Paternity assessment in rhesus macaques (Macaca mulatta): Multilocus DNA fingerprinting and PCR marker typing

Establishing kinship relations in primates using modern molecular genetic techniques has enhanced the ability to scrutinize a number of fundamental biological issues. We screened 51 human short tandem repeats (STRs) for cross-species PCR amplification in rhesus macaques (Macaca mulatta) and identified 11 polymorphic loci with heterozygosity rates of at least 0.6. These markers were used for paternity testing in three social groups (M, R, and S) of rhesus macaques from Cayo Santiago, Puerto Rico. Several consecutive birth cohorts were analyzed in which approximately 200 males were tested for paternity against more than 100 mother/infant pairs. Despite a combined exclusion rate of more than 99.9% in all three groups, some cases could not be solved unequivocally with the STR markers and additional testing of the MHC-associated DQB1 polymorphism. A final decision became possible through multilocus DNA fingerprinting with one or more of the oligonucleotide probes (GATA)₄, (CA)₈, and (CAC)₅. Paternity assessment by multilocus DNA analysis with probe (CAC)₅ alone was found to have limitations in rhesus macaques as regards the number of potential sires which might be involved in a given case. Multilocus DNA fingerprinting requires large amounts of DNA, and the ensuing autoradiographic patterns present difficulties in comparisons across gels and even within the same gel across remote lanes. Computer-assisted image analysis was incapable of eliminating this problem. Therefore, a dual approach to DNA typing has been adopted, using STR markers to reduce the number of potential sires to a level where all remaining candidates can be tested by multilocus DNA fingerprinting on a single gel, preferably in lanes adjacent to the mother/infant pair. Am. J. Primatol. 44:1–18, 1998. © 1998 Wiley-Liss, Inc.     

Power and limits of DNA-profiling in primate populations: Paternity assessment in rhesus macaques from Cayo Santiago

Paternity testing was performed in one social group (S) of rhesus macaques from Cayo Santiago, Puerto Rico. In 11/15 cases, sires could be identified comparing the multilocus DNA profiles of 19 males to those of the corresponding mother/child dyads. All 19 males could be excluded from paternity in the remaining four cases. Decision making was partly based on likelihoods of DNA profiles, and the theoretical model underlying these calculation is described. In a second social group (M), held in captivity, paternity testing was impeded by a deficit of maternal bands and by an increased extent of band sharing of mothers and their infants. Some possible explanations for these findings, including increased homozygosity in group M, are discussed.     

DNA “fingerprinting” and the genetic management of a captive chimpanzee population (Pan troglodytes)

DNA fingerprinting probes are cloned sequences which simultaneously detect a large number of similar hypervariable loci in the target DNA. The resulting highly polymorphic pattern visualized on an autoradiograph allows resolution of questions concerning individual identification and parentage. M13 bacteriophage has been used as a DNA fingerprinting probe for paternity ascertainment among captive chimpanzees housed in multi-male groups as part of the National Chimpanzee Breeding and Research Program. In 31 cases of unknown paternity where DNA samples for mother, offspring, and all potential sires were available, DNA fingerprinting with M13 resulted in the unambiguous assignment of paternity for all 31 infants. Knowledge of pedigrees among the captive-born animals is used to address several issues important in the genetic management of captive breeding colonies, including estimation of effective population size and of the rate of decline in genetic variability, variance in male and female reproduction, and the effect of social dominance on male reproductive success. Our analysis demonstrates the beneficial effects of genetic management by comparing the managed dedicated cohort to the Bastrop colony as a whole.     

Male rank,reproductive behavior,and reproductive success in free-ranging rhesus macaques

Paternity assessment through DNA fingerprinting by synthetic oligonucleotide probes was applied to one birth cohort in a social group of free-ranging rhesus macaques (Macaca mulatta) on Cayo Santiago. The 11 group males and 9 males from other groups were observed mating with the females. Paternity was determined for 11 of the 15 infants. Male dominance rank was not associated with reproductive success. High-ranking resident males (N=5) sired 27% of the infants born during a one-year study. Four of the 11 infants of known paternity were sired by males of other social groups. The four infants of unknown paternity were sired either by males not observed mating with the females or the low-ranking male who was not fingerprinted. Male dominance rank was not associated with reproductive activity during conception cycles. These results suggest that the effect of rank on male reproductive success is not a predictable correlation, but a conditional probability.     

