Restriction Fragment Length Polymorphism Linkage Research Articles

Evidence for mitochondrial pseudogenes (numts) as a source of contamination in the phylogeny of human whipworms.

Trichuris trichiura and T. suis are whipworms of humans and pigs, respectively, but it has recently been suggested that humans may be infected with multiple genotypes or species of Trichuris and cross-infection with Trichuris of pig origin has also been reported. In addition, the species status of Trichuris in non-human primates is unsettled and it is unknown how many whipworm species we share with other primates. Herein, we inferred the phylogeny of Trichuris collected from human, baboon and pig based on nuclear (18S and beta-tubulin) and mitochondrial (cox1) genes and evaluated the use of three PCR linked restriction fragment length polymorphism (PCR-RFLP) to identify worms. We found that all baboon worms clustered with human worms and that all these primate worms are different from T. suis. In general, there was an agreement between the phylogeny established based on the nuclear and mtDNA genes. However, we found evidence for non-targeted cox1 gene amplification for a subset of the human worms and suggest the presence of mitochondrial pseudogenes (numts) of pig cox1 gene in the human Trichuris genome. In conclusion, phylogenetic characterization of human whipworm based on the cox1 gene alone may be problematic without suitable preceded measures to avoid the numts amplification.

Phylogenetic relationships among Toxocara spp. and Toxascaris sp. from different regions of the world

Toxocara and Toxascaris are parasitic nematodes that infect canids and felids although species of the genus Toxocara also infect humans. This work aimed to establish the phylogenetic and phylogeographic relationship between specimens of T. canis, T. cati, T. malaysiensis and Toxascaris leonina and to evaluate the degree of host specificity. In total, 437 samples (adults and pools of eggs) were collected from canids and felids from eight countries. Parasites were identified by morphology, PCR linked Restriction Fragment Length Polymorphism (PCR-RFLP) and partial sequencing of the mitochondrial gene cox1. Phylogenetic trees were constructed and genetic distance among isolates was estimated. Based on the molecular characterization all worms were identified in agreement with their respective hosts with the exception of three samples; two from cats and one from dogs identified as T. canis and T. cati, respectively. There was no clear geographical clustering of the samples despite this study including parasites from three continents. This is the first study, to our knowledge, to use molecular methods to identify T. canis in cats and T. cati in dogs with host specificity being the most common finding. Our developed PCR-RFLP method was found to be a facile and reliable method for identifying Toxocara species.

The application of sequence specific primer and RT-PCR to LRRK2 gene polymorphism typing

Objective: To establish a new detecting method for disease susceptibility loci R1628P and G2385R of Parkinson’s disease (PD) related gene LRRK2.Methods: Sequence specific primers were designed to make a genotyping of DNA markers with known genotypes by use of quantitative fluorescence real-time PCR (RT-PCR). 100 cases of PD samples with unknown genotypes were tested, and verified by use of polymerase chain reaction linked restriction fragment length polymorphism (PCR-RLFP).Results: The genotyping results of DNA markers proved to be correct, and 100 cases of samples to be tested had a completely consistent genotyping result with PCR-RLFP genotyping result.Conclusions: Sequence specific primer and quantitative fluorescence RT-PCR can successfully make a genotyping for disease susceptibility loci R1628P and G2385R of LRRK2.

A novel technique for identification of Ascarissuum cohorts in pigs

The objective of the present study was to develop a fast, cheap and reliable technique for identifying different cohorts of the swine parasite, Ascaris suum. A polymerase chain reaction linked restriction fragment length polymorphism (PCR-RFLP) technique on mt-DNA was used to identify unique haplotypes of four gravid A. suum females on agarose gels after eggs were recovered from each of the worms. Each of four pigs was inoculated with 2000 embryonated eggs originating from one of the four identified Ascaris haplotypes, respectively. Ascaris larvae were isolated from the small intestine at day 14 post-infection using an agar technique. Single larvae from each pig were transferred to 96-well PCR plates and a simple DNA extraction using a worm lysis buffer was carried out and followed by the PCR-RFLP analysis. More than 100 larvae from each of the four pigs were analysed and all were found to have the same haplotype as the parental female. We conclude that unique haplotypes of female A. suum and offspring can be identified by means of PCR-RFLP on mt-DNA and suggest that this method can be used in future research on Ascaris population biology using cohorts with distinct mt-DNA profile.

