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     

Cloning, quantification and characterization of a minisatellite DNA sequence from common bean Phaseolus vulgaris L.

 We describe the cloning and the characterization of a 130-bp DNA fragment, called OPG9-130, amplified from bean (Phaseolus vulgaris L.) genomic DNA. This fragment corresponds to a minisatellite DNA sequence containing seven repeats of 15 bp which differ slightly from each other in their sequence. Southern analysis showed that the core sequence of 15 bp is repeated in clusters dispersed throughout the genome. The use of this fragment as a probe allowed us to identify common bean lines by their DNA fingerprints. We suggest that OPG9-130 will be useful for line identification as well as for the analysis of genetic relatedness between bean species and lines. Received: 14 February 1998 / Accepted: 10 February 1998     

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     

Repeat sequences from complex ds DNA viruses can be used as minisatellite probes for DNA fingerprinting

In a search for new fingerprinting probes for use with sheep, repeat sequences derived from five poxviruses, an iridovirus and a baculovirus were screened against DNA from sheep pedigrees. Probes constructed from portions of the parapox viruses, orf virus and papular stomatitis virus and the baculovirus from the alfalfa looper, Autographa californica, nuclear polyhedrosis virus all gave fingerprint patterns. Probes from three other poxviruses and an iridovirus did not give useful banding patterns.     

Low-Cot DNA sequences for fingerprinting analysis of germplasm diversity and relationships in Amaranthus

We examined genetic diversity and relationships among 24 cultivated and wild Amaranthus accessions using the total low-Cot DNA and five individual repetitive sequences as probes. These low-Cot DNA probes were obtained by the isolation of various classes of repetitive-DNA sequences, including satellites, minisatellites, microsatellites, rDNA, retrotransposon-like sequences, and other unidentified novel repetitive sequences. DNA fingerprints generated by different types of repetitive-DNA probes revealed different levels of polymorphism in the Amaranthus genomes. A repetitive sequence containing microsatellites was found to be a suitable probe for characterizing intraspecific accessions, whereas more conservative sequences (e.g. rDNA) were informative for resolving phylogenetic relationships among distantly related species.Genetic diversity, measured as restriction fragment length polymorphism (RFLP) and the similarity index at the low-Cot DNA level, was equally high among intraspecific accessions between the two species groups: grain amaranths (A. caudatus, A. cruentus, and A. hypochondriacus) and their putative wild progenitors (A. hybridus, A. powellii, and A. quitensis). At the interspecific level, however, the grain amaranth species are less divergent from each other than their wild progenitors. With the rare exceptions of certain A. caudatus accessions, grain amaranths were found to be closely related to A. hybridus. The results based on low-Cot DNA were comparable with previous RAPD and isozyme studies of the same set of species/accessions of Amaranthus, indicating that low-Cot DNA sequences are suitable probes for a fingerprinting analysis of plant germplasm diversity and for determining phylogenetic relationships. Received: 19 October 1998 / Accepted: 8 January 1999     

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     

DNA profiling of cattle using micro- and minisatellite core probes

We have evaluated 15 different micro- and minisatellite core probes for use in identity and paternity testing in cattle, based on Southern blot hybridization analysis. The core probes were tested in animals of different breeds and by analysis of seven two-generation pedigrees. Of the 15 core probes tested, seven were able to detect on average seven variant bands per individual animal. Segregation analysis showed that on average two out of 3 6 variant bands scored per core probe were genetically linked while two out of 12 variant bands correspond to the same allelic pair. The results obtained demonstrate the effectiveness of multilocus core probes for determining identity and paternity in cattle.     

PCR primed with VNTR core sequences yields species specific patterns and hypervariable probes.

The use of genomic DNA-based techniques in ecological and evolutionary studies has been limited by the availability of suitable probes for species of interest due to the technical difficulty of isolating and applying such probes. We have developed a simple technique that directs polymerase chain reaction (PCR) amplification to regions rich in variable number of tandem repeats (VNTRs). By using published VNTR core sequences as primers in PCRs, fragments were amplified that showed little variation within a species, but did show differences between species. When the amplified fragments were used as probes with genomic DNA Southern blots they produced hypervariable single-locus or few-locus patterns in fish, birds, and humans. We have named this procedure as Directed Amplification of Minisatellite-region DNA (DAMD).     

