Generation of selectable marker-free transgenic eggplant resistant to Alternaria solani using the R/RS site-specific recombination system

Key message

Marker-free transgenic eggplants, exhibiting enhanced resistance to Alternaria solani , can be generated on plant growth regulators (PGRs)- and antibiotic-free MS medium employing the multi-auto-transformation (MAT) vector, pMAT21 - wasabi defensin , wherein isopentenyl transferase ( ipt ) gene is used as a positive selection marker.

Abstract

Use of the selection marker genes conferring antibiotic or herbicide resistance in transgenic plants has been considered a serious problem for environment and the public. Multi-auto-transformation (MAT) vector system has been one of the tools to excise the selection marker gene and produce marker-free transgenic plants. Ipt gene was used as a selection marker gene. Wasabi defensin gene, isolated from Wasabia japonica (a Japanese horseradish which has been a potential source of antimicrobial proteins), was used as a gene of interest. Wasabi defensin gene was cloned from the binary vector, pEKH-WD, to an ipt-type MAT vector, pMAT21, by gateway cloning technology and transferred to Agrobacterium tumefaciens strain EHA105. Infected cotyledon explants of eggplant were cultured on PGRs- and antibiotic-free MS medium. Extreme shooty phenotype/ipt shoots were produced by the explants infected with the pMAT21-wasabi defensin (WD). The same PGRs- and antibiotic-free MS medium was used in subcultures of the ipt shoots. Subsequently, morphologically normal shoots emerged from the Ipt shoots. Molecular analyses of genomic DNA from transgenic plants confirmed the integration of the WD gene and excision of the selection marker (ipt gene). Expression of the WD gene was confirmed by RT-PCR and Northern blot analyses. In vitro whole plant and detached leaf assay of the marker-free transgenic plants exhibited enhanced resistance against Alternaria solani.     

High throughput Agrobacterium tumefaciens-mediated germline transformation of mechanically isolated meristem explants of cotton (Gossypium hirsutum L.)

Key message

Agrobacterium tumefaciens mediates high frequency of germline transformation of cotton meristem explants. The meristem transformation system we developed is rapid, high throughput and genotype-flexible.

Abstract

We have developed a high throughput cotton transformation system based on direct Agrobacterium inoculation of mechanically isolated meristem explants of cotton (Gossypium hirsutum L.). The explants were inoculated with a disarmed A. tumefaciens strain, AB33 harboring a 2 T-DNA binary vector pMON114908. This vector contained a gene of interest, an intron-disrupted β-glucuronidase gene in one T-DNA, and a selectable marker gene, aadA in the other T-DNA. Critical factors, such as method of co-culture, culture temperature during selection, composition of selection medium, and selection scheme were found to influence transformation frequency. The cycle time from initial inoculation to the transplanting of transgenic plants to soil was 7–8 weeks. Stable integration of transgenes and their transmission to progeny were confirmed by molecular and genetic analyses. Transgenes segregated in the expected Mendelian fashion in the T1 generation for most of the transgenic events. It was possible to recover marker-free events in the T1 generation when utilizing a binary vector that contained the selectable marker and gene of interest expression cassettes on independent T-DNAs. The procedure presented here has been used to regenerate thousands of independent transgenic events from multiple varieties with numerous constructs, and we believe it represents a major step forward in cotton transformation technology.     

Transcriptional profiling of Actinobacillus pleuropneumoniae under iron-restricted conditions

Background

Toll-like receptors (TLRs) play a central role in innate immunity. TLRs are membrane glycoproteins and contain leucine rich repeat (LRR) motif in the ectodomain. TLRs recognize and respond to molecules such as lipopolysaccharide, peptidoglycan, flagellin, and RNA from bacteria or viruses. The LRR domains in TLRs have been inferred to be responsible for molecular recognition. All LRRs include the highly conserved segment, LxxLxLxxNxL, in which "L" is Leu, Ile, Val, or Phe and "N" is Asn, Thr, Ser, or Cys and "x" is any amino acid. There are seven classes of LRRs including "typical" ("T") and "bacterial" ("S"). All known domain structures adopt an arc or horseshoe shape. Vertebrate TLRs form six major families. The repeat numbers of LRRs and their "phasing" in TLRs differ with isoforms and species; they are aligned differently in various databases. We identified and aligned LRRs in TLRs by a new method described here.

