Multilocus Probes Research Articles

A novel target-enriched multilocus assay for sponges (Porifera): Red Sea Haplosclerida (Demospongiae) as a test case.

With declining biodiversity worldwide, a better understanding of species diversity and their relationships is imperative for conservation and management efforts. Marine sponges are species-rich ecological key players on coral reefs, but their species diversity is still poorly understood. This is particularly true for the demosponge order Haplosclerida, whose systematic relationships are contentious due to the incongruencies between morphological and molecular phylogenetic hypotheses. The single gene markers applied in previous studies did not resolve these discrepancies. Hence, there is a high need for a genome-wide approach to derive a phylogenetically robust classification and understand this group's evolutionary relationships. To this end, we developed a target enrichment-based multilocus probe assay for the order Haplosclerida using transcriptomic data. This probe assay consists of 20,000 enrichment probes targeting 2956 ultraconserved elements in coding (i.e. exon) regions across the genome and was tested on 26 haplosclerid specimens from the Red Sea. Our target-enrichment approach correctly placed our samples in a well-supported phylogeny, in agreement with previous haplosclerid molecular phylogenies. Our results demonstrate the applicability of high-resolution genomic methods in a systematically complex marine invertebrate group and provide a promising approach for robust phylogenies of Haplosclerida. Subsequently, this will lead to biologically unambiguous taxonomic revisions, better interpretations of biological and ecological observations and new avenues for applied research, conservation and managing declining marine diversity.

Structure of genomic DNA in chicken populations revealed by multilocus DNA probe

Molecular genetic technologies are taking an increasing place in breeding work to improve existing breeds and populations of chickens, as well as in programs to preserve a valuable gene pool. Small local breeds are a source of valuable genes that can be used in breeding. The aim of this work was to obtain new knowledge about the structure of genomic DNA of six chicken populations using multilocus analysis with a labeled molecular probe (GTG)5. Multilocus analysis using labeled DNA probes provides working simultaneously with a large number of genetic loci and calculating population genetic parameters both within populations and between them. The data on use of the multilocus probe (GTG)5 in molecular hybridization reaction in six breeds and populations of chickens were analyzed. The results revealed a large genetic distance between Black-and-White Australorp and the Bald-necked chickens (D = 0.155). Bald-necked chickens are bred in isolation from other breeds to maintain the unique trait of naked necks. According to the criterion of average heterozygosity, the population of Bald-necked chickens surpassed the Yurlov Crowers and Black-and-White Australorps. Obviously, this is due to the intensive breeding work carried out in the last two populations, which reduces genetic diversity. Marker DNA fragments specific for individual breeds were identified. The effectiveness of multilocus analysis as a tool for identifying the features of genome organization in chicken breeds and populations was confirmed.

DNA fingerprinting analysis of Eucalyptus species

DNA fingerprinting is a straightforward approach for identifying variations at a locus. The term DNA-fingerprinting was introduced for the first time by Alec Jeffrey2 in 1985 to describe bar-code-like DNA fragment patterns generated by multilocus probes after electrophoretic separation of genomic DNA fragments. The emerging patterns make up an unique feature of the analyzed individual and are currently considered to be the ultimate tool for biological individualization. Recently, the term DNA fingerprinting/profiling is used to describe the combined use of several single locus detection systems and are being used as versatile tools for investigating various aspects of plant genomes. This technique used to detect the Cylindrocladium quinqueseptatum which infects Eucalyptus species, based on DNA based markers such as RAPD, PCR-RFLP, ITS. Eucalyptus is grown in many parts of the country it has become commercially very important as raw material for the pulp, paper and plywood industries of northern India. Some Eucalyptus species have attracted attention from global development researchers and environmentalists. Such species have desirable traits such as being fast-growing sources of wood, producing oil that can be used for cleaning and functions as a natural insecticide.

DNA fingerprinting analysis of subpopulations of the red squirrel (Sciurus vulgaris, L. 1758)

The relationships within two squirrel groups of 7 and 10 individuals living in small isolated forest areas (»20 ha) were studied by DNA fingerprinting. The study area was located in northwestern Germany, close to the town of Osnabruck. The degrees of relationship determined by the multi locus probes 33.15 and MZ1.3 were >20–40% higher in the two subpopulations compared with a group of unrelated squirrels. The band-sharing rates in the latter group varied between 6 and 57% (33.15) and 17 and 63% (MZ1.3), whereas the squirrels living in the two forest islands had band-sharing rates between 13 and 80%, depending on the probe used. Although the forest islands were isolated by fields and roads, there seemed to be a small genetic exchange between the squirrels of the forest islands and those of the next adjacent larger woodland area.

