Intraspecific divergence in wheats of the Emmer group using in situ hybridization with the Spelt-1 family of tandem repeats

Fluorescence in situ hybridization (FISH) was used to study the distribution of Spelt-1 repetitive DNA sequences on chromosomes of 37 accessions representing eight polyploidy wheat species of the Emmer evolutionary lineage: Triticum dicoccoides Körn, T. dicoccum (Schrank) Schuebel, T. durum Desf., T. polonicum L., T. carthlicum Nevski, T. aethiopicum Jakubz., T. aestivum L., and T. spelta L. Substantial polymorphism in the number, distribution, and the sizes of the Spelt-1 loci was revealed. On the chromosomes of the accessions examined, Spelt-1 tandem repeats were found in seven different positions (per haploid chromosome set). These were “potential hybridization sites”, including the subtelomeric regions of either short or long arms of chromosomes 2A and 6B, the short arm of chromosome 1B, and the long arms of chromosomes 2B and 3B. However, in individual genotypes, only from one to three Spelt-1 loci were revealed. Furthermore, no hybridization with Spelt-1 probe was detected on chromosomes from 12 accessions. Thus, the total number of Spelt-1 sites in karyotypes varied from zero to three, with the average number of 1.16. This was substantially lower than in the species of the Timopheevi section and diploid Aegilops speltoides Tausch, a putative donor of the B genome. The decrease of the content of Spelt-1 sequences in the genomes of the Emmer group wheats in comparison with the species of the Timopheevii group and diploid Ae. speltoides was assumed to result from the repetitive sequences reorganization during polyploidization and the repeat elimination during wheat evolution.     

Flow cytometric analysis for ploidy level differentiation of 45 hairy vetch accessions

Determining the ploidy of plant germplasm is a necessary step in breeding or genetic studies in species. The purpose of this research was to determine the presence of ploidy level differentiation of hairy vetch (Vicia villosd) germplasm. Flow cytometry and root tip chromosome squashing methods were employed to assess 45 accessions labeled V. villosa available through the USDA germplasm collection. Flow cytometry determined that 43 of the accessions were 2C, one accession was 4C, and one accession was 6C. Analysis of accessions by root tip chromosome counts indicated that all accessions were diploid. The 2C accession contains 14 chromosomes and their chromosomes were approximately one-half and one-third in size as compared to the chromosomes of the 4C and 6C accessions, respectively. The 4C accession was observed to have 16 chromosomes and the 6C accession was observed to have 14 chromosomes. The large-scale differences in DNA amounts were due to chromosomal size variability as opposed to ploidy differences. This revealed the incidence of species misidentification of these two V. villosa accessions to be Vicia pannonica. All the V. villosa accessions were observed to be diploid and have similar DNA amounts. Flow cytometry proved to be useful in the efficient assessment of these accessions.     

Nuclear DNA content of Pinus sylvestris (L.) as determined by laser flow cytometry

Nuclear DNA content was determined in nuclei isolated from needles, stems and roots of in vitro grown seedlings and from megagametophytes and embryo of mature seeds in three accessions of Pinus sylvestris L. One accession was from Inari, northern Finland at timber line, and two accessions were from the Alpine region in Italy. Nuclei were mechanically isolated by a chopping method, stained with propidium iodide, and DNA content was determined using an EPICS PROFILE laser flow cytometer. Nuclei isolated from leaves of barley (Hordeum vulgare L. cv. Sultan; 2C=11.12 pg) were used as an internal standard for measurement of pine nuclei. Mean 1C nuclear DNA content of P. sylvestris was 27.88 pg as determined from megagametophyte tissue. Mean 2C value was 52.25 pg as determined from stem and root tissue, and 55.58 pg as determined from embryo tissue. The ratio of 2C to 1C value was 1.87 and 1.99, respectively. Extracts of nuclei from needles contained propidium iodide-absorbing debris which may have interfered with measurements and resulted in lower 2C values than those obtained from stem and root.     

