Genetic characterization and fine mapping for multi-inflorescence in <Emphasis Type="Italic">Brassica napus</Emphasis> L.

Key message

A major QTL for multi-inflorescence was mapped to a 27.18-kb region on A05 in Brassica napus by integrating QTL mapping, microarray analysis and whole-genome sequencing.

Abstract

Multi-inflorescence is a desirable trait for the genetic improvement of rapeseed (Brassica napus L.). However, the genetic mechanism underlying the multi-inflorescence trait is not well understood. In the present study, a doubled haploid (DH) population derived from a cross between single- and multi-inflorescence lines was investigated for the penetrance of multi-inflorescence across 3 years and genotyped with 257 simple sequence repeat and sequence-related amplified polymorphism loci. A major quantitative trait locus (QTL) for penetrance of multi-inflorescence was mapped to a 9.31-Mb region on chromosome A05, explaining 45.81% of phenotypic variance on average. Subsequently, 13 single-inflorescence and 15 multi-inflorescence DH lines were genotyped with the Brassica microarray, and the QTL interval of multi-inflorescence was narrowed to a 0.74-Mb region with 37 successive single nucleotide polymorphisms between single- and multi-inflorescence groups. A 27.18-kb QTL interval was detected by screening 420 recessive F₂ individuals with genome-specific markers. These results will be valuable for gene cloning and molecular breeding of multi-inflorescence in rapeseed.

     

Construction of a genome-anchored,high-density genetic map for melon (<Emphasis Type="Italic">Cucumis melo</Emphasis> L.) and identification of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">melonis</Emphasis> race 1 resistance QTL

Key message

Four QTLs and an epistatic interaction were associated with disease severity in response to inoculation with Fusarium oxysporum f. sp. melonis race 1 in a recombinant inbred line population of melon.

Abstract

The USDA Cucumis melo inbred line, MR-1, harbors a wealth of alleles associated with resistance to several major diseases of melon, including powdery mildew, downy mildew, Alternaria leaf blight, and Fusarium wilt. MR-1 was crossed to an Israeli cultivar, Ananas Yok’neam, which is susceptible to all of these diseases, to generate a recombinant inbred line (RIL) population of 172 lines. In this study, the RIL population was genotyped to construct an ultra-dense genetic linkage map with 5663 binned SNPs anchored to the C. melo genome and exhibits the overall high quality of the assembly. The utility of the densely genotyped population was demonstrated through QTL mapping of a well-studied trait, resistance to Fusarium wilt caused by Fusarium oxysporum f. sp. melonis (Fom) race 1. A major QTL co-located with the previously validated resistance gene Fom-2. In addition, three minor QTLs and an epistatic interaction contributing to Fom race 1 resistance were identified. The MR-1 × AY RIL population provides a valuable resource for future QTL mapping studies and marker-assisted selection of disease resistance in melon.

     

Time-Kill Curves Studies with Amphotericin B Against <Emphasis Type="Italic">Cryptococcus neoformans</Emphasis>/<Emphasis Type="Italic">C</Emphasis>. <Emphasis Type="Italic">gattii</Emphasis> Species Complex Clinical Isolates

Purpose of Review

We reviewed data on amphotericin B (AmB) tolerance among Cryptococcus neoformans/C. gattii species complex clinical isolates and present our results of large recent study on this issue.

Recent Findings

The standard method to detect antifungal susceptibility is based on MIC (minimal inhibitory concentration) determination; however, there is no interpretative clinical breakpoints defined for antifungal agents against Cryptococcus species, and to date, there is no correlation of MIC and clinical response. The time-kill curves (TKC) methodology seems to provide some correlation with outcome and it could identify distinct profiles of AmB-fungicidal activity.

Summary

Our group analyzed 83 human isolates from cryptococcosis cases. The isolates were tested by TKC and showed up 8.3% of tolerance to AmB. Importantly, the AmB-MIC was low for all isolates, including tolerant ones. Our findings are similar to other authors, due the ability of TKC to identify distinct AmB-fungicidal activity and detecting low susceptible isolates.

