Cytoembryological and morphometric characteristics of some Armenian grape cultivars

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Armenian grapevines: cytoembryological, morphological and chemical analysis

The objectives of our study was to phenotype Armenian grapevines on the base of cytoembryological, morphometric and phenolic content analysis of 10 wine and 10 table cultivars (Vitis vinifera L.). The presented results suggest that Armenian table grape cultivars have higher level of abnormalities in the process of seed formation than wine cultivars. However, during ovule development, the observed differences between table and wine varieties were not significant. The berry morphometric analysis demonstrated that table cultivars formed significantly larger and heavier berries than wine cultivars. The obtained results show that wine grapes contained significantly higher phenol concentrations than table grapes, in both colored and white grapevine cultivars. The study of phenol composition, an important chemical descriptor in grapevine phenotyping, provides oenological information useful to improve the wine quality. Cultivar characterization could be used as marker for the selection of table and wine grape breeding programs. These results will be upgraded in the database for Armenian grapevine varieties. In future Armenian germplasm will be compared with datasets of neighboring countries, to determine the varietal origins relationships

Description of the Vitis vinifera L. phenotypic variability in eno-carpological traits by a Euro-Asiatic collaborative network among ampelographic collections

The grapevine intra-specific variability captured an increasing interest during the last decades, as demonstrated by the number of recently funded European projects focused on the grapevine biodiversity preservation. However, nowadays, crop plants are mainly characterized by genotyping methods. The present work summarizes the phenotype data collected among 20 ampelographic collections spread in 15 countries, covering mostly of the viticultural areas in the Euro-Asiatic range: from Portugal to Armenia and from Cyprus to Luxembourg. Together with agro-climatic characterization of the experimental site, in two years, about 2400 accessions were described, following a common experimental protocol mainly focused on the carpological and oenological traits, obtaining a general overview of the distribution of the considered phenotypic traits in the cultivated Vitis vinifera species. The most replicated cultivars were selected and, for the subset of these reference cultivars, their behavior in the different environmental conditions over sites and years was described by ANOVA methods

Description of the vitis vinifera L. Phenotypic variability in eno-carpological traits by a Euro-Asiatic collaborative network among ampelographic collections

The grapevine intra-specific variability captured an increasing interest during the last decades, as demonstrated by the number of recently funded European projects focused on the grapevine biodiversity preservation. However, nowadays, crop plants are mainly characterized by genotyping methods. The present work summarizes the phenotype data collected among 20 ampelographic collections spread over 15 countries, covering most of the viticultural areas in the Euro-Asiatic region: from Portugal to Armenia and from Cyprus to Luxembourg. Together with agro-climatic characterization of the experimental site, over two years about 2,400 accessions were described. A common experimental protocol mainly focused on the carpological and oe-nological traits was followed, obtaining a general overview of the distribution of the considered phenotypic traits in the cultivated Vitis vinifera species. The most replicated cultivars were selected and, for the subset of these reference cultivars, their behavior in the different environmental conditions over sites and years was described by ANOVA methods

An optimized histological proceeding to study the female gametophyte development in grapevine

Background: Reproductive success in seed plants depends on a healthy fruit and seed set. Normal seed development in the angiosperms requires the production of functional female gametophytes. This is particularly evident in seedless cultivars where defects during megagametophyte’s developmental processes have been observed through cytohistological analysis. Several protocols for embryo sac histological analyses in grapevine are reported in literature, mainly based on resin- or paraffin-embedding approaches. However their description is not always fully exhaustive and sometimes they consist of long and laborious steps. The use of different stains is also documented, some of them, such as hematoxylin, requiring long oxidation periods of the dye-solution before using it (from 2 to 6 months) and/or with a differentiation step not easy to handle. Paraffin-embedding associated to examination with light microscope is the simplest methodology, and with less requirements in terms of expertise and costs, achieving a satisfactory resolution for basic histological observations. Safranin O and fast green FCF is an easy staining combination that has been applied in embryological studies of several plant species. Results: Here we describe in detail a paraffin-embedding method for the examination of grapevine ovules at different phenological stages. The histological sample preparation process takes 1 day and a half. Sections of 5 μm thickness can be obtained and good contrast is achieved with the safranin O and fast green FCF staining combination. The method allows the observation of megasporogenesis and megagametogenesis events in the different phenological stages examined. Conclusions: The histological sample preparation process proposed here can be used as a routine procedure to obtain embedded ovaries or microscope slides that would require further steps for examination. We suggest the tested staining combination as a simple and viable technique for basic screenings about the presence in grapevine of a normally and fully developed ovule with embryo sac cells, which is therefore potentially functional

