1 University of Nottingham, School of Biosciences, Sutton Bonington Campus, Loughborough, Leicestershire, LE12 5RD, UK. http://www.nottingham.ac.uk/biosciences/ Email firstname.lastname@example.org
2 Institute of Food Research, Colney Lane, Norwich, NR4 7UA, UK. www.ifr.bbsrc.ac.uk/ Email richard.mithen@BBSRC.AC.UK
3 IRD-ICIPE, PO Box 30772, Nairobi, Kenya. www.icipe.org Email email@example.com
Bambara groundnut (Vigna subterranea (L.) Verdc.) is an African legume valued for its drought tolerance and resistance to pests and diseases. It consists of two botanical forms: var. spontanea, comprising the wild forms, restricted to Cameroon; and var. subterranea comprising the cultivated forms found in many parts of the tropics particularly sub-Saharan Africa. To date, there are no established varieties of bambara groundnut, and marginal and subsistence farmers throughout Africa grow locally adapted landraces. A cross was made between wild (VSSP11; V. subterranea var. spontanea) and cultivated (DipC; V. subterranea var. subterranea) accessions (2n = 22), from Cameroon and western Botswana, respectively, and the F1 hybrid self-pollinated. The resultant F2 progeny have been used to study a range of traits of agronomic interest including internode length, Water Use Efficiency (WUE) using Carbon Isotope Discrimination (Δ13C), seed weight and testa colour. Polymorphic AFLP (Amplified Fragment Length Polymorphism) markers, using different primer combinations, have been identified between the two parental lines and their segregation studied in the F2 mapping population, the latter comprising more than 100 plants. In addition to mapping monogenic traits like the presence (DipC) or absence (VSSP11) of an eye pattern around the hilum, QTL (Quantitative Trait Loci) analysis of traits such as internode length, Δ13C, testa colour and seed weight will help to identify the regions of the bambara groundnut genome associated with these important traits.
The first intraspecific hybrid between wild and cultivated bambara groundnut has been developed and the resulting F2 progenies are being used to construct the first AFLP-based genetic linkage map of this species.
Food legume, contrasting traits, genetic linkage, bulked segregant analysis, cowpea
Bambara groundnut is an important food legume cultivated widely in semi-arid Africa by subsistence and small-scale farmers. It consists of two botanical forms; var. spontanea, comprising the wild forms, restricted to Cameroon, and var. subterranea comprising the cultivated forms, found predominantly in sub-Saharan Africa. It is prized for the nutritional value of its seeds and its drought tolerance in semi-arid environments. Despite its potential as a crop, no co-ordinated plant breeding/improvement programmes have been established for bambara groundnut. For many centuries, farmers have grown locally-adapted landraces consisting of a mixture of genotypes and often resulting in low and unpredictable yields.
Molecular mapping has proven to be a powerful tool for gene localization, gene isolation, marker-assisted selection and evolutionary studies. Knowledge of the organisation of genes controlling economically or agronomically important traits can make a considerable contribution to plant improvement programmes. No gene or DNA sequence information is available for bambara groundnut. Consequently, AFLP marker techniques are being used to detect sequence polymorphisms and to monitor the segregation of DNA sequences in mapping populations. Isozyme patterns, as observed in other species of the genus Vigna, such as V. unguiculata (cowpea) (Panella and Gepts, 1992; Pasquet, 1993; Vaillancourt et al., 1993), were found to be very similar to those observed in bambara groundnut (Pasquet et al., 1999). This suggests a genetic synteny between cowpea and bambara groundnut. To construct the first genetic linkage map of V. subterranea a strategy similar to that used for cowpea genome mapping (Menéndez et al., 1997) has been adopted. A cross was made between a wild and a cultivated accession of bambara groundnut, and the F1 progeny self-pollinated. The resultant F2 progeny have been used to study a range of traits of agronomic interest. Polymorphic AFLP markers, using different primer combinations, have been identified between the two parental lines and their segregation studied in the F2 population, the latter comprising more than 100 plants.
An F2 population of 115 plants generated from a cross between a wild bambara groundnut accession VSSP11 (V. subterranea var. spontanea) from Cameroon and a cultivated accession DipC (V. subterranea var. subterranea) from western Botswana was used as a mapping population in the present study.
Genomic DNA was extracted from 0.2 - 0.3 g freeze-dried leaf tissue using the GenElute Plant Genomic DNA Miniprep Kit (Sigma). The concentration of DNA was estimated using the Nanodrop Spectrophotometer.
