DNA fingerprinting in cattle using oligonucleotide probes

Oligonucleotide probes specific for simple tandem repeat sequences produce individual specific DNA fingerprints in man and all animal species tested so far. Here 11 different synthetic probes were hybridized to bovine genomic DNAs which had been digested with the restriction endonucleases HinfI, AluI and HaeIII. Two of these probes gave DNA fingerprint patterns which were analysed for three German breeds. Different parameters were calculated, such as the average number of bands per individual or the probability of finding identical fingerprints in two unrelated individuals. The number of polymorphic bands varies from 11 to 23 in the different breeds and the probability of finding the same banding pattern in two unrelated individuals ranges from 1.5 x 10(-7) to 2.4 x 10(-7). Hence this DNA fingerprinting procedure allows precise identification of individuals. It is also a useful additional method for paternity testing in cattle.     

The efficiency of multilocus DNA fingerprint probes for individualization and establishment of family relationships, determined from extensive casework.

The properties of human DNA fingerprints detected by multilocus minisatellite probes 33.6 and 33.15 have been investigated in 36 large sibships and in 1,702 Caucasian paternity cases involving the analysis of over 180,000 DNA fingerprint bands. The degree of overlap of minisatellite loci detected by these two probes is shown to be negligible (approximately 1%), and the resulting DNA fingerprints are therefore derived from independent sets of hypervariable loci. The level of allelism and linkage between different hypervariable DNA fragments scored with these probes is also low, implying substantial statistical independence of DNA fragments. Variation between the DNA fingerprints of different individuals indicates that the probability of chance identity is very low (much less than 10(-7) per probe). Empirical observations and theoretical considerations both indicate that genetic heterogeneity between subpopulations is unlikely to affect substantially the statistical evaluation of DNA fingerprints, at least among Caucasians. In paternity analysis, the proportion of nonmaternal DNA fragments in a child which cannot be attributed to the alleged father is shown to be an efficient statistic for distinguishing fathers from nonfathers, even in the presence of minisatellite mutation. Band-sharing estimates between a claimed parent and a child can also distinguish paternity from nonpaternity, though with less efficiency than comparison of a trio of mother, child, and alleged father.     

DNA polymorphism in the major histocompatibility complex of man and various farm animals

In the past few years it has been possible by combining enzymatic cleavage of genomic DNA and the Southern blot hybridization technique to explore the endonuclease recognition site polymorphism of the MHC. HLA class I and DR and DQ alpha and beta class II specific probes as well as human C4 and Bf class III probes were used. All these probes were shown to cross-hybridize with DNA from pigs, cattle, sheep and horses. Hybridization of human genomic DNA with a class I probe showed 15-25 bands per genome depending on the enzyme used. Distinct endonucleases generated clusters of restriction fragments (RF) in HLA-informative families which correlated with HLA specificities. While numerous clusters were found associated with HLA-A alleles almost no cluster was related to HLA B or C specificities. Similarly, class II probes provided a large number of clusters. The existence of these clusters suggested that some polymorphic restriction sites are found in strong linkage disequilibrium and that the underlying mechanism might be gene conversion with heteroduplex correction. Since the degree of polymorphism detected by RF appears to be greater than the polymorphism defined by more traditional methods stronger associations between RF and pathological conditions are to be expected. Southern blot analysis was applied to unrelated pigs and sheep, as well as to families. Preliminary studies have also been performed on a few unrelated cattle and horses. Depending on the endonuclease used the HLA class I probe hybridized with around 15 bands in MHC heterozygous pigs and ruminants while up to 20 bands were found in horses. Therefore, a several-fold greater number of potential class I genes exist compared to those actually expressed. With the class II beta probe, cattle and sheep showed around 10 bands whereas 15 were observed in pigs and around 20 in horses. Based on limited results obtained with DQ alpha and beta probes and with the DR alpha probe there appeared to be fewer of these respective genes. Only one C4 gene has been detected in pig and this gene maps within the SLA region. Hybridization with the human C4 probe in cattle, sheep and horses revealed two to four bands which could possibly account for two C4 genes. To date their linkage to the MHC has not been established. The Southern blot hybridization technique represents a powerful tool for future immunogenetic studies.(ABSTRACT TRUNCATED AT 400 WORDS)     