Results

The new method utilizes known LRR structures to recognize and align new LRR motifs in TLRs and incorporates multiple sequence alignments and secondary structure predictions. TLRs from thirty-four vertebrate were analyzed. The repeat numbers of the LRRs ranges from 16 to 28. The LRRs found in TLRs frequently consists of LxxLxLxxNxLxxLxxxxF/LxxLxx ("T") and sometimes short motifs including LxxLxLxxNxLxxLPx(x)LPxx ("S"). The TLR7 family (TLR7, TLR8, and TLR9) contain 27 LRRs. The LRRs at the N-terminal part have a super-motif of STT with about 80 residues. The super-repeat is represented by STTSTTSTT or _TTSTTSTT. The LRRs in TLRs form one or two horseshoe domains and are mostly flanked by two cysteine clusters including two or four cysteine residue.

Conclusion

Each of the six major TLR families is characterized by their constituent LRR motifs, their repeat numbers, and their patterns of cysteine clusters. The central parts of the TLR1 and TLR7 families and of TLR4 have more irregular or longer LRR motifs. These central parts are inferred to play a key role in the structure and/or function of their TLRs. Furthermore, the super-repeat in the TLR7 family suggests strongly that "bacterial" and "typical" LRRs evolved from a common precursor.     

Interval estimation of genetic susceptibility for retrospective case-control studies

Background

This article describes classical and Bayesian interval estimation of genetic susceptibility based on random samples with pre-specified numbers of unrelated cases and controls.

Results

Frequencies of genotypes in cases and controls can be estimated directly from retrospective case-control data. On the other hand, genetic susceptibility defined as the expected proportion of cases among individuals with a particular genotype depends on the population proportion of cases (prevalence). Given this design, prevalence is an external parameter and hence the susceptibility cannot be estimated based on only the observed data. Interval estimation of susceptibility that can incorporate uncertainty in prevalence values is explored from both classical and Bayesian perspective. Similarity between classical and Bayesian interval estimates in terms of frequentist coverage probabilities for this problem allows an appealing interpretation of classical intervals as bounds for genetic susceptibility. In addition, it is observed that both the asymptotic classical and Bayesian interval estimates have comparable average length. These interval estimates serve as a very good approximation to the "exact" (finite sample) Bayesian interval estimates. Extension from genotypic to allelic susceptibility intervals shows dependency on phenotype-induced deviations from Hardy-Weinberg equilibrium.

Conclusions

The suggested classical and Bayesian interval estimates appear to perform reasonably well. Generally, the use of exact Bayesian interval estimation method is recommended for genetic susceptibility, however the asymptotic classical and approximate Bayesian methods are adequate for sample sizes of at least 50 cases and controls.     

Efficient auto-excision of a selectable marker gene from transgenic citrus by combining the Cre/loxP system and ipt selection

Key message

A highly efficient Cre-mediated deletion system, offering a good alternative for producing marker-free transgenic plants that will relieve public concerns regarding GMOs, was first developed in citrus.

