A new SNP genotyping technology Target SNP-seq and its application in genetic analysis of cucumber varieties

Abstract

To facilitate the utility of SNP-based genotyping, we developed a new method called target SNP-seq which combines the advantages of multiplex PCR amplification and high throughput sequencing. Compared with KASP, Microarrays, GBS and other SNP genotyping methods, target SNP-seq is flexible both in SNPs and samples, yields high accuracy, especially when genotyping genome wide perfect SNPs with high polymorphism and conserved flanking sequences, and is cost-effective, requiring 3 days and $7 for per DNA sample to genotype hundreds of SNP loci. The present study established a DNA fingerprint of 261 cucumber varieties by target SNP-seq with 163 perfect SNPs from 4,612,350 SNPs based on 182 cucumber resequencing datasets. Four distinct subpopulations were found in 261 Chinese cucumber varieties: the north China type, the south China type, the Europe type, and the Xishuangbanna type. The north China type and Xishuangbanna type harbored lower genetic diversity, indicating greater risk of genetic erosion in these two subpopulations. Furthermore, a core set of 24 SNPs was able to distinguish 99% of the 261 cucumber varieties. 29 core cucumber backbone varieties in China were identified. Therefore, target SNP-seq provides a new way to screen out core SNP loci from the whole genome for DNA fingerprinting of crop varieties. The high efficiency and low cost of target SNP-seq is more competitive than the current SNP genotyping methods, and it has excellent application prospects in genetic research, as well as in promoting plant breeding processes in the near future.

Agarose-resolvable InDel markers based on whole genome re-sequencing in cucumber

“…SNP genotyping is thereby mostly performed by commercial companies, and this is known to be a time-consuming process for the generation and achievement of a reasonable data. Early this year, Zhang and his team reported a new SNP genotyping in cucumber based on multiplex PCR amplification and high throughput sequencing 33 . InDel molecular markers provide advantages in different fold including high accuracy and high stability which help in deciphering the confusion that may arise in genetic analysis as compared to other length polymorphic markers.…”

K-seq, an affordable, reliable, and open Klenow NGS-based genotyping technology

“…This requirement increases the cost and limits the number of service providers available. GRR technologies can be improved, and new platforms are still being developed (11)(12)(13)(14)(15). It would be ideal to have open, patent-unencumbered technologies that could be easily used by any molecular biology laboratory.…”

Genetic Diversity, Pedigree Relationships, and A Haplotype-Based DNA Fingerprinting System of Red Bayberry Cultivars

“…SNPs are generally more abundant and more stable than SSRs, which are located at simple repeat regions and had mutation rates several orders of magnitude higher than SNPs (Fischer et al, 2017). Other advantages of SNPs include the easy application of automatic analysis and high consistency in genotyping results from different methods (Wu et al, 2014;Zhang et al, 2020). Moreover, methods that could genotype multiple SNPs simultaneously have been well developed (Tsykun et al, 2017), and the lower PIC per marker has become less a problem for SNPs.…”

“…Taking advantage of the next-generation sequencing (NGS), genotyping by sequencing (GBS) could genotype hundreds to millions of SNPs simultaneously and has been widely applied in crop plants (Kim et al, 2016). In cucumber (Cucumis sativus L.), multiplex PCR amplification and GBS were applied for genotyping multiple genomic segments in the variety fingerprinting (Zhang et al, 2020). SNP-based fingerprinting systems have also been constructed recently in oolong tea (Camellia sinensis L.) (Lin et al, 2020), cacao (Theobroma cacao L.) (Mahabir et al, 2020), eggplants (Solanum L.) (Gramazio et al, 2017), and pineapple (Ananas comosus L.) (Zhou et al, 2015).…”