Analysis of root-knot nematode and fusarium wilt disease resistance in cotton (Gossypium spp.) using chromosome substitution lines from two alien species.

Chromosome substitution (CS) lines in plants are a powerful genetic resource for analyzing the contribution of chromosome segments to phenotypic variance. In this study, a series of interspecific cotton (Gossypium spp.) CS lines were used to identify a new germplasm resource, and to validate chromosomal regions and favorable alleles associated with nematode or fungal disease resistance traits. The CS lines were developed in the G. hirsutum L. TM-1 background with chromosome or chromosome segment substitutions from G. barbadense L. Pima 3-79 or G. tomentosum. Root-knot nematode (Meloidogyne incognita) and fusarium wilt (Fusarium oxysporum f. sp. vasinfectum) (races 1 and 4) resistance alleles and quantitative trait loci (QTL) previously placed on cotton chromosomes using SSR markers in two interspecific recombinant inbred line populations were chosen for testing. Phenotypic responses of increased resistance or susceptibility in controlled inoculation and infested field assays confirmed the resistance QTLs, based on substitution with the positive or negative allele for resistance. Lines CS-B22Lo, CS-B04, and CS-B18 showed high resistance to nematode root-galling, confirming QTLs on chromosomes 4 and 22 (long arm) with resistance alleles from Pima 3-79. Line CS-B16 had less fusarium race 1-induced vascular root staining and higher percent survival than the TM-1 parent, confirming a major resistance QTL on chromosome 16. Lines CS-B(17-11) and CS-B17 had high fusarium race 4 vascular symptoms and low survival due to susceptible alleles introgressed from Pima 3-79, confirming the localization on chromosome 17 of an identified QTL with resistance alleles from TM1 and other resistant lines. Analyses validated regions on chromosomes 11, 16, and 17 harboring nematode and fusarium wilt resistance genes and demonstrated the value of CS lines as both a germplasm resource for breeding programs and as a powerful genetic analysis tool for determining QTL effects for disease resistance. CS lines carrying small alien chromosome segments with favorable QTL alleles could be used for effective introgression of biotic stress resistance or many other desirable traits by targeting gene interactions and reducing linkage drag effects.

Interspecific chromosomal effects on agronomic traits in Gossypium hirsutum by AD analysis using intermated G. barbadense chromosome substitution lines.

The untapped potential of the beneficial alleles from Gossypium barbadense L. has not been well utilized in G. hirsutum L. (often referred to as Upland cotton) breeding programs. This is primarily due to genomic incompatibility and technical challenges associated with conventional methods of interspecific introgression. In this study, we used a hypoaneuploid-based chromosome substitution line as a means for systematically introgressing G. barbadense doubled-haploid line '3-79' germplasm into a common Upland genetic background, inbred 'Texas marker-1' ('TM-1'). We reported on the chromosomal effects, lint percentage, boll weight, seedcotton yield and lint yield in chromosome substitution CS-B (G. barbadense L.) lines. Using an additive-dominance genetic model, we studied the interaction of alleles located on two alien substituted chromosomes versus one alien substituted chromosome using a partial diallel mating design of selected CS-B lines (CS-B05sh, CS-B06, CS-B09, CS-B10, CS-B12, CS-B17 and CS-B18). Among these parents, CS-B09 and CS-B10 were reported for the first time. The donor parent 3-79, had the lowest additive effect for all of the agronomic traits. All of the CS-B lines had significant additive effects with boll weight and lint percentage. CS-B10 had the highest additive effects for lint percentage, and seedcotton and lint yield among all of the lines showing a transgressive genetic mode of inheritance for these traits. CS-B09 had greater additive genetic effects on lint yield, while CS-B06, CS-B10 and CS-B17 had superior additive genetic effects on both lint and seedcotton yield compared to TM-1 parent. The 3-79 line had the highest dominance effects for boll weight (0.513 g) and CS-B10 had the lowest dominance effect for boll weight (-0.702). Some major antagonistic genetic effects for the agronomic traits were present with most of the substituted chromosomes and chromosome arms, a finding suggested their recalcitrance to conventional breeding efforts. The results revealed that the substituted chromosomes and arms of 3-79 carried some cryptic beneficial alleles with potential to improve agronomic traits including yield, whose effects were masked at the whole genome level in 3-79.

Delineation of interspecific epistasis on fiber quality traits in Gossypium hirsutum by ADAA analysis of intermated G. barbadense chromosome substitution lines.

