N-banding Technique Research Articles

Comparative analysis of D and R genomes in two lignes (X-Trit-icosecalewittmak) and their genitors (Secale cerealeL.,Triticum aestivumL.) by N-banding

Abstract We were interested in heterochromatic variability analysis of D and R genomes, evidenced by the N-banding technique and the localization of nucleolar organising regions (NOR) in two triticales (2n=8x= 56, AABBDDRR) and their genitors, common wheat (2n=6x=42, AABBDD) and rye (2n=2x=14, RR). The Analysis of N bands of genomes D and R of the hybrids in comparison with their genitors displayed a different marking. In the genome D, hybrid Mahon-demias x RC9 is much less heterochromatic than both genitor Mahon-demias and Mahon-demias x Merced. In the genome R, differencies involved a great deal of heterochromatin in hybrid Mahon-demias x Merced revealed by thick bands. Nevertheless, hybrid Mahon-demias x RC9 is almost similar to its genitor. In the genitors and in the hybrids, the organising regions (NOR) marked on the chromosomes 1R, 2R, 3R and 6R of rye and on the chromosome 1B of wheat and on the chromosomes 1B, 1R, 2R, 3R, 6R of triticales, showed the a same localisation compared with that describe...

Chromosome N-banding polymorphism in Aegilops geniculata Roth.

The chromosome morphology and the variation in the N-band distribution along the chromosomes were studied in 13 accessions of the wild wheat Aegilops geniculata Roth (2n=28, genome formula UUM°M°). The karyotype consisted of four metacentric, five submetacentric, three subtelocentric, and two satellited pairs of chromosomes. The N-banding technique stained differentially all 14 chromosome pairs. The most prominent bands were observed near the centromeres and in the intercalary regions of both arms. Telomeric bands were found in only seven chromosomes. All chromosomes produced a specific banding pattern, permitting their identification. Polymorphism for presence or absence of particular bands was observed among the different accessions. The variable bands were located predominantly at the terminal and subterminal chromosome regions, and were also unevenly distributed over chromosomes. This polymorphism resulted in up to eight distinct banding pattern variants for each of the Ae. geniculata chromosomes. Each accession in this study showed a unique overall karyotype, sharing 0 to 8 chromosomes of identical banding pattern with the other accessions. A generalized idiogram of Aegilops geniculata is presented.

Identification of Aegilops ovata chromosomes added to the wheat (Triticum aestivum L.) genome

A partial amphiploid Triticum aestivum cv. “Chinese Spring” × Aegilops ovata was backcrossed to XThinese Spring’ to produce wheat-Ac. ovata addition lines. Using the chromosome N-banding technique five different Ae. ovata chromosomes were identified as additions to the wheat genome in the backcross-derivatives. Plants with 2n=43 carried either single alien chromosome as monosomic addition, or more than one alien chromosomes as addition-substitution. The 44-chromosome plants were identified as disomic additions, double monosomic additions, or addition-substitutions. Some of the alien chromosomes had modified heterochromatin structure in comparison with the corresponding chromosomes of the Aegilops ovata parent.

Karyotypic analysis of N-banded chromosomes of diploid alfalfa: Medicago sativa ssp. caerulea and ssp. falcata and their hybrid.

Chromosomes of two diploid (2n = 2x = 16) subspecies of Medicago sativa, ssp. caerulea and ssp. falcata, and their hybrid were studied using an N-banding technique. This study was undertaken to develop an N-banded karyotype of ssp. caerulea and ssp. falcata, and determine if the same technique could be used to identify parental chromosomes in hybrids. The chromosomes of ssp. falcata have only centromeric N bands and thus individual chromosomes could not be identified. However, chromosome-specific bands were observed in ssp. caerulea enabling the identification of each of the eight pairs of chromosomes and the development of an idiogram. All chromosomes have a centromeric band and a telomeric band in the short arm. All of the chromosomes, except chromosomes 7 and 8, have interstitial bands in their long arms and chromosome 5 has two faint interstitial bands in its long arm. The satellited chromosome is easily distinguished due to the secondary constriction and characteristic band at the nucleolar organizer region. The differences in banding patterns between these subspecies makes it possible to distinguish chromosomes from each other in their hybrids. This is the first report of the use of N banding to identify the diploid chromosomes of M. sativa ssp. caerulea and ssp. falcata.

Position-effect variegation in Drosophila melanogaster X chromosome inversion with a breakpoint in a satellite block and its suppression in a secondary rearrangement.

