Genome remodelling in three modern S. officinarumxS. spontaneum sugarcane cultivars

This study provides evidence that nuclear and chromosome remodelling has taken place in sugarcane, a vegetative crop with a complex genome derived from interspecific hybridizations between Saccharum officinarum and S. spontaneum. Detailed knowledge on the chromosomal compositions of the three clones analysed was acquired. (1) All hybrid cultivars were found to be aneuploid, affecting both parental genomes (having chromosomes in addition to full genomes), with chromosome numbers from 2n=102-106 in My5514 and up to 2n=113-117 in C236-51. (2) Comparative in situ hybridization showed that about 16% of these chromosomes are inherited from S. spontaneum and less than 5% are recombinant or translocated chromosomes containing sequences of both S. officinarum and S. spontaneum. (3) Differences between the observed DNA contents (estimated by flow cytometry) and those expected from the number of chromosomes, allowed the introgression of additional S. spontaneum or S. officinarum DNA pieces into the B42231 and C236-51 cultivars to be estimated. (4) Size heterogeneity between S. officinarum homologous chromosomes carrying the 18S-5.8S-25S and 5S ribosomal genes (identified by FISH with pTa71 and pTa794, respectively) confirms remodelling occurred by chromosomal interchange events, at least in these homologous chromosomes. (5) Simultaneous visualization of nucleoli and NORs showed that all 18S-5.8S-25S loci were potentially functional in the three clones, independent of their origin and size.     

Lateral root formation in Vicia faba L.

MI was found to decrease in LP with increase in cell number, and reached minimal values just before the emergence of LP to form lateral roots. These changes in MI have been correlated with the accumulation of cells in G1, 24 hours before a lateral root is formed. The durations of C and the various phases of the mitotic cycle were also investigated in LP, and compared with those in small primordia. As lateral root primordia increase in cell number, the durations of C, S and G2 become longer, while G1 becomes shorter. Also MI and GF decrease while the proportion of quiescent cells increases. Thus, there is a gradual decrease in the rate of cell proliferation as primordia increase in size. The changes which take place in these parameters during lateral root primordium development have been compared with the events which occur in seed proliferating tissues at the onset of dormancy.     

Quantitative chromosome map of the polyploid Saccharum spontaneum by multicolor fluorescence in situ hybridization and imaging methods

Somatic chromosomes of a wild relative of sugarcane (Saccharum spontaneum L.) anther culture-derived clone (AP 85-361, 2n=32) were identified and characterized by computer-aided imaging technology and molecular cytological methods. The presence of four satellite chromosomes and four nearly identical chromosome sets suggests that the clone is a tetrahaploid with the basic number x=8. A quantitative chromosome map, or idiogram, was developed using image analysis of the condensation pattern (CP) at the prometaphase stage of somatic chromosomes. The 45S and 5S ribosomal RNA gene (rDNA) loci were simultaneously visualized by multi-color fluorescence in situ hybridization (McFISH) and precisely localized to the regions of 3p3.1 and 6q1.3 on the idiogram. The simultaneous visualization of two sets of four ribosomal RNA genes confirms tetraploidy of this clone. This conclusion is consistent with results of molecular marker mapping. The quantitative chromosome map produced will become the foundation for genome analyses based on chromosome identity and structure. Previously impossible identification of small chromosomes and untestable hypotheses about the polyploid nature of plants can now be settled with these two approaches of quantitative karyotyping and FISH.     

