Genetic variation of the Toll-like receptors in a Swedish allergic rhinitis case population.

BACKGROUND: Variation in the 10 toll-like receptor (TLR) genes has been significantly associated with allergic rhinitis (AR) in several candidate gene studies and three large genome-wide association studies. These have all investigated common variants, but no investigations for rare variants (MAF ≤ 1%) have been made in AR. The present study aims to describe the genetic variation of the promoter and coding sequences of the 10 TLR genes in 288 AR patients. METHODS: Sanger sequencing and Ion Torrent next-generation sequencing was used to identify polymorphisms in a Swedish AR population and these were subsequently compared and evaluated using 1000Genomes and Exome Aggregation Consortium (ExAC) data. RESULTS: The overall level of genetic variation was clearly different among the 10 TLR genes. The TLR10-TLR1-TLR6 locus was the most variable, while the TLR7-TLR8 locus was consistently showing a much lower level of variation. The AR patients had a total of 37 promoter polymorphisms with 14 rare (MAF ≤ 1%) and 14 AR-specific polymorphisms. These numbers were highly similar when comparing the AR and the European part of the 1000Genomes populations, with the exception of TLR10 where a significant (P = 0.00009) accumulation of polymorphisms were identified. The coding sequences had a total of 119 polymorphisms, 68 were rare and 43 were not present in the European part of the 1000Genomes population. Comparing the numbers of rare and AR-specific SNPs in the patients with the European part of the 1000Genomes population it was seen that the numbers were quite similar both for individual genes and for the sum of all 10 genes. However, TLR1, TLR5, TLR7 and TLR9 showed a significant excess of rare variants in the AR population when compared to the non-Finnish European part of ExAC. In particular the TLR1 S324* nonsense mutation was clearly overrepresented in the AR population. CONCLUSIONS: Most TLR genes showed a similar level of variation between AR patients and public databases, but a significant excess of rare variants in AR patients were detected in TLR1, TLR5, TLR7, TLR9 and TLR10. This further emphasizes the frequently reproduced TLR10-TLR1-TLR6 locus as being involved in the pathogenesis of allergic rhinitis.

Characterization of genetic variation in TLR8 in relation to allergic rhinitis.

BACKGROUND: A previous investigation of all 10 TLR genes for associations with allergic rhinitis (AR) detected a number of significant SNPs in the TLR8 locus. The associations indicated that an accumulation of rare variants could explain the signal. This study therefore searches for rare variants in the TLR8 region and also investigates the reproducibility of previous SNP associations. METHODS: The TLR8 gene was resequenced in 288 AR patients from Malmö and the data were compared with publically available data. Seven previously AR-associated SNPs from TLR8 were analyzed for AR associations in 422 AR patients and 859 controls from the BAMSE cohort. The associations detected in present and previous studies were compared. RESULTS: Sequencing detected 13 polymorphisms (three promotor and 10 coding) among 288 AR patients. Four of the coding polymorphisms were rare (MAF < 1%) and three of those were novel. Two coding polymorphisms were benign missense mutations and the rest were synonymous. Comparison with 1000Genomes and Exome Aggregation Consortium data revealed no accumulation of rare variants in the AR cases. The AR association tests made using the BAMSE cohort yielded five P-values <0.05. Tests of IgE levels yielded four significant SNP associations to birch pollen. Comparing results between different populations revealed opposing risk alleles, different gender effects, and response to different allergens in the different populations. CONCLUSIONS: Rare variants in TLR8 are not associated with AR. Comparison of present and previous association studies reveals contradictory results for common variants. Thus, no associations exist between genetic variation in TLR8 and AR.

Mutation analysis of Swedish haemophilia B families - high frequency of unique mutations.

