Deciphering the Mechanism of Tolerance to Apple Replant Disease Using a Genetic Mapping Approach in a Malling 9 × M. × robusta 5 Population Identifies SNP Markers Linked to Candidate Genes.

Apple replant disease (ARD) is a worldwide economic risk in apple production. Although several studies have shown that the wild apple accession Malus × robusta 5 (Mr5) is ARD-tolerant, the genetics of this tolerance have not yet been elucidated. A genetic mapping approach with a biparental population derived from contrasting parents involving molecular markers provides a means for marker-assisted selection of genetically complex traits and for determining candidate genes. In this study, we crossed the ARD-tolerant wild apple accession Mr5 and the ARD-susceptible rootstock 'M9' and analyzed the resultant progeny for ARD tolerance. Hence, a high-density genetic map using a tunable genotyping-by-sequencing (tGBS) approach was established. A total of 4804 SNPs together with 77 SSR markers were included in the parental maps comprising 17 linkage groups. The phenotypic responses to ARD were evaluated for 106 offspring and classified by an ARD-susceptibility index (ASI). A Kruskal-Wallis test identified SNP markers and one SSR marker on linkage groups (LG) 6 and 2 that correlated with ARD tolerance. We found nine candidate genes linked with these markers, which may be associated with plant response to ARD. These candidate genes provide some insight into the defense mechanisms against ARD and should be studied in more detail.

Apple Replant Disease: Causes and Mitigation Strategies.

After replanting apple (Malus domestica Borkh.) on the same site severe growth suppressions, and a decline in yield and fruit quality are observed in all apple producing areas worldwide. The causes of this complex phenomenon, called apple replant disease (ARD), are only poorly understood up to now which is in part due to inconsistencies in terms and methodologies. Therefore we suggest the following definition for ARD: ARD describes a harmfully disturbed physiological and morphological reaction of apple plants to soils that faced alterations in their (micro-) biome due to the previous apple cultures. The underlying interactions likely have multiple causes that extend beyond common analytical tools in microbial ecology. They are influenced by soil properties, faunal vectors, and trophic cascades, with genotype-specific effects on plant secondary metabolism, particularly phytoalexin biosynthesis. Yet, emerging tools allow to unravel the soil and rhizosphere (micro-) biome, to characterize alterations of habitat quality, and to decipher the plant reactions. Thereby, deep insights into the reactions taking place at the root rhizosphere interface will be gained. Counteractions are suggested, taking into account that culture management should emphasize on improving soil microbial and faunal diversity as well as habitat quality rather than focus on soil disinfection.

Genetic and Pomological Determination of the Trueness-to-Type of Sweet Cherry Cultivars in the German National Fruit Genebank.

Genebank collections preserve many old cultivars with ancient breeding history. However, often, cultivars with synonymous or incorrect names are maintained in multiple collections. Therefore, pomological and genetic characterization is an essential prerequisite for confirming trueness-to-type of cultivars in gene bank collections. In our study, 1442 single sweet cherry (Prunus avium L.) trees of the German Fruit Genebank were evaluated according to their trueness-to-type. For this purpose, pomological analysis was performed, in which the accessions were assigned totheir historical cultivar names. The pomological identifications were based on several historical reference sources, such as fruit references from historical cherry cultivar and fruit-stone collections, as well as historical pomological literature sources. In addition, the cherry trees were genetically analyzed for cultivar identity using 16 SSR markers. Based on pomological characterization and genetic analysis for the majority of the trees (86%), cultivar authenticity could be confirmed. Most markers were highly discriminating and powerful for cultivar identification. The cherry collection showed a high degree of genetic diversity, with an expected heterozygosity He = 0.67. Generally, high genetic admixture between cultivars of different geographic origin and year of origin was obtained after STRUCTURE analysis, demonstrating the extensive exchange of genetic information between cherry cultivars in the collection over time. However, the phylogenetic tree calculated by DARwin reflected the geographic origin of selected cherry cultivars. After parentage analysis with CERVUS, paternity could not be confirmed for three cultivars, indicating the necessity of further pedigree analysis for these cultivars. The results of our study underlined the general importance of evaluating the authenticity of cultivars in genebank collections based on genetic and pomological characterization.

Identification of Candidate Genes Associated With Tolerance to Apple Replant Disease by Genome-Wide Transcriptome Analysis.

