Karyotypes,nucleoli, and amphiplasty in hybrids between Hordeum vulgare L. and H. bulbosum L.

Chromosome measurements were carried out in Hordeum vulgare, H. bulbosum, and their diploid, triploid, and tetraploid hybrids. The chromosomes were classified by using relative values, and thus karyotypes were established. For comparison of these karyotypes both relative and absolute values were used. It was concluded that differential amphiplasty occurred, whereas neutral amphiplasty could not be demonstrated. In the hybrids the relative length of the parts of the chromosomes (long arm, short arm, satellite) was not changed in comparison with these lengths in the pure species. The karyotypes of both species had considerable similarities. From comparing the mean absolute genome lengths, it was, however, concluded that in the pure species, as well as in all hybrid types, the chromosomes of H. vulgare were longer than those of H. bulbosum. In the diploid and tetraploid hybrids the mean genome lengths were shorter than those in the pure species and the triploid hybrids. The differential amphiplasty was such that the secondary constriction of chromosome 6 of H. bulbosum, did not show up in the hybrids. This could be related to the suppression of nucleolar formation in the genome of H. bulbosum, because the maximum number of nucleoli in root tip cells equalled the number of satellite chromosomes. Finally it was found that the pattern of nucleolar fusion in diploid and triploid hybrids deviated from the expectation. The results were discussed in relation to chromosomal disturbances that occurred in the hybrid tissues and that resulted in elimination of chromosomes and other effects.     

Nucleolus formation in structurally reconstructed barley karyotypes with six satellite chromosomes

Two structurally reconstructed karyotypes of Hordeum vulgare which, due to appropriate reciprocal translocations, contain three chromosome pairs with nucleolus organizing activity (chromosomes 5⁷, 6 and 7⁷ in translocation line T 21, chromosomes 3⁶, 6³, and 7 in translocation line T 627) have been studied with respect to the position and function of rDNA as inferrred from the pattern of nucleolus formation. The results obtained reveal quantitative relationships between the size of the secondary constriction (the amount of rRNA cistrons) and the number and size of nucleoli being formed. Quantitative and qualitative aspects of rDNA location and expression are being discussed.     

玉米rRNA基因的组织和表达模式

玉米（Zea mays）只有1对45S rDNA位点并在分裂期染色体形成次缢痕,是研究植物细胞rRNA基因组织和表达模式的简单模型。采用荧光原位杂交（fluorescence in situ hybridization,FISH）、CPD（PI与DAPI组合）染色和银染技术,分析了玉米根尖分生细胞rRNA基因的组织和表达模式。45S rDNA探针在所有间期细胞核中显示2种杂交信号：荧光强烈地位于核仁周边的纽,而相对较弱地分布于核仁内的点。在部分细胞中可观察到点与纽相连或从纽发出;点的数目越多,纽变得越小;点的数目多少与细胞的活性呈正相关。研究结果表明,纽代表了处于凝缩状态的非活性的rDNA染色质,纽解凝缩形成的点是rRNA基因活跃转录的细胞学表现;不同阶段间期核的点的数目变化反映了被活化的rRNA基因数目不同。间期和前期细胞的CPD染色和相继的银染结果显示,大部分rDNA染色质没有参与核仁的形成。rDNA FISH显示,同一间期细胞的2个同源rDNA位点的表达水平存在差异,同源染色体次缢痕的长度差异以及Ag-NOR和银染核仁的异态性进一步证实了这种差异的存在。FISH结果显示,早中期细胞的rDNA染色质相对解凝缩,银染在所有早中期细胞和部分中期细胞显示了明显的核仁,表明玉米的rRNA基因在有丝分裂早中期有较活跃的转录,其转录在晚中期才停止。     

