Primer group for detecting parthenocarpy KASP molecular marker of eggplant and application of primer group

Abstract

The invention provides a primer group for detecting parthenocarpy KASP molecular markers of eggplants and application of the primer group, and belongs to the technical field of molecular biology. The primer group or the kit provided by the invention has the following beneficial effects that the selection efficiency in a field verification experiment reaches 63-91%, the selection efficiency of a parthenocarpy material controlled by a recessive gene is greatly improved, the homozygous time of the parthenocarpy material is shortened, and a powerful tool is provided for rapidly obtaining a new parthenocarpy variety; the parthenocarpy of the plant in the later stage can be predicted by detecting the marker genotype in advance in the seedling stage; the parthenocarpy is identified by using marker screening instead of large-scale'stigma removal ', so that the field workload and the plant loss caused by the environment are greatly reduced; the parthenocarpy phenotype of the plant is a comprehensive result obtained after interaction of the genotype and the environment, selection errors are prone to occurring, the genotype is screened in combination with the molecular marker, and the screening accuracy and efficiency can be greatly improved.

Description

Technical Field

[0001] The invention relates to the technical field of molecular biology, and in particular to a primer set for detecting a KASP molecular marker of eggplant parthenocarpy and an application thereof. Background Art

[0002] Eggplant (Solanum melongena L.) is an important vegetable crop in the Solanaceae family, and is deeply loved by consumers. It is an important raw material for common delicacies on the table. Eggplant is a thermophilic crop. If it encounters adverse environments such as low temperature, high temperature, high humidity and drought during the flowering period, it will cause flowers and fruits to fall, and ultimately reduce the yield. Under environmental conditions with increased extreme weather, the characteristic of normal fruiting without pollination (parthenocarpy) plays a key role in maintaining yield. Parthenocarpy refers to the fact that after the plant develops into a flower, the ovary can normally set fruit and form commercial fruit without pollination and fertilization, and there may be a few aborted seed capsules or no seeds in the fruit. In addition, parthenocarpy can also solve the problem of high cost of using insect pollination or artificial pollination in facility production, effectively reduce labor intensity and improve economic benefits. At the same time, due to the lack of seeds, seedless fruits have thicker flesh and better quality, and the storage time and shelf life of the fruit can be effectively extended. Therefore, parthenocarpy has always been an important selection indicator for horticultural plant breeding.

[0003] Eggplant parthenocarpy is a quantitative trait controlled by multiple genes and regulated by a variety of environmental factors and hormones, and its genetic mechanism is extremely complex. Kuno and Liu Fuzhong's research showed that parthenocarpy is controlled by a single dominant nuclear gene (Liu Fuzhong, "Genetic Analysis and AFLP Molecular Markers of Eggplant Parthenocarpy Genes", Acta Horticulturae Sinica, No. 9, 2008), and in 2021, they applied for and passed the invention patent for "a primer pair for identifying dominant parthenocarpy genes in eggplant using KASP molecular markers and its application". This molecular marker is mainly used in the selection and breeding of dominant parthenocarpy materials. However, the research of Restainoe and Tian Shibing et al. showed that the genes controlling eggplant parthenocarpy have epistatic effects between non-allelic genes, are controlled by a group of recessive genes, and are recessively inherited. At present, the eggplant molecular markers controlled by recessive genes are still to be developed.

[0004] The identification of eggplant parthenocarpic materials and variety selection mainly adopts "stigma removal" in the field, and phenotypic identification is carried out by investigating and identifying whether the fruit can set and swell normally without pollination in the later stage. Generally, 3 flower buds are identified for each plant, and at least 1 that can swell normally is considered to have parthenocarpic properties. This method requires a lot of manpower and material resources, and is easily affected by the external environment. The screening efficiency is low, the identification is difficult, and the material creation time is extremely long. In recent years, with the development of molecular technology, more and more analytical markers involving various plant traits have been developed and applied. Molecular marker-assisted selection is not affected by gene expression and environment, and the target traits can be identified in the early stage of planting, thus greatly improving the selection efficiency. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide a primer set for detecting the KASP molecular marker of eggplant parthenocarpy and its application. The primer set provided by the present invention can greatly improve the selection efficiency of parthenocarpy materials controlled by recessive genes, shorten the homozygous time of parthenocarpy materials, and provide a powerful tool for faster acquisition of new parthenocarpy varieties.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] A primer set for detecting eggplant parthenocarpy KASP molecular markers, the primer set comprising a forward primer F-HEX with a HEX connector, a forward primer F-FAM with a FAM connector and a reverse universal primer; the sequences of F-HEX, F-FAM and the reverse universal primer are shown in SEQ ID NO.3 to SEQ ID NO.5 respectively.