Apple replant disease (ARD) is a worldwide economic risk in apple cultivation for fruit tree nurseries and fruit growers. Several studies on the reaction of apple plants to ARD are documented but less is known about the genetic mechanisms behind this symptomatology. RNA-seq analysis is a powerful tool for revealing candidate genes that are involved in the molecular responses to biotic stresses in plants. The aim of our work was to find differentially expressed genes in response to ARD in Malus. For this, we compared transcriptome data of the rootstock 'M9' (susceptible) and the wild apple genotype M. ×robusta 5 (Mr5, tolerant) after cultivation in ARD soil and disinfected ARD soil, respectively. When comparing apple plantlets grown in ARD soil to those grown in disinfected ARD soil, 1,206 differentially expressed genes (DEGs) were identified based on a log2 fold change, (LFC) ≥ 1 for up- and ≤ -1 for downregulation (p < 0.05). Subsequent validation revealed a highly significant positive correlation (r = 0.91; p < 0.0001) between RNA-seq and RT-qPCR results indicating a high reliability of the RNA-seq data. PageMan analysis showed that transcripts of genes involved in gibberellic acid (GA) biosynthesis were significantly enriched in the DEG dataset. Most of these GA biosynthesis genes were associated with functions in cell wall stabilization. Further genes were related to detoxification processes. Genes of both groups were expressed significantly higher in Mr5, suggesting that the lower susceptibility to ARD in Mr5 is not due to a single mechanism. These findings contribute to a better insight into ARD response in susceptible and tolerant apple genotypes. However, future research is needed to identify the defense mechanisms, which are most effective for the plant to overcome ARD.

Corrigendum: Genes Involved in Stress Response and Especially in Phytoalexin Biosynthesis Are Upregulated in Four Malus Genotypes in Response to Apple Replant Disease.

[This corrects the article DOI: 10.3389/fpls.2019.01724.].

Transferability of ISSR, SCoT and SSR Markers for Chrysanthemum × Morifolium Ramat and Genetic Relationships Among Commercial Russian Cultivars.

Characterization of genetic diversity in germplasm collections requires an efficient set of molecular markers. We assessed the efficiency of 36 new SCoT markers, 10 new ISSR markers, and 5 microsatellites for the characterization of genetic diversity in chrysanthemum core collection of 95 accessions (Russian and foreign cultivars). Seven new SCoT (SCoT12, 20, 21, 23, 29, 31, 34) and six new ISSR markers ((GA)8T, (CT)8G, (CTTCA)3, (GGAGA)3, (TC)8C, (CT)8TG) were efficient for the genetic diversity analysis in Chrysanthemum × morifolium collection. After STRUCTURE analysis, most Russian cultivars showed 20-50% of genetic admixtures of the foreign cultivars. Neighbor joining analysis based on the combination of SSR, ISSR, and SCoT data showed the best accordance with phenotype and origin compared to the separate analysis by each marker type. The position of the accessions within the phylogenetic tree corresponded with the origin and with some important traits, namely, plant height, stem and peduncle thickness, inflorescence type, composite flower and floret types, flower color, and disc color. In addition, several SCoT markers were suitable to separate the groups distinctly by the phenotypical traits such as plant height (SCoT29, SCoT34), thickness of the stem and peduncle (SCoT31, SCoT34), and leaf size and the floret type (SCoT31). These results provide new findings for the selection of markers associated with important traits in Chrysanthemum for trait-oriented breeding and germplasm characterization.

Genetic Diversity and Genome Size Variability in the Russian Genebank Collection of Tea Plant [Camellia sinensis (L). O. Kuntze].

The tea collection of the FRC SSC RAS (Sochi, Maykop in Russia) represents one of the northernmost germplasm comprising a number of locally derived cultivars and ɣ-irradiation mutants. The latter are often characterized by larger genome size, which may lead to better adaptation to biotic and abiotic stress. Such genotypes may be a valuable genetic resource for better adaptability to extreme environmental conditions, which could enable tea cultivation outside global growing regions. Microsatellite markers are often the best choice for genetic diversity analysis in genebank collections. However, their use in polyploid species is questionable because simple sequence repeat (SSR) allele dosage cannot be readily determined. Therefore, the efficiency of SSR and start codon targeted (SCoT) markers was investigated using 43 selected cultivars from the Russian genebank collection derived from mutant breeding and clonal selection. Previously, the increase in genome size was confirmed in 18 mutants within this collection. Despite the presence of polyploid tea genotypes, our study revealed higher efficiency of SSR markers than SCoT markers. Subsequent SSR analysis of the 106 genotypes in the Russian genebank collection revealed three distinct genetic clusters after STRUCTURE analysis. Greater genetic variation was observed within genetic clusters than between clusters, indicating low genetic variation between collections. Nevertheless, the northernmost tea collection exhibited a greater genetic distance from the other two clusters than they did from each other. Close genetic relationships were found between many cultivars with particularly large leaves and mutant forms. Pearson's correlation analysis revealed a significant, moderate correlation between genome size and leaf area size. Our study shows that microsatellite fingerprinting is useful to estimate the genetic diversity and genetic background of tea germplasm in Russia despite polyploid tea accessions. Thus, the results of our study contribute to the development of future tea germplasm conservation strategies and modern tea breeding programs.

