Genomic,evolutionary and expression profile analysis of <Emphasis Type="Italic">Hsp70</Emphasis> superfamily in A and D genome of cotton (<Emphasis Type="Italic">Gossypium</Emphasis> spp.) under the challenge of <Emphasis Type="Italic">Verticillium dahliae</Emphasis>

In this study, we comparatively analyzed the 115 Hsp70 genes identified in Gossypium raimondii, Gossypium hirsutum and Gossypium arboreum genomes. Those Hsp70 genes unequally distributed among chromosomes in A and D genome of cotton (Gossypium spp.), and were classified into 29 groups according to the homology of them. Based on the localization information of the orthologs in Arabidopsis, the Hsp70 proteins were predicted to locate in cytosol, endoplasmic reticulum, mitochondrion or chloroplast. Homologous analysis indicated the evolutionary conservation of Hsp70 in cotton. In addition, those Hsp70 genes were differently expressed in Suyuan-045, Hai-7124 and TM-1, which were highly resistant, resistant, and sensitive to Verticillium dahliae respectively. The expressions of 26 Hsp70 genes were induced by Verticillium dahliae except for Hsp70-07/16/25/26, and the result suggested the potential involvement of them in responding to Verticillium wilt. Hsp70-08/30/31 was highly expressed in both Suyuan-045 and Hai-7124, and it was hypothesized that they might be involved in the resistance to the invasion of Verticillium dahliae. 144h after inoculation with Verticillium dahliae, the expression of Hsp70-13/14/15 was only up-regulated in Suyuan-045, and it was assumed that they might be involved in resistance to the extension of Verticillium dahliae. Further study on those Hsp70 genes would be valuable to reveal the role of them in Verticillium wilt resistance.     

MiR395 Overexpression Increases Eggplant Sensibility to <Emphasis Type="Italic">Verticillium dahliae</Emphasis> Infection

Verticillium wilt (V. wilt), a notorious wilt disease caused by Verticillium dahliae, often leads to the reduction of eggplant (Solanum melongena L.) production. MiRNAs, as a class of small RNAs, can regulate gene expression and then affect growth and development in plants. MiR395 has been proven to respond to sulfate-deficient stress in Arabidopsis thaliana and sulfate is well known to have a close relationship with plant disease resistance. To explore the function of eggplant miR395, we examined its expression in V. dahliae-infected eggplant by qRT-PCR and found miR395 exhibited a gradual reduction trend with time after infection. We then expressed pre-miR395 from Arabidopsis thaliana in Suqi eggplant and resistance analysis showed that miR395 overexpressed plants were hypersensitive to V. dahliae infection. We further measured the content of GSH and activities of POD and SOD and the results indicated that the index of GSH/POD/SOD in the overexpressed plants was lower than that of the wild-type control under V. dahliae infection. These results suggest that miR395 plays a negative role in eggplant response to V. dahliae infection.     

Combined use of biocontrol agents and zeolite as a management strategy against <Emphasis Type="Italic">Fusarium</Emphasis> and <Emphasis Type="Italic">Verticillium</Emphasis> wilt

Vascular wilt pathogens, like Fusarium oxysporum and Verticillium dahliae, cause heavy economic loses to a range of crops. The lack of chemical control intensifies the problem. In the present study, the initial in vitro activity of 134 bacterial isolates, originating from various stages of the composting process of cotton residues, against F. oxysporum f. sp. melonis (FOM) and V. dahliae was evaluated. The most efficient strains, named SP10 and C20 M, belong to Bacillus sp. Both strains significantly reduced Fusarium and Vertilicillium wilt in melon and aubergine respectively. Furthermore, zeolite was tested alone or in combination with SP10 against V. dahliae and FOM. It was shown that the combination of zeolite and SP10 in the transplant soil plug was the most disease suppressive treatment. Interestingly the single application of zeolite was also plant-protective. The positive effect of zeolite on plant health could be linked with the recorded up-regulation of plant defense genes.     

