A set of eggplant SNP molecular markers, an SNP liquid chip, a kit and applications thereof

Abstract

The application belongs to the field of molecular genetics breeding, and discloses a combination of eggplant SNP molecular markers, which comprises 1604 SNP molecular markers. The application also discloses an eggplant SNP liquid chip and a kit, which comprise probes or / and primers for detecting the combination of eggplant SNP molecular markers, and discloses application of the combination of eggplant SNP molecular markers, the eggplant SNP liquid chip or the kit in at least one of the following: identification of eggplant germplasm resources, evaluation of genetic diversity, identification of variety purity and improvement breeding of eggplant peel color. The combination of eggplant SNP molecular markers provided by the application has high detection flux, large output result information, high target site detection rate and accurate and reliable typing results.

Description

Technical Field

[0001] This invention belongs to the field of molecular genetics breeding, and particularly relates to an eggplant SNP molecular marker combination, whole genome liquid chip, reagent kit and its application. Background Technology

[0002] eggplant( Solanum melongena Eggplant (L.) is an important vegetable crop with global economic significance. According to the FAO, the total global harvested area is 1,892,798 hectares, with a yield of 59,312,599.76 metric tons. Eggplant is rich in essential nutrients, containing polyphenols such as anthocyanins and chlorogenic acid, which are beneficial to health. Eggplant exhibits significant heterosis, making F1 hybrid varieties a popular choice in cultivation. Selecting superior lines and hybrid parents are key steps in breeding high-yielding and high-quality eggplant varieties. Selecting superior inbred lines is crucial for hybrid breeding, and backcrossing is often used to introduce desirable traits into superior inbred lines. However, traditional backcrossing methods are labor-intensive and time-consuming because they select individuals based on phenotypic characteristics. Marker-assisted selection accelerates this process by using molecular markers to select individuals with high background recovery rates from parents. Compared to random or phenotypic selection, using marker spacing of 10–20 cM provides an advantage of one to two backcross generations. Studies have shown that introducing target genes into superior lines can be achieved in two backcross generations, while restoring more than 96% of the recipient parent's genotype. However, the lack of affordable, high-throughput genotyping tools has hindered related research on eggplant.

[0003] SNP markers are widely used in plant breeding for genetic diversity analysis, marker-assisted selection (MAS), and QTL mapping due to their high polymorphism and ease of high-throughput detection. With the availability of high-quality eggplant genome sequencing, whole-genome SNP detection has become easier. Currently, three main methods are used for SNP detection. The first is genotyping via sequencing by genes (GBS). This has proven successful in genetic diversity analysis and QTL mapping in eggplant. However, its high cost and complex genotyping process hinder its wider application. The second method relies on PCR techniques, such as competitive allele-specific PCR (KASP) and TaqMan, which can effectively identify specific known loci. These methods have been used for fine mapping of genes / QTLs and SNP detection in eggplant. The third method involves SNP arrays, currently constructed using two strategies. Solid-state SNP arrays are based on Illumina or Affymetrix platforms. Solid-state SNP arrays exhibit some limitations, including high equipment cost, low flexibility in marker redesign, and reliance on specific genotyping platforms. The second type is the target sequencing genotyping (GBTS) system based on multiplex PCR or solution capture, which has advantages such as high genotyping accuracy, high throughput, low cost, and flexible design, and has been applied to various crops. Liu et al. identified 219 genome-wide perfect SNPs in eggplant using resequencing data from 45 different lines. They analyzed the genetic diversity and unique DNA fingerprints of 377 eggplant varieties using these 219 SNPs. Five related SNPs were found near SUN and OVATE homologs. However, the number of SNPs included in this method is too low to meet breeding requirements. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the deficiencies and defects mentioned in the background art above, and to provide an eggplant 1.6K molecular marker combination, chip, reagent kit and its application. To solve the above-mentioned technical problems, the technical solution proposed by this invention is as follows: A method for constructing an SNP liquid phase chip for eggplant breeding includes the following steps: (1) providing an eggplant SNP core marker set, wherein the core marker set is obtained through the following steps: (a) Based on whole-genome resequencing data from at least 118 diverse eggplant materials, aligned to the eggplant reference genome SME-HQ_V2.0, 1590 highly polymorphic SNP loci were screened that met the following criteria: minimum allele frequency (MAF) ≥ 0.05, deletion rate < 0.1, heterozygosity < 0.1, sequencing depth ≥ 9×, and evenly distributed across the 12 chromosomes; (b) 1385 lengths related to eggplant flesh and peel color were identified. (a) The outer functional sequence Seq1 of bp was verified by specific primers and sequencing comparison. The single nucleotide deletions at positions 1 and 30 were identified as functional SNP sites related to fruit flesh color. (c) 12 known functional SNP sites related to the number of flowers, fruit length, fruit shape, peel color and flesh color of eggplant were collected. Their chromosome positions, physical coordinates, variation types and associated traits are shown in Table 3 of the specification. (d) The 1590 sites obtained in step (a), the 2 sites obtained in step (b) and the 12 sites obtained in step (c) were merged to obtain a core marker set containing 1604 SNP sites. (2) Based on the flanking sequences of the 1604 SNP sites in the core marker set obtained in step (1) (d), multiplex PCR primers or hybridization probes that can specifically capture or amplify the genomic regions containing each SNP site were designed. (3) The set of multiplex PCR primers or hybridization probes designed in step (2) was applied to a multiplex PCR-based targeted capture sequencing platform to construct the SNP liquid phase chip.

