The 35S ribosomal DNA (rDNA) units, repeated in tandem at one or more chromosomal loci, are separated by an intergenic spacer (IGS) containing functional elements involved in the regulation of ...transcription of downstream rRNA genes. In the present work, we have compared the IGS molecular organizations in two divergent species of Fagaceae, Fagus sylvatica and Quercus suber, aiming to comprehend the evolution of the IGS sequences within the family. Self- and cross-hybridization FISH was done on representative species of the Fagaceae. The IGS length variability and the methylation level of 18 and 25S rRNA genes were assessed in representatives of three genera of this family: Fagus, Quercus and Castanea. The intergenic spacers in Beech and Cork Oak showed similar overall organizations comprising putative functional elements needed for rRNA gene activity and containing a non-transcribed spacer (NTS), a promoter region, and a 5'-external transcribed spacer. In the NTS: the sub-repeats structure in Beech is more organized than in Cork Oak, sharing some short motifs which results in the lowest sequence similarity of the entire IGS; the AT-rich region differed in both spacers by a GC-rich block inserted in Cork Oak. The 5'-ETS is the region with the higher similarity, having nonetheless different lengths. FISH with the NTS-5'-ETS revealed fainter signals in cross-hybridization in agreement with the divergence between genera. The diversity of IGS lengths revealed variants from ∼ 2 kb in Fagus, and Quercus up to 5.3 kb in Castanea, and a lack of correlation between the number of variants and the number of rDNA loci in several species. Methylation of 25S Bam HI site was confirmed in all species and detected for the first time in the 18S of Q. suber and Q. faginea. These results provide important clues for the evolutionary trends of the rDNA 25S-18S IGS in the Fagaceae family.
The first genome of Vitis vinifera vinifera (PN40024), published in 2007, boosted grapevine related studies. While this reference genome is a suitable tool for the overall studies in the field, it ...lacks the ability to unveil changes accumulated during V. v. vinifera domestication. The subspecies V. v. sylvestris preserves wild characteristics, making it a good material to provide insights into V. v. vinifera domestication. The difference in the reproductive strategy between both subspecies is one of the characteristics that set them apart. While V. v. vinifera flowers are hermaphrodite, V. v. sylvestris is mostly dioecious. In this paper, we compare the re-sequencing of the genomes from a male and a female individual of the wild sylvestris, against the reference vinifera genome (PN40024). Variant analysis reveals a low number but with high impact modifications in coding regions, essentially non-synonymous single nucleotide polymorphisms and frame shifts caused by insertions and deletions. The sex-locus was manually inspected, and the results obtained are in line with the most recent works related with wild grapevine sex. In this paper we also describe for the first time RNA editing in transcripts of 14 genes in the sex-determining region, including VviYABBY and VviPLATZ.
Widespread agricultural losses attributed to drought, often combined with high temperatures, frequently occur in the field, namely in Mediterranean climate areas, where the existing scenarios for ...climate change indicate an increase in the frequency of heat waves and severe drought events in summer. Grapevine (
L.) is the most cultivated fruit species in the world and the most valuable one and is a traditional Mediterranean species. Currently, viticulture must adjust to impending climate changes that are already pushing vine-growers toward the use of ancient and resilient varieties. Portugal is very rich in grapevine biodiversity, however, currently, 90% of the total producing area is planted with only 16 varieties. There is a pressing need to understand the existing genetic diversity and the physiological potential of the varieties/genotypes available to be able to respond to climate changes. With the above scenario in mind, an assembly of 65 differentially expresses genes (DEGs) previously identified as responsive to abiotic stresses in two well studied genotypes, 'Touriga Nacional' and 'Trincadeira,' was designed to scan the gene expression of leaf samples from 10 traditional Portuguese varieties growing in two regions with distinct environmental conditions. Forty-five of those DEGs proved to be associated to "abiotic stress" and were chosen to build a custom qPCR array to identify uncharacterized genotypes as sensitive or tolerant to abiotic stress. According to the experimental set-up behind the array design these DEGs can also be used as indicators of the main abiotic stress that the plant is subjected and responding to (drought, heat, or excess light).
