Extraordinarily conserved chromosomal synteny of Citrus species revealed by chromosome‐specific painting

Reliable identification of individual chromosomes in eukaryotic species is the foundation for comparative chromosome synteny and evolutionary studies. Unfortunately, chromosome identification has been a major challenge for plants with small chromosomes, such as the Citrus species. We developed oligonucleotide‐based chromosome painting probes for all nine chromosomes in Citrus maxima (Pummelo). We were able to identify all C. maxima chromosomes in the same metaphase cells using multiple rounds of sequential fluorescence in situ hybridization with the painting probes. We conducted comparative chromosome painting analysis in six different Citrus and related species. We found that each painting probe hybridized to only a single chromosome in all other five species, suggesting that the six species have maintained a complete chromosomal synteny after more than 9 million years of divergence. No interchromosomal rearrangement was identified in any species. These results support the hypothesis that karyotypes of woody species are more stable than herbaceous plants because woody plants need a longer period to fix chromosome structural variants in natural populations.     

Development of Species-Specific InDel Markers in Citrus

Citrus taxonomy is complex owing to the existence of a wide range of species: Poncirus is used mainly for rootstock; Fortunella produces small fruit and edible pericarp; and Citrus comprises the most widespread fruit crop species worldwide. Rapidly increasing genome resources from different citrus species facilitate the development of convenient and genome-wide molecular markers that can be applied to both inter- and intra-species analyses. In this study, by comparing the genome sequences of four citrus species, a set of 1958 InDels were identified and 453 candidate InDels were converted into PCR-based markers. Among these candidate InDels, 268 (65%) exhibited length polymorphisms from 30 bp to 200 bp when applied to seven species from the genera Poncirus, Fortunella and Citrus. Seven InDel markers exhibited high intraspecific polymorphisms in a natural pummelo population. The results showed that the InDel markers are effective for both inter- and intra-specific variation and identification analyses. These InDel markers are expected to be applied to germplasm identification, phylogenetic analysis, genetic diversity evaluation and marker-assisted breeding in citrus.     

Genetic diversity and population structure of pummelo (<Emphasis Type="Italic">Citrus maxima</Emphasis>) germplasm in China

Pummelo (Citrus maxima) is one of the basic species of Citrus. It has been cultivated for about 4000 years in China, and therefore, there are abundant germplasm during the long time of culture. However, there is still a lack of a detailed study of the genetic characteristics of pummelo population. In this study, genetic diversity and population structure among 274 pummelo accessions collected in China were analyzed using 31 nuclear simple sequence repeat (nSSR) markers. The observed heterozygosity was calculated as 0.325 and genetic differentiation Fst as 0.077. Genetic structure analysis divided the whole germplasm into three subpopulations, Pop-a, Pop-b, and Pop-c. Pop-a was composed of accessions mostly from Southeast China, Pop-b was composed of accessions from the central region of South China, and Pop-c was composed of accessions from Southwest China. Meanwhile, the analysis of principal coordinate analysis and neighbor-join tree supported the viewpoint of three subpopulations, and then the possible dispersal routes of pummelos in China were proposed. This study provides an insight into the genetic diversity, facilitates future genome-wide association studies, and promotes the breeding program of pummelo as well.     

Citrus phylogeny and genetic origin of important species as investigated by molecular markers

Citrus phylogeny was investigated using RAPD, SCAR and cpDNA markers. The genotypes analyzed included 36 accessions belonging to Citrus together with 1 accession from each of the related genera Poncirus, Fortunella, Microcitrus and Eremocitrus. Phylogenetic analysis with 262 RAPDs and 14 SCARs indicated that Fortunella is phylogenetically close to Citrus while the other three related genera are distant from Citrus and from each other. Within Citrus, the separation into two subgenera, Citrus and Papeda, designated by Swingle, was clearly observed except for C. celebica and C. indica. Almost all the accessions belonging to subgenus Citrus fell into three clusters, each including 1 genotype that was considered to be a true species. Different phylogenetic relationships were revealed with cpDNA data. Citrus genotypes were separated into subgenera Archicitrus and Metacitrus, as proposed by Tanaka, while the division of subgenera Citrus and Papeda disappeared. C. medica and C. indica were quite distant from other citrus as well from related genera. C. ichangensis appeared to be the ancestor of the mandarin cluster, including C. tachibana. Lemon and Palestine sweet lime were clustered into the Pummelo cluster led by C. latipes. C. aurantifolia was located in the Micrantha cluster. Furthermore, genetic origin was studied on 17 cultivated citrus genotypes by the same molecular markers, and a hybrid origin was hypothesized for all the tested genotypes. The assumptions are discussed with respect to previous studies; similar results were obtained for the origin of orange and grapefruit. Hybrids of citron and sour orange were assumed for lemon, Palestine sweet lime, bergamot and Volkamer lemon, while a citron × mandarin hybrid was assumed for Rangpur lime and Rough lemon. For Mexican lime our molecular data indicated C. micrantha to be the female parent and C. medica as the male one. Received: 5 October 1999 / Accepted: 3 November 1999     