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.

Quantitation of fetal DNA in maternal serum during the first trimester of pregnancy by the use of a DAZ repetitive probe

Cell-free fetal DNA in maternal plasma or serum is at present widely investigated as a source of fetal genetic material, both in studies of pregnancy-related disorders and in planning strategies for non-invasive prenatal diagnosis. Despite the number of trials already performed on the quantitation of fetal DNA, data about the amount of DNA at the beginning of pregnancy, in particular in the first trimester, remain limited. A new probe mapping on the deleted in azoospermia (DAZ) repetitive region of the Yq chromosome was designed for an early assessment of fetal DNA concentration in maternal serum. Among 57 pregnant women prospectively studied in their first trimester, fetal DNA was detected already by the 5th gestational week, with the analysis becoming reliable by the 8th week of gestation when a 100% accuracy in fetal sex determination was achieved. Moreover, in the three cases of pregnancy ending in fetal loss, the amount of fetal DNA apparently decreased before the abortion was diagnosed, whereas it consistently showed an increasing trend in normal pregnancies. Real-time PCR with the use of DAZ multilocus probe can efficiently quantitate free fetal DNA in the maternal serum at the beginning of pregnancy.

A framework linkage map of bermudagrass (Cynodon dactylon × transvaalensis) based on single-dose restriction fragments

This study describes the first detailed linkage maps of two bermudagrass species, Cynodon dactylon (T89) and Cynodon transvaalensis (T574), based on single-dose restriction fragments (SDRFs). The mapping population consisted of 113 F1 progeny of a cross between the two parents. Loci were generated using 179 bermudagrass genomic clones and 50 heterologous cDNAs from Pennisetum and rice. The map of T89 is based on 155 SDRFs and 17 double-dose restriction fragments on 35 linkage groups, with an average marker spacing of 15.3 cM. The map of T574 is based on 77 SDRF loci on 18 linkage groups with an average marker spacing of 16.5 cM. About 16 T89 linkage groups were arranged into four complete and eight into four incomplete homologous sets, while 15 T574 linkage groups were arranged into seven complete homologous sets, all on the basis of multi-locus probes and repulsion linkages. Eleven T89 and three T574 linkage groups remain unassigned. In each parent consensus maps were built based on alignments of homologous linkage groups. Four ancestral chromosomes were inferred after aligning T89 and T574 parental consensus maps using multi-locus probes. The inferred ancestral marker orders were used in comparisons to a detailed Sorghum linkage map using 40 common probes, and to the rice genome sequence using 98 significant BLAST hits, to find regions of colinearity. Using these maps we have estimated the recombinational length of the T89 and T574 genomes at 3,012 and 1,569 cM, respectively, which are 61 and 62% covered by our maps.

Using DNA fingerprinting to establish paternity of Asian elephantsElephas maximusborn at Pinnawela Elephant Orphanage, Sri Lanka

The Asian elephant Elephas maximus is threatened with extinction unless intensive conservation and captive-breeding programmes are implemented without delay. The success of breeding programmes depends critically on maintenance of genetic diversity within captive populations; hence, the accurate assignment of paternity of progeny is of considerable importance. Determination of paternity from field observations is complicated by the fact that oestrous ♀♀ may mate with more than one ♂. The aim of this study was to use DNA fingerprinting to establish the paternity of three Asian elephant calves born at Pin-nawela Elephant Orphanage, Sri Lanka. DNA was extracted from blood samples taken from the calves, their dams and three putative sires, and fingerprinted using the multi-locus DNA probe 33.15. The sire of each calf was identified unambiguously by comparative analysis of the DNA fingerprints of the calf and dam with those of each bull. Results from a pairwise comparison of band-sharing probabilities confirmed the paternity identification.

DNA analysis : Current status in court

AbstractSince the introduction of DNA profiling the methods have change considerably and so have the way that results are expressed. There have been many different techniques used to produce a DNA profile. The starting point was multilocus probes, followed by single locus probes, neither of which are now widely used, and then Short tandem Repeat (STR) analysis. Whatever the next step in DNA analysis is, the methods of interpretation will remain essentially the same. We rely on the concept and reality of the benign state for this to be used for the good of society, not the oppression of individuals or groups.

