Repetitive, genome-specific probes in wheat (Triticum aestivum L. em Thell) amplified with minisatellite core sequences

The detection and analysis of DNA polymorphisms in crops is an essential component of marker-assisted selection and cultivar identification in plant breeding. We have explored the direct amplification of minisatellite DNA by PCR (DAMD-PCR) as a means for generating DNA probes that are useful for detecting DNA polymorphisms and DNA fingerprinting in wheat. This technique was facilitated by high-stringency PCR with known plant and animal minisatellite core sequences as primers on wheat genomic DNA. The products of DAMD-PCR from Triticum aestivum, T. durum, T. monococcum, T. speltoides and T. tauschii showed a high degree of polymorphism and the various genomes could be identified. Cloning of the DAMD-PCR products and subsequent Southern hybridization frequently revealed polymorphic probes showing a good degree of genome specificity. In addition, polymorphic, single locus, and moderately dispersed PCR products were cloned that may have a potential for DNA fingerprinting. Our experiments were limited primarily to diploid wheats and the results indicated that DAMD-PCR may isolate genome-specific probes from wild diploid wheat species that could be used to monitor genome introgression into hexaploid wheat.This paper reports the results of research only. Mention of a proprietary product does not constitute an endorsement or a recommendation for its use by the USDA or the University of Missouri. Contribution from the University of Missouri, the Agricultural Experimental Station and U.S. Department of Agriculture-Agricultural Research Service, Plant Genetics Research Unit, journal series No. 12523     

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     

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.     

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     

DNA fingerprinting: a tool for determining genetic distances between strains of poultry

Summary DNA fingerprinting, a technique based on the detection of hypervariable minisatellite regions in DNA restriction fragments, was tested for its applicability to conduct population genetics in poultry. Using MspI digestion and phage M13 DNA as a probe, between 25 and 35 minisatellite-containing DNA fragments were observed per bird. Comparison of the banding pattern of offspring with their parents revealed that the bands were inherited as stable genetic traits. The variability of the DNA fingerprinting pattern was reduced in inbred strains. DNA fingerprints of chickens from five well-defined populations of known genetic relationships were analyzed and indices of genetic distances were computed. They correctly reflected the history of these strains, indicating that DNA fingerprinting may be a powerful tool to characterize genetic relationships between different breeding populations of the same species.     

Genetic mapping of hypervariable minisatellite sequences in rice (Oryza sativa L.)

Minisatellites, or DNA fingerprinting sequences, have been utilized in animal linkage studies for several years but have not been used as markers for plant genome mapping. In animal genome mapping they have resulted in limited success because they are evenly dispersed in some species but are often clustered near telomeric regions, as observed on human chromosomes. The purpose of the present study was to generate DNA fingerprints utilizing several rice-derived minisatellites containing different core sequences and numbers of repeat units, followed by assessing their potential for use as genetic markers when mapped to a rice recombinant inbred line (RIL) population. Sites of segregating minisatellite loci were mapped onto 11 of the 12 rice RIL linkage maps. The implications for the use of rice minisatellite core sequences as genetic markers on linkage maps in rice are discussed. Received: 1 March 1999 / Accepted: 22 June 1999     

The physical location of fourteen RFLP markers in rice (Oryza sativa L.)

A biotin-labeled in situ hybridization technique was used in order to physically map RFLP markers to the chromosomes of rice (Oryza sativa L.). Fourteen RFLP markers, associated with the ends of the linkage groups on rice chromosomes 7, 8, 11, 12, were physically mapped onto specific regions of the chromosomes. The average detection rate of in situ hybridization was 5.91%. The markers were located on seven different chromosome arms. Ten of the fourteen markers were distributed near the chromosome ends. This demonstrated that the RFLP linkage groups involved covered a wide physical distance and that the centromeric region was bisected by all but one linkage group. Two markers covered a short genetic distance but were physically distant, while two covering a longer genetic distance were physically closer together. This indicates that considerable variation can, and does, exist between genetic and physical maps.This paper is a contribution of the U.S. Department of Agriculture, Agricultural Research Service, and Missouri Agricultural Experiment Station, Journal Series No. 11 882All programs and services of the U.S. Department of Agriculture are offered on a nondiscriminatory basis without regard to race, color, national origin, religion, sex, age, marital status, or handicapThis paper reports the results of research only. Mention of a proprietary product does not constitute an endorsement or a recommendation for its use by the U.S. Department of Agriculture or the University of Missouri     