Silencing of <Emphasis Type="Italic">GbANS</Emphasis> reduces cotton resistance to <Emphasis Type="Italic">Verticillium dahliae</Emphasis> through decreased ROS scavenging during the pathogen invasion process

Anthocyanins are secondary metabolites that play important roles in plant adaption to adverse environments. The anthocyanin biosynthetic pathway is conserved in high plants. Previous studies revealed the significant role of anthocyanins in natural-colorized cotton. However, little is known about the involvement of anthocyanins in the interaction of cotton and pathogen. In this study, a pathogen-induced gene was isolated from Gossypium barbadense that encodes an anthocyanidin synthase protein (GbANS) with dioxygenase structures. GbANS was preferentially expressed in colored tissue. Silencing of GbANS significantly reduced the production of anthocyanins, as well as the cotton’s resistance to Verticillium dahliae. Biochemical studies revealed that GbANS-silenced cotton accumulated more hydrogen peroxide compared to control plants during the V. dahliae invasion process. This accumulation of hydrogen peroxide corresponded with increased cell death around the invasion sites, which in turn accelerated the V. dahliae infection. Taken together, we found that GbANS contributes to the biosynthesis of anthocyanins in cotton and anthocyanins positively regulate cotton’s resistance to V. dahliae.     

Fine mapping of the root-knot nematode resistance gene <Emphasis Type="Italic">Me1</Emphasis> in pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis> L.) and development of markers tightly linked to <Emphasis Type="Italic">Me1</Emphasis>

Root-knot nematodes (RKNs) can severely damage crops, including peppers, worldwide. The application of resistance genes identified in the Capsicum annuum genome may represent a safe and economically relevant strategy for controlling RKNs. Among the Me genes (Me1, Me3, Me7, and N) that have been mapped to a cluster on chromosome P9, Me1 confers a heat-stable and broad-spectrum resistance that is difficult for virulent RKNs to overcome. In this study, we developed several closely linked kompetitive allele-specific PCR (KASPar) markers, simple sequence repeat (SSR) markers, sequence characterized amplified region (SCAR) markers, and high-resolution melting (HRM) markers for the mapping of RKN-resistance genes. Analyses of 948 individuals (BC₁ and F₂ progenies) revealed that Me1 was located between SCAR marker 16880-1-V2 and HRM marker 16830-H-V2, with 13 and 0 recombination events with Me1, respectively. These markers were localized to a 132-kb interval, which included six genes. The development of several PCR-based markers closely linked to Me1 will be useful for the marker-assisted selection of RKN resistance in pepper cultivars. Among these markers, 16830-H-V2 and 16830-CAPS are present in the CA09g16830 gene, which is predicted to be a putative late blight resistance protein homolog R1A-3 gene. This gene appears to be a suitable Me1 candidate gene.     

Rapid identification of QTLs underlying resistance to <Emphasis Type="Italic">Cucumber mosaic virus</Emphasis> in pepper (<Emphasis Type="Italic">Capsicum frutescens</Emphasis>)

Key message

Next-generation sequencing enabled a fast discovery of QTLs controlling CMV resistant in pepper. The gene CA02g19570 as a possible candidate gene of qCmr2.1 was identified for resistance to CMV in pepper.

Abstract

Cucumber mosaic virus (CMV) is one of the most important viruses infecting pepper, but the genetic basis of CMV resistance in pepper is elusive. In this study, we identified a candidate gene for CMV resistance QTL, qCmr2.1 through SLAF-seq. Segregation analysis in F₂, BC₁ and F_2:3 populations derived from a cross between two inbred lines ‘PBC688’ (CMV-resistant) and ‘G29’ (CMV-susceptible) suggested quantitative inheritance of resistance to CMV in pepper. Genome-wide comparison of SNP profiles between the CMV-resistant and CMV-susceptible bulks constructed from an F₂ population identified two QTLs, designated as qCmr2.1 on chromosome 2 and qCmr11.1 on chromosome 11 for resistance to CMV in PBC688, which were confirmed by InDel marker-based classical QTL mapping in the F₂ population. As a major QTL, joint SLAF-seq and traditional QTL analysis delimited qCmr2.1 to a 330 kb genomic region. Two pepper genes, CA02g19570 and CA02g19600, were identified in this region, which are homologous with the genes LOC104113703, LOC104248995, LOC102603934 and LOC101248357, which were predicted to encode N-like protein associated with TMV-resistant in Solanum crops. Quantitative RT-PCR revealed higher expression levels of CA02g19570 in CMV resistance genotypes. The CA02g19600 did not exhibit obvious regularity in expression patterns. Higher relative expression levels of CA02g19570 in PBC688 and F₁ were compared with those in G29 during days after inoculation. These results provide support for CA02g19570 as a possible candidate gene of qCmr2.1 for resistance to CMV in pepper.