     

Identification of candidate genes of QTLs for seed weight in <Emphasis Type="Italic">Brassica napus</Emphasis> through comparative mapping among <Emphasis Type="Italic">Arabidopsis</Emphasis> and <Emphasis Type="Italic">Brassica</Emphasis>species

Background

Map-based cloning of quantitative trait loci (QTLs) in polyploidy crop species remains a challenge due to the complexity of their genome structures. QTLs for seed weight in B. napus have been identified, but information on candidate genes for identified QTLs of this important trait is still rare.

Results

In this study, a whole genome genetic linkage map for B. napus was constructed using simple sequence repeat (SSR) markers that covered a genetic distance of 2,126.4 cM with an average distance of 5.36 cM between markers. A procedure was developed to establish colinearity of SSR loci on B. napus with its two progenitor diploid species B. rapa and B. oleracea through extensive bioinformatics analysis. With the aid of B. rapa and B. oleracea genome sequences, the 421 homologous colinear loci deduced from the SSR loci of B. napus were shown to correspond to 398 homologous loci in Arabidopsis thaliana. Through comparative mapping of Arabidopsis and the three Brassica species, 227 homologous genes for seed size/weight were mapped on the B. napus genetic map, establishing the genetic bases for the important agronomic trait in this amphidiploid species. Furthermore, 12 candidate genes underlying 8 QTLs for seed weight were identified, and a gene-specific marker for BnAP2 was developed through molecular cloning using the seed weight/size gene distribution map in B. napus.

Conclusions

Our study showed that it is feasible to identify candidate genes of QTLs using a SSR-based B. napus genetic map through comparative mapping among Arabidopsis and B. napus and its two progenitor species B. rapa and B. oleracea. Identification of candidate genes for seed weight in amphidiploid B. napus will accelerate the process of isolating the mapped QTLs for this important trait, and this approach may be useful for QTL identification of other traits of agronomic significance.

     

Iridoid and phenylethanoid/phenylpropanoid metabolite profiles of <Emphasis Type="Italic">Scrophularia</Emphasis> and <Emphasis Type="Italic">Verbascum</Emphasis> species used medicinally in North America

Introduction

Botanicals containing iridoid and phenylethanoid/phenylpropanoid glycosides are used worldwide for the treatment of inflammatory musculoskeletal conditions that are primary causes of human years lived with disability, such as arthritis and lower back pain.

Objectives

We report the analysis of candidate anti-inflammatory metabolites of several endemic Scrophularia species and Verbascum thapsus used medicinally by peoples of North America.

Methods

Leaves, stems, and roots were analyzed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) and partial least squares-discriminant analysis (PLS-DA) was performed in MetaboAnalyst 3.0 after processing the datasets in Progenesis QI.

Results

Comparison of the datasets revealed significant and differential accumulation of iridoid and phenylethanoid/phenylpropanoid glycosides in the tissues of the endemic Scrophularia species and Verbascum thapsus.

Conclusions

Our investigation identified several species of pharmacological interest as good sources for harpagoside and other important anti-inflammatory metabolites.

     

New microsatellite markers developed from <Emphasis Type="Italic">Urochloa humidicola</Emphasis> (Poaceae) and cross amplification in different <Emphasis Type="Italic">Urochloa</Emphasis> species

Background

Urochloa humidicola is a forage grass that grows in tropical regions and is recognized for its tolerance to seasonal flooding. It is a polyploid and apomictic species with high phenotypic plasticity. As molecular tools are important in facilitating the development of new cultivars and in the classification of related species, the objectives of this study were to develop new polymorphic microsatellite markers from an enriched library constructed from U. humidicola and to evaluate their transferability to other Urochloa species.