Reduced genetic variation in Norwegian Peregrine Falcons Falco peregrinus indicated by minisatellite DNA fingerprinting

The Scandinavian Peregrine Falcon Falco peregrinus population went through a severe population bottleneck during the second half of the twentieth century, and was almost extinct during the 1970s. This event may have reduced the amount of genetic variation in the population. With this background, a comparative study, using multilocus, minisatellite DNA fingerprinting, was carried out on broods of the Peregrine Falcon, the Merlin Falco columbarius and the Eurasian Hobby Falco subbuteo from south-east Norway. Band-sharing analysis of DNA fingerprints was used to test whether broods of Peregrine Falcons showed a greater between-nest similarity in their fingerprint profiles than did broods of the two congeneric species breeding in the same region, which have not undergone any recent population bottlenecks. The results show that broods of Peregrine Falcons were significantly more similar to each other genetically than were broods of either Merlins or Eurasian Hobbies. Furthermore, there was a positive correlation between the similarity in minisatellite DNA and the similarity in a set of 11 microsatellite loci analysed for a subset of the Peregrine Falcon samples. The correlation supports the assumption that minisatellite fingerprints provide a reliable indicator of overall genetic similarity, i.e. relatedness, between breeding pairs in the population. Hence we can conclude that broods of Peregrine Falcons were genetically more related to each other than were broods of the other two species. The high similarity in minisatellite DNA between broods indicates a loss of genetic variation in the Peregrine Falcon population caused by the bottleneck, but this explanation can only be verified through a comparative genetic study of individuals sampled before and after the bottleneck event.     

DNA fingerprinting of individual species and intergeneric and interspecific hybrids of genera Bos and Bison,subfamily Bovinae

Genome fingerprinting with a hypervariable minisatellite sequence of phage M13 DNA was used to study the genetic variation in individual species of the genera Bos and Bison (subfamily Bovinae) and in their interspecific and intergeneric hybrids. DNA fingerprints were obtained for domestic cow Bos taurus primigenius, vatussy Bos taurus macroceros, banteng Bos javanicus, gaur Bos gaurus, wisent Bison bonasus, bison Bison bison, and for the interspecific and intergeneric hybrids. Compared with the original species, most hybrids showed a greater variation in number and size of hybridization fragments. An association was revealed between the number of hybridization fragments and blood composition of interspecific hybrids resulting from unique crossing of domestic cow and banteng. Pairwise similarity coefficients were calculated to construct a dendrogram of genetic similarity, which reflected the relationships between the parental species and hybrids varying in blood composition. The applicability of the method for identifying interspecific and intergeneric hybrids and for studying the consequences of distant hybridization in the subfamily Bovinae is discussed.     

DNA fingerprint variation in some blackberry species (Rubus subg.Rubus,Rosaceae)

We have analysed samples from Sweden, Denmark, and Germany of six facultatively apomictic blackberry species to investigate the accordance between a taxonomy based on morphological characters on the one hand, and distribution of genetic variation estimated by DNA fingerprinting on the other hand. DNA fingerprint variation was found to be quite restricted in all species investigated. The first taxonomic group included three species related toRubus nessensis, two being characterized by one very widespread DNA fingerprint in all three countries and a few rare ones, whereas the third species differed between Sweden and Germany. The second taxonomic group included species related toR. gracilis. Two of these species exhibited very similar DNA fingerprints, whereas the third species deviated clearly. The utilization of DNA fingerprinting as a tool in taxonomy is discussed; most likely this method could become a useful complement to morphology, especially in plant groups with reduced levels of genetic recombination.     

