Multiple methods for the identification of polymorphic simple sequence repeats (SSRs) in sorghum [Sorghum bicolor (L.) Moench]

Simple sequence repeats (SSRs), also known as microsatellites, are highly variable DNA sequences that can be used as markers for the genetic analysis of plants. Three approaches were followed for the development of PCR primers for the amplification of DNA fragments containing SSRs from sorghum [Sorghum bicolor (L.) Moench]: a search for sorghum SSRs in public DNA databases; the use of SSR-specific primers developed in the Poaceae species maize (Zea mays L.) and seashore paspalum grass (Paspalum vaginatum Swartz); and the screening of sorghum genomic libraries by hybridization with SSR oligonucleotides. A total of 49 sorghum SSR-specific PCR primer pairs (two designed from GenBank SSR-containing sequences and 47 from the sequences of genomic clones) were screened on a panel of 17 sorghum and one maize accession. Ten primer pairs from paspalum and 90 from maize were also screened for polymorphism in sorghum. Length polymorphisms among amplification products were detected with 15 of these primer pairs, yielding diversity values ranging from 0.2 to 0.8 with an average diversity of 0.56. These primer pairs are now available for use as markers in crop improvement and conservation efforts.     

Conserved simple sequence repeats for the Limnanthaceae (Brassicales)

The Limnanthaceae (Order Brassicales) is a family of 18 taxa of Limnanthes (meadowfoam) native to California, Oregon, and British Columbia. Cultivated meadowfoam (L. alba Benth.), a recently domesticated plant, has been the focus of research and development as an industrial oilseed for three decades. The goal of the present research was to develop several hundred simple sequence repeat (SSR) markers for genetic mapping, molecular breeding, and genomics research in wild and cultivated meadowfoam taxa. We developed 389 SSR markers for cultivated meadowfoam by isolating and sequencing 1,596 clones from L. alba genomic DNA libraries enriched for AG _n or AC _n repeats, identifying one or more unique SSRs in 696 clone sequences, and designing and testing primers for 624 unique SSRs. The SSR markers were screened for cross- taxa utility and polymorphisms among ten of 17 taxa in the Limnanthaceae; 373 of these markers were polymorphic and 106 amplified loci from every taxon. Cross-taxa amplification percentages ranged from 37.3% in L. douglasii ssp. rosea (145/389) to 85.6% in L. montana (333/389). The SSR markers amplified 4,160 unique bands from 14 genotypes sampled from ten taxa (10.7 unique bands per SSR marker), of which 972 were genotype-specific. Mean and maximum haplotype heterozygosities were 0.71 and 0.90, respectively, among six L. alba genotypes and 0.63 and 0.93, respectively, among 14 genotypes (ten taxa). The SSR markers supply a critical mass of high-throughput DNA markers for biological and agricultural research across the Limnanthaceae and open the way to the development of a genetic linkage map for meadowfoam (x = 5).Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by O. Savolainen     

Characterization and molecular genetic mapping of microsatellite loci in pepper

Microsatellites or simple sequence repeats are highly variable DNA sequences that can be used as informative markers for the genetic analysis of plants and animals. For the development of microsatellite markers in Capsicum, microsatellites were isolated from two small-insert genomic libraries and the GenBank database. Using five types of oligonucleotides, (AT)₁₅, (GA)₁₅, (GT)₁₅, (ATT)₁₀ and (TTG)₁₀, as probes, positive clones were isolated from the genomic libraries, and sequenced. Out of 130 positive clones, 77 clones showed microsatellite motifs, out of which 40 reliable microsatellite markers were developed. (GA) _n and (GT) _n sequences were found to occur most frequently in the pepper genome, followed by (TTG) _n and (AT) _n . Additional 36 microsatellite primers were also developed from GenBank and other published data. To measure the information content of these markers, the polymorphism information contents (PICs) were calculated. Capsicum microsatellite markers from the genomic libraries have shown a high level of PIC value, 0.76, twice the value for markers from GenBank data. Forty six microsatellite loci were placed on the SNU-RFLP linkage map, which had been derived from the interspecific cross between Capsicum annuum TF68 and Capsicum chinense Habanero. The current SNU2 pepper map with 333 markers in 15 linkage groups contains 46 SSR and 287 RFLP markers covering 1,761.5 cM with an average distance of 5.3 cM between markers.Communicated by J. Dvorak     

