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Prairie cordgrass (Spartina pectinataLink) is a warm season (C4) perennial grass that could be a potential candidate for studying polyploidization and its effects on environmental adaptability. Polyploidy plays an important role in prairie cordgrass's adaptation and distribution on different ecoregions in the North American Prairie. However, the genetic backgrounds of polyploidy prairie cordgrass distributed in the different edaphic regions are different. Thus, to extend our knowledge of the effect of polyploidy on environmental adaptation, it is necessary to have diverse ploidy levels of plants with a similar genetic background. Tetraploid prairie cordgrass, IL‐102 (2n= 4x= 40), was used as explant material to generate octoploids. Germinated seeds were treated with 0.00, 0.03, 0.06, 0.1, and 1% colchicine or 0.000, 0.002, 0.003, 0.005, and 0.007% oryzalin solution with 0.5% dimethyl sulfoxide (DMSO) for 6, 12, or 24 h. Ploidy levels of treated seedlings were determined by flow cytometric analysis and their putative ploidy were confirmed by the chromosome count. Our results show that stable octoploid plants (2n= 8x= 80) were obtained directly by treating with antimitotic agents or by propagating cytochimeric plants. Induced octoploid plants showed increased genome size and stomata size compared with tetraploid plants. By obtaining different cytotypes in the same genetic background, sole effects of ploidy variations on agronomic traits can be determined.

The next generation of bioenergy crops will be grown on marginal lands. Prairie cordgrass (Spartina pectinataLink) is well suited to marginal land because of its tolerance to stresses such as salinity and water fluctuations. Developing prairie cordgrass as an energy crop requires genomic information such as genomic size and ploidy level. The 2C nuclear genome size of 11 prairie cordgrass populations, originating from Illinois, North Dakota, and South Dakota, were determined by flow cytometry using somatic G1 nuclei, and the ploidy variation confirmed by counting the chromosome number. One population from each of North Dakota and South Dakota, as well as the Red River, had genome sizes of 3.1 pg of DNA per G1 somatic nucleus whereas, the majority of the eight populations from Illinois had a genome size of 1.6 pg. One plant from one of the Illinois populations had an estimated genome size of 2.3 pg. These genome sizes of 3.1 and 1.6 pg, along with their respective chromosome numbers of 80 and 40 indicate octoploid and tetraploid plants. The chromosome number of 60 observed in the plant with 2.3 pg indicated that this plant was hexaploid. This study provides the first estimates of genome size for prairie cordgrass and is also the first to report observed hexaploidy in this species. This genomic information provides critical knowledge for designing efficient breeding schemes for plant improvement.

The perennial grass, Miscanthus×giganteusis a sterile triploid, which due to its growth rate and biomass accumulation has significant economic potential as a new bioenergy crop. The sterility associated with the triploid genome of this accession requires labor‐intensive vegetative, instead of seed propagation for potential commercial production. Chromosome doubling was used to produce hexaploid plants in an effort to restore fertility to M×giganteus. Tissue culture derived calli from immature inflorescences were treated with the antimitotic agents, colchicine and oryzalin in liquid and solid media. Calli survival rate decreased with increasing concentrations and durations of colchicine or oryzalin treatments and ranged from 0% to 100%. Nuclear DNA content, as determined by flow cytometry, indicated that the frequency of chromosome‐doubled calli varied between compounds and concentrations with the greatest proportion of callus doubling observed using 2‐day treatments of 15 μmoryzalin (78%) or 939 μmcolchicine (67%). Liquid media treatments were more effective than solid gels for chromosome doubling. Although oryzalin was effective at chromosome doubling, it inhibited callus growth and plant regeneration frequency. Seven hexaploid plants with doubled DNA content were generated, which displayed increased stomata size (30.0±0.2 μm) compared with regenerated triploid M. ×giganteusplants (24.3±1.0 μm). Following clonal replication these plants will be evaluated for growth rate, biomass accumulation, and pollen viability. Successful chromosome doubling and plant regeneration of M.×giganteussuggests that ploidy manipulation of this plant and its parental species (Miscanthus sinensisand Miscanthus sacchariflorus) could be a means to access genetic variability for the improvement of Miscanthusas a biofuel/bioenergy crop.

Weedy Amaranthusspecies pose a serious threat to agriculture. One of the areas that causes the greatest concern is development of herbicide resistance and subsequent transfer of herbicide resistance genes among the species. To determine the potential impact of interspecific hybridization, one must first be able to detect such hybrids. To determine if genome size could be useful in the detection of interspecific hybrids among the weedy Amaranthusspecies, the genome sizes of the weedy Amaranthusspecies sympatric in Illinois were analyzed. In a series of experiments, the genome sizes of these species were determined by flow cytometric analysis. A significant variation was observed with respect to nuclear DNA content in the eight species examined. The genome sizes ranged from ≈0.95 pg in A. palmerito ≈1.4 pg in A. tuberculatusOverlap of genome sizes among the species does exist; however, due to the reproductive biology of the species, this overlap does not preclude the detection of interspecific hybrids. Any dioecious weedy Amaranthusplant in Illinois that has a genome size between 1.3 pg and 1.1 pg is probably a hybrid. Thus, the potential for genome size determination to reveal interspecific hybrids among weedy Amaranthusspecies has been demonstrated.

