Construction and characterisation of a yeast artificial chromosome library containing five haploid sugarbeet (Beta vulgaris L.) genome equivalents

A yeast artificial chromosome (YAC) genomic library of Beta vulgaris was constructed in the pYAC4 vector. High-molecular-weight DNA was prepared from agarose-embedded leaf protoplasts from a triploid cultivar. The library was found to contain 33,500 clones in an ordered array of microtiter plates. Mean size of the inserts was estimated to be 135 kb, and the library should therefore represent the equivalent of five haploid genomes. The library was characterised for the presence of highly repetitive, chloroplast and single-copy sequences. In order to isolate single-copy sequences, 18 pools of DNA, each from 1920 individual YAC clones, were prepared for rapid screening of the library by the polymerase chain reaction. The results of these screenings showed that the number of isolated clones was at or near the frequency expected.     

Chloroplast DNA inheritance in theStellaria longipes complex (Caryophyllaceae)

Inheritance of chloroplast DNA (cpDNA) was examined in F₁ progenies derived from three crosses and three corresponding reciprocal crosses betweenStellaria porsildii andS. longifolia. Chloroplast DNA restriction fragments were analyzed using methods of nonradioactive digoxigenin-11-dUTP labeling and chemiluminescent detection with Lumi-Phos 530. Distinct interspecific restriction fragment polymorphisms were identified and used to demonstrate the mode of cpDNA inheritance. Mode of cpDNA inheritance differed among crosses. Two crosses in whichS. porsildii, SP2920-21, was the maternal parent exhibited three different types of plastids, maternal, paternal and biparental, among the F₁ hybrids, suggesting a biparental cpDNA inheritance and plastid sorting-out inStellaria.     

Genetic analysis of morphological variation in Brassica oleracea using molecular markers

A cross between the open-pollinated Brassica oleracea cabbage cultivar Wisconsin Golden Acre and the hybrid broccoli cultivar Packman was used with molecular markers to investigate the genetic control of morphological variation. Twenty-two traits derived from leaf, stem, and flowering measurements were analyzed in 90 F₂ individuals that were also classified for genotype by restriction fragment length polymorphism (RFLP) markers. Seventy-two RFLP loci, which covered the mapped genome at an average of 10 map-unit intervals on all nine linkage groups, were tested individually for associations to phenotypic measurements by single factor ANOVA, and markers with significant associations (P<0.05) were used to develop multilocus models. These data were utilized to describe the location, parental contribution of alleles, magnitude of effect, and the gene action of trait loci. Single marker loci that were significantly associated (P<0.05) with trait measurements accounted for 6.7–42.7% of the phenotypic variation. Multilocus models described as much as 60.1% of the phenotypic variation for a given trait. In some cases, different related traits had common marker-locus associations with similar gene action and genotypic class ranking. The numbers, action, and linkages, of genes controlling traits estimated with marker loci in this population corresponded to estimates based on classical genetic methods from other studies using similar, or similarly-wide, crosses. There was no evidence that genome duplication accounted for a significant portion of multiple genes controlling trait loci over the entire genome, but possible duplications of trait loci were identified for two regions with linked, duplicated marker loci.     

RFLP tagging of a new semidwarfing gene in rice

A new rice semidwarfing gene which is not allelic tosd1, temporarily designated assdg, might be of use as a new source of semidwarfism in rice breeding programs. We report here the identification of a DNA marker closely linked to this gene. The DNA marker was identified by testing 120 mapped rice RFLP makers as hybridization probes for Southern analysis of a pair of nearly isogenic lines with or withoutsdg. Linkage association of the marker with the gene was verified using a F₂ population segregating for semidwarfism. RFLP analysis showed thatsdg is closely linked to a single-copy DNA clone RZ182 on chromosome 5, with a distance of 4.3 centiMorgans between them. This marker may facilitate early selection for the semidwarfing gene in rice breeding programs     

Growth hormone and insulin-like growth factor I concentrations in bulls of various growth hormone genotypes

A leucine/valine substitution at amino acid position 127 was identified by the polymerase chain reaction and restriction fragment length polymorphism in the bovine growth hormone gene. Genotyping was performed in 84 AI bulls of three different breeds, in which plasma concentrations of growth hormone (GH) and insulin-like growth factor I (IGF-1) were also measured. Gene frequencies of variants L (leucine) und V (valine) were 0.80/0.20 (Black and White), 0.90/0.10 (Brown), 0.71/0.29 (Simmental). Hormone concentrations were measured during different physiological conditions (normal feeding, fasting, realimentation) in the majority of animals. Generally, genotype LL was associated with higher concentrations of GH than LV. This difference was significant in Black and White bulls (P < 0.05). In contrast, IGF-1 concentrations were higher in LV than in LL animals. This was most pronounced in mature, realimented Simmental bulls. We conclude that the various GH alleles influence the circulating concentrations of GH and IGF-1.     