玉米rRNA基因的组织和表达模式

玉米(Zea mays)只有1对45S rDNA位点并在分裂期染色体形成次缢痕, 是研究植物细胞rRNA基因组织和表达模式的简单模型。采用荧光原位杂交(fluorescence in situ hybridization, FISH)、CPD(PI与DAPI组合)染色和银染技术, 分析了玉米根尖分生细胞rRNA基因的组织和表达模式。45S rDNA探针在所有间期细胞核中显示2种杂交信号: 荧光强烈地位于核仁周边的纽, 而相对较弱地分布于核仁内的点。在部分细胞中可观察到点与纽相连或从纽发出; 点的数目越多, 纽变得越小; 点的数目多少与细胞的活性呈正相关。研究结果表明, 纽代表了处于凝缩状态的非活性的rDNA染色质, 纽解凝缩形成的点是rRNA基因活跃转录的细胞学表现; 不同阶段间期核的点的数目变化反映了被活化的rRNA基因数目不同。间期和前期细胞的CPD染色和相继的银染结果显示, 大部分rDNA染色质没有参与核仁的形成。rDNA FISH显示, 同一间期细胞的2个同源rDNA位点的表达水平存在差异, 同源染色体次缢痕的长度差异以及Ag-NOR和银染核仁的异态性进一步证实了这种差异的存在。FISH结果显示, 早中期细胞的rDNA染色质相对解凝缩, 银染在所有早中期细胞和部分中期细胞显示了明显的核仁, 表明玉米的rRNA基因在有丝分裂早中期有较活跃的转录, 其转录在晚中期才停止。     

Trisomic analysis of ribosomal RNA cistron multiplicity in barley (Hordeum vulgare L.)

Ribosomal RNA cistron numbers in all the seven primary trisomics of diploid barley (Hordeum vulgare L.) were determined by DNA-rRNA filter hybridisation. Trisomies for the nucleolus organiser (NO) chromosomes 6 and 7 showed the highest levels of rDNA (DNA complementary to rRNA) indicating the localisation of rRNA cistrons on the NOs. Chromosomes 6 and 7 possessed 1,580 and 2,690 rRNA (18S + 5.8S + 26S) cistrons respectively. Trisomics for the other chromosomes (except for 3) also displayed levels of rDNA significantly higher (22–32%) than the diploid controls although the dosage of NOs was not altered. These non-specific increases were also present in trisomics for 6 and 7 (NOs) which showed further increases equivalent to their respective contributions. The nonspecific increases due to trisomy is indicative of rDNA compensation. Such increases did not persist in diploid sibs of the trisomics, demonstrating the nonheritable nature of the compensation.     

Nucleolar number variation in Hordeum species,their haploids and interspecific hybrids

Nucleolar number variation has been investigated in root tip cells by cytologically determining the number of nucleoli per cell in autoploids and alloploids of Hordeum species, their haploids and interspecific hybrids. The nucleolus organisers in autoploid types of H. vulgare or H. bulbosum did not show any alterations irrespective of the ploidy level. The nucleolar number variation in these species results from a definite pattern of fusion and the maximum number of nucleoli per nucleus corresponds to the number of secondary constrictions. Nucleolus formation in alloploids and interspecific hybrids is impaired on some of the NORs, suggesting differential amphiplasty or nucleolar dominance. A comparison of nucleolar formation in the alloploid species (brachyantherum, arizonicum, procerum and parodii), their haploids, and the interspecific hybrids revealed different degrees of variation from the expected mean and maximum numbers of nucleoli. While the deviations in hybrids between alloploids (H. arizonicum and H. brachyantherum or H. procerum and H. brachyantherum) are marginal, nucleolar dominance is more pronounced in hybrids involving H. vulgare or H. bulbosum as one of the parents and is invariably associated with the disappearance of the secondary constriction(s) from the NOR(s) contributed by one of the parents, and the number of nucleoli is appropriately reduced.     

