Isolation and characterization of microsatellite loci in the intertidal sponge Halichondria panicea

GA‐ and CA‐enriched genomic libraries were constructed for the intertidal sponge Halichondria panicea. Unique repeat motifs identified varied from the expected simple dinucleotide repeats to more complex repeat units. All sequences tended to be highly repetitive but did not necessarily contain the targeted motifs. Seven microsatellite loci were evaluated on sponges from the clone source population. All seven were polymorphic with 5.43 ± 0.92 mean number of alleles. Six of the seven loci that could be resolved had mean heterozygosities of 0.14–0.68. The loci identified here will be useful for population studies.     

Segments containing alternating purine and pyrimidine dinucleotides: patterns of polymorphism in humans and prevalence throughout phylogeny.

Tandem dinucleotide repeats of GT or AC [(GT)n/(AC)n] where n greater than or equal to 14 are highly polymorphic and other simple repeats such as (CT)n/(AG)n and (A)n(T)n are also polymorphic. The uniformity of these sequences precludes a mechanistic differentiation between recombination or polymerase slippage. Since (GT)n/(AC)n or (CT)n/(AG)n segments of desired size were not available in our gene of interest, we analyzed a 187+ bp segment in the factor IX gene with multiple short dinucleotide repeats. This sequence contains a melody of short dinucleotide repeats which includes a 142+ bp segment of alternating purines and pyrimidines. Amplification of this sequence in 167 individuals of different ethnicity and direct sequencing of 106 individuals (23 kb of sequence) failed to reveal simple variation in the number of tandem dinucleotide repeats. However, polymorphism in the 142+ alternating purine and pyrimidine segment (RY)n was detected due to the insertion of two related repeat units of 24 bp (A) and 26 bp (B). Two previously described alleles (AB, A2B2) and two novel presumptive recombinants were found (A2B, A3B2) for a total of four alleles. An analysis of (RY)n segments in GenBank revealed an extraordinary enrichment in the genome of mammals, invertebrates, and yeast and a marked reduction in bacteria. Rodent (RY)n were larger and substantially more frequent than those in primates. When a second (RY)n was examined in the exon 8 of human factor IX gene, it was polymorphic at short repeats of (GT)n/(AC)n (n = 3-6) in Western Europeans and Koreans. In addition, an (RY)n in the dystrophin gene had four polymorphic alleles involving AT and GT dinucleotides. Thus (RY)n segments appear to be abundant and highly polymorphic. The asymmetric patterns of polymorphism and the absence of simple dinucleotide variation in 23 kb of sequence are compatible with recombination or sister chromatid exchange, but not polymerase slippage. By inference, recombination should underlie the polymorphisms at (GT)n/(AC)n since they are a subset of (RY)n and they commonly occur in the context of longer (RY)n.     

A polymorphic (CA)n repeat element maps the human glucokinase gene (GCK) to chromosome 7p.

A compound imperfect dinucleotide repeat element, [CA]4TTTGT[CT]7[CA]9AA[CA]4CCACATA[CA]3, was found approximately 10 kb 3' to the human glucokinase gene (GCK) from analysis of contiguous genomic DNA obtained from a bacteriophage lambda chromosome walk. Direct human genomic sequencing revealed the source of polymorphism to be variable numbers of CT and CA repeats. Altogether six alleles that range in length from +10 to -15 nucleotides compared to the most common (Z) allele have been identified. Alleles Z, Z + 2, and Z + 4 were present in American Blacks, Pima Indians, and Caucasians, with somewhat varied frequencies among the groups. Two alleles, Z + 10 and Z - 15, appear to be unique to American Blacks, while a Z + 6 allele was observed only in the Caucasian population studied. Observed heterozygosity of the polymorphism in the CEPH reference pedigree collection is 44% and the PIC 0.44. The polymorphism is assayed by PCR amplification and resolution of 32P-end-labeled products (ranging in length from 180 to 205 bp) on denaturing polyacrylamide sequencing gels. Using the PCR assay, the human glucokinase gene was physically localized to chromosome 7 in a panel of rodent/human somatic cell lines. Genetic analysis in CEPH pedigrees placed the dinucleotide repeat element, and thereby the human glucokinase gene, on chromosome 7p between TCRG and a RFLP locus D7S57. The glucokinase dinucleotide repeat genetic marker can now be used to assess the role of the glucokinase gene in diabetes by population association studies. In addition, this repeat marker and others flanking it on chromosome 7 can be used in linkage studies with families segregating the disorder.     