DNA fingerprinting in domestic animals using four different minisatellite probes

Four probes known to allow DNA fingerprinting in the human (M13, Jeffreys' core sequence, the human alpha globin hypervariable region [HVR], and a mouse probe related to the Drosophila Per gene) were checked for their ability to reveal "genetic bar codes" in cattle, horses, pigs, dogs, chickens, and a European cyprinid fish, the barbel (Barbus barbus L.). Individual-specific patterns were obtained in cattle using M13, Jeffreys' core sequence, and the alpha globin HVR, in horses, dogs, and pigs using M13, Jeffreys' core sequence, and the Per probe, and in chicken and fish using the four different probes. Although we observed a considerable heterogeneity in the extent of interindividual variation, depending on the particular probe-species combination, the fingerprints are polymorphic enough to be used efficiently in animal identification, paternity testing, and as a source of genetic markers for linkage analysis. These markers should substantially accelerate the mapping of genes affecting economically important traits.     

Identification of molecular markers in soybean comparing RFLP,RAPD and AFLP DNA mapping techniques

Three different DNA mapping techniques—RFLP, RAPD and AFLP—were used on identical soybean germplasm to compare their ability to identify markers in the development of a genetic linkage map. Polymorphisms present in fourteen different soybean cultivars were demonstrated using all three techniques. AFLP, a novel PCR-based technique, was able to identify multiple polymorphic bands in a denaturing gel using 60 of 64 primer pairs tested. AFLP relies on primers designed in part on sequences for endonuclease restriction sites and on three selective nucleotides. The 60 diagnostic primer pairs tested for AFLP analysis each distinguished on average six polymorphic bands. Using specific primers designed for soybean fromEco RI andMse I restriction site sequences and three selective nucleotides, as many as 12 polymorphic bands per primer could be obtained with AFLP techniques. Only 35% of the RAPD reactions identified a polymorphic band using the same soybean cultivars, and in those positive reactions, typically only one or two polymorphic bands per gel were found. Identification of polymorphic bands using RFLP techniques was the most cumbersome, because Southern blotting and probe hybridization were required. Over 50% of the soybean RFLP probes examined failed to distinguish even a single polymorphic band, and the RFLP probes that did distinguish polymorphic bands seldom identified more than one polymorphic band. We conclude that, among the three techniques tested, AFLP is the most useful.     

Paternity determination in the adder (Vipera berus)—DNA fingerprinting or random amplified polymorphic DNA?

We performed breeding experiments with adders (Vipera berus) to determine whether multiple matings may result in multiple paternity. DNA fingerprinting of mothers, their offspring, and possible fathers using a polydinucleotide probe [(TG) _n ] gave a low overall similarity between unrelated individuals (0.18±0.07; SD) and an average of 17 bands that were male-specific. In no cases were there fewer than seven paternal-specific bands present in the fingerprint of an offspring, enabling us unambiguously to identify the biological father among five males. Multiple paternity was detected in the investigated broods with offspring sired exclusively by the captive males. PCR amplification of random amplified polymorphic DNA (RAPD) using 16 decamer primers gave 76 bands and an average similarity of 0.95 (±0.01) between the males, which were collected at different, geographically well-separated localities. Although there were on average 8.3 (±1.9) bands that differ between males in pairwise comparisons, there were only 1.9 (±1.1) bands per male that are specific for a particular individual. Thus, RAPDs are adequate for paternity determination only in experiments with a low number of males, whereas DNA fingerprinting offers sufficient information to discriminate between large numbers of putative fathers.The Swedish Natural Science Research Council, the Nilsson-Ehle Foundation, and the Erik Philip-Sörensen Foundation supported our research.     

