Isolation and characterisation of (AC)n microsatellite genetic markers from human chromosome 16.

A cosmid library of human chromosome 16 has been subcloned, and (AC)n microsatellite positive clones have been identified and sequenced. Oligonucleotide primers flanking the repeat were designed and synthesized for (AC)n microsatellites with n greater than 16. These microsatellite loci were then mapped by PCR using a somatic cell hybrid panel of human chromosome 16, and their heterozygosities and allele frequencies determined. Fourteen (AC)n microsatellites were mapped to discrete physical intervals of human chromosome 16 defined by a mouse/human hybrid panel. Nine of these have expected heterozygosities ranging between 0.60 and 0.79, four have expected heterozygosities between 0.02 and 0.49, and one detected three loci where the alleles could not be resolved.     

Dinucleotide Repeat Loci Contribute Highly Informative Genetic Markers to the Human Chromosome 2 Linkage Map

Microsatellite repeat loci can provide informative markers for genetic linkage. Currently, the human chromosome 2 genetic linkage map has very few highly polymorphic markers. Being such a large chromosome, it will require a large number of informative markers for the dense coverage desired to allow disease genes to be mapped quickly and accurately. Dinucleotide repeat loci from two anonymous chromosome 2 genomic DNA clones were sequenced so that oligonucleotide primers could be designed for amplifying each locus using the polymerase chain reaction (PCR). Five sets of PCR primers were also generated from nucleotide sequences in the GenBank Database of chromosome 2 genes containing dinucleotide repeats. In addition, one PCR primer pair was made that amplifies a restriction fragment length polymorphism on the TNP1 gene (Hoth and Engel, 1991). These markers were placed on the CEPH genetic linkage map by screening the CEPH reference DNA panel with each primer set, combining these data with those of other markers previously placed on the map, and analyzing the combined data set using CRI-MAP and LINKAGE. The microsatellite loci are highly informative markers and the TNP1 locus, as expected, is only moderately informative. A map was constructed with 38 ordered loci (odds 1000:1) spanning 296 cM (male) and 476 cM (female) of chromosome 2 compared with 306 cM (male) and 529 cM (female) for a previous map of 20 markers.     

Population genetic structure and distribution of introduced American mink (<Emphasis Type="Italic">Mustela vison</Emphasis>) in Spain,based on microsatellite variation

The population genetic structure of an invasive species in Spain, the American mink (Mustela vison), was investigated using microsatellite DNA markers. This semi-aquatic carnivore, originating from North America, was imported into Europe for fur farming since the beginning of the 20th century. Due to massive escapes, farm damages, deliberate releases and/or accidents, feral mink populations were established in the aquatic ecosystems of many European countries, including Spain. We genotyped 155 American mink originating from the Spanish regions Basque Country, Catalonia, Castilla-Leon, Aragon, Valencia and Galicia using 10 polymorphic microsatellite loci to highlight population genetic structure, distribution and dispersal. M. vison populations in Spain appear differentiated and not yet connected by gene flow. Bayesian clustering analyses and spatial analyses of molecular variance detected four inferred clusters, overall coinciding with the sampled geographical localities. Preliminary testing shows moderate to large estimated effective population sizes. Molecular analyses result useful to provide baseline data for further research on the evolution of invasive mink populations, as well as support local management strategies and indirectly benefit the conservation of threatened species in Spain, such as the endangered European mink (Mustela lutreola), and the polecat (Mustela putorius), which share the habitat with the American mink. This paper is dedicated to the memory of Xavier Domingo-Roura.     