[0008] In the present invention, the SEQ ID NO.3 to SEQ ID NO.5 are specifically:

[0009] SEQ ID NO.3:

[0010] 5'-GAAGGTGACCAAGTTCATGCTGGTTTTCTACCTTGTAATCCCCT CG-3';

[0011] SEQ ID NO.4:

[0012] 5'-GAAGGTCGGAGTCAACGGATTGGTTTTCTACCTTGTAATCCCCT CT-3';

[0013] SEQ ID NO.5:

[0014] 5'-ACTTAACACCTTTTTTTCTTTTAAATCTTC-3'.

[0015] In the present invention, the KASP molecular marker is located at 89413187 bp of chromosome 6 of the eggplant genome, and the genotype is G / T.

[0016] In the present invention, the sequence of 50 bp before and after the KASP molecular marker is shown as SEQ ID NO.1 or SEQ ID NO.2.

[0017] In the present invention, SEQ ID NO.1 and SEQ ID NO.2 are specifically:

[0018] SEQ ID NO.1: 5'-GGTTTTCTACCTTGTAATCCCCTCG-3';

[0019] SEQ ID NO. 2: 5'-GGTTTTCTACCTTGTAATCCCCTCT-3'.

[0020] In the present invention, when the genotype at 89413187bp of chromosome 6 of the material to be tested is G:G, the material to be tested is parthenocarpy; when the genotype at 89413187bp of chromosome 6 of the material to be tested is T:T, the material to be tested is non-parthenocarpy.

[0021] The present invention also provides a kit, which comprises the primer set described in the above technical solution.

[0022] The present invention also provides an application of the primer set or the kit described in the above technical solution, including but not limited to the following applications:

[0023] (1) Identify or assist in identifying the parthenocarpy of eggplant;

[0024] (2) preparing a product for identifying or assisting in identifying the parthenocarpy of eggplant;

[0025] (3) Breeding or assisting in breeding parthenocarpic eggplant;

[0026] (4) Prepare products for breeding or assisting in breeding parthenocarpic eggplants.

[0027] The present invention also provides a method for identifying or assisting in identifying the parthenocarpy of eggplant, comprising the following steps: extracting genomic DNA of the eggplant material to be tested; performing PCR amplification on the extracted genomic DNA using the primer set described in the above technical solution or the kit described in the above technical solution, and performing genotyping according to the fluorescent signal of the PCR amplification product;

[0028] If the genotype is G:G, the material to be tested is homozygous parthenocarpy; if the genotype is T:T, the material to be tested is non-parthenocarpy; if the genotype is G:T, the material to be tested is heterozygous parthenocarpy.

[0029] The present invention also provides a method for breeding or auxiliary breeding of parthenocarpic eggplants, comprising the following steps: extracting genomic DNA of the eggplant material to be tested; performing PCR amplification on the extracted genomic DNA using the primer set described in the above technical solution or the kit described in the above technical solution, and performing genotyping according to the fluorescent signal of the PCR amplification product;

[0030] If the genotype is G:G, the material to be tested is homozygous parthenocarpy; if the genotype is T:T, the material to be tested is non-parthenocarpy; if the genotype is G:T, the material to be tested is heterozygous parthenocarpy.

[0031] In the present invention, the PCR amplification reaction system is preferably 1.0 μL of 20 ng / μL to 50 ng / μL eggplant genomic DNA, 5.0 μL of KASP Master Mix 2×VV4, 0.5 μL of primer set, and ddH2O supplemented to 10.0 μL; wherein the molar concentration ratio of F-HEX:F-FAM:reverse universal primer is 1:1:3.

[0032] In the present invention, the PCR amplification reaction procedure is preferably: 94°C pre-denaturation, 15 min; 94°C, 20 s, 61°C-55°C, 1 min, during which the temperature is reduced by 0.6°C each cycle, 10 cycles; 94°C, 20 s, 55°C, 60 s, 42 cycles.