DNA fingerprinting in rice using oligonucleotide probes specific for simple repetitive DNA sequences

In this report we describe the use of five oligonucleotide probes, namely (GATA)₄, (GACA)₄, (GGAT)₄, (GAA)₆ and (CAC)₅, to reveal highly polymorphic DNA regions in rice. With each of the oligonucleotide probes, the level of polymorphism was high enough to distinguish several rice genotypes. Moreover, individual plants of one cultivar showed the same cultivar-specific DNA fingerprint. The multilocus fingerprint patterns were somatically stable. Our study demonstrates that microsatellite-derived DNA fingerprints are ideally suited for the identification of rice genotypes. As the majority of the probes detected a high level of polymorphism, they can be very useful in monitoring and aiding gene introgression from wild rice into cultivars.     

DNA fingerprinting of Indian isolates of Xanthomonas oryzae pv. oryzae

 A high level of genetic polymorphism was detected among Indian isolates of Xanthomonas oryzae pv. oryzae using hypervariable probes such as a microsatellite oligonucleotide, probe (TG)₁₀, a human minisatellite probe, pV47, an avirulence gene probe, avrXa10 and a repeat clone, pBS101. These DNA probes detected multiple loci in the bacterial genome generating complex DNA fingerprints and differentiated all of the bacterial isolates. Analysis of fingerprints indicated that pV47, (TG)₁₀ and pBS101 have a lower probability of identical match than avrXa10 and therefore are potential probes for DNA fingerprinting and variability analysis of Xanthomonas oryzae pv. oryzae pathogen populations. Cluster analysis based on hybridization patterns using all of the above probes showed five groups at 56% similarity. Studies on the methylation patterns of isolates representing the three important races of X. oryzae pv. oryzae indicated more methylation in the most virulent isolate, suggesting a possible role of methylation in pathogenicity. Received: 8 December 1996 / Accepted: 20 December 1996     

A new system of comparing PCR primers applied to ISSR fingerprinting of potato cultivars

 Commercial scale fingerprinting of potato cultivars is made difficult by the need for speed, reliability and the ability to distinguish between large numbers of genotypes. There are also problems in extrapolating the results of small experimental studies to predict the performance of techniques or primers for larger applications. The potential of ISSR-PCR for fingerprinting purposes was evaluated using four primers on 34 potato cultivars. The complex band profiles generated were reproducible between repeat PCRs, DNA extractions, electrophoreses and gel scorings. Two primers were each able to distinguish all cultivars. The combined use of any two of the four primers also allowed complete diagnosis. It is concluded that ISSR-PCR provides a quick, reliable and highly informative system for DNA fingerprinting that is amenable for routine applications. Two possible correlates of the ability of primers to distinguish between genotypes were then examined. Marker Index failed to correlate significantly with genotype diagnosis, but a strong and seemingly linear relationship was observed between Resolving Power of a primer and its ability to distinguish genotypes (r²=0.98). Resolving Power of one or a pair of primers was found to provide a moderately accurate estimate of the number of genotypes identified. Possible implications for future studies on DNA fingerprinting are discussed. Received: 7 May 1998 / Accepted: 15 July 1998     

Low levels of DNA sequence variation among adapted genotypes of hexaploid wheat

 PCR products from regions corresponding to sequences hybridising to wheat RFLP probes were sequenced in order to establish the level of DNA sequence variation among adapted wheat genotypes. Hexaploid bread wheat shows a very low rate of nucleotide polymorphism, approximately 1 polymorphic nucleotide per 1000 basepairs. Differences in PCR product length can be exploited to design genome-specific amplicons, which may have use in gene tagging or in diagnostic applications. Interpretation of results may be complicated by the simultaneous amplification of orthologous and paralogous sequences. These findings have significant implications for the use of STS markers in wheat and other polyploid species. Received: 3 October 1998 / Accepted: 28 November 1998     

