Microsatellite markers from a microdissected swine chromosome 6 genomic library

To develop additional microsatellite (MS) markers in the region of the porcine skeletal muscle ryanodine receptor gene (RYR1), a microdissected genomic library was generated from the proximal half of the q arm of swine chromosome 6. Purified DNA was restriction enzyme-digested, ligated to oligonucleotide adaptors and amplified by PCR using primers complementary to the adaptor sequences. The purity of the amplified products and boundaries of the microdissected chromosomal region were verified by fluorescence in situ hybridization. (CA)n-containing sequences were then identified in a small insert genomic library generated from the PCR-amplified microdissected DNA. Oligonucleotide primers were developed for the PCR amplification of 30 of the 46 (CA)n repeat-containing clones, which were subsequently used to amplify DNA isolated from unrelated pigs of different breeds to determine the informativeness of these MS markers. Twenty-two of these MS markers were genotyped on the University of Illinois Yorkshire x Meishan swine reference population. These 22 markers were all assigned within a 50.7-CM region of the swine chromosome 6 linkage map, indicating the specificity of the microdissected library.     

Development of 47 new microsatellite markers from a BTA6 library

Chromosome-specific libraries provide a means to isolate genetic markers from specific chromosomal regions. A small-insert BTA6 library, constructed by microdissection, was screened for dinucleotide repeats (CA)15 and (GA)15. A total of 47 new microsatellite loci were developed and tested for polymorphism and informativeness using the MARC bovine mapping family.     

Mapping of rabbit chromosome 1 markers generated from a microsatellite-enriched chromosome-specific library

A genomic DNA library was produced from flow-sorted rabbit chromosome 1 and enriched for fragments containing CA-repeats. Clones containing CA-repeats were identified and primers for amplification of the microsatellite were developed after sequencing the clone. The degree of polymorphism was tested in rabbits from different breeds. This approach identified 12 microsatellite markers which could be used for studying linkage relationships in the progeny of an F(2)-intercross: (AX/JUxIIIVO/JU) F(2), and two backcrosses: (OS/JxX/J)X/J and (WH/JxX/J)X/J. Seven of these markers were mapped on chromosome 1.     

Characterization and regional mapping of new anonymous chromosome 20-specific DNA markers isolated from a flow-sorted DNA library

Employing the flow-sorted chromosome 20-specific DNA library LL20NS01, we isolated seven novel unique poly- and monomorphic DNA markers specific to human chromosome 20. Initially, 201 phage clones were analyzed regarding insert size and repetitivity. By testing 14 single- and low-copy number clones for their ability to detect RFLPs, three polymorphisms were revealed by two probes, pFMS22-1.4 [D20S22] and pFMS76 [D20S23]. Seven of twenty probes (35%) were assigned to chromosome 20 using a somatic cell hybrid DNA panel. Five of them were regionally mapped by in situ hybridization. Three DNA markers, pFMS51 [D20S29], pFMS76 [D20S23], and pFMS106 [D20S30], were assigned to 20p11.2-p12, and two markers, pFMS22-1.4 [D20S22] and pFMS135 [D20S31], to 20q12-q13.3. Our new chromosome 20-specific DNA markers should be useful for the molecular characterization of this rather underpopulated human chromosome.     

Regional mapping of unique DNA sequences from human chromosome 3 derived from a flow-sorted chromosome library

Eight single-copy DNA probes specific for human chromosome 3 were isolated by screening a human chromosome 3-derived genomic library. Southern blot analyses of DNAs isolated from a panel of somatic cell hybrids allowed us to regionally assign all probes to subregions on chromosome 3. Three clones were localized to the short arm of chromosome 3 (3p21----pter), two to the long arm (3q21----qter), and three to the 3q21----3p21 subregion. Six of these DNA sequences map to regions overlapping a segment of chromosome 3 (3p14----p23) frequently deleted in small cell lung cancer cells. Restriction fragment length polymorphism analyses indicate that at least three of the eight single-copy probes studies show MspI or BglII polymorphisms. This library is a useful source of chromosome 3-specific probes.     

Mapping of 50 cosmid clones isolated from a flow-sorted human X chromosome library by fluorescence in situ hybridization.

Fifty cosmids have been mapped to metaphase chromosomes by fluorescence in situ hybridization under conditions that suppress signals from repetitive DNA sequences. The cosmid clones were isolated from a flow-sorted human X chromosome library. Thirty-eight of the clones were localized to chromosome X and 12 to autosomes such as chromosomes 3, 7, 8, 14, and 17. Although most of the cosmids mapped to the X chromosome appeared to be scattered along both the short and long arms, 10 cosmids were localized to the centromeric region of the chromosome. Southern blot analysis revealed that only two of these clones hybridized to probe pXBR-1, which detects the DXZ1 locus. In addition, 4 out of 5 cosmids mapped on chromosome 8 also localized on the centromeric region. While localization of X-specific cosmids will facilitate the physical mapping of the human X chromosome, cosmids mapped to the centromeric regions of chromosomes X and 8 should be especially useful for studying the structure and organization of these regions.     