Species-specific ‘fingerprints’ of deer in China

State governments in China have laid down laws for protecting rare and endangered species, but the effective protection depends on accurate species identification. Although DNA fingerprinting has appeared in court as legal evidence for many years, it is unsuitable for species identification as previous multilocus probes produce individual-specific ‘fingerprints’. This study outlines the development of a new species-specific probe – the pta2 probe. Using this oligonucleotide probe, the identification of all deer species in China is made possible by species-specific ‘fingerprints’. Material evidence can then be provided to any judicial organization, which will have a profound effect on the criminal activities of poachers, thereby helping to protect wild animals and conserve biodiversity. Interestingly, the pta2 probe can also reveal the gender of detected deer species.

DNA fingerprinting with homologous multilocus probes and search for DNA markers associated with yield attributes in silkworm, Bombyx mori

RFLP clones harbouring multi-copy DNA sequences were isolated from the Pst I sub-genomic library of the indigenous silkworm race, Nistari, and were used for DNA fingerprinting studies in 13 stocks of silkworm, Bombyx mori L. Six multilocus probes produced 180 RFLP markers that showed a high level (98%) of polymorphism and are highly useful in molecular mapping, genotype characterization and marker assisted selection (MAS). The dendrogram derived from UPGMA analysis clearly divides the 13 silkworm stocks into two major clusters: high- and low-yield stocks. Furthermore, adopting multiple regression analyses, the RFLP marker(s) associated with characters of economic importance were identified, a first of its kind for any species of insect of commercial importance. The results obtained create an opportunity of using germplasm stocks directly for isolating specific RFLP band(s) and use it for MAS in breeding programs.

A new oligonucleotide probe for the giant panda

AbstractQuantifying genetic diversity in populations is one of the fundamental measures for species conservation. This is far more important for critically endangered species like giant pandas, where there are few individuals remaining in the population. However, previous multilocus probes could not identify homozygous loci resulting from inbreeding of giant pandas, and produced few polymorphic loci. As a result, we have prepared a new oligonucleotide probe, which had the highest paternity probability and succeeded in identifying the homozygous loci and in discriminating giant panda individuals.

Minisatellite and Hprt mutations in V79 cells irradiated with helium ions and gamma rays

Purpose : To evaluate and compare cytotoxic and mutational effects of graded doses of γ-rays and 4 He ++ ions at different LET values (nominally 80 and 123 keV/ μ m) in V79 cells. Materials and methods : 4 He ++ ion beams at 80 and 123 keV/ μ m were supplied by the 7 MV Van de Graaff CN accelerator of the INFN-LNL in the dose range 0.3-2.4 Gy at a dose rate of 1 Gy/min. Gamma-irradiation was performed by the 60 Co ' γbeam' of CNR-FRAE (at the INFN-LNL) in the dose range 0.5-6.0 Gy at a dose rate of 1 Gy/min. After irradiation, the cells were seeded to measure surviving fraction (SF) and mutant frequency (MF) at the Hprt locus on the basis of 6-thioguanine resistance. Alterations at minisatellite sequences (MS) of clones derived from irradiated and unirradiated cells were detected by Southern blot analysis using a multi-locus probe (DNA fingerprinting). Results : Survival data from 4 He ++ irradiation at two LET values (80 and 123 keV/ μ m) yielded similar results: α =(1.08 ±0.04)/Gy and (0.90 ±0.03)/Gy, respectively. The best fit for mutant induction at the Hprt locus after 80 keV/ μ m 4 He ++ was a linear function of the dose in the dose-interval 0-1.5 Gy: α =(47.77 ±16.01) ×10 -6 /Gy. The best fit for mutant induction after 123 keV/ μ m 4 He ++ in the dose-interval 0-1.2 Gy was a linear-quadratic function: α =(86.01 ±13.80) ×10 -6 /Gy; β =(42.87 ±11.03) ×10 -6 /Gy 2. For γ-irradiation, the best fit of Hprt mutation data gave: α =(4.14 ±2.67) ×10 -6 /Gy; β =(0.63 ±0.86) ×10 -6 /Gy 2. The best fitting of MS alteration data with linear-quadratic or linear relationships gave: for γ-rays, α =0.56 mutants/Gy and β =0.52 mutants/Gy 2 ; for 80 keV/ μ m 4 He ++, α =3.70 mutants/Gy and β =9.00 mutants/Gy 2 ; for 123 keV/ μ m 4 He ++, α =4.36 mutants/Gy. Conclusions : The results reported here confirmed the higher cytotoxic and mutagenic effects of helium ions in comparison with γ-irradiation and the ability of DNA fingerprint analysis to investigate DNA damage induced by different ionizing radiations. The results of the mutagenic effects measured by the two tests are in agreement.

