Meiotic Pachytene Chromosomes Research Articles

Oligo-FISH of Populus simonii Pachytene Chromosomes Improves Karyotyping and Genome Assembly.

Poplar was one of the first woody species whose individual chromosomes could be identified using chromosome specific painting probes. Nevertheless, high-resolution karyotype construction remains a challenge. Here, we developed a karyotype based on the meiotic pachytene chromosome of Populus simonii which is a Chinese native species with many excellent traits. This karyotype was anchored by oligonucleotide (oligo)-based chromosome specific painting probes, a centromere-specific repeat (Ps34), ribosomal DNA, and telomeric DNA. We updated the known karyotype formula for P. simonii to 2n = 2x = 38 = 26m + 8st + 4t and the karyotype was 2C. The fluorescence in situ hybridization (FISH) results revealed some errors in the current P. simonii genome assembly. The 45S rDNA loci were located at the end of the short arm of chromosomes 8 and 14 by FISH. However, they were assembled on pseudochromosomes 8 and 15. In addition, the Ps34 loci were distributed in every centromere of the P. simonii chromosome in the FISH results, but they were only found to be present in pseudochromosomes 1, 3, 6, 10, 16, 17, 18, and 19. Our results reveal that pachytene chromosomes oligo-FISH is a powerful tool for constructing high-resolution karyotypes and improving the quality of genome assembly.

Bulked Oligo-FISH for Chromosome Painting and Chromosome Barcoding.

Recently developed bulked oligo-FISH is a highly versatile method, which is applicable in any plant species with an assembled genome sequence. This technique allows in situ identification of individual chromosomes, large chromosomal rearrangements, comparative karyotype analysis, or even the reconstruction of the three-dimensional organization of the genome. The method is based on the identification of thousands of short oligonucleotides, unique to specific genome regions, which are synthesized in parallel, fluorescently labeled and used as probes for FISH. In this chapter, we propose a detailed protocol for amplification and labeling of single-stranded oligo-based painting probes from so-called MYtags immortal libraries, the preparation of mitotic metaphase and meiotic pachytene chromosome spreads, and a protocol for the fluorescence in situ hybridization procedure using the synthetic oligo probes. The proposed protocols are demonstrated for banana (Musa spp.).

Chromosome Painting Facilitates Anchoring Reference Genome Sequence to Chromosomes In Situ and Integrated Karyotyping in Banana (Musa Spp.).

Oligo painting FISH was established to identify all chromosomes in banana (Musa spp.) and to anchor pseudomolecules of reference genome sequence of Musa acuminata spp. malaccensis “DH Pahang” to individual chromosomes in situ. A total of 19 chromosome/chromosome-arm specific oligo painting probes were developed and were shown to be suitable for molecular cytogenetic studies in genus Musa. For the first time, molecular karyotypes of diploid M. acuminata spp. malaccensis (A genome), M. balbisiana (B genome), and M. schizocarpa (S genome) from the Eumusa section of Musa, which contributed to the evolution of edible banana cultivars, were established. This was achieved after a combined use of oligo painting probes and a set of previously developed banana cytogenetic markers. The density of oligo painting probes was sufficient to study chromosomal rearrangements on mitotic as well as on meiotic pachytene chromosomes. This advance will enable comparative FISH mapping and identification of chromosomal translocations which accompanied genome evolution and speciation in the family Musaceae.

Two jatropha karyotypes constructed from meiotic pachytene chromosomes: Pericentric distribution of heterochromatin and variation in repetitive DNAs.

Jatropha (Jatropha curcas) is an oil-bearing plant used for biodiesel production. Construction of its standard karyotype and identification of the euchromatin/heterochromatin distribution associated with gene expression and meiotic recombination are essential to fully characterize its genome. Here, we developed a J. curcas karyotype based on meiotic pachytene chromosomes. In addition, a karyotype of J. integerrima, a useful species for jatropha breeding, was also constructed. Five out of eleven J. curcas chromosomes were metacentric, but only two were metacentric in J. integerrima. Almost all of the heterochromatin was distributed around the pericentric regions. The interstitial and distal regions were euchromatic without heterochromatic knobs, except for small heterochromatin regions associated with the subtelomeric repeat sequence JcSat1. These pericentric heterochromatin distribution patterns, together with chromosome structure data and the results of FISH probing with rDNA and JcSat1, allowed us to classify all chromosomes of both species. The two species had two 35S rDNA loci and one 5S rDNA locus; one 35S rDNA locus in J. integerrima was located on the interstitial region of the short arms. In addition, JcSat1 was found at only the heterochromatic ends of the J. curcas chromosome, not the J. integerrima chromosome. Despite the same chromosome number, the two pachytene chromosome-based karyotypes suggest variation in chromosome structure and distribution of repetitive DNAs in these two species.

