent; and filtering SNP loci by taking a minimum allele frequency MAF (Minor allele frequency) of more than 0.05 as a screening standard, and finally obtaining 8668967 high-quality SNP loci.
4) GWAS analysis
To avoid the occurrence of false positive results when two subpopulations are mixed for association analysis, population analysis is performed prior to association analysis (see FIG. 1). And carrying out association analysis and significance detection on the SNP locus obtained after screening and the fruit width phenotype, and finally obtaining the SNP locus related to the target character. The specific method is to perform genome-wide association analysis by using GEMMA software, select significant SNP sites with p=1×10 -8 as a threshold level line of significant correlation, identify 5 segments significantly correlated with fruit width in the whole genome (as in fig. 2), wherein the segment on chromosome 4 has the greatest genetic contribution to fruit width. Initially selecting 5 SNP loci with P value (-log 10) more than or equal to 12.0 on chromosome 4, performing KASP genotyping on 22 tomato materials, and further selecting SNP loci with good genotyping effect.
The 5 SNP sites were named: slFW4_3154269, slFW4_2986588, slFW4_2997658, slFW4_3116566, slFW4_3192067;
The specific positions of the 5 SNP loci are as follows:
SlFW _3154269, located at 3154269 th position of full length sequence of chromosome 4 of tomato genome, the variation site of SNP is C/T;
SlFW _2986588, located at 2986588 th position of full length sequence of chromosome 4 of tomato genome, the variation site of SNP is A/C;
SlFW _2997658, located at 2997658 th position of full length sequence of chromosome 4 of tomato genome, the variation site of SNP is A/G;
SlFW _3116566, located at 3116566 th position of full length sequence of chromosome 4 of tomato genome, the variation site of SNP is A/G;
SlFW4_3192067, located at 3192067 th position of full length sequence of chromosome 4 of tomato genome, the variation site of SNP is T/G.
EXAMPLE 2 development of allelic variant site KASP molecular markers related to tomato fruit width
According to the physical position information of the obviously associated SNP locus, referring to the sequence information in Phytozome software, downloading 80bp sequences at the upstream and downstream of the SNP locus for KASP primer design, wherein the primers comprise two specific forward primers and a universal reverse primer, and the primer sequences are synthesized by the division of biological engineering (Shanghai).
The method comprises the following steps:
(1) SlFW4_3154269 KASP molecular marker and KASP primer
The nucleotide sequence of the KASP molecular marker is shown as SEQ ID NO.1 in the sequence table;
the sequence of the KASP primer is as follows:
SlFW4_3154269 specific forward primer 1: the nucleotide sequence is shown as SEQ ID NO.2 in the sequence table;
SlFW4_3154269 specific forward primer 2: the nucleotide sequence is shown as SEQ ID NO.3 in the sequence table;
SlFW 4-3154269 general reverse primer: the nucleotide sequence is shown as SEQ ID NO.4 in the sequence table.
(2) SlFW4_2986588 KASP molecular marker and KASP primer
The nucleotide sequence of the KASP molecular marker is shown as SEQ ID NO.5 in the sequence table;
the sequence of the KASP primer is as follows:
SlFW4_2986588 specific forward primer 1: the nucleotide sequence is shown as SEQ ID NO.6 in the sequence table;
SlFW4_2986588 specific forward primer 2: the nucleotide sequence is shown as SEQ ID NO.7 in the sequence table;
SlFW 4-2986588 general reverse primer: the nucleotide sequence is shown as SEQ ID NO.8 in the sequence table.
(3) SlFW4_2997658 KASP molecular marker and KASP primer
The nucleotide sequence of the KASP molecular marker is shown as SEQ ID NO.9 in the sequence table;
the sequence of the KASP primer is as follows:
SlFW4_2997658 specific forward primer 1: the nucleotide sequence is shown as SEQ ID NO.10 in the sequence table;
SlFW4_2997658 specific forward primer 2: the nucleotide sequence is shown as SEQ ID NO.11 in the sequence table;
SlFW 4-2997658 general reverse primer: the nucleotide sequence is shown as SEQ ID NO.12 in the sequence table.