Findings

Microsatellite sequences were identified from a previously constructed enriched library, and specific primers were designed for 40 loci. Isolated di-nucleotide repeat motifs were the most abundant followed by tetra-nucleotide repeats. Of the tested loci, 38 displayed polymorphism when screened across 34 polyploid Urochloa sp. genotypes, including 20 accessions and six hybrids of U. humidicola and two accessions each from U. brizantha, U. dictyoneura, U. decumbens and U. ruziziensis. The number of bands per Simple Sequence Repeat (SSR) locus ranged from one to 29 with a mean of 11.5 bands per locus. The mean Polymorphism Information Content (PIC) of all loci was 0.7136, and the mean Discrimination Power (DP) was 0.7873. Six loci amplified in all species tested. STRUCTURE analysis revealed six different allelic pools, and the genetic similarity values analyzed using Jaccard's coefficient ranged from 0.000 to 0.913.

Conclusions

This work reports new polymorphic microsatellite markers that will be useful for breeding programs for Urochloa humidicola and other Urochloa species as well as for genetic map development, germplasm characterization, evolutionary and taxonomic studies and marker-assisted trait selection.

     

Disruption of <Emphasis Type="Italic">YLR162W</Emphasis> in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> results in increased tolerance to organic solvents

Objective

To identify a novel gene responsible for organic solvent-tolerance by screening a transposon-mediated deletion mutant library based on Saccharomyces cerevisiae L3262.

Results

One strain tolerant of up to 0.5 % (v/v) n-hexane and cyclohexane was isolated. The determination of transposon insertion site identified one gene, YLR162W, and revealed disruption of the ORF of this gene, indicating that organic solvent tolerance can be conferred. Such a tolerant phenotype reverted to the sensitive phenotype on the autologous or overexpression of this gene. This transposon mutant grew faster than the control strain when cultured at 30 °C in YPD medium containing 0.5 % (v/v) n-hexane and cyclohexane respectively.

Conclusion

Disruption of YLR162W in S. cerevisiae results in increased tolerance to organic solvents.

     

Quantitative analysis of tetrahydrofolate metabolites from <Emphasis Type="Italic">clostridium autoethanogenum</Emphasis>

Introduction

Quantification of tetrahydrofolates (THFs), important metabolites in the Wood–Ljungdahl pathway (WLP) of acetogens, is challenging given their sensitivity to oxygen.

Objective

To develop a simple anaerobic protocol to enable reliable THFs quantification from bioreactors.

Methods

Anaerobic cultures were mixed with anaerobic acetonitrile for extraction. Targeted LC–MS/MS was used for quantification.

Results

Tetrahydrofolates can only be quantified if sampled anaerobically. THF levels showed a strong correlation to acetyl-CoA, the end product of the WLP.

Conclusion

Our method is useful for relative quantification of THFs across different growth conditions. Absolute quantification of THFs requires the use of labelled standards.

     

Fine mapping of a quantitative resistance gene for gray leaf spot of maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.) derived from teosinte (<Emphasis Type="Italic">Z. mays</Emphasis> ssp. <Emphasis Type="Italic">parviglumis</Emphasis>)

Key message

In this study we mapped the QTL Qgls8 for gray leaf spot (GLS) resistance in maize to a ~130 kb region on chromosome 8 including five predicted genes.

Abstract

In previous work, using near isogenic line (NIL) populations in which segments of the teosinte (Zea mays ssp. parviglumis) genome had been introgressed into the background of the maize line B73, we had identified a QTL on chromosome 8, here called Qgls8, for gray leaf spot (GLS) resistance. We identified alternate teosinte alleles at this QTL, one conferring increased GLS resistance and one increased susceptibility relative to the B73 allele. Using segregating populations derived from NIL parents carrying these contrasting alleles, we were able to delimit the QTL region to a ~130 kb (based on the B73 genome) which encompassed five predicted genes.

     

QTL mapping identifies a major locus for resistance in wheat to Sunn pest (<Emphasis Type="Italic">Eurygaster</Emphasis><Emphasis Type="Italic">integriceps</Emphasis>) feeding at the vegetative growth stage

Key message

This research provides the first report of a major locus controlling wheat resistance to Sunn pest. It developed and validated SNP markers that will be useful for marker-assisted selection.

