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Eastern gamagrass (Tripsacum dactyloides L.) belongs to the same tribe of the Poaceae family as maize (Zea mays L.) and grows naturally in the same region where maize is commercially produced in the USA. Although no evidence exists of gene flow from maize to eastern gamagrass in nature, experimental crosses between the two species were produced using specific techniques. As part of environmental risk assessment, the possibility of transgene flow from maize to eastern gamagrass populations in nature was evaluated with the objectives: (1) to assess the seeds of eastern gamagrass populations naturally growing near commercial maize fields for the presence of a transgenic glyphosate-tolerance gene (cp4 epsps) that would indicate cross-pollination between the two species, and (2) to evaluate the possibility of interspecific hybridization between transgenic maize used as male parent and eastern gamagrass used as female parent. A total of 46,643 seeds from 54 eastern gamagrass populations collected in proximity of maize fields in Illinois, USA were planted in a field in 2014 and 2015. Emerged seedlings were treated with glyphosate herbicide and assessed for survival. An additional 48,000 seeds from the same 54 eastern gamagrass populations were tested for the presence of the cp4 epsps transgene markers using TaqMan® PCR method. The results from these trials showed that no seedlings survived the herbicide treatment and no seed indicated presence of the herbicide tolerant cp4 epsps transgene, even though these eastern gamagrass populations were exposed to glyphosate-tolerant maize pollen for years. Furthermore, no interspecific hybrid seeds were produced from 135 hand-pollination attempts involving 1529 eastern gamagrass spikelets exposed to maize pollen. Together, these results indicate that there is no evidence of gene flow from maize to eastern gamagrass in natural habitats. The outcome of this study should be taken in consideration when assessing for environmental risks regarding the consequence of gene flow from transgenic maize to its wild relatives. Electronic supplementary material The online version of this article (doi:10.1007/s11248-017-0020-7) contains supplementary material, which is available to authorized users.

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 (C value) 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(−1) or 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.

The genome size of cultivated Cicer arietinum and 12 wild Cicer sp. including seven annual and five perennial species were analyzed using flow cytometry. A significant 2C genome size variation was observed among the Cicer sp. The 2C genome size ranged from 1.00 pg in wild species, Cicer judaicum, to 1.76 pg in cultivated species, C. arietinum. The wild perennial species all had a genome size of ≈1.6 pg. Most if not all of this genome size variation occurred among wild annual species. A significant positive correlation between 2C genome size and seed mass was observed among 12 wild Cicer sp. at α = 0.05. However, artificial selection appears to decrease nucleotype effects in cultivated C. arietinum, which resulted in no correlation between seed mass and genome size at α = 0.05.

Chloroplast DNA (cpDNA) is most often maternally inherited and highly conserved leading to previous observation of little to no sequence variation. Comparing cpDNA haplotypes have provided valuable insight into the establishment and migration of polyploid populations. However, to use chloroplast haplotypes to their full potential intrapopulational variation needs to be addressed. In this study, cpDNA haplotype variation was surveyed within 16 natural populations of prairie cordgrass (Spartina pectinata Link) located east of the 100th west meridian and north of the 35th north parallel in the U.S.A. using two non-coding, polymorphic chloroplast regions. Two main clades were defined with subclades as follows: haplotype 1 and haplotype 2A and 2B. It was discovered that seven populations showed intrapopulational chloroplast genome variation. Of the total amount of variation, 95.5% occurred within the octoploid populations and 4.5% occurred within the tetraploid populations. Both variant haplotypes, 2A and 2B, were found in a larger sampling of one of the natural populations, but no variation was found in a mixed ploidy population. The intrapopulational cpDNA variation we found in this study cannot directly be related to mechanisms of introduction of the non-native populations into native populations. Therefore, this cpDNA variation could be novel natural variation that has been fixed as the octoploid populations were established and moved northwest. This analysis provides insight into determining the usefulness of indels and single nucleotide polymorphisms for population identification and may provide information in regards to the origin of chloroplast variation and its subsequent fixation and establishment in natural prairie cordgrass populations.

Prairie cordgrass (Spartina pectinata Link) is a productive warm-season, C4 perennial grass native to most of North America having tolerance to wet, cold, and saline growing conditions. Excellent stress tolerance, along with high biomass yields, makes prairie cordgrass a good candidate as a dedicated energy crop on marginal land. However, there is little information available on genetic variation, including yield potential, of native populations in the USA. The objectives of this study were to evaluate biomass yield and to identify the nature and extent of genetic variation in natural populations of prairie cordgrass by comparing endemic strains collected throughout the USA. Forty-two prairie cordgrass populations were collected from prairie-remnant sites in 13 states and evaluated at the University of Illinois in Urbana, IL. The 4-year field study of prairie cordgrass revealed extensive variations in biomass yield and phenotypic traits associated with biomass yield among these populations. Strong correlations were observed between the phenotypic values and origins of the populations. Path coefficient analysis indicated that tiller mass, tiller density, heading date, plant height, and phytomer number positively affected biomass yield directly or indirectly. However, the phenotypic traits including biomass yield showed significant variation among years except for phytomer number and heading date. With the extensive genetic variability and high biomass yield potential demonstrated in this experiment, prairie cordgrass could become a highly productive bioenergy crop by developing a well-planned breeding program.