The use of microsatellite DNA markers for soybean genotype identification

Conventional morphological and pigementation traits, as well as disease resistance, have been used to distinguish the uniqueness of new soybean cultivars for purposes of plant variety protection. With increasing numbers of cultivars and a finite number of conventional characters, it has become apparent that such traits will not suffice to establish uniqueness. The objective of this work was to provide an initial evaluation of microsatellite or simple-sequence-repeat (SSR) DNA markers to develop unique DNA profiles of soybean genotypes. Microsatellites are DNA sequences such as (AT) _n /(TA) _n and (ATT) _n /(TAA) _n that are composed of tandemly repeated 2–5-basepair DNA core sequences. The DNA sequences flanking microsatellites are generally conserved allowing the selection of polymerase chain reaction (PCR) primers that will amplify the intervening SSR. Variation in the number of tandem repeats, n, results in PCR product length differences. The SSR alleles present at three (AT) _n /(TA) _n and four (ATT) _n /(TAA) _n loci were determined in each of 96 diverse soybean genotypes. Between 11 and 26 alleles were found at each of the seven loci. Only two genotypes had identical SSR allelic profiles and these had very similar pedigrees. The gene diversity for the seven markers averaged 0.87 for all 96 genotypes and 0.74 for a subset of 26 North American cultivars. These are much higher than soybean gene diversity values obtained using RFLP markers, and are similar to the average values obtained for human microsatellite markers. SSR markers provide an excellent complement to the conventional markers that are currently used to characterize soybean genotypes.     

Isolation and characterization of microsatellites in trembling aspen (Populus tremuloides)

 We have identified, isolated, and characterized microsatellite/simple sequence repeat (SSR) loci in trembling aspen (Populus tremuloides) by screening partial genomic libraries. We have also examined the compatibility and use of the P. tremuloides SSR primers to resolve microsatellites in other Populus species. Fourteen microsatellites were identified from 1600 clones screened. The TC/AG microsatellites were the most abundant. A total of 29 alleles were detected in 36 P. tremuloides individuals at the four SSR loci (two each of di- and tri-nucleotide repeats) characterized. The number of alleles at the SSR loci ranged from 5 to 11, with an average of 7.25 alleles per locus, and the observed heterozygosity ranged from 0.19 to 0.82, with a mean of 0.46 per locus. Although the highest polymorphism was observed for a dinucleotide SSR locus, the trinucleotide SSR loci showed substantial polymorphism. There were 34 unique multilocus genotypes among the 36 P. tremuloides individuals examined, and 89% of the individuals had unique multilocus genotypes. Two pairs of SSR primers were successful in PCR, amplifying genomic DNA and resolving microsatellites of comparable size from Populus deltoides, P. nigra, P.×canadensis, and P. maximowiczii. The microsatellite DNA markers developed could be used for clonal fingerprinting, certification of controlled crosses, genome mapping, marker-assisted early selection, genetic diversity assessments, and conservation and sustainable management of poplar genetic resources. Received: 14 November 1997 / Accepted: 17 November 1997     

Development and characterisation of simple sequence repeat (SSR) markers for white clover (Trifolium repens L.)

