Transferability of the EST-SSRs developed on Nules clementine (Citrus clementina Hort ex Tan) to other Citrus species and their effectiveness for genetic mapping

Abstract

Background: During the last decade, numerous microsatellite markers were developed for genotyping and to identify closely related plant genotypes. In citrus, previously developed microsatellite markers were arisen from genomic libraries and more often located in non coding DNA sequences. To optimize the use of these EST-SSRs as genetic markers in genome mapping programs and citrus systematic analysis, we have investigated their polymorphism related to the type (di or trinucleotide) or their position in the coding sequences.

Results: Among 11000 unigenes from a Clementine EST library, we have found at least one microsatellite sequence (repeated units size ranged from 2 to 6 nucleotides) in 1500 unigenes (13.6%). More than 95% of these SSRs were di or trinucleotides. If trinucleotide microsatellites were encountered trough all part of EST sequences, dinucleotide microsatellites were preferentially (50%) concentrated in the 5' 100th nucleotides. We assessed the polymorphism of 41 EST-SSR, by PCR amplification droved with flanking primers among ten Citrus species plus 3 from other genera. More than 90% of EST-SSR markers were polymorphic. Furthermore, dinucleotide microsatellite markers were more polymorphic than trinucleotide ones, probably related to their distribution that was more often located in the 5' UnTranslated Region (UTR). We obtained a good agreement of diversity relationships between the citrus species and relatives assessed with EST-SSR markers with the established taxonomy and phylogeny. To end, the heterozygosity of each genotype and all dual combinations were studied to evaluate the percentage of mappable markers. Higher values (> 45%) were observed for putative Citrus inter-specific hybrids (lime lemon, or sour orange) than for Citrus basic true species (mandarin, pummelo and citron) (<30%). Most favorable combinations for genome mapping were observed in those involving interspecific hybrid genotypes. Those gave higher levels of mappable markers (>70%) with a significant proportion suitable for synteny analysis.

Conclusion: Fourty one new EST-SSR markers were produced and were available for citrus genetic studies. Whatever the position of the SSR in the ESTs the EST-SSR markers we developed are powerful to investigate genetic diversity and genome mapping in citrus.

Figure 1

Comparison of the unigenes distribution in MIPs function categories between the citrus EST collection and the ESTs that contain SSR.

Figure 2

Position of the dinucleotide and trinucleotide SSRs from 5' end EST sequences.

Figure 3

Polymorphism of the trinucleotide microsatellite (EST164 STMS) among citrus genotypes detected by silver nitrate staining gel electrophoresis. Below the photography size allelic interpretation of each genotype is detailed. The order of the sample is the following: Invitrogen 10 bp ladder (Lane M), 'Nules' clementine (lane 1), 'Washington Navel' sweet orange (lane 2), 'Cleopatra' mandarin (lane 3), 'Willow leaf' mandarin (lane 4), 'Morocco' sour orange (lane 5), 'Marsh' grapefruit (lane 6), 'Corsican' citron (lane 7), 'Mexican' lime (lane 8), 'Brazil sweet' lime (lane 9), 'Lisbon Foothill' lemon (lane 10), 'Sans pepin' pummelo (lane 11), 'Pink' pummelo (lane 12), 'Kindia' combava (lane 13), 'Rubidoux' trifoliate orange (lane 14), 'Marumi' kumquat (Lane 15).

Figure 4

Dendrogram representing the structure of genetic diversity and relationships observed between the 16 citrus genotypes aimed by the polymorphism of the 39 single locus EST-STMS markers.