Delimiting the Origin of a B Chromosome by FISH Mapping,Chromosome Painting and DNA Sequence Analysis in Astyanax paranae (Teleostei,Characiformes)

Supernumerary (B) chromosomes have been shown to contain a wide variety of repetitive sequences. For this reason, fluorescent in situ hybridisation (FISH) is a useful tool for ascertaining the origin of these genomic elements, especially when combined with painting from microdissected B chromosomes. In order to investigate the origin of B chromosomes in the fish species Astyanax paranae, these two approaches were used along with PCR amplification of specific DNA sequences obtained from the B chromosomes and its comparison with those residing in the A chromosomes. Remarkably, chromosome painting with the one-arm metacentric B chromosome probe showed hybridization signals on entire B chromosome, while FISH mapping revealed the presence of H1 histone and 18S rDNA genes symmetrically placed in both arms of the B chromosome. These results support the hypothesis that the B chromosome of A. paranae is an isochromosome. Additionally, the chromosome pairs Nos. 2 or 23 are considered the possible B chromosome ancestors since both contain syntenic H1 and 18S rRNA sequences. The analysis of DNA sequence fragments of the histone and rRNA genes obtained from the microdissected B chromosomes showed high similarity with those obtained from 0B individuals, which supports the intraspecific origin of B chromosomes in A. paranae. Finally, the population hereby analysed showed a female-biased B chromosome presence suggesting that B chromosomes in this species could influence sex determinism.     

B chromosome ancestry revealed by histone genes in the migratory locust

In addition to the standard set of chromosomes (A), about 15% of eukaryote genomes carry B chromosomes. In most cases, B chromosomes behave as genomic parasites being detrimental for the individuals carrying them and prospering in natural populations because of transmission advantages (drive). B chromosomes are mostly made up of repetitive DNA sequences, especially ribosomal DNA (rDNA), satellite DNA and mobile elements. In only two cases have B chromosomes been shown to carry protein-coding genes. Although some B chromosomes seem to have derived from interspecific hybridisation, the most likely source of B chromosomes is the host genome itself, but the specific A chromosome being the B ancestor has not been identified in any B-containing species. Here, we provide strong evidence for B chromosome ancestry in the migratory locust, based on the location of genes for the H3 and H4 histones in the B chromosome and a single A chromosome pair (i.e. the eighth in order of decreasing size). The high DNA sequence similarity of A and B chromosome H3–H4 genes supports B-origin from chromosome 8. The higher variation shown by B sequences, compared to A sequences, suggests that B chromosome sequences are most likely inactive and thus less subjected to purifying selection. Estimates of time of divergence for histone genes from A and B chromosomes suggest that B chromosomes are quite old (>750,000 years), showing the B-chromosome ability to persist in natural populations for long periods of time.     

B Chromosome Polymorphism in the Fish, Astyanax scabripinnis

Four populations of Astyanax scabripinnis (Pisces, Characidae) were analyzed for B chromosome features. This species contains a metacentric macrochromosome (B_M), similar in size to the first chromosome of the karyotype, besides two variant forms, a large submetacentric (B_SM) and a small metacentric (B_m), both showing a reduced frequency. These variant forms were observed only in the populations from the Campos do Jordão region (São Paulo State, Brazil), although not present in all the populations sampled. The three B chromosome forms are heterochromatic and at least the B_M and B_SM are also GC-rich, as evidenced by C-banding and chromomycin A₃ staining. In spite of the morphological similarity between the B_M form and the first chromosome pair, they differ in the G-banding pattern, which does not favor a possible relationship among these chromosomes. FISH with a telomeric DNA probe evidenced signals on the terminal region of all chromosomes, including the Bs. Interstitial telomeric bands, indicative of some chromosomal rearrangements such as translocation or tandem fusion in the origin of the B chromosomes, were not observed. B_SM and B_m are probable derivative B chromosome forms from an ancestral B_M chromosome, showing a restricted occurrence and low frequency in the populations.     