Genetic Relationships Among Wild and Cultivated Accessions of Curry Leaf Plant (Murraya koenigii (L.) Spreng.), as Revealed by DNA Fingerprinting Methods

Murraya koenigii (L.) Spreng. (Rutaceae), is an aromatic plant and much valued for its flavor, nutritive and medicinal properties. In this study, three DNA fingerprinting methods viz., random amplification of polymorphic DNA (RAPD), directed amplification of minisatellite DNA (DAMD), and inter-simple sequence repeat (ISSR), were used to unravel the genetic variability and relationships across 92 wild and cultivated M. koenigii accessions. A total of 310, 102, and 184, DNA fragments were amplified using 20 RAPD, 5 DAMD, and 13 ISSR primers, revealing 95.80, 96.07, and 96.73% polymorphism, respectively, across all accessions. The average polymorphic information content value obtained with RAPD, DAMD, and ISSR markers was 0.244, 0.250, and 0.281, respectively. The UPGMA tree, based on Jaccard’s similarity coefficient generated from the cumulative (RAPD, DAMD, and ISSR) band data showed two distinct clusters, clearly separating wild and cultivated accessions in the dendrogram. Percentage polymorphism, gene diversity (H), and Shannon information index (I) estimates were higher in cultivated accessions compared to wild accessions. The overall high level of polymorphism and varied range of genetic distances revealed a wide genetic base in M. koenigii accessions. The study suggests that RAPD, DAMD, and ISSR markers are highly useful to unravel the genetic variability in wild and cultivated accessions of M. koenigii.     

Evaluation of Genetic Diversity and Development of a Core Collection of Wild Rice (Oryza rufipogon Griff.) Populations in China

Common wild rice (Oryza rufipogon Griff.), the progenitor of Asian cultivated rice (O. sativa L.), is endangered due to habitat loss. The objectives of this research were to evaluate the genetic diversity of wild rice species in isolated populations and to develop a core collection of representative genotypes for ex situ conservation. We collected 885 wild rice accessions from eight geographically distinct regions and transplanted these accessions in a protected conservation garden over a period of almost two decades. We evaluated these accessions for 13 morphological or phenological traits and genotyped them for 36 DNA markers evenly distributed on the 12 chromosomes. The coefficient of variation of quantitative traits was 0.56 and ranged from 0.37 to 1.06. SSR markers detected 206 different alleles with an average of 6 alleles per locus. The mean polymorphism information content (PIC) was 0.64 in all populations, indicating that the marker loci have a high level of polymorphism and genetic diversity in all populations. Phylogenetic analyses based on morphological and molecular data revealed remarkable differences in the genetic diversity of common wild rice populations. The results showed that the Zengcheng, Gaozhou, and Suixi populations possess higher levels of genetic diversity, whereas the Huilai and Boluo populations have lower levels of genetic diversity than do the other populations. Based on their genetic distance, 130 accessions were selected as a core collection that retained over 90% of the alleles at the 36 marker loci. This genetically diverse core collection will be a useful resource for genomic studies of rice and for initiatives aimed at developing rice with improved agronomic traits.     

Weitere Untersuchungen über Chromosomenverhältnisse und DNS-Gehalt bei Anuren (Amphibia)

The karyotypes of the toad Bufo marinus L. (2n=22) and the frogs Limnodynastes tasmaniensis Gthr. (2n=24), Rana temporaria L., R. esculenta L. (both 2n=26) and R. arvalis Nills. (2n=24) were analysed in colchicine treated leukocyte and spermatogonial metaphases and/or embryonic and larval mitoses. The DNA content of Feulgen stained erythrocyte nuclei was measured microspectrophotometrically. Heteromorphic sex chromosomes are absent in all species. L. tasmaniensis has the lowest DNA content among these species. The south American toad B. marinus shows a karyotype similar to the other known toad species and contains the same amount of DNA as the European species B. calamita with the lowest DNA amount among the European toads. In southern German populations of R. temporaria besides animals with the “standard”-karyotype (2n=26) individuals with 1 or 2, in rare cases with 3 or 4 supernumerary chromosomes have been found. The supernumeraries are heterochromatic and smaller than the smallest chromosome of the “standard”-karyotype. If only 1 or 2 supernumerary chromosomes are present, they seem to show normal mendelian inheritance as a rule. The observation of a few tadpoles with intraindividual different numbers of supernumeraries points to the occurrence of unequal distribution of these chromosomes in individuals containing a higher number of supernumerary chromosomes. The karyotype of R. esculenta is very similar to the “standard”-karyotype of R. temporaria, but the chromosomes of R. esculenta are somewhat longer than those of R. temporaria. R. esculenta contains about 54% more DNA than R. temporaria in the erythrocyte nuclei, so that it must be assumed that all chromosomes of R. esculenta contain more DNA than their homologues in R. temporaria. R. arvalis possesses about 28% more DNA than R. temporaria. It is supposed that these interspecific differences in DNA content of the Rana species — as observed earlier in Bufo species — are not a consequence of differential polyteny but are caused during evolutionary processes by local increase in DNA in the chromosomes of R. esculenta and R. arvalis.     