Genetic Diversity in Diospyros Germplasm in the Western Caucasus Based on SSR and ISSR Polymorphism.

Persimmon germplasm in the Western Caucasus represent one of the most northerly collections. In our study, 51 commercial cultivars of D. kaki, 3 accessions of D. virginiana and 57 D. lotus accessions from six geographically distant populations were investigated using 19 microsatellite and 10 inter simple sequence repeat (ISSR) markers. After STRUCTURE analysis, the single accessions of Diospyros were allocated to three genetic clusters. Genetic admixtures in the important genotypes of D. kaki were revealed, whereas D. lotus accessions showed no admixture with other genetic clusters. The correspondence of genetic data and phenotypical traits was estimated in the D. kaki collection. The most frost tolerant genotypes of the collection, such as "Mountain Rogers", "Nikitskaya Bordovaya", "Rossiyanka", "MVG Omarova", "Meader", "Costata", "BBG", and "Jiro", showed a high percentage of genetic admixtures and were grouped close to D. virginiana. Some of these genotypes are known to be interspecific hybrids with D. virginiana. A low level of genetic diversity between the distant D. lotus populations was revealed and it can be speculated that D. lotus was introduced to the Western Caucasus from a single germplasm source. These results are an important basis for the implementation of conservation measures, developing breeding strategies, and improving breeding efficiency.

Genetic integrity is still maintained in natural populations of the indigenous wild apple species Malus sylvestris (Mill.) in Saxony as demonstrated with nuclear SSR and chloroplast DNA markers.

Malus sylvestris (Mill.) is the only indigenous wild apple species in Central Europe. Agriculture, forestry, and urbanization increasingly endanger Malus sylvestris natural habitats. In addition, the risks of cross-hybridization associated with increase in the cultivation of the domesticated apple Malus × domestica (Borkh.) threaten the genetic integrity of M. sylvestris. The present study investigated the number of hybrids, genetic diversity, and genetic structure of 292 putative M. sylvestris that originate from five different natural M. sylvestris populations in Saxony, Germany. All samples were genetically analyzed using nine nuclear microsatellite markers (ncSSR) and four maternally inherited chloroplast markers (cpDNA) along with 56 apple cultivars commonly cultivated in Saxony. Eighty-seven percent of the wild apple accessions were identified as pure M. sylvestris. The cpDNA analysis showed six private haplotypes for M. sylvestris, whereas three haplotypes were present in M. sylvestris and M. × domestica. The analysis of molecular variance (AMOVA) resulted in a moderate (ncSSR) and great (cpDNA) variation among pure M. sylvestris and M. × domestica individuals indicating a low gene flow between both species. The genetic diversity within the pure M. sylvestris populations was high with a weak genetic structure between the M. sylvestris populations indicating an unrestricted genetic exchange between these M. sylvestris populations. The clear distinguishing of M. sylvestris and M. ×domestica confirms our expectation of the existence of pure M. sylvestris accessions in this area and supports the argument for the implementation of preservation measures to protect the M. sylvestris populations in Saxony.

Diversity-oriented synthesis of 1-hydroxy-2,4-benzodioates by regioselective [3+3] cyclocondensations of 1,3-bis(silyloxy)-1,3-butadienes with 3-alkoxy- and 3-silyloxy-2-alkoxycarbonyl-2-en-1-ones.

1-Hydroxy-3,5-dimethyl-2,4-benzodioates (4-hydroxyisophthalates) were prepared by [3+3] cyclocondensation of 1,3-bis(silyloxy)-1,3-butadienes with 3-ethoxycarbonyl-4-trimethylsilyloxy-3-penten-2-one which is synthesized from (symmetrical) ethyl 2-acetylacetoacetate. The [3+3] cyclization of 1,3-bis(silyloxy)-1,3-butadienes with 3-alkoxy-2-alkoxycarbonyl-2-en-1-ones, readily available by reaction of beta-ketoesters with trialkyl orthoformiates, provide a convenient and regioselective approach to a great variety of 3-substituted 1-hydroxy-2,4-benzodioates that are not readily available by other methods.

Genes Involved in Stress Response and Especially in Phytoalexin Biosynthesis Are Upregulated in Four Malus Genotypes in Response to Apple Replant Disease.

