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.     

Analyses of quantitative trait loci associated with resistance to shape Sclerotinia sclerotiorum in sunflowers (shape Helianthus annuus L.) using molecular markers

Restriction fragment length polymorphism and isoenzyme markers were used to investigate quantitative trait loci involved in sunflower resistance to mycelial extension of Sclerotinia sclerotiorum on leaves and capitula. Seed weight, oil content and flowering data were also evaluated. Four quantitative trait loci were demonstrated for leaf resistance and two for capitulum resistance. One of these zones appears involved in resistance to both types of S. sclerotiorum attack while the others appear specific for resistance of one part of the plant. Two quantitative trait loci were detected for seed weight, three for oil content and three for flowering date. Individual quantitative trait loci explained 9% to 48% of the phenotypic variability, confirming the polygenic basis of the quantitative traits studied. Overall, the quantitative trait loci explain 60% of the genetic variation for leaf resistance and 38% for capitulum resistance to S. sclerotiorum. One linkage group is particularly interesting since it includes quantitative trait loci for all the five quantitative traits measured. Hypotheses for linkage versus pleiotropy and consequences of all the results in resistance breeding are discussed.     

Influence of genotype and gelling agents on in vitro regeneration by organogenesis in sunflower

Fourteen recombinant inbred lines of sunflower (Helianthus annuus L.) and their parents (PAC-2 and RHA-266) were tested for their organogenesis ability. Seeds were surface sterilized and germinated on hormone free half strength MS basal medium containing 10 g l^-1 sucrose solidified with five different gelling agents: Phytagar (Gibco laboratoires) 3 g l^-1, Phytagel (Sigma) 3 g l^-1, Agarose (Sigma) 5 g l^-1, Arcagel (Sigma) 4 g l^-1 and Agar-Agar (Fisher France) 7 g l^-1. Cotyledons from 2-day-old seedlings were split in half and the four explants of each seed were cultived in 55 mm diameter petri dishes containing 10 ml of MS medium supplemented with 50 μM KNO₃, 1 μM myo-inositol, 5 μM casein hydrolysate, 4.4 μM of BA and 5.4 μM of NAA solidified with the same gelling agents. The experimental design was a randomized complete block with 3 replications. A replicate for each genotype consisted of ten petri dishes containing four explants. The statistical analysis showed significant differences among genotypes and gelling agents. Of the fourteen recombinant inbred lines tested `C93' presented the highest values for all regeneration traits in the five different media and it was better than the best parent. Agarose and Agar-Agar were more better than other gelling agents for shoot induction. This revised version was published online in June 2006 with corrections to the Cover Date.     

Genetic determinism of reproductive fitness traits under drought stress in the model legume <Emphasis Type="Italic">Medicago truncatula</Emphasis>

Fitness traits that determine the reproductive ability of individuals and the persistence of populations are affected by drought stress. Medicago truncatula that commonly encounters drought stress in its natural area, and for which large natural diversity and genetic tools are available, is a suitable species to investigate genetic determinism of fitness traits under stress. In a common garden, three successive cycles of short drought stress were applied after flowering, during the reproductive stage that is the most susceptible to drought for that species. Ten genotypes derived from natural populations and a mapping population were used to investigate the genetic determinism of vegetative and reproductive traits as components of fitness. A large genetic variation was observed and transgressive genotypes (more resistant or more susceptible than the parental genotypes) were found in the mapping population. Fitness traits were reduced by 5–74% in drought condition compared to well-watered condition. The most affected characters were total pod number per plant and total pod weight per plant. A total of 49 QTL, explaining between 6 and 38% of phenotypic variation for vegetative and reproductive fitness traits, were detected on all chromosomes except chromosome 6. A major QTL for flowering date (R ² of 19 and 38%) that co-located with QTL for reproductive fitness traits were found on chromosome 7. In this study, no major QTL specific to drought-stressed or well-watered conditions were detected. We, thus, showed that QTL explaining fitness traits were numerous with small effects, in accordance with the genetic determinism of a complex trait.     