GENETIC VARIABILITY AND POPULATION DIFFERENTIATION INFERRED FROM DNA FINGERPRINTING IN SILVEREYES (AVES: ZOSTEROPIDAE)

This study evaluated DNA fingerprinting as a tool for estimating population genetic diversity and differentiation by comparing minisatellite variation in island and mainland populations of silvereyes (Aves: Zosterops lateralis). Three populations with different recent histories were compared: (1) Heron Island and neighboring islands, colonized 3000 to 4000 yr ago; (2) Lady Elliot Island, colonized within the past two decades; and (3) an adjacent mainland population, which presumably has existed for thousands of years. The degree of genetic variability within the three populations reflected both their size and the time since their colonization. Minisatellite diversity was highest in the mainland population, intermediate in the Capricorn Island group (which was shown to represent a single admixture), and lowest in the Lady Elliot Island population, possibly because of a recent population bottleneck during colonization. Mean band sharing between any two populations was less than the mean within either of those populations, and four fingerprint bands common to island birds were rare or absent in the fingerprints of mainland birds. In the absence of significant gene flow between the mainland and the islands, the populations have apparently become distinct at minisatellite loci, as evidenced by differences in both allelic diversity and in the frequencies of specific fragments. Within the Heron Island population, cohort analyses demonstrated the temporal stability of the fingerprint profile over 6 yr. This study demonstrates that length polymorphisms at minisatellite loci may be stable enough over time to retain information about recent historical and demographic effects on the relative genetic variability and differentiation of small, closely related populations.     

Epigenetic changes and transposon reactivation in Thai rice hybrids

Inter- or intraspecific hybridization is the first step in transferring exogenous traits to the germplasm of a recipient crop. One of the complicating factors is the occurrence of epigenetic modifications of the hybrids, which in turn can change their gene expression and phenotype. In this study we present an analysis of epigenome changes in rice hybrids that were obtained by crossing rice cultivars, most of them of indica type and Thai origin. Comparing amplified fragment length polymorphism (AFLP) fingerprints of twenty-four cultivars, we calculated Nei’s indexes for measuring genetic relationships. Epigenetic changes in their hybrids were established using methylation-sensitive AFLP fingerprinting and transposon display of the rice transposable elements (TEs) Stowaway Os-1 and Mashu, leading to the question whether the relationship between parental genomes is a predictor of epigenome changes, TE reactivation and changes in TE methylation. Our study now reveals that the genetic relationship between the parents and DNA methylation changes in their hybrids is not significantly correlated. Moreover, genetic distance correlates only weakly with Mashu reactivation, whereas it does not correlate with Stowaway Os-1 reactivation. Our observations also suggest that epigenome changes in the hybrids are localized events affecting specific chromosomal regions and transposons rather than affecting the genomic methylation landscape as a whole. The weak correlation between genetic distance and Mashu methylation and reactivation points at only limited influence of genetic background on the epigenetic status of the transposon. Our study further demonstrates that hybridizations between and among specific japonica and indica cultivars induce both genomic DNA methylation and reactivation/methylation change in the Stowaway Os-1 and Mashu transposons. The observed epigenetic changes seem to affect the transposons in a clear manner, partly driven by stochastic processes, which may account for a broader phenotypic plasticity of the hybrids. A better understanding of the epigenome changes leading to such transposon activation can lead to the development of novel tools for more variability in future rice breeding.     

Polymorphisms in grapevine DNA detected by the RAPD PCR technique

A sensitivie, reproducible technique is described for detecting polymorphisms between the nuclear DNA of grapevine isolates using the RAPD PCR technique. Unique fingerprints of a number of cultivars were readily distinguished by using either single primers or mixtures of two primers. The method will be used to provide a databank of fingerprints for the rapid identification of grapevine cultivars, and to develop phylogenetic relationships. It will also be extended and modified in an attempt to detect polymorphisms between DNAs of clonal selections of individual cultivars.     