INTRODUCTION: Haemophilia B is caused by a heterogeneous spectrum of mutations. Mutation characterization is important in genetic counselling, prenatal diagnosis and to predict risk of inhibitor development. AIMS: To study the mutation spectrum, frequency of unique recurrent mutations, genotype-phenotype association and inhibitor development in a population-based study of the complete Swedish haemophilia B population. METHODS: The study included, facilitated by centralized DNA diagnostics, the complete registered Swedish haemophilia B population (113 families: 47 severe, 22 moderate and 44 mild), each represented by a single patient. Mutation characterization was performed by conventional sequencing of all exons and haplotyping by genotyping of single nucleotide variants and microsatellites. RESULTS: A mutation was found in every family: eight had large deletions, three had small deletions (<10 base pair) and 102 had single base pair substitutions (69 missense, 26 nonsense, four splice site and three promoter). Ten novel mutations were found and were predicted to be deleterious. Sixteen mutations (one total gene deletion, 14 substitutions and one acceptor splice site) were present in more than one family. Of the single nucleotide mutations (37/102), 36% arose at CpG sites. Haplotyping of families with identical mutations and present analyses showed that the frequency of unique mutations was at least 65%. Inhibitors developed in 9/47 (19%) patients with severe haemophilia B. CONCLUSION: The spectrum of haemophilia B mutations reveals at least 65% of the families carry a unique mutation, but with more inhibitor patients than reported internationally, probably as a result of many 'null' mutations.

Origin of mutation in sporadic cases of severe haemophilia A in Sweden.

UNLABELLED: Many newly diagnosed Swedish severe haemophilia A (HA) patients are sporadic cases. Some genotypically non-carrier mothers have gone on to have two descendants with the same mutation, presumably because of mosaicism. AIMS: To define the origin of mutation in sporadic cases of HA, reveal possible sex-specific differences in mutagenesis and identify potential mosaics among non-carrier mothers. METHOD: Sanger sequencing characterized the mutations and microsatellite haplotyping determined the origin of the X chromosome carrying the mutation in 3 generations of 45 families with sporadic severe HA. Droplet digital polymerase chain reaction (ddPCR) was used in five cases to reveal that mosaicism mutations are not found on conventional DNA sequencing. RESULTS: In 23 out of 45 families, the mother carried the mutation and in 5 out of 28 families, the grandmother was also a carrier. The X chromosome was of grandpaternal origin in 17 out of 23 cases. One of five tested mothers was a mosaic with a mutation frequency of 7%. CONCLUSION: In 40 out of 45 families, the sporadic case resulted from a mutation in the last two generations. In 82% (23/28), the carrier mothers had a de novo mutation where the X chromosome was of paternal origin in 74% (17/23). ddPCR is a potentially powerful and promising analysis for mosaicism in HA.

Replication of genomewide associations with allergic sensitization and allergic rhinitis.

BACKGROUND: Three genomewide metastudies have recently reported associations with self-reported allergic rhinitis and allergic sensitization. The three studies together identified a set of 37 loci but showed low concordance. This study investigates the reproducibility of the detected single nucleotide polymorphism (SNP) associations in an extensively characterized longitudinal cohort, BAMSE. METHODS: Phenotypic evaluation of allergic rhinitis (AR) and allergic sensitization was performed on 2153 children from BAMSE at 8 and 16 years of age. Allele frequencies of 39 SNPs were investigated for association with the exact allergic phenotypes of the metastudies. Odds ratios and false discovery rates were calculated, and the impact of asthma was evaluated. The cases were also evaluated for age at onset effects (≤ or >8 years of age). RESULTS: Association tests of the 39 SNPs identified 12 SNPs with P-values < 0.05 and Q-values < 0.10. Two of the four loci (TLR6-TLR1 and HLA-DQA1-HLA-DQB1) identified in all three original studies were also identified in this study. Three SNPs located in the TLR6-TLR1 locus had the lowest P-values and Q-values < 0.1 when using a well-defined AR phenotype. Two loci showed significant age at onset effects, but the effect of asthma on the associations was very limited. CONCLUSION: The TLR6-TLR1 locus is likely to have a central role in the development of allergic disease. The association between genetic variation in the SSTR1-MIPOL1 and TSLP-SLC25A46 loci and age at onset is the first report of age at onset effects in allergic rhinitis.