Isolation and characterization of highly polymorphic microsatellite loci in Schreibers’ long‐fingered bat,Miniopterus schreibersii (Chiroptera: Vespertilionidae)

Five polymorphic, dinucleotide microsatellite loci have been identified and characterized in Schreibers’ long‐fingered bat, Miniopterus schreibersii (Chiroptera: Vespertilionidae). These include three uninterrupted (CA)_n repeats, ranging in length from (CA)₁₇ to (CA)₁₉, one interrupted repeat (CA)₄(CG)₂(CA)₁₃, and one uninterrupted (GA)₂₇ repeat. All loci were highly polymorphic, with 17–20 alleles identified per locus. Observed heterozygosity levels (0.66–0.82) are lower than expected due to homozygote excess, probably caused by pooling of populations, resulting in population substructuring. All five polymorphic loci were also successfully amplified in the closely related M. fraterculus. Two pairs of primers additionally amplified polymorphic microsatellites in Chaerophon sp.     

One hundred and one new simple sequence repeat-based markers for the canine genome

One hundred and one new dinucleotide repeat polymorphisms specific for the canine genome have been identified and characterized. Screening of both primary libraries and marker selected libraries enriched for simple sequence repeats led to the isolation of large numbers of genomic clones that contained (CA)_n repeats. Over 200 of these clones were sequenced, and PCR primers that bracket the repeat were developed for those that contained ten or more continuous (CA)_n units. This effort led to the production of 101 polymorphic markers, which were assigned to one of four categories depending on their degree of polymorphism. Fiftyfour markers were found to be highly or very highly polymorphic as they had four or more alleles when tested on a panel of unrelated dogs. This group of markers will be useful for following inheritance of traits in crosses between dogs.The nucleotide sequence data reported in this paper have been submitted to GenBank and have been assigned the accession numbers indicated in Table 1.     

Mapping microsatellite markers identified in porcine EST sequences

A sequence search of swine expressed sequence tags (EST) data in GenBank identified over 100 sequence files which contained a microsatellite repeat or simple sequence repeat (SSR). Most of these repeat motifs were dinucleotide (CA/GT) repeats; however, a number of tri-, tetra-, penta- and hexa-nucleotide repeats were also detected. An initial assessment of six dinucleotide and 14 higher-order repeat markers indicated that only dinucleotide markers yielded a sufficient number of informative markers (100% vs. 14% for dinucleotide and higher order repeats, respectively). Primers were designed for an additional 50 di- and one tri-nucleotide SSRs. Overall, 42 markers were polymorphic in the US Meat Animal Research Center (MARC) reference population, 17 markers were uninformative and 12 primer pairs failed to satisfactorily amplify genomic DNA. A comparison of di-nucleotide repeat vs. markers with repeat motifs of three to six bases demonstrated that 72% of dinucleotide markers were informative relative to only 7% of other repeat motifs. The difference was the result of a much higher percentage of monomorphic markers in the three to six base repeat motif markers than in the dinucleotide markers (64% vs. 14%). Either higher order repeat motifs are less polymorphic in the porcine genome or our selection criteria for repeat length of more than 17 contiguous bases was too low. The mapped microsatellite markers add to the porcine genetic map and provide valuable links between the porcine and human genome.     

猪PAC克隆的微卫星DNA分离研究

利用(CA)8核心序列,设计锚定引物,采用直接测序法,从单个猪PAC克隆中分离到一个新微卫星DNA。根据该微卫星DNA的侧翼序列,设计了专一引物,在8个猪种40个个体中检测到3个等位基因,片段长度分别为305 bp、307 bp和309 bp。3种等位基因纯合子个体的PCR产物序列分析表明,这3种等位基因分别有12、13和14次CA双核苷酸重复。 Abstract:A novel microsatellite DNA was isolated from a single porcine PAC clone by sequencing the PAC clone directly with (CA)n repeat motif anchored primer.The specific primer pairs flanking the (CA)n repeat region were used to amplify the genomic DNA of 40 individuals from 8 pig breeds,which detected three alleles with the fragment length of 305 bp,307 bp and 309 bp.The PCR product sequencing results of homozygous animals representing three alleles revealed that those three alleles contained 12,13 and 14 CA dinucleotide repeats respectively.     