An improved method for inducing prometaphase chromosomes in plants

BackgroundDetailed karyotyping using metaphase chromosomes in melon (Cucumis melo L.) remains a challenge because of their small chromosome sizes and poor stainability. Prometaphase chromosomes, which are two times longer and loosely condensed, provide a significantly better resolution for fluorescence in situ hybridization (FISH) than metaphase chromosomes. However, suitable method for acquiring prometaphase chromosomes in melon have been poorly investigated.ResultsIn this study, a modified Carnoy’s solution II (MC II) [6:3:1 (v/v) ethanol: acetic acid: chloroform] was used as a pretreatment solution to obtain prometaphase chromosomes. We demonstrated that the prometaphase chromosomes obtained using the MC II method are excellent for karyotyping and FISH analysis. We also observed that a combination of MC II and the modified air dry (ADI) method provides a satisfactory meiotic pachytene chromosome preparation with reduced cytoplasmic background and clear chromatin spreads. Moreover, we demonstrated that pachytene and prometaphase chromosomes of melon and Abelia × grandiflora generate significantly better FISH images when prepared using the method described. We confirmed, for the first time, that Abelia × grandiflora has pairs of both strong and weak 45S ribosomal DNA signals on the short arms of their metaphase chromosomes.ConclusionThe MC II and ADI method are simple and effective for acquiring prometaphase and pachytene chromosomes with reduced cytoplasm background in plants. Our methods provide high-resolution FISH images that can help accelerate molecular cytogenetic research in plants.

Molecular and cytogenetic analyses provide evidence of the introgression of chromosomal segments from the wild Cucumis hystrix into the cultivated cucumber through the bridge of a synthetic allotetraploid

Cucumis × hytivus (2n = 4× = 38) is a synthetic allotetraploid obtained from interspecific hybridization between the cucumber (2n = 2× = 14) and its wild relative C. hystrix (2n = 2× = 24). The synthesis of this species built a bridge for cucumber improvement through gene introgression. Allotriploid and introgression lines (ILs) have previously been produced and characterized with respect to morphology, cytology, and molecular markers. However, no clear evidence of how the chromosomal segments of C. hystrix were introgressed and inherited was found owing to the small size of chromosomes. In the present study, cucumber-C. hystrix introgression lines were developed by backcrossing the allotriploid to North China cucumber breeding line “P01” followed by self-pollination. The introgressed segments of C. hystrix in the ILs were revealed by meiotic pachytene chromosome analysis. Fluorescence in situ hybridization (FISH) was performed on pachytene chromosomes using fosmid clones from cucumber, which confirmed that introgression occurred in the long arm of chromosome 7. Molecular analysis using a set of 53 simple sequence repeats (SSRs) indicated that the chromosomal segments of C. hystrix were introduced into 4 cucumber chromosomes, the short arms of chromosomes 2 and 6, and long arms of chromosomes 3 and 7. The inheritance of alien sequences in the long arm of chromosome 7 was investigated with 21 SSRs in self-pollinated progenies. C. hystrix-specific bands of several SSRs were still present in some individuals, indicating that the introgressed segment was partially preserved. The first unambiguous identification of alien chromosome segments in cucumber ILs using combined molecular cytogenetics could facilitate the determination of effects of wild alleles and promote cucumber improvement.

