Polymorphism at the Hor 1 locus of barley (Hordeum vulgare L.)

The Hor 1 locus of barley encodes a group of seed storage polypeptides called C hordein. Two-dimensional electrophoretic analysis of C-hordein fractions from six cultivars with different alleles at the Hor 1 locus showed extensive polymorphism. A total of 34 major polypeptides was mapped, with between 4 and 18 present in each cultivar. There was less variation among the same cultivars in the numbers (6 to 10) of restriction fragments of genomic DNA which hybridized to a cDNA clone related to C hordein. The total number of restriction fragments was also lower (22), and most pairs of cultivars had more restriction fragments than polypeptides in common. A total number of about 20–30 C-hordein genes per haploid genome was estimated. The results indicate that cultivars differ mainly in the extent of gene and polypeptide divergence, rather than in the degree of gene reiteration. They are consistent with the proposed origin of the multiple structural genes at the Hor 1 locus by the duplication and divergence of a single ancestral gene.NACB was supported by a grant from the Home Grown Cereals Authority.     

Proteomic and genetic analysis of glycinin subunits of sixteen soybean genotypes.

We investigated proteomic and genomic profiles of glycinin, a family of major storage proteins in 16 different soybean genotypes consisting of four groups including wild soybean (Glycine soja), unimproved cultivated soybean landraces from Asia (G. max), ancestors of N. American soybean (G. max), and modern soybean (G. max) genotypes. We observed considerable variation in all five glycinin subunits, G1, G2 G3, G4 and G5 using proteomics and genetic analysis. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry (MS) analysis showed that the wild genotypes had a range of 25-29 glycinin protein spots that included both acidic and basic polypeptides followed by the ancestors with 24-28, modern cultivars with 24-25, and landraces with 17-23 protein spots. Overall, the wild genotypes have a higher number of protein spots when compared to the other three genotypes. Major variation was observed in acidic polypeptides of G3, G4 and G5 compared to G1 and G2, and minor variation was observed in basic polypeptides of all subunits. Our data indicated that there are major variations of glycinin subunits between wild and cultivated genotypes rather than within the same groups. Based on Southern blot DNA analysis, we observed genetic polymorphisms in group I genes (G1, G2, and G3) between and within the four genotype groups, but not in group II genes (G4 and G5). This is the first study reporting the comparative analysis of glycinin in a diverse set of soybean genotypes using combined proteomic and genetic analysis.     

Identification and mapping of polymorphic RAPD markers of pea (Pisum sativum L.) genome

Various pea cultivars, lines, and mutants were studied by the RAPD method. Polymorphic fragments characteristic of certain pea genotypes and which can be used for identifying genotypes were detected. Inheritance of some polymorphic RAPD fragments was studied. Mendelian inheritance of these fragments was shown. By analyzing the data obtained in studies of RAPD polymorphism, genetic distances between different pea cultivars, lines, and mutants were calculated and a genealogic dendogram showing a varying extent of differences between RAPD patterns was constructed. Ten new RAPD markers linked to various pea genes were detected. Genetic distances between RAPD markers and genes to which they are linked were calculated, and the respective disposition of RAPD markers on chromosomes was established.     

Legumin heterogeneity inPisum

Analyses of heterogeneity in legumin subunit patterns, legumin precursor polypeptides, and restriction fragments containing legumin genes have shown thatPisum (pea) genotypes vary in the extent of gene and polypeptide divergence but not in the degree of gene reiteration. Genotypes containing single and multiple ^M subunits had the same numbers of legumin genes. The potential value of this heterogeneity in genetical analyses is outlined.This work was supported by the Agricultural and Food Research Council via a grant-in-aid to the John Innes Institute. We acknowledge financial support from Agrigenetics Corporation, Boulder, Colorado, and from the CEC Biomolecular Engineering Programme, Contract GBI-4-113-UK.     

Identification and mapping of polymorphic RAPD markers of pea (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.) genome

Various pea cultivars, lines, and mutants were studied by the RAPD method. Polymorphic fragments characteristic of certain pea genotypes and which can be used for identifying genotypes were detected. Inheritance of some polymorphic RAPD fragments was studied. Mendelian inheritance of these fragments was shown. By analyzing the data obtained in studies of RAPD polymorphism, genetic distances between different pea cultivars, lines, and mutants were calculated and a genealogic dendogram showing a varying extent of differences between RAPD patterns was constructed. Ten new RAPD markers linked to various pea genes were detected. Genetic distances between RAPD markers and genes to which they are linked were calculated, and the respective disposition of RAPD markers on chromosomes was established.Translated from Genetika, Vol. 41, No. 3, 2005, pp. 341–348.Original Russian Text Copyright © 2005 by Koveza, Kokaeva, Konovalov, Gostimsky.     

The structural organization and DNA sequence of a wheat histone H4 gene.

Some wheat histone H4 genes have been cloned from a Charon 4 wheat genomic DNA library using sea urchin histone H4 DNA as a probe. DNA sequence analysis of a cloned gene showed that the deduced amino acid sequence of wheat histone H4 protein was identical to that of pea. The 5' end of wheat histone H4 mRNA was mapped on the cloned gene by the S1-procedure. Southern blotting analysis of the genomic DNA indicated that histone H4 genes were reiterated 100 to 125 times per hexaploid wheat genome.     

