Vf Gene Research Articles

Corrected and republished from: "Vibrio fischeri Possesses Xds and Dns Nucleases That Differentially Influence Phosphate Scavenging, Aggregation, Competence, and Symbiotic Colonization of Squid".

Cells of Vibrio fischeri colonize the light organ of Euprymna scolopes, providing the squid bioluminescence in exchange for nutrients and protection. The bacteria encounter DNA-rich mucus throughout their transition to a symbiotic lifestyle, leading us to hypothesize a role for nuclease activity in the colonization process. In support of this, we detected abundant extracellular nuclease activity in growing cells of V. fischeri. To discover the gene(s) responsible for this activity, we screened a V. fischeri transposon mutant library for nuclease-deficient strains. Interestingly, only one strain, whose transposon insertion mapped to nuclease gene VF_1451, showed a complete loss of nuclease activity in our screens. A database search revealed that VF_1451 is homologous to the nuclease-encoding gene xds in Vibrio cholerae. However, V. fischeri strains lacking xds eventually revealed slight nuclease activity on plates upon prolonged incubation. This led us to hypothesize that a second secreted nuclease, identified through a database search as VF_0437, a homolog of V. cholerae dns, might be responsible for the residual nuclease activity. Here, we show that Xds and/or Dns are involved in essential aspects of V. fischeri biology, including natural transformation, aggregation, and phosphate scavenging. Furthermore, strains lacking either nuclease were outcompeted by the wild type for squid colonization. Understanding the specific role of nuclease activity in the squid colonization process represents an intriguing area of future research.IMPORTANCEFrom soil and water to host-associated secretions such as mucus, environments that bacteria inhabit are awash in DNA. Extracellular DNA (eDNA) is a nutritious resource that microbes dedicate significant energy to exploit. Calcium binds eDNA to promote cell-cell aggregation and horizontal gene transfer. eDNA hydrolysis impacts the construction of and dispersal from biofilms. Strategies in which pathogens use nucleases to avoid phagocytosis or disseminate by degrading host secretions are well-documented; significantly less is known about nucleases in mutualistic associations. This study describes the role of nucleases in the mutualism between Vibrio fischeri and its squid host Euprymna scolopes. We find that nuclease activity is an important determinant of colonization in V. fischeri, broadening our understanding of how microbes establish and maintain beneficial associations.

Scab Resistance in some Apples Genotypes from the UASVM Collection Cluj-Napoca

Apple is one of the most consumed fruits in the world and as well one of the most important crops in temperate areas. Apple scab, caused by Venturia inaequalis is damaging diseases that affects apple species and causes up to 70% yield losses depending of cultivar’s resistance. In order to control this disease, costly chemical pesticides are necessary, which increase the pollution of environment by their toxicity. This study is focused on checking the behaviour of certain apple varieties from the pomological collection at UASVM Cluj-Napoca against apple scab infection. Nine apple cultivars ('Fuji Kiku', 'Red Cap', 'Lena', 'Katja', 'Gala Mitchgla', 'Fiesta', 'Yellow', 'Pinova' and 'Reinette du Canada'), were analysed in terms of behaviour to scab attack on leaves and fruits. Visual observations were completed by molecular analyses for the potential presence of the Vf gene in some cases. Combining results from visual observation and molecular assays, the 'Lena' and 'Pinova' cultivars were noted as resistant to scab in our field trial conditions. This indicates a good opportunity to bring them into apple breeding programs for resistance to apple scab.

An apparent breakdown of Vf resistance occurring in an apple orchard in Bistrita area