Behaviour of ribosomal genes and nucleolar domains during activation in sugarcane (Saccharum officinarum L.) root primordia: from the unsoaked quiescent state to the steady state of proliferation

Changes in the organisation of ribosomal genes and nucleolar protein components were analysed in sugarcane (Saccharum officinarum L. cv Cristalina) from the time the quiescent primordia of the radical bands of nodes were stimulated to proliferate by water imbibition, until the meristematic population reached the steady state of proliferation in the growing roots. The kinetics of proliferation was evaluated by flow cytometry, and by the mitotic indexes, in roots of different lengths. All the quiescent cells were in a pre-replicative state (G0), with a 2C DNA content. During their activation process, they progressively reached the steady state of proliferation (mitotic index 7%), with rather fixed frequencies for cells with 2C (G1), 4C (G2), and values between them corresponding to cells replicating their DNA. Decondensation of the ribosomal genes was followed by FISH with probes for the major 25S and 18S rRNAs, and variations in the numbers of nucleoli were recorded in squashed cells after silver staining. The ultrastructure of nucleoli was analysed by electron microscopy, using the EDTA regressive staining for ribonucleoproteins. Quiescent nucleoli showed a clear segregation of their main components: Fibrillar Centre, Dense Fibrillar Component and Cajal's bodies while lacked any Granular Component. However the proliferating ones showed them highly intermingled, except for the Cajal's bodies. Our results revealed a high plasticity of the nucleolar domains in response to cell activation, and allowed to establish a correlation between dispersion of NORs with formation of small fibrillar centers and a nucleolus with all its domains intermingled, and the activation of cell proliferation during root sprouting.     

The response of apical meristems of primary roots of Vicia faba L. to colchicine treatments

The effects of 0.5% and 0.025% solutions of colchicine on the passage of cells through the mitotic cycle in apical meristems of primary roots of Vicia faba have been examined. Both treatments affected cell progression through the mitotic cycle in the same way: S and G1 were shorter, and G2 and mitosis longer, than the corresponding control values. The duration of the various phases of the mitotic cycle were similar to those reported previously for apical meristems of lateral roots though cycle time itself was longer. Recovery of root proliferating tissues from colchicine-induced inhibition of growth is correlated with the presence of quiescent cells. Meristems which have no quiescent cells do not recover from eolchicine treatment, while meristems which contain many quiescent cells recover faster than those which contain few. The growth fraction and the proportion of proliferating cells with a short cycle time are linearly related to the duration of the S period in root meristems.     

Nuclear genome size and genomic distribution of ribosomal DNA in Musa and Ensete (Musaceae): taxonomic implications

Nuclear DNA content and genomic distributions of 5S and 45S rDNA were examined in nineteen diploid accessions of the genus Musa representing its four sections Eumusa, Rhodochlamys, Callimusa and Australimusa, and in Ensete gilletii, which was the outgroup in this study. In the Eumusa (x = 11), 2C DNA content ranged from 1.130 to 1.377 pg, M. balbisiana having the lowest DNA content of all sections. M. beccarii (x = 9), a representative of Callimusa, had the highest 2C nuclear DNA content (1.561 pg). Species belonging to Rhodochlamys (x = 11) and Australimusa (x = 10) had 2C DNA contents ranging from 1.191 to 1.299 pg and from 1.435 to 1.547 pg, respectively. E. gilletii (x = 9) had 2C DNA content of 1.210 pg. The number of 5S rDNA loci in Musa varied from 4 to 8 per diploid cell. While different numbers of 5S rDNA loci were observed within Eumusa and Rhodochlamys, four 5S rDNA loci were observed in all accessions of Australimusa. M. beccarii (Callimusa) and E. gilletii contained 5S rRNA gene clusters on five and six chromosomes, respectively. The number of 45S rDNA loci was conserved within individual sections. Hierarchical cluster analysis of genome size, number of chromosomes and 45S rDNA sites suggested a close relationship between Rhodochlamys and Eumusa; Australimusa was clearly separated as were M. beccarii and E. gilletii. Within the Eumusa-Rhodochlamys group, M. balbisiana, M. schizocarpa and M. ornata formed distinct subgroups, clearly separated from the accessions of M. acuminata, M. mannii, M. laterita and M. velutina, which formed a tight subgroup. The results expand the knowledge of genome size and genomic distribution of ribosomal DNA in Musa and Ensete. They aid in clarification of the taxonomical classification of Musa and show a need to supplement the analyses on the DNA sequence level with cytogenetic studies.     

