Ordering three DNA polymorphisms on human chromosome 3 by sperm typing.

Three loci on the short arm of human chromosome 3 were ordered by sperm typing to expand the limited genetic map of this region. Almost 300 individual sperm from a donor triply heterozygous at D3S2, D3S11, and D3S12 were amplified by PCR using primers flanking the polymorphic site at each locus. Primary PCR product was reamplified using allele-specific primers of different lengths, allowing the allelic state at each locus to be determined by gel electrophoresis. Maximum likelihood analysis of the sperm-typing data showed that the most likely order was D3S2-D3S11-D3S12 with an odds ratio of almost 5000:1 when compared to the next most likely order. This finding should be useful in interpreting loss of heterozygosity on 3p in a variety of cancers. Our results also demonstrate the practicality of ordering DNA polymorphisms using sperm typing.     

Close linkage between bovine prolactin and BoLA-DRB3 genes: genetic mapping in cattle by single sperm typing.

The major histocompatibility complex and prolactin (PRL) genes are syntenic in humans and cattle but the genetic distance between these loci has not been determined for either species. In this study, the sperm typing technique was used to measure the recombination frequency between the bovine lymphocyte antigen (BoLA)-DRB3 and PRL loci. A total of 300 sperm were typed from one doubly heterozygous bull for segregation of DRB3 and PRL alleles. Sperm typing was performed using the polymerase chain reaction (PCR) and restriction enzyme cleavage of the PCR products, followed by resolution of the restriction fragments in polyacrylamide gels. Digestion with the restriction endonuclease RsaI allowed the unambiguous discrimination of alleles for both loci. The maximum likelihood estimation of the recombination fraction theta = 0.04, with a 95% confidence interval of 0.01 to 0.07. Close linkage between PRL and DRB3 has important implications for marker-assisted selection in animal breeding since PRL has been shown to be closely linked to a locus that affects milk yield, and BoLA loci influence susceptibility to a number of infectious diseases. Our results demonstrate the general applicability of the sperm typing procedure for gene mapping in species other than humans and provide an example of how parallel efforts to map the genomes of agriculturally important species of animals can have a positive impact on the development of a primary human linkage map.     

Multipoint linkage map of the human pseudoautosomal region,based on single-sperm typing: do double crossovers occur during male meiosis?

Sperm typing was used to measure recombination fractions among pseudoautosomal markers and the beginning of the X/Y-specific sequences located at the pseudoautosomal boundary. These experiments included primer-extension preamplification and PCR followed by allele typing using gel electrophoresis. A newly developed data-analysis program allowed the construction of the first multipoint-linkage sperm-typing map, using results obtained on seven loci from three individuals. The large sample size not only confirmed the increased recombination activity of the pseudoautosomal region but allowed an estimate of interference of recombination to be made. The coefficient of coincidence was calculated to be .26 over a physical distance of only approximately 1,800 kb. The observation of a few sperm presumably resulting from double recombination argues that more than one crossover event can occur in this region during male meiosis.     

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.     

Sperm typing allows accurate measurement of the recombination fraction between D3S2 and D3S3 on the short arm of human chromosome 3.

The interval between the D3S2 and D3S3 loci on human chromosome 3p is a frequent site of deletions in a number of different cancers and contains the most common fragile site in man. Both loci have been physically mapped to 3p but because heterozygosity for D3S3 is so infrequent, recombination between them could not be determined accurately by using family studies. Sperm typing can measure recombination between DNA polymorphisms even in a single individual and thus can make use of polymorphisms with a low PIC. The recombination fraction between D3S2 and D3S3 was estimated to be 0.28 based on analyzing 189 and 77 sperm from two doubly heterozygous donors, respectively. These results demonstrate one of the ways in which sperm typing can complement pedigree analysis in constructing genetic maps.     

