Determination of DNA methylation associated with Acer rubrum (red maple) adaptation to metals: analysis of global DNA modifications and methylation‐sensitive amplified polymorphism

Red maple (Acer rubum), a common deciduous tree species in Northern Ontario, has shown resistance to soil metal contamination. Previous reports have indicated that this plant does not accumulate metals in its tissue. However, low level of nickel and copper corresponding to the bioavailable levels in contaminated soils in Northern Ontario causes severe physiological damages. No differentiation between metal‐contaminated and uncontaminated populations has been reported based on genetic analyses. The main objective of this study was to assess whether DNA methylation is involved in A. rubrum adaptation to soil metal contamination. Global cytosine and methylation‐sensitive amplified polymorphism (MSAP) analyses were carried out in A. rubrum populations from metal‐contaminated and uncontaminated sites. The global modified cytosine ratios in genomic DNA revealed a significant decrease in cytosine methylation in genotypes from a metal‐contaminated site compared to uncontaminated populations. Other genotypes from a different metal‐contaminated site within the same region appear to be recalcitrant to metal‐induced DNA alterations even ≥30 years of tree life exposure to nickel and copper . MSAP analysis showed a high level of polymorphisms in both uncontaminated (77%) and metal‐contaminated (72%) populations. Overall, 205 CCGG loci were identified in which 127 were methylated in either outer or inner cytosine. No differentiation among populations was established based on several genetic parameters tested. The variations for nonmethylated and methylated loci were compared by analysis of molecular variance (AMOVA). For methylated loci, molecular variance among and within populations was 1.5% and 13.2%, respectively. These values were low (0.6% for among populations and 5.8% for within populations) for unmethylated loci. Metal contamination is seen to affect methylation of cytosine residues in CCGG motifs in the A. rubrum populations that were analyzed.     

Inoculation of susceptible and resistant potato plants with the late blight pathogen Phytophthora infestans: effects on an aphid and its parasitoid

Plants are exposed to microbial pathogens as well as herbivorous insects and their natural enemies. Here, we examined the effects of inoculation of potato plants, Solanum tuberosum L. (Solanaceae), with the late blight pathogen Phytophthora infestans (Mont.) de Bary (Peronosporales: Pythiaceae) on an aphid species commonly infesting potato crops and one of the aphid's major parasitoids. We observed the peach‐potato aphid, Myzus persicae Sulzer (Hemiptera: Aphididae), and its natural enemy, the biocontrol agent Aphidius colemani Viereck (Hymenoptera: Braconidae), on potato either inoculated with water or P. infestans. Population growth of the aphid, parasitism rate of its natural enemy, and other insect life‐history traits were compared on several potato genotypes, the susceptible cultivar Désirée and genetically modified (GM) isogenic lines carrying genes conferring resistance to P. infestans. Effects of P. infestans inoculation on the intrinsic rate of aphid population increase and the performance of the parasitoid were only found on the susceptible cultivar. Insect traits were similar when comparing inoculated with non‐inoculated resistant GM genotypes. We also tested how GM‐plant characteristics such as location of gene insertion and number of R genes could influence non‐target insects by comparing insect performance among GM events. Different transformation events leading to different positions of R‐gene insertion in the genome influenced aphids either with or without P. infestans infection, whereas effects of position of R‐gene insertion on the parasitoid A. colemani were evident only in the presence of inoculation with P. infestans. We conclude that it is important to study different transformation events before continuing with further stages of risk assessment of this GM crop. This provides important information on the effects of plant resistance to a phytopathogen on non‐target insects at various trophic levels.     

Microarray analysis of the interaction between the aphid <Emphasis Type="Italic">Rhopalosiphum padi</Emphasis> and host plants reveals both differences and similarities between susceptible and partially resistant barley lines

The bird cherry-oat aphid (Rhopalosiphum padi L.) is an important pest on cereals causing plant growth reduction without specific leaf symptoms. Breeding of barley (Hordeum vulgare L.) for R. padi resistance shows that there are several resistance genes, reducing aphid growth. To identify candidate sequences for resistance-related genes, we performed microarray analysis of gene expression after aphid infestation in two susceptible and two partially resistant barley genotypes. One of the four lines is a descendant of two of the other genotypes. There were large differences in gene induction between the four lines, indicating substantial variation in response even between closely related genotypes. Genes induced in aphid-infested tissue were mainly related to defence, primary metabolism and signalling. Only 24 genes were induced in all lines, none of them related to oxidative stress or secondary metabolism. Few genes were down-regulated, with none being common to all four lines. There were differences in aphid-induced gene regulation between resistant and susceptible lines. Results from control plants without aphids also revealed differences in constitutive gene expression between the two types of lines. Candidate sequences for induced and constitutive resistance factors have been identified, among them a proteinase inhibitor, a serine/threonine kinase and several thionins. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Resistance evaluation of wheat germplasm containing Dn4 or Dny against Russian wheat aphid biotype RWASA3

