Flow cytometry: a quick method to determine ploidy levels in honeybush (Cyclopia spp.)

Motsa, Mlamuli M.; Bester, C.; Slabbert, M.M.; Hannweg, K.; Booyse, M.

doi:10.1007/s10722-018-0648-z

Cited by 5 publications

(7 citation statements)

References 58 publications

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“…Genetic material from this genebank has recently been made commercially available for the establishment of cultivated Honeybush stands, including in the Langeberg that supports the genetically distinct GAR population (Joubert et al, 2011;Niemandt et al, 2018). The effective population size of the C. subternata nuclear genome is a scale of magnitude larger than the cpDNA due to the species high ploidy level (hexalpoid, 2n = 6x = 54, (Motsa et al, 2018;Schutte, 1997), as such drift may occur more slowly. Additionally, pollen dispersal by carpenter bees (Xylocopa spp) may reduce population divergence through rare long distance dispersal events.…”

Section: Distribution Of C Subternata Genetic Diversitymentioning

confidence: 99%

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

Galuszynski

Potts

2020

PeerJ

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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.

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Section: Distribution Of C Subternata Genetic Diversitymentioning

confidence: 99%

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

Galuszynski

Potts

2020

PeerJ

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“…Buffer selection therefore focused on those previously found suitable for flow cytometry analyses of recalcitrant and/or woody plant species. The Partec buffer, though successfully used on woody and non-woody plant species rich in secondary metabolites, including coconut [28], honeybush [29], Lupinus sp. [30], and Brassica sp.…”

Section: Discussionmentioning

confidence: 99%

Rooibos (Aspalathus linearis) Genome Size Estimation Using Flow Cytometry and K-Mer Analyses

Mgwatyu

Stander

Ferreira³

et al. 2020

Plants

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Plant genomes provide information on biosynthetic pathways involved in the production of industrially relevant compounds. Genome size estimates are essential for the initiation of genome projects. The genome size of rooibos (Aspalathus linearis species complex) was estimated using DAPI flow cytometry and k-mer analyses. For flow cytometry, a suitable nuclei isolation buffer, plant tissue and a transport medium for rooibos ecotype samples collected from distant locations were identified. When using radicles from commercial rooibos seedlings, Woody Plant Buffer and Vicia faba as an internal standard, the flow cytometry-estimated genome size of rooibos was 1.24 ± 0.01 Gbp. The estimates for eight wild rooibos growth types did not deviate significantly from this value. K-mer analysis was performed using Illumina paired-end sequencing data from one commercial rooibos genotype. For biocomputational estimation of the genome size, four k-mer analysis methods were investigated: A standard formula and three popular programs (BBNorm, GenomeScope, and FindGSE). GenomeScope estimates were strongly affected by parameter settings, specifically CovMax. When using the complete k-mer frequency histogram (up to 9 × 105), the programs did not deviate significantly, estimating an average rooibos genome size of 1.03 ± 0.04 Gbp. Differences between the flow cytometry and biocomputational estimates are discussed.

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“…6 en 7), is die effek nie groot genoeg om 'n praktiese verskil aan die probleem om tussen saadkleur te onderskei te maak nie. Genetiese variasie sal moontlik 'n beduidende faktor wees om gedragsverskille in hierdie genus (Cyclopia) van poliploïde spesies te bepaal, maar moederplant-veldomgewing interaksie is ook al met uiteenlopende saadeienskappe in ander spesies gekoppel, en moet daarom in ag geneem word (Carta et al, 2016;Gutterman, 2000;Motsa et al, 2018;Penfield en MacGregor, 2016;Postma en Ågren, 2015;Wulff, 2017). Omgewingsfaktore (nl.…”

Section: Effek Van Saadposisieunclassified

Honeybush ( Cyclopia spp.) mature embryo culture and the influence of pod position on seed colour

Koen¹,

Slabbert

Booyse³

et al. 2021

Suid-Afrikaanse Tydskrif vir Natuurwetenskap en Tegnologie

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Plante (verskillende genotipes) van Cyclopia spesies word gebruik om Suid-Afrikaanse heuningbostee (’n kruietee produk) te produseer en word tans deur teling vir grootskaalse verbouing verbeter. Saaddimorfisme en dormansie beïnvloed egter die ontkiemingspersentasie negatief. Deur gebruik te maak van volwasse embrioweefselkulture is studies om die ontkiemingspersentasie van saad te verhoog op C. longifolia, C. maculata en C. subternata gedoen. Die effek van stratifikasie op saadontkieming deur geen of ’n kouebehandeling toe te pas, is ook ondersoek. Volwasse embrios is hierna uit sade verwyder en vir ’n 24-uur fotoperiode by 25°C ± 1°C op weefselkultuur gekweek. Die kweek van volwasse embrios deur middel van weefselkulture was suksesvol en het deurlopend meer plante vergeleke met konvensionele saaimetode gelewer. Studies om die effek wat saadpeulposisie van die moederplant op saadkleur het, is ook gedoen. Volwasse heuningbospeule is vanaf die boonste, middelste en onderste-derdes van die blaarbedekte deel van die heuningbosplante versamel om die aantal groen/intermediêre/bruin sade wat by elke gedeelte geproduseer is, te bepaal. Die verhouding van saaddimorfisme wat by elke gedeelte geproduseer word, kon egter nie deur peulposisie voorspel word nie.

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Flow cytometry: a quick method to determine ploidy levels in honeybush (Cyclopia spp.)

Cited by 5 publications

References 58 publications

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

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

Rooibos (Aspalathus linearis) Genome Size Estimation Using Flow Cytometry and K-Mer Analyses

Honeybush ( Cyclopia spp.) mature embryo culture and the influence of pod position on seed colour

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