Assessing the Likelihood of Gene Flow from Sugarcane (Saccharum Hybrids) to Wild Relatives in South Africa
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ORIGINAL RESEARCH published: 07 June 2018 doi: 10.3389/fbioe.2018.00072 Assessing the Likelihood of Gene Flow From Sugarcane (Saccharum Hybrids) to Wild Relatives in South Africa Sandy J. Snyman 1,2*, Dennis M. Komape 3, Hlobisile Khanyi 3, Johnnie van den Berg 3, Dirk Cilliers 3, Dyfed Lloyd Evans 1,2,4, Sandra Barnard 3 and Stefan J. Siebert 3 1 Crop Biology Resource Centre, South African Sugarcane Research Institute, Mount Edgecombe, South Africa, 2 Department of Biology, School of Life Sciences, University of KwaZulu-Natal, Westville, South Africa, 3 Unit for Environmental Sciences and Management, North-West University, Potchefstroom, South Africa, 4 BeauSci Ltd., Waterbeach, Cambridge, United Kingdom Pre-commercialization studies on environmental biosafety of genetically modified (GM) crops are necessary to evaluate the potential for sexual hybridization with related plant species that occur in the release area. The aim of the study was a preliminary assessment of factors that may contribute to gene flow from sugarcane (Saccharum Edited by: Andrew F. Roberts, hybrids) to indigenous relatives in the sugarcane production regions of Mpumalanga International Life Sciences Institute, and KwaZulu-Natal provinces, South Africa. In the first instance, an assessment United States of Saccharum wild relatives was conducted based on existing phylogenies and Reviewed by: Graham Bonnett, literature surveys. The prevalence, spatial overlap, proximity, distribution potential, and Commonwealth Scientific and flowering times of wild relatives in sugarcane production regions based on the above, Industrial Research Organisation and on herbaria records and field surveys were conducted for Imperata, Sorghum, (CSIRO), Australia Alan John Gray, Cleistachne, and Miscanthidium species. Eleven species were selected for spatial Centre for Ecology & Hydrology, analyses based on their presence within the sugarcane cultivation region: four species in Edinburgh, United Kingdom the Saccharinae and seven in the Sorghinae. Secondly, fragments of the nuclear internal *Correspondence: Sandy J. Snyman transcribed spacer (ITS) regions of the 5.8s ribosomal gene and two chloroplast genes, [email protected] ribulose-bisphosphate carboxylase (rbcL), and maturase K (matK) were sequenced or assembled from short read data to confirm relatedness between Saccharum hybrids and Specialty section: This article was submitted to its wild relatives. Phylogenetic analyses of the ITS cassette showed that the closest wild Biosafety and Biosecurity, relative species to commercial sugarcane were Miscanthidium capense, Miscanthidium a section of the journal junceum, and Narenga porphyrocoma. Sorghum was found to be more distantly related Frontiers in Bioengineering and Biotechnology to Saccharum than previously described. Based on the phylogeny described in our study, Received: 23 January 2018 the only species to highlight in terms of evolutionary divergence times from Saccharum Accepted: 17 May 2018 are those within the genus Miscanthidium, most especially M. capense, and M. junceum Published: 07 June 2018 which are only 3 million years divergent from Saccharum. Field assessment of pollen Citation: viability of 13 commercial sugarcane cultivars using two stains, iodine potassium iodide Snyman SJ, Komape DM, Khanyi H, van den Berg J, Cilliers D, Lloyd (IKI) and triphenyl tetrazolium chloride, showed decreasing pollen viability (from 85 to 0%) Evans D, Barnard S and Siebert SJ from the north to the south eastern regions of the study area. Future work will include (2018) Assessing the Likelihood of Gene Flow From Sugarcane other aspects influencing gene flow such as cytological compatibility and introgression (Saccharum Hybrids) to Wild Relatives between sugarcane and Miscanthidium species. in South Africa. Front. Bioeng. Biotechnol. 6:72. Keywords: gene flow, hybridization, pollen viability, phytogeography, spatial assessment, phylogeny, doi: 10.3389/fbioe.2018.00072 Miscanthidium Frontiers in Bioengineering and Biotechnology | www.frontiersin.org 1 June 2018 | Volume 6 | Article 72 Snyman et al. Gene Flow Likelihood in Sugarcane INTRODUCTION and herbicide and salt tolerance), creeping bentgrass (herbicide tolerance), and oilseed rape (herbicide tolerance) (Rieger et al., Commercial sugarcane (Saccharum hybrids) was thought to 2002; Warwick et al., 2003; Chen et al., 2004; Watrud et al., 2004; have arisen from an interspecific hybridization event between S. Zapiola et al., 2008). This could lead to the evolution of highly spontaneum and S. officinarum in Java in the late 1800’s (Paterson competitive weeds and the degeneration of the genetic diversity et al., 2013). Recent literature, though, suggests