Date Published: May 22, 2019
Publisher: Public Library of Science
Author(s): Habtamu Ayalew, Pak Wah Tsang, Chenggen Chu, Junzhou Wang, Shuyu Liu, Caifu Chen, Xue-Feng Ma, Guihua Bai.
Advances in high-throughput genotyping enable the generation of genome-scale data much more easily and at lower cost than ever before. However, small-scale and cost-effective high-throughput single-nucleotide polymorphism (SNP) genotyping technologies are still under development. In this study, we compared the performances of TaqMan, KASP and rhAmp SNP genotyping platforms in terms of their assay design flexibility, assay design success rate, allele call rate and quality, ease of experiment run and cost per sample. Fifty SNP markers linked to genes governing various agronomic traits of wheat were chosen to design SNP assays. Design success rates were 39/50, 49/50, and 49/50 for TaqMan, KASP, and rhAmp, respectively, and 30 SNP assays were manufactured for genotyping comparisons across the three platforms. rhAmp showed 97% of samples amplified while TaqMan and KASP showed 93% and 93.5% of amplifications, respectively. Allele call quality of rhAmp was 97%, while it was 98% for both TaqMan and KASP. rhAmp and KASP showed significantly better (p < 0.001) allele discrimination than TaqMan; however, TaqMan showed the most compact cluster. Based on the current market, rhAmp was the least expensive technology followed by KASP. In conclusion, rhAmp provides a reliable and cost-effective option for targeted genotyping and marker-assisted selection in crop genetic improvement.
Plant breeding is one of the applied research areas presumed to benefit greatly from the current advances in molecular marker technologies. Genome-scale sequencing technologies such as genotyping by sequencing (GBS) enable the generation of millions of single-nucleotide polymorphisms (SNPs) that can be used in genome-wide association studies (GWAS) to identify genes or quantitative trait loci (QTL) governing traits of interest [1–3]. Parallel developments in phenomics and statistical genetics enabled the identification of SNPs that are associated with desirable plant traits [4–7]. SNP markers are becoming preferred among breeders and molecular biologists because of their low-cost, high-genomic abundance, locus specificity, co-dominant inheritance, amenability to high-throughput genotyping, and relatively low genotyping error rates [8–10].
The three platforms did significantly differ in assay design success rate, allele call quality, allele cluster separation, cluster compactness and NTC-to-cluster distance (Table 2). Assay designs were started with 50 functional SNPs or INDELs of wheat across the three platforms. KASP and rhAmp both had higher successful design rates (49/50) than that of TaqMan (39/50) mainly because TaqMan probes cannot be designed if INDELs are more than six bps. Thus, only 30 sequences that contain target SNPs were used to design assays for all three platforms for genotyping platform comparisons (S1 Table). One SNP assay failed in all the three platforms. In each platform, 2,784 data points, including NTCs, were generated (96*29). Some samples failed to amplify even though the SNP assays worked well with other samples in the panel. Such failures may be ascribed to pipetting errors or primer mismatch due to mutations in these samples. As a result, TaqMan showed the highest number of unamplified samples (7%), followed by KASP (6.5%) and rhAmp (3%). Seventy-seven amplified samples had very low fluorescence making them difficult to be classified as either of the allele forms (Allele 11, 12 or 22). These alleles were reported as “Invalid” by allele auto calling using the Thermo Fisher Cloud Genotyping application. TaqMan showed 57 invalid allele calls, while KASP and rhAmp showed 13 and seven, respectively.
Small-scale high-throughput genotyping systems are in high demand in current plant breeding programs. TaqMan and KASP chemistries are among the most popular technologies in this regard so far. However, TaqMan is more expensive and less flexible in assay design because it does not accept INDELs longer than 6 bps. KASP requires higher amount of DNA template and yet its allele cluster separation is lower than that of TaqMan and rhAmp due to its low fluorescence signal. rhAmp was developed to fill these gaps at lower cost with more flexibility in assay design compared with TaqMan and better allele discrimination efficiency with less DNA input compared to KASP. Comparative performance of these three technologies in genotyping hexaploid species has never been reported before. This research was conducted to objectively compare the three technologies in terms of ease of assay design, allele call rate and quality, allele cluster compactness and angle of separation between allele clusters (homozygotes from heterozygotes).