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.     

Microdissection and molecular manipulation of single chromosomes in woody fruit trees with small chromosomes using pomelo (<Emphasis Type="Italic">Citrus grandis</Emphasis>) as a model. I. Construction of single chromosomal DNA libraries

Construction of single chromosomal DNA libraries by means of chromosome microdissection and microcloning will be useful for genomic research, especially for those species that have not been extensively studied genetically. Application of the technology of microdissection and microcloning to woody fruit plants has not been reported hitherto, largely due to the generally small sizes of metaphase chromosomes and the difficulty of chromosome preparation. The present study was performed to establish a method for single chromosome microdissection and microcloning in woody fruit species using pomelo as a model. The standard karyotype of a pomelo cultivar (Citrus grandis cv. Guanxi) was established based on 20 prometaphase photomicrographs. According to the standard karyotype, chromosome 1 was identified and isolated with fine glass microneedles controlled by a micromanipulator. DNA fragments ranging from 0.3 kb to 2 kb were acquired from the isolated single chromosome 1 via two rounds of PCR mediated by Sau3A linker adaptors and then cloned into T-easy vectors to generate a DNA library of chromosome 1. Approximately 30,000 recombinant clones were obtained. Evaluation based on 108 randomly selected clones showed that the sizes of the cloned inserts varied from 0.5 kb to 1.5 kb with an average of 860 bp. Our research suggests that microdissection and microcloning of single small chromosomes in woody plants is feasible.Communicated by P. Langridge     

Using chromosomal data in the phylogenetic and molecular dating framework: karyotype evolution and diversification in Nierembergia (Solanaceae) influenced by historical changes in sea level

Karyotype data within a phylogenetic framework and molecular dating were used to examine chromosome evolution in Nierembergia and to infer how geological or climatic processes have influenced in the diversification of this solanaceous genus native to South America and Mexico. Despite the numerous studies comparing karyotype features across species, including the use of molecular phylogenies, to date relatively few studies have used formal comparative methods to elucidate chromosomal evolution, especially to reconstruct the whole ancestral karyotypes. Here, we mapped on the Nierembergia phylogeny one complete set of chromosomal data obtained by conventional staining, AgNOR‐, C‐ and fluorescent chromosome banding, and fluorescent in situ hybridisation. In addition, we used a Bayesian molecular relaxed clock to estimate divergence times between species. Nierembergia showed two major divergent clades: a mountainous species group with symmetrical karyotypes, large chromosomes, only one nucleolar organising region (NOR) and without centromeric heterochromatin, and a lowland species group with asymmetrical karyotypes, small chromosomes, two chromosomes pairs with NORs and centromeric heterochromatin bands. Molecular dating on the DNA phylogeny revealed that both groups diverged during Late Miocene, when Atlantic marine ingressions, called the ‘Paranense Sea’, probably forced the ancestors of these species to find refuge in unflooded areas for about 2 Myr. This split agrees with an increased asymmetry and heterochromatin amount, and decrease in karyotype length and chromosome size. Thus, when the two Nierembergia ancestral lineages were isolated, major divergences occurred in chromosomal evolution, and then each lineage underwent speciation separately, with relatively minor changes in chromosomal characteristics.     

Flow cytometric analysis of the chromosomes and stability of a wheat cell-culture line

A rapidly growing, long-term suspension culture derived from Triticum aestivum L. (wheat) was synchronized using hydroxyurea and colchicine, and a chromosome suspension with chromosomes was made. After staining with the DNA-specific fluorochromes Hoechst 33258 and Chromomycin univariate and bivariate flow-cytometry histograms showed 15 clearly resolved peaks corresponding to individual chromosome types or groups of chromosomes with similar DNA contents. The flow karyotype was closely similar to a histogram of DNA content measurements of Feulgen-stained chromosomes made by microdensitometry. We were able to show the stability of the flow karyotype of the cell line over a year, while a parallel subculture had a slightly different, stable, karyotype following different growth conditions. The data indicate that flow cytometric analysis of plant karyotypes enables accurate, statistically precise chromosome classification and karyotyping of cereals. There was little overlap between individual flow-histogram peaks, so the method is useful for flow sorting and the construction of chromosome specific-recombinant DNA libraries. Using bivariate analysis, the AT:GC ratio of all the chromosomes was remarkably similar, in striking contrast to mammalian flow karyotypes. We speculate about a fundamental difference in organization and homogenization of DNA sequences between chromosomes within mammalian and plant genomes. Received: 24 April 1996 / Accepted: 24 May 1996     