The dinucleotide (CA) repeat polymorphism of estrogen receptor beta but not the dinucleotide (TA) repeat polymorphism of estrogen receptor alpha is associated with venous ulceration

Venous ulcers are the predominant form of chronic wound in the elderly, accounting for around 70% of all cases. The steroid sex hormone estrogen plays a crucial role in normal human skin maintenance and during cutaneous wound repair following injury. Estrogen can reverse age-related impaired wound healing by dampening the inflammatory response and increasing matrix deposition at the wound site. The molecular actions of estrogen are mediated through two nuclear sex steroid hormone receptors, estrogen receptor alpha (ERalpha) and beta (ERbeta). We have conducted a case-control study to investigate whether dinucleotide repeat polymorphisms in the estrogen receptor genes are associated with venous ulceration in the UK Caucasian population. Genomic fragments containing the ERalpha dinucleotide (TA)(n) repeat polymorphism or the ERbeta dinucleotide (CA)(n) repeat polymorphism were amplified by polymerase chain reaction in subject DNA samples and genotyped according to fragment length by capillary electrophoresis. There was no evidence to suggest that the TA repeat polymorphism of ERalpha was associated with venous ulceration. However, the CA*18 allele of the ERbeta CA repeat polymorphism was significantly associated with venous ulceration (n = 120, OR = 1.8, 95% CI = 1.1-2.8, P = 0.02). When the CA repeats alleles were grouped together into either low (L < or = 18) or high (H > 18) numbers of CA repeats, the low (L) repeat allele was significantly associated with venous ulceration (OR = 1.5, 95% CI = 1.0-2.2, P = 0.03). Our results show that a specific ERbeta variant is associated with impaired healing in the elderly, predisposing individuals to venous ulceration.     

Dinucleotide (CA/GT) repeat polymorphism in intron 17B of the cystic fibrosis transmembrane conductance regulator (CFTR) gene

Summary We describe a polymorphic microsatellite (IVS17BCA) in intron 17B of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. It consists of an 11 to 20 CA/GT dinucleotide repeat, located 424 bp from exon 17B.     

CA/GT microsatellite alleles within the cystic fibrosis transmembrane conductance regulator (CFTR) gene are not generated by unequal crossingover.

The gene responsible for cystic fibrosis (CF) has recently been identified, and a three-nucleotide deletion (delta F508 mutation) that results in the loss of a phenylalanine residue in the first putative ATP-binding domain of the predicted protein (CF transmembrane conductance regulator, CFTR) has been found to be the major CF mutation. Although several other mutations have been identified in the CFTR gene, most of them are very rare, making their application to genetic diagnosis difficult. While characterizing the genomic region encompassing the CF locus, we have identified three CA/GT blocks that flank exon 9 of the CF gene. One of the CA/GT blocks exhibits a highly informative variable number of dinucleotide repeats (VNDR) polymorphism. This intragenic VNDR microsatellite should, by itself, provide full information for genetic analysis in approximately 80% of CF families and will help elucidate the associations between DNA polymorphism haplotypes and specific gene mutations. Haplotype analyses of CF chromosomes with and without the delta F508 mutation suggest that the different alleles are generated by slipped-strand mispairing within the dinucleotide repeat during DNA replication, rather than by unequal crossingover within a recombination hot spot.     

A minisatellite and a microsatellite polymorphism within 1.5 kb at the human muscle glycogen phosphorylase (PYGM) locus can be amplified by PCR and have combined informativeness of PIC 0.95.