Host plant resistance can effectively manage Russian wheat aphid (Diuraphis noxia) Kurdjumov (Homoptera: Aphididae) in areas where it is an economically important pest of wheat. However, biotypes of D. noxia virulent on wheat containing resistance gene Dn4 have been reported in both the United States and South Africa. Thirty wheat genotypes, including susceptible Yuma, resistant CItr2401, as well as 25 genotypes containing Dn4 and three genotypes containing Dny were planted under greenhouse conditions in Bethlehem, South Africa, and screened with D. noxia biotype RWASA3. RWASA3 caused susceptible damage symptoms in MTRWA92‐145, Ankor, Halt, Bond CL, 18FAWWON‐SA 262, Prowers99, 18FAWWON‐SA 264, Hatcher, Yumar, Corwa and Thunder CL all reported to contain the Dn4 resistance gene. Genotypes PI586956, Stanton and 18FAWWON‐SA 257, containing the Dny‐resistance gene were susceptible to RWASA3. Similarly, coinciding development of virulence to resistance genes Dn4 and Dny was reported in the United States. However, in this study, 13 Dn4‐containing genotypes showed moderate resistance when screened with RWASA3 alluding to a more complex biotype‐gene‐interaction. These findings could indicate that Dn4 and Dny may be related and possibly share a similar or common resistance factor. Further studies will be aimed at explaining these results investigating the possibility of an allelic cluster or series for Dn4, possibly including Dny.     

Tolerance and overcompensation to infection by Phytophthora infestans in the wild perennial climber Solanum dulcamara

Studies of infection by Phytophthora infestans—the causal agent of potato late blight—in wild species can provide novel insights into plant defense responses, and indicate how wild plants might be influenced by recurrent epidemics in agricultural fields. In the present study, our aim was to investigate if different clones of Solanum dulcamara (a relative of potato) collected in the wild differ in resistance and tolerance to infection by a common European isolate of P. infestans. We performed infection experiments with six S. dulcamara genotypes (clones) both in the laboratory and in the field and measured the degree of infection and plant performance traits. In the laboratory, the six evaluated genotypes varied from resistant to susceptible, as measured by degree of infection 20 days post infection. Two of the four genotypes susceptible to infection showed a quadratic (concave downward) relationship between the degree of infection and shoot length, with maximum shoot length at intermediate values of infection. This result suggests overcompensation, that is, an increase in growth in infected individuals. The number of leaves decreased with increasing degree of infection, but at different rates in the four susceptible genotypes, indicating genetic variation for tolerance. In the field, the inoculated genotypes did not show any disease symptoms, but plant biomass at the end of the growing season was higher for inoculated plants than for controls, in‐line with the overcompensation detected in the laboratory. We conclude that in S. dulcamara there are indications of genetic variation for both resistance and tolerance to P. infestans infection. Moreover, some genotypes displayed overcompensation. Learning about plant tolerance and overcompensation to infection by pathogens can help broaden our understanding of plant defense in natural populations and help develop more sustainable plant protection strategies for economically important crop diseases.     

Application of DNA markers linked to the potato <Emphasis Type="Italic">H1</Emphasis> gene conferring resistance to pathotype Ro1 of <Emphasis Type="Italic">Globodera rostochiensis</Emphasis>

Ninety-one potato genotypes (cultivars and breeding lines) selected as resistant or susceptible to pathotype Ro1 of Globodera rostochiensis were screened for the presence of two PCR markers, 0.14 and 0.76 kb in length. Both PCR markers were linked with the H1 gene, located at the distal end of the long arm of chromosome V, and were present in 88 to 100% of the resistant cultivars and breeding lines. The 0.76 kb PCR marker was detected in all resistant genotypes and in approximately 86% of susceptible breeding lines as well as in all susceptible cultivars. The 0.14 kb marker was detected in 88% of resistant breeding lines and in 94% of resistant cultivars. Most of the susceptible genotypes tested (91% of cultivars, but only 50% of breeding lines) did not show the presence of the 0.14 kb marker. We conclude that the 0.14 kb H1 marker is likely to be useful for the proper selection of potato genotypes resistant to the Ro1 pathotype of G. rostochiensis.     

Role of Pseudomonas fluorescens and INA against Black Rot of Cabbage

Cabbage (Brassica oleracea var. capitata) is an important vegetable crop among crucifers. It is affected by a bacterial disease known as black rot. Black rot is caused by Xanthomonas campestris pv. campestris a disease of worldwide importance. The present study highlights the effect of biotic inducer—Pseudomonas fluorescens—and an abiotic inducer—2,6‐dichloro‐isonicotinic acid—in combating black rot, followed by their effect on the seed treatment and disease incidence, role of antioxidant enzymes followed by validation of the defence‐related genes by quantitative real‐time PCR. The resistant (Pusa mukta) and the highly susceptible (NBH boss) cabbage cultivars were analysed for defence‐related enzymes such as peroxidase and superoxide dismutase. An increase in total peroxidase and superoxide dismutase activity was observed upon inoculation with X. campestris pv. campestris. The activity was greater in resistant cultivar when compared to susceptible ones. Both enzyme activity assays and qPCR analyses for the expression of the defence genes in susceptible and resistant cultivars demonstrated that the peroxidase gene was up‐regulated in resistant cultivar compared to susceptible cultivar. The present study proved that P. fluorescens‐induced resistance against X. campestris pv. campestris in cabbage seedlings is more efficient as compared to the use of INA—abiotic inducer.     