Reports of genome size variation in soybean [Glycine max(L.) Merr.] have ranged from 40 to 0%. This wide range has resulted in doubts of the existence of intraspecific DNA variation in soybean. Eighteen soybean lines were analyzed by flow cytometry to determine their genome size. The lines were selected on the basis of diversity of origin. Intraspecific genome size variation was observed at approximately 4%. To ensure that the variation observed was reproducible and not due to technique error, the two highest and lowest genomes size accessions were reanalyzed. The order and variation observed between the high and low genome size accessions were maintained. To ensure further that the differences were reproducible, seeds from the two highest and lowest genome size accessions were planted in different locations in the USA, grown to maturity, harvested, and the seeds returned to Illinois. The harvested seed was analyzed and again the order and variation in genome size between the high and low genome size accessions were similar to the previous two analyses even though more than 1 yr had passed between the analysis. In addition, two experiments using Amaranthus palmeriS. Wats as an internal standard were conducted. In both of these experiments, the observed variation between the previously reported high and low genome size soybean lines was approximately 1 to 2%. The variation between the high and low genome size soybean lines is reproducible. The variation reported here indicates that the DNA amount variation is between 1 and 4%, lower than was originally reported.

For improvement of hairy vetch (Vicia villosaRoth) as a winter cover crop, it is necessary to gain insight into the magnitude of variability present in the species. This study was conducted to assess the sources of genetic and phenotypic variability of V. villosaaccessions. Thirteen morphological and agronomic traits were measured on 42 populations of V. villosaand two populations of V. pannonicaCrantz in field studies at Urbana, IL, for 2 yr. These measurements include initial seed weight, germination, stem length, stem width, leaf length, and leaf width in the fall and spring, winter survival, biomass, and a ratio of organic C to total N (C:N ratio) of the plants. The multivariate data set was analyzed by canonical discriminant analysis (CDA) in combination with a clustering procedure. In this analysis, the first two canonical variates were significant and accounted for 94% of the among‐accession variability. The canonical variates indicated that fall and spring measurements of the leaf and stem, and C:N ratio are the most differentiating traits among the accessions. The canonical variates were used to cluster the accessions into four subgroups on the basis of the differentiating traits. Canonical discriminant analysis was useful in identifying the genetic variation and the traits that better describe the variation among hairy vetch populations. Cluster analysis was successful in differentiating the accessions into similar subgroups on the basis of the measured traits. Plant breeders can use the information on variation among Viciaaccessions and focus on traits of particular significance.

Toxic agents in the environment pose serious threats to ecosystems and to the public health. The single cell gel electrophoresis (SCGE) or Comet assay quantitatively measures genomic damage as DNA strand breaks. The micronucleus (MCN) test is an established assay that measures chromosomal damage. Micronuclei are formed from chromosome fragments or from whole chromosomes that have not undergone mitosis properly. This test is usually conducted microscopically. However, micronuclei can also be analyzed using flow cytometry. Chinese hamster ovary (CHO) cells were exposed to ethylmethanesulfonate (EMS), for 4 h in a total volume of 25 μl. These cells were immediately analyzed for genomic DNA damage by SCGE. In concurrent parallel experiments, CHO cells were treated with EMS in 6-well plates for 4 h, the cells were washed and fresh medium was added. The cells were allowed to grow for 45 to 48 h to express micronuclei. The data demonstrated that both DNA strand breaks and micronuclei were induced in a significant and concentration-dependent manner. There was a significant and high correlation (r=0.91; P≤0.001) between the acute induction of DNA strand breaks and the subsequent generation of micronuclei. These data indicate that using molecular and computer technologies, the genotoxic impact of toxic and environmental agents can be rapidly and comprehensively evaluated in mammalian cell systems. Teratogenesis Carcinog. Mutagen. Suppl. 2:1–11, 2003. © 2003 Wiley-Liss, Inc.

In several plant species, sticky chromosomes are a consequence of genetic mutations or environmental effects on mitosis and meiosis. Sticky chromosomes result in an unequal distribution of genetic material in daughter cells. This unequal distribution is hypothesized to result in an increase in the coefficient of variation (CV) of the G1 peak of dividing cells. The st1 mutant and a nonmutant line in the same genetic background of maize (Zea mays L.) were planted in a soilless mix. A wheat (Triticum aestivum L. em thell.) line was grown in both low and high aluminum-saturated soil. Both plant species were assessed for sticky chromosomes by Feulgen-stained mitotic analysis and flow cytometric analysis of propidium iodide (PI)-stained G1 nuclei. In the st1 mutant, a significant increase in the number of abnormal anaphase figures was observed. An increase in abnormal mitotic figures was observed in wheat plants grown in aluminum soil. Using flow cytometry, an increase in the CV of the G1/G0 peak was seen in the maize mutant and in wheat grown at high levels of aluminum saturation. This increase correlated with the number of abnormal anaphase cells observed. Flow cytometry was sensitive enough to detect the intraplant nuclear DNA variation associated with sticky chromosomes within a plant. Cytometry 49:36–41, 2002. © 2002 Wiley-Liss, Inc.