Highly informative molecular markers, such as simple sequence repeats (SSRs), can greatly accelerate breeding programs. The aim of this study was to develop and characterise a comprehensive set of SSR markers for white clover (Trifolium repens L.), which can be used to tag genes and quantitative trait loci controlling traits of agronomic interest. Sequence analysis of 1123 clones from genomic libraries enriched for (CA) _n repeats yielded 793 clones containing SSR loci. The majority of SSRs consisted of perfect dinucleotide repeats, only 7% being trinucleotide repeats. After exclusion of redundant sequences and SSR loci with less than 25 bp of flanking sequence, 397 potentially useful SSRs remained. Primer pairs were designed for 117 SSR loci and PCR products in the expected size range were amplified from 101 loci. These markers were highly polymorphic, 88% detecting polymorphism across seven white clover genotypes with an average allele number of 4.8. Four primer pairs were tested in an F₂ population revealing Mendelian segregation. Successful cross-species amplification was achieved in at least one out of eight legume species for 46 of 54 primer pairs. The rate of successful amplification was significantly higher for Trifolium species when compared to species of other genera. The markers developed in this study not only provide valuable tools for molecular breeding of white clover but may also have applications in related taxa. Received: 3 April 2000 / Accepted: 12 May 2000     

Characterization of microsatellite markers in peach [Prunus persica (L.) Batsch]

Microsatellites have emerged as an important system of molecular markers. We evaluated the potential of microsatellites for use in genetic studies of peach [Prunus persica (L.) Batsch]. Microsatellite loci in peach were identified by screening a pUC8 genomic library, a λZAPII leaf cDNA library, as well as through database searches. Primer sequences for the microsatellite loci were tested from the related Rosaceae species apple (Malus×domestica) and sour cherry (Prunus cerasus L.). The genomic library was screened for CT, CA and AGG repeats, while the cDNA library was screened for (CT)_n- and (CA)_n-containing clones. Estimates of microsatellite frequencies were determined from the genomic library screening, and indicate that CT repeats occur every 100 kb, CA repeats every 420 kb, and AGG repeats every 700 kb in the peach genome. Microsatellite- containing clones were sequenced, and specific PCR primers were designed to amplify the microsatellite- containing regions from genomic DNA. The level of microsatellite polymorphism was evaluated among 28 scion peach cultivars which displayed one to four alleles per primer pair. Five microsatellites were found to segregate in intraspecific peach-mapping crosses. In addition, these microsatellite markers were tested for their utility in cross-species amplification for use in comparative mapping both within the Rosaceae, and with the un- related species Arabidopsis thaliana L. Received: 18 June 1999 / Accepted: 6 December 1999     

An integrated genetic linkage map of avocado

 An avocado genomic library was screened with various microsatellite repeats. (A/T)_n and (TC/AG)_n sequences were found to be the most frequent repeats. One hundred and seventy-two positive clones were sequenced successfully of which 113 were found to contain simple sequence repeats (SSR). Polymerase chain reaction primers were designed to the regions flanking the SSR in 62 clones. A GenBank search of avocado DNA sequences revealed 1 sequence containing a (CT)₁₀ repeat. A total of 92 avocado-specific SSR markers were screened for polymorphism using 50 offspring of a cross between the avocado cultivars ‘Pinkerton’ and ‘Ettinger’. Both are standard avocado cultivars which are normally outcrossed and highly heterozygous. Fifty polymorphic SSR loci, 17 random amplified polymorphic DNA (RAPD) and 23 minisatellite DNA Fingerprint (DFP) bands were used to construct the avocado genetic map. The resulting data were analyzed with various mapping programs in order to assess which program best accommodated data from progeny of heterozygous parents. The analyses resulted in 12 linkage groups with 34 markers (25 SSRs, 3 RAPDs and 6 DFP bands) covering 352.6 cM. This initial map can serve as a basis for developing a detailed genomic map and for detection of linkage between markers and quantitative trait loci. Received: 2 April 1996 / Accepted: 28 February 1997     

