Eggplant SNP (Single Nucleotide Polymorphism) molecular marker combination, SNP liquid chip, kit and application thereof

Abstract

The invention belongs to the field of molecular genetic breeding, and discloses an eggplant SNP (Single Nucleotide Polymorphism) molecular marker combination which comprises 1604 SNP molecular markers, and the position information is shown in the specification table 1. The invention also discloses an eggplant SNP liquid phase chip and a kit, which comprise a probe or / and a primer for detecting the eggplant SNP molecular marker combination, the invention also discloses an application of at least one of an eggplant SNP molecular marker combination, an eggplant SNP liquid phase chip or a kit in eggplant germplasm resource identification, genetic diversity evaluation, variety purity identification, eggplant genetic map construction and QTL positioning thereof, molecular marker-assisted selective breeding, whole genome selection association analysis, directional improvement or multi-gene pyramiding breeding. The eggplant SNP molecular marker combination provided by the invention is high in detection flux, large in output result information amount, high in target site detection rate and accurate and reliable in typing result.

Description

Technical Field

[0001] The invention belongs to the field of molecular genetic breeding, and in particular relates to an eggplant SNP molecular marker combination, a whole genome liquid phase chip, a kit and applications thereof. Background Art

[0002] Eggplant (Solanum melongena L) is an important vegetable crop with global economic importance, with a total harvested area of ​​1,892,798 hectares and a production of 59,312,599.76 metric tons reported by FAO. Eggplant is rich in essential nutrients and contains polyphenols such as anthocyanins and chlorogenic acid, which are beneficial to health. Eggplant exhibits great heterosis, making F1 hybrids a popular choice in cultivation. Breeding of superior lines and selection of hybrid parents are key steps in developing high-yielding and high-quality eggplant varieties. Breeding of superior inbred lines is essential for the selection of hybrids, and backcross breeding is often used to introduce desirable traits into superior inbred lines. However, the traditional backcross breeding method is labor-intensive and time-consuming because it selects individuals based on phenotypic characteristics. Molecular marker-assisted selection accelerates this process by selecting individuals with high parental background recovery using molecular markers. Utilizing marker spacing of 10-20 cM provides an advantage of one to two backcross generations compared to random or phenotypic selection. Studies have shown that introgression of target genes into elite lines can be achieved in two backcross generations while recovering more than 96% of the recipient parent genotype. However, the lack of affordable high-throughput genotyping tools has hampered related research in 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, SNP detection across the genome has become easier. Currently, three main approaches are used for SNP detection. Genotyping by sequencing (GBS). It has been shown to be successfully applied in genetic diversity analysis and QTL mapping in eggplant. Nevertheless, the high cost and complex genotyping process hinder its wider application. The second approach relies on PCR techniques such as competitive allele-specific PCR (KASP), Taqman, etc., which can effectively identify specific known loci. These methods have been used for gene / QTL fine mapping and SNP detection in eggplant. The third approach involves SNP arrays, and there are currently two construction strategies. Solid SNP arrays are based on Illumina or Affymetrix platforms. Solid SNP arrays exhibit some limitations, including high equipment cost, low flexibility in marker redesign, and specific genotyping platforms. The second is a target sequencing genotyping (GBTS) system based on multiplex PCR or solution capture, which has the advantages of high genotyping accuracy, high throughput, low cost, and flexible design, and has been applied to various crops. Liu et al. used resequencing data from 45 different lines to identify 219 whole-genome perfect SNPs in eggplant. The genetic diversity and unique DNA fingerprints of 377 eggplant varieties were analyzed using 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 the requirements of breeding. 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 above background technology and provide an eggplant 1.6K molecular marker combination, chip, kit and application thereof.