Homologies in human and Macasa fuscata chromosomes revealed by in situ suppression hybridization with human chromosome specific DNA libraries

We established chromosomal homologies between all chromosomes of the human karyotype and that of an old world monkey (Macaca fuscata) by chromosomal in situ suppression (CISS) hybridization with human chromosome specific DNA libraries. Except for the human chromosome 2 library and limited cross-hybridization of X and Y chromosome libraries all human DNA libraries hybridized to single GTG-banded macaque chromosomes. Only three macaque chromosomes (2, 7, 13) were each hybridized by two separate human libraries (7 and 21, 14 and 15, 20 and 22 respectively). Thus, an unequivocally high degree of synteny between human and macaque chromosomes has been maintained for more than 20 million years. As previously suggested, both Papionini (macaques, baboons, mandrills and cercocebus monkeys, all of which have nearly identical karyotypes) and humans are chromosomally conservative. The results suggest, that CISS hybridization can be expected to become an indispensable tool in comparative chromosome and gene mapping and will help clarify chromosomal phylogenies with speed and accuracy.by E.R. Schmidt     

Chromosomal analysis of twelve species of Microhylidae (Anura) from Madagascar

The karyotypes of four species of Dyscophinae and eight species of Cophylinae were analyzed. The chromosome number was 2n=26 in all cases. Between the two subfamilies a difference in the form of the karyotype was observed; the chromosomes show a gradual decrease in length in the Dyscophinae, whereas in the Cophylinae the karyotype demonstrates a clear discontinuity of size between pairs 5 and 6.Chromosomal polymorphism was found in Plethodontohyla tuberata, the chromosomes of pair 4 were subtelocentric in the homozygous specimens, whereas this pair showed a subtelocentric and a submetacentric chromosome of equal length in the heterozygous one, suggesting a pericentric inversion. Although in the Cophylinae the chromosome number is constant, the number of chromosome arms is variable. Pericentric inversions seem to play an important role in the chromosomal evolution of the Cophylinae.     

Genome-wide Identification and Characterization of a Dehydrin Gene Family in Poplar (Populus trichocarpa)

Dehydrins (DHNs) define a complex group of stress inducible proteins characterized by the presence of one or more lysine-rich motifs. DHNs are present in multiple copies in the genome of plant species. Although genome-wide analysis of DHNs composition and chromosomal distribution has been conducted in herbaceous species, it remains unexplored in woody plants. Here, we report on the identification of ten genes encoding eleven putative DHN polypeptides in Populus. We document that DHN genes occur as duplicated blocks distributed over seven of the 19 poplar chromosomes likely as a result of segmental and tandem duplication events. Based on conserved motifs, poplar DHNs were assigned to four subgroups with the K_n subgroup being the most frequent. One putative DHN polypeptide (PtrDHN-10) with a SKS arrangement could originate from a recombination between SK_n and K_nS genes. In silico analysis of microarray data showed that in unstressed poplar, DHN genes are expressed in all vegetative tissues except for mature leaves. This exhaustive survey of DHN genes in poplar provides important information that will assist future studies on their functional role in poplar.     

The chromosomes of Lepturinae (Coleoptera: Cerambycidae) II. A study of eight more species,with focus on Desmocerus palliatus

Following the study of 28 species of Lepturinae (Coleoptera: Cerambycidae) the karyotypes of seven additional Palaearctic and one Nearctic species are established. The 19,X male karyotypes found in genera Stictoleptura (four species), Vadonia and Judolia (one species each) confirm the loss of Y chromosome in Lepturini. The 22,XY male karyotype of Cortodera humeralis is very close to that of some species of Rhagiini (genera Gaurotes, Acmaeops, Dinoptera, all 22,XY) and Grammoptera ruficornis (24,XY) recently reported. We propose that these taxa could form a monophyletic group within Rhagiini. The karyotype of the Nearctic species Desmocerus palliates (23,neoXneoXneoY) is quite different and characterized by the presence of many acrocentric chromosomes and a complex autosome–gonosome translocation. Its particular karyotype is compatible with its present classification within a separate tribe, the Desmocerini.     

The Giemsa banding patterns of the standard and four reconstructed karyotypes of Vicia faba

The Giemsa banding patterns of the standard karyotype of Vicia faba and of four new karyotypes with easily interdistinguishable chromosomes due to interchanges and inversions are described and compared with the data of other authors on preferential Giemsa staining in Vicia faba. All karyotypes contain 14 easily reproducible marker bands which characterize chromosome segments known to be heterochromatic. It is shown that the preferential Giemsa staining of chromosome regions is a valuable tool for the localization of translocation and inversion points in the chromosomes of the reconstructed Vicia karyotypes. A close correlation exists between banding patterns, segment extension by incorporation into chromosomal DNA of azacytidine and mutagen-specific clustering of induced chromatid aberrations in the new karyotypes.     