The genetic control of nucleolus formation in wheat

The wheat variety Chinese Spring has four pairs of nucleolus organisers of known rDNA content. The genetic control of these has been investigated in root tip cells by cytologically scoring the number of nucleoli per cell in (a) aneuploid derivatives each having a different dosage of a particular chromosome or chromosome arm and (b) in substitution lines where nucleolus organiser chromosomes have been replaced by homologues possessing different amounts of rDNA. It has been assumed that nucleolus organiser activity is correlated with nucleolus size and thus with the presence of a cytologically visible nucleolus. Those nucleolus organisers on chromosomes 1A and 5D, which together possess only 10% of the rDNA form a visible nucleolus only infrequently in the presence of the larger nucleolus organisers on chromosomes 1B and 6B. When a major pair of organisers on chromosomes 1B or 6B is deleted, the smaller nucleolus organisers form a visible nucleolus more frequently. Similarly, when the major nucleolus organisers are replaced by organisers with less rDNA, the smaller nucleolus organisers form visible nucleoli more frequently. When a small nucleolus organiser is replaced by one with much more rDNA, a larger nucleolus is formed. These and other findings lead to the general conclusions that there is a frequently, but not invariably, seen correlation between rRNA gene number and nucleolus size. However the relative size of the nucleolus formed depends principally upon the proportion of the total active rRNA genes in the cell which are localised at the nucleolus organiser in question. Varying the dosage of at least 13 non nucleolus organiser chromosomes also resulted in changes in the number of visible nucleoli per cell. This implies the genetic control of individual nucleolus organisers is complex. Inclusion in the wheat genome of the nucleolus organiser chromosome from Aegilops umbellulata, causes suppression of the wheat nucleolus organisers, the Aegilops umbellulata organiser remaining active. This suppression is similar to that observed in many interspecific plant and animal hybrids.     

Genetic variations in Lycoris radiata var. radiata in Japan

The genetic variations of Lycoris radiata var. radiata, a completely sterile triploid from Japan, were examined by comparing the nucleotide sequences of genomic DNA regions in 11 triploid strains sampled from Japan and four triploid strains sampled from China, and in two diploid strains of Lycoris radiata var. pumila, which is endemic to China and fertile. For this purpose, two genes were analyzed, the lectin gene in the nuclear genome and the maturase gene in the chloroplast genome. A clear genetic constancy was observed in their DNA nucleotide sequences. For both genes, completely identical nucleotide sequences were detected in the 11 Japanese and four Chinese triploid strains and also between the two Chinese diploid strains. However, some genetic variations were observed between the Japanese and Chinese triploid strains, and between the triploid and diploid strains. These results are consistent with the findings obtained from previous chromosome karyotype analyses and allozyme analyses. In addition, in our preliminary FISH analysis of the physical mapping of the rRNA gene family, the 18S-5.8S-26S rRNA and 5S rRNA loci were localized on six and four chromosomes, respectively. Regarding the 18S-5.8S-26S rRNA loci, two were associated with two SAT chromosomes. The remaining four were distinguished by having no secondary constriction. Localization of 5S rRNA loci to chromosome spreads revealed three sites on the proximal part of the long arm of three acrocentric chromosomes and one site on the distal part of the long arm of the SAT chromosome; the latter site was juxtaposed to the 18S-5.8S-26S rRNA loci. These findings indicate that L. radiata var. radiata is not a typical autotriploid. The present paper discusses the possible origin of L. radiata var. radiata from a diploid variety of L. radiata var. pumila, based on the molecular cytogenetic analysis and DNA sequence analysis.     

