International effort toward a SSR-based linkage map for C. clementina : [P128]

Following the difficulties encountered for assembling a 1.2 x sequencing of the highly heterozygous sweet orange genome, the International Citrus Genomic Consortium (ICOC) decided to estab1ish the first reference sequence of a whole nuclear citrus genome from a haploid Clementine. A saturated genetic linkage map of Clementine based on sequence-characterized markers was considered by the ICGC as an important too1 for genome sequence assemb1y. In this framework, CIRAD proposed to use an interspecific population C. maxima x C. clementina to implement the reference Clementine genetic map. A population of 250 hybrids of Chandler pummelo x Clementine was established in Corsica and 190 hybrids were used in this first phase of mapping. Collaboration was established between two French organizations (CIRAD and INRA), two groups from United States (UF-CREC and UCR), one Spanish institute (IVIA), INRA Morocco and Cukurova University from Turkey. Forty markers were found heterozygous in Clementine among a previous set of 90 SSR markers developed by CIRAD from microsatellite-enriched genomic libraries. With the objective to integrate the physical and genetic maps of Clementine, CIRAD and IVIA have developed new SSR markers from microsatellite sequences identified in BAC End Sequences (BES) of diploid Clementine. On hundred and 10 of these new markers were found heterozygous for Clementine or Chandler pummelo and were used for genotyping. INRA France deve1oped 500 SSR markers from ESTs databases and found 170 markers heterozygous for Clementine. INRA Morocco contributed to the genotyping of 112 SSR markers developed from EST databases and genomic libraries, while 50 ESTs SSR were analysed by Cukurova University. SSR markers mainly developed from EST databases and already mapped for sweet orange were genotyped by UF-CREC (70 markers) and UCR (60 markers) to allow comparisons among the C. sinensis. C. maxima and C. c1ementina maps. lndeed, taking advantage of the important allelic differentiation between Clementine and Chandler, two parallel linkage maps can be developed from this population. As perspective, in the framework of the global haploid Clementine sequencing project, a collaboration between the French and Spanish groups plans: (i) to extend the population size to 380 hybrids between Clementine and pummelo. and (ii) to develop an array from SNPs identified in Clementine BES for High- Throughput Genotyping. All genotyping data will be stored in the online TropGene database (http://tropgenedb.cirad.fr/). Additional international groups are very welcome to join the project, using these progenies for genotyping their own markers. This should contribute to a very high density map of Clementine and to comparative mapping studies between citrus species. (Texte intégral

A reference linkage Map of C. clementina based on SNPs, SSRs and indels (P477)

A haploid C. clementina was chosen by the International Citrus Genomic Consortium (ICGC) to establish the reference whole genome sequence for Citrus. Development of a dense clementine linkage map was part of the objectives of this global collaborative project. Two inter-specific populations between C. clementina and C. maxima were used for this purpose. 156 hybrids of Nules clementine x Pink pummelo and 200 hybrids of Chandler pummelo x Nules clementine were genotyped with 1003 markers. 306 were SSRs markers (66 from genomic libraries, 207 from ESTs and 33 from clementine BAC end sequences -BES-), 34 were Indels markers mined from BES and 663 SNPs mined from Clementine BES or identified by candidate gene sequencing. 901 markers were successfully mapped in the 9 clementine linkage groups. Segregation distortion was observed for many loci of clementine when it was used as male parent while it followed Mendelian segregation for most markers when used as female parent. However marker order was mostly conserved between the male and female maps; thus, data of the two populations were joined to establish the reference clementine genetic map. The total clementine linkage map cover 1250 cM with linkage groups from 105 cM until 210 cM. This map is strongly anchored on a large diploid clementine BAC library resource. It is a powerful tool for Citrus genetics and supports the alignment of the haploid clementine whole genome sequence in the framework of the ICGC collaborative project. (Résumé d'auteur

Fortunella margarita Transcriptional Reprogramming Triggered by Xanthomonas citri subsp. citri

<p>Abstract</p> <p>Background</p> <p>Citrus canker disease caused by the bacterial pathogen <it>Xanthomonas citri </it>subsp. <it>citri (</it>Xcc) <it>has </it>become endemic in areas where high temperature, rain, humidity, and windy conditions provide a favourable environment for the dissemination of the bacterium. Xcc is pathogenic on many commercial citrus varieties but appears to elicit an incompatible reaction on the citrus relative <it>Fortunella margarita </it>Swing (kumquat), in the form of a very distinct delayed necrotic response. We have developed subtractive libraries enriched in sequences expressed in kumquat leaves during both early and late stages of the disease. The isolated differentially expressed transcripts were subsequently sequenced. Our results demonstrate how the use of microarray expression profiling can help assign roles to previously uncharacterized genes and elucidate plant pathogenesis-response related mechanisms. This can be considered to be a case study in a citrus relative where high throughput technologies were utilized to understand defence mechanisms in <it>Fortunella </it>and citrus at the molecular level.</p> <p>Results</p> <p><b>cDNAs from sequenced kumquat libraries (ESTs) made from subtracted RNA populations, healthy vs. infected, were used to make this microarray</b>. Of 2054 selected genes on a customized array, 317 were differentially expressed (P < 0.05) in Xcc challenged kumquat plants compared to mock-inoculated ones. This study identified components of the incompatible interaction such as reactive oxygen species (ROS) and programmed cell death (PCD). Common defence mechanisms and a number of resistance genes were also identified. In addition, there were a considerable number of differentially regulated genes that had no homologues in the databases. This could be an indication of either a specialized set of genes employed by kumquat in response to canker disease or new defence mechanisms in citrus.</p> <p>Conclusion</p> <p>Functional categorization of kumquat Xcc-responsive genes revealed an enhanced defence-related metabolism as well as a number of resistant response-specific genes in the kumquat transcriptome in response to Xcc inoculation. Gene expression profile(s) were analyzed to assemble a comprehensive and inclusive image of the molecular interaction in the kumquat/Xcc system. This was done in order to elucidate molecular mechanisms associated with the development of the hypersensitive response phenotype in kumquat leaves. These data will be used to perform comparisons among citrus species to evaluate means to enhance the host immune responses against bacterial diseases.</p

