Clonal variation in the parthenogenetic rock lizard Lacerta armeniaca.

Genetic diversity at 35 allozyme loci was surveyed in seven populations of Lacerta armeniaca. Fixed heterozygotes were present at 16 loci, with homozygotes at 17 loci. Variation occurred at two loci, one in each of two populations, indicating one widespread clone, one restricted clone, and one apparently restricted clone. The low level of variation in this species suggests a recent restricted origin, involving few parental individuals.     

Genetic variation and de novo mutations in the parthenogenetic Caucasian rock lizard Darevskia unisexualis

Unisexual all-female lizards of the genus Darevskia that are well adapted to various habitats are known to reproduce normally by true parthenogenesis. Although they consist of unisexual lineages and lack effective genetic recombination, they are characterized by some level of genetic polymorphism. To reveal the mutational contribution to overall genetic variability, the most straightforward and conclusive way is the direct detection of mutation events in pedigree genotyping. Earlier we selected from genomic library of D. unisexualis two polymorphic microsatellite containing loci Du281 and Du215. In this study, these two loci were analyzed to detect possible de novo mutations in 168 parthenogenetic offspring of 49 D. unisexualis mothers and in 147 offspring of 50 D. armeniaca mothers. No mutant alleles were detected in D. armeniaca offspring at both loci, and in D. unisexualis offspring at the Du215 locus. There were a total of seven mutational events in the germ lines of four of the 49 D. unisexualis mothers at the Du281 locus, yielding the mutation rate of 0.1428 events per germ line tissue. Sequencing of the mutant alleles has shown that most mutations occur via deletion or insertion of single microsatellite repeat being identical in all offspring of the family. This indicates that such mutations emerge at the early stages of embryogenesis. In this study we characterized single highly unstable (GATA)(n) containing locus in parthenogenetic lizard species D. unisexualis. Besides, we characterized various types of mutant alleles of this locus found in the D. unisexualis offspring of the first generation. Our data has shown that microsatellite mutations at highly unstable loci can make a significant contribution to population variability of parthenogenetic lizards.     

The material ancestry and approximate age of parthenogenetic species of Caucasian rock lizards (Lacerta: Lacertidae)

Restriction enzymes were used to assay variation among mitochondrial DNAs from parthenogenetic and sexual species of Lacerta. This permitted identification of the sexual species that acted as the maternal parent of the various hybrid-parthenogenetic lineages. Lacerta mixta was the maternal parent for both L. dahli and L. armeniaca, L. valentini was the maternal parent for L. uzzelli, and L. raddei was the maternal parent of L. rostombekovi. The maternal ancestry of L. unisexualis is not as clear. The sample of L. nairensis was very similar to one from a population of L. raddei and either species could be the maternal parent of L. unisexualis. The parthenogenetic species all had very low nucleotide diversity in absolute terms and in comparison to their sexual relatives. The close similarity between mtDNAs from the parthenogenetic species and their respective sexual maternal ancestor species provides strong evidence for the recent origin of the parthenogens. The low diversity of the parthenogens indicates that few females were involved in their origins; the maternal parents of L. dahli and L. armeniaca could have come from a single population. The patterns of mtDNA variation in Lacerta are very similar to those in Cnemidophorus and Heteronotia, establishing recent and geographically restricted origins as a general feature of parthenogenetic lizards.     

Genomic variation in parthenogenetic lizard Darevskia armeniaca: evidence from DNA fingerprinting data

Microsatellites, or short tandem repeats, are abundant across genomes of most organisms. It is evident that the most straightforward and conclusive way of studying mutations in microsatellite-containing loci is to use clonally transmitted genomes or DNA sequences inherited in multigeneration pedigrees. At present, little is known about the origin of genetic variation in species that lack effective genetic recombination. DNA fingerprinting in 43 families of the parthenogenetic lizard species Darevskia armeniaca (131 siblings), using (GACA)(4), (GGCA)(4), (GATA)(4), and (CAC)(5) probes, revealed mutant fingerprints in siblings that differed from their mothers in several restriction DNA fragments. In some cases, the mutant fingerprints detected in siblings were also found in population samples. The mutation rate for new restriction fragment length estimated by using multilocus probes varied from 0.8 x 10(-2) to 4.9 x 10(-2) per band/per sibling. Probably, the most variations detected as restriction fragment length polymorphism have germ-line origin, but somatic changes of (CAC)(n) fingerprints in adult lizards were also observed. These results provide new evidence of existing unstable regions in genomes of parthenogenetic vertebrate animals, which provide genetic variation in unisexual populations.     

