Genome sequence comparisons reveal interspecific hybridizations and admixture in citrus domestication : [W317]

Citrus is the most globally significant tree fruit, but its history of domestication is not well understood. Cultivated citrus types are believed to be direct selections from, or hybrids of, several progenitor species; however, the identities of these species and their contributions to modern cultivars remain unclear and controversial. A collection of different genomes of mandarins, pummelos, and oranges, including a high quality reference sequence from a haploid Clementine mandarin, has been assembled. By comparative analyses we show that these cultivated types were derived from two progenitor species, C. maxima and C. reticulata. Although it was determined that the sequenced cultivated pummelos represent selections from C. maxima, the cultivated mandarins proved to be introgressions of C. maxima into a distinct second population identified as the ancestral wild mandarin species, C. reticulata. Sweet and sour oranges were found to be hybrids between these two species. Sweet orange, the most widely cultivated citrus, arose as the offspring of previously admixed individuals. Sour (or Seville) orange was found to be an F1 hybrid of pure C. maximaand C. reticulata parents, indicating that wild mandarins were at least a part of the early breeding germplasm. Sequence analysis also revealed that at least one Chinese "wild mandarin" actually represents a citrus species distinct from C. reticulata, suggesting the possibility that other unrecognized wild citrus species may be identified. Understanding the phylogeny of cultivated citrus by comparative genome analysis will clarify taxonomic relationships and potentially lead to previously inconceivable opportunities for genetic improvement. (Texte intégral

Citrus genomics

Citrus fruits (sweet orange, mandarin, pummelo, grapefruit, lemon, lime and assorted hybrids) are among the most widely grown and economically important fruit tree crops in the world. As fresh fruit, they are an important and nutrient dense food source for human diets; as processed juice products, primarily sweet orange juice, they represent a globally traded commodity. To support genetic improvement efforts for this important crop, the international citrus genetics community has collaborated with international sequencing centers in the development of freely available genomic resources, some of which are described herein. Most notable, two full-length annotated genome assemblies have been produced and made available to the global research community. The first genome, based exclusively on Sanger sequencing, is from a haploid plant derived from 'Clementine' mandarin, to serve as the reference genome for citrus. A second genome assembly was produced from the sweet orange clone 'Ridge Pineapple', this was done primarily with 454 technology. Extensive EST datasets and a number of microarray platforms for exploring the transcriptomic responses of citrus species and hybrids to a wide range of conditions have been shared, to support exploitation and utilization of genome sequence information. As many researchers in the citrus genomics community are also actively engaged in genetic improvement programs, there has been a natural integration of improvement efforts with the rapidly evolving genomic tools. Examples described below include work designed to better understand the control of gene expression in citrus polyploids, which are being used in development of seedless triploid selections or as rootstocks; unraveling the molecular mechanisms of host-pathogen interactions to devise novel genetic strategies to overcome a multitude of devastating diseases that currently threaten citrus production globally; and the mining of SSR and SNP markers for linkage studies to enable marker-assisted parental selection and breeding strategies, illustrated bywork on two citrus traits, nucellar embryony and juvenility, which significantly impact breeding approaches. Citrus genome resources are available through publicly available web portals, through the USDOEJGI (phytozome.net) and Tree Fruit Genome Database Resources (tfGDR, citrusgenomedb.org). Work is continuing to expand and improve the citrus genome sequence resources and tools, to enable application of sequence-derived knowledge in improving citrus plants and to managing better their interactions with biotic and abiotic factors. (Résumé d'auteur

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

Sequencing of diverse mandarin, pummelo and orange genomes reveals complex history of admixture during citrus domestication

Cultivated citrus are selections from, or hybrids of, wild progenitor species whose identities and contributions to citrus domestication remain controversial. Here we sequence and compare citrus genomes-a high-quality reference haploid clementine genome and mandarin, pummelo, sweet-orange and sour-orange genomes-and show that cultivated types derive from two progenitor species. Although cultivated pummelos represent selections from one progenitor species, Citrus maxima, cultivated mandarins are introgressions of C. maxima into the ancestral mandarin species Citrus reticulata. The most widely cultivated citrus, sweet orange, is the offspring of previously admixed individuals, but sour orange is an F1 hybrid of pure C. maxima and C. reticulata parents, thus implying that wild mandarins were part of the early breeding germplasm. A Chinese wild 'mandarin' diverges substantially from C. reticulata, thus suggesting the possibility of other unrecognized wild citrus species. Understanding citrus phylogeny through genome analysis clarifies taxonomic relationships and facilitates sequence-directed genetic improvement. (Résumé d'auteur

Genome sequence analysis and comparisons reveal ancestral hybridization and admixture events in the origins of some citrus cultivars : S03O01

