Root Ideotype Influences Nitrogen Transport and Assimilation in Maize.

Maize (Zea mays, L.) yield is strongly influenced by external nitrogen inputs and their availability in the soil solution. Overuse of nitrogen-fertilizers can have detrimental ecological consequences through increased nitrogen pollution of water and the release of the potent greenhouse gas, nitrous oxide. To improve yield and overall nitrogen use efficiency (NUE), a deeper understanding of nitrogen uptake and utilization is required. This study examines the performance of two contrasting maize inbred lines, B73 and F44. F44 was selected in Florida on predominantly sandy acidic soils subject to nitrate leaching while B73 was selected in Iowa on rich mollisol soils. Transcriptional, enzymatic and nitrogen transport analytical tools were used to identify differences in their N absorption and utilization capabilities. Our results show that B73 and F44 differ significantly in their genetic, enzymatic, and biochemical root nitrogen transport and assimilatory pathways. The phenotypes show a strong genetic relationship linked to nitrogen form, where B73 showed a greater capacity for ammonium transport and assimilation whereas F44 preferred nitrate. The contrasting phenotypes are typified by differences in root system architecture (RSA) developed in the presence of both nitrate and ammonium. F44 crown roots were longer, had a higher surface area and volume with a greater lateral root number and density than B73. In contrast, B73 roots (primary, seminal, and crown) were more abundant but lacked the defining features of the F44 crown roots. An F1 hybrid between B73 and F44 mirrored the B73 nitrogen specificity and root architecture phenotypes, indicating complete dominance of the B73 inbred. This study highlights the important link between RSA and nitrogen management and why both variables need to be tested together when defining NUE improvements in any selection program.

A genomic approach to gene fusion technology.

Gene expression profiling provides powerful analyses of transcriptional responses to cellular perturbation. In contrast to DNA array-based methods, reporter gene technology has been underused for this application. Here we describe a genomewide, genome-registered collection of Escherichia coli bioluminescent reporter gene fusions. DNA sequences from plasmid-borne, random fusions of E. coli chromosomal DNA to a Photorhabdus luminescens luxCDABE reporter allowed precise mapping of each fusion. The utility of this collection covering about 30% of the transcriptional units was tested by analyzing individual fusions representative of heat shock, SOS, OxyR, SoxRS, and cya/crp stress-responsive regulons. Each fusion strain responded as anticipated to environmental conditions known to activate the corresponding regulatory circuit. Thus, the collection mirrors E. coli's transcriptional wiring diagram. This genomewide collection of gene fusions provides an independent test of results from other gene expression analyses. Accordingly, a DNA microarray-based analysis of mitomycin C-treated E. coli indicated elevated expression of expected and unanticipated genes. Selected luxCDABE fusions corresponding to these up-regulated genes were used to confirm or contradict the DNA microarray results. The power of partnering gene fusion and DNA microarray technology to discover promoters and define operons was demonstrated when data from both suggested that a cluster of 20 genes encoding production of type I extracellular polysaccharide in E. coli form a single operon.

High-density microarray-mediated gene expression profiling of Escherichia coli.

A nearly complete collection of 4,290 Escherichia coli open reading frames was amplified and arrayed in high density on glass slides. To exploit this reagent, conditions for RNA isolation from E. coli cells, cDNA production with attendant fluorescent dye incorporation, DNA-DNA hybridization, and hybrid quantitation have been established. A brief isopropyl-beta-D-thiogalactopyranoside (IPTG) treatment elevated lacZ, lacY, and lacA transcript content about 30-fold; in contrast, most other transcript titers remained unchanged. Distinct RNA expression patterns between E. coli cultures in the exponential and transitional phases of growth were catalogued, as were differences associated with culturing in minimal and rich media. The relative abundance of each transcript was estimated by using hybridization of a genomic DNA-derived, fluorescently labeled probe as a correction factor. This inventory provided a quantitative view of the steady-state level of each mRNA species. Genes the expression of which was detected by this method were enumerated, and results were compared with the current understanding of E. coli physiology.

New experimental and computational approaches to the analysis of gene expression.