DNA polymorphism in the major histocompatibility complex of man and various farm animals*

Summary. In the past few years it has been possible by combining enzymatic cleavage of genomic DNA and the Southern blot hybridization technique to explore the endonuclease recognition site polymorphism of the MHC. HLA class I and DR and DQ alpha and beta class II specific probes as well as human C4 and Bf class III probes were used. All these probes were shown to cross-hybridize with DNA from pigs, cattle, sheep and horses. Hybridization of human genomic DNA with a class I probe showed 15–25 bands per genome depending on the enzyme used. Distinct endonucleases generated clusters of restriction fragments (RF) in HLA-informative families which correlated with HLA specificites. While numerous clusters were found associated with HLA-A alleles almost no cluster was related to HLA B or C specificities. Similarly, class II probes provided a large number of clusters. The existence of these clusters suggested that some polymorphic restriction sites are found in strong linkage disequilibrium and that the underlying mechanism might be gene conversion with heteroduplex correction. Since the degree of polymorphism detected by RF appears to be greater than the polymorphism defined by more traditional methods stronger associations between RF and pathological conditions are to be expected. Southern blot analysis was applied to unrelated pigs and sheep, as well as to families. Preliminary studies have also been performed on a few unrelated cattle and horses. Depending on the endonuclease used the HLA class I probe hybridized with around 15 bands in MHC heterozygous pigs and ruminants while up to 20 bands were found in horses. Therefore, a several-fold greater number of potential class I genes exist compared to those actually expressed. With the class II beta probe, cattle and sheep showed around 10 bands whereas 15 were observed in pigs and around 20 in horses. Based on limited results obtained with DQ alpha and beta probes and with the DR alpha probe there appeared to be fewer of these respective genes. Only one C4 gene has been detected in pig and this gene maps within the SLA region. Hybridization with the human C4 probe in cattle, sheep and horses revealed two to four bands which could possibly account for two C4 genes. To date their linkage to the MHC has not been established. The Southern blot hybridization technique represents a powerful tool for future immunogenetic studies. This is even more so in large farm animals where for various reasons it is almost impossible to conduct certain types of investigation that are easily performed in rodents or in man. Although the data are still preliminary, they already extend our knowledge of the MHC in domestic animals far beyond what could have been reasonably anticipated using conventional methods.     

Validation of 15 microsatellites for parentage testing in North American bison, Bison bison and domestic cattle

Fifteen bovine microsatellites were evaluated for use in parentage testing in 725 bison from 14 public populations, 178 bison from two private ranches and 107 domestic cattle from five different breeds. The number of alleles per locus ranged from five to 16 in bison and from five to 13 in cattle. On average, expected heterozygosity, polymorphism information content (PIC) and probability of exclusion values were slightly lower in bison than in cattle. A core set of 12 loci was further refined to produce a set of multiplexed markers suitable for routine parentage testing. Assuming one known parent, the core set of markers provides exclusion probabilities in bison of 0.9955 and in cattle of 0.9995 averaged across all populations or breeds tested. Tests of Hardy-Weinberg and linkage equilibrium showed only minor deviations. This core set of 12 loci represent a powerful and efficient method for determining parentage in North American bison and domestic cattle.     

苹果属小金海棠的遗传多样性初步研究

用RAPD技术建立了苹果属小金海棠（Malus xiaojinensis）2个自然分布区的3个群体内随机选取的30株树（10株／每群体）及其相应的子代实生苗共6个群体、60个样本植株的分子标记。通过对15个3承机引物产生的81条RAPD这的统计,计算了不同群体RAPD多态性带的数目。用TREECON软件分析了不同群体及所有个体间的遗传关系,并用AMOVA技术分析了物种的遗传变异。结果是15个引物在全部分析个体中产生了58条多态性带,平均每引物3.8条。现有分布3个群体及其相应的子代实生苗群体的平均多态性带的数目都为1.5条左右。其中平均多态性带的数目最低的群体仅有0.7条,最高的群体也只有2.5条。遗传关系分析表明,2个自然分布区的不同群体间存在遗传分化现象。AMOVA分析显示小金海棠的遗传变异有相当一部分来源于群体间。     