A combination of AFLP and SSR markers provides extensive map coverage and identification of homo(eo)logous linkage groups in a sugarcane cultivar

Sugarcane varieties are complex polyploids carrying in excess of 100 chromosomes and are derived from interspecific hybridisation between the domesticated Saccharum officinarum and the wild relative S. spontaneum. A map was constructed in Denotes variety covered by Australian plant breeding rights., an Australian cultivar, from a segregating F1 population, using 40 amplified fragment length polymorphism (AFLP) primer combinations, five randomly amplified DNA fingerprints (RAF) primers and 72 simple sequence repeat (SSR) primers. Using these PCR-based marker systems, we generated 1,365 polymorphic markers, of which 967 (71%) were single-dose (SD) markers. Of these SD 967 markers, 910 were distributed on 116 linkage groups (LGs) with a total map length of 9,058.3 cM. Genome organisation was significantly greater than observed in previously reported maps for Saccharum spp. With the addition of 123 double-dose markers, 36 (3:1) segregating markers and a further five SD markers, 1,074 markers were mapped onto 136 LGs. Repulsion phase linkage detected preferential pairing for 40 LGs, which formed 11 LG pairs and three multi-chromosome pairing groups. Using SSRs, double-dose markers and repulsion phase linkage, we succeeded in forming 127 of the 136 LGs into eight homo(eo)logy groups (HG). Two HGs were each represented by two sets of LGs. These sets of LGs potentially correspond to S. officinarum chromosomes, with each set aligning to either end of one or two larger LGs. The larger chromosomes in the two HGs potentially correspond to S. spontaneum chromosomes. This suggestion is consistent with the different basic chromosome number of the two species that are hybridised to form sugarcane cultivars, S. spontaneum (x=8) and S. officinarum (x=10), and illustrates the structural relationship between the genomes of these two species. The discrepancy of coverage between HGs highlights the difficulty in mapping large parts of the genome.     

甘蔗细茎野生种云南不同生态类型的RAPD分析

利用25个随机引物对来自云南不同生态类型的82份甘蔗细茎野生种（Saccharnm spontanenm L．）和4份国外种材料进行RAPD标记,结果表明：云南甘蔗细茎野生种不同生态类型的遗传变异较大,具有丰富的遗传多样性,低续度类型的遗传多样性明显高于高纬度类型,在相同的纬度范围内,随着海拔的升高,其多态性逐渐减少,基于分子聚类分析,86份材料被划分为8个不同群体,表现出明显的地理分布的特点,结果初步证明了云南甘蔗细茎野生种可能起源于云南南部低海拔,低纬度地区,而后逐渐向高海拔,高纬度的西北和东北部演化,扩散,提出了云南南部可能是野生甘蔗起源中心之一的观点。     

Asynchrony of DNA replication in the chromosomes of Luzula

Seedlings of Luzula purpurea (2n=6) were placed in contact with H³-thymidine for 30 minutes. After removal of the isotope the roots and leaf primordia were fixed at intervals between 0 and 14 hours. The percentage of labelled mitoses follows a very close curve in roots and leaf primordia. In both tissues the value of G₂ is approximately 3 to 4 hours and of S circa 8 hours. DNA replication in the chromosomes of L. purpurea is asynchronous. The discontinuous DNA synthesis discloses that Luzula chromosomes are composed of many segments replicating independently of each other. The results support a polycentric rather than a completely diffuse kinetochore system in this species.     

Detailed alignment of saccharum and sorghum chromosomes: comparative organization of closely related diploid and polyploid genomes.