The impact of river fragmentation on the population persistence of native and alien mink: an ecological trap for the endangered European mink

The genetic diversity of feral and ranch American mink was studied in order to detect gene flux among rivers, investigate the processes of invasion, and determine the possible effects of river barriers. Tissue samples of 78 feral American mink from 5 different river catchments and 18 ranch mink, collected between 2007 and 2011 in Biscay, northern Spain, were genotyped at 21 microsatellite loci. Lack of genetic differentiation of feral mink among the sites and high differentiation between feral and ranch mink was suggested. These results confirm that the mink population established on Butrón River at the beginning of the 1990s may be the origin of almost all the feral mink population within the study area. Additionally, the occurrence of American and European mink was used to analyse the effect of fragmentation on the population viability. The size and composition of the home range of male European mink was considered to model minimum viable units for presence/absence. Forty-two minimum viable units were randomly distributed among rivers in order to analyse the effect of fragmentation on mink occurrence. Barriers were mapped and classified as slight, moderate or absolute, depending on the effect on mink movement, and were introduced into the models. The presence of European and American mink depended on the non-fragmented main river stretches and the number of tributaries free from barriers. Results showed that fragmented rivers can be temporarily occupied but the likelihood of death means that these areas are only sink patches for mink.     

Homology of G-banded mammalian chromosomes

Linkage relationships of homologous loci and high resolution G-banding patterns of man, mouse, rat, chinese hamster, rabbit, cat, mink, pig, ox and sheep were used for identification of 11 evolutionary conservative autosomal regions. The distributions and inversions of these regions in the ancestor genomes of some phylums have been shown. For example, the regions homologous to human Ip region were detected in cat, mink and rabbit genomes. In the genomes of rodents studied the intraregion inversion was detected. In the ox and sheep genomes the distal end deletions were detected within these regions. In the pig genome these regions were represented solely by disruptions. We supposed that the rapid "catastrophic" chromosomal evolution took place during short time periods of some orders and families separation.     

应用PCR进行水稻染色体末端区域作图

利用RAPD引物介寻的不对称复性温度PCR的方法（RM－PCR）,发展了一种基于端粒重复序列的新型分子标记。并在一个籼粳杂交来源（窄叶青8号×京系17）的水稻双单倍体群体中进行了端粒重复相关顺序的遗传定位。在23个定位位点中,有12个定位在水稻8个染色体臂的最远端,并将所在染色体分别向外延长了7．7～22．6cM。其中有些可能是定位在亚端粒区。有5个位点被定位在着丝粒区,另外6个位点定位于染色体内其他区域。     

Fecal DNA analysis for identifying species and sex of sympatric carnivores: a noninvasive method for conservation on the Tsushima Islands, Japan

Fecal analysis is a useful tool for the investigation of food habits and species identity in mammals. However, it is generally difficult to identify the species based on the morphological features and contents of feces deposited by mammals of similar body size. Therefore we developed noninvasive DNA analysis methods using fecal samples for identification of the species and sex of four small sympatric carnivores living on the Tsushima Islands of Japan: the leopard cat (Felis bengalensis), Japanese marten (Martes melampus), Siberian weasel (Mustela sibirica), and feral cat (Felis catus). Based on DNA sequence data from previous phylogenetic studies, we designed species-specific primers for polymerase chain reaction (PCR) amplification of the partial mitochondrial cytochrome b gene (112-347 bp) to identify the species and primers for the partial SRY gene (135 bp) to determine the sex. Due to the adjustment of PCR conditions, those specific DNA fragments were successfully amplified and then applied for species and sex identification. Nucleotide sequences obtained from the PCR products corresponded with cytochrome b sequences of the carnivore species expected. The protocol developed could be a valuable tool in the management and conservation of the four carnivore species occurring on the Tsushima Islands.     

RAPD markers linked to brown planthopper Nilaparvatha lugens resistance locus in rice

Brown plant hopper, a major pest in rice causes "hopper burn" in the field. The resistance gene for brown planthopper was mapped by using 20 recombinant inbred lines (RIL's) derived from a cross between resistant line Oryza. officinalis derivative (IR 54742-2-21-12-17-6) and a susceptible rice cultivar ASD 16 using bulked segregant analysis. On an average of 4 loci were amplified and two RAPD primers amplified loci that co-segregated with resistance/susceptibility. The segregating RAPD loci were mapped using Mapmaker programme into 13 groups. The expected and the 95% confidence level were found to be 15.2 and 47.7 cM respectively, confirming the location of the brown planthopper resistant gene on the region of chromosome 4. These RAPD markers will accelerate breeding programme for brown planthopper resistance.     

