Tobacco single-copy DNA is highly homologous to sequences present in the genomes of its diploid progenitors

Summary We compared the single-copy DNA sequences of the tetraploid tobacco plant, Nicotiana tabacum, with those of its diploid progenitors N. sylvestris and N. tomentosiformis. We observed that 65% of N. sylvestris and N. tomentosiformis single-copy DNA fragments reacted with each other using moderately stringent hybridization conditions (60° C, 0.18 M Na⁺). An additional 10% sequence homology was detected when the hybridization temperature was reduced by 10° C. The thermal stability of interspecific single-copy DNA duplexes indicated that they were approximately 6% more mispaired than homologous single-copy DNA duplexes. In contrast, we observed almost no single-copy DNA divergence between N. tabacum and its diploid progenitors. Greater than 99% of N. sylvestris and N. tomentosiformis single-copy DNAs reacted with N. tabacum DNA using moderately stringent hybridization conditions. The thermal stability of these duplexes indicated that they contained no more sequence mismatch than homologous single-copy duplexes. Together, our results show that significant single-copy DNA sequence divergence has occurred between the diploid N. sylvestris and N. tomentosiformis genomes. However, by applying our experimental criteria these single-copy DNAs are indistinguishable from their counterparts in the hybrid N. tabacum nucleus.     

Spatial analysis of nuclear and mitochondrial RFLP genotypes in populations of the chestnut blight fungus, Cryphonectria parasitica

Spatial structure of both nuclear and mitochondrial RFLPs were studied in several populations of the chestnut blight fungus, Cryphonectria parasitica, using a variety of spatial autocorrelation tests designed to detect nonrandom patterns. Fungal individuals were sampled from cankers on infected chestnut trees, and the location of each tree was mapped. Single-locus nuclear RFLPs, nuclear fingerprints, and mitochondrial DNA haplotypes were determined for each individual. Individuals with the same DNA fingerprint genotypes occurred closer together than would be expected at random in four of the five plots, while mitochondrial DNA haplotypes were aggregated in all five plots. Genetic distances between individuals, expressed as one minus the proportion of shared restriction fragment size classes for fingerprints and mitochondrial haplotypes, were significantly correlated with Euclidean distances between individuals in four of the five populations, but these correlations were very weak (r < 0.18). The same DNA fingerprint and single-copy nuclear RFLP alleles occurred on the same trees or immediately neighbouring trees more often than would be expected at random. Most of the aggregation for all three genetic markers occurred among individuals within the same cluster of chestnut stems or on neighbouring trees. Lack of spatial autocorrelation in one population was probably due to sampling on a larger scale that was too coarse to detect any patterns. Significant aggregation of genotypes in C. parasitica is most likely caused by some degree of restricted dispersal within populations. The implications of restricted dispersal are discussed in relation to the breeding system and isolation by distance in populations of. C. parasitica.     

Construction of the physical map of the chloroplast DNA of Phaseolus vulgaris and localization of ribosomal and transfer RNA genes

Construction of a physical map of the chloroplast DNA from Phaseolus vulgaris showed that this circular molecule is segmentally organized into four regions. Unlike other chloroplast DNAs which have analogous organization, two single-copy regions that separate two inverted repeats have been demonstrated to exist in both relative orientations, giving rise to two populations of DNA molecules.Hybridization studies using individual rRNA and tRNA species revealed the location of a set of rRNA genes and at least seven tRNA genes in each inverted repeat region, a minimum of 17 tRNA genes in the large single-copy region and one tRNA gene in the small single-copy region. The tRNA genes code for 24 tRNA species corresponding to 16 amino acids. Comparison of this gene map with those of other chloroplast DNAs suggests that DNA sequence rearrangements, involving some tRNA genes, have occurred.     

DNA Sequence Comparison among Closely Related Drosophila Species in the MULLERI Complex

DNA hybridization was used to establish DNA sequence relationships among seven Drosophila species. Single-copy DNA was isolated from four species within the Drosophila mulleri complex, D. mojavensis, D. arizonensis, D. ritae and D. starmeri. These single-copy DNAs were used as tracers to be hybridized with each other and one additional member of the mulleri complex, D. aldrichi, a member of a closely related complex, D. hydei, and a distantly related species, D. melanogaster. Two methods have been used to determine the relatedness between these species: (1) the extent of duplex formed as measured by binding to hydroxyapatite and (2) the thermal stability of the duplexed DNA. Moderately repetitive DNA was purified from these species and used similarly to determine the divergence of this family of sequences. The rate of nucleotide substitution was estimated to be 0.2 +/-, 0.1% base pair change per million years for both single-copy and middle-repetitive DNAs. The size of the D. arizonensis genome, a representative of the mulleri complex, was calculated to be 2.2 X 10(8) base pairs from its kinetic complexity similar to that of D. hydei. The relative amounts (18%) and average reiteration frequency (100 copies) of the middle-repetitive DNA are similar for all Drosophila species studied. Finally, the data are presented in a phylogenetic tree.     