Linkage maps are important tools in evolutionary genetics and in studies of speciation. We performed a karyotyping study and constructed high-density linkage maps for two closely related killifish species, Lucania parva and L. goodei, that differ in salinity tolerance and still hybridize in their contact zone in Florida. Using SNPs from orthologous EST contigs, we compared synteny between the two species to determine how genomic architecture has shifted with divergence. Karyotyping revealed that L. goodei possesses 24 acrocentric chromosomes (1N) whereas L. parva possesses 23 chromosomes (1N), one of which is a large metacentric chromosome. Likewise, high-density single-nucleotide polymorphism−based linkage maps indicated 24 linkage groups for L. goodei and 23 linkage groups for L. parva. Synteny mapping revealed two linkage groups in L. goodei that were highly syntenic with the largest linkage group in L. parva. Together, this evidence points to the largest linkage group in L. parva being the result of a chromosomal fusion. We further compared synteny between Lucania with the genome of a more distant teleost relative medaka (Oryzias latipes) and found good conservation of synteny at the chromosomal level. Each Lucania LG had a single best match with each medaka chromosome. These results provide the groundwork for future studies on the genetic architecture of reproductive isolation and salinity tolerance in Lucania and other Fundulidae.

Information on genome size, ploidy level, and genomic polymorphisms among accessions of the genus Miscanthus can assist in taxonomic studies, help understand the evolution of the genus, and provide valuable information to biomass crop improvement programs. Taxonomic investigation combining variation in plant morphology, genome size, chromosome numbers, and simple sequence repeat (SSR) marker polymorphisms were applied to characterize 101 Miscanthus accessions. A total of 258 amplicons generated from 17 informative SSR primer pairs was subjected to cluster and principal coordinate analysis and used to characterize genetic variation and relationships among 31 Miscanthus accessions, including four interspecific Miscanthus hybrids created from controlled pollinations, and four Saccharum, six Erianthus, and one Sorghum bicolor accessions. Miscanthus accessions were distinct from accessions in the genera Erianthus and Saccharum. Miscanthus accessions fell into five taxonomic groups, including the existing taxonomic section Miscanthus, diploid and tetraploid Miscanthus sacchariflorus, and a fourth (M. × giganteus) and fifth group (Miscanthus ‘purpurascens’); the last two being intermediate forms. In contrast to previous work, our findings suggest diploid and tetraploid M. sacchariflorus are taxonomically different, the latter more closely related to M. sacchariflorus var lutarioriparius. We also suggest that Miscanthus ‘purpurascens’ accessions are interspecific hybrids between Miscanthus sinensis and diploid M. sacchariflorus based on DNA content and SSR polymorphisms. The evolution of Miscanthus and related genera is discussed based on combined analysis and geographical origin.

The existence of neopolyploidy in prairie cordgrass (Spartina pectinata Link) has been documented. The neohexaploid was discovered coexisting with tetraploids in central Illinois, and has been reported to exhibit competitiveness in the natural environment. It is hypothesized that the natural tetraploid cytotype produced the hexaploid cytotype via production of unreduced gametes. Meiosis I chromosome pairing was observed in tetraploid (2n = 4x = 40), hexaploid (2n = 6x = 60), and octoploid (2n = 8x = 80) accessions and the percentage of meiotic abnormality was determined. Significant differences in meiotic abnormality exist between tetraploid, hexaploid, and octoploid cytotypes. An elevated incidence of abnormal, predominantly trivalent pairing in the neohexaploid suggests that it may possess homologous chromosomes in sets of three, in contrast to the tetraploid and octoploid cytotypes, which likely possess homologous chromosomes in sets of two. Abnormal chromosome pairing in the hexaploid may result in unequal allocation of chromosomes to daughter cells during later stages of meiosis. Chromosome pairing patterns in tetraploid, hexaploid, and octoploid cytotypes indicate genome compositions of AABB, AAABBB, and AABBA′A′B′B′, respectively.

