Genetics of resistance in apple against <Emphasis Type="Italic">Venturia inaequalis</Emphasis> (Wint.) Cke

Apple (Malus × domestica) is the third important fruit in terms of production and consumption worldwide. Apple scab caused by Venturia inaequalis is the most devastating disease of apple. In the apple-growing regions, many fungicides are sprayed to control the disease leading to increase in the production cost. Development of scab-resistant cultivars is the long-lasting solution to control the disease. In apples, more than 20 major scab resistance genes have been identified in various cultivars and few wild relatives. Of all these genes, Rvi6 derived from Malus floribunda has been most extensively used in different breeding programs. Gene for gene interactions of these resistance genes with the avirulence genes from V. inaequalis have been understood in many cases. QTL-based polygenic resistance has also been characterized in apple. Nucleotide Binding Site Leucine-Rich Repeats (NBS-LRR) have been identified from the apple genome and many of them have been characterized from the scab resistance region. Molecular markers associated with most of the major scab resistance genes have been identified and their position has been mapped on different linkage groups. Marker-assisted selection (MAS) can be helpful in speeding up and accurately identifying the scab-resistant parents and progeny. Pyramiding of several major resistance genes can be undertaken for more durable resistance against apple scab. The present paper reviews the Malus-Venturia pathosystem, current status of knowledge about scab resistance genes, and their application in breeding against apple scab.     

Biochemical and molecular studies on the resistance mechanisms in tea [<Emphasis Type="Italic">Camellia sinensis</Emphasis> (L.) O. Kuntze] against blister blight disease

Tea (Camellia sinensis) plantations are exposed to biotic and abiotic stresses. Among the biotic factors, blister blight (BB), caused by Exobasidium vexans, affects the quality and quantity of the product and demands high fungicide application. A long term solution for disease resistance would require the knowledge of the basic molecular and biochemical changes occurring in plant as an attempt to resist the pathogen and limit the spread of the disease which can further help in developing resistant cultivars using biotechnological tools. Thus, gene expression studies using the cDNA based suppressive subtractive hybridization library, characterization of genes for pathogenesis related (PR) proteins [chitinase (CsCHIT), glucanase (CsGLUC), phenylalanine ammonia lyase (CsPAL)] and genes in flavonoid pathway were accessed in the BB resistant and susceptible cultivars, SA6 and TES34, respectively. Further, biochemical analysis of PR and antioxidant enzymes (POX, APX, SOD) involved in BB resistance have been carried out to investigate the potential molecular and biochemical changes. Various stages of pathogen development had varied impact on PR protein, flavonoid pathway and anti-oxidative enzymes and indicates the possible role of reactive oxygen species, lignins, flavonoids, anthocyanins and other synthesized compounds in acting as antimicrobial/antifungal agents in tea cultivars.     

The isolation of the <Emphasis Type="Italic">IGT</Emphasis> family genes in <Emphasis Type="Italic">Malus × domestica</Emphasis> and their expressions in four idiotype apple cultivars

IGT family genes share the highly conserved motif GφL-(A/T) IGT in domain II and play an essential role in plant form. The tree architecture of apple (Malus ×?domestica Borkh.) affects fruit quality and yield. However, little information is available regarding IGT family genes in apple. Apple cultivars of four ideotypes (columnar, tip bearer, spur, and standard) were selected to characterize IGT family genes. Four IGT family members named MdoTAC1a, MdoTAC1b, MdoLAZY1, and MdoLAZY2 were found in the apple genome, sharing four conserved domains. In addition, MdoLAZY1 and MdoLAZY2 contain a fifth domain (EAR motif) at the C-terminus. There was no difference in the coding sequences of each gene in the four cultivars, but several mutated sites were found in their promoters. The four genes displayed lower expression levels in all tested tissues and organs of the columnar cultivar than in the other three cultivars, while expression levels of MdoTAC1a and MdoTAC1b in shoot tips and vegetative buds were highest in the standard cultivar, followed by spur, tip bearing, and columnar cultivars in decreasing order. These results indicate that IGT gene promoters are of great importance in the development of apple tree architecture and lay a theoretical basis for developing gene-specific markers for marker-assisted selection in breeding programs.     