Serologic and immunochromatographic detection of oxygenated polyenoic acids in Euglena gracilis var. bacillaris

The extent of evolutionary conservation of DNA complimentary to RNA stored in the mature oocyte of the sea urchin S. purpuratus has been assessed. To do this, such DNA was hybridized with total genomic DNA of S. purpuratus and S. franciscanus and the thermal stability of the resultant duplexes was measured by two methods. In the first method, the duplexes were bound to hydroxylapatite and thermally eluted; the difference in thermal stability between homologous and heterologous duplexes averaged 6.9 degrees C in duplicate determinations. In the second experiment, the same hybrids were thermally melted in 2.4M tetraethylammonium chloride, then assayed with S1 nuclease; the difference in thermal stability of homologous and heterologous duplexes was 4.8 degrees C. Either value is significantly lower than the divergence of total single-copy DNA among these species as measured by the same techniques. This demonstrates that DNA sequences complimentary to maternal RNA are conserved during evolution, and thus that a high fraction of them are likely to be physiologically functional.     

Nuclear DNA sequences from late Pleistocene megafauna

We report the retrieval and characterization of multi- and single-copy nuclear DNA sequences from Alaskan and Siberian mammoths (Mammuthus primigenius). In addition, a nuclear copy of a mitochondrial gene was recovered. Furthermore, a 13,000-year-old ground sloth and a 33,000- year-old cave bear yielded multicopy nuclear DNA sequences. Thus, multicopy and single-copy genes can be analyzed from Pleistocene faunal remains. The results also show that under some circumstances, nucleotide sequence differences between alleles found within one individual can be distinguished from DNA sequence variation caused by postmortem DNA damage. The nuclear sequences retrieved from the mammoths suggest that mammoths were more similar to Asian elephants than to African elephants.      

Evidence that populations of Dictyostelium single-copy mRNA transcripts carry common repeat sequences.

Two recombinant plasmids, M4 and KH10, carrying Dictyostelium DNA inserted into the Eco RI restriction endonuclease site of pMB9 by poly(dA)-poly(dT) tailing, were selected for study because they are complementary to abundant mRNA populations from Dictyostelium. Both plasmids have been shown to hybridize a heterogeneous size class of mRNAs which, in the case of KH10, comprise 5-10% of the pulse-labeled poly(A)+ RNA from vegetative cells. Analysis of the sequence organization of the two pieces of Dictyostelium DNA shows that they consist mostly of single-copy sequences with a short DNA sequence which is repeated in the genome and interspersed with single-copy DNA. These and other results suggest that the majority of the hybridization of pulse-labeled mRNA to M4 and KH10 is to the short "repeated" DNA sequences. In the genome, members of these repeat families appear to be transcribed onto a population of different single-copy mRNAs. Additional results show that M4 DNA contains a sequence which is entirely complementary to a discrete mRNA.     

A study of the evolution of repeated DNA sequences in primates and the existence of a new class of repetitive sequences in primates.

A new class of lowly repetitive DNA sequences has been detected in the primate genome. The renaturation rate of this sequence class is practically indistinguishable from the renaturation rate of single-copy sequences. Consequently, this lowly repetitive sequence class has not been previously observed in DNA renaturation rate studies. This new sequence class is significant in that it might occupy a major fraction of the primate genome.Based on a study of the thermal stabilities of DNA heteroduplexes constructed from human DNA and either bonnet monkey or galago DNAs, we are able to compare the relative mutation rates of repetitive and single-copy sequences in the primate genome. We find that the mutation rate of short, interspersed repetitive sequences is either less than or approximately equal to the mutation rate of single-copy sequences. This implies that the base sequence of these repetitive sequences is important to their biological function.We also find that numerous mutations have accumulated in interspersed repeated sequences since the divergence of galago and human. These mutations are only recognizable because they occur at specific sites in the repeated sequence rather than at random sites in the sequence. Although interspersed repetitive sequences from human and galago can readily cross-hybridize, these site-specific mutations identify them as being two distinct classes. In contrast, far fewer site-specific mutations have occurred since the divergence of human and monkey.     

Population variation of human mtDNA control region sequences detected by enzymatic amplification and sequence-specific oligonucleotide probes.