Satellite DNA content illuminates the ancestry of a supernumerary (B) chromosome

B chromosomes are supernumerary genomic elements most likely derived from the standard (A) chromosomes, whose dispensability has freed their DNA sequences to evolve fast, thus making it difficult to uncover their ancestry. Here, we show the ancestry of a B chromosome in the grasshopper Eumigus monticola by means of the high-throughput analysis of the satellitome, i.e., the whole collection of satellite DNA (satDNA). The satellitome found in this species consists of 27 satDNA families, with monomer length between 5 and 325 nt and A + T content between 42.9 and 83.3 %. Two out of the 20 clustered satDNA families (EmoSat26–41 and EmoSat27–102) were observed only on the B chromosome. The A chromosome carrying the highest number of satDNA families was the megameric S8 (13 families), six of which were also present in the B chromosome, and three of these were exclusive of the S8 and B chromosomes. The absence in the B chromosome of the H3 histone gene cluster (located interstitially on S8) and three satDNA families (located distally on S8) allowed delimiting the possible origin of the B chromosome to the proximal third of the S8 autosome, through a breakpoint between EmoSat11–122 and the H3 cluster. Interestingly, bioinformatic analysis revealed the presence of seeds for the two B-specific satDNAs in the A chromosomes, suggesting their massive amplification in the B chromosome after its origin. Therefore, intraspecifically arisen B chromosomes can harbor DNA sequences apparently being B-specific.     

Satellite DNA and chromosomes in Neotropical fishes: methods,applications and perspectives

Constitutive heterochromatin represents a substantial portion of the eukaryote genome, and it is mainly composed of tandemly repeated DNA sequences, such as satellite DNAs, which are also enriched by other dispersed repeated elements, including transposons. Studies on the organization, structure, composition and in situ localization of satellite DNAs have led to consistent advances in the understanding of the genome evolution of species, with a particular focus on heterochromatic domains, the diversification of heteromorphic sex chromosomes and the origin and maintenance of B chromosomes. Satellite DNAs can be chromosome specific or species specific, or they can characterize different species from a genus, family or even representatives of a given order. In some cases, the presence of these repeated elements in members of a single clade has enabled inferences of a phylogenetic nature. Genomic DNA restriction, using specific enzymes, is the most frequently used method for isolating satellite DNAs. Recent methods such as C₀t–1 DNA and chromosome microdissection, however, have proven to be efficient alternatives for the study of this class of DNA. Neotropical ichthyofauna is extremely rich and diverse enabling multiple approaches with regard to the differentiation and evolution of the genome. Genome components of some species and genera have been isolated, mapped and correlated with possible functions and structures of the chromosomes. The 5SHindIII‐DNA satellite DNA, which is specific to Hoplias malabaricus of the Erythrinidae family, has an exclusively centromeric location. The As51 satellite DNA, which is closely correlated with the genome diversification of some species from the genus Astyanax, has also been used to infer relationships between species. In the Prochilodontidae family, two repetitive DNA sequences were mapped on the chromosomes, and the SATH 1 satellite DNA is associated with the origin of heterochromatic B chromosomes in Prochilodus lineatus. Among species of the genus Characidium and the Parodontidae family, amplifications of satellite DNAs have demonstrated that these sequences are related to the differentiation of heteromorphic sex chromosomes. The possible elimination of satellite DNA units could explain the genome compaction that occurs among some species of Neotropical Tetraodontiformes. These topics are discussed in the present review, showing the importance of satellite DNA analysis in the differentiation and karyotype evolution of Actinopterygii.     

A single, recent origin of the accessory B chromosome of the grasshopper Eyprepocnemis plorans

