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A single nucleotide polymorphism SNP is one-base variation in a single DNA nucleotide that occurs at a specific position in the genome 1. Compared with simple sequence repeats SSRs , SNPs are the most abundant and stable genetic variation in genomes 2 , 3. The wide occurrence of SNPs in a gene or in a regulatory region generally act as biological markers for identified genes associated with important traits 4. In plants, SNPs have a particularly important application in reflecting both natural genetic variability and genetic drift created by breeders during plant improvement 5 , 6.

Thus, SNPs are ideal markers for genetic background analysis due to their advantages in high-throughput detection and easy integration of genotyping data 7. Currently, DNA fingerprints of germplasms or varieties based on SNP markers have been constructed in several crops, such as rice 8 , maize 7 , wheat 9 , cotton 10 , soybean 11 , eggplant 12 and pepper 13 , 14 , most of which are applied with DNA microarray systems.

It is difficult to meet the rapidly increasing need for SNP genotyping The continuous progress in high-throughput sequencing has facilitated the development of some high-throughput SNP genotyping methods, such as the Gene Chip microarray or the KASP platform competitive allele-specific PCR While chip-based platforms require enormous amounts of time for SNP assay designing and are only suitable for genotyping hundreds of samples with thousands of SNPs KASP system also need relatively high cost for genotyping each sample.

These SNP genotyping platforms often find that some SNP loci cannot be well detected and genotyped because the flanking regions of these SNPs are not conserved or there are other hits in the genome due to the sequences homology 18 , 19 , This causes many false positive or false negative results in SNP genotyping. Moreover, the huge cost of one-time investment in instruments or expensive microarrays also limits the use of SNPs in most laboratories.

Therefore, it is necessary to use a new technology that is cost-effective and highly accurate for genotyping middle-scale — markers genome-wide SNPs. At present, next-generation sequencing NGS technology is widely used, and the whole-genome sequences of hundreds of species have been assembled, which has promoted large-scale re-sequencing and produced millions of SNPs for genetic research To our best knowledge, few studies have focused on analyzing extensive variation genomes and developing perfect SNP loci which harbor conserved flanking regions and have unique PCR amplification in the genome.

Here, we developed a new method called target SNP-seq which combines the advantages of high-throughput sequencing and multiplex PCR amplification by using genome-wide perfect SNPs which meet the requirements of harboring conserved flanking sequences and being captured uniquely in PCR amplification. Furthermore, this approach could gain hundreds of SNP genotypes by sequencing the SNP region with an average of times coverage in a short time and at low cost. As is well known, the narrow genetic background and high risk of genetic erosion cucumber has been clearly demonstrated because of hybrid varieties deriving from limited parents and breeders pursing similar breeding goals 27 , The annually increasing number of commercial cucumber varieties presents a major challenge for plant variety management and protection in China.

Although several DNA fingerprinting research in cucumber were reported using simple satellite repeat SSR technology 28 , 29 , 30 , it was still difficult to obtain genotypes in high throughput way. Cucumber genome was the first sequenced and well assembled species in vegetables 31 , and whole genome sequencing in various cucumber germplasms made it possible to discover perfect SSR and SNP for genotyping Our previous research analyzed the genetic diversity of Chinese cucumber varieties with perfect SSRs by target SSR-seq technology In this study, we applied the target SNP-seq genotyping technology with perfect SNPs in establishing the DNA fingerprint of cucumber varieties, and revealed the population structure and genetic features in cucumber varieties.

The first-draft cucumber genome was assembled from the Chinese cucumber inbred line 31 , and its Version 2 was used as a reference genome in the present study. Two units 2U of enzyme were used per reaction. Finally, the constructed library was sequenced on Highseq X platform. In the current study, sequencing reads of varieties were aligned to cucumber reference genome V2 by using BWA to determine the physical location of each target SNP amplicon The maximal numbers reads of SNP allele was taken as the major allele, while the other allele was taken as minor allele.

To ensure the accuracy of SNP genotypes, we also filtered those sequencing reads with major allelic depth less than 20 in a variety. For heterozygous varieties, a ratio of major and minor alleles under 0. Finally, specific SNP variant bases for each variety were identified by analyzing the in-house barcodes assigned raw sequence reads.