AFLP analysis was performed as described by Vos et al. (1995), using the AFLP™ Analysis System I AFLP Starter Primer Kit (Gibco BRL–Life Technologies, Inc.), with minor modifications. Fifty-four EcoRI + MseI selective primer combinations were screened for their ability to detect polymorphisms in the two parents of the mapping population. Selective primers were designated by their restriction site (E, EcoRI; M, MseI) and the number and nature of the additional nucleotides (e.g., E-AAG, M-CTT, etc.). Those combinations that generated at least 8 polymorphic fragments were chosen for use in the analysis of the F2 material. The samples were analysed using CEQTM 8000 Genetic Analysis System from Beckman Coulter.
Carbon isotope discrimination has been well established as a potentially useful surrogate for WUE in several plant species. The Δ13C was determined in dried leaf powder using Isotope Ratio Mass Spectrometer (IRMS), at the National Facility, Dept. of Crop Physiology, University of Agricultural Sciences, Bangalore, India. The IRMS was interfaced with an elemental analyser through a continuous flow device to determine the stable isotope ratios on a continuous flow basis. Carbon isotope fractionation values were computed in relation to PDB (Ehleringer and Osmond 1989) and expressed as per ml (‰).
The first intraspecific hybrid between a wild and cultivated bambara groundnut accession was produced and the F1 hybrid selfed to generate the F2 population for DNA and trait-based mapping.
Several contrasting morphological traits have been identified between the two parents of the hybrid. VSSP11, the wild accession from Cameroon and the male parent, has a spreading habit with a tendency to form pentafoliate leaves, fewer (5-8) branches/plant and poor yields (15-20 seeds/plant). The seeds vary in size and have reddish brown testa with dark brown marks. In contrast, DipC, the female parent from Botswana, has an erect habit with trifoliate leaves, many (20-26) branches/plant and high yields (150-200 seeds/plant). The seeds are uniform with cream testa, each with a dark eye.
Inheritance and segregation of trait based markers
Analysis of F2 data reveals that internode length, used as a measure of spreading and non-spreading growth habit, number of branches per plant, WUE estimated from the stable Δ13C value, expression of trifoliate and pentafoliate leaves, testa colour and 100-seed weight measured from each F2 plant are all quantitatively inherited.
Figure 1: Internode length of F2 plants
Figure 2. 13C values of F2 plants
The Δ13C values used to estimate WUE shows transgressive segregation in the F2 population with the average values of the 2 parents VSSP11 (21.5) and DipC (20.5) differing by 1 unit.
The eye pattern around the hilum is a monogenic trait controlled by a single recessive gene and segregating in a 1:3 ratio.
Based on segregation and recombination observed in the F2 population for internode length and the number of branches, four phenotypic classes have been identified and are now being grown in Swaziland in Southern Africa.
The AFLP marker data obtained from the F2 mapping population, using at least fifteen different primer combinations, are now being analysed in two stages. Firstly, the markers will be assembled into a linkage map using JOINMAP (Kyazma BV, Wageningen, Netherlands) and, secondly, an attempt will be made to correlate the genes for the traits of interest with the markers located on the map. In doing so, we will exploit both traditional mapping and Bulked Segregant Analysis to identify markers related to traits under study and position them on the genetic linkage map.
Ehleringer JR and Osmond CB (1989). Stable isotopes. In ‘Plant physiological ecology: field methods and instrumentation’ (Ed. RW Pearcy, J Ehleringer, HA Mooney and PW Rundel) Chapman & Hall, London, UK. pp. 281-300.
Menéndez CM, Hall AE and Gepts P (1997). A genetic linkage map of cowpea (Vigna unguiculata) developed from a cross between two inbred, domesticated lines. Theoretical and Applied Genetics 95, 1210–1217.
Panella L and Gepts P (1992). Genetic relationships within Vigna unguiculata (L.) Walp. based on isozyme analyses. Genetic Resources and Crop Evolution 39, 71–88.
Pasquet RS (1993). Variation at isozyme loci in wild Vigna unguiculata (L.) Walp. (Fabaceae, Phaseoleae). Plant Systematics and Evolution 186, 157–173.
Pasquet RS, Schwedes S and Gepts P (1999). Isozyme diversity in bambara groundnut. Crop Science 39, 1228-1236.
Vaillancourt RE, Weeden NF and Barnard J (1993). Isozyme diversity in the cowpea species complex. Crop Science 33, 606–613.
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