Parentage analysis within a semi-free-ranging group of Barbary macaques Macaca sylvanus

This study of a group of semi-free-ranging Barbary macaques Macaca sylvanus aimed to determine paternity, to establish whether any individual male achieved prominent mating success and to assess genetic variability. Analyses involved electrophoresis of 15 blood protein systems and multilocus DNA fingerprinting (isotopic and nonisotopic). Genetic variability was low; only two blood protein systems were polymorphic. Although all DNA-fingerprints were individual-specific, they showed a high average band-sharing index value (0.67). Nevertheless, a combination of all methods permitted inference of paternity in 11 out of 15 (73%) cases tested. Several males from different age classes fathered infants.     

Human chromosome variation: the discriminatory power of Q-band heteromorphism (variant) analysis in distinguishing between individuals, with specific application to cases of questionable paternity.

The chromosomes from 57 persons were analyzed by means of quinacrine fluorescent staining in order to assess the amount of variation and the discriminatory power of Q-band heteromorphism analysis. Chromosomes 3, 4, 13, 14, 15, 21, 22, and Y of each person were visually compared to those of 56 others, for a total of 1,596 comparisons. No two persons were found to have the same set of variants. The number of differences between chromosomes for each comparison ranged from 2 to 12 out of a possible total of 14 for females and 15 for males. Relatives were also distinguishable, and differences ranged from two to seven. We used the frequency with which each chromosome was useful for telling two people apart, and estimated the probability of finding two persons with the same set of quinacrine variants as .0003. Distinctly different heteromorphisms were found in the 39 unrelated persons for each of the chromosomes examined. In this small population, the number of different sets of variants observed for chromosomes 3, 4, 13, 14, 15, 21, 22, and Y were six, seven, 27, 16, 20, 15, 24, and five, respectively, for a total number of possible combinations of 1.14 X 10(15). As a test of the usefulness of chromosome heteromorphisms in paternity cases, 12 father-mother-child trios of virtually certain paternity, owing to the father-child segregation of a rare structural rearrangement, were coded and recombined at random to produce 120 cases of uncertain paternity. When the code was broken, 108 "alleged fathers" had been excluded correctly and the 12 biological fathers had been included correctly.     

HLA-DQα allele and genotype frequencies in various human populations determined by using enzymatic amplification and oligonucleotide probes

Allele and genotype frequencies at the HLA-DQ alpha locus have been determined by the use of polymerase chain reaction (PCR) amplification and nonradioactive oligonucleotide probes. The probes define six alleles and 21 genotypes in a dot-blot format. A total of over 1,400 individuals from 11 populations has been typed by two different laboratories using this method. In contrast to some variable-number-of-tandem-repeat markers that have been used for identity determination, DQ alpha genotype frequencies do not deviate significantly from Hardy-Weinberg equilibrium in all populations studied. The distribution of alleles varies significantly between most of these populations. In Caucasians, the allele frequencies range from 4.3% to 28.5%. In this population, the power of discrimination is .94, and, for paternity determination, the power of exclusion is .642. These population data will allow the use of the HLA-DQ alpha marker in paternity determination, the analysis of individual identity in forensic samples, and anthropological studies.     

Nineteen new microsatellite DNA polymorphisms in pigtailed macaques (Macaca nemestrina)

Microsatellite loci known to be polymorphic in baboons (Papio hamadryas) and/or humans were tested in pigtailed macaques (Macaca nemestrina) from the Washington Regional Primate Research Center. Nineteen polymorphisms were identified in the macaques, with an average of 9.2 alleles per locus and an average heterozygosity of 0.76. Seven loci were analyzed using radiolabelled PCR primers and standard gel electrophoresis. Twelve loci were studied using fluorescently labelled primers and the Perkin-Elmer ABI 377 genotyping system. Of these 19 pigtailed macaque polymorphisms, 12 were used to perform paternity testing among captive animals. In a set of 15 infants, this panel of 12 genetic polymorphisms was sufficient to establish paternity in all cases. The number of alleles per locus in pigtailed macaques was compared with the number of alleles in a sample of baboons, and no significant correlation was observed. This indicates that population genetic processes such as genetic drift and recurrent mutation act rapidly enough on these loci to eliminate any relationship in levels of polymorphism across those two species. These 19 loci will be valuable for a range of genetic studies in pigtailed macaques, including paternity testing, analysis of population structure and differentiation among wild populations, and genetic linkage mapping.     

