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Nearly 65,000 citrus EST (Expressed Sequence Tags) have been investigated using the CitEST project database. Microsatellites were investigated in the unigene sequences from Citrus spp. and Poncirus trifoliata. From these sequences, approximately 35% of the non-redundant ESTs contained SSRs. The frequencies of different SSR motifs were similar between Citrus spp and trifoliate orange. In general, mononucleotide repeats appeared to be the most abundant SSRs in the CitEST database, but we also identify di-, tri-, tetra-, penta- and hexanucleotide repeats. The AG/CT and AAG/CTT were the most common dinucleotide and trinucleotide motifs, with frequencies of 54.4% and 25.2%, respectively. Primer sequences flanking SSR motifs were successfully designed and synthesized. After in silico polymorphism analysis, a subset of sixty-eight primers was validated in different Citrus spp. and Poncirus trifoliata. PCR-amplification revealed polymorphism in citrus with all tested primer pairs and showed the potential of these markers for linkage mapping. Our study showed that the CitEST database can be exploited for the development of SSR markers that can amplify Citrus spp. and related genus for comparative mapping and other genetic analyses.

Leprosis, caused by Citrus leprosis virus, cytoplasmic type (CiLV-C), is the main viral disease in the Brazilian citrus industry. This occurs because of the widespread source of inoculum and the year-round presence of the vector, the tenuipalpid mite Brevipalpus phoenicis, in citrus plants. In addition, while some Citrus species are resistant to CiLV-C, C. sinensis, the main cultivated species in the country, is extremely susceptible to the disease. The main objective of this work was to identify genes in C. sinensis cv. Pêra plants that were differentially expressed after the host was challenged with CiLV-C. In order to accomplish that, cDNA libraries were constructed from healthy and CiLV-inoculated sweet orange leaves. Two hundred and fifty-four genes were found to differ significantly in terms of expression, with 193 of them induced and 61 repressed after inoculation. Here we discuss the possible roles of a sub-set of these genes involved in metabolism, energy, signaling and cell rescue, defense and virulence, and indicate which kind of response may take place in the initial steps of the disease. Although the symptoms induced by CiLV-C in its compatible interaction with sweet orange resemble those of hypersensitive response (HR) in incompatible interactions, our data indicate that, apparently, the manifestation of leprosis symptoms should not be considered HR.

The randomly amplified polymorphic DNA (RAPD) technique was used to distinguish nucellar and zygotic seedlings resulting from crosses between the ‘Montenegrina’ (Citrus deliciosa Tenore) and‘ King’ (C. nobilis Loureiro) tangerines. The aim of the present study was to develop tangerine varieties with a reduced number of seeds and organoleptic characteristics similar to the ‘Montenegrina’ tangerine. Embryos were isolated from seeds, cultivated in vitro, and acclimated in a greenhouse. Four random primers were used to identify 54 plants of sexual origin from a total of 202 individuals. The degree of polymorphism of each primer was reflected in the number of zygotic plants obtained per primer. Cluster analysis of parents and progeny separated the individuals into distinct groups with a maximum genetic dissimilarity of 20%.A técnica RAPD (random amplified polymorphic DNA) foi utilizada para distinguir plântulas nucelares e zigóticas resultantes do cruzamento entre as tangerineiras ‘Montenegrina’ (Citrus deliciosa Tenore) e ‘King’ (C. nobilis Loureiro). Este cruzamento foi realizado objetivando a obtenção de variedades de tangerineiras com características organolépticas de fruto semelhantes à tangerina ‘Montenegrina’ e menor número de sementes. Embriões foram isolados das sementes e cultivados in vitro e aclimatizados em casa de vegetação. Utilizando-se de 4 primers de seqüência randômica foram identificadas 54 plantas de origem sexual de um total de 202 indivíduos. O grau de polimorfismo de cada primer refletiu no número de plantas zigóticas obtidas por primer, sendo o total de zigóticos identificados pela soma das informações geradas pelos 4 primers. Análise de agrupamento com os parentais e a progênie separou os indivíduos em grupos distintos com uma dissimilaridade genética máxima de 20%.