High resolution genetic typing of the class II HLA-DRB1 locus using group-specific amplification and SSO-hybridisation in microplates

The HLA-DRB1 locus is one of the most polymorphic HLA class II loci and rapid and accurate typing of this polymorphism is important both in bone-marrow transplantation, analysis of disease association and in forensic medicine. The allelic variation at DRB1 is characterized by combinations of a limited number of amino-acid motifs, reducing the resolution of a typing strategy based on a single PCR and subsequent analysis of polymorphic motifs. In the present paper we describe a strategy for typing of DRB1 based on eight allele-specific PCRs followed by sandwich hybridization to immobilized probes in a microplate format. The combined approach results in a rapid typing system with very high resolution. Using a rapid DNA extraction protocol, a complete HLA-DRB1 typing can be performed in less than a day.     

Linkage analysis by two-dimensional DNA typing.

In two-dimensional (2-D) DNA typing, genomic DNA fragments are separated, first according to size by electrophoresis in a neutral polyacrylamide gel and second according to sequence by denaturing gradient gel electrophoresis, followed by hybridization analysis using micro- and minisatellite core probes. The 2-D DNA typing method generates a large amount of information on polymorphic loci per gel. Here we demonstrate the potential usefulness of 2-D DNA typing in an empirical linkage study on the red factor in cattle, and we show an example of the 2-D DNA typing analysis of a human pedigree. The power efficiency of 2-D DNA typing in general is compared with that of single-locus typing by simulation. The results indicate that, although 2-D DNA typing is very efficient in generating data on polymorphic loci, its power to detect linkage is lower than single-locus typing, because it is not obvious whether a spot represents the presence of one or two alleles. It is possible to compensate for this lower informativeness by increasing the sample size. Genome scanning by 2-D DNA typing has the potential to be more efficient than current genotyping methods in scoring polymorphic loci. Hence, it could become a method of choice in mapping genetic traits in humans and animals.     

INFERENCE OF SPERM COMPETITION FROM BROODS OF FIELD-CAUGHT DROSOPHILA

In field studies of multiple mating and sperm competition there typically is no experimental control over the number of times that a female mates, the interval between matings, or the genetic identity of multiple fathers contributing to a brood. Irrespective of this complexity, high-resolution molecular markers can be used to assign paternity with considerable confidence. This study employed two highly heterozygous microsatellite loci to assess multiple paternity and sperm displacement in a sample of broods taken from a natural population of Drosophila melanogaster. The large number of alleles present at each of the loci makes it difficult to derive explicit maximum-likelihood estimates for multiple paternity and sperm displacement from brood samples. Monte Carlo simulations were used to estimate maximum-likelihood parameters for the distribution of female remating frequency and the proportion of offspring sired by the second or subsequent mating males. Estimates were made based on genotypes scored at two distinct marker loci because they were found to give statistically homogeneous results. Fitting a Poisson distribution of number of matings, the mean number of males mated by a female was 1.82. The sperm displacement parameter estimated from doubly mated females were 0.79 and 0.86 for the two loci (0.83 for the joint estimate). The overall probability that a multiply mated female will be misclassified as singly mated was only 0.006, which indicates that microsatellites can provide excellent resolution for identifying multiple mating. In addition, microsatellites can be used to generate relatively precise estimates of sperm precedence in brood-structured samples from a natural population.     

我国3个民族13个SNPs位点多态性及遗传学关系的比较

采用荧光标记复合扩增毛细管电泳技术, 基于等位基因特异性PCR原理, 通过正交实验法建立了荧光标记复合扩增片段长度差异等位基因特异性SNPs分型体系, 该体系可以根据产物长度和产物峰的数量一次完成13个SNPs分型, 纯合子为单一产物峰, 杂合子为长度相差4 bp的两个产物峰。采用该体系对我国辽宁地区汉族、内蒙古地区蒙古族和广西地区壮族3个民族13个SNPs位点多态性进行群体调查, 获得了3个民族13个SNPs等位基因分布频率, 比较了3个民族等位基因的差异, 并对其遗传学关系进行了研究。结果显示: 3个民族13个SNPs的等位基因分布具有多态性, 多个SNPs等位基因分布具有显著性差异(P≤0.01), 抽样调查结果符合Hardy-Weinberg平衡; 辽南地区汉族人群与内蒙古蒙古族人群的亲缘关系更为接近, 与广西壮族之间的亲缘关系相对较远。     

Linkage relationships and haplotype polymorphism among cichlid Mhc class II B loci.