that the heritage in indigenous grasses. is more complicated, especially when considering the nuclear This study was conducted to assess the likelihood of gene phyologenetic relationships (Lloyd Evans and Joshi, 2016a). The flow from commercial sugarcane to wild relatives in the sugar complex ancestry, the polyploid and aneuploid nature of modern production regions of South Africa. Factors such as spatial sugarcane makes conventional breeding challenging (Butterfield overlap, proximity, flowering synchrony and pollen viability et al., 2001). Notwithstanding these issues, in excess of 60 “N” are prerequisites for hybridization to occur. Therefore, if close sugarcane cultivars have been released in the South African relatives occur in areas where sugarcane is cultivated, then industry since 1955, but environmental constraints affect sexual transgenic sugarcane presents a likelihood for gene flow to hybridization because floral induction, flowering synchronicity these species. To assess this possibility, the objectives are as between selected parental germplasm and pollen fertility are follows: (i) review the literature to identify the wild relatives problematic at sub-tropical latitudes (Brett, 1950; Horsley of Saccharum, collate what is known about gene flow between and Zhou, 2013). Attempts to increase genetic diversity by cultivated Saccharum hybrids and wild relatives in South intergeneric crossing of commercial hybrids and members of the Africa, determine overlapping flowering times and assess pollen “Saccharum complex” have met with either limited or no success, viability of commercial sugarcane; (ii) quantify the distribution even under controlled conditions with human intervention, and of wild Saccharum relatives and assess the spatial overlap there are no reports of such hybridization in the wild (Bonnett of their distributions with commercial sugarcane plantations; et al., 2008; Cheavegatti-Gianotto et al., 2011; Organisation for (iii) determine phylogenetic relationships within the Saccharum Economic Cooperation and Development, 2013). complex to confirm which species are most closely related to Cultivar improvement using genetic modification (GM) cultivated sugarcane; (iv) make an assessment of the likelihood technology is being explored and a range of traits have been of gene flow potential between related species and cultivated introduced to sugarcane (reviews by Lakshmanan et al., 2005; sugarcane. Brumbley et al., 2008; Meyer and Snyman, 2013). Commercial cultivation of GM sugarcane has only been approved in Indonesia (Xue et al., 2014) and more recently, Brazil1, but research of this MATERIALS AND METHODS nature is underway in most sugarcane-producing countries. In South Africa, legislation governs the use and cultivation Phytogeography of Saccharum Wild of GM crops [namely the Genetically Modified Organisms Act Relatives in South Africa (Act 15 of 1997) and the National Environmental Management Wild relatives which diverged from Saccharum <7.3 million Act (Act 107 of 1998)]. One aspect of GM crop cultivation that years ago (based on chloroplast sequence chronograms) were requires assessment prior to commercial release is establishing identified from a global phylogeny based on chloroplast the likelihood of lateral gene flow between related plant genomes/regions for the Poaceae (Skendzic et al., 2007; Soreng species. Hybridization is only possible between a crop plant et al., 2015; Lloyd Evans and Joshi, 2016a). Eleven species of the and a wild relative if a number of barriers to gene flow are Sorghinae and Saccharinae subtribes of the Andropogoneae were traversed (McGeoch et al., 2009). According to den Nijs et al. selected for spatial analyses based on their presence within the (2004), successful gene transfer (barrier crossing) requires plant sugarcane cultivation region of South Africa: four species that populations to: (a) overlap spatially; (b) overlap temporally belong to Saccharinae and seven to Sorghinae (Organisation for (flowering periods); and (c) be sufficiently close biologically Economic Cooperation and Development, 2013; Fish et al., 2015; that the resulting hybrids are fertile, facilitating introgression of Soreng et al., 2015). Grass nomenclature is in accordance with genetic material into a new population. The probability of and The Plant List (2013). extent of gene flow varies according to these limiting factors Herbarium specimens were sourced from 11 South African (Légère, 2005). herbaria. All specimen data were captured and a gap analysis Gene flow from transgenic crops to wild relatives may have conducted for the study area to identify where insufficient negative environmental effects if the hybrid plants inherit an information was available regarding the occurrence of wild increased capacity for invasiveness and weediness of a species relatives. At these sites, sugarcane