[0005] In order to solve the above technical problems, the technical solution proposed by the present invention is:

[0006] An eggplant SNP molecular marker combination includes 1604 SNP molecular markers, the physical positions of the 1604 molecular markers are determined based on the eggplant SME-HQ_V2.0 genome sequence alignment, and the position information is shown in Table 1 of the specification. The genotype information of the 1604 molecular markers is expressed in the form of "ref / alt (reference base / variant base)".

[0007] Based on a general inventive concept, the present invention also provides an eggplant SNP chip, which includes probes and / or primers for detecting the above-mentioned eggplant SNP molecular marker combination.

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

[0009] Based on a general inventive concept, the present invention also provides the use of at least one of the eggplant SNP molecular marker combination, the eggplant SNP chip or the kit in the identification of eggplant germplasm resources.

[0010] Based on a general inventive concept, the present invention also provides the use of at least one of the eggplant SNP molecular marker combination, the eggplant SNP chip or the kit in eggplant genetic diversity assessment.

[0011] Based on a general inventive concept, the present invention also provides the use of at least one of the eggplant SNP molecular marker combination, the eggplant SNP chip or the kit in the purity identification of eggplant varieties.

[0012] Based on a general inventive concept, the present invention also provides the use of at least one of the eggplant SNP molecular marker combination, the eggplant SNP chip or the kit in eggplant genetic map construction or QTL positioning.

[0013] Based on a general inventive concept, the present invention also provides the use of at least one of the above-mentioned eggplant SNP molecular marker combination, the above-mentioned eggplant SNP chip or the above-mentioned kit in eggplant molecular marker-assisted selection breeding, directed improvement or multi-gene aggregation breeding.

[0014] Based on a general inventive concept, the present invention also provides the use of at least one of the eggplant SNP molecular marker combination, the eggplant SNP chip or the kit in whole-genome selection association analysis of eggplant.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] (1) Based on the collected resequencing data of 118 eggplant varieties with rich diversity, the present invention selects 1604 molecular marker sites with strong representativeness, high polymorphism (MAF average value 0.36), good versatility, high coverage rate on the genome (average coverage rate 99.37%) and uniform distribution from millions of molecular markers; and provides a chip for detecting the eggplant SNP molecular marker combination, which has high detection throughput, large output result information volume, high target site detection rate (99.72%), and accurate and reliable typing results (repeat consistency rate 99.86%); it is a high-throughput, high-information, high-accuracy, high-repeatability, low-cost, multi-functional and widely used molecular marker chip.

[0017] (2) The liquid phase chip of the present invention adds 14 functional markers related to the important traits of eggplant, such as flower number, fruit length, fruit shape, peel color, and flesh color, which is beneficial to molecular marker-assisted selection, targeted improvement, multi-gene aggregation breeding, and important trait gene mining and identification and functional analysis, etc., and has great utilization value in the process of eggplant variety improvement and variety breeding.

[0018] (3) Compared with traditional solid-phase chips, the liquid chip involved in the present invention has higher flexibility and can add marker sites at any time according to the application scenario. At the same time, the liquid chip relies on the second-generation sequencing platform, with low typing cost, high detection throughput, and large one-time output data volume. It can cover the detection of nearly a thousand materials at the same time, providing technical means for large-scale typing. Based on targeted capture sequencing technology, the liquid chip can not only type the target site, but also accurately type the genetic variation sites within a certain range around the target site. Therefore, more SNP typing information can be obtained than the expected marker site, providing richer site information support for eggplant molecular breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The distribution of eggplant 1.6K liquid phase chip sites on chromosomes in the embodiment of the present invention;

[0020] Figure 2 This is a MAF distribution diagram of the eggplant 1.6K liquid phase chip site in an embodiment of the present invention;

[0021] Figure 3 The mGPS liquid chip technology principle and process involved in the embodiments of the present invention;

[0022] Figure 4 is the genotype consistency rate of repeated samples of eggplant 1.6K liquid phase chip in the embodiment of the present invention;

[0023] Figure 5 It is the 99 eggplant model structures analyzed based on 1.6K liquid phase chip in the embodiment of the present invention;

