In this study, 25388 non-redundant
P. trifoliata EST sequences (obtained after clustering ESTs from NCBI) were screened for microsatellites using the SSRLocator software
[ 9]. The search was restricted to repeats with a minimum number of 8, 7, 5, 4 and 4 complete repeat units for di-, tri-, tetra-, penta- and hexa-nucleotide motifs, respectively. In total, 976 SSRs were identified from 887 ESTs (Appendix A, Table S3). Thus 3.49% of the
P. trifoliata EST contained one or more microsatellites with one SSR per 4 kb EST (976 SSR in 22.7 Mb). Of the 887 SSR containing EST sequences, 695 (78%) ESTs are suitable for
P. trifoliata EST SSR (PteSR) marker development. These sequences were further compared by blast against the NCBI non-redundant protein database with an
E-value cut-off of 1.e
-3, in order to identify functional EST sequences. As a result, 699 sequences containing di (333), tri (170), tetra (47), penta (60) and hexa (59) microsatellites unites were identified as functional
P. trifoliata specific EST SSR motifs (
a). Microsatellites with higher repeat numbers and having a significant similarity to existing proteins in the NCBI database were favored because they are generally considered to be more polymorphic and functional. With these criteria, 542 primer pairs were selected for the
in silico PCR (VPCR) amplification analysis, which was performed using SSRLocator. Whole genomic sequences of
Citrus sinensis and
C. clementina were used as templates for the virtual PCR amplification analysis and results reveal that 226 (41%) primer pairs had positive VPCR amplification. To determine the genomic distribution of developed PteSR marker, we mapped 542 marker on nine chromosome of
C. sinensis and results revealed that 198 (36%) markers distributed among nine chromosomes of
C. sinensis with an average marker density 0.83 Mb (Figs. 1b, 2). Chromosome mapping of the markers showed highest frequency of markers on chromosome 5 (38 markers, 7%) and lowest on chromosome 1 (8, 1.4%) (
b).
In silico cross genera transferability was performed using an
in silico PCR strategy to select a sub-set of PteSR marker for subsequent estimatation of their utility for genetic diversity of citrus and relative species. The results showed the highest transferability (42%) in
C. sinensis and the lowest (1%) in
Arabidopsis with an average of 12% of PteSR markers transferable in citrus and non citrus species (
c). Based on the
in silico cross-genera transferability results the best 108 primer pairs were selected for subsequent PCR amplification and analysis for transferability to citrus species and for determining their polymorphism in eight
Citrus spp. (
C. sinensis,
C. reticulata,
C. grandis,
C. aurantifolia,
C. limon,
C. medica,
C. ichangensis,
C. paradisi,
P. trifoliata and
Fortunella hindsii). Among all 108 primer pairs tested, 18 (16%) failed to amplify the expected products. The remaining 90 microsatellite loci produced the expected products, of which 61 (56%) were found to be polymorphic and transferable to citrus related species.