Construction of a genetic linkage map and identification of molecular markers in peach rootstocks for response to peach tree short life syndrome

Peach tree short life (PTSL) is a devastating disease syndrome of peach [Prunus persica (L.) Batsch] caused by multiple factors; the molecular biology of its tolerance/susceptibility is unknown. The difficulty of studying PTSL is that tree survival or death is not obvious until 3 to 5 years after planting when the symptoms of PTSL first appear. Tolerance to PTSL was unknown in Prunus until the rootstock Guardian® ‘BY520-9’ was introduced into commercial orchards in 1994. To study the genetics of the response to PTSL, a controlled F₂ cross was made between Guardian® ‘BY520-9’ selection 3-17-7 (PTSL-tolerant) and Nemaguard (PTSL-susceptible). An F₁ hybrid was then selfed to generate an F₂ population expected to segregate for PTSL response. One hundred fifty-one AFLPs and 21 SSRs, including anchor loci from the Prunus reference genetic map, were used to construct a molecular genetic map based on 100 F₂ seedlings. This map covers a genetic distance of 737 cM with an average marker spacing of 4.7 cM and will be used as a framework to construct a highly saturated molecular genetic map. Of the 140 mapped AFLP markers, 38 were associated with PTSL response, as identified previously by bulked segregant analysis. The distribution of the markers associated with PTSL response on the newly constructed genetic map was compared with the recently published Prunus resistance map. This comparison revealed that some resistance gene analogs and several PTSL-associated AFLP markers were located in the same regions in several Prunus linkage groups: G1, G2, G4, G5, and G6. This peach rootstock map can also be viewed and compared with other Prunus maps in comparative map viewer CMap in the Genome Database for Rosaceae (GDR) at http://www.rosaceae.org     

The Balkan chamois,an archipelago or a peninsula? Insights from nuclear and mitochondrial DNA

The Balkan chamois (Rupicapra rupicapra balcanica) is widespread on the Balkan Peninsula, along mountain massifs from Croatia in the north to Greece in the south and Bulgaria in the east. Knowledge on the genetic structure of Balkan chamois populations is limited and restricted to local studies. Therefore, the main objective of this study was to use nuclear (16 microsatellites) and mitochondrial (partial 376 base pairs control region) markers to investigate the genetic structure of this chamois subspecies throughout its distribution range and to obtain information on the degree of connectivity of the different (sub)populations. We extracted DNA from bone, dried skin and muscle tissue and successfully genotyped 92 individuals of Balkan chamois and sequenced the partial control region in 44 individuals. The Bayesian analysis suggested 3 genetic clusters and assigned individuals from Serbia and Bulgaria to two separate clusters, while individuals from the other countries belonged to the same cluster. Thirty new haplotypes were obtained from partial mitochondrial DNA sequences, with private haplotypes in all analyzed populations and only two haplotypes shared among populations, indicating the possibility of past translocations. The subspecies genetic composition presented here provides the necessary starting point to assess the conservation status of the Balkan chamois and allows the development of conservation strategies necessary for its sustainable management and conservation.

     

Bme585 I [5'-CCCGC(4/6)-3'], a new isoschizomer of restriction endonuclease Fau I, isolated from a strain of Bacillus mesentericus

Bme585 I is a new member of the restriction endonuclease type IIS family. It was partially purified from the heterothrophic, mesophilic bacterial strain Bacillus mesentericus 585 by ammonium sulphate precipitation and phosphocellulose column chromatography. Bme585 I is a monomeric protein with a molecular mass of 62 kD. The enzyme is active over a broad pH range from 7.0 to 8.8, has a temperature optimum of 37 degrees C and tolerance of NaCl in reaction buffer from 0 to 400 mM. Bme585 I recognizes the asymmetric sequence 5'-CCCGC(4/6)-3' and is therefore an isoschizomer of restriction endonuclease Fau I.     

In vivo impact of CpG1826 oligodeoxynucleotide on CD8 T cell primary responses and survival

CpG oligodeoxynucleotide (ODN) promotes maturation of APCs in vivo and induces strong type 1 T cell responses in mice. In this study, we have investigated the ability of CpG1826 to modulate peptide-specific CD8 T cell responses in a context where CD4 T cells are likely to play a minor role. The effects of CpG1826 were evaluated in a system where a population of NP68-specific F5 TCR transgenic CD8 T cells is diluted into a polyclonal host following adoptive transfer into C57BL/10 syngeneic recipients. Using this approach, we found that CpG1826 enhanced the ability of F5 CD8 T cells to undergo multiple divisions in vivo, to express IFN-gamma ex vivo, and to up-regulate memory-associated cell surface markers such as CD122 (IL-2Rbeta) and Ly-6C. Moreover, CpG1826 greatly increased in vivo cytotoxic activity. Using tetramer detection, we found that CpG1826 promoted long-term survival of Ag-specific CD8 T cells after immunization while no NP68-specific cells were detected when the cognate peptide was injected alone. These results indicate that CpG1826 acts as an adjuvant which increases CD8 T cell effector responses and promotes long-term survival of NP68 peptide-specific cells in vivo. They also suggest that this adjuvant can modulate CD8 T cell responses in a system which is likely to be independent of CD4 T cell help.     