Characterization of a human chromosome 8 cosmid library constructed from flow-sorted chromosomes.

A cosmid library for human chromosome 8 has been constructed from flow-sorted chromosomes in the vector sCos-1. This library is 85% human and has been arrayed into 210 microtiter plates representing four genome equivalents. Cosmids have been isolated with 10 of 11 probes representing nine different loci from chromosome 8.     

Mouse microsatellites from a flow-sorted 4:6 Robertsonian chromosome

Twenty microsatellites were generated from a previously characterized gt10 library containing C57BL/6J mouse DNA from a flow-sorted 4:6 Robertsonian chromosome. These sequences were analyzed for size variation between different strains of mice with the polymerase chain reaction (PCR) and mapped by use of either strain distribution patterns (SDPs) in recombinant inbred (RI) strains, or intra- and interspecific backcrosses. Eighty-five percent of the sequences showed allelic variations between different inbred strains of mice and the wild mouse, Mus spretus, and 70% were variant between inbred strains. Eight (62%) of the 13 repeats that have been mapped lie on Chromosomes (Chr) 4 and 6. This approach is an effective way of generating informative markers on specific chromosomes.     

Development of bovine microsatellite markers from a microsatellite-enriched library

A bovine genomic library enriched for DNA fragments bearing poly(dC-dA).poly(dG-dT) sequences was prepared and screened. Twenty-four clones bearing microsatellites were subject to sequence analysis and marker development as appropriate. Three of the 24 clones had microsatellites that were not evaluated for marker development owing to presence of a satellite element in two cases and limited repeat length in the third. Of the remaining 21 clones, all but one yielded polymorphic microsatellites. All but two of the polymorphic markers could be assigned to chromosomal locations by either linkage analysis or on the basis of X-linked inheritance as determined by heterozygosity limited to females. An unexpected clustering of markers was observed as 4 of the 20 polymorphic microsatellites mapped to a region of less than approximately 30 cM on Chromosome (Chr) 19, and four markers displayed X-linked inheritance.     

Isolation of anonymous DNA markers for human chromosome 22q11 from a flow-sorted library,and mapping using hybrids from patients with DiGeorge syndrome

Summary DiGeorge syndrome (DGS) is a human developmental defect of the structures derived from the third and fourth pharyngeal pouches. It apparently arises due to deletion of 22q11. We describe a strategy for the isolation of DNA probes for this region. A deleted chromosome 22, which includes 22q11, was flow-sorted from a lymphoblastoid cell line of a patient with cat eye syndrome and used as the source of DNA. A DNA library was constructed from this chromosome by cloning into the EcoR1 site of the vector Lambda gt10. Inserts were amplified by PCR and mapped using a somatic cell hybrid panel of this region. Out of 32 probes, 14 were mapped to 22q11. These probes were further sublocalised within the region by dosage analysis of DGS patients, and by the use of two new hybrid cell lines which we have produced from DGS patients. One of these lines (7939B662) contains the altered human chromosome segregated from its normal homologue. This chromosome 22 contains an interstitial deletion in 22q11, and will be useful for localising further probes to the DGS region.     

Construction and characterization of a YAC library with a lowfrequency of chimeric clones from flow-sorted human chromosome 9

Human chromosome 9 DNA, flow-sorted from somaticcell hybrid PK-87-9, has been used to construct two complete digest YAC libraries. The combined representation of chromosome 9 in these libraries, estimated by hybridization of chromosome 9-specific sequences to YAC colony grids, is 95%. The frequency of chimeric clones, analyzed by fluorescence in situ hybridization of chromosome 9 YACs to human metaphase chromosomes, was estimated to be 4%. These libraries provide a resource for physical mapping and for moving from genetic markers to disease loci on chromosome 9.     

Analysis of four microsatellite markers on the long arm of chromosome 9 by meiotic recombination in flow-sorted single sperm.

Meiotic recombination in flow-sorted single sperm was used to analyze four highly polymorphic microsatellite markers on the long arm of chromosome 9. The microsatellites comprised three tightly linked markers: 9CMP1 (D9S109), 9CMP2 (D9S127), and D9S53, which map to 9q31, and a reference marker, ASS, which is located in 9q34.1. Haplotypes of single sperm were assessed by using PCR in a single-step multiplex reaction to amplify each locus. Recombinant haplotypes were identified by their relative infrequency and were analyzed using THREELOC, a maximum-likelihood-analysis program, and an adaptation of CRI-MAP. The most likely order of these markers was cen-D9S109-D9S127-D9S53-ASS-tel with D9S109, D9S127, and D9S53 being separated by a genetic distance of approximately 3%. The order of the latter three markers did not however achieve statistical significance using the THREELOC program.     