[0005] In the above-described method for constructing SNP liquid-phase chips, preferably, in step (b), the sequences of the specific primers are as shown in SEQ ID NO:2~11.

[0006] Based on a general inventive concept, the present invention also provides an eggplant SNP liquid phase chip, the eggplant SNP chip including probes and / or primers for eggplant SNP molecular marker combination, the eggplant SNP molecular marker combination including 1604 SNP molecular markers, wherein the physical locations of 1602 molecular markers are determined based on eggplant SME-HQ_V2.0 genome sequence alignment, and the other 2 SNP sites are located on the functional sequence Seq1 related to eggplant flesh color. The location information of the 1604 SNP molecular markers is shown in Table 1 of the specification.

[0007] Based on a general inventive concept, the present invention also provides an eggplant SNP liquid phase chip, which is constructed by the above-described method.

[0008] Based on a general inventive concept, the present invention also provides a kit comprising the eggplant SNP liquid phase chip as described above.

[0009] Based on a general inventive concept, the present invention also provides a kit comprising the eggplant SNP chip as described above.

[0010] Based on a general inventive concept, the present invention also provides the application of at least one of the eggplant SNP chip as described above or the kit as described above in the identification of eggplant germplasm resources.

[0011] Based on a general inventive concept, the present invention also provides the application of at least one of the eggplant SNP chip as described above or the kit as described above in the assessment of genetic diversity in eggplant.

[0012] Based on a general inventive concept, the present invention also provides the application of at least one of the eggplant SNP chip as described above or the kit as described above in the identification of eggplant variety purity.

[0013] Based on a general inventive concept, the present invention also provides the application of at least one of the eggplant SNP chips as described above or the kits as described above in the construction of eggplant genetic maps or QTL mapping.

[0014] Based on a general inventive concept, the present invention also provides the application of at least one of the eggplant SNP chips as described above or the kits as described above in eggplant molecular marker-assisted selection breeding, directional improvement or multi-gene aggregation breeding.

[0015] Based on a general inventive concept, the present invention also provides the application of at least one of the eggplant SNP chips as described above or the kits as described above in selection association analysis of the whole genome of eggplant.

[0016] Based on a general inventive concept, the present invention also provides a method for identifying eggplant germplasm resources, including using an eggplant SNP liquid phase chip as described above or a kit as described above to genotype the test sample, and performing population genetic analysis based on the obtained genotype data of 1604 SNP loci to identify its species affiliation and genetic background.

[0017] Based on a general inventive concept, the present invention also provides a method for assisting in the selection of flesh color in eggplant breeding, comprising using an eggplant SNP liquid phase chip as described above or a kit as described above to detect the genotype at position 1 and / or position 30 of SEQ ID NO:1, and screening individual plants carrying the target allele based on the association between the missing allele and the green flesh trait.