Analysis of genomic copy number variation in equine recurrent airway obstruction (heaves)

We explored the involvement of genomic copy number variants (CNVs) in susceptibility to recurrent airway obstruction (RAO), or heaves—an asthmalike inflammatory disease in horses. Analysis of 16 RAO‐susceptible (cases) and six RAO‐resistant (control) horses on a custom‐made whole‐genome 400K equine tiling array identified 245 CNV regions (CNVRs), 197 previously known and 48 new, distributed on all horse autosomes and the X chromosome. Among the new CNVRs, 30 were exclusively found in RAO cases and were further analyzed by quantitative PCR, including additional cases and controls. Suggestive association (P = 0.03; corrected P = 0.06) was found between RAO and a loss on chromosome 5 involving NME7, a gene necessary for ciliary functions in lungs and involved in primary ciliary dyskinesia in humans. The CNVR could be a potential marker for RAO susceptibility but needs further study in additional RAO cohorts. Other CNVRs were not associated with RAO, although several involved genes of interest, such as SPI2/SERPINA1 from the serpin gene family, which are associated with chronic obstructive pulmonary disease and asthma in humans. The SPI2/SERPINA1 CNVR showed striking variation among horses, but it was not significantly different between RAO cases and controls. The findings provide baseline information on the relationship between CNVs and RAO susceptibility. Discovery of new CNVs and the use of a larger population of RAO‐affected and control horses are needed to shed more light on their significance in modulating this complex and heterogeneous disease.     

A genome‐wide SNP scan reveals two loci associated with the chicken resistance to Marek's disease

Marek's disease (MD) is a neoplastic disease in chickens, caused by the Marek's disease virus (MDV). To investigate host genetic resistance to MD, we conducted a genome‐wide association study (GWAS) on 67 MDV‐infected chickens based on a case and control design, including 57 susceptible chickens in the case group and 10 resistant chickens as controls. After searching 38 655 valid genomic markers, two SNPs were found to be associated with host resistance to MD. One SNP, rs14527240, reaching chromosome‐wide significance level (P < 0.01) was located in the SPARC‐related modular calcium‐binding 1 (SMOC1) gene on GGA5. The other one, GGaluGA156129, reaching genome‐wide significance (P < 0.05), was located in the protein tyrosine phosphatase, non‐receptor type 3 (PTPN3) gene on GGA2. In addition, expression patterns of these two genes in spleens were detected by qPCR. The expression of SMOC1 was significantly up‐regulated (P < 0.05), whereas the expression of PTNP3 did not show significance when the case group was compared with the control group. Up‐regulation of SMOC1 in susceptible spleens suggests its important roles in MD tumorigenesis. This is the first study to investigate MD‐resistant loci, and it demonstrates the power of GWASs for mapping genes associated with MD resistance.     

Isolation of a naturally-occurring nickel resistance plasmid from a rare hypersaline estuary (Laguna Madre,Texas, USA) for potential use as a bio-indicator of metal contamination

Metal-resistant bacteria were isolated from sediments of the Laguna Madre, a rare hypersaline estuary impacted by many anthropogenic compounds, including various metals and metalloids. Bacteria were initially isolated on nutrient agar supplemented with NaCl; random isolates (n = 100) were tested for metal resistance toward zinc, nickel, chromium, and cadmium using a pour plate disc assay. Metal-resistant cultures were assayed for plasmids that contained naturally-occurring heavy metal resistance genes. Putative metal-resistance plasmids were tested for metal-resistance efficacy by transforming a metal-sensitive strain of Escherichia coli. Polymerase Chain Reaction (PCR) primers were designed to detect cnrA, part of a nickel–cobalt resistance gene cluster, and restriction endonuclease digests were performed to detect restriction sites within the plasmid. Results showed that many bacterial isolates tested were resistant toward most of the metals used in this study. Among tested bacteria cultures, 34 were resistant to zinc, 64 were resistant to chromium, and 51 resistant to cadmium. Only 8 cultures were resistant to nickel; however, most bacteria were found to be resistant to more than one metal. Several plasmids were found from the bacteria isolates. One plasmid, designated pDZ5, was isolated from a bacterium identified as Bacillus pumilus by 16S rRNA sequencing. Plasmid pDZ5 conferred nickel resistance to the metal-sensitive E. coli strain and was found to contain cnrA as confirmed by PCR amplification. Plasmid pDZ5 was successfully cut with restriction enzymes for potential ligation with reporter genes. The presence, abundance and expression of pDZ5 may prove to be a useful bio-indicator of metal contamination, specifically nickel pollution, in the Laguna Madre due to the fewer number of bacteria that were nickel-resistant compared to other metals.