KARYOTYPIC STUDIES IN SOLANUM SECTION BASARTHRUM (SOLANACEAE)

Mitotic chromosomes of 450 individuals, representing 59 accessions of 18 of the 22 species of Solanum sect. Basarthrum were studied. Statistical analyses of chromosome length, genome length, and centromere position yielded estimates of karyotype composition and asymmetry. A generalized karyotype of the section shows that most of the chromosomes are metacentric (44%) or submetacentric (53%). A general trend emerged: the greater the total genome length, the more asymmetric the karyotype. However, karyotype asymmetry does not seem to be based on major chromosomal rearrangements. There is variation in species groups in the karyotype formula, and chromosome length differences were useful in distinguishing series. Patterns of cytological variation reinforce the systematic arrangement of the taxa based on morphological, chemical, and molecular studies. Chromosomal differences characterize most of the species. Five species bear subtelocentric chromosomes, two have telocentrics, and two have satellites. The genomes of the staminate and pistillate plants of the dioecious S. appendiculatum are identical. Of the species proposed as progenitors of the domesticated cultigen S. muricatum, S. caripense is the most similar karyotypically.     

Multi-directional chromosome painting maps homologies between species belonging to three genera of New World monkeys and humans

We mapped chromosomal homologies in two species of Chiropotes (Pitheciini, Saki Monkeys) and one species of Aotus (Aotinae, Owl Monkey) by multi-directional chromosome painting. Human chromosome probes were hybridized to Chiropotes utahicki, C. israelita and Aotus nancymae metaphases. Wooly Monkey chromosome paints were also hybridized to Owl Monkey metaphases. We established Owl Monkey chromosome paint probes by flow sorting and reciprocally hybridized them to human chromosomes. The karyotypes of the Bearded Saki Monkeys studied here are close to the hypothesized ancestral platyrrhine karytoype, while that of the Owl Monkey appears to be highly derived. The A. nancymae karyotype is highly shuffled and only three human syntenic groups were found conserved coexisting with 17 derived human homologous associations. A minimum of 14 fissions and 13 fusions would be required to derive the A. nancymae karyotype from that of the ancestral New World primate karyotype. An inversion between homologs to segments of human 10 and 16 suggests a link between Callicebus and Chiropotes, while the syntenic association of 10/11 found in Aotus and Callicebus suggests a link between these two genera. Future molecular cytogenetic work will be needed to determine whether these rearrangements represent synapomorphic chromosomal traits.     

Chromosomal fission accounts for small‐scale radiations in Zamia (Zamiaceae; Cycadales)

Zamia is unique among Cycadales in its diversity of morphology, ecology and chromosome numbers. The chromosome numbers in Zamia range from 16 to 28, excluding 20, manifest as both interspecific and intraspecific series. It has long been recognized that Robertsonian transformations (chromosomal fission or fusion) probably dominate karyotype evolution in Zamiaceae, although it has been debated whether chromosome numbers are increasing or decreasing. We re‐analyse published karyotypes of Zamia spp., relating both chromosome forms and sizes to recent phylogenetic data. We show that karyotype evolution is most probably moving towards increased asymmetry, with higher numbers of smaller chromosomes, thus supporting chromosomal fission. We also address additional hypotheses for increasing chromosome numbers, namely pericentric inversions and unequal translocations. Finally, we discuss the role of these chromosomal changes in evolutionary radiations. © 2011 The Linnean Society of London, Botanical Journal of the Linnean Society, 2011, 165 , 168–185.     