B chromosomes are dispensable chromosomes found in >2000 eukaryotic species, usually behaving as genomic parasites. Most B chromosomes seem to be made up of the same kind of DNA sequences present in the A chromosomes. This sequence similarity makes it difficult to obtain specific molecular probes that may permit B-presence diagnosis without cytogenetic analysis. We have developed a sequence-characterized amplified region (SCAR) marker for B chromosomes in the grasshopper Eyprepocnemis plorans, which specifically amplifies a 1510-bp DNA fragment exclusively in B-carrying individuals. Fluorescent in situ hybridization and fiber FISH analyses showed that this marker is a tandemly repeated DNA sequence closely intermingled with 45S rDNA. PCR reactions showed the presence of SCAR-like sequences in the A chromosomes, but in two separate fragments, supporting the intraspecific origin of B chromosomes in this species. SCAR marker DNA sequence showed to be identical in B chromosome variants from several localities from Spain and Morocco, and it was very similar to those found in B chromosome variants from Greece and Armenia. This strongly suggests that this sequence was already present in the ancestral B chromosome of this species. In addition, the scarce sequence variation observed among several B variants from very distant populations suggests either a functional constraint or, more likely, a recent and unique origin for B chromosomes in this species.     

Chromosomal Mapping of Repetitive DNAs in the Grasshopper Abracris flavolineata Reveal Possible Ancestry of the B Chromosome and H3 Histone Spreading

Supernumerary chromosomes (B chromosomes) occur in approximately 15% of eukaryote species. Although these chromosomes have been extensively studied, knowledge concerning their specific molecular composition is lacking in most cases. The accumulation of repetitive DNAs is one remarkable characteristic of B chromosomes, and the occurrence of distinct types of multigene families, satellite DNAs and some transposable elements have been reported. Here, we describe the organization of repetitive DNAs in the A complement and B chromosome system in the grasshopper species Abracris flavolineata using classical cytogenetic techniques and FISH analysis using probes for five multigene families, telomeric repeats and repetitive C₀t-1 DNA fractions. The 18S rRNA and H3 histone multigene families are highly variable and well distributed in A. flavolineata chromosomes, which contrasts with the conservation of U snRNA genes and less variable distribution of 5S rDNA sequences. The H3 histone gene was an extensively distributed with clusters occurring in all chromosomes. Repetitive DNAs were concentrated in C-positive regions, including the pericentromeric region and small chromosomal arms, with some occurrence in C-negative regions, but abundance was low in the B chromosome. Finally, the first demonstration of the U2 snRNA gene in B chromosomes in A. flavolineata may shed light on its possible origin. These results provide new information regarding chromosomal variability for repetitive DNAs in grasshoppers and the specific molecular composition of B chromosomes.     

A cytogenetic analysis in Psophus stridulus (L.) (Orthoptera: Acrididae): B-chromosomes and abnormal spermatid nuclei

The male chromosome complement of Psophus stridulus (L.) (Orthoptera: Acrididae) has been analyzed by using orcein staining, C-banding and silver impregnation. During spermatogenesis only one pair of autosomes (M₉) shows an active nucleolar organizer region located in a C-banded constriction. There are other chromosome pairs with constrictions but these do not show nucleolar activity. The relationship between these constrictions and the C-banding pattern exhibited by this species is analyzed.In a sample of 83 males from five populations, two different supernumerary chromosomes were observed. Four males had a metacentric B-chromosome (B^m) similar in size to the sex chromosome and mitotically stable. Its meiotic behaviour indicates that it is an isochromosome. An additional small B-chromosome (B⁸) was also found in a single follicle of one individual carrying the B^m.A high rate of abnormal spermatids (macrospermatids) was scored in the individuals carrying B's. This proportion is notably higher in the follicle containing both the B^m and the B⁸.     

Centromeric repetitive DNA sequences in the genus Brassica

Representatives of two major repetitive DNA sequence families from the diploid Brassica species B. campestris and B. oleracea were isolated, sequenced and localized to chromosomes by in situ hybridization. Both sequences were located near the centromeres of many chromosome pairs in both diploid species, but major sites of the two probes were all on different chromosome pairs. Such chromosome specificity is unusual for plant paracentromeric repetitive DNA. Reduction of stringency of hybridization gave centromeric hybridization sites on more chromosomes, indicating that there are divergent sequences present on other chromosomes. In tetraploid species derived from the diploids, the number of hybridization sites was different from the sum of the diploid ancestors, and some chromosomes had both sequences, indicating relatively rapid homogenization and copy number evolution since the origin of the tetraploid species.     