Prairie cordgrass plants (Spartina pectinata Bosc ex Link) were examined from 61 locations representing the geographic distribution of prairie cordgrass in the U.S. Using flow cytometry, the genome size of 183 individual plants of prairie cordgrass was determined, and the chromosome counts were obtained. Three distinct ploidy levels were observed: tetraploid ( $$ \overline x = {1}.{56} {\text{pg}},{2}n = {4} \times = {4}0 $$ ), hexaploid ( $$ \overline x { = 2}.{33} {\text{pg}},{2}n = {6} \times = {6}0 $$ ), and octoploid ( $$ \overline {\text{x}} { = 3}.0{6} {\text{pg}},{2}n = {8} \times { = 8}0 $$ ). In the sampled areas, the tetraploid populations extended from the East North Central to the New England regions of the U.S., while the octoploid cytotypes were mostly distributed in the West North Central regions. Populations of the tetraploids and octoploids were found in close proximity in the West North Central (Iowa and Kansas) and the West South Central (Oklahoma) regions. The hexaploid cytotype was found in one mixed population (4x + 6x) occurring in Illinois. No statistically significant intraploidy genome size variation was found in the tetraploid populations, while significant intraploidy genome size variation was found in the octoploid populations. This study precisely defined the geographic distribution of cytotypes in prairie cordgrass throughout the different regions of the U.S. These results provide critical genome size and ploidy distribution information needed to design efficient breeding schemes for high yielding cultivars of prairie cordgrass with local adaption.

Simple sequence repeats (SSRs) were used to assess genetic diversity and study genetic relatedness in a large collection of Malus germplasm. A total of 164 accessions from the Malus core collection, maintained at the University of Illinois, were genotyped using apple SSR markers. Each of the accessions was genotyped using a single robust SSR marker from each of the 17 different linkage groups in Malus. Data were subjected to principal component analysis, and a dendrogram was constructed to establish genetic relatedness. As expected, this diverse core collection showed high allelic diversity; moreover, this allelic diversity was higher than that previously reported. Cluster analysis revealed the presence of four distinct clusters of accessions in this collection.

Identifying bioenergy crops that can be produced successfully on marginal lands, such as those affected by salinity, reduces the pressure to produce energy crops on land that would otherwise be used to produce food crops. In this paper, the degree of salinity tolerance of “Red River” prairie cordgrass (Spartina pectinata Link) and “Cave-in-Rock” switchgrass (Panicum virgatum L.) was determined by evaluating seed germination, plant growth, ion uptake, and leaf anatomical feature responses in saline conditions. Red River seeds retained 50% of germination potential under high salinity up to 300 mM NaCl, whereas Cave-in-Rock seed germination was reduced by 80% at 300 mM NaCl. Red River seedlings survived up to 500 mM NaCl, while more than 30% of Cave-in-Rock did not survive above 100 mM NaCl in greenhouse experiments. Red River produced more biomass and second-generation tillers and had a greater root and shoot biomass ratio than Cave-in-Rock under all salinity ranges. Sodium accumulation in shoots of Cave-in-Rock increased with increasing salinity, whereas Red River maintained a low level of sodium in biomass through salt-gland exclusion. The increasing rate of selectivity coefficient for potassium over sodium in Red River was higher than in Cave-in-Rock with increasing salinity. Although seed germination and plant growth decreased as salinity increased, Red River retained its potential of seed germination and to produce new tillers and biomass under salinity stress.

The next generation of bioenergy crops will be grown on marginal lands. Prairie cordgrass (Spartina pectinata Link) 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 x giganteus is 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 x 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 15μM oryzalin (78%) or 939μM colchicine (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. x giganteus plants (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. x giganteus suggests that ploidy manipulation of this plant and its parental species (Miscanthus sinensis and Miscanthus sacchariflorus) could be a means to access genetic variability for the improvement of Miscanthus as a biofuel/bioenergy crop.

Summary •Amaranthus tuberculatus represents one of the most dramatic cases of weed invasion documented in the midwestern USA. The species is infamous for evolving resistance to multiple herbicides, and predicting whether these resistances may be transferred to widespread weeds of the Amaranthus hybridus aggregate is a matter of epidemiological concern. Here, we explore the patterns of genetic exchange between Amaranthus tuberculatus and A. hybridus in an effort to understand whether allele introgression occurs throughout the genome and if fecundity penalties are associated with genetic exchange. •We evaluated 192 homoploid BC1s at 197 amplified fragment length polymorphism (AFLP) loci, as well as two loci associated with herbicide resistance: ALS and PPO. We also assessed the fecundity of each genotype by evaluation of seed production or pollen development. •It was discovered that genetic exchange between the species is unidirectional. Whereas A. hybridus alleles transfer with little or no penalty to A. tuberculatus, the reciprocal exchange is significantly distorted and potentially of limited evolutionary consequence. •Our previous hypothesis suggesting unidirectional introgression at ALS owing to circumstantial linkage is now modified to account for the more generalized distortion of genetic exchange observed in this study.