In(1LR)pn2a is a pericentric inversion with a euchromatic breakpoint in the 2E polytene region and a heterochromatic breakpoint in the right arm of the X chromosome. It is associated with position-effect variegation (PEV) of the pn, wapl, Pgd and other vital loci of the 2E region, which are relocated near the bulk of the X heterochromatin. Cytological analysis showed that the rearrangement brings the 1A-2E euchromatic segment directly into contact with a major portion of the h34 block, a heterochromatic region that is positively stained by the N-banding technique and contains the AAGAG satellite sequences. Molecular cloning revealed the presence of a new junction between euchromatin and AAGAG satellite sequences and demonstrated that the euchromatic breakpoint of In(1LR)pn2a lies in the vinculin gene. In the X ray-induced secondary rearrangement In(1LR)r30, consisting of a pericentric inversion superimposed on In(1LR)pn2a, the h34 material remains associated with the 2E region but is separated from the rest of the X heterochromatin. In this case, the pn, wapl and Pgd loci no longer variegate, suggesting that the satellite-containing h34 region is not able per se to induce detectable PEV on the adjacent euchromatic genes.

Giemsa N-banding Polymorphism in Six Botanical Varieties and Six Cultivars of Barley, Hordeum vulgare L.

Mitotic chromosomes in six botanical varieties and six cultivars of barley, Hordeum vulgare L. were studied by Giemsa N-banding technique. Chromosomes 1 and 4 are metacentric and thus their short (S) and long (L) arms can not be differentiated through conventional staining method. These were easily differentiated on the basis of the bands on them. The classification of the bands was in accordance to Generalized Cytological Nomenclature of Cereal Chromosomes (GCNCC). Minor differences in the banding pattern among the studied varieties/cultivars were observed. The results indicated that all varieties of Hordeum investigated had characteristic banding pattern and polymorphism was evident on 1, 2, 4 and 6 chromosomes. Among the cultivars N-banding polymorphism was observed in interstitial regions of chromosomes 1, 2, 3 and 6. On the basis of these results it can be concluded that both varieties and cultivars may be identified due to their N-banding patterns.

Variability of Genome B Somatic Karyotype in Triticum aestivum L.

The aim of this study was to assess the degree of variability in the main morphological characteristics of individual B-genome chromosomes and the quantity and distribution of their heterochromatin blocks in nine T. aestivum cultivars, differing in origin, biological and economic characters.The summed up lengths of B-genome chromosomes in the various cultivars were assessed, as well as the degree of variability in individual chromosome sizes and arms, arm ratios, ranking of chromosomes in the genome for these characters and type of heterochromatin region distribution following staining after N-banding technique. Important regularities were revealed in range of chromosome size and arms in respect to summed up chromosome lengths of the genome in the various cultivars. They were of considerable importance for the construction of chromosome arrangement models in the genome. On the basis of these characteristics chromosomes with extreme values occupied most stable positions in chromosome arms with smal-lest or greatest lengths. Such were 5B chromosome in respect to both its arms, 4B and partially 6B chromosome in respect to their long arms, 3B and 4B chromosomes. Despite the stable behaviour of 5B chromosome in the various cultivars, within the framework of a given cultivar it changes most in respect to the long arm.Cultivar variations were found in type of chromosome heterochromatin regions. Chromosomes 5B, 6B and 4B were distinguished by most insignificant changes in the big heterochromatin regions near the centromere. Chromosome 1B was distinguished by its highest variability in type of staining. A hypothetical model has been proposed for ranking the chromosomes in the genome on the basis of the frequency with which a given chromosome occupied a definite position in the genome and its relative values in respect to the summed up lengths of all chromosomes.

Structural differentiation of chromosomes between Triticum dicoccoides Koern. and T. araraticum Jakubz., showing high meiotic pairing homology.

Tetraploid wheats were divided into the Emmer (genomic constitution, AABB) and the Timopheevi (AAGG) groups based on the low level of meiotic chromosome pairing and sterility in their hybrids. Triticum dicocoides lines KU8821A and KU8821C and T. araraticum line KU8821B, which show a high level of meiotic chromosome pairing in the hybrids, have been considered to be intermediate types between the Emmer and the Timopheevi wheats. These intermediate types may play an important role in the differentiation of tetraploid wheats. Chromosome structural differentiation was examined by the N-banding technique. These lines showed either the banding patterns of the Timopheevi or of the Emmer groups, and were not intermediate plants between the two groups based on the chromosome structure. The present results suggest that the Emmer and the Timopheevi groups were derived diphyletically in the evolution of tetraploid wheats.