New universal mitochondrial PCR markers reveal new information on maternal citrus phylogeny

The aim of this work was to provide a set of mitochondrial markers to reveal polymorphism and to study the maternal phylogeny in citrus. We first used 44 universal markers previously described in the literature: nine of these markers produced amplification products but only one revealed polymorphism in citrus. We then designed six conserved pairs of primers using the complete mitochondrial DNA sequences of Arabidopsis thaliana and Beta vulgaris to amplify polymorphic intergenic and intronic regions. From these six pairs of primers, three from introns of genes coding for NADH dehydrogenase subunits 2, 5, and 7, revealed polymorphism in citrus. First, we confirmed that citrus have a maternal mitochondrial inheritance in two populations of 250 and 120 individuals. We then conducted a phylogenic study using four polymorphic primers on 77 genotypes representing the diversity of Citrus and two related genera. Seven mitotypes were identified. Six mitotypes (Poncirus, Fortunella, Citrus medica, Citrus micrantha, Citrus reticulata, and Citrus maxima) were congruent with previous taxonomic investigations. The seventh mitotype enabled us to distinguish an acidic mandarin group (‘Cleopatra’, ‘Sunki’ and ‘Shekwasha’) from other mandarins and revealed a maternal relationship with Citrus limonia (‘Rangpur’ lime, ‘Volkamer’ lemon) and Citrus jambhiri (‘Rough’ lemon). This mitotype contained only cultivated species used as rootstocks due to their good tolerances to abiotic stress. Our results also suggest that two species classified by Swingle and Reece, Citrus limon, and Citrus aurantifolia, have multiple maternal cytoplasmic origins.     

Genetic diversity in the orange subfamily Aurantioideae. II. Genetic relationships among genera and species

Genetic relationships were studied by means of ten isoenzymatic systems, at the genus and species level, using two distances and four methods of aggregation in a germplasm collection of 198 cultivars and accessions of 54 species belonging to Citrus and 13 related genera. The most consistent results were obtained by the chord distance and the neighbor-joining clustering method. Citrus species were distributed in two main groups: the orange-mandarin group and the lime lemon-citron-pummelo group. The species C. halimii and C. tachibana are not included in these groups. Mandarin species fall into three main subgroups: one includes C. sinensis; the second, C. aurantium, the third, small-fruit species. The citron, the pummelo and the ancient lemon subgroups form a cluster to which the species belonging to subgenus Papeda and the cultivated limes, lemons and bergamots are related. Microcitrus spp, to which Severinia buxifolia and Atalantia ceylanica seem to be related, cluster with the lime lemon-citron-pummelo group while Fortunella is close to the orange-mandarin group. Poncirus trifoliata, the most important species for citrus rootstock improvement is located far from Citrus but connected to it through Fortunella spp. A broad distribution of species has been found that should be taken into account to sample new genotypes in the search of desired characters in order to fully and efficiently use genetic resources for citrus improvement.     

Identification and genomic distribution of<Emphasis Type="Italic"> gypsy</Emphasis> like retrotransposons in<Emphasis Type="Italic"> Citrus</Emphasis> and<Emphasis Type="Italic"> Poncirus</Emphasis>

Transposable elements might be importantly involved in citrus genetic instability and genome evolution. The presence of gypsy like retrotransposons, their heterogeneity and genomic distribution in Citrus and Poncirus, have been investigated. Eight clones containing part of the POL coding region of gypsy like retrotransposons have been isolated from a commercial variety of Citrus clementina, one of the few sexual species in Citrus. Four of the eight clones might correspond to active elements given that they present all the conserved motifs described in the literature as essential for activity, no in-frame stop codon and no frame-shift mutation. High homology has been found between some of these citrus elements and retroelements within a resistance-gene cluster from potato, another from Poncirus trifoliata and two putative resistance polyproteins from rice. Nested copies of gypsy like elements are scattered along the Citrus and Poncirus genomes. The results on genomic distribution show that these elements were introduced before the divergence of both genera and evolved separately thereafter. IRAPs based on gypsy and copia types of retrotransposons seem to distribute differently, therefore gypsy based IRAPs prove a new, complementary set of molecular markers in Citrus to study and map genetic variability, especially for disease resistance. Similarly to copia-derived IRAPs, the number of copies and heterozygosity values found for gypsy derived IRAPs are lower in Poncirus than in Citrus aurantium, which is less apomictic and the most usual rootstock for clementines until 1970.Communicated by C. Möllers     