[0018] Based on a general inventive concept, the present invention also provides a method for identifying the purity of eggplant hybrids, including using an eggplant SNP liquid phase chip as described above or a kit as described above to perform genotyping on the parents and hybrid offspring, selecting SNP loci where there is homozygous difference between the parents, and if the offspring is heterozygous at that locus, it is determined to be a true hybrid.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) Based on the collected resequencing data of 118 eggplant varieties with rich diversity, this invention selects 1604 molecular marker sites from millions of molecular markers that are highly representative, highly polymorphic (mean MAF 0.36), have good universality, high coverage on the genome (average coverage 99.37%), and are evenly distributed. It also provides a chip for detecting the combination of SNP molecular markers in eggplant. The chip has high throughput, large output information, high target site detection rate (99.72%), and accurate and reliable genotyping results (reproducibility rate 99.86%). It is a high-throughput, high-information, high-accuracy, high-reproducibility, low-cost, multifunctional molecular marker chip with a wide range of applications.

[0020] (2) The liquid phase chip of the present invention adds 14 functional markers related to important traits of eggplant, such as the number of flowers, fruit length, fruit shape, peel color and flesh color. This is beneficial for molecular marker-assisted selection, targeted improvement, multi-gene aggregation breeding, and the mining, identification and functional analysis of important trait genes. It has great application value in the process of eggplant variety improvement and variety selection.

[0021] (3) Compared with traditional solid-phase chips, the liquid-phase chip involved in this invention has higher flexibility and can add marker sites at any time according to the needs of the application scenario. At the same time, the liquid-phase chip relies on the second-generation sequencing platform, which has lower genotyping cost, higher detection throughput, and a large amount of data output at one time, which can simultaneously cover the detection of nearly a thousand materials, providing a technical means for large-scale genotyping. Based on targeted capture sequencing technology, the liquid-phase chip can not only genotype the target site, but also accurately genotype the genetic variation sites within a certain range around the target site. Therefore, more SNP genotyping information can be obtained than the expected marker sites, providing richer site information support for eggplant molecular breeding. Attached Figure Description

[0022] Figure 1 This is the distribution of eggplant 1.6K liquid phase chip sites on chromosomes in an embodiment of the present invention; Figure 2 This is a MAF distribution map of eggplant 1.6K liquid phase chip sites in an embodiment of the present invention; Figure 3 This describes the principle and process of mGPS liquid phase chip technology involved in the embodiments of the present invention; Figure 4 This refers to the genotypic consistency rate of eggplant 1.6K liquid-phase chip replicate samples in this embodiment of the invention; Figure 5 These are the structures of 99 eggplant models analyzed using a 1.6K liquid phase chip in this embodiment of the invention; Figure 6 This embodiment of the invention uses a 1.6K liquid phase chip to perform principal component analysis on 99 eggplant varieties; Figure 7 This refers to 99 eggplant genetic trees based on a 1.6K liquid phase chip in this embodiment of the invention; Figure 8 In this embodiment of the invention, candidate offspring are screened from the BC1F1 population based on a 1.6K liquid phase chip: A. PRPG statistics of candidate offspring in the BC1F1 population; B. Genotypic visualization of candidate offspring in the BC1F1 population; C. Phenotypic visualization of candidate offspring in the BC1F1 population.

[0023] Figure 9 This is a schematic diagram of the specific primer design for identifying the outer functional sequence Seq1 in an embodiment of the present invention; the diagram shows the full length of Seq1 (SEQ ID NO: 1) of 1385 bp and the positions of the 5 pairs of verification primers designed thereon.

[0024] Figure 10 This is the embodiment of the invention. Figure 9 The image shows the electrophoresis results of PCR verification of eggplant materials with different flesh colors using the primers shown. The first row in the image is the sample number, and the second row is the primer number used. The full-length primer is 23F+28R. "Full-length-high" indicates that PCR amplification of the full-length Seq1 was performed using a high-fidelity enzyme.

[0025] Figure 11 This is a schematic diagram illustrating the verification results of sequencing and comparing PCR amplicon after integrating the Seq1 sequence into the reference genome in an embodiment of the present invention.

[0026] Figure 12 This is a schematic diagram illustrating the results of comparing Seq1 sequences in different eggplant materials and identifying their polymorphic sites in an embodiment of the present invention. Detailed Implementation

[0027] To facilitate understanding of the present invention, the present invention will be described more fully and in detail below with reference to the accompanying drawings and preferred embodiments, but the scope of protection of the present invention is not limited to the following specific embodiments.