Many times small differences in genome size are reported between or within plant species in which no cytologic confirmation is made. Attempts to repeat these studies have met with limited success. The controversy then becomes whether or not these small differences that were not confirmed cytologically are real. The present study was undertaken to determine if the ≈1% nuclear variation seen by flow cytometry among wheat lines selected for aluminum response was due to actual chromatin differences. The three parental wheat cultivars used in the aluminum selection along with the isolines resulting from the selection were analyzed. One parental line had previously been reported to have alien chromatin substituted for the corresponding wheat homoeologous chromatin. Genomic in situ hybridization was used to determine the presence or absence of rye chromatin in three cultivars and six near-isolines of wheat. Upon observing metaphase chromosomes of the Century parent and its isolines, two of the chromosomes were observed to be one-half yellow-orange, indicating rye chromatin with the remaining portion of the chromosomes and the other 40 wheat chromosomes having no label indicating wheat chromatin. In the Chisholm parent and its isolines, none of the chromosomes were labeled, indicating the absence of rye chromatin. In addition, none of the third parents' chromosomes had the rye yellow-orange signal. The wheat lines with the larger DNA contents were observed to have alien DNA present. DNA differences between the normal wheat chromosomes and the substituting alien chromatin were calculated based on total chromosome length. The increase in genome size of the wheat lines containing the alien chromatin appears to be the result of the alien chromatin having ≈43% more DNA than the wheat chromatin it is replacing. Thus, the small DNA difference previously reported by flow cytometry was demonstrated to be a real DNA variation due to the presence of small fragments of alien chromosomes added to the wheat genome. Cytometry 41:36–40, 2000 © 2000 Wiley-Liss, Inc.

Commercial products of agronomic crop plants may become a reliable and inexpensive source of phytonutrients, such as antimutagenic food supplements. We previously demonstrated that PCC, an ethanol extract of a commercial soybean processing by-product, was able to repress induced genomic DNA damage, whole cell clastogenicity, and point mutation in mammalian cells. In this paper we separated PCC into a series of chemically defined fractions and determined their ability to repress induced mutagenic damage in Chinese hamster lung cells, Chinese hamster ovary cells and human lymphocytes. Fraction PCC70 (PCC 70% methanol eluate) contained the flavonoids from PCC and daidzin and genistin repressed 2-acetoxyacetylaminofluorene (2AAAF)-induced DNA damage measured with single cell gel electrophoresis. Genistein, however, enhanced the genotoxic impact of 2AAAF. Fraction PCC100 (PCC 100% methanol eluate) had the greatest level of antigenotoxic activity against 2AAAF in CHO cells and repressed the genotoxic capacity of the dietary carcinogen 2-amino-3-methylimidazo-(4,5-f)quinoline (IQ) in human lymphocytes. These data indicate that commercial soybean products and by-products may be a source of chemoprotective food additives. Teratogenesis Carcinog. Mutagen. 19:121–135, 1999. © 1999 Wiley-Liss, Inc.

Sex of preimplantation porcine embryos was determined by DNA amplification using porcine male(Y chromosome)-specific DNA primers in the polymerase chain reaction (PCR). In order to determine the sensitivity of this sexing method, single porcine embryos ranging from unfertilized ova to the blastocyst stage were amplified in the PCR using the Y-specific primers, and analyzed by ethidium bromide-staining of polyacrylamide gels. The 192 bp product which denotes the presence of the Y chromosome was seen in the embryos. The unfertilized ova which is of female origin gave no product. These results are representative of PCR analysis of a total of 34 swine embryos.Results obtained using the PCR for sexing were validated by karyotyping and confirmed by in situ hybridization with the porcine Y-chromosome-specific probe. In order to confirm the sex of the embryos determined by PCR, 10 day-old porcine preimplantation embryos were biopsied to produce a small number of cells for sex determination via PCR, while the remainder of the embryo was prepared for in situ hybridization using the biotinylated probe. In situ hybridization performed on embryos shown to be male by PCR, showed pinpoint fluorescence within the nuclei, similar to that obtained when male porcine lymphocytes were hybridized. No evidence of fluorescence was seen when in situ hybridization was performed in parallel on embryos determined to be female by the PCR.The PCR was found to be a relatively fast, accurate and reproducible means of sex determination of swine preimplantation embryos. This capability could have significant impact on animal breeding and production programs by using PCR as a screening tool for traits of economic importance.

Germination and emergence in cold conditions (12–15°C) is important for obtaining improved maize (Zea maysL.) yields in low and no‐tillage production systems. The mesocotyl plays a decisive role in determining the ability of seedlings to emerge under adverse conditions such as deep planting or cold soils. The major factor involved in elongation of the mesocotyl is somatic polyploidy, referred to as endopolyploidization. Endopolyploidy of the mesocotyl tissue was determined by flow cytometry during 1992. Nuclei were isolated from mesocotyls of various maize populations grown at 23 and 15°C. Endopolyploidy of populations selected for cold tolerance was compared with endopolyploidy of their original unselected populations. The populations selected for cold tolerance had an endopolyploid distribution similar to their original unselected populations at both temperatures. Only the NA population was observed to have a different distribution of endopolyploidization in the selected population as compared with the original unselected population. The response was speculated to be the result of different mechanisms of cold tolerance in the NA vs. the NB and NS populations. Endopolyploidization was not affected by cold tolerance per se, but may be associated with freeze tolerance. A significant difference with respect to temperature was observed. At 15°C, the number of the highest ploid nuclei (8C) were reduced, while the number of 2C nuclei increased. This change reflects the slowing of cell division at cooler temperatures.