Flow cytometric analysis in Lagenaria siceraria (Cucurbitaceae) indicates correlation of genome size with usage types and growing elevation

The occurrence and extent of genome size variation within species is controversially discussed and thorough analyses are rare. Given the large morphological variation in Lagenaria siceraria (bottle gourds) and its wide distribution in Africa we here analysed (1) the genome size variation within cultivars of L. siceraria, (2) the correlation between genome size and morphological traits, and (3) the geographical patterns of DNA content within the species. We measured 2C-values of 366 individuals from 117 accessions of L. siceraria (2n = 22) from Africa, America and Asia via flow cytometry with propidium iodide as DNA stain. We found that 2C-value in L. siceraria (0.683–0.776 pg/2C) is about two times lower than previously reported and varies by about 12% among all accessions. Moreover, our results indicated a clear correlation of genome size with two different seed or usage types and with growing elevations in West Africa. Within the seed types genome size varies by 6.6 and 7.5%, respectively. The genome size differences in seed types of L. siceraria might indicate differences in their evolutionary history and necessitates a re-evaluation of the phylogenetic relationships within L. siceraria while the correlation between 2C-value and the elevation of the collecting sites might indicate an adaptation of genome size to an unknown ecological parameter connected to altitude.     

Phenolic constituents and genetic profile of Hypericum perforatum L. from India

The content of hypericins (hypericin and pseudohypericin), hyperforin, and flavonoids (rutin, hyperoside, quercitrin, and quercetin) and genetic profiles of eight accessions of Hypericum perforatum L., collected from different locations in India, have been determined. The secondary metabolite content was determined using a highly selective LC/MS/MS method. Pearson and Spearman's correlation coefficient were used to investigate the relationships between the secondary metabolites and a significant positive correlation was found between hypericin and pseudohypericin contents. Genetic profiling was undertaken using the random amplification of polymorphic DNA (RAPD) and single sequence repeat (SSR) methods. Among the 49 random primers used for the initial screening, only nine yielded polymorphic RAPD profiles. The SSR analysis shows that seven out of the 11 primers were polymorphic. There exists only a partial correlation between the chemical content and genetic profiling data among the accessions under study.     

Molecular and Cytogenetic Characterization of Wild Musa Species

The production of bananas is threatened by rapid spreading of various diseases and adverse environmental conditions. The preservation and characterization of banana diversity is essential for the purposes of crop improvement. The world''s largest banana germplasm collection maintained at the Bioversity International Transit Centre (ITC) in Belgium is continuously expanded by new accessions of edible cultivars and wild species. Detailed morphological and molecular characterization of the accessions is necessary for efficient management of the collection and utilization of banana diversity. In this work, nuclear DNA content and genomic distribution of 45S and 5S rDNA were examined in 21 diploid accessions recently added to ITC collection, representing both sections of the genus Musa. 2C DNA content in the section Musa ranged from 1.217 to 1.315 pg. Species belonging to section Callimusa had 2C DNA contents ranging from 1.390 to 1.772 pg. While the number of 45S rDNA loci was conserved in the section Musa, it was highly variable in Callimusa species. 5S rRNA gene clusters were found on two to eight chromosomes per diploid cell. The accessions were genotyped using a set of 19 microsatellite markers to establish their relationships with the remaining accessions held at ITC. Genetic diversity done by SSR genotyping platform was extended by phylogenetic analysis of ITS region. ITS sequence data supported the clustering obtained by SSR analysis for most of the accessions. High level of nucleotide diversity and presence of more than two types of ITS sequences in eight wild diploids pointed to their origin by hybridization of different genotypes. This study significantly expands the number of wild Musa species where nuclear genome size and genomic distribution of rDNA loci is known. SSR genotyping identified Musa species that are closely related to the previously characterized accessions and provided data to aid in their classification. Sequence analysis of ITS region provided further information about evolutionary relationships between individual accessions and suggested that some of analyzed accessions were interspecific hybrids and/or backcross progeny.     