Analysis of genetic diversity in cowpea (Vigna unguiculata L.Walp.) cultivars with random amplified polymorphic DNA markers

The genetic diversity among ten Indian cultivars of cowpea was analyzed using 18 sets of RAPD markers. A total of 181 bands with an average of 15 bands per primer were obtained. Out of 181 bands, 148 showed polymorphism (81.7%). The variation in genetic diversity among these cultivars ranged from 0.1742 to 0.4054. Cluster analysis based on Jaccard’s similarity coefficient using UPGMA with high bootstrap values revealed two distinct clusters I and II comprised of two and seven cultivars, respectively. Cluster II was further differentiated into various subclusters. Cultivar IC-9883 was found to be unique based on its altogether distinct position in the dendrogram and two-dimensional space projections.     

On the use of DNA fingerprints for linkage studies in cattle

To find a marker for the bovine "muscular hypertrophy" gene and for the "roan" locus, we have typed six cattle pedigrees totaling 540 animals for nine blood group systems, for 12 biochemical markers, for RFLPs at four loci, and with five probes revealing multilocus DNA fingerprints. Segregation analysis of the fingerprint bands showed that, in cattle, a fingerprint probe will reveal a mean of 7.6 clearly resolvable bands, behaving as simple, highly informative Mendelian entities characterized by a mean mutation rate of +/- 1/4500 gametes. For one of the bands, we observed a "mutation burst" generating germline mosaicism. Because some of the fingerprint bands were allelic or corresponded to clustered minisatellites, a mean of only 5.7 independent loci is explored per probe. Fingerprint bands revealed by different probes also show a clear propensity for close linkage, pointing toward nonrandom distribution of minisatellite sequences or the existence of minisatellite clusters. Although this reduces the power of fingerprints for linkage analysis substantially, we were able to demonstrate genetic linkage between fingerprint bands and at least three of the classical markers, to exclude the roan locus from 4.5 Morgans of the bovine genome with the DNA fingerprints and for an additional 2.5 Morgans with the classical markers, and to identify a solid candidate marker for the bovine muscular hypertrophy gene, yielding a lod score greater than or equal to 2.84 without any obliged recombinant.     

Qualitative and quantitative characterization of RAPD variation among snap bean (Phaseolus vulgaris) genotypes

Ten snap bean (Phaseolus vulgaris) genotypes were screened for polymorphism with 400 RAPD (random amplified polymorphic DNA) primers. Polymorphic RAPDs were scored and classified into three categories based on ethidium bromide staining intensity. An average of 5.19 RAPD bands were scored per primer for the 364 primers that gave scorable amplification products. An average of 2.15 polymorphic RAPDs were detected per primer. The results show that primer screening may reduce the number of RAPD reactions required for the analysis of genetic relationships among snap-bean genotypes by over 60%. Based on the analysis of the distribution of RAPD amplification, the same number of polymorphic RAPDs were amplified from different genotypes for all RAPD band intensity levels. A comparison of RAPD band amplification frequency among genotypes for the three categories of bands classified by amplification strength revealed a measurable difference in the frequencies of RAPDs classified as faint (weakly amplifying) compared to RAPD bands classified as bold (strongly amplifying) indicating a possible scoring error due to the underscoring of faint bands. Correlation analysis showed that RAPD bands amplified by the same primer are not more closely correlated then RAPD bands amplified by different primers but are more highly correlated then expected by chance. Pairwise comparisons of RAPD bands indicate that the distribution of RAPD amplification among genotypes will be a useful criterion for establishing RAPD band identity. For the average pairwise comparison of genotypes, 50% of primers tested and 15.8% of all scored RAPDs detected polymorphism. Based on RAPD data Nei's average gene diversity at a locus was 0.158 based on all scorable RAPD bands and 0.388 if only polymorphic RAPD loci were considered. RAPD-derived 1 relationships among genotypes are reported for the ten genotypes included in this study. The data presented here demonstrate that many informative, polymorphic RAPDs can be found among snap bean cultivars. These RAPDs may be useful for the unique identification of bean varieties, the organization of bean germplasm, and applications of molecular markers to bean breeding.     