Environmental and economic factors have stimulated research in the area of bioenergy crops. While many plants have been identified as potential energy crops, one species in particular, Miscanthus x giganteus, appears to have the most promise. As researchers attempt to exploit and improve M. x giganteus, genome information is critical. In this study, the genome size of M. x giganteus and its two progenitor species were examined by flow cytometry and stomatal cell analyses. M. x giganteus was found to have genome size of 7.0 pg while Miscanthus sinensis and Miscanthus sacchariflorus were observed to have genome sizes of 5.5 and 4.5 pg respectively with stomatal size correlating with genome size. Upon computing the two tetraploid × diploid hybrids theoretical genome sizes, the data presented in this paper supports the hypothesis of the union of a 2x M. sacchariflorus and a 1x M. sinensis gamete for the formation of the allotriploid, M. x giganteus. Such genomic information provides basic knowledge that is important in M. x giganteus plant improvement.

Large proportions of nonhybrid progeny result from controlled crosses between Palmer amaranth and common waterhemp, both dioecious weeds. Agamospermy was proposed as an explanation for this phenomenon, and here we provide evidence in support of this hypothesis. We evaluated 60 nonhybrid offspring from two interspecific crosses, and all plants were females and had DNA content values similar to the female (Palmer amaranth) parent. Among nine hybrids resulting from these crosses, eight were nonviable (lethal or neuter), and only one hybrid allowed for continued gene movement. Cytogenetic evaluation of this hybrid revealed triploidy, further supporting the occurrence of unreduced gametes in these species. In light of this new evidence, we examine earlier data regarding Palmer amaranth by common waterhemp hybridization and suggest some prior conclusions may be premature. Nomenclature: Palmer amaranth, Amaranthus palmeri S. Wats. AMAPAcommon waterhemp, Amaranthus tuberculatus (Moq.) Sauer var. rudis (S...

In maize, the st1 mutant has been observed to result in chromosomes that stick together during both mitotic and meiotic anaphase. These sticky chromosomes result in abnormal chromosome separation at anaphase. Although the mechanism producing the st1 mutant phenotype is unknown, delayed replication of knob heterochromatin has been implicated in similar phenomena that result in sticky chromosomes. Primed in situ labeling (PRINS) was used to locate the 180-bp knob DNA sequences on mitotic metaphase chromosomes of several maize lines. The chromosomal regions labeled by PRINS corresponded to the reported C bands found in these lines. Additionally, PRINS was used to identify knob-bearing regions in anaphase spreads of a line carrying the st1 mutant and a nonmutant line having a similar number of chromosome knobs. The increase in abnormal anaphase figures in the st1 mutant was not accompanied by an increase in association of knob DNA with abnormal anaphases. Thus, the increase in chromosomal stickiness appears to be due to an increase in stickiness of knob and nonknob chromosomal regions. The mechanism responsible for the st1 mutant, therefore, is hypothesized to be different from that implicated in the other previously described sticky chromosomes situations.

In the state of Illinois, waterhemp and smooth pigweed are among the worst agricultural weeds. Previous research shows high potential for hybridization between these two species. However, the actual occurrence of hybrids in natural settings is still uncertain. Morphological similarity between hybrids and waterhemp makes field surveys of hybrids difficult to conduct. The main purpose of this study was to characterize the morphology of waterhemp × smooth pigweed F1hybrids, emphasizing evaluation of characters that may allow for hybrid discrimination in fieldAmaranthuscommunities. Concurrently, the study characterized hybrid reproductive fitness, chromosome number, and DNA content. To accomplish this, hybrids were obtained from field crosses. A species-specific polymorphism in theALSgene was used to verify hybrid identity. Significant differences (α = 0.05) between hybrids and individuals of the parental species were observed for five staminate and five carpellate characters. Of these, five characters differentiated hybrids from waterhemp. However, clustering analyses using these characters indicated that morphological differences were not reliable enough, by themselves, for unambiguous hybrid identification. Also, hybrid homoploidy (2n= 32) with respect to parental species excluded chromosome counts in hybridity determinations. However, DNA content analysis may be used for such purpose. Hybrids had an average of 1.21 pg of DNA per 2C nucleus, a value intermediate to that of parental species. Hybrids produced 3.3 or 0.7% the seed output of parental and sibling waterhemp individuals, respectively. Percent micropollen in hybrids was 95-times greater than in parental species. Hybrid sterility appears to be the most reliable feature for hybrid discrimination when conducting field surveys. However, molecular and cytogenetic analyses as employed in this study may be desired for ultimate identity corroboration.