Abstract

Sunn pest (Eurygaster integriceps Puton) is the most destructive insect pest of bread wheat and durum wheat in West and Central Asia and East Europe. Breeding for resistance at the vegetative stage of growth is vital in reducing the damage caused by overwintered adult populations that feed on shoot and leaves of seedlings, and in reducing the next generation of pest populations (nymphs and adults), which can cause damage to grain quality by feeding on spikes. In the present study, two doubled haploid (DH) populations involving resistant landraces from Afghanistan were genotyped with the 90k SNP iSelect assay and candidate gene-based KASP markers. The DH lines and parents were phenotyped for resistance to Sunn pest feeding, using artificial infestation cages at Terbol station, in Lebanon, over three years. Quantitative trait locus (QTL) analysis identified a single major locus on chromosome 4BS in the two populations, with the resistance allele derived from the landrace accessions, IG139431 and IG139883. The QTL explained a maximum of 42 % of the phenotypic variation in the Cham6 × IG139431 and 56 % in the Cham6 × IG139883 populations. SNP markers closest to the QTL showed high similarity to rice genes that putatively encode proteins for defense response to herbivory and wounding. The markers were validated in a large, unrelated population of parental wheat genotypes. All wheat lines carrying the ‘C–G’ haplotype at the identified SNPs were resistant, suggesting that selection based on a haplotype of favourable alleles would be effective in predicting resistance status of unknown genotypes.

     

<Emphasis Type="Italic">PiggyBac</Emphasis> transposon-mediated mutagenesis and application in yeast <Emphasis Type="Italic">Komagataella phaffii</Emphasis>

Objective

Around one-fourth of the Komagataella phaffii genes encode hypothetical proteins with unknown functions. However, lack of powerful tools for genetic screening in K. phaffii significantly limits the functional analysis of these unknown genes. Transposon mutagenesis has been utilized as an insertional mutagenesis tool in many other organisms and would be extremely valuable if it could be applied in K. phaffii.

Results

In this study, we investigated in K. phaffii the transposition activity and efficiency of piggyBac (PB) transposon, a DNA transposon from the cabbage looper moth Trichoplusia ni through the integrated-plasmid system. We also designed a binary-plasmid system which could generate stable mutants. Finally we evaluated the quality of this mutagenesis system by a simple screening for functional genes involved in K. phaffii carbon catabolite repression.

Conclusions

Our results demonstrate that PB-mediated mutagenesis could be a feasible and useful tool for functional gene screening in K. phaffii.

     

Deletion of <Emphasis Type="Italic">ldh</Emphasis>A and <Emphasis Type="Italic">ald</Emphasis>H genes in <Emphasis Type="Italic">Klebsiella</Emphasis><Emphasis Type="Italic">pneumoniae</Emphasis> to enhance 1,3-propanediol production

Objectives

To improve 1,3-propanediol (1,3-PD) production and reduce byproduct concentration during the fermentation of Klebsiella pneumonia.

Results

Klebsiella. pneumonia 2-1ΔldhA, K. pneumonia 2-1ΔaldH and K. pneumonia 2-1ΔldhAΔaldH mutant strains were obtained through deletion of the ldhA gene encoding lactate dehydrogenase required for lactate synthesis and the aldH gene encoding acetaldehyde dehydrogenase involved in the synthesis of ethanol. After fed-batch fermentation, the production of 1,3-PD from glycerol was enhanced and the concentrations of byproducts were reduced compared with the original strain K. pneumonia 2-1. The maximum yields of 1,3-PD were 85.7, 82.5 and 87.5 g/l in the respective mutant strains.

Conclusion

Deletion of either aldH or ldhA promoted 1,3-PD production in K. pneumonia.