[0024] Figure 6 It is the principal component analysis of 99 eggplant varieties based on 1.6K liquid phase chip in the embodiment of the present invention;

[0025] Figure 7 It is a 99 eggplant genetic tree based on 1.6K liquid phase chip in the embodiment of the present invention;

[0026] Figure 8This is the screening of candidate offspring from the BC1F1 population based on the 1.6K liquid phase chip in an embodiment of the present invention: A. PRPG statistics of candidate offspring from the BC1F1 population; B. genotype visualization of BC1F1 candidate offspring; C. phenotype visualization of BC1F1 candidate offspring. DETAILED DESCRIPTION

[0027] In order to facilitate the understanding of the present invention, the present invention will be described more comprehensively and meticulously below in conjunction with the accompanying drawings and preferred embodiments of the present invention, but the protection scope of the present invention is not limited to the following specific embodiments.

[0028] Unless otherwise defined, all professional terms used below have the same meanings as those generally understood by those skilled in the art. The professional terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention.

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

[0030] Embodiment 1:

[0031] An eggplant SNP molecular marker combination of the present invention includes 1604 SNP molecular markers, the physical positions of the 1604 molecular markers are determined based on the eggplant SME-HQ_V2.0 genome sequence alignment, the genotype information of the 1604 molecular markers is expressed in the form of "ref / alt (reference base / variant base)", and the position information is shown in Table 1.

[0032] Table 1: Information on 1604 SNP loci of eggplant SNP molecular marker combination

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[0052] The process of obtaining the molecular marker combination is as follows:

[0053] 1. Collection of eggplant germplasm resources

[0054] In order to obtain rich and diverse whole-genome SNP sites, 80 eggplant materials from different regions and varieties were collected for whole-genome resequencing. At the same time, the project team collected 16 eggplant resequencing data from the early stage and 22 eggplant material resequencing data from the NCBI database.

[0055] 2. Whole-genome resequencing of eggplant

[0056] The 80 eggplant materials collected were subjected to DNA extraction and whole genome resequencing. The specific steps included:

[0057] (1) Extract DNA using magnetic beads.

[0058] (2) Using the MGI library standard method, those that passed the quality inspection were used for DNA-seq sequencing library construction.

[0059] (3) After the library passed the quality inspection, it was sequenced using the BGI sequencing platform, with a sequencing strategy of PE150 and a sequencing depth of 10×.

[0060] Sentieon was used to compare and detect variations in all the collected 118 eggplant material resequencing data. The analysis process is as follows:

[0061] (1) Use Sentieon to align reads to the corresponding eggplant reference genome (SME-HQ_V2.0), positionally sort, and mark duplicate reads.

[0062] (2) Use Sentieon to detect the variant sites of each sample and obtain the variant information of each sample.

[0063] (3) Sentieon was used for joint-calling, and the gVCF of all samples was jointly analyzed to obtain the variation results of each individual in the population. To ensure the accuracy of SNP, the SNP sites obtained after the joint analysis were initially hard-filtered (SNP hard-filtering standard: "QD<2.0||FS>60.0||MQ<40.0||SOR>3.0||MQRankSum<-12.5||ReadPosRankSum<-8.0").

[0064] 3. Site screening

[0065] (1) Genome-wide loci

[0066] The MAF value, detection rate, heterozygosity rate and sequencing depth of the loci were calculated and counted from the VCF file containing the SNP variation information of all samples, and polymorphic SNP loci with minimum allele frequency MAF ≥ 0.05, deletion rate < 0.1, heterozygosity rate < 0.1, and sequencing depth ≥ 9× were selected as initial candidate loci. All the above candidate loci were used for primer design. For each SNP locus, the specificity and copy number were analyzed according to the sequences on both sides of the SNP locus, and the primers that could not be uniquely aligned on the genome and contained repeated sequences in the flanking sequences were removed. Based on the principle of uniform distribution, the sites with high MAF values ​​were selected for retention, and 1590 SNP loci with high polymorphism were finally obtained.

