AFLPs are incompatible with RAPD and morphological data in Pennisetum purpureum (Napier grass)

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Abstract

Accessions of Pennisetum purpureum in agricultural research centres in sub-Saharan Africa were genotyped using Amplified Fragment Length Polymorphisms (AFLPs) using primer combinations MluI/MseI. Cluster analyses of resulting bands suggested that the samples could be grouped into five large and five small-sized clusters. Principal coordinate analysis revealed two major clusters being well separated, with bulk of the samples forming one, poorly structured cluster. Our analyses failed to reveal the regional groupings found in previous studies using a subset of our sample universe and based on RAPDs as well as morphological and agronomical data. The inability of the AFLP data to clearly demarcate accessions is ascribed to either the presence of high levels of genetic uniformity, or non-optimal primers and/or bad germplasm management practices. The re-registration of all accessions, based on DNA barcoding, is suggested as a means to resolve the lingering problems regarding the identity of accessions.

Introduction

Pennisetum Rich. (Poaceae) is an important grass genus as it includes two commonly cultivated species Pennisetum purpureum Schumach. and Pennisetum glaucum (L.) R. Br. (Brunken, 1977, Dahlgren et al., 1985). P. purpureum, commonly known as Napier grass, is valued for its high productivity, perennial nature, and pest resistance; it is an important forage crop for dairy cattle in smallholder farming systems of the tropics and subtropics (Lowe et al., 2003, Bhandari et al., 2004). P. glaucum, commonly known as Pearl millet, is a coarse, annual bunch grass (Gupta and Mhere, 1997) with a nutrient value higher than that of rice or wheat (Uprety and Austin, 1972). Both species are indigenous to Africa, however they are now widely grown for grain, feed, and forage in Asia and the Americas (Gupta and Mhere, 1997).

Out-crossing in P. purpureum results in high genetic diversity captured formally in description of numerous cultivars (Augustin and Tcacenco, 1993), difficult to distinguish on morphological grounds (Bhandari et al., 2004). P. purpureum and P. glaucum also hybridize naturally and the resulting hybrids, although sterile, are also utilised by farmers (Burton, 1944). The hybrid resembles P. purpureum because of its larger genomic contribution (Gonzales and Hanna, 1984), but it still retains some of the qualities of P. glaucum (Muldoon and Pearson, 1979). Man-made hybrids between these two species (Gildenhuys, 1950) have become popular under the name “Bana”. However, the name “Bana” is often confusingly used also as a synonym for some Napier grass cultivars.

Stem-boring butterflies Busseola fusca Fuller (Noctuidae) and Chilo partellus Swinhoe (Crambidae) cause great yield loss in maize. Resource-poor farmers in tropical and southern Africa therefore undertake habitat management or ‘push–pull’ strategies in response: Desmodium uncinatum (Jacq.) DC. (Fabaceae) is planted amongst maize to repel moths, and Napier grass is planted on the outer verges of maize fields to attract moths which then lay their eggs on them instead on the maize (Khan et al., 2000, Van den Berg et al., 2001). Different morphological forms of Napier grass in nature had been collected and further developed into cultivars, and these are being maintained as clones for this purpose in sub-Saharan Africa. However, these clonal strains are being exchanged without proper pedigree records (Bhandari et al., 2004) hence great confusion surrounds their true identity (Boonman, 1993, Bhandari et al., 2004).

Due to the rapid expansion of use of Napier grass in the habitat management of stem borers, a need has arisen to unambiguously determine the identity of the different cultivars currently in use. A number of studies have targeted this issue using Random Amplification of Polymorphic DNA (RAPD) as the method of choice. These studies found RAPD to be a useful tool to quantify genetic distances and to distinguish between plant accessions (Smith et al., 1993, Daher et al., 2002, Lowe et al., 2003, Passo et al., 2005). In most cases, however, only a relatively small sample was analysed. RAPDs have the disadvantage of producing a relatively small number of fragments and this becomes problematic when a large number of samples are analysed. In addition, RAPD analyses can produce inconsistent fragment amplification resulting in polymorphisms unsuitable for unbiased and objective scoring which then necessitates costly repeat of the analyses (Smith et al., 1993).

The International Livestock Research Institute (ILRI) in Ethiopia maintains a large collection of P. purpureum and its hybrids. Lowe et al. (2003) used RAPD analysis to analyse 56 of those. Analyses of inter-accession variation revealed that 49 samples had no, or very low, levels of variation, confirming field observations that plants are predominantly propagated clonally. A neighbour-joining dendrogram suggested that the bulk of samples would fall into well-defined regional groups, and the P. glaucum samples were also distinctly separated. Most of these samples were also characterised morphologically and agronomically earlier by Van de Wouw et al. (1999). In addition, while the RAPD analysis could apparently separate hybrids from pure Pennisetum individuals, the morphological and agronomic classifications failed to do so. On the other hand the RAPD study was unable to separate the accessions of dwarf forms from accessions of tall forms.

Amplified Fragment Length Polymorphisms (AFLPs) have lately become a popular method of fingerprinting because of low cost and reliability in reproducing genetic fingerprints of individuals (Meudt and Clarke, 2006, Mueller and Wolfenbarger, 1999). However, here too inconsistent fragment amplification can occur. Notwithstanding the AFLP technique has been in use for more than a decade, and has become automated to a large extent, no work on fingerprinting of Napier grass cultivars thus has been published so far.

In this paper we investigate the utility of AFLPs in fingerprinting Napier grass using the ILRI collection and additional samples, aiming at testing whether Lowe et al. (2003) regional groupings could be recovered.

Section snippets

Plant samples

The origins of most samples used in this study are not well documented (see above) and the presented data are based mostly on personal communication with nursery managers (Table 1). In this regard, we have retained names provided to us and have also included the presumed place of origin.

Different Pennisetum cultivar samples were obtained from several countries in sub-Saharan Africa. The majority of samples were obtained from collections sustained by the ILRI as well as by the Eduardo Mondlana

Results and discussion

The availability of the ILRI samples subject to cluster analyses of morphological, agronomic and RAPD data provided a platform to test the efficacy of AFLPs in P. purpureum and its hybrids. However, a direct comparison between our and the published data was not possible because of different (although largely overlapping) sets of accessions.

The first cluster analysis (with the P. setaceum and P. macrourum samples included in the analysed matrix) separated these two species together with Estcourt

Acknowledgements

We gratefully acknowledge the assistance of the Gatsby Charitable Foundation (UK) for partially funding this project. We thank Prof. János Podani (Budapest, Hungary) for performing the optimal-searching analysis.

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