PCR primed with minisatellite core sequences yields DNA fingerprinting probes in wheat

 Four minisatellite core sequences were used as primers in a polymerase chain reaction (PCR) technique, known as the directed amplification of minisatellite-region DNA (DAMD), to detect polymorphisms in three pairs of hexaploid/tetraploid wheat cultivars. In each pair, the tetraploid cultivar (genomic formula AABB) was extracted from its corresponding hexaploid (genomic formula AABBDD) parent. Reproducible profiles of the amplified products revealed characteristic bands that were present only in the hexaploid wheats but not in their extracted tetraploids. Some polymorphisms were observed among the hexaploid cultivars. Twenty-three DAMD-PCR amplified fragments were isolated and screened as molecular probes on the genomic DNA of wild wheat species, hexaploid wheat and triticale cultivars. Subsequently, 8 of the fragments were cloned and sequenced. The DAMD-PCR clones revealed various degrees of polymorphism among different wild and cultivated wheats. Two clones yielded individual-specific DNA fingerprinting patterns which could be used for species differentiation and cultivar identification. The results demonstrated the use of DAMD-PCR as a tool for the isolation of informative molecular probes for DNA fingerprinting in wheat cultivars and species. Received: 13 May 1996/Accepted: 11 October 1996     

Direct amplification of minisatellite-region DNA with VNTR core sequences in the genus Oryza

 A polymerase chain reaction (PCR) application, involving the directed amplification of minisatellite-region DNA (DAMD) with several minisatellite core sequences as primers, was used to detect genetic variation in 17 species of the genus Oryza and several rice cultivars (O. sativa L.). The electrophoretic analysis of DAMD-PCR products showed high levels of variation between different species and little variation between different cultivars of O. sativa. Polymorphisms were also found between accessions within a species, and between individual plants within an accession of several wild species. The DAMD-PCR yielded genome-specific banding patterns for the species studied. Several DAMD-PCR-generated DNA fragments were cloned and characterized. One clone was capable of detecting multiple fragments and revealed individual-specific hybridization banding patterns using genomic DNA from wild species as well as rice cultivars. A second clone detected only a single polymorphic locus, while a third clone expressed a strong genome specificity by Southern analysis. The results demonstrated that DAMD-PCR is potentially useful for species and genome identification in Oryza. The DAMD-PCR technique also allows for the isolation of informative molecular probes to be utilized in DNA fingerprinting and genome identification in rice. Received: 1 October 1996 / Accepted: 25 April 1997     

An alternative approach to evaluating the intraspecific genetic variability of parasites

Analysis of DNA polymorphisms provides important information for the molecular characterization of parasite strains and clones. Because we still know little about the genomes of parasites, such analysis has to rely on methods applicable to any eukaryotic genome, such as DNA fingerprinting with multilocal minisatellite probes and the polymerase chain reaction (PCR)-based random amplified polymorphic DNA technique (RAPD). However, DNA fingerprinting is cumbersome and needs large amounts of parasite DNA, and RAPD can exhibit low reproducibility and spurious bands, both of which appear to be related to the low stringency of the PCR procedure. Riva Oliveira, Andréa Macedo, Egler Chiari and Sérgio Pena here evaluate the applicability to parasites of a technique described two years ago called simple sequence repeat-anchored PCR amplification (SSR-PCR), in which a single primer is needed [the (CA)(8)RY primer] and highstringency conditions are applied.     

Genetic variation detected by DNA fingerprinting with a rice minisatellite probe in Oryza sativa L.

A rice minisatellite probe detecting DNA fingerprints was used to assess genetic variation in cultivated rice (Oryza sativa L.). Fifty-seven cultivars of rice, including 40 closely related cultivars released in the US, were studied. Rice DNA fingerprinting revealed high levels of polymorphism among distantly related cultivars. The variability of fingerprinting pattern was reduced in the closely related cultivars. A genetic similarity index (S) was computed based on shared fragments between each pair of cultivars, and genetic distance (D) was used to construct the dendrograms depicting genetic relationships among rice cultivars. Cluster analysis of genetic distance tended to group rice cultivars into different units corresponding with their varietal types and breeding pedigrees. However, by comparison with the coefficients of parentage, the criterion of relatedness based on DNA fingerprints appeared to overestimate the genetic relationships between some of the closely related US cultivars. Although this may reduce the power of fingerprints for genetic analysis, we were able to demonstrate that DNA fingerprinting with minisatellite sequences is simpler and more sensitive than most other types of marker systems in detecting genetic variation in rice.This paper reports the results of research only. Mention of a proprietary product does not consititute an endorsement or a recommendation for its use by the USDA or the University of Missouri. Contribution from the US Department of Agriculture, Agricultural Research Service, Plant Genetics Research Unit, and the University of Missouri Agricultural Experiment Station Journal Series No. 12178.     