[0067] (2) Functional sites

[0068] Fourteen functional loci related to important traits of eggplant such as flower number, fruit length, fruit shape, skin color, and flesh color from previous studies were collected.

[0069] 4. Preparation of Eggplant 1.6K Liquid Phase Chip

[0070] All marker sites were obtained by integrating the above-mentioned site screening, namely, 1590 whole genome site markers with strong representativeness, high polymorphism, good versatility and uniform distribution 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, which together constitute the eggplant 1.6K liquid phase chip sites. Specifically, the 1604 selected molecular markers were developed into the eggplant 1.6K liquid phase chip using the multiplex PCR precise positioning sequencing typing technology (Genotyping by Pinpoint Sequencing of multiplex PCR products, mGPS) of Huazhi Biotechnology Co., Ltd. The mGPS liquid phase chip technology is a targeted capture sequencing technology based on multiplex PCR. Specific primers are designed for multiple target intervals in the reference genome, and multiple different target sequences are specifically amplified simultaneously by multiplex PCR. Then, the PCR amplicons are sequenced by library construction and second-generation sequencing, so as to obtain the genotypes of all SNP / InDel sites in the target interval.

[0071] The average coverage of eggplant 1.6K liquid phase chip sites on chromosomes is 99.37%, with an average spacing of 700Kb. The 1.6K sites are evenly distributed on each chromosome. The density distribution diagram is shown in Figure 1 The eggplant 1.6K chip locus has a high polymorphism, with an average minor allele frequency (MAF value) of 0.36. The MAF value distribution diagram is shown in Figure 2 shown.

[0072] The genotyping process of the eggplant 1.6K liquid phase chip specifically includes:

[0073] (1) Extraction and quality control of genomic DNA

[0074] The magnetic bead method was used to extract genomic DNA from fresh eggplant leaves, and the DNA samples were quality tested. The quality test included concentration test and integrity test. The DNA concentration was measured by Qubit fluorescence quantification instrument, and the integrity of DNA was tested by 1% agarose gel electrophoresis. The samples that passed the quality test were used for library preparation.

[0075] (2) Construction and quality control of mGPS library

[0076] (a) Using each genomic DNA as a template and multiple PCR reaction reagents, the first round of PCR amplification is performed to simultaneously perform specific amplification on multiple target intervals. The amplified products are purified using magnetic beads to remove impurities and non-specific amplified products. The concentration of the purified products is detected by Qubit fluorescence quantification instrument, and the fragment size is detected by agarose gel electrophoresis.

[0077] (b) Perform a second round of PCR on the purified amplified product. Through the second round of PCR, the adapter sequence required for the second-generation sequencing is introduced to both sides of the amplicon product, and the product is purified and recovered using magnetic beads. The library concentration is detected by Qubit fluorescence quantification instrument, and the fragment size is detected by agarose gel electrophoresis. Once the concentration and fragment size are qualified, the construction of the mGPS sequencing library is completed.

[0078] 3. Sequencing

[0079] After the library quality inspection is qualified, the sequencing chip is loaded and sequenced on the machine according to the standards of the BGI sequencing platform, and the sequencing strategy is PE150.

[0080] 4. Data Analysis

[0081] The raw data after high-throughput sequencing was processed by quality control filtering, and fastp software was used to remove reads containing adapter contamination and low-quality reads. The obtained clean reads were compared with the target genome using BWA software, and then the sequencing results were analyzed for variant sites using GATK software to obtain the genotyping results of the target sites. The principle and process of mGPS liquid phase chip technology can be found in Figure 3 .