Physical mapping of the 18S-25S and 5S ribosomal RNA genes in diploid bananas

Fluorescent in situ hybridisation (FISH) was used to determine the number and distribution of the 18S-25S and 5S rDNA sites on mitotic chromosomes of 6 wild and 2 edible diploid (2n=22) accessions belonging to the two banana species, Musa acuminata and M. balbisiana. FISH with the 18S-25S probe resulted in signals on one pair of chromosomes, the position of signals corresponded to the secondary constriction at the end of a short arm. The intensity of labelling was different between the homologues and the larger site corresponded to a larger secondary constriction. This labelling pattern was observed consistently in all genotypes. On the other hand, differences in the number of 5S sites were observed between the accessions. While in some of the wild seeded species, the 5S rDNA was localised on two pairs of chromosomes, hybridisation signals appeared on three pairs of chromosomes in other wild accessions. Quite unexpectedly, only five sites of 5S rDNA were reproducibly observed in the two vegetatively propagated diploid edible cultivars, Pisang Mas and Niyarma Yik, evidence for structural heterozygosity. A dual colour FISH showed that in all accessions, the satellite chromosomes carrying the 18S-25S loci did not carry the 5S loci. The results demonstrate that molecular cytogenetics can be applied to Musa and that physical cytogenetic maps can be generated. This revised version was published online in July 2006 with corrections to the Cover Date.     

Variability of ribosomal DNA sites inFestuca pratensis, Lolium perenne, and their intergeneric hybrids, revealed by FISH and GISH

This study focuses on the variability of chromosomal location and number of ribosomal DNA (rDNA) sites in some diploid and autotetraploidFestuca pratensis andLolium perenne cultivars, as well as on identification of rDNA-bearing chromosomes in their triploid and tetraploidF. pratensis ×L. perenne hybrids. The rDNA loci were mapped using fluorescence in situ hybridization (FISH) with 5S and 25S rDNA probes, and the origin of parental genomes was verified by genomic in situ hybridization (GISH) withL. perenne genomicDNAas a probe, andF. pratensis genomic DNA as a block. FISH detected variation in the number and chromosomal location of both 5S and 45S rDNA sites. InF. pratensis mostly additional signals of 5S rDNA loci occurred, as compared with standardF. pratensis karyotypes. Losses of 45S rDNA loci were more frequent inL. perenne cultivars and intergeneric hybrids. Comparison of theF. pratensis andL. perenne genomes approved a higher number of rDNA sites as well as variation in chromosomal rDNA location inL. perenne. A greater instability ofF. pratensis-genome-like andL. perenne-genome-like chromosomes in tetraploid hybrids was revealed, indicating gains and losses of rDNA loci, respectively. Our data indicate that the rDNA loci physically mapped on chromosomes 2 and 3 inF. pratensis and on chromosome 3 inL. perenne are useful markers for these chromosomes in intergenericFestuca ×Lolium hybrids.     

Distribution of 5S and 35S rRNA gene sites in 34 Chenopodium species (Amaranthaceae)

Studies on Chenopodium chromosomes are scarce and restricted mainly to chromosome number estimation. To extend our knowledge on karyotype structure of the genus, the organization of 5S and 35S rRNA genes in Chenopodium chromosomes was studied. The rDNA sites were predominantly located at chromosomal termini, except in a few species where 5S rDNA sites were interstitial. The majority of the diploid species possessed one pair each of 35S and 5S rDNA sites located on separate chromosomes. Slightly higher diversity in rDNA site number was observed in polyploid accessions. One or two pairs of 35S rDNA sites were observed in tetraploids and hexaploids. Tetraploid species had two, four or six sites and hexaploid species had six or eight sites of 5S rDNA, respectively. These data indicate that, in the evolution of some polyploid species, there has been a tendency to reduce the number of rDNA sites. Additionally, polymorphism in rDNA site number was observed. Possible mechanisms of rDNA locus evolution are discussed. © 2012 The Linnean Society of London, Botanical Journal of the Linnean Society, 2012, ??, ??–??.     