大口黑鲈微卫星DNA指纹图谱的构建和遗传结构分析

以国内目前养殖的大口黑鲈[Micropterus salmoides(Lacépède)]群体(CH)、2009年引进的佛罗里达亚种(FL-09)、2010年引进的佛罗里达亚种(FL-10)、2010年引进的北方亚种(NT-10)为实验材料,应用43个微卫星DNA标记对这4个大口黑鲈群体进行遗传检测,构建了各群体的微卫星DNA指纹图谱,并对其遗传结构进行分析。结果显示:CH、FL-09、FL-10和NT-10群体的平均等位基因数(A)分别为2.58、3.74、3.70和4.21,平均期望杂合度(He)分别为0.4549、0.4896、0.5010和0.6138,平均多态信息量(PIC)分别为0.3786、0.4443、0.4566和0.5546,表明国内目前养殖的大口黑鲈群体遗传多样性水平远远低于国外新引进的大口黑鲈群体。利用UPGMA法对4个群体进行聚类,结果 FL-09和FL-10聚为一支,遗传距离为0.0506;NT-10和CH聚为另一支,遗传距离为0.4244,推测FL-09和FL-10两个佛罗里达亚种属于相同的群体,而NT-10和CH两个北方亚种来自不同的群体,甚至不同的水系。同时从指纹图谱中,筛选到5个特异的微卫星标记(JZL114、MiSaTPW11、Lma120、Mdo6和Msal21),可以鉴别FL、NT-10和CH群体,其中MiSaTPW11和Msal21这两个标记组合可以完全鉴别这3个群体。将5个特异性微卫星标记的图谱数据转化成计算机可以识别的数码指纹,可以方便应用于大口黑鲈不同群体及其杂交种的鉴定。研究结果可以为我国大口黑鲈种质资源保存、品种鉴定和良种选育提供理论依据。     

M13 DNA fingerprinting, a new tool for classification and identification of Lactobacillus spp.

The optimal conditions for the application of M13 DNA fingerprinting to the genus Lactobacillus were determined. Comparative fingerprint analysis of representative strains of Lactobacillus delbrueckii subsp. delbrueckii, Lact. delbrueckii subsp. lactis, Lact. delbrueckii subsp. bulgaricus, Lact. helveticus and Lact. casei permitted the differentiation of species, subspecies and individual strains and the quantitative determination of their genetic relatedness. The results confirm the high specificity of M13 DNA fingerprinting and indicate that it might be used in the classification of Lactobacillus spp.     

M13 DNA fingerprinting, a new tool for classification and identification of Lactobacillus spp.

The optimal conditions for the application of M13 DNA fingerprinting to the genus Lactobacillus were determined. Comparative fingerprint analysis of representative strains of Lactobacillus delbrueckii subsp. delbrueckii, Lact. delbrueckii subsp. lactis, Lact. delbrueckii subsp. bulgaricus, Lact. helveticus and Lact. casei permitted the differentiation of species, subspecies and individual strains and the quantitative determination of their genetic relatedness. The results confirm the high specificity of M13 DNA fingerprinting and indicate that it might be used in the classification of Lactobacillus spp.     