The retrotransposon RTip1 is integrated into a novel type of minisatellite, MiniSip1, in the genome of the common morning glory and carries another new type of minisatellite, MiniSip2

 Transposable elements have often been discovered as new insertion sequences in known genes, and minisatellites are often employed as molecular markers in diagnostic and mapping studies. We compared the genes for flower pigmentation in a line of the common morning glory bearing fully colored flowers with those in two anthocyanin ^flaked mutable lines producing variegated flowers and found RFLPs at the region of the ANS gene for anthocyanin biosynthesis. The DNA rearrangements detected by the RFLPs are due to integration of a novel type of minisatellite, MiniSip1, having a long LTR retrotransposon, RTip1, inserted in the mutable lines. The structural analysis of the rearranged region revealed that the 12.4-kb RTip1 element is flanked by 5-bp target duplications within the MiniSip1 sequence and contains two LTR sequences of about 590 bp, a primer binding site for tRNA^Lys, a typical polypurine tract and another new type of minisatellite, MiniSip2. Since no long open reading frame corresponding to the gag and pol genes was found, RTip1 appears to be a defective Ty3/gypsy-like element. Interestingly, the 269-bp-long MiniSip1 element comprises two alternating motifs of 41 bp and 19 bp, whereas the 962 bp long MiniSip2 element consists of two partially alternating motifs of 86 bp and 90 bp which are partially homologous to each other. Possible evolutionary processes that may have generated the rearranged structure at the ANS gene region are also discussed. Received: 25 April 1997 / Accepted: 16 May 1997     

PCR primed with minisatellite core sequences yields species-specific patterns and assessment of population variability in fishes of the genus Brycon

Minisatellite core sequences were used as single primers in polymerase chain reaction (PCR) to amplify genomic DNA in a way similar to the random amplified polymorphic DNA methodology. This technique, known as Directed Amplification of Minisatellite‐region DNA, was applied in order to differentiate three neotropical fish species (Brycon orbignyanus, B. microlepis and B. lundii) and to detect possible genetic variations among samples of the threatened species, B. lundii, collected in two regions with distinct environmental conditions in the area of influence of a hydroelectric dam. Most primers generated species‐specific banding patterns and high levels of intraspecific polymorphism. The genetic variation observed between the two sampling regions of B. lundii was also high enough to suggest the presence of distinct stocks of this species along the same river basin. The results demonstrated that minisatellite core sequences are potentially useful as single primers in PCR to assist in species and population identification. The observed genetic stock differentiation in B. lundii associated with ecological and demographic data constitute a crucial task to develop efficient conservation strategies in order to preserve the genetic diversity of this endangered fish species.     

Identification of DNA amplification fingerprinting (DAF) markers close to the symbiosis-ineffective sym31 mutation of pea (Pisum sativum L.)

 We demonstrate efficient genome mapping through a combination of bulked segregant analysis (BSA) with DNA amplification fingerprinting (DAF). Two sets of 64 octamer DAF primers, along with two PCR programs of low- and high-annealing temperatures (30°C and 55°C, respectively), appeared to be enough to locate molecular markers within 2–5 cM of a gene of interest. This approach allowed the rapid identification of four BSA markers linked to the pea (Pisum sativum L.) Sym31 gene, which is responsible for bacteroid and symbiosome differentiation. Three of these markers are shown to be tightly linked to the sym31 mutation. Two markers flanking the Sym31 gene, A21-310 and B1-277, cover a 4–5 cM interval of pea linkage group 3. Both markers were converted to sequence-characterized amplified regions (SCARs). The flanking markers may be potential tools for marker-assisted selection or for positional cloning of the Sym31 gene. Received: 2 July 1998 / Accepted: 8 October 1998     