An optimized paraffin-embedding method to study embryo sac development in grapevine

Pollination and fertilization are crucial stages in grape development affecting fruit set and, consequently, potential yield. The achievement of these two processes is linked to the successful completion of the events involved in the production of functional male (pollen) and female (embryo sac) gametophytes. This is particularly evident in seedless cultivars where defects during female gametophyte developmental processes have been observed through cytohistological analysis. Plant histological processing includes a wide variety of methods and techniques that can be used in different combinations to achieve the desired result. Various methodologies can be found in literature to study the development of the grapevine female gametophyte, mainly based on resin- or paraffin-embedding techniques. Unlike resin-based techniques, paraffin-embedding approaches contain the costs and, despite that the achieved resolution is not so high, can be an efficient tool if there is no need to observe detailed structure of tissues, cells, organelles and macromolecular complexes. On the other hand, the protocols in the published papers are not always fully exhaustive, they report very long incubation times and also different stains, some of them requiring long ripening periods and/or not easy to handle. Here we present a paraffin-embedding method for the examination of grapevine ovules at different phenological stages. The incubation times at each step (fixation, dehydration, clearing, infiltration, and staining) have been optimized in order to reduce the duration of the entire process. The staining combination used, as far as we know, has never been reported for grapevine’s ovules. Good quality images with adequate contrast were obtained with this methodology allowing the observation of inner and outer integuments, nucellus and embryo sac structures

Developmental, transcriptome, and genetic alterations associated with parthenocarpy in the grapevine seedless somatic variant Corinto bianco

Seedlessness is a relevant trait in grapevine cultivars intended for fresh consumption or raisin production. Previous DNA marker analysis indicated that Corinto bianco (CB) is a parthenocarpic somatic variant of the seeded cultivar Pedro Ximenes (PX). This study compared both variant lines to determine the basis of this parthenocarpic phenotype. At maturity, CB seedless berries were 6-fold smaller than PX berries. The macrogametophyte was absent from CB ovules, and CB was also pollen sterile. Occasionally, one seed developed in 1.6% of CB berries. Microsatellite genotyping and flow cytometry analyses of seedlings generated from these seeds showed that most CB viable seeds were formed by fertilization of unreduced gametes generated by meiotic diplospory, a process that has not been described previously in grapevine. Microarray and RNA-sequencing analyses identified 1958 genes that were differentially expressed between CB and PX developing flowers. Genes downregulated in CB were enriched in gametophyte-preferentially expressed transcripts, indicating the absence of regular post-meiotic germline development in CB. RNA-sequencing was also used for genetic variant calling and 14 single-nucleotide polymorphisms distinguishing the CB and PX variant lines were detected. Among these, CB-specific polymorphisms were considered as candidate parthenocarpy-responsible mutations, including a putative deleterious substitution in a HAL2-like protein. Collectively, these results revealed that the absence of a mature macrogametophyte, probably due to meiosis arrest, coupled with a process of fertilization-independent fruit growth, caused parthenocarpy in CB. This study provides a number of grapevine parthenocarpy-responsible candidate genes and shows how genomic approaches can shed light on the genetic origin of woody crop somatic variants. © 2015 The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved

Caracterización genética y de caracteres reproductivos en variedades de vid sin semilla de Armenia