Assessment of DNA damage and repair in adults consuming allyl isothiocyanate or Brassica vegetables

Allyl isothiocyanate (AITC) is a dietary component with possible anticancer effects, though much information about AITC and cancer has been obtained from cell studies. To investigate the effect of AITC on DNA integrity in vivo, a crossover study was conducted. Adults (n= 46) consumed AITC, AITC-rich vegetables [mustard and cabbage (M/C)] or a control treatment with a controlled diet for 10 days each. On day 11, volunteers provided blood and urine before and after consuming treatments. Volunteers were characterized for genotype for GSTM1 and GSTT1 (glutathione S-transferases) and XPD (DNA repair). DNA integrity in peripheral blood mononuclear cells was assessed by single-cell gel electrophoresis. Urine was analyzed for 8-oxo-7,8-dihydro-2’-deoxyguanosine (8-oxodG) and creatinine. Ten-day intake of neither AITC nor M/C resulted in statistically significant differences in DNA strand breaks [least squares mean (LSmean) % DNA in tail±S.E.M.: 4.8±0.6 for control, 5.7±0.7 for AITC, 5.3±0.6 for M/C] or urinary 8-oxodG (LSmean μg 8-oxodG/g creatinine±S.E.M.: 2.95±0.09 for control, 2.88±0.09 for AITC, 3.06±0.09 for M/C). Both AITC and M/C increased DNA strand breaks 3 h postconsumption (LSmean % DNA in tail±S.E.M.: 3.2±0.7 for control, 8.3±1.7 for AITC, 8.0±1.7 for M/C), and this difference disappeared at 6 h (4.2±0.9 for control, 5.7±1.2 for AITC, 5.5±1.2 for M/C). Genotypes for GSTM1, GSTT1 and XPD were not associated with treatment effects. In summary, DNA damage appeared to be induced in the short term by AITC and AITC-rich products, but that damage disappeared quickly, and neither AITC nor AITC-rich products affected DNA base excision repair.     

Postpartum fertility in beef cattle producing twins

Postpartum fertility was measured in 42 plur iparous Hereford cows and first calf Hereford heifers that calved after embryo transfer to induce twins. Dams were exposed to a Hereford bull from 4: 00 P.M. to 8:00 A.M. each day from 60 days postpartum until pregnancy was confirmed or calves were weaned at 180 days of age. Days open ($\text{X}\text{± SE}$) for dams that produced single and twin calves were 84.1 ± 4.6 (n = 12) and 94.9 ± 6.2 (n = 30), respectively. Corresponding values for dams that nursed one calf, including six females that lost one calf of a twin set at birth, and dams that nursed twins were 89.3 ± 6.4 (n = 18) and 93.4 ± 8.5 (n = 24). No significant differences were observed due to calving or suckling twin calves. Heifers that calved twins had a shorter mean interval to conception than cows that calved twins. These results are interpreted to mean that with proper management during the prepartum and postpartum periods, reduced fertility in beef cattle that produce twins need not occur.     

Paternity determination in captive lowland gorillas and orangutans and wild mountain gorillas by microsatellite analysis

Paternity exclusion studies provide useful information for testing certain theories of behavioral ecology and for the management and conservation of both wild and captive populations of endangered species. This study used eight human nuclear microsatellite loci, in the absence of species-specific PCR primers, to genetically identify the sires of 12 captive lowland gorillas (Gorilla gorilla gorilla) and 2 captive orangutans (Pongo pygmaeus pygmaeus andPongo p. abelii). Parentage assignments were confirmed by excluding all except a single potential sire for each offspring with the least two loci. Sire-offspring relationships were verified in 12 of the 14 cases, and reassigned in the case of two gorilla offspring. The orangutan paternity typing was supplemented by DNA fingerprinting. Additionally, five of the eight microsatellite loci, in conjunction with behavioral data, were used for a non-exhaustive set of paternity exclusions for five wild mountain gorillas (Gorilla g. beringei). The eight loci described in this study should be useful additions to the tools available for the study of genetics in the great apes.     

Mating structure and genetic relatedness among gynes in the primitively eusocial wasp Polistes snelleni (Hymenoptera: Vespidae)

Mating structure and genetic relatedness among gynes (potential reproductive females) of the paper wasp Polistes snelleni were investigated using DNA microsatellite markers. All colonies had one foundress inseminated by a single male, and no sign of inbreeding was detected. The mean genetic relatedness among gynes was 0.734 ± 0.028 (SE), which is not significantly different from the expected value of 0.75 for full sisters.     