Variability of multilocus DNA markers in populations of the Siberian (Capreolus pygargus Pall.) and European (C. capreolus L.) roe deer

Two types of nuclear DNA markers, M13 minisatellites and RAPD, were used to examine intraspecific and interspecific variation in closely related roe deer species, Capreolus capreolus L. and C. pygargus Pall. The roe deer populations studied were highly polymorphic for minisatellite DNA markers (S = 0.12-0.36). Heterozygosities of the RAPD loci were 0.185 (Russian Far East), 0.145, 0.131, 0.088 (Cis-Ural), and 0.06 (France). They correlated with karyotypic variation of B chromosomes (r = 0.975, P < 0.02; Spearman's correlation coefficient r = 1, P = 0.1 x 10(-5)), which indicated a contribution of microchromosomes to genetic variation of the species. The genetic distance D between the closely related species C. capreolus and C. pygargus was 50 times greater than the distance between populations within a species. The estimates of heterozygosity and genetic distance between local populations of Cis-Ural and the Far East suggest their specific spatial organization within this geographical range and reveal features of their historical development.     

Preparation of synthetic tandem-repetitive probes for DNA fingerprinting

DNA fingerprints are generated using probes that hybridize to hypervariable minisatellites, also known as variable number tandem repeat loci. Cloned minisatellites have served as the predominant source of DNA fingerprinting probes. A short segment within the repeat units of minisatellites, called the "core" sequence, is highly conserved within a family of related minisatellites, thereby allowing a single-cloned minisatellite to cross-hybridize to 20 to 40 other minisatellites. In this article, we describe a method for the synthetic preparation of polymeric core sequence probes for DNA fingerprinting. Unlike "monomeric" oligonucleotide probes, the polymeric probes mimic the tandem-repetitive structure of minisatellites, and thus each probe molecule can potentially form many sites of hybridization with a target minisatellite. The synthetic probes are cloned into plasmid DNA to provide a perpetual source of probe material.     

DNA Fingerprinting of Individual Species and Intergeneric and Interspecific Hybrids of the Genera Bos and Bison, Subfamily Bovinae

Genome fingerprinting with a hypervariable minisatellite sequence of phage M13 DNA was used to study the genetic variation in individual species of the genera Bosand Bison(subfamily Bovinae) and in their interspecific and intergeneric hybrids. DNA fingerprints were obtained for domestic cow Bos taurus primigenius, vatussy Bos taurus macroceros, banteng Bos javanicus, gaur Bos gaurus, wisent Bison bonasus, bison Bison bison, and for the interspecific and intergeneric hybrids. Compared with the original species, most hybrids showed a greater variation in number and size of hybridization fragments. An association was revealed between the number of hybridization fragments and degree of consanguinity of interspecific hybrids resulting from unique crossing of domestic cow and banteng. Pairwise similarity coefficients were calculated to construct a dendrogram of genetic similarity, which reflected the relationships between the parental species and hybrids varying in degree of consanguinity. The applicability of the method for identifying interspecific and intergeneric hybrids and for studying the consequences of hybridization in the subfamily Bovinae is discussed.     

Molecular diversity and relationships among <Emphasis Type="Italic">Cymbidium goeringii</Emphasis> cultivars based on inter-simple sequence repeat (ISSR) markers

Spring orchid (Cymbidium goeringii) is a popular flowering plant species. There have been few molecular studies of the genetic diversity and conservation genetics on this species. An assessment of the level of genetic diversity in cultivated spring orchid would facilitate development of the future germplasm conservation for cultivar improvement. In the present study, DNA markers of intersimple sequence repeats (ISSR) were identified and the ISSR fingerprinting technique was used to evaluate genetic diversity in C. goeringii cultivars. Twenty-five ISSR primers were selected to produce a total of 224 ISSR loci for evaluation of the genetic diversity. A wide genetic variation was found in the 50 tested cultivars with Nei’s gene diversity (H = 0.2241) and 93.75% of polymorphic loci. Fifty cultivars were unequivocally distinguished based on ISSR fingerprinting. Cultivar-specific ISSR markers were identified in seven of 50 tested cultivars. Unweighted pair-group mean analysis (UPGMA) and principal coordinates analysis (PCA) grouped them into two clusters: one composed the cultivars mainly from Japan, and the other contained three major subclusters mainly from China. Two Chinese subclusters were generally consistent with horticultural classification, and the third Chinese subcluster contained cultivars from various horticultural groups. Our results suggest that the ISSR technique provides a powerful tool for cultivar identification and establishment of genetic relationships of cultivars in C. goeringii.     