KARYOTYPES OF IRIS PUMILA AND RELATED SPECIES

Randolph , L. F. and Jyotirmay Mitra . (Cornell U., Ithaca.) Karyotypes of Iris pumila and related species. Amer. Jour. Bot. 46(2): 93-102. Illus. 1959.—The karyotypes of 30- and 32-chromosome geographical variants of the amphidiploid I. pumila from Russia and the Balkans were compared with the karyotype of the typical 32-chromosome Austrian forms of this species and with those of the diploid I. attica and I. pseudopumila, previously reported to be the basic species from which I. pumila originated. Plants from 3 collections of a Crimean form of I. pumila with 32 chromosomes had a pair of long chromosomes with submedian centromeres morphologically similar to chromosome 1 of the typical form of I. pumila. In addition, there was another heteromorphic pair of submedian chromosomes with one of the members having a shorter short arm. The manner in which this altered chromosome could have arisen as a result of a heterobrachial inversion is described. Five different collections of I. pumila with 30 chromosomes from Russia differ in several respects from the typical 32-chromosome I. pumila. They have an unusually long pair of chromosomes with a submedian centromere and a secondary constriction in the long arm. This chromosome is the original chromosome 2 which had been altered by the addition of a segment equivalent to the most of the long arm of one of the shorter chromosomes with subterminal centromere. The manner in which this could have occurred as the result of unequal reciprocal translocation is described. Loss of the remaining diminutive portion of the short chromosome with subterminal constriction assumed to have been involved in the unequal interchange of segments producing the modified, longer chromosome 2 would account for the reduction in chromosome number from 32 to 30 in the Russian form of I. pumila. Four pairs of chromosomes with satellites have been found in the 30-chromosome plants whereas 6 pairs of satellited chromosomes are present in the 32-chromosome I. pumila. The spontaneous occurrence of chromosomal alterations of the type here described are considered to be significant factors in the process of chromosomal repatterning resulting in the appearance of new geographical races, and eventually of species of iris, with altered chromosome numbers and modified karyotypes. More specifically it is concluded that amphidiploidy accompanied by chromosomal repatterning resulting from segmental interchange, heterobrachial inversion and related types of chromosomal alterations has played an important role in the evolution of I. pumila and karyological forms of this species occupying different geographical areas.     

国产黄连属植物的染色体核型分析

中国黄连属6种1变种分属叶掌状三全裂和叶掌状五全裂2个类群,前者包括三角叶黄连、峨眉黄连、云南黄连、黄连和其变种短萼黄连;而分布于滇东南的五裂黄连和产于台湾的五叶黄连则归于后者。采用根尖压片法和卡宝品红染色法,对除五叶黄连外的中国黄连属5种1变种,以及日本黄连进行染色体核型比较分析,从细胞学角度为探讨中国黄连属植物的系统分类提供新的线索和证据。结果表明:(1)五裂黄连(2n=2x=18=2M+16m)、短萼黄连(2n=2x=18=8m+10sm)和日本黄连(2n=2x=18=12m+6sm)的染色体数目和核型均为首次报道。(2)7个材料的染色体基数均为9,除三角叶黄连为三倍体外,其余均为二倍体。(3)叶为掌状三全裂的二倍体种核型一致,为2A型,染色体类型以及不对称系数均很相似;叶为掌状五全裂的五裂黄连与五叶黄连的核型更接近,为1A型,核型特征的共性表明了这2个类群的自然属性。(4)三倍体三角叶黄连的不对称程度较高,核型为3A型,其染色体大小与峨眉黄连最接近。(5)根据核型不对称程度和染色体大小,结合地理分布,推测叶掌状五全裂种为本属的原始类群。     

Chromosomal phylogeny and karyotype evolution in x=7 crucifer species (Brassicaceae)

Karyotype evolution in species with identical chromosome number but belonging to distinct phylogenetic clades is a long-standing question of plant biology, intractable by conventional cytogenetic techniques. Here, we apply comparative chromosome painting (CCP) to reconstruct karyotype evolution in eight species with x=7 (2n=14, 28) chromosomes from six Brassicaceae tribes. CCP data allowed us to reconstruct an ancestral Proto-Calepineae Karyotype (PCK; n=7) shared by all x=7 species analyzed. The PCK has been preserved in the tribes Calepineae, Conringieae, and Noccaeeae, whereas karyotypes of Eutremeae, Isatideae, and Sisymbrieae are characterized by an additional translocation. The inferred chromosomal phylogeny provided compelling evidence for a monophyletic origin of the x=7 tribes. Moreover, chromosomal data along with previously published gene phylogenies strongly suggest the PCK to represent an ancestral karyotype of the tribe Brassiceae prior to its tribe-specific whole-genome triplication. As the PCK shares five chromosomes and conserved associations of genomic blocks with the putative Ancestral Crucifer Karyotype (n=8) of crucifer Lineage I, we propose that both karyotypes descended from a common ancestor. A tentative origin of the PCK via chromosome number reduction from n=8 to n=7 is outlined. Comparative chromosome maps of two important model species, Noccaea caerulescens and Thellungiella halophila, and complete karyotypes of two purported autotetraploid Calepineae species (2n=4x=28) were reconstructed by CCP.     