Application of a modified drop method for high-resolution pachytene chromosome spreads in two Phalaenopsis species

BackgroundPreparation of good chromosome spreads without cytoplasmic contamination is the crucial step in cytogenetic mapping. To date, cytogenetic research in the Orchidaceae family has been carried out solely on mitotic metaphase chromosomes. Well-spread meiotic pachytene chromosomes can provide higher resolution and fine detail for analysis of chromosomal structure and are also beneficial for chromosomal FISH (fluorescence in situ hybridization) mapping. However, an adequate method for the preparation of meiotic pachytene chromosomes in orchid species has not yet been reported.ResultsTwo Taiwanese native Phalaenopsis species were selected to test the modified drop method for preparation of meiotic pachytene chromosomes from pollinia. In this modified method, pollinia were ground and treated with an enzyme mixture to completely remove cell walls. Protoplasts were resuspended in ethanol/glacial acetic acid and dropped onto a wet inclined slide of 30° from a height of 0.5 m. The sample was then flowed down the inclined plane to spread the chromosomes. Hundreds of pachytene chromosomes with little to no cytoplasmic contamination were well spread on each slide. We also showed that the resolution of 45S rDNA-containing chromosomes at the pachytene stage was up to 20 times higher than that at metaphase. Slides prepared following this modified drop method were amenable to FISH mapping of both 45S and 5S rDNA on pachytene chromosomes and, after FISH, the chromosomal structure remained intact for further analysis.ConclusionThis modified drop method is suitable for pachytene spreads from pollinia of Phalaenopsis orchids. The large number and high-resolution pachytene spreads, with little or no cytoplasmic contamination, prepared by the modified drop method could be used for FISH mapping of DNA fragments to accelerate the integration of cytogenetic and molecular research in Phalaenopsis orchids.

Molecular cytogenetic analysis and genomic organization of major DNA repeats in castor bean (Ricinus communis L.).

This article addresses the bioinformatic, molecular genetic, and cytogenetic study of castor bean (Ricinus communis, 2n = 20), which belongs to the monotypic Ricinus genus within the Euphorbiaceae family. Because castor bean chromosomes are small, karyotypic studies are difficult. However, the use of DNA repeats has yielded new prospects for karyotypic research and genome characterization. In the present study, major DNA repeat sequences were identified, characterized and localized on mitotic metaphase and meiotic pachytene chromosomes. Analyses of the nucleotide composition, curvature models, and FISH localization of the rcsat39 repeat suggest that this repeat plays a key role in building heterochromatic arrays in castor bean. Additionally, the rcsat390 sequences were determined to be chromosome-specific repeats located in the pericentromeric region of mitotic chromosome A (pachytene chromosome 1). The localization of rcsat39, rcsat390, 45S and 5S rDNA genes allowed for the development of cytogenetic landmarks for chromosome identification. General questions linked to heterochromatin formation, DNA repeat distribution, and the evolutionary emergence of the genome are discussed. The article may be of interest to biologists studying small genome organization and short monomer DNA repeats.

Cytogenetic mapping with centromeric bacterial artificial chromosomes contigs shows that this recombination-poor region comprises more than half of barley chromosome 3H.

Genetic maps are based on the frequency of recombination and often show different positions of molecular markers in comparison to physical maps, particularly in the centromere that is generally poor in meiotic recombinations. To decipher the position and order of DNA sequences genetically mapped to the centromere of barley (Hordeum vulgare) chromosome 3H, fluorescence in situ hybridization with mitotic metaphase and meiotic pachytene chromosomes was performed with 70 genomic single-copy probes derived from 65 fingerprinted bacterial artificial chromosomes (BAC) contigs genetically assigned to this recombination cold spot. The total physical distribution of the centromeric 5.5 cM bin of 3H comprises 58% of the mitotic metaphase chromosome length. Mitotic and meiotic chromatin of this recombination-poor region is preferentially marked by a heterochromatin-typical histone mark (H3K9me2), while recombination enriched subterminal chromosome regions are enriched in euchromatin-typical histone marks (H3K4me2, H3K4me3, H3K27me3) suggesting that the meiotic recombination rate could be influenced by the chromatin landscape.

Evidence for emergence of sex-determining gene(s) in a centromeric region in Vasconcellea parviflora.