Genomic content and new insights on the origin of the B chromosome of the cichlid fish <Emphasis Type="Italic">Astatotilapia latifasciata</Emphasis>

B chromosomes are additional chromosomes widely studied in a diversity of eukaryotic groups, including fungi, plants and animals, but their origin, evolution and possible functions are not clearly understood. To further understand the genomic content and the evolutionary history of B chromosomes, classical and molecular cytogenetic analyses were conducted in the cichlid fish Astatotilapia latifasciata, which harbor 1–2 B chromosomes. Through cytogenetic mapping of several probes, including transposable elements, rRNA genes, a repeated DNA genomic fraction (C ₀ t − 1 DNA), whole genome probes (comparative genomic hybridization), and BAC clones from Oreochromis niloticus, we found similarities between the B chromosome and the 1st chromosome pair and chromosomes harboring rRNA genes. Based on the cytogenetic mapping data, we suggest the B chromosome may have evolved from a small chromosomal fragment followed by the invasion of the proto-B chromosome by several repeated DNA families.     

Segmental Duplications Are Common in Rice Genome

Segmental duplications on rice (Oryza sativa L.) chromosomes 8, 9, 11, and 12 were studied by examining the distributions of sequences resolved by 13 probes detecting multiple copies of DNA sequences. Four of the hybridization bands detected by a repetitive sequence probe, rTRS, were mapped to the ends of all the four chromosomes. Two or three of the bands detected by each of the other 12 probes were also mapped to different chromosomes. The bands detected by the same probe usually occurred in similar locations of different chromosomes. Loci detected by different DNA probes were often similarly arranged on different chromosomes. Chromosomes 8 and 9 showed colinearity of marker loci arrangement indicating a possible common origin. A segment on chromosome 9 was also very similar to the previously reported duplicated fragments on the ends of chromosomes 11 and 12 which were also detected in this study, indicating a likely common origin. Moreover, the various degrees of distributional similarity of the segments suggest a complex relationship among the chromosomes in the evolution of the rice genome. These results support the proposition that chromosome duplication and diversification may be a mechanism for the origin and evolution of the chromosomes in the rice genome.     

水稻基因组中的节段重复

利用１３个多拷贝探针,研究水稻（Ｏｒｙｚａ ｓａｔｉｖａ Ｌ．）基因组第８、９、１１和１２染色体上的节段重复。由同一探针检测到的多拷贝位点通常位于不同染色体的相同部位。不同探针检测到的多拷贝位点在不同染色体上的位置顺序相同。第８种９染色体上的相同多拷贝位点的线性排列,提示这两条染色体在进行上可能来源于同一原始染色体。而第９染色体上的一个节段与前人报道的以及本研究进一步证实的第１１和第１２染色体短臂     

The chromosomes and DNA of Allium cepa

Giemsa C-banded idiograms that allow the identification of all chromosomes have been prepared for Allium cepa, Ornithogalum virens, and Secale cereale. An analysis of A. cepa DNA has determined that: (1) It has the lowest GC content so far reported for an angiosperm (32%). (2) It appears to have no satellite DNA detectable by CsCl or Cs₂SO₄-Ag⁺ density gradient centrifugation. (3) Aside from fold back DNA and unreactable fragments, a C₀t curve indicates that most of the DNA can be adequately described as two major middle repetitive components (Fractions I and II) and a single copy component (Fraction III). And (4) most of the repeated DNA sequences are involved in a short period interspersion pattern with single copy and other repetitive sequences. In situ hybridization of tritiated cRNAs to fold back, long repeated, and Fraction I DNA from A. cepa to squash preparations of chromosomes and nuclei from A. cepa, O. virens, and S. cereale root tips indicates: (1) Sequences complementary to fold back DNA are scattered throughout the genome of A. cepa except for telomeric heterochromatin and nucleolus organizers while they are not detectable in the genomes of O. virens or S. cereale. (2) Although long repeated sequences are scattered throughout the genome of A. cepa, they are concentrated to some extent in telomeric heterochromatin and nucleolus organizers (NOs). Sequences complementary to long repeats of A. cepa occur primarily in chromosome three of O. virens while these sequences are more common in the genome of more distantly related S. cereale. (3) Fraction I DNA is scattered throughout the genome of A. cepa while it is hardly detectable in the genomes of O. virens and S. cereale. These results are discussed in regard to the evolutionary conservation and function of repeated DNA sequences.     