Genome organization of repetitive elements in the rodent,Peromyscus leucopus

To document the frequency and distribution of repetitive elements in Peromyscus leucopus, the white-footed mouse, a cosmid genomic library was examined. Two thousand thirteen randomly chosen recombinants, with an average insert size of 35 kb and representing 2.35% of the haploid genome of P. leucopus, were screened with probes representing microsatellites, tandem repeats, and transposable elements. Of the four dinucleotides, (GT)_n was present in 87% of the clones, (CT)_n was present in 59% of the clones, and (AT)_n and (GC)_n each was represented in our sample by a single clone (0.05%). (TCC)_n was present in 8% of the clones. Of the tandem repeats, the 28S ribosomal probe and the (TTAGGG)_n telomere probe were not represented in the library, whereas a heterochromatic fragment was present in 9% of the clones. A transposable element, mys, was estimated to occur in 4700 copies, whereas a long interspersed element (LINE) was estimated to occur in about 41,000 copies per haploid genome. LINE and mys occurred together in the same clones more frequently than expected on the basis of chance. Hybridizing the library to genomic DNA from P. leucopus, Reithrodontomys fulvescens, Mus musculus, and human produced general agreement between phylogenetic relatedness and intensity of hybridization. However, dinucleotide repeats appeared to account for a disproportionately high number of positive clones in the more distantly related taxa.     

Limited variability of CTG/CAG repeats in <Emphasis Type="Italic">Lycopersicon</Emphasis> nuclear DNA

Seven clones containing (CTG)_n/(CAG)_n repeats (n ≥ 4) were isolated by screening Lycopersicon esculentum genomic DNA. Four of the clones contained more than one simple sequence repeat (SSR). The SSRs were analyzed in several L. esculentum cultivars after polymerase chain reaction (PCR) amplification. No length variations were observed, suggesting considerable locus stability. Five clones are from transcribed regions, which might explain the lack of cultivar variations. However the conservation of CTG repeats was limited as differences in some transcribed loci were registered between L. pennellii and other Lycopersicon species. It is noted that in Lycopersicon trinucleotide repeat variation might be used for species identification.     

Isolation and characterization of polymorphic microsatellites in the genome of Yak (Bos grunniens)

The researches on yak genetics and breeding were extremely restricted due to lacking of reliable DNA molecular markers. The microsatellites with repeat motif (AC)_n/(GT)_n in yak genome were enriched by Dynal magnetic beads and the gene libraries containing (AC)_n/(GT)_n were constructed. Among the 92 identified and sequenced positive clones, 40 contained perfect repeats (43.48 %), 41 contained imperfect repeats (44.57 %) and 11 contained compound repeats (11.96 %). As compared with the percentage of perfect repeats, no significant increases of imperfect repeats were observed in yak genome, which indicated that the level of adaptive evolution of the ability to repair damaged genomic DNA for yaks were high enough to endure the natural pressure of nucleotide substitution resulted from ultraviolet irradiation in high-altitude areas. Totally 19 polymorphic microsatellite loci were screened and genotyped on the basis of electropherograms on an ABI 310 Genetic Analyzer. All the loci exhibited moderate to high-level polymorphisms in a test population of Bos grunniens and the polymorphic information content ranged from 0.299 to 0.861 (mean 0.678). The newly isolated (AC)_n/(GT)_n repeats from yak genome will display their potential values in examining intra-population genetic structure and inter-population relationships, and also in investigating molecular markers for production and adaptive traits of individual/population.     

Abundance and length polymorphism of microsatellite repeats in Beta vulgaris L.