A method for detecting sequence variation of hypervariable segments of the mtDNA control region was developed. The technique uses hybridization of sequence-specific oligonucleotide (SSO) probes to DNA sequences that have been amplified by PCR. The nucleotide sequences of the two hypervariable segments of the mtDNA control region from 52 individuals were determined; these sequences were then used to define nine regions suitable for SSO typing. A total of 23 SSO probes were used to detect sequence variants at these nine regions in 525 individuals from five ethnic groups (African, Asian, Caucasian, Japanese, and Mexican). The SSO typing revealed an enormous amount of variability, with 274 mtDNA types observed among these 525 individuals and with diversity values, for each population, exceeding .95. For each of the nine mtDNA regions significant differences in the frequencies of sequence variants were observed between these five populations. The mtDNA SSO-typing system was successfully applied to a case involving individual identification of skeletal remains; the probability of a random match was approximately 0.7%. The potential useful applications of this mtDNA SSO-typing system thus include the analysis of individual identity as well as population genetic studies.     

Evolution of thermal physiology and growth rate between populations of the western fence lizard (Sceloporus occidentalis)

Summary Hatchling Sceloporus occidentalis from northern populations (central Oregon) grow more slowly than hatchlings from southern populations (southern California) in nature. In this study, I determine whether this difference in growth rate results from differences in thermal environment and/or in thermoregulatory behavior. To determine the degree to which the thermal environment affects growth rate among populations, I reared hatchings from the northern and southern populations in a cycling thermal regime in one of three experimental treatments differing in access to radiant heat (6, 9, or 12 h radiant heat; remainder of 24 h at 15°C). I also measured the body temperature that each individual voluntarily selected over the course of the daily activity cycle. Growth rate varied positively with duration of access to radiant heat. Within the three treatments, individual growth rate was positively correlated with body temperature. Moreover, the difference in growth rate between the northern and southern populations was due in part to differences in behavior — individuals from northern populations selected lower body temperatures. I found that significant variation in body temperature was associated with family membership, suggesting that thermal physiology has a genetic basis. Moreover, growth rate was correlated with body temperature among families in each population suggesting a genetic correlation underlies the phenotypic correlations. Thus, genetically based variation in thermal physiology contributes to differences in growth rate among individuals within a population as well as to differences among populations.     

DNA DIVERGENCE AMONG HOMINOIDS

We have determined the degree of single-copy DNA divergence among the extant members of the Hominoidea employing the technique of DNA-DNA hybridization. The species studied include humans, two species of chimpanzees, gorillas, two subspecies of orangutans, and two species of gibbons; as an outgroup we have used a member of the Old World monkeys (Cercopithecidae), the baboon. Our methods are different from those previously used and allow us to control for two factors other than base-pair mismatch that can affect the thermal stability of DNA duplexes: the base composition and duplex length. In addition, we have studied more than one individual for most species and thus are able to assess the effect of intraspecific variation on phylogenetic conclusions. The results indicate that the closest extant relatives of humans are the chimpanzees. Gorillas are the next closest, followed by orangutans and gibbons. This result is strongly supported statistically, as there is virtually no overlap in measurements between different taxa. Our conclusions are in agreement with a growing amount of molecular evidence supporting this pattern of relatedness. The data behave as a reasonably good molecular clock, and we do not see an indication of slowdown in molecular evolution in the clade containing humans and African apes, contrary to what has been documented for protein-coding regions. Because of the clocklike nature of the results, we have estimated that the divergence of humans and chimpanzees occurred about 6–8 million years ago. Results from orangutans indicate that the Borneo and Sumatra populations are genetically distinct, about as different as the named species of chimpanzees.     

Genetic variation of Hoplolaimus columbus populations in the United States using PCR-RFLP analysis of nuclear rDNA ITS regions

Hoplolaimus columbus is an important nematode pest which causes economic loss of crops including corn, cotton, and soybean in the Southeastern United States. DNA sequences of the ITS1-5.8S-ITS2 region of ribosomal DNA from H. columbus were aligned and analyzed to characterize intraspecific genetic variation between eleven populations collected from Georgia, Louisiana, North Carolina, and South Carolina. In comparative sequence analysis with clones from either one or two individuals obtained from the eleven populations, we found variability existed among clones from an individual and that clonal diversity observed from within individuals was verified by PCR-RFLP. PCR-RFLP analysis with Rsa I and Msp I restriction enzymes yielded several fragments on 3.0% agarose gel that corresponded to different haplotypes in all populations and the sum of digested products exceeded the length of undigested PCR products, which revealed that ITS heterogeneity existed in a genome of H. columbus. This indicates that heterogeneity may play a role in the evolution of this parthenogenetic species.     

Clonal structure of the introduced freshwater snail Potamopyrgus antipodarum (Prosobranchia: Hydrobiidae), as revealed by DNA fingerprinting.