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A total of cucumber varieties were selected in this study to evaluate the novel technique of target SNP-seq in variety identification. Previous studies showed that the genetic purity of hybrid seeds is prone to contamination due to the occurrence of out-crossing with foreign or self-pollinated and physical admixtures In order to increase the genotype accuracy of each cucumber variety, newly expanded young leaves grown from 30 seeds were used for DNA extraction.

The concentration of all extracted DNA was quantified using a Qubit2. All these calculations were performed in Excel using the following formulae:. In order to determine population number K , three parallel runs were performed for each simulated value of K ranging from 2 to 10 with the following parameters: MCMC Markov chain Monte Carlo replicas run for 10, iterations and , generations of burn-in for each K. To verify the optimal number of clusters, principal component analysis PCA was performed in the respective R package.

The neighbor-joining NJ tree generated from MEGA7 was used to analyze the genetic relationship among varieties based on the genetic distance in poppr R package. Additionally, we measured the genetic differentiation between pairs of subpopulations with an analysis of molecular variance AMOVA and computed the pairwise Fst in the poppr R package. To screen out a minimal number of SNPs for distinguishing the maximal number of cucumber varieties, we used a script in Perl based on analyzing the genetic diversity of each SNP site, which was successfully used in selecting core SSR for cucumber identification 33 and core SNP for pepper identification 13 , respectively.

According to the UPOV standard for the authentic testing of crop varieties 42 , two varieties were believed to be identical when they have same genotype in a certain set of molecular markers. We set up a pairwise comparison matrix by calculating the numbers of differential SNP genotypes between each pair of varieties, and the missing genotype was treated as null.

One variety has less differential SNP genotype with other cucumber varieties indicated that it has a closer kinship with others. The top 10 percent of varieties with close kinship in each subpopulation were considered as core varieties. Moreover, to verify that this comparison matrix was able to represent the kinship of cucumber varieties, correlation analysis among three pairwise comparison matrix differential SNP genotypes, genetic distance and the genetic similarity by NTSYSpc2.

This new method combines the advantages of multiplexing PCR and targeted deep sequencing. We could obtain the SNP genotype data by analyzing the specific barcodes assigned to DNA samples from the raw sequence reads. Thus, target SNP-seq is suitable for use in DNA fingerprinting, variety identification, genetic research, and molecular breeding. The SNP genotype of the inbred cucumber line from target SNP-seq was the same as that of the cucumber reference genome, indicating that this technique had good repeatability and high accuracy in SNP genotyping.

A total of 4,, SNPs were detected in the cucumber V2 whole-genome sequence 31 , indicating that every According to the objective in this study, we selected perfect SNPs for multiplex PCR primer designing, which were evenly distributed on the whole genome. The number of SNP loci per chromosome ranged from 21 on chromosome 5 to 26 on chromosome 7. The average marker spacing was 1. Genetic characterization of SNPs in cucumber varieties.

A total of The sequencing depth of This distribution was a comparatively ideal model in high-throughput sequencing, which ensured enough coverage for most SNP data and avoided polarized sequencing on the bulk amplicons According to the major and minor allelic reads ratio, Distribution of sequencing coverage and reads ratios of target SNP-seq. The observed heterozygosity Ho of varieties ranged from 0 to 0. The PIC value ranged from 0. Interestingly, the inbreeding coefficient in three SNP loci was equal to 1, indicating that these SNPs had no heterozygous genotype in all varieties.

The average genetic diversity coefficient for varieties was 0.

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Overall, varieties were assigned to subgroup 1 Pop1 and 81 varieties were assigned to subgroup 2 Pop2. To detect the subpopulation of cucumber varieties, we further investigated the structure changes with the increase of K value. Pair wise estimates of Fst ranged from 0. The same genetic divergence among cucumber varieties was also observed by PCA and neighbor-joining tree.

In PCA analysis, the first axis explained The PCA plot indicated that the Europe type showed a disperse distribution while the other types presented cluster distribution Fig. The dendrogram of cucumber varieties also exhibited four distinct subgroups, and the Xishuangbanna type was first separated from other types, followed by the Europe type, south China type, and north China type Fig. These four subpopulations were also in accordance with the geographical distribution and morphological characteristics of cucumber varieties, which proved that target SNP-seq is a powerful tool for genetic analysis.

Population structure analysis in cucumber varieties.