A polymorphic microsatellite in the limit dextrinase gene of barley (Hordeum vulgare L.)

A DNA fragment containing the exons 16, 17 and intron 16 of the limit dextrinase gene was cloned using a 654 bp cDNA as probe. Intron 16 contained a simple sequence repeat (microsatellite). PCR primers were designed to amplify that microsatellite. Using these primers, the limit dextrinase gene was mapped to the short arm of chromosome 1 (7H) using 150 DH lines from the Steptoe × Morex mapping population. This gene co-segregated with the RFLP marker ABC154A. QTLs for malt extract, -amylase activity, diastatic power and fine-coarse difference previously mapped in the North American Barley Genome Mapping Project have been located in this chromosome region. Five limit dextrinase alleles were detected in 31 barley cultivars with a PIC of 0.75. Ten different alleles/genes were identified in 23 uncultivated Hordeum species or subspecies using these microsatellite primers. The primers also amplified one fragment from wheat and two from oat. This microsatellite should be useful for marker-assisted selection for malting quality.     

马传染性贫血病毒美洲流行株与我国弱毒疫苗株鉴别诊断方法的研究

马传染性贫血病毒(Equine infectious anemia virus,EIAV)是反转录病毒科慢病毒属的成员,是马传染性贫血病的病原。二十世纪七十年代我国就研制出马传染性贫血驴白细胞弱毒疫苗,成为世界第一个成功地应用该疫苗控制了我国的马传贫的发生[1]。而且我国的马传贫弱毒疫苗对异源的美国、古巴和阿根廷等毒株也有很高的保护率[2]。因此将我国的马传贫驴细胞弱毒疫苗推向国际市场成为可能。然而目前制约该苗出口的技术问题是现行的OIE推荐的琼脂扩散实验和ELISA等血清学方法不能鉴别自然感染马与我国弱毒疫苗免疫马,针对这个关键问题,本试验…     

Multicolor-based discrimination of 21 short tandem repeats and amelogenin using four fluorescent universal primers

The aim of this study was to develop a cost-effective genotyping method using high-quality DNA for human identification. A total of 21 short tandem repeats (STRs) and amelogenin were selected, and fluorescent fragments at 22 loci were simultaneously amplified in a single-tube reaction using locus-specific primers with 24-base universal tails and four fluorescent universal primers. Several nucleotide substitutions in universal tails and fluorescent universal primers enabled the detection of specific fluorescent fragments from the 22 loci. Multiplex polymerase chain reaction (PCR) produced intense FAM-, VIC-, NED-, and PET-labeled fragments ranging from 90 to 400 bp, and these fragments were discriminated using standard capillary electrophoretic analysis. The selected 22 loci were also analyzed using two commercial kits (the AmpFLSTR Identifiler Kit and the PowerPlex ESX 17 System), and results for two loci (D19S433 and D16S539) were discordant between these kits due to mutations at the primer binding sites. All genotypes from the 100 samples were determined using 2.5 ng of DNA by our method, and the expected alleles were completely recovered. Multiplex 22-locus genotyping using four fluorescent universal primers effectively reduces the costs to less than 20% of genotyping using commercial kits, and our method would be useful to detect silent alleles from commercial kit analysis.     

Highly polymorphic microsatellite loci for Speyeria idalia (Lepidoptera: Nymphalidae)

Four polymorphic microsatellite loci were identified in the butterfly Speyeria idalia. We constructed a phagemid library and screened approximately 120 000 inserts. Probing with GT15, we identified 36 positives and designed polymerase chain reaction (PCR) primers for 12 potential loci. Of those loci, only four consistently produced polymorphic, diallelic PCR products in the expected size range. These results are consistent with previous studies concerning the low frequency of microsatellite loci in the lepidoptera, although these four loci are highly polymorphic and therefore likely to provide information on the fine scale genetic structure among populations in this species.     