Genetic diversity is the foundation of any crop improvement program, but the most cultivated Upland cotton [Gossypium hirsutum L., 2n = 52, genomic formula 2(AD)(1)] has a very narrow gene pool resulting from its evolutionary origin and domestication history. Cultivars of this cotton species (G. hirsutum L.) are prized for their combination of exceptional yield, other agronomic traits, and good fiber properties, whereas the other cultivated 52-chromosome species, G. barbadense L. [2n = 52, genomic formula 2(AD)(2)], is widely regarded as having the opposite attributes. It has exceptionally good fiber qualities, but generally lower yield and less desirable agronomic traits. Breeders have long aspired to combine the best attributes of G. hirsutum and G. barbadense, but have had limited success. F(1) hybrids are readily created and largely fertile, so the limited success may be due to cryptic biological and technical challenges associated with the conventional methods of interspecific introgression. We have developed a complementary alternative approach for introgression based on chromosome substitution line, followed by increasingly sophisticated genetic analyses of chromosome-derived families to describe the inheritance and breeding values of the chromosome substitution lines. Here, we analyze fiber quality traits of progeny families from a partial diallel crossing scheme among selected chromosome substitution lines (CS-B lines). The results provide a more detailed and precise QTL dissection of fiber traits, and an opportunity to examine allelic interaction effects between two substituted chromosomes versus one substituted chromosome. This approach creates new germplasm based on pair wise combinations of quasi-isogenic chromosome substitutions. The relative genetic simplicity of two-chromosome interactions departs significantly from complex or RIL-based populations, in which huge numbers of loci are segregating in all 26 chromosome pairs. Data were analyzed according to the ADAA genetic model, which revealed significant additive, dominance, and additive-by-additive epistasis effects on all of the fiber quality traits associated with the substituted chromosome or chromosome arm of CS-B lines. Fiber of line 3-79, the donor parent for the substituted chromosomes, had the highest Upper Half Mean length (UHM), uniformity ratio, strength, elongation, and lowest micronaire among all parents and hybrids. CS-B16 and CS-B25 had significant additive effects for all fiber traits. Assuming a uniform genetic background of the CS-B lines, the comparative analysis of the double-heterozygous hybrid combinations (CS-B × CS-B) versus their respective single heterozygous combinations (CS-B × TM-1) demonstrated that interspecific epistatic effects between the genes in the chromosomes played a major role in most of the fiber quality traits. Results showed that fiber of several hybrids including CS-B16 × CS-B22Lo, CS-B16 × CS-B25 and CS-B16 × TM-1 had significantly greater dominance effects for elongation and hybrid CS-B16 × CS-B17 had higher fiber strength than their parental lines. Multiple antagonistic genetic effects were also present for fiber quality traits associated with most of the substituted chromosomes and chromosome arms. Results from this study highlight the vital importance of epistasis in fiber quality traits and detected novel effects of some cryptic beneficial alleles affecting fiber quality on the 3-79 chromosomes, whose effects were not detected in the 3-79 parental lines.

Comprehensive molecular cytogenetic analysis of sorghum genome architecture: distribution of euchromatin, heterochromatin, genes and recombination in comparison to rice.

Cytogenetic maps of sorghum chromosomes 3-7, 9, and 10 were constructed on the basis of the fluorescence in situ hybridization (FISH) of approximately 18-30 BAC probes mapped across each of these chromosomes. Distal regions of euchromatin and pericentromeric regions of heterochromatin were delimited for all 10 sorghum chromosomes and their DNA content quantified. Euchromatic DNA spans approximately 50% of the sorghum genome, ranging from approximately 60% of chromosome 1 (SBI-01) to approximately 33% of chromosome 7 (SBI-07). This portion of the sorghum genome is predicted to encode approximately 70% of the sorghum genes ( approximately 1 gene model/12.3 kbp), assuming that rice and sorghum encode a similar number of genes. Heterochromatin spans approximately 411 Mbp of the sorghum genome, a region characterized by a approximately 34-fold lower rate of recombination and approximately 3-fold lower gene density compared to euchromatic DNA. The sorghum and rice genomes exhibit a high degree of macrocolinearity; however, the sorghum genome is approximately 2-fold larger than the rice genome. The distal euchromatic regions of sorghum chromosomes 3-7 and 10 are approximately 1.8-fold larger overall and exhibit an approximately 1.5-fold lower average rate of recombination than the colinear regions of the homeologous rice chromosomes. By contrast, the pericentromeric heterochromatic regions of these chromosomes are on average approximately 3.6-fold larger in sorghum and recombination is suppressed approximately 15-fold compared to the colinear regions of rice chromosomes.

