Molecular nature of allelic polymorphism of highly variable microsatellite locus Du161(arm) in unisexual lizard Darevskia armeniaca (Lacertidae)

In the genome of unisexual (parthenogenetic) lizard Darevskia armeniaca, highly variable locus Du 161 (arm) was discovered. Analysis of allelic polymorphism was carried out using locus-specific PCR of the lizard DNA specimens from 13 isolated Armenian populations (N = 138). In the sample examined, a total of 12 Du 161(arm) alleles were identified, and their differences at the level of primary DNA structure were determined. Sequence analysis of the Du 161 (arm) alleles showed that their microsatellite clusters contained repeats of one type (GATA repeats). Allelic Du 161 (arm) variants differed in the number of GATA monomers in microsatellite, point mutations of transition and transversion types, located at fixed distances from microsatellite cluster, and by single nucleotide insertions, as well as by longer insertions located within and outside of the microsatellite cluster. Moreover, point mutations formed different combinations (haplotypes), typical of certain alleles. These combinations can be used for the analysis of the origin and inheritance of these alleles in D. armeniaca, as well as for investigation of their interspecific variation in the representatives of the genus Darevskia.     

Nucleotide sequences of the microsatellite locus <Emphasis Type="Italic">Du215 (arm)</Emphasis> allelic variants in the parthenospecies <Emphasis Type="Italic">Darevskia armeniaca</Emphasis> (Lacertidae)

Using monolocus PCR analysis with the pairs of primers designed for the Du215 locus of Darevskia unisexualis, allelic polymorphism at the orthologous locus in the populations of the related parthenospecies D. armeniaca was investigated. It was demonstrated that Du215 (arm) locus was polymorphic and in the populations of parthenospecies D. armeniaca (n = 127) represented by at least three allelic variants, differing from each other by the size and composition of microsatellite cluster, and by single nucleotide substitutions in flanking DNA. Unlike the Du215 locus, Du215 (arm) was shown contain not only GATA, but also (GACA) repeats, which were absent in D. unisexualis. Thus, in this study, the data on the molecular nature of allelic polymorphism at one of the microsatellite loci of the parthenospecies D. armeniaca were reported.     

Molecular structure of allelic variants of microsatellite loci <Emphasis Type="Italic">Du281</Emphasis> and <Emphasis Type="Italic">Du47</Emphasis> in unisexual and bisexual lizard species of the genus <Emphasis Type="Italic">Darevskia</Emphasis>

Experimental data on the molecular structure and variability of microsatellite loci in unisexual and bisexual lizard species of the genus Darevskia were analyzed. The allelic variants of Du281 and Du47 were found to differ in the number of monomers, the structure of microsatellite clusters, and point mutations in these clusters and flanking DNA. Interspecific comparison of alleles of these loci revealed both variable regions in the microsatellite clusters and allele-specific evolutionarily conserved nucleotide groups. In general, the results of comparative structural analysis of allelic variants testify to a high genetic similarity of the unisexual and bisexual lizard species studied and reveals the characteristic features of their interspecies variability.     

Instability of (GATA)<Subscript> n</Subscript> microsatellite loci in the parthenogenetic Caucasian rock lizard<Emphasis Type="Italic"> Darevskia unisexualis</Emphasis> (Lacertidae)