Dissection of Bacterial Wilt on Medicago truncatula Revealed Two Type III Secretion System Effectors Acting on Root Infection Process and Disease Development

Ralstonia solanacearum is the causal agent of the devastating bacterial wilt disease, which colonizes susceptible Medicago truncatula via the intact root tip. Infection involves four steps: appearance of root tip symptoms, root tip cortical cell invasion, vessel colonization, and foliar wilting. We examined this pathosystem by in vitro inoculation of intact roots of susceptible or resistant M. truncatula with the pathogenic strain GMI1000. The infection process was type III secretion system dependent and required two type III effectors, Gala7 and AvrA, which were shown to be involved at different stages of infection. Both effectors were involved in development of root tip symptoms, and Gala7 was the main determinant for bacterial invasion of cortical cells. Vessel invasion depended on the host genetic background and was never observed in the resistant line. The invasion of the root tip vasculature in the susceptible line caused foliar wilting. The avrA mutant showed reduced aggressiveness in all steps of the infection process, suggesting a global role in R. solanacearum pathogenicity. The roles of these two effectors in subsequent stages were studied using an assay that bypassed the penetration step; with this assay, the avrA mutant showed no effect compared with the GMI1000 strain, indicating that AvrA is important in early stages of infection. However, later disease symptoms were reduced in the gala7 mutant, indicating a key role in later stages of infection.     

Genotypic variation and chromosomal location of QTLs for somatic embryogenesis revealed by epidermal layers culture of recombinant inbred lines in the sunflower (Helianthus annuus L.)

The present study was conducted to identify the genetic factors controlling somatic embryogenesis in the sunflower. Two traits, the number of embryogenic explants per 40 explants plated (EE/40 E) and the number of embryos per 40 explants (E/40 E), were scored in 74 recombinant inbred lines (RILs) from a cross between ’PAC-2’ and ’RHA-266’. The experiment was designed as a randomized complete block with 76 genotypes (74 recombinant inbred lines and two parents) and three replications. Each replication consisted of three Erlenmeyer flasks with 40 epidermal layers (explants). Analyses of variance indicated the existence of highly significant differences among parental genotypes and their RILs. Heritabilities for the somatic embryogenesis traits studied, EE/40 E and E/40 E, were high (0.64 and 0.77 respectively) and the genetic gain, in percentage of the best parent for 10% of selected RILs, was significant. Four QTLs for EE/40 E (tee) and seven for E/40 E (ete) were detected using composite interval mapping and AFLP mapping. The QTLs for EE/40 E explained 48% of the phenotypic variation while the QTLs for E/40 E explained about 89% of the variation. Received:14 December 1999 / Accepted:18 May 2000     

Somatic embryogenesis by liquid culture of epidermal layers in sunflower: from genetic control to cell development

Embryos were obtained using liquid medium culture of sunflower hypocotyl epidermis layers according to the Pélissier etal. (1990) method. In the present work we identified genetic factors controlling somatic embryogenesis and we evidenced the role of ionic channels in embryogenic tissues. Two traits, the number of embryogenic explants (EE) and the number of embryos (EM) were scored in 74 recombinant inbred lines (RILs) from a cross between lines PAC-2 and RHA-266. Analysis of variance indicated the existence of highly significant differences among the parental genotypes and their RILs. Heritability for the somatic embryogenesis traits studied were high (0.64 for EE and 0.77 for EM). Four quantitative trait loci (QTLs) for EE and seven for EM were detected using composite interval mapping. The QTLs for EE explained 48% of the phenotypic variation while the QTLs for EM explained about 89% of the variation, thus revealing several genomic regions related to somatic embryogenesis control in sunflower. In order to study the distribution of ion channels in somatic embryos as compared to zygotic ones, we used a fluorescent-labelled phenylalkylamine, DM-Bodipy PAA, as a probe. Fluorescence labelling was determined by confocal microscopy. The probe intensively labelled the protoderm and epidermis cells in both zygotic and somatic embryos. Callus exhibited labelling on sites where somatic embryos developed. Considering that the location of phenylalkylamine (PAA) binding sites is related to the distribution of ion channels, the high intensity in the protoderm and epidermis of embryos, point to similar properties and functions and their key role in embryo development.