Performance of Cryptolaemus montrouzieri feeding on Pulvinaria aurantii ovisacs on citrus plants

We studied the performance of the coccinellid Cryptolaemus montrouzieri on citrus plant species infested with orange pulvinaria scale, Pulvinaria aurantii, under controlled conditions in the laboratory. The experimental host plants were blood orange, grapefruit, lemon, pummelo and kumquat. All life history parameters indicated best performance of C. montrouzieri, when reared on P. aurantii ovisacs on the leaves of grapefruit. Pummelo, blood orange, lemon or kumquat were less suitable.     

Transferability of the EST-SSRs developed on Nules clementine (<Emphasis Type="Italic">Citrus clementina</Emphasis> Hort ex Tan) to other <Emphasis Type="Italic">Citrus</Emphasis> species and their effectiveness for genetic mapping

Background

During the last decade, numerous microsatellite markers were developed for genotyping and to identify closely related plant genotypes. In citrus, previously developed microsatellite markers were arisen from genomic libraries and more often located in non coding DNA sequences. To optimize the use of these EST-SSRs as genetic markers in genome mapping programs and citrus systematic analysis, we have investigated their polymorphism related to the type (di or trinucleotide) or their position in the coding sequences.

Results

Among 11000 unigenes from a Clementine EST library, we have found at least one microsatellite sequence (repeated units size ranged from 2 to 6 nucleotides) in 1500 unigenes (13.6%). More than 95% of these SSRs were di or trinucleotides. If trinucleotide microsatellites were encountered trough all part of EST sequences, dinucleotide microsatellites were preferentially (50%) concentrated in the 5' 100th nucleotides. We assessed the polymorphism of 41 EST-SSR, by PCR amplification droved with flanking primers among ten Citrus species plus 3 from other genera. More than 90% of EST-SSR markers were polymorphic. Furthermore, dinucleotide microsatellite markers were more polymorphic than trinucleotide ones, probably related to their distribution that was more often located in the 5' UnTranslated Region (UTR). We obtained a good agreement of diversity relationships between the citrus species and relatives assessed with EST-SSR markers with the established taxonomy and phylogeny. To end, the heterozygosity of each genotype and all dual combinations were studied to evaluate the percentage of mappable markers. Higher values (> 45%) were observed for putative Citrus inter-specific hybrids (lime lemon, or sour orange) than for Citrus basic true species (mandarin, pummelo and citron) (<30%). Most favorable combinations for genome mapping were observed in those involving interspecific hybrid genotypes. Those gave higher levels of mappable markers (>70%) with a significant proportion suitable for synteny analysis.

Conclusion

Fourty one new EST-SSR markers were produced and were available for citrus genetic studies. Whatever the position of the SSR in the ESTs the EST-SSR markers we developed are powerful to investigate genetic diversity and genome mapping in citrus.

     

Production and molecular characterization of potential seedless cybrid plants between pollen sterile Satsuma mandarin and two seedy <Emphasis Type="Italic">Citrus</Emphasis> cultivars

Cytoplasmic male sterility (CMS) is known to be controlled by mitochondrial genome in higher plants including Satsuma mandarin (Citrus unshiu Marc.). Citrus symmetric fusion experiments often produce diploid cybrids possessing nuclear DNA from the mesophyll parent and mitochondrial DNA (mtDNA) from the embryogenic callus parent. Therefore, it is possible to transfer CMS from Satsuma mandarin as callus parent to seedy citrus cultivars as leaf one by somatic cybridization. Herein, symmetric fusion technique was adopted to create cybrids for potential seedlessness by transferring CMS from Citrus unshiu Marc. cv. Guoqing No. 1 (G1) to two traditional Chinese seedy citrus cultivars, ‘Shatian’ pummelo (C. grandis (L) Osbeck) and ‘Bingtang’ orange (C. sinensis (L) Osbeck). Flow cytometry analysis showed that 19 plants recovered from G1 + ‘Bingtang’ orange and 17 of 35 plants regenerated from G1 + ‘Shatian’ pummelo were diploid. The remaining plants from G1 + ‘Shatian’ pummelo were tetraploid. The diploid plants from the two combinations were confirmed as true cybrids by simple sequence repeat (SSR) and cleaved amplified polymorphic sequence (CAPS) analysis, with nuclear DNA from their corresponding leaf parent and mtDNA from their common suspension parent, G1 Satsuma mandarin. The remaining plants from G1 + ‘Shatian’ pummelo were identified as somatic hybrids with mtDNA from G1. The chloroplast simple sequence repeat (cp-SSR) analysis revealed somatic hybrid/cybrid plants from the two combinations in most cases possessed either of their parental chloroplast type, and two plants from G1 +‘Shatian’ pummelo and all embryoids analyzed from G1 + ‘Bingtang’ orange possessed chloroplast DNA (cpDNA) from both parents. These results demonstrated that we succeeded in introducing mtDNA from G1 Satsuma mandarin into the two target seedy citrus cultivars for potential seedlessness through symmetric fusion.     