Herbicide contamination of natural resources, specifically groundwater, has drawn considerable public attention in recent years. Although deleterious effects of herbicides at large dosages have been well documented, information is lacking about the possible effects of herbicide levels that are found in public water supplies. Chromosomal damage is examined by flow cytometry on Chinese hamster ovary (CHO) cells that are exposed to three herbicides (atrazine, simazine, and bentazon) at concentrations deemed safe by the U.S. Environmental Protection Agency (USEPA) for drinking water. A known clastogen (ara‐C) is also included as a reference for comparing the magnitude of chromosomal damage caused by herbicides. Chromosomal damage was assessed by measuring the coefficient of variation (CV) and percent chromosomes present in the larger chromosome distribution peaks. Exposure to atrazine (6‐chloro‐N‐ethyl‐N′‐(1‐methylethyl)‐1,3,5‐triazine‐2,4‐diamine) increased the CV of the largest chromosome distribution peak at concentrations considered safe by the USEPA. Chromosomes exposed to both simazine (6‐chloro‐N,N′‐diethyl‐1,3,5‐triazine‐2,4‐diamine) and bentazon (3‐(1‐methylethyl)‐(1H)‐2,1,3‐benzothiadiazin‐4(3H)‐one 2,2‐dioxide) did not exhibit chromosomal damage. Further analyses carried out on atrazine concentrations about equal to levels found in public water supplies revealed the potential of atrazine to induce chromosome breakages in CHO cells. Atrazine concentrations that were manyfold greater than the contamination limits exhibited a true clastogenic nature like ara‐C. The results provide evidence for further investigations as to the potential health risk of consuming water contaminated with atrazine.

We investigated the induction of DNA strand breaks in the single cell gel electrophoresis (SCGE or comet) assay with whole cell clastogenicity measured with flow cytometric analysis in cells from an isolated clone of the Chinese hamster ovary (CHO) AS52 cell line. Under identical treatment conditions the responses were compared with forward mutation at gptusing 2‐acetoxyacetylaminofluorene (2AAAF), ultraviolet radiation (UV) and ethyl methanesulfonate (EMS). Cytotoxicity for each agent was evaluated in the SCGE and forward mutation assays. Forward mutation was 4–10‐fold more sensitive than DNA strand breaks detected in the SCGE assay. For 2AAAF and EMS, the kinetics of the induction of genetic damage were similar for the three assays, although there were differences in sensitivity. With UV, the induction kinetics of gptmutation differed from that expressed by SCGE and flow cytometric analysis. With the chemical mutagens 2AAAF and EMS, there was a high correlation between the SCGE assay and forward mutation and also between the SCGE assay and flow cytometry. There was no significant correlation between flow cytometry and forward mutation. With UV, only the SCGE assay and flow cytometry were correlated. Agent‐specific variations in the intragenomic distribution of DNA damage for each mutagen was measured in the SCGE assay. Environ. Mol. Mutagen. 32:360–368, 1998 © 1998 Wiley‐Liss, Inc.

The somatic chromosomes ofTriticum turgicum var.durum cv. Langdon andT. dicoccoides (AABB tetraploids),T. timopheevii, andT. araraticum (AAGG tetraploids) were assayed for distribution patterns of a highly repeated 120bp DNA sequence by in situ hybridization. The repeated sequence appears to be an ancient sequence shared withSecale andAegilops. The distribution patterns of the chromosomes were compared to the patterns of the A and B genome chromosomes ofT. aestivum cv. Chinese Spring (AABBDD hexaploid).T. turgidum andT. dicoccoides were observed to have identical in situ hybridization patterns. In both species, nine chromosomes with a total of 21 sites of hybridization were observed. The pattern, with few exceptions, was identical to that of Chinese Spring.T. araraticum andT. timopheevii were observed to have different patterns. InT. araraticum, six chromosomes with 21 total hybridization sites are present while inT. timopheevii nine chromosomes with 19 total sites exist. Major differences in hybridization patterns were observed between the B and G genomes. The divergence of the tetraploid wheats in this study appears to have resulted in changes in location, not in amount, of the ancient repeated sequence.

The 1RS.1BL translocation is widely used by wheat (Triticum aestivumL.) breeders to enhance agronomic performance and particularly grain yield. Research is lacking that compares agronomic trait expression in closely related stocks with or without the translocated chromosome arm. Our objective was to determine the effects of 1RS.1BL on means and genetic variances for grain yield and other agronomic traits in two hard red winter wheat populations segregating for the ‘Aurora’ translocation. Two field experiments were conducted using different genetic materials derived from two crosses, OK83398 × ‘Chisholm’ and OK83398 × ‘Arkan’. In Experiment 1, 25 pairs of F5‐derived near‐isolines homozygons for either 1B or 1RS.1BL were evaluated in three environments. In Experiment 2, plants homozygons for either 1B or 1RS.1BL were selected in the F2, and further subsampled in the F3; forty F3(F2) lines per chromosome type in each cross were evaluated in four environments. The average effect of 1RS.1BL in Experiment 1 was to increase grain yield by 9 to 10%, increase aerial biomass by 11 to 12%, and increase kernel weight by 4 to 6%, depending on the cross. Differences in kernels per spike, harvest index, and test weight were not detected, whereas differences in plant height and the number of spikes per meter squared were inconsistent between crosses or among environments. The magnitude of the effect of 1RS.1BL varied considerably among the paired near‐isolines, presumably by the effects of genes present on other wheat chromosomes. Heterogeneity of genetic variance in Experiment 2 was usually caused by larger variance among 1RS.1BL genotypes than 1B genotypes. These results encourage further use of 1RS.IBL to improve agronomic traits of wheat.