[0082] In order to verify the genotyping effect of eggplant 1.6K liquid phase chip, the 1.6K liquid phase chip obtained above was used to set up 2 technical replicates for 4 randomly selected materials. By comparing the genotype results of the 4 replicate samples, the genotype consistency rate was between 99.75% and 100.00%, and the average consistency rate was 99.86%. The results are shown in Figure 4 The above data show that when the eggplant 1.6K liquid phase chip is used to perform genotyping on the materials to be tested, the target site detection rate is high, the stability is good, and the typing results are accurate and reliable.

[0083] Embodiment 2:

[0084] This example verifies the application of the eggplant 1.6K liquid phase chip in the identification of eggplant germplasm resources.

[0085] A total of 99 eggplant germplasms were used for genetic diversity analysis. Genomic DNA was isolated from fresh leaves using the magnetic bead method, and SNPs were detected using the eggplant 1.6K liquid phase SNP chip to analyze the genetic diversity of 99 high-generation inbred lines. The change curve of the Bayesian Information Criterion (BIC) determined the optimal K value, and the cross-validation error showed that the optimal K value was K = 2, such as Figure 5The results showed that the 99 eggplant samples consisted of 95 individuals in subgroup 1 and 4 individuals in subgroup 2, the 4 individuals in subgroup 2 were classified as Solanum aethiopicum L., and the remaining 95 materials belonged to Solanum melongena L.

[0086] The genetic structure was analyzed using ADMIXTURE software with 1000 replicates. The genetic tree was constructed using the neighbor-joining method in Phylip, and principal component analysis (PCA) was performed using Plink2, as Figure 6 As shown, the first and second principal components together explained 71.24% of the genetic variance. The results showed that Solanum aethiopicum L. and Solanum melongena L. had obvious clustering characteristics. The materials with a genetic composition of 50.15% Solanum melongena L and 49.85% Solanumaethiopicum L were a separate class.

[0087] In addition, phylogenetic analysis was performed using neighbor-joining trees based on pairwise genetic distances, e.g. Figure 7 The results of the study classified the 99 eggplant samples into six different subgroups, of which four wild eggplant samples were clustered together, while the remaining 95 eggplant samples were divided into another five groups.

[0088] The principal component analysis and phylogenetic analysis above showed that the eggplant 1.6K SNP molecular marker has the ability to distinguish between cultivated and wild eggplants, thereby identifying the hybrid progeny of cultivated and wild eggplants. This result shows that the eggplant 1.6K liquid phase chip can be used for the genetic diversity analysis of eggplant germplasm resources.

[0089] Embodiment 3:

[0090] This example verifies the application of eggplant 1.6K liquid phase array in molecular marker-assisted breeding.

[0091] The current parent (NY) shows 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. In order to enhance the fruit color, backcrossing is required. The fruit color of the donor parent (WT) is purple-red.

[0092] Whole genome resequencing of NY and WT was performed first, and then anthocyanin biosynthesis and regulatory genes associated with eggplant fruit were identified. SNPs identified in the associated genes between NY and WT were used to design PARMS-SNP primers, which were designed using Primer 5.0. The SNP genotyping method was based on 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 the eggplant 1.6K SNP liquid phase chip, and the background recovery rate was calculated using the formula A / (A+B)*100%, where A represents the same number of markers as the parent, and B is the total number of markers evaluated for background selection. The genotype of each individual was visualized using an in-house R script.

[0093] Using four SNP markers that are closely related to eggplant fruit color, a total of 1104 BC1F1 eggplants were screened and 65 target strains were identified. Subsequently, the eggplant 1.6K liquid phase chip was used to genotype the 65 BC1F1 individuals, and the background recovery rate of each individual was determined. The genotyping efficiency of 1603 SNP loci ranged from 99.06% to 99.81%. Among the 1603 SNPs analyzed, 824 SNPs with homozygous differences in WT and NY eggplants were selected for background recovery rate calculation. Among them, the distribution of the 824 SNP markers was mainly observed on chromosome 7, and the SNP density on chromosome 4 was the lowest. The average backcross rate of the 65 individual plants was 41.36%, and the lowest and highest backcross rates were 17.32% and 64.91%, respectively ( Figure 8 A). Further phenotypic evaluation showed that the color of WNBC1F-45 was purple, while the fruit shape was similar to that of NY ( Figure 8 B and 8C). Screening candidate progeny from the BC1F1 population using foreground selection markers and the eggplant 1.6K liquid phase array as background markers can improve eggplant fruit color.