[0033] Beneficial technical effects: The present invention provides a primer set for detecting the KASP molecular marker of eggplant parthenocarpy and its application. The primer set or kit provided by the present invention has the following beneficial effects:

[0034] (1) In the field verification experiment, 77 materials were verified for parthenocarpy, and the selection efficiency reached 63% to 91%, which greatly improved the selection efficiency of parthenocarpy materials controlled by recessive genes, shortened the homozygous time of parthenocarpy materials, and provided a powerful tool for faster acquisition of new parthenocarpy varieties;

[0035] (2) The marker genotype can be detected in advance during the seedling stage to predict the parthenocarpy of the plant in the later stage;

[0036] (3) Using marker screening to replace large-scale “stigma removal” to identify parthenocarpy greatly reduces field workload and plant losses caused by the environment;

[0037] (4) The parthenocarpy phenotype of plants is the comprehensive result of the interaction between genotype and environment. Parthenocarpy based on phenotype may lead to selection errors due to environmental differences. Combining molecular markers to screen genotypes can greatly improve the accuracy and efficiency of screening. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 It is the development of parthenocarpic and non-parthenocarpic fruits; among them, Figure 1 a is the swelling of the parthenocarpic material after removing the stigma. Figure 1 b shows the seed development inside parthenocarpic and non-parthenocarpic fruits;

[0039] Figure 2 This is the marker typing diagram of eggplant in the F2 population of D-7-1*GF5; the blue squares represent homozygous G:G genotypes; the orange circles represent homozygous T:T genotypes; the green triangles represent heterozygous genotypes, the same below;

[0040] Figure 3 This is the marker typing diagram of eggplant of the F2 population of D-6*GF5;

[0041] Figure 4 This is the marker typing diagram of eggplant in the F2 population of D-6*GF8. DETAILED DESCRIPTION

[0042] In order to better understand the present invention, the content of the present invention is further explained in conjunction with the following examples, but the content of the present invention is not limited to the following examples. The materials, reagents, etc. used in the examples and test examples of the present invention, unless otherwise specified, can be obtained from commercial channels; the methods used in the examples and test examples of the present invention, unless otherwise specified, are conventional methods.

[0043] Example 1

[0044] Eggplant (high-generation inbred line strong parthenocarpy material D-7-1) leaf material was selected, and DNA was extracted using a DNA extraction kit for sequencing; the resequencing file (VCF) of the eggplant material was obtained. Through the Bulked Segregant Analysis (BSA) analysis, a QTL related to parthenocarpy was located in the 83874671-89413187bp interval of chromosome 6.

[0045] Example 2 Design of KASP molecular marker Dsnp2

[0046] The molecular marker Dsnp2 related to eggplant parthenocarpy of the present invention is obtained by the following method:

[0047] 1) Use a DNA extraction kit to extract genomic DNA from eggplant parent (high-generation inbred line strong parthenocarpic material D-7-1×non-parthenocarpic material GF5) seedlings and hybrid offspring seedlings;

[0048] 2) Screening of gene markers related to eggplant parthenocarpy using PCR method;

[0049] 3) A KASP marker Dsnp2 was developed. After polymorphism detection, it was found that it was significantly correlated with the parthenocarpy of eggplant. This marker can be used to identify the parthenocarpy of eggplant materials.

[0050] Finally, it was determined that the molecular marker was located at 89413187 on chromosome 6 of the eggplant genome, and the sequence of 50 bp before and after the molecular marker site was:

[0051] SEQ ID NO.1: 5'-GGTTTTCTACCTTGTAATCCCCTCG-3';

[0052] SEQ ID NO. 2: 5'-GGTTTTCTACCTTGTAATCCCCTCT-3'.

[0053] Primer set designed based on KASP molecular markers:

[0054] F-HEX, SEQ ID NO.3:

[0055] 5'-GAAGGTGACCAAGTTCATGCTGGTTTTCTACCTTGTAATCCCCT CG-3';

[0056] F-FAM, SEQ ID NO.4:

[0057] 5'-GAAGGTCGGAGTCAACGGATTGGTTTTCTACCTTGTAATCCCCT CT-3';

[0058] Reverse universal primer, SEQ ID NO.5:

[0059] 5'-ACTTAACACCTTTTTTTCTTTTAAATCTTC-3'.

[0060] Example 3 Identification of sequence differences in eggplant parthenocarpic materials using molecular markers

[0061] The method for identifying the parthenocarpy of eggplant comprises the following steps:

[0062] (1) extracting genomic DNA of the eggplant material to be tested;

[0063] (2) performing PCR amplification on eggplant genomic DNA using the primer set in Example (2);

[0064] (3) Based on the difference in fluorescence signals of PCR products, the genotype of each eggplant material to be tested was identified using Kraken software, that is, the homozygous GG genotype (parthenocarpic type), homozygous TT genotype (non-parthenocarpic type), heterozygous GT genotype ( Figure 2 to Figure 4 ).