Origin of Swedish hemophilia B mutations.

BACKGROUND: More than 1100 mutations that cause hemophilia B (HB) have been identified. At the same time, specific F9 mutations are present at high frequencies in certain populations, which raise questions about the origin of HB mutations. OBJECTIVES: To describe the mutation spectrum of all HB families in Sweden and investigate if mutations appearing in several families are due to independent recurrent mutations (RMs) or to a common mutation event (i.e. are identical by descent (IBD)). PATIENTS/METHODS: The registered Swedish HB population consists of patients from 86 families. Mutations were identified by resequencing and identical haplotypes were defined using 74 markers and a control population of 285 individuals. The ages of IBD mutations were estimated using ESTIAGE. RESULTS: Out of 77 presumably unrelated patients with substitution mutations, 47 patients (61%) had mutations in common with other patients. Haplotyping of the 47 patients showed that 24 patients had IBD mutations (51%) with estimated ages of between two and 23 generations. A majority of these patients had mild disease. Eight of the 15 mutations observed in more than one family were C>T transitions in CpG sites and all eight were RMs. CONCLUSIONS: The association of IBD mutations with a mild phenotype is similar to what has been previously observed in hemophilia A. Noteworthy features of the mutations that are common to more than one family are the equal proportions of patients with RM and IBD mutations and the correlation between the occurrence of RMs and C>T transitions at CpG sites.

Origin of Swedish hemophilia A mutations.

BACKGROUND: Hemophilia A (HA) has a high level of variation within the disease class, with more than 1000 mutations being listed in the HAMSTeRS database. At the same time a number of F8 mutations are present in specific populations at high frequencies. OBJECTIVES: The simultaneous presence of large numbers of rare mutations and a small number of high-frequency mutations raises questions about the origins of HA mutations. The present study was aimed at describing the origins of HA mutations in the complete Swedish population. The primary issue was to determine what proportion of identical mutations are identical by descent (IBD) and what proportion are attributable to recurrent mutation events. The age of IBD mutations was also determined. PATIENTS/METHODS: In Sweden, the care of HA is centralized, and the Swedish HA population consists of ~ 750 patients from > 300 families (35% severe, 15% moderate, and 50% mild). Identical haplotypes were defined by single-nucleotide polymorphism and microsatellite haplotyping, and the ages of the mutations were estimated with estiage. RESULTS: Among 212 presumably unrelated patients with substitution mutations, 97 (46%) had mutations in common with other patients. Haplotyping of the 97 patients showed that 47 had IBD mutations (22%) with estimated ages of between two and 35 generations. The frequency of mild disease increased with an increasing number of patients sharing the mutations. CONCLUSIONS: A majority of the IBD mutations are mild and have age estimates of a few hundred years, but some could date back to the Middle Ages.

Investigation of disease-associated factors in haemophilia A patients without detectable mutations.

To investigate disease causing mechanism in haemophilia A patients without detectable mutation. Screening for F8 mutations in 307 haemophilia A patients using: re-sequencing and inversion PCR, reverse transcription (RT-PCR) of mRNA, MLPA analysis, haplotyping using SNP and microsatellite markers. No F8 mutations were detected in 9 of the 307 patients (2.9%) using re-sequencing and inversion PCR. MLPA analysis detected duplication in exon 6 in one patient and RT-PCR showed no products for different regions of mRNA in four other patients, indicating failed transcription. No obvious associations were observed between the phenotypes of the nine patients, their F8 haplotypes and the putative mutations detected. The mutation-positive patients carrying the same haplotypes as the mutation-negative patients show a multitude of different mutations, emphasizing the lack of associations at the haplotype level. VWF mutation screening and factor V measurements ruled out type 2N VWD and combined factor V and VIII deficiency respectively. To further investigate a possible role for FVIII interacting factors the haplotypes/diplotypes of F2, F9, F10 and VWF were compared. The nine patients had no specific haplotype/diplotype combination in common that can explain disease. Duplications and faulty transcription contribute to the mutational spectrum of haemophilia A patients where conventional mutation screening fail to identify mutations.