 Scab resistance through the Rvi6 gene was largely used in Romanian breeding programs including those from Fruit Research & Development Station (FRDS) Bistrita. The aim of the study was to verify the efficacy of the resistance due to the Vf gene under the current conditions in the Bistrita area. Three scab resistant apple cultivars bred at FRDS Bistrita were monitored in the field, in comparison with the Rvi6 ‘Florina’ and ‘Prima’ cvs., and the sensitive ‘Idared’ and ‘Golden Delicious’ cvs. Molecular markers (AM-19, U1₄₀₀ and AL07) were used in PCR to check the presence of the Rvi6 gene in the genome of the three Romanian varieties. During the years 2019-2021, scab lesions appeared on all the resistant cultivars studied. PCR results showed the presence of Rvi6 gene on ‘Bistritean’, ‘Salva’, ‘Aura’, ‘Prima’ and ‘Florina’ genoms. The results suggest a possible breakdown of the resistance given by the Rvi6 gene in the current ecological conditions in this area. Scab symptoms occurring on cultivars that have the Rvi6 gene could lead to the hypothesis that different scab races are now present in our area or that some genetic variations occurred on the existing races so that they overcome Rvi6 gene.

Molecular Screening of Some Apple Progenies for Detection Vf Gene Using Marker Assisted Selection


 Apple scab, caused by Venturia inaequalis is one of the most damaging pathogens that affects apple species. Cross combinations were made between Auriu de Bistrița cv. (female genitor) - a valuable local variety in terms of fruit quality but only tolerant to scab, and Florina cv. (male genitor) used as a donor of Vf resistance gene. It was first detected in Malus floribunda Clone 821, which was later transferred to commercial varieties by different breeding programs. To confirm the presence of Vf gene, progenies resulting from the mentioned combination were tested with MAS (Marker Assited Selection), using two dominant primers pairs (AM19, U1400), and another one codominant (AL07) used to distinguish homozygous and heterozygous genotypes. After the crossing combination, a number of twenty-six hybrids were obtained, of which 50% (13 hybrids) were clasified as resistant (heterozygous), respectively 50% (13 hybrids) as susceptible (recessive homozygotes), so the Mendelian ratio was confirmed.

Possible step-up in prevalence for Escherichia coli ST131 from fecal to clinical isolates: inferred virulence potential comparative studies within phylogenetic group B2

BackgroundEscherichia coli sequence type (ST)131 is an important urinary tract pathogen, and is responsible for considerable healthcare-associated problems and costs worldwide. A better understanding of the factors that contribute to its rapid worldwide spread may help in arresting its continual spread. We studied a large collection of fecal and urinary E. coli ST131 and E. coli non-ST131 phylogenetic group B2 isolates, from women, men and children, in regional NSW, Australia.ResultsWe found out that there was a step up in ST131 prevalence (and possibly in virulence) from fecal to clinical (urinary) isolates in general, and specifically among ciprofloxacin resistant isolates, in the 3 host groups. Furthermore, our results revealed that the inferred virulence potential of the ST131 isolates (as measured by VF gene scores) was much higher than that of non-ST131 phylogenetic group B2 isolates, and this was much more pronounced amongst the urinary isolates. This finding suggests presence of possible E. coli phylogenetic B2 subgroups with varying levels of virulence, with ST131 being much more virulent compared to others. A strong association between ST131 and fluoroquinolone (FQ) resistance was also demonstrated, suggesting that FQ use is related to ST131 emergence and spread. Specifically, about 77% of ST131 isolates from women and men, and 47% from children, were extended spectrum β- lactamase (ESBL) producers. Moreover, FQ resistant ST131 ESBL isolates on average harbored more VF genes than all other isolates.ConclusionsThe strong association between ST131 prevalence and FQ resistance amongst the studied isolates suggests that FQ use is related to ST131 emergence and spread. Furthermore, our results demonstrate that FQ resistance and a plurality of VF genes can exist together in ST131, something that has traditionally been regarded as being inversely related. This may partly contribute to the emergence and worldwide spread of ST131.

Quorum Sensing and Cyclic di-GMP Exert Control Over Motility of Vibrio fischeri KB2B1.

