Discovery of a seventh <Emphasis Type="Italic">Rpp</Emphasis> soybean rust resistance locus in soybean accession PI 605823

Key message

A novel Rpp gene from PI 605823 for resistance to Phakopsora pachyrhizi was mapped on chromosome 19.

Abstract

Soybean rust, caused by the obligate biotrophic fungal pathogen Phakopsora pachyrhizi Syd. & P. Syd, is a disease threat to soybean production in regions of the world with mild winters. Host plant resistance conditioned by resistance to P. pachyrhizi (Rpp) genes has been found in numerous soybean accessions, and at least 10 Rpp genes or alleles have been mapped to six genetic loci. Identifying additional disease-resistance genes will facilitate development of soybean cultivars with durable resistance. PI 605823, a plant introduction from Vietnam, was previously identified as resistant to US populations of P. pachyrhizi in greenhouse and field trials. In this study, bulked segregant analysis using an F₂ population derived from ‘Williams 82’ × PI 605823 identified a genomic region associated with resistance to P. pachyrhizi isolate GA12, which had been collected in the US State of Georgia in 2012. To further map the resistance locus, linkage mapping was carried out using single-nucleotide polymorphism markers and phenotypic data from greenhouse assays with an F_2:3 population derived from Williams 82 × PI 605823 and an F_4:5 population derived from ‘5601T’ × PI 605823. A novel resistance gene, Rpp7, was mapped to a 154-kb interval (Gm19: 39,462,291–39,616,643 Glyma.Wm82.a2) on chromosome 19 that is different from the genomic locations of any previously reported Rpp genes. This new gene could be incorporated into elite breeding lines to help provide more durable resistance to soybean rust.

     

Single nucleotide polymorphism markers for rapid detection of the <Emphasis Type="Italic">Rsv4</Emphasis> locus for soybean mosaic virus resistance in diverse germplasm

Soybean mosaic virus (SMV) causes a substantial decrease in soybean yield and reduction of seed quality. The most effective management strategy to control the virus is the deployment of host resistance. Seven SMV strains and three independent multi-allelic loci for SMV resistance have been identified previously. The goal of this research was to detect single nucleotide polymorphisms (SNPs) associated with SMV resistance at the Rsv4 locus. Ten soybean accessions, with confirmed resistance genes, were used for sequencing the candidate gene Glyma.02g121400. Alignment of these sequences revealed three SNPs displaying 100% consistency for genotypes carrying the Rsv4 gene. These SNPs were applied for a rapid screen of diverse soybean germplasm using the Sequenom iPLEX Gold platform, phenotyped with SMV-G1 and G7 strains to determine phenotype and classified into several groups carrying the proposed R-gene. The population of V94-5152 (Rsv4) × Lee 68 (rsv) was screened using novel SNPs to create a genetic map with improved resolution to determine the location of the Rsv4. To observe the recombination frequencies within the population, three additional SNPs on both sides of the Glyma.02g121400 gene were added. A linkage map revealed a distance of 3.6 cM between the Rsv4 locus and the closest SNP, thus shifting the putative Rsv4 region downstream on chromosome 2. With this region, five candidate genes have been proposed. The genomic position of the discovered SNPs, linked to the Rsv4, could increase screening precision and accelerate breeding efforts to develop multi-strain-resistant crops.     

Characterization of <Emphasis Type="Italic">Pm59</Emphasis>, a novel powdery mildew resistance gene in Afghanistan wheat landrace PI 181356

Key message

A new powdery mildew resistance gene, designated Pm59, was identified in Afghanistan wheat landrace PI 181356, and mapped in the terminal region of the long arm of chromosome 7A.

