Development of InDel Markers for Brassica rapa Based on a High-resolution Melting Curve

Abstract

Brassica rapa is one of the most important leafy vegetable crops with large cultivated area in China. To increase the availability of DNA markers in B. rapa, we developed insertion-deletion (InDel) markers utilizing high-resolution melting (HRM) curve analysis. We designed primers for 252 InDels (≥ 3 bp) evenly distributed in the genome and tested gene polymorphisms with eight accessions. In total, 208 markers were specifically amplified, and 148 InDels with polymorphism were genotyped successfully using HRM. We further analyzed the correlation with InDel size, GC number, and predicted the difference in Tm values (∆Tm) using 208 markers with specific amplification. We found that the success rate of InDel markers was correlated with the GC number of InDel and the predicted-∆Tm, but not clearly correlated with the length of InDel. When the GC number within InDel was ≥ 8, the successful rate exceeded 90.0%. When the predicted-∆Tm reached 0.5 °C, the success rate was greater than 90.0%, and when it was ≥ 0.6 °C, the rate climbed to 100.0%, indicating their role as the optimal parameter for successful development of an applicable InDel marker. The polymorphic InDel markers can be easily genotyped using HRM. They are of great value in genetic analysis, construction of linkage map, and molecular marker-assisted selection in B. rapa.

Keywords

• Brassica rapa
• InDel marker
• HRM
• genotyping