Application of High Resolution Melt analysis (HRM) for screening haplotype variation in a non-model plant genus: Cyclopia (Honeybush)

Abstract

Aim This study has three broad aims: to (a) develop genus-specific primers for High Resolution Melt analysis (HRM) of members of Cyclopia Vent., (b) test the haplotype discrimination of HRM compared to Sanger sequencing, and (c) provide an example of using HRM to detect novel haplotype variation in wild C. subternata Vogel. populations. Location The Cape Floristic Region (CFR), located along the southern Cape of South Africa. Methods Polymorphic loci were detected through a screening process of sequencing 12 non-coding chloroplast DNA segments across 14 Cyclopia species. Twelve genus-specific primer combinations were designed around variable cpDNA loci, four of which failed to amplify under PCR; the eight remaining were applied to test the specificity, sensitivity and accuracy of HRM. The three top performing HRM Primer combinations were then applied to detect novel haplotypes in wild C. subternata populations, and phylogeographic patterns of C. subternata were explored. Results We present a framework for applying HRM to non-model systems. HRM accuracy varied across the PCR products screened using the genus-specific primers developed, ranging between 56 and 100%. The nucleotide variation failing to produce distinct melt curves is discussed. The top three performing regions, having 100% specificity (i.e. different haplotypes were never grouped into the same cluster, no false negatives), were able to detect novel haplotypes in wild C. subternata populations with high accuracy (96%). Sensitivity below 100% (i.e. a single haplotype being clustered into multiple unique groups during HRM curve analysis, false positives) was resolved through sequence confirmation of each cluster resulting in a final accuracy of 100%. Phylogeographic analyses revealed that wild C. subternata populations tend to exhibit phylogeographic structuring across mountain ranges (accounting for 73.8% of genetic variation base on an AMOVA), and genetic differentiation between populations increases with distance (p < 0.05 for IBD analyses). Conclusions After screening for regions with high HRM clustering specificity—akin to the screening process associated with most PCR based markers—the technology was found to be a high throughput tool for detecting genetic variation in non-model plants.

Applied phylogeography ofCyclopia intermedia(Fabaceae) highlights the need for ‘duty of care’ when cultivating honeybush

“…com., 2019). Furthermore, Honeybush cultivation has been encouraged to take place near to wild Cyclopia populations (Jacobs, 2008) that, based on the evidence presented here and elsewhere (Galuszynski & Potts, 2020), are likely to be genetically distinct-as previously suspected (Potts, 2017a;Schutte, 1997). Commercial populations are therefore likely to pose a threat to the genetic integrity of wild Cyclopia if gene flow occurs.…”

“…As the application of HRM to members of Cyclopia populations has been described elsewhere (Galuszynski & Potts, 2020), only a brief overview of the method is provided here. Two non-coding cpDNA regions (two fragments from the atpI-aptH intergenic spacer and one from the ndhA intron) were amplified using Cyclopia specific primers (provided in Table 2) and subsequently screened for nucleotide variation as detected by HRM curve analysis (i.e., samples with different melt curves).…”

“…Kies, C. genistoides (L) R.Br., C. longifolia Vogel, and C. sessiliflora (L) R.Br.) that were previously sequenced to develop the genus specific HRM markers (Galuszynski & Potts, 2020) were included in the SP network. Default options were used to build the network, although each indel event was reduced to a single base pair (TCS counts each base as an independent evolutionary event).…”

Peer Review #1 of "Applied phylogeography of Cyclopia intermedia (Fabaceae) highlights the need for ‘duty of care’ when cultivating honeybush (v0.2)"

“…This study therefore sets out to test the postulation put forward by Potts (2017a), that -due to the topographic complexity of the Cape landscape coupled with limited seed dispersal by ants -members of Cyclopia will exhibit highly structured chloroplast genetic diversity; such a pattern has already been observed in the preliminary haplotype screening of wild Cyclopia subternata Vogel. populations (Galuszynski and Potts, 2020).…”

“…Spectrophotometer (Thermo Fisher Scientific, Wilmington, DE19810r Scientific, USA) and ng/L DNA dilutions were made for PCR amplification and HRM analysis. As the application of HRM to members of Cyclopia populations has been described elsewhere (Galuszynski and Potts, 2020), only a brief overview of the method is provided here. Two non-coding cpDNA regions (two fragments from the atpI-aptH intergenic spacer and one from the ndhA intron) were amplified using Cyclopia specific primers (provided in Table 2) and subsequently screened for nucleotide variation as detected by HRM curve analysis (i.e.…”