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.

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.

Genetic mapping and QTL analysis of fiber-related traits in cotton ( Gossypium).

Cotton, the leading natural fiber crop, is largely produced by two primary cultivated allotetraploid species known as Upland or American cotton ( Gossypium hirsutum L.) and Pima or Egyptian cotton ( G. barbadense L.). The allotetraploid species diverged from each other and from their diploid progenitors (A or D genome) through selection and domestication after polyploidization. To analyze cotton AD genomes and dissect agronomic traits, we have developed a genetic map in an F2 population derived from interspecific hybrids between G. hirsutum L. cv. Acala-44 and G. barbadense L. cv. Pima S-7. A total of 392 genetic loci, including 333 amplified fragment length polymorphisms (AFLPs), 47 simple sequence repeats (SSRs), and 12 restriction fragment length polymorphisms (RFLPs), were mapped in 42 linkage groups, which span 3,287 cM and cover approximately 70% of the genome. Using chromosomal aneuploid interspecific hybrids and a set of 29 RFLP and SSR framework markers, we assigned 19 linkage groups involving 223 loci to 12 chromosomes. Comparing four pairs of homoeologous chromosomes, we found that with one exception linkage distances in the A-subgenome chromosomes were larger than those in their D-subgenome homoeologues, reflecting higher recombination frequencies and/or larger chromosomes in the A subgenome. Segregation distortion was observed in 30 out of 392 loci mapped in cotton. Moreover, approximately 29% of the RFLPs behaved as dominant loci, which may result from rapid genomic changes. The cotton genetic map was used for quantitative trait loci (QTL) analysis using composite interval mapping and permutation tests. We detected seven QTLs for six fiber-related traits; five of these were distributed among A-subgenome chromosomes, the genome donor of fiber traits. The detection of QTLs in both the A subgenome in this study and the D subgenome in a previous study suggests that fiber-related traits are controlled by the genes in homoeologous genomes, which are subjected to selection and domestication. Some chromosomes contain clusters of QTLs and presumably contribute to the large amount of phenotypic variation that is present for fiber-related traits.

A methodological approach for the construction of a radiation hybrid map of bovine chromosome 5

A bovine 5,000 rad WG-RH panel was used to construct an RH map of bovine chromosome 5 (BTA5). Twenty-one microsatellites and thirteen genes were scored in the panel using PAGE and radioactive labeling. Marker retention ranged from 8.9 percent-25.8 percent and averaged 17.8 percent. Pairwise locus analysis placed all markers in a single syntenic group with a LOD support of 4.0. At a LOD support of 8.0, a centromeric group of 23 syntenic markers was formed. Telomeric groups of 11 and 9 markers were assembled with a LOD support of 6.0 and 8.0, respectively. All markers were ordered by maximum likelihood methods using the program RHMAP. Only 13 markers were ordered with a LOD support of at least 3.0, while 25 and 29 markers were ordered with a support of at least 2.0 and 1.0, respectively. Total length of the comprehensive RH map was 435.9 cR5,000, with an average marker separation of 12.8 cR5,000. The largest gaps in the map were 55.0 and 30.4 cR5,000 in length. The locus orders of markers common to both the RH map and the USDA-MARC linkage map were identical. The relationship between the RH and linkage maps was calculated to be 3.74 cR5,000/cM.

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.

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 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.

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.

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.

Distribution and sequence analysis of the centromere-associated repetitive element CEN38 of Sorghum bicolor (Poaceae).

Fluorescence in situ hybridization (FISH) of a large-insert genomic clone, BAC 22B2, previously suggested that Sorghum bicolor (2n = 20) has the tetraploid architecture A(b)A(b)B(b)B(b). Here, we report on BAC 22B2 subclone pCEN38 (1047-bp insert) as related to sorghum and sugarcane. Mitotic FISH of six different subclones of BAC 22B2 showed that pCEN38 produced the strongest specificity to the A(b) subgenome and signal occurred primarily near centromeres. Southern blots of pCEN38 to 21 crop plants revealed a narrow taxonomic distribution. Meiotic metaphase I FISH positioned pCEN38 sequences near active centromeres. Pachytene FISH revealed that the distributions are trimodal in several B(b) and possibly all sorghum chromosomes. DNA sequencing revealed that the pCEN38 fragment contains three tandemly repeated dimers (<280 bp) of the same sequence family found in sorghum clone pSau3A10, and that each dimer consists of two divergent monomers (<140 bp). Sequence comparisons revealed homology between the pCEN38 monomers and the SCEN 140 bp tandem repeat family of sugarcane. FISH of pCEN38 yielded signal in centromere regions of most but not all sugarcane chromosomes. Results suggest that sugarcane and sorghum share at least one ancestor harboring elements similar to pCEN38 and SCEN and that each species had an ancestor in which the repetitive element was weakly present or lacking.

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.

Cytogenetic alignment of the bovine chromosome 13 genome map by fluorescence in-situ hybridization of human chromosome 10 and 20 comparative markers.

A bovine bacterial artificial chromosome (BAC) library was screened for the presence of six genes (IL2RA, VIM, THBD, PLC-II, CSNK2A1 and TOP1) previously assigned to human chromosomes 10 or 20 (HSA10 or HSA20). Four of the genes were found represented in the bovine BAC library by at least one clone. The identified BAC clones were used as probes in single-color fluorescence in-situ hybridization (FISH) to determine the chromosomal band location of each gene. As predicted by the human/bovine comparative map and comparative chromosome painting analysis, the four genes mapped to bovine chromosome 13 (BTA13). Dual-color FISH was then used to integrate these four type I markers into the existing BTA13 genome map. These FISH results anchor the BTA13 genome map from bands 14-23, and confirm the presence of a conserved HSA10 homologous synteny group on BTA13 centromeric to a HSA20 homologous segment.

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.

Physical assignment of microsatellite- containing BACs to bovine chromosomes.

Here we report the physical assignment of 40 microsatellite markers by fluorescence in situ hybridization to 13 different bovine chromosomes. This information will be valuable in providing physically anchored landmarks for the construction of contigs throughout the bovine genome. It also is useful for the purpose of integrating the linkage maps of these chromosomes to their physical maps and determining the physical coverage of these linkage groups.

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.

Comparative mapping of bovine chromosome 13 by fluorescence in situ hybridization.

We present chromosomal fluorescence in situ hybridization (FISH) results that both extend the HSA20/BTA13 comparative map as well as cytogenetically anchor two microsatellite markers. A bovine bacterial artificial chromosome (BAC) library was screened for conserved genes (type 1 loci) previously assigned to HSA10 or HSA20 and BTA13, and for microsatellites selected from two published BTA13 linkage maps. Clones from six out of nine comparative loci and both microsatellites were found represented in the BAC library. These BAC clones were used as probes in single colour FISH to determine the chromosome band position of each locus. As predicted by the human/bovine comparative map, all type 1 loci mapped to BTA13. Because single colour FISH analysis revealed that the loci were clustered within the distal half of BTA13, dual colour FISH was used to confirm the locus order. Established order was centromere-PRNP-(SOD1L/AVP/OXT)-(BL42/GNAS1)- HCK-CSSM30. The findings confirm the presence of a conserved HSA20 homologous synteny group on BTA13 distal of a HSA10 homologous segment.