Abstract

Powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), is an important foliar disease of wheat worldwide. In the Great Plains of the USA, Bgt isolates virulent to widely used powdery mildew resistance genes, such as Pm3a, were previously identified. The objectives of this study were to characterize the powdery mildew resistance gene in Afghanistan landrace PI 181356, which exhibited high resistance to Bgt isolates collected in southern Great Plains, and identify molecular markers for marker-assisted selection. An F₂ population and F_2:3 lines derived from a cross between PI 181356 and OK1059060-126135-3 were used in this study. Genetic analysis indicated that PI 181356 carries a single dominant gene, designated Pm59, in the terminal region of the long arm of chromosome 7A. Pm59 was mapped to an interval between sequence tag site (STS) markers Xmag1759 and Xmag1714 with genetic distances of 0.4 cM distal to Xmag1759 and 5.7 cM proximal to Xmag1714. Physical mapping suggested that Pm59 is in the distal bin 7AL 0.99–1.00. Pm59 is a novel powdery mildew resistance gene, and confers resistance to Bgt isolates collected from the Great Plains and the state of Montana. Therefore, Pm59 can be used to breed powdery mildew-resistant cultivars in these regions. Xmag1759 is ideal for marker-assisted selection of Pm59 in wheat breeding.

     

A <Emphasis Type="Italic">bean common mosaic virus</Emphasis> (BCMV)-resistance gene is fine-mapped to the same region as <Emphasis Type="Italic">Rsv1</Emphasis>-<Emphasis Type="Italic">h</Emphasis> in the soybean cultivar Suweon 97

Key message

In the soybean cultivar Suweon 97, BCMV-resistance gene was fine-mapped to a 58.1-kb region co-localizing with the Soybean mosaic virus (SMV)-resistance gene, Rsv1-h raising a possibility that the same gene is utilized against both viral pathogens.

Abstract

Certain soybean cultivars exhibit resistance against soybean mosaic virus (SMV) or bean common mosaic virus (BCMV). Although several SMV-resistance loci have been reported, the understanding of the mechanism underlying BCMV resistance in soybean is limited. Here, by crossing a resistant cultivar Suweon 97 with a susceptible cultivar Williams 82 and inoculating 220 F₂ individuals with a BCMV strain (HZZB011), we observed a 3:1 (resistant/susceptible) segregation ratio, suggesting that Suweon 97 possesses a single dominant resistance gene against BCMV. By performing bulked segregant analysis with 186 polymorphic simple sequence repeat (SSR) markers across the genome, the resistance gene was determined to be linked with marker BARSOYSSR_13_1109. Examining the genotypes of nearby SSR markers on all 220 F₂ individuals then narrowed down the gene between markers BARSOYSSR_13_1109 and BARSOYSSR_13_1122. Furthermore, 14 previously established F_2:3 lines showing crossovers between the two markers were assayed for their phenotypes upon BCMV inoculation. By developing six more SNP (single nucleotide polymorphism) markers, the resistance gene was finally delimited to a 58.1-kb interval flanked by BARSOYSSR_13_1114 and SNP-49. Five genes were annotated in this interval of the Williams 82 genome, including a characteristic coiled-coil nucleotide-binding site-leucine-rich repeat (CC-NBS-LRR, CNL)-type of resistance gene, Glyma13g184800. Coincidentally, the SMV-resistance allele Rsv1-h was previously mapped to almost the same region, thereby suggesting that soybean Suweon 97 likely relies on the same CNL-type R gene to resist both viral pathogens.

     

<Emphasis Type="Italic">NmEXT</Emphasis> Extensin Gene: a Positive Regulator of Resistance Response Against the Oomycete <Emphasis Type="Italic">Phytophthora nicotianae</Emphasis>

The oomycete pathogens produce important diseases in many plant species. To identify extensin genes expressed during the oomycete Phytophthora nicotianae-Nicotiana megalosiphon interaction, we used the SuperSAGE technology. Using this approach, we detected a N. megalosiphon extensin gene (NmEXT) triggered during the interaction. The extensin gene accumulation induced by the pathogen correlated with disease resistance in different Nicotiana species. Transient expression of NmEXT gene in susceptible Nicotiana tabacum enhanced the resistance to P. nicotianae. Our date indicated that NmEXT gene served a positive role in N. tabacum resistance against P. nicotianae.     