A candidate recombination modifier gene for Zea mays L.

Maize meiotic mutant desynaptic (dy) was tested as a candidate recombination modifier gene because its effect is manifested in prophase I. Recombination rates for desynaptic (dy) and its wild type were compared in two ways: (1) segregation analysis using six linked molecular markers on chromosome 1L and (2) cytogenetic analysis using fluorescence in situ hybridization (FISH)-aided meiotic configurations observed in metaphase I. Chromosome 1L map lengths among the six linked markers were 45-63 cM for five F2 dy/dy plants, significantly lower than the wild-type F2 map distance of 72 cM. Chromosomes 2 and 6 were marked with rDNA FISH probes, and their map lengths were estimated from FISH-adorned meiotic configurations using the expectation-maximization algorithm. Chiasma frequencies for dy/dy plants were significantly reduced for both arms of chromosome 2, for chromosome arm 6L, and for eight unidentified chromosomes. There was a notable exception for the nucleolus-organizing region-bearing arm chromosome arm 6S, where dy increased chiasma frequency. Maize meiotic mutant desynaptic is a recombination modifier gene based on cytogenetic and segregation analyses.

A detailed RFLP map of cotton, Gossypium hirsutum x Gossypium barbadense: chromosome organization and evolution in a disomic polyploid genome.

We employ a detailed restriction fragment length polymorphism (RFLP) map to investigate chromosome organization and evolution in cotton, a disomic polyploid. About 46.2% of nuclear DNA probes detect RFLPs distinguishing Gossypium hirsutum and Gossypium barbadense; and 705 RFLP loci are assembled into 41 linkage groups and 4675 cM. The subgenomic origin (A vs. D) of most, and chromosomal identity of 14 (of 26), linkage groups is shown. The A and D subgenomes show similar recombinational length, suggesting that repetitive DNA in the physically larger A subgenome is recombinationally inert. RFLPs are somewhat more abundant in the D subgenome. Linkage among duplicated RFLPs reveals 11 pairs of homoelogous chromosomal regions-two appear homosequential, most differ by inversions, and at least one differs by a translocation. Most homoeologies involve chromosomes from different subgenomes, putatively reflecting the n = 13 to n = 26 polyploidization event of 1.1-1.9 million years ago. Several observations suggest that another, earlier, polyploidization event spawned n = 13 cottons, at least 25 million years ago. The cotton genome contains about 400-kb DNA per cM, hence map-based gene cloning is feasible. The cotton map affords new opportunities to study chromosome evolution, and to exploit Gossypium genetic resources for improvement of the world's leading natural fiber.

Bacterial artificial chromosome-based physical map of the rice genome constructed by restriction fingerprint analysis.

Genome-wide physical mapping with bacteria-based large-insert clones (e.g., BACs, PACs, and PBCs) promises to revolutionize genomics of large, complex genomes. To accelerate rice and other grass species genome research, we developed a genome-wide BAC-based map of the rice genome. The map consists of 298 BAC contigs and covers 419 Mb of the 430-Mb rice genome. Subsequent analysis indicated that the contigs constituting the map are accurate and reliable. Particularly important to proficiency were (1) a high-resolution, high-throughput DNA sequencing gel-based electrophoretic method for BAC fingerprinting, (2) the use of several complementary large-insert BAC libraries, and (3) computer-aided contig assembly. It has been demonstrated that the fingerprinting method is not significantly influenced by repeated sequences, genome size, and genome complexity. Use of several complementary libraries developed with different restriction enzymes minimized the "gaps" in the physical map. In contrast to previous estimates, a clonal coverage of 6.0-8.0 genome equivalents seems to be sufficient for development of a genome-wide physical map of approximately 95% genome coverage. This study indicates that genome-wide BAC-based physical maps can be developed quickly and economically for a variety of plant and animal species by restriction fingerprint analysis via DNA sequencing gel-based electrophoresis.

Physical mapping of the liguleless linkage group in Sorghum bicolor using rice RFLP-selected sorghum BACs.