A region of maize chromosome 2 affects response to downy mildew pathogens

Quantitative trait loci (QTLs) for downy mildew resistance in maize were identified based on co-segregation with linked restriction fragment length polymorphisms or simple sequence repeats in 220 F2 progeny from a cross between susceptible and resistant parents. Disease response was assessed on F3 families in nurseries in Egypt, Thailand, and South Texas and after inoculation in a controlled greenhouse test. Heritability of the disease reaction was high (around 93% in Thailand). One hundred and thirty polymorphic markers were assigned to the ten chromosomes of maize with LOD scores exceeding 4.9 and covering about 1,265 cM with an average interval length between markers of 9.5 cM. About 90% of the genome is located within 10 cM of the nearest marker. Three putative QTLs were detected in association with resistance to downy mildew in different environments using composite interval mapping. Despite environmental and symptom differences, one locus on chromosome 2 had a major effect and explained up to 70% of the phenotypic variation in Thailand where disease pressure was the highest. The other two QTLs on chromosome 3 and chromosome 9 had minor effects; each explained no more than 4% of the phenotypic variation. The three QTLs appeared to have additive effects on resistance, identifying one major gene and two minor genes that contribute to downy mildew resistance.

A PCR marker system for monitoring the frequencies of normal and sterility‐inducing cytoplasm types in German chive varieties

AbstractRandom amplified polymorphic DNAs and atp9‐related sequences were amplified in cytoplasmic male sterile (CMS) and maintainer lines drawn from a backcross programme to represent all five known cytoplasm types in chives. From these PCR amplifications, markers associated with CMS‐inducing cytoplasm types, (S1) and (S2), and for two of the three known normal cytoplasm types, (N2) and (N3), were developed. These newly developed PCR markers were used to determine the cytoplasm types in 126 plants representing 12 German chive varieties. The dependability of these PCR markers was confirmed by analysis with previously described and marker‐trait linked restriction fragment length polymorphisms. Two to five cytoplasm types were found in each of the 12 German chive varieties investigated. While the (S1) cytoplasm occurred, on average, at a frequency of 5% and the (S2) cytoplasm at 12%, the three normal cytoplasms (N1), (N2) and (N3) were present at 30, 29 and 24%, respectively. Thus, the prospects of finding maintainers for both CMS systems are relatively high in this population, if the frequency of non‐restoring alleles for the nuclear genes involved is also high enough.

Linkage Equilibrium among Tightly Linked RFLPs and the Ms Locus in Open-pollinated Onion Populations

Cytoplasmic male sterility (CMS) in onion (Allium cepa L.) is conditioned by the interaction of the male-sterile (S) cytoplasm with recessive alleles at a single nuclear male-fertility restoration locus (Ms). In order to seed propagate male-sterile plants (S msms), onion breeders must identify maintainer lines possessing normal (N) male-fertile cytoplasm and homozygous recessive at the Ms locus (N msms). Molecular markers have been identified distinguishing N and S cytoplasms and closely linked to the nuclear Ms locus. In this study, we evaluated testcross progenies from randomly selected N-cytoplasmic plants from three open-pollinated populations for nuclear restoration of male fertility over at least three environments. The Ms locus and linked restriction fragment length polymorphisms (0.9 and 1.7 cM) were at linkage equilibrium in all three open-pollinated onion populations, indicating that these linked markers cannot be used to identify maintaining genotypes in open-pollinated onion populations. However, cytoplasmic evaluations were effective in reducing the number of testcrosses required to identify CMS-maintaining genotypes.