Bacterial motility is critical for symbiotic colonization by Vibrio fischeri of its host, the squid Euprymna scolopes, facilitating movement from surface biofilms to spaces deep inside the symbiotic organ. While colonization has been studied traditionally using strain ES114, others, including KB2B1, can outcompete ES114 for colonization for a variety of reasons, including superior biofilm formation. We report here that KB2B1 also exhibits an unusual pattern of migration through a soft agar medium: whereas ES114 migrates rapidly and steadily, KB2B1 migrates slowly and then ceases migration. To better understand this phenomenon, we isolated and sequenced five motile KB2B1 suppressor mutants. One harbored a mutation in the gene for the cAMP receptor protein (crp); because this strain also exhibited a growth defect, it was not characterized further. Two other suppressors contained mutations in the quorum sensing pathway that controls bacterial bioluminescence in response to cell density, and two had mutations in the diguanylate cyclase (DGC) gene VF_1200. Subsequent analysis indicated that (1) the quorum sensing mutations shifted KB2B1 to a perceived low cell density state and (2) the high cell density state inhibited migration via the downstream regulator LitR. Similar to the initial point mutations, deletion of the VF_1200 DGC gene increased migration. Consistent with the possibility that production of the second messenger c-di-GMP inhibited the motility of KB2B1, reporter-based measurements of c-di-GMP revealed that KB2B1 produced higher levels of c-di-GMP than ES114, and overproduction of a c-di-GMP phosphodiesterase promoted migration of KB2B1. Finally, we assessed the role of viscosity in controlling the quorum sensing pathway using polyvinylpyrrolidone and found that viscosity increased light production of KB2B1 but not ES114. Together, our data indicate that while the two strains share regulators in common, they differ in the specifics of the regulatory control over downstream phenotypes such as motility.

Pyramiding resistance genes and widening the genetic base of the apple (Malus × domestica Borkh.) crop

Apple breeding is active worldwide and yet the apple crop is in a precarious state as it relies on few dominant cultivars and only the Rvi6 (formerly Vf) gene, that confers resistance to scab, has been extensively exploited in the cultivars entered the market in recent years. However, there are some 20 disease resistance genes described in apple and the apple germplasm includes thousands of accessions in the repositories. In this paper, a breeding programme is described, whereby 36 genotypes, including ancient and contemporary apple cultivars, were crossed to produce a new set of selections that combine extensive genetic resources with pyramided resistance genes to several apple diseases, such as scab and powdery mildew. The 110 cross combinations carried out successfully, of the 260 initially planned, produced 7,876 offsprings, reduced to 2,969 after screening with molecular markers associated with five resistance genes. Selections with three or two resistance genes and good agronomic characteristics were kept for further field observations with the aims of creating new cultivars for the market and new parents for future breeding projects

MOLECULAR SCREENING OF SOME ROMANIAN APPLE CULTIVARS FOR SCAB RESISTANCE GENES

Apple scab, incided by the fungus Venturia inaequalis (Cke.) Wint., is a devastating disease of apple reported from almost all apple producing Romanian areas, which causes up to 70% losses of production. Molecular markers were used for detection of scab resistance genes in 22 old and introduced apple cultivars ('Romus 3', 'Romus 5', 'Rebra', 'Rustic', 'Nicol', 'Colmar', 'Colonade', registered by Research Institute for Fruit Growing Pitesti; 'Generos', 'Iris', 'Irisem', 'Luca', 'Ciprian', 'Cezar', 'Remar', 'Valery', 'Real', registered by Research Station for Fruit Growing Voinesti, Dambovita; 'Aura', 'Starkprim', 'Ionaprim', 'Bistritean', registered by Research Station for Fruit Growing Bistrita and old cultivars: 'Domnesc', 'Cretesc'). The presence of scab resistance genes were detected using the molecular markers: AL-07 (SCAR), AM19 (SCAR), VfC for Rvi6 (Vf) gene, AD13 (SCAR) for Rvi4 (Vr1) gene, OPL19 (SCAR) for Rvi2 (Vh2) and Rvi8 (Vh8) genes and OPB12 (STS) for Rvi5 (Vm) gene. The Rvi6 gene was detected in 17 cultivars from different breeding center. The marker AD13 presents in genome of 8 cultivars, such as 'Romus 3', 'Romus 5', 'Generos', 'Iris', 'Irisem', 'Cezar', 'Remar', 'Aura'. The Rvi5 gene was revealed in 3 cultivars ('Nicol', 'Generos', 'Irisem'), only.