     

Identification and molecular genetic mapping of <Emphasis Type="Italic">Chili veinal mottle virus</Emphasis> (ChiVMV) resistance genes in pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis>)

Chili veinal mottle virus (ChiVMV) threatens the agricultural production of peppers (Capsicum annuum) in Asia and Africa. In this study, we evaluated ChiVMV resistance in the four pepper varieties CV3, CV4, CV8, and CV9. Segregation analyses revealed that CV3 and CV8 contain the single dominant resistance gene Cvr1, and CV9 contains the single recessive resistance gene cvr4. SNP markers were developed and used to map the Cvr1 gene in CV3 to the short arm of chromosome 6 where NLR genes are clustered. In CV4 oligogenic resistance loci were detected. A genotyping-by-sequencing (GBS) combined with modified sliding window approach mapped two resistance loci, to chromosomes 6 and 10. The development of SNP markers and the resulting knowledge of genomic positioning will assist in breeding ChiVMV-resistant pepper varieties and in the fine mapping of ChiVMV resistance genes.     

Physical mapping of NBS-coding resistance genes to the <Emphasis Type="Italic">Me</Emphasis>-gene cluster on chromosome P9 reveals markers tightly linked to the <Emphasis Type="Italic">N</Emphasis> gene for root-knot nematode resistance in pepper

Natural root-knot nematode resistance genes are unique resources to control this major pest in pepper (Capsicum annuum). Although four genes (Me1, Me3, Me7 and N) conferring broad-spectrum resistance were mapped to a cluster in a 28-cm interval on chromosome P9, limited markers targeting this region were available. In the present study, the Me-gene cluster was structurally annotated for resistance genes to develop markers targeting the N gene. As a result, the Me-gene cluster (4.07 Mb in size) was found to contain three resistance gene hotspots. In addition, a SSR maker tightly linked to the N gene (0.8 cM away) was developed for marker-assisted selection in pepper.     

Enhanced salinity stress tolerance in transgenic chilli pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis> L.) plants overexpressing the wheat antiporter (<Emphasis Type="Italic">TaNHX2</Emphasis>) gene

Transgenic chilli pepper (Capsicum annuum L.) plants tolerant to salinity stress were produced by introducing the wheat Na⁺/H⁺ antiporter gene (TaNHX2) via Agrobacterium-mediated transformation. Cotyledonary explants were infected with Agrobacterium tumefaciens strain LBA4404 harboring a binary vector pBin438 that contains a wheat antiporter (TaNHX2) gene driven by the double CaMV 35S promoter and NPT II gene as a selectable marker. PCR and semiquantitative RT-PCR analysis confirmed that the TaNHX2 gene had been integrated and expressed in the T₁ generation of transgenic pepper plants as compared to the non-transformed plants. Southern blot analysis further verified the integration and presence of TaNHX2 gene in the genome of chilli pepper plants. Biochemical assays of these transgenic plants revealed enhanced levels of proline, chlorophyll, superoxide dismutase, ascorbate peroxidase, relative water content, and reduced levels of hydrogen peroxide (H₂O₂), malondialdehyde compared to wild-type plants under salt stress conditions. The present investigation clearly showed that overexpression of the TaNHX2 gene enhanced salt stress tolerance in transgenic chilli pepper plants.     

Role of Arabidopsis <Emphasis Type="Italic">ABF1</Emphasis>/<Emphasis Type="Italic">3</Emphasis>/<Emphasis Type="Italic">4</Emphasis> during <Emphasis Type="Italic">det1</Emphasis> germination in salt and osmotic stress conditions

Key message

Arabidopsis det1 mutants exhibit salt and osmotic stress resistant germination. This phenotype requires HY5, ABF1, ABF3, and ABF4.

Abstract

While DE-ETIOLATED 1 (DET1) is well known as a negative regulator of light development, here we describe how det1 mutants also exhibit altered responses to salt and osmotic stress, specifically salt and mannitol resistant germination. LONG HYPOCOTYL 5 (HY5) positively regulates both light and abscisic acid (ABA) signalling. We found that hy5 suppressed the det1 salt and mannitol resistant germination phenotype, thus, det1 stress resistant germination requires HY5. We then queried publically available microarray datasets to identify genes downstream of HY5 that were differentially expressed in det1 mutants. Our analysis revealed that ABA regulated genes, including ABA RESPONSIVE ELEMENT BINDING FACTOR 3 (ABF3), are downregulated in det1 seedlings. We found that ABF3 is induced by salt in wildtype seeds, while homologues ABF4 and ABF1 are repressed, and all three genes are underexpressed in det1 seeds. We then investigated the role of ABF3, ABF4, and ABF1 in det1 phenotypes. Double mutant analysis showed that abf3, abf4, and abf1 all suppress the det1 salt/osmotic stress resistant germination phenotype. In addition, abf1 suppressed det1 rapid water loss and open stomata phenotypes. Thus interactions between ABF genes contribute to det1 salt/osmotic stress response phenotypes.

