Non-random inheritance of mitochondrial genomes in Citrus hybrids produced by protoplast fusion

Somatic hybridization offers the possibility of manipulating chloroplast and mitochondrial genomes and evaluating their role on cultivar qualities in citrus. Numerous associations between Willow-leaf mandarin (Citrus deliciosa Ten.), as embryogenic parent, and sweet orange cv. Valencia (Citrus sinensis (L.) Osb.), as mesophyll parent, and between Willow-leaf mandarin (embryogenic parent) and grapefruit cv. Duncan (Citrus paradisi Macf.) (mesophyll parent) were obtained by the fusion of protoplasts induced by polyethylene glycol. Regenerated plants were characterized by flow cytometry and nuclear and mitochondrial DNA restriction fragment length polymorphism (RFLP). All plants were diploid. Diploid plants with the nuclear RFLP patterns of mandarin or sweet orange were identified in the progeny between these two parents, while only grapefruit nuclear types were found in the mandarin + grapefruit progeny. The diploid plants with the nuclear profile of the mesophyll parent originated systematically from cells formed through spontaneous association of the nuclear genome of the mesophyll parent and the mitochondrial genome of the embryogenic parent. These plants are assumed to be alloplasmic hybrids or cybrids. They were viable and have been propagated for field testing.     

Allotetraploid hybrids between citrus and seven related genera produced by somatic hybridization

We have developed an efficient protoplast-fusion method to produce somatic hybrid allopolyploid plants that combine Citrus with seven related genera, including four that are sexually incompatible. In this paper we report the creation of 18 new allotetraploid hybrids of Citrus, including ten among sexually incompatible related genera, that may have direct cultivar potential as improved citrus rootstocks. All hybrids were confirmed by cytological and RAPD analyses. If fertile, the attributes of these hybrids may be amenable to further genetic manipulation by breeding at the tetraploid level. Wide somatic hybridization of Citrus via protoplast fusion bypasses biological barriers to the natural allopolyploidization of Citrus, and creates new evolutionary opportunities that would be difficult or impossible to achieve by natural or conventional hybridization.Florida Agricultural Experiment Station Journal Series No. R-04520     

Citrus somatic hybridization with potential for improved blight and CTV resistance

Summary The production of five new somatic hybrids with potential for improved disease resistance is reported herein. Protoplast isolation, fusion, and plant regeneration was achieved from Caipira sweet orange (Citrus sinensis L. Osbeck) as an embryogenic parental source and Volkamer lemon (C. volkameriana Pasquale), Cleopatra mandarin (C. reticulata Blanco), and Rough lemon (C. jambhiri Lushington) as non-embryogenic parental sources. Fusion involving Cleopatra mandarin and Rangpur lime (C. limonia L. Osbeck) as embryogenic parental sources with Sour orange (C. aurantium L.) also resulted in somatic hybrid plants. Somatic hybridization was confirmed by leaf morphology evaluation, chromosome counting, and randomly amplified polymorphic DNA (RAPD) analyses. Somatic hybrids may combine complementary characteristics from both parental sources and have potential for tolerance to blight and citrus tristeza virus (CTV).     

Regeneration and molecular characterization of intergeneric somatic hybrids between Citrus reticulata and Poncirus trifoliata

Citrus exocortis viroid (CEV) is widespread in citrus production areas where trifoliate orange [Poncirus trifoliata (L.) Raf.] is used as rootstock. Citrus reticulata Blanco cv. Red tangerine, a different rootstock, is tolerant to CEV. Embryogenic protoplasts of C. reticulata cv. Red tangerine were electrically fused with mesophyll protoplasts from P. trifoliata, and five embryoids were regenerated after 40 days of culture. The embryoids were cut into several pieces and subcultured on shoot induction medium. After 5 months and several subcultures, shoots initially regenerated. The plants grew vigorously with well-developed root systems and exhibited the trifoliate leaf character of P. trifoliata. Chromosome counts on four randomly selected root tips revealed them to be tetraploids (2n=4x=36). RAPD analysis of four randomly selected plants verified their hybridity. This hybridity was further confirmed by AFLP analysis using four primer pairs, from which a total of 65 specific bands were detected. Cytoplasmic genome analysis using universal primers revealed that their chloroplast DNA banding pattern was identical to that of trifoliate orange, while the banding pattern of mitochondrial DNA was identical to that of Red tangerine. The potential of this somatic hybrid as a means to control tree size and provide multi-resistance is discussed.     