Superinstability (TCT/TCC)n of microsatellite DNA in parthenogenetic lizards Darevskia unisexualis (family Lacertidae)

Using multilocus DNA fingerprinting, we have examined variability of (TCT)n microsatellite and M13 minisatellite DNA repeats in populations, families, and tissues of Caucasian parthenogenetic rock lizards Darevskia unisexualis (Lacertidae). It has been shown for the first time that population and family DNA samples of D. unisexualis (75 samples in total) have individually specific DNA fingerprinting patterns of (TCT)n fragments. Analysis of inheritance of (TCT)n microsatellites in 46 first-generation progeny in 17 parthenogenetic D. unisexualis families revealed their extremely high instability. Mutant TCT fingerprint phenotypes were found in virtually each animal of the progeny. Moreover, varying fragments in the progeny and their original variants in the mothers were shown to simultaneously contain (TCT)n and (TCC)n polypyrimidine clusters. At the same time, no variability of (TCT)n fragments has been detected in the tissues and organs of mature parthenogenetic lizards and in the analogous tissues of the two-week-old progeny of this year. This suggests the absence of somatic mosaicism and methylation of the corresponding loci in the samples. Along with the hyperinstability of (TCT/TCC)n polypyrimidine clusters, we have shown that the population and family DNA fingerprinting patterns of M13 minisatellites were invariable and monomorphic in the same DNA samples of D. unisexualis. Our results indicate that mutations at loci containing polypyrimidine microsatellites significantly contribute to the total genomic variability of parthenogenetic lizards D. unisexualis.     

Variability of GATA-microsatellite DNA in populations of the parthenogenetic species of lizard Lacerta unisexualis Darevsky

Multilocus DNA fingerprinting was used to analyze the genome variation of mini- and microsatellite DNA regions in parthenogenetic Caucasian rock lizard Lacerta unisexualis. The DNA fingerprints obtained with probe M13 were nearly identical in all populations examined (the average similarity index S = 0.992). The fingerprints obtained with probe (GATA)4 varied (S = 0.862). Polymorphic fragments were assumed to correspond to allelic variants of genetically unstable GATA loci. Comparison of the fingerprints of animals from four geographically isolated populations revealed several population-specific GATA microsatellite markers. Based on their distribution among the populations, the corresponding alleles were assumed to originate from a common ancestral allele.     

Increased variability of (TCC)n microsatelline loci in populations of the parthenogenetic lizard Lacerta unisexualis Darevsky

In four isolated populations of parthenogenetic Caucasian rock lizard Lacerta unisexualis, variability of (TCC)n loci was examined using multilocus DNA fingerprinting. Unexpectedly high variability of (TCC)n microsatellites was found in all four populations. The mean similarity index was 0.825, which is higher than similarity estimates obtained for other mini- and microsatellite loci in L. unisexualis and parthenogenetic species L. dahli and L. armeniaca studied earlier. The high variation level of (TCC)n loci was shown to be at least partially associated with the presence of a diverged (TCC)n sequence fraction in the L. unisexualis genome. Mutations at some other genetically unstable (TCC)n loci may cause their structural diversity in populations of L. unisexualis.     

Study of allelic polymorphism of (GATA)n-containing loci in parthenogenetic lizards Darevskia unisexualis (Lacertidae)

The genesis of mini- and microsatellite loci, which is under extensive study in humans and some other bisexual species, have been virtually overlooked in species with clonal mode of reproduction. Earlier, using multilocus DNA fingerprinting, we have examined variability of some mini- and microsatellite DNA markers in parthenogenetic lizards from the genus Darevskia. In particular, mutant (GATA)n-restrictive DNA fragments were found in Darevskia unisexualis. In the present study, we examined intraspecific polymorphism of three cloned loci of D. unisexualis--Du323, Du215, and Du281--containing (GATA)7GAT(GATA)2, GAT(GATA)9, and (GATA)10TA(GATA) microsatellite clusters, respectively. Different levels of intrapopulation and interpopulation variability of these loci were found. Locus Du281 showed the highest polymorphism--six allelic variants (in the sample of 68 DNA specimens). Three alleles were found for locus Du215. The Du325 locus was electrophoretically invariant. The primers chosen for loci Du323, Du215, and Du281 were also used for PCR analysis of homologous loci in two presumptive parental bisexual species, D. valentini and D. nairensis. The PCR products of the corresponding loci of the parental species had approximately the same size (approximately 200 bp) as their counterparts in D. unisexualis, but the polymorphism levels of the paternal, maternal, and hybrid species were shown to be somewhat different. These data on the structure of the D. unisexualis loci provide a possibility to study genetic diversity in the parthenogenetic species D. unisexualis and other related unisexual and bisexual species of this genus, which can provide new information on the origin of parthenogenetic species and on the phylogenetic relationships in the genus Darevskia. These data can also be used for resolving problems of marking the lizard genome, which is still poorly studied.     