Checking of individuality by DNA profiling

A review of methods of DNA analysis used in forensic medicine for identification, paternity testing, etc. is provided. Among other techniques, DNA fingerprinting using different probes and polymerase chain reaction-based techniques such as amplified sequence polymorphisms and minisatellite variant repeat mapping are thoroughly described and both theoretical and practical aspects are discussed.     

Sheep linkage mapping: restriction fragment length polymorphism detection with heterologous cDNA probes

Summary. A selection of cattle, human and sheep cDNA probes were screened against sheep genomic DNA, cut with 10 different restriction enzymes, to assess the usefulness of these probes for restriction fragment length polymorphism (RFLP) linkage studies in sheep. Two-thirds of the cattle cDNA probes showed moderate to strong homology with sheep DNA samples, compared with less than half of the human cDNA probes at the final washing stringency chosen for the experiments. The set of probes tested detected a useful frequency of RFLPs. Fifty-seven per cent of probes showing moderate to strong homology identified RFLPs with one or more restriction enzymes. Restriction enzymes that detected RFLPs most frequently in sheep were Taq I and Msp I. The results show that sheep and cattle cDNA probes, including candidate genes for production traits, identified a high frequency of RFLPs suitable for genetic mapping in sheep.     

Assignment of RAPD marker probes designed from 12 linkage groups of Flammulina velutipes to CHEF-separated chromosomal DNAs

Electrophoretic karyotype analyses of Flammulina velutipes FSB and its monokaryotic progeny, omFSB1 and omFSB2, obtained from oidia were performed by contour-clamped homogeneous electric field (CHEF) gel electrophoresis. At least 11 chromosome-sized DNA bands (CB 1 through CB 11) for FSB, 6 bands for omFSB1, and 7 bands for omFSB2, respectively, were resolved on a CHEF gel. Southern hybridization analysis on CHEF-separated chromosomal DNA of FSB was carried out using RAPD marker probes prepared from each of the 12 linkage groups. The bands CB 1, 2, and 4 each hybridized to two or three probes for different linkage groups. The bands CB 5 and 6 both hybridized to a common probe. The bands CB 3, 7, 8, and 9 each hybridized to a single specific probe for different linkage groups. The two smallest bands (CB 10 and 11) did not hybridize with any probes.     

Highly variable SSR markers in Douglas-fir: Mendelian inheritance and map locations

Twenty-two highly variable SSR markers were developed in Douglas-fir [Pseudotsuga menziesii (Mirb.) Franco] from five SSR-enriched genomic libraries. Fifteen PCR primer pairs amplified a single codominant locus, while seven primer pairs occasionally amplified two loci. The Mendelian inheritance of all 22 SSRs was confirmed via segregation analyses in several Douglas-fir families. The mean observed heterozygosity and the mean number of alleles per locus were 0.855 (SE=0.020) and 23 (SE=1.6), respectively. Twenty markers were used in genetic linkage analysis and mapped to ten known linkage groups. Because of their high polymorphism and unambiguous phenotypes, 15 single-locus markers were selected as the most suitable for DNA fingerprinting and parentage analysis. Only three SSRs were sufficient to achieve an average probability of exclusion from paternity of 0.998 in a Douglas-fir seed orchard block consisting of 59 parents.Communicated by O. Savolainen