Transferability and Level of Heterozygosity of Microsatellite Markers in <Emphasis Type="Italic">Citrus</Emphasis> Species

Microsatellite markers are a powerful tool for genetic studies, including germplasm conservation, cultivar identification, and integration of linkage maps. Several works have shown that primer pairs designed for one species can be used in related species to facilitate wider application because it reduces the costs for primer development. The objective of this study was to evaluate the transferability of microsatellite primers which was previously developed from the genomic library of Pêra sweet orange (Citrus sinensis L. Osbeck) and to determine the level of heterozygosity between citrus accessions and related genera. Twenty-four microsatellite loci were evaluated on 12 genotypes of Citrus, Poncirus, and an intergeneric hybrid. All analyzed markers were transferable across all genotypes. Seventeen loci were polymorphic, and the number of alleles per loci ranged from one to six. The lowest level of heterozygosity was observed for Poncirus trifoliata (L.) Raf. cultivars while the highest level was for Swingle citrumelo. In general, microsatellite markers showed wide genetic variation and demonstrated that they can be useful in citrus breeding programs.     

Phylogenetic Relationships of Citrus and Its Relatives Based on matK Gene Sequences

The genus Citrus includes mandarin, orange, lemon, grapefruit and lime, which have high economic and nutritional value. The family Rutaceae can be divided into 7 subfamilies, including Aurantioideae. The genus Citrus belongs to the subfamily Aurantioideae. In this study, we sequenced the chloroplast matK genes of 135 accessions from 22 genera of Aurantioideae and analyzed them phylogenetically. Our study includes many accessions that have not been examined in other studies. The subfamily Aurantioideae has been classified into 2 tribes, Clauseneae and Citreae, and our current molecular analysis clearly discriminate Citreae from Clauseneae by using only 1 chloroplast DNA sequence. Our study confirms previous observations on the molecular phylogeny of Aurantioideae in many aspects. However, we have provided novel information on these genetic relationships. For example, inconsistent with the previous observation, and consistent with our preliminary study using the chloroplast rbcL genes, our analysis showed that Feroniella oblata is not nested in Citrus species and is closely related with Feronia limonia. Furthermore, we have shown that Murraya paniculata is similar to Merrillia caloxylon and is dissimilar to Murraya koenigii. We found that “true citrus fruit trees” could be divided into 2 subclusters. One subcluster included Citrus, Fortunella, and Poncirus, while the other cluster included Microcitrus and Eremocitrus. Compared to previous studies, our current study is the most extensive phylogenetic study of Citrus species since it includes 93 accessions. The results indicate that Citrus species can be classified into 3 clusters: a citron cluster, a pummelo cluster, and a mandarin cluster. Although most mandarin accessions belonged to the mandarin cluster, we found some exceptions. We also obtained the information on the genetic background of various species of acid citrus grown in Japan. Because the genus Citrus contains many important accessions, we have comprehensively discussed the classification of this genus.     

Induction of triploid Citrus plants from endosperm calli in vitro

Summary Triploid hybrid Citrus plants were regenerated by somatic embryogenesis in vitro from endosperm derived calli. A sequence of media formulations was used to induce and support proliferation of primary callus from endosperm, to induce embryogenesis from primary callus, and to allow embryo development leading to viable plantlets. Calli were induced from cellular endosperm of Citrus sinensis (sweet orange), C. Xparadisi (grapefruit), and C. grandis (pummelo) excised 12–14 weeks post-anthesis. Induction of embryogenesis from sweet orange and pummelo primary calli required gibberellic acid and double mineral nutrient concentrations. Embryogenesis was not induced from grapefruit calli in these experiments. Only sweet orange embryos developed sufficiently to allow plant regeneration. Triploid axillary buds were minigrafted onto etiolated diploid rootstock seedlings in vitro in order to transfer triploid regenerants to soil and the external environment. Triploidy (2n = 3x = 27) was observed consistently in all phases of regeneration and in recovered plants. These results demonstrate that triploid hybrid plant recovery from Citrus endosperm can overcome barriers to sexual hybridization resulting from apomixis.Florida Agricultural Experiment Station Journal Series No. R-00627     