Mini- and microsatellites, comprising tandemly repeated short nucleotide sequences, are abundant dispersed repetitive elements that are ubiquitous in eukaryotic genomes. In humans and other bisexual species hypervariable mini- and microsatellite loci provide highly informative systems for monitoring of germline and somatic instability. However, little is known about the mechanisms by which these loci mutate in species that lack effective genetic recombination. Here, multilocus DNA fingerprinting was used to study M13 minisatellite and (GATA) _n microsatellite instability in the parthenogenetic Caucasian rock lizard Darevskia unisexualis (Lacertidae). DNA fingerprinting of 25 parthenogenetic families, from six isolated populations in Armenia (comprising a total of 84 siblings), using the oligonucleotide (GATA)₄ as a hybridization probe, revealed mutant fingerprinting phenotypes in 13 siblings that differed from their mothers in several restriction DNA fragments. In three families (8 siblings), the mutations were present in the germline. Moreover, the mutant fingerprint phenotypes detected in siblings were also present in population DNA samples. No intrafamily variations in DNA fingerprint patterns were observed with the M13 minisatellite probe. Estimates of the mutation rate for (GATA) _n microsatellite loci in D. unisexualis showed that it was as high as that seen in some bisexual species, reaching 15% per sibling or 0.95% per microsatellite band. Furthermore, in one case, a somatic (GATA) _n microsatellite mutation was observed in an adult lizard. These findings directly demonstrate that mutations in (GATA) _n microsatellite loci comprise an important source of genetic variation in parthenogenetic populations of D. unisexualis.Communicated by G. P. Georgiev     

Molecular genetic mapping of the <Emphasis Type="Italic">sy1</Emphasis> and <Emphasis Type="Italic">sy9</Emphasis> asynaptic genes in rye (<Emphasis Type="Italic">Secale cereale</Emphasis> L.) using microsatellite and isozyme markers

Studies of phenotypical expression of synaptic mutations in combination with the localization of corresponding genes on a genetic map permit individual stages of the meiotic process to be differentiated. Two rye asynaptic genes, sy1 and sy9, were mapped with the use of microsatellite markers (SSR) in the pericentromeric regions of the long chromosome arms 7R and 2R, respectively. The sy9 gene cosegregated with two SSR markers Xscm43 and Xgwm132. The asynaptic gene sy1 was mapped within the interval between the isozyme locus Aat2 and two cosegregating loci Xrems1188 and Xrems1135 that are located at a distance of 0.4 cM proximally and 0.1 cM distally with respect to the gene lous. Possible evolutionary relationships of the mapped genes with homeological loci of the Triticeae species and more distant cereal species, such as maize and rice, are discussed.     

Evidence for gametoclonal variation in potato (<Emphasis Type="Italic">Solanum tuberosum</Emphasis> L.)

Gametoclonal variation that occurs in gametic cells in culture and is recovered in their regenerated derivatives has not been reported in potato (Solanum tuberosum L.). Based on a set of 24 differentiating phenotypic traits, canonical variates analysis genetically distinguished the androgenic (di)haploid (2n = 2x = 24) D4 from its tetraploid (2n = 4x = 48) anther-derived sibs and anther donor JTH/C-107. Nuclear microsatellite analysis over six polymorphic loci indicated that meiotic rearrangements and mutant alleles were primarily associated with the release of gametoclonal variation. The incidence of null alleles in D4 at the loci STACCAS3 and STM0031 was also indicative of mutations occurring within the priming sequence. Microsatellite results were supported by random amplified polymorphic DNA (RAPD) assays that characterized a total of 567 loci (bins) representing 4,258 amplified fragments. Sixty-five new RAPDs that were absent in the anther donor and in either of its parents, viz., S. phureja Juz. & Buk. IVP-35 and S. tuberosum cv. Kufri Jyoti were present in D4, indicating the occurrence of extensive recombinational events. The results have been discussed in the context of microsatellite null alleles providing the most conclusive evidence for gametoclonal variation.     

Regions associated with repression of the barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis>) <Emphasis Type="Italic">VERNALIZATION1</Emphasis> gene are not required for cold induction