GENETIC VARIABILITY AND SPATIAL SEPARATION IN THE SEA PALM KELP POSTELSIA PALMAEFORMIS (PHAEOPHYCEAE) AS ASSESSED WITH M13 FINGERPRINTS AND RAPDS1

Postelsia palmaeformis Ruprecht is an annual species, occuring from southern California to Vancouver Island, Canada, in upper intertidal sites exposed to extreme wave shock. Because of its limited spore dispersal, discrete and inbred populations are likely on the local scale, yet dispersal of drifting and fertile thalli raises the possibility of outbred populations on a regional scale. M13 minisatellite DNA fingerprinting and random amplified polymorphic DNA (RAPD) marks were used in a complementary fashion to investigate genetic variability among 24 individuals on scales of clusters (= coalesced holdfasts). < 1 m, 10 m, 25 m, 16 km, and 250 km. Based on M13 fingerprinting, genetic relatedness within clusters was extremely high. Three of six clusters had at hast two identical individuals, and similarity values within five clusters were ≧0.90. Similarities between two of three clusters separated by < 1 m were significantly higher than between cluster pairs separated by 25 m and 250 km: however, the similarity between two clusters separated by 25 m was equivalent to the similarity between two clusters separated by 250 km. Thus, genetic relatedness as determined by M13 fingerprinting generally decreased as distance increased to 25 m. Conversely, RAPD data easily discriminated populations separated by 16 and 250 km but were not useful in discriminating individuals from < 1 to 25 m. Results from the complementary data sets suggest that most dispersal occurs over distances of 1–5 m, individuals within a cluster are siblings, and distinguishable biogeographic populations are present along the coast.     

Cross-amplification tests of ungulate primers in the endangered Neotropical pampas deer (Ozotoceros bezoarticus)

In cross-species amplification tests of 15 ungulate primers in pampas deer, five were retained to form a small panel of highly polymorphic loci that could be used to efficiently screen populations of this endangered species. The polymerase chain reactions were performed incorporating the universal fluorescent labeled M13 (-21) primer. In 69 pampas deer, average allelic diversity was 15, expected heterozygosity was 0.869 and the mean polymorphic information content value was 0.847. Paternity exclusion probabilities over loci were NE-1P = 0.01336 and NE-2P = 0.00135, and combined non-exclusion probability of identity was P(ID) = 3 x 10(-8).     

DNA primase associated with 10 S DNA polymerase α from calf thymus

Among multiple subspecies of DNA polymerase α of calf thymus, only 10 S DNA polymerase α had a capacity to initiate DNA synthesis on an unprimed single-stranded, circular M13 phage DNA in the presence of ribonucleoside triphosphates (DNA primase activity). The primase was copurified with 10 S DNA polymerase α through the purification and both activities cosedimented at 10 S through gradients of either sucrose or glycerol. Furthermore, these two activities were immunoprecipitated at a similar efficiency by a monoclonal antibody directed against calf thymus DNA polymerase α. These results indicate that the primase is tightly bound to 10 S DNA polymerase α. The RNA polymerizing activity was resistant to α-amanitin, required high concentration of all four ribonucleoside triphosphates (800 μM) for its maximal activity, and produced the limited length of oligonucleotides (around 10 nucleotides long) which were necessary to serve as a primer for DNA synthesis. Covalent bonding to RNA to DNA was strongly suggested by the nearest neighbour frequency analysis and the DNAase treatment. The DNA synthesis primed by the RNA oligomers may be carried out by the associating DNA polymerase α because it was strongly inhibited by araCTP, resistant to d₂TTP, and was also inhibited by aphidicolin but at relatively high concentration. The primase preferred single-stranded DNA as a template, but it also showed an activity on the double-stranded DNA from calf thymus at an efficiency of approx. 10% of that with single-stranded DNA.     