(4) SlFW4_3116566 KASP molecular marker and KASP primer
The nucleotide sequence of the KASP molecular marker is shown as SEQ ID NO.13 in the sequence table
SlFW4_3116566 specific forward primer 1: the nucleotide sequence is shown as SEQ ID NO.14 in the sequence table;
SlFW4_3116566 specific forward primer 2: the nucleotide sequence is shown as SEQ ID NO.15 in the sequence table;
SlFW 4-3116566 general reverse primer: the nucleotide sequence is shown as SEQ ID NO.16 in the sequence table.
(5) SlFW4_3192067 KASP molecular marker and KASP primer
The nucleotide sequence of the KASP molecular marker is shown as SEQ ID NO.17 in the sequence table;
SlFW4_3192067 specific forward primer 1: the nucleotide sequence is shown as SEQ ID NO.18 in the sequence table;
SlFW4_3192067 specific forward primer 2: the nucleotide sequence is shown as SEQ ID NO.19 in the sequence table;
SlFW 4-3192067 general reverse primer: the nucleotide sequence is shown as SEQ ID NO.20 in the sequence table.
The 5 'end of the specific forward primer 1 is connected with a fluorescence tag sequence of FAM group, and the 5' end of the specific forward primer 2 is connected with a fluorescence tag sequence of HEX group.
And (3) performing KASP genotyping on 22 parts of tomato materials by adopting the KASP primer pair with 5 SNP loci, and further selecting KASP molecular markers with good genotyping effect.
Table 422 tomato material number
The specific procedure for KASP genotyping is as follows:
Fresh leaves of each strain are taken, genome DNA is extracted by adopting a CTAB method, a IntelliQube genotyping platform is used, and KASP primers are utilized to amplify and fluorescence detect genome DNA samples of the groups.
The PCR system for KASP reaction detection is: DNA 0.8. Mu.l, 2x KASP Master mix 0.75. Mu.l (supplied by LGC Co.), KASP mix primer 0.05. Mu.l.
The preparation method of the KASP mixed primer comprises the steps of respectively diluting the specific forward primer 1, the specific forward primer 2 and the universal reverse primer to 100 mu M by double distilled water, and then mixing the mixed primers with ddH 2 O according to the volume ratio of 12:12:30:46;
the PCR reaction program for KASP reaction detection is 94 ℃,15 min,94 ℃,20 sec, 61-55 ℃ and 60sec, the renaturation temperature is reduced by 0.6 ℃ and 55 ℃ and 60sec in each cycle, and the total cycle is 10 times; 94 ℃,20 sec,55 ℃, 60sec and 26 times of total circulation.
The fluorescence data reading and analysis are carried out by using a IntelliQube machine, the abscissa and the ordinate respectively represent the magnitudes of FAM and HEX fluorescence signals, the signal point close to the vertical axis represents that only HEX fluorescence signals are detected, the signal point close to the horizontal axis represents that only FAM signals are detected, the signal point at the middle position can simultaneously detect FAM and HEX signals, the black dot close to the origin represents the amplified signals of negative control (no DNA sample is added), and the FAM and HEX signals are not obvious. The amplification signals are respectively connected with the origin, and the closer the formed included angle is to the right angle, the better the parting effect is. As shown in fig. 3-7, none of the markers SlFW4_2986588, slFW4_2997658 and SlFW4_3116566 can be typed (see fig. 4-6), only one typing result of the marker SlFW4_3192067 is an intermediate type (see fig. 7), all of the signals with FAM are typed by the marker SlFW4_3154269, the typing efficiency is 91%, and the signal points are concentrated (see fig. 3). Therefore, after eliminating the markers which cannot be typed or have poor typing effect, the SlFW4_3154269 locus and the corresponding molecular markers and primers thereof are selected preferentially finally.
