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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.

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.

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 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.

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.

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.

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.

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.

Aluminum toxicity of acid soils is an important growth limiting factor which can reduce crop yields. Breeders have used various screening techniques for the rapid selection of Al tolerance lines. Al toxicity is complex having multiple effects on plant growth. The question becomes can plant selection for tolerance at one specific Al saturation result in tolerance at multiple aluminum saturation levels. Six near-isolines and three wheat (Triticum aestivum L.) cultivars differing in aluminum response on the basis of Al exclusion in the root cell wall were used in this study. Seeds of each line were planted in three soil treatments, 0.5%, 27% and 65% aluminum saturation respectively. Mitotic analysis was carried out on root tip cells. Soils with aluminum saturation were seen to induce sticky chromosome damage in both tolerant and susceptible lines. Lagging chromosomes, chromosome fragments and anaphase bridges were seen in cells of root tips grown in soils with increased aluminum saturation. The number of cells in susceptible lines exhibited an increase in stickiness as the aluminum saturation of the soil increased. In tolerant lines, the number of cells with stickiness increased at 27% saturation, but then decreased at 65% aluminum saturation. The amount of chromosome stickiness appears to be not only dependent upon the plant response to phytotoxic Al but also the aluminum saturation level at which the plant is grown. Tolerance as measured by reduced chromosomal stickiness did not occur until the plants were grown in very high soil Al saturation, indicating a single selection scheme for Al tolerance may not be adequate to develop the highest degree of tolerance.

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.

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.

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

Prior research indicated that analysis of nuclear DNA content by flow cytometry could be used to distinguish smooth pigweed × tall waterhemp hybrids when the parent lines are known. Flow cytometry was performed on nuclei isolated from several Illinois populations of smooth pigweed and tall waterhemp. The smooth pigweed and tall waterhemp analyzed had nonoverlapping 2C nuclear DNA content values, with mean values of 1.04 and 1.34 pg, respectively. The consistent difference in DNA content observed between the two species indicates that DNA content analysis can be used to distinguish their hybrid progeny in natural populations. Nomenclature: Smooth pigweed, Amaranthus hybridus L. AMACH; tall waterhemp, Amaranthus tuberculatus (Moq.) Sauer AMATU.

Several studies have indicated a possible link between genome size and earliness in maize. In this study, an original maize population, South African photo-period insensitive maize composite II (C0) and several selected generations were analyzed for days to flowering, plant height, ear height, and yield. The selection criterion was earliness. Over six cycles of selection a 14-day decrease in days to flowering was obtained with no significant reduction in grain yield. In the initial population (C0) and the most advanced selection (C6) 101 plants were analyzed for nuclear DNA content. A significant decrease in the mean nuclear DNA content of the C0 population (102 AU) was observed with respect to the C0 population (105 AU). This reduction was the result of a decrease in frequency of plants in the C6 population with large genome sizes. Therefore it was concluded that selection for earliness resulted in selection against plants with large genome sizes. Hence, the nucleotype of a maize plant can be modified by selection such as early flowering time.

SUMMARY Nuclei were isolated from stems, mesocotyls, nodal roots and root tips of two maize hybrids. The fluorochrome, propidium iodide (PI), was used to stain the nuclei that were passed through a flow cytometer-cell sorter. Significant differences in fluorescence intensities were observed among the diploid nuclei from the different plant organs. These observations, in conjunction with previously reported DAPI fluorescence data, reflect the state of chromatin condensation in diploid nuclei isolated from meristematic and differentiating tissues. Nuclei from root meristematic tissues showed no or very little chromatin condensation. Mesocotyls and nodal roots, 1-wk-old, appeared to contain chromatin in a more diffuse state owing to completed differentiation which allows more DNA to be accessible to PI. Mesocotyls, 2-wk-old owing to senescence, contained degraded chromatin in which DNA was more accessible to PI because it lacked associated proteins. The diploid nuclei from stems had the highest amount of chromatin condensation. The results show the usefulness of flow cytometry for analyzing chromatin condensation in plant nuclei. The study also shows the importance of selecting a particular type of fluorochrome in the analysis of nuclear chromatin.