Characterisation of Isolates of Phytophthora infestans From Hungary

A total of 36 single-lesion isolates were collected from 9 crops of potato and 13 of tomato in different regions of Hungary in the past decade, particularly in 1998. These were analysed for mating type, sensitivity to metalaxyl, allozyme genotype at glucose-6-phosphate isomerase and peptidase loci and genotype at 24 loci detected using the multilocus RFLP probe RG57. The ratios of the mating types A1 to A2 were 8 : 9 and 4 : 15 among isolates recovered from potato and tomato, respectively. Resistance to metalaxyl was found more frequently among isolates from potato and in the A1 mating type. The populations were not clearly differentiated on the basis of host origin. All isolates were homozygous (100/100) at the locus for glucose-6-phosphate isomerase. Unlike in other European countries, the most common peptidase allele was 96. Genotypes at the peptidase locus were 96/96 (50%), 96/100 (27.7%) and 100/100 (16.6%). In addition, one isolate from 1993 and another from 1998, were defined as 83/96, a genotype that had not been described elsewhere. The 18 RG57 fingerprints that were observed among 36 isolates, with one exception, seem to be unique to Europe. On the basis of combined genotypic traits, 20 multilocus genotypes were designated. These data, which reveal a remarkable variability with unique genotypes of the late blight pathogen, suggest that migration and sexual and/or asexual recombination have a role in the recent evolution of the pathogen in Hungary.

AFLP and RFLP (RG57) fingerprints can give conflicting evidence about the relatedness of isolates of Phytophthora infestans

Selected isolates of Phytophthora infestans from around England and Wales were fingerprinted using both RG57, a multi-locus RFLP probe, and Amplified Fragment Length Polymorphisms (AFLPs). The larger number of polymorphisms detectable with the AFLP method allowed resolution of several similar AFLP genotypes among isolates with identical RG57 fingerprints. However, some isolates with the same RG57 genotype had remarkably dissimilar AFLP genotypes, suggesting that there has been convergent evolution of some RG57 fingerprints. Also, some isolates with dissimilar RG57 fingerprints had similar or identical AFLP fingerprints. Both techniques distinguished isolates of mitochondrial DNA haplotype la from those of haplotype IIa. However, with AFLPs only, most of the isolates of A2 mating type were very similar and were distinguished from those of A1 mating type, suggesting that gene flow between A1 and A2 genotypes is limited and that sexual recombination is rare.

Genetic diversity in the marine phytoplankton: a review and a consideration of Antarctic phytoplankton

Molecular analysis of phytoplankton population structure has lagged behind other groups and has usually been inferred from physiological data determined from relatively few clones. Nearly every physiological measurement has shown that no single clone of any phytoplankton species can be considered truly representative of that species. One important reason why studies of phytoplankton population structure are perhaps 20 or more years behind those of other organisms is because of the necessity to establish clonal cultures prior to genetic analysis and the inability to perform fine-scale sampling under most conditions. Isozyme analysis, performed for a few species, has revealed heterozygosity between populations. In addition, fingerprinting analyses, such as Random Amplified Polymorphic DNAs (RAPDs) or multi-locus probes, have shown that phytoplankton blooms are not mono-clonal, are highly diverse and isolates are related by geographic origin. In the Southern Ocean, only two studies have been made of the population structure of phytoplankton. The first, based on quantitative genetic analysis of morphometric features, suggests that there is sufficient genetic variation in populations of Thalassiosira tumida to allow speciation in terms of major shifts in morphology under conditions of continued directional selection. The second, using sequence data from the noncoding regions of the internal transcribed spacer region (ITS) in the ribosomal cistron as a molecular marker, shows that populations of Phaeocystis antarctica within continental water masses are homogenous with little evidence of population structure. Populations found within the Antarctic Circumpolar Current are genetically distinct from others, suggesting the currents also play an important role in determining population structure in phytoplankton populations.

Frequencies of multiple paternity in three Acrocephalus species (Aves Sylviidae) with different mating systems (A. palustris, A. arundinaceus, A. paludicola)