[0065] The PCR reaction system was: 1.0 μL of 20-50 ng / μL eggplant genomic DNA, 5.0 μL of KASP Master Mix 2×VV4, 0.5 μL of KASP Assay Mix (molar concentration ratio of F-HEX: F-FAM: R = 1:1:3), and ddH2O, a total of 10.0 μL.

[0066] The amplification program was as follows: pre-denaturation at 94°C for 15 min; 94°C for 20 s (denaturation)—61°C to 55°C for 1 min (renaturation & extension: 10 cycles of amplification with a touch down program, decreasing 0.6°C per cycle); 94°C for 20 s (denaturation)—55°C for 60 s for a further 42 cycles.

[0067] After amplification, use the Stepone fluorescence quantitative instrument to detect the fluorescence signal and check the typing. If the typing is not sufficient, continue amplification and check the typing every 3 cycles until the typing is complete, and export the experimental results from the Kraken software.

[0068] According to the setting of the present invention, 42 cycles of amplification can achieve complete typing.

[0069] Experimental Example 1 Identification of Parthenocarpy of 77 Eggplant Resources

[0070] 77 eggplant materials from different genetic backgrounds (the experimental materials were varieties independently bred by the eggplant breeding team of the Institute of Vegetables and Flowers of Chongqing Academy of Agricultural Sciences) were treated with stigma removal, that is, the petals were pushed aside before flowering and the stigma was removed from the bottom with tweezers. Three flower buds were treated for each plant, 20 plants were treated for each, and the treatment was repeated three times. After the 14th day, the fruit expansion was investigated. If at least one fruit could expand normally, it was identified as a parthenocarpic plant with parthenocarpy. A single plant with three flower buds that could not set fruit normally or did not expand normally was considered a non-parthenocarpic plant and did not have parthenocarpy ( Figure 1 ).

[0071] Parthenocarpy rate = number of normally enlarged fruits / number of treated flower buds*100%.

[0072] Test Example 2

[0073] The different eggplant germplasms in Experimental Example 1 were tested according to the methods of Example 1 and Example 3. The results are shown in Table 1.

[0074] The results showed that the genotyping of the Dsnp2 marker in 77 eggplant materials with different genetic backgrounds was linked to the parthenocarpic phenotype, and the next step of breeding was carried out by selecting offspring individuals with the GG genotype. This indicates that the Dsnp2 marker can assist in the selection breeding of eggplant parthenocarpic lines or their offspring.

[0075] Table 1 Comparison of KASP marker genotypes and phenotypes in 77 samples

[0076]

[0077]

[0078] Note: Phenotype "1" represents parthenocarpy, "0" represents non-parthenocarpy; genotype "1" represents parthenocarpy genotype, "0" represents non-parthenocarpy genotype, and "0.5" represents heterozygous parthenocarpy genotype.

[0079] Experimental Example 3 Analysis of parthenocarpy using Dsnp2 marker

[0080] The test materials are the eggplant parthenocarpic inbred lines D-7-1, D-6 and non-parthenocarpic inbred lines GF5 and GF8 independently bred by the eggplant breeding team of the Institute of Vegetables and Flowers of Chongqing Academy of Agricultural Sciences. The F2 segregation populations obtained by hybridization and self-pollination of D-6*GF8, D-7-1*GF5, and D-6*GF5 contain 156, 185, and 143 plants in 2023, respectively. The field experiment was carried out in the Modern Agricultural Park of Chongqing Academy of Agricultural Sciences. The test materials were sown on October 20, 2023 and planted in a simple greenhouse on March 2 of the following year; the plant spacing was 50 cm, the row spacing was 70 cm, and the field was uniformly managed normally.

[0081] The three flowers of the two eggplants were "stigma removed" to identify parthenocarpy. Plants with at least one fruit that can swell normally are defined as parthenocarpy plants with parthenocarpy. Plants with three flower buds that cannot set fruit normally or swell abnormally are considered non-parthenocarpy plants and do not have parthenocarpy.

[0082] Parthenocarpy rate = number of normally enlarged fruits / number of treated flower buds*100%.

[0083] The method in Example 3 was used to identify the monocotyledonous properties of the tested material. The experimental results were exported from the Kraken software and are shown in Table 2 and Figures 2 to 4 .

[0084] Table 2 The results of the F2 population in Example 1 and Example 3 and the results of parthenocarpy field identification are consistent with the results

[0085]

[0086]

[0087] The results of the identification of the parthenocarpic genotype and phenotype of each individual plant in the association analysis population showed that the genotyping of the Dsnp2 marker in the three F2 populations tested was linked to the parthenocarpic phenotype, and the next step of breeding could be carried out by selecting individuals with the TT genotype, indicating that the Dsnp2 marker can assist in the selection and breeding of eggplant parthenocarpic lines or their offspring.