Molecular phylogenetics and anti-<Emphasis Type="Italic">Pythium</Emphasis> activity of endophytes from rhizomes of wild ginger congener, <Emphasis Type="Italic">Zingiber zerumbet</Emphasis> Smith

Zingiber zerumbet, a perennial rhizomatous herb exhibits remarkable disease resistance as well as a wide range of pharmacological activities. Towards characterizing the endophytic population of Z. zerumbet rhizomes, experiments were carried out during two different growing seasons viz., early-June of 2013 and late-July of 2014. A total of 34 endophytes were isolated and categorized into 11 morphologically distinct groups. Fungi were observed to predominate bacterial species with colonization frequency values ranging from 12.5 to 50 %. Among the 11 endophyte groups isolated, molecular analyses based on ITS/16S rRNA gene sequences identified seven isolate groups as Fusarium solani, two as F. oxysporum and one as the bacterium Rhizobium spp. Phylogenetic tree clustered the ITS sequences from Z. zerumbet endophytes into distinct clades consistent with morphological and sequence analysis. Dual culture assays were carried out to determine antagonistic activity of the isolated endophytes against Pythium myriotylum, an economically significant soil-borne phytopathogen of cultivated ginger. Experiments revealed significant P. myriotylum growth inhibition by F. solani and F. oxysporum isolates with percentage of inhibition (PoI) ranging from 45.17 ± 0.29 to 62.2 ± 2.58 with F. oxysporum exhibiting higher PoI values against P. myriotylum. Using ZzEF8 metabolite extract, concentration-dependent P. myriotylum hyphal growth inhibition was observed following radial diffusion assays. These observations were confirmed by scanning electron microscopy analysis wherein exposure to ZzEF8 metabolite extract induced hyphal deformities. Results indicate Z. zerumbet endophytes as promising resources for biologically active compounds and as biocontrol agents for soft rot disease management caused by Pythium spp.     

Mapping novel aphid resistance QTL from wild soybean, <Emphasis Type="Italic">Glycine soja</Emphasis> 85-32

Key message

Two novel QTLs conferring aphid resistance were mapped and validated on soybean chromosomes 8 and 16, respectively. Closely linked markers were developed to assist breeding for aphid resistance.

Abstract

Soybean aphid, Aphis glycines Matsumura, is a highly destructive pest for soybean production. E08934, a soybean advanced breeding line derived from the wild soybean Glycine soja 85-32, has shown strong resistance to aphids. To dissect the genetic basis of aphid resistance in E08934, a mapping population (070020) consisting of 140 F₃-derived lines was developed by crossing E08934 with an aphid-susceptible line E00003. This mapping population was evaluated for aphid resistance in a greenhouse trial in 2010 and three field trials in 2009, 2010, and 2011, respectively. The broad-sense heritability across the field trials was 0.84. In the mapping population 070020, two major quantitative trait loci (QTL) were detected as significantly associated with aphid resistance, and designated as Rag6 and Rag3c, respectively. Rag6 was mapped to a 10.5 centiMorgan (cM) interval between markers MSUSNP08-2 and Satt209 on chromosome 8, explaining 19.5–46.4% of the phenotypic variance in different trials. Rag3c was located at a 7.5 cM interval between markers MSUSNP16-10 and Sat_370 on chromosome 16, explaining 12.5–22.9% of the phenotypic variance in different trials. Rag3c had less resistance effect than Rag6 across all the trials. Furthermore, Rag6 and Rag3c were confirmed in two validation populations with different genetic backgrounds. No significant interaction was detected between Rag6 and Rag3c in either the mapping population or the validation populations. Both Rag6 and Rag3c were indicated as conferring antibiosis resistance to aphids by a no-choice test. The new aphid-resistance gene(s) derived from the wild germplasm G. soja 85-32 are valuable in improving soybeans for aphid resistance.

     

Molecular evidence for hybridization between invasive <Emphasis Type="Italic">Solidago canadensis</Emphasis> and native <Emphasis Type="Italic">S</Emphasis>. <Emphasis Type="Italic">virgaurea</Emphasis>

Hybridization between alien and native species is biologically very important and could lead to genetic erosion of native taxa. Solidago × niederederi was discovered over a century ago in Austria and described by Khek as a natural hybrid between the alien (nowadays regarded also as invasive) S. canadensis and native S. virgaurea. Although interspecific hybridization in the genus Solidago is considered to be relatively common, hybrid nature of S. × niederederi has not been independently proven using molecular tools, to date. Because proper identification of the parentage for the hybrid Solidago individuals solely based on morphological features can be misleading, in this paper we report an additive polymorphism pattern expressed in the ITS sequences obtained from individuals representing S. × niederederi, and confirm the previous hypothesis that the parental species of this hybrid are S. canadensis and S. virgaurea. Additionally, based on variability at the cpDNA rpl32-trnL locus, we showed that in natural populations hybridization occurs in both directions.     