Concerns have arisen about the possible effects of herbicide contamination in aquatic ecosystems. Crop herbicides are introduced into the aquatic environment both inadvertently through runoff events and intentionally through the use of those registered for use in waterways. Acetochlor and atrazine are two agricultural crop herbicides that have often been reported to contaminate waters. Diquat and fluridone are both registered aquatic management herbicides. In this study, a mammalian in vitro cell cytotoxicity assay was used to evaluate the cytotoxicity of these four commonly used herbicides. The ranked order of the cytotoxicity was: diquat (C(1/2) = 0.036 mM +/- 0.011) > acetochlor (C(1/2) = 0.060 mM +/- 0.010) > fluridone (C(1/2) = 0.172 mM +/- 0.029) atrazine (C(1/2) = 0.581 mM +/- 0.050). In addition, flow cytometric analysis was conducted on CHO cells to investigate the potential impact of these four herbicides on the cell cycle. Acetochlor and diquat had the greatest impact on the cell cycle. Acetochor exposure resulted in a decreased number of cells in the G1 phase of the cell cycle, whereas diquat exposure resulted in a decreased number of cells in both the G1 and G2 phases. Both atrazine and fluridone resulted in a decrease in cells in the G2 phase. The agricultural crop herbicides and aquatic management herbicides gave similar results in cytotoxicity and in the cell-cycle assay at the end points tested.

Weedy Amaranthus species 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 Amaranthus species, the genome sizes of the weedy Amaranthus species 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. palmeri to 1.4 pg in A. tuberculatus. Overlap 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 Amaranthus plant 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 Amaranthus species has been demonstrated.

Mitotic chromosome analysis has proven to be an important tool in monitoring the potential for genetic exchange among related plant species. One major obstacle to using mitotic chromosome analysis in any species is obtaining large numbers of clear, well-spread metaphase chromosomes necessary to perform cytological techniques such as chromosome banding and fluorescent in situ hybridization. The ability to obtain good chromosome spreads is in part determined by the number and morphology of the roots, which contain the metaphase tissue. Many Amaranthus species produce very thin, delicate roots. The technique used in the process described herein provides for much more substantial roots, allowing for higher probability of obtaining well-spread metaphase chromosomes. Seeds were planted in a soilless mixture, and then cuttings and leaves were taken from the plants. The cuttings were sterilized and placed in Murashige and Skoog (MS) media, while leaf tissue was analyzed by flow cytometry, both pre- and post-propagation, to obtain DNA contents. No changes in DNA content were observed. The in vitro procedure produced significantly larger roots than were produced in soilless mix. Furthermore, all of the in vitro roots observed had 32 chromosomes of normal morphology. In vitro root propagation allowed large numbers of roots to be obtained from a single plant, thereby resulting in increased probability of obtaining cells with metaphase chromosomes that reflected the original plants' chromosome numbers and therefore may be used for molecular cytogenetic analysis.

The nuclear DNA content for a group of 40 Malus species and hybrids has been estimated using flow cytometry. Estimates of nuclear DNA content for this germplasm collection range from 1.45 pg for Malus fusca (diploid) to 2.57 pg for Malus ioensis (triploid). Among diploids, the nuclear (2C) DNA ranges from 1.45 pg for M. fusca to 1.68 pg for Malus transitoria. Among triploids, the nuclear (3C) DNA content ranges from 2.37 pg / 3C for Malus sikkimensis to 2.57 pg / 3C for M. ioensis. Given the complexity of the apple genome and its suggested allopolyploid origin, the results obtained in this study confirm earlier reports that polyploids can easily withstand the loss of a certain amount of DNA, and that there is a slight tendency towards diminished haploid nuclear DNA content with increased polyploidy.Key words: apple, DNA content, flow cytometry, Malus, ploidy.

Several populations of different Amaranthus species have been reported resistant to single or multiple herbicides. Interspecific hybridization among amaranths is hypothesized to contribute to the evolution of herbicide resistance. Although other studies have shown the occurrence of interspecific Amaranthus hybrids, little has been done to establish the likelihood of hybridization under field conditions. The main objective of this study was to determine potential field frequencies of hybridization between tall waterhemp females and smooth pigweed. Field hybridization plots were established during each of two growing seasons. Individuals of the two species were transplanted to field plots and arranged at varying distances from each other. Hybrid progeny were detected using the acetolactate synthase (ALS) gene as a marker. Smooth pigweed parents were homozygous for a herbicide-resistance ALS allele, whereas maternal tall waterhemps were homozygous for a herbicide-sensitive ALS form. Heterozygous interspecific progeny were thus detected by means of herbicide selection. Molecular and cytogenetic tools were used to verify the validity of the data obtained. Averaged among female waterhemp plants and across the two field seasons, hybridization occurred at a frequency of 33%. A single tall waterhemp plant was capable of producing more than 200,000 hybrids, suggesting little if any gametic incompatibility. All flowering hybrids obtained from tall waterhemp females were of dioecious condition and female sex. Observed sexual segregation was consistent with previously postulated chromosomal XY-type system in tall waterhemp sex determination, where males are the heterogametic sex.