Abstract

The presence of marker genes in genetically modified crops raises public concerns regarding their safety. The removal of marker genes can prevent the risk of their flow into the environment and hasten the public’s acceptance of transgenic products. In this study, a new construct based on the Cre/loxP site-recombination system was designed to delete marker genes from transgenic citrus. In the construct, the selectable marker gene isopentenyltransferase gene (ipt) from Agrobacterium tumefaciens and the Cre recombinase gene were flanked by two loxP recognition sites in the direct orientation. The green fluorescent protein (gfp) reporter gene for monitoring the transformation of foreign genes was located outside of the loxP sequences. Transformation and deletion efficiencies of the vector were investigated using nopaline synthase gene (NosP) and CaMV 35S promoters to drive expression of Cre. Analysis of GFP activity showed that 28.1 and 13.6 % transformation efficiencies could be obtained by NosP- and CaMV 35S-driven deletions, respectively. Molecular analysis demonstrated that 100 % deletion efficiency was observed in the transgenic plants. The complete excision of the marker gene was found in all deletion events driven by NosP and in 81.8 % of deletion events driven by CaMV 35S. The results showed that Cre/loxP-mediated excision was highly efficient and precise in citrus. This approach provides a reliable strategy for auto-deletion of selectable marker genes from transgenic citrus to produce marker-free transgenic plants.     

Virus-induced gene silencing for comparative functional studies in Gladiolus hybridus

Key message

Virus-induced gene silencing (VIGS) system could be performed successfully in Gladiolus hybridus with vacuum infiltration of cormels and young plants.

Abstract

Functional analysis of genes in gladiolus has previously been impractical due to the lack of an efficient stable genetic transformation method. However, virus-induced gene silencing (VIGS) is effective in some plants which are difficult to transform through other methods. Although the Tobacco rattle virus (TRV)-based VIGS system has been developed and used for verifying gene functions in diverse plants, an appropriate TRV-VIGS approach for gladiolus has not been established yet. In this report we describe the first use of the TRV-VIGS system for gene silencing in gladiolus. Vacuum infiltration of cormels and young plants with the GhPDS-VIGS vector effectively down-regulated the PHYTOENE DESATURASE ortholog GhPDS gene and also resulted in various degrees of photobleaching in Gladiolus hybridus. The reduction in GhPDS expression was tested after TRV-based vector infection using real-time RT-PCR. In addition, the progress of TRV infection was detected by fluorescence visualization using a pTRV2: CP-GFP vector. In conclusion, the TRV-mediated VIGS described here will be an effective gene function analysis mechanism in gladiolus.     

“It is about how the net looks”: a qualitative study of perceptions and practices related to mosquito net care and repair in two districts in eastern Uganda

Background

Although the numbers of malaria cases in China have been declining in recent years, outbreaks of Plasmodium vivax malaria were still being reported in rural areas south of the Yellow River. To better understand the transmission dynamics of P. vivax parasites in China, the extent of genetic diversity of P. vivax populations circulating in Bozhou of Anhui province of China were investigated using three polymorphic genetic markers: merozoite surface proteins 1 and 3α (pvmsp-1 and pvmsp-3α) and circumsporozoite protein (pvcsp).

Methods

Forty-five P. vivax clinical isolates from Bouzhou of Anhui province were collected from 2009 to 2010 and were analysed using PCR/RFLP or DNA sequencing.

Results

Seven and six distinct allelic variants were identified using PCR/RFLP analysis of pvmsp-3α with HhaI and AluI, respectively. DNA sequence analysis of pvmsp-1 (variable block 5) revealed that there were Sal-I and recombinant types but not Belem type, and seven distinct allelic variants in pvmsp-1 were detected, with recombinant subtype 2 (R2) being predominant (66.7%). All the isolates carried pvcsp with VK210 type but not VK247 or P. vivax-like types in the samples. Sequence analysis of pvcsp gene revealed 12 distinct allelic variants, with VK210-1 being predominant (41.5%).

Conclusions

The present data indicate that there is some degree of genetic diversity among P. vivax populations in Anhui province of China. The genetic data obtained may assist in the surveillance of P. vivax infection in endemic areas or in tracking potential future disease outbreak.     

The plant genetic background affects the efficiency of the pepper major nematode resistance genes Me1 and Me3

Key message

The plant genetic background influences the efficiency of major resistance genes to root-knot nematodes in pepper and has to be considered in breeding strategies.