Characterisation of repeated sequences from microdissected B chromosomes of Crepis capillaris

The B chromosome of Crepis capillaris was isolated from the standard chromosomes by microdissection, and the chromosomal DNA amplified using the degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR). The PCR product was cloned and a Bspecific library created and characterised. Southern and in situ hybridisation analyses of the DOP-PCR product from microdissected B chromosomes confirmed that the B chromosome is composed mainly of sequences also present in the A chromosomes but lacks the main repeated DNA families located in the A-chromosomal heterochromatin. From 100 clones analysed, 12% of the generated B-chromosomal library was shown to be composed of dispersed repeats located in both the A and B chromosomes. No B-specific repeated sequence was detected. One of the most abundant repeated DNAs within the library, the family B134, was further characterised. Repeating units show a sequence similarity range from 69% to 90% and are characterised by their richness in (CA)n repeats. In situ hybridisation revealed that members of this family are dispersed throughout the A and B chromosomes but are more concentrated in the pericentromeric heterochromatin of the B, indicating that the molecular organization of B heterochromatin is different from that of the A chromosomes. Compared with the A chromosomes, the Bs contain about 20,000 copies per micron more of the B134 sequence. This indicates that B134 was amplified on the B chromosome after its origin. The B134 sequences in the B chromosomes have also diverged from those on the A chromosomes. Although the DNA composition of A and B chromosomes is similar, Bs are evolving separately from A chromosomes at the molecular level.     

A comparative study of retrotransposons in the centromeric regions of A and B chromosomes of maize

Bacterial Artificial Chromosomes (BACs) derived from the B chromosome, based on homology with the B specific sequence, were subcloned and sequenced. Analysis of DNA sequence data indicated the presence of 23 common retroelements, as well as novel sequences of B chromosome origin. Generally, where the same retrotransposon type was observed in both A and B chromosomes, there were more copies per unit of sequence in the B centromeric region (the major site of B repeat) than in the A centromere, except for Huck-1. Based on previous estimates of the age of the major burst of transposition into the maize genome, the oldest retrotransposons (Ji-6 and Tekay, approximately 5.0 and 5.2 million years ago, respectively) were found in the B centromere region only, while the next two oldest (Huck-1 and Opie-1) were found in both the A and B sequences. Phylogenetic analysis of Opie retroelements from both A and B centromeres indicated that some of the B Opie centromeric sequences share a more recent common ancestor with A Opie retroelements than they do with other B Opie centromeric sequences. These results imply that the supernumerary maize B chromosome has coexisted with the A chromosomes during that period of transposition. They also support the hypothesis that the B chromosome had its origins from A chromosome elements, or that alternative origins, such as being donated to the maize genome in a wide species cross, preceded six million years ago, because the spectrum of retrotransposons in the two chromosomes is quite similar.     

The distribution of repetitive DNAs between regular and supernumerary chromosomes in Species of Glossina (Tsetse): a two-step process in the origin of supernumeraries

Several species of tsetse fly within the Morsitans and Fusca subgenera of Glossina contain supernumerary (B) chromosomes. Previous studies on the meiotic behaviour of chromosomes (Southern and Pell, 1973) and the C-band patterns (Jordan et al., 1977) have indicated a close similarity between the Y chromosome and the supernumeraries. The distributions of the highly abundant families of DNA (satellite DNAs) between the autosomes, sex chromosomes and B chromosomes of G.m. morsitans, G. austeni and G. pallidipes have been examined by in situ hybridisation. In addition, the organisation and sequence homologies of satellite DNAs have been examined by restriction enzymes and heterologous hybridisations in in situ and Southern transfer conditions. The majority of satellite sequences that are homologous between species are distributed in several different arrangements between A and B chromosome telomeres with minority sequences at some centromeric and intercalary locations. There is no extensive satellite DNA similarity between the Y and B chromosomes. We suggest that the Y and B chromosome associations and synchronous allocycly during meiosis are the result of extensive heterochromatinisation of these two chromosome types, that is probably a reflection of two separate stages involved in the generation of the B chromosomes in the genus. The independent evolution of satellites and supernumeraries is discussed.     