Identification of RFLP markers linked to the cereal cyst nematode resistance gene (Cre) in wheat

The cereal cyst nematode (CCN) (Heterodera avenae Woll.) is an economically damaging pest of wheat in many of the worlds cereal growing areas. The development of CCN-resistant cultivars may be accelerated by the use of molecular markers. The Cre gene of the wheat line AUS 10894 confers resistance to CCN. Using a pair of near-isogenic lines (NILs) that should differ only in a small chromosome segment containing the Cre locus, we screened 58 group-2 probes and found two (Tag605 and CDO588) that detect polymorphism between the NILs. Nulli-tetrasomic and ditelosomic lines confirmed that the restriction fragment length polymorphism (RFLP) markers identified were derived from the long arm of wheat chromosome 2. Crosses between AUS 10894 and Spear and the NIL AP and its recurrent parent Prins were used to produce F₂ populations that gave the expected 31 segregation ratio for the resistance gene. Linkage analysis identified two RFLP markers flanking the resistance gene. Xglk605 and Xcdo588 mapped 7.3 cM (LOD=6.0) and 8.4 cM (LOD=6.7), respectively, from the Cre locus.     

Genome size and restriction fragment length polymorphism analysis of Vibrio cholerae strains belonging to different serovars and biotypes

Abstract The genome size of Vibrio cholerae has been determined by pulsed field gel electrophoresis following digestion of chromosomal DNA with endonucleases. The genome size of all the classical strains examined was about 3000 kb and that of El Tor biotype was 2500 kb. The Not I and S fi I digestion patterns of the genomes of several V. cholerae straimns belonging to different serovars and biotypes showed distinct restriction fragment length polymorphism (RFLP). RFLP analysis together with the genome size can be used to differentiate strains of different serovars and biotypes of V. cholerae .     

Restriction Fragment Length Polymorphism (RFLP) of Genomic DNA of Moraxella (Branhamella) catarrhalis Isolates in a Hospital

Epidemiological typing, based on restriction fragment length polymorphism (RFLP) by pulsed-field gel electrophoresis (PFGE), was attempted for the 38 clinical isolates of Moraxella catarrhalis obtained at Shinshu University Hospital during the years 1987 and 1993. Digestion with SmaI or NotI generated well separable, 12 to 5 genomic DNA fragments ranging from 1,000 kb to 30 kb and the strains could be classified into 14 or 13 types, respectively. The electrophoretic profile differed with the strain in most of them and was hence useful to distinguish the each strain. Investigation for their RFLP have, however, suggested that majority of them, including the type strain ATCC25238, may have derived from a common ancestor.     

Enhanced detection of polymorphic DNA by multiple arbitrary amplicon profiling of endonuclease-digested DNA: identification of markers tightly linked to the supernodulation locus in soybean

Multiple endonuclease digestion of template DNA or amplification products can increase significantly the detection of polymorphic DNA in fingerprints generated by multiple arbitrary amplicon profiling (MAAP). This coupling of endonuclease cleavage and amplification of arbitrary stretches of DNA, directed by short oligonucleotide primers, readily allowed distinction of closely related fungal and bacterial isolates and plant cultivars. MAAP analysis of cleaved template DNA enabled the identification of molecular markers linked to a developmental locus of soybean (Glycine max L. Merrill). Ethyl methane sulfonate (EMS)-induced supernodulating, near-isogenic lines altered in the nts locus, which controls nodule formation, could be distinguished from each other and from the parent cultivar by amplification of template pre-digested with 2–3 restriction enzymes. A total of 42 DNA polymorphisms were detected using only 19 octamer primers. In the absence of digestion, 25 primers failed to differentiate these soybean genotypes. Several polymorphic products co-segregated tightly with the nts locus in F₂ families from crosses between the allelic mutants nts382 and nts1007 and the ancestral G. soja Sieb. & Succ. PI468.397. Our results suggest that EMS is capable of inducing extensive DNA alterations, probably around discrete mutational hot-spots. EMS-induced DNA polymorphisms may constitute sequence-tagged markers diagnostic of specific genomic regions.     

Structural difference between polymerized and non-polymerized fragment X, obtained by plasmin digest of fibrinogen

Fragment X (LMrFX) was obtained as low molecular weight preparations from a late stage 2 plasmin digest of human fibrinogen. The thrombin-treated LMrFX preparations, which resulted in impaired polymerization, were further subfractionated into polymerized and non-polymerized components. The fractions were examined by SDS-PAGE and immunochemical methods. In polymerized fractions, more peptide bands were observed on SDS-PAGE in the reduced state than in non-polymerized fractions. Both fractions contained a similar number of internal cleavages in the A, Bβ and γ chains, which are linked by disulfide bonds. Thus, the partial deficiencies in polymerization sites of the carboxy terminal region of the γ chain and the amino terminal portions of the Bβ chain, as well as internal cleavage, were considered to participate in the impairment of the thrombin-induced polymerization of LMrFX.