Abstract

Root-knot nematodes (RKNs), Meloidogyne spp., are extremely polyphagous plant parasites worldwide. Since the use of most chemical nematicides is being prohibited, genetic resistance is an efficient alternative way to protect crops against these pests. However, nematode populations proved able to breakdown plant resistance, and genetic resources in terms of resistance genes (R-genes) are limited. Sustainable management of these valuable resources is thus a key point of R-gene durability. In pepper, Me1 and Me3 are two dominant major R-genes, currently used in breeding programs to control M. arenaria, M. incognita and M. javanica, the three main RKN species. These two genes differ in the hypersensitive response induced by nematode infection. In this study, they were introgressed in either a susceptible or a partially resistant genetic background, in either homozygous or heterozygous allelic status. Challenging these genotypes with an avirulent M. incognita isolate demonstrated that (1) the efficiency of the R-genes in reducing the reproductive potential of RKNs is strongly affected by the plant genetic background, (2) the allelic status of the R-genes has no effect on nematode reproduction. These results highlight the primary importance of the choice of both the R-gene and the genetic background into which it is introgressed during the selection of new elite cultivars by plant breeders.     

From the Western Alps across Central Europe: Postglacial recolonisation of the tufa stream specialist Rhyacophila pubescens (Insecta,Trichoptera)

Background

Hybridization can have complex effects on evolutionary dynamics in ants because of the combination of haplodiploid sex-determination and eusociality. While hybrid non-reproductive workers have been found in a range of species, examples of gene-flow via hybrid queens and males are rare. We studied hybridization in East African army ants (Dorylus subgenus Anomma) using morphology, mitochondrial DNA sequences, and nuclear microsatellites.

Results

While the mitochondrial phylogeny had a strong geographic signal, different species were not recovered as monophyletic. At our main study site at Kakamega Forest, a mitochondrial haplotype was shared between a "Dorylus molestus-like" and a "Dorylus wilverthi-like" form. This pattern is best explained by introgression following hybridization between D. molestus and D. wilverthi. Microsatellite data from workers showed that the two morphological forms correspond to two distinct genetic clusters, with a significant proportion of individuals being classified as hybrids.

Conclusions

We conclude that hybridization and gene-flow between the two army ant species D. molestus and D. wilverthi has occurred, and that mating between the two forms continues to regularly produce hybrid workers. Hybridization is particularly surprising in army ants because workers have control over which males are allowed to mate with a young virgin queen inside the colony.     

Progress in lactic acid bacterial phage research

Background

The cyanobacterium Synechocystis sp. PCC 6803 is widely used for research on photosynthesis and circadian rhythms, and also finds application in sustainable biotechnologies. Synechocystis is naturally transformable and undergoes homologous recombination, which enables the development of a variety of tools for genetic and genomic manipulations. To generate multiple gene deletions and/or replacements, marker-less manipulation methods based on counter-selection are generally employed. Currently available methods require two transformation steps with different DNA plasmids.

Results

In this study, we present a marker-less gene deletion and replacement strategy in Synechocystis sp. PCC 6803 which needs only a single transformation step. The method utilizes an nptI-sacB double selection cassette and exploits the ability of the cyanobacterium to undergo two successive genomic recombination events via double and single crossing-over upon application of appropriate selective procedures.

Conclusions

By reducing the number of cloning steps, this strategy will facilitate gene manipulation, gain-of-function studies, and automated screening of mutants.     

Genetic diversity and population structure in the US Upland cotton (Gossypium hirsutum L.)

Key message

Genetic diversity and population structure in the US Upland cotton was established and core sets of allelic richness were identified for developing association mapping populations in cotton.