Cytological identification of 1B/1R wheat-rye translocations in winter wheat breeding lines

The incorporation of rye (S. cereale L.) chromatin into winter wheat (T. aestivum L.) cultivars is often achieved via hybridization of unadapted wheat-rye translocation lines with adapted wheat germplasm. Identification of progenies possessing the translocated chromosome has traditionally involved phenotypic screening for the desired rye characteristics. In this study, the Giemsa N-banding technique was evaluated as a potential screening tool for detection of 1B/1R wheat-rye translocations. Five breeding lines were examined from the pedigree Aurora/2*TAM W-101. The differential banding patterns of chromosome 1B contributed by TAM W-101 and chromosome 1B/1R contributed by Aurora allowed unequivocal identification of translocation genotypes. Three of the lines were found to be heterogeneous, whereby plants were homozygous for either the normal 1B or the translocated 1B/1R chromosome. The remaining two lines were observed to be homozygous and homogeneous for the translocated 1B/1R chromosome. The implication of N-banding chromosomal analyses to wheat breeding is presented.

Cytogenetic studies in barley chromosome 1 by means of telotrisomic, acrotrisomic and conventional analysis

Genetic and cytogenetic techniques were applied to linkage analysis of chromosome 1. Eight marker genes, including five on the short arm and three on the long arm, were analyzed with two telotrisomic lines, Triplo 1S and 1L, and one acrotrisomic line, Triplo 1L1S. Telotrisomic analysis confirmed the position of ac2, gs3, f3, br, f5, and f8 on the short arm, and 1k2 and n on the long arm of the linkage map of chromosome 1. Conventional three-point tests with two triple genetic marker stocks showed that fcis located between br and gs3, and n is located in the middle of f8 and 1k2. Acrotrisomic for 1L1S was used for cytogenetic linkage mapping. Giemsa N-banding technique showed that the long (1L) and short (1S) arm had deficiencies of 37.5% and 73%, respectively. Genes f5, br, fc, gs3 and f8 in 1S and 1k2 in 1L were located in the deficient segments of 1L1S. A trisomic ratio obtained with n indicated an association of this gene with the long arm of the acrocentric chromosome. Cytological behavior, morphological characteristics, fertility, and transmission in the acrotrisomic 1L1S are also reported.

Acrotrisomic analysis in linkage mapping in barley (Hordeum vulgare L.)

Three acrotrisomic lines, Triplo IL(1S), 3L(3S), and 4L(4S), each carrying an extra acrocentric chromosome, were used for cytogenetic linkage mapping of barley chromosomes. The cytological structures of the acrocentric chromosome of the three acrotrisomic lines were studied with an improved Giemsa N-banding technique. The long (1L) and short arm (1S) of chromosome 1 had deficiencies of approximately 38% and 65%, respectively. The percentages of deficiencies were 0 and 77.8% for 3L and 3S, and 31.7 and 59.3% for 4L and 4S, respectively. All three genes tested (br, f c , gs3) in 1S and all three genes tested, f8, n and 1k2 in 1L showed a disomic ratio indicating that they are located in the deficient segments. Two genes (a c , yst2) located in the middle segment of 3S in linkage map showed a trisomic ratio, and two others a n , x s showed a disomic ratio. The only gene(f9) tested in 4L showed a trisomic ratio. Two genes (1g4, g1) located in the proximal segment of 4S in the linkage map showed a trisomic ratio, whereas two genes (br2, g13) located distally in 4S showed a disomic ratio, indicating that the breakage occurred between g1 and br2. This experiment demonstrates a new method for physical localization of genes on chromosome segments in material such as barley in which pachytene analysis can not be effectively used for accurate determination of break points in structural changes. Problems associated with this new technique are discussed.

An improved Giemsa N-banding technique for the identification of barley chromosomes

An improved Giemsa N-banding technique for the identification of barley chromosomes

Identification and designation of telocentric chromosomes in barley by means of Giemsa N-banding technique