[0094] Embodiment 4:

[0095] This example verifies the application of the eggplant 1.6K liquid phase chip in the identification of true and false eggplant hybrids.

[0096] In this example, a total of 52 F1 hybrids were analyzed, and the parental inbred lines were examined using the eggplant 1.6K liquid phase chip, and the difference and homozygous SNPs in the parental inbred lines were selected to identify true hybrids and false hybrids. If the selected SNP is heterozygous in the detected individual plant, the individual seedling is classified as a true hybrid; on the contrary, if the selected SNP is homozygous, the individual seedling is classified as a false hybrid.

[0097] The results showed that all 408 SNP sites in 51 F1 hybrids showed heterozygosity, confirming that these 51 materials were true hybrids with a seed purity of 98.08%. Only one individual 408SNP was homozygous and was identified as paternal genotyping. This result indicates that the individual is the paternal material. However, the genotype of the pseudo-hybrid plant is the same as that of the paternal inbred line, which may be caused by mechanical contamination during seed harvesting or transportation, rather than by incomplete manual removal of anthers during seed production, which would lead to the existence of pseudo-hybridization. This shows that the eggplant 1.6K liquid phase chip of the present application can detect seed purity at the seedling stage, thereby saving a lot of time.

[0098] The above contents are further detailed descriptions of the present invention in combination with specific preferred embodiments, and it cannot be determined that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the field to which the present invention belongs, several simple deductions or substitutions can be made without departing from the concept of the present invention, which should be regarded as falling within the scope of protection of the present invention.

Claims

1. An eggplant SNP molecular marker combination, characterized in that: It includes 1604 SNP molecular markers, and the physical positions of the 1604 molecular markers are determined based on the eggplant SME-HQ_V2.0 genome sequence alignment, and the position information is shown in Table 1 of the specification.

2. An eggplant SNP liquid phase chip, characterized in that: The eggplant SNP chip comprises probes and / or primers for detecting the eggplant SNP molecular marker combination according to claim 1.

3. A kit, characterized in that: The kit comprises the eggplant SNP liquid phase chip as claimed in claim 2.

4. Use of at least one of the eggplant SNP molecular marker combination according to claim 1, the eggplant SNP liquid phase chip according to claim 2, or the kit according to claim 3 in the identification of eggplant germplasm resources.

5. Use of at least one of the eggplant SNP molecular marker combination according to claim 1, the eggplant SNP liquid phase chip according to claim 2, or the kit according to claim 3 in eggplant genetic diversity assessment.

6. Use of at least one of the eggplant SNP molecular marker combination according to claim 1, the eggplant SNP liquid phase chip according to claim 2, or the kit according to claim 3 in the purity identification of eggplant varieties.

7. Use of at least one of the eggplant SNP molecular marker combination according to claim 1, the eggplant SNP liquid phase chip according to claim 2, or the kit according to claim 3 in eggplant genetic map construction or QTL positioning.

8. Use of at least one of the eggplant SNP molecular marker combination according to claim 1, the eggplant SNP liquid phase chip according to claim 2, or the kit according to claim 3 in eggplant molecular marker-assisted selection breeding, directed improvement, or multi-gene aggregation breeding.

9. Use of at least one of the eggplant SNP molecular marker combination according to claim 1, the eggplant SNP liquid phase chip according to claim 2, or the kit according to claim 3 in whole-genome selection association analysis of eggplant.

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