Permanent Genetic Resources added to Molecular Ecology Resources database 1 January 2009-30 April 2009.

This article documents the addition of 283 microsatellite marker loci to the Molecular Ecology Resources Database. Loci were developed for the following species: Agalinis acuta; Ambrosia artemisiifolia; Berula erecta; Casuarius casuarius; Cercospora zeae-maydis; Chorthippus parallelus; Conyza canadensis; Cotesia sesamiae; Epinephelus acanthistius; Ficedula hypoleuca; Grindelia hirsutula; Guadua angustifolia; Leucadendron rubrum; Maritrema novaezealandensis; Meretrix meretrix; Nilaparvata lugens; Oxyeleotris marmoratus; Phoxinus neogaeus; Pristomyrmex punctatus; Pseudobagrus brevicorpus; Seiridium cardinale; Stenopsyche marmorata; Tetranychus evansi and Xerus inauris. These loci were cross-tested on the following species: Agalinis decemloba; Agalinis tenella; Agalinis obtusifolia; Agalinis setacea; Agalinis skinneriana; Cercospora zeina; Cercospora kikuchii; Cercospora sorghi; Mycosphaerella graminicola; Setosphaeria turcica; Magnaporthe oryzae; Cotesia flavipes; Cotesia marginiventris; Grindelia Xpaludosa; Grindelia chiloensis; Grindelia fastigiata; Grindelia lanceolata; Grindelia squarrosa; Leucadendron coniferum; Leucadendron salicifolium; Leucadendron tinctum; Leucadendron meridianum; Laodelphax striatellus; Sogatella furcifera; Phoxinus eos; Phoxinus rigidus; Phoxinus brevispinosus; Phoxinus bicolor; Tetranychus urticae; Tetranychus turkestani; Tetranychus ludeni; Tetranychus neocaledonicus; Tetranychus amicus; Amphitetranychus viennensis; Eotetranychus rubiphilus; Eotetranychus tiliarium; Oligonychus perseae; Panonychus citri; Bryobia rubrioculus; Schizonobia bundi; Petrobia harti; Xerus princeps; Spermophilus tridecemlineatus and Sciurus carolinensis.

Ty1-copia-retrotransposon behavior in a polyploid cotton.

Retrotransposons constitute a ubiquitous and dynamic component of plant genomes. Intragenomic and intergenomic comparisons of related genomes offer potential insights into retrotransposon behavior and genomic effects. Here, we have used fluorescent in-situ hybridization to determine the chromosomal distributions of a Ty1-copia-like retrotransposon in the cotton AD-genome tetraploid Gossypium hirsutum and closely related putative A- and D-genome diploid ancestors. Retrotransposon clone A108 hybridized to all G. hirsutum chromosomes, approximately equal in intensity in the A- and D-subgenomes. Similar results were obtained by hybridization of A108 to the A-genome diploid G. arboreum, whereas no signal was detected on chromosomes of the D-genome diploid G. raimondii. The significance and potential causes of these observations are discussed.

New ribosomal RNA gene locations in Gossypium hirsutum mapped by meiotic FISH.

In this study we have mapped newly identified rDNA loci in Gossypium hirsutum. Four new minor 18S-26S rDNA loci, in addition to the sites previously identified, were mapped using fluorescence in situ hybridization (FISH) to heterozygous translocation (NT) quadrivalents (IVs). The newly detected 18S-26S rDNA loci were mapped to the right arms of chromosomes 8, 9, 15, 17, 19, 20, and 23 and the left arms of chromosomes 5, 11, 12, and 14. Using the rDNA loci as common reference points, we detected several erroneous arm assignments in the previously published map of NT breakpoints. The data are summarized in the form of an integrated map for all 17 known rDNA loci, relative to centromeres, telomeres, and NT breakpoints. This information will facilitate future locus-specific research on rRNA gene evolution and function.

Dispersed repetitive DNA has spread to new genomes since polyploid formation in cotton.

