Mitochondrial genome size variation and restriction fragment length polymorphisms in threePhaseolus species

Summary Restriction patterns of mitochondrial DNA (mtDNA) from threePhaseolus species were examined to estimate their relative genome sizes and to determine the level of interspecific variability and relatedness. Three restriction endonucleases that produced relatively simple profiles were identified and used to determine the genome size of the three species. Taking into account fragment stoichiometries, the average estimates across enzymes were 456, 324, and 400 kb, respectively, forP. vulgaris, P. coccineus, andP. acutifolius. Restriction fragment length polymorphisms (RFLPs) differentiated the species when the mtDNAs were digested with seven endonucleases and hybridized with five cosmid clones covering ca. 200 kb of mtDNA sequences. Proportions of shared restriction fragments between every two species were computed as F-values and demonstrated thatP. vulgaris andP. coccineus are more related to each other than either is toP. acutifolius, and that the latter has a similar degree of relationship to the other two species.     

Some combinatorial problems of DNA restriction fragment length polymorphisms

Recombinant DNA techniques provide a means of defining new polymorphisms at the DNA sequence level. Polymorphisms arise when individuals differ in the location and number of sites where restriction endonucleases can cleave their DNA. Each such site exhibits two possible states: one for the presence of a specific endonuclease recognition sequence, the other for its absence. The states of a system of adjacent sites can be revealed experimentally by cleaving a person's DNA into a set of fragments. For experimentally well-understood systems of sites, we consider problems of counting numbers of possible fragments, haplotypes, genotypes, and phenotypes, and the means of resolving phenotype-genotype ambiguities. The degree of polymorphism generated by such systems and the importance to gene mapping are discussed.     

Characterization of the Citrus genome through analysis of restriction fragment length polymorphisms

Studies on the nature of restriction fragment length polymorphisms (RFLPs) were undertaken to characterize the Citrus genome. This type of analysis has not been carried out with any other perennial crop. Citrus reticulata Blanco cv Clementine, C. xparadisi Macf. cv Duncan, and an F₁ hybrid (LB 1–21) were used to determine what probe/enzyme combinations revealed polymorphisms in Southern analysis, and a backcross family (LB 1–21xClementine) of 65 randomly selected hybrid seedlings was used for some analyses. A majority (73%) of the clones examined from a PstI genomic library appeared to detect single-copy sequences based on RFLP banding patterns, while clones from a cDNA library revealed a lower percentage of single copy sequences. When hybridization stringencies were lowered, 21% of the genomic clones examined revealed greater copy numbers. PstI digestion of Duncan DNA indicated abundant methylation, so the relatively high frequency of multiple-copy sequences observed at moderate stringency cannot be attributed to a lack of methylation of the Citrus DNA. The polymorphisms in banding patterns observed primarily resulted from insertions and/or deletions rather than from base substitutions, and a model is presented to account for the varying patterns obtained from individual probes with different restriction enzymes. Finally, a model for transposon activity in Citrus is proposed, based on observations made during the course of these studies.     

Chromosome 13 restriction fragment length polymorphisms

Summary The gene locus for hereditary retinoblastoma is on human chromosome 13, band q14. With this gene localization in mind, we cloned DNA fragments from this chromosome. Three of the fragments identify restriction fragment length polymorphisms. These three fragments are from the region 13q12–13q22, the chromosome region which contains the retinoblastoma locus. We expect that these restriction fragment length polymorphisms will be linked to the retinoblastoma locus, and that they will serve in certain retinoblastoma families as predictors of retinoblastoma gene carriers.They will also be useful in studies of other gene loci thought to be on chromosome 13.This research was supported by grants from the National Institutes of Health HD04807, CA29883, and EY04543, by a grant from Fight for Sight, Inc., New York City, and by the Anna Fuller Fund     

DNA restriction fragment length polymorphisms in the rDNA repeat unit of Entomophaga