Public and private EST (Expressed Sequence Tag) programs provide access to a large number of ESTs from a number of plant species, including Arabidopsis, corn, soybean, rice, wheat. In addition to the homology of each EST to genes in GenBank, information about homology to all other ESTs in the data base can be obtained. To estimate expression levels of genes represented in the DuPont EST data base we count the number of times each gene has been seen in different cDNA libraries, from different tissues, developmental stages or induction conditions. This quantitation of message levels is quite accurate for highly expressed messages and, unlike conventional Northern blots, allows comparison of expression levels between different genes. Lists of most highly expresses genes in different libraries can be compiled. Also, if EST data is available for cDNA libraries derived from different developmental stages, gene expression profiles across development can be assembled. We present an example of such a profile for soybean seed development. Gene expression data obtained from Electronic Northern analysis can be confirmed and extended beyond the realm of highly expressed genes by using high density DNA arrays. The ESTs identified as interesting can be arrayed on nylon or glass and probed with total labeled cDNA first strand from the tissue of interest. Two-color fluorescent labeling allows accurate mRNA ratio measurements. We are currently using the DNA array technology to study chemical induction of gene expression and the biosynthesis of oil, carbohydrate and protein in developing seeds.

The STR120 satellite DNA of soybean: organization, evolution and chromosomal specificity.

A highly repeated DNA sequence family, STR120, with tandemly arranged repetitive units (monomers) of approximately 120bp, has been identified in soybean [Glycine max (L.) Merr.]. Five related clones showing tandem repeats of a 120-bp-long monomer were isolated from a soybean genomic library. Results of Southern blotting experiments using three of the clones as probes onto genomic DNA digested with different restriction enzymes were in agreement with a tandem arrangement of these sequences in the genome. A total of 12 monomers were sequenced, showing considerable sequence heterogeneity. A consensus sequence of 126 bp was obtained that exhibits an average similarity of 81% to the sequenced units. In three of the clones identified, neighbouring units are significantly more similar to each other than to units from different clones; in the remaining two clones, however, similarity between the two units observed is low (70%), while the overall similarity between the two clones is high (95%). This indicates that in these cases the repetitive unit may be the dimer rather than the monomer. Based on the presence of direct repeats within each monomer, we suggest that the 120-bp monomer may itself have evolved by duplication of an ancestral 60-bp unit. The STR120 family distribution is limited to annual soybeans and is not found, at least at high-copy number, in related perennial soybeans or other members of the tribe Phaseolae. Fluorescence in situ hybridization (FISH) to metaphase chromosomes using four of the clones as probes shows that the number of chromosomal locations differs depending on the stringency conditions and goes from two to eight when the stringency is progressively lowered. The estimated copy number for one of the clones is from 5000 to 10000, but this may just represent a lower boundary for the whole family in consideration of the high sequence divergence observed within the family. FISH and sequence analysis therefore indicate that different subfamilies as well as higher-order repeat units are present in the STR120 family, very much like those in primate alpha satellite DNA, and that some of the subfamilies seem to exhibit divergence on a chromosomal basis.

Soybean chromosome painting: a strategy for somatic cytogenetics.

Cytological identification of soybean mitotic metaphase chromosomes (2n = 40) has been severely limited by their small size and uniform karyomorphology. We have developed fluorescent in situ hybridization (FISH), PCR-primed in situ labelling (PCR-PRINS) procedures, and molecular probes for routine cytological identification and for the physical mapping of soybean somatic chromosomes. Chromosome preparation has been achieved by modifications of previous protocols and through the preparation of root-tip protoplasts prior to chromosome spreading. Initially our probe selection focused on highly repeated DNAs that provide very intense localized hybridization signals. Repetitive gene probes that have proven valuable include the rDNA loci (5S and 45S) which are chromosome specific. We have also developed satellite DNA probes for two different sequence families: the SB92 and the STR120 satellites. Both of these are tandemly arranged at multiple chromosomal loci. By using different cloned examples of each family, we have been able to selectively label unique subsets of soybean chromosomes. Double hybridization with biotin and digoxigenin labeled probes has allowed us to determine the chromosomal overlap between different probes. In addition, we have joined portions of the metaphase chromosome painting patterns with the genetic map by single-copy FISH and PCR-PRINS detection of the RFLP loci G8.15, G17.3, and A199a and A199b. Total genomic DNA in situ hybridization (GISH) patterns were also used to characterize the soybean chromosomes.

Hypervariable microsatellites provide a general source of polymorphic DNA markers for the chloroplast genome.