[0028] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the invention.

[0029] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0030] Example 1: An eggplant SNP molecular marker combinatorial of the present invention includes 1604 SNP molecular markers. The genotype information of the 1604 molecular markers is represented in the form of "ref / alt (reference base / variant base)", and the position information is shown in Table 1.

[0031] Table 1: Information on 1604 SNP sites in eggplant SNP molecular marker combinations

[0032] The process for obtaining this combination of molecular markers is as follows: 1. Collection of eggplant germplasm resources To obtain a rich variety of whole-genome SNP loci, 80 eggplant accessions from different regions and varieties were collected and subjected to whole-genome resequencing. In addition, 16 existing eggplant resequencing data from previous projects and 22 eggplant accessions from the NCBI database were also collected.

[0033] 2. Eggplant whole genome resequencing DNA extraction and whole-genome resequencing were performed on the 80 collected eggplant samples. The specific steps included: (1) Extract DNA using magnetic beads.

[0034] (2) Using the MGI library standard method, qualified libraries were used for DNA-seq sequencing library construction.

[0035] (3) After the library passed the quality inspection, it was sequenced using the MGI sequencing platform with the PE150 sequencing strategy and a sequencing depth of 10×.

[0036] Sentieon was used to compare and detect variants in the resequencing data of all 118 collected eggplant materials. The analysis process is as follows: (1) Sentieon was used to align reads to the corresponding eggplant reference genome (SME-HQ_V2.0), sort the positions, and mark duplicate reads.

[0037] (2) Use Sentieon to detect variant sites for each sample and obtain variant information for each sample.

[0038] (3) Sentieon was used for joint-calling to perform joint analysis of gVCF for all samples to obtain the variation results of each individual in the population. To ensure the accuracy of SNPs, the SNP sites obtained after joint analysis were initially hard-filtered (SNP hard-filter criteria: "QD<2.0 || FS>60.0 || MQ<40.0 || SOR>3.0 || MQRankSum<-12.5 || ReadPosRankSum<-8.0").

[0039] 3. Site screening (1) Whole genome loci The MAF value, detection rate, heterozygosity, and sequencing depth of the loci were calculated and statistically analyzed from the VCF file containing SNP variation information of all samples. Polymorphic SNP loci with a minimum allele frequency (MAF) ≥ 0.05, a deletion rate < 0.1, a heterozygosity rate < 0.1, and a sequencing depth ≥ 9× were selected as initial candidate loci. All of the above candidate loci were used for primer design. For each SNP locus, specificity and copy number analyses were performed based on the flanking sequences of the SNP locus, and primers that could not be uniquely aligned to the genome or contained repetitive sequences in the flanking sequences were removed. Based on the principle of uniform distribution, loci with high MAF values ​​were preferred for retention, ultimately yielding 1590 highly polymorphic SNP loci.

[0040] (2) Identification of outer edge sequences (Seq1) and discovery of functional sites The applicant discovered that the gene SmPPR2, associated with eggplant peel and flesh color formation, exhibits sequence differences between the SME-HQ_V2.0 reference genome and published literature (Lv et al., 2023; Fang et al., 2003). Based on this, we identified and extracted a specific DNA sequence of 1385 bp (hereinafter referred to as "Seq1"). This sequence was present in materials exhibiting specific flesh colors.

[0041] A specific DNA sequence of 1385 bp (as shown in SEQ ID NO:1):

[0042] To verify the functional relevance and polymorphism of Seq1, five pairs of specific primers were designed (primers and their sequences are shown in the image below). Figure 9 PCR amplification and sequencing verification were performed on eggplant materials with different peel and flesh colors (as shown in Figure 1, Table 2, and SEQ ID NO: 2~11). The results showed that the Seq1 sequence and its fragments were only stably present in green flesh materials (see Figure 2). Figure 10 (Seq1 PCR verification diagram).

[0043] Table 2: 5 pairs of specific primers

[0044] Furthermore, seq1 was integrated into the SME-HQ_V2.0 reference genome to construct a new reference genome. The sample was expanded using the five primer sets designed above, and then sequenced, followed by alignment to the new reference genome. The Seq1 alignment results are as follows: Figure 11 As shown, the results indicate that the five sets of primers designed can amplify the target fragment and can be aligned to seq1.