45S rDNA在小麦及其近缘物种染色体上的分布

将染色体C-分带和原位杂交技术相结合,系统研究了45S rDNA在栽培一粒小麦、野生二粒小麦、普通小麦、大麦、簇毛麦、硬簇麦、六倍体燕麦及鹅观草等物种染色体上的分布情况。这些物种染色体的次缢痕区都有45S rDNA位点, 某些非随体染色体上也有45S rDNA位点分布。以小麦—鹅观草1Rk^#1二体附加系为材料,通过顺序C分带-FISH技术首次将一个45S rDNA定位到1Rk^#1染色体短臂末端。     

Physical mapping of rRNA genes by fluorescent in-situ hybridization and structural analysis of 5S rRNA genes and intergenic spacer sequences in sugar beet (Beta vulgaris)

A digoxigenin-labelled 5S rDNA probe (pTa-794) and a rhodamine-labelled 18S-5.8S-25S rDNA probe (pTa71) were used for double-target in-situ hybridization to root-tip metaphase, prophase and interphase chromosomes of cultivated beet,Beta vulgaris L. After in-situ hybridization with the 18S-5.8S-25S rDNA probe, one major pair of sites was detected which corresponded to the secondary constriction at the end of the short arm of chromosome 1. The two rDNA chromosomes were often associated and the loci only contracted in late metaphase. In the majority of the metaphase plates analyzed, we found a single additional minor hybridization site with pTa71. One pair of 5S rRNA gene clusters was localized near the centromere on the short arm of one of the three largest chromosomes which does not carry the 18S-5.8S-25S genes. Because of the difficulties in distinguishing the very similarly-sizedB. vulgaris chromosomes in metaphase preparations, the 5S and the 18S-5.8S-25S rRNA genes can be used as markers for chromosome identification. TwoXbaI fragments (pXV1 and pXV2), comprising the 5S ribosomal RNA gene and the adjacent intergenic spacer, were isolated. The two 5S rDNA repeats were 349 bp and 351 bp long, showing considerable sequence variation in the intergenic spacer. The use of fluorescent in-situ hybridization, complemented by molecular data, for gene mapping and for integrating genetic and physical maps of beet species is discussed.     

Chromosome instability in Hordeum vulgare x H. bulbosum hybrids

Chromosome number in Hordeum vulgare x H. bulbosum hybrids ranged between the haploid and diploid number but with peaks in frequency occurring at the 14 and 7 chromosome level. This was reflected in a gradual change from hybrid morphology to that of haploid H. vulgare. The rate of chromosome elimination differed significantly between hybrids, while within each hybrid, differences in mean chromosome number were recorded between and within individual tillers. An increase in temperature from 25–30° C caused a significant increase in the rate of elimination of H. bulbosum chromosomes.     

Nucleolar behaviour in Triticum

The maximum number of major nucleoli (macronucleoli) per nucleus of hexaploid, tetraploid and diploid wheat, Aegilops speltoides and Ae. squarrosa corresponded to the number of satellited chromosomes of each species. Smaller nucleoli (micronucleoli) were rare or absent in all of these species except the hexaploid, in which they were predominantly organized on chromosome arm 5D^s. — Fewer than the maximum number of macronucleoli in a mitotic interphase nucleus resulted from fusion of developing nucleoli. Enforced proximity of nucleolus-organizing regions resulted in more frequent fusion of nucleoli. — Analyses of related interphase nuclei showed that nucleoli, and hence probably chromosomes, undergo limited movement during mitotic interphase. These observations also indicate that specific chromosomes do not occupy specific sites in the interphase nucleus.     

Molecular cytogenetics and characterization of a ZZ/ZW sex chromosome system in Triportheus nematurus (Characiformes, Characidae)

Chromosomes of Triportheus nematurus, a fish species from family Characidae, were analyzed in order to establish the conventional karyotype, location of C-band positive heterochromatin, Ag-NORs, GC- and AT-rich sites, and mapping of 18S and 5S rDNA with fluorescence in situ hybridization (FISH). The diploid number found was 2n = 52 chromosomes in both males and females. However, the females presented a pair of differentiated heteromorphic chromosomes, characterizing a ZZ/ZW sex chromosome system. The Z chromosome was metacentric and the largest one in the karyotype, bearing C-positive heterochromatin at pericentromeric and telomeric regions. The W chromosome was middle-sized submetacentric, appearing mostly heterochromatic after C-banding and presenting heterogeneous heterochromatin composed of GC- and AT-rich regions revealed by fluorochrome staining. Ag-NORs were also GC-rich and surrounded by heterochromatic regions, being located at the secondary constriction on the short arms of the second chromosome pair, in agreement with 18S rDNA sites detected with FISH. The 18S and 5S rDNA were aligned in tandem, representing an uncommon situation in fishes. The results obtained reinforce the basal condition of the ZZ/ZW sex system in the genus Triportheus, probably arisen prior to speciation in the group.     