BACKGROUND: The study of plant populations is greatly facilitated by the deployment of chloroplast DNA markers. Asymmetric inheritance, lower effective population sizes and perceived lower mutation rates indicate that the chloroplast genome may have different patterns of genetic diversity compared to nuclear genomes. Convenient assays that would allow intraspecific chloroplast variability to be detected are required. RESULTS: Eukaryote nuclear genomes contain ubiquitous simple sequence repeat (microsatellite) loci that are highly polymorphic in length; these polymorphisms can be rapidly typed by the polymerase chain reaction (PCR). Using primers flanking simple mononucleotide repeat motifs in the chloroplast DNA of annual and perennial soybean species, we demonstrate that microsatellites in the chloroplast genome also exhibit length variation, and that this polymorphism is due to changes in the repeat region. Furthermore, we have observed a nonrandom geographic distribution of variations at these loci, and have examined the number and location of such repeats within the chloroplast genomes of other species. CONCLUSIONS: PCR-based analysis of mononucleotide repeats may be used to detect both intraspecific and interspecific variability in the chloroplast genomes of seed plants. The analysis of polymorphic microsatellites thus provides an important experimental tool to examine a range of issues in plant genetics.

Polymorphic simple sequence repeat regions in chloroplast genomes: applications to the population genetics of pines.

Simple sequence repeats (SSRs), consisting of tandemly repeated multiple copies of mono-, di-, tri-, or tetranucleotide motifs, are ubiquitous in eukaryotic genomes and are frequently used as genetic markers, taking advantage of their length polymorphism. We have examined the polymorphism of such sequences in the chloroplast genomes of plants, by using a PCR-based assay. GenBank searches identified the presence of several (dA)n.(dT)n mononucleotide stretches in chloroplast genomes. A chloroplast (cp) SSR was identified in three pine species (Pinus contorta, Pinus sylvestris, and Pinus thunbergii) 312 bp upstream of the psbA gene. DNA amplification of this repeated region from 11 pine species identified nine length variants. The polymorphic amplified fragments were isolated and the DNA sequence was determined, confirming that the length polymorphism was caused by variation in the length of the repeated region. In the pines, the chloroplast genome is transmitted through pollen and this PCR assay may be used to monitor gene flow in this genus. Analysis of 305 individuals from seven populations of Pinus leucodermis Ant. revealed the presence of four variants with intrapopulational diversities ranging from 0.000 to 0.629 and an average of 0.320. Restriction fragment length polymorphism analysis of cpDNA on the same populations previously failed to detect any variation. Population subdivision based on cpSSR was higher (Gst = 0.22, where Gst is coefficient of gene differentiation) than that revealed in a previous isozyme study (Gst = 0.05). We anticipate that SSR loci within the chloroplast genome should provide a highly informative assay for the analysis of the genetic structure of plant populations.

Genetic diagnostics in plant breeding: RAPDs, microsatellites and machines.

The science of mapping genetic traits, including those of agronomic interest, is well established and many genetic marker systems are available. However, the application of genetic diagnostics in plant breeding is in its infancy. The sample throughput and cost requirements are very different from those of medical DNA diagnostics. It will be necessary to automate the DNA isolation process, DNA amplification-based allele identification and data handling. Here, we discuss recent progress in the development of molecular technology for plant breeding.

Global and local genome mapping in Arabidopsis thaliana by using recombinant inbred lines and random amplified polymorphic DNAs.

A population of Arabidopsis thaliana recombinant inbred lines was constructed and used to develop a high-density genetic linkage map containing 252 random amplified polymorphic DNA markers and 60 previously mapped restriction fragment length polymorphisms. Linkage groups were correlated to the classical genetic map by inclusion of nine phenotypic markers in the mapping cross. We also applied a technique for local mapping that allows targeting of markers to a selected genome region by pooling DNA from recombinant inbred lines based on their genotype. We conclude that random amplified polymorphic DNAs, used in conjunction with a recombinant inbred population, can facilitate the genetic and physical characterization of the Arabidopsis genome and that this method is generally applicable to other organisms for which appropriate populations either are available or can be developed.

DNA polymorphisms amplified by arbitrary primers are useful as genetic markers.

Molecular genetic maps are commonly constructed by analyzing the segregation of restriction fragment length polymorphisms (RFLPs) among the progeny of a sexual cross. Here we describe a new DNA polymorphism assay based on the amplification of random DNA segments with single primers of arbitrary nucleotide sequence. These polymorphisms, simply detected as DNA segments which amplify from one parent but not the other, are inherited in a Mendelian fashion and can be used to construct genetic maps in a variety of species. We suggest that these polymorphisms be called RAPD markers, after Random Amplified Polymorphic DNA.

Two strands of DNA are not equivalent as probes in hybridizations at low stringency.

Only one of the two complementary strands of a restriction fragment hybridizes under low stringency conditions to a cloned Arabidopsis thaliana genomic DNA fragment. We propose that this effect is caused by the energetic nonequivalence of the two possible mismatched duplexes, resulting from the accumulation of mismatches and extrahelical bases. These mismatches will differ between the two duplexes. The choice of probe strand may therefore be important for the success of heterologous hybridizations utilizing single-stranded probes.