The systematic value of nuclear DNA content for all species of Narcissus L. (Amaryllidaceae)

The taxonomy of all species of Narcissus (Amaryllidaceae), an important horticultural crop, has not been investigated recently. As a new approach, genome size was determined by flow cytometry with propidium iodide from 375 accessions. The somatic nuclear DNA contents (2C) were shown to range from 14 to 38 pg for the diploids. Narcissus assoanus and N. gaditanus are, based on their nuclear DNA content, removed from section Apodanthi and placed in a new section Juncifolii. The different ploidy levels and species involved were entangled for N . “fernandesii” s.l. and a new allotetraploid form is named here. Section Pseudonarcissus was much more heterogeneous in nuclear DNA content than expected. Sixty-five accessions of N. pseudonarcissus possessed, with 23.7 pg, similar amounts of DNA. However, several species from this section were clearly distinctive in nuclear DNA content. It runs from the diploid N. primigenius with 21.7 pg to the also diploid N. nevadensis with 38.2 pg. Also N. abscissus and N. moleroi are with about 26 pg clearly different from N. pseudonarcissus. For the first time, in 11 accessions, hexaploidy was found in N. pseudonarcissus ssp. bicolor. A new section Nevadensis with 30–39 pg of nuclear DNA was split off from the section Pseudonarcissus with now 21–27 pg. A nonoploid N. dubius with 96.3 pg has by far the highest amount of nuclear DNA and can be calculated to have the highest ploidy ever reported in Narcisssus. The total number of Narcissus species was determined as 36, nine more than in Flora Europaea and they were divided up in two subgenera and 11 sections. Flow cytometry is shown to produce easily obtainable and original systematic data that lead to new insights. Genome size or C-value turns out to be one of the most salient features to define the status of the species in the genus Narcissus.     

DNS-Gehalt und Chromosomenstruktur bei Amphibien

Comparative karyotype analysis and cytophotometric DNA measurements on further amphibian species (Hyla arborea, Bombina variegata, B. bombina, Triturus vulgaris, T. alpestris, and Salamandra salamandra) were carried out. The relative DNA values of the genomes determined for these species and other amphibians investigated earlier (Ullerich, 1966, 1967), already do nearly exclude the hypothesis that the interspecific differences in DNA content in frogs, toads, and salamanders are caused by differential polynemy of their chromosomes. Electron microscopic investigations on the DNA axes of lampbrush chromosomes of Bufo calamita, B. viridis, B. bufo, Rana esculenta, Bombina variegata, and Triturus alpestris treated with trypsin and ribonuclease confirm that the chromosomes of these species are not polynemic; in all species analysed the lampbrush chromosomes consist of the same number of DNA strands. The double-strandedness observed regularly in several segments of the chromatid axes in the loops as well as in the interchromomeric regions of all species suggest that the chromatids possibly are divided into half-chromatids. The minimum diameter of these two deoxyribonuclease-sensitive fibrills is 20–35 Å, whereas the chromatid axes in those segments which do not show double-strandedness mostly measure 40–65 Å. The high DNA amounts and interspecific differences in DNA content in the amphibian species analysed, probably in all amphibians, therefore must be caused during evolutionary processes by local increase (perhaps in a smaller extent also by-local decrease) in DNA in the chromosomes.     