Preliminary Evidence for Rootstock Effects on HLB Infection Frequency and Disease Severity in Sweet Orange and ‘SugarBelle’ Trees

Evidence is accumulating that root system collapse is involved with HLB-induced tree decline, especially with trees on Swingle and Carrizo.&nbsp; Phytophthora resistance appears to be breaking down in HLB-infected trees on Swingle.&nbsp; Other stresses caused by blight, nematodes, cold, etc. also appear to be interacting with HLB to increase HLB disease frequency and severity.&nbsp; Improved rootstocks could help to mitigate these problems, allowing for sustainable production under appropriate nutrition. We are testing complex hybrid rootstock candidates (diploid and tetraploid) to determine their affect on HLB disease establishment and severity in trees grafted with sweet orange scions; field and greenhouse experiments are underway.&nbsp; Rootstocks differentially translocate nutrients, phytohormones (plant growth regulators), micro-RNAs, small proteins (pathogenesis related?), and other metabolites to the scion.&nbsp; This could have both direct and indirect, quantitative and quantitative affects on scion gene expression, and possibly Liberibacter pathogenesis in citrus – especially with unique complex allotetraploid rootstocks. Data from two young field trials (both with the ‘bad neighbor’ effect) established to evaluate new rootstock candidates, previously not screened for HLB tolerance, will be presented. These include a trial of 3.5 year old trees of ‘SugarBelle’ that is nearly 100% infected with HLB, and a trial of 4.5 year old trees of sweet orange on &gt;50 rootstocks that is approximately 15% infected.&nbsp; Rootstock differences regarding HLB disease frequency and severity are emerging. Complex ‘tetrazyg’ rootstock Orange #19 (Nova+HBPummelo x Cleopatra+Argentine trifoliate orange) is showing more HLB tolerance at both locations.&nbsp;&nbsp; Data on percentages of symptomatic fruit and fruit drop per rootstock will be presented

Preliminary Evidence for Rootstock Effects on HLB Infection Frequency and Disease Severity in Sweet Orange and ‘SugarBelle’ Trees

Evidence is accumulating that root system collapse is involved with HLB-induced tree decline, especially with trees on Swingle and Carrizo.  Phytophthora resistance appears to be breaking down in HLB-infected trees on Swingle.  Other stresses caused by blight, nematodes, cold, etc. also appear to be interacting with HLB to increase HLB disease frequency and severity.  Improved rootstocks could help to mitigate these problems, allowing for sustainable production under appropriate nutrition. We are testing complex hybrid rootstock candidates (diploid and tetraploid) to determine their affect on HLB disease establishment and severity in trees grafted with sweet orange scions; field and greenhouse experiments are underway.  Rootstocks differentially translocate nutrients, phytohormones (plant growth regulators), micro-RNAs, small proteins (pathogenesis related?), and other metabolites to the scion.  This could have both direct and indirect, quantitative and quantitative affects on scion gene expression, and possibly Liberibacter pathogenesis in citrus – especially with unique complex allotetraploid rootstocks. Data from two young field trials (both with the ‘bad neighbor’ effect) established to evaluate new rootstock candidates, previously not screened for HLB tolerance, will be presented. These include a trial of 3.5 year old trees of ‘SugarBelle’ that is nearly 100% infected with HLB, and a trial of 4.5 year old trees of sweet orange on &gt;50 rootstocks that is approximately 15% infected.  Rootstock differences regarding HLB disease frequency and severity are emerging. Complex ‘tetrazyg’ rootstock Orange #19 (Nova+HBPummelo x Cleopatra+Argentine trifoliate orange) is showing more HLB tolerance at both locations.   Data on percentages of symptomatic fruit and fruit drop per rootstock will be presented

Identification of genes associated with low furanocoumarin content in grapefruit

Some furanocoumarins in grapefruit (Citrus paradisi) are associated with the so-called grapefruit juice effect. Previous phytochemical quantification and genetic analysis suggested that the synthesis of these furanocoumarins may be controlled by a single gene in the pathway. In this study, cDNA-amplified fragment length polymorphism (cDNA-AFLP) analysis of fruit tissues was performed to identify the candidate gene(s) likely associated with low furanocoumarin content in grapefruit. Fifteen tentative differentially expressed fragments were cloned through the cDNA-AFLP analysis of the grapefruit variety Foster and its spontaneous low-furanocoumarin mutant Low Acid Foster. Sequence analysis revealed a cDNA-AFLP fragment, Contig 6, was homologous to a substrate-proved psoralen synthase gene, CYP71A22, and was part of citrus unigenes Cit.3003 and Csi.1332, and predicted genes Ciclev10004717m in mandarin and orange1.1g041507m in sweet orange. The two predicted genes contained the highly conserved motifs at one of the substrate recognition sites of CYP71A22. Digital gene expression profile showed the unigenes were expressed only in fruit and seed. Quantitative real-time PCR also proved Contig 6 was down-regulated in Low Acid Foster. These results showed the differentially expressed Contig 6 was related to the reduced furanocoumarin levels in the mutant. The identified fragment, homologs, unigenes, and genes may facilitate further furanocoumarin genetic study and grapefruit variety improvement. </jats:p