[0045] By comparing Seq1 sequences in different materials, a total of 30 polymorphic SNP sites were identified. To efficiently integrate them into the liquid-phase chip, the SNPs at positions 1 and 30 were selected as representative markers for output, i.e., the sites labeled "Seq1" in Table 1 of the final chip site list (see Table 1). Figure 12 (Seq1 polymorphic site output diagram). These two sites are closely related to the color trait of eggplant flesh.

[0046] (3) Functional sites Fourteen functional loci related to important traits of eggplant, such as flower number, fruit length, fruit shape, peel color, and flesh color, were collected from previous studies. Detailed information on these 14 functional loci is summarized in Table 3.

[0047] Table 3: 14 functional marker sites

[0048] 4. Fabrication of an eggplant-based 1.6K liquid phase chip Integrating the aforementioned site screening, all marker loci were obtained, namely 1590 genome-wide markers that are highly representative, polymorphic, universal, and evenly distributed on chromosomes, and 14 functional markers related to important traits of eggplant, such as flower number, fruit length, fruit shape, peel color, and flesh color. These together constitute the eggplant 1.6K liquid-phase chip loci. Specifically, the selected 1604 molecular markers were used for genotyping by Pinpoint Sequencing of multiplex PCR products, developed by Huazhi Biotechnology Co., Ltd. m GPS) was developed into a 1.6K liquid phase chip for eggplant. m GPS liquid-phase chip technology is a targeted capture sequencing technology based on multiplex PCR. It designs specific primers for multiple target regions in the reference genome and simultaneously amplifies multiple different target sequences using multiplex PCR. Then, it constructs sequencing libraries and performs next-generation sequencing on the PCR amplicon to obtain the genotypes of all SNP / InDel sites within the target region.

[0049] The average coverage of 1.6K liquid-phase microarray loci on eggplant chromosomes was 99.37%, with an average spacing of 700 Kb. The 1.6K loci were evenly distributed across each chromosome, as shown in the density distribution diagram below. Figure 1 As shown in the figure, the eggplant 1.6K microarray locus exhibits high polymorphism, with a mean minor allele frequency (MAF value) of 0.36. The MAF value distribution is shown in the figure below. Figure 2 As shown.

[0050] The genotyping process for this eggplant using a 1.6K liquid phase chip specifically includes: (1) Extraction and quality control of genomic DNA Genomic DNA was extracted from fresh eggplant leaves using a magnetic bead method, and the DNA samples underwent quality testing. Quality testing included concentration and integrity testing; DNA concentration was determined using a Qubit quantitative PCR instrument, and DNA integrity was assessed using 1% agarose gel electrophoresis. Samples that passed quality control were used for library preparation.

[0051] (2) m GPS Library Construction and Quality Control (a) Using genomic DNA as templates, a first round of PCR amplification was performed using multiplex PCR reagents to simultaneously and specifically amplify multiple target regions. The amplification products were purified using magnetic beads to remove impurities and non-specifically amplified products. The concentration of the purified products was detected using a Qubit real-time fluorescence instrument, and the fragment size was detected by agarose gel electrophoresis.

[0052] (b) A second round of PCR was performed on the purified amplification products. In the second round of PCR, the adapter sequences required for next-generation sequencing were introduced to both sides of the amplicon products. The products were then purified and recovered using magnetic beads. Library concentration was detected using a Qubit quantitative PCR instrument, and fragment size was detected by agarose gel electrophoresis. Once the concentration and fragment size measurements were satisfactory, the sequencing was complete. m GPS sequencing library construction.

[0053] 3. Sequencing After the library passed quality control, sequencing chip loading and sequencing were performed according to the standards of the BGI sequencing platform, with the sequencing strategy being PE150.

[0054] 4. Data Analysis The raw data after high-throughput sequencing underwent quality control filtering, and FASTP software was used to remove reads containing adapter contamination and low-quality reads. BWA software was used to align the obtained clean reads with the target genome, and then GATK software was used to analyze the sequenced results for variant sites, obtaining the genotyping results for the target loci. m The principles and processes of GPS liquid phase chip technology can be found in [link to documentation]. Figure 3 .