Activity of the VERNALIZATION1 (VRN1) gene is required for flowering in temperate cereals such as wheat and barley. In varieties that require prolonged exposure to cold to flower (vernalization), VRN1 is expressed at low levels and is induced by vernalization to trigger flowering. In other varieties, deletions or insertions in the first intron of the VRN1 gene are associated with increased VRN1 expression in the absence of cold treatment, reducing or eliminating the requirement for vernalization. To characterize natural variation in VRN1, the first intron of the barley (Hordeum vulgare) VRN1 gene (HvVRN1) was assayed for deletions or insertions in a collection of 1,000 barleys from diverse geographical regions. Ten alleles of HvVRN1 containing deletions or insertions in the first intron were identified, including three alleles that have not been described previously. Different HvVRN1 alleles were associated with differing levels of HvVRN1 expression in non-vernalized plants and with different flowering behaviour. Using overlapping deletions, we delineated regions in the HvVRN1 first intron that are associated with low levels of HvVRN1 expression in non-vernalized plants. Deletion of these intronic regions does not prevent induction of HvVRN1 by cold or the maintenance of increased HvVRN1 expression following cold treatment. We suggest that regions within the first intron of HvVRN1 are required to maintain low levels of HvVRN1 expression prior to winter but act independently of the regulatory mechanisms that mediate induction of HvVRN1 by cold during winter. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers 1179825, 1179833, 1179836, 1179858.     

Comparative analysis of <Emphasis Type="Italic">iol</Emphasis> clusters in <Emphasis Type="Italic">Lactobacillus casei</Emphasis> strains

The present study was designed to expand genetic knowledge of myo -inositol (MI) metabolism in Lactobacillus casei. Twenty-four L. casei isolates of dairy origin were tested for the presence of iol cluster. PCR screening revealed eight strains encoded functions involved in MI utilization, of which one strain was able to use MI as carbon source. To gain a deeper understanding of the function of iol genes, four of the eight observed iol clusters were subjected to the full sequencing procedure. The results showed that the iol cluster was not a common feature among dairy L. casei strains. In addition, the four iol clusters were highly similar to one another in terms of sequence similarity and operon architecture. However, abundant polymorphisms that comprised a majority of synonymous mutations were detected throughout the full sequences. Three of them distributed among iolB, iolC, and iolT genes were found in linkage to MI-negative phenotype. Compared with other bacterial iol clusters, the iol cluster of L. casei showed a high similarity with that of Bacillus subtilis.     

Detection of the introgression of genome elements of the <Emphasis Type="Italic">Aegilops cylindrica</Emphasis> host into the <Emphasis Type="Italic">Triticum aestivum</Emphasis> L. genome by ISSR and SSR analysis

To reveal sites of the donor genome in wheat crossed with Aegilops cylindrica, which acquired conferred resistance to fungal diseases, a comparative analysis of introgressive and parental forms was conducted. Two systems of PCR analysis, ISSR and SSR–PCR, were employed. Upon use of 7 ISSR primers in genotypes of 30 individual plants BC₁F₉ belonging to lines 5/55-91 and 5/20-91, 19 ISSR loci were revealed and assigned to introgressive fragments of Aegilops cylindrica genome in Triticum aestivum. The 40 pairs of SSR primers allowed the detection of seven introgressive alleles; three of these alleles were located on common wheat chromosomes in the B genome, while four alleles, in the D genome. Based on data of microsatellite analysis, it was assumed that the telomeric region of the long arm of common wheat chromosome 6A also changed. ISSR and SSR methods were shown to be effective for detecting variability caused by introgression of foreign genetic material into the genome of common wheat.Translated from Genetika, Vol. 40, No. 12, 2004, pp. 1654–1661.Original Russian Text Copyright © 2004 by Galaev, Babayants, Sivolap.     

Molecular Phylogenetics of <Emphasis Type="Italic">Aotus</Emphasis> (Platyrrhini,Cebidae)

