VARIATION OF NUCLEAR DNA CONTENT IN HELIANTHUS ANNUUS (ASTERACEAE)

Nuclear 2C DNA content was determined by laser flow cytometry for 13 diploid (2n = 34) lines (cultivated varieties and inbred lines) of Helianthus annuus. Mean DNA amount of second leaf nuclei varied from 6.01 to 7.95 pg (32%) among lines. Mean DNA content varied up to 19% within lines. Variability in mean DNA content exceeding 27% and 48% was detected among leaves from different nodes of plants of the open-pollinated variety, Californicus, and the inbred line, RHA 299, respectively. The root tip and shoot tip nuclei of H. annuus have been reported to consist of a mixture of aneuploid (17 to 33 chromosomes) and diploid (34 chromosomes) cells, a condition called aneusomaty. Chromosome counts of root tips and an analysis of the distribution of DNA content of large numbers of nuclei from leaves indicate that aneusomaty either does not occur, or is not common, among the lines investigated. The intraspecific, intraline, and intraplant variation in DNA content in H. annuus support the concept that a sizable portion of a plant genome is unstable and subject to rapid changes in DNA amount.     

High-resolution flow karyotyping and chromosome sorting in Vicia faba lines with standard and reconstructed karyotypes

Flow cytometric analysis has been performed on chromosomes isolated from formaldehyde-fixed root tips in a Vicia faba (2n = 12) line with a standard (wild-type) karyotype and in six V. faba translocation lines with reconstructed karyotypes. The resolution of individual chromosome types on histograms of chromosome fluorescence intensity (flow karyotypes) depended on the type of fluorochrome used for chromosome staining. The highest degree of resolution was achieved with 4,6-diamidino-2-phenylindole (DAPI). The lower resolution obtained after staining with mithramycin A (MIT) and propidium iodide (PI) was probably due to the sensitivity of these stains to changes in chromatin structure induced by formaldehyde fixation. After the staining with DAPI, only 1 chromosome type could be discriminated in the line with a standard karyotype. In the translocation lines, the number of chromosome types resolved on flow karyotypes ranged from 2 in the G and the ACB lines to all (6) chromosome types in the EFK and EF lines. Refined flow karyotyping permitted the sorting of a total of 15 different chromosome types from five of the translocation lines. It is expected that flow sorting of chromosomes from reconstructed karyotypes will become a powerful tool in the study of nuclear genome organisation in V. faba.     

Flow Cytometric Analysis and Chromosome Sorting of Barley (Hordeum vulgare L.)

Flow cytometric analysis was systematically performed to optimize the concentration and duration of hydroxyurea (DNA synthesis inhibitor) and trifluralin (metaphase blocking reagent) treatments for synchronizing the cell cycle and accumulating metaphase chromosomes in barley root tips. A high metaphase index (76.5% in the root tip meristematic area) was routinely achieved. Seedlings of about 1.0-cm length were treated with 1.25 mM hydroxyurea for 14 h to synchronize the root tip meristem cells at the S/G2 phase. After rinsing with hydroxyurea, the seedlings were incubated in a hydroxyurea-free solution for 2 h and were treated with 1 microM trifluralin for 4 h to accumulate mitotic cells in the metaphase. The consistent high metaphase index depended on the uniform germination of seeds prior to treatment. High-quality and high-quantity isolated metaphase chromosomes were suitable for flow cytometric analysis and sorting. Flow karyotypes of barley chromosomes were established via univariate and bivariate analysis. A variation of flow karyotypes was detected among barley lines. Two single chromosome types were identified and sorted. Bivariate analysis showed no variation among barley individual chromosomes in AT and GC content.     

Flow Cytometric Analysis and Chromosome Sorting of Barley (Hordeum vulgare L.)