Comparative cytogenetics of six Indo‐Pacific moray eels (Anguilliformes: Muraenidae) by chromosomal banding and fluorescence in situ hybridization

A comparative cytogenetic analysis, using both conventional staining techniques and fluorescence in situ hybridization, of six Indo‐Pacific moray eels from three different genera (Gymnothorax fimbriatus, Gymnothorax flavimarginatus, Gymnothorax javanicus, Gymnothorax undulatus, Echidna nebulosa and Gymnomuraena zebra), was carried out to investigate the chromosomal differentiation in the family Muraenidae. Four species displayed a diploid chromosome number 2n = 42, which is common among the Muraenidae. Two other species, G. javanicus and G. flavimarginatus, were characterized by different chromosome numbers (2n = 40 and 2n = 36). For most species, a large amount of constitutive heterochromatin was detected in the chromosomes, with species‐specific C‐banding patterns that enabled pairing of the homologous chromosomes. In all species, the major ribosomal genes were localized in the guanine‐cytosine‐rich region of one chromosome pair, but in different chromosomal locations. The (TTAGGG)_n telomeric sequences were mapped onto chromosomal ends in all muraenid species studied. The comparison of the results derived from this study with those available in the literature confirms a substantial conservation of the diploid chromosome number in the Muraenidae and supports the hypothesis that rearrangements have occurred that have diversified their karyotypes. Furthermore, the finding of two species with different diploid chromosome numbers suggests that additional chromosomal rearrangements, such as Robertsonian fusions, have occurred in the karyotype evolution of the Muraenidae.     

Karyotype and genome size variation in Ajuga L. (Ajugoideae–Lamiaceae)

Chromosome number changes and karyotype evolution play an important role in plant genome diversification and eventually in speciation. The genus Ajuga L. (Lamiaceae) has approximately 50 species distributed in temperate to subtropical regions. Four of these species are currently recognized in Korea (A. decumbens Thunb., A. multiflora Bunge, A. nipponensis Makino and A. spectabilis Nakai). Understanding the karyotype evolution in Ajuga has been hampered by the small size of their chromosomes and symmetrical karyotypes. Here we used classic Feulgen staining to establish chromosome numbers and construct karyotypes of the four species of Ajuga recognized in Korea and flow cytometry was used to study their variation in genome. The chromosome number of all investigated plants was 2n = 32. Still, the 2C DNA content ranged from 2.18 pg (A. decumbens) to 4.53 pg (A. multiflora). While the chromosome numbers were the same for all investigated species, the genome size variation could potentially be used as a taxonomic marker.     

Cytological study of Tibetia (Fabaceae) in the Hengduan Mountains region, China

The Hengduan Mountains comprise one of the world's most important hot spots of biodiversity. Tibetia (Ali) H.P. Tsui (Fabaceae), which has four or five species in two sections, is one of the genera endemic to the region. This paper describes for the first time the karyotype of three of those species. The chromosome counts of all three are 2n = 16. The karyotypes of the species examined contain chromosomes of variable karyotypic symmetry with centromeres at median and submedian positions that correlate with the morphological characteristics of the species. Karyotypic variation at the diploid level appears to be the predominant feature of chromosome evolution in the genus and may provide a clue to the study of evolutionary patterns of plants in this region. Received: June 12, 2001 / Accepted: September 12, 2001     

Fissions,fusions, and translocations shaped the karyotype and multiple sex chromosome constitution of the northeast‐Asian wood white butterfly,Leptidea amurensis

Previous studies have shown a dynamic karyotype evolution and the presence of complex sex chromosome systems in three cryptic Leptidea species from the Western Palearctic. To further explore the chromosomal particularities of Leptidea butterflies, we examined the karyotype of an Eastern Palearctic species, Leptidea amurensis. We found a high number of chromosomes that differed between the sexes and slightly varied in females (i.e. 2n = 118–119 in females and 2n = 122 in males). The analysis of female meiotic chromosomes revealed multiple sex chromosomes with three W and six Z chromosomes. The curious sex chromosome constitution [i.e. W_1–3/Z_1–6 (females) and Z_1–6/Z_1–6 (males)] and the observed heterozygotes for a chromosomal fusion are together responsible for the sex‐specific and intraspecific variability in chromosome numbers. However, in contrast to the Western Palearctic Leptidea species, the single chromosomal fusion and static distribution of cytogenetic markers (18S rDNA and H3 histone genes) suggest that the karyotype of L. amurensis is stable. The data obtained for four Leptidea species suggest that the multiple sex chromosome system, although different among species, is a common feature of the genus Leptidea. Furthermore, inter‐ and intraspecific variations in chromosome numbers and the complex meiotic pairing of these multiple sex chromosomes indicate the role of chromosomal fissions, fusions, and translocations in the karyotype evolution of Leptidea butterflies.