[0055] To verify the genotyping effect of the 1.6K liquid phase chip in eggplant, two technical replicates were set up for four randomly selected materials using the aforementioned 1.6K liquid phase chip. Genotyping results from the four replicates were compared, and the genotypic concordance rate ranged from 99.75% to 100.00%, with an average concordance rate of 99.86%. The results are as follows: Figure 4 As shown above, the data demonstrates that the eggplant 1.6K liquid phase chip exhibits high target site detection rate, good stability, and accurate and reliable genotyping results when performing genotyping on the sample.

[0056] Example 2: This embodiment verifies the application of the eggplant 1.6K liquid phase chip in the identification of eggplant germplasm resources.

[0057] Genetic diversity analysis was performed on 99 eggplant germplasm accessions. Genomic DNA was isolated from fresh leaves using magnetic beads, and SNPs were detected using an eggplant 1.6K liquid chromatography SNP chip to analyze the genetic diversity of the 99 high-generation inbred lines. The optimal K value was determined by the variation curve of the Bayesian Information Criterion (BIC), and cross-validation error indicated that the optimal K value was K=2. Figure 5 As shown. The results indicated that the 99 eggplant samples consisted of 95 individuals from subgroup 1 and 4 individuals from subgroup 2. The 4 individuals from subgroup 2 were classified as... Solanum aethiopicum L. The remaining 95 materials belong to Solanum melongena L .

[0058] Genetic structure was analyzed using ADMIXTURE software, repeated 1000 times. A genetic tree was constructed using the neighbor-joining method in Phylip, and principal component analysis (PCA) was performed using Plink2. Figure 6 As shown, the first and second principal components together elucidate 71.24% of the genetic variance. The results indicate that... Solanum aethiopicum L. and Solanum melongena L. It exhibits obvious clustering characteristics. Its genetic composition is 50.15%. Solanum melongena L and 49.85% Solanum aethiopicum L The materials are classified as a separate category.

[0059] Furthermore, phylogenetic analysis was performed using a neighbor-joining tree based on pairwise genetic distance, as shown in Figure 7. The results divided 99 eggplant samples into 6 distinct subgroups, with 4 wild eggplant samples clustered together, while the remaining 95 eggplant samples were divided into 5 other groups.

[0060] Principal component analysis and phylogenetic analysis showed that the eggplant 1.6K SNP molecular marker has the ability to distinguish between cultivated and wild eggplant, thus identifying hybrid progeny materials of cultivated and wild eggplant. This result indicates that the eggplant 1.6K liquid-phase chip can be used for genetic diversity analysis of eggplant germplasm resources.

[0061] Example 3: This embodiment verifies the application of eggplant 1.6K liquid phase chip in molecular marker-assisted breeding.

[0062] The current parent (NY) exhibits resistance to bacterial wilt (Xiao, Xo, Lin, W., Nie, H., Duan, Z. and Liu, K., 2024. QTL Mapping for Bacterial Wilt Resistance in Eggplant via Bulked Segregant Analysis Using Genotyping by Sequencing. Agronomy. 14.). However, its fruit color is green. Backcrossing is needed to enhance fruit color. The donor parent (WT) has purplish-red fruit.

[0063] First, whole-genome resequencing was performed on NY and WT crops to identify genes involved in anthocyanin biosynthesis and regulation in eggplant fruit. Four genes (MYB1, ANS, SmF3'5'H, and SmTT8) playing important roles in anthocyanin biosynthesis in eggplant pericarp were screened. Based on the resequencing data of NY and WT, SNPs were screened from these four genes for designing PARMS-SNP primers. PARMS-SNP primers were designed using Primer 5.0 (as shown in Table 3). SNP genotyping was performed using the method outlined by Lu et al. (Lu, J., Hou, J., Ouyang, Y., Luo, H., Zhao, J., Mao, C., et al., 2020. A direct PCR–based SNP marker–assisted selection system (D-MAS) for different crops. Molecular Breeding. 40, 9.). Subsequently, background selection was performed using an eggplant 1.6K SNP liquid phase chip. The background recovery rate was calculated using the formula A / (A+B)*100%, where A represents the number of markers identical to the parent and B is the total number of markers used for background selection. The genotype of each individual was visualized using an internal R script.

[0064] Table 4. Four SNP markers used for eggplant peel color screening.