Field apple scab susceptibility of a diverse Malus germplasm collection identifies potential sources of resistance for apple breeding

BackgroundBreeding for resistance to apple scab (caused by Venturia inaequalis), the most devastating fungal disease of apples, relies on genetic resources maintained in germplasm collections.MethodsTo identify new sources of scab resistance, we evaluated 177 Malus accessions, including 27 primary and 13 hybrid Malus species from diverse geographical origins, in an orchard at Geneva, New York. We also screened a differential host set for 2 years to monitor for changes in the effectiveness of ten known scab resistance genes, which allowed us to confirm the presence of virulent pathogen races in the orchard.ResultsWe found that ~ 37% of the wild Malus accessions and domesticated cultivars were resistant to apple scab in the field. Several of these accessions were unrelated to sources of previously known resistance genes and are promising for apple scab genetic research and resistance breeding. Cultivars carrying the Rvi6 (Vf) gene from Malus floribunda clone 821, e.g. ‘Liberty’ or ‘Florina’, remained resistant despite the breakdown of Rvi6. ‘Demir’, a Malus hybrid from Turkey, and ‘Chisel Jersey’, a traditional English hard cider cultivar, showed fewer symptoms than the Rvi6 resistant cultivar ‘Prima’. Races 1 to 7 and 9 of V. inaequalis were present in the orchard, but no scab was observed on the indicator host accessions for races 11 and 12.ConclusionsDetailed and systematic screening of Malus germplasm identified resistant and moderately resistant donor accessions based on resistance reaction types. These accessions are promising for use in future genetic studies to identify novel sources of scab resistance alleles for apple breeding to develop cultivars with durable apple scab resistance.

Genomic Analysis of Bovine Staphylococcus aureus Isolates from Milk To Elucidate Diversity and Determine the Distributions of Antimicrobial and Virulence Genes and Their Association with Mastitis.

Staphylococcus aureus causes persistent clinical and subclinical bovine intramammary infections (IMI) worldwide. However, there is a lack of comprehensive information regarding genetic diversity, the presence of antimicrobial resistance (AMR), and virulence genes for S. aureus in bovine milk in Canada. Here, we performed whole-genome sequencing (WGS) of 119 Canadian bovine milk S. aureus isolates and determined they belonged to 8 sequence types (ST151, ST352, ST351, ST2187, ST2270, ST126, ST133, and ST8), 5 clonal complexes (CC151, CC97, CC126, CC133, and CC8), and 18 distinct Spa types. Pan-, core, and accessory genomes were composed of 6,340, 1,279, and 2,431 genes, respectively. Based on phenotypic screening for AMR, resistance was common against beta-lactams (19% of isolates) and sulfonamides (7% of isolates), whereas resistance against pirlimycin, tetracycline, ceftiofur, and erythromycin and to the combination of penicillin and novobiocin was uncommon (3, 3, 3, 2, and 2% of all isolates, respectively). We also determined distributions of 191 virulence factors (VFs) in 119 S. aureus isolates after classifying them into 5 functional categories (adherence [n = 28], exoenzymes [n = 21], immune evasion [n = 20], iron metabolism [n = 29], and toxins [n = 93]). Additionally, we calculated the pathogenic potential of distinct CCs and STs and determined that CC151 (ST151 and ST351) had the highest pathogenic potential (calculated by subtracting core-VFs from total VFs), followed by CC97 (ST352 and ST2187) and CC126 (ST126 and ST2270), potentially linked to their higher prevalence in bovine IMI worldwide. However, there was no statistically significant link between the presence of VF genes and mastitis.IMPORTANCE Staphylococcus aureus is a major cause of bovine intramammary infections, leading to significant economic losses to dairy industry in Canada and worldwide. There is a lack of knowledge regarding genetic diversity, the presence of antimicrobial resistance (AMR), and virulence genes for S. aureus isolated from bovine milk in Canada. Based on whole-genome sequencing and genomic analysis, we have determined the phylogeny and diversity of S. aureus in bovine milk and concluded that it had a large accessory genome, limited distribution of AMR genes, variable VF gene profiles and sequence types (ST), and clonal complex (CC)-specific pathogenic potentials. Comprehensive information on the population structure, as well as the virulence and resistance characteristics of S. aureus from bovine milk, will allow for source attribution, risk assessment, and improved therapeutic approaches in cattle.