FIS modulates the kinetics of successive interactions of RNA polymerase with the core and upstream regions of the tyrT promoter

We have applied laser UV photo-footprinting to characterise kinetically complexes involving the activator protein FIS, RNA polymerase and the tyrT promoter of Escherichia coli. FIS photo-footprints strongly to three binding sites upstream of the core promoter. The polymerase photo-footprints in the near-consensus -35 hexamer on the non-template strand of DNA in a fashion similar to that of stable complexes involving the lacUV5 promoter. The kinetics of the interactions of polymerase alone with the tyrT promoter differ from those observed previously at the lacUV5 promoter. In the absence of FIS, we observe an upstream polymerase-induced signal at -122 within FIS site III that occurs subsequent to changes in the core promoter region and is strongly dependent on negative supercoiling. These observations support the proposal that the upstream region of the promoter is wrapped around the polymerase. We propose that the wrapped DNA allows the polymerase to overcome, at least in part, the barrier to DNA untwisting imparted by the G+C-rich discriminator. We further suggest that FIS plays a similar role and may facilitate polymerase escape.     

Genetic mapping of the evergrowing gene in peach [Prunus persica (L.) Batsch

In temperate locations, terminal apices on evergrowing (also called evergreen) peach trees keep growing in winter until killed by low temperatures, while the lateral buds go into dormancy. A recessive allele of a single gene (evergrowing or evg) controls this trait in peach. The amplified fragment length polymorphism (AFLP) technique and bulked segregant analysis were applied to construct a local genetic linkage map for the evg gene from the cross Empress op op dwarf x Evergrowing (P.I. 442380). This map, comprising nine AFLP markers and the evg locus, covers a total genetic distance of 79.3 cM. Four dominant AFLP markers (EAT/MCAC, ETT/MCCA2, EAT/MCTA, and ETT/MACC) were linked to the evg locus at distances of 1, 5.3, 6.7, and 11.7 cM, respectively. EAT/MCAC and EAT/MCTA were converted into polymorphic sequence-tagged sites. Microsatellite markers in the evg region were developed from peach bacterial artificial chromosome (BAC) clones that hybridized to the AFLP marker fragments. Using three microsatellite anchor markers (pchgms12, pchgms17, and pchgms19), the local genetic linkage map was integrated into one minor linkage group of a previously constructed peach rootstock genetic linkage map. Three AFLP markers from the rootstock genetic linkage map were found linked to the evg locus.     

A bacterial artificial chromosome (BAC) library for sunflower, and identification of clones containing genes for putative transmembrane receptors

Sunflower (Helianthus annuus L.) is an economically important oil seed crop with an estimated genome size of 3000 Mb. We have constructed a bacterial artificial chromosome (BAC) library for sunflower, which represents an estimated 4- to 5-fold coverage of the genome. Nuclei isolated from young leaves were used as a source of high-molecular-weight DNA and a partial restriction endonuclease digestion protocol was used to cleave the DNA. A random sample of 60 clones indicated an average insert size of 80 kb, implying a 95% probability of recovering any specific sequence of interest. The library was screened with chloroplast DNA probes. Only 0.1% of the clones were identified to be of chloroplast origin, indicating that contamination with organellar DNAs is very low. The utility of the library was evaluated by screening for the presence of genes for putative transmembrane receptors sharing epidermal growth factor (EGF) and integrin-like domains. First, a homologous sunflower EST (HaELP1) was obtained by degenerate RT-PCR cloning, using Arabidopsis thaliana genes (AtELP) as a source of consensus sequences. Three different BACs yielded positive hybridization signals when HaELP1 was used as a probe. BAC subcloning and sequencing demonstrated the presence of two different loci putatively homologous to genes for transmembrane proteins with EGF- and integrin-like domains from sunflower. This work demonstrates the suitability of the library for homology map-based cloning of sunflower genes and physical mapping of the sunflower genome.