Multi-locus DNA fingerprints were obtained from individuals of the hydrobiid snail, Potamopyrgus antipodarum (= P. jenkinsi), by using an RNA derivative (pSPT 18.15) of Jeffrey's 33.15 minisatellite core sequence. Whole-body homogenization of snails yielded 3.21 +/- 0.09 micrograms DNA per individual, producing complex profiles comprising 12-22 fragments within the 1.0-20.0 kilobase (kb) size range. Fingerprints from natural and experimental populations identified three distinct clonal genotypes corresponding to morphological strains A, B and C, with only rare mutational variants. Mother-offspring comparisons of genetic fingerprints revealed genetic stability during apomictic parthenogenesis. Data support the notion that British populations of P. antipodarum comprise three widespread obligate parthenogenetic clones resulting from a mid-19th Century introduction from Australasia. The present-day low levels of genotypic diversity are discussed in relation to the typical occurrence of P. antipodarum in man-made or immature habitats.     

Numerous group I introns with variable distributions in the ribosomal DNA of a lichen fungus.

The length of the small subunit ribosomal DNA (SSU rDNA) differs significantly among individuals from natural populations of the ascomycetous lichen complex Cladonia chlorophaea. The sequence of the 3' region of the SSU rDNA from two individuals, chosen to represent the shortest and longest sequences, revealed multiple insertions within a region that otherwise aligned with a 520-nucleotide sequence of the SSU rDNA in Saccharomyces cerevisiae. The high degree of variability in SSU rDNA size can be accounted for by different numbers of insertions; one individual had two group I introns and the second had five introns, two of which were clearly related to introns at identical positions in the other individual. Yet, introns in different positions, whether within an individual or between individuals, were not similar in sequence. The distribution of introns at three of the positions is consistent with either intron loss or acquisition, and clearly indicates the dynamic variability in this region of the nuclear genome. All seven insertions, which ranged in size from 210 to 228 nucleotides, had the conserved sequence and secondary structural elements of group I introns. The variation in distribution and sequence of group I introns within a short highly conserved region of rDNA presents a unique opportunity for examining the molecular evolution and mobility of group I introns within a systematics framework.     

Consistent geographic structure among multiple nuclear sequences and cpDNA polymorphisms of Cardamine nipponica Franch. et Savat. (Brassicaceae)

Molecular phylogeography has inferred the history of differentiation between regions and/or among populations following the Pleistocene climatic oscillations, mostly based on the genetic structure of organelle DNA. However, such genetic structure only reflects the history of a single gene, and studies based on single-copy genes of nuclear DNA (nDNA) are required for phylogeography, although their efficiency remains unclear. To examine the utility of nDNA loci, the genetic structures of three genes from Cardamine nipponica, which is closely related to the model species Arabidopsis thaliana, were elucidated: the nDNA genes DET1, PHYA, PHYE, as well as chloroplast DNA (cpDNA). In 279 individuals collected from throughout the range of the species, strong genetic differentiation between northern and central Japan was found for all loci. This result suggested that populations in central Japan experienced a different history from those in northern Japan during the Pleistocene climatic oscillations. In addition, the evidence of refugia at the edges of the distribution, where the genetic structure was less influenced by colonization following range expansion, was shown for several loci. The specific genetic structure within the southernmost populations of northern Japan suggested that this region was also isolated during range expansion. Hence, the consistent history among loci and a more detailed history from several loci indicated that cpDNA can represent the history of vicariance and demonstrated the efficiency of single-copy nuclear genes in phylogeography.     

Mitochondrial phylogeography of the Eurasian beaver Castor fiber L

Nucleotide variation in an approximately 490 bp fragment of the mitochondrial DNA control region (mtDNA CR) was used to describe the genetic variation and phylogeographical pattern in the Eurasian beaver (Castor fiber) over its entire range. The sampling effort was focused on the relict populations that survived a drastic population bottleneck, caused by overhunting, at the end of the 19th century. A total of 152 individuals grouped into eight populations representing all currently recognized subspecies were studied. Sixteen haplotypes were detected, none of them shared among populations. Intrapopulation sequence variation was very low, most likely a result of the severe bottleneck. Extreme genetic structure could result from human-mediated extinction of intermediate populations, but it could also be an effect of prior substantial structuring of the beaver populations with watersheds of major Eurasian rivers acting as barriers to gene flow. Phylogenetic analysis revealed the presence of two mtDNA lineages: eastern (Poland, Lithuania, Russia and Mongolia) and western (Germany, Norway and France), the former comprising more divergent haplotypes. The low level of sequence divergence of the entire cytochrome b gene among six individuals representing six subspecies suggests differentiation during the last glacial period and existence of multiple glacial refugia. At least two evolutionary significant units (ESU) can be identified, the western and the eastern haplogroup. The individual relict populations should be regarded as management units, the eastern subspecies possibly also as ESUs. Guidelines for future translocations and reintroductions are proposed.