The number of mitotic chromosomal C‐bands, the percent of the genome comprised of C‐band heterochromatin, and genome size (4C DNA content) were determined for 22 North American inbred and open‐pollinated lines of Zea mays.The number of C‐bands ranged from 0 in Tama Knobless Flint to 18 in Zapolate Grande. The percent C‐band heterochromatin ranged from 0% in Tama Knobless Flint to 16.9% in Tx601. Genome size varied over 23%: Gaspe Flint had the lowest DNA content (9.82 pg), and Zapolate Grande had the highest (12.12 pg). Genome size and the amount of heterochromatin were significantly correlated. The corn lines were assigned to five maturity zones encompassing a south‐to‐north range from Mexico to Canada. Significant negative correlations were detected between the amount of C‐band heterochromatin and maturity zones, and between DNA content and maturity zones among the lines. It is speculated that the simultaneous selection by man for earlier maturation and plant size may be related to the lower DNA content of corn varieties adapted to higher latitudes. Such selection for larger plants may have been achieved through selection for more cells, which could result from the shorter mitotic cycle time that correlates with reduced DNA amount.

Intra- and inter-specific variation in CO2 assimilation rate (A) in Triticum spp. is well documented for reproductive growth stages. Research was conducted to characterize early vegetative photosynthetic variation in a diverse set of cultivated hexaploid wheat (T. aestivum L.) germplasm and in wild tetraploid (T. dicoccoides Korn) and hexaploid x tetraploid populations. Choice of hexaploid genotypes was based on maximum genetic distance between cultivars within the HRW and SRW wheat classes of the USA. The tetraploid material was produced by hybridizing two accessions of T. dicoccoides previously shown to differ widely in A and A/Chl but with similar leaf morphology. Genetic variability in the HRW and SRW gene pools was attributed to more recently developed descendent lines and unrelated lines rather than parental lines. Phenotypic distributions for A, stomatal conductance (gs), and internal CO2 concentration (Ci) in the F2 tetraploid population were continuous and showed transgressive segregation, reflecting quantitative inheritance with intermediate heritability. Variability in A was not associated with chlorophyll content or CO2 supply to the mesophyll measured as Ci. Genetic variability in A was also observed in the interspecific backcross population, 2*TAM W-101/PI 428109, thereby providing a germplasm pool to select for high A while restoring the D genome of hexaploid wheat. These results suggest that genetic improvement of vegetative assimilation rate is feasible in hexaploid wheat via homologous transfer from an alien source.

Flag leaves of many diploid and tetraploid Tritlcum species are known to have higher photosynthesis rates than flag leaves of cultivated hexaploid wheat (Triticum aestivumL.). Yet these differences are reportedly absent or less pronounced on earlier developed leaves. Our objective was to study the relationship between photosynthesis, stomatal conductance, and water‐use efficiency (WUE) in various Triticumacessions at the tillering stage. Net CO2assimilation per leaf area (A) and associated gas exchange characteristics were measured on newly expanded leaves of 6‐week‐old growth chamber‐grown plants of four wild diploids, six wild tetraploids, and two cultivated hexaploids. Significant variation in A was observed with values ranging from 31.1 μmol CO2m−2s−1in the diploid T. speltoldesto 20.7 in one accession of T. dicoccoides; A for the two hexaploid wheats averaged 22.6. This A range for leaves of plants at tillering is similar to that reported by others measuring A of tlag leaves. Because higher A was associated with higher stomatai conductance (r= 0.86**, significant at the 0.01 probability level), which led to higher transpiration, there was no significant correlation between A and WUE. There was no significant correlation between A and internal CO2concentration (C1), so differences in A among accessions were not explained by differences in CO2supply to the mesophyll. Two T. dicoccoidesaccessions found to differ in A and conductance at tillering also had different flag leaf A and conductance. Comparison of A vs. Ciresponse curves between these two accessions showed that the higher A was related to a higher capacity for mesophyll photosynthesis rather than stomatal conductance per se. The results show that the variation in A among accessions was to a significant extent attributable to nonstomatal factors and that differences in A among Triticumaccessions are detectable at early vegetative growth stages.