We sequenced a genomic clone (pMCMP1), previously reported to detect a VNTR polymorphism at the PYGM locus, and found a dinucleotide repeat segment (CA)14(GA)25 and a complex (AT)-repeat-rich segment containing 63 repeats spanning 160 bp. Resolution of PCR-amplified genomic DNA from the (CA)(GA) repeat region on DNA sequencing gels revealed a highly informative polymorphism with alleles differing by 2-bp intervals and ranging in size from 156 to 190 bp. Among three racial groups, a total of 18 alleles were observed. Fourteen alleles were observed in Caucasians (PIC 0.89), 12 alleles in American Blacks (PIC 0.89), and 9 alleles in Pima Indians (PIC 0.73). PCR amplification of the (AT) repeat region and resolution of the products on DNA sequencing gels revealed a complex variable length polymorphism with alleles distributed in size from 367 to 970 bp. Twenty-eight alleles were found in American Blacks (PIC 0.94), 6 alleles in Pima Indians (PIC 0.70), and 11 alleles in Caucasians (PIC 0.71). Comparison of the previously described VNTR RFLP alleles visualized by Southern hybridization to the PCR products described in this report demonstrated that the polymorphism described in both assays was identical. However, a larger number of alleles could be detected from the PCR-amplified products. Combined informativeness, PIC 0.95, for the two polymorphisms was determined from haplotype analysis of 100 Caucasian chromosomes. Therefore, for genotyping purposes, informativeness is maximized from using both polymorphisms.     

Development and characterization of polymorphic microsatellite markers in taro (Colocasia esculenta).

Microsatellite-containing sequences were isolated from enriched genomic libraries of taro (Colocasia esculenta (L.) Schott). The sequencing of 269 clones yielded 77 inserts containing repeat motifs. The majority of these (81.7%) were dinucleotide or trinucleotide repeats. The GT/CA repeat motif was the most common, accounting for 42% of all repeat types. From a total of 43 primer pairs designed, 41 produced markers within the expected size range. Sixteen (39%) were polymorphic when screened against a restricted set of taro genotypes from Southeast Asia and Oceania, with an average of 3.2 alleles detected on each locus. These markers represent a useful resource for taro germplasm management, genome mapping, and marker-assisted selection.     

Microsatellite DNA markers in Populus tremuloides.

Markers for eight new microsatellite DNA or simple sequence repeat (SSR) loci were developed and characterized in trembling aspen (Populus tremuloides) from a partial genomic library. Informativeness of these microsatellite DNA markers was examined by determining polymorphisms in 38 P. tremuloides individuals. Inheritance of selected markers was tested in progenies of controlled crosses. Six characterized SSR loci were of dinucleotide repeats (two perfect and four imperfect), and one each of trinucleotide and tetranucleotide repeats. The monomorphic SSR locus (PTR15) was of a compound imperfect dinucleotide repeat. The primers of one highly polymorphic SSR locus (PTR7) amplified two loci, and alleles could not be assigned to a specific locus. At the other six polymorphic loci, 25 alleles were detected in 38 P. tremuloides individuals; the number of alleles ranged from 2 to 7, with an average of 4.2 alleles per locus, and the observed heterozygosity ranged from 0.05 to 0.61, with an average of 0.36 per locus. The two perfect dinucleotide and one trinucleotide microsatellite DNA loci were the most informative. Microsatellite DNA variants of four SSR loci characterized previously followed a single-locus Mendelian inheritance pattern, whereas those of PTR7 from the present study showed a two-locus Mendelian inheritance pattern in controlled crosses. The microsatellite DNA markers developed and reported here could be used for assisting various genetic, breeding, biotechnology, genome mapping, conservation, and sustainable forest management programs in poplars.     

Identification of abundant and informative microsatellites from shrimp (Penaeus monodon) genome

Microsatellites were isolated from P. monodon genomic libraries by direct sequencing of recombinant clones without probe screening. Forty-nine out of 83 clones sequenced contained 99 microsatellite arrays of three or more repeats. When five or more and ten or more repeats were considered, 28 and 14 microsatellites were detected, respectively. The 99 microsatellites were classified as perfect (75%), imperfect (6%), compound perfect (3%) and compound imperfect (16%). The abundance of di-, tri-, tetra- and hexanucleotide repeats were 67%, 20%, 9% and 3%, respectively. The dinucleotide repeats included 36 (CT)n, 31 (GT)n, 17(AT)n and 3 (CG)n. One octanucleotide repeat (ATTTATTC)5 was found within a large repeat sequence. Optimal annealing temperatures were determined for PCR using 11 primer sets encompassing 15 microsatellites. Ten primer sets provided successful amplifications with allele sizes generally ranging from 139 to 410 bp. All these primers amplified polymorphic loci with PIC values ranging from 0.63 to 0.96. Two primer sets amplified additional bands which can easily be distinguished from the bands of the main locus. Three out of 10 P. monodon microsatellites also amplified alleles in P. vannamei. The abundance and informative nature of P. monodon microsatellites and their potential for cross-species amplification make them useful for genetic studies.     

