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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. Citrus Research and Development Foundation 108O568 Ministerio de Ciencia e Innovación: AGL2008-00596-MCI, 2008/121, IPT-010000-2010-43, PSE-060000-2009-8, AGL2007-65437-C04-01/AGR European Commission Citrus Research and Development Foundation itl-bio-03-10122 015453 This work was principally funded by the French ANR CITRUSSEQ project. The European Commission, under the FP6-2003-INCO-DEV-2 project CIBEWU (n°015453), the Spanish Ministerio de Ciencia e Innovación grants, AGL2007-65437-C04-01/AGR and AGL2008-00596-MCI, the Spanish PSE-060000-2009-8 and IPT-010000-2010-43 projects, the Prometeo project 2008/121 Generalidad Valenciana, the Turkish TUBITAK Project No: 108O568; the California Citrus Research Board and UC Discovery grant itl-bio-03-10122 and the Florida Citrus Research and Development Foundation (CRDF), grants #67 and 71 also contributed to the work.

The sour taste of lemons (Citrus limon (L.) Burm.) is determined by the amount of citric acid in vacuoles of juice sac cells. Faris is a "sweet" lemon variety since it accumulates low levels of citric acid. The University of California Riverside Citrus Variety Collection includes a Faris tree that produces sweet (Faris non-acid; FNA) and sour fruit (Faris acid; FA) on different branches; it is apparently a graft chimera with layer L1 derived from Millsweet limetta and layer L2 from a standard lemon. The transcription profiles of Faris sweet lemon were compared with Faris acid lemon and Frost Lisbon (L), which is a standard sour lemon genetically indistinguishable from Faris in prior work with SSR markers. Analysis of microarray data revealed that the transcriptomes of the two sour lemon genotypes were nearly identical. In contrast, the transcriptome of Faris sweet lemon was very different from those of both sour lemons. Among about 1,000 FNA-specific, presumably pH-related genes, the homolog of Arabidopsis H(+)-ATPase proton pump AHA10 was not expressed in FNA, but highly expressed in FA and L. Since Arabidopsis AHA10 is involved in biosynthesis and acidification of vacuoles, the lack of expression of the AHA10 citrus homolog represents a very conspicuous molecular feature of the FNA sweet phenotype. In addition, high expression of several 2-oxoglutarate degradation-related genes in FNA suggests activation of the GABA shunt and degradation of valine and tyrosine as components of the mechanism that reduces the level of citric acid in sweet lemon.

A total of 293 putative citrange F 1 seedlings, derived from 13 crosses and visually identified by their trifoliate leaf morphology, were investigated using six characterized expressed sequence tag (EST) microsatellite primers. Their origins and ploidy levels were simultaneously and unambiguously determined by these markers. Besides a majority of diploid zygotic hybrids, as high as 24% triploid hybrids were produced, only by Citrus sinensis (sweet orange) × Poncirus trifoliata (trifoliate orange), but not the reciprocal crosses. The results indicated that 2 n eggs originated only from the maternal sweet orange parent, contributing the extra haploid genome in the triploids. It was further inferred that the 2 n eggs in sweet orange result from first division restitution (FDR), not second division restitution (SDR), according to the marker genotypes of these triploids. High percentages of self-cross and nucellar origin seedlings were found only from P. trifoliata × C. sinensis , but not from the reciprocals, simply due to their different reliabilities in the visual leaf identification of the seedlings. Survey of leaves indicated that adult triploid seedlings had predominantly monofoliate leaves, compared to diploid hybrids with mostly trifoliate leaves. The possible genetic mechanism and potential applications were discussed.

Simple sequence repeat (SSR) markers generated from expressed sequence tag (EST) sequences represent useful tools for genotyping and their development is relatively easy because of the public availability of EST databases. We report design and application of EST–SSRs to assess the level of genetic diversity among thirty-five asparagus cultivars and to fingerprint DePaoli, a new variety released by University of California, Riverside. DNA was isolated from bulks of pooled cladophylls coming from five plants of each variety to reduce the number of DNA extractions and PCR reactions. Allele frequencies were estimated from the intensity of the bands in two bulks and two individual plant samples for each variety. Although asparagus varieties derive from a limited germplasm pool, eight EST–SSR loci differentiated all of the analyzed cultivars. Moreover, UPGMA (unweighted pair group method with arithmetic mean) and neighbor-joining trees, as well as principal components analysis separated the cultivars into clusters corresponding to the geographical areas where they originated.

