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

Abstract

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.

PLASMOLYSIS, HECHTIAN STRAND FORMATION, AND LOCALIZED MEMBRANE‐WALL ADHESIONS IN THE DESMID, CLOSTERIUM ACEROSUM (CHLOROPHYTA)1

“…Integrins are proteins that span the PM and link the cytoskeletal system with components of the ECM of animal cells or CW of plant cells. Evidence for integrins in plants has come from studies using the cross‐reactivity of plant proteins with antibodies raised against subunits of integrin from animals as well as from production of molecular constructs of plant genes that have similarity to animal integrins (Pont‐Lezica et al 1993, Kaminskyj and Heath 1995, Reuzeau and Pont‐Lezica 1995, Gens et al 1996, Faik et al 1998, Nagpal and Quatrano 1999, Sun et al 2000, Gentzbittiel et al 2002). In animals and possibly plants, the major ECM components that interact with integrins are substrate adhesive molecules such as fibronectin and vitronectin (Zhu et al 1993, Reuzeau and Pont‐Lezica 1995).…”

HACRE1, a recently inserted copia-like retrotransposon of sunflower (Helianthus annuus L.)

“…Ten micrograms of total DNA was extracted by 2 consecutive maxipreps from one clone (406J06) of a bacterial artificial chromosome (BAC) library (Gentzbittel et al 2002) and purified of E. coli genomic DNA contamination by Plasmid-Safe ATP-Dependent DNase (Epicentre Biotechnologies). DNA was sheared according to the nebulization protocol of Wilson and Mardis (1997).…”

Molecular advances on agricultural crop improvement to meet current cultivating demands

“…Assessing genetic diversity within a genetic pool of novel breeding germplasm could make crop improvement more effi cient by the directed accumulation of desired alleles (Darvishzadeh et al, 2010). Several bacterial artifi cial chromosome (BAC) libraries have been constructed for sunfl ower (Feng et al, 2006;Gentzbittel et al, 2002;Özdemir et al, 2004). The libraries are equivalent to approximately 8 haploid genomes of sunfl ower and provide a greater than 99% probability of obtaining a clone of interest and they have been employed for isolating and physical mapping of loci such as the FAD2-1 locus (Schuppert et al, 2006) or the fertility restorer Rf1 locus (Hamrit et al, 2008).…”