The majority of DNA that is found in most of the flowering plants appears to be non‐coding DNA. Much of this excess DNA consists of nucleotide sequences which exist as multiple copies throughout the genome and are designated as repetitive sequences. Those sequences which are found in moderately high to high numbers of copies are observed to be of the greatest value as cytological markers. Moderately high copies may exist as sequences which are dispersed throughout the chromosomes of some species and not dispersed in other more distantly related species. By taking advantage of this characteristic and the technique of in situ hybridization with biotinylated probes, breakpoints of chromosomal translocations may be observed between species such as wheat and rye. Many of the high copy number repetitive sequences are organized in a tandem fashion in specific loci in the chromosome. Chromosomal identification may be accomplished by using the in situ hybridization technique. Upon in situ hybridization with a repetitive sequence isolated from Aegilops squarrosa, the patterns of the sites of hybridization allowed the D‐genome chromosomes to be identified. The sequence was also observed only on the D‐genome chromosomes of several polyploid species indicating its usefulness as a genome specific marker. Using this genome specificity, assessment of the orientation of the D‐genome chromosomal segments of hexaploid wheat carrying the sequence during interphase and prophase of mitotic root tip cells was possible. Repetitive DNA sequences, therefore, provide cytological markers necessary for studies of chromosomal identification, genome allocation, and genome orientation. The use of biotin‐labeled DNA probes allows the technique of in situ hybridization to be performed much more rapidly and with a greater degree of safety and reliability.

We investigated the induction of DNA strand breaks in the single cell gel electrophoresis (SCGE or comet) assay with whole cell clastogenicity measured with flow cytometric analysis in cells from an isolated clone of the Chinese hamster ovary (CHO) AS52 cell line. Under identical treatment conditions the responses were compared with forward mutation at gpt using 2-acetoxyacetylaminofluorene (2AAAF), ultraviolet radiation (UV) and ethyl methanesulfonate (EMS). Cytotoxicity for each agent was evaluated in the SCGE and forward mutation assays. Forward mutation was 4–10-fold more sensitive than DNA strand breaks detected in the SCGE assay. For 2AAAF and EMS, the kinetics of the induction of genetic damage were similar for the three assays, although there were differences in sensitivity. With UV, the induction kinetics of gpt mutation differed from that expressed by SCGE and flow cytometric analysis. With the chemical mutagens 2AAAF and EMS, there was a high correlation between the SCGE assay and forward mutation and also between the SCGE assay and flow cytometry. There was no significant correlation between flow cytometry and forward mutation. With UV, only the SCGE assay and flow cytometry were correlated. Agent-specific variations in the intragenomic distribution of DNA damage for each mutagen was measured in the SCGE assay. Environ. Mol. Mutagen. 32:360–368, 1998 © 1998 Wiley-Liss, Inc.

The chromosome of three tetraploid Aegilops L. species containing the D-genome were analyzed by in situ hybridization with a repeated DNA sequence clone pAS1 isolated from Aegilops squarrosa and observed to be D-genome specific. This sequence is found on all seven D-genome chromosome pairs of A. squarrosa and hexaploid wheat. Two distinct D-genome patterns were observed in the tetraploid species. The D-genome of A. cylindrica was similar to hexaploid wheat. Seven pairs of chromosomes having large amounts and numerous sites of the sequence were observed. Five chromosome pairs with fewer and smaller sites of the repetitive sequence were observed in the D-genomes of A. crassa and A. ventricosa. In addition to these major repeated sequence differences, chromosomal modifications appear to have occurred between T. aestivum and A. cylindrica and between A. crassa and A. ventricosa resulting in changes with respect to location of the sequence between the respective species. D-genome divergence with respect to pAS1 sequence appears to have occurred at least in two forms, one characterized by the changes in amount of repetitive sequence and the second by changes in location of the sequence.

Cultivar specific DNA profiles in rye were revealed by polymerase chain reaction (PCR) using randomly amplified polymorphic DNA (RAPD) sequences. Ten base primers were used for the amplification of genomic DNA of rye cultivars by PCR. RAPD analysis was found to be reproducible among samples between PCR runs. When amplification profiles of different rye cultivars were compared using various primers, the overall profiles were cultivar specific. However, not all primers revealed polymorphisms. These primers appear to amplify conserved sequences in all rye cultivars. Intracultivar studies were conducted on two of the cultivars. In the cultivar Imperial, no polymorphisms were observed among ten plants analyzed with five primers. In the cultivar Balboa, polymorphisms were observed among fifty plants with four of the ten primers analyzed. Despite the small amount of intracultivar variability, RAPD analysis has the potential to be a rapid and reliable method of cultivar identification in this outcrossing species.

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.

Correlations between genome size and area of geographic adaptation have been reported in maize (Zea maysL.). Such correlations have been suggested to occur due to lower genome sizes being more favorable where shorter growing seasons exist. The objective of this study was to determine if genome size and relative maturity are correlated in hybrid maize. Thirty hybrids, obtained from Pioneer Hi‐Bred International, Inc. (Johnson, IA), were analyzed. Genome size determinations were made using flow cytometric techniques. No correlation between genome size and relative maturity was observed. Since the area of adaptation of these hybrids encompasses a wide range of latitudes, there appears to be no correlation between genome size and latitude as well. The lack of correlations among these characters may result from the breeding structure of hybrid maize.