SUMMARYAmphibian metamorphosis is a complex process that has been speculated to involve DNA amplification and chromatin rearrangement. While recent studies have concentrated on chromatin rearrangement, only a few studies have dealt with variation in the amount of DNA during amphibian metamorphosis. In this study, nuclei were isolated from Xenopus laevis at various developmental stages. The nuclei were examined in both an unfixed state and a fixed state. The nuclei were stained with propidium iodide and analyzed by flow cytometry to determine their fluorescence intensity. The unfixed nuclei had higher fluorescence variation compared with the fixed nuclei. This increase in variation appeared due to the presence of nuclei of variable fluorescence intensity within the unfixed nuclei. Upon optimum fixation, which has been speculated to result in more homogeneous chromatin conformation and to reduce staining artifacts, the nuclei were observed to have less fluorescence intensity variation. The differential fluorescence observed in this study is consistent with the hypothesis that large-scale intra-individual DNA variation is not associated with amphibian metamorphosis.

Summary 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.

With 1 figure and 2 tables Abstract In vivo doubled haploid (DH) technology provides a means of creating new maize inbred lines relatively quickly; however, productivity is limited by false-positive (FP) plants for haploidy and for dihaploidy, which consume resources of space and labour until detected. This work examines the potential for using stomata guard cell length measurement as a means for early detection of FP plants. We found that the true haploid and DH plants could be differentiated from FP and untreated diploid controls as early as Leaf 2 stage by stomata guard cell length measurement. Furthermore, DH plants were distinguishable from haploid and other diploid plants by the Leaf 7 growth stage. Results suggest that, when used together with screening through the anthocyanin colour marker system and flower fertility, stomata guard cell measurement is an easy, non-destructive, early screening method that may lead to a greater efficiency in DH production systems and optimization of resource allocation for space and labour.

Background 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. Methods 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. Results 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. Conclusions 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.

Flow cytometry was used to characterize isolates of Phialophora gregata, the causal organism of brown stem rot (BSR) in soybean, using the fluorescence intensity of YOYO-1 stained conidia. The conidia were derived from isolates of P. gregata isolated from field-grown soybean vascular tissue from two locations in Wisconsin. At each location, four soybean cultivars were grown. Two cultivars were susceptible to BSR and two cultivars were resistant to BSR. Ninety-six isolates, one per plant, were characterized for DNA content. The stained conidia were analyzed with an EPICS XL (Coulter Corporation, Hialeah, Florida, USA) flow cytometer cell sorter with CICERO software (San Rafael, CA, USA). The isolates differed in their mean DNA content, ranging from 78.0 to 116.8 arbitrary units. Statistical analysis showed a significant difference in the DNA content variability of the isolates between the two locations. A notable difference in DNA content variability was observed between the isolates collected from the resistant cultivars between the two locations. However, no significant variation occurred in the DNA content of the isolates collected from susceptible cultivars within or between locations. The observed variation among the isolates is hypothesized to be due to the occurrence of separate populations of P. gregata and selection for mean DNA content that could colonize specific plant genotypes.

The agronomically important herbicide atrazine has been reported to cause damage to animal chromosomes at levels of atrazine found contaminating drinking water supplies. While documenting potential chromosome damage is important it is equally important to compare the damage with the potential consequences of compounds readily found in our food and water supply. In this study atrazine and caffeine, a ubiquitous food additive, were compared at equal levels and at real exposure levels for their ability to damage animals chromosomes in cell culture. Nuclei and chromosomes from treated and control cells were analyzed by flow cytometry. At extremely low levels, atrazine was found to be a more potent clastogen. Caffeine had no effect on the chromosomes at the lower levels. Both chemicals were genotoxic at the potential exposure levels with caffeine being more disruptive than atrazine. Atrazine appears to be a more potent damaging agent than caffeine at similar levels of exposure; however, the levels of caffeine one is exposed to during everyday life appears to be more damaging on the endpoints analyzed in this study than the levels of atrazine found contaminating water supplies. The advantages and limitations of whole cell clasotgenicity are also presented in light of these results.

Background 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. Methods 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. Results 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. Conclusions 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.

