Population Analysis of Diospyros lotus in the Northwestern Caucasus Based on Leaf Morphology and Multilocus DNA Markers.

Diospyros lotus is the one of the most frost-tolerant species in the Diospyros genera, used as a rootstock for colder regions. Natural populations of D. lotus have a fragmented character of distribution in the Northwestern Caucasus, one of the coldest regions of Diospyros cultivation. To predict the behavior of D. lotus populations in an extreme environment, it is necessary to investigate the intraspecific genetic diversity and phenotypic variability of populations in the colder regions. In this study, we analyzed five geographically distant populations of D. lotus according to 33 morphological leaf traits, and the most informative traits were established, namely, leaf length, leaf width, leaf index (leaf to length ratio) and the length of the fourth veins. Additionally, we evaluated the intraspecific genetic diversity of D. lotus using ISSR and SCoT markers and proposed a new parameter for the evaluation of genetic polymorphism among populations, in order to eliminate the effect of sample number. This new parameter is the relative genetic polymorphism, which is the ratio of polymorphism to the number of samples. Based on morphological and genetic data, the northernmost population from Shkhafit was phenotypically and genetically distant from the other populations. The correspondence between several morphological traits (leaf width, leaf length and first to fifth right vein angles) and several marker bands (SCoT5, SCoT7, SCoT30: 800-1500 bp; ISSR13, ISSR14, ISSR880: 500-1000 bp) were observed for the Shkhafit population. Unique SCoT and ISSR fragments can be used as markers for breeding purposes. The results provide a better understanding of adaptive mechanisms in D. lotus in extreme environments and will be important for the further expansion of the cultivation area for persimmon in colder regions.

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.

Biochemical and Genetic Responses of Tea (Camellia sinensis (L.) Kuntze) Microplants under Mannitol-Induced Osmotic Stress In Vitro.

Osmotic stress is a major factor reducing the growth and yield of many horticultural crops worldwide. To reveal reliable markers of tolerant genotypes, we need a comprehensive understanding of the responsive mechanisms in crops. In vitro stress induction can be an efficient tool to study the mechanisms of responses in plants to help gain a better understanding of the physiological and genetic responses of plant tissues against each stress factor. In the present study, the osmotic stress was induced by addition of mannitol into the culture media to reveal biochemical and genetic responses of tea microplants. The contents of proline, threonine, epigallocatechin, and epigallocatechin gallate were increased in leaves during mannitol treatment. The expression level of several genes, namely DHN2, LOX1, LOX6, BAM, SUS1, TPS11, RS1, RS2, and SnRK1.3, was elevated by 2-10 times under mannitol-induced osmotic stress, while the expression of many other stress-related genes was not changed significantly. Surprisingly, down-regulation of the following genes, viz. bHLH12, bHLH7, bHLH21, bHLH43, CBF1, WRKY2, SWEET1, SWEET2, SWEET3, INV5, and LOX7, was observed. During this study, two major groups of highly correlated genes were observed. The first group included seven genes, namely CBF1, DHN3, HXK2,SnRK1.1, SPS, SWEET3, and SWEET1. The second group comprised eight genes, viz. DHN2, SnRK1.3, HXK3, RS1, RS2,LOX6, SUS4, and BAM5. A high level of correlation indicates the high strength connection of the genes which can be co-expressed or can be linked to the joint regulons. The present study demonstrates that tea plants develop several adaptations to cope under osmotic stress in vitro; however, some important stress-related genes were silent or downregulated in microplants.

Physiological, biochemical and genetic responses of Caucasian tea (Camellia sinensis (L.) Kuntze) genotypes under cold and frost stress.

BACKGROUND: Cold and frost are two serious factors limiting the yield of many crops worldwide, including the tea plant (Camellia sinensis (L.) Kuntze). The acclimatization of tea plant from tropical to temperate climate regions resulted in unique germplasm in the North-Western Caucasus with extremely frost-tolerant genotypes. METHODS: The aim of the current research was to evaluate the physiological, biochemical and genetic responses of tolerant and sensitive tea cultivars exposed to cold (0 to +2 °C for 7 days) and frost (-6 to -8 °C for 5 days). Relative water content, cell membranes integrity, pH of the cell sap, water soluble protein, cations, sugars, amino acids were measured under cold and frost. Comparative expression of the following genes ICE1, CBF1, WRKY2, DHN1, DHN2, DHN3, NAC17, NAC26, NAC30, SnRK1.1, SnRK1.2, SnRK1.3, bHLH7, bHLH43, P5CS, LOX1, LOX6, LOX7 were analyzed. RESULTS: We found elevated protein (by 3-4 times) and cations (potassium, calcium and magnesium) contents in the leaves of both cultivars under cold and frost treatments. Meanwhile, Leu, Met, Val, Thr, Ser were increased under cold and frost, however tolerant cv. Gruzinskii7 showed earlier accumulation of these amino acids. Out of 18 studied genes, 11 were expressed at greater level in the frost- tolerant cultivar comparing with frost-sensitive one: ICE1, CBF1, WRKY2, DHN2, NAC17, NAC26, SnRK1.1, SnRK1.3, bHLH43, P5CS and LOX6. Positive correlations between certain amino acids namely, Met, Thr, Leu and Ser and studied genes were found. Taken together, the revealed cold responses in Caucasian tea cultivars help better understanding of tea tolerance to low temperature stress and role of revealed metabolites need to be further evaluated in different tea genotypes.