The species flocks of cichlid fishes in the Great East African Lakes are paradigms of adaptive radiation and hence, of great interest to evolutionary biologists. Phylogenetic studies of these fishes have, however, been hampered by the lack of suitable polymorphic markers. The genes of the major histocompatibility complex hold the promise to provide, through their extensive polymorphism, a large number of such markers, but their use has been hampered by the complexity of the genetic system and the lack of definition of the individual loci. In this study we take the first substantial step to alleviate this problem. Using a combination of methods, including the typing of single sperm cells, gyno- or androgenetic individuals, and haploid embryos, as well as sequencing of class II B restriction fragments isolated from gels for Southern blots, we identify the previously characterized homology groups as distinct loci. At least 17 polymorphic class II B loci, all of which are presumably transcribed, have been found among the different species studied. Most of these loci are shared across the various cichlid species and genera. The number of loci per haplotype varies from individual to individual, ranging from 1 to 13. A total of 21 distinct haplotypes differing in the number of loci they carry has thus far been identified. All the polymorphic loci are part of the same cluster in which, however, distances between at least some of the loci (as indicated by recombination frequencies) are relatively large. Both the individual loci and the haplotypes can now be used to study phylogenetic relationships among the members of the species flocks and the mode in which speciation occurs during adaptive radiation.     

猪单个精子单倍型分析方法研究

应用引物延伸预扩增,内嵌引物设计策略进行了ＰＣＲ扩增并结合聚丙烯酰胺凝胶电泳及银染技术,对猪单个精子１２号染色体上４个微卫星位点进行了ＰＣＲ扩增。结果表明,上述技术的应用可以清晰地鉴定出每个精子的单倍型,单精子分型技术的应用为猪高精度遗传作图及需要样本量足够的大遗传现象研究提供了独特的工具。     

Development of a feline whole genome radiation hybrid panel and comparative mapping of human chromosome 12 and 22 loci

A 5000-rad whole genome radiation hybrid panel is described for the domestic cat, derived from irradiated male feline fibroblasts fused to a recipient hamster cell line. A panel of 93 cell lines has an estimated retention frequency of 0.39 (range 0.13-0.71) based upon PCR typing of 54 feline markers. To test the panel's utility, we determined the order of 16 Type I (coding gene) loci, 14 Type II (microsatellite) loci, and 1 endogenous retroviral element on feline chromosomes B4 and D3. Assessment of marker order derived from the RH panel was compared to assignments of the same loci using interspecies backcross mapping data, human homologue positions, and human-cat chromosome painting homologies. Assessment of concordant and discordant marker order for these loci provides improved resolution into the evolution of subchromosomal genome organizations and the methods to track them in these species.     

Genome amplification of single sperm using multiple displacement amplification

Sperm typing is an effective way to study recombination rate on a fine scale in regions of interest. There are two strategies for the amplification of single meiotic recombinants: repulsion-phase allele-specific PCR and whole genome amplification (WGA). The former can selectively amplify single recombinant molecules from a batch of sperm but is not scalable for high-throughput operation. Currently, primer extension pre-amplification is the only method used in WGA of single sperm, whereas it has limited capacity to produce high-coverage products enough for the analysis of local recombination rate in multiple large regions. Here, we applied for the first time a recently developed WGA method, multiple displacement amplification (MDA), to amplify single sperm DNA, and demonstrated its great potential for producing high-yield and high-coverage products. In a 50 μl reaction, 76 or 93% of loci can be amplified at least 2500- or 250-fold, respectively, from single sperm DNA, and second-round MDA can further offer >200-fold amplification. The MDA products are usable for a variety of genetic applications, including sequencing and microsatellite marker and single nucleotide polymorphism (SNP) analysis. The use of MDA in single sperm amplification may open a new era for studies on local recombination rates.     

