The spatial organization of replication is determined by cell size independently of chromosome copy number

We have studied how Escherichia coli coordinates replication and division cycles by tracking replisomes in individual cells through thousands of division cycles. By analysing the replication and division of the cells at various growth rates and with different genetic perturbations, we have reached four major conclusions: (1) Initiation is not based on titration of DnaA to the chromosomal binding sites because initiation is still coordinated with the division cycle several generations after DnaA synthesis is turned off. (2) We can rule out that initiation is triggered by a simple DnaA-ADP/ATP switch mechanism because initiation is still triggered at a well-defined cell size in a DARS1/DARS2/datA triple knockout if DnaA is overexpressed. (3) From the spatial organization of replisomes at different growth rates, it appears as if initiation is triggered by termination, but this hypothesis is ruled out by an NGS-based marker frequency analysis. (4) Overlapping replication cycles do not affect the spatial organization of the replication machinery and it appears as if each replication center contains a fixed number of DnaN molecules independently of how many replication forks are being processed.

Characterization of Rph24: A Gene Conferring Adult Plant Resistance to Puccinia hordei in Barley

We identified Rph24 as a locus in barley (Hordeum vulgare L.) controlling adult plant resistance (APR) to leaf rust, caused by Puccinia hordei. The locus was previously reported as a quantitative trait locus in barley line ND24260-1 and named qRphND. We crossed ND24260-1 to the leaf-rust-susceptible standard Gus and determined inheritance patterns in the progeny. For the comparative marker frequency analysis (MFA), resistant and susceptible tails of the F2 were genotyped with Diversity Arrays Technology genotyping-by-sequencing (DArT-Seq) markers. The Rph24 locus was positioned at 55.5 centimorgans on chromosome 6H on the DArT-Seq consensus map. Evaluation of F2:3 families confirmed that a single locus from ND24260-1 conferred partial resistance. The haploblock strongly associated with the Rph24 locus was used to estimate the allele frequency in a collection of 282 international barley cultivars. Rph24 was frequently paired with APR locus Rph20 in cultivars displaying high levels of APR to leaf rust. The markers identified in this study for Rph24 should be useful for marker-assisted selection.

Multiple replication origins with diverse control mechanisms in Haloarcula hispanica

The use of multiple replication origins in archaea is not well understood. In particular, little is known about their specific control mechanisms. Here, we investigated the active replication origins in the three replicons of a halophilic archaeon, Haloarcula hispanica, by extensive gene deletion, DNA mutation and genome-wide marker frequency analyses. We revealed that individual origins are specifically dependent on their co-located cdc6 genes, and a single active origin/cdc6 pairing is essential and sufficient for each replicon. Notably, we demonstrated that the activities of oriC1 and oriC2, the two origins on the main chromosome, are differently controlled. A G-rich inverted repeat located in the internal region between the two inverted origin recognition boxes (ORBs) plays as an enhancer for oriC1, whereas the replication initiation at oriC2 is negatively regulated by an ORB-rich region located downstream of oriC2-cdc6E, likely via Cdc6E-titrating. The oriC2 placed on a plasmid is incompatible with the wild-type (but not the ΔoriC2) host strain, further indicating that strict control of the oriC2 activity is important for the cell. This is the first report revealing diverse control mechanisms of origins in haloarchaea, which has provided novel insights into the use and coordination of multiple replication origins in the domain of Archaea.

Mapping Mendelian traits in asexual progeny using changes in marker allele frequency

SummaryLinkage between markers and genes that affect a phenotype of interest may be determined by examining differences in marker allele frequency in the extreme progeny of a cross between two inbred lines. This strategy is usually employed when pooling is used to reduce genotyping costs. When the cross progeny are asexual, the extreme progeny may be selected by multiple generations of asexual reproduction and selection. We analyse this method of measuring phenotype in asexual progeny and examine the changes in marker allele frequency due to selection over many generations. Stochasticity in marker frequency in the selected population arises due to the finite initial population size. We derive the distribution of marker frequency as a result of selection at a single major locus, and show that in order to avoid spurious changes in marker allele frequency in the selected population, the initial population size should be in the low to mid hundreds.

Multiple Replication Origins of Halobacterium sp. Strain NRC-1: Properties of the Conserved orc7-Dependent oriC1

ABSTRACT The eukaryote-like DNA replication system of the model haloarchaeon Halobacterium NRC-1 is encoded within a circular chromosome and two large megaplasmids or minichromosomes, pNRC100 and pNRC200. We previously showed by genetic analysis that 2 (orc2 and orc10) of the 10 genes coding for Orc-Cdc6 replication initiator proteins were essential, while a third (orc7), located near a highly conserved autonomously replicating sequence, oriC1, was nonessential for cell viability. Here we used whole-genome marker frequency analysis (MFA) and found multiple peaks, indicative of multiple replication origins. The largest chromosomal peaks were located proximal to orc7 (oriC1) and orc10 (oriC2), and the largest peaks on the extrachromosomal elements were near orc9 (oriP1) in both pNRC100 and -200 and near orc4 (oriP2) in pNRC200. MFA of deletion strains containing different combinations of chromosomal orc genes showed that replication initiation at oriC1 requires orc7 but not orc6 and orc8. The initiation sites at oriC1 were determined by replication initiation point analysis and found to map divergently within and near an AT-rich element flanked by likely Orc binding sites. The oriC1 region, Orc binding sites, and orc7 gene orthologs were conserved in all sequenced haloarchaea. Serial deletion of orc genes resulted in the construction of a minimal strain containing not only orc2 and orc10 but also orc9. Our results suggest that replication in this model system is intriguing and more complex than previously thought. We discuss these results from the perspective of the replication strategy and evolution of haloarchaeal genomes.