Simple sequence repeats (SSRs) are known to exhibit high degrees of variability even among closely related individuals. Their usage as nuclear genetic markers requires their conversion into sequence-tagged sites (STSs). In this paper we present the development of simple sequences as STSs for Beta vulgaris. This species comprises wild, cultivated, and weedy forms; the latter are thought to originate from accidental hybridisation between the other two. Two partial genomic libraries were screened with simple sequence motifs (AT, CA, CT, ATT, GTG, and CA, CT, respectively). Clones of 22 CA, nine CT, eight ATT, and one GTG sequence were obtained. AT micro satellites were present in compound motifs, not recognised by the probe. Sequence comparisons revealed that 20 CA clones containing short motifs (<16 bp) were variants of a previously described approximately 320-bp satellite DNA (Schmidt et al. 1991), and hence did not correspond to unique loci. Polymorphism of one (ATT)₁₅ and three (CT)_n, with n=15, 17 and 26, was detected by PCR on a sample of 64 plants from the different forms of B. vulgaris. 13 (ATT), 13 (CT), nine (CT) alleles and one (CT) allele were detected. One of the ATT alleles was much larger than the others (>800 bp). Genetic variability was high among wild beets, lower among cultivated beets, and intermediate among weed beets. One allele of each locus was found at high frequencies in cultivated beets and, to a lower extent, in weed beets. The combination of three polymorphic loci allowed the individual identification of 17/17 wild and 15/15 weed beets, and 21/32, mostly homozygous, cultivated beets.     

Characterization of trinucleotide SSR motifs in wheat

Length differences among trinucleotide-based microsatellite alleles can be more easily detected and frequently produce fewer ”stutter bands” as compared to dinucleotide-based microsatellite markers. Our objective was to determine which trinucleotide motif(s) would be the most-polymorphic and abundant source of trinucleotide microsatellite markers in wheat (Triticum aestivum L.). Four genomic libraries of cultivar ’Chinese Spring’ were screened with nine trinucleotide probes. Based on the screening of 28550 clones, the occurrences of (CTT/GAA) _n , (GGA/CCT) _n , (TAA/ATT) _n , (CAA/GTT) _n , (GGT/CCA) _n , (CAT/GTA) _n , (CGA/GCT) _n , (CTA/GAT) _n , and (CGT/GCA) _n repeats were estimated to be 5.4×10⁴, 3.5×10⁴, 3.2×10⁴, 1.2×10⁴, 6.3×10³, 4.9×10³, 4.5×10³, 4.5×10³ and 3.6×10³, i.e., once every 293 kbp, 456 kbp, 500 kbp, 1.3 Mbp, 2.6 Mbp, 3.2 Mbp, 3.6 Mbp, 3.6 Mbp and 4.5 Mbp in the wheat genome, respectively. Of 236 clones selected for sequencing, 38 (93%) (TAA/ATT) _n , 30 (43%) (CTT/GAA) _n , 16 (59%) (CAA/GTT) _n , 3 (27%) (CAT/GTA) _n and 2 (4%) (GGA/CCT) _n clones contained microsatellites with eight or more perfect repeats. From these data, 29, 27 and 16 PCR primer sets were designed and tested to the (TAA/ATT) _n , (CTT/GAA) _n and (CAA/GTT) _n microsatellites, respectively. A total of 12 (41.4%) primers designed to (TAA/ATT) _n , four (14.8%) to (CTT/GAA) _n , and two (12.5%) to (CAA/GTT) _n resulted in polymorphic markers. The results indicated that (TAA/ATT) _n microsatellites would provide the most-abundant and the most-polymorphic source of trinucleotide microsatellite markers in wheat. Received: 17 February 2001 / Accepted: 31 May 2001     

Characterization of highly variable (GA/CT) n microsatellites in the bur oak,Quercus macrocarpa