Polyploid formation has played a major role in the evolution of many plant and animal genomes; however, surprisingly little is known regarding the subsequent evolution of DNA sequences that become newly united in a common nucleus. Of particular interest is the repetitive DNA fraction, which accounts for most nuclear DNA in higher plants and animals and which can be remarkably different, even in closely related taxa. In one recently formed polyploid, cotton (Gossypium barbadense L.; AD genome), 83 non-cross-hybridizing DNA clones contain dispersed repeats that are estimated to comprise about 24% of the nuclear DNA. Among these, 64 (77%) are largely restricted to diploid taxa containing the larger A genome and collectively account for about half of the difference in DNA content between Old World (A) and New World (D) diploid ancestors of cultivated AD tetraploid cotton. In tetraploid cotton, FISH analysis showed that some A-genome dispersed repeats appear to have spread to D-genome chromosomes. Such spread may also account for the finding that one, and only one, D-genome diploid cotton, Gossypium gossypioides, contains moderate levels of (otherwise) A-genome-specific repeats in addition to normal levels of D-genome repeats. The discovery of A-genome repeats in G. gossypioides adds genome-wide support to a suggestion previously based on evidence from only a single genetic locus that this species may be either the closest living descendant of the New World cotton ancestor, or an adulterated relic of polyploid formation. Spread of dispersed repeats in the early stages of polyploid formation may provide a tag to identify diploid progenitors of a polyploid. Although most repetitive clones do not correspond to known DNA sequences, 4 correspond to known transposons, most contain internal subrepeats, and at least 12 (including 2 of the possible transposons) hybridize to mRNAs expressed at readily discernible levels in cotton seedlings, implicating transposition as one possible mechanism of spread. Integration of molecular, phylogenetic, and cytogenetic analysis of dispersed repetitive DNA may shed new light on evolution of other polyploid genomes, as well as providing valuable landmarks for many aspects of genome analysis.

Use of meiotic FISH for identification of a new monosome in Gossypium hirsutum L.

The extensive use of molecular cytogenetics in human genetics and clinical diagnostics indicates that analogous applications in plants are highly feasible. One sort of application would be the identification of new aneuploids, which traditionally involves either direct karyotypic identification, which is feasible in only a few plant species, or tests with markers (cytogenetic, genetic, or molecular), which require sexual hybridization and at least one subsequent seed or plant generation. We have used meiotic fluorescence in situ hybridization (FISH) to analyze a new monosome of cotton (Gossypium hirsutum L., 2n = 4x = 52, 2(AD)1) that had a phenotype which seemed to be distinct from monosomes in the Cotton Cytogenetic Collection. Painting with A2-genome DNA revealed the monosome's D-subgenome origin. DAPI-PI staining showed that the monosome carries a major NOR, delimiting it to the major NOR-bearing chromosomes of the D-subgenome, i.e., 16 or 23. Dual-color FISH with 5S and 18S-28S rDNAs indicated that the monosome contains separate major clusters of each of these two tandemly repeated rDNA elements, thus delimiting the monosome to chromosome 23, for which the Cotton Cytogenetic Collection has previously been devoid of any sort of deficiency. Of the 26 chromosomes in the cotton genome, the Collection now provides coverage for 16 (70%) in the form of monosomy, and 20 (77%) in the form of monosomy and (or) telosomy. Use of molecular cytogenetic methods to identify a new plant aneuploid in cotton exemplifies the fact that a physicochemical karyotypic chromosome identification system is not required a priori for application of new molecular cytogenetic methods, thus indicating their potential applicability to nearly all plant species.

A rapid stain-clearing method for video based cytological analysis of cotton megagametophytes.

Optical "clearing" is a cost saving method for preparing large numbers of whole, dissected or thickly sectioned cytological specimens such as plant ovules and ovaries. Minimal labor is required and specimens retain three-dimensional integrity. Previous development of high contrast stain-clearing methods using hemalum to impart contrast has facilitated analysis and photography under bright field illumination for small ovules. The deep stain intensity of hemalum, however, often precludes adequate light transmission and contrast within internal focal planes, limiting the applicability of hemalum-based stain-clearing to small specimens. Having encountered this problem for nucelli of cotton (Gossypium barbadense L.), which are roughly 300 microns thick at fertilization, we have developed a modified stain-clearing system. The two key features of these new methods are the use of azure, C, which allows the intensity of staining to be readily regulated, and contrast manipulation via video signal and image processing. Intensity of azure C stain was readily controlled by modifying the staining and/or dehydration media to produce relatively low contrast specimens. Analysis was facilitated by indirect viewing on a video monitor using adjustments of sensitivity, exposure, and contrast of the charge-coupled device (CCD) camera. Digital processing provided further enhancement. Acceptable images were obtained from virtually all specimens. These methods, which combine low contrast (high transmittance) specimens with high contrast imaging, should facilitate data acquisition on reproduction, thus the developmental and genetic characterization of reproductive mutants. Other applications, e.g., in pathology and embryology, are readily envisioned.