The Characterization of Jalapeno (Capsicum annuum) Germplasm Based on RAPD Markers and Morphology

Characterization of germplasm collections is often criticized due to the lack of relevance given to “unadapted” germplasm by commercial breeders. Within Capsicum, only specific pod types are commercially important. Jalapeno peppers are becoming increasingly important due to the increase in sale of Capsicum-based food products. Unfortunately, few Jalapeno cultivars are available to growers. A Capsicum is classified as a Jalapeno based largely on pod shape, rendering a liberal definition of a Jalapeno. Curators and breeders with knowledge of pepper collections submitted accessions characterized as a Jalapeno. These accessions were grown at two locations to cull accessions not included in the Jalapeno market class. Accessions were characterized for traits important to commercial breeders at both locations. In addition, accessions were characterized using a set of RAPD markers dispersed throughout the genome in a separate mapping population. A subset, created from RAPD marker-based estimates of genetic distance, was created to represent the range of genetic diversity available among all Jalapeno accessions analyzed. These accessions will add genetic diversity to a breeding program without changing pod type expectations. The comparison between Jalapeno accessions and currently grown Jalapeno cultivars was examined based on differences in RAPD marker frequency. In addition, differences in marker frequencies were used to compare Jalapenos and other C. annuum market types also characterized with RAPD markers. The characterization of Jalapenos will assist breeders in their future efforts to diversify their Capsicum breeding base.

Analysis of the genetic composition of anther-derived potato by randomly amplified polymorphic DNA and simple sequence repeats

Randomly amplified polymorphic DNA (RAPD) and simple sequence repeat (SSR) analyses were used to characterize the genetic composition of anther-derived plants of a diploid potato clone, CP2 (Solanum chacoense 80-1 × S. phureja 1-3). The ploidy of anther-derived plants was first determined by flow cytometry. A total of 44 decamer primers was screened for RAPD polymorphism. The loci that segregated were selected and scored. The monoploids had less than half as many loci carrying RAPD markers compared with the anther donor. Among 14 anther-derived diploids, 5 were identified as homozygous by marker frequency similar to monoploids and 9 as heterozygous. Five of seven SSRs obtained from published potato sequences were polymorphic in CP2. CP2 was found to be heterozygous with two alleles at four SSR loci (TC/TA, AAG, AGA, CTT) and three alleles at a ACTC locus. Primer pairs flanking each of the five polymorphic SSRs revealed that monoploids had only the allele contributed by S. chacoense 80-1. Homozygous diploids had only one band per SSR locus, whereas heterozygous diploids displayed more than one allele for at least one SSR locus. Results of the SSR analysis supported the findings based on RAPD markers; the same five diploid clones were characterized as homozygous by both SSR and RAPD markers.Key words: androgenesis, anther culture, microsatellites, RAPDs, Solanum phureja, Solanum chacoense, SSRs, short tandem repeats.

Comparison of Genetic Diversity in Core and Whole Germplasm Collections for Phaseolus vulgaris L.

The number of Phaseolus vulgaris germplasm accessions numbers more than 30,000. While the large numbers of accessions increase the probability of preserving genetic variability they simultaneously limit the efficient and routine utilization of this resource. From the approximately 4000 P. vulgaris accessions in the C.I.A.T. whole collection that were collected in Mexico, a core collection of 400 accessions was developed based on variation for agronomic performance, ecological adaptation, and seed characteristics. Random samples of 90 accessions each were drawn from the core and whole collections and evaluated for 224 polymorphic RAPD bands. Based on analysis of the RAPD data there were no significant differences in genetic diversity between the two samples. The correlation of marker frequency for the two samples was 0.984 confirming that the two samples represent the same population.

Directional RAPD Marker Frequency Changes in Two Divergently Selected Beet Populations

In the past 20 years, betalain pigments found in red beet (Beta vulgaris L.) have been adopted for use as natural red food coloring. In an effort to develop red beet populations with elevated levels of betalain pigment, recurrent half-sib family selection for high pigment and both high and low solids was practiced for seven cycles, resulting in the development of a high pigment–high solids (HPHS) and a high pigment–low solids (HPLS) population. Thirty-one randomly selected decamer primers were chosen to assess RAPD marker frequencies on genomic DNA samples isolated from 47 randomly chosen individual plants in each of cycles 1, 3, and 6 in both HPHS and HPLS. A total of 161 RAPD markers were evaluated. Chi-square and regression analyses were performed to determine presence/absence of linear trends in marker frequencies during the selection scheme. Comparisons were made for individual cycles between HPHS and HPLS and among cycles within HPHS and HPLS. Significant linear trends were detected in both cases for key RAPD primers. Chi-square tests revealed a subset of the markers which exhibited significant frequency changes across cycles were associated with selection as opposed to genetic drift. These data demonstrate changes in RAPD marker frequencies with recurrent selection and suggest linkage of RAPD markers to genes controlling pigment and solids in red beet.