Further molecular discrimination of Spanish strains of Echinococcus granulosus

We have designed two polymerase chain reaction (PCR) primer sets (PEg9F1-PEg9R1 and PEg16F1-PEg16R1) and two PCR protocols (Eg9-PCR and Eg16-PCR) for discrimination of Echinococcus granulosus genotypes. The oligonucleotide sequences originate from two E. granulosus DNA multiplex-PCR amplification fragments, previously reported, that allows species-specific discrimination between Taenia saginata, Taenia solium, and E. granulosus. The Eg9-PCR, Eg16-PCR, and Eg9-PCR linked restriction fragment length polymorphism (RFLP) analysis was used to characterize 53 E. granulosus isolates from the central region of Spain, highly endemic for echinococcosis. The analysis resulted in: (i) the discrimination of E. granulosus from Echinococcus multilocularis; (ii) the characterisation and discrimination of discrete E. granulosus strains from Spain; and (iii) the identification of two distinct genotypes within E. granulosus Spanish pig isolates. To further characterize the genetic variants in pigs, fragments of the NADH dehydrogenase I (ND1) and the cytochrome c oxidase subunit I (CO1) genes were amplified from parasite DNA and sequenced. The results again revealed the presence of two distinct genotypes: the G1 (sheep–dog strain) and G7 (pig–dog strain) genotypes. This observation could have important consequences for human health in Spain. Furthermore, the Eg9-PCR, Eg16-PCR, and Eg9-PCR-RFLP protocols can be used as additional methods to discriminate various E. granulosus genotypes.

Molecular characterization of the freshwater snail Lymnaea natalensis (Gastropoda: Lymnaeidae) on Madagascar with an observation of an unusual polymorphism in ribosomal small subunit genes

AbstractRecent characterization of nuclear ribosomal small subunit (SSU) genes has shown that variant nucleotides within this region could be useful for species and species group identification within the genus Lymnaea (Gastropoda: Lymnaeidae). This study aimed to characterize a range of populations of Lymnaea natalensis Krauss, 1848 on Madagascar, and addressed two related questions. First, is there any evidence of intraspecific variation of the SSU and, if so, what might be its significance? Secondly, might this variation jeopardize the use of SSU for lymnaeid taxonomy and phylogeny?Lymnaea natalensis (n= 212) was collected from 17 sampling localities, spanning the northern and southern ends of the island. Variation within a selected region of the SSU known to vary between species, the V1 and V2, was assayed by polymerase chain reaction (PCR) linked restriction fragment length polymorphism (RFLP) and denaturing gradient gel electrophoresis (DGGE) analysis. The PCR‐RFLP profiles indicated a striking dimorphism across populations at two restriction site loci (CfoI & MspI) within the E10‐1 helix of the V2 region. The observed RFLP variation was confirmed by direct sequencing and by genomic digestion with subsequent hybridization. Putative heterozygotes were also encountered and in these individuals the SSU arrays composed of two distinct types approximately 1% divergent. A severe departure from Hardy–Weinberg equilibrium with a highly statistically significant (P < 10−5) heterozygote deficiency was found and genetic variation among populations was highly structured (Fst= 0.53). The geographic distribution of the variants was mapped, revealing that one variant was restricted to higher, predominately colder environments and was thought to be an adaptation. The molecular basis of the SSU variation was caused by single nucleotide polymorphisms (SNPs). To test for the possibility of cryptic taxa, an analysis of individuals representative of the SSU variant types with isoenzyme analysis (ISA), randomly amplified polymorphic DNA (RAPDs) and PCR‐RFLP analysis of the ribosomal Internal Transcribed Spacer (ITS) was performed. Little variation was revealed and none that correlated to the groups suggested by SSU, confirming that the SSU variation was intraspecific. The levels of intraspecific divergence of the V1 and V2 within Lymnaea were not appreciably different (1%) from interspecific and would therefore question the validity of these data for lymnaeid taxonomy and phylogeny.

Molecular characterization of the freshwater snail Lymnaea natalensis (Gastropoda: Lymnaeidae) on Madagascar with an observation of an unusual polymorphism in ribosomal small subunit genes