Founder contribution and pedigree inference in a captive breeding colony of lion-tailed macaques,using mitochondrial DNA and DNA fingerprint analyses

Captive colony genetic management is dependent on knowledge of the colony pedigree. In most zoos, records for colonies held prior to the past decade are incomplete or nonexistent. We have used a nested set of molecular techniques to infer genetic relationships in a colony of 15 lion-tailed macaques after twenty years of unmonitored reproduction. The troop was first divided into lines of matrilineal descent using mitochondrial DNA RFLP patterns. Paternity and maternity were then assigned on the basis of DNA fingerprint analysis. As mtDNA is maternally inherited, it is of no use in paternity assessment, but enabled subdivision of the colony into maternal lines. DNA fingerprinting would have been of little use by itself because of high levels of relatedness in several generations of offspring within the colony. The combination of the two techniques, however, enabled strong inference of both paternity and maternity in all cases. These data were instrumental in the inclusion of this troop in the AAZPA Species Survival Plan.     

M13 phage DNA as a universal marker for DNA fingerprinting of animals, plants and microorganisms

Hypervariable polymorphic patterns were detected with M13 phage DNA as a probe in genomic DNA of organisms belonging to different taxonomic groups including animals (vertebrates and invertebrates), plants and microorganisms. Individual-specific restriction pattern analysis (DNA fingerprinting) with this probe proved to be useful for individual identification, analysis of somatic stability and paternity testing in man. The nuclear type of inheritance indicates that the hypervariable DNA regions in question are located in the chromosomes, not in the mitochondrial DNA. The data obtained also demonstrate a potential range of M13 DNA applications as a probe for DNA fingerprinting of animals, plants and microorganisms, particularly for the determination of inbred lines, identification of bacterial strains and establishing stock, variety and strain distinctions.     

Maternal Behavior Indicates Survival and Cause-Specific Mortality of Moose Calves

Continuing research on cause-specific mortality and annual survival of moose (Alces alces) calves in northeastern Minnesota, USA, is important to understanding the long-term trajectory of the population. In 2013 and 2014, we observed global positioning system (GPS)-collared, female moose exhibit a specific behavior (i.e., mortality movement) associated with the death of their GPS-collared neonate. The females made a rapid, long-distance movement (flee), followed by a return to the calf mortality site. We used characteristics of this movement in 2013–2014 (n = 46) to develop models for assessing calf survival, and then evaluated these models using female movement rates (n = 49) in 2015−2016. Using this behavior as an indicator of calf mortality in 2016, we conducted field investigations, leading to evidence of 15 mortalities at a mean age of 30.6 ± 15.5 (SE) days (range = 3–243 days). We launched 21 investigations in response to a mortality movement and they resulted in confirmation of 11 of the 15 calf mortalities. Specific causes of mortality included 9 wolf (Canis lupus)-kills, 3 black bear (Ursus americanus)-kills, 1 unknown predator-kill, and 2 deaths following vehicle collisions. The mean distance females fled after a mortality was 1,873 ± 412 m (range = 126–5,805 m, n = 14). Females that made return visits returned a mean 2.8 ± 0.5 times (range = 1–5, n = 8) to within a mean 106 ± 22 m (range = 34–230 m, n = 8) of the mortality site. Calf survival to 30 days of age was 67 ± 8% (95% CI = 53–84%, n = 36) but declined to 53 ± 8% (95% CI = 39–72%, n = 36) by 3 months of age. We developed 2 population-level movement models to improve the efficacy of using the mortality movement to identify and locate calf mortalities in real time via field investigations. The first approach, a temporal-based model, used a 3-day average movement velocity threshold (118 m/hr) for all females to indicate calf mortality and accurately predicted survival status in 51% (n = 105) of the cases. The second approach, an age-specific model using different thresholds (28–135 m/hr) for females relative to calf age, was 80% (n = 231) accurate. Using movement behavior of females to assess calf mortality yielded important insights into mechanisms influencing the population decline that will inform future management decisions. © 2019 The Wildlife Society     

Long-term sperm storage in the Siberian crane (<Emphasis Type="Italic">Grus leucogeranus</Emphasis> pallas): Analysis of paternity and relatedness under artificial insemination