A heterologous rDNA probe was used to detect restriction fragment length polymorphisms in Entomophaga rDNA sequences. Six Canadian strains of Entomophaga aulicae, isolated from the spruce bud worm or hemlock looper in Ontario or Newfoundland, showed no detectable rDNA variation at 10 different restriction enzyme loci: BamHI, DraI, EcoRI, EcoRV, HindIII, HinfI, MspI, PstI, RsaI, or TaqI. A total of 14 isolates of E. aulicae representative of several different geographic and host origins were compared at the DraI and HindIII rDNA loci and two different banding patterns were detected. Of these, 12 showed the same patterns and were designated E. aulicae type 1. The two members which differed were designated E. aulicae type 2. The variations in rDNA restriction sites did not appear to be geographically dependent. Entomophaga maimaiga, a recently reclassified species from the E. aulicae complex, displayed an rDNA banding pattern clearly distinguishable from the E. aulicae patterns with DraI, EcoRI, EcoRV, or HindIII. Members of the E. grylli species complex exhibited patterns which clearly differed from the patterns seen with either E. aulicae or E. maimaiga isolates. However, members of the E. grylli species complex appeared to be more heterogeneous than those in the E. aulicae complex. Among four E. grylli members, three different rDNA banding patterns were detected with either HindIII or DraI. These were designated as E. grylli type 1, type 2, and type 3. An undesignated Entomophaga isolate from a dipteran host displayed rDNA polymorphisms not previously noted in either the lepidopteran or orthopteran isolates. Our results suggest that RFLPs in rDNA are useful in the delineation of genera and species within the Entomophthorales, but may not be as useful at lower taxonomic levels. These and other RFLPs can however provide useful information regarding the epidemiology of Entomophaga epizootics.     

Interspecific mitochondrial DNA restriction fragment length polymorphisms in Aspergillus section Flavi

Mitochondrial DNA restriction fragment length polymorphisms (RFLPs) are described for 64 isolates, representing 11 species of Aspergillus section Flavi. Mitochondrial DNA haplotypes were identified following digestion of total cellular DNA with the restriction enzymes HaeIII, AseI, or DraI. In general, isolates of the same species possessed identical mitochondrial DNA haplotypes. Three haplotypes were found in multiple, closely related species: one in A. flavus, A. oryzae, and A. subolivaceus; a second in A. parasiticus and A. sojae; and a third in A. tamarii and A. flavofurcatis. Four distinct haplotypes were each associated with a single species: A. nomius, A. avenaceus, A. leporis, and A. zonatus. Mitochondrial DNA haplotypes complement traditional morphological and growth criteria in making taxonomic decisions within Aspergillus section Flavi.     

Megabase scale restriction fragment length polymorphisms in the human major histocompatibility complex

Pulsed-field gel electrophoresis of human peripheral blood lymphocyte DNA was used to ascertain the extent of megabase restriction fragment length variation in the HLA region of human chromosome 6 and to determine whether previously reported diversity was due to experimental variation or DNA polymorphism. Polymorphism was found to predominate in the vicinity of the class II DR, class III complement, and the class I A genes and to be limited or absent near the class II DP genes, the TNF genes, and the class I B and C genes. Thus, the MHC region is characterized by both fine and large-scale structural diversity.     

Phylogenetic analysis of pines using ribosomal DNA restriction fragment length polymorphisms

Phylogenetic relationships among 30 species of the genusPinus were studied using restriction site polymorphism in the large subunit of nuclear rDNA. Of the 58 restriction sites scored, 48 were phylogenetically informative, and the 30 species reduced to ten taxa when species with identical restriction site patterns were combined. These ten taxa corresponded to the currently recognized subsections of the genus, with the sole exception ofP. leiophylla, which was identical in its pattern of restriction sites to all three species included from subsect.Oocarpae despite its being in a different section of subg.Pinus (Pinea instead ofPinus). A measure of the proportion of phylogenetic information contained within the data set (Homoplasy Excess Ratio, or HER) revealed that the character states were significantly non-randomly distributed among the ten taxa (HER = 0.71, p < 0.01). Branchand-bound searches using either Wagner or Dollo parsimony as the optimization criterion were carried out using PAUP in order to estimate phylogenetic relationships among the ten taxa. Three taxa (Picea pungens, Tsuga canadensis, andLarix decidua) were used independently as outgroups for purposes of rooting the trees. Despite the extreme differences in the assumptions underlying the Wagner and Dollo parsimony, the two gave surprisingly similar estimates of phylogeny, with both analyses supporting the monophyly of the two major subgeneraPinus andStrobus and differing in topology only in the placement of subsect.Ponderosae within subg.Pinus. The likelihood for the Wagner tree was only slightly higher than that computed for the Dollo tree.     

Chloroplast DNA restriction fragment length polymorphisms (RFLPs) in Musa species

Summary Taxonomic and phylogenetic determinations within the genus Musa are established using a numerical, morphology-based scoring system. However, within this system, the classification and relationships of some types are disputed. The application of chloroplast DNA (cpDNA) restriction fragment length polymorphism (RFLP) analysis to Musa taxonomy provided valuable, supplemental information about the classification of, and relationships between, Musa species and subspecies. Whole-cell DNA was extracted from lyophilized Musa leaf-blade tissue and digested with various restriction enzymes, Southern blotted onto nylon membranes, and probed using radioactively labeled heterologous orchid cpDNA fragments. Phylogenies were inferred from cpDNA RFLP patterns using PAUP software. The relationships between most species examined were as expected; however, some species (M. beccarii and M. basjoo) did not conform to the conventional morphology-based phylogeny.     