Fertile fruit trees obtained by somatic hybridization: navel orange (Citrus sinensis) and Troyer citrange (C. sinensis x Poncirus trifoliata)

Summary Nucellar cell suspension protoplasts of navel orange (Citrus sinsensis Osb.) were chemically fused with mesophyll protoplasts of Troyer citrange (C. sinensis x Poncirus trifoliata) and cultured in hormone-free Murashige and Tucker medium containing 0.6 M sucrose. Two types of plant were regenerated through embryogenesis. One type showed intermediate mono-and difoliate leaves and the other types was identical to Troyer citrange. The regenerated plants with intermediate morphology were demonstrated by chromosome counts and rDNA analysis to be amphidiploid somatic hybrids. Five clones of these somatic hybrids were grafted in the field. After 4 years, they set flowers having a morphology intermediate between those of the two parents. The pollen grains showed high stainability and sufficient germinability, and were larger than those of Troyer citrange. The fruits of the somatic hybrids were large and spherical with thick rinds. Most of them contained seeds with normal germinability. These results indicate that somatic hybridization is a useful tool for Citrus breeding.     

Regeneration and characterization of somatic hybrids combining sweet orange and mandarin/mandarin hybrid cultivars for citrus scion improvement

Protoplast fusion between sweet orange and mandarin/mandarin hybrids scion cultivars was performed following the model ??diploid embryogenic callus protoplast?+?diploid mesophyll-derived protoplast??. Protoplasts were isolated from embryogenic calli of ??Pera?? and ??Westin?? sweet orange cultivars (Citrus sinensis) and from young leaves of ??Fremont??, Nules??, and ??Thomas?? mandarins (C. reticulata), and ??Nova?? tangelo [C. reticulata?×?(C. paradisi?×?C. reticulata)]. The regenerated plants were characterized based on their leaf morphology (thickness), ploidy level, and simple sequence repeat (SSR) molecular markers. Plants were successfully generated only when ??Pera?? sweet orange was used as the embryogenic parent. Fifteen plants were regenerated being 7 tetraploid and 8 diploid. Based on SSR molecular markers analyses all 7 tetraploid regenerated plants revealed to be allotetraploids (somatic hybrids), including 2 from the combination of ??Pera?? sweet orange?+???Fremont?? mandarin, 3 ??Pera?? sweet orange?+???Nules?? mandarin, and 2 ??Pera?? sweet orange?+???Nova?? tangelo, and all the diploid regenerated plants showed the ??Pera?? sweet orange marker profile. Somatic hybrids were inoculated with Alternaria alternata and no disease symptoms were detected 96?h post-inoculation. This hybrid material has the potential to be used as a tetraploid parent in interploid crosses for citrus scion breeding.     

Genetic Diversity in Musa acuminata Colla and Musa balbisiana Colla and some of their natural hybrids using AFLP Markers

Genetic diversity and relationships were assessed in 28 accessions of Musa acuminata (AA) Colla and Musa balbisiana (BB) Colla, and some of their natural hybrids, using the amplified fragment length polymorphisms (AFLP) technique. Fifteen AFLP +3 primer pairs produced 527 polymorphic bands among the accessions. Neighbor-joining and principal co-ordinate (PCO) analyses using Jaccard's similarity coefficient produced four major clusters that closely corresponded with the genome composition of the accessions (AA, BB, AAB and ABB). The AFLP data distinguished between the wild diploid accessions and suggested new subspecies relationships in the M. acuminata complex that are different from those based on morphological data. The data suggested that there are three subspecies within the M. acuminata complex (ssp. burmannica Simmonds, malaccensis Simmonds, and microcarpa Simmonds). 'Tjau Lagada' (ssp. microcarpa), 'Truncata' [ssp truncata (Ridl.) Shepherd] and 'SF247' [ssp. banksii (F.Muell) Simmonds] clustered very closely with 'Gros Michel' and 'Km 5', indicating that more than one M. acuminata subspecies may be involved in the origin of triploid AAA bananas. 'Calcutta 4' (ssp. burmannicoides De Langhe &; Devreux) and 'Long Tavoy' (ssp. burmannica) were closely related and could be together in the same subspecies. This study also showed that there is much more genetic diversity within M. balbisiana that was split into two groups: (1) 'I-63' and 'HND' and (2) 'Los Banos', 'MPL' (Montpellier), '10852', 'Singapuri', 'Etikehel', and 'Butohan 1' as the other.     