Flow cytometric analysis was systematically performed to optimize the concentration and duration of hydroxyurea (DNA synthesis inhibitor) and trifluralin (metaphase blocking reagent) treatments for synchronizing the cell cycle and accumulating metaphase chromosomes in barley root tips. A high metaphase index (76.5% in the root tip meristematic area) was routinely achieved. Seedlings of about 1.0-cm length were treated with 1.25 mM hydroxyurea for 14 h to synchronize the root tip meristem cells at the S/G2 phase. After rinsing with hydroxyurea, the seedlings were incubated in a hydroxyurea-free solution for 2 h and were treated with 1 M trifluralin for 4 h to accumulate mitotic cells in the metaphase. The consistent high metaphase index depended on the uniform germination of seeds prior to treatment. High-quality and high-quantity isolated metaphase chromosomes were suitable for flow cytometric analysis and sorting. Flow karyotypes of barley chromosomes were established via univariate and bivariate analysis. A variation of flow karyotypes was detected among barley lines. Two single chromosome types were identified and sorted. Bivariate analysis showed no variation among barley individual chromosomes in AT and GC content.     

Flow sorting and microcloning of maize chromosome 1

Flow sorting maize chromosome 1 and construction of the first chromosome 1 DNA Lambda library are described. Maize metaphase chromosome suspensions were prepared from synchronized seedling root tip cells of the maize hybrid line Seneca 60 and stained with propidium iodide for flow karyotyping and sorting. The observed flow karyotype was very similar to the predicted flow karyotype constructed based on published values for the relative chromosome sizes of Seneca 60. The estimated size of chromosomes from the peak for the chromosome 1 matched the expected size of maize chromosome 1. The peak for the chromosome 1 was well resolved from other peaks on the flow karyotype. An average of 7 x 10(3) chromosomes of chromosome 1 could be produced from 10 root tips. About 0.6 million chromosomes of maize chromosome 1 were sorted and pooled based on the cytogram of fluorescent pulse area Vs fluorescent pulse width and stored at -20 degrees C in the freezer. DNA isolated from sorted chromosomes was good quality of more than 100 kb in size. Chromosome 1 DNA was partially digested with BamHI, dephosphorylated and ligated with arms of BamHI digested Lambda Dash vector. A total of 1.2 x 10(5) independent recombinants with the average insert size 12.6 kb was obtained. This library covered approximately 90% of maize chromosome 1. Hybridization of cloned fragments with labeled maize genomic DNA showed that the high, middle, or low copy number DNA sequences presented in the different phage clones. PCR (polymerase chain reaction) using chromosome-specific primers confirmed the specificity of this library. The individual chromosome library is useful in plant genome mapping and gene isolation.     

Chromosome heteromorphism quantified by high-resolution bivariate flow karyotyping.

Maternal and paternal homologues of many chromosome types can be differentiated on the basis of their peak position in Hoechst 33258 versus chromomycin A3 bivariate flow karyotypes. We demonstrate here the magnitude of DNA content differences among normal chromosomes of the same type. Significant peak-position differences between homologues were observed for an average of four chromosome types in each of the karyotypes of 98 different individuals. The frequency of individuals with differences in homologue peak positions varied among chromosome types: e.g., chromosome 15, 61%; chromosome 3, 4%. Flow karyotypes of 33 unrelated individuals were compared to determine the range of peak position among normal chromosomes. Chromosomes Y, 21, 22, 15, 16, 13, 14, and 19 were most heteromorphic, and chromosomes 2-8 and X were least heteromorphic. The largest chromosome 21 was 45% larger than the smallest 21 chromosome observed. The base composition of the variable regions differed among chromosome types. DNA contents of chromosome variants determined from flow karyotypes were closely correlated to measurements of DNA content made of gallocyanin chrome alum-stained metaphase chromosomes on slides. Fluorescence in situ hybridization with chromosome-specific repetitive sequences indicated that variability in their copy number is partly responsible for peak-position variability in some chromosomes. Heteromorphic chromosomes are identified for which parental flow karyotype information will be essential if de novo rearrangements resulting in small DNA content changes are to be detected with flow karyotyping.     

Analysis and sorting of rye (Secale cereale L.) chromosomes using flow cytometry.