The core SNP set was mainly used in variety identification and could distinguish the maximum number of varieties using the minimum number of SNP loci. A Perl script was developed to calculate the discrimination power of perfect SNPs by comparing 33 pairwise between varieties To assess the ability of the 24 core SNPs to represent SNPs in variety identification, we calculated the correlation coefficients of genetic similarity, genetic distance, and differential SNP markers in cucumber varieties Fig. For all SNPs, the correlation coefficients r of genetic similarity with genetic distance and differential SNP markers were both —0.

This suggests higher values of genetic similarity are associated with lower values of genetic distance and differential SNP markers. Hence, the 24 core SNP loci selected in the present study were able to represent SNPs in the identification of cucumber varieties from Chinese markets. Ellipses obliquing to right and left respectively indicate positive and negative correlations with significant p -values at 0.

As is well known, hybrid cucumber varieties in China were derived from limited germplasms and breeders pursed similar breeding goals, which result in the genetic diversity of cultivated cucumber is obviously narrow. The average differential SNP markers in the north China type was The reason for this is that the north China type has a long history of variety hybridization breeding in China while the Europe type was introduced to China in recent years. For each subpopulation, the top 10 percent of varieties with the minimum number of differential SNP genotypes were determined as core varieties.

Blue to red indicate the increasing number of differential SNP genotypes. Over the past three decades, several methods for SNP genotyping have been described, and the application of SNP technology has accelerated in genetic research However, the high labor cost and low efficiency are still challenges for current SNP genotyping platforms 25 , In the present study, we reported the utility of target SNP-seq—a novel method for SNP genotyping and applied in genetic analysis of cucumber varieties.

We first designed a primer panel consisting of discovered perfect SNPs in cucumber based on resequencing datasets in the multiplex PCR approach of target SNP-seq. Then, the millions of reads were aligned to the cucumber reference genome V2 based on strict alignment parameters, in order to obtain the accurate SNP genotypes Fig. Due to heterozygosis genotype was typically calculated by the ratio between major and minor raw sequencing reads, higher sequencing depth can score higher call rates and accuracy in genotyping and sequencing technologies Cucumber is recognized to have originated in the Himalayan Mountains, and has been domesticated for nearly years According to morphological characteristics and geographical distribution, more than cucumber germplasms and the core set of cucumber accessions were classified the into four types as follows: Indian group, Eurasian group, East Asian group, and Xishuangbanna group The cultivation of cucumber in China has a long history, dating back to the Han dynasty according to historic records, when the diplomat Zhang Qian brought back cucumber through the Silk Route Although the genetic diversity of cucumber germplasm resources in China has been well investigated by molecular markers, relatively few markers or varieties were tested, and a comprehensive study of cultivated varieties in the Chinese market was lacking 27 , Because the assessment of genetic variation and relationship in cultivated varieties is essential for plant variety protection and breeding novel varieties to satisfy customers worldwide 51 , this study analyzed dominant cucumber varieties in the Chinese market using target SNP-seq with genome-wide perfect SNPs, and we found that Chinese cucumber varieties could be classified to four subgroups i.

Our previous study also found that the South China type and Europe type had higher genetic diversity than that in north China type and Xishuangbanna type 33 , indicating the narrow genetic background of the latter two types. The close similarity and narrow genetic diversity of cucumber varieties implies a risk of genetic erosion in the breeding process caused by narrowing the exploitation of new genes Therefore, it is urgent to increase the genetic diversity of varieties in the north China type and Xishuangbanna type by introgressing some favorable genes from natural resources into the breeding system.

In order to protect the intellectual property of crop breeders, each new candidate variety must undergo DUS distinctness, uniformity, and stability 42 testing. It is well known that the molecular markers have advantages in identifying varieties, including their co-dominance, high reproducibility, and the fact that they are free from environmental impact, compared with the phenotypic or morphological characters by traditional field inspection The SNP technology was applied to variety identification and DNA fingerprinting because it is preferable to high-throughput genotyping, which was studied based on microarrays or other systems in several crops such as maize, wheat, etc 7 , 9 , However, the high cost for each variety and the large amount of time consumed make the wide use of this technology difficult in variety identification and management, in addition to the proportion of false positives or negatives caused by SNPs with diverse flanking sequences or non-specific capturing.

Our results are of great importance for the identification of the authenticity and measurement of the purity of cucumber varieties, and for the protection of plant varieties in the future.