The objective of this study was to ascertain the usefulness of polymerase chain reaction (PCR)-based microsatellite analysis for studying pollination and parentage in a wind-pollinated temperate tree. A small insert genomic library of the bur oak (Quercus macrocarpa) was constructed and screened for the presence of (CA/GT) _n and (GA/CT) _n repeats. The proportion of positive clones yielded estimates of 3×10⁵ such dinucleotide repeats per genome, roughly comparable to abundances reported in other eukaryotic genomes. Thirteen positive clones were sequenced. In contrast to mammalian genomes, the (GA/CT) _n motif was more abundant than the (CA/GT) _n motif in these clones. The (GA/CT) _n repeats also showed longer average repeat length (mean n=16.2 versus 7.3), suggesting that they are better candidates for yielding polymorphic genetic markers in oak genomes. Indeed, a survey of adult bur oaks and offspring in a small stand in northern Illinois at 3 of these (GA/CT) _n microsatellite loci revealed Mendelian inheritance and extremely high levels of polymorphism, with the number of alleles at each locus ranging from 11–20 and heterozygosity ranging from 0.66 to 0.75. These results, indicating that (GA/CT) _n microsatellites are both abundant and highly polymorphic in the bur oak genome, suggest that such genetic markers have tremendous potential for applications for studies of parentage, pollination and dispersal in temperate trees.     

Development of microsatellite markers for mango (Mangifera indica L.)

Microsatellite markers for mango (Mangifera indica L.) were developed using a genomic library enriched for (GA)_n and (GT)_n dinucleotide repeats. A subset of 41 positive clones was sequenced and primers were designed. Twenty‐eight primer pairs produced polymorphic amplification products for a diversity sample including 15 mango cultivars and two accessions from the related species Mangifera laurina and Mangifera applanata. Nineteen simple sequence repeat (SSR) loci with clear scorable patterns were chosen to study diversity in the mango germplasm bank of Guadalupe (FWI). The number of alleles ranged from three to 13 with observed levels of heterozygosity ranging from 0.059 to 0.857.     

Genome-wide identification of simple sequence repeats and development of polymorphic SSR markers for genetic studies in tea plant (<Emphasis Type="Italic">Camellia sinensis</Emphasis>)

The tea plant (Camellia sinensis (L.) O. Kuntze) is one of the most popular non-alcoholic beverage crops worldwide. The availability of complete genome sequences for the Camellia sinensis var. ‘Shuchazao’ has provided the opportunity to identify all types of simple sequence repeat (SSR) markers by genome-wide scan. In this study, a total of 667,980 SSRs were identified in the ~?3.08 Gb genome, with an overall density of 216.88 SSRs/Mb. Dinucleotide repeats were predominant among microsatellites (72.25%), followed by trinucleotide repeats (15.35%), while the remaining SSRs accounted for less than 13%. The motif AG/CT (49.96%) and AT/TA (40.14%) were the most and the second most abundant among all identified SSR motifs, respectively; meanwhile, AAT/ATT (41.29%) and AAAT/ATTT (67.47%) were the most common among trinucleotides and tetranucleotides, respectively. A total of 300 primer pairs were designed to screen six tea cultivars for polymorphisms of SSR markers using the five selected repeat types of microsatellite sequences. The resulting 96 SSR markers that yielded polymorphic and unambiguous bands were further deployed on 47 tea cultivars for genetic diversity assessment, demonstrating high polymorphism of these SSR markers. Remarkably, the dendrogram revealed that the phylogenetic relationships among these tea cultivars are highly consistent with their genetic backgrounds or places of origin. The identified genome-wide SSRs and newly developed SSR markers will provide a powerful means for genetic researches in tea plant, including genetic diversity and evolutionary origin analysis, fingerprinting, QTL mapping, and marker-assisted selection for breeding.     

Variable (CA/GT)n simple sequence repeat DNA in the alga Chlamydomonas

A partial genomic DNA library of Chlamydomonas reinhardtii was screened with an (AC)₁₁ probe for the presence of (CA/GT)_n simple sequence repeats (SSRs). Based on the frequency of these repeats in the partial genomic library, we estimate that (CA/GT)_n repeats occur at a rate of about one every 17.7 kb in the C. reinhardtii genome. Ten positive clones were sequenced and four polymerase chain reaction (PCR) primer sets flanking (CA/GT)_n sequences were constructed for four loci. The PCR was used to specifically amplify these regions from multiple isolates of C. reinhardtii. All four loci were highly polymorphic in the C. reinhardtii isolates. A simple Mendelian inheritance pattern was found for all four loci, which showed 2:2 segregation in the tetrads resulting from a cross between C. reinhardtii and C. smithii. Our results suggest that these simple sequence repeat DNA loci will be useful for identity testing, population studies, linkage analysis, and genome mapping in Chlamydomonas.     

