Chromosomal markers distinguish hybrids and non-hybrid accessions of mandarin

Mandarin is the common name of a heterogeneous group of Citrus species with a large range of variation in morphological and molecular characters as well as in number of species. Aiming to identify chromosome markers and to clarify the relationship within this group, the karyotype of 13 mandarin accessions were analyzed using CMA/DAPI staining and in situ hybridization with 5S and 45S rDNA probes. The CMA band pattern together with the position of rDNA sites revealed that mandarins can be separated karyologically into three groups: a) C. sunki and C. reshni; b) the Mediterranean mandarin, C. deliciosa, and the closely related C. tangerina cv. Dancy and C. reticulata cv. Cravo; c) the remaining cultivars, which are cytologically heterozygous and most probably interspecific hybrids. The former two groups are assumed to be pure species together with C. medica and C. grandis. A chromosome marker for mandarin species was identified and the relationship among the pure species and some hybrids is discussed.     

A Numerical Taxonomic Study of Citrus and Its Close Relatives

Fifty nine taxa of Citrus, Fortunella and Poncirus were studied by hierarchical agglomerative clustering analysis and quantas type 3 analysis using 86 morphological characters. Five affinity groups were obtained in Citrus. The first group includes C. medica, C. limon, C. limonia, C. aurantifolia and C. iambhiri; the second includes C. grandis, C. aurantium, C. sinensis and C. paradisi; the third is mandarin (C. reticulata); the forth is C. ichangensis; The last is C. hongheensis. Mandarin oranges show two subgroups: one comprise satsuma, King, Shagan, Tankan and Bendiguangju, and the other consists of the remaining typical mandrins. The two papeda oranges, honghe papeda and ichang papeda, could not be clustered into the same group, indicating that they might have evolved from two compeletly different ancestors. Pumelos(C. grandis) show some relationships to honghe papeda orange. C. limonia is assumed to be a hybrid between rough lemon and mandarin. The sweet orange(C. sinensis) is considered to be the offspring of pomelo and mandarin. Zhekiang jinju a small mandarin generally considered similar to calamondin in China, should be a true mandarin orange based on our results. Poncirus was well distinguished from the other two genera, Citrus and Fortunella, by the numerical methods used in this study. Our results show that citron (C. medica), mandarin (C. reticulata) and pumelo (C. grandis) are the original species of the subgenus Citrus, which was congruent with the re-sults obtained by Barrett and Soost (1976), Potvin (1983) and Handa (1985).     

Cytogenetics of Manihot esculenta Crantz (cassava) and eight related species

Thirty-nine cultivars of cassava and eight related wild species of Manihot were analyzed in this work for number, morphology and size of chromosomes, prophase condensation pattern and the structure of the interphase nucleus. In four accessions, the chromosome size was measured and in some others, the number of secondary constrictions, meiotic behavior, C-band pattern, CMA/DAPI bands, nucleoli number and the location of 5S and 18S-5.8S-28S rDNA sites were also observed. All investigated accessions showed a similar karyotype with 2n = 36, small metacentric to submetacentric chromosomes. Two pairs of terminal secondary constrictions were observed in the chromosome complement of each accession except Manihot sp. 1, which presented two proximal secondary constrictions. The prophase chromosome condensation pattern was proximal and the interphase nuclei structure was areticulate to semi-reticulate. The meiosis, investigated in seven cultivars and four wild species, was regular, displaying 18 bivalents. C-banding revealed heterochromatin in 9 or 10 chromosomes. The analysis with fluorochromes frequently showed four chromosome pairs with a single CMA+ terminal or subterminal band and a few other chromosomes with DAPI+ unstable bands. Six 45S rDNA sites were revealed by FISH, which seemed to colocalize with six CMA+ bands. Only one chromosome pair presented a 5S rDNA site. The maximum nucleoli number observed per nucleus was also six. These data suggest that all Manihot species present a very similar chromosome complement.     

