The domestication of citrus, is poorly understood. Cultivated types are selections from, or hybrids of, wild progenitor species, whose identities and contributions remain controversial. By comparative analysis of a collection of citrus genomes, including a high quality haploid reference, we show that cultivated types were derived from two progenitor species. Though cultivated pummelos represent selections from a single progenitor species, C. maxima, cultivated mandarins are introgressions of C. maxima into the ancestral mandarin species, C. 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, implying that wild mandarins were part of the early breeding germplasm. A wild “mandarin” from China exhibited substantial divergence from C. reticulata, suggesting the possibility of other unrecognized wild citrus species. Understanding citrus phylogeny through genome analysis clarifies taxonomic relationships and enables sequence-directed genetic improvement.

BackgroundMost modern citrus cultivars have an interspecific origin. As a foundational step towards deciphering the interspecific genome structures, a reference whole genome sequence was produced by the International Citrus Genome Consortium from a haploid derived from Clementine mandarin. The availability of a saturated genetic map of Clementine was identified as an essential prerequisite to assist the whole genome sequence assembly. Clementine is believed to be a ‘Mediterranean’ mandarin × sweet orange hybrid, and sweet orange likely arose from interspecific hybridizations between mandarin and pummelo gene pools. The primary goals of the present study were to establish a Clementine reference map using codominant markers, and to perform comparative mapping of pummelo, sweet orange, and Clementine.ResultsFive parental genetic maps were established from three segregating populations, which were genotyped with Single Nucleotide Polymorphism (SNP), Simple Sequence Repeats (SSR) and Insertion-Deletion (Indel) markers. An initial medium density reference map (961 markers for 1084.1 cM) of the Clementine was established by combining male and female Clementine segregation data. This Clementine map was compared with two pummelo maps and a sweet orange map. The linear order of markers was highly conserved in the different species. However, significant differences in map size were observed, which suggests a variation in the recombination rates. Skewed segregations were much higher in the male than female Clementine mapping data. The mapping data confirmed that Clementine arose from hybridization between ‘Mediterranean’ mandarin and sweet orange. The results identified nine recombination break points for the sweet orange gamete that contributed to the Clementine genome.ConclusionsA reference genetic map of citrus, used to facilitate the chromosome assembly of the first citrus reference genome sequence, was established. The high conservation of marker order observed at the interspecific level should allow reasonable inferences of most citrus genome sequences by mapping next-generation sequencing (NGS) data in the reference genome sequence. The genome of the haploid Clementine used to establish the citrus reference genome sequence appears to have been inherited primarily from the ‘Mediterranean’ mandarin. The high frequency of skewed allelic segregations in the male Clementine data underline the probable extent of deviation from Mendelian segregation for characters controlled by heterozygous loci in male parents.

Citrus medica contributed to all limes and lemons and was the direct male parent for the main sub-groups in combination with C. micrantha or close papeda species (for C. aurata, C. excelsa, C. macrophylla and C. aurantifolia--'Mexican' lime types of Tanaka's taxa), C. reticulata(for C. limonia, C. karna and C. jambhiri varieties of Tanaka's taxa, including popular citrus rootstocks such as 'Rangpur' lime, 'Volkamer' and 'Rough' lemons), C. aurantium (for C. limetta and C. limon--yellow lemon types--varieties of Tanaka's taxa) or the C. maxima × C. reticulate hybrid (for C. limettioides--'Palestine sweet' lime types--and C. meyeri). Among triploid limes, C. latifolia accessions ('Tahiti' and 'Persian' lime types) result from the fertilization of a haploid ovule of C. limon by a diploid gamete of C. aurantifolia, while C. aurantifolia triploid accessions ('Tanepao' lime types and 'Madagascar' lemon) probably result from an interspecific backcross (a diploid ovule of C. aurantifolia fertilized by C. medica). As limes and lemons were vegetatively propagated (apomixis, horticultural practices) the intra-sub-group phenotypic diversity results from asexual variations.

Citrus trees have a long juvenile phase that delays their reproductive development by between 6 and 20 years, depending on the species. With the aim of accelerating their flowering time, we transformed juvenile citrus seedlings to constitutively express the Arabidopsis LEAFY (LFY) or APETALA1 (AP1) genes, which promote flower initiation in Arabidopsis. Both types of transgenic citrus produced fertile flowers and fruits as early as the first year, notably through a mechanism involving an appreciable shortening of their juvenile phase. Furthermore, expression of AP1 was as efficient as LFY in the initiation of flowers, and did not produce any severe developmental abnormality. Both types of transgenic trees flowered in consecutive years, and their flowering response was under environmental control. In addition, zygotic and nucellar derived transgenic seedlings had a very short juvenile phase and flowered in their first spring, demonstrating the stability and inheritance of this trait. These results open new possibilities for domestication, genetic improvement, and experimental research in citrus and other woody species.