<Emphasis Type="Italic">Aeromonas</Emphasis> and <Emphasis Type="Italic">Plesiomonas</Emphasis> species from scarlet ibis (<Emphasis Type="Italic">Eudocimus ruber</Emphasis>) and their environment: monitoring antimicrobial susceptibility and virulence

The present study aimed at evaluating the role of captive scarlet ibises (Eudocimus ruber) and their environment as reservoirs of Aeromonas spp. and Plesiomonas spp., and analyzing the in vitro antimicrobial susceptibility and virulence of the recovered bacterial isolates. Thus, non-lactose and weak-lactose fermenting, oxidase positive Gram-negative bacilli were recovered from cloacal samples (n = 30) of scarlet ibises kept in a conservational facility and from water samples (n = 30) from their environment. Then, the antimicrobial susceptibility, hemolytic activity and biofilm production of the recovered Aeromonas spp. and Plesiomonas shigelloides strains were assessed. In addition, the virulence-associated genes of Aeromonas spp. were detected. Ten Aeromonas veronii bv. sobria, 2 Aeromonas hydrophila complex and 10 P. shigelloides were recovered. Intermediate susceptibility to piperacillin-tazobactam and cefepime was observed in 2 Aeromonas spp. and 1 P. shigelloides, respectively, and resistance to gentamicin was observed in 4 P. shigelloides. The automated susceptibility analysis revealed resistance to piperacillin-tazobactam and meropenem among Aeromonas spp. and intermediate susceptibility to gentamicin among P. shigelloides. All Aeromonas isolates presented hemolytic activity, while 3 P. shigelloides were non-hemolytic. All Aeromonas spp. and 3/10 P. shigelloides were biofilm-producers, at 28 °C, while 10 Aeromonas spp. and 6/10 P. shigelloides produced biofilms, at 37 °C. The most prevalent virulence genes of Aeromonas spp. were asa1 and ascV. Scarlet ibises and their environment harbour potentially pathogenic bacteria, thus requiring monitoring and measures to prevent contamination of humans and other animals.     

A major QTL (<Emphasis Type="Italic">qFT12.1</Emphasis>) allele from wild soybean delays flowering time

Soybean is highly sensitive to photoperiod. To improve the adaptability and productivity of soybean, it is essential to understand the molecular mechanisms regulating flowering time. To identify new flowering time QTLs, we evaluated a BC₃F₅ population consisting of 120 chromosome segment substitution lines (CSSLs) over 2 years under field conditions. CSSLs were derived from a cross between the cultivated soybean cultivar Jackson and the wild soybean accession JWS156-1, followed by continuous backcrossing using Jackson as the recurrent parent. Four QTLs (qFT07.1, qFT12.1, qFT12.2, and qFT19.1) were detected on three chromosomes. Of these, qFT12.1 showed the highest effect, accounting for 36.37–38.27% of the total phenotypic variation over 2 years. This QTL was further confirmed in the F₇ recombinant inbred line population (n?=?94) derived from the same cross (Jackson × JWS156-1). Analysis of the qFT12.1 BC₃F₅ residual heterozygous line RHL509 validated the allele effect of qFT12.1 and revealed that the recessive allele of qFT12.1 resulted in delayed flowering. Evaluating the qFT12.1 near-isogenic lines (NILs) under different growth conditions showed that NILs with the wild soybean genotype always showed later flowering than those with the cultivated soybean genotype. qFT12.1 was delimited to a 2703-kb interval between the markers BARCSOYSSR_12_0220 and BARCSOYSSR_12_0368 on chromosome 12. qFT12.1 may be a new flowering time gene locus in soybean.     

Discovery and introgression of the wild sunflower-derived novel downy mildew resistance gene <Emphasis Type="Italic">Pl</Emphasis><Subscript><Emphasis Type="Italic">19</Emphasis></Subscript> in confection sunflower (<Emphasis Type="Italic">Helianthus annuus</Emphasis> L.)

Key message

A new downy mildew resistance gene, Pl ₁₉ , was identified from wild Helianthus annuus accession PI 435414, introduced to confection sunflower, and genetically mapped to linkage group 4 of the sunflower genome.