Molecular cytogenetic analysis of Leymus racemosus chromosomes added to wheat

Five disomic, two double-disomic, and two ditelosomic addition lines and one disomic substitution line derived from the crosses of Triticum aestivum (2n=6x=42, AABBDD)×Leymus racemosus (2n= 4x=28, JJNN) were identified by C-banding analysis. The homoeology of the added Leymus chromosomes was determined by RFLP analysis. Four of five disomic addition lines belonged to group 2, 5, 6 and 7 chromosomes of L. racemosus; these were designated as 2Lr?1(NAU516), 5Lr?1(NAU504, NAU514), 6Lr?1 (NAU512), and 7Lr?1(NAU501). Two additional chromosomes, 1Lr?1 and 3Lr?1, were present in double-disomic addition lines 1Lr?1+5Lr?1 (NAU525) and 3Lr?1+7Lr?1(NAU524), respec-tively. In the disomic substitution line wheat chromosome 2B was replaced by L. racemosus chromosome 2Lr?1 (NAU551). Two telocentric chromosomes, 2Lr?2S (NAU509) and 7Lr?1S (NAU511), were isolated as ditelosomic addition lines. The study presented here provides the first evidence of homoeology of the added L. racemosus chromosomes with wheat chromosomes using DNA markers. Our data provide the basis for further directed chromosome engineering aimed at producing compensating wheat-L. racemosus translocation lines.     

THE DNA BASE COMPOSITION OF INDIVIDUAL CHROMOSOMES AND CHROMOSOME SEGMENTS FROM CHIRONOMUS TENTANS

The base composition of DNA was determined for individual chromosomes from the dipteran Chironomus tentans and for each one of six different segments of one of the chromosomes. The isolations were carried out by micromanipulation and the DNA purines were first extracted from the isolated components and afterwards separated by means of microelectrophoresis on a cellulose fiber. It was found that DNA from this material has an unusual composition corresponding to a guanine + cytosine content of about 30%. This composition was not a function of the polytenic condition but was also found for DNA from testis tissue. Furthermore Drosophila has a more traditional base composition for the bulk of DNA. Statistically significant variations in base data were found between whole chromosomes as well as between the segments from one of the chromosomes.     

Development and use of novel SSR markers for molecular genetic diversity in Italian millet (Setaria italica L.)

Italian millet is a commercially important grain crop. Nineteen polymorphic simple sequence repeat (SSR) markers, developed through construction of an SSR-enriched library from genomic DNA of Italian millet (Setaria italica L., P. Beauv.), were used for assessment of molecular genetic diversity against 40 accessions of S. italica. In total, 85 alleles were detected, with an average of 4.5 alleles per locus. The average gene diversity and polymorphism information content (PIC) values were 0.412 and 0.376, ranging from 0.02 to 0.88 and from 0.02 to 0.87, respectively. Values for observed (H _O) and expected (H _E) heterozygosities ranged from 0 to 0.73 and from 0.03 to 0.89, respectively. Nine loci deviated from Hardy-Weinberg equilibrium. The mean similarity coefficient among accessions was 0.6593. Based on the UPGMA algorithm, six different groups were successfully identified. In this clustering analysis, all Korean accessions grouped in one cluster, indicating that Korean accessions are genetically quite distinct from other introduced accessions. These newly developed microsatellite markers should be very useful tools for several genetic studies, including an assessment of diversity and population structure in Italian millet.     

FISHIS: Fluorescence In Situ Hybridization in Suspension and Chromosome Flow Sorting Made Easy