Mapping the chromosomes of Poncirus trifoliata Raf. by BAC-FISH

In spite of the importance of Citrus in agriculture and recent progress in genetic mapping and cytogenetics of this group, chromosome mapping of Citrus species is still limited to rDNA probes. In order to obtain a better chromosome characterization of one species from this group, CMA/DAPI double staining followed by in situ hybridization using 45S rDNA and 24 BACs (BAC-FISH) were used on Poncirus trifoliata. The BACs used were obtained from a genomic library of this species and were selected by membrane hybridization using genomic DNA. Four of them were isolated from the Citrus tristeza virus (Ctv) resistance gene region. The P. trifoliata karyotype is composed of two chromosome pairs with one terminal and one proximal CMA(+) band (B type chromosomes), four chromosome pairs with a single CMA(+) band (D type) and three chromosome pairs without bands (F type). In situ hybridization with 13 of the BACs gave single copy signals on seven chromosome pairs. At least one BAC was mapped on each arm of the two B chromosome pairs. Among the four D chromosome pairs, two were identified by BACs mapped on the long arms, one has a 45S rDNA site and the other had no signal. Six BACs allowed identification of the three F chromosome pairs, with one pair hybridizing with four BACs from the Ctv resistance gene region. In summary, all nine chromosome pairs could be differentiated, seven of them by BAC-FISH, while the other two chromosomes could be recognized by the CMA(+) band pattern and 45S rDNA sites. This first BAC-FISH map gives a general framework for comparative genome structure and evolutionary studies in Citrus and Poncirus, allowing the integration of genetic and physical maps when these BACs are included.     

A Study on Chemical Components of Essential Oils from Citrus bergamia and Its Close Relatives and Its Taxonomy

Citrus bergamia Risso. is a rare perfumery plant. Taxonomists have different views on the taxonomy of C. bergamia. Chemical components of leaf and peel essential oils from C. bergamia, and its close relatives, C. limon, C. aurantifolia and three varieties of C. aurantium, were analyzed by GC and GC-MS. The analytical result shows that the chemical compositions of the leaf essential oils from C. bergamia are basically the same as those from three varieties of C. aurantium. Their main components are linalool (29.19-39.75% )and linalyl acetate (24.73-30.24% )etc., and contents of other components are also similar. But their peel essential oils are different. The peel essential oils from C. bergamia contain less limonene (29.94%) than those from C. aurantium (92.55-94.31% ) and less beta-pinene (3.00%) and y-terpinene(3.48% )than those from C. limon or C. aurantifolia (respectiyely 9.16% and 10.42% ) . The peel essential oils from C. bergamia contain not only as much linalool (22.20%) and linalyl acetate (32.66%)as those in the leaf essential oils from C. aurantium, but also as much limonene(29.94% )as that in the peel essential oils from C. limon or C. aurantifolia . The contents of limonene are close to those of the essential oils from C. aurantifolia. This result shows that C. bergamia may be a natural hybrid between C.aurantium and C. aurantifolia, as proposed by Sinclair W. B.     

The introduction of cultivated citrus to Europe via Northern Africa and the Iberian Peninsula

The circumstances concerning the diffusion of the main cultivated citrus from their places of origin in Asia are studied here, showing that the citron (’Citrus medica L.) was the only one knew in Ancient times in Europe, while the lemon (C. limon [L.] Osbeck), lime (C. aurantiifolia [Christm.] Swingle), pomelo (C. maxima [Burm.] Merr.) and sour orange (C. x aurantium L.) were introduced to Europe by the Muslims via the Iberian Peninsula and Sicily, and that the grapefruit (C. paradisi Macfad.), mandarin (C. reticulata Blanco) and sweet orange (C. x aurantium L.) arrived to the West between the fifteenth and nineteenth centuries as a result of the trade with the British and Portuguese colonies.     

Phylogenetic origin of limes and lemons revealed by cytoplasmic and nuclear markers

Background and Aims The origin of limes and lemons has been a source of conflicting taxonomic opinions. Biochemical studies, numerical taxonomy and recent molecular studies suggested that cultivated Citrus species result from interspecific hybridization between four basic taxa (C. reticulata, C. maxima, C. medica and C. micrantha). However, the origin of most lemons and limes remains controversial or unknown. The aim of this study was to perform extended analyses of the diversity, genetic structure and origin of limes and lemons.Methods The study was based on 133 Citrus accessions. It combined maternal phylogeny studies based on mitochondrial and chloroplastic markers, and nuclear structure analysis based on the evaluation of ploidy level and the use of 123 markers, including 73 basic taxa diagnostic single nucleotide polymorphism (SNP) and indel markers.Key Results The lime and lemon horticultural group appears to be highly polymorphic, with diploid, triploid and tetraploid varieties, and to result from many independent reticulation events which defined the sub-groups. Maternal phylogeny involves four cytoplasmic types out of the six encountered in the Citrus genus. All lime and lemon accessions were highly heterozygous, with interspecific admixture of two, three and even the four ancestral taxa genomes. Molecular polymorphism between varieties of the same sub-group was very low.Conclusions 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. reticulata 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.     