Revealing of allelic polymorphism in the populations of parthenogenetic lizards Darevskia dahli (Lacertidae) using locus-specific PCR

Locus-specific PCR was used to study the genetic polymorphism in three populations of parthenogenetic lizard species Darevskia dahli. The analysis was carried at the two (GATA)n-containing loci (Du215 and Du281) using the sample of 26 individuals. A total of eight Du215 and three Du281 allelic variants were detected. It was demonstrated that all the lizards examined were heterozygous at these loci. In 12 animals, unusual Du215 allelic variant was revealed, the origin of which was thought to be associated with different types of genomic rearrangements, or segmental duplication. The populations studied were substantially different relative to the levels of allelic polymorphism, which could be explained by different habitation conditions, leading to accumulation of mutations in noncoding genome regions.     

Molecular characterization of allelic variants of (GATA)n microsatellite loci in parthenogenetic lizards Darevskia unisexualis (Lacertidae)

Populations of parthenogenetic lizards of the genus Darevskia consist of genetically identical animals, and represent a unique model for studying the molecular mechanisms underlying the variability and evolution of hypervariable DNA repeats. As unisexual lineages, parthenogenetic lizards are characterized by some level of genetic diversity at microsatellite loci. We cloned and sequenced a number of (GATA)n microsatellite loci of Darevskia unisexualis. PCR products from these loci were also sequenced and the degree of intraspecific polymorphism was assessed. Among the five (GATA)n loci analysed, two (Du215 and Du281) were polymorphic. Cross-species analysis of Du215 and Du281 indicate that the priming sites at the D. unisexualis loci are conserved in the bisexual parental species, D. raddei and D. valentini. Sequencing the PCR products amplified from Du215 and Du281 and from monomorphic Du323 showed that allelic differences at the polymorphic loci are caused by microsatellite mutations and by point mutations in the flanking regions. The haplotypes identified among the allelic variants of Du281 and among its orthologues in the parental species provide new evidence of the cross-species origin of D. unisexualis. To our knowledge, these data are the first to characterize the nucleotide sequences of allelic variants at microsatellite loci within parthenogenetic vertebrate animals.     

Variation of mini- and microsatellite DNA repeats in parthenogenetic lizard Darevskia armeniaca as revealed by DNA fingerprinting analysis

Population and family samples of two morphological forms (mutant and normal with respect to dorsal color) of pathogenetic lizard Darevskia armeniaca were examined by means of DNA fingerprinting using M13 mini- and (GATA)n and (TCC)n microsatellite DNA markers. The morphological forms examined were characterized by clonally inherited, species-specific patterns of the DNA markers, which were different from the species-specific DNA fingerprints of the other parthenogenetic species of the genus Darevskia (D. dahli. D. unisexualis, and D. rostombekovi). The mean index of similarity (S) obtained for a sample of 36 individuals from three isolated populations using three types of DNA markers was 0.966. This was similar to the variability level observed in D. dahli (0.962) (P > 0.05), but higher than that in D. unisexualis (0.950) (P < 0.05) and D. rostombekovi (0.875) (P < 0.01). Inheritance of M13 minisatellite and (TCC)n microsatellite DNA markers in the F1 offspring of parthenogenetic lizards was examined. It was shown that variability and clonal diversity of the fingerprint phenotypes observed in the populations and families of D. armeniaca could be at least partly explained by RFLP mutations in microsatellite repeats.     

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

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

Differentiation in DNA fingerprinting among species of the genus Acer L. in Campania (Italy)

Abstract

Natural populations of species in the Acer genus occurring in Campania (southern Italy) were surveyed by screening seven microsatellite loci. Primer pairs for Acer pseudoplatanus L. microsatellite loci were analysed in six different species: Acer lobelii Ten., Acer campestre L., Acer pseudoplatanus L., Acer obtusatum W. et K., Acer neapolitanum Ten. and Acer monspessulanum L. The aim of the present study was to survey the genetic variability and genetic structure of natural populations of the Acer genus in Campania. The high degree of polymorphism observed in six different species of Acer makes these markers useful for investigating genetic variation at various spatial scales, and for the analysis of gene flow and of the mating system.     

Detection of genetically unstable loci in parthenogenic families of lizards of theLacerta genus by DNA fingerprinting

Two parthenogenic families of unisexual species of Caucasian rock lizards of genusLacerta, L. armeniaca andL. unisexualis, were analyzed by DNA fingerprinting. Inheritance of M13 minisatellite and of (GACA) _n , (GATA) _n , and (TCC) _n microsatellite loci in the first generation of the lizards was studied. M13, (GACA) _n , and (TCC) _n loci in the families ofL. armeniaca were strictly inherited, as well as M13 and (GACA) _n loci in the families ofL. unisexualis: each DNA fragment in the fingerprint patterns of progeny could be detected in the maternal pattern. However, when a (TCC)₅₀ microsatellite probe was applied in the study ofL. unisexualis families, specific DNA fragments with altered mobility were revealed in the progeny patterns, and the frequency of such events was rather high. It might be hypothesized that some of the (TCC) _n loci inL. unisexualis genome are highly mutable. Hence, the family analysis allowed us to demonstrate experimentally the presence of genetically unstable loci in genomes of parthenogenic species of vertebrates. The nature and mechanism of the instability of these loci in parthenogenesis remain obscure.     