Relationships among 88 accessions representing 45 Citrus species, three man-made hybrids, and six related genera were examined for restriction fragment length polymorphisms (RFLP). Thirty-two Citrus and three Microcitrus accessions were also examined by random amplified polymorphic DNA (RAPD) analysis. A measure of relative heterozygosity was estimated based on the mean of the number of fragments per individual per probe-enzyme combination (PEC) divided by total number of fragments per PEC for all non-hybrid Citrus individuals. The presence in a Citrus species of a rare band found also in a related genus was taken as an indication of possible introgression, while the presence of several fragments unique to 1 species was used to indicate non-involvement of that species in hybridization events. Most species that have been described in the literature as hybrids had high heterozygosity indices and no unique fragments. Distance matrices and dendrograms were generated using simple matching coefficient and neighbor-joining cluster analysis. RFLP and RAPD data gave approximately the same results. These data showed C. maxima was affiliated with the papedas C. hongheensis and C. latipes. C. medica clustered with C. indica when only non-hybrid taxa were examined, or among limes, lemons, and relatives when all species were considered. Mandarins did not show strongly supported groupings among themselves, nor with other species. These data showed that several accessions were probably assigned to the wrong species.

Trifoliate orange [Poncirus trifoliata (L.) Raf.] is frequently used as a parent in citrus rootstock breeding, but the origin and amount of genetic diversity in germ plasm collections are poorly understood. Most accessions are self-compatible, but produce a mixture of sexual and apomictic seedlings. Variation among 48 vegetatively propagated trifoliate orange accessions was assessed at seven isozyme loci, together with the restriction fragment length polymorphisms (RFLPs) detected by 38 probe-enzyme combinations and the inter-simple sequence repeat (ISSR) markers generated by 11 primers. Isozymes and RFLPs detected few polymorphisms among accessions, although genetic analysis has shown that the common phenotype is heterozygous for four isozyme and at least four RFLP loci. ISSR amplification generated multiple banding profiles with an average of 58 fragments/primer/accession. These fragments were repeatable across DNA samples extracted from different trees of the same accession or extracted at different times, and across separate PCR runs. Seventeen unique marker phenotypes were identified. The 48 trifoliate orange accessions were classified into four major groups based on polymorphic ISSR markers. All large-flowered accessions are in group 4, while small-flowered accessions are in group 3. Many ISSR markers segregated in progeny derived by open-pollination (probably mostly selfing) of a common accession, indicating that these ISSR markers are also heterozygous. Accessions having identical genotypes for a large number of heterozygous markers are unlikely to have diverged by recombination. Thus the limited divergence we detected among most accessions most likely originated by mutation. ‘Monoembryonic’ and ‘Simmons’ differed from other accessions only in the loss of specific markers, indicating that they originated as zygotic seedlings of individuals similar to the common genotype. Three accessions recently introduced from China have relatively different fingerprints with 3–14 unique ISSR markers, and probably represent a much more divergent germ plasm that may be a valuable breeding resource.

Background Three gametoclonal plants of Citrus clementina Hort. ex Tan., cv. Nules, designated ESP, FRA, and ITA (derived from three labs in Spain, France, and Italy, respectively), were selected for cytological and molecular characterization in order to elucidate genomic rearrangements provoked by haploidization. The study included comparisons of their ploidy, homozygosity, genome integrity, and gene dosage, using chromosome counting, flow cytometry, SSR marker genotyping, and array-Comparative Genomic Hybridization (array-CGH). Results Chromosome counting and flow cytometry revealed that ESP and FRA were haploid, but ITA was tri-haploid. Homozygous patterns, represented by a single peak (allele), were observed among the three plants at almost all SSR loci distributed across the entire diploid donor genome. Those few loci with extra peaks visualized as output from automated sequencing runs, generally low or ambiguous, might result from amplicons of paralogous members at the locus, non-specific sites, or unexpected recombinant alleles. No new alleles were found, suggesting the genomes remained stable and intact during gametogenesis and regeneration. The integrity of the haploid genome also was supported by array-CGH studies, in which genomic profiles were comparable to the diploid control. Conclusions The presence of few gene hybridization abnormalities, corroborated by gene dosage measurements, were hypothetically due to the segregation of hemizygous alleles and minor genomic rearrangements occurring during the haploidization procedure. In conclusion, these plants that are valuable genetic and breeding materials contain completely homozygous and essentially intact genomes.