Physical mapping of BACs by fluorescent in situ hybridization (FISH) was used to analyze the liguleless (lg-1) linkage group in sorghum and compare it to the conserved region in rice and maize. Six liguleless-associated rice restriction fragment length polymorphism (RFLP) markers were used to select 16 homeologous sorghum BACs, which were in turn used to physically map the liguleless linkage group in sorghum. Results show a basic conservation of the liguleless region in sorghum relative to the linkage map of rice. One marker which is distal in rice is more medial in sorghum, and another marker which is found within the linkage group in rice is on a different chromosome in sorghum. BACs associated with linkage group I hybridize to chromosome It, which was identified by using FISH in a sorghum cytogenetic stock trisomic for chromosome I (denoted It), and a BAC associated with linkage group E hybridized to an unidentified chromosome. Selected BACs, representing RFLP loci, were end-cloned for RFLP mapping, and the relative linkage order of these clones was in full agreement with the physical data. Similarities in locus order and the association of RFLP-selected BAC markers with two different chromosomes were found to exist between the linkage map of the liguleless region in maize and the physical map of the liguleless region in sorghum.

A molecular cytogenetic map of sorghum chromosome 1. Fluorescence in situ hybridization analysis with mapped bacterial artificial chromosomes.

We used structural genomic resources for Sorghum bicolor (L.) Moench to target and develop multiple molecular cytogenetic probes that would provide extensive coverage for a specific chromosome of sorghum. Bacterial artificial chromosome (BAC) clones containing molecular markers mapped across sorghum linkage group A were labeled as probes for fluorescence in situ hybridization (FISH). Signals from single-, dual-, and multiprobe BAC-FISH to spreads of mitotic chromosomes and pachytene bivalents were associated with the largest sorghum chromosome, which bears the nucleolus organizing region (NOR). The order of individual BAC-FISH loci along the chromosome was fully concordant to that of marker loci along the linkage map. In addition, the order of several tightly linked molecular markers was clarified by FISH analysis. The FISH results indicate that markers from the linkage map positions 0.0-81.8 cM reside in the short arm of chromosome 1 whereas markers from 81.8-242.9 cM are located in the long arm of chromosome 1. The centromere and NOR were located in a large heterochromatic region that spans approximately 60% of chromosome 1. In contrast, this region represents only 0.7% of the total genetic map distance of this chromosome. Variation in recombination frequency among euchromatic chromosomal regions also was apparent. The integrated data underscore the value of cytological data, because minor errors and uncertainties in linkage maps can involve huge physical regions. The successful development of multiprobe FISH cocktails suggests that it is feasible to develop chromosome-specific "paints" from genomic resources rather than flow sorting or microdissection and that when applied to pachytene chromatin, such cocktails provide an especially powerful framework for mapping. Such a molecular cytogenetic infrastructure would be inherently cross-linked with other genomic tools and thereby establish a cytogenomics system with extensive utility in development and application of genomic resources, cloning, transgene localization, development of plant "chromonomics," germplasm introgression, and marker-assisted breeding. In combination with previously reported work, the results indicate that a sorghum cytogenomics system would be partially applicable to other gramineous genera.

Resistance gene candidates identified by PCR with degenerate oligonucleotide primers map to clusters of resistance genes in lettuce.

The recent cloning of genes for resistance against diverse pathogens from a variety of plants has revealed that many share conserved sequence motifs. This provides the possibility of isolating numerous additional resistance genes by polymerase chain reaction (PCR) with degenerate oligonucleotide primers. We amplified resistance gene candidates (RGCs) from lettuce with multiple combinations of primers with low degeneracy designed from motifs in the nucleotide binding sites (NBSs) of RPS2 of Arabidopsis thaliana and N of tobacco. Genomic DNA, cDNA, and bacterial artificial chromosome (BAC) clones were successfully used as templates. Four families of sequences were identified that had the same similarity to each other as to resistance genes from other species. The relationship of the amplified products to resistance genes was evaluated by several sequence and genetic criteria. The amplified products contained open reading frames with additional sequences characteristic of NBSs. Hybridization of RGCs to genomic DNA and to BAC clones revealed large numbers of related sequences. Genetic analysis demonstrated the existence of clustered multigene families for each of the four RGC sequences. This parallels classical genetic data on clustering of disease resistance genes. Two of the four families mapped to known clusters of resistance genes; these two families were therefore studied in greater detail. Additional evidence that these RGCs could be resistance genes was gained by the identification of leucine-rich repeat (LRR) regions in sequences adjoining the NBS similar to those in RPM1 and RPS2 of A. thaliana. Fluorescent in situ hybridization confirmed the clustered genomic distribution of these sequences. The use of PCR with degenerate oligonucleotide primers is therefore an efficient method to identify numerous RGCs in plants.