用SSR标记研究柑橘属及其近缘属植物的亲缘关系

用SSR标记分析了29份柑橘属及近缘属植物的亲缘关系。7对SSR引物在29个样品中扩增得到114个等位基因,平均每个位点有16.3个等位基因。计算匹配系数后用邻接法进行聚类,结果表明,澳洲指橘与柑橘属的亲缘关系很近;SSR位点的高纯合频率支持富民枳种的地位;枳与柑橘属的关系较远,枳不大可能是从柑橘属衍生而来;Swingle的亚属的划分以及田中的原生柑橘类和后生柑橘类的划分界线不清晰;现代栽培柑橘的起源与大翼橙关系密切;柑橘属的枸橼、柚和宽皮橘都很好地分离,支持其为现代栽培柑橘的3个基本种的观点。     

Intra- and interspecific chromosome polymorphisms in cultivated Cichorium L. species (Asteraceae)

Endive (Cichorium endivia L.) and chicory (C. intybus L.) both have 2n = 18, but until now, there has been no detailed karyomorphological characterization. The present work evaluated five accessions of each species using FISH with rDNA probes and fluorochrome staining with CMA and DAPI. Both species presented distinct banding patterns after fluorochrome staining: while endive had proximal CMA⁺⁺/DAPI⁻ bands in the short arms of pairs 1, 2 and 3, chicory had proximal CMA-positive bands in chromosomes 1 and 3 and interstitial in the short arm of chromosome 8. Among endive accessions, FISH procedures revealed conserved position and number of 5S and 45S rDNA sites (two and three pairs, respectively), associated with the CMA-positive bands. Notwithstanding, polymorphisms were detected within chicory accessions regarding the number and the distribution of rDNA sites in relation to the most frequent karyotype (two pairs with 45S and one with 5S rDNA). The karyological markers developed allowed karyotypic differentiation between both species, uncovering peculiarities in the number and position of rDNA sites, which suggest chromosome rearrangements, such as translocations in chicory cultivars. The interspecific and intraspecific polymorphisms observed emphasize the potential of karyomorphological evaluations, helping our understanding of the relationships and evolution of the group.     

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.     

Evaluation of Genetic Diversity in Lemons and Some of Their Relatives Based on SRAP and SSR Markers

Genetic diversity was evaluated by sequence-related amplified polymorphism (SRAP) and simple sequence repeat (SSR) markers among 45 lemons (Citrus limon (L.) Burm. f.), five citrons (Citrus medica L.), four rough lemons (Citrus jambhiri Lush), and two Citrus volkameriana accessions. Twenty-one SRAP primer combinations produced a total of 141 (77%) polymorphic fragments with an average of 6.7 fragments per primer combinations whereas 13 SSR primers produced a total of 26 (76%) polymorphic fragments with an average of 2.0 per primer. The unweighted pair-group method arithmetic average analysis as assessed with combined SRAP and SSR data demonstrated that the accessions had a similarity range from 0.65 to 1.00. Rough lemons and C. volkameriana accessions were relatively closely related. In lemon group, accessions from hybrid origin were distant from the others. We also applied principal components analysis (PCA) for a better presentation of relation among the accessions studies. Using PCA, 88.7% of the total variation in the original dimensions could be represented by just the two dimensions defined by the first two PCs. Although nearly all accessions could be distinguished, there was a low level of genetic diversity detected among lemon cultivars.     

Development of SSR markers from Citrus clementina (Rutaceae) BAC end sequences and interspecific transferability in Citrus

? Premise of the study: Microsatellite primers were developed from bacterial artificial chromosome (BAC) end sequences of Citrus clementina and their transferability and polymorphism tested in the genus Citrus for future anchorage of physical and genetic maps and comparative interspecific genetic mapping. ? Methods and Results: Using PAGE and DNA silver staining, 79 primer pairs were selected for their transferability and polymorphism among 526 microsatellites mined in BES. A preliminary diversity study in Citrus was conducted with 18 of them, in C. reticulata, C. maxima, C. medica, C. sinensis, C. aurantium, C. paradisi, C. lemon, C. aurantifolia, and some papedas (wild citrus), using a capillary electrophoresis fragment analyzer. Intra- and interspecific polymorphism was observed, and heterozygous markers were identified for the different genotypes to be used for genetic mapping. ? Conclusions: These results indicate the utility of the developed primers for comparative mapping studies and the integration of physical and genetic maps.     