Hybridization,polyploidy and clonality influence geographic patterns of diversity and salt tolerance in the model halophyte seashore paspalum (Paspalum vaginatum)

In plant species, variation in levels of clonality, ploidy and interspecific hybridization can interact to influence geographic patterns of genetic diversity. These factors commonly vary in plants that specialize on saline habitats (halophytes) and may play a role in how they adapt to salinity variation across their range. One such halophyte is the turfgrass and emerging genomic model system seashore paspalum (Paspalum vaginatum Swartz). To investigate how clonal propagation, ploidy variation, and interspecific hybridization vary across ecotypes and local salinity levels in wild P. vaginatum, we employed genotyping‐by‐sequencing, cpDNA sequencing and flow cytometry in 218 accessions representing > 170 wild collections from throughout the coastal southern United States plus USDA germplasm. We found that the two morphologically distinct ecotypes of P. vaginatum differ in their adaptive strategies. The fine‐textured ecotype is diploid and appears to reproduce in the wild both sexually and by clonal propagation; in contrast, the coarse‐textured ecotype consists largely of clonally‐propagating triploid and diploid genotypes. The coarse‐textured ecotype appears to be derived from hybridization between fine‐textured P. vaginatum and an unidentified Paspalum species. These clonally propagating hybrid genotypes are more broadly distributed than clonal fine‐textured genotypes and may represent a transition to a more generalist adaptive strategy. Additionally, the triploid genotypes vary in whether they carry one or two copies of the P. vaginatum subgenome, indicating multiple evolutionary origins. This variation in subgenome composition shows associations with local ocean salinity levels across the sampled populations and may play a role in local adaptation.     

Informativeness of human (dC-dA)n · (dG-dT)n polymorphisms

Abundant human interspersed repetitive DNA sequences of the form (dC-dA)_n · (dG-dT)_n have been shown to exhibit length polymorphisms. Examination of over 100 human (dC-dA)_n · (dG-dT)_n sequences revealed that the sequences differed from each other both in numbers of repeats and in repeat sequence type. Using a set of precise classification rules, the sequences were divided into three categories: perfect repeat sequences without interruptions in the runs of CA or GT dinucleotides (64% of total), imperfect repeat sequences with one or more interruptions in the run of repeats (25%), and compound repeat sequences with adjacent tandem simple repeats of a different sequence (11%). Informativeness of (dC-dA)_n · (dG-dT)_n markers in the perfect sequence category was found to increase with increasing average numbers of repeats. PIC values ranged from 0 at about 10 or fewer repeats to above 0.8 for sequences with about 24 or more repeats. (dC-dA)_n · (dG-dT)_n polymorphisms in the imperfect sequence category showed lower informativeness than expected on the basis of the total numbers of repeats. The longest run of uninterrupted CA or GT repeats was found to be the best predictor of informativeness of (dC-dA)_n · (dG-dT)_n polymorphisms regardless of the repeat sequence category.     

Characterization of porcine polymorphic microsatellite loci

Twenty-seven (CA)_n and two (GA)_n microsatellite clones were isolated out of a size-selected genomic pig library. These were sequenced and the number of uninterrupted dinucleotides was found to range from 12 to 26. Flanking primers were chosen for 11 dinucleotide repeats and optimal conditions for polymerase chain reaction (PCR) amplifications were established. Different microsatellite loci were amplified simultaneously by combining primer sets. Related and unrelated pigs were screened for length polymorphisms of the different microsatellite loci. The polymorphic information content (PIC) of these loci ranged between 0.62 and 0.83. Segregation studies in pig reference families established Mendelian inheritance. Locus S0022 was found to be X-linked.