The most discriminating characteristic between the cultivated
subsp.
and the wild-form
subsp.
is their sexual system. Flowers of cultivars are mainly hermaphroditic, whereas wild plants have female ...and male individuals whose flowers follow a hermaphroditic pattern during early stages of development and later develop non-functional reproductive organs. In angiosperms, the basic developmental system for floral organ identity is explained by the ABCDE model. This model postulates that regulatory gene functions work in a combinatorial way to confer organ identity in each whorl. In wild
nothing is known about the function and expression profile of these genes. Here we show an overall view of the temporal and spatial expression pattern of the ABCDE genes as well as the pattern of
that establishes a boundary between the stamen and the carpel whorls, in the male, female and complete flower types. The results show a similar pattern in
species suggesting that the pathway leading to unisexuality acts independently and/or downstream of B- and C- function genes.
Polyploidization is a major evolutionary process in plants where hybridization and chromosome doubling induce enormous genomic stress and can generate genetic and epigenetic modifications. However, ...proper evaluation of DNA sequence restructuring events and the precise characterization of sequences involved are still sparse.
Inter Retrotransposons Amplified Polymorphism (IRAP), Retrotransposons Microsatellite Amplified Polymorphism (REMAP) and Inter Simple Sequence Repeat (ISSR) largely confirmed the absence of any intraspecific variation in wheat, rye and triticale. The comparative analysis of banding profiles between wheat and rye inbred lines revealed 34% of monomorphic (common to both parental species) bands for the ten different primer combinations used. The analysis of triticale plants uncovered nearly 51% of rearranged bands in the polyploid, being the majority of these modifications, due to the loss of rye bands (83%). Sequence analysis of rye fragments absent in triticale revealed for instance homology with hydroxyproline-rich glycoproteins (HRGP), a protein that belongs to a major family of inducible defence response proteins. Conversely, a wheat-specific band absent in triticale comprises a nested structure of copia-like retrotransposons elements, namely Claudia and Barbara. Sequencing of a polyploid-specific band (absent in both parents) revealed a microsatellite related sequence. Cytological studies using Fluorescent In Situ Hybridization (FISH) with REMAP products revealed a widespread distribution of retrotransposon and/or microsatellite flanking sequences on rye chromosomes, with a preferential accumulation in heterochromatic sub-telomeric domains.
Here, we used PCR-based molecular marker techniques involving retrotransposons and microsatellites to uncover polyploidization induced genetic restructuring in triticale. Sequence analysis of rearranged genomic fragments either from rye or wheat origin showed these to be retrotransposon-related as well as coding sequences. Further FISH analysis revealed possible chromosome hotspots for sequence rearrangements. The role of chromatin condensation on the origin of genomic rearrangements mediated by polyploidization in triticale is also discussed.
Grapevine is an extremely important crop worldwide. In southern Europe, post-flowering phases of the growth cycle can occur under high temperatures, excessive light, and drought conditions at soil ...and/or atmospheric level. In this study, we subjected greenhouse grown grapevine, variety Aragonez, to two individual abiotic stresses, water deficit stress (WDS), and heat stress (HS). The adaptation of plants to stress is a complex response triggered by cascades of molecular networks involved in stress perception, signal transduction, and the expression of specific stress-related genes and metabolites. Approaches such as array-based transcript profiling allow assessing the expression of thousands of genes in control and stress tissues. Using microarrays, we analyzed the leaf transcriptomic profile of the grapevine plants. Photosynthesis measurements verified that the plants were significantly affected by the stresses applied. Leaf gene expression was obtained using a high-throughput transcriptomic grapevine array, the 23K custom-made Affymetrix Vitis GeneChip. We identified 1,594 genes as differentially expressed between control and treatments and grouped them into ten major functional categories using MapMan software. The transcriptome of Aragonez was more significantly affected by HS when compared with WDS. The number of genes coding for heat-shock proteins and transcription factors expressed solely in response to HS suggesting their expression as unique signatures of HS. However, a cross-talk between the response pathways to both stresses was observed at the level of AP2/ERF transcription factors.