Procedures for chromosome analysis and sorting using flow cytometry (flow cytogenetics) were developed for rye (Secale cereale L.). Suspensions of intact chromosomes were prepared by mechanical homogenization of synchronized root tips after mild fixation with formaldehyde. Histograms of relative fluorescence intensity obtained after the analysis of DAPI-stained chromosomes (flow karyotypes) were characterized and the chromosome content of the DNA peaks was determined. Chromosome 1R could be discriminated on a flow karyotype of S. cereale 'Imperial'. The remaining rye chromosomes (2R-7R) could be discriminated and sorted from individual wheat-rye addition lines. The analysis of lines with reconstructed karyotypes demonstrated a possibility of sorting translocation chromosomes. Supernumerary B chromosomes could be sorted from an experimental rye population and from S. cereale 'Adams'. Flow-sorted chromosomes were identified by fluorescence in situ hybridization (FISH) with probes for various DNA repeats. Large numbers of chromosomes of a single type sorted onto microscopic slides facilitated detection of rarely occurring chromosome variants by FISH with specific probes. PCR with chromosome-specific primers confirmed the identity of sorted fractions and indicated suitability of sorted chromosomes for physical mapping. The possibility to sort large numbers of chromosomes opens a way for the construction of large-insert chromosome-specific DNA libraries in rye.     

中国17个优良玉米自交系的分子标记杂合性及其与杂交种性状的关系研究

在玉米单交种育种中 ,鉴定高产杂交种和具有优良特性的自交系是一个重要的问题。研究以 1 7个优良玉米自交系为亲本 ,按照双列杂交配组合 ,利用 RAPD技术分析了 1 7个自交系的多态性以及 RAPD标记与 9个重要农艺性状 (包括产量 )的关系。基于 RAPD标记计算的相似系数聚类将 1 7个自交系分为 5个类群 ,经分析与系谱亲缘关系基本一致。杂交种性状及其特殊配合力与亲本间的遗传距离是高度相关的 ,与聚类前比较 ,聚类后平均遗传距离与平均产量、平均特殊配合力的相关系数显著提高 ,类间平均产量高于类内平均产量。RAPD技术可揭示优良玉米自交系的系谱亲缘关系 ,将自交系划分成不同的类群 ,从而为选择类间自交系杂交 ,进行亲本选配和分子标记辅助育种提供一种方法。     

Application of flow karyotyping in prenatal detection of chromosome aberrations

This paper describes the application of bivariate flow karyotyping to (1) classification of chromosomes isolated from cultures of cells taken by amniocentesis and (2) detection of numerical and structural aberrations. Chromosomes were isolated from primary cultures 2-5 wk after amniocentesis, stained with Hoechst 33258 and chromomycin A3, and analyzed using dual beam flow cytometry. Information about chromosome DNA content and DNA base composition was derived from the locations of the peaks in the flow karyotypes, each peak being produced by one or more chromosome types with similar DNA content and DNA base composition. Information about the relative frequency of each chromosome type was determined on the basis of the relative volume of the peak for that chromosome type. Cytogenetic information determined on the basis of flow karyotypes was compared with that obtained by visual analysis following G-banding. Variability among the peak means and volumes in flow karyotypes was determined from analyses of 50 normal amniocyte cultures. Numerical aberrations involving chromosomes 21, 18, and Y were detected correctly in all of 28 analyses, including eight in a blind study. Structural aberrations involving chromosomes 1, 2, 3, 6, 9-12, 13, 14, 15, 21, and 22 were detected in all of seven cultures in a blind study. Flow karyotypes proved to be insensitive to small, normally occurring chromosome polymorphisms detected by banding analysis. In addition, a few samples were erroneously scored as having numerical aberrations.     

Flow sorting of mitotic chromosomes in common wheat (Triticum aestivum L.)

The aim of this study was to develop an improved procedure for preparation of chromosome suspensions, and to evaluate the potential of flow cytometry for chromosome sorting in wheat. Suspensions of intact chromosomes were prepared by mechanical homogenization of synchronized root tips after mild fixation with formaldehyde. Histograms of relative fluorescence intensity (flow karyotypes) obtained after the analysis of DAPI-stained chromosomes were characterized and the chromosome content of all peaks on wheat flow karyotype was determined for the first time. Only chromosome 3B could be discriminated on flow karyotypes of wheat lines with standard karyotype. Remaining chromosomes formed three composite peaks and could be sorted only as groups. Chromosome 3B could be sorted at purity >95% as determined by microscopic evaluation of sorted fractions that were labeled using C-PRINS with primers for GAA microsatellites and for Afa repeats, respectively. Chromosome 5BL/7BL could be sorted in two wheat cultivars at similar purity, indicating a potential of various wheat stocks for sorting of other chromosome types. PCR with chromosome-specific primers confirmed the identity of sorted fractions and suitability of flow-sorted chromosomes for physical mapping and for construction of small-insert DNA libraries. Sorted chromosomes were also found suitable for the preparation of high-molecular-weight DNA. On the basis of these results, it seems realistic to propose construction of large-insert chromosome-specific DNA libraries in wheat. The availability of such libraries would greatly simplify the analysis of the complex wheat genome.     