Example 3 application of Kasp molecular markers for tomato fruit width selection
Firstly, a natural population material containing 109 parts of tomatoes is constructed by using varieties of wild tomatoes, cherry tomatoes, large tomatoes and the like, population phenotype identification is carried out in Hangzhou Zhejiang in 2022 in tables 5-6, a field test adopts a completely random block design, 3 holes are repeatedly planted in each material, and 6 strains of materials are reserved in each hole. Referring to tomato germplasm resource description Specification, 6 tomatoes in commodity maturity are randomly selected from each material, the width is accurately measured by using a vernier caliper, the average value is taken as an identification result, and finally phenotype data of the fruit width is obtained, wherein 16 strains of materials do not obtain the fruit width data due to cultivation management.
TABLE 51 st to 54 th parts tomato natural population materials
Table 6, 55 th to 109 th portions of tomato natural population material
The specific application method for selecting tomato fruit width by using KASP molecular marker is as follows:
And taking seedling leaves of each strain growing for two weeks, extracting genome DNA by adopting a CTAB method, and amplifying and fluorescence detecting genome DNA samples of the population by using a IntelliQube genotyping platform and using KASP molecular markers of SlFW4_ 3154269. A PCR reaction system containing 0.8. Mu.l of template DNA, 2x KASP Master mix 0.75. Mu.l (supplied by LGC company) and 0.05. Mu.l of KASP mixed primer.
The preparation method of the KASP mixed primer comprises the steps of respectively diluting the specific forward primer 1, the specific forward primer 2 and the universal reverse primer to 100 mu M by double distilled water, and then mixing the mixed primers with ddH 2 O according to the volume ratio of 12:12:30:46;
The PCR reaction program is 94 ℃,15 min,94 ℃, 20sec, 61-55 ℃ and 60sec, the renaturation temperature of each cycle is reduced by 0.6 ℃,55 ℃ and 60sec, and the total cycle is 10 times; 94 ℃, 20sec,55 ℃, 60sec and 26 times of total circulation.
Performing fluorescence data reading and analysis by using a IntelliQube machine, wherein the abscissa and the ordinate respectively represent the magnitudes of FAM and HEX fluorescence signals, and signal points close to the position of the vertical axis represent that only HEX fluorescence signals are detected, and the corresponding tomato genotype is TT; signal points near the horizontal axis represent that only FAM signals are detected, and the corresponding tomato genotype is CC; the black dots near the origin indicate the amplified signal of the negative control (no DNA sample added), neither FAM nor HEX signal is evident.
The KASP molecular markers gave a highly consistent and well-defined population typing effect in 109 tomato populations (as shown in figure 8). In the parting result of SlFW-3154269, 2 genotypes can be obviously distinguished and clustered, and two amplification signals of FAM and HEX form right angles with the original point line, so that the parting effect is good.
The genotype of tomato fruits and the data of the fruit width resistance phenotype of field statistics detected by the KASP molecular markers are shown in tables 7-8; combining genotype and fruit width resistance phenotypes, the average fruit width of the CC (HapI) carrying genotype line was found to be 61.13mm, while the average fruit width of the TT (HapII) carrying genotype line was found to be 35.49mm, the fruit widths of both genotype lines reached very significant levels (p < 0.0001) (as shown in fig. 9), the allele C/T at SlFW4_3154269 locus correlated very significantly with tomato fruit width, indicating that this marker could indicate selection of fruit width.
TABLE 7 strains with genotype CC and phenotypic data
TABLE 8 strains with genotype TT and phenotypic data
。
Finally, while the invention has been described in detail in the foregoing general description and with reference to specific embodiments, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Claims (7)
1. The KASP molecular marker closely linked with the width of tomato fruits is characterized in that: amplifying the primer of the KASP molecular marker, wherein the primer is used for specifically detecting SNP locus at 3154269 th site of the full-length sequence of chromosome 4 of the tomato genome, and the variation locus of the SNP is C/T;
the nucleotide sequence of the KASP molecular marker is shown as SEQ ID NO.1 in the sequence table.
2. The KASP molecular marker closely linked to tomato fruit width of claim 1, wherein: the primers for amplifying the KASP molecular marker are a specific forward primer 1, a specific forward primer 2 and a universal reverse primer respectively; the nucleotide sequence of the specific forward primer 1 is shown as SEQ ID NO.2 in a sequence table; the nucleotide sequence of the specific forward primer 2 is shown as SEQ ID NO.3 in a sequence table; the nucleotide sequence of the universal reverse primer is shown as SEQ ID NO.4 in the sequence table.