The agronomic advantage of the T1BL. 1RS translocation and the potential importance of chromosome 1BS to end‐use quality of bread wheat (Triticum aestivumL.) warrant a more precise definition of the relationship of T1BL‐1RS to bread wheat quality. This study was conducted to determine the effect of TIBL. 1RS from ‘Aurora’ on grain and flour quality in multiple near‐isoline pairs with varying genetic background, and to examine the genetic variation among random inbred lines selected only for 1B or T1BL 1RS. Two experiments were conducted, each with a different set of genetic materials derived from the same pair of crosses. In one experiment, 25 pairs of F5‐derived near‐isolines homozygous for either 1B or T1BL‐ 1RS were evaluated in at least two environments; in the second experiment, plants homozygous for either 1B or T1BL‐1RS were selected in the F2, and 40 inbred lines per chromosome type in each cross were evaluated in four environments. The near‐isolines showed high isogenecity for all traits based on comparison of duplicate lines within pairs. The translocated chromosome, on the average, increased grain protein concentration and decreased mixogram rating, mlxogram band width, and sedimentation volume in both crosses. Kernel hardness, flour yield, and mixing time either decreased or did not change in the TIBL 1RS isolines depending on the cross. Water absorption did not change. The T1BL‐1RS and 1B isolines showed similar mixograph properties in eight of the 25 pairs. Genetic variance of TIBL IRS random inbred lines was not reduced for those traits where deleterious effects were most notable, e.g., mixing tolerance and sedimentation volume. Hence, the detrimental effect of T1BL‐ 1RS on dough properties might be mitigated, relative to non‐TIBL 1RS genotypes with equivalent pedigree, by rigorous selection of background genotype. Special attention must be given to mixing tolerance and sedimentation.

The nuclear DNA content was determined in two separate experiments for 19 maize inbred lines and 26 maize F1hybrids. Ten inbred lines were initially screened in the first experiment. An 11 per cent difference in nuclear DNA amount was observed between the two lines with the lowest amounts of DNA and two lines with the largest genome sizes. All possible hybrid combinations were made among these four lines. In all cases, the genome sizes of the F1hybrids were not significantly different from their expected parental means. In several cases, however, F1plants of specific crosses were not uniform in genome size. In these crosses, genome sizes observed ranged from the genome size of the low parental genome to the largest parental genome. These results indicate an instability in F1genome sizes in certain maize crosses. In order to corroborate this hypothesis, a second experiment was performed. To remove any biases with respect to genome size, 14 F1maize hybrids were selected solely on the basis of their heterotic response with no regard for their genome sizes or the genome sizes of their parental inbred lines. The nuclear DNA content of the nine parental lines and 14 hybrids was determined. In most of the crosses, the nuclear DNA content of the F1hybrids was not significantly different from their respective parental means. However, in five parental combinations, the mean nuclear DNA content of the F, hybrids was significantly higher than their respective parental means. The combined results of this study support the hypothesis of instability in nuclear DNA content in F1hybrids of maize. This instability appears not to be universal in all maize hybrids but is restricted to specific parental combinations.Heredity (1993) 70, 294–300; doi:10.1038/hdy.1993.42

Extensive nuclear DNA content variation has been observed inZea mays. Of particular interest is the effect of this variation on the agronomic potential of maize. In the present study, yield and growth data were collected on 12 southwestern US maize open-pollinated populations. These populations, originally cultivated by the Indians of the southwestern US for both human and animal consumption and adapted to various altitudes, were grown in replicated plots at the University of Illinois Agronomy-Plant Pathology South Farm. All growth and yield parameters were found to be negatively correlated with nuclear DNA amount. The negative correlations of nuclear DNA amount and growth parameters were more pronounced at 60 days after planting (DAP) than 30 DAP. Agronomically-important yield parameters, such as ear or seed weight and seed number per plant, also exhibited a significant negative correlations with nuclear DNA amount. These correlations demonstrate how the nucleotype may exhibit a high degree of influence on the agronomic phenotype. Although the results presented here represent only three replications at one location in 1 year, the observations noted suggest that nucleotype plays an integral role in determining the agronomic performance of maize. Further studies are needed to fully document this role.

Previous studies have indicated a positive correlation between genome size and altitude among plant species. It has been hypothesized that increasing genome size occurs due to increasing C-banded heterochromatin. In corn, increasing altitude has been correlated with decreasing knob (C-banded) heterochromatin, suggesting that DNA content may decrease with increasing altitude. In this study, nuclear DNA content of 12 southwestern United States Indian maize populations, collected at various altitudes, was determined. The significant positive correlation observed between genome size and altitude suggests that corn follows the trend of increasing DNA content with increasing altitude observed in other plant species. Whether this correlation is due to increasing knob heterochromatin or additional intra- or supernumerary chromosomal DNA sequences has yet to be determined.

Enhanced NH4 nutrition causes thicker roots (higher g/m root length) than NO3 nutrition. This study examined the underlying anatomy of N-form altered root morphology. Corn (B73xLH51) was cultured in complete nutrient solutions containing NO3- or NH4-N. Plants were harvested four times between 13 and 20 days after germination. Root lengths were measured and photomicrographs taken of primary and nodal root cross-sections. Plants grew better under the NH4 regime. This was probably due to controlling acidity in the pH 6.0 to 6.5 range using 2.0 mM MES. The primary root system was 16% thicker under NH4-N. No difference due to N form in the cross-sectional area of the primary axis base was detected, suggesting that NH4-N caused thicker laterals. Cross sectional areas of the first tier nodal roots were 27%- and 2nd tier roots 10.5%- greater under NH4-N. Changes in the stelar and cortical regions were proportional in 1st tier nodal roots, but the cortex accounted for most of the NH4-induced thickening in 2nd tier nodal roots. The number of late metaxylem elements was not affected by N form. Possible implications of these morphological and anatomical differences are discussed.