Chromosomal relationships among cultivars of Citrus reticulata Blanco, its hybrids and related species

Karyotypes of 93 individuals belonging to 18 accessions of mandarins, mandarin hybrids and two related species were analysed with the fluorochromes CMA and DAPI, to identify marker chromosomes. The karyotypes revealed highly differentiated banding patterns and could be classified in four groups (I–IV) according to the presence/absence of chromosomes with three bands (type A) or with two bands (one proximal and one terminal, type B, or both terminal, type C). The accessions of group I exhibited the simplest and homozygous karyotypes (lacking chromosome types A, B and C), represented by `Sunki' and `Cleopatra'. Group II (lacking chromosome types A and B) included three accessions of Mediterranean mandarins and `Cravo' mandarin, all of them with very similar and almost completely homozygous karyotypes. All other karyotypes of groups II and III (lacking chromosome type A) were heterozygous for one or more chromosome pairs and most of them seemed to be hybrid derivatives from non-mandarin accessions. Group IV (with chromosome types A and B) was represented only by two heterozygous hybrids (`Murcott' and `King'). The karyotype of most hybrids agrees with one of the possible combinations resulting from chromosome types segregation from their putative ancestor karyotypes, but at least `Orlando' seemed to be a more complex hybrid. Comparing with banding patterns of other Citrus species, those of group I and the Mediterranean mandarins (group II) are the best candidates to represent C. reticulata (sensu Swingle) as a true species.     

The plastome of Citrus. Physical map,variation among Citrus cultivars and species and comparison with related genera

Summary A physical plastome map was constructed for Citrus aurantium, and the plastomes of species and cultivars of Citrus and of two Citrus relatives were analysed by Southern blot-hybridisation of labelled total tobacco cpDNA to digests of total Citrus DNA. A resemblance was found between the plastomes of cultivars of C. limon (lemon), C. sinensis (orange), C. aurantium (sour orange), C. paradisii (grapefruit) and C. grandis (pomello). The plastomes of other Citrus types such as mandarin (C. reticulata) and citron (C. medico) differed from each other as well as from the plastomes of the aforementioned group. The plastomes of Poncirus trifoliata and Microcitrus sp. are distinct from each other as well as from the Citrus types.     

Transmission of herbicide resistance from a monoecious to a dioecious weedy Amaranthus species

The genus Amaranthus includes several important monoecious and dioecious weed species, and several populations of these species have developed resistance to herbicides. These species are closely related and two or more species often coexist in agricultural settings. Collectively, these attributes raise the concern that herbicide resistance might transfer from one weedy Amaranthus species to another. We performed research to determine if a dominant allele encoding a herbicide-insensitive form of acetolactate synthase (ALS) could be transferred from a monoecious species, A. hybridus, to a dioecious species, A. rudis. Numerous F₁ hybrids were obtained from controlled crosses in a greenhouse between A. rudis and herbicide-resistant A. hybridus, and most (85%) of these hybrids were herbicide-resistant. Molecular analysis of the ALS gene was used to verify that herbicide-resistant hybrids contained both an A. rudis and an A. hybridus ALS allele. Although hybrids had greatly reduced fertility, 42 BC₁ plants were obtained by backcrossing 33 hybrids with male A. rudis. Fertility was greatly restored in BC₁ progeny, and numerous BC₂ progeny were obtained from a second backcross to A. rudis. The herbicide-resistance allele from A. hybridus was transmitted to 50% of the BC₁ progeny. The resistance allele was subsequently transmitted to and conferred herbicide resistance in 39 of 110 plants analyzed from four BC₂ families. Parental species, hybrids, and BC₂ progeny were compared for 2C nuclear DNA contents. The mean hybrid 2C nuclear DNA content, 1.27 pg, was equal to the average between A. rudis and A. hybridus, which had 2C DNA contents of 1.42 and 1.12 pg, respectively. The mean 2C DNA content of BC₂ plants, 1.40 pg, was significantly (! < 0.01) less than that of the recurring A. rudis parent and indicated that BC₂ plants were not polyploid. This report demonstrates that herbicide resistance can be acquired by A. rudis through a hybridization event with A. hybridus.     