Sex chromosomes have been studied in many plant and animal species. However, few species are suitable as models to study the evolutionary histories of sex chromosomes. We previously demonstrated that papaya (Carica papaya) (2n = 2x = 18), a fruit tree in the family Caricaceae, contains recently emerged but cytologically heteromorphic X/Y chromosomes. We have been intrigued by the possible presence and evolution of sex chromosomes in other dioecious Caricaceae species. We selected a set of 22 bacterial artificial chromosome (BAC) clones that are distributed along the papaya X/Y chromosomes. These BACs were mapped to the meiotic pachytene chromosomes of Vasconcellea parviflora (2n = 2x = 18), a species that diverged from papaya ∼27 million years ago. We demonstrate that V. parviflora contains a pair of heteromorphic X/Y chromosomes that are homologous to the papaya X/Y chromosomes. The comparative mapping results revealed that the male-specific regions of the Y chromosomes (MSYs) probably initiated near the centromere of the Y chromosomes in both species. The two MSYs, however, shared only a small chromosomal domain near the centromere in otherwise rearranged chromosomes. The V. parviflora MSY expanded toward the short arm of the chromosome, whereas the papaya MSY expanded in the opposite direction. Most BACs mapped to papaya MSY were not located in V. parviflora MSY, revealing different DNA compositions in the two MSYs. These results suggest that mutation of gene(s) in the centromeric region may have triggered sex chromosome evolution in these plant species.

High resolution molecular cytogenetic techniques in plants: Pachytene- and fibre-FISH

Fluorescencein situhybridization (FISH) is the most versatile and accurate molecular cytogenetic technique for determining euchromatic-heterochromatic boundaries and the locations of repetitive and single-copy DNA sequences and of chromosome-specific BAC clones on chromosomes. The combination of cytogenetic and genetic methods yields a highresolution physical map. FISH allows direct mapping of specific DNA sequences inside the cell (interphase nuclei), along meiotic pachytene chromosomes and isolated chromatin (DNA fibres). The increased sensitivity of the technique and its ability to detect gene locations provide a powerful research tool for genetic and pre-breeding studies. FISH-based physical mapping plays an important role and is increasingly used for studies at the cytological level on the chromatin organization that controls gene expression and regulation. The present minireview describes some of the benefits of alternative FISH-based techniques and their application for studying plant chromosomes and genomes.

Preparations of Meiotic Pachytene Chromosomes and Extended DNA Fibers from Cotton Suitable for Fluorescence In Situ Hybridization

Fluorescence in situ hybridization (FISH) has become one of the most important techniques applied in plant molecular cytogenetics. However, the application of this technique in cotton has lagged behind because of difficulties in chromosome preparation. The focus of this article was FISH performed not only on cotton pachytene chromosomes, but also on cotton extended DNA fibers. The cotton pollen mother cells (PMCs) instead of buds or anthers were directly digested in enzyme to completely breakdown the cell wall. Before the routine acetic acid treatment, PMCs were incubated in acetic acid and enzyme mixture to remove the cytoplasm and clear the background. The method of ice-cold Carnoy's solution spreading chromosome was adopted instead of nitrogen removed method to avoid chromosomes losing and fully stretch chromosome. With the above-improved steps, the high-quality well-differentiated pachytene chromosomes with clear background were obtained. FISH results demonstrated that a mature protocol of cotton pachytene chromosomes preparation was presented. Intact and no debris cotton nuclei were obtained by chopping from etiolation cotyledons instead of the conventional liquid nitrogen grinding method. After incubating the nuclei with nucleus lysis buffer on slide, the parallel and clear background DNA fibers were acquired along the slide. This method overcomes the twist, accumulation and fracture of DNA fibers compared with other methods. The entire process of DNA fibers preparation requires only 30 min, in contrast, it takes 3 h with routine nitrogen grinding method. The poisonous mercaptoethanol in nucleus lysis buffer is replaced by nonpoisonous dithiothreitol. PVP40 in nucleus isolation buffer is used to prevent oxidation. The probability of success in isolating nuclei for DNA fiber preparation is almost 100% tested with this method in cotton. So a rapid, safe, and efficient method for the preparation of cotton extended DNA fibers suitable for FISH was established.