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.

 Premise of the study : Looming petroleum shortages and projected negative impacts of human-induced climate change may be partly alleviated by the development and use of bioenergy feedstock crops. Miscanthus × giganteus , a highly productive sterile triploid hybrid grass that was discovered in Japan several decades ago, has considerable potential as an alternative source of energy. Given the risks, however, involved in the reliance upon production of one clone of this hybrid, which is a natural cross between Miscanthus saccharifl orus and Miscanthus sinensis , for lignocellulosic bioenergy production, natural occurrences of triploidy were investigated in sympatric populations of tetraploid M. saccharifl orus and diploid M. sinensis in Japan.  Methods : Seeds were counted and DNA content was estimated by flcytometry for plants of M. saccharifl orus and M. sinensis in several sympatric populations throughout Japan. Chromosomes were also counted for select plants.  Key results : Based on seed-set data, M. saccharifl orus has signifi cantly lower seed set than M. sinensis in Japan. Putative triploid seeds were found on M. saccharifl orus plants in southern Japan.  Conclusions : This is the fi rst report of the natural occurrence of Miscanthus triploid plants in several decades. If found to be sterile and similar in productivity to the commonly cultivated clone of M. × giganteus , these triploid plants might serve as additional sources of genetic variation for bioenergy production. Seed set data also indicates that other triploid plants might be found in more northern regions of Japan.

Nuclei from different tissues such as stem, mesocotyl, nodal root and root tip of diploid and tetraploid maize were isolated, stained with propidium iodide and passed through an EPICS-751 flow-cytometer cell sorter. Variations in flow histograms were observed in different tissues. Stem tissues of both the diploid and tetraploid had two peaks representing G1 and G2 somatic nuclei. The remaining tissues in both the diploids and tetraploids exhibited three peaks. The first peak observed in these tissues represents G1 somatic nuclei of the lowest ploidy level. The second peak represent G2 somatic nuclei of the lowest ploidy level+G1 somatic nuclei of the next ploidy level. The third peak represents G2 of the higher ploidy level+G1 somatic nuclei of the next higher ploidy level. Statistically significant differences were observed between the diploid and tetraploid maize tissues with respect to nuclei distribution in the higher ploidy level peaks implying variation in the degree of endopolyploidy in the diploid and tetraploid maize. The results of this study suggest that the amount of endopolyploid observed in maize genotypes has an effect on their overall agronomic performance under the field conditions.

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

Previous studies have indicated a relationship between genome size and cold tolerance in plants. Many species adapted to growth in cool environments have large genome sizes. These studies are based on interspecific DNA content variation. In this study, the nuclear DNA content of eight maize populations was determined. These populations were obtained from the University of Nebraska and represent populations selected for cold tolerance and their respective unselected original populations. Intraspecific DNA content variation was observed between the selected and unselected populations. Upon assessing the data based solely on cold tolerance, no clear relationship between genome size and cold tolerance was apparent. When both freeze tolerance and cold tolerance were considered, populations which were cold tolerant and exhibited a certain degree of freeze tolerance were observed to have significantly larger genomes relative to the unselected populations. Thus, it appears that the relationship between intraspecific genome size variation and cold tolerance is similar to the relationship between interspecific genome size variation and growth at cooler temperatures in plants.

Fifteen maize populations were analyzed with respect to their genome size and chloroplast number per guard cell. The reported genome sizes of these populations range from 9.82 pg to 13.8 pg. When all the populations were analyzed, a positive correlation between genome size and chloroplast number was observed. Upon removal of two of the populations from the analyses, a significant positive correlation was observed with an r value of 0.72. These results are consistent with published data which demonstrate a positive correlation between interspecific genome size and chloroplast number. Genome size does appear to influence the number of chloroplasts per guard cell within the species Zea mays ssp. mays . Two of the populations have chloroplast numbers indicative of large genome size, yet have small genome sizes. In these populations, genetic factors appear to regulate chloroplast number per guard cell. Nucleotypic factors, in addition to genetic factors, should be included in those mechanisms which regulate chloroplast number within as well as among species.