The accurate identification of taxa of Aotus is essential for 1) the development of precise biomedical assays, 2) the determination of potential illegal traffic of this genus, and 3) conservation. Although many studies have contributed to what we know about the phylogenetics of Aotus, none used a sufficiently large number of samples to clarify its complexity. To address this need, we sequenced 696 base pairs of the mitochondrial cytochrome-oxidase II gene (mtCOII) in 69 specimens of 7 taxa of Aotus. We also analyzed 8 microsatellite loci in 136 individuals of 6 taxa. In contrast to previous studies, we sampled only wild individuals and have a precise geographical origin for each one. The mtDNA results showed that: 1) the northern gray-necked group of Aotus is genetically more homogeneous than the polyphyletic red-necked group of Aotus; 2) the ancestors of Aotus vociferans seem to be the original species candidate for the current Aotus; 3) Aotus azarae azarae and A. a. boliviensis are the most differentiated taxa, likely a result of extreme genetic drift during stasipatric speciation; 4) the first genetic splits found among taxa of Aotus occurred during the Pliocene (or even Miocene) while the most recent ones happened during the Pleistocene, when forest refugia may have played an important role in speciation. The mean number of microsatellite alleles was 3–5.33 alleles per locus. We found some private alleles that could be useful in helping to identify illegal trade, although a larger sample size is needed to ensure that these alleles are really private to the relevant taxa. These new findings increase our understanding of the phylogeny of Aotus and the level of genetic diversity within different taxa of Aotus.     

Development and characterization of 12 compound microsatellite markers in <Emphasis Type="Italic">Platypus </Emphasis><Emphasis Type="Italic">quercivorus</Emphasis> (Murayama) (Coleoptera: Platypodidae)

Twelve compound microsatellite loci were isolated from the ambrosia beetle Platypus quercivorus. Every locus was polymorphic among 50 individuals from two localities, with two to six alleles per locus, without linkage disequilibrium. The observed and expected heterozygosities ranged from 0.140 to 0.740 and from 0.255 to 0.731, respectively. These markers will be available for population genetic studies and parentage analysis of this beetle.     

Microsatellite mutation rates in the eastern tiger salamander (<Emphasis Type="Italic">Ambystoma tigrinum tigrinum</Emphasis>) differ 10-fold across loci

Microsatellites are commonly used for mapping and population genetics because of their high heterozygosities and allelic variability (i.e., polymorphism). Microsatellite markers are generally more polymorphic than other types of molecular markers such as allozymes or SNPs because the insertions/deletions that give rise to microsatellite variability are relatively common compared to nucleotide substitutions. Nevertheless, direct evidence of microsatellite mutation rates (MMRs) is lacking in most vertebrate groups despite the importance of such estimates to key population parameters (e.g., genetic differentiation or θ = 4N _e μ). Herein, we present empirical data on MMRs in eastern tiger salamanders (Ambystoma tigrinum tigrinum). We conducted captive breeding trials and genotyped over 1,000 offspring at a suite of microsatellite loci. These data on 7,906 allele transfers provide the first direct estimates of MMRs in amphibians, and they illustrate that MMRs can vary by more than an order of magnitude across loci within a given species (one locus had ten mutations whereas the others had none).     

Thermodynamic characteristization of Di-, Tri-, and tetranucleotide loci in parthenogenetic lizards <Emphasis Type="Italic">Darevskia unisexualis</Emphasis>

Allelic polymorphism of three microsatellite loci from the genome of parthenogenetic lizard Darevskia unisexualis was characterized using analysis of free energy (Gibbs energy) of the DNA/DNA duplex formation within the stepwise mutational model. It was demonstrated that the number of microsatellite cluster monomericic units would change to decrease the mean free energy of the locus. In addition, based on the analysis of nucleotide composition, the GC content of each locus was evaluated, and belonging of the loci examined to certain isochore families was suggested.     

Development and characterization of microsatellite markers for parentage analyses of the coral reef damselfish (<Emphasis Type="Italic">Pomacentrus amboinensis</Emphasis>: Pomacentridae)

Five new polymorphic microsatellite loci were developed for the coral reef damselfish Pomacentrus amboinensis. Twenty-four individuals from two Great Barrier Reef populations were genotyped at the five loci, with numbers of alleles per locus ranging from 6–23 and observed heterozygosity between 0.42–0.92. In addition, the cross-species testing of six primers developed for Stegastes partitus revealed one primer (SpGATA40) that was also polymorphic for P. amboinensis. Due to high levels of polymorphism (≥14 alleles) in at least four of the six loci and a high proportion of tetranucleotide repeats, these microsatellite markers should be useful for parentage assignment as well as other investigations of individual relatedness.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of ten threatened species of <Emphasis Type="Italic">Mammillaria</Emphasis> (Cactaceae)