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.

Chromosomal assignment of RFLP linkage groups harboring important QTLs on an intraspecific cotton (Gossypium hirsutum L.) Joinmap.

Chromosome identities were assigned to 15 linkage groups of the RFLP joinmap developed from four intraspecific cotton (Gossypium hirsutum L.) populations with different genetic backgrounds (Acala, Delta, and Texas Plains). The linkage groups were assigned to chromosomes by deficiency analysis of probes in the previously published joinmap, based on genomic DNA from hypoaneuploid chromosome substitution lines. These findings were integrated with QTL identification for multiple fiber and yield traits. Overall results revealed the presence of 63 QTLs on five different chromosomes of the A subgenome (chromosomes-03, -07, -09, -10, and -12) and 29 QTLs on the three different D subgenome (chromosomes-14 Lo, -20, and the long arm of -26). Linkage group-1 (chromosome-03) harbored 26 QTLs, covering 117 cM with 54 RFLP loci. Linkage group-2, (the long arm of chromosome-26) harbored 19 QTLs, covering 77.6 cM with 27 RFLP loci. Approximately 49% of the putative 92 QTLs for agronomic and fiber quality traits were placed on the above two major joinmap linkage groups, which correspond to just two different chromosomes, indicating that cotton chromosomes may have islands of high and low meiotic recombination like some other eukaryotic organisms. In addition, it reveals highly recombined and putative gene abundant regions in the cotton genome. QTLs for fiber quality traits in certain regions are located between two RFLP markers with an average of less than one cM (approximately 0.4-0.6 Mb) and possibly represent targets for map-based cloning. Identification of chromosomal location of RFLP markers common to different intra- and interspecific-populations will facilitate development of portable framework markers, as well as genetic and physical mapping of the cotton genome.

Tests of six cotton (Gossypium hirsutum L.) mutants for association with aneuploids.

Genetic mutants are useful tools for basic and applied research to elucidate the developmental and regulatory processes of the cotton plant (Gossypium hirsutum L.). Their value is enhanced with knowledge of their location in the genome. The results of aneuploid tests used to locate mutant loci on specific chromosomes in G. hirsutum L. are reported. Thirty-four monosomes and telosomes, representing 18 of the 26 chromosomes, were used in combination with six mutants that were associated with nine loci. The mutant loci were glandless stem and boll (gl1gl6), immature fiber (im), Ligon lintless-2 (Li2), methylation (me), nonpinking (np1np2), and Raimondal (Ra1Ra2). We found that im was associated with chromosome 3 that contains linkage group VI (accessory involucre and frego bract); Li2 was associated with chromosome 18 that contains linkage group XVI (open bud and yellow pollen-2); and me was associated with chromosome 9. The remaining three mutants were not associated with the aneuploids in the tests. Knowledge of these chromosome assignments provides a valuable reference for specific studies of mutants and for further genome mapping efforts.

A maximum likelihood algorithm for genome mapping of cytogenetic loci from meiotic configuration data.

Frequencies of meiotic configurations in cytogenetic stocks are dependent on chiasma frequencies in segments defined by centromeres, breakpoints, and telomeres. The expectation maximization algorithm is proposed as a general method to perform maximum likelihood estimations of the chiasma frequencies in the intervals between such locations. The estimates can be translated via mapping functions into genetic maps of cytogenetic landmarks. One set of observational data was analyzed to exemplify application of these methods, results of which were largely concordant with other comparable data. The method was also tested by Monte Carlo simulation of frequencies of meiotic configurations from a monotelodisomic translocation heterozygote, assuming six different sample sizes. The estimate averages were always close to the values given initially to the parameters. The maximum likelihood estimation procedures can be extended readily to other kinds of cytogenetic stocks and allow the pooling of diverse cytogenetic data to collectively estimate lengths of segments, arms, and chromosomes.

Simple sequence repeat (SSR) markers linked to the Ligon lintless (Li(1)) mutant in cotton.