Seven complete chromosomes and nine telocentric chromosomes in telotrisomics of barley (Hordeum vulgare L.) were identified and designated by an improved Giemsa N-banding technique. Karyotype analysis and Giemsa N-banding patterns of complete and telocentric chromosomes at somatic late prophase, prometaphase and metaphase have shown the following results: Chromosome 1 is a median chromosome with a long arm (Telo 1L) carrying a centromeric band, while short arm (Telo 1S) has a centromeric band and two intercalary bands. Chromosome 2 is the longest in the barley chromosome complement. Both arms show a centromeric band, an intercalary band and two faint dots on each chromatid at middle to distal regions. The banding pattern of Telo 2L (a centromeric and an intercalary band) and Telo 2S (a centromeric, two intercalary and a terminal band) corresponded to the banding pattern of the long and short arm of chromosome 2. Chromosome 3 is a submedian chromosome and its long arm is the second longest in the barley chromosome complement. Telo 3L has a centromeric (fainter than Telo 3S) and an intercalary band. It also shows a faint dot on each chromatid at distal region. Telo 3S shows a dark centromeric band only. Chromosome 4 is the most heavily banded one in barley chromosome complement. Both arms showed a dark centromeric band. Three dark intercalary bands and faint telomeric dot were observed in the long arm (4L), while two dark intercalary bands in the short arm (4S) were arranged very close to each other and appeared as a single large band in metaphase chromosomes. A faint dot was observed in each chromatid at the distal region in the 4S. Chromosome 5 is the smallest chromosome, which carries a centromeric band and an intercalary band on the long arm. Telo 5L, with a faint centromeric band and an intercalary band, is similar to the long arm. Chromosomes 6 and 7 are satellited chromosomes showing mainly centromeric bands. Telo 6S is identical to the short arm of chromosome 6 with a centromeric band. Telo 3L and Telo 4L were previously designated as Telo 3S and Telo 4S based on the genetic/linkage analysis. However, from the Giemsa banding pattern it is evident that these telocentric chromosomes are not correctly identified and the linkage map for chromosome 3 and 4 should be reversed. One out of ten triple 2S plants studied showed about 50% deficiency in the distal portion of the short arm. Telo 4L also showed a deletion of the distal euchromatic region of the long arm. This deletion (32%) may complicate genetic analysis, as genes located on the deficient segment would show a disomic ratio. It has been clearly demonstrated that the telocentric chromosomes of barley carry half of the centromere. Banding pattern polymorphism was attributed, at least partly, to the mitotic stages and differences in techniques.

Characterization of heterochromatin in Schistocerca gregaria by C- and N-banding methods

Mitotic and meiotic chromosomes of Schistocerca gregaria were C-, mild N- and strong N-banded. After C-banding, three out of eleven autosomes show, in addition to the centromeric C-bands, a second C-band. — The mild N-banding method produces a single N-band in each of only four chromosomes. With the exception of one N-band these mild N-bands correspond to the non-centromeric, second C-bands, indicating the heterochromatic character of at least three mild N-band regions. — The strong N-banding technique produces bands both at the C- and mild N-band positions and additionally a third band in one chromosome (M8), not present after C- or mild N-banding. — The N-bands do not correspond to the nucleolus organizer regions. Because of the mechanisms of the N-banding methods, it is concluded that the centromeric heterochromatin, as well as the non-centromeric N-band regions, contain high quantities of non-histone proteins. Presumably a specific difference exists between the non-histone proteins in the centromeric and non-centromeric N-band regions because the centromeres are banded by the strong N-banding technique, but not after mild N-banding. It is concluded that the N-band regions (two exceptions) contain a heterochromatin type which has the following features in common with the α-heterochromatin of Drosophila: C- as well as N-banding positive, high nonhistone protein content, repetitive and late replicating DNA. It is discussed whether the N-banded heterochromatin regions of Schistocerca contain that DNA fraction which is, like the Drosophila α-heterochromatin, underreplicated in polyploid nuclei.

N-banding and nucleolus organisers in Asellus aquaticus (Crust. Isop.).

The N-banding technique was used to stain the nucleolus organiser of the karyotype of Asellus aquaticus (crust. Isop.). Observations were made on the morphological expression of nucleolus organisers as secondary constrictions and the presence of nucleoli in mitotic prophase. An attempt was made to correlate the various results and it seems likely that N-banding is not a reflection of NO activity.

Location of nucleolar organizers in animal and plant chromosomes by means of an improved N-banding technique.

With an improved N-banding technique, the location of nucleolar organizing region was determined in 27 kinds of material including mammals, a marsupial, birds, amphibians, fishes, an insect and plants. In most cases the N-bands were clearly located on certain specific regions of chromosomes, such as the secondary constriction, satellite, centromere, telomere and heterochromatic segment, while in some species they were detected as very minute bodies distributed over many chromosomes. From the available cytological and biochemical data it was suggested that the N-bands represent certain structural non-histone proteins specifically linked to nucleolar organizers in various eukaryotic chromosomes.