Abstract

Elite plant breeding programs could likely benefit from the unexploited standing genetic variation of obsolete cultivars without the yield drag typically associated with wild accessions. A set of 381 accessions comprising 378 Upland (Gossypium hirsutum L.) and 3 G. barbadense L. accessions of the United States cotton belt were genotyped using 120 genome-wide SSR markers to establish the genetic diversity and population structure in tetraploid cotton. These accessions represent more than 100 years of Upland cotton breeding in the United States. Genetic diversity analysis identified a total of 546 alleles across 141 marker loci. Twenty-two percent of the alleles in Upland accessions were unique, specific to a single accession. Population structure analysis revealed extensive admixture and identified five subgroups corresponding to Southeastern, Midsouth, Southwest, and Western zones of cotton growing areas in the United States, with the three accessions of G. barbadense forming a separate cluster. Phylogenetic analysis supported the subgroups identified by STRUCTURE. Average genetic distance between G. hirsutum accessions was 0.195 indicating low levels of genetic diversity in Upland cotton germplasm pool. The results from both population structure and phylogenetic analysis were in agreement with pedigree information, although there were a few exceptions. Further, core sets of different sizes representing different levels of allelic richness in Upland cotton were identified. Establishment of genetic diversity, population structure, and identification of core sets from this study could be useful for genetic and genomic analysis and systematic utilization of the standing genetic variation in Upland cotton.     

Increased polyclonal immunoglobulin reactivity toward human and bacterial proteins is associated with clinical protection in human Plasmodium infection

Introduction

By 2030, more than 50% of the African population will live in urban areas. Controlling malaria reduces the disease burden and further improves economic development. As a complement to treated nets and prompt access to treatment, measures targeted against the larval stage of Anopheles sp. mosquitoes are a promising strategy for urban areas. However, a precise knowledge of the geographic location and potentially of ecological characteristics of breeding sites is of major importance for such interventions.

Methods

In total 151 km² of central Dar es Salaam, the biggest city of Tanzania, were systematically searched for open mosquito breeding sites. Ecologic parameters, mosquito larvae density and geographic location were recorded for each site. Logistic regression analysis was used to determine the key ecological factors explaining the different densities of mosquito larvae.

Results

A total of 405 potential open breeding sites were examined. Large drains, swamps and puddles were associated with no or low Anopheles sp. larvae density. The probability of Anopheles sp. larvae to be present was reduced when water was identified as "turbid". Small breeding sites were more commonly colonized by Anopheles sp. larvae. Further, Anopheles gambiae s.l. larvae were found in highly organically polluted habitats.

Conclusions

Clear ecological characteristics of the breeding requirements of Anopheles sp. larvae could not be identified in this setting. Hence, every stagnant open water body, including very polluted ones, have to be considered as potential malaria vector breeding sites.     

A little help from my friends: caring for premature babies in a war zone

Background

Too much or too little milk production are common problems in a lactation consultant's practice. Whereas underproduction is widely discussed in the lactation literature, overabundant milk supply is not. In my practice I work with women who experience moderate to severe oversupply syndrome. In most cases the syndrome can be successfully treated with full removal of milk followed by unilateral breastfeeding ad lib with the same breast offered at every breastfeed in a certain time block ("block feeding").

Case presentations

Four cases of over-supply of breast milk are presented. The management and outcome of each case is described.

Conclusion

Overabundant milk supply is an often under-diagnosed condition in otherwise healthy lactating women. Full drainage and "block feeding" offer an adequate and userfriendly way to normalize milk production and treat symptoms in both mother and child.     

Analysis of IL-12 p40 subunit gene and IFN-γ G5644A polymorphisms in Idiopathic Pulmonary Fibrosis

Background

Genes encoding cytokine mediators are prime candidates for genetic analysis in conditions with T-helper (Th) cell disease driven imbalance. Idiopathic Pulmonary Fibrosis (IPF) is a predominantly Th2 mediated disease associated with a paucity of interferon-gamma (IFN-γ). The paucity of IFN-γ may favor the development of progressive fibrosis in IPF. Interleukin-12 (IL-12) plays a key role in inducing IFN-γ production. The aim of the current study was to assess whether the 1188 (A/C) 3'UTR single nucleotide polymorphism (SNP) in the IL-12 p40 subunit gene which was recently found to be functional and the 5644 (G/A) 3' UTR SNP of the IFN-γ gene were associated with susceptibility to IPF.