Ligon lintless (Li(1)) is a monogenic, dominant mutant in cotton, whose expression results in extreme reductions in fiber length on mature seed. The objectives of this research were to compare fiber initiation between the Li(1) mutant and TM-1 to reveal the fiber initiation differences between normal and mutant phenotypes, to develop a linkage map of simple sequence repeat (SSR) markers with the Li(1) locus, and to identify the chromosomal location of the Li(1) locus. Comparative scanning electron microscopy studies of fiber development in a normal TM-1 genotype and the near-isogenic Li(1) mutant at 1 and 3 days postanthesis revealed little differences between the two during early stages of development, suggesting that Li(1) gene expression occurs later, probably during the elongation phase. Thirty-eight SSR loci were found to be polymorphic between TM-1 and Li(1) and were used for mapping in an F(2) population. Twenty-two SSR loci, along with Li(1), were located on eight linkage groups, covering a total genetic distance of 218.3 cM. Analysis of individual monosomic and monotelodisomic plants indicated that two SSR loci (MP4030 and MP673) from the Li(1) linkage group were located on chromosome 22.

Macromolecular organization and genetic mapping of a rapidly evolving chromosome-specific tandem repeat family (B77) in cotton (Gossypium).

Isolation and characterization of the most prominent repetitive element families in the genome of tetraploid cotton (Gossypium barbadense L; [39]) revealed a small subset of families that showed very different properties in tetraploids than in their diploid progenitors, separated by 1-2 million years. One element, B77, was characterized in detail, and compared to the well-conserved 5S and 45S rRNA genes. The 572 bp B77 repeat was found to be concentrated in several discontinuous tandem arrays confined to a single 550 kb SalI fragment in tetraploid cotton. Genetic mapping based on the absence of the pentameric 'rung' in the G. barbadense 'ladder' showed that B77 maps to a D-subgenome chromosome. In situ hybridization supports the contention that the array is confined largely to a single chromosomal site in the D-subgenome. The B77 repeat has undergone a substantial increase in copy number since formation of tetraploid cotton from its diploid relatives. RFLPs observed among tetraploid cotton species suggest that amplification and/or rearrangement of the repeat may have continued after divergence of the five tetraploid cotton species. B77 contains many short direct repeats and shares significant DNA sequence homology with a Nicotiana alata retrotransposon Tna1-2 integrase motif. The recent amplification of B77 on linkage group D04 suggests that the D-subgenome of tetraploid cotton may be subject to different evolutionary constraints than the D-genome diploid chromosomes, which exhibit few genome-specific elements. Further, the abundance of B77 in G. gossypioides supports independent evidence that it may be the closest extant relative of the D-genome ancestor of cotton.

In situ hybridization of biotinylated DNA probes to cotton meiotic chromosomes.

A modified procedure for in situ hybridization of biotinylated probes to meiotic chromosomes of cotton has been developed with high retention of squashed cells on slides, preservation of acid-fixed chromosome morphology, exceptionally low levels of background precipitate at nonspecific hybridization sites and improved photomicrographic recording. Salient features of the techniques include pretreatment of slides before squashing, cold storage of squash preparations, and use of interference filters for distinguishing precipitate from chromatin. A cloned 18S/28S ribosomal DNA fragment from soybean was biotinylated via nick-translation and hybridized to microsporocyte meiotic chromosomes of cotton (Gossypium hirsutum L. and G. hirsutum L. X G. barbadense L.). Enzymatically formed precipitate from streptavidin-bound peroxidase marked the in situ hybridization. In situ hybridization of biotinylated probes to cotton meiotic chromosomes adds the specificity and resoltion of in situ hybridization to the chromosomal and genomic perspectives provided by meiotic cytogenetic analyses. Molecular cytogenetic analyses of meiotic cells offer certain inherent analytical advantages over analyses of somatic cells, e.g., in terms of mapping, and for studying fundamental biological and genetic problems, particularly for organisms that are not amenable to somatic karyotypic analysis.

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.

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.

The assignment of PRKCI to bovine chromosome 1q34-->q36 by FISH suggests a new assignment to human chromosome 3.

Two bovine BAC clones were identified by PCR as containing the bovine gene PRKCI. Both clones were assigned by FISH to bands q34-->q36 on BTA1. The sequence information derived from genomic DNA and from both clones was identical and showed a high degree of homology to human PRKCI (HSAXq21.3, 95.5% homology), and mouse Prkcl (MMU3, 13.8 cM, 87.6% homology) and rat Prkcl (88.8% homology). This assignment could suggest a disruption of the synteny conservation of mammalian X-linked genes, but most likely suggests a misassignment of this gene to the human X.