Since its inception, the International Citrus Genome Consortium (ICGC) has pursued development of freely available genomics resources and tools for the benefit of the citrus research community; these efforts were conducted by citrus scientists in concert with researchers from some of the world's major genome sequencing centers and corporations. An early ICGC goal was to produce a full-length genome assembly. Two reference genome sequences were made publicly available in January 2011, from a 'Clemenules' clementine-derived haploid and diploid 'Ridge Pineapple' sweet orange, produced using Sanger and 454 sequencing technologies respectively. Subsequent work has proceeded to improve the assemblies and their annotation; a brief summary of their characteristics and status of the assemblies will be presented. As sequencing technology has evolved and costs have dramatically decreased, other citrus genomes have been sequenced (the diploid 'Clemenules', 'Ponkan', 'Willowleaf', 'W. Murcott' mandarins; 'Siamese Sweet' and 'Chandler' pummelo; and sour orange, among others). A comparative analysis of these genomes has made it possible to describe the genomic contributions from ancestral outbred populations to major cultivar types, and to propose likely underlying models. The results confirmed previous reports that clementine arose from a hybridization event between 'Willowleaf' mandarin and sweet orange. Further, introgressions of Citrus reticulata and Citrus maxima germplasm, long implicated in its ancestry, clearly defined the segmented mosaic genome of sweet orange, through the ancient admixture of these species. Finally, sour orange was found to be a direct hybrid of a pummelo and mandarin. Interestingly, although the two pummelos sequenced appear to have their derivations from a single wild species (C. maxima), the mandarins sequenced reveal ancestral admixture of C. maxima with C. reticulata. Implications and future directions for collaborative research will be discussed. (Texte intégral

Construction of High-Density Genetic Maps and Detection of QTLs Associated With Huanglongbing Tolerance in Citrus

Huanglongbing (HLB), or citrus greening, is the most devastating disease in citrus worldwide. Commercial citrus varieties including sweet orange (Citrus sinensis) are highly susceptible to HLB, and trifoliate orange (Poncirus trifoliata, a close Citrus relative) is widely considered resistant or highly tolerant to HLB. In this study, an intergeneric F1 population of sweet orange and trifoliate orange was genotyped by Genotyping-by-Sequencing, and high-density SNP-based genetic maps were constructed separately for trifoliate orange and sweet orange. The two genetic maps exhibited high synteny and high coverage of the citrus genome. Progenies of the F1 population and their parents were planted in a replicated field trial, exposed to intense HLB pressure for 3 years, and then evaluated for susceptibility to HLB over 2 years. The F1 population exhibited a wide range in severity of HLB foliar symptom and canopy damage. Genome-wide QTL analysis based on the phenotypic data of foliar symptom and canopy damage in 2 years identified three clusters of repeatable QTLs in trifoliate orange linkage groups LG-t6, LG-t8 and LG-t9. Co-localization of QTLs for two traits was observed within all three regions. Additionally, one cluster of QTLs in sweet orange (linkage group LG-s7) was also detected. The majority of the identified QTLs each explained 18–30% of the phenotypic variation, indicating their major role in determining HLB responses. These results show, for the first time, a quantitative genetic nature yet the presence of major loci for the HLB tolerance in trifoliate orange. The results suggest that sweet orange also contains useful genetic factor(s) for improving HLB tolerance in commercial citrus varieties. Findings from this study should be very valuable and timely to researchers worldwide as they are hastily searching for genetic solutions to the devastating HLB crisis through breeding, genetic engineering, or genome editing

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

Comparative genetic mapping between clementine, pummelo and sweet orange and the interspecicic structure of the clementine genome

The availability of a saturated genetic map of Clementine was identified by the International Citrus Genome Consortium as an essential prerequisite to assist the assembly of the reference whole genome sequence based on a Clementine derived haploid. The primary goals of the present study were to establish a Clementine reference map, and to perform comparative mapping with pummelo and sweet orange. Five parental genetic maps were established with SNPs, SSRs and InDels. A medium density reference map (961 markers for 1084.1 cM) of Clementine was established and used by the ICGC to facilitate the chromosome assembly of the haploid genome sequence. Comparative mapping with pummelo and sweet orange revealed that the linear order of markers was highly conserved. Reasonable inferences of most citrus genomes should be obtained by mapping next-generation sequencing data against the haploid reference genome sequence. Skewed segregations were frequent and higher in the male than female Clementine potentially leading to false interpretation of the genetic determinism of phenotypic traits. The mapping data confirmed that Clementine arose from hybridization between 'Mediterranean' mandarin and sweet orange and identified nine recombination break points for the sweet orange gamete that contributed to the Clementine genome. Introgression of pummelo genome fragments were identified in heterozygosity in each chromosome. Moreover, it appeared that the genome of the haploid Clementine used to establish the citrus reference genome sequence was inherited primarily from the 'Mediterranean' mandarin. The usefulness of this genetic map, anchored in the reference whole genome sequence, is discussed. (Résumé d'auteur

A new evolutionary framework for the genus Citrus: Its origin, evolution and dispersal [W183]

We present first solid insights on the origin, evolution and dispersal of citrus and elucidate the genealogy of the most important wild and cultivated varieties. These findings draw a new evolutionary framework for these fruit crops, a scenario that challenges current taxonomic and phylogenetic thoughts and points towards a reformulation of the genus Citrus. Based on genomic, phylogenetic and biogeographical analyses of the genus Citrus we propose that the center of origin of citrus was the Southeast foothills of the Himalayas, in a region including the eastern area of Assam, northern Myanmar and western Yunnan. Our analyses suggest that the ancestral citrus species underwent a sudden speciation during late Miocene and that the new species dispersed from there to surrounding regions, coinciding with a drastic transition from wetter monsoonal conditions to a drier climate. The Australian citrus and Tachibana mandarin split later from mainland citrus during the early Pliocene and Pleistocene, respectively. (Résumé d'auteur