Recent characterization of nuclear ribosomal small subunit (SSU) genes has shown that variant nucleotides within this region could be useful for species and species group identification within the genus Lymnaea (Gastropoda: Lymnaeidae). This study aimed to characterize a range of populations of Lymnaea natalensis Krauss, 1848 on Madagascar, and addressed two related questions. First, is there any evidence of intraspecific variation of the SSU and, if so, what might be its significance? Secondly, might this variation jeopardize the use of SSU for lymnaeid taxonomy and phylogeny?Lymnaea natalensis (n= 212) was collected from 17 sampling localities, spanning the northern and southern ends of the island. Variation within a selected region of the SSU known to vary between species, the V1 and V2, was assayed by polymerase chain reaction (PCR) linked restriction fragment length polymorphism (RFLP) and denaturing gradient gel electrophoresis (DGGE) analysis. The PCR-RFLP profiles indicated a striking dimorphism across populations at two restriction site loci (CfoI & MspI) within the E10-1 helix of the V2 region. The observed RFLP variation was confirmed by direct sequencing and by genomic digestion with subsequent hybridization. Putative heterozygotes were also encountered and in these individuals the SSU arrays composed of two distinct types approximately 1% divergent. A severe departure from Hardy–Weinberg equilibrium with a highly statistically significant (P < 10−5) heterozygote deficiency was found and genetic variation among populations was highly structured (Fst= 0.53). The geographic distribution of the variants was mapped, revealing that one variant was restricted to higher, predominately colder environments and was thought to be an adaptation. The molecular basis of the SSU variation was caused by single nucleotide polymorphisms (SNPs). To test for the possibility of cryptic taxa, an analysis of individuals representative of the SSU variant types with isoenzyme analysis (ISA), randomly amplified polymorphic DNA (RAPDs) and PCR-RFLP analysis of the ribosomal Internal Transcribed Spacer (ITS) was performed. Little variation was revealed and none that correlated to the groups suggested by SSU, confirming that the SSU variation was intraspecific. The levels of intraspecific divergence of the V1 and V2 within Lymnaea were not appreciably different (1%) from interspecific and would therefore question the validity of these data for lymnaeid taxonomy and phylogeny.

Development of PCR markers for wheat leaf rust resistance gene Lr47

The leaf rust resistance gene Lr47 confers resistance to a wide spectrum of leaf rust strains. This gene was recently transferred from chromosome 7S of Triticum speltoides to chromosome 7A of hexaploid wheat Triticum aestivum. To facilitate the transfer of Lr47 to commercial varieties, the completely linked restriction fragment length polymorphism (RFLP) locus Xabc465 was converted into a PCR-based marker. Barley clone ABC465 is orthologous to the type-I wheat sucrose synthase gene and primers were designed for the conserved regions between the two sequences. These conserved primers were used to amplify, clone and sequence different alleles from T. speltoides and T. aestivum. This sequence information was used to identify the T. speltoides sequence, detect allele-specific mutations, and design specific primers. Cosegregation of the PCR product of these primers and the T. speltoides chromosome segment was confirmed in four backcross-populations. To complement this dominant marker, a cleavage amplified polymorphic sequence (CAPS) was developed for the 7A allele of Xabc465. This CAPS marker is useful to select homozygous Lr47 plants from F2 or backcross-F2 segregating populations, and in combination with the T- speltoides specific primers is expected to facilitate the deployment of Lr47 in new bread wheat varieties.

Sequence Tagged Site Marker-Assisted Selection for Three Bacterial Blight Resistance Genes in Rice

IR65598-112 and the two sister lines IR65600-42 and IR65600-96 are promising new plant type (NPT) rice lines with high yield potential. However, these lines are susceptible to bacterial blight caused by Xanthomonas oryzae pv. oryzae (Xoo). To improve the resistance of the three NPT lines to Xoo, three bacterial blight (BB) resistance genes, xa5, xa13, and Xa21, were successfully transferred to the NPT lines via a marker-aided backcrossing procedure. Sequence tagged site (STS) markers for the two resistance genes were developed based on DNA sequences of their linked restriction fragment length polymorphism (RFLP) markers (RG556 and RG207 for xa5 and RG136 for xa13). Marker polymorphism for xa5 was detected after digestion of RG556 polymerase chain reaction (PCR) products with MaeII enzyme and digestion of RG136 PCR product with HinfI enzyme for xa13. The STS marker for Xa21 was designed previously from the sequence of a genomic clone RAPD248. Fifty-nine BC 3 F 2 near-isogenic lines (NILs) in the three NPT backgrounds containing one to three BB resistance genes in various combinations were developed through marker-assisted selection (MAS) for the resistance genes and phenotypic selection for the NPT. The BC 3 F 3 NILs having more than one BB resistance gene showed a wider resistance spectrum and manifested increased levels of resistance to the Xoo races, as compared with those having a single BB resistance gene. Results for two F 2 populations and the progeny testing of their F 3 lines showed that MAS reached an accuracy of 95 and 96% of identifying homozygous resistant plants for xa5 and xa13, respectively. These NPT NILs for BB resistance genes provided valuable materials for breeding and genetic studies of single-gene effects and interaction of several resistance genes. The results demonstrate the usefulness of MAS in gene pyramiding for BB resistance, particularly for recessive genes, such as xa5 and xa13, that are difficult to select through conventional breeding in the presence of a dominant gene such as Xa21.