Repetitive DNA of grapevine: classes present and sequences suitable for cultivar identification

Summary Repetitive DNA sequences present in the grapevine genome were investigated as probes for distinguishing species and cultivars. Microsatellite sequences, minisatellite sequences, tandemly arrayed genes and highly repetitive grapevine sequences were studied. The relative abundance of microsatellite and minisatellite DNA in the genome varied with the repeat sequence and determined their usefulness in detecting RFLPs. Cloned Vitis ribosomal repeat units were characterised and showed length heterogeneity (9.14–12.15 kb) between and within species. A highly repetitive DNA sequence isolated from V. vinifera was found to be specific only to those species classified as Euvitis. DNA polymorphisms were found between Vitis species and between cultivars of V. vinifera with all classes of repeat DNA sequences studied. DNA sequences suitable for DNA fingerprinting gave genotype-specific patterns for all of the cultivars and species examined. The DNA polymorphisms detected indicates a moderate to high level of heterozygosity in grapevine cultivars.On leave from the Biochemical Research Institute, Nippon Menard Cosmetic Co, Ltd, Ogaki Gifuken, 503 Japan     

An integrated molecular linkage map of diploid wheat based on a <Emphasis Type="Italic">Triticum boeoticum</Emphasis> × <Emphasis Type="Italic">T. monococcum</Emphasis> RIL population

Diploid A genome species of wheat harbour immense variability for biotic stresses and productivity traits, and these could be transferred efficiently to hexaploid wheat through marker assisted selection, provided the target genes are tagged at diploid level first. Here we report an integrated molecular linkage map of A genome diploid wheat based on 93 recombinant inbred lines (RILs) derived from Triticum boeoticum × Triticum monococcum inter sub-specific cross. The parental lines were analysed with 306 simple sequence repeat (SSR) and 194 RFLP markers, including 66 bin mapped ESTs. Out of 306 SSRs tested for polymorphism, 74 (24.2%) did not show amplification (null) in both the parents. Overall, 171 (73.7%) of the 232 remaining SSR and 98 (50.5%) of the 194 RFLP markers were polymorphic. Both A and D genome specific SSR markers showed similar transferability to A genome of diploid wheat species. The 176 polymorphic markers, that were assayed on a set of 93 RILs, yielded 188 polymorphic loci and 177 of these as well as two additional morphological traits mapped on seven linkage groups with a total map length of 1,262 cM, which is longer than most of the available A genome linkage maps in diploid and hexaploid wheat. About 58 loci showed distorted segregation with majority of these mapping on chromosome 2A^m. With a few exceptions, the position and order of the markers was similar to the ones in other maps of the wheat A genome. Chromosome 1A^m of T. monococcum and T. boeoticum showed a small paracentric inversion relative to the A genome of hexaploid wheat. The described linkage map could be useful for gene tagging, marker assisted gene introgression from diploid into hexaploid wheat as well as for map based cloning of genes from diploid A genome species and orthologous genes from hexaploid wheat.     

Identification of repetitive, genome-specific probes in crucifer oilseed species.

Direct amplification of minisatellite DNA by PCR (DAMD PCR) was used to amplify and subsequently clone several fragments of DNA from crucifer species. The PCR-derived fragments of DNA were generated using known minisatellite core sequences as PCR primers. Southern hybridization of these putative minisatellite DNA fragments revealed that many were genome-specific; they hybridized with high affinity only to the genomic DNA of the species from which they were cloned. The DNA fragments were believed to be dispersed in the genome, based on smear-like hybridization signals on EcoRI-, BamHI-, and HindIII-digested genomic DNA. Genome-specific probes were specifically isolated from Brassica rapa (A genome), Brassica nigra (B genome), and Sinapis alba in addition to several other crucifer species. The sequence of a B. rapa specific probe (pBr17.1.3A) contained a minisatellite region that could be divided into three tandem repeats; each repeat contained between two and five subrepeats and each subrepeat shared a highly conserved core region of 29 bp. This minisatellite sequence also hybridized with high affinity to the A genome species B. napus and B. juncea. This research showed that dispersed, genome-specific probes can be isolated using DAMD PCR and that these probes could be used to detect and quantify alien DNA present in progeny from intergeneric or interspecific crosses.     