ISH-facilitated analysis of meiotic bivalent pairing.

Chiasmata constitute one of the cornerstones of sexual reproduction in most eukaryotes. They mediate the reciprocal genetic exchange between homologues and are essential to the proper orientation of the homologous centromeres in meiosis I. As markers of recombination, they offer a cytological means of mapping. Rather than trying to accurately count individual chiasmata, we have examined properties of the mathematical relationship between frequencies of nonadorned disomic configurations in meiosis (ring, rods, and univalents) and the probabilities at which arms of the respective chromosomes are chiasmate (one or more chiasma per arm). Numerical analyses indicated that conventionally analyzed bivalents with nonidentified arms yield statistically biased estimates of chiasma probabilities under a broad range of circumstances. We subsequently analyzed estimators derived from adorned configurations with ISH-marked arms, which were found to be statistically far superior, and with no assumptions concerning interference across the centromere. We applied this methodology in the study of chromosomes 16 and 23 of cotton (Gossypium hirsutum), and estimated their arm lengths in centimorgans. The results for chromosome 23, the only one of the two chromosomes with a documented RFLP map, were consistent with the literature. Similar molecular-meiotic configuration analyses can be used for a wide variety of eukaryotic organisms and purposes: for example, providing far more powerful meiotic comparisons of genomes of chromosomes, and a rapid means of evaluating effects on recombination. Key words : meiotic configurations, chiasma frequencies, in situ hybridization, cotton.

Distribution of 5S and 18S-28S rDNA loci in a tetraploid cotton (Gossypium hirsutum L.) and its putative diploid ancestors.

The most widely cultivated species of cotton, Gossypium hirsutum, is a disomic tetraploid (2n=4x=52). It has been proposed previously that extant A- and D-genome species are most closely related to the diploid progenitors of the tetraploid. We used fluorescent in situ hybridization (FISH) to determine the distribution of 5S and 18S-28S rDNA loci in the A-genome species G. herbaceum and G. arboreum, the D-genome species G. raimondii and G. thurberi, and the AD tetraploid G. hirsutum. High signal-to-noise, single-label FISH was used to enumerate rDNA loci, and simultaneous, dual-label FISH was used to determine the syntenic relationships of 5S rDNA loci relative to 18S-28S rDNA loci. These techniques provided greater sensitivity than our previous methods and permitted detection of six new G. hirsutum 18S-28S rDNA loci, bringing the total number of observed loci to 11. Differences in the intensity of the hybridization signal at these loci allowed us to designate them as major, intermediate, or minor 18S-28S loci. Using genomic painting with labeled A-genome DNA, five 18S-28S loci were localized to the G. hirsutum A-subgenome and six to the D-subgenome. Four of the 11 18S-28S rDNA loci in G. hirsutum could not be accounted for in its presumed diploid progenitors, as both A-genome species had three loci and both D-genome species had four. G. hirsutum has two 5S rDNA loci, both of which are syntenic to major 18S-28S rDNA loci. All four of the diploid genomes we examined contained a single 5S locus. In g. herbaceum (A1) and G. thurberi (D1), the 5S locus is syntenic to a major 18S-28S locus, but in G. arboreum (A2) and G. raimondii (D5), the proposed D-genome progenitor of G. hirsutum, the 5S loci are syntenic to minor and intermediate 18S-28S loci, respectively. The multiplicity, variation in size and site number, and lack of additivity between the tetraploid species and its putative diploid ancestors indicate that the behavior of rDNA loci in cotton is nondogmatic, and considerably more complex and dynamic than previously envisioned. The relative variability of 18S-28S rDNA loci versus 5S rDNA loci suggests that the behavior of tandem repeats can differ widely.

Identification of a homeologous chromosome pair by in situ DNA hybridization to ribosomal RNA loci in meiotic chromosomes of cotton (Gossypium hirsutum).