Ribosomal RNA genes in soybean and common bean: chromosomal organization, expression, and evolution

Ribosomal RNA (5S and 45S) genes were investigated by FISH in two related legumes: soybean [Glycine max (L.) Merr.] and common bean (Phaseolis vulgaris L.). These species are both members of the same tribe (Phaseoleae), but common bean is diploid while soybean is a tetraploid which has undergone diploidization. In contrast to ploidy expectations, soybean had only one 5S and one 45S rDNA locus whereas common bean had more than two 5S rDNA loci and two 45S rDNA loci. Double hybridization experiments with differentially labelled probes indicated that the soybean 45S and 5S rDNA loci are located on different chromosomes and in their distal regions. Likewise, the common bean 45S and 5S rDNA loci were on unique chromosomes, though two of the 5S rDNA loci were on the same chromosome. FISH analysis of interphase nuclei revealed the spatial arrangement of rDNA loci and suggested expression patterns. In both species, we observed one or more 5S rDNA hybridization sites and two 45S rDNA hybridization sites associated with the nucleolar periphery. The 45S rDNA hybridization patterns frequently exhibited gene puffs as de-condensed chromatin strings within the nucleoli. The other condensed rDNA sites (both 5S and 45S) were spatially distant from the nucleolus in nucleoplasmic regions containing heterochromatin. The distribution of rDNA between the nucleoplasm and the nucleoli is consistent with differential gene expression between homologous alleles and among homoeologous loci.     

The time rate and mechanism of chromosome elimination in Hordeum hybrids

Seed development at 20±1° C in continuous light was studied during the first 5 days after pollination in diploid Hordeum vulgare, diploid H. bulbosum and the cross, H. vulgare x H. bulbosum, where H. bulbosum chromosomes were eliminated. Developing seeds were fixed and stained at known intervals after pollination and the embryo sac contents dissected out for cytological examination. — In all cases, the pattern of development was similar to that previously described for the Triticeae. After intraspecific pollination, the rate of endosperm and embryo development was significantly faster in H. vulgare than in H. bulbosum. In hybrid tissues, the rate was intermediate, but often much nearer to that of H. vulgare at first. Elimination of H. bulbosum chromosomes occurred only during endosperm and embryo mitoses. Usually, 0–3 chromosomes were lost at any one division but up to 7 were lost at some. Elimination, which occurred as early as zygotic anaphase, was nearly or quite complete in all dividing cells in both embryo and endosperm after 5 days. The mean number of chromosomes lost per nucleus per nuclear cycle was low at first but rose rapidly and stayed high for about a day in each tissue before falling quickly. The rate of elimination in each tissue was maximal when that tissue first synthesized significant amounts of new cytoplasm (day 2 after pollination in the endosperm and day 3 in the embryo). At mitosis, chromosomes being eliminated differed from others only in failing to congress at metaphase or to reach a pole at anaphase or both. — It is noted that in several widely different examples where either haploids are produced when only hybrids are expected, or where chromosomes of one species are preferentially eliminated from hybrid cells, nucleolar activity was suppressed in chromosomes of the genome which was selectively or preferentially eliminated. Consequently, it is suggested that chromosome elimination in Hordeum hybrids may be caused by a disturbed control of protein metablism in hybrid seeds and perhaps H. bulbosum chromosomes are selectively eliminated because they are less efficient than H. vulgare chromosomes at forming normal attachments to spindle protein.