     

Identification and validation of QTL and their associated genes for pre-emergent metribuzin tolerance in hexaploid wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

Background

Herbicide tolerance is an important trait that allows effective weed management in wheat crops. Genetic knowledge of metribuzin tolerance in wheat is needed to develop new cultivars for the industry. Here, we evaluated metribuzin tolerance in a recombinant inbred line (RIL) mapping population derived from Synthetic W7984 and Opata 85 over two consecutive years to identify quantitative trait loci (QTL) contributing to the trait. Herbicide tolerance was measured by two chlorophyll traits, SPAD chlorophyll content index (CCI) and visual senescence score (SNS). The markers associated with major QTL from Synthetic W7984, positively contributing to reduced phytotoxic effects under herbicide treatment were validated in two F_3/4 recombinant inbred populations developed from crosses of Synthetic W7984?×?Westonia and Synthetic W7984?×?Lang.

Results

Composite interval mapping (CIM) identified four QTL, two on chromosome 4A and one each on chromosomes 2D and 1A. The chromosomal position of the two QTL mapped on 4A within 10 cM intervals was refined and validated by multiple interval mapping (MIM). The major QTL affecting both measures of tolerance jointly explained 42 and 45% of the phenotypic variation by percentage CCI reduction and SNS, respectively. The identified QTL have a pure additive effect. The metribuzin tolerant allele of markers, Xgwm33 and Xbarc343, conferred lower phytotoxicity and explained the maximum phenotypic variation of 28.8 and 24.5%, respectively. The approximate physical localization of the QTL revealed the presence of five candidate genes (ribulose-bisphosphate carboxylase, oxidoreductase (rbcS), glycosyltransferase, serine/threonine-specific protein kinase and phosphotransferase) with a direct role in photosynthesis and/or metabolic detoxification pathways.

Conclusion

Metribuzin causes photo-inhibition by interrupting electron flow in PSII. Consequently, chlorophyll traits enabled the measure of high proportion of genetic variability in the mapping population. The validated molecular markers associated with metribuzin tolerance mediating QTL may be used in marker-assisted breeding to select metribuzin tolerant lines. Alternatively, validated favourable alleles could be introgressed into elite wheat cultivars to enhance metribuzin tolerance and improve grain yield in dryland farming for sustainable wheat production.

     

<Emphasis Type="Italic">Rhizobium</Emphasis> strains in the biological control of the phytopathogenic fungi <Emphasis Type="Italic">Sclerotium</Emphasis> (<Emphasis Type="Italic">Athelia</Emphasis>) <Emphasis Type="Italic">rolfsii</Emphasis> on the common bean

Aims

To identify Rhizobium strains’ ability to biocontrol Sclerotium rolfsii, a fungus that causes serious damage to the common bean and other important crops, 78 previously isolated rhizobia from common bean were assessed.

Methods

Dual cultures, volatiles, indole-acetic acid (IAA), siderophore production and 16S rRNA sequencing were employed to select strains for pot and field experiments.

Results

Thirty-three antagonistic strains were detected in dual cultures, 16 of which were able to inhibit ≥84% fungus mycelial growth. Antagonistic strains produced up to 36.5 μg mL^?1 of IAA, and a direct correlation was verified between IAA production and mycelium inhibition. SEMIA 460 inhibited 45% of mycelial growth through volatile compounds. 16S rRNA sequences confirmed strains as Rhizobium species. In pot condition, common bean plants grown on S. rolfsii-infested soil and inoculated with SEMIA 4032, 4077, 4088, 4080, 4085, or 439 presented less or no disease symptoms. The most efficient strains under field conditions, SEMIA 439 and 4088, decreased disease incidence by 18.3 and 14.5% of the S. rolfsii-infested control.

Conclusions

Rhizobium strains could be strong antagonists towards S. rolfsii growth. SEMIA 4032, 4077, 4088, 4080, 4085, and 439 are effective in the biological control of the collar rot of the common bean.

     

Evidence of intralocus recombination at the <Emphasis Type="Italic">Glu-3</Emphasis> loci in bread wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

Key message

Recombination at the Glu-3 loci was identified, and strong genetic linkage was observed only between the amplicons representing i-type and s-type genes located, respectively, at the Glu-A3 and Glu-B3 loci.