Mammillaria species are the most numerous within Cactaceae family, and some of them are threatened with extinction as a result of human activities. In this work, results of in vitro propagation are presented for ten Mammillaria species, testing 20 combinations of indole-3-acetic acid (IAA) and kinetin. Best results on shoot formation were obtained using kinetin at two levels: 27.9 and 46.5 μM. All IAA levels tested were able to induce de novo shoot formation in M. bocasana, M. densispina, M. hahniana, M. hutchisoniana, M. orcutii, M. pectinifera, M. perbella, M. picta, M. rhodantha, and M. zephyranthoides. Depending on the IAA level tested, four responding groups were observed concerning their highest shoot-formation number. For all species, the highest average of shoot formation was achieved with 5.7:46.5 or 11.4:46.5 μM IAA/kinetin, yielding 4.8 and 4.7 shoots per explant, respectively, in 60 d. Rooting of regenerated shoots was achieved by leaving the explants in their shoot-induction medium or transferring them to half-strength MS medium. Hardening of regenerated plants was successfully achieved by planting them in peat moss substrate after a desiccation treatment at room temperature for 3 d.     

Osmotic shock improves <Emphasis Type="Italic">Tnt1</Emphasis> transposition frequency in <Emphasis Type="Italic">Medicago truncatula</Emphasis> cv Jemalong during in vitro regeneration

Insertion mutant collections are powerful tools for genetic studies in plants. Although large-scale insertional mutagenesis using T-DNA is not feasible in legumes, the Tnt1 tobacco retrotransposon can be used as a very efficient mutagen in the Medicago truncatula R108 genotype. In this article, we show that Tnt1 can also be exploited to create insertional mutants via transformation and/or regeneration in the reference cultivar Jemalong. Tnt1 insertional mutagenesis in Jemalong following Agrobacterium tumefaciens-mediated transformation was found to be very efficient, with an average of greater than 15 insertions/line. In contrast, regeneration using low-copy transgenic starter lines resulted in a highly variable rate of new Tnt1 insertions. With the goal of increasing the number of additional Tnt1 insertions during regeneration of starter lines, we have compared the insertion frequencies for a number of different regeneration protocols. In addition, we have been able to show that sucrose-mediated osmotic shock preceding regeneration significantly increases the transposition frequency. Under optimal conditions, 95% of the regenerated Jemalong plants possess new insertions.     

<Emphasis Type="Italic">Pm37</Emphasis>, a new broadly effective powdery mildew resistance gene from <Emphasis Type="Italic">Triticum </Emphasis><Emphasis Type="Italic">timopheevii</Emphasis>

Powdery mildew is an important foliar disease in wheat, especially in areas with a cool or maritime climate. A dominant powdery mildew resistance gene transferred to the hexaploid germplasm line NC99BGTAG11 from T. timopheevii subsp. armeniacum was mapped distally on the long arm of chromosome 7A. Differential reactions were observed between the resistance gene in NC99BGTAG11 and the alleles of the Pm1 locus that is also located on chromosome arm 7AL. Observed segregation in F_2:3 lines from the cross NC99BGTAG11 × Axminster (Pm1a) demonstrate that germplasm line NC99BGTAG11 carries a novel powdery mildew resistance gene, which is now designated as Pm37. This new gene is highly effective against all powdery mildew isolates tested so far. Analyses of the population with molecular markers indicate that Pm37 is located 16 cM proximal to the Pm1 complex. Simple sequence repeat (SSR) markers Xgwm332 and Xwmc790 were located 0.5 cM proximal and distal, respectively, to Pm37. In order to identify new markers in the region, wheat expressed sequence tags (ESTs) located in the distal 10% of 7AL that were orthologous to sequences from chromosome 6 of rice were targeted. The two new EST-derived STS markers were located distal to Pm37 and one marker was closely linked to the Pm1a region. These new markers can be used in marker-assisted selection schemes to develop wheat cultivars with pyramids of powdery mildew resistance genes, including combinations of Pm37 in coupling linkage with alleles of the Pm1 locus.     