Abstract

Wild Helianthus annuus accession PI 435414 exhibited resistance to downy mildew, which is one of the most destructive diseases to sunflower production globally. Evaluation of the 140 BC₁F_2:3 families derived from the cross of CMS CONFSCLB1 and PI 435414 against Plasmopara halstedii race 734 revealed that a single dominant gene controls downy mildew resistance in the population. Bulked segregant analysis conducted in the BC₁F₂ population with 860 simple sequence repeat (SSR) markers indicated that the resistance derived from wild H. annuus was associated with SSR markers located on linkage group (LG) 4 of the sunflower genome. To map and tag this resistance locus, designated Pl ₁₉ , 140 BC₁F₂ individuals were used to construct a linkage map of the gene region. Two SSR markers, ORS963 and HT298, were linked to Pl ₁₉ within a distance of 4.7 cM. After screening 27 additional single nucleotide polymorphism (SNP) markers previously mapped to this region, two flanking SNP markers, NSA_003564 and NSA_006089, were identified as surrounding the Pl ₁₉ gene at a distance of 0.6 cM from each side. Genetic analysis indicated that Pl ₁₉ is different from Pl ₁₇ , which had previously been mapped to LG4, but is closely linked to Pl ₁₇ . This new gene is highly effective against the most predominant and virulent races of P. halstedii currently identified in North America and is the first downy mildew resistance gene that has been transferred to confection sunflower. The selected resistant germplasm derived from homozygous BC₂F₃ progeny provides a novel gene for use in confection sunflower breeding programs.

     

Genetic mapping and development of co-segregating markers of <Emphasis Type="Italic">RpsQ</Emphasis>, which provides resistance to <Emphasis Type="Italic">Phytophthora sojae</Emphasis> in soybean

Key message

The RpsQ Phytophthora resistance locus was finely mapped to a 118-kb region on soybean chromosome 3. A best candidate gene was predicted and three co-segregating gene markers were developed.

Abstract

Phytophthora root rot (PRR), caused by Phytophthora sojae, is a major threat to sustainable soybean production. The use of genetically resistant cultivars is considered the most effective way to control this disease. The Chinese soybean cultivar Qichadou 1 exhibited a broad spectrum resistance, with a distinct resistance phenotype, following inoculation with 36 Chinese P. sojae isolates. Genetic analyses indicated that the disease resistance in Qichadou 1 is controlled by a single dominant gene. This gene locus was designated as RpsQ and mapped to a 118-kb region between BARCSOYSSR_03_0165 and InDel281 on soybean chromosome 3, and co-segregated with Insert11, Insert144 and SNP276. Within this region, there was only one gene Glyma.03g27200 encoding a protein with a typical serine/threonine protein kinase structure, and the expression pattern analysis showed that this gene induced by P. sojae infection, which was suggested as a best candidate gene of RpsQ. Candidate gene specific marker Insert144 was used to distinguish RpsQ from the other known Rps genes on chromosome 3. Identical polymerase chain reaction amplification products were produced for cultivars Qichadou 1 (RpsQ) and Ludou 4 (Rps9). All other cultivars carrying Rps genes on chromosome 3 produced different PCR products, which all lacked a 144-bp fragment present in Qichadou 1 and Ludou 4. The phenotypes of the analyzed cultivars combined with the physical position of the PRR resistance locus, candidate gene analyses, and the candidate gene marker test revealed RpsQ and Rps9 are likely the same gene, and confer resistance to P. sojae.

     

Functional characterization of the powdery mildew susceptibility gene <Emphasis Type="Italic">SmMLO1</Emphasis> in eggplant (<Emphasis Type="Italic">Solanum melongena</Emphasis> L.)