The Molecular Genetics of Self-incompatibility in Petunia hybrida

Abstract The cultivated petunia ( Petunia hybrida ) has been a popular system in which to study genetic, physiological and biochemical aspects of gametophytic self-incompatibility. As with other members of the Solanaceae a number of S-RNase genes have been isolated for functional S -alleles. We have identified S-RNase sequences for two additional functional S -alleles, S v and S 3 . These alleles are more similar to alleles from other families of the Solanaceae ( Nicotiana and Solanum ) than to any petunia alleles reported previously. The total number of S -alleles in P. hybrida is at least ten in spite of its cultivated origin. However, most cultivars of P. hybrida are in fact self-compatible and this appears to arise from the prominence of a single previously described allele S o . The implications of this observation for the origin of self-compatibility in P. hybrida are discussed. The S -locus of P. hybrida has recently been mapped using an indirect method involving T-DNA insertions. Seven T-DNA insertions that were previously shown to be closely linked to the S -locus were physically mapped on the long arm of chromosome III using fluorescent in-situ hybridization. The most tightly linked T-DNA insertions are in a sub-centromeric position. This is consistent with the centric fragments of P. inflata obtained by irradiation mutagenesis that carry additional S -loci and confer a pollen-part mutant phenotype. An S -linked restriction fragment length polymorphism (RFLP) marker, CP100 was used to confirm this chromosomal assignment and has provided evidence for S -locus synteny in the Solanaceae.

BUNDLE SHEATH DEFECTIVE2, a novel protein required for post-translational regulation of the rbcL gene of maize.

The Bundle sheath defective2 (Bsd2) gene is required for ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco) accumulation in maize. Using a Mutator transposable element as a molecular probe, we identified a tightly linked restriction fragment length polymorphism that cosegregated with the bsd2-conferred phenotype. This fragment was cloned, and sequences flanking the Mutator insertion were used to screen a maize leaf cDNA library. Using a full-length cDNA clone isolated in this screen, we show that an abundant 0.6-kb transcript could be detected in wild-type plants but not in bsd2-m1 plants. This 0.6-kb transcript accumulated to low levels in plants carrying an allele derived from bsd2-m1 that conditions a less severe mutant phenotype. Taken together, these data strongly suggest that we have cloned the Bsd2 gene. Sequence analysis of the full-length cDNA clone revealed a chloroplast targeting sequence and a region of homology shared between BSD2 and the DnaJ class of molecular chaperones. This region of homology is limited to a cysteine-rich Zn binding domain in DnaJ believed to play a role in protein-protein interactions. We show that BSD2 is targeted to the chloroplast but is not involved in general photosynthetic complex assembly or protein import. In bsd2 mutants, we could not detect the Rubisco protein, but the chloroplast-encoded Rubisco large subunit transcript (rbcL) was abundant and associated with polysomes in both bundle sheath and mesophyll cells. By characterizing Bsd2 expression patterns and analyzing the bsd2-conferred phenotype, we propose a model for BSD2 in the post-translational regulation of rbcL in maize.

Identification of Quantitative Loci for Tolerance to Barley Yellow Dwarf Virus in Oat

ABSTRACT Molecular markers linked to quantitative trait loci conditioning tolerance to barley yellow dwarf virus (BYDV) were identified in oat (Avena sativa) using amplified fragment length polymorphism (AFLP) analysis. Near-isogenic and recombinant inbred lines (NILs and RILs, respectively) derived from a cross of Clintland64 (BYDV-sensitive) and IL86-5698 (BYDV-tolerant) were evaluated for their responses to an Illinois isolate of the PAV strain of BYDV. Individual markers identified in the analysis of the NILs explained up to 35% of the variability seen in the tolerance response. Single-point analysis of the marker data from the RIL population identified 24 markers in three linkage groups that were associated with tolerance to BYDV infection at P </= 0.001. These markers defined three major loci, A, C, and E, that were contributed by the tolerant parent (IL86-5698) and explained 35.0, 20.6, and 17.0% of the variability, respectively. Three minor loci G, H(1), and R) were identified at P </= 0.01. These loci were contributed by the sensitive parent (Clintland64) and explained 5.8, 5.6, and 5.6% of the variability, respectively. Interval analysis showed that only the loci A, C, and E are associated significantly with BYDV tolerance at log of the likelihood ratio >/= 3.0. These loci explained about 50% total of the variation in BYDV tolerance in multimarker regression analysis in both years. The BYDV tolerance loci A, C, E, and R were mapped to hexaploid oat restriction fragment length polymorphism linkage groups 2, 8, 36, and 5, respectively, by analyzing the segregation of the AFLP markers in the Kanota x Ogle RIL population.