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     

The carotenoid pigments in the juice and flavedo of a mandarin hybrid (Citrus reticulata) cv michal during ripening

The carotenoid pigments ofthe mandarin hybrid (Citrus reticulata) cv Michal, in the juice and flavedo were characterized at three ripening stages, before, during and after colour break. During ripening the characteristic mandarin pattern was formed in the juice, which contained cryptoxanthin as the principal pigment. In the flavedo the chloroplast carotenoid pattern of the green fruit changed into the characteristic pattern of C. reticulata with a high level of citraurin which, together with cryptoxanthin, imparts an intensive reddish tint to the hybrid. The flavedo contained an unusual C₃₀ apocarotenoid, β-citraurinene (8′-apo-β-caroten-3-ol). The flavedo carotenoids of this accidental hybrid were compared with the carotenoids of the presumed parents Dancy tangerine and Clementine. The hybrid resembles more the second parent, from which it inherited the ability to biosynthesize a higher amount of citraurin as well as citraurinene. Citraurinene, considered a Citrus hybrid-specific pigment, was found for the first time in a Citrus variety. A possible biosynthetic pathway of the Citrus C₃₀ -apocarotenoids is proposed.     

Production of fertile intergeneric somatic hybrids between Brassica napus and Sinapis arvensis for the enrichment of the rapeseed gene pool

Somatic hybrids between Brassica napus (oilseed rape) and its wild relative Sinapis arvensis (Xinjiang wild mustard) from northwestern China were produced by fusing mesophyll protoplasts. Fifty-four plants were identified as symmetric hybrids and four as asymmetric hybrids by random amplified polymorphic DNA analysis and nuclear DNA content. The morphology of investigated 58 hybrid plants resembled characters from both parental species. Highly fertile hybrids were recovered where the fertility was associated with the choice of B. napus genotype. Enhanced disease resistance to Leptosphaeria maculans was found in S. arvensis and in the hybrid offspring. This plant material has great potentials not only for use as a bridge for the introduction of a number of valuable traits from the wild species to Brassica crops but also for breeding new varieties with improved blackleg resistance.     

Phylogenetic relationships within the genus Citrus (Rutaceae) and related genera as revealed by RFLP and RAPD analysis

 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. Received: 30 September 1997 / Accepted: 29 October 1997     

A simple chromosomal marker can reliably distinguishes <Emphasis Type="Italic">Poncirus</Emphasis> from <Emphasis Type="Italic">Citrus</Emphasis> species

Several chromosome types have been recognized in Citrus and related genera by chromomycin A₃ (CMA) banding patterns and fluorescent in situ hybridization (FISH). They can be used to characterize cultivars and species or as markers in hybridization and backcrossing experiments. In the present work, characterization of six cultivars of P. trifoliata (“Barnes”, “Fawcett”, “Flying Dragon”, “Pomeroy”, “Rubidoux”, “USDA”) and one P. trifoliata × C. limonia hybrid was performed by sequential analyses of CMA banding and FISH using 5S and 45S rDNA as probes. All six cultivars showed a similar CMA⁺ banding pattern with the karyotype formula 4B + 8D + 6F. The capital letters indicate chromosomal types: B, a chromosome with one telomeric and one proximal band; D, with only one telomeric band; F, without bands. In situ hybridization labeling was also similar among cultivars. Three chromosome pairs displayed a closely linked set of 5S and 45S rDNA sites, two of them co-located with the proximal band of the B type chromosomes (B/5S-45S) and the third one co-located with the terminal band of a D pair (D/5S-45S). The B/5S-45S chromosome has never been found in any citrus accessions investigated so far. Therefore, this B chromosome can be used as a marker to recognize the intergeneric Poncirus × Citrus hybrids. The intergeneric hybrid analyzed here displayed the karyotype formula 4B + 8D + 6F, with two chromosome types B/5S-45S and two D/5S-45S. The karyotype formula and the presence of two B/5S-45S chromosomes clearly indicate that the plant investigated is a symmetric hybrid. It also demonstrates the suitability of karyotype analyses to differentiate zygotic embryos or somatic cell fusions involving trifoliate orange germplasm. During the submission of this paper, we analyzed 25 other citrus cultivars with the same methodology and we found that the chromosome marker reported here can indeed distinguish Poncirus trifoliata from grapefruits, pummelos, and one variegated access of Citrus, besides the previously reported access of limes, limons, citrons, and sweet-oranges. However, among 14 mandarin cultivars, two of them displayed a single B/5S-45S chromosome, whereas in Citrus hystrix D.C., a far related species belonging to the Papeda subgenus, this chromosome type was found in homozygosis. Since these two mandarin cultivars are probably of hybrid origin, we assume that for almost all commercial cultivars and species of the subgenus Citrus this B type chromosome is a useful genetic marker.     