Somatic embryogenesis (SE) is a propagation tool of particular interest for accelerating the deployment of new high-performance planting stock in multivarietal forestry. However, genetic conformity ...in in vitro propagated plants should be assessed as early as possible, especially in long-living trees such as conifers. The main objective of this work was to study such conformity based on genetic stability at simple sequence repeat (SSR) loci during somatic embryogenesis in maritime pine (Pinus pinaster Ait.). Embryogenic cell lines (ECLs) subjected to tissue proliferation during 6, 14 or 22 months, as well as emblings regenerated from several ECLs, were analyzed. Genetic variation at seven SSR loci was detected in ECLs under proliferation conditions for all time points, and in 5 out of 52 emblings recovered from somatic embryos. Three of these five emblings showed an abnormal phenotype consisting mainly of plagiotropism and loss of apical dominance. Despite the variation found in somatic embryogenesis-derived plant material, no correlation was established between genetic stability at the analyzed loci and abnormal embling phenotype, present in 64% of the emblings. The use of microsatellites in this work was efficient for monitoring mutation events during the somatic embryogenesis in P. pinaster. These molecular markers should be useful in the implementation of new breeding and deployment strategies for improved trees using SE.
The sweet chestnut tree (Castanea sativa Mill.) is one of the most significant Mediterranean tree species, being an important natural resource for the wood and fruit industries. It is a monoecious ...species, presenting unisexual male catkins and bisexual catkins, with the latter having distinct male and female flowers. Despite the importance of the sweet chestnut tree, little is known regarding the molecular mechanisms involved in the determination of sexual organ identity. Thus, the study of how the different flowers of C. sativa develop is fundamental to understand the reproductive success of this species and the impact of flower phenology on its productivity. In this study, a C. sativa de novo transcriptome was assembled and the homologous genes to those of the ABCDE model for floral organ identity were identified. Expression analysis showed that the C. sativa B- and C-class genes are differentially expressed in the male flowers and female flowers. Yeast two-hybrid analysis also suggested that changes in the canonical ABCDE protein–protein interactions may underlie the mechanisms necessary to the development of separate male and female flowers, as reported for the monoecious Fagaceae Quercus suber. The results here depicted constitute a step towards the understanding of the molecular mechanisms involved in unisexual flower development in C. sativa, also suggesting that the ABCDE model for flower organ identity may be molecularly conserved in the predominantly monoecious Fagaceae family.
Vitis vinifera can be divided into two subspecies, V. vinifera subsp. vinifera, one of the most important agricultural crops in the world, and its wild ancestor, V. vinifera subsp. sylvestris. Three ...flower types can be observed: hermaphrodite and female (on some varieties) in vinifera, and male or female flowers in sylvestris. It is assumed that the different flower types in the wild ancestor arose through specific floral patterns of organ abortion. A considerable amount of data about the diversity of sexual systems in grapevines has been collected over the past century. Several grapevine breeding studies led to the hypothesis that dioecy in vinifera is derived from a hermaphrodite ancestor and could be controlled by either, one or two linked genetic determinants following Mendelian inherence. More recently, experiments using molecular approaches suggested that these loci were located in a specific region of the chromosome 2 of vinifera. Based on the works published so far, its seems evident that a putative sex locus is present in chromosome 2. However, it is still not fully elucidated whether flower types are regulated by two linked loci or by one locus with three alleles. Nevertheless, several genes could contribute to sex determination in grapevine. This review presents the results from early studies, combined with the recent molecular approaches, which may contribute to the design of new experiments towards a better understanding of the sex inheritance in grapevine.
Since the last decade of the last century, it is known that many old grapevine varieties are descendants of other varieties through natural crossing. Portugal has an important program for the ...conservation of representative samples of intra-varietal variability of all autochthonous varieties, managed by the Portuguese Association for Grapevine Diversity (PORVID), which makes looking for genotypes with dubious identification an important activity from a perspective of its valorisation. This communication presents the results of the molecular analysis of 5,000 samples (accessions) from the PORVID’s collection, using nine microsatellite
loci
currently recommended by the International Organization of Vine and Wine (OIV) for genetic grapevine identification. The results obtained confirmed the molecular identity of 4,220 samples corresponding to 214 varieties present in the official list of Portuguese varieties. In 780 samples, 95 profiles with a plural number of accessions revealed not to be listed in the Vitis International Variety Catalogue (VIVC) database, corresponding to possible varieties either descendent from natural crossing from at least one known parental variety, or from undetermined origin. Furthermore, the need for a comprehensive strategy aimed at uncovering other hidden varieties is discussed to prevent their imminent loss, deepen understanding of their origin, and add economic value and sustainability to the vine and wine sector.
PDF
