Development of a controlled-environment assay to induce iron deficiency chlorosis in soybean by adjusting calcium carbonates, pH, and nodulation.

BACKGROUND: Soybean iron deficiency chlorosis (IDC) is an important nutrient stress frequently found in high pH and/or soils high in calcium carbonates. To advance the understanding of IDC resistance in soybean, a rapid (21-day) controlled-environment assay was developed to investigate the effects of nodulation, pH, and calcium carbonate levels on soybean iron deficiency traits. This system was tested on four genotypes known to exhibit differences in iron efficiency, including two standard IDC check cultivars and a pair of near-isogenic lines exhibiting variation at an IDC resistance quantitative trait locus. Visual score, chlorophyll content, plant height, root dry mass, and shoot dry mass were measured to quantify iron stress. RESULTS: Calcium carbonate levels and nodulation were found to have the greatest effects on IDC severity. Increasing calcium carbonate levels worsened IDC symptoms, while nodulation reduced symptoms in all genotypes. Higher pH levels increased iron deficiency symptoms in check genotypes 'Corsoy 79' and 'Dawson', but did not induce iron deficiency symptoms in near-isogenic lines. A significant interaction was observed between genotype, nodulation, and calcium carbonate level, indicating that a specific treatment level could discern IDC symptoms between genotypes differing in resistance to IDC. CONCLUSIONS: IDC symptoms were successfully induced in the Check Genotypes Experiment as well as the NIL Experiment, indicating the success of using this assay for inducing IDC in controlled environments. However, our results suggest that treatment levels that best differentiate genotypes for their IDC resistance may need to be determined for each experiment because of the unique way in which different genotypes display symptoms and respond to iron deficiency conditions.

Instability of bacterial artificial chromosome (BAC) clones containing tandemly repeated DNA sequences.

The cloning and propagation of large DNA fragments as bacterial artificial chromosomes (BACs) has become a valuable technique in genome research. BAC clones are highly stable in the host, Escherichia coli, a major advantage over yeast artificial chromosomes (YACs) in which recombination-induced instability is a major drawback. Here we report that BAC clones containing tandemly repeated DNA elements are not stable and can undergo drastic deletions during routine library maintenance and DNA preparation. Instability was observed in three BAC clones from sorghum, rice, and potato, each containing distinct tandem repeats. As many as 46% and 74% of the single colonies derived from a rice BAC clone containing 5S ribosomal RNA genes had insert deletions after 24 and 120 h of growth, respectively. We also demonstrated that BAC insert rearrangement can occur in the early stage of library construction and duplication. Thus, a minimum growth approach may not avoid the instability problem of such clones. The impact of BAC instability on genome research is discussed.

A tandemly repeated DNA sequence is associated with both knob-like heterochromatin and a highly decondensed structure in the meiotic pachytene chromosomes of rice.

Highly repetitive tandem DNA sequence repeats are often associated with centromeric and telomeric regions of eukaryotic chromosomes. The rice tandem repeat Os48 is organized as long arrays of a 355 bp monomer and is mainly located in the telomeric regions. The chromosomal locations of the Os48 sequence were determined by fluorescence in situ hybridization (FISH) on rice pachytene chromosomes. The majority of the Os48 loci are associated with brightly 4',6-diamidino-2-phenylindole (DAPI)-stained and knob-like heterochromatin in rice pachytene chromosomes. As with other DNA sequences located in the heterochromatic regions, the cytosines of the CG and C(A/T)G sites within the Os48 repeat are heavily methylated. Surprisingly, a proportion of the FISH signals are highly decondensed and deviate significantly from the DAPI-stained periphery of the pachytene chromosomes. This highly decondensed chromatin structure has not been reported in pachytene chromosomes prepared from alcohol/acid-fixed meiotic samples in any other eukaryotic species. The condensation of the Os48 sequences is dynamic during prophase I of meiosis. The FISH signals derived from the Os48 repeat progress from a condensed configuration between leptonema and early pachynema into a decondensed structure from middle pachynema to diakinesis, and then return to a condensed form at metaphase I.

Complex mtDNA constitutes an approximate 620-kb insertion on Arabidopsis thaliana chromosome 2: implication of potential sequencing errors caused by large-unit repeats.

Previously conducted sequence analysis of Arabidopsis thaliana (ecotype Columbia-0) reported an insertion of 270-kb mtDNA into the pericentric region on the short arm of chromosome 2. DNA fiber-based fluorescence in situ hybridization analyses reveal that the mtDNA insert is 618 +/- 42 kb, approximately 2.3 times greater than that determined by contig assembly and sequencing analysis. Portions of the mitochondrial genome previously believed to be absent were identified within the insert. Sections of the mtDNA are repeated throughout the insert. The cytological data illustrate that DNA contig assembly by using bacterial artificial chromosomes tends to produce a minimal clone path by skipping over duplicated regions, thereby resulting in sequencing errors. We demonstrate that fiber-fluorescence in situ hybridization is a powerful technique to analyze large repetitive regions in the higher eukaryotic genomes and is a valuable complement to ongoing large genome sequencing projects.