Effect of androgen deprivation therapy on the expression of prostate cancer biomarkers MSMB and MSMB-binding protein CRISP3.

We have investigated the effects of short-term neoadjuvant and long-term androgen deprivation therapies (ADTs) on ß-microseminoprotein (MSMB) and cysteine-rich secretory protein-3 (CRISP3) expression in prostate cancer patients. We also studied if MSMB expression was related to genotype and epigenetic silencing. Using an Affymetrix cDNA microarray analysis, we investigated the expression of MSMB, CRISP3, androgen receptor (AR), KLK3 and Enhancer of Zeste Homologue-2 (EZH2) in tissue from prostate cancer patients receiving (n=17) or not receiving (n=23) ADT before radical prostatectomy. MSMB, CRISP3 and AR were studied in tissue from the same patients undergoing TURP before and during ADT (n=16). MSMB genotyping of these patients was performed by TaqMan PCR. MSMB and KLK3 expression levels decreased during ADT. Expression levels of AR and CRISP3 were not affected by short-term ADT but were high in castration-resistant prostate cancer (CRPC) and metastases. Levels of EZH2 were also high in metastases, where MSMB was low. Genotyping of the MSMB rs10993994 polymorphism showed that the TT genotype conveys poor MSMB expression. MSMB expression is influenced by androgens, but also by genotype and epigenetic silencing. AR and CRISP3 expression are not influenced by short-term ADT, and high levels were found in CRPC and metastases.

CLC- a novel susceptibility gene for allergic rhinitis?

BACKGROUND: Studies of the nasal lavage fluid proteome have previously identified proteins differently expressed in patients with symptomatic allergic rhinitis, e.g. S100A7, prolactin-inducible protein (PIP), wingless-type MMTV integration site family, member 2B (WNT2B), Charcot-Leyden crystal protein (CLC) and palate lung nasal epithelial clone (PLUNC). The aim of the present study was to investigate if genetic variation associated with allergic rhinitis can be found in these genes. METHODS: Peripheral blood was collected from 251 patients with birch and/or grass pollen-induced allergic rhinitis and 386 nonatopic healthy controls. A total of 39 single nucleotide polymorphisms (SNPs) distributed over the genes PIP, WNT2B, CLC and PLUNC were selected from dbSNP, genotyped and investigated for associations with allergic rhinitis. Twelve additional SNPs were subsequently analysed for CLC. RESULTS: All 22 investigated SNPs in CLC were polymorphic. Ten SNPs yielded significant differences between cases and controls with respect to genotype frequencies. Homozygotes for the minor allele were more common in allergic individuals compared to healthy controls. The minor alleles of these SNPs were all located on the same haplotype. Furthermore, homozygotes for the minor allele of two of the promoter SNPs had higher average scores for birch in skin prick test. In contrast, for seven SNPs within the gene, heterozygotes and homozygotes for the major allele had higher average scores for grass. None of the other three genes showed association. CONCLUSION: Genetic variation in CLC was found to be associated with allergic rhinitis. The pattern of variation is compatible with a recessive inheritance model and the previously observed altered protein levels detected in patients with allergic rhinitis.

Important research questions in allergy and related diseases: 3-chronic rhinosinusitis and nasal polyposis - a GALEN study.