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.     

ZZ/ZW sex chromosome system in an undescribed species of the genus Apareiodon (Characiformes, Parodontidae)

The chromosomes of an undescribed species of the genus Apareiodon (Characiformes, Parodontidae) from the Verde River, a headwater affluent of the Tibagi River (Paraná State, Brazil), were investigated using conventional Giemsa and Ag stainings, C-banding, CMA(3) fluorescence and fluorescent in situ hybridization (FISH) using 18S and 5S rDNA probes. The diploid chromosome number was 2n = 54, with the karyotype composed of 48 meta/submetacentric and six subtelocentric chromosomes in males, and 47 meta/submetacentric + seven subtelocentric chromosomes in females. The difference is hypothesized to be due to a ZZ/ZW heteromorphic sex chromosome system, a cytotaxonomic characteristic previously observed only in some species of the genus Parodon (family Parodontidae). The presence of similar and/or identical heteromorphic sex chromosome systems might suggest that species of the genera Parodon and Apareiodon bearing ZZ/ZW heteromorphic sex chromosomes likely constitute a monophyletic group, a hypothesis to be tested by a robust phylogeny of the family.     

Oranges and lemons: clues to the taxonomy of Citrus from molecular markers

Go into any grocery store and one is confronted with an array of Citrus fruit: oranges, grapefruit, mandarins (tangerines), lemons and limes. This is rich bounty for the shopper, but taxonomists are perplexed as to how to classify the various kinds of Citrus that have existed since antiquity. Now, thanks to new genetic and molecular biological techniques, the relationships between these fruit are being unraveled and show that there are probably only three true species.     

无量山种子植物的区系平衡点

在较为完善的分类学处理后所得的包含207科,1026属,2540种植物的名录基础上,本文初步报道了地处滇中南的无量山的种子植物区系组成的初步结果,并在山体各海拔段进行了属的分布区类型每一类分布的详尽统计,得出了热带和温带两大基本成分在无量山达到平衡（各占50％）海拔点,称之为区系平衡点。该点从历史角度看可以反映无量山种子植物区系成分随自然历史变迁所发生的演变,从现实看表明了该地植物区系的过渡性质,同时,寓意着我国亚热带地区客观存在着一条区系平衡线,并且,该点还是热带成分的一个重要性限制点;另一方面可以作为植物引种和选择引种驯化锻炼基地的一个参考指标。     

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.     

The karyotype of Nothoscordum arenarium Herter (Gilliesioideae, Alliaceae): A populational and cytomolecular analysis

The genus Nothoscordum Kunth comprises approximately 20 species native to South America. Karyologically, the genus is remarkable for its large chromosomes and Robertsonian translocations. Variation in chromosome number has been recorded in a few polyploid species and it is unknown among diploids. This study presents the chromosome number and morphology of 53 individuals of seven populations of N. arenarium Herter (2n = 10). In addition, karyotype analyses after C-banding, staining with CMA and DAPI, and in situ hybridization with 5S and 45S rDNA probes were performed in six individuals from one population. All individuals exhibited 2n = 10 (6M + 4A), except for one tetraploid (2n = 20, 12M + 8A) and one triploid (2n = 15, 9M + 6A) plant. C-banding revealed the presence of CMA(+) /DAPI (-) heterochromatin in the short arm and in the proximal region of the long arm of all acrocentric chromosomes. The 45S rDNA sites co-localized with the CMA (+) regions of the acrocentrics short arms, while the 5S rDNA probe only hybridized with the subterminal region of a pair of metacentric chromosomes. A change in the pattern of CMA bands and rDNA sites was observed in only one individual bearing a reciprocal translocation involving the long arm of a metacentric and the long arm of an acrocentric chromosome. These data suggest that, despite isolated cases of polyploidy and translocation, the karyotype of N. arenarium is very stable and the karyotypic instability described for other species may be associated with their polyploid condition.