Nucleotypic variation has been speculated to play a role in the adaptation of crop species to environmental stress. The objective of this study was to determine if nuclear DNA content variability was associated with aluminium (Al) tolerance in wheat. Six wheat (Triticum aestivumL.) near isolines (differing in Al response), two recurrent parents (Al sensitive), and one donor parent (Al tolerant) were all analysed for nuclear DNA content using flow cytometry. A 1.7% variation in nuclear DNA content was observed among the nine wheat lines. No association between Al response and nuclear DNA content was observed. All of the wheat near isolines had a nuclear DNA content similar to their recurrent parent. The wheat genome appears to be stable with no unusual inheritance of nuclear DNA content observed. Flow cytometric analysis proved to be sensitive enough to detect nuclear DNA content variability at the level of 0.5% variation among wheat lines.

Flow cytometry was used to compare 14 potential reference standards for plant DNA content determination. Both chicken and plant internal standards were used, as were propidium iodide (PI) and 4'-6-diamidino-2-phenylindole (DAPI) as fluorochromes. Means and standard errors of the means are presented for the 14 potential reference standards, and the means are compared to those obtained by Feulgen densitometry. Five species are recommended as an initial set of international standards for future plant DNA content determinations: Sorghum bicolor cv. Pioneer 8695 (2C = 1.74 pg), Pisum sativum cv. Minerva Maple (2C = 9.56 pg), Hordeum vulgare cv. Sultan (2C = 11.12 pg), Vicia faba (2C = 26.66 pg), and Allium cepa cv. Ailsa Craig (2C = 33.55 pg). It is recommended that the reference standard of choice be one with 2C and 4C nuclear DNA content peaks similar to, but not overlapping, the 2C and 4C peaks of the target species. We recommend PI as the fluorochrome of choice for flow cytometric determination of plant DNA content. DAPI should be used only if the estimated DNA value is corroborated by using a second stain that has no bias for AT- or GC-rich sequences within genomes.

Genome size has recently been reported to vary 16% in pumpkins (Cucurbita spp.). The majority of this variation can be attributed to genome size differences in pumpkins of various taxonomical classes. The purpose of this study was to determine if intraspecific genome size variability could be detected by flow cytometry in Cucurbita pepo subsp. pepo pumpkin cultivars with similar fruit morphology. The pie pumpkins group was chosen for this study because of their similar fruit size, shape, and color. Genome sizes ranged from 1.109 pg in Spooktacular to 1.064 pg in Small Sugar. Spooktacular had a genome size larger than Small Sugar in all three experiments. Therefore, intraspecific genome size variation does exist in C. pepo subsp. pepo among pumpkin cultivars of similar fruit morphology.

The induction kinetics of genetic damage were measured in one clone of a mammalian cell line (CHO AS52) with three genotoxicity assays, the single cell gel electrophoresis (Comet) assay, laser beam flow cytometry and forward mutation. The first two assays allow for the rapid analysis of genotoxic damage in individual nuclei. The alkaline Comet assay detects DNA strand breaks, alkali-labile sites and incomplete excision repair sites. Flow cytometry measures chromosome damage that results in an unequal distribution of nuclear DNA in daughter cells. We calibrated these assays to compare acute DNA damage and longer term clastogenicity with forward mutation at the gpt locus using ethyl methanesulfonate (EMS). The EMS treatments were conducted in F12 medium for 2 h. AS52 cells carry a single functional gpt gene which provides for quantitation of gpt mutants by selecting for 6-thioguanine resistance. EMS induced a concentration-dependent response with median Comet tail moment values of 1.06 microns for the negative control and 64.6 microns with 20 mM. The coefficient of variation (CV) of the negative-control with flow cytometry was 2.33; the CV value increased to 4.87 in cells treated with 20 mM EMS, EMS (8 mM) induced a mutant frequency of 779.8 x 10(-6) at a relative survival of 64.4%. Genetic response factors were calculated and the data demonstrate that the induction kinetics of genetic damage as measured by the Comet assay (15.6) and flow cytometry (14.2) were more closely related than that determined for mutation induction (7.9). These three assays measure a wide spectrum of genetic events at the level of DNA, the gene and the chromosome and demonstrate the usefulness of the Comet assay and flow cytometry as two relatively rapid procedures to detect genotoxic damage in mammalian cells.

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.

Intraspecific nuclear DNA content has been documented in various angiosperm species. The purpose of this study was to determine the nuclear DNA content variation in soybean (Glycine max(L.) Merr.). Several studies have suggested that DNA content variation exists in soybean. Ninety soybean lines representing diverse geographic locations in China were analysed by flow cytometry. Nuclei were isolated and stained with either the fluorochrome DAPI or PI. After analysis, it was determined that PI stained nuclei more accurately assessed the total DNA content in soybean. A 12% variation in nuclear DNA content was observed among the 90 lines. The amount of nuclear DNA in the lines was within the DNA range of United States cultivars previously examined. Nuclear DNA content variation in soybean is much less than the variation reported in maize. These results could be due to the lack of polymorphism in large chromosomal elements containing large blocks of repetitive DNA. In addition, unlike maize, the amounts of DNA variation did not decrease as a result of more intensive breeding in United States cultivars. Intraspecific nuclear DNA content variability is very different between these two major agronomic species.