DNA fingerprinting of red clover (Trifolium pratense L.) with Jeffrey's probes: detection of somaclonal variation and other applications

Summary DNA fingerprints generated by the Jeffreys' probes, 33.6 and 33.15, indicated the presence of minisatellite-like sequences in the red clover genome. The fingerprints generated by probe 33.6 gave less background and fewer but better defined bands than those obtained with probe 33.15. Assay of a regenerative somaclonal variant (F49R) by DNA fingerprinting with probe 33.6 detected mutation that was unlinked to the regenerative trait. The fingerprints obtained under the applied conditions also demonstrated genetic stability of consecutive generations of the regenerants in tissue culture. DNA fingerprints of F₁ plants revealed that each polymorphic band was inherited from either one or the other parent. Both probes distinguished individual-specific genotypes in seven cultivars of red clover. Greater variability in DNA fingerprints was detected between (V=0.899) than within (0.417≤V≤0.548) cultivars.     

牛鞭草品种EST-SSR指纹图谱构建及遗传多样性分析

为探讨牛鞭草(Hemarthria spp.)品种间的遗传多样性,从86对EST-SSR引物中筛选出8对引物对牛鞭草属6个品种进行指纹图谱的构建及遗传多样性分析。结果表明,8对EST-SSR引物对牛鞭草属6个品种共扩增出193条清晰条带,多态性条带161条,多态性比例为83.4%。每条引物的多态信息含量(PIC)为0.480~0.695,平均为0.602。UPGMA聚类分析表明,牛鞭草属6个品种在相似系数为0.652处可分为两大类群。8对EST-SSR引物均能将6个品种完全区分开,以3对EST-SSR引物扩增的电泳图谱为基础,建立了牛鞭草属6个品种的指纹图谱标准模式图,每个品种都有唯一的指纹图谱。牛鞭草属6个品种的平均Nei’s基因多样性指数为0.333,平均Shannon信息指数为0.496,品种间的相似系数介于0.399~0.782之间。可见,牛鞭草属植物品种的遗传多样性较丰富,种间差异明显。     

Fingerprinting closely related xanthomonas pathovars with random nonamer oligonucleotide microarrays

Current bacterial DNA-typing methods are typically based on gel-based fingerprinting methods. As such, they access a limited complement of genetic information and many independent restriction enzymes or probes are required to achieve statistical rigor and confidence in the resulting pattern of DNA fragments. Furthermore, statistical comparison of gel-based fingerprints is complex and nonstandardized. To overcome these limitations of gel-based microbial DNA fingerprinting, we developed a prototype, 47-probe microarray consisting of randomly selected nonamer oligonucleotides. Custom image analysis algorithms and statistical tools were developed to automatically extract fingerprint profiles from microarray images. The prototype array and new image analysis algorithms were used to analyze 14 closely related Xanthomonas pathovars. Of the 47 probes on the prototype array, 10 had diagnostic value (based on a chi-squared test) and were used to construct statistically robust microarray fingerprints. Analysis of the microarray fingerprints showed clear differences between the 14 test organisms, including the separation of X. oryzae strains 43836 and 49072, which could not be resolved by traditional gel electrophoresis of REP-PCR amplification products. The proof-of-application study described here represents an important first step to high-resolution bacterial DNA fingerprinting with microarrays. The universal nature of the nonamer fingerprinting microarray and data analysis methods developed here also forms a basis for method standardization and application to the forensic identification of other closely related bacteria.