Background Most 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. Results Five 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. Conclusions A 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.

Twenty-four simple sequence repeat (SSR) markers were used to detect molecular polymorphisms among 370 mostly sexually derived Citrus accessions from the collection of citrus germplasm maintained at the University of California, Riverside. A total of 275 alleles were detected with an average of 11.5 alleles per locus and an average polymorphism information content of 0.625. Genetic diversity statistics were calculated for each individual SSR marker, the entire population, and for specified Citrus groups. Phylogenetic relationships among all citrus accessions and putative non-hybrid Citrus accessions were determined by constructing neighbor-joining trees. There was strong support for monophyly at the species level when hybrid taxa were removed from the data set. Both of these trees indicate that Fortunella clusters within the genus Citrus but Poncirus is a sister genus to Citrus. Additionally, Citrus accessions were probabilistically assigned to populations or multiple populations if their genotype indicated an admixture by a model-based clustering approach. This approach identified five populations in this data set. These separate analyses (distance and model based) both support the hypothesis that there are only a few naturally occurring species of Citrus and most other types of Citrus arose through various hybridization events between these naturally occurring forms.

Sixty-five microsatellite alleles amplified from ancestral citrus accessions classified in three separate genera were evaluated for sequence polymorphism to establish the basis of inter- and intra-allelic genetic variation, evaluate the extent of size homoplasy, and determine an appropriate model (stepwise or infinite allele) for analysis of citrus microsatellite alleles. Sequences for each locus were aligned and subsequently used to determine relationships between alleles of different taxa via parsimony. Interallelic size variation at each SSR locus examined was due to changes in repeat copy number with one exception. Sequencing these alleles uncovered new distinct point mutations in the microsatellite region and the region flanking the microsatellite. Several of the point mutations were found to be genus, species, or allele specific, and some mutations were informative about the inferred evolutionary relationships among alleles. Overall, homoplasy was observed in alleles from all three loci, where the core microsatellite repeat was changed causing alleles of the same size class to be identical in state but not identical by descent. Because nearly all changes in allele size (with one exception) were due to expansion or contraction of the repeat motif, this suggests that a stepwise mutation model, which assumes homoplasy may occur, would be the most appropriate for analyzing Citrus SSR data. The collected data indicate that microsatellites can be a useful tool for evaluating Citrus species and two related genera since repeat motifs were reasonably well retained. However, this work also demonstrated that the number of microsatellite alleles is clearly an underestimate of the number of sequence variants present.

Citron (Citrus medica L.) is one of the three basic species of the genus Citrus L. that have contributed to the development of cultivated citrus. We analyzed the genetic diversity of 47 citrons (32 from Yunnan Province, China; and 15 of Mediterranean origin) to understand their diversity and relationships within the species. Genetic analysis was conducted using data from microsatellite markers, single nucleotide polymorphisms generated from sequences of a nuclear malate dehydrogenase gene and a chloroplast gene, rps16. Neighbor joining and maximum parsimony analyses were conducted. All three approaches found citron to be monophyletic. Population structure analysis clustered the 47 citrons into three distinct groups. The first group consisted of wild, non-fingered citrons generally having locules, juice sacs and seeds within the fruit. The second cluster consisted mostly of fingered citrons that lacked locules, juice sacs or seeds, and some non-fingered types with smaller locules and vestigial juice sacs, but with seeds. All accessions that clustered in groups I and II originated in China. The third cluster consisted of citrons cultivated in the Mediterranean region. Genetic distance between the clusters from population structure analysis indicated considerable diversity within the species. A citron-specific microsatellite marker was identified and characterized. We observed considerable heterozygosity in certain citrons, contrary to previous reports.