An integrated molecular cytogenetic map of Cucumis sativus L. chromosome 2

BackgroundIntegration of molecular, genetic and cytological maps is still a challenge for most plant species. Recent progress in molecular and cytogenetic studies created a basis for developing integrated maps in cucumber (Cucumis sativus L.).ResultsIn this study, eleven fosmid clones and three plasmids containing 45S rDNA, the centromeric satellite repeat Type III and the pericentriomeric repeat CsRP1 sequences respectively were hybridized to cucumber metaphase chromosomes to assign their cytological location on chromosome 2. Moreover, an integrated molecular cytogenetic map of cucumber chromosomes 2 was constructed by fluorescence in situ hybridization (FISH) mapping of 11 fosmid clones together with the cucumber centromere-specific Type III sequence on meiotic pachytene chromosomes. The cytogenetic map was fully integrated with genetic linkage map since each fosmid clone was anchored by a genetically mapped simple sequence repeat marker (SSR). The relationship between the genetic and physical distances along chromosome was analyzed.ConclusionsRecombination was not evenly distributed along the physical length of chromosome 2. Suppression of recombination was found in centromeric and pericentromeric regions. Our results also indicated that the molecular markers composing the linkage map for chromosome 2 provided excellent coverage of the chromosome.

Integration of Genetic and Cytological Maps and Development of a Pachytene Chromosome-based Karyotype in Papaya

A significant amount of genetic and genomic resources have been developed in papaya (Carica papaya, $$ {\hbox{2n = 2}} \times { = 18} $$ ), including genetic linkage maps consisting of nine major and three minor linkage groups. However, the 12 genetic linkage groups have not been integrated with the nine chromosomes of papaya. Bacterial artificial chromosome (BAC) clones associated with each linkage group were recently isolated. These linkage group-specific BACs were mapped to meiotic pachytene chromosomes of papaya using fluorescence in situ hybridization (FISH). The FISH mapping results integrated the 12 linkage groups into the nine papaya chromosomes. We developed a pachytene chromosome-based high resolution karyotype for the hermaphrodite plant genome of papaya cultivar SunUp. The chromosomal distribution of heterochromatin in the papaya genome is provided in the karyotype with the X chromosome representing the most euchromatic chromosome in the papaya genome. FISH mapping also revealed a significant amplification of sequences related to the 5S ribosomal RNA genes, which was detected in the male-specific region of the Y chromosome, but not in the corresponding region in the X chromosome.

Molecular Analysis of a Large Subtelomeric Nucleotide-Binding-Site–Leucine-Rich-Repeat Family in Two Representative Genotypes of the Major Gene Pools of Phaseolus vulgaris

In common bean, the B4 disease resistance gene cluster is a complex cluster localized at the end of linkage group (LG) B4, containing at least three R specificities to the fungus Colletotrichum lindemuthianum. To investigate the evolution of this R cluster since the divergence of Andean and Mesoamerican gene pools, DNA sequences were characterized from two representative genotypes of the two major gene pools of common bean (BAT93: Mesoamerican; JaloEEP558: Andean). Sequences encoding 29 B4-CC nucleotide-binding-site-leucine-rich-repeat (B4-CNL) genes were determined-12 from JaloEEP558 and 17 from BAT93. Although sequence exchange events were identified, phylogenetic analyses revealed that they were not frequent enough to lead to homogenization of B4-CNL sequences within a haplotype. Genetic mapping based on pulsed-field gel electrophoresis separation confirmed that the B4-CNL family is a large family specific to one end of LG B4 and is present at two distinct blocks separated by 26 cM. Fluorescent in situ hybridization on meiotic pachytene chromosomes revealed that two B4-CNL blocks are located in the subtelomeric region of the short arm of chromosome 4 on both sides of a heterochromatic block (knob), suggesting that this peculiar genomic environment may favor the proliferation of a large R gene cluster.

Imaging of Silkworm Meiotic Chromosome by Atomic Force Microscopy

Although structural information of mitotic chromosomes has been accumulated, little information is available for meiotic chromosome structures. Here, we applied atomic force microscopy (AFM) to investigate the ultrastructures of the silkworm, Bombyx mori, meiotic pachytene chromosome in its native state with nanometer scale resolution. Two levels of DNA folding were observed on the meiotic chromosome surface, 50-70 nm granules, which were considered to be 30 nm chromatin fibers, and spherical protrusions of 400-600 nm, which were considered to be chromomeres and arranged on the surface of the chromosome parallel to the chromosome longitudinal axis. These observations suggested that AFM study is an excellent approach for obtaining information concerning the silkworm pachytene chromosome higher order structure.