The complex polyploid genomes of three Saccharum species have been aligned with the compact diploid genome of Sorghum (2n = 2x = 20). A set of 428 DNA probes from different Poaceae (grasses) detected 2460 loci in F1 progeny of the crosses Saccharum officinarum Green German x S. spontaneum IND 81-146, and S. spontaneum PIN 84-1 x S. officinarum Muntok Java. Thirty-one DNA probes detected 226 loci in S. officinarum LA Purple x S. robustum Molokai 5829. Genetic maps of the six Saccharum genotypes, including up to 72 linkage groups, were assembled into "homologous groups" based on parallel arrangements of duplicated loci. About 84% of the loci mapped by 242 common probes were homologous between Saccharum and Sorghum. Only one interchromosomal and two intrachromosomal rearrangements differentiated both S. officinarum and S. spontaneum from Sorghum, but 11 additional cases of chromosome structural polymorphism were found within Saccharum. Diploidization was advanced in S. robustum, incipient in S. officinarum, and absent in S. spontaneum, consistent with biogeographic data suggesting that S. robustum is the ancestor of S. officinarum, but raising new questions about the antiquity of S. spontaneum. The densely mapped Sorghum genome will be a valuable tool in ongoing molecular analysis of the complex Saccharum genome.     

Lack of Mitotic Delays at the Onset of Proliferation in Dormant Root Primordia Challenged by Ionizing Radiation

X-rays at doses between 2.5 and 20 Gy were applied to Allium cepa L. bulbs containing either dormant root primordia (before water imbibition) or actively proliferating meristems. Irradiation of the primordia that were enriched in G₀ cells neither delayed proliferation onset nor root sprouting. Under both protocols, irradiation decreased the final length of the roots to about 60 % (at 20 Gy) of that reached by the unirradiated controls. Irradiation of the proliferating meristems increased the mitotic index at some fixation times. This could not be due to a rise in the cell entry into mitosis, as the rate of root growth decreased simultaneously. The increased mitotic index should be the consequence of a delay in the relative time taken by mitosis in the whole cycle time. Lengthened mitosis probably allows the post-replicative repair of most DNA lesions, as the frequency of interphases with micronuclei was higher in the cells which were irradiated when still dormant than in those irradiated when cycling. Thus, the mitotic delays should be the consequence of a checkpoint pathway activated by the presence of DNA damage. This feedback mechanism seems only to develop after cell proliferation is restored. This revised version was published online in July 2006 with corrections to the Cover Date.     

Inference of subgenomic origin of BACs in an interspecific hybrid sugarcane cultivar by overlapping oligonucleotide hybridizations

Sugarcane (Saccharum spp.) breeders in the early 20th century made remarkable progress in increasing yield and disease resistance by crossing Saccharum spontaneum L., a wild relative, to Saccharum officinarum L., a traditional cultivar. Modern sugarcane cultivars have approximately 71%-83% of their chromosomes originating from S. officinarum, approximately 10%-21% from S. spontaneum, and approximately 2%-13% recombinant or translocated chromosomes. In the present work, C(0)t-based cloning and sequencing (CBCS) was implemented to further explore highly repetitive DNA and to seek species-specific repeated DNA in both S. officinarum and S. spontaneum. For putatively species-specific sequences, overlappping oligonucleotide probes (overgos) were designed and hybridized to BAC filters from the interspecific hybrid sugarcane cultivar 'R570' to try to deduce parental origins of BAC clones. We inferred that 12?967 BACs putatively originated from S. officinarum and 5117 BACs from S. spontaneum. Another 1103 BACs were hybridized by both species-specific overgos, too many to account for by conventional recombination, thus suggesting ectopic recombination and (or) translocation of DNA elements. Constructing a low C(0)t library is useful to collect highly repeated DNA sequences and to search for potentially species-specific molecular markers, especially among recently diverged species. Even in the absence of repeat families that are species-specific in their entirety, the identification of localized variations within consensus sequences, coupled with the site specificity of short synthetic overgos, permits researchers to monitor species-specific or species-enriched variants.     

Molecular recombination and the repair of DNA double-strand breaks in CHO cells.

