With the advancement of genomics and molecular biology techniques, high-throughput molecular marker technologies such as SNP chips and Genotyping-By-Sequencing (GBS) based on second-generation sequencing have been developed. These high-throughput technology platforms have significant applications in various research areas, including the discovery of SNP loci among different genetic materials, selection of genetic backgrounds in crop breeding processes, and genome-wide association studies. However, due to their relatively high costs and complex experimental procedures, it is challenging to employ these high-throughput technology platforms in molecular marker-assisted breeding for crops.
In practical applications of molecular marker-assisted breeding for crops, several functional polymorphic loci associated with important agronomic traits of interest are typically utilized. Molecular markers based on PCR technology are designed for these loci, facilitating the assisted selection of genetic lineages.
In this approach, with the aid of Taq DNA ligase, a ligation reaction takes place only when both the left and right oligonucleotide probes perfectly complement the target DNA sequence, leaving no gaps between them. If there are any gaps between these two probes, the ligation reaction is impeded. Subsequently, through fluorescence scanning and the alignment of gel positioning based on shifts in sequencing peaks, variations in genotypes at the same locus are ascertained by considering the last three unmarked probe base differences. By examining the last seven unmarked probe base differences, distinct loci are identified, thereby enabling the precise detection of SNP sites.
Multiplex PCR Amplification: Obtaining the fragment where the target locus is located.
Multiplex LDR: Producing detection products of varying lengths.
Sequencing: Different peaks are obtained based on sequencing electrophoresis.
Interpretation of SNP results and data analysis.
Universality: Applicable for the genotyping of any polymorphic locus and various SNP loci, without the need for the introduction of restriction enzyme sites.
Accurate Typing with High Detection Rates: Accuracy can exceed 99.2%.
Rapid Detection: The most significant advantage of LDR technology is its short experimental time. Compared to Taqman, RFLP, SSCP, and DHPLC, LDR technology allows for easier control of detection conditions, resulting in an overall experimental duration of only a few weeks.
Flexible Experimentation, High Detection Rates, and Short Experimental Cycles.
Double assurance based on hybridization and connection reactions, ensuring accurate results.
Stringent quality control during the experimental process and a 10% replicate control, ensuring result accuracy.
Suitable for medium to low SNP typing scales, recommended for use with up to 30 SNP loci and sample sizes of fewer than a thousand.
This detection technology is suitable for various medium to low-throughput SNP testing scales, such as QTL localization studies, candidate gene or locus association analysis, molecular breeding, and more.