Mining new scab resistance alleles in apple (Malus × domestica Borkh.) germplasm of Kashmir: Towards breeding scab free apple cultivars

Apple an important fruit crop in Kashmir ranks first in all the fruits grown here. It is commercially the most important horticultural crop grown in temperate parts of the world. To control scab in Apple a lot of fungicides are being used however, an alternative approach to fungicide application is the use of scab resistant cultivars as a genetic stock. Therefore, there is an urgent need to identify more cultivars resistant to scab with maximum genes and can be used for apple breeding. In view of this, one hundred apple cultivars grown in Kashmir Valley were screened for the presence of Vf gene conferring resistance against apple scab. Eight trait linked markers were used so for and among them three markers like AL07, AM19, and CH05e03 proved to be useful to distinguish the resistant cultivars from the other apple germplasm. These three markers have amplified resistant fragments in five cultivars. Thus out of the total of 100 cultivars investigated in the present study, only five cultivars namely Shalimar I, Shalimar II, Dubhour, Gold Spur, and Crab apple were found to harbour resistant alleles which may be resistant to some of the races of V. inaequalis. Among the five cultivars harbouring the scab resistant alleles for some races of V. inaequalis, three cultivars namely, Shalimar I, Shalimar II, and Dubhour, which contain the maximum number of scab resistance alleles can be used as parents in further breeding programms to develop more resistant apple cultivars with high market acceptance.

Interdisciplinary teams in apple breeding and co-authors of varieties

The article presents the results of 64 years apple breeding work at the Russian Research Institute of Fruit Crop Breeding (RRIFCB) and shows the high role of the interdisciplinary team. It is noted that at the RRIFCB, leading institute for fruit crop breeding in Russia, over the past period 54 apple varieties of different ripening periods have been created and included in the State Register of breeding achievements admitted for use. Among them 19 are first in Russia and in the world triploid varieties derived from intervalent crossings of type 2 × 4; 24 immune to scab (with the Vf gene), including the first immune to scab domestic variety Imrus (immune Russian) and 5 columnar scab immune varieties. Triploid apple varieties are characterized by more regular fruiting by years, high fruit marketability and partial self-fertility. Scab immune apple varieties when introduced into orchards contribute to a more favourable environmental situation in the orchards and their surroundings and reduce the cost of additional chemical treatments. Columnar varieties make it possible to create super-intensive orchards with an early abundant yield and to simplify and reduce labor-intensive costs for the formation and care of trees. The creation of all these types of varieties was possible thanks to a large interdisciplinary team, which in addition to the main breeder includes specialists of many other specialties: variety specialists, phytopathologists, biochemists, geneticists (about 20 researchers). Among the 54 new apple varieties created at the RRIFCB, only one variety Pamyat Voinu was created by one breeder and two varieties (Orlik and Orlovskoye Polosatoye) were created by the main author and one co-author. The rest of the varieties were created by three and more co-authors. Specialists of different specialties (interdisciplinary teams) are needed for successful breeding work. The success of breeding work also largely depends on the volume of the hybrid fund. With a large amount of material in the plant nursery and breeding orchard, a more rigorous selection for the desired characteristics is possible.

Allelic Diversity in Apple Germplasm for Fruit Quality and Scab Resistance Using SSR Markers

The present investigation entitled “Allelic diversity in apple germplasm for fruit quality and scab resistance using SSR markers” was conducted in division of plant biotechnology, SKUAST-K, Shalimar during the year 2018. In the present study, four SSR markers namely Md-ACS-1, Md-ACO-1, ALO-7 and AM-19 were used for screening of 40 apple cultivars presently under cultivation in SKUAST-K for quality and scab resistance. The results revealed that out of 40 cultivars evaluated, 3 were found homozygous (ACS-1-2/2), 17 were heterozygous (ACS-1-1/2) and 10 were homozygous (ACS-1-1/1), while as no amplification of ACS-1 was found for 10 cultivars. Similarly, for Md-ACO-1, 3 cultivars were found homozygous, 35 as heterozygous and 2 did not show any amplification. As far as presence of Vf gene conferring resistance against scab disease, ALO-7 amplified at 820bp and 570bp alleles, 570 bp fragment was observed in resistant genotypes and was absent in susceptible genotypes, while the 820 amplicon was observed in both resistant and susceptible genotypes. The markers AM19 led to the amplification of 520 bp fragment in few cultivars.