Towards improvement of marker assisted selection of apple scab resistant cultivars: <Emphasis Type="Italic">Venturia inaequalis</Emphasis> virulence surveys and standardization of molecular marker alleles associated with resistance genes

Molecular breeding for pathogen resistance faces two major problems that delay its widespread adoption, resistance breakdown and difficulties in unambiguously identifying the alleles of the markers associated with specific resistance genes. Since the breakdown of the Rvi6 (Vf) gene in the Northern part of Europe breeders have intensified the search for new resistance sources to be introduced into their breeding programs. Alternative major genes to Rvi6 are available (e.g. Rvi2, Rvi4, Rvi5, Rvi10; Rvi11, Rvi12, Rvi13, and Rvi15, respectively Vh2, Vh4, Vm, Va, Vbj, Vb, Vd, Vr2 according to the old apple scab resistance gene nomenclature) but, with few exceptions (i.e., Rvi4, Rvi5 and, Rvi13), they have so far not been incorporated in commercial varieties. Pyramiding, i.e., combining several of these major resistance genes (R-genes) in individual plants, is one of the most promising strategies currently available to develop apple cultivars with durable apple scab resistance. But, which genes are the best suited to produce such new cultivars? Although the most interesting genes are surely those whose resistance so far has not been broken by the pathogen, genes with resistance that has been overcome coupled with only limited spread of the virulence may also be used in the pyramiding process. However, obtaining information on whether an R-gene is overcome and if so, the extent of the spread of the virulence is difficult and time consuming. Furthermore, often such reports are not up-to-date and the correctness of the data is difficult to verify. To solve these problems, the initiative “Monitoring of Venturia inaequalis virulences” has been proposed. The monitoring is based on a network of orchards of selected differential hosts. Incidence and severity of scab on these genotypes will be collected yearly; and after validation, the data will be published through the homepage of the project (www.vinquest.ch). Here, we present an outline of this initiative. A second major obstacle for broad adoption of marker assisted selection is the lack of tools to align marker analyzes performed in different laboratories to unambiguously identify the alleles linked to specific resistances. The identification of the alleles of the markers in coupling with the resistance genes is often very difficult, if the same genotype used to develop the markers is not simultaneously analyzed. In this paper we present an approach to standardize the size of the alleles in coupling with the resistance genes, using easily accessible cultivars. The proposed procedure has been applied to selected markers for the apple scab resistance genes Rvi2, Rvi4, Rvi5, Rvi6, Rvi11, Rvi12, Rvi13, Rvi14 and Rvi15 (respectively Vh2, Vh4, Vm, Vf, Vbj, Vb, Vd, Rvi14 and Vr2 according to the old nomenclature).     

Identification of a leucine-rich repeat receptor-like serine/threonine-protein kinase as a candidate gene for <Emphasis Type="Italic">Rvi12 (Vb)</Emphasis>-based apple scab resistance

Apple scab caused by Venturia inaequalis is the most important fungal disease of apples (Malus × domestica). Currently, the disease is controlled by up to 15 fungicide applications to the crop per year. Resistant apple cultivars will help promote the sustainable control of scab in commercial orchards. The breakdown of the Rvi6 (Vf) major-gene based resistance, the most used resistance gene in apple breeding, prompted the identification and characterization of new scab resistance genes. By using a large segregating population, the Rvi12 scab resistance gene was previously mapped to a genetic location flanked by molecular markers SNP_23.599 and SNP_24.482. Starting from these markers, utilizing chromosome walking of a Hansen’s baccata #2 (HB2) BAC-library; a single BAC clone spanning the Rvi12 interval was identified. Following Pacific Biosciences (PacBio) RS II sequencing and the use of the hierarchical genome assembly process (HGAP) assembly of the BAC clone sequence, the Rvi12 resistance locus was localized to a 62.3-kb genomic region. Gene prediction and in silico characterization identified a single candidate resistance gene. The gene, named here as Rvi12_Cd5, belongs to the LRR receptor-like serine/threonine-protein kinase family. In silico comparison of the resistance allele from HB2 and the susceptible allele from Golden Delicious (GD) identified the presence of an additional intron in the HB2 allele. Conserved domain analysis identified the presence of four additional LRR motifs in the susceptible allele compared to the resistance allele. The constitutive expression of Rvi12_Cd5 in HB2, together with its structural similarity to known resistance genes, makes it the most likely candidate for Rvi12 scab resistance in apple.     