3. The KASP molecular marker closely linked to tomato fruit width of claim 2, wherein: the specific forward primer 1 and the specific forward primer 2 are connected with two different fluorescent tag sequences.
4. The KASP molecular marker closely linked to tomato fruit width of claim 2, wherein: the 5' end of the specific forward primer 1 is connected with a fluorescence tag sequence of FAM group; and the 5' end of the specific forward primer 2 is connected with a fluorescent tag sequence of HEX group.
5. A detection kit or detection reagent prepared by amplifying a KASP molecule-labeled KASP primer of claim 1.
6. Use of a primer for amplifying a KASP molecular marker according to claim 4 for identifying tomato fruit width.
7. The use according to claim 6, characterized in that: the method comprises the following steps:
(1) Extracting genome DNA of a tomato plant to be detected;
(2) Taking genomic DNA of tomato plants as a template, and carrying out PCR amplification and KASP reaction detection by using primers for amplifying KASP molecular markers;
(3) Reading a fluorescent signal detected by KASP reaction, and judging that the sample carries genotype CC when the genomic DNA of the tomato plant sample shows a fluorescent group signal carried by the specific forward primer 1, wherein the fruit width is larger; if the genomic DNA of the tomato plant sample shows the signal of the fluorescent group carried by the specific forward primer 2, the sample can be judged to carry genotype TT, and the fruit width is smaller.
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| CN111406588A (en) * | 2020-05-08 | 2020-07-14 | 中国农业科学院蔬菜花卉研究所 | Method for promoting fruit expansion and ripening of tomatoes |
| CN115960953A (en) * | 2022-12-29 | 2023-04-14 | 河南农业大学 | Application of SlEIN4 gene in regulation and control of tomato fruit characters, vector and application of vector |
| CN116410986A (en) * | 2023-02-15 | 2023-07-11 | 浙江大学 | Gene involved in regulating and controlling plant height, fruit shape and yield of tomatoes and application thereof |
| WO2023208078A1 (en) * | 2022-04-27 | 2023-11-02 | 中国农业科学院农业基因组研究所 | Genome structure variation for regulating tomato fruit soluble solid content, related product, and application |
| CN117144046A (en) * | 2023-09-20 | 2023-12-01 | 江苏省农业科学院 | SNP molecular marker related to tomato fruit glossiness and application thereof |
| WO2023236840A1 (en) * | 2022-06-07 | 2023-12-14 | 江苏省农业科学院 | Snp site closely associated with anthocyanin content in vigna unguiculata, kasp molecular marker primer, and use thereof |
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| CA3134097A1 (en) * | 2019-06-13 | 2020-12-17 | Wim VRIEZEN | Tomato plant producing fruit having improved ripening characteristics |
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| CN111406588A (en) * | 2020-05-08 | 2020-07-14 | 中国农业科学院蔬菜花卉研究所 | Method for promoting fruit expansion and ripening of tomatoes |
| WO2023208078A1 (en) * | 2022-04-27 | 2023-11-02 | 中国农业科学院农业基因组研究所 | Genome structure variation for regulating tomato fruit soluble solid content, related product, and application |
| WO2023236840A1 (en) * | 2022-06-07 | 2023-12-14 | 江苏省农业科学院 | Snp site closely associated with anthocyanin content in vigna unguiculata, kasp molecular marker primer, and use thereof |
| CN115960953A (en) * | 2022-12-29 | 2023-04-14 | 河南农业大学 | Application of SlEIN4 gene in regulation and control of tomato fruit characters, vector and application of vector |
| CN116410986A (en) * | 2023-02-15 | 2023-07-11 | 浙江大学 | Gene involved in regulating and controlling plant height, fruit shape and yield of tomatoes and application thereof |
| CN117144046A (en) * | 2023-09-20 | 2023-12-01 | 江苏省农业科学院 | SNP molecular marker related to tomato fruit glossiness and application thereof |
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