Working collection formation of apple-tree (Malus domestica Mill.) gene pool for priority breeding trends

Aim. To define the priority trends of apple breeding in the southern steppe of Ukraine and to identify varieties – sources of high valuable-for-breeding indices for building up a working collection. Results and Discussion. Spring frosts and wet weather in May-June contributing to development of the scab pathogen (Venturia inaequalis), high temperature and water deficit in the 2nd half of the growing period, when fruits grow and ripen, are the major stress weather/climatic factors for apple trees in the southern steppe of Ukraine. Therefore, the priority in breeding is given to developing varieties that would be resistant to several unfavorable factors. At the same time, commercial use of new varieties is possible provided high commercial quality indicators of fruits. High resistance of buds to spring frosts was observed in varieties Vechirnia Zoria, Moldavskoye Krasnoye, and Prima. Oligogenic varieties (genes Vm and Vf), including Harant, Skifske Zoloto, and Liberty, showed no signs of scab development. Varieties with polygenic resistance to this disease were identified; they included Vechirnia Zoria, Ornament, Carola. Drought-tolerant varieties with high water-holding capacity of leaves and their turgor restoration after wilting, including Carola, Florina, and Prima, were selected by a laboratory method. Assessment of drought tolerance in the field made it possible to enrich this group with numerous varieties. Varieties giving fruits with high commercial qualities on insufficient water availability, such as Vechirnia Zoria, Harant, Moldavskoye Krasnoye, Ornament and others, were distinguished. Varieties combining resistance to several unfavorable abiotic and biotic factors with high marketability traits of fruits were singled out. Among them. Harant, Delicious Spur, Liberty, and Prima should be mentioned. Conclusions. The development of varieties with complex tolerance to spring frosts, drought, scab pathogen and high qualities of fruits is the priority trend in the breeding of apple trees in the southern steppe of Ukraine. Varieties - sources of individual valuable traits and their various combinations were identified. Varieties Vechirnia Zoria, Moldavskoye Krasnoye, Ornament, and Golden Resistant combine the maximum number of valuable-for-breeding features. It is varieties-sources of several traits that should make up a working collection of apple trees to increase the breeding efficiency.

КОМПЛЕКСНЫЙ ПОДХОД В СОЗДАНИИ УСТОЙЧИВЫХ К ПАРШЕ ФОРМ ЯБЛОНИ: ФИТОПАТОЛОГИЧЕСКОЕ ТЕСТИРОВАНИЕ И МАРКЕР-ОПОСРЕДОВАННЫЙ ОТБОР

The creation of highly scab-resistant apple genotypes increases the profitability and environmental friendliness of the production of this fruit crop. Early stage evaluation of the resistance using phytopathological testing and DNA markers allows to accelerate the process of breeding for this trait allows the use. The purpose of the study was to comprehensively assess resistance to the causative agent of scab of hybrid seedlings of apple obtained from crossing susceptible (Rennet Simirenko) and resistant (Modi) cultivars for the Rvi6 gene, using infectious background and marker assisted selection. A phytopathological analysis of 207 hybrid apple seedlings against the natural background of apple scab pathogen revealed 117 (56%) plants without scab lesions (0 points). The remaining seedlings had lesions of varying degrees. DNA marker analysis of the Rvi6 gene allowed identification of 105 plants (51%) with the Rvi6rvi6 genotype and 102 (49%) – rvi6rvi6 , which is close to a theoretical segregation ratio 1:1 in the crosses of this type. Comparison of the results of infectious evaluation and DNA-marker analysis showed 98% coincidence of the presence of the resistance gene with no lesions. In general, the complex use of the phenotypic and molecular markers evaluation of the hybrid family for resistance to Venturia inaequalis (Cooke) G. Winter shows a high degree of agreement between the results of the methods. However, the discrepancy between the qualitative classes of reactions to infection and the results of molecular genetic analysis indicates an insufficient strength of the natural infectious background of the study year. The low efficiency of the development of the disease was due to the unfavorable weather and climatic conditions at the beginning of the vegetative period and the formation of infection, which were expressed in a higher average monthly temperature and a small amount of precipitation compared to the norm. Our results suggest an advantage, a comprehensive assessment of resistance to the scab pathogen using an infectious background and marker-assisted selection for the Vf gene ( Rvi6 ) in which the first stage involves the selection of resistant samples against a natural infectious background, followed by confirmation of the presence of the desired gene using a DNA marker analysis.