Eggplant (Solanum melongena L.) is one of the most important vegetables among the Solanaceae and can be a host to fungal species causing powdery mildew (PM) disease. Specific homologs of the plant Mildew Locus O (MLO) gene family are PM susceptibility factors, as their loss of function results in a recessive form of resistance known as mlo resistance. In a previous work, we isolated the eggplant MLO homolog SmMLO1. SmMLO1 is closely related to MLO susceptibility genes characterized in other plant species. However, it displays a peculiar non-synonymous substitution that leads to a T → M amino acid change at protein position 422, in correspondence of the MLO calmodulin-binding domain. In this study, we performed the functional characterization of SmMLO1. Transgenic overexpression of SmMLO1 in a tomato mlo mutant compromised resistance to the tomato PM pathogen Oidium neolycopersici, thus indicating that SmMLO1 is a PM susceptibility factor in eggplant. PM susceptibility was also restored by the transgenic expression of a synthetic gene, named s-SmMLO1, encoding a protein identical to SmMLO1, except for the presence of T at position 422. This indicates that the T → M polymorphism does not affect the protein role as PM susceptibility factor. Overall, the results of this work are of interest for the functional characterization of MLO proteins and the introduction of PM resistance in eggplant using reverse genetics.     

Fine mapping of the soybean aphid-resistance genes <Emphasis Type="Italic">Rag6</Emphasis> and <Emphasis Type="Italic">Rag3c</Emphasis> from <Emphasis Type="Italic">Glycine soja</Emphasis> 85-32

Key message

Rag6 and Rag3c were delimited to a 49-kb interval on chromosome 8 and a 150-kb interval on chromosome 16, respectively. Structural variants in the exons of candidate genes were identified.

Abstract

The soybean aphid, an invasive species, has significantly threatened soybean production in North America since 2000. Host-plant resistance is known as an ideal management strategy for aphids. Two novel aphid-resistance loci, Rag6 and Rag3c, from Glycine soja 85-32, were previously detected in a 10.5-cM interval on chromosome 8 and a 7.5-cM interval on chromosome 16, respectively. Defining the exact genomic position of these two genes is critical for improving the effectiveness of marker-assisted selection for aphid resistance and for identification of the functional genes. To pinpoint the locations of Rag6 and Rag3c, four populations segregating for Rag6 and Rag3c were used to fine map these two genes. The availability of the Illumina Infinium SoySNP50K/8K iSelect BeadChip, combined with single-nucleotide polymorphism (SNP) markers discovered through the whole-genome re-sequencing of E12901, facilitated the fine mapping process. Rag6 was refined to a 49-kb interval on chromosome 8 with four candidate genes, including three clustered nucleotide-binding site leucine-rich repeat (NBS–LRR) genes and an amine oxidase encoding gene. Rag3c was refined to a 150-kb interval on chromosome 16 with 11 candidate genes, two of which are a LRR gene and a lipase gene. Moreover, by sequencing the whole-genome exome-capture of the resistant source (E12901), structural variants were identified in the exons of the candidate genes of Rag6 and Rag3c. The closely linked SNP markers and the candidate gene information presented in this study will be significant resources for integrating Rag6 and Rag3c into elite cultivars and for future functional genetics studies.

     

<Emphasis Type="Italic">qRfg3</Emphasis>, a novel quantitative resistance locus against <Emphasis Type="Italic">Gibberella</Emphasis> stalk rot in maize

Key message

A quantitative trait locus  qRfg3 imparts recessive resistance to maize Gibberella stalk rot. qRfg3 has been mapped into a 350-kb interval and could reduce the disease severity index by ~26.6%.

Abstract

Gibberella stalk rot, caused by the fungal pathogen Fusarium graminearum, severely affects maize yield and grain quality worldwide. To identify more resistance quantitative trait loci (QTLs) against this disease, we analyzed a recombinant inbred line (RIL) population derived from a cross between resistant H127R and susceptible C7-2 inbred lines. Within this population, maize resistance to Gibberella stalk rot had high broad-sense heritability. A major QTL, qRfg3, on chromosome 3 was consistently detected across three field trials, accounting for 10.7–19.4% of the total phenotypic variation. Using a progeny-based sequential fine-mapping strategy, we narrowed qRfg3 down to an interval of ~350 kb. We further demonstrated that qRfg3 is a recessive resistance locus to Gibberella stalk rot that reduced the disease severity index by ~26.6%. Both the gene location and recessive genetic mode distinguish qRfg3 from other stalk rot resistance loci. Hence, qRfg3 is valuable as a complement to existing resistance QTLs to improve maize resistance to Gibberella stalk rot.

