Iplex application guide

In our study, two different platforms were used for single nucleotide polymorphism (SNP) genotyping. For stage 1, genotyping was performed by using the Illumina Omni 1 platform. The Sequenom iPLEX system (Sequenom, Inc., San Diego, CA, USA) was used in stage 2. Polymerase chain reaction and extension primers were designed using Mass ARRAY Assay Design 3.1 software (Sequenom, Inc.). Manufacture’s iPLEX Application Guide (Sequenom, Inc.) was performed for genotyping procedures. All of the genotyping reactions were performed in 384-well plates. Each plate included a duplicate for three or four participants selected at random, as well as six to nine negative controls in which water was substituted for DNA. The average concordance rate was 99.8%.

A Sequenom MassARRAY analysis was performed to validate the significant GWAS-derived SNPs located within the major QTL intervals in the hybrid population (289 individuals) and the landrace population (69 accessions) using a MassARRAY compact system (Sequenom, San Diego, CA). Moreover, QTL-derived markers were validated in a landrace population (99 accessions) using the same methods. Primers for these markers (flanking the SNP site for PCR amplification and extension) were designed for genotyping. The primers and genomic sequences are listed in Table S1 and Appendix S1, respectively. PCR was performed in 384-well plates using HotStart Taq DNA polymerase (Qiagen). The steps of the PCR program and application of shrimp alkaline phosphatase (SAP) were conducted in accordance with the protocols in the Sequenom iPLEX Application Guide (Version 1, Sequenom, San Diego, CA). Primer extension was performed using an iPLEX^TM Reagent Kit (Sequenom), and the genotype was detected using a matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometer for SNP genotyping.

SNP genotyping was performed using the mass array time-of-flight mass spectrometer (Sequenom Company, USA) technique. Polymerase chain reaction (PCR) and extension primers were designed by the Mass ARRAY Assay Design 3.1 software (Sequenom Company, USA). The genotyping procedures were performed using the manufacturer's iPLEX Application Guide (Sequenom Company, USA). The PCR program for amplification conditions was 94°C, 15 min; 45 cycles × (94°C, 20 s; 56°C, 30 s; 72°C, 1 min); 72°C, 5 min. All the genotyping reactions were performed in 384-well plates, and each plate included four randomly selected duplicates and 6 negative controls; double distilled water was used as the negative controls. The average concordance rate of the genotype was 99.5%.