In situ DNA hybridization with 18S-28S and 5S ribosomal DNA probes was used to map 18S-28S nucleolar organizers and tandem 5S repeats to meiotic chromosomes of cotton (Gossypium hirsutum L.). Mapping was performed by correlating hybridization sites to particular positions in translocation quadrivalents. Arm assignment required translocation quadrivalents with at least one interstitial chiasma and sufficient distance between the hybridization site and the centromere. We had previously localized a major 18S-28S site to the short arm of chromosome 9; here we mapped two additional major 18S-28S sites to the short arm of chromosome 16 and the left arm of chromosome 23. We also identified and mapped a minor 18S-28S site to the short arm of chromosome 7. Two 5S sites of unequal size were identified, the larger one near the centromere of chromosome 9 and the smaller one near the centromere of chromosome 23. Synteny of 5S and 18S-28S sites indicated homeology of chromosomes 9 and 23, while positions of the other two 18S-28S sites supplement genetic evidence that chromosomes 7 and 16 are homeologous.

Aneuploidy, structural chromosome changes, and DNA amounts in the annual taxa of the Haplopappus spinulosus complex.

Haplopappus gracilis (n = 2), Haplopappus ravenii (n = 4), and Haplopappus wigginsii (n = 4) are isolated by F1 hybrid sterility due mainly to translocation heterozygosity. There is no evidence that this can be overcome at the diploid level so that introgression can occur among them. They are also separated geographically, but occasional populations of H. gracilis and H. ravenii may be brought together along roadways to form sterile hybrids. There were no statistically significant differences in nuclear DNA content among the same or structurally different aneuploid n = 2 and n = 3 chromosome races or ecotypes of H. gracilis. Some of the H. gracilis races were not significantly different from one race of the ancestral H. ravenii, and these samples of both species were from plants growing on poor soils in contrast to accessions from normal habitats. How much and which classes of DNA in these species are subject to changes induced by environmental effects is not known. There were no correlations between DNA amounts and altitude, latitude, and longitude. H. wigginsii had a greater amount of DNA per nucleus than either H. ravenii or H. gracilis, and its increased DNA content may reflect a more rapid accumulation of noncoding sequences due to facultative self-compatibility not found in the other two species.

Chromosome pairing relationships among the A, B, and D genomes of bread wheat.

Chromosome pairing and chiasma frequency were studied in bread wheat euhaploids (2n = 3x = 21; ABD genomes) with and without the major pairing regulatorPh1. This constitutes the first report of chromosome pairing relationships among the A, B, and D genomes of wheat without the influence of an alien genome. AllPh1 euhaploids had very little pairing, with 0.62-1.05 rod bivalents per cell; ring bivalents were virtually absent and mean arm-binding frequency (c) values ranged from 0.050 to 0.086. In contrast, theph1b euhaploids had extensive homoeologous pairing, with chiasma frequency 7.5-11.6 times higher than that in thePh1 euhaploids. They had 0.53-1.16 trivalents, 1.53-1.74 ring bivalents, and 2.90-3.57 rod bivalents, withc from 0.580 to 0.629. N-banding of meiotic chromosomes showed strongly preferential pairing between chromosomes of the A and D genomes; 80% of the pairing was between these genomes, especially in the presence of theph1b allele. The application of mathematical models to unmarked chromosomes also supported a 2∶1 genomic structure of theph1b euhaploids. Numerical modeling suggested that about 80% of the metaphase I association was between the two most related genomes in the presence ofph1b, but that pairing under Ph1 was considerably more random. The data demonstrate that the A and D genomes are much more closely related to each other than either is to B. These results may have phylogenetic significance and hence breeding implications.

Mayer's hemalum-methyl salicylate: a stain-clearing technique for observations within whole ovules.

Nondissected ovaries of tuber-bearing Solanum sp. were stained with Mayer's hemalum, a positive stain for chromatin and nucleoli, and then optically cleared with methyl salicylate, a clearing agent. Clarity, resolution and contrast within the ovules dissected from ovaries were comparable to those of sectioned, paraffin embedded ovaries. Contrast within ovules greatly exceeded that of unstained and nonspecifically stained clearings, and eliminated the need of special optics, i.e., Nomarski interference-contrast optics, for optimal viewing and photography. Much less time and labor were required than for embedded specimens. Usefulness of the technique for cytogenetic and cytological research was verified by analyzing meiosis and other features of megasporogenesis and megagametogenesis in normal, and in two meiotic mutants, of Solanum. The results illustrate the usefulness of combined Mayer's hemalum staining and methyl salicylate clearing, and suggest additional stain-clearing agent combinations have potential for cytological and cytogenetic research. Preliminary results with other species suggest the technique may also be useful for classroom instruction.