High-resolution recombination patterns in a region of human chromosome 21 measured by sperm typing

For decades, classical crossover studies and linkage disequilibrium (LD) analysis of genomic regions suggested that human meiotic crossovers may not be randomly distributed along chromosomes but are focused instead in "hot spots." Recent sperm typing studies provided data at very high resolution and accuracy that defined the physical limits of a number of hot spots. The data were also used to test whether patterns of LD can predict hot spot locations. These sperm typing studies focused on several small regions of the genome already known or suspected of containing a hot spot based on the presence of LD breakdown or previous experimental evidence of hot spot activity. Comparable data on target regions not specifically chosen using these two criteria is lacking but is needed to make an unbiased test of whether LD data alone can accurately predict active hot spots. We used sperm typing to estimate recombination in 17 almost contiguous ~5 kb intervals spanning 103 kb of human Chromosome 21. We found two intervals that contained new hot spots. The comparison of our data with recombination rates predicted by statistical analyses of LD showed that, overall, the two datasets corresponded well, except for one predicted hot spot that showed little crossing over. This study doubles the experimental data on recombination in men at the highest resolution and accuracy and supports the emerging genome-wide picture that recombination is localized in small regions separated by cold areas. Detailed study of one of the new hot spots revealed a sperm donor with a decrease in recombination intensity at the canonical recombination site but an increase in crossover activity nearby. This unique finding suggests that the position and intensity of hot spots may evolve by means of a concerted mechanism that maintains the overall recombination intensity in the region.     

Minisatellites show rare and simple intra-allelic instability in the mouse germ line

Minisatellites provide very informative systems for analyzing processes of tandem repeat DNA turnover in humans. The mouse genome also contains authentic minisatellites, but none has yet been found to show high levels of instability. Indirect evidence using minisatellite variant repeat mapping by PCR in Mus musculus subspecies suggested that mouse minisatellites mutate at a rate below 10(-3) per gamete and mainly by intra-allelic events. This is in sharp contrast to the complex interallelic mutations observed at high frequency at some human loci. To define more directly the turnover mechanisms and rates of instability at one of the most variable mouse minisatellites (MMS80), we used size-enrichment small-pool PCR (SESP-PCR) to recover de novo mutant alleles from sperm DNA from homozygous BALB/cJ mice and from strain DHA heterozygotes. The sperm mutation rate at MMS80 was extremely low, at or below 5 x 10(-6) per sperm. Comparison of progenitor and mutant allele structures showed that these rare mutants had arisen by simple and primarily, if not exclusively, intra-allelic mutation events. These results suggest a fundamental difference in turnover mechanisms at minisatellites between mice and human.     

Variation in the number of sperm transferred during mating among males of the Colorado potato beetle (Coleoptera: Chrysomelidae)

We suggest an explanation for the bimodal distribution in the number of sperm transferred into the female during mating by males of the Colorado potato beetle (Leptinotarsa decemlineata). While in most matings females receive 20,000 sperm, in others they receive less than 1000. A variety of potential proximate causes for this bimodal distribution including experimental procedures and several physiological and behavioral causes were examined: male body size, female body size, male mating history, socio-sexual environments, duration and number of mounts, and duration and number of copulations. Surprisingly, none of these explained the observed distribution. However, we did find that the total time spent in copulation was significantly less variable among males that transferred more than 20,000 sperm than among males that passed less than 1000 sperm. In addition, males transferring large amounts of sperm resulted in proportionally more sperm inside the female’s spermatheca within a short period of time than males passing less than 1000 sperm. The lack of pattern in male sperm ejaculate suggests definite differences in sperm delivery tactics and may be related to the female condition rather than the male.     