Cytoplasmic diversity,phylogenetic relationships and molecular evolution of Tunisian Citrus species as inferred from mutational events and pseudogene of chloroplast trnL-trnF spacer

The genus Citrus L. is among the most important fruit trees in the world. In this report, cytoplasmic polymorphism of twenty seven Tunisian Citrus cultivars was explored using the chloroplast trnL-trnF intergenic spacer. Chloroplast sequences showed variation in length and nucleotide content. Haplotype and nucleotide diversity showed low variations. Molecular phylogenetic tree identifies Citrus maternal origins and demonstrates two major groups distinguishing between mandarin and pummelo groups. The trnL-trnF intergenic spacer showed one copy of pseudogene of the original trnF gene in 27 Citrus species at position 275 bp with a size varying from 49 to 63 bp. The anticodon domain was identified as the most conserved element, but one transversion (T−>C) was found in the D-domain. Meanwhile, one transversion (T−>A) and one transition (T−>G) were found in the T-domain. Neutrality tests (Tajima, Fu & Li and Fu) which revealed positive and non-significant values and Pi and θ_W assume a neutral model of evolution and advocated a constant population size. The study demonstrates the resolving power of trnL-trnF sequence data to prove both pummelo and mandarin gene pool’s contribution in the development of Tunisian secondary species and inferring their genetic and phylogenetic relationships.     

Production of additional allotetraploid somatic hybrids combining mandarings and sweet orange with pre-selected pummelos as potential candidates to replace sour orange rootstock

Summary Sour orange (Citrus aurantium L.) rootstock has historically been a widely utilized eitrus rootstock throughout the world due to its wide soil adaptability and superior horticultural performance. However, quick-decline isolates of citrus tristeza virus (CTV) have demolished entire industries of sour orange rootstock in some countries, including Brazil and Venezuela. CTV is presently destroying millions of trees of sour orange rootstock in Florida and threatens the citrus industries of Texas and Mexico, where sour orange is the predominant rootstock. Efforts to replace sour orange rootstock are combining traditional breeding and biotechnology approaches, including somatic hybridization and transformation. Molecular techniques have confirmed that sour orange is probably a hybrid of mandarin and pummelo. A major focus of our program continues to be the somatic hybridization of superior mandarins with pre-selected pummelo parents. Here, we report the regeneration of allotetraploid somatic hybrid plants from seven new mandarin+pummelo combinations and one new sweet orange+pummelo combination. All new somatic hybrids were confirmed by leaf morphology, ploidy analysis via flow cytometry, and random amplified polymorphic DNA analysis to show nuclear contributions from both parents in corresponding hybrids. These new somatic hybrids are being propagated by tissue culture and/or rooted cuttings for further evaluation of disease resistance and horticultural performance in field trials.     

Production of thermostable β-amylase and pullulanase by Clostridium thermosulfurogenes SV2 in solid-state fermentation: Screening of nutrients using Plackett-Burman design

Screening of fifteen nutrients belonging to four categories, viz., carbon, nitrogen, salt and complex organic sources was carried out using Plackett-Burman design for the production of thermostable #-amylase and pullulanase by Clostridium thermosulfurogenes SV2 in solid-state fermentation (SSF). This design involves screening of up to `nу' variables in just `n' number of experiments. Regression co-efficients and t-values were calculated by subjecting the experimental data to statistical analysis. Lactose, diammonium hydrogen phosphate, calcium chloride and casein hydrolysate showed higher regression co-efficients in the biomass formation. Among the fifteen nutrients screened, based on their performance in terms of product promoting ability, availability and cost, magnesium chloride, potato starch, ferrous sulphate, pearl millet flour and corn steep liquor were identified as most effective and, therefore, selected for inclusion in further optimization studies.     

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.