Single-copy,species-transferable microsatellite markers developed from loblolly pine ESTs

Microsatellites, or simple sequence repeats (SSRs), are usually regarded as the markers of choice in population genetics research because they exhibit high variability. The development cost of these markers is usually high. In addition, microsatellite primers developed for one species often do not cross-amplify in related species, requiring separate development for each species. However, microsatellites found in expressed sequence tags (ESTs) might better cross-amplify as they reside in or near conserved coding DNA. In this study, we identified 14 Pinus taeda (loblolly pine) EST-SSRs from public EST databases and tested for their cross-species transferability to P. contorta ssp. latifolia, P. ponderosa, and P. sylvestris. As part of our development of a P. contorta microsatellite set, we also compared their transferability to that of 99 traditional microsatellite markers developed in P. taeda and tested on P. contorta ssp. latifolia. Compared to traditional microsatellites, EST-SSRs had higher transfer rates across pine species; however, the level of polymorphism of microsatellites derived from ESTs was lower. Sequence analyses revealed that the frequencies of insertions/deletions and base substitutions were lower in EST-SSRs than in other types of microsatellites, confirming that EST-SSRs are more conserved than traditional SSRs. Our results also provide a battery of 23 polymorphic, robust microsatellite primer pairs for lodgepole pine.Communicated by O. Savolainen     

Construction of a chromosome-enriched HpaII library from flow-sorted wheat chromosomes.

We report here the first successful generation of a chromosome-enriched library from flow sorted plant chromosomes. Chromosomes with a characteristic DNA content (a peak) were sorted from a synchronized cell culture (TaKB1, derived from Triticum aestivum). A HpaII library was constructed from the sorted chromosomes and half of the cloned DNA sequences analysed are unique or low copy. Approximately half of these sequences when used as probes detect sequences on wheat chromosome 4A. The generation and analysis of the chromosome library is described in detail and the prospects of using flow-sorted plant chromosomes discussed.     

Characterization of 30 new microsatellite markers,developed from enriched genomic DNA library of zoysiagrass Zoysia japonica Steud.

The present study reports the development of 30 polymorphic microsatellite markers for zoysiagrass Zoysia japonica Steud. The 30 markers produced a total of 125 alleles with an average of 4.2 alleles per locus. Values for observed and expected heterozygosities ranged from 0.10 to 0.95 and from 0.15 to 0.81, respectively. At significance threshold (P < 0.05), 11 loci deviated from Hardy–Weinberg equilibrium, whereas significant linkage disequilibrium values were observed between 16 pairs of loci. These markers may provide information needed to select genetically diverse parents for developing breeding and mapping populations of zoysiagrass.     

Mapping of 55 new rat microsatellite markers from chromosome-specific libraries

Fifty-five novel rat microsatellite markers were isolated from libraries specific for rat chromosomes (Chrs) 1, 2, and 7. The markers were mapped in three backcross rat populations. Thirty of these markers mapped to Chrs 1, 2, or 7, while the other 25 mapped to other chromosomes. New markers for two genes, liver-specific transporter gene (Livtr) and insulin-responsive glucose transporter (Glut4), were also mapped to rat Chrs 9 and 10, respectively. Three provisionally assigned markers from previous studies were also confirmed. Detailed methodologies for the generation and enrichment of clones containing repeat sequences and for the isolation of chromosome-specific markers are presented, since they represent unique combinations and modifications of previous protocols. Such methods and the newly presented markers should be useful for both specific and general mapping studies in the rat. Received: 11 February 1998 / Accepted: 7 April 1998     

Direct construction of a chromosome-specific NotI linking library from flow-sorted chromosomes.

A linking library consists of genomic DNA fragments which contain a specific rare restriction enzyme site; such clones are very useful as probes in pulsed field gel electrophoresis and in mapping and cloning large regions of DNA. However, identifying those linking clones which map to a certain chromosomal region can be laborious. Therefore, we have developed a straightforward procedure for constructing a linking library directly from flow-sorted chromosomes. As a test of the approach, a NotI linking library was constructed from the chromosome 17 fraction of a flow-sort of human chromosomes, using only 70 ng of DNA. Thirteen of sixteen linking clones were mapped to chromosome 17, suggesting that the library is highly enriched for this chromosome. This method should be generally applicable to other chromosomes and enzymes as well.