Whole-genome duplication, or polyploidy, is common in many plant species and often leads to better adaptation to adverse environmental condition. However, little is known about the physiological and molecular determinants underlying adaptation. We examined the drought tolerance in diploid (2x) and autotetraploid (4x) clones of Rangpur lime (Citrus limonia) rootstocks grafted with 2x Valencia Delta sweet orange (Citrus sinensis) scions, named V/2xRL and V/4xRL, respectively. Physiological experiments to study root-shoot communication associated with gene expression studies in roots and leaves were performed. V/4xRL was much more tolerant to water deficit than V/2xRL. Gene expression analysis in leaves and roots showed that more genes related to the response to water stress were differentially expressed in V/2xRL than in V/4xRL. Prior to the stress, when comparing V/4xRL to V/2xRL, V/4xRL leaves had lower stomatal conductance and greater abscisic acid (ABA) content. In roots, ABA content was higher in V/4xRL and was associated to a greater expression of drought responsive genes, including CsNCED1, a pivotal regulatory gene of ABA biosynthesis. We conclude that tetraploidy modifies the expression of genes in Rangpur lime citrus roots to regulate long-distance ABA signalling and adaptation to stress.

BackgroundWith the increasing availability of EST databases and whole genome sequences, SNPs have become the most abundant and powerful polymorphic markers. However, SNP chip data generally suffers from ascertainment biases caused by the SNP discovery and selection process in which a small number of individuals are used as discovery panels. The ongoing International Citrus Genome Consortium sequencing project of the highly heterozygous Clementine and sweet orange genomes will soon result in the release of several hundred thousand SNPs. The primary goals of this study were: (i) to estimate the transferability within the genus Citrus of SNPs discovered from Clementine BACend sequencing (BES), (ii) to estimate bias associated with the very narrow discovery panel, and (iii) to evaluate the usefulness of the Clementine-derived SNP markers for diversity analysis and comparative mapping studies between the different cultivated Citrus species.ResultsFifty-four accessions covering the main Citrus species and 52 interspecific hybrids between pummelo and Clementine were genotyped on a GoldenGate array platform using 1,457 SNPs mined from Clementine BES and 37 SNPs identified between and within C. maxima, C. medica, C. reticulata and C. micrantha. Consistent results were obtained from 622 SNP loci. Of these markers, 116 displayed incomplete transferability primarily in C. medica, C. maxima and wild Citrus species. The two primary biases associated with the SNP mining in Clementine were an overestimation of the C. reticulata diversity and an underestimation of the interspecific differentiation. However, the genetic stratification of the gene pool was high, with very frequent significant linkage disequilibrium. Furthermore, the shared intraspecific polymorphism and accession heterozygosity were generally enough to perform interspecific comparative genetic mapping.ConclusionsA set of 622 SNP markers providing consistent results was selected. Of the markers mined from Clementine, 80.5% were successfully transferred to the whole Citrus gene pool. Despite the ascertainment biases in relation to the Clementine origin, the SNP data confirm the important stratification of the gene pools around C. maxima, C. medica and C. reticulata as well as previous hypothesis on the origin of secondary species. The implemented SNP marker set will be very useful for comparative genetic mapping in Citrus and genetic association in C. reticulata.

Numerous molecular polymorphisms (SNPs and indels), which are potentially useful for the analysis of interspecific genetic structures, have been identified. The nuclear phylogenetic network for Citrus and its sexually compatible relatives was consistent with the geographical origins of these genera. The positive selection observed for a few genes will help further works to analyse the molecular basis of the variability of the associated traits. This study presents new insights into the origin of C. sinensis.

Very little is known about the molecular origin of the large phenotypic differentiation between genotypes arising from somatic chromosome set doubling and their diploid parents. In this study, the anatomy and physiology of diploid (2x) and autotetraploid (4x) Rangpur lime (Citrus limonia Osbeck) seedlings has been characterized. Growth of 2x was more vigorous than 4x although leaves, stems, and roots of 4x plants were thicker and contained larger cells than 2x that may have a large impact on cell-to-cell water exchanges. Leaf water content was higher in 4x than in 2x. Leaf transcriptome expression using a citrus microarray containing 21 081 genes revealed that the number of genes differentially expressed in both genotypes was less than 1% and the maximum rate of gene expression change within a 2-fold range. Six up-regulated genes in 4x were targeted to validate microarray results by real-time reverse transcription-PCR. Five of these genes were apparently involved in the response to water deficit, suggesting that, in control conditions, the genome expression of citrus autotetraploids may act in a similar way to diploids under water-deficit stress condition. The sixth up-regulated gene which codes for a histone may also play an important role in regulating the transcription of growth processes. These results show that the large phenotypic differentiation in 4x Rangpur lime compared with 2x is not associated with large changes in genome expression. This suggests that, in 4x Rangpur lime, subtle changes in gene expression may be at the origin of the phenotypic differentiation of 4x citrus when compared with 2x.