BglII restriction fragment length polymorphisms at the human p53 gene locus

Summary A new restriction fragment length polymorphism detected with the restriction endonuclease BglII (AGATCT) is presented.     

Restriction maps and restriction fragment length polymorphisms of the human 21-hydroxylase genes

Restriction maps were constructed for the two human 21-hydroxylase genes (21-OHA and 21-OHB) by using DNA from subjects homozygous for a deletion of each gene. Comparing the patterns of these two genes, a KpnI restriction site occurred in the 21-OHA gene in place of a TaqI site in the 21-OHB gene about 1-kb from the 5' end of the gene, and an extra EcoRI site was located 500 bp 5' to the common EcoRI site. The DNA of fourteen unrelated normal subjects was digested with nine restriction endonucleases (AccI, BamHI, BgIII, EcoRI, HindIII, KpnI, MspI, SacI and TaqI). Restriction fragment length polymorphisms were found with EcoRI, HindIII and AccI that resulted from polymorphic endonuclease sites outside the genes.     

Novel detection of restriction fragment length polymorphisms in the human major histocompatibility complex

Cosmid genomic DNA clones have been used as hybridization probes in genomic Southern blot analysis to define restriction fragment length polymorphisms (RFLPs) in the major histocompatibility complex (MHC). Using 14 different enzymes and three overlapping cosmid clones we have detected six RFLPs in a 100 kilobase (kb) segment of DNA in the class III region extending centromeric of theTNFA gene towardHLA-DR. Four of the five RFLPs, defined using the enzymesTaqI,Rsa I,Hinc II, andHind III, and detected by the cosmid clone cosM7B, map to a 29 kb segment of DNA that includes all of the recently described G2 (BAT2) gene and a large portion of the 3 end of the G3 (BAT3) gene. The different RFLP variants were established by analyzing the DNA from three informative families and a panel of 51HLA-homozygous typing cell lines. CosM7B detectsTaq I variants of 4.3 kb, and 2.9 kb or 2.8 kb, Rsa I variants of 2.9 kb or 2.4 kb,Hinc II variants of 5.8 kb or 3.8 kb and 1.4 kb, and aHind III variant of 4.8 kb, while cosOT2 detects Taq I variants of 4.5 kb or 4 kb. The distribution of theRsa 1, Hinc II and Taq I RFLPs detected by cosM7B, and theTaq I RFLP detected with cosOT2, within the panel of cell line DNAs was assessed by Southern blotting. The 4.3 kbTaq I variant was observed in only one cell line with the extended haplotypeHLA-A29, C-, B44, SC30, DR4. The other RFLPs, however, occurred much more frequently. The 2.8 kb Taq I variant was observed in 20 % of haplotypes, the 2.9 kbRsa I variant was observed in 42% of haplotypes, and the 5.8 kbHinc I variant was observed in 12 % of haplotypes analyzed. The 4.5 kbTaq I variant detected by the overlapping cosmid cosOT2 was present in 21 % of haplotypes. Analysis of the RFLP variants with each other revealed seven different haplotypic combinations. Three of the haplotypic combinations were each subdivided into two subsets on the basis of the Nco I RFLP variant they carried at theTNF-B locus. These haplotypic combinations potentially allow differentiation among different extended haplotypes such asHLA-B8, SC01, DR3, HLA-B18, F1 C30, DR3, andHLA-B44, FC31, DR7. The RFLPs detected by the cosmid clones thus provide new tools which will be useful in the further genetic analysis of the MHC class III region.     

Comparison of DNA restriction fragment length polymorphisms of Nostoc strains in and from cycads

DNA was prepared from cyanobacteria freshly isolated from coralloid roots of natural populations of five cycad species: Ceratozamia mexicana mexicana (Mexico), C. mexicana robusta (Mexico), Dioon spinulosum (Mexico), Zamia furfuraceae (Mexico) and Z. skinneri (Costa Rica). Using the Southern blot technique and cloned Anabaena PCC 7120 nifK and glnA genes as probes, restriction fragment length polymorphisms of these cyanobacterial symbionts were compared. The five cyanobacterial preparations showed differences in the sizes of their DNA fragments hybridizing with both probes, indicating that different cyanobacterial species and/or strains were in the symbiotic associations. On the other hand, a similar comparison of cyanobacteria freshly collected from a single Encephalartos altensteinii coralloid root and from three independently subcultured isolates from the same coralloid root revealed that these were likely to be one and the same organism. Moreover, the complexity of restriction patterns shows that a mixture of Nostoc strains can associate with a single cycad species although a single cyanobacterial strain can predominate in the root of a single cycad plant. Thus, a wide range of Nostoc strains appear to associate with the coralloid roots of cycads.Non-standard abbreviations bp base pairs - kbp kilobase pairs - RFLP's restriction fragment length polymorphisms     