Diagnosis of alpha 1-antitrypsin deficiency by enzymatic amplification of human genomic DNA and direct sequencing of polymerase chain reaction products.

We have compared sequencing of cloned "polymerase chain reaction" (PCR) products and the direct sequencing of PCR products in the examination of individuals from six families affected with alpha 1-antitrypsin (AAT) deficiency. In families where paternity was in question we confirmed consanguinity by DNA fingerprinting using a panel of locus-specific minisatellite probes. We demonstrate that direct sequencing of PCR amplification products is the method of choice for the absolutely specific diagnosis of AAT deficiency and can distinguish normals, heterozygotes and homozygotes in a single, rapid and facile assay. Furthermore, we demonstrate the reproducibility of the PCR and a rapid DNA isolation procedure. We have also shown that two loci can be simultaneously amplified and that the PCR product from each locus can be independently examined by direct DNA sequencing.     

Phylogeny of tall fescue and related species using RFLPs

The wild species of tall fescue (Festuca arundinacea var.genuina Schreb.) represent a wide range of genetic variation and constitute potential germplasm for tall fescue improvement. Our objective was to evaluate genome specificity of the previously-identified DNA probes and to examine the phylogenetic relationship of tall fescue with six related species by using RFLP data. A total of 29 DNA probes from aPstI-genomic library of tall fescue were hybridized toEcoRI-orHindIII-digested DNA of 32 plants from sixFestuca species and fromLolium perenne L. Fifteen probes hybridized to all seven species. The remaining 14 probes showed differential hybridization patterns (i.e., ±), especially at the diploid and tetraploid levels. This hybridization pattern reflected genome divergence in these species. The DNA probes will be useful markers in breeding programs involving interspecific and intergeneric hybridization. Cluster analyses were performed using the average genetic distances calculated with the RFLP data from 53 probe-enzyme combinations. Generally, genotypes from the same species were grouped in the same cluster. These data indicated that tall fescue has a close relationship withF. pratensis Huds. (diploid),F. arundinacea var.glaucescens Boiss. (tetraploid), andL. perenne L. (diploid) and thatFestuca pratensis andL. perenne had the closest degree of relationship.This paper is a contribution of the Missouri Agricultural Experimental Station, Journal Series no. 11,798     

Fingerprinting genomes by use of PCR with primers that encode protein motifs or contain sequences that regulate gene expression

PCR primers of arbitrary nucleotide sequence have identified DNA polymorphisms useful for genetic mapping in a large variety of organisms. Although technically very powerful, the use of arbitrary primers for genome mapping has the disadvantage of characterizing DNA sequences of unknown function. Thus, there is no reason to anticipate that DNA fragments amplified by use of arbitrary primers will be enriched for either transcribed or promoter sequences that may be conserved in evolution. For these reasons, we modified the arbitrarily primed PCR method by using oligonucleotide primers derived from conserved promoter elements and protein motifs. Twenty-nine of these primers were tested individually and in pairwise combinations for their ability to amplify genomic DNA from a variety of species including various inbred strains of laboratory mice and Mus spretus. Using recombinant inbred strains of mice, we determined the chromosomal location of 27 polymorphic fragments in the mouse genome. The results demonstrated that motif sequence-tagged PCR products are reliable markers for mapping the mouse genome and that motif primers can also be used for genomic fingerprinting of many divergent species.     

Hypervariable single and multi-locus DNA polymorphisms for genetic typing of non-human primates

The series of hypervariable, “minisatellite” loci characterized byJeffreys and coworkers in the human myoglobin gene have proved to be DNA sequences highly conserved throughout the eukaryotic genome, and hence the methodology developed for human DNA “fingerprinting” has found immediate application in an ever expanding number of species. Primatologists have not been slow to profit from a method which predicts individual recognition to a very high degree of probability, and initial studies have focused on paternity allocation (rather than paternity exclusion, as designated by the classical biochemical markers), adaptive aspects of socio-sexual behaviour patterns and mating systems. A number of probes with sequences corresponding to the common minisatellite core sequences have been used for probing genomic DNA, and synthetic, G-rich oligonucleotides (15 – 37 bases), corresponding to the core sequence of the minisatellite repeat unit, or simply di-, tri-, or tetranucleotide repeats, appear to be equally discriminatory. The multiple banding patterns produced on hybridization of these probes to restriction enzyme digests of DNA provide an advantage in that the probability of two unrelated individuals sharing the same banding pattern will be low. However, the uncertainty of linkage of the multiple loci identified precludes genotyping and population genetic analyses based on allele frequencies. In contrast, single locus analysis allows DNA typing using variable number tandem repeat (VNTR) or restriction fragment length (RFLP) DNA polymorphisms, and the merits and drawbacks relative to DNA fingerprinting are discussed. For the behavioural primatologists dealing with defined, accessible troops of primates, the value of multilocus DNA fingerprinting, in terms of established methodology and availability of probes applicable to species as phylogenetically wide-ranging as apes and prosimians, may well outweigh the loss of genotypic and population structure data.     