In the Great Plains, winter wheat (Triticum aestivumL.) lines carrying the 1BS/1RL wheat‐rye translocated chromosome are being used extensively in wheat breeding programs. This study was conducted to determine the influence of the 1BL/1RS chromosomone gametic transmission in adapted winter wheat backgrounds. Oklahoma experimental line OK83398 (‘Aurora’/2*TAM W‐101) was crossed to both ‘Arkan’ and ‘Chisholm’. The F2plants were analyzed as to their chromosomal composition using the chromosomal N‐banding procedure. In both crosses a significant deviation of a 1 homozygous 1B:2 heterozygous:1 homozygous 1BL/1RS ratio at the P= 0.05 level was observed. The transmission of gametes containing the 1BL/1RS translocated chromosome was observed to be reduced in F1hybrids in winter wheat backgrounds.

Date palm is an important fruit crop and member of palm family, reported with varied ploidy levels, i.e., 14 (2n= 28) to 18 (2n= 36) pairs of chromosomes and genomic size due to the limited work done on its cytological aspect. The amount of nuclear DNA content is extremely important to understand the hereditary constituent of any species. Hence, the present study was conducted with the aim to estimate the ploidy level and especially the genomic size (Cvalue) of date palm by studying fifty date palm cultivars with flow cytometry using propidium iodide (PI) as the fluorescent dye. The maize genome size (5.14 pg) was used as the internal reference. The results obtained regarding genomic size of date palm cultivars significantly varied and ranged from 1.59 to 1.84 pg 2C−1or 780.11 to 903.06 Mbp when converted into base pairs of DNA. The average genome size of studied cultivars was observed to be 1.726 2C−1, while, that of nuclei significantly varied from cultivar to cultivar and appeared independent to the genomic size of the studied cultivars. Intraspecific variations were not detected among five exotic cultivars (Amber, Sugae, Medjool, Safawi and Ajwa) grown in Pakistan in relation to their place of origin. The cluster analysis exhibited two main groups of cultivars, the first group comprised 65.3% (33 cultivars) and the second group contained 35.7% (17 cultivars). In addition, no ploidy was observed among all the studied cultivars. The findings regarding the estimated genomic sizes of studied cultivars may be helpful in understanding date palm genetics, breeding and genome sequencing programs further.

A highly repeated DNA sequence isolated from Aegllops squarrosa was used to Identify specific chromosomes in hexaploid wheat. The DNA sequence found In clone pAS1 appears D-genome specific and, therefore, allows for the unambiguous identification of the D-genome chromosomes. In situ hybridization using blotin labeled DNA probes was found to be a rapid and reliable technique to identify sites of hybridization on D-genome chromosomes. Multiple sites of hybridization of the sequence allowed the Identification of all seven pairs of the D-genome chromosomes.

Flow cytometry was used to determine quantitative intraspecific DNA content variation in Zea mays. Previous studies using flow cytometry had indicated that intraspecific variation in corn was beyond the resolution of the method. The DNA content variation among corn lines observed in this study was in agreement with the amount of variation observed using microdensitometry. In order to observe intraspecific variation, the fluorochrome DAPI was shown to be superior to mithramycin. The fluorochrome: nuclei ratio was found to be critical when DAPI was used because of self absorption of the fluorescence. Flow cytometry with the fluorochrome DAPI was found to be a rapid and reliable alternative to microdensitometry in examining intraspecific DNA content variation in corn.

A wide breadth of DNA content variation has been reported among maize lines. While the extent of this variation has been well documented, few studies have focused on its cause. Some of the nuclear DNA content variation has been explained by the presence of B chromosomes or knobs. However, variation in these two structures does not account for all of the observed variation. In order to identify other fluctuating DNA sequences, a rapid and reliable method of estimating relative abundance of DNA sequences needed to be developed. The potential of flow cytometry in conjunction with spot hybridization for determining relative abundance of specific DNA sequences in maize was studied. Different numbers of G1 phase nuclei were sorted on nitrocellulose filters and non-radioactive hybridization and signal detection performed. Results from these experiments revealed a significant, positive linear correlation between the amount of target sequence and signal density using both knob (R = 0.98) and ribosomal spacer (R =0.99) DNA sequences. In addition, G1 phase nuclei of eight inbred lines differing in the amount of knob heterochromatin, were sorted on to filters and the non-radioactive hybridization and signal detection performed. A significant, positive linear correlation between C-band number and signal density (R =0.83; P = 0.0051) as well as between per cent heterochromatin and signal density (R=0.96;P = 0.0002) was observed. These results indicate the usefulness of flow cytometry for spot hybridization in determining the relative abundance of DNA sequences in the maize genome.

Diploid nuclei from stem, mesocotyl, nodal root and root tip tissue of two maize hybrids were examined with respect to their DNA content. The nuclei were isolated and stained with DAPI and passed through a flow cytometer-cell sorter. The titration curve for each tissue was determined. Significant variation was observed among nuclei of different tissue types. Stem and root tips had the highest diploid nuclear DNA amounts while 2-week-old mesocotyl had the lowest diploid nuclear DNA amount. These results provide evidence that during plant development and differentiation, the amount of DNA within a diploid nucleus changes through loss of specific DNA sequences. This study also demonstrates the sensitivity of flow cytometry in detecting small intraplant variation in nuclear DNA.