Using ten microsatellite loci, paternity analysis has been conducted for 71 individuals of the Siberian crane (Grus leucogeranus Pallas) obtained under artificial insemination in Oka Crane Breeding Center in 2001–2014. The fathers of 39 chicks were the sires whose sperm was used for insemination directly before fertilized egg laying. Paternity of 23 fertilizations belonged to the sires whose sperm was used in the beginning or middle of insemination cycle. Nine cases of fertilization resulted from natural copulation of artificially inseminated females with their social partners. The terms of sperm storage in the female’s reproductive ducts before fertilization were 0–6 days in the case of paternity of the last sperm donor and 2–15 days in the case of competing sperm by previous donors. Genetic relatedness by microsatellite loci between breeders of the captive Siberian crane population does not prevent fertilization and does not always lead to inbreeding depression.     

DNA relatedness among some thermophilic members of the genus Methanobacterium: emendation of the species Methanobacterium thermoautotrophicum and rejection of Methanobacterium thermoformicicum as a synonym of Methanobacterium thermoautotrophicum.

DNA reassociation was used to determine levels of relatedness among four thermophilic Methanobacterium strains that are able to use formate and between these organisms and two representative strains of Methanobacterium thermoautotrophicum, strain delta HT (= DSM 1053T = ATCC 29096T) (T = type strain) and strain Marburg (= DSM 2133). Three homology groups were delineated, and these groups coincided with the clusters identified by antigenic fingerprinting. The first group, which had levels of cross hybridization that ranged from 73 to 99%, included M. thermoautotrophicum delta HT, Methanobacterium thermoformicicum Z-245, Methanobacterium sp. strain THF, and Methanobacterium sp. strain FTF. The second and third groups were each represented by only one strain, Methanobacterium sp. strain CB-12 and M. thermoautotrophicum Marburg, respectively (cross-hybridization levels, 13 to 30 and 29 to 33%, respectively). Our results indicate that the name M. thermoformicicum should be rejected as it is a synonym of M. thermoautotrophicum. The taxonomic positions of strains Marburg and CB-12 need further investigation.     

Decrease of plasma taurine in Gaucher disease and its sustained correction during enzyme replacement therapy

Summary. Gaucher disease is caused by an autosomal-recessive deficiency of glucocerebrosidase. Cells of monocytic/macrophagic origin accumulate glucosylceramide. This leads to hepatosplenomegaly, bone destruction, thrombocytopenia and anemia. Enzyme replacement therapy (ERT) with macrophage-targeted glucocerebrosidase leads to normalization of these parameters. The way of macrophage activation in Gaucher disease is not known. Recently, the osmolytes taurine, betaine and inositol were identified as important regulators of macrophage function in liver. Therefore, the role of plasma taurine in Gaucher disease as a primarily macrophage-derived disease was studied. Fasting plasma levels were measured from blood samples of healthy control subjects (n = 29, m : f = 11 : 18, mean age 37 ± 3 years), from un-treated Gaucher patients (n = 16, m : f = 7 : 9, mean age 44 ± 4 years) and those treated for 37 ± 2 months (n = 54, m : f = 19 : 35, mean age 47 ± 2 years). Amino acid analysis was carried out in a BioChrom amino acid analyzer. In the untreated patients, plasma taurine was 45 ± 3 μM, as compared to the controls with a plasma taurine of 63 ± 4 μM (p < 0.01). The aver-age increase of plasma taurine during the first year of ERT was 18 ± 8 μM (n = 10). Patients treated for an average of 37 months (range 1–9 years of ERT) had a plasma taurine of 65 ± 4 μM (n = 54), which was not different from the controls. It is concluded that Gaucher patients show decreased plasma taurine levels and that therapy of Gaucher disease might correct this. It has to be established, whether decreased taurine availability is a cofactor of the permanent activation of glucosylceramide-storing monocytes/macrophages in this disease. Received January 25, 2000/Accepted January 31, 2000