Chronic rhinosinusitis is one of the most common health care challenges, with significant direct medical costs and severe impact on lower airway disease and general health outcomes. The diagnosis of chronic rhinosinusitis (CRS) currently is based on clinical signs, nasal endoscopy and CT scanning, and therapeutic recommendations are focussing on 2 classes of drugs, corticosteroids and antibiotics. A better understanding of the pathogenesis and the factors amplifying mucosal inflammation therefore seems to be crucial for the development of new diagnostic and therapeutic tools. In an effort to extend knowledge in this area, the WP 2.7.2 of the GA(2)LEN network of excellence currently collects data and samples of 1000 CRS patients and 250 control subjects. The main objective of this project is to characterize patients with upper airway disease on the basis of clinical parameters, infectious agents, inflammatory mechanisms and remodeling processes. This collaborative research will result in better knowledge on patient phenotypes, pathomechanisms, and subtypes in chronic rhinosinusitis. This review summarizes the state of the art on chronic rhinosinusitis and nasal polyposis in different aspects of the disease. It defines potential gaps in the current research, and points to future research perspectives and targets.

A haplotype in the inducible T-cell tyrosine kinase is a risk factor for seasonal allergic rhinitis.

BACKGROUND: Identification of disease-associated single nucleotide polymorphisms (SNPs) in seasonal allergic rhinitis (SAR) may be facilitated by focusing on genes in a disease-associated pathway. OBJECTIVE: To search for SNPs in genes that belong to the T-cell receptor (TCR) pathway and that change in expression in allergen-challenged CD4+ cells from patients with SAR. METHODS: CD4+ cells from patients with SAR were analysed with gene expression microarrays. Allele, genotype and haplotype frequencies were compared in 251 patients and 386 healthy controls. RESULTS: Gene expression microarray analysis of allergen-challenged CD4+ cells from patients with SAR showed that 25 of 38 TCR pathway genes were differentially expressed. A total of 62 SNPs were analysed in eight of the 25 genes; ICOS, IL4, IL5, IL13, CSF2, CTLA4, the inducible T-cell tyrosine kinase (ITK) and CD3D. Significant chi-squared values were identified for several markers in the ITK kinase gene region. A total of five SNPs were nominally significant at the 5% level. Haplotype analysis of the five significant SNPs showed increased frequency of a haplotype that covered most of the coding part of ITK. The functional relevance of ITK was supported by analysis of an independent material, which showed increased expression of ITK in allergen-challenged CD4+ cells from patients, but not from controls. CONCLUSION: Analysis of SNPs in TCR pathway genes revealed that a haplotype that covers a major part of the coding sequence of ITK is a risk factor for SAR.

Association between polymorphisms in the prostate-specific antigen (PSA) promoter and release of PSA.

Variations in serum prostate-specific antigen (PSA) have been ascribed to A/G nucleotide polymorphisms located at -158 bp (rs266882) and -4643 bp (rs925013), relative to the transcription start site within the promoter of the PSA gene. PSA is also an androgen receptor target (AR) gene and polymorphisms in AR gene are known to affect AR function. Our objective was to compare the impact of these A/G polymorphisms separately or in combination with AR CAG micro satellite on regulation of PSA secretion into seminal plasma and blood in young men. Leukocyte DNA was extracted from 291 conscripts and genotyping performed with the Sequenom Mass Array System. PSA was measured with an immunofluorometric assay. Linear regression analysis was used to test the association of polymorphism frequencies with serum and seminal plasma levels of PSA. PSA gene polymorphisms at -158 bp or -4643 bp did not alone influence total PSA (tPSA) levels in seminal plasma or in blood. Homozygotes for the A-allele at -158 bp in combination with CAG > 22 had significantly higher serum levels of tPSA than subjects carrying the G-allele (p = 0.01). In conclusion, the PSA gene polymorphisms did not importantly influence the levels of tPSA in seminal plasma or in blood. tPSA in serum was influenced by interactions between PSA promoter variants and AR CAG polymorphism.

The evolutionary history of the common chloroplast genome of Arabidopsis thaliana and A. suecica.