Genetic parentage was determined by DNA-fingerprinting with multilocus probes and microsatellite PCR in three Acrocephalus species with different mating systems: the socially monogamous marsh warbler (A. palustris), the polygynous great reed warbler (A. arundinaceus) and the promiscuous aquatic warbler (A. paludicola). The amount of extra-pair paternity (EPP)/multiple paternity (MP) was then related to ecological parameters (increasing productivity) and parental care (decreasing male participation) of Acrocephalus species. Marsh warblers (33 broods, 131 young plus putative parents) were studied over 3 years in a German population. No case of social polygyny but four extrapair young (EPY) were detected in three socially monogamous broods. The low frequency of extra-pair fertilization (EPF, 9.1% of broods, 3.1% of offspring) may be best attributed to intense male mate-guarding and restrictions on mate assessment due to an extremely short breeding window. The rate of social polygyny in a German great reed warbler population varied between 8 and 35% of males during a 5-year period. In 48 broods (27 monogamous and 21 polygynous males) with 194 young, 5 nests (3 of polygynous, 2 of monogamous males) with 19 EPY were recorded: EPF-rates are 10.4% of broods and 9.8% of offspring. In none of the nests were neighbouring males the genetic fathers, but in 2 nests, “bachelor” males from distant territories had sired the offspring. The EPP-rate in this species seems to be related to an incomplete correlation between male and territory characteristics leaving some females to correct their choice. Aquatic warblers were studied during three years in a Polish population. In each of the years, MP was recorded in more than half of a total of 75 broods (mean 76%). The proportion out of 340 young sired by one to five “additional” males varied between 25 and 57% (mean 39.1%). Food resources are neither critical nor economically defendable in the species' habitat and paternity assurance behaviours of males and female choice are yet not fully understood.

Locus-specific contig assembly in highly-duplicated genomes, using the BAC-RF method

Polyploidy, the presence of multiple sets of chromosomes that are similar but not identical, complicates both chromosome walking and assembly of sequence-ready contigs for many plant taxa including a large number of economically-significant crops. Traditional 'dot-blot hybridization' or PCR-based assays for identifying BAC clones corresponding to a mapped DNA landmark usually do not provide sufficient information to distinguish between allelic and non-allelic loci. A restriction fragment matching method using pools of BAC DNA in combination with dot-blots reveals the locus specificity of individual BACs that correspond to multi-locus DNA probes, in a manner that can efficiently be applied on a large scale. This approach also provides an alternative means of mapping DNA loci that exploits many advantages of 'radiation hybrid' mapping in taxa for which such hybrids are not available. The BAC-RF method is a practical and reliable approach for using high-density RFLP maps to anchor sequence-ready BAC contigs in highly-duplicated genomes, provides an alternative to high-density robotic gridding for screening BAC libraries when the necessary equipment is not available, and permits the expedient isolation of individual members of multigene or repetitive DNA families for a wide range of genetic and evolutionary investigations.

DNA fingerprinting reveals polygyny in the subterranean rodent Ctenomys talarum.

DNA fingerprinting was used to characterize patterns of paternity in two populations of Ctenomys talarum from Buenos Aires Province, Argentina. The multilocus probe PV47-2 was used to detect variation in genomic DNA extracted from 12 females, their 32 offspring, and 14 putative sires. For 11 out of 12 litters examined, a single male capable of providing all nonmaternal bands was identified. Within each study population, individual males sired more than one litter, suggesting that C. talarum is polygynous. No evidence of multiple paternity of litters was found. High band-sharing values among females suggest that further research is needed to assess the population genetic structure of this species.

Population structure and dynamics of Magnaporthe grisea in the Indian Himalayas.

The population genetics of Magnaporthe grisea, the rice blast pathogen, were analyzed in a center of rice diversity (the Uttar Pradesh hills of the Indian Himalayas) using multilocus and single-, or low-copy, DNA markers. Based on DNA fingerprinting with the multilocus probe MGR586 and single-locus probes, 157 haplotypes clustered into 56 lineages (at >/=70% MGR586 band similarity, each with unique single-locus profiles) and high diversity indices were detected among 458 isolates collected from 29 sites during 1992-1995. Most valleys sampled had distinct populations (73% of the lineages were site specific) with some containing one or a few lineages, confirming the importance of clonal propagation, and others were very diverse. Widely distributed lineages suggested that migration occurs across the region and into the Indo-Gangetic plains. Repeated sampling at one site, Matli, (170 isolates, 1992-1995) yielded 19 lineages and diversity significantly greater than that reported from similar samples from Colombia and the Philippines. Analysis of allelic associations using pairwise comparisons and multilocus variance analysis failed to reject the hypothesis of gametic phase equilibrium. The Matli population shifted from highly diverse in 1992 to almost complete dominance by one lineage in 1995. Such population dynamics are consistent with recombination followed by differential survival of clonal descendants of recombinant progeny. At another site, Ranichauri, population (n = 84) composition changed from 2 to 11 lineages over 2 yr and yielded additional evidence for equilibrium. Sexually fertile and hermaphrodite isolates of both mating types were recovered from rice in both Matli and Ranichauri. We demonstrate that Himalayan M. grisea populations are diverse and dynamic and conclude that the structure of some populations may be affected to some extent by sexual recombination.