Involvement of knob heterochromatin in mitotic abnormalities in germinating aged seeds of maize.

Seeds of three inbred lines of maize, with contrasting numbers of heterochromatic knobs and stored under two different ageing treatments, were analyzed for the occurrence of abnormalities at mitotic anaphase in root meristems 3, 7, 21,42, and 56 days after germination, and in root meristems of their freshly harvested selfed progeny. The largest category of detectable aberrations involved breakage of knobbed chromosome arms. We have obtained evidence that knob heterochromatin plays a central role in the origin of primary chromosome bridges. The initial event responsible for the occurrence of breakages and lagging chromosomes was characterized by the nondisjunction of newly replicated sister chromatids, which was observed to occur preferentially at the knob level. Such configurations, and all the other types of abnormalities (as for example, lagging chromosomes, typical chromosome bridges, fragments, and micronuclei), were observed at decreasing frequencies throughout root growth. Nevertheless, we have detected the occurrence of breakage-fusion-bridge cycles that were initiated by broken chromosomes. The relationship between late-replicating DNA in maize knob heterochromatin and the vulnerability of such regions to breakage is discussed. Our observations suggest a similarity between the mechanisms involved or associated with the origin of the described abnormalities and those reported to occur in cultured maize cells.     

DNA content of wheat monosomics at interphase estimated by flow cytometry

 Two complete, independently maintained sets of 21 monosomic wheat lines derived from cv. ‘Chinese Spring’ were analyzed for their DNA content at the G1 stage with flow cytometry. The DNA content of individual chromosomes was estimated by subtracting the value of a monosomic line from that of euploid wheat. Our data show that the estimated 2C DNA of individual wheat chromosomes in 21 monosomics at the G1 stage ranges from about 0.58 pg in chromosome 1D to approximately 1.12 pg in chromosome 3A. The A genome (2C=6.15 pg) seems to contain more DNA than the B (2C=6.09 pg) and D (2C=5.05 pg) genomes. Analysis of variance showed significant differences (α=0.01) in DNA content both among homoeologous groups and among genomes. Our estimates of interphase DNA content of wheat chromosomes from monosomic lines were poorly correlated to the chromosome sizes at metaphase (r=0.622, P≤0.01). This poor correlation might be due to differential coiling among chromosomes during cell division, possible bias of fluorochrome binding to heterochromatin, or heterogeneity among monosomic lines. Finally, flow cytometry may aid but cannot replace cytological checks in aneuploid maintenance. Received: 21 January 1997 / Accepted: 23 June 1997     

Intraplant Nuclear DNA Content Variation in Diploid Nuclei of Maize (Zea mays L.)

Diploid nuclei from stem, mesocotyl, nodal root and root tiptissue of two maize hybrids were examined with respect to theirDNA content. The nuclei were isolated and stained with DAPIand passed through a flow cytometer-cell sorter. The titrationcurve for each tissue was determined. Significant variationwas observed among nuclei of different tissue types. Stem androot tips had the highest diploid nuclear DNA amounts while2-week-old mesocotyl had the lowest diploid nuclear DNA amount.These results provide evidence that during plant developmentand differentiation, the amount of DNA within a diploid nucleuschanges through loss of specific DNA sequences. This study alsodemonstrates the sensitivity of flow cytometry in detectingsmall intraplant variation in nuclear DNA. Key words: Flow cytometry, fluorochrome DAPI, DNA content, tissue differentiation, plant development     