DNA isolated from Black Mexican sweet maize populations segregating for the presence of B chromosomes was used for PCR amplifications of 320 RAPD primers. Seventeen primers produced detectable polymorphisms between populations known to carry B chromosomes and populations devoid of B chromosomes. To ensure that the primers which generated a polymorphic band were indeed identifying B chromosomes, five Arizona Indian corn populations, three tetraploid corn populations and two High Loss corn lines differing in their B chromosome composition were analysed. Four primers were selected to screen these maize populations. Root tip counts were performed on the lines to confirm the association of the RAPD markers to the presence of B chromosomes.

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-metbylethyl)-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'-dietbyl-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.

The agronomic advantage of the T1BL. 1RS translocation and the potential importance of chromosome 1BS to end-use quality of bread wheat (Triticum aestivum L.) 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. Published with the approval of the Director, Oklahoma Agric. Exp. Stn.

Randomly amplified polymorphic DNA (RAPD) markers have the potential to reveal genetic similarity among plants. RAPD profiles of thirteen rhododendron hybrids, species and cultivars were analyzed to study their genetic relationships. The cluster analysis grouped together varieties and/or hybrids in accordance with their known genetic relationship. For instance, two varieties of Rhododendron yakushimanum , var. Mist Maiden and var. Ken Janeck, clustered together while R. yakushimanum var. Pink Parasol clustered with a hybrid of Pink Parasol × Rhododendron smirnowii . These three varieties and the hybrid then clustered with R. smirnowii . Similarly, the other hybrids with common parents showed a closer genetic relatedness with each other than with other rhododendrons. The sequence homology of two amplification products was studied by Southern hybridization. The amplification fragment OPL07 660 of four Rhododendron species, Rhododendron Anna Baldsiefens, Rhododendron arborescens , Rhododendron atlanticum , and Rhododendron poukhanense were homologous to each other. In contrast to this, amplification product OPL03 900 of R. Anna Baldsiefens has no homology to any other amplification product. The genetic relationship revealed from cluster analysis on the basis of RAPD profiles was similar to their known genetic makeup.

Amplification profiles produced by polymerase chain reaction (PCR) using randomly amplified polymorphic DNA sequences (RAPD) have the potential for species and cultivar identification. Since most rhododendron plants are vegetatively propagated, it is imperative that RAPD profiles be stable during this propagation. Three species of rhododendron, Rhododendron arborescens, R. atlanticum and R. yedoense var. poukhanense were used to produce species specific amplification profiles. Stability of amplification profiles among individually cloned plants of each species were studied. Ten plants of R. atlanticum, 9 of R. arborescens, and 10 of R. yedoense var. poukhanense were studied with 10 random primers. No polymorphism was observed among individual plants of R. atlanticum and R. arborescens with all the primers. The amplification product of one plant of R. yedoense var. poukhanense showed a difference of one band with one primer. The rest of the profiles with 9 primers were identical in all plants of this species. In order to ascertain that RAPD markers can indeed reveal real genetic differences among plants, F2 plants of two hybrids were analyzed. In contrast to the clonally propagated plants, extensive polymorphisms were observed among the individual F2 plants. Thus, RAPD analysis can be used to detect genetic variability. This stability of RAPD profiles in clonally propagated rhododendron indicates the usefulness of these markers in plant identification.

Prairie cordgrass (Spartina pectinata), a perennial C4 grass native to the North American prairie, has several distinctive characteristics that potentially make it a model crop for production in stressful environments. However, little is known about the transcriptome dynamics of prairie cordgrass despite its unique freezing stress tolerance. Therefore, the purpose of this work was to explore the transcriptome dynamics of prairie cordgrass in response to freezing stress at -5°C for 5 min and 30 min. We used a RNA-sequencing method to assemble the S. pectinata leaf transcriptome and performed gene-expression profiling of the transcripts under freezing treatment. Six differentially expressed gene (DEG) groups were categorized from the profiling. In addition, two major consecutive orders of gene expression were observed in response to freezing; the first being the acute up-regulation of genes involved in plasma membrane modification, calcium-mediated signaling, proteasome-related proteins, and transcription regulators (e.g., MYB and WRKY). The follow-up and second response was of genes involved in encoding the putative anti-freezing protein and the previously known DNA and cell-damage-repair proteins. Moreover, we identified the genes involved in epigenetic regulation and circadian-clock expression. Our results indicate that freezing response in S. pectinata reflects dynamic changes in rapid-time duration, as well as in metabolic, transcriptional, post-translational, and epigenetic regulation.

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