La apirenia de la mayoría de las variedades comerciales de la vid (Vitis vinifera L.)procede de 'Sultanina', una variedad con origen en Asia Menor. El principal objetivo deeste trabajo ha sido la caracterización de posibles fuentes alternativas de apirenia en elgermoplasma armenio. Se han estudiado 40 accesiones apirenas de las coleccionesarmenias de vid en Echmiadzin (ARM006) y en Nalbandyan (ARM011), así como deexplotaciones privadas de la región de Armavir (Armenia). El análisis fenotípico debayas se realizó de acuerdo con los descriptores de la OIV, y el análisis genéticomediante el estudio de la mutación causal de apirenia en Sultanina en el gen VviAGL11y del marcador VviAPT3 ligado al locus del sexo. El análisis de viabilidad y morfologíade los granos de polen se visualizó por microscopía óptica y electrónica de barrido. Elanálisis fenotípico de bayas reveló una amplia variación en el peso de las mismas, asícomo en la formación de rudimentos seminales. Las flores de nueve cultivares sonhermafroditas con un alto nivel de viabilidad del polen. La accesión 'Karmir kishmish'se caracterizó por tener flores funcionalmente femeninas con baja viabilidad de polen yse confirmó genéticamente con VviAPT3. El análisis de microscopía mostró que losgranos de polen de las flores hermafroditas tienen forma esferoidal con 3 colporacionesy numerosas perforaciones, mientras que el de la variedad 'Karmir kishmish' estambién esferoidal, pero acolporado y con menos perforaciones. El análisis genéticoreveló que todas las accesiones portan la mutación puntual dominante en VviAGL11 quecausa la estenospermocarpia en 'Sultanina'. De hecho, el análisis de 7 marcadores SSRy 48 SNPs demostró que todas son descendientes de la misma. Este estudio confirmaque las variedades apirenas armenias descienden de 'Sultanina', y motiva la búsquedade otros determinantes genéticos que causen variación en el contenido de semillas de lasuvas para utilizar como fuentes alternativas en programas de mejora de uva de mesa

Identification and characterization of grapevine Genetic Resources maintained in Eastern European collections

The Near East and the Caucasus regions are considered as gene and domestication centre for grapevine. In an earlier project "Conservation and Sustainable Use of Grapevine Genetic Resources in the Caucasus and Northern Black Sea Region" (2003-2007) it turned out that 2,654 accessions from autochthonous cultivars maintained by Armenia, Azerbaijan, Georgia, Moldova, Russian Federation and Ukraine in ten grapevine collections may belong to 1,283 cultivars. But trueness to type assessment by morphology and genetic fingerprinting still needed to be done. In COST Action FA1003 a first step in that direction was initiated. The following countries participated: Albania, Armenia, Austria, Azerbaijan, Bulgaria, Croatia, Georgia, Hungary, Latvia, Moldova, Romania, Slovakia, Slovenia and Ukraine. Mainly Vitis vinifera accessions (1098 samples) and 76 Vitis sylvestris individuals were analyzed by nine SSR-markers (VVS2, VVMD5, VVMD7, VVMD25, VVMD27, VVMD28, VVMD32, VrZag62, VrZag79). Cultivar identity confirmation/rejection was attempted for 306 genotypes/cultivars by comparison of the generated genetic profiles with international SSR-marker databases and ampelographic studies. The outcome proved unambiguously the necessity of morphologic description and photos (a) for comparison with bibliography, (b) for a clear and explicit definition of the cultivar and (c) the detection of sampling errors and misnomers. From the 1,098 analyzed accessions, 997 turned out to be indigenous to the participating countries. The remaining 101 accessions were Western European cultivars. The 997 fingerprints of indigenous accessions resulted in 658 unique profiles/cultivars. From these 353 (54 %) are only maintained in the countries of origin and 300 (46 %) unique genotypes exist only once in the Eastern European collections. For these 300 genotypes duplicate preservation needs to be initiated. In addition, the high ratio of non redundant genetic material of Eastern European origin suggests an immense unexplored diversity. Documentation of the entire information in the European Vitis Database will assist both germplasm maintenance and documentation of cultivar specific data