Identification of (CA/GT)n polymorphisms within the X-linked chronic granulomatous disease (X-CGD) gene: utility for prenatal diagnosis.

The majority of known female carriers of X-linked chronic granulomatous disease (X-CGD), a deficiency of the gp91-phox (phagocyte oxidase) subunit and the most common genetic subtype of CGD, are not informative for the linked restriction fragment length polymorphisms (RFLPs) described to date. The isolation and characterization of two polymorphic (CA/GT)n repeats that lie within the X-CGD gene are reported, which are a useful linked marker for prenatal diagnosis. cDNA for gp91-phox was used to probe a genomic library. Genomic clones were isolated and screened for (CA/GT)n repeats. The repeats were isolated and sequences surrounding the repeats were determined. Oligonucleotide primer pairs surrounding the repeats were chosen to facilitate polymerase chain reaction (PCR) across the repeat. Analysis of DNA derived from over 100 individuals shows both markers to be highly polymorphic with a resultant high proportion of heterozygosity in females. Several kindreds affected by X-CGD were studied and the (CA/GT)n length polymorphisms were shown to segregate with the clinical syndrome or biochemical carrier status. The technique was prospectively applied to several kindreds containing a carrier mother and an affected child. In a case where a male fetus was shown to carry the unaffected allele, the pregnancy was carried to term and the child was not affected. This approach is highly informative in a multiple allele system, can provide a technical analysis in just hours, requires only a ng of DNA, and permits the transport of diagnostic samples. Therefore, this method can be used early in pregnancy on a chorionic villus biopsy sample for prenatal diagnosis.

Autosomal dominant nocturnal frontal lobe epilepsy. A video-polysomnographic and genetic appraisal of 40 patients and delineation of the epileptic syndrome.

A number of clinical and aetiological studies have been performed, during the last 30 years, on patients with abnormal nocturnal motor and behavioural phenomena. The aetiological conclusions of these studies were often conflicting, suggesting either an epileptic or a non-epileptic origin. Among the clinical characteristics of these patients, the familial clustering was one thoroughly accepted. A nocturnal familial form of frontal lobe epilepsy (autosomal dominant nocturnal frontal lobe epilepsy, ADNFLE), often misdiagnosed as parasomnia, has been recently described in some families. In one large Australian kindred, a missense mutation in the second transmembrane domain of the neuronal nicotinic acetylcholine receptor alpha 4 subunit (CHRNA4) gene, located on chromosome 20 q13.2-13.3, has been reported to be associated with nocturnal frontal lobe epilepsy. We performed an extensive clinical and video-polysomnographic study in 40 patients complaining of repeated abnormal nocturnal motor and/or behavioural phenomena, from 30 unrelated Italian families. Thirty-eight patients had an electroclinical picture strongly suggesting the diagnosis of ADNFLE. They had a wide clinical spectrum, ranging from nocturnal enuresis to sleep-related violent behaviour, thus including all the main features of the so-called 'typical' parasomnias. The video-polysomnographic recording confirmed the wide spectrum of abnormal manifestations, including sudden awakenings with dystonic/ dyskinetic movements (in 42.1% of patients), complex behaviours (13.2%) and sleep-related violent behaviour (5.3%). The EEG findings showed ictal epileptiform abnormalities predominantly over frontal areas in 31.6% of patients. In another 47.4% of patients the EEG showed ictal rhythmic slow activity over anterior areas. Only 18.4% of the patients had already received a correct diagnosis of epilepsy. In 73.3% of the patients treated with anti-epileptic drugs the seizures were readily controlled. Pedigree analysis on 28 of the families was consistent with autosomal dominant transmission with reduced penetrance (81%). DNAs from 20 representative affected individuals were sequenced in order to check for the presence of the missense mutation in the CHRNA4 gene found in the Australian kindred affected by ADNFLE. Nucleotide sequence analysis did not reveal the presence of this mutation, but it did confirm the presence of two other base substitutions, not leading to amino acid changes. These two intragenic polymorphisms, together with a closely linked restriction fragment length polymorphism at the D20S20 locus, have been used for linkage analysis of ADNFLE to the terminal region of the long arm of chromosome 20 in five compliant families. The results allowed us to exclude linkage of ADNFLE to this chromosomal region in these families, thus confirming the locus heterogeneity of the disorder. Large and full video-polysomnographical studies are of the utmost importance in order to clarify the real prevalence of both nocturnal frontal lobe epilepsy and parasomnias, and to provide a correct therapy.