RNA sequencing analysis provides new insights into dynamic molecular responses to <Emphasis Type="Italic">Valsa mali</Emphasis> pathogenicity in apple ‘Changfu No. 2’

Valsa canker caused by the necrotrophic pathogen Valsa mali (Vm) severely affects apple production in Eastern Asia. The molecular basis underlying the apple response to Vm infection is poorly understood. Hence, we performed RNA sequencing (RNA-seq) to investigate the dynamic gene expression profiles of a major apple cultivar, ‘Changfu No.2’, during Vm infection. Compared with the control (C), 104, 313, and 1059 differentially expressed genes (DEGs) were detected from the phloem tissue within the range of 0.9–1.3 cm (T1), 0.5–0.9 cm (T2), and 0.1–0.5 cm (T3) beyond the lesion periphery, respectively. Gene ontology (GO) enrichment analysis revealed that the DEGs associated with plant growth and development were down-regulated, whereas those related to defense responses were up-regulated. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that hormonal and Ca²⁺ signaling and phenylpropanoid biosynthesis were involved in the defense responses. In conclusion, multiple defense responses associated with ABA, JA, ET, Ca²⁺, and cell wall signals contributed to the defense against Vm infection in ‘Changfu No.2’. In contrast, the DEGs with inhibited expression were involved in plant growth and development; auxin signaling and several resistance genes might weaken the resistance of ‘Changfu No.2’ to pathogens. Our results offer a new insight into plant responses against necrotrophs and could benefit programs aimed at breeding for Vm resistance.     

Frequency of a natural truncated allele of <Emphasis Type="Italic">MdMLO19</Emphasis> in the germplasm of <Emphasis Type="Italic">Malus domestica</Emphasis>

Podosphaera leucotricha is the causal agent of powdery mildew (PM) in apple. To reduce the amount of fungicides required to control this pathogen, the development of resistant apple cultivars should become a priority. Resistance to PM was achieved in various crops by knocking out specific members of the MLO gene family that are responsible for PM susceptibility (S-genes). In apple, the knockdown of MdMLO19 resulted in PM resistance. However, since gene silencing technologies such as RNAi are perceived unfavorably in Europe, a different approach that exploits this type of resistance is needed. This work evaluates the presence of non-functional naturally occurring alleles of MdMLO19 in apple germplasm. The screening of the re-sequencing data of 63 apple individuals led to the identification of 627 single nucleotide polymorphisms (SNPs) in five MLO genes (MdMLO5, MdMLO7, MdMLO11, MdMLO18, and MdMLO19), 127 of which were located in exons. The T-1201 insertion of a single nucleotide in MdMLO19 caused the formation of an early stop codon, resulting in a truncated protein lacking 185 amino acids, including the calmodulin-binding domain. The presence of the insertion was evaluated in 115 individuals. It was heterozygous in 64 and homozygous in 25. Twelve of the 25 individuals carrying the insertion in homozygosity were susceptible to PM. After barley, pea, cucumber, and tomato, apple would be the fifth species for which a natural non-functional mlo allele has been found.     

Fine mapping of the rosy apple aphid resistance locus <Emphasis Type="Italic">Dp-fl</Emphasis> on linkage group 8 of the apple cultivar ‘Florina’