Development of a MLST-biased SNP microarray for Candida albicans

We have developed a single nucleotide polymorphism (SNP) detection microarray for the human pathogenic yeast, Candida albicans, consisting of synthetic oligonucleotides bound to microscope slides. The array consists of multiple replicates of 79 SNPs, derived from 19 discrete loci located on all eight chromosomes. These loci include seven genes consisting of 57 SNPs that comprise a multi-locus sequence typing (MLST) consensus scheme for the species. The remaining 22 SNPs are from 12 additional loci located at intervals on the remaining chromosomes. In order to include highly informative polymorphisms from the MLST set on the array we performed a linkage analysis of major genotypes between the two pairs of MLST-linked genes. In addition, we performed a matched-set analysis for each SNP located within individual MLST loci. This analysis resulted in the reduction of informatively redundant mutations in the array for a large percentage of strains. We believe that a SNP array will be helpful in extending our knowledge of the epidemiology and genetics of C. albicans as a supplement to MLST typing.     

Sperm transfer and paternity in the scorpionfly <Emphasis Type="Italic">Panorpa cognata</Emphasis>: large variance in traits favoured by post-copulatory episodes of sexual selection

Post-copulatory episodes of sexual selection can be a powerful selective force influencing the reproductive success of males. In order to understand variation in male fertilisation success, we first need to consider the pattern of sperm utilisation by females following matings with more than one male. Second, we need to study those traits responsible for male success in sperm competition. Here we study both male sperm transfer characteristics as well as offspring paternity of females mated to two males in the scorpionfly Panorpa cognata. By repeatedly mating males to virgin females and interrupting copulation at defined time points, we found for all males that sperm transfer set off after approximately 40 min. During the remaining copulation, sperm transfer of individual males was continuous and with constant rate. Yet the rate of sperm transfer differed between individual males from about one sperm per minute to more than eight sperm per minute for the most successful males. In addition, we measured the fertilisation success in sperm competition of males with known sperm transfer capability. The relative number of sperm transferred by males during copulation, estimated from copulation duration and the males’ individual sperm transfer rate, explained a large proportion of variation in offspring paternity. The mode of sperm competition in this species, thus, conforms largely to a fair raffle following complete mixing of sperm prior to fertilisation. Hence, male differences in both the ability to copulate for long and of rapid sperm transfer will translate directly into differences in reproductive success.     

Single paternity of clutches and sperm storage in the promiscuous green turtle (Chelonia mydas)

Paternity of 22 green turtle ( Chelonia mydas ) clutches from 13 females of the southern Great Barrier Reef breeding population was determined through microsatellite analyses at five loci, including the analysis of successive clutches for nine of the females. A large number of alleles per locus (10–40) provided probabilities of detecting multiple paternity that were quite high, particularly at all loci combined (99.9%). Although green turtles are promiscuous breeders and there was an expectation of finding extensive multiple paternity, only two clutches were multiply sired and, in these, very few eggs had been fertilized by a secondary male. The rarity of multiple paternity may reflect either a low proportion of multiple matings by females in this population, or sperm competition, possibly resulting from a first-male sperm preference. Additionally, the analysis of > 900 offspring provided data on mutations, which included 20 mutation events that were observed in 27 offspring and involved both maternal and paternal lineages. Most mutations ( n = 16) occurred at a single highly variable locus and their presence emphasizes the need to use multiple loci in paternity studies.     

Evaluation of VNTR typing for the identification of Mycobacterium ulcerans in environmental samples from Victoria, Australia

Reliable molecular detection of Mycobacterium ulcerans in environmental samples is essential to study the ecology and transmission of this important human pathogen. Variable number tandem repeat (VNTR) typing is a valuable method for distinguishing M. ulcerans isolates from different geographic regions and for distinguishing M. ulcerans from other members of the Mycobacterium marinum/M. ulcerans complex, but its application to environmental samples has not yet been evaluated systematically. This study compares the sensitivity and specificity of PCR detection of 13 VNTR loci to determine the best loci for the analysis of environmental samples. This study demonstrates that VNTR typing using selected loci can be a useful addition to established molecular methods for detecting M. ulcerans in the environment and highlights some of the issues encountered when using molecular methods to detect microorganisms in environmental samples. When applied to environmental samples collected from an endemic region in Victoria, Australia, VNTR typing confirmed that the strain of M. ulcerans being detected was indistinguishable from the strain causing disease in humans in that region.