The evolutionary history of the common chloroplast (cp) genome of the allotetraploid Arabidopsis suecica and its maternal parent A. thaliana was investigated by sequencing 50 fragments of cpDNA, resulting in 98 polymorphic sites. The variation in the A. suecica sample was small, in contrast to that of the A. thaliana sample. The time to the most recent common ancestor (T(MRCA)) of the A. suecica cp genome alone was estimated to be about one 37th of the T(MRCA) of both the A. thaliana and A. suecica cp genomes. This corresponds to A. suecica having a MRCA between 10 000 and 50 000 years ago, suggesting that the entire species originated during, or before, this period of time, although the estimates are sensitive to assumptions made about population size and mutation rate. The data was also consistent with the hypothesis of A. suecica being of single origin. Isolation-by-distance and population structure in A. thaliana depended upon the geographical scale analysed; isolation-by-distance was found to be weak on the global scale but locally pronounced. Within the genealogical cp tree of A. thaliana, there were indications that the root of the A. suecica species is located among accessions of A. thaliana that come primarily from central Europe. Selective neutrality of the cp genome could not be rejected, despite the fact that it contains several completely linked protein-coding genes.

Genetic analysis of 31 Swedish type 1 von Willebrand disease families reveals incomplete linkage to the von Willebrand factor gene and a high frequency of a certain disease haplotype.

BACKGROUND: The most common type of von Willebrand disease (VWD), type 1, has in only a few cases been explained by an identified causative mutation in the von Willebrand factor (VWF) gene. The ABO blood group and other modifier loci outside the VWF gene may contribute to the development of type 1 VWD. OBJECTIVES AND METHODS: Our aim was to determine whether there was genetic linkage to the VWF gene in 31 Swedish type 1 VWD families. Stringent diagnostic criteria in accordance with ISTH guidelines were used. Genetic linkage was investigated by using two highly informative dinucleotide microsatellite markers, which we have recently identified, located in introns six and 15 of the VWF gene. We also investigated the existence of common disease haplotypes and the relation between type 1 VWD and ABO blood group. RESULTS: We found genetic linkage to the VWF gene in 27 (87%) of the families. However, in four (13%) of the families, there was clearly no genetic linkage. We found the 4751A>G (Tyr1584Cys) sequence variation in exon 28, which is a common mutation in the Canadian VWD population (14.3%), in only one of the 31 families (3.2%). A possible common mutation was identified in six of the 27 (22%) families with genetic linkage. Blood group O was over-represented among type 1 VWD patients. CONCLUSION: We conclude that there is linkage between the VWF gene and hereditary type 1 VWD in a majority of families.

Co-segregation of the PROS1 locus and protein S deficiency in families having no detectable mutations in PROS1.

Inherited deficiency of protein S constitutes an important risk factor of venous thrombosis. Many reports have demonstrated that causative mutations in the protein S gene are found only in approximately 50% of the cases with protein S deficiency. It is uncertain whether the protein S gene is causative in all cases of protein S deficiency or if other genes are involved in cases where no mutation is identified. The aim of the current study was to determine whether haplotypes of the protein S gene cosegregate with the disease phenotype in cases where no mutations have been found. Eight protein S-deficient families comprising 115 individuals where previous DNA sequencing had failed to detect any causative mutations were analyzed using four microsatellite markers in the protein S gene region. Co-segregation between microsatellite haplotypes and protein S deficiency was found in seven of the investigated families, one family being uninformative. This suggests that the causative genetic defects are located in or close to the protein S gene in a majority of such cases where no mutations have been found.

Chloroplast DNA indicates a single origin of the allotetraploid Arabidopsis suecica.

