High resolution melting analysis as a rapid and highly sensitive method for Cichorium plasmotype characterization

Somatic hybridization in Cichorium species has already been extensively investigated. Hybrid or cybrid characterization requires an effective plasmotype screening method. We evaluated high resolution melting (HRM) analysis for the detection of specific mitochondrial (mt) and chloroplast (cp) markers to distinguish two industrial chicory (Cichorium intybus var. sativum) plasmotypes from five wild type chicory (C. intybus) and two endive (C. endivia) plasmotypes. Three mt (coxII-2, cob-1, and cob-2) and three cp HRM markers (trnL-trnF, trnL-trnF-2, and ndhF-1) were successfully developed. Two markers (coxII-2 and trnL-trnF) were additionally able to discriminate heterozygous plasmotypes containing at least 25 % of one parental plasmotype. Moreover, the technique was successfully used to characterize the cytoplasms of 50 simple sequence repeats (SSR) confirmed somatic hybrids of C. intybus var. sativum U+2018VL52U+2019 and C. endivia var. crispum U+2018Wallone DespaU+2019. HRM enables a rapid (less than 2 h) and efficient high-throughput scanning of multiple fragments due to the 384-well plates and is cost efficient because of its small reaction volume of 10 U+03BCl. This is the first report on the use of HRM analysis on Cichorium species. The technique is a fast and simple alternative for laborious and costly sequencing in plasmotyping regenerants obtained after somatic fusion.

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