Human restriction fragment length polymorphisms and cancer risk assessment

The polymorphic restriction fragments of the human Ha-ras locus, produced by the variable tandem repetition (VTR) of a short consensus sequence, fall into three classes based on allelic frequencies. Alleles of the "rare" class (individual frequencies less than 0.5%) have been detected only in white blood cell and tumor DNA of cancer patients. This phenomenon is independent of ethnic origin. No significant association of rare alleles with cancer patients has been demonstrated at an independent tandem repeat locus, VTR4.1. The results suggest that the Ha-ras restriction fragment length polymorphism is useful in cancer risk assessment.     

Intrageneric relationships of maple trees based on the chloroplast DNA restriction fragment length polymorphisms

A maple tree genus,Acer is the largest genus in broad-leaved deciduous trees and contains about 200 species. A delimitation of the genus is clear but the intrageneric classification was controversial because of homoplasies in morphological characters. In this study, a phylogenetic relationship inAcer was inferred based on chloroplast DNA restriction site polymorphisms with 17 restriction endonucleases and previously proposed intrageneric classifications were evaluated. The phylogenetic tree showed that (1) sectionsArguta, Cissifolia, Lithocarpa, Macrantha, Palmata, Spicata, Tataricum, Trifoliata sensu Ogata (1967: Bull. Tokyo Univ. Forests 63: 89–206) were monophyletic groups respectively, (2) sectionsCampestria, Goniocarpa, Platanoidea sensu Ogata (1967) were polyphyletic respectively, and (3) sectionsDistyla andParviflora formed a sister group. An average of estimated nucleotide substitution rates of Acer chloroplast DNA was calculated as 7.9×10⁻¹¹±1.4×10⁻¹¹ nucleotide substitutions par site par year, which coincides well with previously reported rates of perennial plants. Divergence eras of eastern Asia and North American species in both sectionsSpicata andRubra were estimated to be late Miocene. In consideration with previous data, multiple migrations and disjunctions are likely to have formed the eastern Asian and North American disjunct distribution.     

Linkage of restriction fragment length polymorphisms and isozymes in Citrus

Summary Genetic linkage analysis was performed using two segregating populations of citrus. One population arose from an intergeneric backcross of Citrus grandis (L.) Osb. cv Thong Dee and Poncirus trifoliata (L.) Raf. cv Pomeroy, using the former as the recurrent (female) parent. The other population came from an interspecific backcross of C. reticulata Blanco cv Clementine and C. x paradisi Macf. cv Duncan, using the former as the recurrent (male) parent. A total of 11 isozyme and 58 restriction fragment length polymorphisms were found to segregate in a monogenic fashion in one or both populations. Linkage analysis revealed that 62 of the loci examined mapped to 11 linkage groups, while 7 loci segregated independently from all other markers. Gene order was highly conserved between the maps generated from the two divergent segregating populations. Possible applications of the use of such maps in tree fruit breeding are discussed.     

DNA restriction fragment length polymorphisms correlate with isozyme diversity in Phaseolus vulgaris L.

Summary Genetic variation in Phaseolus vulgaris L. (P. vulgaris) was investigated at the isozyme and DNA levels. We constructed a library of size-selected Pst I clones of P. vulgaris nuclear DNA. Clones from this library were used to examine 14 P. vulgaris accessions for restriction fragment length polymorphisms (RFLPs). DNAs from each accession were analyzed with three restriction enzymes and 18 single copy probes. The same accessions were also examined for variability at 16 isozyme loci. Accessions included four representatives of the T phaseolin group and five representatives each of the C and S phaseolin groups. One member of the S group (the breeding line XR-235-1-1) was derived from a cross between P. vulgaris and P. coccineus. Isozymes and RFLPs revealed very similar patterns of genetic variation. Little variation was observed among accessions with C and T phaseolin types or among those with the S phaseolin type. However, both isozyme and RFLP data grouped accessions with S phaseolin separately from those accessions with C or T phaseolin. The highest degree of polymorphism was observed between XR-235-1-1 and members of the C/T group. RFLP markers will supplement isozymes, increasing the number of polymorphic loci that can be analyzed in breeding, genetic, and evolutionary studies of Phaseolus.