     

First study on characterization of virulence and antibiotic resistance genes in verotoxigenic and enterotoxigenic <Emphasis Type="Italic">E</Emphasis>. <Emphasis Type="Italic">coli</Emphasis> isolated from raw milk and unpasteurized traditional cheeses in Romania

The study focused on the incidence of enterotoxigenic Escherichia coli (ETEC) and verotoxigenic E. coli (VTEC) in raw milk and traditional dairy cheeses marketed in Romania, characterizing the virulence and antibiotic resistance genes of these isolates. One hundred and twenty samples of raw milk and 80 samples of unpasteurized telemy cheese were collected and cultured according to the international standard protocol. All the characteristic E. coli cultures were analyzed for the presence of STa, STb, LT, stx1, and stx2 toxicity genes. The ETEC/VTEC strains were tested for the presence of antibiotic resistance genes, such as aadA1, tetA, tetB, tetC, tetG, dfrA1, qnrA, aaC, sul1, bla _SHV , bla _CMY , bla _TEM , and ere(A), using PCR. The results showed that 27 samples (18.62%) were positive for one of the virulence genes investigated. 48.1% (n = 13) tested positive at the genes encoding for tetracycline resistance, tetA being the most prevalent one (61.5%; n = 8). A high percent (33.3%; n = 9) revealed the beta-lactamase (bla _TEM ) resistance gene, and none of the samples tested positive for bla _CMY and bla _SHV genes. The genes responsible for resistance to sulfonamides (sul1) and trimethoprim (dfrA1) were detected in rates of 14.8% (n = 4) and 7.4% (n = 2), respectively. E. coli is highly prevalent in raw milk and unpasteurized cheeses marketed in Romania. These strains might represent an important reservoir of resistance genes which can easily spread into other European countries, given the unique market.     

Molecular mapping of a rust resistance gene <Emphasis Type="Italic">R</Emphasis><Subscript><Emphasis Type="Italic">14</Emphasis></Subscript> in cultivated sunflower line PH 3

Sunflower, the fifth largest oilseed crop in the world, plays an important role in human diets. Recently, sunflower production in North America has suffered serious yield losses from newly evolved races of sunflower rust (Puccinia helianthi Schwein.). The rust resistance gene, designated R ₁₄ , in a germplasm line PH 3 originated from a wild Helianthus annuus L. population resistant to 11 rust races. PH 3 has seedling with an extraordinary purple hypocotyl color. The objectives of this study were to map both the R ₁₄ rust resistance gene and the purple hypocotyl gene-designated PHC in PH 3, and to identify molecular markers for marker-assisted breeding for sunflower rust resistance. A set of 517 mapped SSR/InDel and four SNP markers was used to detect polymorphisms between the parents. Fourteen markers covering a genetic distance of 17.0 cM on linkage group (LG) 11 were linked to R ₁₄ . R ₁₄ was mapped to the middle of the LG, with a dominant SNP marker NSA_000064 as the closest marker at a distance of 0.7 cM, and another codominant marker ORS542 linked at 3.5 cM proximally. One dominant marker ZVG53 was linked on the distal side at 6.9 cM. The PHC gene was also linked to R ₁₄ with a distance of 6.2 cM. Chi-squared analysis of the segregation ratios of R ₁₄ , PHC, and ten linked markers indicated a deviation from an expected 1:2:1 or 3:1 ratio. The closely linked molecular or morphological markers could facilitate sunflower rust-resistant breeding and accelerate the development of rust-resistant hybrids.     

QTL-seq and marker development for resistance to <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">niveum</Emphasis> race 1 in cultivated watermelon

Fusarium wilt, caused by the fungus Fusarium oxysporum f. sp. niveum (Fon), is one of the predominant diseases of watermelon. Resistance to Fon race 1 is conferred by a single major quantitative trait locus (QTL), Fo-1.1, but resolution of this region has been poor due to low marker density. In this study, a combination of whole genome resequencing of bulked segregants (QTL-seq analysis) followed by QTL mapping with kompetitive allele specific PCR (KASP) markers developed across Fo-1.1 successfully increased the resolution from 2.03 to 1.56 Mb and 315 kb, respectively. The linkage of the KASP markers to Fon race 1 resistance across a wide range of watermelon germplasm was validated in a set of elite watermelon cultivars. The linked markers described here provide a breeder-friendly toolkit immediately available for high-throughput genotyping in large-scale breeding programs for fine mapping and incorporation of Fon race 1 resistance in watermelon.