Current status and the future of fluorescence in situ hybridization (FISH) in plant genome research

Fluorescence in situ hybridization (FISH), which allows direct mapping of DNA sequences on chromosomes, has become the most important technique in plant molecular cytogenetics research. Repetitive DNA sequence can generate unique FISH patterns on individual chromosomes for karyotyping and phylogenetic analysis. FISH on meiotic pachytene chromosomes coupled with digital imaging systems has become an efficient method to develop physical maps in plant species. FISH on extended DNA fibers provides a high-resolution mapping approach to analyze large DNA molecules and to characterize large genomic loci. FISH-based physical mapping provides a valuable complementary approach in genome sequencing and map-based cloning research. We expect that FISH will continue to play an important role in relating DNA sequence information to chromosome biology. FISH coupled with immunoassays will be increasingly used to study features of chromatin at the cytological level that control expression and regulation of genes.

Predicting Chromosomal Locations of Genetically Mapped Loci in Maize Using the Morgan2McClintock Translator

The Morgan2McClintock Translator permits prediction of meiotic pachytene chromosome map positions from recombination-based linkage data using recombination nodule frequency distributions. Its outputs permit estimation of DNA content between mapped loci and help to create an integrated overview of the maize nuclear genome structure.

An integrated map of Oryza sativa L. chromosome 5

The developments of molecular marker-based genetic linkage maps are now routine. Physical maps based on contigs of large insert genomic clones have been established in several plant species. However, integration of genetic, physical, and cytological maps is still a challenge for most plant species. Here we present an integrated map of rice (Oryza sativa L.) chromosome 5, developed by fluorescence in situ hybridization mapping of 18 bacterial artificial chromosome (BAC) clones or PI-derived artificial chromosome (PAC) clones on meiotic pachytene chromosomes. Each BAC/PAC clone was anchored by a restriction fragment length polymorphism marker mapped to the rice genetic linkage map. This molecular cytogenetic map shows the genetic recombination and sequence information of a physical map, correlated to the cytological features of rice chromosome 5. Detailed comparisons of the distances between markers on genetic, cytological, and physical maps, revealed the distributions of recombination events and molecular organization of the chromosomal features of rice chromosome 5 at the pachytene stage. Discordance of distances between the markers was found among the different maps. Our results revealed that neither the recombination events nor the degree of chromatin condensation were evenly distributed along the entire length of chromosome 5. Detailed comparisons of the correlative positions of markers on the genetic, cytological, and physical maps of rice chromosome 5 provide insight into the molecular architecture of rice chromosome 5, in relation to its cytological features and recombination events on the genetic map. The prospective applications of such an integrated cytogenetic map are discussed.

A high-resolution karyotype of cucumber (Cucumis sativusL. 'Winter Long') revealed by C-banding, pachytene analysis, and RAPD-aided fluorescence in situ hybridization

Using molecular cytogenetic DNA markers, C-banding, pachytene analysis, and fluorescence in situ hybridization (FISH), a high-resolution karyotype was established in the cucumber. C-banding showed distinct hetero chromatic bands on the pericentromeric, telomeric, and intercalary regions of the chromosomes. The C-banding patterns were also consistent with the morphology of 4'-6-diamino-2-phenylindole dihydrochloride (DAPI)-stained pachytene chromosomes. Two repetitive DNA fragments, CsRP1 and CsRP2, were obtained by PCR and localized on the mitotic metaphase and meiotic pachytene chromosomes. CsRP1 was detected on the pericentromeric heterochromatic regions of all chromosomes, except chromosome 1. CsRP2 was detected on 5 (chromosomes 1, 2, 3, 4, and 7) of 7 chromosomes. All homologous chromosome pairs could be distinguished by FISH using 2 RAPD markers. This is the first report on molecular karyotyping of mitotic and meiotic spreads of cucumber.