Apple replant disease (ARD) is a soil-borne disease, which is of particular importance for fruit tree nurseries and fruit growers. The disease manifests by a poor vegetative development, stunted growth, and reduced yield in terms of quantity and quality, if apple plants (usually rootstocks) are replanted several times at the same site. Genotype-specific differences in the reaction of apple plants to ARD are documented, but less is known about the genetic mechanisms behind this symptomatology. Recent transcriptome analyses resulted in a number of candidate genes possibly involved in the plant response. In the present study, the expression of 108 selected candidate genes was investigated in root and leaf tissue of four different apple genotypes grown in untreated ARD soil and ARD soil disinfected by γ-irradiation originating from two different sites in Germany. Thirty-nine out of the 108 candidate genes were differentially expressed in roots by taking a p-value of < 0.05 and a fold change of > 1.5 as cutoff. Sixteen genes were more than 4.5-fold upregulated in roots of plants grown in ARD soil. The four genes MNL2 (putative mannosidase); ALF5 (multi antimicrobial extrusion protein); UGT73B4 (uridine diphosphate (UDP)-glycosyltransferase 73B4), and ECHI (chitin-binding) were significantly upregulated in roots. These genes seem to be related to the host plant response to ARD, although they have never been described in this context before. Six of the highly upregulated genes belong to the phytoalexin biosynthesis pathway. Their genotype-specific gene expression pattern was consistent with the phytoalexin content measured in roots. The biphenyl synthase (BIS) genes were found to be useful as early biomarkers for ARD, because their expression pattern correlated well with the phenotypic reaction of the Malus genotypes investigated.

Synthesis and solution structure of 3,5-dioxopimelic acid diesters--stable 1,3,5,7-tetracarbonyl derivatives.

A variety of 3,5-dioxopimelic acid diesters, stable 1,3,5,7-tetracarbonyl derivatives, were prepared by catalytic condensation of 1,3-bis(trimethylsilyloxy)-1,3-butadienes with methyl malonyl chloride. The keto-enol tautomerization of these compounds has been investigated by NMR spectroscopy. One keto and up to four enolic tautomers could be detected in chloroform solution and the influence of the substituents on the tautomeric equilibria has been studied.

Assessing gene flow in apple using a descendant of Malus sieversii var. sieversii f. niedzwetzkyana as an identifier for pollen dispersal.

The release of genetically engineered apple trees raises the question of their potential environmental impact, and the transfer via pollen of transgenes to cross-compatible cultivars of Malus domestica and Malus species is deemed to be the greatest source for environmental exposure. The hybrid TNR 31-35, a descendant of Malus sieversii var. sieversii f. niedzwetzkyana, carrying a homozygous, dominant gene responsible for red pigmentation in all plant parts, was used to assess gene flow in an apple scion repository of genetic resources. The red pigmentation provides a morphological marker that enables large-scale evaluation of cross-fertilization under natural conditions. In two consecutive years, 60 and then 56 apple trees of 38 different Malus domestica cultivars were selected to serve as pollen-receptor trees. In these two years, 6876 and then 5513 seeds, respectively, were gathered from pollen-receptor trees located at different distances, 2-100 m from 15 pollen-dispenser trees. In total, 11 797 seedlings were examined. An average of 1.8% and 1.4%, respectively, of all seedlings obtained showed red-colored leaves. Considering both years of sampling, 69% of the seeds fertilized by TNR 31-35 were found at less than 10 m from the nearest pollen-dispenser tree. Almost 91% of all seeds fertilized by TNR 31-35 were found at less than 60 m from the nearest pollen-dispenser tree, which is equal to 30 adjacent trees along the row. In this study, pollen was dispersed at least 104 m. After phenotypical evaluation, seedlings selected as red-colored were investigated by simple sequence repeat (SSR) analysis. Each seedling was tested with at least one heteromorphic SSR-marker, which allows the verification of TNR 31-35 as the male parent. All but four seedlings showed one allele specific for the appropriate fruiting tree and the second allele specific for the pollen-dispenser TNR 31-35.

Evaluation of the uniformity and stability of T-DNA integration and gene expression in transgenic apple plants

The generation of transgenic apple plants relies on the molecular analysis of transgene integration and expression based on polymerase chain reaction (PCR) analysis, blotting techniques and enzymatic assays on vitro leaves of putative transgenic regenerates. In order to assess the uniformity and the stability of transfer DNA (T-DNA) integration and gene expression, we studied 26 transgenic apple lines carrying the attacin E gene from Hyalophora cecropia, the beta-glucuronidase gene, and the nptII gene. Plants were evaluated using standard molecular techniques, such as PCR, Southern blot, reverse transcription PCR (RT-PCR) and Enzyme Linked Immunosorbent Assay (ELISA), and propagated in vitro on non-selective antibiotic-free media for four years to mimic natural conditions in the field. In some T-lines transgene integration and expression did not remain stable; differences were also found between distinct plants of a single T-line. Individual plants with partially or completely silenced transgenes were identified as well as plants with non-detectable T-DNA. Several lines appeared chimeric or partially silenced. Although most molecular techniques can reliably detect the presence of transgenic cells, they often fail to detect mixtures of transformed and non-transformed cells, or cells with silenced transgenes. This should be taken into consideration, especially in the case of vegetatively propagated trees, where non-transformed or silenced plant parts could mistakenly be used as propagation material.