Evaluation of “sequence-tagged-site” PCR products as molecular markers in wheat

The polymerase chain reaction (PCR) is an attractive technique for many genome mapping and characterization projects. One PCR approach which has been evaluated involves the use of randomly amplified polymorphic DNA (RAPD). An alternative to RAPDs is the sequence-tagged-site (STS) approach, whereby PCR primers are designed from mapped low-copy-number sequences. In this study, we sequenced and designed primers from 22 wheat RFLP clones in addition to testing 15 primer sets that had been previously used to amplify DNA sequences in the barley genome. Our results indicated that most of the primers amplified sequences that mapped to the expected chromosomes in wheat. Additionally, 9 of 16 primer sets tested revealed polymorphisms among 20 hexaploid wheat genotypes when PCR products were digested with restriction enzymes. These results suggest that the STS-based PCR analysis will be useful for generation of informative molecular markers in hexaploid wheat.Contribution no. J-2833 of the Montana Agric Exp Stn     

Detection of DNA sequence polymorphisms among wheat varieties

Summary A DNA marker detection strategy that allows the rapid, efficient resolution of high levels of polymorphism among closely related lines of common wheat (Triticum aestivum) has been developed to circumvent the apparent lack of restriction fragment length polymorphism in many important self-pollinated crop species. The technique of randomly amplified polymorphic DNA (RAPD) was combined with a denaturing gradient gel electrophoresis system (DGGE) to explore DNA sequence polymorphisms among different genotypes of wheat. Of the 65 primer combinations used for the polymerase chain reaction (PCR) amplifications, over 38% of them produced readily detectable and reproducible DNA polymorphisms between a spring wheat line, SO852, and a winter wheat variety, Clark. A high level of polymorphism was observed among a number of commercial varieties and breeding lines of wheat. This procedure was also used to detect polymorphisms in a recombinant inbred population to test the feasibility of its application in genome mapping. This DNA polymorphism detection system provides an opportunity for pedigree analysis and fingerprinting of developed wheat lines as well as construction of a high density genetic map of wheat. Without the need for ³²P and sophisticated DNA extraction procedures, this approach should make it feasible to utilize marker-based selection in a plant breeding program.     

Minisatellite DNA fingerprints of salmonid fishes

The human minisatellite probes 33.6 and 33.15 cross-hybridized to DNA digests of Atlantic salmon, brown trout and rainbow trout revealing complex multi-banded patterns. These DNA fingerprints (in excess of 40 resolvable fragments in some cases) were highly polymorphic, individual specific and found to be stable, both somatically and in the germline. Pedigree analysis of an Atlantic salmon family confirmed that the minisatellite fragments showed Mendelian inheritance. With only a single occurrence of linkage and allelism being observed it is likely the minisatellite loci are widely distributed throughout the salmonid genome. The potential applications for both multi- and single locus minisatellite probes in salmonid research are discussed.     

Checking of individuality by DNA profiling

A review of methods of DNA analysis used in forensic medicine for identification, paternity testing, etc. is provided. Among other techniques, DNA fingerprinting using different probes and polymerase chain reaction-based techniques such as amplified sequence polymorphisms and minisatellite variant repeat mapping are thoroughly described and both theoretical and practical aspects are discussed.     

DNA “fingerprints” detect genetic variation inAcer negundo (Aceraceae)

Genomic DNA samples from 21 box elder plants collected in Missouri (U.S.A.) were digested with restriction enzyme and southern blot hybridized with the M13 minisatellite probe. Each plant was found to have a unique DNA fragment pattern. Moreover, levels of genetic variation estimated from a similarity index appear to be related to sampling distances. However, size of the fragments utilized in the analysis affects the estimates of genetic variation to a considerable degree.     

