654 Table 1 Summary on identification and validation of SFP in pigeonpea Parental genotype combination SFPs identification SFPs validation Funct Integr Genomics (2011) 11:651–657 ICP 28×ICPW 94 ICPL 151×ICPL 87 ICPL 8755×ICPL 227 Total No. of probes Present Containing SFPs 5,405 5,455 4,989 5,810 850 854 780 5,692 38 58 43 99 No. of selected probes No. of probes with sequence variation in Full length amplicon sequence Probe sequence 34 37 34 75 31 20 18 67 of 454/FLX transcript reads and Sanger ESTs of pigeonpea (Raju et al. 2010). By applying e-value≤10−5 and sequence similarity≥80%, 2,745 (48.2%) out of 5,692 SFP containing probes identified homologues in pigeonpea. These pigeon- pea sequences were further examined for the presence of interrogation position for the corresponding soybean SFP probe. As a result, 1,815 pigeonpea TUSs were found positive and used for primer designing with an expected amplicon size of 200–500 bp. In summary, primer pairs could be designed for 1,131 TUSs containing SFP probe target regions (Electronic Supplementary Material Table 1). In other cases, primers could not be designed, this was mainly due to either the probe target region being very near (≤20 bp) the end of sequence or inability to fit the default parameters of the Primer3 software (Nayak et al. 2010). In order to investigate whether the identified SFPs were related to sequence variations, 179 SFPs were randomly selected for validation. PCR with these 179 primer pairs on the same set of six genotypes used for SFP discovery provided strong and prominent amplicons in 102 (56.98%) cases. In the remaining cases, either no amplification or nonspecific amplification was observed. Amplicons gener- ated for the 102 primers were sequenced using Sanger sequencing methodology. As a result, good quality se- quence data were obtained for 99 primer pairs, which were further analyzed to identify the SNPs and INDELs. Most sequences were 250–650 bp, but some were as short as 207 bp or as long as 1,336 bp. Analysis of the sequence data with “Divest” tool (Jayashree et al. 2009) showed a total of 7,535 sequence polymorphisms (including SNPs Fig. 1 A snapshot of SFP anal- ysis in pigeonpea for the probe set GmaAffx.5953.1.A1_at of Affymetrix Soybean Genome Array. Two replicates of genotype ICPL 8755 and genotype ICPL 227 has been shown in dark and dotted lines, respectively. The top panel shows log intensities (left side) and hybridization affinities (right side) for all probes of the probe set for two genotypes. The bottom panel shows the difference in hybridization affinities (left side) and the individual outlying scores (right side) for each probe between two genotypes