The large size and complex polyploid nature of many genomes has often hampered genomics development, as is the case for several plants of high agronomic value. Isolating single chromosomes or chromosome arms via flow sorting offers a clue to resolve such complexity by focusing sequencing to a discrete and self-consistent part of the whole genome. The occurrence of sufficient differences in the size and or base-pair composition of the individual chromosomes, which is uncommon in plants, is critical for the success of flow sorting. We overcome this limitation by developing a robust method for labeling isolated chromosomes, named Fluorescent In situ Hybridization In suspension (FISHIS). FISHIS employs fluorescently labeled synthetic repetitive DNA probes, which are hybridized, in a wash-less procedure, to chromosomes in suspension following DNA alkaline denaturation. All typical A, B and D genomes of wheat, as well as individual chromosomes from pasta (T. durum L.) and bread (T. aestivum L.) wheat, were flow-sorted, after FISHIS, at high purity. For the first time in eukaryotes, each individual chromosome of a diploid organism, Dasypyrum villosum (L.) Candargy, was flow-sorted regardless of its size or base-pair related content. FISHIS-based chromosome sorting is a powerful and innovative flow cytogenetic tool which can develop new genomic resources from each plant species, where microsatellite DNA probes are available and high quality chromosome suspensions could be produced. The joining of FISHIS labeling and flow sorting with the Next Generation Sequencing methodology will enforce genomics for more species, and by this mightier chromosome approach it will be possible to increase our knowledge about structure, evolution and function of plant genome to be used for crop improvement. It is also anticipated that this technique could contribute to analyze and sort animal chromosomes with peculiar cytogenetic abnormalities, such as copy number variations or cytogenetic aberrations.     

Differential rRNA Genes Expression in Hexaploid Wheat Related to NOR Methylation

Ribosomal RNA genes expression was analysed in 18 Portuguese bread wheat accessions (Triticum aestivum L. em Thell.), and the number of argyrophilic-nucleolar organiser regions (Ag-NORs) per cultivar was scored. Ten accessions presented six Ag-NORs per metaphase and six nucleoli per interphase, and eight accessions presented four Ag-NORs per metaphase and four nucleoli per interphase. Fluorescent in situ hybridisation with the 45S rDNA sequence pTa71 and genomic DNA from Aegilops tauschii (2n = 2x= 14, DD) confirmed the Ag-NOR location and identified the six satellited chromosomes as being the chromosome pairs 1B, 6B and 5D. The methylation pattern of the NOR region was studied by Southern blot using pTa71 as probe, which represented a complete rDNA unit of bread wheat. DNA digestions performed by MspI and HpaII resulted in different patterns revealing the high level of cytosine methylation at their recognition sequences. The total percentage values of NOR methylation indicated that wheat accessions with a maximum of four Ag-NORs were more heavily methylated at the NOR region than accessions with a maximum of six Ag-NORs.     

Sports and hybrids of triploid Hosta ‘Sum and Substance’ reveal chromosome losses and gains in all three apical layers

Ploidy distributions in L1, L2, and L3 apical or meristematic layers of 56 different plants (79 accessions) from vegetative and sexual progeny of the triploid Hosta ‘Sum and Substance’ were determined. Nuclear DNA contents (2C) of each apical layer were measured by flow cytometry with propidium iodide, and inferred ploidies are calculated. During tissue culture, the triploid (L1–L2–L3?=?3–3–3) Hosta ‘Sum and Substance’ exhibited chromosome losses resulting in somaclonal variants such as DNA aneuploids (e.g., 2.7–2.7–2.7) and aneuploidy chimeras (e.g., 3–2.7–2.7). Most interestingly, some chimeras exhibited even an increase in genome size as in plants with 3.5–3–3 configuration. Hybrids of H. ‘Sum and Substance’ show only losses of nuclear DNA compared with the original triploid. This gives rise to fully aneuploid plants and no chimeras. The measurements of Hosta ‘Sum and Substance’ lineages of sports and hybrids indicate that chromosome losses or gains are an important source of new cultivars. The complexity of chromosomal distribution in derivatives from the triploid Hosta ‘Sum and Substance’ is discussed.     

The DNA content of the individual chromosomes of rye

Summary The relative DNA content of individual chromosomes of Secale cereale L. was determined in 25 cells by microdensitometry of Feulgen stained preparations. The correlation value between relative DNA content and relative chromosome length was r=0.61 for all 328 chromosomes measured. However, the correlation coefficients calculated for individual cells as well as for mean values always approached 1. Taking into account the structure of rye chromosomes, this indicates that microdensitometric results may not be accurate when large quantities of heterochromatic DNA sequences are present in analyzed material.