Generating single-copy nuclear gene data for a recent adaptive radiation

Recent adaptive radiations provide an exceptional opportunity to understand the processes of speciation and adaptation. However, reconstructing the phylogenetic history of recent and rapidly evolving clades often requires the use of multiple, independent gene genealogies. Nuclear introns are an obvious source of the necessary data but their use is often limited because degenerate primers can amplify paralogous loci. To identify PCR primers for a large number of loci in an especially rapid adaptive radiation, that of the flowering plant genus Aquilegia (Ranunculaceae), we developed an efficient method for amplifying multiple single-copy nuclear loci by sequencing a modest number of clones from a cDNA library and designing PCR primers; with one primer anchored in the 3' untranslated region (3'-UTR) and one primer in the coding region of each gene. Variation between paralogous loci evolves more quickly in 3'-UTR regions compared to adjacent exons, and therefore we achieved high specificity for isolating orthologous loci. Furthermore, we were able to identify genes containing large introns by amplifying genes from genomic DNA and comparing the PCR product size to that predicted from their cDNA sequence. In Aquilegia eight out of eleven loci were isolated with this method and six of these loci had introns. Among four genes sequenced for samples spanning the phylogenetic breadth of the genus, we found sequence variation at levels similar to that observed in ITS, further supporting the recent and rapid radiation in Aquilegia. We assessed the orthology of amplification products by phylogenetic congruence among loci, the presence of two well established phylogenetic relationships, and similarity among loci for levels of sequence variation. Higher levels of variation among samples for one locus suggest possible paralogy. Overall, this method provides an efficient means of isolating predominantly single-copy loci from both low and high-copy gene families, providing ample nuclear variation for reconstructing species-level phylogenies in non-model taxa.     

New murine polymorphisms detected by random amplified polymorphic DNA (RAPD) PCR and mapped by use of recombinant inbred strains

Oligonucleotide primers of random sequence that were 12 bases in length, 58% in GC content, and lacking internal palindromes were designed. By random amplified polymorphic DNA (RAPD) PCR, these primers were used to survey for DNA variations between the progenitors of the mouse AXB and BXA recombinant inbred sets (A/J and C57BL/6J). We identified 17 DNA variants detected by 10 primers. Map positions for these variants were determined by comparing their strain distribution patterns in the AXB, BXA recombinant inbred sets with strain distribution patterns of previously published loci. When necessary, BXD and NXSM recombinant inbred sets were also used. These 17 new loci mapped to 12 chromosomes. The 10 primers were also used to survey 20 inbred mouse strains including the progenitors of other recombinant inbred sets and four mouse strains recently inbred from the wild (CAST/Ei, MOLF/Ei, PERA/Ei, and SPRET/Ei).     

Genetic and physical mapping of Lrk10-like receptor kinase sequences in hexaploid oat (Avena sativa L.).

Oat receptor-like kinase gene sequences, homologous to the Lrk10 gene from wheat (Triticum aestivum L.), were mapped in oat (Avena sativa L.). PCR primers designed from the wheat Lrk10 were used to produce ALrk10 from oat. Two DNA sequences, ALrk1A1 and ALrk4A5, were produced from primers designed from coding and noncoding regions of ALrk10. Their use as RFLP probes indicated that the kinase genes mapped to four loci on different hexaploid oat 'Kanota' x 'Ogle' linkage groups (4_12, 5, 6, and 13) and to a fifth locus unlinked to other markers. Three of these linkage groups contain a region homologous to the short arm of chromosome I of wheat and the fourth contains a region homologous to chromosome 3 of wheat. Analysis with several nullisomics of oat indicated that two of the map locations are on satellite chromosomes. RFLP mapping in a 'Dumont' x 'OT328' population indicated that one map location is closely linked to Pg9, a resistance gene to oat stem rust (Puccinia graminis subsp. avenae). Comparative mapping indicates this to be the region of a presumed cluster of crown rust (Puccinia coronata subsp. avenae) and stem rust resistance genes (Pg3, Pg9, Pc44, Pc46, Pc50, Pc68, Pc95, and PcX). The map position of several RGAs located on KO6 and KO3_38 with respect to Lrk10 and storage protein genes are also reported.     