Conserved Plasmid Hydrogen-Uptake (hup)-Specific Sequences within HupRhizobium leguminosarum Strains

Thirteen Rhizobium leguminosarum strains previously reported as H(2)-uptake hydrogenase positive (Hup) or negative (Hup) were analyzed for the presence and conservation of DNA sequences homologous to cloned Bradyrhizobium japonicum hup-specific DNA from cosmid pHU1 (M. A. Cantrell, R. A. Haugland, and H. J. Evans, Proc. Natl. Acad. Sci. USA 80:181-185, 1983). The Hup phenotype of these strains was reexamined by determining hydrogenase activity induced in bacteroids from pea nodules. Five strains, including H(2) oxidation-ATP synthesis-coupled and -uncoupled strains, induced significant rates of H(2)-uptake hydrogenase activity and contained DNA sequences homologous to three probe DNA fragments (5.9-kilobase [kb] HindIII, 2.9-kb EcoRI, and 5.0-kb EcoRI) from pHU1. The pattern of genomic DNA HindIII and EcoRI fragments with significant homology to each of the three probes was identical in all five strains regardless of the H(2)-dependent ATP generation trait. The restriction fragments containing the homology totalled about 22 kb of DNA common to the five strains. In all instances the putative hup sequences were located on a plasmid that also contained nif genes. The molecular sizes of the identified hup-sym plasmids ranged between 184 and 212 megadaltons. No common DNA sequences homologous to B. japonicum hup DNA were found in genomic DNA from any of the eight remaining strains showing no significant hydrogenase activity in pea bacteroids. These results suggest that the identified DNA region contains genes essential for hydrogenase activity in R. leguminosarum and that its organization is highly conserved within Hup strains in this symbiotic species.     

The molecular karyotype of Leishmania major and mapping of alpha and beta tubulin gene families to multiple unlinked chromosomal loci.

The arrangement of tubulin genes in the genome of the protozoan parasite Leishmania major was studied by genomic Southern blot analysis and mapping of genes to chromosomes fractionated by pulsed field gradient gel (PFG) electrophoresis. alpha-tubulin genes exist as a tandem array of 2.4 kb PstI fragments. beta-tubulin genes are found as a tandem array of 3.9 kb AvaI or PvuI fragments, but additional genes are also found on other genomic DNA fragments. Chromosome-sized DNA molecules released from promastigotes of L. major were fractionated into at least 17 chromosome bands of approximate size 400-4000 kb by PFG gel electrophoresis. Some bands may be present in non-equimolar amounts suggesting that there may be more than 17 chromosomes. All alpha-tubulin genes were localized to a single band (chromosome 7). beta-tubulin genes were localized to four bands (chromosomes 6, 10, 16 and 17). This shows that the alpha- and beta- tubulin gene families are unlinked in L. major. There is a single chromosomal locus for the alpha-tubulin tandem array whereas beta-tubulin genes exist both as a tandem array and as dispersed genes at four chromosomal loci.     

Development and study of SCAR markers in pea (Pisum sativum L.)

In order to develop more specific markers that characterize particular regions of the pea genome, the data on nucleotide sequences of RAPD fragments were used for choosing more extended primers, which may be helpful in amplifying a fragment corresponding to the particular DNA region. Of the 14 STS markers obtained from 14 polymorphic RAPD fragments, 12 were polymorphic, i.e., they are SCAR markers that can be used in genetic analysis. The transition from complex RAPD spectra to amplification of a particular SCAR marker substantially facilitates analysis of large samples for the presence or absence of the examined fragment. Inheritance of the developed SCAR markers was studied in F1 and F2. SCAR markers were used to identify various pea lines, cultivars, and mutants. It was established that the study of amplification of STS markers in various pea genotypes at varying temperatures of annealing and the comparison with amplification of the original RAPD fragments in the same genotypes provide an approach for analysis of RAPD polymorphism type.     

High-resolution DNA fingerprinting of parthenogenetic root-knot nematodes using AFLP analysis

Amplified fragment length polymorphism (AFLP) analysis has been used to characterize 15 root-knot nematode populations belonging to the three parthenogenetic species Meloidogyne arenaria , M. incognita and M. javanica. Sixteen primer combinations were used to generate AFLP patterns, with a total number of amplified fragments ranging from 872 to 1087, depending on the population tested. Two kinds of polymorphic DNA fragments could be distinguished: bands amplified in a single genotype, and bands polymorphic between genotypes (i.e. amplified in not all but at least two genotypes). Based on presence/absence of amplified bands and pairwise similarity values, all the populations tested were clustered according to their specific status. Significant intraspecific variation was revealed by AFLP, with DNA fragments polymorphic among populations within each of the three species tested. M. arenaria appeared as the most variable species, while M. javanica was the least polymorphic. Within each specific cluster, no general correlation could be found between genomic similarity and geographical origin of the populations. The results reported here showed the ability of the AFLP procedure to generate markers useful for genetic analysis in root-knot nematodes.     

The single-copy gene encoding high-mobility-group protein HMG-I/Y from pea contains a single intron and is expressed in all organs

The coding and 3-downstream regions of the gene encoding the high mobility group protein HMG-I/Y from pea have been isolated, sequenced and characterised. A 795 bp pea genomic fragment containing the coding region of the pea HMG-I/Y gene with a single intron of 201 bp was isolated by PCR. The gene encodes a protein of 197 amino acid residues with four copies of the AT-hook DNA-binding motif encoded by exon 2. Southern blot analysis on genomic DNA revealed the presence of a single copy of the HMG-I/Y gene in the haploid genome. The pea HMG-I/Y gene is expressed in all organs of pea including roots, stems, leaves, flowers, tendrils and developing seeds, as determined by northern blot analysis.