Freely available computer programs were arranged in a pipeline to extract microsatellites from public citrus EST sequences, retrieved from the NCBI. In total, 3,278 bi- to hexa-type SSR-containing sequences were identified from 56,199 citrus ESTs. On an average, one SSR was found per 5.2 kb of EST sequence, with the tri-nucleotide motifs as the most abundant. Primer sequences flanking SSR motifs were successfully identified from 2,295 citrus ESTs. Among those, a subset (100 pairs) were synthesized and tested to determine polymorphism and heterozygosity between/within two genera, sweet orange (C. sinensis) and Poncirus (P. trifoliata), which are the parents of the citrus core mapping population selected for an international citrus genomics effort. Eighty-seven pairs of primers gave PCR amplification to the anticipated SSRs, of which 52 and 35 appear to be homozygous and heterozygous, respectively, in sweet orange, and 67 and 20, respectively, in Poncirus. By pairing the loci between the two intergeneric species, it was found that 40 are heterozygous in at least one species with two alleles (9), three alleles (28), or four alleles (3), and the remaining 47 are homozygous in both species with either one allele (31) or two alleles (16). These EST-derived SSRs can be a resource used for understanding of the citrus SSR distribution and frequency, and development of citrus EST-SSR genetic and physical maps. These SSR primer sequences are available upon request.

The segregation of 141 polymorphic expressed sequence tag-simple sequence repeat (EST-SSR) markers in an F1 intergeneric citrus population was studied to build the first extensive EST maps for the maternal sweet orange and paternal Poncirus genomes. Of these markers, 122 were found segregating in sweet orange, 59 in Poncirus, and 40 in both. Eleven linkage groups with 113 markers in sweet orange, 8 with 45 markers in Poncirus, and 13 with 123 markers in the cross pollinator (CP) consensus of both, were constructed. About 775.8 cM of sweet orange genome and 425.7 cM of Poncirus genome were covered. Through comparison of shared markers, three cases were found where two linkage groups in one map apparently were colinear with one group of the other map; Poncirus linkages Ar1a and Ar1b and consensus linkages CP1a and CP1b, were both collinear with one sweet orange linkage, Sa1, as were sweet orange Sa3a and Sa3b with Poncirus Ar3 and consensus CP3, and sweet orange Sa7a and Sa7b, and consensus CP7a and CP7b with Poncirus Ar7. These EST-SSR markers are particularly useful for constructing comparative framework maps for related genera because they amplify orthologous genes to provide anchor points across taxa. All SSR primers are freely available to the citrus community.

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