Molecular recombination and the repair of DNA double-strand breaks (DSB) have been examined in the G-0 and S phase of the cell cycle using a temperature-sensitive CHO cell line to test i) if there are cell cycle restrictions on the repair of DSB's' ii) the extent to which molecular recombination can be induced between either sister chromatids or homologous chromosomes and iii) whether repair of DSB's involves recombination (3). Mitomycin C (1-2 micrograms/ml) or ionizing radiation (50 krad) followed by incubation resulted in molecular recombination (hybrid DNA) in S phase cells. Approximately 0.03 to 0.10% of the molecules (number average molecular weight: 5.6 x 10(6) Daltons after shearing) had hybrid regions for more than 75% of their length. However, no recombination was detected in G-0 cells. Since the repair of DSB was observed in both stages with more than 50% of the breaks repaired in 5 hours, it appears that DSB repair in G-0 cells does not involve recombination between homologous chromosomes. The possibility is not excluded that repair in G-0 cells involves only small regions (less than 4 x 10(6) Daltons).     

割手密2C型蛋白磷酸酶ScPP2C基因的克隆与表达分析

割手密由于具有抗病、抗虫、抗逆等潜在的有利基因,历来被用于甘蔗的抗性遗传改良。在割手密中克隆编码2C型蛋白磷酸酶(protein phosphatase 2C, PP2C)的基因,并分析其在干旱胁迫下的差异表达。本研究利用RT-PCR方法从割手密叶片中克隆得到PP2C基因的CDS全长序列,利用生物信息学在线软件对PP2C蛋白的理化性质、蛋白结构进行预测分析,并采用实时荧光定量的方法分析该基因在不同干旱处理下的差异表达。结果显示从割手密中克隆到一个全长951 bp编码316个氨基酸的2C型蛋白磷酸酶基因,命名为ScPP2C,GenBank登录号为MG322120。荧光定量分析表明,随着干旱胁迫时间的延长,其表达量呈先上调后下调的表达模式,说明该基因是干旱胁迫诱导型基因。本研究通过挖掘甘蔗野生种割手密中的抗旱新基因ScPP2C,为甘蔗野生种质资源开发利用、转基因抗旱甘蔗新品种的选育提供了参考依据。     

Cell-specific endopolyploidy in developing Artemia

Summary Cells in developing Artemia franciscana SFB demonstrated tissue-specific differences in DNA content, as determined by fluorescence intensity of bisbenzimide-stained nuclei and by nuclear area. The general epidermis comprised proliferating diploid (2C) cells. The setal cells had 4C–8C DNA content and did not divide during the first two instars. Salt gland cells were polyploid (>8C) and also did not undergo mitosis. Neural cells in the brain were diploid and were replicating. Cells in the thorax region of the gut had a 4C–8C DNA content and were proliferating. The muscle cells in the cephalic appendages contained 2C non-replicating nuclei. Only diploid epidermal cells were involved in segment morphogenesis. There was no difference in number of chromosomes (n=42) in the epidermal cells and the gut cells, indicating that the tissue-specific endopolyploidy was due to endoreduplication.     

Studies on the Growth in Culture of Plant Cells: XVIII. NUCLEAR CYTOLOGY OF ACER PSEUDOPLATANUS SUSPENSION CULTURES

Established suspension culture strains of Acer pseudoplatanuswere analysed by chromosome counting and microdensitometricDNA measurements of individual nuclei. Comparison with the root-tipcomplement (2n=4x=52) showed the cultures to be entirely aneuploidwith modal chromosome numbers of approximately 75 and 135 plussome cells with 250–350. Analysis of the frequency distributionsof nuclear DNA values through the growth cycle of a batch cultureshowed that stationary phase cells accumulated in G1 of thecell cycle. Stationary phase DNA distributions could thus beused to indicate the chromosomal status of a culture withoutthe complication of G1 and G2 DNA values for each chromosomenumber. Root-tip and cultured cells showed a close correlationbetween DNA content and chromosome number indicating that structuralchanges and loss of chromosomes had been at random.     

RFLP linkage map and genome analysis of Saccharum spontaneum.