[0065] Based on Table 4, four SNP markers closely related to eggplant fruit color were designed, and a total of 1104 BC1F1 eggplant plants were screened, from which 65 target plants were identified. Subsequently, the genotypes of these 65 BC1F1 individuals were performed using an eggplant 1.6K liquid chromatography-mass spectrometry (LC-MS) chip, and the background recovery rate for each individual was determined. The genotyping efficiency of the 1604 SNP loci ranged from 99.06% to 99.81%. Of the 1604 SNPs analyzed, 824 homozygous SNPs showing differences in WT and NY eggplants were selected for background recovery rate calculation. The distribution of these 824 SNP markers was mainly observed on chromosome 7, with the lowest SNP density on chromosome 4. The average backcross rate of the 65 individual plants was 41.36%, with the lowest and highest backcross rates being 17.32% and 64.91%, respectively. Figure 8 A). Further phenotypic evaluation showed that WNBC1F-45 was purple in color, while the fruit shape was similar to NY ( Figure 8 Using foreground selection markers and an eggplant 1.6K liquid phase chip as background markers to screen candidate progeny from the BC1F1 population can improve eggplant fruit color.

[0066] Example 4: This embodiment verifies the application of the 1.6K liquid phase chip for eggplant in the identification of true and false hybrids in eggplant.

[0067] This embodiment analyzed 52 F1 hybrids. The parental inbred lines were examined using an eggplant 1.6K liquid chromatography-mass spectrometry (LC-MS) chip. Differential and homozygous SNPs were selected from the parental inbred lines to identify true and false hybrids. If the selected SNP was heterozygous in the tested individual plant, the individual seedling was classified as a true hybrid; conversely, if the selected SNP was homozygous, the individual seedling was classified as a false hybrid.

[0068] The results showed that among the 1604 SNP loci, 408 were homozygous and differentially expressed in both parents. All 408 SNP loci in the 51 F1 hybrids exhibited heterozygosity, confirming that these 51 materials were true hybrids with a seed purity of 98.08%. Only one individual showed homozygosity in 408 SNP loci and was identified as the paternal genotype. This result indicates that this individual was the paternal material. However, the genotype of the false hybrid was identical to that of the paternal inbred line. This is likely due to mechanical contamination during seed harvesting or transportation, rather than incomplete manual removal of anthers during seed production, which leads to false hybridization. This demonstrates that the eggplant 1.6K liquid phase chip proposed in this application can detect seed purity at the seedling stage, thus saving significant time.

[0069] The above description, in conjunction with specific preferred embodiments, provides a further detailed explanation of the present invention. However, it should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, various simple deductions or substitutions can be made without departing from the concept of the present invention, and all such modifications and substitutions should be considered within the scope of protection of the present invention.

Claims

1. An eggplant SNP liquid phase chip, characterized in that, The eggplant SNP liquid-phase chip includes probes and / or primers for eggplant SNP molecular marker assemblies. The eggplant SNP molecular marker assemblies include 1604 SNP molecular markers. The physical locations of 1602 molecular markers were determined based on eggplant SME-HQ_V2.0 genome sequence alignment. The other two SNP sites are located on a functional sequence Seq1 associated with eggplant flesh color. The sequence of Seq1 is shown in SEQ ID NO:

1. The location information of the 1604 SNP molecular markers is shown in the table below. 。 2. A reagent kit, characterized in that, Including the eggplant SNP liquid phase chip as described in claim 1.

3. The application of the eggplant SNP liquid phase chip of claim 1 or the kit of claim 2 in at least one of the following: eggplant germplasm resource identification, eggplant genetic diversity assessment, eggplant variety purity identification, and eggplant pericarp color improvement breeding.

4. A method for identifying eggplant germplasm resources, characterized in that, This includes using the eggplant SNP liquid phase chip as described in claim 1 or the kit as described in claim 2 to genotype the test sample, and performing population genetic analysis based on the obtained genotype data of 1604 SNP loci to identify the species affiliation and genetic background of the test sample.

5. A method for identifying the purity of eggplant hybrids, characterized in that, This includes using the eggplant SNP liquid phase chip as described in claim 1 or the kit as described in claim 2 to perform genotyping on the parents and hybrid offspring, selecting SNP sites where there are homozygous differences between the parents, and determining that the offspring is a true hybrid if it is heterozygous at that site.

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