Genetic diversity of African maize inbred lines revealed by SSR markers

Knowledge of genetic diversity (GD) and relationships among maize inbred lines is indispensable in a breeding program. Our objectives were to (1) investigate the level of genetic diversity among maize inbred lines and (2) assess their genetic structures by applying simple sequence repeat (SSR) markers. Fifty-six highland and mid-altitude maize inbred lines obtained from CIMMYT programs in Ethiopia and Zimbabwe were genotyped using 27 SSR loci. All of the genotypes studied could unequivocally be distinguished with the combination of the SSRs used. In total, 104 SSR alleles were identified, with a mean of 3.85 alleles per locus. The average polymorphism information content (PIC) was 0.58. GD expressed as Euclidean distance, varied from 0.28 to 0.73 with an average of 0.59. Cluster analysis using unweighted pair group method with arithmetic average (UPGMA) suggested five groups among the inbred lines. Most of the inbred lines adapted to the highlands and the mid-altitudes were positioned in different clusters with a few discrepancies. The pattern of groupings of the inbred lines was mostly consistent with available pedigree information. The variability detected using SSR markers could potentially contribute towards effective utilization of the inbred lines for the exploitation of heterosis and formation of genetically diverse source populations in Ethiopian maize improvement programs.     

玉米茎秆高糖种质资源筛选与评价

以151个玉米自交系为材料,对玉米茎秆高糖种质资源进行了筛选与评价。结果表明,自交系茎秆含糖量(B rix)变化范围为3.5%～16.9%,能为茎秆高糖玉米新品种选育所利用的自交系(B rix≥10%)种质资源比较丰富,约占测定自交系总数43.1%。通过聚类分析,151个玉米自交系按茎秆含糖量差异可分为3大类,第3类属于茎秆高糖类型(12.3%～16.9%),共有22个自交系,这一类群种质可能是培育茎秆高糖玉米新品种的重要亲本材料,其中78599-1-550、78599-2、YXD053-646、Y53-245、预CY509等5个自交系尤其适合作为茎秆高糖玉米育种亲本。     

Genetic diversity and its relationship to hybrid performance in maize as revealed by RFLP and AFLP markers

 The challenge to maize breeders is to identify inbred lines that produce highly heterotic hybrids. In the present study we surveyed genetic divergence among 13 inbred lines of maize using DNA markers and assessed the relationship between genetic distance and hybrid performance in a diallel set of crosses between them. The parental lines were assayed for DNA polymorphism using 135 restriction fragment length polymorphisms (RFLPs) and 209 amplified-fragment polymorphisms (AFLPs). Considerable variation among inbreds was detected with RFLP and AFLP markers. Moreover AFLPs detect polymorphisms more efficiently in comparison to RFLPs, due to the larger number of loci assayed in a single PCR reaction. Genetic distances (GDs), calculated from RFLP and AFLP data, were greater among lines belonging to different heterotic groups compared to those calculated from lines of the same heterotic group. Cluster analysis based on GDs revealed associations among lines which agree with expectations based on pedigree information. The GD values of the 78 F₁ crosses were partioned into general (GGD) and specific (SGD) components. Correlations of GD with F₁ performance for grain yield were positive but too small to be of predictive value. The correlations of SGDs, particularly those based on AFLP data, with specific combining-ability effects for yield may have a practical utility in predicting hybrid performance. Received: 15 August 1997 / Accepted: 19 September 1997     

Flow cytometric sorting of maize chromosome 9 from an oat-maize chromosome addition line

Large numbers of maize chromosome 9 can be collected with high purity by flow cytometric sorting of chromosomes isolated from a disomic maize chromosome addition line of oat. Metaphase chromosome suspensions were prepared from highly synchronized seedling root-tips of an oat-maize chromosome-9 addition line (OM9) and its parental oat and maize lines. Chromosomes were stained with propidium iodide for flow cytometric analysis and sorting. Flow-karyotypes of the oat-maize addition line showed an extra peak not present in the parental oat line. This peak is due to the presence of a maize chromosome-9 pair within the genome of OM9. Separation of maize chromosome 9 by flow cytometric sorting of a chromosome preparation from a normal maize line was not possible because of its size similarity (DNA content) to maize chromosomes 6, 7 and 8. However, it is possible to separate maize chromosome 9 from oat chromosomes and chromatids. An average of about 6×10³ chromosomes of maize chromosome 9 can be collected by flow-sorting from chromosomes isolated from 30 root tips (ten seedlings) of the oat-maize addition line. Purity of the maize chromosome 9, sorted from the oat-maize chromosome addition line, was estimated to be more than 90% based on genomic in situ hybridization analysis. Sorting of individual chromosomes provides valuable genomic tools for physical mapping, library construction, and gene isolation. Received: 28 February 2000 / Accepted: 14 July 2000     