PCR detection of genes encoding nitrite reductase in denitrifying bacteria

Using consensus regions in gene sequences encoding the two forms of nitrite reductase (Nir), a key enzyme in the denitrification pathway, we designed two sets of PCR primers to amplify cd1- and Cu-nir. The primers were evaluated by screening defined denitrifying strains, denitrifying isolates from wastewater treatment plants, and extracts from activated sludge. Sequence relationships of nir genes were also established. The cd1 primers were designed to amplify a 778 to 799-bp region of cd1-nir in the six published sequences. Likewise, the Cu primers amplified a 473-bp region in seven of the eight published Cu-nir sequences. Together, the two sets of PCR primers amplified nir genes in nine species within four genera, as well as in four of the seven sludge isolates. The primers did not amplify genes of nondenitrifying strains. The Cu primers amplified the expected fragment in all 13 sludge samples, but cd1-nir fragments were only obtained in five samples. PCR products of the expected sizes were verified as nir genes after hybridization to DNA probes, except in one case. The sequenced nir fragments were related to other nir sequences, demonstrating that the primers amplified the correct gene. The selected primer sites for Cu-nir were conserved, while broad-range primers targeting conserved regions of cd1-nir seem to be difficult to find. We also report on the existence of Cu-nir in Paracoccus denitrificans Pd1222.     

Comparative mapping of mink (Mustela vison) chromosome 8p: localization of three human BAC clones

Using fluorescent in situ hybridization (FISH), three human BAC clones, localized in the terminal region of human chromosome 17p (HSA17p13; 1.44--3.68 Mp), were mapped to chromosome 8p of American mink (MVI8p). It was demonstrated that in MVI8p the region, homeologous to HSA17p13, was split into three fragments, which were detected within terminal, pericentric, and probably nucleolus-organizing regions. Using human BAC clones as heterologous markers for mapping of the gene sequences to the chromosomes of American mink, regional localization of eight sequences (PRPF8, SLC43A2, and RILP in MVI8p25; C17orf31 in MVI8p21-22; and CTNS, TAX1BP3, and P2RX5 in MVI8p11) was deduced.     

Comparative mapping of mink (<Emphasis Type="Italic">Mustela vision</Emphasis>) chromosome 8p: Localization of three human BAC clones

Using fluorescent in situ hybridization (FISH), three human BAC clones, localized in the terminal region of human chromosome 17p (HSA17p13; 1.44–3.68 Mp), were mapped in chromosome 8p of American mink (MVI8p). It was demonstrated that in MVI8p the region, homeologous to HSA17p13, was split into three fragments, which were detected within terminal, pericentric, and probably nucleolus-organizing regions. Using human BAC clones as heterologous markers for mapping of the gene sequences to the chromosomes of American mink, regional localization of eight sequences (PRPF8, SLC43A2, and RILP in MVI8p25; C17orf31 in MVI8p21-22; and CARKL, CTNS, TAX1BP3, and P2RX5 in MVI8p11) was deduced.     

Hybridization between escaped domestic and wild American mink (Neovison vison)

The release of domesticated organisms into natural populations may adversely affect these populations through predation, resource competition, and the introduction of disease. Additionally, the potential for hybridization between wild and domestic conspecifics is of great concern because it can alter the evolutionary integrity of the affected populations. Wild American mink ( Neovison vison ) populations may be threatened not only by competition for resources with domestic mink originating from farms, but by breeding with such escapees. Using 10 microsatellite loci, we genotyped mink from Ontario, Canada, sampled from two farms, two putatively mixed populations in regions surrounding the mink farms, and two wild populations with no recent history of mink farming. Using individual-based Bayesian population assignment, we identified four population clusters, including one wild, and three domestic populations. The latter were not clustered by farm but rather by distinct line-bred colour phases. Population clustering also identified domestic and hybrid mink in the free-ranging populations. Nearly two-thirds of the mink sampled in the two putatively mixed populations (78% and 43%) were either farm escapees or descendants of escapees. Principal components analysis of allele frequencies supported our Bayesian assignment results. The power of our assignment test was assessed using simulated hybrid genotypes which suggested that our overall correct classification rate was 96.2%. The overwhelming presence of domestic animals and their hybridization with mink in natural populations is of great concern for the future sustainability of wild mink populations.