DNA sequencing was performed on up to 12 chloroplast DNA regions [giving a total of 4288 base pairs (bp) in length] from the allopolyploid Arabidopsis suecica (48 accessions) and its two parental species, A. thaliana (25 accessions) and A. arenosa (seven accessions). Arabidopsis suecica was identical to A. thaliana at all 93 sites where A. thaliana and A. arenosa differed, thus showing that A. thaliana is the maternal parent of A. suecica. Under the assumption that A. thaliana and A. arenosa separated 5 million years ago, we estimated a substitution rate of 2.9 x 10(-9) per site per year in noncoding single copy sequence. Within A. thaliana we found 12 substitution (single bp) and eight insertion/deletion (indel) polymorphisms, separating the 25 accessions into 15 haplotypes. Eight of the A. thaliana accessions from central Sweden formed one cluster, which was separated from a cluster consisting of central European and extreme southern Swedish accessions. This latter cluster also included the A. suecica accessions, which were all identical except for one 5 bp indel. We interpret this low level of variation as a strong indication that A. suecica effectively has a single origin, which we dated at 20 000 years ago or more.

Genetic variation in Arabidopsis suecica and its parental species A. arenosa and A. thaliana.

Random amplified polymorphic DNA (RAPD) markers were used to estimate the level of genetic variation in Swedish accessions of the allopolyploid Arabidopsis suecica and its parental species A. thaliana and A. arenosa. The results showed clear differences among the three species with respect to the level of variation. A. arenosa was highly variable, A. thaliana showed a moderate level of variation whereas A. suecica was much less variable than the two other species. An extended analysis covering 19 Swedish populations of A. suecica corroborated the low level of variation in this species, yet 16 unique phenotypes were observed. No isolation by distance was observed. When the genetic variation was partitioned among and within populations of A. suecica, the results showed that the majority of the variation (81%) occurred among populations. This result is interpreted as a strong indication that A. suecica is autogamous in nature.

Primer mixtures in RAPD analysis.

RAPD (random amplified polymorphic DNA) is a multiplex marker system that conventionally uses single-primer PCR to amplify random DNA fragments. Because of its multiplex nature, it is frequently used in bulked segregant analysis (BSA). In view of the very large numbers of markers BSA often requires, we investigated the use of mixtures of primers as a method of increasing the number of markers available. Theoretically, if a single-primer reaction produces x bands on average, an unrestrained PCR process using a primers should produce xa2 bands. Initially, we investigated mixtures containing from one to five primers. The average number of products increased slightly from the single-primer to the multiple-primer case, whereas it was rather constant for the different multi-primer combinations. This deviation from the theoretical expectations, which we attribute to the effects of competition, shows mixtures of more than two primers to be inefficient. The properties of two-primer mixtures in which the proportions of the two primers were varied were also investigated. The intensities of most of the products were influenced by the proportions of the primers used to create the mixture. A good fit was obtained to a model in which the average competitive ability of a band is directly proportional to the probability of randomly obtaining the band-producing primer combination from the pool of primers. Using two-primer mixtures, a(a-1)/2 different two-primer mixtures can be produced. A comparison of different schemes for constructing the two-primer mixtures indicates that the degree of resampling is similar for all schemes. In conclusion, the use of two-primer mixtures is a simple but very powerful strategy in BSA as it can generate an extremely large number of markers.

Positive correlation between recombination rates and levels of genetic variation in natural populations of sea beet (Beta vulgaris subsp. maritima).

The relation between the level of genetic variation and the rate of recombination per physical unit was investigated in sea beet (Beta vulgaris subsp. maritima). The rate of recombination per physical unit was estimated indirectly through marker density in an RFLP linkage map of sugar beet. From this map, we also selected RFLP markers covering two of the nine chromosomes in Beta. The markers were used to estimate the level of genetic variation in three populations of sea beet, two from Italy and one from England. Two estimates of genetic variation were employed, one based on the number of alleles in the sample and the other on heterozygosity. A statistically significant positive correlation was found between recombination rate and genetic variation. Several theoretical explanations for this are discussed, background selection being one. A correlation similar to this has been observed previously in Drosophila, one that was higher than what we obtained for Beta. This is consistent with various biological differences between the two species.