An interspecific backcross of Lycopersicon esculentum x L. hirsutum: linkage analysis and a QTL study of sexual compatibility factors and floral traits.

A BC1 population of the self-compatible tomato Lycopersicon esculentum and its wild self-incompatible relative L. hirsutum f. typicum was used for restriction fragment length polymorphism linkage analysis and quantitative trait loci (QTL) mapping of reproductive behavior and floral traits. The self-incompatibility locus, S, on chromosome 1 harbored the only QTL for self-incompatibility indicating that the transition to self-compatibility in the lineage leading to the cultivated tomato was primarily the result of mutations at the S locus. Moreover, the major QTL controlling unilateral incongruity also mapped to the S locus, supporting the hypothesis that self-incompatibility and unilateral incongruity are not independent mechanisms. The mating behavior of near-isogenic lines carrying the L. hirsutum allele for the S locus on chromosome 1 in an otherwise L. esculentum background support these conclusions. The S locus region of chromosome 1 also harbors most major QTL for several floral traits important to pollination biology (e.g., number and size of flowers), suggesting a gene complex controlling both genetic and morphological mechanisms of reproduction control. Similar associations in other flowering plants suggest that such complex may have been conserved since early periods of plant evolution or else reflect a convergent evolutionary process.

Pod Dehiscence of Soybean: Identification of Quantitative Trait Loci

The dehiscence of pods (shattering) prior to harvest is an undesirable trait of soybean, Glycine max (L.) Merr. Pod dehiscence (PD) is relatively uncommon in modern North American soybean cultivars, but is often observed when unimproved germplasm or the wild species, G. soja Siebold & Zucc., are used as parents to introgress useful genes or to develop genetically diverse breeding populations. In light of the potential for efficient selection using DNA markers, the objective of this study was to identify quantitative trait loci (QTL) that condition resistance to PD. A map of 140 linked restriction fragment length polymorphism (RFLP) markers was constructed using 120 F4-derived lines from a soybean population (Young x PI 416937) that segregated for resistance to PD. These lines were scored for PD on a visual scale of 1 to 10 at both Athens, Georgia, and Windblow, North Carolina, in 1994. Heritability of pod dehiscence was 92%. Associations of marker loci with QTL that condition resistance to PD were tested using homozygous RFLP class means in a single-factor ANOVA. A total of five putatively independent RFLP markers were associated with PD at both locations and in a combined analysis over locations. A single RFLP locus on linkage group J of the USDA/Iowa State University map accounted for 44% of the variation in PD score. Epistasis was observed between one pair of significant marker loci. These results establish the genomic location of one major and a few minor QTL, identify an epistatic interaction, and indicate transgressive segregation which is plausibly the result of susceptibility alleles contributed by the resistant parent.

Identification of RFLP markers linked to a gene for downy mildew resistance (Sdm) in sorghum

The Sdm locus in sorghum accession SC325 confers resistance to downy mildew caused by pathotypes 1 and 3 of Peronosclerospora sorghi. Restriction fragment length polymorphism linkage analysis placed the Sdm locus near loci detected by probes pSbTXS552 and pSbTXS361 at 5.0 and 7.9 cM, respectively. Fragment patterns for the cross segregating for disease resistance differed from those for the original mapping cross, preventing assignment of Sdm to a linkage group. Keywords: Peronosclerospora sorghi, resistance, RFLP, linkage, hybrid breeding.