Toward the phylogeny of Caucasian rock lizards: implications from mitochondrial DNA gene sequences (Reptilia: Lacertidae)

A maximum parsimony phylogeny of 14 Caucasian species of rock lizards, genus Ijicerta , subgenus Archaeolacerta , was constructed from mitochondrial cytochrome b and ATPase 6 partial gene sequences. Congruence analyses were carried out between the two genes. A synthesis of the data sets reveals three well supported monophyletic groups: (1) the caucasica group including ( Lacerta derjugini (( Lalpina, L. clarkorum ) ( L. caucasica, L. daghestanica ))); (2) the rudis group including ( L. parvula ( L. portschinskii ( L. vakntini, L. rudis ))); and (3) the saxicola group including ( L. mixta ( L. nairensis ( L. saxicola, L. raddeijj ). Despite the diagnosis of three groups, the placement of L. praticola as a basal taxon is uncertain, as are the relationships among the three groups. The mitochondrial DNA sequence data suggested prior hybridization between L. mixta and L. alpina and possibly between L. saxicola and L. nairensis. Lacerta raddei was resolved as a paraphyletic species on the mtDNA tree; this may result from either hybridization or random gene sorting.     

Genetic variation and species identification of Thai Boesenbergia (Zingiberaceae) analyzed by chloroplast DNA polymorphism

Genetic variation and molecular phylogeny of 22 taxa representing 14 extant species and 3 unidentified taxa of Boesenbergia in Thailand and four outgroup species (Cornukaempferia aurantiflora, Hedychium biflorum, Kaempferia parviflora, and Scaphochlamys rubescens) were examined by sequencing of 3 chloroplast (cp) DNA regions (matK, psbA-trnH and petA-psbJ). Low interspecific genetic divergence (0.25-1.74%) were observed in these investigated taxa. The 50% majority-rule consensus tree constructed from combined chloroplast DNA sequences allocated Boesenbergia in this study into 3 different groups. Using psbA-1F/psbA-3R primers, an insertion of 491 bp was observed in B. petiolata. Restriction analysis of the amplicon (380-410 bp) from the remaining species with Rsa I further differentiated Boesenbergia to 2 groupings; I (B. basispicata, B. longiflora, B. longipes, B. plicata, B.pulcherrima, B. tenuispicata, B. thorelii, B. xiphostachya, Boesenbergia sp.1 and Boesenbergia sp.3; phylogenetic clade A) that possesses a Rsa I restriction site and II (B.curtisii, B. regalis, B. rotunda and Boesenbergia sp.2; phylogenetic clade B and B. siamensis; phylogenetic clade C) that lacks a restriction site of Rsa I. Single nucleotide polymorphism (SNP) and indels found can be unambiguously applied to authenticate specie-origin of all investigated samples and revealed that Boesenbergia sp.1, Boesenbergia sp.2 and B. pulcherrima (Mahidol University, Kanchanaburi), B. cf. pulcherrima1 (Prachuap Khiri Khan) and B. cf. pulcherrima2 (Thong Pha Phum, Kanchanaburi) are B. plicata, B. rotunda and B. pulcherrima, respectively. In addition, molecular data also suggested that Boesenbergia sp.3 should be further differentiated from B. longiflora and regarded as a newly unidentified Boesenbergia species.     

Genetic variation of the St. Lawrence beluga whale population assessed by DNA fingerprinting

Recent surveys suggest that the endangered St. Lawrence beluga ( Delphinapterus leucas ) population is not recovering significantly despite 20 years of protection. Dead individuals that have been autopsied show high levels of tumours and infections. This situation could be a result of pollution, loss of genetic variation, inbreeding depression or a combination of these factors. Analyses of DNA fingerprints from St. Lawrence belugas with three minisatellite probes (Jeffreys 33.6, 33.15 and M13) indicate a reduced level of genetic variation compared to Beaufort Sea animals. The average band-sharing between individuals of the St. Lawrence beluga population for the three probes (0.534, 0.573 and 0.478, respectively) was significantly higher than that of the Beaufort Sea beluga population (0.343, 0.424, 0.314, respectively). Higher levels of mean allele frequency in the St. Lawrence belugas (0.33 vs. 0.21) suggest that this population is composed of individuals which are related. Inbreeding depression could therefore be a factor in the lack of recovery of the St. Lawrence beluga population.