Development of 12 polymorphic microsatellite markers in <Emphasis Type="Italic">Coilia ectenes</Emphasis> Jordan and Seale, 1905 (Clupeiformes: Engraulidae) and cross-species amplification in <Emphasis Type="Italic">Coilia mystus</Emphasis> Linnaeus, 1758

In this study, twelve polymorphic microsatellite markers were developed for Coilia ectenes (synonym C. nasus). In a sample of 30 C. ectenes individuals in a Wuhan population from Yangtze River, a total of 78 alleles were detected, and the number of alleles per locus ranged from 3 to 12. The observed and expected heterozygosities ranged from 0.100 to 1.000 and from 0.098 to 0.899, respectively. Six loci significantly deviated from the Hardy-Weinberg equilibrium after Bonferroni correction (P < 0.004), and no significant linkage disequilibrium between pairs of loci was found. The applicability of these markers in a closely related species, C. mystus, was evaluated by cross-species amplification. These microsatellite markers will be useful in studies on population genetics, conservation genetics, and fishery management and in the construction of genetic linkage maps of C. ectenes and C. mystus.     

Isolation of a <Emphasis Type="Italic">Ve</Emphasis> homolog, <Emphasis Type="Italic">mVe1</Emphasis>, and its relationship to verticillium wilt resistance in <Emphasis Type="Italic">Mentha longifolia</Emphasis> (L.) Huds

As a step toward greater understanding of the genetics of verticillium wilt resistance in plants, we report the sequencing of a candidate wilt resistance gene, mVe1, from the mint diploid model species, Mentha longifolia (Lamiaceae). mVe1 is a putative homolog of tomato (Solanum lycopersicum L.) verticillium wilt (Ve) resistance genes. The mVe1 gene has a coding region of 3,051 bp. The predicted mVe1 protein contains a leucine-rich repeat domain, a common feature of plant disease resistance proteins. We compared 13 mVe1 alleles from three mint species. These alleles shared 96.2–99.6% nucleotide identity. We analyzed four M. longifolia populations segregating with respect to mVe1 alleles and wilt resistance versus susceptibility and found one association between mVe1 genotype and wilt phenotype. We conclude that mVe1 may play a role in mint verticillium wilt resistance, but variation for resistance in our segregating progenies is likely polygenic. Therefore, further investigations of mVe1 and identification of additional candidate genes are both warranted.     

Assessment of genetic diversity and relationships among <Emphasis Type="Italic">Coix lacryma-jobi</Emphasis> accessions using microsatellite markers

The present study describes the assessment of genetic diversity and relationships among 79 Job’s tears (Coix lacrymajobi L.) accessions collected from China and Korea using 17 microsatellite markers. A total of 57 alleles were detected with an average of 3.4 alleles per locus. A high frequency of rare alleles (36.3 %) was observed within the collection. Values for observed (H_O), expected heterozygosity (H_E) and Shannon’s information index (I) within the analysis ranged from 0.00 (GBssrJT183) to 0.81 (GBssrJT130), from 0.01 (GBssrJT170) to 0.65 (GBssrJT130) and from 0.034 (GBssrJt170) to 1.13 (GBssrJT130), respectively. The locus GBJT130 was the most informative marker with the highest values for observed and effective alleles as well as for H_O, H_E and I. Based on the UPGMA algorithm, the majority of the Chinese accessions grouped in one cluster, whereas all the Korean accessions grouped together in a separate cluster, indicating that Chinese accessions are genetically quite distinct from Korean accessions. No relation between genetic relatedness among Job’s tears accessions and their place of collection was observed. Chinese accessions exhibited greater within population polymorphism (P = 95 %, H_E = 0.30, I = 0.52) than the accessions from Korea (P = 68 %, H_E = 0.13, I =0.24), indicating their potentiality as a reservoir of novel alleles for crop improvement. However, in general the low diversity within each population indicates a narrow genetic base within our collection.