An RFLP linkage map of the wild sugarcane species Saccharum spontaneum L. (2n = 8x = 40-128) was constructed, comprising 216 loci, detected by 116 DNA probes, and distributed over 44 linkage groups. At a density of at least one marker every 25-cM interval, the coverage of the genome was estimated as 86%. For the generation of RFLP markers, probes were surveyed from seven DNA libraries: three sugarcane cDNA, one oat cDNA, one rice cDNA, and one barley cDNA, as well as one sugarcane genomic. Sixty-two maize genomic clones that were previously mapped on maize were used to initiate a comparative map between the sugarcane, sorghum, and maize genomes. Based on the RFLP segregation data, we conclude that this species is an autopolyploid, with an estimated genome size of 2107 cM.     

Genetic segregation of microsatellite markers in Saccharum officinarum and S. spontaneum

Genetic mapping techniques can be used to study the interaction between two different genomes after hybridization. This study investigated a Saccharum officinarum (Green German or GG, 2n approximately 11x approximately 110) x S. spontaneum (IND 81-146 or IND, 2n approximately 7x approximately 56) interspecific cross. Segregation of 193 microsatellite (SSR) loci was evaluated in the F(1) progeny of 169 full-sibs of the cross. Following the two-way pseudo-testcross strategy and 'cross pollination' population type, linkage groups (LG) and phases were established for each parent map, using the criteria of LOD score > or = 3.0 and a maximum recombination frequency of 0.35. Of the 193 markers analyzed, 61 were IND-specific, 106 were GG-specific, and 26 were heterozygous in both parents. About 78% of the markers segregated in a Mendelian fashion and 22% were distorted (as evaluated by chi(2)-tests, P < or = 0.01). The GG map included 91 marker loci arranged into 25 LG covering 1180 cM of the officinarum genome. The IND map consisted of 46 marker loci assembled into 10 LG, which spanned 614 cM of the spontaneum genome. A specific chromosome associated with segregation distortion was detected in the female (GG) genome only, probably as a result of double reduction. The segregation patterns of the marker loci indicated a centromere-driven distortion process with the shared allelic markers (as putative centromeres) regulating the placement and association of markers with opposite phase (coupling vs repulsion) and dosage on either side. Although incomplete, the framework maps were informative with respect to segregation distortion, chromosome fusion, rearrangements, and translocations, observed in both parental genomes as a result of their merger.     

The amounts of nuclear DNA and the duration of DNA synthetic period (S) in related diploid and autotetraploid species of oats

The relative amounts of nuclear DNA of root meristematic cells of two related diploid Avena species, A. strigosa 2x and A. pilosa, which have different karyotypes, and an autotetraploid of one, A. strigosa 4x, were measured by Feulgen microspectrophotometry. The durations of various periods of their mitotic cycles were studied by autoradiography of cells pulse-labeled with tritiated thymidine. The results show that the autotetraploid, with twice the amount of nuclear DNA of its diploid, has the same duration of S period as the diploid, while A. pilosa, with intermediate nuclear DNA content, has a longer S period. These results support the hypothesis that homologous chromosomes or genomes require similar duration for their DNA synthesis and suggest that the structures of chromosomes are involved in temporal control of the DNA synthesis in cells.     

DNA amounts and chromatin compactness in Vicia

2C DNA amounts and areas of chromatin were determined with a M 86 Vickers microdensitometer in 56 species of Vicia (x=5, 6, 7), exhibiting large differences in chromosome size. There were significant differences between the species both in DNA content and chromatin area. The nuclear DNA amounts range from 3.85 to 27.07 pg. DNA distribution appears discontinuous; species cluster into distinct groups and the average nuclear DNA amount separating each successive pair is approximately the same (2.23 pg). The compaction of DNA in interphase nuclei increases with increasing DNA amount, which is, at least partly, due to a disproportionate increase in the heterochromatin relative to the euchromatin component of DNA. Comparisons of DNA readings at various stages of the cell cycle show that the DNA amounts are underestimated by microdensitometry in nuclei with high DNA density. Estimation of relative DNA content and area of individual chromosomes were made in twelve species. The results show that changes in DNA content within chromosomes affect the degree of metaphase coiling in an orderly fashion.