Abstract

The low-molecular weight glutenin subunits (LMW-GSs) are one of the major components of wheat seed storage proteins and play a critical role in the determination of wheat end-use quality. The genes encoding this class of proteins are located at the orthologous Glu-3 loci (Glu-A3, Glu-B3, and Glu-D3). Due to the complexity of these chromosomal regions and the high sequence similarity between different LMW-GS genes, their organization and recombination characteristics are still incompletely understood. This study examined intralocus recombination at the Glu-3 loci in two recombinant inbred line (RIL) and one doubled haploid (DH) population, all segregating for the Glu-A3, Glu-B3, and Glu-D3 loci. The analysis was conducted using a gene marker system that consists of the amplification of the complete set of the LMW-GS genes and their visualization by capillary electrophoresis. Recombinant marker haplotypes were detected in all three populations with different recombination rates depending on the locus and the population. No recombination was observed between the amplicons representing i-type and s-type LMW-GS genes located, respectively, at the Glu-A3 and Glu-B3 loci, indicating tight linkage between these genes. Results of this study contribute to better understanding the genetic linkage and recombination between different LMW-GS genes, the structure of the Glu-3 loci, and the development of more specific molecular markers that better represent the genetic diversity of these loci. In this way, a more precise analysis of the contribution of various LMW-GSs to end-use quality of wheat may be achieved.

     

Expression of <Emphasis Type="Italic">Ciona intestinalis</Emphasis> AOX causes male reproductive defects in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

Background

Mitochondrial alternative respiratory-chain enzymes are phylogenetically widespread, and buffer stresses affecting oxidative phosphorylation in species that possess them. However, they have been lost in the evolutionary lineages leading to vertebrates and arthropods, raising the question as to what survival or reproductive disadvantages they confer. Recent interest in using them in therapy lends a biomedical dimension to this question.

Methods

Here, we examined the impact of the expression of Ciona intestinalis alternative oxidase, AOX, on the reproductive success of Drosophila melanogaster males. Sperm-competition assays were performed between flies carrying three copies of a ubiquitously expressed AOX construct, driven by the α-tubulin promoter, and wild-type males of the same genetic background.

Results

In sperm-competition assays, AOX conferred a substantial disadvantage, associated with decreased production of mature sperm. Sperm differentiation appeared to proceed until the last stages, but was spatially deranged, with spermatozoids retained in the testis instead of being released to the seminal vesicle. High AOX expression was detected in the outermost cell-layer of the testis sheath, which we hypothesize may disrupt a signal required for sperm maturation.

Conclusions

AOX expression in Drosophila thus has effects that are deleterious to male reproductive function. Our results imply that AOX therapy must be developed with caution.

     

Inhibitory effects of YCW and MOS from <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> on <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Salmonella pullorum</Emphasis> adhesion to Caco-2 cells

Background

For many years, yeast cell walls (YCW) and mannan oligosaccharides (MOS) have been used as alternatives to antibiotics and health feed additives to enhance the growth performance and health of food animals. In the present study, the inhibitory effects of YCWand MOS on the adhesion of enteropathogenic bacteria to intestinal epithelial cells were tested.

Methods

YCW and MOS were extracted from Saccharomyces cerevisiae (XM 0315), and the morphology of YCW and MOS bound to pathogenic bacteria was observed by scanning electron microscopy (SEM). Real-time fluorescent quantitative PCR was used to quantitatively analyze the effects of YCW and MOS on the adhesion of Escherichia coli (CVCC3367) and Salmonella pullorum (CVCC520) to Caco-2 cells.

Results

The results showed that YCW inhibited E. coli and S. pullorum binding to Caco-2 cells by 95% and 74%, respectively, whereas MOS prevented E. coli and S. pullorum binding by 67% and 50%, respectively.

Conclusions

These data suggest that YCW has a stronger ability than MOS to inhibit pathogenic bacteria from adhering to Caco-2 cells in vitro.