Molecular cytogenetic identification of B genome chromosomes linked to blackleg disease resistance in Brassica napus × B. carinata interspecific hybrids

Key message

Provide evidence that the Brassica B genome chromosome B3 carries blackleg resistance gene, and also the B genome chromosomes were inherited several generations along with B. napus chromosomes.

Abstract

Blackleg disease caused by fungus Leptosphaeria maculans causes significant yield losses in Brassica napus. Brassica carinata possesses excellent resistance to this disease. To introgress blackleg resistance, crosses between B. napus cv. Westar and B. carinata were done. The interspecific-hybrids were backcrossed twice to Westar and self-pollinated three times to produce BC₂S₃ families. Doubled haploid lines (DH1) were produced from one blackleg resistant family. SSR markers were used to study the association between B genome chromosome(s) and blackleg resistance. The entire B3 chromosome of B. carinata was associated with blackleg resistance in DH1. A second DH population (DH2) was produced from F₁s of resistant DH1 lines crossed to blackleg susceptible B. napus cv. Polo where resistance was found to be associated with SSR markers from the middle to bottom of the B3 and top of the B8 chromosomes. The results demonstrated that the B3 chromosome carried gene(s) for blackleg resistance. Genomic in situ hybridization (GISH) and GISH-like analysis of the DH2 lines revealed that susceptible lines, in addition to B. napus chromosomes, possessed one pair of B genome chromosomes (2n = 40), while resistant lines had either one (2n = 40) or two pairs (2n = 42) of B chromosomes. The molecular and GISH data suggested that the B chromosome in the susceptible lines was B7, while it was difficult to confirm the identity of the B chromosomes in the resistant lines. Also, B chromosomes were found to be inherited over several generations along with B. napus chromosomes.     

Identification of the Genomic Constitution ofMusaL. Lines (Bananas, Plantains and Hybrids) Using Molecular Cytogenetics

The genomic constitutions of someMusaL. lines (bananas, plantainsand artificial hybrids) were identified using molecular cytogenetictechniques. Double targetin situDNA:DNA hybridization to chromosomespreads using as probes, total genomic DNA isolated from diploidMusalinesof known AA (labelled with biotin-11-dUTP) and BB (labelledwith digoxigenin-11-dUTP) genome constitution was carried out.The use of 60% acetic acid combined with heating over a flamegave high quality chromosome spreads free of cytoplasm forinsituhybridization. Total genomic A DNA labelled broad centromericregions of all 22 chromosomes of the diploid line, Calcutta4 (M. acuminataColla. ssp.burmanniccoides; A genome) with somechromosomes showing stronger hybridization. Labelled DNA fromthe B genome hybridized strongly to the centromeric regionsof all 22 chromosomes of Butohan 2 (M. balbisianaColla; B genome).The two satellited chromosomes of genome B labelled stronglywith genomic A DNA.In situhybridization of labelled A and Bgenomic DNA to metaphase chromosomes of triploid AAB and ABBcultivars discriminated between A and B genome chromosomes.The plantains Agbagba, Obino l'Ewai and Mbi Egome showed 22genome A and 11 genome B chromosomes while the cooking bananasBluggoe and Fougamou showed 11 genome A and 22 genome B chromosomes.Hybridization of labelled A and B genomic DNA to chromosomesof the hybrids showed that TMP2x 2829-62 has all 22 genome Achromosomes while TMPx 4698-1 has 33 genome A and 11 genomeB chromosomes.In situhybridization of labelled total genomicDNA to chromosomes has immense potential for identificationof chromosome origin and can be used to characterize cultivarsand hybrids produced inMusabreeding.Copyright 1997 Annals ofBotany Company Genomicin situhybridization; banana; plantain; hybrids; plant breeding; genome organization; biodiversity