Three repeated sequence clones, pAS1(1.0 Kb), pAS2(1.8 Kb) and pAS12(2.5 Kb), were isolated fromAegilops squarrosa (Triticum tauschii). The inserts of the three clones did not hybridize to each other. Two of the clones, pAS2 and pAS12, contain repeated sequences which were distributed throughout the genome. The clone pAS1 sequence was more restricted and was located in specific areas on telomeres and certain interstitial sites along the chromosome length. This cloned sequence was also found to be restricted to the D genome at the level ofin situ hybridization. The pAS1 sequence will be useful in chromosomal identification and phylogenetic analysis. All three clones will allow assessment of genome plasticity inAegilops squarrosa.

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.

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 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.

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 ofhexaploid 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. IN THE EARLY 1960's, studies were underway in which the DNA duplex of various organisms was disassociated into two strands and then restored by incubation under proper conditions. Before these studies were undertaken in higher organisms, the speculation was that the large dilution ofnucleotides in the vast amounts of DNA per cell would require restoration (renaturation) to proceed for months. However, certain classes ofDNA which rapidly renatured were observed in eukaryotic organisms (Britten and Kohne, 1968). These classes of DNA represent sequences which are reiterated throughout the genome and are designated as repeated DNA sequences. The most highly repeated DNA sequences may consist of simple sequence or satellite DNA. Satellite DNA is defined as DNA which can be separated from the bulk of the main DNA by ultracentrifiguation in a neutral cesium chloride density gradient. 'Received for publication 29 June 1986; revision accepted 24 November 1986. 2 Current address is Department of Agronomy, Oklahoma State University, Stillwater, OK 74078. In addition, species containing cryptic or hidden satellite sequences have been observed (Comeo, Ginelli and Polli 1970; Bendich and Anderson, 1974; Peacock et al., 1981). These cryptic sequences require changes in the conditions of centrifugation, yet still represent highly repeated DNA sequences. Triticum aestivum L. has been reported to have a 2C DNA content of approximately 34.6 picogranis (pg) (Bennett, Smith and HeslopHarrison, 1982). A genome size of this magnitude would require less than 1% of the total DNA to carry out coding and other sequencerelated functions (Flavell, 1980). Much of the excess DNA appears to be composed of repeated DNA sequences. Estimates of the amount of repeated DNA sequences in plants have ranged from 35% to 95% (Flavell et al., 1974; Murray et al., 1979; Nagl et al., 1983). In T. aestivum, the proportion of repeated DNA sequences has been estimated at 83% and has been speculated to be much higher (Flavell et al., 1974; Flavell, 1980). Repeated sequences in plants appear to be undergoing constant amplification, deletion, and sequence divergence

The chromosomes of polyploid Triticum species and the putative donor diploid species were analyzed by in situ hybridization with a repeated DNA sequence clone pSc 119 isolated from rye and also found in wheat. In Triticum aestivum cv. Chinese Spring, chromosome 4A showed one terminal site in the short arm and one terminal and two interstitial sites of hybridization in the long arm. Triticum turgidum contained a 4A chromosome identical to 'Chinese Spring' with respect to hybridization sites. Chromosome 4A of the timopheevi wheats differed from 4A of 'Chinese Spring' in that the site of the sequence on the short arm was subterminal rather than terminal. Of the A-, B-, and D-genome progenitor species, only potential B-genome donors Aegilops speltoides and Aegilops sharonensis each showed a chromosome with hybridization sites similar to 4A. This suggested that 4A belongs to the B genome. Moreover, with regard to this sequence, chromosome 4A has undergone only minor changes during the evolution of the polyploid wheats.Key words: wheat evolution, in situ hybridization, biotin labeling.

The number of mitotic chromosomal C-bands, the percent ofthe 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 thehighest (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.