A comprehensive assessment of source material for apple breeding for immunity and fruit quality

The results of years of studying new and promising varieties of Apple trees (Malus domestica Borkh) grown in Southern Russia. Evaluation of Apple cultivars was carried out for resistance to the major fungal pathogens scab (Venturia inaequalis (Cooke) G. Winter) and powdery mildew (Podosphaera leucotricha Salm.), the quality and marketability of fruits, the content of biologically active substances underlying the antioxidant properties of fruits. An advanced method of polyploidy based on inclusion of parental forms with immunity to scab was applied in Apple selection. Integrated assessment of key biological traits of varieties and forms of Apple with diverse ploidy in unstable environments resulted in a selection of donors and sources of significant characteristics for Apple breeding such as resistance and immunity to major fungal pathogens, large fruit, bright red color of the fruit, improved chemical composition. High-quality varieties of Apple breeding in SKZNIISiV together with VNIISPK combining immunity to scab (the Vf gene) with high resistance to powdery mildew were selected: Fortune, Amulet, Carmen, Nika, Margo, Orfei, Souyz and others. Apple-tree grades, immune to scab, with a high viability of pollen (83–98%) were revealed: Azimuth, Vasilisa, Carmen, Lubava, Margo, Fortune, Liberty, Prima, Florina, Freedom. The promising parental forms passing to most of their hybrid progeny good tastes of fruits are Golden Delicious tetraploid, Wealthy tetraploid, Belle de Boskoop, Balsgard 0247 E, Papirovka tetraploid, and diploidy grades to Alkmene, Prima, Redfree and Kuban. Apple cultivars were revealed with high chemical indicators including the content of sugars: Orfei (10.8 %), Champion (10.5 %); vitamin C: Vasilisa and Souyz (more than 9.0 mg/100 g), hybrid form 44-24-49-uy (more than 11.4 mg/100 g); and P-active substances – Vasilisa, Lubava, Margo (more than 110.0 mg/100 g), whose use in breeding programs encourages the rapid creation of new genotypes with improved biochemical composition of fruits.

Cisgenic Rvi6 scab-resistant apple lines show no differences in Rvi6 transcription when compared with conventionally bred cultivars.

The expression of the apple scab resistance gene Rvi6 in different apple cultivars and lines is not modulated by biotic or abiotic factors. All commercially important apple cultivars are susceptible to Venturia inaequalis, the causal organism of apple scab. A limited number of apple cultivars were bred to express the resistance gene Vf from the wild apple genotype Malus floribunda 821. Positional cloning of the Vf locus allowed the identification of the Rvi6 (formerly HcrVf2) scab resistance gene that was subsequently used to generate cisgenic apple lines. It is important to understand and compare how this resistance gene is transcribed and modulated during infection in conventionally bred cultivars and in cisgenic lines. The aim of this work was to study the transcription pattern of Rvi6 in three classically bred apple cultivars and six lines of 'Gala' genetically modified to express Rvi6. Rvi6 transcription was analyzed at two time points using quantitative real-time PCR (RT-qPCR) following inoculation with V. inaequalis conidia or water. Rvi6 transcription was assessed in relation to five reference genes. β-Actin, RNAPol, and UBC were the most suited to performing RT-qPCR experiments on Malus × domestica. Inoculation with V. inaequalis conidia under conditions conducive to scab infection failed to produce any significant changes to the transcription level of Rvi6. Rvi6 expression levels were inconsistent in response to external treatments in the different apple cultivars, and transgenic, intragenic or cisgenic lines.