Methods

We investigated the allelic distribution in these loci in UK white Caucasoid subjects comprising 73 patients with IPF and 157 healthy controls. The SNPs were determined using the polymerase chain reaction in association with sequence-specific primers incorporating mismatches at the 3'-end.

Results

Our results showed that these polymorphisms were distributed similarly in the IPF and control groups

Conclusion

We conclude that these two potentially important candidate gene single nucleotide polymorphisms are not associated with susceptibility to IPF.     

The relationship between reported fever and Plasmodium falciparum infection in African children

Background

The genetic diversity of Plasmodium falciparum has been extensively studied in various parts of the world. However, limited data are available from Pakistan. This study aimed to establish molecular characterization of P. falciparum field isolates in Pakistan measured with two highly polymorphic genetic markers, i.e. the merozoite surface protein 1 (msp-1)and 2 (msp-2).

Methods

Between October 2005 and October 2007, 244 blood samples from patients with symptomatic blood-slide confirmed P. falciparum mono-infections attending the Aga Khan University Hospital, Karachi, or its collection units located in Sindh and Baluchistan provinces, Pakistan were collected. The genetic diversity of P. falciparum was analysed by length polymorphism following gel electrophoresis of DNA products from nested polymerase chain reactions (PCR) targeting block 2 of msp-1 and block 3 of msp-2, including their respective allelic families KI, MAD 20, RO33, and FC27, 3D7/IC.

Results

A total of 238/244 (98%) patients had a positive PCR outcome in at least one genetic marker; the remaining six were excluded from analysis. A majority of patients had monoclonal infections. Only 56/231 (24%) and 51/236 (22%) carried multiple P. falciparum genotypes in msp-1 and msp-2, respectively. The estimated total number of genotypes was 25 msp-1 (12 KI; 8 MAD20; 5 RO33) and 33 msp-2 (14 FC27; 19 3D7/IC).

Conclusions

This is the first report on molecular characterization of P. falciparum field isolates in Pakistan with regards to multiplicity of infection. The genetic diversity and allelic distribution found in this study is similar to previous reports from India and Southeast Asian countries with low malaria endemicity.     

Inhibitory activity of Indian spice plant Cinnamomum zeylanicum extracts against Alternaria solani and Curvularia lunata, the pathogenic dematiaceous moulds

Background

Enteric fever is an endemic problem in Nepal and Widal agglutination test is widely used for its diagnosis but a normal baseline titer in healthy population and cutoff values have not been established.

Methods

We measured average baseline antibody titers against "O" and "H" antigens of Salmonella enterica serotype Typhi and "H" antigens of serotypes Paratyphi A and Paratyphi B among apparently healthy blood donors in Nepal. The antibody titers were measured using Standard Widal Confirmatory Quantitative Tube test.

Results

Among the 100 blood samples collected from healthy volunteers, 62 individuals had significant antibody titers (≥ 1:20) against one of the four antigens against S. enterica. Among 54 samples with an anti-O titer against serotype Typhi, 15 and 36 samples had titers of ≥ 1:60 and ≥ 1:40, respectively. A significant proportion (12% of all) had anti-O titer of ≥ 1:80. Similarly, among the 59 samples demonstrating anti-H titers of ≥ 1:20 to S. enterica serotype Typhi, 29 had a titer of ≥ 1:80 and 12 had 1:160. For S. enterica serotypes Paratyphi A and B, anti-H titers of ≥ 1:20 were found only in 12% and 3%, respectively, of all samples tested.

Conclusion

When a single Widal agglutination titer is used for the diagnosis of enteric fever, it will be more appropriate to change the currently used cutoff levels against S. enterica serotype Typhi to > 1:80 for anti-O and > 1:160 for anti-H titers for Nepal.