Mapping of RFLP probes characterized for their polymorphism on wheat

 Wheat anonymous probes were selected for their efficiency for providing a readable hybridization pattern and revealing RFLP among wheat varieties. We report the mapping of 132 such probes (20 wheat-leaf cDNA, 28 wheat-root cDNA and 84 genomic DNA) on the reference population of the International Triticeae Mapping Initiative (ITMI) derived from the cross W-7984 with Opata85. Each probe has been characterized for its polymorphism information content. The 132 probes allowed us to map 160 loci. Received: 7 July 1998 / Accepted: 19 October 1998     

Single-stranded DNA in the genetic transformation of wheat (Triticum aestivum L.): transformation frequency and integration pattern

Two non-linked marker genes (gus and bar) were co-introduced by microprojectile bombardment into wheat cells. Four different DNA structures were compared with respect to ability to integrate into the wheat genome: circular or linear (l) DNA as a single- or double-stranded plasmid (ss and ds, respectively). In eight independent experiments, linearized DNA integrated in the ds or ss form with a high efficiency of up to 14% for l-ssDNA. Molecular analyses by Southern blotting showed that all DNA forms gave a similar complicated integration pattern of the bar gene. Received: 20 July 1998 / Accepted: 30 January 1999     

Amplified fragment length polymorphisms as a tool for DNA fingerprinting sunflower germplasm: genetic diversity among oilseed inbred lines

 Amplified fragment length polymorphism (AFLP) analysis is a rapid and efficient method for producing DNA fingerprints. The AFLP diversity of sunflower has not been described, and much of the public germ plasm of sunflower has not yet been fingerprinted. Our objectives were to: (1) estimate genetic similarities, polymorphism rates, and polymorphic information contents (PICs) for AFLP markers among elite public oilseed inbred lines, and (2) assess the genetic diversity of inbred lines using genetic similarities estimated from AFLP fingerprints. We produced fingerprints for 24 public inbred lines of sunflower (Helianthus annuus L.) using six AFLP primer combinations. These primers produced a total of 359 AFLP markers or about 60 markers per primer combination. Genetic similarities ranged from 0.70 to 0.91, polymorphism rates ranged from 7 to 24%, and PICs ranged from 0.0 to 0.5. Genetic similarities were lower overall for maintainer (B)×restorer (R) crosses than for B×B or R×R crosses. Principal-coordinate and cluster analyses separated lines into two groups, one for B-lines and another for R-lines. These groupings illustrate the breeding history and basic heterotic pattern (B×R) of sunflower and the widespread practice of using B×B and R×R crosses to develop new lines. There were, nevertheless, distinct subgroups within these groups. These subgroups may represent unique heterotic groups and create a basis for formally describing heterotic patterns in sunflower. Received: 10 June 1996 / Accepted: 4 April 1997     

Repetitive plasmid sequences generate DNA fingerprinting patterns in mammals

Bacterial plasmids with stringently regulated copy numbers have directly repeated DNA sequences, termed iterons, in the vicinity of their replication origins. These sequences bind a specific protein exerting a key role in the initiation of plasmid replication. Plasmids P1, pSC101 and RFS1010 have different iteron sequences and belong to three different incompatibility groups. Used as DNA probes each of these plasmids generates specific patterns in mammals similar to those obtained by the DNA fingerprinting technique. The iteron-containing regions were identified as the part of the plasmids responsible for those patterns by using polymerase chain reaction (PCR) amplified DNA segments that contained the iteron regions as probes.     

Mammalian species identification by interspersed repeat PCR fingerprinting

Most DNA methods for species identification of animal tissues test the presence/absence of one species per assay, requiring several tests for a complete analysis and prior knowledge of the species that are potentially present in the sample. Here we demonstrate that PCR with fluorescently labeled MIR (mammalian-wide interspersed repeat) primers generate fingerprints that are suitable for rapid identification of known and unknown species on an automatic sequencing apparatus and with computer-assisted data processing. The method allows the analysis of processed meat samples and offers a convenient alternative to sequencing of mitochondrial DNA. Received 19 December 1997/ Accepted in revised form 15 June 1998