Detection and validation of single feature polymorphisms in cowpea (Vigna unguiculata L. Walp) using a soybean genome array

Abstract

Background: Cowpea (Vigna unguiculata L. Walp) is an important food and fodder legume of the semiarid tropics and subtropics worldwide, especially in sub-Saharan Africa. High density genetic linkage maps are needed for marker assisted breeding but are not available for cowpea. A single feature polymorphism (SFP) is a microarray-based marker which can be used for high throughput genotyping and high density mapping.

Results: Here we report detection and validation of SFPs in cowpea using a readily available soybean (Glycine max) genome array. Robustified projection pursuit (RPP) was used for statistical analysis using RNA as a surrogate for DNA. Using a 15% outlying score cut-off, 1058 potential SFPs were enumerated between two parents of a recombinant inbred line (RIL) population segregating for several important traits including drought tolerance, Fusarium and brown blotch resistance, grain size and photoperiod sensitivity. Sequencing of 25 putative polymorphism-containing amplicons yielded a SFP probe set validation rate of 68%.

Conclusion: We conclude that the Affymetrix soybean genome array is a satisfactory platform for identification of some 1000's of SFPs for cowpea. This study provides an example of extension of genomic resources from a well supported species to an orphan crop. Presumably, other legume systems are similarly tractable to SFP marker development using existing legume array resources.

Figure 1

Schematic diagram of SFP validation protocol.

Figure 2

Plots of signal intensities, affinity differences and individual outlying scores. Left panels: log intensities (PM, perfect match) for a representative probe set (Gma.1863.1.S1_at) from two genotypes. Middle panels: the differences of average log intensities between two genotypes. Right panel: individual outlying scores for each probe. Dotted lines indicate IT93K-503-1 and solid lines indicate CB46. This SFP was identified both in stress (upper layer) and non-stress (lower layer) datasets.

Figure 3

A representative gel image of PCR amplification. Aliquots of the PCR products of ten (GS1 to GS10) representative primer pairs were loaded and separated on a 1.2% agarose gel. 1, CB46; 2, IT93K-503-1; M, size marker. The arrows indicate non-specific amplification.

Figure 4

Alignment of cowpea amplicon sequences related to a probe set (Gma.1863.1.S1_at) and its target sequence from the soybean SIF. Polymorphic residues between CB46 and IT93K-503-1 are highlighted in grey, polymorphic residues between cowpea methyl filtered sequence and soybean SIF are in black. The position of SFP probe number 2 detected by the RPP method is underlined. Arrows indicate SNPs. I, IT93K-503-1; C, CB46; G, cowpea methyl filtered sequence; S, target sequence from soybean SIF.

Figure 5

Alignments of validated SFPs. Grey background indicates SNP and black background indicates INDEL. I, IT-93K-503-1; C, CB46; G, methyl filtered cowpea sequence; S, target sequence from soybean SIF. The target SFP probe number is given in parenthesis.

Figure 6

Useful byproducts of genomic amplicon sequencing. Residues highlighted in grey indicate polymorphism between two cowpea genotypes though no polymorphism was detected in the SFP probe region detected by RPP (underlined). I, IT93K-503-1; C, CB46; G, cowpea methyl filtered sequence; S, target sequence from soybean SIF.