Suitability of old apple varieties in organic farming, based on their resistance against apple scab and powdery mildew

Apple scab and powdery mildew are the two most destructive fungal diseases of apple, especially in organic orchards, where synthetic pesticides are prohibited. Only a limited choice of natural substances for control of pests and diseases allowed to be used. Growing resistant/tolerant cultivars can highly reduce the number of the necessary plant protection treatments resulting in lower costs and environmental impact. Besides the newly developed cultivars, several old apple cultivars are thought to be resistant/tolerant to the main fungal diseases of apple; however, in most cases, the degree and type of their resistance is not well characterized. To evaluate the resistance of ten old cultivars against apple scab and powdery mildew, field evaluations were carried out for 6 years. Molecular marker analysis was also carried out: six different markers were used for the detection of three major scab resistance genes (Vf, Vh4, Vh2). The resistance gene Vf could not be found in any old cultivar while the resistance genes Vh2, Vh4 are possibly present in several cultivars. The results are explaining the good disease tolerance of several cultivars (e.g., ‘Batul’), and also suggest their relevance in the breeding as well as in organic farming. Some old cultivars showed good field resistance against fungal diseases, even though their resistance against scab could not be explained with the presence of the investigated major scab resistance genes. This suggests that a few cultivars might possess polygenic resistance, or maybe new major scab resistance genes.

Fine mapping of the gene Rvi18 (V25) for broad-spectrum resistance to apple scab, and development of a linked SSR marker suitable for marker-assisted breeding

Apple scab, caused by the fungal pathogen Venturia inaequalis, is one of the most devastating diseases for the apple growing industry in temperate zones with humid springs and summers. Breeding programs around the world have identified several sources of resistance, of which the Rvi6 (Vf) gene from Malus floribunda 821 has been the most widely used. The appearance of Rvi6-virulent strains of V. inaequalis in several European countries have underlined the necessity of pyramiding different effective resistance genes for durably resistant cultivars. Here we report the mapping of the new apple scab resistance gene Rvi18 (V25) from the selection 1980-015-025 of the apple breeding program at Wageningen University and Research Centre, The Netherlands. This gene was fine mapped on the proximal part of LG11 to a region of 34 Kb in the apple genome sequence of ‘Golden Delicious’, using 894 progeny plants, and SSR, DArT, AFLP, and SNP markers. One gene on the ‘Golden Delicious’ reference genome was identified as the potential susceptibility allele of the resistance gene. Moreover, an SSR marker has been developed of which one of its amplicons sizes is highly specific for Rvi18, thus facilitating the directed pyramiding of resistance genes through marker assisted breeding.

Apple-Tree Resistance to Abiotic Factors in Winter

Data on the reaction of apple cultivars to unfavorable autumn-winter conditions were analysed and summarised. New scab resistant apple cultivars (gene Vf) obtained from the hybridisation of local adapted varieties with a donor of scab immunity also displayed hardiness to unfavourable winter conditions. As a result of artificial freezing, the cultivars resistant to spring frost (-3.5 oC) during flowering were identified. The mechanism of adaptation to unfavourable winter conditions, and the possibility of preliminary prediction of winter hardiness according to some indicators of the physiological state of tissues in wintering trees were found. In all apple cultivars, increase of the concentrations of sucrose, anthocyanin and cyanidin was noted during the autumn-winter period, but in winter-hardy cultivars this process was more intensive; the amount of cyanidin in the bark of shoots was 6-8 times higher in comparison with non-winter-hardy cultivars. Higher water loss in non-winter-hardy cultivars was observed under conditions of extreme temperatures, which can lead to significant freezing and sunburns late in winter. In winter-hardy cultivars, peroxidase activity decreased in winter, while in the non-winter-hardy cultivars it remains high, due to incomplete hardening of tissues.