Comparison of photosynthesis and antioxidant performance of several Citrus and Fortunella species (Rutaceae) under natural chilling stress

Citrus plants originate from southeastern Asia, in a large area with various climates characterized by a broad range of temperatures. Some species have been diversified in temperate climates, others in subtropical climates. Temperature is assumed to be a key factor in citrus species adaptation and diversification of basic cellular functions. In a field experiment, the tolerance of the three fundamental Citrus species C. medica L., C. reticulata Blanco and C. maxima (Burm.) Merr., and Fortunella japonica (Thunb.) Swing. to photooxidative stress caused by seasonal climatic changes was evaluated on adult trees by measuring net photosynthesis (Pnet), stomatal conductance (Gs), maximum photosynthesis (Pmax) and chlorophyll fluorescence (Fv/Fm). In addition, seasonal changes in oxidative status, antioxidant enzymes (superoxide dismutase, catalase, ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase and glutathione reductase) and antioxidant metabolites (ascorbate and glutathione) were monitored. Mandarin and pummelo appeared to be the most tolerant, showing the lowest down-regulation of photosynthetic parameters, and the lowest accumulation of oxidized compounds associated with efficiency of their antioxidant system. Kumquat showed intermediate behaviour, with a large diminution of photosynthetic parameters and marked accumulation of hydrogen peroxide, whereas the malondialdehyde content remained low, with a strong induction of glutathione synthesis. Finally, citron appeared to be the most sensitive genotype with a marked decrease in photosynthetic performance, the largest accumulation of oxidative parameters, insufficient induction of antioxidant enzymes and down-regulation of ascorbate and glutathione synthesis.     

Flavanone Glycoside Biosynthesis in Citrus: Chalcone Synthase, UDP-Glucose:Flavanone-7-O-Glucosyl-Transferase and -Rhamnosyl-Transferase Activities in Cell-Free Extracts

Previous indirect evidence suggested that the biosynthesis of flavonoids in Citrus may not proceed via the usual chalcone synthase reaction and that glycosylation occurs during chalcone formation and not afterward, as has been reported in other species. We detected chalcone-synthase and UDP-glucose:flavanone-7-O-glucosyl-transferase activities in cell-free extracts of Citrus. The glucosylated flavanone was further rhamnosylated when exogenous UDP-glucose and NADPH were added to the extract. Chalcone-synthase activity was detected in cell-free extracts derived from young leaves and fruits. Young fruits (2 millimeter diameter) had the highest chalcone synthase activity. UDP-glucose:flavanone-7-O-glucosyl-transferase activity was measured in cell-free extracts derived from young leaves and fruits of Citrus mitis and Citrus maxima. The highest UDP-glucose:flavanone-7-O-glucosyl-transferase activity was found in young C. maxima leaves. These data indicate that Citrus contains a flavonoid pathway similar to that studied in other species.     

Isolation,Characterization, and Expression Analysis of an <Emphasis Type="Italic">SKP1-</Emphasis>like Gene from ‘Shatian’ Pummelo (<Emphasis Type="Italic">Citrus grandis</Emphasis> Osbeck)

Plant s-phase kinase-associated protein 1 (SKP1) genes have diverse functions in plant developmental and physiological activities. Herein, we described a novel SKP1 gene, designated as CgSKP1, from ‘Shatian’ pummelo (Citrus grandis Osbeck). The cDNA sequence of CgSKP1 was 603 bp and contained an open reading frame of 477 bp. Genomic sequence of the CgSKP1 gene contained two exons and one intron. The predicted amino acid sequence of this gene is consisted of 158 amino acids with theoretical proteins size of 17.9 kDa. CgSKP1 had high identity with SKP1 genes from other plant species within two conserved region. Full-length cDNAs were also amplified and cloned from six citrus varieties, with 95% nucleotide identity and about 98% amino acid similarity among them. Gel blot analysis suggested that CgSKP1 existed as a single locus in the ‘Shatian’ pummelo genome. The expression of CgSKP1 was gradually increased during flower developmental stages in ‘Shatian’ pummelo. Moreover, expression analysis by RT-PCR, qRT-PCR and in situ hybridization of CgSKP1 showed that it was highly expressed in the leaf, petal, anther and ovary, but lowly in the style. These findings indicated that CgSKP1 was closely related to ‘Shatian’ pummelo flower development.