     

Evidence of genetically diverse virulent mating types of <Emphasis Type="Italic">Phytophthora capsici</Emphasis> from <Emphasis Type="Italic">Capsicum annum</Emphasis> L.

Chili pepper (Capsicum annum L.) is an important economic crop that is severely destroyed by the filamentous oomycete Phytophthora capsici. Little is known about this pathogen in key chili pepper farms in Punjab province, Pakistan. We investigated the genetic diversity of P. capsici strains using standard taxonomic and molecular tools, and characterized their colony growth patterns as well as their disease severity on chili pepper plants under the greenhouse conditions. Phylogenetic analysis based on ribosomal DNA (rDNA), β-tubulin and translation elongation factor 1α loci revealed divergent evolution in the population structure of P. capsici isolates. The mean oospore diameter of mating type A1 isolates was greater than that of mating type A2 isolates. We provide first evidence of an uneven distribution of highly virulent mating type A1 and A2 of P. capsici that are insensitive to mefenoxam, pyrimorph, dimethomorph, and azoxystrobin fungicides, and represent a risk factor that could ease outpacing the current P. capsici management strategies.     

Host preferences and biotic potential of <Emphasis Type="Italic">Trialeurodes vaporariorum</Emphasis> and <Emphasis Type="Italic">Bemisia tabaci</Emphasis> (Hemiptera: Aleyrodidae) in tomato and pepper

Whiteflies Bemisia tabaci (Gennadius) and Trialeurodes vaporariorum (Westwood) (Hemiptera: Aleyrodidae) are important pests in pepper (Capsicum annuum L.) and tomato (Solanum lycopersicum L.) crops in many countries. Contrary to what is observed for all other countries, in Uruguay, B. tabaci is mainly found on pepper and rarely on tomato, while T. vaporariorum is exclusively found on tomato. This study tested the oviposition preferences and biotic potential of these two whiteflies reared on both host plants. The developmental time, survival rates, longevity, fecundity and main population parameters were characterized. Both whitefly species showed different preference patterns regarding their host plants. T. vaporariorum preferred tomato instead of pepper to oviposit. Their developmental time is longer on pepper. B. tabaci preferred pepper, but the difference from tomato was not very strong. Pepper affects the biotic expression of T. vaporariorum negatively, while B. tabaci is able to develop equally on both host plants. These results show that the distribution differences of both whiteflies observed on both host plants could have a biological basis.     

QTL mapping for resistance to Ceratocystis wilt in <Emphasis Type="Italic">Eucalyptus</Emphasis>

Ceratocystis wilt caused by the fungus Ceratocystis fimbriata, is currently one of the major diseases in commercial plantations of Eucalyptus trees in Brazil. Deployment of resistant genotypes has been the main strategy for effective disease management. The present study aimed at identifying genomic regions underlying the genetic control of resistance to Ceratocystis wilt in Eucalyptus by quantitative trait loci (QTL) mapping in an outbred hybrid progeny derived from a cross between (Eucalyptus dunnii × Eucalyptus grandis) × (Eucalyptus urophylla × Eucalyptus globulus). A segregating population of 127 individuals was phenotyped for resistance to Ceratocystis wilt using controlled inoculation under a completely randomized design with five clonal replicates per individual plant. The phenotypic resistance response followed a continuous variation, enabling us to analyze the trait in a quantitative manner. The population was genotyped with 114 microsatellite markers and 110 were mapped with an average interval of 12.3 cM. Using a sib-pair interval-mapping approach five QTLs were identified for disease resistance, located on linkage groups 1, 3, 5, 8, and 10, and their estimated individual heritability ranged from 0.096 to 0.342. The QTL on linkage group 3 overlaps with other fungal disease-resistance QTLs mapped earlier and is consistent with the annotation of several disease-resistance genes on this chromosome in the E. grandis genome. This is the first study to identify and attempt to quantify the effects of QTLs associated with resistance to Ceratocystis wilt in Eucalyptus.