[0088] Experimental Example 4: Using Dsnp2 molecular marker to carry out eggplant parthenocarpy assisted selection breeding

[0089] Using parthenocarpic inbred lines with different genetic backgrounds and non-parthenocarpic inbred lines for hybridization, and then combining them with marker-assisted selection. The F2 population obtained by hybridization is first identified by markers, such as Figures 2 to 4 As shown, the blue squares represent the homozygous GG genotype; the orange dots represent the homozygous TT genotype; and the green triangles represent the heterozygous GT. (The typing results are clearly distinguished by color, indicating that the KASP marker typing is reliable); it was found that the parthenocarpy rate of all pure GG genotype eggplant F2 offspring groups was significantly higher than that of the homozygous TT gene eggplant F2 offspring group. This result once again verifies the high linkage between the Dsnp2 molecular marker and parthenocarpy, and can be used in molecular assisted breeding of eggplant parthenocarpy.

[0090] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A primer set for detecting the KASP molecular marker of eggplant parthenocarpy, characterized in that: The primer set includes a forward primer F-HEX with a HEX connector, a forward primer F-FAM with a FAM connector and a reverse universal primer; the sequences of F-HEX, F-FAM and the reverse universal primer are shown in SEQ ID NO.3 to SEQ ID NO.5 respectively.

2. The primer set according to claim 1, characterized in that The KASP molecular marker is located at 89413187 bp of chromosome 6 of the eggplant genome, and the genotype is G / T.

3. The primer set according to claim 1 or 2, characterized in that: When the genotype at 89413187bp of chromosome 6 of the material to be tested is G:G, the material to be tested is parthenocarpy; when the genotype at 89413187bp of chromosome 6 of the material to be tested is T:T, the material to be tested is non-parthenocarpy.

4. A kit, characterized in that: The invention comprises the primer set according to any one of claims 1 to 3.

5. Use of the primer set according to any one of claims 1 to 3 or the kit according to claim 4, characterized in that: Including but not limited to the following applications: (1) Identify or assist in identifying the parthenocarpy of eggplant; (2) preparing a product for identifying or assisting in identifying the parthenocarpy of eggplant; (3) Breeding or assisting in breeding parthenocarpic eggplant; (4) Prepare products for breeding or assisting in breeding parthenocarpic eggplants.

6. A method for identifying or assisting in identifying the parthenocarpy of eggplant, characterized in that: The following steps are involved: Extracting genomic DNA from eggplant materials to be tested; Performing PCR amplification on the extracted genomic DNA using the primer set described in any one of claims 1 to 3 or the kit described in claim 4, and performing genotyping according to the fluorescent signal of the PCR amplification product; If the genotype is G:G, the material to be tested is homozygous parthenocarpy; if the genotype is T:T, the material to be tested is non-parthenocarpy; if the genotype is G:T, the material to be tested is heterozygous parthenocarpy.

7. A method for breeding or assisting breeding of parthenocarpic eggplants, characterized in that: The following steps are involved: Extracting genomic DNA from eggplant materials to be tested; Performing PCR amplification on the extracted genomic DNA using the primer set described in any one of claims 1 to 3 or the kit described in claim 4, and performing genotyping according to the fluorescent signal of the PCR amplification product; If the genotype is G:G, the material to be tested is homozygous parthenocarpy; if the genotype is T:T, the material to be tested is non-parthenocarpy; if the genotype is G:T, the material to be tested is heterozygous parthenocarpy.

8. The use according to claim 5 or the method according to any one of claims 6 to 7, characterized in that: The PCR amplification reaction system was 1.0 μL of 20-50 ng / μL eggplant genomic DNA, 5.0 μL of KASP Master Mix 2×VV4, 0.5 μL of primer set, and ddH2O supplemented to 10.0 μL; among them, the molar concentration ratio of F-HEX: F-FAM: reverse universal primer was 1:1:

3.

9. The use according to claim 5 or the method according to any one of claims 6 to 7, characterized in that: The PCR amplification reaction program was as follows: pre-denaturation at 94°C for 15 min; 94°C for 20 s, 61°C-55°C for 1 min, with the temperature decreasing by 0.6°C during each cycle, for 10 cycles; 94°C for 20 s, 55°C for 60 s, for 42 cycles.

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