Structure of the yeast HOM3 gene which encodes aspartokinase.

The yeast HOM3 gene has been cloned molecularly by complementation of a HOM3 mutant. The gene is located about 8 kilobase pairs from HIS1 and is present as a single copy in the yeast genome. Mutations in HOM3 result in a requirement for threonine and methionine (or homoserine) for growth and a lack of detectable aspartokinase activity. The nucleotide sequence of HOM3 predicts an enzyme 414 amino acids long that shows homology to the three Escherichia coli aspartokinases, indicating that it is the structural gene for yeast aspartokinase. An approximately 1800-base pair mRNA is transcribed from the HOM3 gene, initiating at several start sites, 80 and 70 base pairs downstream, respectively, from two TATA boxes. Upstream of the TATA boxes is a single TGACTC sequence. This sequence has been shown to be essential for regulation of several genes that encode amino acid biosynthetic enzymes by the general control system. However, no increase in aspartokinase mRNA is observed under general control derepressing conditions.

Structure of wheat gamma-gliadin genes.

We have cloned and sequenced two linked members of the wheat (Triticum aestivum) gliadin multigene family. One gene encodes a gamma-gliadin which is 292 amino acids (aa) long. S1 mapping indicates that this gene is transcriptionally active. The second gene, which is only marginally active by S1 mapping, is closely related to the first gene. Moreover, it is probably incapable of encoding a full-length gamma-gliadin due to the presence of two premature in-frame stop codons. Neither gene contains introns. Nucleotide sequences of the two genes show the presence of characteristic repeats which have a derived aa consensus sequence Pro-Gln-Gln-Pro-Gln-Gln-Pro-Phe-Pro-Gln. A comparison of the promoter regions of these gamma-gliadin genes with those of the alpha-gliadin genes shows the presence of a highly conserved region that could be involved in tissue specific and developmental regulation.

Conservation and variability of wheat alpha/beta-gliadin genes.

We have sequenced two genomic clones for wheat alpha/beta-gliadin storage protein genes. Comparison with a known sequence reveals close homology between the three and confirms the previously suspected evolutionary relatedness of members of this gliadin family. The coding region can be divided into six domains. Two unusual structures were found within this region: (i) The P-boxes which are composed of 12 codons, six of which are for proline, that are tandemly repeated four or five times; and (ii) Two polyglutamine stretches which consist of 18-22 tandemly repeated glutamine codons in one case, and 7-28 in the second. Analysis of the P-box structures revealed that certain mutations were probably present in the hypothetical ancestral alpha/beta-gliadin gene prior to gene multiplication. None of the genes have introns. All of the genes appear to contain typical eukaryotic promoters and also possess the double polyadenylation signal of plants.

Complete nucleotide sequence of the AIDS virus, HTLV-III.

The complete nucleotide sequence of two human T-cell leukaemia type III (HTLV-III) proviral DNAs each have four long open reading frames, the first two corresponding to the gag and pol genes. The fourth open reading frame encodes two functional polypeptides, a large precursor of the major envelope glycoprotein and a smaller protein derived from the 3'-terminus long open reading frame analogous to the long open reading frame (lor) product of HTLV-I and -II.

Developmentally regulated plant genes: the nucleotide sequence of a wheat gliadin genomic clone.

Gliadins, the major wheat seed storage proteins, are encoded by a multigene family. Northern blot analysis shows that gliadin genes are transcribed in endosperm tissue into two classes of poly(A)+ mRNA, 1400 bases (class I) and 1600 bases (class II) in length. Using poly(A)+ RNA from developing wheat endosperm we constructed a cDNA library from which a number of clones coding for alpha/beta and gamma gliadins were identified by hybrid-selected mRNA translation and DNA sequencing. These cDNA clones were used as probes for the isolation of genomic gliadin clones from a wheat genomic library. One such genomic clone was characterized in detail and its DNA sequence determined. It contains a gene for a 33-kd alpha/beta gliadin protein (a 20 amino acid signal peptide and a 266 amino acid mature protein) which is very rich in glutamine (33.8%) and proline (15.4%). The gene sequence does not contain introns. A typical eukaryotic promoter sequence is present at -104 (relative to the translation initiation codon) and there are two normal polyadenylation signals 77 and 134 bases downstream from the translation termination codon. The coding sequence contains some internal sequence repetition, and is highly homologous to several alpha/beta gliadin cDNA clones. Homology to a gamma-gliadin cDNA clone is low, and there is no homology with known glutenin or zein cDNA sequences.