Mitochondrial DNA transfer to the nucleus generates extensive insertion site variation in maize

Mitochondrial DNA (mtDNA) insertions into nuclear chromosomes have been documented in a number of eukaryotes. We used fluorescence in situ hybridization (FISH) to examine the variation of mtDNA insertions in maize. Twenty overlapping cosmids, representing the 570-kb maize mitochondrial genome, were individually labeled and hybridized to root tip metaphase chromosomes from the B73 inbred line. A minimum of 15 mtDNA insertion sites on nine chromosomes were detectable using this method. One site near the centromere on chromosome arm 9L was identified by a majority of the cosmids. To examine variation in nuclear mitochondrial DNA sequences (NUMTs), a mixture of labeled cosmids was applied to chromosome spreads of ten diverse inbred lines: A188, A632, B37, B73, BMS, KYS, Mo17, Oh43, W22, and W23. The number of detectable NUMTs varied dramatically among the lines. None of the tested inbred lines other than B73 showed the strong hybridization signal on 9L, suggesting that there is a recent mtDNA insertion at this site in B73. Different sources of B73 and W23 were examined for NUMT variation within inbred lines. Differences were detectable, suggesting either that mtDNA is being incorporated or lost from the maize nuclear genome continuously. The results indicate that mtDNA insertions represent a major source of nuclear chromosomal variation.     

Organ-specific invertase deficiency in the primary root of an inbred maize line

An organ-specific invertase deficiency affecting only the primary root system is described in the Oh 43 inbred maize (Zea mays). Invertases (acid and neutral/soluble and insoluble) were assayed in various tissues of hybrid (NK 508) and inbred (Oh 43, W 22) maize lines to determine the basis for an early report that Oh 43 root tips were unable to grow on sucrose agar (27). Substantial acid invertase activity (7.3 to 16.1 micromoles of glucose per milligram of protein per hour) was evident in extracts of all tissues tested except the primary root system of Oh 43. This deficiency was also evident in lateral roots arising from the primary root. In contrast, morphologically identical lateral roots from the adventitious root system had normal invertase levels. These results suggest that ontogenetic origin of root tissues is an important determinant of invertase expression in maize. Adventitious roots (including the seminals) arise above the scutellar node and are, therefore, of shoot origin. The Oh 43 deficiency also demonstrated that invertase activity was not essential for maize root growth. Sucrose synthase was active in extracts from all root apices and theoretically provided the only available avenue for sucrose degradation in primary root tips of Oh 43. The deficiency described here will provide a useful avenue of investigation into the expression and significance of root invertase.     

Genetic distance of inbred lines and prediction of maize single-cross performance using RAPD markers

 To evaluate the genetic diversity of 18 maize inbred lines, and to determine the correlation between genetic distance and single-cross hybrid performance, we have used random amplified polymorphic DNA (RAPD), a PCR-based technique. Eight of these lines came from a Thai synthetic population (BR-105), and the others derived from a Brazilian composite population (BR-106). Thirty two different primers were used giving a total of 325 reproducible amplification products, 262 of them being polymorphic. Genetic divergence was determinated using Jaccard’s similarity coefficient, and a final dendrogram was constructed using an unweighted pair-group method with arithmetical averages (UPGMA). Cluster analysis divided the samples into three distinct groups (GI, GII and GIII) that were confirmed by principal-coordinate analysis. The genetic distances (GD) were correlated with important agronomic traits for single-cross hybrids and heterosis. No correlation was found when group division was not considered, but significant correlations were detected between GI×GII and GI×GIII GDs with their respective single-cross hybrid grain-yield values. Three groups were identified; that is, the BR-106 population was divided in two different groups and the BR-105 population remained mostly as one group. The results indicated that RAPD can be used as a tool for determining the extent of genetic diversity among tropical maize inbred lines, for allocating genotypes into different groups, and also to aid in the choice of the superior crosses to be made among maize inbred lines, so reducing the number of crosses required under field evaluation. Received: 24 May 1996 / Accepted: 22 November 1996