Improved fidelity of thermostable ligases for detection of microsatellite repeat sequences using nucleoside analogs

Microsatellite repeats consisting of dinucleotide sequences are ubiquitous in the human genome and have proven useful for linkage analysis, positional cloning and forensic identification purposes. In this study, the potential of utilizing the ligase detection reaction for the analysis of such microsatellite repeat sequences was investigated. Initially, the fidelity of thermostable DNA ligases was measured for model dinucleotide repeat sequences. Subsequently, the effect of modified oligonucleotides on ligation fidelity for dinucleotide repeats was determined using the nucleoside analogs nitroimidazole, inosine, 7-deazaguanosine and 2-pyrimidinone, as well as natural base mismatches. The measured error rates for a standard dinucleotide template indicated that the nitroimidazole nucleoside analogs could be used to increase the fidelity of ligation when compared to unmodified primers. Furthermore, use of formamide in the ligation buffer also increased ligation fidelity for dinucleotide repeat sequences. Using ligation-based assays to detect polymorphic alleles of microsatellite repeats in the human genome opens the possibility of using array-based typing of these loci for human identification, loss-of-heterozygosity studies and linkage analysis.     

Characterization of the alternate allelic forms of human PAX7

Six different allelic forms of the human neurogenic and myogenic developmental gene, PAX7, have been identified. They are distinguished by the number of tandem tetranucleotide, GAAG, repeats at a polymorphic site within the second intron of the paired box. Within the same intron, a second polymorphic site was found to have variable numbers of a dinucleotide TG repeat. The alleles are identified by a PCR-based method with oligo primers that span the variable regions of the intron. Several of the alleles include a duplicate copy of the entire paired box. Segregation studies demonstrate that the PAX7 alleles are inherited in a Mendelian fashion and that the duplicate copies of the PAX7 paired box region present in some of the alleles are closely linked. This initial study identified differences in the distribution of PAX7 alleles in DNA from patients with the skeletal muscle myopathy, dermatomyositis. Recognition of genetic polymorphism of PAX7 allows new approaches to understanding the role of PAX7 in myogenesis, neurogenesis, and neuromuscular disorders. Received: 13 July 1999 / Accepted: 29 November 1999     

Microsatellite DNA markers in the giant freshwater prawn, Macrobrachium rosenbergii: a tool for genetic analysis

Eight microsatellite loci were identified and characterized in the commercially important giant freshwater prawn, Macrobrachium rosenbergii. The microsatellite loci were detected by the random screening for dinucleotide repeat units within a partial genomic library developed for the species with biotinylated probes (CA)(15) , (AT)(15) and (GA)(15) . All the eight loci were found to be polymorphic. The number of alleles and observed heterozygosities per locus ranged between three to 16 and 0.22 to 0.71, respectively. These microsatellite markers will be useful for the conservation and management of wild and cultured stocks and population genetic studies of freshwater prawn.     

Abundance and polymorphism of microsatellite markers in the tea tree (Melaleuca alternifolia, Myrtaceae)

 The sequencing of 831 clones from an enriched microsatellite library of Melaleuca alternifolia (Myrtaceae) yielded 715 inserts containing repeat motifs. The majority of these (98%) were dinucleotide repeats or trinucleotide repeats averaging 22 and 8 repeat motifs respectively. The AG/GA motif was the most common, accounting for 43% of all microsatellites. From a total of 139 primer pairs designed, 102 produced markers within the expected size range. The majority of these (93) were polymorphic. Primer pairs were tested on five selected M. alternifolia genotypes. Loci based on dinucleotide repeats detected on average a greater number of alleles (4.2) than those based on trinucleotide repeats (2.9). The loci described will provide a large pool of polymorphisms useful for population studies, genetic mapping, and possibly application in other Myrtaceae. Received: 28 July 1998 / Accepted: 8 October 1998