Rosy apple aphid (Dysaphis plantaginea), is one of the major insect pests of apple, causing serious physical and economic damage to fruit production. A dominant resistance gene Dp-fl was previously mapped at the bottom of linkage group LG8 from the cultivar ‘Florina’, linked to the SSR CH01h10. The development of additional genetic markers mapping closer to Dp-fl was needed to position the gene accurately and to improve the effectiveness of marker-assisted breeding (MAB). The aims of this study were to identify single nucleotide polymorphisms (SNPs) in the region of Dp-fl and to position these SNPs relative to Dp-fl. To generate a fine map of the Dp-fl interval, a total of 191 plants segregating for resistance and derived from four different populations were tested with temperature-switch PCR (TSP) markers developed for SNPs located in the region of CH01h10. All the plants were phenotypically evaluated for aphid resistance and those data compared with the genetic data. These efforts resulted in positioning the Dp-fl resistance locus in a genetic interval corresponding to a physical distance of about 330 kb on the ‘Golden Delicious’ genome. The new markers were tested on several apple founder cultivars in order to test the specificity of the SNPs and, thus, the best markers for the MAB were identified. Finally, the 330-kb interval was analyzed for the identification of coding sequences and putative candidate genes for D. plantaginea resistance were identified.     

Combination of newly developed SNP and InDel markers for genotyping the <Emphasis Type="Italic">Cf-9</Emphasis> locus conferring disease resistance to leaf mold disease in the tomato

The Cf-9 gene in the tomato is known to confer resistance against leaf mold disease caused by Cladosporium fulvum, and a gene-based marker targeted to the Cf-9 allele has been widely used as a crop protection approach. However, we found this marker to be misleading in genotyping. Therefore, we developed new single-nucleotide polymorphism (SNP) and insertion and deletion (InDel) markers targeted to the Cf-9 allele in order to increase genotyping accuracy and facilitate high-throughput screening. The DNA sequences of reported Cf-9, cf-9, Cf-0, and closely related Cf-4 alleles were compared, and two functional and non-synonymous SNPs were found to distinguish the Cf-9 resistance allele from the cf-9, Cf-0, and Cf-4 alleles. An SNP marker including these two SNPs was developed and applied to the genotyping of 33 tomato cultivars by high-resolution melting analysis. Our SNP marker was able to select all three Cf-9 genotypes (resistant, heterozygous, and susceptible alleles). Interestingly, two cultivars were grouped separately from these three genotypes. To further examine this outgroup, we preformed polymerase chain reaction (PCR) on two InDel regions identified by sequence comparison of the Cf-9 and Cf-4 genes. The band patterns revealed that these two cultivars carried Cf-4 rather than Cf-9 alleles and that three cultivars classified in the Cf-9 resistance group actually carried both Cf-9 and Cf-4 genes. To determine whether these genotyping results were consistent with disease resistance phenotypes, we examined the induction of a hypersensitive response by transiently expressing the corresponding effector genes, and found that the results matched perfectly with the genotyping results. These findings indicate that the combination of our SNP and InDel markers allows resistant Cf-9 alleles to be distinguished from cf-9 and Cf-4 alleles, which will be useful for marker-assisted selection of tomato cultivars resistant to C. fulvum.     

Selection of reliable reference genes for RT-qPCR during methyl jasmonate,salicylic acid and hydrogen peroxide treatments in <Emphasis Type="Italic">Ganoderma lucidum</Emphasis>

This study aimed to identify suitable reference genes under three chemical inducers, methyl jasmonate (MeJA), salicylic acid (SA) and hydrogen peroxide (H₂O₂) in Ganoderma lucidum. In this study, expression stabilities of 14 candidate reference genes had been validated. Four algorithms were used: geNorm, NormFinder, BestKeeper, and RefFinder. Our results showed that, in short time, UCE2 (ubiquitin conjugating enzyme) was the most stable gene both in MeJA and H₂O₂ treatments, ACTIN (beta-actin) was the most suitable reference gene for SA treatment. ACTIN/UCE2 were considered the most suitable genes to normalize in MeJA, SA and H₂O₂ conditions. In long time, PP2A (protein phosphatase 2A regulatory subunit) was the most stable gene in MeJA and SA treatments, UCE2 was the most suitable reference gene for H₂O₂ treatment. PP2A/UBQ1 (polyubiquitin 1) were considered the most suitable genes to normalize in MeJA, SA and H₂O₂ conditions. Furthermore, target gene, oxidosqualene cyclase (osc), was selected to validate the most and least stable reference genes under different treatments. Our work provided a better support to study the regulatory mechanism of MeJA, SA and H₂O₂ on biological functions.