Atrazine-resistant cytoplasmic male-sterile-nigra broccoli obtained by protoplast fusion between cytoplasmic male-sterile Brassica oleracea and atrazine-resistant Brassica campestris

Summary By using restriction endonuclease digestion patterns, the degree of intraspecific polymorphism of mitochondrial DNA in four diploid species of wheat and Aegilops, Ae. speltoides, Ae. longissima, Ae. squarrosa, and Triticum monococcum, was assessed. The outbreeding Ae. speltoides was found to possess the highest degree of variability, the mean number of nucleotide substitutions among conspecific individuals being 0.027 substitutions per nucleotide site. A very low degree of mtDNA variation was detected among Ae. longissima accessions, with most of the enzyme-probe combinations exhibiting uniform hybridization patterns. The mean number of substitutions among Ae. longissima individuals was 0.001 substitutions per nucleotide site. The domesticated diploid wheat T. monococcum var. monococcum and its conspecific variant T. monococcum var. boeoticum seem to lack mitochondrial DNA variability altogether. Thus, the restriction fragment pattern can be used as a characteristic identifier of the T. monococcum cytoplasmic genome. Similarly, Ae. squarrosa accessions were found to be genetically uniform. A higher degree of variation among accessions is observed when noncoding sequences are used as probes then when adjacent coding regions are used. Thus, while noncoding regions may contain regulatory functions, they are subject to less stringent functional constraints than protein-coding regions. Intraspecific variation in mitochondrial DNA correlates perfectly with the nuclear variability detected by using protein electrophoretic characters. This correlation indicates that both types of variation are selectively neutral and are affected only by the effective population size.     

Minisatellite DNA profiles: rapid sample identification in linkage analysis

Locus-specific minisatellite probes detect multiple alleles with heterozygosities of greater than 90% when hybridised to HinfI and AluI restriction digests of human DNA. We have hybridised 4 of these probes to a panel of DNAs digested with 6 of the restriction enzymes which commonly reveal di-allelic polymorphisms in the human genome. All 6 enzymes detected multiple resolvable alleles with at least 1 of the 4 minisatellite probes tested, thus providing a rapid and efficient means to check family relationships and paternity on filters already being used for linkage analysis.     

Transferability of wheat microsatellites to diploid Triticeae species carrying the A, B and D genomes

Hexaploid wheat (Triticum aestivum L em Thell) is derived from a complex hybridization procedure involving three diploid species carrying the A, B and D genomes. In this study, we evaluated the ability of microsatellite sequences from T. aestivum to be revealed on different ancestral diploid species more or less closely related, i.e. to test for their transferability. Fifty five primer pairs, evenly distributed all over the genome, were investigated. Forty three of them mapped to single loci on the hexaploid wheat genetic map although only 20 (46%) gave single PCR products; the 23 others (54%) gave more than one band with either only one being polymorphic, the others remaining monomorphic, or with several co-segregating polymorphic bands. The other 12 detected two (9) or three (3) different loci. From the 20 primer pairs which gave one amplification pro- duct on hexaploid wheat, nine (45%) also amplified products on only one of the diploid species, and seven (35%) on more than one. Four microsatellites (20%) which mapped to chromosomes from the B genome of wheat, did not give any amplification signal on any of the diploid species. This suggests that some regions of the B genome have evolved more rapidly compared to the A or D genomes since the emergence of polyploidy, or else that the donor(s) of this B genome has(have) not yet been identified. Our results confirm that Triticum monococcum ssp. urartu and Triticum tauschii were the main donors of the A and D genomes respectively, and that Aegilops speltoides is related to the ancestor(s) of the wheat polyploid B genome. Received: 21 June 2000 / Accepted: 15 November 2000     

"Major minisatellite loci" detected by minisatellite clones 33.6 and 33.15 correspond to the cognate loci D1S111 and D7S437

G. Chimini et al. (1989, Genomics 5: 316-324) have recently reported that the two multilocus DNA fingerprinting probes 33.6 and 33.15 each detect a single major site in the human genome, at 1q23 and 7q35-q36, respectively, and speculate that these sites represent particularly large loci homologous to these probes. However, the human minisatellite loci cloned in 33.6 and 33.15 can themselves be assigned by somatic cell hybrid analysis to 1cen-q24 and 7q31.3-qter, respectively, corresponding to the "major loci" of Chimini et al. Furthermore, under their hybridization conditions, both 33.6 and 33.15 act largely as locus-specific minisatellite probes. The "major minisatellite loci" postulated by Chimini et al. do not therefore appear to represent major localized clusters of minisatellites in the human genome, but rather the loci cloned in 33.6 and 33.15.